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Novel Multiplex Droplet Digital PCR Assays to Monitor Minimal Residual Disease in Chronic Myeloid Leukemia Patients Showing Atypical BCR-ABL1 Transcripts.

ABSTRACT: BCR-ABL1 fusion transcript is the minimal residual disease marker in chronic myeloid leukemia; 2% of patients show unusual breakpoints generating atypical transcripts, not quantifiable by standardized real-time PCR (RT-PCR). Response monitoring is performed by non-quantitative NESTED PCR, useless for evaluating patients' molecular remission, excluding them from treatment-free-remission protocols. Droplet digital PCR (ddPCR) is highly sensitive technology, allowing an absolute quantification independent of standard curves. Based on this, we have developed assays able to evaluate the molecular response in atypical patients. We designed new ddPCR-based molecular assays able to quantify atypical BCR-ABL1 transcripts, with a detection limit of 0.001%, validated in a cohort of 65 RNA from 11 patients. Fifty samples were identified congruently by ddPCR and NESTED PCR (40 positives and 10 negatives for atypical BCR-ABL1 transcript), while 11 positive samples were detected only by ddPCR. Our results highlight ddPCR usefulness, primarily when the BCR-ABL1/ABL1 level is less than 1.5% and NESTED PCR results are often inaccurate. Furthermore, we identified 3 patients who maintained a deep molecular response for at least one year, who could be considered good candidates for treatment-free remission approaches. Here, we describe a new promising molecular approach, highly sensitive, to monitor atypical BCR-ABL1 patients, paving the foundation to include them in treatment-free remission protocols.

SUBMITTER: Petiti J

PROVIDER: S-EPMC7290999 | biostudies-literature |

REPOSITORIES: biostudies-literature

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A new highly sensitive real-time quantitative-PCR method for detection of BCR-ABL1 to monitor minimal residual disease in chronic myeloid leukemia after discontinuation of imatinib.

Project description:Tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL1 fusion protein, encoded by the Philadelphia chromosome, have drastically improved the outcomes for patients with chronic myeloid leukemia (CML). Although several real-time quantitative polymerase chain reaction (RQ-PCR) kits for the detection of BCR-ABL1 transcripts are commercially available, their accuracy and efficiency in laboratory practice require reevaluation. We have developed a new in-house RQ-PCR method to detect minimal residual disease (MRD) in CML cases. MRD was analyzed in 102 patients with CML from the DOMEST study, a clinical trial to study the rationale for imatinib mesylate discontinuation in Japan. The BCR-ABL1/ABL1 ratio was evaluated using the international standard (IS) ratio, where IS < 0.1% was defined as a major molecular response. At enrollment, BCR-ABL1 transcripts were undetectable in all samples using a widely-applied RQ-PCR method performed in the commercial laboratory, BML (BML Inc., Tokyo, Japan); however, the in-house method detected the BCR-ABL1 transcripts in five samples (5%) (mean IS ratio: 0.0062 ± 0.0010%). After discontinuation of imatinib, BCR-ABL1 transcripts were detected using the in-house RQ-PCR in 21 patients (21%) that were not positive using the BML method. Nineteen samples were also tested using a commercially available RQ-PCR assay kit with a detection limit of IS ratio, 0.0032 (ODK-1201, Otsuka Pharmaceutical Co., Tokyo, Japan). This method detected low levels of BCR-ABL1 transcripts in 14 samples (74%), but scored negative for five samples (26%) that were positive using the in-house method. From the perspective of the in-house RQ-PCR method, number of patients confirmed loss of MMR was 4. These data suggest that our new in-house RQ-PCR method is effective for monitoring MRD in CML.

| S-EPMC6400442 | biostudies-literature

Clinical Utility of Droplet Digital PCR to Monitor BCR-ABL1 Transcripts of Patients With Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia Post-chimeric Antigen Receptor19/22 T-Cell Cocktail Therapy.

Project description:Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) accounts for 20-30% of adult patients with ALL, characterized by translocation of t (9, 22). Tyrosine kinase inhibitors (TKIs) have significantly improved the outcome even though there are still some problems including relapse due to drug-resistant mutations and suboptimal molecular remission depth. Previously, we reported the safety and efficacy of sequential infusion of CD19/22 chimeric antigen receptor T-cell (CAR-T) immunotherapy in the treatment of relapsed/refractory (R/R) B-cell neoplasms including cases with Ph+ ALL. Given possible deeper reaction, more patients were expected to reach optimal minimal residual disease (MRD) response. An alternative method, duplex droplet digital PCR (ddPCR) with high sensitivity was established, which could provide absolute quantification of MRD without the need for calibration curves. Here, we retrospectively collected 95 bone marrow samples from 10 patients with R/R Ph+, who received 19/22 CAR-T-cell cocktail therapy. Notably, sequential molecular remission for more than 3 months (SMR3), a significant indicator based on ddPCR after CAR-T infusion was established, which was defined as a sequential molecular remission for not <3 months with negative MRD. In this cohort, no recurrence was observed in six patients achieving SMR3, where four of whom accepted allogeneic hematopoietic stem cell transplantation (allo-HSCT) after CAR-T cell regimen. Unfortunately, the other four patients who did not reach SMR3 relapsed, and did not receive extra specific treatment except CAR-T regimen. To sum up, ddPCR may be an alternative, especially when nucleic acid was insufficient in clinical practice. No achievement of SMR3 may be an early warning of potential relapse after CAR-T and indicating the initiation of other therapies including allo-HSCT.

| S-EPMC8059437 | biostudies-literature

Assessment of individual molecular response in chronic myeloid leukemia patients with atypical BCR-ABL1 fusion transcripts: recommendations by the EUTOS cooperative network.

Project description:PurposeApproximately 1-2% of chronic myeloid leukemia (CML) patients harbor atypical BCR-ABL1 transcripts that cannot be monitored by real-time quantitative PCR (RT-qPCR) using standard methodologies. Within the European Treatment and Outcome Study (EUTOS) for CML we established and validated robust RT-qPCR methods for these patients.MethodsBCR-ABL1 transcripts were amplified and sequenced to characterize the underlying fusion. Residual disease monitoring was carried out by RT-qPCR with specific primers and probes using serial dilutions of appropriate BCR-ABL1 and GUSB plasmid DNA calibrators. Results were expressed as log reduction of the BCR-ABL1/GUSB ratio relative to the patient-specific baseline value and evaluated as an individual molecular response (IMR).ResultsIn total, 330 blood samples (2-34 per patient, median 8) from 33 CML patients (19 male, median age 62 years) were analyzed. Patients expressed seven different atypical BCR-ABL1 transcripts (e1a2, n = 6; e6a2, n = 1; e8a2, n = 2; e13a3, n = 4; e14a3, n = 6; e13a3/e14a3, n = 2; e19a2, n = 12). Most patients (61%) responded well to TKI therapy and achieved an IMR of at least one log reduction 3 months after diagnosis. Four patients relapsed with a significant increase of BCR-ABL1/GUSB ratios.ConclusionsCharacterization of atypical BCR-ABL1 transcripts is essential for adequate patient monitoring and to avoid false-negative results. The results cannot be expressed on the International Scale (IS) and thus the common molecular milestones and guidelines for treatment are difficult to apply. We, therefore, suggest reporting IMR levels in these cases as a time-dependent log reduction of BCR-ABL1 transcript levels compared to baseline prior to therapy.

| S-EPMC8397658 | biostudies-literature

Outcomes of children with BCR-ABL1–like acute lymphoblastic leukemia treated with risk-directed therapy based on the levels of minimal residual disease.

Project description:PurposeBCR-ABL1–like acute lymphoblastic leukemia (ALL) is a recently identified B-cell ALL (B-ALL)subtype with poor outcome that exhibits a gene expression profile similar to BCR-ABL1-positive ALL but lacks the BCR-ABL1 fusion protein. We examined the outcome of children with BCR-ABL1–like ALL treated with risk-directed therapy based on minimal residual disease (MRD) levels during remission induction.Patients and methodsAmong 422 patients with B-ALL enrolled onto the Total Therapy XV study between 2000 and 2007, 344 had adequate samples for gene expression profiling. Next-generation sequencing and/or analysis of genes known to be altered in B-ALL were performed in patients with BCR-ABL1–likeALL who had available material. Outcome was compared between patients with and those without BCR-ABL1–like ALL.ResultsForty (11.6%) of the 344 patients had BCR-ABL1–like ALL. They were significantly more likely to be male, have Down syndrome, and have higher MRD levels on day 19 and at the end of induction than did other patients with B-ALL. Among 25 patients comprehensively studied for genetic abnormalities, 11 harbored a genomic rearrangement of CRLF2, six had fusion transcripts responsive to ABL tyrosine kinase inhibitors or JAK inhibitors, and seven had mutations involving the Ras signaling pathway. There were no significant differences in event-free survival (90.0% +/- 4.7% [SE] v. 88.4% +/- .9% at 5 years; P = .41or in overall survival (92.5% +/- 4.2% v. 95.1% +/- 1.3% at 5 years; P = .41) between patients with and without BCR-ABL1–like ALL.ConclusionPatients who have BCR-ABL1–like ALL with poor initial treatment response can be salvaged with MRD-based risk-directed therapy and may benefit from identification of kinase-activating lesions for targeted therapies.

| S-EPMC4162497 | biostudies-literature

Application of droplet digital PCR in minimal residual disease monitoring of rare fusion transcripts and mutations in haematological malignancies.

Project description:Leukaemia of various subtypes are driven by distinct chromosomal rearrangement or genetic abnormalities. The leukaemogenic fusion transcripts or genetic mutations serve as molecular markers for minimal residual disease (MRD) monitoring. The current study evaluated the applicability of several droplet digital PCR assays for the detection of these targets at RNA and DNA levels (atypical BCR::ABL1 e19a2, e23a2ins52, e13a2ins74, rare types of CBFB::MYH11 (G and I), PCM1::JAK2, KMT2A::ELL2, PICALM::MLLT10 fusion transcripts and CEBPA frame-shift and insertion/duplication mutations) with high sensitivity. The analytical performances were assessed by the limit of blanks, limit of detection, limit of quantification and linear regression. Our data demonstrated serial MRD monitoring for patients at molecular level could become "digitalized", which was deemed important to guide clinicians in treatment decision for better patient care.

| S-EPMC10944481 | biostudies-literature

Targeting the PTP1B-Bcr-Abl1 interaction for the degradation of T315I mutant Bcr-Abl1 in chronic myeloid leukemia.

Project description:Small-molecule-induced degradation of mutant Bcr-Abl1 provides a potential approach to overcome Bcr-Abl1 tyrosine kinase inhibitor (TKI)-resistant chronic myeloid leukemia (CML). Our previous study reported that a synthetic steroidal glycoside SBF-1 showed remarkable anti-CML activity by inducing the degradation of native Bcr-Abl1 protein. Here, we observed the comparable growth inhibition for SBF-1 in CML cells harboring T315I mutant Bcr-Abl1 in vitro and in vivo. SBF-1 triggered its degradation through disrupting the interaction between protein-tyrosine phosphatase 1B (PTP1B) and Bcr-Abl1. Using SBF-1 as a tool, we found that Tyr46 in the PTP1B catalytic domain and Tyr852 in the Bcr-Abl1 pleckstrin-homology (PH) domain are critical for their interaction. Moreover, the phosphorylation of Tyr1086 within the Bcr-Abl1 SH2 domain recruited the E3 ubiquitin ligase c-Cbl to catalyze K27-linked ubiquitin chains, which serve as a recognition signal for p62-dependent autophagic degradation. PTP1B dephosphorylated Bcr-Abl1 at Tyr1086 and prevented the recruitment of c-Cbl, leading to the stability of Bcr-Abl1. This study unravels the action mechanism of PTP1B in stabilizing Bcr-Abl1 protein and indicates that the PTP1B-Bcr-Abl1 interaction might be one of druggable targets for TKI-resistant CML with point mutations.

| S-EPMC9807508 | biostudies-literature

Molecular dynamics reveal BCR-ABL1 polymutants as a unique mechanism of resistance to PAN-BCR-ABL1 kinase inhibitor therapy.

Project description:The acquisition of mutations within the BCR-ABL1 kinase domain is frequently associated with tyrosine kinase inhibitor (TKI) failure in chronic myeloid leukemia. Sensitive sequencing techniques have revealed a high prevalence of compound BCR-ABL1 mutations (polymutants) in patients failing TKI therapy. To investigate the molecular consequences of such complex mutant proteins with regards to TKI resistance, we determined by cloning techniques the presence of polymutants in a cohort of chronic-phase patients receiving imatinib followed by dasatinib therapy. The analysis revealed a high frequency of polymutant BCR-ABL1 alleles even after failure of frontline imatinib, and also the progressive exhaustion of the pool of unmutated BCR-ABL1 alleles over the course of sequential TKI therapy. Molecular dynamics analyses of the most frequent polymutants in complex with TKIs revealed the basis of TKI resistance. Modeling of BCR-ABL1 in complex with the potent pan-BCR-ABL1 TKI ponatinib highlighted potentially effective therapeutic strategies for patients carrying these recalcitrant and complex BCR-ABL1 mutant proteins while unveiling unique mechanisms of escape to ponatinib therapy.

| S-EPMC3948238 | biostudies-literature

GATA1 Expression in BCR/ABL1-negative Myeloproliferative Neoplasms.

Project description:This study aimed to determine GATA1 expression levels to better characterize subgroups in BCR/ABL1-negative myeloproliferative neoplasms (MPNs).This study enrolled 49 patients diagnosed as having BCR/ABL1-negative MPN on the basis of the 2016 World Health Organization classification : nine polycythemia vera (PV), 17 essential thrombocythemia (ET), 12 prefibrotic primary myelofibrosis (prePMF), and 11 overt primary myelofibrosis (PMF). Relevant clinical and laboratory data were retrieved from the medical records. The molecular analysis of CALR and MPL mutations and quantification of JAK2 V617F allele burden were performed. GATA1 expression was assessed by an immunohistochemical assay on bone marrow biopsy. GATA1 expression was analyzed serially in 18 patients.GATA1 expression decreased significantly in PMF compared with that in other subtypes, while no statistical difference was identified between ET and prePMF. GATA1 expression did not differ according to the mutation profiles or the allele burden of JAK2 V617F, but it decreased significantly in patients with overt fibrosis or leukemic transformation.Our results suggest that GATA1 expression is significantly low in PMF and decreases with progressive fibrosis and possibly with leukemic transformation, although our attempt to accurately distinguish between subgroups using GATA1 immunohistochemical approach did not achieve statistical significance. A large patient cohort with long term follow-up is required to evaluate the prognostic value of GATA1 expression.

| S-EPMC5895858 | biostudies-other

Development and evaluation of armored RNA-based standards for quantification of BCR-ABL1<sup>p210/p190</sup> fusion gene transcripts.

Project description:BackgroundStandards play an important role in detection of the BCR-ABL1 fusion gene (FG) transcript. However, the standards widely used in laboratories are mainly based on plasmids or cDNA, which cannot accurately reflect the process of RNA extraction and cDNA synthesis. Therefore, we aimed to develop armored RNA-based standards for p210 and p190 BCR-ABL1FG transcripts' quantification.MethodsUsing overlapping polymerase chain reaction (PCR) technology, we first linked a segment of the p210 or p190 BCR-ABL1FG transcript with four control genes (CGs; ABL1, BCR, GUSB, and B2M) to form p210FG-CG and p190FG-CG. Subsequently, using armored RNA technology, we prepared p210FG-CG- and p190FG-CG-armored RNAs and the p210FG-CG and p190FG-CG standards, the values of which were assigned by digital PCR (dPCR).ResultsThe p210FG-CG and p190FG-CG standards were stable and homogeneous, and were significantly linear with r2 > 0.98. A field trial including 52 laboratories across China showed that the coefficient of variation (CV%) of BCR-ABL1 values among samples was in the range of 58.6%-129.6% for p210 samples and 73.2%-194.0% for p190 samples when using local standards. By contrast, when using the p210FG-CG and p190FG-CG standards, the CV% of BCR-ABL1 values was decreased to 35.6%-124.9% and 36.6%-170.6% for p210 and p190 samples, respectively. In addition, 33.3% (3/9) of the p210 and p190 samples had CV% values <50.0%, whereas 44.4% (4/9) and 77.8% (7/9) of the samples had lower CV% values when using the p210FG-CG and p190FG-CG standards.ConclusionThe overall variability of detection of BCR-ABL1 transcripts decreased significantly when using the p210FG-CG or p190FG-CG standards, especially the p190FG-CG standard.

| S-EPMC6816845 | biostudies-literature

Assessing Measurable Residual Disease in Chronic Myeloid Leukemia. BCR-ABL1 IS in the <i>Avant-Garde</i> of Molecular Hematology.

Project description:Chronic myelogenous leukemia (CML) is a malignancy of the myeloid cell lineage characterized by a recurrent chromosomal abnormality: the Philadelphia chromosome, which results from the reciprocal translocation of the chromosomes 9 and 22. The Philadelphia chromosome contains a fusion gene called BCR-ABL1. The BCR-ABL1 codes for an aberrantly functioning tyrosine kinase that drives the malignant proliferation of the founding clone. The advent of tyrosine kinase inhibitors (TKI) represents a landmark in the treatment of CML, that has led to tremendous improvement in the remission and survival rates. Since the introduction of imatinib, the first TKI, several other TKI have been approved that further broadened the arsenal against CML. Patients treated with TKIs require sensitive monitoring of BCR-ABL1 transcripts with quantitative real-time polymerase chain reaction (qRT-PCT), which has become an essential part of managing patients with CML. In this review, we discuss the importance of the BCR-ABL1 assay, and we highlight the growing importance of BCR-ABL1 dynamics. We also introduce a mathematical correction for the BCR-ABL1 assay that could help homogenizing the use of the ABL1 as a control gene. Finally, we discuss the growing body of evidence concerning treatment-free remission. Along with the continuous improvement in the therapeutic arsenal against CML, the molecular monitoring of CML represents the avant-garde in the struggle to make CML a curable disease.

| S-EPMC6768007 | biostudies-literature

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