Mutational profile of endometrial hyperplasia and risk of progression to endometrioid adenocarcinoma.

BACKGROUND: Endometrial hyperplasia is a precursor to endometrioid adenocarcinoma (EMC), the most common uterine cancer. The likelihood of progression to carcinoma may be evaluated by histologic subclassification of endometrial hyperplasia, although these subclasses are subjective and only modestly reproducible among pathologists. Patient care would be improved by a more objective test to predict the risk of cancer progression. METHODS: Next-generation sequencing was performed on archived endometrial biopsy specimens from a retrospective cohort of women with endometrial hyperplasia. Cases were considered to be either progressing if the patient subsequently developed EMC or resolving if the patient had a subsequent negative tissue sampling or no cancer during medium-term follow-up (32 patients: 15 progressing and 17 resolving). Somatic mutations in endometrial hyperplasia were assessed for enrichment in progressing cases versus resolving cases, with an emphasis on genes commonly mutated in EMC. RESULTS: Several mutations were more common in progressing hyperplasia than resolving hyperplasia, although significant overlap was observed between progressing and resolving cases. Mutations included those in PTEN, PIK3CA, and FGFR2, genes commonly mutated in EMC. Mutations in ARID1A and MYC were seen only in progressing hyperplasia, although these were uncommon; this limited diagnostic sensitivity. Progressing hyperplasia demonstrated an accumulation of mutations in oncogenic signaling pathways similarly to endometrial carcinoma. CONCLUSIONS: Because of mutational differences between progressing and nonprogressing hyperplasia, mutational analysis may predict the risk of progression from endometrial hyperplasia to EMC.

Female adnexal tumors of probable Wolffian origin: morphological, immunohistochemical, and molecular analysis of 15 cases.

Female adnexal tumors of probable Wolffian origin are rare and present a diagnostic challenge due to their morphological and immunohistochemical overlap with more common ovarian and broad ligament entities. We evaluated the morphological, immunohistochemical, and molecular features of 15 tumors of probable Wolffian origin. Patients ranged from 32 to 69 (mean 47) years and tumors from 1.8 to 30 (mean 10) cm. All except one arose in para-adnexal soft tissues. Follow-up was available for six patients, five of whom were alive and well, while the sixth, who had extra-adnexal disease at diagnosis, died from unrelated causes. The following patterns were noted: tubular (all tumors), solid 11/15 (73%), sieve-like 7/15 (47%), and reticular 1/15 (7%). A myxoid background was present in 3/15 (20%) of tumors and eosinophilic luminal secretions in 11/15 (73%). Most tumors (12/15, 80%) had low-grade nuclear atypia, while three showed foci with scattered high-grade atypia. Mitotic index ranged from 0 to 17 (mean 4) per ten high-power fields. Tumors were positive for pankeratin and negative for TTF-1. EMA, GATA3, and PAX8 were positive in 2/10 (20%; focal), 3/15 (20%; focal), and 1/15 (7%; focal) of tumors, respectively. CD10, SF-1, calretinin, inhibin, ER, PR, cytokeratin 7, and WT1 were variably expressed. Pathogenic mutations were rare and included STK11 (n = 3), APC (n = 1), and MBD4 (n = 1). Copy number variations were detected in the three tumors with STK11 mutations and a myxoid background. These data demonstrate that female adnexal tumors of probable Wolffian origin are morphologically and immunohistochemically diverse, but infrequently harbor pathogenic mutations. However, their lack of mutations in contrast to their mimickers may be a valuable tool in diagnostically difficult cases.

Appendiceal Endometriosis: Is Diagnosis Dependent on Pathology Evaluation? A Prospective Cohort Study.

STUDY OBJECTIVE: To evaluate the diagnosis of appendiceal endometriosis (AppE) in coincidental appendectomy specimens using standard versus modified histopathologic analysis. DESIGN: Prospective analysis of 300 consecutive patients undergoing coincidental appendectomy at the time of a primary gynecologic procedure. SETTING: Academic tertiary referral hospital in the northeastern United States. PATIENTS: Women aged 22 to 52 years undergoing gynecologic surgery for the management of endometriosis or chronic pelvic pain between 2013 and 2015. INTERVENTIONS: Each appendix specimen underwent standard pathologic analysis with 4 sections performed. Modified pathologic analysis, consisting of standard analysis plus serial sectioning and complete evaluation of the appendix and mesoappendix, was then performed. The first pathologist reviewed all the slides to render a diagnosis. The slides of the subjects with abnormal pathology were rereviewed. On rereview, the diagnosis was confirmed, and the data on which protocol, standard or modified, achieved the diagnosis was rendered. The pathologist performing the second review was blinded to whether the slides from the standard or modified histopathology protocol achieved the original diagnosis. This allowed each specimen to serve as its own control. MEASUREMENTS AND MAIN RESULTS: The primary outcome is the detection of AppE. The standard analysis identified endometriosis in 7.7% (n = 23) of appendiceal specimens, whereas the modified analysis identified endometriosis in 10.0% (n = 30; odds ratio 1.3; confidence interval, 1.1-1.7; p = .01). When all pathology findings were combined, the standard analysis identified abnormal pathology in 9.3% (n = 28) of the specimens, whereas the modified analysis identified abnormal pathology in 12.3% (n = 37; odds ratio 1.4; confidence interval, 1.1-1.7; p <.01). Other abnormal appendiceal pathology identified in this study included polyps, neuroendocrine tumors, and acute appendicitis. The average number of slides required for the standard analysis was 1.4 compared with 4.9 slides for the modified analysis. At this institution, the average increase in the cost of slide production for the modified protocol was $12.07. CONCLUSION: Modified pathologic analysis resulted in a significantly higher rate of diagnosis of endometriosis and abnormal pathology in coincidental appendectomy performed during a primary gynecologic procedure for endometriosis and/or chronic pelvic pain. The use of a standard pathologic protocol likely contributes to underdiagnosis of AppE. The implementation of a modified histopathologic protocol should be considered for improving diagnosis rates of appendiceal pathology in coincidental appendectomy specimens.

Robotic-assisted Laparoscopic Management of Chemoresistant Myoinvasive Complete Molar Pregnancy.

Postmolar malignant conditions are rare after evacuation of a complete molar pregnancy. Both medical and surgical management have a role in the treatment of persistent gestational trophoblastic neoplasia. Treatment decisions must account for the natural history of the disease, previous therapies, site of disease, and the patient's desire for uterine preservation. We report on a woman who presented with chemotherapy-refractory persistent gestational trophoblastic disease (GTD). She was found to have isolated, persistent trophoblastic tissue within the uterine myometrium. She underwent a robotic-assisted laparoscopic hysterectomy with curative results. Minimally invasive surgical management may be an option for treatment of women with isolated myoinvasive GTD.

Analytical Validation of Cxbladder® Detect, Triage, and Monitor: Assays for Detection and Management of Urothelial Carcinoma.

Background: Cxbladder® assays are reverse transcription-quantitative polymerase chain reaction (RT-qPCR) tests incorporating five genetic biomarkers (CDK1, MDK, IGFBP5, HOXA13, and CXCR2) to provide risk stratification for urothelial carcinoma (UC) in patients with hematuria or undergoing surveillance for recurrent disease. This study evaluated the analytical validity of the Cxbladder Detect, Triage, and Monitor assays. Methods: Pre-specified acceptance criteria, including the assays' fundamental aspects (sample and reagent stability, RNA extraction quality, RT-qPCR linearity, and analytical sensitivity and specificity), accuracy and precision, and reproducibility between laboratories. Results: Cxbladder had an analytical sensitivity of 12.5-31.1 RNA copies/mL urine for the CDK1, MDK, IGFBP5, and HOXA13 UC biomarkers and 68.9 RNA copies/mL for the inflammatory biomarker CXCR2. All the pre-specified analytical criteria were met. Cxbladder had diagnostic sensitivity, specificity, positive predictive value, and negative predictive values of 77%, 94%, 68%, and 96%, respectively, for Detect; 95%, 46%, 20%, and 98% for Triage; and 91%, 39%, 21%, and 96% for Monitor. Cxbladder had high analytical accuracy (≤10.63% inaccuracy across all biomarkers) and good reproducibility (>85% concordance between laboratories). Conclusions: Cxbladder accurately and reproducibly detects UC biomarker expression and can aid clinicians in risk stratification of hematuria patients or those undergoing surveillance for recurrent UC.

Cyclosporin A impairs the secretion and activity of ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeat).

The protease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeat) cleaves multimers of von Willebrand factor, thus regulating platelet aggregation. ADAMTS13 deficiency leads to the fatal disorder thrombotic thrombocytopenic purpura (TTP). It has been observed that cyclosporin A (CsA) treatment, particularly in transplant patients, may sometimes be linked to the development of TTP. Until now, the reason for such a link was unclear. Here we provide evidence demonstrating that cyclophilin B (CypB) activity plays an important role in the secretion of active ADAMTS13. We found that CsA, an inhibitor of CypB, reduces the secretion of ADAMTS13 and leads to conformational changes in the protein resulting in diminished ADAMTS13 proteolytic activity. A direct, functional interaction between CypB (which possesses peptidyl-prolyl cis-trans isomerase (PPIase) and chaperone functions) and ADAMTS13 is demonstrated using immunoprecipitation and siRNA knockdown of CypB. Finally, CypB knock-out mice were found to have reduced ADAMTS13 levels. Taken together, our findings indicate that cyclophilin-mediated activity is an important factor affecting secretion and activity of ADAMTS13. The large number of proline residues in ADAMTS13 is consistent with the important role of cis-trans isomerization in the proper folding of this protein. These results altogether provide a novel mechanistic explanation for CsA-induced TTP in transplant patients.

The PCOS GWAS Candidate Gene ZNF217 Influences Theca Cell Expression of DENND1A.V2, CYP17A1, and Androgen Production.

Polycystic ovary syndrome (PCOS), a common endocrine disorder of women, is characterized by increased ovarian androgen production and anovulatory infertility. Genome-wide association studies (GWAS) have identified more than 20 PCOS candidate loci. One GWAS candidate locus encompasses ZNF217, a zinc finger transcription factor. Immunohistochemical staining of ovarian tissue demonstrated significantly lower staining intensity for ZNF217 protein in PCOS theca interna compared to ovarian tissue from normal ovulatory women. Immunofluorescence staining of normal and PCOS theca cells demonstrated nuclear localization of ZNF217, with lower intensity in PCOS cells. Western blotting showed reduced ZNF217 protein in PCOS theca cells compared to normal theca cells, and that treatment with forskolin, which mimics the action of luteinizing hormone (LH), reduces ZNF217 expression. Lower ZNF217 expression in PCOS theca cells was confirmed by quantitative reverse transcription polymerase chain reaction. Notably, there was an inverse relationship between ZNF217 messenger RNA (mRNA) levels and theca cell androgen (dehydroepiandrosterone; DHEA) synthesis. The abundance of mRNA encoding a splice variant of DENND1A (DENND1A.V2), a PCOS candidate gene that positively regulates androgen biosynthesis, was also inversely related to ZNF217 mRNA levels. This relationship may be driven by increased miR-130b-3p, which targets DENND1A.V2 transcripts and is directly correlated with ZNF217 expression. Forced expression of ZNF217 in PCOS theca cells reduced androgen production, CYP17A1 and DENND1A.V2 mRNA, while increasing mIR-130b-3p. Conversely, knockdown of ZNF217 in normal theca cells with short hairpin RNA-expressing lentivirus particles increased DENND1A.V2 and CYP17A1 mRNA. These observations suggest that ZNF217 is part of a network of PCOS candidate genes regulating thecal cell androgen production involving DENND1A.V2 and miR-130b-3p.

Multisite Quality Improvement Study of a Patient-Pathologist Consultation Program.

OBJECTIVES: The aim of this multisite quality improvement study was to evaluate patients' experiences with the patient-centered pathology (PCP) consultation program and to determine whether PCP enhanced their care experience. METHODS: Patients were invited to attend PCP consultations to review their pathology report and slides and have their questions answered by the pathologist privately, with the option to attend the appointment with family members or friends for support. A patient experience questionnaire (PEQ) was administered to patients, who participated voluntarily in the PCP, and survey data were collected and stored in REDCap. Statistical analysis was performed using SAS 9.4 (SAS Institute). RESULTS: Sixty-seven patients (95.5% female) aged 18 to 84 years across 4 institutions completed the PEQ. Overall, 58% and 15.8% of patients had breast and brain tumors, respectively, and 59.7% of tumors were newly diagnosed. Most patients thought it was important for them to learn as much as they could about their health condition. However, the majority of patients reported some degree of difficulty learning about their health condition based on written information, despite 97% having completed high school and/or further education. The majority of patients rated their pathologist as "excellent" across communication metrics. Ultimately, 100% of respondents were satisfied, found their visits to be useful, and would recommend the PCP to other patients. CONCLUSIONS: Patients found that personalized clinical encounters with pathologists improved their understanding of their health condition and their satisfaction with their care experience. Patients thought pathologists communicated respectfully, effectively, and empathetically.

Targeting IDH1 as a Prosenescent Therapy in High-grade Serous Ovarian Cancer.

Epithelial ovarian cancer (EOC) is the deadliest gynecologic cancer. High-grade serous carcinoma (HGSC) is the most frequently diagnosed and lethal histosubtype of EOC. A significant proportion of patients with HGSC relapse with chemoresistant disease. Therefore, there is an urgent need for novel therapeutic strategies for HGSC. Metabolic reprogramming is a hallmark of cancer cells, and targeting metabolism for cancer therapy may be beneficial. Here, we found that in comparison with normal fallopian tube epithelial cells, HGSC cells preferentially utilize glucose in the TCA cycle and not for aerobic glycolysis. This correlated with universally increased TCA cycle enzyme expression in HGSC cells under adherent conditions. HGSC disseminates as tumor cell spheroids within the peritoneal cavity. We found that wild-type isocitrate dehydrogenase I (IDH1) is the only TCA cycle enzyme upregulated in both adherent and spheroid conditions and is associated with reduced progression-free survival. IDH1 protein expression is also increased in patients with primary HGSC tumors. Pharmacologic inhibition or knockdown of IDH1 decreased proliferation of multiple HGSC cell lines by inducing senescence. Mechanistically, suppression of IDH1 increased the repressive histone mark H3K9me2 at multiple E2F target gene loci, which led to decreased expression of these genes. Altogether, these data suggest that increased IDH1 activity is an important metabolic adaptation in HGSC and that targeting wild-type IDH1 in HGSC alters the repressive histone epigenetic landscape to induce senescence. IMPLICATIONS: Inhibition of IDH1 may act as a novel therapeutic approach to alter both the metabolism and epigenetics of HGSC as a prosenescent therapy.

Adventitial Cystic Disease of the Radial Artery.

Upper extremity adventitial cystic disease is rare, but the characteristic findings of this lesion should be known to the hand surgeon and used to guide treatment. We present a case of a young adult male who developed a painless mass in his distal forearm. Diagnostic imaging workup revealed a cystic mass that extended within and encased the radial artery. Both MRI and direct intraoperative visualization confirmed the presence of a stalk connecting the intra-mural radial artery mass to the radiocarpal joint. The mass and stalk were excised en bloc with fenestration of the volar capsule to prevent recurrence. This case demonstrates a less common example of upper extremity adventitial cystic disease and supports the articular theory of origin of these lesions. When surgical excision is performed, an attempt should be made to identify and excise the articular stalk in an effort to minimize risk of recurrence.

Tumor lysis syndrome in a patient with ovarian yolk sac tumor.

•Tumor lysis syndrome is an oncologic emergency with profound metabolic derangements.•Germ cell tumors with large disease burden increase the risk for tumor lysis syndrome.•Herein we present a case of tumor lysis syndrome prior to initiation of cytotoxic chemotherapy for ovarian yolk sac tumor.

Clonal evolution in paired endometrial intraepithelial neoplasia/atypical hyperplasia and endometrioid adenocarcinoma.

Endometrial intraepithelial neoplasia (EIN) and atypical endometrial hyperplasia (AH) are histomorphologically defined precursors to endometrioid adenocarcinoma, which are unified as EIN/AH by the World Health Organization. EIN/AH harbors a constellation of molecular alterations similar to those found in endometrioid adenocarcinoma. However, the process of clonal evolution from EIN/AH to carcinoma is poorly characterized. To investigate, we performed next-generation sequencing, copy number alteration (CNA) analysis, and immunohistochemistry for mismatch repair protein expression on EIN/AH and endometrioid adenocarcinoma samples from 6 hysterectomy cases with spatially distinct EIN/AH and carcinoma. In evaluating all samples, EIN/AH and carcinoma did not differ in mutational burden, CNA burden, or specific genes mutated (all P>.1). All paired EIN/AH and carcinoma samples shared at least one identical somatic mutation, frequently in PI(3)K pathway members. Large CNAs (>10 genes in length) were identified in 83% of cases; paired EIN/AH and carcinoma samples shared at least one identical CNA in these cases. Mismatch repair protein expression matched in all paired EIN/AH and carcinoma samples. All paired EIN/AH and carcinoma samples had identical The Cancer Genome Atlas subtype, with 3 classified as "copy number low endometrioid" and 3 classified as "microsatellite instability hypermutated." Although paired EIN/AH and carcinoma samples were clonal, private mutations (ie, present in only one sample) were identified in EIN/AH and carcinoma in all cases, frequently in established cancer-driving genes. These findings indicate that EIN/AH gives rise to endometrioid adenocarcinoma by a complex process of subclone evolution, not a linear accumulation of molecular events.

The effects of removing the GAT domain from E. coli GMP synthetase.

E. coli GMP synthetase (GMPS) catalyzes the conversion of XMP to GMP. Ammonia, generated in the amino-terminal glutamine amidotransferase (GAT) domain, is transferred by an unknown mechanism to the ATP-pyrophosphatase (ATPP) domain, where it attacks a highly reactive adenyl-XMP intermediate, leading to GMP formation. To study the structural requirements for the activity of E. coli GMPS, we used PCR to generate a protein expression construct that contains the ATPP domain as well as the predicted dimerization domain (DD). The ATPP/DD protein is active in solution, utilizing NH (4) (+) as an NH(3) donor. Size-exclusion chromatography demonstrates a dimeric mass for the ATPP/ DD protein, providing the first evidence in solution for the structural organization of the intact GMPS. Kinetic characterization of the ATPP/DD domain protein provides evidence that the presence of the GAT domain can regulate the activity of the ATPP domain.

Characterization of coding synonymous and non-synonymous variants in ADAMTS13 using ex vivo and in silico approaches.

Synonymous variations, which are defined as codon substitutions that do not change the encoded amino acid, were previously thought to have no effect on the properties of the synthesized protein(s). However, mounting evidence shows that these "silent" variations can have a significant impact on protein expression and function and should no longer be considered "silent". Here, the effects of six synonymous and six non-synonymous variations, previously found in the gene of ADAMTS13, the von Willebrand Factor (VWF) cleaving hemostatic protease, have been investigated using a variety of approaches. The ADAMTS13 mRNA and protein expression levels, as well as the conformation and activity of the variants have been compared to that of wild-type ADAMTS13. Interestingly, not only the non-synonymous variants but also the synonymous variants have been found to change the protein expression levels, conformation and function. Bioinformatic analysis of ADAMTS13 mRNA structure, amino acid conservation and codon usage allowed us to establish correlations between mRNA stability, RSCU, and intracellular protein expression. This study demonstrates that variants and more specifically, synonymous variants can have a substantial and definite effect on ADAMTS13 function and that bioinformatic analysis may allow development of predictive tools to identify variants that will have significant effects on the encoded protein.

A splice variant of ADAMTS13 is expressed in human hepatic stellate cells and cancerous tissues.

Although ADAMTS13, the von Willebrand factor (VWF)-cleaving protease, is expressed in a range of tissues, the physiological significance of tissue-specific ADAMTS13 alternative splicing isoforms have yet to be clarified. Screening a panel of human tissues and cell lines revealed a spliced ADAMTS13 transcript in hepatic stellate cells and a hepatoma cell line that retains the 25th intron. A nonsense codon within the intron truncates the protease, which gains 64 novel amino acids in lieu of both CUB domains. This isoform, while retaining VWF-cleaving capability, accumulates intracellularly and its biological inaccessibility may prevent its participation in regulating haemostasis and other physiologic functions.