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Analysis of SF3B1 mutations in monoclonal B‐cell lymphocytosis

2012; Wiley; Volume: 31; Issue: 1 Linguagem: Inglês

10.1002/hon.2013

1099-1069

Mariangela Greco, Daniela Capello, Alessio Bruscaggin, Valeria Spina, Silvia Rasi, Sara Monti, Carmela Ciardullo, Stefania Cresta, Marco Fangazio, Gianluca Gaïdano, Robin Foà, Davide Rossi,

Immunodeficiency and Autoimmune Disorders

Clinical monoclonal B-cell lymphocytosis (cMBL) represents an asymptomatic monoclonal B-cell expansion with less than 5.0 × 109/L circulating cells that is occasionally identified during a diagnostic workup of an otherwise asymptomatic lymphocytosis 1-5. Most cMBLs are characterized by a chronic lymphocytic leukaemia (CLL) phenotype and might represent the initial phases of CLL 2-5. At the molecular level, cMBL with a CLL phenotype carry CLL-related abnormalities in ~50% cases, which are mainly represented by the 13q14 deletion 2-4. Consistent with the indolent phenotype of this condition, genetic lesions predicting poor prognosis in CLL, such as disruption of TP53 and ATM as well as mutations of NOTCH1, are rare in cMBL with a CLL phenotype 2-4, 6. Recent investigations of the CLL coding genome have revealed recurrent mutations of the SF3B1 splicing gene 7-9. In CLL, SF3B1 mutations occur at a low rate (~5%) at presentation, where they identify poor survival patients, while they are recurrent (~17%) in more advanced phases of the disease, thus suggesting a relationship between SF3B1 mutations and CLL aggressiveness 7-9. Here, we investigated the occurrence of SF3B1 mutations in 63 consecutive cMBL with a CLL phenotype presenting at our clinic for the initial evaluation of an asymptomatic lymphocytosis. Peripheral blood mononuclear cell samples drawn at cMBL presentation were analysed for SF3B1, NOTCH1, TP53 and IGHV mutations by DNA Sanger sequencing, and for 13q14 deletion, +12, 11q22–q23 deletion and 17p13 deletion by FISH 3, 6, 8. An SF3B1 mutation (K700E) that is known to be highly recurrent in CLL was also independently investigated by amplification refractory mutation system (ARMS) PCR 7-9. Patients provided informed consent in accordance with local Institutional Review Board requirements and the Declaration of Helsinki. The study was approved by the Ethical Committee of the Ospedale Maggiore della Carità di Novara associated with the Amedeo Avogadro University of Eastern Piedmont (Protocol Code 59/CE; Study Number CE 8/11). The clinical profile of the cMBL cohort was representative of this condition 2-4. Median age was 68 years (range: 40–88 years). The male : female ratio was 33:30. The median absolute lymphocyte count and median B-cell count were 5.0 × 109/L (range: 1.7–7.6 × 109/L) and 2.9 × 109/L (range: 0.2–4.9 × 109/L), respectively. The median value of Hb was 14.1 g/dL (range: 11.0–16.9 g/dL) and of platelets 220 × 109/L (range: 148–420). The biological profile of cMBL was consistent with the indolent nature of this condition. Expression on CD38 and ZAP70 occurred in 8/60 (13.3%) and in 9/53 (17.0%) cases, respectively. An IGHV identity >= 98% was present in 9/59 (15.3%) cMBLs and stereotyped VH CDR3 in 9/59 (15.3%). Deletion of 13q14 was observed in 29/63 (46.0%) cases, trisomy 12 in 0/63 and 11q22–q23 deletion in 2/63 (3.2%). One of 63 (1.5%) cMBLs harboured a biallelic TP53 disruption by the deletion of one allele and mutation of the second allele. NOTCH1 mutations were present in 2/63 (3.2%) cMBL 6. One single SF3B1 mutation occurred in 1/63 (1.5%) cMBL and was represented by a missense mutation of somatic origin leading to the K666N substitution (Figure 1). The cMBL case harbouring this SF3B1 mutation presented with an absolute lymphocyte count of 4.7 × 109/L and a B-cell count of 2.3 × 109/L, was CD38 and ZAP70 negative, expressed a mutated IGHV3-48 gene (IGHV identity 95.15%), lacked a stereotyped VH CDR3, showed a normal FISH karyotype and lacked mutations of TP53 and NOTCH1. It progressed to CLL (absolute lymphocyte count of 16.3 × 109/L and a B-cell count of 10.2 × 109/L, Rai stage 0) after 44 months of follow-up. Analysis of a sequential sample collected at cMBL progression to CLL confirmed the SF3B1 K666N substitution 8. The sensitivity of DNA Sanger sequencing does not allow the identification of a mutation whose allelic representation is <10%. Because the peripheral blood monoclonal B-cell content in cMBL is lower than in CLL, we specifically designed a high sensitivity ARMS PCR assay 10 for detecting the SF3B1 K700E mutation, that accounts for ~60% SF3B1 substitutions in CLL 7-9. Despite the high sensitivity (1%) of this assay, ARMS did not identify additional K700E mutations in cMBL. The low prevalence of SF3B1 mutations observed in cMBL is consistent with the rarity in this indolent condition of other genetic lesions otherwise associated with high-risk CLL. The authors have no conflict of interest to disclose. This study was supported by AIRC, Special Program Molecular Clinical Oncology, 5 × 1000, No. 10007, Milan, Italy (to G.G. and to R.F.); Progetto FIRB-Programma 'Futuro in Ricerca' 2008 (to D.R.), PRIN 2008 (to G.G.) and 2009 (to D.R.), MIUR, Rome, Italy; Progetto Giovani Ricercatori 2008 (to D.R.) and Ricerca Sanitaria Finalizzata 2008 (to G.G.) and 2009 (to D.C.), Ministero della Salute, Rome, Italy; Ricerca Sanitaria Finalizzata 2009 (to D.C.), Regione Piemonte, Turin, Italy; Novara-AIL Onlus, Novara, Italy (to G.G. and D.R.); Compagnia di San Paolo, Turin, Italy (to R.F.). S.M. is being supported by fellowships from Novara-AIL Onlus, Novara. S.R. is being supported by a fellowship from Società Italiana di Ematologia Sperimentale. M.G., A.B., V.S., S.R., C.C., S.C. and M.F. performed molecular studies and interpreted data; D.C. contributed to study design and data interpretation; S.M. performed and interpreted FISH analysis; G.G., R.F. and D.R. designed the study and wrote the manuscript.