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Introduction Immunotherapy with checkpoint inhibitors (CPIs) has revolutionised cancer treatment but has no convincing effect in metastatic castration-resistant prostate cancer (mCRPC). It has been suggested that a combination of CPI and hypofractionated stereotactic body radiotherapy (SBRT) may work synergistically, and recent trials have supported this. We hypothesise that adding SBRT to CPI treatment can improve response rates in patients with mCRPC. Methods and analysis The CheckPRO trial is an open-label, randomised, two-stage, phase II trial. We aim to enrol and randomise 80 evaluable patients with mCRPC who progressed following ≥2 lines of treatment. Enrolment started in November 2019 with 38 months expected enrolment period. The participants receive treatment for 52 weeks including four cycles of ipilimumab and nivolumab with or without concomitant SBRT (24 Gray in three fractions) to a single soft tissue or bone metastasis, followed by 10 cycles of nivolumab. Participants are followed until progression, death, or for 12 months after the end of treatment. Co-primary endpoints are the objective response rate and prostate-specific antigen (PSA) response rate. Secondary endpoints include safety, radiographic progression-free survival, clinical benefit rate, duration of response, PSA-progression-free survival beyond 12 weeks, quality of life and overall survival. Exploratory endpoints include translational analyses of tumour biopsies and consecutive blood samples. Biopsies from metastatic sites are collected at baseline, before the third treatment and at the end of treatment. Blood sampling for immune monitoring and circulating tumour DNA is performed consecutively at baseline and every radiographic assessment. Ethics and dissemination This study follows the Helsinki Declaration and is approved by the Danish Ethics Committee System (journal no. H-19016100). All participants must receive written and oral information and provide a signed informed consent docume

Background/Aim: MicroRNAs (miRNAs) are small noncoding RNAs involved in gene expression regulation and have been investigated as potential biomarkers for various diseases, including ovarian cancer (OC). However, lack of standardized protocols regarding e.g., RNA isolation, cDNA synthesis, spike-in controls for experimental steps, and data normalization, impacts cross validation of results across research groups and hinders implementation of miRNAs as clinical biomarkers. Materials and Methods: RNA was isolated from matching fresh-frozen tissue (FF), formalin-fixed paraffin embedded (FFPE) tissue, and plasma samples from twenty women diagnosed with OC using three commercial kits: miRNeasy Tissue/Cells, miRNeasy FFPE, and miRNeasy Serum/Plasma (Qiagen, Copenhagen, Denmark). RNA isolation, cDNA synthesis, and PCR performance were tested using miRCURY LNA miRNA Quality Control PCR (QC) Panels (Qiagen). Finally, miRNA stability was assessed using five algorithms: BestKeeper, Normfinder, GeNorm, comparative delta-Ct and comprehensive ranking provided by a web-based RefFinder tool. Results: RNA from FF, FFPE and plasma was extracted using commercially available kits and the differences in yield and purity were examined. We developed a simple method for identifying and potentially excluding samples based on their crossing point values from RT-qPCR data, which could improve existing manufacture guidelines. Moreover, we discussed how assessment of miRNA stability differs between algorithms, possibly leading to inconsistent results. Conclusion: We present guidelines for RNA isolation, cDNA synthesis, and data normalization for successful miRNA expression profiling using RT-qPCR in corresponding biological OC specimens. We recommend QC panels in combination with spike-in controls and interplate controls to monitor process efficiencies.

Ovarian cancer (OC) is the eighth most common type of cancer for women worldwide. The current diagnostic and prognostic routine available for OC management either lack specificity or are very costly. Gene expression profiling has shown to be a very effective tool in exploring new molecular markers for patients with OC, although association of such markers with patient survival and clinical outcome is still elusive. Here, we performed gene expression profiling of different subtypes of OC to evaluate its association with patient overall survival (OS) and aggressive forms of the disease. By global mRNA microarray profiling in a total of 196 epithelial OC patients (161 serous, 15 endometrioid, 11 mucinous, and 9 clear cell carcinomas), we found four candidates-HSPA1A, CD99, RAB3A and POM121L9P, which associated with OS and poor clinicopathological features. The overexpression of all combined was correlated with shorter OS and progression-free survival (PFS). Furthermore, the combination of at least two markers were further associated with advanced grade, chemotherapy resistance, and progressive disease. These results indicate that a panel comprised of a few predictors that associates with a more aggressive form of OC may be clinically relevant, presenting a better performance than one marker alone.

Ovarian cancer (OC), the eighth-leading cause of cancer-related death among females worldwide, is mainly represented by epithelial OC (EOC) that can be further subdivided into four subtypes: Serous (75%), endometrioid (10%), clear cell (10%), and mucinous (3%). Major reasons for high mortality are the poor biological understanding of the OC mechanisms and a lack of reliable markers defining each EOC subtype. MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression primarily by targeting messenger RNA (mRNA) transcripts. Their aberrant expression patterns have been associated with cancer development, including OC. However, the role of miRNAs in tumorigenesis is still to be determined, mainly due to the lack of consensus regarding optimal methodologies for identification and validation of miRNAs and their targets. Several tools for computational target prediction exist, but false interpretations remain a problem. The experimental validation of every potential miRNA-mRNA pair is not feasible, as it is laborious and expensive. In this study, we analyzed the correlation between global miRNA and mRNA expression patterns derived from microarray profiling of 197 EOC patients to identify the signatures of miRNA-mRNA interactions associated with overall survival (OS). The aim was to investigate whether these miRNA-mRNA signatures might have a prognostic value for OS in different subtypes of EOC. The content of our cohort (162 serous carcinomas, 15 endometrioid carcinomas, 11 mucinous carcinomas, and 9 clear cell carcinomas) reflects a real-world scenario of EOC. Several interaction pairs between 6 miRNAs (hsamiR- 126-3p, hsa-miR-223-3p, hsa-miR-23a-5p, hsa-miR-27a-5p, hsa-miR-486-5p, and hsamiR- 506-3p) and 8 mRNAs (ATF3, CH25H, EMP1, HBB, HBEGF, NAMPT, POSTN, and PROCR) were identified and the findings appear to be well supported by the literature. This indicates that our study has a potential to reveal miRNA-mRNA signatures relevant for EOC. Th

The usage of next generation sequencing in combination with targeted gene panels has enforced a better understanding of tumor compositions. The identification of key genomic bi-omarkers underlying a disease are crucial for diagnosis, prognosis, treatment and therapeutic re-sponses. The Oncomine™ Comprehensive Assay v3 (OCAv3) covers 161 cancer‐associated genes and is routinely employed to support clinical decision making for a therapeutic course. An improved version, Oncomine™ Comprehensive Assay Plus (OCA‐Plus), has been recently developed, covering 501 genes (144 overlapping with OCAv3) in addition to microsatellite instability (MSI) and tumor mutational burden (TMB) assays in one workflow. The validation of MSI and TMB was not addressed in the present study. However, the implementation of new assays must be validated and confirmed across multiple samples before it can be introduced into a clinical setting. Here, we report the comparison of DNA sequencing results from 50 ovarian cancer formalin‐fixed, paraffin‐embed-ded samples subjected to OCAv3 and OCA‐Plus. A validation assessment of gene mutations identified using OCA‐Plus was performed on the 144 overlapping genes and 313,769 intersecting nucle-otide positions of the OCAv3 and the OCA‐Plus. Our results showed a 91% concordance within variants classified as likely‐pathogenic or pathogenic. Moreover, results showed that a region of PTEN is poorly covered by the OCA‐Plus assay, hence, we implemented rescue filters for those variants. In conclusion, the OCA‐Plus can reflect the mutational profile of genomic variants com-pared with OCAv3 of 144 overlapping genes, without compromising performance.

Ovarian clear cell carcinoma (OCCC) is characterized by dismal prognosis, partially due to its low sensitivity to standard chemotherapy regimen. It is also well-known for presenting unique molecular features in comparison to other epithelial ovarian cancer subtypes. Here, we aim to identify potential subgroups of patients in order to (1) determine their molecular features and (2) characterize their mutational signature. Furthermore, we sought to perform the investigation based on a potentially clinically relevant setting. To that end, we assessed the mutational profile and genomic instability of 55 patients extracted from the Gynecologic Cancer Database (DGCD) by using a panel comprised of 409 cancer-associated genes and a microsatellite assay, respectively; both are currently used in our routine environment. In accordance with previous findings, ARID1A and PIK3CA were the most prevalent mutations, present in 49.1% and 41.8%, respectively. From those, the co-occurrence of ARID1A and PIK3CA mutations was observed in 36.1% of subjects, indicating that this association might be a common feature of OCCC. The microsatellite instability frequency was low across samples. An unbiased assessment of signatures identified the presence of three subgroups, where “PIK3CA” and “Double hit” (with ARID1A and PIK3CA double mutation) subgroups exhibited unique signatures, whilst “ARID1A” and “Undetermined” (no mutations on ARID1A nor PIK3CA) subgroups showed similar profiles. Those differences were further indicated by COSMIC signatures. Taken together, the current findings suggest that OCCC presents distinct mutational landscapes within its group, which may indicate different therapeutic approaches according to its subgroup. Although encouraging, it is noteworthy that the current results are limited by sample size, and further investigation on a larger group would be crucial to better elucidate them.

Data analysis has become a crucial aspect in clinical oncology to interpret output from next-generation sequencing-based testing. NGS being able to resolve billions of sequencing reactions in a few days has consequently increased the demand for tools to handle and analyze such large data sets. Many tools have been developed since the advent of NGS, featuring their own peculiarities. Increased awareness when interpreting alterations in the genome is therefore of utmost importance, as the same data using different tools can provide diverse outcomes. Hence, it is crucial to evaluate and validate bioinformatic pipelines in clinical settings. Moreover, personalized medicine implies treatment targeting efficacy of biological drugs for specific genomic alterations. Here, we focused on different sequencing technologies, features underlying the genome complexity, and bioinformatic tools that can impact the final annotation. Additionally, we discuss the clinical demand and design for implementing NGS.

Biliary tract cancers (BTC) are a rare heterogeneous disease group with a dismal prognosis and limited treatment options. The mutational landscape consists of genetic aberrations both shared by and characteristic for anatomical location. Here, we present exome sequencing data on 22 genes from a phase 2 trial using a clinically validated panel used in patients with colorectal cancer. A total of 56 patients were included in a one-armed phase 2 trial investigating the treatment combination of capecitabine, gemcitabine, oxaliplatin, and cetuximab. Tissue DNA yield and quality allowed analysis of 30 patients on our panel including 22 genes. ARID1A (33%) and TP53 (33%) were found to be most frequently mutated followed by KRAS mutations found in 20% of the patients. Mutational aberrations in ARID1A were found more prevalent than expected, whereas TP53 and KRAS were in concordance with earlier reported data. Mutation in CTNNB1 was significantly associated with poor prognosis. Our panel is clinically validated and suitable for a high volume of samples to detect mutations in patients with BTC. However, it is reasonable to assume that the clinical utility could be optimized in this patient group by extending the panel to include BTC specific mutations with potential therapeutic consequences such as IDH1/2, FGFR fusions, ERBB3, and BRCA1/2.

Background Ovarian cancer is the fifth most common cancer in women worldwide. Moreover, there are no reliable minimal invasive tests to secure the diagnosis of malignant pelvic masses. Cell-free, circulating microRNAs have the potential as diagnostic biomarkers in cancer. Here, we performed and validated a miRNA panel with the potential to distinguish OC from benign pelvic masses. Methods The profile of plasma microRNA was determined with a panel of 46 candidates in a discovery group and a validation group, each consisting of 190 pre-surgery plasma samples from age-matched patients with malignant (n = 95) and benign pelvic mass (n = 95), by real time RT-qPCR. Results Four up-regulated (miR-200c-3p, miR-221-3p, miR-21-5p, and miR-484) and two down-regulated (miR-195-5p and miR-451a) microRNAs were discovered. From those, miR-200c-3p and miR-221-3p were further confirmed in a validation cohort. A combination of these 2 microRNAs together with CA-125 yielded an overall diagnostic accuracy of AUC = 0.96. Conclusions We showed consistent plasma microRNA profiles that provide independent diagnostic information of late stage OC.

Ovarian cancer is a silent killer and, due to late diagnosis and frequent chemo resistance in patients, the primary cause of fatality amongst the various types of gynecological cancer. The discovery of a specific and sensitive biomarker for ovarian cancer could improve early diagnosis, thereby saving lives. Biomarkers could also improve treatment, by predicting which patients will benefit from specific treatment strategies. DNA methylation is an epigenetic mechanism, and 'methylation imbalance' is characteristic of cancer. Previous research suggests that changes in DNA methylation can be used diagnostically, and that they may predict resistance to treatment. This paper gives an up-to-date overview of research investigating the potential of DNA methylation-based markers for diagnostics, prognostics, screening and prediction of drug resistance for ovarian cancer patients. DNA methylation cancer-biomarkers may be useful for cancer treatment, particularly since they are chemically stable and since cancer-associated changes in methylation typically precedes tumor growth. DNA methylation markers could improve diagnosis and treatment and might even be used for screening in the future. Furthermore, DNA methylation biomarkers could facilitate the development of precision medicine. However, at this point no biomarkers for ovarian cancer have a sufficient combination of sensitivity and specificity in a clinical setting. A reason for this is that most studies have focused on a single or a few methylation sites. More large screenings and genome-wide studies must be performed to increase the chance of identifying a DNA methylation marker which can identify ovarian cancer.

Annexin A2 is a 36-kDa protein interfering with multiple cellular processes especially in cancer progression. The present review aimed to show the relations between Annexin A2 and cancer. A systematic search for studies investigating cancer and Annexin A2 expression was conducted using PubMed. Acute lymphoblastic leukaemia, acute promyelocytic leukaemia, clear cell renal cell carcinoma, breast, cervical, colorectal, endometrial, gastric cancer, glioblastoma, hepatocellular carcinoma, lung, multiple myeloma, oesophageal squamous cell carcinoma, ovarian cancer, pancreatic duct adenocarcinoma, prostate cancer and urothelial carcinoma were evaluated. Annexin A2 expression correlates with resistance to treatment, binding to the bone marrow, histological grade and type, TNM-stage and shortened overall survival. The regulation of Annexin A2 is of interest due to its potential as target for a more individualized cancer management.

BACKGROUND: The precise mechanism by which the immune system is adversely affected in cancer patients remains poorly understood, but the accumulation of immune suppressive/pro-tumorigenic myeloid-derived suppressor cells (MDSCs) is thought to be one prominent mechanism contributing to immunologic tolerance of malignant cells in epithelial ovarian cancer (EOC). To this end, we hypothesized genetic variation in MDSC pathway genes would be associated with survival after EOC diagnoses.METHODS: We measured the hazard of death due to EOC within 10 years of diagnosis, overall and by invasive subtype, attributable to SNPs in 24 genes relevant in the MDSC pathway in 10,751 women diagnosed with invasive EOC. Versatile Gene-based Association study (VEGAS) and the Admixture Likelihood method (AML), were used to test gene and pathway associations with survival.RESULTS: We did not identify individual SNPs that were significantly associated with survival after correction for multiple testing (p<3.5 x 10-5), nor did we identify significant associations between the MDSC pathway overall, or the 24 individual genes and EOC survival.CONCLUSIONS: In this well-powered analysis, we observed no evidence that inherited variations in MDSC-associated SNPs, individual genes, or the collective genetic pathway contributed to EOC survival outcomes.IMPACT: Common inherited variation in genes relevant to MDSCs were not associated with survival in women diagnosed with invasive EOC.

To examine serum YKL-40 in women developing gestational diabetes mellitus (GDM). In the present large observational cohort study of 1179 pregnant women, we determined serum YKL-40 four times during pregnancy (at gestational age 12, 20, 25, and 32 weeks). Pregnancy outcome was obtained from medical records. Sixty-eight women (5.8%) developed GDM. Serum YKL-40 increased from gestational age (GA) 12 weeks and the following weeks in the women who developed GDM and was independent of BMI, parity, and maternal age (OR = 2.69, 95% CI: 1.45-5.00, p = 0.002). No association was found between serum YKL-40 and the oral glucose tolerance test results. In conclusion, YKL-40 significantly increased in pregnant women with GDM compared with women without GDM, probably reflecting the low-grade inflammation of GDM. However, we did not find an association between serum concentrations of YKL-40 in early pregnancy and the development of GDM and thus we conclude that YKL-40 alone is not usable as a biomarker for early prediction of GDM.

AIM: To develop and validate a biomarker-based index to optimize referral and diagnosis of patients with suspected ovarian cancer. Furthermore, to compare this new index with the Risk of Malignancy Index (RMI) and Risk of Ovarian Malignancy Algorithm (ROMA).PATIENTS AND METHODS: A training study, consisting of patients with benign ovarian disease (n=809) and ovarian cancer (n=246), was used to develop the Copenhagen Index (CPH-I) utilizing the variables serum HE4, serum CA125 and patient age. Eight international studies provided the validation population; comprising 1060 patients with benign ovarian masses and 550 patients with ovarian cancer.RESULTS: Overall, 2665 patients were included. CPH-I was highly significant in discriminating benign from malignant ovarian disease. At the defined cut-off of 0.070 for CPH-I the sensitivity and specificity were 95.0% and 78.4% respectively in the training cohort and 82.0% and 88.4% in the validation cohort. Comparison of CPH-I, ROMA and RMI demonstrated area-under-curve (AUC) at 0.960, 0.954 and 0.959 respectively in the training study and 0.951, 0.953 and 0.935 respectively in the validation study. Using a sensitivity of 95.0%, the specificities for CPH-I, ROMA and RMI in the training cohort were 78.4%, 71.7% and 81.5% respectively, and in the validation cohort 67.3%, 70.7% and 69.5% respectively.CONCLUSION: All three indices perform well at the clinically relevant sensitivity of 95%, but CPH-I, unlike RMI and ROMA, is independent of ultrasound and menopausal status, and may provide a simple index to optimize referral of women with suspected ovarian cancer.

The primary objective of this study was to analyse Tetranectin (TN) expression in tumour tissues and TN serum concentration in 758 women with epithelial ovarian tumours. The second was to evaluate, whether TN tissue expression levels correlate with clinico-pathological parameters and prognosis of the disease. Using tissue arrays we analysed the expression levels in tissues from 166 women with borderline ovarian tumours (BOTs) and 592 women with ovarian cancer (OC). A panel of three antibodies was used for immunohistochemistry: a polyclonal and two monoclonal antibodies. Serum TN was measured using the polyclonal antibody A-371. Univariate survival analyses stratified for chemotherapy showed that positive tissue TN as demonstrated by the polyclonal antibody indicated a significantly longer overall survival (OS) (p = 0.0001) as well as cancer specific survival (CSS) (p < 0.0001). High serum TN was likewise found to imply longer OS (p < 0.0001) and CSS (p < 0.0001), whereas tissue staining with the two monoclonal antibodies failed to demonstrate any significant correlation with either survival type. Univariate Kaplan-Meier survival analysis performed on all OC cases showed a significantly longer OS (p = 0.0009) and CSS (p = 0.0006) for women with TN positive tumour tissue and in women with high serum TN levels (p < 0.0001 for both). However, in the multivariate Cox regression analysis, only serum TN was found to be an independent prognostic factor for OS (p = 0.01) and not for CSS (p = 0.08). In conclusion, our results predict that a positive TN expression of both tumour tissue and serum points to a more favourable outcome for OC patients.

We compared morphologic computed tomography (CT)-based to metabolic fluoro-deoxy-glucose (FDG) positron emission tomography (PET)/CT-based response evaluation in patients with metastatic colorectal cancer and correlated the findings with survival and KRAS status. From 2006 to 2009, patients were included in a phase II trial and treated with cetuximab and irinotecan every second week. They underwent FDG-PET/CT examination at baseline and after every fourth treatment cycle. Response evaluation was performed prospectively according to Response Evaluation Criteria in Solid Tumors (RECIST 1.0) and retrospectively according to Positron Emission Tomography Response Criteria in Solid Tumors (PERCIST). Best overall responses were registered. Sixty-one patients were eligible for response evaluation. Partial response (PR) rate was 18%, stable disease (SD) rate 64%, and progressive disease (PD) rate 18%. Partial metabolic response (PMR) rate was 56%, stable metabolic disease rate 33%, and progressive metabolic disease (PMD) rate 11%. Response agreement was poor, κ-coefficient 0.19. Hazard ratio for overall survival for responders (PR/PMR) versus nonresponders (PD/PMD) was higher for CT- than for FDG-PET/CT evaluation. Within patients with KRAS mutations, none had PR but 44% had PMR. In conclusion, morphologic and metabolic response agreement was poor primarily because a large part of the patients shifted from SD with CT evaluation to PMR when evaluated with FDG-PET/CT. Furthermore, a larger fraction of the patients with KRAS mutations had a metabolic treatment response.

OBJECTIVE: To investigate the influence of handling and storage on HE4 and CA125 serum and EDTA plasma levels to clarify any important consequences for a clinical setting.METHODS: Blood samples from 13 ovarian cancer (OC) patients were collected and allowed to clot or sediment for up to 72 hours at 4 °C or 20 °C, then processed into serum and EDTA plasma. Furthermore, the effects of up to eight repetitive cycles of freeze/thaw were investigated. HE4 and CA125 were analyzed using a Chemiluminescent Microparticle Immunoassay on the Architect i2000sr System.RESULTS: No significant effect of processing time for HE4 could be shown. HE4 EDTA plasma levels were insignificantly lower (3%) than serum levels (p = 0.41). Similarly, no significant effect of processing time for CA125 could be demonstrated. CA125 levels at 4 °C were significantly reduced compared to levels at 20 °C (p = 0.024). No significant difference between CA125 serum and plasma levels were found (p = 0.46). Serum and EDTA plasma samples were stable during the eight cycles of freezing and thawing (CA125: all p > 0.2; HE4: all p > 0.5).CONCLUSION: No systematic difference could be demonstrated for HE4. CA125 is not dependent on processing time, EDTA plasma or serum. Levels of CA125 are significantly reduced at 4 °C compared to levels at 20°C, but this difference was less than 6% and is not considered clinically relevant.

Human epididymis protein 4 (HE4) has received major attention as a potential tumor marker in epithelial ovarian cancer; however, evidence of significant overexpression of HE4 in several other human cancers is expanding. To assess the possible limitations or benefits of HE4 in a clinical setting, this review aims to systematically outline published results of HE4 tissue expression and serum HE4 levels in healthy individuals and patients with benign or malignant tumors. Our findings suggest scientific basis for a potential diagnostic ability of HE4 in gynecologic cancer and lung cancer, and further research is needed regarding other cancers. Yet, it is important to recognize that other malignancies can cause increased HE4 levels. Furthermore, attention should be paid to the influence of age and renal function on HE4 serum levels in future studies as well as in the clinic for proper interpretation of serum HE4 test results. Cancer Epidemiol Biomarkers Prev; 23(11); 2285-95. ©2014 AACR.

PURPOSE: We tested the hypothesis that expression of microRNAs (miRNAs) in cancer tissue can predict effectiveness of bevacizumab added to capecitabine and oxaliplatin (CAPEOX) in patients with metastatic colorectal cancer (mCRC).EXPERIMENTAL DESIGN: Patients with mCRC treated with first line CAPEOX and bevacizumab (CAPEOXBEV): screening (n = 212) and validation (n = 121) cohorts, or CAPEOX alone: control cohort (n = 127), were identified retrospectively and archival primary tumor samples were collected. Expression of 754 miRNAs was analyzed in the screening cohort using polymerase chain reaction (PCR) arrays and expression levels were related to time to disease progression (TTP) and overall survival (OS). Significant miRNAs from the screening study were analyzed in all three cohorts using custom PCR arrays. In situ hybridization (ISH) was done for selected miRNAs.RESULTS: In the screening study, 26 miRNAs were significantly correlated with outcome in multivariate analyses. Twenty-two miRNAs were selected for further study. Higher miR-664-3p expression and lower miR-455-5p expression were predictive of improved outcome in the CAPEOXBEV cohorts and showed a significant interaction with bevacizumab effectiveness. The effects were strongest for OS. Both miRNAs showed high expression in stromal cells. Higher expression of miR-196b-5p and miR-592 predicted improved outcome regardless of bevacizumab treatment, with similar effect estimates in all three cohorts.CONCLUSIONS: We have identified potentially predictive miRNAs for bevacizumab effectiveness and additional miRNAs that could be related to chemotherapy effectiveness or prognosis in patients with mCRC. Our findings need further validation in large cohorts, preferably from completed randomized trials.