An Immunogenetic Imprint of Ongoing Antigen Interactions in Hairy Cell Leukemia Expressing IGHV4-34 Receptors

Hairy cell leukemia (HCL) is a rare indolent B-cell malignancy thought to derive from a BRAF-mutant antigen-experienced B cell. Supporting evidence includes a transcriptional profile that is most similar to post-germinal center (GC) memory B cells; the presence of somatic hypermutation (SHM) in the majority of HCL cases; and, the preferential usage of the IGHV4-34 gene in the minority of classic HCL (HCLc) cases with low or no SHM. IGHV4-34-expressing HCLc is clinically more aggressive than other HCLc; moreover, it is enriched for BRAF-wildtype cases while it displays frequent MAP2K1 mutations. Overall, this constellation of features raises questions regarding the precise nature and ontogeny of IGHV4-34-expressing HCLc. Here we addressed this issue through investigating the subclonal architecture of the B cell receptor immunoglobulin (BcR IG) gene repertoire in a series of 17 cases with a typical phenotype of HCLc expressing the IGHV4-34 gene. Previous Sanger analysis had documented 10 cases carrying IGHV genes with 100% germline identity (GI), 2 cases with minimal SHM (GI>99%) and 5 with a more significant SHM load (GI<98%). Total RNA was isolated from peripheral blood mononuclear cells and IGHV-IGHD-IGHJ rearrangements were RT-PCR amplified and subjected to NGS sequencing using a paired-end protocol. Quality filtering of NGS raw reads was performed with a purpose-built bioinformatics pipeline and downstream analysis was performed with the IMGT/HighV-QUEST, tripr and IgIDivA softwares. Clonotypes were defined as clusters of rearrangement sequences expressing the IGHV4-34 gene and bearing identical variable heavy complementarity-determining region 3 (VH CDR3) amino acid (aa) sequences. Variants of a given clonotype i.e. gene rearrangement sequences with a VH CDR3 of identical length differing in up to 2 aa positions were defined as sub-clonotypes; these are likely to have differentiated from the main clonotype due to ongoing SHM. For the analysis of intraclonal diversification (ID) within the clonotypic rarranged IGHV genes, we used the metrics 'convergence score', describing the tendency of the BcR IG to acquire more mutations, and 'maximal pathway length', showing whether ongoing SHM acquisition takes place. Overall, we obtained 3,238,715 raw reads (median 178,446 reads/sample). Of these, 2,226,444 reads corresponded to productive IGHV4-34 gene rearrangements (median 127,647 sequences/sample) that were assigned to a total of 10,532 clonotypes (median 593 clonotypes/sample). The median frequency of the dominant clonotype was 64.4%, indicating a significant subclonal branching due to ID. Indeed, overall, we detected 5,267 subclonotypes (median 213 subclonotypes/sample) corresponding to 242,271 unique nucleotide (nt) variants (median 8098 nt variants/sample). ID was observed in both IG-mutated (<100% GI) and IG-unmutated (100% GI) cases, albeit was significantly (p=0.01) more extensive in the former (median number of nucleotide variants/sample: 21,306 vs 7,099, respectively). Next, we compared the present HCLc dataset against a corresponding dataset produced following the same approach from 11 IG-mutated CLL (M-CLL) cases expressing the IGHV4-34 gene, of which 5 belonged to stereotyped subset #4, notable for a high degree of ID. No statistically significant differences were identified in convergence scores between HCLc vs CLL, indicating that the former also displays considerable ID despite an overall lower number of clonal SHMs (underlying the higher GI at the clonal level i.e. by Sanger sequencing analysis). Moreover, HCLc exhibited significantly (p=0.002) higher maximal pathway lengths compared to CLL, suggesting that acquisition of subclonal SHM in IGHV4-34 expressing HCLc is an ongoing process that takes place progressively in many steps. Also relevant to mention, different recurrent replacement SHMs were seen in IGHV4-34 expressing HCLc vs CLL: indicatively, G36S in VH CDR1 was present in subclones of 11/17 HCLc cases whereas it was totally absent in CLL. In conclusion, we report a complex immunogenetic architecture for IGHV4-34 expressing HCLc with distinctive, disease-biased features and extensive ID, likely mediated by ongoing antigen interactions. This finding questions a post-GC origin for at least a subgroup of HCLc, while also highlighting the need to reappraise the ontogenetic relationship of this particular HCLc subgroup vs all other HCLc.

Kreitman:NIH: Patents & Royalties: Co-Inventor for Moxetumomab Pasudotox Patent; AstraZeneca: Other: Clinical Testing, Research Funding; Pfizer: Other: Receive drugs for clinical testing; Teva: Other: Receive drugs for clinical testing; Genentech: Other: Drugs for clinical testing, Research Funding; Novartis: Other: Drugs for Clinical testing, Research Funding. Chatzidimitriou:Janssen: Research Funding; AbbVie: Research Funding. Stamatopoulos:Janssen: Research Funding; AbbVie: Research Funding.