Genetic Lesions in Russian CLL Patients with the Most Common Stereotyped Antigen Receptors.

Chronic lymphocytic leukemia (CLL) is one of the most common B-cell malignancies in Western countries. IGHV mutational status is the most important prognostic factor for this disease. CLL is characterized by an extreme narrowing of the IGHV genes repertoire and the existence of subgroups of quasi-identical stereotyped antigenic receptors (SAR). Some of these subgroups have already been identified as independent prognostic factors for CLL. Here, we report the frequencies of TP53, NOTCH1, and SF3B1 gene mutations and chromosomal aberrations assessed by NGS and FISH in 152 CLL patients with the most common SAR in Russia. We noted these lesions to be much more common in patients with certain SAR than average in CLL. The profile of these aberrations differs between the subgroups of SAR, despite the similarity of their structure. For most of these subgroups mutations prevailed in a single gene, except for CLL#5 with all three genes affected by mutations. It should be noted that our data concerning the mutation frequency in some SAR groups differ from that obtained previously, which could be due to the population differences between patient cohorts. The research in this area should be important for better understanding the pathogenesis of CLL and therapy optimization.

Repertoire of Rearranged Immunoglobulin Heavy Chain Genes in Russian Patients With B-Cell Lymphoproliferative Diseases.

INTRODUCTION: Immunoglobulin heavy chain variable region (IGHV) repertoire narrowing could be an evidence for the involvement of a limited set of antigens in the development of lymphomas. For chronic lymphocytic leukemia (CLL) the existence of more than 200 subgroups of tumor IGHV antigen-binding sites, so called "stereotypical" antigen receptors (SAR) has been shown. For others lymphomas the possibility of SARs is also suggested. The aim of this study is to compare the tumor IGHVs and possible SARs in various B-cell malignancies in Russia and other countries. MATERIALS AND METHODS: The study included samples of 1800 CLL patients, 52 patients with mantle cell lymphoma, 48 patients with hairy cell lymphoma and 37 patients with splenic marginal cell lymphoma. The nucleotide sequences of the IGHV genes were determined according to ERIC protocol. RESULTS: In CLL most common IGHV genes were IGHV1-69, IGHV1-2, IGHV3-30 and IGHV4-34. The most common SARs were CLL#1, CLL#6, CLL#2, CLL#3. In MCL the most common genes were IGHV4-34, IGHV3-21, IGHV3-23. In 5 MCL patients CDR3 sequences were identified matching definitions of a stereotyped. In the half of SMZL patients was identified gene IGHV1-2. Other IGHV genes were much less common. Two pairs of SMZL patients have motives similar to each other. In HCL IGHV repertoire was the most variable, no trends for antigen receptor stereotypy were observed. It was found that SARs are highly disease-specific both at the level of nucleotide and amino acid sequences. CONCLUSION: Our results suggest that antigens crucial for the pathogenesis of B-cell malignancies could be disease-specific. Further studies on extended samples of non-CLL patients concerning the role of SARs in pathogenesis of these diseases may also contribute to the development of new diagnostic and prognostic markers.

A simple and efficient method for DNA extraction from skin and paraffin-embedded tissues applicable to T-cell clonality assays.

PCR-based clonality assay of rearranged T-cell receptor genes gamma and beta (TCRG and TCRB) in a number of cases could be essential to discriminate between cutaneous T-cell lymphomas and reactive lymphoproliferative lesions in the skin. However, extraction of good-quality DNA from skin specimens (especially formalin-fixed paraffin-embedded) remains a challenge. Common procedures, being labour-intensive and time-consuming and requiring toxic solvents such as phenol and chloroform, still may end up with DNA sample of insufficient quality. We herewith present a simple and efficient method for DNA isolation based on ammonia extraction of tissue, followed by neutralization and simultaneous salting out of proteins with acetic acid. We have analysed 30 samples - 24 fresh (16 skin, two spleen and six lymph node) and six paraffin-embedded. Standard procedure (proteinase K digestion, followed by phenol/chloroform extraction) has been carried out simultaneously. We observed good PCR signal for TCRG rearrangements in 30 samples processed with the new protocol and only in 20 extracted with proteinase K/phenol/chloroform. For TCRB, the success rate was 29 of 30 with the new protocol, compared to 11 of 30 with conventional protocol. The proposed method of DNA extraction should improve the value of T-cell clonality assay, because insufficient DNA quality and quantity may bias analysis towards monoclonality and therefore cause false-positive results.