Increased HER2 Gene Copy Number Is Associated With Response to Gefitinib Therapy in Epidermal Growth Factor Receptor–Positive Non–Small-Cell
http://www.100md.com
《临床肿瘤学》
the Department of Medicine/Medical Oncology and Pathology, University of Colorado Cancer Center, Aurora, CO
Department of Medical Oncology, Bellaria Hospital
CINECA-Interuniversity Consortium, Bologna
Department of Medical Oncology, Scientific Institute University Hospital San Raffaele, Milano
Department of Medical Oncology, Policlinico Monteluce, Perugia, Italy
Hamon Center for Therapeutic Oncology Research and Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX
ABSTRACT
PURPOSE: In non-small-cell lung cancer (NSCLC), response to tyrosine kinase inhibitors (TKIs) is significantly associated with the presence of increased copy number and/or activating mutations of the epidermal growth factor receptor gene (EGFR). Preclinical data indicate that HER2, a member of the EGFR family, could enhance TKI sensitivity.
PATIENTS AND METHODS: HER2 gene copy numbers per cell were evaluated by fluorescent in situ hybridization (FISH) in 102 NSCLC patients treated with gefitinib, and previously evaluated for EGFR status by FISH, immunohistochemistry, and presence of mutations.
RESULTS: Patients with HER2 high copy number (high polysomy and gene amplification [HER2 FISH positive]) represented 22.8% of patients, and compared with patients with no or low gain (HER2 FISH negative), had significantly better objective response (OR, 34.8% v 6.4%; P = .001), disease control rate (DCR, 56.5% v 33.3%; P = .04), time to progression (TTP, 9.05 v 2.7 months; P = .02), and a trend toward longer overall survival (OS, 20.8 v 8.4 months; P = .056). HER2 protein expression investigated by immunohistochemistry was positive in only five of 72 (7%) patients analyzed and all 89 patients tested by DNA sequencing were negative for mutations in HER2 exon 20. Patients with HER2 FISH-positive tumors displaying increased expression of EGFR protein, gene gain, or mutations (EGFR positive) had a significantly better OR, DCR, TTP, and OS than patients negative for both receptors.
CONCLUSION: Increased copy number of the HER2 gene is associated with gefitinib sensitivity in EGFR-positive patients, supporting use of HER2 FISH analysis for selection of patients for TKI therapy.
INTRODUCTION
Non–small-cell lung cancer (NSCLC) has been the leading cause of cancer deaths in the world.1 Two-drug combination chemotherapy is the standard of care for advanced NSCLC, but even with newly developed chemotherapy strategies, the median survival rarely exceeds 8 to 9 months and the fraction of patients alive after 1 year is approximately 30%.2-4 The epidermal growth factor receptor (EGFR) is a prototypical member of the EGFR family that also includes HER2/neu (Erb-B2), HER3 (Erb-B3), and HER4 (Erb-B4).5-7 These receptors represent ideal therapeutic targets in cancer because they play a critical role in tumor proliferation and patient survival, and specific mutations and activation may cause tumor addition.7 EGFR and HER2 are highly expressed in many solid tumors,8 and increased EGFR and/or HER2 expression has been associated with poor prognosis.9-11 In NSCLC, we have previously reported on the prognostic aspects of EGFR and HER2 gene and protein overexpression12,13 and found that increased EGFR or HER2 gene copy numbers had a tendency to be associated with short survival. Using quantitative real-time polymerase chain reaction (PCR) systems, Brabender et al9 demonstrated that a high level of HER2 mRNA expression was associated with inferior survival in NSCLC patients and coexpression of HER2 and EGFR mRNAs had an additive prognostic impact.
Several new therapeutic agents that interfere with the EGFR signaling have been developed.14 Among the most promising of these new drugs are gefitinib (ZD 1839 [Iressa]; AstraZeneca, Macclesfield, United Kingdom), and erlotinib (OSI 774 [Tarceva], Genentech, San Francisco, CA). Both are orally active, selective EGFR tyrosine kinase inhibitors (TKIs) that demonstrated antitumoral activity against a variety of human cancer cell lines expressing EGFR,15 and both are approved for use in NSCLC in the United States. These drugs produced a response and stable disease in pretreated NSCLC patients,16-18 and the use of erlotinib resulted in a significant survival benefit when compared with placebo.19
Recently, several groups reported a strong association between response to TKIs and specific mutation in the tyrosine kinase domain of the EGFR gene and/or gain of the EGFR genomic sequences.20-22 In our previous study, increased EGFR gene copy number evaluated by fluorescent in situ hybridization (FISH), a high level of protein expression evaluated by immunohistochemistry (IHC), and the presence of EGFR gene mutations were all significantly associated with higher response rates and lower progression rates.23 Each of these three EGFR markers was also associated with a significantly longer time to disease progression (TTP), whereas only increased gene copy number was significantly related to survival in multivariate analyses. A strong association between increased EGFR gene copy number and clinical outcome has also been reported in patients with bronchioloalveolar carcinoma subtypes.24 Moreover, EGFR-TKI sensitivity depends on the activated status of the EGFR-related pathways, particularly of the antiapoptotic PI3K-AKT pathway.24-27
It is known that EGFR family members exist as monomers spanning the plasma membrane, and the monomeric receptors dimerize and become functionally active after binding to the appropriate ligand. HER2 is the preferred EGFR partner,27 and preclinical data have shown that tumors that overexpress HER2 are the most sensitive to gefitinib, possibly because this drug induces sequestration of HER2 and HER3 receptors in an inactive heterodimer configuration with the EGFR.29-31 In addition, patients with bronchioloalveolar carcinomas are more sensitive to TKIs,18 and this particular lung cancer subtype frequently shows a high level of EGFR and HER2 coexpression.32
Although preclinical and clinical data suggest that HER2 is relevant for TKI sensitivity,28-32 its role in NSCLC patients has been investigated only marginally, and association between level of HER2 protein expression evaluated by IHC and gefitinib sensitivity was not found in 43 NSCLC patients.33 In addition, a missense substitution and in-frame insertions were reportedly found in five of 120 primary lung tumors, all confined to the tyrosine kinase domain of the HER2 gene.34 These mutations were found in current or former smokers (in tumors with adenocarcinoma histology) and were not associated with mutation in the EGFR tyrosine kinase domain or HER2 gene amplification.
On the basis of these available preclinical and clinical data, we performed this study to investigate whether genomic gains, mutations, and overexpression of HER2 is associated with sensitivity to gefitinib in NSCLC patients, and whether EGFR and HER2 have an additive effect.
PATIENTS AND METHODS
Patient Population
This study was conducted in a cohort of advanced NSCLC patients treated at three Italian institutions: Bellaria Hospital (Bologna), Scientific Institute University Hospital San Raffaele (Milan), and Policlinico Monteluce (Perugia). The cohort included 102 consecutive NSCLC patients, 80 of whom were accrued onto the AKT prospective clinical trial25 and 22 of whom were consecutively accrued from the Expanded Access Study (EAS) of gefitinib that followed the AKT trial. Eligibility for both studies included histologically confirmed NSCLC with measurable locally advanced or metastatic disease, which progressed or relapsed after chemotherapy, or medical contraindications for chemotherapy. Patients were classified as nonsmokers (never smoker), former smokers (quit smoking > 6 months before starting gefitinib therapy), or current smokers (quit smoking < 6 months before starting gefitinib therapy or active smokers). The AKT study was approved by the Bellaria Hospital Institutional Ethical Review Board and written informed consent was obtained from each patient before enrollment. In the subgroup of EAS patients, the Institutional Review Board approval was obtained according to Good Clinical Practice, and a specific written informed consent was obtained from each patient (EAS consent form, Italian version). No clinical or biologic characteristic was considered for patient selection; the single criterion considered was the availability of tumor tissue.
Patients received gefitinib (250 mg/d) and were evaluated for response after 2 months according to the Response Evaluation Criteria in Solid Tumors Group criteria.35 Tumor response was assessed by computed tomography scan, with a confirmatory evaluation repeated in patients with complete response, partial response, and stable disease at least 4 weeks after the initial determination of response. Outcome in these patients was previously reported and associated with EGFR status.23 Among the patients' characteristics, female sex and never smoking status were significantly associated with better response; and female sex, adenocarcinoma and bronchioloalveolar histology, and performance status 0 to 1 were significantly associated with longer survival.
Tissue Preparation and FISH and IHC Analyses
Sections from paraffin-embedded tissue blocks containing representative malignant cells and obtained at the time of diagnosis were used for the FISH and IHC analyses. Histopathologic classification was determined on a slide section stained with hematoxylin and eosin based on WHO criteria.36 Dual-target, dual-color FISH assays were performed using the PathVysion HER-2 DNA probe kit (Vysis/Abbott Laboratories, Downers Grove, IL), which includes the LSI HER-2 SpectrumOrange and the CEP 17 SpectrumGreen probes, as described elsewhere.13 The reference slide (stained with hematoxylin and eosin) was the adjacent section on which the dominant tumor foci were identified, and copy numbers of the HER2 gene and chromosome 17 centromere probes were assessed and recorded independently in at least 100 nonoverlapping nuclei with intact morphology. Analysis was performed independently by two observers (F.C., M.V.G.) blinded to the patients' clinical characteristics. According to the frequency of tumor cells with a specific number of copies of the HER2 gene and chromosome 17 centromere, patients were classified into two strata: FISH negative, with no or low genomic gain ( four copies of the gene in > 40% of cells) and FISH positive, with a high level of polysomy ( four copies of the gene in 40% of cells) or gene amplification, defined by presence of tight gene clusters and a ratio of gene/chromosome per cell 2, or 15 copies of the genes per cell in 10% of analyzed cells.
IHC assessment for HER2 was done using the HercepTest (DAKO, Glostrup, Denmark) as described previously.13 Analysis was performed by two microscopists (W.A.F., F.R.H.) blinded to the clinical histories, using a x10 magnification objective. Only clear staining of the tumor cell membranes was considered positive, whereas diffuse cytoplasmic or granular staining was diagnosed as negative. A semiquantitative approach was used to generate a score for each tissue core, similar to that previously described for EGFR.13 The percentage of positive cells (0% to 100%) was multiplied by the dominant staining intensity pattern (1, negative or trace; 2, weak; 3, moderate; 4, intense), producing scores from 0 to 400. Specimens with low levels of expression were re-evaluated for the presence or absence of membranous or cytoplasmic staining using a x40 objective. Specimens with scores 0 to 200 and 201 to 400 were respectively classified as having low (IHC negative) and high (IHC positive) levels of expression.
Mutation Analysis
DNA for mutation analysis was obtained from tumors of 89 patients. In 22 patients, tumor cells were microdissected manually or by laser capture technique using the PALM instrument (P.A.L.M. Microlaser Technologies AG Inc, Bernried, Germany), according to the manufacturer's guidelines. We recently demonstrated that HER2 mutations were limited to exon 20 in NSCLC36; therefore, the analysis in this cohort was limited to this exon. One hundred nanograms of genomic DNA was amplified for HER2 exon 20 using primers as follows: 5'-GCC ATG GCT GTG GTT TGT GAT GG–3' and 5'-ATC CTA GCC CCT TGT GGA CAT AGG-3'. All PCR products were incubated using exonuclease I and shrimp alkaline phosphatase (Amersham Biosciences Corp, Piscataway, NJ) and sequenced directly using Applied Biosystems PRISM dye terminator cycle sequencing method (Perkin-Elmer Corp, Foster City, CA). All sequence variants were confirmed by independent PCR amplifications and sequenced in both directions.
Statistical Analysis
Differences between the FISH groups were compared by Fisher's exact test or 2 test for qualitative variables and by Student's t test. Normality of the distribution was assessed by Kolmogorov-Smirnov test. TTP, overall survival (OS), and the 95% CIs were evaluated by the Kaplan-Meier method, 38 comparing the FISH groups by log-rank test. Risk factors associated with response (such as those that were evaluated in a multivariable analysis) and only those variables with significant results in univariate analysis were included in the multivariate model using Cox and Snell R2 and Nagelkerke R2.39 The criteria for variable removal was the likelihood ratio statistic based on the estimated maximum partial likelihood (default P = 0.10 for removal from the model).
RESULTS
HER2 FISH, IHC, and Mutation Analyses
FISH results were obtained in 101 patients: 77.2% had no or low gain of HER2 genomic sequences (HER2 FISH negative) and 22.8% had high levels of HER2 genomic gain (HER2 FISH positive). Figure 1 illustrates tumors categorized as FISH negative (Fig 1A) and FISH positive (Figs 1B and 1C) for the HER2 gene. There was no association between HER2 FISH status and tumor histology. HER2 FISH-positive status was more frequently observed in female and never smoking patients, although the association was not statistically significant (P = .10 and P = .32, respectively). If we consider only the 10 patients who displayed HER2 gene amplification, those associations were statistically significant (female sex, P = .03; never smoking history, P = .04).
Table 1 shows the association between HER2 FISH patterns and response to treatment, TTP, and survival after treatment. In the HER2-positive group, the overall response rate (OR) was 34.8% and the disease control rate (DCR) was 56.5%, which was significantly better than that observed in the HER2-negative group (OR, 6.4%; P = .001 v DCR, 33.3%; P = .04). With respect to TTP, HER2-positive patients also did better than the HER2-negative group (Fig 2). At 12 months, 9.0% of HER2-negative patients had not experienced disease progression compared with 34.8% of HER2-positive patients (P = .005). TTP was significantly longer in HER2-positive (median, 9.05 months) than in HER2-negative patients (median, 2.7 months; P = .02). Median survival was longer in HER2-positive patients (20.8 v 8.4 months; P = .056), and the fraction of patients alive at 12 months also favored HER2-positive patients (60.9% v 37.2; P = .056; Fig 2).
HER2 protein levels were successfully assessed by IHC in 72 patients of this cohort, and only five patients (7%) were classified as HER2 IHC positive (score 200). There was no significant association between protein expression and response rate, disease control rate, median TTP, and median OS. Mutation analysis was successfully performed in 89 specimens and none showed changes in exon 20. Therefore, HER2 protein levels and mutation were not included in additional analyses.
Association Among HER2 FISH and EGFR FISH, IHC, and Mutation
Response analysis. Table 2 summarizes the results of EGFR FISH and mutation analyses, tumor histology, and response to treatment for all HER2 FISH-positive patients. HER2 gene gain (high polysomy and gene amplification) was significantly associated with EGFR gene gain (P = .004) and with EGFR gene mutations (P = .003). In tumors from 17 patients with gene amplification, six (35%) had coamplification of both EGFR and HER2 genes, whereas four (24%) had only HER2 gene amplification and seven (41%) had only EGFR gene amplification.
Among the 23 HER2 FISH-positive patients, 19 had EGFR mutation analyses performed and eight had EGFR mutations (four tumors with L858R and four with deletion in exon 19), of whom all but one responded to gefitinib. Among the 11 HER2 FISH-positive patients without EGFR mutation, three had stable disease and eight had progressive disease. Seven patients with tumors positive for at least one of the EGFR markers (FISH, IHC, or mutation) who were also HER2 FISH positive (HER2 FISH positive/EGFR positive) responded to treatment. The tumor histology was adenocarcinoma in four of those patients, bronchioalveolar carcinoma in two of those patients, and undifferentiated carcinoma in one of those patients. Three HER2 FISH-positive/EGFR-positive patients had stable disease; the tumor histology was adenocarcinoma in one patient and squamous cell carcinoma in two patients. Three HER2-positive/EGFR-positive patients had progressive disease; the tumor histology was adenocarcinoma in two and squamous cell carcinoma in one patient.
Table 3 shows the association between HER2 and EGFR status and treatment response. Patients with a high copy number of both genes (HER2 FISH positive/EGFR FISH positive) had the highest OR (53.8%) and DCR (76.9%), and these results were significantly better than those observed in patients with HER2 FISH-negative and/or EGFR FISH-negative tumors (OR, 6.8%; P < .001 and DCR, 33.0%; P = .002). The HER2 FISH-positive/EGFR FISH-negative patients had lower OR than double-positive patients, although the difference was not statistically significant (OR, 21.0%; P = .07). No difference in response was observed between HER2 FISH-negative/EGFR FISH-positive patients and the double-negative HER2 FISH-negative/EGFR FISH-negative patients (OR, 10.0% v 1.6%; P = .27 and DCR, 30.0%; v 25.4%; P = .71), although the latter group had a significantly worse outcome when compared with HER2 FISH-positive and/or EGFR FISH-positive patients (OR, 1.6% v 28.6%; P < .001 and DCR, 25.4% v 57.1%; P = .001).
Similar findings were observed when EGFR was evaluated by IHC. OR and DCR were significantly better in double-positive HER2 FISH-positive/EGFR IHC-positive patients when compared with all other groups of patients (OR, 53.8% v 7.1%; P < .001 and DCR, 76.9 v 34.5; P = .004). A significant difference in OR was observed between double-positive and HER2 FISH-negative/EGFR IHC-positive patients (OR, 11.1%; P = .003). No difference was found between HER2 FISH-positive/EGFR IHC-negative and double-negative HER2 FISH-negative/EGFR IHC-negative patients, in which OR and DCR were significantly worse than in the other three groups combined (OR, 0% v 19.1%; P = .009 and DCR, 13.7% v 51.5%; P = .001).
Patients with HER2 FISH-positive/EGFR-positive mutation tumors had the best OR and DCR (87.5% for both), which were significantly higher than in patients with HER2 FISH negative and/or EGFR mutation-negative status (OR, 5.0%; P < .001 and DCR, 31.3%; P = .003). Among the seven patients with HER2 FISH-negative/EGFR-positive mutation, one patient responded (OR, 14.2%) and one patient had disease stabilization (DCR, 28.5%). In the HER2 FISH-positive/EGFR-negative mutation group, no patient responded and DCR was 27.2%. These results were not different from those observed in double-negative HER2 FISH-negative/EGFR-negative mutation patients (OR, 4.8%; P = 1.0 and DCR, 32.2%; P = 1.0), in whom OR was significantly worse than in the other groups combined (OR, 30.8%; P = .002).
To define which of the variables could independently predict response to gefitinib, factors with statistically significant impact in the univariate analysis (sex, smoking history, and HER2 and EGFR status) were included into a multivariable model. Smoking history was significantly related to a lower probability of response (hazard ratio [HR], 7.95; 95% CI, 1.87 to 33.86; P = .005), whereas the presence of high HER2 gene copy number (HER2 positive) was significantly associated with an increased probability to respond (HR, 0.12; 95% CI, 0.03 to 0.48; P = .003). Sex was immediately removed at the first step of the backward elimination. HER2-positive status remained an independent variable for response also when the model was adjusted for EGFR assessed by FISH (HR, 0.19; 95% CI, 0.04 to 0.82; P = .026) and by IHC (HR, 0.09; 95% CI, 0.02 to 0.41; P = .002), and showed a nonsignificant trend when the model was adjusted for EGFR mutation (HR, 0.22; 95% CI, 0.04 to 1.21; P = .08).
TTP and Survival
TTP and survival data according to the combination of HER2 and EGFR status are summarized in Table 4. In the double-positive HER2 FISH-positive/EGFR FISH-positive patients, the median TTP and OS was 9.8 and 20.8 months, respectively, which was significantly longer than those observed in the HER2 FISH-negative and/or EGFR FISH-negative groups (TTP, 2.6 months; P = .007 and OS, 8.3 months; P = .04; Figs 3A and 3B), and with a nonsignificant trend when compared with the HER2 FISH-negative/EGFR FISH-positive patients (TTP, 5.3 months; P = .20 and OS, 9.3 months; P = .13). Patients with HER2 FISH-positive/EGFR FISH-negative tumors had the same poor outcome as the double-negative group (TTP, 2.3 v 2.6 months; P = .4 and OS, 6.0 v 7.3 months; P = .4).
Similar results were observed when EGFR was evaluated by IHC. TTP and survival were significantly longer in double-positive patients (HER2 FISH-positive/EGFR IHC-positive) when compared with the other three group of patients combined (TTP, 12.3 v 2.6 months; P = .006 and OS, 20.8 v 8.4 months; P = .030; Figs 3C and 3D) and with a statistically significant longer TTP and trend toward better survival when compared with patients with HER2 FISH-negative/EGFR IHC-positive tumors (TTP, 4.2 months; P = .046 and OS, 11.3; P = .12). The patients with HER2 FISH-positive/EGFR IHC-negative tumors had similarly poor outcome compared with the double-negative group (TTP, 2.3 v 2.1 months; P = .06 and OS, 3.3 v 5.0 months; P = .39).
When EGFR was evaluated for the presence of mutations, patients with HER2 FISH-positive/EGFR-positive mutation tumors had a significantly longer TTP and OS when compared with other patients combined (TTP, 15.5 v 2.6 months; P = .003 and OS, not reached v 8.3 months; P = .001; Figs 3E and 3F), but also when compared with HER2 FISH-negative/EGFR-positive mutation (TTP, 2.8 months; P = .004 and OS, 5.7 months; P = .030) patients. The group of patients EGFR-negative/HER2-positive mutation had the worst outcome in terms of TTP (2.3 months) and OS (6.5 months).
DISCUSSION
TKIs are offering new hope for patients with NSCLC. Identification of biologic processes underlying the sensitivity to these drugs is an important step, not only for proper selection of patients for therapy, but also for development of new therapeutic strategies. We previously demonstrated that increased EGFR gene copy number, high EGFR protein expression, and/or specific EGFR mutations all predict high sensitivity to gefitinib treatment of NSCLC, whereas increased gene copy number also predicted prolonged survival in multivariate analysis.23 In the current study we demonstrated that, independently of the method for EGFR assessment, EGFR-positive patients who also had increased copy numbers of the HER2 gene had a better response rate, disease control rate, TTP, and survival. The outcome of double-positive patients was significantly better than that of the HER2 FISH-negative/EGFR-positive or HER2-negative and/or EGFR-negative patients. Of particular interest, among the seven patients with EGFR mutations but negative for FISH HER2, only one patient achieved objective response, and this group of patients had a poor outcome similar to that of the EGFR-negative mutation groups. The current findings suggest that high copy numbers of the HER2 gene increase sensitivity to gefitinib therapy. Conversely, in the absence of EGFR, HER2 alone is not able to drive gefitinib sensitivity. In fact, the outcome of EGFR-negative/HER2-positive patients was not different from the outcome of patients negative for both receptors, and this double-negative group of patients had the worst outcome for all of the clinical end points.
The important role of HER2 for gefitinib sensitivity is strongly supported by preclinical data. Several groups demonstrated that gefitinib has a marked antiproliferative effect on tumors that express high levels of HER2.29-31,40 Anido et al30 recently demonstrated that only HER2-overexpressing cells that have significant levels of EGFR are inhibited by gefitinib. In these cells, gefitinib not only directly inhibits EGFR, but also inhibits HER2 and HER3 by sequestration of these receptors into inactive EGFR heterodimers. These data are concordant with our findings, in which the benefit was enhanced in HER2 FISH-positive/EGFR-positive patients, with no difference between HER2 FISH-positive/EGFR-negative and HER2 FISH-negative/EGFR-negative patients.
The prognostic role of EGFR in lung cancer is not well defined, but studies have shown that patients overexpressing EGFR and HER2 have poor prognosis.9-12 Few studies have addressed survival of NSCLC patients in association with HER2 gene amplification, and conclusions remain unclear. In 345 patients with stage I NSCLC41 no association between HER2 gene amplification determined by FISH and survival was detected. Conversely, in stage I to IIIA NSCLC patients, a high level of HER2 mRNA expression was significantly associated with poor survival in 83 patients9 and HER2 gene amplification determined by FISH was associated with shorter survival in 140 patients.42 We have previously reported a study comparing HER2 protein expression by IHC and gene copy number by FISH in resected NSCLC and found good correlation in two thirds of the tumors investigated.43 Although the number of patients with strong protein expression (3+) was limited, they had a tendency toward shorter survival.43 Interestingly, Brabender et al9 showed that high coexpression of EGFR and HER2 mRNAs had additive impact on survival after EGFR TKI therapy, as shown in our study. Therefore, it might be expected that HER2-positive/EGFR-positive patients had the best survival outcome as shown in our study, confirming the positive impact of gefitinib in this group of patients.
Another interesting finding of this study is the association of HER2 and EGFR gene amplifications and mutations in the tyrosine kinase domain of the EGFR gene. This is the first time that such association is reported in lung cancer. Coamplification of oncogenes has been described only rarely. However, a recent investigation of HER2, EGFR, CCDN1, CMYC, and MDM2 in breast tumors concluded that oncogene coamplifications were more prevalent than expected based on the individual frequencies. These data are consistent with the hypothesis that tumors that are sufficiently genetically unstable to develop one gene amplification have an increased probability to develop multiple amplifications.44
Female sex, adenocarcinoma and bronchioloalveolar histology, and never smoking history are among clinical characteristics significantly related to response to gefitinib therapy.16,17,45 It is known that these characteristics are associated with the presence of EGFR mutations20-22,46 and EGFR gene gain.23 We currently showed that gain in copy number of the HER2 gene is significantly associated to female sex and never smoking history, giving another biologic explication for the high sensitivity observed in patients with these characteristics. In this study, only five of 72 patients (7%) were found to have a high level of HER2 protein expression, and no significant association was detected with outcome to gefitinib therapy. Using a different scoring system, we and others have showed previously that high expression of HER2 protein (score 3+) is relatively infrequent in NSCLC patients.13,43,47 However, the few patients with high protein expression reportedly have a good response to trastuzumab,47,48 and the ultimate clinical role of HER2 protein expression in relation to gefitinib needs to be investigated in a larger study population.
Finally, our findings provide a strong rationale to explore the use of gefitinib or other TKIs in combination with anti-HER2 agents that may exert their activity by complementary mechanisms. In breast cancer, clinical trials are currently exploring the combination of gefitinib and trastuzumab, a monoclonal antibody against the extracellular domain of HER2,30 supported by the preclinical evidence of the strong antitumor effects of this combination.29,40
In conclusion, this study showed that, independently of the method for EGFR assessment, increased copy numbers of the HER2 gene enhance sensitivity to gefitinib therapy in NSCLC patients with EGFR-positive tumors. These results support the use of HER2 FISH assay as a complementary test to EGFR assay for selection of patients for TKIs therapy. Mutations in the tyrosine kinase domain of the HER2 gene seem to be infrequent and not clinically relevant. Therapeutic strategies against both EGFR and HER2 deserve additional evaluation in lung cancer. Prospective trials are warranted to validate these data.
Authors' Disclosures of Potential Conflicts of Interest
Although all authors have completed the disclosure declaration, the following authors or their immediate family members have indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.
Acknowledgment
We thank the Department of Pathology of the Bellaria Hospital-Bologna, Institute San Raffaele-Milano, and Policlinico Monteluce-Perugia for providing patient tissues; AstraZeneca Pharmaceuticals for providing the drug; and the Cytogenetics and Tissue Procurement Cores of the University of Colorado Cancer Center for technical assistance.
NOTES
Supported by National Cancer Institute grants Cancer Center shared grant P30-CA46934 and Specialized Program of Research Excellence P01-CA58187. F.C. was a Visiting Professor at the University of Colorado Health Sciences Center sponsored by the Department of Medical Oncology of the Bellaria Hospital, Bologna, Italy. Supported in part by a grant from the Associazione Italiana per la Ricerca sul Cancro.
Both F.C. and M.V.-G. contributed equally to this work.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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Cappuzzo F, Magrini E, Ceresoli GL, et al: AKT phosphorylation and gefitinib efficacy in patients with advanced Non-Small Cell Lung Cancer. J Natl Cancer Inst 6: 1133-1141, 2004
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Tzahar E, Waterman H, Chen X, et al: A hierarchical network of interreceptor interactions determines signal transduction by Neu differentiation factor/neuregulin and epidermal growth factor. Mol Cell Biol 16: 5276-5287, 1996
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Moulder SL, Yakes FM, Muthuswamy SK, et al: Epidermal growth factor receptor (HER1) tyrosine kinase inhibitor ZD1839 (Iressa) inhibits HER2/neu (erbB2)-overexpressing breast cancer cells in vitro and in vivo. Cancer Res 61: 8887-8895, 2001
Anido J, Matar P, Albanell J, et al: ZD1839, a specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, induces the formation of inactive EGFR/HER2 and EGFR/HER3 heterodimers and prevents heregulin signaling in HER2-overexpressing breast cancer cells. Clin Cancer Res 9: 1274-1283, 2003
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Department of Medical Oncology, Bellaria Hospital
CINECA-Interuniversity Consortium, Bologna
Department of Medical Oncology, Scientific Institute University Hospital San Raffaele, Milano
Department of Medical Oncology, Policlinico Monteluce, Perugia, Italy
Hamon Center for Therapeutic Oncology Research and Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX
ABSTRACT
PURPOSE: In non-small-cell lung cancer (NSCLC), response to tyrosine kinase inhibitors (TKIs) is significantly associated with the presence of increased copy number and/or activating mutations of the epidermal growth factor receptor gene (EGFR). Preclinical data indicate that HER2, a member of the EGFR family, could enhance TKI sensitivity.
PATIENTS AND METHODS: HER2 gene copy numbers per cell were evaluated by fluorescent in situ hybridization (FISH) in 102 NSCLC patients treated with gefitinib, and previously evaluated for EGFR status by FISH, immunohistochemistry, and presence of mutations.
RESULTS: Patients with HER2 high copy number (high polysomy and gene amplification [HER2 FISH positive]) represented 22.8% of patients, and compared with patients with no or low gain (HER2 FISH negative), had significantly better objective response (OR, 34.8% v 6.4%; P = .001), disease control rate (DCR, 56.5% v 33.3%; P = .04), time to progression (TTP, 9.05 v 2.7 months; P = .02), and a trend toward longer overall survival (OS, 20.8 v 8.4 months; P = .056). HER2 protein expression investigated by immunohistochemistry was positive in only five of 72 (7%) patients analyzed and all 89 patients tested by DNA sequencing were negative for mutations in HER2 exon 20. Patients with HER2 FISH-positive tumors displaying increased expression of EGFR protein, gene gain, or mutations (EGFR positive) had a significantly better OR, DCR, TTP, and OS than patients negative for both receptors.
CONCLUSION: Increased copy number of the HER2 gene is associated with gefitinib sensitivity in EGFR-positive patients, supporting use of HER2 FISH analysis for selection of patients for TKI therapy.
INTRODUCTION
Non–small-cell lung cancer (NSCLC) has been the leading cause of cancer deaths in the world.1 Two-drug combination chemotherapy is the standard of care for advanced NSCLC, but even with newly developed chemotherapy strategies, the median survival rarely exceeds 8 to 9 months and the fraction of patients alive after 1 year is approximately 30%.2-4 The epidermal growth factor receptor (EGFR) is a prototypical member of the EGFR family that also includes HER2/neu (Erb-B2), HER3 (Erb-B3), and HER4 (Erb-B4).5-7 These receptors represent ideal therapeutic targets in cancer because they play a critical role in tumor proliferation and patient survival, and specific mutations and activation may cause tumor addition.7 EGFR and HER2 are highly expressed in many solid tumors,8 and increased EGFR and/or HER2 expression has been associated with poor prognosis.9-11 In NSCLC, we have previously reported on the prognostic aspects of EGFR and HER2 gene and protein overexpression12,13 and found that increased EGFR or HER2 gene copy numbers had a tendency to be associated with short survival. Using quantitative real-time polymerase chain reaction (PCR) systems, Brabender et al9 demonstrated that a high level of HER2 mRNA expression was associated with inferior survival in NSCLC patients and coexpression of HER2 and EGFR mRNAs had an additive prognostic impact.
Several new therapeutic agents that interfere with the EGFR signaling have been developed.14 Among the most promising of these new drugs are gefitinib (ZD 1839 [Iressa]; AstraZeneca, Macclesfield, United Kingdom), and erlotinib (OSI 774 [Tarceva], Genentech, San Francisco, CA). Both are orally active, selective EGFR tyrosine kinase inhibitors (TKIs) that demonstrated antitumoral activity against a variety of human cancer cell lines expressing EGFR,15 and both are approved for use in NSCLC in the United States. These drugs produced a response and stable disease in pretreated NSCLC patients,16-18 and the use of erlotinib resulted in a significant survival benefit when compared with placebo.19
Recently, several groups reported a strong association between response to TKIs and specific mutation in the tyrosine kinase domain of the EGFR gene and/or gain of the EGFR genomic sequences.20-22 In our previous study, increased EGFR gene copy number evaluated by fluorescent in situ hybridization (FISH), a high level of protein expression evaluated by immunohistochemistry (IHC), and the presence of EGFR gene mutations were all significantly associated with higher response rates and lower progression rates.23 Each of these three EGFR markers was also associated with a significantly longer time to disease progression (TTP), whereas only increased gene copy number was significantly related to survival in multivariate analyses. A strong association between increased EGFR gene copy number and clinical outcome has also been reported in patients with bronchioloalveolar carcinoma subtypes.24 Moreover, EGFR-TKI sensitivity depends on the activated status of the EGFR-related pathways, particularly of the antiapoptotic PI3K-AKT pathway.24-27
It is known that EGFR family members exist as monomers spanning the plasma membrane, and the monomeric receptors dimerize and become functionally active after binding to the appropriate ligand. HER2 is the preferred EGFR partner,27 and preclinical data have shown that tumors that overexpress HER2 are the most sensitive to gefitinib, possibly because this drug induces sequestration of HER2 and HER3 receptors in an inactive heterodimer configuration with the EGFR.29-31 In addition, patients with bronchioloalveolar carcinomas are more sensitive to TKIs,18 and this particular lung cancer subtype frequently shows a high level of EGFR and HER2 coexpression.32
Although preclinical and clinical data suggest that HER2 is relevant for TKI sensitivity,28-32 its role in NSCLC patients has been investigated only marginally, and association between level of HER2 protein expression evaluated by IHC and gefitinib sensitivity was not found in 43 NSCLC patients.33 In addition, a missense substitution and in-frame insertions were reportedly found in five of 120 primary lung tumors, all confined to the tyrosine kinase domain of the HER2 gene.34 These mutations were found in current or former smokers (in tumors with adenocarcinoma histology) and were not associated with mutation in the EGFR tyrosine kinase domain or HER2 gene amplification.
On the basis of these available preclinical and clinical data, we performed this study to investigate whether genomic gains, mutations, and overexpression of HER2 is associated with sensitivity to gefitinib in NSCLC patients, and whether EGFR and HER2 have an additive effect.
PATIENTS AND METHODS
Patient Population
This study was conducted in a cohort of advanced NSCLC patients treated at three Italian institutions: Bellaria Hospital (Bologna), Scientific Institute University Hospital San Raffaele (Milan), and Policlinico Monteluce (Perugia). The cohort included 102 consecutive NSCLC patients, 80 of whom were accrued onto the AKT prospective clinical trial25 and 22 of whom were consecutively accrued from the Expanded Access Study (EAS) of gefitinib that followed the AKT trial. Eligibility for both studies included histologically confirmed NSCLC with measurable locally advanced or metastatic disease, which progressed or relapsed after chemotherapy, or medical contraindications for chemotherapy. Patients were classified as nonsmokers (never smoker), former smokers (quit smoking > 6 months before starting gefitinib therapy), or current smokers (quit smoking < 6 months before starting gefitinib therapy or active smokers). The AKT study was approved by the Bellaria Hospital Institutional Ethical Review Board and written informed consent was obtained from each patient before enrollment. In the subgroup of EAS patients, the Institutional Review Board approval was obtained according to Good Clinical Practice, and a specific written informed consent was obtained from each patient (EAS consent form, Italian version). No clinical or biologic characteristic was considered for patient selection; the single criterion considered was the availability of tumor tissue.
Patients received gefitinib (250 mg/d) and were evaluated for response after 2 months according to the Response Evaluation Criteria in Solid Tumors Group criteria.35 Tumor response was assessed by computed tomography scan, with a confirmatory evaluation repeated in patients with complete response, partial response, and stable disease at least 4 weeks after the initial determination of response. Outcome in these patients was previously reported and associated with EGFR status.23 Among the patients' characteristics, female sex and never smoking status were significantly associated with better response; and female sex, adenocarcinoma and bronchioloalveolar histology, and performance status 0 to 1 were significantly associated with longer survival.
Tissue Preparation and FISH and IHC Analyses
Sections from paraffin-embedded tissue blocks containing representative malignant cells and obtained at the time of diagnosis were used for the FISH and IHC analyses. Histopathologic classification was determined on a slide section stained with hematoxylin and eosin based on WHO criteria.36 Dual-target, dual-color FISH assays were performed using the PathVysion HER-2 DNA probe kit (Vysis/Abbott Laboratories, Downers Grove, IL), which includes the LSI HER-2 SpectrumOrange and the CEP 17 SpectrumGreen probes, as described elsewhere.13 The reference slide (stained with hematoxylin and eosin) was the adjacent section on which the dominant tumor foci were identified, and copy numbers of the HER2 gene and chromosome 17 centromere probes were assessed and recorded independently in at least 100 nonoverlapping nuclei with intact morphology. Analysis was performed independently by two observers (F.C., M.V.G.) blinded to the patients' clinical characteristics. According to the frequency of tumor cells with a specific number of copies of the HER2 gene and chromosome 17 centromere, patients were classified into two strata: FISH negative, with no or low genomic gain ( four copies of the gene in > 40% of cells) and FISH positive, with a high level of polysomy ( four copies of the gene in 40% of cells) or gene amplification, defined by presence of tight gene clusters and a ratio of gene/chromosome per cell 2, or 15 copies of the genes per cell in 10% of analyzed cells.
IHC assessment for HER2 was done using the HercepTest (DAKO, Glostrup, Denmark) as described previously.13 Analysis was performed by two microscopists (W.A.F., F.R.H.) blinded to the clinical histories, using a x10 magnification objective. Only clear staining of the tumor cell membranes was considered positive, whereas diffuse cytoplasmic or granular staining was diagnosed as negative. A semiquantitative approach was used to generate a score for each tissue core, similar to that previously described for EGFR.13 The percentage of positive cells (0% to 100%) was multiplied by the dominant staining intensity pattern (1, negative or trace; 2, weak; 3, moderate; 4, intense), producing scores from 0 to 400. Specimens with low levels of expression were re-evaluated for the presence or absence of membranous or cytoplasmic staining using a x40 objective. Specimens with scores 0 to 200 and 201 to 400 were respectively classified as having low (IHC negative) and high (IHC positive) levels of expression.
Mutation Analysis
DNA for mutation analysis was obtained from tumors of 89 patients. In 22 patients, tumor cells were microdissected manually or by laser capture technique using the PALM instrument (P.A.L.M. Microlaser Technologies AG Inc, Bernried, Germany), according to the manufacturer's guidelines. We recently demonstrated that HER2 mutations were limited to exon 20 in NSCLC36; therefore, the analysis in this cohort was limited to this exon. One hundred nanograms of genomic DNA was amplified for HER2 exon 20 using primers as follows: 5'-GCC ATG GCT GTG GTT TGT GAT GG–3' and 5'-ATC CTA GCC CCT TGT GGA CAT AGG-3'. All PCR products were incubated using exonuclease I and shrimp alkaline phosphatase (Amersham Biosciences Corp, Piscataway, NJ) and sequenced directly using Applied Biosystems PRISM dye terminator cycle sequencing method (Perkin-Elmer Corp, Foster City, CA). All sequence variants were confirmed by independent PCR amplifications and sequenced in both directions.
Statistical Analysis
Differences between the FISH groups were compared by Fisher's exact test or 2 test for qualitative variables and by Student's t test. Normality of the distribution was assessed by Kolmogorov-Smirnov test. TTP, overall survival (OS), and the 95% CIs were evaluated by the Kaplan-Meier method, 38 comparing the FISH groups by log-rank test. Risk factors associated with response (such as those that were evaluated in a multivariable analysis) and only those variables with significant results in univariate analysis were included in the multivariate model using Cox and Snell R2 and Nagelkerke R2.39 The criteria for variable removal was the likelihood ratio statistic based on the estimated maximum partial likelihood (default P = 0.10 for removal from the model).
RESULTS
HER2 FISH, IHC, and Mutation Analyses
FISH results were obtained in 101 patients: 77.2% had no or low gain of HER2 genomic sequences (HER2 FISH negative) and 22.8% had high levels of HER2 genomic gain (HER2 FISH positive). Figure 1 illustrates tumors categorized as FISH negative (Fig 1A) and FISH positive (Figs 1B and 1C) for the HER2 gene. There was no association between HER2 FISH status and tumor histology. HER2 FISH-positive status was more frequently observed in female and never smoking patients, although the association was not statistically significant (P = .10 and P = .32, respectively). If we consider only the 10 patients who displayed HER2 gene amplification, those associations were statistically significant (female sex, P = .03; never smoking history, P = .04).
Table 1 shows the association between HER2 FISH patterns and response to treatment, TTP, and survival after treatment. In the HER2-positive group, the overall response rate (OR) was 34.8% and the disease control rate (DCR) was 56.5%, which was significantly better than that observed in the HER2-negative group (OR, 6.4%; P = .001 v DCR, 33.3%; P = .04). With respect to TTP, HER2-positive patients also did better than the HER2-negative group (Fig 2). At 12 months, 9.0% of HER2-negative patients had not experienced disease progression compared with 34.8% of HER2-positive patients (P = .005). TTP was significantly longer in HER2-positive (median, 9.05 months) than in HER2-negative patients (median, 2.7 months; P = .02). Median survival was longer in HER2-positive patients (20.8 v 8.4 months; P = .056), and the fraction of patients alive at 12 months also favored HER2-positive patients (60.9% v 37.2; P = .056; Fig 2).
HER2 protein levels were successfully assessed by IHC in 72 patients of this cohort, and only five patients (7%) were classified as HER2 IHC positive (score 200). There was no significant association between protein expression and response rate, disease control rate, median TTP, and median OS. Mutation analysis was successfully performed in 89 specimens and none showed changes in exon 20. Therefore, HER2 protein levels and mutation were not included in additional analyses.
Association Among HER2 FISH and EGFR FISH, IHC, and Mutation
Response analysis. Table 2 summarizes the results of EGFR FISH and mutation analyses, tumor histology, and response to treatment for all HER2 FISH-positive patients. HER2 gene gain (high polysomy and gene amplification) was significantly associated with EGFR gene gain (P = .004) and with EGFR gene mutations (P = .003). In tumors from 17 patients with gene amplification, six (35%) had coamplification of both EGFR and HER2 genes, whereas four (24%) had only HER2 gene amplification and seven (41%) had only EGFR gene amplification.
Among the 23 HER2 FISH-positive patients, 19 had EGFR mutation analyses performed and eight had EGFR mutations (four tumors with L858R and four with deletion in exon 19), of whom all but one responded to gefitinib. Among the 11 HER2 FISH-positive patients without EGFR mutation, three had stable disease and eight had progressive disease. Seven patients with tumors positive for at least one of the EGFR markers (FISH, IHC, or mutation) who were also HER2 FISH positive (HER2 FISH positive/EGFR positive) responded to treatment. The tumor histology was adenocarcinoma in four of those patients, bronchioalveolar carcinoma in two of those patients, and undifferentiated carcinoma in one of those patients. Three HER2 FISH-positive/EGFR-positive patients had stable disease; the tumor histology was adenocarcinoma in one patient and squamous cell carcinoma in two patients. Three HER2-positive/EGFR-positive patients had progressive disease; the tumor histology was adenocarcinoma in two and squamous cell carcinoma in one patient.
Table 3 shows the association between HER2 and EGFR status and treatment response. Patients with a high copy number of both genes (HER2 FISH positive/EGFR FISH positive) had the highest OR (53.8%) and DCR (76.9%), and these results were significantly better than those observed in patients with HER2 FISH-negative and/or EGFR FISH-negative tumors (OR, 6.8%; P < .001 and DCR, 33.0%; P = .002). The HER2 FISH-positive/EGFR FISH-negative patients had lower OR than double-positive patients, although the difference was not statistically significant (OR, 21.0%; P = .07). No difference in response was observed between HER2 FISH-negative/EGFR FISH-positive patients and the double-negative HER2 FISH-negative/EGFR FISH-negative patients (OR, 10.0% v 1.6%; P = .27 and DCR, 30.0%; v 25.4%; P = .71), although the latter group had a significantly worse outcome when compared with HER2 FISH-positive and/or EGFR FISH-positive patients (OR, 1.6% v 28.6%; P < .001 and DCR, 25.4% v 57.1%; P = .001).
Similar findings were observed when EGFR was evaluated by IHC. OR and DCR were significantly better in double-positive HER2 FISH-positive/EGFR IHC-positive patients when compared with all other groups of patients (OR, 53.8% v 7.1%; P < .001 and DCR, 76.9 v 34.5; P = .004). A significant difference in OR was observed between double-positive and HER2 FISH-negative/EGFR IHC-positive patients (OR, 11.1%; P = .003). No difference was found between HER2 FISH-positive/EGFR IHC-negative and double-negative HER2 FISH-negative/EGFR IHC-negative patients, in which OR and DCR were significantly worse than in the other three groups combined (OR, 0% v 19.1%; P = .009 and DCR, 13.7% v 51.5%; P = .001).
Patients with HER2 FISH-positive/EGFR-positive mutation tumors had the best OR and DCR (87.5% for both), which were significantly higher than in patients with HER2 FISH negative and/or EGFR mutation-negative status (OR, 5.0%; P < .001 and DCR, 31.3%; P = .003). Among the seven patients with HER2 FISH-negative/EGFR-positive mutation, one patient responded (OR, 14.2%) and one patient had disease stabilization (DCR, 28.5%). In the HER2 FISH-positive/EGFR-negative mutation group, no patient responded and DCR was 27.2%. These results were not different from those observed in double-negative HER2 FISH-negative/EGFR-negative mutation patients (OR, 4.8%; P = 1.0 and DCR, 32.2%; P = 1.0), in whom OR was significantly worse than in the other groups combined (OR, 30.8%; P = .002).
To define which of the variables could independently predict response to gefitinib, factors with statistically significant impact in the univariate analysis (sex, smoking history, and HER2 and EGFR status) were included into a multivariable model. Smoking history was significantly related to a lower probability of response (hazard ratio [HR], 7.95; 95% CI, 1.87 to 33.86; P = .005), whereas the presence of high HER2 gene copy number (HER2 positive) was significantly associated with an increased probability to respond (HR, 0.12; 95% CI, 0.03 to 0.48; P = .003). Sex was immediately removed at the first step of the backward elimination. HER2-positive status remained an independent variable for response also when the model was adjusted for EGFR assessed by FISH (HR, 0.19; 95% CI, 0.04 to 0.82; P = .026) and by IHC (HR, 0.09; 95% CI, 0.02 to 0.41; P = .002), and showed a nonsignificant trend when the model was adjusted for EGFR mutation (HR, 0.22; 95% CI, 0.04 to 1.21; P = .08).
TTP and Survival
TTP and survival data according to the combination of HER2 and EGFR status are summarized in Table 4. In the double-positive HER2 FISH-positive/EGFR FISH-positive patients, the median TTP and OS was 9.8 and 20.8 months, respectively, which was significantly longer than those observed in the HER2 FISH-negative and/or EGFR FISH-negative groups (TTP, 2.6 months; P = .007 and OS, 8.3 months; P = .04; Figs 3A and 3B), and with a nonsignificant trend when compared with the HER2 FISH-negative/EGFR FISH-positive patients (TTP, 5.3 months; P = .20 and OS, 9.3 months; P = .13). Patients with HER2 FISH-positive/EGFR FISH-negative tumors had the same poor outcome as the double-negative group (TTP, 2.3 v 2.6 months; P = .4 and OS, 6.0 v 7.3 months; P = .4).
Similar results were observed when EGFR was evaluated by IHC. TTP and survival were significantly longer in double-positive patients (HER2 FISH-positive/EGFR IHC-positive) when compared with the other three group of patients combined (TTP, 12.3 v 2.6 months; P = .006 and OS, 20.8 v 8.4 months; P = .030; Figs 3C and 3D) and with a statistically significant longer TTP and trend toward better survival when compared with patients with HER2 FISH-negative/EGFR IHC-positive tumors (TTP, 4.2 months; P = .046 and OS, 11.3; P = .12). The patients with HER2 FISH-positive/EGFR IHC-negative tumors had similarly poor outcome compared with the double-negative group (TTP, 2.3 v 2.1 months; P = .06 and OS, 3.3 v 5.0 months; P = .39).
When EGFR was evaluated for the presence of mutations, patients with HER2 FISH-positive/EGFR-positive mutation tumors had a significantly longer TTP and OS when compared with other patients combined (TTP, 15.5 v 2.6 months; P = .003 and OS, not reached v 8.3 months; P = .001; Figs 3E and 3F), but also when compared with HER2 FISH-negative/EGFR-positive mutation (TTP, 2.8 months; P = .004 and OS, 5.7 months; P = .030) patients. The group of patients EGFR-negative/HER2-positive mutation had the worst outcome in terms of TTP (2.3 months) and OS (6.5 months).
DISCUSSION
TKIs are offering new hope for patients with NSCLC. Identification of biologic processes underlying the sensitivity to these drugs is an important step, not only for proper selection of patients for therapy, but also for development of new therapeutic strategies. We previously demonstrated that increased EGFR gene copy number, high EGFR protein expression, and/or specific EGFR mutations all predict high sensitivity to gefitinib treatment of NSCLC, whereas increased gene copy number also predicted prolonged survival in multivariate analysis.23 In the current study we demonstrated that, independently of the method for EGFR assessment, EGFR-positive patients who also had increased copy numbers of the HER2 gene had a better response rate, disease control rate, TTP, and survival. The outcome of double-positive patients was significantly better than that of the HER2 FISH-negative/EGFR-positive or HER2-negative and/or EGFR-negative patients. Of particular interest, among the seven patients with EGFR mutations but negative for FISH HER2, only one patient achieved objective response, and this group of patients had a poor outcome similar to that of the EGFR-negative mutation groups. The current findings suggest that high copy numbers of the HER2 gene increase sensitivity to gefitinib therapy. Conversely, in the absence of EGFR, HER2 alone is not able to drive gefitinib sensitivity. In fact, the outcome of EGFR-negative/HER2-positive patients was not different from the outcome of patients negative for both receptors, and this double-negative group of patients had the worst outcome for all of the clinical end points.
The important role of HER2 for gefitinib sensitivity is strongly supported by preclinical data. Several groups demonstrated that gefitinib has a marked antiproliferative effect on tumors that express high levels of HER2.29-31,40 Anido et al30 recently demonstrated that only HER2-overexpressing cells that have significant levels of EGFR are inhibited by gefitinib. In these cells, gefitinib not only directly inhibits EGFR, but also inhibits HER2 and HER3 by sequestration of these receptors into inactive EGFR heterodimers. These data are concordant with our findings, in which the benefit was enhanced in HER2 FISH-positive/EGFR-positive patients, with no difference between HER2 FISH-positive/EGFR-negative and HER2 FISH-negative/EGFR-negative patients.
The prognostic role of EGFR in lung cancer is not well defined, but studies have shown that patients overexpressing EGFR and HER2 have poor prognosis.9-12 Few studies have addressed survival of NSCLC patients in association with HER2 gene amplification, and conclusions remain unclear. In 345 patients with stage I NSCLC41 no association between HER2 gene amplification determined by FISH and survival was detected. Conversely, in stage I to IIIA NSCLC patients, a high level of HER2 mRNA expression was significantly associated with poor survival in 83 patients9 and HER2 gene amplification determined by FISH was associated with shorter survival in 140 patients.42 We have previously reported a study comparing HER2 protein expression by IHC and gene copy number by FISH in resected NSCLC and found good correlation in two thirds of the tumors investigated.43 Although the number of patients with strong protein expression (3+) was limited, they had a tendency toward shorter survival.43 Interestingly, Brabender et al9 showed that high coexpression of EGFR and HER2 mRNAs had additive impact on survival after EGFR TKI therapy, as shown in our study. Therefore, it might be expected that HER2-positive/EGFR-positive patients had the best survival outcome as shown in our study, confirming the positive impact of gefitinib in this group of patients.
Another interesting finding of this study is the association of HER2 and EGFR gene amplifications and mutations in the tyrosine kinase domain of the EGFR gene. This is the first time that such association is reported in lung cancer. Coamplification of oncogenes has been described only rarely. However, a recent investigation of HER2, EGFR, CCDN1, CMYC, and MDM2 in breast tumors concluded that oncogene coamplifications were more prevalent than expected based on the individual frequencies. These data are consistent with the hypothesis that tumors that are sufficiently genetically unstable to develop one gene amplification have an increased probability to develop multiple amplifications.44
Female sex, adenocarcinoma and bronchioloalveolar histology, and never smoking history are among clinical characteristics significantly related to response to gefitinib therapy.16,17,45 It is known that these characteristics are associated with the presence of EGFR mutations20-22,46 and EGFR gene gain.23 We currently showed that gain in copy number of the HER2 gene is significantly associated to female sex and never smoking history, giving another biologic explication for the high sensitivity observed in patients with these characteristics. In this study, only five of 72 patients (7%) were found to have a high level of HER2 protein expression, and no significant association was detected with outcome to gefitinib therapy. Using a different scoring system, we and others have showed previously that high expression of HER2 protein (score 3+) is relatively infrequent in NSCLC patients.13,43,47 However, the few patients with high protein expression reportedly have a good response to trastuzumab,47,48 and the ultimate clinical role of HER2 protein expression in relation to gefitinib needs to be investigated in a larger study population.
Finally, our findings provide a strong rationale to explore the use of gefitinib or other TKIs in combination with anti-HER2 agents that may exert their activity by complementary mechanisms. In breast cancer, clinical trials are currently exploring the combination of gefitinib and trastuzumab, a monoclonal antibody against the extracellular domain of HER2,30 supported by the preclinical evidence of the strong antitumor effects of this combination.29,40
In conclusion, this study showed that, independently of the method for EGFR assessment, increased copy numbers of the HER2 gene enhance sensitivity to gefitinib therapy in NSCLC patients with EGFR-positive tumors. These results support the use of HER2 FISH assay as a complementary test to EGFR assay for selection of patients for TKIs therapy. Mutations in the tyrosine kinase domain of the HER2 gene seem to be infrequent and not clinically relevant. Therapeutic strategies against both EGFR and HER2 deserve additional evaluation in lung cancer. Prospective trials are warranted to validate these data.
Authors' Disclosures of Potential Conflicts of Interest
Although all authors have completed the disclosure declaration, the following authors or their immediate family members have indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.
Acknowledgment
We thank the Department of Pathology of the Bellaria Hospital-Bologna, Institute San Raffaele-Milano, and Policlinico Monteluce-Perugia for providing patient tissues; AstraZeneca Pharmaceuticals for providing the drug; and the Cytogenetics and Tissue Procurement Cores of the University of Colorado Cancer Center for technical assistance.
NOTES
Supported by National Cancer Institute grants Cancer Center shared grant P30-CA46934 and Specialized Program of Research Excellence P01-CA58187. F.C. was a Visiting Professor at the University of Colorado Health Sciences Center sponsored by the Department of Medical Oncology of the Bellaria Hospital, Bologna, Italy. Supported in part by a grant from the Associazione Italiana per la Ricerca sul Cancro.
Both F.C. and M.V.-G. contributed equally to this work.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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