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Germ line mutations in the Adenomatous polyposis colitumor suppressor gene cause a hereditary form of intestinal tumorigenesis in both mice and man. Here we show that in ApcMin/+mice, which carry a heterozygous germ line mutation at codon 850 of Apc, there is progressive loss of immature and mature thymocytes from approximately 80 days of age with complete regression of the thymus by 120 days. In addition, ApcMin/+mice show parallel depletion of splenic natural killer (NK) cells, immature B cells, and B progenitor cells in bone marrow due to complete loss of interleukin 7 (IL-7)-dependent B-cell progenitors. Using bone marrow transplantation experiments into wild-type recipients, we have shown that the capacity of transplanted ApcMin/+bone marrow cells for T- and B-cell development appears normal. In contrast, although the ApcMin/+bone marrow microenvironment supported short-term reconstitution with wild-type bone marrow, ApcMin/+animals that received transplants subsequently underwent lymphodepletion. Fibroblast colony-forming unit (CFU-F) colony assays revealed a significant reduction in colony-forming mesenchymal progenitor cells in the bone marrow of ApcMin/+mice compared with wild-type animals prior to the onset of lymphodepletion. This suggests that an altered bone marrow microenvironment may account for the selective lymphocyte depletion observed in this model of familial adenomatous polyposis. (Blood. 2004;103:1050-1058)

Diffuse large B-cell lymphoma (DLBCL) is the most frequently aggressive Non-Hodgkin's lymphoma (NHL). DLBCL first line therapy consists of R-CHOP regimen, which combines classical chemotherapeutical drugs and corticosteroids with Rituximab. Rituximab is a chimeric anti-CD20 monoclonal antibody which targets the CD20 surface antigen expressed on both normal and malignant B cells. Rituximab triggers antibody- and complement-dependent cytotoxicity, growth inhibition and apoptosis. Rituximab inhibits the expression of the anti-apoptotic proteins Bcl-2/Bcl-XL, down-modulating different survival pathways. Clusterin (CLU) is an ubiquitary protein involved in many physiological and pathological processes such as apoptosis and cancer. CLU has different transcriptional isoforms, which share exons from 2 to 9 but have a unique exon 1 and at least two protein forms: The cytoplasmatic precursor of about 60 kDa is secreted outside the cell after post-traslational modifications (sCLU), whereas a truncated protein (50–55 kDa) is produced only in particular conditions and localizes in the nucleus (nCLU). CLU expression is disregulated in almost all cancers and its role has been deeply investigated in solid tumors. Only limited information is currently available about its function in lymphomas.Toledo, a DLBCL-derived cell line and normal B lymphocytes were chosen as experimental models. B lymphocytes were purified and immuno-magnetically sorted from peripheral blood of 4 healthy volunteers. Toledo and B lymphocytes were treated with 10 or 100 μg/ml Rituximab or doxorubicin (from 0.05 to 1 μM) for 48 hours. Phenotype analysis was performed by flow cytometry; cell viability was assessed by Trypan blue exclusion; apoptosis was evaluated by Annexin V/propidium iodide. CLU expression and localization were evaluated by real time PCR, Western Blot (WB) and immunocytochemistry (ICC). Primers for the real time PCR were design in order to distinguish the unique exon 1 of each CLU transcriptional isoforms or exons common to all variants (CLU3-4).Rituximab treatment doesn't significantly inhibit Toledo proliferation and does not induce apoptosis despite the fact that these cells are CD20+. This suggests that Toledo are resistant to Rituximab. As expected, doxorubicin causes a marked growth inhibition by necrosis and apoptosis. Toledo cells have a very low basal level of the 60 kDa CLU protein form. Both Rituximab and doxorubicin up-regulate the 60 kDa form with 1 μM doxorubicin causing the most dramatic increase. The analysis of transcriptional isoforms by real-time PCR shows that CLU mRNA is almost undetectable in Toledo. CLU transcriptional variant 2 (CLU2) and regions common to all CLU transcripts (CLU3-4) are significantly up-regulated only after 1 μM doxorubicin but not after Rituximab. Fluorescence microscopy clearly shows a cytoplasmic localization of CLU under normal growth conditions. Interestingly, the treatment with both Rituximab and doxorubicin causes the appearance of a nuclear staining. This fact might be consistent with the onset of a process of cell death. 10 and 100 μg/ml Rituximab inhibits B lymphocytes cell survival in a dose-dependent manner (39.7% and 44% respectively after 48 hours). Interestingly, B lymphocytes clearly express high basal levels of the 60 kDa CLU and produce an additional form of about 55 kDa only after Rituximab but not after doxorubicin. Furthermore Rituximab-treated B lymphocytes display a Bcl-2 protein decrease while Toledo cells show high expression of Bcl-2 that doesn't seem to change in response to Rituximab.Toledo are resistant to Rituximab up to 100 μg/ml and express very low levels of CLU under normal growth conditions. 60 kDa CLU is partially up-regulated and Bcl-2 expression is not significantly affected by Rituximab treatment. Doxorubicin strongly up-regulates the 60 kDa CLU isoform. At the transcriptional level CLU2 shows the same expression profile of the 60 kDa protein form. On the contrary, Rituximab changes the CLU profile of responsive B lymphocytes in that leads to the appearance of a 55 kDa form that is not visible after doxorubicin treatment. Rituximab also causes a reduction in the expression of the antiapoptotic protein Bcl-2. The biological role of CLU during the response of peripheral B lymphocytes to Rituximab treatment is yet to be determined and is currently being investigated.No relevant conflicts of interest to declare.

Chronic lymphocytic leukemia (CLL) patients bearing 13q14 deletion are known to experience a more favorable clinical course. Recent studies, focusing on patients with loss of 13q as the sole cytogenetic aberration at diagnosis (del13q-only cases), showed that the number of malignant cells carrying this genetic lesion correlates with a more aggressive clinical behavior. However, whether the size of the 13q deletion may also influence the clinical outcome remains to be elucidated.Probes for chromosome 13q (LSI-RB1, LSI-D13S319), 11q (LSI-ATM), 17p (LSI-p53) and chromosome 12 (CEP12) were utilized on nuclei collected at diagnosis from: i) a multi-institutional CLL cohort (342 del13q-only cases) and ii) a consecutive unselected single-institution cohort of 265 cases. RB1 deleted cases (delRB1) were defined as having at least 5% of deleted nuclei. Time to treatment (TTT) intervals, as well as Rai staging, IGHV mutational status, CD38 and ZAP70 expression, B2-microglobulin levels, all evaluated at diagnosis, were also available for all cases that entered the study. Genome wide DNA profile was performed in a pilot series of 90 CLL samples using Affymetrix GeneChip Human SNP6 arrays.According to genome wide DNA analysis, delRB1 occurred in a proportion of del13q-only cases (36/90; 40%), always comprising the deleted region detected with the LSI-D13S319 probe (that covers the miR-15a/16-1 cluster and the DLEU2 gene) and characterized by a larger chromosome loss (median size 2.07 Mb vs. a median size of 0.86 Mb for the canonical del13S319). Maximally selected log-rank statistics identified the 70% of nuclei bearing del13S319 as the most appropriate cut-off value capable of separating del13q-only cases into two subgroups with different TTT distributions. Consistently, del13q-only cases with at least 70% of nuclei bearing del13S319 showed a significantly shorter TTT than del13q-only cases with less than 70% deleted nuclei (p=0.0001). Del13q-only cases were then divided in four subsets according to the percentage of nuclei bearing del13S319 with or without a concomitant delRB1: del13S319 <70% (group 1), 144 cases; del13S319 <70% + delRB1 (group 2), 95 cases; del13S319 >70% (group 3), 64 cases; del13S319 >70% + delRB1 (group 4), 39 cases. The median TTT of group 1 (not reached) was significantly longer than the median TTT of group 2 (92 months, p=0.012), group 3 (68 months, p<0.0001), and group 4 (82 months, p=0.0025; see Fig. 1A). Multivariate Cox proportional hazard analyses selected the presence of delRB1 (p=0.029), along with the IGHV mutational status (p<0.0001), as an independent negative prognosticator in the context of del13q-only cases with low/intermediate Rai risk (Rai stage of 0/I at diagnosis) and <70% of del13S319. Cases belonging to the consecutive unselected single-institution CLL cohort were divided into subsets according to the classification proposed by Döhner et al (NEJM, 2000). Notably, the presence of del13S319 in <70% of cells in the absence of delRB1 identified a patient subset with particularly stable and benign clinical course (group A in Fig. 1B, 48 cases; median TTT not reached). Conversely, patients characterized by del13S319 in <70% of cells but with a larger deletion, as determined by concomitant delRB1 (group B, 24 cases), or del13S319 in >70% of cells (with or without delRB1, group C, 25 cases) or a normal karyotype (group D, 75 cases) had shorter median TTT intervals (ranging from 105 to 129 months, p<0.01 in all the comparisons). Finally, patients affected by CLL bearing trisomy 12 (group E, 48 cases) and del11q or del17p (group F, 45 cases) experienced the worst clinical courses (p<0.0001).In the context of del13q-only cases, different clinical outcomes were associated to the percentage of 13q14 deleted cells, as well as to the size of the 13q14 deletion, as detected by the LSI-RB1 probe. Moreover, the presence of delRB1 emerged as a feature capable of refining the prognostic assessment in the context of CLL cases with <70% del13S319. The underlying genetic mechanisms correlated with the different clinical outcomes and associated with the size of the 13q deletion are presently under investigation.No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

The main objective in the intent to “cure” chronic myeloid leukemia (CML) is to obtain complete cytogenetic remission (CCR) and molecular remission. Tyrosin kinase inhibitors (TKI) treated patients (pts) achieve CCR, but BCR-ABL transcripts can still be detectable, and complete molecular remission (CMR), intended as undetectable transcript, are rare. Moreover about 10% of up-front treated patients show resistance to Imatinib, that reaches 30% in late chronic phase, loss of response during treatment is not negligible, and treatment cannot be stopped. Thus the eradication of residual disease still appears a difficult goal for a TKI alone. An alternative approach to target the residual disease is an active specific immunotherapy. We associated TKI therapy and immunogenic peptides derived from the p210 b3a2 and b2a2 fusion protein (developed by M. Bocchia et al. University of Siena) in pts with chronic phase (CP) CML with stable disease in trying to obtain a specific immunologic activation able to induce a measurable clinical response.17 pts (11 M:6 F) with CP CML, median age 55,5 (range 30-68) treated with Imatinib (16) or Dasatinib (1) were enrolled in the studies. All patients but one were in late chronic phase and had been treated with 2 (2 pts), 3 (11 pts) or >3 (4 pts) lines of therapies. Median time from diagnosis was 64.1(16-143) months, and patients were treated for a median of 30.8 months with TKI before peptide vaccination. 15 pts had b3a2 and 2 a b2a2 transcript. Pts presented with stable, measurable disease at cytogenetic or molecular level from at last 6 months. Vaccination included GM-CSF pre treatment and administration of 5 p210 b3a2 (CMLVAX100) or 1 b2a2 (CMLVAX25) derived peptides. Treatment plan consisted of an induction plan of 6 vaccinations every two weeks, followed by additional boosts every 3-6 months for responding patients. During vaccination, patients continued their conventional treatment with Imatinib/Dasatinib. Prior to vaccine all patients were tested with an intradermal injection of peptides (DTH) to evaluate their sensitivity to the CML antigens, and all of them resulted negative. Cytogenetics, FISH and molecular biology, peptide-specific immune responses (DTH, CD4+ proliferation, immunophenotype) were analyzed before and during treatment.15/17 pts are evaluable (2 patients had just completed the first 3 months and were not considered for their short follow up), and all patients but one showed a variable degree of response. All patients presented with some degree of DTH indicating the “recognition” of peptides by effector T cells (biologic response). 5/9 pts with positive cytogenetic (2-66% Ph+) reached CCR, and 3 also CMR, while 1 patient did not respond (the one with high tumor burden, 66% Ph+). 3/6 pts in CCR at time of vaccination reached CMR. The majority of responses were rapidly reached (after the induction) and were long lasting. After 69 month follow up 6/15 patients are still treated. Patients suspended vaccination due to: no response (1), lost CCR (5), progression (1), 2nd neoplasm (1), allergic reaction (1). One patient that reached CCR and MCR after vaccination stopped imatinib and was closely monitored thereafter. She is now treated with only vaccine boosts twice/year and still in CMR after 28 month. Specific immune response will be described.These data suggest that addition of b3a2 or b2a2-specific vaccine may have synergistc effect with TKI favouring reduction of residual disease and increasing the number of patients that reach CMR. A multicentric trial is ongoing through the GIMEMA CML study group, and a pilot study to stop TKI in long lasting CMR is in preparation.No relevant conflicts of interest to declare.

Figure Figure The immunoglobulin (Ig) beta protein (CD79b) dimerizes with Ig alpha (CD79a) to form the signaling component of the B-cell antigen receptor complex (BCR). Given the critical role of Ig beta in BCR signaling, it has been suggested that its variable expression in CLL may induce either cell proliferation or apoptosis. High CD79b expression has been associated with atypical morphology, strong expression of surface Ig, advanced clinical stage and short overall survival in B-CLL (Garcia Vela, 1999; Zucchetto, 2006). Moreover, high CD38 expression was correlated with high CD40, CD69 and CD79b which are consistent with an activation phenotype (Damle, 2002). Furthermore, we have demonstrated that the ZAP-70+ CLL subgroup shows a rapid disease progression and an inferior overall survival (Del Principe, 2006). Since it has been described that BCR engagement has significant effects on B-CLL cell survival, activation and cell cycle progression (Deglesne, 2006), we decided to evaluate the real impact of CD79b expression on B-CLL prognosis. The primary aims of our research were: 1) to determine progression-free survival (PFS) and overall survival (OS) upon CD79b expression; 2) whether CD79b could predict varied outcome within ZAP-70+ and ZAP-70 negative subgroups; and finally 3) whether CD79b was an independent prognostic factor. Therefore, we investigated 401 patients (pts), median age 65 years (range 33–89), 213 males and 188 females. With regard to modified Rai stages, 123 pts had a low stage, 261 an intermediate stage and 17 a high stage. CD79b was determined by multicolor flow cytometry, using the monoclonal antibody anti-CD79 beta-FITC (clone SN8, Dako) and fixing a cut-off value of 30%. CD79b+ B-CLL pts were 207/401 (52%). CD79b>30% was significantly associated with the intermediate/high Rai stage (p=0.00001), beta-2 microglobulin >2.2 mg/dl (p<0.0001) and with multiple intrathoracic/abdominal lymphadenopathies and/or splenomegaly (p<0.0001). Low CD79b was significantly correlated either with IgVH mutated status (>2%) (86/105; p<0.0001) or lower soluble CD23 levels (125/161; p<0.00001). Significant associations were found either between CD38<30% and lower CD79b (172/194; p<0.0001) or ZAP- 70<20% and CD79b<30% (145/193; p<0.0001). With regard to cytogenetics, there were strict correlations either between high CD79b and trisomy 12 (25/33; p=0.001) or high CD79b and del17p (16/20; p=0.001). Median follow up duration from diagnosis was 68 months (range 6–230). Concerning clinical outcome, both a shorter PFS (Figure) and OS were observed in CD79b+ pts (12% vs 58% at 16 years; p<0.0001 and 49% vs 85% at 16 years; p<0.0001) as well as in ZAP-70+ pts (5% vs 52% at 11 years; p<0.0001 and 33% vs 87% at 18 years; p<0.0001). To further explore the prognostic value of CD79b, we investigated its expression within ZAP-70+ (159 pts) and ZAP-70 negative (242 pts) subsets. As a matter of fact, CD79b+ pts showed a shorter PFS and OS both within the ZAP-70+ subset (7% vs 19% at 10 years; p=0.00006 and 25% vs 65% at 16 years; p=0.006) and within the ZAP-70 negative subset (29% vs 67% at 14 years, p<0.00001 and 86% vs 100% at 14 years; p=0.01). In multivariate analysis of PFS and OS, in which age, Rai stages, CD38, CD69, CD79b and ZAP-70 entered, ZAP-70 (p=0.001 and p=0.001), CD69 (p=0.001 and p=0.01) and CD79b (p=0.0007 and p=0.03) resulted to be independent prognostic factors. Therefore CD79b, easily determined by flow cytometry, should be considered another important prognostic parameter and could be used to early identify and stratify B-CLL progressive pts.

CD69 membrane protein is expressed early in the activation of lymphoid cells and is inducible by B-cells through cross-linking of surface immunoglobulins. Moreover, B-CLL cells exhibit features of activated and of antigen-experienced B lymphocytes overexpressing activation markers such as CD23, CD25, CD69 and CD71 (Damle, 2002). Furthermore, we have demonstrated that ZAP-70+ CLL subgroup shows a rapid disease progression and an inferior overall survival (Del Principe, 2006). The generation of antimurine CD69 monoclonal antibodies able to induce down-modulation or partial depletion of CD69+ cells (Sancho, 2006), prompted us to evaluate the real impact of CD69 expression on B-CLL prognosis. The primary aims of our study were: to determine progression-free survival (PFS) and overall survival (OS) upon CD69 expression; whether CD69 could predict varied outcome within ZAP-70+ and ZAP-70 negative subgroups; and finally whether CD69 was an independent prognostic factor. Therefore, we investigated 247 pts, median age 65 years, 131 males and 116 females. With regard to modified Rai stages, 69 pts had a low stage, 167 an intermediate stage and 11 a high stage. CD69 was determined by multicolor flow cytometry fixing a cut-off value of 30%. CD69+ B-CLL pts were 73/247 (30%). CD69 >30% was significantly associated with an intermediate/high Rai stage (p=0.001), lymphocyte doubling time (LDT) <12 months (p=0.00005) and beta-2 microglobulin >2.2 mg/dl (p=0.002). Lower CD69 expression and IgVH mutated status (>2%) were significantly correlated (73/92; p=0.0003). Furthermore, we found significant associations between lower CD69 and lower CD38 (137/184; p=0.02) or lower ZAP-70 (110/144; p=0.01). Lower levels of soluble CD23 (sCD23) were strongly associated with lower CD69 (127/158; p<0.00001). With regard to clinical outcome, both a significant shorter PFS (Figure) and OS were observed in CD69+ pts (5% vs 56% at 14 years; p<0.00001 and 44% vs 66% at 14 years; p=0.00001) as well as in ZAP-70+ pts (7% vs 62% at 12 years; p<0.00001 and 26% vs 90% at 14 years; p<0.00001). To further explore the prognostic impact of CD69, we investigated its expression within ZAP-70+ (103 pts) and ZAP-70 negative (144 pts) subsets. CD69+ pts showed a shorter PFS both within the ZAP-70+ subset (11% vs 27% at 8 years; p=0.004) and within the ZAP-70 negative subset (21% vs 79% at 12 years, p<0.00001). In multivariate analysis of PFS and OS, in which Rai modified stages, CD38, sCD23, LDT, CD69 and ZAP-70 entered, both ZAP-70 (p=0.0003 and p=0.0002) and CD69 (p=0.005 and p=0.0004) resulted to be independent prognostic factors. Therefore, CD69, determined by flow cytometry, could be considered as a new promising immunologic prognostic parameter in B-CLL. Furthermore, since the ZAP-70 negative subgroup consists of a heterogeneous population presenting variable outcome, CD69 might better stratify B-CLL subsets and early identify progressive pts in order to take timely therapeutic decisions. Figure Figure