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Prognostic Significance of K-ras Codon 12 Mutations in Patients With Resected Stage I and II Non–Small-Cell Lung Cancer

From the Departments of Medicine, Surgery, and Pathology, State University of New York Health Science Center and Veterans Affairs Medical Center, Syracuse, NY.

Address reprint requests to Stephen L. Graziano, MD, Veterans Affairs Medical Center, 800 Irving Ave, Syracuse, NY 13210

	ABSTRACT

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PURPOSE: The aim of this study was to investigate the prognostic importance of codon 12 K-ras mutations in patients with early-stage non–small-cell lung cancer (NSCLC).

PATIENTS AND METHODS: We identified 260 patients with surgically resected stage I (n = 193) and stage II (n = 67) NSCLC with at least a 5-year follow-up. We performed polymerase chain reaction analysis of DNA obtained from paraffin-embedded NSCLC tissue, using mutation-specific probes for codon 12 K-ras.

RESULTS: K-ras mutations were detected in 35 of 213 assessable specimens (16.4%). K-ras mutations were detected in 27 of 93 adenocarcinomas (29.0%), one of 61 squamous cell carcinomas (1.6%), five of 39 large-cell carcinomas (12.8%), and two of 20 adenosquamous carcinomas (10%) (P = .001). G to T transversions accounted for 71% of the mutations. There was no statistically significant difference in overall survival for all patients with K-ras mutations (median survival, 39 months) compared with patients without K-ras mutations (median survival, 53 months; P = .33). There was no statistically significant difference in overall or disease-free survival for subgroups with stage I disease, adenocarcinoma, or non–squamous cell carcinoma or for specific amino acid substitutions. The median survival time for stage II patients with K-ras mutations was 13 months, compared with 38 months for patients without K-ras mutations (P = .03).

CONCLUSION: Codon 12 K-ras mutations were more common in adenocarcinomas than in squamous cell carcinomas. For the subgroup with stage II NSCLC, there was a statistically significant adverse effect on survival for the presence of K-ras mutations. However, when the entire group was considered, the presence of K-ras mutations was not of prognostic significance in this cohort of patients with resected early-stage NSCLC.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
THE WELL-ESTABLISHED negative prognostic factors for patients with non–small-cell lung cancer (NSCLC) include higher tumor-node-metastasis system stage, weight loss, poor performance status, and presence of systemic symptoms.¹ Other studies have reported the possible prognostic impact of age, histologic subtype (squamous v nonsquamous),² degree of differentiation,³ and vascular invasion.³ Interest in the biology of lung cancer has prompted studies of other potentially useful prognostic markers, such as aneuploidy,^4,5 mutation and activation of proto-oncogenes,^6-8 loss of blood group antigens on tumor cells,⁹ deletion of tumor suppressor genes,¹⁰ tumor angiogenesis,¹¹ as well as others.¹²

The ras family of oncogenes (N-ras, H-ras, K-ras) encode for a 21-kd protein (p21), which has both guanosine 5'-triphosphate (GTP) binding and GTPase activity. ras p21 is a signal transducer located on the inner surface of the plasma membrane. Hyperexpression of ras p21 results in growth stimulation, whereas point mutations at codons 12, 13, and 61 alter its structure, preventing inactivation and causing cell transformation.

ras mutations have been reported in NSCLC, particularly adenocarcinoma, as opposed to small-cell lung cancer.^13,14 Studies have suggested that the majority of these mutations were present in codon 12 of K-ras and that G to T transversions were most common.^15,16 Rodenhuis et al¹⁷ and Bos¹⁶ have reported that about 30% of adenocarcinomas of the lung have K-ras mutations at codon 12. In addition, an association between smoking and the presence of ras mutations has been seen.¹⁷ A study by Slebos et al⁶ detected point mutations in codon 12 of K-ras in 19 of 69 completely resected patients with adenocarcinoma of the lung (28%). Tumors positive for ras mutations tended to be smaller and less well differentiated than those without mutations. With a median follow-up period of 3 years, 12 of the 19 patients with mutations died, compared with 16 of 50 patients without mutations (P = .002). The presence of K-ras mutations was associated with shortened disease-free and overall survival.⁶

The purpose of the present study was to investigate the prognostic significance of codon 12 K-ras mutations in alarge cohort of well-characterized patients with resected stage I and II NSCLC and to compare these results to those present in the literature.

	PATIENTS AND METHODS

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Patient Population
Two hundred seventy-six patients with stage I and II NSCLC who had undergone curative-intent surgery and for whom follow-up information covering at least 5 years was available were identified for study from four hospitals in Syracuse, NY. These included University Hospital (State University of New York Health Science Center), Veterans Affairs Medical Center, Crouse Hospital, and St. Joseph's Hospital. Local institutional review board approval was obtained for all studies. The patients were identified from the tumor registries, operating room records, and records of individual thoracic surgeons. Cases were excluded if there were two primary tumors at the time of diagnosis or if the patient did not leave the hospital after surgery, died within 30 days of surgery, or did not meet the definition of stage I (T1 or T2N0M0) or II (T1 or T2N1M0), as defined by the International Staging System.^18,19 This study was performed before the revisions in the International Staging System, and we therefore did not include T3N0N0 (stage IIB) patients. Because this was a community-based cohort of patients, no uniform mediastinal staging was carried out.

Once identified, the patient charts, including pathology and operative reports, were reviewed, and the data were coded. One representative formalin-fixed paraffin-embedded block of the primary tumor was obtained for each case. Sixteen cases were excluded for the following reasons: unable to obtain tissue block (eight cases), insufficient tumor in the tissue block (five cases), and diagnosis of small-cell lung cancer after review (three cases). The characteristics of the remaining 260 patients are listed in Table 1.

Cell Lines
Cell lines that each have one of the six K-ras point mutations in codon 12 were used as positive controls. Calu-1, A427, and A549 (American Type Culture Collection, Rockville, MD) were derived from lung carcinomas and have cysteine, aspartic acid, and serine mutations, respectively.^14,20 SW480 and SW1116 (American Type Culture Collection) were derived from adenocarcinomas of the colon and contain valine and alanine mutations, respectively.¹⁴ Finally, the cell line A2182, derived from a metastatic lung carcinoma and containing an arginine mutation, was a gift of Stuart Aaronson, MD, Mount Sinai Hospital, New York, NY.²¹

Also included as normal controls were five Epstein-Barr virus (EBV) immortalized B-lymphoblastoid cell lines, UMC-EBV31, UMC-EBV36, UMC-EBV46, UMC-EBV57, and UMC-EBV59, which were established by culturing the peripheral-blood lymphocytes of five small-cell lung cancer patients with EBV-containing conditioned media from the marmoset cell line B-95-8 (a gift of Berton Zbar, MD, National Cancer Institute, Frederick Cancer Research Facility, Frederick, MD).²²

Cell Line and Sample Processing
Fifty million viable cells, as determined by trypan blue exclusion, from each of the control cell lines were centrifuged at 2,000 x g for 15 minutes. The cell pellets were fixed in 10% formalin for 15 minutes, transferred to Histowrap (Obex, Inc, Amherst, NY), and placed in cassettes. The cassettes were successively treated with 10% formalin, 50% ethanol, 80% ethanol, 95% ethanol twice, absolute ethanol twice, xylene twice, and then paraffin three times in a Tissue Tek VIP Automated Processor (Ames Division, Miles Laboratories, Inc, Elkhart, IN). The fixed cell pellets were then placed in a tissue mold and embedded with Paraplast X-tra (Oxford Labware, St. Louis, MO) and then frozen to let the paraffin harden. Blocks containing 100%, 50%, 20%, 10%, 1%, 0.1%, or 0% of particular K-ras mutant carcinoma cells mixed with K-ras wild-type EBV-transformed B lymphocytes were prepared.

Sections were prepared from each of the paraffin-embedded samples using a microtome (Reichert-Jung, Heidelberg, Germany). Disposable microtome blades (Baxter Scientific, Edison, NJ) were changed, and the disposable microtome blade adaptor was washed with 1 N HCl, then 1 N NaOH, and finally RNase-free water after each block was cut to prevent contamination of samples. The first and last sections of each sample were stained with hematoxylin and eosin and reviewed by a pathologist (M.R.) to verify the diagnosis and to determine the percentage of tumor in each sample. Only those specimens containing at least 10% tumor were considered for evaluation.

Three 10-µm-thick sections of each sample were deparaffinized using xylene (two 30-minute washes) and absolute alcohol (two 5-minute washes). The samples were then allowed to dry at room temperature for at least 1 hour. After drying, 460 µL of lysis solution (150 mM NaCl, 25 mM EDTA, and 1% sodium dodecyl sulfate) and 40 µL of Proteinase K (5 mg/mL) were added to each sample. The samples were then incubated overnight on a rotating platform shaker at 37°C. DNA was organically extracted as previously described²³ and precipitated in cold 95% ethanol at -20°C overnight. The ethanol was decanted, and the DNA pellets were dried for 1 hour at room temperature. The DNA was then resuspended in 10 mM Tris-HCl (pH 7.0) at 60°C for 1 hour. DNA content was determined spectrophotometrically.

DNA-PCR
In order to determine whether amplifiable DNA was present, a 0.5-µg aliquot of each of the DNA samples was then amplified with the beta-globin primers PCO₃ and PCO₄ and analyzed by Southern blot hybridization with probe RS06, as previously described.²⁴

Polymerase chain reaction (PCR) analysis was performed using oligonucleotide primers that flank codon 12 of the wild-type and mutant K-ras oncogene (Clontech Laboratories, Inc, Palo Alto, CA). An oligonucleotide hybridization probe panel was used to identify the wild-type and all six known K-ras codon 12 mutants (Clontech Laboratories).

An optimized PCR protocol was used, as described below. MgCl concentrations were varied in a range of 1.5 to 3.5 mM, and pH was varied from 8.5 to 10. We found that 2.0 mM MgCl₂ and pH 9.0 provided the best sensitivity and specificity (data not shown). We also optimized the ratio of the nucleoside triphosphates deoxyuridine triphosphate (dUTP) and deoxyribothymidine 5'-triphosphate (dTTP) in order to use uracil-N-glycosylase (Perkin-Elmer, Norwalk, CT) to prevent "carryover" contamination. Testing four different concentrations of dTTPs and dUTPs (75% dTTPs + 25% dUTPs, 50% dTTPs + 50% dUTPs, 25% dTTPs + 75% dUTPs, and 100% dUTPs), we found that the mixture of 75% dTTPs + 25% dUTPs provided a hybridization signal of maximum intensity and still allowed for more than 99.9% degradation of K-ras PCR product (data not shown). Anti-Taq antibodies (a gift from Ortho Clinical Diagnostics, Inc., Rochester, NY) were used to prevent nonspecific amplification at low temperatures²⁵ and also to improve assay performance (data not shown).

A 0.5-µg aliquot of each of the DNA samples was amplified in duplicate in a Gene Amp PCR system 9600 (Perkin-Elmer). After an initial warming of the DNA at 95°C for 3 minutes, the thermocycler program consisted of 60 cycles of the following parameters: 96°C for 1 minute, 56°C for 1 minute, and 74°C for 1 minute. After the last cycle, the temperature was held at 72°C until the amplified DNA was further processed. The hybridization and wash conditions were optimized using three different hybridization temperatures (37°C, 63°C, and 65°C) in combination with three different wash temperatures (58°C, 61°C, and 63°C) for the wild-type and K-ras mutant control targets. Hybridization and wash temperatures of 65°C and 63°C, respectively, were the most sensitive and specific conditions for all targets.

Each sample was analyzed on four sets of Southern blots. The first set was hybridized with the wild-type probe, whereas the remaining three sets were hybridized with a different mutant-specific probe. After initial overnight autoradiography, these latter membranes were then "stripped" using a solution containing 290 mM NaCl, 20 mM NaH₂PO₄, 1.9 mM EDTA, and 1.0% sodium dodecyl sulfate and boiling for 10 minutes. Autoradiography was then performed to ensure that all signals had been removed from the membranes. The three membranes were then hybridized with one each of the remaining mutant K-ras probes. To ascertain the relative sensitivity and specificity of this hybridization schema, multiple replicates were performed for each hybridization with the 100%, 50%, 20%, 10%, 1%, 0.1%, and 0% mutant and wild-type control specimens. An input concentration dose-response curve was observed for each mutant, with 100% sensitivity and specificity being observed down to the 10% concentration. Below that concentration, sensitivity deteriorated. Hence, in the comparative analyses performed on the data, only those samples having a signal greater than the 10% mutant control were considered positive for that mutant.

Statistical Considerations
Survival time was defined as the time from the date of surgery until death from any cause. Disease-free survival was defined as the time from surgery until the time of recurrence or date of last follow-up. An observation was censored for disease-free survival at the date of death due to other causes or date of last follow-up. Life probability calculations were performed using the Kaplan-Meier method²⁶ and the log-rank test to detect differences in survival curves.²⁷ Mutations of K-ras as well as the clinical, pathologic, and biologic variables were tested for association with overall survival and disease-free survival.²⁸ Cox's proportional hazard models were used to determine the impact of patient characteristics on overall survival and disease-free survival.²⁹ For purposes of analysis, categorical variables were broken into dichotomous groups. The Mantel-Haenszel ² statistic was used to determine differences in categorical variables.³⁰

	RESULTS

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The characteristics of the 260 patients are listed in Table 1. The median age was 64 years, and 71% were males. One hundred ninety-three patients (74%) had stage I disease, and 67 patients (26%) had stage II disease. The histologic subtypes were adenocarcinoma (42.3%), squamous cell (28.1%), large-cell anaplastic (16.2%), adenosquamous (10.4%), and bronchioloalveolar (1.9%). Sixty-six percent of patients underwent preoperative mediastinoscopy, and 232 patients (89%) had at least one bronchial, hilar, or mediastinal node noted in the pathology report. Of the stage II patients, 19 were treated with postoperative radiation therapy, and one received adjuvant chemotherapy. The median survival times for the stage I and II patients were more than 60 and 32 months, respectively. The 3- and 5-year survival rates were 67% and 53% for stage I patients and 49% and 25% for stage II patients, respectively.

Of the 260 specimens, 213 yielded interpretable results for codon 12 K-ras mutations. The characteristics of the 213 assessable patients did not differ from those of the entire cohort (Table 1). Forty-seven could not be analyzed because there was less than 10% tumor in the specimen (33) or there was no wild-type K-ras signal (14). Of the 213 patients analyzed, 35 (16.4%) had a codon 12 K-ras mutation, including 27 of 93 patients with adenocarcinomas (29.0%), one of 61 patients with squamous cell carcinomas (1.6%), five of 39 patients with large-cell carcinomas (12.8%), and two of 20 patients with adenosquamous carcinomas (10%). The difference in detection rates between squamous cell carcinoma (one of 61 patients) and non–squamous cell carcinoma (34 of 152 patients) was statistically significant (P < .001).

Figure 1 shows an example of a Southern blot for the detection of glycine (wild-type K-ras) and the cysteine mutation. K-ras mutations were observed in 34 of 198 smokers (17.2%) and one of 14 nonsmokers (7.1%) (P = .33). Table 2 lists the specific codon 12 K-ras mutations that were observed. G to T transversions were the most common, with 18 of 35 cysteine (51%) and seven of 35 valine (20%) mutations detected.

View larger version (85K): [in this window] [in a new window]   Fig 1. Southern blot analysis of K-ras wild-type (A) and cysteine mutation (B). Controls include primer only, B-lymphoblastoid cell lines, and cell lines with known K-ras mutations. Two definite mutations (100P, 124P) and one borderline mutation (12P) are seen (B). Repeat analysis of 12P was positive.

When the total group was analyzed, there was no significant difference in overall or disease-free survival for patients with or without codon 12 K-ras mutations (Table 3 and Fig 2). The median survival time for all patients with codon 12 K-ras mutations was 39 months, compared with 53 months for those without mutations (P = .33). When analyzed by subgroups, there was no significant difference seen for overall survival or disease-free survival for patients with or without codon 12 K-ras mutations for those with stage I disease, adenocarcinoma alone, or nonsquamous carcinomas (Table 3). However, for patients with stage II disease, the median survival was 13 months for those with K-ras mutations (n = 13) and 38 months for those without K-ras mutations (n = 49) (P = .03) (Table 3 and Fig 3). There was no statistically significant difference in survival when analyzed by the specific K-ras mutations (Table 2 and Fig 4). A Cox proportional hazards regression model which included age, N-stage, T-stage, and K-ras showed a significant association with overall survival for all variables except K-ras mutation. The relative risks and 95% confidence intervals for each were as follows: age, 1.03 (1.01 to 1.05); N-stage, 1.97 (1.36 to 2.83); T-stage, 1.89 (1.25 to 2.94); and K-ras, .76 (.50 to 1.20).

View larger version (50K): [in this window] [in a new window]   Fig 2. (A) Overall survival curves for patients with and without codon 12 K-ras mutation (P = .33). (B) Disease-free survival curves for patients with and without codon 12 K-ras mutations (P = .90).

View larger version (26K): [in this window] [in a new window]   Fig 3. Overall survival for stage II patients with and without codon 12 K-ras mutations (P = .03).

View larger version (27K): [in this window] [in a new window]   Fig 4. Overall survival for patients with specific codon 12 K-ras mutations compared with wild-type (K-RAS-). CYS, Cysteine (n = 18); VAL, valine (n = 7); ASP, aspartic acid (n = 4); ALA, alanine (n = 3); ARG, arginine (n = 2).

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
If reliable prognostic factors for recurrence and death could be found, then patients identified as being at high risk could be offered innovative systemic therapies. The present study was designed to test the prognostic significance of codon 12 K-ras mutations in patients with resected stage I and II NSCLC. We chose to limit our study to early-stage NSCLC, because the utility of such a prognostic marker is greater in a group of patients treated with potentially curative therapy. A sensitive, allele-specific codon 12 K-ras PCR assay was developed, and considerable effort was taken to avoid false-positives. When the entire group of patients was considered, codon 12 K-ras mutations were not of prognostic significance except for the subgroup of patients with stage II disease. The other major finding was that codon 12 K-ras mutations were more common in adenocarcinomas than in squamous cell carcinomas.

Table 4 lists the studies that have investigated K-ras mutations in patients with NSCLC. Of 13 studies, five examined patients with all histologies of NSCLC and eight confined their analysis to patients with adenocarcinoma.^6,15,31-41 When all patients with NSCLC are considered, it seems that about 20% have a mutation of K-ras. For the subgroup with adenocarcinoma, the incidence of K-ras mutations is higher, about 30%. In our study, only one of 61 patients with squamous cell carcinoma (1.6%) had a K-ras mutation. The low frequency of K-ras mutations in patients with squamous cell carcinoma has been seen in other series. Rodenhuis and Slebos⁴² found no K-ras mutations in 43 squamous cell carcinomas and confined subsequent work to adenocarcinomas. Keohavong et al³⁷ reported one of 37 squamous cell carcinomas (3%) and Kwiatkowski et al⁴¹ found only three of 62 squamous cell carcinomas (5%) to have a mutation.

In contrast, two non-U.S. studies reported a significant percentage of squamous cell carcinomas with mutations at K-ras. Rosell et al³⁵ reported an 18% incidence of K-ras mutations in patients with squamous cell carcinoma. In their study, squamous cell carcinoma made up 59% of the entire group, a percentage higher than seen in most United States series. In addition, G to A transitions accounted for 37% of the mutations. Cho et al³⁸ reported similar findings in a South Korean study. By contrast, in most United States series, including ours, G to T transversions predominate.

Although early studies suggested that K-ras mutations were an important negative prognostic factor, several more recent and larger studies cast doubt on this (Table 4). The interpretation of many of these studies is limited by small numbers and the inclusion of heterogeneous groups of patients. Often, patients with stages ranging from I to IV are included, groups with vastly differing prognosis based on stage alone. In the recent large series of 275 patients reported by Rosell et al,³⁶ ras mutation was not a significant prognostic factor for the group as a whole. A recent study by Keohavong et al³⁷ detected K-ras mutations in 39 of 126 adenocarcinomas (31%) but found no correlation with survival for the entire group. For the 63 stage I patients, there was a trend for shorter survival in the patients with K-ras mutations (P = .14). They also found a strong association between smoking and the presence of K-ras mutations. A second study, reported by Siegfried et al,³⁹ in 181 patients with adenocarcinoma showed no statistically significant effect on survival for the entire group (stages I to IV) or for the subgroups with earlier stage (stage I and II). Likewise, Kwiatkowski et al,⁴¹ in a study of 244 patients with stage I disease, showed that codon 12 K-ras mutations were not prognostic for cancer-free or overall survival on univariate analysis. However, on multivariate analysis, codon 12 K-ras mutations were a statistically significant predictor of recurrence but not overall survival. Thus, the present study is in agreement with several recent large studies which have shown either no statistically significant effect on survival or an effect on survival only for a limited subgroup. Effects that are seen only in subgroups may not be reliable and need to be confirmed by other studies.

Since there is evidence that certain K-ras are more oncogenic than others,⁴³ several investigators have suggested an effect for specific K-ras mutations on survival in NSCLC.^36,39 In the present study, we found no statistically significant differences in survival on the basis of specific amino acid substitutions. This is not surprising, considering the small numbers of individual mutations. Currently, there are not sufficient data to conclude that specific codon 12 K-ras mutations are prognostic.

In summary, in a large cohort of patients with resected stage I and II NSCLC, we found that codon 12 K-ras mutations were common in patients with adenocarcinoma (29%) but infrequent in squamous cell carcinoma (1.6%) and that the majority of mutations (71%) were G to T transversions. Although an adverse effect on survival was seen for the subgroup of patients with stage II disease, codon 12 K-ras mutations were not of prognostic significance for the entire group.

	ACKNOWLEDGMENTS

 
Supported by the Veterans Affairs Research Service

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Submitted June 4, 1998; accepted October 22, 1998.

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