Craig M. Hooker

The prevalence of psychological distress by cancer site

The goal of this project was to determine the prevalence of psychological distress among a large sample of cancer patients (n=4496). In addition, variations in distress among 14 cancer diagnoses were examined.

DNA Methylation Markers and Early Recurrence in Stage I Lung Cancer

BACKGROUND Despite optimal and early surgical treatment of non-small-cell lung cancer (NSCLC), many patients die of recurrent NSCLC. We investigated the association between gene methylation and recurrence of the tumor. METHODS Fifty-one patients with stage I NSCLC who underwent curative resection but who had a recurrence within 40 months after resection (case patients) were matched on the basis of age, NSCLC stage, sex, and date of surgery to 116 patients with stage I NSCLC who underwent curative resection but who did not have a recurrence within 40 months after resection (controls). We investigated whether the methylation of seven genes in tumor and lymph nodes was associated with tumor recurrence. RESULTS In a multivariate model, promoter methylation of the cyclin-dependent kinase inhibitor 2A gene p16, the H-cadherin gene CDH13, the Ras association domain family 1 gene RASSF1A, and the adenomatous polyposis coli gene APC in tumors and in histologically tumor-negative lymph nodes was associated with tumor recurrence, independently of NSCLC stage, age, sex, race, smoking history, and histologic characteristics of the tumor. Methylation of the promoter regions of p16 and CDH13 in both tumor and mediastinal lymph nodes was associated with an odds ratio of recurrent cancer of 15.50 in the original cohort and an odds ratio of 25.25 when the original cohort was combined with an independent validation cohort of 20 patients with stage I NSCLC. CONCLUSIONS Methylation of the promoter region of the four genes in patients with stage I NSCLC treated with curative intent by means of surgery is associated with early recurrence.

Combination Epigenetic Therapy Has Efficacy in Patients with Refractory Advanced Non Small Cell Lung Cancer

UNLABELLED Epigenetic alterations are strongly associated with the development of cancer. We conducted a phase I/II trial of combined epigenetic therapy with azacitidine and entinostat, inhibitors of DNA methylation and histone deacetylation, respectively, in extensively pretreated patients with recurrent metastatic non-small cell lung cancer. This therapy is well tolerated, and objective responses were observed, including a complete response and a partial response in a patient who remains alive and without disease progression approximately 2 years after completing protocol therapy. Median survival in the entire cohort was 6.4 months (95% CI 3.8-9.2), comparing favorably with existing therapeutic options. Demethylation of a set of 4 epigenetically silenced genes known to be associated with lung cancer was detectable in serial blood samples in these patients and was associated with improved progression-free (P = 0.034) and overall survival (P = 0.035). Four of 19 patients had major objective responses to subsequent anticancer therapies given immediately after epigenetic therapy. SIGNIFICANCE This study demonstrates that combined epigenetic therapy with low-dose azacitidine and entinostat results in objective, durable responses in patients with solid tumors and defines a blood-based biomarker that correlates with clinical benefit.

Elevated Incidence of Lung Cancer Among HIV-Infected Individuals

PURPOSE People with HIV infection in the United States frequently smoke tobacco. We sought to characterize lung cancer incidence among HIV-infected individuals, examine whether cancer risk was related to HIV-induced immunosuppression, and assess whether the high prevalence of smoking explained elevated risk. METHODS We conducted a retrospective cohort study at an HIV specialty clinic in Baltimore, MD (1989-2003). Incident lung cancers were identified using hospital records. We used negative binomial regression to compare incidence across subgroups defined by demographics, use of highly active antiretroviral therapy (HAART), and HIV markers. Standardized incidence ratios (SIRs) compared incidence with an urban reference population (Detroit, MI). We adjusted SIRs for the effect of smoking, using smoking prevalences estimated from part of the cohort and the general population. 95% CIs and P values were two sided. RESULTS Thirty-three lung cancers were observed among 5,238 HIV-infected patients (incidence: 170 per 100,000 person-years). Incidence increased with age (P < .0001), but did not differ by sex, race, or CD4 count. Incidence tended to increase with calendar year (P = .09) and HAART use (P = .10), and was inversely related to HIV viral load (P = .03), but these associations were attenuated with age adjustment. The SIR was 4.7 (95% CI, 3.2 to 6.5) versus the general population. Twenty-eight lung cancer patients (85%) and 69% of the cohort were smokers. After smoking adjustment, risk remained elevated (SIR, 2.5; 95% CI, 1.6 to 3.5). CONCLUSION Lung cancer risk was substantially elevated in HIV-infected individuals. Incidence was unrelated to HIV-induced immunosuppression. Notably, incidence remained high after adjustment for smoking, suggesting the involvement of additional factors.

Promoter Hypermethylation of Resected Bronchial Margins: A Field Defect of Changes?

Purpose: Histologically positive bronchial margins after resection for non-small cell lung cancer are associated with shortened patient survival due to local recurrence. We hypothesized that DNA promoter hypermethylation changes at bronchial margins could be detected in patients with no histological evidence of malignancy and that they would reflect epigenetic events in the primary tumor. Experimental Design: Bronchial margins, primary tumor, bronchoalveolar fluid, and paired nonmalignant lung were obtained from 20 non-small cell lung cancer patients who underwent a lobectomy or greater resection. Disease-specific recurrence was the primary end point. The methylation status of p16, MGMT, DAPK, SOCS1, RASSF1A, COX2, and RARβ was examined using methylation-specific polymerase chain reaction. Results: All malignancies had methylation in at least one locus. Concordance of one gene with an identical epigenetic change in the tumor or bronchial margin was observed in 85% of patients. Only one patient had methylation at the bronchial margin for a gene that was not methylated in the corresponding tumor. Median time to recurrence was 37 months (range, 5–71 months). There were two local recurrences and five metastases. There were no significant correlations between DNA methylation in tumor, margins, or bronchoalveolar fluid specimens and either regional recurrence or distant metastases. Conclusions: Histologically negative bronchial margins of resected non-small cell lung cancer exhibit frequent hypermethylation changes in multiple genes. These hyper-methylation abnormalities are also present in the primary tumor and thus may represent a field defect of preneoplastic changes that occurs early in carcinogenesis.

Aberrant Hypermethylation Tumor Suppressor Genes in Pancreatic Endocrine Neoplasms

Objective Because histologic features can be unreliable in determining the malignant potential of pancreatic endocrine neoplasms (PENs), we characterized the methylation patterns of PENs to develop a molecular marker system useful for clinical prognosis. Summary Background Data Aberrant promoter methylation of tumor suppressor genes is associated with a loss of gene function that can afford selective growth advantages to neoplastic cells. Gene hypermethylation, coupled with sporadic genetic mutations, defines the heterogeneous biology of human neoplasms. Methods Forty-eight well-differentiated PENs were subjected to methylation-specific polymerase chain reaction to detect aberrant methylation associated with 11 candidate tumor suppressor genes (INK4a/p16, APC, O6-MGMT, hMLH1, p73, E-cadherin, RAR-&bgr;, p14ARF, GST-&pgr;, TIMP3, and RASSF1A). Methylation differences among PENs, subdivided according to tumor size, lymph node status, or liver metastasis, were analyzed, and the association of gene methylation with tumor recurrence and patient survival was evaluated. Results Aberrant hypermethylation of any of the 11 tumor suppressor genes was detected in 87% of the PENs. In decreasing order of frequency, the 5 most commonly methylated genes were: RASSF1A (75%), INK4a/p16 (40%), O6-MGMT (40%), RAR-&bgr; (25%), and hMLH1 (23%). In general, tumors larger than 5 cm and those associated with lymph node or hepatic metastases exhibited a higher frequency of methylation at each promoter site compared with PENs without malignant histologic features. The methylation of specific tumor suppressor genes was an independent predictor of early PEN recurrence and decreased 5-year survival following surgical resection. The accumulation of methylation of multiple tumor suppressor genes was associated with early tumor recurrence and reduced survival among a subpopulation of patients with lymph node-negative PENs. Conclusions Aberrant methylation of multiple tumor suppressor genes is associated with advanced tumor stage and identifies molecularly distinct PENs with identical histologic characteristics. The methylation status of specific tumor suppressor genes is predictive of PEN behavior.

Pulmonary resection in octogenarians with stage I nonsmall cell lung cancer: a 22-year experience.

BACKGROUND Recent reports indicate that age is not a contraindication to pulmonary resection for octogenarians with nonsmall cell lung cancer (NSCLC), but other data are lacking. The purpose of this study was to determine outcomes in these patients, particularly short- and long-term survival with stage I disease. METHODS A retrospective cohort of 68 octogenarians with NSCLC who underwent curative resection from 1980 to 2002 was followed-up for outcomes. RESULTS Median age was 82 years old (range, 80-87 years old) consisting of 44 males (65%), with a mean follow-up of 32 months (range, 1-178 months). Operations included: 47 lobectomies (69%), 11 wedge resections (16%), 5 segmentectomies (8%), 4 bilobectomies (6%), and 1 pneumonectomy (1%). There were 31 adenocarcinomas (46%), 18 squamous carcinomas (26%), 12 bronchioalveolar carcinomas (18%), 4 large cell carcinomas (6%), and 3 miscellaneous malignant neoplasms (4%). Median hospital stay was 7 days (range, 3-53 days). Thirty-day mortality was 8.8% (n = 6) with 83% developing cardiopulmonary complications. Overall actuarial survival at 1, 3, and 5 years was 73%, 51%, and 34%, respectively. Of 41 patients (60%) with stage I disease, 23 were T1 lesions. Five-year survival was significantly different between stages Ia and Ib patients (61% and 10%, respectively, p = 0.001). Patients in more advanced stages had a 5-year survival of 3/27 (11%). Multivariate analysis identified advanced tumor stage, lower ASA physical status, and low FEV(1) as factors associated with poorer long-term survival. CONCLUSIONS The 5-year survival, particularly in patients with stage Ia tumors with favorable ASA and FEV(1), supports the notion that health status and tumor stage outweigh chronologic age in determining surgical candidates.

Hypermethylation of the GATA Genes in Lung Cancer

Purpose: In lung cancer, DNA hypermethylation is known to be a common event. Experimental Design: Gene expression and methylation status of GATA-4, GATA-5, and GATA-6 were analyzed with cell lines and primary human lung cancers. Methylation profiles of primary lung cancers were analyzed and correlated with clinical as well as histopathological data. Results: Complete methylation was detected by methylation-specific PCR for both GATA-4 and GATA-5 in four cell lines (H358, DMS-53, A549, and H1299), all of which had no expression by reverse transcription-PCR analysis. Demethylation with 5-aza-2′deoxycytidine restored expression in each case. GATA-6 was ubiquitously expressed in all of the six cell lines. GATA-4 bisulfite sequencing revealed complete methylation of the GATA-4 promoter in H358 cells, correlating well with its lack of expression at the mRNA level. Only a few CpG sites showed methylation by bisulfite sequencing within the GATA-4 promoter in a cell line that expressed the gene. In 63 cases of primary lung cancers, GATA-4 and GATA-5 promoter methylation was detected in (42 of 63) 67% and (26 of 63) 41%, respectively. GATA-6 remained unmethylated both in cell lines and primary tumors. Six autopsy specimens of normal lung tissue showed no aberrant promoter hypermethylation for the GATA genes. Correlation of concomitant GATA-4 and GATA-5 methylation with clinicopathological parameters only found a statistically significant increase in methylation frequency with increasing patient age (P < 0.001). Conclusions: These epigenetic changes in the GATA genes in lung cancer are tumor-specific, relate to the loss of GATA gene expression, and occur increasingly in the elderly.

Genomic and Epigenomic Integration Identifies a Prognostic Signature in Colon Cancer

Purpose: The importance of genetic and epigenetic alterations maybe in their aggregate role in altering core pathways in tumorigenesis. Experimental Design: Merging genome-wide genomic and epigenomic alterations, we identify key genes and pathways altered in colorectal cancers (CRC). DNA methylation analysis was tested for predicting survival in CRC patients using Cox proportional hazard model. Results: We identified 29 low frequency-mutated genes that are also inactivated by epigenetic mechanisms in CRC. Pathway analysis showed the extracellular matrix (ECM) remodeling pathway is silenced in CRC. Six ECM pathway genes were tested for their prognostic potential in large CRC cohorts (n = 777). DNA methylation of IGFBP3 and EVL predicted for poor survival (IGFBP3: HR = 2.58, 95% CI: 1.37–4.87, P = 0.004; EVL: HR = 2.48, 95% CI: 1.07–5.74, P = 0.034) and simultaneous methylation of multiple genes predicted significantly worse survival (HR = 8.61, 95% CI: 2.16–34.36, P < 0.001 for methylation of IGFBP3, EVL, CD109, and FLNC). DNA methylation of IGFBP3 and EVL was validated as a prognostic marker in an independent contemporary-matched cohort (IGFBP3 HR = 2.06, 95% CI: 1.04–4.09, P = 0.038; EVL HR = 2.23, 95% CI: 1.00–5.0, P = 0.05) and EVL DNA methylation remained significant in a secondary historical validation cohort (HR = 1.41, 95% CI: 1.05–1.89, P = 0.022). Moreover, DNA methylation of selected ECM genes helps to stratify the high-risk stage 2 colon cancers patients who would benefit from adjuvant chemotherapy (HR: 5.85, 95% CI: 2.03–16.83, P = 0.001 for simultaneous methylation of IGFBP3, EVL, and CD109). Conclusions: CRC that have silenced genes in ECM pathway components show worse survival suggesting that our finding provides novel prognostic biomarkers for CRC and reflects the high importance of integrative analyses linking genetic and epigenetic abnormalities with pathway disruption in cancer. Clin Cancer Res; 17(6); 1535–45. ©2011 AACR.

Epigenetic alteration of Wnt pathway antagonists in progressive glandular neoplasia of the lung

BACKGROUND Atypical adenomatous hyperplasia (AAH) is now recognized as a precursor lesion from which lung adenocarcinomas arise and thus represents an ideal target for studying the early genetic and epigenetic alterations associated with lung tumorigenesis such as alterations of the Wnt pathway. METHODS We assessed the level of Wnt signaling activity in lung cancer cell lines by determining the level of active beta-catenin and determined the level of expression of Wnt antagonists APC, DKK1, DKK3, LKB1, SFRP1, 2, 4, 5, WIF1 and RUNX3 using reverse transcription-polymerase chain reaction. Using multiplex nested methylation-specific polymerase chain reaction, we analyzed promoter region methylation of these genes in resected lung tissue in the histopathologic sequence of glandular neoplasia (normal lung parenchyma, low-grade and high-grade AAH, adenocarcinoma). RESULTS The majority of non-small cell lung cancer cell lines (11 of 16, 69%) have evidence of active Wnt signaling and silencing of Wnt antagonists correlated with promoter hypermethylation. Promoter region methylation of Wnt antagonists was common in primary lung adenocarcinoma and there was a significant increase in the frequency of methylation for Wnt antagonist genes and the number of genes methylated with each stage of tumorigenesis (test for rend P <or= 0.01). Additionally, odds ratios for promoter hypermethylation of individual or multiple Wnt antagonist genes and adenocarcinomas were statistically significantly elevated and ranged between 3.64 and 48.17. CONCLUSION These results show that gene silencing of Wnt antagonists by promoter hypermethylation occurs during the earliest stages of glandular neoplasia of the lung and accumulates with progression toward malignancy.