Despite its indolent nature, chronic lymphocytic leukemia (CLL) remains an incurable disease. To establish the potential pathogenic role of miRNAs, the identification of deregulated miRNAs in CLL is crucial.
A risk score based on three biological features (CD38, ZAP-70, and IGHV mutational status) was previously developed to predict progression-free survival (PFS) in untreated Binet A CLL patients. Here we perform a score validation analysis in a prospective and independent cohort of patients. Biological markers (CD38, ZAP-70, and IGHV mutational status) and gene expression profiles (GEP) of leukemic cells from CLL patients included in a prospective multicenter observational study (O-CLL1-GISL protocol, clinicaltrial.gov ID:NCT00917549) were used to assess the value and reproducibility of this score. To date, 468 Binet A patients were classified as low- (0 positive marker), intermediate- (1 positive marker), or high-risk (2 or 3 positive markers) using the progression risk score. The 3-year PFS probability was 91.7%, 82.9%, and 57.4% for low-, intermediate-, and high-risk (P?0.0001) cases, respectively. These values were similar to those found in the original cohort. At Cox multivariate analysis, Rai stage, absolute lymphocyte count, progression risk score, and ?-2 microglobulin maintained an independent prognostic impact on PFS. This score remained a predictor of progression when analysis was limited to 371 Rai 0 cases (P?0.0001). Finally, the cells from the different CLL risk groups showed differences in their gene expression patterns. These results confirm the ability of this progression risk score to predict PFS among Binet A patients. The utility of the score was also extended by demonstrating that it retains prognostic value when applied exclusively to Rai 0 patients. Specific transcriptional patterns were significantly associated with risk groups.
NOTCH1 mutations have recently emerged as new genetic lesions significantly correlated with survival in chronic lymphocytic leukaemia (CLL). We performed deep next generation sequencing of the NOTCH1 mutation hotspot in 384 cases at diagnosis, including 100 monoclonal B cell lymphocytosis (MBL) and 284 Binet stage A CLL cases, enrolled in the Gruppo Italiano Studio Linfomi O-CLL1 multicentre trial. The NOTCH1 c.7541_7542delCT dinucleotide deletion was detected and confirmed by an extremely sensitive polymerase chain reaction-based approach in 11% of MBL and 13·4% of CLL patients. Remarkably, the NOTCH1 mutation was often observed at low clonal level, mainly in MBL patients. Sequential analyses in a fraction of cases showed that the NOTCH1 mutation generally does not occur during the disease course and that the mutational load in positive cases tends to be stable over time. NOTCH1-mutated cases, even at low clonal level, displayed a significant reduction in median progression-free survival, although NOTCH1 mutation lost its prognostic impact in a multivariate analysis including 11q and/or 17p deletion, IGHV mutational status, and MBL or CLL status. Our data highlight the importance of using highly sensitive methods to measure NOTCH1 mutations, in order to improve prognostic stratification and obtain useful information for potential therapeutic approaches.
To investigate the incidence and clinical relevance of classic and new prognostic markers, IGHV gene mutational status, and chromosomal abnormalities in clinical monoclonal B lymphocytosis (cMBL) compared with Rai stage 0 chronic lymphocytic leukemia (Rai0-CLL).
Small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs are non-coding RNAs involved in the maturation of other RNA molecules. Alterations of sno/scaRNA expression may play a role in cancerogenesis. This study elucidates the patterns of sno/scaRNA expression in 211 chronic lymphocytic leukemia (CLL) patients (Binet stage A) also in comparison with those of different normal B-cell subsets.
Many squamous cell carcinomas (SCCs) are characterized by high levels of EGFR and by overexpression of the ?Np63? isoform. Here, we investigated the regulation of ?Np63? expression upon EGFR activation and the role of the EGFR-?Np63? axis in proliferation of SCC tumor-initiating cells (TICs). SCC cell lines A-431, Cal-27, and SCC-25 treated with EGF showed a time-dependent increase in ?Np63? expression at the protein and mRNA levels, which was blocked by the tyrosine kinase inhibitor (TKI) Lapatinib. RNA interference experiments suggested the role of STAT3 in regulating ?Np63? expression downstream of EGFR. Inactivation of EGFR by the monoclonal antibody Cetuximab and RNA interference against STAT3 or ?Np63? impaired the TICs ability to grow under non-differentiating conditions. Radiation treatment, which triggers EGFR activation, induced ?Np63? accumulation without affecting TICs proliferation, whereas the combination Cetuximab plus radiation significantly reduced TICs growth under non-differentiating conditions. Together, our findings provide evidence that ?Np63? expression is regulated by EGFR activation through STAT3 and that the EGFR-?Np63? axis is crucial for proliferation of TICs present in SCCs.
Primary plasma cell leukemia (pPCL) is a rare and very aggressive form of plasma cell dyscrasia. To date, no information on microRNA (miRNA) expression in pPCL has been reported. This study aimed at investigating the involvement of miRNAs in pPCL and their possible relationship with higher tumor aggressiveness.
Plasma cell leukemia (PCL) is a rare form of plasma cell dyscrasia that presents either as a progression of previously diagnosed multiple myeloma, namely secondary PCL, or as initial manifestation of disease, namely primary PCL (pPCL). Although the presenting signs and symptoms include those seen in multiple myeloma, pPCL is characterized by several aspects that define a more aggressive course. Here, we have investigated the transcriptome of pPCLs and correlated differential expression profiles with outcome to provide insights into the biology of the disease.
This study investigated the efficiency of Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) repair systems in rejoining DNA double-strand breaks (DSB) induced in CCD-34Lu cells by different ?-ray doses. The kinetics of DNA repair was assessed by analyzing the fluorescence decrease of ?-H2AX foci measured by SOID (Sum Of Integrated Density) parameter and counting foci number in the time-interval 0.5-24 hours after irradiation. Comparison of the two methods showed that the SOID parameter was useful in determining the amount and the persistence of DNA damage signal after exposure to high or low doses of ionizing radiation. The efficiency of DSB rejoining during the cell cycle was assessed by distinguishing G1, S, and G2 phase cells on the basis of nuclear fluorescence of the CENP-F protein. Six hours after irradiation, ?-H2AX foci resolution was higher in G2 compared to G1 cells in which both NHEJ and HR can cooperate. The rejoining of ?-H2AX foci in G2 phase cells was, moreover, decreased by RI-1, the chemical inhibitor of HR, demonstrating that homologous recombination is at work early after irradiation. The relevance of HR in DSB repair was assessed in DNA-PK-deficient M059J cells and in CCD-34Lu treated with the DNA-PKcs inhibitor, NU7026. In both conditions, the kinetics of ?-H2AX demonstrated that DSBs repair was markedly affected when NHEJ was absent or impaired, even in G2 phase cells in which HR should be at work. The recruitment of RAD51 at DSB sites was, moreover, delayed in M059J and in NU7026 treated-CCD-34Lu, with respect to DNA-PKcs proficient cells and continued for 24 hours despite the decrease in DNA repair. The impairment of NHEJ affected the efficiency of the HR system and significantly decreased cell survival after ionizing radiation, confirming that DSB rejoining is strictly dependent on the integrity of the NHEJ repair system.
Highly homologous B-cell receptors, characterized by non-random combinations of immunoglobulin heavy-chain variable (IGHV) genes and heavy-chain complementarity determining region-3 (HCDR3), are expressed in a recurrent fraction of patients affected by chronic lymphocytic leukemia (CLL). We investigated the IGHV status of 1131 productive IG rearrangements from a panel of 1126 CLL patients from a multicenter Italian study group, and correlated the presence and class of HCDR3 stereotyped subsets with the major cytogenetic alterations evaluated by FISH, molecular prognostic factors, and the time to first treatment (TTFT) of patients with early stage disease (Binet A). Stereotyped HCDR3 sequences were found in 357 cases (31.7%), 231 of which (64.7%) were unmutated. In addition to the previously described subsets, 31 new putative stereotypes subsets were identified. Significant associations between different stereotyped HCDR3 sequences and molecular prognostic factors, such as CD38 and ZAP-70 expression, IGHV mutational status and genomic abnormalities were found. In particular, deletion of 17p13 was significantly represented in stereotype subset #1. Notably, subset #1 was significantly correlated with a substantially reduced TTFT compared to other CLL groups showing unmutated IGHV, ZAP-70 or CD38 positivity and unfavorable cytogenetic lesions including del(17)(p13). Moreover, subset #2 was strongly associated with deletion of 13q14, subsets #8 and #10 with trisomy 12, whereas subset #4 was characterized by the prevalent absence of the common cytogenetic abnormalities. Our data from a large and representative panel of CLL patients indicate that particular stereotyped HCDR3 sequences are associated with specific cytogenetic lesions and a distinct clinical outcome.
Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease characterized by recurrent chromosomal aberrations of prognostic significance. We aimed to evaluate the potential of the multiplex ligation-dependent probe amplification (MLPA) assay to detect genomic alterations in CLL. Highly purified (>90%) peripheral mononuclear CD19+ cell populations from 100 untreated CLL patients (pts) in early stage disease (Binet stage A) were included in this study. All samples were investigated by fluorescence in situ hybridization (FISH) for the presence of trisomy 12 and 17p13.1, 11q22.3, and 13q14.3 deletions. For MPLA analysis, DNA was amplified by means of two commercially available probes sets allowing the simultaneous screening of 56 genomic sequences. Overall, a high degree of concordance (95%) between MPLA and FISH results was found, if the abnormal clone was present in more than 30% of the leukemic cell population. The use of multiple MPLA probes allowed the fine-mapping of the 13q14 deletion and the identification of intragenic or small alterations undetected by FISH. Moreover, additional alterations in 2p24 (MYCN) (3 pts), 8q24 (MYC) (1 pt), 9p21 (CDKN2A2B) (1 pt), 1q21 (LMNA) (1 pt), and 6q25-26 (1 pt) regions not covered by a standard FISH assay were detected and all confirmed by FISH. Our data extend previously limited evidence that MLPA may represent a useful technique for the characterization of well-known lesions as well as the investigation of additional genomic changes in CLL.
The mutational status of the immunoglobulin heavy-chain variable region (IGHV) genes utilized by chronic lymphocytic leukemia (CLL) clones defines two disease subgroups. Patients with unmutated IGHV have a more aggressive disease and a worse outcome than patients with cells having somatic IGHV gene mutations. Moreover, up to 30% of the unmutated CLL clones exhibit very similar or identical B cell receptors (BcR), often encoded by the same IG genes. These "stereotyped" BcRs have been classified into defined subsets. The presence of an IGHV gene somatic mutation and the utilization of a skewed gene repertoire compared with normal B cells together with the expression of stereotyped receptors by unmutated CLL clones may indicate stimulation/selection by antigenic epitopes. This antigenic stimulation may occur prior to or during neoplastic transformation, but it is unknown whether this stimulation/selection continues after leukemogenesis has ceased. In this study, we focused on seven CLL cases with stereotyped BcR Subset #8 found among a cohort of 700 patients; in six, the cells expressed IgG and utilized IGHV4-39 and IGKV1-39/IGKV1D-39 genes, as reported for Subset #8 BcR. One case exhibited special features, including expression of IgM or IgG by different subclones consequent to an isotype switch, allelic inclusion at the IGH locus in the IgM-expressing cells and a particular pattern of cytogenetic lesions. Collectively, the data indicate a process of antigenic stimulation/selection of the fully transformed CLL cells leading to the expansion of the Subset #8 IgG-bearing subclone.
Global DNA hypomethylation affecting repeat sequences has been reported in different cancer types. Herein, we investigated the methylation levels of repetitive DNA elements in chronic lymphocytic leukemia (CLL), their correlation with the major cytogenetic and molecular features, and clinical relevance in predicting therapy-free survival (TFS). A quantitative bisulfite-PCR Pyrosequencing method was used to evaluate methylation of Alu, long interspersed nuclear elements-1 (LINE-1) and satellite-? (SAT-?) sequences in 77 untreated early-stage (Binet A) CLL patients. Peripheral B-cells from 7 healthy donors were used as controls. Methylation levels (median %5mC) were lower in B-CLLs compared with controls (21.4 vs. 25.9; 66.8 vs. 85.7; 84.0, vs. 88.2 for Alu, LINE-1 and SAT-?, respectively) (p < 0.001). Among CLL patients, a significant association was observed with 17p13.1 deletion (16.8 vs. 22.4; 51.2 vs. 68.5; 52.6 vs. 85.0, for Alu, LINE-1 and SAT-?) but not with other major genetic lesions, IgVH mutation status, CD38 or ZAP-70 expression. Follow-up analyses showed that lower SAT-? methylation levels appeared to be an independent prognostic marker significantly associated with shorter TFS. Our study extended previous limited evidences in methylation of repetitive sequences in CLL suggesting an important biological and clinical relevance in the disease.
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.
To describe the clinical and biological features of a series of immunophenotypic variant of B-CLL (v-CLL) characterised by intermediate RMH score, in the absence of t(11;14)(q13;q32) in FISH analysis in comparison with a series of typical CLL.
To date, little evidence of miRNA expression/deregulation in multiple myeloma has been reported. To characterize miRNA in the context of the major multiple myeloma molecular types, we generated miRNA expression profiles of highly purified malignant plasma cells from 40 primary tumors. Furthermore, transcriptional profiles, available for all patients, were used to investigate the occurrence of miRNA/predicted target mRNA pair anticorrelations, and the miRNA and genome-wide DNA data were integrated in a subset of patients to evaluate the influence of allelic imbalances on miRNA expression. Differential miRNA expression patterns were identified, which were mainly associated with the major IGH translocations; particularly, t(4;14) patients showed specific overexpression of let-7e, miR-125a-5p, and miR-99b belonging to a cluster at 19q13.33. The occurrence of other lesions (ie, 1q gain, 13q and 17p deletions, and hyperdiploidy) was slightly characterized by specific miRNA signatures. Furthermore, the occurrence of several allelic imbalances or loss of heterozygosity was found significantly associated with the altered expression of miRNAs located in the involved regions, such as let-7b at 22q13.31 or miR-140-3p at 16q22. Finally, the integrative analysis based on computational target prediction and miRNA/mRNA profiling defined a network of putative functional miRNA-target regulatory relations supported by expression data.
Multiple myeloma (MM) is characterized by a wide spectrum of genetic changes. Global hypomethylation of repetitive genomic sequences such as long interspersed nuclear element 1 (LINE-1), Alu and satellite alpha (SAT-alpha) sequences has been associated with chromosomal instability in cancer. Methylation status of repetitive elements in MM has never been investigated. In the present study, we used a quantitative bisulfite-polymerase chain reaction pyrosequencing method to evaluate the methylation patterns of LINE-1, Alu and SAT-alpha in 23 human myeloma cell lines (HMCLs) and purified bone marrow plasma cells from 53 newly diagnosed MM patients representative of different molecular subtypes, 7 plasma cell leukemias (PCLs) and 11 healthy controls. MMs showed a decrease of Alu [median: 21.1 %5-methylated cytosine (%5mC)], LINE-1 (70.0%5mC) and SAT-alpha (77.9%5mC) methylation levels compared with controls (25.2, 79.5and 89.5%5mC, respectively). Methylation levels were lower in PCLs and HMCLs compared with MMs (16.7 and 14.8%5mC for Alu, 45.5 and 42.4%5mC for LINE-1 and 33.3 and 43.3%5mC for SAT-alpha, respectively). Notably, LINE-1 and SAT-alpha methylation was significantly lower in the non-hyperdiploid versus hyperdiploid MMs (P = 0.01 and 0.02, respectively), whereas Alu and SAT-alpha methylation was significantly lower in MMs with t(4;14) (P = 0.02 and 0.004, respectively). Finally, we correlated methylation patterns with DNA methyltransferases (DNMTs) messenger RNA levels showing in particular a progressive and significant increase of DNMT1 expression from controls to MMs, PCLs and HMCLs (P < 0.001). Our results indicate that global hypomethylation of repetitive elements is significantly associated with tumor progression in MM and may contribute toward a more extensive stratification of the disease.
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.
Multiple myeloma (MM) is characterized by marked genomic heterogeneity. Beyond structural rearrangements, a relevant role in its biology is represented by allelic imbalances leading to significant variations in ploidy status. To elucidate better the genomic complexity of MM, we analyzed a panel of 45 patients using combined FISH and microarray approaches. We firstly generated genome-wide profiles of 41 MMs and four plasma cell leukemias, using a self-developed procedure to infer exact local copy numbers (CNs) for each sample. Our analysis allowed the identification of a significant fraction of patients showing near-tetraploidy. Furthermore, a conventional hierarchical clustering analysis showed that near-tetraploidy, 1q gain, hyperdiploidy, and recursive deletions at 1p and chromosomes 13, 14, and 22 were the main aberrations driving samples grouping. Moreover, mapping information was integrated with gene expression profiles of the tumor samples. A multiclass analysis of transcriptional profiles characterizing the different clusters showed marked gene-dosage effects, particularly concerning 1q transcripts; this finding was also confirmed by a nonparametric analysis between normalized gene expression levels and local CN variations (1027 highly-significant correlated genes). Finally, we identified several loci in which gene expression correlated with the occurrence of loss of heterozygosity. Our results provide insights into the composite network linking genome structure and transcriptional features in MM.
It is thought that altered microRNA (miRNA) expression due to various mechanisms plays a critical role in the pathogenesis of most human cancers. Notably, about half of the known miRNAs are intragenic and frequently coexpressed with their host genes. To date there is little evidence concerning miRNA expression in multiple myeloma (MM). In an attempt to provide insights into miRNA deregulation in MM, we profiled global miRNA expression in a panel of molecularly well-characterized human myeloma cell lines (HMCLs) using high-resolution microarrays, and then used integrative analyses to identify altered patterns correlated with DNA copy number (CN) or gene expression profiles. We identified 16 miRNAs mapped to chromosomal regions frequently involved in numerical imbalances in MM, whose expression significantly correlated with the CN of the corresponding miRNA genes; among these, miR-22 expression was also affected by chromosome arm 17p loss in a representative panel of primary MM tumors. The expression of 32 intronic miRNAs significantly correlated with that of their host transcripts, some of which were highly deregulated in MM patients. The expression of some of the miRNAs was validated by quantitative RT-PCR. Finally, a number of the identified miRNAs have previously been reported to play important roles in tumorigenesis. Overall, our data highlight that genomic alterations may significantly affect miRNA expression in HMCLs and demonstrate a frequent coexpression of intronic miRNAs with their host genes that may have a pathogenetic relevance in plasma cell transformation.
The 6-minute walk test (6MWT) is a well-known instrument for assessing the functional capacity of a variety of groups, including the obese. It is a simple, low-cost and easily applied method to objectively assess the level of exercise capacity. The aim of the present study was to study the functional capacity of a severely obese population before and after bariatric surgery.
CD26 (dipeptidyl peptidase IV, DPP IV) is widely expressed by T and natural killer (NK) cells, epithelial and endothelial cells of different tissues, and it is strongly upregulated in activated B-cells; moreover it plays a regulatory role in the neoplastic transformation and progression of various types of tumours. CD26 expression was evaluated by means of flow cytometry in various peripheral B-cell lymphoid tumours: 12 follicular and 12 mantle cell lymphomas, 20 multiple myelomas (MMs), 12 hairy cell leukaemias (HCLs), 112 chronic lymphocytic leukaemias (CLLs), 20 CD5(negative) B-cell chronic lymphoproliferative diseases (CD5(neg) B-CLPDs) and 12 diffuse large cell lymphomas (DLCLs). CD26 expression was absent or barely detectable in follicular and mantle cell lymphomas, high in MMs and HCLs, and variable in CLLs, in CD5(neg) B-CLPDs and in DLCLs. CD26 significantly correlated with CD49d and CD38 expressions (p < 0.0001) in B-CLLs, and there was a significant correlation between CD26 and ZAP-70 expressions or IgVH mutational status (p < 0.0001). After a median follow-up of 36 months, 65 B-CLL patients were treated; taking 10% as the best CD26 cut-off value, Kaplan-Meier curves revealed a significantly shorter time to treatment in the CD26-positive cases (p < 0.0001). Overall, our data indicate that CD26 expression may identify subsets of B-CLL patients with an unfavourable clinical outcome in terms of therapeutic need, thus suggesting its potential role as a marker (together with CD38 and CD49d) in a future routine cytofluorimetric panel to be validated for the prognostic stratification of B-CLLs.
Cell response to ionising radiation depends, besides on genetic and physiological features of the biological systems, on environmental conditions occurring during DNA repair. Many data showed that microgravity, experienced by astronauts during space flights or modeled on Earth, causes apoptosis, cytoskeletal alteration, cell growth inhibition, increased frequency of mutations and chromosome aberrations. In this study, we analysed the progression of the rejoining of double strand breaks (DSBs) in human peripheral blood lymphocytes (PBLs) irradiated with gamma-rays and incubated in static condition (1 g) or in modeled microgravity (MMG). gamma-H2AX foci formation and disappearance, monitored during the repair incubation, showed that the kinetics of DSBs rejoining was different in the two gravity conditions. The fraction of foci-positive cells decreased slower in MMG than in 1 g at 6 and 24 h after irradiation (P<0.01) and the mean number of gamma-H2AX foci per nucleus was significantly higher in MMG than in 1g at the same time-points (P<0.001). In the same samples we determined apoptotic level and the rate of DSB rejoining during post-irradiation incubation. A significant induction of apoptosis was observed in MMG at 24 h after irradiation (P<0.001), whereas at shorter times the level of apoptosis was slightly higher in MMG respect to 1 g. In accordance with the kinetics of gamma-H2AX foci, the slower rejoining of radiation-induced DSBs in MMG was observed by DNA fragmentation analyses during the repair incubation; the data of pulsed-field gel electrophoresis assay showed that the fraction of DNA released in the gel was significantly higher in PBL incubated in MMG after irradiation with respect to cells maintained in 1 g. Our results provide evidences that MMG incubation during DNA repair delayed the rate of radiation-induced DSB rejoining, and increased, as a consequence, the genotoxic effects of ionising radiation.
The development of autoimmune hemolytic anemia (AIHA) in patients with chronic lymphocytic leukemia (CLL) is associated with specific biological features. The occurrence of AIHA was hereby investigated in a retrospective series of 585 CLL patients with available immunoglobulin heavy chain variable (IGHV) gene status. AIHA occurred in 73 patients and was significantly associated with an IGHV unmutated (UM) status (P < 0.0001) and unfavorable [del(17)(p13) and del(11)(q23)] cytogenetic lesions (P < 0.0001). Stereotyped HCDR3 sequences were identified in 29.6% of cases and were similarly represented among patients developing or not AIHA; notably, subset #3 was associated with a significantly higher risk of AIHA than the other patients (P = 0.004). Multivariate analysis showed that UM IGHV, del(17)(p13) and del(11)(q23), but not stereotyped subset #3, were the strongest independent variables associated with AIHA. Based on these findings, we generated a biological risk score for AIHA development according to the presence of none (low risk), one (intermediated risk), or two (high risk) of the independent risk factors. Overall, our data indicate that UM IGHV status and/or unfavorable cytogenetic lesions are associated with the risk of developing secondary AIHA in CLL patients and suggest a possible role of specific stereotyped B-cell receptor subsets in a proportion of cases.
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.
Primary plasma cell leukemia (pPCL) is a rare, yet aggressive form of de novo plasma cell tumor, distinct from secondary PCL (sPCL) which represents a leukemic transformation of pre-existing multiple myeloma (MM). Herein, we performed a comprehensive molecular analysis of a prospective series of pPCLs by means of FISH, single nucleotide polymorphism (SNP) array and gene expression profiling (GEP). IGH@ translocations were identified in 87% of pPCL cases, with prevalence of t(11;14) (40%) and t(14;16) (30.5%), whereas the most frequent numerical alterations involved 1p (38%), 1q (48%), 6q (29%), 8p (42%), 13q (74%), 14q (71%), 16q (53%), and 17p (35%). We identified a minimal biallelic deletion (1.5 Mb) in 8p21.2 encompassing the PPP2R2A gene, belonging to a family of putative tumor suppressors and found to be significantly down-regulated in deleted cases. Mutations of TP53 were identified in four cases, all but one associated with a monoallelic deletion of the gene, whereas activating mutations of the BRAF oncogene occurred in one case and were absent in N- and K-RAS. To evaluate the influence of allelic imbalances in transcriptional expression we performed an integrated genomic analysis with GEP data, showing a significant dosage effect of genes involved in transcription, translation, methyltransferase activity, apoptosis as well as Wnt and NF-kB signaling pathways. Overall, we provide a compendium of genomic alterations in a prospective series of pPCLs which may contribute to improve our understanding of the pathogenesis of this aggressive form of plasma cell dyscrasia and the mechanisms of tumor progression in MM.
The European Myeloma Network has organized two workshops on fluorescence in situ hybridization in multiple myeloma. The first aimed to identify specific indications and consensus technical approaches of current practice. A second workshop followed a quality control exercise in which 21 laboratories analyzed diagnostic cases of purified plasma cells for recurrent abnormalities. The summary report was discussed at the EHA Myeloma Scientific Working Group Meeting 2010. During the quality control exercise, there was acceptable agreement on more than 1,000 tests. The conclusions from the exercise were that the primary clinical applications for FISH analysis were for newly diagnosed cases of MM or frank relapse cases. A range of technical recommendations included: 1) material should be part of the first draw of the aspirate; 2) samples should be sent at suitable times to allow for the lengthy processing procedure; 3) most importantly, PCs must be purified or specifically identified; 4) positive cut-off levels should be relatively conservative: 10% for fusion or break-apart probes, 20% for numerical abnormalities; 5) informative probes should be combined to best effect; 6) in specialist laboratories, a single experienced analyst is considered adequate; 7) at least 100 PC should be scored; 8) essential abnormalities to test for are t(4;14), t(14;16) and 17p13 deletions; 9) suitable commercial probes should be available for clinically relevant abnormalities; 10) the clinical report should be expressed clearly and must state the percentage of PC involved and the method used for identification; 11) a retrospective European based FISH data bank linked to clinical data should be generated; and 12) prospective analysis should be centralized for upcoming trials based on the recommendations made. The European Myeloma Network aims to build on these recommendations to establish standards for a common European data base to define subgroups with prognostic significance.
To assess biologic features related to the development of immune thrombocytopenia (ITP) in patients with chronic lymphocytic leukemia (CLL).
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