Development of a prognostic risk model for clear cell renal cell carcinoma by systematic evaluation of DNA methylation markers.

BACKGROUND: Current risk models for renal cell carcinoma (RCC) based on clinicopathological factors are sub-optimal in accurately identifying high-risk patients. Here, we perform a head-to-head comparison of previously published DNA methylation markers and propose a potential prognostic model for clear cell RCC (ccRCC). PATIENTS AND METHODS: Promoter methylation of PCDH8, BNC1, SCUBE3, GREM1, LAD1, NEFH, RASSF1A, GATA5, SFRP1, CDO1, and NEURL was determined by nested methylation-specific PCR. To identify clinically relevant methylated regions, The Cancer Genome Atlas (TCGA) was used to guide primer design. Formalin-fixed paraffin-embedded (FFPE) tissue samples from 336 non-metastatic ccRCC patients from the prospective Netherlands Cohort Study (NLCS) were used to develop a Cox proportional hazards model using stepwise backward elimination and bootstrapping to correct for optimism. For validation purposes, FFPE ccRCC tissue of 64 patients from the University Hospitals Leuven and a series of 232 cases from The Cancer Genome Atlas (TCGA) were used. RESULTS: Methylation of GREM1, GATA5, LAD1, NEFH, NEURL, and SFRP1 was associated with poor ccRCC-specific survival, independent of age, sex, tumor size, TNM stage or tumor grade. Moreover, the association between GREM1, NEFH, and NEURL methylation and outcome was shown to be dependent on the genomic region. A prognostic biomarker model containing GREM1, GATA5, LAD1, NEFH and NEURL methylation in combination with clinicopathological characteristics, performed better compared to the model with clinicopathological characteristics only (clinical model), in both the NLCS and the validation population with a c-statistic of 0.71 versus 0.65 and a c-statistic of 0.95 versus 0.86 consecutively. However, the biomarker model had limited added prognostic value in the TCGA series with a c-statistic of 0.76 versus 0.75 for the clinical model. CONCLUSION: In this study we performed a head-to-head comparison of potential prognostic methylation markers for ccRCC using a novel approach to guide primers design which utilizes the optimal location for measuring DNA methylation. Using this approach, we identified five methylation markers that potentially show prognostic value in addition to currently known clinicopathological factors.

Multitarget Stool DNA Test Performance in an Average-Risk Colorectal Cancer Screening Population.

INTRODUCTION: We set out to evaluate the performance of a multitarget stool DNA (MT-sDNA) in an average-risk colonoscopy-controlled colorectal cancer (CRC) screening population. MT-sDNA stool test results were evaluated against fecal immunochemical test (FIT) results for the detection of different lesions, including molecularly defined high-risk adenomas and several other tumor characteristics. METHODS: Whole stool samples (n = 1,047) were prospectively collected and subjected to an MT-sDNA test, which tests for KRAS mutations, NDRG4 and BMP3 promoter methylation, and hemoglobin. Results for detecting CRC (n = 7), advanced precancerous lesions (advanced adenoma [AA] and advanced serrated polyps; n = 119), and non-AAs (n = 191) were compared with those of FIT alone (thresholds of 50, 75, and 100 hemoglobin/mL). AAs with high risk of progression were defined by the presence of specific DNA copy number events as measured by low-pass whole genome sequencing. RESULTS: The MT-sDNA test was more sensitive than FIT alone in detecting advanced precancerous lesions (46% (55/119) vs 27% (32/119), respectively, P < 0.001). Specificities among individuals with nonadvanced or negative findings (controls) were 89% (791/888) and 93% (828/888) for MT-sDNA and FIT testing, respectively. A positive MT-sDNA test was associated with multiple lesions (P = 0.005), larger lesions (P = 0.03), and lesions with tubulovillous architecture (P = 0.04). The sensitivity of the MT-sDNA test or FIT in detecting individuals with high-risk AAs (n = 19) from individuals with low-risk AAs (n = 52) was not significantly different. DISCUSSION: In an average-risk screening population, the MT-sDNA test has an increased sensitivity for detecting advanced precancerous lesions compared with FIT alone. AAs with a high risk of progression were not detected with significantly higher sensitivity by MT-sDNA or FIT.

Molecular profiling of longitudinally observed small colorectal polyps: A cohort study.

BACKGROUND: Knowledge of the natural history of colorectal adenomas is limited because these lesions are removed upon detection. The few studies in which small adenomas have been left in situ for a limited period of time, have shown that most lesions remain stable or even completely regress. Specific DNA copy number changes ('cancer associated events' or CAEs) are associated with progression of adenomas to cancer. In this study we evaluated whether molecular features of progression correlated with growth of small polyps. METHODS: Small (6-9 mm) colorectal precursor lesions detected on CT-colonography (CTC) were left in situ and re-evaluated with CTC after three years. Based on volumetric change, polyps were classified as either grown, stable or regressed. Surveillance CTC was followed by colonoscopy, during which all lesions were resected. Using DNA isolated from FFPE polyp tissues, low-coverage whole genome sequencing was performed to determine DNA copy number profiles, as well as target enrichment mutation analysis and CpG island methylation phenotype (CIMP) analysis. Expression of DNA mismatch repair (MMR) proteins was determined by immunohistochemistry. Samples were marked as MMR proficient if all MMR proteins were expressed. FINDINGS: Out of 68 polyps resected at colonoscopy, for 65 (96%) material was available. Of these, 31 (48%) had grown, 27 (41%) remained stable and 7 (11%) regressed. Polyps with at least one CAE had higher growth rates compared to polyps without CAEs (difference 91% growth (95% CI 13-169), p = .023). CAEs were absent in lesions that had partially regressed. Mutations occurred in 94% of the polyps, with higher growth rates being associated with polyps having ≥2 mutations compared to lesions with only 0-1 mutations (difference 99% growth (95% CI 9-189), p = .032). All samples were MMR proficient. No relation between growth and CIMP was observed. INTERPRETATION: Molecular alterations associated with colorectal cancer, correlated with growth of small polyps and were absent in polyps that regressed. Therefore, this longitudinal study provides in vivo support in the human setting for the functional role of these molecular alterations, that have mostly been identified by cross sectional observations in tissue samples of colorectal adenomas and cancers. FUND: Alpe d'Huzes- Dutch Cancer Society (project number NKI2013-6338).

NDRG4, an early detection marker for colorectal cancer, is specifically expressed in enteric neurons.

BACKGROUND: Promoter methylation of N-myc Downstream-Regulated Gene 4 (NDRG4) in fecal DNA is an established early detection marker for colorectal cancer (CRC). Despite its connection to CRC, NDRG4 is predominantly studied in brain and heart, with little to no knowledge about its expression or role in other organs. In this study, we aimed to determine the whole-body expression of NDRG4, with a focus on the intestinal tract. METHODS: We investigated NDRG4 expression throughout the body by immunohistochemistry, Western Blotting and in situ mRNA hybridization using tissues from NDRG4 wild-type, heterozygous and knockout mice and humans. In addition, we explored cell-specific expression of NDRG4 in murine whole-mount gut preparations using immunofluorescence and confocal microscopy. KEY RESULTS: NDRG4 is specifically expressed within nervous system structures throughout the body. In the intestinal tract of both mouse and man, NDRG4 immunoreactivity was restricted to the enteric nervous system (ENS), where it labeled cell bodies of the myenteric and submucosal plexuses and interconnecting nerve fibers. More precisely, NDRG4 expression was limited to neurons, as NDRG4 always co-localized with HuC/D (pan-neuronal marker) but never with GFAP (an enteric glial cell marker). Furthermore, NDRG4 was expressed in various neuropeptide Y positive neurons, but was only found in a minority (~10%) of neurons expressing neuronal nitric oxide synthase. CONCLUSIONS AND INFERENCES: NDRG4 is exclusively expressed by central, peripheral and enteric neurons/nerves, suggesting a neuronal-specific role of this protein. Our findings raise the question whether NDRG4, via the ENS, an understudied component of the tumor microenvironment, supports CRC development and/or progression.

Body size, physical activity, genetic variants in the insulin-like growth factor pathway and colorectal cancer risk.

Insulin-like growth factors (IGFs) have been associated with growth, body size, physical activity and colorectal cancer (CRC). We hypothesized that variants in IGF-related genes increase the CRC susceptibility associated with a larger body size and a lack of physical activity. We assessed this in The Netherlands Cohort Study. Participants (n = 120852) completed a baseline questionnaire on diet and cancer. ~75% returned toenail clippings. Using a case-cohort approach and 16.3 years of follow-up, toenail DNA from 3768 subcohort members and 2580 CRC cases was genotyped. We aggregated unfavorable alleles (potentially increasing CRC risk) for 18 single nucleotide polymorphisms in 8 genes into a sum score. The sum score (in tertiles) and an IGF1 19-CA repeat polymorphism (19/19, 19/non-19 and non-19/non-19 repeats) in combination with body size (mostly in tertiles) and (non-)occupational physical activity (>12, 8-12 and <8 kJ/min in the job and >90, >60-90, >30-60 and ≤30 min/day) were analyzed by Cox regression. Increasingly higher hazard ratios (HRs) for CRC were observed for a larger adult body mass index, larger trouser size and tallness in the presence of more unfavorable alleles in men. HRs (95% confidence intervals) for joint effects were 1.55 (1.06-2.25), 1.78 (1.29-2.46) and 1.48 (1.01-2.17), respectively. In women, variant repeat alleles halved CRC risk irrespective of body size and physical activity. Almost no interactions tested significant. To conclude, a larger body size was a CRC risk factor in men in the presence of an accumulation of unfavorable alleles in IGF-related genes, but interactions were generally nonsignificant.

Resistance to sunitinib in renal cell carcinoma: From molecular mechanisms to predictive markers and future perspectives.

The introduction of agents that inhibit tumor angiogenesis by targeting vascular endothelial growth factor (VEGF) signaling has made a significant impact on the survival of patients with metastasized renal cell carcinoma (RCC). Sunitinib, a tyrosine kinase inhibitor of the VEGF receptor, has become the mainstay of treatment for these patients. Although treatment with sunitinib substantially improved patient outcome, the initial success is overshadowed by the occurrence of resistance. The mechanisms of resistance are poorly understood. Insight into the molecular mechanisms of resistance will help to better understand the biology of RCC and can ultimately aid the development of more effective therapies for patients with this infaust disease. In this review we comprehensively discuss molecular mechanisms of resistance to sunitinib and the involved biological processes, summarize potential biomarkers that predict response and resistance to treatment with sunitinib, and elaborate on future perspectives in the treatment of metastasized RCC.

Prognostic value of CpG island methylator phenotype among colorectal cancer patients: a systematic review and meta-analysis.

BACKGROUND: Divergent findings regarding the prognostic value of CpG island methylator phenotype (CIMP) in colorectal cancer (CRC) patients exist in current literature. We aim to review data from published studies in order to examine the association between CIMP and CRC prognosis. MATERIALS AND METHODS: A comprehensive search for studies reporting disease-free survival (DFS), overall survival (OS), or cancer-specific mortality of CRC patients stratified by CIMP is carried out. Study findings are summarized descriptively and quantitatively, using adjusted hazard ratios (HRs) as summary statistics. RESULTS: Thirty-three studies reporting survival in 10 635 patients are included for review. Nineteen studies provide data suitable for meta-analysis. The definition of CIMP regarding gene panel, marker threshold, and laboratory method varies across studies. Pooled analysis shows that CIMP is significantly associated with shorter DFS (pooled HR estimate 1.45; 95% confidence interval (CI) 1.07-1.97, Q = 3.95, I(2) = 0%) and OS (pooled HR estimate 1.43; 95% CI 1.18-1.73, Q = 4.03, I(2) = 0%) among CRC patients irrespective of microsatellite instability (MSI) status. Subgroup analysis of microsatellite stable (MSS) CRC patients also shows significant association between shorter OS (pooled HR estimate 1.37; 95% CI 1.12-1.68, Q = 4.45, I(2) = 33%) and CIMP. Seven studies have explored CIMP's value as a predictive factor on stage II and III CRC patient's DFS after receiving adjuvant 5-fluorouracil (5-FU) therapy: of these, four studies showed that adjuvant chemotherapy conferred a DFS benefit among CIMP(+) patients, one concluded to the contrary, and two found no significant correlation. Insufficient data was present for statistical synthesis of CIMP's predictive value among CRC patients receiving adjuvant 5-FU therapy. CONCLUSION: CIMP is independently associated with significantly worse prognosis in CRC patients. However, CIMP's value as a predictive factor in assessing whether adjuvant 5-FU therapy will confer additional survival benefit to CRC patients remained to be determined through future prospective randomized studies.

Long-term dietary sodium, potassium and fluid intake; exploring potential novel risk factors for renal cell cancer in the Netherlands Cohort Study on diet and cancer.

BACKGROUND: As sodium, potassium and fluid intake are related to hypertension, an established risk factor for renal cell cancer (RCC), they may be independent risk factors for RCC. METHODS: The Netherlands Cohort Study (NLCS) with case-cohort design included 120,852 participants aged 55-69 years. At baseline, diet and lifestyle were assessed with questionnaires. After 17.3 years of follow-up, 485 RCC cases and 4438 subcohort members were available for analyses. RESULTS: Sodium intake increased RCC risk (P-trend=0.03), whereas fluid and potassium intake did not. For high sodium and low fluid intake, the RCC risk additionally increased (P-interaction=0.02). CONCLUSION: Sodium intake is a potential risk factor for RCC, particularly if fluid consumption is low.

A novel classification of colorectal tumors based on microsatellite instability, the CpG island methylator phenotype and chromosomal instability: implications for prognosis.

BACKGROUND: We studied the overlap between the major (epi)genomic events microsatellite instability (MSI), the CpG island methylator phenotype (CIMP) and chromosomal instability (CIN) in colorectal cancer (CRC), and whether specific (epi)genotypes were associated with CRC-related deaths. PATIENTS AND METHODS: Molecular analyses using tumor DNA were successful in 509 CRC cases identified within the Netherlands Cohort Study in the period 1989-1993. Follow-up for the vital status until May 2005 was 100%. RESULTS: MSI (12.6%), CIMP-only (5.3%), CIMP + CIN (13.4%), CIN-only (58.2%) and triple-negative tumors (10.6%) differed significantly regarding tumor localization, differentiation grade, initial adjuvant therapy (AT) use and genetic characteristics (P ≤ 0.03). CIMP-only, CIMP + CIN and triple-negative tumors, compared with CIN-only tumors, were significantly associated with a 3.67, 2.44 and 3.78-fold risk of CRC-related deaths after 2-year follow-up (95% confidence intervals, CIs, 1.70-7.91, 1.35-4.41 and 1.97-7.25, respectively), but not after late follow-up. MSI tumors were borderline significantly associated with a 0.40-fold risk of CRC-related deaths after late follow-up (95% CI 0.15-1.03). CONCLUSION(S): This is the first study to show that specific (epi)genotypes may hold a differential prognostic value that may vary over time. Although no specific treatment data were available, an explanation for the differential findings over time might be that (epi)genotypes modify therapy response.

The influence of choice of therapy on quality of life in patients with neurogenic thoracic outlet syndrome.

OBJECTIVES: We aimed to compare quality of life (QoL) after surgical decompression of the thoracic outlet versus non-operative therapy in patients with neurogenic thoracic outlet syndrome (N-TOS). DESIGN AND METHODS: We retrospectively identified 46 patients, diagnosed with N-TOS between 1999 and 2008. Twenty-four operated and 22 conservatively treated patients were sent questionnaires on their current symptoms and QoL. A matched control group (n = 24) of healthy individuals was selected for QoL comparison. Statistics were performed with linear and logistic regression analysis. RESULTS: ANOVA revealed a significant QoL difference between the three groups (p = 0.001). Separate analysis between groups demonstrated that all patients with N-TOS-like symptoms have a lower QoL than healthy controls (p = 0.001 resp. p < or = 0.000). No difference was found between conservatively and surgically treated patients (p = 0.26). EQ-5D response rate was 83%. Of the 24 surgically treated patients, 15 would choose surgery again in a similar situation, although 4 did not benefit in terms of symptom reduction. Symptom relief and VAS pain scores in the conservatively and surgically treated patients did not show significant differences (p = 0.95 resp. p = 0.40). CONCLUSIONS: All patients with N-TOS have a significantly decreased QoL compared with healthy individuals, regardless of the type of therapy they received. In this small study, surgical decompression fails to improve QoL in patients with N-TOS to the level measured in the healthy control group, despite symptom reduction consistent with previous reports. Variables significantly associated with outcome were duration of symptoms and localisation (variables included in the prediction model: age, sex, duration of symptoms, presence of paraesthesias, localisation, Adson's, Wright's and Roos' test, history of trauma, cervical arthrosis). In the perspective of QoL, the benefit of decompressive surgery is questionable. Improving patient selection seems imperative in order to achieve better results in our surgically treated patients.

MGMT and MLH1 promoter methylation versus APC, KRAS and BRAF gene mutations in colorectal cancer: indications for distinct pathways and sequence of events.

BACKGROUND: To study how caretaker gene silencing relates to gatekeeper mutations in colorectal cancer (CRC), we investigated whether O6-methylguanine DNA methyltransferase (MGMT) and Human Mut-L Homologue 1 (MLH1) promoter hypermethylation are associated with APC, KRAS and BRAF mutations among 734 CRC patients. METHODS: We compared MGMT hypermethylation with G:C > A:T mutations in APC and KRAS and with the occurrence of such mutations in CpG or non-CpG dinucleotides in APC. We also compared MLH1 hypermethylation with truncating APC mutations and activating KRAS and BRAF mutations. RESULTS: Only 10% of the tumors showed both MGMT and MLH1 hypermethylation. MGMT hypermethylation occurred more frequently in tumors with G:C > A:T KRAS mutations (55%) compared with those without these mutations (38%, P < 0.001). No such difference was observed for G:C > A:T mutations in APC, regardless of whether mutations occurred in CpG or non-CpG dinucleotides. MLH1 hypermethylation was less common in tumors with APC mutations (P = 0.006) or KRAS mutations (P = 0.001), but was positively associated with BRAF mutations (P < 0.001). CONCLUSIONS: MGMT hypermethylation is associated with G:C > A:T mutations in KRAS, but not in APC, suggesting that MGMT hypermethylation may succeed APC mutations but precedes KRAS mutations in colorectal carcinogenesis. MLH1-hypermethylated tumors harbor fewer APC and KRAS mutations and more BRAF mutations, suggesting that they develop distinctly from an MGMT methylator pathway.

E2Fs mediate a fundamental cell-cycle deregulation in high-grade serous ovarian carcinomas.

Several studies described a role for the E2F/Rb pathway in ovarian serous carcinomas (SCAs). Since E2F/Rb pathway deregulation is a general hallmark of human cancer, it remains unclear whether this deregulation is of particular importance in SCAs or whether it reflects a common oncological feature. Here, we have clarified this issue by the examination of microarray expression profiles of SCAs and particularly by the comparison with another, less malignant, ovarian cancer type, serous borderline tumours (SBTs). Results were validated by quantitative RT-PCR, both on the microarray samples and on an independent panel, and TP53 mutation analysis was performed. This integrated analysis revealed a significant increase in the expression of the transcription factors E2F1 and E2F3 in SCAs, when compared to SBTs. This was associated with vast overexpression of E2F target genes in SCAs compared to SBTs. High-grade SCAs in particular exhibited a major deregulated E2F target expression pattern. Generally, overexpression of E2F targets in SCAs appeared to be well structured since those targets considered negative regulators of the cell cycle or promoters of apoptosis were usually not overexpressed in SCAs. Similar to E2F target deregulation, TP53 mutations were identified in SCA3s, to a lesser extent in SCA1s, and not in SBTs. These results suggest that a structured, generally up-regulated E2F transcription factor activity is associated with a global cell-cycle disturbance in high-grade SCAs and exceeds typical E2F/Rb pathway disruption in tumours, at least compared with SBTs.

Aberrant methylation of the Adenomatous Polyposis Coli (APC) gene promoter is associated with the inflammatory breast cancer phenotype.

Aberrant methylation of the adenomatous polyposis coli (APC) gene promoter occurs in about 40% of breast tumours and has been correlated with reduced APC protein levels. To what extent epigenetic alterations of the APC gene may differ according to specific breast cancer phenotypes, remains to be elucidated. Our aim was to explore the role of APC methylation in the inflammatory breast cancer (IBC) phenotype. The status of APC gene promoter hypermethylation was investigated in DNA from normal breast tissues, IBC and non-IBC by both conventional and real-time quantitative methylation-specific PCR (MSP). APC methylation levels were compared with APC mRNA and protein levels. Hypermethylation of the APC gene promoter was present in 71% of IBC samples (n=21) and 43% of non-IBC samples (n=30) by conventional MSP (P=0.047). The APC gene also showed an increased frequency of high methylation levels in IBC (in 74% of cases, n=19) vs non-IBC (in 46% of cases, n=35) using a qMSP assay (P=0.048). We observed no significant association between APC methylation levels by qMSP and APC mRNA or protein expression levels. In conclusion, for the first time, we report the association of aberrant methylation of the APC gene promoter with the IBC phenotype, which might be of biological and clinical importance.

Poorer outcome in stromal HIF-2 alpha- and CA9-positive colorectal adenocarcinomas is associated with wild-type TP53 but not with BNIP3 promoter hypermethylation or apoptosis.

Stromal expression of hypoxia inducible factor 2 alpha (HIF-2 alpha) and carbonic anhydrase 9 (CA9) are associated with a poorer prognosis in colorectal cancer (CRC). Tumour cell death, regulated by a hypoxic stromal microenvironment, could be of importance in this respect. Therefore, we correlated apoptosis, TP53 mutational status and BNIP3 promoter hypermethylation of CRC cells with HIF-2 alpha- and CA9-related poor outcome. In a series of 195 CRCs, TP53 mutations in exons 5-8 were analysed by direct sequencing, and promoter hypermethylation of BNIP3 was determined by methylation-specific PCR. Expressions of HIF-2 alpha, CA9, p53, BNIP3 and M30 were analysed immunohistochemically. Poorer survival of HIF-2 alpha and CA9 stromal-positive CRCs was associated with wild-type TP53 (P=0.001 and P=0.0391), but not with BNIP3 methylation. Furthermore, apoptotic levels were independent of the TP53 status, but lower in unmethylated BNIP3 CRCs (P=0.004). It appears that wild-type TP53 in CRC cells favours the progression of tumours expressing markers for hypoxia in their stroma, rather than in the epithelial compartment. Preserved BNIP3 function in CRC cells lowers apoptosis, and may thus be involved in alternative cell death pathways, such as autophagic cell death. However, BNIP3 silencing in tumour cells does not impact on hypoxia-driven poorer prognosis. These results suggest that the biology of CRC cells can be modified by alterations in the tumour microenvironment under conditions of tumour hypoxia.

RASSF1A methylation and K-ras and B-raf mutations and recurrent endometrial cancer.

BACKGROUND: Aberrations in mediators of Ras signaling may increase the risk of developing recurrent endometrial carcinoma. PATIENTS AND METHODS: Primary tumors of patients with (n = 44) and without (n = 44) recurrent stage I endometrioid endometrial carcinoma were compared regarding the presence of K-ras mutations (codons 12 and 13), B-raf mutations (V599), and RASSF1A gene promoter methylation. RESULTS: K-ras mutations were present in 18% of the patients independent of recurrent disease. No B-raf mutations were found. RASSF1A methylation was demonstrated in 85% of endometrial carcinomas, independent of recurrence. The presence of K-ras mutations and RASSF1A promoter methylation were not related, either directly or inversely. Analysis in premenopausal endometrial carcinomas demonstrated K-ras mutations in 40%, no B-raf mutations, and RASSF1A promoter methylation in 70% of the cases. RASSF1A methylation was also observed in samples of cyclic (n = 14), hyperplastic (n = 8), and atrophic (n = 13) endometrial tissues in 21%, 50% and 38%, respectively. CONCLUSIONS: RASSF1A methylation was observed in a high frequency in endometrioid endometrial carcinoma whereas K-ras and B-raf mutations were observed in a low frequency. No association was observed with the development of recurrent disease. High-frequency RASSF1A methylation in premenopausal carcinomas and an increased frequency in endometrial hyperplasia indicate that this may be an early event in endometrial carcinogenesis.

Clonal analysis favours a monoclonal origin for serous borderline tumours with peritoneal implants.

Serous borderline tumours (SBTs) of the ovary were originally classified as such because the vast majority behave in a remarkably indolent manner, even in the presence of widespread tumour deposits, termed implants, and/or lymph node involvement. The pathogenesis of the implants is currently unknown. Two major hypotheses have been proposed: the first favours a monoclonal origin, arguing that the peritoneal lesions derive from neoplastic cells that are shed from the primary ovarian tumour. The second hypothesis favours a polyclonal origin as a result of a field defect of susceptible Müllerian cells from which multiple independent tumours arise. To test both hypotheses, genome-wide allelotyping and B-RAF/K-RAS mutation analyses were employed to assess clonality in 25 metachronous or synchronous tumours from ten SBT patients. Loss of heterozygosity (LOH) profiling and K-RAS/B-RAF mutation analysis showed concordance of the genetic changes in all sites in 21 tumours from eight patients who were informative. These results favour a common origin, underscored by a likelihood ratio (probability of common origin/probability of independent origin) ranging from 2.43 to 7,662,850. In conclusion, this study strongly supports the hypothesis that both non-invasive and invasive implants arise as a consequence of spread from a single ovarian site.

APC, beta-catenin, and E-cadherin and the development of recurrent endometrial carcinoma.

Endometrial carcinoma, generally, has a good prognosis. However, in some patients, the tumor appears to behave very aggressively, a course that cannot be explained with histopathological characteristics. More insight into the molecular background can be valuable to clarify these differences in tumor behavior. The three components associated with the Wnt pathway--i.e., adenomatous polyposis coli (APC), beta-catenin, and E-cadherin--were evaluated in a case-control study of 28 patients with stage-I endometrial carcinomas to determine their involvement in the development of recurrent disease. Mutation analysis of the mutation cluster region of the APC gene, determination of gene promoter methylation status of the APC-1A and E-cadherin genes, and immunohistochemical analysis of APC, E-cadherin, and beta-catenin were performed using paraffin-embedded tumor tissue. Twenty-one APC gene mutations were detected in 12 of 28 (43%) patients. Only three mutations would result in a stopcodon in the APC gene. APC gene promoter methylation was assessed in 12 of 28 (43%) patients. APC immunostaining was absent in two of 24 (8.3%) patients. The occurrence of APC mutations, APC gene promoter methylation, and APC immunostaining were not predictive for recurrence. No E-cadherin expression was observed in four of 24 patients (17%). E-cadherin gene promoter methylation could not be detected in any of the patients. The absence of E-cadherin expression was predictive for distant metastases, but not for local recurrence. Nuclear localization of beta-catenin was present in nine of 24 (38%) patients and was not predictive for recurrent disease. Involvement of epigenetic and genetic aberrations in APC and beta-catenin genes seems to be of minor importance for the development of local recurrences and distant metastases. Although the number of patients is limited, E-cadherin expression appears to be predictive for the development of distant metastases in endometrial carcinoma.

Defective mismatch repair and the development of recurrent endometrial carcinoma.

OBJECTIVE: To investigate whether defective DNA mismatch repair (MMR) defines a subgroup at risk for recurrence in sporadic endometrial carcinoma patients. METHODS: Primary tumors from 44 patients with recurrent stage I endometrial carcinoma were compared after matching, with tumors of 44 patients being free of recurrence for minimal 3 years. Paraffin-embedded primary tumors (n = 88) and recurrent tumors (n = 32) were subjected to immunohistochemical analysis for hMSH2 and hMLH1 expression. Subsequently, a staining index (SI = 0-9) was calculated based on staining intensity and quantity. DNA was extracted from paraffin-embedded tissues, and promoter methylation of hMLH1 was determined by nested methylation-specific PCR (MSP). Microsatellite instability (MSI) was assessed by BAT-26 or BAT-25. RESULTS: Low hMSH2 expression was observed in 2% of primary tumors of control patients without recurrence, 14% of primary tumors of patients with recurrence, and 0% of recurrent tumors. Low hMLH1 expression was observed in 32%, 19%, and 22%, respectively. hMLH1 gene promoter methylation was detected in 50%, 47%, and 32%, and MSI was found in 16%, 14%, and 30%, respectively. No significant differences were found between primary tumors of patients with and without recurrence with respect to hMSH2 and hMLH1 expression, hMLH1 promoter methylation, and MSI. When primary and recurrent tumors were compared, there was an increased correlation of hMLH1 methylation with low hMLH1 expression and MSI in recurrent tumors. CONCLUSION: MSI, hMLH1 promoter methylation, and the expression of hMLH1 and hMSH2 are not predictive for the development of recurrent stage I endometrial carcinoma. In the progression of tumor, "de novo" hMLH1 methylation rarely occurs, instead there is further derailment of the MMR pathway in affected tumors.

The effect of the cyclin-dependent kinase inhibitor olomoucine on cell cycle kinetics.

The effect of the cyclin-dependent (CDK) inhibitors olomoucine and roscovitine on cell kinetics was studied. To this end, nonsmall cell lung cancer (NSCLC) cell line MR65 and neuroblastoma cell line CHP-212 were pulse labeled with bromodeoxyuridine (BrdUrd) and chased in culture medium, to which various concentrations of olomoucine or roscovitine were added. A dose-dependent inhibition of the G1/S-phase and G2/ M-/G1 transitions was observed. Furthermore, S-phase progression was also inhibited in a dose-dependent manner. Similarly, roscovitine, another CDK inhibitor with a 10-fold higher efficiency for both CDK1 and CDK2 as compared to olomoucine, showed the same effects at a 10-fold lower concentration. At the highest tested doses both olomoucine (200 microM) and roscovitine (40 microM) induced a complete cell cycle block in both cell lines, paralleled by the appearance of apoptotic figures. In these cultures a decrease in CDK1 protein level was found as shown by Western blotting. Bivariate CDK1/DNA analysis confirmed these observations and showed that a subpopulation of cells with characteristics of apoptosis became CDK1 negative. The presented data suggest that cyclins and CDKs are involved at an important nodal point shared by pathways regulating cellular proliferation and apoptosis.