Lori S. Tillmans

Cigarette Smoking and Colorectal Cancer Risk by Molecularly Defined Subtypes

BACKGROUND Cigarette smoking is an established risk factor for colorectal cancer. Because colorectal carcinogenesis is a heterogeneous process, we investigated whether cigarette smoking is differentially associated with molecularly defined subtypes of colorectal cancer. METHODS We evaluated associations between smoking and incident colorectal cancer, overall and by microsatellite instability (MSI) phenotype (MSI-high vs MSI-low or microsatellite stable), CpG island methylator phenotype (CIMP positive or CIMP negative), and BRAF mutation status (BRAF mutation positive or BRAF mutation negative), among 37 399 participants in a population-based cohort study (the Iowa Womens Health Study). Cigarette smoking (and other exposures) was assessed by self-report at baseline in 1986, including smoking status (never and ever [former or current]), age at initiation, total duration, average number of cigarettes smoked per day, cumulative pack-years, and induction period. Vital status and state of residence were determined by mailed follow-up questionnaires in 1987, 1989, 1992, and 1997 and by linkage to Iowa death certificate records. Nonrespondents were checked via the National Death Index to identify descendants. Participants with newly diagnosed (ie, incident) colorectal cancer were identified through annual linkage with the Iowa Cancer Registry. Archived paraffin-embedded tumor tissue specimens were obtained for 555 patients with colorectal cancer who were diagnosed from January 1, 1986, through December 31, 2002, and MSI status, CIMP status, and BRAF status were determined. Multivariable Cox regression models were fit to estimate relative risks (RRs) and 95% confidence intervals (CIs). RESULTS Ever-smokers were at moderately increased risk for incident colorectal cancer (RR = 1.19, 95% CI = 1.05 to 1.35) compared with never-smokers. Higher risk estimates were observed for current smokers with MSI-high tumors (RR = 1.99, 95% CI = 1.26 to 3.14), CIMP-positive tumors (RR = 1.88, 95% CI = 1.22 to 2.90), and BRAF mutation-positive tumors (RR = 1.92, 95% CI = 1.22 to 3.02). Other smoking-related variables (ie, age at initiation, total duration, average number of cigarettes smoked per day, cumulative pack-years, and induction period) were also associated with MSI-high, CIMP-positive, and BRAF mutation-positive tumor subtypes. Conversely, cigarette smoking status (ever vs never) was not associated with the MSI-low or microsatellite stable (RR = 1.00, 95% CI = 0.79 to 1.25), CIMP-negative (RR = 1.02, 95% CI = 0.81 to 1.30), or BRAF mutation-negative subtypes (RR = 1.00, 95% CI = 0.65 to 1.27). CONCLUSIONS In this prospective study of older women, cigarette smoking was associated with the MSI-high, CIMP-positive, and BRAF mutation-positive colorectal cancer subtypes, which indicates that epigenetic modification may be functionally involved in smoking-related colorectal carcinogenesis.

Anaplastic lymphoma kinase immunoreactivity correlates with ALK gene rearrangement and transcriptional up-regulation in non–small cell lung carcinomas

Recently, the fusion gene EML4-ALK was identified in non-small cell lung carcinoma, which could be a potential therapeutic target. We investigated the prevalence of anaplastic lymphoma kinase protein expression in these tumors by immunohistochemistry and correlated the results with data from ALK molecular studies. Gene expression profiling was performed on 35 adenocarcinomas to identify cases with ALK gene up-regulation, which was correlated with protein overexpression by immunohistochemistry. Immunohistochemistry was also performed on an independent cohort consisting of 150 adenocarcinomas and 150 squamous cell carcinomas to evaluate the utility of anaplastic lymphoma kinase immunostaining as a screening tool. Florescence in situ hybridization for the ALK locus and reverse transcriptase-polymerase chain reaction for EML4-ALK were performed on tumors positive for anaplastic lymphoma kinase by immunohistochemistry. Transcriptional up-regulation of ALK was identified in 2 (6%) of 35 adenocarcinomas by gene expression profiling. These 2 cases were positive for anaplastic lymphoma kinase by immunohistochemistry, whereas the remaining 33 cases were completely negative. In the independent cohort, anaplastic lymphoma kinase immunostaining was positive in 1 of 150 squamous cell carcinomas and in 3 of 150 adenocarcinomas. The 6 cases positive for anaplastic lymphoma kinase by immunohistochemistry showed evidence of ALK locus rearrangement by florescence in situ hybridization but were negative for EGFR and KRAS mutation. The presence of EML4-ALK fusion transcript was confirmed in 2 cases by reverse transcriptase-polymerase chain reaction. In conclusion, anaplastic lymphoma kinase immunoreactivity in non-small cell lung carcinomas was associated with transcriptional up-regulation, ALK locus rearrangement, and the presence of EML4-ALK fusion transcript. Anaplastic lymphoma kinase immunohistochemistry may have utility as a screening tool or as a surrogate marker for the molecular techniques to detect the EML4-ALK fusion gene in these tumors.

Gene Panel Model Predictive of Outcome in Men at High-Risk of Systemic Progression and Death From Prostate Cancer After Radical Retropubic Prostatectomy

PURPOSE In men who are at high-risk of prostate cancer, progression and death from cancer after radical retropubic prostatectomy (RRP), limited prognostic information is provided by established prognostic features. The objective of this study was to develop a model predictive of outcome in this group of patients. METHODS Candidate genes were identified from microarray expression data from 102 laser capture microdissected prostate tissue samples. Candidates were overexpressed in tumor compared with normal prostate and more frequently in Gleason patterns 4 and 5 than in 3. A case control study of 157 high-risk patients, matched on Gleason score and stage with systemic progression or death of prostate cancer as the end point, was used to evaluate the expression of candidate genes and build a multivariate model. Tumor was collected from the highest Gleason score in paraffin-embedded blocks and the gene expression was quantified by real-time reverse transcription polymerase chain reaction. Validation of the final model was performed on a separate case-control study of 57 high-risk patients who underwent RRP. RESULTS A model incorporating gene expression of topoisomerase-2a, cadherin-10, the fusion status based on ERG, ETV1, and ETV4 expression, and the aneuploidy status resulted in a 0.81 area under the curve (AUC) in receiver operating characteristic statistical analysis for the identification of men with systemic progression and death from high grade prostate cancer. The AUC was 0.79 in the independent validation study. CONCLUSION The model can identify men with high-risk prostate cancer who may benefit from more intensive postoperative follow-up and adjuvant therapies.

Identification of Prognostic Biomarkers for Prostate Cancer

Purpose: This paper describes a process for the identification of genes that can report on the aggressiveness of prostate tumors and thereby add to the information provided by current pathologic analysis. Materials and Methods: Expression profiling data from over 100 laser capture microdissection derived samples from nonneoplastic epithelium; Gleason patterns 3, 4, and 5 and node metastasis prostate cancer were used to identify genes at abnormally high levels in only some tumors. These variably overexpressed genes were stratified by their association with aggressive phenotypes and were subsequently filtered to exclude genes with redundant expression patterns. Selected genes were validated in a case-control study in which cases (systemic progression within 5 years) and controls (no systemic progression at 7 years of follow-up) were matched for all clinical and pathologic criteria from time of prostatectomy (n = 175). Both cases and controls, therefore, could have nodal invasion or seminal vesicle involvement at the time of initial treatment. Results: A number of candidate variably overexpressed genes selected for their association with aggressive prostate cancer phenotype were evaluated in the case control study. The most prominent candidates were SSTR1 and genes related to proliferation, including TOP2A. Conclusions: The process described here identified genes that add information not available from current clinical measures and can improve the prognosis of prostate cancer.

Prostate cancer risk locus at 8q24 as a regulatory hub by physical interactions with multiple genomic loci across the genome

Chromosome 8q24 locus contains regulatory variants that modulate genetic risk to various cancers including prostate cancer (PC). However, the biological mechanism underlying this regulation is not well understood. Here, we developed a chromosome conformation capture (3C)-based multi-target sequencing technology and systematically examined three PC risk regions at the 8q24 locus and their potential regulatory targets across human genome in six cell lines. We observed frequent physical contacts of this risk locus with multiple genomic regions, in particular, inter-chromosomal interaction with CD96 at 3q13 and intra-chromosomal interaction with MYC at 8q24. We identified at least five interaction hot spots within the predicted functional regulatory elements at the 8q24 risk locus. We also found intra-chromosomal interaction genes PVT1, FAM84B and GSDMC and inter-chromosomal interaction gene CXorf36 in most of the six cell lines. Other gene regions appeared to be cell line-specific, such as RRP12 in LNCaP, USP14 in DU-145 and SMIN3 in lymphoblastoid cell line. We further found that the 8q24 functional domains more likely interacted with genomic regions containing genes enriched in critical pathways such as Wnt signaling and promoter motifs such as E2F1 and TCF3. This result suggests that the risk locus may function as a regulatory hub by physical interactions with multiple genes important for prostate carcinogenesis. Further understanding genetic effect and biological mechanism of these chromatin interactions will shed light on the newly discovered regulatory role of the risk locus in PC etiology and progression.

Associations Between Intake of Folate and Related Micronutrients with Molecularly Defined Colorectal Cancer Risks in the Iowa Women's Health Study

Folate and related micronturients may affect colorectal cancer (CRC) risk, but the molecular mechanism(s) remain incompletely defined. We analyzed associations between dietary folate, vitamin B6, vitamin B12, and methionine with incident CRC, overall and by microsatellite instability (MSS/MSI-L or MSI-H), CpG island methylator phenotype (CIMP-negative or CIMP-positive), BRAF mutation (negative or positive), and KRAS mutation (negative or positive) status in the prospective, population-based Iowa Womens Health Study (IWHS; 55–69 years at baseline; n = 41,836). Intake estimates were obtained from baseline, self-reported food frequency questionnaires. Molecular marker data were obtained for 514 incident CRC cases. Folate intake was inversely associated with overall CRC risk in age-adjusted Cox regression models, whereas methionine intake was inversely associated with overall CRC risk in multivariable-adjusted models [relative risk (RR) = 0.81; 95% CI = 0.69–0.95; P trend = 0.001 and RR = 0.72; 95% CI = 0.54–0.96; P trend = 0.03 for highest vs. lowest quartiles, respectively]. None of the dietary exposures were associated with MSI, CIMP, BRAF, or KRAS defined CRC subtypes. These data provide minimal support for major effects from the examined micronutrients on overall or molecularly defined CRC risks in the IWHS cohort.

Cigarette smoking and colorectal cancer risk by KRAS mutation status among older women

OBJECTIVES:Existing data support a modest association between cigarette smoking and incident colorectal cancer (CRC) overall. In this study, we evaluated associations between cigarette smoking and CRC risk stratified by KRAS mutation status, using data and tissue resources from the Iowa Womens Health Study (IWHS).METHODS:The IWHS is a population-based cohort study of cancer incidence among 41,836 randomly selected Iowa women, ages 55–69 years of age at enrollment (1986). Exposure data, including cigarette smoking, were obtained by self-report at baseline. Incident CRCs (n=1,233) were ascertained by annual linkage with the Iowa Cancer Registry. Archived tissue specimens from CRC cases recorded through 2002 were recently requested for molecular epidemiology investigations. Tumor KRAS mutation status was determined by direct sequencing of exon 2, with informative results in 507/555 (91%) available CRC cases (342 mutation negative and 165 mutation positive). Multivariate Cox regression models were fit to estimate relative risks (RRs) and 95% confidence intervals (CIs) for associations between cigarette smoking variables and KRAS-defined CRC subtypes.RESULTS:Multiple smoking variables were associated with increased risk for KRAS mutation-negative tumors, including age at initiation (P=0.02), average number of cigarettes per day (P=0.01), cumulative pack-years (P=0.05), and induction period (P=0.04), with the highest point estimate observed for women who smoked ≥40 cigarettes per day on average (RR=2.38; 95% CI=1.25–4.51; compared with never smokers). Further consideration of CRC subsite suggested that cigarette smoking may be a stronger risk factor for KRAS mutation-negative tumors located in the proximal colon than in the distal colorectum. None of the smoking variables were significantly associated with KRAS mutation-positive CRCs (overall or stratified by anatomic subsite).CONCLUSIONS:Data from this prospective study of older women demonstrate differential associations between cigarette smoking and CRC subtypes defined by KRAS mutation status, and are consistent with the hypothesis that smoking adversely affects the serrated pathway of colorectal carcinogenesis.

Postmenopausal hormone therapy and colorectal cancer risk by molecularly defined subtypes among older women

Background Postmenopausal hormone (PMH) therapy may reduce colorectal cancer (CRC) risk, but existing data are inconclusive. Objectives To evaluate associations between PMH therapy and incident CRC, overall and by molecularly defined subtypes, in the population-based Iowa Womens Health Study of older women. Methods Exposure data were collected from Iowa Womens Health Study participants (55–69 years) at baseline (1986). Archived, paraffin-embedded tissue specimens for 553 CRC cases were collected and analysed to determine microsatellite instability (MSI-L/MSS or MSI-H), CpG island methylator phenotype (CIMP-negative or CIMP-positive) and BRAF mutation (BRAF-wildtype or BRAF-mutated) status. Multivariable Cox regression models were fit to estimate RRs and 95% CIs. Results PMH therapy (ever vs never use) was inversely associated with incident CRC overall (RR=0.82; 95% CI 0.72 to 0.93), with a significantly lower risk for MSI-L/MSS tumours (RR=0.75; 95% CI 0.60 to 0.94), and borderline significantly lower risks for CIMP-negative (RR=0.79; 95% CI 0.63 to 1.01) and BRAF-wildtype (RR=0.83; 95% CI 0.66 to 1.04) tumours. For PMH therapy >5 years, the subtype-specific risk estimates for MSI-L/MSS, CIMP-negative and BRAF-wildtype tumours were: RR=0.60, 95% CI 0.40 to 0.91; RR=0.68, 95% CI 0.45 to 1.03; and RR=0.70, 95% CI 0.47 to 1.05, respectively. PMH therapy was not significantly associated with the MSI-H, CIMP-positive or BRAF-mutated CRC subtypes. Conclusions In this prospective cohort study, PMH therapy was inversely associated with distinct molecularly defined CRC subtypes, which may be related to differential effects from oestrogen and/or progestin on heterogeneous pathways of colorectal carcinogenesis.

Impact of sample acquisition and linear amplification on gene expression profiling of lung adenocarcinoma: laser capture micro-dissection cell-sampling versus bulk tissue-sampling

BackgroundThe methods used for sample selection and processing can have a strong influence on the expression values obtained through microarray profiling. Laser capture microdissection (LCM) provides higher specificity in the selection of target cells compared to traditional bulk tissue selection methods, but at an increased processing cost. The benefit gained from the higher tissue specificity realized through LCM sampling is evaluated in this study through a comparison of microarray expression profiles obtained from same-samples using bulk and LCM processing.MethodsExpression data from ten lung adenocarcinoma samples and six adjacent normal samples were acquired using LCM and bulk sampling methods. Expression values were evaluated for correlation between sample processing methods, as well as for bias introduced by the additional linear amplification required for LCM sample profiling.ResultsThe direct comparison of expression values obtained from the bulk and LCM sampled datasets reveals a large number of probesets with significantly varied expression. Many of these variations were shown to be related to bias arising from the process of linear amplification, which is required for LCM sample preparation. A comparison of differentially expressed genes (cancer vs. normal) selected in the bulk and LCM datasets also showed substantial differences. There were more than twice as many down-regulated probesets identified in the LCM data than identified in the bulk data. Controlling for the previously identified amplification bias did not have a substantial impact on the differences identified in the differentially expressed probesets found in the bulk and LCM samples.ConclusionLCM-coupled microarray expression profiling was shown to uniquely identify a large number of differentially expressed probesets not otherwise found using bulk tissue sampling. The information gain realized from the LCM sampling was limited to differential analysis, as the absolute expression values obtained for some probesets using this studys protocol were biased during the second round of amplification. Consequently, LCM may enable investigators to obtain additional information in microarray studies not easily found using bulk tissue samples, but it is of critical importance that potential amplification biases are controlled for.

Tumor budding in colorectal carcinoma: Confirmation of prognostic significance and histologic cutoff in a population-based cohort

Tumor budding in colorectal carcinoma has been associated with poor outcome in multiple studies, but the absence of an established histologic cutoff for “high” tumor budding, heterogeneity in study populations, and varying methods for assessing tumor budding have hindered widespread incorporation of this parameter in clinical reports. We used an established scoring system in a population-based cohort to determine a histologic cutoff for “high” tumor budding and confirm its prognostic significance. We retrieved hematoxylin and eosin–stained sections from 553 incident colorectal carcinoma cases. Each case was previously characterized for select molecular alterations and survival data. Interobserver agreement was assessed between 2 gastrointestinal pathologists and a group of 4 general surgical pathologists. High budding (≥10 tumor buds in a ×20 objective field) was present in 32% of cases, low budding in 46%, and no budding in 22%. High tumor budding was associated with advanced pathologic stage (P<0.001), microsatellite stability (P=0.005), KRAS mutation (P=0.010), and on multivariate analysis with a >2 times risk of cancer-specific death (hazard ratio=2.57 [1.27, 5.19]). After multivariate adjustment, by penalized smoothing splines, we found increasing tumor bud counts from 5 upward to be associated with an increasingly shortened cancer-specific survival. By this method, a tumor bud count of 10 corresponded to approximately 2.5 times risk of cancer-specific death. The interobserver agreement was good with weighted &kgr; of 0.70 for 2 gastrointestinal pathologists over 121 random cases and 0.72 between all 6 pathologists for 20 random cases. Using an established method to assess budding on routine histologic stains, we have shown that a cutoff of 10 for high tumor budding is independently associated with a significantly worse prognosis. The reproducibility data provide support for the routine widespread implementation of tumor budding in clinical reports.