Multiplex profiling identifies distinct local and systemic alterations during intraperitoneal chemotherapy for ovarian cancer: An NRG Oncology/Gynecologic Oncology Group Study.

OBJECTIVES: Ovarian cancer leads to abdominal carcinomatosis and late stage (III/IV) diagnosis in 75% of patients. Three randomized phase III trials have demonstrated that intraperitoneal (IP) chemotherapy improves outcomes in epithelial ovarian cancer. While IP treatment is validated by clinical trials, there is a poor understanding of the mechanism(s) leading to the survival advantage other than the increased concentration of cytotoxic drugs within the tumor microenvironment. A better understanding of this process through analysis of dynamic biomarkers should promote novel approaches that may enhance tumor clearance. We propose this pilot study to confirm the feasibility of collecting serial peritoneal samples from implanted catheters in women receiving IP chemotherapy. We believe these specimens may be used for multiplex analysis to reveal unique biomarker fluctuations when compared to peripheral blood. METHODS: From 13 women participating on GOG 252, 30 whole blood, 12 peritoneal fluid (PF), and 20 peritoneal wash (PW) with 30mL saline were obtained. Samples were requested prior to the first three chemotherapy cycles. Samples were assessed for volume, cell populations, protein, RNA, and miRNA content changes. RESULTS: Median volume for PF was 1.6mL and 3.1mL for PW. PW is a dilution of PF capable of capturing measurable biomarkers. Peritoneal aspirates contain a unique profile of biomarkers distinct from blood. miRNA undergo earlier alteration with chemotherapy than genes. Flow cytometry does not adequately capture biomarker fluctuations. CONCLUSIONS: As a proof of principle study, this trial provides evidence that sampling the peritoneal cavity can be adapted for biomarker analysis.

Anti-PD-L1 prolongs survival and triggers T cell but not humoral anti-tumor immune responses in a human MUC1-expressing preclinical ovarian cancer model.

Monoclonal antibodies that block inhibitory immune checkpoint molecules and enhance anti-tumor responses show clinical promise in advanced solid tumors. Most of the preliminary evidence on therapeutic efficacy of immune checkpoint blockers comes from studies in melanoma, lung and renal cancer. To test the in vivo potential of programmed death-ligand 1 (PD-L1) blockade in ovarian cancer, we recently generated a new transplantable tumor model using human mucin 1 (MUC1)-expressing 2F8 cells. The MUC1 transgenic (MUC1.Tg) mice develop large number of intraperitoneal (IP) tumors following IP injection of 8 × 10(5) syngeneic 2F8 cells. The tumors are aggressive and display little T cell infiltration. Anti-PD-L1 antibody was administered IP every 2 weeks (200 µg/dose) for a total of three doses. Treatment was started 21 days post-tumor challenge, a time point which corresponds to late tumor stage. The anti-PD-L1 treatment led to substantial T cell infiltration within the tumor and significantly increased survival (p = 0.001) compared to isotype control-treated mice. When the same therapy was administered to wild-type mice challenged with 2F8 tumors, no survival benefit was observed, despite the presence of high titer anti-MUC1 antibodies. However, earlier treatment (day 11) and higher frequency of IP injections restored the T cell responses and led to prolonged survival. Splenocyte profiling via Nanostring using probes for 511 immune genes revealed a treatment-induced immune gene signature consistent with increased T cell-mediated immunity. These findings strongly support further preclinical and clinical strategies exploring PD-L1 blockade in ovarian cancer.

Genomic imprinting at a boundary element flanking the SDHD locus.

Germline mutations in SDHD, a mitochondrial complex II (succinate dehydrogenase) subunit gene at chromosome band 11q23, cause highly penetrant paraganglioma (PGL) tumors when transmitted through fathers. In contrast, maternal transmission rarely, if ever, leads to tumor development. The mechanism underlying this unusual monogenic tumor predisposition pattern is poorly understood. Here, we describe identification of imprinted methylation within an alternative promoter for a large intergenic non-coding RNA located at a distant gene desert boundary flanking SDHD. Methylation at this site primarily occurs within two consecutive HpaII restriction enzyme sites in a tissue-specific manner, most commonly in the adrenal gland. Informative fetal tissues and PGL tumors demonstrate maternal allelic hypermethylation. While a strong binding site for the enhancer-blocking protein CTCF within the alternative promoter shows no evidence of methylation, hyper-methylated adrenal tissues show increased binding of the chromatin-looping factor cohesin relative to the hypo-methylated tissues. These results suggest that the differential allelic methylation we observe at this locus is associated with altered chromatin architectures. These results provide molecular evidence for imprinting at a boundary element flanking the SDHD locus and suggest that epigenetic suppression of the maternal allele is the underlying mechanism of the imprinted penetrance of SDHD mutations.

Identification of a 4.9-kilo base-pair Alu-mediated founder SDHD deletion in two extended paraganglioma families from Austria.

Hereditary paraganglioma (PGL) is characterized by the development of highly vascularized paraganglionic tumors as a result of germline mutations in the SDHB, SDHC or SDHD subunit genes of succinate dehydrogenase (SDH; mitochondrial complex II), or in the Von Hippel-Lindau tumor-suppressor gene. Although many PGL mutations have been described, gross SDHD deletions have not yet been implicated as founder mutations and are rarely characterized at the DNA sequence level. We investigated the genetic basis of head and neck PGLs observed in 20 subjects from two unrelated multiplex pedigrees from Austria and identified a 4944-base pair partial SDHD deletion, which escaped PCR-based detection methods. The deletion occurred between Alu elements and was present within the same haplotype context in both pedigrees, indicating a founder effect. The deletion caused tumors only after a paternal transmission similar to other conventional SDHD mutations, suggesting preservation of genomic imprinting mechanisms operating at this locus. These data describe a large SDHD deletion at the genomic sequence level and indicate that gross SDHD deletions could be a founder PGL mutation in certain populations.

Cisplatin-induced immune modulation in ovarian cancer mouse models with distinct inflammation profiles.

The backbone of ovarian cancer treatment is platinum-based chemotherapy and aggressive surgical debulking. New therapeutic approaches using immunotherapy via immune checkpoint blockade, which have demonstrated clinical efficacy in other tumor types, have been less promising in ovarian cancer. To increase their clinical efficacy, checkpoint inhibitors are now being tested in clinical trials in combination with chemotherapy. Here, we evaluated the impact of cisplatin on tumor immunogenicity and its in vivo roles when used alone or in combination with anti-PD-L1, in two novel murine ovarian cancer cell models. The 2F8 and its platinum-resistant derivative 2F8cis model, display distinct inflammatory profiles and chemotherapy sensitivities, and mirror the primary and recurrent human disease, respectively. Acute and chronic exposure to cisplatin enhances tumor immunogenicity by increasing calreticulin, MHC class I, antigen presentation and T-cell infiltration. Cisplatin also upregulates PD-L1 expression in vitro and in vivo, demonstrating a dual, paradoxical immune modulatory effect and supporting the rationale for combination with immune checkpoint blockade. One of the pathways activated by cisplatin treatment is the cGAS/STING pathway. Chronic cisplatin treatment led to upregulation of cGAS and STING proteins in 2F8cis compared to parental 2F8 cells, while acute exposure to cisplatin further increases cGAS and STING levels in both 2F8 and 2F8cis cells. Overexpression of cGAS/STING modifies tumor immunogenicity by upregulating PD-L1, MHC I and calreticulin in tumor cells. Anti-PD-L1 alone in a platinum-sensitive model or with cisplatin in a platinum-resistant model increases survival. These studies have high translational potential in ovarian cancer.

Coordinate up-regulation of TMEM97 and cholesterol biosynthesis genes in normal ovarian surface epithelial cells treated with progesterone: implications for pathogenesis of ovarian cancer.

BACKGROUND: Ovarian cancer (OvCa) most often derives from ovarian surface epithelial (OSE) cells. Several lines of evidence strongly suggest that increased exposure to progesterone (P4) protects women against developing OvCa. However, the underlying mechanisms of this protection are incompletely understood. METHODS: To determine downstream gene targets of P4, we established short term in vitro cultures of non-neoplastic OSE cells from six subjects, exposed the cells to P4 (10-6 M) for five days and performed transcriptional profiling with oligonucleotide microarrays containing over 22,000 transcripts. RESULTS: We identified concordant but modest gene expression changes in cholesterol/lipid homeostasis genes in three of six samples (responders), whereas the other three samples (non-responders) showed no expressional response to P4. The most up-regulated gene was TMEM97 which encodes a transmembrane protein of unknown function (MAC30). Analyses of outlier transcripts, whose expression levels changed most significantly upon P4 exposure, uncovered coordinate up-regulation of 14 cholesterol biosynthesis enzymes, insulin-induced gene 1, low density lipoprotein receptor, ABCG1, endothelial lipase, stearoyl- CoA and fatty acid desaturases, long-chain fatty-acyl elongase, and down-regulation of steroidogenic acute regulatory protein and ABCC6. Highly correlated tissue-specific expression patterns of TMEM97 and the cholesterol biosynthesis genes were confirmed by analysis of the GNF Atlas 2 universal gene expression database. Real-time quantitative RT-PCR analyses revealed 2.4-fold suppression of the TMEM97 gene expression in short-term cultures of OvCa relative to the normal OSE cells. CONCLUSION: These findings suggest that a co-regulated transcript network of cholesterol/lipid homeostasis genes and TMEM97 are downstream targets of P4 in normal OSE cells and that TMEM97 plays a role in cholesterol and lipid metabolism. The P4-induced alterations in cholesterol and lipid metabolism in OSE cells might play a role in conferring protection against OvCa.

Common variations in ALG9 are not associated with bipolar I disorder: a family-based study.

BACKGROUND: A mannosyltransferase gene (ALG9, DIBD1) at chromosome band 11q23 was previously identified to be disrupted by a balanced chromosomal translocation t(9;11)(p24;q23) co-segregating with bipolar affective disorder in a small family. Inborn ALG9 deficiency (congenital disorders of glycosylation type IL) is associated with progressive microcephaly, seizures, developmental delay, and hepatomegaly. It is unknown whether common variations of ALG9 predispose to bipolar affective disorder. METHODS: We tested five polymorphic markers spanning ALG9 (three intragenic and one upstream microsatellite repeats and one common missense variation, V289I (rs10502151) for their association with bipolar I disorder in two pedigree series. The NIMH (National Institute of Mental Health) pedigrees had a total of 166 families showing transmissions to 250 affected offspring, whereas The PITT (The University of Pittsburgh) pedigrees had a total of 129 families showing transmissions to 135 cases. We used transmission disequilibrium test for the association analyses. RESULTS: We identified three common and distinct haplotypes spanning the ALG9 gene. We found no statistically-significant evidence of transmission disequilibrium of marker alleles or multi-marker haplotypes to the affected offspring with bipolar I disorder. CONCLUSION: These results suggest that common variations in ALG9 do not play a major role in predisposition to bipolar affective disorder.

Predictors and prevalence of paraganglioma syndrome associated with mutations of the SDHC gene.

CONTEXT: Paraganglioma syndrome includes inherited head and neck paragangliomas (HNPs) and adrenal or extra-adrenal pheochromocytomas and are classified according to the susceptibility genes SDHB, SDHC, and SDHD. In contrast with those with germline mutations of the SDHB and SDHD genes, clinical and genetic data on patients with mutations of SDHC are scarce. OBJECTIVE: To determine the prevalence and clinical characteristics of SDHC mutation carriers compared with patients with SDHB and SDHD mutations and with sporadic cases. DESIGN, SETTING, AND PATIENTS: Genetic screening for SDHC mutations in an international HNP registry of 121 unrelated index cases and in 371 sporadic cases from a pheochromocytoma registry, conducted January 1, 2001, until December 31, 2004. Identified index cases and affected relatives were clinically evaluated. MAIN OUTCOME MEASURES: Prevalence of and clinical findings for SDHC mutation-associated HNPs vs those with SDHB and SDHD mutations. RESULTS: The prevalence of SDHC carriers was 4% in HNP but 0% in pheochromocytoma index cases. None of the SDHC mutation carriers had signs of pheochromocytoma. We compared HNPs in 22 SDHC mutation carriers with the HNPs of SDHB (n = 15) and SDHD (n = 42) mutation carriers and with 90 patients with sporadic HNPs. Location, number of tumors, malignancy, and age were different: more carotid body tumors were found in SDHC (13/22 [59%]) than in sporadic HNPs (29/90 [32%], P = .03), as well as fewer instances of multiple tumors in SDHC (2/22) than in SDHD (24/42; P<.001), 0 malignant tumors in SDHC vs 6/15 in SDHB (P = .002), and younger age at diagnosis in SDHC than in sporadic HNPs (45 vs 52 years; P = .03). CONCLUSIONS: Patients with HNP, but not those with pheochromocytoma, harbor SDHC mutations in addition to those in SDHB and SDHD. In total, more than one quarter of HNP patients carry a mutation in 1 of these 3 genes. Head and neck paragangliomas associated with SDHC mutations are virtually exclusively benign and seldom multifocal. Analysis for germline mutations of SDHC is recommended in apparently sporadic HNP to identify risk of inheritance.

MUC1 positive, Kras and Pten driven mouse gynecologic tumors replicate human tumors and vary in survival and nuclear grade based on anatomical location.

Activating mutations of Kras oncogene and deletions of Pten tumor suppressor gene play important roles in cancers of the female genital tract. We developed here new preclinical models for gynecologic cancers, using conditional (Cre-loxP) mice with floxed genetic alterations in Kras and Pten. The triple transgenic mice, briefly called MUC1KrasPten, express human MUC1 antigen as self and carry a silent oncogenic KrasG12D and Pten deletion mutation. Injection of Cre-encoding adenovirus (AdCre) in the ovarian bursa, oviduct or uterus activates the floxed mutations and initiates ovarian, oviductal, and endometrial cancer, respectively. Anatomical site-specific Cre-loxP recombination throughout the genital tract of MUC1KrasPten mice leads to MUC1 positive genital tract tumors, and the development of these tumors is influenced by the anatomical environment. Endometrioid histology was consistently displayed in all tumors of the murine genital tract (ovaries, oviducts, and uterus). Tumors showed increased expression of MUC1 glycoprotein and triggered de novo antibodies in tumor bearing hosts, mimicking the immunobiology seen in patients. In contrast to the ovarian and endometrial tumors, oviductal tumors showed higher nuclear grade. Survival for oviduct tumors was significantly lower than for endometrial tumors (p = 0.0015), yet similar to survival for ovarian cancer. Oviducts seem to favor the development of high grade tumors, providing preclinical evidence in support of the postulated role of fallopian tubes as the originating site for high grade human ovarian tumors.

Altitude is a phenotypic modifier in hereditary paraganglioma type 1: evidence for an oxygen-sensing defect.

Hereditary paraganglioma type 1 (PGL1) is characterized by slow-growing and vascularized tumors that often develop in the carotid body (CB) and is caused by mutations in the gene for succinate dehydrogenase D ( SDHD) of mitochondrial complex II. The mechanisms of tumorigenesis and the factors affecting penetrance and expressivity are unknown. Because chronic hypoxic stimulation at high altitudes causes sporadic CB paragangliomas, it has been hypothesized that the SDHD gene product may be involved in oxygen sensing. On this background, we examined genotype-phenotype-environment relationships and tested whether higher altitudes adversely affect the phenotype in PGL1. An analysis of 58 subjects from 23 families revealed that nonsense/splicing mutation carriers developed symptoms 8.5 years earlier than missense mutation carriers ( P<0.012). We also found that subjects who were diagnosed with single tumors at their first clinical evaluation lived at lower average altitudes and were exposed to lower altitude-years than those with multiple tumors ( P<0.012). Pheochromocytomas developed in six subjects (approximately 10%), five of whom had nonsense mutations ( P=0.052). Subjects with pheochromocytomas also lived at higher average altitudes and were exposed to higher altitude-years than those without them ( P=0.026). To test whether altitude is also associated with the more frequent detection of germ-line founder mutations among sporadic cases in The Netherlands than in the USA ( P=0.00033), we calculated population-weighted elevations of the two countries. We found that the population-weighted elevations were approximately 260 m for the US and 2 m for the central-western Netherlands ( P~0), where three Dutch founder mutations were discovered. This finding suggests that low altitudes in The Netherlands reduce penetrance and relax the natural selection on SDHD mutations. Collectively, these data suggest that higher altitudes and nonsense/splicing mutations are associated with phenotypic severity in PGL1 and support the hypothesis that SDHD mutations impair oxygen sensing.

A mannosyltransferase gene at 11q23 is disrupted by a translocation breakpoint that co-segregates with bipolar affective disorder in a small family.

Bipolar affective disorder (BPAD) is a complex neuropsychiatric disease characterized by extreme mood swings. Genetic influences affect the disease susceptibility substantially, yet the underlying mechanisms are unknown. We previously described a pedigree in which all five individuals with BPAD and one individual with recurrent major depression were carriers of a reciprocal chromosomal translocation t(9;11)(p24;q23). Gene content analyses of the breakpoint junctions revealed disruption of a gene (DIBD1 ) at 11q23, a genomic region that has also been implicated in schizophrenia and Tourette syndrome. DIBD1 is predicted to encode a mannosyltransferase similar to Saccaromyces cerevisiae Alg9p of the protein N-glycosylation pathway. The in-born errors of protein N-glycosylation cause congenital disorders of glycosylation in humans. DIBD1 shows uniform expression in the tested subregions of the brain by Northern analysis. Sequence analysis revealed four intragenic single nucleotide polymorphisms. The valine residue at V289I was conserved in other eukaryotic species, whereas its frequency was approximately 65% in humans. We performed linkage and linkage disequilibrium analyses in two NIMH bipolar pedigree series using four tightly linked simple tandem repeat polymorphisms (STRPs) and the V289I. These analyses overall failed to support a role for DIBD1 in disease susceptibility. The most-significant finding was a lod score of 1.18 (P=0.0098), obtained by an intronic STRP D11S5025, in the subset of 22 multiplex pedigrees. In conclusion, we found that a mannosyltransferase gene at 11q23 is disrupted by a translocation breakpoint co-segregating with BPAD in a family. However, its role in the disease susceptibility remains unconfirmed.

Analysis of CHEK2 gene for ovarian cancer susceptibility.

OBJECTIVES: A deletion variant in the CHEK2 gene (del1100C) has been implicated as a low-penetrance risk factor for breast cancer. We sought to determine contribution of CHEK2 mutations to the etiology of ovarian cancer (OvCa). METHODS: We used cases ascertained from the United States through Gynecologic Oncology Group (GOG) protocols 172, 182, and 144, the University of Hawaii Cancer Research Center, and Creighton University. Control women were recruited from Pittsburgh and Hawaii. Denaturing high-performance liquid chromatography, sequence analysis, and single nucleotide polymorphism genotyping by Pyrosequencing were employed to analyze the CHEK2 gene. RESULTS: Mutation screening of the CHEK2 gene in 48 cases who had a first-degree relative with OvCa uncovered only del1100C and A252G variants. Altogether, the del1100C variant was detected in none of 751 unselected cases, in 1 of 52 (1.9%) cases who had a first-degree relative with OvCa, and in 3 of 521 (0.6%) unselected controls. The frequencies of del1100C and A252G variants did not show statistically significant differences between the cases and the controls. CONCLUSIONS: These results suggest that variations in CHEK2 do not make a significant contribution to the pathogenesis of OvCa in the U.S. population.