BCR Gene Research Articles

Complex variant of Philadelphia translocation involving chromosomes 9, 12, and 22 in a case with chronic myeloid leukaemia.

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder included in the broader diagnostic category of myeloproliferative neoplasms, associated with fusion by BCR gene at chromosome 22q11 to ABL1 gene at chromosome 9q34 with the formation of the Philadelphia (Ph) chromosome. In 2–10% of CML cases, the fusion gene arises in connection with a variant translocation, involving chromosomes 9, 22, and one or more different chromosomes; consequently, the Ph chromosome could be masked within a complex chromosome rearrangement. In cases with variant Ph translocation a deletion on der(9) may be more frequently observed than in cases with the classical one. Herein we describe a novel case of CML with complex variant Ph translocation involving chromosomes 9, 12, and 22. We present the hematologic response and cytogenetic response after Imatinib treatment. We also speculated the mechanism which had originated the chromosome rearrangement.

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

Atypical BCR-ABL fusion transcript (e6a2) in pediatric acute lymphoblastic leukemia

Introduction: Among precursor-b-acute lymphoblastic leukemia cases, bcr-AbL translocation occurs in around 20–30% of adults and in ≤5% of children. Minor breakpoint transcripts (e1a2) are found in about 70% of positive bcr-AbL cases and major breakpoint transcripts (e13a2, e14a2) in about 30% cases. However, other atypical transcripts are sometimes observed. Case Report: A rare form of chimeric bcr-AbL fusion transcript (e6a2) was detected in a pediatric patient with precursor-b-acute lymphoblastic leukemia by reverse transcriptase polymerase chain reaction. Sequence analysis of the fusion region of the amplified cDNA fragment showed an inframe joining of exon 6 of the bcr gene and exon 2 of the AbL gene, giving rise to an e6a2 BCR-ABL transcript. This finding was also confirmed by fluorescent in situ hybridization. Conclusion: The findings in this case shows that

Copy Number Variations and IKZF1 Mutations In Pediatric CML

Chronic myeloid leukemia (CML) occurring in children and adolescents is associated with a number of clinical and molecular features that differ from the correspondent disease in patients in older age. Detailed molecular analyses of the genomic BCR-ABL1 breakpoints in a cohort of 60 pediatric individuals from the German CML-paed I and CML-paed II trial revealed a significantly different breakpoints distribution compared to adult CML. Especially the observed bimodal breakpoint distribution pattern in the BCR gene and a higher proportion of breakpoints within Alu repeat regions vary between pediatric CML and adult CML and resembles the pattern observed in adult BCR-ABL1-postitive acute lymphoblastic leukemia.To identify additional genetic variations, in addition to the BCR-ABL1 fusion gene, high resolution whole-genome microarray analyses using Affymetrix Cytogenetics Whole-Genome 2.7M or CytoScan® HD arrays were performed in a selected sub-cohort of pediatric CML patients from the German CML-paed II trial; twenty individuals diagnosed in chronic phase (CP) and two individuals diagnosed in blast crisis (BC) were screened. In contrast to adult CML patients in whom about 25% of individuals in CP exhibit detectable CNVs, nearly 60% of pediatric patients have CNVs (1.9 CNVs per case) in this initial stage of CML. All identified CNVs were private and no recurrent genetic aberration was associated with early disease manifestation in children. Two patients in BC showed an increased number of CNVs (6.5 CNVs per case) which is in accordance with data from adult patients, indicating that additional secondary genetic events are associated with disease progression. One of the patients in lymphoid CML-BC harbored a deletion involving the IKZF1 gene, an aberration frequently observed in advanced disease stages in adult CML and ALL.To further evaluate the incidence of IKZF1 mutations and their impact on the disease progression in pediatric CML patients, deep sequencing of IKZF1 was performed in 55 patients; 52 individuals in CP and 3 individuals in BC. Previously performed whole genome array analyses showed a deletion of exon 4 to 7 in the IKZF1 gene in one of the three analyzed patients in BC. Deep sequencing identified no additional mutation within IKZF1 in both remaining individuals. Interestingly, two patients in CP showed a single nucleotide insertion resulting in a premature stop codon, E35 and Y348 respectively. One patient developed imatinib resistantance 1.5 years after treatment onset. The second patient underwent stem cell transplantation 1 year after initial diagnosis. To date, with a follow-up of 7 and 6 years respectively, no patient progressed to advanced disease. These findings are in line with findings in adult CML, where IKZF1 aberrations are preferentially observed in advanced disease stages or imatinib resistant patients. Disclosures:No relevant conflicts of interest to declare.

A High Throughput Exome Sequencing Approach To Analyse Events Within a Good Responder CML Patient Under Imatinib At Diagnosis and Under Remission

BackgroundGenome sequencing has emerged recently as a technology that can be used to address questions regarding the clonal evolution of cancers. This has the potential to be translated into practical applications in a clinical setting. We have undertaken a case study by carrying out whole exome sequencing of a patient with CML since its clinical outcome varies amongst patients during the progression of disease.Our study is an attempt for a better understanding of why patients differ in their response to different dose of drug regimen and to determine its role in clinical outcome. Our case involves a patient with BCR–ABL positive CML. She has been responding to Imatinib, at 200 mg once a day, a dosage lower than the recommended 400mg /day. She has been taking 200 mg / day irregularly for two years and there were frequent interruptions of regular dosage schedule because of severe symptomatic cytopenias requiring blood transfusions. At the end of 2 years, there has been no progression of disease. Her bone marrow aspirate and biopsy have been normal and the BCR-ABL transcript has been below detectable levels.It was by serendipity that we happened to study the whole exome of this patient before we started her on Imatinib. As her dose response was erratic, we decided to do a longitudinal study. We examined the whole exome of the patient with CML at different points during the progression of disease. The aim of this study is to identify novel polymorphisms or variants which might be associated with the case, there by resisting the disease to progress. We were interested in the process of understanding the variability of the clinical outcome to standard treatment from a genomics perspective. ResultsThe exome sequencing of the bone marrow aspirate was performed at the time of diagnosis and two years post treatment with Imatinib. Matched skin biopsy was used as a control. This study has been approved by the Institutional Ethical Review Board of St. John's Medical college and Hospital.The SNVs of the skin data were used as the control in this experiment to account for germ line mutations. We observe severe genome instability in terms of single nucleotide variants (SNVs) at the time of diagnosis. The counts of SNVs are observed to be drastically reduced in the treated bone marrow sample than at the time of diagnosis. No insertions or deletions were observed in this longitudinal case study. It was also observed that the SNV counts were not dominated in any particular chromosome.The SNVs picked up by exome sequencing were hence contrasted at the stage of diagnosis and post treatment. These SNVs were compared to dbSNP to retain only those which are novel and not previously reported. This finally gave rise to three discreet sets to consider: 1). The SNVs present in the genome of the patient at diagnosis 2). Post treatment, and 3). a subset common to both of them. The non-synonymous coding mutations with single base substitutions were 1,08,436 at the time of diagnosis and reduced to 163 in a stage of remission. The overlaps between these two categories were 169 in number.These hits in respective categories were further filtered based on the score of SNV call, relevance to the disease, presence within domain boundaries and final predicted impact on the function of the coded protein. We have identified novel mutations within the ABL, BCR, Kit and NOTCH genes with high probability of impact on function in the bone marrow sample at diagnosis. These will further be validated experimentally to be confirmed as a probable marker for screening patients. This study will be extended to screen a cohort of patients with similar prognosis as the patient in our case study. The novel mutations will be mapped on the protein structure to show which functional domains could be affected and to provide a structural basis for the deleterious effects of such mutations. We hence attempt to understand this clinical case with an integrated view involving basic experimental biology, bio-informatics and structural biology.These polymorphisms would provide insights on the evolution of CML and eventually allow us to use such readouts as a screen across CML patients to identify cases with better disease prognosis. With the distinct population groups and diverse spectrum of diseases, we believe that our work lays the foundation for larger studies in both CML and other diseases requiring such approaches. Disclosures:No relevant conflicts of interest to declare.

NGS-Assisted DNA-Based Digital qPCR Facilitates Stratification Of CML Patients In Long-Term Molecular Remission Based On The Presence Of Detectable BCR-ABL1 DNA

Recent studies indicate that 40% of CML patients on imatinib who achieve complete molecular remission (CMR; equivalent to MR4 or better in current terminology) remain disease-free after drug discontinuation, raising the possibility of an “operational cure”. However, the safe introduction of TKI withdrawal into clinical practice would require a method of evaluating risk of relapse, which is likely to be related to the presence of residual disease undetectable by RT-qPCR. A PCR method based on the use of genomic DNA has been shown to be more sensitive for the detection of residual disease than one that relies on cDNA and may, therefore, help to predict outcome post-withdrawal. However, the former method is arduous requiring a customised patient-specific assay. We describe here a method based on targeted next-generation sequencing (NGS) which allows the rapid identification of BCR-ABL1 breakpoints and the generation of DNA-based qPCR assays. The location of the BCR-ABL1 fusion junction was mapped in disease samples from 32 CML patients using Illumina's MiSeq platform. A custom TruSeq DNA target enrichment system (Illumina) was used to enrich for fragments containing sequences from the BCR and ABL1 genes using probes covering both genes including an additional 50kb of sequence in both 5' and 3' directions. All 32 patients' t(9;22) translocation junctions were successfully mapped using a custom designed bioinformatics algorithm. To date we have designed and validated DNA qPCR assays for 26 of these.On testing consecutive clinical samples from all 26 patients after achievement of MR4 or better (previously defined as CMR; mean 6 samples per patient), the DNA-qPCR assays detected residual disease in 8 out of 26 patients (31%), demonstrating that a DNA-based method is capable of identifying the presence of residual leukaemia in a proportion of patients who would be defined as “disease-free” using conventional RNA-based methodology.One area in which inaccuracy may be introduced into the DNA qPCR technique is in the use of a standard curve generated using serial dilutions of patient's diagnostic material. We therefore sought to further enhance the sensitivity of a DNA-based approach by optimising this technique for use on a digital quantitative PCR (dqPCR) platform, which provides absolute molecular quantification without the need for standard curve. Using DNA dqPCR on a Fludigm BioMark HD instrument, we measured the level of BCR-ABL1 in 48 samples from 6 CML patients after achievement of a sustained response of MR4 or better. At the time point at which patients were first determined to have achieved at least MR4, all samples tested positive by DNA dqPCR. However, in subsequent samples collected two years following achievement of at least an MR4 (the point of entry into ongoing withdrawal trials, at which point all six patients had achieved MR4.5 or better), we found that patients fell into two distinct categories: 1) those whose disease continued to be detectable by DNA dqPCR (n=3), and 2) those whose disease had fallen to a level that was no longer detectable by DNA dqPCR (n=3). In contrast the conventional BCR-ABL1 DNA qPCR method detected disease in only one of these six patients at this time point, suggesting superior sensitivity of a digital PCR-based screening platform.From this pilot study we conclude that NGS-facilitated DNA dqPCR may be used to stratify patients with RT-qPCR-undetectable disease according to residual disease burden and may therefore prove valuable in the identification of patients for whom TKI therapy could be safely reduced or stopped. Disclosures:Milojkovic:BMS: Honoraria; Pfizer: Honoraria; Ariad: Honoraria; Novartis: Honoraria. Apperley:Novartis: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria; Pfizer, Ariad: Honoraria (not direct from company), Honoraria (not direct from company) Other.

Interference in Pheromone-Responsive Conjugation of a High-Level Bacitracin Resistant Enterococcus faecalis Plasmid of Poultry Origin

The current study reports on contact interference of a high-level bacitracin- resistant pheromone-responsive plasmid of Enterococcus faecalis strain 543 of poultry origin during conjugative transfer of bcr antimicrobial resistance genes using a polyclonal antiserum aggregation substance44–560 (AS). After induction with pheromones produced by the recipient strain E. faecalis JH2-2, clumping of the donor E. faecalis strain 543 was observed as well as high transfer frequencies of bcr in short time broth mating. Filter mating assays from donor strain E. faecalis 543 to the recipient strain E. faecalis JH2-2 revealed conjugative transfer of asa1 (AS), bcrRAB and traB (negative regulator pheromone response) genes. The presence of these genes in transconjugants was confirmed by antimicrobial susceptibility testing, PCR, Southern hybridization and sequencing. A significant reduction in formation of aggregates was observed when the polyclonal anti-AS44–560 was added in the pheromone-responsive conjugation experiments as compared to the induced state. Moreover, interference of anti-AS44–560 antibodies in pheromone-responsive conjugation was demonstrated by a reduction in horizontal transfer of asa1 and bcr genes between E. faecalis strain 543 and E. faecalis JH2-2. Reducing the pheromone-responsive conjugation of E. faecalis is of interest because of its clinical importance in the horizontal transfer of antimicrobial resistance.

Overcoming resistance in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is characterized by the BCR–ABL fusion protein produced as a result of the reciprocal translocation between a portion of the ABL tyrosine kinase gene on chromosome 9 and a region within the BCR gene on chromosome 22. The BCR–ABL oncoprotein displays constitutively elevated tyrosine kinase activity that drives the pathogenesis of CML by activating multiple signaling pathways including the RAS/MAPK, PI3K/AKT and JAK2/STAT5 pathways. The development of tyrosine kinase inhibitors (TKIs) aimed at BCR–ABL revolutionized the treatment of CML. Imatinib was the first of the TKIs to become available after it was approved by the US FDA in 2002 based upon data from the IRIS. Indeed, imatinib was associated with unprecedented rates of hematologic, cytogenetic and molecular responses in comparison with IFN-a with complete hematologic response (CHR) rates of 97%, complete cytogenetic response (CCyR) rates of 82% and major molecular response (MMR) rates on the international scale (IS) of 86% [1]. Treatment with imatinib also resulted in significantly improved rates of freedom from progression in comparison with IFN-a with an 8-year freedom from progression to accelerated or blast phase of 92% and afforded patients who were able to remain on treatment an 8-year overall survival (OS) of 85% [1]. However, imatinib use has been complicated by the development of resistance. Resistance led to 24% of patients in IRIS failing to achieve a CCyR at 18 months, which represented treatment failure [2]. Resistance also led 17% of patients on imatinib in IRIS to develop relapsed disease and 7% to develop progressive disease [2]. These numbers are, in fact, likely to be underestimates given the number of patients in the IRIS study who came off the trial and were not ultimately included in analysis. Treatment failure and disease progression due to resistance is also seen in patients treated with the subsequently approved second-generation TKIs, dasatinib, nilotinib and bosutinib. The etiology of resistance to TKIs is multifactorial and involves BCR–ABL independent and dependent mechanisms. Insufficient plasma levels of imatinib due to noncompliance, drug–drug interactions or binding to acute-phase inflammatory proteins can all result in resistance. It is known that trough plasma levels of imatinib are important for good clinical outcomes, and it has been demonstrated that patients with high imatinib exposure have better rates of CCyR, MMR and event-free survival [3]. Imatinib and all three of the second-generation TKIs are substrates for, and are extensively metabolized by, the cytochrome P450 enzymes including CYP3A4. As there are multiple drugs, food products and supplements that either induce or inhibit CYP3A4 activity, the potential for drug interactions is high. Alpha 1 acid glycoprotein has been identified as an acutephase inflammatory product that binds directly to imatinib and is frequently “As the number of drugs available for the treatment of chronic myeloid leukemia has expanded, so have the strategies for overcoming or avoiding resistance.”

Role of Methylenetetrahydrofolate Reductase (Mthfr), Glutathione S-transferases (Gsts M1 and T1) and Haptoglobin (Hp) Gene Polymorphisms in Susceptibility to Chronic Myeloid Leukemia (Cml)

Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasm derived from an abnormal pluripotent hematopoietic stem cell that underwent a chromosomal translocation t(9;22)(q34;q11) acquiring the BCR-ABL1 fusion gene, also known as the Philadelphia chromosome. Although the clinical and biological aspects are well documented, little is known about individual susceptibility to CML. There are no known hereditary, familial, geographic, ethnic, or economic associations with CML, and the mechanisms behind disease progression are not fully understood. However, while a specific chromosomal translocation is the common oncogenetic mechanism of CML, it may have environmental causes such as irradiation and chemical (e.g., benzene) exposure, which exert toxicity via pro-oxidant mechanisms. Oxidative stress has been observed in several hematopoietic malignancies, including acute and chronic myeloid leukemias. It is known that BCR/ABL expression stimulates reactive oxygen species (ROS) production in hematopoietic progenitor cells, and studies have suggested that ROS may contribute to increased DNA damage in CML cells. Leukemia cell lines expressing BCR-ABL1 kinase accumulate ROS and oxidative DNA damage, resulting in genomic instability, which leads to the disease relapse and/or malignant progression to a fatal blast phase. These suggest a probable genetic susceptibility to this disease related to the body’s ability to defend itself from external aggression such as chemicals, ionizing radiation and others, through functional polymorphic variant enzymes that metabolize toxicants and/or protect against oxidative stress. Thus, this review aims to address a comprehensive summary of CML, including the physiological function of the normal proteins encoded by the ABL1 and BCR genes and the oncogenetic mechanism of the BCR-ABL1 fusion proteins, as well as to discuss the role of polymorphisms in methylenetetrahydrofolate reductase (MTHFR), glutathione S-transferases (GSTs M1 and T1) and haptoglobin (Hp) genes in the susceptibility to Chronic Myeloid Leukemia (CML).

A novel cytogenetic abnormality r(7)(::p11.2->q36.3::) in a Philadelphia-positive chronic myeloid leukemia case

The so-calledPhiladelphia(Ph) chromosome is present in more than 90% of chronic myeloid leukemia (CML) cases. It results in juxtaposition of the 5' part of the BCR gene on chromosome 22 and the 3' part of the ABL1 gene on chromosome 9. An additional acquired monosomy 7 or deletion of 7q is associated with poor prognosis in a variety of myeloid disorders. Here we report a novel Ph chromosome positive CML case with a ring chromosome 7 [r(7)]. Immunophenotyping was compatible with CML, although 4.5% of total leucocytes appeared like acute myelogeneous leukemia (AML) subtype M2. The r(7) was characterized in detail by array-proven multicolor banding (aMCB), the latter being of enormous significance to characterize breakpoint regions in detail. Underlying mechanisms and prognostic are discussed, as ring chromosomes are rare cytogenetic abnormalities in hematopoietic malignancies.

Analysis of the clinico-hematological relevance of the breakpoint location within M-BCR in chronic myeloid leukemia

The Philadelphia chromosome (Ph) derives from the balanced translocation between chromosomes 9 and 22. This chromosomal translocation results in the fusion between the 5' part of the BCR gene, normally located on chromosome 22, and the 3' part of the ABL gene on chromosome 9 giving origin to a BCR-ABL fusion gene which is transcribed and then translated into a hybrid protein. In general, three breakpoint cluster regions in the BCR gene have been described: major (M-BCR), minor (m-BCR) and micro (μ-BCR). Three main variants of the BCR-ABL gene have been described depending on the length of the sequence of the BCR gene included that encode for the P190, P210, P230 proteins. Most patients (95 %) were found to have P210 protein that resulted from rearrangement in the M-BCR region in the BCR gene and thus gives rise to b2a2 or b3a2 variants. The incidence of one or other rearrangement in chronic myeloid leukemia (CML) patients varies in different reported series. These two variants are associated with distinct clinical types of human leukemias. In this study, we report the frequencies of M-BCR-ABL fusion transcripts in 44 CML patients and we review the data on the correlations between the type of M-BCR/ABL variant and the corresponding sex, age and biological features. Forty-four untreated chronic phase CML patients were studied. The type of BCR-ABL fusion transcripts was determined by reverse transcriptase polymerase chain reaction (RT-PCR). More than half of them showed b3a2 fusion transcript (64 %), while (36 %) showed b2a2 transcript. No patients coexpressed b3a2/b2a2. Correlation between biological data demonstrated that: (a) M-BCR rearrangements were not associated with the sex of the patients. (b) Patients with b3a2 rearrangements were older than patients with b2a2 transcripts. (c) M-BCR rearrangements were influenced neither by the white blood count (WBC) nor with hemoglobin levels. However, platelet level is more elevated in patients with b3a2 transcript (681.2/L vs. 207/L; P = 0.001). In conclusion, we observed significant correlations between age, platelet level and M-BCR-ABL transcript, these observations deserve further investigations.

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.

ETV6-ABL1 CML Like Disorder with Complex Translocation Involving Chromosome 9, 12 and 14

AbstractAbstract 4438A rare group of myeloproliferative disorders has been described associated with gene rearrangements producing novel tyrosine kinases other than BCR/ABL.A rearrangement involving the ETV6 and ABL1 genes, associated with t(9;12)(q34;p13) translocation, has been detected in Ph-negative chronic myeloproliferative disorders and has been demonstrated to have tyrosine kinase activity in signal transduction pathways similar to the BCR/ABL fusion protein. The ETV6 gene is a member of the E26 transformation-specific family (ETS) of transcription factors located at 12p13. It has been implicated in the rearrangement of over 48 different chromosome bands, ultimately playing a role in leukemogenesis. Thus far, only eleven BCRABL1-negative CML patients with a variant ABL1 gene (9q34) rearrangement, involving fusion to ETV6 aka TEL (12p13) have been reported. ETV6-ABL1 rearrangement have also been implicated in other hematologic diseases including 6 acute B lymphoid leukemia, 1 acute T lymphoid leukemia, 3 acute myeloid leukemia, 1 myelodysplastic syndrome, and 3 chronic myeloproliferative disorders.We present a 62 year old male who sought medical attention for acute coronary syndrome with bone pain and asthenia in May 2011. Physical exam showed a splenomegaly. He was found to have hemoglobin of 9.9g/dL, WBC 90G/L and platelets 105G/L. The peripheral blood differential count revealed 67% segmented neutrophils, 2% eosinophils, 0 basophils, 3% monocyte, 28% immature stages and 5% lymphocytes per 100 white blood cells. Bone marrow biopsy revealed myeloid hyperplasia suggestive of a myeloproliferative disorder. The genetic characteristic of this patient is to present an exceptional chromosomal complex rearrangement according to Pellestor classification, involving 3 chromosomes 9, 14, 12 with five breakpoints: 9q, 12p, 12q, and two different on 14q. More precisely, it probably compounds of a three way translocation t(9;12;14)(q34,p13,q?) associated with a independent translocation between both long arms of chromosomes 12 and 14. FISH analysis using BCR/ABL TC DFusion (Kreatech) shows involvement of ABL1 probe in a translocation with short arm of chromosome 12 and no evidence of rearrangement of BCR gene suggesting the possibility of juxtaposition of ABL1 gene with a gene different than the BCR gene. Moreover whole chromosome 14 painting confirms various involvements of chromosome 14. Molecular biology confirmed the suspected ETV6-ABL1 fusion gene and allows us to follow minimal residual disease. It should be noted that a t(9;12;14)(q34;p13;q22) has already been described as a variant of the t(9;12)(q34;p13) with complex insertions of ETV6 into ABL1. Other chromosome than the 14 has known to be implicated in t(9;12) variant as chromosome 17.First, he was treated with imatinib with progression despite the addition of hydroxyurea. Treatment was switched at 3 months for dasatinib. At 3 months, ETV6-ABL1/ABL1 is 0.32% and at 6 months he obtains a Major Molecular Response (defined as MRD under 0.1% like in BCR-ABL measurement).In conclusion, we present a new case of ETV6-ABL1 myeloproliferative disorder due to complex rearragement which experiments good response to dasatinib suggesting that second generation TKI are a good option for treatment of such disease. Disclosures:No relevant conflicts of interest to declare.

Fostamatinib Inhibits BCR Signaling, and Reduces Tumor Cell Activation and Proliferation in Patients with Relapsed Refractory Chronic Lymphocytic Leukemia.

Abstract 2882B-cell receptor (BCR) signaling contributes to the pathogenesis of chronic lymphocytic leukemia (CLL). Spleen tyrosine kinase (SYK) activated directly downstream of the BCR is essential for the induction of proliferation and survival pathways. The SYK inhibitor fostamatinib disrupts BCR signaling and was the first such inhibitor to show significant clinical activity in patients with mature B-cell malignancies. Fostamatinib has been shown to both induce apoptosis in unstimulated CLL cells as well as to inhibit BCR induced anti-apoptotic signals in vitro (Gobessi et al., 2009; Quiroga et al., 2009). Similarly, using the Eμ-TCL1 transgenic mouse model, fostamatinib has been shown to inhibit the growth of malignant B-cells without significant alteration of normal B-cells (Suljagic et al., 2010). In the first phase I/II clinical trial investigating fostamatinib in relapsed B-cell non-Hodgkin’s lymphoma (NHL) and CLL, clinical efficacy was observed in a variety of histologies with the highest response rate in CLL/SLL patients (Friedberg et al., 2010). Eleven CLL/SLL patients enrolled in this trial donated cellular material for correlative studies. Using these primary tumor samples, we evaluated the effects of fostamatinib on CLL cells in vivo after one cycle of treatment.We first validated the on-target effect of fostamatinib by using quantitative RT-PCR to measure expression of validated pathway specific gene signatures. Fostamatinib greatly down-regulated 12/12 evaluated BCR signature genes and significantly reduced the BCR gene signature score (computed as the average expression of the pathway specific genes; p=.002) Effective inhibition of BCR signaling was confirmed by a significant reduction in the phosphorylation of both BTK and ERK; two key BCR signaling molecules located downstream of SYK activation. Interestingly, BCR signaling was inhibited in CLL cells from all patients regardless of response to therapy. We next expanded our analysis to look at NF-κB and MYC gene signatures. We found that 11/11 representative NF-κB signature genes and 5/5 MYC signature genes were also down-regulated resulting in a significant reduction in both gene signature scores (p=.004 and p=.020, respectively). Confirming these results, we also observed a significant reduction in JUNB (p<.001) and MYC (p=.026) at the protein level. Interestingly, the reduction in NF-κB and MYC signature scores was highly correlated with the degree of reduction in BCR signaling suggesting that these pathways are linked. In addition to changes in the gene signatures we also observed a significant reduction in the cellular activation immunephenotype; CD69 and CD86 expression were significantly reduced by fostamatinib (p=.033 and p=.004, respectively). Further, we found that CD38 (an activation marker with prognostic significance) was also reduced on treatment although not to a significant extent. Finally, fostamatinib significantly reduced tumor proliferation as determined by the percentage of CLL cells expressing Ki67 (p=.005). Eight of the 11 patients in this study achieved a clinically significant response; interestingly however, the 3 non-responders demonstrated significantly brighter CD38 expression with an MFI up to 9-times the CD38 MFI in responders. A possible role of CD38 as a biomarker for response should be further explored in patients treated with BCR directed kinase inhibitors.In conclusion, fostamatinib and other inhibitors of BCR-related kinases constitute a major advance in the treatment of CLL. In vitro data with these compounds suggests that interruption of BCR signaling and survival pathways activated in the tissue microenvironments are likely responsible for the observed clinical response as only a moderate direct induction of apoptosis is seen in vitro. Here we demonstrate that inhibition of BCR-mediated signaling by fostamatinib results in a reduction in CLL proliferation and activation in vivo. Together these data provide a blueprint to further study the mechanism of action and resistance mechanisms of not only fostamatinib but also other BCR targeted therapeutics.This work was supported in part by the Intramural Research Program of the National, Heart, Lung and Blood Institute and by the University of Rochester SPORE in lymphoma P50 CA13080503, Rigel and the James P. Wilmot Foundation. Disclosures:Friedberg:Rigel: Research Funding.

Myeloproliferative Neoplasm with BCR-JAK2 Fusion Gene As the Result of t(9;22)(p24,11.2) in a Brazilian Patient

AbstractAbstract 4808 IntroductionChronic Myeloid Leukemia (CML) is the most well described disease result of t(9;22)(q34,q11.2). This chromosomal rearrangement leads to well-know BCR-ABL fusion that promotes tyrosine kinase activity. There are others oncogenic BCR fusion found such as PDGFRA (4q12), FGFR1(8p12) that causes myeloproliferative disorders (MD).JAK2 gene is one of the 4 genes members of JAK family. The JAK2 V617F mutation which results from a G –>T transversion at nucleotide 1849 in exon 14 of the JAK2 gene, the consequence of which is substitution of valine by phenylalanine at codon 617 is associated with MD and it is a major diagnosis criterion for Primary Myeloficrosis, Polycythaemia vera and Essential thrombocytemia. There are described a lot chromosomal translocations involving the JAK2 locus.We report an extremely rare case with BCR-JAK2 fusion gene as the result of t(9;22)(p24,q11.2) for the first time in Brazilian people, and it is the 6thcase all of the world. Case ReportIn April 2010, a 54 years old male patient presented fatigue, abdominal pain and splenomegaly. A blood count revealed leukocytosis 93.380/mm3 with a predominance of neutrophils and left shift. Conventional cytogenetic analysis was performed and it was evidenced 46,XY, t(9;22)(p24;q11.2) in 90% metaphases examined, due to expected association it was promoted BCR-ABL1 fusion gene and it was not detected by using RT-PCR. He was treated with imatinib 400mg/day because the involvement of BCR gene. After three months he presented weight loss, progressive splenomegaly without hematologic response and it was modified to Dasatinib 150mg/day plus hydroxyureia 3g/day. In August 2011, due to not hematologic response, it was stopped Dasatinib treatment and nowadays patient has been treating with hydroxyureia 1.5g/day. His last follow up in May 2012, blood count was abnormal Hb 16.8g/dl leukocytes 7730/mm3 and low platelets count 32.000/mm3. The differential count showed 65.3% segmented granulocytes, 13.6% eosinophlis, 1.6% basophil, 2.6% monocytes, 16.9% lymphocytes.It was repeated conventional Karyotyping and it was evidenced 46,XY, t(9;22)(p24;q11.2) in all of metaphases examined. The presence of BCR-ABL rearrangement was excluded by using the fluorescence in situ hybridization (FISH) using a BCR-ABL probe. In addition, it was not evidenced FIP1L1-PDGFRa fusion gene and JAK2 V617F mutation by using RT-PCR. DiscussionWe have described a male patient with MD with t(9;22)(p24;q11.2) wich leads to the BCR-JAK2 fusion and it was not evidenced BCR-ABL1, FIP1L1-PDGFRa fusion genes and JAK2 V617F mutation by using RT-PCR. Moreover, patient has not been achieved hematologic response with tyrosine kinase inhibitors: imatinib and dasatinib. In the five cases reported three presented MD, one Acute Myeloid Leukemia and one Acute Lymphoblastic Leukemia. Only in one case report it was prescribed imatinib and the patient lost the follow up (Table1). The BCR-JAK2 fusion protein contain the coiled-coil dimerization domain of BCR and the protein tyrosine Kinase domain (JH1) of JAK2. It was not possible to define what would be the best therapy, because tyrosine kinase inhibitors may not be effective to the BCR-JAK2 fusion. Maybe in MD presentation, we could return to pre- tyrosine kinase inhibitors era based on treatments with hydroxyureia, subcutaneous cytarabine and interferon for patients that were not potential candidates for allogeneic transplant.ReferencesAgesextranslocationClinical presentationTreatmentFollow upGriesing F et al63Ft(9;22)(p24;q11.2)MDHy; Cy; MitDeathCirmena G et al67Ft(9;22)(p24;q11)AMLHD + allo BMTDeathLane SW et al44Mt(9;22)(p24;q11.2)MDNot describedNot describedElnaggar MM et al84Mt(9;22)(p24;q11.2)MDHy; ImaLost follow upTirado CA et al14Mt(9;22)(p24;q11.2)ALLPolychemotherapyRemissionHy: Hydroxyurea; Cy: Cytarabine; Mit: Mitoxantrone; HD: High dose chemotherapy; allo BMT allogeneic Bone Marrow Transplantation; Ima: imatinib; ALL: Acute lymphoblastic leukemia. Disclosures:No relevant conflicts of interest to declare.

Renal Medullary Carcinoma in an Adolescent with Homozygous Hemoglobin SS

Abstract 4774Renal medullary carcinoma (RMC) is a highly aggressive malignant neoplasm originally thought to be unique to individuals with sickle cell trait (SCT). Common symptoms include hematuria, abdominal pain, and weight loss. The disease is usually metastatic at presentation and is almost universally fatal within months of diagnosis. Herein, we describe an adolescent with homozygous hemoglobin SS (Hgb SS) and RMC. This is the third case of RMC reported in Hgb SS, and the first case with prior hydroxyurea therapy (Dimashkieh Arch Pathol Lab Med 2003 and Swartz Urology 2002). In addition to five reported cases in individuals with hemoglobin SC, it now appears that RMC is not unique to individuals with SCT and should instead be considered more inclusively related to the family of sickle hemoglobinopathies (Davis Am J Surg Pathol 1995, Luthra Intern Med 2010, Baig J Natl Med Assoc 2006, Swartz Urology 2002).A 13 year-old African American male with Hgb SS, treated with hydroxyurea since 2 months of age, presented with a several month history of recurrent abdominal pain and microscopic hematuria. An abdominal ultrasound demonstrated normal appearing kidneys that measured 8.4 cm and 9.7 cm on the right and left sides, respectively. He continued to have abdominal pain, often associated with intermittent sickle cell extremity pain. A repeat sonogram was obtained 9 months after the initial ultrasound due to a new complaint of scrotal pain. The formerly normal left kidney now had a 5 cm mass in the mid-to-lower pole, along with moderate caliceal dilation and cortical thinning of the superior pole. Computed tomography (CT) and positron emission tomography scans confirmed the renal mass along with local invasion into the psoas muscle, liver metastases, pulmonary metastases, and retroperitoneal lymphadenopathy.Multiple core needle biopsy specimens were obtained from the left kidney, diagnostic of RMC. Histopathology showed widespread areas of necrosis with only a few areas of viable tumor. The viable tumor cells formed gland-like structures and had infiltrating sheets with rhabdoid features, eosinophilic cytoplasm, and prominent nucleoli. Immunohistochemical analysis demonstrated strong positivity for EMA, P53 (>95%), and PAX8. CD117 was mostly negative, however there were focal areas of weak cytoplasmic reactivity. ALK-1 immunostain, INI-1, p63, and hepar-1 were all negative. Fluorescence in situ hybridization showed no evidence of t(9;22) BCR;ABL rearrangement but did show extra copies of the BCR gene in 73% of cells.Neoadjuvant chemotherapy with 3 cycles of carboplatin, gemcitabine, and paclitaxel and one cycle of carboplatin, gemcitabine and bortezomib resulted in an improvement on imaging in the primary tumor and metastatic sites. A left radical nephrectomy and lymph node dissection were then performed. Pathology showed 10–20% focally viable renal medullary carcinoma within the kidney with evidence of vascular invasion and positive margins. The sampled lymph nodes were free of tumor. Several weeks later he underwent thoracoscopic resection of left pleural and pulmonary nodules, both of which were positive for metastatic disease.Over the next 11 months, he received 5 cycles of chemotherapy consisting of alternating cycles of carboplatin, gemcitabine, paclitaxel and carboplatin, gemcitabine and bortezomib, followed by 5 additional cycles of methotrexate, vinblastine, doxorubicin and cisplatin. Palliative radiation therapy delivered to the psoas muscle for pain management was beneficial. After a brief initial response to therapy, interval imaging studies demonstrated stable disease followed by subsequent florid progression. Given disease progression, his therapy was changed to the tyrosine kinase and vascular endothelial growth factor receptor antagonist, axitinib. Sixteen months from the time diagnosis, he is ambulatory but with widespread metastatic disease.This case highlights the need for a heightened index of suspicion for RMC in all patients with sickle hemoglobinopathies, not just those with SCT. The lack of sensitivity of renal ultrasound in this case and two others suggests that CT may be required for early diagnosis of RMC (Blitman AJR 2005 and Wesche Pediatr Pathol Lab Med 1998). While hydroxyurea is an effective medication with an excellent safety margin in sickle cell disease, the carcinogenic risk of long-term exposure in sick cell disease remains unknown. Disclosures:Off Label Use: Hydroxyurea for sickle cell disease modulation.

1093P - BCR ABL Fusion Protein Detection by a Modern Approach: Fluroscent Immuno Bead Assay

ABSTRACT Introduction Chronic Myeloid Leukemia (CML) is probably the most extensively studied human malignancy. CML patients (90-95%) harbor the Philadelphia (Ph) chromosome, a shortened chromosome 22, resulting from a reciprocal translocation t (9; 22) (q34; q11) between the long arms of chromosome 9 and 22, fusion ABL proto-oncogene on chromosome 9 with the BCR gene on chromosome 22. BCR-ABL fusion gene plays a key role in pathogenesis of the disease. Approximately 15-30% of adult and 2-5% of childhood ALL and Materials and methods Study Period: June 2009- February 2012 Test Specimen Peripheral blood or Bone marrow from 242 CML patient (from hospital registry of a tertiary cancer institute of Eastern India) Normal (as negative control): Peripheral blood from 20 individuals BCR-ABL positive cell line: K562 FISH was performed using (for 100 samples) directly labeled dual color prob for detection of BCR-ABL as standard laboratory protocol. Isolation of WBC were done by lysis buffer. Then lysate treatment were done to release the intracellular BCR-ABL protein. After fluorescent immune bead assay were performed by captured bead and detector reagent. After forming a sandwich complex (if sample contain targeted protein), they were analysed by flowcytometry method. Result In 242 cases 166 (68.6%) were positive for BCR ABL fusion protein by Flowcytometric Bead Assay. Approximately 96% cases were observed as positive by both techniques FISH and Flowcytometric Bead Assay. Conclusion We conclude that the Flowcytometry Immuno Bead Assay is a faster, easier and reliable technique for detection of BCR ABL protein in leukemic cells and shows promise for serial evaluation of patients undergoing treatment. The main advantage of immune bead assay is not dependence on the breakpoint position in the BCR gene. Moreover this technique is based on protein translation label. Disclosure All authors have declared no conflicts of interest.

Analysis and Identification of Mutation in Chronic Myelogenous Leukemia (Cml) and Bcr_Abl in Leukemia Patients aged 40 to 50 Year

Chronic Myelogenous Leukemia (CML) is one of the few cancers that is known to caused by a single, specific genetic mutation in more than 90 of cases. The transformation to CML is caused by a reciprocal translocation of the BCR gene on chromosome 22 (at q11) and the ABL gene on chromosome 9 (at q34), resulting in a fused BCR-ABL gene dubbed the chromosome, approximately 44,000 people in the United States are diagnosed with some form of leukemia. The tyrosine kinase inhibitors (TKIs) Imatinib, Dasatinib, and Nilotinib inhibit activity of the BCR-ABL fusion protein, resulting in both hematologic responses. The aim of my study was to determine and confirm presence of Philadelphia chromosome and presence of BCR-ABL in patient with CML. Determination of the presence of the Ph chromosome by karyotyping at diagnosis is necessary as part of differential diagnosis and as prognosis. The standard methods of banding are the Q, G, R, and C banding techniques. In this study chromosomes have been treated to trypsin for taking different bands, karyotyping and FISH were equal, as shown in the present work, except for the fact that FISH was faster. In cases that did not present metaphases by karyotyping, FISH was extremely useful in providing a result. In daily practice, FISH can be very helpful since index of karyotyping, when the patient is receiving myelotoxic drugs, there may not be enough metaphases for analysis. RT-PCR is still more sensitive but its clinical meaning has to be clarified. Most of PCR include the detection of the translocation at the level of the RNA transcript. The method usually consists of two steps. The first reaction is that of obtaining cDNA from RNA by means of reverse transcription (RT). Subsequently, an aliquot of cDNA is transferred to another tube and subjected to the second reaction, that is, PCR amplification. Two different enzymes are used, namely, reverse transcriptase for the RT step and Taq polymerase for the PCR. Flow cytometry is a technique for analyzing large populations of single cells. For this method, the diagnostician has to analyze multiple bivariate dot plots from the cytometer. The flow cytometric immune bead assay is a fast and easy technique for specific detection of BCR-ABL proteins in leukemic cells. It is not dependent of the breakpoint position in the BCR gene, does not need special laboratory facilities other then a routine flow cytometer, provides results within several hours and it can be run in parallel to routine immunophenotyping.

Genotyping of Chimerical BCR-ABL1 RNA in Chronic Myeloid Leukemia by Integrated DNA Chip

Chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive (Ph(+)) acute lymphoblastic leukemia (ALL) are associated with fusion of the BCR and ABL1 genes by chromosome translocation. The chimerical BCR-ABL1 gene encodes different fusion proteins that vary in size, depending on the breakpoint in the BCR region. Different types of fusion genes in CML and Ph(+) ALL are thought to be related to the clinical course and outcome of each patient. Currently, the genotypes are determined by PCR, followed by gel electrophoresis or DNA sequencing (among other methodologies). Our major aim was to develop a diagnostic method for CML genotyping by means of an integrated process of DNA microarray. Here, we describe a method of integrated multiplex reverse transcription, asymmetric PCR, and hybridization, all in the same reaction mixture in a chamber assembled on the surface of capture oligonucleotide probes immobilized on a glass slide. The integrated system successfully identified the four predominant types of chimerical BCR-ABL1 RNA (b3a2, b2a2, e1a2, and c3a2), which together account for 98% of CML cases. The integrated multiplex system also had a high sensitivity of detection (as little as 200 molecules of target RNA molecules). The integrated process saves time and effort, and it also the advantage of minimizing the steps needed for automated detection of different types of chimerical CML RNA.

The importance of molecular cytogenetic analysis prior to using cell lines in research: The case of the KG-1a leukemia cell line

KG-1 and its less differentiated subline KG-1a are leukemia cell lines used in research in a number of laboratories. The karyotypes of the two lines were initially identical. In the following years, further analysis revealed that the cell lines had acquired additional karyotypical abnormalities and differed in the presence of certain typical chromosomal rearrangements. To obtain cytogenetic authentication prior to the use of the two cell lines, we analyzed their karyotype by combining DAPI- and CMA-chromosome bandings and a fluorescence in situ hybridization (FISH)-based approach by using BAC clones useful for the identification of chromosome regions of interest. Sequences of the MYC, PLZF, RARA and BCR genes, that are known to play a critical role in leukemogenesis, and certain BAC clones mapped to five known common fragile sites (CFS) were used for the FISH analysis. A telomeric probe (TTAGGG)n and a set of BAC clones were used to characterize the marker chromosome der(1) that was observed in the cell line KG-1a. The existence of notable differences between the karyotype of the KG-1a cell line previously described, and that described in this study, demonstrate that the use of established cancer cell lines should be preceded by cytogenetic and/or molecular characterization.