Major Breakpoint Cluster Region Research Articles

Update in Childhood Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) in childhood represents only 3% of newly diagnosed pediatric leukemia. The diagnostic hallmark of CML is the Philadelphia (Ph) chromosome, which derives from the fusion of the ABL1-oncogene located on chromosome 9 to the breakpoint cluster region (BCR) gene on chromosome 22, resulting in a constitutively dysregulated ABL1 tyrosine kinase, either as 210 kDa or 190 kDa. Depending on the localization of the breakpoint site within the major BCR region, the majority of CML patients exhibit transcripts with either the b3a2 or b2a2 junction, or both. Several questions are still open with regard to childhood CML, especially concerning the biologic and clinical features of the disease, and the treatment of choice for pediatric patients with CML. Moreover, over the last few years, several tyrosine kinase inhibitors (TKIs) have been available for children and adolescents with CML, and current clinical practice investigates what the effective and optimal doses of TKIs are in these two categories of patients. The use of TKIs in pediatric patients with CML has also opened up questions on the following items: (1) the long-term effects of these drugs on children; (2) the management of pediatric CML forms resistant or intolerant to TKIs; (3) the monitoring of disease outcomes during treatment; (4) and the right timing to discontinue therapy. Despite the efficacy of TKIs also in the pediatric population, the potential late adverse effects, and the drug resistance, leave open the possibility of allogeneic hematopoietic stem cell transplantation as a treatment option in pediatric CML. Published data and personal experiences regarding these issues will be analyzed and discussed.

Targeted Next Generation Sequencing Reveals a Third Breakpoint Cluster Region and New Partner Genes in the KMT2A Recombinome

Chromosomal rearrangements of the KMT2A gene are associated with acute leukemias and myelodysplastic syndromes. The large number of known KMT2A fusions (>100) renders a precise diagnosis a demanding task. More than 50% of all KMT2A partner genes have been analyzed at the DCAL, including the novel partner genes BCAS4, FAM13A, RANBP3, and STK4. Even though all KMT2A rearrangements are associated with high-risk acute leukemia, the outcome (poor or very poor) is influenced by the partner gene. So far, we have analyzed more than 3,200 patients positive for a KMT2A rearrangement. The breakpoints of these cases are located mainly in the major breakpoint cluster region (bcr1) and to a small extent in the recently described minor bcr (bcr2). A small number of breakpoints were also found outside of these two bcrs.Most of these patients were analyzed by long distance inverse (LDI)- or multiplex-PCR which only cover bcr1. More recently, we used targeted KMT2A-NGS with whole gene coverage in over 450 patients, which was initially applied selectively in patients negative by LDI- and multiplex-PCR and then used more widely.Within the KMT2A-NGS group, 410 patients had bcr1 breakpoints mainly between the KMT2A exons 7 and 13, while 46 patients bcr2 breakpoints mainly between exons 20 and 24. Of note, five patients had their breakpoint outside of these two bcrs: three of them within intron 2 and no functional KMT2A rearrangement; the other two within intron 35 and intron 36, fusing almost the whole KMT2A gene in frame to the respective partner genes ARHGEF12 and MLLT4. These two breakpoints may define a third and rare bcr (bcr3), although further cases are needed to support this hypothesis. Interestingly, 70 patients displayed a 3‘-KMT2A deletion, indicating that the number of terminal deletions is higher than described previously. Two patients had a 5‘-KMT2A deletion. All deletions started or ended in bcr1 and bcr2.We also observed a striking difference in the distribution of partner genes between bcr1 and bcr2. The most frequent translocation partners fused to bcr1 sites are transcription factors, while the partner genes linked to bcr2 sites generally code for cytosolic proteins. In bcr1, the 4 most frequent partner genes AFF1, MLLT3, MLLT1, and MLLT10, found in 80% of cases, all code for transcription factors that are part of the super elongation complex (SEC). These fusions therefore all lead to disruption of the hematopoietic lineage commitment.In contrast in bcr2, 3 partner genes USP2, MLLT4, and USP8 account for 85% of the cases. USP2 and USP8 are ubiquitin specific peptidases involved in cell signaling and exclusively fused to bcr2 in KMT2A. While MLLT4 is found as a partner in bcr1, bcr2 and bcr3 fusions; unlike other recurrent KMT2A partners linked to bcr1, it is not a transcription factor and it exerts oncogenic potential via dimerization like other cytosolic partners. We hypothesize that the oncogenic properties of USP2 and USP8 are dependent on dimerization like MLLT4 and that the most frequent fusions involving at different bcrs favor different oncogenic mechanisms: bcr1 transactivation and bcr2 dimerization. Further studies are needed to explain why USP2 and USP8 are exclusively associated with bcr2, and why the most frequent partner genes AFF1 and MLLT3 of the bcr1 are less frequent in bcr2.In conclusion, targeted NGS combined with bioinformatic analysis has expanded our knowledge of the KMT2A recombinome to include more fusion partners and has generated new hypotheses for future research on oncogenic mechanisms. DisclosuresNo relevant conflicts of interest to declare.

Importance of BCR/ABL1 Transcript Type in CML

<h3>Introduction</h3> The <i>BCR/ABL1</i> fusion exists in several different isoforms, depending on the precise position of the t(9;22)(q34;q11) genomic breakpoints in each case. In chronic myeloid leukemia (CML), the two most common isoforms are known as e13a2 and e14a2, which are shorthand for mRNA fusions of <i>BCR</i> exon 13 or <i>BCR</i> exon 14, respectively, to <i>ABL1</i> exon 2. Historically, these two fusions were referred to as b2a2 and b3a2, with b2 (<i>BCR</i> exon 13) and b3 (<i>BCR</i> exon 14) corresponding to the second and third exons within the classically-defined 5.8-kb major breakpoint cluster region (M-BCR) within <i>BCR.</i>¹ Both e13a2 and e14a2 encode a 210-kDa BCR/ABL1 protein (p210), but e14a2 encodes a chimeric protein that is 25 amino acids larger than e13a2, corresponding to the 75 bp of <i>BCR</i> exon 14. At the genomic DNA level, the chromosome 22 breakpoint typically occurs in <i>BCR</i> intron 13 for e13a2 and <i>BCR</i> intron 14 for e14a2. Collectively, e13a2 and e14a2 <i>BCR/ABL1</i> account for 98% of CML cases, with the majority of these expressing e14a2. Approximately 10% of cases express both isoforms.^1,2 A large international survey of CML cases found small but significant differences in the relative prevalence of the two fusions in relation to age and gender, with e13a2 being more frequent in males and less common in the elderly.²

The role of reciprocal fusions in MLL-r acute leukemia: studying the chromosomal translocation t(6;11)

Leukemia patients bearing t(6;11)(q27;q23) translocations can be divided in two subgroups: those with breakpoints in the major breakpoint cluster region of MLL (introns 9–10; associated mainly with AML M1/4/5), and others with breakpoints in the minor breakpoint cluster region (introns 21–23), associated with T-ALL. We cloned all four of the resulting fusion genes (MLL-AF6, AF6-MLL, exMLL-AF6, AF6-shMLL) and subsequently transfected them to generate stable cell culture models. Their molecular function was tested by inducing gene expression for 48 h in a Doxycycline-dependent fashion. Here, we present our results upon differential gene expression (DGE) that were obtained by the “Massive Analyses of cDNA Ends” (MACE-Seq) technology, an established 3′-end based RNA-Seq method. Our results indicate that the PHD/BD domain, present in the AF6-MLL and the exMLL-AF6 fusion protein, is responsible for chromatin activation in a genome-wide fashion. This led to strong deregulation of transcriptional processes involving protein-coding genes, pseudogenes, non-annotated genes, and RNA genes, e.g., LincRNAs and microRNAs, respectively. While cooperation between the MLL-AF6 and AF6-MLL fusion proteins appears to be required for the above-mentioned effects, exMLL-AF6 is able to cause similar effects on its own. The exMLL-AF6/AF6-shMLL co-expressing cell line displayed the induction of a myeloid-specific and a T-cell specific gene signature, which may explain the T-ALL disease phenotype observed in patients with such breakpoints. This again demonstrated that MLL fusion proteins are instructive and allow to study their pathomolecular mechanisms.

Biology of t(6;11) Fusion Proteins and Their Role in MLL-Rearranged Acute Leukemia Lineage Determination

Chromosomal translocations are genetic rearrangements where a chromosomal segment is transferred to a non-homologous chromosome which give rise to novel chimeras. Chromosomal rearrangements play a significant role in the development of acute leukemias (acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML)). Chromosomal translocation events occurring at 11q23 involving the KMT2A or Mixed-Lineage Leukemia (MLL) gene (n=102) can be diagnosed in about 5-10% of all acute leukemia patients (Marschalek Ann Lab Med 2016), especially prevalent in infant acute leukemias (up to 70% of cases). Different chromosomal translocation partner genes (such as AF4, AF6, AF9orENL and ELL) account for the majority of leukemia cases and have their genomic breakpoints within a major breakpoint cluster region (BCR intron 9-11; Meyer et. al. Leukemia 2018). Some rearrangements are specifically associated with particular disease phenotype e.g. the majority of ALL patients (~ 90%) are mainly caused by the following gene fusions, MLL-AF4, MLL-AF9, MLL-ENL. We are interested in a rare but yet drastic chromosomal translocation t(6;11)(q27;q23) which fuses KMT2A/MLL to Afadin (AFDN/AF6) gene. This chromosomal rearrangement has a very poor prognosis (survival-rate is ~10%) and is predominantly diagnosed in patients with high-risk AML. In this project, we investigate the molecular consequences of two different MLL-AF6 fusions and their corresponding reciprocal AF6-MLL fusions. MLL-AF6 fusions are mainly occurring within MLL intron 9 to 11 and are associated with an AML disease phenotype, while the same fusion occurring within the minor breakpoints region in MLL intron 21 until exon (ex) 24 are mainly diagnosed with T-ALL (T-cell acute lymphoblastic leukemia) disease phenotype. The molecular mechanism that determines the resulting disease phenotype is yet unknown. Therefore, we cloned all of these t(6;11) fusion proteins in order to investigate the functional consequences of the two different breakpoints (MLLex1-9::AF6ex2-30, AF6ex1::MLLex10-37; MLLex1-21::AF6ex2-30, AF6ex1::MLLex22-37). All 4 fusion genes were introduced into our inducible Sleeping Beauty system (Ivics et. al. Mobile DNA 2010; Kowarz et. al. Biotechnol J. 2015) and stably transfected reporter cell lines. Basically, these 4 fusion proteins differ only in the presence or absence of their Plant homeodomain 1-3/Bromodomain (PHD1-3/BD) domain (see Figure 1). The PHD domain regulates the epigenetic and transcriptional regulatory functions of wildtype MLL. Subsequently, we analyzed gene expression differences by the MACE-Seq (Massive Analyses of cDNA Ends). MACE data revealed fundamental differences in gene expression profiles when analyzing the two different sets of t(6;11) fusion genes. The resulting profiles have similarities to either AML or T-ALL and might give a rational explanation for the different lineages in these t(6;11) patients. Altogether, these results notably indicate that our study will provide a novel insight into this type of high-risk leukemia and subsequently will be useful for developing of novel and appropriate therapeutic strategies against acute leukemia. Disclosures No relevant conflicts of interest to declare.

277P - Characteristics of BCR-ABL rearrangement variants in Pakistani patients with chronic myeloid leukemia and acute lymphocytic leukemia

BackgroundA reciprocal translocation between the long arms of chromosome 9 and 22 [t (9;22) (q 34; q 1.1)] results in the formation of a fusion hybrid gene also called Philadelphia chromosome encodes proteins with a constitutively activated tyrosine kinase activity [1,2]. These proteins being leukemogenic result in Chronic Myeloid leukemia (CML) and Acute lymphoid leukemia (ALL). Breakage in the long arms of chromosome 9 (ABL1 gene) can occur anywhere within a > 300-kb segment at the 5’end of the gene [1]. Site of breakage in chromosome 22 (BCR gene) can occur in different regions within a 5.8kb region known as the major breakpoint cluster region (M-bcr), spanning 5 exons previously known as b1 to b5 but now as exon 12 to 16 [1]. In CML, the breakpoint in the BCR gene mostly (95%) falls within the major break point cluster region with b2a2 at 40 % and b3a2 at 55% [1]. MethodsThe number of reported patients was 686 over a period of 2 years and 10 months (from October 2016 to July 2019). Males comprised 58% of the total while females were 42%. The male to female ratio was 1.4:1. The tested patients were divided into 3 major groups according to their age i.e. x < 20, 20>x<50 and x > 50.The mean age was 40.2 years. ResultsOut of the tested 686 patients, 303 were reported to have the BCRABL1 transcript off which 54.2% were males and 45.9% were females. The ratio of patients diagnosed with Chronic Myeloid Leukemia to Acute Lymphoid leukemia was 9.8:1. 9% of the patients had the mutation e1a2 while 32% had b2a2 and 59% were reported with b3a2. ConclusionsWe conclude that the distribution of BCR/ABL transcript types in patients with CML and ALL differs from most, but not all other populations studied. Currently there are no data from studies of CML and ALL in analyzing a comparable number of cases. The underlying mechanism and causes for the sex-depending distribution of the BCR/ABL transcript types remains open to speculation. Legal entity responsible for the studyZeeshan Ansar Ahmed. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Chronic myeloid leukemia: the concepts of resistance and persistence and the relationship with the BCR-ABL1 transcript type.

Chronic myeloid leukemia is driven by a hybrid gene, BCR-ABL1, that codes for a leukemogenic tyrosine kinase (TK) protein of 210 KDa (p210BCR-ABL1). Resistance to TK inhibitor (TKI) therapy occurs in relatively few patients, no more than 10%, while persistence of minimal residual disease during TKI therapy occurs in the great majority of patients. Resistance is a cause of death, persistence is compatible with a fairly normal length and quality of life, but may require lifelong treatment. The causes of resistance are heterogeneous, including the development of other genomic abnormalities or the altered expression of other genes, requiring different treatments. The causes of persistence may not be the same as those of resistance. We hypothesize that the variability in breakpoint position within the Major-breakpoint cluster region (M-bcr), resulting in two different messenger RNAs that may or may not include exon 14 of BCR (e13a2 and e14a2, respectively), and, as a consequence, in two p210BCR-ABL1 proteins that differ by 25 amino acids, may be a cause of persistence. The hypothesis is based on a critical review of the relationships between the BCR-ABL1 transcript types, the response to TKIs, the outcome of treatment, and the immune response, suggesting that the e14a2 transcript is associated with more and deeper molecular responses, hence with a higher probability of achieving treatment-free remission (TFR). Investigating this putative cause of persistence may help bringing more patients into stable TFR.

How to detect the rare BCR-ABL (e14a3) transcript: A case report and literature review.

The Philadelphia (Ph; BCR-ABL) chromosome originates from a translocation event between chromosomes 9 and 22, and results in the BCR-ABL fusion gene. In chronic myelogenous leukemia (CML), the BCR-ABL gene is mainly coded for by a major breakpoint cluster region (M-bcr, e13a2 and e14a2). However, in some patients, BCR-ABL genes are encoded by a minor (m)-bcr, e1a2, and a micro (µ)-bcr region, e19a2. These transcripts revealed a different clinical course. The present study described a CML patient whose cytogenetics and FISH analyses of bone marrow revealed a karyotype of 46, XY t(9,22) (q34;q11), while the commercial kits of quantitative PCR (qPCR) failed to detect the BCR-ABL fusion gene. Further multiplex Reverse transcription-PCR (RT-PCR) and sequencing analyses identified a rare e14a3 (b3a3) fusion transcript.

Application of Real-time Quantitative PCR in Detecting Atypical BCR/ABL mRNA Transcripts in Chronic Myelocytic Leukemia

To detect atypical BCR/ABL mRNA transcript by real-time quantitative PCR in CML patients without e13a2/e14a2,e19a2 or e1a2 transcripts, and investigate its value of clinical application. Twelve cases of CML with positive for t(9;22) translocation, but negative for common major and minor breakpoint cluster regions comfirmed by chromosome karyotyping or FISH analysis, were collected from July 2012 to December 2015. These 12 cases were then detected for b2a3(e13a3), b3a3(e14a3), e6a2, e8a2 and e1a3 fusion variants by real-time quantitative PCR. Among 12 cases 4 variant transcripts were detected, including e1a3 in 1 case (8.33%), e8a2 in 2 cases (16.67%), b2a3 in 5 cases (41.67%) and b3a3 in 4 cases (33.33%), with total positivity of 100%, moreover b2a3 and b3a3 were predominant. The detecting atypical BCR/ABL mRNA transcripts by real-time quantitative PCR is suitable for the diagnosis of CML negative for P210, P190 and P230 by standard real-time PCR test, and this detection is still the standard and economic method for monitoring minimal residual disease in CML patients with variants of BCR/ABL fusion gene.

No influence of BCR-ABL1 transcript types e13a2 and e14a2 on long-term survival: results in 1494 patients with chronic myeloid leukemia treated with imatinib.

The genomic break on the major breakpoint cluster region of chromosome 22 results in two BCR-ABL1 transcripts of different sizes, e14a2 and e13a2. Favorable survival probabilities of patients with chronic myeloid leukemia (CML) in combination with too small patient samples may yet have obstructed the observation of differences in overall survival of patients according to transcript type. To overcome potential power problems, overall survival (OS) probabilities and probabilities of CML-related death were analyzed in 1494 patients randomized to first-line imatinib treatment. OS probabilities and probabilities of dying of CML were compared using the log-rank or Gray test whichever was appropriate. Both tests were stratified for the EUTOS long-term survival score. Between the groups with a single transcript, neither OS probabilities (stratified log-rank test: p=0.106) nor probabilities of CML-related death were significantly different (stratified Gray test: p=0.256). Regarding OS, the Cox hazard ratio (HR) of transcript type e13a2 (n=565) to type e14a2 (n=738) was 1.332 (95% CI 0.940-1.887). Considering probabilities of leukemia-related death, the corresponding subdistribution HR resulted in 1.284 (95% CI 0.758-2.176). Outcome did not change if patients with both transcripts (n=191) were added to the 738 with type e14a2 only. The prognostic association of transcript type and long-term survival outcome was weak and without clinical relevance. However, earlier reported differences in the rate and the depth of molecular response could be relevant for the chance of successfully discontinuing TKI treatment. The effect of transcript type on molecular relapse after discontinuation is unknown, yet.

Prevalence of BCL-2/J(H) Translocation in Healthy African Americans.

The translocation t(14;18)(q32;q21) (BCL-2/J(H)) is present in over 80% of all follicular lymphomas and is detectable in peripheral blood lymphocytes (PBL) of healthy individuals. The prevalence of this translocation has not been studied in African Americans (AAs). Given the higher incidence of follicular lymphomas in whites compared to AAs in the United States (USA), we hypothesized that the translocation prevalence in the blood of AAs would be lower. DNA was isolated from PBL from blood samples collected from participants from FL. Polymerase chain reaction was performed on the BCL-2/J(H) major (MBC) and minor breakpoint cluster (mBC) regions. Eight of the 77 (10.4%) blood samples from AA participants were positive for MBC (95% CI, 4.6-19.5%), and three (3.9%) were positive for mBC (95% CI, 0.81-10.97%) of BCL-2/J(H), with a total of 11 (14.3%) participants with positive samples (95% CI, 7.35-24.13%). In 167 white patient samples, 22 (13.2%; 95% CI, 8.44-19.26%) were positive for MBC, and five (3.0%; 95% CI, 0.98-6.85%) were positive for mBC, with a total of 25 (15%) participants with positive samples (CI, 9.93-21.30%). The prevalence of t(14;18)(q32;q21) is not significantly different among AAs and whites from the USA. The lower prevalence of follicular lymphomas in AAs compared with whites is likely a result of differences in secondary molecular alterations involved in follicular lymphoma development. This study is the first report of prevalence of t(14;18) in an AA cohort.

Novel e8a2 BCR/ABL Fusion Transcript in Case of a Myeloproliferative Neoplasm.

Main objective of this work was to confirm the occurrence of rare BCR-ABL fusion variant involving the a2 region of the ABL gene and e8 of BCR gene in a patient of Myeloproliferative neoplasm positive for t(9;22) translocation but negative for common major and minor breakpoint cluster regions. A patient with elevated white blood cell count was subjected to classical cytogenetics, FISH as well as RT-PCR testing using commercial kits as well as published primers and in house testing protocol. The translocation event in chromosome 9 and 22 could be successfully detected. BCR/ABL dual color, dual fusion probe generated a classical balanced translocation scenario within the nucleus. In RT-PCR we found an unexpectedly large amplification band around 1700bp, which is consistent with e8a2 transcript. Nine metaphases showed 46,XY,t(9;22)(q34;q11.2)[9] by cytogenetic. A rare e8a2 break point in the BCR-ABL gene in myeloproliferative neoplasm disease detected in India. It also emphasizes the utility of cytogenetic and FISH for primary diagnosis of any neoplasm in blood. Our Patient detected rare BCR-ABL fusion variant e8a2 was on imatinib 400mg since last 3months. After 3months fluorescent in situ analysis and reverse transcriptase pcr analysis showed negative results.

Abstract B48: Similar prevalence of t(14,18) in African American and Caucasian subjects

Abstract Background: It has been shown that t(14;18), a translocation involving IgH and BCL-2 genes that is present in over 80% of all cases of follicular lymphomas, is detectable in the peripheral blood of up to 52% of normal Caucasian patients from Germany and up to 16% of normal individuals from Japan (Yasukawa et al, Blood 2001). The fact that follicular lymphoma is one of the two most common lymphomas in the USA and Germany and much less common in Japan, demonstrates that this molecular alteration is perhaps an early event in the development of this malignancy which is not sufficient for its causation. There have not been any studies evaluating the prevalence of this translocation in healthy African American (AA) subjects. We speculated that the prevalence of this translocation will be lower in healthy AA subjects given the findings of lower prevalence of IgH translocations in AA patients with multiple myeloma compared with Caucasian patients, which may explain the better outcome of myeloma in AA subjects (Baker et al, Blood 2013). This effort to evaluate the prevalence of t (14; 18) in AA subjects, was part of a pilot study in which we brought an educational program on the importance of cancer research together with concomitant access to a translational research project to AA churches utilizing a mobile research unit in an attempt to overcome some of the known barriers to AA subject participation in clinical research (Colon-Otero et al, Cancer2008; Colon-Otero et al, J Cancer Educ 2012). Methods: IRB approval of the project was obtained and peripheral blood samples collected from consented subjects during educational sessions in Northeast Florida AA Churches. Caucasian subjects were identified from the Mayo Clinic Bio-bank. DNA was isolated from the buffy coat fractions and a PCR quality control assay targeting a 293bp fragment of an HLA class II gene was used to determine acceptable DNA quality and quantity. Next, a two-step semi-nested PCR was performed on the t (14;18) major and minor breakpoint cluster regions using ~500ng of DNA for each assay. A consensus JH primer was used in combination with primers complementary to BCL-2 sequences for both, the major and minor breakpoint cluster regions. PCR products were run on a 1% agarose gel. In order to confirm the positive results, positive PCR products were analyzed by direct sequence by Sanger method and subsequently assembled to the human genome using BLAST tool. Results: A total of 110 blood samples from AA subjects were analyzed for the presence of t (14;18). A total of 13 of the samples were positive for the major breakpoint site (12%) and 4 were positive for the minor breakpoint site (4%), for a total of 17 positive samples (15%). In 167 Caucasian patients samples, a total of 22 (13%) were positive for the major breakpoint site and 5 (3%) were positive for the minor breakpoint site for a total of 27 positive samples (16%). All cases were confirmed by Sanger sequencing. Conclusions: The prevalence of t(14;18) is not significantly different among African American and Caucasian subjects from the USA based on our findings. This represents the first report of the prevalence of t(14;18) in an African American population. The prevalence found in our Caucasian subjects is significantly lower than what had been described in previous population series. A plausible explanation is the high heterogeneity of approaches used across studies, significantly affecting the assay sensitivity. Additional studies with larger series may reveal minor differences in the prevalence of t(14;18) among these populations. Citation Format: Scott Van Wier, Gerardo Colon-Otero, Gregory Ahmann, Esteban Braggio, Monica Albertie, Sikander Ailawadhi, Rafael Fonseca. Similar prevalence of t(14,18) in African American and Caucasian subjects. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B48.

Effectiveness of First and Second Line Nilotinib in Chronic Myeloid Leukemia (CML) Patients with Atypical e1a2 BCR-ABL Transcripts

▪IntroductionChronic myeloid leukemia (CML) is characterized by a reciprocal translocation between chromosome 9 and 22, resulting in a BCR-ABL fusion gene. In CML, over 95% of the breakpoints involve the major breakpoint cluster region (M-bcr) of BCR introns downstream of either exon 13 or 14 and introns downstream of ABL exon 2, resulting in fusion transcripts e13a2 (b2a2) and e14a2 (b3a2). The minor breakpoint cluster region (m-bcr) in BCR gene is a less common breakpoint involved the exon 1, fused to ABL a2, resulting in e1a2 fusion transcript. Other transcripts such as e19a2, e1a3, e6a2, e13a3, and e14a3 occur less frequently.The e1a2 transcripts exist in the most of acute lymphoblastic leukemia, and CML expressing only e1a2 transcripts is rare. Although anecdotal reports of patients with the e1a2 transcripts had a good prognosis, to our knowledge, the outcome of most of patients with this transcript alone in the era of imatinib is very poor. We performed this study to investigate the frequency of e1a2BCR-ABL CML and mutations in the kinase domain of BCR-ABL after imatinib failure, and to evaluate their treatment prognosis.ObjectiveTo investigate the frequency of e1a2BCR-ABL CML and mutations in the kinase domain of BCR-ABL after imatinib failure, and to evaluate their treatment prognosis.Patients and MethodsThe records of all CML patients at our hospital from January 2004 to March 2014 were reviewed to identify patients with only e1a2BCR-ABL fusion transcripts consequent to breakpoints in minor BCR. Patients with e1a2 coexisting with e13a2 and/or e14a2 were excluded from this analysis. BCR-ABL transcripts were revealed at diagnosis by quantitative PCR followed by conventional agarose electrophoresis of PCR products. Molecular follow-up of BCR-ABL transcripts throughout treatment was performed by quantitative PCR and cytogenetic follow-up was performed by karyotype analysis following the guidelines of the European Leukemia Net. BCR-ABL kinase domain point mutations have been assessed using Sanger sequencing in patients with imatinib failure and progression.Event-free survival (EFS) was measured from the start of each therapy until progression to the BP or death from any cause during treatment. Overall survival was defined from date of CML diagnosis to date of death or last follow-up.ResultsNineteen (0.6%) of the 3172 CML patients, during the study period had e1a2BCR-ABL CML. At the time of diagnosis, 16 patients were in CP, 2 in BP, and 1 in AP. We only retrospectively study 12 CML patients. The median follow-up since the diagnosis of CML is 35 months (range, 5-149 months).9 of 12 patients received TKI as their initial therapy (7 imatinib, 1 nilotinib;Table 1). Among patients in CP, 6 received imatinib as frontline therapy and 3 received imatinib after Hu or in combination with IFN, and after a median follow-up of 28 months (range, 9-145 months), 2 patients had no response to imatinib, and the best response for the others was complete hematologic response (CHR) in 5, mCyR in 1 and MR4.5 in 1. Notably, only 1 of 9 patients receiving imatinib achieved MR4.5. 2 patients who was not found imatinib-resistant mutation was changed to nilotinib as second line TKI and 2 patients was changed to alloSCT. Only 1 who underwent alloSCT achieved MR4.5 and the others retained a hematologic response. 2 of the patients in CP progressed to BP (including 1 with H396R mutation) after a median of 27 months (range, 12-41 months) and died at 61 and 48 months after diagnosis, respectively.2 patients in BP (including 1 after CDOP+IA failure) received imatinib . The two patients had no response to imatinib and 1 died after 8 months. 1 in AP received nolitinib as frontline therapy. The patient achieved MR4.5 after 3 months and returned to CP.Overall, 8 patients (7 CP, 1 AP) were alive at a median of 26 months (range, 5-149 months) after diagnosis: 2 with ＞MR4.5 on TKI (1 on imatinib, 1 nilotinib) and 1 with ＞MR4.5 after alloSCT. Median survival was 28 months for patients in CP at the start of therapy, and 5 months for the patient in AP.ConclusionWe conclude that the frequency of e1a2BCR-ABL CML is only 0.6%. It suggests an inferior outcome to imatinib, and the mutations in the kinase domain of BCR-ABL have been detected in 20% of imatinib-resistant patients. Perhaps nilotinib as first line treatment has a high rate of MR4.5 in newly diagnosed CML and should be considered firstly, but the role of alloSCT for these patients is irreplaceable. DisclosuresNo relevant conflicts of interest to declare.

A case of chronic myeloid leukemia with the m-bcr (p190) molecular rearrangement identified during treatment

Chronic myeloid leukemia (CML) is a hematological malignancy in which a high percentage of patients cytogenetically express the Philadelphia chromosome. This chromosome results from the reciprocal translocation, t(9;22)(q34;q11.2). During this exchange, the Abelson gene (abl) on chromosome 9 and the breakpoint cluster region gene (bcr) on chromosome 22 generate the breakpoint cluster region-Abelson murine leukemia (BCR-ABL) fusion gene. The most frequent breakpoint on chromosome 22, located between exons 12 and 16, is called the major breakpoint cluster region (M-bcr) and with its counterpart in exon 2 of chromosome 9 results in the b2a2 and b3a2 transcripts that code for a 210 kDa protein. Less frequently, a second rearrangement, minor breakpoint cluster

Chronic myeloid leukemia with an e1a3 BCR-ABL fusion protein: transformation to lymphoid blast crisis

Chronic myelogenous leukemia (CML) results from the neoplastic transformation of a hematopoietic stem cell. CML is cytogenetically characterized by the presence of the Philadelphia chromosome (Ph’). Most patients with CML express e13a2 or e14a2 mRNAs that result from a rearrangement of the major breakpoint cluster regions (M-BCR) generating the 210-kDa (p210BCR-ABL) fusion proteins b2a2 or b3a2 respectively. The e1a3 CML-related atypical translocation has been reported with an indolent clinical course, low leukocyte count, long chronic phase even without treatment and good response to therapy. We report the case of a patient initially diagnosed as CML in chronic phase whose cells expressed the e1a3 variant. The patient readily responded to imatinib 400 mg with the achievement of a rapid complete cytogenetic response and the normalization of the blood count values, but after 5 months transformed into lymphoid blast crisis.

Nilotinib for the Frontline Treatment of Chronic Myeloid Leukemia Carrying the p230 Transcript: Dream or Reality?

OPINION article Front. Oncol., 03 February 2014Sec. Hematologic Malignancies https://doi.org/10.3389/fonc.2014.00017

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.

Chronic Myeloid Leukemia (CML) Patients With Atypical e1a2 P190 BCR-ABL Translocation Show a Poor Response To Therapy With Tyrosine Kinase Inhibitors (TKI)

IntroductionP210 BCR-ABL translocation resulting from rearrangements within the major breakpoint cluster region (M-BCR), either e13a2 or e14a2, is the molecular hallmark of chronic myeloid leukemia (CML). However, some CML patients may harbor atypical BCR-ABL rearrangements such e1a2 P190 BCR-ABL which involves the minor breakpoint cluster region (m-BCR). Response to therapy with tyrosine kinase inhibitors (TKI) and outcome of such atypical patients is not well defined. ObjectiveTo evaluate response to TKI therapy of CML patients with the atypical e1a2 P190 BCR-ABL translocation. Patients and MethodsSince 2009, 4 patients with CML in chronic phase and with atypical e1a2 P190 BCR-ABL rearrangement have been recruited in various institutions belonging to the Hematological Molecular Biology Group (GBMH) of the Spanish Society of Hematology (SEHH). Patient characteristics, treatments administered and response to therapy for the 4 patients is shown in Table 1. BCR-ABL transcripts were revealed at diagnosis by quantitative PCR followed by conventional agarose electrophoresis of PCR products. Molecular follow-up of BCR-ABL transcripts throughout treatment was performed by quantitative PCR following the guidelines of the European Leukemia Net. ResultsOne patient received treatment (HU and INF+araC) prior to TKI (Pat. 1; Table 1). All 4 patients received Imatinib as initial TKI treatment. Two of the patients treated with Imatinib (Pat. 1,2) obtained a complete molecular response (CMR) and the other 2 (Pat. 3,4) only achieved a complete hematological response (CHR) as best response (Table 1). All patients had to switch to a second generation TKI (3 Nilotinib and 1 Dasatinib) due to intolerance to Imatinib (n=1; Pat. 1) or resistance (n=3; Pat. 2-4). The patient who received Dasatinib as second line TKI (Pat. 3) only achieved a partial hematologic response (PHR) and was changed to Nilotinib as third line TKI, achieving CHR after which the patient entered in blast crisis and died 36 months after diagnosis (Table 1). Overall, only 1 (Pat. 1) out of the 4 patients included in the present study achieved a sustained molecular response with Imatinib. At last follow-up, among the 4 patients included in the study, all 4 had needed a change of TKI, 1 had died due to disease progression (Pat. 3) and only 2 of them retained a molecular response (Pat. 1,2). [Display omitted] ConclusionCML patients harboring atypical e1a2 P190 BCR-ABL transcripts show a poor response and short-lived responses to TKI therapy and therefore should be identified as high-risk patients at diagnosis. These patients must be closely monitored during therapy with TKI and should be treated upfront with a second generation TKI or even be considered for allogeneic SCT in the early phase of the disease.Paper presented on behalf of the Hematological Molecular Biology Group (GBMH) of the Spanish Society of Hematology (SEHH). AJ-V and IB contributed equally to this work. Disclosures:No relevant conflicts of interest to declare.

Rare e14a3 (b3a3) BCR-ABL fusion in chronic myeloid leukemia in India: the threats and challenges in monitoring minimal residual disease (MRD).

Objective: The primary objective of this work was to confirm the occurrence of rare BCR ABL fusion variant involving the a3 region of the ABL gene in a patient positive for t(9;22) translocation but negative for common major and minor breakpoint cluster regions and the challenges and threats that it poses in a routine laboratory setting which use commercial kits for monitoring the minimal residual disease.Methods: A patient with elevated white blood cell count was subjected to classical cytogenetics, FISH as well as RT-PCR testing using commercial kits as well as published primers and in house testing protocol. PCR amplicon generated from in the process was sequenced and analyzed.Results: The translocation event in chromosome 9 and 22 could be successfully detected. BCR/ABL dual color, dual fusion probe generated a classical balanced translocation scenario within the nucleus of affected cells and presented a ‘1O1G2F’ signal pattern. RT-PCR with probes from commercial kit designed to detect common breakpoints within the M- and m-BCR regions involving e13a2, e14a2 and e1a2 fusion variants respectively failed to generate any signal. Further investigation revealed presence of the rare e14a3 (b3a3) fusion.Discussion: This is the first report of rare e14a3 fusion in the BCR ABL gene in a CML patient from India. The observation indicates the need for interrogating rare BCR ABL fusions when common breakpoint cluster regions are absent such that minimal residual disease (MRD), critical for disease monitoring, can be performed and false positive remission cases can be avoided. It also emphasizes the utility and significance of cytogenetics and FISH techniques in primary diagnosis of CML and use of RT-PCR based assays only for generating secondary information within special reference to MRD.Conclusions: The rare e14a3 (b3a3) fusion of the BCR ABL gene is present in Indian population as demonstrated from this first report and clinical laboratories using commercial kit that do not cover such rare fusions are likely to generate false result thereby declaring complete molecular remission in CML patients under therapy while conducting MRD assay using RT-PCR technology.