Increased Epidermal Growth Factor Receptor Gene Copy Number Detected by Fluorescence In Situ Hybridization Associates With Increased Sensiti
http://www.100md.com
《临床肿瘤学》
the University of Colorado Health Sciences Center, Denver, CO
Southwest Oncology Group Statistical Center, San Antonio, TX
Puget Sound Oncology Consortium, Seattle, WA
University of California, Davis Cancer Center, Sacramento, CA
ABSTRACT
PURPOSE: Bronchioloalveolar carcinoma (BAC) and adenocarcinomas with BAC features seem to be increasing in incidence, particularly in younger, never-smoking women. Epidermal growth factor receptor (EGFR) inhibitors demonstrated response rates of 20% to 30% in patients with advanced BAC subtypes, but selection methods for patient therapy are not established.
PATIENTS AND METHODS: EGFR and HER2 gene copy numbers were assessed by fluorescence in situ hybridization (FISH) in 81 patients treated with gefitinib 500 mg/d (Southwest Oncology Group protocol S0126) and were correlated to treatment outcome. Tumors were classified into two main strata: FISH-positive (high polysomy/gene amplification) and FISH-negative (disomy/low polysomy).
RESULTS: In 81 patients, the median survival time for EGFR/FISH-negative patients was 8 months and not yet reached for FISH-positive patients (but approaching 18 months; hazard ratio [HR] = 2.02; P = .042). Median progression-free survival time for EGFR/FISH-positive patients was 9 months versus 4 months for the FISH-negative patients (HR = 1.67; P = .072). In multivariate analysis, EGFR copy number by FISH remained a significant predictive factor for survival after accounting for smoking status, sex, histology, and performance status. Fifty-five patients were evaluated for response using Response Evaluation Criteria in Solid Tumors Group, and 12 of 19 EGFR/FISH-positive patients (63%) demonstrated disease control versus 14 (39%) of 36 patients in the FISH-negative group (P = .087). No association was found between HER2 gene copy number and response (n = 39 patients) or survival (n = 56 patients; P > .10).
CONCLUSION: Increased EGFR gene copy number detected by FISH is associated with improved survival after gefitinib therapy in patients with advanced BAC, suggesting FISH methodology can be used to assess survival potential in patients treated with EGFR tyrosine kinase inhibitors.
INTRODUCTION
Non–small-cell lung cancer (NSCLC) is the leading cause of cancer death in the world.1 Although platinum-based chemotherapy results in palliation of symptoms and increased survival in patients with advanced-stage disease, benefits are modest and new approaches are clearly needed.2,3 Recently, improved understanding of molecular biology has led to development of a wide variety of inhibitors of target proteins involved in proliferation, apoptosis, and angiogenesis. Clinical application of agents such as imatinib in chronic myeloid leukemia4 and gastrointestinal stromal tumors (GIST)5 and trastuzumab in breast cancers6 has resulted in dramatic and often sustained response in tumors expressing the target proteins.
The epidermal growth factor receptor (EGFR), a member of the HER or Erb-B family of type I receptor tyrosine kinases, is implicated in the development and progression of cancer7-10 and is expressed in many human epithelial malignancies, including NSCLC.8,10 Several small molecules have been synthesized to inhibit the tyrosine kinase domain of EGFR,11,12 including gefitinib (Iressa; AstraZeneca, Macclesfield, United Kingdom) and erlotinib (Tarceva; Roche, Basche, Switzerland/OSI Pharmaceuticals Inc, Melville, NY). Both are orally active, selective EGFR tyrosine-kinase inhibitors (EGFR-TKI) that produce objective response rates of 9% to 26% in previously untreated or previously treated patients with advanced-stage NSCLC.13-15 Recently a phase III trial (BR21) comparing erlotinib with placebo as a second- or third-line therapy reported a survival benefit for the EGFR inhibitor (hazard ratio [HR] = 0.73).16 Importantly, this survival benefit was not confined to objective responders, nor to a single sex or histology.
These data emphasize the need to identify predictive factors of benefit from EGFR–TKI that could be used to select patients most likely to benefit. Although it has been reported that EGFR protein expression assessed by immunohistochemistry (IHC) is not correlated with gefitinib response, few studies have directly addressed this question.17-19 Recent reports have instead turned attention to specific missense mutations and deletions in the tyrosine kinase domain of the EGFR gene,20,21 which are highly associated with gefitinib response. However, the low frequency of these mutations (four of 81) in United States lung cancer patients who are current or former smokers,22 and the results of the BR21 study described above, suggest that a substantial subset of patients without these mutations also derive a survival benefit. Furthermore, although these activating mutations identify patients with high response rates, they cannot account for the high stable disease rates, reported to occur in approximately 30% of NSCLC patients treated with gefitinib.13,14 Furthermore, there are no published data available describing the relationship between EGFR mutations and survival, and no larger clinical cohort studies have so far been reported on the association between EGFR mutations and treatment response and survival.
Increased EGFR gene copy number detected by fluorescence in situ hybridization (FISH) is associated with a poor prognosis in NSCLC,23 similar to the assessment of HER2 by FISH in breast cancer patients.24 Moreover, among patients with breast cancer, FISH is reported to be a more reliable predictor of treatment benefit with the HER2 inhibitor trastuzumab (Herceptin; Genentech, San Francisco, CA) than is protein expression by IHC, especially in the patients with intermediate protein expression (2+).25 We have previously evaluated EGFR gene copy number in a cohort of 102 NSCLC patients treated with gefitinib and found high copy number or gene amplification in approximately 30% and a significant correlation between gene EGFR gene status and outcome.26
Bronchioalveolar carcinoma (BAC) subtypes of NSCLC are characterized by unique pathologic, radiographic, and clinical features27 and seem to be increasing in incidence, particularly in younger nonsmoking women.28,29 BAC and adenocarcinoma with BAC features have been reported to be particularly sensitive to EGFR tyrosine kinase inhibitors, with response rates of 25% to 30%30 and prolonged survival in a subset of patients. We have previously reported the efficacy of gefitinib in a large cohort of advanced-stage BAC patients treated on a prospective clinical trial of the Southwest Oncology Group (SWOG; S0126).31 Because archival tumor tissue was collected from the great majority of patients enrolled, the S0126 trial represents a unique pathologic resource for study of EGFR pathways. On the basis of our prior experience with NSCLC patients treated with gefitinib, we hypothesized that increased EGFR and/or HER2 gene copy numbers detected by FISH would be associated with increased efficacy of gefitinib in the subset of patients with NSCLC who have BAC or adenocarcinoma with BAC features. Here we report the results of this analysis in patient tumor tissue from the S0126 study, correlated with clinical outcome.
PATIENTS AND METHODS
All patients enrolled were required to have histologically proven, stage IIIB (by pleural effusion) or IV BAC or adenocarcinoma with BAC features. Pathologic eligibility was based on an institutional definition of BAC, although a central review was subsequently carried out using the WHO Classification.27 Histopathologic subtypes in this report are based on this central pathology review. Cytologic specimens were not accepted for the BAC diagnosis, and patients with only cytologic diagnosis were not eligible for S0126.
Patients were required to have an SWOG performance status of 0 to 2. Prestudy evaluation included history and physical examination; complete blood cell count with differential and platelets, serum chemistries of alkaline phosphatase, AST or ALT, lactate dehydrogenase and albumin; chest radiograph; and computed tomography of chest, liver, and adrenal glands. Bone scan and/or brain computed tomography or magnetic resonance imaging were required only if clinically indicated based on symptoms and physician judgment. Patients with a history of brain metastases were ineligible for the present study. Pregnant or nursing women were ineligible, and women and men of reproductive potential were unable to participate unless they agreed to use an effective contraceptive method. Eligible patients had no other prior malignancy except for adequately treated basal cell or squamous cell skin cancer, in situ cervical cancer, adequately treated stage I or II cancer from which the patient was in complete remission, or any other cancer from which the patient was disease-free for at least 5 years.
All patients were informed of the investigational nature of this study and signed a written informed consent in accordance with local institutional review board and federal guidelines. All patients had measurable or nonmeasurable but assessable disease.
The study consisted of 137 eligible patients divided into two cohorts: chemotherapy-naive patients (n = 101) and those with previous chemotherapy (n = 36); one patient died before initiation of treatment. Patients were treated with daily oral gefitinib at a dose of 500 mg/d until progression or prohibitive toxicity. Patient characteristics were median age 68 years (range, 34 to 88 years), male and female distribution of 45% and 51%, respectively, performance status of 0 versus 1/2 (89% v 11%), and stage IIIB versus IV (11% v 89%).
Histopathologic diagnosis and subtyping of BAC was performed on hematoxylin-eosin stained sections by consensus reading by two of the authors (W.A.F. and F.R.H.) using the WHO criteria.27 For each patient, serial 4-μm paraffin-embedded tissue sections containing representative malignant cells were sliced. Cell copy numbers were investigated by FISH using the LSI EGFR SpectrumOrange/CEP 7 SpectrumGreen probe and the PathVysion HER-2 DNA probe Kit (Vysis; Abbott Laboratories, Downers Grove, IL) according to protocols described elsewhere.23,32 Using the reference hematoxylin-eosin–stained slide of the adjacent section where the dominant tumor foci were identified, copy numbers of the EGFR and HER2 genes and chromosome 7 and 17 probes as controls were assessed and recorded independently in at least 100 nonoverlapping nuclei with intact morphology. The FISH analysis was performed independently by two observers (M.V.G., A.C.X.) blinded to the patients' clinical characteristics. According to the frequency of tumor cells with specific number of copies of the EGFR or HER2 genes and chromosome 7 and 17 centromeres, 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 high level of polysomy ( four copies of the gene in 40% of cells), or gene amplification, defined by presence of tight gene clusters, a gene/chromosome per cell ratio 2, or 15 copies of the genes per cell in 10% of analyzed cells. Figure 1 shows tumors categorized as FISH-negative (A, B) and FISH-positive (C, D) for the EGFR gene.
Statistical Methods
Outcome definitions. Response evaluation was performed by Response Evaluation Criteria in Solid Tumors.33 Only patients with measurable disease were included in the response evaluation, whereas the survival analysis included all the patients. Survival data were analyzed from the day the patient started gefitinib treatment until death. Overall survival was calculated as the time from registration to S0126 to death from any cause or last contact. Progression-free survival was calculated as the time from registration to S0126 to either progression of disease or death from any cause or last contact.
Analysis methods. Survival curves were estimated by the product-limit method34 and compared using the log-rank test.35 Cox proportional hazards regression was used to assess the influence of EGFR FISH and standard prognostic factors on survival outcomes and to estimate hazard ratios.36 Multivariate models were constructed using backward stepwise regression methods. All univariately significant covariates were included in the stepwise selection.
RESULTS
Protocol S0126 enrolled 145 patients, of whom eight patients were ineligible and one did not receive protocol treatment, leaving 136 eligible patients for analysis. Among those, 81 patients had tumor tissue available for EGFR gene analysis by FISH analysis (Table 1), and 56 patients had tissue available for HER2 gene analysis by FISH.
There were no statistical differences in sex, smoking status, performance status, and histology between the total S0126 cohort and the subcohort of 81 patients who underwent EGFR FISH analysis (Table 1). Similarly, no statistical difference in survival outcome between the total S0126 population and the EGFR FISH subcohort was observed (Fig 2A). Thus the EGFR FISH subcohort seemed representative of the total S0126 population.
The number of patients in each EGFR FISH category is shown in Table 2. Altogether, 26 (32%) of 81 patients were positive for EGFR FISH, and there were no significant differences between the EGFR FISH-positive and -negative groups in terms of sex, histology, smoking status, or performance status (Table 1). For response analysis, 55 of the 81 EGFR FISH patients had measurable disease. In the FISH-positive group, five (26%) of 19 patients had objective response and 12 patients (63%) had disease control (objective response or stable disease), whereas in the FISH negative group, four (11%) of 36 patients had objective response (P = .14) and 14 patients (39%) had disease control (P = .087; Table 2).
All 81 eligible patients with assessable tumor tissue for EGFR FISH analysis were included in the survival analysis. The progression-free survival and overall survival curves for patients with FISH-positive and -negative tumors are shown in Figures 2B and C, respectively. The median progression-free survival time for the FISH-negative patients was 4 months (95% CI, 2 to 5 months) versus 9 months (95% CI, 3 to 20 months) for the FISH-positive patients with an HR of 1.67 (P = .072; 95% CI, 0.96 to 2.91; Fig 2B). The median survival time for the FISH-negative patients was 8 months (95% CI, 6 to 15 months). Although the median survival for the FISH-positive patients has not yet been reached, it is approaching 18 months, with an HR of 2.02 (95% CI, 1.03 to 3.99; P = .042; Fig 2C).
For the HER2 analyses, 17 (30%) of 56 patients were HER2 FISH-positive. No statistically significant associations were observed between the gene copy numbers and tumor response and survival. Response assessment was achieved in 39 patients with HER2 analysis, and four (36%) of 11 patients in the HER2 FISH-positive group had response or stable disease, compared with 13 (46%) of 28 patients in the HER2 FISH-negative group (P > .05; Table 2). All the HER2-positive responders were also EGFR FISH-positive. The median survival in the HER2-positive and -negative group was 16 months (95% CI, 2 to 18 months) and 13 months (95% CI, 8 to 19 months), respectively (Fig 2D; log-rank P = .80).
The response rates and survival were also analyzed with respect to histologic subtypes. Among the eight patients with adenocarcinoma, no responders were observed, but two patients had stable disease (disease control rate, two of eight patients = 25%). However, among 27 patients with adenocarcinoma with BAC features, five patients (19%) achieved response and 12 patients (44%) achieved stable disease (DCR, 17 of 27 patients = 63%). In the BAC nonmucinous group, six (30%) of 20 patients had response and eight patients (40%) had stable disease (DCR, 14 of 20 patients = 70%), whereas in the BAC mucinous group, none of the 11 patients had response or stable disease (2 P = .0004).
A multivariate Cox regression model (Table 3) was used to assess the possibility that the effect of EGFR copy number by FISH on survival could be explained by other standard prognostic factors. EGFR copy number by FISH remained a significant prognostic factor for both overall (P = .0261) and progression-free survival (P = .034) after accounting for smoking status, sex, histology, and performance status.
DISCUSSION
This study demonstrates that increased EGFR gene copy number detected by FISH is associated with improved survival after gefitinib therapy in patients with advanced-stage BAC and adenocarcinoma with BAC features, a subset of NSCLC that may serve as a model for study of EGFR pathways because of its underlying biologic characteristics.31 In the current study, approximately one third of the patients had increased EGFR gene copy number, and these patients also had a trend for higher response rates and a longer time to progression after gefitinib therapy. Although RECIST response assessment is commonly not applicable in patients with BAC because the diffuse pulmonary infiltration cannot be measured, we believe that the significant difference in survival between patients with EGFR FISH-positive and -negative tumors strongly supports the hypothesis that increased gene copy number associates with increased efficacy of gefitinib. There is very little information in the literature regarding survival for patients with advanced BAC. In a study by Breathnach et al,37 28 patients with advanced BAC treated with chemotherapy or radiotherapy were analyzed. The median survival time from start of initial treatment was 11.7 months (95% CI, 8.7 to 16.7 months). In our previous SWOG trial (S9714) evaluating paclitaxel in advanced BAC, the median survival was 12 months.38 In the current study, the median survival time for the FISH-positive group has not yet been reached but is approaching 18 months, versus 8 months for the FISH-negative group. We have previously reported that increased EGFR gene copy number was associated with a poor prognosis in patients with surgically resected NSCLC.23 In this study, we verify that increased EGFR gene copy number is a positive predictive marker for improved survival under the influence of gefitinib therapy. These observations are similar to data reported for breast cancer patients with HER2 gene amplification, who have a poor prognosis but a greater likelihood of benefiting from trastuzumab (Herceptin; Genentech).6
Demographic and survival data were compared between the EGFR FISH-positive subpopulation and the total study population, and no differences were observed in terms of known prognostic factors such as sex, smoking status, performance status, or histology. In addition, there was no difference in overall survival between the total population and the FISH-tested cohort. We did not demonstrate any differences in treatment outcomes related to HER2 gene copy numbers However, the relatively small number of patients (n = 56) who had tumor tissue available for HER2 analysis might have contributed to the lack of statistical difference.
Although the focus of the current report is the predictive value of EGFR FISH for survival in patients with advanced stage BAC, these data will ultimately require correlation with other methods of assessing the biologic viability of EGFR and associated signal transduction pathways, such as EGFR protein levels, EGFR mutation analysis, and measurement of downstream markers like Akt and MAPK. For example, we have previously reported that MAPK levels, as assessed by immunohistochemistry (IHC), are predictive of sensitivity to gefitinib in BAC tumors.31
It has recently been indicated that from the completed TRIBUTE (Tarceva Responses in Conjunction with Paclitaxel and Carboplatin) trial that the EGFR mutant tumors per se represent a more indolent biology than the nonmutant NSCLC tumors.39 A question can be raised whether increased EGFR gene copy number is a surrogate marker for EGFR mutation, which has been beyond the scope of the present study and the limited size of this patient population. However, in our previous study for unselected NSCLC patients, we found that among eight patients with EGFR mutations who responded to therapy, seven patients were also FISH-positive, and among the six nonresponding patients with EGFR mutations, four presented with normal disomic FISH pattern. Although EGFR mutations in the Italian cohort were associated with a high response rate (53%), only 60% had disease control. Thus 40% (six of 15) of the patients with EGFR mutations had no clinical benefit from gefitinib treatment. In multivariate analysis, only FISH was an independent prognostic/predictive factor.26 Furthermore, if the FISH was a surrogate marker for EGFR mutations, the previous described result from the TRIBUTE trial would indicate a better prognosis for the EGFR FISH-positive patients independent of gefitinib therapy, which would be in contrast to our previous finding that the EGFR FISH-positive patients seem to have a more aggressive biology.23 However, a definitive assessment of EGFR gene copy number by FISH, EGFR mutations, and sensitivity to EGFR TKIs will require additional preclinical studies and much larger clinical data sets than that provided by the current study population.
Nevertheless, the clinical implications of our findings are considerable in regard to patient selection for therapy with EGFR TKIs. BAC is a disease entity that seems to be increasing in incidence.28,29 Although preliminary studies have demonstrated relatively high response rates for EGFR inhibitors in patients with BAC and its histologic subtypes,38,40,41 no studies have yet demonstrated survival benefit from these agents in this patient population. The current study demonstrated a significant survival benefit in EGFR FISH-positive patients, suggesting that increased EGFR gene copy numbers detected by FISH can be used as a marker to assess survival potential in patients to be treated with EGFR TKIs. FISH technology is applicable for clinical use, as analysis is performed on routine paraffin-embedded material. Future prospective larger clinical studies should be encouraged to validate the current findings, to compare the EGFR FISH analysis with other clinical and molecular markers (eg, IHC markers and EGFR and HER2 gene mutations), and to identify an optimal panel of markers for selection of patients to EGFR-TKI therapy.
Authors' Disclosures of Potential Conflicts of Interest
Although all authors completed the disclosure declaration, the following authors or their immediate family members 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 ASCOapos;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.
NOTES
Supported in part by the following Public Health Service Cooperative Agreement Grant No. awarded by the National Cancer Institute, United States Department of Health and Human Services: NCI CCSG P30-CA46934, Lung SPORE P50 CA058187, CA38926, and CA32102.
F.R.H. 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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Bailey R, Kris M, Wolf M, et al: Gefitinib (Iressa, ZD 1839) monotherapy for pretreated advanced non-small cell lung cancer in IDEAL 1 and 2: Tumor response is not clinically relevantly predictable from tumor EGFR membrane staining alone. Lung Cancer 41:S71, 2003 (abstr O-242)
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Lynch TJ, Bell DW, Sordella R, et al: Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 350:2129-2139, 2004
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Southwest Oncology Group Statistical Center, San Antonio, TX
Puget Sound Oncology Consortium, Seattle, WA
University of California, Davis Cancer Center, Sacramento, CA
ABSTRACT
PURPOSE: Bronchioloalveolar carcinoma (BAC) and adenocarcinomas with BAC features seem to be increasing in incidence, particularly in younger, never-smoking women. Epidermal growth factor receptor (EGFR) inhibitors demonstrated response rates of 20% to 30% in patients with advanced BAC subtypes, but selection methods for patient therapy are not established.
PATIENTS AND METHODS: EGFR and HER2 gene copy numbers were assessed by fluorescence in situ hybridization (FISH) in 81 patients treated with gefitinib 500 mg/d (Southwest Oncology Group protocol S0126) and were correlated to treatment outcome. Tumors were classified into two main strata: FISH-positive (high polysomy/gene amplification) and FISH-negative (disomy/low polysomy).
RESULTS: In 81 patients, the median survival time for EGFR/FISH-negative patients was 8 months and not yet reached for FISH-positive patients (but approaching 18 months; hazard ratio [HR] = 2.02; P = .042). Median progression-free survival time for EGFR/FISH-positive patients was 9 months versus 4 months for the FISH-negative patients (HR = 1.67; P = .072). In multivariate analysis, EGFR copy number by FISH remained a significant predictive factor for survival after accounting for smoking status, sex, histology, and performance status. Fifty-five patients were evaluated for response using Response Evaluation Criteria in Solid Tumors Group, and 12 of 19 EGFR/FISH-positive patients (63%) demonstrated disease control versus 14 (39%) of 36 patients in the FISH-negative group (P = .087). No association was found between HER2 gene copy number and response (n = 39 patients) or survival (n = 56 patients; P > .10).
CONCLUSION: Increased EGFR gene copy number detected by FISH is associated with improved survival after gefitinib therapy in patients with advanced BAC, suggesting FISH methodology can be used to assess survival potential in patients treated with EGFR tyrosine kinase inhibitors.
INTRODUCTION
Non–small-cell lung cancer (NSCLC) is the leading cause of cancer death in the world.1 Although platinum-based chemotherapy results in palliation of symptoms and increased survival in patients with advanced-stage disease, benefits are modest and new approaches are clearly needed.2,3 Recently, improved understanding of molecular biology has led to development of a wide variety of inhibitors of target proteins involved in proliferation, apoptosis, and angiogenesis. Clinical application of agents such as imatinib in chronic myeloid leukemia4 and gastrointestinal stromal tumors (GIST)5 and trastuzumab in breast cancers6 has resulted in dramatic and often sustained response in tumors expressing the target proteins.
The epidermal growth factor receptor (EGFR), a member of the HER or Erb-B family of type I receptor tyrosine kinases, is implicated in the development and progression of cancer7-10 and is expressed in many human epithelial malignancies, including NSCLC.8,10 Several small molecules have been synthesized to inhibit the tyrosine kinase domain of EGFR,11,12 including gefitinib (Iressa; AstraZeneca, Macclesfield, United Kingdom) and erlotinib (Tarceva; Roche, Basche, Switzerland/OSI Pharmaceuticals Inc, Melville, NY). Both are orally active, selective EGFR tyrosine-kinase inhibitors (EGFR-TKI) that produce objective response rates of 9% to 26% in previously untreated or previously treated patients with advanced-stage NSCLC.13-15 Recently a phase III trial (BR21) comparing erlotinib with placebo as a second- or third-line therapy reported a survival benefit for the EGFR inhibitor (hazard ratio [HR] = 0.73).16 Importantly, this survival benefit was not confined to objective responders, nor to a single sex or histology.
These data emphasize the need to identify predictive factors of benefit from EGFR–TKI that could be used to select patients most likely to benefit. Although it has been reported that EGFR protein expression assessed by immunohistochemistry (IHC) is not correlated with gefitinib response, few studies have directly addressed this question.17-19 Recent reports have instead turned attention to specific missense mutations and deletions in the tyrosine kinase domain of the EGFR gene,20,21 which are highly associated with gefitinib response. However, the low frequency of these mutations (four of 81) in United States lung cancer patients who are current or former smokers,22 and the results of the BR21 study described above, suggest that a substantial subset of patients without these mutations also derive a survival benefit. Furthermore, although these activating mutations identify patients with high response rates, they cannot account for the high stable disease rates, reported to occur in approximately 30% of NSCLC patients treated with gefitinib.13,14 Furthermore, there are no published data available describing the relationship between EGFR mutations and survival, and no larger clinical cohort studies have so far been reported on the association between EGFR mutations and treatment response and survival.
Increased EGFR gene copy number detected by fluorescence in situ hybridization (FISH) is associated with a poor prognosis in NSCLC,23 similar to the assessment of HER2 by FISH in breast cancer patients.24 Moreover, among patients with breast cancer, FISH is reported to be a more reliable predictor of treatment benefit with the HER2 inhibitor trastuzumab (Herceptin; Genentech, San Francisco, CA) than is protein expression by IHC, especially in the patients with intermediate protein expression (2+).25 We have previously evaluated EGFR gene copy number in a cohort of 102 NSCLC patients treated with gefitinib and found high copy number or gene amplification in approximately 30% and a significant correlation between gene EGFR gene status and outcome.26
Bronchioalveolar carcinoma (BAC) subtypes of NSCLC are characterized by unique pathologic, radiographic, and clinical features27 and seem to be increasing in incidence, particularly in younger nonsmoking women.28,29 BAC and adenocarcinoma with BAC features have been reported to be particularly sensitive to EGFR tyrosine kinase inhibitors, with response rates of 25% to 30%30 and prolonged survival in a subset of patients. We have previously reported the efficacy of gefitinib in a large cohort of advanced-stage BAC patients treated on a prospective clinical trial of the Southwest Oncology Group (SWOG; S0126).31 Because archival tumor tissue was collected from the great majority of patients enrolled, the S0126 trial represents a unique pathologic resource for study of EGFR pathways. On the basis of our prior experience with NSCLC patients treated with gefitinib, we hypothesized that increased EGFR and/or HER2 gene copy numbers detected by FISH would be associated with increased efficacy of gefitinib in the subset of patients with NSCLC who have BAC or adenocarcinoma with BAC features. Here we report the results of this analysis in patient tumor tissue from the S0126 study, correlated with clinical outcome.
PATIENTS AND METHODS
All patients enrolled were required to have histologically proven, stage IIIB (by pleural effusion) or IV BAC or adenocarcinoma with BAC features. Pathologic eligibility was based on an institutional definition of BAC, although a central review was subsequently carried out using the WHO Classification.27 Histopathologic subtypes in this report are based on this central pathology review. Cytologic specimens were not accepted for the BAC diagnosis, and patients with only cytologic diagnosis were not eligible for S0126.
Patients were required to have an SWOG performance status of 0 to 2. Prestudy evaluation included history and physical examination; complete blood cell count with differential and platelets, serum chemistries of alkaline phosphatase, AST or ALT, lactate dehydrogenase and albumin; chest radiograph; and computed tomography of chest, liver, and adrenal glands. Bone scan and/or brain computed tomography or magnetic resonance imaging were required only if clinically indicated based on symptoms and physician judgment. Patients with a history of brain metastases were ineligible for the present study. Pregnant or nursing women were ineligible, and women and men of reproductive potential were unable to participate unless they agreed to use an effective contraceptive method. Eligible patients had no other prior malignancy except for adequately treated basal cell or squamous cell skin cancer, in situ cervical cancer, adequately treated stage I or II cancer from which the patient was in complete remission, or any other cancer from which the patient was disease-free for at least 5 years.
All patients were informed of the investigational nature of this study and signed a written informed consent in accordance with local institutional review board and federal guidelines. All patients had measurable or nonmeasurable but assessable disease.
The study consisted of 137 eligible patients divided into two cohorts: chemotherapy-naive patients (n = 101) and those with previous chemotherapy (n = 36); one patient died before initiation of treatment. Patients were treated with daily oral gefitinib at a dose of 500 mg/d until progression or prohibitive toxicity. Patient characteristics were median age 68 years (range, 34 to 88 years), male and female distribution of 45% and 51%, respectively, performance status of 0 versus 1/2 (89% v 11%), and stage IIIB versus IV (11% v 89%).
Histopathologic diagnosis and subtyping of BAC was performed on hematoxylin-eosin stained sections by consensus reading by two of the authors (W.A.F. and F.R.H.) using the WHO criteria.27 For each patient, serial 4-μm paraffin-embedded tissue sections containing representative malignant cells were sliced. Cell copy numbers were investigated by FISH using the LSI EGFR SpectrumOrange/CEP 7 SpectrumGreen probe and the PathVysion HER-2 DNA probe Kit (Vysis; Abbott Laboratories, Downers Grove, IL) according to protocols described elsewhere.23,32 Using the reference hematoxylin-eosin–stained slide of the adjacent section where the dominant tumor foci were identified, copy numbers of the EGFR and HER2 genes and chromosome 7 and 17 probes as controls were assessed and recorded independently in at least 100 nonoverlapping nuclei with intact morphology. The FISH analysis was performed independently by two observers (M.V.G., A.C.X.) blinded to the patients' clinical characteristics. According to the frequency of tumor cells with specific number of copies of the EGFR or HER2 genes and chromosome 7 and 17 centromeres, 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 high level of polysomy ( four copies of the gene in 40% of cells), or gene amplification, defined by presence of tight gene clusters, a gene/chromosome per cell ratio 2, or 15 copies of the genes per cell in 10% of analyzed cells. Figure 1 shows tumors categorized as FISH-negative (A, B) and FISH-positive (C, D) for the EGFR gene.
Statistical Methods
Outcome definitions. Response evaluation was performed by Response Evaluation Criteria in Solid Tumors.33 Only patients with measurable disease were included in the response evaluation, whereas the survival analysis included all the patients. Survival data were analyzed from the day the patient started gefitinib treatment until death. Overall survival was calculated as the time from registration to S0126 to death from any cause or last contact. Progression-free survival was calculated as the time from registration to S0126 to either progression of disease or death from any cause or last contact.
Analysis methods. Survival curves were estimated by the product-limit method34 and compared using the log-rank test.35 Cox proportional hazards regression was used to assess the influence of EGFR FISH and standard prognostic factors on survival outcomes and to estimate hazard ratios.36 Multivariate models were constructed using backward stepwise regression methods. All univariately significant covariates were included in the stepwise selection.
RESULTS
Protocol S0126 enrolled 145 patients, of whom eight patients were ineligible and one did not receive protocol treatment, leaving 136 eligible patients for analysis. Among those, 81 patients had tumor tissue available for EGFR gene analysis by FISH analysis (Table 1), and 56 patients had tissue available for HER2 gene analysis by FISH.
There were no statistical differences in sex, smoking status, performance status, and histology between the total S0126 cohort and the subcohort of 81 patients who underwent EGFR FISH analysis (Table 1). Similarly, no statistical difference in survival outcome between the total S0126 population and the EGFR FISH subcohort was observed (Fig 2A). Thus the EGFR FISH subcohort seemed representative of the total S0126 population.
The number of patients in each EGFR FISH category is shown in Table 2. Altogether, 26 (32%) of 81 patients were positive for EGFR FISH, and there were no significant differences between the EGFR FISH-positive and -negative groups in terms of sex, histology, smoking status, or performance status (Table 1). For response analysis, 55 of the 81 EGFR FISH patients had measurable disease. In the FISH-positive group, five (26%) of 19 patients had objective response and 12 patients (63%) had disease control (objective response or stable disease), whereas in the FISH negative group, four (11%) of 36 patients had objective response (P = .14) and 14 patients (39%) had disease control (P = .087; Table 2).
All 81 eligible patients with assessable tumor tissue for EGFR FISH analysis were included in the survival analysis. The progression-free survival and overall survival curves for patients with FISH-positive and -negative tumors are shown in Figures 2B and C, respectively. The median progression-free survival time for the FISH-negative patients was 4 months (95% CI, 2 to 5 months) versus 9 months (95% CI, 3 to 20 months) for the FISH-positive patients with an HR of 1.67 (P = .072; 95% CI, 0.96 to 2.91; Fig 2B). The median survival time for the FISH-negative patients was 8 months (95% CI, 6 to 15 months). Although the median survival for the FISH-positive patients has not yet been reached, it is approaching 18 months, with an HR of 2.02 (95% CI, 1.03 to 3.99; P = .042; Fig 2C).
For the HER2 analyses, 17 (30%) of 56 patients were HER2 FISH-positive. No statistically significant associations were observed between the gene copy numbers and tumor response and survival. Response assessment was achieved in 39 patients with HER2 analysis, and four (36%) of 11 patients in the HER2 FISH-positive group had response or stable disease, compared with 13 (46%) of 28 patients in the HER2 FISH-negative group (P > .05; Table 2). All the HER2-positive responders were also EGFR FISH-positive. The median survival in the HER2-positive and -negative group was 16 months (95% CI, 2 to 18 months) and 13 months (95% CI, 8 to 19 months), respectively (Fig 2D; log-rank P = .80).
The response rates and survival were also analyzed with respect to histologic subtypes. Among the eight patients with adenocarcinoma, no responders were observed, but two patients had stable disease (disease control rate, two of eight patients = 25%). However, among 27 patients with adenocarcinoma with BAC features, five patients (19%) achieved response and 12 patients (44%) achieved stable disease (DCR, 17 of 27 patients = 63%). In the BAC nonmucinous group, six (30%) of 20 patients had response and eight patients (40%) had stable disease (DCR, 14 of 20 patients = 70%), whereas in the BAC mucinous group, none of the 11 patients had response or stable disease (2 P = .0004).
A multivariate Cox regression model (Table 3) was used to assess the possibility that the effect of EGFR copy number by FISH on survival could be explained by other standard prognostic factors. EGFR copy number by FISH remained a significant prognostic factor for both overall (P = .0261) and progression-free survival (P = .034) after accounting for smoking status, sex, histology, and performance status.
DISCUSSION
This study demonstrates that increased EGFR gene copy number detected by FISH is associated with improved survival after gefitinib therapy in patients with advanced-stage BAC and adenocarcinoma with BAC features, a subset of NSCLC that may serve as a model for study of EGFR pathways because of its underlying biologic characteristics.31 In the current study, approximately one third of the patients had increased EGFR gene copy number, and these patients also had a trend for higher response rates and a longer time to progression after gefitinib therapy. Although RECIST response assessment is commonly not applicable in patients with BAC because the diffuse pulmonary infiltration cannot be measured, we believe that the significant difference in survival between patients with EGFR FISH-positive and -negative tumors strongly supports the hypothesis that increased gene copy number associates with increased efficacy of gefitinib. There is very little information in the literature regarding survival for patients with advanced BAC. In a study by Breathnach et al,37 28 patients with advanced BAC treated with chemotherapy or radiotherapy were analyzed. The median survival time from start of initial treatment was 11.7 months (95% CI, 8.7 to 16.7 months). In our previous SWOG trial (S9714) evaluating paclitaxel in advanced BAC, the median survival was 12 months.38 In the current study, the median survival time for the FISH-positive group has not yet been reached but is approaching 18 months, versus 8 months for the FISH-negative group. We have previously reported that increased EGFR gene copy number was associated with a poor prognosis in patients with surgically resected NSCLC.23 In this study, we verify that increased EGFR gene copy number is a positive predictive marker for improved survival under the influence of gefitinib therapy. These observations are similar to data reported for breast cancer patients with HER2 gene amplification, who have a poor prognosis but a greater likelihood of benefiting from trastuzumab (Herceptin; Genentech).6
Demographic and survival data were compared between the EGFR FISH-positive subpopulation and the total study population, and no differences were observed in terms of known prognostic factors such as sex, smoking status, performance status, or histology. In addition, there was no difference in overall survival between the total population and the FISH-tested cohort. We did not demonstrate any differences in treatment outcomes related to HER2 gene copy numbers However, the relatively small number of patients (n = 56) who had tumor tissue available for HER2 analysis might have contributed to the lack of statistical difference.
Although the focus of the current report is the predictive value of EGFR FISH for survival in patients with advanced stage BAC, these data will ultimately require correlation with other methods of assessing the biologic viability of EGFR and associated signal transduction pathways, such as EGFR protein levels, EGFR mutation analysis, and measurement of downstream markers like Akt and MAPK. For example, we have previously reported that MAPK levels, as assessed by immunohistochemistry (IHC), are predictive of sensitivity to gefitinib in BAC tumors.31
It has recently been indicated that from the completed TRIBUTE (Tarceva Responses in Conjunction with Paclitaxel and Carboplatin) trial that the EGFR mutant tumors per se represent a more indolent biology than the nonmutant NSCLC tumors.39 A question can be raised whether increased EGFR gene copy number is a surrogate marker for EGFR mutation, which has been beyond the scope of the present study and the limited size of this patient population. However, in our previous study for unselected NSCLC patients, we found that among eight patients with EGFR mutations who responded to therapy, seven patients were also FISH-positive, and among the six nonresponding patients with EGFR mutations, four presented with normal disomic FISH pattern. Although EGFR mutations in the Italian cohort were associated with a high response rate (53%), only 60% had disease control. Thus 40% (six of 15) of the patients with EGFR mutations had no clinical benefit from gefitinib treatment. In multivariate analysis, only FISH was an independent prognostic/predictive factor.26 Furthermore, if the FISH was a surrogate marker for EGFR mutations, the previous described result from the TRIBUTE trial would indicate a better prognosis for the EGFR FISH-positive patients independent of gefitinib therapy, which would be in contrast to our previous finding that the EGFR FISH-positive patients seem to have a more aggressive biology.23 However, a definitive assessment of EGFR gene copy number by FISH, EGFR mutations, and sensitivity to EGFR TKIs will require additional preclinical studies and much larger clinical data sets than that provided by the current study population.
Nevertheless, the clinical implications of our findings are considerable in regard to patient selection for therapy with EGFR TKIs. BAC is a disease entity that seems to be increasing in incidence.28,29 Although preliminary studies have demonstrated relatively high response rates for EGFR inhibitors in patients with BAC and its histologic subtypes,38,40,41 no studies have yet demonstrated survival benefit from these agents in this patient population. The current study demonstrated a significant survival benefit in EGFR FISH-positive patients, suggesting that increased EGFR gene copy numbers detected by FISH can be used as a marker to assess survival potential in patients to be treated with EGFR TKIs. FISH technology is applicable for clinical use, as analysis is performed on routine paraffin-embedded material. Future prospective larger clinical studies should be encouraged to validate the current findings, to compare the EGFR FISH analysis with other clinical and molecular markers (eg, IHC markers and EGFR and HER2 gene mutations), and to identify an optimal panel of markers for selection of patients to EGFR-TKI therapy.
Authors' Disclosures of Potential Conflicts of Interest
Although all authors completed the disclosure declaration, the following authors or their immediate family members 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 ASCOapos;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.
NOTES
Supported in part by the following Public Health Service Cooperative Agreement Grant No. awarded by the National Cancer Institute, United States Department of Health and Human Services: NCI CCSG P30-CA46934, Lung SPORE P50 CA058187, CA38926, and CA32102.
F.R.H. 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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