This work was supported by the National Nature Science Funds in China (Fund No. 81071805) and Dalian Merricon Gene Diagnosis Technology Co., Ltd. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.
Conceived and designed the experiments: ML SL. Performed the experiments: ML WL HW. Analyzed the data: ML SL. Contributed reagents/materials/analysis tools: ML QZ LL SL. Wrote the manuscript: ML SL RWL.
Mismatch repair (MMR) plays a pivotal role in keeping the genome stable. MMR dysfunction can lead to carcinogenesis by gene mutation accumulation. HMSH2 and hMLH1 are two key components of MMR. High or low expression of them often mark the status of MMR function. Mutations (EGFR, KRAS, etc) are common in non-small cell lung cancer (NSCLC). However, it is not clear what role MMR plays in NSCLC gene mutations. The expression of MMR proteins hMSH2 and hMLH1, and the proliferation markers PCNA and Ki67 were measured by immunohistochemistry in 181 NSCLCs. EGFR and KRAS mutations were identified by high resolution melting analysis. Stronger hMLH1 expression correlated to a higher frequency of EGFR mutations in exon 19 and 21 (p<0.0005). Overexpression of hMLH1 and the adenocarcinoma subtype were both independent factors that related to EGFR mutations in NSCLCs (p=0.013 and p<0.0005). The expression of hMLH1, hMSH2 and PCNA increased, while Ki67 expression significantly decreased (p=0.030) in NSCLCs with EGFR mutations. Overexpression of hMLH1 could be a new molecular marker to predict the response to EGFR-TKIs in NSCLCs. Furthermore, EGFR mutations might be an early event of NSCLC that occur before MMR dysfunction.
Lung cancer is the most frequent and deadly malignant tumor worldwide, with non-small cell lung cancer (NSCLC) being the predominant form. Carcinogenesis of NSCLC is a multistep process involving alterations of multiple genes including oncogene activation and tumor suppressor gene inactivation [
Mismatch repair (MMR) is an important type of DNA repair, playing a pivotal role in maintaining genome stability [
The study was approved by the Ethics Committee of the Second Hospital of Dalian Medical University. All specimens in the research were from tissue surgically removed without affecting the diagnosis and treatment. They were collected with the written informed consent of the patients or families before surgery. The data were analyzed anonymously. All procedures were in accordance with the Declaration of Helsinki.
A total of 181 tumor specimens were collected from NSCLC patients who underwent surgical procedures at the affiliated hospitals of Dalian Medical University from 2007 to 2009. Of these, there were 112 adenocarcinomas, 58 squamous cell carcinomas, 4 adeno-squamous cell carcinomas, 5 large cell carcinomas and 2 sarcomatoid carcinomas. Two certified pathologists independently diagnosed and classified all the patients according to the WHO classification (2004). Of the 181 patients studied, 109 were men and 72 were women with a mean ± SD age of 62.0 ± 9.3 years (36-80 years). None of the patients received radio- or chemotherapy before their operations. The patients' information and histopathological features of the tumors in this cohort are presented in
Age | ||||||||||||
≤60 | 79 | 59.5 | 68.4 | 88.6 | 57.0 | 12.7 | 25.3 | 5.1 | ||||
>60 | 102 | 54.9 | 73.5 | 88.2 | 65.7 | 13.7 | 21.6 | 5.9 | ||||
Gender | ||||||||||||
Female | 72 | 54.2 | 79.2 | 90.3 | 55.6 | 20.8 | a | 37.5 | c | 0.0 | b | |
Male | 109 | 58.7 | 66.1 | 87.2 | 66.1 | 8.3 | 13.8 | 9.2 | ||||
Pathology | ||||||||||||
Adc | 112 | 56.3 | 77.7 | a |
90.2 | 56.3 | 21.4 | c | 32.1 | c | 5.4 | |
SCC | 58 | 56.9 | 62.1 | 87.9 | 70.7 | 0.0 | 5.2 | 6.9 | ||||
Smoking | ||||||||||||
Non-smoking | 115 | 56.5 | 78.3 | b |
88.7 | 60.0 | 17.4 | a | 30.4 | c | 3.5 | |
Smoking | 66 | 57.6 | 59.1 | 87.9 | 65.2 | 6.1 | 10.6 | 9.1 | ||||
Tumor site | ||||||||||||
Left lung | 85 | 55.3 | 76.5 | 88.2 | 57.6 | 17.6 | 18.8 | 8.2 | ||||
Right lung | 96 | 58.3 | 66.7 | 88.5 | 65.6 | 9.4 | 27.1 | 3.1 | ||||
LN metastasis | ||||||||||||
No | 86 | 61.6 | 72.1 | 88.4 | 65.1 | 12.8 | 19.8 | 8.1 | ||||
Yes | 95 | 52.6 | 70.5 | 88.4 | 58.9 | 13.7 | 26.3 | 3.2 | ||||
Stage | ||||||||||||
I & II | 111 | 61.3 | 74.8 | 91.0 | 61.3 | 16.2 | 23.4 | 5.4 | ||||
III & IV | 70 | 50.0 | 65.7 | 84.3 | 62.9 | 8.6 | 22.9 | 5.7 |
Adc: adenocarcinoma; SCC: squamous cell carcinoma; LN: lymph node.
a p<0.05, bp<0.01, cp<0.0005 (Pearson chi-square test).
* When smoking history was controlled, hMLH1 expression is not significantly different between Adc and SCC, p=0.267; when pathological classification was controlled, it is different between non-smokers than smokers, p= 0.009 (CMH test).
Monoclonal antibodies against human hMSH2 (1:250, clone FE11, Invitrogen, Life technologies, USA), hMLH1 (1:50, clone 14, Invitrogen, Life technologies, USA), PCNA (1:400, clone PC10, Thermo scientific, USA) and Ki67 (1:100, clone K-2, Invitrogen, Life technologies, USA) were used as primary antibodies. Biotin-streptavidin-peroxidase staining with 3, 3’-diaminobenzidine-tetrahydrochloride (DAB) detection were used. Immunohistochemistry was performed as previously described [
Tumor enriched areas were selected and cut from the stained frozen sections marked by two pathologists. Genomic DNA was extracted from these areas and purified according to the manufacturer’s protocol (Tiangen, Beijing, China) [
The Pearson chi-square test and Fisher’s exact test were used to compare the difference of protein expression between clinicopathological parameters. Spearman’s correlation analysis was used to test the correlation between protein expression. The Cochran’s and Mantel-Haenszel (CMH) test was used to compare the difference of hMLH1 expression between smoking status and between the tumor classifications, with the other variable controlled. Logistic regression was used to analyze the factors related to EGFR mutations. All of the analyses were performed with SPSS 13.0 at the significance level of p<0.05.
All of the proteins, hMSH2, hMLH1, PCNA and Ki67, were expressed in the nuclei of tumor cells (
Immunohistochemical profiling of hMLH1 protein positive expression (A), hMSH2 protein positive expression (B), PCNA protein positive expression (C) and Ki67 protein positive expression (D). (×200).
There were 81 cases with hMSH2 and hMLH1 co-expression, 22 cases with only hMSH2 expression, 48 cases with only hMLH1 expression and 30 cases without a positive expression of either hMSH2 or hMLH1. The hMSH2 expression was significantly correlated to the hMLH1 expression (p=0.038; r=0.155). The expression of hMLH1 was stronger in the cases with PCNA expression (p=0.005), but not in those with Ki67 expression (p=0.495). There was a trend of hMLH1 expression increasing with PCNA expression (p=0.056). Expression of hMSH2 was not correlated to the expression of either PCNA or Ki67 (p=0.802; p=0.099) (
hMSH2 |
PCNA |
Ki67 |
||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
- | + | ++ | - | + | ++ | - | + | ++ | ||||
hMLH1 | - | 31 | 4 | 18 | a | 12 | 21 | 20 | b | 9 | 28 | 6 |
+ | 21 | 6 | 35 | 6 | 40 | 16 | 26 | 31 | 5 | |||
++ | 26 | 4 | 36 | 3 | 34 | 29 | 24 | 30 | 12 | |||
hMSH2 | - | 9 | 39 | 30 | 34 | 36 | 8 | |||||
+ | 3 | 7 | 4 | 7 | 5 | 2 | ||||||
++ | 9 | 49 | 31 | 28 | 48 | 13 |
a p<0.05 (Spearman’s correlation analysis), Spearman’s rank correlation coefficient (r) is 0.155.
b p=0.056 (Spearman’s correlation analysis), p=0.005 (Pearson chi-square test).
Out of the 181 patients with NSCLCs, there were 10 cases (5.5%) with a KRAS mutation and 66 cases (36.5%) with an EGFR mutation (24 cases in exon 19 and 42 cases in exon 21) (
Different melting curves showing mutation type (red line) relative to wild type (grey line) of KRAS exon 2 (a), EGFR exon 19 (b) and EGFR exon 21 (c). Every sample was analyzed in triplicate. The data was plotted directly (A) or the wild type was chosen as a horizontal base line (B).
There was no significant difference in the frequency of Ki67 or PCNA expression between NSCLCs with and without EGFR mutation in exon 19 or exon 21 (p>0.05,
KRAS | M | 10 | 60.0 | 80.0 | 80.0 | 40.0 | |
W | 171 | 56.7 | 70.8 | 88.9 | 63.2 | ||
EGFR exon 19 | M | 24 | 70.8 | 91.7 | a | 95.8 | 45.8 |
W | 157 | 54.8 | 68.2 | 87.3 | 64.3 | ||
EGFR exon 21 | M | 42 | 52.4 | 88.1 | b | 92.9 | 54.8 |
W | 139 | 58.3 | 66.2 | 87.1 | 64.0 |
M: mutation, W: wild type.
a p<0.05, b p<0.01 (Pearson chi-square test or Fisher’s exact test).
The frequency of hMLH1 expression was higher in NSCLCs with an EGFR exon 19 mutation than in those without the mutation (91.7% to 68.2%, p=0.018) and in NSCLCs with an EGFR exon 21 mutation than in those without the mutation (88.1% to 66.2%, p=0.006). As hMLH1 expression increases (from -, + to ++), the frequency of EGFR mutations (exon 19 and 21) were 13.2%, 38.7% and 53.0% respectively (p<0.0005). Similar correlations were not found with hMSH2 expression (
Molecular targeting of drugs is beginning to play a more important role in tumor treatment. To improve clinical results for patients with NSCLC, targeted therapies are increasingly being used with encouraging outcomes, particularly in patients with specific molecular features [
To the best of our knowledge, we report here for the first time that hMLH1 expression is related to EGFR mutations in both exon 19 and exon 21, but hMSH2 expression is not. Generally, women and non-smoking patients with adenocarcinoma have a relatively high probability of EGFR mutations. But lung adenocarcinoma is common in women and non-smokers, and most women in East Asia are non-smokers. Therefore, clinicopathological characteristics do not predict EGFR mutations very well. We found hMLH1 expression and adenocarcinoma were independent factors related to EGFR mutations. Moreover, the stronger the hMLH1 expression, the higher EGFR mutation frequency. Gender and smoking history were not independently correlated to EGFR mutation frequency. It would be interesting to study the value of hMLH1 overexpression as a marker to predict the response of NSCLC patients to EGFR-TKIs.
In previous studies, Xinarianos et al. reported that lower hMLH1 expression was more frequent in heavy smokers [
Both PCNA and Ki67 can be used to indicate the status of cell proliferation. PCNA is stimulated in the process of MMR as a necessary component [
In addition, Kouso et al. demonstrated the independence of hMSH2 and hMLH1 expression with different roles in NSCLC [
In summary, EGFR mutations in exon 19 and 21 correlate with MMR dysfunction in NSCLC. Overexpression of hMLH1 could be a new marker for patient sensitivity to EGFR-TKIs. In the past, MMR dysfunction has been assumed to cause EGFR mutations. However, EGFR mutations could also increase hMLH1 overexpression as a compensatory mechanism. A cause-effect relationship has not been established either way. Further studies would be required to provide further insight into which event occurs first. In other case, the possibility of using hMLH1 as an indicator of TKI responses may prove useful.
The authors thank Professor Shichang Yue for his assistance with patient recruitment.