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rearrangements in 69 patients reported by other investigators. Most patients had significant frequencies of somatic hypermutation (SHM), with a mean homology to germline of 93.9% [6]. The SHMs were analyzed for whether they replaced the amino acid (R) or were silent (S), and whether replacement mutations occurred in the complementarity determining regions (CDRs) more than in framework regions (FRs). Several patients were identified with a low probability of
mutations being random (p¼0.001–0.003), hence suggestive of antigen-driven mutation. In that study,
two patients were VH4-34þ, both unmutated, defined as 498% homology to the germline sequence, and both presentedwith elevated tumor burden, likeHCLv, but only one of the two patients had HCLv based on immunophenotype [6]. Other reports included several
VH4-34þ patients with HCL, including two unmu- tatedHCLvcases outof38[7],and fiveHCLccases out of 83, three of which were unmutated [8]. The following will review a more recently published study associating VH4-34+ HCL with presenting features and prognosis, and will include previously unpublished clinical observations in several of these patients [9].
Variable heavy gene usage in classic hairy cell leukemia versus hairy cell leukemia variant
To determine VH usage of the expressed VDJ rearrangement in the patients with HCL, total RNA was prepared from HCL cells using either PAXgene tubes if the HCL clone was prominent relative to normal B-cells, or CD11c-sorted HCL
cells that were obtained from heparinized blood by Ficoll centrifugation [9]. A total of 22 rearrange- ments were analyzed from 20 patients with HCLv and 63 from 62 with HCLc. A total of 30 different VH genes were cloned. In HCLc, the most common
VH genes were VH3-23 (n¼13, 21%), VH4-34 (n¼6, 10%), and VH3-30 (n¼5, 8%). In HCLv, the most common VH genes were VH4-34 (n¼8, 36%), and then VH4-39 and VH4-61 (each n¼2, 9%). Thus, while VH4-34 was the second most commonly
used gene in HCLc, the largest usage difference between HCLc and HCLv was with VH4-34 (p¼0.007).
Degree of homology to germline VH in classic hairy cell leukemia versus hairy cell leukemia variant
To determine whether the mutation frequency differed by HCLc vs. HCLv, or by VH gene usage, the cloned sequences were compared to the closest germline sequence on the ImMunoGeneTics (IMGT) database [9]. As shown in Figure 1(A), 13
(93%) out of 14 VH4-34þ rearrangements were unmutated, compared to 11 (15%) out of 71 of the
others (p¼461078). The median homology to germline was 99.4% vs. 95.6% for VH4-34 positive vs. negative rearrangements, and the homologies were significantly different by rank order (Wilcoxon) analysis (p50.0001). As can be seen in Figure 1(A), there was no difference in homology between HCLv and HCLc (median 97.6% vs. 95.9%, respectively,
Figure 1. (A) Homology to germline sequence for HCLv (green) or HCLc (red) rearrangements based on VH4-34 status. (B) White blood cell counts at diagnosis for patients with HCLv (green) or HCLc (red) based on VH4-34 status. (C–F) Overall survival in patients with respect to VH4-34 status and variant phenotype. (C, D) Data for all 82 patients with respect to VH4-34 (C) or variant (D) status, (E) data for only patients with HCLc, (F) data for only VH4-34-negative patients.
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