The purpose of this study was to characterize insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF-1R) expression in patients with nonsmall cell lung cancer (NSCLC).
The purpose of this study was to characterize insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF-1R) expression in patients with nonsmall cell lung cancer (NSCLC).
A total of 459 patients who underwent curative resection of NSCLC were studied (median follow-up duration, 4.01 years). Expression of the IR and IGF-1R protein in tumor specimens was assessed immunohistochemically using tissue microarrays.
The cytoplasmic IR score was higher in patients with adenocarcinoma (ADC) than in those with squamous cell carcinoma (SCC), whereas cytoplasmic IGF-1R score was higher in patients with SCC than those with ADC. Neither IR nor IGF-1R expression was associated with sex, smoking history, or clinical stage. Patients with positive IR or IGF-1R expression levels had poor recurrence-free (RFS) (3.8 vs 3.3 years; 3.8 vs 2.0 years, respectively), but similar overall survival (OS). Patients with high expression levels of IR and IGF-1R had shorter RFS and OS compared with those with low levels of IR and/or IGF-1R expression. Finally, a multivariate analysis revealed the impact of IR, but not of IGF-1R, as an independent predictive marker of NSCLC survival: hazard ratio (HR) for OS, 1.005 (95% confidence interval [CI], 1.001-1.010], HR for RFS, 1.005 (95% CI, 1.001-1.009), when IR score was tested as a continuous variable.
Overexpression of IR predicts a poor survival among patients with NSCLC, especially those with SCC. These results might serve as future guidance to the clinical trials involving IR or IGR-1R targeting agents. Cancer 2012;. © 2011 American Cancer Society.
Nonsmall cell lung cancer (NSCLC) is the leading cause of cancer-related deaths in the United States, and current systemic therapies for NSCLC have limited efficacy, indicating the need for novel treatment strategies.1 One potentially effective strategy is targeting the insulin-like growth factor (IGF)/insulin signaling pathway. The IGF/insulin system is composed of ligands (insulin, IGF-I, and IGF-II), receptors (insulin receptor [IR], IGF-1R, and IGF-2R), and IGF-binding proteins 1-6. The IR and IGF-1R are highly homologous heterotetrameric molecules composed of 2 extracellular α-subunits with binding capacity and 2 transmembrane β-subunits with tyrosine kinase activity that are activated in response to ligand binding.2 The IR and IGF-1R can homodimerize as well as heterodimerize with each other and have selective affinities to ligands, such as IGF-I, IGF-II, and insulin.3 Whereas IGF-I and insulin bind to IGF-1R and the IR, respectively, with high selectivity, IGF-II binds to IGF-1R as well as an alternative splicing variant of the IR, termed IR-A.4 Also, whereas insulin has direct access to its receptors, the bioavailability of IGF-I is influenced by IGF-binding proteins.2
The IGF-1R and IR interact and share similar downstream signaling pathways, including mitogen-activated protein kinase and phosphatidylinositol 3-kinase. The IGF-1R and IR participate in mitogenic and antiapoptotic signaling in normal and neoplastic epithelia. In mammals, the IR and IGF-1R have also evolved to exert different biological functions. Specifically, the IR plays a central role in glucose homeostasis, whereas IGF-IR is a regulator of body growth.5 Interestingly, expression of IR-A, often referred to as the fetal isoform of the IR, is elevated in several human cancers.6 In patients with breast cancer, total level of the IR expression was indicative of poor survival, whereas total IGF-IR expression was not.7, 8 Furthermore, IR down-regulation has shown to inhibit polyoma virus middle T antigen-induced tumor growth in vivo.9 Insulin may be involved in the growth of malignancies.5 Aberrant IR expression sensitized cancer cells to the pleiotropic effects of circulating insulin, leading to acquire resistance to both conventional and targeted therapies, especially in hyperinsulinemic patients.10, 11 Given the impact of IGF-1R/IR signaling on the development and progression of several types of cancer, researchers have long studied the prognostic value of IGF-1R protein expression in patients with NSCLC12-14 or small-cell lung cancer.15 Researchers have also studied the impact of the epidermal growth factor receptor (EGFR) pathway, which is suggested to crosstalk with the IGF-1R pathway, on survival in patients with lung cancer.16, 17 Despite the data indicating the potential prognostic significance of IR overexpression, information about IR expression in lung cancer cases is very limited.
The aim of this study was to analyze IR and IGF-1R expression in correlation with clinicopathological parameters in NSCLC patients and to evaluate the prognostic impact of the expression on the survival of these patients.
The study patients' baseline characteristics are listed in Table 1. Detailed clinical and pathological information was available for most of the patients and included their demographic data, smoking history (never-smokers va ever-smokers [patients who had smoked at least 100 cigarettes in their lifetimes]), pathological TNM stage, and overall survival (OS) and recurrence-free survival (RFS) duration. The numbers of patients per sex were well balanced, and almost two-thirds of the patients had adenocarcinoma (ADC). Most of the patients had early-stage NSCLC and were ever-smokers. Tissue banking and research conduct were approved by The University of Texas MD Anderson Cancer Center institutional review board. All patients provided their informed consent to participate in the study, and all identifying information was removed from the database.
|Feature||NSCLC Histologic Typea|
|Adenocarcinoma (n = 298)||Squamous Carcinoma (n = 161)||Total (n = 459)|
|Median age (range), y||66 (32-88)||66 (43-89)||66 (32-89)|
|Male||133 (44.6%)||96 (59.6%)||231 (50.3%)|
|Female||165 (55.4%)||65 (40.4%)||228 (49.7%)|
|Never||47 (15.8%)||1 (0.6%)||48 (10.5%)|
|Former||132 (44.3%)||76 (47.2%)||208 (45.3%)|
|Current||118 (39.6%)||83 (51.6%)||201 (43.8%)|
|Unknown||1 (0.3%)||1 (0.6%)||2 (0.4%)|
|Caucasian||272 (91.3%)||146 (90.7%)||418 (91.1%)|
|Others||26 (8.7%)||15 (9.3%)||41 (8.9%)|
|1||120 (40.3%)||57 (35.4%)||177 (38.6%)|
|2||145 (48.7%)||83 (51.6%)||228 (49.7%)|
|3||11 (3.7%)||11 (6.8%)||22 (4.8%)|
|4||22 (7.4%)||10 (6.2%)||32 (7%)|
|0||212 (71.1%)||104 (64.6%)||316 (68.8%)|
|1||42 (14.1%)||37 (23.0%)||79 (17.2%)|
|2||43 (14.4%)||19 (11.8%)||62 (13.5%)|
|x||1 (0.3%)||1 (0.6%)||2 (0.4%)|
|I||184 (61.7%)||90 (55.9%)||274 (59.7%)|
|II||83 (27.9%)||58 (36.0%)||141 (30.7%)|
|III||20 (6.7%)||10 (6.2%)||30 (6.5%)|
|IV||11 (3.7%)||3 (1.9%)||14 (3.1%)|
For this study, archived formalin-fixed, paraffin-embedded specimens of NSCLC were obtained from previously described tissue banks at MD Anderson.18 The tissue specimens were originally collected from 1997 to 2005 and were classified using the 2004 World Health Organization classification system.19 A tissue microarray (TMA) set comprising 511 NSCLC specimens (334 ADCs and 177 squamous cell carcinomas [SCCs]) obtained from patients who underwent surgery at MD Anderson from 2003 to 2005 was constructed. Only patients with available staging information were included in our analysis (n = 459). After histological examination of the NSCLC specimens, the NSCLC TMAs were constructed by obtaining 3 1-mm-diameter cores from each tumor at 3 different sites (periphery, intermediate, and central tumor). The TMAs were prepared using a manual tissue arrayer (Advanced Tissue Arrayer ATA100; Chemicon International, Tumecula, Calif).
Expression of IGF-1R, and IR in TMAs was measured using immunostaining. Primary antibodies used in immunohistochemical (IHC) analysis were purchased from Cell Signaling Technology (Danvers, Mass) (IGF-1R) or Santa Cruz Biotechnology (Santa Cruz, Calif) (IR). Details of the IHC analysis were described previously.18 The expression of IGF-1R and IR was quantified by 2 independent observers (C.B. and I.I.W.) who were unaware of the patients' outcomes. Cytoplasmic, membranous, and nuclear expression was quantified using a 4-value intensity scoring system (0, 1+, 2+, and 3+) and the percentage (0%-100%) of the extent of reactivity. Next, the expression score was calculated by multiplying the intensity value by the reactivity extension value (range, 0-300). Tumors with IHC score equal to or higher than 1 were considered positive staining for the markers.
Summary statistical analysis of biomarker expression levels according to patient baseline characteristics was performed. Wilcoxon rank sum and Kruskal-Wallis tests were performed to compare the biomarker expression in different subgroups defined by categorical variables, such as histology and smoking history. Spearman rank correlation coefficients were used to determine the correlation among biomarkers. Student t test and Pearson correlation coefficient were used to analyze gene expression data. The OS and RFS durations in each subgroup of patients were then determined using the Kaplan-Meier method and compared using the log-rank test. Cox proportional hazards models were used for multivariate analysis. All statistical tests were 2 sided, and P values of no more than .05 were considered statistically significant.
IHC staining of the tissue specimens obtained from the 459 patients revealed quantifiable expression of the IR and IGF-1R in most of the NSCLCs (Fig. 1). The expression of the IR, IGF-1R was mainly cytoplasmic with modest membraneous staining; we did not detect nuclear staining of these proteins. The expression levels at cytoplasmic and membranous locations for each marker were well correlated with each other (data not shown). Cytoplasmic IR expression levels were significantly higher in ADC specimens than in SCC specimens (P = 2.4 E-4, Fig. 2A), but the membranous IR expression levels were similar. The levels of IR expression according to sex, stage, and smoking history were similar (Table 2). Cytoplasmic and membraneous expressions of IGF-1R (P = 4.7 E-05, Fig. 2B) were significantly associated with SCC. Expression of the IR and that of IGF-1R were not correlated with each other, and expression of pIGF-1R/IR was not significantly correlated with that of the IR and IGF-1R (data not shown). When we compared expression of IR and IGF-1R using gene expression and clinical data retrieved from the Gene Expression Omnibus (GSE3141; http://www.ncbi.nlm.nih.gov/geo/),20 which include 58 patients with ADC and 53 with SCC, normalized expression of IR (probe ID: 213792_s_at and 226450_at) were significantly higher in ADC than in SCC (P = .057 and .0010, respectively, Fig. 2C), whereas those of IGF-1R (probe ID: 225330_at and 203627 at) were significantly higher in SCC than in ADC (P = 3.5 E-7 and 1.3 E-9, respectively, Fig. 2D). The expression of 2 probes in each genes were strongly correlated with each other (Pearson correlation coefficient [RP]: 0.773, P = 2.6 E-23 for IR, and RP: 0.830, P = 2.1 E-29 for IGF-1R). We confirmed these differential expression patterns using an independent dataset with 138 patients (GSE8894).21
|Negative IR (N = 274)||Positive IR (N = 180)||Pa|
|Age Median age (range), y||66 (32-88)||66 (42-89)||.810|
|Gender||Female||130 (47.4%)||97 (53.9%)||.174|
|Male||144 (52.6%)||83 (46.1%)|
|Race||Caucasian||245 (89.4%)||168 (93.3%)||.154|
|Noncaucasian||29 (10.6%)||12 (6.7%)|
|Histology||Adenocarcinoma||172 (62.8%)||123 (68.3%)||.224|
|Squamous cell carcinoma||102 (37.2%)||57 (31.7%)|
|Smoking||Never smoker||34 (12.4%)||14 (7.8%)||.265|
|Former smoker||119 (43.4%)||86 (47.8%)|
|Current smoker||120 (43.8%)||79 (43.9%)|
|Unknown||1 (0.4%)||1 (0.6%)|
|T categoryb||T1||100 (36.5%)||72 (40.0%)||.892|
|T2||140 (51.1%)||88 (48.9%)|
|T3||14 (5.1%)||8 (4.4%)|
|T4||20 (7.3%)||12 (6.7%)|
|N categoryb||N0||191 (69.7%)||122 (67.8%)||.861|
|N1||45 (16.4%)||33 (18.3%)|
|N2||37 (13.5%)||25 (13.9%)|
|Nx||1 (0.4%)||0 (0%)|
|Final stageb||I||162 (59.1%)||109 (60.6%)||.930|
|II||86 (31.4%)||54 (30.0%)|
|III and IV||26 (8.5%)||17 (9.4%)|
After a median follow-up duration of 4.1 years for the censored observations (data cutoff: September 2010), RFS durations in patients with positive membraneous IR expression levels were significantly shorter than those in patients with negative IR expression levels (median: 3.8 years vs 3.3 years, P = .044 [log-rank test]) (Fig 3A). In contrast, the OS durations were similar in patients with positive versus negative IR expression levels (P = .430 [log-rank test]) (Fig 3B). Cytoplasmic IGF-1R and IR expression did not have impacts on RFS and OS in univariate analysis. Although membraneous IR expression levels were not significantly different between ADC and SCC, SCC patients with positive IR expression had shorter RFS and OS durations than those with negative IR expression (Fig. 3C and D), whereas IR expression in ADC patients did not make any difference in survival (Fig. 3E and F). When we performed a subgroup analysis by gender, there were similar OS in both IR positive and negative groups, whereas we observed prolonged RFS in IR negative groups in both gender (log rank test, P = .085 and .047 for male and female, respectively). Patients with positive membraneous IGF-1R expression showed poor RFS compared with those with negative expression (P= .044), whereas there was a similar OS between these groups (Fig. 4A and B). In the subgroup analysis by histology, SCC patients with positive or negative IGF-1R had a similar OS and RFS (Fig. 4C and D), whereas ADC patients with positive IGF-1R expression (N = 11) had poor OS and RFS compared with those with negative IGF-1R expression (Fig. 4E and F). Interestingly, patients with positive membraneous IGF-1R and IR expression levels (N = 23) had shorter RFS and OS durations than did all of the other patients (Fig. 5). When tested as continuous variables, the IGF-1R scores were not associated with differences in survival duration (data not shown). In contrast, the IR IHC score significantly impacted RFS and OS durations when tested as a continuous variable (hazard ratio [HR] for OS, 1.0045 [95% confidence interval [CI], 1.0001-1.0089]; P = .0470; HR for RFS, 1.0052 [95% CI, 1.0005-1.0100]; P = .0311). We confirmed the prognostic value of the IR IHC score regarding RFS and OS in the multivariate analysis (Table 3). When we performed the multivariate analysis in each histology subgroup, the negative impact of IR was significant only in patients with SCC (HR for OS: 1.009 [1.002-1.016], P = .014 and HR for RFS: 1.007 [1.001-1.014], P = .027) (Table 4). In the multivariate model with IGF-1R, IR only retained the significance. Unfortunately, we were not able to show the survival difference from gene expression databases, mainly because of lack of statistical power from small sample size.
|Variable||HR for OS (95% CI)||P||HR for RFS (95% CI)||P|
|Age||1.013 (0.998-1.029)||.089||1.009 (0.996-1.022)||.169|
|(Male vs female)||1.577 (1.172-2.123)||.003||1.439 (1.110-1.866)||.006|
|Histology (SCC vs ADC)||1.279 (0.947-1.726)||.109||1.179 (0.904-1.538)||.224|
|II vs I||1.837 (1.330-2.537)||<.001||1.846 (1.392-2.449)||<.001|
|III/IV vs I||2.294 (1.471-3.576)||<.001||2.214 (1.478-3.315)||<.001|
|IR expressiona||1.005 (1.001-1.010)||.028||1.005 (1.001-1.009)||.019|
|Variable||HR for OS (95% CI)||P Value||HR for RFS (95% CI)||P Value|
|Age||1.005 (0.980-1.029)||.718||1.002 (0.981-1.024)||.850|
|(Male vs Female)||1.133 (0.713-1.801)||.598||1.125 (0.736-1.718)||.586|
|II vs I||1.536 (0.920-2.563||.101||1.701 (1.075-2.691)||.023|
|III/IV vs I||2.561 (1.258-5.213)||.010||2.874 (1.472-5.611)||.002|
|IR expressiona||1.009 (1.002-1.016)||.014||1.007 (1.001-1.014)||.027|
Recent evidences imply that the IR expression has a role in cancer cell proliferation, angiogenesis, lymphangiogenesis, and metastasis in patients with various cancers.5, 22-24 However, firm studies of IR expression in primary human cancers are surprisingly scarce. In this article, we have demonstrated, to our knowledge for the first time, the survival impact of IR expression in patients with NSCLC. The results described herein provide evidence that IR expression in tumor specimen is an independent prognostic factor for OS and RFS in patients with resected NSCLC. The impact of IR expression on survival is substantial, even though the HRs for OS and RFS look small. Considering the IHC scores were put as a continuous variable in the multivariate model, the increase of HRs per 1 IR score is almost comparable to those per 1 year of age (Tables 3 and 4). Our data suggest that IR is a potential target in cancer therapy and that patients with NSCLC should be stratified based on IR expression in future clinical trials with IGF-1R/IR-targeted agents. Additional information can be retrieved from completed clinical trials of IGF-1R- or IR-targeted agents via retrospective analysis to determine whether differences in IR expression level among treatment groups affect outcomes and thus have therapeutic implications.
Because the IGF-1R pathway is frequently deregulated in human tumors, including NSCLC, investigators have assessed the role of IGF-1R expression as a prognostic factor. Previous studies have shown that IGF-1R expression is consistently associated with SCC.12-14 However, these previous studies do not consistently support a definite impact of IGF-1R protein expression on survival of NSCLC.12-14 Because it is well known that IGF-1R and IR are structurally similar and functionally interact to promote cell proliferation and survival,5 we sought to determine whether expression of IGF-1R, IR, or their co-expression have prognostic impacts in NSCLC. Our data confirm previous findings of IGF-1R expression in association with histological feature of SCC.12-14 We also show previously unidentified results, including: 1) IR expression was consistently associated with ADC; 2) patients with IGF-1R-positive or IR-positive tumors showed significantly shorter RFS than those with IGF-1R-negative tumors or IR-negative tumors, respectively (Figs. 3A and 4A); 3) IGF-1R and IR expressions were negatively correlated with survival duration of patients with ADC and SCC, respectively (Figs. 3C and D and 4E and F), although the number of patients with ADC was not enough to draw a firm conclusion about the correlation; and 4) patients with positive IR and IGF-1R expression levels had significantly shorter survival durations than did patients with negative IR and/or negative IGF-1R expression levels, although the number of patients in the positive expression group was relatively small. Interestingly, we observed similar survival durations in patients with negative IR and/or negative IGF-1R (Fig. 5). These data suggest that IR and IGF-1R could have independent roles in the prognosis of lung cancer patients. Further, these 2 receptors could function as potential prognostic factors dependent on the histological feature of patients. In a multivariate analysis, however, IGF-1R did not retain a significant hazard ratio for RFS and OS, whereas IR did, suggesting a definite impact of IR expression, not IGF-1R expression, on survival of NSCLC.
Based on our findings that IGF-1R expression was greater in ever-smokers than in never-smokers25 and in patients with SCC than in those with ADC and whereas IR expression was higher in patients with ADC than in those with SCC, we could suggest that cotargeting of the IR and IGF-1R may have broader coverage than targeting of IGF-1R alone. Of interest, some anti-IGF-1R antibody-based clinical trials, which have shown initial benefit in patients with SCC, revealed limitations in therapeutic efficacy.26 More importantly, recent phase III trials of the anti-IGF-1R antibody figitumumab were terminated prematurely because of an increase in the number of serious adverse events and excess mortality in the experimental arm. The resistance to anti-IGF-1R antibody could be explained by the possibility that reduced IGF-1R availability in hybrid IGF-1R/IR receptors may result in increased numbers of IR homodimers, leading to unwanted enhanced IR signaling. Also, anti-IGF-1R antibodies may not effectively block signaling from hybrid IGF-1R/IR. In previous studies, down-regulation of IGF-1R expression did not affect the IR expression levels but, interestingly, sensitized cells to insulin activation of downstream signaling pathways in several breast cancer cell lines27 or osteoblasts.28 In a pancreatic neuroendocrine tumor model in which the researchers treated tumors with the anti-IGF-1R antibody IMC-A12, elevated IR signaling before and after treatment may have manifested as intrinsic and adaptive resistance to anti-IGF-1R Ab-based therapies.29 Also, authors have noted that hyperinsulinemia is a compensatory mechanism to IGF-1R inhibitors,30, 31 which may directly activate IR signaling pathways. In these regard, small-molecule tyrosine kinase domain inhibitors (TKI) of IGF-1R, which are under investigation in clinical trials, could be a better strategy to target IGF-1R/IR signaling, as IGF-1R monoclonal antibody induced a compensatory increase of IR phosphorylation, which could be abolished by IGF-1R TKI.32 A challenge to the development of IGF-1R-targeted kinase inhibitors may be the similarity of the catalytic domain to that of the IR.33-35 Inhibition of the IR in normal cells could have brought significant undesired effects on glucose homeostasis, and chronic treatment may result in symptoms of diabetes.36 However, little data on the tolerability and efficacy of the small-molecule inhibitors of IGF-1R are currently available.37 Further investigation of the role of targeting IR in lung cancer is warranted.
A recent study examined the role of insulin and phosphorylated IR (pIR) in patients with NSCLC.38 The researchers confirmed the intracellular presence of insulin in 30 of 112 patients using IHC analysis and observed a significant correlation with pIR expression. Although the results of both DFS and OS analyses were not statistically significant, the investigators observed a trend of longer survival durations in patients with insulin-positive tumors than in those with insulin-negative tumors. One of the possible explanations for the increased survival durations in these patients is the close relationship of insulin reactivity with unaggressive, highly differentiated forms in the study population; however, we cannot conclude the significance of the survival difference because of the lack of statistical significance and relatively small number in the study. Furthermore, the researchers in this previous study correlated intracellular insulin levels, not pIR expression levels, with patient survival, which only suggested that noncirculatory ligands impacted survival.
In conclusion, we demonstrated that IGF-1R expression was higher in patients with SCC or ever-smokers than in those with ADC or never-smokers. On the other hand, IR expression was higher in patients with ADC than those with SCC. In addition, we show the prognostic impact of IR expression on survival in patients with resected NSCLC. Given our current observations and the previous reports supporting a possible role of IR expression in the resistance mechanisms to IGF-1R–targeted agents, clinical trials are warranted to assess the therapeutic efficacy of co-targeting both IGF-1R and IR. We believe that the roles of the IR in lung cancer biology and tumorigenesis should be important subjects of future research.
This work was supported by National Institutes of Health Grant R01 CA100816 and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2011-0017639) (to H.-Y.L.) and by Department of Defense Grant W81XWH-04-1-0142-01-VITAL and W8XWH-06-1-0303 BATTLE (to W.K.H.).
The authors made no disclousres