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Idiopathic pulmonary fibrosis (IPF) is a progressive, irreversible, and rapidly fatal interstitial lung disease marked by the replacement of lung alveoli with dense fibrotic matrices. Although the mechanisms initiating IPF remain unclear, rare and common alleles of genes expressed in lung epithelia, combined with aging, contribute to the risk for this condition. Consistently, single-cell RNA sequencing (scRNA-seq) studies have identified lung basal cell heterogeneity in IPF that might be pathogenic. We used single-cell cloning technologies to generate “libraries” of basal stem cells from the distal lungs of 16 patients with IPF and 10 controls. We identified a major stem cell variant that was distinguished from normal stem cells by its ability to transform normal lung fibroblasts into pathogenic myofibroblasts in vitro and to activate and recruit myofibroblasts in clonal xenografts. This profibrotic stem cell variant, which was shown to preexist in low quantities in normal and even fetal lungs, expressed a broad network of genes implicated in organ fibrosis and showed overlap in gene expression with abnormal epithelial signatures identified in previously published scRNA-seq studies of IPF. Drug screens highlighted specific vulnerabilities of this profibrotic variant to inhibitors of epidermal growth factor and mammalian target of rapamycin signaling as prospective therapeutic targets. This profibrotic stem cell variant in IPF was distinct from recently identified profibrotic stem cell variants in chronic obstructive pulmonary disease and may extend the notion that inappropriate accrual of minor and preexisting stem cell variants contributes to chronic lung conditions.

Supplementary Table 4

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Supplementary Table 1

Supplementary Table 3

The current theory of carcinogenesis for the deadliest of 'ovarian' cancers-high-grade serous carcinoma (HGSC)-holds that the malignancy develops first in the fallopian tube and spreads to the ovaries, peritoneum, and/or regional lymph nodes. This is based primarily on the observation of early forms of serous neoplasia (serous tubal intraepithelial lesions [STILs], and serous tubal intraepithelial carcinomas [STICS]) in the fimbria of women undergoing risk reduction surgery. However, these lesions are uncommon in the general population, confer a low risk (5%) of HGSC following their removal in at-risk women with germ-line BRCA1/2 mutations, and require 4 or more years to recur as intraperitoneal HGSC. These features suggest that isolated STILs and STICs behave as precursors, with uncertain cancer risk rather than carcinomas. Their evolution to HGSC within, or after, escape from the tube could proceed stepwise with multiple biologic events; however, it is unclear whether tubal or ovarian HGSCs encountered in the setting of advanced disease evolved in the same fashion. The latter scenario could also be explained by a 'catastrophic' model in which STICs suddenly develop with invasive and metastatic potential, overwhelming or obscuring the site of origin. Moreover, a similar model might explain the sudden emergence of HGSC in the peritoneal cavity following escape of precursor cells years before. Long-term follow-up data from opportunistic or prophylactic salpingectomy should shed light on where malignant transformation occurs, as well as the timeline from precursor to metastatic HGSC. © 2022 The Pathological Society of Great Britain and Ireland.

Table S1. Differential expression analysis for CD8 T cells.

Table S5. List of Genes Used to Generate NK Cell Activation Scores.

Supplementary Figure 1 from p53 Autoantibodies as Potential Detection and Prognostic Biomarkers in Serous Ovarian Cancer

Table S2. Differential expression analysis for Treg CD4 T cells.

Table S3. Differential expression analysis for non-Treg CD4 T cells.

Immune therapies have had limited efficacy in high-grade serous ovarian cancer (HGSC), as the cellular targets and mechanism(s) of action of these agents in HGSC are unknown. Here we performed immune functional and single-cell RNA sequencing transcriptional profiling on novel HGSC organoid/immune cell co-cultures treated with a unique bispecific anti-programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) antibody compared with monospecific anti-PD-1 or anti-PD-L1 controls. Comparing the functions of these agents across all immune cell types in real time identified key immune checkpoint blockade (ICB) targets that have eluded currently available monospecific therapies. The bispecific antibody induced superior cellular state changes in both T and natural killer (NK) cells. It uniquely induced NK cells to transition from inert to more active and cytotoxic phenotypes, implicating NK cells as a key missing component of the current ICB-induced immune response in HGSC. It also induced a subset of CD8 T cells to transition from naïve to more active and cytotoxic progenitor-exhausted phenotypes post-treatment, revealing the small, previously uncharacterized population of CD8 T cells responding to ICB in HGSC. These state changes were driven partially through bispecific antibody-induced downregulation of the bromodomain-containing protein BRD1. Small-molecule inhibition of BRD1 induced similar state changes in vitro and demonstrated efficacy in vivo, validating the co-culture results. Our results demonstrate that state changes in both NK and a subset of T cells may be critical in inducing an effective anti-tumor immune response and suggest that immune therapies able to induce such cellular state changes, such as BRD1 inhibitors, may have increased efficacy in HGSC.Significance:This study indicates that increased efficacy of immune therapies in ovarian cancer is driven by state changes of NK and small subsets of CD8 T cells into active and cytotoxic states.

Background: This study examined the value of serum p53 autoantibodies (p53-AAb) as detection and prognostic biomarkers in ovarian cancer.Methods: p53-AAb were detected by ELISA in sera obtained preoperatively from women undergoing surgery for a pelvic mass. This group included women subsequently diagnosed with invasive serous ovarian cancer (n = 60), nonserous ovarian cancers (n = 30), and women with benign disease (n = 30). Age-matched controls were selected from the general population (n = 120). Receiver operating characteristic curves were constructed to compare the values of p53-AAb, CA 125, and HE4 as a screening biomarker. Kaplan-Meier curves and Cox proportional hazards modeling were used to assess its prognostic value on survival.Results: p53-AAb were detected in 25 of 60 (41.7%) of serous cases, 4 of 30 (13.3%) nonserous cases, 3 of 30 (10%) benign disease cases, and 10 of 120 (8.3%) controls (combined P = 0.0002). p53-AAb did not significantly improve the detection of cases [area under the curve (AUC), 0.69] or the discrimination of benign versus malignant disease (AUC, 0.64) compared with CA 125 (AUC, 0.99) or HE4 (AUC, 0.98). In multivariate analysis among cases, p53-AAb correlated only with a family history of breast cancer (P = 0.01). Detectable p53 antibodies in pretreatment sera were correlated with improved overall survival (P = 0.04; hazard ratio, 0.57; 95% confidence interval, 0.33-0.97) in serous ovarian cancer.Conclusions: Antibodies to p53 are detected in the sera of 42% of patients with advanced serous ovarian cancer.Impact: Although their utility as a preoperative diagnostic biomarker, beyond CA 125 and HE4, is limited, p53-AAb are prognostic for improved overall survival. Cancer Epidemiol Biomarkers Prev; 19(3); 859–68

Table S4. Differential expression analysis for NK cells.

Supplementary Materials and Methods, Figures, and Figure and Table legends

Supplementary Table 2 from Human Epididymis Protein 4 (HE4) Is a Secreted Glycoprotein that Is Overexpressed by Serous and Endometrioid Ovarian Carcinomas

Supplementary Table 1 from Human Epididymis Protein 4 (HE4) Is a Secreted Glycoprotein that Is Overexpressed by Serous and Endometrioid Ovarian Carcinomas

Among the genes most commonly identified in gene expression profiles of epithelial ovarian carcinomas (EOC) is the gene for human epididymis protein 4 (HE4). To ascertain its clinical utility, we did a comprehensive assessment of HE4 protein expression in benign and malignant ovarian and nonovarian tissues by immunohistochemistry. In comparison with normal surface epithelium, which does not express HE4, we found that cortical inclusion cysts lined by metaplastic Mullerian epithelium abundantly express the protein. Its expression in tumors was restricted to certain histologic subtype: 93% of serous and 100% of endometrioid EOCs expressed HE4, whereas only 50% and 0% of clear cell carcinomas and mucinous tumors, respectively, were positive. Tissue microarrays revealed that the majority of nonovarian carcinomas do not express HE4, consistent with our observation that HE4 protein expression is highly restricted in normal tissue to the reproductive tracts and respiratory epithelium. HE4 is predicted to encode a secreted protein. Using reverse transcription-PCR, we identified ovarian cancer cell lines that endogenously overexpress HE4. Cultured medium from these cells revealed a secreted form of HE4 that is N-glycosylated. This observation is consistent with the recent report that HE4 circulates in the bloodstream of patients with EOC. Therefore, HE4 is a secreted glycoprotein that is overexpressed by serous and endometrioid EOCs. Its expression in cortical inclusion cysts suggests that formation of Mullerian epithelium is a prerequisite step in the development of some types of EOCs.

AIMS Most vulvar squamous cell carcinomas are human papillomavirus (HPV)-associated or TP53-mutant. A third category of HPV-independent TP53-wild-type lesions is uncommon and not fully understood. Differentiated exophytic vulvar intraepithelial lesion (DEVIL) has been characterised as a precursor of this latter category. The reproducibility of the diagnosis of DEVIL and its distinction from lesions with overlapping morphology has not been studied. Our aim was to establish the interobserver agreement in the diagnosis of DEVIL and its distinction from neoplastic and reactive conditions of the vulva on haematoxylin and eosin evaluation. METHODS AND RESULTS A set of 35 slides was evaluated by eight reviewers (two trainees and six practising gynaecological pathologists). The set included DEVIL, condyloma, established vulvar precursors [high-grade squamous intraepithelial lesion (HSIL) and differentiated vulvar intraepithelial neoplasia (dVIN)] with superimposed acanthosis or verruciform growth, lichen simplex chronicus (LSC), and psoriasis. Kappa (κ) values were calculated. Overall, interobserver agreement was moderate (κ = 0.56), improving to substantial (κ = 0.7) when evaluation was performed by practising pathologists. Agreement was strong for the diagnosis of HSIL (κ = 0.88), and substantial for the diagnosis of DEVIL (κ = 0.61), condyloma (κ = 0.79), and LSC (κ = 0.72). Agreement was moderate for the diagnosis of dVIN (κ = 0.59) and psoriasis (κ = 0.53). Perfect agreement (6/6) among practising pathologists was observed in 43% of cases, and majority agreement (5/6 or 4/6) was observed in 48% of cases. CONCLUSIONS Reproducibility in the diagnosis of verruciform vulvar lesions, including the novel DEVIL, is acceptable overall. Reproducibility is higher for well-known lesions such as HSIL and condyloma than for more challenging diagnoses such as DEVIL, dVIN, and psoriasis. Agreement is higher among practising gynaecological pathologists, suggesting that training and experience improve reproducibility. Our findings support the inclusion of DEVIL as a diagnostic entity in the classification of vulvar squamous lesions.

Mucinous ovarian tumors rarely harbor mural nodules, which have historically been classified as sarcoma-like, anaplastic carcinomatous, or sarcomatous on the basis of predominant morphologic features. The molecular relationship between mural nodules and associated mucinous ovarian tumors remains poorly characterized, as does the molecular pathogenesis of these mural nodules. Thus, we analyzed the morphological, immunohistochemical, and genetic features of 13 mucinous ovarian tumors and associated mural nodule(s). Three harbored sarcoma-like mural nodules and ten contained anaplastic carcinomatous nodules, including 1 tumor with spatially discrete anaplastic carcinomatous and sarcomatous nodules. Twelve of 13 cases showed genetic evidence of clonality between the mural nodule(s) and associated mucinous ovarian tumor, including all three tumors with sarcoma-like morphology. Mural nodules were genetically identical in the five cases in which there were multiple discrete mural nodules that were sequenced separately. MTAP and p53 immunohistochemistry confirmed the distribution of neoplastic cells in a subset of sarcoma-like and anaplastic carcinomatous nodules. No single recurrent genetic alteration was associated with mural nodule development. No recurrent genetic differences were identified between mural nodules with sarcoma-like, anaplastic carcinomatous, and sarcomatous morphology. Of 11 patients with clinical follow-up, three died of disease 3, 8, and 9 months after diagnosis, but no recurrent genetic events were associated with poor outcome. These molecular data suggest that sarcoma-like, anaplastic carcinomatous, and sarcomatous nodules represent a morphologic spectrum of clonal neoplasms arising in mucinous ovarian tumors rather than three discrete biological entities.

Immune therapies have had limited efficacy in high-grade serous ovarian cancer (HGSC), as the cellular targets and mechanism(s) of action of these agents in HGSC are unknown. Here we performed immune functional and single-cell RNA sequencing transcriptional profiling on novel HGSC organoid/immune cell co-cultures treated with a unique bispecific anti-programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) antibody compared with monospecific anti-PD-1 or anti-PD-L1 controls. Comparing the functions of these agents across all immune cell types in real time identified key immune checkpoint blockade (ICB) targets that have eluded currently available monospecific therapies. The bispecific antibody induced superior cellular state changes in both T and natural killer (NK) cells. It uniquely induced NK cells to transition from inert to more active and cytotoxic phenotypes, implicating NK cells as a key missing component of the current ICB-induced immune response in HGSC. It also induced a subset of CD8 T cells to transition from naïve to more active and cytotoxic progenitor-exhausted phenotypes post-treatment, revealing the small, previously uncharacterized population of CD8 T cells responding to ICB in HGSC. These state changes were driven partially through bispecific antibody-induced downregulation of the bromodomain-containing protein BRD1. Small-molecule inhibition of BRD1 induced similar state changes in vitro and demonstrated efficacy in vivo, validating the co-culture results. Our results demonstrate that state changes in both NK and a subset of T cells may be critical in inducing an effective anti-tumor immune response and suggest that immune therapies able to induce such cellular state changes, such as BRD1 inhibitors, may have increased efficacy in HGSC. Significance: This study indicates that increased efficacy of immune therapies in ovarian cancer is driven by state changes of NK and small subsets of CD8 T cells into active and cytotoxic states.

Malignant peritoneal mesothelioma is a rare aggressive tumor that arises from the peritoneal lining. While recurrent BAP1 mutations have been identified in a subset of mesotheliomas, molecular characteristics of peritoneal mesotheliomas, including those lacking BAP1 alterations, remain poorly understood. Using targeted next-generation sequencing, we examined the molecular features of 26 diffuse malignant peritoneal mesotheliomas. As part of an exploratory analysis, we analyzed an additional localized peritoneal mesothelioma and one well-differentiated papillary mesothelioma with invasive foci. Genomic characterization identified categories of diffuse malignant peritoneal mesotheliomas: The first group included 18 (69%) tumors with recurrent BAP1 alterations, with eight (31%) having more than one BAP1 alterations, and concomitant alterations in PBRM1 (46%) and SETD2 (35%). All tumors with complete loss of BAP1 expression by immunohistochemistry harbored BAP1 molecular alterations. PBRM1 alterations were significantly enriched in the BAP1-altered cohort. Frequent copy number loss of BAP1, ARID1B, PRDM1, PBRM1, SETD2, NF2, and CDKN2A was noted. The second group included eight (31%) BAP1-wild-type tumors: two with TP53 mutations, one with a TRAF7 activating mutation, one with a SUZ12 inactivating mutation, and three with ALK rearrangements that we previously published. One TP53-mutant biphasic mesothelioma showed evidence of genomic near-haploidization showing loss of heterozygosity of all chromosomes except 5, 7, 16, and 20. The localized peritoneal mesothelioma harbored a nonsense CHEK2 mutation, and the well-differentiated papillary mesothelioma with invasive foci harbored no reportable variants. In conclusion, we described the genetic categories of diffuse malignant peritoneal mesotheliomas, with BAP1-mutant and BAP1-wild-type groups. Our findings implicated DNA repair, epigenetics, and cell cycle regulation in the pathogenesis of peritoneal mesotheliomas, with identification of potential therapeutic targets.

Prostatic-type differentiation in the lower female genital tract is encountered rarely and its causes and clinical associations are not well established. Within the vagina, reports to date have invariably described ectopic prostatic-type differentiation as restricted to the lamina propria. We recently encountered a patient receiving testosterone for gender dysphoria whose vaginectomy specimen showed a prostatic glandular proliferation within the surface epithelium. To elucidate its potential association with androgen exposure, we sought similar lesions, resected over a 26-year period, from patients with exogenous or endogenous androgen excess. Thirteen cases were identified, involving the vagina (n=12) and exocervix (n=1). The most common clinical context was gender dysphoria with long-term testosterone therapy; the lesion was present in 7 of 8 gender-dysphoric patients examined. Four other patients had congenital disorders of sexual development associated with endogenous androgen excess (congenital adrenal hyperplasia, 46,XY disorder of sexual development, and ovotesticular disorder of sexual development). Two had no known exposure to androgen excess. Immunohistochemically, glands stained for NKX3.1 (100% of cases), androgen receptor (100%), CK7 (92%), and prostate-specific antigen (69%). Follow-up (median duration, 11 mo) showed no masses or neoplasia. We propose the designation "androgen-associated prostatic metaplasia" for this form of prostate tissue with distinctive clinical, histologic and immunohistochemical features. It is novel and previously unrecognized within the vagina. It is strikingly prevalent among patients undergoing gender-affirming surgery, an increasingly common procedure. Recognition is important to distinguish it from other potentially neoplastic glandular lesions and facilitate accrual of more follow-up data to better understand its natural history.

Epidemiologic and histopathologic findings and the laying hen model support the long-standing incessant ovulation hypothesis and cortical inclusion cyst involvement in sporadic ovarian cancer development. MicroRNA-200 (miR-200) family is highly expressed in ovarian cancer. Herewith, we show that ovarian surface epithelial (OSE) cells with ectopic miR-200 expression formed stabilized cysts in three-dimensional (3D) organotypic culture with E-cadherin fragment expression and steroid hormone pathway activation, whereas ovarian cancer 3D cultures with miR-200 knockdown showed elevated TGF-β expression, mitotic spindle disorientation, increased lumenization, disruption of ROCK-mediated myosin II phosphorylation, and SRC signaling, which led to histotype-dependent loss of collective movement in tumor spread. Gene expression profiling revealed that epithelial-mesenchymal transition and hypoxia were the top enriched gene sets regulated by miR-200 in both OSE and ovarian cancer cells. The molecular changes uncovered by the in vitro studies were verified in both human and laying hen ovarian cysts and tumor specimens. As miR-200 is also essential for ovulation, our results of estrogen pathway activation in miR-200-expressing OSE cells add another intriguing link between incessant ovulation and ovarian carcinogenesis.

Epithelial proliferations in the fallopian tube have been characterized by some as stem cell outgrowths (SCOUTs) and divided into type I and type II. Type II SCOUTs exhibit diffuse cellular beta-catenin nuclear staining (β-catenin+), implying a CTNNB1 mutation. SCOUTs are more common in perimenopausal and postmenopausal women and are associated with ovarian cancer but have not been linked directly to malignancy. We analyzed type II SCOUTs in various gynecologic conditions, and searched for endometrioid atypical hyperplasias (tubal endometrioid intraepithelial neoplasia) or adenocarcinomas in the tube. β-catenin+ SCOUT frequency in cases of neoplasia was 66.7% per case and 30.7% per nonfimbrial cross-section for uterine endometrioid carcinomas versus 25% and 13.3% for controls, respectively (P=0.02 and 0.09). Multiple (3 or more) β-catenin+ SCOUTs in a single section were uncommon; 6 of 9 were associated with a carcinoma or proliferative lesion in the endometrium. Tubal endometrioid intraepithelial neoplasia/atypical hyperplasia displayed complex growth, including focal cribriform growth patterns and squamous morules. Two cases of type II SCOUTs associated with tubal endometrioid intraepithelial neoplasia/atypical hyperplasia and/or adenocarcinomas in the fallopian tube were identified, both of which coexisted with a separate endometrioid adenocarcinoma, one with bilateral ovarian endometrioid adenocarcinomas. Both benign and neoplastic tubal lesions were β-catenin+. This report is the first to link components of a unique β-catenin+ endometrioid carcinogenic sequence in the fallopian tube. It further emphasizes the multifocal nature of endometrioid neoplasia in the female genital tract and poses questions regarding the frequency and biologic underpinnings of β-catenin+ proliferations in the oviduct.

In most cases of suspected endometrial neoplasia tumor origin can be correctly assigned according to a combination of clinical, radiologic, and pathologic features, even when the latter are based upon the examination of relatively small biopsy samples. However there are well-recognized exceptions to this rule which continue to create diagnostic difficulty, and sometimes difficulties persist even after the detailed examination of resection specimens. Among the most common problems encountered in practice are the distinction of primary endometrial and primary endocervical adenocarcinomas, and the determination of tumor origin when there is synchronous, multifocal involvement of gynecologic tract sites, for example the endometrium and the ovary. However, accurate diagnosis in these cases is important because this has significant staging, management and prognostic implications. In this review we discuss the value and limitations of key morphologic, immunophenotypic and molecular findings in these diagnostic scenarios.

Simple Summary There is limited information about genomic markers, especially for cervical adenocarcinoma treatment decisions. In this prospective study, it was found that nonsynonymous PIK3CA mutation detected in the patient’s circulating DNA collected before treatment or during follow-up was significantly associated with decreased progression-free survival or overall survival. It is the first indication of the predictive power of PIK3CA aberration in cervical adenocarcinoma. The work contributes to the development of liquid biopsies for the prolonged strategy of surveillance and indicates the possibility of tailoring management of this particular women’s cancer. Abstract Personalized treatment of genetically stratified subgroups has the potential to improve outcomes in many malignant tumors. This study distills clinically meaningful prognostic/predictive genomic marker for cervical adenocarcinoma using signature genomic aberrations and single-point nonsynonymous mutation-specific droplet digital PCR (ddPCR). Mutations in PIK3CA E542K, E545K, or H1047R were detected in 41.7% of tumors. PIK3CA mutation detected in the patient’s circulating DNA collected before treatment or during follow-up was significantly associated with decreased progression-free survival or overall survival. PIK3CA mutation in the circulating DNA during follow-up after treatment predicted recurrence with 100% sensitivity and 64.29% specificity. It is the first indication of the predictive power of PIK3CA mutations in cervical adenocarcinoma. The work contributes to the development of liquid biopsies for follow up surveillance and a possibility of tailoring management of this particular women’s cancer.

Uterine carcinoma (UC) is the most common gynecologic malignancy in the United States. TP53 mutant UCs cause a disproportionate number of deaths due to limited therapies for these tumors and the lack of mechanistic understanding of their fundamental vulnerabilities. Here we sought to understand the functional and therapeutic relevance of TP53 mutations in UC. We functionally profiled targetable TP53 dependent DNA damage repair and cell cycle control pathways in a panel of TP53 mutant UC cell lines and patient-derived organoids. There were no consistent defects in DNA damage repair pathways. Rather, most models demonstrated dependence on defective G2/M cell cycle checkpoints and subsequent upregulation of Aurora kinase-LKB1-p53-AKT signaling in the setting of baseline mitotic defects. This combination makes them sensitive to Aurora kinase inhibition. Resistant lines demonstrated an intact G2/M checkpoint, and combining Aurora kinase and WEE1 inhibitors, which then push these cells through mitosis with Aurora kinase inhibitor-induced spindle defects, led to apoptosis in these cases. Overall, this work presents Aurora kinase inhibitors alone or in combination with WEE1 inhibitors as relevant mechanism driven therapies for TP53 mutant UCs. Context specific functional assessment of the G2/M checkpoint may serve as a biomarker in identifying Aurora kinase inhibitor sensitive tumors.

Simple Summary Cancers arising from the lining of the uterus, endometrial cancers, are the most common gynecologic malignancy in the United States. Once endometrial cancer escapes the uterus and grows in distant locations, there are limited therapeutic options. The most aggressive and lethal endometrial cancers carry alterations in the protein p53, which is a critical guardian of many cellular functions. The role of these p53 alterations in endometrial cancer is not well understood. The goal of this work was to use p53 altered models of endometrial cancer to understand which, if any, therapeutically targetable vulnerabilities these p53 alterations may confer in endometrial cancer. Here we show that many of these p53 altered cells have problems with cell division which can be targeted with novel single and combination therapies. These discoveries may lead to relevant new therapies for difficult to treat advanced stage endometrial cancers. Abstract Uterine carcinoma (UC) is the most common gynecologic malignancy in the United States. TP53 mutant UCs cause a disproportionate number of deaths due to limited therapies for these tumors and the lack of mechanistic understanding of their fundamental vulnerabilities. Here we sought to understand the functional and therapeutic relevance of TP53 mutations in UC. We functionally profiled targetable TP53 dependent DNA damage repair and cell cycle control pathways in a panel of TP53 mutant UC cell lines and patient-derived organoids. There were no consistent defects in DNA damage repair pathways. Rather, most models demonstrated dependence on defective G2/M cell cycle checkpoints and subsequent upregulation of Aurora kinase-LKB1-p53-AKT signaling in the setting of baseline mitotic defects. This combination makes them sensitive to Aurora kinase inhibition. Resistant lines demonstrated an intact G2/M checkpoint, and combining Aurora kinase and WEE1 inhibitors, which then push these cells through mitosis with Aurora kinase inhibitor-induced spindle defects, led to apoptosis in these cases. Overall, this work presents Aurora kinase inhibitors alone or in combination with WEE1 inhibitors as relevant mechanism driven therapies for TP53 mutant UCs. Context specific functional assessment of the G2/M checkpoint may serve as a biomarker in identifying Aurora kinase inhibitor sensitive tumors.

Endometriosis is generally histopathologically defined as the presence of at least 2 of the following: endometrial stroma, Mullerian epithelium, and/or hemosiderin-laden macrophages (HLM). Despite clinically evident endometriotic lesions, biopsies are frequently nondiagnostic. In this study, we conducted a large-scale review of biopsies of lesions clinically thought to represent endometriosis and correlate the histologic findings with clinical appearance to expand sensitivity of the pathologic definition of endometriosis, particularly in patients on hormonal therapy. In all, 112 biopsies from 78 patients (mean age=25, range 18-39 yr) were reviewed for histopathologic features suggestive of or diagnostic for endometriosis including the presence of endometrial stroma, Mullerian epithelium, dystrophic calcifications, HLM, chronic inflammation, adhesions, and vascular proliferation. Endometriosis was confirmed by pathologic criteria in 37 of 78 patients (47%). Biopsies from patients on hormonal therapy (n=62, 80%) were significantly less likely to meet pathologic criteria for endometriosis (P=0.01). Nondiagnostic biopsies (70/112; 63%) frequently displayed HLM (20%), chronic inflammation (29%), dystrophic calcifications (26%), vascular proliferation (20%), or adhesions (20%) and were significantly more likely to have a vascular clinical appearance (P=0.01). Diagnostic biopsies (42/112; 38%) were more likely to have a blue/black clinical appearance (P=0.03), demonstrate HLM (P=0.004), and display pseudodecidualization (P=0.05). Patients with a high clinical suspicion of endometriosis have a range of histologic findings, with less than half meeting the current histopathologic criteria for diagnosing endometriosis. Given the heterogeneous histopathologic appearance, revision of the histologic criteria may be warranted with further exploration, particularly for lesions with predominantly vascular features.

Synovial sarcoma most commonly occurs in the extremities but has rarely been described in the female genital tract. In this series, we describe the clinical, morphologic, immunohistochemical, and molecular features of 7 cases of vulvovaginal synovial sarcoma (vulva, n=6; vagina, n=1). We emphasize their wide morphologic spectrum, which can overlap significantly with other more common tumors at these sites, as highlighted by 2 cases initially diagnosed as other entities (endometrioid carcinoma and malignant peripheral nerve sheath tumor). The average patient age was 41 (range: 23 to 62) years and tumor size ranged from 0.8 to 7 cm. Histologically, the tumors were biphasic (n=6) and monophasic (n=1). All cases were confirmed with fluorescence in situ hybridization or sequencing, and 5/5 cases were positive for the novel immunohistochemical markers SSX and SS18-SSX. In 3 cases with follow-up, 2 patients died of disease and 1 was alive with no evidence of disease. Previously described cases arising in the female genital tract are also reviewed. Vulvovaginal monophasic synovial sarcoma raises a broad differential diagnosis, including smooth muscle tumors, spindled carcinomas, and melanoma. Biphasic synovial sarcoma may mimic Mullerian carcinosarcoma, endometrioid carcinoma with spindled, corded, and hyalinized elements, and mesonephric-like adenocarcinoma. Awareness that synovial sarcoma can occur in the female genital tract with a wide variety of histologic appearances is critical for correctly diagnosing this rare entity. In particular, synovial sarcoma should be considered for any deeply situated "adenocarcinoma" in the vulva, with attention to subtle spindle cell differentiation.

Mucinous ovarian tumors rarely harbor mural nodules, which have historically been classified as sarcoma-like, anaplastic carcinomatous, or sarcomatous on the basis of predominant morphologic features. The molecular relationship between mural nodules and associated mucinous ovarian tumors remains poorly characterized, as does the molecular pathogenesis of these mural nodules. Thus, we analyzed the morphological, immunohistochemical, and genetic features of 13 mucinous ovarian tumors and associated mural nodule(s). Three harbored sarcoma-like mural nodules and ten contained anaplastic carcinomatous nodules, including 1 tumor with spatially discrete anaplastic carcinomatous and sarcomatous nodules. Twelve of 13 cases showed genetic evidence of clonality between the mural nodule(s) and associated mucinous ovarian tumor, including all three tumors with sarcoma-like morphology. Mural nodules were genetically identical in the five cases in which there were multiple discrete mural nodules that were sequenced separately. MTAP and p53 immunohistochemistry confirmed the distribution of neoplastic cells in a subset of sarcoma-like and anaplastic carcinomatous nodules. No single recurrent genetic alteration was associated with mural nodule development. No recurrent genetic differences were identified between mural nodules with sarcoma-like, anaplastic carcinomatous, and sarcomatous morphology. Of 11 patients with clinical follow-up, three died of disease 3, 8, and 9 months after diagnosis, but no recurrent genetic events were associated with poor outcome. These molecular data suggest that sarcoma-like, anaplastic carcinomatous, and sarcomatous nodules represent a morphologic spectrum of clonal neoplasms arising in mucinous ovarian tumors rather than three discrete biological entities.

Uterine tumors resembling ovarian sex cord tumor (UTROSCT) are rare mesenchymal neoplasms, of uncertain biologic potential, that were recently reported to exhibit recurrent gene fusions involving NCOA2–3. The purpose of this study was to, using a larger sample size, better characterize the histopathologic and molecular diversity of UTROSCT. Twenty-six cases of UTROSCT from five institutions were selected for further study. Fluorescence in situ hybridization for NCOA1, NCOA2, NCOA3, ESR1 and GREB1, and targeted RNA sequencing was performed on seventeen and eight UTROSCTs, respectively. Eight cases underwent massively parallel sequencing to detect single nucleotide variants (SNV), copy number variations (CNV), and structural variants using a targeted hybrid-capture based assay. NCOA1–3 rearrangement was identified in 81.8% (18/22) of cases. The most common fusion was ESR1-NCOA3, occurring in 40.9% (9/22). GREB1-NCOA1 (n=4), ESR1-NCOA2 (n=3), and GREB1-NCOA2 (n=1) rearrangements were also identified. No recurrent SNVs were identified and no tumor had SNVs in FOXL2, DICER1, STK11, or AKT1, which can be seen in ovarian sex cord-stromal tumors. CNVs were infrequent. Clinical follow-up was available for eleven cases with a mean follow-up interval of 94.4 (range 1 – 319) months. Only one case had a recurrence 66 months after the initial diagnosis and this was the single case with a with a GREB1-NCOA2 fusion. This study reports the morphologic spectrum of UTROSCT and confirms the recently reported recurrent NCOA2/3 gene fusions, in addition to identifying novel rearrangements involving NCOA1 in these tumors.

This article summarizes the pathogenesis of ovarian carcinoma, focusing on the paradox of high-grade serous carcinogenesis. The fallopian tube is the prime site of origin in early serous cancers. Because a subset of serous cancers is associated with early serous proliferations absent intramucosal carcinomas, "precursor escape" is emerging, whereby some advanced cancers trace their roots to early serous proliferations. This has parallels in the endometriosis model and opens up a novel mechanism by which advanced malignancy could emerge without an obvious tubal carcinoma. The impact of this concept on classification of serous cancer and expectations from preventive strategies are discussed.

Background Primary adenocarcinoma of the anal canal is a rare and aggressive gastrointestinal disease with unclear pathogenesis. Because of its rarity, no clear clinical practice guideline has been defined and a targeted therapeutic armamentarium has yet to be developed. The present article aimed at addressing this information gap by in-depth characterising the anal glandular neoplasms at the histologic, immunologic, genomic and epidemiologic levels. Methods In this multi-institutional study, we first examined the histological features displayed by each collected tumour (n = 74) and analysed their etiological relationship with human papillomavirus (HPV) infection. The intratumoural immune cell subsets (CD4, CD8, Foxp3), the expression of immune checkpoints (PD-1, PD-L1), the defect in mismatch repair proteins and the mutation analysis of multiple clinically relevant genes in the gastrointestinal cancer setting were also determined. Finally, the prognostic significance of each clinicopathological variable was assessed. Results Phenotypic analysis revealed two region-specific subtypes of anal canal adenocarcinoma. The significant differences in the HPV status, density of tumour-infiltrating lymphocytes, expression of immune checkpoints and mutational profile of several targetable genes further supported the separation of these latter neoplasms into two distinct entities. Importantly, anal gland/transitional-type cancers, which poorly respond to standard treatments, displayed less mutations in downstream effectors of the EGFR signalling pathway (i.e., KRAS and NRAS) and demonstrated a significantly higher expression of the immune inhibitory ligand-receptor pair PD-1/PD-L1 compared to their counterparts arising from the colorectal mucosa. Conclusions Taken together, the findings reported in the present article reveal, for the first time, that glandular neoplasms of the anal canal arise by HPV-dependent or independent pathways. These etiological differences leads to both individual immune profiles and mutational landscapes that can be targeted for therapeutic benefits.

Mesonephric adenocarcinoma most commonly arises in the cervix and is presumed to be derived from normal or hyperplastic mesonephric remnants. It is characterized by recurrent KRAS mutations and lack of PIK3CA/PTEN alterations. Adenocarcinomas of the uterine corpus and ovary characterized by morphologic and immunophenotypic similarities to mesonephric adenocarcinoma have been reported. The pathogenesis of these tumors, which have been designated "mesonephric-like adenocarcinomas" is unknown, and it has been debated whether these represent mesonephric adenocarcinomas that arise in the endometrium/ovary or endometrioid adenocarcinomas that closely mimic mesonephric adenocarcinoma. The relationship at the molecular level between mesonephric adenocarcinomas and mesonephric-like adenocarcinomas is unknown. The aim of this study was to examine the molecular alterations in mesonephric-like adenocarcinomas to identify driver mutations and potential therapeutically targetable mutations, and to determine the relationship between mesonephric-like adenocarcinomas and mesonephric adenocarcinomas using targeted next-generation sequencing. Seven mesonephric-like adenocarcinomas (4 ovarian, 3 uterine corpus) underwent targeted next-generation sequencing to detect mutations, copy number variations and structural variants in exonic regions of 300 cancer genes, and 113 selected intronic regions across 35 genes. All 7 tumors (100%) harbored canonical activating KRAS mutations (4 G12D, 3 G12V). PIK3CA activating mutations were identified in 3 of 7 (43%) cases. There were no alterations in PTEN, ARID1A, or TP53 in any of the tumors. In copy number analysis, 5 of 7 (71%) tumors exhibited 1q gain, which was accompanied by 1p loss in 2 cases. In addition, 4 of 7 (57%) tumors had chromosome 10 gain, which was accompanied by gain of chromosome 12 in 3 cases. Mesonephric-like adenocarcinomas, similar to mesonephric adenocarcinomas, are characterized by recurrent KRAS mutations, gain of 1q, lack of PTEN mutations, and gains of chromosomes 10 and 12. PIK3CA mutations, which have not previously been identified in mesonephric adenocarcinoma, were found in 3 of 7 (43%) mesonephric-like adenocarcinomas in our study. Mesonephric-like adenocarcinomas exhibit strikingly similar molecular aberrations to mesonephric adenocarcinomas, but also frequently harbor PIK3CA mutations, demonstrating biological overlap with carcinomas of both mesonephric and Mullerian (endometrioid) differentiation. Given the previously documented association with endometriosis (ovarian neoplasms) and the prominent endometrial involvement (uterine corpus neoplasms), we believe these are best regarded as of Mullerian origin and representing adenocarcinomas which differentiate along mesonephric lines; as such, we propose the term mesonephric-like Mullerian adenocarcinoma.

A rare but serious complication of endometriosis is the development of carcinoma, and clear cell and endometrioid carcinomas of the ovary are the two most common malignancies which arise from endometriosis. They are distinct diseases, characterized by unique morphologies, immunohistochemical profiles, and responses to treatment. However, both arise in endometriosis and can share common mutations. The overlapping mutational profiles of clear cell and endometrioid carcinomas suggest that their varied histologies may be due to a different cell of origin which gives rise to each type of cancer. Cochrane and colleagues address this question in a recent article in this journal. They show that a marker of ovarian clear cell carcinoma, cystathionine gamma lyase, is expressed in ciliated cells. Similarly, they show that markers of secretory cells (estrogen receptor and methylenetetrahydrofolate dehydrogenase 1) are expressed in ovarian endometrioid carcinoma. Taken together, they suggest that endometrioid and clear cell carcinomas arise from cells related to secretory and ciliated cells, respectively. We discuss Cochrane et al's work in the context of other efforts to determine the cell of origin of gynecological malignancies, with an emphasis on recent developments and challenges unique to the area. These limitations complicate our interpretation of tumor differentiation; does it reflect nature imposed by a specific cell of origin or nurture, by either mutation(s) or environment? Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Objective The purpose of this study was to determine the prevalence of incidentally discovered serous tubal intraepithelial carcinoma in women without a genetic risk for or history of high grade serous carcinoma (HGSC) in the gynecologic tract. Methods All pathology reports at our institution that included bilateral salpingectomies from January 2006–December 2011 were examined in women >50years old in which the entire tube or the distal one-third was examined histologically with the complete (proximal and distal fallopian tube) or modified (distal one third of the tube) SEE-FIM protocol. Cases were divided into: Group 1, a history of or known risk factors (BRCA1 or BRCA2 mutations) for HGSC and Group 2, those without these attributes for whom a STIC would be unexpected (incidental). Women undergoing unspecified "risk-reducing" procedures were included in Group 1. Results Of 4051 identified total, 2268 had complete examination of the distal fallopian tube and were age 50 or above. Of these, 1747 were in group 2. Two STICs were identified (0.1%), one associated with a grade 2 endometrial endometrioid adenocarcinoma and one with a low-grade ovarian serous carcinoma in the setting of a serous borderline tumor. Conclusions Incidental STICs in women over age 50 are uncommon. However, the significance of lesser tubal atypias (0.3% in this study), risk of STIC in women with no epithelial pathology and the risk imposed by coexisting endometrioid neoplasia are unclear and require further study.

In recent years it has become clear that many extra-uterine (pelvic) high-grade serous carcinomas (serous carcinomas) are preceded by a precursor lesion in the distal fallopian tube. Precursors range from small self-limited 'p53 signatures' to expansile serous tubal intraepithelial neoplasms that include both serous tubal epithelial proliferations (or lesions) of uncertain significance and serous tubal intraepithelial carcinomas. These precursors can be considered from three perspectives. The first is biologic underpinnings, which are multifactorial, and include the intersection of DNA damage with Tp53 mutations and disturbances in transcriptional regulation that increase with age. The second perspective is the morphologic discovery and classification of intraepithelial neoplasms that are intercepted early in their natural history, either incidentally or in risk-reduction surgeries for germline mutations. For the practicing pathologist, as well as the investigators, a distinction between a primary intraepithelial neoplasm and an intramucosal carcinoma must be made to avoid misinterpreting (or underestimating) the significance of these proliferations. The third perspective is the application of this information to intervention, devising strategies that will actually lower the ovarian cancer death rate by opportunistic salpingectomy, widespread comprehensive genetic screening and early detection. Central to this issue are the questions of (1) whether some STICs are metastatic, (2) whether lower-grade epithelial proliferations can invade prior to evolving into intraepithelial carcinoma, or (3) metastasize and become malignant elsewhere ('precursor escape'). An important caveat is the persistent and unsettling reality that many high-grade serous carcinomas are not associated with an obvious point of initiation in the fallopian tube. The pathologist sits squarely in the midst of all of these issues, and has a pivotal role in managing expectations for stemming the death rate from this lethal disease.