Prognostic Impact of EGFR Amplification and Visceral Pleural Invasion in Early Stage Pulmonary Squamous Cell Carcinomas Patients after Surgical Resection of Primary Tumor

Abstract

Over the last few decades, an increasing amount of information has been accumulated on biomarkers in non-small cell lung cancer (NSCLC). Despite these advances, most biomarkers have been identified in the adenocarcinoma histological subtype (AC). However, the application of molecular-targeted therapies in the prognosis and treatment of SCC in the clinical setting is very limited, becoming one of the main focus areas in research. Here, we prospectively analyzed the frequency of numerical/structural abnormalities of chromosomes 5, 7, 8, 9, 13 and 22 with FISH in 48 pulmonary SCC patients. From a total of 12 probes, only abnormalities of the 7p12 and 22q12 chromosomal regions were identified as unique genetic variables associated with the prognosis of the disease. The study for these two chromosomal regions was extended to 108 patients with SCC. Overall, chromosome losses were observed more frequently than chromosome gains, i.e., 61% versus 19% of all the chromosome abnormalities detected. The highest levels of genetic amplification were detected for the 5p15.2, 7p12, 8q24 and 22q11 chromosome bands, of which several genes are potentially involved in the pathogenesis of SCC, among others, include the EGFR gene at chromosome 7p12. Patients who displayed EGFR amplification (n = 13; 12%) were mostly older than 65 years (p = 0.07) and exclusively patients in early T-primary tumor stage (pT1−pT2; p = 0.03) with a significantly shortened overall survival (OS) (p ≤ 0.001). Regarding prognosis, the clinical, biological, and histopathologic characteristics of the disease that displayed a significant adverse influence on OS in the univariate analysis included patients older than 65 years (p = 0.02), the presence of lymph node involvement (p = 0.005), metastasis (p = 0.01) and, visceral pleural invasion (VPI) at diagnosis (p = 0.04). EGFR amplification also conferred an adverse impact on patient OS in the whole series (p = 0.02) and especially in patients in early stages (pT1−pT2; p = 0.01). A multivariate analysis of the prognostic factors for OS showed that the most informative combination of independent variables to predict an adverse outcome was the presence of VPI and/or EGFR amplification (p < 0.001). Based on these two variables, a scoring system was built to stratify patients into low- (no adverse features: score 0; n = 69), intermediate- (one adverse feature: score 1; n = 29) and high-risk (two adverse features: score 2; n = 5) groups, with significantly different (p = 0.001) OS rates at 50 months, which were as following: 32%, 28% and 0%, respectively. In the present study, we show that the presence of a high level of 7p12 (EGFR) amplification, exclusively detected in early stage SCC (pT1−pT2), is an independent adverse prognostic factor for OS. The identification of the EGFR gene copy number using FISH techniques may provide a more accurate diagnosis of high-risk populations after the complete resection of the primary tumor. When combined with VPI, three groups of pulmonary SCC were clearly identified that show the extent of the disease. This is of such importance that further prospective studies are necessary in larger series of SCC patients to be classified at the time of diagnosis. This could be achieved with the combined assessment of 7p12 amplification and VPI in primary tumor samples.

Keywords: EGFR amplification; early stage; prognosis; squamous cell lung carcinoma.

Figure 1

Chromosomal abnormalities detected in pulmonary squamous cell carcinomas (SCC) patients by FISH techniques (n = 48). This summary plot illustrates the frequency of copy number gains (plotted in white above zero values in the x-axis) and losses (plotted in black below zero values in the x-axis) identified in patients with SCC of the lung. The blue colour indicates the accumulated frequency of the amplification of the chromosomal region analysed, being able to discriminate the polysomies of the amplification of the gene.

Figure 2

Interphase nuclei with the amplified EGFR gene detected by FISH techniques from a biopsy sample of a patient with squamous cell carcinoma of the lung (probes for identifying chromosome 7 centromere (7p11; green spots) and EGFR gene (7p11.2; red spots)).

Figure 3

Clinic, histopathologic and biologic-disease characteristics of pulmonary SCC patients which showed a significant impact on overall survival in the univariate analysis: (A) Age, (B) lymph node status, (C) presence of distant metastasis and (D) pleural invasion. Survival information was available for 103 cases.

Figure 4

EGFR amplification detected in primary tumors from patients with pulmonary SCC by FISH techniques which showed a significant impact on overall survival in the univariate analysis: (A) all patients and (B) only in early stages: pT1–pT2 patients. Survival information was available for 103 cases.

Figure 5

Prognostic score proposed for pulmonary SCC patients, based on the two most informative prognostic factors: pleural invasion, and EGFR status; p < 0.001. Survival information was available from 103 cases.