Evgeny Arons

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

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.

High prevalence of MAP2K1 mutations in variant and IGHV4-34-expressing hairy-cell leukemias.

To understand the genetic mechanisms driving variant and IGHV4-34–expressing hairy-cell leukemias, we performed whole-exome sequencing of leukemia samples from ten affected individuals, including six with matched normal samples. We identified activating mutations in the MAP2K1 gene (encoding MEK1) in 5 of these 10 samples and in 10 of 21 samples in a validation set (overall frequency of 15/31), suggesting potential new strategies for treating individuals with these diseases.

VH4-34+ hairy cell leukemia, a new variant with poor prognosis despite standard therapy

Hairy cell leukemia variant (HCLv) presents with high disease burden, lack of typical antigens like CD25, and poor response to standard treatments like cladribine. Occasionally, patients with classic HCL respond poorly. Clinical and molecular features of HCL and HCLv has not been compared. Rearrangements expressing immunoglobulin VH chain were sequenced, including 22 from 20 patients with HCLv and 63 from 62 patients with classic HCL. Most patients were seeking relapsed/refractory trials, representing a poor-prognosis population. VH4-34, a gene commonly used in autoimmune disorders, was observed in 8 (40%) HCLv and 6 (10%) classic (P = .004) HCL patients. Compared with 71 VH4-34(-) rearrangements, 14 VH4-34(+) rearrangements were more frequently (P < .001) unmutated, defined as greater than 98% homologous to germline sequence. VH4-34(+) patients had greater white blood cell counts at diagnosis (P = .002), lower response rate (P < .001) and progression-free survival (P = .007) after initial cladribine, and shorter overall survival from diagnosis (P < .001). Response and survival were more closely related to VH4-34 status than to whether or not patients had HCLv. VH4-34(+) HCL is an important disorder that only partly overlaps with the previously described HCLv. Response to initial single-agent cladribine therapy is suboptimal; these patients should be considered for alternative approaches, including antibody-related therapy.

The bruton tyrosine kinase inhibitor ibrutinib (PCI‐32765) blocks hairy cell leukaemia survival, proliferation and B cell receptor signalling: a new therapeutic approach

B cell receptor (BCR) signalling plays a critical role in the progression of several B‐cell malignancies, but its role in hairy cell leukaemia (HCL) is ambiguous. Bruton tyrosine kinase (BTK), a key player in BCR signalling, as well as B cell migration and adhesion, can be targeted with ibrutinib, a selective, irreversible BTK inhibitor. We analysed BTK expression and function in HCL and analysed the effects of ibrutinib on HCL cells. We demonstrated uniform BTK protein expression in HCL cells. Ibrutinib significantly inhibited HCL proliferation and cell cycle progression. Accordingly, ibrutinib also reduced HCL cell survival after BCR triggering with anti‐immunoglobulins and abrogated the activation of kinases downstream of the BCR (PI3K and MAPK). Ibrutinib also inhibited BCR‐dependent secretion of the chemokines CCL3 and CCL4 by HCL cells. Interestingly, ibrutinib inhibited also CXCL12‐induced signalling, a key pathway for bone marrow homing. Collectively, our data support the clinical development of ibrutinib in patients with HCL.

Minimal Residual Disease in Hairy Cell Leukemia Patients Assessed by Clone-Specific Polymerase Chain Reaction

Cladribine induces long-term complete remission in hairy cell leukemia (HCL) patients but does not clear minimal residual disease (MRD) according to high-sensitivity PCR assays. To quantify MRD in patients after anti-CD22 recombinant immunotoxin BL22 and other agents, we used a relative quantitative PCR (RQ-PCR) assay using a primer and probe, both patient specific for the immunoglobulin heavy chain rearrangement. Using this method, we were able to detect one Bonna 12 HCL cell in either 106 Jurkat cells or in 106 normal mononuclear cells. We studied 84 samples from 10 patients, taken before or after treatment with BL22 and other agents. Patient-specific RQ-PCR was much more sensitive than flow cytometry, which in turn was (as recently reported) more sensitive than PCR using consensus primers. RQ-PCR was positive in 62 of 62 (100%) flow-positive samples in 10 patients and in 20 of 22 (91%) flow-negative samples in six patients. The relative level of MRD as quantified by RQ-PCR correlated with disease status and remission. Thus, patient-specific RQ-PCR is the most sensitive test for MRD in HCL patients and could be used to determine maximal response in patients obtaining multiple cycles of nonmyelotoxic biological treatment for this disease.

Cladribine with Immediate Rituximab for the Treatment of Patients with Variant Hairy Cell Leukemia

Purpose: In contrast with the classic form, variant hairy cell leukemia (HCLv) responds poorly to single-agent purine analogs, expresses unmutated BRAF, has shorter overall survival, and lacks effective standard therapy. No treatment has achieved a high complete remission (CR) rate even in small series, and of 39 reported cases from six studies, overall response rate after cladribine was 44% with 8% CRs. Rituximab has been found to increase the sensitivity of malignant cells to cladribine, suggesting that combination with cladribine might improve response in HCLv. To test this hypothesis, patients with HCLv were treated with simultaneous cladribine and rituximab. Experimental Design: Patients with HCLv with 0 to 1 prior courses of cladribine received cladribine 0.15 mg/kg for days 1 to 5, with eight weekly doses of rituximab 375 mg/m2 beginning day 1. Restaging was performed, and minimal residual disease (MRD) in blood and marrow was quantified using PCR, immunohistochemistry, and flow cytometry. Results: By 6 months, 9 (90%) of 10 patients achieved CR, compared with 3 (8%) of 39 reported cases treated with cladribine alone (P < 0.0001). Of the 9 CRs, 8 remain free of MRD at 12 to 48 (median 27) months of follow-up. No dose-limiting toxicities were observed when beginning cladribine and rituximab on the same day, although most patients required short-term steroids to prevent and treat rituximab infusion reactions. Cytopenias in CRs resolved in 7 to 211 (median 34) days without major infections. Conclusion: Although cladribine alone lacks effectiveness for early or relapsed HCLv, cladribine with immediate rituximab achieves CRs without MRD and is feasible to administer. Clin Cancer Res; 19(24); 6873–81. ©2013 AACR.

Evidence of canonical somatic hypermutation in hairy cell leukemia

To compare hairy cell leukemia (HCL) with chronic lymphocytic leukemia (CLL) and normal B cells with respect to their B-cell receptors, somatic hypermutation (SHM) features in HCL were examined in a series of 130 immunoglobulin gene heavy chain rearrangements, including 102 from 100 classic (HCLc) and 28 from 26 variant (HCLv) patients. The frequency of unmutated rearrangements in HCLc was much lower than that in HCLv (17% vs 54%, P < .001) or historically in CLL (17% vs 46%, P < .001), but HCLv and CLL were similar (P = .45). As previously reported for CLL, evidence of canonical SHM was observed in HCLc rearrangements, including: (1) a higher ratio of replacement to silent mutations in the complementarity determining regions than in the framework regions (2.83 vs 1.41, P < .001), (2) higher transition to transversion ratio than would be expected if mutations were random (1.49 vs 0.5, P < .001), and (3) higher than expected concentration of mutations within RGYW hot spots (13.92% vs 3.33%, P < .001). HCLv met these 3 criteria of canonical SHM to a lesser extent. These data suggest that, whereas HCLc cells may recognize antigen-like CLL and normal B cells before malignant transformation, HCLv cells from some patients may originate differently, possibly without undergoing antigen recognition.

Characterization of T-cell repertoire in hairy cell leukemia patients before and after recombinant immunotoxin BL22 therapy

We previously reported that hairy cell leukemia (HCL) patients have high percentages of CD56+/CD57+/CD3+ large granular lymphocytes consistent with cytotoxic T-lymphocytes (CTLs), and other investigators have reported skewing of the T-cell repertoire. In previous studies of up to seven HCL patients, many of the 22 established T-cell receptor (TCR) beta variable region (TRBV) families showed mono- or oligoclonal restriction. To determine whether percentages of CTLs are correlated with TRBV clonal excess, we studied 20 HCL patients with flow cytometry, PCR of TCR gamma and TRBV regions, and fractional gel electrophoresis of PCR-amplified TRBV CDR3 domains (CDR3 spectratyping). Increased percentages of CD3+/CD8+/CD57+ CTLs correlated with more mono/oligoclonal and fewer polyclonal TRBV families (r=0.53; P=0.016). Age correlated with number of mono/oligoclonal TRBV families (r=0.51; P=0.022). Time since last purine analog therapy correlated with number of polyclonal TRBV families (r=0.46; P=0.040), but treatment with the anti-CD22 recombinant immunotoxin BL22 was not related to clonal excess. We conclude that abnormalities in the T-cell repertoire in HCL patients may represent deficient immunity, and may be exacerbated by purine analogs. Increased CD3+/CD57+ T-cells may be a useful marker of abnormal TRBV repertoire in HCL patients, and might prove useful in deciding whether patients should receive biologic antibody-based treatment rather than repeated courses of purine analog for relapsed disease.

Response of hairy cell leukemia to bendamustine

A 47-year-old male was diagnosed with hairy cell leukemia (HCL) in 1998 after presenting with pancytopenia and splenomegaly. Peripheral blood smear, bone marrow, and flow cytometry were diagnostic of HCL, with bright positivity for CD20, CD22, CD11c, CD25, and FMC7, and positive staining for CD19, CD123, CD103, and lambda light chain. He did not respond to a 7-day cycle of cladribine and subsequently underwent splenectomy followed by rituximab, both without response. He achieved a clinical remission with pentostatin lasting 5 years. He relapsed and received multiple courses of pentostatin over several years before progressing and becoming refractory to pentostatin, 10 years after the initial diagnosis. He became transfusion-dependent for both blood and platelets, with neutrophil count 400/mm, platelet count 13 000/mm, and hemoglobin 8.7 g/dL, and bone marrow replaced with HCL. Computed tomography (CT) showed no peripheral but extensive retroperitoneal lymphadenopathy. He began eight cycles of bendamustine 90 mg/m days 1 and 2, every 4–7 weeks. Delays of retreatment were related to infections but not cytopenias worse than baseline. He became transfusion-independent after cycle 3, and achieved a partial response lasting 6 months. Bone marrow biopsy after cycle 7 showed about one-third of the marrow involved. The circulating HCL cell count decreased from 730/mm just after cycle 1 to 12/mm prior to cycle 3, and was 2/mm at 1 month after cycle 8. Although this patient did not respond to initial cladribine, characterization of his HCL cells, including positivity for B-cell markers, CD103, CD25, and CD123, was consistent with classic HCL based on the World Health Organization (WHO) definition [1]. Using 1:1 conjugates assessed by flow cytometry and control beads as described [2], the numbers of sites/cell on the HCL cells were 70 000 for CD20, 30 000 for CD22, and 4300 for CD25. Molecular characterization of the HCL cells [3] showed them to express the VH3-30-3 immunoglobulin heavy chain variable (IGHV) rearrangement, with 96.28% homology to germline. Analysis in the region of the third complementarity determining region (CDR3) of the heavy chain showed usage of IGHD3-22 and IGHJ4. To determine whether bendamustine would resolve the patient’s cytopenias or only worsen them by further myelosuppression, blood counts were assessed before each cycle and at multiple time points in between. As shown in Figure 1(B), the platelet count appeared erratic at first due to multiple platelet transfusions required, but platelet transfusions were not needed after cycle 3, and the platelet count rose to a maximum of 193 prior to cycle 7. Similarly, as shown in Figure 1(C), red cell transfusions were not needed after cycle 3 day 2 (C3D2) and the hemoglobin (Hgb) exceeded 10.0 after cycle 7. As shown in Figure 1(A), the neutrophil count did not resolve to the normal range during treatment with bendamustine, but did meet established criteria for partial response (PR) with respect to 50% improvement from baseline independent of transfusions or colony stimulating factors [4,5].

PRAME expression in hairy cell leukemia

PRAME has been proposed as a useful marker for solid tumors and acute B-cell malignancies. Several studies demonstrate expression in CLL. To further examine its B-cell tumor distribution, we studied PRAME in both CLL and hairy cell leukemia (HCL). While by conventional PCR only 8% of 37 HCL and 27% of 22 CLL patients were positive, nearly all patients and normal donors expressed PRAME by real-time quantitative (TaqMan) PCR. We conclude that HCL and CLL differ in PRAME overexpression, and that basal normal expression of PRAME may limit its usefulness for following patients with minimal residual CLL or HCL.