Chemoprevention with Cyclooxygenase and Epidermal Growth Factor Receptor Inhibitors in Familial Adenomatous Polyposis Patients: mRNA Signatures of Duodenal Neoplasia

Familial adenomatous polyposis (FAP) is an autosomal dominant inherited disorder due to germline mutations in the APC (adenomatous polyposis coli) gene (1, 2). FAP is characterized by the formation of hundreds to thousands of adenomatous polyps in the colorectum and a nearly 100% lifetime risk of colorectal cancer if left untreated (3). Prophylactic colectomy has become the standard of care once the extent of colorectal polyposis is beyond endoscopic control. FAP patients are also at greatly increased risk for duodenal neoplasia, with duodenal adenomas eventually forming in >50% of FAP patients and duodenal adenocarcinoma occurring in up to 12% (3–8). As mutations in the APC gene are central to the initiation and development of colorectal cancer with 80% of sporadic colorectal cancers having APC loss or inactivation, FAP is an excellent model to study the molecular events leading to development of colorectal cancer and other intestinal cancers.

Chemoprevention studies in FAP patients can provide clues as to how drugs modify tumor progression. Multiple studies have shown that sulindac, a cyclooxygenase (COX) inhibitor and NSAID, significantly inhibits colorectal adenomatous polyps in FAP patients (9, 10). However, sulindac has failed to show a significant reduction in duodenal adenomas in FAP patients (11, 12). This is thought to be due to increased COX2 expression in the duodenal tissue compared with colonic tissue of FAP patients (13). Studies have suggested that APC inactivation and EGFR signaling promote COX2 expression, leading to the development of intestinal neoplasms (14, 15). The convergence between the WNT and EGFR signaling pathways and COX2 activity was demonstrated in a mouse model of FAP in which a combination of a COX and an EGFR inhibitor diminished small intestinal adenoma development by 87% (16). These results led us to test the hypothesis that a combination of COX and EGFR inhibition would impede adenoma formation in the duodenum of subjects with FAP. We recently reported on a positive clinical study where patients with FAP were treated with either placebo or sulindac and erlotinib (sulindac–erlotinib). At 6 months, the median total duodenal polyp burden had increased by 6 mm from baseline in the placebo arm and decreased by 9 mm in the sulindac–erlotinib arm (P < 0.001; ref. 17).

Here, we report the gene expression analyses from duodenal tissue at endpoint compared with baseline for those subjects enrolled in the trial. We show that the EGFR and COX2 pathways are activated in duodenal polyps and that the drug combination of sulindac–erlotinib blocks this activation. In addition, we found evidence for activation of IFNγ and IL12 signaling pathways, suggesting that the recruitment of both Th1 and natural killer (NK) T cells may have contributed to the polyp regression (size and number) observed in the drug-treated arm (18, 19).

This study was approved by the University of Utah Institutional Review Board (IRB#39278), conducted in accordance with recognized ethical guidelines, and informed consent was obtained from each subject. A randomized, two-arm chemoprevention trial was conducted between July 2010 and June 2014 in which FAP patients were treated either with placebo or with sulindac (150 mg twice daily) plus erlotinib (75 mg/day) for 6 months (registered with ClinicalTrials.gov as NCT 01187901). Seventy-three individuals with FAP completed the study. For patients with significant discomfort or evidence of toxicity, the dose was lowered during the course of the study as described previously (17). Endoscopic duodenal biopsies were taken for grossly uninvolved tissue at baseline and endpoint, while polyps were obtained at endpoint only. Tissues were placed in RNAlater Stabilization Solution (Thermo Fisher Scientific).

A subset of tissue from 10 research participants who responded to the drug combination (sulindac–erlotinib) and 10 research participants who progressed on placebo was selected for molecular analyses (Table 1). Total RNA was prepared from biopsies using a Qiagen RNeasy Mini Kit (Qiagen #74106) following the manufacturer's instructions and including the on-column RNase-free DNase treatment. RNA quantity and quality was determined using a Thermo Fisher Scientific NanoDrop Spectrophotometer and Agilent Bioanalyzer.

Fifty-two endpoint (20 uninvolved, 32 adenomas) and 17 baseline (all uninvolved) total RNA samples were treated with RiboZero Gold (Illumina) to remove ribosomal RNA prior to cDNA library preparation using the Illumina TruSeq Stranded Total RNA Sample Prep Protocol. PCR-amplified libraries were sequenced on Illumina HiSeq 2500 instrument using 50-cycle single-read chemistry. Sequencing datasets are deposited to NCBI GEO submission #GSE94919. Sequencing reads were aligned to the RefSeq Hg38 human genome using the Novoalign application (Novocraft). Differentially expressed genes were calculated using the USeq application “DefinedRegionsDifferentialSeq” (DRDS) as described previously (20, 21). The DRDS application uses DESeq2 negative binomial statistics together with a Benjamini and Hochberg FDR to identify differentially expressed genes (22). For paired sample comparisons, the DESeq2 paired analysis application in R was used.

Total RNA from 12 endpoint polyps (6 from placebo and 6 from drug endpoint) of similar 3 to 4 mm size, noted in Table 1, was further evaluated by targeted gene expression using the Human Inflammation and Immunity Transcriptome 475 gene panel (Qiagen # RHS-005Z). This targeted approach improves sequencing depth and reduces biases in amplification. The panel includes a diverse set of cytokines, growth factors, and transcription factors important in mediating general and specialized immune responses. RNA is converted to cDNA and amplified with a multiplex primer panel that labels each cDNA molecule with a unique molecular tag. The quantified indexed library DNA was pooled to an equimolar concentration alongside other samples. The pooled library DNA was amplified by emulsion PCR and enriched for positive ion sphere particles (ISP) using the Ion Torrent One Touch System II (Life Technologies) and the Ion PI Hi-Q OT2 Kit (Life Technologies).Templated ISPs were sequenced on a PIv3 micro-chip using the Ion Torrent Proton Machine (Life Technologies) and the Ion PI Hi-Q Sequencing 200 Kit (Life Technologies) for 130 cycles (520 flows). Sequence reads were aligned to the RefSeq Hg38 human genome using the STAR RNA read mapper. Reads that aligned to more than 3 sites in the human genome or did not have at least 60 bp aligned were excluded. Differentially expressed genes between treatment groups were determined using the QIASeq secondary data analysis tool (Qiagen). Student t test was applied after data normalization using total molecular tag counts.

The expression of MMP7, CD44, FOS, TM4SF5, EGFR, and PTGS2 was confirmed by qRT-PCR. cDNA was synthesized using the SuperScript VILO cDNA Synthesis Kit (Invitrogen #11754250) following the manufacturer's instructions. Predesigned IDT primer-probe sets were used together with PrimeTime Gene Expression Master Mix (IDT #1055771) to measure expression of the following genes: CD44 (IDT #Hs.PT.58.2004193), EGFR (IDT #Hs.PT.58.20590781), FOS (IDT #Hs.PT.58.15540029), PTGS2 (#Hs.PT.58.77266), MMP7 (IDT #Hs.PT.58.40068681), and TM4SF5 (IDT #Hs.PT.58.39684117). Relative gene expression was determined after normalization to the geometric mean expression of the internal control genes UBC (IDT #Hs.PT.39a.22214853), GAPDH (IDT #Hs.PT.58.40035104), and KRR1 (IDT #Hs.PT.58.4223891). Each gene was run in triplicate on a Bio-Rad CFX96 Real-Time PCR System. Data analysis was performed with the Bio-Rad CFX Manager software. GraphPad Prism 7 was used for plotting qRT-PCR results and for statistical analysis.

Tumor purity was assessed using the R application Estimation of Stromal and Immune cells in Malignant Tumors using Expression data (ESTIMATE) as described previously (23, 24). Normalized RPKM values from all expressed genes (≥10 reads) were used to determine RNA expression from immune and stromal cells in each of the 32 duodenal polyp RNA sequencing (RNA-Seq) datasets. Percent tumor purity was then calculated by combining immune and stromal scores.

Principal component analysis (PCA) and hierarchical clustering was used to identify sets of differentially expressed genes that separate samples based on placebo versus drug treatment (25, 26). Ingenuity Pathway Analysis (IPA) was used to predict signaling pathways changing between the groups. Signaling pathways regulated by known transcription factors, cytokines, growth factors, and kinases (upstream regulators) were identified using a Fisher exact test.

We determined a z-score using IPA that infers the activation state of an upstream regulator based on the direction of fold change of its target genes (27). A z-score ≥2 was considered statistically significant.

Formalin-fixed paraffin-embedded (FFPE) polyp tissue from 10 patients on placebo (19 duodenum, 3 colon) and 10 patients on drug (7 duodenum and 3 colon) resected for clinical pathologic evaluation were stained for CD56 (a marker for NK cells) according to ARUP clinical laboratory test number 2003589. Briefly, sections were cut at 4μm, melted at 60°C for 30 minutes, stained on the BenchMark Ultra (Ventana Medical Systems) using the CD56 mouse mAb, clone 123C3.D5 (Abgent, catalog # AH10009) at a dilution of 1:40, and detected using the UltraView Universal DAB Detection Kit (Roche). The sections were counterstained with hematoxylin. CD56⁺ cells were counted in up to 10 high-power fields (HPF, 40×). Some polyp tissue had less than 10 HPF of dysplastic tissue. Associations of CD56 counts with receiving drug treatment were estimated through Poisson regression, offset by the log of HPFs present. An alpha level of 0.05 was used to determine statistical significance. SAS 9.4 was used for statistical analysis. Using a likelihood ratio test comparing Poisson and negative binomial models, we found that our data are overdispersed using the Poisson model, so a negative binomial model was used.

The primary objective of this study was to determine whether the sulindac–erlotinib drug combination was affecting the intended molecular targets/pathways in duodenal polyps. Duodenal tissue from 20 individuals, 10 from each arm of the trial, was selected on the basis of change in duodenal polyp burden over the 6-month investigation period. Some patients underwent dose reduction, so the average consumed daily dose is reported (Table 1). In addition, we report the reported adverse events, the individual polyps used from the patients, reported as size in mm, and the corresponding calculated tumor purity.

The average number of unique aligned reads per sample was 14.8 million. Approximately 74% of human RefSeq genes (18,698/25,199) had a minimum of 10 exonic reads in one or more samples and were therefore considered expressed in the human duodenum. Figure 1 is a schematic showing the number of subjects and samples used to identify differences in gene expression between: (i) baseline uninvolved and endpoint uninvolved; (ii) baseline uninvolved and endpoint polyp; and (iii) endpoint uninvolved and endpoint polyp. Overall, the dynamic range of gene expression observed across these relatively small adenomas (<10 mm) was less than typically seen in invasive cancers (28). Thus, a 2.0-fold change cutoff was used to select genes that distinguish between subjects treated with placebo versus drug. A 1.5-fold change was used for analyses involving pathway discovery.

PCA of differentially expressed genes from endpoint versus baseline comparisons showed clear separation of polyps from patients on drug versus polyps from patients on placebo (Fig. 2A). Principal component 1 (PC1) accounted for 20% of the variability observed in the data and separated polyps from patients on drug from patients on placebo. Principal component 2 (PC2) accounted for 14% of the variation in the data and separated 2 polyps (6mmA_017 and 6mmB_017), bottom center, from patient 2 on drug, from the other 11 polyps from patients on drug. These two polyps may represent acquired resistance to the drug. Principal component 3 accounted for 8% of the variation in the gene expression data.

Hierarchical clustering analysis of the top genes that compose principal components 1 and 2 and are in the WNT signaling pathway (14 genes), PGE2 pathway (10 genes), or EGFR pathway (10 genes) are shown in Fig. 2B. Endpoint versus baseline comparisons show a separation of polyps from patients on drug versus polyps from patients on placebo. The gene expression patterns of these genes suggest that the drug combination blocks adenoma progression through WNT signaling and specifically blocks the targeted PGE2 and EGFR signaling processes. Two outlier polyps identified from patient 2 (D6mmB_017 and D6mmA_017) in PCA showed high expression of a subset of genes (MMP7, AXIN2, CXCL5, EGR1, FOS; Fig. 2B), suggesting a lack of drug effect, and possible drug resistance in these polyps.

When comparing endpoint samples with paired baseline uninvolved duodenal tissue, 3 patients (one on drug and two on placebo) were excluded due to a lack of baseline tissue (Table 1). Five polyps were also excluded from the final analysis because tumor purity was estimated at <50% (Table 1; Supplementary Fig. S1). Differential gene expression with the <50% tumor purity included and excluded is presented in Supplementary Table S1. When comparing endpoint polyp samples with paired baseline uninvolved duodenum from patients randomized to placebo, we identified 977 differentially expressed genes (fold change ≥ 2.0; FDR < 0.05) by DESeq2 analysis (Supplementary Table S1). The same comparison from patients on drug yielded only 51 differentially expressed genes, suggesting that the drug combination restores the normal duodenal biology (Supplementary Table S1). Comparing endpoint uninvolved duodenum with paired baseline uninvolved duodenum, we identified 1 differentially expressed gene in patients on sulindac–erlotinib and 11 differentially expressed genes in patients on placebo (Supplementary Table S1).

To evaluate whether there were drug-specific effects on normal tissue, we compared endpoint uninvolved duodenum between patients on sulindac–erlotinib and patients on placebo (Supplementary Table S2). No differentially expressed genes (fold change ≥ 2.0; FDR < 0.05) were found. Only one differentially expressed gene, NANOS3, was found comparing endpoint adenomas with paired endpoint uninvolved duodenum from drug-treated patients. In contrast, 493 differentially expressed genes were found comparing endpoint adenomas with paired endpoint uninvolved duodenum from patients on placebo (Supplementary Table S2). Again, this set of differentially expressed genes includes multiple known genes involved in adenoma polyp progression, including CD44, MMP7, and CEMIP (also known as KIAA1199; ref. 29).

CD44, MMP7, FOS, TM4SF5, EGFR, and PTGS2 gene expression, representing the three target signaling pathways WNT, EGFR, and COX2, were evaluated by qRT-PCR using PrimeTime gene expression assays (IDT; Fig. 3). Relative gene expression was determined by the 2^−ΔΔC_t method using GAPDH, KRR1, and UBC as the internal control genes (30). CD44 and MMP7 show a significant increase in gene expression in polyp as compared with normal from the placebo group (P < 0.05), consistent with previous reports (Fig. 3; refs. 29, 31). In polyps from subjects on placebo, we observe upregulation of EGFR mRNA, a major effector after APC transformation (32, 33). We also observe downregulation of TM4SF5, which has been reported to be associated with elevated TM4SF5 protein expression, suggesting activation of a previously observed feedback mechanism involving proteasome inhibition in response to elevated TM4SF5 protein levels (34). Notably for all six genes, there is no significant change in gene expression of normal versus polyp from the drug group. When comparing gene expression between polyps from subjects on placebo versus subjects on drug, the FOS gene is the only one to show a significant difference (P < 0.05). The two polyps from patient 2 may represent acquired resistance to the drug, as both had higher FOS expression and were outliers (Fig. 2). Certain genes, such as PTGS2, had wide variation in gene expression; thus, additional confirmation was performed using qRT-PCR. The different platforms had high correlation in measurement, suggesting biologic (and not experimental) variability (Supplementary Fig. S2). Previous studies evaluating the levels of PTGS2 mRNA in intestinal polyps have been mixed, with some studies showing elevated RNA levels (35, 36) and others showing no change compared with uninvolved tissue (37), which is consistent with the major regulation of COX2 being posttranslational (38). Even in studies showing statistical differences in the level of PTGS2, induction is significantly varied across subjects, which is similar to our findings in duodenal polyps from FAP patients.

The differentially expressed list of 2,637 genes representing 1.5-fold differential expression and FDR <0.05 (Supplementary Table S1) were uploaded into IPA software (2,591 of the genes annotated in IPA) to identify the pathways affected in duodenal tissue when normal epithelia becomes a polyp. The software also identified which of these pathway changes were repressed by sulindac–erlotinib treatment. Multiple known signaling pathways important in adenoma development were predicted to be activated in adenomas from patients on placebo, including CTNNB1 (WNT), EGFR, TNF, and PGE2 pathways (Table 2; Supplementary Table S3). In contrast, adenomas from patients on drug showed almost complete loss of cancer pathway signaling in agreement with the reduction in polyp burden observed in these patients. The z-score predicts the activation state of the upstream regulator based on the direction of fold change of its gene targets. A z-score ≥2 suggests the upstream regulator is significantly activated. The Fisher exact P value is based on the overlap of differentially expressed genes and known regulator targets with no information regarding activation state. We also observed inhibition of multiple signaling pathways in polyps from patients on placebo that include pathways related to intestinal development and tumor suppression (CDX2) (39, 40), heterochromatin assembly (CBX5), cell adhesion (CTNNA) and IFN signaling (IFNα and IFNγ).

IPA analysis also revealed immune signaling pathways that are downregulated in placebo polyps and not downregulated in drug polyps, most notably IFNα and IFNγ. Because PGE2, a product of COX2 enzyme, is potent inducer of IL10, which in turn suppresses IFNγ signaling (41), this observation would be consistent with increased COX2 expression in placebo polyps. Similarly, polyps from patients treated with sulindac–erlotinib would not have COX2 overexpression and would not have suppression of IFNγ. To further explore this finding, an inflammation and immunity transcriptome panel was run on 6 placebo polyps and 6 drug polyps to enhance coverage. These results confirmed that IFNα, IFNγ, and IL12 are more active in polyps from patients on drug, whereas PGE2 is less active in polyps from patients on drug (Table 3; Supplementary Tables S4 and S5).

The RNA expression data suggest activation of the specific T-cell and NK-mediated immune response through IL12 and IFNγ. We thus stained FFPE tissues from the clinical trial for the presence of NK cells using IHC for CD56 (neural cell adhesion molecule; NCAM). We observe an increase in NK cells in polyps from patients on drug versus placebo, but the observation is not statistically significant. Polyps from the group treated with drug had an average CD56 count of 1.18 per HPF ranging from 0 to 3.57 per HPF. The placebo group had an average CD56 count of 0.846 per HPF, ranging from 0 to 1.83. Upon fitting the data to a negative binomial model, we find a 1.43 increase in count per HPF in the polyps from patients on drug (95% CI, 0.77–2.66; P = 0.2641). Although there is a slight increase in NK cells, which may represent activation of T-cell–mediated immune response, it is not statistically different from the polyps from patients on placebo.

We recently completed and reported the clinical findings of a chemoprevention trial aimed at preventing progression of duodenal adenomas in patients with FAP (17). Combined treatment with sulindac–erlotinib resulted in a 56% reduction in duodenal polyp burden after 6 months, whereas the placebo arm had a 31% increase in duodenal polyp burden. The goal of this study was to characterize the molecular changes associated with adenomatous polyp regression in FAP patients treated with sulindac–erlotinib. RNA-Seq technology was used to define the human duodenum transcriptome in FAP patients treated with sulindac–erlotinib or placebo.

One of the challenges resulting from the success of the clinical trial was that there was very limited polyp tissue available from patients on drug. Consequently, the power to discover molecular changes was limited, ranging from 51% to 80% depending on the number of paired comparisons (Supplementary Data). Even with this limitation, the robustness of these gene expression–based signatures provided convincing evidence that the sulindac–erlotinib drug combination was inhibiting the intended WNT, EGFR, and PGE2 pathways. This fits with our current biologic and genetic understanding of the progression of colon cancer, expands our understanding of how these drugs drive the regression of duodenal polyps in FAP patients, and identifies new biomarkers for diagnostics and therapeutics.

Adenomas from patients on placebo displayed a wide range of gene expression changes, including changes in RNA transcripts associated with increased WNT, EGFR, and PGE2 signaling. Increased expression of many WNT signaling targets, including AXIN2, LGR5, MMP7, and MYC observed in our study are in agreement with previously published gene expression studies from sporadic and/or FAP patient cohorts (29, 31, 42). These genes play an important role in regulating cell proliferation and tumor progression in colon adenomas. AXIN2 and LGR5 are both considered negative regulators of WNT signaling, while MMP7 and MYC are positive regulators. AXIN2 or conductin protein is part of the multiprotein APC complex that regulates the stability of β-catenin, and LGR5 is an established cell surface protein marker of intestinal stem cells (43, 44). MMP7 protein plays a role in the breakdown of extracellular matrix, T-cell migration, and tumor metastasis (45). MYC protein plays multiple roles in the development of cancer, including the regulation of cell proliferation, apoptosis, and epithelial-to-mesenchymal transition (46). The upregulation of these genes provides strong support of WNT activation in FAP duodenal adenomas in our study similar to previous gene expression studies.

The RNA levels of many target genes of both EGFR and PGE2 signaling were also significantly increased in duodenal adenomas from patients on placebo but not on drug. The increased expression of immediate early genes FOS and EGR1 in duodenal adenomas is consistent with increased EGFR signaling and cell proliferation. Increased PGE2 signaling was associated with increased mRNA levels of the stem cell marker CD44. PGE2 increases the number and survival of CD44⁺ stem cells in human colorectal cancer and animal models of colon tumor metastasis (47). The increased activation of EGFR and PGE2 pathways in adenomatous polyps from FAP patients in our study further supports the use of the combined sulindac–erlotinib therapy in our phase II clinical trial for the treatment of duodenal adenomas in FAP patients.

Pathways selectively downregulated in placebo polyps but not drug polyps compared with paired uninvolved duodenal tissue included α-catenin (CTNNA) and chromobox 5 (CBX5). α-Catenin is an actin-binding protein whose cellular distribution is regulated by β-catenin (48). Reciprocally α-catenin inhibits Wnt/β-catenin–mediated transcription through dual binding of β-catenin and actin. The observed decrease in α-catenin signaling in duodenal polyps observed in our study may be the result of altered α-catenin localization and/or reduced transcriptional regulation through α-catenin. Chromobox 5, also known as heterochromatin protein 1 alpha (HP1α), is a nuclear protein that mediates transcriptional silencing through interactions with H3K9 methyltransferase and DNA methyltransferases 1 and 3 (49). HP1α is also important in accurate chromosomal segregation during mitosis (50). Decreased HP1α signaling observed in placebo polyps may reflect an increase in transcriptional activity and/or decrease in chromosomal stability necessary for duodenal polyp development and progression. Together, the reduced activity of α-catenin and HP1α are both suggestive of increased transcriptional activity associated with tumor development and growth.

Following sulindac–erlotinib therapy, a pronounced reduction in the number of genes differentially expressed in adenomas was observed in the patients on drug. The average size of adenomas collected from patients on drug was smaller than those collected from patients on placebo, yet retained their adenomatous appearance upon pathologic examination. The pronounced regression of duodenal adenomas in our phase II clinical trial was surprising based on previous trials using sulindac therapy alone (11, 12). At best, we anticipated an inhibition of further duodenal polyp growth in our FAP patient cohort. Although this is clearly speculative, and further work will be required, our results may suggest that the combination therapy of sulindac–erlotinib is restoring some level of innate immunosurveillance and adenoma cell killing in the duodenum of FAP patients on drug. The derepression of IFNα signaling and presence of CD56⁺ NK cells support this idea. It has been shown that increased levels of PGE2 cause a decrease IL12 signaling, which is important in the recruitment of NK cells and cytotoxic T lymphocytes (51). Both mouse and human cancer cells deficient in COX or PGE2 show increased immune signaling and T-cell–dependent growth control compared with cancer cells expressing COX (52). These findings are consistent with our studies that show sulindac–erlotinib restores the expression of genes important in the innate immune response and NK-cell surveillance and function.

On the basis of RNA analysis, we observed potential resistance to the drug combination in multiple polyps from patient 2. This chemoprevention trial, however, was limited in its ability to evaluate resistance by the minimal polyp tissue that was from patients on drug and available at the end of the trial. It will be important for future work to examine the molecular changes in polyps that are persistent, do not regress, and likely represent resistance to EGFR and COX inhibition. Future chemoprevention trials with this drug combination will benefit by extending the treatment period beyond 6 months by which time-resistant polyps would be more established and evident. In addition, sampling for future studies should consider use of highly sensitive new technologies, such as single-cell RNA and DNA sequencing, liquid biopsy for circulating tumor DNA and cytokines in blood, single-cell mass cytometry for protein expression, and metabolomics. These other endpoints would enable one to overcome limitations of sample number as well as detecting distinct responsive and resistant cells within a single polyp. In a follow-up clinical trial (NCT02961374), efficacy and tolerability of erlotinib alone, given once weekly, in FAP patients with Spigelman stage II to III duodenal polyposis is being examined. Secondary aims will similarly evaluate the gene expression profiles of duodenal tissues. These analyses will be important to examine whether inhibition of the COX2 pathway is required to derepress IFNα signaling.

In summary, our analysis of gene expression in duodenal tissue from patients on drug compared with patients on placebo describes key changes in cancer, inflammation, and innate immunity signaling pathways. Many of the genes and pathways described in our study support previous findings of molecular changes observed in colon adenomas from FAP patients. The observed reduction in duodenal polyp number and size together with the inhibition of WNT, EGFR, and PGE2 signaling and increase in IFNγ signaling provide important insights into the mechanisms of duodenal polyp regression in FAP patients treated with sulindac–erlotinib.

N.J. Samadder has received speakers bureau honoraria from Cook Medical, Inc., is a consultant/advisory board member for Janssen, and has provided expert testimony for Medico-Legal Consulting. R. Burt is a consultant/advisory board member for Thetis Pharma. No potential conflicts of interest were disclosed by the other authors.

Conception and design: D.A. Delker, N.J. Samadder, R.W. Burt, D.W. Neklason

Development of methodology: D.A. Delker, A.C. Wood, N.J. Samadder, D.W. Neklason

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A.C. Wood, A.K. Snow, N.J. Samadder, K.E. Affolter, I.J. Stijleman, P. Kanth, K.R. Byrne, R.W. Burt, D.W. Neklason

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): D.A. Delker, A.C. Wood, A.K. Snow, K.M. Boucher, L.M. Pappas, P.S. Bernard, D.W. Neklason

Writing, review, and/or revision of the manuscript: D.A. Delker, A.K. Snow, N.J. Samadder, W.S. Samowitz, K.E. Affolter, K.M. Boucher, P. Kanth, K.R. Byrne, R.W. Burt, P.S. Bernard, D.W. Neklason

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): D.A. Delker, R.W. Burt, D.W. Neklason

Study supervision: N.J. Samadder, R.W. Burt, D.W. Neklason

Other (evaluated pathology): W.S. Samowitz

We thank Dr. Matt Topham for helpful discussions of signaling pathways, Michelle W. Done, Megan Keener, Therese Berry, and Danielle Sample for study coordination effort, and the research participants who are committed to finding solutions for managing their condition.

This work was supported by NI HHHSN2612012000131 (to P.S. Bernard, D.W. Neklason, D.A. Delker, A.C. Wood, and I.J. Stijleman), NCIPO1-CA073992 (to R.W. Burt, D.W. Neklason, A.K. Snow, N.J. Samadder, W.S. Samowitz, K. M. Boucher, L.M. Pappas, P. Kanth, and K.R. Byrne), National Cancer Institute Cancer Center Support Grant P30-CA042014 (to K.M. Boucher and L.M. Pappas), National Center for Advancing Translational Sciences of the NIH under Award Number UL1TR00106, and Huntsman Cancer Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.