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Editor—PTEN, a tumour suppressor gene located in chromosome 10q23, is homologous to tyrosine and dual specificity phosphatases with a high degree of substrate specificity. This enzymatic activity is necessary forPTEN/MMAC1 tumour suppressor function.1-4 Mutations of this gene have been identified in many glioma, glioblastoma, prostate, kidney, and breast carcinoma cell lines and in primary tumours including gliomas, and breast, thyroid, and kidney carcinomas.1 2 5 Germline mutations of the PTEN gene underlie Cowden disease, an autosomal dominant disorder associated with an increased risk of breast and thyroid cancer and possibly endometrial malignancy. Benign tumours of the intestine (hamartomas) and skin (such as trichilemmomas) also occur.6 7
LKB1, a candidate tumour suppressor gene, encodes a serine/threonine kinase which is highly homologous (84%) to Xenopus serine/threonine kinase XEEK1.8 Germline mutations in LKB1 have been associated with Peutz-Jeghers syndrome (PJS).9 10 Jenneet al 10 report that their success in identifying the gene by analysing only two candidate sequences was based on strong linkage disequilibrium. This is surprising, as while linkage disequilibrium is a powerful tool in disease gene identification, no linkage disequilibrium has been reported in PJS and the patients typically display different mutations.9 10 PJS is characterised by hamartomatous intestinal polyposis, mucocutaneous pigmentation, and increased risk of cancer of multiple organ systems.6 LKB1 germline mutations are typically of inactivating nature, often causing truncation of the protein product.9-12 Genes involved in hereditary cancer syndromes are often targets of somatic mutations. In the case of theLKB1 gene, several studies, with the exception of one study on colorectal cancer,13 have reported a low frequency of LKB1 somatic mutations in colorectal, testicular, breast, and gastric cancers.14-16
The aetiology of laryngeal cancer is considered to be multifactorial and genetic alterations are likely to play a role in it. ThePTEN and LKB1genes have been recently characterised and appear to cause somewhat similar phenotypes when mutated in the germline.6 Because of their tumour suppressor function, bothPTEN and LKB1 are possible candidates as genes which could be involved in laryngeal tumorigenesis. To test this hypothesis, we evaluated the role ofPTEN and LKB1somatic mutations in malignant and premalignant laryngeal tumours.
This study was based on laryngeal tumour specimens collected from 16 patients treated at Helsinki University Central Hospital, Department of Otorhinolaryngology and Head and Neck Surgery. Thirteen of these patients had laryngeal carcinoma and three had laryngeal papilloma. Of the patients with papilloma, one had juvenile onset and two had adult onset disease. One of these adult onset patients had a malignant transformation of his laryngeal tumour into epidermoid carcinoma (table1). The tumour samples were resected during operations and fresh frozen. A 5 μm section of each tumour was stained with haematoxylin to ensure that the sample contained at least 60% of tumour tissue. DNA was extracted from the laryngeal tumour samples with phenol-chloroform according to the standard procedure.
The nine exons of the PTEN gene were sequenced by means of nested primers designed within the flanking intronic sequence. PCR conditions and primers have been described previously.7 17 After PCR, 5 μl of each amplicon was run on a 2% agarose gel to verify the specificity of the PCR reaction. The rest was purified with QIAquick PCR purification kit (QIAGEN GmbH, Hilder, Germany). Direct sequencing of PCR products was performed with the ABI PRISM Dye Terminator or ABI PRISM dRhodamine cycle sequencing kits (Perkin-Elmer Applied Biosytems Division, Forster City, CA). Cycle sequencing products were analysed on an Applied Biosytems model 377 DNA sequencer (PE/ABI) or 310 Genetic Analyzer (PE/ABI).
Mutation analysis of the LKB1 gene was performed by single strand conformation polymorphism (SSCP) analysis. Primer sequences and PCR conditions used in this study have been described previously.18 After PCR, 5 μl of each reaction product was mixed with 5 μl of denaturing loading buffer (98% formamide, 20 mmol/l EDTA, 0.05% bromphenol blue, and 0.05% xylene cyanol), denatured for five minutes at 94°C, and subjected to electrophoresis on 0.4 mm × 30 cm × 45 cm gels containing 0.6 × mutation detection enhancement solution (AT Biochem, Malvern, PA) and 0.6 × TBE buffer. Electrophoresis was conducted at 4 W overnight. PCR fragments were visualised in gels by silver staining. In order to validate the SSCP results, amplicon of exon 1 in theLKB1 gene from all samples were directly sequenced (as described above).
To determine whether the PTEN andLKB1 genes are mutated in laryngeal tumours, we screened 16 laryngeal tumour samples by genomic sequencing and SSCP assay, respectively. We performed direct sequencing of amplicon forPTEN mutation analysis; no mutations in the coding sequences and exon/intron boundaries were found. To screen forLKB1 mutations, SSCP assay was applied in conjunction with PCR. Difference in mobility pattern on the SSCP assay usually suggests a variant, the nature of which should be examined by sequencing. In our hands SSCP analysis ofLKB1 has always shown known mutations and it is reasonable to assume a 70% to 80% sensitivity, which is commonly reported for the method.19 To validate the SSCP results, exon 1 was directly sequenced from each sample. No mutations in exon 1 and the respective exon/intron boundaries were observed. The mutation detection methods used in this study would not have detected some mutation types, such as large genomic deletions or alterations in the promoter region. Yet the complete absence of changes indicates that somatic mutations of PTEN andLKB1 are not frequent in laryngeal tumours.
The molecular events which induce laryngeal tumorigenesis, especially laryngeal carcinogenesis, are not well characterised. Proto-oncogenes seem to be the target of the risk factors (cigarette smoking, alcohol abuse, ionising radiation, and human papillomavirus infection) that are commonly considered to be associated with laryngeal squamous cell carcinoma. Several tumour suppressor genes have been shown to play important roles in human tumours, including head and neck cancers. Mutations of the p53 gene are frequent events in primary squamous cell carcinomas of the head and neck as well as in SCCHN cell lines. While many cancer genes are associated with laryngeal tumorigenesis,20 our study suggests thatPTEN and LKB1 are not among them.
The authors are grateful to Annukka Nyholm, Tiiu Arumäe, and Irina Suomalainen for excellent technical assistance. This work was supported by a grant from Helsinki University Central Hospital Research Fund.