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Clinical Cancer Research Vol. 6, 1639-1646, May 2000
© 2000 American Association for Cancer Research

Advances in Brief

Correlation of Genetic Instability with Mismatch Repair Protein Expression and p53 Mutations in Non-Small Cell Lung Cancer1

Jer-Wei Chang, Yi-Chun Chen, Chih-Yi Chen, Jung-Ta Chen, Shin-Kuang Chen and Yi-Ching Wang2

Institute of Toxicology, Chung Shan Medical College, Taichung [J-W. C., Y-C. C., S-K. C.]; Departments of Thoracic Surgery [C-Y. C.] and Pathology [J-T. C.], Veterans General Hospital–Taichung, Taichung; and Department of Biology, National Taiwan Normal University, Taipei [Y-C. W.], Taiwan, Republic of China


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
To examine the etiological association of genetic instability in lung tumorigenesis, we investigated the frequency of microsatellite instability (MI) of eight dinucleotide repeat markers in 68 patients with non-small cell lung cancer. Twenty-eight patients (41.2%) evidenced instability in multiple tested microsatellite markers ranging from 3–7 and were defined as MI-positive patients. MI occurred more frequently in patients suffering from squamous cell lung carcinoma (P = 0.004). We examined the association between MI and expression of hMLH1 mismatch repair protein by immunohistochemical analysis of hMLH1 protein in paraffin-embedded tumors from 64 patients. Twenty MI-positive patients (76.9%) had no expression of hMLH1 protein. The data showed that MI was associated with altered hMLH1 expression (P = 0.03). To examine the role of genetic instability in the previous identified small intragenic deletion of the p53 gene, we explored the association between MI and p53 gene mutations. All patients, except one, containing small intragenic deletion in p53 gene showed MI (P = 0.018). In addition, we found that MI was not associated with the prognosis. Our data suggest that MI plays a significant role in non-small cell lung cancer tumorigenesis in Taiwan and that MI is associated with the altered expression of hMLH1 mismatch repair protein. In addition, MI may be involved in frequent small intragenic deletions of p53 gene.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
MI3 represents expansions or contractions of the short-tandem repeat sequences (microsatellites) in one or both alleles in tumor DNA as compared with matching normal DNA. Microsatellites are prone to strand-slippage during replication and defective mismatch repair (1) . MI has been shown to be a marker for genetic instability in HNPCC and is thought to reflect multiple replication errors from abnormalities of the mismatch repair genes, such as hMSH2 and hMLH1 (2, 3, 4) . The MI phenotype has also been found in a substantial number of sporadic colon carcinomas (5, 6, 7) and in tumors of several other organs (reviewed in Ref. 8 ). However, the cause of MI in sporadic tumors is less clear. Somatic mutations of mismatch repair genes have been shown in only a proportion of colon and endometrial tumors with MI (9 , 10) . However, immunohistochemical analysis has demonstrated that loss of mismatch repair protein (mainly hMLH1) occurs frequently in sporadic colon cancer and gastric cancer with MI (6 , 7 , 11 , 12) .

MI has also been described in LC. In SCLC, 45–76% of primary tumors are found to have MI in the form of deletion or expansion of dinucleotide or tetranucleotide repeats (13 , 14) . However, in the other major subtype of LC, NSCLC, there have been conflicting data regarding MI frequencies (ranging from 0–69%) and patterns (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) . For example, no MI was found in 87 LC patients in Finland (21) . Significantly different results were reported, however, in another study of 35 NSCLC patients in whom instabilities occurred at a rate of 69% and affected several microsatellite markers concurrently (22) . In addition, there has been little evidence in NSCLC that the MI is associated with an alteration of mismatch repair genes.

In our previous study on the mutation spectrum of the p53 tumor suppressor gene in LC patients, distinct patterns of p53 gene mutation were observed (26) . Seven of the 11 mutations detected (64%) were deletions of 1–12 bp at G:C bp, or at bp in the immediate vicinity of repetitive sequences and/or tandem repeat sequences. Our data suggest that a distinct environmental factor(s) and/or genetic factor(s) that specifically induces short deletions in repeat sequences is involved in lung tumorigenesis in Taiwan. These deletion mutations may be produced during the progression of lung tumorigenesis resulting from endogenous mechanisms, such as DNA polymerase replication errors and/or mismatch DNA repair deficiencies. To examine the etiological association of MI in lung tumorigenesis and the possible involvement of genetic instability of patients with small intragenic deletions from the p53 gene, we investigated the frequency of the MI of eight polymorphic markers in 68 NSCLC patients. We also explored the association between acquisition of a replication error phenotype, mismatch repair alteration, and p53 mutation. The clinical importance of MI was also investigated with regard to prognosis.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Samples and Preparation of DNA.
Surgical specimens were obtained from 68 patients with NSCLC (36 SQs, 25 ADs, 5 adenosquamous carcinomas, and 2 large cell carcinomas). The tumor types and stages were determined according to the WHO classification method (27) and the tumor-node-metastasis system (28) , respectively. The duration and amount of smoking before diagnosis of LC patients were obtained from hospital records. A cumulative cigarette pack-year history for each patient was then calculated. The patients were classified into smoking and nonsmoking groups. The number of years since last smoking for all ex-smokers was <10 yr. Therefore, the smoking group included both current smokers and ex-smokers. Follow-up of all patients was performed at 2-month intervals in the 1st yr after surgery and at 3-month intervals thereafter, at outpatient clinics or by routine phone calls. The end of the follow-up period was defined as November 15, 1998, for all patients. The mean follow-up period for all patients was 18.9 months (range, 0.5–51). For the 22 patients who survived the follow-up period (censored patients), the mean follow-up time was 24.7 months. For the 46 patients who died during the follow-up period, the mean follow-up time was 11.9 months. Representative proportions of well-separated normal lung tissues and tumoral lung tissues were taken after surgical resection, immediately snap-frozen, and subsequently stored in liquid nitrogen. Genomic DNA was prepared using proteinase K digestion and phenol/chloroform extraction, followed by ethanol precipitation.

Analysis of MI.
MI was analyzed in all 68 patients by PCR using eight microsatellite markers on five different chromosomes obtained as MAPPAIRS primers from Research Genetics (Huntsville, AL): D3S1215 (3q13), D3S1292 (3q21–25.2), D9S126 (9p21), D9S162 (9p22–23), D10S185 (10q23–24), D13S170 (13q22–31), D17S5 (17p13.3), and D17S786 (17p13.1). These markers were selected on the basis of the fact that they had been analyzed in previous studies (e.g., D3S1215, D3S1292, D9S162, D10S185, D13S170, and D17S5) and/or were located near the known tumor suppressor genes (e.g., D9S162 and D17S786 are located near the CDKN2 and p53 tumor suppressor gene, respectively). For all markers, the following PCR conditions were used: 0.5 µM of each primer, 200 µM dNTP, 2.5 units of Taq polymerase, a standard polymerase buffer supplied with enzyme (1.5 mM MgCl2), and 150 ng of genomic DNA. The total volume of the PCR mix was 25 µl. The PCR temperature program was: 95°C denaturation for 5 min; 35 cycles of 1 min each at 95°C, 1.75 min at 55°C, and 1.75 min at 72°C; and a final extension run at 72°C for 10 min. The PCR products were denatured for 3 min at 95°C and run on a 6% polyacrylamide gel containing 7 M urea at 20 W for 4–5 h. The gels were dried and exposed to radiographic film (X-OMAT; Kodak). MI was revealed by the presence of one or more novel bands (expansions or contractions of repeats) in the tumors, but absent in the paired normal DNA. MI positive was defined as instability in three or more markers. This definition can be used to score MI positive unequivocally (29) .

Analysis of hMLH1 Protein Expression–Immunohistochemistry Assay.
Paraffin blocks of 64 tumors were cut into 5-µm slices and then processed using standard deparaffinization and rehydration techniques. A monoclonal antibody, G168-278 (1:250; PharMingen), was used as the primary antibody to detect hMLH1 protein expression. The primary antibody was detected using biotinylated secondary antibody (DAKO LSAB Kit K675), used as recommended by the manufacturer. The sections were then counterstained with hematoxylin. The normal staining pattern for hMLH1 was nuclear. Tumor cells that exhibited an absence of nuclear staining in the presence of nonneoplastic cells and infiltrating lymphocytes with nuclear staining were considered to have an abnormal pattern. In addition, a section of normal colon was served as a positive control for immunohistochemical assay. The samples were assayed in batches, including both MI-positive and MI-negative patients. The staining results were examined without knowledge of the MI status. Each sample was assayed by repeating analyses at least twice.

Analysis of p53 Mutation.
PCR/single-stranded conformational polymorphism was used to detect the presence of mutations in the p53 tumor suppressor gene of 64 patients. Oligodeoxynucleotide primers and thermocycle PCR conditions designed to produce DNA fragments of p53 gene exons 4–11 are described in Ref. 30 . PCR products were subjected to electrophoresis at 30 W for 4–5 h in a 6% nondenaturing polyacrylamide gel with 5% glycerol and fan-cooled at room temperature. Abnormal DNA fragments detected during PCR/single-stranded conformational polymorphism analysis were sequenced using the dideoxy chain termination method, with {alpha}-35S-dATP, PCR-amplified primers, and a sequenase II kit (United States Biochemical Corporation).

Statistical Analysis.
The Pearson {chi}2 test was used to compare the possibility of MI among cases and between various clinicopathological parameters. Type III censoring was performed on subjects who were still alive at the end of the study (31) . The Kaplan-Meier method was used to estimate the probability of survival as a function of time and the median survival (32) . The log rank test was used to assess the significance of the difference between pairs of survival probabilities (33) .


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
MI in Lung Tumors.
We examined MI in tumor DNA from 68 NSCLC patients using eight dinucleotide repeat markers. Table 1Citation lists the results of MI of analyzed lung tumors and includes relevant clinical information. Twenty-eight patients (41.2%) evidenced instability in multiple-tested microsatellite markers ranging from 3–7 and are defined as MI-positive patients. Patients 1–3 and 9–14 showed MI in five or more markers. Microsatellite marker alterations were mostly observed at D13S170 (45.6%), D9S162 (39.7%), D17S786 (33.8%), D10S185 (32.4%), and D3S1292 (30.9%). The frequencies of MI in the remaining markers were 11.8% for D3S1215, 20.6% for D9S126, and 0% for D17S5. Representative results of the MI analysis are shown in Fig. 1Citation . The instability seemed as either expansion or compression of a single band or a ladder of bands.


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  		Table 1

Clinicopathological and molecular data of 68 NSCLC patients

 


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  		Fig. 1. Representative figures of microsatellite analysis of normal (N) and tumor (T) DNA of LC patients at the D3S1215, D3S1292, D9S126, D9S162, D10S185, D13S170, D17S5, and D17S786 loci. The patient number (Pt’s no) is designated above each block. MI is shown as the contraction or expansion of a single band or a ladder of bands of the tumor DNA compared with the normal DNA. Loss of heterozygosity can be seen in the tumor DNA of patient 26 at D3S1215 and of patients 49 and 24 at D17S5.

 
To examine the association of MI with the clinical characteristics of patients, the occurrence of MI was compared with the patients’ clinicopathological parameters (Table 2)Citation . The incidence of MI positive was higher in SQ patients (61.1%, 22 of 36) than in AD patients (24.0%, 6 of 25; P = 0.004). However, similar ages, sexes, smoking histories, and clinical stages were noted in both groups of patients, those with and without instability.


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  		Table 2

Clinical features of MI-positive and -negative patients

 
Correlation with Altered Expression of hMLH1 Protein.
The paraffin-embedded tumors were available from 64 patients. The expression of hMLH1 mismatch repair protein was examined in these patients, including 26 MI-positive patients and 38 MI-negative patients (Table 1)Citation . The incidence of negative hMLH1 expression was higher in MI-positive patients (76.9%, 20 of 26) than in MI-negative patients (50.0%, 19 of 38; Table 2Citation ). The data show that MI was associated with an altered hMLH1 expression (P = 0.03). Representative results of the immunohistochemistry analysis are shown in Fig. 2Citation .



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  		Fig. 2. Representative figures of immunohistochemical analysis of hMLH1 protein in paraffin sections of lung tumor specimens. Nuclear immunoreactivity was found in patient 48 (A) and patient 49 (B). C, negative tumor was from patient 2 with MI in multiple markers. Original magnification: A, x200; B and C, x100.

 
Correlation with p53 Gene Mutations.
Tumors from 64 patients were analyzed for both MI and p53 gene mutation. Four patients had a point mutation, and seven had a small intragenic deletion in the p53 gene (Table 1)Citation . Fig. 3Citation shows the representative autographs of 6 of the 7 deletion mutations detected, which were deletions of 1–12 bp at G:C bp, or at bp in the immediate vicinity of repetitive sequences and/or tandem repeat sequences. Of these seven deletion patients, six (85.7%) evidenced instability at three or more markers. There was a significant association between p53 small intragenic deletion and MI (P = 0.018, Table 2Citation ).



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  		Fig. 3. Autoradiographs of sequencing analyses of patients exhibiting deletion mutations of the p53 tumor suppressor gene. All sequences shown are of the sense strand. Some patients also exhibited wild-type alleles, which are also shown in the figure. A-F are from patients 1, 4, 8, 6, 7, and 35, respectively. Bases encoded by same amino acid are in brackets. {square}, bases deleted. The areas around deleted bases are also shown. These data are taken from Ref. 26 .

 
Correlation with Cancer Prognosis.
The survival of patients whose tumors showed MI positive was similar to those of MI negative (median survival times of 26 months for patients with and without MI; P > 0.05 by the log rank test; Fig. 4Citation ). In the SQ group, survival was also not influenced by MI (data not shown).



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  		Fig. 4. The Kaplan-Meier survival curves with respect to MI. Ps were obtained using the log rank test. The MI-positive group had similar prognoses as the MI-negative group (P = 0.81). Median survival times were both 26 months for patients with and without MI.

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
In this study, 28 patients (41.2%) evidenced instability in tested microsatellite markers ranging from 3–7 and were defined as MI-positive patients. The data indicated that Taiwanese NSCLC patients had high frequencies of MI in multiple loci. In NSCLCs, the data on MI have been conflicting in regard to frequencies and patterns (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) . One proposed explanation for the conflicting results is that MI is associated with specific tumor type and/or stage and that cancers of different types and stages were analyzed in the contrasting studies. It is also possible that there are geographical and/or ethnic factors that cause MI. In addition, the varying results may be due, in part, to differences in the types and number of primers analyzed, as well as to the differences in the definition of MI. Microsatellite loci on various chromosomes may have differing frequencies of instability. For instance, the frequencies of MI in the various loci examined ranged from 0–45.6% in the present study. However, when comparing the MI frequency between different studies, the existence of common microsatellite marker(s) should be taken into consideration. For example, the frequency of instability of D9S162 was 39.7% in our study, whereas this frequency was 0% and 1.8% in two previous studies on NSCLC (16 , 21) . Therefore, MI plays a significant role in NSCLC tumorigenesis in Taiwan.

We found that LC patients with p53 small intragenic deletion had frequent MI. To our knowledge, this is the first study to demonstrate that MI occurs frequently in patients with small intragenic deletion in the p53 gene. The high frequency of small intragenic deletions in repetitive sequences and/or tandem repeats is probably conservative in other regions of the genome of these patients. We propose that the relatively high frequency of instability of the polymorphic microsatellite sequences indicates a destabilization of the genome that may affect other genes preferentially in repetitive sequences and/or tandem repeats. In a subgroup of NSCLCs, MI may predispose to further genetic alterations. The mutation spectrum analysis of other genes involved in tumorigenesis was required to confirm this hypothesis.

A higher frequency of MI in SQ patients compared with AD patients suggests a difference in the genetic changes contributing to the tumorigenesis of SQ and AD in Taiwan. In fact, our analysis also indicated a higher frequency of p53 mutation in SQ compared with that in AD (26) . Furthermore, the frequency of MI was similar between early and advanced stages of LC, whereas the p53 mutations occurred more frequently in patients with advanced stages of LC (26) . In addition, MI occurs more often than does the p53 mutation. These observations suggest that MI may be an earlier molecular event compared with the p53 mutation during the pathogenesis of lung tumorigenesis in Taiwan.

Changes in the length of certain microsatellites have been correlated with changes in the expression of mismatch repair genes in HNPCC and in other somatic tumors (6 , 7 , 11 , 12) . However, the data on mismatch repair protein expression in LC is scarce. To our knowledge, this is the first report of a high frequent alteration of hMLH1 mismatch repair protein in LC. We found a close relationship between hMLH1 mismatch repair protein expression and MI status. Twenty (76.9%) MI-positive patients showed no expression of hMLH1 mismatch repair protein. However, six MI-positive patients showed a positive nuclear stain for hMLH1 protein. It is possible that there is altered expression or mutation in one of the other mismatch repair genes (hMSH2, hMSH3, hMSH6, or hPMS2) in these patients (34) . Note that all patients, except two containing deletion of the p53 gene, had a negative expression of hMLH1 mismatch repair protein. However, due to the small number analyzed, no statistical correlation could be made regarding the effect of hMLH1 alteration on p53 small intragenic deletion.

The association of MI with the altered expression of hMLH1 mismatch repair protein and the p53 small intragenic deletion may be unique to NSCLC in Taiwan. We are currently investigating the mRNA expression and the methylation status of several mismatch repair genes, including hMLH1 and hMSH2, in NSCLC patients. It is also very important to clarify whether somatic mutations of known mismatch repair genes or components involved in the replication system are responsible for the acquisition of MI in NSCLC in Taiwan. Alterations of hMSH2 and hMLH1 mismatch repair genes would point to an acquired mutator phenotype (3 , 4) . Mutations in the RII gene have also been shown to preferentially occur at microsatellite regions and in tumors with genomic instability (34) . In the search for genes and mechanisms involved in the instability of lung tumors, we have also started a mutation analysis of hMSH2 and hMLH1 genes, as well as the RII gene.


   ACKNOWLEDGMENTS
 
We are grateful to Profs. Yuh-Shan Jou and Ming-Chei Maa for critical revision of the manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by Grant DOH86-HR-611 from the National Health Research Institute (Department of Health, The Executive Yuan, Republic of China) and by Grants NSC 87-2314-B-040-024 and NSC 89-2318-B-003-001-M51 from the National Science Council (The Executive Yuan, Republic of China). Back

2 To whom requests for reprints should be addressed, at Department of Biology, National Taiwan Normal University, No. 88, Sec. 4, Tingchou Road, Taipei 116, Taiwan, Republic of China. Phone: 886-2-29336876, ext. 211; Fax: 886-2-29312904; E-mail: t43017{at}cc.ntnu.edu.tw Back

3 The abbreviations used are: MI, microsatellite instability; SQ, squamous cell carcinoma; AD, adenocarcinoma; LC, lung cancer; NSCLC, non-small-cell LC; HNPCC, hereditary nonpolyposis colorectal cancer; RII gene, transforming growth factor ß type II receptor gene. Back

Received 10/20/99; revised 2/23/00; accepted 2/24/00.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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