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Keywords:

  • v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS);
  • lung adenocarcinoma;
  • mutant allele-specific imbalance;
  • prognosis;
  • prediction

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SOURCES
  8. REFERENCES

BACKGROUND

The prognostic and therapeutic implications of the spectrum of v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) oncogene substitutions in lung cancer remain poorly understood. The objective of this study was to determine whether KRAS oncogene substitutions differed with regard to prognosis or predictive value in lung adenocarcinoma.

METHODS

KRAS oncogene substitutions and mutant allele-specific imbalance (MASI) were determined in patients with lung adenocarcinoma, and the associations with overall survival (OS), recurrence-free survival (RFS), and chemotherapy interactions were assessed.

RESULTS

KRAS mutational analysis was performed on 988 lung adenocarcinomas, and 318 KRAS mutations were identified. In this predominantly early stage cohort (78.6% of patients had stage I-III disease), OS and RFS did not differ according to the type of KRAS substitution (OS, P = .612; RFS, P = .089). There was a trend toward better OS in the subset of patients with KRAS codon 13 mutations (P = .052), but that trend was not significant in multivariate analysis (P = .076). RFS did not differ according to codon type in univariate analysis (P = .322). There was a marked difference in RFS based on the presence of MASI in univariate analysis (P = .004) and multivariate analysis (P = .009). A test for interaction was performed to determine whether the effect of chemotherapy on OS and RFS differed based on KRAS substitution type, codon type, or the presence of MASI. That test indicated that there were no differences in the effects of chemotherapy for any of variables examined.

CONCLUSIONS

KRAS codon 13 mutations and MASI were identified as candidate biomarkers for prognosis, and it may be useful to incorporate them into prospective studies evaluating novel therapies in KRAS-mutant lung adenocarcinoma. Cancer 2013;119:2268–2274. © 2013 American Cancer Society.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SOURCES
  8. REFERENCES

Lung cancer is the leading cause of cancer-related mortality in the United States.[1] Nonsmall cell lung cancer (NSCLC) accounts for 85% of all lung cancers, whereas small ell lung cancer accounts for about 15%.[2] Historically treated as a single disease entity, the identification of driver mutations and the development of molecularly targeted agents against the epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) fusion oncogene have permanently shifted the landscape of NSCLC therapy toward a personalized approach.

Data from the Lung Cancer Mutation Consortium indicate that, of 1000 tumors from patients with lung adenocarcinoma, mutations in the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) were the most prevalent of the driver mutations, identified in 25% of all cases.[3] The presence of a KRAS mutation is thought to have prognostic implications with regard to survival; in a meta-analysis of studies assessing RAS mutations in lung adenocarcinomas, the presence of a RAS mutation was associated with a 50% relative increase in the risk of death.[4] However, the prognostic implications of RAS mutations have not been validated in a prospective fashion.

The predictive properties of KRAS mutations were explored in a molecular analysis of the patients included in the JBR.10 clinical trial, which evaluated the role of cisplatin and vinorelbine in the adjuvant setting.[5] In that analysis, KRAS mutations were neither prognostic of survival nor predictive of a differential benefit from adjuvant chemotherapy. Retrospective studies in the metastatic setting have failed to demonstrate a differential chemotherapy effect based on the presence or absence of a KRAS mutation.[6, 7] The predictive value of individual KRAS oncogene substitutions was explored in a pooled analysis of trials evaluating the addition of cetuximab to chemotherapy in the treatment of metastatic colorectal cancer.[8] In that study, patients with a G13D KRAS oncogene substitution derived greater benefit from cetuximab-based chemotherapy compared with patients with G12D and other KRAS oncogene substitutions, suggesting clinical heterogeneity among subtypes of KRAS mutations.

Furthermore, the significance of KRAS gene copy number change is uncertain. It has been observed that KRAS mutations may be associated with a higher KRAS gene copy number.[9] The combination of mutations and copy number gain may result in an imbalance between the wild-type allele (W) and the mutant allele (M), which may results in a situation in which M is predominant over W, a scenario defined as mutant allele-specific imbalance (MASI).[12] The incomplete dominance of M over W is most frequently a result of selective amplification of M, but it may also be because of the presence only of M in the absence of W, as in acquired uniparental disomy, which frequently leads to complete MASI.[13] Recent reports suggest that the combination of a KRAS mutation and copy number gain may be associated with adverse outcomes in patients with lung and colon adenocarcinoma.[12, 14, 15]

The objective of the current study was to assess whether the spectrum of KRAS oncogene substitutions differs with regard to their clinical behavior, specifically examining the type of KRAS amino acid (AA) substitution present, the presence of a codon 12 versus codon 13 mutation, and the presence or absence of MASI.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SOURCES
  8. REFERENCES

This was a retrospective analysis of banked tumor specimens that were collected from patients with newly diagnosed lung adenocarcinoma at the University of Pittsburgh Medical Center (UPMC) between 2005 and 2011. All formalin-fixed, paraffin-embedded specimens that remained after complete pathologic sign out of the case were considered for inclusion in the study and were selected on the basis of meeting a minimum tissue requirement of 300 cells per sample. Guided by hematoxylin and eosin (H&E)-stained slides, tumor targets that contained >70% tumor cells were manually microdissected from the 4-μm unstained histologic sections. Mutational analysis of KRAS codons 12 and 13 was then performed as previously described.[16] Briefly, DNA was isolated from each target using the DNeasy tissue kit (Qiagen, Valencia, Calif) according to the manufacturer's instructions. Polymerase chain reaction products were sequenced in both the sense and antisense directions using the BigDye Terminator version 3.1 cycle-sequencing kit on an ABI 3130 automated sequencer (Applied Biosystems, Inc., Foster City, Calif) according to the manufacturer's instructions. The sequences were analyzed using Mutation Surveyor software (SoftGenetics, LLC, State College, Penn). Samples were classified as mutated or wild type for KRAS based on the sequencing results. MASI was determined in a semiquantitative fashion on sequencing electropherograms and was defined as a KRAS mutant peak greater than the wild-type peak on (M > W) or a mutant peak equal to the wild-type peak (M = W), and no MASI was defined as a mutant peak less than a wild-type peak (M < W) (Fig. 1A).

image

Figure 1. (A) Recurrence-free survival and mutant allele-specific imbalance (MASI) are defined on sequencing electropherograms as either a KRAS-mutant (M) peak equal to the wild-type (W) peak (M = W) or a KRAS-mutant peak greater than the wild-type peak (M > W). (B) These Kaplan-Meier survival curves illustrate recurrence-free survival and KRAS MASI (to indicates time zero [baseline]). (C) Multivariate analysis of recurrence-free survival and MASI was adjusted for stage. HR indicates hazard ratio; CI, confidence interval.

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Baseline demographics, staging, and smoking history were obtained through a review of the medical record in the UPMC Medical Archival System. First-line treatment data, follow-up data with regard to first recurrences, and survival were collected through the UPMC Network Cancer Registry. Recurrence-free survival (RFS) and overall survival (OS) were calculated either from the date of surgery (in patients with stage I to III disease) or from the date of a diagnostic biopsy (in patients with stage IV disease) to the date of recurrence or death, respectively. Patients who had <30 days of follow-up were excluded from the survival analyses. Those patients who did not experience the event of interest were censored at the date of their last follow-up. Survival probabilities were estimated using the Kaplan-Meier method. Cox proportional hazard models were used to examine the effects of KRAS mutations and MASI on OS and RFS while controlling for age, sex, race, smoking history, disease stage, and receipt of chemotherapy. Only significant factors were left in the final model with the type of KRAS mutation or MASI. A test for interaction was used to determine whether the effect of the KRAS mutation or MASI was modified by the receipt chemotherapy both in patients with resected stage I to III disease (either neoadjuvant or adjuvant chemotherapy) and in the metastatic setting. This study was conducted under an exemption approved by the University of Pittsburgh Institutional Review Board.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SOURCES
  8. REFERENCES

By using the inclusion criterion of 300 cells per sample, 988 tumor specimens that were collected between 2005 and 2011 were sequenced for KRAS mutations. Of those tumors, 318 (32.2%) harbored KRAS mutations, the majority of which were codon 12 mutations (298 tumors; 93.7%). Glycine-to-cysteine (GLY[RIGHTWARDS ARROW]CYS) AA substitutions were the most common codon 12 (139 tumors; 46.6%) and codon 13 (10 tumors; 58.8%) oncogene substitutions (Table 1). This was predominantly an early stage cohort, consisting of 143 patients (45%) with stage I disease, 52 patients (16.4%) with stage II disease, 55 patients (17.3%) with stage III disease, and 64 patients (20.1%) with stage IV disease (Table 1). The median follow-up was 24.3 months (range, 0-6.5 years).

Table 1. Clinical and Pathologic Features of Patients With KRAS-Mutant Lung Adenocarcinoma
CharacteristicNo. of Patients (%)
  1. Abbreviations: ASP, aspartic acid; CYS, cysteine; GLY, glycine; KRAS, v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog; M, mutant allele; MASI, mutant allele-specific imbalance; VAL, valine; W, wild-type allele.

  2. a

    All patients with stage I through III disease underwent a surgical resection with the exception of 7 patients (2.2%) who received concurrent chemoradiotherapy and who were not included in interaction models for chemotherapy benefit. Chemotherapy for those with stage I through III disease includes both adjuvant and neoadjuvant chemotherapy. An additional 11 patients (3.5%) had an unknown treatment history.

  3. b

    All patients who had stage IV disease treated with chemotherapy in the first-line setting received conventional chemotherapy with the exception of 1 patient who received erlotinib.

Age: Median [range], y67 [39-92]
Men129 (40.6)
Women189 (59.4)
Race 
White291 (91.5)
Black21 (6.6)
Smoking status 
Current smoker102 (32.1)
Former smoker143 (45)
Never smoker18 (5.7)
Unknown55 (17.3)
Stage 
I143 (45)
II52 (16.3)
III55 (17.3)
IV64 (20.9)
KRAS mutation 
Codon 12298 (93.7)
Codon 1317 (5.3)
Codon 12 
GLY[RIGHTWARDS ARROW]CYS139 (46.6)
GLY[RIGHTWARDS ARROW]VAL59 (19.8)
GLY[RIGHTWARDS ARROW]ASP46 (15.4)
Other40 (13.4)
Unknown14 (4.7)
Codon 13 
GLY[RIGHTWARDS ARROW]CYS10 (58.8)
GLY[RIGHTWARDS ARROW]ASP6 (35.3)
GLY[RIGHTWARDS ARROW]VAL1 (5.9)
MASI 
M>W19 (6)
M=W17 (5.3)
M<W180 (56.6)
Unknown102 (32.1)
Stage I-IIIa 
Chemotherapy72 (22.6)
No chemotherapy160 (50.3)
Stage IV first-line therapyb 
Chemotherapy56 (17.6)
No chemotherapy12 (3.8)

OS did not differ according to the type of KRAS AA substitution either in univariate analysis (P = .612) (Fig. 2A) or in multivariate analysis adjusting for age and stage (P = .287). Similarly, RFS did not differ according to the type of KRAS AA substitution either in univariate analysis (P = .089) (Fig. 2B) or in multivariate analysis adjusting for stage (P = .126). It is noteworthy that there was a trend toward better OS in the subset of patients with KRAS codon 13 mutations (P = .052) (Fig. 2C); however, this trend was not statistically significant in multivariate analysis adjusting for age and stage (P = .076). In addition, RFS did not differ according to codon type either in univariate analysis (P = .322) (Fig. 2D) or in multivariate analysis adjusting for stage (P = .318). However, RFS differed significantly according to MASI status both in univariate analysis (P = .004) (Fig. 1B) and in multivariate analysis controlling for stage (P = .009). Controlling for stage, MASI, defined as M > W (hazard ratio, 2.42; 95% confidence interval, 1.06-5.52) or M = W (hazard ratio, 3.96; 95% confidence interval, 1.37-11.44), was associated with a greater risk of recurrence compared with no MASI (M < W) (Fig. 1C).

image

Figure 2. Kaplan-Meier survival curves for patients with v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations illustrate (A) overall survival according to KRAS amino acid substitution, (B) recurrence-free survival according to KRAS amino acid substitution, (C) overall survival according to KRAS codon type, and (D) recurrence-free survival according to KRAS codon type. GLY indicates glycine; t0, time zero (baseline); ALA, alanine; ASP, aspartic acid; CYS, cysteine; VAL, valine.

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Next, we examined whether KRAS mutation subtypes differed with regard to the predictive value of a chemotherapy benefit. A test for interaction was performed to determine whether the effect of chemotherapy on OS differed based on the type of KRAS AA substitution present, the codon type, or the presence or absence of MASI. Adjusting for age and stage, there were no differences in the effects of chemotherapy for any of the variables examined (AA substitution, P = .795; codon type, P = .438; and MASI, P = .598) on OS (Table 2). A similar test for interaction was performed for RFS among patients who had resected stage I through III disease to explore whether there were any differences with regard to the effects of chemotherapy in the neoadjuvant and adjuvant settings. In that analysis, no interaction was observed between the AA substitution (P = .357), the codon type (P = .790), or MASI status (P = .392) and a chemotherapy benefit (Table 3).

Table 2. The Effect of Chemotherapy on Overall Survival According to KRAS Oncogene Substitution and Mutant Allele-Specific Imbalance Adjusting for Age and Stage
 No. of Deaths/No. of Patients 
Onogene SubstitutionChemotherapyNo ChemotherapyHR (95% CI)
  1. Abbreviations: AA, amino acid; ALA, alanine; ASP, aspartic acid; CI, confidence interval; CYS, cysteine; GLY, glycine; HR, hazard ratio; KRAS, v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog; M, mutant allele; MASI, mutant allele-specific imbalance; VAL, valine; W, wild-type allele.

GLY[RIGHTWARDS ARROW]CYS32/6324/710.53 (0.27-1.05)
GLY[RIGHTWARDS ARROW]VAL8/1910/340.62 (0.23-1.72)
GLY[RIGHTWARDS ARROW]ASP11/245/220.64 (0.21-2.00)
GLY[RIGHTWARDS ARROW]ALA8/102/141.56 (0.30-8.25)
Other4/64/80.75 (0.17-3.26)
Test for interaction: AA substitution × treatment  P = .795
Codon 1265/12048/1470.58 (0.33-1.02)
Codon 131/61/111.69 (0.10-28.03)
Test for interaction: Codon × treatment  P = .438
M<W32/7016/940.83 (0.35-1.97)
M=W5/64/80.66 (0.15-2.82)
M>W5/85/100.41 (0.10-1.63)
Test for interaction MASI × treatment  P = .598
Table 3. The Effect of Chemotherapy on Recurrence-Free Survival According to KRAS Oncogene Substitution and MASI in Patients With Resected Stage I to III Disease Adjusted for Stage
 No. of Recurrences/No. of Patients 
Oncogene SubstitutionChemotherapyNo ChemotherapyHR (95% CI)
  1. Abbreviations: AA, amino acid; ALA, alanine; ASP, aspartic acid; CI, confidence interval; CYS, cysteine; GLY, glycine; HR, hazard ratio; KRAS, v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog; M, mutant allele; MASI, mutant allele-specific imbalance; VAL, valine; W, wild-type allele.

GLY[RIGHTWARDS ARROW]CYS9/2718/640.47 (0.17-1.30)
GLY[RIGHTWARDS ARROW]VAL5/107/321.06 (0.28-3.94)
GLY[RIGHTWARDS ARROW]ASP3/122/221.00 (0.16-7.60)
GLY[RIGHTWARDS ARROW]ALA1/11/147.65 (0.45-130.19)
Other3/53/71.19 (0.23-6.24)
Test for interaction: AA substitution × treatment  P = .357
Codon 1223/5633/1410.90 (0.41-1.97)
Codon 131/31/81.33 (0.08-22.18)
Test for interaction: Codon × treatment  P = .790
M<W17/3616/911.17 (0.46-2.96)
M=W0/04/80.53 (0.10-2.95)
M>W2/36/100.53 (0.10-2.95)
Test for interaction: MASI × treatment  P = .392

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SOURCES
  8. REFERENCES

In the largest published series of KRAS mutant lung adenocarcinomas to date, we report no differences in prognosis based on the type of KRAS AA substitution present, a trend toward better survival among patients with KRAS codon 13 mutations compared with codon 12 mutations, and a markedly negative prognosis associated with the presence of KRAS MASI. It is important to note that this was largely an early stage cohort, which is likely a reflection of the minimum tissue requirement needed for inclusion in the analysis.

In cell line models, KRAS codon 12 mutations appear to be a more potent oncogenic driver compared with codon 13 mutations, inducing a higher level of resistance to apoptosis and a predisposition toward anchorage-independent growth.[17] Remarkably, in the small number of patients with codon 13 mutations in the current study, there was a trend toward better OS in univariate analysis, which did not remain significant in multivariate analysis adjusting for age and stage. In a molecular analysis of 1532 patients who were included in the Lung Adjuvant Cisplatin Evaluation (LACE) meta-analysis, in total, 300 KRAS mutations were identified. KRAS mutations, considered neither as a whole nor as subsets of AA substitutions or codon types, had prognostic value in the resected early stage setting.[18, 19] Ultimately, KRAS codon 13 mutations are infrequent in number (there were 24 codon 13 mutations in the LACE meta-analysis and 17 in the current study). Thus, a robust analysis to further delineate the true prognostic value of KRAS codon 13 in lung adenocarcinoma will require pooling of codon 13 mutations across institutions to obtain an adequate number of patients.

In the relapsed metastatic setting, an analysis of KRAS AA substitutions in patients who were treated on the Biomarker-integrated Approach of Targeted Therapy for Lung Cancer Elimination (BATTLE) trial demonstrated 48 KRAS mutations among 268 tumors profiled. The presence of a GLY[RIGHTWARDS ARROW]CYS or GLY[RIGHTWARDS ARROW]VAL substitution at codon 12 was associated with significantly worse progression-free survival (PFS) compared with the other KRAS AA substitutions or wild-type KRAS.[20] Sixty-four patients with KRAS-mutant stage IV disease were included in the current analysis, and we were unable to demonstrate an association between KRAS AA substitution subtypes and survival (both OS and RFS) in a multivariate analysis controlling for stage. It should be noted that the BATTLE clinical trial reflects a refractory NSCLC population as distinct from the current population, which represents a predominantly early stage and first-line metastatic cohort. Cell line data indicate that codon 12 GLY[RIGHTWARDS ARROW]CYS and GLY[RIGHTWARDS ARROW]VAL mutations are associated with activated Ral signaling and decreased factor-dependent Akt activation.[20] The difference in the prognostic significance of individual AA substitutions between the current study and the BATTLE analysis may be a reflection of a distinct biology between the relapsed metastatic setting and the adjuvant or first-line metastatic setting.

Soh et al and others previously demonstrated that direct sequencing is a valid method for quantifying MASI using several techniques, including subcloning, plasmid mixture experiments, and restriction fragment length polymorphism combined with band intensity measurement on gel electrophoresis.[11, 12, 15, 21, 22] Stromal contamination is of concern in the determination of MASI in tumor specimens; however, review of H&E slides and manual microdissection limits the proportion of nontumor DNA in a given sample. The presence of remaining nontumor DNA would randomly affect all specimens, and nontumor DNA increases the wild-type peak and decreases the mutant:wild-type peak ratio, thereby decreasing the ability to detect MASI on sequencing electropherograms.

We demonstrated that the presence of KRAS mutant MASI was associated with a markedly inferior RFS compared with the absence of MASI. In prior studies, we demonstrated that the presence of MASI also was associated with worse OS, mirroring the adverse RFS with MASI observed in the current study.[21] The presence of MASI is frequently associated with KRAS amplification, as demonstrated by fluorescent in situ hybridization studies.[21] This suggests that the KRAS mutant:wild-type peak ratio observed on sequencing electropherograms is representative of the actual KRAS mutant allele:wild-type allele ratio within the tumor. This allelic imbalance is a reflection both of the post-transcriptional dosage of the KRAS allele and its concomitant kinase activity. Although amplification may account for KRAS MASI, other potential mechanisms may include uniparental disomy, chromosome 12 hyperdiploidy, or KRAS homozygous mutation. The implications of KRAS MASI will require prospective validation as a biomarker of prognosis.

In the current study, there was no suggestion of a predictive value for chemotherapy benefit among the subtypes of KRAS AA substitutions, among the subtypes of KRAS codons, or among the subpopulations with or without MASI. In both the JBR.10 clinical trial and the LACE meta-analysis, KRAS mutations considered as a whole were not associated with differential chemotherapy effectiveness.[5, 18] In an analysis of patients who were included in the LACE meta-analysis according to KRAS mutation subtypes, the subset of patients with KRAS codon 13 mutations fared worse with adjuvant chemotherapy relative to the observation arm.[19] In drawing a comparison with the LACE meta-analysis, KRAS codon 13 mutations in the current study were associated with a nonstatistically significant trend toward worse OS and RFS with chemotherapy versus no chemotherapy. However, the number of patients in those subanalyses was small, and the resultant confidence intervals were wide.

The major limitation of these predictive analyses (which also applies to the prognostic analyses) is that they represent a biomarker analysis that was not conducted in the context of a prospective randomized clinical trial. Comparisons in this study were made between KRAS mutation subtypes and not relative to a wild-type population, limiting our ability to draw conclusions from our predictive analyses. However, the aims of this study were to ascertain whether there were differences in prognosis and predictive benefit between different types of KRAS mutations and not necessarily between KRAS-mutant and KRAS wild-type lung adenocarcinomas. The KRAS wild-type population itself is a molecularly heterogeneous population; that is, it includes a significant proportion of patients who are KRAS wild-type but whose tumors harbor other oncogenic drivers, such as EGFR mutations or ALK translocations, among others.[3] Therefore, we chose to focus the current analyses on a purely KRAS-mutant cohort. This study is meant to be hypothesis-generating, and the incorporation of these candidate biomarkers in a prospective fashion into clinical trial design will be necessary to define the true predictive value of KRAS mutation subtypes.

FUNDING SOURCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SOURCES
  8. REFERENCES

This study was supported by National Institutes of Health/National Cancer Institute (NIH/NCI) Specialized Research Excellence in Lung Cancer Grant (P50-CA090440) and by an NIH/NCI Cancer Center Support Grant (5 P30 CA047904).

CONFLICT OF INTEREST DISCLOSURES

M. A. Socinski is a consultant for Celgene and Teva and currently receives research funding from Genentech, GSK, Merrimack, and Synta.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SOURCES
  8. REFERENCES
  • 1
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    Tejpar S, Celik I, Schlichting M, Sartorius U, Bokemeyer C, Van Cutsem E.Association of KRAS G13D tumor mutations with outcome in patients with metastatic colorectal cancer treated with first-line chemotherapy with or without cetuximab.J Clin Oncol.2012;30:35703577.
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    Engel E.A new genetic concept: uniparental disomy and its potential effect, isodisomy.Am J Med Genet.1980;6:137143.
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    Sasaki H, Hikosaka Y, Kawano O, Moriyama S, Yano M, Fujii Y.Evaluation of Kras gene mutation and copy number gain in non-small cell lung cancer.J Thorac Oncol.2011;6:1520.
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    Hartman DJ, Davison JM, Foxwell TJ, Nikiforova MN, Chiosea SI.Mutant allele-specific imbalance modulates prognostic impact of KRAS mutations in colorectal adenocarcinoma and is associated with worse overall survival.Int J Cancer.2012;131:18101817.
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    Chiosea S, Shuai Y, Cieply K, Nikiforova MN, Dacic S.EGFR fluorescence in situ hybridization-positive lung adenocarcinoma: incidence of coexisting KRAS and BRAF mutations.Hum Pathol.2010;41:10531060.
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    Guerrero S, Casanova I, Farre L, Mazo A, Capella G, Mangues R.K-ras codon 12 mutation induces higher level of resistance to apoptosis and predisposition to anchorage-independent growth than codon 13 mutation or proto-oncogene overexpression.Cancer Res.2000;60:67506756.
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    Tsao MS, Hainaut P, Bourredjem A, et al.LACE-Bio pooled analysis of the prognostic and predictive value of KRAS mutation in completely resected non-small cell lung cancer (NSCLC) [abstract].Ann Oncol.2010;21( suppl). Abstract 4218.
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    Shepherd FA, Bourredjem A, Brambilla E, et al.Prognostic and predictive effects of KRAS mutation subtype in completely resected non-small cell lung cancer (NSCLC): a LACE-Bio study [abstract].J Clin Oncol.2012;30( suppl). Abstract 7007.
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    Ihle NT, Byers LA, Kim ES, et al.Effect of KRAS oncogene substitutions on protein behavior: implications for signaling and clinical outcome.J Natl Cancer Inst.2012;104:228239.
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    Chiosea SI, Sherer CK, Jelic T, Dacic S.KRAS mutant allele-specific imbalance in lung adenocarcinoma.Mod Pathol.2011;24:15711577.
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    Oakley GJ 3rd, Chiosea SI.Higher dosage of the epidermal growth factor receptor mutant allele in lung adenocarcinoma correlates with younger age, stage IV at presentation, and poorer survival.J Thorac Oncol.2011;6:14071412.