Correction: Parashar et al. Patient-Derived Ovarian Cancer Spheroids Rely on PI3K-AKT Signaling Addiction for Cancer Stemness and Chemoresistance. Cancers 2022, 14, 958.

The authors would like to correct the author byline to include Dr [...].

Patient-Derived Ovarian Cancer Spheroids Rely on PI3K-AKT Signaling Addiction for Cancer Stemness and Chemoresistance.

Ovarian cancer is the most lethal gynecological malignancy among women worldwide and is characterized by aggressiveness, cancer stemness, and frequent relapse due to resistance to platinum-based therapy. Ovarian cancer cells metastasize through ascites fluid as 3D spheroids which are more resistant to apoptosis and chemotherapeutic agents. However, the precise mechanism as an oncogenic addiction that makes 3D spheroids resistant to apoptosis and chemotherapeutic agents is not understood. To study the signaling addiction mechanism that occurs during cancer progression in patients, we developed an endometrioid subtype ovarian cancer cell line named 'MCW-OV-SL-3' from the ovary of a 70-year-old patient with stage 1A endometrioid adenocarcinoma of the ovary. We found that the cell line MCW-OV-SL-3 exhibits interstitial duplication of 1q (q21-q42), where this duplication resulted in high expression of the PIK3C2B gene and aberrant activation of PI3K-AKT-ERK signaling. Using short tandem repeat (STR) analysis, we demonstrated that the cell line exhibits a unique genetic identity compared to existing ovarian cancer cell lines. Notably, the MCW-OV-SL-3 cell line was able to form 3D spheroids spontaneously, which is an inherent property of tumor cells when plated on cell culture dishes. Importantly, the tumor spheroids derived from the MCW-OV-SL-3 cell line expressed high levels of c-Kit, PROM1, ZEB1, SNAI, VIM, and Twist1 compared to 2D monolayer cells. We also observed that the hyperactivation of ERK and PI3K/AKT signaling in these cancer cells resulted in resistance to cisplatin. In summary, the MCW-OV-SL3 endometrioid cell line is an excellent model to study the mechanism of cancer stemness and chemoresistance in endometrioid ovarian cancer.

Comprehensive serum proteomic analysis in early endometrial cancer.

OBJECTIVE: Endometrial cancer is the most common gynecologic cancer and yet much is still unknown about this disease. Our goal was to identify unique biomarkers of disease by performing a comprehensive proteomic analysis of early stage, low-grade endometrial cancer through analysis of serum collected from patients pre- and post-definitive surgery. METHODS: We used mass spectrometry (MS)-based proteomics to identify serum proteins from these patients. Serum samples from women undergoing hysterectomy with bilateral salpingo-oophorectomy for benign reasons served as control samples for the correlative studies. We then correlated our findings with The Cancer Genome Atlas (TCGA) database for additional confirmation. RESULTS: The Ingenuity Pathway Analysis of proteins that were differentially expressed in endometrial cancer showed increased cell survival and decreased organismal death, the most common hallmarks of cancer. We identified over expression of FAM83D (family with sequence similarity 83, member D) in the serum of patients with early stage low-grade endometrial cancer and verified the same in the endometrial cancer cell lines and patient tumors. We also confirmed our hypothesis that FAM83D may serve as a biomarker for endometrial cancer in a cohort of patients with endometrial cancer from The Cancer Genome Atlas (TCGA) project. CONCLUSION: Comprehensive proteomic analysis is a feasible strategy for potential biomarker identification. Using this technique, FAM83D was identified as a candidate biomarker in early endometrial cancer in our patient samples and was not present in benign control samples. FAM83D has been associated with poor clinical outcomes in several human malignancies. SIGNIFICANCE: Our manuscript describes an alternative approach to comprehensive protein analysis in a model pre and post tumor removal for a sample of patients with early endometrial cancer. The model is innovative and the findings of over expression FAM83D in this population of early cancer may be useful in the study of a disease where there are few biomarkers or targetable therapies.

Peritoneal Spread of Ovarian Cancer Harbors Therapeutic Vulnerabilities Regulated by FOXM1 and EGFR/ERBB2 Signaling.

Peritoneal spread is the primary mechanism of metastasis of ovarian cancer, and survival of ovarian cancer cells in the peritoneal cavity as nonadherent spheroids and their adherence to the mesothelium of distant organs lead to cancer progression, metastasis, and mortality. However, the mechanisms that govern this metastatic process in ovarian cancer cells remain poorly understood. In this study, we cultured ovarian cancer cell lines in adherent and nonadherent conditions in vitro and analyzed changes in mRNA and protein levels to identify mechanisms of tumor cell survival and proliferation in adherent and nonadherent cells. EGFR or ERBB2 upregulated ZEB1 in nonadherent cells, which caused resistance to cell death and increased tumor-initiating capacity. Conversely, Forkhead box M1 (FOXM1) was required for the induction of integrin ß1, integrin-α V, and integrin-α 5 for adhesion of cancer cells. FOXM1 also upregulated ZEB1, which could act as a feedback inhibitor of FOXM1, and caused the transition of adherent cells to nonadherent cells. Strikingly, the combinatorial treatment with lapatinib [dual kinase inhibitor of EGFR (ERBB1) and ERBB2] and thiostrepton (FOXM1 inhibitor) reduced growth and peritoneal spread of ovarian cancer cells more effectively than either single-agent treatment in vivo. In conclusion, these results demonstrate that FOXM1 and EGFR/ERBB2 pathways are key points of vulnerability for therapy to disrupt peritoneal spread and adhesion of ovarian cancer cells. SIGNIFICANCE: This study describes the mechanism exhibited by ovarian cancer cells required for adherent cell transition to nonadherent form during peritoneal spread and metastasis. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/24/5554/F1.large.jpg.

SOX11 hypermethylation as a tumor biomarker in endometrial cancer.

We previously reported that SOX4 is overexpressed in endometrial cancer and that it partially contributes to hypermethylation of miR-129-2 and miR-203. The current study seeks to identify methylation and expression levels of the SOX gene family in endometrial carcinomas. Methylation levels of the 16 SOX gene family members were measured by combining bisulfite restriction analysis (COBRA), MassARRAY, and pyrosequencing assays of cell lines and endometrial cancer samples. Gene expression was determined by RT-qPCR. The methylation level of the SOX11 locus was correlated with clinicopathologic factors in primary endometrial tumors and in TCGA endometrial cohort. It was also examined in DNA of serum and endometrial specimens from a longitudinal cohort of early stage endometrial cancer patients. COBRA assays indicated that hypermethylation of SOX1, SOX2, SOX11, SOX14, SOX15, SOX17, and SOX18 was present in endometrial cancer cell lines and not in the normal control. SOX11 expression was reactivated only by a DNA methylation inhibitor. Moreover, aberrant DNA methylation of SOX11 was detected in the majority of endometrioid endometrial carcinomas (n=114) and none of the 22 adjacent normal endometrial samples (P<0.0001). The methylation status of SOX11 associated significantly with microsatellite instability and MLH1 methylation in endometrial tumors (P<0.0001), and this finding was validated in TCGA endometrial cohort. Furthermore, SOX11 was not hypermethylated in serum DNA from early stage endometrial cancer patients. This study found that hypermethylation of SOX11 is common in endometrial carcinomas and strongly associates with microsatellite instability and MLH1 methylation.

Phase II study of single-agent cabozantinib in patients with recurrent clear cell ovarian, primary peritoneal or fallopian tube cancer (NRG-GY001).

OBJECTIVE: To evaluate the efficacy and tolerability of cabozantinib in recurrent clear cell ovarian, primary peritoneal or fallopian tube cancer. METHODS: Patients with recurrent ovarian, fallopian or primary peritoneal tumors with at least 50% clear cell histomorphology, measurable disease, one or two prior regimens and ECOG performance status 0-2 received cabozantinib 60 mg orally once daily continuously, in 4-week cycles until disease progression or unacceptable toxicity. Primary endpoints were progression-free survival (PFS) at six months and complete or partial tumor response (as assessed by RECIST 1.1). Secondary endpoints included toxicity, PFS, and overall survival (OS). RESULTS: Over 19 months, 13 patients were accrued. Fifty-four percent of patients were ≥60 years of age. Performance statuses of 0 and 1 comprised 8 and 5 patients. No objective tumor responses were seen. Three (23% [95% CI: 5%, 54%]) of 13 patients had PFS ≥6 months, including one patient who received cabozantinib for 23 cycles and was still on treatment as of the data cut-off date. Median PFS and OS were 3.6 and 8.1 months, respectively. There was one patient with a grade 5 event: a thromboembolic event considered possibly related to study therapy; patient's cause of death was determined to be due to disease and protocol treatment. Four other patients had thromboembolic events (two grade 3 and one each grade 1 and grade 2). Other grade 3 or higher events reported in two or more patients were nausea, vomiting, fatigue, dyspnea, and dehydration. CONCLUSIONS: Cabozantinib demonstrated minimal activity in the second- and third-line treatments of clear cell ovarian, fallopian tube or primary peritoneal carcinoma.

A prospective pilot study on the incidence of post-operative lymphedema in women with endometrial cancer.

To determine the incidence of lower-extremity lymphedema after surgical therapy including lymphadenectomy in endometrial cancer patients using standardized leg measurements. Also, to determine additional risk factors for the development of lymphedema and to study the effect of lymphedema on one's quality of life. In this prospective cohort study, patients with the diagnosis of endometrial cancer who were to undergo definitive surgical management were evaluated pre-operatively and followed post-operatively over the course of two years. Standardized leg measurements were performed by the same individuals at six time-points. Subjects also completed a standardized quality-of-life survey at each time-point. The incidence of lymphedema in 39 women with endometrial cancer using a standardized leg measurement protocol was 12.8% with lymphedema defined as a 20% increase in post-operative leg measurements. There was no significant association between the development of lymphedema and the number of pelvic or para-aortic lymph nodes removed, medical comorbidities, or surgical approach (p > 0.05). Of the five patients who met criteria for lymphedema, only one had worsening quality-of-life concerns post-operatively on the FACT-En, version 4, survey. This is the first prospective study using standardized leg measurements to calculate the incidence of post-operative lymphedema in endometrial cancer. Medical comorbidities, surgical approach, number of lymph nodes removed, and location of lymph nodes removed did not appear to affect the development of lymphedema in this cohort. A prospective, multicenter trial is needed to confirm these findings and to further assess the impact of lymphedema on one's quality of life.

Hypermethylation of miR-203 in endometrial carcinomas.

OBJECTIVES: Aberrant expression of SOX4 in endometrial cancer has been identified and partially was contributed to hypermethylation of miR-129-2. Other miRNAs are suspected to influence SOX 4 as well. The current study seeks to identify other hypermethylated miRNAs that regulate SOX4 in endometrial carcinomas. METHODS: Methylation levels of miRNA promoter regions were measured by combined bisulfite restriction analysis (COBRA) and pyrosequencing assays. Gene expression was determined by RT-qPCR. Methylation level of a miRNA locus was corrected with clinicopathologic factors for 252 gynecological specimens. RESULTS: In silico analysis identified 13 miRNA loci bound on the 3'-UTR of SOX4. Using COBRA assays, increased methylation of miR-203, miR-219-2, miR-596, and miR-618 was detected in endometrial cancer cells relative to those seen in a normal cell line and in normal endometrium. Transfection of a miR-203 mimic decreased SOX4 gene expression. Hypermethylation of miR-203 was detected in 52% of type I endometrioid endometrial carcinomas (n=131) but was not seen in any of 10 uninvolved normal endometria (P<0.001). Methylation status of miR-203 was significantly associated with microsatellite instability and MLH1 methylation in endometrial tumors (P<0.001). Furthermore, hypermethylation of miR-203 was found in endometrioid and clear endometrial subtype tumors, but not in cervical squamous cell and ovarian carcinomas. CONCLUSIONS: Hypermethylation of miR-203 is a frequent event in endometrial carcinomas and is strongly associated with microsatellite instability and MLH1 methylation status. Thus, miR-203 methylation level might represent a marker for patients with endometrioid and clear endometrial sub-cancers.

Positive predictive value of liquid-based and conventional cervical Papanicolaou smears reported as malignant.

OBJECTIVES: The predictive value of cervical Papanicolaou (Pap) smears reported as "positive for malignancy," especially those obtained by the liquid-based method, has not been adequately assessed. The objectives of this study are to determine the positive predictive value of Papanicolaou smears with features of malignancy, to compare the accuracy of Papanicolaou smears obtained by the liquid-based method to those obtained by the conventional technique in this setting, and to study the factors influencing a false-positive cytologic diagnosis of malignancy. MATERIALS AND METHODS: Pap smears significant for malignant cytology were identified at Fletcher Allen Health Care Hospital in Burlington, VT, from May 1, 1995, to April 30, 2001. A retrospective review of the hospital records and pathology reports was performed documenting patient characteristics, the collection technique, and the final histology. An independent review of the cytology and histology was performed. The positive predictive value and false-positive rate of malignant cytology were calculated for the liquid-based and conventional Pap smear techniques. RESULTS: A total of 472,743 Pap smears were performed during the period specified. One hundred four Pap smears were reported as positive for malignancy, yielding a prevalence rate of 0.02%. A total of 68 patients had paired cytology and histology specimens. Malignant cytology was identified in 36 smears obtained by the liquid-based technique and 32 smears obtained by the conventional technique. A true-positive result, meaning malignant cytology confirmed by the presence of invasive carcinoma on histology, was obtained in 61 of 68 (89.7%) patients. A false-positive result, meaning malignant cytology not confirmed by histology, was obtained in 7 of the 68 (10.3%) patients. The false-positive rate of malignant cytology was 8.4% for the liquid-based technique and 12.5% for the conventional technique. All 7 false-positive smears were diagnosed with high-grade dysplasia by histology. Three of the 7 patients with high-grade dysplasia had previous treatment for dysplasia, one of whom was also pregnant at the time of the smear. CONCLUSIONS: Malignant cervical Papanicolaou smear cytology has a high positive predictive value in the setting of gynecologic and nongynecologic malignancies. Previous treatment for cervical dysplasia or pregnancy may influence the false-positive rate of malignant cytology.