Daniela Furlan

CHK1 frameshift mutations in genetically unstable colorectal and endometrial cancers.

The protein encoded by the CHK1 gene plays an important role in the G2 checkpoint in mammalian cells. In its coding region it presents a sequence of nine consecutive adenines that are a potential site of mutations in tumors with microsatellite instability (MSI). We analyzed the presence of frameshift mutations in the CHK1 gene in human colon and endometrial cancer samples. In the same cancer samples genes known to be altered in these tumors (BAX, TGFBRII, and IGFIIR) were also analyzed. CHK1 frameshfit mutations were found in 1 out 10 colon cancers and 2 out of 7 endometrial cancers showing MSI. CHK1 alterations were associated with the presence of a high degree of MSI. No alterations were found in patients with tumors showing low frequency or lacking instability (microsatellite stable). The same was true for the other four genes analyzed. The insertion or deletion of one A in the poly A tract resulted in a truncated protein. Alterations of the CHK1 gene could represent an alternative way of cancer cells to escape from cell cycle control. Genes Chromosomes Cancer 26:176–180, 1999.

Immunohistochemical pattern of hMSH2/hMLH1 in familial and sporadic colorectal, gastric, endometrial and ovarian carcinomas with instability in microsatellite sequences

Abstract. Alterations of DNA mismatch repair (MMR) genes are involved in carcinogenesis of sporadic and inherited human cancers characterised by instability of DNA microsatellite sequences (MSI). MSI tumours are usually identified using molecular analysis. In the present investigation, hMLH1 and hMSH2 immunohistochemistry was tested in order to evaluate the utility of this method in predicting MMR deficiency. Colorectal (72), gastric (68), endometrial (44) and ovarian (17) carcinomas were independently evaluated for familial history, histological type of tumour, MSI status and immunohistochemical results. Loss of expression of either hMLH1 or hMSH2 was observed in 51 of 55 (92.8%) MSI tumours, while 145 of 146 microsatellite stable (MSS) tumours expressed both the hMLH1 and hMSH2 gene products. Independently of tumour site, an overall agreement between immunohistochemical and molecular results was observed in 15 hereditary non-polyposis colorectal cancer-related tumours. Among sporadic tumours, only 2 of 60 colorectal and 2 of 66 gastric carcinomas, displaying MSI, expressed both hMLH1 and hMSH2 gene products. All 39 endometrial and 16 ovarian tumours presented a concordant molecular and immunohistochemical profile. These data show that immunohistochemistry is an accurate and rapid method to predict the presence of defective DNA MMR genes and to identify both sporadic and familial MSI tumours.

Colorectal poorly differentiated neuroendocrine carcinomas and mixed adenoneuroendocrine carcinomas: insights into the diagnostic immunophenotype, assessment of methylation profile, and search for prognostic markers.

Colorectal poorly differentiated neuroendocrine carcinomas (NECs) and mixed adenoneuroendocrine carcinomas (MANECs) are well-recognized entities generally known to be associated with biological aggressiveness and poor patient survival. However, a few published papers have highlighted the existence of a subgroup of tumors with a better survival than expected; however, to date, there are no established parameters that usefully identify this category. In the present study we have investigated the morphologic features, the CpG methylator phenotype (CIMP), microsatellite instability (MSI), and the immunohistochemical profile, including the expression of transcription factors (TTF1, ASH1, CDX2, and PAX5), stem cell markers (CD117 and CD34), and cytokeratins 7 and 20, in a series of 39 carcinomas (27 NECs and 12 MANECs) to better characterize such neoplasms and to search for prognostic indicators. No different patient survival was observed between NECs and MANECs. Neoplasms showed a heterogenous spectrum of morphologic and immunohistochemical features; however, only large-cell subtype, significant peritumoral lymphoid reaction, CD117 immunoreactivity, vascular invasion, and MSI/CIMP+ status were significantly correlated with prognosis on univariable analysis. Furthermore, vascular invasion and CD117 immunoreactivity were independent prognostic markers on multivariable analysis. In addition to these prognostic features, neoplasms showed different expression of transcription factors, stem cell markers, and cytokeratins that should be considered for diagnostic purposes and, especially, for discriminating among possible differential diagnoses.

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.

Prognostic factors for ampullary adenocarcinomas: tumor stage, tumor histology, tumor location, immunohistochemistry and microsatellite instability.

Prognostic factors for ampullary carcinomas (ACs) are poorly defined. Fifty three resected ACs were analyzed for CDX2, MUC1, MUC5AC, MUC6, MUC2, and for mismatch repair proteins (hMLH1, hMSH2, PMS2, hMSH6) using immunohistochemistry. Microsatellite instability (MSI) status was evaluated by fluorescently labeled PCR using an automated sequencer. Univariate and multivariate analysis was performed for clinicopathological, immunohistochemical and molecular parameters. CDX2 was found in 32 out of 53 (60%) ACs with a significantly higher frequency among intestinal ACs compared with biliopancreatic (BP) ACs. The MUC1, MUC5AC, MUC6, MUC2 apomucins were expressed in 75, 43, 39, and 28% of ACs, respectively, with a significantly higher coexpression of MUC1/MUC5AC in BP ACs. MSI and loss of expression of hMLH1/PMS2 or hMSH2/hMSH6 proteins were observed only in intestinal ACs. Factors significantly correlated with improved survival in the univariate analysis were: low stage, absence of lymph nodes metastases, negative surgical margins (R0 status), and presence of MSI. In the multivariate analysis, stage was the only independent prognostic factor of survival. We conclude that stage is the only independent prognostic factor of survival in the multivariate analysis, whereas histological criteria and the immunohistochemical expression of apomucins and CDX2 are helpful in the classification and understanding of the histogenesis of ACs.

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.

Two PMS2 Mutations in a Turcot Syndrome Family with Small Bowel Cancers

We report the clinicopathological, genetic, and immunohistochemical characterization of an atypical Turcot syndrome (TS) family with small bowel cancer. The tumor family history of a patient with cafè-au-lait spots (CALS) and early onset adenomas, duodenal cancer, and glioblastoma was positive for colonic adenoma (mother), jejunal (maternal grandfather), lung (father), and colorectal (paternal uncle) cancers. PMS2 genetic testing identified the nonsense 1951C>T (Q643X) and the missense 161C>T (S46I) mutations. PMS2 expression was absent in the probands duodenal cancer with high microsatellite instability. The normal cells also displayed no PMS2 expression and some degree of instability. Our findings point out the association between PMS2 and TS, and support the hypothesis that patients with a few polyps, small bowel tumors with a very early onset, glioblastoma, and CALS should be considered as a variant of hereditary nonpolyposis colorectal cancer. A recessive model of inheritance caused by compound heterozygous mutations was consistent with the observed severe clinical phenotype and has important implications for predicting cancer risk in both the proband and his relatives.

BRCA1 and BRCA2 genes: Role in hereditary breast and ovarian cancer in Italy

The heritable defects of BRCA1 and BRCA2 genes have been shown to predispose to breast and ovarian cancers. In a previous report, we analyzed 46 Italian families with breast and/or ovarian cancer for BRCA1 mutations. In the present study, those families and 11 others were screened for BRCA2 mutations; the newly enrolled families were also analyzed for the BRCA1 gene. The coding region and splice boundaries of BRCA2 and BRCA1 genes were assessed by the protein‐truncation test and single‐strand conformational polymorphism. A total of 20 different mutations were found in 21 families (37%). A total of 9 families (16%) showed mutations in the BRCA1 gene, including the one new mutation identified in this study (5382insC), and 12 families (21%) presented mutations in the BRCA2 gene. BRCA2‐mutated families presented breast and ovarian cancers or breast cancers only, whereas most BRCA1‐mutated families presented ovarian cancer alone or in association with breast cancer. All the BRCA2 mutations led to a truncated protein: 6 were frameshift mutations, 4 were non‐sense mutations and 2 involved the intronic invariant region leading to splice variants. Therefore, in the Italian population, the cumulative proportion of BRCA1 and BRCA2 mutations was within the range observed in other studies (37%), with higher involvement of BRCA2 than of BRCA1. Many families in which no mutations were found presented a very high incidence of breast and/or ovarian cancer. Among the 36 BRCA1 and BRCA2 wild‐type families, 24 presented at least 4 cancer cases, indicating the existence of other important predisposing genes. Int. J. Cancer 83:5–9, 1999.

The role of histological investigation in prognostic evaluation of advanced gastric cancer. Analysis of histological structure and molecular changes compared with invasive pattern and stage.

Abstract. The relative contribution of tumour histology or molecular changes, compared with invasion pattern or stage, to prognostic assessment of gastric cancer was investigated in a series of 185 advanced (T2 to T4, stage IB to IV) cancers that had undergone intentionally curative surgery at Varese General Hospital. Survival analysis of the histological types considered in commonly used classifications, such as Lauren, Kubo, the World Health Organization (WHO) and related classifications, allowed separation of a small high-grade (Hg, 12 cases) group of adenosquamous, anaplastic and small cell endocrine carcinomas from a large cohesive group (C, 86 glandular or solid cancers) and from another large (87 cases) group of tumours with dissociated cells [29 diffuse (D) and 58 mixed (M) tumours]. Univariate and multivariate analysis showed the independent prognostic value of this C/M+D/Hg classification approach, which proved superior to other classifications and to cell dissociation at the growing front or angio, lympho and neuroinvasion. Expression of sialyl Lewisc, the DUPAN-2 antigen, proved to be an independent predictor of worse survival among tumours beyond stage I, showing an exclusively or predominantly cohesive structure. Microsatellite instability (MSI) predicted favourable survival in purely cohesive tumours of intermediate (II) stage, especially of solid/medullary and lymphoid stroma/lymphoepithelioma-like structure, among which two distinct tumour subsets were characterised, one MSI-positive and the other Epstein-Barr virus positive. T2N0M0 (stage IB) tumours showed mostly favourable survival independently from histological type, invasive pattern, DUPAN-2 or MSI status. It is concluded that an appropriate histological evaluation, coupled with sialylated glycoproteins histochemistry and, for stage-II tumours, MSI tests may contribute significantly to prognostic assessment of tumours beyond stage I. However, the stage itself, with special reference to lymph-node metastases and invasion level beyond subserosa, remains the most important prognostic clue for gastric cancer.

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.