Microsatellite Instability at Selected Tetranucleotide Repeats Is Associated with p53 Mutations in Non-Small Cell Lung Cancer¹

MSI³was initially identified in colorectal cancer and was immediately clinically significant because of its association with HNPCC(1). MSI is thought to be caused by a failure of the mismatch repair system to repair errors that occur during DNA replication (1). Several mismatch repair genes have been identified (hMLH1, hMSH2, hPMS1, hPMS2, and hMSH6), and their inactivation has been well-characterized in HNPCC and occasionally in other noncolonic tumors with MSI (1, 2). A genetically and clinically distinct form of MSI has also been described in which microsatellite alterations are present at selected tri- and tetranucleotide repeats in noncolonic, non-HNPCC tumors (1). In these tumors, larger repeats are more commonly altered than smaller repeats. This finding is in marked contrast to observations in HNPCC and most sporadic gastric and endometrial tumors, which display an elevated level of instability at mono- and dinucleotide repeats (1). This distinct subtype of MSI has been termed EMAST (1). These observations in NSCLC have recently been expanded and described in greater detail.⁴

Although the genetic basis for MSI associated with defective mismatch repair is being increasingly clarified, the mechanism underlying microsatellite alterations in EMAST tumors has yet to be identified(1). The lack of overlap between genome wide MSI and EMAST(+) tumors suggests that a nonmismatch repair pathway might be involved (1). The p53 protein is a significant component of the cellular response to DNA damage (3). Accumulation of p53 protein leads to cell cycle arrest to prevent the replication of damaged DNA, and if the DNA is not repaired, it can lead to apoptosis in cells with severe DNA damage (3, 4). The p53gene is frequently mutated in NSCLC, and similar to EMAST(+)status, these mutations occur more commonly in squamous cell lung cancer than in adenocarcinoma of the lung (5). Therefore,we examined the relationship between p53 gene mutations and EMAST in NSCLC.

Primary tumor, normal lung, and blood were collected from 88 patients undergoing surgical resection of NSCLC at The Johns Hopkins Hospital or the Johns Hopkins Bayview Medical Center. Pathological stage was determined using the revised International System for Staging Lung Cancer (6). Lymphocytes were collected from blood and used a source of normal DNA. Tumor samples and normal lung tissue were promptly frozen at −80°C after initial gross pathological examination. The Joint Committee on Clinical Investigation of The Johns Hopkins School of Medicine approved this research protocol, and written informed consent was obtained from all patients.

Portions of the primary tumor were cut into 7-μm sections, stained with H&E, and examined by light microscopy. Additional 12-μm sections were cut and placed in a mixture of 1% SDS and proteinase K at 48°C overnight. Tumors with a low neoplastic cellularity (<70%) were further microdissected to remove contaminating normal cells. DNA was extracted with phenol/chloroform and precipitated with ethanol.

Microsatellite analysis was performed on all 88 tumors using a panel of 13 markers (L17686, D20S82, UT5320, D8S321, L17835,D20S85, UT5307, D9S242, G29028, D11S488, ACTβ2, G08460, and CSFIR1) previously shown to demonstrate frequent microsatellite alterations (expansion or deletion of a repeat unit) at tetranucleotide repeats in NSCLC (5). Results of microsatellite analysis using these markers on 50 of these tumors have been reported previously (5). In addition, tumors harboring microsatellite alterations with the 13-marker panel were also analyzed using the Bethesda Consensus Conference reference panel of five mono- and dinucleotide markers (BAT-25, BAT-26, D2S123,D5S346, D17S250) for determining MSI in colorectal cancer (no similar panel has been selected to date for NSCLC; Ref.1). Using this reference panel, MSI-H tumors are defined as having instability in two or more markers, low frequency MSI tumors are defined as having instability in one marker, and microsatellite stable tumors demonstrate no instability (1). These reference markers have been shown to identify those tumors with defective mismatch repair, primarily in the two major mismatch repair genes, hMSH2 and hMLH1 (1). Primers were obtained from Research Genetics (Huntsville, AL) or synthesized based on sequences in the Genome Database. One marker from each primer pair was labeled with T4 polynucleotide kinase (New England Biolabs). PCR amplification was performed in separate reactions with 60 ng of DNA isolated from the tumor, and control nonneoplastic DNA as described above. Products were separated in 8% denaturing urea-polyacrylamide-formamide gels followed by autoradiography.

Mutation analysis of the p53 gene was performed on all 88 lung cancers by both direct dideoxy nucleotide sequencing and using the GeneChip p53 assay (Affymetrix Inc., Santa Clara, CA) as reported previously (7). A 1.8-kb fragment of the p53 gene (exons 5–9) was amplified from primary tumor DNA in all 88 patients by PCR (8, 9). The PCR products were purified and sequenced directly using cycle sequencing (Amplicycle sequencing kit, Perkin-Elmer, Branchburg, NJ), and the products of the sequencing reactions were then separated by electrophoresis in 8 m urea and 6% polyacrylamide gels, fixed, and exposed to film. In addition, exons 2–11 of the p53 gene from all 88 tumors were sequenced using the GeneChip p53 assay as described (7).

Comparisons between groups were performed using the Fisher’s exact test (two-tailed).

Pathological characteristics from 88 patients undergoing pulmonary resection for NSCLC were reviewed. Forty-four patients had stage I disease (26 stage IA and 18 stage IB), 24 patients had stage II NSCLC(3 stage IIa and 21 stage IIb), and 20 patients had stage III disease. Mean tumor size was 3.8 cm. The cell type of the 88 NSCLCs included squamous cell cancer (n = 37), adenocarcinoma(n = 44), adenosquamous carcinoma(n = 1), large cell carcinoma(n = 4), and poorly differentiated NSCLC(n = 2).

Thirty-one of these 88 cancers (35%) demonstrated a novel allele[EMAST(+)] with at least 1 of 13 microsatellite markers (Fig. 1). Twenty-two of 31 EMAST(+) tumors had MSI at 1 marker, whereas 9 of the EMAST(+) tumors demonstrated microsatellite alterations at ≥2 of 13 markers. These microsatellite “shifts” were seen in 12 tumors(14%) at L17686, 7 tumors (8%) at D20S82, 6(7%) tumors at UT5320, 4 (4%) tumors at 8S321,3 (3%) tumors at L17835 and D20S85, 2 (2%)tumors at two markers (UT5307 and D9S242), and in 1 tumor (1%) at four markers (G29028, D11S488, ACTβ2, and G08460). Squamous cell cancers were significantly more likely to be EMAST(+) than adenocarcinoma of the lung (51% versus 20%; P = 0.006). No significant association between patient age, race, gender,tumor size, tumor stage, and tumor differentiation and EMAST status was observed.

All 31 EMAST(+) tumors were examined with a reference panel of five mono- and dinucleotide markers used for determining MSI. Only 1 of 31 EMAST(+) tumors (3%) had instability at a single marker (low frequency microsatellite instability), and the remaining 30 tumors were microsatellite stable.

p53 mutations were detected in 50 of 88 (57%) patients with NSCLC. Mutations of the p53 gene were found significantly(P = 0.006) more often in squamous cell cancer [27 of 37 (73%)] than in adenocarcinoma [18 of 44 (41%)]. Thirty-three (66%) of the p53 mutant tumors contained a missense mutation, eight (16%) contained a nonsense mutation, five (10%)contained a deletion, and four (8%) contained a splice site mutation.

The relationship between EMAST positivity and mutation of the p53 gene is shown in Table 1. p53 mutations were significantly more common in EMAST(+)than in EMAST(−) NSCLC (81% versus 44%; P = 0.001). Among squamous cell cancers, p53 mutations were also significantly(P = 0.04) more common in EMAST(+) than in EMAST(−) tumors (89% versus 55%). A similar trend was observed with adenocarcinoma of the lung, but this difference did not reach statistical significance (67% versus 35%; P = 0.15). Seven of nine tumors (78%) with MSI at two or more markers contained a p53 mutation.

Eighty % (4 of 5) of the tumors containing a deletion in the p53 gene were EMAST(+), whereas only 47% (21 of 45) of tumors containing a single bp mutation were also EMAST(+). Only 16% of the tumors with wild-type p53 were EMAST(+).

Although widespread genome wide MSI is rare in NSCLC,microsatellite alterations at selected tetranucleotide repeats are not uncommon in primary lung cancer. Interestingly, these microsatellite alterations were more common in squamous cell cancer than in adenocarcinoma of the lung. Furthermore, EMAST(+) tumors were significantly more likely to harbor a p53 mutation than EMAST(−) cancers.

Microsatellite alterations have become useful clonal markers for the early detection of cancer. The generation of novel alleles through the insertion or deletion of a short tandem repeat creates a new DNA marker present only in neoplastic and not in nonneoplastic cells. These markers are easier to detect in clinical samples than subtle changes in loss of heterozygosity and have shown promise in the early detection of bladder, lung, and head and neck cancers in preliminary studies (5, 10, 11, 12, 13, 14, 15). The detection of novel microsatellite alleles in the urine has been used to identify both primary and recurrent bladder cancer (11, 12). In several cases, these microsatellite alterations have preceded any clinically detectable(urine cytology, cystoscopy) signs of cancer. In addition,tumor-specific microsatellite alterations have been detected in the bronchoalveolar lavage fluid, sputum, and plasma of patients with lung cancer, suggesting that these alterations may also be potential markers for the early detection of this common malignancy (5, 14, 15).

The association between gene alterations and MSI has been evaluated previously in several tumor types. Frameshift mutations within mononucleotide repeat sequences in several genes(TGFβ-II, BAX, IGFRII, E2F-4, and TCF-4) have been identified in MSI-H colon cancer (16, 17, 18, 19, 20). The human BAX gene,which promotes apoptosis, is inactivated by frameshift mutation in∼50% of HNPCC syndrome colon cancers (17, 19). Rampino et al. (17) have suggested that BAXgene inactivation downstream from p53 in the apoptotic pathway would eliminate the selective pressure for p53 gene mutations in colorectal tumorigenesis. Indeed, the incidence of p53 gene mutations is lower in MSI-H colon cancer than in sporadic colon cancer (17, 19).

In contrast to colon cancer, a strong correlation between MSI and p53 mutations has been reported in ovarian cancer(21). MSI was more frequent in tumors containing an insertion or deletion mutation than in tumors with a missense mutation,and 50% of the deletions occurred at iterated sequences or direct repeats (16). Sood et al. (21)concluded that the pattern of p53 mutations(insertions/deletions at iterated bases) observed in ovarian cancer was consistent with DNA strand slippage during replication, and therefore, p53 mutations were likely caused by a generalized genomic instability rather than being the cause of genomic instability. In contrast to these findings, no association between MSI and p53 mutations has been found in gastric cancer, and it was also not observed in the present series of NSCLCs using the Bethesda Conference criteria for defining MSI (22, 23).

MSI attributable to a defect in a DNA repair pathway distinct from mismatch repair has been hypothesized but remains undiscovered. The p53 protein plays a central role in the repair of genetically damaged cells. In response to DNA damage, p53 protein levels rapidly increase by a posttranscriptional mechanism (24). The induction of p53 also results in the transcriptional activation of other cell cycle regulatory genes, such as p21^WAF-1 leading to arrest of the cell cycle at the G₁-S checkpoint (24). Loss of functional p53 protein leads to a failure of cell cycle arrest and genomic instability, as evidenced by inopportune gene amplification, aneuploidy, and/or chromosome loss through DNA strand breakage and rejoining (4, 24, 25). The inability to delay cell division and allow adequate DNA repair to take place increases the probability that DNA damage will remain uncorrected during DNA replication, leading to the proliferation of genetically damaged cells(4). The failure to repair deletions or mutations in critical tumor suppressor genes can provide affected clones with a growth advantage. In this study, microsatellite alterations at selected tetranucleotide repeat markers were more commonly present in tumors containing a p53 mutation. These clonal alterations in the noncoding DNA at the microsatellite markers examined are unlikely to provide any growth advantage to the tumor and may simply reflect the defective DNA repair capability of p53 mutant cells. However, an increased mutation rate at tri- or tetranucleotide repeat sequences within genes regulating cell growth may provide a growth advantage to p53 mutant cells. Germ-line studies have documented a higher background rate of instability in larger (tri- or tetranucleotide) microsatellites, which perhaps explains why these particular microsatellites are commonly altered in p53mutant primary tumors (26, 27).

Microsatellite alterations at selected tetranucleotide markers are common in non-small cell lung, head and neck, and bladder cancer. This study suggests that the underlying mechanism leading to EMAST(+)tumors differs from MSI attributable to defective mismatch repair and is more likely related to abrogation of a p53-dependent repair pathway. A better understanding of this pathway leading to the EMAST phenotype in primary tumors may help further define the role of this diagnostic strategy in cancer screening. The widespread presence of p53 mutations in human cancer suggests that these markers may also be useful in the molecular detection of other tumor types.