Spatial genomic diversity associated with APOBEC mutagenesis in squamous cell carcinoma arising from ovarian teratoma.

Although the gross and microscopic features of squamous cell carcinoma arising from ovarian mature cystic teratoma (MCT-SCC) vary from case to case, the spatial spreading of genomic alterations within the tumor remains unclear. To clarify the spatial genomic diversity in MCT-SCCs, we performed whole-exome sequencing by collecting 16 samples from histologically different parts of two MCT-SCCs. Both cases showed histological diversity within the tumors (case 1: nonkeratinizing and keratinizing SCC and case 2: nonkeratinizing SCC and anaplastic carcinoma) and had different somatic mutation profiles by histological findings. Mutation signature analysis revealed a significantly enriched apolipoprotein B mRNA editing enzyme catalytic subunit (APOBEC) signature at all sites. Intriguingly, the spread of genomic alterations within the tumor and the clonal evolution patterns from nonmalignant epithelium to cancer sites differed between cases. TP53 mutation and copy number alterations were widespread at all sites, including the nonmalignant epithelium, in case 1. Keratinizing and nonkeratinizing SCCs were differentiated by the occurrence of unique somatic mutations from a common ancestral clone. In contrast, the nonmalignant epithelium showed almost no somatic mutations in case 2. TP53 mutation and the copy number alteration similarities were observed only in nonkeratinizing SCC samples. Nonkeratinizing SCC and anaplastic carcinoma shared almost no somatic mutations, suggesting that each locally and independently arose in the MCT. We demonstrated that two MCT-SCCs with different histologic findings were highly heterogeneous tumors with clearly different clones associated with APOBEC-mediated mutagenesis, suggesting the importance of evaluating intratumor histological and genetic heterogeneity among multiple sites of MCT-SCC.

Biological significance of KRAS mutant allele expression in ovarian endometriosis.

KRAS is the most frequently mutated in ovarian endometriosis. However, it is unclear whether the KRAS mutant allele's mRNA is expressed and plays a biological role in ovarian endometriosis. Here, we performed mutation-specific RNA in situ hybridization to evaluate mutant allele expression of KRAS p.G12V, the most frequently detected mutation in ovarian endometriosis in our previous study, in formalin-fixed paraffin-embedded tissue (FFPE) samples of ovarian endometriosis, cancer cell lines, and ovarian cancers. First, we verified that mutant or wild-type allele of KRAS were expressed in all 5 cancer cell lines and 9 ovarian cancer cases corresponding to the mutation status. Next, we applied this assay to 26 ovarian endometriosis cases, and observed mutant allele expression of KRAS p.G12V in 10 cases. Mutant or wild-type allele of KRAS were expressed in line with mutation status in 12 available endometriosis cases for which KRAS gene sequence was determined. Comparison of clinical features between ovarian endometriosis with KRAS p.G12V mutant allele expression and with KRAS wild-type showed that KRAS p.G12V mutant allele expression was significantly associated with inflammation in ovarian endometriosis. Finally, we assessed the spatial distribution of KRAS mutant allele expression in 5 endometriosis cases by performing multiregional sampling. Intratumor heterogeneity of KRAS mutant allele expression was observed in two endometriosis cases, whereas the spatial distribution of KRAS p.G12V mutation signals were diffuse and homogenous in ovarian cancer. In conclusion, evaluation of oncogene mutant expression will be useful for clarifying the biological significance of oncogene mutations in benign tumors.

Proposing a molecular classification associated with hypercoagulation in ovarian clear cell carcinoma.

BACKGROUND: Although ovarian clear cell carcinoma (CCC) is associated with high incidence of thromboembolism, the clinicopathological and biological significance of hypercoagulable status in CCC remains unclear. MATERIALS AND METHODS: We retrospectively analyzed pretreatment D-dimer levels, thromboembolic status, and clinical outcome of 125 CCCs in the discovery set and 143 CCCs in two other independent validation sets. Next, we performed RNA sequencing of 93 CCCs and compared coagulation-related gene profiles with 2492 pan-cancer data. We investigated differences in molecular characteristics of CCC subclasses based on coagulation status. RESULTS: In the discovery dataset, D-dimer elevation above the normal range was significantly associated with shorter progression-free and overall survival, irrespective to thromboembolic status. Multivariate analysis identified D-dimer elevation and clinical stage as an independent prognostic factors. We confirmed the prognostic significance of D-dimer elevation in the validation sets. Tissue factor and IL6, which are considered key elements of cancer-induced hypercoagulation, were highly expressed in CCC than in other cancers regardless of D-dimer level. Higher activity of various oncogenic pathways was observed in CCC with compared to without D-dimer elevation. Moreover, hierarchical cluster analysis divided 57 CCCs with D-dimer elevation into immunologically hot and cold tumor subtypes. Hot tumors were characterized by enrichment of T-cell inflamed phenotype, inflammation, the epithelial-mesenchymal transition, and high serum levels of CRP, and cold tumors by enrichment of cell cycle and MYC pathways. CONCLUSIONS: CCC represents hypercoagulable disease and elevate D-dimer is a prognostic factor for decreased survival in CCC. D-dimer high CCC has distinct molecular characteristics into the inflammatory-driven pathway (hot tumor) and the immune-suppressive pathway (cold tumor). Treatment implication of our proposed molecular classification merits further investigation.

Clonal lineage from normal endometrium to ovarian clear cell carcinoma through ovarian endometriosis.

Clear cell carcinoma of the ovary is thought to arise from endometriosis. In addition, retrograde menstruation of shed endometrium is considered the origin of endometriosis. However, little evidence supports cellular continuity from uterine endometrium to clear cell carcinoma through endometriosis at the genomic level. Here, we performed multiregional whole-exome sequencing to clarify clonal relationships among uterine endometrium, ovarian endometriosis and ovarian clear cell carcinoma in a 56-year-old patient. Many somatic mutations including cancer-associated gene mutations in ARID1A, ATM, CDH4, NRAS and PIK3CA were shared among epithelium samples from uterine endometrium, endometriotic lesions distant from and adjacent to the carcinoma, and the carcinoma. The mutant allele frequencies of shared mutations increased from uterine endometrium to distant endometriosis, adjacent endometriosis, and carcinoma. Although a splice site mutation of ARID1A was shared among the four epithelium samples, a frameshift insertion in ARID1A was shared by adjacent endometriosis and carcinoma samples, suggesting that the biallelic mutations triggered malignant transformation. Somatic copy number alterations, including loss of heterozygosity events at PIK3CA and ATM, were identified only in adjacent endometriosis and carcinoma, suggesting that mutant allele-specific imbalance is another key factor driving malignant transformation. By reconstructing a clonal evolution tree based on the somatic mutations, we showed that the epithelium samples were derived from a single ancestral clone. Although the study was limited to a single patient, the results from this illustrative case could suggest the possibility that epithelial cells of ovarian endometriosis and clear cell carcinoma were descendants of uterine endometrial epithelium.

Different mutation profiles between epithelium and stroma in endometriosis and normal endometrium.

STUDY QUESTION: Are there common mutation profiles between epithelial and stromal cells in ovarian endometriotic tissue and the normal endometrium? SUMMARY ANSWER: Our study revealed no common mutations between epithelial and stromal cells in ovarian endometriotic tissue and the normal endometrium. WHAT IS KNOWN ALREADY: Epithelial cells in both ovarian endometriotic tissue and the normal endometrium harbor somatic mutations in cancer-associated genes such as phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) and KRAS proto-oncogene, GTPase (KRAS). STUDY DESIGN, SIZE, DURATION: We performed a retrospective study to identify the mutation profiles of stromal cells in endometriotic tissue and the normal endometrium. We collected 11 endometriotic stroma samples and 10 normal endometrial stroma samples between 2013 and 2017 at a tertiary care center. PARTICIPANTS/MATERIALS, SETTING, METHODS: The laser microdissection method was used to obtain stromal cells in ovarian endometriotic and normal endometrial tissues from patients with ovarian endometriosis and/or other non-invasive gynecological diseases. Target gene sequencing was performed to assess and compare the mutation profiles of stromal cells with those of epithelial cells obtained in our previous study. For target gene sequencing, 76 genes were selected based on previous genomic analyses for ovarian endometriosis, normal endometrium, endometriosis-related ovarian cancer and endometrial cancer. MAIN RESULTS AND THE ROLE OF CHANCE: Stromal samples in ovarian endometrioma and normal endometrium harbor somatic mutations (18 mutations in 11 endometriosis samples and 16 mutations in 10 normal endometrial samples) but did not share any mutations with paired epithelial samples. The mutant allele frequency of stromal samples was significantly lower than that of epithelial samples in ovarian endometrioma (P = 6.0 × 10-11) and normal endometrium (P = 1.4 × 10-7). LIMITATIONS, REASONS FOR CAUTION: The number of genes evaluated in the mutational analysis was limited. Additionally, the functional roles of somatic mutations in stromal cells remain unclear. WIDER IMPLICATIONS OF THE FINDINGS: Different mutation profiles between paired epithelial and stromal cells in both ovarian endometrioma and normal endometrium suggest that origins of epithelial and stromal cells would be independent of each other in both normal endometrium and ovarian endometrioma; however, the theory of epithelial-mesenchymal transition is proposed in ovarian endometrioma. STUDY FUNDING/COMPETING INTEREST(S): This work was supported in part by the Japan Society for the Promotion of Science KAKENHI grant number JP15H02373 (Grant-in-Aid for Scientific Research A for I.I.), JP16H06267 (Grant-in-Aid for Young Scientists A for K.Y.), JP17K08688 (Grant-in-Aid for Scientific Research C for H.N.) and JP16H06279 (Grant-in-Aid for Scientific Research on Innovative Areas-Platforms for Advanced Technologies and Research Resources for H.N. and K.Y). There are no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: Not applicable.

Novel MXD4-NUTM1 fusion transcript identified in primary ovarian undifferentiated small round cell sarcoma.

Primary ovarian sarcomas are extremely rare tumors, and their genomic and transcriptomic alterations remain to be elucidated. We performed whole exome sequencing of primary tumor and matched normal blood samples derived from one patient with ovarian undifferentiated small round cell sarcoma. We identified 8 nonsynonymous somatic mutations, and all mutations were missense or nonsense changes. Next, we performed RNA sequencing of the tumor sample and identified two in-frame fusion transcripts: MXD4-NUTM1 and ARL6-POT1. Most NUTM1 exons were retained in the MXD4-NUTM1 fusion transcript, and we confirmed an increase in NUTM1 mRNA and protein expression in tumor tissue. Further genomic and transcriptomic analyses might lead to the development of new therapeutic strategies based on the molecular characteristics of ovarian undifferentiated small round cell sarcoma.

Recurrent ovarian undifferentiated carcinoma resembling hepatoid morphology treated with pegylated liposomal doxorubicin and bevacizumab.

Hepatoid carcinomas are undifferentiated epithelial carcinomas that are pathologically similar to hepatocellular carcinoma, but occur in a variety of organs. Hepatoid carcinomas, as strictly defined, typically produce α-fetoprotein. In addition, a standard effective chemotherapy regimen for hepatoid carcinoma has yet to be established. We present a case of advanced primary ovarian cancer that was pathologically similar to hepatoid carcinoma without staining for α-fetoprotein or hepatocyte paraffin 1. The primary ovarian, metastatic, and recurrent tumors shared similar pathological characteristics. Fourth-line chemotherapy with pegylated liposomal doxorubicin and bevacizumab was effective in treating the recurrent tumor, even though this disease had recurred three times.

Performance of p16INK4a/Ki-67 immunocytochemistry for identifying CIN2+ in atypical squamous cells of undetermined significance and low-grade squamous intraepithelial lesion specimens: a Japanese Gynecologic Oncology Group study.

BACKGROUND: p16(INK4a) immunohistochemistry has revealed a high rate of positivity in cervical intraepithelial neoplasia grade 2 (CIN2) and more severe conditions (CIN2+). The Lower Anogenital Squamous Terminology Standardization project proposed p16(INK4a) immunohistochemistry as an ancillary test for CIN. Immunocytochemistry involving dual staining for p16(INK4a) and Ki-67 in the triage of atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesions (LSIL) is reported to be useful in the identification of CIN2+. However, it is unclear whether p16(INK4a)/Ki-67 immunocytochemistry is of practical relevance for the triage of ASCUS and LSIL in the Japanese screening system. METHODS: From 427 women fulfilling the eligibility criteria, 188 ASCUS and 239 LSIL specimens were analyzed. The accuracy of p16(INK4a)/Ki-67 immunocytochemistry and genotyping of high-risk human papillomaviruses (HPVs) in detecting CIN2+ were compared. RESULTS: p16(INK4a)/Ki-67 immunocytochemistry was positive in 33.5 % (63/188) of ASCUS, and 36.8 % (88/239) of LSIL specimens. The sensitivity and specificity of p16(INK4a)/Ki-67 immunocytochemistry was 87.3 % (95 % confidence interval 78.0-93.8 %) and 76.4 % (71.6-80.8 %), respectively. The positive and negative predictive values were 45.7 % (37.6-54.0 %) and 96.4 % (93.4-98.3 %), respectively; positive and negative likelihood ratios were 3.71 and 0.17, respectively. Using the McNemar test, p16(INK4a)/Ki-67 immunocytochemistry showed equivalent sensitivity but higher specificity than the HPV genotyping test CONCLUSIONS: Compared with high-risk HPV genotyping, p16(INK4a)/Ki-67 immunocytochemistry was a more accurate triage test for identifying CIN2+ in ASCUS and LSIL specimens.

Proteomics identified nuclear N-myc downstream-regulated gene 1 as a prognostic tissue biomarker candidate in renal cell carcinoma.

The aim of this study was to identify proteins with aberrant expression in clear cell renal cell carcinoma (ccRCC), and elucidate their clinical utilities. The protein expression profiles of primary ccRCC tumor tissues and neighboring non-tumor tissues were obtained from 9 patients by two-dimensional difference gel electrophoresis and mass spectrometry. Comparative analysis of 3771 protein spots led to the identification of 73 proteins that were expressed at aberrant levels in tumor tissues compared with non-tumor tissues. Among these 73 proteins, we further focused on N-myc downstream-regulated gene 1 protein (NDRG1). NDRG1 expression is regulated by members of myc family as well as by p53, HIF1A, and SGK1. The biological and clinical significance of NDRG1 is controversial for various malignancies and no detailed studies on NDRG1 have been reported in ccRCC until our study. For the 82 newly enrolled ccRCC patients, immunohistochemical analysis revealed a significant association between nuclear NDRG1 and favorable prognosis (p<0.05). Multivariate analysis demonstrated the role of NDRG1 as an independent factor of progression-free survival (p=0.01). Subsequent in vitro gene suppression assay demonstrated that NDRG1 silencing significantly enhanced cell proliferation and invasion of RCC cells. The cytotoxic effects of NDRG1 up-regulation induced by an iron chelator were also confirmed. These findings suggest that nuclear NDRG1 has tumor suppressive effects, and the NDRG1 expression may have clinical values in ccRCC. Nuclear NDRG1 may provide additional insights on molecular backgrounds of ccRCC progression, and contribute to the development of novel therapeutic strategy.

Refractory ovarian squamous cell carcinoma arising from a seromucinous borderline tumor with squamous overgrowth: A case report.

Ovarian squamous cell carcinoma (SCC) is rare, and most cases arise from ovarian teratomas. Herein, we present a case of ovarian SCC arising from an ovarian seromucinous borderline tumor (SMBT) with squamous overgrowth. A 71-year-old woman an underwent emergency laparotomy due to the rupture of a right ovarian tumor suspected to be a borderline or malignant tumor. We performed a total abdominal hysterectomy, bilateral salpingo-oophorectomy, and partial omentectomy. The postoperative diagnosis was stage IC3 ovarian SCC arising from the SMBT with a squamous overgrowth. Subsequently, she underwent six cycles of combination therapy comprising paclitaxel and carboplatin. Two months after the last chemotherapy treatment, she presented with back pain. A CT scan showed a 14 mm pelvic tumor affecting the ureter, leading to right hydronephrosis. The patient underwent tumor resection and ureteroureterostomy. The pathological diagnosis was keratinizing SCC, representing ovarian cancer recurrence. Eight months after the removal of the recurrent tumor, we found a 35 mm recurrent pelvic tumor causing right hydronephrosis. Additionally, a 20 mm pleural dissemination was identified. Comprehensive genome profiling of recurrent tumor revealed genomic abnormalities in TP53, ARID1A, PTEN, PIK3R1, and CDKN2A/2B. Regarding immunotherapy biomarkers, the microsatellite instability test result was negative, the tumor mutation burden was low, and PD-L1 was highly expressed. The patient was referred to another hospital for participation in an immunotherapy clinical trial for ovarian SCC. This case indicates that refractory ovarian SCC can arise from SMBT. Further evaluation of additional cases is required to identify the molecular biological characteristics of ovarian SCC.

The enhancer of zeste homolog 2 (EZH2), a potential therapeutic target, is regulated by miR-101 in renal cancer cells.

We investigated a prognostic significance and the mechanism of aberrant nuclear expression of EZH2, a histone methyltransferase, in human renal cell carcinoma (RCC). We found nuclear EZH2 in 48 of 100 RCCs and it was significantly correlated with worse survival in RCC patients. We detected a decreased expression of miR-101 in 15 of 54 RCCs. We found that re-expression of miR-101 resulted in EZH2 depletion and decreased renal cancer cell proliferation. Our results show nuclear EZH2 as a prognostic marker of worse survival in human RCC, and identify miR-101 as a negative regulator of EZH2 expression and renal cancer cell proliferation.

Spatiotemporal dynamics of clonal selection and diversification in normal endometrial epithelium.

It has become evident that somatic mutations in cancer-associated genes accumulate in the normal endometrium, but spatiotemporal understanding of the evolution and expansion of mutant clones is limited. To elucidate the timing and mechanism of the clonal expansion of somatic mutations in cancer-associated genes in the normal endometrium, we sequence 1311 endometrial glands from 37 women. By collecting endometrial glands from different parts of the endometrium, we show that multiple glands with the same somatic mutations occupy substantial areas of the endometrium. We demonstrate that "rhizome structures", in which the basal glands run horizontally along the muscular layer and multiple vertical glands rise from the basal gland, originate from the same ancestral clone. Moreover, mutant clones detected in the vertical glands diversify by acquiring additional mutations. These results suggest that clonal expansions through the rhizome structures are involved in the mechanism by which mutant clones extend their territories. Furthermore, we show clonal expansions and copy neutral loss-of-heterozygosity events occur early in life, suggesting such events can be tolerated many years in the normal endometrium. Our results of the evolutionary dynamics of mutant clones in the human endometrium will lead to a better understanding of the mechanisms of endometrial regeneration during the menstrual cycle and the development of therapies for the prevention and treatment of endometrium-related diseases.

RUNX2 expression in developing human bones and various bone tumors.

The heterozygous germline mutation of runt-related protein 2 (RUNX2) causes cleidocranial dysplasia. To clarify the involvement of RUNX2 in human osteogenesis, fetal bones and various bone tumors were immunohistochemically examined. During both membranous and endochondral ossification in the fetus (n= 8), RUNX2 was expressed not only in osteoblastic cells but also in surrounding mesenchymal cells and early stage chondrocytes. Such an expression pattern was recapitulated in bone tumors: RUNX2 was unequivocally expressed in osteosarcoma (n= 20) and fibrous dysplasia (n= 10), regardless of the site of occurrence, cell morphology or amount of neoplastic osteoid. RUNX2 expression was limited to less differentiated cells in chondrogenic tumors (n= 20). We further analyzed whether RUNX2 expression was regulated by bone morphogenetic protein-2 (BMP-2), which is critical for osteoblastic differentiation. With real-time polymerase chain reaction, the RUNX2 mRNA level was correlated with BMP-2 mRNA level, and both levels were significantly higher in three osteosarcoma cell lines than in three chondrosarcoma cell lines. With treatment of recombinant BMP-2, the RUNX2 mRNA level was significantly altered in these cell lines. RUNX2 expression is constitutive in developing and neoplastic human osteogenesis, and is most likely to be regulated by BMP-2.

Pyloric gland metaplasia/differentiation in multiple organ systems in a patient with Peutz-Jegher's syndrome.

Peutz-Jegher's syndrome (PJS) involves multiple organ systems and the development of hamartomatous, metaplastic, or neoplastic lesions of different cell lineages. Among them, glandular lesions are the most common, but their properties are obscure. We report here a 53-year-old woman with PJS who developed multiple hamartomatous polyps in the jejunum and mucinous glandular lesions in multiple organ systems: glandular metaplasia in the urinary bladder; lobular endocervical glandular hyperplasia in the uterine cervix; mucinous metaplasia in the right fallopian tube; mucinous adenoma in the left ovary. Histological and immunohistochemical analyses disclosed that all of the intestinal and extra-intestinal lesions were associated with pyloric gland metaplasia/differentiation across the organ systems. In the general population, the organs described above rarely or infrequently show pyloric gland phenotype, to say nothing of trans-organ involvement. It is strongly suggested that commitment to pyloric gland metaplasia/differentiation is closely associated with PJS.

Identifying prognostic factors in Japanese women with pseudomyxoma peritonei: a retrospective clinico-pathological study of the Tohoku Gynecologic Cancer Unit.

Pseudomyxoma peritonei (PMP) is a rare condition of mucinous ascites associated mainly with mucinous tumors of appendix or ovary. PMP often recurs after treatment and may eventually cause death by abdominal visceral dysfunction via compression with mucinous ascites. Although radical peritonectomy and hyperthermic intra-peritoneal chemotherapy are becoming popular globally, the optimal treatment of PMP has not been established in Japan. We conducted a retrospective multicenter study to clarify the optimal treatment and the prognostic factors of PMP. A total of 23 patients with PMP were analyzed in the Tohoku Gynecologic Cancer Unit (TGCU). Clinical and follow-up data were retrieved and a central pathology review was performed. The median follow-up period was 46 months. Eleven patients underwent complete resection. There were 7 deaths out of 13 recurrences/progressions in this period. All the recurrence/progression was confined to the abdomen. Unexpectedly, neither radical peritonectomy nor hyperthermic intra-peritoneal chemotherapy had been performed, indicating that both radical peritonectomy and hyperthermic intra-peritoneal chemotherapy are not yet popular in Japan. The medians of overall survival and disease-free period were 166 months and 30 months, respectively. Univariate and multivariate analyses revealed that the only prognostic factor was macroscopic residual tumor (P=0.022). Although chemotherapy was not a prognostic factor (P=0.16), those who received intra-peritoneal chemotherapy tended to have a better prognosis than those who received systemic or no chemotherapy (P=0.064). In conclusion, the macroscopic residual tumor is an important prognostic factor in Japanese patients with PMP.

Three-dimensional understanding of the morphological complexity of the human uterine endometrium.

The fundamental morphology of the endometrial glands is not sufficiently understood by 2D observation because these glands have complicated winding and branching patterns. To construct a large picture of the endometrial gland structure, we performed tissue-clearing-based 3D imaging of human uterine endometrial tissue. Our 3D immunohistochemistry and layer analyses revealed that the endometrial glands form a plexus network in the stratum basalis and expand horizontally along the muscular layer, similar to the rhizome of grass. We then extended our method to assess the 3D morphology of tissue affected by adenomyosis, a representative "endometrium-related disease," and observed its 3D morphological features, including the direct invasion of endometrial glands into the myometrium and an ant colony-like network of ectopic endometrial glands within the myometrium. Thus, further understanding of the morphology of the human endometrium based on 3D analysis will lead to the identification of the pathogenesis of endometrium-related diseases.

Alternate mucoid and hyalinized stroma in clear cell carcinoma of the ovary: manifestation of serial stromal remodeling.

The stroma in ovarian clear cell carcinoma often shows alternate mucoid and hyalinized change. The hyalinized stroma is recognized to be an aberrant deposition of basement membrane material produced by tumor cells. The mucoid stroma, however, has drawn far less attention, and its significance remains unclear. We examined 60 ovarian clear cell carcinomas for the distribution and nature of the mucoid stroma. For comparison, 125 other surface epithelial ovarian tumors were examined. Twenty-nine of 60 (48%) clear cell carcinomas showed a mucoid stroma, either focally (21 cases) or diffusely (8 cases). The mucoid stroma in clear cell carcinomas was distinct from that in other surface epithelial tumors as follows: it showed a compact spherule-like appearance, commonly occupying the cores of small papillae. It also exhibited a cribriform pattern, resembling that of adenoid cystic carcinoma. It was rarely associated with stromal cells, despite the presence of abundant glycosaminoglycan including hyaluronan. Alternatively, it was strongly associated with hyalinized stroma. Among 40 clear cell carcinomas that had at least one type of stroma, 26 (65%) had both, either concomitantly or separately. The mucoid stroma tended to attenuate if the hyalinized stroma developed. In vitro, a clear cell carcinoma cell line, HAC-2, formed a spherule-like structure containing hyaluronan in the center, and a significant amount of hyaluronan was detected by latex agglutination immunoturbidimetry, indicating that HAC-2 itself has the potential to produce hyaluronan. All of these facts indicate that the spherule-like mucoid stroma and hyalinized stroma represent different phases of the stromal remodeling process, which is promoted by the deposition of different extracellular matrices produced by clear cell carcinoma cells. The spherule-like mucoid stroma and hyalinized stroma are considered complementary diagnostic signatures of ovarian clear cell carcinoma.

Primary strumal carcinoid tumor of the ovary with multiple bone and breast metastases.

Although primary carcinoid tumor of the ovary is an extremely rare neoplasm, survival is excellent if the disease is confined to one ovary. Herein, we present a case of primary strumal carcinoid tumor of the ovary, stage IA, borderline malignancy, in a 34-year-old woman. Histological findings of the right ovary indicated higher atypical nuclei, higher mitotic rate and focal necrosis of tumorous cells in some areas, findings that are compatible with atypical carcinoid of the lung. Immunohistochemical staining was positive for synaptophysin, neuron-specific enolase, chromogranin A, Ki-67, topoisomerase IIalpha, peptide YY, and thyroglobulin. Three and a half years postoperatively, multiple bone and breast metastases were found and anticancer chemotherapy was ineffective. The results in the present case indicate that an ovarian carcinoid tumor found to be 'atypical carcinoid' according to pulmonary carcinoid criteria or immunohistochemical staining (i.e. highly positive for topoisomerase IIalpha and Ki-67) may have a poor prognosis.

ARID1A protein expression is retained in ovarian endometriosis with ARID1A loss-of-function mutations: implication for the two-hit hypothesis.

ARID1A loss-of-function mutation accompanied by a loss of ARID1A protein expression is considered one of the most important driver events in endometriosis-associated ovarian cancer. Although our recent genomic study clarified that ARID1A loss-of-function mutations were detected in 13% of ovarian endometriosis, an association between the ARID1A mutation status and ARID1A protein expression in ovarian endometriosis remains unclear. We performed immunohistochemical staining for ARID1A in 78 ovarian endometriosis samples and 99 clear cell carcinoma samples. We revealed that not only 70 endometriosis samples without ARID1A mutations but also eight endometriosis samples with ARID1A loss-of-function mutations retained ARID1A protein expression. On the other hand, most of clear cell carcinomas with ARID1A loss-of-function mutations showed a loss of ARID1A protein expression. In particular, clear cell carcinoma samples which harbor multiple ARID1A loss-of-function mutations or both a single ARID1A loss-of-function mutation and ARID1A allelic imbalance lost ARID1A protein expression. However, ARID1A protein expression was retained in seven clear cell carcinomas with ARID1A loss-of-function mutations. These results suggest that a single ARID1A loss-of-function mutation is insufficient for ARID1A loss in ovarian endometriosis and some clear cell carcinoma. Further driver events may be needed for the malignant transformation of ovarian endometriosis with ARID1A loss-of-function mutations.

XCL1 expression correlates with CD8-positive T cells infiltration and PD-L1 expression in squamous cell carcinoma arising from mature cystic teratoma of the ovary.

Molecular characteristics of carcinoma arising from mature cystic teratoma of the ovary (MCT) remain unclear due to its rarity. We analyzed RNA-sequencing data of 2322 pan-cancer [1378 squamous cell carcinomas (SCC), 6 adenosquamous carcinomas (ASC), and 938 adenocarcinomas (AC)] including six carcinomas arising from MCT (four SCCs, one ASC, and one AC). Hierarchical clustering and principal component analysis showed that gene expression profiles of carcinomas arising from MCT were different between each histological type and that gene expression profiles of SCCs arising MCT (MCT-SCCs) was apparently similar to those of lung SCCs. By epidermis-associated pathways activity based on gene set enrichment analysis, 1030 SCCs were divided into two groups: epidermis-signature high (head and neck, esophagus, and skin) and low (cervix, lung, and MCT). In addition to pan-SCC transcriptome analysis, cytokeratin profiling based on immunohistochemistry in the independent samples of 21 MCT-SCCs clarified that MCT-SCC dominantly expressed CK18, suggesting the origin of MCT-SCC was columnar epithelium. Subsequently, we investigated differentially expressed genes in MCT-SCCs compared with different SCCs and identified XCL1 was specifically overexpressed in MCT-SCCs. Through immunohistochemistry analysis, we identified XCL1 expression on tumor cells in 13/24 (54%) of MCT-SCCs but not in MCTs. XCL1 expression was also significantly associated with the number of tumor-infiltrating CD8-positive T cells and PD-L1 expression on tumor cells. XCL1 produced by tumor cells may induce PD1/PD-L1 interaction and dysfunction of CD8-positive T cells in tumor microenvironment. XCL1 expression may be a novel biomarker for malignant transformation of MCT into SCC and a biomarker candidate for therapeutic response to an anti-PD1/PD-L1 therapy.