Increased expression of neurotensin in high grade serous ovarian carcinoma with evidence of serous tubal intraepithelial carcinoma.

High grade serous ovarian carcinoma (HGSC) without identifiable serous tubal intraepithelial carcinoma (STIC) within the fallopian tube (FT) occurs in approximately 50% of patients. The objective of this study was to use a multisite tumor sampling approach to study HGSC with and without STIC. RNAseq analysis of HGSC samples collected from multiple sites e.g. ovary, FT and peritoneum, revealed moderate levels of intrapatient heterogeneity in gene expression that could influence molecular profiles. Mixed-model ANOVA analysis of gene expression in tumor samples from patients with multiple tumor sites (n = 13) and patients with a single site tumor sample (n = 11) to compare HGSC-STIC to HGSC-NOSTIC identified neurotensin (NTS) as significantly higher (> two-fold change, False Discovery Rate (FDR) < 0.10) in HGSC-STIC. This data was validated using publicly available RNA-Seq datasets. Concordance between higher NTS gene expression and NTS peptide levels in HGSC-STIC samples was demonstrated by immunohistochemistry. To determine the role of NTS in HGSC, five ovarian cancer (OvCa) cell lines were screened for expression of NTS and its receptors, NTSR1 and NTSR3. Increased expression of NTS and NSTR1 was observed in several of the OvCa cells, whereas the NTSR3 receptor was lower in all OvCa cells, compared to immortalized FT epithelial cells. Treatment with NTSR1 inhibitor (SR48692) decreased cell proliferation, but increased cell migration in OvCa cells. The effects of SR48692 were receptor mediated, since transient RNAi knockdown of NTSR1 mimicked the migratory effects and knockdown of NTSR3 mimicked the anti-proliferative effects. Further, knockdown of NTSR1 or NTSR3 was associated with acquisition of distinct morphological phenotypes, epithelial or mesenchymal, respectively. Taken together, our results reveal a difference in a biologically active pathway between HGSC with and without STIC. Furthermore, we identify neurotensin signaling as an important pathway involved in cell proliferation and epithelial-mesenchymal transition in HGSC-STIC which warrants further study as a potential therapeutic target. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Roles of the C-terminal domains of topoisomerase IIα and topoisomerase IIß in regulation of the decatenation checkpoint.

Topoisomerase (topo) IIα and IIß maintain genome stability and are targets for anti-tumor drugs. In this study, we demonstrate that the decatenation checkpoint is regulated, not only by topo IIα, as previously reported, but also by topo IIß. The decatenation checkpoint is most efficient when both isoforms are present. Regulation of this checkpoint and sensitivity to topo II-targeted drugs is influenced by the C-terminal domain (CTD) of the topo II isoforms and by a conserved non-catalytic tyrosine, Y640 in topo IIα and Y656 in topo IIß. Deletion of most of the CTD of topo IIα, while preserving the nuclear localization signal (NLS), enhances the decatenation checkpoint and sensitivity to topo II-targeted drugs. In contrast, deletion of most of the CTD of topo IIß, while preserving the NLS, and mutation of Y640 in topo IIα and Y656 in topo IIß inhibits these activities. Structural studies suggest that the differential impact of the CTD on topo IIα and topo IIß function may be due to differences in CTD charge distribution and differential alignment of the CTD with reference to transport DNA. Together these results suggest that topo IIα and topo IIß cooperate to maintain genome stability, which may be distinctly modulated by their CTDs.

Characterisation of homologous recombination deficiency in paired primary and recurrent high-grade serous ovarian cancer.

BACKGROUND: Homologous recombination deficiency (HRD) is shown to predict response to DNA-damaging therapies in patients with high-grade serous ovarian cancer (HGSOC); however, changes in HRD during progression remains unknown. METHODS: HRD scores were evaluated in paired primary and/or recurrent HGSOC samples (N = 107) from 54 patients with adjuvant platinum-based chemotherapy. BRCA1/2 mutation, BRCA1 methylation, loss of heterozygosity (LOH), and HRD scores were characterised using tumour DNA-based next-generation sequencing assays. RESULTS: Among 50 evaluable pairs (N = 100 samples), high intra-patient correlation in HRD score was observed (r2 = 0.93). BRCA1/2 mutations, BRCA1/2 LOH, and HRD were maintained between primary and recurrent samples, except for one pair in which a BRCA1 reversion mutation was identified in the recurrent sample. Despite the reversion, both samples were classified as having high HRD scores ( ≥ 42). All samples with BRCA1/2 mutations exhibited high HRD scores; however, high HRD scores were more prevalent than BRCA1/2 mutations (55% vs. 30%, respectively). CONCLUSION: Markers of HRD were maintained between the primary and recurrent samples, regardless of other genomic changes that occurred during recurrence. HRD score/markers in primary tumours may be valuable and adequate for selection of platinum-based therapy and/or poly-ADP-ribose-polymerase (PARP) inhibitors in recurrent HGSOC.

HOXA4/HOXB3 gene expression signature as a biomarker of recurrence in patients with high-grade serous ovarian cancer following primary cytoreductive surgery and first-line adjuvant chemotherapy.

OBJECTIVES: Aberrant homeobox (HOX) gene expression is reported in high-grade serous ovarian carcinoma (HGSOC), however, its prognostic significance remains unclear. METHODS: HOX genes associated with progression-free survival (PFS) in a discovery cohort of primary HGSOC samples with RNA sequencing data, and those previously reported to be associated with clinical outcomes, were selected for qPCR testing in an independent training cohort of primary HGSOC samples (n=71). A prognostic model for PFS was developed using univariate and multivariate Cox regression. Patients were stratified into risk groups that optimized the test statistic. The model was tested in an independent HGSOC cohort from The Cancer Genome Atlas (TCGA) (n=320). The effect of selected HOX genes on drug sensitivity and reactive oxygen species (ROS) accumulation was examined in vitro. RESULTS: Of 23 HOX genes tested in the training cohort, HOXA4 (HR=1.20, 95% CI=1.07-1.34, P=0.002) and HOXB3 (HR=1.09, 95% CI=1.01-1.17, P=0.027) overexpression were significantly associated with shorter PFS in multivariate analysis. Based on the optimal cutoff of the HOXA4/HOXB3 risk score, median PFS was 16.9months (95% CI=14.6-21.2months) and not reached (>80months) for patients with high and low risk scores, respectively (HR=8.89, 95% CI=2.09-37.74, P<0.001). In TCGA, the HOXA4/HOXB3 risk score was significantly associated with disease-free survival (HR=1.44, 95% CI=1.00-2.09, P=0.048). HOXA4 or HOXB3 overexpression in ovarian cancer cells decreased sensitivity to cisplatin and attenuated the generation of cisplatin-induced ROS (P<0.05). CONCLUSIONS: HOXA4/HOXB3 gene expression-based risk score may be useful for prognostic risk stratification and warrants prospective validation in HGSOC patients.

Expression profile of COL2A1 and the pseudogene SLC6A10P predicts tumor recurrence in high-grade serous ovarian cancer.

Tumor recurrence, following initial response to adjuvant chemotherapy, is a major problem in women with high-grade serous ovarian cancer (HGSOC). Microarray analysis of primary tumors has identified genes that may be useful in risk stratification/overall survival, but are of limited value in predicting the >70% rate for tumor recurrence. In this study, we performed RNA-Seq analysis of primary and recurrent HGSOC to first identify unique differentially expressed genes. From this dataset, we selected 21 archetypical coding genes and one noncoding RNA, based on statistically significant differences in their expression profile between tumors, for validation by qPCR in a larger cohort of 110 ovarian tumors (71 primary and 39 recurrent) and for testing association of specific genes with time-to-recurrence (TTR). Kaplan-Meier tests revealed that high expression of collagen type II, alpha 1 (COL2A1) was associated with delayed TTR (HR = 0.47, 95% CI: 0.27-0.82, p = 0.008), whereas low expression of the pseudogene, solute carrier family 6 member 10 (SLC6A10P), was associated with longer TTR (HR = 0.53, 95% CI: 0.30-0.93, p = 0.027). Notably, TTR was significantly delayed for tumors that simultaneously highly expressed COL2A1 and lowly expressed SLC6A10P (HR = 0.21, 95% CI: 0.082-0.54, p = 0.0011), an estimated median of 95 months as compared to an estimated median of 16 months for subjects expressing other levels of COL2A1 and SLC6A10P. Thus, evaluating expression levels of COL2A1 and SLC6A10P at primary surgery could be beneficial for clinically managing recurrence of HGSOC.

Biological role and clinical implications of homeobox genes in serous epithelial ovarian cancer.

Homeobox (HOX) genes are a family of transcription factors that are essential regulators of development. HOX genes play important roles in normal reproductive physiology, as well as in the development and progression of serous carcinomas, the predominant and most aggressive subtype of epithelial ovarian cancer (EOC). This review discusses aberrant HOX gene expression in serous EOC and its impact on tumor development and progression. Further identification of HOX target genes may facilitate the development of novel diagnostic and therapeutic strategies to improve the prognosis of patients with serous EOC.

Targeting Aurora A Kinase (AAK) in Platinum-Resistant High Grade Serous Ovarian Cancer.

Aurora A kinase (AAK) involved in G2-M transition is functionally involved in centrosome maturation and maintaining an active spindle assembly checkpoint. We tested the hypothesis that in platinum-taxane resistant high grade serous ovarian cancer (HGSOC) inhibition of AAK involved in G2-M transition would enhance the anti-tumor activity of cisplatin (CP) or paclitaxel (PT). Using HGSOC cell lines from platinum-taxane refractory patients that do not harbor BRCA1/2 mutations, we tested the anti-tumor activity of CP, or PT alone or in combination with the AAK inhibitor alisertib (AL). Treatment with CP for 3 h or PT for 6 h followed sequentially by AL for 48 h led to a significant decrease in cell survival (p < 0.001) compared to treatment with either drug alone in HGSOC cells but not in immortalized normal human ovarian surface epithelium or normal human fallopian tube secretory epithelium cells. The treatment with CP or PT followed by AL also led to a significant increase in reactive oxygen species (p < 0.05), apoptosis (p < 0.001) and accumulation of cells in G2/M that was accompanied by a modest increase in expression of AAK. Downregulation of AAK, but not aurora B kinase, with targeted siRNAs also significantly enhanced apoptosis by CP or PT, suggesting that AL specifically targeted AAK. In summary, in HGSOC without BRCA1/2 mutations, CP, or PT resistance can potentially be circumvented by sequential treatment with AL that inhibits AAK involved in G2-M transition.

RNA Immune Signatures from Pan-Cancer Analysis Are Prognostic for High-Grade Serous Ovarian Cancer and Other Female Cancers.

Immune cell infiltrates within the tumor microenvironment can influence treatment response and outcome in several cancers. In this study, we developed RNA-based immune signatures from pan-cancer analysis that could serve as potential markers across tumor types and tested them for association with outcome in high-grade serous ovarian cancer (HGSOC) and other female cancers. Pan-cancer RNA-Seq cluster analysis of immune-related gene expression profiles in The Cancer Genome Atlas (TCGA) from 29 different solid tumors (4446 specimens) identified distinct but concordant gene signatures. Among these immune signatures, Cytotoxic Lymphocyte Immune Signature (CLIS), T-cell trafficking (TCT), and the TCT to M2 tumor-associated macrophage (M2TAM) ratio (TCT:M2TAM) were significantly (p < 0.05) associated with overall survival (OS), using multivariable Cox proportional hazards regression models, in a discovery cohort and two independent validation cohorts of HGSOC patients. Notably, the TCT:M2TAM ratio was highly significant (p ≤ 0.000001) in two HGSOC cohorts. Immune signatures were also significant (p < 0.05) in the presence of tumor cytoreduction, BRCA1/2 mutation, and COL2A1 expression. Importantly, the CLIS and TCT signatures were also validated for prognostic significance (p < 0.05) in TCGA cohorts for endometrial and high tumor mutational burden (Hi-TMB) breast cancer. These immune signatures also have the potential for being predictive in other cancers and for patients following different treatment strategies.

Association of immunophenotype with expression of topoisomerase II α and ß in adult acute myeloid leukemia.

Anthracyclines used in the treatment of acute myelogenous leukemia (AML) inhibit the activity of the mammalian topoisomerase II (topo II) isoforms, topo II α and topo IIß. In 230 patients with non-M3 AML who received frontline ara-C/daunorubicin we determined expression of topo IIα and topo IIß by RT-PCR and its relationship to immunophenotype (IP) and outcomes. Treatment outcomes were analyzed by logistic or Cox regression. In 211 patients, available for analysis, topo IIα expression was significantly lower than topo IIß (P < 0.0001). In contrast to topo IIα, topo IIß was significantly associated with blast percentage in marrow or blood (P = 0.0001), CD7 (P = 0.01), CD14 (P < 0.0001) and CD54 (P < 0.0001). Event free survival was worse for CD56-negative compared to CD56-high (HR = 1.9, 95% CI [1.0-3.5], p = 0.04), and overall survival was worse for CD-15 low as compared to CD15-high (HR = 2.2, 95% CI [1.1-4.2], p = 0.02). Ingenuity pathway analysis indicated topo IIß and immunophenotype markers in a network associated with cell-to-cell signaling, hematological system development/function and inflammatory response. Topo IIß expression reflects disease biology of highly proliferative disease and distinct IP but does not appear to be an independent variable influencing outcome in adult AML patients treated with anthracycline-based therapy.

Clonal lineage of high grade serous ovarian cancer in a patient with neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is caused by mutations in the NF1 gene encoding neurofibromin, which negatively regulates Ras signaling. NF1 patients have an increased risk of developing early onset breast cancer, however, the association between NF1 and high grade serous ovarian cancer (HGSOC) is unclear. Since most NF1-related tumors exhibit early biallelic inactivation of NF1, we evaluated the evolution of genetic alterations in HGSOC in an NF1 patient. Somatic variation analysis of whole exome sequencing of tumor samples from both ovaries and a peritoneal metastasis showed a clonal lineage originating from an ancestral clone within the left adnexa, which exhibited copy number (CN) loss of heterozygosity (LOH) in the region of chromosome 17 containing TP53, NF1, and BRCA1 and mutation of the other TP53 allele. This event led to biallelic inactivation of NF1 and TP53 and LOH for the BRCA1 germline mutation. Subsequent CN alterations were found in the dominant tumor clone in the left ovary and nearly 100% of tumor at other sites. Neurofibromin modeling studies suggested that the germline NF1 mutation could potentially alter protein function. These results demonstrate early, biallelic inactivation of neurofibromin in HGSOC and highlight the potential of targeting RAS signaling in NF1 patients.

Dynamics of the Intratumoral Immune Response during Progression of High-Grade Serous Ovarian Cancer.

PURPOSE: Tumor-infiltrating lymphocytes (TILs) have an established impact on the prognosis of high-grade serous ovarian carcinoma (HGSOC), however, their role in recurrent ovarian cancer is largely unknown. We therefore systematically investigated TIL densities and MHC class I and II (MHC1, 2) expression in the progression of HGSOC. EXPERIMENTAL DESIGN: CD3+, CD4+, CD8+ TILs and MHC1, 2 expression were evaluated by immunohistochemistry on tissue microarrays in 113 paired primary and recurrent HGSOC. TILs were quantified by image analysis. All patients had been included to the EU-funded OCTIPS FP7 project. RESULTS: CD3+, CD4+, CD8+ TILs and MHC1 and MHC2 expression showed significant correlations between primary and recurrent tumor levels (Spearman rho 0.427, 0.533, 0.361, 0.456, 0.526 respectively; P<.0001 each). Paired testing revealed higher CD4+ densities and MHC1 expression in recurrent tumors (Wilcoxon P=.034 and P=.018). There was also a shift towards higher CD3+ TILs levels in recurrent carcinomas when analyzing platinum-sensitive tumors only (Wilcoxon P=.026) and in pairs with recurrent tumor tissue from first relapse only (Wilcoxon P=.031). High MHC2 expression was the only parameter to be significantly linked to prolonged progression-free survival after first relapse (PFS2, log-rank P=.012). CONCLUSIONS: This is the first study that analyzed the development of TILs density and MHC expression in paired primary and recurrent HGSOC. The level of the antitumoral immune response in recurrent tumors was clearly dependent on the one in the primary tumor. Our data contribute to the understanding of temporal heterogeneity of HGSOC immune microenvironment and have implications for selection of samples for biomarker testing in the setting of immune-targeting therapeutics.

Exploring the clonal evolution of CD133/aldehyde-dehydrogenase-1 (ALDH1)-positive cancer stem-like cells from primary to recurrent high-grade serous ovarian cancer (HGSOC). A study of the Ovarian Cancer Therapy-Innovative Models Prolong Survival (OCTIPS) Consortium.

BACKGROUND: High-grade serous ovarian cancer (HGSOC) causes 80% of all ovarian cancer (OC) deaths. In this setting, the role of cancer stem-like cells (CSCs) is still unclear. In particular, the evolution of CSC biomarkers from primary (pOC) to recurrent (rOC) HGSOCs is unknown. Aim of this study was to investigate changes in CD133 and aldehyde dehydrogenase-1 (ALDH1) CSC biomarker expression in pOC and rOC HGSOCs. METHODS: Two-hundred and twenty-four pOC and rOC intrapatient paired tissue samples derived from 112 HGSOC patients were evaluated for CD133 and ALDH1 expression using immunohistochemistry (IHC); pOCs and rOCs were compared for CD133 and/or ALDH1 levels. Expression profiles were also correlated with patients' clinicopathological and survival data. RESULTS: Some 49.1% of the patient population (55/112) and 37.5% (42/112) pOCs were CD133+ and ALDH1+ respectively. CD133+ and ALDH1+ samples were detected in 33.9% (38/112) and 36.6% (41/112) rOCs. CD133/ALDH1 coexpression was observed in 23.2% (26/112) and 15.2% (17/112) of pOCs and rOCs respectively. Pairwise analysis showed a significant shift of CD133 staining from higher (pOCs) to lower expression levels (rOCs) (p < 0.0001). Furthermore, all CD133 + pOC patients were International Federation of Gynaecology and Obstetrics (FIGO)-stage III/IV (p < 0.0001) and had significantly worse progression-free interval (PFI) (p = 0.04) and overall survival (OS) (p = 0.02). On multivariate analysis, CD133/ALDH1 coexpression in pOCs was identified as independent prognostic factor for PFI (HR: 1.64; 95% CI: 1.03-2.60; p = 0.036) and OS (HR: 1.71; 95% CI: 1.01-2.88; p = 0.045). Analysis on 52 pts patients with known somatic BRCA status revealed that BRCA mutations did not influence CSC biomarker expression. CONCLUSIONS: The study showed that CD133/ALDH1 expression impacts HGSOC patients' survival and first suggests that CSCs might undergo phenotypic change during the disease course similarly to non stem-like cancer cells, providing also a first evidence that there is no correlation between CSCs and BRCA status.

Mechanisms regulating resistance to inhibitors of topoisomerase II.

Inhibitors of topoisomerase II (topo II) are clinically effective in the management of hematological malignancies and solid tumors. The efficacy of anti-tumor drugs targeting topo II is often limited by resistance and studies with in vitro cell culture models have provided several insights on potential mechanisms. Multidrug transporters that are involved in the efflux and consequently reduced cytotoxicity of diverse anti-tumor agents suggest that they play an important role in resistance to clinically active drugs. However, in clinical trials, modulating the multidrug-resistant phenotype with agents that inhibit the efflux pump has not had an impact. Since reduced drug accumulation per se is insufficient to explain tumor cell resistance to topo II inhibitors several studies have focused on characterizing mechanisms that impact on DNA damage mediated by drugs that target the enzyme. Mammalian topo IIα and topo IIß isozymes exhibit similar catalytic, but different biologic, activities. Whereas topo IIα is associated with cell division, topo IIß is involved in differentiation. In addition to site specific mutations that can affect drug-induced topo II-mediated DNA damage, post-translation modification of topo II primarily by phosphorylation can potentially affect enzyme-mediated DNA damage and the downstream cytotoxic response of drugs targeting topo II. Signaling pathways that can affect phosphorylation and changes in intracellular calcium levels/calcium dependent signaling that can regulate site-specific phosphorylation of topoisomerase have an impact on downstream cytotoxic effects of topo II inhibitors. Overall, tumor cell resistance to inhibitors of topo II is a complex process that is orchestrated not only by cellular pharmacokinetics but more importantly by enzymatic alterations that govern the intrinsic drug sensitivity.

Predicting responses to sunitinib using single nucleotide polymorphisms: Progress and recommendations for future trials.

Targeted therapy with tyrosine kinase inhibitors has led to a substantial improvement in the standard of care for patients with advanced or metastatic clear cell renal cell carcinoma. Because the mechanism of action, metabolism and transport of tyrosine kinase inhibitors can affect outcome and toxicity, several investigators have pursued the identification of single nucleotide polymorphisms (SNPs) in genes associated with these actions. We discuss SNPs associated with outcome and toxicity following sunitinib therapy and provide recommendations for future trials to facilitate the use of SNPs in personalized therapy for this disease.