CD49d promotes disease progression in chronic lymphocytic leukemia: new insights from CD49d bimodal expression.

CD49d is a remarkable prognostic biomarker of chronic lymphocytic leukemia (CLL). The cutoff value for the extensively validated 30% of positive CLL cells is able to separate CLL patients into 2 subgroups with different prognoses, but it does not consider the pattern of CD49d expression. In the present study, we analyzed a cohort of 1630 CLL samples and identified the presence of ∼20% of CLL cases (n = 313) characterized by a bimodal expression of CD49d, that is, concomitant presence of a CD49d+ subpopulation and a CD49d- subpopulation. At variance with the highly stable CD49d expression observed in CLL patients with a homogeneous pattern of CD49d expression, CD49d bimodal CLL showed a higher level of variability in sequential samples, and an increase in the CD49d+ subpopulation over time after therapy. The CD49d+ subpopulation from CD49d bimodal CLL displayed higher levels of proliferation compared with the CD49d- cells; and was more highly represented in the bone marrow compared with peripheral blood (PB), and in PB CLL subsets expressing the CXCR4dim/CD5bright phenotype, known to be enriched in proliferative cells. From a clinical standpoint, CLL patients with CD49d bimodal expression, regardless of whether the CD49d+ subpopulation exceeded the 30% cutoff or not, experienced clinical behavior similar to CD49d+ CLL, both in chemoimmunotherapy (n = 1522) and in ibrutinib (n = 158) settings. Altogether, these results suggest that CD49d can drive disease progression in CLL, and that the pattern of CD49d expression should also be considered to improve the prognostic impact of this biomarker in CLL.

TP53 Mutations with Low Variant Allele Frequency Predict Short Survival in Chronic Lymphocytic Leukemia.

PURPOSE: In chronic lymphocytic leukemia (CLL), TP53 mutations are associated with reduced survival and resistance to standard chemoimmunotherapy (CIT). Nevertheless, the clinical impact of subclonal TP53 mutations below 10% to 15% variant allele frequency (VAF) remains unclear. EXPERIMENTAL DESIGN: Using a training/validation approach, we retrospectively analyzed the clinical and biological features of TP53 mutations above (high-VAF) or below (low-VAF) the previously reported 10.0% VAF threshold, as determined by deep next-generation sequencing. Clinical impact of low-VAF TP53 mutations was also confirmed in a cohort (n = 251) of CLL treated with fludarabine-cyclophosphamide-rituximab (FCR) or FCR-like regimens from two UK trials. RESULTS: In the training cohort, 97 of 684 patients bore 152 TP53 mutations, while in the validation cohort, 71 of 536 patients had 109 TP53 mutations. In both cohorts, patients with the TP53 mutation experienced significantly shorter overall survival (OS) than TP53 wild-type patients, regardless of the TP53 mutation VAF. By combining TP53 mutation and 17p13.1 deletion (del17p) data in the total cohort (n = 1,220), 113 cases were TP53 mutated only (73/113 with low-VAF mutations), 55 del17p/TP53 mutated (3/55 with low-VAF mutations), 20 del17p only, and 1,032 (84.6%) TP53 wild-type. A model including low-VAF cases outperformed the canonical model, which considered only high-VAF cases (c-indices 0.643 vs. 0.603, P < 0.0001), and improved the prognostic risk stratification of CLL International Prognostic Index. Clinical results were confirmed in CIT-treated cases (n = 552) from the retrospective cohort, and the UK trials cohort. CONCLUSIONS: TP53 mutations affected OS regardless of VAF. This finding can be used to update the definition of TP53 mutated CLL for clinical purposes.

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 (>or=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.

5-aza-2'-deoxycytidine-induced expression of functional cancer testis antigens in human renal cell carcinoma: immunotherapeutic implications.

PURPOSE: Limited therapeutic options are presently available for advanced renal cell carcinoma (RCC). This study was designed to define the clinical potential of the DNA hypomethylating agent 5-aza-2'-deoxycytidine (5-AZA-CdR) in human RCC, through its control of the expression of "therapeutic targets" of the cancer testis antigen (CTA) family, and of the tumor-associated antigen RAGE-1, in RCC cells. EXPERIMENTAL DESIGN: Reverse transcription (RT)-PCR assays of a panel of RCC cells treated with 5-AZA-CdR, investigated the induction of the expression of several CTAs and of RAGE-1. Immunoprecipitation and Western blotting assessed whether the expression of CTA-specific mRNA induced by 5-AZA-CdR resulted in a translated protein of appropriate molecular weight. The functional activity of de novo expressed CTA was evaluated using (51)Cr release cytotoxicity assays of 5-AZA-CdR-treated HLA-A2-positive RCC cells using HLA-A2-restricted NY-ESO-1-specific CTLs. RESULTS: Exposure to 5-AZA-CdR invariably induced the expression of the CTA MAGE-1, -2, -3, and -4, GAGE 1-6, and NY-ESO-1 in all of the RCC cells investigated. De novo expression of NY-ESO-1 was persistent, being still detectable 60 days after the end of treatment, and generated a functional protein efficiently recognized by HLA-A2-restricted NY-ESO-1-specific CTLs. 5-AZA-CdR also induced RAGE-1 expression in RAGE-1-negative RCC and sarcoma cells but not in neoplastic cells of different histology. CONCLUSIONS: This study provides the scientific rationale to establish new strategies of chemoimmunotherapy in RCC patients. The well-defined immunogenicity of the investigated CTAs and of RAGE-1 suggest that systemic administration of 5-AZA-CdR represents a promising strategy to enhance the constitutively poor immunogenic potential of RCC cells, and to propose that virtually all RCC patients receive active and/or adoptive CTA- or RAGE-1-based immunotherapy.

Cluster analysis of immunophenotypic data: the example of chronic lymphocytic leukemia.

Studies of gene expression profiling have been successfully used for the identification of molecules to be employed as potential prognosticators. In analogy with gene expression profiling, we have previously proposed an original method to identify the immunophenotypic signature of chronic lymphocytic leukemia (CLL) subsets with different prognosis, named surface-antigen expression profiling. According to this method, expression data for surface markers can be successfully analyzed by data mining tools identical to those employed in gene expression profiling studies, including unsupervised and supervised algorithms, with the aim to identify the immunophenotypic signature of CLL subsets with different prognosis. By employing an identical approach for investigating the reactivity of a wide panel of monoclonal antibodies provided by the "Ninth International Workshop on Leukocyte Differentiation Antigens", we were able to identify some of them (i.e. TCL1, CCR7, FCRL2, FCRL3, and CD150) as additional potential markers with prognostic relevance in CLL. These suggestions need to be confirmed: (i) in a new set of clinically characterized CLL cases; (ii) in combination with other prognostic markers in the context of comprehensive scoring systems for clinical outcome prediction.

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.

Promoter methylation controls the expression of MAGE2, 3 and 4 genes in human cutaneous melanoma.

Cancer-testis antigens expressed by different-histotype transformed cells are suitable targets for tumor immunotherapy. However, their heterogeneous expression in neoplastic lesions limits the eligibility of patients for cancer-testis antigen-directed vaccination, and low levels of cancer-testis antigens' expression may impair immune recognition of malignant cells. Because of the primary clinical relevance of cancer-testis antigens' expression in neoplastic tissues, 68 unrelated or sequential metastatic lesions from 56 patients were used to characterize the molecular mechanisms regulating the presence and levels of expression of different cancer-testis antigens of the MAGE family (i.e., MAGE2, 3 and 4) in cutaneous melanoma. Polymerase chain reaction-based methylation analyses showed that methylation status of specific cytosine-guanine dinucleotides in the promoters of investigated cancer-testis antigens correlated with their heterogeneous expression within unrelated metastatic melanoma lesions, and with their homogeneous expression among sequential metastases from three patients with melanoma. Unlike methylated promoters, unmethylated promoters of MAGE2, 3 and 4 genes drove the expression of reporter gene-enhanced green fluorescent protein after transient transfection of cancer-testis antigen-positive Mel 142 melanoma cells. Furthermore, de novo expression of MAGE3 gene induced by the treatment of Mel 195 melanoma cells with the DNA hypomethylating agent 5-aza-2'-deoxycytidine was associated with a 6%-12% demethylation of selected cytosine-guanine dinucleotides in its promoter. Finally, 5-aza-2'-deoxycytidine induced a 16-fold increase of MAGE3 expression in Mel 313 melanoma cells expressing constitutively low levels of the antigen, but did not affect that of Mel 275 melanoma cells expressing high baseline levels of MAGE3. Overall, these findings identify promoter methylation as a shared mechanism directly regulating the expression of therapeutic cancer-testis antigens in metastatic melanomas, and foresee the clinical use of 5-aza-2'-deoxycytidine to design new chemoimmunotherapeutic strategies in patients with melanoma.

Vaccination of stage IV patients with allogeneic IL-4- or IL-2-gene-transduced melanoma cells generates functional antibodies against vaccinating and autologous melanoma cells.

The antibody (Ab) response to allogeneic Me14932 and autologous melanoma cells was analyzed in 13 Stage IV (AJCC) melanoma patients immunized with Me14932 cells transduced with the IL-4 (Me14932/IL-4) ( n=10) or IL-2 (Me14932/IL-2) ( n=3) gene. No Ab response was observed before the 4th vaccination. Among 8 patients that received four vaccinations, 3/5 patients vaccinated with Me14932/IL-4 cells developed Ab (IgG and/or IgM) to Me14932 ( n=3) and to autologous ( n=2) melanoma cells, and 2/3 patients vaccinated with Me14932/IL-2 cells developed Ab (IgG) to Me14932, but not to autologous melanoma cells. Further, among these 5 responding patients, circulating Ab against the HLA-A3 allele, expressed only on vaccinating cells, were identified in the immune sera of 4 patients immunized with Me14932/IL-4 ( n=2) or Me14932/IL-2 ( n=2) cells. These sera mediated antibody-dependent cell cytotoxicity (ADCC) of Me14932 cells, and a direct correlation ( r=0.85; P=0.03) between intensity of staining (IgG) and extent of lysis was found. Immune serum of one of these patients also induced ADCC of autologous melanoma cells, and serum from another patient mediated complement cytotoxicity of Me14932, but not of autologous melanoma cells. Thus, Abs against vaccinating and autologous melanoma cells were generated in 62% of patients after four vaccinations with cytokine-transduced melanoma cells. These findings demonstrate that the identification and titration of alloreactive Ab helps to monitor the extent of immunization against cellular vaccines, while the induction of Ab reactive to antigens shared between vaccinating and autologous melanoma cells may contribute to their therapeutic efficacy.