Abstract
Peutz-Jeghers syndrome (PJS) is characterized by multiple gastrointestinal hamartomatous polyps, mucocutaneous melanin deposition, and increased risk of cancer, mainly in the gastrointestinal tract. We examined mutations of the LKB1, β-catenin, APC, K-ras, and p53 genes in 27 gastrointestinal hamartomatous polyps from 10 patients in nine PJS families. Of these hamartomatous polyps, one intestinal polyp had an adenomatous lesion, and one gastric polyp contained adenomatous and carcinomatous lesions. Germ-line mutations of the LKB1 gene were detected in six PJS families. Somatic mutations of the LKB1 gene were found in 5 polyps, whereas loss of heterozygosity (LOH) at the LKB1 locus at 19p was seen in 14 other polyps. In adenomatous lesions microdissected from hamartomatous polyps, both β-catenin mutation and 19p LOH were detected. Furthermore, a carcinomatous lesion in a gastric hamartomatous polyp was found to contain a mutation of the p53 gene and LOH at the p53 locus in addition to LOH at the LKB1 locus and a β-catenin mutation. K-ras mutations were detected in a few polyps, whereas no APC mutation or 5q LOH was detected in hamartomatous polyps. These results suggest that gastrointestinal hamartomatous polyps in PJS patients develop through inactivation of the LKB1 gene by germ-line mutation plus somatic mutation or LOH of the unaffected LKB1 allele, and that additional mutations of the β-catenin gene and p53 gene convert hamartomatous polyps into adenomatous and carcinomatous lesions.
Introduction
PJS 3 ,(1 , 2) is an autosomal dominant disease that is characterized by multiple gastrointestinal hamartomatous polyps and melanin spots on the lips and buccal mucosa at a young age. Patients are at increased risk of forming carcinomas in the gastrointestinal tract, uterus, ovary, breast, and other extragastrointestinal organs (3, 4, 5) . The LKB1 (STK11) gene at chromosome 19p13.3 was identified in 1998 (6 , 7) as the main causative gene for PJS, and germ-line mutations of this gene have been detected in about 60 PJS families. However, mechanisms underlying the development of hamartomas and carcinomas are still not fully understood. Hamartomas are assumed to progress to carcinomas because some hamartomas have been reported to contain adenomatous and/or carcinomatous lesions (8, 9, 10) , but it is not known whether carcinomas that develop from hamartomas acquire genetic alterations similar to those seen in sporadic colon carcinomas. Although LOH at 19p near the LKB1 gene has been demonstrated in hamartomatous polyps (11 , 12) , and 17p LOH has been detected in colon carcinomas (12) , other somatic alterations in tumors from PJS patients remain unclear. In the present study, we examined alterations in LKB1 and other genes in gastrointestinal polyps from PJS patients. We found somatic mutations of the LKB1 gene and the β-catenin gene, which are probably causative alterations for hamartomas and/or carcinomas in PJS.
Materials and Methods
Materials.
Twenty-seven gastrointestinal polyps and corresponding normal tissues were obtained from 10 patients in nine Japanese PJS families after obtaining informed consent. Polyp samples (5–55 mm in diameter) included 14 freshly frozen specimens and 13 formalin-fixed, paraffin-embedded specimens. These polyps were diagnosed histopathologically as hamartomatous polyps. One intestinal polyp had an adenomatous lesion, and one gastric polyp contained adenomatous and intestinal-type carcinomatous lesions. Genomic DNA was extracted from each specimen using proteinase K, SDS, and phenol-chloroform.
Mutation Analysis.
DNA samples were amplified for SSCP analysis of the LKB1, APC, β-catenin, K-ras, and p53 genes using PCR. The primers used to analyze germ-line and somatic mutation of LKB1 gene were the same as those reported previously (7 , 13) . Primers for somatic mutations of the APC, β-catenin, and p53 genes were the same as those described in previously published reports (14, 15, 16) , and primers for the K-ras mutation were the ras Gene Primer set (Takara Biochemicals, Kyoto, Japan). Conditions for PCR were the same as those described previously (14) . When abnormal bands were detected in the SSCP analysis, single-stranded DNA fragments were extracted, amplified by asymmetrical PCR, and then subjected to direct sequencing by a dideoxy chain termination reaction (14) .
Results
Germ-line Mutation of the LKB1 Gene.
Germ-line mutations of the LKB1 gene were identified in six patients in independent Japanese PJS families (Table 1) ⇓ . Five of the six patients had frameshift mutations resulting in a truncated LKB1 protein, and one patient had a mutation at the 5′ splice site of exon 2. Of these frameshift mutations, 1 was a 25-bp insertion that was a repeat of the sequence from the third base of codon 189 to the third base of codon 197. All six of these mutations were novel mutations.
Germ-line mutations of the LKB1 gene in Japanese PJS patients
Somatic Mutation and LOH of the LKB1 Gene in Hamartomatous Polyps.
Somatic mutations of the LKB1 gene were found in 5 of 27 (19%) intestinal hamartomatous polyps (Fig. 1 ⇓ ; Table 2 ⇓ ). TTGT deletions at codons 263–265 were detected in four polyps with an identified germ-line mutation, and a C insertion at codons 216–217 was detected in one polyp, all of which caused truncation of the LKB1 protein. LOH of the LKB1 locus was analyzed using microsatellite markers of 19p near to the LKB1 gene. Fourteen of 27 (52%) hamartomatous polyps without somatic mutation of the LKB1 gene showed 19p LOH. In total, 19 of 27 (70%) hamartomatous polyps showed possible inactivation of the LKB1 gene. In PCR-SSCP analysis, one intestinal hamartomatous polyp, PJR4 P1, showed a decreased intensity of the normal bands (corresponding to the normal allele) compared with the mutant bands (corresponding to germ-line mutation; data not shown), indicating inactivation of the LKB1 gene through germ-line mutation and loss of the normal allele. K-ras mutations of GGC (Gly) to GAC (Asp) at codon 13 were detected in 3 of 27 polyps, but no APC mutation or 5q LOH was detected in hamartomatous polyps.
Nucleotide sequence of somatic mutation of the LKB1 gene in DNA from an intestinal hamartomatous polyp from a PJS patient. TTGT deletion at codons 263–265 is present in polyp P1 from PJS patient PLK76, with a germ-line mutation at codon 37 of this gene.
Somatic mutations of the LKB1 gene in intestinal hamartomatous polyps from PJS patients
Genetic Alterations in Adenomatous Legions in PJS Polyps.
In one hamartomatous polyp, PLK396P6 (35 mm in size), a somatic β-catenin gene mutation was present in a small (3-mm) adenomatous legion that was dissected from a paraffin-embedded specimen (Fig. 2) ⇓ . This lesion also had 19p LOH. Mutations of the β-catenin gene were also detected in five other large (25–50-mm) intestinal polyps (Table 3) ⇓ . In a specimen of gastric hamartomatous polyp PLK276–3GaP1 (55 mm), an adenomatous lesion was present between the hamartomatous and carcinomatous areas. This adenomatous lesion was found to have β-catenin gene mutation and 19p LOH. Mutations detected in adenomatous lesions of intestinal polyps were ACC (Thr) to GCC (Ala) at codon 41, and a mutation in an adenomatous lesion of gastric polyp was TCT (Ser) to CCT (Pro) at codon 37.
β-Catenin gene mutation in DNA from an adenomatous lesion within an intestinal hamartomatous polyp from a PJS patient. The adenomatous area was dissected and analyzed for β-catenin mutation. Mutation from ACC to GCC at codon 41 is present in DNA from the adenomatous lesion of hamartomatous polyp PLK396P6.
β-Catenin gene mutations in gastrointestinal polyps from PJS patients
Genetic Alterations in a Carcinomatous Lesion in a PJS Polyp.
A carcinomatous lesion of gastric polyp PLK276–3GaP1 had a p53 gene mutation from CAT (His) to GAT (Asp) at codon 193 and LOH at TP53. The carcinomatous lesion exhibited the same β-catenin gene mutation as that in an adenomatous lesion of this polyp. 19p LOH near the LKB1 locus was detected in this carcinoma as well as in the adenomatous and hamartomatous areas of the same gastric polyp.
Discussion
In hamartomatous polyps from PJS patients, the present study demonstrated somatic mutations as well as germ-line mutations of the LKB1 gene, suggesting inactivation of the both alleles. Somatic mutations of the LKB1 gene were present in 5 of 27 (19%) hamartomatous polyps, and LOH of 19p near the LKB1 locus was detected in 14 of 27 (52%) hamartomatous polyps. Loss of the normal allele of LKB1 was confirmed in a hamartoma (PJR4P1) by SSCP analysis. These results suggest that although a certain percentage of hamartomatous polyps of PJS patients develop by inactivation of the LKB1 gene through germ-line mutation plus loss of the normal allele, additional mutation in the normal allele facilitates development of this disease.
With respect to the further development of hamartomatous polyps, adenomatous and carcinomatous lesions are often found within some gastrointestinal hamartomatous polyps from PJS patients (8 , 9 , 10) . Because gastrointestinal carcinomas of PJS are adenocarcinomas, precursors of these carcinomas are assumed to be adenomas. Histopathological observations indicated that adenomatous epithelium evolves within preexisting hamartomatous polyps, and carcinomatous changes were seen in the adenomatous area (8) . It has also been reported that PJS patients have such adenomatous lesions in the colon, small intestine, pancreatic duct, and stomach (8) .
In the present study, we detected β-catenin gene mutations in six large intestinal polyps, with the position and direction of these β-catenin mutations being similar to those observed frequently in sporadic colorectal adenomas and adenocarcinomas. Moreover, we found that a small adenomatous lesion within one large hamartomatous polyp had both β-catenin gene mutation and 19p LOH. This result seems to be consistent with previous histopathological observations. In five other intestinal polyps with β-catenin mutation, the intensity of mutant bands of the β-catenin gene in SSCP was rather weak compared with that of the normal bands, which indicated that the extent of cells having β-catenin mutation within hamartomatous polyps was small, although the adenomatous areas could not be clearly identified in these five polyps. In the gastric polyp, β-catenin gene mutation and 19p LOH were detected in an adenomatous lesion between the carcinomatous and the hamartomatous area. The carcinomatous lesion of this polyp had p53 gene mutation and 17p LOH, in addition to 19p LOH and the same β-catenin gene mutation as that seen in the adenomatous lesion. These data suggest the following mechanisms: inactivation of the LKB1 gene causes the formation of a hamartomatous polyp, and during growth of the hamartoma, β-catenin gene mutation occurs, resulting in a change of the hamartoma to an adenoma because the activating β-catenin mutation has been known to be associated with adenoma formation (18 , 19) . Such an adenomatous lesion may progress to carcinoma through additional genetic alterations, such as p53 mutation, as seen in sporadic colorectal carcinogenesis (16) . The contribution of APC mutation to PJS carcinogenesis appears to be small.
Considering the present and previous observations, we propose a possible hamartoma-adenoma-carcinoma sequence for gastrointestinal carcinogenesis in PJS patients, rather than the direct change of a hamartoma into an adenocarcinoma. PJS patients develop not only gastrointestinal carcinomas but also various extragastrointestinal carcinomas, which are assumed to be formed by inactivation of the LKB1 gene, as evidenced by breast carcinomas from our PJS patients showing 19p LOH (data not shown). To clarify the mechanism of increased risk of carcinogenesis in PJS patients, a more detailed analysis of genetic changes in both gastrointestinal and extragastrointestinal tumors is necessary.
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 the Project“ High-Technology Research Center” and a Grant from the Ministry of Education, Science, Sports and Culture of Japan.
-
↵2 To whom requests for reprints should be addressed, at Hereditary Tumor Research Project, Tokyo Metropolitan Komagome Hospital, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8677, Japan. Phone: 81-3-3823-2101, ext. 4425; Fax: 81-3-3823-5433; E-mail: mmiyaki{at}opal.famille.ne.jp
-
↵3 The abbreviations used are: PJS, Peutz-Jeghers syndrome; LOH, loss of heterozygosity; SSCP, single-strand conformation polymorphism.
- Received July 5, 2000.
- Accepted September 26, 2000.
- ©2000 American Association for Cancer Research.
References
Volume 60, Issue 22
