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Purpose: Gene expression–based molecular subtypes of high-grade serous tubo-ovarian cancer (HGSOC), demonstrated across multiple studies, may provide improved stratification for molecularly targeted trials. However, evaluation of clinical utility has been hindered by nonstandardized methods, which are not applicable in a clinical setting. We sought to generate a clinical grade minimal gene set assay for classification of individual tumor specimens into HGSOC subtypes and confirm previously reported subtype-associated features. Experimental Design: Adopting two independent approaches, we derived and internally validated algorithms for subtype prediction using published gene expression data from 1,650 tumors. We applied resulting models to NanoString data on 3,829 HGSOCs from the Ovarian Tumor Tissue Analysis consortium. We further developed, confirmed, and validated a reduced, minimal gene set predictor, with methods suitable for a single-patient setting. Results: Gene expression data were used to derive the predictor of high-grade serous ovarian carcinoma molecular subtype (PrOTYPE) assay. We established a de facto standard as a consensus of two parallel approaches. PrOTYPE subtypes are significantly associated with age, stage, residual disease, tumor-infiltrating lymphocytes, and outcome. The locked-down clinical grade PrOTYPE test includes a model with 55 genes that predicted gene expression subtype with >95% accuracy that was maintained in all analytic and biological validations. Conclusions: We validated the PrOTYPE assay following the Institute of Medicine guidelines for the development of omics-based tests. This fully defined and locked-down clinical grade assay will enable trial design with molecular subtype stratification and allow for objective assessment of the predictive value of HGSOC molecular subtypes in precision medicine applications. See related commentary by McMullen et al., p. 5271

PURPOSE Germline BRCA1 and/or BRCA2 mutations (gBRCAms) are risk factors for pancreatic cancer. The extent to which demographic and geographic factors affect the uptake of gBRCAm testing in pancreatic cancer (PC) is unknown. METHODS We conducted a retrospective, descriptive analysis of demographic/geographic data from the first 2,206 patients with metastatic PC (mPC) screened for eligibility to enter the phase III POLO trial of maintenance olaparib. No formal statistical tests were performed. RESULTS Of 2,167 patients with previously unknown gBRCAm status, 128 (5.9%) had a newly identified gBRCAm; rates were highest in the United States, France, and Israel (9.5%, 7.6%, and 7.4%, respectively). When including patients with a previously known gBRCAm, prevalence rose to 7.2% (or 5.8% after excluding populations enriched in Ashkenazi Jews, who are known to have a high rate of BRCA1 and BRCA2 founder mutations). Patients with a gBRCAm were slightly younger (57.9 v 61.1 years) and more likely to have early-onset mPC than those without. Higher newly identified gBRCAm prevalence was observed among African American (n = 28) versus white (n = 1,808), Asian (n = 218), and other (n = 61) patients (10.7% v 6.1%, 5.0%, and 1.6%, respectively). Of 139 white patients with a gBRCAm, 110 were newly identified during screening; the majority of gBRCAms in African American, Asian, and Hispanic patients (n = 3, n = 11, and n = 5, respectively) were newly identified. CONCLUSION We identified substantial geographic and some racial variability in gBRCAm prevalence among patients with mPC, an important consideration given the increased use of familial screening and possible future use of targeted therapies in this setting. Although our study included small numbers of nonwhite patients, prior knowledge of their gBRCAm status was limited compared with their white counterparts, which suggests disparities in genetic testing uptake.

High grade serous ovarian cancer (HGSOC) is a highly heterogeneous disease that often presents at an advanced, metastatic state. The multi-scale complexity of HGSOC is a major obstacle to measuring response to neoadjuvant chemotherapy (NACT) and understanding its determinants. Here we propose a radiogenomic framework integrating clinical, radiomic, and blood-based biomarkers to measure and predict the response of HGSOC patients to NACT, showing how quantitative imaging data can serve as the backbone of multi-scale data integration. We developed and validated our approach in two independent highly-annotated multi-omic multi-lesion data sets. In a discovery cohort (n=72) we found that different tumour sites present distinct response patterns, and identified volumetric response assessment as a better predictor of overall survival (OS) than RECIST 1.1 status. We trained an ensemble machine learning approach to predict tumour volume response to NACT from data obtained prior to treatment, and validated the model in an internal hold-out cohort (n=20) and an independent external patient cohort (n=42). Benchmarking integrated models against models built on single data types highlighted the importance of comprehensive patient characterisation. Our study sets the foundation for developing new clinical trials of NACT in HGSOC.

Background: High grade serous carcinoma (HGSC) is the most common and poorest prognosis subtype of ovarian cancer. The large majority of patients present with advanced (stage IIIC or IV) disease with a median survival of only 3-4 years. By contrast, approximately 10% patients are diagnosed with early stage disease, confined to the ovary/fallopian tube, of whom 90% are cured with surgery and platinum-based chemotherapy. Beyond TP53 mutation, classic activating oncogene mutations are rare in HGSC. Rather, HGSC is marked by extreme copy number (CN) abnormalities, and this complexity has prevented detailed understanding of the mutational processes underpinning outcomes in HGSC. We have used shallow whole genome sequencing (sWGS) to develop novel CN signatures that are able to deconvolute the complexity of HGSC genomes. However, nearly all genomics studies in HGSC have examined advanced stage disease, and little is known about the genomics of early stage HGSC - specifically, it is unclear whether these tumors are identified purely by chance or whether they represent a specific subset that does not metastasize. Hypotheses: We hypothesized that early stage HGSC has a distinct copy number landscape compared to stage IIIC/IV disease. Methods and results: We have identified 43 cases of FIGO stage I-IIA HGSC from the pathology archives of three large London Gynaecological Cancer Centres and 52 late-stage (stage IIIC-IV) cases from BriTROC-1 cohort. Median age at diagnosis was 61.3 years vs 62.3 years respectively. There were no significant differences in mutation rates of TP53 and BRCA1/2, and TP53 mutations were near-universal in both cohorts. We also did not find cohort-specific focal SCNA that could explain biological behavior. However, ploidy was significant higher in late-stage (median 3.0) than early-stage (median 1.9) samples. CN signature exposures were significantly different between early and late stage cohorts. Relative exposure of signature 3 was greater in early-stage and greater signature 4 in late-stage. Unsupervised clustering based on CN signatures identified three clusters that were prognostic. Summary:This project identifed that early stage and late stage HGSC have highly similar patterns of mutation and focal SCNA. However, genome-wide analysis indicates that the abnormalities seen in advanced disease are not present in early stage disease, which may represent a discrete subset with reduced metastatic potential. By identifying and characterizing the copy number signature changes, these data suggest that diagnosis at early-stage might reflect biological differences and not fortuitous chance. These data improve understanding of HGSC biology and may reveal potential new treatment strategies. Citation Format: Zhao Cheng, Hasan B. Mirza, Darren P. Ennis, Philip Smith, Lena Morrill Gavarró, Chishimba Sokota, Gaia Giannone, Teodora Goranova, Thomas Bradley, Anna Piskorz, Michelle Lockley, Baljeet Kaur, Naveena Singh, Laura A. Tookman, Jonathan Krell, Jackie McDermott, Geoff Macintyre, Florian Markowetz, James D. Brenton, Iain A. McNeish. The copy number landscape of early stage ovarian high grade serous carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 53.

PurposeHigh grade serous carcinoma (HGSC) is the commonest type of ovarian cancer. Nearly all HGSC cases are diagnosed at late stage and it is not clear whether early stage HGSC has unique characteristics compared to late stage tumours.Experimental DesignWe analysed samples from 45 patients with FIGO stage I - IIA HGSC - 40 from the pathology archives of three large UK cancer centres and 5 from the BriTROC-1 study. We performed shallow whole genome sequencing (sWGS) and targeted next generation sequencing to investigate somatic mutations and copy number alterations. We compared results to 51 stage IIIC/IV HGSC patients from the BriTROC-1 study.ResultsThere was no difference in median age between the early stage (median 61.3 years, range 40-84) and late stage (median 62.3 years, range 34-76) patients at diagnosis. TP53 mutations were near-universal (92% early stage, 100% late stage samples) and there were no significant differences in the rates of other somatic mutations, including BRCA1 and BRCA2, or focal copy number alterations between early- and late-stage cohorts. There were also no unique amplifications or deletions in either cohort. However, median ploidy was greater in late stage (median 3.1) than early stage (median 2.0) samples. In addition, there were higher numbers of breakpoints per 10MB and per chromosome arm and higher absolute copy number in late stage than early stage cohorts; early stage samples had longer segment length. Overall copy number signature exposures were significantly different between early and late stage samples with greater signature 3 exposure in early stage and greater signature 4 in late stage. Both simplex plot and unsupervised hierarchical clustering suggested that a subset of late stage samples retain early stage appearances with high signature 3 and co-clustering with the early stage samplesConclusionsThese data suggest that there are no unique mutations or focal copy number alterations in early stage HGSC. However, whole genome duplication is significantly more common in late-stage disease, suggesting evolution during disease progression. However, a subset of late stage HGSC retains early-stage features, which are associated with improved overall survival.

PURPOSE Chromosomal aberration and DNA copy number change are robust hallmarks of cancer. The gold standard for detecting copy number changes in tumor cells is fluorescence in situ hybridization (FISH) using locus-specific probes that are imaged as fluorescent spots. However, spot counting often does not perform well on solid tumor tissue sections due to partially represented or overlapping nuclei. MATERIALS AND METHODS To overcome these challenges, we have developed a computational approach called FrenchFISH, which comprises a nuclear volume correction method coupled with two types of Poisson models: either a Poisson model for improved manual spot counting without the need for control probes or a homogeneous Poisson point process model for automated spot counting. RESULTS We benchmarked the performance of FrenchFISH against previous approaches using a controlled simulation scenario and tested it experimentally in 12 ovarian carcinoma FFPE-tissue sections for copy number alterations at three loci (c-Myc, hTERC, and SE7). FrenchFISH outperformed standard spot counting with 74% of the automated counts having < 1 copy number difference from the manual counts and 17% having < 2 copy number differences, while taking less than one third of the time of manual counting. CONCLUSION FrenchFISH is a general approach that can be used to enhance clinical diagnosis on sections of any tissue by both speeding up and improving the accuracy of spot count estimates.

GermlineWe conducted a retrospective, descriptive analysis of demographic/geographic data from the first 2,206 patients with metastatic PC (mPC) screened for eligibility to enter the phase III POLO trial of maintenance olaparib. No formal statistical tests were performed.Of 2,167 patients with previously unknown gBRCAm status, 128 (5.9%) had a newly identified gBRCAm; rates were highest in the United States, France, and Israel (9.5%, 7.6%, and 7.4%, respectively). When including patients with a previously known gBRCAm, prevalence rose to 7.2% (or 5.8% after excluding populations enriched in Ashkenazi Jews, who are known to have a high rate ofWe identified substantial geographic and some racial variability in gBRCAm prevalence among patients with mPC, an important consideration given the increased use of familial screening and possible future use of targeted therapies in this setting. Although our study included small numbers of nonwhite patients, prior knowledge of their gBRCAm status was limited compared with their white counterparts, which suggests disparities in genetic testing uptake.

BACKGROUND: Investigating tumour evolution and acquired chemotherapy resistance requires analysis of sequential tumour material. We describe the feasibility of obtaining research biopsies in women with relapsed ovarian high-grade serous carcinoma (HGSC).METHODS: Women with relapsed ovarian HGSC underwent either image-guided biopsy or intra-operative biopsy during secondary debulking, and samples were fixed in methanol-based fixative. Tagged-amplicon sequencing was performed on biopsy DNA.RESULTS: We screened 519 patients in order to enrol 220. Two hundred and two patients underwent successful biopsy, 118 of which were image-guided. There were 22 study-related adverse events (AE) in the image-guided biopsies, all grades 1 and 2; pain was the commonest AE. There were pre-specified significant AE in 3/118 biopsies (2.5%). 87% biopsies were fit-for-purpose for genomic analyses. Median DNA yield was 2.87 μg, and was higher in biopsies utilising 14 G or 16 G needles compared to 18 G. TP53 mutations were identified in 94.4% patients.CONCLUSIONS: Obtaining tumour biopsies for research in relapsed HGSC is safe and feasible. Adverse events are rare. The large majority of biopsies yield sufficient DNA for genomic analyses-we recommend use of larger gauge needles and methanol fixation for such biopsies, as DNA yields are higher but with no increase in AEs.British Journal of Cancer advance online publication, 30 March 2017; doi:10.1038/bjc.2017.86 www.bjcancer.com.

PurposeThe known EOC susceptibility genes account for less than 50% of the heritable risk of ovarian cancer suggesting other susceptibility genes exist. The aim of this study was to evaluate the contribution to ovarian cancer susceptibility of rare deleterious germline variants in a set of candidate genes.MethodsWe sequenced the coding region of 54 candidate genes in 6385 invasive EOC cases and 6115 controls of broad European ancestry. Genes with an increased frequency of putative deleterious variants in cases verses controls were further examined in an independent set of 14,146 EOC cases and 28,661 controls from the ovarian cancer association consortium and the UK Biobank. For each gene, we estimated the EOC risks and evaluated associations between germline variant status and clinical characteristics.ResultsThe odds ratios (OR) associated for high-grade serous ovarian cancer were 3.01 for PALB2 (95% CI 1.59 – 5.68; P = 0.00068), 1.99 for POLK (95% CI 1.15 – 3.43; P = 0.014), and 4.07 for SLX4 (95% CI 1.34-12.4; P = 0.013). Deleterious mutations in FBXO10 were associated with a reduced risk of disease (OR 0.27, 95% CI 0.07 −1.00, P=0.049). However, based on the Bayes false discovery probability, only the association for PALB2 in high-grade serous ovarian cancer is likely to represent a true positive.ConclusionsWe have found strong evidence that carriers of PALB2 deleterious mutations are at increased risk of high-grade serous ovarian cancer. Whether the magnitude of risk is sufficiently high to warrant the inclusion of PALB2 in cancer gene panels for ovarian cancer risk testing is unclear; much larger sample sizes will be needed to provide sufficiently precise estimates for clinical counselling.

The known epithelial ovarian cancer (EOC) susceptibility genes account for less than 50% of the heritable risk of ovarian cancer suggesting that other susceptibility genes exist. The aim of this study was to evaluate the contribution to ovarian cancer susceptibility of rare deleterious germline variants in a set of candidate genes.We sequenced the coding region of 54 candidate genes in 6385 invasive EOC cases and 6115 controls of broad European ancestry. Genes with an increased frequency of putative deleterious variants in cases versus controls were further examined in an independent set of 14 135 EOC cases and 28 655 controls from the Ovarian Cancer Association Consortium and the UK Biobank. For each gene, we estimated the EOC risks and evaluated associations between germline variant status and clinical characteristics.The ORs associated for high-grade serous ovarian cancer were 3.01 forWe have found strong evidence that carriers of

Chromosomal aberration and DNA copy number change are robust hallmarks of cancer. Imaging of spots generated using fluorescence in situ hybridisation (FISH) of locus specific probes is routinely used to detect copy number changes in tumour nuclei. However, it often does not perform well on solid tumour tissue sections, where partially represented or overlapping nuclei are common. To overcome these challenges, we have developed a computational approach called FrenchFISH, which comprises a nuclear volume correction method coupled with two types of Poisson models: either a Poisson model for improved manual spot counting without the need for control probes; or a homogenous Poisson Point Process model for automated spot counting. We benchmarked the performance of FrenchFISH against previous approaches in a controlled simulation scenario and exemplify its use in 12 ovarian cancer FFPE-tissue sections, for which we assess copy number alterations in three loci (c-Myc, hTERC and SE7). We show that FrenchFISH outperforms standard spot counting approaches and that the automated spot counting is significantly faster than manual without loss of performance. FrenchFISH is a general approach that can be used to enhance clinical diagnosis on sections of any tissue.

Existing methods to improve detection of circulating tumor DNA (ctDNA) have focused on genomic alterations but have rarely considered the biological properties of plasma cell-free DNA (cfDNA). We hypothesized that differences in fragment lengths of circulating DNA could be exploited to enhance sensitivity for detecting the presence of ctDNA and for noninvasive genomic analysis of cancer. We surveyed ctDNA fragment sizes in 344 plasma samples from 200 patients with cancer using low-pass whole-genome sequencing (0.4×). To establish the size distribution of mutant ctDNA, tumor-guided personalized deep sequencing was performed in 19 patients. We detected enrichment of ctDNA in fragment sizes between 90 and 150 bp and developed methods for in vitro and in silico size selection of these fragments. Selecting fragments between 90 and 150 bp improved detection of tumor DNA, with more than twofold median enrichment in >95% of cases and more than fourfold enrichment in >10% of cases. Analysis of size-selected cfDNA identified clinically actionable mutations and copy number alterations that were otherwise not detected. Identification of plasma samples from patients with advanced cancer was improved by predictive models integrating fragment length and copy number analysis of cfDNA, with area under the curve (AUC) >0.99 compared to AUC 0.91 compared to AUC < 0.5 without fragmentation features. Fragment size analysis and selective sequencing of specific fragment sizes can boost ctDNA detection and could complement or provide an alternative to deeper sequencing of cfDNA.

420 Background: The POLO study (NCT02184195) showed that mPaC patients (pts) with a deleterious or suspected deleterious gBRCAm, and whose disease had not progressed during ≥16 weeks of first-line platinum-based chemotherapy, had significantly longer progression-free survival (PFS, primary endpoint) with maintenance olaparib vs placebo: median 7.4 vs 3.8 months, hazard ratio (HR) 0.53; P= 0.004. PFS benefit was observed in pts with g BRCA1m (HR 0.40) and g BRCA2m (HR 0.63). The POLO study represents the largest BRCAm prevalence study in pancreatic cancer . We report additional exploratory analysis to further characterize patient gBRCAm profiles, including the relationship with efficacy. Methods: Pts were enrolled based on either a previously identified gBRCAm status from a local test result and subsequently confirmed by central testing, or a prospectively identified gBRCAm. Pts received maintenance olaparib 300 mg twice daily (tablet) or placebo. PFS was assessed by blinded independent central review (modified RECIST v1.1). Results: Of 3194 prospectively screened pts, a valid BRCA test result was obtained for 3175 (99%) from 12 countries; gBRCAm prevalence was 6.2% in pts not previously known to harbor a gBRCAm (196/3175; 1.6% g BRCA1m, 4.5% g BRCA2m). In countries (n = 8) with > 100 pts prospectively tested, highest gBRCAm prevalence was 9.2% (USA) and lowest 4.0% (Spain). Prevalence by race ( > 100 pts); 6.4% Caucasian, 4.6% Asian. In total, 154 pts with a gBRCAm satisfied all eligibility criteria and were randomized (106 prospectively tested and 48 by local test [44/48 subsequently confirmed by Myriad testing]). 37/154 (24%) randomized pts carried a common Ashkenazi Jewish founder mutation, the majority being from Israel (21 pts). From a total of 151 variants, frameshift mutations were most frequent (g BRCA1m 69.6%, g BRCA2m 71.4%) followed by nonsense mutations (g BRCA1m 6.5%, g BRCA2m 17.1%). The efficacy (PFS) of olaparib vs placebo in the different subgroups are shown in the table. Conclusions: In pts with mPaC enrolled in POLO, g BRCA2m were more prevalent than g BRCA1m and mutation type was predominantly frameshift. PFS benefit was consistent across a heterogenous spectrum of gBRCAm and with the previously reported full analysis set. Clinical trial information: NCT02184195. [Table: see text]

4621 Background: The POLO study (NCT02184195) showed that mPaC patients (pts) with a deleterious or suspected deleterious gBRCAm, and whose disease had not progressed during ≥16 weeks of first-line platinum-based chemotherapy, had significantly longer progression-free survival (PFS, primary endpoint) with maintenance olaparib vs placebo: median 7.4 vs 3.8 months, hazard ratio (HR) 0.53; P=0.004. PFS benefit was observed in pts with g BRCA1m (HR 0.40) and g BRCA2m (HR 0.63). The POLO study represents the largest BRCAm prevalence study in pancreatic cancer. We report additional exploratory analysis to further characterize patient gBRCAm profiles, including the relationship with efficacy. Methods: Pts were enrolled based on either a previously identified gBRCAm status from a local test result and subsequently confirmed by central testing, or a prospectively identified gBRCAm. Pts received maintenance olaparib 300 mg twice daily (tablet) or placebo. PFS was assessed by blinded independent central review (modified RECIST v1.1). Results: Of 3194 prospectively screened pts, a valid BRCA test result was obtained for 3175 (99%) from 12 countries; gBRCAm prevalence was 6.2% in pts not previously known to harbor a gBRCAm (196/3175; 1.6% g BRCA1m, 4.5% g BRCA2m). In countries (n=8) with >100 pts prospectively tested, highest gBRCAm prevalence was 9.2% (USA) and lowest 4.0% (Spain). Prevalence by race (>100 pts); 6.4% Caucasian, 4.6% Asian. In total, 154 pts with a gBRCAm satisfied all eligibility criteria and were randomized (106 prospectively tested and 48 by local test [44/48 subsequently confirmed by Myriad testing]). 37/154 (24%) randomized pts carried a common Ashkenazi Jewish founder mutation, the majority being from Israel (21 pts). From a total of 151 variants, frameshift mutations were most frequent (g BRCA1m 69.6%, g BRCA2m 71.4%) followed by nonsense mutations (g BRCA1m 6.5%, g BRCA2m 17.1%). The efficacy (PFS) of olaparib vs placebo in the different subgroups are shown in the table. Conclusions: In pts with mPaC enrolled in POLO, g BRCA2m were more prevalent than g BRCA1m and mutation type was predominantly frameshift. PFS benefit was consistent across a heterogenous spectrum of gBRCAm and with the previously reported full analysis set. Clinical trial information: NCT02184195 . [Table: see text]

Tumours with profound copy-number aberration elude molecular stratification due to their genomic complexity. By representing this complexity as a mixture of copy-number signatures, we provide molecular explanations for differing clinical outcomes. Here we present a method for copy-number signature identification, deriving eight signatures in 117 shallow whole-genome sequenced high-grade serous ovarian cancers (HGSOC), which validated on independent cohorts of 95 deep whole-genome sequenced, and 402 SNP array-profiled cases. Three copy-number signatures predicted longer overall survival, while the others predicted poorer outcome. We found evidence for the mutational processes giving rise to copy-number change for six of the eight signatures via correlations with other genomic features. Our results provide insights into the pathogenesis of HGSOC by uncovering multiple mutational processes that shape genomes following TP53 mutation. Importantly, our work shows that most HGSOC have a mixture of mutational processes suggesting that targeting a single mutator phenotype may be therapeutically suboptimal.

L’angiogenesi è uno dei sei principali meccanismi alla base del cancro, ed è stato studiato approfonditamente negli ultimi 20 anni. L’obiettivo del presente studio è stato quello di determinare sia la densità capillare sia l’infiltrato macrofagico nei campioni di carcinoma laringeo e di determinarne la correlazione con gli aspetti clinici e patologici. Sia la densità capillare (CD34) sia l’infiltrato macrofagico (CD68) sono stati determinati con metodiche immunoistochimiche mediante microarray. Il nostro campione ha mostrato una densità capillare media di 14,27 ± 12,92 vasi su campo ingrandito a 200×, e l’infiltrato macrofagico medio è stato di 5,19 ± 4,32. La densità capillare si è dimostrata superiore nei pazienti metastatici. Inoltre uno studio di regressione lineare ha mostrato che l’entità dell’infiltrato macrofagico poteva predire la densità capillare del campione di carcinoma laringeo preso in esame. Non abbiamo invece individuato una correlazione fra ambo i fattori studiati e l’incidenza delle recidive o gli altri fattori clinici presi in esame. Il nostro studio aggiunge dati ad un problema che per quanto studiato a fondo negli ultimi 20 anni resta nella sostanza controverso.

The anticoagulant therapy with acenocoumarol is generally associated with a high risk of bleeding and thromboembolic events. We applied eight already existing acenocoumarol dosing algorithms to Bulgarian patients with low acenocoumarol dose requirements and investigated which of these algorithms would predict most precisely the dose anticoagulant. Two patients with Bulgarian origin were referred to the outpatient clinical laboratory of “St. Ekaterina” University Hospital for Cardiovascular Surgery and Cardiology, Sofia, Bulgaria. After obtaining written informed consent, both patients were genotyped for polymorphisms in genes for Cytochrome P450 2C9 (CYP2C9), Vitamin K epoxide reductase (VKORC1), Apolipoprotein E (APOE), and Cytochrome P450 4F2 (CYP4F2). All applied acenocoumarol dosing algorithms predicted relatively similar doses of coumarin anticoagulant in both patients. However, van Schie et al.’s algorithm allowed more accurate calculation of the optimal dose in our patients with extremely low acenocoumarol requirements. Genotyping of selected polymorphic variants in CYP2C9 and VKORC1 showed that both patients were compound heterozygotes for CYP2C9 (CYP2C9*2/*3) and homozygotes for both variants in VKORC1 (VKORC1 1173 T/T, and VKORC1-1639 A/A). This combination of genotypes suggested high sensitivity to acenocoumarol leading to the low anticoagulant dose requirements (0.25 and 1 mg/day, respectively) needed to reach the target International Normalized Ratio of 2.5–3.5. The genotyping of polymorphic variants in VKORC1 and CYP2C9, together with clinical and demographic parameters, can serve for more precise definition of the individual starting and maintenance doses of coumarin derivatives in each patient.

Pathogenic mutations in BRCA1/2 tumor suppressor genes increase the lifetime risk for developing breast and ovarian cancer. The aim of the present study was to evaluate the sensitivity and specificity of the Ion Torrent PGM™ for diagnostic mutation screening of BRCA1/2 genes. In the current study we included a cohort of 58 Bulgarian high-risk breast cancer patients to validate a next-generation sequencing approach for diagnostic mutation screening of the BRCA1/2 genes using the Ion Torrent Personal Genome Machine® (PGM™) platform. We have also optimized the workflow by comparing two different library preparation methods and using three software packages: NextGENE, Torrent Suite variantCaller, and Samtools/BCFtools to achieve detection of all variants with high specificity and sensitivity. We have validated a quick and accurate diagnostic test, with an overall specificity of 95.9 % and sensitivity of up to 100 %, which can be the first method of choice, followed by confirmation of the identified variants by Sanger sequencing. Our results prove that the Ion AmpliSeq™ BRCA1/2 Community Panel used with the PGM™ platform, and coupled with our variant selection pipeline, is able to detect all sequence variants discovered by Sanger sequencing. The application of the new test which outperforms the classical approach in turn-around time and price will have great impact in the clinical practice to identify the mutation carriers and guide the better personalized treatment of the patients, as well as to contribute for the improved prophylaxis in hereditary breast and ovarian cancer families.

The aim of the present study was to determine the relative quantitative expression of hypoxia-inducible factor (HIF)-1α, −2α and −3α, and VEGF-A in laryngeal carcinoma. A total of 63 patients with carcinoma of the larynx were enrolled in the study. Total RNA was isolated from fresh, frozen normal and tumor tissues of each patient, and quantitative polymerase chain reaction was performed. HIF-1α was upregulated in the majority of patients (44 patients; 69.84%). By contrast, only 7 (11.11%) patients from the whole group displayed HIF-2α overexpression, while the HIF-3α isoform was silenced in the majority of patients (48 patients, 76.19%). A small group of 5 (7.94%) patients exhibited significant overexpression of the HIF-3α isoform. VEGF-A expression was significantly higher (P

UNLABELLED Saponins are natural substances produced by a large number of plants, one of which is Tribulus terrestris L. (TT). They have been reported to possess an antitumor activity exerted by regulating various signaling pathways in the cell. Although the mechanisms of action of saponin extracts from various plants have been widely studied, limited data are available about TT. The present study aimed to analyze the impact of saponin extract from TT on cell processes in breast carcinoma cell lines. The variations in expression of a group of 32 selected genes were examined by real-time PCR after saponin treatment of MCF7 and MCF10A cell lines. Only three genes - CXCR4, CCR7 and BCL2, showed changes in their mRNA levels after the application of the herb extract. While CXCR4 expression was reduced in both cell lines, CCR7 and BCL2 levels decreased only in tumorigenic MCF7 cells, implying cell-specificity of the saponin action. Our results suggested that TT extract containing saponins was likely to affect the processes of apoptosis and metastasizing of cancer cells. Further in vivo studies will show its applicability as an anticancer therapeutic agent. KEYWORDS saponins, Tribulus terrestris, breast cancer, CXCR4, CCR7, BCL2.

Introductory paragraphNon-invasive analysis of cancer genomes using cell-free circulating tumour DNA (ctDNA) is being widely implemented for clinical indications. The sensitivity for detecting the presence of ctDNA and genomic changes in ctDNA is limited by its low concentration compared to cell-free DNA of non-tumour origin. We studied the feasibility for enrichment of ctDNA by size selection, in plasma samples collected before and during chemotherapy treatment in 13 patients with recurrent high-grade serous ovarian cancer. We evaluated the effects using targeted and whole genome sequencing. Selecting DNA fragments between 90-150 bp before analysis yielded enrichment of mutated DNA fraction of up to 11-fold. This allowed identification of adverse copy number alterations, including MYC amplification, otherwise not observed. Size selection allows detection of tumour alterations masked by non-tumour DNA in plasma and could help overcome sensitivity limitations of liquid biopsy for applications in early diagnosis, detection of minimal residual disease, and genomic profiling.

The OCAC OncoArray genotyping project: et al., To identify common alleles associated with different histotypes of epithelial ovarian cancer (EOC), we pooled data from multiple genome-wide genotyping projects totaling 25,509 EOC cases and 40,941 controls. We identified nine new susceptibility loci for different EOC histotypes: six for serous EOC histotypes (3q28, 4q32.3, 8q21.11, 10q24.33, 18q11.2 and 22q12.1), two for mucinous EOC (3q22.3 and 9q31.1) and one for endometrioid EOC (5q12.3). We then performed meta-analysis on the results for high-grade serous ovarian cancer with the results from analysis of 31,448 BRCA1 and BRCA2 mutation carriers, including 3,887 mutation carriers with EOC. This identified three additional susceptibility loci at 2q13, 8q24.1 and 12q24.31. Integrated analyses of genes and regulatory biofeatures at each locus predicted candidate susceptibility genes, including OBFC1, a new candidate susceptibility gene for low-grade and borderline serous EOC., The OCAC OncoArray genotyping project was funded through grants from the US National Institutes of Health (CA1X01HG007491-01 (C.I.A.), U19-CA148112 (T.A.S.), R01-CA149429 (C.M.P.) and R01-CA058598 (M.T.G.)); Canadian Institutes of Health Research (MOP-86727 (L.E.K.)); and the Ovarian Cancer Research Fund (A.B.). Funding for the CIMBA OncoArray genotyping was provided by the Government of Canada through Genome Canada and the Canadian Institutes of Health Research, the Ministère de l’Économie, de la Science et de l’Innovation du Québec through Génome Québec, the Quebec Breast Cancer Foundation for the PERSPECTIVE project, the US National Institutes of Health (CA1X01HG007491-01 (C.I.A.)), the Odense University Hospital Research Foundation (M.T.), the National R&D Program for Cancer Control, Ministry of Health and Welfare, Republic of Korea (1420190 (S.K.P.)), the Italian Association for Cancer Research (IG16933 (L.O.)) and German Cancer Aid (110837 (R.K.S.).

BACKGROUND: The genomic complexity of profound copy-number aberration has prevented effective molecular stratification of high grade serous ovarian carcinoma (HGSOC). Recent algorithmic advances have enabled interpretation of complex genomic changes by identifying mutational signatures—genomic patterns that are the imprint of mutagenic processes accumulated over the lifetime of a cancer cell. We hypothesized that specific features of copy-number (CN) abnormalities could represent the imprints of distinct mutational processes, and developed methods to identify signatures from copy-number features in HGSOC. METHODS: We derived copy-number signatures from absolute copy number profiles from 253 primary and relapsed HGSOC samples from 132 patients in the BriTROC-1 cohort using low-cost shallow whole-genome sequencing (sWGS; 0.1×). A subset of 56 of these cases had deep whole-genome sequencing (dWGS) performed for mutation analysis and comparison with sWGS data. Independent validation was performed using 112 dWGS HGSOC cases from PCAWG and 415 HGSOC cases with SNP array and whole exome sequence from TCGA. CN signature exposures were correlated with mutation data, SNV signatures, and other measures derived from deep WGS and exome sequencing to identify statistically significant genomic associations using a false discovery rate RESULTS: We identified 7 CN signatures that provided a molecular framework to rederive the major defining elements of HGSOC genomes, including defective homologous recombination (HRD), tandem duplication, amplification of CCNE1 and amplification-associated fold-back inversions. Almost all patients with HGSOC demonstrated a mixture of signatures indicative of combinations of mutational processes, including those with early driver events such as BRCA2 mutation (in addition to HRD signatures). High exposure to CN signature 3, characterised by BRCA1/2-related HRD, was associated with improved overall survival. Conversely, high exposure to signature 1, which was characterised by oncogenic RAS signaling (including NF1, KRAS and NRAS mutation), predicted platinum-resistant relapse and poor survival. CONCLUSIONS: HGSOC lacks clinically-relevant patient stratification, which is reflected in poor survival and is a significant barrier to precision medicine. Copy-number signature exposures at diagnosis predict both overall survival and the probability of platinum-resistant relapse. Our results suggest that early TP53 mutation, the ubiquitous initiating event in HGSOC, may permit multiple mutational processes to co-evolve, potentially simultaneously and that additional signature exposures may alter the risk of developing therapeutic resistance. Thus, our results suggest that HGSOC is a continuum of genomes. We derived signatures using inexpensive sWGS of DNA from core biopsies. These approaches are rapid and cost effective, thus providing a clear path to clinical implementation. By dissecting the mutational forces shaping HGSOC genomes, our study paves the way to understanding extreme genomic complexity, as well as revealing the evolution of tumors as they relapse and acquire resistance to therapy. Citation Format: Geoff Macintyre, Teodora E. Goranova, Dilrini De Silva, Darren Ennis, Anna M. Piskorz, Matthew Eldridge, Daoud Sie, Liz-Anne Lewsley, Aishah Hanif, Cheryl Wilson, Suzanne Dowson, Rosalind M. Glasspool, Michelle Lockley, Elly Brockbank, Ana Montes, Axel Walther, Sudha Sundar, Richard Edmondson, Geoff D. Hall, Andrew Clamp, Charlie Gourley, Marcia Hall, Christina Fotopoulou, Hani Gabra, James Paul, Anna Supernat, David Millan, Aoisha Hoyle, Gareth Bryson, Craig Nourse, Laura Mincarelli, Luis Navarro Sanchez, Bauke Ylstra, Mercedes Jimenez-Linan, Luiza Moore, Oliver Hofmann, Florian Markowetz, Iain A. McNeish, James D. Brenton. COPY-NUMBER SIGNATURES AND MUTATIONAL PROCESSES IN HIGH GRADE SEROUS OVARIAN CARCINOMA [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr AP09.

Background Laryngeal squamous cell carcinoma (laryngeal SCC) is a frequently occurring cancer of the head and neck area. Epigenetic changes of tumor-related genes contribute to its genesis and progression. Methods We assessed promoter methylation status of the selected genes (CDKN2A, MGMT, MLH1, and DAPK) using methylation-sensitive high resolution melting (MS-HRM) in 100 patients with laryngeal SCC and studied the correlations with clinical characteristics. Results The prevalence of promoter methylation in MGMT, CDKN2A, MLH1, and DAPK was 59 of 97 (60.8%), 46 of 97 (47.4%), 45 of 97 (46.4%), and 41 of 97 patients (42.3%), respectively. Significantly increased methylation of CDKN2A was observed in heavy smokers. Epigenetic inactivation of CDKN2A and MLH1 were found to be associated with lymph node involvement. An inverse correlation was present between MLH1 methylation and alcohol consumption. Conclusion Our results strongly suggest that deregulation of p16-associated, and MLH1-associated pathways, because of promoter hypermethylation, is associated with increased cancer cell migration, tumor invasiveness, and, thus, aggressive phenotype. © 2013 Wiley Periodicals, Inc. Head Neck 36: 1103–1108, 2014

We analyzed whole exome sequencing data in germline DNA from 412 high grade serous ovarian cancer (HGSOC) cases from The Cancer Genome Atlas Project and identified 5,517 genes harboring a predicted deleterious germline coding mutation in at least one HGSOC case. Gene-set enrichment analysis showed enrichment for genes involved in DNA repair (p = 1.8×10-3). Twelve DNA repair genes - APEX1, APLF, ATX, EME1, FANCL, FANCM, MAD2L2, PARP2, PARP3, POLN, RAD54L and SMUG1 – were prioritized for targeted sequencing in up to 3,107 HGSOC cases, 1,491 cases of other epithelial ovarian cancer (EOC) subtypes and 3,368 unaffected controls of European origin. We estimated mutation prevalence for each gene and tested for associations with disease risk. Mutations were identified in both cases and controls in all genes except MAD2L2, where we found no evidence of mutations in controls. In FANCM we observed a higher mutation frequency in HGSOC cases compared to controls (29/3,107 cases, 0.96 percent; 13/3,368 controls, 0.38 percent; P=0.008) with little evidence for association with other subtypes (6/1,491, 0.40 percent; P=0.82). The relative risk of HGSOC associated with deleterious FANCM mutations was estimated to be 2.5 (95% CI 1.3 – 5.0; P=0.006). In summary, whole exome sequencing of EOC cases with large-scale replication in case-control studies has identified FANCM as a likely novel susceptibility gene for HGSOC, with mutations associated with a moderate increase in risk. These data may have clinical implications for risk prediction and prevention approaches for high-grade serous ovarian cancer in the future and a significant impact on reducing disease mortality.

The hypoxia that arises due to the rapid proliferation of tumor cells is a fundamental driving force for the canonical pathway of neovascularization. In the current study we report a very strong correlation between mRNA expression levels of HIF-2α (but not HIF-1α), VEGFR-1, VEGFR-2 and MMP2 in ex vivo samples from laryngeal carcinoma. Sixty-three samples from patients with histopathologically verified carcinoma of the larynx were examined in this study. Total RNA was isolated from both normal and tumor fresh frozen tissues of each patient and real-time quantitative PCR reactions were performed. The mRNA expression levels of HIF-1α, HIF-2α, VEGFR1, VEGFR2 and MMP2 were acquired. We found strong positive correlations between mRNA expression levels of HIF-2α and VEGFR-1, r s (98) = .671, p

Intratumor heterogeneity—heterogeneity of cancer cells within a single tumor—is considered one of the most problematic factors of treatment. Genetic heterogeneity, such as in somatic mutations and chromosome aberrations, is a common characteristic of human solid tumors and is probably the basis of biological heterogeneity. Using mutations in APC, TP53 and KRAS as markers to identify distinct colorectal cancer subpopulations, we analyzed a total of 42 primary colorectal cancer tissues and six paired liver metastases with multipoint microsampling, which enabled analysis of mutation patterns and allelic imbalances with a resolution of 0.01 mm2 (about 200 cells). There was usually more than one subpopulation in each primary tumor. Only two of 15 (13.3%) cases with three gene mutations and eight of 27 (29.6%) cases with two gene mutations had a single subpopulation. Cells with mutations in all of the examined genes usually constituted the major population. Multipoint microsampling of six primary and metastatic tumor pairs revealed that the majority of discrepancies in mutation patterns found with the bulk tissue analysis were due to loss of subpopulations in the metastatic tissues. In addition, multipoint microsampling uncovered substantial changes in subpopulations that were not detected with bulk tissue analysis. Specifically, the proportion of KRAS mutation-negative subpopulations increased in the metastatic tumors of four cases. Because KRAS mutation status is linked to cetuximab/panitumumab efficacy, subpopulation dynamics could lead to differences in response to cetuximab/panitumumab in primary versus metastatic tumors. Electronic supplementary material The online version of this article (doi:10.1007/s10585-011-9381-0) contains supplementary material, which is available to authorized users.

Introduction Large screening studies in ovarian cancer (OC) using CA125 and transvaginal ultrasound have not shown a significant improvement in mortality. There is a need for new diagnostic biomarkers in OC. High-grade serous ovarian cancer (HGSOC) is characterised by ubiquitous TP53 mutations and extreme genomic rearrangement. Circulating tumour DNA (ctDNA) can be detected in plasma in >80% of women with relapsed HGSOC. However, detection of ctDNA in untreated, early stage patients is more challenging due to small disease volume and low levels of ctDNA. We aim to assess the proportion of women with newly diagnosed HGSOC with detectable ctDNA using a combination of sequencing assays including a new, sensitive, whole genome sequencing analysis method. Material and methods Targeted sequencing for TP53 and shallow whole genome sequencing (sWGS) were performed on 148 plasma samples collected from women with newly diagnosed, untreated HGSOC. Results and discussions Using targeted sequencing for TP53 ctDNA can be detected in 50% of newly diagnosed HGSOC. The addition of a genome wide measure of copy number aberration (t-MAD) and in-silico size selection of DNA fragments between 90–150 bp increases rates of ctDNA detection. Using the size selected t-MAD score we see a sensitivity of 80% for a specificity of 95% to discriminate HGSOC cases from healthy controls. Conclusion Plasma ctDNA can be detected in women with newly diagnosed HGSOC using targeted sequencing for TP53 and sWGS. By combining these low cost, high throughput sequencing assays we show that ctDNA has the potential be used in a diagnostic setting in HGSOC.

Introduction High-grade serous ovarian carcinoma (HGSOC) is characterised by ubiquitous TP53 mutations and significant copy-number alterations. Current ctDNA assays for HGSOC have focused on detection of TP53 mutations. In patients with low-stage disease or during therapy, the amount of ctDNA may be below the threshold of reliable detection. We tested whether sensitivity for ctDNA detection could be increased by combining TP53 mutations with focal genomic amplifications. Material and methods Targeted TP53 sequencing was performed on 264 plasma samples of 137 patients from the BriTROC-1 study with relapsed HGSOC tissue samples. Four patients had no somatic TP53 mutation detected and were excluded from analysis. Shallow WGS (0.1x) was performed on 256 plasma samples and matched tumour samples. The length-adjusted Z-score was calculated for each genomic segment and was used to distinguish significant focal amplifications (FA). Results and discussions TP53 mutations were detected in 180 plasma samples with median mutant allele fraction (MAF) of 5% (IQR 1.4%–16%). Twenty-five patients had at least one plasma sample with MAF >20%, which could be used for genomic profiling. Focal amplifications were observed in 151 plasma samples with a median of 4 amplifications per sample. The FA with the maximum observed Z-score per sample was significantly correlated with TP53 MAF. Both approaches detected ctDNA in about half of the samples. However, in some plasma samples ctDNA could be detected only by one of the used methods. Conclusion Combining targeted TP53 sequencing with shallow WGS may be a more sensitive approach for ctDNA detection.

Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant disease, caused by germline mutations in DNA mismatch-repair genes (MMR). These mutations lead to microsatellite instability (MSI). It has been found that the MSI is not confined to the setting of hereditary disease and may be seen in approximately 12-17% of the sporadic CRCs. In 1998 a National Registry for CRC was instituted in Queen Giovanna Hospital, Sofia. A total of 150 patients have been selected for MSI analysis and 25 tumors showed to be unstable, 14 with loss of heterozygosity (LOH). These tumors were further analyzed for MLH1 promoter hypermethylation and a significant association between this epigenetic change and MSI/LOH sporadic cases. We proposed this method as a step that follows the analysis for MSI and prior to the screening for MMR mutations. The mutation screening detected four known and two novel mutations, one unpublished and four known intronic polymorphisms in both hMLH1 and hMSH2 genes. The use of IHC analysis has been found effective in the investigation of some unclear molecular variations. We developed an efficient diagnostic strategy for HNPCC testing and the mutation status of 80% MSI HNPCC cases could be detected.

Laryngeal squamous cell carcinoma (LSCC) is the second most common tumour of the head and neck. It is characterized by frequent aberrations in two cell-cycle regulators--CDKN2A and TP53. However, LSCC has been often studied as a part of the group of head and neck cancers and not as an individual entity. In the current study we aimed to examine mutation status of CDKN2A and TP53 genes in 108 LSCC patients. DNA was extracted from fresh-frozen tumour tissues; exons 1-3 of CDKN2A and exons 5-8 of TP53 were screened for mutations by direct sequencing. Genetic aberrations in CDKN2A were found in 16 (14.2%) and those in TP53--in 56/108 (51.9%) tumours. Seven mutations (two insertions, three deletions, one missense and one silent) detected in CDKN2A were not described previously. Also, we found seven novel deletions and a novel indel in TP53. No significant associations with clinical features were found. However, TP53 mutations were predominantly observed in smokers with advanced stage tumours. Screening for genetic aberrations in a defined group of LSCC contributes to the knowledge about laryngeal carcinogenesis. Further investigations are required to confirm the observed trends in associations with clinical features.

Objective van't Veer and colleagues developed a 70-gene prognosis profile known as MammaPrint to identify breast cancer patients who were at low risk of developing metastases. We evaluated the prognostic value of the 70-gene MammaPrint profile in Japanese women with node-negative breast cancer. Methods Frozen tumour samples from 102 eligible node-negative breast cancer patients aged 70 or younger were characterized with the MammaPrint array. The patients were treated with breast-conserving therapy or mastectomy with axillary lymph node dissection between December 1998 and August 2001. About 73 percent received adjuvant hormonal therapy and 28 percent received adjuvant chemotherapy. The gene expression profiles obtained by MammaPrint classified the patients as high- or low-genomic risk. The median follow-up was 7.1 years. Results Among the 102 patients, 20 (20%) were classified as low-genomic risk and 82 (80%) were classified as high-genomic risk. The probability of distant metastasis-free survival at five years was 100% for the low-risk group and 94% for the high-risk group. Conclusions The 70-gene MammaPrint prognosis profile accurately identified Japanese breast cancer patients at low risk of developing recurrences. In fact, 100% of the individuals in the low-risk category remained metastasis-free for the duration of the observation period.

Laryngeal cancer is the most common type of malignant head and neck tumor worldwide. A lot of factors turn it from a disease with very good opportunities for prevention, early diagnosis, successful treatment and good prognosis into a disabling one with high death rate. The five years survival from 52% for the period from 1950 to 1954 has changed to only 64.4% for the period from 2003 to 2009. What underlies this unsatisfactory progress? As a matter of fact while the etiological factors are well known, the molecular basis of laryngeal carcinogenesis remains unrevealed

Aim: Laryngeal squamous cell carcinomas (LSCC) are frequently occurring cancers with a high risk of local recurrences or second primary tumours developing within the first two years of diagnosis. A common feature of LSCC is the epigenetic inactivation of certain genes by promoter hypermethylation. In a previous study we found associations of promoter methylation with some clinical features of LSCC. The present study aimed at assessing promoter hypermethylation of CDKN2A, MGMT and MLH1 as prognostic factors in LSCC patients. Methods: We examined the methylation status of the selected three genes in 61 laryngeal tumours by Methylation-specific High-Resolution Melting and analyzed its association with the overall survival of the patients. Results: We observed promoter hypermethylation of MGMT, CDKN2A and MLH1 genes in 31 (50.8%), 25 (40.9%) and 24 (39.3%) patients, respectively. Kaplan-Meier survival analysis showed significant association of methylation status of MGMT gene with overall survival (p = 0.018). Patients with unmethylated MGMT had longer median survival (35.1 months) compared to those with methylation (25.7 months). Multivariate Cox regression confirmed that the association was independent of other clinical factors and/or smoking history of the patients. Conclusions: Our results showed that MGMT promoter methylation is a promising prognostic factor for LSCC patients. Disclosure: All authors have declared no conflicts of interest.

Background: Median overall survival (OS) for women with high-grade serous ovarian cancer (HGSOC) is similar to 4 years, yet survival varies widely between patients. There are no well-established, gene expression signatures associated with prognosis. The aim of this study was to develop a robust prognostic signature for OS in patients with HGSOC. Patients and methods: Expression of 513 genes, selected from a meta-analysis of 1455 tumours and other candidates, was measured using NanoString technology from formalin-fixed paraffin-embedded tumour tissue collected from 3769 women with HGSOC from multiple studies. Elastic net regularization for survival analysis was applied to develop a prognostic model for 5-year OS, trained on 2702 tumours from 15 studies and evaluated on an independent set of 1067 tumours from six studies. Results: Expression levels of 276 genes were associated with OS (false discovery rate < 0.05) in covariate-adjusted single-gene analyses. The top five genes were TAP1, ZFHX4, CXCL9, FBN1 and PTGER3 (P < 0.001). The best performing prognostic signature included 101 genes enriched in pathways with treatment implications. Each gain of one standard deviation in the gene expression score conferred a greater than twofold increase in risk of death [hazard ratio (HR) 2.35, 95% confidence interval (CI) 2.02-2.71; P < 0.001]. Median survival [HR (95% CI)] by gene expression score quintile was 9.5 (8.3 to -), 5.4 (4.6-7.0), 3.8 (3.3-4.6), 3.2 (2.9-3.7) and 2.3 (2.1-2.6) years. Conclusion: The OTTA-SPOT (Ovarian Tumor Tissue Analysis consortium - Stratified Prognosis of Ovarian Tumours) gene expression signature may improve risk stratification in clinical trials by identifying patients who are least likely to achieve 5-year survival. The identified novel genes associated with the outcome may also yield opportunities for the development of targeted therapeutic approaches.

Multistage carcinogenesis is an important concept in cancer biology. Each new stage is triggered by the acquisition of an additional genetic aberration, leading to clonal expansion of the cancer cell. The resulting tumor mass consists of cancer cells with all genetic aberrations, but may include precursor cells at some point of carcinogenesis. We analyzed six colorectal cancer tissues with APC, K-ras, and p53 mutations. From each sample, 40-50 areas (100x100x40microm) consisting only of cancer cells were microdissected, and genomic DNA was purified. Ratios of mutated and normal alleles were quantitated by the SNaPshot assay, a primer extension assay. In five tumor tissues, we identified cancer cell subpopulations corresponding to putative precursors, i.e., cells with mutations in one or two of the three genes. All samples were likely to be of monoclonal origin, and temporal sequences of the mutations could be deduced from the mutation patterns of putative precursors. The orders of mutation events were variable. However, the two carcinoma tissues accompanying adenoma regions started with the APC mutation, not contradicting the previous studies. The analysis also revealed considerable heterogeneity in allele ratios of one or two of the chromosomes. The current findings are promising to uncover the process of carcinogenesis directly from the tumor tissue of the patient.