Silvia Pizzi

RASSF1A promoter methylation and 3p21.3 loss of heterozygosity are features of foregut, but not midgut and hindgut, malignant endocrine tumours.

The Ras‐association domain family 1A (RASSF1A) tumour suppressor gene is inactivated in a variety of solid tumours, usually by epigenetic silencing of the promoter and/or allelic loss of its locus at 3p21.3. RASSF1A induces cell cycle arrest through inhibition of cyclin D1 accumulation. In this work, 62 endocrine tumours from different sites in the gut were investigated for methylation of the RASSF1A promoter using the polymerase chain reaction, the presence of 3p21.3 deletions by loss of heterozygosity analysis, and cyclin D1 expression by immunohistochemistry. Methylation was found in 20/62 (32%) cases and was restricted to foregut tumours; deletion at 3p21.3 was found in 15/58 (26%) informative cases and restricted to malignant foregut tumours; cyclin D1 hyperexpression was found in 31/58 (53%) cases and correlated with RASSF1A methylation. Our data suggest that RASSF1A is involved in the development of endocrine tumours derived from the foregut only, and that the presence of both RASSF1A methylation and 3p21.3 deletion is associated with malignancy. These results may provide a rationale for foregut‐targeted therapy for aggressive endocrine carcinomas entailing the use of demethylating agents. Copyright

Genetic alterations in poorly differentiated endocrine carcinomas of the gastrointestinal tract.

The molecular pathogenesis of poorly differentiated endocrine carcinomas of the gastrointestinal tract (GI PDECs) remains unclear. It has been suggested that these lesions either originate from multipotent stem cells that also can serve as the origin of nonendocrine adenocarcinomas or arise due to the dedifferentiation of well‐differentiated endocrine carcinomas (WDECs).

Malignancy-associated allelic losses on the X-chromosome in foregut but not in midgut endocrine tumours

Information on genetic changes involved in the progression of gastroenteropancreatic (GEP) endocrine tumours is scanty. On the other hand, the identification of molecular markers of malignancy could be crucial for the prognostic evaluation of these neoplasms, which is hardly predictable on the basis of conventional histological criteria. An association of X‐chromosome deletions with malignancy has already been found in gastric endocrine tumours. To investigate this further, a comparative loss of heterozygosity (LOH) analysis was performed on 17 pancreatic endocrine tumours (PETs) and 17 intestinal (ten ileal, six appendiceal, and one rectal) carcinoids from female patients. The relationship of X‐chromosome LOH with the ploidy status of the neoplasms was also investigated. LOH was found in six of eight malignant PETs (60% of the informative markers), but was infrequent in the nine benign ones (4.5%). In contrast, although retention of heterozygosity was consistently observed in benign midgut tumours, LOH was infrequent in malignant carcinoids (15%). No correlation was found between LOH and the ploidy status. These results indicate an association between X‐chromosome LOH and malignancy in foregut endocrine tumours. The lack of such an association in midgut carcinoids suggests that different molecular mechanisms are involved in the progression of these two categories of endocrine neoplasms, which are otherwise considered to be closely related. These findings emphasize the need for further molecular studies on GEP endocrine tumours, carefully subdivided according to their anatomical site of origin. Copyright

Association Between Recurrence of Sporadic Colorectal Cancer, High Level of Microsatellite Instability, and Loss of Heterozygosity at Chromosome 18q

PURPOSE:Microsatellite instability and loss of heterozygosity of chromosomes 18q, 8p, and 4p are genetic alterations commonly found in colorectal cancer. We investigated whether these genetic markers allow for the stratification of patients with Stage II to III colorectal cancer into groups with different recurrence risks, and with different prognoses.METHODS:Tumors of 113 patients were evaluated for loss of heterozygosity of chromosomes 18q, 8p, and 4p and for microsatellite instability by use of six microsatellite markers. Genetic alterations involving each of these genetic markers were examined for association with disease recurrences and survival.RESULTS:Loss of heterozygosity of chromosomes 18q, informative in 96 percent of cases, in Stage III tumors was associated with higher risk of overall recurrence (P < 0.001), local recurrence (P < 0.001), distant metastases (P < 0.001), decreased overall survival (P = 0.002), and disease-free survival (P < 0.001). The recurrence rates and survival rates among patients with Stage II colorectal cancer were independent of loss of heterozygosity of chromosome 18q. Stage III and loss of heterozygosity of chromosome 8p also were associated with a higher risk of recurrences when these factors were considered individually. In multivariate analysis, only loss of heterozygosity of chromosome 18q was independently associated with risk of recurrences (P < 0.001) and with disease-free survival (P = 0.001). No correlation was observed between microsatellite instability and recurrence rates. However, microsatellite instability was associated with improved overall survival (P = 0.04) and with a longer disease-free interval (P = 0.002). Only in five cases (16.7 percent) was it possible to perform resection of recurrences; two of these patients had microsatellite instability tumor. In no cases was it possible to resect recurrence of tumors with loss of heterozygosity of chromosome 18q.CONCLUSIONS:Loss of heterozygosity of chromosome 18q is an informative genetic marker, which in resected Stage III colorectal cancer can be used to predict recurrences and survival. Microsatellite instability identified cases that, even in the case of recurrence, have a more favorable prognosis.

Malignancy-associated X chromosome allelic losses in foregut endocrine neoplasms: further evidence from lung tumors.

Association of X chromosome allelic losses with tumor malignancy has been identified in foregut but not in midgut endocrine neoplasms. The aim of this study was to investigate the association of deletions on X chromosome with malignancy in lung neuroendocrine tumors, another family of foregut neoplasms comprising four categories with increased malignancy: typical and atypical carcinoids, large cell neuroendocrine and small cell lung carcinomas. To evaluate loss of heterozygosity, DNA extracted from nine typical carcinoids, 17 atypical carcinoids, six large cell neuroendocrine carcinomas and five small cell lung carcinomas was PCR-amplified for 18 microsatellite markers spanning the whole X chromosome. All tissue samples were formalin-fixed and paraffin-embedded. X chromosome losses were absent in typical carcinoids, whereas they were found in nine out of 17 atypical carcinoids and in five out of six large cell neuroendocrine carcinomas (involving 28 and 70% of informative loci, respectively). On the contrary, deletions on X chromosome were an extremely rare event in small cell lung carcinomas. In atypical carcinoids, the presence of losses was associated with larger tumor size, higher pT status and advanced stage. No death occurred in atypical carcinoid patients without deletions on X chromosome, whereas all atypical carcinoid patients who had died from disease showed allelic losses. In conclusion, X chromosome allelic losses, absent in benign ‘typical’ carcinoids, progressively increased in frequency from intermediate-grade ‘atypical’ carcinoids to high-grade large cell neuroendocrine carcinomas. These results extend the association of deletions on X chromosome with malignancy, already demonstrated in other foregut endocrine neoplasms, to lung neuroendocrine tumors. The absence of X chromosome allelic losses in small cell lung carcinomas underlines a striking difference from large cell neuroendocrine carcinomas, possibly linked to different pathogenetic mechanisms of these two highly aggressive neuroendocrine lung tumors.

Sex Chromosome Alterations Associate with Tumor Progression in Sporadic Colorectal Carcinomas

Purpose: The X and Y chromosomes have been associated with malignancy in different types of human tumors. This study attempts to determine the involvement of X chromosome and pseudoautosomal regions (PAR) in sporadic colorectal carcinogenesis. Experimental Design: An allelotyping of X chromosome in 20 premalignant and 22 malignant sporadic colorectal tumors (CRC) from female patients and an analysis of losses [loss of heterozygosity (LOH)] on PARs from 44 CRCs and 12 adenomas of male patients were carried out. In male tumors, a fluorescence in situ hybridization analysis was done to identify which sex chromosome was possibly lost. Results: The LOH frequency in female CRCs was 46% with higher incidence in patients with tumor recurrence than in those who were disease-free (P < 0.01) and with a significant difference from adenomas (11%; P < 0.0001). The LOH rate of PARs in male CRCs was 37% with a frequency significantly higher in patients with recurrence (P < 0.03). These results were maintained also when data from PARs of all 66 male and female patients were cumulated (P < 0.05). LOH in PARs was significantly correlated with LOH at 5q (P < 0.01) and 18q (P < 0.01), early and late events, respectively, in colorectal carcinogenesis. Fluorescence in situ hybridization analysis in male patients with extensive PAR LOH revealed a preferential loss of the Y chromosome. Conclusions: Our data suggest a role for sex chromosome deletions in the malignant progression of sporadic CRCs and support the presence in the PARs of putative tumor suppressor genes involved in the progression of human sporadic CRCs.

Genetic alterations in combined neuroendocrine neoplasms of the lung.

Large-cell neuroendocrine and small-cell lung carcinomas are highly aggressive neuroendocrine tumors that can be associated in a variant of ‘small-cell lung carcinoma combined with large-cell neuroendocrine carcinoma’. Little is known about this rare tumor type with biphenotypic neuroendocrine differentiation. The aim of the present study was to genetically characterize each component of a series of combined small-cell/large-cell neuroendocrine carcinomas, to gain information on their histogenesis and to compare the alterations observed with those found in their respective pure forms. To this end, 22 formalin-fixed, paraffin-embedded lung neuroendocrine tumors obtained from surgical resections were investigated: six combined small-cell/large-cell carcinomas, eight pure large-cell carcinomas and eight pure small-cell carcinomas. For the combined neuroendocrine neoplasms, DNA was extracted separately from each of the two cytologically different populations. Allelic imbalance was investigated by PCR amplification of 30 highly polymorphic microsatellite markers located at 11 different chromosomal regions. A common background of genetic alterations, similar in both components of the combined neoplasms, was demonstrated at 17p13.1, 3p14.2–3p21.2, 4q12–4q24, 5q21 and 9p21. In fact, the two components appeared to be more similar to each other than to their respective pure forms. In addition, allelic imbalances preferentially involving one of the two components were found. These alterations often appeared to be specific for this histological variant, as compared with those observed in pure forms or in the literature. In conclusion, this is the first report in which a molecular characterization of the variant of small-cell lung carcinoma combined with large-cell neuroendocrine carcinoma was performed. The finding of common alterations in the two phenotypically different neuroendocrine cell components suggests a close genetic relationship and supports the hypothesis of a monoclonal origin from a common ancestor. The genetic differences observed provide the basis for the divergent differentiation and parallel the morphological differences in the two components of these combined neuroendocrine neoplasms.

Pleomorph poorly differentiated endocrine carcinoma of the rectum

We present a case of poorly differentiated endocrine carcinoma (PDEC) of the rectum identified immunohistochemically and characterized by a high degree of cellular pleomorphism, including bizarre giant cells. This case indicates that gastrointestinal PDECs are not restricted to small cell carcinomas. Among the multiple genes investigated, loss of heterozygosity (LOH) of the p53 locus without p53 immohistochemical accumulation, overexpression of c-kit and absent expression of p16 were seen.

Xq25 and Xq26 identify the common minimal deletion region in malignant gastroenteropancreatic endocrine carcinomas

Loss of heterozygosity (LOH) for markers on X chromosome are associated with malignancy in endocrine tumors of the stomach and pancreas. The aim of this work is to investigate low-grade, well-differentiated endocrine carcinomas (WDEC) vs high-grade, poorly differentiated endocrine carcinomas (PDEC) of the gastroenteropancreatic (GEP) tract for common deletion regions on X chromosome. We performed a comparative allelotyping analysis with 24 highly polymorphic markers for the X chromosome in 12 WDECs and 5 PDECs. Overall, the LOH frequency in all informative loci investigated was 59% in primary and 61% in metastasis, with a significantly higher rate in PDECs than in WDECs (p<0.015 for primary and p<0.00005 for metastasis). In both WDECs and PDECs, the small Xq25 region as defined by DXS8059, DXS8098, and DXS8009 markers showed higher LOH rate as compared to the rest of the chromosome markers (p<0.04). In addition, LOH was very frequently elevated also in DXS294 and in DXS102 loci mapping the chromosomal region Xq26. In no instances differences were found between primary tumors and metastases. Methylation analysis revealed that Xq25 loss preferentially occurred on the inactive X chromosome, a feature in agreement with findings from other human cancers suggesting escape of tumor suppressor genes to X chromosome inactivation at this region. Overall, our data indicate that the two chromosomal regions, Xq25 and Xq26, may participate to the malignant progression of GEP endocrine carcinomas.

Adenomatous polyposis coli alteration in digestive endocrine tumours: correlation with nuclear translocation of β-catenin and chromosomal instability

The role of Wnt pathway in digestive endocrine tumours is debated. The aim of this work is to investigate key players in Wnt pathway by a multimodal approach. Sixty cases (49 well-differentiated and 11 poorly differentiated) were investigated for methylation of adenomatous polyposis coli (APC) and E-cadherin promoters, the loss of heterozygosity (LOH) at APC locus and beta-catenin and E-cadherin expression by immunohistochemistry. Tumours showing altered beta-catenin localization were tested for beta-catenin and APC mutations. APC promoter methylation was restricted to gastroduodenal tumours (21 out of 59, 36%), prevalent in poorly differentiated carcinomas (P=0.042) and correlating with aggressive features (high histology grade, P<0.02; tumour death, P=0.026; high fractional allelic loss, P=0.002, in turn correlating with short survival, P=0.017). LOH at APC locus was found in 14 out of 53 cases (26%, 10 gastroduodenal and 4 colorectal), prevalent in poorly differentiated carcinomas (P=0.002) and correlating with histology grade (P=0.012). beta-catenin abnormal expression was found in 41 out of 54 cases (76%), with nuclear staining correlating with APC alteration (P=0.047) and short survival (P=0.006). APC, but not beta-catenin, gene mutations were found (7 out of 35 tumours), 4 of which in the midgut. E-cadherin promoter methylation was rarely detected (2 out of 52 cases), with cytoplasmic expression in 18 out of 43 cases (42%), not correlating with any clinico-pathological feature. In conclusion, Wnt pathway alterations, as represented by abnormal beta-catenin localization, are common events in digestive endocrine tumours, but only nuclear expression correlates with tumour aggressiveness. Though with different alteration mechanisms according to anatomical site, APC plays a major role in Wnt pathway activation and in determining the high chromosomal instability observed in aggressive endocrine carcinomas.