Single-Cell RNA Sequencing of Ovarian Cancer: Promises and Challenges.

Ovarian cancer remains the leading cause of death from gynecologic malignancy in the Western world. Tumors are comprised of heterogeneous populations of various cancer, immune, and stromal cells; it is hypothesized that rare cancer stem cells within these subpopulations lead to disease recurrence and treatment resistance. Technological advances now allow for the analysis of tumor genomes and transcriptomes at the single-cell level, which provides the resolution to potentially identify these rare cancer stem cells within the larger tumor.In this chapter, we review the evolution of next-generation RNA sequencing techniques, the methodology of single-cell isolation and sequencing, sequencing data analysis, and the potential applications in ovarian cancer. We also summarize the current published work using single-cell sequencing in ovarian cancer.By utilizing this novel technique to characterize the gene expression of rare subpopulations, new targets and treatment pathways may be identified in ovarian cancer to change treatment paradigms.

Multiomic Analysis of Subtype Evolution and Heterogeneity in High-Grade Serous Ovarian Carcinoma.

Multiple studies have identified transcriptome subtypes of high-grade serous ovarian carcinoma (HGSOC), but their interpretation and translation are complicated by tumor evolution and polyclonality accompanied by extensive accumulation of somatic aberrations, varying cell type admixtures, and different tissues of origin. In this study, we examined the chronology of HGSOC subtype evolution in the context of these factors using a novel integrative analysis of absolute copy-number analysis and gene expression in The Cancer Genome Atlas complemented by single-cell analysis of six independent tumors. Tumor purity, ploidy, and subclonality were reliably inferred from different genomic platforms, and these characteristics displayed marked differences between subtypes. Genomic lesions associated with HGSOC subtypes tended to be subclonal, implying subtype divergence at later stages of tumor evolution. Subclonality of recurrent HGSOC alterations was evident for proliferative tumors, characterized by extreme genomic instability, absence of immune infiltration, and greater patient age. In contrast, differentiated tumors were characterized by largely intact genome integrity, high immune infiltration, and younger patient age. Single-cell sequencing of 42,000 tumor cells revealed widespread heterogeneity in tumor cell type composition that drove bulk subtypes but demonstrated a lack of intrinsic subtypes among tumor epithelial cells. Our findings prompt the dismissal of discrete transcriptome subtypes for HGSOC and replacement by a more realistic model of continuous tumor development that includes mixtures of subclones, accumulation of somatic aberrations, infiltration of immune and stromal cells in proportions correlated with tumor stage and tissue of origin, and evolution between properties previously associated with discrete subtypes. SIGNIFICANCE: This study infers whether transcriptome-based groupings of tumors differentiate early in carcinogenesis and are, therefore, appropriate targets for therapy and demonstrates that this is not the case for HGSOC.

Incisional carcinoma of Mullerian Origin: A case report and review of literature.

Primary incisional carcinoma (PIC) is a rare, delayed complication of surgery, usually attributed to the malignant transformation of endometriosis. We report a case of incisional carcinoma with nodal metastases in a 55-year-old woman, 18 years after cesarean section. She underwent extirpative surgery, including hysterectomy and bilateral salpingo-oophorectomy, without intraperitoneal disease identifed. Adjuvant treatment included sandwiched platinum-based chemotherapy (carboplatin and paclitaxel) and radiation. She remains disease-free 8 months after completing therapy. We identified 46 additional reported cases. Of these, >90% had undergone an "endometrium-exposing" surgery, most commonly cesarean section; while no cases followed adnexal-only surgery. The median time between antecedent surgery and presentation was 18 years. At presentation, tumors were often large (median 8 cm), and symptomatic with pain (63%) and/or mass (26%). Serum CA125 levels were commonly, albeit slightly, elevated (median 57U/ml (IQR 22-96, Range 6-1690)). Lymph node metastases were common (35%), with most following a vulvar-type spread pattern (inguinal first). Most patients (63%) were treated with chemotherapy +/- radiation. Approximately 50% of patients recurred promptly (median < 6 months), but long-term survival was reported following combined chemotherapy/radiation. Lymph node metastases portended a shorter disease-free interval, with 73% of cases recurring (median 5 months) despite chemotherapy-based treatment. These data suggest that some incisional carcinomas may result from displacement of healthy endometrium followed by delayed malignant transformation. Chemotherapy-only and radiation-only treatments are attended by modest prognosis. Taken together, these data suggest there is both need and potential avenues for improved prevention, detection, and treatment of this condition.

Targeting Mitochondria for Treatment of Chemoresistant Ovarian Cancer.

Ovarian cancer is the leading cause of death from gynecologic malignancy in the Western world. This is due, in part, to the fact that despite standard treatment of surgery and platinum/paclitaxel most patients recur with ultimately chemoresistant disease. Ovarian cancer is a unique form of solid tumor that develops, metastasizes and recurs in the same space, the abdominal cavity, which becomes a unique microenvironment characterized by ascites, hypoxia and low glucose levels. It is under these conditions that cancer cells adapt and switch to mitochondrial respiration, which becomes crucial to their survival, and therefore an ideal metabolic target for chemoresistant ovarian cancer. Importantly, independent of microenvironmental factors, mitochondria spatial redistribution has been associated to both tumor metastasis and chemoresistance in ovarian cancer while specific sets of genetic mutations have been shown to cause aberrant dependence on mitochondrial pathways in the most aggressive ovarian cancer subtypes. In this review we summarize on targeting mitochondria for treatment of chemoresistant ovarian cancer and current state of understanding of the role of mitochondria respiration in ovarian cancer. We feel this is an important and timely topic given that ovarian cancer remains the deadliest of the gynecological diseases, and that the mitochondrial pathway has recently emerged as critical in sustaining solid tumor progression.

Molecular characterization of endometrial cancer and therapeutic implications.

PURPOSE OF REVIEW: The present article reviews genomic subtyping of endometrial carcinoma and new molecular markers with therapeutic and prognostic implications. RECENT FINDINGS: Endometrial cancer has historically been classified through histology into endometrioid (type 1) and nonendometrioid (type II, mainly serous) subtypes. Molecular classification through genomic analysis now allows for a major advance in characterization; four distinct subgroups have been identified: polymerase ε (POLE) ultramutated, microsatellite unstable, copy number low/microsatellite stable, and copy number high/'serous-like'. These subtypes have prognostic implications and may aid in the identification of early-stage patients who are at high risk for recurrence. Through analysis of surrogate markers (POLE, MSI, and p53) and other validated molecular alterations (L1CAM), it may be possible to obtain an integrated molecular risk profile. Ongoing studies are utilizing this risk profile in order to identify patients who may benefit from additional treatment for early-stage disease. SUMMARY: Molecular characterization of endometrial cancer into subgroups has prognostic and therapeutic implications. Further development of an integrated molecular risk profile may identify patients who could benefit from additional treatment because of a higher risk of recurrence.

Single-cell sequencing in ovarian cancer: a new frontier in precision medicine.

PURPOSE OF REVIEW: This article discusses the advances, applications and challenges of using single-cell RNA sequencing data in guiding treatment decisions for ovarian cancer. RECENT FINDINGS: Genetic heterogeneity is a hallmark of ovarian cancer biology and underlies treatment resistance. Defining the different cell types present within a single ovarian cancer is difficult, but could ultimately lead to improvements in diagnosis and treatment. Next-generation sequencing technologies have rapidly increased our understanding of the molecular landscape of epithelial ovarian cancers, but the majority of these studies are conducted on bulk samples, resulting in data that represents an 'average' of all cells present. Single-cell sequencing provides a means to characterize heterogeneity with a tumor tissue in ovarian cancer patients and opens up opportunity to determine key molecular properties that influence clinical outcomes, including prognosis and treatment response. SUMMARY: Single-cell sequencing provides a powerful tool in improving our understanding of tumor cell heterogeneity for the purpose of informing personalized cancer treatment.

RNA Sequencing of Carboplatin- and Paclitaxel-Resistant Endometrial Cancer Cells Reveals New Stratification Markers and Molecular Targets for Cancer Treatment.

Despite advances in surgical technique and adjuvant treatment, endometrial cancer has recently seen an increase in incidence and mortality in the USA. The majority of endometrial cancers can be cured by surgery alone or in combination with adjuvant chemo- or radiotherapy; however, a subset of patients experience recurrence for reasons that remain unclear. Recurrence is associated with chemoresistance to carboplatin and paclitaxel and consequentially, high mortality. Understanding the pathways involved in endometrial cancer chemoresistance is paramount for the identification of biomarkers and novel molecular targets for this disease. Here, we generated the first matched pairs of carboplatin-sensitive/carboplatin-resistant and paclitaxel-sensitive/paclitaxel-resistant endometrial cancer cells and subjected them to bulk RNA sequencing analysis. We found that 45 genes are commonly upregulated in carboplatin- and paclitaxel-resistant cells as compared to controls. Of these, the leukemia inhibitory factor, (LIF), the protein tyrosine phosphatase type IVA, member 3 (PTP4A3), and the transforming growth factor beta 1 (TGFB1) showed a highly significant correlation between expression level and endometrial cancer overall survival (OS) and can stratify the 545 endometrial cancer patients in the TCGA cohort into a high-risk and low-risk-cohorts. Additionally, four genes within the 45 upregulated chemoresistance-associated genes are ADAMTS5, MICAL2, STAT5A, and PTP4A3 codes for proteins for which small-molecule inhibitors already exist. We identified these proteins as molecular targets for chemoresistant endometrial cancer and showed that treatment with their correspondent inhibitors effectively killed otherwise chemoresistant cells. Collectively, these findings underline the utility of matched pair of chemosensitive and chemoresistant cancer cells to identify markers for endometrial cancer risk stratification and to serve as a pharmacogenomics model for identification of alternative chemotherapy approaches for treatment of patients with recurrent disease.

Anakinra use during pregnancy in patients with cryopyrin-associated periodic syndromes (CAPS).

Objective: To describe the pregnancy course and outcome, and use of anakinra, a recombinant selective IL-1 receptor blocker, during pregnancy in patients with cryopyrin-associated periodic syndromes (CAPS), including familial cold auto-inflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS) and neonatal onset multi-system inflammatory disease (NOMID). Methods: Women currently enrolled in natural history protocols (NCT00059748, and/or NCT00069329 under IND) who have been pregnant were included. Subjects underwent a structured, standardized interview with regards to maternal health, pregnancy and fetal outcomes. Medical records were reviewed. Results: Nine women (four with FCAS, one with MWS and four with NOMID) reported one to four pregnancies, each resulting in a total of fifteen FCAS, three MWS, and six NOMID pregnancies. Six births from FCAS mothers and three births from NOMID mothers occurred while patients were receiving anakinra. If a woman became pregnant while taking anakinra, the pre-pregnancy anakinra dose was continued. Anakinra dose was increased during one twin pregnancy. No preterm births or serious complications of pregnancy were observed. One fetus of the twin pregnancy had renal agenesis and suffered fetal demise. Genetic testing showed the deceased twin carried the same NLRP3 c.785T>C, p.V262A mutation as the mother. The other twin is healthy and mutation negative. Conclusions: Anakinra was continued during pregnancy in women with CAPS and provided significant, persistent CAPS symptom relief while continuing to prevent the long-term sequelae of CAPS. Anakinra was well tolerated. Although a causal relation between anakinra and renal agenesis seems unlikely, further safety data are needed.

Identification of virulence properties in Salmonella Typhimurium DT104 using Caenorhabditis elegans.

Salmonella enterica serover Typhimurium definitive phage type DT104, resistant to multiple antibiotics, is one of the most widespread Salmonella species in human infection worldwide. Although several cohort studies indicate that DT104 carrying the multidrug resistance (MDR) locus on salmonella genomic island 1 is a possible hyper-virulent strain compared to DT104 strains without MDR, or other Salmonella enterica serotypes, existing experimental evidence regarding virulence properties associated with the MDR region is controversial. To address this question, we constructed an isogenic MDR deletion (∆MDR) mutant strain of DT104, SNS12, by allelic exchange and used Caenorhabditis elegans as a host model to assess differences in virulence between these two strains. SNS12 exhibited decreased virulence in C. elegans, and we observed increased colonization and proliferation of the intestine of C. elegans by DT104. The immune response against MDR-carrying DT104 appears to function through a non-canonical Unfolded Protein Response (UPR) pathway, namely prion-like-(QN-rich)-domain-bearing protein pathway (PQN), in a ced-1 dependent manner in C. elegans. Further, we also demonstrate that genes of the PQN pathway and antimicrobial peptide gene abf-2, are expressed at higher transcriptional levels in worms immediately following exposure to DT104, in comparison with worms exposed to SNS12. Altogether, our results suggest that the MDR region of Salmonella Typhimurium DT104 has a direct role in virulence against Caenorhabditis elegans.