Single-cell and bulk RNA sequencing reveal ligands and receptors associated with worse overall survival in serous ovarian cancer.

BACKGROUND: Serous ovarian carcinoma is the most frequent histological subgroup of ovarian cancer and the leading cause of death among gynecologic tumors. The tumor microenvironment and cancer-associated fibroblasts (CAFs) have a critical role in the origin and progression of cancer. We comprehensively characterized the crosstalk between CAFs and ovarian cancer cells from malignant fluids to identify specific ligands and receptors mediating intercellular communications and disrupted pathways related to prognosis and therapy response. METHODS: Malignant fluids of serous ovarian cancer, including tumor-derived organoids, CAFs-enriched (eCAFs), and malignant effusion cells (no cultured) paired with normal ovarian tissues, were explored by RNA-sequencing. These data were integrated with single-cell RNA-sequencing data of ascites from ovarian cancer patients. The most relevant ligand and receptor interactions were used to identify differentially expressed genes with prognostic values in ovarian cancer. RESULTS: CAF ligands and epithelial cancer cell receptors were enriched for PI3K-AKT, focal adhesion, and epithelial-mesenchymal transition signaling pathways. Collagens, MIF, MDK, APP, and laminin were detected as the most significant signaling, and the top ligand-receptor interactions THBS2/THBS3 (CAFs)-CD47 (cancer cells), MDK (CAFs)-NCL/SDC2/SDC4 (cancer cells) as potential therapeutic targets. Interestingly, 34 genes encoding receptors and ligands of the PI3K pathway were associated with the outcome, response to treatment, and overall survival in ovarian cancer. Up-regulated genes from this list consistently predicted a worse overall survival (hazard ratio > 1.0 and log-rank P < 0.05) in two independent validation cohorts. CONCLUSIONS: This study describes critical signaling pathways, ligands, and receptors involved in the communication between CAFs and cancer cells that have prognostic and therapeutic significance in ovarian cancer. Video abstract.

Penile Cancer-Derived Cells Molecularly Characterized as Models to Guide Targeted Therapies.

Penile cancer (PeCa) is a common disease in poor and developing countries, showing high morbidity rates. Despite the recent progress in understanding the molecular events involved in PeCa, the lack of well-characterized in vitro models precludes new advances in anticancer drug development. Here we describe the establishment of five human primary penile cancer-derived cell cultures, including two epithelial and three cancer-associated fibroblast (CAF) cells. Using high-throughput genomic approaches, we found that the epithelial PeCa derived- cells recapitulate the molecular alterations of their primary tumors and present the same deregulated signaling pathways. The differentially expressed genes and proteins identified are components of key oncogenic pathways, including EGFR and PI3K/AKT/mTOR. We showed that epithelial PeCa derived cells presented a good response to cisplatin, a common therapeutic approach used in PeCa patients. The growth of a PeCa-derived cell overexpressing EGFR was inhibited by EGFR inhibitors (cetuximab, gefitinib, and erlotinib). We also identified CAF signature markers in three PeCa-derived cells with fibroblast-like morphology, indicating that those cells are suitable models for PeCa microenvironment studies. We thus demonstrate the utility of PeCa cell models to dissect mechanisms that promote penile carcinogenesis, which are useful models to evaluate therapeutic approaches for the disease.

Clinical and Genetic Investigations of 109 Index Patients With Dilated Cardiomyopathy and 445 of Their Relatives.

BACKGROUND: It was the aim to investigate the frequency and genetic basis of dilated cardiomyopathy (DCM) among relatives of index patients with unexplained heart failure at a tertiary referral center. METHODS: Clinical investigations were performed in 109 DCM index patients and 445 of their relatives. All index patients underwent genetic investigations of 76 disease-associated DCM genes. A family history of DCM occurred in 11% (n=12) while clinical investigations identified familial DCM in a total of 32% (n=35). One-fifth of all relatives (n=95) had DCM of whom 60% (n=57) had symptoms of heart failure at diagnosis, whereas 40% (n=38) were asymptomatic. Symptomatic relatives had a shorter event-free survival than asymptomatic DCM relatives (P<0.001). RESULTS: Genetic investigations identified 43 pathogenic (n=27) or likely pathogenic (n=16) variants according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology criteria. Forty-four percent (n=48/109) of index patients carried a pathogenic/likely pathogenic variant of whom 36% (n=27/74) had sporadic DCM, whereas 60% (21/35) were familial cases. Thirteen of the pathogenic/likely pathogenic variants were also present in ≥7 affected individuals and thereby considered to be of sufficient high confidence for use in predictive genetic testing. CONCLUSIONS: A family history of DCM identified only 34% (n=12/35) of hereditary DCM, whereas systematic clinical screening identified the remaining 66% (n=23) of DCM families. This emphasized the importance of clinical investigations to identify familial DCM. The high number of pathogenic/likely pathogenic variants identified in familial DCM provides a firm basis for offering genetic investigations in affected families. This should also be considered in sporadic cases since adequate family evaluation may not always be possible and the results of the genetic investigations may carry prognostic information with an impact on individual management.

Germline Mutation in MUS81 Resulting in Impaired Protein Stability is Associated with Familial Breast and Thyroid Cancer.

Multiple primary thyroid cancer (TC) and breast cancer (BC) are commonly diagnosed, and the lifetime risk for these cancers is increased in patients with a positive family history of both TC and BC. Although this phenotype is partially explained by TP53 or PTEN mutations, a significant number of patients are negative for these alterations. We judiciously recruited patients diagnosed with BC and/or TC having a family history of these tumors and assessed their whole-exome sequencing. After variant prioritization, we selected MUS81 c.1292G>A (p.R431H) for further investigation. This variant was genotyped in a healthy population and sporadic BC/TC tissues and investigated at the protein level and cellular models. MUS81 c.1292G>A was the most frequent variant (25%) and the strongest candidate due to its function of double-strand break repair. This variant was confirmed in four relatives from two families. MUS81 p.R431H protein exhibited lower expression levels in tumors from patients positive for the germline variant, compared with wild-type BC, and normal breast and thyroid tissues. Using cell line models, we showed that c.1292G>A induced protein instability and affected DNA damage response. We suggest that MUS81 is a novel candidate involved in familial BC/TC based on its low frequency in healthy individuals and proven effect in protein stability.

Phenotypic presentations of Hajdu-Cheney syndrome according to age - 5 distinct clinical presentations.

We present five Danish individuals with Hajdu-Cheney syndrome (HJCYS) (OMIM #102500), a rare multisystem skeletal disorder with distinctive facies, generalised osteoporosis and progressive focal bone destruction. In four cases positive genetic screening of exon 34 of NOTCH2 supported the clinical diagnosis; in one of these cases, mosaicism was demonstrated, which, to our knowledge, has not previously been reported. In one case no genetic testing was performed since the phenotype was definite, and the diagnosis in the mother was genetically confirmed. The age of the patients differs widely from ten to 57 years, allowing a natural history description of the phenotype associated with this ultra-rare condition. The evolution of the condition is most apparent in the incremental bone loss leading to osteoporosis and the acro-osteolysis, both of which contribute significantly to disease burden.

Pathogenic RBM20-Variants Are Associated With a Severe Disease Expression in Male Patients With Dilated Cardiomyopathy.

Background As pathogenic variants in the gene for RBM20 appear with a frequency of 6% among Danish patients with dilated cardiomyopathy (DCM), it was the aim to investigate the associated disease expression in affected families. Methods and Results Clinical investigations were routinely performed in DCM index-patients and their relatives. In addition, ≥76 recognized and likely DCM-genes were investigated. DNA-sequence-variants within RBM20 were considered suitable for genetic testing when they fulfilled the criteria of (1) being pathogenic according to the American College of Medical Genetics and Genomics-classification, (2) appeared with an allele frequency of <1:10.000, and (3) segregated with DCM in ≥7 affected individuals. A total of 80 individuals from 15 families carried 5 different pathogenic RBM20-variants considered suitable for genetic testing. The penetrance was 66% (53/80) and age-dependent. Males were both significantly younger and had lower ejection fraction at diagnosis than females (age, 29±11 versus 48±12 years; P<0.01; ejection fraction, 29±13% versus 38±9%; P<0.01). Furthermore, 11 of 31 affected males needed a cardiac transplant while none of 22 affected females required this treatment ( P<0.001). Thirty percent of RBM20-carriers with DCM died suddenly or experienced severe ventricular arrhythmias although no adverse events were identified among healthy RBM20-carriers with a normal cardiac investigation. The event-free survival of male RBM20-carriers was significantly shorter compared with female carriers ( P<0.001). Conclusions The disease expression associated with pathogenic RBM20-variants was severe especially in males. The findings of the current study suggested that close clinical follow-up of RBM20-carriers is important which may ensure early detection of disease development and thereby improve management.

Post-mortem testing; germline BRCA1/2 variant detection using archival FFPE non-tumor tissue. A new paradigm in genetic counseling.

Accurate estimation of cancer risk in HBOC families often requires BRCA1/2 testing, but this may be impossible in deceased family members. Previous, testing archival formalin-fixed, paraffin-embedded (FFPE) tissue for germline BRCA1/2 variants was unsuccessful, except for the Jewish founder mutations. A high-throughput method to systematically test for variants in all coding regions of BRCA1/2 in archival FFPE samples of non-tumor tissue is described, using HaloPlex target enrichment and next-generation sequencing. In a validation study, correct identification of variants or wild-type was possible in 25 out of 30 (83%) FFPE samples (age range 1-14 years), with a known variant status in BRCA1/2. No false positive was found. Unsuccessful identification was due to highly degraded DNA or presence of large intragenic deletions. In clinical use, a total of 201 FFPE samples (aged 0-43 years) were processed. Thirty-six samples were rejected because of highly degraded DNA or failed library preparation. Fifteen samples were investigated to search for a known variant. In the remaining 150 samples (aged 0-38 years), three variants known to affect function and one variant likely to affect function in BRCA1, six variants known to affect function and one variant likely to affect function in BRCA2, as well as four variants of unknown significance (VUS) in BRCA1 and three VUS in BRCA2 were discovered. It is now possible to test for germline BRCA1/2 variants in deceased persons, using archival FFPE samples from non-tumor tissue. Accurate genetic counseling is achievable in families where variant testing would otherwise be impossible.

Browning of human adipocytes requires KLF11 and reprogramming of PPARγ superenhancers.

Long-term exposure to peroxisome proliferator-activated receptor γ (PPARγ) agonists such as rosiglitazone induces browning of rodent and human adipocytes; however, the transcriptional mechanisms governing this phenotypic switch in adipocytes are largely unknown. Here we show that rosiglitazone-induced browning of human adipocytes activates a comprehensive gene program that leads to increased mitochondrial oxidative capacity. Once induced, this gene program and oxidative capacity are maintained independently of rosiglitazone, suggesting that additional browning factors are activated. Browning triggers reprogramming of PPARγ binding, leading to the formation of PPARγ "superenhancers" that are selective for brown-in-white (brite) adipocytes. These are highly associated with key brite-selective genes. Based on such an association, we identified an evolutionarily conserved metabolic regulator, Kruppel-like factor 11 (KLF11), as a novel browning transcription factor in human adipocytes that is required for rosiglitazone-induced browning, including the increase in mitochondrial oxidative capacity. KLF11 is directly induced by PPARγ and appears to cooperate with PPARγ in a feed-forward manner to activate and maintain the brite-selective gene program.