Cancer Letters

Cancer Letters

Volume 254, Issue 2, 8 September 2007, Pages 265-273
Cancer Letters

Origin and prognostic value of circulating KRAS mutations in lung cancer patients

https://doi.org/10.1016/j.canlet.2007.03.008Get rights and content

Abstract

Because of the current controversy on the origin and clinical value of circulating KRAS codon 12 mutations in lung cancer, we screened 180 patients using a combined restriction fragment-length polymorphism and polymerase chain reaction (RFLP–PCR) assay. We detected KRAS mutations in 9% plasma samples and 0% matched lymphocytes. Plasma KRAS mutations correlated significantly with poor prognosis. We validated the positive results in a second laboratory by DNA sequencing and found matching codon 12 sequences in blood and tumor in 78% evaluable cases. These results support the notion that circulating KRAS mutations originate from tumors and are prognostically relevant in lung cancer.

Introduction

Globally, lung cancer is the leading cause of cancer-related death, and approximately 80% of lung cancers are of the non-small cell (NSCLC) subtype [1]. Even when taking into consideration multiple established prognostic factors, including tumor stage, histology, patient age, gender, co-morbidity and therapy, the determination of prognosis in NSCLC patients is unreliable. A number of prognostic serum tumor markers, including lactate dehydrogenase (LDH) and carcinoembryonic antigen (CEA) have been introduced, but their accuracy can be perturbed by inflammation, smoking and other factors [2].

Several studies have reported on the presence in body fluids of cell-free deoxyribonucleic acid (DNA) in patients with lung cancer [3]. We and others have demonstrated that elevated concentrations of cell-free DNA in serum and/or plasma (circulating DNA) are associated with tumor stage and patient survival, and that changes may correlate with response to chemotherapy [4], [5], [6], [7], [8], [9]. Perhaps even more importantly, circulating DNA from lung cancer patients was shown to contain tumor-specific genetic and epigenetic alterations, including mutations, loss of heterozygosity (LOH), and gene promoter hypermethylation [10]. While many of these alterations may potentially be useful in the clinic, mutations of the KRAS (Kirsten rat sarcoma viral oncogene homolog) gene have garnered particular attention in NSCLC for a number of reasons. RAS mutations are a key element of lung carcinogenesis; resulting in constitutive activation of RAS, which drives cells into a state of deregulated, growth factor-independent proliferation [11]. RAS mutations have been detected in up to 40% of NSCLC tumor tissues, which is a considerable rate in view of the heterogeneity of this disease. Up to 60% of all RAS mutations are confined to codon 12 of KRAS, simplifying detection [12]. Such mutations, overall, appear to be associated with poor prognosis in NSCLC patients and with resistance to gefitinib or erlotinib, suggesting that circulating KRAS mutations may be similarly informative. KRAS mutations have been readily detected in the sputum, bronchoalveolar lavage fluid, pleural effusion, serum and plasma of NSCLC patients [13], [14], [15], [16]. Because of an ongoing controversy in this field, we decided to further evaluate the prognostic value and reliability of plasma KRAS mutation analysis in lung cancer patients.

Section snippets

Patients

Blood samples from 180 consecutive patients with newly diagnosed or relapsed, histologically confirmed NSCLC were collected at two Swiss University Hospitals (Bern and Zurich) between April 2001 and June 2003 [8]. The study was approved by the local ethic committees, and all patients gave written informed consent prior to sampling. By December 2004, survival data were collected by reviewing internal documents and contacting primary care physicians.

DNA extraction from blood samples

As described previously, 10 ml of peripheral

Patient characteristics

Over a period of 25 months, blood samples were drawn from 180 consecutive NSCLC patients (Table 1) [8]. Sixty-nine percent were male, and at least 69% had a current or past history of cigarette smoking. The median age at diagnosis was 61 years (range 36–81 years), and median survival was 12 months (range 1–61 months). Histopathological diagnoses comprised adenocarcinoma (44%), squamous cell carcinoma (26%), large cell carcinoma (20%), and undifferentiated carcinoma (10%). Seventy percent of the

Discussion

There are two widely used methods to screen for KRAS mutations in circulating DNA, allele-specific PCR, and RFLP–PCR. We gave precedence to the latter method, because of the potential enrichment for mutant alleles, and its feasibility in the clinical setting, as demonstrated in prior studies [18], [19]. Using an established patient sample collection and database, we screened for circulating KRAS codon 12 mutations in the largest (n = 180) group of NSCLC patients reported so far [8]. We detected

Acknowledgments

We thank our patients and the following persons and institutions for their contribution: our nursing staff for collection of blood samples; Pathology Institute Laenggasse (Bern), Pathology Institute Boss and Spichtin (Basel), and Institute of Clinical Pathology (University Hospital Zurich) for tumor tissue samples; V. Kocher (Pathology, University of Bern), C. Bigosch (University Hospital Zurich), W.S. Holland and H.L. Chen (University of California Davis Cancer Center) for technical

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