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

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 5 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 MIpositive patients (76.9%) had no expression of hMLH1 protein. The data showed that MI was associated with altered hMLH1 expression (P 5 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 5 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 MI represents expansions or contractions of the shorttandem 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 (2–4). The MI phenotype has also been found in a substantial number of sporadic colon carcinomas (5–7) 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 (15–25). 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 Received 10/20/99; revised 2/23/00; accepted 2/24/00. 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 89-2318B-003-001-M51 from the National Science Council (The Executive Yuan, Republic of China). 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: [email protected]. 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 b type II receptor gene. 1639 Vol. 6, 1639–1646, May 2000 Clinical Cancer Research Research. on April 2, 2017. © 2000 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Table 1 Clinicopathological and molecular data of 68 NSCLC patients Case Sex Age Smoking habits Type Stage hMLH1 expression p53 mutation Microsatellite markers D3S1215 D3S1292 D9S126 D9S162 D10S185 D13S170 D17S5 D17S786 MI-positive patients 1 M 75 Y SQ IIIa Yes Del 1 1 1 1 1 2 M 66 Y SQ IIIa – Pt 1 1 1 1 1 3 M 72 Y AD IIIa – Pt 1 1 1 1 1 4 M 82 Y SQ I Yes Del LOH 1 1 1 1 5 M 68 Y SQ IIIa – Del 1 1 1 1 6 F 40 N SQ I – Del 1 1 1 7 M 73 Y SQ I – Del LOH 1 1 1 8 M 73 Y SQ IIIa – Del LOH 1 1 1 9 M 70 Y SQ I – – 1 1 1 1 1 1 1 10 M 76 Y SQ I Yes – 1 1 1 1 1 1 11 M 70 Y SQ II – – 1 1 1 1 1 1 12 F 46 N AD IIIa – – 1 1 1 1 1 1 13 M 68 Y SQ II Yes – 1 1 1 1 1 14 F 59 N LC IIIa ND – LOH 1 1 1 1 1 15 M 68 Y SQ I – – 1 1 1 1 16 M 72 Y SQ I – – 1 1 1 1 17 M 69 Y SQ I – – 1 1 1 1 18 M 66 Y SQ II – – 1 1 1 1 19 M 73 Y SQ IIIa – – 1 1 1 1 20 F 68 N AD I – – 1 1 1 1 21 M 70 Y AD II ND – 1 1 1 1 22 M 75 Y AD IIIa – – 1 1 1 1 23 M 57 Y SQ I Yes – LOH 1 1 1 24 M 75 Y SQ I Yes – 1 1 1 LOH 25 M 50 Y SQ II – – 1 1 1 26 M 76 Y SQ II – – LOH 1 1 1 27 M 77 Y SQ II – – 1 1 1 28 F 76 N AD I – – 1 1 1 MI-negative patients 29 M 69 Y SQ IIIa – – 1 1 30 M 67 N AD I Yes – 1 1 31 M 76 Y AD I Yes – 1 1 32 M 67 Y AD II – – LOH 1 1 33 M 67 Y AS II Yes – 1 1 34 M 72 Y AS IIIa Yes – 1 1 35 M 75 Y SQ IIIa ND Del 1 1 36 M 69 N AS IIIa – Pt 1 1 37 M 72 N SQ II – Pt 1 38 M 67 Y SQ I Yes – 1 39 M 74 Y SQ II – – 1 40 M 70 Y SQ IIIa – – 1 41 M 86 Y AD I – – 1 42 M 68 Y AD I Yes – 1 43 M 69 N AD I Yes – 1 44 M 69 Y AD II Yes – 1 45 M 68 Y AD III – – 1 46 M 51 N AD IIIa Yes – 1 47 F 64 N AD IIIa – – 1 48 M 61 Y AS IIIa Yes – 1 49 M 68 Y AS IIIa Yes – 1 LOH 50 M 70 Y LC I Yes – 51 M 80 Y SQ II – – 52 M 67 Y AD I – – 53 F 74 N SQ I Yes – 54 M 70 Y AD I – – 55 M 78 Y SQ II – – 56 M 70 N SQ II – – 57 F 43 N AD IIIa Yes – 58 M 80 Y AD IIIa – – 59 M 73 Y AD I – – 60 M 68 Y SQ I Yes – 61 M 69 Y AD IIIa – – 62 M 62 Y SQ IV – – 63 M 70 Y SQ IIIb Yes ND 64 M 68 N AD I Yes – 65 F 69 N AD I ND ND 66 M 72 Y SQ IIIa – ND 67 M 48 N AD IIIa Yes – 68 M 76 Y SQ I Yes ND a LC, large cell carcinoma; AS, adenosquamous carcinoma. b Yes, with hMLH1 expression; –, without hMLH1 expression. c Del, small intragenic deletion; Pt, point mutation; –, without p53 gene mutation; ND, not determined. d 1, patients with instability at the indicated marker; LOH, loss of heterozygosity. e MI-positive patients were defined as instability in three or more markers. 1640 Genetic Instability and p53 Mutation in NSCLC Research. on April 2, 2017. © 2000 American Association for Cancer clincancerres.aacrjournals.org Downloaded from 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 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 exsmokers 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 mM of each primer, 200 mM 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 ml. 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 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. 1641 Clinical Cancer Research Research. on April 2, 2017. © 2000 American Association for Cancer clincancerres.aacrjournals.org Downloaded from 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 unequivo-