Akihiro Nyuya

Clinical impact of endometrial cancer stratified by genetic mutational profiles, POLE mutation, and microsatellite instability

Background The molecular characterization of endometrial cancer (EC) can facilitate identification of various tumor subtypes. Although EC patients with POLE mutations reproducibly demonstrate better prognosis, the outcome of patients with microsatellite instability (MSI) remains controversial. This study attempted to interrogate whether genetic stratification of EC can identify distinct subsets with prognostic significance. Materials and methods A cohort of 138 EC patients who underwent surgical resection with curative intent was enrolled. Sanger sequencing was used to evaluate mutations in the POLE and KRAS genes. MSI analysis was performed using four mononucleotide repeat markers and methylation status of the MLH1 promoter was measured by a fluorescent bisulfite polymerase chain reaction (PCR). Protein expression for mismatch repair (MMR) proteins was evaluated by immunohistochemistry (IHC). Results Extensive hypermethylation of the MLH1 promoter was observed in 69.6% ECs with MLH1 deficiency and 3.5% with MMR proficiency, but in none of the ECs with loss of other MMR genes (P < .0001). MSI-positive and POLE mutations were found in 29.0% and 8.7% EC patients, respectively. Our MSI analysis showed a sensitivity of 92.7% for EC patients with MMR deficiency, and a specificity of 97.9% for EC patients with MMR proficiency. In univariate and multivariate analyses, POLE mutations and MSI status was significantly associated with progression-free survival (P = 0.0129 and 0.0064, respectively) but not with endometrial cancer-specific survival. Conclusions This study provides significant evidence that analyses of proofreading POLE mutations and MSI status based on mononucleotide repeat markers are potentially useful biomarkers to identify EC patients with better prognosis.

Clinical outcomes of women with ovarian metastases of colorectal cancer treated with oophorectomy with respect to their somatic mutation profiles

We clarified the clinical prevalence of ovarian metastases from colorectal cancers (CRCs) in 296 female patients with CRC and evaluated clinical outcomes with relation to their mutational profiles, such as BRAF/KRAS mutation and microsatellite instability (MSI) status. The female CRCs were categorised into three subsets: CRCs with ovarian metastases [6.4% (n = 19), 5-year overall survival (OS) = 24.7%], CRCs with extra-ovarian metastases only [32.4% (n = 96), 5-year OS = 34.5%] and CRCs without any recurrence or metastasis [61.2% (n = 181), 5-year OS = 91.3%]. All patients with ovarian metastases underwent oophorectomy; of these, 9 who received preoperative chemotherapy had measurable metastases to extra-ovarian sites and the ovaries. Although 5 of 9 (56%) achieved partial response or complete response at extra-ovarian sites, no patient archived objective response at ovarian sites. Regarding the mutation profiles, in CRCs with extra-ovarian metastases only, the median survival time (MST) after initial treatments to progression to stage IV or recurrence was 13 [95% confidence interval (CI): 7–16 months] in BRAF-mutant and 34 months (95% CI: 22–58 months) in BRAF wild-type (P = 0.0033). Although ovarian metastases demonstrated poor response to systemic chemotherapy in CRCs with ovarian metastases, the MST after initial treatments to progression to stage IV or recurrence was 22 (95% CI: 21–25 months) in BRAF-mutant and 38 months (95% CI: 24–42 months) in BRAF wild-type (P = 0.0398). The outcomes of patients with ovarian metastases could be improved by oophorectomy regardless of their mutation profiles.

Heterogeneity of Epigenetic and Epithelial Mesenchymal Transition Marks in Hepatocellular Carcinoma with Keratin 19 Proficiency

Objective: Keratin 19 (K19) expression is a potential predictor of poor prognosis in patients with hepatocellular carcinoma (HCC). To clarify the feature of K19-proficient HCC, we traced epigenetic footprints in cultured cells and clinical materials. Patients and Methods: In vitro, KRT19 promoter methylation was analyzed and 5-aza-2’-deoxycytidine with trichostatin A (TSA) treatment was performed. Among 564 surgically resected HCCs, the clinicopathological relevance of K19-proficent HCCs was performed in comparison with hepatocytic (HepPar-1 and arginase-1), epithelial-mesenchymal transition (E-cadherin and vimentin), biliary differentiation-associated (K7 and NOTCH-1) markers, and epigenetic markers (KRT19 promoter/long interspersed nucleotide element-1 [LINE-1] methylation status). Results: KRT19 promoter methylation was clearly associated with K19 deficiency and 5-aza-2’-deoxycytidine with TSA treatment-stimulated K19 re-expression, implicating DNA methylation as a potential epigenetic process for K19 expression. After excluding HCCs with recurrence, TNM stage as IIIB or greater, preoperative therapy, transplantation, and combined hepatocellular cholangiocarcinoma, we assessed 125 of 564 HCC cases. In this cohort, K19 expression was found in 29 HCCs (23.2%) and corresponded with poor survival following surgery (p = 0.025) and extrahepatic recurrence-free survival (p = 0.017). Compared with K19-deficient HCCs, lower KRT19 promoter methylation level was observed in K19-proficient HCCs (p < 0.0001). Conversely, HCC with genome-wide LINE-1 hypermethylation was frequently observed in K19-proficient HCCs (p = 0.0079). Additionally, K19 proficiency was associated with K7 proficiency (p = 0.043), and reduced E-cadherin and HepPar-1 expression (p = 0.043 and p < 0.0001, respectively). Conclusions: K19-proficient HCC exhibited poor prognosis owing to extrahepatic recurrence, with molecular signatures differing from those in conventional cancer stem cells, providing novel insights of the heterogeneity underlying tumor development.

Su1995 Genetic and Epigenetic Alterations in the Netrin-1 Receptors, UNC5c and DCC, Constitutes a Previously Unrecognized Pathway in Gastric Cancer Progression

Background: Downregulated expression of the netrin-1 dependence receptors, UNC5C and DCC, is a frequent event in significant proportion of colorectal tumors. However, the tumor suppressive role of this important cancer-related pathway in other gastrointestinal cancers remains unclear. We hypothesized that similar to colorectal cancer, down-regulation of UNC5C and DCC may have an important growth-regulatory function in gastric tumorigenesis as well. Methods: We performed a series of genetic (loss of heterozygosity, LOH) and epigenetic analysis (aberrant DNA methylation) for UNC5C and DCC in a cohort of 105 sporadic gastric cancers and the corresponding normal gastric mucosa. Results: In this study, the distribution of gastric cancers as categorized by TNM staging system (UICC 7th) was as follows; 20, 24, 28, and 29 cases in stages I, II, III, and IV, respectively. Overall, 58% (48 out of 83 informative cases) and 76% (65 out of 85 informative cases) of gastric cancers demonstrated either a genetic or epigenetic alteration in the UNC5C and DCC genes, respectively. Interestingly, although epigenetic mechanisms were the primary cause for UNC5C inactivation, methylation-induced transcriptional silencing served also served as the second hit responsible for the DCC gene loss. Taken together, 52% (36 out of 69 gastric cancers with informative data) of gastric cancers showed cumulative defects in these two dependence receptors. The frequency of LOH and aberrant methylation of both dependence receptors gradually increased with progressive tumor stages. Conclusions: This is the first study that unravels the observation that majority of gastric cancers harbor defects in netrin1 receptors. Our observation that both genetic and epigenetic alterations are present early, and continue to escalate in a stepwise manner with the disease progression, highlights the fundamental importance of this growth regulatory pathway in gastric carcinogenesis.