Cancer Tumor Samples Research Articles

TAF1B depletion leads to apoptotic cell death by inducing nucleolar stress and activating p53-miR-101 circuit in hepatocellular carcinoma.

TAF1B (TATA Box Binding Protein (TBP)-Associated Factor) is an RNA polymerase regulating rDNA activity, stress response, and cell cycle. However, the function of TAF1B in the progression of hepatocellular carcinoma (HCC) is unknown. In this study, we intended to characterize the crucial role and molecular mechanisms of TAF1B in modulating nucleolar stress in HCC. We analyzed the differential expression and prognostic value of TAF1B in hepatocellular carcinoma based on The Cancer Genome Atlas (TCGA) database, tumor and paraneoplastic tissue samples from clinical hepatocellular carcinoma patients, and typical hepatocellular carcinoma. We detected cell proliferation and apoptosis by lentiviral knockdown of TAF1B expression levels in HepG2 and SMMC-7721 cells using clone formation, apoptosis, and Western blotting (WB) detection of apoptosis marker proteins. Simultaneously, we investigated the influence of TAF1B knockdown on the function of the pre-initiation complex (PIC) by WB, and co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP) assays verified the interaction between the complexes and the effect on rDNA activity. Immunofluorescence assays measured the expression of marker proteins of nucleolus stress, fluorescence in situ hybridization (FISH) assays checked the rDNA activity, and qRT-PCR assays tested the pre-rRNA levels. Regarding molecular mechanisms, we investigated the role of p53 and miR-101 in modulating nucleolar stress and apoptosis. Finally, the impact of TAF1B knockdown on tumor growth, apoptosis, and p53 expression was observed in xenograft tumors. We identified that TAF1B was highly expressed in hepatocellular carcinoma and associated with poor prognosis in HCC patients. TAF1B depletion modulated nucleolar stress and apoptosis in hepatocellular carcinoma cells through positive and negative feedback from p53-miR-101. RNA polymerase I transcription repression triggered post-transcriptional activation of miR-101 in a p53-dependent manner. In turn, miR-101 negatively feeds back through direct inhibition of the p53-mediated PARP pathway. These findings broaden our comprehension of the function of TAF1B-mediated nucleolar stress in hepatocellular carcinoma and may offer new biomarkers for exploring prospective therapeutic targets in HCC.

Cancer Stem Cell Markers-Clinical Relevance and Prognostic Value in High-Grade Serous Ovarian Cancer (HGSOC) Based on The Cancer Genome Atlas Analysis.

Cancer stem cells (CSCs) may contribute to an increased risk of recurrence in ovarian cancer (OC). Further research is needed to identify associations between CSC markers and OC patients' clinical outcomes with greater certainty. If they prove to be correct, in the future, the CSC markers can be used to help predict survival and indicate new therapeutic targets. This study aimed to determine the CSC markers at mRNA and protein levels and their association with clinical presentation, outcome, and risk of recurrence in HGSOC (High-Grade Serous Ovarian Cancer). TCGA (The Cancer Genome Atlas) database with 558 ovarian cancer tumor samples was used for the evaluation of 13 CSC markers (ALDH1A1, CD44, EPCAM, KIT, LGR5, NES, NOTCH3, POU5F1, PROM1, PTTG1, ROR1, SOX9, and THY1). Data on mRNA and protein levels assessed by microarray and mass spectrometry were retrieved from TCGA. Models to predict chemotherapy response and survival were built using multiple variables, including epidemiological data, expression levels, and machine learning methodology. ALDH1A1 and LGR5 mRNA expressions indicated a higher platinum sensitivity (p = 3.50 × 10-3; p = 0.01, respectively). POU5F1 mRNA expression marked platinum-resistant tumors (p = 9.43 × 10-3). CD44 and EPCAM mRNA expression correlated with longer overall survival (OS) (p = 0.043; p = 0.039, respectively). THY1 mRNA and protein levels were associated with worse OS (p = 0.019; p = 0.015, respectively). Disease-free survival (DFS) was positively affected by EPCAM (p = 0.004), LGR5 (p = 0.018), and CD44 (p = 0.012). In the multivariate model based on CSC marker expression, the high-risk group had 9.1 months longer median overall survival than the low-risk group (p < 0.001). ALDH1A1, CD44, EPCAM, LGR5, POU5F1, and THY1 levels in OC may be used as prognostic factors for the primary outcome and help predict the treatment response.

S100s and HMGB1 Crosstalk in Pancreatic Cancer Tumors.

Pancreatic cancer remains a disease that is very difficult to treat. S100 proteins are small calcium binding proteins with diverse intra- and extracellular functions that modulate different aspects of tumorigenesis, including tumor growth and metastasis. High mobility group box 1 (HMGB1) protein is a multifaceted protein that also actively influences the development and progression of tumors. In this study, we investigate the possible correlations, at the transcript level, between S100s and HMGB1 in pancreatic cancer. For this purpose, we calculated Pearson's correlations between the transcript levels of 13 cancer-related S100 genes and HMGB1 in a cDNA array containing 19 pancreatic cancer tumor samples, and in 8 human pancreatic cancer cell lines. Statistically significant positive correlations were found in 5.5% (5 out of 91) and 37.4% (34 of 91) of the possible S100/S100 or S100/HMGB1 pairs in cells and tumors, respectively. Our data suggest that many S100 proteins crosstalk in pancreatic tumors either with other members of the S100 family, or with HMGB1. These newly observed interdependencies may be used to further the characterization of pancreatic tumors based on S100 and HMGB1 transcription profiles.

Nuclear receptor co-repressor NCOR2 and its relation to GPER with prognostic impact in ovarian cancer.

The significance of the non-classical G-protein-coupled estrogen receptor (GPER) as positive or negative prognostic factor for ovarian cancer patients remains still controversial. Recent results indicate that an imbalance of both co-factors and co-repressors of nuclear receptors regulates ovarian carcinogenesis by altering the transcriptional activity through chromatin remodeling. The present study aims to investigate whether the expression of the nuclear co-repressor NCOR2 plays a role in GPER signaling which thereby could positively impact overall survival rates of ovarian cancer patients. NCOR2 expression was evaluated by immunohistochemistry in a cohort of 156 epithelial ovarian cancer (EOC) tumor samples and correlated with GPER expression. The correlation and differences in clinical and histopathological variables as well as their effect on prognosis were analyzed by Spearman's correlation, Kruskal-Wallis test and Kaplan-Meier estimates. Histologic subtypes were associated with different NCOR2 expression patterns. More specifically, serous and mucinous EOC demonstrated a higher NCOR2 expression (P = 0.008). In addition, high nuclear NCOR2 expression correlated significantly with high GPER expression (cc = 0.245, P = 0.008). A combined evaluation of both high NCOR2 (IRS > 6) and high GPER (IRS > 8) expression revealed an association of a significantly improved overall survival (median OS 50.9 versus 105.1months, P = 0.048). Our results support the hypothesis that nuclear co-repressors such as NCOR2 may influence the transcription of target genes in EOC such as GPER. Understanding the role of nuclear co-repressors on signaling pathways will allow a better understanding of the factors involved in prognosis and clinical outcome of EOC patients.

Actionable spontaneous antibody responses antagonize malignant progression in ovarian carcinoma

ObjectiveTo demonstrate that shared antibody responses in endometriosis and endometriosis-associated ovarian cancer spontaneously antagonize malignant progression and can be leveraged to develop future immunotherapies. MethodsB cells from cyopreserved clear cell ovarian carcinoma (CCC, n = 2), endometrioid ovarian carcinoma (EC, n = 2), and endometriomas (n = 2) were isolated, activated, and EBV-immortalized. Antibodies were purified from B cell supernatants and used for screening arrays containing most of the human proteome. Targets were prioritized based on accessibility (transmembrane or secreted proteins), expression in endometriosis and cancer, and concurrent IgA and IgG responses. We focused on antibodies targeting tumor-promoting syndecan binding protein (SDCBP) to demonstrate anti-tumor activity. Immunoblots and qPCR were performed to assess SDCBP expression in ovarian cancer and endometriosis cell lines and tumor samples. Recombinant IgG4 was generated using the variable heavy and light chains of dominant B cell receptors (BCRs) reacting against the extracellular domain of SDCBP, and used in in vivo studies in human CCC- and high-grade serous ovarian carcinoma (HGSOC)-bearing immunodeficient mice. ResultsNine accessible proteins detected by both IgA and IgG were identified in all samples - including SDCBP, which is expressed in ovarian carcinomas of multiple histologies. Administration of α-SDCBP IgG4 in OVCAR3 (HGSOC), TOV21G and RMG-I (CCC) tumor-bearing mice significantly decreased tumor volume compared to control irrelevant IgG4. ConclusionsSpontaneous antibody responses exert suboptimal but measurable immune pressure against malignant progression in ovarian carcinomas. Using tumor-derived antibodies for developing novel immunotherapeutics warrants further investigation.

Mutation Hotspots Found in Bladder Cancer Aid Prediction of Carcinogenic Risk in Normal Urothelium.

More than 80,000 new cases of bladder cancer are estimated to be diagnosed in 2023. However, the 5-year survival rate for bladder cancer has not changed in decades, highlighting the need for prevention. Numerous cancer-causing mutations are present in the urothelium long before signs of cancer arise. Mutation hotspots in cancer-driving genes were identified in non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC) tumor samples. Mutation burden within the hotspot regions was measured in normal urothelium with a low and high risk of cancer. A significant correlation was found between the mutation burden in normal urothelium and bladder cancer tissue within the hotspot regions. A combination of measured hotspot burden and personal risk factors was used to fit machine learning classification models. The efficacy of each model to differentiate between adjacent benign urothelium from bladder cancer patients and normal urothelium from healthy donors was measured. A random forest model using a combination of personal risk factors and mutations within MIBC hotspots yielded the highest AUC of 0.9286 for the prediction of high- vs. low-risk normal urothelium. Currently, there are no effective biomarkers to assess subclinical field disease and early carcinogenic progression in the bladder. Our findings demonstrate novel differences in mutation hotspots in NMIBC and MIBC and provide the first evidence for mutation hotspots to aid in the assessment of cancer risk in the normal urothelium. Early risk assessment and identification of patients at high risk of bladder cancer before the clinical presentation of the disease can pave the way for targeted personalized preventative therapy.

CircPVT1 promotes ER‐positive breast tumorigenesis and drug resistance by targeting ESR1 and MAVS

The molecular mechanisms underlying estrogen receptor (ER)‐positive breast carcinogenesis and endocrine therapy resistance remain incompletely understood. Here, we report that circPVT1, a circular RNA generated from the lncRNA PVT1, is highly expressed in ERα‐positive breast cancer cell lines and tumor samples and is functionally important in promoting ERα‐positive breast tumorigenesis and endocrine therapy resistance. CircPVT1 acts as a competing endogenous RNA (ceRNA) to sponge miR‐181a‐2‐3p, promoting the expression of ESR1 and downstream ERα‐target genes and breast cancer cell growth. Furthermore, circPVT1 directly interacts with MAVS protein to disrupt the RIGI–MAVS complex formation, inhibiting type I interferon (IFN) signaling pathway and anti‐tumor immunity. Anti‐sense oligonucleotide (ASO)‐targeting circPVT1 inhibits ERα‐positive breast cancer cell and tumor growth, re‐sensitizing tamoxifen‐resistant ERα‐positive breast cancer cells to tamoxifen treatment. Taken together, our data demonstrated that circPVT1 can work through both ceRNA and protein scaffolding mechanisms to promote cancer. Thus, circPVT1 may serve as a diagnostic biomarker and therapeutic target for ERα‐positive breast cancer in the clinic.

Immature natural killer cells promote progression of triple-negative breast cancer.

Natural killer (NK) cells are cytotoxic lymphocytes that accumulate within the tumor microenvironment and are generally considered to be antitumorigenic. Using single-cell RNA sequencing and functional analysis of multiple triple-negative breast cancer (TNBC) and basal tumor samples, we observed a unique subcluster of Socs3highCD11b-CD27- immature NK cells that were present only in TNBC samples. These tumor-infiltrating NK cells expressed a reduced cytotoxic granzyme signature and, in mice, were responsible for activating cancer stem cells through Wnt signaling. NK cell-mediated activation of these cancer stem cells subsequently enhanced tumor progression in mice, whereas depletion of NK cells or Wnt ligand secretion from NK cells by LGK-974 decreased tumor progression. In addition, NK cell depletion or inhibition of their function improved anti-programmed cell death ligand 1 (PD-L1) antibody or chemotherapy response in mice with TNBC. Furthermore, tumor samples from patients with TNBC and non-TNBC revealed that increased numbers of CD56bright NK cells were present in TNBC tumors and were correlated to poor overall survival in patients with TNBC. Together, our findings identify a population of protumorigenic NK cells that may be exploited for both diagnostic and therapeutic strategies to improve outcomes for patients with TNBC.

Living on the edge: how stromal cells restrict liver cancer invasion

The global incidence of primary liver cancer is increasing and currently ranks amongst the top-five causes of premature death from cancer annually. Like in many solid tumours, liver cancer cells are closely associated with stromal cells, known as non-parenchymal cells (NPCs), in the liver, including fibroblasts and endothelial cells, which normally support liver function and structure. Although single-cell RNA sequencing has begun to unpick the interactions between tumour cells and this non-epithelial microenvironment, these -omic approaches cannot mechanistically resolve how the non-parenchymal stroma interacts with tumour cells, nor can they resolve whether a fibroblast exerts differential outcomes on mutant versus healthy epithelial cells.In this issue of Disease Models & Mechanisms, Liqin Zhu and colleagues describe a novel in vitro microwell culture system to co-culture NPCs with either healthy hepatocytes or tumour cells of intrahepatic cholangiocarcinoma (iCCA), an aggressive type of primary liver cancer. When the authors co-cultured NPCs with primary hepatocytes, the NPCs supported hepatocyte spheroid growth, maintaining the phenotype of the healthy hepatocytes in culture. Interestingly, when cultured with mutant iCCA cells, NPCs supressed the growth of cancer spheroids, suggesting a divergent role of NPCs in tumour growth.To understand the role of these stromal cells in tumour growth further, the authors then co-cultured healthy hepatocytes and tumour cells in their microwell system and found that tumour cells invade and kill healthy hepatocytes. The addition of NPCs to this system significantly limited this process, and in vitro cancer cells were constrained, no longer killing healthy hepatocytes. Following on from this, the authors used an orthotopic transplantation mouse model of liver cancer and liver tumour samples from patients to show that activated myofibroblasts localised at the interface between tumour and normal tissue. Together, the data suggest that, in liver cancer, NPCs, in particular myofibroblasts, form a physical barrier that limits the ability of cancer cells to invade and damage normal tissue.Targeting the tumour microenvironment to limit disease progression is an attractive therapeutic prospect that could be agnostic to the underlying genetic and molecular heterogeneity seen in cancer. However, as NPCs have previously been shown to provide pro-proliferative signals in cancer and modify the intratumoral immune landscape, more research is required to understand the nuanced NPC-epithelial cell relationship in cancer. We should carefully consider the diverse and potentially conflicting roles of NPCs in tumour progression, and advanced in vitro systems, such as those developed by Zhu and colleagues, will help us decipher this.

Development of a Sensitive Digital Droplet PCR Screening Assay for the Detection of GPR126 Non-Coding Mutations in Bladder Cancer Urine Liquid Biopsies.

Recent whole-genome sequencing studies identified two novel recurrent mutations in the enhancer region of GPR126 in urothelial bladder cancer (UBC) tumor samples. This mutational hotspot is the second most common after the TERT promoter in UBC. The aim of the study was to develop a digital droplet PCR screening assay for the simultaneous detection of GPR126 mutations in a single tube. Its performance combined with TERT promoter mutation analysis was evaluated in urine of healthy volunteers (n = 50) and patients with cystitis (n = 22) and UBC (n = 70). The developed assay was validated using DNA constructs carrying the studied variants. None of the mutations were detected in control and cystitis group samples. GPR126 mutations were observed in the urine of 25/70 UBC patients (area under the ROC curve (AUC) of 0.679; mutant allele fraction (MAF) of 21.61 [8.30-44.52] %); TERT mutations-in 40/70 (AUC of 0.786; MAF = 28.29 [19.03-38.08] %); ≥1 mutation-in 47/70 (AUC of 0.836)). The simultaneous presence of GPR126 and TERT mutations was observed in 18/70 cases, with no difference in MAFs for the paired samples (31.96 [14.78-47.49] % vs. 27.13 [17.00-37.62] %, p = 0.349, respectively). The combined analysis of these common non-coding mutations in urine allows the sensitive and non-invasive detection of UBC.

Systemic structural analysis of alterations reveals a common structural basis of driver mutations in cancer.

A major effort in cancer research is to organize the complexities of the disease into fundamental traits. Despite conceptual progress in the last decades and the synthesis of hallmark features, no organizing principles governing cancer beyond cellular features exist. We analyzed experimentally determined structures harboring the most significant and prevalent driver missense mutations in human cancer, covering 73% (n = 168178) of theCatalog of Somatic Mutation in Cancer tumor samples(COSMIC). The results reveal that a single structural element-κ-helix (polyproline II helix)-lies at the core of driver point mutations, with significant enrichment in all major anatomical sites, suggesting that a small number of molecular traits are shared by most and perhaps all types of cancer. Thus, we uncovered the lowest possible level of organization at which carcinogenesis takes place at the protein level. This framework provides an initial scheme for a mechanistic understanding underlying the development of tumors and pinpoints key vulnerabilities.

Machine learning-based ovarian cancer prediction with XGboost and stochastic gradient boosting models

Ovarian cancer is one of the most common types of gynecological malignancies with its high mortality rate, silent and occult tumor growth, late onset of symptoms and diagnosis in advanced stages. Therefore, the need to develop new diagnostic techniques to predict the course of the disease and the prognosis of this malignancy has increased. In this study, ovarian cancer and benign ovarian tumor samples will be classified to create an accurate diagnostic predictive model using the machine learning method XGBoost and Stochastic Gradient Boosting and disease-related risk factors will be determined. This current study considered the open-access ovarian cancer and benign ovarian tumor samples data set. For this purpose, data from 349 patients were included. The data set was divided as 80:20 as a training and test dataset. XGBoost and Stochastic Gradient Boosting were constructed for the classification via five-fold cross-validation. Accuracy, balanced accuracy, sensitivity, specificity, positive predictive value, and negative predictive value performance metrics were evaluated for model performance. Among the performance criteria in the test stage obtained from the XGBoost model that has the best classification result; accuracy, balanced accuracy, sensitivity, specificity, positive predictive value, negative predictive value, and F1 score were obtained as 89.5%, 88.7%, 85.7%, 91.7%, 85.7%, 91.7%, and 85.7%, respectively. According to the variable importance obtained as a result of the model, the variables most associated with the diagnosis were CA72-4, HE4, LYM%, ALB, EO%, BUN, RBC, NEU, and MCV, respectively. The applied machine learning model successfully classified ovarian cancer and created a highly accurate diagnostic prediction model. The results from the study revealed effective parameters that can diagnose ovarian cancer with high accuracy. With the parameters determined as a result of the modeling, the clinician will be able to simplify and facilitate the decision-making process for the diagnosis of ovarian cancer.

Immune infiltration could predict the efficacy of short-term radiotherapy in patients with cervical cancer.

Radiotherapyis the main treatment forcervical cancer. It is usually applied alone or in combination with surgery and/or chemotherapy. To explore the association between immune microenvironment of cervical cancer and radiotherapy response, we collected 20 paired cervical cancer tumor samples before and after radiotherapy and partial clinical information. With paired-end RNA-seq, we quantified the immune infiltration and tumor purity of these samples, and obtained 6350 differentially expressed genes before and after radiotherapy. With the help of R language, the function enrichment analysis and 22 immune cells infiltration analysis were carried out. Moreover, we built a random forest model based on the immune microenvironment to predict the short-term efficacy of radiotherapy. We found that the effect of radiotherapy on the immune microenvironment of stage III and IV cervical cancer patients was weaker than that of stage I and II cervical cancer patients. Radiotherapy can significantly reduce the tumor purity and increase immune infiltration. The proportions of the immune infiltrating cells are predictive of the radiotherapy efficacy. In addition, the local mucositis caused by radiotherapy can improve the curative effect of radiotherapy.

Feasibility of a novel non-invasive swab technique for serial whole-exome sequencing of cervical tumors during chemoradiation therapy.

BackgroundClinically relevant genetic predictors of radiation response for cervical cancer are understudied due to the morbidity of repeat invasive biopsies required to obtain genetic material. Thus, we aimed to demonstrate the feasibility of a novel noninvasive cervical swab technique to (1) collect tumor DNA with adequate throughput to (2) perform whole-exome sequencing (WES) at serial time points over the course of chemoradiation therapy (CRT).MethodsCervical cancer tumor samples from patients undergoing chemoradiation were collected at baseline, at week 1, week 3, and at the completion of CRT (week 5) using a noninvasive swab-based biopsy technique. Swab samples were analyzed with whole-exome sequencing (WES) with mutation calling using a custom pipeline optimized for shallow whole-exome sequencing with low tumor purity (TP). Tumor mutation changes over the course of treatment were profiled.Results216 samples were collected and successfully sequenced for 70 patients (94% of total number of tumor samples collected). A total of 33 patients had a complete set of samples at all four time points. The mean mapping rate was 98% for all samples, and the mean target coverage was 180. Estimated TP was greater than 5% for all samples. Overall mutation frequency decreased during CRT but mapping rate and mean target coverage remained at >98% and >180 reads at week 5.ConclusionThis study demonstrates the feasibility and application of a noninvasive swab-based technique for WES analysis which may be applied to investigate dynamic tumor mutational changes during treatment to identify novel genes which confer radiation resistance.

A pan-cancer analysis of matrisome proteins reveals CTHRC1 and a related network as major ECM regulators across cancers.

The extracellular matrix in the tumour microenvironment can regulate cancer cell growth and progression. A pan-cancer analysis of TCGA data from 30 cancer types, identified the top 5% of matrisome genes with amplifications or deletions in their copy number, that affect their expression and cancer survival. A similar analysis of matrisome genes in individual cancers identified CTHRC1 to be significantly altered. CTHRC1, a regulator of collagen synthesis, was identified as the most prominently upregulated matrisome gene of interest across cancers. Differential gene expression analysis identified 19 genes whose expression is increased with CTHRC1. STRING analysis of these genes classified them as ‘extracellular’, involved most prominently in ECM organization and cell adhesion. KEGG analysis showed their involvement in ECM-receptor and growth factor signalling. Cytohubba analysis of these genes revealed 13 hub genes, of which MMP13, POSTN, SFRP4, ADAMTS16 and FNDC1 were significantly altered in their expression with CTHRC1 and seen to affect survival across cancers. This could in part be mediated by their overlapping roles in regulating ECM (collagen or fibronectin) expression and organisation. In breast cancer tumour samples CTHRC1 protein levels are significantly upregulated with POSTN and MMP13, further supporting the need to evaluate their crosstalk in cancers.

CYP1B1 Augments the Mesenchymal, Claudin-Low, and Chemoresistant Phenotypes of Triple-Negative Breast Cancer Cells.

Cytochrome P4501B1 (CYP1B1) is elevated in breast cancer. Studies indicate a relationship between CYP1B1 and aggressive cancer phenotypes. Here, we report on in vitro studies in triple-negative breast cancer cell lines, where knockdown (KD) of CYP1B1 was used to determine the influence of its expression on invasive cell phenotypes. CYP1B1 KD in MDA-MB-231 cells resulted in the loss of mesenchymal morphology, altered expression of epithelial–mesenchymal genes, and increased claudin (CLDN) RNA and protein. CYP1B1 KD cells had increased cell-to-cell contact and paracellular barrier function, a reduced rate of cell proliferation, abrogation of migratory and invasive activity, and diminished spheroid formation. Analysis of clinical breast cancer tumor samples revealed an association between tumors exhibiting higher CYP1B1 RNA levels and diminished overall and disease-free survival. Tumor expression of CYP1B1 was inversely associated with CLDN7 expression, and CYP1B1HI/CLDN7LOW identified patients with lower median survival. Cells with CYP1B1 KD had an enhanced chemosensitivity to paclitaxel, 5-fluorouracil, and cisplatin. Our findings that CYP1B1 KD can increase chemosensitivity points to therapeutic targeting of this enzyme. CYP1B1 inhibitors in combination with chemotherapeutic drugs may provide a novel targeted and effective approach to adjuvant or neoadjuvant therapy against certain forms of highly metastatic breast cancer.

Breaking satellite silence: human satellite II RNA expression in ovarian cancer.

Multiple cancer types demonstrate abnormal expression of repetitive RNA sequences as a form of epigenetic instability. There is growing interest in understanding the role of repetitive RNAs in cancer pathogenesis and immunogenicity and in their potential role as diagnostic or therapeutic biomarkers. In this issue of the JCI, Porter and colleagues report on satellite RNA in a subset of ovarian cancers. The authors found that high expression of human satellite (HSAT) repeats — but not other families of repeats — was associated with an immunosuppressive phenotype in ovarian cancer cell lines and tumor samples. Further induction of HSAT RNA levels in vitro, surprisingly, leads to innate immune activation, suggesting a potential therapeutic strategy. This work highlights the expanding role of repetitive RNAs in tumor biology and the need to better define specific classes of repetitive elements expressed in cancer — as well as their role in tumorigenesis, tumor immunity, and the host response to cancer.

Predictive value of the Leuven HRD test compared with Myriad myChoice PLUS on 468 ovarian cancer samples from the PAOLA-1/ENGOT-ov25 trial (LBA 6)

Objectives: The phase III PAOLA-1/ENGOT-ov25 trial (NCT02477644) evaluated first-line standard therapy including bevacizumab in advanced ovarian cancer with the addition of maintenance olaparib or placebo. Significant improved progression-free survival (PFS) was observed in homologous recombination deficient (HRD) tumors, with or without BRCA mutation (BRCAm), tested with Myriad myChoice PLUS (Myriad test) in contradiction to homologous recombination proficient (HRP) tumors, revealing the need for HRD testing in first line. As part of the ENGOT European HRD initiative, we developed the ‘Leuven’ HRD test. To the best of our knowledge, this is the first presentation of an alternative academic laboratory-developed HRD test compared with the Myriad test including an analysis of the predictive value for olaparib efficacy (PFS) in first-line ovarian cancer. Methods: The Leuven HRD test was first developed on ovarian cancer tumor samples of the biobank of the University Hospitals Leuven. Then, we analyzed formalin-fixed paraffin-embedded (FFPE) derived DNA from 468 available ovarian cancer samples of the PAOLA-1/ENGOT-ov25 trial. We performed capture-based targeted resequencing of ±90,000 genome-wide single nucleotide polymorphisms (SNPs) at ±40x coverage, and coding exons of BRCA1, BRCA2, RAD51C, RAD51D, PALB2, BLM, BARD1, BRIP1 and TP53 at ±400x coverage. All samples were analyzed using the Leuven HRD and the Myriad test. The BRCAm status, genomic instability score (GIS) and HRD status (comprising both the BRCAm and GIS status) were compared between both tests. The main objective was to compare the predictive value of both tests for predicting PFS in the olaparib versus placebo arm. The Hazard Ratio (HR) and associated 95% confidence interval (CI) were calculated with the use of a Cox proportional hazards model. Conclusions: A robust correlation between the Leuven HRD and Myriad myChoice PLUS test was observed. The Leuven HRD test showed in the PAOLA-1 trial a similar impact on PFS as the Myriad test. Subgroup analyses confirmed the PFS benefit with olaparib in patients with Leuven test BRCAm and HRD-positive/BRCAwt tumors. Objectives: The phase III PAOLA-1/ENGOT-ov25 trial (NCT02477644) evaluated first-line standard therapy including bevacizumab in advanced ovarian cancer with the addition of maintenance olaparib or placebo. Significant improved progression-free survival (PFS) was observed in homologous recombination deficient (HRD) tumors, with or without BRCA mutation (BRCAm), tested with Myriad myChoice PLUS (Myriad test) in contradiction to homologous recombination proficient (HRP) tumors, revealing the need for HRD testing in first line. As part of the ENGOT European HRD initiative, we developed the ‘Leuven’ HRD test. To the best of our knowledge, this is the first presentation of an alternative academic laboratory-developed HRD test compared with the Myriad test including an analysis of the predictive value for olaparib efficacy (PFS) in first-line ovarian cancer. Methods: The Leuven HRD test was first developed on ovarian cancer tumor samples of the biobank of the University Hospitals Leuven. Then, we analyzed formalin-fixed paraffin-embedded (FFPE) derived DNA from 468 available ovarian cancer samples of the PAOLA-1/ENGOT-ov25 trial. We performed capture-based targeted resequencing of ±90,000 genome-wide single nucleotide polymorphisms (SNPs) at ±40x coverage, and coding exons of BRCA1, BRCA2, RAD51C, RAD51D, PALB2, BLM, BARD1, BRIP1 and TP53 at ±400x coverage. All samples were analyzed using the Leuven HRD and the Myriad test. The BRCAm status, genomic instability score (GIS) and HRD status (comprising both the BRCAm and GIS status) were compared between both tests. The main objective was to compare the predictive value of both tests for predicting PFS in the olaparib versus placebo arm. The Hazard Ratio (HR) and associated 95% confidence interval (CI) were calculated with the use of a Cox proportional hazards model. Conclusions: A robust correlation between the Leuven HRD and Myriad myChoice PLUS test was observed. The Leuven HRD test showed in the PAOLA-1 trial a similar impact on PFS as the Myriad test. Subgroup analyses confirmed the PFS benefit with olaparib in patients with Leuven test BRCAm and HRD-positive/BRCAwt tumors.

Molecular analysis of endometrioid and non-endometrioid uterine cancer and response to treatment (246)

<b>Objectives:</b> Carboplatin and Paclitaxel are the recommended first-line regimens for uterine cancer patients requiring systemic treatment. In light of promising new alternative systemic therapies, we aimed to identify biomarkers that may predict response to carboplatin. <b>Methods:</b> Uterine cancer tumor samples were analyzed using next-generation sequencing (NGS: NextSeq, 592 Genes) and immunohistochemistry (IHC) (Caris Life Sciences, Phoenix, AZ). Real-world overall survival (OS) was obtained from insurance claims data, and Kaplan-Meier estimates were calculated for molecularly defined patient cohorts from the first treatment of carboplatin to the last day of contact. The median survival of carboplatin-associated survival (CAS) was determined after the first carboplatin treatment. Two cohorts were created and compared: <i>1)</i> OS after carboplatin-based treatment that was less than the median CAS of the whole cohort (Non-Responders, "NR"); and <i>2)</i> OS after carboplatin-based treatment that was longer than the median CAS (Responders, "R"). NGS and IHC results of the tumor samples of these cohorts were compared. Statistical significance was calculated using Mann-Whitney U and Chi-square test and adjusted for multiple comparisons (adjusted p-value = q-value). <b>Results:</b> Total 2436 uterine cancer patients who received carboplatin treatment with molecular data were included in this analysis: 578 (23.7%) were of endometrioid histology, and 1051 (43.1%) serous, carcinosarcoma, or clear cell histology. Irrespective of histology, 20 genes were significantly altered (q<0.05) between R and NR, including genes related to the pathways involved in Base/Nucleotide Excision Repair, Cell Cycle Control, Chromatin Remodeling, RTK RAS, HR, PI3K, VEGF, and WNT signaling (Table 1). Of note, <i>KMT2C,</i> a histone methyltransferase, was more frequently mutated in Rs compared to NRs (8.44% vs 3.73% mutated, p=0.01). But <i>HOOK3</i> and <i>NSD3</i> were more frequently amplified in NRs compared to Rs (1.19% vs 0.12%, q=0.047; 2.14% vs 0.73%, q=0.11). <i>NSD3,</i> a gene involved in the regulation of tumorigenesis, was amplified in 1.07% of patients and was associated with a median difference in survival of 661 fewer days (HR: 2.75, 95% CI: 1.70-4.45, p<0.00001). Among endometrioid and serous uterine histology, ER positivity was seen significantly more frequently in Rs compared to NRs (83% vs 66.2%, q=0.03; 60.1% vs 45.8%, q=0.03). However, <i>CCNE1</i> amplification was less frequent in Rs compared to NRs (4.80% vs 16.4%, q=0.01). There were no molecular differences seen between Rs and NRs in carcinosarcoma and clear cell histologies treated with carboplatin. <b>Conclusions:</b> There are significant differences in the molecular profiles of endometrial cancers that have longer median CAS compared to those with shorter median CAS. Further investigation into biomarkers that may predict response to carboplatin-based therapy in endometrial cancer is needed.

The Potential Tumor-Suppressor DHRS7 Inversely Correlates with EGFR Expression in Prostate Cancer Cells and Tumor Samples.

Simple SummaryProstate cancer is one of the most common malignancies in men. Current therapies are initially effective but resistance often develops, leading to tumor recurrence and death. Further research on new players, mechanisms involved in prostate cancer, and therapy resistance is needed. We studied the role of DHRS7, a potential tumor suppressor with currently unknown physiological function, in prostate cancer cells using proteome and gene expression analyses. Despite the fact that DHRS7 can inactivate 5α-dihydrotestosterone, its effect on prostate cancer cells seems to be unrelated to androgen metabolism. When comparing three widely studied prostate cancer cell lines, we observed a negative correlation between DHRS7 and EGFR expression. DHRS7 knockdown enhanced EGFR expression, while knockdown of EGFR tended to increase DHRS7 expression. Importantly, DHRS7 expression negatively correlates with EGFR expression and positively with survival rates in prostate cancer patients. This study suggests a tumor-suppressor role for DHRS7 by modulating EGFR expression in prostate cancer.Prostate cancer (PCa), one of the most common malignancies in men, typically responds to initial treatment, but resistance to therapy often leads to metastases and death. The dehydrogenase/reductase 7 (DHRS7, SDR34C1) is an “orphan” enzyme without known physiological function. DHRS7 was previously found to be decreased in higher-stage PCa, and siRNA-mediated knockdown increased the aggressiveness of LNCaP cells. To further explore the role of DHRS7 in PCa, we analyzed the proteome of LNCaP cells following DHRS7 knockdown to assess potentially altered pathways. Although DHRS7 is able to inactivate 5α-dihydrotestosterone, DHRS7 knockdown did not affect androgen receptor (AR) target gene expression, and its effect on PCa cells seems to be androgen-independent. Importantly, proteome analyses revealed increased expression of epidermal growth factor receptor (EGFR), which was confirmed by RT-qPCR and Western blotting. Comparison of AR-positive LNCaP with AR-negative PC-3 and DU145 PCa cell lines revealed a negative correlation between DHRS7 and EGFR expression. Conversely, EGFR knockdown enhanced DHRS7 expression in these cells. Importantly, analysis of patient samples revealed a negative correlation between DHRS7 and EGFR expression, both at the mRNA and protein levels, and DHRS7 expression correlated positively with patient survival rates. These results suggest a protective role for DHRS7 in PCa.