Bostjan Humar

Hereditary diffuse gastric cancer: translation of CDH1 germline mutations into clinical practice

Hereditary diffuse gastric cancer (HDGC) is the only known cancer syndrome that is dominated by gastric adenocarcinoma. HDGC is caused by germline mutation of the CDH1 gene that encodes the cell adhesion protein E-cadherin. Mutation carriers have a more than 70% lifetime risk of developing DGC and an elevated risk of lobular breast cancer. Intestinaltype gastric cancer is not part of the syndrome. Clinical management of HDGC involves predictive genetic testing beginning at or near 16 years of age. It is recommended that mutation carriers undergo prophylactic gastrectomy after about 20 years of age. Anatomical mapping has demonstrated that mutation carriers develop multifocal stage T1a signet ring cell carcinomas, with up to several hundred foci being observed in single stomachs. These foci develop following the somatic inactivation of the second CDH1 allele by mechanisms that include DNA promoter hypermethylation.

E-Cadherin Deficiency Initiates Gastric Signet-Ring Cell Carcinoma in Mice and Man

The importance of loss of the cell-cell adhesion molecule E-cadherin (encoded by CDH1) to tumor progression is well established. However, CDH1 germ-line mutations predispose to the cancer susceptibility syndrome hereditary diffuse gastric cancer (HDGC), suggesting a role for E-cadherin in tumor initiation. The earliest indications of cancer in the stomachs of CDH1 mutation carriers are microscopic foci of intramucosal signet-ring cell carcinoma (SRCC; designated eHDGC). Here, we used N-methyl-N-nitrosourea (MNU) to promote gastric carcinogenesis in wild-type (wt) and cdh1(+/-) mice. MNU induced a variety of gastric tumors; however, intramucosal SRCC developed with an 11 times higher incidence in cdh1(+/-) mice compared with wt mice. The murine SRCC resembled the human eHDGCs in that they were hypoproliferative, lacked nuclear beta-catenin accumulation, and had reduced membrane localization of E-cadherin and its interacting junctional proteins. The down-regulation of E-cadherin in the murine SRCCs confirmed the importance of the second CDH1 hit to the initiation of diffuse gastric cancer. CDH1 promoter hypermethylation has been proposed to be a major second hit in advanced HDGC; however, its contribution to eHDGC was unknown. We thus examined a series of human eHDGC and detected CDH1 promoter methylation in 50% of foci. Promoter methylation was accompanied by reduced wt CDH1 mRNA levels in the foci and had a monoclonal pattern, consistent with an epigenetic initiation of disease. Together, these findings provide compelling evidence for a deficiency in cell-to-cell adhesion being sufficient to initiate diffuse gastric cancer in the absence of hyperproliferation and beta-catenin activation.

Prognostic analysis of E-cadherin gene promoter hypermethylation in patients with surgically resected, node-positive, diffuse gastric cancer.

Purpose: Recent investigations have demonstrated that hypermethylation is a frequent mechanism for silencing tumor suppressor genes. This is a potentially reversible epigenetic change, and it is the target of a novel class of anticancer compounds with demethylating activity. Better understanding of the clinical implications of hypermethylation will allow the optimal planning of future trials with demethylating drugs. In this perspective, we investigated whether hypermethylation in the CDH1 promoter region is correlated with poor prognosis of patients with surgically resected, node-positive, diffuse gastric cancer. Experimental Design: Consecutive cases of diffuse gastric cancer were considered eligible for study entry. Additional inclusion criteria were radical surgery with a minimum of D1 lymphadenectomy, complete follow-up information, and availability of tumor specimens for methylation-specific PCR and immunohistochemistry analyses. Results: CDH1 promoter hypermethylation was found in 40 of 73 cases (54%), and it was significantly associated with worse prognosis. In patients with and without hypermethylation, the 5-year event-free survival rate was 30% and 62%, respectively, and the 5-year overall survival rate was 35% and 67%, respectively. CDH1 promoter hypermethylation retained its prognostic role for disease-free survival (P < 0.001) and overall survival (P < 0.001) in multivariate analysis. Immunohistochemistry showed a significant association between CDH1 methylation and E-cadherin expression (P < 0.001). Conclusions: This study shows adverse prognostic effect of CDH1 promoter hypermethylation in patients with diffuse gastric cancer. This form of cancer, and other types with frequent hypermethylation and silencing of critical tumor suppressor genes, would make appropriate targets for the testing of novel compounds with demethylating activity.

Association of CDH1 haplotypes with susceptibility to sporadic diffuse gastric cancer.

Truncating mutations in the gene for the cell to cell adhesion protein E-cadherin are the most consistent genetic alterations observed in sporadic and hereditary diffuse gastric cancer (DGC). In addition to these inactivating mutations, a CDH1 promoter polymorphism at position −160 has been reported to lead to transcriptional downregulation of the gene in vitro. We therefore performed a case–control study to investigate whether this variant is associated with an increased susceptibility to DGC. The frequency of the −160A allele was significantly higher (P<0.005) in 53 diffuse gastric cancer cases compared to 70 matched controls. The odds ratio associated with the A-allele was 2.27 for CA-heterozygotes (95%CI 1.16–4.44) and 7.84 for AA-homozygotes (95%CI 2.89–21.24). Two additional polymorphisms (the 48+6T→C and the 2076C→T variant) were genotyped and shown to be equally distributed among cases and controls. Haplotype analysis with the three polymorphisms confirmed an association with disease (P<0.004). However, this analysis suggested the −160C→A CDH1 promoter polymorphism may be in linkage disequilibrium with a distinct aetiological locus or acts in combination with other functional variants in or near the CDH1 region.

Destabilized Adhesion in the Gastric Proliferative Zone and c-Src Kinase Activation Mark the Development of Early Diffuse Gastric Cancer

The initial development of diffuse gastric cancer (DGC) is poorly understood. The study of E-cadherin (CDH1) germ line mutation carriers predisposed to DGC provides a rare opportunity to elucidate the genetic and biological events surrounding disease initiation. Samples from various stages of hereditary and sporadic DGC were investigated to determine general mechanisms underlying early DGC development. Paraffin-embedded tissues from 13 CDH1 mutation carriers and from 10 sporadic early DGC cases were analyzed. Immunofluorescence and immunohistochemistry using differentiation, proliferation, and adhesion markers showed that DGC initiation seems to occur at the proliferative zone (the upper neck) of the gastric epithelium and correlates with absent or reduced expression of junctional proteins (beta-actin, p120, Lin-7). Slow proliferation of neoplastic cells at the upper gastric neck leads to the formation of intramucosal signet-ring cell carcinoma (SRCC) displaying differentiated features. As shown by immunolabeling, invasion from SRCC lesions beyond the gastric mucosa is associated with poor differentiation, increased proliferation, activation of the c-Src system, and an epithelial-mesenchymal transition. Our results provide a molecular description of the early development of DGC and explain the relationship between the two main DGC types, poorly differentiated carcinoma and SRCC: both share their origin, but SRCC develops following cancer cell differentiation and seems relatively indolent in its intramucosal stage.

Prognostic Role of Interleukin-1β Gene and Interleukin-1 Receptor Antagonist Gene Polymorphisms in Patients With Advanced Gastric Cancer

PURPOSEnA high interleukin-1beta (IL-1B) and interleukin-1 receptor antagonist (IL-RN) ratio underlies an unfavorable proinflammatory status. Also, it seems to be involved in the mechanisms of cancer cachexia and tumor angiogenesis and metastasis. Two single nucleotide polymorphisms in IL-1B gene (IL-1B-511C/T,IL-1B-31T/C) and a variable number of tandem repeat polymorphisms in IL-RN gene (IL-1RNlong/2) enhance the circulating levels of the two cytokines. The prognostic role of IL-1B/IL-1RN genotypes was investigated in patients with relapsed and metastatic gastric cancer treated with palliative chemotherapy.nnnPATIENTS AND METHODSnBefore starting palliative chemotherapy, 123 prospectively enrolled patients supplied peripheral-blood samples for DNA extraction. Survival data were analyzed according to IL-1RN/IL-1B genotypes.nnnRESULTSnForty-two patients showed wild-type genotypes (IL-1RNlong/long, IL-1B-511C/C, and IL-1B-31T/T; group A). Forty-five patients showed the IL-1RN2 polymorphism, with wild-type IL-1B genotypes in seven patients and with IL-1B-511C/T and/or IL-1B-31T/C polymorphisms in 38 patients (group B). The remaining 36 patients demonstrated wild-type IL-1RN, with IL-1B-511C/T and/or IL-1B-31T/C polymorphisms (group C). In group A and B patients, the median progression-free survival (PFS) was 25 and 26 weeks, respectively, and median overall survival (OS) was 42 and 43 weeks, respectively. Group C patients showed worse PFS (median, 16 weeks) and OS (median, 28 weeks) than group A (P = .006 for PFS; P = .0001 for OS) and group B patients (P = .01 for PFS; P = .0001 for OS). The long/T/C haplotype was overrepresented in patients with shortened PFS (P = .001) and OS (P = .0005).nnnCONCLUSIONnIn patients with advanced gastric cancer, IL-1B polymorphisms showed adverse prognostic influence when coupled with wild-type IL-1RN genotype. These findings deserve further investigation for potential anticancer activity of recombinant IL-RN.

ASSOCIATION OF THYMIDYLATE SYNTHASE POLYMORPHISMS WITH GASTRIC CANCER SUSCEPTIBILITY

We investigated in a case‐control study a possible role of thymidylate synthase gene (TS) polymorphisms for gastric cancer susceptibility. Lymphocyte genomic DNA from 134 Italian gastric cancer patients and 139 controls was used for genotyping two polymorphisms in the TS 5′‐untranslated region (5′‐UTR); a double (2R) or triple (3R) 28‐bp repeat and a G/C polymorphism within the triple repeats allele (3G allele). Samples were also genotyped at a 6‐bp deletion/insertion (del6 or ins6) polymorphism at position 1494 in the TS 3′‐untranslated region (3′‐UTR). Unconditional regression with odd ratios (OR) and 95% confidence intervals (CI), haplotype and linkage disequilibrium analyses were used to investigate the association of the polymorphisms with the disease. The global allelic distribution was in Hardy‐Weinberg equilibrium. Genotypes with the 3G allele (2R/3G, 3C/3G, 3G/3G) were significantly more frequent in patients than controls and were associated with gastric cancer risk (OR = 2.06; 95% CI = 1.26–3.35). A significant risk was also observed for carriers of the del6 allele in the 3′‐UTR. Odds ratios for combined 3G‐del6/ins6 and 3G‐del6/del6 genotypes were 2.59 (95% CI = 1.36–4.94) and 2.81 (95% CI = 1.22–6.64), respectively. The 3G‐del6 haplotype showed a significant association with the disease (p = 0.01). Polymorphisms in the TS gene may contribute to gastric cancer susceptibility and this finding deserve further investigation in the context of novel strategies for gastric cancer prevention. In vitro, 3G genotypes have been related to high TS mRNA expression, which may underlie one of the possible etiologic mechanisms.

Epigenetic silencing in non-neoplastic epithelia identifies E-cadherin (CDH1) as a target for chemoprevention of lobular neoplasia.

Invasive lobular carcinoma (ILC) of the breast is believed to develop from in situ lesions, atypical lobular hyperplasia (ALH), and lobular carcinoma in situ (LCIS). Down‐regulation of the cell–cell adhesion protein E‐cadherin is a defining feature of lobular breast cancer (LBC) and already occurs in ALH and LCIS. Apart from mutational mechanisms, epigenetic silencing of the E‐cadherin gene (CDH1) is thought to be involved in E‐cadherin down‐regulation and has been observed at a high frequency in ILC. Whether CDH1 promoter methylation is already present in in situ lesions and thus contributes to the initiation of LBC is not established. We thus examined microdissected archived tissue from 20 LBCs by methylation‐specific PCR to determine the CDH1 methylation status of lobular lesions. Nineteen of the 20 LBCs had a hypermethylated CDH1 promoter, including 13/14 ILCs and 13/13 ALHs or LCIS. Bisulphite sequencing indicated that methylation was complete within the investigated promoter fragment. Intriguingly, CDH1 methylation was likewise present in 8/8 adjacent non‐neoplastic epithelia, but not in 6/6 mammary epithelia from healthy subjects. E‐cadherin protein and mRNA were down‐regulated in in situ lesions relative to adjacent epithelia. Together, these results indicate that CDH1 promoter methylation occurs in LBC prior to E‐cadherin down‐regulation and neoplastic formation. We thus propose that epigenetic silencing represents the first of the two hits required to silence both CDH1 alleles for LBC to develop. Because promoter methylation is in principle reversible, our findings suggest that chemoprevention of LBC by epigenetic drugs should be feasible. Furthermore, the presence of CDH1 methylation in pre‐neoplastic epithelia suggests the existence of mammary regions with increased disease susceptibility, providing an explanation for the often multifocal presentation of LBC. Copyright

Slow proliferation as a biological feature of colorectal cancer metastasis.

Background:We have recently reported an inverse relationship between colon cancer progression and tumour proliferative activity. Here, we extend our findings by evaluating the proliferative activity of liver metastatic lesions and primary colorectal cancers (CRC) that differ in their metastatic potential.Methods:Using an earlier established multi-gene proliferation signature (GPS), proliferative levels were analysed in 73 primary CRCs and 27 liver metastases.Results:Compared with primary CRCs, we observed a significantly lower expression of the GPS in liver metastases and confirmed their lower proliferative levels by quantitative RT–PCR and Ki-67 immunostaining. No difference could be detected in apoptotic indices as assessed by M30 immunostaining, indicating that the net growth rate is lower in metastases relative to primary tumours. Notably, relapsed primaries or those with established metastases had significantly lower proliferative activity than CRCs that were non-metastatic and did not relapse.Conclusion:Our results suggest that slow proliferation is a biological characteristic of both liver metastases and those primary tumours with the ability to metastasise. The delineation of the mechanisms underlying the inverse association between proliferation and CRC aggressiveness may be important for the development of new therapeutic strategies.

Reduced expression of a gene proliferation signature is associated with enhanced malignancy in colon cancer.

The association between cell proliferation and the malignant potential of colon cancer is not well understood. Here, we evaluated this association using a colon-specific gene proliferation signature (GPS). The GPS was derived by combining gene expression data obtained from the analysis of a cancer cell line model and a published colon crypt profile. The GPS was overexpressed in both actively cycling cells in vitro and the proliferate compartment of colon crypts. K-means clustering was used to independantly stratify two cohorts of colon tumours into two groups with high and low GPS expression. Notably, we observed a significant association between reduced GPS expression and an increased likelihood of recurrence (P<0.05), leading to shorter disease-free survival in both cohorts. This finding was not a result of methodological bias as we verified the well-established association between breast cancer malignancy and increased proliferation, by applying our GPS to public breast cancer data. In this study, we show that reduced proliferation is a biological feature characterizing the majority of aggressive colon cancers. This contrasts with many other carcinomas such as breast cancer. Investigating the reasons underlying this unusual observation may provide important insight into the biology of colon cancer progression and putative novel therapy options.