Pancreatic Cancer Patient-derived Organoids Can Predict Response to Neoadjuvant Chemotherapy.

OBJECTIVE: To evaluate if patient-derived organoids (PDOs) may predict response to neoadjuvant (NAT) chemotherapy in patients with pancreatic adenocarcinoma. BACKGROUND: PDOs have been explored as a biomarker of therapy response and for personalized therapeutics in patients with pancreatic cancer. METHODS: During 2017-2021, patients were enrolled into an IRB-approved protocol and PDO cultures were established. PDOs of interest were analyzed through a translational pipeline incorporating molecular profiling and drug sensitivity testing. RESULTS: One hundred thirty-six samples, including both surgical resections and fine needle aspiration/biopsy from 117 patients with pancreatic cancer were collected. This biobank included diversity in stage, sex, age, and race, with minority populations representing 1/3 of collected cases (16% Black, 9% Asian, 7% Hispanic/Latino). Among surgical specimens, PDO generation was successful in 71% (15 of 21) of patients who had received NAT prior to sample collection and in 76% (39 of 51) of patients who were untreated with chemotherapy or radiation at the time of collection. Pathological response to NAT correlated with PDO chemotherapy response, particularly oxaliplatin. We demonstrated the feasibility of a rapid PDO drug screen and generated data within 7 days of tissue resection. CONCLUSION: Herein we report a large single-institution organoid biobank, including ethnic minority samples. The ability to establish PDOs from chemotherapy-naive and post-NAT tissue enables longitudinal PDO generation to assess dynamic chemotherapy sensitivity profiling. PDOs can be rapidly screened and further development of rapid screening may aid in the initial stratification of patients to the most active NAT regimen.

Multi-disciplinary summit on genetics services for women with gynecologic cancers: A Society of Gynecologic Oncology White Paper.

OBJECTIVE: To assess current practice, advise minimum standards, and identify educational gaps relevant to genetic screening, counseling, and testing of women affected by gynecologic cancers. METHODS: The Society of Gynecologic Oncology (SGO) organized a multidisciplinary summit that included representatives from the American College of Obstetricians and Gynecologists (ACOG), the American Society Clinical Oncology (ASCO), the National Society of Genetic Counselors (NSGC), and patient advocacy groups, BrightPink and Facing our Risk of Cancer Empowered (FORCE). Three subject areas were discussed: care delivery models for genetic testing, barriers to genetic testing, and educational opportunities for providers of genetic testing. RESULTS: The group endorsed current SGO, National Comprehensive Cancer Network (NCCN), and NSGC genetic testing guidelines for women affected with ovarian, tubal, peritoneal cancers, or DNA mismatch repair deficient endometrial cancer. Three main areas of unmet need were identified: timely and universal genetic testing for women with ovarian, fallopian tube, and peritoneal cancers; education regarding minimum standards for genetic counseling and testing; and barriers to implementation of testing of both affected individuals as well as cascade testing of family members. Consensus building among all stakeholders resulted in an action plan to address gaps in education of gynecologic oncology providers and delivery of cancer genetics care.

A New Role for ERα: Silencing via DNA Methylation of Basal, Stem Cell, and EMT Genes.

Resistance to hormonal therapies is a major clinical problem in the treatment of estrogen receptor α-positive (ERα+) breast cancers. Epigenetic marks, namely DNA methylation of cytosine at specific CpG sites (5mCpG), are frequently associated with ERα+ status in human breast cancers. Therefore, ERα may regulate gene expression in part via DNA methylation. This hypothesis was evaluated using a panel of breast cancer cell line models of antiestrogen resistance. Microarray gene expression profiling was used to identify genes normally silenced in ERα+ cells but derepressed upon exposure to the demethylating agent decitabine, derepressed upon long-term loss of ERα expression, and resuppressed by gain of ERα activity/expression. ERα-dependent DNA methylation targets (n = 39) were enriched for ERα-binding sites, basal-up/luminal-down markers, cancer stem cell, epithelial-mesenchymal transition, and inflammatory and tumor suppressor genes. Kaplan-Meier survival curve and Cox proportional hazards regression analyses indicated that these targets predicted poor distant metastasis-free survival among a large cohort of breast cancer patients. The basal breast cancer subtype markers LCN2 and IFI27 showed the greatest inverse relationship with ERα expression/activity and contain ERα-binding sites. Thus, genes that are methylated in an ERα-dependent manner may serve as predictive biomarkers in breast cancer. IMPLICATIONS: ERα directs DNA methylation-mediated silencing of specific genes that have biomarker potential in breast cancer subtypes. Mol Cancer Res; 15(2); 152-64. ©2016 AACR.

Autophagy orchestrates adaptive responses to targeted therapy in endometrial cancer.

Targeted therapies in endometrial cancer (EC) using kinase inhibitors rarely result in complete tumor remission and are frequently challenged by the appearance of refractory cell clones, eventually resulting in disease relapse. Dissecting adaptive mechanisms is of vital importance to circumvent clinical drug resistance and improve the efficacy of targeted agents in EC. Sorafenib is an FDA-approved multitarget tyrosine and serine/threonine kinase inhibitor currently used to treat hepatocellular carcinoma, advanced renal carcinoma and radioactive iodine-resistant thyroid carcinoma. Unfortunately, sorafenib showed very modest effects in a multi-institutional phase II trial in advanced uterine carcinoma patients. Here, by leveraging RNA-sequencing data from the Cancer Cell Line Encyclopedia and cell survival studies from compound-based high-throughput screenings we have identified the lysosomal pathway as a potential compartment involved in the resistance to sorafenib. By performing additional functional biology studies we have demonstrated that this resistance could be related to macroautophagy/autophagy. Specifically, our results indicate that sorafenib triggers a mechanistic MAPK/JNK-dependent early protective autophagic response in EC cells, providing an adaptive response to therapeutic stress. By generating in vivo subcutaneous EC cell line tumors, lung metastatic assays and primary EC orthoxenografts experiments, we demonstrate that targeting autophagy enhances sorafenib cytotoxicity and suppresses tumor growth and pulmonary metastasis progression. In conclusion, sorafenib induces the activation of a protective autophagic response in EC cells. These results provide insights into the unopposed resistance of advanced EC to sorafenib and highlight a new strategy for therapeutic intervention in recurrent EC.

Comprehensive Genomic Characterization of Long Non-coding RNAs across Human Cancers.

The discovery of long non-coding RNA (lncRNA) has dramatically altered our understanding of cancer. Here, we describe a comprehensive analysis of lncRNA alterations at transcriptional, genomic, and epigenetic levels in 5,037 human tumor specimens across 13 cancer types from The Cancer Genome Atlas. Our results suggest that the expression and dysregulation of lncRNAs are highly cancer type specific compared with protein-coding genes. Using the integrative data generated by this analysis, we present a clinically guided small interfering RNA screening strategy and a co-expression analysis approach to identify cancer driver lncRNAs and predict their functions. This provides a resource for investigating lncRNAs in cancer and lays the groundwork for the development of new diagnostics and treatments.

Molecular subtypes of uterine leiomyosarcoma and correlation with clinical outcome.

The molecular etiology of uterine leiomyosarcoma (ULMS) is poorly understood, which accounts for the wide disparity in outcomes among women with this disease. We examined and compared the molecular profiles of ULMS and normal myometrium (NL) to identify clinically relevant molecular subtypes. Discovery cases included 29 NL and 23 ULMS specimens. RNA was hybridized to Affymetrix U133A 2.0 transcription microarrays. Differentially expressed genes and pathways were identified using standard methods. Fourteen NL and 44 ULMS independent archival samples were used for external validation. Molecular subgroups were correlated with clinical outcome. Pathway analyses of differentially expressed genes between ULMS and NL samples identified overrepresentation of cell cycle regulation, DNA repair, and genomic integrity. External validation confirmed differential expression in 31 genes (P < 4.4 × 10(-4), Bonferroni corrected), with 84% of the overexpressed genes, including CDC7, CDC20, GTSE1, CCNA2, CCNB1, and CCNB2, participating in cell cycle regulation. Unsupervised clustering of ULMS identified two clades that were reproducibly associated with progression-free (median, 4.0 vs 26.0 months; P = .02; HR, 0.33) and overall (median, 18.2 vs 77.2 months; P = .04; HR, 0.33) survival. Cell cycle genes play a key role in ULMS sarcomagenesis, providing opportunities for therapeutic targeting. Reproducible molecular subtypes associated with clinical outcome may permit individualized adjuvant treatment after clinical trial validation.

A functional genomic approach identifies FAL1 as an oncogenic long noncoding RNA that associates with BMI1 and represses p21 expression in cancer.

In a genome-wide survey on somatic copy-number alterations (SCNAs) of long noncoding RNA (lncRNA) in 2,394 tumor specimens from 12 cancer types, we found that about 21.8% of lncRNA genes were located in regions with focal SCNAs. By integrating bioinformatics analyses of lncRNA SCNAs and expression with functional screening assays, we identified an oncogene, focally amplified lncRNA on chromosome 1 (FAL1), whose copy number and expression are correlated with outcomes in ovarian cancer. FAL1 associates with the epigenetic repressor BMI1 and regulates its stability in order to modulate the transcription of a number of genes including CDKN1A. The oncogenic activity of FAL1 is partially attributable to its repression of p21. FAL1-specific siRNAs significantly inhibit tumor growth in vivo.

Interplay between oxygen and Fe-S cluster biogenesis: insights from the Suf pathway.

Iron-sulfur (Fe-S) cluster metalloproteins conduct essential functions in nearly all contemporary forms of life. The nearly ubiquitous presence of Fe-S clusters and the fundamental requirement for Fe-S clusters in both aerobic and anaerobic Archaea, Bacteria, and Eukarya suggest that these clusters were likely integrated into central metabolic pathways early in the evolution of life prior to the widespread oxidation of Earth's atmosphere. Intriguingly, Fe-S cluster-dependent metabolism is sensitive to disruption by oxygen because of the decreased bioavailability of ferric iron as well as direct oxidation of sulfur trafficking intermediates and Fe-S clusters by reactive oxygen species. This fact, coupled with the ubiquity of Fe-S clusters in aerobic organisms, suggests that organisms evolved with mechanisms that facilitate the biogenesis and use of these essential cofactors in the presence of oxygen, which gradually began to accumulate around 2.5 billion years ago as oxygenic photosynthesis proliferated and reduced minerals that buffered against oxidation were depleted. This review highlights the most ancient of the Fe-S cluster biogenesis pathways, the Suf system, which likely was present in early anaerobic forms of life. Herein, we use the evolution of the Suf pathway to assess the relationships between the biochemical functions and physiological roles of Suf proteins, with an emphasis on the selective pressure of oxygen toxicity. Our analysis suggests that diversification into oxygen-containing environments disrupted iron and sulfur metabolism and was a main driving force in the acquisition of accessory Suf proteins (such as SufD, SufE, and SufS) by the core SufB-SufC scaffold complex. This analysis provides a new framework for the study of Fe-S cluster biogenesis pathways and Fe-S cluster-containing metalloenzymes and their complicated patterns of divergence in response to oxygen.

Whole exome sequence analysis of serous borderline tumors of the ovary.

OBJECTIVE: Serous borderline tumor (SBT) is a unique histopathologic entity of the ovary, believed to be intermediate between benign cystadenoma and invasive low-grade serous carcinoma. While somatic mutations in the KRAS or BRAF, and rarely ERBB2, genes have been well characterized in SBTs, other genetic alterations have not been described. Toward a more comprehensive understanding of the molecular genetic architecture of SBTs, we undertook whole exome sequencing of this tumor type. METHODS: Following pathologic review and laser capture microdissection to enrich for tumor cells, whole exomes were prepared from DNA of two independent SBTs and subjected to massively parallel DNA sequencing. RESULTS: Both tumors contained an activating mutation of the BRAF gene. A total of 15 additional somatic mutations were identified, nine in one tumor and six in the other. Eleven were missense mutations and four were nonsense or deletion mutations. Fourteen of the 16 genes found to be mutated in this study have been reported to be mutated in other cancers. Furthermore, 12 of these genes are mutated in ovarian cancers. The FBXW7 and KIAA1462 genes are noteworthy candidates for a pathogenic role in serous borderline tumorigenesis. CONCLUSIONS: These findings suggest that a very small number of somatic genetic mutations are characteristic of SBTs of the ovary, thus supporting their classification as a relatively genetically stable tumor type. The mutant genes described herein represent novel candidates for the pathogenesis of ovarian SBT.

Analysis of PSPHL as a Candidate Gene Influencing the Racial Disparity in Endometrial Cancer.

Endometrial cancer is the most commonly diagnosed gynecologic malignancy in the United States. A well recognized disparity by race in both incidence and survival outcome exists for this cancer. Specifically Caucasians are about two times more likely to develop endometrial cancer than are African-Americans. However, African-American women are more likely to die from this disease than are Caucasians. The basis for this disparity remains unknown. Previous studies have identified differences in the types and frequencies of gene mutations among endometrial cancers from Caucasians and African-Americans suggesting that the tumors from these two groups might have differing underlying genetic defects. We performed a gene expression microarray study in an effort to identify differentially expressed transcripts between African-American and Caucasian women's endometrial cancers. Our gene expression screen identified a list of potential biomarkers that are differentially expressed between these two groups of cancers. Of these we identified a poorly characterized transcript with a region of homology to phospho serine phosphatase (PSPH) and designated phospho serine phosphatase like (PSPHL) as the most differentially over-expressed gene in cancers from African-Americans. We further clarified the nature of expressed transcripts. Northern blot analysis confirmed the message was limited to a transcript of under 1 kB. Sequence analysis of transcripts confirmed two alternate open reading frame (ORF) isoforms due to alternative splicing events. Splice specific primer sets confirmed both isoforms were differentially expressed in tissues from Caucasians and African-Americans. We further examined the expression in other tissues from women to include normal endometrium, normal and malignant ovary. In all cases PSPHL expression was more often present in tissues from African-Americans than Caucasians. Our data confirm the African-American based expression of the PSPHL transcript in endometrial cancer and also identify its expression in other tissues from African-Americans including ovary and ovarian cancer. PSPHL represents a candidate gene that might influence the observed racial disparity in endometrial and other cancers.

A phase 2 trial of erlotinib in patients with previously treated squamous cell and adenocarcinoma of the esophagus.

BACKGROUND: Tyrosine kinase inhibitors (TKIs) of the epidermal growth factor receptor (EGFR) have activity in solid tumors. The authors evaluated an oral EGFR TKI, erlotinib, in patients with previously treated esophageal cancer. METHODS: Thirty patients with measurable, metastatic cancer of the esophageal and gastroesophageal junction received 150 mg erlotinib daily. EGFR-negative tumors (6 patients; 20%) and EGFR-over expressing tumors (24 patients; 80%) were treated. Most patients were men (70%) with adenocarcinoma (57%) and had received previous chemotherapy (97%). RESULTS: Two partial responses were observe d in the EGFR-positive cohort (2 of 24 patients; 8%), and no responses were observed in the EGFR-negative cohort (0 of 6 patients). Reponses were limited to patients who had squamous cell carcinoma (2 of 13 patients; 15%; response duration, 5.5-7 months). The time to tumor progression was longer in patients who had squamous cell carcinoma (3.3 months; range, 1-24 months) compared with patients who had adenocarcinoma (1.6 months; range, 1-6 months; P = .026). Therapy was tolerable with the expected toxicity of skin rash (grade 1-2, 67%; grade 3, 10%). CONCLUSIONS: Erlotinib had limited activity in patients with esophageal cancer, and responses and some protracted stable disease were observed in those with squamous cell carcinoma. Efficacy according to EGFR status could not be assessed given the rarity of EGFR-negative tumors. The current results indicated that further evaluation of this agent in squamous cell carcinoma is warranted. Cancer 2011. © 2010 American Cancer Society.

Genetic analysis of the early natural history of epithelial ovarian carcinoma.

BACKGROUND: The high mortality rate associated with epithelial ovarian carcinoma (EOC) reflects diagnosis commonly at an advanced stage, but improved early detection is hindered by uncertainty as to the histologic origin and early natural history of this malignancy. METHODOLOGY/PRINCIPAL FINDINGS: Here we report combined molecular genetic and morphologic analyses of normal human ovarian tissues and early stage cancers, from both BRCA mutation carriers and the general population, indicating that EOCs frequently arise from dysplastic precursor lesions within epithelial inclusion cysts. In pathologically normal ovaries, molecular evidence of oncogenic stress was observed specifically within epithelial inclusion cysts. To further explore potential very early events in ovarian tumorigenesis, ovarian tissues from women not known to be at high risk for ovarian cancer were subjected to laser catapult microdissection and gene expression profiling. These studies revealed a quasi-neoplastic expression signature in benign ovarian cystic inclusion epithelium compared to surface epithelium, specifically with respect to genes affecting signal transduction, cell cycle control, and mitotic spindle formation. Consistent with this gene expression profile, a significantly higher cell proliferation index (increased cell proliferation and decreased apoptosis) was observed in histopathologically normal ovarian cystic compared to surface epithelium. Furthermore, aneuploidy was frequently identified in normal ovarian cystic epithelium but not in surface epithelium. CONCLUSIONS/SIGNIFICANCE: Together, these data indicate that EOC frequently arises in ovarian cystic inclusions, is preceded by an identifiable dysplastic precursor lesion, and that increased cell proliferation, decreased apoptosis, and aneuploidy are likely to represent very early aberrations in ovarian tumorigenesis.

Implication of snoRNA U50 in human breast cancer.

Deletion of chromosome 6q is frequent in breast cancer, and the deletion often involves a region in 6q14-q16. At present, however, the underlying tumor suppressor gene has not been established. Based on a recent study identifying snoRNA U50 as a candidate for the 6q14-16 tumor suppressor gene in prostate cancer, we investigated whether U50 is also involved in breast cancer. PCR-based approaches showed that U50 underwent frequent genomic deletion and transcriptional downregulation in cell lines derived from breast cancer. Mutation screening identified the same 2-bp deletion of U50 as in prostate cancer in both cell lines and primary tumors from breast cancer, and the deletion was both somatic and in germline. Genotyping of a cohort of breast cancer cases and controls for the mutation demonstrated that, while homozygous genotype of the mutation was rare, its heterozygous genotype occurred more frequently in women with breast cancer. Functionally, re-expression of U50 resulted in the inhibition of colony formation in breast cancer cell lines. These results suggest that noncoding snoRNA U50 plays a role in the development and/or progression of breast cancer.

Intraepithelial T cells and tumor proliferation: impact on the benefit from surgical cytoreduction in advanced serous ovarian cancer.

BACKGROUND: The aim of the study was to determine whether tumor-infiltrating lymphocytes and/or tumor mitotic activity could identify subgroups of patients with advanced serous epithelial ovarian cancer who would maximally benefit from aggressive surgical cytoreduction. METHODS: Snap-frozen specimens from 134 consecutive patients with stage III or IV serous or poorly differentiated ovarian adenocarcinoma undergoing primary debulking surgery from a single US institution were characterized based on CD3(+), CD8(+), FoxP3(+) tumor-infiltrating lymphocytes, and Ki67 expression. Kaplan-Meier survival curves were estimated and compared using a log-rank statistic. A multivariate Cox model was used to estimate adjusted hazard ratios. Interactions were modeled using recursive partitioning based on maximal prognostic differentiation. RESULTS: Brisk intraepithelial CD8(+) cells (P = .035) and low Ki67 expression (P = .042) portended prolonged survival. The T-cell infiltration was more likely to occur in tumors with high proliferation index. Patients whose tumors exhibited low Ki67 expression and high intraepithelial CD8(+) frequency had a 5-year survival rate of 73.3%. Patients with aggressive tumor behavior, that is, whose tumors exhibited low frequency of intraepithelial CD8(+) T cells or high Ki67 expression were more likely to draw benefit from aggressive surgical cytoreduction. Survival was similar for patients with brisk CD8(+) T cells who had optimal or suboptimal debulking. Likewise, survival was similar for patients with low Ki67 expression who had optimal or suboptimal debulking. CONCLUSIONS: For the first time, these novel interactions of T cells, tumor proliferation index, and surgical treatment reveal that biological prognosticators may be useful for surgical decision making in ovarian cancer.

Microarray analysis of early stage serous ovarian cancers shows profiles predictive of favorable outcome.

PURPOSE: Although few women with advanced serous ovarian cancer are cured, detection of the disease at an early stage is associated with a much higher likelihood of survival. We previously used gene expression array analysis to distinguish subsets of advanced cancers based on disease outcome. In the present study, we report on gene expression of early-stage cancers and validate our prognostic model for advanced-stage cancers. EXPERIMENTAL DESIGN: Frozen specimens from 39 stage I/II, 42 stage III/IV, and 20 low malignant potential cancers were obtained from four different sites. A linear discriminant model was used to predict survival based upon array data. RESULTS: We validated the late-stage survival model and show that three of the most differentially expressed genes continue to be predictive of outcome. Most early-stage cancers (38 of 39 invasive, 15 of 20 low malignant potential) were classified as long-term survivors (median probabilities 0.97 and 0.86). MAL, the most differentially expressed gene, was further validated at the protein level and found to be an independent predictor of poor survival in an unselected group of advanced serous cancers (P = 0.0004). CONCLUSIONS: These data suggest that serous ovarian cancers detected at an early stage generally have a favorable underlying biology similar to advanced-stage cases that are long-term survivors. Conversely, most late-stage ovarian cancers seem to have a more virulent biology. This insight suggests that if screening approaches are to succeed it will be necessary to develop approaches that are able to detect these virulent cancers at an early stage.

A phase II trial of erlotinib in combination with bevacizumab in patients with metastatic breast cancer.

PURPOSE: To evaluate the efficacy and toxicity of erlotinib plus bevacizumab in patients with metastatic breast cancer (MBC), targeting the epidermal growth factor receptor (EGFR/HER1) and the vascular endothelial growth factor (VEGF) pathway. EXPERIMENTAL DESIGN: Thirty-eight patients with MBC were enrolled and treated at two institutions with erlotinib, a small molecule EGFR tyrosine kinase inhibitor (150 mg p.o. daily) plus bevacizumab, an anti-VEGF antibody (15 mg/kg i.v. every 3 weeks). Patients had one to two prior chemotherapy regimens for metastatic disease. The primary end point was response rate by Response Evaluation Criteria in Solid Tumors criteria using a Simon 2-stage design. Secondary end points included toxicity, time to progression, response duration, and stabilization of disease of > or = 26 weeks. Correlative studies were done on tumor tissue, including EGFR expression and mutation analysis. RESULTS: One patient achieved a partial response for 52+ months. Fifteen patients had stable disease at first evaluation at 9 weeks; 4 of these patients had stable disease beyond 26 weeks. Median time to progression was 11 weeks (95% confidence interval, 8-18 weeks). Diarrhea of any grade was observed in 84% of patients (grade 3 in 3%); 76% experienced grade 1 or 2 skin rash, and 18% developed hypertension (grade 3 in 11%). The level of EGFR expression was not predictive of response to therapy. CONCLUSIONS: The combination of erlotinib and bevacizumab was well-tolerated but had limited activity in unselected patients with previously treated MBC. Biomarkers are needed to identify those MBC patients likely to respond to anti-EGFR/HER1 plus anti-VEGF therapy.