Journal articles on the topic 'Predictive DNA typing'
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Li, Xin, Xuan Zhang, Xiangyu Lin, Liting Cai, Yan Wang, and Zhiqiang Chang. "Classification and Prognosis Analysis of Pancreatic Cancer Based on DNA Methylation Profile and Clinical Information." Genes 13, no. 10 (October 21, 2022): 1913. http://dx.doi.org/10.3390/genes13101913.
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Pancreatic adenocarcinoma (PAAD) has a poor prognosis with high individual variation in the treatment response among patients; however, there is no standard molecular typing method for PAAD prognosis in clinical practice. We analyzed DNA methylation data from The Cancer Genome Atlas database, which identified 1235 differentially methylated DNA genes between PAAD and adjacent tissue samples. Among these, 78 methylation markers independently affecting PAAD prognosis were identified after adjusting for significant clinical factors. Based on these genes, two subtypes of PAAD were identified through consistent clustering. Fourteen specifically methylated genes were further identified to be associated with survival. Further analyses of the transcriptome data identified 301 differentially expressed cancer driver genes between the two PAAD subtypes and the degree of immune cell infiltration differed significantly between the subtypes. The 14 specific genes characterizing the unique methylation patterns of the subtypes were used to construct a Bayesian network-based prognostic prediction model for typing that showed good predictive value (area under the curve value of 0.937). This study provides new insight into the heterogeneity of pancreatic tumors from an epigenetic perspective, offering new strategies and targets for personalized treatment plan evaluation and precision medicine for patients with PAAD.
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Titov, Sergei E., Evgeniya S. Kozorezova, Pavel S. Demenkov, Yulia A. Veryaskina, Irina V. Kuznetsova, Sergey L. Vorobyev, Roman A. Chernikov, Ilya V. Sleptsov, Nataliya I. Timofeeva, and Mikhail K. Ivanov. "Preoperative Typing of Thyroid and Parathyroid Tumors with a Combined Molecular Classifier." Cancers 13, no. 2 (January 11, 2021): 237. http://dx.doi.org/10.3390/cancers13020237.
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In previous studies, we described a method for detecting and typing malignant tumors of the thyroid gland in fine-needle aspiration biopsy samples via analysis of a molecular marker panel (normalized HMGA2 mRNA level; normalized microRNA-146b, -221, and -375 levels; mitochondrial-to-nuclear DNA ratio; and BRAFV600E mutation) in cytological preparations by quantitative PCR. In the present study, we aimed to estimate the specificity of the typing of different thyroid tumors by the proposed method. Fine-needle aspiration cytological preparations from 278 patients were used. The histological diagnosis was known for each sample. The positive and negative predictive values of the method assessed in this study were, respectively, 100% and 98% for papillary thyroid carcinoma (n = 63), 100% and 100% for medullary thyroid carcinoma (n = 19), 43.5% and 98% for follicular carcinoma (n = 15), and 86% and 100% for Hürthle cell carcinoma (n = 6). Thus, we demonstrate that the diagnostic panel, including the analysis of microRNA expression, mRNA expression, the BRAFV600E mutation, and the mitochondrial-to-nuclear DNA ratio, allows the highly accurate identification of papillary thyroid carcinoma, medullary thyroid carcinoma, and Hürthle cell carcinoma but not malignant follicular tumors (positive predictive value was below 50%).
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Titov, Sergei E., Evgeniya S. Kozorezova, Pavel S. Demenkov, Yulia A. Veryaskina, Irina V. Kuznetsova, Sergey L. Vorobyev, Roman A. Chernikov, Ilya V. Sleptsov, Nataliya I. Timofeeva, and Mikhail K. Ivanov. "Preoperative Typing of Thyroid and Parathyroid Tumors with a Combined Molecular Classifier." Cancers 13, no. 2 (January 11, 2021): 237. http://dx.doi.org/10.3390/cancers13020237.
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In previous studies, we described a method for detecting and typing malignant tumors of the thyroid gland in fine-needle aspiration biopsy samples via analysis of a molecular marker panel (normalized HMGA2 mRNA level; normalized microRNA-146b, -221, and -375 levels; mitochondrial-to-nuclear DNA ratio; and BRAFV600E mutation) in cytological preparations by quantitative PCR. In the present study, we aimed to estimate the specificity of the typing of different thyroid tumors by the proposed method. Fine-needle aspiration cytological preparations from 278 patients were used. The histological diagnosis was known for each sample. The positive and negative predictive values of the method assessed in this study were, respectively, 100% and 98% for papillary thyroid carcinoma (n = 63), 100% and 100% for medullary thyroid carcinoma (n = 19), 43.5% and 98% for follicular carcinoma (n = 15), and 86% and 100% for Hürthle cell carcinoma (n = 6). Thus, we demonstrate that the diagnostic panel, including the analysis of microRNA expression, mRNA expression, the BRAFV600E mutation, and the mitochondrial-to-nuclear DNA ratio, allows the highly accurate identification of papillary thyroid carcinoma, medullary thyroid carcinoma, and Hürthle cell carcinoma but not malignant follicular tumors (positive predictive value was below 50%).
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Martí-Carreras, Joan, and Piet Maes. "BKTyper—Web Application for VP1 and NCCR Polyoma BK Typing." Proceedings 50, no. 1 (June 9, 2020): 25. http://dx.doi.org/10.3390/proceedings2020050025.
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Human polyoma BK virus (BKV) prevalence has been increasing due to the introduction of more potent immunosuppressive agents, mostly in immunocompromised patients. BKV has been linked mostly to polyomavirus-associated hemorrhagic cystitis, and polyomavirus-associated nephropathy. BKV is a circular double stranded DNA virus (cdsDNA) with an average genome size of 5100 bp and an average GC content of 40%. Its genome codifies for five proteins: VP1, VP2, VP3, Angio gene, and the antigen T (which includes an event of alternative splicing, yielding a short and a large antigen T transcript). Additionally, it contains the non-coding control region (NCCR), known to be highly repetitive and to vary in number, length, and location of the repeats. Subtyping of BKV has been mainly studied in VP1 and the NCCR. Subtyping and subgrouping of BKV is conducted routinely in diagnostic assays and in epidemiological studies. Recently, Morel et al. published (Journal of Clinical Microbiology 2017; 55, 4) a strategy to subtype BKV through 100 bp VP1 amplicon. NCCR diversity is more complex than VP1, as it is configured by five repeat blocks (O, P, Q, R, and S). NCCR blocks can vary in number and length, resulting in a gradient of infectivity and replication. Rearranged NCCR have been linked to diverse patient etiologies, although any specific arrangement has failed to correlate with disease outcome or to have any predictive value. Due to the high abundance of BKV individuals and the clinical implications for human health that may represent BKV typing, a reliable, automatic, and free typing tool would be of great interest. Here, BKTyper is presented, a whole genome genotyper for polyoma BKV, based on a VP1 typing by Morel’s algorithm and NCCR block identification. BKTyper can accept both whole BKV genome or regions of interest in fasta format to generate the typing profile and phylogenetic analysis.
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Rietveld, MJH, JC van der Valk, IL Bongers, TM Stroet, PE Slagboom, and DI Boomsma. "Zygosity diagnosis in young twins by parental report." Twin Research 3, no. 3 (June 1, 2000): 134–41. http://dx.doi.org/10.1375/twin.3.3.134.
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AbstractThis study reports on zygosity determination in twins of childhood age. Parents responded to questionnaire items dealing with twin similarity in physical characteristics and frequency of mistaking one twin for another by parents, relatives and strangers. The accuracy of zygosity diagnosis was evaluated across twins aged 6, 8, and 10 and across parents. In addition, it was examined whether the use of multiple raters and the use of longitudinal data lead to an improvement of zygosity assignment. Complete data on zygosity questions and on genetic markers or blood profiles were available for 618 twin pairs at the age of 6 years. The method used was predictive discriminant analyses. Agreement between zygosity assigned by the replies to the questions and zygosity determined by DNA markers/blood typing was around 93%. The accuracy of assignment remained constant across age and parents. Analyses of data provided by both parents and collected over multiple ages did not result in better prediction of zygosity. Details on the discriminant function are provided. Twin Research (2000) 3, 134–141.
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Melsheimer, Peter, R. Klaes, M. v. Knebel Doeberitz, and G. Bastert. "Prospective clinical study comparing DNA flow cytometry and HPV typing as predictive tests for persistence and progression of CIN I/II." Cytometry 46, no. 3 (2001): 166–71. http://dx.doi.org/10.1002/cyto.1101.
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Almeida, Nalvo F., Shuangchun Yan, Rongman Cai, Christopher R. Clarke, Cindy E. Morris, Norman W. Schaad, Erin L. Schuenzel, et al. "PAMDB, A Multilocus Sequence Typing and Analysis Database and Website for Plant-Associated Microbes." Phytopathology® 100, no. 3 (March 2010): 208–15. http://dx.doi.org/10.1094/phyto-100-3-0208.
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Although there are adequate DNA sequence differences among plant-associated and plant-pathogenic bacteria to facilitate molecular approaches for their identification, identification at a taxonomic level that is predictive of their phenotype is a challenge. The problem is the absence of a taxonomy that describes genetic variation at a biologically relevant resolution and of a database containing reference strains for comparison. Moreover, molecular evolution, population genetics, ecology, and epidemiology of many plant-pathogenic and plant-associated bacteria are still poorly understood. To address these challenges, a database with web interface was specifically designed for plant-associated and plant-pathogenic microorganisms. The Plant-Associated Microbes Database (PAMDB) comprises, thus far, data from multilocus sequence typing and analysis (MLST/MLSA) studies of Acidovorax citrulli, Pseudomonas syringae, Ralstonia solanacearum, and Xanthomonas spp. Using data deposited in PAMDB, a robust phylogeny of Xanthomonas axonopodis and related bacteria has been inferred, and the diversity existing in the Xanthomonas genus and in described Xanthomonas spp. has been compared with the diversity in P. syringae and R. solanacearum. Moreover, we show how PAMDB makes it easy to distinguish between different pathogens that cause almost identical diseases. The scalable design of PAMDB will make it easy to add more plant pathogens in the future.
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Roth, A. D., S. Tejpar, P. Yan, R. Fiocca, D. Dietrich, G. Bodoky, R. Labianca, D. Cunningham, E. Van Cutsem, and F. Bosman. "Tissue biomarkers (BIOM) in colon cancer (COC): The translational study on the randomized phase III trial comparing infused irinotecan/5-fluorouracil (5-FU)/folinic acid (FA) to 5-FU/FA in stage II-III COC patients (pts)." Journal of Clinical Oncology 25, no. 18_suppl (June 20, 2007): 4022. http://dx.doi.org/10.1200/jco.2007.25.18_suppl.4022.
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4022 Background and Aims: PETACC 3 is a large adjuvant trial with 3,005 COC pts. The value of BIOM in COC in adjuvant setting is still a matter of debate because of lack of large data sets. We took advantage of PETACC 3 to assess P53, SMAD4, thymidylate synthetase (TS), telomerase (HTERT) expressions, UGT1A1 genotype, KRAS and BRAF mutations, microsatellite instability (MSI), 18q and 8p LOH with regard to their prognostic and predictive value and their individual interactions on a very large homogeneous cohort of COC pts. In addition we investigated the association between UGT1A1 genotype and occurrence of diarrhoea and Gd 4 neutropenia. Methods: 1,564 formalin fixed paraffin embedded (FFPE) tissue blocks of PETACC 3 pts were prospectively collected and 5–20μ sections cut. DNA from normal (Nor) and tumoral (Tu) tissue was extracted after section microdissection. P53, SMAD4, TS and HTERT were assessed by immunohistochemistry (IHC); MSI was typed with 10 markers, KRAS exon 2 and BRAF exon 15 mutations by allele specific real time PCR on Tu DNA; 18q and 8p LOH by typing multiple SNPs by pyrosequencing on Nor/Tu DNA; UGT1A1 genotypes by PCR and fragment sizing on Nor DNA. Prognostic/predictive value of each BIOM is analysed by Cox regression for disease free survival and by logistic regression for specific toxicity. Associations between any 2 categorized BIOM and between each BIOM and each known prognostic variable are tested by chi-square tests. Results: DNA of 1405 pts was extracted and successfully analyzed in 97.1% for KRAS, 98.6% for BRAF, 94% for 18q LOH, 93.6% for MSI, 86% for UGT1A1, 8p LOH is still ongoing. Of 1530 pts slides IHC analysis was successful in 94.5% for P53, 94.2% for SMAD4, 82.9% for TS, 53.9% for HTERT. The clinical database was made available in Nov 06 and statistical analysis started on Dec 11th 2006. Conclusion: This is the largest multicenter centrally coordinated tissue BIOM study performed in COC to date. The high success rate of analysis shows that large prospective BIOM studies can be performed on routine FFPE material. Final results on the prognostic/predictive value of each molecular BIOM will be available at the meeting. No significant financial relationships to disclose.
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Chapman, Rebecca, David R. Ghasemi, Pierre LeBlond, Hendrik Witt, Jacques Grill, John-Paul Kilday, Piergiorgio Modena, et al. "EPEN-24. Biological markers of ependymoma in children and adolescents (BIOMECA): Systematic comparison of methods for the precise evaluation of biomarkers for ependymoma diagnosis and prognostication." Neuro-Oncology 24, Supplement_1 (June 1, 2022): i44. http://dx.doi.org/10.1093/neuonc/noac079.160.
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Abstract The identification and validation of prognostic and diagnostic biomarkers is a key element of The SIOP Ependymoma II trial, realised through the Biomarkers of Ependymoma in Children and Adolescents study (BIOMECA). BIOMECA aims to identify and validate biomarkers for prediction of outcome whilst enhancing stratification for the next generation of ependymoma trials. We outline our findings from the first 147 consecutive BIOMECA cases (posterior fossa, PF=111; supratentorial, ST=32; spinal, SP=4). We compared various methods for biomarker assessment, across six European laboratories to determine key analysis methods. Methods included: methylation-based classification (EPIC 850K DNA methylation array) (n=141); immunohistochemistry (IHC) for nuclear p65-RELA (n=32), H3K27me3 (n=115), and Tenascin-C (TNC) (n=147); copy number (CN) analysis by FISH, MLPA (1q, CDKN2A) (n=147), and MIP (molecular inversion probe) and DNA methylation array (1q, CDKN2A, 6q, 11q, 13q, 22q) (n=141); analysis of ZFTA- and YAP1-fusions by RT-PCR, sequencing, Nanostring assays and break-apart FISH (n=32). Using DNA methylation-based classification, 91% (n=101/111) of PF cases classified as PF ependymoma group A (PFA) and 69% (n=22/32) of ST cases as ST ependymoma, ZFTA fusion-positive (ZFTA). Most PFAs demonstrated inter-centre agreement for loss of H3K27me3, and were TNC positive, representing surrogate markers for PFA identification. Combinations of p65-RELA IHC, FISH analysis, and RNA-based methods were suitable to identify ZFTA- and YAP1- fused ST ependymomas. Predictive CN alterations were identified by high-resolution, quantitative MIP technology.The integration of histopathology assessment and molecular typing is now critical as the updated 2021 WHO CNS5 classification of ependymomas lists seven molecularly distinct entities. This study highlights the importance of evaluating different methods in a prospective trial cohort. Here, advanced molecular techniques represent powerful tools for the classification of ependymoma entities (DNA methylation array) and for the detection of CN alterations (MIP) and specific fusions, enabling the correct classification and identification of prognostic markers.
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Volante, Marco, Alfred K. Lam, Mauro Papotti, and Giovanni Tallini. "Molecular Pathology of Poorly Differentiated and Anaplastic Thyroid Cancer: What Do Pathologists Need to Know?" Endocrine Pathology 32, no. 1 (February 4, 2021): 63–76. http://dx.doi.org/10.1007/s12022-021-09665-2.
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AbstractThe molecular characterization of poorly and anaplastic thyroid carcinomas has been greatly improved in the last years following the advent of high throughput technologies. However, with special reference to genomic data, the prevalence of reported alterations is partly affected by classification criteria. The impact of molecular pathology in these tumors is multifaceted and bears diagnostic, prognostic, and predictive implications although its use in the clinical practice is not completely assessed. Genomic profiling data claim that genetic alterations in poorly differentiated and anaplastic thyroid carcinomas include “Early” and “Late” molecular events, which are consistent with a multi-step model of progression. “Early” driver events are mostly RAS and BRAF mutations, whereas “Late” changes include above all TP53 and TERT promoter mutations, as well as dysregulation of gene involved in the cell cycle, chromatin remodeling, histone modifications, and DNA mismatch repair. Gene fusions are rare but represent relevant therapeutic targets. Epigenetic modifications are also playing a relevant role in poorly differentiated and anaplastic thyroid carcinomas, with altered regulation of either genes by methylation/deacetylation or non-coding RNAs. The biological effects of epigenetic modifications are not fully elucidated but interfere with a wide spectrum of cellular functions. From a clinical standpoint, the combination of genomic and epigenetic data shows that several molecular alterations affect druggable cellular pathways in poorly differentiated and anaplastic thyroid carcinomas, although the clinical impact of molecular typing of these tumors in terms of predictive biomarker testing is still under exploration.
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Shaffer, Brian C., Christine Tremblay, Daniel Peaceman, Jennifer Hsu, Seth M. Steinberg, Marcela Uribe, Sharon Adams, Willy A. Flegel, and Steven Z. Pavletic. "Minor Histocompatibility Antigen Mismatch and Incidence of Graft Versus Host Disease, Event-Free, and Overall Survival in Patients Undergoing Unrelated Donor Allogeneic Hematopoietic Cell Transplantation." Blood 120, no. 21 (November 16, 2012): 4201. http://dx.doi.org/10.1182/blood.v120.21.4201.4201.
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Abstract Abstract 4201 Graft versus Host Disease (GvHD) is a potentially devastating complication of allogeneic hematopoietic cell transplantation (HCT) initiated in part due to donor-recipient disparities in immunoreactive proteins, or minor histocompatibility antigens (mHAgs). There are no established prognostic tools to predict which patients will get acute or chronic GvHD. Analysis of mHAg mismatch is a potential predictive tool for GvHD; however, previous studies attempting to establish a relationship between mHAg mismatch and GvHD have been largely equivocal. Here we tested the hypothesis that analysis of an expanded set of mHAgs for mismatch in the GvH direction can be predictive of acute or chronic GvHD by NIH criteria. We additionally analyzed event-free (EFS) and overall survival (OS) in the mHAg matched and mismatched subgroups. Recipient/donor pairs from 45 HLA-A, -B, -C, and DRB1 matched unrelated donor transplants from 2007–2011 were retrospectively typed for 19 mHAgs using an SSP-PCR typing kit (Minor Histocompatibility Antigen Typing Kit; Life Technologies, Carlsbad, CA). Genomic DNA was obtained from peripheral blood mononuclear cells. EFS and OS were estimated using the Kaplan-Meier method. The relationship between mismatch and acute or chronic GvHD was described using Fischer's Exact Test. Cumulative incidence of treatment related mortality (TRM) was calculated using the Gooley Method. Two patients expired of early TRM and were excluded from the GvHD analyses. The rate of acute GvHD grades II-IV was 6 of 14 in those without a mHAg mismatch and 21 of 29 in those with a mismatch (43% versus 72%, P = 0.062). The rate of chronic GvHD was 8 of 14 in those without a mismatch and 10 of 29 in those with a mismatch (53% versus 34%, P = 0.140). The presence of a mismatch did not significantly impact EFS (P = 0.42) or OS (P = 0.26). The cumulative incidence of TRM at 24 months post transplantation was greater in patients with a mismatch (36% versus 13%). Two year OS was superior in patients who were conditioned with alemtuzumab (N = 24) and had a lower degree of mismatch (0–1 mismatch = 72% versus 2+ mismatches = 38%, P = 0.038). This study suggests the possibility of a relationship between mHAg mismatch and acute GvHD and TRM. Further study using this expanded mHAg analysis on a larger cohort of individuals would more adequately define the potential benefit of mHAg mismatch analysis in the context of unrelated donor HCT. Disclosures: No relevant conflicts of interest to declare.
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Park, Jae H., Andrew J. Yee, Thomas R. Spitzer, Susan L. Saidman, David T. Ting, Andrea Pannone, Jessica Reid, Steven McAfee, and Bimalangshu R. Dey. "KIR Ligand Incompatibility in HLA-Identical Sibling Nonmyeloablative Hematopoietic Stem Cell Transplantation for Hematologic Malignancies." Blood 108, no. 11 (November 16, 2006): 5371. http://dx.doi.org/10.1182/blood.v108.11.5371.5371.
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Abstract Natural killer (NK) cell alloreactivity based on inhibitory killer immunoglobulin-like receptor (KIR)-ligand incompatibility (i.e., missing KIR ligand) in the graft-vs-host (GVH) direction has been described to favorably influence engraftment, GVHD, and graft-vs-tumor (GVT) effect following haploidentical or matched unrelated HSCT in patients with hematologic malignancies. The degree and effect of KIR-ligand incompatibility has recently been explored following HLA-identical sibling HSCT (Hsu et al., Blood2005;105:4878). Based on a murine model, we developed a clinical trial with a goal of deliberately inducing a GVHD-free mixed chimeric platform following nonmyeloablative conditioning (consisting of equine ATG, cyclophosphamide, thymic irradiation, and a brief course of cyclosporine) and HLA-matched sibling HSCT. Donor leukocyte infusions (DLI) were given as early as five weeks post-HSCT to patients without GVHD in an attempt to achieve a conversion to full donor chimerism (FDC) with the goal of fully capturing GVT effect with little or no GVHD. In this study we hypothesized that KIR-ligand incompatibility in the GVH direction and KIR-ligand compatibility in the host-vs-graft (HVG) direction would reduce the rate of graft failure, decrease the incidence of GVHD, and improve overall survival following HLA-matched sibling HSCT. Fourteen transplant recipients (bone marrow, n=9; peripheral blood stem cell, n=5) with refractory hematologic malignancies (NHL, n=7; HD, n=3; MM, n=2; CLL, n=1; AML, n=1) were analyzed. KIR typing was accomplished using PCR amplified DNA from both donor and recipient patient samples. Typing of the amplified DNA was performed using the Lifecodes KIR-SSO typing kits (Tepnel Lifecodes Corporation). By using the SSO (sequence-specific oligonucleotides) technology in conjunction with the Luminex Instrument, KIR loci were identified for each patient and donor sample. KIR-ligand (HLA) incompatibility in the GVH and HVG directions were assessed based on HLA and KIR genotyping (KIR2DL1, KIR2DL2, KIR2DL3 and KIR3DL1) of 14 donors and 12 of the 14 recipients. Six of the 14 patients eventually lost their grafts despite DLI. Seven patients spontaneously achieved FDC and one converted to FDC following DLI. The missing KIR ligand analysis showed 12 patients (86%, n=12/14) with KIR-ligand incompatibility in the GVH direction (1 missing ligand, n=9; 2 missing ligands, n=3) and 10 patients (83%, n=10/12) with KIR-ligand incompatibility in the HVG direction (1 missing ligand, n=9; 2 missing ligands n=1). The presence or the degree of KIR-ligand incompatibility in GVH or HVG direction was not found to be predictive of spontaneous FDC or graft rejection. There was no significant relationship between the number of missing KIR ligands in either direction and the development of acute or chronic GVHD. This study shows a higher rate of KIR-ligand incompatibility in the GVH (86%) and HVG (83%) directions in the setting of HLA-matched sibling HSCT than previously reported. Although the small number of patients does not allow for statistically meaningful conclusions regarding clinical outcome, the observation of a high incidence of KIR-ligand incompatibility in this population justifies the study of larger patient cohorts to determine the influence of NK cell alloreactivity on transplant outcomes.
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Eing, Bodo R., Andrea Becker, Arthur Sohns, and Ronald Ringelmann. "Comparison of Roche Cobas Amplicor Mycobacterium tuberculosis Assay with In-House PCR and Culture for Detection of M. tuberculosis." Journal of Clinical Microbiology 36, no. 7 (1998): 2023–29. http://dx.doi.org/10.1128/jcm.36.7.2023-2029.1998.
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The new Roche Cobas Amplicor Mycobacterium tuberculosisassay, which is a semiautomated version of the manually performed Roche Amplicor M. tuberculosis test, was compared to culture and an IS6110-based in-house PCR protocol. A total of 1,681 specimens from 833 patients, including specimen types other than sputum, were tested in parallel by both the in-house PCR and the Cobas Amplicor M. tuberculosis assay. After we resolved discrepant PCR results, the sensitivity, specificity, and positive and negative predictive values for the Cobas Amplicor M. tuberculosis assay were 66.33, 99.71, 94.36, and 97.66%, respectively. The corresponding values for the in-house PCR were 91.08, 99.85, 97.87, and 99.37%, respectively. For culture- and smear-positive specimens, the sensitivity of the Cobas AmplicorM. tuberculosis test was 96.42% (in-house PCR, 100%). If only smear-negative sputum specimens were considered, the Cobas Amplicor M. tuberculosis assay exhibited a sensitivity of 45.45% (in-house PCR, 63.63%) relative to that of culture. With a modified protocol for DNA extraction (washing of samples plus ultrasonication), both PCR methods performed better with gastric aspirates than with sputum samples (sensitivity of the Cobas AmplicorM. tuberculosis assay with smear-negative gastric aspirates, 70.00%; sensitivity of in-house PCR, 90.00%). With dithiothreitol being used for liquefaction of specimens in this study, the Cobas Amplicor M. tuberculosis assay exhibited an inhibition rate of 9.16%. In our view, the new Cobas Amplicor M. tuberculosis test (i) is well suited for typing of smear-positive specimens, (ii) may also be applied to gastric aspirates and other types of specimens if DNA extraction methods are modified appropriately, and (iii) exhibits a sensitivity with smear-negative sputum specimens which makes it recommendable that a minimum of three samples from the same patient be tested.
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Nessa, Khairun, Dilruba Ahmed, Johirul Islam, FM Lutful Kabir, and M. Anowar Hossain. "Usefulness of a Multiplex PCR for Detection of Diarrheagenic Escherichia coli in a Diagnostic Microbiology Laboratory Setting." Bangladesh Journal of Medical Microbiology 1, no. 2 (May 25, 2016): 38–42. http://dx.doi.org/10.3329/bjmm.v1i2.21506.
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A multiplex PCR assay was evaluated for diagnosis of diarrheagenic Escherichia coli in stool samples of patients with diarrhoea submitted to a diagnostic microbiology laboratory. Two procedures of DNA template preparationproteinase K buffer method and the boiling method were evaluated to examine isolates of E. coli from 150 selected diarrhoeal cases. By proteinase K buffer method, 119 strains (79.3%) of E. coli were characterized to various categories by their genes that included 55.5% enteroaggregative E. coli (EAEC), 18.5% enterotoxigenic E. coli (ETEC), 1.7% enteropathogenic E. coli (EPEC), and 0.8% Shiga toxin-producing E. coli (STEC). Although boiling method was less time consuming (<24 hrs) and less costly (<8.0 US $/ per test) but was less efficient in typing E. coli compared to proteinase K method (41.3% vs. 79.3% ; p<0.001). The sensitivity and specificity of boiling method compared to proteinase K method was 48.7% and 87.1% while the positive and negative predictive value was 93.5% and 30.7%, respectively. The majority of pathogenic E. coli were detected in children (78.0%) under five years age with 53.3% under one year, and 68.7% of the children were male. Children under 5 years age were frequently infected with EAEC (71.6%) compared to ETEC (24.3%), EPEC (2.7%) and STEC (1.4%). The multiplex PCR assay could be effectively used as a rapid diagnostic tool for characterization of diarrheagenic E. coli using a single reaction tube in the clinical laboratory setting.Bangladesh J Med Microbiol 2007; 01 (02): 38-42
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Kahn, Ryan, Sushmita Gordhandas, Brandon Paul Maddy, Becky Baltich Nelson, Gulce Askin, Paul J. Christos, Thomas A. Caputo, Eloise Chapman-Davis, Kevin Holcomb, and Melissa Kristen Frey. "Universal endometrial cancer tumor typing: How much has immunohistochemistry, microsatellite instability, and MLH1 methylation improved the diagnosis of Lynch syndrome across the population?" Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): e17119-e17119. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.e17119.
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e17119 Background: Universal tumor testing for defective DNA mismatch repair (MMR) is recommended for all women diagnosed with endometrial cancer (EC) to identify those with underlying Lynch syndrome (LS). However, since its implementation in 2013, the effectiveness of this screening method on identifying individuals with LS across the population has not been well studied. The aim of this study was to evaluate outcomes of MMR immunohistochemistry (IHC), MLH1 methylation, and microsatellite instability (MSI) analysis among EC patients. Methods: We conducted a complete systematic search of online databases PubMed, Embase, Medline, and Cochrane Library between 1990-2018. A DerSimonian–Laird random-effects model meta-analysis was utilized to estimate the weighted prevalence of LS diagnoses. Results: The comprehensive search produced 3,427 publications. 29 peer-review studies met the inclusion criteria. 6,649 EC patients were identified, 206 (3%) were confirmed to have LS following positive universal tumor molecular screening.5,917 patients underwent tumor IHC, 28% had abnormal staining. 3,140 patients underwent MSI analysis, 31% had MSI instability. Among EC patients with deficient IHC staining or positive MSI analysis, the weighted prevalence of LS was 15% and 19% respectively. 1159 patients exhibited loss of MLH1 staining, 143 (13.7%) were found to be MLH1 methylation negative, 32 demonstrated a germline MLH1 mutation (2.8% of all MLH1 absent staining; 22.4% of all MLH1 methylation negative). 43% of EC patients diagnosed with LS via tumor typing would have been missed by family history-based screening alone. Conclusions: Despite widespread implementation of universal tumor testing in EC, data regarding results have previously been limited. For the first time, this study provides large-scale predictive values that will help practitioners evaluate abnormal results in the context of LS and aid in patient counseling. [Table: see text]
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Bastos-Oreiro, Mariana, Javier Anguita, Carolina Martínez-Laperche, Almudena Navarro, Lucía Fernandez, Pascual Balsalobre, Cristina Muñoz, et al. "Mismatches In Killer Immunoglobulin Receptor (KIR) Ligands and Inhibitory KIR Receptors Between Donor and Recipients Improve Survival After Non T Cell Depleted Haploidentical Transplantation." Blood 122, no. 21 (November 15, 2013): 2009. http://dx.doi.org/10.1182/blood.v122.21.2009.2009.
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Abstract Introduction Alloreactivity triggered by interaction of Killer immunoglobulin-like receptors (KIRs) on donor Natural killer (NK) cells and their ligands on recipients plays a role in the graft-versus-tumour effect. Different predictive models have been postulated for measuring alloreactivity: ligand incompatibility model, receptor-ligand model, missing ligand model and KIR gene-gene model. Our aim in this study is to evaluate the importance of differences in KIR and HLA genotype between donor and recipient (missing ligand model and KIR gene-gene model) in the setting of non T cell depleted haploidentical haematopoietic stem cell transplantation (HSCT) with high-dose, post-infusion cyclophosphamide (Cy) Patients and Methods 33 consecutive patients with haematological malignancies who received haploidentical HSCT with high-dose Cy post-transplantation between 2007 and 2013, and their donors were included for analysis (Table 1). HLA typing was used to identify KIR ligands HLA-B and HLA-C. KIR genotype was analyzed by PCR (KIR Typing, Miltenyi Biotec) on genomic DNA (Maxwell 16 Blood DNA Kit, Promega) from peripheral blood samples, and revealed with ethidium bromide after agarose gel electrophoresis. Results Demographic data and KIR genotype characteristics of donors and recipients are listed in Table 1. We found that KIR ligands mismatch between donor and recipient is related with improve of OS (52% vs. 82%; p=0,041) and DFS (63% vs. 22%; p=0,037) a year after transplant (Figure 1). Mismatch of inhibitors KIR receptors (iKIR) gene content between donors and recipients is also related with an improvement of OS (94% vs. 75%; p=0,049) and DFS (13% vs. 61%; p= 0,028) compared with no mismatch pairs. In the multivariable analysis, both KIR ligands mismatches (HR=4,38 CI:0.9-21; p=0,061) as well as iKIR mismatches (HR=4,15 CI:1-16; p=0,047), show a tendency to be independent variables for the reduction of disease relapse rate. Cumulative incidence of relapse for both variables studied is shown in Figure 2. Conspicuously, a sub-analysis in Hodgkin Lymphoma patients group shows and improve in DFS a year after transplant in patients with KIR ligands mismatches ( 100% vs. 48% p: 0,006) and iKIR mismatches (100% vs. % 33% p: 0,049), despite the small number of patients. Contrary to data published by other groups, patients receiving donor’s progenitors with Bx haplotype with centromeric genes (Cen-BB) did not show a benefit in survival (SG 85% vs. 90%, p= 0,05 and DFS 66% vs. 10%, p=0,047) a year after transplant Conclusion Our data suggest that in the setting of non T cell depleted haploidentical HSCT with high dose Cy post-infusion, KIR ligands and iKIR mismatch are related with an improve in survival, specially in the sub-group of patients with Hodgkin disease Disclosures: No relevant conflicts of interest to declare.
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Tota, Joseph, Salaheddin M. Mahmud, Alex Ferenczy, François Coutlée, and Eduardo L. Franco. "Promising strategies for cervical cancer screening in the post-human papillomavirus vaccination era." Sexual Health 7, no. 3 (2010): 376. http://dx.doi.org/10.1071/sh10022.
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Human papillomavirus (HPV) vaccination is expected to reduce the burden of cervical cancer in most settings; however, it is also expected to interfere with the effectiveness of screening. In the future, maintaining Pap cytology as the primary cervical screening test may become too costly. As the prevalence of cervical dysplasias decreases, the positive predictive value of the Pap test will also decrease, and, as a result, more women will be referred for unnecessary diagnostic procedures and follow-up. HPV DNA testing has recently emerged as the most likely candidate to replace cytology for primary screening. It is less prone to human error and much more sensitive than the Pap smear in detecting high-grade cervical lesions. Incorporating this test would improve the overall quality of screening programs and allow spacing out screening tests, while maintaining safety and lowering costs. Although HPV testing is less specific than Pap cytology, this issue could be resolved by reserving the latter for the more labour-efficient task of triaging HPV-positive cases. Because most HPV-positive smears would contain relevant abnormalities, Pap cytology would be expected to perform with sufficient accuracy under these circumstances. HPV Pap triage would also provide a low-cost strategy to monitor long-term vaccine efficacy. Although demonstration projects could start implementing HPV testing as a population screening tool, more research is needed to determine the optimal age to initiate screening, the role of HPV typing and other markers of disease progression, and appropriate follow-up algorithms for HPV-positive and Pap-negative women.
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Duncan, Daniel, Venkata Thodima, Jack Yen, Samir Parekh, Alessandro Lagana, and Rita Shaknovich. "Development of a Neoantigen Prediction Tool for Patient Stratification in Immuno-Oncology Trials." Blood 132, Supplement 1 (November 29, 2018): 2215. http://dx.doi.org/10.1182/blood-2018-99-119094.
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Abstract Immunotherapeutic agents are quickly becoming a routine aspect of treatment paradigms. However, despite clinical successes, cancer immunotherapy faces major challenges. One such challenge is that efficacy in most patients is unpredictable. Many immunotherapy treatments have demonstrated efficacy in only select cancer types. Variability in patient response indicates that immunotherapy needs to be patient-specific in order to be most effective. Identifying biomarkers that have value in predicting benefit from treatment with immunotherapy has been difficult. Few predictive biomarkers for immunotherapy treatments are robustly validated for use in clinical trials. Pivotal trials reveal that treatment benefits with checkpoint blockers is not solely restricted to PD-L1-positive patients, indicating the existence of other unknown biomarkers that could be predictive of response. Similarly, tumor mutational burden (TMB) has limitations, as it is not able to effectively segregate patients that are likely to respond to immunotherapeutic agents in tumor types that are relatively mutationally dormant. Increasingly, evidence suggests that tumor immunogenicity may be a strong biomarker for immunotherapy patient selection. In short, the abundance of predicted immunogenic mutations may be useful in predicting patients likely to benefit from checkpoint blockade and related immunotherapies. To address this need for a more specific biomarker, we have designed an assay and bioinformatic work-flow utilizing a multimodal neo-antigen prediction approach that combines data on somatic variants, RNA expression, and compatibility of resultant epitope with host HLA type. In summary, whole exome and whole transcriptome sequencing are performed on a patient tumor sample, and HLA typing is performed on a matched germline patient sample. The somatic variants (tumor-specific mutations) identified by exome sequencing are compared to the RNA sequencing data to identify the most prevalent variants in the transcriptome occurring in the most highly expressed regions. These highly expressed mutations are most likely to be translated into mutant peptides that can interact with MHC molecules and be subsequently presented on the tumor cell surface as neoantigens. The subject's HLA type is then determined using the seq2hla computational tool. Next, a molecular modeling tool, NetMHC4.0, compares the structures of the candidate mutant peptides to the HLA molecule structures and generates a goodness of fit prediction. A higher binding affinity between mutant peptide and HLA molecule corresponds to a greater likelihood of this complex existing on the cell surface as a neoantigen. This data - the DNA sequencing, RNA expression and binding affinity calculation - is combined via a series of filters to generate an immunogenicity score associated with each tumor mutation / predicted mutant peptide. These candidate neoantigens are then returned as a rank order list for each case. This information then can be used to guide targeted therapies and to stratify patients with higher immunogenicity scores for immunotherapy. To test our bioinformatic pipeline, we utilized a subset of multiple myeloma samples. Such analysis yielded a rank list of predicted neoantigens for each tumor sample, with associated immunogenicity scores for each prediction. Additionally, TMB was calculated for these samples. We compared the number of predicted neoantigens from our workflow to the TMB of the tumors as a proxy for this assay's performance against a current clinically utilized biomarker (TMB). The numbers of predicted neoantigens for the samples ranged from 19 to 61 (Average number of 41 neoantigens per sample), and the TMB scores for these samples respectively were between 7 and 13 mutations per megabase. Comparing these results using Pearson Correlation method yields a strong R squared value of 0.91. Among top ranking neoantigens were peptides associated with TP53, SIK3, ATM and NOTCH2 genes among others, and representing known frequently mutated genes in multiple myeloma. Therefore, our neoantigen predictor demonstrates promise as a reliable tool to identify markers of tumor immunogenicity. These preliminary results suggest that further validation of our process is warranted and may yield a new method for use in patient stratification and response prediction in immuno-oncology trials. Disclosures No relevant conflicts of interest to declare.
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Pang, Jungang, Jun Liu, Wantian Liang, Lijun Yang, and Liangyin Wu. "High Neutrophil-to-Platelet Ratio Is Associated with Poor Survival in Patients with Acute Aortic Dissection." Disease Markers 2022 (June 21, 2022): 1–8. http://dx.doi.org/10.1155/2022/5402507.
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Background. Acute aortic dissection (AAD), a serious and fatal cardiovascular disease, is characterized by inflammation that may further aggravate the condition. We evaluated the value of the neutrophil-to-platelet ratio (NPR) in the prognosis of AAD. Methods. We collected records of patients with AAD and clinical data from 2010 to 2020 and followed up on the relevant information for 136 months. The Kaplan–Meier (K–M) survival along with the univariate and multivariate Cox analyses was used to examine the prognostic value of NPR in AAD. In addition, nomograms were constructed by combining NPR, age, Stanford typing, and treatment methods. The accuracy of nomograms was evaluated using calibration plots, and the prediction efficiency of nomograms was evaluated by receiver operating characteristic curve analysis and decision curve analysis (DCA). Results. The K–M analysis showed that AAD patients with higher NPR exhibited worse prognosis. In addition, different Stanford typing and treatment methods produced varied prognosis results. Univariate and multivariate Cox analyses showed that NPR value, age, classification, and treatment were independent prognostic factors for the overall survival time of patients with AAD. Nomograms constructed by combining NPR, age, Stanford typing, and treatment methods showed good predictive efficacy, and the AUC values for 1-, 3-, and 5-year predicting were 0.82, 0.79, and 0.74, respectively. Conclusions. Our results suggest that pretreatment NPR can be used as a potential prognostic marker of overall survival time in patients with AAD.
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Flegel, Wagner, Muller, and Gassner. "Rh phenotype prediction by DNA typing and its application to practice." Transfusion Medicine 8, no. 4 (October 1998): 281–302. http://dx.doi.org/10.1046/j.1365-3148.1998.00173.x.
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Strauß, Lena, Ulla Ruffing, Salim Abdulla, Abraham Alabi, Ruslan Akulenko, Marcelino Garrine, Anja Germann, et al. "Detecting Staphylococcus aureus Virulence and Resistance Genes: a Comparison of Whole-Genome Sequencing and DNA Microarray Technology." Journal of Clinical Microbiology 54, no. 4 (January 27, 2016): 1008–16. http://dx.doi.org/10.1128/jcm.03022-15.
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Staphylococcus aureusis a major bacterial pathogen causing a variety of diseases ranging from wound infections to severe bacteremia or intoxications. Besides host factors, the course and severity of disease is also widely dependent on the genotype of the bacterium. Whole-genome sequencing (WGS), followed by bioinformatic sequence analysis, is currently the most extensive genotyping method available. To identify clinically relevant staphylococcal virulence and resistance genes in WGS data, we developed anin silicotyping scheme for the software SeqSphere+(Ridom GmbH, Münster, Germany). The implemented target genes (n= 182) correspond to those queried by the IdentibacS. aureusGenotyping DNA microarray (Alere Technologies, Jena, Germany). Thein silicoscheme was evaluated by comparing the typing results of microarray and of WGS for 154 humanS. aureusisolates. A total of 96.8% (n= 27,119) of all typing results were equally identified with microarray and WGS (40.6% present and 56.2% absent). Discrepancies (3.2% in total) were caused by WGS errors (1.7%), microarray hybridization failures (1.3%), wrong prediction of ambiguous microarray results (0.1%), or unknown causes (0.1%). Superior to the microarray, WGS enabled the distinction of allelic variants, which may be essential for the prediction of bacterial virulence and resistance phenotypes. Multilocus sequence typing clonal complexes and staphylococcal cassette chromosomemecelement types inferred from microarray hybridization patterns were equally determined by WGS. In conclusion, WGS may substitute array-based methods due to its universal methodology, open and expandable nature, and rapid parallel analysis capacity for different characteristics in once-generated sequences.
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Lee, Ji Eun, Jeong Min Lee, Jana Naue, Jan Fleckhaus, Ana Freire-Aradas, Jacqueline Neubauer, Ewelina Pośpiech, et al. "A collaborative exercise on DNA methylation-based age prediction and body fluid typing." Forensic Science International: Genetics 57 (March 2022): 102656. http://dx.doi.org/10.1016/j.fsigen.2021.102656.
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Church, SE, J. Reeves, DR Zollinger, J. McKay-Fleisch, AJ Bahrami, M. Holpert, AM White, et al. "P03.05 Deep spatial profiling of the immune landscape of MSI and MSS colorectal tumors." Journal for ImmunoTherapy of Cancer 8, Suppl 2 (October 2020): A24.1—A24. http://dx.doi.org/10.1136/jitc-2020-itoc7.45.
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IntroductionIn colorectal cancer (CRC) there have been many recent advances in immune related biomarkers that are both prognostic and predictive of response to immunotherapy. Microsatellite instability (MSI)/mismatch repair deficiency dMMR is present in 15–20% of CRCs and correlates with increased immunogenic mutations that often augment lymphocyte infiltration into the tumor microenvironment (TME). Additionally, location of tumor infiltrating T cells in two areas of the TME, the tumor center (CT) and invasive margin (IM) has also been shown to be prognostic and predictive of response to immunotherapy. Here we use multiplexed protein and RNA digital spatial profiling to elicit the immune landscape of MSI-MSS characterized CRC tumors.MethodsForty-eight CRC tumors were analyzed for gene expression using the NanoString® nCounter® PanCancer IO 360™ Research Use Only (RUO) Gene Expression Panel and assessed for 48 cell typing and biological signatures, including MMR loss/MSI predictor and the Tumor Inflammation Signature (TIS). A subset of 18 CRC tumors (6 MSI-TIS-hi, 6 MSS-TIS-hi, 6 MSS-TIS-lo) was selected for analysis with the RUO GeoMx™ Digital Spatial Profiler (DSP) using 40 antibodies (human IO protein panel), or 84 RNA probes (human IO RNA panel). Selection of regions of interest (ROIs) in two locations, CT and IM were guided by staining with fluorescent markers (CD45, CD3, pan-CK, DNA). 300–600 µM diameter circle ROIs were selected, and in some cases segmented by pan-CK+/pan-CK-. For 2 immune hot samples contour profiling at the IM into stromal and tumor regions was performed using 1400+ RNA probes with NGS readout.SummaryUsing whole tissue gene expression analysis, we determined the TIS and IO 360 signature scores for 48 CRC tumors using PanCancer IO 360 assay. 18 tumors within this cohort were selected based on TIS status to further dissect the location-dependent immune contexture of the TME. Protein DSP confirmed loss of dMMR markers (MSH2/MLH1) and identified an increased amount of potentially suppressive macrophages (CD163+PD-L1+) in MSI-TIS-hi versus MSS-TIS-hi tumors. Segmentation of ROIs based on tumor versus stroma (pan-CK±) identified samples with high proportions of tumor-invading TILs. Two MSI-TIS-hi profiled using probes against 1400+ mRNA targets confirmed protein results (CD163 in IM) and identified tumor-related signatures corresponding to the inside of the tumor (Cytokeratins, HER2/ERBB2, MET).ConclusionsHere we show the use of novel high-plex spatial profiling to profile location and pathways in the TME of MSI and MSS CRC tumors. These findings elicit unique biology related to the location and signaling of immune cells, which have the potential to unveil targets for therapeutic combinations.Disclosure InformationS.E. Church: A. Employment (full or part-time); Significant; NanoString Technologies. J. Reeves: A. Employment (full or part-time); Significant; NanoString Technologies. D.R. Zollinger: A. Employment (full or part-time); Significant; NanoString Technologies. J. McKay-Fleisch: A. Employment (full or part-time); Significant; NanoString Technologies. A.J. Bahrami: A. Employment (full or part-time); Significant; NanoString Technologies. M. Holpert: A. Employment (full or part-time); Significant; NanoString Technologies. A.M. White: A. Employment (full or part-time); Significant; NanoString Technologies. M.D. Bailey: A. Employment (full or part-time); Significant; NanoString Technologies. C.R. Merritt: A. Employment (full or part-time); Significant; NanoString Technologies. M. Hoang: A. Employment (full or part-time); Significant; NanoString Technologies. S. Warren: A. Employment (full or part-time); Significant; NanoString Technologies. J.M. Beechem: A. Employment (full or part-time); Significant; NanoString Technologies.
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Rajasagi, Mohini, Derin Keskin, Jintaek Kim, Wandi Zhang, Sachet Shukla, David DeLuca, John Sidney, et al. "Systematic Identification of Personal Mutated Tumor-Specific Neoantigens in CLL." Blood 120, no. 21 (November 16, 2012): 954. http://dx.doi.org/10.1182/blood.v120.21.954.954.
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Abstract Abstract 954 A major goal of cancer immunotherapy is to target tumor cells with high specificity and minimal toxicity. Tumor neoantigens are attractive targets for immunotherapy as they are generated from cancer-driven gene mutations and are expressed exclusively in tumor cells. Neoantigens have not previously been systematically investigated in human tumors due to technical challenges in identifying them. To address this, and to identify immunogenic epitopes personal to an individual tumor, we sought to exploit results from next-generation DNA sequencing to detect somatic mutations together with algorithms that predict peptide binding to HLA class I. We tested this approach in chronic lymphocytic leukemia (CLL), a B cell malignancy associated with a rich source of mutations (Wang et al, NEJM 2011). In a published study, we sequenced 91 CLL samples using whole-genome (n=3) or whole-exome (n=88; WES) DNA sequencing. To detect mutations, sequences in each tumor sample were compared with corresponding normal sequences, and the frequency of various mutation classes that lead to amino acid alterations were determined. On a per-tumor level, we detected a median of 17 (range: 2–72) missense mutations; 1 (range: 0–6) splice-site mutation; 1 (range: 0–1) read-through mutation, and 1 (range: 0–5) frameshift mutation. Frameshift mutations were predicted to generate potential neo-open reading frames with a median length of 40 amino acids (range: 0–180). Only peptides binding HLA molecules have the potential to elicit immune responses. We therefore focused on mutations generating autologous HLA binding peptides. Using a well-validated MHC class I prediction algorithm, pan-NetMHC, we systematically evaluated the binding potential of 9- and 10-mers tiled around each mutation. For 31 of 91 samples with available HLA typing, a median of 22 strongly binding peptides (range: 9–75) was predicted to be generated from a median of 26 (range: 7–87) mutations per sample. Using a competitive HLA binding assay, we experimentally validated the predicted high-binding capacity of 60 of 112 (53.5%) synthesized peptides, generated from 3 patients. To determine if tumor neoantigens are naturally recognized by cytotoxic T lymphocytes, we focused our analysis on CLL patients who had undergone successful hematopoietic stem cell transplantation (HSCT) followed by repeated vaccinations with irradiated autologous whole CLL cells, as normal donor T cell reconstitution following HSCT can overcome endogenous immune defects of the host and since donor T cells in this setting are already primed against host leukemia cells in vivo. Analysis of one patient identified 31 coding mutations, which generated 47 peptides predicted to bind autologous HLA. Of these, 24 were experimentally confirmed to bind autologous HLA alleles and thereafter were screened for inducing T cell reactivity. Using IFN-g ELISPOT reactivity following 2 ex vivo peptide stimulations, memory CD8+ T cell responses could be detected against a HLA A2-binding peptide derived from mutated FNDC3B (VVMSWAPPV). These cells were preferentially reactive to the mutated (but not wildtype) peptide. Results were confirmed with peptide-reactive T cell clones and staining with HLA-A2+/mutated FNDC3B peptide-specific tetramers. Mutated FNDC3B-reactive T cells also recognized HLA-A2+ expressing cells transfected with a minigene encompassing 300 base pairs surrounding the FNDC3B mutation, consistent with endogenous processing and presentation of the peptide. Ongoing studies focus on exploring the tumor cytolytic potential of mutation-specific T cells, and the kinetics of developing neoantigen-specific T cell reactivity in relation to immune reconstitution. Our studies provide proof-of-concept for systematic identification of tumor neoantigens by integrating information from next-generation sequencing of tumors with predictive HLA binding algorithms. These studies now set the stage for the implementation of clinical studies to explore the therapeutic potential of targeting tumor neoantigens. Disclosures: No relevant conflicts of interest to declare.
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McFarland, JG, RH Aster, JB Bussel, JG Gianopoulos, RS Derbes, and PJ Newman. "Prenatal diagnosis of neonatal alloimmune thrombocytopenia using allele- specific oligonucleotide probes." Blood 78, no. 9 (November 1, 1991): 2276–82. http://dx.doi.org/10.1182/blood.v78.9.2276.2276.
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Abstract The prediction of neonatal alloimmune thrombocytopenia (NATP) in affected families has, in the past, been based on information about gene frequencies of the antigen systems involved, parental phenotyping, and fetal platelet counts. We explored the feasibility of allele- specific oligonucleotide probe typing for PIA antigens to determine the risk of second or subsequent fetuses in families where one infant had a diagnosis of anti-PIA1-mediated NATP. A total of eight families at risk for delivering an affected fetus were studied with both serologic and oligonucleotide typing. The correlation between serologic and oligonucleotide PIA types was 100%. Similarly, in an additional eight families not at risk for PIA1-mediated NATP, serologic and oligonucleotide typing maintained a perfect correlation. DNA isolated from fetal leukocytes as well as fetal amniocytes was successfully typed using this technology. Oligonucleotide-based typing of fetuses at risk for NATP whose fathers are heterozygous for the PIA antigens allows early recognition of affected fetuses so that prenatal therapy of mothers can be instituted if necessary. When fetuses are found to be unaffected, invasive, and/or expensive, prenatal interventions can be avoided.
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McFarland, JG, RH Aster, JB Bussel, JG Gianopoulos, RS Derbes, and PJ Newman. "Prenatal diagnosis of neonatal alloimmune thrombocytopenia using allele- specific oligonucleotide probes." Blood 78, no. 9 (November 1, 1991): 2276–82. http://dx.doi.org/10.1182/blood.v78.9.2276.bloodjournal7892276.
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The prediction of neonatal alloimmune thrombocytopenia (NATP) in affected families has, in the past, been based on information about gene frequencies of the antigen systems involved, parental phenotyping, and fetal platelet counts. We explored the feasibility of allele- specific oligonucleotide probe typing for PIA antigens to determine the risk of second or subsequent fetuses in families where one infant had a diagnosis of anti-PIA1-mediated NATP. A total of eight families at risk for delivering an affected fetus were studied with both serologic and oligonucleotide typing. The correlation between serologic and oligonucleotide PIA types was 100%. Similarly, in an additional eight families not at risk for PIA1-mediated NATP, serologic and oligonucleotide typing maintained a perfect correlation. DNA isolated from fetal leukocytes as well as fetal amniocytes was successfully typed using this technology. Oligonucleotide-based typing of fetuses at risk for NATP whose fathers are heterozygous for the PIA antigens allows early recognition of affected fetuses so that prenatal therapy of mothers can be instituted if necessary. When fetuses are found to be unaffected, invasive, and/or expensive, prenatal interventions can be avoided.
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Irshaid, N. M., S. Ramadan, E. S. Wester, P. Olausson, Å. Hellberg, J. Y. Merza, and M. L. Olsson. "Phenotype prediction by DNA-based typing of clinically significant blood group systems in Jordanian blood donors." Vox Sanguinis 83, no. 1 (July 2002): 55–62. http://dx.doi.org/10.1046/j.1423-0410.2002.00182.x.
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Gross, Theresa E., Jan Fleckhaus, and Peter M. Schneider. "Progress in the implementation of massively parallel sequencing for forensic genetics: results of a European-wide survey among professional users." International Journal of Legal Medicine 135, no. 4 (April 13, 2021): 1425–32. http://dx.doi.org/10.1007/s00414-021-02569-0.
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AbstractA European-wide online survey was conducted to generate an overview on the state-of-the-art using massively parallel sequencing (MPS) platforms for forensic DNA analysis and DNA phenotyping among forensic practitioners in Europe. The survey was part of the dissemination activities of the “VISible Attributes through GEnomics – VISAGE” Horizon 2020 funded European research project [30], in preparation of a series of educational training activities. A total of 105 replies from 32 European countries representing participants from police, governmental, academic, and private laboratories providing professional services in the field of forensic genetics were included in the final analysis. Of these, 73% already own an MPS platform or are planning to acquire one within the next 1–2 years. One-third of the participants have already carried out MPS-based STR sequencing, identity, or ancestry SNP typing. A total of 23–40% of participants are planning to explore all FDP applications showing the overall very high interest in using MPS for the whole range of forensic MPS markers and applications. About 50% of the participants have previously gathered experience using forensic DNA phenotyping (FDP) markers based on conventional (i.e., not MPS-based) DNA typing methods. A total of 55% of the participants have attended training on the general use of MPS technology, but 36% have received no training whatsoever. Accordingly, 90% have expressed high or medium interest to attend training on the analysis and interpretation of DNA phenotyping data for predicting appearance, ancestry, and age. The results of our survey will provide valuable information for organizing relevant training workshops on all aspects of MPS-based DNA phenotyping for the forensic genetics scientific community.
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van der Schoot, C. Ellen, Florentine Thurik, Peter G. Scheffer, Frithjofna Abbink, van der Ploeg P.B. Catharina, Barbera Veldhuisen, and Masja de Haas. "Prenatal Fetal DNA Testing for Predicting HDFN, FNAIT, and RhIG Candidacy." Blood 122, no. 21 (November 15, 2013): SCI—51—SCI—51. http://dx.doi.org/10.1182/blood.v122.21.sci-51.sci-51.
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Abstract Red cell blood group antigen and platelet antigen incompatibility between a pregnant women and her fetus can result in maternal alloimmunisation and, consequently, hemolytic disease of the fetus and newborn (HDFN) and fetal neonatal alloimmune thrombocytopenia (FNAIT), respectively. For a long time, immunization against D-antigen has been the major cause of HDFN, but postnatal immune-prophylaxis and more recently antenatal immunoprophylaxis, has successfully decreased its incidence. The majority of severe FNAIT cases are caused by antibodies against HPA-1a, and a comparable immunoprophylaxis program is presently investigated. Postnatal immunoprophylaxis is traditionally given based on the fetal blood group determined on cord blood, since only 60 percent of the newborns of a D-negative mother are D-positive. But as long as the fetal blood group is not known, antenatal prophylaxis is administered to all D-negative women. Also in alloimmunised pregnant women, knowledge of the fetal antigen status is beneficial to tailor pregnancy management. In case of red cell alloantibodies, the antibody titers have to be followed during pregnancy and the fetus is careful monitored to recognize fetuses needing intrauterine blood transfusions. In many countries, pregnant women with anti-HPA-1a alloantibodies are treated with intravenous IgG during the last trimester of pregnancy to prevent intracranial hemorrhages. Traditionally, fetal blood group genotyping has been performed through amniocentesis. This invasive procedure carries a small risk of miscarriage (especially in FNAIT cases) and could potentially enhance maternal sensitization. The discovery of cell-free fetal DNA in the blood of pregnant women in 1997 presented a noninvasive, and thus safe, method to determine the fetal blood group genotype. Cell-free fetal DNA is released from trophoblastic cells undergoing apoptosis. Within the maternal circulation, cell-free fetal DNA is present among an overwhelming background of maternal cell-free DNA, predominately of hematopoietic origin. It can be detected at as early as five weeks of gestation, and gradually increases during pregnancy, from 10 genome equivalents (GE) per ml to around 300 GE late in pregnancy, although quantities vary between pregnancies. After birth, cell-free fetal DNA is cleared from the maternal circulation within several hours, with an observed half-life of 16 minutes. Since 2000, many laboratories have developed assays that are sensitive and specific enough to genotype the fetus reliably using cell-free DNA isolated from maternal plasma. This assay is relatively simple for RHD, because in Caucasians D-negativity is caused by the complete absence of the RHD gene. However, due to the high frequency of variant RHD genes, especially in African blacks and Asians, the interpretation of the assay can be difficult. Most other blood groups are caused by SNPs, and the design of those assays is, therefore, technically more challenging. However, nowadays for most clinical blood group antigens (D, c, C, E, e, K, HPA-1a), reliable, non-invasive genotyping assays are offered worldwide on a routine basis, and invasive procedures for fetal blood group typing have become obsolete. In several European countries, large-scale feasibility studies have been performed to investigate whether non-invasive fetal RHD genotyping could be safely applied to restrict antenatal immunoprophylaxis to those D-negative women carrying D-positive fetuses. This would prevent the unnecessarily exposure of 40 percent of D-negative women to the small but non-negligible risk of infection with a blood-borne disease, as anti-D is still produced from plasma of hyperimmunised donors. Furthermore, worldwide supplies of RhD immunoglobulin are limited. Based on the promising outcomes of these studies, the Danish and Dutch government decided in 2010 and 2011, respectively, to implement fetal RHD typing. With our fully automated assay we encountered only eight false negative results in more than 25,000 tested pregnancies (0.03%, 95% CI: 0.01-0.05%). In the Netherlands, postnatal immunoprophylaxis is also now given based on the PCR result in week 27 of pregnancy, and no routine cord blood serology is performed anymore. Overall, this program is cost-effective in the Dutch setting. In several other European countries (e.g., Sweden, UK, France) studies are ongoing on the implementation of fetal RHD genotyping to guide prophylaxis. Disclosures: No relevant conflicts of interest to declare.
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Zupanič Pajnič, Irena, Tomaž Zupanc, Tamara Leskovar, Matija Črešnar, and Paolo Fattorini. "Eye and Hair Color Prediction of Ancient and Second World War Skeletal Remains Using a Forensic PCR-MPS Approach." Genes 13, no. 8 (August 12, 2022): 1432. http://dx.doi.org/10.3390/genes13081432.
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To test the usefulness of the forensic PCR-MPS approach to eye and hair color prediction for aged skeletons, a customized version of the PCR-MPS HIrisPlex panel was used on two sets of samples. The first set contained 11 skeletons dated from the 3rd to the 18th centuries AD, and for each of them at least four bone types were analyzed (for a total of 47 samples). In the second set, 24 skeletons from the Second World War were analyzed, and only petrous bones from the skulls were tested. Good-quality libraries were achieved in 83.3% of the cases for the ancient skeletons and in all Second World War petrous bones, with 94.7% and 100% of the markers, respectively, suitable for SNP typing. Consensus typing was achieved for about 91.7% of the markers in 10 out of 11 ancient skeletons, and the HIrisPlex-S webtool was then used to generate phenotypic predictions. Full predictions were achieved for 3 (27.3%) ancient skeletons and 12 (50%) Second World War petrous bones. In the remaining cases, different levels of AUC (area under the receiver operating curve) loss were computed because of no available data (NA) for 8.3% of markers in ancient skeletons and 4.2% of markers in Second World War petrous bones. Although the PCR-based approach has been replaced with new techniques in ancient DNA studies, the results show that customized forensic technologies can be successfully applied to aged bone remains, highlighting the role of the template in the success of PCR-MPS analysis. However, because several typical errors of ancient DNA sequencing were scored, replicate tests and accurate evaluation by an expert remain indispensable tools.
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Wen, Xiaosha, Huijie Pu, Quan Liu, Zifen Guo, and Dixian Luo. "Circulating Tumor DNA—A Novel Biomarker of Tumor Progression and Its Favorable Detection Techniques." Cancers 14, no. 24 (December 7, 2022): 6025. http://dx.doi.org/10.3390/cancers14246025.
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Cancer is the second leading cause of death in the world and seriously affects the quality of life of patients. The diagnostic techniques for tumors mainly include tumor biomarker detection, instrumental examination, and tissue biopsy. In recent years, liquid technology represented by circulating tumor DNA (ctDNA) has gradually replaced traditional technology with its advantages of being non-invasive and accurate, its high specificity, and its high sensitivity. ctDNA may carry throughout the circulatory system through tumor cell necrosis, apoptosis, circulating exosome secretion, etc., carrying the characteristic changes in tumors, such as mutation, methylation, microsatellite instability, gene rearrangement, etc. In this paper, ctDNA mutation and methylation, as the objects to describe the preparation process before ctDNA analysis, and the detection methods of two gene-level changes, including a series of enrichment detection techniques derived from PCR, sequencing-based detection techniques, and comprehensive detection techniques, are combined with new materials. In addition, the role of ctDNA in various stages of cancer development is summarized, such as early screening, diagnosis, molecular typing, prognosis prediction, recurrence monitoring, and drug guidance. In summary, ctDNA is an ideal biomarker involved in the whole process of tumor development.
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Shen, Kai, Patricia Antalis, John Gladitz, Sameera Sayeed, Azad Ahmed, Shujun Yu, Jay Hayes, et al. "Identification, Distribution, and Expression of Novel Genes in 10 Clinical Isolates of Nontypeable Haemophilus influenzae." Infection and Immunity 73, no. 6 (June 2005): 3479–91. http://dx.doi.org/10.1128/iai.73.6.3479-3491.2005.
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ABSTRACT We hypothesize that Haemophilus influenzae, as a species, possesses a much greater number of genes than that found in any single H. influenzae genome. This supragenome is distributed throughout naturally occurring infectious populations, and new strains arise through autocompetence and autotransformation systems. The effect is that H. influenzae populations can readily adapt to environmental stressors. The supragenome hypothesis predicts that significant differences exist between and among the genomes of individual infectious strains of nontypeable H. influenzae (NTHi). To test this prediction, we obtained 10 low-passage NTHi clinical isolates from the middle ear effusions of patients with chronic otitis media. DNA sequencing was performed with 771 clones chosen at random from a pooled genomic library. Homology searching demonstrated that ∼10% of these clones were novel compared to the H. influenzae Rd KW20 genome, and most of them did not match any DNA sequence in GenBank. Amino acid homology searches using hypothetical translations of the open reading frames revealed homologies to a variety of proteins, including bacterial virulence factors not previously identified in the NTHi isolates. The distribution and expression of 53 of these genes among the 10 strains were determined by PCR- and reverse transcription PCR-based analyses. These unique genes were nonuniformly distributed among the 10 isolates, and transcription of these genes in planktonic cultures was detected in 50% (177 of 352) of the occurrences. All of the novel sequences were transcribed in one or more of the NTHi isolates. Seventeen percent (9 of 53) of the novel genes were identified in all 10 NTHi strains, with each of the remaining 44 being present in only a subset of the strains. These genic distribution analyses were more effective as a strain discrimination tool than either multilocus sequence typing or 23S ribosomal gene typing methods.
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Wheeler, Marsha, Chris Frazar, Kerry Lannert, Shelley N. Fletcher, Haley Huston, Samantha Harris, Meghan Delaney, Deborah Nickerson, and Jill Johnsen. "Prediction of MNS Blood Group Antigens Using Next Generation Sequencing." Blood 128, no. 22 (December 2, 2016): 1458. http://dx.doi.org/10.1182/blood.v128.22.1458.1458.
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Abstract Background The MNS blood group system is second in diversity only to the RH blood group system, with 46 described antigens. MNS system antigens are carried on glycophorins GPA and GPB that are products of the GYPA and GYPB genes, respectively. GYPA and GYPB are homologous paralogs which lie adjacent to each other on chromosome 4 in tandem with a third GYP paralog, GYPE. Current DNA-based testing methods for predicting MNS can be confounded by all types of genetic variation at the GYP locus, particularly in individuals of non-European ancestry. We sought to develop a next generation sequencing (NGS) approach for the systematic characterization of the GYP locus to accurately predict the common M/N and S/s blood group antigens and simultaneously identify other clinically relevant GYP DNA variants. Study Methods A total of 1139 samples were DNA sequenced; 1135 were from a previous study of blood donors self-identified to be of Asian American or Native American descent, and were 4 WHO reference DNAs (NIBSC). Blood donors had been tested for M and N by serology and for M/N and S/s using a single nucleotide variant (SNV) blood group genotyping platform (Bioarray). Samples were selected to enrich for MNS serology-SNV discrepancies or indeterminate results. BloodSeq is a NGS targeted panel that includes capture of 97.4kb over 3 genomic regions including the exons, pseudo-exons, introns, and proximal intergenic regions of GYPA, GYPB, and GYPE. This custom capture was used to generate Illumina, paired-end 100 bp DNA sequence reads. Raw sequence data was aligned to the human reference genome (hg19) and SNVs assessed using standard calling methods (GATK HaplotypeCaller). To predict MNS blood group system antigens, we determined variants which identified ISBT alleles; M/N antigens were defined as codominant alleles with multiple variant sites present in GYPA exon 2, while S/s antigens were defined by GYPB c.143T>C (p.Thr48Met) and 2 known GYPB silencing SNVs. Other DNA variants were cross-referenced with ISBT to predict associated blood group antigens. Results In a preliminary analyses, standard DNA variant calling methods predicted S/s (GYPB) SNVs accurately. However, alleles with M (GYPA) blood group variants exhibited a low call rate. Visualization of aligned reads indicated alleles corresponding to the M blood group sequence align poorly to the reference GYPA sequence. We traced the origin of these poor quality alignments to the presence of a region in GYPE with high sequence homology to the GYPA M allele. Notably, in the reference genome the GYPA gene has DNA variants indicative of theN genotype. With this knowledge, we developed a new approach which considers alignments of all 3 genes (GYPA, GYPB, GYPE) to predict M/N and S/s blood group antigens. Applying this method, BloodSeq predicted M with high concordance with serology (99.2%) and SNV genotype (99.6%), similar to the SNV genotype-serology concordance for M (98.9%). BloodSeq also predicted S/s in high concordance with the SNV predicted genotype (99.4% and 99.8%, respectively for S and s). Prediction of N by both BloodSeq and SNV genotype were similar to each other (99.6%) but exhibited lower accuracy (86.1% and 85.6%, respectively) when compared to serology. Interestingly, most (90%) of the N discrepancies were genetic prediction of absent N antigen but a positive N result by serology. We suspect these discrepancies result from cross-reactivity of reagent antibodies with "N" (an N-like antigen encoded by GYPB), which would require additional DNA sequence curation, or other underlying genetic variation. Additionally, 9 GYPA and GYPB variants indicative of other named ISBT alleles were detected, as well as a novel predicted frameshift variant in GYPA. Conclusion Our results demonstrate that a targeted NGS approach followed by an analysis pipeline customized for the GYP locus can simultaneously predict M/N and S/s blood groups and detect other GYP variants of known clinical significance. We propose that use of GYP locus-specific DNA sequence analysis strategies, such as addition of alternative reference sequences, should allow for automated and reliable classification of the M/N, S/s, and other variants in the MNS blood group system using next generation DNA sequencing. This work provides the foundation for a DNA-based, high resolution blood-typing method for the detection of clinically relevant MNS blood group system genetic variation. Disclosures Johnsen: CSL Behring: Consultancy; Octapharma: Consultancy.
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Chen, Minchun, Winfried März, Klaus H. Usadel, Ernst H. Scheuermann, and Bernhard O. Boehm. "Typing of the HLA-DRB3 gene by temperature gradient gel electrophoresis prediction of the resolution of four allelic fragments by computational simulation of DNA melting." Journal of Immunological Methods 168, no. 2 (February 1994): 257–65. http://dx.doi.org/10.1016/0022-1759(94)90063-9.
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Brambati, Chiara, Cristina Toffalori, Elisabetta Xue, Lara Crucitti, Raffaella Greco, Alessandra Crippa, Benedetta Mazzi, et al. "Droplet Digital PCR for DNMT3A and IDH1/2 Mutations to Improve Early Diagnosis of Acute Myeloid Leukemia Relapse after Allogeneic HSCT." Blood 124, no. 21 (December 6, 2014): 3951. http://dx.doi.org/10.1182/blood.v124.21.3951.3951.
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Abstract INTRODUCTION:Despite the considerable improvement documented over the last two decades in the outcome of allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) for Acute Myeloid Leukemia (AML), primary disease relapse still represents the main cause of mortality in transplanted patients. Since most of the available therapies for post-transplantation relapse display very limited activity when enacted in overt hematologic recurrence, efforts are aimed to anticipate relapse detection and treatment to the Minimal Residual Disease (MRD) stage. Still, the genetic heterogeneity and extensive clonal evolution which are distinctive features of AML hinder the identification of reliable MRD markers. Recent studies demonstrated that mutations in the DNMT3A and IDH1/2 genes occur very early during the step-wise process of leukemogenesis, possibly representing disease founder mutations, shared by all disease subclones and maintained throughout the patient longitudinal history. Moreover, by being present both in full-fledged transformed cells and their progenitors, their tracking might provide a wider scope on the efficacy of allo-HSCT in eradicating preleukemic stem cells. METHODS: We took advantage of ultra-sensitive droplet digital PCR (ddPCR) to test a total of 52 bone marrow samples collected longitudinally over time from 17 patients who received myeloablative allo-HSCT for AML. All patients carried at least one mutation amongst DNMT3A R882H, IDH1 R132C, IDH1 R132H, IDH2 R140Q and IDH2 R172K, documented at diagnosis by conventional Sanger sequencing. As controls, we tested bone marrow samples collected at diagnosis from 7 patients typing negative for the mutations, and peripheral blood samples from 8 healthy individuals. ddPCR assays were performed using the Bio-Rad QX100 system: each sample was tested in duplicates, employing 25 ng of genomic DNA in each reaction well and using as reference for each mutation-specific assay the respective wild-type allele. Samples with a mutant-to-wild-type ratio above 0.1% were considered positive. ddPCR results were compared to those obtained testing the same samples by quantitative PCR (qPCR) assessment of the WT1 gene transcript (considering as threshold for relapse prediction 250 copies of WT1/104 copies of ABL) and by qPCR-based hematopoietic chimerism assessment (employing the AlleleSEQR Chimerism Assay and considering as threshold for relapse prediction a host-specific signal above 1%). RESULTS:All the 17 samples collected at diagnosis and typing positive for the mutations of interest by conventional Sanger sequencing resulted positive also for the corresponding ddPCR assay. None of the samples from healthy individuals or from patients typing negative for the mutations resulted positive by ddPCR. All the samples tested at post-transplantation relapse remained positive for the mutations present at diagnosis, except for one case, originally carrying both DNMT3A and IDH2 mutations and typing negative for the latter at relapse. This observation might argue against the putative role of IDH mutations as leukemia-founder events, and suggests that, when present, DNMT3A could represent a more reliable MRD marker. In samples harvested in overt leukemia, the population carrying the mutant allele, quantified by ddPCR, consistently exceeded the morphological count of leukemic blasts. When post-transplantation remission samples were tested, 32/32 (100%) of those harvested from patients who remained long-term leukemia-free (median follow-up after allo-HSCT: 19 months) resulted negative for the mutations of interest, whereas 3/5 (60%) of those from patients who subsequently relapsed resulted positive. Of notice, only 1 of those 5 samples displayed WT1 transcript overexpression and host chimerism above the 1% threshold, whereas the remaining 4 resulted negative by both qPCR-based techniques. CONCLUSIONS: Although the very small number of patients included in this preliminary analysis warrants for caution, ddPCR for DNMT3A and IDH1/2 mutations appears extremely promising, displaying optimal specificity and very high sensitivity in relapse prediction, and comparing favorably with our present and historical results obtained by qPCR-based post-transplantation monitoring techniques. Disclosures Bonini: MolMed S.p.A.: Consultancy.
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Holmes, David S., Juan Pablo Cárdenas, Jorge H. Valdés, Raquel Quatrini, M. Esparza, Hector Osorio, F. Duarte, C. Lefimil, and Eugenia Jedlicki. "Comparative Genomics Begins to Unravel the Ecophysiology of Bioleaching." Advanced Materials Research 71-73 (May 2009): 143–50. http://dx.doi.org/10.4028/www.scientific.net/amr.71-73.143.
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The metabolic potential of 16 bioleaching microorganisms (Eubacteria and Archaea) has been investigated, allowing the prediction of potential inter- and intra-species physiological interactions (ecophysiology) during spatial and temporal changes that are known to occur within industrial bioleaching heaps. Genome analysis has allowed preliminary models to be built for genes and pathways involved in key processes such as nitrogen and carbon cycling, sulfur and iron uptake and homeostasis, extra-cellular polysaccharide biosynthesis, heavy metal resistance and energy metabolism. This paper will focus on the diverse ways that microorganisms obtain carbon from their environment with a particular emphasis on elucidating how these processes might be expected to vary over space and time during the lifetime of a bioleaching operation. It is anticipated that this knowledge will improve our understanding of fundamental biological processes in extremely acidic environments and it is hoped that it will capture usable knowledge that can be applied to bioleaching. Comparative genomics between two strains of Acidthiobacillus ferrooxidans highlights the importance of lateral gene transfer in increasing genetic and metabolic potential and suggests that classical molecular DNA techniques, such as rDNA typing, significantly underestimate the microbial diversity of bioleaching heaps.
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Pang, Xionghao, Juanjuan Qian, Hua Jin, Lei Zhang, Lin Lin, Yuli Wang, Yi Lei, Zeqiang Zhou, Meixiang Li, and Henghui Zhang. "Durable benefit from immunotherapy and accompanied lupus erythematosus in pancreatic adenocarcinoma with DNA repair deficiency." Journal for ImmunoTherapy of Cancer 8, no. 2 (July 2020): e000463. http://dx.doi.org/10.1136/jitc-2019-000463.
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BackgroundClinical trials showed limited benefit of anti-PD-1 (programmed cell death 1) monotherapy in pancreatic adenocarcinoma patients and immune-related adverse events caused by immune checkpoint inhibitors were rarely reported in pancreatic adenocarcinoma. Here, we report the first case of durable benefit along with systemic lupus erythematosus following immunotherapy in mismatch repair-proficient pancreatic cancer.Case presentationWe describe a 57-year-old woman with resected stage ⅢB pancreatic cancer who underwent several lines of conventional chemotherapy after multiple lymph node metastases. When the disease progressed again, the patient received an off-label treatment with pembrolizumab (100 mg every 3 weeks). After four cycles of immunotherapy treatment, CA19-9 level rapidly decreased to normal and the lymph node metastases reduced dramatically in volume, demonstrating a partial response to the therapy by RECIST 1.1 criteria. She continued on pembrolizumab and a total of eight cycles of administration she had received. Her lesions showed consistent reduction in size even when the medication had been stopped. Actually the patient experienced durable benefit from anti-PD-1 therapy for more than 4 years and she is still in good condition without tumor relapses to date. Besides, she was diagnosed with systemic lupus erythematosus 2 months after the last dose of pembrolizumab. Molecular profiling identified two deleterious PALB2 alterations including a germline mutation (PALB2 c.3114–1G>A) and a somatic mutation (PALB2 c.2514+1G>C) in this patient, suggesting the potential of DNA homologous recombination deficiency. Multiplex immunohistochemistry and RNA-seq results revealed a brisk immune cell infiltration in her resected primary lesion. Additionally, humanleukocyte antigen (HLA) typing assay identified two previously reported systemic lupus erythematosus risk alleles HLA-DRB1*15:01 and HLA-DQB1*06:02 in this patient.ConclusionsThe deleterious mutations of PALB2 closely related to homologous recombination deficiency or alterations of DNA damage response and repair genes might be promising biomarkers for predicting efficacy of immune checkpoint inhibitors in pancreatic adenocarcinoma. Genetic correlation behind immunotherapy-induced systemic lupus erythematosus and associated mechanism remain to be elucidated.
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Kolesnikov, Yevgeniy, Aleksey Maksimov, Oleg Kit, and Denis Kutilin. "DEPENDENCE OF OVERALL AND RELAPSE-FREE PATIENTS SURVIVAL FROM MOLECULAR GENETIC SUBTYPE OF ESOPHAGEAL SQUAMOUS CELL CANCER." Problems in oncology 65, no. 5 (May 1, 2019): 691–700. http://dx.doi.org/10.37469/0507-3758-2019-65-5-691-700.
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Esophageal squamous cell carcinoma (ESCC) is a group of heterogeneous tumors with a different prognosis. In recent years, various molecular signatures of ESCC have been identified, which are different in different populations. The aim of the study was the molecular typing of ESCC patients in southern Russia and the assessment of patient survival, taking into account the identified tumor molecular genetic subtype. The material for the study was the sections of FFPE-blocks of 124 patients with ESCC. Tumor and non-tumor esophagus cells isolation was carried out by laser microdissection with contactless capture. 248 DNA samples were extracted from the cells by the phenol-chloroform method. For molecular typing of ESCC, the relative copy number variation (CNV) of 8 genes (CUL3, ATG7, SOX2, TP63, YAP1, VGLL4, CDK6, KDM6A) was determined by Real-Time qPCR and 7 single nucleotide polymorphisms (SNP) (NFE2L2 (c.85G> A), NOTCH1 ( c.1379C> T), NOTCH1 (c.1451G> T), ZNF750 (c.414C> A), ZNF750 (c..1621G> A), SMARCA4 (p.Q758 *, c.2272C> T), KMT2D (Q5170 *, c.15508C> T)) were determined by the method of Sanger direct sequencing. During the study, in ESCC patients of the Southern Russia population identified SNP in genes NFE2L2, NOTCH1, SMARCA4, KMT2D and CNV of genes CUL3, ATG7, SOX2, TP63, YAP1, VGLL4, CDK6 and KDM6A, earlier described for populations of Eastern Europe, Canada and the USA. Three molecular genetic subtypes of ESCC were verified, based on the differences in SNP and CNV of these genes: ESCC1 was verified in 31.5%, ESCC2 in 66.1%, and ESCC3 in 2.4% of patients. At the same time, higher survival rates were established in ESCC patients with the molecular genetic subtype ESCC2, as compared with ESCC1 and ESCC3. Differences in survival between the three groups were statistically significant (p = 0.00001). Thus, the determination of the molecular genetic subtype of ESCC is an important approach to improve the prediction of the course of this disease and the possibility of adjusting the appropriate therapy.
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Howson, Emma L. A., Richard J. Orton, Valerie Mioulet, Tiziana Lembo, Donald P. King, and Veronica L. Fowler. "GoPrime: Development of an In Silico Framework to Predict the Performance of Real-Time PCR Primers and Probes Using Foot-and-Mouth Disease Virus as a Model." Pathogens 9, no. 4 (April 20, 2020): 303. http://dx.doi.org/10.3390/pathogens9040303.
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Real-time PCR (rPCR) is a widely accepted diagnostic tool for the detection and quantification of nucleic acid targets. In order for these assays to achieve high sensitivity and specificity, primer and probe-template complementarity is essential; however, mismatches are often unavoidable and can result in false-negative results and errors in quantifying target sequences. Primer and probe sequences therefore require continual evaluation to ensure they remain fit for purpose. This paper describes the development of a linear model and associated computational tool (GoPrime) designed to predict the performance of rPCR primers and probes across multiple sequence data. Empirical data were generated using DNA oligonucleotides (n = 90) that systematically introduced variation in the primer and probe target regions of a diagnostic assay routinely used to detect foot-and-mouth disease virus (FMDV); an animal virus that exhibits a high degree of sequence variability. These assays revealed consistent impacts of patterns of substitutions in primer and probe-sites on rPCR cycle threshold (CT) and limit of detection (LOD). These data were used to populate GoPrime, which was subsequently used to predict rPCR results for DNA templates (n = 7) representing the natural sequence variability within FMDV. GoPrime was also applicable to other areas of the FMDV genome, with predictions for the likely targets of a FMDV-typing assay consistent with published experimental data. Although further work is required to improve these tools, including assessing the impact of primer-template mismatches in the reverse transcription step and the broader impact of mismatches for other assays, these data support the use of mathematical models for rapidly predicting the performance of rPCR primers and probes in silico.
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James, C. Alston, Peter Ronning, Darren Cullinan, Kelsy C. Cotto, Erica K. Barnell, Katie M. Campbell, Zachary L. Skidmore, et al. "In Silico Epitope Prediction Analyses Highlight the Potential for Distracting Antigen Immunodominance with Allogeneic Cancer Vaccines." Cancer Research Communications 1, no. 2 (November 2021): 115–26. http://dx.doi.org/10.1158/2767-9764.crc-21-0029.
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Allogeneic cancer vaccines are designed to induce antitumor immune responses with the goal of impacting tumor growth. Typical allogeneic cancer vaccines are produced by expansion of established cancer cell lines, transfection with vectors encoding immunostimulatory cytokines, and lethal irradiation. More than 100 clinical trials have investigated the clinical benefit of allogeneic cancer vaccines in various cancer types. Results show limited therapeutic benefit in clinical trials and currently there are no FDA-approved allogeneic cancer vaccines. We used recently developed bioinformatics tools including the pVACseq suite of software tools to analyze DNA/RNA-sequencing data from the The Cancer Genome Atlas to examine the repertoire of antigens presented by a typical allogeneic cancer vaccine, and to simulate allogeneic cancer vaccine clinical trials. Specifically, for each simulated clinical trial, we modeled the repertoire of antigens presented by allogeneic cancer vaccines consisting of three hypothetical cancer cell lines to 30 patients with the same cancer type. Simulations were repeated ten times for each cancer type. Each tumor sample in the vaccine and the vaccine recipient was subjected to human leukocyte antigen (HLA) typing, differential expression analyses for tumor-associated antigens (TAA), germline variant calling, and neoantigen prediction. These analyses provided a robust, quantitative comparison between potentially beneficial TAAs and neoantigens versus distracting antigens present in the allogeneic cancer vaccines. We observe that distracting antigens greatly outnumber shared TAAs and neoantigens, providing one potential explanation for the lack of observed responses to allogeneic cancer vaccines. This analysis provides additional rationale for the redirection of efforts toward a personalized cancer vaccine approach. Significance: A comprehensive examination of allogeneic cancer vaccine antigen repertoire using large-scale genomics datasets highlights the large number of distracting antigens and argues for more personalized approaches to immunotherapy that leverage recent strategies in tumor antigen identification.
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Kardaun, Jan W. P. F., Lon White, Helaine E. Resnick, Helen Petrovitch, Santica M. Marcovina, Ann M. Saunders, Dan J. Foley, and Richard J. Havlik. "Genotypes and Phenotypes for Apolipoprotein E and Alzheimer Disease in the Honolulu-Asia Aging Study." Clinical Chemistry 46, no. 10 (October 1, 2000): 1548–54. http://dx.doi.org/10.1093/clinchem/46.10.1548.
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Abstract Background: The utility of apolipoprotein E (ApoE) type as an indicator of genetic susceptibility to Alzheimer disease (AD) depends on the reliability of typing. Although ApoE protein isoform phenotyping is generally assumed equivalent to genotyping from DNA, phenotype-genotype differences have been reported. Methods: ApoE genotype and phenotype results were examined for 3564 older (ages 71–93 years) Japanese-American male participants of the Honolulu-Asia Aging Study, an ongoing population-based study of aging and dementia. Results: Both methods demonstrated similar associations of ApoE type with AD: a direct association with ApoE4 and a less dramatic inverse association ApoE2. Advanced age did not appear to influence the ApoE4-AD association. The association with AD among ApoE4 homozygotes [odds ratio (OR) = 14.7] was higher than expected based on an observed OR of 2.0 in heterozygotes. Phenotype-genotype nonconcordance was more frequent for ApoE2 than for ApoE4. The ApoE2 phenotype occurred at a frequency of 7.9% vs a genotype frequency of 4.9%, corresponding to a probability of 56% that an individual with ApoE2 phenotype had the same genotype. Conclusions: Whereas E4 and E2 phenotypes and genotypes were comparably associated with AD, neither method would be expected to substantially improve the efficiency of case finding in the context of population screening beyond prediction based on age and education. Nonconcordance of phenotype and genotype was substantial for E2 and modest for E4 in this population. The ApoE4-AD association was independent of age.
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Noris, P., S. Simsek, LG de Bruijne-Admiraal, L. Porcelijn, E. Huiskes, GJ van der Vlist, EF van Leeuwen, CE van der Schoot, and AE von dem Borne. "Max(a), a new low-frequency platelet-specific antigen localized on glycoprotein IIb, is associated with neonatal alloimmune thrombocytopenia." Blood 86, no. 3 (August 1, 1995): 1019–26. http://dx.doi.org/10.1182/blood.v86.3.1019.1019.
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Abstract We have identified a new platelet-specific alloantigen, Max(a), responsible for a typical case of neonatal alloimmune thrombocytopenic purpura. The maternal serum reacted strongly with paternal platelets in the platelet immunofluorescence test, whereas platelet alloantigen typing showed that no known human platelet antigen (HPA)-system was involved. In the monoclonal antibody (MoAb)-specific immobilization of platelet antigens (MAIPA) assay, the new antigen was located on the platelet membrane glycoprotein (GP) IIb-IIIa complex, but immunoprecipitation and immunoblot experiments to further localize the antigen failed. However, in the MAIPA assay, the binding of the anti- Max(a) antibodies from the maternal serum was blocked by two anti-GPIIb MoAbs. Thus, the antigen appeared to be located on GPIIb. Analysis of the family lead to the identification of six additional Max(a+) individuals. Three of these six individuals and the father were tested in the platelet aggregation test and were found to be normal. In the analysis of normal donors, three of 500 were typed positive for the new platelet-specific antigen, indicating a phenotype frequency of 0.6% in the normal population. Platelet RNA was isolated from the newborn's Max(a)+ father and from a healthy donor phenotyped as Max(a-), reverse- transcribed, and the entire GPIIb coding region was amplified by polymerase chain reaction. Subsequent nucleotide sequence analysis showed a single G-->A substitution at position 2,603, predicting a valine-->methionine amino acid substitution at position 837 of the mature glycoprotein. This mutation abolished a BsiYI restriction site at the cDNA level and a BstNI restriction site at genomic DNA level, respectively. The genetic association between the new antigen and this point mutation was confirmed by allele-specific restriction analysis on cDNA and on genomic DNA, as well as by allele-specific primer amplification on genomic DNA. The new mutation is 19 bp upstream of the mutation underlying the HPA-3 system. Therefore, we also evaluated the association between Mas and the HPA-3 polymorphism. So far, all Max(a+) individuals were also found to be HPA-3b, whereas 50 HPA-3a individuals were all Max(a-). This may indicate that Max(a) is a variant of the HPA- 3 allele.
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Noris, P., S. Simsek, LG de Bruijne-Admiraal, L. Porcelijn, E. Huiskes, GJ van der Vlist, EF van Leeuwen, CE van der Schoot, and AE von dem Borne. "Max(a), a new low-frequency platelet-specific antigen localized on glycoprotein IIb, is associated with neonatal alloimmune thrombocytopenia." Blood 86, no. 3 (August 1, 1995): 1019–26. http://dx.doi.org/10.1182/blood.v86.3.1019.bloodjournal8631019.
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We have identified a new platelet-specific alloantigen, Max(a), responsible for a typical case of neonatal alloimmune thrombocytopenic purpura. The maternal serum reacted strongly with paternal platelets in the platelet immunofluorescence test, whereas platelet alloantigen typing showed that no known human platelet antigen (HPA)-system was involved. In the monoclonal antibody (MoAb)-specific immobilization of platelet antigens (MAIPA) assay, the new antigen was located on the platelet membrane glycoprotein (GP) IIb-IIIa complex, but immunoprecipitation and immunoblot experiments to further localize the antigen failed. However, in the MAIPA assay, the binding of the anti- Max(a) antibodies from the maternal serum was blocked by two anti-GPIIb MoAbs. Thus, the antigen appeared to be located on GPIIb. Analysis of the family lead to the identification of six additional Max(a+) individuals. Three of these six individuals and the father were tested in the platelet aggregation test and were found to be normal. In the analysis of normal donors, three of 500 were typed positive for the new platelet-specific antigen, indicating a phenotype frequency of 0.6% in the normal population. Platelet RNA was isolated from the newborn's Max(a)+ father and from a healthy donor phenotyped as Max(a-), reverse- transcribed, and the entire GPIIb coding region was amplified by polymerase chain reaction. Subsequent nucleotide sequence analysis showed a single G-->A substitution at position 2,603, predicting a valine-->methionine amino acid substitution at position 837 of the mature glycoprotein. This mutation abolished a BsiYI restriction site at the cDNA level and a BstNI restriction site at genomic DNA level, respectively. The genetic association between the new antigen and this point mutation was confirmed by allele-specific restriction analysis on cDNA and on genomic DNA, as well as by allele-specific primer amplification on genomic DNA. The new mutation is 19 bp upstream of the mutation underlying the HPA-3 system. Therefore, we also evaluated the association between Mas and the HPA-3 polymorphism. So far, all Max(a+) individuals were also found to be HPA-3b, whereas 50 HPA-3a individuals were all Max(a-). This may indicate that Max(a) is a variant of the HPA- 3 allele.
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Oyervides-Muñoz, Mariel A., Antonio A. Pérez-Maya, Celia N. Sánchez-Domínguez, Anais Berlanga-Garza, Mauro Antonio-Macedo, Lezmes D. Valdéz-Chapa, Ricardo M. Cerda-Flores, Victor Trevino, Hugo A. Barrera-Saldaña, and María L. Garza-Rodríguez. "Multiple HPV Infections and Viral Load Association in Persistent Cervical Lesions in Mexican Women." Viruses 12, no. 4 (March 31, 2020): 380. http://dx.doi.org/10.3390/v12040380.
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Persistent high-risk human papillomavirus (HR-HPV) infections play a major role in the development of invasive cervical cancer (CC), and screening for such infections is in many countries the primary method of detecting and preventing CC. HPV typing can be used for triage and risk stratification of women with atypical squamous cells of undetermined significance (ASC-US)/low-grade cervical lesions (LSIL), though the current clinical practice in Mexico is to diagnose CC or its preceding conditions mainly via histology and HR-HPV detection. Additional information regarding these HPV infections, such as viral load and co-infecting agents, might also be useful for diagnosing, predicting, and evaluating the possible consequences of the infection and of its prevention by vaccination. The goal of this follow-up hospital case study was to determine if HPV types, multiple HPV infections, and viral loads were associated with infection persistence and the cervical lesion grade. A total of 294 cervical cytology samples drawn from patients with gynecological alterations were used in this study. HPV types were identified by real-time PCR DNA analysis. A subset of HPV-positive patients was reevaluated to identify persistent infections. We identified HPV types 16, 18, and 39 as the most prevalent. One hundred five of the patients (59%) were infected with more than one type of HPV. The types of HPV associated with multiple HPV infections were 16, 18, and 39. In the follow-up samples, 38% of patients had not cleared the initially detected HPV infection, and these were considered persistent. We found here an association between multiple HPV infections and high viral loads with and infection persistence. Our findings suggest there are benefits in ascertaining viral load and multiple HPV infections status of HR-HPV infections for predicting the risk of persistence, a requirement for developing CC. These findings contribute to our understanding of HPV epidemiology and may allow screening programs to better assess the cancer-developing risks associated with individual HR-HPV infections.
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Zheng, Yan, Ti-Cheng Chang, Gang Wu, Jane S. Hankins, Mitchell J. Weiss, Connie M. Westhoff, and Stella T. Chou. "Accurate Prediction of RH Genotypes Using Whole Genome Sequencing Data." Blood 132, Supplement 1 (November 29, 2018): 2332. http://dx.doi.org/10.1182/blood-2018-99-119681.
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Abstract Introduction RBC alloimmunization is common in patients with sickle cell disease (SCD). Despite serological matching RBCs for major Rh antigens, Rh alloimmunization remains problematic. The Rh blood group is encoded by two genes RHD and RHCE, which exhibit extensive nucleotide polymorphism and chromosome structural changes, resulting in the formation of Rh variant antigens. Rh variants can result in loss of protein epitopes or expression of neo-epitopes, and are common in SCD patients. Hence SCD patients harboring Rh variants can be predisposed to Rh alloimmunization. Given the limitation of traditional serologic antigen typing for detection of Rh variants, molecular genotyping has become required. A DNA microarray-based platform, BioArray RHCE and RHD BeadChip (Immuncor) is available for RH genotyping. However, it detects the most common, but not all, variants. Whole exome sequence data have been used for prediction of Rh variants (Chou, et. al, Blood Adv., 2017), offer some advantages, including detection of rare variants, structural rearrangements and copy number variation. However, whole genome sequence (WGS) analysis of RHD/RHCE is challenging due to difficulties in mapping next generation sequencing (NGS) reads to this duplicated gene family. We developed a computational algorithm to identify RH variants using WGS data. Methods The pipeline included three major components, RH allele database construction, RH variant calling, and classification of Rh blood group according the identified variants. The RH allele database was built based on NCBI Blood Group Antigen Gene Mutation (BGMUT) and International Society of Blood Transfusion (ISBT) database. Since the alleles in the BGMUT and ISBT databases were specified according to conventional RH genes (RHD, L08429; RHCE, DQ322275) that are different from those on reference human genome, we first called the variations based on the reference human genome. The positions of the identified variations were subsequently corrected to match with the BGMUT and ISBT annotation system. Next, the NGS reads with low base quality and/or mapping quality were discarded during the variation calling step. Synonymous and non-synonymous amino acid changes were characterized for each polymorphism. Haplotypes were constructed for the segments with NGS read support. Gene sequencing coverage was calculated to determine gene deletions or amplifications. Lastly, we implemented an algorithm to predict RH genotypes based on a selection of candidate alleles by read-mapping profile which considers both sequence variations and sequence consistency followed by a likelihood-based ranking of all pairwise combinations of the selected alleles. The allele combination with the highest likelihood is considered the most likely pair of alleles at a given locus. Patient specimens used in this study were from participants of the Sickle Cell Clinical Research and Intervention Program (SCCRIP, Hankins et al. Pediatr Blood Cancer. 2018). Results We validated our method in a cohort of 58 SCD patients whose RH genotypes had been determined by BioArray RhCE and RhD BeadChip and supplementary molecular tests that identify the most common variants among individuals of African descent. In this validation cohort including a total of 11 RHD and 13 RHCE alleles, our approach achieved a concordance rate of 85.85% (91 of 106 alleles) for RHD and 83.02% (88 of 106 alleles) for RHCE genotyping. WGS was highly sensitive in distinguishing homozygosity from heterozygosity of genes. By comparing the numbers of NGS reads on RH regions and whole genome average coverage, heterozygous deletion can be determined. Since WGS provides comprehensive genotyping, our analysis identified single nucleotide polymorphisms that were not identified by the BeadChip and supplemental molecular testing. The final source of discordance was likely due to the short read length of NGS such that haplotype phases cannot be correctly predicted if the variations are separated by thousands of base pairs, for which long read DNA sequencing or RNA/cDNA sequencing are required. Evaluation of the identified discrepancies is ongoing. Conclusions We developed and validated a diagnostic method for RH genotyping that leveraged the accuracy and flexibility of RH genotyping based on WGS data. With further optimization of our method, this may be useful for RBC genotype matching sickle cell patients to blood donors in the future. Disclosures Hankins: Novartis: Research Funding; Global Blood Therapeutics: Research Funding; NCQA: Consultancy; bluebird bio: Consultancy.
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Hughes, Andrew E. O., Maureen C. Montgomery, Chang Liu, and Eric T. Weimer. "Quantification of Allele-Specific HLA Expression with Nanopore Long-Read Sequencing." Blood 136, Supplement 1 (November 5, 2020): 42–43. http://dx.doi.org/10.1182/blood-2020-140902.
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Human leukocyte antigen (HLA) typing plays a critical role in evaluating donor-recipient compatibility prior to hematopoietic cell transplantation (HCT) to minimize the risk of rejection and graft versus host disease (GVHD). Compared to traditional sequence-based methods for HLA typing, next-generation sequencing offers significant advantages in terms of accuracy, turnaround time, and cost (Weimer et al., JMD, 2016). Nevertheless, an intrinsic limitation of DNA-based typing is that it does not quantify HLA gene expression, which has been implicated in clinical outcomes (Petersdorf et al., Blood, 2014; Petersdorf et al., NEJM, 2015). Previously, we demonstrated simultaneous HLA class I genotyping and gene-level expression analysis by RNA-seq using nanopore long-read sequencing (Montgomery et al., JMD, 2020). Given that mismatches in both class I and class II HLA genes-as well as the relative expression of individual alleles-impact donor-recipient compatibility, we sought to build on our previous work by quantifying allele-specific expression of both class I and class II HLA loci in donor lymphocytes. For this study, mRNA was isolated from peripheral blood lymphocytes from 12 donors. Barcoded cDNA libraries were prepared and sequenced on MinION flow cells (R9.4.1) using MinKNOW (v3.1.13) to a median depth of 1.6x106reads. Basecalling and demultiplexing were performed with Albacore (v2.3.4) or Guppy (v2.3.1), and adapter trimming was performed with Porechop (v0.2.3). Processed reads were aligned to the international ImMunoGeneTics project (IMGT) HLA database (v3.41.0) using minimap2 (v2.17). Reads mapping to individual HLA loci were realigned to allele-specific references using subject HLA types determined by Athlon (v1.0) or Illumina sequencing. In parallel, library size factors were estimated by aligning reads to GRCh38, counting reads in genes with HTseq (v0.12.4), and using trimmed mean of M-values normalization. As shown in Fig. 1, we observed higher expression of HLA class I genes compared to class II (median 593 vs. 150, p < 0.001, Mann-Whitney U test), a pattern consistent with a mixture of primarily T cells, which express class I genes, as well as B cells, which express both class I and II. Within class I genes, we observed the highest expression of HLA-B, followed by HLA-A, and HLA-C (median 663, 578, and 459, respectively). Within class II, we observed the highest expression of HLA-DPB1, followed by HLA-DRB1, and HLA-DQB1 (median 281, 266, and 104, respectively). Importantly, we observed significant variation in expression both between and within alleles of individual HLA genes, suggesting that HLA type alone does not accurately predict HLA expression. We next analyzed HLA-DPB1 specifically, given reports that the risk of GVHD in HCT recipients with HLA-DPB1mismatched donors is modulated by HLA-DPB1 expression (Petersdorf et al., NEJM, 2015). Of note, HLA-DPB1 expression is linked to a single nucleotide polymorphism, rs9277534, which can be imputed from HLA-DPB1 type (Meurer et al., Front Immunol, 2018). Accordingly, we analyzed HLA-DPB1 expression conditioned on rs9277534 genotype. Although we observed lower HLA-DPB1 expression for the 'A' allele compared to 'G' (median 220 vs. 265), consistent with the reported association, this difference was not statistically significant (p = 0.22, Mann-Whitney U test). Furthermore, we observed significant variation in expression among 'A' alleles, with normalized counts ranging from 57 to 408 (vs. 191 to 367 for 'G' alleles). In this study, we demonstrate the feasibility of quantifying allele-specific expression of both class I and class II HLA genes with nanopore long-read sequencing. Taken together, our results reveal extensive variation in the expression of class I and class II HLA loci, even after accounting for individual allele types and known markers of expression. These results emphasize the potential value of methods, such as nanopore sequencing, for directly quantifying allele-specific HLA expression to develop improved risk prediction models that can inform the evaluation of donor-recipient immunocompatibility. Disclosures No relevant conflicts of interest to declare.
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Correale, Pierpaolo, Rita Emilena Saladino, Giovanna Bianco, Alessia Stranges, Diana Giannarelli, Andrea Sergi, Maria Antonietta Mazzei, et al. "Correlation of HLA-B*35 and DRB1*11 alleles with a high risk of interstitial aseptic pneumonitis in cancer patients receiving PD-1/PDL1 immune-checkpoint blockade." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): e15094-e15094. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.e15094.
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e15094 Background: Tumor infiltrating CTL-rescue by PD-1/PDL1 immune-checkpoint blockade is a recommended treatment for several malignancies including non-small-cell-lung-cancer (NSCLC), malignant melanoma, head and neck, kidney, and urothelial cancer. MAbs to either PD-1 (Nivolumab and Pembrolizumab) or PDL1 (Atezolizumab and Durvalumab) are considered as active drugs in these patients, however, their use may be complicated by unpredictable immune-related adverse events (irAEs) and in less extent by a dreaded interstitial aseptic pneumonitis (IAP). Methods: We carried out a retrospective multi-institutional analysis aimed to investigate possible clinical and biological parameters correlated with the occurrence of IAP in a cohort of 256 cancer patients (188 with mNSCLC and 78 with other malignancies) who received PD-1/PDL-1 blockade since November 2015. Association of IAP with clinical/biological factors was assessed by the chi-square test. Statistics were performed by the SPSS software 23.0. HLA molecular analysis was performed by reverse SSO DNA typing assays on patients’ PBMCs. Results: A centralised radiological review recorded a IAP in 29 patients (11.3%) whose 15 (5.8%) were free of symptoms. There was no correlation of IAP risk with tumor type, specific PD-1 or PDL1 blocking mAb, radiation therapy, inflammatory markers, and other irAEs. IAP was more frequent in males than females [RR = 2.03, (95% CI: 0.63-5.61) p > 0.05] and occurred more often in mNSCLC patients who had received metronomic chemotherapy +/- bevacizumab [RR = 3.05 (95% CI: 1.32-7.06),P = 0.005] or TKI [RR = 1.73 (95% CI: 0.75-4.00),P > 0.05] compared with other treatments prior PD-1/PDL1 blockade. Finally, IAP occurrence was strictly correlated to the expression of HLA-B*35 [RR = 1.73 (95% CI: 0.81-3.71) P < 0.05] and DRB1*11 [RR = 2.34 (95% CI: 1.02-5.39); P = 0.03], two alleles associated to common autoimmune diseases. Conclusions: Taken together, our findings may have relevant implications in predicting the risk of IAP in mNSCLC receiving PD-1/PDL1 blockade and provide the rationale for further immunological and clinical investigations.
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Wang, Di, Jie Wang, Xiaohui Niu, Zhen Huang, Zhijie Wang, Qing Zhang, Jianchun Duan, Hua Bai, and Lin Hao. "Clone evolution and genomic alteration analysis of osteosarcoma and matched lung metastasis." Journal of Clinical Oncology 35, no. 15_suppl (May 20, 2017): 11032. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.11032.
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11032 Background: Lung metastasis (LM), as the most common metastatic site, is the main reason resulting in treatment failure and death of osteosarcoma (OS). But there was no report about the clone evolution and genomic alteration in the process of LM of OS. Methods: Multiregion whole-genome sequencing and whole-exome sequencing were performed on ten patients with primary OS and matched lung metastatic tumors. A set of high confident somatic nucleotide variants (SNV), small insertion and deletion (indel) and copy number variation (CNV) in each sample were identified, then the n-dimensional Bayesian Dirichlet process was applied to define the constituent mutation clusters as clone or subclone. Neoantigen prediction and HLA typing were performed using NetMHC3.0 and POLYSOLVER algorithm, respectively. Results: There were diversified metastatic progression during lung metastasis of OS including linear evolution (7/10) and parallel evolution (3/10), and metastasis-to-metastasis spread was also found in a patient with multiple metastasis; Mutation accumulative effect during the metastasis of OS was evident, LM had much higher mutation load (fold change = 4.1, P-value < 0.01) and neoantigen burden (fold change = 4.5, P-value < 0.001) than the primary tumor; DNA mismatch repair (MMR) genes relevant deleterious mutation events were found in germline in 8/10 metastatic OS cases and MMR genes mutation were much more in LM than primary cancer; The genome instability of LM was prevalently much more significant than primary tumor, with higher copy number variation frequency (fold change = 7.01, P-value < 0.01). Conclusions: The evolution of OS during LM was very complex with diversified metastatic progression. For the LM of OS, the novel therapy should be considering the much higher mutation load, especially those causing tumor neoantigens, higher genomic instability which were associated with tumor immune therapy. Furthermore, the findings of germline MMR genes mutation in primary OS which had occurred LM and more MMR genes mutation in LM of OS indicated potential clinical benefit on immune checkpoint inhibitor therapy and other small molecular drugs for MMR genes.
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Nakaseko, Chiaki, Miki Nishimura, Shinnichi Ozawa, Ryuko Cho, Chikako Ohwada, Atsuo Maruta, Hisashi Sakamaki, et al. "Risk Factors and Outcome of Chronic GVHD: Analysis of 5,660 Patients Undergoing Unrelated Bone Marrow Transplantation through the Japan Marrow Donor Program." Blood 106, no. 11 (November 16, 2005): 139. http://dx.doi.org/10.1182/blood.v106.11.139.139.
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Abstract Background: Chronic GVHD (cGVHD) remains the major cause of late morbidity and mortality after allogeneic stem cell transplantation. However, there are limited data available on cGVHD after unrelated BMT (UR-BMT). We retrospectively analyzed the data of 5,660 patients who underwent UR-BMT through the Japan Marrow Donor Program (JMDP) between January 1993 and June 2004. Methods: Data were collected by the JMDP using a standard report form. Follow-up reports were submitted at 100 days, 1 year, and then annually after transplantation. Overall survival (OS) was estimated by the Kaplan-Meier method and patients surviving beyond day 100 after transplant were analyzed for the development of cGVHD. The log-rank test was used for univariate analysis and time-dependent Cox proportional hazards modeling was used for multivariate analysis. The cumulative incidence of cGVHD and of relapse was calculated using the Gray method considering death without cGVHD and death without relapse as respective competing risks. Results: The median age of all patients was 28 years and the median follow-up was 433.5 days after transplant. Estimated 5-year OS of all patients and those with hematological malignancies was 47.4% and 45.5%, respectively. A total of 3,974 patients survived beyond day 100 after transplant and their cumulative incidence of cGVHD was 43.2% at day 500 and 44.9% at day 2,000 post-transplant. The cumulative incidence of extensive cGVHD was 28.8% at day 2,000 post-transplant. In multivariate analysis, variables predicting cGVHD were recipient age (p=0.000), donor age (p=0.002), diagnosis of hematological malignancy (HR=1.99, p=0.000), HLA class I mismatch by either serology or DNA typing (HR=1.24, p=0.020), acute GVHD (I: HR=1.50, p=0.000; II: HR=2.07, p=0.000; III and IV: HR=2.25, p=0.000) and no platelet recovery over 50,000/mm3 before day 100 (HR=1.36, P=0.002). There was a significant difference between patients <20 and ≥20 years old (HR=1.27, p=0.000). However, there were no significant differences between any adults grouped by age decade (p=0.894). OS at 5 years in patients surviving >100 days post-transplant was 62.4% without cGVHD, 68.0% with limited cGVHD, and 55.4% with extensive cGVHD (p=0.000). In the patients with hematological malignancies, OS at 5 years was 58.8%, 67.3% and 55.8%, respectively (p=0.000). Cumulative incidence of relapse of hematological malignancies at day 2,000 in patients surviving >100 days post-transplant was 17.6% with limited cGVHD, 18.4% with extensive cGVHD and 27.1% without cGVHD (P=0.000). Conclusions: This study provides strong evidence of risk factors for developing cGVHD after UR-BMT and suggests that limited cGVHD provides a survival benefit to patients with hematological malignancies by reducing the risk of relapse without increasing the risk of death from cGVHD. There was a significant difference in occurrence of cGVHD between patients <20 and ≥20 years old but no differences comparing any age ≥20 years.
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Satbir, Thakur, Son Tran, Mohit Jain, Austin Lewis, Luis Murguia-Favela, Faisal M. Khan, Kevin J. Bielamowicz, et al. "A Novel Anti-Cancer Vaccine Approach for the Treatment of High-Risk Leukemia in Children." Blood 136, Supplement 1 (November 5, 2020): 25. http://dx.doi.org/10.1182/blood-2020-143381.
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Introduction: There is strong experimental and clinical data to indicate the critical involvement of immune evasion in relapsed leukemia in children. A well-defined characteristic of refractory leukemia is the accumulation of genetic aberrations and mutations that may act as drivers or passengers in the process of tumor recurrence. Many of these mutations get translated into proteins that contain tumor-specific immune-stimulatory epitopes (neoantigens) that can elicit host antitumor immune responses. Although, in general, the mutation rate is lower in pediatric tumors, recent studies have shown that almost 90% of pediatric leukemias carry potentially actionable neoepitopes. In this study, we describe the results from a comprehensive experimental approach of neoantigen prediction coupled with antigen processing and HLA-binding prediction algorithms with in vitro validation assays for the generation of neoantigen vaccines against high-risk leukemias in children. Methods: DNA and RNA from leukemia cells and matched fibroblasts were obtained. Raw reads were aligned to human reference genome and somatic variants (SNVs) were called using Strelka v1.0.1441. RNA-seq data from leukemic cells were used to predict neoantigen expression levels resulting from SNVs using STAR (2.4.1)12 and Cufflinks v2.2.1. Normalized expression data were then cross-referenced with the list of SNVs to identify leukemia-specific mutant proteins. HLA typing for each sample was carried out from RNA-seq data using seq2HLA v2.2. Using the patient's HLA phenotype, we then used NetMHCons v1.1 to predict short peptides derived from leukemia-specific mutant proteins that will bind to autologous HLA Class I molecules. These 8/9-mers were filtered to predict a high likelihood of proteasomal or immune-proteasomal processing and transporter associated with antigen processing (TAP) using NetChop v3.1 and the immune epitope database (IEDB), respectively. The peptides identified were rank-ordered based on the composite immunogenicity score derived from MHC class I binding affinities, proteasomal processing and TCR binding predictions and synthesized accordingly. Peripheral blood derived dendritic cells (DCs) and CD8+ T-cells were isolated and expanded in culture with relevant cytokines. The DCs were pulsed with peptides and then co-cultured with CD8+ T-cells. After five days, the primed CD8+ T-Cells were separated, washed and exposed to the patient's leukemic cells at varying ratios and the leukemia specific CD8+ T-cell activation was quantified by IFN gamma secretion using ELISpot assays. Results: In the leukemia specimen studied, approximately 5% of all on-target germline mutations were found only in leukemic cells. Tumor mutational burden was, on average, 0.34 mut/Mb. Analysis of the highest ranking synthetic peptides (approximately 10 per leukemia sample) showed leukemia-specific activation of patient's T-cells as measured by the mean number of spots observed in ELISpot assays. For example, in patient one (15 year old male, high-risk ALL, one year off therapy), 14 individual short sequences were identified and corresponding peptides were synthesized. Among these, three peptides were not soluble and three peptides showed significant activity above controls. Maximum leukemia specific T-cell activation was noted with peptide #7 QQSALVLL (mean 135 ELISpots compared to 72 in controls, p<0.05, triplicate) indicating a strong nonantigenic potential in this region. Furthermore, this activity was significantly diminished when an extra amino acid was added to this peptide (LQQSALVLL, mean 79 spots) showing the specificity of the approach. A number of other peptides and combinations in non-overlapping regions gave intermediate activities. Discussion: Completed data, including the vaccine peptide sequences and corresponding activities showed the feasibility of identifying pediatric leukemia neoantigen sequences in personalized mutational landscapes of these patients. In addition, we have provided an in vitro experimental approach to validate the potential of such vaccines in future clinical studies and this methodology can also be used to identify agents for effective combinations such as immune checkpoint inhibitors. A clinical trial using these strategies is in development for the treatment of high-risk leukemia in children. Disclosures No relevant conflicts of interest to declare.