Gianluca Festini

Molecular and Transcriptional Characterization of 17p Loss in B-Cell Chronic Lymphocytic Leukemia

Distinct genetic abnormalities, such as TP53 deletion at 17p13.1, have been identified as having adverse prognostic relevance in B‐cell chronic lymphocytic leukemia (B‐CLL), and conventional cytogenetic studies have shown that TP53 deletion in B‐CLL is mainly associated with the loss of 17p due to complex chromosomal rearrangements. We used an integrative genomic approach to investigate the significance of 17p loss in 18 B‐CLLs in Binet stage A, carrying a TP53 monoallelic deletion detected by means of fluorescence in situ hybridization (FISH). Genome‐wide DNA analysis using single nucleotide polymorphism (SNP) arrays of 12 of 18 samples showed 17p loss in 11 cases, with breakpoints scattered along the 17p11.2 region. FISH analysis confirmed these findings and revealed 17p loss in a small fraction of leukemic cells in the remaining TP53‐deleted case, and it also indicated 17p loss in the six cases not investigated by means of SNP arrays. Mutations in exons 2–11 of the remaining TP53 allele were found in 9 of 12 deleted samples. Gene‐expression profiling of 60 B‐CLLs, including seven patients with 17p loss, identified 40 differentially expressed genes in 17p‐ versus 17p normal samples, 35 of which were downregulated in 17p‐tumors. The majority (30 of 35) of these transcripts, including putative tumor suppressor genes, mapped to 17p, thus indicating a remarkable gene‐dosage effect. Our data provide evidence that 17p loss may play an additional pathogenetic role in B‐CLL and suggest that the concomitant loss of multiple tumor suppressor genes could be responsible for the highly adverse prognostic relevance associated with TP53 loss.

Definition of progression risk based on combinations of cellular and molecular markers in patients with Binet stage A chronic lymphocytic leukaemia

IGHV mutational status and ZAP‐70 or CD38 expression correlate with clinical course in B‐cell chronic lymphocytic leukaemia (CLL). The three markers may be discordant in the single case and there is no consensus on their combined use in clinical practise. This multicenter study investigated this issue. Two‐hundred and sixty‐two Binet stage A patients were studied for the three markers. Sixty patients were profiled with HG‐U133A gene expression chips. Disease progression was determined by time from diagnosis to treatment (TTT). The probability of being treatment‐free at 3 years was significantly shorter in patients with unmutated IGHV genes (IGHVunmut 66% vs. 93%, chi square of log‐rank = 30, P < 0·0001), ZAP‐70 positive (ZAP‐70pos 73% vs. 96%, chi square of log‐rank = 8·2, P = 0·004) or CD38‐positive cells (CD38pos 68% vs. 91%, chi square of log‐rank = 21, P < 0·0001). Cox multivariate regression analysis showed that the three markers had an independent predictive value for TTT of similar power. A prognostic system based on presence of none (low‐risk), one (intermediate‐risk) or two or three (high‐risk) markers was generated. Based on such criteria, 56%, 23% and 21% of cases were clustered in low (HR = 1), intermediate [HR = 2·8, 95% confidence interval (CI) 2·4–5·8] and high‐risk group (HR = 8·0, 95% CI 3·9–16·2). Specific transcriptional patterns were significantly associated with risk groups.

Pegylated-interferon plus ribavirin for HCV-positive indolent non-Hodgkin lymphomas.

Hepatitis C virus (HCV) causes not only chronic liver disease, but is also implicated in several lymphoproliferative disorders (Pozzato et al, 1994). The identification of CD81 as the main HCV receptor could explain this lymphotropism because this receptor is expressed in B lymphocytes (Pileri et al, 1998). The CD81 engagement determines cell activation with overexpression of CD69, CD71, CD86, and the chemokinereceptor CXCR3 (Rosa et al, 2005). In addition, an increased prevalence of t(14,18) translocation has been reported in HCV-patients, even in the absence of lymphoproliferative disorders (Zignego et al, 2002). This interaction of HCV with the immune system might explain the role of HCV in several extra-hepatic diseases, such as non-Hodgkin lymphoma (NHL). As HCV-positive lymphoproliferative disorders have been successfully treated with antiviral therapy (Gisbert et al, 2005), we compared the traditional treatment of interferon plus ribavirina (IFN + RIBA) with the newest antiviral therapy i.e. pegylated interferon alpha plus ribavirina (PEG-IFN +RIBA) in a small group of indolent NHL. Eighteen patients affected by HCV-related B-NHL were consecutively included in the study from February 1998 to January 2004. Criteria for patient’s selection were: (i) lowgrade B-NHL at first diagnosis or relapse, (ii) indolent course, (iii) HCV infection (HCV-RNA positivity in serum). Patients that carried the hepatitis B surface antigen (HBsAg) or were positive for anti-human immunodeficiency virus antibodies were excluded. All patients gave their informed consent before entry in the study. B cell monoclonality was analysed in the peripheral blood mononuclear cells and bone marrow samples of four cases by the previously described isotype-specific IGH VDJ Gene Scanning technique: briefly, a forward primer labelled with 6-carboxyfluorescein (FAM) fluorochrome in the second VDJ polymerase chain reaction (PCR) round was used. The VDJ PCR product and the internal size standard GeneScan 400 HD (Applied Biosystem, Forster City, CA, USA) were added to deionized formamide, and then subjected to capillary electrophoresis. The detection of IGHV gene segments was performed using VDJ FR1 PCR protocol, in which a second multiplexing PCR, IGHV3, IGHV4, IGHV5, IGHV6 primers and the IGHJ region were added. IGHV1-IGHV4, and IGHV3-IGHV6 primers were each labelled with a different fluorescent dye: FAM, HEX, NED or ROX. PCR products were added to the internal GeneScan 500 LIZ size standard. This mixture was subjected to capillary electrophoresis. When a monoclone was found, the PCR product was sequenced by standard methods. Eight patients (Group A) received 3 MU of IFN alpha-2b three times a week for 48 weeks. Ten patients (Group B) received PEG-IFN alpha-2b 1Æ5 lg/kg once a week for 48 weeks. Both groups received ribavirin 1000 mg or 1200 mg daily according to body weight (lower or greater than 75 kg) for 48 weeks (Manns et al, 2001). The patients were randomized on the basis of a computer-generated random list. The patient diagnoses were: one follicular lymphoma, one splenic lymphoma with villous lymphocytes, and 16 lymphoplasmacytoid lymphomas. The follicular lymphoma and the splenic lymphoma had node involvement (axillary and mediastinal/retroperitoneal, respectively). HCV genotyping showed genotype 1b in 11 cases (61%) while genotype 2a/2c was detected in the others. Eleven cases had chronic hepatitis at liver biopsy. Thirteen patients had dosable levels of cryoglobulins (from 2% to 35%) and high rheumatoid factor levels (from 30 to 21 700 U/l). Therapy outcome (Table I) was as follows:

Integrative Genomics Analyses Reveal Molecularly Distinct Subgroups of B-Cell Chronic Lymphocytic Leukemia Patients with 13q14 Deletion

Purpose: Chromosome 13q14 deletion occurs in a substantial number of chronic lymphocytic leukemia (CLL) patients and it is believed to play a pathogenetic role. The exact mechanisms involved in this lesion have not yet been fully elucidated because of its heterogeneity and the imprecise knowledge of the implicated genes. This study was addressed to further contribute to the molecular definition of this lesion in CLL. Experimental Design: We applied single-nucleotide polymorphism (SNP)-array technology and gene expression profiling data to investigate the 13q14 deletion occurring in a panel of 100 untreated, early-stage (Binet A) patients representative of the major genetics, molecular, and biological features of the disease. Results: Concordantly with FISH analysis, SNP arrays identified 44 patients with del(13)(q14) including 11 cases with a biallelic deletion. The shorter monoallelic deletion was 635-kb long. The loss of the miR-15a/16-1 cluster occurred in all del(13)(q14) cases except in 2 patients with a monoallelic deletion, who retained both copies. MiR-15a/16 expression was significantly downregulated only in patients with the biallelic loss of the miRNA cluster compared to 13q normal cases. Finally, the natural grouping of SNP profiles by nonnegative matrix factorization algorithm showed that patients could be classified into 2 separate clusters, mainly characterized by short/biallelic versus wide/monoallelic 13q14 deletions. Supervised analyses of expression data showed that specific transcriptional profiles are correlated with these 2 genomic subgroups. Conclusions: Overall, our data highlight the presence of 2 distinct molecular types of 13q14 deletions, which may be of clinical relevance in CLL. Clin Cancer Res; 16(23); 5641–53. ©2010 AACR.

Chromosome 2p gain in monoclonal B-cell lymphocytosis and in early stage chronic lymphocytic leukemia.

Recent studies have described chromosome 2p gain as a recurrent lesion in chronic lymphocytic leukemia (CLL). We investigated the 2p gain and its relationship with common prognostic biomarkers in a prospective series of 69 clinical monoclonal B‐cell lymphocytosis (cMBL) and 218 early stage (Binet A) CLL patients. The 2p gain was detected by FISH in 17 patients (6%, 16 CLL, and 1 cMBL) and further characterized by single nucleotide polymorphism‐array. Overall, unfavorable cytogenetic deletions, i.e., del(11)(q23) and del(17)(p13) (P = 0.002), were significantly more frequent in 2p gain cases, as well as unmutated status of IGHV (P < 1 × 10−4) and CD38 (P < 1 × 10−4) and ZAP‐70 positive expression (P = 0.003). Furthermore, 2p gain patients had significantly higher utilization of stereotyped B‐cell receptors compared with 2p negative patients (P = 0.009), and the incidence of stereotyped subset #1 in 2p gain patients was significantly higher than that found in the remaining CLLs (P = 0.031). Transcriptional profiling analysis identified several genes significantly upregulated in 2p gain CLLs, most of which mapped to 2p. Among these, NCOA1 and ROCK2 are known for their involvement in tumor progression in several human cancers, whereas among those located in different chromosomes, CAV1 at 7q31.1 has been recently identified to play a critical role in CLL progression. Thus, 2p gain can be present since the early stages of the disease, particularly in those cases characterized by other poor prognosis markers. The finding of genes upregulated in the cells with 2p gain provides new insights to define the pathogenic role of this lesion. Am. J. Hematol. 2013.

A progression-risk score to predict treatment-free survival for early stage chronic lymphocytic leukemia patients

A progression-risk score to predict treatment-free survival for early stage chronic lymphocytic leukemia patients

Excellent outcomes of 2G-TKI therapy after imatinib failure in chronic phase CML patients

Second-generation tyrosine kinase inhibitors (2G-TKIs) dasatinib and nilotinib produced historical rates of about 50% complete cytogenetic response (CCyR) and about 40% major molecular response (MMR) in chronic myeloid leukaemia (CML) patients failing imatinib. Direct comparisons between dasatinib and nilotinib are lacking, and few studies addressed the dynamics of deep molecular response (DMR) in a “real-life” setting. We retrospectively analyzed 163 patients receiving dasatinib (n = 95) or nilotinib (n = 68) as second-line therapy after imatinib. The two cohorts were comparable for diseases characteristics, although there was a higher rate of dasatinib use in imatinib-resistant and of nilotinib in intolerant patients. Overall, 75% patients not in CCyR and 60% patients not in MMR at 2G-TKI start attained this response. DMR was achieved by 61 patients (37.4%), with estimated rate of stable DMR at 5 years of 24%. After a median follow-up of 48 months, 60% of patients persisted on their second-line treatment. Rates and kinetics of cytogenetic and molecular responses, progression-free and overall survival were similar for dasatinib and nilotinib. In a “real-life” setting, dasatinib and nilotinib resulted equally effective and safe after imatinib failure, determining high rates of CCyR and MMR, and a significant chance of stable DMR, a prerequisite for treatment discontinuation.