E1a2 Transcripts Research Articles

Characterization and Quantification Of Rare BCR-ABL1 Fusion Transcripts

BackgroundIn chronic myeloid leukemia (CML) treatment with tyrosine kinase inhibitors induces extremely good responses in most of the patients (pts). To control for treatment responses molecular monitoring by quantitative real time PCR has become a standard procedure. Many international efforts have been done to standardize PCR assays, an international conversion factor has been introduced which allows comparison of results between laboratories, and response criteria have been defined based on %BCR-ABL1/ABL1 values. A drawback of the current state is that standardization, so far, addressed only the most common BCR-ABL1 fusion transcripts e13a2 and e14a2. For rare fusion types response criteria have not been applied and biological and prognostic significance are widely unknown. Aim1) Analyze incidence and associations of rare BCR-ABL1 fusion types with other biological parameters. 2) Develop assays for rare BCR-ABL1 fusions to allow molecular monitoring and analysis of therapy response in CML patients with rare fusion types. Patients and MethodsIn 5,730 individual CML pts a detailed molecular characterization of the BCR-ABL1 rearrangement was performed between 8/2005 and 6/2013. Rare fusion transcripts were further characterized by Sanger sequencing, and quantification was performed by a LightCycler based real time assay using hybridization probes. Probes and reverse primers delevoped for M-bcr transcripts were used (Emig et al., Leukemia, 1999). Solely the forward primer was modified and adapted to the BCR breakpoint. Assays were optimized to obtain similar efficiencies and sensitivities (1:10,000) compared to the standard p210 assay. ResultsIn the entire cohort of 5,730 CML pts rare fusion types were detected in 112 cases (2.0%). In detail, of the rare types 49/112 (43.8%) had breakpoints in the m-bcr (minor breakpoint cluster region, corresponding to p190): e1a2 (n=46, 41.1%) and e1a3 (n=3, 2.7%). 26 cases (23.2%) had rare M-bcr types (major breakpoint cluster region, corresponding to p210): e13a3 (n=8), e14a3 (n=15), e13e14a3 (n=2) and one case with e14a3 and insertion of 45 nucleotides. 20 pts (17.9%) had a typical µ-bcr breakpoint with e19a2 (corresponding to p230), including one case with an additional alternative splicing event that leads to an e13a2 fusion with 19 bp insertion. Further very rare fusions were: e4a2 (n=2), e6a2 (n=5), e16a2 (n=1), e18a2 (n=2), and e8a2 (n=7) (e8a2 fusions had in frame additions or deletions of 39-70 bp). The male/female ratio in this cohort was 65/47 (1.4), and median age was 66.9 years (y) (range: 20-87y). In 77 of the 112 cases a sample at diagnosis was available. In the remaining 35 pts monitoring started later. Of the 61 cases with available cytogenetics 38 pts had t(9;22) sole and 23 (37.7%) had additional chromosomal aberrations (ACA): -7 (n=2), +8 (n=4), i(17q) (n=1), dup(17)(q23q25) (n=1), -Y (n=7), +der(22)t(9;22) (n=3), rare translocations (n=5). Thus, the frequency of ACA was significantly higher than has been described for CML in general (6.9%, Fabarius et al., Blood, 2011).Of the 112 patients 714 individual samples were quantified (e1a2 and e1a3: n=237, e13a3 and e14a3: n=191, e19a2: n=144, e16a2: n=24, e18a2: n=21, e8a2: n=60, e6a2: n=28 e4a2: n=9). Regular monitoring at every 3 months was available in 76 pts who were treated with firstline imatinib (n=64), followed by second line nilotinib (n=4) or dasatinib (n=3); firstline dasatinib (n=7), followed by nilotinib (n=1), or firstline nilotinib (n=5). Response was evaluated after 1 year and subdivided as follows: 1) CMolR (complete molecular response): undetectable BCR-ABL1, 2) MMolR (major molecular response): ≤0.1% BCR-ABL1/ABL1, 3) no response: no or less that 1 log decrease, 4) suboptimal response: > 0.1% BCR-ABL1/ABL1. In 15 pts (19.7%) a CMolR and in 20 pts (26.3%) a MMolR was reached (in 1 case after switch from imatinib to nilotinib and in 1 from imatinib to dasatinib). However, there was a large number of cases with no response: n=19 (25.0%) or suboptimal response: n=22 (28.9%). In 55 of the 76 continuously monitored pts cytogenetics at time of diagnosis was available. In the poor responders slightly more cases had ACA (12/28; 42.9%) compared to the good responders (7/27; 25.9%) (n.s.). ConclusionRare BCR-ABL1 fusion types 1) occur in 2% of all CML, 2) are associated with higher number of ACA, 3) have no or suboptimal response to TKI treatment in more than 50% of pts. Disclosures:Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Dicker:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Fasan:MLL Munich Leukemia Laboratory: Employment. Weber:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Clinical activity of ponatinib in patients with chronic myeloid leukemia in chronic phase with e1a2 transcripts

The two major types of transcripts[1][1] of the BCR-ABL1 oncoprotein are b2a2 (e13a2) and b3a2 (e14a2). These transcripts are observed in over 95% of patients with chronic myeloid leukemia in chronic phase (CML-CP) at the time of initial presentation. Very rarely (<1%), CML patients are seen

Simultaneous occurrence of ETV6-RUNX1 and BCR-ABL1 (e1a2) transcripts in a child with B-cell acute lymphoblastic leukemia

We report on a rare case of a 3-year-old boy with B-cell acute lymphoblastic leukemia (B-ALL), which was characterized simultaneously with two different fusion transcripts: ETV6-RUNX1 and BCR-ABL1 (e1a2). The patient presented with fever, diarrhea, normal white blood cell counts of 5.9×10(9)/L without circulating abnormal cells, anemia, and thrombocytopenia, as well as an enlarged liver without splenomegaly. The bone marrow was markedly hypercellular with a total infiltration of agranular lymphoid blast cells with a B-II (pre-B) lymphoblastic phenotype: cyCD79α(+), CD19(+), sCD22(+), CD10(+), CD20(-), CD34(+), and sIgM(-), with dim aberrant co-expression of the myeloid-associated markers CD13(+) and CD33(+). Conventional cytogenetic analysis was unsuccessful; however, molecular analysis revealed the BCR-ABL1 (p190) and ETV6-RUNX1 transcripts. A diagnosis of BCR-ABL1 (p190)-positive and ETV6-RUNX1-positive B-ALL was made, and treatment was initiated according to the AIEOP-BFM-ALL2000 protocol. A complete remission was achieved after the first induction course of chemotherapy. Twelve months after the diagnosis, the child is alive with levels of residual disease of <0.05% estimated both by 8-color flow cytometry and real-time quantitative reverse transcription polymerase chain reaction.

The BCR-ABL1 Transcript Type Does Not Influence the Response and the Outcome of Chronic Myeloid Leukemia Patients Treated Frontline with Nilotinib

AbstractAbstract 1680 Background.Chronic myeloid leukemia (CML) is characterized by the presence of the BCR-ABL1 hybrid gene. Different types of BCR-ABL1 fusion transcripts can be found, but the most frequent are the e13a2 (b2a2) and the e14a2 (b3a2). In the tyrosine kinase inhibitors (TKIs) era, few data about the prognostic significance of the transcript type in early chronic phase (ECP) CML are available. Three larger studies suggested that the e13a2 transcript may have an adverse prognostic impact in ECP CML patients treated with imatinib (IM): Vega-Ruiz et al. (251 patients, ASH 2007) reported inferior molecular responses; Lucas et al. (71 patients, Haematologica 2009) reported lower cytogenetic response rates and lower event-free survival (EFS); the GIMEMA CML WP (493 patients, EHA 2011) reported a slower time to major molecular response (MMR) with inferior EFS and progression-free survival (PFS). To our knowledge this is the first evaluation of the prognostic influence of the BCR-ABL1 transcript type on the responses and the outcome of ECP CML treated frontline with nilotinib (NIL). Methods.The CML Italian Registry of Nilotinib includes 215 patients treated with NIL-based regimens. The patients were enrolled within 2 multicenter phase II studies conducted by the GIMEMA CML WP (ClinicalTrials.gov. NCT00481052 and NCT00769327) or treated at the “S. Orsola-Malpighi” University Hospital (Bologna, Italy), with NIL 300 mg BID or 400 mg BID as initial treatment. All the registered patients were analyzed. Patients expressing rare transcripts and patients with both b2a2 and b3a2 transcripts were excluded: 201 out of 215 patients were evaluable, 81 (40%) with e13a2 transcript and 120 (60%) with e14a2 transcript. Differences between groups were tested using χ2 test, Fisher exact test or t-test, as appropriate. Response monitoring: conventional cytogenetic examination (bone marrow) and QPCR (peripheral blood). Definitions: MMR: BCR-ABLIS ratio <0.1% (International Scale); failures: according to 2009 ELN recommendations; events: failure or treatment discontinuation for any reason. The time-to-response and the outcome were estimated using the Kaplan-Meier method, and compared by log-rank test. Results.The baseline characteristics of the 2 groups were comparable (no significant differences in age, Sokal/Hasford/EUTOS score distribution, clonal chromosomal abnormalities in Ph+ cells, NIL dose), except for the percentage of basophils in the peripheral blood, higher in patients with e14a2 transcript (3.4% vs 2.3%, p=0.01). The median observation was 29 months (range 18–47); 92% of the patients had at least 2 year observation. The CCgR and MMR rates at 12 months were comparable in the 2 groups. The time to MMR was longer for patients with e13a2 transcript (6 months vs 3 months, p=0.04), but the overall CCgR rates (93.8 vs 91.7, p=0.79) and the overall MMR rates (85.1 vs 90.0, p=0.38) were not significantly different in patients with e13a2 or e14a2 transcript, respectively. The probability of Overall Survival (OS), Progression-Free Survival (PFS) and Failure-Free Survival (FFS) were comparable: 91.4% vs 95.8% (p=0.61), 90.7% vs 95.0% (p=0.51), and 90.7% vs 88.7% (p=0.40) in patients with e13a2 and e14a2 transcript, respectively. Conclusions.In our experience, based on 201 early CP CML patients treated frontline with NIL with a minimum follow-up of 18 months, the BCR-ABL transcript type did not show any relevant prognostic impact. The time to MMR was longer in patients with e13a2 transcript, but no response and outcome differences have been observed so far. The number of observed events was low and a longer observation is required. Acknowledgments.European LeukemiaNet, COFIN, Bologna University, BolognAIL Disclosures:Castagnetti:Novartis Pharma: Consultancy, Honoraria, Speakers Bureau; Bristol Myers Squibb: Consultancy, Honoraria, Speakers Bureau. Gugliotta:Novartis: Consultancy, Honoraria; Bristol-Myers-Squibb: Consultancy, Honoraria. Breccia:Bristol Myers Squibb: Consultancy; Novartis: Consultancy. Cavazzini:Novartis Pharma: Honoraria; Bristol Myers Squibb: Honoraria. Turri:Novartis: Consultancy, Novartis Other; Bristol Myers Squibb: Bristol Myers Squibb, Bristol Myers Squibb Other, Consultancy. Soverini:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Saglio:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy. Martinelli:Novartis: Consultancy, Honoraria, Speakers Bureau; Bristol-Myers-Squibb: Consultancy, Honoraria, Speakers Bureau. Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Rosti:Novartis Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Speakers Bureau; Pfizer: Speakers Bureau.

Response to Frontline Therapy with Second Generation Tyrosine Kinase Inhibitors in Chronic Myeloid Leukemia: Analysis of Outcome for b3a2 Vs. b2a2 Fusion Transcripts

AbstractAbstract 3781 Background:CML is characterized by the reciprocal translocation between chromosomes 9 and 22 [t(9;22)(q34;q11.2)], also known as the Philadelphia chromosome. This results in the formation of the BCR-ABL fusion gene, which is translated to a protein with increased tyrosine kinase activity. The breakpoint in the BCR gene can occur in different sites, most commonly between exons 13 and 15 which fuse to exon 2 of the ABL gene creating the b2a2 and b3a2 junctions. Previous studies have reported a better outcome for patients (pts) having a b2a2 transcript when treated with imatinib. To our knowledge there are no published reports analyzing the outcome of pts treated with a 2ndgeneration TKI as initial therapy for CML according to the fusion transcript. We analyzed the significance of these variations among pts with CML in chronic phase (CP) enrolled in 2 parallel trials of dasatinib and nilotinib as initial therapy. Patients and Methods:A total of 204 pts with CML in CP, treated at our institution between 2005 and 2012 with nilotinib or dasatinib as frontline therapy were included in this analysis. Among them 88 (43%) had the b2a2 transcript (median age of 47 years; range 18–80) at the start of therapy, 74 (36%) showed the b3a2 variant (median age of 47; range 17–82 years), and 42 (21%) presented both (median age 53; range 27–81). One patient had a b3a3 transcript and 2 pts showed an e1a2 transcript. Forty-two (48%) of those with b2a2 were treated with dasatinib and 46 (52%) with nilotinib; of the pts with b3a2 34 (46%) were treated with dasatinib and 40 (54%) with nilotinib, in the group of pts with both transcripts 24 (57%) received dasatinib 24, and 18 (43%) of them nilotinib (Table 1). Results:The Sokal risk group for those with the b2a2 transcript was high in 10%, intermediate in 29%, and low in 61%. For pts with b3a2 the risk classification was 5%, 25, and 70%, respectively. The group with both transcripts had 10%, 31% and 69%, respectively. Significant difference was observed in the time of molecular response and overall outcome between pts with b2a2 and those with b3a2 (CCyR 91% vs. 96%; MMR 75% vs. 91%, respectively). The 3-year probability of overall survival (OS) was 100% for both groups. The 3-year probability of event-free survival (EFS) was 93% and 99%, and the transformation-free survival (TFS) 99% and 98% for the b2a2 and the b3a2 group respectively (Table 2). On the 12 mo. follow-up one of the pts with e1a2 transcript achieved CCyR (treated with nilotinib), and the second patient (pt) major cytogenetic response (receiving dasatinib). The pt presenting with b3a3 was treated with dasatinib and achieved MMR by 3 mo. and CCyR at the 12 months follow-up. Conclusions:Although all pts whether they express b3a2 or b2a2 at diagnosis, have an excellent overall survival, those with b3a2 have a significantly higher rate of molecular responses and a trend for better EFS. These results mirror what we have previously reported for pts treated with imatinib as initial therapy. The biologic characteristics for this difference warrant further investigation.Table 1Treatment distribution in analyzed groupsTranscriptDasatinibNilotinibb2a242 (48%)46 (52%)b3a234 (46%)40 (54%)b2a2 and b3a224 (57%)18 (43%)Table 2Results of patients with variant BCR-ABL transcripts treated with 2ndgeneration TKIsb2a2b3a2b2a2+b3a2Overall CCyR90.4%95.7%95.1%Overall MMR75%91.5%95%12 mo. CCyR93.8%100%100%12 mo. MR66.7%98.184.7%3 years O.S.100%100%94%3 years EFS93%99%98%3 years TFS99%98%100% Disclosures:Kantarjian:Novartis: Research Funding; Bristol Myers Squibb: Research Funding; Ariad: Research Funding; Pfizer: Research Funding. Jabbour:Bristol Myers Squibb: Honoraria; Pfizer: Honoraria; Novartis: Honoraria. Cortes:Novartis: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Ariad: Consultancy, Research Funding.

Rare Blast Phase (BP) Phenotypes of Chronic Myeloid Leukemia (CML)

AbstractAbstract 4434Approximately 65% of patients (pts) with CML BP exhibit a myeloid phenotype, 30% a lymphoid, and 5% of cases exhibit other phenotypes. The vast majority of lymphoid BP cases are of B-cell origin. Other phenotypes of CML BP are very infrequent and poorly characterized. To evaluate the incidence and outcome of unusual BP phenotypes, we reviewed 1143 pts with CML BP diagnosed and treated at M.D. Anderson Cancer Center between November 1973 and February 2012. Thirteen (1%) pts had non-B-cell/non-myeloid CML BP: 8 pts with T-cell differentiation (T-BP) (including 4 pts with biphenotypic T-cell/myeloid), 2 pts with basophilic (B-BP), and 3 pts with megakaryoblastic phenotype (M-BP) (Table 1). Five pts presented initially in BP, whereas 7 other cases evolved to BP from chronic phase (CP) and 1 pt from accelerated phase (AP). The median time from diagnosis to transformation was 15.2 months (mos) (range, 0–103). The median age was 50 years (range, 21–74), median WBC count at presentation 40.2×109/L (range, 2.6–236), hemoglobin 10.7 g/dL (range, 7.1–13.6), platelet count 171×109/L (range, 17–383), peripheral blood blasts 9% (range, 0–100), and bone marrow blasts 20% (range, 0–88). Nine (69%) pts developed extramedullary disease: 4 with lymphadenopathy (LAN), 1 with LAN and mediastinal mass, 1 with pericardial tamponade and mediastinal mass, 1 with central nervous system disease, 1 with facial muscle involvement and 1 with splenomegaly, LAN, and granulocytic sarcoma of the breast. Four pts expressed a b2a2 BCR-ABL1 transcript (p210), whereas 3 pts expressed b3a2 (p210), 3 expressed e1a2 (p190), 2 expressed b2a2+b3a2 (p210), and 1 expressed an e13b2+e14a2 (p210) transcript at transformation that switched to e1a2 (p190) during dasatinib (DAS) therapy. Median number of treatments received prior to BP was 2 (range, 1 to 12): 6 pts received interferon (IFN), 5 imatinib (IMA), 3 allogenetic stem cell transplant (ASCT), 3 Ara-C, 1 DAS and 1 hyper-CVAD (HCVAD). Initial therapy consisted of HCVAD (n=4, combined with IMA 600 mg/d in 1 pt and with DAS 70 mg/d in 1 pt), VAD (n=1), nilotinib 400 mg twice daily (n=1), DAS 50 mg twice daily (n=1), troxicitabine (n=1), gemtuzumab (n=1), idarubicin (IDA) and ara-C (n=3; 1 with IMA 600 mg/d and 1 with IMA 400 mg/d), and ara-C (n=1). Four pts responded: 3 with chemotherapy and IMA, achieving a complete cytogenetic response (CCyR) that lasted 22, 26 and 79 mos, respectively; 1 pt achieved a major molecular response (MMR) after receiving gemtuzumab followed by haploidenticial SCT, lasting for 24 mos. Median duration of response was 25 mos (range, 22–79). Subsequent therapy consisted of a tyrosine kinase inhibitor (TKI) in 6 out of 13 pts. Two pts treated with DAS at 140 mg/d and 70 mg twice daily achieved a CCyR and 1 pt achieved MMR on DAS 140 mg/d. Of the 7 pts treated with DAS, 2 had BCR-ABL1 mutations: 1 carried K271R, Y232C/K271R, and M343Y/K271R prior to DAS, then acquired V299L and F317I on DAS, while the other pt carried Y264S prior to DAS, then developed V299L on DAS. After a median follow up of 4.2 years (range, 0.2 to 14.4), 4/13 (30%) pts are alive: 1 in MMR on IMA 400 mg/d, 1 in complete molecular response (CMR) on DAS 100 mg/d, 1 not in remission (non-compliance to DAS 140 mg/d), and 1 currently receiving HCVAD and DAS. By phenotype, 5/8 pts with T-BP responded to TKIs: 1 CMR, 1 CCyR, 1 MMR, 1 partial cytogenetic response (PCyR) and 1 hematologic response (HR); 2/3 pts with M-BP responded: 1 CCyR and 1 CHR. None of the B-BP pts responded. In conclusion, non-B-cell/non-myeloid CML BP occurs in 1% of pts with CML, frequently infiltrates extramedullary sites, and exhibits high resistance to conventional chemotherapy. Long-term responses can be achieved with ABL1 TKIs (preferably in combination with chemotherapy), which can be used as a bridge to allogeneic SCT. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Patterns of BCR breakpoints in patients with coexisting inv(16)(p13.1q22) and t(9;22)(q34;q11.2)

We read with great interest ‘‘Coexistence of inversion 16 and the Philadelphia chromosome comprising P190 BCR/ ABL in chronic myeloid leukemia blast crisis’’ by Ninomiya et al. [1]. As the authors note, their case represents the first reported usage of the minor BCR breakpoint in the setting of chronic myelogenous leukemia (CML) in myeloid blast phase with a concomitant inv(16)(p13.1q22). All previously investigated similar cases show usage of the major BCR breakpoint, while all investigated cases of de novo acute myeloid leukemia (AML) with coexisting inv(16)(p13.1q22) and t(9;22)(q34;q11.2) use the minor BCR breakpoint. Indeed, this pattern is reinforced by several patients described in the literature but not listed in the authors’ Table 1 ‘‘Summary of reported cases of Ph? myeloid leukemia with inv(16)’’ [2–4]. In addition, we have recently seen a patient who conforms to this apparent pattern. A 49-year-old woman developed persistent leukopenia (white blood cell count 1.7 9 10/L) and mild thrombocytopenia (platelet count 113 9 10/L) 12 months after the initiation of therapy for infiltrating ductal carcinoma of the breast. Her treatment regimen had included four cycles of adriamycin and cyclophosphamide, followed by paclitaxel and trastuzumab. She had also undergone a bilateral mastectomy, and radiation therapy to the chest wall had been initiated 8 months after her original diagnosis. Other than the leukopenia and thrombocytopenia, her CBC parameters were unremarkable. There were no physical or symptomatic findings of note. Review of the peripheral blood smear identified rare circulating blasts (1–2%) with fine chromatin, irregular nuclear contours and conspicuous nucleoli. A bone marrow evaluation revealed hypercellularity with increased blasts (10–15%) and abnormal eosinophils with basophilic granules. By flow cytometric analysis, the blasts showed a myeloid immunophenotype (positive for CD13, dim CD33, HLADR, CD117, and myeloperoxidase; negative for CD2, surface CD3, CD4, CD5, CD8, CD10, CD19, CD20, TdT, CD64, CD14, and CD34). Cytogenetic analysis revealed a karyotype of 46,XX,inv(16)(p13.1q22)[5]/46,idem,t(9;22)(q34;q11.2) [7]/46,XX[8], and fluorescence in situ hybridization confirmed the CBFB (16q22) rearrangement. Thus, both the inv(16) and the Philadelphia chromosome were present in the same metaphase cells, and did not represent biclonal populations. RT-PCR analysis revealed e1-a2 BCR-ABL1 transcripts, corresponding to the p190 minor form, but no e13-a2 or e14-a2 (p210, major form) transcripts (Figs. 1, 2, 3). Given the patient’s history, our diagnosis was therapy-related myeloid neoplasm. The overall findings, including the usage of the minor BCR breakpoint, correlate well with the previously documented pattern of de novo AML with coexisting inv(16)(p13.1q22) and t(9;22)(q34;q11.2). The present case is the second reported therapy-related myeloid neoplasm with coexisting inv(16) and t(9;22), and is similar in many respects to the previously described case [2]. Somewhat remarkably, our patient showed no evidence of the BCR-ABL1 fusion by FISH or RT-PCR after only 3 weeks of single agent treatment with imatinib mesylate. Thus, including our patient, there appear to be at least 18 patients described in the literature with simultaneous D. Bustamante (&) D. R. Czuchlewski Department of Pathology, University of New Mexico, TriCore Reference Laboratories, 1001 Woodward Place NE, Albuquerque, NM 87102, USA e-mail: DCzuchlewski@salud.unm.edu

BCR-ABL rearrangement and HLA antigens: a possible link to leukemia pathogenesis and immunotherapy

BCR-ABL rearrangement and HLA antigens: a possible link to leukemia pathogenesis and immunotherapy

Quantitative Multiplex Measurement of BCR-ABL1 Expression on the International Scale Using the BCR/ABL1 Quant™ RUO Reagents and the LightCycler® 480

Abstract 4430 Introduction:BCR-ABL1 e13a2 or e14a2 fusion transcripts corresponding to the translocation (9;22) are present in >95% of chronic myeloid leukemia (CML) patients. This molecular signature is thus useful to monitor tumor burden and tyrosine kinase inhibitor treatment response in CML. The e1a2 variant is found in about 5% of children with acute lymphoblastic leukemia (ALL) and 20–35% of adult ALL. Growing evidence indicates that the e1a2 variant is associated with inferior outcome and higher risk of relapse in CML. It is therefore also important to study e1a2 expression levels in these individuals. The objective of this study was to evaluate the use of a single multiplex assay (BCR/ABL1 Quant™ RUO) for the quantitative detection of e1a2, e13a2, and e14a2 fusion transcripts. These reagents were initially optimized for use with the ABI 7500 series of instruments (Brown et al., Blood Cancer Journal 2011). Here we investigated their compatibility and performance on the LightCycler® 480 System (LC480). Method:Control RNAs containing or lacking BCR-ABL1 transcripts were obtained from commercial sources. The kit's Calibrator standard curve samples and the ARQ IS Calibrator Panel were both prepared by heat lysis prior to reverse transcription (RT). The RNA was then reverse transcribed and then amplified by multiplex, real-time quantitative PCR on the ABI 7500 Fast Dx or LC480 using target-specific primers for e1a2, e13a2, e14a2, and ABL1 as an endogenous internal control. Runs on the LC480 used a ramp rate of 1.6 degrees/second to mimic that of the ABI 7500. Also, color compensation reagents (positive simplex reactions) were used on the LC480 per manufacturer's recommendations to generate a color compensation profile to enhance the performance of the multiplex reactions. Four-point standard curves were generated in each run using the included multiplex ARQ Calibrators (each calibrator sample contains each target: BCR/ABL1, ABL1, and BCR/ABL1 Quant Norm, an exogenous internal control). Fusion transcript identity was determined by capillary electrophoresis on an ABI 3130xl series instrument with 36 cm capillaries using POP7. Data were analyzed using either instrument's software (SDS v1.4 for the ABI 7500 Fast Dx and LightCycler software v1.5 for the LC480) or proprietary Excel-based software developed by Arista Molecular. Result:Analytical performance assessed using cell line dilutions showed a lower limit of detection equivalent to at least one translocation-positive cell in 100,000 normal cells. Linearity of >5 logs was observed with these samples. Breakpoints e13a2, e14a2, and e1a2 were all detected with various control materials. Negative specimens did not generate signal. ANOVA of multiple independent runs of controls sample replicates in randomly assigned plate locations on the LC480 showed excellent inter- and intra-run reproducibility and no detectable spatial bias across the plate. Side-by-side runs of the samples on both the 7500 and LC480 showed excellent correspondence of the results. Finally, testing of residual clinical specimens and prototype secondary IS reference materials (ARQ IS Calibrator Panel) linked to the WHO 1st International Genetic Reference Panel for the quantitation of BCR-ABL translocation indicated that the assay has the performance required to report on the International Scale (IS) when performed on the LC480. Conclusion:The BCR/ABL1 Quant RUO reagents are compatible with use on the LC480 instrument. They have the advantage of both quantifying and distinguishing e1a2, e13a2, and e14a2 fusion transcripts in a single reaction. Streamlined reagent formulation, multiplex assay format, and inclusion of ARQ calibrators improve the workflow and increase the number of specimens that can be tested per run. Our results suggest that this research assay has the performance required for reporting on the IS. Further evaluation with a larger set of clinical specimens is warranted. Disclosures:Brown:Asuragen: Employment. Laosinchai-Wolf:Asuragen: Employment. Labourier:Asuragen: Employment.

Analysis of the GeneXpert System on the International Multicentre ICORG 08–02 Phase II Study of Nilotinib 300mg BID as Frontline Treatment in Patients with Early Chronic Phase Chronic Myeloid Leukemia (ECPCML),

Abstract 3774 BACKGROUND/METHODS:Data from the ENESTnd phase III trial showed superiority of nilotinib over imatinib leading to accelerated approval of nilotinib as initial treatment of ECPCML at a dose of 300mg BID. ICORG, the All-Ireland Cooperative Oncology Research Group, has been coordinating an international open-label, single stage, multicenter, investigator-initiated phase II study (ClinicalTrials.gov NCT00809211) of the safety and efficacy of nilotinib 300 mg BID in previously untreated patients with ECPCML. The primary study endpoint is the complete cytogenetic response (CCyR) rate at 6 months; secondary endpoints include the kinetics of molecular response, determined by RQ-PCR at baseline and 3 monthly from start of treatment as well as an evaluation of a novel rapid turnaround PCR system “GeneXpert” with IS BCR-ABL1/ABL1 RQ-PCR. The study closed to accrual with 61 patients (median age 54 years [range 20 –77]) enrolled; 53% have low risk Sokal score, 25% intermediate and 22% high risk. Median follow up is currently 12 months (range 1–30) and by November 2011 all patients will have had at least 6 months follow up. RESULTS:At the time of this analysis, 6 and 12 month follow up data were available in 43 and 31 patients, respectively. By intention to treat analysis, 41/43 (95%) have achieved CCyR within 6 months with all patients still on treatment at 12 months achieving CCyR. 26/43 (60%) and 25/31 (81%) of patients have achieved a major molecular response (MMR) (BCR-ABL1/ABL1 IS ≤ 0.1%) within 6 and 12 months, respectively, with 16/31 (52%) and 11/31 (35%) patients on treatment at least 12 months achieving BCR-ABL1/ABL1 IS ≤ 0.01% and ≤ 0.0032%, respectively. Sokal risk had no apparent effect on response with 12/16 (75%) low risk and 13/15 (87%) intermediate/high risk patients achieving MMR within 12 months, p=0.65. Thus far 4/6 (67%) high risk Sokal patients achieved BCR-ABL1/ABL1 IS ≤ 0.0032% within 12 months. No patients have suffered CML progression to date. 53 patients remain on study. Six patients have been removed from study due to adverse events: 4 patients due to persistent drug-related toxicity; 2 patients non drug-related events (death due to progressive multiple system atrophy and colorectal carcinoma, respectively). In addition, one patient was enrolled but never received study drug and an additional patient was lost to follow up. Treatment was generally well tolerated and toxicities easily managed. Haematologic toxicity was minimal with grade III/IV thrombocytopenia seen in 3 (5%) patients and only a single patient (2%) each with grade III/IV neutropenia and anemia, respectively. The most common grade III/IV non-haematologic toxicity was lipase elevation, seen in 14/61 (23%). No cases of acute pancreatitis were seen. Other grade III/IV non-hematologic toxicities were uncommon (< 5% patients). While treatment was interrupted at least once in 36/59 (61%) patients, the median duration of interruptions was short (5 days). At last follow up 49/59 (83%) of patients were taking ≥ 300mg BID. The study evaluation of the “GeneXpert” PCR system consisted of paired BCR-ABL1/ABL1 measurements performed by RQ-PCR methodologies aligned to International Standard (IS) and by the Xpert BCR-ABL Monitor system on 36 evaluable patients at diagnosis and at 128 subsequent three-monthly time-points. In patients expressing e13a2 or e14a2 BCR-ABL1 transcripts, both techniques had comparable results at diagnosis: IS median BCR-ABL1/ABL1 41.0% vs 44.0% median BCR-ABL1/ABL1 on the Xpert BCR-ABL Monitor. In the 122 paired analyses, correlation between methodologies, without automated system IS conversion, over a five log range (IS BCR-ABL1/ABL1 100–0.001%) was favourable (r2=0.845), however a progressive decline in correlation was noted with each decreasing log IS BCR-ABL1/ABL1 level. No significant difference was observed between the Xpert BCR-ABL Monitor and IS RQ-PCR in identifying MMR (44.3% vs 41.8%). CONCLUSION:In ECPCML, nilotinib 300mg BID induces high rates of CCyR and MMR with a substantial fraction of patients achieving responses in the range of complete molecular response (CMR). This regimen's toxicity is modest. The GeneXpert system provides a reliable and rapid means of assessing CML patients' response to tyrosine kinase inhibitor (TKI) therapy. Development of a reagent lot-specific conversion factor to the IS would enhance GeneXpert's applicability in monitoring TKI therapy in patients with CML. Disclosures:O'Dwyer:Novartis: Honoraria, Research Funding. Giles:Novartis: Consultancy, Honoraria, Research Funding. le Coutre:Novartis: Honoraria, Research Funding, Speakers Bureau. Nagler:Novartis: Honoraria, Research Funding. Egan:Novartis: Employment. Conneally:Novartis: Honoraria.

Detection of Minimal Residual Leukemia Predicts the Outcome of Patients with Philadelphia-Chromosome Positive Acute Lymphoblastic Leukemia Treated with Tyrosine Kinase Inhibitors Plus Chemotherapy

Abstract 1453 Background:Combination of the hyperCVAD regimen and imatinib or dasatinib has produced long-term leukemia free responses in patients (pts) with Philadelphia (Ph) chromosome positive (Ph+) acute lymphoblastic leukemia (ALL) even without an allogeneic stem cell transplant. Identifying high risk pts can allow for a better selection of pts to undergo a transplant procedure in first complete remission (CR). Methods:From April 2001 to March 2011, 122 pts with newly diagnosed Ph+ ALL were treated on sequential frontline regimens of hyperCVAD + imatinib (n= 54) and hyperCVAD + dasatinib (n= 68). Among these, 101 pts achieved complete response (CR) with one induction course and had at least one minimal residual disease (MRD) assessment; 25 pts underwent an allogeneic stem cell transplant in first remission and were excluded from this analysis. MRD monitoring by multi-parameter flow cytometry (MFC) was performed using 4 or 6 color combinations of antibodies to lymphoblast and myeloid antigens (e.g., CD10, CD13, CD15, CD19, CD20, CD22, CD25, CD33, CD34, CD38, CD58, CD66c, and CD81), with a sensitivity of 0.01%. RQ-PCR for BCR-ABL was performed using TaqMan primer/probes for the e1a2, e13a2 (b2a2), and e14a2 (b3a2) BCR-ABL transcripts in a single tube with normalization to total ABL transcripts. All values were standardized to the international scale. Result:The median age for the cohort was 54 years (range, 21 – 84 years). Cytogenetics were Ph alone (n=13; 17%) and Ph with another abnormality (n=50; 66%). The other 13 pts (17%) were positive for the BCR-ABL fusion gene. There was no difference in survival by achievement of a major molecular response (MMR, BCR-ABL/ABL < 0.1%) at CR (p=.22). However, pts achieving a MMR, at 3, 6, 9, and 12 months had a significantly better survival than those not achieving MMR at these time points (p=.02, p=.04, and p=.05, and p=.01, respectively). Similarly, achievement of a negative MFC at CR did not predict for improved survival (p=.2). At 3 and12 months (insufficient data at 6 and 9 months), achieving a negative MRD status by MFC was associated with a significantly improved survival (p=.04 and p=.001). Figures represent data for landmark analysis at 3 months. Conclusion:MRD monitoring by PCR and MFC can identify pts who may benefit from intensification of treatment with an allogeneic stem cell transplant in first CR. [Display omitted] [Display omitted] Disclosures:Ravandi:Novartis: Honoraria; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding. Off Label Use: First line treatment of Philadelphia-chromosome positive ALL with dasatinib. Burger:Cellgene: Consultancy; Pharmacyclics: Consultancy, Research Funding; Genzyme: Consultancy; Calistoga: Research Funding; Noxxon: Consultancy, Research Funding. Cortes:Novartis: Consultancy; Novartis: Research Funding; BMS: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding. Kantarjian:Novartis: Consultancy; Novartis: Research Funding; Pfizer: Research Funding; BMS: Research Funding.

Fine structure of translocation breakpoints within the major breakpoint region in BCR-ABL1-positive leukemias

The chromosomal translocation t(9;22)(q34;q22), with expression of the BCR-ABL1 fusion gene is the cytogenetic and molecular hallmark of chronic myeloid leukemia (CML) and a subset of acute lymphoblastic leukemia (ALL). Basically two types of BCR-ABL1 chimeric mRNA transcripts have been observed: (1) e13a2/e14a2 transcripts in CML and ALL, resulting from chromosomal breaks in the major breakpoint cluster region (M-bcr) of the BCR gene and (2) e1a2 transcripts in ALL resulting from breaks in the minor breakpoint cluster region (m-bcr) of the BCR gene. To gain a better understanding of this molecular alteration, we developed a long-distance inverse PCR (LDI PCR) method for M-bcr breakpoint identification in BCR-ABL1-positive cases and were thus able to identify the chromosomal breakpoints within the M-bcr in 62 BCR-ABL1-positive samples. The corresponding reciprocal breakpoints were identified and molecularly characterized in 45 of these cases. In 2 samples, the breaks were located 5′ to the ABL1 locus and in one case, the der(9) break was identified on 9q34.13 several hundred kB 3′ telomeric to ABL1. The analysis of breaks revealed no significant clustering and no association with repetitive elements (Alu, L1, L2) or recombination signal sequence sites. The established LDI PCR permits fast, relatively easy and unbiased identification of breakpoints in the M-bcr region of BCR and also enables the molecular analysis of more complex translocations with breakpoints outside the ABL1 gene locus or other BCR fusion genes.

CML with e6a2 BCR-ABL1 transcript: an aggressive entity?

Dear Editor, The BCR-ABL1 fusion gene is characteristic of CML. Four different breakpoint cluster regions (bcr) have been described in BCR. The most common BCR breakpoints, in 95% of CML cases, occur in M-BCR, i.e., introns 13 or 14, resulting in an e13a2 (b2a2) or an e14a2 (b3a2) BCR-ABL1 transcript. “Atypical” transcripts, e8a2, e19a2, e13a3, e14a3, e1a3, and e6a2 BCR-ABL1, account for less than 1% of CML cases [1, 2]. Only 11 CML patients have been reported with the e6a2 variant. A male predominance and a worse outcome have been suggested for this variant [3]. Here, we describe an additional CML case with an e6a2 fusion transcript. This 57-year-old male patient presented with hepatosplenomegaly, weight loss, cough, and fever. Laboratory examination showed a hemoglobin level of 12.1 g/dL, a white blood cell count of 43.5×10*9/L with 5% myeloblasts and 10% eosinophils, a platelet count of 129×10*9/L, and an LDH of 3,363 U/L (normal value, 313–618 U/L). An abdominal CT scan confirmed the hepatomegaly (craniocaudal diameter of 24 cm) and splenomegaly (bipolar diameter of 20 cm). Sokal score was 2.11, and Hasford score, 2,211.6 (both high risk). Bone marrow aspirate showed a hypocellular marrow with 10% myeloblasts and marked eosinophilia. On trephine biopsy, a myeloproliferative disease with striking fibrosis was seen. Taqman RQ-PCR was negative for e13a2/e14a2 transcripts and weakly positive for the e1a2 transcript in peripheral blood. A subsequent cytogenetic analysis showed the t(9;22)(q34;q11) translocation in all metaphases and trisomy of chromosome 8 in 6 out of 10 metaphases, indicating clonal evolution. Fluorescent in situ hybridization (FISH), using the Vysis LSI BCR/ABL1 extra signal dual color translocation probe, revealed, in addition to the expected fusion signal on der(22), a small fusion on der(9), further suggesting the presence of a variant breakpoint, located centromeric to M-BCR (Fig. 1). Qualitative reverse transcription polymerase chain reaction (RT-PCR), using forward primers in BCR exon 1 and reverse primers in ABL1 exon 2, confirmed the presence of a variant transcript of 502 bp. This was identified as e6a2 after Sanger sequencing. Based on these findings, a diagnosis of CML in an accelerated phase was made, and the patient was started on imatinib 600 mg daily. Three months later, while still on imatinib, he was readmitted with CML in blast crisis. He had cutaneous chloromas, severe abdominal pain, a productive cough, and life-threatening dyspnea, necessitating intubation and mechanical ventilation. Peripheral leukocyte count was 465× 10*9/L with 29% myeloblasts; he had severe anemia (Hb, 6.5 g/L), thrombocytopenia (75×10*9/L), and an LDH of 10,612 U/L. Chest CT scan revealed multiple pathologically enlarged mediastinal and hilar lymph nodes. Fundoscopic examination showed features of hyperviscosity. Peripheral blood karyotype and FISH now showed an extra derivative der(22)t(9;22) in ~50% of cells. Following leukapheresis and PV is a senior clinical investigator of the Fund for Scientific ResearchFlanders (Belgium) (FWO Vlaanderen). K. A. Beel : J. Maertens Department of Hematology, Internal Medicine, University Hospital Leuven, Leuven, Belgium

Additional chromosome abnormalities in chronic myeloid leukemia

The Philadelphia (Ph) chromosome and/or Breakpoint cluster region-Abelson leukemia virus oncogene transcript are unique markers for chronic myeloid leukemia (CML). However, CML demonstrates heterogeneous presentations and outcomes. We analyzed the cytogenetic and molecular results of CML patients to evaluate their correlation with clinical presentations and outcome. A total of 84 newly diagnosed CML patients were enrolled in the study. Patients were treated according to disease status. Bone marrow samples were obtained to perform cytogenetic and molecular studies. Clinical presentations, treatment courses, and survival were reviewed retrospectively. Among 84 patients, 72 had chronic phase and 12 had accelerated phase CML. Cytogenetic study showed 69 (82.1%) with the classic Ph chromosome, 6 (7.2%) with a variant Ph chromosome, and 9 (10.7%) with additional chromosome abnormalities. Fifty-four (64.3%) cases harbored b3a2 transcripts, 29 (34.5%) had b2a2 transcript, and 1 had e19a2 transcript. There was no difference in clinical presentations between different cytogenetic and molecular groups; however, additional chromosome abnormalities were significantly associated with the accelerated phase. Imatinib therapy was an effective treatment, as measured by cytogenetic response, when administered as first- and second-line therapy in chronic phase patients. Survival analysis showed that old age, additional chromosome abnormalities, high Sokal score, and no cytogenetic response in second-line therapy had a significant poor impact ( p < 0.05). In conclusion, we presented the cytogenetic and molecular pattern of CML patients and demonstrated that the additional chromosome abnormality was associated with poor outcome.

Clinical Phenotype and Response to Imatinib of Chronic Myelogenous Leukemia Patients Harbouring Atypical BCR-ABL Transcripts. A Retrospective Analysis From the French Group of CML (Fi-LMC) and the French Group of Molecular Biologists for Hematological Malignancies (GBMHM).

AbstractAbstract 3378Chronic Myelogenous Leukemia (CML) originates in the Philadelphia chromosome, a reciprocal translocation creating the fusion oncogene BCR-ABL. In 1–2% of CML cases, breakpoints fall outside the M-BCR gene on chromosome 22, leading to the synthesis of a variety of atypical BCR-ABL transcripts [shortened: e1a2 (m-BCR), e6a2, e8a2, b2a3 (e13a3), b3a3 (e14a3), or elongated transcripts: e19a2 (m-BCR)] and to the synthesis of different molecular weight BCR-ABL proteins that might have different tyrosine kinase activities. Thus, clinical phenotypes and BCR-ABL inhibition by tyrosine kinase inhibitors might be different and lead to different prognostic features. We retrospectively analysed at the national level, the clinical characteristics and the responses to imatinib (IM) of 63 patients with CML harbouring atypical BCR-ABL transcripts: 22 e1a2 [Group 1 (G1)], 20 e19a2 [Group 2 (G2)], 5 e8a2 [Group 3 (G3)], 4 e6a2 [Group 4 (G4)], 5 b2a3 [Group 5 (G5)], and 3 b3a3 [Group 6 (G6)] BCR-ABL transcripts. The general characteristics of the patients and their best response to IM are depicted in Table 1:Surprisingly, e1a2 and e19a2 transcripts seem significantly more frequent in females than in males conversely to typical BCR-ABL transcripts (p=0.01) and occurring more often in the elderly (p=0.05). The majority of the patients presented with typical cytological CML features, however, a significant monocytosis was observed in e1a2 and e8a2 atypical transcripts (p=0.0002). The median time on IM and the interval between diagnosis and IM were not statistically different between the 6 groups. Overall, there was no significant difference in the (hematologic, cytogenetic, molecular) responses to IM, but e1a2 transcripts seem less sensitive to this agent. The overall survival since diagnosis or since IM initiation was not different between atypical transcripts (p=0.55 and p=0.73 respectively), however, the progression-free survival (PFS) since diagnosis with e1a2 transcripts was significantly worse than for all other atypical transcripts (p=0.02) as shown in Figure 1: [Display omitted] The PFS since IM initiation was somewhat worse for e1a2 transcripts, but close to significance (p=0.09), but the follow-up is not very long yet. Fifteen patients among 63 had second generation TKIs (TKI2), 7 in group 1, 3 in group 2, 1 in groups 3, 4, 5, and 2 in group 6. Only one patient (b3a3 transcript) developed a MBC being on IM. Two patients developed a T315I BCR-ABL mutation (1 e1a2, and 1 e6a2). Two patients got allo-transplanted (1 e1a2 alive and well at last follow-up, 1 e19 a2 died from GVHD).In conclusion, atypical BCR-ABL transcripts induce a particular molecular and subsequent clinical phenotypes, particularly e1a2 transcripts showing in this study poor prognosis features. The response of atypical BCR-ABL transcripts to IM might vary from that what it is for classical M-BCR transcripts, but a longer follow-up is needed. Disclosures:No relevant conflicts of interest to declare.

Dynamics of Minimal Residual Leukemia After Combinations of the HyperCVAD Regimen with Imatinib or Dasatinib In Patients with Philadelphia-Chromosome Positive Acute Lymphoblastic Leukemia

AbstractAbstract 2127 Background:The addition of tyrosine kinase inhibitors imatinib and dasatinib to combination chemotherapy regimens has improved the outcome of patients (pts) with Philadelphia-chromosme-positive (Ph+) acute lymphoblastic leukemia (ALL). We examined whether the dynamics of minimal residual disease (MRD) measured by multiparameter flow cytometry (MFC) or reverse transcription quantitative polymerase chain reaction (RQ-PCR) was different among pts treated with the combination of hyperCVAD with imatinib or dasatinib. Materials and Methods:From April 2001 to September 2006, 54 pts with newly diagnosed Ph+ ALL were treated with the combination of hyperCVAD and imatinib; from October 2006 to July 2009, 42 pts were treated with the hyperCVAD and dasatinib regimen. The median ages for the two groups were 50 and 51 years [ranges, (27 - 84) and (21 - 78)]. Fifty one (94%) and 40 (95%) achieved complete remission (CR) on the two regimen and were followed by serial bone marrow assessments for MRD. MFC was performed using 4 or 6 color combinations of antibodies to lymphoblast and myeloid antigens (e.g., CD10, CD13, CD15, CD19, CD20, CD22, CD25, CD33, CD34, CD38, CD58, CD66c, and CD81), with a sensitivity of 0.01%. RQ-PCR for BCR-ABL was performed using TaqMan primer/probes for the e1a2, e13a2 (b2a2), and e14a2 (b3a2) BCR-ABL transcripts in a single tube with normalization to total ABL transcripts. Results:The dynamics of the MRD by RQ-PCR and MFC over the initial 18 month period after achieving CR are shown in the attached figures 1 and 2, respectively. The decline in the median value of BCR-ABL/ABL was more rapid with dasatinib, with more patients reaching the level of <0.01 at 3 and 6 months (p <0.05 and p=0.05, respectively). Similarly, a higher proportion of patients receiving dasatinib were negative by MFC at 3 and 6 months after therapy; 82% and 92% were negative at 3 months, and 94% and 100% negative at 6 months for imatinib and dasatinib, respectively. Conclusions:Dasatinib containing regimen produces a deeper and more rapid decline in the BCR-ABL and MRD by MFC in the first 6 months of treatment. Whether this would translate to an improved long-term disease-free and overall survival remains to be investigated. [Display omitted] [Display omitted] Disclosures:Ravandi: Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Speakers Bureau. Off Label Use: frontline use of dasatinib in acute lymphoblastic leukemia. Thomas: Novartis: Honoraria; Bristol-Meyer-Squibb: Honoraria; Pfizer:; Amgen: Research Funding. Cortes: Bristol Myers Squibb: Research Funding; Novartis: Research Funding. Kantarjian: Novartis Pharmaceuticals: Research Funding; Bristol Myers Squibb: Research Funding.

Results of An International Study of Quantitative BCR/ABL Assays Using Defined RNA Samples

AbstractAbstract 2742Molecular monitoring of BCR/ABL transcripts by real time quantitative reverse transcription PCR (qRT-PCR) is an essential technique for clinical management of patients with BCR/ABL-positive CML and ALL. Though quantitative BCR/ABL assays are performed in hundreds of laboratories worldwide, results among these laboratories cannot be reliably compared due to heterogeneity in test methods, data analysis, reporting, and lack of quantitative standards. Recent efforts towards standardization have been limited in scope. Aliquots of RNA were sent to clinical test centers worldwide in order to evaluate methods and reporting for e1a2, b2a2, and b3a2 transcript levels using their own qRT-PCR assays. Total RNA was isolated from tissue culture cells that expressed each of the different BCR/ABL transcripts. Serial log dilutions were prepared, ranging from 100 to 10−5, in RNA isolated from HL60 cells. Laboratories performed 5 independent qRT-PCR reactions for each sample type at each dilution. In addition, 15 qRT-PCR reactions of the 10−3 b3a2 RNA dilution were run to assess reproducibility within and between laboratories. Participants were asked to run the samples following their standard protocols and to report cycle threshold (Ct), quantitative values for BCR/ABL and housekeeping genes, and ratios of BCR/ABL to housekeeping genes for each sample RNA. Thirty-seven (n=37) participants have submitted qRT-PCR results for analysis (36, 37, and 34 labs generated data for b2a2, b3a2, and e1a2, respectively). The limit of detection for this study was defined as the lowest dilution that a Ct value could be detected for all 5 replicates. For b2a2, 15, 16, 4, and 1 lab(s) showed a limit of detection at the 10−5, 10−4, 10−3, and 10−2 dilutions, respectively. For b3a2, 20, 13, and 4 labs showed a limit of detection at the 10−5, 10−4, and 10−3 dilutions, respectively. For e1a2, 10, 21, 2, and 1 lab(s) showed a limit of detection at the 10−5, 10-4, 10-3, and 10-2 dilutions, respectively. Log %BCR/ABL ratio values provided a method for comparing results between the different laboratories for each BCR/ABL dilution series. Linear regression analysis revealed concordance among the majority of participant data over the 10-1 to 10-4 dilutions. The overall slope values showed comparable results among the majority of b2a2 (mean=0.939; median=0.9627; range (0.399 – 1.1872)), b3a2 (mean=0.925; median=0.922; range (0.625 – 1.140)), and e1a2 (mean=0.897; median=0.909; range (0.5174 – 1.138)) laboratory results (Fig. 1–3)). Thirty-four (n=34) out of the 37 laboratories reported Ct values for all 15 replicates and only those with a complete data set were included in the inter-lab calculations. Eleven laboratories either did not report their copy number data or used other reporting units such as nanograms or cell numbers; therefore, only 26 laboratories were included in the overall analysis of copy numbers. The median copy number was 348.4, with a range from 15.6 to 547,000 copies (approximately a 4.5 log difference); the median intra-lab %CV was 19.2% with a range from 4.2% to 82.6%. While our international performance evaluation using serially diluted RNA samples has reinforced the fact that heterogeneity exists among clinical laboratories, it has also demonstrated that performance within a laboratory is overall very consistent. Accordingly, the availability of defined BCR/ABL RNAs may facilitate the validation of all phases of quantitative BCR/ABL analysis and may be extremely useful as a tool for monitoring assay performance. Ongoing analyses of these materials, along with the development of additional control materials, may solidify consensus around their application in routine laboratory testing and possible integration in worldwide efforts to standardize quantitative BCR/ABL testing. [Display omitted] [Display omitted] [Display omitted] Disclosure:Sher:Invivoscribe Technologies Inc: Employment. Miller:Invivoscribe Technologies Inc: Employment. Huang:Invivoscribe Technologies Inc: Employment. Shaw:Invivoscribe Technologies Inc: Employment.

Development and Evaluation of a Quantitative Multiplex Assay for the Harmonization of BCR-ABL1 Measurement on the International Reporting Scale.

AbstractAbstract 3416 Introduction:Sensitive and standardized methods are required for the monitoring of BCR-ABL1 fusion transcripts in chronic myeloid leukemia (CML) patients treated with tyrosine kinase inhibitors (TKI). Large multi-site studies have led to establishment of the International Scale (IS) for consistent interpretation of individual patient response and comparison between patients and laboratories. On this scale, the baseline of BCR-ABL1 (b2a2 or b3a2) to control gene ratio is defined as 100% IS with a 3-log reduction indicative of major molecular response (MMR; 0.1% IS or lower). Growing evidence indicates that the e1a2 variant is associated with inferior outcome and higher risk of relapse. It is therefore also important to closely monitor e1a2 expression levels during the course of TKI treatment. The objective of this study was to evaluate the use of a single multiplex assay (BCR/ABL1 Quant™, for Research Use Only) for the quantitative and qualitative detection of e1a2, b2a2, and b3a2 fusion transcripts and to compare its performance against existing laboratory-developed tests (LDT) and the IS. Methods:Total RNA was reverse transcribed and then amplified by multiplex, real-time quantitative PCR on the ABI 7500 or ABI 7500 Fast Dx system using target-specific primers for e1a2, b2a2, b3a2 and ABL1 as an endogenous control. Four-point standard curves were generated in each run using the included multiplex Armored RNA Quant® Calibrators. Fusion transcript identity was determined by capillary electrophoresis. Archived total RNA samples from two independent sites were purified from white blood cells enriched from bone marrow or whole blood specimens with the respective laboratory validated extraction methods and tested with each site's LDT. Establishment and validation of a conversion factor (CF) was performed as described in Branford et al. (Blood, 2008). Results:Analytical performance assessed using cell line dilutions and synthetic in vitro transcript RNA showed a limit of quantitation of 10–15 copies of BCR-ABL1 per PCR reaction and a limit of detection of 2–5 copies. The linear dynamic range of the assay reached the equivalent of at least one translocation-positive cell in 100,000 normal cells. Analysis of 115 residual clinical specimens showed an excellent correlation between each LDT and the BCR/ABL1 Quant assay for the quantitative measurement of BCR-ABL1 to ABL1 ratio (paired correlation of 0.97 for both LDTs). There was 100% agreement for the qualitative detection of major and minor fusion transcripts. The assay was compared to an IS reference laboratory (IMVS, Australia) by testing 20 specimens covering ratios of about 0.005 to 10%. The mean bias between methods corresponded to a CF of 1.04 (BCR/ABL1 Quant measured % ratio × 1.04 = IS % ratio). Fourteen months later the process was repeated with an independent lot of reagents. The difference between methods after conversion was again minimal (bias of -0.019) validating that the % ratio values obtained with the BCR/ABL1 Quant (RUO) assay are comparable to the IS. Overall, the 95% limit of agreement between methods was less than plus or minus 5 fold and 87.5% of the specimens were correctly classified in terms of MMR status (above or below 0.1% IS). Conclusions:The BCR/ABL1 Quant (RUO) assay has the advantage of both quantifying in a single reaction and distinguishing fusion transcripts e1a2, b2a2, and b3a2. Our results indicate that this research assay also has the performance required for reporting on the IS. Validation of this multiplex assay format might therefore improve laboratories' workflow and facilitate harmonization of BCR-ABL1 quantitative measurement, increasing adherence to the current recommendations for reporting on the IS. Disclosures:No relevant conflicts of interest to declare.

A unique BCR–ABL1 transcript with the insertion of intronic sequence from BCR and ABL1 genes in a patient with Philadelphia-positive chronic myeloid leukemia: a case study

The BCR–ABL1 fusion gene results from a reciprocal translocation rearrangement, t(9;22)(q34;q11.2), and is a hallmark of chronic myeloid leukemia (CML). The breakpoint on chromosome 9 is mostly 5′ to ABL1 exon 2, whereas on chromosome 22, the breakpoint can occur in various regions involving the major breakpoint cluster region (M-bcr) in CML and the minor breakpoint cluster region (m-bcr) in acute lymphoblastic leukemia. Described here is a rare case of Philadelphia-positive CML with intronic splice sites. This atypical BCR–ABL1 transcript was detected along with a classic e13a2 transcript, using reverse transcription polymerase chain reaction (RT-PCR). Nucleotide sequence analysis revealed a joining of BCR intron 13 with ABL1 intron 1a. Both transcripts were detected when the patient was on hydroxyurea treatment; with imatinib mesylate therapy, the atypical transcript disappeared. To our knowledge, this is the first report of BCR–ABL1 transcript with breakpoint occurring within both BCR and ABL1 introns and fusion of intronic sequences from both BCR and ABL1 genes.

Clonal evolution with double Ph followed by tetraploidy in imatinib-treated chronic myeloid leukemia with e19a2 transcript in transformation

Chronic myeloid leukemia (CML) with the e19a2 transcript coding for p230 is typically associated with a benign clinical course unless accompanied at presentation with additional chromosomal abnormalities. We report here a case of CML with e19a2 who did not show additional chromosomal abnormalities at diagnosis, but progressed to the fatal advanced stage in approximately 2 years. The patient was initially treated with imatinib, which, however, could be administered only intermittently at reduced doses because of recurrent thrombocytopenia and fluid retention. Nine months after starting imatinib, fluorescence in situ hybridization (FISH) with the BCR/ABL-ES fusion probe revealed 96% and 3% of bone marrow cells with one and two BCR/ABL1 fusion signals, respectively. Two years after starting therapy, leukocytosis recurred and the bone marrow contained 8.2% large and bizarre myeloblasts. Cytogenetic analysis revealed double Ph clones as well as tetraploid cells with four to five Ph chromosomes. FISH analysis confirmed the presence of cells with two to five BCR/ABL1 fusion signals. The patient died of disease progression in 2 months. No point mutation was detected in the region coding for the BCR/ABL tyrosine kinase domain by sequence analysis. It is speculated that the amplification of the BCR/ABL1 fusion gene by duplication of Ph and tetraploidy led to the progression of CML with the e19a2 transcript.