TP53 variants in p53 signatures and the clonality of STICs in RRSO samples.

OBJECTIVE: Precursor lesions may be identified in fallopian tube tissue after risk-reducing salpingo-oophorectomy (RRSO) in patients with pathogenic variants of BRCA1/2. Serous tubal intraepithelial carcinoma (STIC) is considered a precursor of high-grade serous carcinoma, whereas the significance of the p53 signature remains unclear. In this study, we investigated the relationship between the p53 signature and the risk of ovarian cancer. METHODS: We analyzed the clinicopathological findings and conducted DNA sequencing for TP53 variants of p53 signatures and STIC lesions isolated using laser capture microdissection in 13 patients with pathogenic variants of BRCA1/2 who underwent RRSO and 17 control patients with the benign gynecologic disease. RESULTS: TP53 pathogenic variants were detected significantly higher in RRSO group than control (p<0.001). No difference in the frequency of p53 signatures were observed between groups (53.8% vs 29.4%; p=0.17). TP53 sequencing and next-generation sequencing analysis in a patient with STIC and occult cancer revealed 2 TP53 mutations causing different p53 staining for STICs and another TP53 mutation shared between STIC and occult cancer. CONCLUSION: The sequence analysis for TP53 revealed 2 types of p53 signatures, one with a risk of progression to STIC and ovarian cancer with pathological variants in TP53 and the other with a low risk of progression without pathological variants in TP53 as seen in control.

Germline multigene panel testing revealed a BRCA2 pathogenic variant in a patient with suspected Lynch syndrome.

There has been a rapid advance in germline multigene panel testing by next-generation sequencing, and it is being widely used in clinical settings. A 56-year-old woman suspected of having Lynch syndrome was identified as a BRCA2 pathogenic variant carrier by multigene panel testing. The patient was diagnosed with endometrial cancer at the age of 39 years, and total laparoscopic hysterectomy and bilateral salpingectomy were performed at the age of 49 years; however, bilateral oophorectomy was not performed at that time. As she had a family history of colorectal cancer and a history of endometrial cancer, Lynch syndrome was suspected. However, germline multigene panel testing revealed a pathogenic BRCA2 variant rather than pathogenic variants in mismatch repair genes. In this case, with conventional genetic risk assessment, we were unable to determine whether the patient had a high risk of hereditary breast and ovarian cancer; thus, germline multigene panel testing may provide valuable information to improve disease management strategies for patients in clinical settings. Particularly, germline multigene panel testing may be useful for detecting hereditary tumor syndromes if a patient does not present with a typical family history of cancer.

Patient-derived ovarian cancer organoids capture the genomic profiles of primary tumours applicable for drug sensitivity and resistance testing.

The use of primary patient-derived organoids for drug sensitivity and resistance testing could play an important role in precision cancer medicine. We developed expandable ovarian cancer organoids in < 3 weeks; these organoids captured the characteristics of histological cancer subtypes and replicated the mutational landscape of the primary tumours. Seven pairs of organoids (3 high-grade serous, 1 clear cell, 3 endometrioid) and original tumours shared 59.5% (36.1-73.1%) of the variants identified. Copy number variations were also similar among organoids and primary tumours. The organoid that harboured the BRCA1 pathogenic variant (p.L63*) showed a higher sensitivity to PARP inhibitor, olaparib, as well as to platinum drugs compared to the other organoids, whereas an organoid derived from clear cell ovarian cancer was resistant to conventional drugs for ovarian cancer, namely platinum drugs, paclitaxel, and olaparib. The overall success rate of primary organoid culture, including those of various histological subtypes, was 80% (28/35). Our data show that patient-derived organoids are suitable physiological ex vivo cancer models that can be used to screen effective personalised ovarian cancer drugs.

Establishment and characterization of a new malignant peritoneal mesothelioma cell line, KOG-1, from the ascitic fluid of a patient with pemetrexed chemotherapy resistance.

Malignant peritoneal mesothelioma (MPeM) is a rare and aggressive form of malignant mesothelioma. Sufficient biological tools for studying the functional characteristics of this cancer have not been developed. Therefore, in this study, a novel human cancer cell line, KOG-1, was established from ascites fluids isolated from a 39-year-old Japanese woman with pemetrexed-resistant MPeM. Cells were dendritic or linear immediately after thawing, showed a jigsaw puzzle-like and spindle arrangement during growth, and formed monolayers without contact inhibition in two-dimensional (2D) culture. The population doubling time was 13.7 h. Karyotypic and molecular genetic analyses showed that chromosome numbers ranged from 62 to 142, with a peak of 73 with complicated copy number alterations. No germline BAP1 pathogenic variant was detected. Cells expressed various tumor markers of mesothelioma, such as calretinin, podoplanin, and Wilms tumor 1 (WT-1). Drug sensitivity and resistance testing with a set of 36 drugs using 2D and three-dimensional (3D) culture models demonstrated that KOG-1 cells showed high and low sensitivity to pemetrexed under 2D and 3D culture conditions, respectively, whereas control ovarian cancer cell lines showed low sensitivity to pemetrexed under both culture conditions. This newly established cell line will be a valuable biological resource to expand the feasibility of functional studies as well as drug testing for potential therapeutic purposes in MPeM.

GSTP1 rs1695 is associated with both hematological toxicity and prognosis of ovarian cancer treated with paclitaxel plus carboplatin combination chemotherapy: a comprehensive analysis using targeted resequencing of 100 pharmacogenes.

PURPOSE: To find genetic variants that predicted toxicity and/or efficacy of paclitaxel plus carboplatin combination therapy (TC therapy). PATIENTS AND METHODS: In a retrospective case-control study, we analyzed 320 patients who had received TC therapy for gynecological cancers (ovarian, fallopian tube, peritoneal, uterine, and cervical cancers) and collected their germline DNA. We performed a comprehensive pharmacogenomic analysis using a targeted resequencing panel of 100 pharmacogenes. For 1,013 variants passing QC, case-control association studies and survival analyses were conducted. RESULTS: GSTP1 rs1695 showed the smallest p value for hematotoxicity association, and the 105Ile wild type allele had a significantly higher risk of severe hematotoxicity (neutropenia G4, thrombocytopenia ≥ G3 and anemia ≥ G3) than the 105Val allele (p=0.00034, odds ratio=5.71 (95% confidence interval:1.77-18.44)). Next, we assessed 5-year progression-free survival (PFS) and overall survival (OS) in 56 advanced ovarian cancer patients who received tri-weekly TC as a first-line chemotherapy. Patients with the 105Ile/105Ile genotype showed significantly better PFS (p=0.00070) and OS (p=0.0012) than those with the 105Ile/105Val or 105Val/105Val genotype. CONCLUSION: Our study indicates that the GSTP1 rs1695 105Ile/105Ile genotype is associated with both severe hematotoxicity and high efficacy of TC therapy, identifying a possible prognostic indicator for patients with TC therapy.

Clinical utility of a self-administered questionnaire for assessment of hereditary gynecologic cancer.

BACKGROUND: A patient's medical history and familial cancer history are important information for assessing the risk of hereditary cancer. We have generated a self-administered questionnaire for patients with gynecologic cancer. This pilot study analyzed the usefulness of this questionnaire and the rates of patients that meet the Society of Gynecologic Oncology criteria in ovarian cancer and endometrial cancer patients. METHOD: Ovarian or endometrial cancer patients were recruited for this study. After informed consent was obtained, participants completed the questionnaire. Genetic risks were assessed from the data of each patient's questionnaire by Society of Gynecologic Oncology guideline. Clinical and pathological findings were compared between the genetic risk groups. RESULTS: A total of 105 patients were identified with ovarian cancer and 56 patients with endometrial cancer eligible for this study. According to the Society of Gynecologic Oncology guideline, of the 105 ovarian cancer patients, 25 patients (23%) had a 20-25% risk and three patients (2.9%) had a 5-10% risk of hereditary breast and ovarian cancer syndrome. A further 22 patients (21%) had a 5-10% risk of Lynch syndrome. Two patients (1.9%) met the Amsterdam criteria II. Of 56 endometrial cancer patients, 24 patients (42.9%) had a 5-10% risk of Lynch syndrome. The endometrial cancer patients with genetic risk of Lynch syndrome were younger (mean age: 47.79) at diagnosis compared to patients without a genetic risk of Lynch syndrome (mean age: 57.91). CONCLUSIONS: In this study, we were able to show that the newly designed questionnaire is a useful tool for evaluating cancer family history along with Society of Gynecologic Oncology criteria or Amsterdam criteria II. When considering the risk of Lynch syndrome for a patient with ovarian cancer, it is important to collect a second and third relative's family history.

UGT1A1 polymorphism as a prognostic indicator of stage I ovarian clear cell carcinoma patients treated with irinotecan.

We investigated whether UGT1A1 polymorphisms are associated with the prognosis of ovarian cancer patients treated with irinotecan. UGT1A1 genotypes were analyzed in 11 stage I ovarian clear cell carcinoma patients who received irinotecan as first-line therapy. Progression-free survival, overall survival and adverse events were also assessed for each genotype. Three patients harbored UGT1A1*1/*6 while another three harbored UGT1A1*1/*28. Two patients with a wild-type genotype experienced recurrence and one died, whereas no recurrence or death was observed in patients with heterozygous genotypes. Adverse events tended to be more severe in patients with UGT1A1*6 and *28, although progression-free survival and overall survival rates tended to be better than in wild-type; the differences were not significant. We conclude that UGT1A1 polymorphisms have the potential to be a prognostic marker of irinotecan treatment.

Prevalence of pathogenic germline variants detected by multigene sequencing in unselected Japanese patients with ovarian cancer.

Pathogenic germline BRCA1, BRCA2 (BRCA1/2), and several other gene variants predispose women to primary ovarian, fallopian tube, and peritoneal carcinoma (OC), although variant frequency and relevance information is scarce in Japanese women with OC. Using targeted panel sequencing, we screened 230 unselected Japanese women with OC from our hospital-based cohort for pathogenic germline variants in 75 or 79 OC-associated genes. Pathogenic variants of 11 genes were identified in 41 (17.8%) women: 19 (8.3%; BRCA1), 8 (3.5%; BRCA2), 6 (2.6%; mismatch repair genes), 3 (1.3%; RAD51D), 2 (0.9%; ATM), 1 (0.4%; MRE11A), 1 (FANCC), and 1 (GABRA6). Carriers of BRCA1/2 or any other tested gene pathogenic variants were more likely to be diagnosed younger, have first or second-degree relatives with OC, and have OC classified as high-grade serous carcinoma (HGSC). After adjustment for these variables, all 3 features were independent predictive factors for pathogenic variants in any tested genes whereas only the latter two remained for variants in BRCA1/2. Our data indicate similar variant prevalence in Japanese patients with OC and other ethnic groups and suggest that HGSC and OC family history may facilitate genetic predisposition prediction in Japanese patients with OC and referring high-risk patients for genetic counseling and testing.

Polymorphisms of estrogen metabolism-related genes ESR1, UGT2B17, and UGT1A1 are not associated with osteoporosis in surgically menopausal Japanese women.

INTRODUCTION: Bilateral salpingo-oophorectomy (BSO) is a risk factor for osteoporosis. Previous studies have reported an association between genetic polymorphisms and the risk of developing osteoporosis. However, the relationship between osteoporosis and genetic polymorphisms in Japanese women treated with BSO is not well understood. To improve the quality of life for post-BSO patients, it is important to determine the genetic factors that influence their risk for osteoporosis. The aim of this study was to investigate the association between gene variations of estrogen metabolism-related genes and osteoporosis in surgically menopausal patients, which may improve their quality of life. MATERIAL AND METHODS: This study included 203 menopausal women treated with BSO because of gynecologic disorders. One hundred and twenty-six women with artificial (surgical) menopause, who had undergone BSO in the premenopausal period, were compared with 77 women with natural menopause, who had undergone BSO in the postmenopausal period. The women were tested for bone mineral density to diagnose osteoporosis. Polymorphisms of estrogen receptor 1 (ESR1) and UDP-glucuronosyl transferase (UGT) genes UGT2B17 and UGT1A1 were analyzed, and their association with bone mass and osteoporosis was statistically evaluated. RESULTS: No significant association was found between osteoporosis and polymorphisms in ESR1, UGT2B17, or UGT1A1 in both groups, suggesting that BSO might be a more significant physiological factor in influencing bone mass density compared to genetic variations. CONCLUSIONS: These results suggest that the ESR1, UGT2B17, and UGT1A1 polymorphisms are not genetic factors affecting osteoporosis in postmenopausal Japanese women.

Osteoporosis is less frequent in endometrial cancer survivors with hypertriglyceridemia.

We previously reported an association between dyslipidemia and endometrial cancers. Osteoporosis is also reported to relate with some cancers. A common etiologic event has been proposed between dyslipidemia and osteoporosis. However, the pattern of interrelationships among dyslipidemia, osteoporosis and endometrial cancer is not well understood. To improve the quality of life of endometrial cancer survivors, these relationships should be determined. This study included 179 Japanese menopausal women who underwent bilateral salpingo-oophorectomy, including 114 women with incident endometrial cancer and 65 without endometrial cancer. The women were categorized according to dyslipidemia status. Bone mineral density was measured and compared between groups. Osteoporosis was statistically more frequent in women with hypertriglyceridemia who did not have endometrial cancer. In contrast, osteoporosis was statistically less frequent in women with hypertriglyceridemia who had endometrial cancer. In this cross-sectional study in a Japanese population, osteoporosis was associated with hypertriglyceridemia in post-menopausal women without endometrial cancer, but was less frequent in endometrial cancer survivors with hypertriglyceridemia.

Family history and BRCA1/BRCA2 status among Japanese ovarian cancer patients and occult cancer in a BRCA1 mutant case.

BACKGROUND: This study aimed to examine family history among Japanese ovarian cancer patients and to investigate the TP53 status of fallopian tube epithelial and ovarian cancer cells in a Japanese BRCA1 mutant case that may be associated with the transformed state in hereditary ovarian cancer. METHODS: One hundred and two primary ovarian cancer patients were retrospectively evaluated in this cross-sectional study. The family history of cancer was determined in probands. In a BRCA1 mutant case, p53 immunostaining and direct sequencing, followed by laser-capture microdissection, were performed for the fallopian tube, considered the origin of ovarian cancer. RESULTS: Nine of 102 (8.8%) families were regarded as having hereditary breast-ovarian cancer syndrome, two families (2.0%) were diagnosed with Lynch syndrome and six patients harbored BRCA1 or BRCA2 mutations. One case underwent risk-reductive salpingo-oophorectomy as a BRCA1 mutant carrier was retrospectively diagnosed as occult cancer. Common TP53 mutations were detected in cancer and fallopian tube epithelial cells in the case. CONCLUSIONS: Here, we integrate family cancer history and histology in ovarian cancer cases as well as TP53 status in a BRCA1 mutant case into a discussion regarding carcinogenesis in a Japanese population. The TP53 status for the BRCA1 mutant case examined here supports the recently proposed theory that ovarian cancer develops because of BRCA1 or BRCA2 inactivation and/or TP53 mutations.

Polymorphisms in the UGT1A1 gene predict adverse effects of irinotecan in the treatment of gynecologic cancer in Japanese patients.

Irinotecan is a key chemotherapeutic drug used to treat many tumors, including cervical and ovarian cancers; however, irinotecan can cause toxicity, particularly in the presence of uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) gene polymorphisms, which are associated with reduced enzyme activity. Here, we investigated the prevalence of three different variants of UGT1A1 (UGT1A1*6, UGT1A1*27 and UGT1A1*28) and their relationships with irinotecan-induced adverse events in patients with gynecologic cancer, who are treated with lower doses of irinotecan than patients with other types of solid tumors. Fifty-three female patients treated with irinotecan and 362 female patients not treated with irinotecan were screened for UGT1A1*6, UGT1A1*27 and UGT1A1*28. Homozygosity for UGT1A1*6 or heterozygosity for UGT1A1*6/*28 was associated with a high risk of severe absolute neutrophil count decrease or diarrhea (odds ratios: 16.03 and 31.33, respectively). In contrast, serum bilirubin levels were not associated with irinotecan toxicity. Homozygosity for UGT1A1*6/*6 and heterozygosity for UGT1A1*6/*28 were associated with an increased risk of absolute neutrophil count and/or diarrhea in Japanese gynecologic cancer patients, despite the lower doses of irinotecan used in these patients. UGT1A1*6 and UGT1A1*28 are potential predictors of severe absolute neutrophil decrease and diarrhea caused by low-dose irinotecan in gynecologic cancer patients.

Hypertriglyceridemia is frequent in endometrial cancer survivors.

OBJECTIVE: Previous studies have reported an association between endometrial cancer and the risk of metabolic syndrome; however, the pattern of endometrial cancer-associated dyslipidemia is not well understood. The standard therapy for endometrial cancer is total abdominal hysterectomy and bilateral salpingo-oophorectomy. Premenopausal bilateral salpingo-oophorectomy may cause adverse events, including dyslipidemia. Gynecologists have to care dyslipidemia in endometrial cancer survivors at cancer follow-up clinic. METHODS: This study included 693 patients who had undergone bilateral salpingo-oophorectomy, and included 412 women with incident endometrial cancer and 281 controls. We divided the patients into two categories according to whether they had a premenopausal or postmenopausal bilateral oophorectomy. Serum lipid levels were measured and statistically analyzed. RESULTS: Hypertriglyceridemia was statistically more frequent in patients who had undergone bilateral salpingo-oophorectomy both before and after menopause than in the corresponding non-endometrial cancer controls. High levels of low-density lipoprotein cholesterol and a high low-density lipoprotein cholesterol/high-density lipoprotein cholesterol ratio were statistically more frequent in patients who had undergone bilateral salpingo-oophorectomy before menopause than in non-endometrial cancer controls. CONCLUSIONS: Our report highlights the importance of the relationship between endometrial cancer and lipid metabolism, which may aid in preventing cerebrovascular or cardiovascular diseases due to dyslipidemia and improving the quality of life in endometrial cancer survivors.

Experience of risk-reducing salpingo-oophorectomy for a BRCA1 mutation carrier and establishment of a system performing a preventive surgery for hereditary breast and ovarian cancer syndrome in Japan: our challenges for the future.

BACKGROUND: Risk-reducing salpingo-oophorectomy is currently regarded as the most certain primary method for preventing ovarian cancer among BRCA1/2 mutation carriers with hereditary breast and ovarian cancer syndrome. However, risk-reducing salpingo-oophorectomy has rarely been performed in Japan. METHODS: We developed the first system in Japan for performing risk-reducing salpingo-oophorectomy for BRCA1/2 mutation carriers at our university hospital in 2008. RESULTS: The indication for risk-reducing salpingo-oophorectomy for patients with hereditary breast/ovarian cancer syndrome is currently limited in Japan. This situation may be because of the limited number of genetic counseling units, the limited number of facilities that can perform BRCA1/2 genetic testing and the fact that prophylactic surgery is not covered by health insurance in Japan. CONCLUSIONS: Recent treatment guidelines for breast cancer in Japan recommended risk-reducing salpingo-oophorectomy for BRCA1/2 mutation carriers. Risk-reducing salpingo-oophorectomy should be performed in the framework of the standard therapeutic modality for BRCA1/2 mutation carriers in the near future.

The origin of stroma surrounding epithelial ovarian cancer cells.

Cancer stroma is thought to play an important role in tumor behavior, including invasion or metastasis and response to therapy. Cancer stroma is generally thought either to be non-neoplastic cells, including tissue-marrow or bone-marrow-derived fibroblasts, or to originate in epithelial mesenchymal transition of cancer cells. In this study, we evaluated the status of the p53 gene in both the cancer cells and the cancer stroma in epithelial ovarian cancer (EOC) to elucidate the origin of the stroma. Samples from 16 EOC patients were included in this study. Tumor cells and adjacent nontumor stromal cells were microdissected and DNA was extracted separately. We analyzed p53 sequences (exons 5-8) of both cancer and stromal tissues in all cases. Furthermore, we examined p53 protein expression in all cases. Mutations in p53 were detected in 9 of the 16 EOCs: in 8 of these cases, the mutations were detected only in cancer cells. In 1 case, the same mutation (R248Q) was detected in both cancer and stromal tissues, and p53 protein expression was detected in both the cancer cells and the cancer stroma. Most cancer stroma in EOC is thought to originate from non-neoplastic cells, but some parts of the cancer stroma might originate from cancer cells.

EGRI and FOSB gene expressions in cancer stroma are independent prognostic indicators for epithelial ovarian cancer receiving standard therapy.

Stromal components interact with cancer cells to promote growth and metastasis. The purpose of this study was to identify genes expressed in stroma, which could provide prognostic information in epithelial ovarian cancer (EOC). Seventy-four patients were included. We performed gene expression profiling and confirmed array data using RT-PCR and immunohistochemistry. By microarray analysis, 52 candidate genes associated with progression free survival (PFS) were identified (P < 0.005). Expression of the early growth response 1 (EGR1) and FBJ murine osteosarcoma viral oncogene homolog B (FOSB) genes was further analyzed. Array data were confirmed by RT-PCR and multivariate analysis demonstrated that both EGR1 and FOSB expression in cancer stroma, and EGR1 expression in cancer are independent prognostic factors in EOC. Immunohistochemically, EGR1 protein is localized in cancer cells and α-smooth muscle actin positive stromal fibroblasts. The EGR1 and FOSB expression in stromal cells and EGR1 expression in cancer cells are prognostic indicators in EOC.

Blood-direct InvaderPlus® as a new method for genetic testing.

AIM: This study compared the efficiency of Blood-direct InvaderPlus® with existing methods for detecting genetic polymorphisms by using clinical samples, and to determine whether this new method can be used for future clinical studies. MATERIALS & METHODS: We developed a new method, called Blood-direct InvaderPlus, for detecting genetic polymorphisms. Whole-blood samples were subjected to genetic analysis with the new method and also the conventional method. RESULTS: The results obtained with the new method were consistent with those of direct sequencing. Blood-direct InvaderPlus was able to detect gene polymorphisms of UGT1A1*6, *27, *28, *60, VKORC1 -1639G>A, VKORC1 1173T>C and CYP2C9 *2 and *3 much faster than the conventional methods. CONCLUSION: Blood-direct InvaderPlus allowed accurate, simple and rapid detection of genetic polymorphisms.

A genome-wide association study identifies genetic variants in the CDKN2BAS locus associated with endometriosis in Japanese.

Although the pathogenesis of endometriosis is not well understood, genetic factors have been considered to have critical roles in its etiology. Through a genome-wide association study and a replication study using a total of 1,907 Japanese individuals with endometriosis (cases) and 5,292 controls, we identified a significant association of endometriosis with rs10965235 (P = 5.57 x 10(-12), odds ratio = 1.44), which is located in CDKN2BAS on chromosome 9p21, encoding the cyclin-dependent kinase inhibitor 2B antisense RNA. By fine mapping, the SNP showing the strongest association was located in intron 16 of CDKN2BAS and was implicated in regulating the expression of p15, p16 and p14. A SNP, rs16826658, in the LD block including WNT4 on chromosome 1p36, which is considered to play an important role in the development of the female genital tract, revealed a possible association with endometriosis (P = 1.66 x 10(-6), odds ratio = 1.20). Our findings suggest that these regions are new susceptibility loci for endometriosis.