Elisa Sozzi

The Prognostic Value of TP53 Mutations in Chronic Lymphocytic Leukemia Is Independent of Del17p13: Implications for Overall Survival and Chemorefractoriness

Purpose: Del17p13 predicts poor outcome and chemorefractoriness in chronic lymphocytic leukemia (CLL). Conversely, it is unknown whether TP53 mutations carry any prognostic value independent of del17p13. We tested the independent prognostic value of TP53 mutations in CLL. Experimental Design: The study was based on a consecutive series of 308 CLL. DNA sequencing of TP53 exons 2 to 10 and del17p13 interphase fluorescence in situ hybridization were done at CLL diagnosis. Study end points were survival and chemorefractoriness. Results: At diagnosis, TP53 mutations (n = 32) occurred in 31 of 308 (10.0%) patients. Of all CLL showing TP53 disruption by either mutation and/or deletion (n = 44), 10 cases (22.7%) showed TP53 mutations in the absence of del17p13. Multivariate analysis selected TP53 mutations (hazard ratio, 3.20; P = 0.002) as an independent predictor of overall survival after adjustment for del17p13. Also, multivariate analysis selected TP53 mutations (hazard ratio, 3.97; P < 0.001) as an independent predictor of chemorefractoriness after adjustment for del17p13. Compared with cases without TP53 alterations, CLL harboring any type of TP53 disruption (mutation only, del17p13 only, or both mutation and del17p13) uniformly displayed a high prevalence of unfavorable prognosticators and poor outcome. Analysis of sequential CLL samples showed the acquisition of new or additional TP53 alterations at the time of chemorefractoriness. Conclusions: These data show that (a) TP53 mutations are an independent predictor of short survival and chemorefractoriness, and (b) that CLL presenting with TP53 mutations without del17p13 fare as poorly as CLL carrying del17p13. Because CLL harboring TP53 mutations without del17p13 are currently not recognized by conventional diagnostic strategies, these results may be relevant for a comprehensive prognostic characterization of CLL.

Simple genetic diagnosis of hairy cell leukemia by sensitive detection of the BRAF-V600E mutation

Hairy cell leukemia (HCL) is a distinct clinicopathologic entity that responds well to purine analogs but is sometimes difficult to differentiate from HCL-like disorders (e.g., splenic marginal zone lymphoma and HCL variant). We recently identified the BRAF-V600E mutation as the disease-defining genetic event in HCL. In this study, we describe a new, simple, and inexpensive test for genetics-based diagnosis of HCL in whole-blood samples that detects BRAF-V600E through a sensitive allele-specific PCR qualitative assay followed by agarose-gel electrophoresis. This approach detected BRAF-V600E in all 123 leukemic HCL samples investigated containing as few as 0.1% leukemic cells. BRAF-V600E was detected at different time points during the disease course, even after therapy, pointing to its pivotal role in HCL pathogenesis and maintenance of the leukemic clone. Conversely, 115 non-HCL chronic B-cell neoplasms, including 79 HCL-like disorders, were invariably negative for BRAF-V600E. This molecular assay is a powerful tool for improving the diagnostic accuracy in HCL.

The prognosis of clinical monoclonal B cell lymphocytosis differs from prognosis of Rai 0 chronic lymphocytic leukaemia and is recapitulated by biological risk factors

Monoclonal B‐cell lymphocytosis (MBL) is an asymptomatic monoclonal expansion of <5·0 × 109/l circulating CLL‐phenotype B‐cells. The relationship between MBL and Rai 0 CLL, as well as the impact of biological risk factors on MBL prognosis, are unknown. Out of 460 B‐cell expansions with CLL‐phenotype, 123 clinical MBL (cMBL) were compared to 154 Rai 0 CLL according to clinical and biological profile and outcome. cMBL had better humoral immune capacity and lower infection risk, lower prevalence of del11q22‐q23/del17p13 and TP53 mutations, slower lymphocyte doubling time, and longer treatment‐free survival. Also, cMBL diagnosis was a protective factor for treatment risk. Despite these favourable features, all cMBL were projected to progress, and lymphocytes <1·2 × 109/l and >3·7 × 109/l were the best thresholds predicting the lowest and highest risk of progression to CLL. Although IGHV status, CD38 and CD49d expression, and fluorescence in situ hybridization (FISH) karyotype individually predicted treatment‐free survival, multivariate analysis identified the presence of +12 or del17p13 as the sole independent predictor of treatment requirement in cMBL (Hazard ratio: 5·39, 95% confidence interval 1·98–14·44, P = 0·001). Overall, these data showed that cMBL has a more favourable clinical course than Rai 0 CLL. Given that the biological profile can predict treatment requirement, stratification based on biological prognosticators may be helpful for cMBL management.

Molecular and clinical features of chronic lymphocytic leukaemia with stereotyped B cell receptors: Results from an Italian multicentre study

A fraction of chronic lymphocytic leukaemia (CLL) cases carry highly homologous B‐cell receptors (BCR), i.e. characterized by non‐random combinations of immunoglobulin heavy‐chain variable (IGHV) genes and heavy‐chain complementarity determining region‐3 (HCDR3), often associated with a restricted selection of IGVK/L light chains. Such ‘stereotyped’ BCR occur more frequently in CLL with unmutated (UM) than mutated (M) IGHV genes. We analysed 1426 IG rearrangements (from 1398 CLL cases) by a clustering driven by HCDR3 similarities. Molecular findings were correlated to time‐to‐treatment (TTT) and presence of known prognosticators. Sixty‐nine clusters (319 IG‐rearrangements, 22·4%) with stereotyped BCR were identified. Among 30 confirmed clusters (≥3 IG‐rearrangements/cluster), we found 14 novel clusters, of which 11 had M IG rearrangements (M clusters) and predominantly (8/11) used IGHV3 subgroup genes. Recurrent cluster‐biased amino acid changes were found throughout IGHV sequences of these ‘M clusters’. Regarding clinical outcome: (i) UM CLL from the IGHV1‐2/1‐3/1‐18/1‐46/7‐4‐1/IGKV1‐39 cluster had poorer prognosis than UM/M cases, or UM cases using the same IGHV genes but not in clusters; (ii) M CLL from the IGHV3‐21/IGLV3‐21 cluster had TTT similar to UM CLL, and shorter than M CLL expressing IGHV3‐21 but not in cluster. Altogether, our analysis identified additional molecular and clinical features for CLL expressing stereotyped BCR.

The normal IGHV1-69-derived B-cell repertoire contains stereotypic patterns characteristic of unmutated CLL

The cell of origin of chronic lymphocytic leukemia (CLL) has long been sought, and immunoglobulin gene analysis provides new clues. In the unmutated subset (U-CLL), there is increased usage of the 51p1-related alleles of the immunoglobulin heavy chain variable 1-69 gene, often combined with selected genes and with immunoglobulin heavy chain diversity IGHJ6. Stereotypic characteristics of the HCDR3 result and suggest antigen selection of the leukemic clones. We have now analyzed 51p1/IGHJ6 combinations in normal blood B cells from 3 healthy persons for parallel sequence patterns. A high proportion (33.3% of sequences) revealed stereotypic patterns, with several (15.0%) being similar to those described in U-CLL. Previously unreported CLL-associated stereotypes were detected in 4.8%. Stereotypes (13.6%) not detected in CLL also were found. The HCDR2-IGHJ6 sequences were essentially unmutated. Junctional amino acids in normal B cells were heterogeneous, as in cases of stereotyped CLL. Phenotypically, normal B cells expressing 51p1-derived immunoglobulin M were naive. This snapshot of the naive B-cell repertoire reveals subsets of B cells closely related to those characteristic of CLL. Conserved patterns in the 51p1-encoded immunoglobulin M of normal B cells suggest a restricted sequence repertoire shaped by evolution to recognize common pathogens. Proliferative pressure on these cells is the likely route to U-CLL.

Hairy cell leukemias with unmutated IGHV genes define the minor subset refractory to single agent cladribine and with more aggressive behavior

Hairy cell leukemia (HCL) is generally responsive to single-agent cladribine, and only a minority of patients are refractory and with poor prognosis. HCLs generally express mutated (M) and, in a minority, unmutated (UM) IGHV. In a multicenter clinical trial in newly diagnosed HCL, we prospectively investigated clinical and molecular parameters predicting response and event-free survival after single-agent cladribine. Of 58 HCLs, 6 expressed UM-IGHV (UM-HCL) and 52 M-IGHV (M-HCL). Beneficial responses were obtained in 53 of 58 patients (91%), whereas treatment failures were observed in 5 of 58 patients (9%). Failures were associated significantly with UM-IGHV (5 of 5 failures vs 1 of 53 beneficial responses had UM-IGHV, P < .001), leukocytosis (3 of 5 vs 3 of 53, P = .006), and bulky spleen (4 of 5 vs 4 of 53, P < .001). The UM-HCL not benefiting from cladribine characteristically had bulky spleen (4 of 5, 80%), leukocytosis (3 of 5, 60%), and TP53 defects (2 of 5, 40%), and progressed rapidly after first treatment (median event-free survival, 7.5 months). Our data suggest that UM-HCLs identify the minor subgroup failing cladribine treatment and with more aggressive disease. High incidence of TP53 dysfunction indicates a potential mechanism of resistance to cladribine in the UM-HCL group. Overall, our data provide new molecular elements relevant for treatment concerns in HCL.

Genome‐wide DNA analysis identifies recurrent imbalances predicting outcome in chronic lymphocytic leukaemia with 17p deletion

Deletion of 17p (TP53) identifies a rare subset of chronic lymphocytic leukaemia (17p‐ CLL) with aggressive behaviour. Genome‐wide DNA‐profiling was performed to investigate 18 patients with 17p‐ CLL. All cases had multiple copy‐number (CN) changes. Among the several recurrent CN changes identified, 8q24.13‐q24.1‐gain (MYC), 8p‐loss (TNFRSF10A/B, also known as TRAIL1/2) and 2p16.1‐p14‐gain (REL/BCL11A) appeared frequently represented. 8p‐loss and 2p16.1‐p14‐gain also appeared clinically relevant and predicted significant shorter time from diagnosis to treatment (8p‐loss) and overall survival (8p‐loss and 2p16.1‐p14‐gain, P < 0·05). These observations document a highly unstable genome in 17p‐ CLL and suggest that additional genes outside the TP53 locus may be important for tumour behaviour.

Angiopoietin-2 plasma dosage predicts time to first treatment and overall survival in chronic lymphocytic leukemia

The clinical relevance of angiopoietin-2 (Ang2) in chronic lymphocytic leukemia (CLL) was previously suggested by the association between high Ang2, and shorter progression-free survival reported in small series of patients. Here, we evaluated Ang2 glycoprotein levels in plasma samples collected from a multicentric cohort of CLL patients (n = 316) using an enzyme-linked immunosorbent assay method, and we investigated its prognostic role in relation to time to first treatment (TTFT) and overall survival. Based on a cutoff equal to 2459 pg/mL, we divided our cohort in 2 subsets (high and low Ang2) composing 100 (31.6%) and 216 (68.4%) patients, respectively. High Ang2 was predictive of reduced TTFT (P < .001) and overall survival (P = .002). Multivariate analysis confirmed that high Ang2 was an independent prognosticator for TTFT (hazard ratio = 1.739; 95% confidence interval, 1.059-2.857; P = .029). Significant associations were found between high Ang2 and advanced Binet stages (P < .001), high beta(2)-microglobulin (P < .001), unmutated variable region of immunoglobulin heavy chain gene status (P < .001), high CD38 and zeta-chain-associated protein kinase 70 expression (P < .001 and P = .003), and intermediate/high cytogenetic risk (P = .005). Moreover, Ang2 added prognostic power to other conventional prognosticators and helped to refine prognosis among CLL subsets with both high and low vascular endothelial growth factor plasma levels. Ang2 plasma level may be a useful independent prognosticator for CLL.

Expression of Mutated IGHV3-23 Genes in Chronic Lymphocytic Leukemia Identifies a Disease Subset with Peculiar Clinical and Biological Features

Purpose: B-cell chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease whose outcome can be foreseen by investigating the mutational status of immunoglobulin heavy chain variable (IGHV) genes. Moreover, a different prognosis was reported for CLL expressing specific IGHV genes in the context or not of stereotyped B-cell receptors. Here we investigated novel associations between usage of specific IGHV genes and clinical features in CLL. Experimental Design: Among 1,426 CLL-specific IG-rearrangements, stereotyped B-cell receptor clusters never utilized the IGHV3-23 gene. Given this notion, this study was aimed at characterizing the IGHV3-23 gene in CLL, and identifying the properties of IGHV3-23–expressing CLL. Results: IGHV3-23 was the second most frequently used (134 of 1,426) and usually mutated (M; 109 of 134) IGHV gene in our CLL series. In the vast majority of M IGHV3-23 sequences, the configuration of the 13 amino acids involved in superantigen recognition was consistent with superantigen binding. Clinically, M IGHV3-23 CLL had shorter time-to-treatment than other M non–IGHV3-23 CLL, and multivariate analyses selected IGHV3-23 gene usage, Rai staging, and chromosomal abnormalities as independent prognosticators for M CLL. Compared with M non–IGHV3-23 CLL, the gene expression profile of M IGHV3-23 CLL was deprived in genes, including the growth/tumor suppressor genes PDCD4, TIA1, and RASSF5, whose downregulation is under control of miR-15a and miR-16-1. Accordingly, relatively higher levels of miR-15a and miR-16-1 were found in M IGHV3-23 compared with M non–IGHV3-23 CLL. Conclusions: Altogether, expression of the IGHV3-23 gene characterizes a CLL subset with distinct clinical and biological features. Clin Cancer Res; 16(2); 620–8

Low-dose oral fludarabine plus cyclophosphamide in elderly patients with untreated and relapsed or refractory chronic lymphocytic Leukaemia†

Fludarabine plus cyclophosphamide (FC) at conventional doses is an effective treatment for chronic lymphocytic leukaemia (CLL). However, FC at standard doses may give hematological and non‐hematological toxicity, predominantly in the elderly. Intravenous or oral low‐dose FC regimens remain highly effective in elderly patients with Low‐Grade Lymphomas other than CLL and are well tolerated. We tested efficacy and toxicity of oral FC at reduced doses in 26 elderly patients (median 71 years) with previously untreated (UT‐CLL, n = 14) or relapsed/refractory CLL (R‐CLL, n = 12), unfit for conventional treatments. Twentyfour‐of‐26 (92%) patients (14/14, 100% UT‐CLL; 10/12, 83.5% R‐CLL) obtained a response, with 12/26 (46%) complete responses (9/14, 64.2% in UT‐CLL; 3/12, 25% in R‐CLL). Non‐hematological toxicity was mild and myelosuppression was documented in 8/26 (31%) patients (4/14, 28% UT‐CLL; 4/12, 33% R‐CLL). With a median follow‐up of 24 months, median event‐free survival was 48 months with no differences between UT‐CLL and R‐CLL and all responders were alive. Low‐dose oral FC treatment showed good efficacy in both untreated and refractory/relapsed CLL. The treatment is useful in elderly patients who cannot benefit of more aggressive schedules and is easy to administer on an outpatient basis. Copyright