ALBERT

Description: Abstract 262 Hairy cell leukemia (HCL) is an indolent neoplasm of small mature B-lymphoid cells, which are found in peripheral blood and bone marrow (BM), and are characterized by hairy projections of their abundant cytoplasm. In clinical practice, HCL needs to be differentiated from similar indolent lymphoid neoplasms. In a study based on massively parallel sequencing of the whole exome of leukemic and matched normal cells from a HCL patient and subsequent targeted resequencing in additional patients, Tiacci et al (N Engl J Med. 2011 Jun 16;364:2305–15) have recently identified the BRAF V600E mutation as a genetic alteration associated with this disease. This somatic mutation was previously detected in diverse human cancers, with a particularly high frequency in melanoma (Nature. 2002 Jun 27;417:949–54; N Engl J Med. 2005 Nov 17;353:2135–47). In order to develop a reliable molecular diagnostic tool and verify its sensitivity and specificity in the diagnosis of HCL, we developed an allele-specific PCR for the BRAF V600E mutation, and searched for this molecular lesion in a series of 239 patients with mature B-cell lymphoid neoplasms. The study population included 62 patients with HCL, 91 with splenic marginal zone lymphoma (SMZL), 29 with Waldenström macroglobulinemia (WM), and 57 with B-cell chronic lymphoproliferative disorders (B-CLPD). Genomic DNA was extracted from bone marrow (BM) biopsies in 61 cases of HCL, from BM in 90 patients with diverse lymphoid neoplasms (33 SMZL, 29 WM, 28 B-CLPD), and from peripheral blood (PB) in the remaining 88 patients (1 HCL, 58 SMZL, 29 B-CLPD). The BRAF V600E mutation was detected in all patients with HCL (62/62) and in none of those with SMZL or WM. Two of the 57 patients with B-CLPD carried the mutation, and their clinical features are as follows. Case #1. This 41 year-old woman presented in November 2008 with asymptomatic lymphocytosis, without any evidence of lymphadenopathy, splenomegaly or hepatomegaly. Laboratory data showed: Hb 12.9 g/dL, WBC count 16 × 109/L (62% lymphoid cells), and PLT count 283 × 109/L. On BM biopsy, an interstitial lymphoid infiltrate (60% of the whole cellularity) composed by small, lymphocyte/centrocyte-like cells was found. By immunohistochemistry, neoplastic cells showed expression of CD20, CD79a and cyclin-D1, but were uniformly negative for CD5, CD10, CD23, CD25 and DBA44, and annexin A1. At flow cytometry analysis, they were CD20 and FMC7 positive and CD10, CD38, CD5, CD23, CD11c, CD25, DBA44 and CD103 negative. FISH for t(11;14), performed for cyclin D1 expression, was negative. Immunoglobulin rearrangement was IGHV3-48*02, IGHD7-27*01 IGHJ4*02. So far, lymphocytosis has remained stable and the patient is regularly followed without any need for treatment. Case #2. This 62 year-old male presented in 2006 with thrombocytopenia and splenomegaly, and was diagnosed with HCL was established in another hospital (no additional data are available). He was treated with cladribine with a partial response. In May 2008, we evaluated this patient in Pavia. The spleen was palpable 3 cm under the costal margin, and laboratory data showed: Hb 15.4 g/dL, WBC count 3.9 × 109/L, and PLT vount 95 × 109/L. BM biopsy showed a 20% lymphoid infiltrate with interstitial and sinusoidal pattern, composed by small to medium sized cells with evident nucleoli, resembling pro-lymphocytes. By immunohistochemistry, cells were positive for CD20 and negative for CD5, CD23, cyclin-D1, CD25, DBA44, and annexin A1. Flow cytometry demonstrated the expression of CD20, FMC7 and CD11c, partial expression (25%) of CD103, and negativity for CD5, CD10, CD38, CD23, DBA44, CD11c and CD25. This patient was asymptomatic and a watch-and wait-policy was adopted. These findings indicate that the allele-specific PCR we developed is able to detect the mutation in the bone marrow of all patients with HCL, and confirm that the BRAF V600E mutation is highly specific for HCL within mature B-cell neoplasms. Only 2/177 (1.1%) patients with lymphoid neoplasms other than HCL (2/57 or 3.5% of patients with B-CLPD) were positive for BRAF V600E. This is in agreement with a previous study that found BRAF mutations in 2.4% of patients with non-Hodgkin's lymphoma (Br J Cancer. 2003 Nov 17;89:1958–60). The detection of the BRAF V600E mutation in the clone (or, at least, in a subclone) of mature B-cell lymphoid neoplasms without typical HCL features might help to define their biology. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3680 In B-cell malignancies, biased usage of immunoglobulin heavy chain variable (IGHV) genes and stereotyped clusters of immunoglobulin receptor suggest that a limited set of antigens or superantigens are involved in malignant transformation. In the present study we analyzed the IGH rearrangements of a large series of patients with Waldenström Macroglobulinemia (WM), IgM-monoclonal gammopathies of undetermined significance (IgM-MGUS) and IgM-related disorders (IgM-RD), with the aim to characterize IGH repertoire and to search for clusters of stereotyped receptors. A total of 123 patients with IgM monoclonal gammopathies, including 59 WM, 55 IgM-MGUS and 9 IgM-RD underwent IGHV amplification and direct sequencing. The median age of patients was 63 years (range: 32–86). Diagnosis of WM, IgM-MGUS and IgM-RD was made according to the consensus criteria proposed at the 2nd International Workshop on WM. All IGHV-D-J rearrangements were analyzed using the IMGT databases and the IMGT/V-QUEST tool to identify HCDR3 aminoacid (AA) sequences. HCDR3 sequences were aligned with each other and compared to 28,400 HCDR3 sequences from public databases (EMBL, NCBI, IMGT/LIGM-DB) and from unpublished multi-laboratory databases. Alignments were performed using the multiple sequence alignment software ClustalX (2.0). Criteria for including sequences in a cluster were: 〉60% aminoacid identity, IGHV gene belonging to the same clan, identical HCDR3 length and same junction residues and/or same IGHD-J genes. A productive monoclonal IGHV-D-J rearrangement was obtained in 99/123 patients, including 55 WM, 37 IgM-MGUS and 7 IgM-RD. IGHV genes were mutated in 94/99 patients (95%). The median somatic hypermutation rate was 93.3% (range 85.5%-97.9%). Four cases showed 〉98% identity with the germline sequences, whereas one single WM sample showed 100% identity with the germline sequence. When compared with the normal mature B-cell repertoire, a significant over-representation of the IGHV3 family and a significant under-representation of the IGHV1 and IGHV4 families were observed both in WM (87%, 7% and 2% respectively) and in IgM-MGUS (78%, 8% and 8% respectively). IGHV3-23 was the gene most commonly used (29%). Analysis of IGHV3-23 sequences for AA changes in the positions involved in superantigen recognition by IGHV3 subgroup genes showed that the majority of cases had two or more non-permissive AA changes that compromise the capacity to mediate superantigen recognition and binding. The frequency of novel N-glycosylation sites, introduced by the somatic hypermutation process, in this series of patients (12%) was not statistically different from that observed in normal memory B-cells, suggesting that WM, IgM-MGUS and IgM-RD cells do not need to interact with stromal GC environmental elements. The median HCDR3 length was 13 AA (range: 5–29) and was similar in WM, IgM-MGUS and IgM-RD. Intra-WM/IgM-MGUS/IgM-RD search for HCDR3 similarity showed no association that met minimal requirements to define stereotyped receptors. The comparison of WM/IgM-MGUS/IgM-RD sequences with non WM/IgM-MGUS/IgM-RD database showed that WM/IgM-MGUS/IgM-RD sequences are unrelated to known CLL or SMZL subsets. Only one IgM-MGUS occurring in a HCV-positive patient formed a novel subset with other 4 HCV-related lymphoproliferative disorders. The findings of this analysis of WM, IgM-MGUS and IgM-RD IGH sequences indicate that: i) family-usage in the WM, IgM-MGUS and IgM-RD showed a skewed usage compared to normal B-cell repertoire; ii) WM, IgM-MGUS and IgM-RD-specific HCDR3 clusters do not occur to a frequency detectable with currently available databases; iii) WM, IgM-MGUS and IgM-RD sequences are not related to known CLL and SMZL HCDR3 clusters; iv) one IgM-MGUS showed similarities with sequences derived from pathological HCV-related lymphoproliferations, suggesting that, at least in a fraction of cases, HCV may have a pathogenetic role. For the large majority of IgM-related disorders, however, there is little evidence in favor of a B-cell receptor-driven pathogenesis. Disclosures: No relevant conflicts of interest to declare.