Digital RNA sequencing using unique molecular identifiers enables ultrasensitive RNA mutation analysis.

Mutation analysis is typically performed at the DNA level since most technical approaches are developed for DNA analysis. However, some applications, like transcriptional mutagenesis, RNA editing and gene expression analysis, require RNA analysis. Here, we combine reverse transcription and digital DNA sequencing to enable low error digital RNA sequencing. We evaluate yield, reproducibility, dynamic range and error correction rate for seven different reverse transcription conditions using multiplexed assays. The yield, reproducibility and error rate vary substantially between the specific conditions, where the yield differs 9.9-fold between the best and worst performing condition. Next, we show that error rates similar to DNA sequencing can be achieved for RNA using appropriate reverse transcription conditions, enabling detection of mutant allele frequencies <0.1% at RNA level. We also detect mutations at both DNA and RNA levels in tumor tissue using a breast cancer panel. Finally, we demonstrate that digital RNA sequencing can be applied to liquid biopsies, analyzing cell-free gene transcripts. In conclusion, we demonstrate that digital RNA sequencing is suitable for ultrasensitive RNA mutation analysis, enabling several basic research and clinical applications.

Repurposing proteasome inhibitors for improved treatment of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is associated with poor prognosis and limited treatment options due to the lack of important receptors (estrogen receptor [ER], progesterone receptor [PR], and human epidermal growth factor receptor 2 [HER2]) used for targeted therapy. However, high-throughput in vitro drug screening of cell lines is a powerful tool for identifying effective drugs for a disease. Here, we determine the intrinsic chemosensitivity of TNBC cell lines to proteasome inhibitors (PIs), thereby identifying potentially potent 2-drug combinations for TNBC. Eight TNBC cell lines (BT-549, CAL-148, HCC1806, HCC38, HCC70, MDA-MB-436, MDA-MB-453, and MDA-MB-468) and two controls (MCF-10A and MCF-7) were first exposed to 18 drugs (11 PIs and 7 clinically relevant chemotherapeutic agents) as monotherapy, followed by prediction of potent 2-drug combinations using the IDACombo pipeline. The synergistic effects of the 2-drug combinations were evaluated with SynergyFinder in four TNBC cell lines (CAL-148, HCC1806, HCC38, and MDA-MB-468) and three controls (BT-474, MCF-7, and T47D) in vitro, followed by further evaluation of tumor regression in zebrafish tumor models established using HCC1806 and MCF-7 cells. Monotherapy identified nine effective drugs (bortezomib, carfilzomib, cisplatin, delanzomib, docetaxel, epoxomicin, MLN-2238, MLN-9708, and nedaplatin) across all cell lines. PIs (e.g., bortezomib, delanzomib, and epoxomicin) were highly potent drugs in TNBC cells, of which bortezomib and delanzomib inhibited the chymotrypsin-like activity of the 20 S proteasome by 100% at 10 µM. Moreover, several potent 2-drug combinations (e.g., bortezomib+nedaplatin and epoxomicin+epirubicin) that killed virtually 100% of cells were also identified. Although HCC1806- and MCF-7-derived xenografts treated with bortezomib+nedaplatin and carboplatin+paclitaxel were smaller, HCC1806 cells frequently metastasized to the trunk region. Taken together, we show that PIs used in combination with platinum agents or topoisomerase inhibitors exhibit increased efficiency with almost 100% inhibition in TNBC cell lines, indicating that PIs are therefore promising compounds to use as combination therapy for TNBC.

Cancer stem cells are prevalent in the basal-like 2 and mesenchymal triple-negative breast cancer subtypes in vitro.

Background: Triple-negative breast cancer (TNBC) is an aggressive subtype with the most unfavorable clinical outcomes, in part due to tumor heterogeneity, treatment resistance, and tumor relapse. The TNBC subtypes [basal-like 1 (BL1), basal-like 2 (BL2), mesenchymal (M), and luminal androgen receptor (LAR)] are biologically and clinically distinct entities that respond differently to local and systemic therapies. Therefore, we need to have a better understanding of cancer stemness relating to drug-resistant populations in the TNBC subtypes. Methods: Breast cancer stem cell (BCSC) distribution was investigated using an integrated flow cytometry approach with the ALDEFLUOR™ assay (ALDH) and CD24/CD44 antibodies. In total, 27 commercially available cell lines derived from normal and malignant mammary tissue were characterized into differentiated tumor cells and/or BCSC subpopulations (ALDH-CD44+CD24-/low enriched mesenchymal-like BCSCs, ALDH+non-CD44+CD24-/low enriched epithelial-like BCSCs, and highly purified ALDH+CD44+CD24-/low BCSCs). Results: BCSCs were not only enriched in estrogen receptor (ER) negative (mean, 49.6% versus 6.9% in ER+) and TNBC cell lines (51.3% versus 2.1% in Luminal A), but certain BCSC subpopulations (e.g., enriched mesenchymal-like BCSCs) were also significantly more common in the M (64.0% versus 6.2% in BL1; 64.0% versus 0% in LAR) and BL2 (77.4% versus 6.2% in BL1; 77.4% versus 0% in LAR; 77.4% versus 10.4% in TNBC UNS) TNBC subtypes. In contrast, ALDH status alone was not indicative of ER status or BC subtype. Conclusion: Taken together, these findings demonstrate the enrichment of potentially treatment-resistant BCSC subpopulations in the M and BL2 triple-negative breast cancer subtypes.

Clinical relevance of biomarker discordance between primary breast cancers and synchronous axillary lymph node metastases.

Clinical decision-making for patients with breast cancer (BC) is still primarily based on biomarker characteristics of the primary tumor, together with the evaluation of synchronous axillary lymph node metastasis (LNM). In this study, we investigated the prevalence of discordance in the biomarkers and surrogate subtyping between the primary BC and the LNM, and whether subsequent changes would have altered clinical treatment recommendations. In this retrospective study, 94 patients treated for unifocal primary BC and synchronous LNM at Sahlgrenska UniversityHospital during 2018 were included. Estrogen (ER) and progesterone (PR) receptor, Ki67, and HER2 status were assessed in the primary tumor and LNM using immunohistochemistry. Discordances between the primary tumor and the LNM were analyzed for each individual biomarker and surrogate subtyping. The concordance between the primary tumor and the LNM for ER, PR, Ki67, and HER2 status was 98.9%, 89.4%, 72.3%, and 95.8%, respectively. Discordance in surrogate subtyping was found in 28.7% of the tumors and matched LNMs, the majority (81.5%) of which changed to a more favorable subtype in the LNM; most commonly from Luminal B to Luminal A (48.6%). No changes in surrogate subtyping were detected where ER or HER2 status changed from negativity in the BC to positivity in the LNM, thereby showing no additional value in performing immunohistochemistry on the LNM from a treatment decision-making perspective. However, large studies need to be performed that test both the primary BCs and synchronous LNMs for more accurate diagnostics.

Optimization of Cell Viability Assays for Drug Sensitivity Screens.

During the preclinical stages of the drug discovery process, cell viability assays are fundamental tools for studying the phenotypic properties and overall health of cells following in vitro drug sensitivity screens. Therefore, it is important to optimize your viability assay of choice to obtain reproducible and replicable results, as well as use relevant drug response metrics (e.g., IC50, AUC, GR50, and GRmax) to identify candidate drugs for further evaluation in vivo. Herein, we used the resazurin reduction assay which is a quick, cost-effective, simple-to-use, and sensitive method for examining the phenotypic properties of cells. Using the MCF7 breast cancer cell line, we provide a detailed step-by-step protocol for optimizing drug sensitivity screens using the resazurin assay.

Clinical evaluation of molecular surrogate subtypes in patients with ipsilateral multifocal primary breast cancer.

BACKGROUND: When ipsilateral multifocal primary breast cancer (IMBC) is detected, standard routine is to evaluate the largest tumor with immunohistochemistry (IHC). As all foci are not routinely characterized, many patients may not receive optimal adjuvant treatment. Here, we assess the clinical relevance of examining at least two foci present in patients with IMBC. METHODS: Patients diagnosed and treated for IMBC at Sahlgrenska University Hospital (Gothenburg, Sweden) between 2012 and 2017 were screened. In total, 180 patients with ≥ 2 invasive foci (183 specimens) were assessed with IHC and included in this study. Expression of the estrogen (ER) and progesterone (PR) receptors, Ki67, HER2, and tumor grade were used to determine the molecular surrogate subtypes and discordance among the foci was recorded. An additional multidisciplinary team board was then held to re-assess whether treatment recommendations changed due to discordances in molecular surrogate subtype between the different foci. RESULTS: Discordance in ER, PR, HER2, and Ki67 was found in 2.7%, 19.1%, 7.7%, and 16.9% of invasive foci, respectively. Discordance in the molecular surrogate subtypes was found in 48 of 180 (26.7%) patients, which resulted in therapy changes for 11 patients (6.1%). These patients received additional endocrine therapy (n = 2), chemotherapy (n = 3), and combined chemotherapy and trastuzumab (n = 6). CONCLUSION: Taken together, when assessing at least two tumor foci with IHC, regardless of shared morphology or tumor grade between the different foci, 6.1% of patients with IMBC were recommended additional adjuvant treatment. A pathologic assessment using IHC of all foci is therefore recommended to assist in individualized treatment decision making.

Pan-cancer analysis of genomic and transcriptomic data reveals the prognostic relevance of human proteasome genes in different cancer types.

BACKGROUND: The human proteasome gene family (PSM) consists of 49 genes that play a crucial role in cancer proteostasis. However, little is known about the effect of PSM gene expression and genetic alterations on clinical outcome in different cancer forms. METHODS: Here, we performed a comprehensive pan-cancer analysis of genetic alterations in PSM genes and the subsequent prognostic value of PSM expression using data from The Cancer Genome Atlas (TCGA) containing over 10,000 samples representing up to 33 different cancer types. External validation was performed using a breast cancer cohort and KM plotter with four cancer types. RESULTS: The PSM genetic alteration frequency was high in certain cancer types (e.g. 67%; esophageal adenocarcinoma), with DNA amplification being most common. Compared with normal tissue, most PSM genes were predominantly overexpressed in cancer. Survival analysis also established a relationship with PSM gene expression and adverse clinical outcome, where PSMA1 and PSMD11 expression were linked to more unfavorable prognosis in ≥ 30% of cancer types for both overall survival (OS) and relapse-free interval (PFI). Interestingly, PSMB5 gene expression was associated with OS (36%) and PFI (27%), and OS for PSMD2 (42%), especially when overexpressed. CONCLUSION: These findings indicate that several PSM genes may potentially be prognostic biomarkers and novel therapeutic targets for different cancer forms.

Pan-cancer analysis identifies BIRC5 as a prognostic biomarker.

BACKGROUND: The BIRC5 gene encodes for the Survivin protein, which is a member of the inhibitor of apoptosis family. Survivin is found in humans during fetal development, but generally not in adult cells thereafter. Previous studies have shown that Survivin is abundant in most cancer cells, thereby making it a promising target for anti-cancer drugs and a potential prognostic tool. METHODS: To assess genetic alterations and mutations in the BIRC5 gene as well as BIRC5 co-expression with other genes, genomic and transcriptomic data were downloaded via cBioPortal for approximately 9000 samples from The Cancer Genome Atlas (TCGA) representing 33 different cancer types and 11 pan-cancer organ systems, and validated using the ICGC Data Portal and COSMIC. TCGA BIRC5 RNA sequencing data from 33 different cancer types and matching normal tissue samples for 16 cancer types were downloaded from Broad GDAC Firehose and validated using breast cancer microarray data from our previous work and data sets from the GENT2 web-based tool. Survival data were analyzed with multivariable Cox proportional hazards regression analysis and validated using KM plotter for breast-, ovarian-, lung- and gastric cancer. RESULTS: Although genetic alterations in BIRC5 were not common in cancer, BIRC5 expression was significantly higher in cancer tissue compared to normal tissue in the 16 different cancer types. For 14/33 cancer types, higher BIRC5 expression was linked to worse overall survival (OS, 4/14 after adjusting for both age and tumor grade and 10/14 after adjusting only for age). Interestingly, higher BIRC5 expression was associated with better OS in lung squamous cell carcinoma and ovarian serous cystadenocarcinoma. Higher BIRC5 expression was also linked to shorter progressive-free interval (PFI) for 14/33 cancer types (4/14 after adjusting for both age and tumor grade and 10/14 after adjusting only for age). External validation showed that high BIRC5 expression was significantly associated with worse OS for breast-, lung-, and gastric cancer. CONCLUSIONS: Our findings suggest that BIRC5 overexpression is associated with the initiation and progression of several cancer types, and thereby a promising prognostic biomarker.

Age-related long-term response in rat thyroid tissue and plasma after internal low dose exposure to 131I.

131I is used clinically for therapy, and may be released during nuclear accidents. After the Chernobyl accident papillary thyroid carcinoma incidence increased in children, but not adults. The aims of this study were to compare 131I irradiation-dependent differences in RNA and protein expression in the thyroid and plasma of young and adult rats, and identify potential age-dependent biomarkers for 131I exposure. Twelve young (5 weeks) and twelve adult Sprague Dawley rats (17 weeks) were i.v. injected with 50 kBq 131I (absorbed dose to thyroid = 0.1 Gy), and sixteen unexposed age-matched rats were used as controls. The rats were killed 3-9 months after administration. Microarray analysis was performed using RNA from thyroid samples, while LC-MS/MS analysis was performed on proteins extracted from thyroid tissue and plasma. Canonical pathways, biological functions and upstream regulators were analysed for the identified transcripts and proteins. Distinct age-dependent differences in gene and protein expression were observed. Novel biomarkers for thyroid 131I exposure were identified: (PTH), age-dependent dose response (CA1, FTL1, PVALB (youngsters) and HSPB6 (adults)), thyroid function (Vegfb (adults)). Further validation using clinical samples are needed to explore the role of the identified biomarkers.

Trefoil factor family proteins as potential diagnostic markers for mucinous invasive ovarian carcinoma.

Introduction: Ovarian cancer (OC) is the leading cause of gynecological cancer-related death. Of the main OC histologic subtypes, invasive mucinous carcinomas (MC) account for only 3% of OC cases and are frequently associated with favorable prognosis. Nevertheless, MCs differ greatly from the other OC histotypes in clinical, pathological, and biological behavior. However, the origin and molecular pathogenesis of MC are not yet fully understood. Therefore, identification of novel diagnostic markers could potentially facilitate early diagnosis of OC, particularly the MC histotype, thereby leading to the development of histotype-specific treatment regimens and improved survival rates. Methods: In the present study, Trefoil factor gene family members (TFF1, TFF2 and TFF3) were identified as MC histotype-specific biomarkers using RNA sequencing (RNA-seq) data for 95 stage I-II OCs. The diagnostic value of TFF1, TFF2 and TFF3 was then evaluated by immunohistochemistry on 206 stage I-II OCs stratified by histotype (high-grade serous carcinoma [HGSC], endometrioid carcinoma [EC], clear cell carcinoma [CCC], and MC). Results: We showed significantly elevated intracytoplasmic protein expression levels for TFF1, TFF2 and TFF3 in MC samples, thereby revealing an association between expression of Trefoil factor gene family members and the MC histotype. Taken together, these findings suggest that the TFF proteins may play a pivotal role in tumor initiation and progression for the MC histotype. Conclusion: Taken together, these findings suggest that the TFF proteins may play a pivotal role in tumor initiation and progression for the MC histotype. Moreover, these novel histotype-specific diagnostic biomarkers may not only improve patient stratification of early-stage ovarian carcinomas but may also be candidates for the development of molecular targeted therapies.

Correction: Long-term transcriptomic and proteomic effects in Sprague Dawley rat thyroid and plasma after internal low dose 131I exposure.

[This corrects the article DOI: 10.1371/journal.pone.0244098.].

Genetic alterations associated with multiple primary malignancies.

Breast cancer (BC) patients are frequently at risk of developing other malignancies following treatment. Although studies have been conducted to elucidate the etiology of multiple primary malignancies (MPM) after a BC diagnosis, few studies have investigated other previously diagnosed primary malignancies (OPPM) before BC. Here, genome-wide profiling was used to identify potential driver DNA copy number alterations and somatic mutations that promote the development of MPMs. To compare the genomic profiles for two primary tumors (BC and OPPM) from the same patient, tumor pairs from 26 young women (≤50 years) diagnosed with one or more primary malignancies before breast cancer were analyzed. Malignant melanoma was the most frequent OPPM, followed by gynecologic- and hematologic malignancies. However, significantly more genetic alterations were detected in BC compared to the OPPM. BC also showed more genetic similarity as a group than the tumor pairs. Clonality testing showed that genetic alterations on chromosomes 1, 3, 16, and 19 were concordant in both tumors in 13 patients. TP53 mutations were also found to be prevalent in BC, MM, and HM. Although all samples were classified as genetically unstable, chromothripsis-like patterns were primarily observed in BC. Taken together, few recurrent genetic alterations were identified in both tumor pairs that can explain the development of MPMs in the same patient. However, larger studies are warranted to further investigate key driver mutations associated with MPMs.

Comparison of breast cancer surrogate subtyping using a closed-system RT-qPCR breast cancer assay and immunohistochemistry on 100 core needle biopsies with matching surgical specimens.

BACKGROUND: Routine clinical management of breast cancer (BC) currently depends on surrogate subtypes according to estrogen- (ER) and progesterone (PR) receptor, Ki-67, and HER2-status. However, there has been growing demand for reduced immunohistochemistry (IHC) turnaround times. The Xpert® Breast Cancer STRAT4* Assay (STRAT4)*, a standardized test for ESR1/PGR/MKi67/ERBB2 mRNA biomarker assessment, takes less than 2 hours. Here, we compared the concordance between the STRAT4 and IHC/SISH, thereby evaluating the effect of method choice on surrogate subtype assessment and adjuvant treatment decisions. METHODS: In total, 100 formalin-fixed paraffin-embedded core needle biopsy (CNB) samples and matching surgical specimens for 98 patients with primary invasive BC were evaluated using the STRAT4 assay. The concordance between STRAT4 and IHC was calculated for individual markers for the CNB and surgical specimens. In addition, we investigated whether changes in surrogate BC subtyping based on the STRAT4 results would change adjuvant treatment recommendations. RESULTS: The overall percent agreement (OPA) between STRAT4 and IHC/SISH ranged between 76 and 99% for the different biomarkers. Concordance for all four biomarkers in the surgical specimens and CNBs was only 66 and 57%, respectively. In total, 74% of surgical specimens were concordant for subtype, regardless of the method used. IHC- and STRAT4-based subtyping for the surgical specimen were shown to be discordant for 25/98 patients and 18/25 patients would theoretically have been recommended a different adjuvant treatment, primarily receiving more chemotherapy and trastuzumab. CONCLUSIONS: A comparison of data from IHC/in situ hybridization and STRAT4 demonstrated that subsequent changes in surrogate subtyping for the surgical specimen may theoretically result in more adjuvant treatment given, primarily with chemotherapy and trastuzumab.

Long-term transcriptomic and proteomic effects in Sprague Dawley rat thyroid and plasma after internal low dose 131I exposure.

BACKGROUND: Radioiodide (131I) is commonly used to treat thyroid cancer and hyperthyroidis.131I released during nuclear accidents, have resulted in increased incidence of thyroid cancer in children. Therefore, a better understanding of underlying cellular mechanisms behind 131I exposure is of great clinical and radiation protection interest. The aim of this work was to study the long-term dose-related effects of 131I exposure in thyroid tissue and plasma in young rats and identify potential biomarkers. MATERIALS AND METHODS: Male Sprague Dawley rats (5-week-old) were i.v. injected with 0.5, 5.0, 50 or 500 kBq 131I (Dthyroid ca 1-1000 mGy), and killed after nine months at which time the thyroid and blood samples were collected. Gene expression microarray analysis (thyroid samples) and LC-MS/MS analysis (thyroid and plasma samples) were performed to assess differential gene and protein expression profiles in treated and corresponding untreated control samples. Bioinformatics analyses were performed using the DAVID functional annotation tool and Ingenuity Pathway Analysis (IPA). The gene expression microarray data and LC-MS/MS data were validated using qRT-PCR and ELISA, respectively. RESULTS: Nine 131I exposure-related candidate biomarkers (transcripts: Afp and RT1-Bb, and proteins: ARF3, DLD, IKBKB, NONO, RAB6A, RPN2, and SLC25A5) were identified in thyroid tissue. Two dose-related protein candidate biomarkers were identified in thyroid (APRT and LDHA) and two in plasma (DSG4 and TGM3). Candidate biomarkers for thyroid function included the ACADL and SORBS2 (all activities), TPO and TG proteins (low activities). 131I exposure was shown to have a profound effect on metabolism, immune system, apoptosis and cell death. Furthermore, several signalling pathways essential for normal cellular function (actin cytoskeleton signalling, HGF signalling, NRF2-mediated oxidative stress, integrin signalling, calcium signalling) were also significantly regulated. CONCLUSION: Exposure-related and dose-related effects on gene and protein expression generated few expression patterns useful as biomarkers for thyroid function and cancer.

Identification of candidate genetic variants and altered protein expression in neural stem and mature neural cells support altered microtubule function to be an essential component in bipolar disorder.

Identification of causative genetic variants leading to the development of bipolar disorder (BD) could result in genetic tests that would facilitate diagnosis. A better understanding of affected genes and pathways is also necessary for targeting of genes that may improve treatment strategies. To date several susceptibility genes have been reported from genome-wide association studies (GWAS), but little is known about specific variants that affect disease development. Here, we performed quantitative proteomics and whole-genome sequencing (WGS). Quantitative proteomics revealed NLRP2 as the most significantly up-regulated protein in neural stem cells and mature neural cells obtained from BD-patient cell samples. These results are in concordance with our previously published transcriptome analysis. Furthermore, the levels of FEZ2 and CADM2 proteins were also significantly differentially expressed in BD compared to control derived cells. The levels of FEZ2 were significantly downregulated in neural stem cells (NSC) while CADM2 was significantly up-regulated in mature neuronal cell culture. Promising novel candidate mutations were identified in the ANK3, NEK3, NEK7, TUBB, ANKRD1, and BRD2 genes. A literature search of candidate variants and deregulated proteins revealed that there are several connections to microtubule function for the molecules putatively involved. Microtubule function in neurons is critical for axon structure and axonal transport. A functional dynamic microtubule is also needed for an advocate response to cellular and environmental stress. If microtubule dynamics is compromised by mutations, it could be followed by deregulated expression forming a possible explanation for the inherited vulnerability to stressful life events that have been proposed to trigger mood episodes in BD patients.

Male Breast Carcinoma after Irradiation and Long-Term Phenothiazine Exposure: A Case Report.

We present a young male patient with breast cancer having several risk factors likely acting in consort: irradiation of the breast for gynecomastia in adolescence and a life-long administration of phenothiazine for schizophrenia from the age of 16 years, with elevated serum prolactin level resulting in breast cancer development 24 years after irradiation.

Previously diagnosed multiple primary malignancies in patients with breast carcinoma in Western Sweden between 2007 and 2018.

PURPOSE: Multiple primary malignancies (MPMs) caused by breast cancer treatment are well described, but only few studies to date describe which other previous primary malignancies (OPPMs) occur before breast cancer. The purpose of the present study was to evaluate the prevalence of OPPMs in patients with breast cancer between 2007 and 2018 in Western Sweden. METHODS: Patient selection was performed using both pathology reports at Sahlgrenska University Hospital (Sweden) and the Swedish Cancer Registry. All newly diagnosed breast cancer patients were screened for presence of OPPM. RESULTS: In total, 8031 breast cancer patients were diagnosed at Sahlgrenska University Hospital between 2007 and 2018. The prevalence of breast cancer patients with OPPMs (n = 414) increased from on average 2.6% to 8.2% during this 12-year period and ranged from 17 to 59 patients annually. The most striking increase in prevalence was found among the gynecological tumors (endometrium and ovarian adenocarcinomas), malignant melanomas and gastrointestinal malignancies. These findings were validated using data of the Swedish Cancer Registry. CONCLUSIONS: The overall survival rates for cancer patients have improved tremendously during the past 40 years, in part due to individually tailored therapies and screening programs. Our study revealed an increasing trend of OPPMs in breast cancer patients.

Integrative genomics approach identifies molecular features associated with early-stage ovarian carcinoma histotypes.

Ovarian cancer comprises multiple subtypes (clear-cell (CCC), endometrioid (EC), high-grade serous (HGSC), low-grade serous (LGSC), and mucinous carcinomas (MC)) with differing molecular and clinical behavior. However, robust histotype-specific biomarkers for clinical use have yet to be identified. Here, we utilized a multi-omics approach to identify novel histotype-specific genetic markers associated with ovarian carcinoma histotypes (CCC, EC, HGSC, and MC) using DNA methylation, DNA copy number alteration and RNA sequencing data for 96 primary invasive early-stage (stage I and II) ovarian carcinomas. More specifically, the DNA methylation analysis revealed hypermethylation for CCC in comparison with the other histotypes. Moreover, copy number imbalances and novel chromothripsis-like rearrangements (n = 64) were identified in ovarian carcinoma, with the highest number of chromothripsis-like patterns in HGSC. For the 1000 most variable transcripts, underexpression was most prominent for all histotypes in comparison with normal ovarian samples. Overall, the integrative approach identified 46 putative oncogenes (overexpressed, hypomethylated and DNA gain) and three putative tumor suppressor genes (underexpressed, hypermethylated and DNA loss) when comparing the different histotypes. In conclusion, the current study provides novel insights into molecular features associated with early-stage ovarian carcinoma that may improve patient stratification and subclassification of the histotypes.

Optimization of cell viability assays to improve replicability and reproducibility of cancer drug sensitivity screens.

Cancer drug development has been riddled with high attrition rates, in part, due to poor reproducibility of preclinical models for drug discovery. Poor experimental design and lack of scientific transparency may cause experimental biases that in turn affect data quality, robustness and reproducibility. Here, we pinpoint sources of experimental variability in conventional 2D cell-based cancer drug screens to determine the effect of confounders on cell viability for MCF7 and HCC38 breast cancer cell lines treated with platinum agents (cisplatin and carboplatin) and a proteasome inhibitor (bortezomib). Variance component analysis demonstrated that variations in cell viability were primarily associated with the choice of pharmaceutical drug and cell line, and less likely to be due to the type of growth medium or assay incubation time. Furthermore, careful consideration should be given to different methods of storing diluted pharmaceutical drugs and use of DMSO controls due to the potential risk of evaporation and the subsequent effect on dose-response curves. Optimization of experimental parameters not only improved data quality substantially but also resulted in reproducible results for bortezomib- and cisplatin-treated HCC38, MCF7, MCF-10A, and MDA-MB-436 cells. Taken together, these findings indicate that replicability (the same analyst re-performs the same experiment multiple times) and reproducibility (different analysts perform the same experiment using different experimental conditions) for cell-based drug screens can be improved by identifying potential confounders and subsequent optimization of experimental parameters for each cell line.

Validation of Novel Prognostic Biomarkers for Early-Stage Clear-Cell, Endometrioid and Mucinous Ovarian Carcinomas Using Immunohistochemistry.

Early-stage (I and II) ovarian carcinoma patients generally have good prognosis. Yet, some patients die earlier than expected. Thus, it is important to stratify early-stage patients into risk groups to identify those in need of more aggressive treatment regimens. The prognostic value of 29 histotype-specific biomarkers identified using RNA sequencing was evaluated for early-stage clear-cell (CCC), endometrioid (EC) and mucinous (MC) ovarian carcinomas (n = 112) using immunohistochemistry on tissue microarrays. Biomarkers with prognostic significance were further evaluated in an external ovarian carcinoma data set using the web-based Kaplan-Meier plotter tool. Here, we provide evidence of aberrant protein expression patterns and prognostic significance of 17 novel histotype-specific prognostic biomarkers [10 for CCC (ARPC2, CCT5, GNB1, KCTD10, NUP155, RPL13A, RPL37, SETD3, SMYD2, TRIO), three for EC (CECR1, KIF26B, PIK3CA), and four for MC (CHEK1, FOXM1, KIF23, PARPBP)], suggesting biological heterogeneity within the histotypes. Combined predictive models comprising the protein expression status of the validated CCC, EC and MC biomarkers together with established clinical markers (age, stage, CA125, ploidy) improved the predictive power in comparison with models containing established clinical markers alone, further strengthening the importance of the biomarkers in ovarian carcinoma. Further, even improved predictive powers were demonstrated when combining these models with our previously identified prognostic biomarkers PITHD1 (CCC) and GPR158 (MC). Moreover, the proteins demonstrated improved risk prediction of CCC-, EC-, and MC-associated ovarian carcinoma survival. The novel histotype-specific prognostic biomarkers may not only improve prognostication and patient stratification of early-stage ovarian carcinomas, but may also guide future clinical therapy decisions.