Molecular analysis for ovarian cancer detection in patient-friendly samples.

BACKGROUND: High ovarian cancer mortality rates motivate the development of effective and patient-friendly diagnostics. Here, we explored the potential of molecular testing in patient-friendly samples for ovarian cancer detection. METHODS: Home-collected urine, cervicovaginal self-samples, and clinician-taken cervical scrapes were prospectively collected from 54 patients diagnosed with a highly suspicious ovarian mass (benign n = 25, malignant n = 29). All samples were tested for nine methylation markers, using quantitative methylation-specific PCRs that were verified on ovarian tissue samples, and compared to non-paired patient-friendly samples of 110 age-matched healthy controls. Copy number analysis was performed on a subset of urine samples of ovarian cancer patients by shallow whole-genome sequencing. RESULTS: Three methylation markers are significantly elevated in full void urine of ovarian cancer patients as compared to healthy controls (C2CD4D, P = 0.008; CDO1, P = 0.022; MAL, P = 0.008), of which two are also discriminatory in cervical scrapes (C2CD4D, P = 0.001; CDO1, P = 0.004). When comparing benign and malignant ovarian masses, GHSR shows significantly elevated methylation levels in the urine sediment of ovarian cancer patients (P = 0.024). Other methylation markers demonstrate comparably high methylation levels in benign and malignant ovarian masses. Cervicovaginal self-samples show no elevated methylation levels in patients with ovarian masses as compared to healthy controls. Copy number changes are identified in 4 out of 23 urine samples of ovarian cancer patients. CONCLUSIONS: Our study reveals increased methylation levels of ovarian cancer-associated genes and copy number aberrations in the urine of ovarian cancer patients. Our findings support continued research into urine biomarkers for ovarian cancer detection and highlight the importance of including benign ovarian masses in future studies to develop a clinically useful test.

Ovarian cancer is often found late with limited treatment options. Currently, it is difficult to diagnose ovarian cancer correctly and no recommended early detection or screening methods exist. Our aim was to explore the use of DNA-based tests in patient-friendly samples for ovarian cancer detection. Patient-friendly samples are patient materials that can be collected from home without pain or discomfort, such as self-collected vaginal swabs and urine. Using DNA-based tests, we found that urine of women with ovarian cancer contains ovarian cancer-associated signals. Our findings encourage further development of a potential urine test for ovarian cancer detection. This approach could aid early detection and guide women with ovarian masses to appropriate specialist care.

Evaluation of the prognostic potential of histopathological subtyping in high-grade serous ovarian carcinoma.

High-grade serous ovarian carcinoma (HGSOC) can be categorized into four gene expression-based subtypes, with supposedly distinct prognoses and treatment responses. Murakami et al. translated these gene expression-based subtypes into the histopathological mesenchymal, immunoreactive, solid and proliferative, and papilloglandular subtypes, showing differences in survival outcomes. Miyagawa et al. refined these criteria to improve the interobserver concordance. The current retrospective study evaluated the interobserver variability and the prognostic differences between the histopathologic subtypes using the criteria of both Murakami et al. and Miyagawa et al. in 208 HGSOC cases. The mesenchymal subtype was considered first, followed by the immunoreactive subtype. Non-conforming cases were categorized as solid and proliferative or papilloglandular. The mesenchymal subtype was identified in 122 patients (58.7%) for both criteria. Using the criteria of Murakami et al., 10 cases (4.8%) were immunoreactive, 26 (12.5%) solid and proliferative, and 50 (24%) papilloglandular, with a concordance rate of 62.5% (κ = 0.34, p < .001). Using the Miyagawa et al. criteria, 23 cases (11%) were immunoreactive, 20 (9.6%) solid and proliferative, and 43 (20.7%) papilloglandular. No survival differences were observed between the subtypes. The fair reproducibility of the histopathological subtype classification of HGSOC and the lack of survival differences among these subtypes indicate the need for further refinement of the criteria and exploration of their correlation with overall survival outcomes before clinical application.

Kinase activities in pancreatic ductal adenocarcinoma with prognostic and therapeutic avenues.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a limited number of known driver mutations but considerable cancer cell heterogeneity. Phosphoproteomics provides a direct read-out of aberrant signaling and the resultant clinically relevant phenotype. Mass spectrometry (MS)-based proteomics and phosphoproteomics were applied to 42 PDAC tumors. Data encompassed over 19 936 phosphoserine or phosphothreonine (pS/T; in 5412 phosphoproteins) and 1208 phosphotyrosine (pY; in 501 phosphoproteins) sites and a total of 3756 proteins. Proteome data identified three distinct subtypes with tumor intrinsic and stromal features. Subsequently, three phospho-subtypes were apparent: two tumor intrinsic (Phos1/2) and one stromal (Phos3), resembling known PDAC molecular subtypes. Kinase activity was analyzed by the Integrative iNferred Kinase Activity (INKA) scoring. Phospho-subtypes displayed differential phosphorylation signals and kinase activity, such as FGR and GSK3 activation in Phos1, SRC kinase family and EPHA2 in Phos2, and EGFR, INSR, MET, ABL1, HIPK1, JAK, and PRKCD in Phos3. Kinase activity analysis of an external PDAC cohort supported our findings and underscored the importance of PI3K/AKT and ERK pathways, among others. Interestingly, unfavorable patient prognosis correlated with higher RTK, PAK2, STK10, and CDK7 activity and high proliferation, whereas long survival was associated with MYLK and PTK6 activity, which was previously unknown. Subtype-associated activity profiles can guide therapeutic combination approaches in tumor and stroma-enriched tissues, and emphasize the critical role of parallel signaling pathways. In addition, kinase activity profiling identifies potential disease markers with prognostic significance.

The Information Technology (IT) Infrastructure of the Multicenter Archipelago of Ovarian Cancer Research Biobank: A Potential Blueprint for Other Biobanks.

Objective: Biobanks play a crucial role in fundamental and translational research by storing valuable biomaterials and data for future analyses. However, the design of their information technology (IT) infrastructures is often customized to specific requirements, thereby lacking the ability to be used for biobanks comprising other (types of) diseases. This results in substantial costs, time, and efforts for each new biobank project. The Dutch multicenter Archipelago of Ovarian Cancer Research (AOCR) biobank has developed an innovative, reusable IT infrastructure capable of adaptation to various biobanks, thereby enabling cost-effective and efficient implementation and management of biobank IT systems. Methods and Results: The AOCR IT infrastructure incorporates preexisting biobank software, mainly managed by Health-RI. The web-based registration tool Ldot is used for secure storage and pseudonymization of patient data. Clinicopathological data are retrieved from the Netherlands Cancer Registry and the Dutch nationwide pathology databank (Palga), both established repositories, reducing administrative workload and ensuring high data quality. Metadata of collected biomaterials are stored in the OpenSpecimen system. For digital pathology research, a hematoxylin and eosin-stained slide from each patient's tumor is digitized and uploaded to Slide Score. Furthermore, adhering to the Findable, Accessible, Interoperable, and Reusable (FAIR) principles, genomic data derived from the AOCR samples are stored in cBioPortal. Conclusion: The IT infrastructure of the AOCR biobank represents a new standard for biobanks, offering flexibility to handle diverse diseases and types of biomaterials. This infrastructure bypasses the need for disease-specific, custom-built software, thereby being cost- and time-effective while ensuring data quality and legislative compliance. The adaptability of this infrastructure highlights its potential to serve as a blueprint for the development of IT infrastructures in both new and existing biobanks.

Polymorphisms in transcription factor binding sites and enhancer regions and pancreatic ductal adenocarcinoma risk.

Genome-wide association studies (GWAS) are a powerful tool for detecting variants associated with complex traits and can help risk stratification and prevention strategies against pancreatic ductal adenocarcinoma (PDAC). However, the strict significance threshold commonly used makes it likely that many true risk loci are missed. Functional annotation of GWAS polymorphisms is a proven strategy to identify additional risk loci. We aimed to investigate single-nucleotide polymorphisms (SNP) in regulatory regions [transcription factor binding sites (TFBSs) and enhancers] that could change the expression profile of multiple genes they act upon and thereby modify PDAC risk. We analyzed a total of 12,636 PDAC cases and 43,443 controls from PanScan/PanC4 and the East Asian GWAS (discovery populations), and the PANDoRA consortium (replication population). We identified four associations that reached study-wide statistical significance in the overall meta-analysis: rs2472632(A) (enhancer variant, OR 1.10, 95%CI 1.06,1.13, p = 5.5 × 10-8), rs17358295(G) (enhancer variant, OR 1.16, 95%CI 1.10,1.22, p = 6.1 × 10-7), rs2232079(T) (TFBS variant, OR 0.88, 95%CI 0.83,0.93, p = 6.4 × 10-6) and rs10025845(A) (TFBS variant, OR 1.88, 95%CI 1.50,1.12, p = 1.32 × 10-5). The SNP with the most significant association, rs2472632, is located in an enhancer predicted to target the coiled-coil domain containing 34 oncogene. Our results provide new insights into genetic risk factors for PDAC by a focused analysis of polymorphisms in regulatory regions and demonstrating the usefulness of functional prioritization to identify loci associated with PDAC risk.

A scan of all coding region variants of the human genome, identifies 13q12.2-rs9579139 and 15q24.1-rs2277598 as novel risk loci for pancreatic ductal adenocarcinoma.

Coding sequence variants comprise a small fraction of the germline genetic variability of the human genome. However, they often cause deleterious change in protein function and are therefore associated with pathogenic phenotypes. To identify novel pancreatic ductal adenocarcinoma (PDAC) risk loci, we carried out a complete scan of all common missense and synonymous SNPs and analysed them in a case-control study comprising four different populations, for a total of 14 538 PDAC cases and 190 657 controls. We observed a statistically significant association between 13q12.2-rs9581957-T and PDAC risk (P = 2.46 × 10-9), that is in linkage disequilibrium (LD) with a deleterious missense variant (rs9579139) of the URAD gene. Recent findings suggest that this gene is active in peroxisomes. Considering that peroxisomes have a key role as molecular scavengers, especially in eliminating reactive oxygen species, a malfunctioning URAD protein might expose the cell to a higher load of potentially DNA damaging molecules and therefore increase PDAC risk. The association was observed in individuals of European and Asian ethnicity. We also observed the association of the missense variant 15q24.1-rs2277598-T, that belongs to BBS4 gene, with increased PDAC risk (P = 1.53 × 10-6). rs2277598 is associated with body mass index and is in LD with diabetes susceptibility loci. In conclusion, we identified two missense variants associated with the risk of developing PDAC independently from the ethnicity highlighting the importance of conducting reanalysis of genome-wide association studies (GWASs) in light of functional data.

Human ovarian aging is characterized by oxidative damage and mitochondrial dysfunction.

STUDY QUESTION: Are human ovarian aging and the age-related female fertility decline caused by oxidative stress and mitochondrial dysfunction in oocytes? SUMMARY ANSWER: We found oxidative damage in oocytes of advanced maternal age, even at the primordial follicle stage, and confirmed mitochondrial dysfunction in such oocytes, which likely resulted in the use of alternative energy sources. WHAT IS KNOWN ALREADY: Signs of reactive oxygen species-induced damage and mitochondrial dysfunction have been observed in maturing follicles, and even in early stages of embryogenesis. However, although recent evidence indicates that also primordial follicles have metabolically active mitochondria, it is still often assumed that these follicles avoid oxidative phosphorylation to prevent oxidative damage in dictyate arrested oocytes. Data on the influence of ovarian aging on oocyte metabolism and mitochondrial function are still limited. STUDY DESIGN, SIZE, DURATION: A set of 39 formalin-fixed and paraffin-embedded ovarian tissue biopsies were divided into different age groups and used for immunofluorescence analysis of oxidative phosphorylation activity and oxidative damage to proteins, lipids, and DNA. Additionally, 150 immature oocytes (90 germinal vesicle oocytes and 60 metaphase I oocytes) and 15 cumulus cell samples were divided into different age groups and used for targeted metabolomics and lipidomics analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian tissues used for immunofluorescence microscopy were collected through PALGA, the nationwide network, and registry of histo- and cytopathology in The Netherlands. Comprehensive metabolomics and lipidomics were performed by liquid-liquid extraction and full-scan mass spectrometry, using oocytes and cumulus cells of women undergoing ICSI treatment based on male or tubal factor infertility, or fertility preservation for non-medical reasons. MAIN RESULTS AND THE ROLE OF CHANCE: Immunofluorescence imaging on human ovarian tissue indicated oxidative damage by protein and lipid (per)oxidation already at the primordial follicle stage. Metabolomics and lipidomics analysis of oocytes and cumulus cells in advanced maternal-age groups demonstrated a shift in the glutathione-to-oxiglutathione ratio and depletion of phospholipids. Age-related changes in polar metabolites suggested a decrease in mitochondrial function, as demonstrated by NAD+, purine, and pyrimidine depletion, while glycolysis substrates and glutamine accumulated, with age. Oocytes from women of advanced maternal age appeared to use alternative energy sources like glycolysis and the adenosine salvage pathway, and possibly ATP which showed increased production in cumulus cells. LIMITATIONS, REASONS FOR CAUTION: The immature oocytes used in this study were all subjected to ovarian stimulation with high doses of follicle-stimulating hormones, which might have concealed some age-related differences. WIDER IMPLICATIONS OF THE FINDINGS: Further studies on how to improve mitochondrial function, or lower oxidative damage, in oocytes from women of advanced maternal age, for instance by supplementation of NAD+ precursors to promote mitochondrial biogenesis, are warranted. In addition, supplementing the embryo medium of advanced maternal-age embryos with such compounds could be a treatment option worth exploring. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by the Amsterdam UMC. The authors declare to have no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Polymorphic variants involved in methylation regulation: a strategy to discover risk loci for pancreatic ductal adenocarcinoma.

INTRODUCTION: Only a small number of risk factors for pancreatic ductal adenocarcinoma (PDAC) has been established. Several studies identified a role of epigenetics and of deregulation of DNA methylation. DNA methylation is variable across a lifetime and in different tissues; nevertheless, its levels can be regulated by genetic variants like methylation quantitative trait loci (mQTLs), which can be used as a surrogate. MATERIALS AND METHODS: We scanned the whole genome for mQTLs and performed an association study in 14 705 PDAC cases and 246 921 controls. The methylation data were obtained from whole blood and pancreatic cancer tissue through online databases. We used the Pancreatic Cancer Cohort Consortium and the Pancreatic Cancer Case-Control Consortium genome-wide association study (GWAS) data as discovery phase and the Pancreatic Disease Research consortium, the FinnGen project and the Japan Pancreatic Cancer Research consortium GWAS as replication phase. RESULTS: The C allele of 15q26.1-rs12905855 showed an association with a decreased risk of PDAC (OR=0.90, 95% CI 0.87 to 0.94, p=4.93×10-8 in the overall meta-analysis), reaching genome-level statistical significance. 15q26.1-rs12905855 decreases the methylation of a 'C-phosphate-G' (CpG) site located in the promoter region of the RCCD1 antisense (RCCD1-AS1) gene which, when expressed, decreases the expression of the RCC1 domain-containing (RCCD1) gene (part of a histone demethylase complex). Thus, it is possible that the rs12905855 C-allele has a protective role in PDAC development through an increase of RCCD1 gene expression, made possible by the inactivity of RCCD1-AS1. CONCLUSION: We identified a novel PDAC risk locus which modulates cancer risk by controlling gene expression through DNA methylation.

Whole exome sequencing of low grade serous ovarian carcinoma identifies genomic events associated with clinical outcome.

OBJECTIVES: Low-grade serous ovarian carcinoma (LGSOC) is a distinct, rare, ovarian cancer type characterised by younger patient age and intrinsic chemoresistance. Understanding the molecular landscape is crucial for optimising targeted therapy. METHODS: Genomic data from whole exome sequencing of tumour tissue was analysed in a LGSOC cohort with detailed clinical annotation. RESULTS: 63 cases were analysed and three subgroups identified based on single nucleotide variants: canonical MAPK mutant (cMAPKm: 52%, KRAS/BRAF/NRAS), MAPK-associated gene mutation (MAPK-assoc: 27%) and MAPK wild-type (MAPKwt: 21%). NOTCH pathway disruption occurred across all subgroups. Tumour mutational burden (TMB), mutational signatures and recurrent copy number (CN) changes varied across the cohort with co-occurrence of chromosome 1p loss and 1q gain (CN Chr1pq) a recurrent feature. Low TMB and CN Chr1pq were associated with inferior disease-specific survival (HR 6.43; p < 0.001 and HR 3.29, p = 0.011 respectively). Stepwise genomic classification in relation to outcome resulted in four groups (TMB low; CN Chr1pq; MAPKwt/MAPKassoc; cMAPKm). 5 year disease-specific survival was 46%, 55%, 79% and 100% respectively for these groups. The two most favourable genomic subgroups were enriched for the SBS10b mutational signature, particularly the cMAPKm subgroup. CONCLUSIONS: LGSOC comprises multiple genomic subgroups with distinct clinical and molecular features. Chr1pq CN arm disruption and TMB represent promising methods to identify individuals with poorer prognosis. Further investigation of the molecular basis for these observations is required. MAPKwt cases represent around a fifth of patients. NOTCH inhibitors represent a candidate therapeutic strategy worthy of exploration across these cases.

Distinct histopathological features are associated with molecular subtypes and outcome in low grade serous ovarian carcinoma.

Low grade serous ovarian carcinoma (LGSOC) demonstrates unique clinical and molecular features compared to other ovarian cancer types. The relationship between common histological features of LGSOC and molecular events, such as hormone receptor expression patterns and MAPK gene mutation status, remains poorly understood. Recent data suggest some of these molecular features may be biomarkers of response to recently introduced biologically-targeted therapies, namely endocrine therapy and MEK inhibitors. We utilize a cohort of 63 pathologically-confirmed LGSOC cases with whole exome sequencing and hormone receptor expression data to investigate these relationships. LGSOC cases demonstrated uniformly high oestrogen receptor (ER) expression, but variable progesterone receptor (PR) expression intensity. 60% and 37% of cases demonstrated micropapillary and macropapillary patterns of stromal invasion, respectively. 63% of cases demonstrated desmoplasia, which was significantly associated with advanced disease stage and visible residual disease after cytoreductive surgery. MAPK-mutant cases (KRAS, BRAF, NRAS) more frequently demonstrated macropapillary stromal invasion, while Chr1p loss was associated with desmoplasia and low PR expression. Presence of micropapillary stromal invasion and low PR expression were associated with significantly poorer survival after accounting for stage and residual disease status. Together, these data identify novel relationships between histopathological features and molecularly-defined subgroups in LGSOC.

C/EBP-Family Redundancy Determines Patient Survival and Lymph Node Involvement in PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is a dismal disease with a poor clinical prognosis and unsatisfactory treatment options. We previously found that the transcription factor CCAAT/Enhancer-Binding Protein Delta (C/EBPδ) is lowly expressed in PDAC compared to healthy pancreas duct cells, and that patient survival and lymph node involvement in PDAC is correlated with the expression of C/EBPδ in primary tumor cells. C/EBPδ shares a homologous DNA-binding sequence with other C/EBP-proteins, leading to the presumption that other C/EBP-family members might act redundantly and compensate for the loss of C/EBPδ. This implies that patient stratification could be improved when expression levels of multiple C/EBP-family members are considered simultaneously. In this study, we assessed whether the quantification of C/EBPß or C/EBPγ in addition to that of C/EBPδ might improve the prediction of patient survival and lymph node involvement using a cohort of 68 resectable PDAC patients. Using Kaplan-Meier analyses of patient groups with different C/EBP-expression levels, we found that both C/EBPß and C/EBPγ can partially compensate for low C/EBPδ and improve patient survival. Further, we uncovered C/EBPß as a novel predictor of a decreased likelihood of lymph node involvement in PDAC, and found that C/EBPß and C/EBPδ can compensate for the lack of each other in order to reduce the risk of lymph node involvement. C/EBPγ, on the other hand, appears to promote lymph node involvement in the absence of C/EBPδ. Altogether, our results show that the redundancy of C/EBP-family members might have a profound influence on clinical prognoses and that the expression of both C/EPBß and C/EBPγ should be taken into account when dichotomizing patients according to C/EBPδ expression.

Validation of combined carcinoembryonic antigen and glucose testing in pancreatic cyst fluid to differentiate mucinous from non-mucinous cysts.

BACKGROUND: More accurate diagnosis of mucinous cysts will reduce the risk of unnecessary pancreatic surgery. Carcinoembryonic antigen (CEA) and glucose in pancreatic cyst fluid (PCF) can differentiate mucinous from non-mucinous pancreatic cystic neoplasms (PCN). The current study assessed the value of combined CEA and glucose testing in PCF. METHODS: Cross-sectional validation study including prospectively collected PCF from patients undergoing endoscopic ultrasonography-guided fine-needle aspiration (EUS-FNA) and pancreatic surgery. We performed laboratory measurements for CEA and glucose and measured glucose levels by a hand glucometer. Primary outcome was diagnostic accuracy evaluated by receiver operator curves (ROC), sensitivity, specificity, positive, and negative predictive value (PPV, NPV). RESULTS: Overall, PCF was collected from 63 patients, including 33 (52%) with mucinous and 30 (48%) with non-mucinous PCN. Histopathology (n = 36; 57%), cytopathology (n = 2; 3%), or clinical and/or radiological diagnosis (n = 25; 40%) was used as reference standard. Combined CEA (cut-off ≥ 192 ng/ml) and laboratory glucose testing (cut-off ≤ 50 mg/dL) reached 92% specificity and 48% sensitivity, whereas either positive CEA (cut-off ≥ 20 ng/ml) or glucose testing (cut-off ≤ 50 mg/dL) showed 97% sensitivity and 50% specificity. Sensitivity and specificity were 80% and 68% for CEA ≥ 20 ng/mL versus 50% and 93% for CEA ≥ 192 ng/mL (the conventional cut-off level). Laboratory and glucometer glucose both reached 100% sensitivity and 60% and 45% specificity, respectively. None of the biomarkers and cut-offs reached a PPV exceeding 90%, whereas both glucose measurements had a NPV of 100% (i.e., high glucose excludes a mucinous cyst). CONCLUSION: Combined CEA and glucose testing in PCF reached high specificity and sensitivity for differentiating mucinous from non-mucinous PCN. Glucose testing, whether alone or combined with the new CEA cut-off (≥ 20 ng/mL), reached > 95% sensitivity for mucinous cysts, whereas only glucose reached a NPV > 95%.

Establishment of the Dutch Nationwide, Interdisciplinary Infrastructure and Biobank for Fundamental and Translational Ovarian Cancer Research: Archipelago of Ovarian Cancer Research.

OBJECTIVES: Ovarian cancer has the worst overall survival rate of all gynecologic malignancies. For the majority of patients, the 5-year overall survival rate of less than 50% has hardly improved over the last decades. To improve the outcome of patients with all subtypes of ovarian cancer, large-scale fundamental and translational research is needed. To accommodate these types of ovarian cancer research, we have established a Dutch nationwide, interdisciplinary infrastructure and biobank: the Archipelago of Ovarian Cancer Research (AOCR). The AOCR will facilitate fundamental and translational ovarian cancer research and enhance interdisciplinary, national, and international collaboration. DESIGN: The AOCR biobank is a prospective ovarian cancer biobank in which biomaterials are collected, processed, and stored in a uniform matter for future (genetic) scientific research. All 19 Dutch hospitals in which ovarian cancer surgery is performed participate and collaborate in the AOCR biobank. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients of 16 years and older with suspected or diagnosed ovarian, fallopian tube, or primary peritoneal cancer are recruited for participation. Patients who agree to participate give written informed consent for collection, storage, and issue of their biomaterials for future studies. After inclusion, different blood samples are taken at various predefined time points both before and during treatment. In case of a diagnostic paracentesis or biopsy, the residual biomaterials of these procedures are stored in the biobank. During surgery, primary tumor tissue and, if applicable, tissue from metastatic sites are collected and stored. From each patient, a representative histological hematoxylin and eosin stained slide is digitalized for research purposes, including reassessment by a panel of gynecologic pathologists. Clinical and pathological data are obtained on a per-study basis from Dutch registries. Research proposals for the issue of biomaterials and data are evaluated by both the Archipelago Scientific Committee and the Steering Committee. Researchers using the biomaterials from the AOCR biobank are encouraged to enrich the biobank with data and materials resulting from their analyses and experiments. LIMITATIONS: The implementation and first 4 years of collection are financed by an infrastructural grant from the Dutch Cancer Society. Therefore, the main limitation is that the costs for sustaining the biobank after the funding period will have to be covered. This coverage will come from incorporation of budget for biobanking in future grant applications and from fees from external researchers and commercial parties using the biomaterials stored in the AOCR biobank. Moreover, we will apply for grants aimed at sustaining and improving research infrastructures and biobanks. CONCLUSIONS: With the establishment of the Dutch nationwide, interdisciplinary Archipelago of Ovarian Cancer Research infrastructure and biobank, fundamental and translational research on ovarian cancer can be greatly improved. The ultimate aim of this infrastructure is that it will lead to improved diagnostics, treatment, and survival of patients with ovarian cancer.

A Paradoxical Role for Regulatory T Cells in the Tumor Microenvironment of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is considered to be a poorly immunogenic cancer type that combines a low mutation burden with a strong immunosuppressive tumor microenvironment. Regulatory T cells (Tregs) are major drivers of immune suppression but their prognostic role, particularly in gastrointestinal malignancies, remains controversial. Lymphocytic infiltration in 122 PDAC samples was assessed by multispectral immunofluorescence with anti-Keratin, -CD3, -CD8, -FOXP3 and -CD163 antibodies. Differential infiltration by Tregs was analyzed in the context of transcriptomic profiles that were available for 65 tumors. High infiltration of CD3+CD8- (mainly CD4+) T cells and, especially, of the subset expressing FOXP3 (Tregs) was associated with improved patient survival, whilst cytotoxic CD3+CD8+ T cell infiltration did not have an impact on overall survival. Transcriptomic analysis revealed three signatures in PDAC tumors comprising of epithelial-mesenchymal transition (EMT)/stromal, metabolic, and secretory/pancreatic signature. However, none of these signatures explained differences in Treg infiltration. We show that Tregs associate with improved overall survival in PDAC patients. This effect was independent of cytotoxic T cell infiltration and the transcriptomic profiles of their respective tumors. These findings provide a new layer of complexity in the study of PDAC tumor microenvironment that must be considered when developing immunotherapeutic interventions for this disease.

Impact of classical and basal-like molecular subtypes on overall survival in resected pancreatic cancer in the SPACIOUS-2 multicentre study.

BACKGROUND: The recently identified classical and basal-like molecular subtypes of pancreatic cancer impact on overall survival (OS). However, the added value of routine subtyping in both clinical practice and randomized trials is still unclear, as most studies do not consider clinicopathological parameters. This study examined the clinical prognostic value of molecular subtyping in patients with resected pancreatic cancer. METHODS: Subtypes were determined on fresh-frozen resected pancreatic cancer samples from three Dutch centres using the Purity Independent Subtyping of Tumours classification. Patient, treatment, and histopathological variables were compared between subtypes. The prognostic value of subtyping in (simulated) pre- and postoperative settings was assessed using Kaplan-Meier and Cox regression analyses. RESULTS: Of 199 patients with resected pancreatic cancer, 164 (82.4 per cent) were classified as the classical and 35 (17.6 per cent) as the basal-like subtype. Patients with a basal-like subtype had worse OS (11 versus 16 months (HR 1.49, 95 per cent c.i. 1.03 to 2.15; P = 0.035)) than patients with a classical subtype. In multivariable Cox regression analysis, including only clinical variables, the basal-like subtype was a statistically significant predictor for poor OS (HR 1.61, 95 per cent c.i. 1.11 to 2.34; P = 0.013). When histopathological variables were added to this model, the prognostic value of subtyping decreased (HR 1.49, 95 per cent c.i. 1.01 to 2.19; P = 0.045). CONCLUSION: The basal-like subtype was associated with worse OS in patients with resected pancreatic cancer. Adding molecular classification to inform on tumor biology may be used in patient stratification.

Circulating tumor DNA predicts outcome in metastatic gastroesophageal cancer.

BACKGROUND: Circulating tumor DNA (ctDNA) has predictive and prognostic value in localized and metastatic cancer. This study analyzed the prognostic value of baseline and on-treatment ctDNA in metastatic gastroesophageal cancer (mGEC) using a region-specific next generation sequencing (NGS) panel. METHODS: Cell free DNA was isolated from plasma of patients before start of first-line palliative systemic treatment and after 9 and 18 weeks. Two NGS panels were designed comprising the most frequently mutated genes and targetable mutations in GEC. Tumor-derived mutations in matched metastatic biopsies were used to validate that the sequencing panels assessed true tumor-derived variants. Tumor volumes were calculated from baseline CT scans and correlated to variant allele frequency (VAF). Survival analyses were performed using univariable and multivariable Cox-regression analyses. RESULTS: ctDNA was detected in pretreatment plasma in 75% of 72 patients and correlated well with mutations in metastatic biopsies (86% accordance). The VAF correlated with baseline tumor volume (Pearson's R 0.53, p < 0.0001). Detection of multiple gene mutations at baseline in plasma was associated with worse overall survival (OS, HR 2.16, 95% CI 1.10-4.28; p = 0.027) and progression free survival (PFS, HR 2.71, 95% CI 1.28-5.73; p = 0.009). OS and PFS were inferior in patients with residual detectable ctDNA after 9 weeks of treatment (OS: HR 4.95, 95% CI 1.53-16.04; p = 0.008; PFS: HR 4.08, 95% CI 1.31-12.75; p = 0.016). CONCLUSION: Based on our NGS panel, the number of ctDNA mutations before start of first-line chemotherapy has prognostic value. Moreover, residual ctDNA after three cycles of systemic treatment is associated with inferior survival.

Analysis of the glyco-code in pancreatic ductal adenocarcinoma identifies glycan-mediated immune regulatory circuits.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most aggressive malignancies with a 5-year survival rate of only 9%. Despite the fact that changes in glycosylation patterns during tumour progression have been reported, no systematic approach has been conducted to evaluate its potential for patient stratification. By analysing publicly available transcriptomic data of patient samples and cell lines, we identified here two specific glycan profiles in PDAC that correlated with progression, clinical outcome and epithelial to mesenchymal transition (EMT) status. These different glycan profiles, confirmed by glycomics, can be distinguished by the expression of O-glycan fucosylated structures, present only in epithelial cells and regulated by the expression of GALNT3. Moreover, these fucosylated glycans can serve as ligands for DC-SIGN positive tumour-associated macrophages, modulating their activation and inducing the production of IL-10. Our results show mechanisms by which the glyco-code contributes to the tolerogenic microenvironment in PDAC.

Bayesian log-normal deconvolution for enhanced in silico microdissection of bulk gene expression data.

Deconvolution of bulk gene expression profiles into the cellular components is pivotal to portraying tissue's complex cellular make-up, such as the tumor microenvironment. However, the inherently variable nature of gene expression requires a comprehensive statistical model and reliable prior knowledge of individual cell types that can be obtained from single-cell RNA sequencing. We introduce BLADE (Bayesian Log-normAl Deconvolution), a unified Bayesian framework to estimate both cellular composition and gene expression profiles for each cell type. Unlike previous comprehensive statistical approaches, BLADE can handle > 20 types of cells due to the efficient variational inference. Throughout an intensive evaluation with > 700 simulated and real datasets, BLADE demonstrated enhanced robustness against gene expression variability and better completeness than conventional methods, in particular, to reconstruct gene expression profiles of each cell type. In summary, BLADE is a powerful tool to unravel heterogeneous cellular activity in complex biological systems from standard bulk gene expression data.

Minimally invasive versus open distal pancreatectomy for pancreatic ductal adenocarcinoma (DIPLOMA): study protocol for a randomized controlled trial.

BACKGROUND: Recently, the first randomized trials comparing minimally invasive distal pancreatectomy (MIDP) with open distal pancreatectomy (ODP) for non-malignant and malignant disease showed a 2-day reduction in time to functional recovery after MIDP. However, for pancreatic ductal adenocarcinoma (PDAC), concerns have been raised regarding the oncologic safety (i.e., radical resection, lymph node retrieval, and survival) of MIDP, as compared to ODP. Therefore, a randomized controlled trial comparing MIDP and ODP in PDAC regarding oncological safety is warranted. We hypothesize that the microscopically radical resection (R0) rate is non-inferior for MIDP, as compared to ODP. METHODS/DESIGN: DIPLOMA is an international randomized controlled, patient- and pathologist-blinded, non-inferiority trial performed in 38 pancreatic centers in Europe and the USA. A total of 258 patients with an indication for elective distal pancreatectomy with splenectomy because of proven or highly suspected PDAC of the pancreatic body or tail will be randomly allocated to MIDP (laparoscopic or robot-assisted) or ODP in a 1:1 ratio. The primary outcome is the microscopically radical resection margin (R0, distance tumor to pancreatic transection and posterior margin ≥ 1 mm), which is assessed using a standardized histopathology assessment protocol. The sample size is calculated with the following assumptions: 5% one-sided significance level (α), 80% power (1-ß), expected R0 rate in the open group of 58%, expected R0 resection rate in the minimally invasive group of 67%, and a non-inferiority margin of 7%. Secondary outcomes include time to functional recovery, operative outcomes (e.g., blood loss, operative time, and conversion to open surgery), other histopathology findings (e.g., lymph node retrieval, perineural- and lymphovascular invasion), postoperative outcomes (e.g., clinically relevant complications, hospital stay, and administration of adjuvant treatment), time and site of disease recurrence, survival, quality of life, and costs. Follow-up will be performed at the outpatient clinic after 6, 12, 18, 24, and 36 months postoperatively. DISCUSSION: The DIPLOMA trial is designed to investigate the non-inferiority of MIDP versus ODP regarding the microscopically radical resection rate of PDAC in an international setting. TRIAL REGISTRATION: ISRCTN registry ISRCTN44897265 . Prospectively registered on 16 April 2018.