CCNE1 and survival of patients with tubo-ovarian high-grade serous carcinoma: An Ovarian Tumor Tissue Analysis consortium study.

BACKGROUND: Cyclin E1 (CCNE1) is a potential predictive marker and therapeutic target in tubo-ovarian high-grade serous carcinoma (HGSC). Smaller studies have revealed unfavorable associations for CCNE1 amplification and CCNE1 overexpression with survival, but to date no large-scale, histotype-specific validation has been performed. The hypothesis was that high-level amplification of CCNE1 and CCNE1 overexpression, as well as a combination of the two, are linked to shorter overall survival in HGSC. METHODS: Within the Ovarian Tumor Tissue Analysis consortium, amplification status and protein level in 3029 HGSC cases and mRNA expression in 2419 samples were investigated. RESULTS: High-level amplification (>8 copies by chromogenic in situ hybridization) was found in 8.6% of HGSC and overexpression (>60% with at least 5% demonstrating strong intensity by immunohistochemistry) was found in 22.4%. CCNE1 high-level amplification and overexpression both were linked to shorter overall survival in multivariate survival analysis adjusted for age and stage, with hazard stratification by study (hazard ratio [HR], 1.26; 95% CI, 1.08-1.47, p = .034, and HR, 1.18; 95% CI, 1.05-1.32, p = .015, respectively). This was also true for cases with combined high-level amplification/overexpression (HR, 1.26; 95% CI, 1.09-1.47, p = .033). CCNE1 mRNA expression was not associated with overall survival (HR, 1.00 per 1-SD increase; 95% CI, 0.94-1.06; p = .58). CCNE1 high-level amplification is mutually exclusive with the presence of germline BRCA1/2 pathogenic variants and shows an inverse association to RB1 loss. CONCLUSION: This study provides large-scale validation that CCNE1 high-level amplification is associated with shorter survival, supporting its utility as a prognostic biomarker in HGSC.

Differences in the Active Endometrial Microbiota across Body Weight and Cancer in Humans and Mice.

Obesity is a risk factor for endometrial cancer. The aim of this study was to determine whether actively replicating microbiota in the endometrium differ between obese vs. lean and cancer vs. benign states. We performed 16S rRNA amplicon sequencing on endometrial tissues from lean and obese women with and without endometrial cancer, and lean and obese mice. Results displayed human endometrial microbiota clustered into three community types (R = 0.363, p = 0.001). Lactobacillus was dominant in community type 1 (C1) while community type 2 (C2) had high levels of Proteobacteria and more cancer samples when compared to C1 (p = 0.007) and C3 (p = 0.0002). A significant increase in the prevalence of the C2 community type was observed across body mass index and cancer (χ2 = 14.24, p = 0.0002). The relative abundance of Lactobacillus was lower in cancer samples (p = 0.0043), and an OTU with 100% similarity to Lactobacillus iners was enriched in control samples (p = 0.0029). Mouse endometrial microbiota also clustered into three community types (R = 0.419, p = 0.001) which were not influenced by obesity. In conclusion, obesity and cancer are associated with community type prevalence in the human endometrium, and Lactobacillus abundance is associated with normal uterine histologies in humans and mice.

MCM3 is a novel proliferation marker associated with longer survival for patients with tubo-ovarian high-grade serous carcinoma.

Tubo-ovarian high-grade serous carcinomas (HGSC) are highly proliferative neoplasms that generally respond well to platinum/taxane chemotherapy. We recently identified minichromosome maintenance complex component 3 (MCM3), which is involved in the initiation of DNA replication and proliferation, as a favorable prognostic marker in HGSC. Our objective was to further validate whether MCM3 mRNA expression and possibly MCM3 protein levels are associated with survival in patients with HGSC. MCM3 mRNA expression was measured using NanoString expression profiling on formalin-fixed and paraffin-embedded tissue (N = 2355 HGSC) and MCM3 protein expression was assessed by immunohistochemistry (N = 522 HGSC) and compared with Ki-67. Kaplan-Meier curves and the Cox proportional hazards model were used to estimate associations with survival. Among chemotherapy-naïve HGSC, higher MCM3 mRNA expression (one standard deviation increase in the score) was associated with longer overall survival (HR = 0.87, 95% CI 0.81-0.92, p < 0.0001, N = 1840) in multivariable analysis. MCM3 mRNA expression was highest in the HGSC C5.PRO molecular subtype, although no interaction was observed between MCM3, survival and molecular subtypes. MCM3 and Ki-67 protein levels were significantly lower after exposure to neoadjuvant chemotherapy compared to chemotherapy-naïve tumors: 37.0% versus 46.4% and 22.9% versus 34.2%, respectively. Among chemotherapy-naïve HGSC, high MCM3 protein levels were also associated with significantly longer disease-specific survival (HR = 0.52, 95% CI 0.36-0.74, p = 0.0003, N = 392) compared to cases with low MCM3 protein levels in multivariable analysis. MCM3 immunohistochemistry is a promising surrogate marker of proliferation in HGSC.

Restoring Tumour Selectivity of the Bioreductive Prodrug PR-104 by Developing an Analogue Resistant to Aerobic Metabolism by Human Aldo-Keto Reductase 1C3.

PR-104 is a phosphate ester pre-prodrug that is converted in vivo to its cognate alcohol, PR-104A, a latent alkylator which forms potent cytotoxins upon bioreduction. Hypoxia selectivity results from one-electron nitro reduction of PR-104A, in which cytochrome P450 oxidoreductase (POR) plays an important role. However, PR-104A also undergoes 'off-target' two-electron reduction by human aldo-keto reductase 1C3 (AKR1C3), resulting in activation in oxygenated tissues. AKR1C3 expression in human myeloid progenitor cells probably accounts for the dose-limiting myelotoxicity of PR-104 documented in clinical trials, resulting in human PR-104A plasma exposure levels 3.4- to 9.6-fold lower than can be achieved in murine models. Structure-based design to eliminate AKR1C3 activation thus represents a strategy for restoring the therapeutic window of this class of agent in humans. Here, we identified SN29176, a PR-104A analogue resistant to human AKR1C3 activation. SN29176 retains hypoxia selectivity in vitro with aerobic/hypoxic IC50 ratios of 9 to 145, remains a substrate for POR and triggers γH2AX induction and cell cycle arrest in a comparable manner to PR-104A. SN35141, the soluble phosphate pre-prodrug of SN29176, exhibited superior hypoxic tumour log cell kill (>4.0) to PR-104 (2.5-3.7) in vivo at doses predicted to be achievable in humans. Orthologues of human AKR1C3 from mouse, rat and dog were incapable of reducing PR-104A, thus identifying an underlying cause for the discrepancy in PR-104 tolerance in pre-clinical models versus humans. In contrast, the macaque AKR1C3 gene orthologue was able to metabolise PR-104A, indicating that this species may be suitable for evaluating the toxicokinetics of PR-104 analogues for clinical development. We confirmed that SN29176 was not a substrate for AKR1C3 orthologues across all four pre-clinical species, demonstrating that this prodrug analogue class is suitable for further development. Based on these findings, a prodrug candidate was subsequently identified for clinical trials.

The Clinical Relevance of p16 and p53 Status in Patients with Squamous Cell Carcinoma of the Vulva.

OBJECTIVE: To investigate the prognostic significance of HPV status in vulvar squamous cell carcinomas (VSCC) and to determine whether preoperative determination of p16 or p53 status would have clinical relevance. METHODS: Patients treated for VSCC at a tertiary hospital in Sydney, Australia, from 2002 to 2014, were retrospectively evaluated (n = 119). Histological specimens were stained for p53 and p16 expression, and HPV status was determined by PCR detection of HPV DNA. RESULTS: HPV DNA was detected in 19%, p16 expression in 53%, and p53 expression in 37% of patients. Kaplan-Meier survival estimates indicated that p16/HPV-positive patients had superior five-year disease-free survival (76% versus 42%, resp., p = 0.004) and disease-specific survival (DSS) (89% versus 75% resp., p = 0.05) than p53-positive patients. In univariate analysis, nodal metastases (p < 0.001), tumor size >4 cm (p = 0.03), and perineural invasion (p = 0.05) were associated with an increased risk of disease progression and p16 expression with a decreased risk (p = 0.03). In multivariable analysis, only nodal metastases remained independent for risk of disease progression (p = 0.01). For DSS, lymph node metastases (p < 0.001) and tumor size (p = 0.008) remained independently prognostic. CONCLUSION: The p16/HPV and p53 status of VSCC allows separation of patients into two distinct clinicopathological groups, although 10% of patients fall into a third group which is HPV, p16, and p53 negative. p16 status was not independently prognostic in multivariable analysis. Treatment decisions should continue to be based on clinical indicators rather than p16 or p53 status.

A combination of the immunohistochemical markers CK7 and SATB2 is highly sensitive and specific for distinguishing primary ovarian mucinous tumors from colorectal and appendiceal metastases.

Primary ovarian mucinous tumors can be difficult to distinguish from metastatic gastrointestinal neoplasms by histology alone. The expected immunoprofile of a suspected metastatic lower gastrointestinal tumor is CK7-/CK20+/CDX2+/PAX8-. This study assesses the addition of a novel marker SATB2, to improve the diagnostic algorithm. A test cohort included 155 ovarian mucinous tumors (105 carcinomas and 50 borderline tumors) and 230 primary lower gastrointestinal neoplasms (123 colorectal adenocarcinomas and 107 appendiceal neoplasms). All cases were assessed for SATB2, PAX8 CK7, CK20, and CDX2 expression on tissue microarrays. Expression was scored in a 3-tier system as absent, focal (1-50% of tumor cells) and diffuse ( >50% of tumor cells) and then categorized into either absent/present or nondiffuse/diffuse. SATB2 and PAX8 expression was further evaluated in ovarian tumors from an international cohort of 2876 patients (expansion cohort, including 159 mucinous carcinomas and 46 borderline mucinous tumors). The highest accuracy of an individual marker in distinguishing lower gastrointestinal from ovarian mucinous tumors was CK7 (91.7%, nondiffuse/diffuse cut-off) followed by SATB2 (88.8%, present/absent cut-off). The most effective combination was CK7 and SATB2 with accuracy of 95.3% using the 3-tier interpretation, absent/focal/diffuse. This combination outperformed the standard clinical set of CK7, CK20 and CDX2 (87.5%). Re-evaluation of outlier cases confirmed ovarian origin for all but one case. The accuracy of SATB2 was confirmed in the expansion cohort (91.5%). SATB2 expression was also detected in 15% of ovarian endometrioid carcinoma but less than 5% of other ovarian histotypes. A simple two marker combination of CK7 and SATB2 can distinguish lower gastrointestinal from ovarian primary mucinous tumors with greater than 95% accuracy. PAX8 and CDX2 have value as second-line markers. The utility of CK20 in this setting is low and this warrants replacement of this marker with SATB2 in clinical practice.

Degenerate-disc infection study with contaminant control (DISC): Application of a proposed histological scoring system.

Recent evidence into an infectious etiology of discogenic back pain/leg pain are ongoing with contradictory data in literature. We sought to validate the clinical relevance of histopathological evidence of inflammation through a previously proposed histological grading system. In this prospective cohort study, 124 consecutive patients undergoing an elective spinal decompression and/or fusion procedure involving discectomy were selected with intraoperative tissue sampling of intervertebral disc and paraspinal tissue at a single institution. The histological domains were correlated with positive disc cultures to assist in identifying relevant positive infections. Inter-observer analysis of the scoring system was also performed. There were 124 samples (36 cervical and 88 lumbar) obtained. 29 (23.4%) disc specimens and 37 (29.8%) of ligament samples demonstrated growth of C. acnes. In total, 38/124 (30.6%) of disc specimens were positive for growth of any species. There was poor association between positive disc culture and the presence of neutrophilia (p = 0.123) or chronic inflammatory changes (p = 0.092) on histopathological assessment. There was no statistical significance noted across all histological domains examined within the finalised scoring system and positive culture across disc specimens. There was moderate agreement in between observers (kappa range: 0.41-0.60) in the assessment of inflammatory changes using the proposed scoring system. The current study suggests poor correlation between histopathological evidence of chronic or acute inflammation and positive disc cultures questioning the idea that disc infection is the root cause of acute or chronic back pain/leg pain.

Atypical Teratoid Rhabdoid Tumor: Two Case Reports and an Analysis of Adult Cases with Implications for Pathophysiology and Treatment.

We present the first quantitative analysis of atypical teratoid rhabdoid tumors (ATRT) in adults, including two patients from our own institutions. These are of interest as one occurred during pregnancy and one is a long-term survivor. Our review of pathological findings of 50 reported cases of adult ATRT leads us to propose a solely ectodermal origin for the tumor and that epithelial-mesenchymal transition (EMT) is a defining feature. Thus, the term ATRT may be misleading. Our review of clinical findings shows that ATRT tends to originate in mid-line structures adjacent to the CSF, leading to a high rate of leptomeningeal dissemination. Thus, we hypothesize that residual undifferentiated ectoderm in the circumventricular organs, particularly the pituitary and pineal glands, is the most common origin for these tumors. We note that if growth is not arrested soon after diagnosis, or after the first relapse/progression, death is almost universal. While typically rapidly fatal (as in our first case), long-term remission is possible (as in our second). Significant predictors of prognosis were the extent of resection and the use of chemotherapy. Glial differentiation (GFAP staining) was strongly associated with leptomeningeal metastases (chi-squared p = 0.02) and both predicted markedly worse outcomes. Clinical trials including adults are rare. ATRT is primarily a disease of infancy and radiotherapy is generally avoided in those aged less than 3 years old. Treatment options in adults differ from infants in that cranio-spinal irradiation is a viable adjunct to systemic chemotherapy in the adult population. Given the grave prognosis, this combined approach appears reasonable. As effective chemotherapy is likely to cause myelosuppression, we recommend that stem-cell rescue be available locally.

Looking beyond human papillomavirus (HPV) genotype 16 and 18: Defining HPV genotype distribution in cervical cancers in Australia prior to vaccination.

Australia has implemented a high-coverage HPV vaccination program but has not, to date, established the distribution of HPV types that occur in cervical cancers in Australia. This information is important for determining the potential for cervical cancer prevention with both current and broader spectrum HPV vaccines. We analysed 847 cervical cancers diagnosed 2005 to 2015 in tertiary centres in the three most populous Australian states with resolution of specimens containing multiple HPV types using laser-capture microdissection. Archived FFPE tissue was reviewed by specialist pathologists, sandwich sectioned, and initially whole-tissue sections genotyped for HPV. Samples were first genotyped using SPF10-LiPA25 (version 1). Negative samples were screened with DNA ELISA kit HPV SPF10, followed by genotyping with SPF+ LiPA if ELISA positive. If still negative, samples were tested on a qPCR assay targeting the E6 region of HPV16, 18, 45 and 33. Of the 847 cancers (65.1% squamous, 28.7% adenocarcinoma, 4.3% adenosquamous, 2.0% other), 92.9% had HPV detected. Of the HPV-positive cancers, 607 of 787 (77.1%) contained HPV16 or 18, 125 of 787 (15.9%) contained HPV31/33/45/52 or 58, and 55 (7.0%) another HPV type. There was a strong correlation between HPV type and age, with younger women most likely to have HPV16/18 detected and least likely HPV negative. Our findings indicate that cervical cancers diagnosed in Australia more frequently contain HPV16/18 than in international series. This could be due to cervical screening in Australia increasing the proportion of adenocarcinomas, in which types 18 and 16 more strongly predominate, due to prevention of squamous cancers.

Histological analysis of surgical samples and a proposed scoring system for infections in intervertebral discs.

Back pain remains one the most prevalent types of pain and disability worldwide. Infection is estimated to be the underlying cause in approximately 0.01% of patients. Despite recent evidence demonstrating prominent infection rates, a standardised algorithm for diagnosis of disc infection is lacking. Histopathological evaluation can aid in confirming inflammatory changes and also in identifying degenerative changes. Hence, standardising practice through a clear scoring system with regards to inflammation and degeneration may have some utility in the clinical setting. To our knowledge no such systems exist specifically for intervertebral disc infection. A literature review of current methods of scoring inflammation and degeneration in spine surgery and orthopaedic surgery was performed. Based on the current evidence, a scoring system for disc inflammatory and degenerative changes was proposed. We propose four domains for consideration: (1) granulation tissue, (2) dense fibrosis, (3) chronic inflammatory cells, and (4) neutrophil count. The non-standardised nature of diagnosing infections and degeneration in the spinal surgery literature means that this scoring system is currently of particular value. Based on a literature review, our proposed method for diagnosis incorporates a combination of histopathological criteria expected to increase diagnostic sensitivity in the setting of disc infection. Overall, scoring can be applied to surgically obtained material and integrated directly into routine pathological practice.

Identification of one-electron reductases that activate both the hypoxia prodrug SN30000 and diagnostic probe EF5.

SN30000 is a second-generation benzotriazine-N-oxide hypoxia-activated prodrug scheduled for clinical trial. Previously we showed that covalent binding of the hypoxia probe EF5 predicts metabolic activation of SN30000 in a panel of cancer cell lines under anoxia, suggesting that they are activated by the same reductases. However the identity of these reductases is unknown. Here, we test whether forced expression of nine oxidoreductases with known or suspected roles in bioreductive prodrug metabolism (AKR1C3, CYB5R3, FDXR, MTRR, NDOR1, NOS2A, NQO1, NQO2 and POR) enhances oxic or anoxic reduction of SN30000 and EF5 by HCT116 cells. Covalent binding of (14)C-EF5 and reduction of SN30000 to its 1-oxide and nor-oxide metabolites was highly selective for anoxia in all lines, with significantly elevated anoxic metabolism of both compounds in lines over-expressing POR, MTRR, NOS2A or NDOR1. There was a strong correlation between EF5 binding and SN30000 metabolism under anoxia across the cell lines (R(2)=0.84, p=0.0001). Antiproliferative potency of SN30000 under anoxia was increased most strongly by overexpression of MTRR and POR. Transcript abundance in human tumours, evaluated using public domain mRNA expression data, was highest for MTRR, followed by POR, NOS2A and NDOR1, with little variation between tumour types. Immunostaining of tissue microarrays demonstrated variable MTRR protein expression across 517 human cancers with most displaying low expression. In conclusion, we have identified four diflavin reductases (POR, MTRR, NOS2A and NDOR1) capable of reducing both SN30000 and EF5, further supporting use of 2-nitroimidazole probes to predict the ability of hypoxic cells to activate SN30000.

Diflavin oxidoreductases activate the bioreductive prodrug PR-104A under hypoxia.

The clinical agent PR-104 is converted systemically to PR-104A, a nitrogen mustard prodrug designed to target tumor hypoxia. Reductive activation of PR-104A is initiated by one-electron oxidoreductases in a process reversed by oxygen. The identity of these oxidoreductases is unknown, with the exception of cytochrome P450 reductase (POR). To identify other hypoxia-selective PR-104A reductases, nine candidate oxidoreductases were expressed in HCT116 cells. Increased PR-104A-cytotoxicity was observed in cells expressing methionine synthase reductase (MTRR), novel diflavin oxidoreductase 1 (NDOR1), and inducible nitric-oxide synthase (NOS2A), in addition to POR. Plasmid-based expression of these diflavin oxidoreductases also enhanced bioreductive metabolism of PR-104A in an anoxia-specific manner. Diflavin oxidoreductase-dependent PR-104A metabolism was suppressed >90% by pan-flavoenzyme inhibition with diphenyliodonium chloride. Antibodies were used to quantify endogenous POR, MTRR, NDOR1, and NOS2A expression in 23 human tumor cell lines; however, only POR protein was detectable and its expression correlated with anoxic PR-104A reduction (r(2) = 0.712). An anti-POR monoclonal antibody was used to probe expression using human tissue microarrays; 13 of 19 cancer types expressed detectable POR with 21% of cores (185 of 874) staining positive; this heterogeneity suggests that POR is a useful biomarker for PR-104A activation. Immunostaining for carbonic anhydrase 9 (CAIX), reportedly an endogenous marker of hypoxia, revealed only moderate coexpression (9.6%) of both CAIX and POR across a subset of five cancer types.

Dermatofibrosarcoma protuberans: report of a case with a variant ring chromosome and metastases following pregnancy.

BACKGROUND: The most frequent molecular abnormality observed in dermatofibrosarcoma protuberans (DFSP) is the formation of a supernumerary ring chromosome or translocation resulting in fusion of the gene encoding the alpha-chain of type 1 collagen, COL1A1 from 17q22, to the platelet-derived growth factor beta-chain, PDGFB gene from 22q13. Rare cases documenting variant ring or marker chromosomes involving regions other than 17q22 and 22q13 have been reported. Further analysis in three of these cases demonstrated the presence of the COL1A1 and PDGFB genes. METHODS: We report a further case of DFSP with a rare variant ring chromosome. The tumor appeared to undergo accelerated growth during pregnancy, then metastasized following pregnancy. We describe the clinical, histological, immunohistochemical, and cytogenetic features. RESULTS: The metastatic tumor showed a variant r(17;?) chromosome. A locus-specific probe was required to demonstrate presence of the PDGFB gene within the ring, indicating cryptic molecular rearrangement between chromosomes 17 and 22, and recombination with an unknown chromosome. CONCLUSIONS: Cryptic rearrangement of chromosomes 17 and 22 should be suspected in variant ring chromosomes and translocations. Pregnancy may contribute to accelerated growth of DFSP, and delay in surgical resection should be avoided.

Inflammatory myofibroblastic tumor of the left ventricle in an older adult.

We report an unusual study of a 72-year-old woman who presented with a symptomatic space-occupying lesion in the left ventricle. The lesion was surgically excised with a good long-term result. It was histologicaly diagnosed as an inflammatory myofibroblastic tumor. Also known as an inflammatory pseudotumor, these tumors affecting the heart are extremely rare and occur mainly in children and adolescents. We review the literature and highlight the unique nature of the lesion.

Gram stain, culture, and histopathological examination findings for heart valves removed because of infective endocarditis.

Retrospective chart review was undertaken for 480 patients who underwent a total of 506 valve replacements or repair procedures for infective endocarditis. The influence of preoperative antimicrobial treatment on culture, Gram stain, and histopathological examination findings for resected valve specimens was examined. When valves were removed before the end of treatment, organisms were seen on the Gram stain of ground valve material performed in the microbiology laboratory and on Gram-stained histopathological sections in 231 (81%) of 285 and 140 (67%) of 208 specimens, respectively (P=.0007). Gram-positive cocci were either cultured from or observed in excised valve tissue in 42 (67%) of 63 episodes involving negative preoperative blood cultures. Positive Gram stain results for microbiological specimens should be reintroduced into the definite pathological criteria for infective endocarditis. When deciding on how long to continue antimicrobial therapy after valve replacement for endocarditis, valve culture results should be the only laboratory finding taken into account, because it takes months for dead bacteria to be removed from sterile vegetations.