Roberta Cerutti

Microallelotyping defines the monoclonal or the polyclonal origin of mixed and collision endocrine-exocrine tumors of the gut.

Mixed endocrine-exocrine tumors of the gut are a heterogeneous group of neoplasms with uncertain histogenesis showing different morphologic and clinical features. The aim of this work is to clarify the histogenesis of these tumors by studying the genetic profile of both the endocrine and exocrine components. We performed an allelotyping analysis of five mixed endocrine-exocrine tumors (two gastric and three colonic) and one rectal collision tumor, using 35 polymorphic microsatellite markers covering a total of six chromosomes, including 3, 5q, 6, 11, 17, and 18. The loss of heterozygosity (LOH) analysis showed concurrent losses of the same allele in both the endocrine and exocrine components in all of the five mixed tumors composed by a poorly differentiated endocrine carcinoma or a well differentiated endocrine carcinoma associated with adenocarcinoma or adenoma. Among these tumors an identical LOH pattern was frequently found on chromosomes 17p, 18q, and 5q. Additional allelic losses restricted to the poorly differentiated endocrine carcinoma were often observed. On the contrary, in the only collision tumor composed by a well differentiated endocrine carcinoma associated with adenocarcinoma, completely different allelotypes between the two components were detected. These findings confirm a close genetic relationship between the two distinct histologic components within mixed endocrine-exocrine tumors, supporting the hypothesis that a monoclonal mechanism of tumorigenesis is the most frequent genetic event in mixed exocrine-endocrine tumors. The clonal divergence observed in the only collision tumor, composed by a well differentiated endocrine carcinoma associated with an adenocarcinoma, confirms the existence of double tumors growing next to each other coincidentally but showing different histogenesis and different tumorigenetic pathways.

The High Frequency of De novo Promoter Methylation in Synchronous Primary Endometrial and Ovarian Carcinomas

Purpose: The methylation status of hMLH1, CDKN2A, and MGMT was investigated in a panel of synchronous cancers of the ovary and endometrium, fulfilling the clinicopathologic criteria for independent primary tumors to define the possible role of epigenetic mechanisms in the development of these cancers. Experimental Design: Bisulfite-converted DNA from 31 tumors (13 endometrial and 18 ovarian carcinomas) and from matched normal tissue of 13 patients was analyzed by a methylation-specific PCR assay at the CpG-rich 5′ regions of all three genes. In all tumors, we also investigated the presence of microsatellite instability and hMLH1 immunohistochemical expression in relation to hMLH1 hypermethylation status. Results: Methylation of hMLH1, CDKN2A, and MGMT was detected in 39%, 41%, and 48% of endometrial and ovarian tumors, respectively. hMLH1 hypermethylation was observed in all tumors of five patients, and it was invariably associated with loss of hMLH1 protein and presence of microsatellite instability. CDKN2A and MGMT methylation was randomly detected among both endometrial (45% and 24% of cases, respectively) and ovarian carcinomas (39% and 39% of cases, respectively). Concordant methylation at two or three genes was observed in 35% of cases. Conclusions: Epigenetic inactivation of hMLH1, CDKN2A, and MGMT may be a common and early event in the development of synchronous primary endometrial and ovarian carcinomas and may qualify as a marker of a field cancerization encompassing the ovary and endometrium. Detection of MGMT hypermethylation may be useful to define a set of gynecologic malignancies with a specific sensitivity to alkylating chemotherapy.

Different Molecular Profiles Characterize Well-Differentiated Endocrine Tumors and Poorly Differentiated Endocrine Carcinomas of the Gastroenteropancreatic Tract

Purpose: The molecular pathogenesis of gastroenteropancreatic endocrine tumors (ETs) is still largely unknown. The purpose of this work was a molecular characterization of 38 gastroenteropancreatic ETs with respect to the primary site and to the morphofunctional profile, pointing out useful diagnostic or prognostic molecular markers. Experimental Design: Twenty-four well-differentiated ETs or carcinomas (WDET/Cs; 11 pancreatic, 3 gastric, and 10 intestinal) and 14 poorly differentiated endocrine carcinomas (1 pancreatic, 6 gastric, and 7 colorectal) were microallelotyped using 38 polymorphic microsatellite markers covering chromosomes 1, 3, 5q, 6, 11, 17, and 18. Results: Regardless of the primary site, a significantly higher percentage of allelic imbalances (AIs) was observed in poorly differentiated endocrine carcinomas than in WDET/Cs (P = 0.012), except for 3 of 8 nonfunctioning pancreatic endocrine tumors and 1 colorectal WDEC, exhibiting multiple AIs on chromosomes 1, 3, 6, and 11. A strong positive correlation between AI percentage and Ki-67 proliferation index was detected considering both the whole series of ETs (P = 0.004) and the group of WDET/Cs alone (P = 0.011). The survival analysis showed a positive correlation between low percentage of AI and longer survival (P = 0.01). No recurrent AIs at specific chromosomal regions were identifiable with respect to the primary site. Conclusions: The malignant progression of endocrine tumors seems to be associated with complex allelotypes and chromosomal instability. Although no specific molecular markers of malignancy can be defined with certainty, the ploidy status and the degree of chromosomal derangements appear to be the most informative genetic factors with prognostic significance.

Genetic progression in sporadic endometrial and gastrointestinal cancers with high microsatellite instability.

This study selected a series of 136 MSI‐H (microsatellite instability at high frequency) gastric, colorectal, and endometrial carcinomas combining immunohistochemical analysis for hMLH1 or hMSH2 gene products and microsatellite study. The clinico‐pathological profile of all tumours was correlated with the overall instability rates at coding and non‐coding repeats, in order to clarify the role and the mutation timing of seven target genes (TGFβRII, IGFIIR, BAX, hMSH3, hMSH6, CHK1, and BRCA2) in the progression of an MSI‐H neoplasm. Regardless of the primary site, the results confirm a model of oncogenesis in which inactivation of hMLH1, or less frequently hMSH2, may initiate a pathway culminating in a progressive accumulation of frameshifts in coding region (CDR) microsatellites. Comparing gastrointestinal and endometrial tumours, significantly lower levels of microsatellite instability at both coding and non‐coding repeats were observed. Among gastric and colorectal tumours, the detection of small shortening within Bat‐26 and Bat‐25 markers defines a subgroup of MSI‐H gastrointestinal tumours invariably characterized by early stage at diagnosis. In these tumours, mutations of TGFβRII or BAX genes precede frameshifts in the other tested genes. The analysis of correlations between the mutational and clinico‐pathological profiles of advanced gastrointestinal tumours revealed that the higher levels of microsatellite instability at both coding and non‐coding repeats were not associated with a more advanced clinico‐pathological stage or a less favourable outcome. A significant association was observed between a low number of CDR frameshifts and the presence of lymph‐node metastasis in advanced gastrointestinal tumours. The existence of advanced MSI‐H tumours with more aggressive behaviour and a ‘mild mutator phenotype’ could be explained by hypothesizing an overlapping of different mechanisms of tumourigenesis, including both the mutator and the suppressor pathways; this should be tested by further studies. Copyright

Intratumour T cells, their activation status and survival in gastric carcinomas characterised for microsatellite instability and Epstein–Barr virus infection

Gastric carcinomas (GCs) with high microsatellite instability (MSI) or an Epstein–Barr virus (EBV) infection are prevalently poorly differentiated adenocarcinomas with abundant lymphoid infiltration. The aims of the study were to clarify (1) if tumour-infiltrating lymphocytes (TILs) and cytotoxic-activated TILs are associated with a better clinical outcome in patients with GCs characterised for the presence of MSI and EBV; (2) if the nature and the activation status of TILs are involved in tumour cell apoptosis, evaluated using the M30 antibody, directed against a fragment of cytokeratin-18 caspase-cleaved during early steps of epithelial cell apoptosis. The immunophenotype of TILs and the tumour cell apoptosis were analysed with immunohistochemistry in 96 GCs, including 35 MSI GCs, and 61 GCs without MSI [microsatellite stable (MSS)], 17 of which were EBV+. MSI and MSS/EBV+ GCs displayed a significantly higher mean number of cytotoxic-activated TILs and apoptotic tumour cells than MSS/EBV− GCs (CD8+ TILs/HPF, 21.7 and 69.6 vs 6.4; T-cell intracellular antigen (TIA)-1+ TILs/HPF, 16.7 and 32.05 vs 5.2; granzyme B+ TILs/HPF, 7.5 and 8.6 vs 0.8; perforin+ TILs/HPF, 5.9 and 9.2 vs 0.9; and M30 IR tumour cells, 5.9 and 2.9 vs 2.3%). In addition to the most reliable clinico-pathological parameters (lymph node status, depth of tumour invasion and tumour stage), a univariate analysis showed that the presence of CD3+ TILs higher than 14.9 (p=0.01), CD8+ TILs higher than 9.5 (p<0.05) and MSI (p=0.02) were associated with better overall patient survival. Using a Cox regression model, only a high number of CD3+ TILs (p=0.02) and a low tumour stage (p=0.00001) were identified as independent prognostic factors. In conclusion, our study demonstrates that a high number of CD3+ and CD8+ TILs is a characteristic of MSI- and EBV-associated GCs and represents a favourable prognostic factor, independently of the pathogenesis of GCs.

Histidine Decarboxylase, DOPA Decarboxylase, and Vesicular Monoamine Transporter 2 Expression in Neuroendocrine Tumors: Immunohistochemical Study and Gene Expression Analysis:

Histidine decarboxylase (HDC) and vesicular monoamine transporter 2 (v-MAT2) are involved in the biosynthesis and storage of histamine. DOPA decarboxylase (DDC) is involved in the biosynthesis of a variety of amines and shares a high degree of homology with HDC. HDC and v-MAT2 immunoreactivities (IR) have recently been detected in well-differentiated neuroendocrine tumors (WDNETs) and poorly differentiated neuroen-docrine carcinomas (PDNECs) of various sites and have been proposed as general endocrine markers. We evaluated HDC and v-MAT2 IR in a series of 117 WDNETs and PDNECs from different sites. Western blotting analysis was performed to verify the specificity of anti-DDC and anti-HDC antibodies. Real-time RT-PCR was performed using specific probes for HDC and DDC on 42 cases, examined also for DDC IR. HDC and v-MAT2 IR were observed in the majority of WDNETs and PDNECs of all sites and HDC-IR cases were always also DDC-IR. In contrast, high levels of HDC mRNA were detected only in the gastroenteropancreatic WDNETs, which did not show increased DDC mRNA levels. On the other hand, bronchial carcinoids and lung PDNECs showed high DDC mRNA levels, but nearly undetectable HDC mRNA levels. Western blotting analysis showed a cross-reaction between anti-HDC and anti-DDC antibodies. HDC should not be considered as a general endocrine marker and HDC IR in bronchial carcinoids and PDNECs of the lung can probably be attributed to a cross-reaction with DDC.

Up-regulation of the hypoxia-inducible factor–1 transcriptional pathway in colorectal carcinomas

The aim of the study was to identify the impact on prognosis of hypoxia-inducible factor-1 genetic program in colorectal carcinomas and to develop an experimental procedure that would allow a reliable quantitative gene expression analysis in formalin-fixed and paraffin-embedded tissue. The expression of hypoxia-inducible factor-1alpha and 13 hypoxia-inducible factor-1 target genes (AMF, CAIX, VEGF, VEGFR1, VEGFR2, HGF, MET, TGFalpha, EGFR, IGF2, MMP2, PLAUR, NIX) was quantified by real-time polymerase chain reaction in 18 colorectal, poorly differentiated neuroendocrine carcinomas and in 60 invasive colorectal carcinomas. Moreover, hypoxia-inducible factor-1alpha protein expression was evaluated by immunohistochemistry. High levels of hypoxia-inducible factor-1alpha were positively associated with poorly differentiated neuroendocrine carcinoma histology (P < .005), poor differentiation (P < .025), presence of necrosis, and presence of microsatellite instability (P < .05). AMF, TGFalpha, IGF2, NIX, VEGF, and VEGFR2 transcripts were significantly higher in the very aggressive poorly differentiated neuroendocrine carcinomas than in exocrine colorectal carcinomas and TGFalpha expression was significantly associated with presence of lymph nodal metastases (P < .05). High levels of TGFalpha and NIX were significantly associated with decreased overall survival (P < .001; P < .01). The multivariate analysis showed that advanced stage, presence of lymph node metastases, and high TGFalpha expression had an independent effect on survival (P < .006; P < .01; P < .0006). Our study suggests an up-regulation of the hypoxia-inducible factor-1 transcriptional pathway in colorectal carcinomas. hypoxia-inducible factor-1alpha overexpression alone, has no impact on the prognosis of colorectal carcinomas likely because the consequences of hypoxia-inducible factor-1alpha expression/stabilization strongly depend on the genetic background of the transformed cells. Mechanisms leading to increased synthesis of hypoxia-inducible factor-1alpha mRNA via autocrine growth factor loops may play a crucial role in invasive growth in this site.

Allelotypes and Fluorescence In situ Hybridization Profiles of Poorly Differentiated Endocrine Carcinomas of Different Sites

Purpose: The aim of this work was to investigate the genotypic profiles of 36 poorly differentiated endocrine carcinoma (PDEC) of different sites to verify if their very similar phenotype may reflect similar pattern of genetic anomalies and if useful diagnostic or prognostic markers may be pointed out. Experimental Design: All tumors were microallelotyped at 57 microsatellite on 11 autosomes and the allelotypes of a selected panel of tumors were validated by interphasic fluorescence in situ hybridization with centromeric probes for chromosomes 1, 3, 6, 11, 17, and 18 and a probe specific for p53. Results: Regardless of the primary sites, PDECs exhibit very complex allelotypes (86%) and TP53 allelic imbalance (89%). Among these cases, fluorescence in situ hybridization analysis confirmed the presence of multiple aneusomies and a chromosome instability phenotype. Very low percentage of allelic imbalance (AI) and few aneuploidies were detected in only five PDECs for which an overall longer survival was observed. We found recurrent AI on 3p, 5, and 11q13 in lung PDECs, on 5q21, 8p, and 18q21 in colorectal PDECs and on 7 and 11q22 in gastric PDECs. Significantly better outcome was observed in patients with PDEC exhibiting 8q AIs and absence of AI at chromosome regions 6q25 and 6p. Conclusions: The concurrence of p53 inactivation and aneuploidies or chromosome instability are the main features of PDECs. However, the specific allelotypes observed in relation to primary site support the hypothesis that PDECs and exocrine carcinomas of all sites may share early pathogenetic mechanisms. Molecular markers of potential diagnostic and prognostic values for PDECs of different sites have been identified.

Hierarchical clustering analysis of pathologic and molecular data identifies prognostically and biologically distinct groups of colorectal carcinomas

This work has evaluated the potential superiority of a morphomolecular classification based on the combination of clinicopathologic and molecular features of colorectal cancers. A cohort of 126 colorectal carcinomas was investigated by unsupervised hierarchical clustering analysis to combine 13 routinely assessed clinicopathologic features and all five molecular markers recently suggested by Jass’ classification to distinguish four molecular subtypes of sporadic colorectal carcinomas. Survival analysis was assessed by a Cox proportional hazards model. A clear separation into three prognostically significant groups was identified: cluster A and cluster C were associated with good prognosis and cluster B with poor prognosis (P=0.006). Clinicopathologic and molecular features of cluster A and cluster B tumors were strongly concordant with colorectal cancer profiles characterized by microsatellite instability or by chromosomal instability, respectively. The clinicopathologic features of cluster C tumors were suggestive of a less aggressive disease than cluster B tumors. Genetically, they appeared intermediate between cluster A and cluster B tumors, as they were mainly microsatellite stable tumors showing high levels of both MGMT methylation and loss of heterozygosity. Chromosomal instability was significantly lower in cluster C than in cluster B tumors. A more accurate tumor classification should combine the prognostic power of clinicopathologic parameters with molecular biomarkers that provide information regarding the natural history of the cancer. Hierarchical clustering seems to be a useful, promising and powerful tool for further translational studies and should lead us to define a diagnostic and prognostic signature for different carcinomas.

MGMT methylation in diffuse large B-cell lymphoma: validation of quantitative methylation-specific PCR and comparison with MGMT protein expression

Aims: (1) To validate a quantitative real time methylation specific PCR assay (MethyLight) for the detection of O6-methylguanine-DNA methyltransferase (MGMT) gene methylation status (MS) in diffuse large B-cell lymphoma (DLBCL). (2) To determine the immunohistochemical (IHC) expression of the MGMT protein and correlate it with MS. Both IHC and MethyLight results were compared with patient’s outcome. Methods: 71 patients with primary nodal DLBCL were studied. MGMT immunoreactivity was detected using a specific monoclonal antibody. The MS of MGMT gene was analysed in 52/71 DLBCL using MethyLight. A selected subset of 40 DLBCL was also analysed using qualitative methylation-specific PCR (MSP). Statistical analysis of overall survival (OS), lymphoma-specific survival (LSS) and progression free survival (PFS) was performed according to IHC and MS results. Results: 19/71 DLBCLs (27%) were MGMT-negative at IHC; all were analysed, together with 33/52 MGMT-positive DLBCLs. MethyLight showed a better performance than MSP. There was a good correlation between the presence of MGMT expression and the unmethylated status; the absence of IHC expression was poorly correlated with the presence of methylation. Better OS, LSS and PFS was found in DLBCLs with MGMT gene methylation. DLBCLs not expressing MGMT at IHC showed a longer PFS. Conclusions: The quantitative real-time methylation-specific PCR assay for the detection of MGMT gene hypermethylation has been validated for the first time in DLBCL. Immunohistochemistry seems to represent an useful preliminary test to identify unmethylated cases; MS analysis may be performed in non-immunoreactive cases to identify truly methylated DLBCLs, which bear a better prognosis.