Edna Gordian

Serum Free Circulating DNA Is a Useful Biomarker to Distinguish Benign versus Malignant Prostate Disease

Background: Free circulating DNA (fcDNA) has been shown to be elevated in serum of prostate cancer patients compared with benign controls. However, studies evaluating the role of fcDNA as a biomarker in a “representative” patient group who have undergone prostate cancer screening are lacking. Our study examined the use of serum fcDNA levels as a biomarker of prostate cancer in such a setting. Methods: The study included 252 men, with prostate-specific antigen (PSA) levels >4 ng/mL and/or abnormal digital rectal exam. fcDNA levels in serum before prostate biopsy were quantitated by real-time PCR amplification of the glutathione S-transferase, pi, gene. Results: Patients with PSA ≤ 10 ng/mL with fcDNA > 180 ng/mL were at increased risk for prostate cancer compared with those with fcDNA ≤180 ng/mL (odds ratio, 4.27; 95% confidence interval, 2.05-8.88; P < 0.001; area under the curve, 0.742). The multivariate model including age, race, PSA, fcDNA, and interaction between fcDNA and PSA yielded a high negative predictive value of 93.1% and increased specificity of 33.1% compared with negative predictive value of 73.3% and specificity of 6.7% in the model excluding fcDNA. Conclusions: Our results indicate that fcDNA may improve the specificity of prostate cancer screening. Impact: Our study shows that adding fcDNA to prostate cancer screening can reduce the number of unnecessary prostate biopsies. Cancer Epidemiol Biomarkers Prev; 19(8); 1984–91. ©2010 AACR.

Phase I/II Study of Azacitidine, Docetaxel, and Prednisone in Patients With Metastatic Castration-Resistant Prostate Cancer Previously Treated With Docetaxel-Based Therapy

INTRODUCTION Methylation-mediated silencing of genes contributes to docetaxel resistance in prostate cancer. We propose that azacitidine, a demethylating agent, can reverse docetaxel resistance. PATIENTS AND METHODS Metastatic castration-resistant prostate cancer (mCRPC) patients, who progressed during or within 6 months of docetaxel chemotherapy, were eligible. Fifteen and 7 patients were treated in phase I and II, respectively. In phase I, azacitidine and docetaxel were alternately escalated in a standard 3 + 3 design. All patients received prednisone 5 mg twice daily continuously. Patients were evaluated for toxicity and efficacy. Growth arrest and DNA damage-inducible alpha (GADD45A) methylation was measured before and after azacitidine treatment in the first cycle in phase I patients. RESULTS In phase I, no dose-limiting toxicity was observed. At the highest dose (azacitidine 150 mg/m(2) daily for 5 days followed by docetaxel 75 mg/m(2) on day 6), Grade 4 neutropenia was frequent, but infrequent with growth factor. Six patients in the phase II study received the highest dose including growth factor support. The sixth phase II patient died because of neutropenic sepsis. After data and safety monitoring board review, the phase II dose was reduced to azacitidine 75 mg/m(2) daily for 5 days followed by docetaxel 75 mg/m(2) on day 6 with growth factor support. Prostate-specific antigen response was seen in 10 of 19 evaluable patients and objective response was observed in 3 of 10 evaluable patients. Significant demethylation of GADD45A was observed with azacitidine treatment. CONCLUSION The combination of azacitidine, docetaxel, and prednisone with growth factor support is active in mCRPC patients.

Serum GADD45a methylation is a useful biomarker to distinguish benign vs malignant prostate disease

Background:Prostate-specific antigen (PSA) screening for prostate cancer results in a large number of unnecessary prostate biopsies. There is a need for specific molecular markers that can be used in combination with PSA to improve the specificity of PSA screening. We examined GADD45a methylation in blood DNA as a molecular marker for prostate cancer diagnosis.Methods:The study included 82 men, with PSA levels >4 ng ml−1 and/or abnormal digital rectal exam, who underwent prostate biopsy. We compared GADD45a methylation in DNA from serum and buffy coat in 44 patients (22 prostate cancer and 22 benign). GADD45a methylation in serum DNA was examined in 82 patients (34 cancer and 48 benign).Results:There was no significant difference in buffy coat GADD45a methylation between cancer and benign patients. Serum GADD45a methylation was significantly higher in cancer than in benign patients. Classification and regression tree predictive model for prostate cancer including risk groups defined by PSA, free circulating DNA (fcDNA) level and GADD45a methylation yielded specificity of 87.5%, sensitivity of 94.1% and receiver operator characteristic curve area of 0.937.Conclusions:Serum GADD45a methylation in combination with PSA and fcDNA level was useful in distinguishing benign from prostate cancer patients.

Transforming Growth Factor β Signaling Overcomes Dasatinib Resistance in Lung Cancer

Lung cancer is the second most common cancer and the leading cause of cancer-related deaths. Despite recent advances in the development of targeted therapies, patients with advanced disease remain incurable, mostly because metastatic non-small cell lung carcinomas (NSCLC) eventually become resistant to tyrosine kinase inhibitors (TKIs). Kinase inhibitors have the potential for target promiscuity because the kinase super family is the largest family of druggable genes that binds to a common substrate (ATP). As a result, TKIs often developed for a specific purpose have been found to act on other targets. Drug affinity chromatography has been used to show that dasatinib interacts with the TGFβ type I receptor (TβR-I), a serine-threonine kinase. To determine the potential biological relevance of this association, we studied the combined effects of dasatinib and TGFβ on lung cancer cell lines. We found that dasatinib treatment alone had very little effect; however, when NSCLC cell lines were treated with a combination of TGFβ and dasatinib, apoptosis was induced. Combined TGFβ-1 + dasatinib treatment had no effect on the activity of Smad2 or other non-canonical TGFβ intracellular mediators. Interestingly, combined TGFβ and dasatinib treatment resulted in a transient increase in p-Smad3 (seen after 3 hours). In addition, when NSCLC cells were treated with this combination, the pro-apoptotic protein BIM was up-regulated. Knockdown of the expression of Smad3 using Smad3 siRNA also resulted in a decrease in BIM protein, suggesting that TGFβ-1 + dasatinib-induced apoptosis is mediated by Smad3 regulation of BIM. Dasatinib is only effective in killing EGFR mutant cells, which is shown in only 10% of NSCLCs. Therefore, the observation that wild-type EGFR lung cancers can be manipulated to render them sensitive to killing by dasatinib could have important implications for devising innovative and potentially more efficacious treatment strategies for this disease.

Phase I study of azacitidine, docetaxel, and prednisone in patients with metastatic castration-resistant prostate cancer (CRPC) previously treated with docetaxel-based therapy.

DNA methylation of genes contributes to resistance to docetaxel in prostate cancer. We investigated the combination of azacitidine (demethylating agent), docetaxel, and prednisone with the aim of reversing doce- taxel resistance. We treated 22 patients in a phase I/II study. This combination with growth factor support was active in metastatic prostate cancer patients previously treated with docetaxel. Introduction: Methylation-mediated silencing of genes contributes to docetaxel resistance in prostate cancer. We propose that azacitidine, a demethylating agent, can reverse docetaxel resistance. Patients and Methods: Metastatic castration-resistant prostate cancer (mCRPC) patients, who progressed during or within 6 months of docetaxel chemotherapy, were eligible. Fifteen and 7 patients were treated in phase I and II, respectively. In phase I, azacitidine and docetaxel were alternately escalated in a standard 3 þ 3 design. All patients received prednisone 5 mg twice daily continuously. Patients were evaluated for toxicity and efficacy. Growth arrest and DNA damage-inducible alpha (GADD45A) methylation was measured before and after azacitidine treatment in the first cycle in phase I patients. Results: In phase I, no dose-limiting toxicity was observed. At the highest dose (azacitidine 150 mg/m 2 daily for 5 days followed by docetaxel 75 mg/m 2 on day 6), Grade 4 neutropenia was frequent, but infrequent with growth factor. Six patients in the phase II study received the highest dose including growth factor support. The sixth phase II patient died because of neutropenic sepsis. After data and safety monitoring board review, the phase II dose was reduced to azacitidine 75 mg/m 2 daily for 5 days followed by docetaxel 75 mg/m 2 on day 6 with growth factor support. Prostate- specific antigen response was seen in 10 of 19 evaluable patients and objective response was observed in 3 of 10 evaluable patients. Significant demethylation of GADD45A was observed with azacitidine treatment. Conclusion: The combination of azacitidine, docetaxel, and prednisone with growth factor support is active in mCRPC patients.

Abstract PR03: Somatic mutations and ancestry markers in Hispanic lung cancer patients

Introduction: Hispanics are projected to constitute 23% of the U.S. population by 2050. However, large-scale sequencing projects, such as The Cancer Genome Atlas (TCGA), provide little information on this ethnic population. In fact, only seven out of over 500 lung adenocarcinoma tumors sequenced in the TCGA database are reported to be Hispanic. To address the lack of genomic data from Hispanic/Latino patients with lung cancer, the Latino Lung Cancer Registry was established to collect patient data and biospecimens from these patients. Methods: This retrospective observational study examined lung cancer tumor samples from 163 Hispanic/Latino patients, and tumor-derived DNA was subjected to targeted-exome sequencing (>1000 genes, including EGFR, KRAS, STK11, and TP53) and ancestry analysis. Mutation frequencies in this Hispanic/Latino cohort were compared with those in a similar cohort of non-Hispanic white (NHW) patients. Novel mutations in EGFR were functionally characterized, and mutation rates were correlated with ancestry, patient sex, smoking status, and tumor histology. Results: Among adenocarcinomas (n=120) in the Hispanic/Latino cohort, 31% had EGFR mutations versus 17% in the NHW control group (p Conclusions: Driver mutations in Hispanic/Latino lung adenocarcinoma patients differ in frequency from those in NHWs associated with their Indigenous American ancestry. The spectrum of driver mutations needs to be further assessed in the Hispanic/Latino population. Citation Format: William D. Cress, Nicholas T. Gimbrone, Bhaswati Sarcar, Edna Gordian, Jason I. Rivera, Christian Lopez, Jamie K. Teer, Eric A. Welsh, Alberto A. Chiappori, Matthew B. Schabath, Gary W. Reuther, Pedro G. Santiago-Cardona. Somatic mutations and ancestry markers in Hispanic lung cancer patients [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr PR03.

Abstract 4456: Novel oncogenic function of Notch4 in Hispanic lung cancer

Background: Molecular drivers of ~40% of lung adenocarcinomas are still unknown. We reasoned that since the frequencies of driver mutations have been shown to be different between ethnic groups, comparison of targeted exome sequencing data would identify genes with significant alteration frequencies between White Non-Hispanics (WNH) and other racial/ethnic groups. Notch signaling in cancer is context- and tissue-specific and may be both oncogenic and tumor suppressive. The mechanisms of action and genetic alterations involved in Notch oncogenesis have been studied for Notch1 and Notch3; specifically in Non-Small Cell Carcinoma (NSCLC), translocations in Notch3, and gain of function Notch1 mutations have been found in 1 in 10 patients. Notch4 is a mammary oncogene, but little is known about its role in NSCLC tumorigenesis. Truncations in Notch4 have been reported in lung cancer cell lines, and in public databases the frequency of Notch4 alterations in WNH adenocarcinoma is ~5.5%. However, little is known about the mechanisms by which Notch4 acts in lung oncogenesis. Methods: Using data from the TCCTM protocol, we analyzed targeted sequencing of 1,321 genes of interest to determine the mutations driving lung cancer in Hispanic (H/L) patients. The functional relevance of the Notch4 mutations was tested by cloning the intracellular domain (ICD) of Notch4 containing one of the mutations identified in the ANK domain (P1663Q) into pcDNA3.1. Plasmids expressing either the WT or a mutated Notch 4 (P1663Q) were transfected into A549 cells and the effect of this mutation tested on the ability to induce the expression of Notch4 expression genes. Results: In our H/L cohort we see Notch4 altered in about 20% of our samples, and 7/12 of the Notch4 single amino acid substitutions are in the Negative Regulatory Region (NRR). PROVEAN protein analysis of the Notch4 mutations shows that more than half of the mutations would disrupt the NRR. Introduction of the P1663Q mutation in the ANK repeats of the Notch4 ICD, which mediates binding to CSL and is essential for Notch transcriptional activity, resulted in a shift in the amount of transcript of known Notch4 target genes, such as Hes1 and Snai1. This effect is specific, as it is not observed with other genes, previously reported to be targets for other Notch paralogs such as Smad3 and Zeb1. A similar effect of the P1663Q Notch4 mutation on Hes1 transcript levels was observed in breast cancer cells, suggesting that this is not lung cancer specific. Transcriptional activity of the Notch-responsive luciferase construct (pHESLuc) was increased in the presence of Wt Notch4, and reduced in the presence of the P1663Q Notch4 Mu. Conclusion: The nature and high number of genetic alterations in Notch4 in H/L lung cancer patients, suggests that Notch4 may play a critical role in NSCLC tumorigenesis. Our data suggests that mutations in the ANK domain of Notch4 can interfere with its transcriptional activity and function, and should be further characterized. Citation Format: Edna Gordian, Nicholas Gimbrone, Antonio Pannuti, Lucio Miele, W. Douglas Cress, Teresita Munoz-Antonia. Novel oncogenic function of Notch4 in Hispanic lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4456. doi:10.1158/1538-7445.AM2017-4456

Abstract 1787A: Puerto Rico BioBank: The first cancer tissue biobank at a US Hispanic-serving institution

There are currently 580 million people worldwide considered to be of Hispanic origin. In the US Hispanics represent 17% of the population and its largest ethnic minority. Funded by NIH, Moffitt Cancer Center (Florida) and Ponce School of Medicine (Puerto Rico) initiated a partnership that conducts high-quality research, training, and community outreach focusing on cancer health disparities among Hispanics. A central component to this partnership is the establishment of the first cancer-focused biobank in the island, the Puerto Rico BioBank (PRBB). The PRBB has actively recruited cancer patients since 2009; over these years we have established essential collaborations and a functional infrastructure. Eligible cancer patients provide informed consent and agree to donate blood samples (for DNA) and solid tissue(fresh-frozen & paraffin embedded). Additionally, patients complete a comprehensive questionnaire related to clinical and lifestyle factors. Biospecimen collection at the PHSU follow standard operating procedures that mirror and often exceed NCI biobaking best practices for biospecimens and ensuring patient data confidentiality. Currently, the PRBB has over 1,100 consented patients (65.8% females, 34.2% males) all of Hispanic origin. The most common banked tumors are Breast, Prostate, Lung and Endometrium, although other tumors are represented. All the information and data are entered into a biospecimen management system (BMS) developed by the partnership, which allows for real-time biospecimen inventory management and reporting functionality. In conclusion, the Puerto Rican patient samples banked in the PRBB are a unique resource that can support multiple molecular and population studies. Also, a Hispanic-specific biobank can foster collaborations among local and international researchers that may facilitate the application of novel molecular techniques to solving cancer health disparities among Hispanics. Citation Format: Patricia Casbas-Hernandez, Idhaliz Flores, Edward Seijo, Domenico Coppola, Steve Eschrich, Rodrigo Carvajal-Pelaez, Sonia Abac, Dagmar Correa, Edna Gordian, Teresita Munoz-Antonia. Puerto Rico BioBank: The first cancer tissue biobank at a US Hispanic-serving institution. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1787A.

Abstract 1050: Alvespimycin (17-DMAG) blocks TGFβ-induced EMT and migration in A549 lung cancer cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Transforming Growth Factor β (TGFβ) can induce Epithelial-to-Messenchymal Transition (EMT) in Non-Small Cell Lung Cancer (NSCLC) leading to increased potential to migrate and metastasize. A unique Tumor Promoting signature (TGFβ-TP97) with genes highly associated with TGFβ-induced EMT was derived from human lung cancer cell line microarray experiments. The TGFβ-TP97 EMT signature was then searched against the Connectivity Map (CMAP) dataset for drugs that induce gene expression changes opposite to those of the TGFβ-TP97 signature, and identified 17-DMAG as a compound that could potentially inhibit EMT in NSCLC cells. 17-DMAG is an Hsp90 inhibitor recently described to have a stronger antiproliferative effect in NSCLC harboring EGFR mutations over EGFR wild type lung cancers cells. Furthermore, 17-DMAG has been recently shown to prevent TGFβ induced fibrosis. Methods: A549 and A549T (A549 cells grown in the presence of TGFβ for over two weeks, that have acquired a mesenchymal/fibroblast-like phenotype) were treated with increasing concentrations of 17-DMAG, both in the absence and presence of 5ng/mL TGFβ, and proliferation was measured through GLO assay after 72 hours. The effects of 17-DMAG on TGFβ signaling was tested by Western Blotting for phosphorylated Smads and markers of TGFβ-induced EMT. 17-DMAG effects on morphology and migration were also determined through wound healing and invasion assays. Results: 1) A549T cells grown in TGFβ are more sensitive to the effects of 17-DMAG than cells grown without TGFβ; 2) Pre-treatment of A549 cells with 17-DMAG blocks the induction of EMT markers such as Snail and the downregulation of Ecadherin; 3) 17-DMAG blocks TGFβ-induced phosphorylation of SMAD2, SMAD3; and 4) 17-DMAG inhibits migration and decreases motility of A549 cells in the presence of TGFβ. Conclusion: These results show that 17-DMAG is a potent inhibitor of the effects of TGFβ-induced EMT in A549 cells. It interferes with the regulated process of TGFβ signaling by its ability to block Smad activation and induction of EMT proteins. These findings suggest that 17-DMAG can potentially be used in the treatment of NSCLC cells to prevent disease dissemination and invasion. Citation Format: Edna Gordian, Eric Welsh, Teresita Munoz-Antonia. Alvespimycin (17-DMAG) blocks TGFβ-induced EMT and migration in A549 lung cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1050. doi:10.1158/1538-7445.AM2014-1050

Abstract 3395: TGFβ response signature in non-small cell lung carcinoma

Background: Transforming growth factor beta (TGFβ) is a cytokine involved in numerous cellular processes that include proliferation, migration and apoptosis. TGFβ is intriguing in that it plays a dual role in cancer; in some cases it functions as a tumor suppressor while in others it acts as a tumor promoter. Genetic alterations in the TGFβ signaling pathway have been reported in many cancers including breast, colon, gastric, lung, head and neck and pancreatic cancers. Correlations between a TGFβ induced gene expression and clinical outcomes have been described in breast and liver cancers, but not in non-small cell lung cancer (NSCLC). Because TGFβ has been shown to induce an epithelial-to-mesenchymal transition (EMT) in NSCLC that may lead to increased potential to invade and disseminate, we hypothesized that a TGFβ-induced gene expression signature might correlate with this transition and might predict prognosis in NSCLC patients. We therefore decided to attempt to identify gene expression changes induced by TGFβ that correlated with induced proliferation, migration, and EMT. We further investigated whether these TGFβ induced genes might be useful in the classification of NSCLC tumors. Methods: Ten different NSCLC cell lines were assessed for the ability of TGFβ-1 to induce migration, proliferation, and EMT. Cell lines were treated with TGFβ-1 for various time periods and the RNA collected for microarray analysis. Results: In nine out of the ten cell lines the TGFβ pathway was activated after treatment with TGFβ-1, but these lines differ in their TGFβ proliferation, migration and EMT responses. The H1944, H358, and A549 cell lines respond by shifting from an epithelial to a mesenchymal phenotype. Using the before and after treatment samples of these cell lines we looked for genes correlated with this transition. Initially, 632 probesets (534 genes) were identified that changed similarly in these 3 cell lines. Many of these changes seen after 120 hours of treatment were also seen at 48 hours but to a lesser degree. After Principal Component Analysis of the 534 genes in tumor sample a core set of 70 genes were retained and used to analyze lung cancer tumor samples. Using two independently derived sets of lung tumors the EMT signature could be used to identify 3 classes of tumors: tumors with high EMT gene expression, tumors with low EMT expression, and a distinct group of tumors that do not coordinately express the EMT genes identified in this study. Conclusion: Analysis of the differential responses of NSCLC cell lines to TGFβ-1 allows us to group NSCLC cell lines into at least three groups based on their response to TGFβ treatment. Using an EMT signature developed from the cell lines it appears that tumor samples can also be classified into three or more distinct groups that may differ in their in vivo responses to TGFβ-1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3395. doi:1538-7445.AM2012-3395