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Objective POLE-mutant, microsatellite-instable (MSI), p53-mutant and non-specific molecular profile (NSMP) are TCGA-defined molecular subgroups of endometrial cancer (EC). Hypothesizing that morphology and tumor immunology might differ depending on molecular background concerning composition and prognostic impact, we aimed to comprehensively interconnect morphologic, immunologic and molecular data. Methods TCGA-defined molecular groups were determined by immunohistochemistry and sequencing in n = 142 endometrioid EC. WHO-defined histopathological grading was performed. The immunologic microenvironment (iTME) was characterised by the quantification of intraepithelial and stromal populations of tumor-infiltrating lymphocytes (TIL: overall T-cells; T-Killer cells; regulatory T-cells (Treg)). Immunologic parameters were correlated with WHO-grading, TCGA-subgroups and prognosis. Results High density TIL were significantly more frequent in high-grade (G3) compared to low-grade (G1/2) EC in the whole cohort and in the subgroup of POLE-wildtype-/microsatellite-stable-EC. MSI was associated with high-level TIL-infiltration when taking into account the type of mismatch repair defect (MLH1/PMS2; MSH2/MSH6). Prognostic impact of biomarkers depended on molecular subgroups: In p53-mutant EC, Treg were independently prognostic, in NSMP, the unique independently prognostic biomarker was WHO-grading. Conclusions EC morphology and immunology differ depending on genetics. Our study delineated two molecularly distinct subgroups of immunogenic EC characterized by high-density TIL-infiltration: MSI EC and high-grade POLE-wildtype/microsatellite-stable-EC. Prognostic impact of TIL-populations relied on TCGA-subgroups indicating specific roles for TIL depending on molecular background. In NSMP, histopathological grading was the only prognostic biomarker demonstrating the relevance of WHO-grading in an era of molecular subtyping.

Background The Cancer Genome Atlas (TCGA) consortium described EBV positivity(+), high microsatellite instability (MSI-H), genomic stability (GS) and chromosomal instability (CIN) as molecular subtypes in gastric carcinomas (GC). We investigated the predictive and prognostic value of these subtypes with emphasis on CIN in the context of neoadjuvant chemotherapy (CTx) in GC. Methods TCGA subgroups were determined for 612 resected adenocarcinomas of the stomach and gastro-oesophageal junction (291 without, 321 with CTx) and 143 biopsies before CTx. EBV and MSI-H were analysed by standard assays. CIN was detected by multiplex PCRs analysing 22 microsatellite markers. Besides the TCGA classification, CIN was divided into four CIN-subgroups: low, moderate, substantial, high. Mutation profiling was performed for 52 tumours by next-generation sequencing. Results EBV(+) (HR, 0.48; 95% CI, 0.23–1.02), MSI-H (HR, 0.56; 95% CI, 0.35–0.89) and GS (HR, 0.72; 95% CI, 0.45–1.13) were associated with increased survival compared to CIN in the resected tumours. Considering the extended CIN-classification, CIN-substantial was a negative prognostic factor in uni- and multivariable analysis in resected tumours with CTx (each p p = 0.026), but was not prognostically relevant. Conclusion A refined CIN classification reveals tumours with different biological characteristics and potential clinical implications.

The lancet / Digital health 3(10), e654-e664 (2021). doi:10.1016/S2589-7500(21)00133-3, Published by The Lancet, London

Background Even though the post-operative outcome varies greatly among patients with nodal positive colon cancer (UICC stage III), personalized prediction of systemic disease recurrence is currently insufficient. We investigated in a retrospective setting whether genetic and immunological biomarkers can be applied for stratification of distant metastasis occurrence risk. Methods Eighty four patients with complete resection (R0) of stage III colon cancer from two clinical centres were analysed for genetic biomarkers: microsatellite instability, oncogenic mutations in KRAS exon2 and BRAF exon15, expression of osteopontin and the metastasis-associated genes SASH1 and MACC1. Tumor-infiltrating CD3 and CD8 positive T-cells were quantified by immunocytochemistry. Results were correlated with outcome and response to 5-FU based adjuvant chemotherapy, using Cox’s proportional hazard models and integrative two-step cluster analysis. Results Distant metastasis risk was significantly correlated with oncogenic KRAS mutations (p = 0.015), expression of SASH1 (p = 0.016), and the density of CD8-positive T-cells (p = 0.007) in Kaplan-Meier analysis. Upon multivariate Cox-regression analysis, KRAS mutation (p = 0.008) and density of CD8-positive TILs (p = 0.009) were retained as prognostic parameters for metachronous distant metastasis. Integrative two-step cluster analysis was used to combine all genetic markers, allowing stratification of patient subgroups. Post-operative distant metastasis risk ranged from 31% (low-risk) to 41% (intermediate), and 57% (high-risk) (p = 0.032). Increased expression of osteopontin (p = 0.019) and low density of CD8-positive T-cells (p = 0.043) were significantly associated with unfavourable response to 5-FU. Conclusions Integrative biomarker analysis allows stratification of stage III colon cancer patients for the risk of metastatic disease recurrence and may indicate response to 5-FU. Thus, biomarker analysis might facilitate the use of adjuvant therapy for high risk patients. Electronic supplementary material The online version of this article (10.1186/s12885-018-4940-2) contains supplementary material, which is available to authorized users.

Genetic associations between variants on chromosome 5p13 and 8q24 and gastric cancer (GC) have been previously reported in the Asian population. We aimed to replicate these findings and to characterize the associations at the genome and transcriptome level. We performed a fine-mapping association study in 1926 GC patients and 2012 controls of European descent using high dense SNP marker sets on both chromosomal regions. Next, we performed expression quantitative trait locus (eQTL) analyses using gastric transcriptome data from 143 individuals focusing on the GC associated variants. On chromosome 5p13 the strongest association was observed at rs6872282 (P = 2.53 x 10(-04)) and on chromosome 8q24 at rs2585176 (P = 1.09 x 10(-09)). On chromosome 5p13 we found cis-eQTL effects with an up-regulation of PTGER4 expression in GC risk allele carrier (P = 9.27 x 10(-11)). On chromosome 8q24 we observed cis-eQTL effects with an upregulation of PSCA expression in GC risk allele carrier (P = 2.17 x 10(-47)). In addition, we found trans-eQTL effects for the same variants on 8q24 with a downregulation of MBOAT7 expression in GC risk allele carrier (P = 3.11 x 10(-09)). In summary, we confirmed and refined the previously reported GC associations at both chromosomal regions. Our data point to shared etiological factors between Asians and Europeans. Furthermore, our data imply an upregulated expression of PTGER4 and PSCA as well as a downregulated expression of MBOAT7 in gastric tissue as risk-conferring GC pathomechanisms.

Lynch Syndrome (LS) is the most common dominantly inherited colorectal cancer (CRC) predisposition and is caused by a heterozygous germline defect in one of the DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6, or PMS2. High microsatellite instability (MSI-H) and loss of MMR protein expression in tumours reflecting a defective MMR are indicators for LS, as well as a positive family history of early onset CRC. MSH2 and MSH6 form a major functional heterodimer, and MSH3 is an alternative binding partner for MSH2. So far, the role of germline MSH3 variants remains unclear, as to our knowledge heterozygous truncating variants are not regarded causative for LS, but were detected in patients with CRC, and recently biallelic MSH3 defects have been identified in two patients with adenomatous polyposis. By gene screening we investigated the role of MSH3 in 11 LS patients with truncating MSH6 germline variants and an unexplained MSH2 protein loss in their corresponding MSI-H tumours. We report the first two LS patients harbouring heterozygous germline variants c.1035del and c.2732T>G in MSH3 coincidentally with truncating variants in MSH6. In the patient with truncating germline variants in MSH3 and MSH6, two additional somatic second hits in both genes abrogate all binding partners for the MSH2 protein which might subsequently be degraded. The clinical relevance of MSH3 germline variants is currently under re-evaluation, and heterozygous MSH3 defects alone do not seem to induce a LS phenotype, but might aggravate the MSH6 phenotype in affected family members.

Colorectal mixed adenoneuroendocrine carcinomas are rare and clinically aggressive neoplasms with considerable morphological heterogeneity. Data on their genomic characteristics and molecular associations to either conventional colorectal adenocarcinomas or poorly differentiated neuroendocrine neoplasms is still scarce, hampering optimized patient treatment and care. Tissue from 19 colorectal mixed adenoneuroendocrine carcinomas and eight colorectal poorly differentiated neuroendocrine neoplasms (neuroendocrine carcinomas) was microdissected and subjected to next-generation sequencing using a colorectal adenocarcinoma-specific panel comprising 196 amplicons covering 32 genes linked to colorectal adenocarcinoma, and poorly differentiated neuroendocrine neoplasm tumorigenesis. Mixed adenoneuroendocrine carcinomas were also examined for microsatellite instability and MLH-1 promoter methylation status. In three mixed adenoneuroendocrine carcinomas, exocrine and endocrine components were analyzed separately. Genetic testing of colorectal mixed adenoneuroendocrine carcinomas identified 43 somatic mutations clustering in 13/32 genes. Sixteen (84%) tumors harbored at least one somatic mutation, two tumors (11%) displayed high microsatellite instability. Compared with colorectal adenocarcinomas, mixed adenoneuroendocrine carcinomas were more frequently BRAF (37%; P=0.006), and less frequently KRAS (21%; P=0.043) and APC (16%; P=0.001) mutated. Point mutations in neuroendocrine neoplasm-related genes like RB1 or RET were not detected, but one tumor harbored a heterozygous RB1 deletion. Separately analyzed adenocarcinoma and neuroendocrine carcinoma components revealed a shared mutational trunk of driver genes involved in colorectal adenocarcinoma carcinogenesis. Colorectal neuroendocrine carcinomas were similar in their mutation profile to colorectal adenocarcinomas, but compared with mixed adenoneuroendocrine carcinomas, had a higher rate of APC mutations (P=0.027). Our data indicate that colorectal mixed adenoneuroendocrine carcinomas and neuroendocrine carcinomas are genetically closely related to colorectal adenocarcinomas, suggesting that the cells giving rise to these tumors primarily have an intestinal coinage. The identification of BRAF mutations and the frequently present KRAS wild-type status principally render some mixed adenoneuroendocrine carcinomas eligible to targeted treatment strategies used for colorectal adenocarcinomas.

Perioperative chemotherapy is an established treatment of advanced gastric cancer patients. Treatment selection is based on clinical staging (cT). We aimed to establish and validate a prognostic score including clinical and molecular factors, to optimize treatment decisions for these patients.We analyzed 626 carcinomas of the stomach and of the gastro-esophageal junction from two academic centers including primarily resected and pre-/perioperatively treated patients. Patients were divided into a training (N = 269) and validation (N = 357) set. Expression of 11 target genes was measured by quantitative PCR in resected tumors. A risk score to predict overall survival (OS) was generated and validated. Intra-tumoral heterogeneity was assessed by analyzing 50 tumor areas from 10 patients.A risk score including the expression of CCL5, CTNNB1, EXOSC3 and LZTR1 and the clinical parameters cT, tumor localization and histopathologic type suggested two groups with a significant difference in OS [hazard ratio (HR) 0.30; 95% confidence interval (CI) 0.17-0.52]. The risk score was successfully validated in an independent cohort (HR 0.32; 95% CI 0.21-0.51; P 0.001) as well as in subgroups of primarily resected (HR 0.30; 95% CI 0.17-0.54; P 0.001) and pre-/perioperatively treated patients (HR 0.37; 95% CI 0.17-0.81; P = 0.009). A significant difference in OS of high- and low-risk patients was also found in primarily resected patients with intestinal (HR 0.45; 95% CI 0.23-0.90; P = 0.020) and nonintestinal-type carcinomas (HR 0.1; 95% CI 0.02-0.42; P 0.001). Intra-tumor heterogeneity analysis indicated a classification reliability of 95% for a supposed analysis of three biopsies.The identified risk score could substantially contribute to an improved management of gastric cancer patients in the context of perioperative chemotherapy.

Objectives: The aim of this study was to independently validate a genomic signature developed both to assess recurrence risk in stage II patients and to assist in treatment decisions. Background: Adjuvant therapy is recommended for high-risk patients with stage II colon cancer, but better tools to assess the patients’ prognosis accurately are still required. Methods: Previously, an 18-gene signature had been developed and validated on an independent cohort, using full genome microarrays. In this study, the gene signature was translated and validated as a robust diagnostic test (ColoPrint), using customized 8-pack arrays. In addition, clinical validation of the diagnostic ColoPrint assay was performed on 135 patients who underwent curative resection (R0) for colon cancer stage II in Munich. Fresh-frozen tissue, microsatellite instability status, clinical parameters, and follow-up data for all patients were available. The diagnostic ColoPrint readout was determined blindly from the clinical data. Results: ColoPrint identified most stage II patients (73.3%) as at low risk. The 5-year distant-metastasis free survival was 94.9% for low-risk patients and 80.6% for high-risk patients. In multivariable analysis, ColoPrint was the only significant parameter to predict the development of distant metastasis with a hazard ratio of 4.28 (95% confidence interval, 1.36–13.50; P = 0.013). Clinical risk parameters from the American Society of Clinical Oncology (ASCO) recommendation did not add power to the ColoPrint classification. Technical validation of ColoPrint confirmed stability and reproducibility of the diagnostic platform. Conclusions: ColoPrint is able to predict the development of distant metastasis of patients with stage II colon cancer and facilitates the identification of patients who may be safely managed without chemotherapy.

Resistance to chemotherapy is a major obstacle for curative treatment of human gastric cancer (GC). However, the underlying molecular mechanisms are largely unknown. Wingless-type MMTV integration site family members (WNTs) are secreted glycoproteins involved in embryogenesis and, on inappropriate expression in the adult, in cancer. Here, we show expression of WNT6 in GC patient specimens, human GC cell lines and in a mouse model of GC. In human GC cells, WNT6 expression was enhanced by caveolin-1 (Cav1), a scaffold protein of plasma membrane caveolae. WNT6 knock-down and overexpression experiments demonstrated that WNT6 increased the resistance to apoptotic cell death induced by the anthracycline chemotherapeutics epirubicin (Epi) and doxorubicin (Dox). Epi increased the activity of the human WNT6 promoter through Cav1-dependent binding of β-catenin to the proximal WNT6 promoter. Epi increased both WNT6/Wnt6 and Cav1 expression in human GC cells and within the tumor area of a murine model of GC (CEA424-SV40 TAg). In GC patients, WNT6 expression was positively associated with the tumor stage and the nodal status, and inversely correlated with the response to ECF (Epi, cisplatin, 5-fluorouracil) chemotherapy. These results showed that WNT6 and Cav1 are upregulated by chemotherapeutics and enhance the resistance of GC cells to anthracycline drugs. Understanding the molecular mechanisms driving WNT6/Cav1-induced drug resistance will provide benefits in developing new therapies for GC.

Background Mantle cell lymphoma is a clinically heterogeneous disease characterized by overexpression of cyclin D1 protein. Blastoid morphology, high proliferation, and secondary genetic aberrations are markers of aggressive behavior. Expression profiling of mantle cell lymphoma revealed that predominance of the 3'UTR-deficient, short cyclin D1 mRNA isoform was associated with high cyclin D1 levels, a high "proliferation signature" and poor prognosis. Design and Methods Sixty-two cases of mantle cell lymphoma were analyzed for cyclin D1 mRNA isoforms and total cyclin D1 levels by real-time reverse transcriptase polymerase chain reaction, and TP53 alterations were assessed by immunohistochemistry and molecular analysis. Results were correlated with proliferation index and clinical outcome. Results Predominance of the short cyclin D1 mRNA was found in 14 (23%) samples, including four with complete loss of the standard transcript. TP53 alterations were found in 15 (24%) cases. Predominance of 3'UTR-deficient mRNA was significantly associated with high cyclin D1 mRNA levels (P=0.009) and more commonly found in blastoid mantle cell lymphoma (5/11, P=0.060) and cases with a proliferation index of >20% (P=0.026). Both blastoid morphology (11/11, P

Purpose We evaluated DNA polymorphisms in genes related to DNA repair, cell-cycle control and tumour microenvironment to determine possible associations with response and survival in neoadjuvant-treated gastric cancer patients. Patients and methods One hundred and seventy eight patients who received platinum/5FU-based chemotherapy were genotyped for 10 polymorphisms in nine genes ( ERCC1 : Asn118Asn, C > T ; ERCC1 : 8092C>A; TP53 : Arg72Pro, G ; cyclinD1 : Pro241Pro, G > A ; STK15 : Phe31Ile, A > T ; VEGF : 936 C > T ; TNF-α : -308 G > A ; interleukin-1b (IL-1B) : -511 C > T ; IL-1 receptor antagonist (IL-1RN) : variable tandem repeat; IL-8 : -251 T > A ). Genotypes were correlated with histopathological and clinical response and overall (OS) and progression-free survival (PFS). Results Only the cyclinD1 genotypes were associated with clinical response ( P χ 2 = 0.044 ). Significantly worse survival rates were noted in patients homozygous for the G-allele as compared to patients with the AG or AA genotypes of the cyclinD1 polymorphism (OS: P log-rank =0.024; PFS: P log-rank =0.007) and in patients homozygous for the short allele compared to all other genotypes at the IL-1RN polymorphic locus (OS: P log-rank =0.026; PFS: P log-rank =0.013). The combination of both unfavourable genotypes demonstrated strong prognostic relevance (OS: P log-rank =0.006; PFS: P log-rank =0.001). Multivariate analysis for OS in the group of completely resected patients ( n =139) revealed statistical significance for ypM ( P P cyclinD1/IL-1RN genotypes ( P =0.043). Conclusion The cyclinD1 and IL-1RN polymorphisms were associated with survival. The combination of specific cyclinD1 and IL-1RN genotypes showed a particular prognostic relevance and should be considered an independent prognostic marker for neoadjuvant-treated gastric cancer patients.

Promoter hypermethylation occurs in various tumors and leads to silencing of tumor-relevant genes. Thus, promoter methylation analysis (MA) has been established as an important tool in cancer research and diagnostics. Here we present MethyQESD (methylation-quantification of endonuclease-resistant DNA) as a fast, easy, precise and reliable method for quantitative MA without the need of bisulfite-treatment or fluorescent probes. Though MethyQESD principally works with any gene promoter we established MethyQESD for the mismatch repair gene MLH1 and tested its utility to differentiate between sporadic microsatellite unstable (MSI-H) colorectal cancer and hereditary nonpolyposis colorectal cancer (HNPCC) by quantitative MLH1 MA. We investigated formalin-fixed and paraffin-embedded tissue samples from a previously published, well-characterized tumor collective comprising 25 HNPCC, 14 sporadic MSI-H CRC and 16 sporadic microsatellite stable (MSS) CRC. We found a high accuracy of MethyQESD by spiking experiments with dilution series of methylated (SW48 cancer cell line) and unmethylated (blood) DNA (Pearson's r=0.9997 (proximal MLH1 promoter region), r=0.9976 (distal MLH1 promoter region)). MethyQESD and conventional quantitative MA using of 96 formalin-fixed and paraffin-embedded CRC showed a high degree of concordance of both methods (Pearson's r=0.885). HNPCC tumors showed either null MLH1 methylation or a significantly lower degree of MLH1 methylation than sporadic MSI-H CRC (P

Neoadjuvant chemotherapy in gastric cancer is now standard in the Western world; however, only 30–40% of the patients respond to induction therapy. Pretherapeutic predictors of response and prognosis would be of utmost interest to individualize treatment. Glutathione-S-transferase enzymes detoxify therapeutic drugs such as platin derivates and may influence outcome of the treated patients. Therefore, glutathione-S-transferase (GST) polymorphisms were assessed as predictive markers in cisplatinum-based neoadjuvant-treated gastric cancer. DNA was isolated from 139 patients with locally advanced gastric cancer (cT3/4 anyN cM0) before chemotherapy. Multiplex polymerase chain reaction was used for GSTT1 and GSTM1 genes, and allelic discrimination assay with the TaqMan system for the GSTP1 gene. One hundred ten patients could be analyzed for GSTT1 (T-:23; T + 87), 112 for GSTM1 (M-:52; M +:60) and 132 for GSTP1 (Ile/Ile: 55; Ile/Val: 59; Val/Val: 18). There was no significant correlation between any of the GSTT1, GSTM1, or GSTP1 genotypes and patients’ characteristics or histopathological data; only the GSTM1+ genotype was associated with the non-intestinal subtype of the Lauren classification (p = 0.045). GSTT1, GSTM1, and GSTP1 genotypes were not correlated with response to chemotherapy (p = 0.57, p = 0.38, p = 0.33). In R0 resected patients, we found an improved survival for patients with the GSTM1-present genotype compared to patients with the GSTM1-null genotype (p = 0.017). Moreover, the GSTM1-present genotype showed a significantly better tumor-related (p = 0.017) and disease-free survival (p = 0.029). None of the common GST polymorphisms predicts response in our study, but the GSTM1+ genotype was associated with a better prognosis in completely resected patients. Further investigations on chemotherapy-associated gene polymorphisms are warranted.

The EPH/EFN family of receptor tyrosine kinases regulates cell adhesion and migration and has an important role in controlling cell positioning in the normal intestinal epithelium. Inactivation of EPHB2 has recently been shown to accelerate tumorigenesis in the colon and rectum, and we have previously demonstrated frequent frameshift mutations (41%) in an A9 coding microsatellite repeat in exon 17 of EPHB2 in colorectal tumors with microsatellite instability (MSI). In this study, we extended these analyses to extracolonic MSI cancers, and found frameshift EPHB2 mutations in 39% (25/64) of gastric tumors and 14% (8/56) of endometrial tumors. Regression analysis of these EPHB2 mutation data on the basis of our previously proposed statistical model identified EPHB2 as a selective target of frameshift mutations in MSI gastric cancers but not in MSI endometrial carcinomas. These results suggest a functional role for EPHB2 in gastric tumor progression, and emphasize the differences between the tumorigenic processes in MSI gastrointestinal and endometrial cancer.

BACKGROUND NOTCH signaling can exert oncogenic or tumor suppressive functions and can contribute to chemotherapy resistance in cancer. In this study, we aimed to clarify the clinicopathological significance and the prognostic and predictive value of NOTCH1 and NOTCH2 expression in gastric cancer (GC). METHODS NOTCH1 and NOTCH2 expression was determined immunohistochemically in 142 primarily resected GCs using tissue microarrays and in 84 pretherapeutic biopsies from patients treated by neoadjuvant chemotherapy. The results were correlated with survival, response to therapy, and clinico-pathological features. RESULTS Primarily resected patients with NOTCH1-negative tumors demonstrated worse survival. High NOTCH1 expression was associated with early-stage tumors and with significantly increased survival in this subgroup. Higher NOTCH2 expression was associated with early-stage and intestinal-type tumors and with better survival in the subgroup of intestinal-type tumors. In pretherapeutic biopsies, higher NOTCH1 and NOTCH2 expression was more frequent in non-responding patients, but these differences were statistically not significant. CONCLUSION Our findings suggested that, in particular, NOTCH1 expression indicated good prognosis in GC. The close relationship of high NOTCH1 and NOTCH2 expression with early tumor stages may indicate a tumor-suppressive role of NOTCH signaling in GC. The role of NOTCH1 and NOTCH2 in neoadjuvantly treated GC is limited.

Purpose Lynch syndrome is linked to germline mutations in mismatch repair genes. We analyzed the genotype-phenotype correlations in the largest cohort so far reported. Patients and Methods Following standard algorithms, we identified 281 of 574 unrelated families with deleterious germline mutations in MLH1 (n = 124) or MSH2 (n = 157). A total of 988 patients with 1,381 cancers were included in this analysis. Results We identified 181 and 259 individuals with proven or obligatory and 254 and 294 with assumed MLH1 and MSH2 mutations, respectively. Age at diagnosis was younger both in regard to first cancer (40 v 43 years; P < .009) and to first colorectal cancer (CRC; 41 v 44 years; P = .004) in MLH1 (n = 435) versus MSH2 (n = 553) mutation carriers. In both groups, rectal cancers were remarkably frequent, and the time span between first and second CRC was smaller if the first primary occurred left sided. Gastric cancer was the third most frequent malignancy occurring without a similarly affected relative in most cases. All prostate cancers occurred in MSH2 mutation carriers. Conclusion The proportion of rectal cancers and shorter time span to metachronous cancers indicates the need for a defined treatment strategy for primary rectal cancers in hereditary nonpolyposis colorectal cancer patients. Male MLH1 mutation carriers require earlier colonoscopy beginning at age 20 years. We propose regular gastric surveillance starting at age 35 years, regardless of the familial occurrence of this cancer. The association of prostate cancer with MSH2 mutations should be taken into consideration both for clinical and genetic counseling practice.

CCND1 encodes cyclin D1, which plays an important role in the G 1 to S phase transition of the cell cycle. A common polymorphism (c.G870A) increases alternate splicing. Hereditary nonpolyposis colorectal cancer (HNPCC) is caused by mutations in mismatch repair (MMR) genes, mainly MSH2 and MLH1 , and shows a wide range in the age of its onset (AO), suggesting the existence of other modifying genetic factors. To date, two studies have investigated the association between CCND1 G/A variation and AO in HNPCC with contradictory results in 86 and 146 MMR mutation carriers, respectively. To clarify the role of the CCND1 G/A variation in HNPCC, we performed a study in 406 individuals carrying exclusively clear cut pathogenic mutations in MSH2 or MLH1 . We did not observe a significant difference in genotype frequencies of affected and unaffected mutation carriers and healthy controls. A significant association between CCND1 genotypes and AO was found neither in the global comparison (log-rank, P =0.2981; Wilcoxon, P =0.2567) nor in a multivariate Cox regression analysis (hazard ratios 1.111, 95%CI 0.950–1.299, P =0.188 and 1.090, 95%CI 0.868–1.369, P =0.459 for the additive and dominant effect, respectively). We conclude, that the CCND1 G870A sequence variation is not a genetic modifier of the phenotype of HNPCC.

In hereditary nonpolyposis colorectal cancer (HNPCC) syndrome, more than 90% of the carcinomas show microsatellite instability (MSI) due to a loss of mismatch repair (MMR) function. Although adenomas are very common in HNPCC and demonstrate an accelerated adenoma-carcinoma sequence, data about the prevalence and development of MSI in these early neoplastic lesions are lacking. To determine whether MSI and loss of MMR-protein expression are already present in early stages of tumorigenesis and could therefore be used as a screening tool to identify HNPCC patients before they develop an invasive carcinoma, we analyzed 71 adenomas of 36 HNPCC patients during a 5-year follow-up study. These 36 patients were part of a cohort of 122 HNPCC patients who were investigated at the Institute of Pathology, Klinikum Kassel, as part of the multicentric German HNPCC Consortium, which currently serves more than 2,880 registered families. The diagnosis of HNPCC was based either on the detection of a pathogenic germline mutation in the MSH2, MLH1, or MSH6 genes or in cases where a pathogenic mutation was not found; diagnosis of HNPCC was made, because all patients fulfilled the Amsterdam or Bethesda criteria and revealed a high degree of MSI (MSI-H) as well as loss of one of the MMR proteins by IHC in the cancer tissue. We found that most adenomas (58/71) were MSI-H and had loss of MMR-protein expression. Of the 71 adenomas, 3 were MSI-H with expression of all MMR proteins, and 3 out of 71 displayed loss of a MMR protein with the microsatellites being classified as microsatellite stable (MSS). However, 7 of the 31 adenomas that were located more than 5 cm away from the carcinoma revealed an MSS status (n=6) or low in MSI (n=1) and expressed all MMR proteins. In summary, a significant percentage of HNPCC-associated adenomas (7/31, 22.6%) developing at a distance of more than 5 cm from the corresponding carcinoma did not show the MSI-H MMR-deficient phenotype and expressed all MMR genes. To our knowledge, this is the first study that shows that in most HNPCC patients, the mutator pathway is already detectable in adenomas, but MMR-proficient adenomas can also be found. Therefore, screening for MMR deficiency should not be applied routinely in adenomas with the goal to identify HNPCC patients.

We evaluated DNA polymorphisms in the thymidylate synthase (TS) and 5,10-methylene-tetrahydrofolate reductase (MTHFR) genes for an association with response and survival in locally advanced gastric cancer treated with 5-FU based preoperative chemotherapy (CTx). DNA of 238 patients (CTx-group: total n = 135, completely resected (R0) n = 102; without CTx: R0 n = 103) was isolated from blood or from nontumorous tissues. In the CTx-group, genotyping of the tandem repeat and the G/C polymorphism in the triple repeat in the promoter region of the TS gene and of the C677T polymorphism of the MTHFR gene was performed. None of the TS or MTHFR genotypes were associated with histopathological response and only the TS tandem repeat polymorphism was significantly related to survival (all patients n = 135, p = 0.002; R0 resected patients n = 102, p = 0.007; log-rank test). Multivariate analysis revealed ypN (p0.001) and the TS tandem repeat polymorphism as independent prognostic factors in the CTx-R0-group (p = 0.003). Analyzing the prognostic significance of the TS polymorphisms in the R0-group without CTx, TS genotypes were not significantly associated with survival. Comparing survival between R0 patients with and without CTx in the respective TS genotype groups of the tandem repeat polymorphism, a significant survival benefit for the patients with CTx was found for the 2rpt/2rpt (n = 49; p = 0.002) and 2rpt/3rpt genotypes (n = 99; p = 0.004), but not for the 3rpt/3rpt genotype (n = 57; p = 0.93). Patients' survival after CTx was associated with the TS tandem repeat polymorphism. CTx did not improve survival of patients with the 3rpt/3rpt genotype. Thus, a different therapy might be more appropriate for these patients.

Hereditary non-polyposis colorectal cancer (HNPCC) is clinically defined by familial clustering of colorectal cancer and other associated tumours.By thorough molecular and clinical evaluation of 41 families, two different groups were characterised: group 1, 25 families with truncating mutations in MLH1 or MSH2 (12 novel mutations); and group 2, 16 Amsterdam positive families without mutations in these genes and without microsatellite instability in their corresponding tumours.Significant clinical differences between these two groups were found. Firstly, earlier age of onset for all colorectal cancers (median 41 v 55 years; p0.001) and all tumours (median 43 v 56 years; p = 0.022) was observed, comparing groups 1 and 2. Secondly, 68% of the index colorectal cancers were localised proximally of the splenic flexure in group 1 compared with 14% in group 2 (p0.010). Thirdly, more synchronous and metachronous colorectal (p = 0.017) and extracolorectal tumours (p0.001) were found in group 1. Fourthly, a higher colorectal adenoma/carcinoma ratio (p = 0.030) and a tendency towards more synchronous or metachronous adenomas in group 2 (p = 0.084) was observed, indicating a slower progression of adenomas to carcinomas. As three mutation negative tumours revealed chromosomal instability after comparative genomic hybridisation, these tumours may be caused by one or more highly penetrant disease alleles from the chromosomal instability pathway.These data show that HNPCC includes at least two entities with clinical and molecular differences. This will have implications for surveillance programmes and for cancer research.

Alterations of the cell adhesion molecule E-cadherin have been demonstrated in sporadic and hereditary gastric carcinomas. A cell adhesion molecule with functional similarity to E-cadherin is desmoglein 2 (Dsg2), a major component of the desmosomes. In this study, we investigated whether alterations of Dsg2 are involved in gastric carcinogenesis and whether germline mutations contribute to a genetic predisposition in familial gastric cancer patients with no germline mutations in the E-cadherin gene. Seventy-five formalin-fixed, paraffin-embedded tissues from 37 familial and 38 sporadic gastric carcinomas were analysed for Dsg2 expression by immunohistochemistry. DNA from 31 familial gastric cancer patients was analysed for germline mutations and five sporadic tumours were analysed for somatic mutations by DHPLC. Of the 75 tumours, 25 (33%) demonstrated abnormal (reduced and/or non-membrane-associated) Dsg2 expression. There was a trend towards more frequent abnormal expression in diffuse type (42%) than in intestinal type tumours (18%) (p = 0.066). One germline missense variant leading to a non-conservative amino acid change (c. 2810 C > A, Thr 937 Asn) was found in a familial gastric cancer patient with a diffuse type tumour. No somatic mutations were identified. The observed abnormal expression of Dsg2 protein suggests that this molecule is involved in the carcinogenesis of a subset of gastric carcinomas, in particular of the diffuse type. Somatic mutations in the gene do not seem to be a very frequent inactivation event and the finding of no clear pathogenic germline mutation rules out Dsg2 as a major gastric cancer predisposition gene.

Clinical criteria, microsatellite analysis (MSA) and immunohistochemistry (IHC) are important diagnostic tools for identification of hereditary nonpolyposis colorectal cancer (HNPCC) patients who are likely to carry pathogenic germline mutations in mismatch repair genes. Based on MSA and IHC results and subsequent mutation analyses of 1,119 unrelated index patients meeting the Amsterdam II criteria or the classical Bethesda guidelines, we analyzed the value of these tools to predict MLH1 and MSH2 mutations with the aim of establishing optimal strategies for their most efficient sequential use. The overall prevalence of pathogenic germline mutations in our cohort was 20.6% (95% CI = 18.3-23.0%) and 61.8% (95% CI = 56.8-66.6%), respectively, after MSA/IHC-based preselection. IHC was highly predictive (99.1%) and specific (99.6%) with regard to MSA. However, 14 out of 230 mutations (6%) escaped detection by IHC. Thus, IHC cannot be recommended to substitute MSA fully. Nonetheless, IHC is important to indicate the gene that is likely to be affected. To combine both methods efficiently, we propose a novel screening strategy that provides 2 alternative ways of sequential IHC and MSA application, either using IHC or MSA in the first place. A logistic regression model based on the age of the index patient at first tumor diagnosis and the number of fulfilled HNPCC criteria is used to allocate individual patients to that alternative pathway that is expected to be least expensive. A cost analysis reveals that about 25% of the costs can be saved using this strategy.

Although twin studies indicate that inherited genetic factors contribute to about 35% of colorectal cancers (CRC), the exact genetic background has currently been elucidated in only 5-10% of cases. These comprise several hereditary cancer predisposition syndromes that present with a high number of syn- or metachronous neoplasms within an affected person and/or family. Many of these tumors exhibit typical histopathological changes. In general, one should discriminate between cancer syndromes associated with adenomatous and non-adenomatous (i.e., hamartomatous) polyps, the latter being quite rare. The patient's age often serves as a substantial hint to hereditary cancer. The next step of diagnostic work-up includes analysis of microsatellite instability (MSI) together with immunohistochemical detection of a loss of expression in one of the most frequently affected mismatch repair genes (MSH2, MSH6; MLH1, PMS2). Finally, the molecular demonstration of a gene mutation in the blood or germline is the most expensive and tedious procedure. This requires a signed informed consent from the patient after appropriate genetic counseling.

E-cadherin mutations are found in 50% of diffuse-type gastric carcinoma, but not in intestinal gastric carcinoma. Because cell-cell adhesion mediated by E-cadherin plays an important role in epithelial cell survival, E-cadherin mutations could alter the apoptotic behavior of tumor cells. p53 and Bcl-2 family members are also important regulators of cellular apoptosis. This is the first study that investigates the relationship between E-cadherin gene mutation and p53 gene mutation, p53 accumulation, Bcl-2 expression, and Ki-67 expression in diffuse-type gastric carcinoma (24 cases, E-cadherin mutation status: wild-type in 8 patients and mutant in 16 patients). The mutation status of exons 5-8 of p53 was analyzed by denaturing high pressure liquid chromatography (DHPLC) in formalin-fixed, paraffin-embedded tumor sections, followed by direct sequencing of cases with aberrant chromatographic patterns. p53 mutations were found in 1 of 8 tumors without E-cadherin mutation (12.5%) and in 1 of 16 tumors with E-cadherin mutation (6.3%), a difference that was not statistically significant (p = 1.00). p53 accumulation was found in 8 of 24 tumors (33.3%) by immunohistochemical staining. p53 accumulation was significantly more frequent in tumors without E-cadherin mutations (5 of 8 tumors, 62.5%) than in gastric carcinoma tissues with E-cadherin mutations (3 of 16 tumors, 18.8%, p = 0.03). Bcl-2 staining was not observed in gastric carcinoma cells without E-cadherin mutations, but was detectable in 5 of 16 tumors with E-cadherin mutations (31.3%), a difference that was not statistically significant (p = 0.13). No relationship was observed between Ki-67 staining and the E-cadherin mutation status (p = 1.00). These data suggest that the presence of E-cadherin mutations can significantly alter the accumulation of the apoptosis-regulating p53 protein, whereas no correlation with the p53 mutation status or with Ki-67 staining was observed.

Background Methylentetrahydrofolate reductase (MTHFR) plays a major role in folate metabolism and consequently could be an important factor for the efficacy of a treatment with 5-fluorouracil. Our aim was to evaluate the prognostic and predictive value of two well characterized constitutional MTHFR gene polymorphisms for primarily resected and neoadjuvantly treated esophagogastric adenocarcinomas. Methods 569 patients from two centers were analyzed (gastric cancer: 218, carcinoma of the esophagogastric junction (AEG II, III): 208 and esophagus (AEG I): 143). 369 patients received neoadjuvant chemotherapy followed by surgery, 200 patients were resected without preoperative treatment. The MTHFR C677T and A1298C polymorphisms were determined in DNA from peripheral blood lymphozytes. Associations with prognosis, response and clinicopathological factors were analyzed retrospectively within a prospective database (chi-square, log-rank, cox regression). Results Only the MTHFR A1298C polymorphisms had prognostic relevance in neoadjuvantly treated patients but it was not a predictor for response to neoadjuvant chemotherapy. The AC genotype of the MTHFR A1298C polymorphisms was significantly associated with worse outcome (p = 0.02, HR 1.47 (1.06-2.04). If neoadjuvantly treated patients were analyzed based on their tumor localization, the AC genotype of the MTHFR A1298C polymorphisms was a significant negative prognostic factor in patients with gastric cancer according to UICC 6th edition (gastric cancer including AEG type II, III: HR 2.0, 95% CI 1.3-2.0, p = 0.001) and 7th edition (gastric cancer without AEG II, III: HR 2.8, 95% CI 1.5-5.7, p = 0.003), not for AEG I. For both definitions of gastric cancer the AC genotype was confirmed as an independent negative prognostic factor in cox regression analysis. In primarily resected patients neither the MTHFR A1298C nor the MTHFR C677T polymorphisms had prognostic impact. Conclusions The MTHFR A1298C polymorphisms was an independent prognostic factor in patients with neoadjuvantly treated gastric adenocarcinomas (according to both UICC 6th or 7th definitions for gastric cancer) but not in AEG I nor in primarily resected patients, which confirms the impact of this enzyme on chemotherapy associated outcome.

To characterize cytogenetic alterations found in Barrett's adenocarcinoma (BA) and, more importantly, its premalignant stages, we studied chromosomal imbalances in various lesions in the histologically proposed metaplasia-dysplasia-carcinoma sequence using comparative genomic hybridization (CGH). Using 30 esophageal adenocarcinoma resection specimens, we were able to study 30 areas of Barrett's adenocarcinoma and 8 lymph node metastases (LN). In addition, we investigated 25 premalignant lesions adjacent to BA derived from a subset of 14 resection specimens including 11 areas of high grade dysplasia (HGD), 8 areas of low grade dysplasia (LGD), and 6 areas of intestinal metaplasia (IM), which were laser-microdissected and studied with CGH. To validate the CGH findings, fluorescence in situ hybridization analysis on 13 BA with probes specific for HER-2/neu and 20q13.2 were performed. The chromosomal alterations most often identified in BA were: gains on 8q (80%), 20q (60%), 2p, 7p and 10q (47% each), 6p (37%), 15q (33%) and 17q (30%). Losses were observed predominantly on the Y-chromosome (76%), 4q (50%), 5q and 9p (43% each), 18q (40%), 7q (33%) and 14q (30%). High-level amplifications were observed on 8q23-qter, 8p12-pter, 7p11-p14, 7q21-31, 17q11-q23. Recurrent chromosomal changes were also identified in metaplastic (gains on 8q, 6p, 10q, losses on 13q, Y, 9p) and dysplastic epithelium (gains on 8q, 20q, 2p, 10q, 15q, losses on Y, 5q, 9p, 13q, 18q). Novel amplified chromosomal regions on chromosomes 2p and 10q were detected in both Barrett's adenocarcinoma and premalignant lesions. An increase of the average number of detected chromosomal imbalances from IM (7.0 +/- 1.7), to LGD (10.8 +/- 2.2), HGD (13.4 +/- 1.1), BA (13.3 +/- 1.4), and LN (22 +/- 1.2) was seen. Although the detection of common chromosomal alterations in premalignant lesions and adjacent carcinomas suggest a process of clonal expansion, the occurrence of several chromosomal changes in an apparently random order relative to one another is striking evidence that clonal evolution is more complex than would be predicted by linear models. This is probably a reflection of the existence of many divergent neoplastic subpopulations and highlights one of the main problems associated with surveillance of Barrett's patients, namely sampling error.

BACKGROUND The somatogenetic alterations that lead to the development of benign adenomas, borderline tumors, and invasive ovarian carcinomas are not well understood. This study investigated allelic losses in these three types of ovarian tumors. METHODS Twelve genetic regions on chromosomes 2q, 5q, 6p, 6q, 9p, 11q, 17p, 17q, 18q, and 22q were analyzed by polymerase chain reaction for loss of heterozygosity (LOH). The study was performed on formalin fixed, paraffin embedded tissue samples from 72 invasive ovarian carcinomas, 35 ovarian tumors of borderline malignancy, and 25 benign ovarian adenomas. RESULTS LOH was found in only 8% of the analyzed adenomas (at 2q21 and 17p13) and 11% of the borderline tumors (at 2q21, 5q21, 6p21, 17p13, and 17q21). Allelic losses were noted for 77.7% of all of the invasive carcinomas analyzed. The frequency of LOH was 56% at a locus near 17p13 (TP53) and 40.5% at 17q21 (BRCA1). LOH was observed in 30.4% of informative cases at 5q21 and in 21.4% at chromosome 18q21. The chromosomal regions 2q21-22 and 9p21 had deletions at frequencies of 32.4% and 25%, respectively. International Federation of Gynecology and Obstetrics stage and the presence of LOH correlated significantly with survival but were not independent predictors of survival. Serous subtypes of invasive carcinoma were significantly more prone to deletions than nonserous tumors. CONCLUSIONS The results of this study suggest that LOH occurs only occasionally in adenomas and borderline tumors, whereas it is frequently observed in invasive ovarian carcinomas. Therfore, other genetic events seem to be involved in early ovarian tumor development. However, the presence of multiple allelic losses is an indicator of higher stages of invasive carcinoma. Cancer 1997; 80:1241-9. © 1997 American Cancer Society.

Muir-Torre syndrome (MTS) is an autosomal dominant disorder characterized by the presence of at least one sebaceous gland tumour and a minimum of one visceral malignant tumour. Recently, microsatellite instability (MSI) has been detected in the tumours of patients with MTS and germline mutations of the hMSH2 and hMLH1 mismatch repair genes have been detected in some patients with this syndrome. To determine if the tumours of patients with MTS have widespread genomic instability and whether loss of heterozygosity (LOH) in the chromosomal regions containing hMSH2 and hMLH1 is detectable, MSI and LOH were examined at 10 dinucleotide repeats on chromosomes 2p, 3p, 5q, 9p, 17p and 18q. Data were obtained from six sebaceous gland tumours and two adenocarcinomas of the colon from three patients of two Muir-Torre families. MSI was detected at more than half of the loci tested in all sebaceous tumours examined. In addition, there was LOH at D2S119 in one sebaceoma and one sebaceous carcinoma from one patient. The colon carcinomas from two patients showed MSI at five of the 10 loci analysed. These results show that widespread MSI is a feature of tumours in patients with MTS. In addition, the finding of LOH at D2S119, a marker located in the vicinity of hMSH2, in sebaceous tumours of one patient indicates that this gene may have a pathogenetic role in this patient.

We analyzed microsatellite instability (MSI) and loss of heterozygosity (LOH) at 17 microsatellite markers located on chromosomes 2p, 3p, 5q, 6q, 9p, 9q, 17p and 18q in 19 randomly selected keratoacantomas (KAS), in one cutaneous lesion that histologically could not unequivocally be differentiated from squamous cell carcinoma, and in one patient with multiple KAs of longstanding duration. The goals of our study were to determine whether, in a similar manner to some visceral carcinomas, genomic instability could be detected in KAs and to clarify whether molecular analysis might be useful to further characterize KA. MSI was observed in 2 of 21 cases (9.5%) at 5 of 17 loci examined. In one patient with a solitary KA, the presence of MSI and a family history of visceral malignant tumours suggested that the patient might have belonged to a family with Muir-Torre syndrome. In one other MSI+ KA, a definite differential diagnosis in relation to squamous cell carcinoma could not be established. In addition, one sample displayed LOH at 2 of 17 loci analysed whereas in the patient with multiple KAs, LOH at one locus was the only alteration found. In conclusion, the low frequency of MSI and LOH detected in our study suggests that these genetic events are uncommon in KA unless it is associated with a familial disease (e.g. Muir-Torre syndrome) or it has more aggressive histological features.

The hallmarks of Lynch syndrome (LS) include a positive family history of colorectal cancer (CRC), germline mutations in the DNA mismatch repair (MMR) genes, tumours with high microsatellite instability (MSI-H) and loss of MMR protein expression. However, in ∼10–15% of clinically suspected LS cases, MMR mutation analyses cannot explain MSI-H and abnormal immunohistochemistry (IHC) results. The highly variable phenotype of MUTYH-associated polyposis (MAP) can overlap with the LS phenotype, but is inherited recessively. We analysed the MUTYH gene in 85 ‘unresolved’ patients with tumours showing IHC MMR-deficiency without detectable germline mutation. Biallelic p.(Tyr179Cys) MUTYH germline mutations were found in one patient (frequency 1.18%) with CRC, urothelial carcinoma and a sebaceous gland carcinoma. LS was suspected due to a positive family history of CRC and because of MSI-H and MSH2–MSH6 deficiency on IHC in the sebaceous gland carcinoma. Sequencing of this tumour revealed two somatic MSH2 mutations, thus explaining MSI-H and IHC results, and mimicking LS-like histopathology. This is the first report of two somatic MSH2 mutations leading to an MSI-H tumour lacking MSH2–MSH6 protein expression in a patient with MAP. In addition to typical transversion mutations in KRAS and APC, MAP can also induce tumourigenesis via the MSI-pathway.

Somatic CDH1 mutations are found in sporadic diffuse type gastric and lobular breast carcinomas. Germline mutations of the CDH1 gene are the molecular genetic cause of hereditary diffuse gastric cancer. CDH1 in this context acts as a classical tumor suppressor gene and lobular breast carcinomas are observed in association with CDH1 germline mutations in hereditary diffuse gastric cancer ­families. Although some families with a preponderance or even single occurrence of breast carcinomas and a CDH1 germline mutation have been described, the ­incidence of CDH1 germline mutations in breast cancer families without the ­presentation of diffuse type gastric cancer is low.

We examined 30 gastric-cancer patients with a varying degree of family history of stomach cancer and/or synchronous gastric tumors for microsatellite instability. We observed microsatellite instability at at least 1 of 8 loci tested in tumors of 14/30 patients; of these 14, 8 had single locus alterations and 6 had alterations at at least half of the 8 loci. Among the patients with microsatellite instability at > or = 4 loci, 3 patients showed a strong familial clustering of gastric cancer. Mutation analysis of the DNA mismatch repair gene hMLHl on paired non-tumorous and tumor DNA from 10 patients, 6 with microsatellite instability at > or = 4 loci and 4 with an alteration at one locus, revealed a novel missense mutation, present in the normal and tumor DNA of one patient with microsatellite instability at multiple loci in his tumor. His family history of cancer included one second-degree relative affected with gastric cancer. These data suggest that germline mutations in the hMLHl gene occur in some gastric-cancer patients and that in the majority of cases microsatellite instability in gastric tumors may be due to defects in other genes responsible for DNA replication fidelity than the hMLHl.

Cancer stem cell (CSC) based gene expression signatures are associated with prognosis in various tumour types and CSCs are suggested to be particularly drug resistant. The aim of our study was first, to determine the prognostic significance of CSC-related gene expression in residual tumour cells of neoadjuvant-treated gastric cancer (GC) patients. Second, we wished to examine, whether expression alterations between pre- and post-therapeutic tumour samples exist, consistent with an enrichment of drug resistant tumour cells. The expression of 44 genes was analysed in 63 formalin-fixed, paraffin embedded tumour specimens with partial tumour regression (10-50% residual tumour) after neoadjuvant chemotherapy by quantitative real time PCR low-density arrays. A signature of combined GSK3B(high), beta-catenin (CTNNB1)(high) and NOTCH2(low) expression was strongly correlated with better patient survival (p < 0.001). A prognostic relevance of these genes was also found analysing publically available gene expression data. The expression of 9 genes was compared between pretherapeutic biopsies and post-therapeutic resected specimens. A significant post-therapeutic increase in NOTCH2, LGR5 and POU5F1 expression was found in tumours with different tumour regression grades. No significant alterations were observed for GSK3B and CTNNB1. Immunohistochemical analysis demonstrated a chemotherapy-associated increase in the intensity of NOTCH2 staining, but not in the percentage of NOTCH2. Taken together, the GSK3B, CTNNB1 and NOTCH2 expression signature is a novel, promising prognostic parameter for GC. The results of the differential expression analysis indicate a prominent role for NOTCH2 and chemotherapy resistance in GC, which seems to be related to an effect of the drugs on NOTCH2 expression rather than to an enrichment of NOTCH2 expressing tumour cells.

The therapeutic activity of the epidermal growth factor receptor (EGFR)-directed monoclonal antibody cetuximab in gastric cancer is currently being investigated. Reliable biomarkers for the identification of patients who are likely to benefit from the treatment are not available. The aim of the study was to examine the drug sensitivity of five gastric cancer cell lines towards cetuximab as a single agent and to establish predictive markers for chemosensitivity in this cell culture model. The effect of a combination of cetuximab with chemotherapy was compared between a sensitive and a nonsensitive cell line.EGFR expression, activation and localisation, the presence and subcellular localisation of the cell adhesion molecule E-cadherin as well as MET activation were examined by Western blot analysis, flow cytometry and immunofluorescence staining. Cells were treated with varying concentrations of cetuximab and cisplatin and 5-fluorouracil in tumour-relevant concentrations. The biological endpoint was cell viability, which was measured by XTT cell proliferation assay. Response to treatment was evaluated using statistical methods.We assessed the activity of cetuximab in five gastric cancer cell lines (AGS, KATOIII, MKN1, MKN28 and MKN45). The viability of two cell lines, MKN1 and MKN28, was significantly reduced by cetuximab treatment. High EGFR expression and low levels of receptor activation were associated with cetuximab responsiveness. MET activation as well as mutations of KRAS and CDH1 (gene encoding E-cadherin) was associated with cetuximab resistance.These data indicate that our examinations may be clinically relevant, and the candidate markers should therefore be tested in clinical studies.

BACKGROUND: Chemotherapy for advanced colorectal cancer leads to improved survival; however, predictors of response to systemic treatment are not available. Genomic and epigenetic alterations of the gene encoding transcription factor AP-2 epsilon (TFAP2E) are common in human cancers. The gene encoding dickkopf homolog 4 protein (DKK4) is a potential downstream target of TFAP2E and has been implicated in chemotherapy resistance. We aimed to further evaluate the role of TFAP2E and DKK4 as predictors of the response of colorectal cancer to chemotherapy. METHODS: We analyzed the expression, methylation, and function of TFAP2E in colorectal-cancer cell lines in vitro and in patients with colorectal cancer. We examined an initial cohort of 74 patients, followed by four cohorts of patients (total, 220) undergoing chemotherapy or chemoradiation. RESULTS: TFAP2E was hypermethylated in 38 of 74 patients (51%) in the initial cohort. Hypermethylation was associated with decreased expression of TFAP2E in primary and metastatic colorectal-cancer specimens and cell lines. Colorectal-cancer cell lines overexpressing DKK4 showed increased chemoresistance to fluorouracil but not irinotecan or oxaliplatin. In the four other patient cohorts, TFAP2E hypermethylation was significantly associated with nonresponse to chemotherapy (P

The therapeutic activity of the epidermal growth factor receptor (EGFR)-directed monoclonal antibody cetuximab in gastric cancer is currently being investigated in clinical studies. Reliable biomarkers for the identification of patients who are likely to benefit from this treatment are not available. In this study, we assessed the activity of cetuximab in five gastric cancer cell lines (AGS, AZ521, Hs746T, LMSU and MKN1). The viability of two of these cell lines, AZ521 and MKN1, was significantly reduced by cetuximab treatment. High expression and secretion levels of the EGFR-binding ligand, amphiregulin (AREG), were associated with cetuximab responsiveness. MET activation and mutations in Kirsten-Ras gene (KRAS) were associated with cetuximab resistance. By introducing a hierarchy between these markers, we established a model that facilitated the correct classification of all five gastric cancer cell lines as cetuximab responsive or non-responsive. The highest priority was allocated to activating KRAS mutations, followed by MET activation and finally by the levels of secreted AREG. In order to validate these results, we used three additional human gastric cancer cell lines (KATOIII, MKN28 and MKN45). In conclusion, we propose that our model allows the response of gastric cancer cell lines to cetuximab treatment to be predicted.

DNA repair plays an important role in chemoresistance to platinum-based therapy, and therefore polymorphisms in the genes may modulate therapeutic response. We assessed 12 polymorphisms in 7 DNA repair genes and 2 polymorphisms in the MTHFR gene for association with disease response and prognosis. A total of 258 patients included in the study had adenocarcinoma of the esophagus (n = 114) or gastric cancer (n = 144), at stage cT3/4 and cM0, and had been treated with platinum-based neoadjuvant polychemotherapy. The patients were genotyped for polymorphisms in the XPC, XPD, XPG, APEX, XRCC1, NBS1, XRCC3, and MTHFR genes by the allelic discrimination method and the data correlated with various clinical parameters. None of the investigated polymorphisms was associated with histopathological response. XRCC3 polymorphisms rs861539 (P = 0.02) and rs861530 (P = 0.05) showed association with clinical response in gastric cancer. The variants in XRCC3 (rs861539, P = 0.05; rs1799794, P = 0.03) and MTHFR (rs1801131, P = 0.02) were associated with survival in esophageal and gastric cancer, respectively. In R0 resected patients, XRCC3 variants (rs861539, P = 0.04; rs861530, P = 0.02) in esophageal cancer, and XRCC3 (rs1799794, P = 0.02) and MTHFR (rs1801131, P = 0.005) in gastric cancer predicted survival. Cox regression revealed ypT category (P = 0.001) and lymphatic vessel invasion (P = 0.03) to be independent prognostic factors for esophageal cancer, and histopathological response (P = 0.01), MTHFR variant (rs1801131, P = 0.002), and ypN category (P = 0.02) to be prognostic factors for gastric cancer. In gastric cancer patients, MTHFR variant (rs1801131) could serve as a potential prognostic marker. In esophageal cancer patients, none of the polymorphisms studied had conclusive results in multivariate analysis, although XRCC3 variant (rs861539) showed an effect on survival in Kaplan–Meier univariate analysis.

The MUNICON trial confirmed prospectively the usefulness of early response evaluation by 2-deoxy-2-[F-18]fluoro-D-glucose-positron emission tomography (FDG-PET) . Metabolic responders (R) showed initially a higher FDG uptake compared with nonresponders (p = 0.018). An association of the vascular endothelial growth factor (VEGF) 936C>T polymorphism and FDG uptake was reported for breast cancer. Therefore, we investigated the VEGF 936C>T polymorphism for an association with response and survival. The study was based on 110 patients included in the MUNCON trial (103 male, seven female; 75 AEG I, 35 AEG II, event-free survival (EFS) median 21.1 ± 4.6 months). Response was significantly associated with EFS. The VEGF 936C>T polymorphism was determined by PCR and restriction fragment length polymorphism analysis. For analysis, the T-variants were combined. One hundred two patients were evaluable. Seventy-two patients showed the CC, 24 the CT, and six the TT genotype. Median EFS was 29.3 months for CC and 11.7 months for CT/TT (p = 0.04). No association of the genotypes (CC or CT/TT) with the SUV or response was found. Multivariate analysis revealed histopathological regression (p = 0.003) and genotype (p = 0.04) as independent prognostic factors. A combination of genotype and PET response (Gen-PET) defines three prognostic groups early in the course of treatment (p = 0.002). Cox regression analysis including clinical and histopathological response and Gen-PET reveals Gen-PET as independent prognostic factor (p = 0.003). The VEGF 936C>T polymorphism is a prognostic factor in patients undergoing neoadjuvant chemotherapy, although it is not associated with FDG uptake and response. The combination of metabolic response and VEGF 936C>T polymorphism defines three different prognostic groups. These findings need to be confirmed prospectively. This study has been registered in the European Clinical Trials Database as trial 2007-003356-11.

Background: Cetuximab enhances the efficacy of chemotherapy in several cancer types. This trial assessed the activity of cetuximab and chemotherapy in advanced gastric cancer. Methods: Patients with previously untreated, metastatic, gastric cancer received cetuximab 400 mg m−2 at first infusion followed by weekly infusions of 250 mg m−2 combined with FUFOX (oxaliplatin 50 mg m−2, 5-FU 2000 mg m−2, and DL-folinic acid 200 mg m−2 d1, 8, 15 and 22 qd36). The primary endpoint was tumour response. Results: Overall, 52 patients were enrolled. The most common grade 3/4 toxicities were diarrhoea (33%), and skin toxicity (24%). Efficacy was evaluable in 46 patients who showed a response rate of 65% (CI 95%: 50–79%) including four complete responses. Time to progression (TTP) was 7.6 months (CI 95%: 5.0–10.1 months) and overall survival (OS) was 9.5 months (CI 95%: 7.9–11.1 months). Epidermal growth factor receptor (EGFR) was detectable in 60% of tumours but showed no correlation with treatment outcome. A KRAS mutation was found in only 1 of 32 (3%) tumour samples analysed. Conclusion: Cetuximab plus FUFOX showed an interesting high response rate in metastatic gastric cancer. Cetuximab plus platinum–fluoropyrimidine chemotherapy is at present being investigated in a phase III randomised controlled trial.

Contains fulltext : 88919.pdf (Publisher’s version ) (Closed access) Microsatellite instability (MSI) testing in clinics is becoming increasingly widespread; therefore, there is an urgent need for methodology standardization and the availability of quality control. This study is aimed to assess the interlaboratory reproducibility of MSI testing in archive samples by using a panel of 5 recently introduced, mononucleotide repeats (MNR). The quality control involved 8 European institutions. Participants were supplied with DNA extracted from 15 archive colon carcinoma samples and from the corresponding normal tissues. Every group was asked to assess the MSI status of the samples by using the BAT25, BAT26, NR21, NR24, and NR27 mononucleotide markers. Four institutions repeated the analysis using the NCI reference panel to confirm the results obtained with the MNR markers. The overall concordance among institutions for MSI analyses at single locus level was 97.7% when using the MNR panel and 95.0% with the NCI one. The laboratories obtained a full agreement in scoring the MSI status of each patient sample, both using the mononucleotide and the NCI marker sets. With the NCI marker set, however, concordance was lowered to 85.7% when considering the MSI-Low phenotype. Concordance between the 2 panels in scoring the MSI status of each sample was complete if no discrimination was made between MSI-Stable and MSI-L, whereas it dropped to 76.7% if MSI-L was considered. In conclusion, the use of the MNR panel seems to be a robust approach that yields a very high level of reproducibility. The results obtained with the 5 MNR are diagnostically consistent with those obtained by the use of the NCI markers, except for the MSI-Low phenotype. 01 december 2010

Background and aims Germline mutations in the E-cadherin (CDH1) gene have been found in families with hereditary diffuse gastric cancer (HDGC). These families are characterized by a highly penetrant susceptibility to diffuse gastric cancer with an autosomal dominant pattern of inheritance. We describe the clinical presentation of three sibling cases with advanced gastric cancer, the way of confirming the suspicion of underlying HDGC and the clinical management of the other healthy family members. Methods Screening for CDH1 germline mutation was carried out by denaturing high-performance liquid chromatography and automated DNA sequencing. The clinical suspicion of HDGC has been confirmed by identifying a frameshift mutation in exon 9 (1302_1303insA, 1306_1307delTT) of the E-cadherin gene. Results Eight of nine tested family members were positive for the CDH1 germline mutation. Prophylactic laparoscopic gastrectomies were performed in five mutation carriers. After pathological examination, we could identify intramucosal malignant signet-ring cell carcinoma in all resected stomachs. Conclusion This report underlines that prophylactic gastrectomy remains the only option to eliminate the high risk for gastric cancer in CDH1 mutation carriers.

Germline CDH1 point or small frameshift mutations can be identified in 30-50% of hereditary diffuse gastric cancer (HDGC) families. We hypothesized that CDH1 genomic rearrangements would be found in HDGC and identified 160 families with either two gastric cancers in first-degree relatives and with at least one diffuse gastric cancer (DGC) diagnosed before age 50, or three or more DGC in close relatives diagnosed at any age. Sixty-seven carried germline CDH1 point or small frameshift mutations. We screened germline DNA from the 93 mutation negative probands for large genomic rearrangements by Multiplex Ligation-Dependent Probe Amplification. Potential deletions were validated by RT-PCR and breakpoints cloned using a combination of oligo-CGH-arrays and long-range-PCR. In-silico analysis of the CDH1 locus was used to determine a potential mechanism for these rearrangements. Six of 93 (6.5%) previously described mutation negative HDGC probands, from low GC incidence populations (UK and North America), carried genomic deletions (UK and North America). Two families carried an identical deletion spanning 193 593 bp, encompassing the full CDH3 sequence and CDH1 exons 1 and 2. Other deletions affecting exons 1, 2, 15 and/or 16 were identified. The statistically significant over-representation of Alus around breakpoints indicates it as a likely mechanism for these deletions. When all mutations and deletions are considered, the overall frequency of CDH1 alterations in HDGC is approximately 46% (73/160). CDH1 large deletions occur in 4% of HDGC families by mechanisms involving mainly non-allelic homologous recombination in Alu repeat sequences. As the finding of pathogenic CDH1 mutations is useful for management of HDGC families, screening for deletions should be offered to at-risk families.

Germline mutations in mismatch repair (MMR) genes, tumours with high microsatellite instability (MSI-H) and loss of MMR protein expression are the hallmarks of HNPCC (Lynch syndrome). While somatic MLH1 promoter hypermethylation is generally accepted in the tumorigenesis of sporadic tumours, abnormal MLH1 promoter methylation in normal body cells is controversially discussed as a mechanism predisposing patients to HNPCC. In all 94 patients suspected of HNPCC-syndrome with a mean age of onset of 45.5 years, MLH1-deficiency in their tumours but no germline mutation, underwent methylation-specific PCR-screening for MLH1 promoter methylation. In peripheral blood cells of 12 patients an MLH1 promoter methylation, in seven informative cases allele-specific, was found. Normal colonic tissue, buccal mucosa, and tumour tissue available from three patients also presented abnormal methylation in the MLH1 promoter. The heredity of aberrant methylation is questionable. Pro: MLH1 promoter methylation was found in a patient and his mother giving evidence for a familial predisposition for an epimutation in MLH1. Contra: a de novo set-up of methylation in one patient, a mosaic or incomplete methylation pattern in six patients, and no evidence for inheritance of MLH1 promoter methylation in the remaining families. Our findings provide strong evidence that MLH1 promoter methylation in normal body cells mimics HNPCC and constitutes a pathogenic pre-lesion in MLH1. The identification of hypermethylation as an epigenetic defect has important implications for surveillance recommendations, as these patients should be treated like Lynch syndrome patients, whereas the heritability of methylation is still under investigation.

Ten non-synonymous single nucleotide polymorphisms (nsSNPs), which were recently associated with colorectal cancer risk in a comprehensive, array based study (AKAP9 M463I, DKK3 G335R, AMPD1 Q12X, LIPC L356F, PSMB9 V32I, THBS1 N700S, CA6 S90G, ASCC3 C1995S, DHX36 S416C and CPA4 G303C) were re-evaluated in the present study based on 626 German familial non-HNPCC colorectal cancer patients and 736 healthy controls. No associations of any of the 10 nsSNPs with colorectal cancer could be replicated. The combined analyses indicated that further research based on additional independent samples is required.

Purpose: The objective of this study was to analyze the hypermethylation of tumor-related gene promoters for an association with therapy response and clinicopathologic features of neoadjuvant-treated gastric cancer patients. Furthermore, we analyzed the relationship of promoter hypermethylation with microsatellite instability and loss of heterozygosity (LOH) of the tumors.Experimental Design: Pretherapeutic biopsies of 61 patients, subsequently treated with cisplatin and 5-fluorouracil, were studied. Methylation analysis of six gene promoters was done using MethyLight technology. Microsatellite analysis was mainly done in previous studies.Results: The methylation frequencies for the analyzed genes were MGMT, 44%; LOX, 53%; p16, 46%, E-cadherin, 30%; 14-3-3σ, 69%; and HPP1, 82%. Concordant methylation of more than three genes was found in 46% of the tumors and was inversely correlated with the LOH rate (P = 9 × 10−5) and associated with female gender (P = 0.049), nonintestinal type tumors (P = 0.04), and a nonproximal tumor location (P = 0.003). No statistically significant association between the methylation of a single gene or the concordant methylation of multiple genes was found with response or survival. However, patients with none or only one methylated gene showed a trend for an increase in survival (5-year survival rate, 83% versus 35%; P = 0.067).Conclusion: The highly significant inverse correlation of promoter methylation and LOH rate reflects major alternative molecular pathways in gastric carcinogenesis. Methylation was not statistically significantly associated with the response to cisplatin/5-fluorouracil–based therapy. However, a concordant methylation of more than three genes defines subgroups of gastric cancer with distinct biological and genetic characteristics.