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Objectives: Eosinophilic esophagitis (EoE) is a chronic, antigen-mediated disease in children and adults associated with substantial esophageal remodeling and fibrosis. The expression of the remodeling-associated matrix metalloproteinases (MMPs) has not been previously detailed in EoE. Methods: MMP-2 and-14 expression and cellular localization were assessed using real-time quantitative polymerase chain reaction and immunohistochemistry/immunofluorescence in EoE fibroblasts, active and inactive pediatric EoE biopsies, and nondiseased control biopsies. The effect of transforming growth factor (TGF)-b1 treatment on MMP-2 expression in cultured esophageal epithelial (HET1A) cells was analyzed. Results: MMP-2 and-14 mRNA were expressed in EoE fibroblasts and biopsies. Proliferating epithelial cells produced MMP-14 more abundantly in EoE than in controls (P

Background: Cytogenetic analysis encompasses a suite of standard-of-care diagnostic testing methods that is routinely applied in cases of acute myeloid leukemia (AML) to assess chromosomal changes that are clinically relevant for risk classification and treatment decisions., Objective: In this study, we assess the use of Genomic Proximity Mapping (GPM) for cytogenomic analysis of AML diagnostic specimens for detection of cytogenetic risk variants included in the European Leukemia Network (ELN) risk stratification guidelines., Methods: Archival patient samples (N=48) from the Fred Hutchinson Cancer Center leukemia bank with historical clinical cytogenetic data were processed for GPM and analyzed with the CytoTerra ® cloud-based analysis platform., Results: GPM showed 100% concordance for all specific variants that have associated impacts on risk stratification as defined by ELN 2022 criteria, and a 72% concordance rate when considering all variants reported by the FH cytogenetic lab. GPM identified 39 additional variants, including variants of known clinical impact, not observed by cytogenetics., Conclusions: GPM is an effective solution for the evaluation of known AML-associated risk variants and a source for biomarker discovery., Competing Interests: Conflict of Interest Disclosure IL, MM, and SME are employees of Phase Genomics, Inc. A company developing the GPM technology. CY consults for TwinStrand Biosciences.

The presence of measurable residual disease (MRD) is strongly associated with treatment outcomes in acute myeloid leukemia (AML). Despite the correlation with clinical outcomes, MRD assessment has yet to be standardized or routinely incorporated into clinical trials and discrepancies have been observed between different techniques for MRD assessment. In 62 patients with AML, aged 18-60 years, in first complete remission after intensive induction therapy on the randomized phase III SWOG-S0106 clinical trial (clinicaltrials gov. Identifier: NCT00085709), MRD detection by centralized, high-quality multiparametric flow cytometry was compared with a 29-gene panel utilizing duplex sequencing (DS), an ultrasensitive next-generation sequencing method that generates double-stranded consensus sequences to reduce false positive errors. MRD as defined by DS was observed in 22 (35%) patients and was strongly associated with higher rates of relapse (68% vs. 13%; hazard ratio [HR] =8.8; 95% confidence interval [CI]: 3.2-24.5; P<0.001) and decreased survival (32% vs. 82%; HR=5.6; 95% CI: 2.3-13.8; P<0.001) at 5 years. DS MRD strongly outperformed multiparametric flow cytometry MRD, which was observed in ten (16%) patients and marginally associated with higher rates of relapse (50% vs. 30%; HR=2.4; 95% CI: 0.9-6.7; P=0.087) and decreased survival (40% vs. 68%; HR=2.5; 95% CI: 1.0-6.3; P=0.059) at 5 years. Furthermore, the prognostic significance of DS MRD status at the time of remission for subsequent relapse was similar on both randomized arms of the trial. These findings suggest that next-generation sequencing-based AML MRD testing is a powerful tool that could be developed for use in patient management and for early anti-leukemic treatment assessment in clinical trials.

Recurrent gene fusions are common drivers of disease pathophysiology in leukemias. Identifying these structural variants helps stratify disease by risk and assists with therapy choice. Precise molecular diagnosis in low-and-middle-income countries (LMIC) is challenging given the complexity of assays, trained technical support, and the availability of reliable electricity. Current fusion detection methods require a long turnaround time (7-10 days) or advance knowledge of the genes involved in the fusions. Recent technology developments have made sequencing possible without a sophisticated molecular laboratory, potentially making molecular diagnosis accessible to remote areas and low-income settings. We describe a long-read sequencing DNA assay designed with CRISPR guides to select and enrich for recurrent leukemia fusion genes, that does not need a priori knowledge of the abnormality present. By applying rapid sequencing technology based on nanopores, we sequenced long pieces of genomic DNA and successfully detected fusion genes in cell lines and primary specimens (e.g., BCR::ABL1, PML::RARA, CBFB::MYH11, KMT2A::AFF1) using cloud-based bioinformatics workflows with novel custom fusion finder software. We detected fusion genes in 100% of cell lines with the expected breakpoints and confirmed the presence or absence of a recurrent fusion gene in 12 of 14 patient cases. With our optimized assay and cloud-based bioinformatics workflow, these assays and analyses could be performed in under 8 hours. The platform's portability, potential for adaptation to lower-cost devices, and integrated cloud analysis make this assay a candidate to be placed in settings like LMIC to bridge the need of bedside rapid molecular diagnostics., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: LHH and KYY have equity interest in Biodepot LLC, which receives compensation from NCI SBIR contract numbers 75N91020C00009 and 75N91021C00022. The terms of this arrangement have been reviewed and approved by the University of Washington in accordance with its policies governing outside work and financial conflicts of interest in research. This work is related to the following two provisional patent applications: “Optimized assay and efficient computational workflow to deliver same-day leukemia diagnostics” (patent application 63/416,888) “Detecting Leukemia Specific Gene Fusions” (patent application 63/309261), (Copyright: © 2023 Sala-Torra et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Chronic myelogenous leukemia (CML) is a hematopoietic stem cell malignancy that accounts for 15-20% of all cases of leukemia. CML is caused by a translocation between chromosomes 9 and 22 which creates an abnormal fusion gene, BCR::ABL1. The amount of BCR::ABL1 transcript RNA is a marker of disease progression and the effectiveness of tyrosine kinase inhibitor (TKI) treatment. This study determined the analytical and clinical performance of a droplet digital PCR based assay (QXDx BCR-ABL %IS Kit; Bio-Rad) for BCR::ABL1 quantification. The test has a limit of detection of MR4.7 (0.002%) and a linear range of MR0.3-4.7 (50-0.002%IS). Reproducibility of results across multiple sites, days, instruments, and users was evaluated using panels made from BCR::ABL1 positive patient samples. Clinical performance of the assay was evaluated on patient samples and compared to an existing FDA-cleared test. The reproducibility study noted negligible contributions to variance from site, instrument, day, and user for samples spanning from MR 0.7-4.2. The assay demonstrated excellent clinical correlation with the comparator test using a Deming regression with a Pearson R of 0.99, slope of 1.037 and intercept of 0.1084. This data establishes that the QXDx™ BCR-ABL %IS Kit is an accurate, precise, and sensitive system for the diagnosis and monitoring of CML., Competing Interests: Bio-Rad provided reagents to each site to support this study. D.N.S, P.B., N.S., and S.H. are employees of Bio-Rad. Bio-Rad employees have or may have stock in Bio-Rad. In addition to providing employment, the stated Bio-Rad employees have or may have patents, products in development or marketed products in Bio-Rad. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Measurement of BCR activator of RhoGEF and GTPase -ABL proto-oncogene 1, non-receptor tyrosine kinase (BCR-ABL1) mRNA levels by reverse transcription quantitative polymerase chain reaction (RTqPCR) has been critical to treatment protocols and clinical trials in chronic myeloid leukaemia; however, interlaboratory variation remains a significant issue. Reverse transcriptase droplet digital PCR (RTddPCR) has shown potential to improve testing but a large-scale interlaboratory study is required to definitively establish this. In the present study, 10 BCR-ABL1-positive samples with levels ranging from molecular response (MR) 1·0 -MR 5·0 were tested by 23 laboratories using RTddPCR with the QXDX BCR-ABL %IS kit. A subset of participants tested the samples using RTqPCR. All 23 participants using RTddPCR detected BCR-ABL1 in all samples to MR 4·0 . Detection rates for deep-response samples were 95·7% at MR 4·5 , 78·3% at MR 4·7 and 87·0% at MR 5·0 . Interlaboratory coefficient of variation was indirectly proportional to BCR-ABL1 level ranging from 29·3% to 69·0%. Linearity ranged from 0·9330 to 1·000 (average 0·9936). When results were compared for the 11 participants who performed both RTddPCR and RTqPCR, RTddPCR showed a similar limit of detection to RTqPCR with reduced interlaboratory variation and better assay linearity. The ability to detect deep responses with RTddPCR, matched with an improved linearity and reduced interlaboratory variation will allow improved patient management, and is of particular importance for future clinical trials focussed on achieving and maintaining treatment-free remission., (© 2021 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Purpose: Sequential treatment with targeted therapies can result in complex combinations of resistance mutations in drug targets. This mutational complexity has spurred the development of pan-target inhibitors, i.e., therapies for which no single target mutation can cause resistance. Because the propensity for on- versus off-target resistance varies across cancer types, a deeper understanding of the mutational burden in drug targets could rationalize treatment outcomes and prioritize pan-target inhibitors for indications where on-target mutations are most likely. Experimental Design: To measure and model the mutational landscape of a drug target at high resolution, we integrated single-molecule Duplex Sequencing of the ABL1 gene in Philadelphia-positive (Ph + ) leukemias with computational simulations. Results: A combination of drug target mutational burden and tumor-initiating cell fraction is sufficient to predict that most patients with chronic myeloid leukemia are unlikely to harbor ABL1 resistance mutations at the time of diagnosis, rationalizing the exceptional success of targeted therapy in this setting. In contrast, our analysis predicts that many patients with Ph + acute lymphoblastic leukemia (Ph + ALL) harbor multiple preexisting resistant cells with single mutants. The emergence of compound mutations can be traced to initial use of an ABL1 inhibitor that is susceptible to resistance from single point mutations. Conclusions: These results argue that early use of therapies that achieve pan-inhibition of ABL1 resistance mutants might improve outcomes in Ph + ALL. Our findings show how a deep understanding of the mutational burden in drug targets can be quantitatively coupled to phenotypic heterogeneity to rationalize clinical phenomena. Clin Cancer Res; 24(21); 5321-34. ©2018 AACR ., (©2018 American Association for Cancer Research.)

We used next-generation sequencing (NGS) of the immunoglobulin genes to evaluate residual disease in 153 specimens from 32 patients with adult B cell acute lymphoblastic leukemia enrolled in a single multicenter study. The sequencing results were compared with multiparameter flow cytometry (MFC) data in 66 specimens (25 patients) analyzed by both methods. There was a strong concordance (82%) between the methods in the qualitative determination of the presence of disease. However, in 17% of cases, leukemia was detected by sequencing but not by MFC. In 54 bone marrow (BM) and peripheral blood (PB) paired specimens, the burden of leukemia detected by NGS was lower in PB than in BM, although it was still detectable in 68% of the 28 paired specimens with positive BM. Lastly, patients without disease detected by NGS or MFC had a 5-year relapse free survival of > 80%. The results suggest that residual disease detection by immunoglobulin gene sequencing is an extremely sensitive technique and may identify patients that might benefit from transplantation. Moreover, the increased sensitivity of the method may allow frequent peripheral blood testing to supplement marrow sampling to measure disease response., (Copyright © 2017 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Despite the successes of tyrosine kinase inhibitors (TKIs) in improving outcomes in patients with chronic myeloid leukemia (CML) and Philadelphia-positive acute lymphoblastic leukemia (Ph + ALL), allogeneic hematopoietic stem cell transplantation (HSCT) continues to be an important and potentially curative option for selected patients with either disease. After HSCT, TKIs are increasingly being used to treat or prevent disease relapse, and practice patterns suggest that these TKIs are often chosen empirically without regard to pre-HSCT mutation status. We investigated whether ABL kinase domain mutations persist after transplantation and, thus, whether pre-HSCT mutation status should inform the selection of post-HSCT TKIs in these patients. We retrospectively analyzed adults who underwent allogeneic HSCT for CML and Ph + ALL at our institution between 2000 and 2010, and we identified subjects who had detectable BCR-ABL transcripts by polymerase chain reaction (PCR), as well as available RNA for Sanger sequencing of the ABL kinase domain, in both the pre- and post-HSCT settings. In total, 95 CML and 20 Ph + ALL patients with positive PCR transcripts were identified, of which 10 (10.5%) and 4 (20.0%), respectively, were found to have pre-HSCT ABL kinase mutations known to confer TKI resistance. In 9 (64.2%) of these 14 patients, the same kinase mutation was also detectable at an average time of 191 days after HSCT. Seven (50.0%) of the 14 harboring mutations had relapsed/refractory disease by last follow-up, of which, in retrospect, 6 had received a predictably ineffective TKI within the first 100 days after transplantation based on our mutation analysis. These data support the idea that pre-existing mutations in the ABL kinase domain, frequently associated with resistance to TKIs and prevalent in a transplantation population, are persistently detectable in the majority of patients after transplantation. We propose that such resistance patterns should be considered when selecting TKIs in the post-HSCT setting, including clinical trials of post-HSCT TKI prophylaxis., (Published by Elsevier Inc.)

Background: We evaluated BCR-ABL1 kinetics in patients treated with nilotinib and analyzed whether a dynamic model of changes in BCR-ABL1 levels over time could be used to predict long-term responses., Methods: Patients from the nilotinib registration trial (CAMN107A2101; registered at http://www.clinicaltrials.gov as NCT00109707) who had imatinib-resistant or -intolerant Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML) in chronic phase (CP) or accelerated phase with BCR-ABL1 > 10% (on the international scale [IS]) at baseline and, in the first 6 months, had at least three BCR-ABL1 transcript measurements and an average daily dose of at least 720 mg were included in this analysis (N = 123)., Results: More than half of patients (65/123; 53%) had a slow monophasic response and the remainder (58/123; 47%) had a biphasic response, in which patients had a rapid initial decrease in BCR-ABL1 transcripts followed by a more gradual response. The biphasic response type strongly correlated with improved event-free survival (EFS). Data in the first 6 months of follow-up were sufficient to predict EFS at 24 months., Conclusions: Unlike newly diagnosed patients with Ph+ CML-CP-in whom the majority had a biphasic response-approximately half of patients with imatinib-resistant or -intolerant CML had a slower, monophasic response. Second-line patients who did have a biphasic response had an EFS outlook similar to that of newly diagnosed patients treated with imatinib. Our model was comparable to using BCR-ABL1 (IS) ≤ 10% at 6 months as a threshold for predicting EFS.

Background: Current practice guidelines for managing patients with chronic myelogenous leukemia (CML) call for monitoring BCR-ABL transcript concentrations with a quantitative reverse transcription-PCR (qRT-PCR) assay. Because the available laboratory-developed assays lack consensus on the appropriate design, reporting of results, and reference intervals, we developed and evaluated an integrated BCR-ABL assay that yields standardized results for any laboratory and can be performed by technicians with no specialized training., Methods: We used the Cepheid Xpert BCR-ABL Monitor assay to measure both BCR-ABL and ABL (endogenous control) transcripts in blood samples from CML patients and healthy individuals. The assay involves 8 manual pipetting steps, fully automated nucleic acid purification, a nested qRT-PCR step, and data analysis., Results: The BCR-ABL assay requires approximately 2 h 20 min and covers a 5-log concentration range with a lower detection limit for the BCR-ABL:ABL ratio of approximately 0.005%. Assay results were negative for 100% of the 56 known CML-negative samples (12 patients with other hematologic disorders and 44 healthy blood donors). Testing of CML-positive patients undergoing disease monitoring showed 85% agreement with negative results (17 of 20) and 100% agreement with positive results (26 of 26). An imprecision/portability study revealed no differences in performance between sites, days, instruments, and operators., Conclusions: The Xpert BCR-ABL Monitor assay provides a robust and reproducible alternative to laboratory-developed assays. Its ease of use may allow more laboratories to offer BCR-ABL testing for patients, and the short assay time enables same-day results for treating physicians.

Allogeneic transplantation offers a potential cure for patients with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). We performed a retrospective analysis examining pretransplantation and posttransplantation prognostic factors in 90 patients with Ph+ ALL. The median age of the patients was 33 years, with slightly more than half of the patients (58%) in clinical remission at the time of transplantation. Overall, patients had a nonrelapse mortality rate of 30%, a relapse percentage of 34%, and an estimated 5-year disease-free survival rate of 30%. Pretransplantation risk factors for relapse included the expression of the p190 transcript (relative risk [RR] = 5.1; P =.037), evidence of morphologic disease at the time of transplantation (RR = 3.9; P <.001), and type of donor (RR = 2.5; P =.015), with patients receiving autologous or matched related transplants having the highest risk of relapse. The detection of minimal residual disease by reverse transcription polymerase chain reaction for bcr-abl transcripts was a significant posttransplantation risk factor for relapse (RR = 4.4; P =.001), with posttransplantation patients expressing the p190 transcript having the highest risk of relapse (RR = 8.7; P =.0001). In addition, patients with chronic extensive graft-versus-host disease showed a significantly lower risk of relapse (RR = 0.33; P =.038). Thus, these findings indicate that several pretransplantation and posttransplantation risk factors exist for patients with Ph+ ALL. Together, these factors can be used to improve our risk stratification of patients with Ph+ ALL who undergo transplantation, which will greatly enhance our ability to counsel these patients and potentially lead to the development of more specific treatment plans for them., (Copyright 2003 American Society for Blood and Marrow Transplantation)

The bcr-abl chimeric messenger RNA is frequently detected in chronic myeloid leukemia (CML) patients after bone marrow transplantation. It was previously reported that the relapse risk of bcr-abl detection 6 to 12 months after transplantation was greater than 40%. This risk decreased as the time between transplantation and detection increased. To further define the relapse risk associated with bcr-abl molecular detection in "late" CML survivors, 379 consecutive CML patients alive at 18 months after transplantation or later were studied. Ninety of 379 patients (24%) had at least one positive bcr-abl test 18 months after transplantation or later; 13 of 90 bcr-abl-positive patients (14%) and 3 of 289 bcr-abl-negative patients (1.0%) relapsed. The median time from bcr-abl detection to relapse was 916 days (range, 251-2654 days). The hazard ratio of relapse associated with bcr-abl detection was 19.2 (P <.0001). The stage of disease, chronic graft-versus-host disease, and the donor type did not alter the association between bcr-abl and relapse. Quantification of bcr-abl was performed on 344 samples from 85 bcr-abl-positive patients by means of a real-time quantitative reverse transcriptase-polymerase chain reaction assay. The median bcr-abl change of patients who relapsed was significantly greater than those that remained in remission (P =.002). The median bcr-abl level at relapse was 40 443 bcr-abl copies per microg RNA (range, 960-299 552). Of 73 bcr-abl-positive patients who failed to relapse, 69% had only one positive test at a median of 24 copies bcr-abl per microg RNA. The detection of bcr-abl is common following transplantation. The prognostic significance of a qualitative bcr-abl can be refined by quantitative assays and thus may target patients who would benefit from early intervention.