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Department of Hematology, Oncology and Respiratory Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan
Department of Hematology, Oncology and Respiratory Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan
Department of Hematology, Oncology and Respiratory Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan
Department of Hematology, Oncology and Respiratory Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan
Department of Hematology, Oncology and Respiratory Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan
Address for correspondence: Katsuyuki Hotta, MD, PhD, Department of Hematology, Oncology and Respiratory Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1, Shikatacho, Okayama 700-8558, Japan
Affiliations
Department of Hematology, Oncology and Respiratory Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan
Department of Hematology, Oncology and Respiratory Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan
It has not been fully evaluated whether both HER2 gene copy number and HER2 protein expression are related to the outcome of chemoradiotherapy in patients with locally advanced non-small cell lung cancer (LA-NSCLC). The aim of this study was to evaluate their relationships.
Methods
HER2 gene copy number determined by fluorescence in situ hybridization (FISH) and HER2 protein expression determined by immunohistochemistry (IHC) were assessed in 68 patients with LA-NSCLC enrolled in our previous phase II trials of concurrent cisplatin-based chemoradiotherapy, and a multivariate analysis was conducted for response and survival.
Results
HER2-IHC-positive tumors were detected in 23 patients (34%), and the median ratio of HER2 to chromosome 17 copy number was 0.93 (range, 0.55–2.00). The HER2-FISH results were marginally correlated with the IHC results (p = 0.0715). When the median ratio in the FISH analysis was used as a cut-off level for its positivity, there was no association between either HER2-FISH or IHC status and objective response to chemoradiotherapy. Contrary, a multivariate analysis revealed HER2-FISH result but not IHC result was an independent poor prognostic factor for both overall survival and progression-free survival (hazard ratio = 2.568, 95% confidence interval [CI] = 1.117–5.903, p = 0.0264 and hazard ratio = 2.283, 95% CI = 1.005–5.189, p = 0.0487, respectively).
Conclusions
Patients with HER2 FISH-positive LA-NSCLC had a poorer outcome even when treated with cisplatin-based chemoradiotherapy, despite the strong need for validation assessment of these observations. Development of more effective treatment for these high-risk patients is needed to improve their poor prognosis.
Locally advanced non-small cell lung cancer (LA-NSCLC) is characterized by widespread primary tumor and/or extensive nodal involvement, and improving the clinical outcome of LA-NSCLC is one of the major challenges for medical oncologists. Based on randomized controlled trials,
Phase III study of concurrent versus sequential thoracic radiotherapy in combination with mitomycin, vindesine, and cisplatin in unresectable stage III non-small-cell lung cancer.
Long-term benefit is observed in a phase III comparaison of sequential vs concurrent chemoradiation for patients with unresectable stage III NSCLC: RTOG 94-10.
Proc Am Soc Clin Oncol.2003; 22(abstr 2499).: 621a
cisplatin-based chemotherapy and concurrent thoracic irradiation is now considered the standard treatment for LA-NSCLC, and it has yielded a response rate of 80%. Nevertheless, despite the initial response, the majority of patients experience recurrence, and the 5-year survival rate is still around 20%. As a first step in improving the unsatisfactory treatment outcome it would seem reasonable to select LA-NSCLC patients who would benefit least from the standard treatment consisting of cisplatin-based chemotherapy and concurrent thoracic irradiation and then develop novel treatment strategies for this high-risk patient population.
HER2/neu is tyrosine kinase receptor having a molecular weight of 185,000 and is a member of the ErbB protein family. HER2/neu signaling is known to be associated with the proliferation and survival of cancer cells, and it is overexpressed in a variety of tumors, including breast cancer and NSCLC.
HER2 overexpression has been reported to be associated with resistance to both radiation and chemotherapeutic agents, including cisplatin and 5-fluorouracil, in preclinical studies.
Monoclonal antibody to HER-2/neureceptor modulates repair of radiation-induced DNA damage and enhances radiosensitivity of human breast cancer cells overexpressing this oncogene.
Indeed, in several clinical studies of locally advanced cancers, including breast, esophageal, and head and neck cancers, combined modality treatment consisting of systemic chemotherapy and irradiation has been found to be less effective in patients whose tumors overexpress HER2 than in patients whose tumors do not.
Low HER2/neu gene expression is associated with pathological response to concurrent paclitaxel and radiation therapy in locally advanced breast cancer.
Quantitative c-erbB-2 but not c-erbB-1 mRNA expression is a promising marker to predict minor histopathologic response to neoadjuvant radiochemotherapy in oesophageal cancer.
Patient survival is undoubtedly the principal goal of chemoradiotherapy, but the role of HER2 protein expression determined by immunohistochemistry (IHC) in the survival of NSCLC patients has long been a matter of controversy. Two recent meta-analyses have shown a significant association between HER2 protein overexpression and poor outcome in NSCLC patients.
Nevertheless, it seems problematic that the analyses included very heterogeneous studies, with various disease stages, various treatments, and different methodologies for assessment of HER2 status in each study. In addition, it has not yet been determined whether the association holds for LA-NSCLC, because the meta-analyses rarely included relevant studies of LA-NSCLC.
Regarding HER2 gene copy number, fluorescence in situ hybridization (FISH) is another commonly used method of determining the HER2 status of tumors, and its clinical usefulness for predicting survival has been extensively evaluated, mainly in breast cancer.
Assessment of methods for tissue-based detection of the HER-2/neu alteration in human breast cancer: a direct comparison of fluorescence in situ hybridization and immunohistochemistry.
Nevertheless, few studies have formally assessed the association in an LA-NSCLC setting, and the aforementioned meta-analyses included hardly any studies that assessed HER2 gene amplification status and patient outcome.
Thus, in this study, we investigated the association between both HER2-FISH status and high HER2 protein levels and the clinical outcome of LA-NSCLC patients by using the long-term follow-up data sets of two prospective phase II studies that we conducted to evaluate the efficacy of cisplatin-based concurrent chemoradiotherapy against LA-NSCLC.
MATERIALS AND METHODS
Patients and Treatment
This study was a laboratory companion study to the two phase II concurrent chemoradiotherapy studies for locally advanced unresectable stage IIIA and IIIB NSCLC conducted by the Okayama Lung Cancer Study Group (OLCSG) between January 1994 and December 1999. The details of these prospective studies have already been reported.
A phase II study of cisplatin and 5-fluorouracil with concurrent hyperfractionated thoracic radiation for locally advanced non-small-cell lung cancer: a preliminary report from the Okayama Lung Cancer Study Group.
In brief, both studies had similar eligibility criteria: pathologically confirmed NSCLC, previously untreated disease, measurable lesions, age ≤75 years, and IIIA or IIIB clinical stage. Institutional review boards approved the study protocols, and the patients provided written informed consent. Mediastinoscopy was not included in the staging work-up examination. Treatment in the first study (OLCSG 9404) consisted of three cycles of 5-fluorouracil (500 mg/m2, days 1–5) and cisplatin (20 mg/m2, days 1–5) every 4 weeks, and concurrent hyperfractionated radiotherapy (1.25 Gy twice daily, total dose: 62.5–70 Gy). In the second study (OLCSG 9707), treatment comprised a combination of docetaxel 40 mg/m2 and cisplatin 40 mg/m2 given on days 1, 8, 29, and 36 with concurrent radiotherapy (2 Gy daily, total 60 Gy). Patients from whom appropriate tumor specimens for the molecular analyses with providing informed consents had been obtained were included in this study.
FISH and Immunohistochemistry
Sections from paraffin-embedded tissue blocks containing representative malignant cells prepared from tissue obtained at the time of diagnosis were used for the FISH and IHC analyses. The level of HER2 protein expression was determined by IHC using the Dako Herceptest kit (DAKO, Copenhagen, Denmark).
Gefitinib in pretreated non-small-cell lung cancer (NSCLC): analysis of efficacy and correlation with HER2 and epidermal growth factor receptor expression in locally advanced or metastatic NSCLC.
Staining was performed according to the protocol described in the manufacturer's guide. Membrane staining intensity and patterns were evaluated by using the 0 to 3+ scale according to the HercepTest kit scoring guidelines. Scores of 0 or 1+ were considered negative for HER2 overexpression, a score of 2 was considered weakly positive, and a score of 3+ was considered strongly positive. To qualify for 2+ and 3+ scoring, complete membrane staining of more than 10% of tumor cells had to be observed. The results were reported as negative (score 0 to 1+) or positive (score 2+ to 3+).
Dual-target, dualcolor 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.
The reference slide (stained with hematoxylin and eosin) was the section adjacent to the section on which the dominant tumor focus had been identified, and the 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 while constantly refocusing the microscope to detect signals located on different focal planes. The results were reported as negative or positive with the median ratio of HER2 to chromosome 17 copy numbers as the cut-off value.
All molecular analyses were performed independently by 2 observers (S.K. and M.T.) blinded to the patients’ clinical information, and discrepant scores were resolved by consensus.
Radiologic Evaluation and Statistical Analysis
Responses were assessed by using the World Health Organization criteria for OLCSG 9404 and by using Eastern Cooperative Oncology Group criteria for OLCSG 9707. The response to treatment was determined for each patient by extramural reviewers. The Mann-Whitney U test and χ2 or Fisher exact test were used for comparisons among groups. The relationship between HER2 status and response was assessed by logistic regression analysis with adjustment for the impact of the following potential confounders: HER2-FISH status, HER2-IHC status, nodal metastasis (N3 versus others), age (<70 versus ≥70), histology (squamous cell carcinoma versus others), chemotherapy regimens (cisplatin and docetaxel versus cisplatin and 5-fluorouracil), and pretreatment performance status (0 versus 1 or worse). Progression-free survival time was defined as the interval between the start of therapy and the day of documented disease progression or any deaths. Overall survival time was defined as the interval between the start of chemoradiotherapy and the time of death from any cause or the most recent date the patient was known to be alive. The survival curves were constructed by the Kaplan-Meier product-limit method. Differences between the curves were evaluated by the log-rank test. Proportional hazard regression models were used to evaluate the above confounding factors. All p-values were two-sided, and significance was set at a p value of ≤0.05.
RESULTS
Patient Characteristics, Response, and Survival
Because tumor samples were available for 40 (80%) of the 50 patients enrolled for the cisplatin and 5-fluorouracil regimen and for 28 (67%) of the 42 patients enrolled for the cisplatin and docetaxel regimen, a total of 68 patients were included in this study (Table 1). The majority of the patients were male (87%) and had stage IIIB disease (76%).
Combination chemotherapy with concurrent thoracic irradiation (cisplatin and 5-flu-orouracil regimen in the Okayama Lung Cancer Study Group Protocol 9404) and cisplatin and docetaxel regimen in the Okayama Lung Cancer Study Group Protocol 9707).
a Eastern Cooperative Oncology Group performance status.
b Combination chemotherapy with concurrent thoracic irradiation (cisplatin and 5-flu-orouracil regimen in the Okayama Lung Cancer Study Group Protocol 9404) and cisplatin and docetaxel regimen in the Okayama Lung Cancer Study Group Protocol 9707).
An objective response to therapy was observed in 45 (66.2%) patients. The 5-year overall survival rate, median survival time, and median progression-free survival time were 33.8%, 24.0 months, and 12.2 months, respectively, and the median follow-up time in the surviving patients was 87.8 months.
Correlation Between the IHC Results and the FISH Results
According to the HercepTest kit scoring guidelines, 45 (66%), 16 (24%), and 7 (10%) of the tumors were scored 0 to 1+, 2+, and 3+, respectively. The results of the FISH analysis yielded a median ratio of HER2 to chromosome 17 copy numbers of 0.93 (range, 0.55–2.00) that was used as a cut-off value for the FISH positivity in this study.
We investigated the relationship between HER2 copy number and HER2 protein expression, because it is still the subject of controversy in NSCLC.
HER-2/neu protein expression and gene alteration in stage I–IIIA non-small-cell lung cancer: a study of 140 cases using a combination of high throughput tissue microarray, immunohistochemistry, and fluorescent in situ hybridization.
The ratios of HER2 to chromosome 17 copy numbers were higher in the patients with high HER2-IHC scores (2+ to 3+) than in those with low scores (0 to 1+) (median ratio: 0.89 and 0.97, respectively; Mann-Whitney U test; p = 0.0715; Figure 1).
FIGURE 1Association between the HER2 immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) assay results. The FISH results were expressed as the ratio of HER2 to chromosome 17 copy number in each patient. The IHC results were divided into two categories according to the HercepTest kit scoring guidelines: negative (score 0 to 1+) and positive (score 2+ to 3+).
Neither HER2-FISH nor IHC statuses were associated with objective response to the chemoradiotherapy with the objective response rates of 68.2% versus 54.5% in FISH-positive and negative tumors, and 65.2% and 66.7% in HER2-protein positive (score 2+ to 3+) and negative (score 0 to 1+) tumors, respectively. Regarding overall survival, patients with HER2-FISH-positive tumors had a poorer outcome than those with FISH-negative tumors, despite the absence of a statistically significant difference (log-rank test; p = 0.1352), and they had a 5-year overall survival rate of 22.7% and 40.9%, respectively, and a median survival time of 15.5 months and 29.7 months, respectively (Figure 2A). The multivariate analysis revealed that HER2-FISH status was an independent prognostic factor for poor overall survival (hazard ratio [HR] = 2.568, 95% confidence interval [CI] = 1.117–5.903, p = 0.0264; Table 2). In addition, progression-free survival was different in patients with and without HER2-FISH-positive tumors in the univariate analysis (me-dian progression-free survival time of 10.0 months and 14.1 month, respectively, log-rank test; p = 0.0871; Figure 2B) and in the multivariate analysis (HR = 2.283, 95% CI = 1.005–5.189, p = 0.0487; Table 2). Nevertheless, there was no significant association between HER2-IHC status and either overall survival or progression-free survival (Table 2 and Figures 3A,B).
FIGURE 2Survival curves stratified by HER2-fluorescence in situ hybridization (FISH) results. Bold and nonbold lines represent patients with positive and negative FISH results, respectively. A, Overall survival Curves (5-year overall survival rate and median survival time of patients with HER2-FISH positive and negative tumors of 22.7% and 40.9%, respectively, and 15.5 months and 29.7 months, respectively; log-rank test; p = 0.1352). B, Progression-Free survival Curves (median progression-free survival times of 10.0 months and 14.1 month in those with the positive and negative HER2-FISH results, respectively, log-rank test; p = 0.0871).
Combination chemotherapy with concurrent thoracic irradiation (cisplatin and 5-fluorouracil regimen in the Okayama Lung Cancer Study Group Protocol 9404) and cisplatin and docetaxel regimen in the
a Eastern Cooperative Oncology Group performance status.
b Combination chemotherapy with concurrent thoracic irradiation (cisplatin and 5-fluorouracil regimen in the Okayama Lung Cancer Study Group Protocol 9404) and cisplatin and docetaxel regimen in the
FIGURE 3Survival curves stratified by HER2-FISH results. Bold and nonbold lines represent patients with HER2-protein positive (score 2+ to 3+) and negative (score 0 to 1+) tumors, respectively. A, Overall survival Curves (5-year overall survival rate and median survival time of patients with HER2-FISH positive and negative tumors of 39.1% and 31.1%, respectively, and 28.3 months and 23.6 months, respectively; log-rank test; p = 0.4986). B, Progression-Free survival Curves (median progression-free survival times of 14.1 month and 10.6 months in those with the positive and negative HER2-FISH results, respectively, log-rank test; p = 0.8843).
This is the first study to show a significant correlation between HER2-FISH status and both progression-free survival and overall survival of LA-NSCLC patients after the start of cisplatin-based concurrent chemoradiotherapy. The results of this study showed that HER2-FISH-positive tumors are very aggressive even when treated with the current standard chemoradiotherapy (HR for overall survival and median progression-free survival of 2.568 [95% CI = 1.117–5.903] and 2.283 [95% CI = 1.005–5.189], respectively). In addition, since LA-NSCLC patients constitute a heterogeneous population, it seems important to select these patients stratified by HER2-FISH status, and investigate and develop novel treatment strategies especially for those expected to have a poor outcome even if treated by standard chemoradiotherapy.
No previous studies have evaluated the role of HER2-FISH in the survival of LA-NSCLC patients after chemoradiotherapy, although HER2-FISH is associated with treatment outcome neither in patients with more advanced NSCLC receiving chemotherapy alone,
The differences in results between previous studies and our own may be attributable to the selection of patients with different disease stages, differences in treatments and differences in definition of HER2-FISH-positive.
We used the frequently used definition of HER2-positive in the HercepTest kit scoring guidelines and failed to find any association between HER2-IHC status and outcome. Previous studies reported similar results in LA-NSCLC patients treated with combination therapy consisting of thoracic irradiation and cisplatin-based chemotherapy.
Analysis of neuroendocrine markers, HER2 and CEA before and after chemotherapy in patients with stage IIIA non-small cell lung cancer: a Cancer and Leukemia Group B study.
Use of neuroendocrine markers, p53, and HER2 to predict response to chemotherapy in patients with stage III non-small cell lung cancer: a Cancer and Leukemia Group B study.
Some studies in breast cancer have demonstrated that IHC does not consistently discriminate patients likely to have a poor outcome, whereas FISH provides superior prognostic information for segregating high-risk from low-risk breast cancers.
Assessment of methods for tissue-based detection of the HER-2/neu alteration in human breast cancer: a direct comparison of fluorescence in situ hybridization and immunohistochemistry.
These results indicate that the FISH results are more useful for predicting patient outcome than IHC results. Nevertheless, our data need to be interpreted with caution, because the definition of HER2-FISH positivity is still undetermined. Lung cancer is likely to express lower levels of HER2 protein than breast cancer.
Because there is controversy as to whether the criteria for HER2 positivity in breast cancer can be applied to lung cancer, a new definition should be established for NSCLC. We conducted the FISH analysis early after the completion of the original phase II trials, according to the criteria by Hirsch et al
Increased epidermal growth factor receptor gene copy number detected by fluorescence in situ hybridization associates with increased sensitivity to gefitinib in patients with bronchioloalveolar carcinoma subtypes: a Southwest Oncology Group Study.
Thus, we need to further clarify which of the criteria would be better in lung cancer. A validation analysis in a large cohort of lung cancer patients is also strongly recommended to determine whether our definition is appropriate for predicting outcome in LA-NSCLC.
In our study, there was no association between HER2 status and objective response to the combination therapy. This discordance in results for response and survival may simply be attributable to the small sample size, inconsistent response criteria, and definition for HER2-positive results. As another explanation, disease stabilization as well as objective response would be an indicator of a benefit from this combined treatment, as some investigators reported the role of stable disease achieved by chemotherapy in survival advantage.
Continued gefitinib treatment after disease stabilisation prolongs survival of patients with non-small-cell lung cancer: Okayama Lung Cancer Study Group Experience.
In addition, the better survival after chemoradiotherapy in the patients with HER2-FISH-positive results may essentially be attributable to a poor natural history, irrespective of therapeutic efficacy. Several studies in breast cancer have shown that the HER2 status would represent a distinct tumor group with a more aggressive natural history and a worse outcome.
Therefore, determining whether NSCLC with HER2-FISH-positive status has a poorer prognosis regardless of therapy awaits further carefully designed population studies.
As a limitation, we obtained in this study the limited information about the patient demographics from the case report forms of the original phase II trials. Therefore, our results from the multivariate analyses should be cautiously interpreted because we failed to include several important clinical factors (i.e., smoking history) to be adjusted in the model.
In conclusion, we first showed that HER2-FISH status is correlated with the outcome of LA-NSCLC patients after first-line concurrent chemoradiotherapy. This observation supports the notion that LA-NSCLC patients constitute a heterogeneous population with various clinical outcomes that would potentially be stratified by HER2-FISH status, although validation studies are needed in a different LA-NSCLC cohort to confirm our data.
ACKNOWLEDGMENTS
The authors thank Drs. Keisuke Aoe, Noriko Kawata, Keisuke Sugimoto, Hideki Katayama, Haruhito Kamei, Toshiro Yonei, Tomonori Moritaka, Yoichi Watanabe, Akihiro Bessho, Niro Okimoto, Shingo Harita, Mitsuhiro Takemoto, Katsumi Sakae, Kenichi Gemba, Katsuhiko Shiomi, Kenichi Chikamori, Niro Okimoto, Takuo Shibayama, Ichiro Kawada, Ichiro Takata, and Shunkichi Hiraki for their data provision. We are also deeply grateful for all participants and investigators who made this study possible.
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Phase III study of concurrent versus sequential thoracic radiotherapy in combination with mitomycin, vindesine, and cisplatin in unresectable stage III non-small-cell lung cancer.
Long-term benefit is observed in a phase III comparaison of sequential vs concurrent chemoradiation for patients with unresectable stage III NSCLC: RTOG 94-10.
Proc Am Soc Clin Oncol.2003; 22(abstr 2499).: 621a
Monoclonal antibody to HER-2/neureceptor modulates repair of radiation-induced DNA damage and enhances radiosensitivity of human breast cancer cells overexpressing this oncogene.
Low HER2/neu gene expression is associated with pathological response to concurrent paclitaxel and radiation therapy in locally advanced breast cancer.
Quantitative c-erbB-2 but not c-erbB-1 mRNA expression is a promising marker to predict minor histopathologic response to neoadjuvant radiochemotherapy in oesophageal cancer.
Assessment of methods for tissue-based detection of the HER-2/neu alteration in human breast cancer: a direct comparison of fluorescence in situ hybridization and immunohistochemistry.
A phase II study of cisplatin and 5-fluorouracil with concurrent hyperfractionated thoracic radiation for locally advanced non-small-cell lung cancer: a preliminary report from the Okayama Lung Cancer Study Group.
Gefitinib in pretreated non-small-cell lung cancer (NSCLC): analysis of efficacy and correlation with HER2 and epidermal growth factor receptor expression in locally advanced or metastatic NSCLC.
HER-2/neu protein expression and gene alteration in stage I–IIIA non-small-cell lung cancer: a study of 140 cases using a combination of high throughput tissue microarray, immunohistochemistry, and fluorescent in situ hybridization.
Analysis of neuroendocrine markers, HER2 and CEA before and after chemotherapy in patients with stage IIIA non-small cell lung cancer: a Cancer and Leukemia Group B study.
Use of neuroendocrine markers, p53, and HER2 to predict response to chemotherapy in patients with stage III non-small cell lung cancer: a Cancer and Leukemia Group B study.
Increased epidermal growth factor receptor gene copy number detected by fluorescence in situ hybridization associates with increased sensitivity to gefitinib in patients with bronchioloalveolar carcinoma subtypes: a Southwest Oncology Group Study.
Continued gefitinib treatment after disease stabilisation prolongs survival of patients with non-small-cell lung cancer: Okayama Lung Cancer Study Group Experience.
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