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

Blood ◽  
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.

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

Blood ◽  
2012 ◽  
Vol 119 (14) ◽  
pp. 3330-3332 ◽  
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.

scholarly journals Immunoconjugates and new molecular targets in hairy cell leukemia

Hematology ◽  
2012 ◽  
Vol 2012 (1) ◽  
pp. 660-666 ◽  
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.

scholarly journals Genomics of Hairy Cell Leukemia

2017 ◽  
Vol 35 (9) ◽  
pp. 1002-1010 ◽  
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).

Molecular Characteristic of Variant and Classic Hairy Cell Leukemia.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1063-1063
Author(s):  
Evgeny Arons ◽  
Tara Suntum ◽  
Joel Sunshine ◽  
Anna Orthwein ◽  
Inger Margulies ◽  
...  
Keyword(s):  
Hairy Cell Leukemia ◽  
Rank Order ◽  
Tumor Burden ◽  
Hairy Cell ◽  
Cell Leukemia ◽  
Monoclonal Immunoglobulin ◽  
Purine Analogs ◽  
Purine Analog ◽  
Vh Gene ◽  
Germline Sequence

Abstract 10–20% of patients with hairy cell leukemia (HCL) have a variant (HCLv) and present with high tumor burden in spleen and peripheral blood, and are less responsive to purine analogs. HCLv cells lack CD25 and sometimes CD103. Differentiating HCLv from classic HCL (HCLc) is sometimes difficult and it is unclear whether HCLv and HCLc are different disorders. Detailed molecular distinctions between HCLv and HCLc have not been reported. Patients with HCL were studied by flow cytometry and PCR to sequence the monoclonal immunoglobulin heavy chain rearrangements. 50 and 21 VH-D-JH rearrangements were obtained from 49 HCLc and 19 HCLv patients, respectively. All rearrangements except 2 each for HCLc and HCLv were productive. The incidence of VH4 usage was higher in HCLv than in HCLc (57% vs 20%, p=0.0041). VH4-34 was the most common VH gene in HCLv and was more common in HCLv than in HCLc (33% vs 8%, p=0.012). The percentage of rearrangements which were unmutated (defined as &lt; 2% somatic mutations) was higher in HCLv than in HCLc (43% vs 18%, p=0.038), but % homology was similar in both groups by rank order (94.9 vs 94.3% p=0.22). However, in comparing 11 VH4-34 with 60 other rearrangements, homology was higher with VH4-34 (median 99.2 vs 95.2%, p &lt; 0.0001). In fact, the higher frequency of unmutated rearrangements in HCLv vs HCLc was due to VH4-34 cases, since the 7 HCLv VH4-34 rearrangements were all unmated and had higher homology than the other 14 HCLv rearrangements (median 99.6 vs 94.2%, p=0.006). Moreover, unmutated rearrangement incidence was similar between HCLv and HCLc for non-VH4-34 cases (14 vs 13%, p=1.0, median homology 94.2% vs 95.3%, p=0.5). Of the 4 VH4-34+ HCLc (CD25+) patients, 3 (75%) had unmutated rearrangements, and these 3 all had clinical features of HCLv, including presenting with lymphocytosis, large splenomegaly, absent cytopenias, and primary failure of purine analog treatment. Our data shown that homology of monoclonal immunoglobulin rearrangement to the germline sequence of HCL patients appears more related to VH4-34 status than to whether patients have HCLv or HCLc. In fact, no molecular distinctions between HCLv and HCLc were observed in VH4-34-negative patients. Our data suggest VH4-34-positive HCL is itself a variant of HCL affecting ~15% of our patients. Such patients have features of HCLv, but their HCL cells can be CD25+.

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

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5112-5112
Author(s):  
Evgeny Arons ◽  
Hong Zhou ◽  
Yonghong Wang ◽  
Daniel Edelman ◽  
Robert J. Kreitman ◽  
...  
Keyword(s):  
B Cell ◽  
Hairy Cell Leukemia ◽  
Poor Prognosis ◽  
Braf V600e ◽  
Leukemic Cells ◽  
Annexin A1 ◽  
Expression Data ◽  
Hairy Cell ◽  
Cell Leukemia ◽  
Wild Type

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.

Co-Treatment Of Hairy Cell Leukemia and Melanoma With The BRAF Inhibitor Dabrafenib

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5311-5311 ◽  
Author(s):  
Leslie A. Andritsos ◽  
James S. Blachly ◽  
Kari Kendra ◽  
Gerard Lozanski ◽  
Michael R. Grever
Keyword(s):  
Bone Marrow ◽  
Malignant Melanoma ◽  
Hairy Cell Leukemia ◽  
Braf Mutation ◽  
Braf Inhibitor ◽  
Braf V600e Mutation ◽  
Braf V600e ◽  
Hairy Cell ◽  
Cell Leukemia ◽  
Marrow Cellularity

Abstract The activating BRAF mutation V600E has been identified in many human cancers, including colon and lung adenocarcinoma, papillary thyroid cancer, malignant melanoma, and hairy cell leukemia. Here we report for the first time treatment of hairy cell leukemia and malignant melanoma both harboring the BRAF V600E mutation with the BRAF inhibitor dabrafenib. The patient is a 67-year-old man with a history of classic hairy cell leukemia (immunophenotype CD11c, CD19, CD20 (bright), CD25, and CD103). At the time of diagnosis he had pancytopenia and received therapy with cladribine 0.12 mg/kg/day as a 2 hour infusion daily for 5 days, achieving a complete hematologic remission (CHR). His disease recurred 2 years later and he was again treated with cladribine 0.9 mg/kg/day as a 7 day continuous infusion, achieving a CHR. He remained in remission for 5 years, and this time received salvage therapy with pentostatin 4 mg/m2 every 2 weeks for a total of 12 doses. He achieved a CHR with minimal residual disease on bone marrow biopsy (0.3% of lymphocytes). He also had dyserythropoiesis concerning for myelodysplastic syndrome in addition to neurologic toxicity with gait imbalance. He was managed expectantly for the next 2 years, during which time he developed an 8 mm red nodule on the extensor surface of his right forearm, and a shave biopsy showed nodular melanoma, Clark’s level IV. He underwent a wide local excision with a negative axillary sentinel lymph node biopsy followed by adjuvant sargramostim (GM-CSF) for 12 months. His melanoma then recurred at the site of the prior excision. A PET scan showed an additional lesion in the midportion of the right arm. The excised solitary recurrence was sent for BRAF V600E mutation testing, which was positive. He received a course of radiotherapy (30 Gy over 14 days) to the affected limb, after which he had no evidence of disease. During this time, he was found to have worsening thrombocytopenia and splenomegaly, as well as a rising IL2 receptor level (peak 3952 U/mL; normal < 970), while bone marrow biopsy showed a 20% cellular marrow with 40% involvement by classic HCL. BRAF testing of the bone marrow by Sanger sequencing was positive for the V600E mutation. Because both his HCL and melanoma harbored the BRAF V600E mutation, the patient was eligible for and enrolled on a phase I clinical trial of dabrafenib for BRAF V600E mutant malignancies. Dabrafenib was initiated orally at a dose of 150 mg twice daily. Each cycle was 28 days. After 3 cycles, the bone marrow cellularity had improved to 30% with a decrease in the leukemic content to 10-15% of marrow cellularity. After 6 cycles, the bone marrow cellularity was normal for age with no residual HCL detectable by immunohistochemical stains or flow cytometric immunophenotyping. PET/CT scan at this time demonstrated no FDG avid lesions and no splenomegaly. Toxicites have consisted of characteristic RAF-associated skin changes and one instance of squamous cell carcinoma, which was excised. He has otherwise had no side effects from therapy. The patient has now completed 12 cycles of therapy with dabrafenib without evidence of either HCL or melanoma. He will remain on therapy as long as he is deriving clinical benefit per protocol. Given the increased risk of second primary malignancies in HCL, BRAF mutation testing should be considered for patients developing solid tumors in which this has been described, as co-treatment may be possible. Disclosures: Off Label Use: Dabrafenib for treatment of hairy cell leukemia. Kendra:Glaxo Smith-Kline: Research Funding.

Evaluation of the Interest of BRAF –V600E Mutation Detection By Arms-qPCR in Hairy Cell Leukemia Diagnosis and Treatment Monitoring

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2956-2956
Author(s):  
Ouda Ghoual ◽  
Stéphanie Jouve ◽  
Véronique Salaun ◽  
Stéphane Cheze ◽  
Michele Malet ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Hairy Cell Leukemia ◽  
Braf Mutation ◽  
Hematologic Malignancies ◽  
Braf V600e ◽  
Hairy Cell ◽  
Cell Leukemia ◽  
Prolymphocytic Leukemia ◽  
Number Of Patients ◽  
Cell Proliferations

Abstract Background: The discovery of BRAF V600E (Tiacci et al. N.Eng J Med, 2011), has introduced molecular biology in the management of Hairy cell leukemia (HCL). Various techniques for BRAF detection with a specificity of 100% were developed. According to literature, BRAF mutation has been reported in one case of CLL and one B-prolymphocytic leukemia (Langabeer et al. Leukemia research, 2012). The development of BRAF inhibitors for refractory HCL to purine nucleoside analogues (PNA) renders the detection of BRAF mutation indispensable for the diagnosis and monitoring of the minimal residual disease (MRD). Objectives: To test a quantitative PCR on HCL patients at diagnosis, during relapse and at MRD monitoring compared to flow cytometry (FCM), then evaluate its usage on peripheral blood (PB) versus bone marrow (BM) sampling. Methods: We developed a relative quantitative amplification refractory mutation PCR technique (ARMS-qPCR). A retrospective study on a total of 99 samples between 1998 and 2014 was conducted. These samples were previously analyzed by a 4 colors FCM. 38 patients with HCL were tested at diagnosis (21 samples from PB and 17 from BM). 36 patients with other hematologic malignancies were studied, 5 other hairy cell proliferations (3 HCL-Variant (HCL-V) and 2 splenic lymphocyte villous lymphomas SLVL) and 31 non hairy cell proliferations (13 chronic lymphocytic leukemias (CLL), 13 atypical CLL, 3 large B-cell non Hodgkin lymphomas, 2 mantle cell lymphomas, 1 marginal zone lymphoma, 1 acute myeloid leukemia, 1 mastocytosis). Subsequently, we studied 14 patients in relapse (8 samples from PB, 5 from BM and 1 from a diaphragm nodule). Among these patients, 9 had received PNA, 3 interferon (IFN), 1 PNA + rituximab and 1 patient with unknown treatment. We monitored 11 patients for MRD (6 samples from PB and 5 from BM), 6 were treated with PNA, 1 with IFN and 1 with PNA + rituximab and 3 patients with unknown treatment. Results: The sensitivity of our ARMS-qPcr technique attained 0.001%. At diagnosis, the tumor cells ranged from 0.5% to 91% in PB and 3% to 73% in BM. All patients diagnosed as HCL by FCM were also detected by our PCR technique. The average mutated BRAF allele was 8% (0.02-18%) at diagnosis in the PB and 6% (2.5 -14%) in BM. The mutation was not detected for any of the patients harboring other hairy cell proliferations. Of the 36 patients with other hematologic malignancies, no signal was detected except for a weak one for 2 CLL patients with atypical morphology and 1 mixed type CLL/prolymphocytic leukemia (mutated allele: 0.03%, 0.21% and 1% respectively). This tempers the total specificity of BRAF detection in HCL described in earlier publications. At relapse, tumor cells ranged from 0.3% to 81% in PB and 3% to 30% in BM. The average mutated BRAF allele was 5.4% (0.09% -20%) in PB and 3.4% (0.6% -7.5%) in BM. Concerning evaluation of MRD, tumor cells ranged from 0% to 3% in PB and 0% to 2% in BM. All patients having malignant cells detected by FCM were detected by ARMS-qPCR. 2 patients were undetectable by both methods, 1 was treated by PNA + rituximab and the treatment was unknown for the other. The average mutated BRAF allele was 0.5% (0% -1%) in PB and 0.3% (0% -1%) in BM. Patients treated by PNA or IFN and tested in our study were positive for the MRD. DNA was obtained simultaneously from PB and BM for 3 patients (2 diagnosis and 1 relapse). The BRAF mutated allele was similar in PB and BM for 2 patients (4% and 11% in PB, 4% and 14% in BM respectively) and the circulating tumor cells were 60% and 61%. As for the 3rd patient, a lower mutated allele percentage (0.09%) was detected with 1% of tumor cells in blood, where 4% of the mutated allele was found in BM with 25% of tumor cells. Conclusion: This ARMs-qPcr allowed the HCL diagnosis of 100% of tested patients and the differential diagnosis with other hairy cell proliferations though a weak signal was detected in 3 atypical CLL cases. Using this PCR for relapse detection and MRD monitoring is as performing as the FCM. PB sampling which is less invasive than BM puncture seems to be adequate for HCL molecular study. BM aspirate may be considered in case of diagnostic difficulties or very few tumor cells. All patients treated by PNA or IFN and tested in our study were positive for the MRD suggesting that a complete molecular response is hardly achieved with these drugs. This should be confirmed on a larger number of patients as well with the emerging therapies. Disclosures No relevant conflicts of interest to declare.

scholarly journals Concomitant BRAF Mutation in Hairy Cell Leukemia and Papillary Thyroid Cancer

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5140-5140
Author(s):  
Shehab Mohamed ◽  
Mohamed A Yassin ◽  
Abdulqadir Jeprel Nashwan ◽  
Halima El Omri ◽  
Firyal Ibrahim ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Thyroid Cancer ◽  
Papillary Thyroid Cancer ◽  
Hairy Cell Leukemia ◽  
Braf Mutation ◽  
Braf V600e ◽  
Hairy Cell ◽  
Papillary Thyroid ◽  
Cell Leukemia ◽  
Cell Neoplasm

Abstract Hairy cell leukemia (HCL) is an uncommon but distinct form of mature B-cell neoplasm that originates from activated late B-cells. It represents only 2% of all adult lymphoid leukemia; patients are predominantly middle-aged to elderly males with a median age of 50 years and is characterized by pancytopenia, monocytopenia and usually associated with massive splenomegaly. HCL associated with BRAF mutation 100% of cases, it's associated with hematological and oncological malignancies such as melanoma and papillary thyroid cancer with positive BRAF in 40 % of cases. Although the association of both cancers (HCL & papillary thyroid cancer) with BRAF mutation is well established in the literature, up to our knowledge, this specific combination has not been previously reported in one patient. Here we report a case of 48-year old Lebanese male, who presented to with bilateral hip pain and found to have lytic bone lesions on both x-ray and MRI. HIS CBC were normal and abdominal US didn't show any splenomegaly. Work-up for myeloma were negative. Bone marrow examination and flow cytometry results confirmed the diagnosis of hairy cell leukemia. The patient treated with cladrabine. Patient responded but have continues fever, PUO included Piston tomography showed abnormal uptake in thyroid. Ultrasound and final needle aspiration diagnose him as case of papillary thyroid cancer. He was treated with total thyroidectomy and followed up with RAI 30 micori. We sent BRAF from both bone marrow biopsy and thyroid tissue which turn out positive in both. The mutation results in substitution of adenine for thymine at position 1799 in exon 15 of the BRAF that replaces Valine (V) by glutamate (E) at amino acid 600(BRAF V600E). Although the BRAF V600E mutation is frequently present in different neoplasms, such as melanoma, papillary thyroid cancer, non-small cell lung cancer, colorectal cancer and Langerhans cell histiocytosis (X), within the lymphoid neoplasms, the BRAFV600E mutation is found to be highly specific for HCL and testing for this mutation is particularly useful in differentiating classic HCL from other B- cell neoplasm with overlapping features, such as HCL variant Mutation in BRAF (particularly V600E) in HCL remarkably increase the BRAF kinase activity renders the protein constitutively active, phosphorylating then ERK as a monomers independent from upstream regulatory signals or in a RAS-independent manner leading to constitutive activation of RAF-MEK-ERK signaling pathway and enhanced survival of leukemic hairy cells, similar to what occurs in other BRAF-mutated tumors as papillary thyroid carcinomas Other BRAF mutations outside exon 15 were rarely reported as exon 11 F468C and D449E mutations. We emphasize on the link of BRAF mutation in HCL and papillary thyroid cancer. The biology has been established but never in real clinical case. We recommend having high clinical suspicion and sending BRAF mutation in those types of cancers and link it with other possible abnormal findings, as might detect more cases of similar association. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Treatment of Hairy Cell Leukemia with a Focus on Long Lasting Responders to Cladribine: A Thirty-Year Experience from the Institute of Hematology of Bologna

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4005-4005
Author(s):  
Alessandro Broccoli ◽  
Carolina Terragna ◽  
Vittorio Stefoni ◽  
Cinzia Pellegrini ◽  
Beatrice Casadei ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Peripheral Blood ◽  
Hairy Cell Leukemia ◽  
Braf V600e Mutation ◽  
Braf V600e ◽  
Hairy Cell ◽  
Cell Leukemia ◽  
First Line ◽  
Advisory Committees ◽  
Purine Analogs

The treatment of hairy cell leukemia (HCL) has deeply changed over years. Purine analogs, namely cladribine (2CdA) now represent the treatment of choice. The BRAF V600E mutation is now regarded as the pathogenic event. One hundred and eighty-four patients were followed between 1986 and 2018 and treated according to era-specific guidelines. This is the largest monocentric series reported. Responses were classified by combining Consensus Resolution criteria and marrow immunohistochemistry. Patients were grouped according to the number of treatment lines they received (table). Ten patients treated with frontline 2CdA and in complete response (CR) for at least 5 years were tested for the presence of the BRAF V600E mutation in peripheral blood by droplet digital PCR as a molecular marker for active disease. Patients treated first line responded in 86% of cases, with 44% CR. Response rates remained high throughout the first 4 lines (84%, 81%, 79% for the second line onward, with CR in 38%, 37%, 15% of cases respectively), although decreasing progressively with the number of treatments received. One hundred and twenty-two patients received 2CdA as first line treatment, with a response rate of 86% and a CR rate of 54%. Among the 66 CR patients, 45 (68%) have never received further therapy: 11 patients are in continuous CR between 5 and 10 years after treatment, 14 between 10 and 20 years and 3 patients at more than 20 years. Median time-to-next treatment (TTNT) for patients after receiving 2CdA was 8.2 years: partial responders had a significantly shorter median TTNT than CR patients (5.3 years versus median not reached at 25.8 years, p=0.0001) (figure). Seven patients in CR for more than 5 years after front line 2CdA were BRAF V6500E negative in peripheral blood. One of these displayed disease recurrence and required further treatment roughly 2 years later. Three patients were positive for the BRAF V600E mutation at 6.5, 8.4 and 13.7 years after treatment and developed an overt disease relapse between 4 months and 2 years. Patients with HCL require subsequent lines of therapy in more than 50% of cases. Purine analogs allow significant response rates when applied first line and upon retreatment. Some patients may enjoy long lasting responses after one course of 2CdA and display no evidence of BRAF V600E mutation in peripheral blood. A PCR-based evaluation of the allelic burden in peripheral blood may provide information regarding disease activity over time. Figure Disclosures Cavo: Celgene, Janssen, Amgen, BMS, Abbvie, Takeda: Honoraria; Janssen, Celgene: Other: Travel Accommodations; Janssen, Celgene: Speakers Bureau; Janssen, Celgene, Amgen, Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees. Zinzani:TG Therapeutics: Honoraria, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kyowa Kirin: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Immune Design: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sandoz: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Verastem: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; MSD: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Eusapharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Consultancy; Celltrion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Portola: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

scholarly journals Vemurafenib Plus Rituximab in Hairy Cell Leukemia: A Promising Chemotherapy-Free Regimen for Relapsed or Refractory Patients

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1214-1214 ◽  
Author(s):  
Enrico Tiacci ◽  
Luca De Carolis ◽  
Francesco Zaja ◽  
Achille Ambrosetti ◽  
Eugenio Lucia ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Hairy Cell Leukemia ◽  
Limit Of Detection ◽  
Single Agent ◽  
Braf Inhibitor ◽  
Braf V600e ◽  
Phase 2 ◽  
Hairy Cell ◽  
Cell Leukemia ◽  
Purine Analogs

Abstract BACKGROUND: Hairy cell leukemia (HCL) responds well to purine analogs, but up to 50% of patients relapse. We previously identified the BRAF-V600E mutation as the genetic lesion underlying HCL (NEJM 364:230-2315, 2011), and successfully targeted this mutation in the clinic with the oral BRAF inhibitor vemurafenib through an academic phase-2 multi-center Italian trial in HCL patients relapsed after or refractory to purine analogs (NEJM 373:1733-1747, 2015). In these heavily pre-treated patients, vemurafenib given for a median of 16 weeks produced 96% of responses, including 9/26 (35%) complete remissions (CR) and 16/26 (61%) partial remissions (PR), which were obtained after a median of 8 weeks of treatment. Even in complete responders, immunohistochemistry showed residual (~10%) bone marrow HCL cells at the end of treatment, and relapses were common, occurring at a median of 19 months and 6 months in CR and PR patients respectively. Residual HCL cells resisting vemurafenib treatment might be targeted by concomitant immunotherapy with an anti-CD20 monoclonal antibody, an attractive strategy to potentially achieve a more profound response and a better clinical outcome through a chemotherapy-free approach. METHODS: We started an academic, phase-2, single-center trial (EudraCT 2014-003046-27) in relapsed/refractory HCL, which tests vemurafenib in combination with rituximab, another targeted non-myelotoxic drug with known single-agent activity in HCL. Eligibility was extended to patients relapsed also after monotherapy with a BRAF inhibitor. Vemurafenib was given at its standard dose (960 mg twice daily orally) for 8 weeks. Rituximab infusions (375 mg/m2intravenously) were given concomitantly with vemurafenib every 2 weeks, as well as sequentially (after the end of vemurafenib dosing) four times every 2 weeks. RESULTS: We have so far enrolled 22 patients in 16 months. Adverse reactions were reversible, usually mild and consistent with the known toxicity profile of the two drugs when used alone. Notably, a CR was achieved by all 14 patients already evaluable for efficacy (100%), including 4 who had relapsed after a BRAF inhibitor and 1 previously refractory to rituximab. Furthermore, 12/14 patients (86%) obtained the CR as early as after 4 weeks of vemurafenib and 2 concomitant rituximab infusions. This CR rate appears higher than that observed by us and others using vemurafenib alone in BRAF inhibitor-naive patients relapsed after or refractory to purine analogs (CR rate 35-42%; NEJM 373:1733-1747, 2015). Moreover, minimal residual disease (MRD) was undetectable in the bone marrow biopsy and aspirate of 8/11 patients evaluated (73%), both by immunophenotyping and by allele-specific PCR (limit of detection: 0.05% BRAF-V600E copies). In 5 of these 8 patients, MRD clearing was reached even before sequential rituximab dosing post-vemurafenib. In the remaining 3/11 patients, MRD was at most 5% in 2 vemurafenib-naive patients, and 10% in 1 patient relapsed after prior BRAF-inhibitor treatment. In contrast, residual bone marrow disease was a constant feature of all 26 patients treated by us with vemurafenib alone for a longer time period (NEJM 373:1733-1747, 2015). CONCLUSIONS: This study - which is the first one combining vemurafenib and rituximab in relapsed/refractory HCL - suggests that this non-myelotoxic regimen produces more numerous, faster and deeper CRs than vemurafenib alone. Enrollment continues. Disclosures Gaidano: Karyopharm: Consultancy, Honoraria; Morphosys: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau.

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