Lynch, Charles F.

Abstract Background: The immune response has been shown to impact the course of colorectal cancer (CRC). Our goal was to examine the survival of CRC patients in relation to tumor-infiltrating cytotoxic (CD8+) T lymphocytes (CTL). We quantified CTLs in tumor epithelial and stromal tissues and correlated them with clinicopathological characteristics and survival of CRC patients in the Iowa Women's Health Study. Methods: Paraffin-embedded tissue samples were available from 465 post-menopausal women diagnosed with incident CRC in 1986-2002. Tumor microarrays were constructed and immunostained with a CD8 antibody (Clone 144B; Dako). CTLs in tumor epithelial and stromal tissues were categorized by an experienced pathologist into non-detected; mild (1-10 cells per core); moderate (11-29 cells); and strong infiltration (≥30 cells per core), and averaged over cores per each person. We used Cox regression to estimate the hazard ratio (HR) and 95% CI for total and CRC death in relation to each CTL score after adjusting for age of diagnosis, SEER stage, grade, body mass, smoking history and integrated molecular pathway for CRC. The tumors were categorized by integrated pathway (traditional, alternate, serrated, unassigned) based on the combination of phenotypes: microsatellite instability (MSI) status, CpG island methylator (CIMP) phenotype; and mutations in BRAF and/or KRAS genes (described in Samadder, 2013). Results: During follow-up until 2011, 31% of participants died from CRC, 36% from all other causes, and 33% were alive (median follow-up: 8.4 y). The Spearman correlation coefficient between epithelial and stromal CTL scores was 0.52. Both scores were inversely correlated with stage at diagnosis and were higher in tumors characterized by MSI-high, CIMP-high and BRAF mutation-positive status. The highest categories in the epithelial and stromal scores were associated with statistically significant decreases in total death (by 44% and 40%, respectively) and CRC death (by 68% and 56%, respectively) compared to lowest categories. After summing epithelial and stromal CTL scores, the combined HRs (95% CI) in the highest versus lowest category were 0.55 (0.38-0.78) for total death (p-trend = 0.0002) and 0.36 (0.20-0.63) for CRC death (p-trend<0.0001). Additional adjustment for surgery, chemotherapy, radiation, and comorbidities did not markedly change the associations. Finally, there was a statistically significant quantitative interaction between stromal score and MSI status in relation to total and CRC death (P for interaction were 0.02 and 0.0001, respectively); HRs were lower for MSI-high than for MS-stable tumors. There was also a significant interaction between smoking status (current versus never) and epithelial CTL score: the HRs for total and CRC death were lowest among never smokers with the highest score. Conclusions. Our study further supports infiltration of tumors with CTLs as an important prognostic factor in CRC. Citation Format: Anna E. Prizment, Robert A. Vierkant, Thomas C. Smyrk, Lori S. Tillmans, Heather H. Nelson, Charles F. Lynch, Stephen N. Thibodeau, Timothy R. Church, James R. Cerhan, Kristin E. Anderson, Paul J. Limburg. Tumor-infiltrating cytotoxic T-cells are associated with improved survival of colorectal cancer patients. [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 2636.

Abstract Background: Cigarette smoking (smoking), hormone therapy (MHT), and folate intake (folate) are each thought to influence colorectal cancer risk, but the underlying molecular mechanisms remain incompletely defined. Expression of estrogen receptor β (ESR2) has been associated with colorectal cancer stage and survival. Methods: In this prospective cohort study, we examined smoking, MHT, and folate-associated colorectal cancer risks by ESR2 protein expression level among participants in the Iowa Women's Health Study (IWHS). Self-reported exposure variables were assessed at baseline. Archived, paraffin-embedded colorectal cancer tissue specimens were collected and evaluated for ESR2 protein expression by IHC. Multivariate Cox regression models were fit to estimate relative risks (RR) and 95% confidence intervals (CI) for associations between smoking, MHT, or folate and ESR2-defined colorectal cancer subtypes. Results: Informative environmental exposure and protein expression data were available for 491 incident colorectal cancer cases. Positive associations between ESR2-low and -high tumors and several smoking-related variables were noted, most prominently with average number of cigarettes per day (RR, 4.24; 95% CI, 1.81–9.91 for ESR2-low and RR, 2.15; 95% CI, 1.05–4.41 for ESR2-high for ≥40 cigarettes compared with nonsmokers). For MHT, a statistically significant association with ESR2-low tumors was observed with longer duration of exposure (RR, 0.54; 95% CI, 0.26–1.13 for >5 years compared with never use). No associations were found for folate. Conclusions: In this study, smoking and MHT were associated with ESR2 expression patterns. Impact: These data support possible heterogeneous effects from smoking and MHT on ERβ-related pathways of colorectal carcinogenesis in older women. Cancer Epidemiol Biomarkers Prev; 24(4); 713–9. ©2015 AACR.

Abstract Background: Hormone therapy (HT) may prevent colorectal cancer (CRC), but the molecular mechanisms have not been fully defined. Expression of estrogen receptor beta (ERβ) has been associated with CRC stage and survival and may influence HT-related prevention pathways. In this prospective cohort study, we examined HT-associated CRC risk by ERβ protein expression levels in the population-based Iowa Women's Health Study (IWHS). Methods: The IWHS recruited 41,836 randomly selected Iowa women, ages 55-69 years, with a valid driver's license at study entry (1986). Exposure variables were assessed at baseline, by self-report. Incident CRC cases were ascertained by annual linkage with the Iowa Cancer Registry. Archived, paraffin-embedded tissue specimens were collected from 732 CRC cases, diagnosed through 12/31/2002, for molecular epidemiology studies. HT was categorized as never, former, and current and with durations of <=5 years or >5 years. ERβ protein expression was determined by immunohistochemistry on tissue microarrays. For each tumor core, both staining intensity (0-3) and proportion of cells stained (0-5) were scored. These two scores were added to determine negative (0), low (1-5) or high (6-8) ERβ protein expression levels. Multivariate Cox regression models were fit to estimate relative risks (RRs) and 95% confidence intervals (CIs). Results: Complete HT and ERβ data were available from 484 CRC cases. Associations between HT and CRC subtypes defined by ERβ expression level are shown in the Table. Conclusions: Data from the IWHS cohort suggest that current and longer-duration HT exposure favorably influence the risk for ERβ-neg or ERβ-low CRC cases to a greater degree than ERβ-high CRC cases. Further investigation is required to confirm these observations, and to clarify the underlying mechanisms. ERβ Neg or Low ERβ High Hormone Therapy Person years Events, N RR* (95% CI) Events, N RR* (95% CI) Never 341377 128 1.00 (Ref.) 196 1.00 (Ref.) Ever 212696 62 0.74 (0.54,1.03) 98 0.82 (0.63,1.07) Former 151535 48 0.79 (0.56,1.12) 67 0.73 (0.54,0.99) Current 61161 14 0.60 (0.33,1.09) 31 1.11 (0.74,1.64) P-trend 0.05 0.52 Duration < = 5yrs 148704 47 0.84 (0.59,1.19) 68 0.80 (0.60,1.08) >5yrs 60064 14 0.5 (0.26,0.93) 28 0.89 (0.59,1.34) P-trend 0.03 0.25 Citation Format: Lori S. Tillmans, Robert A. Vierkant, Alice H. Wang, Charles F. Lynch, Kristin E. Anderson, Amy J. French, Robert W. Haile, John D. Potter, Susan L. Slager, Thomas C. Smyrk, Stephen N. Thibodeau, James R. Cerhan, Paul J. Limburg. Hormone therapy and incident colorectal cancer risk by ER beta protein expression level in a population-based cohort of older women. [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 3652. doi:10.1158/1538-7445.AM2013-3652

Abstract Genome-wide association studies (GWAS) of 10 different cancers have identified pleiotropic cancer predisposition loci across a region of chromosome 5p15.33 that includes the TERT and CLPTM1L genes. Of these, susceptibility alleles for pancreatic cancer have mapped to the CLPTM1L gene, thus prompting an investigation of the function of CLPTM1L in the pancreas. Immunofluorescence analysis indicated that CLPTM1L localized to the endoplasmic reticulum where it is likely embedded in the membrane, in accord with multiple predicted transmembrane domains. Overexpression of CLPTM1L enhanced growth of pancreatic cancer cells in vitro (1.3–1.5–fold; PDAY7 < 0.003) and in vivo (3.46-fold; PDAY68 = 0.039), suggesting a role in tumor growth; this effect was abrogated by deletion of two hydrophilic domains. Affinity purification followed by mass spectrometry identified an interaction between CLPTM1L and non-muscle myosin II (NMM-II), a protein involved in maintaining cell shape, migration, and cytokinesis. The two proteins colocalized in the cytoplasm and, after treatment with a DNA-damaging agent, at the centrosomes. Overexpression of CLPTM1L and depletion of NMM-II induced aneuploidy, indicating that CLPTM1L may interfere with normal NMM-II function in regulating cytokinesis. Immunohistochemical analysis revealed enhanced staining of CLPTM1L in human pancreatic ductal adenocarcinoma (n = 378) as compared with normal pancreatic tissue samples (n = 17; P = 1.7 × 10−4). Our results suggest that CLPTM1L functions as a growth-promoting gene in the pancreas and that overexpression may lead to an abrogation of normal cytokinesis, indicating that it should be considered as a plausible candidate gene that could explain the effect of pancreatic cancer susceptibility alleles on chr5p15.33. Cancer Res; 74(10); 2785–95. ©2014 AACR.

Abstract Cigarette smoking (CS), hormone therapy (HT), and folate intake (FI) are each thought to influence colorectal cancer risk, but the underlying molecular mechanisms remain incompletely defined. The TP53 (p53) protein, encoded by the TP53 tumor-suppressor gene that is commonly mutated in colorectal cancer, can be readily assessed to differentiate biologically distinct colorectal cancer subtypes. In this prospective cohort study, we examined CS-, HT-, and FI-associated colorectal cancer risks by TP53 protein expression level among Iowa Women's Health Study (IWHS) participants. The IWHS recruited 41,836 randomly selected Iowa women, ages 55 to 69 years, with a valid driver's license at study entry in 1986. Self-reported exposure variables were assessed at baseline. Incident colorectal cancer cases were ascertained by annual linkage with the Iowa Cancer Registry. Archived, paraffin-embedded tissue specimens were collected and evaluated for TP53 protein expression by immunohistochemistry. Multivariate Cox regression models were fit to estimate relative risks (RR) and 95% confidence intervals (CI) for associations between CS, HT, or FI and TP53-defined colorectal cancer subtypes. Informative environmental exposure and protein expression data were available for 492 incident colorectal cancer cases: 222 (45.1%) TP53 negative, 72 (14.6%) TP53 low, and 198 (40.2%) TP53 high. Longer duration (>5 years) of HT was inversely associated with TP53 high colorectal cancers (RR, 0.50; 95% CI, 0.27–0.94). No other statistically significant associations were observed. These data support possible heterogeneous effects from HT on TP53-related pathways of colorectal carcinogenesis in older women. Cancer Epidemiol Biomarkers Prev; 23(2); 350–5. ©2013 AACR.

Abstract Background: Cigarette smoking (CS) is an established colorectal cancer (CRC) risk factor. The p53 protein, encoded by the TP53 tumor suppressor gene that is commonly mutated in CRC, is important in cell cycle arrest needed to repair DNA damage or induce apoptosis to prevent tumor propagation. In this prospective cohort study, we examined CS-associated CRC risks by p53 protein expression level in the population-based Iowa Women's Health Study (IWHS). Methods: The IWHS recruited 41,836 randomly selected Iowa women, ages 55-69 years, with a valid driver's license at study entry (1986). CS and other exposure variables were assessed at baseline, by self-report. Incident CRC cases were ascertained by annual linkage with the Iowa Cancer Registry. Archived, paraffin-embedded tissue specimens were collected from 732 CRC cases diagnosed through 12/31/2002. CS was categorized by smoking status (never, current, former), average number of cigarettes per day (1-19, 20-39, > 40) and cumulative pack-years (1-19, 20-39, > 40). P53 protein expression was determined by immunohistochemistry on tissue microarrays. Tumor cores were scored by combining both staining intensity and percent of cells stained to determine negative, low or high p53 protein expression levels. Multivariate Cox regression models were fit to estimate relative risks (RRs) and 95% confidence intervals (CIs). Results: Of the 732 available CRC cases, 486 (66%) had complete CS and p53 data. Associations between CS and CRC subtypes defined by p53 expression level are shown in the Table. Conclusions: These data demonstrate no clear heterogeneity of CS with CRC subtypes defined by p53 expression levels among older women. 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 1676. doi:1538-7445.AM2012-1676

Abstract Background: Colorectal cancer (CRC) is a molecularly heterogeneous disease. Three molecular pathways (traditional, serrated and alternate) have been recently proposed based on integrated combinations of microsatellite instability (MSI), CpG island methylation phenotype (CIMP), BRAF mutation, and KRAS mutation status (Gastroenterol 2010;138:2088-2100). To our knowledge, clinicopathologic associations with these CRC pathways have not been previously described. Methods: Data and tissue resources were obtained from the population-based Iowa Women's Health Study, which recruited 41,836 randomly selected women, ages 55-69 years, at study entry (1986). Incident CRC cases were ascertained by annual linkage with the Iowa Cancer Registry. Archived, paraffin-embedded tissue specimens were collected from 732 CRC cases diagnosed through 12/31/2002. Established laboratory methods were used to characterize CRC cases by MSI (MSS/MSI-L or MSI-H), CIMP (neg, low, or high), BRAF mutation (wt or mut), and KRAS mutation (wt or mut) status. Molecular pathways were defined as: traditional (MSS/MSI-L, CIMP-neg, BRAF wt, and KRAS wt); serrated (any MSI, CIMP-high, BRAF mut, and KRAS wt), alternate (MSS/MSI-L, CIMP-low, BRAF wt, and KRAS mut), or other pathway. Chi-square tests and analyses of variance were used to compare clinicopathologic features at CRC presentation (age, anatomic subsite, tumor grade, and SEER stage) across the molecular pathways. Results: Comlete and informative MSI, CIMP, BRAF and KRAS data were obtained for 491/732 (67%) CRC cases. Clinicopathologic associations are shown in the Table. Conclusions: These data suggest that distributions of age at diagnosis, subsite, and tumor grade, but not SEER stage, differ across the traditional, serrated, and alternate carcinogenic pathways, which may have implications for molecularly-targeted CRC prevention and/or therapy strategies. 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 5518. doi:1538-7445.AM2012-5518

Abstract Background: Despite recent advances in prevention and therapy, colorectal cancer (CRC) continues to account for nearly 50,000 deaths per year in the US. TNM stage is the current CRC prognostic standard, yet within-stage prognostic heterogeneity is well documented. Microsatellite instability (MSI), CpG island methylation phenotype (CIMP), and BRAF- and KRAS-mutation status represent candidate prognostic markers. However, associations between these markers and CRC survival have not been described in a population-based setting. Methods: Data and tissue resources were obtained from the Iowa Women's Health Study (IWHS), which recruited 41,836 women, ages 55-69 years, at study entry (1986). Incident CRC cases were ascertained by annual linkage with the Iowa Cancer Registry. Archived, paraffin-embedded tissue specimens were collected from 732 representative CRC cases diagnosed through 12/31/2002. Established laboratory methods were used to characterize CRC cases: MSI-H or MSS/MSI-L (by instability in β3 vs. <3 out of 10 markers, respectively); CIMP+ or CIMP− (by promoter hypermethylation in β3 vs. 0-2 out of 5 genes, respectively); BRAFwt or BRAFmut (by fluorescent allele specific PCR to detect the V600E point mutation in exon 15); and KRASwt or KRASmut (by direct sequence analysis of codons 12 and 13 in exon 2). Cox regression models were fit to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for time to death of any cause adjusted for age, stage, grade, and treatment status. Results: CRC cases with informative molecular marker data ranged from 515/732 (70%) for KRAS to 548/732 (75%) for MSI status. Mean follow-up was 4.4 years for deceased individuals and 11.2 years for women alive at last contact. After multivariable adjustment, both MSS/MSI-L (p=0.004) and KRASmut (p=0.048) tumors were associated with poorer survival. Survival did not significantly differ across levels of CIMP and BRAF status. Conclusions: MSI and KRAS status may influence CRC survival among older women. 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 4492. doi:1538-7445.AM2012-4492

Abstract Background: Folate intake (FI) appears to affect colorectal cancer (CRC) risk, but the molecular mechanism(s) involved are incompletely defined. In this study, we analyzed associations between FI and incident CRC, overall and by KRAS mutation status, in the population-based Iowa Women's Health Study (IWHS). Methods: The IWHS recruited 41,836 randomly selected Iowa women, ages 55-69 years, with a valid driver's license at study entry (1986). FI and other exposure variables were assessed at baseline, by self-report. Incident CRC cases were ascertained by annual linkage with the Iowa Cancer Registry. Archived, paraffin-embedded tissue specimens were recently collected from 732 representative CRC cases diagnosed through 12/31/2002. FI (including supplements) was categorized by quartiles. KRAS mutation status (wildtype [wt] vs. mutated [mut]) was determined by direct sequence analysis of codons 12 and 13 (exon 2). Multivariate Cox regression models were fit to estimate relative risks (RRs) and 95% confidence intervals (CIs). Results: Of the 732 available CRC cases, 470 (64%) had complete FI and KRAS data. FI was not associated with incident CRC overall (for quartile 4 vs. quartile 1, RR = 1.03; 95% CI = 0.82-1.28; p trend = 0.90), or with the KRAS-wt or KRAS-mut subtypes (see Table). Conclusions: These data do not support an association between FI and incident CRC, overall or by KRAS-defined subtypes, among older, predominately white, non-Hispanic women. 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 1892. doi:10.1158/1538-7445.AM2011-1892

Abstract Background: Cigarette smoking (CS) is an established, although relatively modest, colorectal cancer (CRC) risk factor. Emerging data suggest that CS might affect distinct pathways of colorectal carcinogenesis. In this study, we examined CS-associated CRC risks by KRAS mutation status in the population-based Iowa Women's Health Study (IWHS). Methods: The IIWHS recruited 41,836 randomly selected Iowa women, ages 55-69 years, with a valid driver's license at study entry (1986). CS and other exposure variables were assessed at baseline, by self-report. Incident CRC cases were ascertained by annual linkage with the Iowa Cancer Registry. Archived, paraffin-embedded tissue specimens were collected from 732 representative CRC cases diagnosed through 12/31/2002. CS was categorized by smoking status (current, former), average number of cigarettes per day (1-19, 20-39, > 40) and pack-years (1-19, 20-39, > 40); never smokers were defined as the reference group. KRAS mutation status (wildtype [wt] vs. mutated [mut]) was determined by direct sequence analysis of codons 12 and 13 (exon 2). Multivariate Cox regression models were fit to estimate relative risks (RRs) and 95% confidence intervals (CIs). Results: Of the 732 available CRC cases, 507 (69%) had complete CS and KRAS data. Each of the CS-related variables was more strongly associated with KRAS-wt vs. KRAS-mut tumors (see Table). However, event rates were relatively small in some cells, and tests for heterogeneity between KRAS-defined subtypes were not statistically significant (p > 0.05 for each comparison). Conclusions: These findings suggest that CS may affect colorectal carcinogenesis through KRAS-independent pathways, but additional data are needed from larger, more diverse study populations. 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 1891. doi:10.1158/1538-7445.AM2011-1891