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Address for correspondence: Francesca Toffalorio, Medical Oncology Department, Thoracic Oncology Division, European Institute of Oncology, Via Ripamonti 435, 20141, Milan, Italy
Affiliations
Medical Oncology Department, Thoracic Oncology Division, European Institute of Oncology, Milan, Italy
More than even before, the efficacy of epidermal growth factors (EGFRs) tyrosine kinase inhibitors in non–small-cell lung cancer patients carrying EGFR wild-type tumors has been under investigation. EGFR wild-type patients represent a large and heterogeneous group of patients. In this setting, the role played by high polysomy of chromosome 7 still remains controversial. Indeed, previous reports did not discriminate between chromosome 7 high polysomy and EGFR amplification and/or did not investigate the concurrent presence of EGFR and KRas mutations.
Methods
We retrospectively collected data from 163 patients analyzed for EGFR status (mutation, amplification, chromosome 7 trysomy, and polysomy), in addition to KRas mutation, between 2000 and 2010 in our institute. Erlotinib was administered to 73 of them. Objective responses and progression-free survivals to erlotinib were evaluated.
Results
High polysomy of chromosome 7 characterized 17% (28 of 163) of EGFR/KRas wild-type tumors, independently of smoking status. In this group, 13 patients received erlotinib at progression. The treatment led one complete and four partial responses, and five stable diseases. Two patients progressed. One patient was lost to follow-up. The mean time to progression was 9 months.
Conclusion
Among the EGFR wild-type population, when analyzed separately, high polysomy of chromosome 7 was the only molecular feature conferring clear signs of sensitivity to erlotinib. Therefore, the evaluation of high polysomy of chromosome 7 could become a helpful tool to predict for the benefit from epidermal growth factors tyrosine kinase inhibitors in selected cases.
More than even before, the efficacy of epidermal growth factors tyrosine kinase inhibitors (EGFR-TKI) in non–small-cell lung cancer (NSCLC) patients carrying EGFR wild-type tumors has been under investigation. A small proportion (1–20%, depending on the trial) of patients with no detectable EGFR-activating mutations show a radiographic response when treated with EGFR-TKI.
Specific EGFR mutations predict treatment outcome of stage IIIB/IV patients with chemotherapy-naive non-small-cell lung cancer receiving first-line gefitinib monotherapy.
Nevertheless, it is possible that other genetic alterations may activate the same pathway. Indeed, EGFR wild-type patients represent a large and heterogeneous group of patients. Within this group are included patients whose tumors carry multiple EGFR gene copy number (amplification) or multiple copy of chromosome 7, where the EGFR gene is located (trysomy-polysomy). Gain of EGFR copy number (EGFR amplification or high polysomy of chromosome 7) was shown to be associated to EGFR-TKI sensitivity in retrospective studies, but the lack of paired analysis with EGFR and KRas mutations limited data interpretation.
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.
Moreover, data related to EGFR amplification or chromosome 7 high polysomy have never been analyzed separately.
Therefore, we retrospectively collected all the samples analyzed for EGFR mutation and amplification, KRas mutation, and chromosome 7 copy number variations. Among the entire cohort of patients included within the database, we searched for patients who counted EGFR-TKI in their medical history and focused on those with EGFR wild-type tumors.
MATERIALS AND METHODS
Data Collection
Database was created by retrospectively searching for tumor samples analyzed for EGFR/Kras mutations and EGFR fluorescent in situ hybridization (FISH) at the European Institute of Oncology, Milan, between 2000 and 2010. Corresponding tumor-related data and medical history of the patients were recorded as well. Particular attention was used toward EGFR-TKI treatments, for which the dates of progression and treatment durations were accurately estimated. Survival data were obtained through direct patient or family contact or, in the cases in which this was not possible, from the local registry office services. Data dealing with patient habits such as the smoking status came from the physician notes. Former smokers were considered as individuals who had smoked at least 100 cigarettes in their lifetime and had quit more than 12 months ago.
All the patients gave their informed consent for the utilization of data for scientific purposes.
Pathological Evaluations
Molecular analyses were performed on formalin-fixed paraffin-embedded tumor specimens derived from radical surgeries or diagnostic procedures. No ad hoc rebiopsies before erlotinib were carried out.
DNA was extracted using a commercially available kit (QIAamp DNA FFPE Tissue kit, Qiagen, Netherlands) and used to amplify exons 18 through 21 of EGFR and exon 2 and 3 of KRas. DNA was sequenced on both forward and reverse directions by capillary electrophoresis using 3500Dx Genetic Analyzer (Applied Biosystems, CA).
EGFR of 32 of 47 specimens of the same tumor series were re-evaluated by the allele-specific polymerase chain reaction test (cobas). DNA was re-extracted with cobas DNA sample preparation kit and analyzed with cobas 4800 EGFR mutation test (Roche Molecular System Inc., Branchburg, NJ).
FISH assays were performed using the Vysis LSI EGFR SpectrumOrange/CEP7 SpectrumGreen Probe (Abbott Molecular Inc., Des Plaines, IL). The results were evaluated according to the guidelines proposed by Cappuzzo et al.
A detailed description of the methodology is provided in Supplementary Material and Methods (Supplemental Digital Content 1, http://links.lww.com/JTO/A705) and Supplemental Table 4 (Supplemental Digital Content 5, http://links.lww.com/JTO/A709).
Statistical Methods
Clinical and molecular characteristics for both the whole population and erlotinib subset were tabulated as counts and percentages. Enumeration of response of the patients and the time to progression were tabulated as well. Differences in clinical and molecular characteristics both for wild-type and mutated EGFR and chromosome 7 polysomy groups were compared using the chi-square test or the Kruskal-Wallis test as appropriate.
RESULTS
Clinical and Molecular Characteristics of the Entire Cohort
One hundred sixty-three patients were identified whose tumors were analyzed for EGFR and KRas mutation, EGFR amplification, and chromosome 7 copy number variations. For clinicopathological characteristics see Table 1. Molecular analysis revealed that EGFR mutations were present in at least 20% of the samples (36 of 163), but only in one patient EGFR mutation was not associated to EGFR amplifications or chromosome 7 copy number variations (Table 2).
TABLE 1Clinical Characteristics of the Entire Cohort
One patient harbors concurrent EGFR mutation, EGFR amplification, and chr.7 high trysomy; two patients concurrent EGFR mutation, EGFR amplification, and chr.7 high polysomy, and one patient with EGFR amplification, chr.7 high polysomy, and KRas mutation.
One patient harbors concurrent EGFR mutation, EGFR amplification, and chr.7 high trysomy; two patients concurrent EGFR mutation, EGFR amplification, and chr.7 high polysomy, and one patient with EGFR amplification, chr.7 high polysomy, and KRas mutation.
One patient harbors concurrent EGFR mutation, EGFR amplification, and chr.7 high trysomy; two patients concurrent EGFR mutation, EGFR amplification, and chr.7 high polysomy, and one patient with EGFR amplification, chr.7 high polysomy, and KRas mutation.
One patient harbors concurrent EGFR mutation, EGFR amplification, and chr.7 high trysomy; two patients concurrent EGFR mutation, EGFR amplification, and chr.7 high polysomy, and one patient with EGFR amplification, chr.7 high polysomy, and KRas mutation.
One patient harbors concurrent EGFR mutation, EGFR amplification, and chr.7 high trysomy; two patients concurrent EGFR mutation, EGFR amplification, and chr.7 high polysomy, and one patient with EGFR amplification, chr.7 high polysomy, and KRas mutation.
a One patient harbors concurrent EGFR mutation, EGFR amplification, and chr.7 high trysomy; two patients concurrent EGFR mutation, EGFR amplification, and chr.7 high polysomy, and one patient with EGFR amplification, chr.7 high polysomy, and KRas mutation.
Patients without EGFR mutations displayed low trysomy in 20.8% (34 of 163), high trysomy in 11.6% (19 of 163), low polysomy in 10.4% (17 of 163), and high polysomy in 17.1% (28 of 163) of the cases (Table 2).
In addition, correlations between the EGFR mutation and other molecular alterations were evaluated. A strong correlation with presence of EGFR amplifications (p ⩽ 0.001) and absence of KRas mutations (p = 0.005) emerged (Supplementary Table 1, Supplemental Digital Content 2, http://links.lww.com/JTO/A706). An association between EGFR mutation and chromosome 7 trysomy (p = 0.019) was also observed. Chromosome 7 polysomy was not correlated with any of the clinicopathological characteristics analyzed (Supplementary Table 2, Supplemental Digital Content 3, http://links.lww.com/JTO/A707).
Clinical and Molecular Features of the Erlotinib Population
Seventy-three of the 163 patients received EGFR inhibitors (Fig. 1). All the patients had advanced disease when treated with EGFR-TKI and most of them (94.5%) previously received at least one line of chemotherapy (Table 3). In terms of molecular features, we observed that 47 (64.4%) patients without EGFR mutations were treated (Table 5). Most of them had chromosome 7 copy number variations. For a complete description of patients see Tables 3 and 4.
One patient harbors concurrent EGFR mutation, EGFR amplification, and chr.7 high trysomy and another patient concurrent EGFR mutation, EGFR amplification, and chr.7 high polysomy.
One patient harbors concurrent EGFR mutation, EGFR amplification, and chr.7 high trysomy and another patient concurrent EGFR mutation, EGFR amplification, and chr.7 high polysomy.
One patient harbors concurrent EGFR mutation, EGFR amplification, and chr.7 high trysomy and another patient concurrent EGFR mutation, EGFR amplification, and chr.7 high polysomy.
One patient harbors concurrent EGFR mutation, EGFR amplification, and chr.7 high trysomy and another patient concurrent EGFR mutation, EGFR amplification, and chr.7 high polysomy.
a One patient harbors concurrent EGFR mutation, EGFR amplification, and chr.7 high trysomy and another patient concurrent EGFR mutation, EGFR amplification, and chr.7 high polysomy.
All the patients received erlotinib and computed tomography scan evaluations were done every 2 to 3 months according to the international guidelines. The extent of the disease response was determined on the basis of the Response Evaluation Criteria in Solid Tumors criteria.
Among the 13 patients with EGFR wild-type and chromosome 7 high polysomy, one complete and four partial responses were observed, with a median progression-free survival (PFS) of 9 months (Table 5). Interestingly, only one patient carrying concurrent chromosome 7 high polysomy and KRas mutation progressed after 2 months. On the contrary, low and high trysomy and low polysomy did not show remarkable signs of erlotinib activity, with the overall response rates of 7% in the patients with low trysomy and 0% in those with high trysomy and low polysomy. Logrank analyses revealed a correlation between longer PFS and a presence of high polysomy (p = 0.004), in an EGFR wild-type background (Supplementary Table 3, Supplemental Digital Content 4, http://links.lww.com/JTO/A708).
TABLE 5Tumor Response and Median PFS of Patients Treated with Erlotinib
Since their identification in 2004, activating EGFR gene mutations have emerged as the most relevant predictor of response to gefitinib or erlotinib. On the contrary, the proven EGFR-TKI activity in the EGFR wild-type population remains to be addressed. Recently two phase III trials examined efficacy of erlotinib compared with second-line mono-chemotherapy in EGFR wild-type NSCLC. In the Tarceva Italian Lung Optimization Trial (TAILOR) trial patients treated with erlotinib, a worse outcome was obtained compared with patients in the chemotherapy arm.
Erlotinib versus docetaxel as second-line treatment of patients with advanced non-small-cell lung cancer and wild-type EGFR tumours (TAILOR): a randomised controlled trial.
In the second trial (PROteomic SElection [PROSE]), the authors have prospectively validated a serum proteomic test able to identify two subgroups of patients with respectively good and poor prognosis. In the first group, erlotinib has shown to be not inferior to standard second-line chemotherapy. On the contrary, survival of the patients with the test signature associated with poor prognosis was superior for those treated with chemotherapy.
Predictive value of a proteomic signature in patients with non-small-cell lung cancer treated with second-line erlotinib or chemotherapy (PROSE): a biomarker-stratified, randomised phase 3 trial.
Taken together, these data suggest that an activity of EGFR-TKI in further lines of therapy restricted only to a not yet well-identified subgroup of EGFR wild-type patients. Indeed EGFR “wild-type” is clearly not a uniform entity as terms really define what a disease is not, as opposed to what a disease is. We know that, unless otherwise specified, the EGFR wild-type lung cancer population must contain anaplastic lymphoma kinase (ALK) gene rearranged, KRas mutant, and a whole host of other molecular subpopulations with different oncogenic drivers that are still being discovered. Consequently, a diffuse consistent benefit of EGFR-TKIs seems unlikely in the EGFR wild-type population. As, in general, this population has few therapeutic options after first-line therapy, defining additional biomarkers that could be used together with EGFR mutation testing to help identify the group of patients deriving even minimal survival benefit from an EGFR-TKI therapy is important. In this context, high polisomy of chromosome 7 might be the best biomarker.
Using the Colorado scoring system described by Cappuzzo et al. almost 30 to 50% of NSCLC have a gain copy number (GCN) of EGFR (FISH-positive NSCLC including EGFR gene amplification or high polysomy of chromosome 7).
A meta-analysis of 22 studies comparing the outcome of EGFR FISH+ and FISH− patients treated with EGFR-TKI confirms that an increased EGFR gene copy number is associated with a moderate overall survival benefit and a substantial PFS benefit.
EGFR gene copy number as a predictive biomarker for patients receiving tyrosine kinase inhibitor treatment: a systematic review and meta-analysis in non-small-cell lung cancer.
However given the association between GCN and EGFR mutations, it is critical to analyze GCN data in a direct comparison with EGFR mutation status. In the majority of studies, this information is not available. Iressa Pan-Asia study trial (IPASS) suggested that the apparent PFS benefit of gefitinib in patients with a high EGFR copy number was due to overlapping EGFR mutations. However, this analysis has been conducted on only 55 patients and compared EGFR-TKI efficacy with that of first-line platinum-based doublets. Furthermore, EGFR amplification and high polysomy were not analyzed separately.
Biomarker analyses and final overall survival results from a phase III, randomized, open-label, first-line study of gefitinib versus carboplatin/paclitaxel in clinically selected patients with advanced non-small-cell lung cancer in Asia (IPASS).
Here, we report the data suggesting the need to better explore the role of chromosome 7 high polysomy as a helpful tool to predict for EGFR-TKI benefit.
In our sample, 17% of the tumors without EGFR mutations are characterized by a high polysomy of chromosome 7. Erlotinib led to a disease control in 80% of the patients with a 30% of partial response and to a median PFS longer than 9 months in a heavily treated group of patients.
Data appeared very interesting when compared with overall response rate and PFS obtained with erlotinib in EGFR wild-type patients not otherwise selected and to chemotherapy in the same line of treatment.
Our data are certainly limited by the very small sample size and by the retrospective nature of analysis but suggest a possible role for chromosome 7 high polysomy as a useful tool to select EGFR wild-type patients who could possibly benefit from an EGFR-TKI treatment.
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Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma.
Specific EGFR mutations predict treatment outcome of stage IIIB/IV patients with chemotherapy-naive non-small-cell lung cancer receiving first-line gefitinib monotherapy.
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.
Erlotinib versus docetaxel as second-line treatment of patients with advanced non-small-cell lung cancer and wild-type EGFR tumours (TAILOR): a randomised controlled trial.
Predictive value of a proteomic signature in patients with non-small-cell lung cancer treated with second-line erlotinib or chemotherapy (PROSE): a biomarker-stratified, randomised phase 3 trial.
EGFR gene copy number as a predictive biomarker for patients receiving tyrosine kinase inhibitor treatment: a systematic review and meta-analysis in non-small-cell lung cancer.
Biomarker analyses and final overall survival results from a phase III, randomized, open-label, first-line study of gefitinib versus carboplatin/paclitaxel in clinically selected patients with advanced non-small-cell lung cancer in Asia (IPASS).
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