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 progressionfree 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. IntroductionHairy cell leukemia variant (HCLv) is a B-cell disorder, recognized for nearly 30 years, which accounts for 10% of hairy-cell leukemia (HCL) cases. Morphology of variant cells was reported to be intermediate between that of classic HCL and prolymphocytic leukemias. [1][2][3] Patients typically present with leukocytosis rather than leukopenia and often lack the neutropenia, anemia, and/or thrombocytopenia with which classic HCL patients present. 1,2,4 By flow cytometry, B-cell antigens FMC7, CD11c, CD20, CD22, and surface immunoglobulin are strongly positive in both classic HCL and HCLv, whereas HCLv differs from classic HCL by lack of CD25, HC-2, and CD123 and by expression of CD27. [3][4][5][6][7] CD103 is usually positive in both but can be negative in HCLv. 2 HCLv lacking both CD25 and CD103 may be difficult to differentiate from splenic marginal zone lymphoma (SMZL)/splenic lymphoma with villous lymphocytes without also relying on morphologic differences between HCL and SMZL. 2,6,7 In contrast to the high complete remission and overall response rates of classic HCL to the administration of purine analogs pentostatin and cladribine, [8][9][10] response in patients with HCLv is limited to partial responses in approximately 50% of patients. [2][3][4]11,12 Several complete responses of HCLv to monoclonal antibody-based therapy with and without chemotherapy have been reported. [13][14][15][16][17] Like other mature B lymphocytes, the malignant cells in HCL patients have 1 or sometimes 2 different rearrangements for immunoglobulin heavy chain. Significant variability can be observ...

Quantitative relationship between the magnitude of variation in minor histocompatibility antigens (mHA) and graft versus host disease (GVHD) pathophysiology in stem cell transplant (SCT) donor-recipient pairs (DRP) is not established. In order to elucidate this relationship, whole exome sequencing (WES) was performed on 27 HLA matched related (MRD), & 50 unrelated donors (URD), to identify nonsynonymous single nucleotide polymorphisms (SNPs). An average 2,463 SNPs were identified in MRD, and 4,287 in URD DRP (p<0.01); resulting peptide antigens that may be presented on HLA class I molecules in each DRP were derived in silico (NetMHCpan ver2.0) and the tissue expression of proteins these were derived from determined (GTex). MRD DRP had an average 3,670 HLA-binding-alloreactive peptides, putative mHA (pmHA) with an IC50 of <500 nM, and URD, had 5,386 (p<0.01). To simulate an alloreactive donor cytotoxic T cell response, the array of pmHA in each patient was considered as an operator matrix modifying a hypothetical cytotoxic T cell clonal vector matrix; each responding T cell clone’s proliferation was determined by the logistic equation of growth, accounting for HLA binding affinity and tissue expression of each alloreactive peptide. The resulting simulated organ-specific alloreactive T cell clonal growth revealed marked variability, with the T cell count differences spanning orders of magnitude between different DRP. Despite an estimated, uniform set of constants used in the model for all DRP, and a heterogeneously treated group of patients, higher total and organ-specific T cell counts were associated with cumulative incidence of moderate to severe GVHD in recipients. In conclusion, exome wide sequence differences and the variable alloreactive peptide binding to HLA in each DRP yields a large range of possible alloreactive donor T cell responses. Our findings also help understand the apparent randomness observed in the development of alloimmune responses.

SummaryTo examine the usage and mutational status of VH genes in hairy cell leukaemia (HCL), we analysed 24 immunoglobulin heavy chain (IgH) sequences expressed in 23 patients. None had premature stop codons. VH3-23 was the most common gene and a VH6 gene was observed for the first time in HCL. Although the mean mutation frequency was 6AE1%, slightly higher than in previous HCL series, four patients had 99AE6-100% homology to germline sequences, three of whom had high tumour burdens and poor outcomes. Despite the high mutation frequency, only two of 24 rearrangements had clear statistical evidence of antigen-dependent somatic mutation. Our results increase the database of reported functional HCL rearrangements to 94 in 92 patients. Overall, both gene usage and mutation frequency are very similar to mucosa-associated lymphoid tissue-type marginal zone lymphoma. The data are consistent with HCL originating from post-germinal centre marginal zone B cells, although the heterogeneity observed suggests that HCL may originate differently in some patients, and this could have implications for prognosis and treatment.

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