Citations

Circulating tumor cells (CTCs) in blood are associated with poor survival of patients with breast, prostate, or colon cancer. We hypothesized that CTCs are associated with poor survival of patients with cholangiocarcinoma (CCA). Eighty‐eight patients with CCA were prospectively enrolled at Mayo Clinic Rochester between June 2010 and September 2014. The CellSearch system by Veridex was used for detection of CTCs in peripheral blood. Associations between CTC, patient and tumor characteristics, and survival were examined using the Cox's proportional hazards model. Fifteen patients (17%) were positive for CTC ≥2 and 8 patients (9%) for CTC ≥5. CTCs were associated with tumor extent. CTC ≥2 (hazard ratio [HR]: 2.5; 95% confidence interval [CI]: 1.1‐5.4; P = 0.02) and CTC ≥5 (HR, 4.1; 95% CI: 1.4‐10.8; P = 0.01) were both independent predictors of survival. In subgroup analyses, CTC ≥2 (HR, 8.2; 95% CI: 1.8‐57.5; P < 0.01) and CTC ≥5 (HR, 7.7; 95% CI: 1.4‐42.9; P = 0.02) were both associated with shorter survival among patients with metastasis. There was a trend toward association of CTC ≥5 with shorter survival in patients with nonmetastatic CCA (HR, 4.3; 95% CI: 1.0‐13.8; P = 0.06). CTC ≥2 (HR, 10.5; 95% CI: 2.2‐40.1; P < 0.01) and CTC ≥5 (HR, 10.2; 95% CI: 1.5‐42.3; P = 0.02) were both associated with shorter survival among patients with perihilar/distal CCA. CTC ≥5 was associated with shorter survival of patients with intrahepatic CCA (HR, 4.2; 95% CI: 1.1‐14.1; P = 0.04). Conclusion: CTCs were associated with more‐aggressive tumor characteristics and independently associated with survival in patients with CCA. Assessment of CTCs may be useful for identifying CCA patients at risk of early mortality. (Hepatology 2016;63:148–158)

Abstract Children who are exposed to secondhand smoke (SHS) are faced with an increased risk of nicotine dependence later in life. We have previously shown that children exposed to SHS have an increased risk of lung cancer as adults. Furthermore, genetic variation plays a role in the susceptibility of individuals to lung cancer. The gene DRD1 mediates dopamine signaling following nicotine exposure, thus we reasoned that DRD1 may be related to lung cancer via modulation of the nicotine reward pathway. Our previous studies identified 4 SNPs (rs4809294, rs2292975, rs4861327, rs686) that were predicted to modulate a microRNA binding site and we assessed the association of these SNPs with lung cancer risk. Our current study investigated whether these SNPs were associated with nicotine dependence and whether this had a subsequent effect on lung cancer risk. We used the National Cancer Institute-Maryland lung cancer case-control study. Cases and controls were recruited from the greater Baltimore metropolitan area. We used ordinal logistic regression to examine the association between SNPs and nicotine dependence. Logistic regression adjusted for age, gender and smoking was used to assess the association between SNPs and lung cancer risk. No significant associations were found between genotypes in the 4 SNPs and nicotine dependence levels (n=1,450). Individuals with the heterozygous allele (AG) exposed to SHS during childhood had increased odds of lung cancer compared to those with the homozygous wild type allele (AA) for rs686 (OR=1.52, CI= 1.12, 2.07). Individuals with the homozygous variant allele (GG) exposed to childhood SHS had increased odds of lung cancer compared to individuals with the homozygous wild type allele (AA) (OR=2.11, CI= 1.45, 3.10). Individuals with the heterozygous allele (GA) and the highest levels of nicotine dependence who were also exposed to childhood SHS, had higher odds of lung cancer than those with the homozygous recessive allele (AA) (OR=3.50, CI=1.37, 8.96). Consistent with earlier findings, heterozygotes (AG) had increased odds of lung cancer but this was not statistically significant (OR=1.90, CI= 0.82, 4.40). To further explore this association, we examined the effect of race on this relationship. Caucasians with the heterozygous allele (AG), the highest levels of nicotine dependence and childhood SHS, had increased odds of lung cancer compared to those with the homozygous recessive allele (AA) for rs686 (OR=1.47, CI=1.06, 2.05). Caucasians with the homozygous allele (GG) who were exposed to childhood SHS, had higher odds of lung cancer compared to individuals with the homozygous recessive allele (AA) for rs686 (OR=1.84, CI=1.15, 2.98). No association was found among African Americans. Although none of the SNPs examined were found to be associated with nicotine dependence, it appears that variations in rs686 may modify an individual's susceptibility to lung cancer due to SHS exposure. Citation Format: Victoria A. Zigmont, Brid M. Ryan, Jin Jen, Ana I. Robles, Cain McClary, Kara Calhoun, Elise D. Bowman, Kirsi Vähäkangas, K. Leigh Greathouse, Wang Yi, Angela S. Wenzlaff, Bo Deng, Ping Yang, Ann G. Schwartz, Curtis C. Harris, Susan Olivo Marston. Childhood exposure to secondhand smoke, nicotine dependence, and DRD1 are associated with lung cancer risk. [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 2156. doi:10.1158/1538-7445.AM2014-2156

Purpose To develop a genomic signature that predicts benefit from trastuzumab in human epidermal growth factor receptor 2–positive breast cancer. Patients and Methods DASL technology was used to quantify mRNA in samples from 1,282 patients enrolled onto the Combination Chemotherapy With or Without Trastuzumab in Treating Women With Breast Cancer (North Central Cancer Treatment Group N9831 [NCCTG-N9831]) adjuvant trastuzumab trial. Cox proportional hazard ratios (HRs), adjusted for significant clinicopathologic risk factors, were used to determine the association of each gene with relapse-free survival (RFS) for 433 patients who received chemotherapy alone (arm A) and 849 patients who received chemotherapy plus trastuzumab (arms B and C). Network and pathway analyses were used to identify key biologic processes linked to RFS. The signature was built by using a voting scheme. Results Network and functional ontology analyses suggested that increased RFS was linked to a subset of immune function genes. A voting scheme model was used to define immune gene enrichment based on the expression of any nine or more of 14 immune function genes at or above the 0.40 quantile for the population. This model was used to identify immune gene–enriched tumors in arm A and arms B and C. Immune gene enrichment was linked to increased RFS in arms B and C (HR, 0.35; 95% CI, 0.22 to 0.55; P < .001), whereas arm B and C patients who did not exhibit immune gene enrichment did not benefit from trastuzumab (HR, 0.89; 95% CI, 0.62 to 1.28; P = .53). Enriched immune function gene expression as defined by our predictive signature was not associated with increased RFS in arm A (HR, 0.90; 95% CI, 0.60 to 1.37; P = .64). Conclusion Increased expression of a subset of immune function genes may provide a means of predicting benefit from adjuvant trastuzumab.

Abstract Lung cancer has a familial component which suggests a genetic contribution to its etiology. Given the strong evidence linking smoking with lung cancer, we studied miRNA-related loci in genes associated with smoking behavior. CHRNA, CHRNB gene families, CYP2A6, and DRD1 (dopamine receptor D1) were mined for SNPs that fell within the seed region of miRNA binding sites and then tested for associations with risk in a three-stage validation approach. A 3′UTR (untranslated region) SNP in DRD1 was associated with a lower risk of lung cancer among individuals exposed to secondhand smoke during childhood [OR, 0.69; 95% confidence interval (CI), 0.60–0.79; P &lt; 0.0001]. This relationship was evident in both ever (OR, 0.74; 95% CI, 0.62–0.88; P = 0.001) and never smokers (OR, 0.61; 95% CI, 0.47–0.79; P &lt; 0.0001), European American (OR, 0.65; 95% CI, 0.53–0.80; P &lt; 0.0001), and African American (OR, 0.73; 95% CI, 0.62–0.88; P = 0.001) populations. Although much remains undefined about the long-term risks associated with exposure to secondhand smoke and heterogeneity between individuals in regard to their susceptibility to the effects of secondhand smoke, our data show an interaction between an SNP in the 3′UTR of DRD1 and exposure to secondhand smoke during childhood. Further work is needed to explore the mechanistic underpinnings of this SNP and the nature of the interaction between DRD1 and exposure to secondhand smoke during childhood. Cancer Prev Res; 7(12); 1210–8. ©2014 AACR.

Abstract In searching for a role for genetic variation in susceptibility to lung cancer, we postulated that miRNA modulating SNPs within smoking behavior associated genes, such as the nicotinic acetylcholine and dopamine receptor families, could modulate susceptibility to tobacco carcinogens and lung cancer risk. Bioinformatic analyses of these gene families identified 5 SNPs predicted to modulate a miRNA binding site. We assessed these SNPs in the National Cancer Institute-MD case-control study (European American [EA] & African Americans [AA] n=1,438). rs686 in DRD1 (dopamine receptor D1) was associated with a reduced risk of lung cancer after adjustment for age, gender and race (OR 0.63, 95% C.I. 0.47-0.85; P=0.002). As DRD1 mediates dopamine signaling following nicotine exposure, we reasoned that the rs686 variant associates with lung cancer risk via modulation of the nicotine reward pathway. However, adjustment for smoking both in terms of smoking status and pack-years did not significantly modulate the association (OR 0.57, 95% C.I. 0.40-0.81, P=0002). In addition, age at smoking initiation, pack-years of smoking and levels of urinary nicotine metabolites (cotinine and hydroxycotinine) did not vary across rs686 genotypes. Moreover, in an analysis of a never smoking cohort from Mayo Clinic, rs686 was also associated with a reduced risk of lung cancer (OR 0.77, 95% C.I. 0.62-0.97; P=0.027; n=629) (pooled analysis OR 0.61, 95% C.I. 0.47-0.78; P&lt;0.0001; n=2,067). Intriguingly, the association between rs686 and lung cancer risk was only observed in subjects exposed to tobacco smoke during childhood (EA smokers: [OR 0.63, 95% C.I. 0.45-0.88; P=0.007] EA never smokers [OR 0.64, 95% C.I. 0.49-0.85; P=0.002] AA smokers OR 0.31, 95% C.I. 0.11-0.89; P=0.029) (model adjusted as above and for education, adult-second-hand smoke exposure). To further explore a relationship between tobacco and DRD1, we exposed lung cancer cell lines to increasing doses of nicotine and observed a significant increase in DRD1 expression (P=0.009). As DRD1 lies within a CpG island, we are currently testing if this regulation occurs via epigenetic modulation of the DRD1 promoter. While a direct role for DRD1 in lung cancer has not previously been reported, a recent report linked DRD to the cancer stem cell hypothesis. Indeed, our work demonstrates that DRD1 is significantly upregulated in lung cells with cancer stem-like properties. Furthermore, miR-296, predicted to bind to variant rs686 allele, is increased in paired lung cancer tissues (P=0.0003), thus linking a decrease in DRD1 with reduced lung cancer risk. Collectively, our work suggests a novel lung cancer pathway whereby rs686 in DRD1 counteracts the increase in DRD1 imposed by exposure to tobacco smoke in both current and never smokers. Furthermore, it hints at a novel role for the dopamine receptor/signaling pathway in lung carcinogenesis. Citation Format: Brid M. Ryan, Ana I. Robles, Andrew C. McClary, Elise Bowman, Kirsi Vahakangas, Susan Olivo-Marston, Ping Yang, Jin Jen, Curtis C. Harris. Interaction between DRD1 and childhood exposure to environmental tobacco smoke modulates lung cancer risk in smokers and never smokers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4581. doi:10.1158/1538-7445.AM2013-4581

Abstract Next Generation Sequencing (NGS) technologies provide rapid genomic analyses of single nucleotide variants, RNA expression and DNA copy number. Application of these technologies to material isolated from formalin fixed paraffin embedded (FFPE) tissue and even degraded frozen material could provide powerful replication samples but remains challenging. We tested the nanoString platform to validate deep sequence analysis of gene expression and DNA copy number in degraded and FFPE material. Firstly, RNA from the Universal Human Reference RNA and a breast cancer cell line (MDA-MB-436) was artificially degraded to different degrees (RIN 1.2-6.8). We used the nanoString platform to simultaneously measure RNA expression across 226 genes in each degraded sample and the corresponding undegraded RNA. Secondly we isolated RNA and DNA from matched fresh frozen and FFPE tissues from nine breast cancer patients (3 HER2+/ER+/PR+, 2 HER2+/ER-/PR-, 2 HER2+/ER+/PR-, 2 HER2-/ER+/PR+) using the nanoString platform to compare expression and copy number across 226 and 86 genes respectively. Finally, we correlated expression and copy number data generated by nanoString with Illumina transcriptome and whole genome sequencing (WGS). NanoString log2 expression fold-change between all artificially degraded samples and their undegraded counterpart showed extremely high correlation (r2&gt;0.91). NanoString DNA copy number between matched fresh-frozen and FFPE showed a high degree of correlation (r2=0.71). All gene amplifications with copy number ≥ 5 in DNA from fresh-frozen material (N=9) were successfully identified in DNA from FFPE material. We also observed good correlation of gene expression between whole transcriptome sequencing and the nanoString platform (r2 0.59 - 0.72) in FFPE and artificially degraded material and for DNA copy number between WGS and nanoString in DNA isolated from cancer cell lines (r2=0.96). The nanoString platform provides reliable data from highly degraded and FFPE material and correlates with sequence analysis of both expression and copy number from NGS platforms demonstrating potential for large-scale replication studies in FFPE material. Citation Format: Nadine Norton, Edith A. Perez, Yan W. Asmann, Jennifer M. Carr, Brian M. Necela, Jennifer M. Kachergus, Jin Jen, Bruce W. Eckloff, E Aubrey Thompson. Analysis of gene expression and copy number variation in breast tumors using both sequencing and hybridization-based platforms. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2005. doi:10.1158/1538-7445.AM2013-2005

Abstract Background: The EML4-ALK fusion gene is more frequently found in younger, never smoking patients with lung cancer. Meanwhile, never smokers exposed to secondhand tobacco smoke (SHS) during childhood are diagnosed at a younger age compared with never smoking patients with lung cancer who are not exposed. We, therefore, hypothesized that SHS, which can induce DNA damage, is associated with the EML4-ALK fusion gene. Methods: We compared the frequency of the EML4-ALK fusion gene among 197 never smoker patients with lung cancer with and without a history of exposure to SHS during childhood at Mayo Clinic. Results: The EML4-ALK fusion gene was detected in 33% of cases from never smokers with a history of SHS exposure during childhood, whereas 47% of never smoking lung cancer cases without a history of childhood SHS exposure tested positive for the fusion gene. Conclusions: The EML4-ALK fusion gene is not enriched in tumors from individuals exposed to SHS during childhood. Impact: These data suggest that childhood exposure to SHS is not a significant etiologic cause of the EML4-ALK fusion gene in lung cancer. Cancer Epidemiol Biomarkers Prev; 23(7); 1432–4. ©2014 AACR.

Abstract Lung cancer is the most common cause of smoking-related mortality in worldwide. However, approximately 10 to 25 % cases are not attributable to smoking. Cumulative evidence suggests that lung cancer from never smokers (&lt;100 cigarettes lifetime) may develop via molecular mechanisms that are different from lung cancers in smokers. Recently, miRNAs have been identified as a new class of regulatory molecules that participate in a variety of biological processes. Because each miRNAs can potentially target a large set of mRNA making and therefore may play a critical role in lung tumorigenesis and patient outcome, we sought to examine the role of miRNA in lung adenocarcinoma among never smokers. A total of 298 samples were analyzed in this study including 168 fresh frozen (FF) tissues from 88 lung adenocarcinomas and 80 adjacent non-involved lung tissues; and 132 formalin-fixed paraffin embedded (FFPE) tissues from 68 tumors and 64 normal lung samples. Gene expression profiling was performed for miRNA and mRNA using Illumina Whole Genome DASL platform. We identified 20 miRNAs with the most significant expression changes between the tumor and normal in both FF and FFPE samples (Fold change &gt; 1.5, p &lt;0.01, FDR &lt;0.05). We also examined mRNA expression signature using 56 matched FF tumors and normal lung tissues. Cox proportional hazard analysis was performed independently for both FF and FFPE cohorts and showed that increased expression of mir-708 was strongly associated with an increased risk of patient death after adjusting for age, sex and tumor stage (FF cohort, HR =1.92; 95% CI =1.02–3.63; p &lt; 0.05 and FFPE cohort, HR=1.90; 95% CI=1.08–3.34; P &lt; 0.05). Candidate biological targets of mir-708 were identified using TargetScan and MicroCosm Targets databases. A total of 13 candidate genes were found and one of these genes, CMT11 (Candidate Mir-708 Target), was down-regulated 1.6 fold on average in tumors that were high in mir-708 expression. Based on the sequence, CMT11 is a novel transmembrane protein. In Cox analysis, reduced CMT11 expression was strongly correlated with an increased risk of death in patients (HR = 2.43, 95% CI = 1.17 − 5.26; P &lt;0.01). Functionally, overexpression of mir-708 in A549 and HEK293T cells significantly increased the rate of cellular proliferation and migration. Over expression of mir-708 in HEK293T cells also repressed ∼70% of the luciferase activity when the reporter RNA contained the 3′ UTR of CMT11. Recently, several studies have showed mir-708 is a potential squamous cell lung cancer marker and one of miRNAs that may predict the recurrence of Stage I lung cancer after surgical resection. Additional evidence also suggests that CMT11 is a negative regulator of Wnt-signaling pathway. Our data provides clinical and functional evidence supporting an important role of mir-708 in lung cancer progression and identifies CMT11 as a down stream target gene mediating this process. 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 LB-346. doi:10.1158/1538-7445.AM2011-LB-346