Claudia Parolini

Phospho-proteomic analysis of mantle cell lymphoma cells suggests a pro-survival role of B-cell receptor signaling.

BackgroundMantle cell lymphoma (MCL) is currently an incurable entity, and new therapeutic approaches are needed. We have applied a high-throughput phospho-proteomic technique to MCL cell lines to identify activated pathways and we have then validated our data in both cell lines and tumor tissues.MethodsPhosphoScan analysis was performed on MCL cell lines. Results were validated by flow cytometry and western blotting. Functional validation was performed by blocking the most active pathway in MCL cell lines.ResultsPhosphoScan identified more than 300 tyrosine-phosporylated proteins, among which many protein kinases. The most abundant peptides belonged to proteins connected with B-cell receptor (BCR) signaling. Active BCR signaling was demonstrated by flow cytometry in MCL cells and by western blotting in MCL tumor tissues. Blocking BCR signaling by Syk inhibitor piceatannol induced dose/time-dependent apoptosis in MCL cell lines, as well as several modifications in the phosphorylation status of BCR pathway members and a collapse of cyclin D1 protein levels.ConclusionOur data support a pro-survival role of BCR signaling in MCL and suggest that this pathway might be a candidate for therapy. Our findings also suggest that Syk activation patterns might be different in MCL compared to other lymphoma subtypes.

The impact of P53 and P21(waf1) expression on the survival of patients with the germinal center phenotype of diffuse large B-cell lymphoma

Purpose: To prospectively investigate the prognostic significance of p21 and p53 expression in diffuse large B-cell lymphoma in the context of the U.S. Intergroup trial comparing conventional cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy to rituximab-CHOP (R-CHOP) induction, with or without maintenance rituximab. Experimental Design: Immunohistochemical staining of 197 paraffin-embedded biopsy specimens was scored by an independent panel of experts. Results: The cyclin-dependent kinase inhibitor, p21, was expressed in 55% of cases examined. In a multivariable analysis adjusting for International Prognostic Index score and BCL2 status, p21 expression was a significant, independent, favorable predictive factor for failure-free survival (relative risk, 0.3; P = 0.001) and overall survival (relative risk, 0.3; P = 0.003) for patients treated with R-CHOP. Expression of p21 was not predictive of outcome for CHOP-treated patients. Only p21-positive cases benefited from the addition of rituximab to CHOP. Among p21-positive patients, treatment with R-CHOP was associated with a higher failure-free survival rate at 5 years compared with CHOP (61% versus 24%; P = 0.01). In contrast, no significant differences were detected in failure-free survival according to treatment arm for p21-negative patients. Expression of p53, alone or in combination with p21, did not predict for outcome in univariable or multivariable analyses. Conclusions: In this study, p21 protein expression emerged as an important independent predictor of a favorable clinical outcome when rituximab was added to CHOP therapy. These data suggest that rituximab-related effects on lymphoma survival pathways may be functionally linked to p21 activity. Clin Cancer Res; 16(8); 2435–42. ©2010 AACR.Immunohistochemically detected over-expression of P53-related protein (P53+++) and absence of P21(waf1) expression (P21-) correspond to loss of function of the P53-gene in diffuse large B-cell lymphoma (DLBCL) patients. Using immunohistochemistry we examined 80 patients with DLBCL and found that 23% had the P53+++/P21- phenotype while 51% had a germinal center (GC) pattern. Both the P53+++/P21- phenotype and the non-GC pattern were associated with inferior outcome. Notably, the prognostic power of the P53+++/P21- phenotype was restricted to patients with a GC pattern, without effect on outcome of patients with a non-GC phenotype. Our results show that immunohistochemistry can parallel gene expression profiling in addressing clinical variability of DLBCL patients.

True 3q Chromosomal Amplification in Squamous Cell Lung Carcinoma by FISH and aCGH Molecular Analysis: Impact on Targeted Drugs

Squamous lung carcinoma lacks specific “ad hoc” therapies. Amplification of chromosome 3q is the most common genomic aberration and this region harbours genes having role as novel targets for therapeutics. There is no standard definition on how to score and report 3q amplification. False versus true 3q chromosomal amplification in squamous cell lung carcinoma may have tremendous impact on trials involving drugs which target DNA zones mapping on 3q. Forty squamous lung carcinomas were analyzed by FISH to assess chromosome 3q amplification. aCGH was performed as gold-standard to avoid false positive amplifications. Three clustered patterns of fluorescent signals were observed. Eight cases out of 40 (20%) showed ≥8 3q signals. Twenty out of 40 (50%) showed from 3 to 7 signals. The remaining showed two fluorescent signals (30%). When corrected by whole chromosome 3 signals, only cases with ≥8 signals maintained a LSI 3q/CEP3 ratio >2. Only the cases showing 3q amplification by aCGH (+3q25.3−3q27.3) showed ≥8 fluorescent signals at FISH evidencing a 3q/3 ratio >2. The remaining cases showed flat genomic portrait at aCGH on chromosome 3. We concluded that: 1) absolute copy number of 3q chromosomal region may harbour false positive interpretation of 3q amplification in squamous cell carcinoma; 2) a case results truly “amplified for chromosome 3q” when showing ≥8 fluorescent 3q signals; 3) trials involving drugs targeting loci on chromosome 3q in squamous lung carcinoma therapy have to consider false versus true 3q chromosomal amplification.

Diagnostic usefulness of fluorescent cytogenetics in differentiating chromophobe renal cell carcinoma from renal oncocytoma: a validation study combining metaphase and interphase analyses.

We investigated the usefulness of interphase fluorescence in situ hybridization (FISH) analysis to differentiate between 11 chromophobe renal carcinomas and 12 renal oncocytomas, showing different clinical outcomes, when compared with conventional metaphase cytogenetics by karyotyping. Karyotypically, 3 chromophobe renal cell carcinomas showed losses of chromosomes, 3 were polyploid, 1 was normal, and 4 failed to grow. Of 12 oncocytomas, 5 showed a normal numeric karyotype and 6 additional structural rearrangements. FISH on chromophobe renal cell carcinomas showed a high percentage of cases (10/11 [91%]) with multiple numeric losses among chromosomes 1, 2, 6, 10, and 17; this interphase pattern was observed irrespective of the 3 different metaphase karyotypes. Of 12 oncocytomas, 11 (92%) revealed a normal numeric chromosomal status showing at least 2 chromosomes without aneusomy by interphase FISH. The study demonstrates that indeed FISH performed on formalin-fixed, paraffin-embedded tissue can provide clinically useful information more reliably than karyotyping of most of these tumors.

Induction of apoptosis in Jeko-1 mantle cell lymphoma cell line by resveratrol: a proteomic analysis.

Therapies for mantle cell lymphoma (MCL) are clinically unsatisfactory, and the search for effective drugs in vitro might foster the evaluation of their activity in vivo. We have investigated the effects of the polyphenolic compound resveratrol on the MCL cell line Jeko-1 using a combination of flow cytometry, Western blotting and two-dimensional electrophoresis to identify the molecules involved in the induction of apoptosis and cell growth regulation. We show that resveratrol induces apoptosis in Jeko-1 cells and modulates several key molecules, including cyclin D1 (CCND1), p53 (TP53), p21 (CDKN1A), BCL2, BAX, Bcl XL (BCL2L1), caspase 9 (CASP9) and p27 (CDKN1B). By high-resolution 2D-PAGE and nano-reverse phase-high performance liquid chromatography coupled with tandem mass spectrometry, we identified 32 differentially expressed proteins in response to resveratrol treatment that belong to important cell death related networks (including c-myc, NF-kappaB and the mitochondrial apoptotic pathway). These findings may improve the understanding of mechanisms mediating the pro-apoptotic effects of resveratrol on MCL cells, and form the basis for its potential use as a therapeutic agent.

CD71 in Gestational Pathology: A Versatile Immunohistochemical Marker With New Possible Applications.

Transferrin receptor/CD71 is a membrane protein expressed on nucleated red blood cells (NRBCs) and trophoblasts. Here, we propose the first study to evaluate the usefulness of CD71 immunolabeling in the main fields of gestational pathology. To this aim, formalin-fixed, paraffin-embedded samples of 45 orthotopic (23 spontaneous abortive and 22 molar pregnancies) and 11 ectopic pregnancies were immunostained for CD71. NRBCs were morphologically evident in 23 cases: 12/23 abortive, 4/11 ectopic, and 7/10 partial molar pregnancies. CD71 immunolabeling detected NRBCs in all 23 previous cases and in 8 new cases: 2 partial moles and 6 spontaneous abortive pregnancies. No NRBCs were detected in complete moles by means of either morphology or immunohistochemistry (IHC). In 4 cases with extensive necrotic changes, CD71 marked NRBCs and a few ghost villi, which were not certainly identifiable with standard histological evaluation. Furthermore, there was an inversely proportional relationship between total percentage of CD71-positive NRBCs and gestational age (R2=0.69; P<0.0001). We conclude that CD71 is a robust IHC marker for the detection of NRBCs and chorionic villi, especially in the presence of necrosis. The demonstration of NRBCs can be important in molar pathology, helping to exclude a complete mole. The application of CD71 could improve the diagnosis of this pathology in selected cases in which diffuse necrotic or hemorrhagic aspects may hinder the interpretation of the conventional approach with histology, IHC for p57, and ploidy analysis. Finally, the inverse correlation between the total percentage of CD71-positive NRBCs and gestational age suggests that this analysis may help in pregnancy dating.

Specific expression patterns of epithelial to mesenchymal transition factors in gestational molar disease.

INTRODUCTION The epithelial to mesenchymal transition, a well-known and re-emerging model in pathology, has not been completely investigated in the field of gestational pathology. This study aims at improving the comprehension of this process in molar disease, even looking for new possible immunohistochemical markers. MATERIALS AND METHODS We have analysed the immunohistochemical expression of Twist1 and Snai2, two of the most important transcription factors involved in epithelial to mesenchymal transition, in formalin-fixed paraffin-embedded samples of 23 spontaneous abortive pregnancies, 22 molar pregnancies (10 partial and 12 complete) and 7 term placentas. RESULTS Twist1 and Snai2 were highly expressed in stromal villi cells of molar disease. Particularly, Twist1 was highly expressed in complete moles compared to both abortive pregnancies (p < 0.001) and partial moles (p < 0.05). Also Snai2 was more expressed by complete moles, differentiating them from non-molar abortions (p < 0.05). DISCUSSION On the basis of the known cadherins and claudins expression in these pathologies, our new findings reinforce the hypothesis of the involvement of epithelial to mesenchymal transition in early molar pregnancies and above all in complete moles. Furthermore, we highlighted that in molar disease not only the trophoblast, but even the villi stromal cells, are involved. Thanks to their specificity, furthermore, these Twist1 and Snai2 could be used as additional immunohistochemical tool in the diagnosis of complete molar disease, with Twist1 as the first choice.

Cellular Senescence Markers p16INK4a and p21CIP1/WAF Are Predictors of Hodgkin Lymphoma Outcome

Purpose: There is evidence that Hodgkin Reed-Sternberg (HRS) cells in classical Hodgkin lymphoma (cHL) could display some molecular and morphologic markers of cellular senescence (CS). We hypothesized that CS mechanisms may have potential prognostic relevance in cHL and investigated whether the expression of the well-established CS biomarkers p21CIP1/WAF1 and p16INK4a by HRS cells might be predictive of the probability of event-free survival (EFS). Experimental Design: The study analyzed a retrospective cohort of 147 patients and the results were validated on a cohort of 91 patients independently diagnosed and treated in a different institution. p16INK4a and p21CIP1/WAF1 were categorized as dichotomous variables (< or ≥ 30% of HRS cells at diagnosis) and evaluated in univariate and multivariate analysis. Results: Both molecules were independent prognostic factors. A positive staining of one of the two molecules in more than 30% HRS cells predicted a better EFS (P < 0.01). p16INK4a/p21CIP1/WAF1 together as a unique categorical variable (both <30%, either <30%, both ≥ 30%) sorted out three prognostic groups with better, intermediate, or worse outcome either overall or within I–II, bulky and advanced stages. The presence or the lack of the robust expression of p21CIP1/WAF1 and/or p16INK4a defined the prognosis in our series. Conclusions: These findings point to (i) the relevance of CS-related mechanisms in cHL, and to (ii) the prognostic value of a simple, reproducible, and low-cost immunohistochemical evaluation of p16INK4a and p21CIP1/WAF1 expression. Clin Cancer Res; 21(22); 5164–72. ©2015 AACR.

CD200 expression is a feature of solid pseudopapillary neoplasms of the pancreas

CD200 has been recently indicated as a robust marker of well-differentiated neuroendocrine neoplasms. Here, we evaluate its role in differential diagnosis of solid pancreatic neoplasms. We immunostained for CD200 22 solid pseudopapillary neoplasms (SPNs), 8 acinar carcinomas (ACs), 2 pancreatoblastomas (PBs), 138 neuroendocrine tumors (PanNETs), and 48 ductal adenocarcinomas. All SPNs showed strong cytoplasmic and membranous staining for CD200, while only one case of AC had focal positivity. The two PBs showed focal CD200 positivity, mainly located in squamoid nests. The vast majority of PanNETs (96%) showed strong cytoplasmic and membranous staining for CD200, whereas all PDACs were negative. As both PanNETs and SPNs express CD200, it has no role in the differential diagnosis between these two entities.

PD-1, PD-L1, and CD163 in pancreatic undifferentiated carcinoma with osteoclast-like giant cells: expression patterns and clinical implications

Undifferentiated carcinoma with osteoclast-like giant cells (UCOGC), a variant of pancreatic ductal adenocarcinoma (PDAC), has a striking genetic similarity to PDAC but a significantly improved overall survival. We hypothesize that this difference could be due to the immune response to the tumor, and as such, we investigated the expression of PD-1, PD-L1, and CD163 in a series of UCOGC. To this aim, 27 pancreatic UCOGCs (11 pure and 16 PDAC-associated), 5 extrapancreatic tumors with osteoclast-like giant cells and 10 pancreatic anaplastic carcinomas were immunostained using antibodies against PD-1, PD-L1, and CD163. In pancreatic UCOGCs, PD-L1 was expressed in neoplastic cells of 17 (63%) of 27 cases, more often in cases with an associated PDAC (P = .04). Expression of PD-L1 was associated with poor prognosis, confirmed by multivariate analysis: patients with PD-L1-positive UCOGCs had a risk of all-cause mortality that was 3 times higher than did patients with PD-L1-negative UCOGCs (hazard ratio, 3.397; 95% confidence interval, 1.023-18.375; P = .034). PD-L1 expression on tumor cells was also associated with aberrant P53 expression (P = .035). PD-1 was expressed on rare lymphocytes in 12 UCOGCs (44.4%), mainly located at the tumor periphery. CD163 was expressed on histiocytes, with a diffuse and strong staining pattern in all UCOGCs. Extrapancreatic tumors with osteoclast-like giant cells showed very similar staining patterns for the same proteins. Anaplastic carcinomas have some similarities to UCOGCs, but PD-L1 has no prognostic roles. Our results may have important implications for immunotherapeutic strategies in UCOGCs; these tumors may also represent a model for future therapeutic approaches against PDAC.