Elsevier

Lung Cancer

Volume 44, Issue 1, April 2004, Pages 23-32
Lung Cancer

K-ras and p16INK4Aalterations in sputum of NSCLC patients and in heavy asymptomatic chronic smokers

https://doi.org/10.1016/j.lungcan.2003.10.002Get rights and content

Abstract

NSCLC rates among the most frequent and lethal neoplasm world-wide and a significant decrease in morbidity and mortality relies only upon effective early diagnostic strategies. We investigated K-ras mutations and p16INK4A hypermethylation in tumor tissue and sputum of 50 patients with NSCLC and correlated them with sputum cytology and with tumor staging, grading and location, to ascertain, in sputum, their potential diagnostic impact. The same genetic/epigenetic abnormalities and cytological features were also evaluated in sputum from 100 chronic heavy smokers. Genetic analysis identified molecular abnormalities in 64% tumors (14/50 K-ras mutations and 24/50 p16INK4A hypermethylation) and in 48% sputum (11/50 K-ras mutations and 16/50 p16INK4A hypermethylation). In tumors K-ras mutations and p16INK4A hypermethylation were mostly mutually exclusive, being found in the same patients in 3 cases only. Genetic abnormalities in sputum were detected only in molecular abnormal tumors. Molecular changes in sputum had rates of detection similar to cytology (42%) but the cyto-molecular combination increased the diagnostic yield up to 60%. Interestingly, the rate of detection of genetic changes in sputum of tumors at early stage (T1) was not significantly different from that of tumors at more advanced stage (T2–T4). In fact K-ras point mutations were frequently recognised in tumors at early stage while p16INK4A inactivation prevailed in tumors at advanced stage (P=0.0063). As expected, diagnostic cytological findings were more frequently found in tumors at advanced stage (P=0.004). No correlation was found between tumor grading and location (central versus peripheral) and molecular changes. p16INK4A hypermethylation, but not K-ras mutations, was documented in sporadic cases of asymptomatic heavy smokers (4%) where it was uncoupled from cytological abnormalities. In conclusion the cyto-molecular diagnostic strategy adopted in this study was able to detect the majority of tumors but in order to be proposed as effective and early diagnostic tool, this molecular panel needs to be tested in prospective studies with adequate follow-up.

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).

References (42)

  • K. Husgafvel-Pursiainen et al.

    K-ras mutations in human adenocarcinoma of the lung: association with smoking and occupational exposure to asbestos

    Int. J. Cancer

    (1993)
  • S. Rodenhuis et al.

    The ras oncogenes in human lung cancer

    Am. Rev. Respir. Dis.

    (1990)
  • R. Rosell et al.

    Prognostic impact of mutated K-ras gene in surgically resected non-small cell lung cancer patients

    Oncogene

    (1993)
  • L. Mao et al.

    Detection of oncogene mutations in sputum precedes diagnosis of lung cancer

    Cancer Res.

    (1994)
  • N.E. Mills et al.

    Increased prevalence of K-ras oncogene mutations in lung adenocarcinoma

    Cancer Res.

    (1995)
  • V.A. Somers et al.

    Detection of K-ras point mutations in sputum from patients with adenocarcinoma of the lung by point-EXACCT

    J. Clin. Oncol.

    (1998)
  • F.S. Steven et al.

    Evidence for the induction of a tumour associated cell surface protease on cytologically normal epithelial cells present in the sputum of patients possessing lung tumours

    Anticancer Res.

    (1999)
  • A. Kamb et al.

    A cell cycle regulator potentially involved in genesis of many tumor types

    Science

    (1994)
  • R.A. Kratzke et al.

    Rb and p16INK4a expression in resected non-small cell lung tumors

    Cancer Res.

    (1996)
  • M. Sanchez-Cespedes et al.

    Inactivation of the INK4A/ARF locus frequently coexists with TP53 mutations in non-small cell lung cancer

    Oncogene

    (1999)
  • S. Vonlanthen et al.

    Expression of p16INK4a/p16alpha and p19ARF/p16beta is frequently altered in non-small cell lung cancer and correlates with p53 overexpression

    Oncogene

    (1998)
  • Cited by (60)

    • Methylation analysis in spontaneous sputum for lung cancer diagnosis

      2014, Lung Cancer
      Citation Excerpt :

      This indicates that sensitivity is higher for tumour DNA compared to cytology [5,6]. It is now generally accepted that DNA hypermethylation of various genes has been associated with cancer [7], which can be detected in sputum of lung cancer patients [6,8–17]. Several sputum studies using methylation-specific PCR (MSP) reported a slightly increased risk for lung cancer for subjects with DNA hypermethylation in more than one gene [10,18].

    • EGFR mutation analysis in sputum of lung cancer patients: A multitechnique study

      2013, Lung Cancer
      Citation Excerpt :

      In sputum, tumour cells can be detected by sputum cytology with a sensitivity of around 66% [6]. Tumour specific molecular alterations, such as mutations in KRAS and p53 [7–9], and DNA promoter hypermethylation of genes [8,10–16] have already been identified in sputum with different molecular techniques. Although EGFR mutation analysis has previously been performed in some sputum samples as part of a larger series of other cytological samples, mostly the outcomes were not compared to the original tumour or detailed information was not given [17–19].

    • Complete surgical resection of lung tumor decreases exhalation of mutated KRAS oncogene

      2012, Respiratory Medicine
      Citation Excerpt :

      They occur frequently in the adenocarcinoma of the lung and to a lesser extent in squamous cell carcinoma.15 DNA isolated from sputum and bronchoalveolar lavage fluid (BAL) of lung cancer patients disclosed the KRAS abnormalities.17–19 Therefore, at least part of EBC specimens obtained from patients with lung cancer should contain mutant KRAS DNA.

    View all citing articles on Scopus
    View full text