K-ras and p16INK4Aalterations in sputum of NSCLC patients and in heavy asymptomatic chronic smokers
Introduction
Lung carcinoma is the leading cause of cancer-related death in male patients. Prognosis of non-small cell lung carcinoma (NSCLC) patients is largely dependent on the stage of the disease at the time of diagnosis: in patients with operable stage I NSCLC, the 5-years survival rate is 65–75% [1], [2], compared to 15% of stage II–IV. However with current standard methods (chest radiography and sputum cytological analysis), it is often difficult to detect early-stage tumors; thus the development of more efficient diagnostic methods, by enabling early diagnosis and therapy, may be able to increase patient survival.
Documented abnormalities in proto-oncogenes and in tumor suppressor genes in NSCLC raised the hope to use molecular markers to increase the sensitivity of early-stage NSCLC detection. A prerequisite for such an approach is that molecular markers have to be present in biological samples and to be obtained through non-invasive procedures (e.g. sputum).
Among molecular alterations identified so far in NSCLC there are point mutations of the ras proto-oncogene and p16INK4A promoter hypermethylation. K-ras mutations are mainly found in adenocarcinoma, with rates ranging from 15 to 50% depending on the material and on the sensitivity of the assay [3], [4], [5], [6], [7]. The vast majority of K-ras mutations affect codon 12 and G to T transversion was the most common [8]. Furthermore, several studies have indicated that lung cancer patients whose tumors were found to harbour K-ras mutations had poorer survival rates [9]. Mao et al. and subsequently other groups described K-ras point mutations in tumor and in sputum specimens or bronchoalveolar lavage fluids from patients with NSCLC [10], [11], [12], [13].
Inactivation of the suppressor p16INK4A has been detected in more than 70% of cell lines from human NSCLC [14] and in 50% of primary tumors [15], [16], [17]. Specifically, homozygous deletions and mutations of p16INK4 have been reported in NSCLCs with highly variable frequency [18], [19], [20], [21], [22], [23], [24]. More recently, methylation at the 5′-CpG islands of the p16INK4A gene was identified as an alternative mechanism of p16INK4A silencing in NSCLCs [25], [26], [27]. Aberrant methylation of the p16INK4 gene promoter was detected both in sputum samples from patients with NSCLC and, interestingly, in sputum samples from cancer-free individuals at high risk [28], [29], [30].
Molecular studies on exfoliative material from patients with NSCLC demonstrated the potential use of different biomarkers in early detection of lung cancer. Nevertheless, to now, there is a lack of solid data on the sensitivity of combined molecular and cytological analysis in sputum for NSCLC detection and their correlation with tumor staging and grading.
In this study we evaluated the prevalence of molecular abnormalities such as K-ras mutations and p16INK4A hypermethylation in sputum collected from NSCLC patients before surgery. Data were correlated: (a) with the molecular pattern of the same genes in tumors, as referenced standard of the molecular profile; (b) with sputum cytology to compare the molecular and cytological diagnostic yield; and (c) with tumor grading, staging and location to test whether these markers are useful for early diagnosis.
Section snippets
Patients and samples
Under visual medical inspection, three spontaneous and noninduced sputum samples were obtained from 50 NSCLC patients immediately prior resectional surgery. The main clinico-phatologic features of NSCLC patients are reported in Table 1. Histology, stage, grading and location of tumors were determined according to the WHO classification [31] and the TNM system [32]. Three sputum samples were also collected from 100 asymptomatic heavy smokers enrolled in our institution in a screening program for
Overall molecular profile in tumor and sputum samples
Genetic analysis identified molecular abnormalities in 32/50 (64%) tumors, 22 from patients with adenocarcinoma (66.7%, n=33) and 10 from patients with squamous cell carcinoma (58.8%, n=17). Twenty-four of 32 molecular abnormal tumors showed genetic alterations in sputum samples, with 75% (24/32) sensitivity of the sputum molecular assay. Eighteen tumors and corresponding sputum samples (36%) did not show any molecular alterations.
K-ras mutations
Mutations in the K-ras gene codon 12 were found in 14 tumor
Discussion
In this study, we analysed K-ras and p16INK4A gene abnormalities in sputum samples and in matched tumor tissues of patients with NSCLC and correlated them with sputum cytology and tumor staging, grading and location, to document the molecular and cytological diagnostic yield in the detection of NSCLC. We also studied the same genes in a homogeneous cohort of asymptomatic heavy smokers.
To analyse p16INK4A hypermethylation we used a nested, two-stage PCR approach which allows to detect 1
Acknowledgements
This paper was partially supported by Cofinanziamento MURST (Cofin, 1999).
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