Academic literature on the topic 'BCR-ABL, LAMP, Molecular Diagnostic, CML'

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.

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.

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.

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.

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.

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

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%.

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.

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.

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.

Leukemias are diseases characterized by an uncontrolled proliferation of malignant hematopoietic stem cells. The fusion gene BCR-ABL is the result of a reciprocal translocation between chromosome 9 and chromosome 22 t (9;22) and encodes an oncogenic tyrosine kinase protein responsible for the neoplastic transformation observed in chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). The isoform p210 is the hallmark of CML, detectable in more than 95% of cases, while in LAL, the fusion gene BCR-ABL may be present in both isoforms p190 (60% of cases) and p210 (40 % of cases). The molecular detection of BCR-ABL is essential to diagnose CML and Philadelphia positive ALL, making possible the administration of proper treatment. Moreover, the discrimination of the isoform, only possible using molecular methods, facilitates the choice of the specific quantitative test for the molecular monitoring during the treatment. To date, the most widely used molecular techniques for the detection of the transcripts are based on the RT-PCR method (Reverse Transcription-Polymerase Chain Reaction). It is a time-consuming procedure consisting of several steps, retro transcription - amplification - gel detection, which must be performed by skilled personnel and in equipped laboratories. The risk of cross-contamination, due to the multistep feature of the technique, and the absence of an internal reaction control, may also lead to false positive or false negative signals generation. The PhD project presented in this thesis describes the development and optimization of an innovative molecular diagnostic test, based on RT-QLAMP® technology (Reverse Transcription Loop-Mediated Isothermal Amplification), and its application in the diagnostic field for the simultaneous detection of BCR-ABL p210 and p190 transcripts. The optimization was performed on both plasmid controls and RNA extracted from cell lines. Moreover the final assay was validated on clinical samples. The LAMP technology has proven to be very sensitive and specific, simple and rapid. The amplification and detection of the transcripts occur in real-time, inside a single tube and starting directly from RNA. The negative clinical samples were validated by the amplification of the internal control and the possibility of cross-contamination of the sample was dramatically decreased. In addition, the method has proved to be very robust due to the insensitivity to the major PCR inhibitors. The new diagnostic assay overcomes all the limitations of the currently used diagnostic methods and represents a valid alternative for the molecular diagnosis of chronic myeloid leukemia and acute lymphoblastic leukemia.