Yasuko Kikuchi

Classification of Epstein–Barr Virus–Positive Gastric Cancers by Definition of DNA Methylation Epigenotypes

Epstein-Barr virus (EBV) is associated with Burkitt lymphoma, nasopharyngeal carcinoma, opportunistic lymphomas in immunocompromised hosts, and a fraction of gastric cancers. Aberrant promoter methylation accompanies human gastric carcinogenesis, though the contribution of EBV to such somatic methylation changes has not been fully clarified. We analyzed promoter methylation in gastric cancer cases with Illuminas Infinium BeadArray and used hierarchical clustering analysis to classify gastric cancers into 3 subgroups: EBV(-)/low methylation, EBV(-)/high methylation, and EBV(+)/high methylation. The 3 epigenotypes were characterized by 3 groups of genes: genes methylated specifically in the EBV(+) tumors (e.g., CXXC4, TIMP2, and PLXND1), genes methylated both in EBV(+) and EBV(-)/high tumors (e.g., COL9A2, EYA1, and ZNF365), and genes methylated in all of the gastric cancers (e.g., AMPH, SORCS3, and AJAP1). Polycomb repressive complex (PRC) target genes in embryonic stem cells were significantly enriched among EBV(-)/high-methylation genes and commonly methylated gastric cancer genes (P = 2 × 10(-15) and 2 × 10(-34), respectively), but not among EBV(+) tumor-specific methylation genes (P = 0.2), suggesting a different cause for EBV(+)-associated de novo methylation. When recombinant EBV was introduced into the EBV(-)/low-methylation epigenotype gastric cancer cell, MKN7, 3 independently established subclones displayed increases in DNA methylation. The promoters targeted by methylation were mostly shared among the 3 subclones, and the new methylation changes caused gene repression. In summary, DNA methylation profiling classified gastric cancer into 3 epigenotypes, and EBV(+) gastric cancers showed distinct methylation patterns likely attributable to EBV infection.

Aberrantly methylated genes in human papillary thyroid cancer and their association with BRAF/RAS mutation

Cancer arises through accumulation of epigenetic and genetic alteration. Aberrant promoter methylation is a common epigenetic mechanism of gene silencing in cancer cells. We here performed genome-wide analysis of DNA methylation of promoter regions by Infinium HumanMethylation27 BeadChip, using 14 clinical papillary thyroid cancer samples and 10 normal thyroid samples. Among the 14 papillary cancer cases, 11 showed frequent aberrant methylation, but the other three cases showed no aberrant methylation at all. Distribution of the hypermethylation among cancer samples was non-random, which implied existence of a subset of preferentially methylated papillary thyroid cancer. Among 25 frequently methylated genes, methylation status of six genes (HIST1H3J, POU4F2, SHOX2, PHKG2, TLX3, HOXA7) was validated quantitatively by pyrosequencing. Epigenetic silencing of these genes in methylated papillary thyroid cancer cell lines was confirmed by gene re-expression following treatment with 5-aza-2′-deoxycytidine and trichostatin A, and detected by real-time RT-PCR. Methylation of these six genes was validated by analysis of additional 20 papillary thyroid cancer and 10 normal samples. Among the 34 cancer samples in total, 26 cancer samples with preferential methylation were significantly associated with mutation of BRAF/RAS oncogene (P = 0.04, Fishers exact test). Thus, we identified new genes with frequent epigenetic hypermethylation in papillary thyroid cancer, two subsets of either preferentially methylated or hardly methylated papillary thyroid cancer, with a concomitant occurrence of oncogene mutation and gene methylation. These hypermethylated genes may constitute potential biomarkers for papillary thyroid cancer.

Serum TFF1 and TFF3 but not TFF2 are higher in women with breast cancer than in women without breast cancer

Breast cancer remains a common malignancy in women, but the take-up for breast cancer screening programs in Japan is still low, possibly due to its perceived inconvenience. TFF1 and TFF3 are expressed in both breast cancer tissue and normal breast. Serum trefoil proteins were reported as cancer screening markers for gastric, prostate, lung, pancreatic cancer and cholangio carcinoma. The purpose of this study was to examine whether serum trefoil proteins could be screening biomarkers for breast cancer. Serum trefoil proteins in 94 breast cancer patients and 84 health check females were measured by ELISA. Serum TFF1 and TFF3 were significantly higher and serum TFF2 was significantly lower in breast cancer patients. Area under the curve of receiver operating characteristic of TFF1, TFF2, and TFF3 was 0.69, 0.83, and. 0.72, respectively. AUC of the combination of TFF1, TFF2, and TFF3 was 0.96. Immunohistochemically, TFF1 expression was positive in 56.5% and TFF3 was positive in 73.9% of breast cancers, while TFF2 was negative in all tumors. Serum TFF1 had positive correlation with expression of TFF1 in breast cancer tissue. Serum concentrations of TFF1 and TFF3 but not TFF2 are higher in women with breast cancer than in women without breast cancer.

Intratumoral bidirectional transitions between epithelial and mesenchymal cells in triple‐negative breast cancer

Epithelial–mesenchymal transition (EMT) and its reverse process, mesenchymal–epithelial transition MET, are crucial in several stages of cancer metastasis. Epithelial–mesenchymal transition allows cancer cells to move to proximal blood vessels for intravasation. However, because EMT and MET processes are dynamic, mesenchymal cancer cells are likely to undergo MET transiently and subsequently re‐undergo EMT to restart the metastatic process. Therefore, spatiotemporally coordinated mutual regulation between EMT and MET could occur during metastasis. To elucidate such regulation, we chose HCC38, a human triple‐negative breast cancer cell line, because HCC38 is composed of epithelial and mesenchymal populations at a fixed ratio even though mesenchymal cells proliferate significantly more slowly than epithelial cells. We purified epithelial and mesenchymal cells from Venus‐labeled and unlabeled HCC38 cells and mixed them at various ratios to follow EMT and MET. Using this system, we found that the efficiency of EMT is approximately an order of magnitude higher than that of MET and that the two populations significantly enhance the transition of cells from the other population to their own. In addition, knockdown of Zinc finger E‐box‐binding homeobox 1 (ZEB1) or Zinc finger protein SNAI2 (SLUG) significantly suppressed EMT but promoted partial MET, indicating that ZEB1 and SLUG are crucial to EMT and MET. We also show that primary breast cancer cells underwent EMT that correlated with changes in expression profiles of genes determining EMT status and breast cancer subtype. These changes were very similar to those observed in EMT in HCC38 cells. Consequently, we propose HCC38 as a suitable model to analyze EMT–MET dynamics that could affect the development of triple‐negative breast cancer.

Post-mastectomy radiation therapy in breast cancer with 1–3 involved lymph nodes: the Pros

In 2014, the Early Breast Cancer Trialists’ Collaborative Group (EBCTCG) reported that post-mastectomy radiation therapy (PMRT) for breast cancer patients with 1–3 cancer-positive lymph nodes is associated with a survival benefit. However, it is not known whether this applies to Japanese patients in daily clinical practice, because this conclusion was based on the results of older, western trials. Therefore, we studied the differences between PMRT results in western breast cancer patients and current practice in Japanese patients. Although we identified three differences, they do not appear to strongly impact the results of EBCTCG. We conclude that Japanese breast cancer patients with 1–3 positive lymph nodes should receive PMRT in daily clinical practice.

A multicenter, observational study of metastatic breast cancer patients who were treated with eribulin mesylate or taxane-based regimens

This multicenter, observational study aimed to investigate the survival benefit of eribulin as well as that of taxane‐based regimens in Japanese patients with metastatic breast cancer (MBC) in a real‐world setting.

PO54 A RETROSPECTIVE MULTICENTER OBSERVATION STUDY IN METASTATIC BREAST CANCER PATIENTS: COMPARATIVE ANALYSIS ON EFFICACY OF ERIBULIN MESYLATE WITH TAXANE REGIMENS (INCLUDING COMBINATION WITH BEVACIZUMAB)

Background: Interest in oral agents for the treatment of metastatic breast cancer (MBC) has increased due to patients’ preference for oral therapy over intravenous (IV) therapy. However, although the quality of life is better with oral therapy, patients are generally unwilling to sacrifice efficacy when prioritizing oral over IV therapy. Capecitabine (X) and cyclophosphamide (C) can be orally administered and have synergistic effects with non-overlapping toxicities in patients with MBC. Clinically, we have observed heavily pretreated patients with MBC with excellent efficacy with XC therapy and less toxicity. In this study, we retrospectively evaluated the efficacy and safety of an oral combination of XC therapy. Methods: A retrospective review was conducted of human epidermal growth factor receptor (HER)2-negative patients with MBC who received XC therapy at Hiroshima City Hiroshima Citizens Hospital; adverse events, time to progression (TTP), and overall survival (OS) were used to evaluate the clinical response to XC therapy. Doses of X and C were determined according to the phase I results from the Kyushu Breast Cancer Study Group (Ohno S et al. Anticancer Research, 2007). A dose of 1657 mg/m2/day capecitabine and 65 mg/m2/day cyclophosphamide was given orally twice daily for 14 days. The treatment was repeated at 3-week intervals until disease progression or treatment interruption due to adverse events. Results: Between 2009 and 2015, we analyzed 71 patients with MBC (median age, 56; range, 27–85 years), with a median previous chemotherapy regimen (range, 0–7). The overall response rate was 40.3%; the response rate (RR) was 41.2% in hormone receptor (HR)positive cancers and 35.0% in HR-negative cancers. The median TTP was 273 days [95% confidence interval (CI), 224–363 days) and median OS was 1045 days (95% CI, 665–1749 days). The median TTP was 197 days in patients who required extended interval or dose reduction and 326 days in patients on standard treatment regimes (p = 0.0047). Further, median TTP was 197 days in patients who suffered from hand-foot syndrome (HFS), whereas 284 days in patients without HFS (p = 0.0362). Grade 3 or 4 leukopenia was observed in 12 cases (16.9%), neutropenia in 10 (14.1%), anemia in 3 (4.2%), and thrombocytopenia in 0 cases (0%). Non-hematological toxicities were mild. HFS was observed in 14 cases (19.7%), although no cases of grade 4 HFS occurred. Only two patients with grade 3 hemorrhagic cystitis interrupted therapy due to adverse events (cyclophosphamide side-effects). Conclusion: Oral XC therapy was very effective with less toxicity in HER2-negative MBC. XC therapy may be a promising oral chemotherapy regime in light of its cost-effectiveness, quality of life, and preference of patients with MBC.