James Shin

Comprehensive genomic analysis identifies SOX2 as a frequently amplified gene in small-cell lung cancer

Small-cell lung cancer (SCLC) is an exceptionally aggressive disease with poor prognosis. Here, we obtained exome, transcriptome and copy-number alteration data from approximately 53 samples consisting of 36 primary human SCLC and normal tissue pairs and 17 matched SCLC and lymphoblastoid cell lines. We also obtained data for 4 primary tumors and 23 SCLC cell lines. We identified 22 significantly mutated genes in SCLC, including genes encoding kinases, G protein–coupled receptors and chromatin-modifying proteins. We found that several members of the SOX family of genes were mutated in SCLC. We also found SOX2 amplification in ∼27% of the samples. Suppression of SOX2 using shRNAs blocked proliferation of SOX2-amplified SCLC lines. RNA sequencing identified multiple fusion transcripts and a recurrent RLF-MYCL1 fusion. Silencing of MYCL1 in SCLC cell lines that had the RLF-MYCL1 fusion decreased cell proliferation. These data provide an in-depth view of the spectrum of genomic alterations in SCLC and identify several potential targets for therapeutic intervention.

Neoadjuvant chemoradiation therapy is beneficial for clinical stage T2 N0 esophageal cancer patients due to inaccurate preoperative staging.

BACKGROUND It remains unclear if patients with clinical stage T2 N0 (cT2 N0) esophageal cancer should be offered induction therapy vs surgical intervention alone. METHODS This was a retrospective cohort study of cT2 N0 patients undergoing induction therapy, followed by surgical resection, or resection alone, at the Johns Hopkins Hospital from 1989 to 2009. Kaplan-Meier analysis was used to compare all-cause mortality in cT2 N0 patients who had resection alone vs those who had induction chemoradiation therapy, followed by resection. RESULTS A study cohort of 69 patients was identified and divided into two groups: 55 patients (79.7%) received induction therapy and 14 (20.3%) did not. No statistically significant difference in 5-year survival rate was observed for the two groups: 49.5% for the resection-only group and 53.8% for the induction group. More than 50% of cT2 N0 patients were understaged. CONCLUSIONS For cT2 N0 esophageal cancer patients, the benefit of neoadjuvant therapy is still unclear. Induction therapy for cT2 N0 did not translate into a statistically significant improvement in survival. However, due to the significant understaging of T2 N0 patients, we recommend neoadjuvant therapy to all cT2N0 patients before operation.

Long-term Survival Outcomes by Smoking Status in Surgical and Nonsurgical Patients With Non-small Cell Lung Cancer: Comparing Never Smokers and Current Smokers

BACKGROUND Survival outcomes of never smokers with non-small cell lung cancer (NSCLC) who undergo surgery are poorly characterized. This investigation compared surgical outcomes of never and current smokers with NSCLC. METHODS This investigation was a single-institution retrospective study of never and current smokers with NSCLC from 1975 to 2004. From an analytic cohort of 4,546 patients with NSCLC, we identified 724 never smokers and 3,822 current smokers. Overall, 1,142 patients underwent surgery with curative intent. For survival analysis by smoking status, hazard ratios (HRs) were estimated using Cox proportional hazard modeling and then further adjusted by other covariates. RESULTS Never smokers were significantly more likely than current smokers to be women (P < .01), older (P < .01), and to have adenocarcinoma (P < .01) and bronchioloalveolar carcinoma (P < .01). No statistically significant differences existed in stage distribution at presentation for the analytic cohort (P = .35) or for the subgroup undergoing surgery (P = .24). The strongest risk factors of mortality among patients with NSCLC who underwent surgery were advanced stage (adjusted hazard ratio, 3.43; 95% CI, 2.32-5.07; P < .01) and elevated American Society of Anesthesiologists classification (adjusted hazard ratio, 2.18; 95% CI, 1.40-3.40; P < .01). The minor trend toward an elevated risk of death on univariate analysis for current vs never smokers in the surgically treated group (hazard ratio, 1.20; 95% CI, 0.98-1.46; P = .07) was completely eliminated when the model was adjusted for covariates (P = .97). CONCLUSIONS Our findings suggest that smoking status at time of lung cancer diagnosis has little impact on the long-term survival of patients with NSCLC, especially after curative surgery. Despite different etiologies between lung cancer in never and current smokers the prognosis is equally dismal.

Cigarette smoke induces epithelial to mesenchymal transition and increases the metastatic ability of breast cancer cells

BackgroundRecent epidemiological studies demonstrate that both active and involuntary exposure to tobacco smoke increase the risk of breast cancer. Little is known, however, about the molecular mechanisms by which continuous, long term exposure to tobacco smoke contributes to breast carcinogenesis because most previous studies have focused on short term treatment models. In this work we have set out to investigate the progressive transforming effects of tobacco smoke on non-tumorigenic mammary epithelial cells and breast cancer cells using in vitro and in vivo models of chronic cigarette smoke exposure.ResultsWe show that both non-tumorigenic (MCF 10A, MCF-12A) and tumorigenic (MCF7) breast epithelial cells exposed to cigarette smoke acquire mesenchymal properties such as fibroblastoid morphology, increased anchorage-independent growth, and increased motility and invasiveness. Moreover, transplantation experiments in mice demonstrate that treatment with cigarette smoke extract renders MCF 10A cells more capable to survive and colonize the mammary ducts and MCF7 cells more prone to metastasize from a subcutaneous injection site, independent of cigarette smoke effects on the host and stromal environment. The extent of transformation and the resulting phenotype thus appear to be associated with the differentiation state of the cells at the time of exposure. Analysis by flow cytometry showed that treatment with CSE leads to the emergence of a CD44hi/CD24low population in MCF 10A cells and of CD44+ and CD49f + MCF7 cells, indicating that cigarette smoke causes the emergence of cell populations bearing markers of self-renewing stem-like cells. The phenotypical alterations induced by cigarette smoke are accompanied by numerous changes in gene expression that are associated with epithelial to mesenchymal transition and tumorigenesis.ConclusionsOur results indicate that exposure to cigarette smoke leads to a more aggressive and transformed phenotype in human mammary epithelial cells and that the differentiation state of the cell at the time of exposure may be an important determinant in the phenotype of the final transformed state.

Human immunodeficiency virus infection as a prognostic factor in surgical patients with non-small cell lung cancer

BACKGROUND The purpose of this study was to assess the effect of human immunodeficiency virus (HIV) infection on postoperative survival among non-small cell lung cancer (NSCLC) patients. METHODS A retrospective cohort study compared 22 HIV-infected lung cancer patients to 2,430 lung cancer patients with HIV-unspecified status who underwent resection at Johns Hopkins Hospital from 1985 to 2009. Subcohort comparative analyses were performed using individual matching methods. RESULTS Thirty-day mortality rates did not differ between HIV-infected and HIV-unspecified patients. Survival rates for HIV-infected lung cancer patients were significantly shorter than for HIV-unspecified patients (median, 26 versus 48 months; p=0.001). After adjustment, the relative hazard of mortality among HIV-infected NSCLC patients was more than threefold that of HIV-unspecified patients (adjusted hazard ratio, 3.08; 95% confidence interval: 1.85 to 5.13). When additional surgical characteristics were modeled in a matched subcohort, the association remained statistically significant (adjusted hazard ratio, 2.31; 95% confidence interval: 1.11 to 4.81). Moreover, HIV-infected lung cancer patients with CD4 counts less than 200 cells/mm3 had shortened median survival compared with patients whose CD4 counts were 200 cells/mm3 or greater (8 versus 40 months; p=0.031). Postoperative pulmonary and infectious complications were also elevated in the HIV-infected group (p=0.001 and p<0.001, respectively). After surgery, median time to cancer progression was shorter among HIV-infected patients (20.4 months) versus HIV-unspecified patients (p=0.061). CONCLUSIONS The HIV-infected NSCLC patients have more postoperative complications, rapid progression to disease recurrence, and poorer postoperative survival. Optimizing immune status before surgery and careful patient selection based on diffusion capacity of lung for carbon monoxide may improve patient outcomes.

A murine xenograft model of spontaneous metastases of human lung adenocarcinoma.

BACKGROUND The flank is commonly used for primary xenografts in mice, but it is rare for these tumors to metastasize. Tail vein injection creates a pattern of metastases, but is artificial. We hypothesized that the liver is a convenient alternative xenograft site and that metastases would gradually proceed spontaneously. MATERIALS AND METHODS Using 15 NOD.CB17-Prkdc(scid)/NcrCrl (NOD/SCID) mice, 10,000 A549 cells were xenografted into the liver while a second group of five mice were xenografted in the flank with 100,000 A549 cells as a control. Mice were euthanized and grossly dissected at 7 wk. A third group of seven mice received liver xenografts with A549 and a mouse each week was euthanized for 7 wk and evaluated. The liver, lung, and spleen were examined histologically. RESULTS At 7 wk, 15/15 liver xenografted mice had gross primary tumor in the liver. Histologic review confirmed multiple microscopic foci of metastatic disease in all mice (15/15) throughout the lungs, mediastinal nodes, and spleen. The control group had primary tumor in the flank (4/5), but none had histologic evidence of metastases. Serially euthanized liver xenografted mice revealed evidence of a gradual spontaneous metastatic model system with the first histologic findings of micrometastases appearing in the lungs by wk 5, which became wide spread by wk 7. Splenic and mediastinal lymph node metastases developed in wk 6 and 7. CONCLUSIONS Liver xenografting of A549 cells into NOD/SCID mice is a reliable way of developing widespread micrometastases. This model allows the study of a gradually developing solid tumor with subsequent metastatic spread.

Abstract 3679: Preliminary results from a patient group, excluded from the National Lung Cancer Screening Trial, who are at high risk for lung cancer- heavy smokers with HIV

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Purpose: Within the HIV population, the incidence of lung cancer is estimated to be 2-4 times that of the general population. Of the non AIDS associated malignancies, lung cancer is the leading cause of death among HIV patients who develop malignancy because of its advanced stage of presentation. The National Lung Cancer Screening Trial recently reported a 20% reduction in mortality for patients undergoing low dose computed tomography (CT) screening, but individuals with HIV diagnosis were excluded. To report the results of an observational pilot study of lung cancer CT screening in a cohort of HIV heavy smokers. Materials and Methods: A prospective cohort study was performed with 185 asymptomatic individuals with confirmed HIV diagnosis who had smoked 20 pack-years or more. All participants had baseline (prevalence) chest CT scanning and 107 patients (58.2%) also received at least one subsequent annual (incidence) examination of the chest and upper abdomen. All patients underwent hand held spirometry testing, cytologic analysis of sputa, and detailed health status questioning. Results: Of the 185 individuals, there were 31.4% females, 68.6% males, 90.3% African-Americans, 8.7% Caucasian, and 1.1% Hispanic. The median age at enrolment was 48 years, and the median number of smoking pack-years was 34. Non-calcified nodules were detected in 4 participants (2.1%) by low-dose prevalent CT compared to no nodules on incident CT scans. Only one patient with malignancy was detected (0.54%) on prevalent scanning and none on incident scanning. Stage of malignancy was advanced. No biopsies were undertaken for benign disease. CT evidence of COPD and coronary artery disease were present in 24% and 27% of patients, respectively. Additional CT findings of clinical importance were noted in the chest in 132 (71.7%) patients and extrathoracically in 30 (16.3%) of patients, respectively. Conclusion: The prevalence results of this pilot study are in line with other observational studies of greater sample size for HIV indeterminate patients. Although the sample size is small, it suggests no elevated incidence of lung cancer in HIV heavy smokers. Although the rate of incident scanning is low, CT screening of HIV patients is feasible. This justifies accruing a larger sample of HIV patients perhaps from an already well defined, large, prospective cohort of HIV heavy smokers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3679. doi:10.1158/1538-7445.AM2011-3679

Abstract 1600: A xenograft model of spontaneous metastases in NOD SCID mice of human non-small cell lung cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction: Xenograft models of human non small cell lung cancer cell lines have been unreliable in studying distal organ metastases. Although the flank, as a primary xenograft site, is typically used for the convenience of measurement, rarely do even the most aggressive cell lines develop metastases. Methods: Using 15 anesthetized NOD.CB17-Prkdcscid/NcrCrl (NOD/SCID) mice, we xenografted 10,000 A549 cells into the liver under direct visualization and allowed the tumors to grow for 7 weeks. The mice were then euthanized, perfused with formalin, and grossly dissected. The liver, lung and spleen were processed, embedded in paraffin, sectioned and stained. A second group of 7 NOD/SCID mice were subsequently xenografted in the liver with A549, and a mouse each week was euthanized for 7 weeks and evaluated. Results: At 7 weeks, all 15 mice had gross primary tumor in the liver as well as grossly evident splenic (10/15) and mediastinal metastases (5/15). There were no gross pulmonary metastases observed (0/15). Histologic review, however, confirmed multiple microscopic foci of metastatic disease in all mice (15/15) throughout the lungs bilaterally, the mediastinal lymph nodes and spleen. Serially euthanized mice revealed histological evidence of a gradual spontaneous metastatic model system. Histologically evident primary tumor was apparent by week 1 with gross primary tumor development in the liver by week 2. Weeks 3 and 4 showed gross and histologic progression of primary tumor size with no apparent evidence of microscopic distal metastases. By week 5, the first histological findings of micrometastases appeared in the lungs which became wide spread in all the pulmonary parenchyma by week 7. Splenic and mediastinal lymph node involvement were observed histologically by week 6. Conclusions: Liver xenografting of A549 into NOD/SCID mice is a reliable way of developing widespread micrometastases to the lungs, mediastinal lymph nodes, and spleen. This model is clinically relevant since, unlike tail vein injection, it allows the study of the biology and treatment of a gradually developing primary lung tumor with subsequent metastatic spread. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1600. doi:10.1158/1538-7445.AM2011-1600

Abstract 2618: DNMT1 as a marker of differential sensitivities to epigenetic therapy of a Kras mutant and Kras wild type human non small cell lung cancer cell line

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL INTRODUCTION: Epigenetic therapy with a demethylating agent and an histone deacetylase inhibitor (HDACi) is now being pursued in hematological as well as solid tumor malignancies. Clinical responses have been varied with complete responses in some patients and no efficacy of the drugs in others. We investigated the effect of this therapy on two targets, DNA methyltransferase1 (DNMT1) and H3K4 modified histones, respectively. METHODS: Two human lung adenocarcinoma cell lines, A549 (Kras mutant) and H838 (Kras wild type) were pre-exposed for 72 hours with daily treatment of 500nM azacitidine alone or 72 hours of daily 500nM azacitidine followed subsequently with 10 nM, 100nM, 1000nM entinostat at 96 hours. DNMT1 and H3K4 protein levels were assayed by Western blot analysis at various time points after treatment. Cells were also re-plated after treatment to assess differential growth characteristics. RESULTS: In vitro growth of H838 cells is sensitive to azacitidine alone and in combination with entinostat whereas growth of the A549 cell line is only sensitive to azacitidine and entinostat in combination. Treatment with azacitidine, severely depleted DNMT1 protein expression in H838 cells both when given alone and in combination with most dose levels of entinostat. This decrease was transient and within 4 days, the protein was fully replenished. Azacitidine alone did not cause an appreciable decrease in DNMT1 protein expression of A549 cells but, when combined with increasing levels of entinostat, DNMT1 levels were significantly depleted and did not replenish for 7 days. Re-plating after treatment showed growth inhibition versus mock of all regimens containing azacitidine in the H838 cell line only whereas growth inhibition in the A549 cells was observed only in the combinatory therapy arms. Furthermore, in the A549 cell line, H3K4 protein levels (a marker of active gene expression) peaked at 7 days after treatment versus a peak in the first 24 hours in the H838 cell line. CONCLUSIONS: Differing sensitivities to epigenetic therapy in lung adenocarcinoma cell lines may reflect underlying differences in molecular phenotypes. Large decreases in DNMT1 expression are associated with sensitivity to azacitidine. In a cell line insensitive to azacidine alone, the addition of entinostat may accentuate DNMT1 protein depletion and delay its reexpression. This effect on DNMT1 may render this latter cancer cell line more sensitive to epigenetic therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2618. doi:10.1158/1538-7445.AM2011-2618