Increased Expression of Myc in Hairy Cell Leukemia, and Cell-Sensitivity to JQ1

Abstract Classic hairy cell leukemia (HCL), comprising 2% of leukemias, is an indolent B-cell malignancy with malignant lymphocytes expressing B-cell antigens CD20 and CD22, CD103, CD11c, CD25, Annexin A1, BRAF V600E mutation, and monoclonal immunoglobulin (Ig) rearrangement. HCL variant (HCLv), which is ~10% as common as HCL, has much poorer response to therapy and more aggressive course, lacks CD25 and Annexin A1, and is wild-type for BRAF. HCLv is considered separate from HCL by the World Health Organization in the unclassifiable splenic B-cell leukemia/lymphomas. Another poor-prognosis group overlaps HCL and HCLv in which unmutated IGHV4-34 Ig rearrangement is expressed. IGHV4-34+ leukemic cells can resemble classic HCL with CD25 and Annexin A1 expression, but are BRAF wild-type. No uniform mutation has been identified for HCLv and IGHV4-34+ HCL, although MAP2K1 (MEK1) mutations have recently been identified in half of cases. Thus HCLv and IGHV4-34+ HCL are less indolent leukemias with few therapy options and no known molecular target. To study HCLv and IGHV4-34+ HCL, leukemic samples were purified by negative B-cell isolation followed by positive CD11c sorting. Following extraction (Qiagene, AllPrep DNA/RNA Kit), RNA samples from patients were analyzed in microarray studies (Human HT-12 v4 BeadChips, Illumina, Inc.). Expression data were compared by unpaired nonparametric analysis using Mann-Whitney. MYC expression using one probe (log2 values, mean +/- standard deviation) was 7.30 +/- 1.51 for 37 HCL vs 9.77 +/- 1.15 for 32 HCLv or IGHV4-34+ HCL (2-sided p<0.0001). For the other probe, expression was 7.07 +/- 1.51 vs 9.44 +/- 1.17 (p<0.0001). Expression data for MYC had previously been submitted for 31 chronic lymphocytic leukemia (CLL) and 16 HCL samples (Dataset GSE2350, Basso et al, Nat Genet, 37:382, 2005). By 1 probe for MYC, expression was 8.07 +/- 0.55 for CLL vs 9.31 +/- 1.19 for HCL (p=0.0023). By another MYC probe, expression was 9.28 +/- 0.47 for CLL vs 10.22 +/- 0.98 for HCL (p=0.0032). To investigate potential therapeutic relevance of aberrant MYC expression in HCL, HCLv and IGHV4-34+ HCL, the bromodomain and extra terminal (BET) protein inhibitor JQ1, which has been associated with down-regulation of c-Myc via Brd4, was incubated with primary leukemic cells and ATP incorporation was measured. JQ1 inhibited 12 samples of HCL (IC50s 214 +/- 217 nM) more potently than 14 samples of CLL (IC50s 1.77uM +/-2.62 uM, p=0.020), and also inhibited 14 samples of HCLv or IGHV4-34+ HCL (IC50s 221 +/- 234 nM) more potently than the 14 CLL samples (p=0.0079). However, JQ1 inhibition was similar comparing HCL and HCLv or IGHV4-34+ HCL (p=0.89). To exclude non-specific inhibition of the cells, the inactive control molecule JQ1R was tested and was only 6.0% +/- 4.0% as active as JQ1 toward HCL or HCLv or IGHV4-34+ HCL samples. Normal peripheral blood mononuclear cells were resistant (IC50 > 20 uM). In conclusion, our results show that MYC expression is higher in HCLv and IGHV4-34+ HCL than in classic HCL and higher in classic HCL than CLL. Moreover, JQ1 inhibits HCL or its variants more potently than CLL, although the inhibition assay used does not detect a difference between the variants and classic HCL. Further experiments with other inhibitors will be needed to determine if the increased expression of MYC in HCL and its poor-prognosis variants can be exploited for treatment. Disclosures No relevant conflicts of interest to declare.

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Both variant and IGHV4-34–expressing hairy cell leukemia lack the BRAF V600E mutation

Blood ◽

10.1182/blood-2011-09-379339 ◽

2012 ◽

Vol 119 (14) ◽

pp. 3330-3332 ◽

Cited By ~ 138

Author(s):

Liqiang Xi ◽

Evgeny Arons ◽

Winnifred Navarro ◽

Katherine R. Calvo ◽

Maryalice Stetler-Stevenson ◽

...

Keyword(s):

B Cell ◽

Hairy Cell Leukemia ◽

Poor Prognosis ◽

Braf V600e Mutation ◽

Braf V600e ◽

Hairy Cell ◽

Cell Leukemia ◽

Wild Type ◽

Ighv Gene ◽

Gene Usage

Abstract Recently, the BRAF V600E mutation was reported in all cases of hairy cell leukemia (HCL) but not in other peripheral B-cell neoplasms. We wished to confirm these results and assess BRAF status in well-characterized cases of HCL associated with poor prognosis, including the immunophenotypically defined HCL variant (HCLv) and HCL expressing the IGHV4-34 immunoglobulin rearrangement. Fifty-three classic HCL (HCLc) and 16 HCLv cases were analyzed for BRAF, including 5 HCLc and 8 HCLv expressing IGHV4-34. BRAF was mutated in 42 (79%) HCLc, but wild-type in 11 (21%) HCLc and 16 (100%) HCLv. All 13 IGHV4-34+ HCLs were wild-type. IGHV gene usage in the 11 HCLc BRAF wild-type cases included 5 IGHV4-34, 5 other, and 1 unknown. Our results suggest that HCLv and IGHV4-34+ HCLs have a different pathogenesis than HCLc and that a significant minority of other HCLc are also wild-type for BRAF V600.

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Genomics of Hairy Cell Leukemia

Journal of Clinical Oncology ◽

10.1200/jco.2016.71.1556 ◽

2017 ◽

Vol 35 (9) ◽

pp. 1002-1010 ◽

Cited By ~ 25

Author(s):

Enrico Tiacci ◽

Valentina Pettirossi ◽

Gianluca Schiavoni ◽

Brunangelo Falini

Keyword(s):

B Cell ◽

Hairy Cell Leukemia ◽

Ex Vivo ◽

Braf Inhibitor ◽

Braf V600e ◽

Leukemic Cells ◽

Splenic Marginal Zone Lymphoma ◽

Hairy Cell ◽

Cell Leukemia ◽

Purine Analogs

Hairy cell leukemia (HCL) is a chronic mature B-cell neoplasm with unique clinicopathologic features and an initial exquisite sensitivity to chemotherapy with purine analogs; however, the disease relapses, often repeatedly. The enigmatic pathogenesis of HCL was recently clarified by the discovery of its underlying genetic cause, the BRAF-V600E kinase-activating mutation, which is somatically and clonally present in almost all patients through the entire disease spectrum and clinical course. By aberrantly activating the RAF-MEK-ERK signaling pathway, BRAF-V600E shapes key biologic features of HCL, including its specific expression signature, hairy morphology, and antiapoptotic behavior. Accompanying mutations of the KLF2 transcription factor or the CDKN1B/p27 cell cycle inhibitor are recurrent in 16% of patients with HCL and likely cooperate with BRAF-V600E in HCL pathogenesis. Conversely, BRAF-V600E is absent in other B-cell neoplasms, including mimickers of HCL that require different treatments (eg, HCL-variant and splenic marginal zone lymphoma). Thus, testing for BRAF-V600E allows for a genetics-based differential diagnosis between HCL and HCL-like tumors, even noninvasively in routine blood samples. BRAF-V600E also represents a new therapeutic target. Patients’ leukemic cells exposed ex vivo to BRAF inhibitors are spoiled of their HCL identity and then undergo apoptosis. In clinical trials of patients with HCL who have experienced multiple relapses after purine analogs or who are refractory to purine analogs, a short course of the oral BRAF inhibitor vemurafenib produced an almost 100% response rate, including complete remission rates of 35% to 42%, without myelotoxicity. To further improve on these results, it will be important to clarify the mechanisms of incomplete leukemic cell eradication by vemurafenib and to explore chemotherapy-free combinations of a BRAF inhibitor with other targeted agents (eg, a MEK inhibitor and/or an anti-CD20 monoclonal antibody).

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BRAF mutation in hairy cell leukemia

Oncology Reviews ◽

10.4081/oncol.2014.253 ◽

2014 ◽

Cited By ~ 6

Author(s):

Ahmad Ahmadzadeh ◽

Saeid Shahrabi ◽

Kaveh Jaseb ◽

Fatemeh Norozi ◽

Mohammad Shahjahani ◽

...

Keyword(s):

B Cell ◽

Hairy Cell Leukemia ◽

Mapk Signaling ◽

Mitogen Activated Protein Kinase ◽

Braf V600e ◽

Common Mutation ◽

Hairy Cell ◽

Cell Leukemia ◽

Mitogen Activated Protein ◽

Almost All

BRAF is a serine/threonine kinase with a regulatory role in the mitogen-activated protein kinase (MAPK) signaling pathway. A mutation in the RAF gene, especially in BRAF protein, leads to an increased stimulation of this cascade, causing uncontrolled cell division and development of malignancy. Several mutations have been observed in the gene coding for this protein in a variety of human malignancies, including hairy cell leukemia (HCL). BRAF V600E is the most common mutation reported in exon15 of BRAF, which is observed in almost all cases of classic HCL, but it is negative in other B-cell malignancies, including the HCL variant. Therefore it can be used as a marker to differentiate between these B-cell disorders. We also discuss the interaction between miRNAs and signaling pathways, including MAPK, in HCL. When this mutation is present, the use of BRAF protein inhibitors may represent an effective treatment. In this review we have evaluated the role of the mutation of the BRAF gene in the pathogenesis and progression of HCL.

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Immunoconjugates and new molecular targets in hairy cell leukemia

Hematology ◽

10.1182/asheducation.v2012.1.660.3798659 ◽

2012 ◽

Vol 2012 (1) ◽

pp. 660-666 ◽

Cited By ~ 19

Author(s):

Robert J. Kreitman

Keyword(s):

Hairy Cell Leukemia ◽

Poor Prognosis ◽

Residual Disease ◽

Single Agent ◽

Braf V600e ◽

Late Relapse ◽

Hairy Cell ◽

Cell Leukemia ◽

Purine Analogs ◽

Purine Analog

Abstract Hairy cell leukemia (HCL) is a B-cell malignancy that in its classic form is exquisitely sensitive to single-agent purine analog therapy, but that is associated in many patients with late relapse and eventual purine analog resistance. Minimal residual disease, which is present in most patients achieving complete remission with purine analogs, retains Ags that are ideal for targeted therapy. Rituximab, which targets CD20, is active as a single agent, particularly if combined with purine analogs. Recombinant immunotoxins targeting either CD25 or CD22 and containing truncated Pseudomonas exotoxin have achieved major responses in relapsed/refractory HCL. Moxetumomab pasudotox in phase 1 testing achieved responses in 86% of such patients (complete in 46%) without dose limiting toxicity and often without MRD. Soluble CD22 has been used for improved detection and monitoring of HCL, particularly the poor-prognosis variant that lacks CD25. Ig rearrangements unique for each HCL patient have been cloned, sequenced, and followed by real-time quantitative PCR using sequence-specific reagents. Analysis of these rearrangements has identified an unmutated IGVH4-34–expressing poor-prognosis variant with immunophenotypic characteristics of either classic or variant HCL. The BRAF V600E mutation, reported in 50% of melanomas, is present in > 85% of HCL cases that are both classic and express rearrangements other than IGVH4-34, making HCL a potential target for specific inhibitors of BRAF V600E. Additional targets are being defined in both classic and variant HCL, which should improve both detection and therapy.

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Evaluation of BRAFV600E, NRAS and KRAS Mutations As Well As IGHV Usage for Diagnostic Use in Hairy Cell Leukemia, Hairy Cell Leukemia-Variant and Splenic Marginal Zone Lymphoma.

Blood ◽

10.1182/blood.v120.21.2679.2679 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2679-2679

Author(s):

Susanne Schnittger ◽

Frank Dicker ◽

Christiane Eder ◽

Sabine Jeromin ◽

Tamara Alpermann ◽

...

Keyword(s):

Flow Cytometry ◽

Hairy Cell Leukemia ◽

Braf V600e Mutation ◽

Braf V600e ◽

Leukemic Cells ◽

Multiparameter Flow Cytometry ◽

Splenic Marginal Zone Lymphoma ◽

Hairy Cell ◽

Cell Leukemia ◽

Equity Ownership

Abstract Abstract 2679 Background: The BRAF V600E mutation has recently been discovered in nearly all cases of hairy cell leukemia (HCL), but not in cases of HCL-variant (HCL-v). However, this perfect correlation has been challenged by studies reporting HCL cases without BRAF V600E. Interestingly, the immunoglobulin heavy chain variable region gene IGHV4–34, which has been associated with poor prognosis in HCL, appeared exclusively and to a high percentage in these BRAF V600E-negative cases of classic HCL and also in HCL-v (Xi et al., Blood, 2011). Further, splenic marginal zone lymphoma (SMZL) is a disease closely related to HCL and HCL-v and BRAF has been shown to be unmutated in this entity. Aims: 1. To characterize our cohorts of HCL, HCL-v and SMZL for the presence of BRAF V600E and to correlate the results with IGHV gene usage. 2. We hypothesized that other genes of the RAF/RAS pathway might be affected. Thus we analysed NRAS, and KRAS in addition to BRAF for mutations in all three entities. Methods: We analyzed the bone marrow or peripheral blood of 314 cases (182 cases with HCL, 49 cases with HCL-v, and 83 cases with SMZL) at diagnosis as confirmed by multiparameter flow cytometry and cytomorphology. The BRAF V600E mutation was analyzed by an mRNA-based reverse transcription allele-specific real-time quantification (RQ-PCR) assay. The BRAF V600E expression was calculated as %BRAF V600E/BRAF wt. NRAS and KRAS were analyzed by melting curve analysis and subsequent Sanger sequencing. IGHV genes and mutation status were analyzed by the use of Biomed-2 primers. An identity of ≥98% of the analyzed IGHV sequence compared to published germline sequences was considered an unmutated IGHV status. Results: In our cohort the median percent leukemic cells was 16% (range 0.2–74%) for HCL, 33% (range 5–59%) for HCL-v and 29% (range: 1–84%) for SMZL as determined by multiparameter flow cytometry. The BRAF V600E mutation was detected in 178/182 (97.8%) of HCL cases, whereas 0/49 of HCL-v and 0/83 SMZL were positive. Thus, the BRAF V600E mutation is 100% specific for HCL regarding these three entities. The median BRAF V600E expression ratio of positive cases was 14.2 (range 0.22 – 280.3). After normalization to % pathological cells as assessed by multiparameter flow cytometry the median ratio was 173 (range:22–1,788). However, in 4 cases with 4%, 8%, 28% and 66% percent leukemic cells by multiparameter flow cytometry, which is within the clone size that can be clearly detected by the BRAF V600E-specific RQ-PCR assay, no mutation was detected. Thus, BRAF V600E detection used for the identification of HCL has a sensitivity of 97.8%. Further, NRAS and KRAS mutation screening in all cases with HCL, HCL-v, and SMZL did not detect any mutation except for one case with SMZL that harboured an NRAS Gly12Asp mutation. This case was found to have an MDS in parallel and thus the mutation more likely belongs to the MDS clone. Thus, analysis of NRAS and KRAS mutations does not further improve diagnostics in these diseases. Further, we analyzed the IGHV usage in all 4 BRAF unmutated HCL and in additional 60 cases (total n=64) with HCL and 41 cases with HCL-v. IGHV4–34 usage was very frequent in HCL-v with 14/41 (34.1%). In contrast, it was never detected in HCL including the BRAF wildtype cases. Thus, we were not able to confirm the usage of the IGHV4–34 gene, which was previously suggested for BRAF V600E negative HCL. On the other hand IGHV5–51 was most frequently found in HCL (9/64, 14.1%) but never detected in HCL-v. We detected an unmutated IGHV status in 12/62 (19.4%) of HCL, which was less frequent compared to 14/40 (35.0%) in HCL-v (p = 0.095). The IGHV mutation status was unmutated in 9/11 (81.8%) IGHV4–34 cases (100% identity to germline each). The four cases of HCL, which lacked BRAF V600E mutation, expressed the IGHV genes IGHV1–3*01 (96.5% identity), IGHV1–69*02 (94.0% identity), IGHV3–9*01 (96.9% identity) and IGHV6-1*01 (99.0% identity), which were also expressed by various BRAF V600E positive HCL cases in our cohort. Conclusions: 1) In our cohort of 314 cases with HCL, HCL-v, and SMZL we confirm a high specificity (100%) and sensitivity (97.8%) for BRAF V600E mutations to detect HCL. 2) Other RAS pathway mutations (NRAS, KRAS) were not detected in any of the three analysed entities. 3) In the 4 rare cases of HCL with BRAF wt we were not able to confirm the previously postulated IGHV4–34 usage. 4) IGHV4–34 further delineates classic HCL from HCL-v. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Equity Ownership. Dicker:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Equity Ownership.

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Hairy cell leukemia cell lines expressing annexin A1 and displaying B-cell receptor signals characteristic of primary tumor cells lack the signature BRAF mutation to reveal unrepresentative origins

Leukemia ◽

10.1038/leu.2012.163 ◽

2012 ◽

Vol 27 (1) ◽

pp. 241-245 ◽

Cited By ~ 16

Author(s):

N J Weston-Bell ◽

D Hendriks ◽

G Sugiyarto ◽

N A Bos ◽

H C Kluin-Nelemans ◽

...

Keyword(s):

B Cell ◽

Tumor Cells ◽

Hairy Cell Leukemia ◽

Leukemia Cell ◽

Cell Receptor ◽

B Cell Receptor ◽

Annexin A1 ◽

Hairy Cell ◽

Cell Leukemia ◽

Leukemia Cell Lines

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A Rare Case of Hairy Cell Leukemia with Unusual Loss of CD123 Associated with COVID-19 at the Time of Presentation

Case Reports in Oncology ◽

10.1159/000512830 ◽

2020 ◽

Vol 13 (3) ◽

pp. 1430-1440

Author(s):

Samah Kohla ◽

Feryal A. Ibrahim ◽

Mahmood B. Aldapt ◽

Hesham ELSabah ◽

Shehab Mohamed ◽

...

Keyword(s):

Bone Marrow ◽

Cancer Patients ◽

Hairy Cell Leukemia ◽

Bone Marrow Examination ◽

Assisted Ventilation ◽

Braf V600e ◽

Leukemic Cells ◽

Hairy Cell ◽

Cell Leukemia ◽

First Case

Coronavirus disease 2019 (COVID-19) pandemic has been a serious threat and has been reported with different presentations and complications. Older age, along with comorbidities such as diabetes, hypertension, or cardiac disease, increases the risk factors for COVID-19 severity and death [N Engl J Med. 2020;382(18):1708–20 and Lancet Respir Med. 2020 05;8(5):475–81]. It is proposed that cancer patients have a significantly higher incidence of severe incidents including admission to the intensive care unit, the necessity for assisted ventilation, and even death after catching the virus compared with non-cancer patients [Lancet Oncol. 2020;21(3):335–7]. It is also described that cancer patients appear to be twice as likely to contract infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) [JAMA Oncol. 2020;6(7):1108–10]. Hairy cell leukemia (HCL) is a rare B-cell lymphoproliferative disorder, with patients typically presenting with cytopenias, marked splenomegaly in 80–90% of patients, circulating leukemia cells, bone marrow infiltration and the presence of BRAF V600E somatic mutation [Indian J Hematol Blood Transfus. 2014;30(Suppl 1):413–7]. Leukemic cells classically have central nuclei and abundant cytoplasm with hairy-like projections and express CD11c, CD25, CD103, and CD123 [Indian J Hematol Blood Transfus. 2014;30(Suppl 1):413–7]. Loss of CD123 in HCL has been rarely reported in the literature [Am J Hematol. 2019;94(12):1413–22]. We describe a unique case of a COVID-19-positive male who presented with severe respiratory symptoms, deteriorated quickly, and was intubated. Workup of severe progressive pancytopenia and bone marrow examination revealed HCL without splenomegaly and with atypical unusual loss of CD123. To our knowledge, this is the first case of CD123-negative HCL without splenomegaly associated with COVID-19 infection as the initial presentation.

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Presence and Absence of the BRAF V600E Mutation in Hairy Cell Leukemia and Its Variants

Blood ◽

10.1182/blood.v118.21.931.931 ◽

2011 ◽

Vol 118 (21) ◽

pp. 931-931

Author(s):

Robert J. Kreitman ◽

Liqiang Xi ◽

Winnifred Navarro ◽

Maryalice Stetler-Stevenson ◽

Evgeny Arons ◽

...

Keyword(s):

Hairy Cell Leukemia ◽

Braf Mutation ◽

Braf V600e ◽

Hairy Cell ◽

Cell Leukemia ◽

Wild Type ◽

Purine Analogs ◽

Ighv Gene ◽

Purine Analog ◽

Gene Usage

Abstract Abstract 931 Background: Hairy cell leukemia (HCL) is a B-cell malignancy with distinctive immunophenotype. Purine analog therapy achieves durable complete remissions in 65–90% of patients. HCL variant (HCLv), recognized by the World Health Organization (WHO) as a different disease, lacks CD25, annexin A1 and/or TRAP, and responds poorly to purine analogs with only partial responses (PR) in <50% and lower overall survival (OS) from diagnosis. The recently described HCL variant expressing the immunoglobulin rearrangement IGHV4-34 also has poor response to purine analogs and OS, but can resemble HCL or HCLv immunophenotypically. The V600E BRAF mutation was recently reported present in 100% of 48 patients with HCL and absent in 16 with related disorders including at least 1 case of HCLv. We wished to confirm these results and test well-characterized cases of HCLv and IGHV4-34+ HCL. Methods: DNA was prepared from the blood of 70 patients with HCL and HCLv, 64 of whom were molecularly characterized with respect to IGHV gene usage. The mutation analysis of BRAF c.1799T>A (V600E) and other variants among codons 599–601 within exon 15 was performed using a target-specific mutant allele enriching COLD-PCR technique followed by pyrosequencing. The apparent percentage of mutant versus wild-type alleles was calculated with allele quantification (AQ) mode using PyroMark Software. The threshold AQ value for classifying samples as positive as a mutation was calculated as 3 standard deviations above the mean value of 24 normal blood samples. Results: Out of 70 total patients tested, 16 (23%) were diagnosed as HCLv based on WHO criteria, and the other 54 were classic HCL. Thirteen (19%) of the 70 cases expressed IGHV4-34, 5 classic HCL and 8 HCLv immunophenotypically. All 6 cases not characterized for IGHV gene usage were classic HCL. The analytic sensitivity of the pyrosequencing assay using cell line controls containing BRAF mutations was <5% tumor cells, and all cases were required to have ≥10% of total white blood cells as HCL. As shown in the table, 28 (40%) of the cases were wild-type with respect to BRAF, including all cases of HCLv. In addition, all 13 cases of IGHV4-34+ HCL, including 5 with classic immunophenotype, were negative for the V600E mutation. Moreover, 7 classic HCL cases were wild-type at V600 of BRAF, including 1 with unknown IGHV and 6 expressing IGHV2-70, IGHV3-15, IGHV3-23, IGHV3-48, IGHV4-39 and IGHV4-59. These 7 cases were relatively resistant to purine analog therapy although numbers were too few for statistical comparisons. In one of these 7 classic HCL cases, CD25 expression had decreased over time. Conclusions: The V600E BRAF mutation is not present in HCLv or in HCL cases with typical immunophenotype expressing IGHV4-34. A significant minority of other classic HCL cases, 7 (14%) of 49, were negative for the V600E BRAF mutation. It is possible that the V600E BRAF mutation is related to factors other than those affecting immunophenotype, including those influencing prognosis. Additional studies will be needed to better understand the role of V600E-mutated BRAF in HCL and the molecular basis of variants of this disease (Supported in part by NCI, intramural research program, NIH). Disclosures: No relevant conflicts of interest to declare.

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Molecular Evidence of Ongoing Antigen Driven Somatic Hypermutation in the Natural History of Classic and Variant Hairy Cell Leukemia

Blood ◽

10.1182/blood.v126.23.2648.2648 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2648-2648

Author(s):

Evgeny Arons ◽

Sarah Davies ◽

Katherine Still ◽

Nathan Chai ◽

Katherine Potocka ◽

...

Keyword(s):

B Cell ◽

Hairy Cell Leukemia ◽

Conflicts Of Interest ◽

Somatic Hypermutation ◽

Braf V600e ◽

Molecular Evidence ◽

Malignant Cells ◽

Hairy Cell ◽

Cell Leukemia ◽

Normal Peripheral Blood

Abstract Classic hairy cell leukemia (HCL) is a clonal chronic B-cell malignancy representing 2% of all leukemias, with neoplastic B lymphocytes in the blood, bone marrow and organs of the classic reticuloendothelial system. HCL variant (HCLv) is less common, typically has inferior prognosis, and usually lacks several HCL-related features including BRAF V600E, CD25, and Annexin A1. The malignant cells in HCL and HCLv patients have one or in unusual cases two different functional immunoglobulin heavy chain rearrangements. Significant variability can be observed in the 3rd complementarity determining region (CDR3), comprising the variable heavy (IGHV), diversity (IGHD), and joining (IGHJ) domains. A role for continued antigen selection in the pathogenesis and evolution of HCL and HCLv has been suggested but limited evidence has been found. We examined HCL and HCLv-derived IGHV genes by massive parallel deep sequencing in 15 HCL and 7 HCLv cases. In all studied samples, a productive monoclonal tumor-derived IGH sequence was detected. Analysis of all reads sharing clonal CDR3 motifs revealed the existence of dominant subclone expansion and multiple subclonal variants. Analysis of phylogenetic trees indicated multiple branching and therefore exposure to multiple rounds of somatic hypermutation (SHM) in the evolution of clonally-related malignant cells. To investigate a possible cause of the high SHM activity and the large number of subclones in HCL and HCLv, we studied the expression of activation induced cytidine deaminase (AID), an enzyme essential for somatic hypermutations, expressed by germinal center (GC) B-cells where SHM occurs. In 5 HCL and 3 HCLv cases we detected a high expression level of AID mRNA, including wild-type and 2 splice variants; in 10 HCL, 4 HCLv, and 3 normal peripheral blood samples, AID expression was not detected using standard end-point PCR conditions. However, more sensitive real-time quantitative PCR detected AID transcripts in virtually all tested HCL and HCLv cases, although the range of transcript levels was large between different cases and varied with individual cases over time. The data obtained for both HCL and HCLv fit a model of tumorigenesis in which the BRAF V600E mutation (or another event in HCLv) initiates neoplastic transformation in a GC B-cell committed to terminal differentiation, but still thereafter still targeted by ongoing SHM Disclosures No relevant conflicts of interest to declare.

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