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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.

Introduction

Infection of peripheral blood mononuclear cells (PBMNCs) has been demonstrated to be a crucial event in the vertical transmission of viruses, and it is known that hepatitis C virus (HCV) can infect PBMNCs. The relationship between vertical transmission of HCV and the presence of positive and negative strands of HCV-RNA in the PBMNCs of HCV-carrier mothers was investigated using reverse transcriptase–polymerase chain reaction (RT-PCR). During the study, 13 consecutive mothers who transmitted infection to their offspring and 53 consecutive mothers who did not were examined. The positive strand of HCV-RNA was identified in the PBMNCs of all mothers who transmitted the infection and in 13 of 53 mothers who did not (P?

Clonal, pre-clinical expansions of hematopoietic cells are increasingly recognized. Clonal hematopoiesis of indeterminate potential (CHIP) has been recently described. It is characterized by the presence of mutations usually involved in myeloid neoplasia in people without any sign of hematological malignancy. Well before CHIP, another pre-clinical clonal expansion has been described for plasma cells (PC) based on the presence of a serum monoclonal protein, namely monoclonal gammopathy of undetermined significance (MGUS). Despite both being frequent, the association between these two bone marrow (BM) clonal entities has not been determined yet. Since the incidence of MGUS and CHIP rises with age reaching >10% of people >70y, we analyzed a unique oldest-old population dataset to define a possible association between these two different BM clonal disorders.

ATP depletion of erythrocytes is associated with discocyte-echinocyte transformation and accumulation of calcium. This study was undertaken to evaluate whether this shape transformation results from interaction of Ca2+, Mg2+, and ATP with structural proteins of the membrane. ATP, Mg2+, or Ca2+ were incorporated into ghosts during hypotonic hemolysis followed by restoration of isotonicity with NaCl and incubation. Reconstituted ghosts were examined by electron microscope after osmium tetroxide fixation followed by platinum and carbon shadowing and in-phase contrast microscope. Four ghost configurations were observed: (1) flat, discoidal electron-penetrable ghosts were produced when ghosts were prepared without any additives, or with ATP alone, Mg2+ alone, or EDTA; (2) biconcave and cup-shaped less electron-penetrable ghosts predominated when equimolar concentrations of Mg2+ and ATP (0.5-2 mM) were introduced into ghosts during hemolysis; (3) spherical, spiculated, or markedly deformed electron-dense ghosts of smaller volume were the prevalent shape forms when Ca2+ alone was introduced during hemolysis; (4) small, smooth, electron-dense spheroids were present when both Ca2+ and Mg ATP (2 mM) were added during hemolysis. The above shapes were independent of ouabain, monovalent cations (NaCl, KCl, or choline chloride), transmembrane osmotic gradient, and ghost volume. The shape effects of Ca2+ and Mg ATP were prevented by preincubation of red cells with SH-blocking agent N-ethylmaleimide (4 mM) and by repeated ghost washing, which was associated with removal of water-soluble fibrous proteins from membranes. It is concluded that the interaction of Ca2+, Mg2+, and ATP with membrane structural proteins exerts a significant role in the control of shape of human erythrocytes.

Heterogeneity within ghosts prepared from stored erythrocytes was studied by multichannel particle analysis of size distributions of reconstituted ghosts. On restoration of isotonicity of hemolysates from fresh erythrocytes, ghosts were smaller than the corresponding red cells. After subsequent incubation for 15 min, they swelled to a final stable volume. The hemolysis of stored erythrocytes and restoration of isotonicity yielded ghosts similar to those from fresh cells, but, after subsequent incubation, a second population of small ghosts appeared. This population resulted from markedly diminished ghost swelling, that is attributed to decreased sodium permeability and increased membrane rigidity, causing resistance to changes in volume. Erythrocyte ATP depletion during storage of ACD blood was associated with an increase in the percentage of small ghosts. Similar ghosts were produced from fresh cells when CaCl2 (1 x 10-6M) was introduced into ghosts during hemolysis. The erythrocyte calcium content increased during prolonged storage from 11.3 ± 6.3 µg atoms/liter of cells (stored for 1-2 wk) to 22.0 ± 7.0 µg atoms/liter of cells, after 7-9 wk of storage. The introduction of nucleotides (2 x 10-3M) into ghosts during hemolysis of stored cells prevented the appearance of small ghosts. The inhibitory effect was greater than predicted from stability constants of calcium nucleotide complexes, indicating the specific interaction of nucleotides with the membrane. The percentage of small ghosts from red cells stored for various times agreed with the percentage of nonviable cells derived from the literature. It is concluded that the decrease in ATP and the accumulation of calcium in stored erythrocytes induce conformational changes of membrane fibrous (contractile) proteins that results in decreases in membrane elasticity and permeability, which is in turn reflected by formation of small ghosts.

In multiple myeloma (MM), whole exome sequencing (WES) studies have revealed four mutational signatures: two associated with aberrant activities of APOBEC cytidine deaminases (Signatures #2 and #13) and two clock-like signatures associated with “cancer age” (Signatures #1 and #5). Mutational signatures have not been investigated systematically in larger series, nor in other primary plasma cell dyscrasias such as monoclonal gammopathy of unknown significance (MGUS) or primary plasma cell leukemia (pPCL). Finally, while APOBEC activity has been correlated to increased mutational burden and poor-prognosis MAF/MAFBtranslocations in MM at diagnosis, this has never been confirmed in multivariate analysis in an independent series.

Introduction

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NOTCH1mutations have recently emerged as new genetic lesions significantly correlated with survival in chronic lymphocytic leukemia (CLL). NOTCH1c.7541_7542delCT is by far the most frequently observed NOTCH1mutation in the disease.

Munshi: Celgene: Consultancy; Millenium: Consultancy; Merck: Consultancy; Onyx: Consultancy.

Biological features related to the development of autoimmune hemolytic anemia (AHIA) in patients with chronic lymphocytic leukemia (CLL) are crucial insights in the understanding of the pathogenesis of autoimmune phenomena in the course of the disease.We retrospectively analyzed 585 CLL patients with available immunoglobulin heavy-chain variable (IGHV) gene status and B-cell receptor (BCR) configuration (HCDR3). Of them, 73 developed AIHA. The clinical characteristics at CLL diagnosis and follow-up were available in all patients, while cytogenetic analysis at the time of diagnosis was available in 409 patients.Occurrence of AIHA was significantly associated with an IGHV unmutated (UM) status (p<0.0001) and unfavorable cytogenetic lesions [del(17)(p13) and del(11)(q23)] (p<0.0001). Stereotyped HCDR3 sequences were identified in 173 of 585 patients (29.6%) and were similarly represented among patients developing AIHA (28,7%) or not (29.6%). Of the stereotyped subsets, subset #3 was associated with a significantly higher risk of AIHA occurrence than the other HCDR3 configurations (p=0.004). Restricting the analysis to UM patients, a strong association was found between AIHA and “truly” UM patients, defined as patients carrying a 100% identity with the germ line configuration. Multivariate analysis showed that “truly” UM IGHV, del(17)(p13) and del(11)(q23) were the strongest independent variables associated with risk of developing AIHA (p=0.02, p=0.0002 and p=0.01, respectively).Based on the results of the multivariate analysis, we constructed a risk score of developing AIHA during time, according to the presence of none (low risk = favorable cytogenetics and mutated (M) IGHV), one (intermediated risk = unfavorable cytogenetic or UM), or two (high risk = unfavorable cytogenetic and UM) risk factors. This scoring system allowed a significant patient risk stratification (Figure 1).Taken together, our data indicate that an 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 BCR subsets in a proportion of cases.No relevant conflicts of interest to declare.

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Abstract 3955

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Anderson: Millennium Pharmaceuticals, Inc.: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Onyx: Consultancy; Merck: Consultancy; Bristol-Myers Squibb: Consultancy; Actelion: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

No relevant conflicts of interest to declare.

Recently in large randomized studies, the addition of Rituximab to standard chemotherapy in the first-line treatment of follicular lymphoma (FL) demonstrated improvements in long-term outcome in FL.We compared the outcome of 111 naive FL patients (pts) treated at our institution from 1995 to 2009 with a single alkylating agent in combination or not with Rituximab: 58 pts received Chlorambucil plus Rituximab (R-Chl) and 53 pts Chlorambucil and prednisone (Chl+PDN). The 2 schedules included an induction phase, where Chlorambucil was given at 6mg/mq for 6 consecutive weeks in both groups and the Rituximab in 4 weekly administrations. The maintenance phase was longer in the Chl+PDN group: Chlorambucil was administered 6mg/mq daily 14 days each month for a total of 12-months of treatment versus 14 days with monthly-Rituximab for 4 consecutive months in the R-Chl group.The demographic and prognostic factors are reported in Table 1.At the end of treatment the ORR was 98% in the R-Chl pts and 77% in the Chl+PDN group, with a percentage of complete response of 79% and 55% respectively. No significant incidence of adverse events were reported and only one HBsAg positive patient in R-Chl group discontinued the therapy. One case of myelodysplastic syndrome was described in a Chl+PDN relapsed patient. With a median follow up of 34 months, the OS and the EFS in R-Chl pts is 95% and 76% respectively. Otherwise, with a median observation time of 82 months in the Chl+PDN group 73% are alive and only 28% of pts maintained the response. The median time to subsequent therapy (TTST) is 21 and 15 months respectively in the 2 groups.The addition of Rituximab to alkylating in first-line treatment in previously untreated FL pts improves significantly the EFS and prolongs the time to next antilymphoma therapy compared with alkylating alone. Because of its low toxicity profile, our combination with Rituximab and Chlorambucil could be considered a first-line therapy, especially in FL patients not eligible for aggressive chemotherapy regimens.No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

High-dose chemotherapy followed by autologous Peripheral Blood Stem Cell (PBSC) transplantation represents an effective option in relapsing/refractory malignant lymphoma as well as in selected solid tumors. Collection of a sufficient amount of stem cells (CD34+ ≥ 2.0×106/kg) by apheresis is mandatory for the procedure. However many factors could influence the mobilization of PBSC and about 30% of patients eligible for this therapeutic option fail stem cell mobilization. Peripheral CD34+ counts of 20/μL is conventionally considered the cut-off for a successful collection.With the aim to identify those factors affecting PBSC mobilization, we retrospectively reviewed data about our experience at European Institute of Oncology from 01/2005 to 05/2009. By evaluating the number of CD34+cells after mobilization, patients were considered as good mobilizers (peripheral CD34+ ≥ 20/μL, group A), relative poor mobilizers (peripheral CD34+ counts <20 and ≥8/ μL, group B) and absolute poor mobilizers (peripheral CD34+ counts <8/μL, group C). A total of 248 patients were enrolled into a mobilizing PBSC program; apheresis was performed when the CD34+ cell count was >5/μL. Patients characteristics were: 140 male, 108 female; median age was 51 yrs; diagnosis included: 124 Non Hodgkin Lymphoma (NHL), 50 Hodgkin Lymphoma (HL), 35 Multiple Myeloma (MM), 5 Acute Leukemia (AL) and 33 solid tumors, 1 Chronic Lymphocitic Leukemia (CLL); mean number of previous chemotherapy lines was 1 (0-9). The main mobilization regimens in hematological patients were cyclophosphamide 4g/mq (n=118) and ESHAP either followed by G-CSF (n=27) or pegylated G-CSF (n=48); the majority of patients affected by solid tumors received ICE regimen plus G-CSF (n=26).By evaluating the number of CD34+cells after mobilization, 163 (65.7%) patients resulted good mobilizers, 43 (17.3%) patients relative poor mobilizers and 42 (17%) patients absolute poor mobilizers. All patients in group A, 31 patients in group B (72%) and 8 patients in group C (19%) collected >2.0×106 CD34+cells/kg. According the Two-sided Fisher's exact test, more than 3 previous chemotherapy lines before mobilization (p<0.001), pretreatment with purine analogues (p=0.007) or 90Y-Ibritumomab Tiuxetan (p=0.002) were found to be independent factors able to affect PBSC mobilization. In a multivariate analysis, these factors were confirmed as detrimental (purine analogs p=0.019, 90Y-Ibritumomab Tiuxetan p=0.003).These results could help to identify those factors affecting PBSC mobilization. To reduce poor mobilizers we suggest to anticipate mobilization program and to evaluate the opportunity to reserve purine analogs or radioimmunotherapy after collection. About 20% of patients defined as absolute poor mobilizers might successfully mobilize PBSC by lowering the CD34+ cut-off before apheresis.No relevant conflicts of interest to declare.

Follicular lymphoma (FL), one of the most common B-cell non-Hodgkin's lymphoma (NHL), is an indolent disease characterized by a continuous relapse pattern. In the last years the treatment have changed with the introduction of Rituximab, that in combination with chemotherapy prolongs the progression-free survival (PFS) and the overall survival (OS) of FL pts. We report here the results of a regimen containing Rituximab and Chlorambucil (Chl) in untreaed FL pts.Since December 2001 48 pts (22 male and 26 female) with naive FL were treated in first-line with R-Chl. The schedule consisted of an induction phase with four weekly infusion of Rituximab at standard dose and 6 consecutive weeks of Chl at 6mg/mq/daily. Pts were restaged and in absence of disease progression received four cycles of consolidation with a monthly Rituximab infusion and 14 days of Chl each month.Median age at diagnosis was 56 years (range 29-79). Ann Arbor stage was I-II in 13 pts and III-IV in 35 pts (73%); 15 pts had bulky disease and 14 pts presented an extranodal localization. B symptoms were present in 8 pts. Histological grading was available in 42 cases, and was 1 in 10 pts, 2 in 26 pts and 3 in 6 pts. FLIPI was evaluable in 46 pts, and was 0-1 in 25 pts (52%), 2 in 12 pts (25%), and >2 in 9 pts (18%). After the induction phase overall response rate (ORR) was 98% with 14 pts in complete remission (CR). After the consolidation phase 39 pts (81%) obtained a CR and eight (17%) a partial response (PR); the remaining patient had a stable disease and underwent high-dose chemotherapy and autologous transplantation. All pts but one concluded the planned treatment. The mean daily dose of Chl administered during the induction phase was 10 mg, while 8 mg in the prolonged treatment. Twenty-eight pts (58%) experienced a neutropenia during treatment, and Chl dose was reduced in 22 pts (46%); however, only 11 pts (23%) had a G3-4 neutropenia. The Chl was stopped only in a patient because of a persistent G3 neutropenia after the first consolidation cycle. After a median observation time of 42.5 months (range 7-94) nine pts relapsed (19%), with a median time to relapse of 20 months (range 7-66). Two pts died, one of disease progression and one of a pre-existing ovarian cancer.Our results described a safe and feasible combination of immuno-chemotherapy in untreated FL pts. With respect to traditional treatments, our regimen shows a similar efficacy and a lower toxicity, that leads to an easier handling. The schedule including four monthly additional Rituximab administrations confirms the superiority of a prolonged exposure to Rituximab over the standard 4-weekly administrations. In conclusion, our data suggest that this combination may be considered as a valid first-line treatment of FL patients, especially in those not eligible for more aggressive chemotherapy regimens.No relevant conflicts of interest to declare.

Abstract 3210

The recent discovery of microRNAs (miRNAs), small noncoding RNAs involved in the regulation of cell cycle, survival, and differentiation programmes, has added a further level of complexity to normal and cancer cell biology. Loss or amplification of miRNA genes by broad cytogenetic abnormalities or minute molecular aberrations has been observed in a variety of human malignancies, with the consequent altered expression of these regulatory genes. Additionally, approximately one third of miRNAs are located within the intronic regions of coding transcription units, and recent evidence indicates that the expression of these miRNAs largely coincides with the transcription of the corresponding host genes. To date, no evidence of deregulated miRNA expression has been reported in multiple myeloma (MM). To provide insights into miRNA biology in MM, we performed an integrative analysis of genome-wide, gene expression and miRNA expression profilings in a panel of 16 human myeloma cell lines (HMCLs). Global miRNA and mRNA expression data were generated on Agilent miRNA microarrays (representing 470 human mature miRNAs) and GeneChip® HG-U133A arrays, respectively, and both quantile-normalized. Genome-wide profiling data were generated on GeneChip® Human Mapping 250K NspI arrays and copy number (CN) values were inferred using the circulary binary segmentation (DNAcopy R Bioconductor package). To measure the correlation between the expression levels of each miRNA and the corresponding CN value or host gene expression, conventional non-parametric analyses were performed (Kendall’s tau and Wilcoxon rank-sum tests). As regards miRNA gene CN, the most frequent alteration identified was represented by gain/amplification (for all miRNA genes investigated, an increased CN was present in at least 3 HMCLs, with an average frequency of 58%), followed by loss (5%) and biallelic deletion (0.3%). Our analysis revealed that 14 different miRNA transcripts (miR-15a, miR-19a, miR-21, miR-22, miR-30d, miR-99b, miR-130b, miR-132, miR-140, miR-185, miR-339, miR-491, miR-503, miR-768-3p) had concordant levels with the inferred CN value of the corresponding miRNA gene. Notably, the identified miRNAs mapped to different genomic regions, some of which are involved in recurrent CN alterations in MM, such as 8q24, 19q13.33, or chromosome arms 13q, 16q, 17p, 17q, 22q, and for some of the miRNAs a role in other types of cancer has already been suggested. As regards intragenic miRNAs, 187 miRNA/host gene pairs were obtained after localizing miRNAs within the absolute 5′ and 3′ regions of genes represented on the HG-U133A arrays; 25 of these showed a significant correlation between miRNA and mRNA levels. Among the most correlated miRNA/hostgene pairs we identified miR-152/COPZ2, miR-342-3p/EVL, miR-335/MEST, miR-25 and miR-106b/MCM7. For some of the identified pairs, miRNA expression levels were validated by means of Q-RT-PCR. In conclusion, we showed that miRNA expression in HMCLs could be affected by the presence of genomic lesions or may correlate with host-gene modulation, suggesting a possible role in the molecular pathogenesis of MM. Our integrative approach represents the basis for further investigations, also in primary tumors, aimed at functionally characterizing specific miRNAs in MM.

Here we present the clinical results and the outcomes of 143 consecutive newly-diagnosed follicular lymphoma (FL) patients (pts) treated with 3 different modalities of treatment in our institution from 1994 until 2007. During this period, from 1994 to 2002, 55 pts received Chlorambucil and Prednisone (Chl+PDN), subsequently 31 pts Rituximab with Chlorambucil (R-Chl) and 57 pts a 4-weekly standard-dose of Rituximab alone (R). Chlorambucil was given in an induction phase at 10mg/day for 6 consecutive weeks followed by a longer maintenance phase in the first group: 2-week pulses of 10 mg daily with 2-week intervals for a total of 12-months of treatment versus four 2-week pulses with monthly-Rituximab. In the single agent R group 36 pts received maintenance with at least 4-bimonthly administrations. The 3 groups were comparable for demographic and prognostic factors: median age at diagnosis was 55 yrs, over 60% of pts were in advanced stages and mainly asyptomatic (about 90%) and over 50% of pts in each group were low-risk FLIPI. A third of pts in both Rituximab-based treatment groups had bone marrow involvement and bulky disease. In terms of ORR 78% of pts treated with Chl+PDN, 97% with R-Chl and 72% with R single agent, obtained a clinical response, with a percentage of complete response of 58%, 90% and 47% respectively. No significant incidence of adverse events were reported and none discontinued the therapy because of toxicity. One case of myelodysplatic syndrome was described in a relapsed patient of the Chl-PDN group. With a median follow up of 72, 56 and 27 months in the 3 groups evaluated, the CR rate was maintained in 29%, 74% and 37% of pts respectively with a median duration of response of 36, 40 and 24 months. Rituximab as first-line in FL pts seems safe, feasible and able to induce a high rate of clinical response similar to those achieved with standard chemotherapy alone. On the other hand, the immunochemotherapy seems to confirm its superiority in terms of response rate and duration of response.

B-cell chronic lymphocytic leukemia (B-CLL) is a heterogeneous disease; some patients have a rapidly progressing disease and others exhibit an indolent course and survive for many years without treatment. Mutation status of IgVH genes utilized by CLL cells represents a very reliable predictor of clinical outcome in B-CLL, but its analysis is expensive and beyond the capacities of most diagnostic laboratories. To identify surrogate markers we performed a gene expression profiling analysis of CD19+ purified cells from 80 B-CLL untreated patients in Binet stage A, by means of Affymetrix GeneChip® HGU133A arrays. The comparison of 46 IgVH-unmutated versus 34 mutated samples using the Prediction Analysis of Microarrays software identified 78 differentially expressed probes, specific for 59 well-characterized genes. Specifically, 43 genes had a higher and 16 genes a lower average expression in the IgVH unmutated group. These genes are involved in cellular functions, including cell cycle regulation (SEPT7, SEPT10, CDK2AP1), cell proliferation (SLAMF1, LDOC1), apoptosis (CD63, IFT57, P2RX1, RNF130, TNFRSF1B), cell adhesion (CNTNAP2, C1orf38, PCDH9), immune response (ZAP70, IFI44), signal transduction (AKAP13, RASGRP1, USP6NL, TGFBR3, AKAP12), lipid metabolism and fatty-acid degradation (FADS3, LPL, LASS6), cell-cell signalling (FCRL2), phospholipid biosynthetic process (AYTL2), regulation of circadian rhythm (EGR3, CRY1, OPN3), DNA-dependent regulation of transcription (MYBL1, NR4A2, NRIP1, ZBTB20), muscle development (VAMP5, SRI, DMD). The expression signature identified in the proprietary database was then validated by means of a meta-analysis of a publicly available gene expression dataset of 100 B-CLL (Haslinger et al., 2005), showing classification accuracy measures leading to a global classification rate of 82.93% of the test set and thus suggesting the strength of the identified expression signature. The expression levels of 11 genes (LPL, ZBTB20, ZAP70, CRY1, COBLL1, SEPT10, LDOC1, TNFRSF1B, DMD, SRI, NRIP1) were confirmed by means of quantitative real-time PCR (Q-RT-PCR) in a subset of 40 CLL patients. The prognostic impact for Time To Treatment (TTT) of the 59 candidate genes of our classifier model was investigated in 77 patients. Forty-nine (36.4%) of these received treatment after a median follow up of 4 years. As expected, patients with unmutated IgVH genes had a risk of therapy requirement that was about 3 times higher (HR: 3.1,95% C.I. 1.6–5.8, p<0.0001) than those with mutated IgVH. Based on microarray expression levels, 43/59 genes significantly predicted TTT with a HR ranging from 1.5 (LPL gene) to 4.2 (SRI gene) (value for ZAP-70 = HR: 1.9, 95% C.I. 1.0–3.4, p=0.039). The same analysis performed in the panel of the 11 genes validated by Q-RT-PCR revealed 4 candidate genes which significantly predicted TTT. Specifically, Cox univariate analysis confirmed ZAP-70 as a predictor of disease outcome and underscored the prognostic role of the LPL, TNFRSF1B and CRY1 genes. The predictive power of the novel putative surrogate markers for the IgVH mutation status is now being further validated at protein expression level.

CD26 (dipeptidyl peptidase IV, DPP IV) is a multifunctional type II cell surface glycoprotein that is widely expressed on T and natural killer cells, as well as on epithelial, endothelial and acinar cells of different tissues; its expression on B cells is very low but it is greatly upregulated following activation. We evaluated, by means of flow cytometry, the expression of CD26 in various types of B-cell lymphoid tumors: Follicular Lymphoma (Fo-Ly, 12 cases), Mantle cell Lymphoma (MCL, 12 cases) Multiple Myeloma (MM, 20 cases), Hairy cell Leukemia (HCL, 12 cases), B-cell Chronic Lymphocytic Leukemia (B-CLL, 112 cases), CD5 negative B-cell Chronic Lymphoproliferative Diseases (CD5neg-B-CLPD, 20 cases) and Diffuse Large cell Lymphoma (DLCL, 12 cases). CD26 expression was absent or low of Fo-Ly and MCL, high on MM and HCL, variable on B-CLL, CD5neg-B-CLPD and DLCL. Fluorescence intensity of positive cells was dim in B-CLL and CD5neg-B-CLPD, heterogeneous in DLCL, and bright in HCL and MM. Interestingly, in CLL patients, CD26 expression was significantly correlated with CD49d and CD38 expressions (p<0.0001); moreover Spearman’s test revealed a significant correlation between CD26, CD38 or ZAP-70 expression and IgVH mutational status (p<0.0001). We also found a significant correlation between CD26 expression and the presence of FISH chromosomal abnormalities grouped on the basis of their prognostic relevance (p<0.0001). After a median follow-up of 36 months, 65/107 CLL pts were treated; taking 10% of positive cells as best cut-off, Kaplan-Meier curves showed that the time without therapy was significantly longer for CD26 negative cases compared with CD26 positive cases (P <0.0001). Moreover the concordant negativity of CD26 and CD38, or ZAP-70 and the combination of CD26 negativity with IgVH mutated status, identified subsets of CLL patients with a favourable outcome in terms of need of therapy.

Multiple myeloma (MM) is characterized by a high genomic instability that involves both ploidy and structural rearrangements. Nearly half of MM tumors are non-hyperdiploid and are frequently associated with 13q deletion and chromosomal translocations involving the immunoglobulin heavy chain (IGH) locus on chromosome 14q32. The remaining tumors are hyperdiploid, showing low prevalence of both IGH translocations and chromosome 13 deletions. Our study was aimed at providing insights into the genomic heterogeneity associated with plasma cell neoplasms by defining the genome-wide pattern of genetic lesions in a representative and stratified panel of MM patients. To this end, genome-wide profiling data of 45 plasma cell dyscrasia patients (41 MM and 4 plasma cell leukemia) were generated on GeneChip® Human Mapping 50K Xba SNP arrays, and the local DNA copy number variations were calculated using the DNAcopy Bioconductor package. The patients were clustered using the non-negative matrix factorization (NMF) algorithm to identify, within the natural grouping of profiles, the strongest clusters on the basis of their genomic characteristics. We identified three consistent clusters, characterized byrecurrent gains of odd-chromosomes, suggestive of the hyperdiploid status (Group A),high frequency of chromosome 13 deletion and 1q gains (Group B), orhigh frequency of chromosomes 13, 14, 16 and 22 deletions and losses of 1p and 4p regions, together with some cases showing 1q gains (Group C). To determine whether peculiar transcription fingerprints characterized these groups, gene expression profiles of 40 out of 45 corresponding samples generated on GeneChip® HG-U133A arrays were analyzed using the Prediction Analysis of Microarray (PAM) software. The multi-class analysis identified 229 transcripts (corresponding to 195 genes), which specifically marked the three groups. In particular, Group A was characterized by the overexpression of genes involved in the translational machinery or thought to be involved in MM pathogenesis such as the HGF, the tumor necrosis factor ligand TRAIL, DKK1, and c-KIT. Upregulation of the CKS1B gene was present in Group B and C, most likely reflecting the high frequencies of 1q gains in tumors within group B and C and its consequent deregulation. Group C was marked by the specific downregulation of genes mainly mapped to 1p arm: AMPD1, CSDE1, AKR1A1 and the PRKACB kinase, suggesting a relationship with the recurrent 1p loss within the group. Our data further supported the notion that structural abnormalities in multiple myeloma are associated with gene expression imbalances, and provide novel analytical approaches for the identification of genetic lesions and molecular patterns of the disease.

The clinical course of CLL is highly heterogeneous: some patients progress rapidly thus requiring early chemotherapy whereas others exhibit a stable disease over years. Gene expression studies have identified a relatively small number of genes that are differentially expressed between these subsets. Resistance to programmed cell death seems to be one of the preferential pathways of neoplastic B cells to survive and to develop resistance to therapy. We investigated by MicroFluidic Card™ technology patients affected by untreated CLL cells for alterations of agonist and antagonist apoptosis genes. Our aim was to shed light on programmed cell death pathway in this particular subset of patients.

Multiple myeloma (MM) is a fatal malignancy of clonal bone marrow plasma cells characterized by a high genomic instability increasing with disease progression. We describe here a genomic amplification at 17p11.2–p12, an unstable chromosomal region characterized by a large number of low copy repeats which have been proven to mediate deletion and duplication in several genomic disorders and amplifications in solid tumors. An approximately 5 Mb 17p11.2–p12 amplified region was detected in KMS-26 myeloma cell line by SNP microarray analysis. Further FISH mapping showed two unidentified amplified chromosomes as well as a complex pattern of rearranged chromosomes 17. The analysis of transcriptional profiles in a proprietary data base of myeloma cell lines, identified 12 significantly overexpressed genes in KMS–26 amplified region, including TNFRSF13B/TACI, COPS3, and NCOR1. The evaluation of their expression levels in a data base including 11 monoclonal gammopathy of uncertain significance, 121 MM and 9 plasma cell leukaemia, showed a significant overexpression of at least one gene in 13 patients. FISH analyses of these patients identified one MM carrying a 3.8 Mb amplified region and two MMs with gains specifically involving the TACI locus. Interestingly, the complete inactivation of TP53 at 17p13.1 was found in KMS–26 whereas a monoallelic loss was identifiable in two of the three patients carrying gain/amplification. Our data suggest that, as in the case of solid tumors, amplification/gain of the 17p11.2–p12 in MM could be mediated by the presence of repeats located in this region and may provide insights for defining novel candidate myeloma-associated genes.

In the present study, we describe the clinico-biological features of 63 cases of variant B-CLL (v-B-CLL) defined according to a previously described diagnostic algorithm for CD5+ mature B-cell leukemias, based on the combined use of Cytofluorimetric (CFM) analysis and t(11;14)(q13;q32) detection by means of fluorescence in situ hybridisation (FISH). These leukemias were characterized by SIgbright/CD23+ or CD23+/− , CD79b/CD20 bright, and negativity for t(11;14)(q13;q32). A historical series of 112 classical B-CLL was used as comparison. The mean age was 61 years (33–85) and M/F ratio 1.9. The v-B-CLL cases were significantly different from the B-CLL cases in terms of the following clinico-hematological variables: age <70 yrs, lymphocytosis <20 ×109/L (P <.0001), lymphocyte doubling time ≤ 12 months (P=.04), high serum ß2-microglobulin levels (P=.003) and presence of splenomegaly (P=.007). No differences were found in terms of sex, Hb levels, platelets count, presence of serum monoclonal component, serum LDH levels and HCV-Ab positivity. In v-B-CLL and B-CLL pts the median follow-up was respectively 56 and 84 months. The median overall survival was 80 months in v-B-CLL pts while it was not reached in B-CLL pts; 37 pts (58.7%) in the first group started chemotherapy vs 84 pts (56.4%) in the latter. The analysis of leukemic cell biological characteristics related to the prognosis was also made in part of the cases. A classic or mixed-CLL cytomorphology was seen only in 22/61 pts (36.1%). The percentage of CD38 positive cases was higher in v-B-CLL (60.3) than in B-CLL (44.6; P=.028); the IgVH mutation status, evaluated in 30/63 cases, was more frequently hypermutated in v-B-CLL group (23/30) than B-CLL (P=.03); the percentage of CFM Zap-70 positivity, evaluated in 42/63 cases, was similar in two groups (47.6 vs 57.4%, respectively). Interestingly, significant differences were found about the frequence of the recurrent chromosome alterations, evaluated in 48/63 cases, by means of FISH analysis: trisomy 12 was more frequent in v-B-CLL (37.5 vs 18.4%, p=0.02), while del13q14 was more frequent in B-CLL (14.5 vs 37.8%, P=0.007). In both groups, del 11q22.3 and del 17p13.1 were relatively rare (4 vs 8%, and 2 vs 7.7%, respectively). In conclusion, our study identifies, on the basis of a defined CFM-FISH diagnostic approach, a variant form of B-CLL that shows significant differences in terms of genetic and clinical features.

Karyotypic instability is strongly associated with multiple myeloma (MM). According to the chromosome number pattern, two major groups are recognized: hyperdiploid (H) tumors, associated with recurrent trisomies involving non-random chromosomes (3, 5, 7, 9, 11, 15, 19 and 21); and non hyperdiploid (NH) tumors associated with hypodiploid, pseudodiploid or near-tetraploid karyotypes. MM patients are approximately equally distributed between the two categories; notably, the most recurrent IGH translocations and chromosome 13 deletion appear to be prevalently associated with NH-MM, whereas recent evidences have suggested that H-MM correlates with a favorable prognosis. To molecularly characterize these two genetic categories, we performed a gene expression profiling analysis on 66 newly-diagnosed MM, characterized by FISH analyses for IGH translocations, 13q14 deletions and additional copies of chromosomes 1, 11 and 19. The ploidy status was investigated by combining two recently proposed FISH approaches (Wuilleme S. et al., 2004; Chng W.J. et al., 2005). The gene expression profiles of highly purified MM plasma cells have been generated by means of high-density oligonucleotide arrays (Affymetrix GeneChip U133A) and subsequently analyzed using unsupervised and supervised approaches (two-dimensional hierarchical clustering and SAM, respectively). The differential expression of 229 genes distinguished the 28 H-MM from the 38 NH-MM cases. The 208 upregulated genes in H-MM mapped mainly on the chromosomes involved in hyperdiploidy, while a significant percentage (29%) of the 21 genes upregulated in NH-MM were localized on 16q. The identified transcripts have been further validated on a publicly available gene expression dataset of an independent cohort of 64 MM patients (Carrasco et al., 2005). Notably, the global classification rate for the 64 cases resulted of 81%, confirming the validity of the identified transcriptional fingerprint. A functional analysis revealed a significant fraction of genes involved in protein biosynthesis (38%), transcriptional machinery and oxidative phosphorylation. Furthermore, an integrative genomic approach using a model-free statistical method (LAP, locally adaptive statistical procedure) supported these findings, allowing the identification in H-MM of globally upregulated regions on the chromosomes 3, 5, 9, 15 and 19, along with the downregulation of a region on 16q arm. Remarkably, two sub-groups are clearly distinguishable within H-MM group, one associated with chromosome 11 gain and the other showing 1q gain and chromosome 13 deletion. A supervised analysis of the H-11 vs H-13/1 patients identified 57 differentially expressed genes. Eleven of the 18 genes up-regulated in the H-11 group mapped to chromosome 11, whereas 21 of the 39 genes up-regulated in the H-13/1 group mapped to the 1q region. Notably, CCND2 resulted the most significantly upregulated gene in H-13/1 group. Our data reinforce the importance of combining cytogenetics and gene expression approaches for a better definition of the genetic alterations in MM and provide a molecular and genomic framework for dissection of disease pathogenesis and clinical management.