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Address for correspondence: Hidefumi Sasaki, MD, PhD, Department of Oncology, Immunology and Surgery, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan
Recent studies for the characterization of the lung cancer genome have suggested that Kras gene was frequently amplified and correlated with activating mutations of Kras, which occur in approximately 5 to 10% of Japanese lung cancers.
Methods:
We analyzed Kras mutation and Kras copy number in 172 Japanese non-small cell lung cancer (NSCLC) cases and their relation to the survival of patients. We also studied using fluorescence in situ hybridization to provide direct evidence of Kras amplification in 40 clinical specimens.
Results:
In 172 NSCLC cases, increased Kras copy number existed in 19 (11.0%) cases. Increased Kras gene copy number was correlated with Kras mutation. Nevertheless, Kras gene copy number gain was not correlated with gender, pathological subtypes, stages, and smoking status. Increased Kras copy number was not associated with overall survival in these 172 cases; however, patients with increased Kras copy number and Kras mutant had significantly worse prognosis, when compared with patients with Kras wild type and Kras not increased. From the fluorescence in situ hybridization analysis, Kras polysomy or amplified patients showed significantly worse prognosis, when compared with Kras disomy patients.
Conclusion:
Kras mutation plus increased copy number was a predictor of poor clinical outcome in patients with NSCLC.
Lung cancer is the deadliest cancer in many developed countries. Mutations of Kras gene occur in approximately 5 to 10% of Japanese non-small cell lung cancers (NSCLCs),
and these mutations are associated with a distinct clinicopathologic subset of NSCLC marked by a strong association with smokers, patients with adenocarcinomas, poor prognosis, ethnics, and in resistance to treatment with tyrosine kinase inhibitors.
On the other hand, early studies in cell lines and murine models established the possibility that amplification of wild-type Ras genes could lead to malignant transformation.
Recent studies using low-resolution, conventional comparative genome hybridization have identified frequent copy number gains of the 12p12. 1 region, including Kras from several cancers.
Comparative genomic hybridization analysis detects frequent, often high-level, overrepresentation of DNA sequences at 3q, 5p, 7p, and 8q in human non-small cell lung carcinomas.
Moreover, in recent large-scale systematic assessments of the lung adenocarcinoma genome, analysis of single-nucleotide polymorphism arrays has identified Kras copy number gains as one of the most common focal amplification events in adenocarcinoma of the lung.
Nevertheless, Kras copy number status at Japanese population using FISH has not been well reported. Because we previously reported the epidermal growth factor receptor (EGFR) copy number gains in NSCLC using FISH
we also evaluated to determine the Kras gene mutation status, and Kras gene amplification may bring important information for the surgically treated Japanese patients with NSCLC. The findings were compared with the clinicopathologic features of NSCLC in this study. FISH analysis was also performed.
PATIENTS AND METHODS
Patients
NSCLC tissues were obtained by surgical excision from 172 patients with NSCLC at Nagoya City University Hospital. EGFR mutation
were already analyzed and reported. The patients were biased population, as mutation centric. The research was approved by the Institutional Review Board of the hospital. All the patients consented to the use of their tissues for the present analysis. The tissues were placed in liquid nitrogen immediately after resection (macrodissected) or fixed by formalin and paraffin embedded. Genomic DNA was extracted using Wizard SV Genomic DNA Purification System (Promega) according to the manufacturer's instructions. Within the patients, we have also analyzed the Kras amplified status for 40 NSCLC cases using FISH methods.
Analysis of Kras Copy Number
Kras copy number was analyzed for 172 patients with NSCLC by qPCR, performed on 7500 Real-Time PCR System (Applied Biosystems) by using a QuantiTect SYBR Green kit (Qiagen, Inc., Valencia, CA).
The run per each patient was triplicate. We quantified each tumor DNA by comparing the target locus to the reference line 1, a repetitive element for which copy number per haploid genome is similar among all the human normal and neoplastic cells. Quantification is based on standard curves from a serial dilution of human normal genomic DNA. The relative Kras copy number was also normalized to the normal human genomic DNA as a calibrator. Copy number change of Kras gene relative to the line 1 and the calibrator was determined by using the formula (TKras/Tline 1)/(CKras/Cline 1), where TKras and Tline 1 are quantities from tumor DNA by using Kras and line 1, and CKras and Cline 1 are quantities from calibrator by using Kras and line 1. Conditions for qPCR reaction were as follows: one cycle of 50°C for 2 minutes; one cycle of 95°C for 15 minutes; 40 cycles of 95°C for 15 seconds; 56°C for 30 seconds; and 72°C for 34 seconds. At the end of the PCR reaction, samples were subjected to a melting analysis to confirm specificity of the amplicon. Kras primers were 5′GGCCTGCTGAAAATGACT-3′ and 5′GAATGGTCCTGCACCAGTA-3′ and they amplified. Total DNA content was estimated by assaying line 1 elements for each sample using the primers 5′-AAAGCCGCTCAACTACATGG-3′ and 5′-TGCTTTGAATGCGTCCCAGAG-3′. Kras increased copy number (ICN) was determined as positive when the copy number was more than 3, according to the Takano et al.
Tumor specimens were obtained at surgical operation and embedded in paraffin. Serial sections (6 mm) containing representative malignant cell were stained with hematoxylin and eosin. Gene copy number per cell was investigated by FISH using the GPS KRAS (TexRed)/CEN12q(FITC) Dual Color FISH probe (GPS laboratories, Kawasaki, Japan) according to a published protocol.
Epidermal growth factor receptor in non-small cell lung carcinomas: correlation between gene copy number and protein expression and impact on prognosis.
Sections were incubated at 56°C overnight, deparaffinized, and dehydrated. After incubation in 2× saline sodium citrate buffer (2×SSC; pH, 7.0) at 75°C for 15 to 25 minutes, sections were digested with protein K (0.25 mg/ml in 2×SSC; pH, 7.0) at 37°C for 15 to 25 minutes, rinsed in 2×SSC at room temperature for 5 minutes, and dehydrated using ethanol in a series of increasing concentrations. The KRAS/CEN 12q probe set was applied per the manufacturer's instructions onto the selected area based on the presence of tumor foci on each slide. The slides were incubated at 80°C for 8 to 10 minutes for codenaturation of chromosomal and probe DNA and then placed in a humidified chamber at 37°C for 20 to 24 hours to allow hybridization to occur. Posthybridization washes were performed in 1.5 M urea and 0.1×SSC at 45°C for 30 minutes and in 2×SSC for 2 minutes at room temperature. Pathologist who was blinded to the patients' clinical characteristics and all other molecular variables performed FISH analysis independently. Patients were classified according to the criteria by Cappuzzo et al.
with ascending number of copies of the Kras gene per cell and the frequency of tumor cells with specific number of copies of the Kras gene and chromosome 12q centromere: gene amplification (defined by presence of tight Kras gene clusters and a ratio of Kras gene to chromosome of ≥2 or ≥15 copies of Kras per cell in ≥10% of analyzed cells), high polysomy (≥4 copies in ≥40% of cells), and low polysomy (≥4 copies in 10–40% of cells) were considered as ICN in our analysis. Disomy (≤2 copies in >90% of cells), low trisomy (≤2 copies in ≥40% of cells, three copies in 10–40% of cells, and 4≥ copies in <10% of cells), and high trisomy (≤2 copies in ≥40% of cells, three copies in ≥40% of cells, and ≥4 copies in <10% of cells) were considered as normal copy number.
Statistical Analysis
For comparisons of proportions, Fisher's exact test was used. The overall survival was examined by the Kaplan-Meier methods, and differences were examined by the log-rank test. Association of risk factors associated with survival was evaluated using Cox proportional hazards regression model. Only those variables with significant results in univariate analysis were included in the multivariate analysis. All analysis was done using a Stat View (version 5, SAS Institute Inc., Cary, NC) software and was considered significant when the p value was less than 0.05.
RESULTS
Kras Mutation Status in 172 Patients
In the previous report, we detected 11% of Kras mutation in our lung cancer cohort.
From the same genotyping assay using the LightCycler, 36 cases were detected to have a Kras mutation in this cohort. We have mainly focused on Kras mutant patients for further copy number and FISH analyses, and this Kras mutation rate did not reflect actual mutation rate. Kras mutation was found in 25 of 106 (23.6%) males and 11 of 66 (16.7%) females; 10 of 67 (14.9%) patients who were aged 64 years and younger and 26 of 105 (24.8%) patients who were older than 65 years; 34 of 137 (24.8%) adenocarcinoma and 2 of 35 (5.7%) nonadenocarcinoma; 11 of 68 (16.2%) never smokers and 25 of 104 (24.0%) smokers; and 15 of 96 (15.6%) of stage I and 21 of 76 (13.2%) of stages II to IV NSCLC. Adenocarinoma showed significantly higher Kras mutation rate. Advanced stage trends shown higher Kras mutation rate.
Kras Copy Number in 172 Patients with Lung Cancer
Kras copy numbers of 172 samples from patients with lung cancer were analyzed by qPCR. Nineteen of 172 cases were found to have increased Kras copy number (>3.0). Only two cases had more than four Kras copy number. Relationship between Kras copy number and clinical-pathologic factors in patients with lung cancer was shown in Table 1. Increased Kras copy number was found in 11 of 106 (10.4%) males and 8 of 66 (12.1%) females; 5 of 75 (6.7%) of patients who were aged 65 years and younger and 14 of 97 (14.4%) of those who were older than 65 years; 13 of 137 (9.5%) of adenocarcinoma and 7 of 36 (19.4%) of nonadenocarcinoma; and 6 of 68 (8.9%) never smokers and 13 of 104 (12.5%) ever smokers. Kras copy number was not significantly correlated with any of the clinical-pathologic factors. In the 36 cases with Kras mutation in this cohort, eight cases had increased Kras copy number. Kras copy number was more frequently increased within Kras mutant type patients (p = 0.0312). In the 46 cases with EGFR mutation in this cohort, only one case had increased Kras copy number. Kras copy number was more frequently increased within EGFR wild-type patients (p = 0.0216).
TABLE 1Clinicopathological Data of 172 Patients with Lung Cancer
We identified only one case of amplification of Kras from 40 cases using FISH analysis. We also detected one high-polysomy case (Figure 1). Using the criteria by Cappuzzo et al., two cases were FISH positive. We have also detected six low-polysomy cases. In this cohort, these eight were evaluated as ICN cases. ICN patients were 5 of 26 (19.2%) males and 3 of 14 (21.4%) females; 2 of 17 (11.8%) patients who were aged 65 years and younger and 6 of 23 (26.1%) of those who were older than 65 years; 5 of 37 (13.5%) of adenocarcinoma and 3 of 3 (100%) of nonadenocarcinoma; 1 of 10 (10.0%) never smokers and 7 of 30 (23.3%) of ever smokers; and 2 of 17 (11.8%) of stage I and 6 of 23 (26.1%) of stages II to IV. Again, we have mainly focused on Kras mutant patients for FISH analyses (mutation centric), and there was no significant correlation between the Kras ICN and these clinical-pathologic factors, except for pathological bias (Table 2). Thirty-nine patients were examined both by PCR and FISH. The average Kras copy number of eight ICN patients (2.664 ± 1.145) was significantly higher than that of 31 patients (1.872 ± 0.845) (p = 0.0357, Student's t test) (p = 0.0164, Spearman's rank correlation).
FIGURE 1Fluorescence in situ hybridization (FISH) analysis for lung cancer tissues. Left upper, disomy case; right upper, low polysomy case; left lower, high polysomy case; right lower, amplification case.
Relationships among Kras Mutation, Increased Kras Copy Number, and Clinical Outcome
Within 172 patients, Kras mutant patients (15/36 were dead, median survival = 41.3 months) had worse prognosis than Kras wild-type patients (38/136 were dead, median survival = 77.3 months, p = 0.0256). Then, we classified into three groups: N (Kras normal copy number and wild type), M or A (Kras mutation or Kras copy number >3), and MA (Kras mutation and Kras copy number >3). The Kaplan-Meier curves are shown in Figure 2. N patients showed best prognosis, M or A patients showed intermediate prognosis, and MA patients showed worst prognosis. Patients with Kras mutation and copy number more than 3 had a statistically significantly worse prognosis compared with those with wild-type Kras and normal copy number (log-rank p = 0.0294).
FIGURE 2Kaplan-Meier curves of overall survival according to Kras mutation and copy number in 172 patients. N patients (Kras copy number < 3 and Kras wild type) showed best prognosis, M (Kras mutation) or A (Kras increased copy number > 3) patients showed intermediate prognosis, and MA patients showed worst prognosis. Patients with Kras mutation and copy number > 3 had a statistically significantly worse prognosis compared with those with wild-type Kras and normal copy number (log-rank p = 0.0294).
Kras ICN Status in the FISH Analysis in 40 Patients with NCSLC and Clinical Outcome
Within 40 patients, Kras ICN patients (6/8 were dead, median survival = 15.7 months) had worse prognosis than Kras normal copy number patients (9/32 were dead, median survival = 41.3 months, p = 0.062) (Figure 3). Although clinical background was imbalanced in this cohort, multivariate analysis also showed that FISH status (ICN) (hazard ratio = 3.092, 1.034–9.247, p = 0.0434) but not pathological stage (p = 0.4319) was an independent prognostic factor.
FIGURE 3Kaplan-Meier curves of overall survival according to Kras copy number in 40 patients according to the fluorescence in situ hybridization (FISH) status. Within 40 patients, Kras increased copy number (ICN) patients (6/8 were dead, median survival = 15.7 months) had worse prognosis than Kras normal copy number patients (9/32 were dead, median survival = 41.3 months, p = 0.062).
In our study, Kras mutation, but not Kras copy number itself, correlated with survival of patients with lung cancer. Nevertheless, Kras mutation plus increased Kras copy number correlated with poor prognosis. In addition, from our FISH analysis, Kras polysomy or amplified patients showed poor prognosis. Thus, Kras copy number gain somehow plays an important role in tumor progression of lung cancers.
Oncogene activation is one of the key processes underlying the development of cancer. Oncogenes can be activated by a variety of mechanisms, such as overexpression and protein mutation leading to elevated or altered activity. Ras genes (including Kras and Nras) encode founding members of an extensive family of small guanosine triphosphate (GTP)ases, which regulates a variety of key cellular processes through their function as molecular switches.
Wild-type Ras proteins cycle between guanosine diphosphate (GDP)- and GTP-bound forms, regulated by GTPase-activating protein, which activate their intrinsic catalytic activity and by guanine nucleotide exchange factors, which stimulate the exchange of GDP for GTP. In their GTP-bound forms, these proteins engage a variety of effectors, such as RAF and phoshoinositide 3-kinase, which mediate both normal and oncogenic signaling effects. Common Ras-activating mutations are located at codons 12 and 13, rendering the proteins insensitive to the action of GTPase-activating proteins and leading to constitutively GTP-bound, actively signaling forms. Ras genes have also been found to undergo copy gains at low frequencies in several cancer types.
Alteration of chromosomal copy number during progression of diffuse-type gastric carcinomas: métaphase and array based comparative genomic hybridization analyses of multiple samples from individual tumors.
A recent high-resolution study of copy number alterations in NSCLC revealed recurrent copy number gains centered on the Kras locus, providing evidence that these gains are functionally significant and under positive selection.
Although Ras proteins mediate a variety of oncogenic stimuli (including proliferative, survival, and antiapoptotic signals), they also have context-dependent activities that can oppose the requirements for tumor growth. In particular, the activity of oncogenic mutant Ras can induce growth arrest or apoptosis, for example, in primary cells,
These diverse, context-dependent activities make Ras proteins a particularly interesting subject for the study of oncogenic alterations.
Clonally chromosomal aberrations are characteristics of tumor cells and have been found in major tumor types. Many such aberrations are established as prognostic and predictive biomarkers including lung cancers.
Epidermal growth factor receptor in non-small cell lung carcinomas: correlation between gene copy number and protein expression and impact on prognosis.
In this report, we provide direct in situ evidence that Kras ICN might somehow correlate with prognosis of NSCLC.
The Ras/Raf pathway links the receptors for growth factors with their downstream effects on gene transcription, cellular proliferation, and survival. Although some overlap with Kras-activating mutations occurred, our findings indicated that Kras ICN itself is exited in some population of NSCLC. Pulciani et al.
reported that transduction of a cell line with multiple copies of a wild-type Ras protooncogene was sufficient to induce malignant transformation in the absence of activating mutations. The author analyzed 35 lung carcinomas for Ras gene amplification using Southern blotting and reported a single case in which Kras amplification was believed to have occurred.
FISH analysis may allow for assessment of the presence and pattern of amplification of a specific gene in individual tumor cells. Thus, we were able to directly observe Kras amplification and to distinguish low-level copy number gains from high-level gains.
FISH also allowed us to detect amplification even when present in a subgroup of cells or multifocally. Our observations suggest that in some tumors, most cells contain many copies of Kras, whereas other tumors consist of a few cells containing many copies of Kras in the midst of a majority of cells containing low-level amplification. The latter pattern raises the possibility that accumulation of Kras in NSCLC could occur in a stepwise manner, beginning with low-level copy number gains and progressing through stages in which some cells continue to accumulate Kras copies. This process of progressive Kras accumulation has been observed during tumor progression in a murine model.
divided Kras mutant population into two groups, Kras-dependent and Kras-independent group. These might be explanation for our results: a part of Kras mutant patients had poor prognosis with increased Kras copy number.
In summary, Kras mutation was significantly associated with worse clinical outcome in patients with NSCLC, but increased Kras gene copy number itself was not significantly associated. In the respect of this result, the populations with Kras mutation may be different from the populations with increased Kras copy number, and the effect of increased Kras copy number to prognosis may be different from that of Kras mutation. Nevertheless, for our analysis using FISH, Kras polysomy or amplification may still be a predictor of lung cancers. In most studies, analysis of Kras copy number has been performed by FISH
FISH analysis was relatively expensive and usually performed for one or few sliced sections. Standardization of copy number analysis should be required to evaluate the copy number accurately. In our analysis, only one case had FISH amplification or high polysomy. This is why we included six polysomy cases as ICN, and using qPCR, more than 3 was determined as ICN. The determination might affect well correlation with prognosis in our study. Actually, Takano et al.
determined 3 as increased MET copy number. Thus, ICN of Kras in NSCLC is moderate.
ACKNOWLEDGMENTS
Supported by Grants-in-Aid for Scientific Research, Japan Society for the Promotion of Science (JSPS) (Nos. 19390367, 21390394, and 21591820) and a grant for cancer research of Program for Developing the Supporting System for Upgrading the Education and Research (2009) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers.
Comparative genomic hybridization analysis detects frequent, often high-level, overrepresentation of DNA sequences at 3q, 5p, 7p, and 8q in human non-small cell lung carcinomas.
Epidermal growth factor receptor in non-small cell lung carcinomas: correlation between gene copy number and protein expression and impact on prognosis.
Alteration of chromosomal copy number during progression of diffuse-type gastric carcinomas: métaphase and array based comparative genomic hybridization analyses of multiple samples from individual tumors.
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