Epithelial-mesenchymal transition in undifferentiated carcinoma of the pancreas with and without osteoclast-like giant cells.

Undifferentiated carcinoma (UC) and undifferentiated carcinoma with osteoclast-like giant cells (UCOGC) are peculiar variants of pancreatic ductal adenocarcinoma (PDAC), characterized by hypercellularity and absence of glandular patterns. The inflammatory microenvironment is peculiar in UCOGC, since it is dominated by macrophages and osteoclast-like giant cells. However, from a molecular point of view, both UC and UCOGC are very similar to conventional PDAC, sharing alterations of the most common genetic drivers. Clinically, UC usually show a worse prognosis, whereas UCOGC may show a better prognosis if it is not associated with a PDAC component. To highlight potential biological differences between these entities, we investigated the role of the epithelial to mesenchymal transition (EMT) in UC and UCOGC. Specifically, we analyzed the immunohistochemical expression of three well-known EMT markers, namely Twist1, Snai2, and E-cadherin, in 16 cases of UCOGC and 10 cases of UC. We found that EMT is more frequently activated in UC (10/10 cases) than in UCOGC (8/16 cases; p = 0.05). Furthermore, in UCOGC, EMT was activated with a higher frequency in cases with an associated PDAC component. Snai2 was the most frequently and strongly expressed marker in both tumor types (10/10 UC, 8/16 UCOGC), and its expression was higher in UC than in UCOGC (mean immunohistochemical score: 4.8 in UC vs. 2.1 in UCOGC, p < 0.01). Our results shed new light on the biology of UC and UCOGC: EMT appeared as a more important process in UC, and Snai2 emerged as a central EMT effector in this setting.

CD117 Is a Specific Marker of Intraductal Papillary Mucinous Neoplasms (IPMN) of the Pancreas, Oncocytic Subtype.

The intraductal oncocytic papillary neoplasm (IOPN) of the pancreas has been recognized by WHO classification as a unique intraductal papillary mucinous neoplasm (IPMN) category. IOPN is composed of oxyphil cells, usually expressing MUC5AC, MUC6, and Hep Par-1, and harboring PRKACA/B fusion genes as their genetic hallmark. Although IOPNs are associated with an infiltrative adenocarcinoma in up to 30% of cases, the survival rate after surgical resection approaches 100%. This highlights the importance of the correct IOPN diagnosis, above all in cases with an associated invasive component. In this study, the immunohistochemical expression of CD117 was investigated in 111 IPMNs, including 17 oncocytic, 45 gastric, 20 pancreatico-biliary, and 29 intestinal IPMNs. We also tested the expression of MUC5AC, MUC6, and Hep Par-1 in the IOPN cohort. CD117 positivity was significantly more frequent in IOPNs compared to the other IPMN subtypes (p < 0.0001). Furthermore, within IOPN, a lower or absent CD117, MUC5AC, MUC6, and Hep Par-1 expression tended to be associated with the presence of an infiltrative component. Our findings shed light into the biology of these complex lesions, which are confirmed to be a distinctive IPMN subtype; notably, CD117 emerged as a potential, additional tool in the differential diagnosis of IPMNs.

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.

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 (R=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 p21(CIP1/WAF1) and p16(INK4a) 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. p16(INK4a) and p21(CIP1/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). p16(INK4a)/p21(CIP1/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 p21(CIP1/WAF1) and/or p16(INK4a) 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 p16(INK4a) and p21(CIP1/WAF1) expression.

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.

Refractory hypertension and rapidly progressive renal failure due to bilateral renal artery stenosis: case report.

INTRODUCTION: We report a case of refractory hypertension and acute renal failure with mild proteinuria due to an unreported bilateral Renal Artery Stenosis, who underwent renal biopsy in the suspicion of rapidly progressive glomerulonephritis. CASE PRESENTATION: A 51-year-old Caucasian male was admitted with refractory hypertension of recent onset and acute renal failure. Duplex Doppler Ultrasonography was performed and provided images highly suggestive for bilateral renal artery stenosis. The patient was referred to the department of interventional radiology, where bilateral selective renal angiography and percutaneous endovascular angioplasty and stenting were performed successfully. CONCLUSION: Duplex Doppler Ultrasonography is thus suggested in patients presenting with refractory hypertension and acute renal failure, especially if atherosclerotic disease and clinical clues of RAS are present. Renal revascularisation with bilateral angioplasty and stenting may play a key role in the treatment of bilateral Renal Artery Stenosis, especially in patients unable to maintain renal function as systemic blood pressure is lowered.

FISH scoring on paraffin sections versus single-cell suspension for chromophobe renal carcinoma and renal oncocytoma.

BACKGROUND: Sectioning of the nuclei on tissue sections may give an overestimate of monosomy, a feature diagnostic of chromophobe renal cell carcinoma versus renal oncocytoma. The aim of the study was to assess whether or not nuclear sectioning may distort the results obtained from interphase fluorescence in situ hybridization (FISH) comparing the data obtained from analysis of isolated nuclei derived from formalin-fixed, paraffin-embedded sections with histological sections from the adjacent sections from the same tumors. PATIENTS AND METHODS: Five chromophobe renal cell carcinomas and five renal oncocytomas were recruited. Sections of 5 µm and 30 µm were cut for FISH to investigate chromosomes 1, 2, 6 10 and 17. RESULTS: FISH of isolated nuclei from renal oncocytomas showed a mean increase of 3.0% for nuclei with two signals when compared to tissue sections. For chromosomes 2, 6, 10 and 17, isolated nuclei showed a mean increase of 4.9% of fluorescent signals over nuclei from tissue sections. FISH analysis of isolated nuclei from chromophobe renal cell carcinoma showed a similar counts. CONCLUSION: When a tumor section exhibits a borderline percentage of nuclei with single signals around the cut-off level on tissue sections, the test should be repeated on isolated nuclei to confirm chromosomal loss, diagnostic of chromophobe renal carcinoma.

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

BACKGROUND: Mantle 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. METHODS: PhosphoScan 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. RESULTS: PhosphoScan 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. CONCLUSION: Our 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.

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.

Renal resistive index and long-term outcome in chronic nephropathies.

PURPOSE: To assess the clinical validity of renal resistive index (RI) to determine prognosis and guide therapy over a long-term follow-up in patients with chronic nephropathies and to verify the commonly used threshold value of 0.70. MATERIALS AND METHODS: Of patients referred to the nephrology center since 1995, 177 were initially enrolled and 86 were followed up for RI and renal function annually for 2-11 years (mean, 5.93 years +/- 2.92 [standard deviation]). All patients gave informed consent for the institutional review board-approved study. Correlations were determined between initial RI and age, estimated glomerular filtration rate (eGFR), proteinuria, hematuria, blood pressure, and biopsy scores. The sample was categorized in four groups on the basis of whether initial values of RI and eGFR were normal, and progression to renal failure was compared. With grouping of the sample by using initial RI (< or =0.61, 0.62-0.69, and > or =0.70), Kaplan-Meier analysis was used to obtain survival curves. RESULTS: Initial RI correlated with final eGFR (R = -0.4, P < .001), systolic blood pressure (R = 0.39, P < .001), proteinuria (R = 0.28, P = .009), and age (R = 0.28, P = .007). In stepwise multiple regression analysis, RI emerged as the only independent risk factor for the progression to renal failure (P < .001). Among the four groups of patients with different initial RIs and eGFRs, the group with an initial RI of 0.70 or higher showed a worse outcome, independent of initial eGFR. In the Kaplan-Meier analysis by using initial RI, only the group with a value of 0.70 or higher showed a rapid decline of renal function (>50% decrease in eGFR in 6 years). CONCLUSION: An RI of 0.70 or higher is predictive of an unfavorable outcome in patients with chronic nephropathies.

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.

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

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.

Establishment of the MAVER-1 cell line, a model for leukemic and aggressive mantle cell lymphoma.

BACKGROUND AND OBJECTIVES: Mantle cell lymphoma (MCL) cell lines are difficult to generate; only nine lines have been described so far and few of them have been thoroughly characterized. DESIGN AND METHODS: We established MAVER-1, a new MCL cell line, obtained from a leukemic MCL harboring both a t(11;14) translocation and a MYC rearrangement, and used immunohistochemistry, flow cytometry, molecular biology and cytogenetic techniques in order to characterize the cell line precisely. RESULTS: By immunohistochemistry and flow cytometry MAVER-1 displayed a classical MCL phenotype (IgM+, l+, CD5+, CD10-, CD19+, CD20+, CD23-, CD79a+, cyclin D1+) and genetic analysis showed a typical V/D/J rearrangement with naïve mutational status. According to both classic cytogenetic analysis and spectral karyotyping, MAVER-1 harbored the t(11;14) translocation associated with a complex karyotype. Molecular analysis by polymerase chain reactions showed that the t(11;14) breakpoint is within the major translocation cluster. Other important abnormalities of MAVER-1 include TP53 gene inactivation by a combined mutation of exon 8 and chromosome 17p13 deletion, ATM deletion, 8q24 (MYC) rearrangement and 8p22 deletion. INTERPRETATION AND CONCLUSIONS: The new cell line will be useful for in vitro studies regarding MCL pathogenesis and drug sensitivity, as well as a diagnostic control material.

Two-dimensional molecular profiling of mantle cell lymphoma.

The present research establishes standard two-dimensional (2-D) maps for control, reactive lymph node and non-Hodgkin's lymphoma (mantle cell lymphoma, MCL). Medium sensitivity, mass spectrometry compatible colloidal Coomassie has revealed a total of ca. 750 spots in each of the maps. Comparison of 2-D maps by statistical packages, such as the PDQuest, established up- and downregulation of a total of ca. 145 spots, with positive variations of up to 10-folds and negative variations of up to 13-folds in both MCL biopsies' protein extracts. Qualitative and quantitative variations in the two lymphoma samples are consistent. More than 20 proteins have been so far identified by matrix assisted laser desorption/ionisation-time of flight (MALDI-TOF)-mass spectrometry, with an additional five spots, which gave very good spectra but could not be matched to any of the presently available databases. Some of the spots, such as the 78 kDa glucose-regulated protein precursor and the glutathione S-transferase P, appear to be in common with other tumors, such as lung adenocarcinoma. Others may simply reflect overall changes in cellular metabolism and growth rate that occur during malignancy and thus might turn out to be in common with any cell population receiving any kind of stress. Some (notably T-cell leukemia/lymphoma protein 1A, TCL1, found to be 10-fold overexpressed) appear to be specific of the non-Hodgkin's lymphoma here studied. Western blot and immunohistochemical analyses were applied to obtain further information about stathmin (Op18) and TCL1, respectively.