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1
Hazazi, Ali, Mohammed Albayedh, Fawaz Albloui, and Mishal Alsulami. "Overview of Molecular Quantification of the BCR-ABL Oncogene in CML Patients." Biosciences Biotechnology Research Asia 19, no. 3 (September 29, 2022): 693–98. http://dx.doi.org/10.13005/bbra/3021.
Full textAbstract:
Chronic myeloid leukemia (CML) is considered a common blood cancers and accounts for approximately 15–20% of the total cases of leukemia. Recent studies indicated that above 95% of patients suffering of CML have been found with a distinctive Philadelphia chromosome that originates from a mutual translocation between both arms of chromosomes 9 and 22. During this mutation the translocation of the ABL gene located on chromosome 9 get transferred to the breakpoint cluster region (BCR) of chromosome 22 as an effect of a joined BCR-ABL gene. Furthermore, BCR-ABL oncogene is characteristically found in CML, causing cells to divide uncontrollably and inducing severe consequences among CML patients. In line with this, applying quantification technique of the BCR-ABL gene using molecular approaches is crucial for patient controlling, initiation of the proper treatment, measurement of response to therapy, and prediction of relapse. Of greater significance, molecular assay and monitoring of the BCR-ABL gene in CML using quantitative RT-PCR provides physicians with essential diagnostic and prognostic information.
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Garuti, Anna, Adalberto Ibatici, Gabriella Cirmena, Maurizio Miglino, Riccardo Varaldo, Colombo Nicoletta, Roberta Grasso, et al. "The Persistence of p190 BCR-ABL Transcripts Is Associated with Lower Probability of Molecular Response to Imatinib in Early and Late Chronic Phase CML Patients." Blood 106, no. 11 (November 16, 2005): 3282. http://dx.doi.org/10.1182/blood.v106.11.3282.3282.
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Abstract Background. It has been demonstrated that about 70% of patients with CML in chronic phase (CP) at diagnosis co-expressed p210 and p190 BCR/ABL transcripts, although at a much lower level (Blood1996;87:5213–17). In previous studies, the co-expression of p210 and p190 BCR-ABL transcripts at diagnosis was considered as indicative of higher tumor burden. However, the clinical relevance of p190 BCR-ABL mRNA monitoring in CML pts under Imatinib on bone marrow (BM) samples is not known. Materials and Methods. BM samples were obtained from 83 pts with CP-CML treated with Imatinib at a daily oral dose ranging between 300–500mg. These included 192 samples from 43 pts with late CP-CML (post-IFN failure) and 140 samples from 40 pts with early CP-CML who received Imatinib as first line therapy. Median follow-up was 18 (3–58) and 39 (12–58) months for early and late CP-CML, respectively. As part of a diagnostic work-up, BM samples from each patient were assessed for expression of both p210 and p190 BCR/ABL levels by real-time quantitative reverse transcription PCR (QRT-PCR) using a TAQ-Man system (ABI Prism 7700 Perkin Elmer) for BCR-ABL and ABL genes. The median number of BM assessment was 3 (2–6) for early CP-CML and 4 (2–10) for late CP-CML. A major molecular response (MMR) was defined as BCR-ABL/ABL ratios less than 0.05%. A specific nested RT-PCR screening was assessed for detection of p210 (b2a2, b3a2) and p190 (e1a2) BCR-ABL transcripts to confirm the negative data of p210 and p190 in QRT-PCR. Results. A MMR was obtained in 20 pts (50%) and 20 pts (46%) in early and late CP-CML respectively. However, early CP-CML pts showed a significantly greater reduction in p210 BCR-ABL levels compared to late CP-CML after 12 months of Imatinib therapy (p=0.006), indicating a different kinetic of molecular response. Co-expression of p210 and p190 BCR-ABL transcripts at diagnosis was 73% for early CP-CML, whereas it was not available for late CP-CML. To test whether the persistence of p190 BCR-ABL transcript was predictive of MMR, we divided CML pts in 2 groups, those with 0 or 1 p190 BCR-ABL positive samples (group 1) and those with 2 or more positive samples (group 2) during the follow-up. We found that CP-CML pts of the group 2 showed a significant lower probability to obtain MMR molecular response compared to pts of group 1 both for late and early CML patients respectively [17/24 (71%) vs 5/19 (26%) with p=0.0039)], [15/21 (71%) vs 6/18 (33%) with p=0.017)]. This correlation holds also for complete cytogenetic response (data not shown). Conclusions. In this study, approximately 50% of pts reached a MMR; half of them had undetectable values of p210 BCR-ABL transcripts. However, in a proportion of pts with complete cytogenetic response and low level of p210 BCR-ABL transcript, the expression of p190 is still detectable. The persistence of p190 signal despite the 2–3log fall in p210 BCR-ABL levels, may be of prognostic significance and may disclose unfolded concepts of biological relevance.
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Giustacchini, Alice, Supat Thongjuea, Petter S. Woll, Paul Sopp, Ana Duenas Perez, Nikolaos Barkas, Christopher Booth, et al. "Single Cell Whole Transcriptome Analysis Reveals Distinct Molecular Signatures of Therapy-Resistant Chronic Myeloid Leukemia Stem Cells." Blood 126, no. 23 (December 3, 2015): 13. http://dx.doi.org/10.1182/blood.v126.23.13.13.
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Abstract Molecularly targeted therapy frequently induces remissions in cancer, but rarely achieves complete disease eradication, with resulting risk of disease relapse/progression. Chronic myeloid leukemia (CML) is a good example, with rare, propagating stem cells (SCs) that are incompletely eradicated by BCR-ABL directed tyrosine kinase inhibitor (TKI) therapy in most cases. Consequently, CML relapse usually occurs following treatment discontinuation. Further, some patients fail to achieve a satisfactory response to TKI therapy and are at risk of disease progression. Ultimately, the depth of response to TKIs is dictated by CML-SCs, however, it has proven challenging to characterise this crucial population of cells as they reside in the same compartment as their normal hematopoietic stem cell (HSC) counterparts from which they cannot be reliably distinguished throughout the disease course. Advances in single cell transcriptomics are opening up unprecedented opportunities to unravel heterogeneity in cell populations. However, to date, this technology has primarily been used to analyse normal tissues, partly reflecting the lack of sensitivity for detection of somatic mutations using current techniques. Herein, we developed a novel protocol allowing targeted amplification of BCR-ABL during single cell cDNA library generation using a modification of a published protocol (PMID:24385147). We then applied this method to analyse 1082 bone marrow SCs (defined as Lin-CD34+CD38-) from a cohort of 10 patients with CML and 3 normal controls. We first validated our method using the BCR-ABL +ve K562 cell line. Surprisingly, using standard methods, BCR-ABL was only detected in a small minority of cells. With our modified technique, we demonstrate robust detection of BCR-ABL in 100% of single K562 cells with parallel whole transcriptome amplification from the same cell. Further, using a plasmid Òspike-inÓ, we demonstrate sensitivity to detect a single molecule of BCR-ABL per cell, without false +ve BCR-ABL amplification in 142 cells tested. We then analysed 40 SCs from a patient with CML who was in a cytogenetic remission (CyR) following 3 months of TKI therapy. BCR-ABL was detected in 16 cells and was absent in 24 cells. With an average of 3.3 million mapped reads per single cell, we detected over 4600 genes per cell with good correlation of single cell ensemble data with bulk analysis. Using this approach we were uniquely able to compare BCR-ABL +ve and -ve cells and identify a large number of differentially expressed genes, which would not have been detected through bulk analysis. We selected a number of genes of biologic interest for validation by Q-PCR and 80% of them were confirmed as differentially expressed, a number of these are highlighted in the heatmap (see Figure). We then analysed 717 single SCs from 8 CML diagnostic samples. This analysis identified marked heterogeneity of BCR-ABL expression with frequencies comparable to the published literature (9% to 92%). Unsupervised clustering analysis revealed many genes that were differentially expressed between BCR-ABL +ve and -ve SCs e.g. CML-SCs showed upregulation of cell cycle signatures and decreased expression of HSC-affiliated genes. We next analysed 223 single SCs from a series of 5 patients in CyR following TKI initiation. BCR-ABL positive SCs could still be detected, albeit at a reduced frequency (0.5% to 45%). In contrast to the diagnostic samples, CML-SCs showed quiescence and HSC signatures that were comparable to normal HSCs. Furthermore, through analysis of serial samples from diagnosis to remission from the same patient, this single-cell approach uniquely allowed analysis of gene expression specifically of the CML-SCs which evade TKI therapy, facilitating the identification of a number of candidate gene-sets including cell surface markers and potentially ÒdruggableÓ targets. Finally, through analysis of a patient with early blast crisis transformation of CML during TKI therapy, we illustrate how single cell RNA sequencing might be applied to predict such early disease progression events. We herein describe a new approach for single cell RNA sequencing of CML-SCs that might be applied to fate-map persistent CML-SCs during and following treatment for discovery research and also to refine precision medicine in CML. This approach could be applied across a range of clonal disorders with potential broader relevance for cancer research. Disclosures No relevant conflicts of interest to declare.
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Pane, F., F. Frigeri, M. Sindona, L. Luciano, F. Ferrara, R. Cimino, G. Meloni, G. Saglio, F. Salvatore, and B. Rotoli. "Neutrophilic-chronic myeloid leukemia: a distinct disease with a specific molecular marker (BCR/ABL with C3/A2 junction) [see comments]." Blood 88, no. 7 (October 1, 1996): 2410–14. http://dx.doi.org/10.1182/blood.v88.7.2410.bloodjournal8872410.
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Neutrophilic-chronic myeloid leukemia (CML-N) is a rare myeloproliferative disorder that runs a much more benign course than chronic myeloid leukemia, and for which no specific underlying molecular lesion has been described so far. We have analyzed the genomic DNA by Southern blotting and the BCR/ABL hybrid gene transcripts by reverse transcriptase-polymerase chain reaction in three patients with clinical findings of CML-N, who did have a t(9;22) chromosomal translocation. In all patients we have found a rare type of BCR/ABL rearrangement, with a breakpoint between exons c3 and c4 of the BCR gene (corresponding to BCR exons 19 and 20). This was confirmed by hybridization with an oligonucleotide probe spanning the c3/a2 region. This type of junction causes almost the entire BCR gene to fuse with ABL. The junction is in frame and it gives rise to a fusion protein of predicted 230 kD. Our data now provide a molecular diagnostic marker for CML-N, and they are consistent with the notion that the inclusion or exclusion of BCR exons in the fusion protein affects dramatically its capacity to derange myeloid proliferation and differentiation, leading to the appearance of different disease phenotypes.
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Liu, Xiaohu, Clark Fruhstorfer, Katharina Rothe, and Xiaoyan Jiang. "A New AHI-1-DNM2-BCR-ABL Complex Regulates Leukemic Properties of Primitive CML Cells through Mediation of Cellular Endocytosis and ROS-Induced Autophagy." Blood 126, no. 23 (December 3, 2015): 52. http://dx.doi.org/10.1182/blood.v126.23.52.52.
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Abstract Tyrosine kinase inhibitor (TKI) therapies have been introduced into clinical practice with remarkable effects on chronic phase CML. However, early relapses, acquired drug resistance, and persistence of leukemic stem cells (LSCs) remain problematic. Improved treatment approaches to specifically target key molecular elements active in CML LSCs are needed. One candidate is the adaptor protein AHI-1 (Abelson helper integration site-1), an oncogene that is highly deregulated in LSCs. An AHI-1-mediated protein complex containing BCR-ABL and JAK2 has been shown to modulate transforming activity and TKI-response/resistance of CML LSCs. We have recently identified the large GTPase Dynamin-2 (DNM2) as another AHI-1 interacting protein using the AHI-1 SH3 domain as protein "bait" in immunoprecipitation (IP)/mass spectrometry. DNM2 plays key roles in the regulation of trafficking processes such as endocytosis, and is activated through tyrosine phosphorylation. However, its role in CML pathogenesis is unknown. We have now demonstrated that transcript levels of DNM2 are significantly increased in treatment-naive CD34+ cells from CML patients who were classified retrospectively, after Imatinib (IM) therapy, as IM-responders (n=11) and IM-nonresponders (n=15) in comparison to CD34+ normal bone marrow cells (n=7, p=0.013 and 0.037). In particular, DNM2 is more highly expressed in CML stem-enriched cells (lin-CD34+CD38-) and progenitor cells (lin-CD34+CD38+) than more mature cells (lin+CD34-, 2-fold). Interestingly, BCR-ABL+ human cells with stable knockdown of DNM2 exhibited significantly reduced cell growth and increased apoptosis as compared to control cells. They also showed enhanced sensitivity to single TKIs or MitMAB (DNM2 inhibitor) and these effects were greatly enhanced by combination treatments (2-3 fold). Mechanistically, co-IP and confocal co-localization analysis with mutant forms of AHI-1 and DNM2 (HA-AHI-1, HA-AHI-1 SH3Δ, Myc-DNM2 and Myc-DNM2 PRDΔ) in 293T cells indicated that the PRD domain of DNM2 is mainly responsible for the interaction between the SH3 domain of AHI-1 and DMN2. More importantly, we identified a new protein interaction between DNM2 and BCR-ABL in both BCR-ABL and BCR-ABL/AHI-1 co-transduced hematopoietic cells using co-IP/Western analysis; this interaction is enhanced in BCR-ABL/AHI-1 co-transduced cells. Moreover, DNM2 phosphorylation was decreased upon IM treatment in BCR-ABL-transduced cells, but remained unchanged in control and T315I mutant cells, suggesting that DNM2 is a direct target of BCR-ABL. Interestingly, we further observed that AHI-1 co-localizes with EEA-1 (early endosome marker) and LAMP-1 (late endosome marker) in cells co-transfected with full-length AHI-1 and DNM2, but not in AHI-1 and DNM2 mutant cells. Using transferrin uptake assays, increased transferrin signals were observed in BCR-ABL/AHI-1 co-transduced cells compared to BCR-ABL- and BCR-ABL/AHI-1 SH3Δ-transduced cells, while transferrin signals were much lower in DNM2-knockdown cells than control cells. These results suggest that the AHI-1-DNM2-BCR-ABL complex indeed improves the kinetics and increases efficiency of endocytosis, which may contribute to response/resistance of primitive CML cells to TKIs. This is further supported by observation of increased surface expression levels of CXCR4 in DNM2 knockdown cells, a key endocytotic target and known mediator of TKI response in CML. Moreover, reactive oxygen species (ROS) signals were found to be higher in BCR-ABL/AHI-1 co-transduced cells than BCR-ABL- and BCR-ABL/AHI-1 SH3Δ-transduced cells, while ROS accumulation was significantly decreased in DNM2-knockdown cells compared to control cells. More interestingly, ROS-induced autophagy was further observed in BCR-ABL/AHI-1 co-transduced cells, but reduced in DNM-2-knockdown cells, with protein expression changes in key autophagy proteins, including ULK-1, Beclin-1, LC3 and p62. To the best of our knowledge, this is the first study to implicate a new AHI-1-DNM2-BCR-ABL complex in the deregulation of endocytosis signaling and ROS production/autophagy in CML. This may play a unique and important role in the regulation of cellular properties of primitive CML cells, including their response/resistance to TKI. Disclosures No relevant conflicts of interest to declare.
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Hossain, Md Imran, Md Jaki Yamani, Md Golzar Hossain, Mohammed Murad Hossain, Md Rafiquzzaman Khan, and Md Abdul Aziz. "Molecular Response of Chronic Myeloid Leukaemia Patients to Tyrosine Kinase Inhibitors." Haematology Journal of Bangladesh 3, no. 02 (January 8, 2020): 42–46. http://dx.doi.org/10.37545/haematoljbd201942.
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Background: The goal of therapy in Chronic Myeloid Leukaemia (CML) with tyrosine kinase inhibitors (TKIs) has been to achieve the molecular responses, as measured by the reduction or elimination of BCR-ABLl transcript. Objective: The aim of the study is to observe the molecular response status of CML patients to TKIs. Methodology: This is an observational study and was conducted in the department of Haematology, Bangabandhu Sheikh Mujib Medical University (BSMMU) from February 2018 to January 2019. A total of 30 diagnosed CML patients on TKIs therapy was checked for quantitative BCR-ABL1 (by qRT-PCR) level. The laboratory monitoring of BCR-ABL1 RNA with RQ-PCR was likely become the model for successful molecular diagnostic monitoring of CML patients to both assess and prognosticate the efficacy of these targeted treatments. Result: Among 30 diagnosed CML patients, the mean age was found 36.1±11.7 years with range from 20 to 70 years. Males were predominant 19 (63.3%), male: female ratio was 1.7:1. Almost two third (63.3%) patients were found optimal (?10%) after 3-month BCR ABL. Age, sex, religion, marital status, occupational status, BMI, monthly income, number of family member, symptoms and sings were not statistically significant (p>0.05) when compared between BCR ABL optimal (?10%) and warning (>10%) group. Mean haemoglobin, total leukocyte count, platelet count, basophil, myelocyte and baseline BCR
ABL1 were not statistically significant (p>0.05) however atypical cells was found statistically significant
(p
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Paschka, Peter, Susan Branford, Christian Lorentz, Rüdiger Hehlmann, Tim Hughes, and Andreas Hochhaus. "Comparison of "Log Reduction from Median Pretherapeutic Value" vs Ratio BCR-ABL/ABL to Express the Therapeutic Response in CML Patients." Blood 104, no. 11 (November 16, 2004): 1013. http://dx.doi.org/10.1182/blood.v104.11.1013.1013.
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Abstract During the last decade, several studies have shown that quantification of residual tumor cells significantly correlates with clinical outcome in chronic myelogenous leukemia (CML). Detection of minimal residual disease (MRD) is now becoming routinely implemented in protocols for guiding therapy and for evaluation of new treatment modalities. The lack of standardization of the methodology represents a major barrier in the comparison of data generated in different studies. Therapeutic response can be expressed in 3 ways: (i) Calculation of the ratio of mRNA transcripts of target to reference gene, e.g. ratio BCR-ABL/ABL, (ii) Individual calculation of the relative molecular response: i.e. comparison of the MRD level after therapy vs pretherapeutic level, and (iii) use of a lab-specific reference point, e.g. a pool of diagnostic samples for calculation of the log reduction (Δlog). In the IRIS trial, a Δlog=3 after 12 mo of imatinib therapy was accompanied by a 100% relapse free survival after 30 mo and defined as "major molecular response" (MMR, Hughes et al. NEJM, 2003). The reference sample, however, is not available for widespread distribution. We sought to establish a relationship between the Δlog approach and the ratio of BCR-ABL to total ABL transcripts. To compare scales, 134 samples (68 RNA, 66 cDNA) of CML pts on imatinib therapy were exchanged between labs in Mannheim and Adelaide. 8 samples represented neg controls and 8 were degraded during shipment. Thus, 118 samples were eligible for analysis. 46 samples were pos for b3a2, 41 for b2a2, and 31 for b3a2&b2a2 BCR-ABL transcripts. In Adelaide, TaqMan PCR was performed for BCR-ABL and BCR transcripts. BCR-ABL and BCR plasmid dilutions were employed as standards, the ratio BCR-ABL/BCR was compared to the ratio of a pooled sample of 30 CML pts at diagnosis (IRIS approach). In Mannheim, quantitative LightCycler PCR was performed for BCR-ABL and total ABL transcripts with a single BCR-ABL plasmid dilution as standard curve for either transcript. The ratio BCR-ABL/ABL was calculated for the individual sample. In 4 samples, BCR-ABL was not detectable in one of the labs, 114 were pos in both labs and are eligible for comparison. None of the neg controls was tested pos in either lab. Δlog and ratio BCR-ABL/ABL correlated with r=−0.87 (p<0.0001). Regression analysis revealed a relationship between Δlog and ratio BCR-ABL/ABL according to the formula: Δlog = −0.91*[log (BCR-ABL/ABL in %)]+2.15. To demonstrate the variability of the reference point 17 paired samples were investigated from CML patients at diagnosis and prior to the start of imatinib after a brief period (10–274 days, median 53) of hydroxyurea therapy. During this interval, median leukocyte counts changed from 138/nl (range 21–605) to 7.8/nl (3.2–41, p=0.086), blasts in peripheral blood from 1% (0–12) to 0% (0–1, p=0.0085), and ratio BCR-ABL/G6PD from 5.5% (2.0–24.6) to 4.3% (1.3–17.1, p=0.036) with a Δlog of 0.2 (−0.3 to 0.8). We conclude that the calculation of the ratio BCR-ABL/ABL is a rational approach to express MRD levels in CML patients after therapy. The ability to calculate the Δlog depends on the access to a reference sample which is not currently available worldwide. We demonstrate here the option to convert results calculated as ratios of BCR-ABL/ABL into Δlog from a pre-therapy median and vice-versa. A 3-log reduction, which has been established as MMR, represents a ratio BCR-ABL/ABL of 0.12%.
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Liu, Xiaohu, Clark Fruhstorfer, Katharina Rothe, and Xiaoyan Jiang. "A New AHI-1-DNM2-BCR-ABL Complex Regulates Endocytosis Processes in Chronic Myeloid Leukemia." Blood 124, no. 21 (December 6, 2014): 896. http://dx.doi.org/10.1182/blood.v124.21.896.896.
Full textAbstract:
Abstract Tyrosine kinase inhibitors (TKIs) have been introduced into clinical practice with remarkable effects on chronic phase CML. However, early relapses, acquired drug resistance, and persistence of leukemic stem cells remain problematic. Improved treatment approaches to target other key molecular elements active in CML stem/progenitor cells are needed. One candidate is AHI-1 (Abelson helper integration site-1), an oncogene that is highly deregulated in CML stem cells. It harbors two key domains, SH3 and WD40-repeat, which are known important mediators of protein-protein interactions. The AHI-1-mediated protein complex containing BCR-ABL and JAK2 has been shown to mediate transforming activity and TKI-response/resistance of CML stem/progenitor cells. We have recently identified Dynamin-2 (DNM2) as another AHI-1 interacting protein using the AHI-1 SH3 domain as protein ‘bait’ in immunoprecipitation/mass spectrometry. DNM2, a large GTPase, is mainly involved in the trafficking processes such as endocytosis, and is activated through tyrosine phosphorylation. Its role in the mediation of CML stem cell functions is unknown. We have now demonstrated that transcript levels of DNM2 are significantly increased in pre-treatment CD34+ stem/progenitor cells from CML patients who were classified retrospectively, after IM therapy, as IM-responders (n=11) and IM-nonresponders (n=15) as compared to CD34+ normal bone marrow cells (n=7, p=0.013 and 0.037). In addition, DNM2 is more highly expressed in CML stem cells (CD34+CD38-) and progenitor cells (CD34+CD38+) than more mature cells (CD34- , 2-fold). Co-immunopreciptation with mutant forms of AHI-1 and DNM2 (HA-AHI-1, HA-AHI-1 SH3Δ, Myc-DNM2 and Myc-DNM2 PRDΔ) in 293T cells indicated that the PRD domain of DNM2 is mainly responsible for the interaction between AHI-1 and DMN2. Co-localization analysis using confocal microscopy further demonstrated that the interaction between full-length AHI-1 and DNM2 occurs in a “punctate dot” pattern throughout the whole cytoplasm; in contrast, the co-localization signals were significantly disrupted in cells co-transfected with AHI-1 and DNM2 mutants. Interestingly, in AHI-1 SH3D mutant cells, AHI-1was located in the nucleus, suggesting that the SH3 domain of AHI-1 is required for AHI-1 cytoplasmic retention. Moreover, AHI-1 was observed to co-localize with EEA-1 (early endosome marker) and LAMP-1 (late endosome marker) in cells co-transfected with full-length AHI-1 and DNM2, but not in AHI-1 and DNM2 mutant cells. Increased transferrin signals were also demonstrated in AHI-1 and DNM2 co-transfected cells compared to their mutant cells, using transferrin uptake assays, suggesting that the interaction between DNM2 and AHI-1 indeed increases the kinetics and efficiency of endocytosis. These results were further confirmed in BCR-ABL-transduced and BCR-ABL/AHI-1 co-transduced hematopoietic cells that are relatively resistant to TKI-induced apoptosis. Particularly, the co-localization signals between DNM2 and AHI-1 were stronger in BCR-ABL/AHI-1 co-transduced cells, and the transferrin uptake was also more efficient as compared to BCR-ABL-transduced cells. On the other hand, transferrin uptake was reduced in CML cells with knockdown of DNM2 and these cells also demonstrated reduced proliferation and increased sensitivity to IM treatment compared to control cells. Importantly, we further identified a new protein interaction between DNM2 and BCR-ABL in both BCR-ABL and BCR-ABL/AHI-1 co-transduced cells and this interaction is enhanced in BCR-ABL/AHI-1 co-transduced cells using co-IP/Western analysis. To the best of our knowledge, this is the first study to implicate this new AHI-1-DNM2-BCR-ABL complex in the deregulation of endocytosis signaling in CML, which may play an unusual role in regulation of the cellular properties of primitive CML cells, including their response/resistance to TKI, by aberrantly disrupting critical endocytosis processes in CML. Disclosures No relevant conflicts of interest to declare.
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Solarska, Iwona, Miroslaw Majewski, Barbara Nasilowska-Adamska, Jan Zaucha, Iwona Kania, Hanna Makuch-Lasica, Monika Lewandowska, et al. "Prognostic Value of Real-Time PCR BCR-ABL Transcript Monitoring after Allogeneic Stem Cell Transplantation in Chronic Myeloid Leukemia Patients." Blood 108, no. 11 (November 16, 2006): 4824. http://dx.doi.org/10.1182/blood.v108.11.4824.4824.
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Abstract Allogeneic hematopoietic stem cell transplantation (alloHSCT) in chronic phase of chronic myeloid leukemia (CML) is associated with long-term disease-free survival and potentially eradication of leukemic cells. The goal of early MRD detection is to allow timely therapeutic intervention before hematologic relapse. The concomitant detection of BCR-ABL mRNA following alloHSCT is strongly associated with relapse, though not absolutely predictive. The relapse risk decreases with increased time after alloHSCT. The detection of BCR-ABL mRNA is the most strongly associated with relapse shortly after HSCT but all patients need to be monitored indefinitely after transplantation by molecular techniques presumably at 3–6 months intervals. Real-time quantitative PCR (RQ-PCR) for BCR-ABL mRNA provides an accurate and reliable measure of response to therapy in CML. In this study we evaluated 412 available samples from 75 patients at 1 month to 10 years after allo HSCT. Quantification of BCR-ABL was performed by RQ-PCR assay according to the Europe Against Cancer (EAC) protocol. Peripheral blood/bone marrow samples were studied every 3–6 months after alloHSCT for the presence of BCR-ABL transcripts using RT-PCR/nested PCR and RQ-PCR. RT-PCR positive patients were analyzed further at monthly intervals. RNA isolation from mononuclear cells was performed by column method. Reverse transcription was performed using Super Script II and random hexamers. BCR-ABL level was normalized with control ABL gene and expressed as the ratio of BCR-ABL/ABL compared to diagnostic sample or median expression values of BCR-ABL/ABL from EAC protocol. In our group BCR-ABL/ABL ratio decreased at least 1000-fold in all patients after alloHSCT. RT-PCR became negative in 64.7% patients after first 90 days. In the group of 65 patients with RQ-PCR tests performed at least 1 year after alloHSCT, 12 (18.5%) patients were always negative (no BCR-ABL/ABL transcripts detected, at least 10−5 test sensitivity), 40 (61.5%) were persistently low-level positive (with the BCR-ABL/ABL ratio less than 0.02%) and 13 (20.0%) patients were stable, high-level positive with transcript levels exceeded 0.02% threshold. The molecular relapse was observed in three patients with 15–80 fold increased of BCR-ABL expression in the first year after SCT. Decreased immunosuppressive therapy allowed achieving molecular remission in two patients. One patient developed hematological relapse despite donor lymphocyte infusion (DLI). One patient developed cytogenetic relapse and was successfully treated with 400 mg imatinib daily dose and achieved molecular remission with a low-level BCR-ABL expression. Standard imatinib therapy was applied in seven patients prior to SCT without negative transplant outcome. We conclude that RQ-PCR is valuable method to quantitate BCR-ABL expression in CML patients after alloHSCT and allows monitoring the kinetics of BCR-ABL mRNA transcipts and is useful in prediction of the hematologic relapse.
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Paiva, Aldair Sousa, Hugo Diogenes De Oliveira Paiva, Geraldo Barroso Cavalcanti, Gioconda DR Leão, Marcos Dias Leão, Roberto C. Vasconcelos, Andrea LAC Fernandes, et al. "Detection of the BCR-ABL Gene By the Real-Time PCR Method in Patients with Chronic Myeloid Leukemia in Rio Grande Do Norte, Brazil." Blood 132, Supplement 1 (November 29, 2018): 5432. http://dx.doi.org/10.1182/blood-2018-99-118853.
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Abstract Background: The Philadelphia chromosome is a cytogenetic change resulting from a reciprocal translocation of genetic material between ABL genes from chromosome 9 and BCR from chromosome 22 or t(9; 22) (q34; 11), forming the chimeric gene BCR- ABL, being associated with chronic myeloid leukemia (CML), acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML). The p190 variant is usually associated with acute forms of leukemia, including AML and ALL, whereas the p210 variant is associated with the chronic phases of CML. Due to the high sensitivity and specificity, nucleic acid amplification techniques by real-time PCR have replaced the conventional cytogenetic techniques for the identification of the Philadelphia chromosome and its p190 and p210 variants. Molecular analysis has been indicated in the initial diagnostic phase and also for the therapeutic monitoring defining the percentage of neoplastic cells present in the patients during the different phases of the treatment (Minimum Residual Disease or MRD).The aim of this study was the transcript BCR-ABL identification in patients with suspected of CML and evaluation of the gene frequency in these patients. Methods: The presence of BCR-ABL gene was investigated in blood samples from 42 patients with suspected CML. The RNA extraction was performed by phenol/chloroform method. The cDNA was submitted to PCR, using specific primers for and BCR-ABL genes by Real time PCR. Results: From all studied patients, 16 (38.10%) were negative, and 26 (59.09%) positive for one of rearrangements: p210 b3a2 and b2a2 in 18 cases (40.91%) and p190 a1a2 in 2 cases (4,76%) and double positive p120/190 in 6 cases (14,28%). We observed that the most common rearrangement was the p210 b3a2, and the molecular results were compatible with clinical and hematologic suspicion. Conclusions: The Real-timePCR, because of its specificity and sensitivity, can be considered the most used technique in routine diagnosis and investigation of MRD of CML patients. Disclosures No relevant conflicts of interest to declare.
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Stangler Herodež, Špela, Nadja Kokalj-Vokač, Zlatko Roškar, and Mojca Dreisinger. "Accuracy and speed of molecular response reporting with Xpert BCR-ABL Ultra in vitro diagnostic test in CML patients." Acta Medico-Biotechnica 14, no. 1 (November 23, 2021): 61–67. http://dx.doi.org/10.18690/actabiomed.215.
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Purpose: The objective of this study was to introduce the GeneXpert Ultra cartridge-based BCR-ABL diagnostic test into routine molecular monitoring of minimal residual disease for chronic myeloid leukemia (CML) patients who are on tyrosine kinase inhibitors (TKIs).Methods: BCR-ABL1 transcript quantification was performed on 100 peripheral blood samples from 33 CMLpatients at different stages of the disease using automated GeneXpert BCR-ABL Ultra assay and following a standardized laboratory protocol (SP).Results: Analysis results were expressed as % on the International Scale (IS) for BCR-ABL1 real-time PCR quantification and estimated using molecular response (MR) according to the laboratory recommendations for scoring deep MRs following CML treatment. A comparison of the two methods showed that there was no statistically significant difference in the results expressed as % on the IS (p = 0.098). Samples were divided into six groups (MR1, MR2, MR3, MR4, MR4.5, and MR5) according to the estimated MR. A total of 74/100 samples had a completely identical MR with both methods, 12/100 samples had a higher MR obtained with GeneXpert, and 14/100 samples had a lower MR compared to the SP. In addition, 2/74 samples had a different MR for two classes obtained with GeneXpert compared to the SP, while the others 72/74 samples differed in one MR class. Analysis of the major molecular response resulted in 82% agreement. Minimal amounts of BCR-ABL1 copies were detected with GeneExpert in eight samples while the SP did not detect them. None of the GeneXpert results had any effect on the clinical decision for treatment with TKIsobtained using the SP.Conclusion: The Xpert BCR-ABL Ultra assay can be considered a useful and very fast clinical tool for molecular follow up of patients on TKIs.
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Toplin, Julie, Courtney Fuller, Linda Fletcher, Stephane Wong, Peter Maslak, Jorge Cortes, Rosemary Mazanet, et al. "An MMR Control RNA for Reliable Monitoring of BCR-ABL Transcripts in Treated CML Patients." Blood 110, no. 11 (November 16, 2007): 2939. http://dx.doi.org/10.1182/blood.v110.11.2939.2939.
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Abstract Introduction: Major Molecular Response (MMR) is defined as a three-log reduction from a standardized baseline of BCR-ABL/control gene transcript ratio in CML patients at diagnosis. MMR has prognostic significance for progression-free survival for patients on Imatinib® therapy. Day-to-day monitoring of the MMR value in clinical laboratories is challenging due to the absence of a commercially available standardized MMR control RNA. To improve the reliability of BCR-ABL quantitation, MolecularMD has evaluated the feasibility of a single MMR control RNA valid for blood samples drawn in EDTA or PAXgene™ tubes. Material and Methods: Patient sample RNAs were interchanged between our laboratory and an International Randomized Interferon versus STI571 study (IRIS) laboratory, which had established an MMR value and international scale reporting. This exchange enabled our laboratory to establish an MMR value and reporting on an international scale using a validated conversion factor. A serial dilution of a BCR-ABL positive cell line into a human BCR-ABL negative cell line was prepared. These dilutions were tested in IRIS laboratories with established MMR value and international scale reporting and at our laboratory by QRT-PCR to determine the BCR-ABL/control gene ratio using respectively BCR and ABL control genes. We compared the BCR-ABL/ABL ratio in 104 paired PB CML patient samples drawn either in EDTA and PAXgene tubes and the BCR-ABL/BCR ratio in 32 patient samples. Stability studies were performed to evaluate the degradation of liquid and dried forms of the MMR RNA. Results: We established a conversion factor (CF) of 0.81 with an MMR value of 0.123%. Using this CF and MMR value, we created appropriate RNA dilutions that matched the MMR value using ABL as a control gene. Repeated analyzes of this MMR control RNA confirmed the accuracy of the sample with a median value of 0.124%, very close to the MMR value defined previously (0.123%). Stability studies demonstrated that the dried RNA samples could be stored several days at 37°C and freeze-thawed up-to 10 times without significant degradation. These RNA samples once reconstituted with water could also be used several times for BCR-ABL monitoring without any significant degradation. Comparison of BCR-ABL/ABL ratio between EDTA and PAXgene tubes revealed differences unlikely to have clinical impact on disease management suggesting that the MMR RNA created would be suitable under both EDTA and PAXgene extraction methodologies. Conclusions: We produced a stable MMR control RNA in large quantity for accurate monitoring of the MMR value. This MMR control RNA is now be tested in several laboratories to confirm the stability and reliability of this reagent. The MMR control RNA will be an important tool for standardizing MMR value in laboratories, and an integral part of a BCR-ABL QRT-PCR diagnostic kit.
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Lundan, Tuija, Franz Gruber, Aleksandra Silye, Henrik Hjorth-Hansen, Ingvild Mikkola, Kari Remes, Bengt Simonsson, Tobias Gedde-Dahl, and Kimmo Porkka. "The Mutation Spectrum of CML Patients Resistant to Imatinib: More Heterogeneous Than Just BCR-ABL Kinase Domain Point Mutations?." Blood 110, no. 11 (November 16, 2007): 1948. http://dx.doi.org/10.1182/blood.v110.11.1948.1948.
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Abstract Cells harboring BCR-ABL kinase domain (KD) mutations are the major source for resistance to tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML). A second and third generation of TKIs are in development for inhibition of mutated forms of BCR-ABL. At present, clinical treatment strategies are based on molecular response and mutational profiling. Profiling is usually performed using unbiased methods (sequencing and D-HPLC of PCR products), but mutation specific methods are upcoming (ARMS-PCR, ASO-PCR, ligation-PCR). Here, we describe molecular findings resulting from a collaborative Nordic approach for molecular characterization of imatinib resistant Norwegian (N) and Finnish (F) CML patients. Complementary DNA derived from a total of 87 subjects (60 N and 27 F) with suboptimal or failed response to imatinib (less than 3 log reduction in tumor load after 12 months of treatment or loss of any response) were subjected to BCR-ABL KD sequencing of amino acid residues 240 to 420 (N) and 209 to 498 (F). In addition, single samples were analyzed by multiplex PCR for detection of transcript variants as well as cloning and sequencing of single clones. BCR-ABL KD mutations were found in 35 patients (20 N, 15 F, 40% of the total material). The most frequent findings were ABL KD point mutations with M351T and G250E representing the single most frequent mutations, 25 and 16%, respectively. The most frequently mutated cluster was in the catalytic loop (40% of all mutations), followed by P-loop (38%) and other regions (22%). Mutations in the gatekeeper cluster were extremely rare in our material (one case of a F317L). Additional sequence variations were observed in 6 patients: the L248V associated deletion of exon 5 (n=1), smaller insertion or deletion variants (2–4 basepairs) resulting in truncated proteins (n=2) and ABL exon 7 splicing (n=3). Finally, three cases with imatinib resistance expressed rare BCR-ABL transcript types without any evidence for KD mutations (two cases of e6a2 and one with e19a2 transcript). Imatinib resistance due to T315I BCR-ABL is extremely rare in Nordic CML patients. In addition to known hotspot mutations we identified novel sequence irregularities in association to imatinib resistance. We hypothesize that resistance-associated BCR-ABL isoforms are more heterogeneous than previously thought. Consequently, diagnostic procedures aiming in comprehensive molecular verification of TKI resistance should be designed as unbiased, since methods solely targeting known mutated BCR-ABL variants will miss many patients. However, an early detection of low-level mutated clones will require sensitive, mutation-specific analyses.
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D'agostini, Elena, Giulia Minnucci, Giulia Amicarelli, Cinzia Pultrone, Veronica Tettamanzi, Chiara Boroni, Silvia Salmoiraghi, Orietta Spinelli, Francesco Colotta, and Alessandro Rambaldi. "Ultra Rapid, One Step Molecular Detection of BCR-ABL Major and Minor Transcripts by Isothermal RT-Loop Mediated Amplification Reaction." Blood 120, no. 21 (November 16, 2012): 2515. http://dx.doi.org/10.1182/blood.v120.21.2515.2515.
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Abstract Abstract 2515 Background: The molecular identification of the BCRABL transcripts in clinical samples is actually based on a conventional Reverse Transcription Polymerase Chain reaction (RT-PCR). Here we present a novel molecular method, based on Loop Mediated Amplification assay that, starting from RNA (RT-LAMP) in one single tube reaction ensures a rapid and simultaneous detection of either the BCR-ABL p190 or p210 fusion transcript as well as the housekeeping gene used as internal quality control. Methods: The BCR-ABL RT-LAMP is a multiplex, isothermal method for retro-transcription, amplification and detection of the Minor (p190) and Major (p210) t(9;22) transcripts and the endogenous Gusb RNA, as internal control for validation of negative results. The employment of fluorescent specific probes allows real-time monitoring of the reaction, so that the test result is obtained in a single, homogeneous step. RT-LAMP is carried out on the Liaison IAM, a 8-wells manageable instrument suitable for isothermal reactions. Liaison IAM incubates at constant temperature, monitors the fluorescent signals and the data produced can be analyzed, upon connection to up to 6 other instruments, for a throughput of 8 to 48 samples. Thanks to the three channels of fluorescence, it can monitor multiplex assays, providing elaborated final objective results with no need of further data analysis by the operator. Results: The level of sensitivity of the triplex BCR-ABL RT-LAMP has been analytically evaluated directly on serial dilutions of RNA extracted respectively from t(9;22) positive cell lines (TOM-1 for p190 or K-562 for p210) into wild type RNA from HL-60 cell line (30 replicates). The p190 and p210 transcripts have been detected and distinguished down to 10−4 and 10−5respectively within 50 minutes. The assay demonstrated 100% specificity since 70 replicates of wild type RNA from 7 cell lines resulted BCR-ABL negative and GUSb positive (internal amplification control). This assay was validated on 60 clinical samples (30 white blood cells RNA from Chronic Myeloid Leukemia, 30 mononuclear cells RNA from B-lineage Acute Lymphoblastic Leukemia). All these samples were obtained at diagnosis and were previously analyzed by conventional RT-PCR. RT-LAMP detected and identified the BCR-ABL fusion transcripts correctly in all cases with a 100% concordance with the reference method. Fully concordant results were obtained also on 30 RNA samples from patients affected by t(9;22) negative hematologic malignancies and on 30 RNA samples obtained from healthy donors in which the RT-LAMP amplified exclusively the housekeeping GUSb transcript. Conclusions: The triplex p190-p210-GUSb RT-LAMP is a one-step procedure for specific, highly sensitive and rapid molecular detection of the BCR-ABL fusion transcripts. The semi-automatic instrument Liaison IAM, simplifies the entire procedure, reduces the contamination risks deriving from the conventional, multi step RT-PCR and significantly improve the diagnostic lab routine. Disclosures: D'agostini: DIASORIN S.p.A: Employment. Minnucci:Diasorin S.p.A.: Employment. Amicarelli:DIASPRIN S.p.A.: Employment. Pultrone:DIASORIN S.p.A.: Employment. Tettamanzi:DIASORIN S.p.A.: Employment. Salmoiraghi:DIASORIN S.p.A.: Consultancy. Spinelli:DIASORIN S.p.A: Consultancy. Colotta:DIASORIN S.p.A: Employment. Rambaldi:DIASORIN S.p.A: Consultancy.
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Ernst, Thomas, Mathias Schmidt, Jenny Rinke, Vivien Schäfer, Anja Waldau, Ellen Obstfelder, Nils Winkelmann, et al. "Molecularly Defined Clonal Evolution in Patients with Chronic Myeloid Leukemia Independent of the BCR-ABL Status." Blood 124, no. 21 (December 6, 2014): 4513. http://dx.doi.org/10.1182/blood.v124.21.4513.4513.
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Abstract Molecularly defined clonal evolution has been identified as a key phenomenon in the biology of acute myeloid leukemia. Molecular aberrations may be markers of individual subclones of the disease, but also responsible for treatment resistance. In most patients with chronic myeloid leukemia (CML), treatment with tyrosine kinase inhibitors (TKI) induces complete cytogenetic remissions (CCyR) characterized by polyclonal hematopoiesis. However, some CML patients show emergence of clonal cytogenetic abnormalities (CCA) in Philadelphia (Ph) negative cells during treatment indicating clonal hematopoiesis. We searched for BCR-ABL independent gene mutations in both Ph-negative and Ph-positive clones in 29 chronic phase CML patients (male, n=16; median age 58 years, range 29-73 years) using targeted deep next-generation sequencing of 25 genes frequently mutated in myeloid disorders: ASXL1, BRAF, CBL, DNMT3A, ETV6, EZH2, FLT3, IDH1, IDH2, JAK2, KIT, KRAS, LNK, MPL, NPM1, NRAS, RUNX1, SF3B1, SRSF2, TP53, TET2, U2AF1, UTX, WT, and ZRSR2. Ph-negative clones were analyzed in 14 CML patients who developed CCA in Ph-negative cells (trisomy 8, n=10; nonrecurrent reciprocal translocations, n=2; monosomy 7, n=2). At time of analysis, patients were in CCyR (n=7), partial CyR (n=6), or minimal CyR (n=1) after a median of 21 months (range 7-114 months) of TKI treatment with imatinib, and/or nilotinib, and/or dasatinib. After exclusion of known polymorphisms, mutations were detected in 6/14 patients (43%) affecting the genes DNMT3A, EZH2, RUNX1, TET2, TP53, U2AF1, and ZRSR2. These patients were in CCyR (n=2), partial CyR (n=3), or minimal CyR (n=1), respectively, indicating BCR-ABL independent mutations in both Ph-negative and Ph-positive subclones. In two patients, the mutations were also found in corresponding diagnostic samples at higher or lower mutation level. To further investigate BCR-ABL independent gene mutations in Ph-positive clones, 15 randomly selected CML patients at diagnosis were analyzed. Mutations additional to BCR-ABL were found in 5/15 CML patients (33%) affecting ASXL1, DNMT3A, RUNX1, and TET2. None of the mutations were recognized in corresponding constitutional DNA specimens indicating that all mutations had been somatically acquired. Deep-sequencing of subsequent samples obtained in early CCyR after three months of TKI treatment revealed one DNMT3A mutation in Ph-negative cells which was also present in Ph-positive cells at diagnosis. Follow-up investigation showed that the mutation persisted in Ph-negative cells throughout CCyR and deep molecular remission (MR4.5) up to month 36 thereby not significantly changing its mutation level of approximately 15% implying a clonal hematopoiesis before the acquisition of the BCR-ABL rearrangement. In summary, BCR-ABL independent gene mutations were frequently found in Ph-negative and Ph-positive clones of CML patients and may be considered as important cofactors in the evolution of CML. Additional mutations acquired in the Ph-positive clone may impact on response to TKI treatment. Mutations preexisting to the occurrence of the BCR-ABL rearrangement may predispose patients to secondary hematological neoplasms. Our findings provide novel genetic information regarding CML biology and warrant further studies and impact on the design and performance of discontinuation trials. Disclosures Schnittger: MLL Munich Leukemia Laboratory: Other. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Hochhaus:Novartis: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; ARIAD: Honoraria, Research Funding; Pfizer: Consultancy, Research Funding.
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Sala Torra, Olga, Lan Beppu, Susan Branford, Linda Fletcher, Gooley Ted, Amy L. Paguirigan, Jordan Smith, and Jerald P. Radich. "Paper or Plastic: RT-PCR of BCR-ABL from Blood Spots Stored and Shipped on Paper." Blood 124, no. 21 (December 6, 2014): 4566. http://dx.doi.org/10.1182/blood.v124.21.4566.4566.
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Abstract In many parts of the world, diagnosis and monitoring of CML patients is limited by the availability and cost of molecular testing. In countries without molecular diagnostic capabilities, blood samples can be shipped to central labs, but this is both hampered by sample degradation, and the high costs of shipping. This study explores the method of directly spotting peripheral blood onto a paper template (dried blood spots), with subsequent shipping, RNA extraction, and BCR-ABL testing. Methods: Blood Spots and Shipment. We received dried blood spots from Australia and African countries by mail or courier, and blood from CML patients from our institution were also used for these experiments. 200μL of blood (PB) was pipetted onto Whatman 503 Protein Saver Cards (PSC; Sigma-Aldrich), where each card contains four 50μL spots. Cards were allowed to dry for at least 24 hours at room temperature. For mailing, PSCs were sealed into glassine envelopes with a packet of desiccant, and then placed inside a mailing envelope following DOT and IATA regulation for shipping non-regulated, exempt human specimens. RNA Extraction from Cards and %BCR-ABL determination. Blood spots were incubated with proteinase K followed by RNA isolation using RNeasy Mini Kits (Qiagen). Extracted RNA was quantified using a NanoDrop spectrometer (Thermo Scientific). %BCR-ABL was determined using the automated Cepheid GeneXpert platform or manual two-step quantitative RT-PCR on the 7900HT Fast Real-Time PCR System (Applied Biosystems). Results: Bench top time course: To test for effects of long transit times on RNA quality, we performed a time course study of cards at room temperature (RT) with 5 samples. For each sample, multiple cards were spotted with PB. The cards were then allowed to sit at RT for predetermined amounts of time, up to 42 days, before extracting RNA. We measured RNA integrity for one of the specimens (CML # 5) and found rapid degradation with the RIN number going from 8.7 for the fresh blood to 2.8 after 28 days on the card. However the amplification for both BCR-ABL and ABL differed less than one cycle between the fresh blood and the last time point by manual qRT-PCR (BCR-ABL Ct = 23.63 for fresh blood and 24.06 for day 28 PSC; ABL Ct = 26.69 for fresh blood and 27.64 for day 28 PSC). Figure 1 shows the results of the time course experiment for the 5 samples as a plot of ΔCt versus time in days. BCR-ABL qRT-PCR concordance studies: We compared the %BCR-ABL results obtained in fresh specimen at the institution sending the sample with the %BCR-ABL results we obtained from RNA extracted from PSC using the Cepheid GeneXpert. Paired evaluable results were available for 9 samples with a median WBC = 9.8 x 109/L (range: 3.37x109/L – 85.5x109/L). Samples were 8 to 49 days old at the time of extraction. The amount of RNA input into the GeneXpert reaction ranged from 38.75ng to 1μg. The %BCR-ABL detected ranged from 0.37% to 27% (see Table). The mean absolute difference between fresh blood and PSC BCR-ABL% is 2%; the relative mean percent change for BCR-ABL, using fresh blood as the reference is 13.1% (S.D., 31.2), P = 0.24. Conclusions and future directions: Dried blood spots are relatively inexpensive method to transport blood that preserves enough RNA stability to allow highly accurate BCR-ABL detection, when compared to results performed on an identical platform using fresh peripheral blood samples. Further studies are undergoing to accurately determine the sensitivity of this method and the feasibility of using regular mail for inexpensive transport of specimens. Table 1IDWBC (1000/μL)Sample Age at Spotting (Days)Sample Age at RNA extraction (Days)RNA ng/μlVolume GeneXPert (μL)Paper %BCR-ABL (IS)GeneXpertFresh Blood % BCR-ABL (IS) GeneXpertI1na010426349naI224.101311092745I38009181544naI47.4285102.4*3.1I55.50495241.92I63.61307.4225912I785.5130102102439I812.212912.415128.8I9na1281.5250.37*0.71I103.370273257.85.7I1115.912731102325I126.612714.415na2.3 *%BCR-ABL was manually calculated due to late ABL Cts because of low starting material. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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Bochicchio, Maria Teresa, Jessica Petiti, Paola Berchialla, Barbara Izzo, Emilia Giugliano, Emanuela Ottaviani, Santa Errichiello, et al. "Droplet Digital PCR for BCR–ABL1 Monitoring in Diagnostic Routine: Ready to Start?" Cancers 13, no. 21 (October 30, 2021): 5470. http://dx.doi.org/10.3390/cancers13215470.
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BCR–ABL1 mRNA levels represent the key molecular marker for the evaluation of minimal residual disease (MRD) in chronic myeloid leukemia (CML) patients and real-time quantitative PCR (RT-qPCR) is currently the standard method to monitor it. In the era of tyrosine kinase inhibitors (TKIs) discontinuation, droplet digital PCR (ddPCR) has emerged to provide a more precise detection of MRD. To hypothesize the use of ddPCR in clinical practice, we designed a multicentric study to evaluate the potential value of ddPCR in the diagnostic routine. Thirty-seven RNA samples from CML patients and five from healthy donors were analyzed using both ddPCR QXDxTMBCR-ABL %IS Kit and LabNet-approved RT-qPCR methodologies in three different Italian laboratories. Our results show that ddPCR has a good agreement with RT-qPCR, but it is more precise to quantify BCR–ABL1 transcript levels. Furthermore, we did not find differences between duplicate or quadruplicate analysis in terms of BCR–ABL1% IS values. Droplet digital PCR could be confidently introduced into the diagnostic routine as a complement to the RT-qPCR.
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Djimadoum Mbanga, Bessimbaye Nadlaou, Issakou Bakarnga-Via, Josué Mbairané, Choua Ouchemi, Abdelsalam Tidjani, and Avocksouma Djona Atchenemou. "Epidemiological, therapeutic, cytogenetic, and molecular profile of chronic myeloid leukemia at the national reference university hospital of N’Djamena from 2010-2020, Chad." World Journal of Advanced Research and Reviews 10, no. 3 (June 30, 2021): 127–40. http://dx.doi.org/10.30574/wjarr.2021.10.3.0269.
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The aim is to determine the epidemiological and cytogenetic profile (Philadelphia chromosome: Ph1) and the bcr-abl gene in patients with chronic myeloid leukemia (CML) and to assess the therapeutic response of patients to hydroxy-urea and Imatinib treatment. From January 1, 2010 to December 30, 2020, an observational study for diagnostic and analytical purposes was carried out in 54 cases of CML at the National Reference University Hospital (CHU-RN) of N’Djamena. All selected patients presented with splenomegaly on physical examination. Significant differences were observed between the proportion of housewives (31.48%) and civil servants (3.70%), emaciated patients (27.84%) and those with fever (3.70%) with the probabilities of 0.001 and 0.001 respectively. Hyperleukocytosis ranged from 100,000-149,000 GB / mm3 (42.30%) and the platelet count ranged from 20,000 / mm3 to 611,000 / mm3. Polymorphic and massive myeloma was 30-80% of cases, a medullary karyotype with 33.33% Ph1 + chromosome and 66.66% Ph1-, 33.33% bcr-abl transcript and 33.33% transcribed b3 a2 respectively. The treatment regimens were: Hydroxy-Urea (HU) + prednisolone (60%), and Imatinib + prednisolone (40%). Partial hematological remission was obtained in 40% of cases. CML is a condition that occurs at all ages and especially in men in our region. The continuation of this study at the national level will allow the public authorities to achieve national prevalence and to organize effective prevention and early management.
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Press, Richard D., Zac Love, Ashlie A. Tronnes, Gwen Kurilik, Michael J. Mauro, Michael W. Deininger, and Brian J. Druker. "BCR-ABL RNA Levels at the Time of a Complete Cytogenetic Response (CCR) Predict the Duration of CCR in Imatinib-Treated Chronic Myeloid Leukemia Patients." Blood 104, no. 11 (November 16, 2004): 1098. http://dx.doi.org/10.1182/blood.v104.11.1098.1098.
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Abstract Background : Imatinib induces a complete cytogenetic response (CCR) in the majority of patients with chronic phase CML. CCR is durable in the majority of patients, but relapse occurs in a subset. To determine the potential of quantitative RT-PCR (qPCR) of BCR-ABL to predict cytogenetic relapse, we serially monitored residual disease in 90 CML patients with an imatinib-induced CCR. Methods and patients : mRNA was prepared from total nucleated cells from blood or bone marrow, and cDNA was synthesized using random hexamer primers. Relative BCR-ABL expression was then measured by real-time fluorescent PCR normalized for G6PDH expression. This assay has a detection limit of 1 CML cell in 100,000 and an analytical precision of 6% (CV). At the start of imatinib therapy, 85% of patients were in chronic phase, at a median 9.5 months after diagnosis. Patients were treated with imatinib alone (64%) or in combination with interferon or cytarabine (32%). One patient each was treated with imatinib in combination with either the farnesyltransferase inhibitor tipifarnib, donor leukocytes (after allogeneic BMT), or an experimental heat shock protein (hsp70) vaccine. During the imatinib follow-up time of 28 months (median), disease monitoring occurred by cytogenetics and qPCR (median 6 samples per patient). The CCR was achieved after 9.7 months (median) of imatinib therapy. Results : At the time of first achieving CCR, BCR-ABL RNA levels had decreased by a median of 1.8 logs below the median baseline level. During further follow-up, 26 patients (29%) experienced cytogenetic relapse (defined as any Ph-positive metaphase cell) at a median 6.0 months after CCR and a median 20 months after starting imatinib. There was no difference in the imatinib treatment time, the time to achieve CCR, or the post-CCR follow-up period between the patients with and without subsequent cytogenetic progression. qPCR data at the time of first CCR were available for 78 patients, including 25 of 26 with a subsequent cytogenetic relapse. The reduction of BCR-ABL RNA at the time of first achieving CCR was significantly less in those patients with a subsequent cytogenetic relapse (median 1.4 log) compared to those with a sustained CCR (median 2.0 log) (P=0.002). In the 64 patients with a sustained CCR, the molecular response progressively improved over time to reach a median reduction of 4.0 log at 15 months after CCR. Of the 29 patients achieving at least a 2 log reduction of BCR-ABL RNA at the time of first reaching CCR, only 3 (10%) had a subsequent cytogenetic relapse. In comparison, 22 of 49 patients (45%) with a less than 2 log reduction at the time of achieving CCR had a subsequent cytogenetic relapse (odds ratio = 7.1; 95% CI 1.9–26). At the time of first achieving CCR, a reduction in BCR-ABL RNA of less than 2 logs thus had a diagnostic sensitivity of 88% and a diagnostic specificity of 49% for predicting subsequent cytogenetic relapse. Conclusions : We conclude that, in the majority of imatinib-treated CML patients reaching CCR, the level of BCR-ABL RNA at the time that the CCR is first achieved is a sensitive predictor of the durability of the CCR. The availability of a laboratory marker capable of stratifying the subsequent risk of disease progression (early in remission) will be useful in targeting additional (or alternative) therapies to those patients with the highest risk.
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Villela, Luis, Melani Otañez Arce, Carlos Best, Alicia Guzman, Elva Gabriela Estrada, Laura Rivera Mendoza, Juan Carlos Lopez Hernandez, Arianna Robles, and David Gomez-Almaguer. ""Ultrasensitive Reactive C-Protein As an Affordable Biomarker of Quantitative Expression of the BCR-ABL Transcript in Chronic Myeloid Leukemia. an Exploratory Study."." Blood 132, Supplement 1 (November 29, 2018): 5450. http://dx.doi.org/10.1182/blood-2018-99-119356.
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Abstract INTRODUCTION Real-time PCR (PCRq) quantification of the BCR-ABL transcript is the most reliable response marker in Chronic Myeloid Leukemia (CML), its levels have proven prognostic implications. However, in some centers it is still a difficult diagnostic tool to access. The ultrasensitive C reactive protein (pcrU) is an exquisite marker of inflammation and contextually the increased activity of the BCR-ABL transcript is associated with an inflammatory state mediated among others by IL-6. Here we explore the association between the molecular response (RM) induced with Imatinib mesylate and the serum levels of pcrU as a possible biomarker of leukemic activity. OBJECTIVE To explore the association between the quantitative expression of the BCR-ABL transcript and the serum levels of pcrU by turbidimetry. PATIENTS AND METHODS 15 patients with CML were studied, in 14 of them the BCR-ABL transcript was determined after 1 year of treatment by PCRq together with the serum levels of pcrU, any inflammatory state present during the sampling was ruled out. As a control 1 patient was studied at diagnosis as described. RESULTS There is a significant concordance between molecular status and serum levels of pcrU in 13 of the 15 patients studied (87% true negatives and 13% false positives). Table 1. When the diagnostic test was performed to evaluate pcrU as a possible MRI biomarker, we observed that it presents a sensitivity of 100%, specificity 84.6% (in an area under the curve of 93%), Positive Predictive Value (PPV) 50% and Negative Predictive Value (NPV) 100%. The probability index (+) = 6.5 (IC95%: 1.8 to 23.2), while the probability index (-) = 0. DISCUSSION The inflammation promoted by the tumor and the escape of the immunologically mediated tumor destruction, have been recognized as the hallmark of cancer and the myeloid cells are key players in this process. Therefore, the detection of an ultrasensitive biomarker of inflammation theoretically has the potential to detect leukemic activity. In this exploratory study a remarkable association was found between the molecular status and the levels of pcrU, making an extension of this study a goal to be pursued. Disclosures Best: Novartis: Consultancy; AbbVie: Consultancy. Gomez-Almaguer:AbbVie: Consultancy; Novartis: Consultancy.
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Reddy, Poluru L., Paul Choppa, Mike M. Moradian, Nicholas T. Potter, Michael M. Quigley, Christopher D. Watt, Holger Höfling, et al. "Evaluation of MMR Concordance Based on Either International Scale (IS) or National Comprehensive Cancer Network (NCCN) Criteria Using Reconstructed “Virtual” CML Patient Profiles From the REVEAL BCR-ABL Methods Comparison Study,." Blood 118, no. 21 (November 18, 2011): 3542. http://dx.doi.org/10.1182/blood.v118.21.3542.3542.
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Abstract Abstract 3542 Background: Achieving major molecular response (MMR) is an important milestone in chronic myeloid leukemia (CML) therapy. MMR has been defined as a 3-log reduction in BCR-ABL transcript levels from a standardized baseline (BL) established in the IRIS trial (Hughes TP, N Engl J Med. 2003). Standardization has been achieved through the development of an IS, which defines MMR as BCR-ABLIS = 0.1%. In contrast, the NCCN defines MMR as a 3-log reduction in BCR-ABL transcript levels but is indefinite on the definition of BL. Here, using reconstructed samples emulating CML patient BCR-ABL levels, the pairwise concordance of MMR determination was examined within and between 3 labs using the IS-standardized GeneXpert® (GX) system and 3 labs using laboratory-developed tests (LDTs). For comparative purposes, this analysis assumes BL is established at the time of diagnosis. Methods: 100 virtual patients (VPs) were emulated based on data from the REVEAL BCR-ABL Methods Comparison Study, in which 8 discrete levels of blinded K562 cell–spiked blood corresponding to BCR-ABLIS ratios ranging from ∼10% to ∼0.01% were analyzed by 3 labs using the IS-standardized GX system and 3 labs using non-IS LDTs. VP emulations were guided by actual patient outcomes in landmark analyses of 7- treatment response (Hughes TP, Blood. 2010). Treatment response profiles over an 18-month time horizon were modeled by assigning one of the 8 BCR-ABL levels ranging from approximately 10%-0.01% IS sampled in the REVEAL study to each of 4 virtual time points (eg, 3, 6, 12, and 18 months). BL levels were selected from quartiles representing pretreatment BCR- ABL ratios between 50–150%; results based on BL levels observed in the IRIS clinical trial will also be presented. 600 VP transcript profiles (VTPs) were then reconstructed using data from each of the 6 laboratories for all 100 VPs. The final 18-month time point in each VTP provided the BCR-ABL level against which the IS or NCCN objective criterion was applied to make MMR determinations. MMR concordance was evaluated by inspecting all possible inter-lab pairwise comparisons among the 100 VPs. Results: Pairwise concordance in MMR as determined by NCCN criterion among all 6 labs is shown in Fig 1A. MMR determinations among the 3 GX labs were concordant in 88% to 93% of VPs. In contrast, MMR determinations among the LDTs were concordant in 43% to 80% of VPs, and MMR determinations were concordant in 53% to 91% of VPs when compared between GX labs and LDTs. When MMR determination based on IS criterion for GX was considered, MMR concordance improved to 93% to 96% among the GX labs in contrast to 51% to 92% concordance observed between the GX and LDT sites (Fig 1B). It is noteworthy that Lab D results more closely approximated the IS than results from the other LDTs examined in the REVEAL study (data not shown). Although Lab D does not report results per the IS, it does report results relative to a median diagnostic BL, similar to the approach used in the IRIS trial. A healthcare system based on LDTs without any attempted IS standardization resulted in MMR concordance of only 43%. Potential sources of discordance among tests will be discussed in detail. Conclusions: These results illustrate that the NCCN criterion for MMR determination is not adequate for inter-lab comparisons of BCR-ABL transcript levels near the clinically important level of MMR. In contrast, standardization to the IS improves inter-lab concordance in MMR determination. Taken together, these results highlight the discrepancies that may result when comparing molecular responses between labs not standardized to the IS. As attainment of MMR is a critical milestone of CML therapy, errors in MMR determination may have an adverse impact on CML disease management. Disclosures: Reddy: Novartis: Research Funding, as Presenting Author, sponsorship to attend ASH. Höfling:Novartis: Employment. Manning:Novartis: Employment. Mignault:Novartis: Employment. Mullaney:Novartis: Employment. Ossa:Novartis: Employment. Stein:Novartis: Employment. Wang:Novartis: Employment. Yang:Novartis: Employment.
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Laudadio, Jennifer, Michael W. N. Deininger, Michael J. Mauro, Brian J. Druker, and Richard D. Press. "An Intron-Derived Insertion/Truncation Mutation in the BCR-ABL Kinase Domain in Three CML Patients Undergoing Kinase Inhibitor Therapy." Blood 110, no. 11 (November 16, 2007): 1953. http://dx.doi.org/10.1182/blood.v110.11.1953.1953.
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Abstract Although targeted inhibition of BCR-ABL with imatinib is an effective therapy for patients with chronic myeloid leukemia, a minority acquire mutations in the kinase domain (KD) that cause imatinib resistance. The spectrum of KD mutations thus far discovered, although quite heterogeneous, includes almost exclusively single nucleotide substitutions in key amino acids regulating drug binding or BCR-ABL function. Here, we describe a KD insertion/truncation mutation in 3 CML patients undergoing kinase inhibitor therapy. Two of these patients were being treated with imatinib (for 12 and 17 months), and one with dasatinib (for 13 months after a prior relapse while on imatinib). Suspected drug resistance was assessed by direct DNA sequencing of a BCR-ABL PCR product extending to the end of the kinase domain. Each of these 3 patients had 35 nucleotides from ABL intron 8 inserted at the normal exon 8–9 splice junction, after nucleotide 1423 (amino acid 475) of Genbank cDNA clone NM_005157. In all 3 cases, the mutation was co-expressed with wild type BCR-ABL sequence. The inserted sequence is derived from intron 8, beginning 1151 bp downstream from the normal splice donor site at the end of exon 8. This 35 bp intronic sequence is flanked by excellent consensus splice donor and acceptor sequences, suggesting alternative splicing as the likely mutational mechanism. The insertion creates a premature translational stop codon after 10 intron-encoded amino acids (figure), thus truncating 653 C-terminal amino acids including part of the KD and the entire last exon region - including a proline-rich domain, 3 nuclear localization signals, a DNA-binding domain, an actin-binding domain, and a nuclear export signal. These 3 insertion mutation cases were detected in our diagnostic clinical molecular pathology laboratory after sequencing 174 cases referred to us for suspected kinase inhibitor resistance, 78 of which contained a detectable mutation. The estimated prevalence of the exon 8/9 insertion/truncation mutation is then approximately 1.7% among patients with suspected drug resistance, and this mutation constitutes approximately 3.8% of all mutations. Conclusion: Kinase domain insertions are an alternative (and not entirely uncommon) mutational mechanism in CML patients undergoing kinase inhibitor therapy. The functional significance in terms of kinase activity and drug resistance remains to be addressed. Figure: Amino acid sequence of the C-terminus of the BCR-ABL kinase domain for the wild type and insertion/truncation mutant (with numbering as per GenBank cDNA clone NM_005157). Figure: Amino acid sequence of the C-terminus of the BCR-ABL kinase domain for the wild type and insertion/truncation mutant (with numbering as per GenBank cDNA clone NM_005157).
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Asif, Muhammad, Abrar Hussain, Abdul Wali, Nazeer Ahmed, Irfan Ali, Zafar Iqbal, Muhammad Amir, Muhammad Shafiq, and Mahmood Rasool. "Molecular, Cytogenetic, and Hematological Analysis of Chronic Myeloid Leukemia Patients and Discovery of Two Novel Translocations." Analytical Cellular Pathology 2021 (August 12, 2021): 1–19. http://dx.doi.org/10.1155/2021/4909012.
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Chronic myeloid leukemia (CML) is a disease of hematopoietic stem cells and is caused by the balanced translocations among the long arms of chromosomes 9 and 22, which are called the Philadelphia (Ph) chromosome. In this study, 131 CML patients were enrolled. Complete blood cell count was performed at the time of diagnosis for all the patients. Cytogenetic (karyotyping) examination using bone marrow samples was conducted on 76 CML patients for the confirmation of Ph-positive (9;22)(q34;q11) standard translocation, complex variant translocation, and additional chromosome abnormalities. FISH was performed on 38 patients for diagnostic purposes and on 39 patients for monitoring purposes. Twenty-two samples of CML patients were evaluated by reverse transcriptase PCR and real-time PCR for the patients who failed to respond against imatinib mesylate. In this study, 72 (54.96%) were males and 59 (45.03%) were females with a median age of 38.5 years. CBC values in the diagnosis process showed that 75 patients had high values of WBC being > 100 × 10 3 / μ l , while 71 (58.01) patients exhibited reduced values of hemoglobin, i.e., <10.00 mg/dl, and high values of PLTs > 100 were observed in 40 (30.53%) patients. Cytogenetic results show that standard translocation was developed in 63 (82.89%), development of complex variant translocations in 4 (5.32%), additional chromosomal abnormalities (ACAs) in 3 (3.94%), and ACAs together with complex variant translocations in 1 (1.31%) patient. At the time of diagnosis, 61 (92.95%) patients were in the chronic phase, 4 (5.63%) were in the accelerated phase, and only 1 (1.40%) was in the blast crisis. Out of twenty-two patients, only 6 CML patients who were shifted from imatinib mesylate to nilotinib showed BCR-ABL-positive amplification. However, only 7 out of twenty-one patients exhibit BCR-ABL gene values ≥ 1 after three months of follow-up when analyzed by the quantitative real-time PCR. In conclusion, we found a novel five-way translocation 46XX,t(1;2;2;17;9;22)(p36.3,q21;q11.2,q21,q34,q11.2) and a novel four-way complex variant translocation 48XY,+8(8;17)(9;22),+der(22)(q11.2;q23)(q34;q11.2) in the accelerated phase.
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Falchi, Lorenzo, Giorgia Desantis, Fabrizio Liberati, Roberta Pace, Maria G. Morandi, Patrizia Scaravaglio, Giuseppe Saglio, and Anna M. Liberati. "Ph’-Positive (Ph’+) Chronic Myeloid Leukemia (CML) Presenting as Ph’+ T-Lymphoblastic Lymphoma (LL) Resistant to High-Dose Chemotherapy and Gleevec." Blood 106, no. 11 (November 16, 2005): 4869. http://dx.doi.org/10.1182/blood.v106.11.4869.4869.
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Abstract BACKGROUND Extramedullary blast crisis (BC) of Ph’+ CML is infrequent and commonly affects bone, lymphoid tissue, skin and soft tissues and central or peripheral nervous system. Most of nodal lymphoid tumors occuring in the setting of CML derive from T-cell precursors and represent the evolution of CML to a lymphoid nodal BC. CASE HISTORY In February 2004, a 52 year-old patient underwent diagnostic wide biopsy of a nasopharingeal mass that caused severe acute respiratory symptoms. At the histological examination, the nasopharyngeal mucosa exhibited a diffuse pattern of infiltration by neoplastic cells with a characteristic single-file arrangement. The cells showed typical convoluted nuclei with one or two nucleoli and abundant cytoplasm (L2, lymphoblasts). The malignant cell population expressed a preT-cell immunophenotype: cytoplasmic CD3(+), CD43 (+), TdT (+/−), CD34(+), CD4(−), CD8(−). Thus, the diagnosis of T-cell LL was formulated. Whole-body CT scan revealed nasopharingeal mass, retropharyngeal, laterocervical, axillary, inguinal enlarged nodes and splenomegaly. Laboratory tests indicated leukocytosis (58,000/μl) with a differential WBC count typical of CML in chronic phase (myeloblasts <1%). This diagnosis was confirmed by a low alkaline phosphatase value (score: 1), histological features of bone marrow (BM) biopsy, classical cytogenetics (presence of the Ph’ in 100% of 25 metaphases analysed and absence of other cytogenetic abnormalities) and FISH evaluation (D-FISH bcr-abl in interphase and metaphase, Oncor probe). The nested RT-PCR (JQ Guo et al., Leukemia;2002,15:2447) disclosed the presence of the hybrid protein p210 (b2,a2), but not that of p190. A laterocervical node was excised to perform cytogenetic and molecular analyses in order to determine whether the T-cell LL was an unrelated disease or the expression of an extramedullary BC. The histology confirmed the presence of a uniform population of T-lymphoblasts in which classical cytogenetic analysis disclosed the following kariotype: 49,XY,t(9;22)(q34;q11),+9,+19,+der(22)t(9;22)(q34;q11) and 50–52,XY t(9;22)(q34;q11),+6+9,+9q+,+19,+20,+22q−. The FISH analysis showed the presence of bcr-abl gene in all cells analyzed and the presence of multiple copies of this gene as well as of double Ph’ chromosome. Nested RT-PCR showed the presence of both p210 and p190 transcripts. These findings indicated that the T-cell LL was an extramedullary BC of a CML simultaneously diagnosed in chronic phase in BM and peripheral blood. The patient was initially treated with vincristine, daunomicine, asparaginase and prednisone combined with Gleevec (800mg/die). Consolidation therapy, consisting of high-dose Ara-C (4g/m2 for 4 days) and mithoxantrone (10mg/m2 for 2 days), followed by a mieloablative course with mithoxantrone (60 mg/m2) and melphalan (180 mg/m2) and autologous stem cell support (ASCT) was administered after an initial clinical response. Gleevec was given during the entire treatment period. A fugacious complete clinical-hematological and a partial cytogenetic (FISH: 12% of cells bcr-abl+) and molecular (number of bcr-abl/104 ABL copies= 184, real-time quantitative RT-PCR: J Gabert et al., Leukemia;2003:1) remission was documented after ASCT. The patient refused further consolidation treatment with Gleevec and died, due to progressive disease, in December 2004.
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Lopez, Jose Luis, and Hector Joel Rico. "Long Term Molecular Response To Triple Therapy With High Doses Imatinib In Patients With Chronic Myeloid Leukemia." Blood 122, no. 21 (November 15, 2013): 5199. http://dx.doi.org/10.1182/blood.v122.21.5199.5199.
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Abstract Introduction Imatinib 400 mg daily is considered the best initial therapy for patients with chronic myeloid leukemia (CML) in the chronic phase (CP). However, only minorities of patients have a complete molecular remission (CMR) Another agent has antileukemic activity against Bcr-Abl-positive cells like Ara-C and interferon, the association of this drugs and imatinib in CML was evaluated in several trials with an increase in molecular response. The aim of this study was to evaluate the major molecular response (MMR) at 12 months with triple treatment schedule, analyze the evolution of these patients and general and hematologic toxicity. Material and Methods Patients diagnosed with CML at the Hospital General de Zona #35 in Juarez, Mexico were included. Eligibility criteria were adults with diagnosis of CML chronic phase on triple regimen for at least 12 months: Pegylated interferon-α 2a 90mcgrs via subcutaneous / week for 4 weeks + PO imatinib 800 mgs a day for 30 days + 20 mgs/mt2 cytarabine from day 1 to 10 subcutaneus. Patients were stratified according to Sokal score at diagnosis. The molecular analysis was performed in Quest diagnostic laboratory by means of real-time quantitative polymerase-chain-reaction (RT-PCR) results are expressed as a percent ratio of BCR-ABL1 to ABL1 and further adjusted to the international scale (IS) since august 2012. Patients could have received previous treatment for CML, with the exception of bone marrow transplantation. All patients provided written informed consent. This study was conducted in accordance with the Declaration of Helsinki. Molecular and adverse events were assessed. An analysis of molecular response at 12 months was planned and follows up patients with MMR every year. A MMR was defined a Bcr-Abl 0.1% or less and complete molecular response (CMR) as undetectable. Hematological toxicity was assessed according WHO scale. Results 41 patients completed the first 12 months in therapy, with a mean age of 44.4 years (17 to 71) 51% male and 49% female, the median and ranges of hemoglobin levels, leukocyte and platelet counts at diagnosis were 10.2 g/dl (5.1-16.0), 209.000 μL3 (10,600 - 529.000) and 565.500 μL (130.000 to 4,272,000) respectively. The percentages of cases by Sokal risk group were 70.7% low, 24.4% intermediate and 4.9% high risk. The Median follow up time was 58 months (range 14 to 120). At 12 months the number of patients who were in MMR was 27 (65.9%) including 8 (19.5%) with no BCR-ABL detectable. Median duration of triple therapy exposure at first year was 24 Weeks (range 12 to 32) Responses by Sokal score were 62%, 70% and 100% for low, intermediate and high respectively. Adverse events occurred in 88% cases; 33% of patients has at least one adverse event (AE) 42% 2 EA and 28% 3 EA, the most important EA was gastrointestinal. (table 1) 43.9% of patients has Hematological toxicity III-IV Median follow up time of patients in RMM was 64 months (range16-120) 2 patients were no evaluable. Patients who have RMM at 12 months 50% achieve a CMR at last follow up, 33% continues in RMM and 17% loss molecular response. Patients with CMR 72% have undetectable bcr-abl, 14% have loss molecular response and 14% in MMR Conclusions In this group of patients MMR was achieved in a higher proportion of cases at 12 months of treatment which is important in the long-term prognosis. Side effects grade 3 and 4 hematologic and non-hematologic were significant in this series of cases appearing in 44 and 88% respectively, which requires close monitoring of patients. The combination of interferon α2a, cytarabine and high-dose imatinib induces a MMR of 66% at 12 months of treatment, a 28%, 56% and 16% in MMR, CMR and loss molecular response respectively at last follow up. Clinical files n =36 Disclosures: No relevant conflicts of interest to declare.
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Preuner, Sandra, Margit Bernroitner, Gerlinde Mitterbauer, Christine Mannhalter, Susanne Herndlhofer, Wolfgang R. Sperr, Gerald Webersinke, Peter Valent, and Thomas Lion. "Proliferation Kinetics of Subclones Carrying Point Mutations In the BCR-ABL TKD During TKI Treatment In CML Patients: Quantitative Monitoring by LD-PCR." Blood 116, no. 21 (November 19, 2010): 2269. http://dx.doi.org/10.1182/blood.v116.21.2269.2269.
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Abstract Abstract 2269 Point mutations in the BCR-ABL tyrosine kinase domain (TKD) are an important mechanism of resistance to treatment with tyrosine kinase inhibitors (TKIs). However, detection of mutant subclones may be difficult to interpret with regard to the risk of imminent onset of resistant disease. The biological relevance of several mutations is not well understood, and some may indeed be biologically neutral (bystander mutations). Moreover, even subclones carrying mutations known to confer resistant disease (driver mutations) may disappear below the detection limit without any alteration of treatment. Surveillance of the size of mutant subclones during treatment could therefore provide better understanding of their biological behavior in vivo, and facilitate timely assessment of impending resistant disease. To address this issue, we have established a ligase-dependent (LD) PCR technique permitting quantitative monitoring of point-mutated subclones (Preuner et al. Leukemia 2008). The LD-PCR approach displays a detection limit ≥1%, and permits reproducible assessment of changes in the size of mutant subclones exceeding ±5%. We have investigated a series of prospectively collected PB specimens derived from CML patients displaying a variety of mutations including M244V, L248V, G250E, Q252H, E255K, T315I, F317L-A/G, M351T, and F359V with the aim to monitor the proliferation kinetics of mutant leukemic cells during the course of treatment. Our observations revealed that a) the appearance of new mutant subclones at a level detectable by LD-PCR occurred as early as a few weeks after initiation of treatment with a TKI, b) the time span until mutant leukemic cells became the dominant BCR-ABL positive clone was variable, ranging between 1–18 months following first detection, c) different mutant subclones appeared sequentially in individual patients upon changes of treatment (Fig.), d) disappearance of a dominant subclone carrying the highly resistant mutation T315I could be documented, e) the response of mutant subclones to treatment and the rise of new mutant subclones could be readily documented by the quantitative LD-PCR approach, f) changes in total BCR-ABL transcripts assessed by RQ-PCR mostly occurred in parallel to corresponding kinetics of mutant subclones, but expanding mutant clones could also be observed at a time when BCR-ABL transcripts were still decreasing, and g) documentation of expanding mutant subclones occasionally preceded the rise in BCR-ABL transcripts by several weeks. Quantitative mutational screening revealed the appearance and expansion of expected mutations, such as G250E, M244V during treatment with imatinib, the F317L on dasatinib or E255K on nilotinib, but also unexpected findings such as selection and outgrowth of a subclone carrying the L248V mutation upon onset of nilotinib. Our observations indicate that quantitative monitoring of mutant subclones during treatment with TKIs provides information on their responsiveness to therapy and the imminent onset of resistant disease. Judicious implementation of quantitative diagnostic approaches such as the LD-PCR technique in the surveillance of CML patients can improve our current options for timely treatment decisions, and could help optimizing disease management in patients displaying point mutations in the BCR-ABL TKD or other sites of potential relevance. After inadequate molecular response to different doses of imatinib (solid line=BCR-ABL1IS transcript levels),a subclone carrying a F317L-A mutation (dashed line) appeared after onset of dasatinib. This subclone disappeared upon switch to nilotinib, but concomitantly, a subclone carrying the T315I mutation (dotted line) evolved rapidly and became the dominant BCR-ABL positive clone. Fig. Example of consecutive occurrence of mutant subclones during treatment with TK inhibitors. Fig. Example of consecutive occurrence of mutant subclones during treatment with TK inhibitors. Disclosures: No relevant conflicts of interest to declare.
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Mori, Silvia, Elisabetta Vagge, Philipp le Coutre, Elisabetta Abruzzese, Bruno Martino, Ester Pungolino, Chiara Elena, et al. "Validation of Digital-PCR Analysis through Programmed imatinib Interruption in Q-RT-PCR Negative Chronic Myeloid Leukemia Patients." Blood 122, no. 21 (November 15, 2013): 4040. http://dx.doi.org/10.1182/blood.v122.21.4040.4040.
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Abstract Introduction Imatinib induces complete cytogenetic response (CCyR) in up to 80% of chronic myeloid leukemia (CML) patients (pts) and major molecular response (MMR) in 33-60% of them. These patients enjoy life expectancy similar to general population. However even undetectable BCR-ABL may not equate to eradication of the disease because the sensitivity of the standard diagnostic method, the Q-RT-PCR, is limited. A new diagnostic method, the digital-PCR (dPCR), able to detect 1 BCR-ABL+ cell out of 107 cells, corresponding to a 100 times increased sensitivity as compared to conventional Q-RT-PCR, was developed (Goh HG et al., Leuk Lymphoma 52(5): 896-904. 2011). Therefore dPCR, assessing with more sensitivity the presence of minimal residual disease, could potentially identify pts in whom CML is eradicated. The Imatinib Suspension And Validation (ISAV) study is aimed at assessing the capability of dPCR to predict relapses after imatinib discontinuation in CML pts with negative Q-RT-PCR results. Methods This study involves 15 sites, 10 in Italy and 5 in each of the following countries: Germany, Spain, The Netherlands, Canada and Israel. In this study CML patients (Chronic Phase or Accelerated Phase) under imatinib therapy since more than 2 years and in complete molecular remission (CMR) were eligible. Patients had to be in CMR for at least 18 months, with a minimum of 3 Q-RT-PCR performed in their own centers. After signing the informed consent, pts were tested for dPCR and discontinued imatinib therapy. They are being monitored by standard Q-RT-PCR for 36 months to assess the maintenance of the molecular remission. At the end of this period, a peripheral blood sample for dPCR analysis will be obtained from those pts who will still have undetectable BCR-ABL transcripts by Q-RT-PCR, to verify CML eradication. The loss of molecular remission is defined as two consecutive positive Q-RT-PCR tests with at least one BCR-ABL/ABL value above 0.1%. Patients losing molecular remission resumed imatinib treatment at the same dosage used before interruption. Patients’ quality of life during imatinib discontinuation/resumption is being evaluated trough the EORTC – C30 Quality of Life questionnaire. Results The enrollment in the ISAV study began in November 2011 and ended in June 2013. The study enrolled 112 pts: Italy 69.6%, Berlin 21.4%, Montreal 5.3%, Zaragoza 2.6% and Tel Hashomer 0.9%. Sixty-one percent of the pts were male and 38% were aged 65 or older; median duration of imatinib treatment is 102 months with median duration of CMR of 32 months before imatinib discontinuation. To date, the median follow-up (FUP) time is 4.6 months [95% CI: 4.1-5.8] and 92 pts out of 112 (82%) had at least one Q-RT-PCR performed after imatinib discontinuation. The following analysis is restricted to 48 pts with a minimum of 6 months of FUP. Of these 48 pts, 20 remained Q-RT-PCR negative (42%, 95% CI:29-56%, median duration of negativity after imatinib discontinuation: 10.3 months). Nineteen pts (40%, 95% CI:27-53%) relapsed and resumed imatinib. All relapses occurred in the first 10 months and all but 3 of them in the first 6 months. A loss of CCyR happened in 5 pts out of 19 (26%): 1 pt regained CCyR after 3 months of re-treatment and is now in CMR, 1 pt died shortly after the diagnosis of relapse because of lung adenocarcinoma and 3 pts are now being monitored after imatinib resumption. No case of progression of CML was observed. After the resumption of imatinib the median time to either MMR or CMR, whichever came first, was 2.1 [95% CI: 0.9-5.8] months. Finally, nine pts (18%, 95% CI:10-31%) regained Q-RT-PCR positivity but never lost MMR. The median time to Q-RT-PCR positivity in this group of pts was 2.92 months (range 1-5 months), and the range of duration of Q-RT-PCR positivity (below 0.1%) is between 2 and 14 months. No significant correlation between relapse and previous duration of imatinib treatment, time to CCyR or duration of CMR was present. Patients previously treated with interferon showed a trend toward lower risk of relapse which is not significant so far. Finally, 19% of pts complained of musculoskeletal/articular pain after imatinib discontinuation. Conclusions After 21 months from the beginning of the study with a median follow-up of 4.6 months, 40% of pts relapsed; the majority of relapses happened in the first 6 months after imatinib discontinuation. The correlation of dPCR results with clinical outcomes will be presented at the meeting. Disclosures: le Coutre: Novartis: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Novartis: Research Funding. Gozzini:Novartis: Consultancy; Bristol Myers Squibb: Consultancy.
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Baba, Shahid Mudasir, Manzoor Ahmad Malik, Rafia Anjum Baba, Javid Rasool Bhat, M. Aleem Jan, Sajad Ahmad Geelani, and Zafar Amin Shah. "A case series of patients with de novo Philadelphia-positive chronic myelogenous leukemia at tertiary care center SKIMS." JMS SKIMS 19, no. 2 (July 2, 2018): 82–84. http://dx.doi.org/10.33883/jms.v19i2.293.
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The well-established molecular pathogenesis of chronic myelogenous leukemia (CML) and its consequences for laboratory testing and clinical management illustrate a classic paradigm for the importance of molecular diagnostics in targeted drug therapy. The success of the tyrosine kinase inhibitor (TKI), Imatinib, as the currently recommended first-line treatment of early chronic phase CMLhas both fueled the need for timely and reproducible molecular testing of the BCR-ABL fusion transcript in diagnosis and monitoring. As ongoing research continues to refine guidelines for monitoring residual disease in patients undergoing TKI therapy, an understanding of molecular technologies and their interpretation is critical. In this regard, we at Cancer Diagnostic and Research Centre, Immunology and Molecular Medicine established a gold standard RT-PCR and RQ-PCR technique to diagnose and monitor residual disease in CMLpatients. JMS 2016; 19(2):82-84
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Jovanovic, J., A. Reiter, D. Cilloni, E. Gottardi, J. Score, H. Popp, G. Metzgeroth, et al. "Sensitive Detection of FIP1L1-PDGFRA Fusion Transcripts by Real-Time Quantitative RT-PCR (RQ-PCR) Reveals Achievement of Molecular Remission in Chronic Eosinophilic Leukemia Treated with Low-Dose Imatinib Therapy." Blood 106, no. 11 (November 16, 2005): 483. http://dx.doi.org/10.1182/blood.v106.11.483.483.
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Abstract The FIP1L1-PDGFRA fusion gene generated by a cryptic interstitial deletion at 4q12 is a recurrent molecular lesion in idiopathic hypereosinophilic syndrome (HES), that forms a basis for diagnosis of chronic eosinophilic leukemia (CEL). This disease entity is particularly important to recognize, since presence of the FIP1L1-PDGFRA fusion predicts a favorable response to molecularly targeted therapy in the form of imatinib, with clinical remissions being achieved with lower doses than are required in BCR-ABL+ chronic myeloid leukemia (CML). In order to improve our understanding of the biology of CEL and to provide a tool to improve the management of patients with this disorder we have developed real-time quantitative reverse transcriptase PCR (RQ-PCR) assays for the FIP1L1-PDGFRA fusion. Taking into account the marked heterogeneity in upstream breakpoints within the FIP1L1 gene, RQ-PCR assays were designed for cases with FIP1L1 breakpoints leading to fusion of exon 9, 10, 11, 12 or 13 to PDGFRA exon 12. FIP1L1-PDGFRA expression was compared to that of validated Europe Against Cancer endogenous control genes - β2microglobulin (B2M) and ABL. Serial dilution of the FIP1L1-PDGFRA+ EOL1 cell-line in fusion gene negative filler cells (HL60) revealed an assay sensitivity of 1 in 105. While identification of the FIP1L1-PDGFRA fusion using conventional RT-PCR approaches can be problematic, RQ-PCR analysis undertaken in diagnostic samples from 31 patients with FIP1L1-PDGFRA+ CEL (median age 53, 31–64 years) revealed that, in the majority, the fusion transcript was expressed at relatively high level (median deltaCt FIP1L1-PDGFRA - B2M, 12.2; median deltaCt FIP1L1-PDGFRA - ABL, 2.3). The FIP1L1-PDGFRA fusion was expressed at comparable level in blood and marrow at diagnosis of CEL. Serial monitoring was undertaken in 17 patients following initiation of imatinib 100mg/d. Overall, 8/8 evaluable patients achieved at least a 3-log reduction in FIP1L1-PDGFRA fusion transcript level within the first year of therapy. In follow-up samples affording a sensitivity of at least 1 in 1000, PCR negativity by quantitative and conventional nested RT-PCR was documented in 8/17 patients following a median of 21 weeks of imatinib (4–64 weeks); in two cases profound PCR negativity (i.e. at a sensitivity level of at least 1 in 105) was documented following 13 weeks and 2 years of imatinib, respectively. Overall, these data demonstrate that CEL with the FIP1L1-PDGFRA fusion is uniquely sensitive to imatinib therapy. This contrasts with BCR-ABL+ CML, in which molecular remission is generally not achieved - a phenomenon that has been postulated to reflect the persistence of a primitive quiescent stem cell population that is resistant to this agent. Understanding the biological basis for the differences in molecular response to imatinib in CML and CEL, may yield further improvements in molecularly-targeted therapeutic approaches.
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Shuvaev, Vasily, Mikhail Fominykh, Vera Udaleva, Irina I. Zotova, Regina Golovchenko, Dzhariyat Shikhbabaeva, Irina Martynkevich, et al. "The Devil Is Not As Black As He Is Painted - 3-Year Experience of Treating Newly Diagnosed CML Patients with Imatinib Generics." Blood 128, no. 22 (December 2, 2016): 5441. http://dx.doi.org/10.1182/blood.v128.22.5441.5441.
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Abstract Introduction. A generic drug is a pharmaceutical drug considered to be equivalent to a brand-name product. A generic drug has to contain the same active ingredients as those of the original formulation. Regulatory agencies used to require that generics be identical to their brand-name counterparts with regards to pharmacokinetic properties. In most cases, generic products are available after the patent protection given to a drug's original developer expires. In Russia, a patent protection lasts for a 10-year period from registration of the original drug. To this day, twelve Imatinib generics have been registered in Russia. Aim. To assess the safety and efficacy of Imatinib generics for treatment of newly diagnosed Chronic myelogenous leukemia patients that have been in our center since August 2012. Materials and methods. 30 newly diagnosed CML patients were started on generics. The drugs: 1) GenericPh 100 mg, in capsules (Ph-Syntez, Russia); 2) GenericG 100 mg, in tablets (Laboratorio TUTEUR S.A.C.I.F.I.A., Argentina); 3) GenericIm 100 mg, in tablets (Sandoz d.d. (Slovenia). Switching from one generic to another was done due to intolerance. We analyzed the range and frequencies of adverse events (AE), cumulative incidences of complete hematologic (CHR), major cytogenetic (MCyR), complete cytogenetic (CCyR), and early molecular responses (BCR-ABL<10% by IS), as well as the rate of BCR-ABL<1% by IS, major molecular (MMR) and molecular 4.0 log (MR4.0, BCR-ABL<0.01% by IS) responses at time-points according to the National CML diagnostic and treatment guidelines. The response rates were assessed only in regard to the generic treatment (with death, progression and switching to second-generation inhibitors as competing risks). Statistical analysis included descriptive statistics and cumulative incidence function. Results. Duration of the treatment with generics was 7-45 months, with a median of 13 months (GenericPh (27) + GenericG (2) + GenericIm (1)). No unexpected adverse events were observed during the Imatinib generics treatment. The generics tolerance did not differ from that of the original brand-name drug. Six patients were switched to second-generation tyrosine kinase inhibitors (TKI2) due to Imatinib intolerance. One patient progressed to blastic phase at 3 months after diagnosis. Three deaths were registered (1 - due to CML and 2 due to concomitant diseases). Overall survival rate was 90% and CML-related mortality - 3%. CHR at 3 months of the treatment was achieved in 93% of the patients. Cumulative response rates for cytogenetic and molecular responses are presented in Table 1. MR4.0 was registered in 23% of patients during overall treatment. Seven patients were switched to TKI2 due to insufficient efficacy of Imatinib. At the time of analysis 13 patients remained on Imatinib generics treatment: 12 patients with CCyR and 1 with PCyR, including 10 patients with MMR. Conclusion. Use of generics demands evaluation of its equivalency and control during its adoption into clinical practice. In terms of efficacy or tolerance no significant differences between the Imatinib generics studied and the original brand-name drug in newly diagnosed CML patients were found. Disclosures Shuvaev: Pfizer: Honoraria; BMS: Honoraria; Novartis pharma: Honoraria. Fominykh:BMS: Honoraria; Novartis Pharma: Honoraria.
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Schenk, Thomas, Martin C. Müller, Alice Fabarius, Philipp Erben, Thomas Ernst, Claudia Haferlach, and Andreas Hochhaus. "Association of Interphase Fluorescence in Situ Hybridization (IP-FISH) Response Criteria with Conventional Metaphase Cytogenetics in CML Patients On Therapy." Blood 114, no. 22 (November 20, 2009): 4707. http://dx.doi.org/10.1182/blood.v114.22.4707.4707.
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Abstract Abstract 4707 The Philadelphia (Ph) chromosome and its molecular equivalent, the BCR-ABL fusion gene, represent the pathogenetic cause and a useful marker for diagnosis and follow up monitoring of chronic myeloid leukemia (CML). Cytogenetic analysis of bone marrow metaphases (Cy) has been established as the standard method. In contrast, interphase fluorescence in situ hybridization (IP-FISH) has been increasingly applied in many studies due to recent optimization of the technique but is not represented in current treatment guidelines. We therefore sought to define IP-FISH response criteria which correspond best with complete (CCyR) and major cytogenetic responses (MCyR). In order to quantitatively compare results of both methods 1,749 consecutive non selected bone marrow samples from 748 CML patients at different stages of CML were analyzed in parallel with Cy and IP-FISH. 5 patients with Ph negative/BCR-ABL positive CML were excluded from the analysis. 643 patients in chronic phase (CP) were analyzed during imatinib based therapy, ten patients received interferon alpha. 74 patients at different stages of the disease received 2nd generation tyrosine kinase inhibitors: nilotinib, n=18 (CP, n=13; accelerated phase, AP, n=2; blast crisis, BC, n=3); dasatinib, n=56 (CP, n=41; AP, n=4; BC, n=11). 21 patients received no therapy or the therapy was not evaluable. The correlation between Ph positive metaphases and the proportion of FISH positive interphase cells was determined using the Spearman's rank correlation coefficient. The chi-square test was used to compare IP-FISH and Cy data. The optimally separating threshold value between Cy and IP-FISH was chosen as cut-off point. Cy and IP-FISH data correlated well (r=0.89; p<0.0001). The following cut-off values were defined: '30% IP-FISH positivity was found to correspond best with MCyR ('35% Ph+ metaphases); <6% IP-FISH positivity was concordant with CCyR (0% Ph+ metaphases). Of 1,163 samples of patients in CCyR, 99.1% showed a percentage of <6% IP-FISH positive cells. 82 of 1,163 samples (7.0%) with 0% Ph+ metaphases by Cy were IP-FISH positive (median 3%, range, 1-21% positive interphases). IP-FISH showed false negative results in 10 of 1,090 samples (0.9%) with a median of 8% Ph+ metaphases (range, 4-40%). Using these IP-FISH cut-off points, the diagnostic specificity for the definition of CCyR was 93.8% for all patients and 93.7% for CP pts only and for the definition of MCyR 89.4% for all patients and 88.4% for CP patients only, respectively. In conclusion, BCR-ABL FISH data derived from bone marrow interphase cells are comparable with metaphase cytogenetics but the cut-off points differ. IP-FISH might be used instead of Cy in order to assess the achievement of response milestones in CML patients during therapy. The prognostic value of IP-FISH data, however, should be analyzed in prospective controlled trials. Disclosures: No relevant conflicts of interest to declare.
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Wang, Jishi, Sixi Wei, Yating Wang, Qixiang Chai, Qin Fang, Yaming Zhang, and Yinghao Lu. "Over-Expression of Heme Oxygenase-1 in Peripheral Blood Predicts the Progression and Relapse Risk of Chronic Myeloid leukemia." Blood 124, no. 21 (December 6, 2014): 5181. http://dx.doi.org/10.1182/blood.v124.21.5181.5181.
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Abstract Background There are limited eligible clinical markers at present to monitor the progress of chronic myeloid leukemia (CML). Heme oxygenase-1 (HO-1), as one of the most important oxidation-regulating enzymes in vivo, suggests the onset and progression of cancer when highly expressed. Furthermore, HO-1 level is related with the occurrence and development of hematological diseases. But the relationship between HO-1 expression and progression/relapse of CML has seldom been studied hitherto. This study aimed to investigate the relationship between them to find out a new molecular marker for prediction. Methods A total of 60 peripheral blood and bone marrow (BM) samples from 25 CML patients in different phases were collected respectively to detect the expressions of HO-1 and bcr/abl using real-time PCR. Routine blood test was performed to detect the changes of leukocyte and platelet counts. The proportion of primitive cells in BM was detected by flow cytometry. The relationship between high HO-1 expression and CML progression and relapse was explored by the analysis of variance by Wilcoxon test and linear regression analysis. The diagnostic accuracy and cutoff values were determined by receiver operating characteristic curve. Results Relative expression of HO-1 mRNA in CML patients peripheral blood was significantly higher than that of donors (P <0.0001), which were 0.57±3.78 and (1.417±1.125)×10–6, respectively. HO-1 expression level in CML patients was 0.061 5±0.062 4, which decreased to 0.009 4±0.006 7 upon CMoR, and remained remarkably higher 0.016 3±0.017 5 than that of normal donors (1.417±1.125)×10–6, P <0.001. When relapse occurred, HO-1 expression significantly increased from 0.020 6±0.021 0 to 3.852±10.285 in CMoR stage and undergoing relapse. According to progression of CML, HO-1 expression level in CML patients increased from CP (0.009 5±0.017 6) to AP (0.028 0±0.055 7) and then to BP (0.276 7± 0.447 0) . And there was a linear correlation between HO-1 expression and proportion of primitive CML cells. The diagnostic accuracies and cutoff values of HO-1 expression for CML-CP, CML-AP, and CML-BP were 1.0, 0.748, and 0.965, respectively, as well as 0.000 070, 0.001 917, and 0.020 696, respectively. Conclusion HO-1 may be a potential molecular indicator for the progression and relapse of CML. Disclosures No relevant conflicts of interest to declare.
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Kulikov, Sergey M., Olga Yu Vinogradova, Ekaterina Yu Chelysheva, Marya V. Galayko, Irina A. Titshenko, Eduard G. Gendgan, Olga M. Senderova, et al. "First Results of Russian Multicenter Population Base Study of the Incidence of Chronic Myeloid Leukemia." Blood 120, no. 21 (November 16, 2012): 4431. http://dx.doi.org/10.1182/blood.v120.21.4431.4431.
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Abstract Abstract 4431 The European Treatment Outcome Study (EUTOS) is register based international investigation started in June 2007. [1] The aim is to study the epidemiology of CML and to gain insight into the ‘real world’ treatment of patients with CML. Population base section (EUTOS-PBS) is the prospective study directed mostly to epidemiology aims. Russian part of the EUTOS-PBS registry collect data of newly diagnosed patients lived in 7 large regions of about 10 millions of population totally. EUTOS-PBS inclusion criteria are following: newly diagnosed CML (Ph +/BCR-ABL) started form 1st October 2009, age: older than 18. Russian CML group includes additional protocol for collection data for patients with clinical symptoms of CML. These patients are included into the roster table and after laboratory confirmation are enrolled into the main phase of the study. Thus, 174 patients were included in pre-phase, 142 (82%) had the diagnosis of CML which was confirmed by cytogenetic/molecular-genetic tests (Ph +/BCR-ABL +), 32 (18%) was not confirmed as CML. Among them 87% (n = 20) - have other Ph–negative chronic myeloproliferative diseases, and also acute leukemia (n = 1), cancer (n = 1), chronic inflammatory processes (n = 1). 142 patients with CML are 73 men, 69 women have the age from 18 to 82 (Me 49) years. 136 (96%) of patients are in the chronic phase, 6 (4%) -in the phase of acceleration, nobody in a blast ñrisis. The standard frequency analysis with adjustment to the standard population of WHO was carried out to estimate distribution. The results was presented in the table 1. As shown registered morbidity in 6 regions is not varied so much: source incidence is 0,58 (0,44–0,69); standardized on WHO incidence is: 0,7 (0,57 – 0,85); per 100 thousands per year. Estimated registered morbidity of CML in Russian regions are in 1.5–2 times less, than published morbidity in western countries. The analysis of the incidence in age stratums (table 2) shows that there is no much growth of age morbidity as expected. It obviously points to low detectability of new CML incidents in senior age categories (are more senior 60 years). This fact is probably the main reason of low total registered morbidity. Tabl.1. Incidence rate of new CML cases in 6 regions of Russia Region population (mln.) N CML for 100000. in year Standard of WHO Mordovia Republic 0.87 14 0.69 0.85 Kirov region 1.46 18 0.53 0.6 Perm territory 2.77 45 0.68 0.8 Bryansk region 1.35 17 0.53 0.65 Irkutsk region 2.55 36 0.56 0.68 Zabaikal's territory 1.36 12 0.44 0.57 Total 10.13 142 0.58 0.7 Table 2.CML incidence in age groups Age groups Male Female Maleandfemale 18–29 0.65 0.57 0.61 30–39 0.86 0.39 0.62 40–49 0.50 0.57 0.54 Conclusion: The CML incidence in Russia regions is underestimated. The main reason is an insufficient CML diagnostic screening in the senior age groups of population. References. 1. http://www.eutos.org/content/registry/documents/documents/e940/infoboxContent941/CML-Registry_February09.pdf Disclosures: Vinogradova: BMS: Consultancy, Speakers Bureau; Novartis: Consultancy, Research Funding, Speakers Bureau. Chelysheva:Novartis Pharma: Research Funding, Speakers Bureau; Bristol Myers Squibb: Research Funding, Speakers Bureau; MSD: Speakers Bureau. Senderova:Novartis: Consultancy. Turkina:Novartis Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.
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James, Chloé, François Delhommeau, Christophe Marzac, Irène Teyssandier, Jean-Pierre Le Couédic, Stéphane Giraudier, Lydia Roy, et al. "Detection of JAK2 V617F in the Diagnosis of Erythrocytosis: Feasibility and Diagnostic Value in Clinical Practice." Blood 106, no. 11 (November 16, 2005): 2595. http://dx.doi.org/10.1182/blood.v106.11.2595.2595.
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Abstract Polycythemia Vera (PV) is a myeloproliferative disorder (MPD), whose diagnosis is currently based on an association of clinical and biological criteria following the WHO or the PVSG classification. It is characterized by a primitive absolute erythrocytosis, and formation of endogenous erythroid colonies (EEC). Recently, we described a point mutation in JAK2 (JAK2 V617F) and showed that this activating mutation was the cause of the disease (James et al, Nature, 2005). This molecular abnormality was therefore likely to be a diagnostic marker of PV, as bcr-abl for chronic myeloid leukemia (CML). Nevertheless, JAK2 V617F is also found in other MPDs, sharing some common features with PV, as essential thrombocythemia, idiopathic myelofibrosis, and other rare MPDs. One major criterion for the diagnosis of PV requires the demonstration of increased red cell mass as measured by isotopic methods. We assessed the value of detection of JAK2 V617F as a first intention diagnostic test in 88 patients with hematocrit values above 51% (=erythrocytosis) and showed that the mutation correlated with the diagnosis of PV according to the WHO (R=0.879) and the PVSG (R=0.717) criteria, with a positive predictive value of 100% in the context of erythrocytosis. Besides, the presence of the mutation strongly correlated with EEC formation in 81/87 patients (R=0.824) and only weakly with the serum erythropoietin level (R=0,416). PCR-based genotyping techniques are less time-consuming, less expensive, than DNA sequencing and are easier to perform in hematology diagnostic laboratories. Therefore, we studied the feasibility of the detection of JAK2 V617F with widespread instruments commonly used in routine. We analyzed 119 samples from patients with a suspicion of myeloproliferative disease and showed that JAK2 V617F was efficiently detected by LightCycler® and TaqMan® genotyping technologies, these latter being a little more sensitive than sequencing. For 50 patients, peripheral blood and bone marrow samples were both available. In all cases (34 mutated, 16 non-mutated) the mutation was identically detected. Based on these results, we propose that the detection of JAK2 V617F in granulocytes has a first place in the diagnostic chart of an erythrocytosis, as bcr-abl in CML, avoiding, if positive, an isotopic red cell mass measurement and bone marrow EEC assays. The presence of JAK2 V617F in a patient with erythrocytosis would then lead to the diagnosis of MPD of PV type. Further prospective studies will be necessary to assess if all the MPDs patients bearing JAK2 V617F can be grouped in a new subset within the MPD entity, especially in term of thrombotic and neoplasic risk.
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Markert, Eva, Udo Siebolts, Margarete Odenthal, Karl-Anton Kreuzer, and Claudia Wickenhauser. "High diagnostic value of morphologic examination and molecular analysis of bone marrow biopsies in a case of BCR-ABL+ CML with clusters of blasts." International Journal of Hematology 89, no. 3 (February 20, 2009): 294–97. http://dx.doi.org/10.1007/s12185-009-0257-x.
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Dolata, MD, Wojciech K. "Megakariocytic Hypoplasia Due To TKIs In a CML Patient Can Be Overcome By TPO Receptor Agonist Eltrombopag." Blood 122, no. 21 (November 15, 2013): 5178. http://dx.doi.org/10.1182/blood.v122.21.5178.5178.
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Background Patients who develop severe thrombocytopenia while treated with TK inhibitors may be forced to stop them, face a worse prognosis, and eventually require a treatment with a bone marrow transplant. Eltrombopag is a TPO receptor agonist that stimulates megakaryocytic proliferation. Case 62-year-old man presented in October 2008 with splenomegaly and leukocytosis. The bone marrow biopsy was diagnostic of CML. Imatinib was started at 400 mg daily resulting in hematological response and resolution of splenomegaly, but severe thrombocytopenia necessitated 50 % dose reduction. Thrombocytopenia was not responsive to Prednisone, and a trial of platelets transfusions with the standard dose of Imatinib had to be stopped because of thrombocytopenia below 10 K and the risk of bleeding – reducing the dose of the TKI was improving the platelet count. Treatment with Dasatinib and with Nilotinib also necessitated 50 % dose reductions because of thrombocytopenia. These reduced TKI dosing resulted in the best Quantitative RT-PCR for bcr/abl product reduction level at – 1.01. Bone marrow biopsy in December 2010 showed no morphologic evidence of CML, and no dysplasia; it was normocellular marrow with erythroid hyperplasia and megakaryocytic hypoplasia. Cytogenetic analysis and FISH showed presence of the Philadelphia chromosome rearrangement. The patient declined the treatment with a Bone marrow transplant. In April 2011 a TPO agonist Eltrombopag 50 mg daily was started, given on two weeks on two weeks off schedule. As platelets remained adequate since then, the Nilotinib was escalated to the full dose of 400 mg twice daily. The patient tolerated the treatment well. Results The patient required about half-a-year support with Eltrombopag; after that it was discontinued as the platelet count remained above 40 K. No dose reduction of Nilotinib was necessary. In January 2012 the patient reached the milestone of Complete Cytogentic Response with the level of bcr/abl transcript minus 2 or less, which continued to decrease and now the patient is achieving a Major Molecular Response. Conclusion Treatment of CML with Tyrosine Kinase Inhibitors – Imatinib, Dasatinib, or Nilotinib in our patient – can be complicated by severe thrombocytopenia. These patients’ next treatment option may be a Bone marrow transplant. Limited use of Eltrombopag can improve thrombocytopenia, and allow these patients to continue TKIs with attainment of the treatment milestons. Disclosures: Off Label Use: Eltrombopag. It is used in thrombocytopenia, in ITP. In this presentation it was used to treat thrombocytopenia secondary to Thyrosine Kinase Inhibitors in Chronic Myelogenous Leukemia.
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Marum, Justine E., Paul PS Wang, Doris Stangl, David T. Yeung, Martin C. Mueller, Christian T. Dietz, Ieuan Walker, et al. "Novel Fusion Genes at CML Diagnosis Reveal a Complex Pattern of Genomic Rearrangements and Sequence Inversions Associated with the Philadelphia Chromosome in Patients with Early Blast Crisis." Blood 128, no. 22 (December 2, 2016): 1219. http://dx.doi.org/10.1182/blood.v128.22.1219.1219.
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Abstract Introduction The emergence of next generation RNA sequencing (RNA-Seq) technologies will likely advance diagnostic, prognostic and therapeutic strategies for patients (pts) with various cancers.Novel fusions have recently been described in AML and solid tumors using RNA-Seq, and many were out-of-frame.It is not known whether novel fusions are generated at diagnosis (Dx) of CML and if so, their impact on treatment outcome. We used RNA-Seq coupled with whole exome sequencing to identify and characterize novel fusions at Dx of CML and at blast crisis (BC). A highly complex pattern of genomic rearrangements of chromosome (chr) 9 and 22 was found in some pts at Dx that generated novel fusions associated with multiple genomic breaks, multiple non-contiguous deletionsand inversion of genomic sequences, including BCR and ABL. Method RNA-Seqwas performed on Dx samples of chronic phase pts treated with first line TKI representing 2 extreme response groups: 14 pts with BC at a median of 6 months (mos), range 3-25 (group A, poor response), and 16 pts with rapid major molecular response by 3mosof imatinib (group B, optimal response). RNA-Seqwas also performed for 9 of 14 pts at BC (group C).The TruSeq Stranded Total RNA-RiboZero Gold Sample Prep Kit (Illumina) was used. This method enables computation of transcription direction and detection of genomic breaks from precursor RNA. Fusions were identified usingthe STAR algorithm and those detected in 4 normal controls were filtered out. Fusions with a high unique read count, supporting genomic breaks or detection atDxand BC for individual pts were prioritized for validation and their somatic status confirmed by RT-PCR.Correspondingwhole exome sequencingwas conducted for 30 samples. Copy number variation was detected usingSequenzaand exon level resolution ofdeletionswas achieved using an in-house sequence read normalization method. Results BCR-ABL fusions were detected by RNA-Seq in 29/30 pts at Dx and all pts at BC. In addition, novel fusions were identified in eachptgroup. GroupA(poor response). AtDx, 8 cytogenetically cryptic novel fusion transcripts were detected in 4/14 pts, Fig A pts1-4. All fusions involved genes or sequences onchr9 and/or 22 and all 4 pts had concomitant genomic inversion events. Fusion partners included inverted ABL intronic sequences and an inverted intergenic region on chr 22, potentially derived from the generation and activation of cryptic splice sites. BCR was a frequent fusion partner (5/8 fusion transcripts). Genomicdeletionswere detected adjacent to some fusions (3deletionsin 1pt),indicatingdeletionsmay have contributed to fusion formation, Fig B. All 4 pts with novel fusions and inversions had very rapid BC (within 5mosofDx). Group B (optimal response).AtDx, only 1/16 pts had a fusion detected in addition to BCR-ABL: TNRC6B (chr22)-NEK6 (chr9), Fig Apt5. Thisptalso had multiple non-contiguousdeletions: 2 each onchr9 and 22 associated with fusion formation, but no inversions, Fig B. Group C (BC). At BC, 3/9 pts gained fusions. No inversions were detected. Two pts had MLL fusions; MLL-BCAT1 (novel) and MLL-MLLT6. The MLL gene is a known fusion partner in acute leukemia, associated with poor prognosis. Both pts had sudden onset BC after a complete cytogenetic response. These fusions were supported by translocation events detected by cytogenetic analysis;t(11;12)(q23;p12) and t(11;17)(q23;q21). The thirdptgained an out-of-frame ANKRD11-UBQLN1 fusion at BC. Indeed, ANKRD11 expression was reduced by 3-fold at BC. Interestingly, thispthad a germline gain of function TP53 mutation. ANKRD11 is a p53 coactivator and loss of expression defined poor prognosis in breast cancer pts that harbored gain of function p53 mutations (Noll, 2012). The ANKRD11 fusion detected at BC in CML may have been selected with disease progression in the context of mutant p53. Conclusion We identified a subset of pts with novel fusions and inversion events at Dx involvingchr9 and 22. These inversions were detected among the pts studied with very rapid BC. The biological effects of the novel fusions remain to be determined. Our data support the presence of novel fusions, additional to BCR-ABL in CML and add a further layer of genetic heterogeneity associated with the Philadelphia translocation. Whether genomic inversions identify a small subset of CML pts with very poor prognosis requires expanded analysis. Disclosures Yeung: BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Research Funding. Mueller:Ariad: Honoraria; Institute for Hematology and Oncology GmbH: Employment; Bristol-Myers Squibb: Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Dietz:Institute for Hematology and Oncology GmbH: Employment. Ross:Novartis Pharmaceuticals: Honoraria, Research Funding; BMS: Honoraria. Hughes:Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Branford:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Research Funding; Bristol Myers Squibb: Research Funding; Qiagen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Cepheid: Consultancy.
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Lopotova, Tereza, Jaroslav Polak, Hana Klamova, Jiri Schwarz, and Jana Moravcova. "Expression Analysis of WT1 Splicing Variants In CML and AML: −5/+KTS as a Novel Candidate for Early Marker of Relapse." Blood 116, no. 21 (November 19, 2010): 2749. http://dx.doi.org/10.1182/blood.v116.21.2749.2749.
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Abstract Abstract 2749 Total WT1 expression is currently used for monitoring of AML patients and is of prognostic value also for CML patients according to our experience. Total WT1 level brings together levels of enormous amount of WT1 variants. Four major splicing variants reffered to as −5/−KTS, −5/+KTS, +5/−KTS and +5/+KTS are produced by combining two main independent splicing sites – exon 5 and KTS sequence. Short WT1 (sWT1) variant originates from usage of an alternative first exon E1a (Dalloso et al., 2001). Interestingly, exon 5 splicing was associated with resistance to apoptosis (Pritchard et al., 2006). The sWT1 was reported as overexpressed specifically in leukemias (Hossain et al., 2006). In view of literature data, we have suggested that splicing variant ratio or particular variant levels might have impact to leukemia pathogenesis, influence responsiveness to therapy and thus be a candidate for novel leukemic markers giving additional or more precise information about the disease state besides total WT1 levels. This idea was enhanced by our previous observation of differences of the four major variants levels associated with risk groups and different achieved responses to therapy among diagnostic AML patients samples (ASH 2009). In the current study, we extended our field of interest also on sWT1 vs. full length WT1. We aimed to find out whether there might be any advantage of monitoring of any of the WT1 variant in AML and CML patients undergoing therapy for more precise, sure and optionally earlier relapse detection/prediction. We have designed a novel method for quantification of sWT1 and full length WT1 separately. Quantification is performed by two separated real-time PCRs based on discriminating forward primers. One of those primers hybridizes onto exon 1 the other onto alternative exon 1a which enables to quantify full length WT1 and sWT1, respectively. For quantification of the four major variants, we used a method that we previously developed and reported (ASH, 2009). By introducing LNA nucleotides into the sequence of the primers with lowest hybridization efficiency we further improved reaction sensitivity, efficiency and robustness. Fourteen CML and ten AML patients undergoing therapy and developing haematological or molecular relapse were retrospectively analysed so far. We found out that the ratio of the four variants was changed in relapse as compared to diagnosis. The most abundant variant at diagnosis was +5/−KTS. In relapse, it became to be −5/+KTS in both AML and CML. The −5/+KTS was the first one to be increased in most of the cases and usually its expression kinetics was the most expressive of all four variants. As compared to BCR-ABL transcript, both total WT1 and −5/+KTS increases were more expressive or even earlier in some of CML patients and thus clearly predicted upcoming relapse. Short WT1 was detected in all AML samples, in CML we found it only in blast crisis not in the chronic phase samples. In all cases the proportion of sWT1 from total WT1 was very low, the majority of total WT1 was represented by full length WT1. Taken together, we showed that mainly −5/+KTS WT1 variant is a good marker of CML and AML and well characterizes the course of the diseases. Our preliminary data indicated that both kinetics and actual levels of −5/+KTS might specify information done by total WT1 levels and in case of CML by BCR-ABL making relapse prediction more sure or possibly even earlier. Supported by MZOUHKT2005. Disclosures: No relevant conflicts of interest to declare.
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Pimentel, Agustin, Daniel A. Forman, Sharon L. Swierczynski, Anthony A. Donato, and Brian Le. "Intracerebellar Granulocytic Sarcoma Presenting as a First Manifestation of Relapse in Chronic Myelogenous Leukemia (CML) During Imatinib Mesylate Therapy: A Case Report and Literature Review." Blood 114, no. 22 (November 20, 2009): 4289. http://dx.doi.org/10.1182/blood.v114.22.4289.4289.
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Abstract Abstract 4289 Introduction Granulocytic sarcoma (GS) is an unusual solid tumor of extramedullary localization consisting of immature granulocytic precursors. It is mostly encountered in the course of acute myelogenous leukemia (AML), myelodysplastic or myeloproliferative disorders, with an estimated incidence of 7.9% in CML (Terjanian et al). GS can occur before, concomitantly, or after the overt development of these hematologic disorders. Most common localizations are lymph nodes, bones, skin and soft tissues, but there is paucity of published data of isolated intracranial GS manifesting as relapse in CML. Case report A 34-year-old male with Philadelphia chromosome-positive (Ph+) CML diagnosed in August 2005 in the chronic phase of the disease. After initiation of imatinib therapy at doses of 400 mg/d he achieved complete hematologic, cytogenetic and molecular responses. He remained in clinical remission for 3 years when he presented with bifrontal headache and dizziness. Physical exam revealed no abnormalities. Peripheral blood count showed WBC 9,200/mm3 with normal differential and no immature forms, platelet 180,000/mm3, and hemoglobin 14.4 g/dl. Magnetic resonance imaging of the brain revealed a 3.8 cm enhancing mass lesion within the left cerebellar hemisphere. He underwent posterior fossa exploration with resection of the solid tumor. Microscopic examination of the cerebellar mass demonstrated a diffuse cellular infiltrate of atypical mononuclear cells with high nuclear to cytoplasmic ratios and fine, immature chromatin, consistent with blasts. Flow cytometric analysis confirmed that the majority of the cells in the specimen were composed of blasts which coexpressed the CD33 and CD4 markers. Fluorescence in situ hybridization analysis on the cerebellar tissue detected the presence of BCR/ABL rearrangement in 60% of nuclei. Taken together, the morphologic, immunophenotypic, and molecular features are diagnostic of a myeloid sarcoma representing transformation of chronic myeloid leukemia. Discussion A literature search was performed to identify all cases of intracranial GS in patients with diagnosis of CML reported in medical literature. Only 3 cases were identified in the literature to date. This case appears to be the first described in the imatinib mesylate era. Table 1 summarizes the clinical data of these previously recorded cases and the present case. Pharmacokinetic analyses (Pfeifer et al, Leis at al, Wolff et al) have demonstrated that imatinib poorly penetrates the blood–brain barrier, with resultant CNS drug levels significantly lower than corresponding plasma levels. Measured CSF levels were below the concentration required for 50% inhibition of the BCR–ABL tyrosine kinase activity, suggesting that Ph+ leukemic patients treated with this selective tyrosine kinase inhibitor are at high risk of CNS relapse even in the setting of complete remission of systemic disease. This case illustrates an unusual site of recurrence of CML. To our knowledge, this is the first case of a granulocytic sarcoma presenting as a solitary parenchymal brain tumor in a patient receiving imatinib for CML. Noticeably, CNS relapse occurred despite lack of systemic evidence of disease. As described in human and animal model studies this may be the result of effective systemic antileukemic activity of imatinib therapy, but with limited activity in the CNS. Poor penetration of imatinib into the cerebrospinal fluid may allow the CNS to become a sanctuary reservoir for relapse of Ph+ CML. Disclosures: No relevant conflicts of interest to declare.
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Mori, Silvia, Elisabetta Vagge, Philipp le Coutre, Elisabetta Abruzzese, Bruno Martino, Ester Pungolino, Chiara Elena, et al. "The Risk of Relapse in CML Patients Who Discontinued imatinib Can Be Predicted Based on Patients Age and the Results of dPCR Analysis." Blood 124, no. 21 (December 6, 2014): 813. http://dx.doi.org/10.1182/blood.v124.21.813.813.
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Abstract Introduction. Chronic myeloid leukemia (CML) patients (pts) treated with imatinib first line achieve complete cytogenetic response (CCyR) in > 70% of cases and major molecular response (MMR) in 18-58%. These pts have a life expectancy similar to the general population. However even undetectable BCR-ABL may not equate to eradication of the disease because of the sensitivity of Q-RT-PCR. A new diagnostic method, the digital-PCR (dPCR), able to detect 1 BCR-ABL+ cell out of 107 cells, has been recently developed (Goh HG et al., 2011). dPCR corresponds to a 100 fold increase in sensitivity as compared to Q-RT-PCR. Therefore, dPCR by assessing the presence of minimal residual disease with higher sensitivity, could potentially identify pts in whom CML has been eradicated. Aims. The Imatinib Suspension And Validation (ISAV) study is aimed at validating the capability of dPCR to predict relapses after imatinib discontinuation in CML pts with negative Q-RT-PCR results. Methods. This study involves 15 sites, 10 in Italy and 1 in each of the following countries: Germany, Spain, The Netherlands, Canada and Israel. CML pts (Chronic or Accelerated Phase) under imatinib therapy since more than 2 years and in complete molecular remission (CMR) were eligible for this study. Patients had to be in CMR for at least 18 months (mts), with a minimum of 3 Q-RT-PCR performed at their own sites. After signing the informed consent, blood samples are obtained for dPCR and the pts discontinue imatinib therapy. Standard Q-RT-PCR is performed monthly (mts 1-6) and then bimonthly for 36 mts to assess the maintenance of the molecular remission. The loss of molecular remission is defined as two consecutive positive Q-RT-PCR tests with at least one BCR-ABL/ABL value above 0.1%. Patients losing molecular remission resume imatinib treatment at the same dosage used before interruption. Patients’ quality of life during imatinib discontinuation/resumption is evaluated through the EORTC – C30 Quality of Life questionnaire. Results. The enrolment in ISAV began in November 2011 and ended in July 2013. The study enrolled 112 pts: Italy 69.6%, Germany 21.4%, Canada 5.3%, Spain 2.6% and Israel 0.9%. Among the 112 pts, 59.3% were male and 37.0% were aged 65 or older; median duration of imatinib treatment was 103.1 mts with median duration of CMR of 25.8 mts before imatinib discontinuation. To date, the median follow-up (FUP) time is 16.6 mts [95% CI: 14.9-18.2]. Forty-seven pts (43.5%, 95% CI: 34.0-53.4) of the 108 eligible pts relapsed and resumed imatinib; 38/47 (80.9%) of them relapsed in the first 9 mts and the last relapse occurred 19.6 mts after imatinib discontinuation. A loss of CCyR occurred in 11 pts (23.4%): 10/11 CCyR losses were recovered; 1 patient withdrew the consent shortly after obtaining a partial cytogenetic response. No case of CML progression was observed. After the resumption of imatinib the median time to either MMR or CMR was 1.9 [95% CI: 1.2-2.4] mts. Of the 61 not-relapsed pts, 43 (39.8% of the total) regained Q-RT-PCR positivity but never lost MMR. The median time to Q-RT-PCR positivity was 3.6 mts [95% CI: 3.0-4.8] and the range of duration of Q-RT-PCR positivity (below 0.1%) was between 5.7 and 29.2 mts. No significant correlation between relapse and previous duration of imatinib treatment, use of interferon, time to CCyR or duration of CMR was identified. An inverse relationship between pts age and risk of relapse is evident: 90% of pts < 45 years relapsed vs 37.5% in the class ≥ 45 - < 65 years and 27.5% of pts ≥ 65 years, p(χ2)<0.0001. dPCR results showed that 23.4% of pts were positive and 76.6% negative, with a dPCR Negative Predictive Value (NPV) of 63.4% (Tab.1) and a significant NPV ratio (dPCR/Q-RT-PCR) of 1.131 [95% CI: 1.032-1.239]. Age and dPCR results predicted the risk of relapse: pts with less than 45 years and with a positive dPCR had the highest risk of relapse (100%) as opposed to pts ≥ 45 years and with negative dPCR (30.6%; Fig.1). Conclusions. After 32 mts from the beginning of the study, with a median FUP of 16.6 mts, 43.5% of pts relapsed; the majority of relapses developed in the first 9 months after imatinib discontinuation. Age < 45 years and dPCR positivity are significantly associated with relapses. Funded by Regione Lombardia. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.
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Bocchia, Monica, Anna Sicuranza, Paola Pacelli, Alessandra Iurlo, Elisabetta Abruzzese, Sara Galimberti, Patrizia Pregno, et al. "Peripheral Blood CD26+ Leukemia Stem Cells Monitoring in Chronic Myeloid Leukemia Patients from Diagnosis to Response to TKIs: Interim Results of a Multicenter Prospective Study (PROSPECTIVE FLOWERS)." Blood 136, Supplement 1 (November 5, 2020): 45–46. http://dx.doi.org/10.1182/blood-2020-139306.
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Background We already showed that in CML pts peripheral blood CD34+/CD38-/CD26+ cell population represent a "CML specific" leukemia stem cell (LSC) circulating compartment. Indeed, we demonstrated that CD26+LSCs are measurable by flow-cytometry in 100% of CML pts at diagnosis the latter representing an alternative and rapid diagnostic tool. In addition, in a cross-sectional study we were able to spot peripheral blood CD26+LSCs also in about 65% of CML during TKI treatment regardless of type and length of TKI treatment and degree of molecular response. However, no prospective data are available regarding the behavior of PB CD26+LSCs in terms of rate and timing of reduction during TKI therapy and the correlation, if any, with the attainment of response according to ELN guidelines. Interestingly, even CML patients in stable TFR may harbor circulating CD26+LSCs thus suggesting a probable active role of the immune system in the control of residual disease. One hypothesis could reside in the presence or absence on the LSCs of molecules (such as PD-L1) able to hamper an anti-leukemic T cell response. From Jan 2018 we conducted a prospective multicenter Italian study including CML pts at diagnosis treated and managed by each of 15 participating center according to ELN guidelines. We here present the first interim analysis after a median time of treatment of 12 mos. Aims The main goals of this study were to prospectively monitoring PB CD26+LScs in CML pts during TKI treatment and to correlate the behavior of LSCs with molecular response. In a proportion of pts PD-L1 expression on CD26+ LSCs at diagnosis was also evaluated. Methods At diagnosis and during TKI treatment, pts have been centrally evaluated in Siena lab for flow-cytometry PB CD26+ LSCs (+3, +6, +12, +18, +24 mos) and PD-L1 expression (at diagnosis). At each time point molecular BCR-ABL/ABLIS ratio was monitored locally in each center. Results 176 consecutive CML pts (IMA 92; NILO 61; DASA 23) were enrolled in the study so far (table 1). PB CD26+LSCs were measured at time 0 (baseline) in all 176 CML pts and in 165/176 (94%), 142/176 (81%) and 112/176 (71%) at +3, +6, +12 mos of TKI treatment, respectively. Median CD26+LSCs absolute number/µL at baseline was 6.96/µL (range 0.0126-64429), at +3 mos 0.0137/µL (range 0-6,49), at +6 mos 0.0056/µL (range 0-1.188), and at +12 mos 0.0112/µL (range 0-0.1824). No significant correlation between number of CD26+LSC, degree of response and BCR-ABL copies was found (Table 2). Interestingly, median CD26+LSCs at diagnosis was found significantly higher in NILO and DASA treated pts (12, 48/µL and 17,48/µL, respectively) than in IMA pts (4,58/µL). So far, 20/176 (11.4%) pts switched to different TKIs, due to failure/suboptimal response: of note, median CD26+ LSCs of this cohort at diagnosis was the highest (23.12/µL). Starting from Jun 2019, 44/176 (25%) CML pts have been evaluated also for PD-L1 expression at diagnosis: of these, 23/44 (52%) resulted PD-L1 positive and 21/44 (48%) resulted negative with a median of CD26+LSCs of 15.39/µL (range 1.28-635.5) and 4.45/µL (range 0.234-113.9), respectively. Conclusions After a sensible drop observed at 3 mos of any TKI treatment, CD26+LSCs are fluctuating and measurable at low level in most of pts (> 65%) even at 18 and 24 mos. We confirmed no correlation between the absolute number of persisting CD26+LSCs and BCR-ABL copies. However, pts with failure or suboptimal response showed the highest level of CD26+ at diagnosis. CD26+LSCs were found PD-L1+ in about half of 44 pts tested. At diagnosis higher CD26+LSCs number, PD-L1 positivity or both may correlate with a lower probability to achieve an optimal response; interim data of this first report will be presented; enrolment and follow up are ongoing. Disclosures Bocchia: Incyte: Honoraria; CELGENE: Honoraria. Abruzzese:Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bms: Honoraria. Galimberti:Novartis: Speakers Bureau; Incyte: Honoraria. Pregno:Incyte-Italy,: Membership on an entity's Board of Directors or advisory committees, Other: conference reports; Novartis-Italy: Membership on an entity's Board of Directors or advisory committees, Other: conference reports; Pfizer-Italy: Membership on an entity's Board of Directors or advisory committees, Other: conference reports. Crugnola:BMS: Honoraria; Janssen: Honoraria; Celgene: Honoraria; Novartis: Honoraria. Liberati:MORPHOSYS: Honoraria, Research Funding; ONCONOVA: Honoraria, Research Funding; INCYTE: Honoraria; VERASTEM: Honoraria, Research Funding; ROCHE: Honoraria, Research Funding; PFIZER: Honoraria, Research Funding; ONCOPEPTIDES AB: Honoraria, Research Funding; TAKEDA: Honoraria, Research Funding; FIBROGEN: Honoraria; BIOPHARMA: Honoraria; ARCHIGEN: Honoraria; BEIGENE: Honoraria; BMS: Honoraria; AMGEN: Honoraria; CELGENE: Honoraria; JANSSEN: Honoraria; ABBVIE: Honoraria, Research Funding; NOVARTIS: Honoraria, Research Funding; KARYOPHARM: Honoraria, Research Funding.
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Fava, Carmen, Paola Berchialla, Jessica Petiti, Maria Teresa Bochicchio, Barbara Izzo, Emilia Giugliano, Emanuela Ottaviani, et al. "A Comparison of Droplet Digital PCR and RT-qPCR for BCR-ABL1 Monitoring in Chronic Myeloid Leukemia." Blood 134, Supplement_1 (November 13, 2019): 2092. http://dx.doi.org/10.1182/blood-2019-125614.
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Background: Monitoring of BCR-ABL1 molecular levels is essential for the management of Chronic Myeloid Leukemia (CML) patients treated with Tyrosine Kinases Inhibitors (TKIs). Real Time Quantitative PCR (RT-qPCR) is currently the standard method for assessing molecular remission (MR) in patients with CML. Recently, droplet digital PCR (ddPCR) has emerged to provide a more accurate detection of minimal residual disease (MRD). In order to hypothesize the use of this new technology in the clinical practice, in the era of TKI discontinuation, we designed a multi-centric study to evaluate the potential value of ddPCR in diagnostic routine. Aims of the study were:1) the evaluation of the agreement between the measures obtained by ddPCR and RT-qPCR and 2) the assessment of the repeatability of the two methods. Methods: Total RNA was extracted from 37 CML patients using Maxwell 16 LEV simplyRNA Blood kit (Promega), following the manufacturer's instructions. Samples were divided in 5 groups based on molecular response (MR) as follow: group 1, MR<3, n=5; group 2, MR 3, n=5; group 3, MR 4, n=9; group 4, MR 4.5, n=9 and group 5, MR 5, n=9. BCR-ABL1 p210 was quantified by RT-qPCR and ddPCR in 3 different Italian laboratories namely Lab A, Lab B and Lab C. Lab B and C performed 1 amplification session each, while Lab A performed 3 independent sessions. All ddPCR experiments were performed using Kit QXDxTM BCR-ABL%IS (Bio-Rad) on the QX200 system (Bio-Rad), according to the manufacturer's instructions. For RT-qPCR experiments, BCR-ABL1 p210 was quantified with three different methods: by using the One-Step BCR-ABL P210 ELITe MGB Kit (ELITech Group), according to manufacturer's protocol (Lab A); by using the One-Step Philadelphia SensiQuant Kit (Bioclarma), according to the manufacturer's instructions (Lab B) and as described in Gabert et al (Lab C). The results obtained were corrected for the laboratory-specific conversion factor, as recommended by Standard Operating Procedures of Labnet CML network (GIMEMA group). The target quantification for each sample was expressed as BCR-ABL1/ABL1 %IS for both RT-qPCR and ddPCR. Statistical analysis: Bland-Altman analysis was performed. For the measurement of the agreement we reported the bias, which is the mean of the difference between the methods, the 95% limits of agreement and the coefficient of variation. Residual variance and coefficients of repeatability (i.e. the upper limits of a prediction interval for the absolute difference between two measurements by the same method on the same item) were computed to achieve the second endpoint. An analysis of sensitivity on the labs was also carried out. All analyses were stratified by the level of disease. Results: A total of 370 measures were included in the analysis, 185 for ddPCR and 185 for RT-qPCR divided as follow: 50 for group 1 and for group 2, 90 for group 3, 4 and 5. In Table 1 we reported the median and interquartile range (IQR) for all levels of disease. Results of the Bland-Altman analysis are shown in Table 2. The coefficients of variation, which expresses the standard deviation as a percentage of the mean, were 2.35, 2.31, 1.10, 1.34, 39.12 in group 1, 2, 3, 4 and 5 respectively. The repeatability coefficients of ddPCR were smaller than qRT-PCR across all the levels of disease, showing a slightly better precision of ddPCR (Table 2). Conclusions: Higher coefficients of variation in group 1 and 2 were probably due to a greater heterogeneity of the patients. In fact, BCR-ABL1/ABL1 levels by RT-qPCR ranged between 1.43 and 6.94 and between 0.10 and 0.25 in group 1 and in group 2 respectively (Table 1). Sensitivity analysis showed that the high coefficient of variation in group 5 can be explained almost all by the variability observed in Lab B. Coefficient of repeatability of ddPCR was always smaller than RT-qPCR for all level of disease showing a slightly better precision. Our results showed that ddPCR has a good agreement with RT-qPCR and it is more precise to quantify BCR-ABL1 transcript levels, particularly for MR 4 and MR 4.5. Thus, ddPCR may be valuable in diagnostic routine. Disclosures Fava: Novartis: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Incyte: Honoraria. Martino:Bio-Rad: Employment. Saglio:Ariad: Consultancy; Incyte: Consultancy; Pfizer: Consultancy; Jansen: Consultancy; Celgene: Consultancy; Novartis: Consultancy; BMS: Consultancy. Martinelli:Roche: Consultancy; BMS: Consultancy; Novartis: Consultancy; ARIAD: Consultancy; Pfizer: Consultancy. Pane:Novartis: Membership on an entity's Board of Directors or advisory committees, Other: research founding; Janssen: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Cilloni:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees.
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Suttorp, Meinolf, Josephine Tabea Tauer, Manuela Krumbholz, and Markus Metzler. "Spleen Size at Diagnosis and Genomic BCR-ABL1 Breakpoint Distribution Differ Age-Dependently in Pediatric Patients with Chronic Myeloid Leukemia (CML)." Blood 126, no. 23 (December 3, 2015): 4827. http://dx.doi.org/10.1182/blood.v126.23.4827.4827.
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Abstract Background: Childhood and adolescence is characterized by significant physiological alterations including host factors relevant for CML disease course and therapy, e.g. maturation of the immune system and skeletal growth. When compared to adults, minors with CML present with higher disease burden at diagnosis, in particular more frequently in advanced stage of CML, with larger spleen size, and higher leukocyte number. These features are still evident in young adults with CML. Also the genomic breakpoint in the BCR-gene differs when adult and pediatric data are compared. We analyzed pediatric data on clinical and laboratory findings including molecular data from children diagnosed with CML with an emphasis on correlation to age and sex. Methods: Data on WBC and spleen size were depicted from the database of 90 German children with CML in CP enrolled into trial CML-PAED II. Taking into account the age-dependent increase of body height during childhood, absolute spleen size below the costal margin was categorized into four categories of relative spleen size: not palpable (I); enlarged moderately (II): age 1-5y <=2cm, age 6-10y <=4cm, age 11-15y <=5cm, age 16-18y <=5cm; enlarged highly (III): age 1-5y >2 - <=7cm, age 6-10y >4 - <=9 cm, age 11-15y >5 - <=13cm, age 16-18 >5 - <=15cm; enlarged excessively (IV): age 1-5y >7cm, age 6-10y >9cm, age 11-15y >13cm, age 16-18 >15cm. DNA was extracted from diagnostic peripheral blood specimen followed by identification of the genomic BCR-ABL breakpoint using long-range PCR. Breakpoint distribution was investigated by Kernel density analysis with respect to age and gender (for details see Krumbholz M et al Genes Chrom Cancer 2012). Results: WBC (mean 242,000/µl; range 20,000 - 1,150,000/µl) showed a peak at age 10y with a non-significant decline in younger and older children and no significant changes in distribution by gender. Age-related categorization of spleen size showed that the proportion of patients exhibiting a moderately or highly enlarged spleen, respectively, was more ore less stable in the range of 20 - 25% from age 6 - 15 y while excessively enlarged spleen size was present in one third of 6 - 10y old children and declined with increasing age (see Table 1). Table 1. Distribution of spleen size in 90 pediatric patients with CML Age [years]No of patients [N] 0 - 510 6 - 10 27 11 - 1539 16 - 18 14 Absolute spleen size andclassified relative organenlargement N (%) N (%) N (%) N (%) Spleen not palpable 6 (60) 5 (19) 13 (33) 7 (50) Spleen <= 5 cm b.c.m.Enlarged moderately 2 (20) 0 ( 0) 9 (33) 7 (26) 10 (26) 10 (26) 3 (21) 7 (50) Spleen >5 - <=15 cm b.c.m. Enlarged highly 2 (20) 2 (20) 9 (33) 5 (18) 10 (26) 8 (20) 3 (21) 3 (21) Spleen > 15 cm b.c.m.Enlarged excessively 0 ( 0) 2 (20) 4 (15) 10 (37) 6 (15) 8 (20) 1 ( 8) 1 ( 7) b.c.m. : below costal margin The molecular footprint regarding BCR breakpoint distribution shifted from childhood to adult pattern at the age of 13 years. Also a difference between boys and girls was more pronounced at the prepubertal age (see Fig. 1). Conclusion: Host factors in growing children are distinct from those of adults. Combined evaluation of adolescent and young adult patients with CML may generate a better understanding of the role of modifying host factors contributing to pathogenesis and development of clinical features of CML. This might form a future basis for age-adapted therapeutic strategies in young individuals with CML. Disclosures Suttorp: Novartis, BMS, Pfizer: Consultancy, Honoraria, Research Funding.
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Enrico, Alicia Inés, Beatriz Moiraghi, Graciela Klein, Maria del Rosario Cabrejo, Renee Crisp, Georgina Bendek, Mariana Gil, et al. "Argentine Registry of Hematologic Disease (RAEH) Chronics Myeloid Leukemia." Blood 118, no. 21 (November 18, 2011): 4455. http://dx.doi.org/10.1182/blood.v118.21.4455.4455.
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Abstract Abstract 4455 Introduction: The CML Registry was developed by the Argentine Society of Hematology as a part of the centralized RAEH. Results from epidemiologic and clinical data related to CML, will enable to determine the geographic distribution of the target population, to establish associated environmental causes, and mainly to rationalize resources supply. Objective: a) To analyze characteristics of CML patients registered in the RAEH; b) To evaluate the CML Registry performance through its first year. Materials and Methods: Patients with CML registered in the RAEH from January 1st, 2011 up to July 31st, 2011. The protocol allowed to enroll de novo patients as well as patients diagnosed from 2000 on. Result: Data reported by 15 hospitals were included: 224 patients were registered. Mean age was 50 (18–86 y) and gender distribution was female: 102, male: 122. Occupational data showed no a characteristic pattern. 96.5% of patients were diagnosed in chronic phase, while 3.5% were diagnosed in accelerated phase/blast crisis. In 6% of patients cytogenetic tests detected 8 abnormalities besides t(9,22): double Philadelphia chromosome and monosomy 12 were the most frequent findings. FISH tests were recorded for 18% of patients at the time of diagnosis. Bone marrow biopsy was reported as a diagnostic procedure in 51%. Qualitative BCR/ABL was recorded for 31% of patients at the time of diagnosis. Molecular RQ-PCR tests for follow-up of treatment response were reported for 51% of patients. Of the registered population, 21% received interferon as previous therapy to Imatinib (IM); 89% received IM 400mg daily; 6% required dose increase. Second line treatment with dasatinib or nilotinib was recorded in 34% and 14% of patients, respectively. At 60 months mean follow-up 8% of the registered population had developed blast crisis and 6% had died. Conclusions. The RAEH’s first year of performance in CML was assessed. This only reflects the experience with 15 sites. Data registered will allow us in the following years to learn about disease epidemiology and available resources to improve patient accessibility. Disclosures: No relevant conflicts of interest to declare.
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Mori, Silvia, Philipp le Coutre, Elisabetta Abruzzese, Bruno Martino, Ester Pungolino, Chiara Elena, Ivana Pierri, et al. "Imatinib Suspension and Validation (ISAV) Study: Results at 24 Months." Blood 126, no. 23 (December 3, 2015): 2775. http://dx.doi.org/10.1182/blood.v126.23.2775.2775.
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Abstract Introduction. A substantial proportion of patients (pts) affected by Chronic Myeloid Leukemia (CML) achieve complete negativity in Q-RT-PCR. In this situation, as already demonstrated in other STOP trials, it is possible to safely discontinue imatinib treatment but it is still not clear how to discriminate subjects who will relapse. In fact even undetectable BCR-ABL may not equate to eradication of the disease because of the sensitivity of Q-RT-PCR. A new diagnostic method, the digital-PCR (dPCR), able to detect 1 BCR-ABL+ cell out of 107 cells, has been developed. Therefore, dPCR by assessing the presence of minimal residual disease with higher sensitivity, could potentially identify pts in whom CML has been eradicated. Aims. The ISAV study is aimed at validating the capability of dPCR to predict relapses after imatinib discontinuation in CML pts with negative Q-RT-PCR results and to evaluate relapse rate and timing of recurrence, survival and the impact of imatinib treatment on Quality of Life (QoL). Methods. This study involves 15 sites, 10 in Italy and 1 in each of the following countries: Germany, Spain, The Netherlands, Canada and Israel. CML pts (Chronic or Accelerated Phase) under imatinib therapy since more than 2 years and in complete molecular remission (CMR) were eligible. Patients had to be in CMR for at least 18 months (mts), with a minimum of 3 Q-RT-PCR performed at their own sites. After signing the informed consent, blood samples were obtained for dPCR and the pts discontinued imatinib therapy. Standard Q-RT-PCR is performed monthly (mts 1-6) and then bimonthly for 36 mts to assess the maintenance of the molecular remission. The loss of molecular remission is defined as two consecutive positive Q-RT-PCR tests with at least one BCR-ABL/ABL value above 0.1%. Patients losing molecular remission resume imatinib treatment at the same dosage used before interruption. Patients' QoL during imatinib discontinuation/resumption is evaluated through the EORTC QLQ-C30 questionnaire. Results. The study enrolled 112 pts with a median follow-up (FUP) time of 28.0 mts [95% CI: 25.5-30.1]. The 59.3% of pts were male and 37.0% were aged 65 or older; median duration of imatinib treatment was 103.1 mts with median duration of CMR of 25.7 mts before imatinib discontinuation. The cumulative probability of survival is 97.8% [95% CI: 91.4-99.5]. dPCR results showed that 23.1% of pts were positive and 75.9% negative, with a significant Negative Predictive Value ratio (dPCR/Q-RT-PCR) of 1.112 [95% CI: 1.009-1.225]. At 24 mts from imatinib discontinuation, 53 pts (49.1%, 95% CI: 39.3-58.9) of the 108 eligible pts relapsed and resumed imatinib; 73.6% of them relapsed in the first 9 mts and the last relapse occurred 21.8 mts after imatinib discontinuation. A loss of CCyR occurred in 13 pts (34.2% of those tested): 10/13 CCyR losses were recovered, the remaining 3 were not assessed for response. No case of CML progression or resistance to imatinib was observed. After the resumption of imatinib the median time to MMR/CMR was 1.9 [95% CI: 1.2-2.4] mts. Of the 55 not-relapsed pts, 42 (38.9% of the total) regained Q-RT-PCR positivity but never lost MMR. The median time to Q-RT-PCR positivity was 2.9 mts [95% CI: 2.0-3.1] in the relapsed pts and 4.5 mts [95% CI: 2.9-6.9] in pts who developed only PCR positivity. No significant correlation between relapse and previous duration of imatinib treatment, use of interferon, time to CCyR, Sokal score or duration of CMR was identified, while an inverse relationship between pts age and risk of relapse is evident. Moreover, age and dPCR results together can predict the risk of relapse: pts with less than 45 years and with a positive dPCR had the highest risk of relapse (100%) as opposed to pts ≥ 45 years and with negative dPCR (36.1%). The analysis of QoL evidenced a statistically significant improvement in the general well-being and symptoms scales at 1 month after imatinib discontinuation and in particular nausea, diarrhea and fatigue (p<0.01). An inverse and transient trend toward increased pain emerged at mts 1 and 3. Conclusions. At 45 mts from the beginning of the study, with a median FUP of 28.0 mts, 49.1% of pts relapsed; the majority of relapses developed in the first 9 mts after imatinib discontinuation. Age < 45 years and dPCR positivity are significantly associated with relapses. QoL analysis showed a significant decrease in symptoms after imatinib discontinuation. Funded by Regione Lombardia. Disclosures Abruzzese: BMS, Novartis, Pfizer, Ariad: Consultancy. Assouline:BMS: Consultancy; Pfizer: Consultancy; Novartis: Consultancy. D'Emilio:Celgene: Research Funding. Dong-Wook:ll-Yang: Consultancy, Honoraria, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis Pharmaceuticals: Consultancy, Honoraria, Research Funding, Speakers Bureau.
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Guilhot, Francois. "Sustained Durability of Responses Plus High Rates of Cytogenetic Responses Result in Long-Term Benefit for Newly Diagnosed Chronic-Phase Chronic Myeloid Leukemia (CML-CP) Treated with Imatinib (IM) Therapy: Update from the IRIS Study." Blood 104, no. 11 (November 16, 2004): 21. http://dx.doi.org/10.1182/blood.v104.11.21.21.
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IM has proven to be superior to IFN+Ara-C for newly diagnosed pts with CML-CP (O’Brien et al, NEJM, 2003). At 30-months of follow-up, 79% of pts randomized to IM remain on IM with 83% of them now treated for > 24 months. Therefore, this analysis is focused on first-line IM pts (n=553). Evaluation included complete hematologic response (CHR), major/complete cytogenetic response (MCyR/CCyR), major molecular response (≥3 log reduction in BCR-ABL/BCR vs. pooled diagnostic samples) time to progression (TTP) - defined as loss of CHR/MCyR or evolution to accelerated phase/blast crisis [AP/BC] or death, time to AP/BC, and overall survival. Median follow-up for first-line IM pts is 31.1. Summary of results are the following: 1st-line imatinib * 95% CI Best observed CHR/MCyR/CCyR (%) 95/87/79 Estimated major molecular response at 12 mos. (%) 40 Estimated cumulative MCyR % at 30 mos. 90 Estimated cumulative CCyR % at 30 mos. 82 Estimated % free of progression at 30 mos. 88 (85–91)* Estimated % free of AP/BC at 30 mos. 95 (93–97)* Estimated % survival at 30 mos. 95 (93–97)* The additional follow-up confirmed durable responses with first-line therapy while also demonstrating the effect of cytogenetic response on long-term outcomes. The estimated rate of confirmed responders remaining in response after achieving a CCyR at 30 months is >92%. Similarly, the estimated rate of patients still in response at 30 months after achieving either a MCyR or CHR is also >92% for both groups. Only 2% of pts with a confirmed loss of MCyR and 1% of the pts with a confirmed loss of CCyR subsequently progressed to AP/BC. Of these pts, 3 of 5 benefited from a dose increase to 600 or 800 mg of IM, while only 1 of 7 pts with a confirmed loss subsequently achieved a MCyR without a dose escalation. A total of 75 pts received a marrow transplant (BMT) after discontinuation from the study; 30 in the IM arm (2 after crossover from IFN) and 45 pts who were randomized to IFN (21 after crossover to IM). There was no difference in survival after BMT between pts who received first-line IM treatment (8 deaths, 1 after crossover) and pts who had received IFN+Ara–C (7 deaths) or IFN+Ara–C followed by IM (7 deaths) (p=0.78). The estimated survival at 12 months after BMT is 70%, 75%, and 68% respectively in the three groups. A landmark analysis showed that for 407 pts who achieved MCyR within 6 months, their estimated rate free of AP/BC at 30 months is 97% vs. 89% for the 124 pts who did not achieve this level of response at 6 months (p<0.001). Additionally, the estimated survival at 30 months for the same pts is 97% vs. 92% (p=0.0162). The achievement of a major molecular response at 12 months was also associated with improved progression-free survival. For patients who had achieved CCyR and a reduction in BCR-ABL transcript level ≥ 3 log at 12 months, the probability of remaining progression free was 100% at 30 months compared to 93% for such patients with reduction in BCR-ABL transcript level < 3 log and 82% for patients who were not in CCyR at 12 months (p<0.0001). These results will be further updated using a data cut-off of 31-July-04 to reflect additional 12-months of data (i.e., 42-month follow-up).
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Nievergall, Eva, John Reynolds, Chung H. Kok, Dale Watkins, Mark Biondo, Samantha J. Busfield, Gino Vairo, et al. "High Plasma Levels of TGF-α and IL-6 at Diagnosis Predict Early Molecular Response Failure and Transformation in CML." Blood 124, no. 21 (December 6, 2014): 1788. http://dx.doi.org/10.1182/blood.v124.21.1788.1788.
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Abstract Introduction: Early molecular response (EMR, BCR-ABL (IS) ≤ 10% at 3 months) is a strong predictor of outcome in imatinib-treated chronic phase chronic myeloid leukemia (CP-CML) patients, but for patients who transform early 3 months may be too late for effective therapeutic intervention. Thus, alternative approaches are required to identify poor responders at the time of diagnosis. The aim of this study was to identify plasma biomarkers at diagnosis that will predict for subsequent EMR failure, early transformation or the development of BCR-ABL1 kinase domain mutations. Cytokine profiling has proven valuable in identifying prognostic factors in myelofibrosis and myelodysplastic syndromes; however, similar comprehensive studies are lacking to date in CML. Methods: Plasma samples from CP-CML patients enrolled to the TIDEL II trial were collected prior to starting imatinib treatment (n=186) and after 6 months on TKI (n=17); and compared to those of healthy donors (n=19). The levels of 39 cytokines, chemokines and growth factors (CC&GF) were measured using a Luminex multiplex assay. To identify potential biomarkers to predict EMR failure, random forest analysis and recursive partitioning techniques in R were applied as statistical methods. Results: Plasma concentrations of 13/39 CC&GF were significantly elevated at CP-CML diagnosis compared to healthy donor samples. Most (EGF, bFGF, VEGF, TGF-α, CXCL1, CCL4, sCD40L and IL-4) werenormalized after 6 months of TKI treatment while others (TNF-α, sIL-2Ra, IL-8, IL-10, IL-1a) remained at higher levels, possibly reflecting persistent disease-induced alterations within the microenvironment. A third subset of CC&GF, such as CCL2, CCL3 and CCL22, showed higher circulation levels only in TKI-treated patients but not at diagnosis, suggesting that changes in these CC&GF could be treatment-related. 183/186 patients had BCR-ABL1 assessments available at 3 months, and 23/183 (13%) did not achieve EMR. Random forest analysis identified TGF-α, IL-6 and IFN-α as the most important CC&GF associated with EMR failure. Recursive partitioning incorporating these three variables produced a classification tree based only on TGF-α and IL-6, and demonstrated that 12/20 (60%) of patients who were TGF-αhi/IL-6hi failed to achieve EMR (Table 1). Importantly, this group contained 3/3 (100%) patients who transformed within the first 12 months of TKI treatment. Both TGF-α (7.99 vs. 60.57 pg/ml, p<0.001) and IL-6 (0.26 vs. 8.35 pg/ml, p=0.004) were increased in plasma samples of EMR failure patients compared to those who achieved EMR, and this was independent of white cell count (both for TGF-α and IL-6) and Sokal Score (TGF-α only). While less compelling, high TGF-α alone was associated with EMR failure in 15/39 (38%) patients compared to 8/144 (6%) in the TGF-αlo group (p<0.001) and reduced transformation-free survival (92% vs. 99%, p=0.029). Interestingly, the TGF-αlo group also had higher rates of complete molecular response (38% vs. 8%, p=0.005). While the relevance of IL-6 in CML pathogenesis has been previously proposed the association of the EGFR ligand TGF-α with CML treatment outcomes is novel. Conclusion: These data highlight for the first time the prognostic value of cytokine profiling in CML patients. The combination of TGF-α and IL-6 plasma levels at CML diagnosis is strongly predictive for EMR, transformation and CMR. While TGF-α alone delineates a poor response group of patients, the addition of IL-6 provides significant additional power and strongly selects for high-risk patients who may benefit significantly from very early proactive intervention. Thus, incorporation of these simple measurements to the diagnostic work-up of CP-CML patients may enable therapy intensity to be individualized according to the risk profile of the patient. Abstract 1788. Table 1: Treatment outcomes in patients grouped according to predictive plasma biomarkers N(of 183 total patients) EMR failure Trans-formation Mutation MMR by 24 months CMR by 24 months + TGF-αhi/IL-6hi 20 12 (60%) 3# 5 9 (45%) 1 (5%) TGF-αhi/IL-6lo 19 3 (16%) 0 0 12 (63%) 2 (11%) TGF-αlo 144 8 (6%) 2## 6 117 (81%) 55 (38%) Overall p value (log-rank) < 0.001* <0.001 <0.001 0.004 0.004 MMR – major molecular response CMR – complete molecular response (undetectable BCR-ABL1) * p value is only indicative as the groups were formed based on EMR # all early transformations (< 1 year of TKI treatment) ## all late transformations Disclosures Reynolds: Novartis: Shareholder Other. Biondo:CSL Limited: Employment. Busfield:CSL Limited: Employment. Vairo:CSL Limited: Employment. Yeung:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. White:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Ariad: Research Funding. Hughes:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees.
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Pasquini, Ricardo, Jorge E. Cortes, Hagop M. Kantarjian, David Joske, Luis A. Meillon, Oleg Zernovak, Ramsathish Sivarathinasami, LaTonya Collins, Timothy P. Hughes, and Dong-Wook Kim. "Survey of the Frontline Treatment and Management of Chronic Myeloid Leukemia (CML) in a Real-Word Setting: The 3rd Annual Update of the Worldwide Observational Registry Collecting Longitudinal Data on Management of Chronic Myeloid Leukemia Patients (The WORLD CML Registry)." Blood 118, no. 21 (November 18, 2011): 1695. http://dx.doi.org/10.1182/blood.v118.21.1695.1695.
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Abstract Abstract 1695 Background: A global, prospective registry was established to document the frequency of diagnostic testing, management (mgmt) strategies, and outcomes of patients (pts) with CML. Here, we summarize the reported deviations from published disease mgmt recommendations and the overall efficacy achieved by pts. Methods: 1853 pts (≥ 16 years of age) within 6 months (mo) + 2 weeks of CML diagnosis were enrolled from Latin America (LA; n = 497), United States (US; n = 379), Asia Pacific (AP; n = 465), Middle East and Africa (MEA; n = 209), and Russia and Turkey (RT; n = 303). Baseline demographics and medical history were collected at enrollment; current disease status and mgmt information were collected at approximately 6-mo intervals or with a change in disease status or mgmt. Results: From February 2008 to June 2011, data were available for 1831 (99%) pts. Across all regions, nearly all (93.8%) screened pts were in chronic phase CML. Regardless of the time of evaluation (eval), disease burden was mostly assessed through the use of hematologic counts (Table 1). Cytogenetic testing and molecular monitoring were used in a minority of pts at any timepoint. Hydroxyurea (HU) and imatinib were the first agents used in 61.9% and 29.5% of pts, respectively (Table 2). Overall, 81.1% of pts received imatinib therapy at some time and it was the most common second agent (48.1%) pts received. Among the 49% of pts who had response assessments, subsequent treatment changes occurred most frequently (23.9% of pts) at the 3-mo timepoint (Table 1). The median time from disease eval to dose/regimen modification was 3 days. Of those who received imatinib, 32% had dose modifications primarily for: lack of efficacy (20%), physician request (20%), and adverse events (19%). Of the pts with a corresponding eval at 12 mo after diagnosis, 88% had a CHR, 65.4% had a CCyR, and 42.5% had a MMR (BCR-ABLIS ≤.1%). These data are preliminary; response assessments by treatment, as well as further efficacy analyses, are ongoing. Conclusions: Overall, the majority of pts did not have cytogenetic or BCR-ABL transcript level testing performed per the European LeukemiaNet recommendations. Furthermore, despite availability of more effective therapies for the treatment of CML, HU is still used as a primary therapy in a substantial proportion of pts. Based on this analysis, pts outside the US primarily receive HU as initial therapy rather than tyrosine kinase inhibitors (TKIs). Overall, second-generation TKIs, such as nilotinib and dasatinib, are infrequently used. These results illustrate the need for continuing education on the mgmt of CML in order to improve outcomes for all pts. Disclosures: Pasquini: Bristol Myers Squibb: Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Cortes:Bristol Myers Squibb: Consultancy, Research Funding; Novartis Pharmaceuitcals: Consultancy, Research Funding. Kantarjian:Pfizer: Research Funding; Novartis: Research Funding; Novartis: Consultancy; BMS: Research Funding. Zernovak:Novartis: Employment, Equity Ownership. Sivarathinasami:Novartis: Employment. Collins:Novartis: Employment. Hughes:Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees. Kim:BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding.
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Denson, Ariel, Kristyn Y. DiSogra, Justin Arnall, Srinivasa R. Sanikommu, Nilay A. Shah, Brittany K. Ragon, Thomas G. Knight, et al. "Characteristics Affecting Durability and Tolerability of Imatinib in Patients Switched from Brand to Generic and Newly Diagnosed Patients Initially Prescribed Generic Imatinib for Chronic Myeloid Leukemia." Blood 136, Supplement 1 (November 5, 2020): 14–17. http://dx.doi.org/10.1182/blood-2020-137250.
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Introduction Gleevec, Imatinib mesylate, is the first in class BCR-ABL tyrosine kinase inhibitor initially approved to treat CML. In February 2016, generic imatinib products became available. As generic products are not required to offer comparative efficacy and safety data, differences may arise. Small reports have found no significant differences in response durability and tolerability in patients transitioned from Gleevec to generic imatinib. Further, lower cost of generic products often influence treatment decisions and patient compliance. We sought to evaluate response durability, tolerability, financial costs, and adherence in patients with chronic phase CML (cpCML) who switched from Gleevec to generic imatinib and newly diagnosed cpCML patients initiated on generic imatinib. Methods We conducted a single-center, retrospective chart review of adult patients who received imatinib therapy for cpCML between June 1, 2015 to November 14, 2019. Patients who received ≥6 months of brand through the Specialty Pharmacy Service (SPS) at Atrium Health prior to switching to generic were included in Group 1 (Switch). Patients who initiated therapy with generic imatinib dispensed from SPS were included in Group 2 (New Start). Durability of response was described determined via peripheral blood BCR-ABL transcripts by PCR and reported major molecular response (MMR) after 12 months generic imatinib therapy. Additional factors characterizing the durability and tolerability of therapy included adverse effects due to drug, dose modifications, adherence rate, prescription cost per month, and frequency of switch between generic products. Results Of 298 patients assessed, 12 patients were evaluable. There were 7 Switch patients and 5 New Start patients. Figure 1. All 12 patients met WHO diagnostic cpCML criteria. No patients in either group had accelerated or blast phase CML, no patients received maintenance imatinib following allogeneic HCT. In the Switch Group, 4 patients (57%) achieved MMR after 12 months of generic therapy. Of the 3 patients that did not achieve MMR, 1 patient relocated prior to 12-month assessment, 1 patient was noted to be non-compliant, and 1 patient had several treatment delays and dose reductions due to toxicities. 1 New Start patient achieved MMR at 12 months. Of those not achieving MMR, 1 was started on a reduced dose (100 mg /day) due to renal dysfunction, 1 had a PDC of 49.10% due to treatment delays while receiving treatment for a different malignancy, and 2 patients had logarithmic decreases in BCR-ABL but had not crossed the MMR threshold after 12 months of therapy. 5 Switch patients (71.4%) reported at least 1 adverse effect related to therapy, 3 of these (42.9%) required dose reduction. The adverse effects requiring dose reductions in the New Start patients included thrombocytopenia (n=2) and myalgia (n=1). All New Start patients reported at least 1 adverse effect with none of these patients requiring a dose reduction. Cost stayed the same or was reduced for 85.7% of the Switch patients, 1 patient experienced a cost increase and did not have co-pay assistance, and 2 patients received copay assistance. Cost of generic therapy was <$10/month in 85.7% of the Switch and 80% of the New Start patients. No patients experienced disease progression and PDC was >90% after 12 months on generic therapy for 71.4% Switch patients and 80% New Start patients. Table 1. and Table 2. Conclusion Patients with cpCML switched from brand to generic imatinib and patients newly started on generic imatinib appear to have durable responses and tolerance to generic imatinib. Dose reductions and non-adherence may have contributed to inadequate disease control in patients not achieving MMR in both groups. Patients switched from brand to generic imatinib may develop new side effects necessitating dose reduction. Thrombocytopenia may be more common in patients switched from brand to generic imatinib. Adherence to brand and generic imatinib is high and medication is affordable with most patients paying <$10/month. Our study is limited by a small sample size and retrospective nature. Prospective large studies are needed to compare tolerability and durability differences between brand and generic imatinib and available imatinib generic products. Disclosures Knight: Foundation for Financial Planning: Research Funding. Ai:Celgene: Speakers Bureau; Incyte: Speakers Bureau. Grunwald:Premier: Consultancy; Astellas: Consultancy; Janssen: Research Funding; Merck: Research Funding; Janssen: Research Funding; Forma Therapeutics: Research Funding; Forma Therapeutics: Research Funding; Astellas: Consultancy; Premier: Consultancy; Trovagene: Consultancy; Trovagene: Consultancy; Daiichi Sankyo: Consultancy; Astellas: Consultancy; Daiichi Sankyo: Consultancy; Trovagene: Consultancy; Abbvie: Consultancy; Abbvie: Consultancy; Agios: Consultancy; Daiichi Sankyo: Consultancy; Agios: Consultancy; Abbvie: Consultancy; Merck: Consultancy; Merck: Consultancy; Agios: Consultancy; Amgen: Consultancy; Amgen: Consultancy; Cardinal Health: Consultancy; Merck: Consultancy; Cardinal Health: Consultancy; Pfizer: Consultancy; Amgen: Consultancy; Pfizer: Consultancy; Cardinal Health: Consultancy; Celgene: Consultancy; Celgene: Consultancy; Pfizer: Consultancy; Incyte: Consultancy, Research Funding; Incyte: Consultancy, Research Funding; Celgene: Consultancy; Incyte: Consultancy, Research Funding; Genentech/Roche: Research Funding; Premier: Consultancy; Genentech/Roche: Research Funding; Genentech/Roche: Research Funding; Forma Therapeutics: Research Funding. Avalos:Juno: Membership on an entity's Board of Directors or advisory committees; Best Practice-Br Med J: Patents & Royalties: receives royalties from a coauthored article on evaluation of neutropenia. Copelan:Amgen: Membership on an entity's Board of Directors or advisory committees. Chojecki:Novartis: Other: Investigator Meeting Attendance; Incyte: Research Funding.
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Kumar, Ashwini, Muntasir Mamun Majumder, Jesus María Lopez Martí, Alun Parsons, Pirkko Mattila, Kimmo Porkka, Olli Kallioniemi, and Caroline A. Heckman. "The Use of RNA Sequencing to Identify Disease-Specific Gene Expression Signatures and Critical Regulatory Networks Across Hematologic Malignancies." Blood 124, no. 21 (December 6, 2014): 2203. http://dx.doi.org/10.1182/blood.v124.21.2203.2203.
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Abstract Introduction Transcriptome analysis by next-generation sequencing (RNA-seq) allows investigation of hematologic malignancies at unsurpassed resolution and provides promising insights into their molecular etiology. Dissecting the underlying biology depends on specific molecular signatures deregulated in the disease subtypes. In addition, gene expression profiles may potentially be used to identify driver genetic alterations and stratify patients based on molecular subtype. In this study, we generated gene expression profiles from RNA-seq data derived from patients with hematologic disease, including acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myelomonocytic leukemia (CMML), myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML) and multiple myeloma (MM). Using these data, we aimed to identify disease-specific differentially expressed genes and gain better understanding of the biological functions of these genes for the development of biomarkers and therapeutic strategies in these malignancies. Methods Mononuclear cells (MNCs) were isolated from bone marrow or peripheral blood by Ficoll separation from AML (n=24), ALL (n=5), CMML (n=10), MDS (n=4), CML (n=2) and MM (n=7) patients. For MM, CD138+ cells were enriched from MNCs by immunomagnetic bead selection. Total RNA from isolated cells was depleted of ribosomal RNA and reverse transcribed for cDNA. RNA-seq libraries were prepared and sequenced on the Illumina HiSeq instrument. Reads were filtered and aligned to the hg19 human reference genome using TopHat. Second step normalization was carried out to compare gene expression values (FPKM) across samples using a normalization factor derived from 18 reference genes. A log normalized relative expression value for each gene was calculated compared to the median value across all samples. Average linkage based hierarchical clustering was performed and then visualized with TreeView. Network and pathway analyses were performed with IPA (www.ingenuity.com) and Cytoscape¨. Results Unsupervised hierarchical clustering of all samples resulted in grouping based on clinical phenotypes with a unique gene signature characteristic for each group (Figure). Analyses of different hematologic malignancies ensured credibility of the classification and highlighted differences in underlying cell signaling networks of each disease. Group I consisted of the multiple myeloma samples, where we identified 25 frequently upregulated genes. Network analyses revealed expression of the upregulated molecules is controlled by two major transcription factors, IRF4 and JUN, which represent the major hubs of the gene signature network. Group II consisted of samples with the BCR-ABL1 fusion and BCR-ABL-like ALLs. Enriched genes in this subgroup included regulators of B-cell development and maturation, plus genes involved in antigen presentation including TCL1A, CD19, CD79, HLA-DQA1, HLA-DQB1 and HLA-DRB1. Group III represented myelodysplastic and myeloproliferative neoplasms and included the MDS and CMML samples. A set of 14 genes differentially enriched in this group formed a unique pro-inflammatory signature. This included TNF-α and IL1B, which act as major regulators of smoldering inflammation driving NF-κB activity and orchestrating downstream activation of signature genes. While MM is known to have activated NF-κB signaling, the gene expression signatures of the MM and MDS/MPN groups were distinct from each other, and included activation of separate sets of cytokines and chemokines. AML samples exhibited heterogeneity in gene expression and formed two groups (IVA, IVB). A HOX gene family expression signature was observed in the FLT3-ITD positive AML samples. Summary Our results show that RNA-seq can be used to identify dominant gene expression patterns characterizing different hematologic disease samples, including those sharing a common genetic base (e.g. BCR-ABL, FLT3-ITD) or clinical phenotype (e.g. MDS/MPN, MM). Based on our results, IRF4 may be an attractive therapeutic target for MM. CMML is difficult to diagnose, however, it can be defined by a set of differentially expressed genes that could potentially be used as diagnostic markers. We also show a pivotal role for NF-κB and TNF-α signaling in the pathogenesis of MDS/MPN suggesting that drugs targeting these factors may be useful for the treatment of these diseases. Figure 1 Figure 1. Disclosures Porkka: BMS: Honoraria; BMS: Research Funding; Novartis: Honoraria; Novartis: Research Funding; Pfizer: Research Funding. Kallioniemi:Medisapiens: Consultancy, Membership on an entity's Board of Directors or advisory committees. Heckman:Celgene: Research Funding.