Plasma Protein Biomarkers in Advanced or Metastatic Colorectal Cancer Patients Receiving Chemotherapy With Bevacizumab or Cetuximab: Results from CALGB 80405 (Alliance).

PURPOSE: CALGB 80405 compared the combination of first-line chemotherapy with cetuximab or bevacizumab in the treatment of advanced or metastatic colorectal cancer (mCRC). Although similar clinical outcomes were observed in the cetuximab-chemotherapy group and the bevacizumab-chemotherapy group, biomarkers could identify patients deriving more benefit from either biologic agent. PATIENTS AND METHODS: In this exploratory analysis, the Angiome, a panel of 24 soluble protein biomarkers were measured in baseline plasma samples in CALGB 80405. Prognostic biomarkers were determined using univariate Cox proportional hazards models. Predictive biomarkers were identified using multivariable Cox regression models including interaction between biomarker level and treatment. RESULTS: In the total population, high plasma levels of Ang-2, CD73, HGF, ICAM-1, IL6, OPN, TIMP-1, TSP-2, VCAM-1, and VEGF-R3 were identified as prognostic of worse progression-free survival (PFS) and overall survival (OS). PlGF was identified as predictive of lack of PFS benefit from bevacizumab [bevacizumab HR, 1.51; 95% confidence interval (CI), 1.10-2.06; cetuximab HR, 0.94; 95% CI, 0.71-1.25; Pinteraction = 0.0298] in the combined FOLFIRI/FOLFOX regimens. High levels of VEGF-D were predictive of lack of PFS benefit from bevacizumab in patients receiving FOLFOX regimen only (FOLFOX/bevacizumab HR, 1.70; 95% CI, 1.19-2.42; FOLFOX/cetuximab HR, 0.92; 95% CI, 0.68-1.24; Pinteraction = 0.0097). CONCLUSIONS: In this exploratory, hypothesis-generating analysis, the Angiome identified multiple prognostic biomarkers and two potential predictive biomarkers for patients with mCRC enrolled in CALGB 80405. PlGF and VEGF-D predicted lack of benefit from bevacizumab in a chemo-dependent manner. See related commentaries by Mishkin and Kohn, p. 2722 and George and Bertagnolli, p. 2725.

Cabozantinib and Panitumumab for RAS Wild-Type Metastatic Colorectal Cancer.

LESSONS LEARNED: Antitumor activity was observed in the study population. Dose modifications of cabozantinib improve long-term tolerability. Biomarkers are needed to identify patient populations most likely to benefit. Further study of cabozantinib with or without panitumumab in patients with metastatic colorectal cancer is warranted. BACKGROUND: The epidermal growth factor receptor (EGFR) antibody panitumumab is active in patients with RAS wild-type (WT) metastatic colorectal cancer (mCRC), but nearly all patients experience resistance. MET amplification is a driver of panitumumab resistance. Cabozantinib is an inhibitor of multiple kinases, including vascular endothelial growth factor receptor 2 (VEGFR2) and c-MET, and may delay or reverse anti-EGFR resistance. METHODS: In this phase Ib clinical trial, we established the maximum tolerated dose (MTD) and recommended phase II dose (RP2D) of cabozantinib and panitumumab. We then treated an expansion cohort to further describe the tolerability and clinical activity of the RP2D. Eligibility included patients with KRAS WT mCRC (later amended to include only RAS WT mCRC) who had received prior treatment with a fluoropyrimidine, oxaliplatin, irinotecan, and bevacizumab. RESULTS: Twenty-five patients were enrolled and treated. The MTD/RP2D was cabozantinib 60 mg p.o. daily and panitumumab 6 mg/kg I.V. every 2 weeks. The objective response rate (ORR) was 16%. Median progression free survival (PFS) was 3.7 months (90% confidence interval [CI], 2.3-7.1). Median overall survival (OS) was 12.1 months (90% CI, 7.5-14.3). Five patients (20%) discontinued treatment due to toxicity, and 18 patients (72%) required a dose reduction of cabozantinib. CONCLUSION: The combination of cabozantinib and panitumumab has activity. Dose reductions of cabozantinib improve tolerability.

Prognostic and Predictive Biomarkers in Patients with Metastatic Colorectal Cancer Receiving Regorafenib.

Regorafenib is a tyrosine kinase inhibitor approved by the FDA for the treatment of patients with chemotherapy refractory metastatic colorectal cancer (mCRC). Regorafenib inhibits signaling through multiple receptors associated with angiogenesis, metastasis, and tumor immunity. Here, we report biomarker results from LCCC1029, a randomized, placebo-controlled, phase II trial of chemotherapy ± regorafenib in patients with second-line mCRC. A panel of 20 soluble protein biomarkers (termed the Angiome) was assessed in the plasma of 149 patients from the LCCC1029 trial both at baseline and along the treatment continuum. Baseline protein levels were analyzed for prognostic and predictive value for progression-free survival (PFS) and overall survival (OS). Changes in protein levels during treatment were analyzed for potential pharmacodynamic effects. Six markers (HGF, IL6, PlGF, VEGF-R1, OPN, and IL6R) were found to be prognostic for PFS. Nine markers (IL6, TIMP-1, PlGF, VCAM-1, ICAM-1, OPN, TSP-2, HGF, and VEGF-R1) were prognostic for OS. Higher baseline levels of OPN (P intx = 0.0167), VCAM-1 (P intx = 0.0216), and PDGF-AA (P intx = 0.0435) appeared to predict for PFS benefit from regorafenib compared with placebo. VCAM-1 was also potentially predictive of OS benefit from regorafenib compared with placebo (P intx = 0.0124). On-treatment changes of six markers reflected potential on-target effect of regorafenib. Consistent results were observed in an Italian cohort where 105 patients with late-stage mCRC received regorafenib monotherapy. The key findings of this study suggest that VCAM-1 may be a predictive biomarker for regorafenib benefit, while multiple protein markers may be prognostic of outcome in patients with mCRC.

Dynamic Changes in Circulating Tumor DNA During Chemoradiation for Locally Advanced Lung Cancer.

PURPOSE: Concurrent chemoradiation therapy (CRT) is the principal treatment modality for locally advanced lung cancer. Cell death due to CRT leads to the release of cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA) into the bloodstream, but the kinetics and characteristics of this process are poorly understood. We hypothesized that there could be clinically meaningful changes in cfDNA and ctDNA during a course of CRT for lung cancer. METHODS AND MATERIALS: Multiple samples of plasma were obtained from 24 patients treated with CRT for locally advanced lung cancer to a mean dose of 66 Gy (range, 58-74 Gy) at the following intervals: before CRT, at weeks 2 and 5 during CRT, and 6 weeks after treatment. cfDNA was quantified, and a novel next generation sequencing (NGS) technique using enhanced tagged/targeted-amplicon sequencing was performed to analyze ctDNA. RESULTS: Patients for whom specific mutations in ctDNA were undetectable at the baseline time point had improved survival, and potentially etiologic driver mutations could be tracked throughout the course of CRT via NGS in multiple patients. We quantified the levels of cfDNA from patients before CRT, at week 2, week 5, and at 6 weeks after treatment. No differences were observed at weeks 2 and 5 of therapy, but we noted a significant increase in cfDNA in the posttreatment follow-up samples compared with samples collected before CRT (P = .05). CONCLUSIONS: Dynamic changes in both cfDNA and ctDNA were observed throughout the course of CRT in patients with locally advanced lung cancer. Specific mutations with therapeutic implications can be identified and tracked using NGS methodologies. Further work is required to characterize the changes in cfDNA and ctDNA over time in patients treated with CRT and to assess the predictive and prognostic potential of this powerful technology.

A phase Ib study of capecitabine and ziv-aflibercept followed by a phase II single-arm expansion cohort in chemotherapy refractory metastatic colorectal cancer.

BACKGROUND: Patients with chemotherapy refractory metastatic colorectal cancer (CRC) have a poor prognosis and limited therapeutic options. In this phase Ib/II clinical trial, we established the maximum tolerated dose (MTD) and recommended phase II dose (RPTD) for the combination of capecitabine and ziv-aflibercept, and then we evaluated the efficacy of the combination in patients with chemotherapy refractory metastatic CRC. METHODS: All patients were required to have a Karnofsky Performance Status > 70% and adequate organ function. The phase Ib dose escalation cohort included patients with advanced solid tumors who had progressed on all standard therapies. Using a standard 3 + 3 design, we identified the MTD and RPTD for the combination. Fifty patients with metastatic CRC who had progressed on or were intolerant of a fluoropyrimidine, oxaliplatin, irinotecan, and bevacizumab were then enrolled in a single-arm phase II expansion cohort, and were treated at the RPTD. Prior EGFR antibody therapy was required for subjects with RAS wildtype tumors. The primary endpoint for the expansion cohort was progression-free survival (PFS) at two months. Secondary endpoints included objective response rate (ORR) and overall survival (OS). RESULTS: A total of 63 patients were enrolled and evaluable for toxicity (13 dose escalation; 50 expansion). The MTD and RPTD were: capecitabine 850 mg/m2, P.O. bid, days 1-14, and ziv-aflibercept 6 mg/kg I.V., day 1, of each 21-day cycle. In the expansion cohort, 72% of patients were progression-free at two months (95% confidence interval [CI], 60-84%). Median PFS and OS were 3.9 months (95% CI, 2.3-4.5) and 7.1 months (95% CI: 5.8-10.0), respectively. Among all patients evaluable for toxicity, the most common treatment related adverse events (all grade [%]; grade ≥ 3 [%]) included palmar-plantar erythrodysesthesia (41%; 6%), hypertension (33%; 22%), and mucositis (19%; 5%). RNA was isolated from archived tumor specimens and gene expression analyses revealed no association between angiogenic biomarkers and clinical outcomes. CONCLUSION: The combination of capecitabine and ziv-aflibercept at the RPTD demonstrated acceptable safety and tolerability. PFS at 2 months in patients with chemotherapy refractory metastatic CRC was significantly greater than that in historical controls, indicating that this combination warrants further study. TRIAL REGISTRATION: This clinical trial was registered in the www.clinicaltrials.gov system as NCT01661972 on July 31, 2012.

RasGRP1 is a potential biomarker to stratify anti-EGFR therapy response in colorectal cancer.

Colorectal cancer (CRC) is the third most frequent neoplastic disorder and is a main cause of tumor-related mortality as many patients progress to stage IV metastatic CRC. Standard care consists of combination chemotherapy (FOLFIRI or FOLFOX). Patients with WT KRAS typing are eligible to receive anti-EGFR therapy combined with chemotherapy. Unfortunately, predicting efficacy of CRC anti-EGFR therapy has remained challenging. Here we uncover that the EGFR-pathway component RasGRP1 acts as CRC tumor suppressor in the context of aberrant Wnt signaling. We find that RasGRP1 suppresses EGF-driven proliferation of colonic epithelial organoids. Having established that RasGRP1 dosage levels impacts biology, we focused on CRC patients next. Mining five different data platforms, we establish that RasGRP1 expression levels decrease with CRC progression and predict poor clinical outcome of patients. Lastly, deletion of one or two Rasgrp1 alleles makes CRC spheroids more susceptible to EGFR inhibition. Retrospective analysis of the CALGB80203 clinical trial shows that addition of anti-EGFR therapy to chemotherapy significantly improves outcome for CRC patients when tumors express low RasGRP1 suppressor levels. In sum, RasGRP1 is a unique biomarker positioned in the EGFR pathway and of potential relevance to anti-EGFR therapy for CRC patients.

An initial genetic analysis of gemcitabine-induced high-grade neutropenia in pancreatic cancer patients in CALGB 80303 (Alliance).

OBJECTIVES: One of the standard of care regimens for advanced pancreatic cancer is gemcitabine-based chemotherapy. The efficacy of gemcitabine is limited by dose-limiting hematologic toxicities especially neutropenia. Uncovering the variability of these toxicities attributed to germline DNA variation is of great importance. PATIENTS AND METHODS: CALGB 80303 was a randomized study in advanced pancreatic cancer patients treated with gemcitabine with or without bevacizumab. The study protocol included genotyping of genes of gemcitabine disposition (CDA, DCTD, SLC29A1, SLC28A1, and SLC29A2), as well as a genome-wide analysis. The clinical phenotype was time to early high-grade neutropenia event accounting for progression or death or other treatment-terminating adverse events as competing for informative events. The inference was carried out on the basis of the association between genotype and cause-specific hazard of a neutropenic event. RESULTS: The primary analyses were carried out on the basis of 294 genetically estimated European pancreatic cancer patients. For CDA rs2072671 (A>C), AC and CC patients had a lower risk of neutropenia than AA patients (P=0.01, hazard ratio: 0.61, 95% confidence interval: 0.41-0.89). For SLC28A1 rs3825876 (G>A), AA patients have a higher risk of neutropenia than GA and GG patients (P=0.02, hazard ratio: 1.51, 95% confidence interval: 1.06-2.16). CDA rs2072671 was associated with increased mRNA expression in whole blood in three studies (P=2.7e-14, 6.61e-62, and 9.70e-65). In the genome-wide analysis, variants in TGFB2 were among the top hits (lowest P=1.62e-06) but had no effect in luciferase assays. CONCLUSION: This is the first genetic analysis of gemcitabine-induced neutropenia using a competing risk model in a prospective randomized clinical study has proposed a potentially novel mechanism of the protective effect of the CDA rs2072671 variant. Further confirmation is needed.

Genetic variation determines VEGF-A plasma levels in cancer patients.

Angiogenesis is essential in tumor biology and is regulated by vascular endothelial growth factor (VEGF) ligands and receptors. Here we aimed to discover genetic variants associated with levels of circulating angiogenic proteins in cancer patients. Plasma was collected at baseline in 216 pancreatic and 114 colorectal cancer patients. Thirty-one angiogenic proteins were measured by ELISA. 484,523 Single Nucleotide Polymorphisms (SNP) were tested for association with plasma levels for each protein in pancreatic cancer patients. Three top-ranked hits were then genotyped in colorectal cancer patients, where associations with the same proteins were measured. The results demonstrated rs2284284 and MCP1 (P-value = 6.7e-08), rs7504372 and VEGF-C (P-value = 9.8e-09), and rs7767396 and VEGF-A (P-value = 5.8e-09) were SNP-protein pairs identified in pancreatic cancer patients. In colorectal cancer patients, only rs7767396 (A > G) and VEGF-A was validated (P-value = 5.18e-05). The AA genotype of rs7767396 exhibited 2.04-2.3 and 2.7-3.4-fold higher VEGF-A levels than those with AG and GG genotypes. The G allele of rs7767396 reduces binding of the NF-AT1 transcription factor. In conclusion, a common genetic variant predicts the plasma levels of VEGF-A in cancer patients through altered binding of NF-AT1.

A Phase I Trial of the IGF-1R Antibody Ganitumab (AMG 479) in Combination with Everolimus (RAD001) and Panitumumab in Patients with Advanced Cancer.

PURPOSE: This study evaluated the maximum tolerated dose or recommended phase II dose (RPTD) and safety and tolerability of the ganitumab and everolimus doublet regimen followed by the ganitumab, everolimus, and panitumumab triplet regimen. MATERIALS AND METHODS: This was a standard 3 + 3 dose escalation trial. Doublet therapy consisted of ganitumab at 12 mg/kg every 2 weeks; doses of everolimus were adjusted according to dose-limiting toxicities (DLTs). Panitumumab at 4.8 mg/kg every 2 weeks was added to the RPTD of ganitumab and everolimus. DLTs were assessed in cycle 1; toxicity evaluation was closely monitored throughout treatment. Treatment continued until disease progression or undesirable toxicity. Pretreatment and on-treatment skin biopsies were collected to assess insulin-like growth factor 1 receptor and mammalian target of rapamycin (mTOR) target modulation. RESULTS: Forty-three subjects were enrolled. In the doublet regimen, two DLTs were observed in cohort 1, no DLTs in cohort -1, and one in cohort -1B. The triplet combination was discontinued because of unacceptable toxicity. Common adverse events were thrombocytopenia/neutropenia, skin rash, mucositis, fatigue, and hyperglycemia. In the doublet regimen, two patients with refractory non-small cell lung cancer (NSCLC) achieved prolonged complete responses ranging from 18 to >60 months; one treatment-naïve patient with chondrosarcoma achieved prolonged stable disease >24 months. In dermal granulation tissue, the insulin-like growth factor receptor and mTOR pathways were potently and specifically inhibited by ganitumab and everolimus, respectively. CONCLUSION: The triplet regimen of ganitumab, everolimus, and panitumumab was associated with unacceptable toxicity. However, the doublet of ganitumab at 12 mg/kg every 2 weeks and everolimus five times weekly had an acceptable safety profile and demonstrated notable clinical activity in patients with refractory NSCLC and sarcoma. IMPLICATIONS FOR PRACTICE: This trial evaluated the maximum tolerated dose or recommended phase II dose and safety and tolerability of the ganitumab and everolimus doublet regimen followed by the ganitumab, everolimus, and panitumumab triplet regimen. Although the triplet regimen of ganitumab, everolimus, and panitumumab was associated with unacceptable toxicity, the doublet of ganitumab at 12 mg/kg every 2 weeks and everolimus at five times weekly had an acceptable safety profile and demonstrated notable clinical activity in patients with refractory non-small cell lung cancer and sarcoma.

Association of Tumor HER3 Messenger RNA Expression With Panitumumab Efficacy in Advanced Colorectal Cancer.

IMPORTANCE: Epidermal growth factor receptor (EGFR) (HER1) signaling depends on ligand binding and dimerization with itself or other HER receptors. We previously showed in a randomized trial that high EGFR ligand expression is predictive of panitumumab benefit in advanced colorectal cancer. Tumor expression of HER3 may further refine the RAS wild-type (wt) population benefitting from anti-EGFR agents. OBJECTIVE: To examine HER3 messenger RNA expression as a prognostic and predictive biomarker for anti-EGFR therapy in a randomized clinical trial of panitumumab. DESIGN, SETTING, AND PARTICIPANTS: The study was a prospectively planned retrospective biomarker study of pretreatment samples from the PICCOLO trial that tested the addition of panitumumab to irinotecan therapy in patients with KRAS wt advanced colorectal cancer who experienced failure with prior fluoropyrimidine treatment. HER3 was assessed as a prognostic marker, then as a predictive biomarker in patients with RAS wt, first as a continuous variable and then as a binary (high vs low) variable. Relationship with MEK-AKT pathway mutations and EGFR ligands epiregulin and amphiregulin (EREG/AREG) were also assessed. MAIN OUTCOMES AND MEASURES: Primary end point was progression-free survival (PFS); secondary end points were response rate and overall survival (OS). RESULTS: In 308 patients (mean age at randomization, 61.6 years; 193 men) higher HER3 was weakly prognostic for OS (hazard ratio [HR] per 2-fold change, 0.91; 95% CI, 0.83-0.99; P = .04) but not PFS (HR, 0.93; 95% CI, 0.83-1.05; P = .25). Higher HER3 was predictive, being associated with prolonged PFS on irinotecan plus panitumumab (IrPan) (HR, 0.71; 95% CI, 0.61-0.82; P < .001), but not irinotecan (HR, 0.96; 95% CI, 0.82-1.13; P = .65) in patients with RAS wt, with significant interaction between biomarker and treatment (P = .001). Similar interaction was seen for OS (P = .004). In an exploratory binary model, dividing the population at the 66th percentile, HER3 was predictive of panitumumab benefit: in patients with high HER3 expression, median PFS was 8.2 months (IrPan) vs 4.4 months (irinotecan) (HR, 0.33; 95% CI, 0.19-0.58; P < .001). Patients with low HER3 expression gained no benefit in PFS: 3.3 months (IrPan) vs 4.3 months (irinotecan) (HR, 0.96; 95% CI, 0.67-1.38; P = .84), with significant interaction (P = .002). The binary model was also predictive for OS, with significant interaction (P = .01). Combining HER3 and ligand data, patients with HER3-high, AREG/EREG-high tumors gained markedly from panitumumab (PFS HR, 0.24; 95% CI, 0.11-0.51; P < .005 and OS HR, 0.36; 95% CI, 0.18-0.73; P = .004). Conversely, patients with HER3-low, AREG/EREG-low tumors did not benefit (PFS HR, 1.14; 95% CI, 0.73-1.79; P = .57 and OS HR, 1.44; 95% CI, 0.92-2.26; P = .11). CONCLUSIONS AND RELEVANCE: High HER3 expression identified patients with RAS wt who gained markedly from panitumumab, and those who did not, with statistically significant biomarker-treatment interactions for PFS and OS. This finding provides insight into the mechanism of anti-EGFR agents and is of potential clinical utility.

Inositol phosphate multikinase dependent transcriptional control.

Production of lipid-derived inositol phosphates including IP4 and IP5 is an evolutionarily conserved process essential for cellular adaptive responses that is dependent on both phospholipase C and the inositol phosphate multikinase Ipk2 (also known as Arg82 and IPMK). Studies of Ipk2, along with Arg82 prior to demonstrating its IP kinase activity, have provided an important link between control of gene expression and IP metabolism as both kinase dependent and independent functions are required for proper transcriptional complex function that enables cellular adaptation in response to extracellular queues such as nutrient availability. Here we define a promoter sequence cis-element, 5'-CCCTAAAAGG-3', that mediates both kinase-dependent and independent functions of Ipk2. Using a synthetic biological strategy, we show that proper gene expression in cells lacking Ipk2 may be restored through add-back of two components: IP4/IP5 production and overproduction of the MADS box DNA binding protein, Mcm1. Our results are consistent with a mechanism by which Ipk2 harbors a dual functionality that stabilizes transcription factor levels and enzymatically produces a small molecule code, which together coordinate control of biological processes and gene expression.

Blood-based markers of efficacy and resistance to cetuximab treatment in metastatic colorectal cancer: results from CALGB 80203 (Alliance).

Circulating protein markers were assessed in patients with colorectal cancer (CRC) treated with cetuximab in CALGB 80203 to identify prognostic and predictive biomarkers. Patients with locally advanced or metastatic CRC received FOLFOX or FOLFIRI chemotherapy (chemo) or chemo in combination with cetuximab. Baseline plasma samples from 152 patients were analyzed for six candidate markers [epidermal growth factor (EGF), heparin-binding EGF (HBEGF), epidermal growth factor receptor (EGFR), HER2, HER3, and CD73]. Analyte levels were associated with survival endpoints using univariate Cox proportional hazards models. Predictive markers were identified using a treatment-by-marker interaction term in the Cox model. Plasma levels of EGF, HBEGF, HER3, and CD73 were prognostic for overall survival (OS) across all patients (KRAS mutant and wild-type). High levels of EGF predicted for lack of OS benefit from cetuximab in KRAS wild-type (WT) patients (chemo HR = 0.98, 95% CI = 0.74-1.29; chemo+cetuximab HR = 1.54, 95% CI = 1.05-2.25; interaction P = 0.045) and benefit from cetuximab in KRAS mutant patients (chemo HR = 1.72, 95% CI = 1.02-2.92; chemo+cetuximab HR = 0.90, 95% CI = 0.67-1.21; interaction P = 0.026). Across all patients, higher HER3 levels were associated with significant OS benefit from cetuximab treatment (chemo HR = 4.82, 95% CI = 1.68-13.84; chemo+cetuximab HR = 0.95, 95% CI = 0.31-2.95; interaction P = 0.046). CD73 was also identified as predictive of OS benefit in KRAS WT patients (chemo HR = 1.28, 95% CI = 0.88-1.84; chemo+cetuximab HR = 0.60, 95% CI = 0.32-1.13; interaction P = 0.049). Although these results are preliminary, and confirmatory studies are necessary before clinical application, the data suggest that HER3 and CD73 may play important roles in the biological response to cetuximab.

Identifying Blood-Based Protein Biomarkers for Antiangiogenic Agents in the Clinic: A Decade of Progress.

Agents that inhibit tumor angiogenesis are widely used and have provided meaningful survival benefits to patients in multiple disease settings. However, these agents differ significantly in their mechanisms of action and potential toxicities, and there are currently no prospectively validated biomarkers to guide the selection of agents for individual patients. Blood-based protein biomarkers are well suited for trials investigating antiangiogenic agents for multiple reasons. Many elements of the molecular pathways that antiangiogenic agents target are present and detectable in the circulation, sample collection is minimally invasive, and samples can be collected throughout the course of treatment. Blood-based biomarkers for antiangiogenic therapies are urgently needed to guide the development of therapeutic strategies. This review provides a brief summary of the current blood-based protein biomarkers for antiangiogenic therapies.

Gene expression markers of efficacy and resistance to cetuximab treatment in metastatic colorectal cancer: results from CALGB 80203 (Alliance).

PURPOSE: Formalin-fixed, paraffin-embedded tumor samples from CALGB 80203 were analyzed for expression of EGFR axis-related genes to identify prognostic or predictive biomarkers for cetuximab treatment. PATIENTS AND METHODS: Patients (238 total) with first-line metastatic colorectal cancer (mCRC) were randomized to FOLFOX or FOLFIRI chemotherapy ± cetuximab. qRT-PCR analyses were conducted on tissues from 103 patients at baseline to measure gene expression levels of HER-related genes, including amphiregulin (AREG), betacellulin (BTC), NT5E (CD73), DUSP4, EGF, EGFR, epigen (EPGN), epiregulin (EREG), HBEGF, ERBB2 (HER2), ERBB3 (HER3), ERBB4 (HER4), PHLDA1, and TGFA. The interactions between expression levels and treatment with respect to progression-free survival (PFS) and overall survival (OS) were modeled using multiplicative Cox proportional hazards models. RESULTS: High tumor mRNA levels of HER2 [hazard ratio (HR), 0.64; P = 0.002] and EREG (HR, 0.89; P = 0.016) were prognostic markers associated with longer PFS across all patients. HER3 and CD73 expression levels were identified as potential predictive markers of benefit from cetuximab. In KRAS wild-type (WT) tumors, low HER3 expression was associated with longer OS from cetuximab treatment, whereas high HER3 expression was associated with shorter OS from cetuximab treatment (chemo + cetuximab: HR, 1.15; chemo-only: HR, 0.48; Pinteraction = 0.029). High CD73 expression was associated with longer PFS from cetuximab treatment in patients with KRAS-WT (chemo + cetuximab: HR, 0.91; chemo-only: HR, 1.57; Pinteraction = 0.026) and KRAS-mutant (Mut) tumors (chemo + cetuximab: HR, 0.80; chemo-only: HR, 1.29; P = 0.025). CONCLUSIONS: Gene expression of HER3 and CD73 was identified as a potential predictive marker for cetuximab. These data implicate HER axis signaling and immune modulation as potential mechanisms of cetuximab action and sensitivity.