Correlation Between Gene Expression of IGF-1R Pathway Markers and Cetuximab Beneﬁt in Metastatic Colorectal Cancer

Published OnlineFirst January 31, 2012; DOI: 10.1158/1078-0432.CCR-11-1135

Clinical Cancer Predictive Biomarkers and Personalized Medicine Research

Correlation between Gene Expression of IGF-1R Pathway Markers and Cetuximab Beneﬁt in Metastatic Colorectal Cancer

Fei Huang, Li-an Xu, and Shirin Khambata-Ford

Abstract Purpose: This study examined potential correlations between markers related to the insulin-like growth factor-1 receptor (IGF-1R) pathway and clinical benefit from the anti–epidermal growth factor receptor (EGFR) monoclonal antibody cetuximab in metastatic colorectal cancer (mCRC). Experimental Design: Gene expression profiles for 70 pretreatment specimens from metastatic lesions of patients with chemorefractory mCRC receiving cetuximab monotherapy were analyzed using 74 predefined Gene-Chip probesets representing 33 unique IGF-1R pathway markers to determine correlations with progression-free survival (PFS) and disease control rate.

Results: Higher IGF-1R, higher GRB7, and lower INSIG2 expression were associated with longer PFS with cetuximab in univariate analyses, particularly in patients with wild-type K-Ras tumors: median, 122 versus 60 days (P ¼ 0.01), 122 versus 57 days (P ¼ 0.011), and 57 versus 156 days (P < 0.0001), favoring higher IGF-

1R, higher GRB7, and lower INSIG2 expression, respectively. Lower IGF-1 expression was associated with a

PFS beneﬁt with cetuximab, whereas lower IGFBP3 and INSR expression levels showed trends for a PFS

beneﬁt. Lower INSIG2 expression (vs. higher expression) was associated with greater PFS in the high epiregulin-expressing group (P ¼ 0.001), but not in the low-expressing cohort suggesting an effect

independent from the previously reported effect of epiregulin expression. Lower INSIG2 expression was also associated with higher disease control rate in the overall population (51.4% vs. 11.4%; P ¼ 0.001) and wild-type K-Ras subset (76.2% vs. 18.2%; P < 0.0001). Conclusions: These results suggest that markers of the IGF-1R pathway may play a role in predicting beneﬁt from cetuximab therapy in mCRC. Additional clinical studies are warranted to validate these ﬁndings. Clin Cancer Res; 18(4); 1156–66. 2012 AACR.

Introduction that the presence of K-Ras exon-2 mutations (codons 12 and 13) predicts lack of cetuximab benefit in mCRC, both as Cetuximab is an anti–epidermal growth factor receptor single-agent therapy in patients who are refractory to che- (EGFR) monoclonal antibody that has antitumor efficacy in motherapy and in combination with chemotherapy in metastatic colorectal cancer (mCRC; refs. 1, 2); locoregion- treatment-na€ve patients (2, 6–8). Whereas these original ally advanced recurrent or metastatic squamous cell carci- studies bundled all known exon-2 mutations as one single noma of the head and neck (3, 4); and advanced non–small "resistance genotype," more recent studies have indicated cell lung cancer (NSCLC; ref. 5). In unselected patients with that patients with tumors carrying the G13D mutation may mCRC, cetuximab significantly improved survival as a sin- benefit from therapy, suggesting that this mutation may not gle agent compared with best supportive care in patients have the same effect as the rest on exon-2 (9, 10). These previously treated with fluoropyrimidine, irinotecan, and findings represent a significant breakthrough in mCRC oxaliplatin (1), and when added to first-line FOLFIRI com- treatment because they allow cetuximab to be directed to pared with FOLFIRI alone (6). Multiple studies have shown patients who are most likely to benefit. However, not all patients with wild-type K-Ras benefit from cetuximab-based therapy, and further refinement of patient selection is Authors' Affiliation: Bristol-Myers Squibb Co., Princeton, New Jersey desirable. Note: Supplementary data for this article are available at Clinical Cancer In chemotherapy (particularly irinotecan)-refractory Research Online (http://clincancerres.aacrjournals.org/). patients, there is a growing list of candidate predictive Corresponding Author: Fei Huang, Bristol-Myers Squibb Co., Route 206 markers under investigation. These include EGFR gene copy and Province Line Rd., Room E1.293, Princeton, NJ 08453. Phone: 609- number measured by FISH, ligand gene expression (amphir- 252-4891; Fax: 1-609-818-5839; E-mail: [email protected] egulin and epiregulin [EREG]) or polymorphisms (EGF), doi: 10.1158/1078-0432.CCR-11-1135 mutations in components of the MAP-Ras-Raf pathway, 2012 American Association for Cancer Research. loss of the PTEN gene, and PIK3CA expression levels or

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IGF-1R Pathway Markers and Cetuximab Beneﬁt in CRC

activated protein kinase (MAPK) and phosphoinositide Translational Relevance 3-kinase (PI3K), and may work collaterally to drive tumor growth and survival (reviewed in ref. 30). The IGF-1R Patients with metastatic colorectal cancer (mCRC) pathway appears to regulate EGFR signaling and, recipro- whose tumors harbor the wild-type K-Ras gene may cally, EGFR ligands may promote tumor survival through derive benefit from treatment with the anti–epidermal IGF-1R (31–33). Indeed, overexpression of the EGFR ligand growth factor receptor (EGFR) monoclonal antibody amphiregulin in NSCLC cell lines stimulates IGF-1R, lead- cetuximab, whereas those patients with tumors bearing ing to IGF-1 and amphiregulin secretion and inhibition of certain K-Ras mutations do not benefit. However, not all apoptosis (32). EGFR tyrosine kinase inhibitors (TKI) can patients with wild-type K-Ras tumors benefit from cetux- induce EGFR/IGF-1R heterodimerization and alter the imab, underscoring the need for additional markers to interactions between IGF-1R and insulin receptor (INSR) help in patient selection. Preclinical evidence suggests substrate (IRS)-1 in cancer cells, activating downstream that the EGFR and insulin-like growth factor-1 receptor signaling and resulting in resistance to further TKI treatment (IGF-1R) pathways interact to drive tumor growth and (34–36). Conversely, coinhibition of both pathways results survival. This study evaluated whether expression levels in synergistic antitumor effects in several preclinical cancer of the IGF-1R pathway markers correlate with cetuximab models (36–41). benefit in patients with chemorefractory mCRC, includ- Therefore, it is reasonable to hypothesize that molecular ing the subset of patients with wild-type K-Ras. The markers associated with the IGF-1R pathway may affect the results provide initial evidence that markers of the antitumor activity of EGFR inhibitors. In this study, we IGF-1R pathway may predict benefit from cetuximab analyzed the gene expression levels of IGF-1R pathway therapy in mCRC, but this preliminary finding requires components previously identified as potential points of validation in additional clinical studies. EGFR-IGF-1R cross-talk (15) and investigated their potential correlation with cetuximab benefit in patients with chemorefractory mCRC, both in the overall population and in the group with K-Ras wild-type tumors. mutations. All have been reported to affect the clinical activity of cetuximab (11–25), mostly in relatively small Methods patient cohorts, often without randomized controls. Com- bining the testing for K-Ras mutation with one or more of Patients and study design these markers could further enhance patient selection and The CA225-045 trial was a phase II pharmacogenomic the overall therapeutic index of cetuximab in mCRC. The study in which 110 patients with chemorefractory mCRC advantages of this approach have been illustrated by the received single-agent cetuximab after failing at least one enrichment in responding patients achieved with nested chemotherapeutic regimen for advanced disease or refusing mutational screening of tumors for K-Ras, B-Raf, N-Ras, prior treatment. Details of the study design and patient PIK3CA (26), or with the combimarker of K-Ras mutation cohort have been described previously (13). Patients plus EREG expression (16). received cetuximab at its standard dose (400 mg/m2 loading In previously untreated patients with mCRC receiving dose; then 250 mg/m2 weekly) for 3 weeks and then were first-line therapy, however, data from randomized con- eligible for dose-escalation every 3 weeks to a maximum of trolled studies are lacking for most of the candidate predic- 400 mg/m2 weekly, provided they had not experienced tive markers for anti-EGFR therapy. One in particular, B-Raf greater than grade II skin rashes. Tumor responses were V600E mutation, has not upheld the potentially predictive evaluated every 9 weeks according to modified WHO cri- value consistently reported in noncontrolled retrospective teria (42). The disease control rate (DCR) was defined as the series of irinotecan-refractory patients (23, 25, 26), showing proportion of patients with a complete response (CR), instead a notable prognostic effect in controlled studies partial response (PR), or stable disease (SD). Progression- of untreated patients, regardless of cetuximab treatment free survival (PFS) was defined as the time from enrollment (6, 27). The data are therefore conflicting and preclude firm until disease progression or death. The study protocol was practical recommendations for any predictive marker other approved by the Institutional Review Board at each partic- than K-Ras exon-2 mutations. Whether the apparent dis- ipating center and all patients provided written informed crepancies are because of the limitations of uncontrolled consent before study procedures were conducted. data sets, where prognostic and predictive effects may be confounded, or to intrinsic biologic differences between Gene expression profiling and K-Ras mutational status tumors exposed to their first- versus later-lines of therapy, A biopsy specimen for use in predictive marker discovery remains to be elucidated. was collected before cetuximab therapy by 3 passes with an Alterations in the insulin-like growth factor (IGF) system 18-gauge needle through a single metastatic lesion. Total have been associated with CRC tumorigenesis and can be RNA was isolated from these tumor samples and 1 mg RNA found in a substantial number of CRC tumors (reviewed in was used for gene profiling on U133Av2.0 GeneChips refs. 28 and 29). The IGF-1 receptor (IGF-1R) and the EGFR according to the manufacturer’s instructions (Affymetrix). pathways share downstream effectors, such as mitogen- K-Ras exon-2 and B-Raf V600E mutation analyses were

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conducted using genomic DNAs isolated from these tumors Table 1. Demographic and baseline as described previously (13). characteristics Gene expression data analysis of preselected genes A total of 74 preselected probesets, representing 33 All patients K-Ras WT N ¼ n ¼ unique IGF-1R pathway–related genes were evaluated, Characteristic ( 70) patients ( 43) including those encoding for ligands (IGF-1, IGF-2, and Sex, n (%) INS), receptors (IGF-1R, INSR, and IGF-2R), binding pro- Male 39 (55.7) 25 (58.1) teins (IGFBP1-7, IGF2BP2, and IGF2BP3), insulin-induced Female 31 (44.3) 18 (41.9) genes (INSIG1 and INSIG2), insulin receptor substrates Age, y, median (range) 59 (25–89) 61 (25–89) (IRS1-4), adapters and downstream effectors (GRB2, GRB7, ECOG performance GRB10, GRB14, SHC1-3, and AKT1-3), and related genes status, n (%) INSRR and IDE. 0 26 (37.1) 17 (39.5) The Affymetrix gene expression data were normalized 1 32 (45.7) 18 (41.9) using a Robust Multichip Average (RMA) method (43, 2 10 (14.3) 6 (14.0) 44). Thresholds for high versus low marker expression level Not reported 2 (2.9) 2 (4.7) were established on the basis of whether the signal intensity Prior chemotherapy was above or below the median value across all subjects if regimens, n (%) the signal intensities of a given marker displayed a normal 1 4 (5.7) 2 (4.7) distribution. However, for IGF-1R and IGF-1, the distribu- 2 23 (32.9) 13 (30.2) tion of signal intensities appeared to be a mixture of 2 3 22 (31.4) 14 (32.6) components; therefore, for these 2 markers, mixture models 4 or more 21 (30.0) 12 (32.6) of 2 normal distributions were used to determine the cutoff Prior surgery, n (%) 3 (4.3) 1 (2.3) points for classifying expression as high versus low. Prior radiation 25 (35.7) 16 (37.2) therapy, n (%) Statistical analysis Abbreviations: ECOG, Eastern Cooperative Oncology Gene expression profiles were compared with clinical Group; WT, wild-type. benefit parameters by univariate analyses. A log-rank test with a 2-sided a ¼ 0.05 was used to compare differences in PFS between patients having high versus low marker expression in the overall population and wild-type K-Ras subpop- PD in 18 patients. Therefore, the DCR was 31.4% in ulation. A Cox proportional hazards model was also used to the overall population and 46.5% in the wild-type K-Ras assess the effect of marker status on PFS in both populations population. The corresponding median PFS values were 60 separately. For these analyses, the HR of the marker effect, its days (95% CI, 59–67) and 61 days (95% CI, 58–56), 2-sided 95% confidence interval (CI), and the P value were respectively. determined. The DCR and its 95% CI were calculated for high and low marker expression groups using Fisher’s exact Distribution of gene expression signal intensity in method in both the overall population and wild-type K-Ras study population subset. Each candidate marker was analyzed separately, and For most genes, the median expression value was used as reported P values were not adjusted for multiplicity. How- the threshold to classify the tumors as high or low expres- ever, a false discovery rate (FDR) method was used for sing for the study population. Therefore, for each of these multiplicity control in the identification of significant mar- candidate predictive markers, 50% of the overall popula- kers predicting clinical benefit of cetuximab therapy in the tion was classified as having high expression. As exceptions, overall population. cutoff values between high and low expression for IGF-1R and IGF-1 expression were determined using mixture mod- > Results els of 2 normal distributions (log2 expression level 7.15 for IGF-1R defining 62.9% of the population as high, and log2 > Patient characteristics and clinical outcome expression level 5.82 for IGF-1 defining 40% of the pop- Correlations between markers from the IGF-1R pathway ulation as high; Fig. 1). and clinical benefit from cetuximab therapy were determined for the overall population of 70 patients with known Correlation of candidate marker expression with DCR K-Ras mutational status and the subset of 43 patients with and PFS wild-type K-Ras tumors. Demographic and clinical charac- Overview of the analyses. Analyses were conducted for teristics are shown in Table 1. Of the 70 patients, the the 74 probesets (representing 33 unique markers) specified objective best response was CR/PR in 5 patients, SD in in the Methods section (see Supplementary Table). With an 17 patients, and progressive disease (PD) in 48 patients. For FDR control at the 0.15 level, only INSIG2 expression was the subgroup with wild-type K-Ras, the objective best found to be significantly correlated with DCR and PFS. response was CR/PR in 5 patients, SD in 15 patients, and However, given the small sample size in this study, a liberal

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IGF-1R Pathway Markers and Cetuximab Beneﬁt in CRC

ABCIGF-1R IGF-1 GRB7 Density Density Density

Expression (log2) Expression (log2) Expression (log2)

DEF INSIG2 IGFBP3 INSR Density Density Density

Expression (log2) Expression (log2) Expression (log2)

Figure 1. Distribution of signal intensities of gene expression in the overall study population for IGF-1R (A), IGF-1 (B), GRB7 (C), INSIG2 (D), IGFBP3 (E), and INSR (F). The vertical line identifies the cutoff for high versus low expression. High expression for IGF-1R and IGF-1 is defined by signal intensity log2 >7.15 and >5.82, respectively, whereas for the other markers, high expression is defined by signal intensity above the median value for the study population.

FDR control at 0.4 was applied to evaluate the overall 0.01; Table 2). This difference in PFS between the high and results. With this control, significant correlations were low IGF-1R expression groups was particularly evident for found for 6 markers, detailed below. For the remaining the patients with longer PFS times. The PFS benefit associ- 27 unique markers, correlations were not significant. For the ated with higher IGF-1R expression in the wild-type K-Ras 6 candidate markers with significant correlations, an addi- subpopulation was not evident in patients with K-Ras tional analysis was conducted to evaluate their effect in mutant tumors, whereas lower IGF-1R expression was asso- patients with high versus low EREG expression within the ciated with a lack of clinical benefit with cetuximab regard- population with K-Ras wild-type tumors, in light of the less of K-Ras mutation status (Fig. 3A). predictive effect that this ligand marker had shown in a data GRB7. Higher GRB7 expression was associated with set generated from this same study (13). In addition, muta- higher DCR rates in response to cetuximab, but differences tional analysis for B-Raf V600E was carried out in 44 of 70 compared with lower GRB7 expression did not reach sta- samples with available material; given the low sample tistical significance in either the overall population (40.0% number, the results of this co-analysis were not informative vs. 22.9%; P ¼ 0.197) or the subpopulation with tumors (not shown). bearing wild-type K-Ras (58.3% vs.31.6%; P ¼0.125; Fig.2). IGF-1R. Higher IGF-1R expression was significantly Nevertheless, higher GRB7 expression was associated with a associated with a DCR benefit during cetuximab therapy PFS benefit in both cohorts (Table 2): median PFS for the in the wild-type K-Ras subgroup (62.5% vs. 26.3%; P ¼ high and low GRB7 expression groups were 65 versus 59 0.031), but not in the overall population (36.4% vs. 23.1%; days, respectively, in the overall population (HR ¼ 0.484; P ¼ 0.295) when compared with lower IGF-1R expression 95% CI, 0.276–0.849; P ¼ 0.01), and 122 versus 57 days, (Fig. 2). Higher IGF-1R was also associated with a PFS respectively, in the wild-type K-Ras subpopulation (HR ¼ benefit, particularly in the wild-type K-Ras subgroup where 0.396; 95% CI, 0.19–0.828; P ¼ 0.011). The PFS benefit median PFS was 122 days compared with 60 days for low associated with higher GRB7 expression was not evident in IGF-1R expression (HR ¼ 0.406; 95% CI, 0.202–0.823; P ¼ K-Ras mutant tumors, whereas lower GRB7 expression was

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A Overall population 80 High 70 P = 0.001 Low 60 P = 0.197 51.4 P = 0.066 P = 0.197 P = 0.197 50 P = 0.295 40.0 40.5 40.0 40.0 40 36.4

DCR (%) 30 23.1 22.9 22.9 22.9 20 17.9 11.4 10

0 Figure 2. DCRs with cetuximab in IGF-1R GRB7 INSIG2 IGF-1 IGFBP3 INSR the overall population (A) and in the subpopulation with wild-type (WT) B K- Ras WT subpopulation K-Ras (B). P < 0.0001 80 76.2 P = 0.031 P = 0.031 P P P 70 = 0.125 = 0.223 = 0.223 62.5 62.5 60 58.3 56.5 56.5

40 35.0 35.0 31.6

DCR (%) 30 26.3 26.3

20 18.2

IGF-1R GRB7 INSIG2 IGF-1 IGFBP3 INSR

associated with a lack of clinical benefit with cetuximab harboring wild-type K-Ras. Lower expression (compared regardless of K-Ras mutation status (Fig. 3B). with higher expression) was associated with signifi- INSIG2. Lower INSIG2 expression was associated with cantly better DCR rates in both the overall population cetuximab benefit, particularly in patients with tumors (51.4% vs. 11.4%; P ¼ 0.001) and the wild-type K-Ras

Table 2. Correlation of candidate marker expression with median PFS (days)

Marker [Probe ID] Overall population K-Ras WT population

High Low HR (95% CI), P High Low HR (95% CI), P

IGF-1R [203628_at] (n ¼ 44) 62 (n ¼ 26) 59 0.546 (0.314–0.949); P ¼ 0.03 (n ¼ 24) 122 (n ¼ 19) 60 0.406 (0.202–0.823); P ¼ 0.01

GRB7 [210761_s_at] (n ¼ 35) 65 (n ¼ 35) 59 0.484 (0.276–0.849); P ¼ 0.01 (n ¼ 24) 122 (n ¼ 19) 57 0.396 (0.19–0.828); P ¼ 0.011

INSIG2 [209566_at] (n ¼ 35) 58 (n ¼ 35) 115 2.21 (1.297–3.765); P ¼ 0.003 (n ¼ 22) 57 (n ¼ 21) 156 4.08 (1.945–8.559); P < 0.0001 IGF-1 [211577_s_at] (n ¼ 28) 58 (n ¼ 42) 62 1.773 (1.032–3.045); P ¼ 0.036 (n ¼ 19) 57 (n ¼ 24) 121 1.813 (0.906–3.63); P ¼ 0.089

IGFBP3 [210095_s_at] (n ¼ 35) 59 (n ¼ 35) 63 1.605 (0.94–2.741); P ¼ 0.081 (n ¼ 20) 61 (n ¼ 23) 120 1.535 (0.768–3.067); P ¼ 0.222 INSR [207851_s_at] (n ¼ 35) 59 (n ¼ 35) 62 1.618 (0.96-2.717); P ¼ 0.066 (n ¼ 20) 61 (n ¼ 23) 121 1.331 (0.683–2.595); P ¼ 0.399

NOTE: For each marker, the Affymetrix probeset ID is listed; for a complete list of probesets for each gene, see the Supplementary Table. Abbreviation: WT, wild-type.

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A IGF- 1R Overall population According to K-Ras status

(n = 44) (n = 20) (n = 26) (n = 23) (n = 3) (n = 24) Proportion free progression Proportion free progression Log-rank P = 0.0299

Time to events, d Time to events, d

B GRB7 Overall population According to K-Ras status (n = 35) (n = 24) (n = 35) (n = 19) (n = 16) Figure 3. Effect of marker (n = 9) expression levels on PFS with cetuximab therapy in the overall population (left) and within the subpopulations with wild-type and mutant K-Ras (right): IGF-1R (A), GRB7 (B), and INSIG2 (C), Proportion free progression Proportion free progression

Log-rank P = 0.01

Time to events, d Time to events, d

C INSIG2 Overall population According to K-Ras status (n = 35) (n = 22) (n = 35) (n = 21) (n = 14) (n = 13) Proportion free progression Proportion free progression Log-rank P = 0.0029

Time to events, d Time to events, d

subpopulation (76.2% vs. 18.2%; P < 0.0001; Fig. 2). than those with high expression (P ¼ 0.001). No Similarly, lower INSIG2 expression was associated with a INSIG2-related effect was observed in the low EREG– PFS benefit in the overall population (median: 115 vs. expressing cohort, suggesting that the effect of INSIG2 is 58 days; HR ¼ 2.21; 95% CI, 1.297–3.765; P ¼ 0.003), independent from the ligand effect (Fig. 4). INSIG2 was and an even greater benefit in the wild-type K-Ras sub- the only one of the 6 markers evaluated in conjunction population (median: 156 vs. 57 days; HR ¼ 4.08; 95% CI, with EREG to show such independence; for the 5 addi- 1.945–8.559; P < 0.0001; Table 2). The PFS benefit tional markers, PFS curves separated between high- and associated with lower INSIG2 expression was not evident low-expressers in the K-Ras wild-type, high EREG–expres- in patients with K-Ras mutant tumors, whereas higher sing population, but the differences did not reach statis- INSIG2 expression was associated with a lack of clinical tical significance (data not shown). benefit regardless of K-Ras mutation status (Fig. 3C). IGF-1. Lower IGF-1 expression was associated with a Interestingly, within the K-Ras wild-type, high EREG– significantly higher DCR with cetuximab in the subpopu- expressing population (n ¼ 31), the patients with low lation with tumors containing wild-type K-Ras (62.5% vs. INSIG2–expressing tumors had significantly better PFS 26.3%; P ¼ 0.031) and showed a trend for a higher DCR in

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D IGF-1 Overall population According to K-Ras status

(n = 28) (n = 19) (n = 42) (n = 24) (n = 18) (n = 9) Proportion free progression Proportion free progression

Log-rank P = 0.0358

Time to events, d Time to events, d

E IGFBP3 Overall population According to K-Ras status (n = 35) (n = 20) (n = 35) (n = 23) (n = 12) (n = 15)

Figure 3. (Continued) IGF-1 (D), IGFBP3 (E), and INSR (F) Proportion free progression Proportion free progression

Log-rank P = 0.0807

Time to events, d Time to events, d F INSR Overall population According to K-Ras status (n = 35) (n = 20) (n = 35) (n = 23) (n = 12) (n = 15) Proportion free progression Proportion free progression

Log-rank P = 0.0647

Time to events, d Time to events, d

the overall population (40.5% vs. 17.9%; P ¼ 0.066) Discussion compared with higher expression levels (Fig. 2). Lower IGF-1 expression was also associated with a PFS benefit, This study indicates that there is a potential correlation which reached statistical significance in the overall popu- between cetuximab benefit in mCRC and expression of lation (median: 62 vs. 58 days; HR ¼ 1.773; 95% CI, 1.032– certain markers in the IGF-1R pathway. The IGF-1R and 3.045; P < 0.036), and a similar trend in the subgroup with its ligand IGF-1 are the main activity drivers of this wild-type K-Ras (median: 121 vs. 57 days; HR ¼ 1.813; 95% signaling axis; however, other components, such as the CI, 0.0906–3.63; P<0.089; Fig. 3D and Table 2). IGF-binding proteins (IGFBP), have a dual function IGFBP3 and INSR. Lower expression levels of IGFBP3 acting as ligand sinks that inhibit signaling and as ligand and INSR were associated with numerically higher DCR reservoirs that maintain signaling. Downstream effectors, rates and showed trends for longer PFS compared with such as GRBs, connect IGF-1R signaling to the prolifer- higher expression levels of these genes (Fig. 3E and F ative MAPK pathway. In parallel, the INSR and its output and Table 2). (such as the INSIG proteins) cross-talk with the IGF-1R,

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n Low expression INSIG2; high expression ereg ( = 20) n Low expression INSIG2; low expression ereg ( = 1) n High expression INSIG2; low expression ereg ( = 11) n High expression INSIG2; high expression ereg ( = 11)

Figure 4. Effect of INSIG2 expression in the group of patients with K-Ras wild-type tumors, according to EREG expression. Proportion free progression

Time to events, d

and probably contribute to regulate its overall activity IGF-1 expression was even more pronounced in the (28). subset of 69 patients with wild-type K-Ras tumors (10.0 vs. In this study, higher IGF-1R, higher GRB7, and lower 3.2 months; P ¼ 0.002; ref. 31). Further indication of the INSIG2 expression were associated on univariate analyses relevance of this pathway for cetuximab clinical benefit has with longer PFS, particularly in patients with wild-type come from the observation that 5 IGF-1/IGF-1R pathway K-Ras tumors. Conversely, lower gene expression levels of polymorphisms were significantly associated with PFS or IGF-1R and GRB7 and higher expression levels of INSIG2 OS in a cohort of 130 patients with mCRC who received were associated with a lack of cetuximab benefit regardless cetuximab monotherapy, with several appearing indepen- of K-Ras mutation status. Lower IGF-1 expression was also dent of K-Ras mutation status in the multivariate analysis associated with a PFS benefit with cetuximab, whereas lower (47). IGFBP3 and INSR expression levels showed trends for a PFS Taken together, these results indicate that markers of the benefit. These observations would point to a potential link IGF-1R pathway are of interest for predicting cetuximab between markers suggestive of high IGF-1R signaling and benefit in mCRC; in our study, the lack of control group low INSR activity and greater sensitivity to EGFR inhibition. and the lax significance threshold used necessitate further Furthermore, at least for one of these markers, INSIG2, this validation in well-controlled and appropriately powered effect would appear to be independent of the association studies before these findings could be considered for use between EREG expression and cetuximab activity. in clinical practice. Without having a control arm (that These observations are consistent with a study of a cohort received no cetuximab therapy), it is not possible to dis- of 85 patients with chemorefractory mCRC who received a criminate whether these candidate markers are truly pre- cetuximab-based regimen, in which higher IGF-1R protein dictive for cetuximab benefit or rather prognostic for disease expression detected by immunohistochemistry was associ- outcome. In fact, several of the markers that were associated ated with significantly longer median overall survival (OS) with clinical benefit in our study have already been compared with low IGF-1R (16.1 vs. 6.7 months; P ¼ 0.006; shown to be prognostic in CRC. For example, higher plasma ref. 45). However, IGF-1R expression was not related to levels of IGF-1 and IGFBP3 were associated with improved response rate or disease progression. Other studies have OS in all 3 arms of a randomized trial of patients with reported trends that diverge from our results. In a study of mCRC treated with different first-line chemotherapy regi- 113 patients with chemorefractory mCRC treated with mens (48). Adjustments for both factors showed that cetuximab or panitumumab (a fully human monoclonal IGFBP3, but not IGF-1, was also independently associated anti-EGFR IgG2), tumor IGF-1R activation, measured by with prolonged time to disease progression. In addition, phosphorylation status, correlated with inferior response upregulation of INSIG2 gene expression has been associ- to both chemotherapy and anti-EGFR treatment (46). In a ated with poor prognosis on initial staging of CRC and cohort of 112 patients with irinotecan-refractory mCRC may act by suppressing chemotherapy-induced, Bax-medi- treated with cetuximab plus irinotecan, negative IGF-1 ated apoptosis (49, 50). This observation is in line with protein expression (score of 0 or 1 by immunohistochem- this study, where low expression of INSIG2 is correlated istry) was associated with longer median PFS compared with prolonged survival and improved disease control, thus with positive (score of 2–4) IGF-1 expression (7.5 vs. 3.0 reinforcing the caveat of whether our findings are truly months; P ¼ 0.002; ref. 31). This PFS benefit with negative predictive or prognostic.

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In previous reports from other solid tumors, high GRB7 primary to metastatic sites, particularly for components of expression was independently associated with reduced OS the IGF-1R pathway. Furthermore, the expression levels of in primary breast cancer, particularly in the subset with members of the IGF-1R pathway can be affected by node-positive disease (51). Although high GRB7 and HER2 exposure to chemotherapy or radiotherapy (57–59), so expression were strongly correlated, coexpression of HER2 it is pertinent to note that the samples in this study were together with GRB7 was associated with a significantly collected from metastatic sites and immediately prior to poorer prognosis than expression of HER2 alone. GRB7 cetuximab treatment without any intervening therapy. upregulation has also been associated with resistance to Any validation process wouldhavetoaddressthepre- the dual EGFR/HER2 tyrosine kinase inhibitor lapatinib dictive robustness of these candidate markers in samples (52). Notably, these negative prognostic correlations from primary and metastatic sites, samples collected at oppose the results reported herein, suggesting that the different points in the course of treatment throughout the specific association between high GRB7 expression and disease, and when cetuximab is used in combination with cetuximab PFS benefit may indeed be predictive. However, chemotherapy or as single agent. the positive correlation with DCR did not reach significance Finally, the link between the EGFR and the IGF/IGF-1R in our study, further indicating the necessity of validation in pathways provides a rationale for the development of adequate and well-controlled trials. combined therapeutic strategies, a promising approach in Future development of these observations will have to preclinical models (36–41). However, clinical data with the consider the integration of these or any other candidate combination of cetuximab or panitumumab with the anti- predictive marker with the patient selection platforms IGF-1R monoclonal antibodies cixutumumab, dalotuzu- already existing (i.e., K-Ras mutation–based selection). The mab, or ganitumumab have been underwhelming (60– CA225-045 study generated the initial results indicating 62). It is possible that a better insight on mechanisms of that EREG expression could be predictive of cetuximab tumor dependence specific for these 2 pathways could benefit (15). This marker was combined with K-Ras muta- enhance the chances of success for such strategies; in that tion, as combimarker, in an exploratory analysis of the regard, studies such as these could help improve patient randomized controlled data set from the NCIC-C017 trial selection strategies for the development of these regimens. of cetuximab versus best supportive care in chemorefractory On the other hand, blocking the IGF-1R pathway with TKIs, patients, to significantly enhance patient selection (16). whose inhibitory spectrum is different from antibodies More recently, Baker and colleagues (53) have reported on (because, for instance, they can potentially inhibit the IR; a predictive 4-gene score derived from a pooled data set ref. 28), could yield better clinical results, given the extent of including CA225-045 samples. transversal cross-talk in this signaling network. It would be of interest to establish how the most relevant In summary, this study provides preliminary evidence candidate markers described here could be used concur- that IGF-1R pathway–related markers may play a role in rently with those previously characterized in this data set. predicting benefit from cetuximab therapy in mCRC. Addi- For instance, our analysis suggests that low INSIG2 expres- tional studies are warranted to validate these findings and to sion could act independently from high EREG expression determine which candidate predictive marker(s) can best be to refine patient selection within the group of patients with integrated into clinical practice for selecting patients most K-Ras wild-type tumors, although the limited size of our likely to benefit from cetuximab. sample warrants cautious interpretation of these results. Unfortunately, the limited availability of sample material Disclosure of Potential Conflicts of Interest for co-analysis makes a full examination of our IGF-related expression markers and the 4 predictive markers from the S. Khambata-Ford is a former employee of Bristol-Myers Squibb. Baker score unfeasible; the same is true for the interesting F. Huang and L-a. Xu are current employees of Bristol-Myers Squibb. analysis that would result from the combination of mutational testing, as reported by Spindler and colleagues (24), Grant Support and expression analysis. An additional question that remains for these and any The editorial assistance for the preparation of this manuscript was provided by Clinical Insights Inc., supported by Bristol-Myers Squibb. other candidate predictive markers, particularly those that The costs of publication of this article were defrayed in part by the are gene expression related, is that of stability and status payment of page charges. This article must therefore be hereby marked concordance between the different potential sources of advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. sample specimens. Although mutational concordance has been the object of prior investigations (54–56), little is Received April 29, 2011; revised October 27, 2011; accepted November 13, known on the concordance in gene expression levels from 2011; published OnlineFirst January 31, 2012.

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1166 Clin Cancer Res; 18(4) February 15, 2012 Clinical Cancer Research

Correlation between Gene Expression of IGF-1R Pathway Markers and Cetuximab Benefit in Metastatic Colorectal Cancer

Fei Huang, Li-an Xu and Shirin Khambata-Ford

Clin Cancer Res 2012;18:1156-1166. Published OnlineFirst January 31, 2012.

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