KRAS mutation and DNA repair and synthesis genes in non‑small‑cell lung cancer

  • Authors:
    • Vienna Ludovini
    • Biagio Ricciuti
    • Francesca R. Tofanetti
    • Clelia Mencaroni
    • Diana Giannarelli
    • Angelo Sidoni
    • Maria S. Reda
    • Annamaria Siggillino
    • Sara Baglivo
    • Lucio Crinò
    • Guido Bellezza
    • Rita Chiari
    • Giulio Metro
  • View Affiliations

  • Published online on: October 1, 2018     https://doi.org/10.3892/mco.2018.1731
  • Pages: 689-696
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Abstract

The aim of the present study was to assess the expression of select DNA repair and synthesis genes in non-small-cell lung cancer (NSCLC) according to KRAS mutation status. ERCC1, TS, RRM1, and BRCA1 mRNA expression levels were assessed from either primary or metastatic tumor specimens of patients diagnosed with epidermal growth factor receptor (EGFR) wild‑type (WT) advanced NSCLC. Total RNA was isolated from paraffin‑embedded tumor specimens using the RNeasy FFPE kit and automatically purified using a QiaCube instrument. Quantification levels were analyzed by real‑time one‑step RT‑PCR using QuantiFast technology, and the results were compared considering β‑actin as the internal reference gene. One hundred and eighty‑four patients with advanced NSCLC were evaluated for the analysis, of which 92 were KRAS‑mutants. Nearly all patients had adenocarcinoma histology (96.7%). Among KRAS‑mutants, the majority had a KRAS codon 12 mutation (88%), the most common being G12C (44.4% of cases). Mean ERCC1 levels were indicated to be significantly higher in KRAS‑mutants when compared with KRAS WT patients (3,234±6.63 vs. 184±1.24; P=0.05). However, mean TS levels were significantly lower in the KRAS‑mutant subgroup compared with the KRAS WT subgroup (4,481±3.756 vs. 5,941±6.4; P=0.039). KRAS‑mutant NSCLCs are more likely to express high ERCC1 and low TS levels. This finding may suggest different sensitivity to cytotoxic chemotherapy according to KRAS mutation status.

Introduction

In the last decade, research in the field of cytotoxic chemotherapy for non-small-cell lung cancer (NSCLC) has considerably slowed down, mainly due to the clinical development of targeted therapies for use in patients whose disease harbors driver genetic alterations, as well as immunotherapy for patients without actionable mutations (1). Nevertheless, the majority of newly diagnosed advanced NSCLCs do not harbor actionable genetic mutations, and only 15–20% derive clinical benefit from immunotherapy, which still makes conventional chemotherapy a fundamental treatment option for a number of patients. In this context, the identification of patients who will benefit from cytotoxic treatment would help physicians to deliver effective treatment to sensitive patients, while preventing others from suffering the side effects of inactive drugs. In recent years, several predictive markers of sensitivity to chemotherapy have been investigated with this purpose, including excision repair cross-complementation 1 (ERCC1), thymidylate synthase (TS), ribonucleotide reductase M1 (RRM1), and breast cancer susceptibility 1 (BRCA1), which are DNA synthesis and repair genes that can potentially predict sensitivity to platinum agents, pemetrexed, gemcitabine, and taxanes, respectively (2,3).

Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation represents the most common genetic alteration in NSCLC, being found in approximately 20–30% of patients (4). Although KRAS acts as a driver mutation in NSCLC, it is not yet an actionable target, since clinical trials with targeted therapies aimed at blocking the RAS pathway have invariably led to disappointing results. On the other hand, recent data have unveiled a negative predictive role for KRAS mutation with regard to cytotoxic treatment, particularly platinum-based chemotherapy, which still represents the standard of care for a few KRAS-mutant advanced NSCLCs (5,6).

Against this background, we investigated the mRNA expression levels of ERCC1, TS, RRM1, and BRCA1 in KRAS-mutant advanced NSCLC patients in order to provide a plausible explanation to the clinical observation that has linked KRAS mutation to poor sensitivity to cytotoxic chemotherapy.

Materials and methods

Study design and patients

Patients diagnosed with epidermal growth factor receptor (EGFR) wild type (WT) advanced NSCLC at the Medical Oncology of Perugia Hospital from January 2006 and November 2016 were eligible for this study. EGFR and KRAS mutation tests were performed on tumor tissue (either primary or metastic, if both tissues were available metastatic cancer specimen was preferred) following physician's request in patients who were eligible to receive cytotoxic treatment for advanced disease. The mRNA expression levels of ERCC1, TS, RRM1, and BRCA1 were assessed through reverse transcription-polymerase chain reaction (RT-PCR) in patients with available tumor tissue. If there was not enough tissue for the evaluation of all markers, the analysis was sequentially conducted according to the following order: ERCC1, TS, RRM1, and BRCA1. In case further tissue was available, anaplastic lymphoma kinase (ALK) gene status was performed.

This retrospective study was approved by local Ethics Committee (Comitato Etico Aziende Sanitarie Umbria), waiving patient consent.

Assessment of EGFR and KRAS mutation status

Formalin-fixed paraffin-embedded (FFPE) tumor blocks were reviewed for quality and tumor content. Tumor cells (≥70%) were macrodissected, and genomic DNA was isolated using QIAmp DNA extraction kit and automatically purified by BioRobot EZ1 instrument (Qiagen S.p.A., Milan, Italy) according to the manufacturer's instructions. Nested polymerase chain reactions (PCRs) were carried out using primers to amplify exons 18 to 21 of EGFR and exons 2 to 3 of KRAS. To facilitate sequencing, internal primers incorporated an M13Tag. PCR products were purified with Exonuclease 1 and Shrimp Alkaline Phosphatase (ExoSAP-IT) a 37°C for 15 min followed by heating at 80°C for 15 min to stop the enzymatic reaction. After purification, the PCR products were sequenced with forward and reverse M13 primers and Big Dye Terminator v1.1 Cycle Sequencing Kit. Sequencing fragments were detected by capillary electrophoresis using 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Electropherograms were analyzed for the presence of mutations using SeqScape v2.7 Software. In all cases, samples harboring mutations were reamplified and resequenced using the same experimental conditions.

Assessment of the mRNA expression levels of ERCC1, TS, RRM1 and BRCA1

RNA was extracted and purified from five consecutive 8-µM slides of microdissected FFPE tumor tissue, using RNeasy FFPE Kit on QIAcube instrument (Qiagen, Milan, Italy). Expression levels of ERCC1, TS, RRM1, and BRCA1 were evaluated on 100 ng RNA of each tumor sample and compared to synthetic healthy lung RNA, using QuantiFast Probe Duplex Assays and QuantiFast Probe RT-PCR Plus Kit (Qiagen, Milan, Italy). This kit offers an integrated genomic DNA removal step to avoid false-positive signals. Reverse transcription PCR and RT-PCR took place in the same tube (One Step RT-PCR). QuantiFast Probe Assays were predesigned to enable amplification and detection of specific gene targets (ERCC1 cat. no. QF00270641; TS cat. no. QF00102375; RRM1 cat. no. QF00452382; BRCA1 cat. no. QF0043126 by Qiagen). These assays were based on dual-labeled hydrolysis probe detection using two different dyes, FAM binds the specific gene of interest and MAX binds selected reference gene (duplex PCR). The reaction probe mix was aliquoted into specific PCR tubes for Rotor-Gene Q Instrument and the cDNA samples were then added. Cycling conditions for one-step RT-PCR included: denaturation at 95°C for 20 min and the PCR conditions included an initial denaturation at 95°C for 5 min followed by 45 cycles of denaturation at 95°C for 15 sec and annealing at 60°C for 30 sec.

All runs included a calibrator sample and a one no-template control, and all samples were measured in triplicate. Comparative Cq method was used for gene expression quantification using β-actin as internal reference gene and commercial RNA control (mRNA from lung, Stratagene, La Jolla, CA, USA) as calibrator. Final expression values were determined as follows: 2−(ΔCt sample-ΔCt calibrator), where ΔCt values of the sample and the calibrator are estimated by subtracting the Cq value of the target gene from the median of the reference genes values as reported by Livak and Schmittgen (7).

Assessment of ALK gene rearrangement

ALK immunohistochemistry based on a 4-tiered score (clone D5-F3, cat. no. 3633, Cell Signaling Technology, dilution 1:250, incubation time: 30 min/room temperature) according to a laboratory developed test was used as screening method for the assessment of ALK status. For each stain positive controls, represented by inflammatory myofibroblastic tumour, were used. Based on the mutual exclusiveness of driver mutations, only patients who were KRAS WT were tested for ALK. In case of any ALK IHC positivity, specimens underwent confirmatory FISH (Break Apart Vysis Probe kit). ALK FISH-positive patients were excluded from the present analysis.

Statistical analysis

Statistical analysis was conducted using the SPSS statistical software package (version 24; SPSS, Inc., Chicago, IL, USA). Chi-square tests or Fisher exact tests were used to analyze correlations between EGFR mutation or ALK rearrangement status and clinico-pathologic variables. Data related to mRNA expression levels of ERCC1, TS, RRM1 and BRCA1 were presented as mean and standard deviation. Student's t test was used for comparison of two groups. All statistical tests were conducted at a 2-sided level of significance of P<0.05.

Results

Patients characteristics

From January 2006 and November 2016 184 EGFR WT advanced NSCLC patients were evaluable for the analysis of at least one DNA repair gene, of which 92 were KRAS-mutants. Table I lists patients characteristics. Overall, the median age was 62 years, 60.3% of patients were male, and 89.7% of patients were current/former smokers. Virtually all patients had adenocarcinoma histology. Among KRAS-mutants, the majority had a KRAS codon 12 mutation (88%), the most common being G12C (44.4% of cases). When KRAS-mutant patients were compared with those who were KRAS WT, significantly more individuals in the KRAS-mutant group were current/former smokers (P=0.003) and males (P=0.023) (Table I). No other statistically significant differences were observed.

Table I.

Patients characteristics.

Table I.

Patients characteristics.

VariableAll patients KRAS-mutantKRAS wild typeP-value
Number of patients, n1849292
Median age, year (range)62 (23–85)63 (42–82)62 (23–85)0.589
Sex, n (%)
  Male111 (60.3)63 (68.5)48 (52.2)0.023
  Female73 (39.7)29 (31.5)44 (47.8)
Performace status, n (%)
  0–1174 (94.5)88 (95.7)86 (93.5)0.474
  ≥210 (5.5)4 (4.3)6 (6.5)
Stage IV
  De novo129 (70.1)65 (70.7)64 (69.6)0.871
  Recurrent55 (29.9)27(29.3)28 (30.4)
Smoking history, n (%)
  Nevera19 (10.3)4 (4.3)15 (16.3)0.003
  Current/former165 (89.7)88 (95.7)77 (83.7)
Histology, n (%)
  Adenocarcinoma178 (96.7)91 (98.9)87 (94.6)0.090
  Squamous cell carcinoma6 (3.3)1 (1.1)5 (5.4)
ALK gene status
  Negative57 (31.0)57 (62.0)
  Not assessed127 (69.0)92 (100.0)b35 (38.0)c
KRAS mutations, n (%)
  Codon 12 81 (88.0)
  G12C 36 (44.5)
  G12V 18 (22.2)
  G12D 10 (12.3)
  G12A 8 (9.9)
  G12F6 (7.4)
  G12R 3 (3.7)
  Codon 13 6 (6.6)
  G13C 3 (50.0)
  G13D 3 (50.0)
  Codon 59 1 (1.1)
  Codon 61 4 (4.3)

a <100 cigarettes in a lifetime.

b Not assessed because of mutual exclusiveness of driver mutations.

c Not assessed due to unavailable tissues for ALK gene assessment. Statistically significant P-values are provided in bold. KRAS, Kirsten rat sarcoma viral oncogene homolog; ALK, anaplastic lymphoma kinase.

ERCC1, TS, RRM1 and BRCA1. Overall, TS and RRM1 expression levels were significantly lower in patients with non-squamous NSCLC as compared with squamous patients (P=0.032 and P=0.017, respectively) (Table II). Similarly, never smokers had significantly lower levels of TS expression vs. smokers (P=0.021). No significant differences were noted for any characteristics according to the type of KRAS mutation (data not shown). Likewise, no significant differences were noted with regard to the distribution of ERCC1, TS, RRM1, and BRCA1 in relation to the type of KRAS mutation (G12C vs. other) (Table III).

Table II.

Patients characteristics and expression levels of ERCC1, TS, RRM1, and BRCA1.

Table II.

Patients characteristics and expression levels of ERCC1, TS, RRM1, and BRCA1.

VariableERCC1 (mean)TS (mean)RRM1 (mean)BRCA1 (mean)
Sex, n (%)
  Male2.415.4212.1511.07
  Female2.734.913.8211.17
  P-valueNSNSNSNS
Performace status, n (%)
  0–12.595.3112.7211.07
  ≥21.162.613.7310.3
  P-valueNSNSNSNS
Stage IV
  De novo2.124.8811.8511.16
  Recurrent3.375.8714.7810.99
  P-valueNSNSNSNS
Smoking history, n (%)
  Never2.665.413.0211.55
  Current/former1.362.8810.757.14
  P-valueNSP=0.021NSNS
Histology, n (%)
  Adenocarcinoma2.535.0712.3910.87
  Squamous cell carcinoma2.689.2824.9817.65
  P-valueNS P=0.032a P=0.017aNS

{ label (or @symbol) needed for fn[@id='tfn4-mco-0-0-1731'] } Statistically significant P-values are provided in bold.

a Although statistically significant, this comparison should be interpreted with caution due to the low number of squamous cell carcinoma. ERCC1, excision repair cross-complementation 1; TS, thymidylate synthase; RRM1, ribonucleotide reductase M1; BRCA1, breast cancer susceptibility 1; n, number; NS, not significant.

Table III.

Median and mean expression levels of TS, ERCC1, RRM1, and BRCA1 according to KRAS codon 12 mutations.

Table III.

Median and mean expression levels of TS, ERCC1, RRM1, and BRCA1 according to KRAS codon 12 mutations.

VariableAll patientsG12COther codon 12 mutationP-value
ERCC
  N813645
  Mean ± SD3.37±6.934.98±102.08±2.030.062
  Median (range)1.69 (0.43–55.02)1.91 (0.43–11.4)1.38 (0.53–55.02)0.074
TS
  N793544
  Mean ± SD4.25±3.383.92±3.054.52±3.450.423
  Median (range)1.73 (0.04–14.7)3.03 (0.04–11.8)3.48 (0.25–14.7)0.415
RRM1
  N773443
  Mean ± SD12.87±14.8314.64±19.211.47±8.990.343
  Median (range)8.84 (1.04–93.98)8.89 (1.04–93.98)8.02 (1.35–38.29)0.516
BRCA1
  N723339
  Mean ± SD9.54±8.459.74±8.879.9±8.270.863
  Median (range)6.66 (0.01–35.21)5.73 (0.01–32.18)7.05 (0.45–35.21)0.921

[i] ERCC1, excision repair cross-complementation 1; TS, thymidylate synthase; RRM1, ribonucleotide reductase M1; BRCA1, breast cancer susceptibility 1; SD, standard deviation; N, number.

Table IV lists the mRNA expression levels of ERCC1, TS, RRM1, and BRCA1 in all patients and according to KRAS mutation status. ERCC1 levels ranged from 0.07 to 55.02, with a mean of 2.54 and median of 1.55; as for TS, the levels ranged from 0.04 to 35.0, with a mean of 5.21 and median of 3.78. RRM1 levels ranged from 0.001 to 93.9, with a mean of 12.82 and a median of 6.86; BRCA1 levels ranged from 0.01 to 64.3, with a mean of 11.11 and median of 7.62. When analyzed according to KRAS mutation status, KRAS-mutant patients had significantly higher mean levels of ERCC1 as compared with KRAS WT patients (3.23±6.63 vs. 1.84±1.24; P=0.052, Table IV, Fig. 1A). On the other hand, mean expression levels of TS were significantly lower in patients who were KRAS-mutant as compared with KRAS WT patients (4.48±3.75 vs. 5.94±6.4; P=0.039, Table IV, Fig. 1B). No statistically significant differences were noted for the median expression levels of ERCC1 and TS according to KRAS mutation status (Table IV). Similarly, no significant differences were observed neither for mean nor for median levels of RRM1 and BRCA1 according to KRAS mutation status.

Table IV.

Median and mean expression levels of TS, ERCC1, RRM1, and BRCA1.

Table IV.

Median and mean expression levels of TS, ERCC1, RRM1, and BRCA1.

VariableAll patients KRAS-mutantKRAS WTP-value
ERCC1
  N1849292
  Mean ± SD2.54±4.823.23±6.631.84±1.240.052
  95% CI1.83–3.241.86–4.61.58–2.1
  Median (range)1.55 (0.07–55.02)1.61 (0.1–55.02)1.54 (0.07–7.12)0.623
TS
  N1808991
  Mean ± SD5.21±4.754.48±3.755.94±6.40.039
  95% CI4.51–5.913.79–5.354.87–7.51
  Median (range)3.78 (0.04–35.0)3.34 (0.05–17.6)3.91 (0.04–35.3)0.133
RRM1
  N1768789
  Mean ± SD12.82±12.0512.82±14.0212.82±9.820.991
  95% CI11.03–14.619.83–15.8110.75–14.89
  Median (range)6.86 (0.001–93.9)8.84 (1.04–93.9)9.1 (0.001–56.8)0.332
BRCA1
  N1718289
  Mean ± SD11.11±10.0310.16±8.9811.99±10.880.234
  95% CI9.6–12.638.18–12.139.7–14.29
  Median (range)7.62 (0.01–64.3)7.58 (0.01–44.5)7.99 (0.1–64.3)0.223

[i] Statistically significant P-values are provided in bold. ERCC1, excision repair cross-complementation 1; TS, thymidylate synthase; RRM1, ribonucleotide reductase M1; BRCA1, breast cancer susceptibility 1; SD, standard deviation; CI, confidence interval; N, number.

Discussion

The aim of this study was to associate the mRNA expression levels of ERCC1, TS, RRM1 and BRCA1 with KRAS mutation status in patients with advanced NSCLC. In light of the mutual exclusiveness existing between EGFR mutation and KRAS mutation, and the evidence that EGFR-mutant patients belong to a biologically distinct subset of patients, we considered it was important to exclude from the analysis EGFR-mutant patients, thus focusing only on individuals whose tumor had a documented EGFR WT status.

Interestingly, we showed for both ERCC1 and TS a similar range of expression levels and median values as compared with a previous report from Maus et al, which analyzed the distribution of ERCC1, TS, and RRM1 in >2,000 NSCLC specimens (8). Likewise, similarly to the Maus et al study, we found that these markers were expressed at a lower level in adenocarcinoma histology as compared with squamous cell carcinoma, which was statistically significant only for TS and RRM1 in our study (Table II). However, it should be noted that the present study was not powered for addressing differences according to histology owing to the small number of squamous cell carcinomas that were included. Therefore, these results should be interpreted very cautiously.

Importantly, we observed that KRAS-mutant patients had significantly higher mean ERCC1 expression levels as compared with KRAS WT patients (P=0.052, Table IV, Fig. 1A). Accordingly, our group and others have previously reported that KRAS-mutant advanced NSCLCs perform poorly on platinum-based chemotherapy (9,10). This finding has been further corroborated by two metanalyses, in which KRAS-mutant advanced NSCLCs treated with platinum-based chemotherapy appeared to experience significantly lower response rates and progression-free survival as compared with the KRAS WT counterpart (5,6). Therefore, the higher mean levels of ERCC1 that have been found in KRAS-mutant patients provide a molecularly plausible explanation for the poor sensitivity to platinum-based chemotherapy observed in patients whose tumor harbors a KRAS mutation.

On the other hand, we reported significantly lower mean levels of TS expression in KRAS-mutant patients as compared with KRAS WT patients (P=0.039, Table IV, Fig. 1B). This finding might imply an increased sensitivity to pemetrexed-based chemotherapy in KRAS-mutant advanced NSCLCs, which would be of clinical relevance owing to the fact that KRAS mutation are mainly found in adenocarcinoma patients, and pemetrexed is approved for clinical use in this histological subset only (4,11). Previously, Moran and colleagues have already reported that KRAS-mutant cell line depend on enhanced folate metabolism in functional experiments (12). Accordingly, a small retrospective analysis suggested that KRAS mutation might predict sensitivity to pemetrexed (13). Moreover, several clinical studies have already reported that TS levels may represent a predictive biomarker for antifolate agents in NSCLC (2). Against this well-established background, we found that patients with KRAS-mutant NSCLC had lower levels of TS, which might explain the enhanced sensitivity to pemetrexed reported in literature. Although preliminary, these findings suggest that the KRAS-mutant subset of patients, for whom no targeted therapies are available, may exhibit a particular sensitivity to pemetrexed, which could represent the basis for the design of future clinical studies aimed to further address this issue.

Importantly, KRAS-mutant NSCLC is a heterogeneous disease, as many type of different type amino-acid substitutions result into several types of KRAS gene mutations. On this basis, in order to exclude an imbalance in terms of expression levels of either ERCC1 and TS, we performed an analysis in KRAS-mutant codon 12 patients according to the type of KRAS mutation (other KRAS codon 12 mutations excluded due to the low number). However, we were not able to identify any differential mRNA expression levels for any markers (Table III). Therefore, we conclude that ERCC1 and TS expression levels were homogeneously distributed in KRAS-mutant codon 12 patients, regardless of the KRAS mutation variant.

Of note, our findings further enlarge the evidence indicating that each molecularly defined subgroup of NSCLC is associated with a different of expression of DNA synthesis and repair genes. In fact, some authors have previously reported that EGFR-mutant NSCLCs express lower ERCC1 expression levels as compared with EGFR WT patients, which might account for the increased sensitivity to platinum-based chemotherapy in EGFR-mutant NSCLCs (1416). Likewise, lower TS expression levels were observed in ALK-positive NSCLCs, which results into greater benefit from pemetrexed-based chemotherapy in ALK-positive patients (1719). Therefore, we provide evidence that, despite being a heterogeneous disease, also KRAS-mutant NSCLC is associated with a peculiar pattern of expression of DNA synthesis and repair genes, mainly ERCC1 and TS, which, in turn, could account for a different sensitivity to platinum agents and pemetrexed, respectively.

Certainly, our study is affected by some limitations, including the retrospective design, the relatively small sample size of patients, the lack of clinical outcome information, and the absence of confirmation by additional functional experiments. In addition, ALK status was assessed only in 62% of KRAS WT patients. However, as ALK rearrangements have been associated with low levels of TS, it is very unlikely that this could have affected the results of this study in terms of TS levels (17). On the other hand, it could be argued that immunohistochemistry (IHC) rather than RT-PCR would provide a direct measure of the acting element, namely the protein. However, there are several arguments against using an IHC-based technique, including the need for highly specific antibodies, the lack of standardized tissue fixation and staining protocols, difficulty in finding a consensual standard for microscopic evaluation, and universal cutoff value (20). For the same reason, some researchers have previously attempted to develop a more reliable and reproducible IHC method based on a fully automated and quantitative immunofluorescence technique (AQUA) (21).

In conclusion, to the best of our knowledge, this is the first study that has evaluated the expression of DNA synthesis and repair genes according to KRAS mutation status in advanced NSCLC patients, which suggests significantly higher mRNA expression levels for ERCC1 and lower for TS in KRAS-mutant patients, but no difference for either RRM1 and BRCA1. As for ERCC1, these results provide a rationale behind the poor sensitivity to platinum-based chemotherapy of KRAS-mutant advanced NSCLCs. On the other hand, whether lower TS expression levels translate into enhanced clinical efficacy of pemetrexed-based chemotherapy in KRAS-mutant patients remains to be determined in prospective clinical trials.

Acknowledgements

Not applicable.

Funding

The study was supported by Associazione Italiana per la Ricerca contro il Cancro (AIRC) (grant no. 15713 AIRC 2014).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

VL, LC and GM designed the research. FRT, CM, ASid, ASig, MSR, RC, SB and GB performed the experiments. BR, DG and GM analyzed the data. BR and GM wrote the paper. VL and GM critically revised the manuscript for important intellectual content.

Ethics approval and consent to participate

Comitato Etico Aziende Sanitarie (CEAS) Umbria approved the study (approval no. 4796/15/AV).

Patient consent for publication

CEAS Umbria approval waived patient consent.

Competing interests

The authors declare that they have no competing interests.

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December-2018
Volume 9 Issue 6

Print ISSN: 2049-9450
Online ISSN:2049-9469

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Spandidos Publications style
Ludovini V, Ricciuti B, Tofanetti FR, Mencaroni C, Giannarelli D, Sidoni A, Reda MS, Siggillino A, Baglivo S, Crinò L, Crinò L, et al: KRAS mutation and DNA repair and synthesis genes in non‑small‑cell lung cancer. Mol Clin Oncol 9: 689-696, 2018
APA
Ludovini, V., Ricciuti, B., Tofanetti, F.R., Mencaroni, C., Giannarelli, D., Sidoni, A. ... Metro, G. (2018). KRAS mutation and DNA repair and synthesis genes in non‑small‑cell lung cancer. Molecular and Clinical Oncology, 9, 689-696. https://doi.org/10.3892/mco.2018.1731
MLA
Ludovini, V., Ricciuti, B., Tofanetti, F. R., Mencaroni, C., Giannarelli, D., Sidoni, A., Reda, M. S., Siggillino, A., Baglivo, S., Crinò, L., Bellezza, G., Chiari, R., Metro, G."KRAS mutation and DNA repair and synthesis genes in non‑small‑cell lung cancer". Molecular and Clinical Oncology 9.6 (2018): 689-696.
Chicago
Ludovini, V., Ricciuti, B., Tofanetti, F. R., Mencaroni, C., Giannarelli, D., Sidoni, A., Reda, M. S., Siggillino, A., Baglivo, S., Crinò, L., Bellezza, G., Chiari, R., Metro, G."KRAS mutation and DNA repair and synthesis genes in non‑small‑cell lung cancer". Molecular and Clinical Oncology 9, no. 6 (2018): 689-696. https://doi.org/10.3892/mco.2018.1731