ALBERT

Description: 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

Description: Advanced tumor-stage mycosis fungoides (MF) and Sézary syndrome (SS) are cutaneous T-cell lymphomas (CTCL) characterized by very poor prognosis. Allogeneic haematopoietic stem cell transplantation (allo-SCT) represents an experimental treatment which has been shown to be very effective in achieving long lasting complete remission, possibly leading to cure in selected patients. However, high transplant-related mortality (TRM) limit its feasibility in the vast majority of patients with CTCL. Reduced-intensity conditioning regimens have been demonstrated to decrease TRM, allowing gradual establishment of full donor chimerism and possible graft-versus-lymphoma effect. In this setting, evaluation of minimal residual disease (MRD) is particularly useful to guide post-transplant strategies such as donor lymphocyte infusion (DLI). Detection of TCRgamma-chain gene rearrangements, owing to the relatively limited complexity of its genetic elements, is routinely used to detect MRD in T-cell malignancies. Nonetheless, due to the extensive combinatorial repertoire and the large hypervariable regions, TCRbeta represents the best target for MRD monitoring, allowing more sensitive detection of patient-specific rearrangements. With this study, we aimed to identify patient-specific TCRbeta rearrangements to monitor MRD in patients enrolled in a clinical phase II trial of reduced intensity allo-SCT for advanced stage refractory MF/SS. Skin biopsy and peripheral blood samples at diagnosis and at different time points after transplant were obtained from 6 out of 9 evaluable patients, all having achieved clinical complete remission (still enduring in 5). The BIOMED-2 multiplex TCRbeta PCR heteroduplex assay (InVivoScribe Technologies, USA) was used for identification of monoclonal TCRbeta rearrangements, clonal PCR products were directly sequenced in both directions using the complete set of V or J primers, and V, D, J segments identified using the ImMunoGeneTics database (http://imgt.cines.fr). Then, clonespecific PCR assays were performed on samples collected from every single patient and specificity tested by parallel amplification of normal polyclonal DNA samples. In all patients a monoclonal TCRbeta rearrangement has been sequenced allowing to obtain clone-specific primers for PCR assays. In 2 patients we identified the presence of a MRD at the early controls after transplant (+2 and +3 months, respectively) when both were polyclonal by standard TCRgamma rearrangement; clone-specific PCR assays for TCRbeta became negative afterwards, in concomitance with the achievement of full donor chimerism. In 3 patients polyclonality of TCRbeta was observed in all post-transplant controls. One developed chronic cutaneous GvHD and skin biopsies verified the absence of clone-specific T-lymphocytes. In another patient, all post-transplant assays performed by the standard TCRgamma-rearrangement were persistently positive, suggesting the presence of a non disease-specific monoclonality. The last of the 6 patients relapsed 52 months after transplant. Retrospective clone-specific analysis of TCRbeta rearrangements in DNA from skin biopsies unveiled the presence of molecular relapse already 24 months before, whereas standard TCRgamma assays became positive only after clinical relapse. Altogether, we observed good stability of monoclonal TCRbeta rearrangements over time. Our results suggest that the analysis of TCRbeta is more sensitive and more specific than the analysis of standard TCRgamma for detection of MRD in CTCLs, allowing earlier identification of relapses and adopatiention of pre-empatientive treatment such as DLI. With this method, we also demonstrated disappearance of MRD in concomitance with the achievement of full donor chimerism after allo-SCT. Finally, disease clone-specific TCRbeta rearrangement detection might be helpful in distinguishing cutaneous manifestations of acute GvHD from early relapse of MF. TCRbeta analyses of samples from the remaining 3 evaluable patients are ongoing and will be included in the final presentation.

Description: Bone marrow biopsies (BMB) taken at the time of diagnosis of 108 patients with chronic lymphocytic leukemia (B-CLL) (median age: 62 years, range: 36–81; M/F ratio: 2/1) were collected between 1985 and 2004, and tested immunohistochemically for the expression of ZAP-70 (zeta-associated protein 70), “Anti-ZAP-70 (human), clone 2F3.2 (mouse monoclonal IgG2a)”. There were degrees of ZAP-70 expression: “negative (a)” (58 patients), “weak (b)” (20 patients) and “intense (c)” (30 patients)” in comparison with natural killer (NK) and T-cell ZAP-70 expression (BMB positive when 20% of B-CLL cells express ZAP-70). The cases were analysed separately by three different experts. In terms of BMB morphology, proliferative centres were 36% in (a) 35% in (b) and 47% in (c); a diffuse pattern: 8.6% in (a), 10% in (b) and 13% in (c); a nodular pattern: 66% in (a), 55% in (b) and 37% in (c): (a) vs (b+c) (p=0.024), (a+b) vs (c) (p=0.014). The different degrees of ZAP-70 expression correlated with two groups of patients according to the Rai and Binet criteria: “0-I-II, A” and “II-III-IV, B, C”. Cases (a) correspond more frequently to the first, and (b+c) to the second (p=0.013); moreover, cases (a+b) correspond more frequently to the first group, and (c) to the second (p=0.0026). A lymphocyte doubling time (LDT) of less than six months and less than one year was most frequent in (c) (p=0.00044) (p=0.0065). An LDT of less than one year was more frequent in (b+c) vs (a) (p=0,01). Patients receiving no therapy were more frequent in (a) or (a+b) (p=0.00011) (p=0.00045). Patients receiving no therapy or only one line of treatment were more frequent in (a) or (a+b) (p=3.2x10–5) (p=0.0073). Cytogenetic abnormalities were more frequent in (a) or (a+b) (p=0.017) (p=0.022), and complex caryotypes more frequent in (a+b), (p=0.016). The incidence of death was lower in (a+b) than (c) (8% vs 27%) (p=0.041). Mann-Whitney analysis showed a statistical difference between (a+b) and (c) for the following variables at diagnosis: PLT (p=0.02); WBC (p=0.05); Ly (p=0.055); LDH (p=0.015); B2m (p=0.0005); splenomegaly (p=0.03); and a statistical difference between (a) vs (b+c) for the following variables at diagnosis: PLT (p=0.044); LDH (p=0.006); B2m (p=0.003); percentage of bone marrow infiltrate (p=0.055); CD 38 flow-cytometry (p=0.0009). Kruskall-Wallis analysis of (a) vs (b) vs (c) showed a statistical difference for the following variables at diagnosis: LDH (p=0.01); B2m (p=0.0017); CD 38 flow-cytometry (p=0.0051). These data confirm the prognostic significance of ZAP-70 expression in patients with B-CLL, and its good correlation with clinical and cytogenetic parameters and the Rai-Binet classification.

Description: 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.

Description: Multiple myeloma (MM) is a malignant proliferation of bone marrow plasma cells characterized by a wide spectrum of genetic and epigenetic changes. Global hypomethylation of repetitive genomic sequences such as long interspersed nuclear elements-1 (LINE-1) and Alu repetitive elements (approximately 500.000 and 1.4 million in the human genome) has been associated with chromosomal instability. Additionally, satellite alfa DNA (SAT-alpha DNA) hypomethylation has been reported to be associated to karyotypic instability in human cancer, possibly playing a role in centromere function. So far, the LINE-1/Alu and centromeric SAT-alpha DNA methylation patterns have not been investigated in the context of the different clinical and molecular MM subtypes. Global DNA methylation changes were investigated in a panel of 53 newly diagnosed, untreated MMs, 7 plasma cell leukemias (PCL) and 11 healthy subjects as controls. DNA was extracted from purified plasma cells, treated with bisulfite and analyzed by bisulfite-PCR and Pyrosequencing. Methylation of LINE-1 and Alu elements was shown to correlate with total 5mC content and thus used to estimate global DNA methylation. MMs showed a decrease of Alu (21.1%) and LINE-1 (70.0%) methylation average levels compared with controls (25.2% and 79.5% respectively). Lower median methylation levels were also found in centromeric SAT-alpha DNA of MMs (77.95%) compared to controls (89.5%). The median methylation level of PCLs was lower than MMs (16.7% versus 21.1% for Alu; 45.5% versus 70.0% for LINE-1; and 33.3% versus 77.9% for SATalpha DNA). Notably, a statistically significant association between SAT-alpha DNA and LINE-1 methylation (Spearman’s rank correlation, ρ = 0.94; P 〈 0.001) was found in MM. The comparison between methylation pattern and different molecular MM subgroups by means of non parametric tests, revealed that LINE-1 and SAT-alpha DNA methylation was significantly lower in the nonhyperdiploid versus hyperdiploid (HD) tumors (P = 0.01 and 0.02 respectively). Alu and SAT-alpha were significantly lower in the MMs with t(4;14) (P = 0.02 and 0.004 respectively). Finally, in the context of translocation/cyclin D (TC) classification, a statistically significant differences inside the five different groups were found for SAT-alpha DNA methylation (P = 0.008, Kruskal-Wallis test). These findings may provide insights into the molecular mechanisms of MM pathogenesis and suggest that our approach may contribute toward a more exhaustive stratification of the disease.

Description: 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.

Description: Indolent Non-follicular non-Hodgkin Lymphoma (NFo-NHL) is a group of relatively frequent lymphoproliferative diseases, nevertheless extended clinical and prognostic studies are still lacking. In 2002 the Gruppo Italiano Studio Linfomi (GISL) initiated a LL02 prospective multicenter phase II trial, with the aim to evaluate the efficacy and safety of FC combination in the first-line therapy of NFo-NHL patients younger than 70 years. Between July 2002 and September 2006, 58 adult patients (35 males and 23 females, median age 64 yrs, range 40–75) affected by NFo-NHL in active disease phase, were consecutively enrolled in 12 GISL Hematological Centres. Patients were treated with a dose of 25 mg/mq Fludarabine plus 250 mg/mq Cyclophosphamide administred intravenously daily for 3 days; each cycle was repeated every 28 days for 6 courses. During the treatment patients received oral thrimethoprim-sulphametoxazole prophylaxis. After the intermediate evaluation, 48/58 patients (82.8%) had an objective response (ORR) with a 20.7% of complete remission (CR) plus 62.1% of partial remission (PR); at the final evaluation the ORR percentage was 84.5% with a 41.4% of CR (24 pts) and 43.1% of PR (25 pts); three patients were in progressive disease (5.2%) and one in stable disease (1.7%). The median overall survival (OS) was not reached with an 88% and 84% at 12 and 24 months; the progression free survival (PFS) was 89% and 77% and the event free survival (EFS) was 81% and 66% at 12 and 24 months respectively.About the toxicity profile, the major toxicity was hematological with a 18% cases of WHO grade III or IV anemia, 40% leucopenia, 33% neutropenia and 10% piastrinopenia. The 12% of patients had an infective episode wich a 7.7% of WHO grade III–IV.In conclusion the FC chemotherapy is a useful chance for advanced untreated non follicular low-grade NHL, with an optimal ORR, CR and PFS. The crucial point of FC remains OS, that not seems to be significantly improved in comparison with fludarabine alone or with standard therapy, even though the better quality of responses; Rituximab plus FC association is growing in literature as the probably key to find a real improvement also in this aspect.

Description: Abstract 4030 INTRODUCTION: An increased bone marrow angiogenesis might be involved in the pathophysiology of a number of haematological malignancies, including myelodysplasia (MDS). Many angiogenic factors have been investigated in MDS, and vascular endothelial growth factor (VEGF) and its significance has been evaluated the most. VEGF mediates its biological effects by binding to two transmembrane tyrosine kinase receptors, VEGFR-1 and VEGFR-2. The VEGF/receptor signaling system is involved in the regulation of two fundamental processes: the formation of blood vessels (angiogenesis) and of blood cells (hematopoiesis). VEGF regulates the two processes by different mechanisms: hematopoietic stem cell survival and repopulation mainly via an autocrin loop, and angiogenesis via a paracrine loop. PATIENTS: The study population included 79 patients (pts) (48 men and 31 women; median age, 70 years). The patients were given a diagnosis of 10 RA, 6 RARS, 17 RCMD, 7 5q-syndrome, 25 RAEB-1 e 14 RAEB-2 according to the WHO classification (2008). In agreement with the International Prognostic Scoring System (IPSS), 28 pts were graded as Low, 28 as Int-1, 17 as Int-2 and 6 as High. According to the WHO classification based prognostic scoring system (WPSS), 14 pts were graded as Very Low, 17 as Low, 11 as Int, 25 as High and 12 Very High. The leukemic evolution occurred in 23 pts. The median leukemia free survival (LFS) was 825 days. Exitus occurred in 49 pts. The median overall survival (OS) was 902 days. We included in the study 20 controls (NC). METHODS: The MicroVessel Density (MVD) was evaluated through CD34 immuno-histochemical reactivity. Each BMB was first totally scanned at low magnification (10×) in order to identify 5 “hot spot” zones and then examined at high magnification (40×). MVD was estimated by hot-spot “method” (MVD-HS) by counting all of the positive-endothelial cells. Lumens were required to consider a structure as microvessel. Sinusoid-like structures, with the exception of arterioles, were also included in the count. To avoid any bias related to BMB cellularity, we calculated a VEGF expression index (VEGFi), defined as the cellularity of the BMBs multiplied by the fraction of VEGF positive cells and expressed as a number between 0 and 1 [(% of BMB cellularity × %VEGF-positive cells)/10⋀4]. Two VEGFi expression classes, respectively Low and High (L-VEGFi and H-VEGFi), were determined considering as cut-off level the mean VEGF(i) expression value + 2 standard deviations. RESULTS: MVD-HS analysis showed increased levels in MDS in comparison to NCs (p=.009). MVD did not differ among IPSS and WPSS categories. MVD did not predicted leukemia free and overall survival (LFS and OS). VEGF expression in bone marrow cells was found as a weak cytoplasmic granular protein expression in proeritroblasts; normoblasts were not immunoreactive. In the myeloid lineage VEGF expression was more intense in immature cells. The macrophages and megakaryocytes showed a granular weak cytoplasmic positivity. The plasmacells showed an intense granular cytoplasmic positivity. VEGFi expression was evenly distributed among the different IPSS and WPSS categories and was higher in MDS in comparison to NCs (p=.006). L-VEGFi MDS pts presented a significant lower transfusional need in the first years from diagnosis (p=.02). We observed that L-VEGFi MDS pts had a longer LFS compared with H-VEGFi ones (p=.006). Moreover, L-VEGFi MDS pts had a significant better OS compared to H-VEGFi pts (p=.03). At multivariate analysis IPSS and WPSS, as expected, predicted OS (p