Overcoming adaptive resistance to anti-VEGF therapy by targeting CD5L.

Antiangiogenic treatment targeting the vascular endothelial growth factor (VEGF) pathway is a powerful tool to combat tumor growth and progression; however, drug resistance frequently emerges. We identify CD5L (CD5 antigen-like precursor) as an important gene upregulated in response to antiangiogenic therapy leading to the emergence of adaptive resistance. By using both an RNA-aptamer and a monoclonal antibody targeting CD5L, we are able to abate the pro-angiogenic effects of CD5L overexpression in both in vitro and in vivo settings. In addition, we find that increased expression of vascular CD5L in cancer patients is associated with bevacizumab resistance and worse overall survival. These findings implicate CD5L as an important factor in adaptive resistance to antiangiogenic therapy and suggest that modalities to target CD5L have potentially important clinical utility.

Clinical analysis of pathologic complete responders in advanced-stage ovarian cancer.

OBJECTIVE: To determine the clinical characteristics of patients who attained pathologic complete response (pCR) after neoadjuvant chemotherapy (NACT) and to identify specific predictive or prognostic factors associated with pCR. METHODS: Two distinct populations of patients who underwent NACT followed by interval tumor reductive surgery (TRS) were used in this retrospective study. The first contained 472 patients from a single institution. The second contained only pCR patients (67); those identified from population one, plus 44 obtained through collaborative institutions. Cox analysis and log-rank tests were performed to assess associations between clinical characteristics and pCR outcome, recurrence-free survival (RFS), and overall survival (OS). RESULTS: The median RFS and OS in our pCR-only population was 24.2 and 80.8 months, respectively, with a median follow-up time of 32.4 months. In our single institution population, 23 patients attained pCR (4.9%) and had longer RFS compared to non-pCR patients with viable microscopic, optimal, or suboptimal residual disease (24.3 vs. 12.1 vs. 11.6 vs. 9.6 months, p = 0.025, 0.012, 0.008, respectively), and longer OS compared to those with optimal or suboptimal residual disease (54.5 vs. 29.4 vs. 25.7 months, p = 0.027, 0.007, respectively). Patients were more than three-fold likely to attain pCR if their CA125 value was normal at the time of surgery (OR 3.54, 95% CI: 1.14-11.05, p = 0.029). CONCLUSIONS: Women with pCR after NACT have significantly longer RFS compared to those with residual viable tumor at the time of interval tumor-reductive surgery, and CA125 is plausible biomarker for identifying these extreme responders preoperatively.

CD63-mediated cloaking of VEGF in small extracellular vesicles contributes to anti-VEGF therapy resistance.

Despite wide use of anti-vascular endothelial growth factor (VEGF) therapy for many solid cancers, most individuals become resistant to this therapy, leading to disease progression. Therefore, new biomarkers and strategies for blocking adaptive resistance of cancer to anti-VEGF therapy are needed. As described here, we demonstrate that cancer-derived small extracellular vesicles package increasing quantities of VEGF and other factors in response to anti-VEGF therapy. The packaging process of VEGF into small extracellular vesicles (EVs) is mediated by the tetraspanin CD63. Furthermore, small EV-VEGF (eVEGF) is not accessible to anti-VEGF antibodies and can trigger intracrine VEGF signaling in endothelial cells. eVEGF promotes angiogenesis and enhances tumor growth despite bevacizumab treatment. These data demonstrate a mechanism where VEGF is partitioned into small EVs and promotes tumor angiogenesis and progression. These findings have clinical implications for biomarkers and therapeutic strategies for ovarian cancer.

Poly-ADP-ribose polymerase inhibitor use in ovarian cancer: expanding indications and novel combination strategies.

The use of poly(ADP-ribose) polymerase (PARP) inhibition is transforming care for the treatment of ovarian cancer, with three different PARP inhibitors (PARPi) gaining US Food and Drug Administration approval since 2014. Given the rapidly expanding use of PARPi, this review aims to summarize the key evidence for their use and therapeutic indications. Furthermore, we provide an overview of the development of PARPi resistance and the emerging role of PARPi combination therapies, including those with anti-angiogenic and immunotherapeutic agents.

Exploring and comparing adverse events between PARP inhibitors.

Ovarian cancer remains one of the most challenging malignancies to treat. Targeted therapies such as poly (ADP-ribose) polymerase (PARP) inhibitors have emerged as one of the most exciting new treatments for ovarian cancer, particularly in women with BRCA1 or BRCA2 mutations or those without a functional homologous recombination repair pathway. Perhaps the most advantageous characteristic of PARP inhibitors is their mechanism of action, which targets cancer cells on the basis of their inherent deficiencies while seemingly avoiding normally functioning cells. Although health-care providers might assume a low toxicity profile because of their specific mechanism of action, PARP inhibitors are not completely benign and overall show a class effect adverse-event profile. Further complicating this situation, three different PARP inhibitors have been approved by the US Food and Drug Administration since 2014, each with their own specific indications and individual toxicity profiles. The diversity of adverse events seen both within and across this class of drug underscores the importance of having a comprehensive reference to help guide clinical decision making when treating patients. This Review characterises and compares all toxicities associated with each PARP inhibitor, both in monotherapy and in novel combinations with other drugs, with a particular focus on potential management strategies to help mitigate toxic effects. Although the excitement surrounding PARP inhibitors might certainly be warranted, a thorough understanding of all associated toxicities is imperative to ensure that patients can achieve maximal clinical benefit.

When Ovarian Cancer Is Not: Characterizing Nonovarian Cancer Pathology in a Laparoscopy-Based Triage System.

OBJECTIVE: Establishing an accurate histologic diagnosis is essential for determining the appropriate course of therapy for ovarian cancer. This study sought to investigate and describe nonovarian cancer pathologies discovered during the systematic laparoscopic workup of presumed advanced ovarian cancer. METHODS: A retrospective cohort of patients with presumed advanced ovarian cancer (based on elevated CA125 and/or imaging) presenting to our center without confirmed pathologic diagnosis were identified and characterized. Patients without ovarian cancer on final pathology were described and compared with those with confirmed epithelial ovarian cancer using standard statistical methods. RESULTS: Nonovarian cancer was found in 26 (7.1%) of 365 cases over 3.5 years of study, and included benign ovarian pathology, and metastatic uterine, breast, and gastrointestinal cancers. Most nonovarian cancer cases could not be diagnosed with percutaneous biopsy, and instead used diagnostic laparoscopy or assessment at the time of laparotomy for diagnosis (58%). No patient received inappropriate treatment. Nonovarian cancer cases were more likely to be nonwhite (P = 0.003), have a better Eastern Cooperative Oncology Group performance status (P < 0.001), and have a lower CA125 value (P < 0.001), and were less likely to have pleural effusions (P = 0.04). CONCLUSIONS: A systematic laparoscopic triage approach to advanced-stage ovarian cancer eliminates incorrect neoadjuvant chemotherapy administration and inappropriate laparotomy. This algorithm identified a population of women who are more likely to have nonovarian cancer pathology. Increasing screening efforts should be focused on conclusive diagnosis with the least invasive testing possible.

Extended left upper quadrant resection during primary cytoreductive surgery for Stage IV ovarian cancer.

OBJECTIVE: The completeness of primary cytoreductive surgery for Stage IV epithelial ovarian cancer is associated with greater progression free survival and overall survival Winter et al. (2008) [1]. Cytoreduction to no gross residual disease in patients with bulky upper abdominal disease presents significant surgical challenges, highlighting the importance of specialized and comprehensive surgical training in the treatment of advanced ovarian cancers Zivanovic et al. (2008) [2]. Extensive upper abdominal surgical procedures have shown to improve the ability to achieve cytoreduction to no gross residual disease Chi et al. (2004) [3]. This film displays an extended left upper quadrant resection in one of our recent patients. METHODS: The patient was a 62-year-old female with a CA-125 of 2,577U/mL, abdominal ascites, and a preoperative CT showing carcinomatosis with a left upper quadrant infiltration. Primary cytoreductive surgery was undertaken with exploratory laparotomy, type 2 radical oophorectomy (en bloc modified radical abdominal hysterectomy, bilateral salpingo-oophorectomy, pan-pelvic peritonectomy, distal colectomy, retosigmoid colectomy), with en bloc omentectomy, transverse colectomy, splenectomy, distal pancreatectomy, and diaphragm peritonectomy. RESULTS: Operative time was 337min with an estimated blood loss of 900mL. The patient was discharged home on post-operative day 10 after a standard prolongation in hospitalization required to meet milestones after extensive upper quadrant cytoreductive surgery. CONCLUSION: Bulky upper abdominal disease can present significant surgical challenges. This film illustrates obtaining cytoreduction to no gross residual disease is feasible. We show transection of the pancreas by reinforced linear staple closure due to its ease of use and surgeon preference, although controversy remains regarding the ideal technique.

Recent Patents for Homologous Recombination Deficiency Assays Among Women with Ovarian Cancer.

Ovarian cancer continues to present a significant health challenge with little progress being made over the past two decades in reducing the incidence and mortality. More recently, novel therapeutics have emerged as a potential way of improving outcomes for women with advanced ovarian cancer who harbor mutations in genes involved in homologous recombination (HR), most notably BRCA1 and BRCA2. In the United States, Olaparib, a PARP inhibitor, has been recently approved for ovarian cancer patients treated with three or more lines of prior chemotherapy who harbor germline mutations in BRCA1 or BRCA2. As a caveat to Olaparib's FDA approval, BRACAnalysis CDx(®) was approved as a companion diagnostic test for women with ovarian cancer to determine their BRCA1/2 mutation status and eligibility for treatment. This review article will provide essential background information on hereditary breast and ovarian cancer (HBOC), describe the therapeutic mechanism of PARP inhibitors, and will chronicle the current and emerging homologous recombination deficiency (HRD) assays and their associated patents.

Routine cystoscopy after robotic gynecologic oncology surgery.

BACKGROUND AND OBJECTIVES: Our aim was to determine whether the use of routine cystoscopy increases lower urinary tract injury detection (bladder and/or ureter) after robotic surgery performed by gynecologic oncologists. METHODS: A retrospective chart review of patients who presented for robotic hysterectomy from 2009-2012 was performed at 2 separate academic medical centers, one that performed routine cystoscopy and one that did not. Statistical analysis was performed with t tests and χ2 tests. RESULTS: We identified 140 cases without cystoscopy and 109 cases with routine cystoscopy. There were no intraoperative or postoperative urinary injuries detected in either group. There were no significant differences in age and body mass index. In the non-cystoscopy group, a larger specimen size (P<.001), less blood loss (P=.013), and a longer mean operative time were observed (P<.0001). In the routine cystoscopy group, more lymphadenectomies were performed with hysterectomy (P=.007) and more patients underwent hysterectomy for ovarian cancer (P=.0192). There were no differences in surgical indications or secondary procedures including bilateral salpingo-oophorectomy, radical hysterectomy, ureterolysis, and pelvic organ prolapse-related procedures. The minimum follow-up period was 30 days in both groups. CONCLUSION: Routine use of cystoscopy did not appear to affect the detection rate of intraoperative lower urinary tract injury during robotic gynecologic surgery because this rate was zero in both groups. However, cystoscopy is relatively simple to perform and can be efficiently incorporated into robotic surgery to avoid the severe morbidity and possible litigation surrounding a urinary tract injury.

Grade 1 endometrioid endometrial carcinoma presenting with pelvic bone metastasis: a case report and review of the literature.

Most grade 1 endometrioid endometrial cancers are confined to the uterus at the time of diagnosis and confer a good prognosis. Rarely will a grade 1 endometrioid endometrial carcinoma present with distant metastasis, especially to the bone. We present the case of a 56-year-old woman with postmenopausal bleeding and right hip pain due to metastatic grade 1 endometrioid uterine cancer invading into the right ischium. We discuss treatment options as well as provide a review of prior published reports on bony metastasis in grade 1 endometrioid endometrial cancers. To date, this case is one of 10 others which demonstrates that even a well-differentiated, low-grade endometrioid endometrial carcinoma can progress in a highly aggressive manner.

Testing mutual exclusivity of ETS rearranged prostate cancer.

Prostate cancer is a clinically heterogeneous and multifocal disease. More than 80% of patients with prostate cancer harbor multiple geographically discrete cancer foci at the time of diagnosis. Emerging data suggest that these foci are molecularly distinct consistent with the hypothesis that they arise as independent clones. One of the strongest arguments is the heterogeneity observed in the status of E26 transformation specific (ETS) rearrangements between discrete tumor foci. The clonal evolution of individual prostate cancer foci based on recent studies demonstrates intertumoral heterogeneity with intratumoral homogeneity. The issue of multifocality and interfocal heterogeneity is important and has not been fully elucidated due to lack of the systematic evaluation of ETS rearrangements in multiple tumor sites. The current study investigates the frequency of multiple gene rearrangements within the same focus and between different cancer foci. Fluorescence in situ hybridization (FISH) assays were designed to detect the four most common recurrent ETS gene rearrangements. In a cohort of 88 men with localized prostate cancer, we found ERG, ETV1, and ETV5 rearrangements in 51% (44/86), 6% (5/85), and 1% (1/86), respectively. None of the cases demonstrated ETV4 rearrangements. Mutual exclusiveness of ETS rearrangements was observed in the majority of cases; however, in six cases, we discovered multiple ETS or 5' fusion partner rearrangements within the same tumor focus. In conclusion, we provide further evidence for prostate cancer tumor heterogeneity with the identification of multiple concurrent gene rearrangements.

ERG cooperates with androgen receptor in regulating trefoil factor 3 in prostate cancer disease progression.

To elucidate the role of ETS gene fusions in castration-resistant prostate cancer (CRPC), we characterized the transcriptome of 54 CRPC tumor samples from men with locally advanced or metastatic disease. Trefoil factor 3 (TFF3) emerged as the most highly differentially regulated gene with respect to ERG rearrangement status and resistance to hormone ablation therapy. Conventional chromatin immunoprecipitation (ChIP)-polymerase chain reaction and ChIP followed by DNA sequencing (ChIP-seq) revealed direct binding of ERG to ETS binding sites in the TFF3 promoter in ERG-rearranged prostate cancer cell lines. These results were confirmed in ERG-rearranged hormone-naive prostate cancer (HNPC) and CRPC tissue samples. Functional studies demonstrated that ERG has an inhibitory effect on TFF3 expression in hormone-naive cancer but not in the castration-resistant state. In addition, we provide evidence suggesting an effect of androgen receptor signaling on ERG-regulated TFF3 expression. Furthermore, TFF3 overexpression enhances ERG-mediated cell invasion in CRPC prostate cancer cells. Taken together, our findings reveal a novel mechanism for enhanced tumor cell aggressiveness resulting from ERG rearrangement in the castration-resistant setting through TFF3 gene expression.

Rearrangements of the RAF kinase pathway in prostate cancer, gastric cancer and melanoma.

Although recurrent gene fusions involving erythroblastosis virus E26 transformation-specific (ETS) family transcription factors are common in prostate cancer, their products are considered 'undruggable' by conventional approaches. Recently, rare targetable gene fusions involving the anaplastic lymphoma receptor tyrosine kinase (ALK) gene, have been identified in 1-5% of lung cancers, suggesting that similar rare gene fusions may occur in other common epithelial cancers, including prostate cancer. Here we used paired-end transcriptome sequencing to screen ETS rearrangement-negative prostate cancers for targetable gene fusions and identified the SLC45A3-BRAF (solute carrier family 45, member 3-v-raf murine sarcoma viral oncogene homolog B1) and ESRP1-RAF1 (epithelial splicing regulatory protein-1-v-raf-1 murine leukemia viral oncogene homolog-1) gene fusions. Expression of SLC45A3-BRAF or ESRP1-RAF1 in prostate cells induced a neoplastic phenotype that was sensitive to RAF and mitogen-activated protein kinase kinase (MAP2K1) inhibitors. Screening a large cohort of patients, we found that, although rare, recurrent rearrangements in the RAF pathway tend to occur in advanced prostate cancers, gastric cancers and melanoma. Taken together, our results emphasize the key role of RAF family gene rearrangements in cancer, suggest that RAF and MEK inhibitors may be useful in a subset of gene fusion-harboring solid tumors and demonstrate that sequencing of tumor transcriptomes and genomes may lead to the identification of rare targetable fusions across cancer types.

SOX2 is an amplified lineage-survival oncogene in lung and esophageal squamous cell carcinomas.

Lineage-survival oncogenes are activated by somatic DNA alterations in cancers arising from the cell lineages in which these genes play a role in normal development. Here we show that a peak of genomic amplification on chromosome 3q26.33 found in squamous cell carcinomas (SCCs) of the lung and esophagus contains the transcription factor gene SOX2, which is mutated in hereditary human esophageal malformations, is necessary for normal esophageal squamous development, promotes differentiation and proliferation of basal tracheal cells and cooperates in induction of pluripotent stem cells. SOX2 expression is required for proliferation and anchorage-independent growth of lung and esophageal cell lines, as shown by RNA interference experiments. Furthermore, ectopic expression of SOX2 here cooperated with FOXE1 or FGFR2 to transform immortalized tracheobronchial epithelial cells. SOX2-driven tumors show expression of markers of both squamous differentiation and pluripotency. These characteristics identify SOX2 as a lineage-survival oncogene in lung and esophageal SCC.

In situ evidence of KRAS amplification and association with increased p21 levels in non-small cell lung carcinoma.

Recent advances in the characterization of the lung cancer genome have suggested that KRAS may frequently be amplified, although little is known regarding the significance of this finding. This is in contrast with activating mutations of KRAS, which occur in approximately 20% of non-small cell lung carcinomas (NSCLCs). We used fluorescence in situ hybridization to provide direct evidence of KRAS amplification for the first time in clinical specimens. We detected amplification in 7 of 100 consecutive NSCLCs, with a concurrent activating KRAS mutation in 4 cases. KRAS amplification was associated with greater expression of p21 as assessed by quantitative immunohistochemical analysis (P = .015). Our data indicate that a sizable subgroup of NSCLCs harbor KRAS amplification, some of which also contain point mutations, and suggest that an increased KRAS copy number may drive p21 overexpression. KRAS amplification may define a unique clinicopathologic subset of NSCLCs with potentially altered responsiveness to targeted therapies.

Amplification of chromosomal segment 4q12 in non-small cell lung cancer.

In cancer, proto-oncogenes are often altered by genomic amplification. Here we report recurrent focal amplifications of chromosomal segment 4q12 overlapping the proto-oncogenes PDGFRA and KIT in non-small cell lung cancer (NSCLC). Single nucleotide polymorphism (SNP) array and fluorescent in situ hybridization (FISH) analysis indicate that 4q12 is amplified in 3-7% of lung adenocarcinomas and 8-10% of lung squamous cell carcinomas. In addition, we demonstrate that the NSCLC cell line NCI-H1703 exhibits focal amplification of PDGFRA and is dependent on PDGFRalpha activity for cell growth. Treatment of NCI-H1703 cells with PDGFRA-specific shRNAs or with the PDGFRalpha/KIT small molecule inhibitors imatinib or sunitinib leads to cell growth inhibition. However, these observations do not extend to NSCLC cell lines with lower-amplitude and broader gains of chromosome 4q. Together these observations implicate PDGFRA and KIT as potential oncogenes in NSCLC, but further study is needed to define the specific characteristics of those tumors that could respond to PDGFRalpha/KIT inhibitors.

N-myc downstream regulated gene 1 (NDRG1) is fused to ERG in prostate cancer.

A step toward the molecular classification of prostate cancer was the discovery of recurrent erythroblast transformation-specific rearrangements, most commonly fusing the androgen-regulated TMPRSS2 promoter to ERG. The TMPRSS2-ERG fusion is observed in around 90% of tumors that overexpress the oncogene ERG. The goal of the current study was to complete the characterization of these ERG-overexpressing prostate cancers. Using fluorescence in situ hybridization and reverse transcription-polymerase chain reaction assays, we screened 101 prostate cancers, identifying 34 cases (34%) with the TMPRSS2-ERG fusion. Seven cases demonstrated ERG rearrangement by fluorescence in situ hybridization without the presence of TMPRSS2-ERG fusion messenger RNA transcripts. Screening for known 5' partners, we determined that three cases harbored the SLC45A3-ERG fusion. To discover novel 5' partners in these ERG-overexpressing and ERG-rearranged cases, we used paired-end RNA sequencing. We first confirmed the utility of this approach by identifying the TMPRSS2-ERG fusion in a known positive prostate cancer case and then discovered a novel fusion involving the androgen-inducible tumor suppressor, NDRG1 (N-myc downstream regulated gene 1), and ERG in two cases. Unlike TMPRSS2-ERG and SCL45A3-ERG fusions, the NDRG1-ERG fusion is predicted to encode a chimeric protein. Like TMPRSS2, SCL45A3 and NDRG1 are inducible not only by androgen but also by estrogen. This study demonstrates that most ERG-overexpressing prostate cancers harbor hormonally regulated TMPRSS2-ERG, SLC45A3-ERG, or NDRG1-ERG fusions. Broader implications of this study support the use of RNA sequencing to discover novel cancer translocations.

Oncosome formation in prostate cancer: association with a region of frequent chromosomal deletion in metastatic disease.

Oncosomes have recently been described as membrane-derived microvesicles secreted by cancer cells, which transfer oncogenic signals and protein complexes across cell boundaries. Here, we show the rapid formation and secretion of oncosomes from DU145 and LNCaP human prostate cancer cells. Oncosome formation was stimulated by epidermal growth factor receptor activation and also by overexpression of membrane-targeted Akt1. Microvesicles shed from prostate cancer cells contained numerous signal transduction proteins and were capable of activating rapid phospho-tyrosine and Akt pathway signaling, and stimulating proliferation and migration, in recipient tumor cells. They also induced a stromal reaction in recipient normal cells. Knockdown of the actin nucleating protein Diaphanous Related Formin 3 (DRF3/Dia2) by RNA interference enhanced rates of oncosome formation, indicating that these structures resemble, and may be identical to, nonapoptotic membrane blebs, a feature of the amoeboid form of cell motility. Analysis of primary and metastatic human prostate tumors using 100K single nucleotide polymorphism arrays revealed a significantly higher frequency of deletion of the locus encoding DRF3 (DIAPH3) in metastatic tumors (P = 0.001) in comparison with organ-confined tumors. Fluorescence in situ hybridization confirmed increased chromosomal loss of DIAPH3 in metastatic tumors in a different cohort of patients (P = 0.006). These data suggest that microvesicles shed from prostate cancer cells can alter the tumor microenvironment in a manner that may promote disease progression. They also show that DRF3 is a physiologically relevant protein that seems to regulate this process.

Distinct genomic aberrations associated with ERG rearranged prostate cancer.

Emerging molecular and clinical data suggest that ETS fusion prostate cancer represents a distinct molecular subclass, driven most commonly by a hormonally regulated promoter and characterized by an aggressive natural history. The study of the genomic landscape of prostate cancer in the light of ETS fusion events is required to understand the foundation of this molecularly and clinically distinct subtype. We performed genome-wide profiling of 49 primary prostate cancers and identified 20 recurrent chromosomal copy number aberrations, mainly occurring as genomic losses. Co-occurring events included losses at 19q13.32 and 1p22.1. We discovered three genomic events associated with ERG rearranged prostate cancer, affecting 6q, 7q, and 16q. 6q loss in nonrearranged prostate cancer is accompanied by gene expression deregulation in an independent dataset and by protein deregulation of MYO6. To analyze copy number alterations within the ETS genes, we performed a comprehensive analysis of all 27 ETS genes and of the 3 Mbp genomic area between ERG and TMPRSS2 (21q) with an unprecedented resolution (30 bp). We demonstrate that high-resolution tiling arrays can be used to pin-point breakpoints leading to fusion events. This study provides further support to define a distinct molecular subtype of prostate cancer based on the presence of ETS gene rearrangements.