Title: Germline Inactivation of PTEN and Dysregulation of the Phosphoinositol-3-Kinase/Akt Pathway Cause Human Lhermitte-Duclos Disease in Adults
Abstract: Lhermitte-Duclos disease (LDD), or dysplastic gangliocytoma of the cerebellum, is an unusual hamartomatous overgrowth disorder. LDD can be familial or, more commonly, sporadic. It has been only recently recognized that LDD may be associated with Cowden syndrome (CS). Over 80% of patients with CS carry germline mutations in PTEN. It remains unclear whether all cases of LDD, even without features of CS, are caused by germline PTEN mutation and whether somatic PTEN mutation occurs in sporadic LDD. We obtained paraffin-embedded LDD lesions from 18 unselected, unrelated patients and performed mutational analysis of PTEN. Overall, 15 (83%) of 18 samples were found to carry a PTEN mutation. All individuals with mutations were adult-onset patients, but the three without mutations were diagnosed at the ages of 1, 3, and 11 years. Germline DNA was available from six adult-onset cases, and all had germline PTEN mutations. Of these six, two had CS features, one did not have CS features, and three were of unknown CS status. Immunohistochemistry revealed that 75% of the LDD samples had complete or partial loss of PTEN expression accompanied by elevated phosphorylated Akt, specifically in the dysplastic gangliocytoma cells. These data suggest that the loss of PTEN function is sufficient to cause LDD. The high frequency and spectrum of germline PTEN mutations in patients ascertaining by LDD alone confirm that LDD is an important defining feature of CS. Individuals with LDD, even without apparent CS features, should be counseled as in CS. Lhermitte-Duclos disease (LDD), or dysplastic gangliocytoma of the cerebellum, is an unusual hamartomatous overgrowth disorder. LDD can be familial or, more commonly, sporadic. It has been only recently recognized that LDD may be associated with Cowden syndrome (CS). Over 80% of patients with CS carry germline mutations in PTEN. It remains unclear whether all cases of LDD, even without features of CS, are caused by germline PTEN mutation and whether somatic PTEN mutation occurs in sporadic LDD. We obtained paraffin-embedded LDD lesions from 18 unselected, unrelated patients and performed mutational analysis of PTEN. Overall, 15 (83%) of 18 samples were found to carry a PTEN mutation. All individuals with mutations were adult-onset patients, but the three without mutations were diagnosed at the ages of 1, 3, and 11 years. Germline DNA was available from six adult-onset cases, and all had germline PTEN mutations. Of these six, two had CS features, one did not have CS features, and three were of unknown CS status. Immunohistochemistry revealed that 75% of the LDD samples had complete or partial loss of PTEN expression accompanied by elevated phosphorylated Akt, specifically in the dysplastic gangliocytoma cells. These data suggest that the loss of PTEN function is sufficient to cause LDD. The high frequency and spectrum of germline PTEN mutations in patients ascertaining by LDD alone confirm that LDD is an important defining feature of CS. Individuals with LDD, even without apparent CS features, should be counseled as in CS. PTEN/MMAC1/TEP1 (MIM 601728), on 10q23.3, encodes a lipid and protein phosphatase (Li and Sun Li and Sun, 1997Li DM Sun H TEP1, encoded by a candidate tumor suppressor locus, is a novel protein tyrosine phosphatase regulated by transforming growth factor beta.Cancer Res. 1997; 57: 2124-2129PubMed Google Scholar; Li et al. Li et al., 1997Li J Yen C Liaw D Podsypanina K Bose S Wang SI Puc J Miliaresis C Rodgers L McCombie R Bigner SH Giovanella BC Ittmann M Tycko B Hibshoosh H Wigler MH Parsons R PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer.Science. 1997; 275: 1943-1947Crossref PubMed Scopus (4079) Google Scholar; Steck et al. 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Germline mutations in PTEN cause Cowden syndrome (CS [MIM 158350]), an autosomal-dominant condition with age-related penetrance characterized by multiple hamartomas affecting derivatives of all three germ layers and by a high risk of breast, thyroid, and endometrial cancers (Liaw et al. Liaw et al., 1997Liaw D Marsh DJ Li J Dahia PL Wang SI Zheng Z Bose S Call KM Tsou HC Peacocke M Eng C Parsons R Germline mutations of the PTEN gene in Cowden disease, an inherited breast and thyroid cancer syndrome.Nat Genet. 1997; 16: 64-67Crossref PubMed Scopus (1617) Google Scholar; Marsh et al. 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A subset of seemingly unrelated clinical conditions, such as Proteus syndrome (PS [MIM 176920]), Proteus-like syndromes, and VATER association with macrocephaly and ventriculomegaly, have been associated with germline PTEN mutations (Zhou et al. Zhou et al., 2000bZhou XP Marsh DJ Hampel H Mulliken JB Gimm O Eng C Germline and germline mosaic PTEN mutations associated with a Proteus-like syndrome of hemihypertrophy, lower limb asymmetry, arteriovenous malformations and lipomatosis.Hum Mol Genet. 2000; 9: 765-768Crossref PubMed Scopus (167) Google Scholar, Zhou et al., 2001Zhou XP Hampel H Thiele H Gorlin RJ Hennekam RC Parisi M Winter RM Eng C Association of germline mutation in the PTEN tumour suppressor gene and Proteus and Proteus-like syndromes.Lancet. 2001; 358: 210-211Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar; Reardon et al. 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LDD is believed to be a hamartomatous overgrowth of hypertrophic ganglion cells that replace the granular-cell layer and Purkinje cells of the cerebellum, resulting in global thickening of the cerebellar folia (Ambler et al. Ambler et al., 1969Ambler M Pogacar S Sidman R Lhermitte-Duclos disease (granule cell hypertrophy of the cerebellum) pathological analysis of the first familial cases.J Neuropathol Exp Neurol. 1969; 28: 622-647Crossref PubMed Scopus (103) Google Scholar; Russell-Jones et al. Russell-Jones et al., 1981Russell-Jones R O’Brien M Wells RS Cowden syndrome.Br J Dermatol. 1981; 105: 57-58Google Scholar; Albrecht et al. 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Nelen et al., 1999Nelen MR Kremer H Konings IB Schoute F van Essen AJ Koch R Woods CG Fryns JP Hamel B Hoefsloot LH Peeters EA Padberg GW Novel PTEN mutations in patients with Cowden disease: absence of clear genotype-phenotype correlations.Eur J Hum Genet. 1999; 7: 267-273Crossref PubMed Scopus (215) Google Scholar; Sutphen et al. Sutphen et al., 1999Sutphen R Diamond TM Minton SE Peacocke M Tsou HC Root AW Severe Lhermitte-Duclos disease with unique germline mutation of PTEN.Am J Med Genet. 1999; 82: 290-293Crossref PubMed Scopus (39) Google Scholar; Robinson and Cohen Robinson and Cohen, 2000Robinson S Cohen AR Cowden disease and Lhermitte-Duclos disease: characterization of a new phakomatosis.Neurosurgery. 2000; 46: 371-383Crossref PubMed Scopus (78) Google Scholar). Of these, 67 cases had features suggestive or diagnostic of CS, although a definitive diagnosis of CS in the majority of these cases was not established. Prior to the identification of PTEN as the CS-susceptibility gene (Liaw et al. Liaw et al., 1997Liaw D Marsh DJ Li J Dahia PL Wang SI Zheng Z Bose S Call KM Tsou HC Peacocke M Eng C Parsons R Germline mutations of the PTEN gene in Cowden disease, an inherited breast and thyroid cancer syndrome.Nat Genet. 1997; 16: 64-67Crossref PubMed Scopus (1617) Google Scholar), the co-occurrence of documented CS cases and families with LDD (Padberg et al. Padberg et al., 1991Padberg GW Schot JD Vielvoye GJ Bots GT de Beer FC Lhermitte-Duclos disease and Cowden disease: a single phakomatosis.Ann Neurol. 1991; 29: 517-523Crossref PubMed Scopus (161) Google Scholar; Albrecht et al. Albrecht et al., 1992Albrecht S Haber RM Goodman JC Duvic M Cowden syndrome and Lhermitte-Duclos disease.Cancer. 1992; 70: 869-876Crossref PubMed Scopus (100) Google Scholar; Eng et al. Eng et al., 1994Eng C Murday V Seal S Mohammed S Hodgson SV Chaudary MA Fentiman IS Ponder BA Eeles RA Cowden syndrome and Lhermitte-Duclos disease in a family: a single genetic syndrome with pleiotropy?.J Med Genet. 1994; 31: 458-461Crossref PubMed Scopus (88) Google Scholar) suggested, but did not show conclusively, that LDD and CS share a common etiology. Subsequently, germline PTEN mutations have been identified in three well-documented CS kindreds segregating LDD (Liaw et al. Liaw et al., 1997Liaw D Marsh DJ Li J Dahia PL Wang SI Zheng Z Bose S Call KM Tsou HC Peacocke M Eng C Parsons R Germline mutations of the PTEN gene in Cowden disease, an inherited breast and thyroid cancer syndrome.Nat Genet. 1997; 16: 64-67Crossref PubMed Scopus (1617) Google Scholar; Nelen et al. Nelen et al., 1997Nelen MR van Staveren WC Peeters EA Hassel MB Gorlin RJ Hamm H Lindboe CF Fryns JP Sijmons RH Woods DG Mariman EC Padberg GW Kremer H Germline mutations in the PTEN/MMAC1 gene in patients with Cowden disease.Hum Mol Genet. 1997; 6: 1383-1387Crossref PubMed Scopus (395) Google Scholar; Marsh et al. Marsh et al., 1998Marsh DJ Coulon V Lunetta KL Rocca-Serra P Dahia PL Zheng Z Liaw D Caron S Duboue B Lin AY Richardson AL Bonnetblanc JM Bressieux JM Cabarrot-Moreau A Chompret A Demange L Eeles RA Yahanda AM Fearon ER Fricker JP Gorlin RJ Hodgson SV Huson S Lacombe D Eng C et al.Mutation spectrum and genotype-phenotype analyses in Cowden disease and Bannayan-Zonana syndrome, two hamartoma syndromes with germline PTEN mutation.Hum Mol Genet. 1998; 7: 507-515Crossref PubMed Scopus (521) Google Scholar), thus lending some molecular credence to the previous clinical hypothesis. Conditional Pten disruption in the mouse cerebellum resulted in a LDD-like phenotype, further suggesting that loss of function of PTEN might be sufficient to cause human LDD (Backman et al. Backman et al., 2001Backman SA Stambolic V Suzuki A Haight J Elia A Pretorius J Tsao MS Shannon P Bolon B Ivy GO Mak TW Deletion of Pten in mouse brain causes seizures, ataxia and defects in soma size resembling Lhermitte-Duclos disease.Nat Genet. 2001; 29: 396-403Crossref PubMed Scopus (390) Google Scholar; Kwon et al. Kwon et al., 2001Kwon CH Zhu X Zhang J Knoop LL Tharp R Smeyne RJ Eberhart CG Burger PC Baker SJ Pten regulates neuronal soma size: a mouse model of Lhermitte-Duclos disease.Nat Genet. 2001; 29: 404-411Crossref PubMed Scopus (349) Google Scholar). However, it is not known if all cases of human LDD, even without features of CS, are caused by germline PTEN mutation and if somatic PTEN mutation can account for sporadic LDD. To this end, we have accrued 18 unselected, unrelated patients with LDD, without regard to the presence or absence of other features and have performed an extensive analysis for the presence of PTEN mutations. We also examined the expression levels of PTEN and phosphorylated Akt (P-Akt) in archived affected cerebellar tissues using immunohistochemistry. Archived paraffin-embedded tissue from 18 unselected, anonymized patients with LDD (with linked demographic, clinical, and pathologic information) was accrued from multiple centers in the United States and Europe, in accordance with protocols approved by the Dana-Farber Cancer Institute (1996–1998) and The Ohio State University (1998–present) institutional review boards. For all patients, the diagnosis of LDD was confirmed by pathology after surgical resection or biopsy. All histopathologic sections were subjected to centralized re-review by C.D.M. and A.R.C. Available clinical information from all 18 patients was reviewed to determine if any of them met the operational diagnostic criteria for CS, as delineated by the International Cowden Consortium or the U.S. National Comprehensive Cancer Network (Eng Eng, 2000Eng C Will the real Cowden syndrome please stand up: revised diagnostic criteria.J Med Genet. 2000; 37: 828-830Crossref PubMed Scopus (435) Google Scholar) (table 1). Of the 18 patients reviewed, 4 individuals (LDD-7, LDD-8a, LDD-8b, and LDD-14), all female, could be clinically classified as having CS. In 3 patients (LDD-1, LDD-5, and LDD-17), the diagnostic features of CS were absent. Insufficient clinical information was available to conclusively determine CS status in the remaining 11 patients.Table 1Summary of Patients with LDD and PTEN and P-Akt Status by ImmunohistochemistryCharacteristics of Patients with LDDResults of AnalysisPatientSex/Age (years)CS/Features of CSPTEN MutationsGermline/SomaticLOHaLOH=loss of heterozygosity./ROHbROH=retention of heterozygosity.PTEN LevelsP-Akt LevelsLDD-G1F/53?16–18 del AA++++LDD-G2M/38?381–4 del AAAG−++LDD-G3F/1?−++−LDD-G4F/55?R130X+−LDD-G5M/27?99 del TGermlineLOH−++LDD-1F/51No758 ins A+++LDD-2F/11No−LDD-5F/35NoP246LGermlineROH+++LDD-7F/50Yes53 del AGermlineLOH+LDD-8aF/43YesY16X+++LDD-8bF/34YesQ110X+++LDD-8cF/??R130X−, ++++, −LDD-9a??Y88HGermlineLDD-11??G36R−LDD-12??K80E++LDD-14F/43Likely347–51del ACAATGermline−−LDD-17M/3Unlikely−LDD-19M/28?Y88HGermlineNIcNI=not informative.+++H141RSomaticNote.—Blank=not done because of lack of sample material.a LOH=loss of heterozygosity.b ROH=retention of heterozygosity.c NI=not informative. Open table in a new tab Note.— Blank=not done because of lack of sample material. DNA was extracted from archival paraffin-embedded cerebellar tissue sections with lesions using a QIAamp DNA Mini Kit (Qiagen). The procedure was performed according to the manufacturer’s instructions, except that a prolonged (2-d) proteinase-K digestion at 65°C was added. The samples were then subjected to PTEN mutation analysis, which was performed without knowledge of other clinical information. The entire coding sequence, the exon-intron boundaries, and the flanking sequences of PTEN were analyzed for mutations using PCR-based denaturing gradient gel electrophoresis (DGGE) and sequencing, as described elsewhere (Marsh et al. Marsh et al., 1998Marsh DJ Coulon V Lunetta KL Rocca-Serra P Dahia PL Zheng Z Liaw D Caron S Duboue B Lin AY Richardson AL Bonnetblanc JM Bressieux JM Cabarrot-Moreau A Chompret A Demange L Eeles RA Yahanda AM Fearon ER Fricker JP Gorlin RJ Hodgson SV Huson S Lacombe D Eng C et al.Mutation spectrum and genotype-phenotype analyses in Cowden disease and Bannayan-Zonana syndrome, two hamartoma syndromes with germline PTEN mutation.Hum Mol Genet. 1998; 7: 507-515Crossref PubMed Scopus (521) Google Scholar; Mutter et al. Mutter et al., 2000Mutter GL Lin MC Fitzgerald JT Kum JB Baak JP Lees JA Weng LP Eng C Altered PTEN expression as a diagnostic marker for the earliest endometrial precancers.J Natl Cancer Inst. 2000; 92: 924-930Crossref PubMed Google Scholar). A mutation-positive result led us to obtain germline DNA from paraffin-embedded adjacent normal tissue, if present, using laser capture microdissection (LCM) (Kurose et al. Kurose et al., 2002Kurose K Gilley K Matsumoto S Watson PH Zhou XP Eng C Frequent somatic mutations in PTEN and TP53 are mutually exclusive in the stroma of breast carcinomas.Nat Genet. 2002; 32: 355-357Crossref PubMed Scopus (344) Google Scholar); LCM-derived germline DNA was then subjected to PTEN mutation analysis. A mutation-negative result in tissue denotes the absence of PTEN mutation in both the germline and in the tissue. Samples that were mutation negative in the coding and flanking intronic regions were subjected to PTEN promoter mutation analysis, as described elsewhere (Zhou et al. Zhou et al., 2003Zhou XP Waite KA Pilarski R Hampel H Fernandez MJ Bos C Dasouki M Feldman GL Greenberg LA Ivanovich J Matloff E Patterson A Pierpont ME Russo D Nassif NT Eng C Germline PTEN promoter mutations and deletions in Cowden/Bannayan-Riley-Ruvalcaba syndrome result in aberrant PTEN protein and dysregulation of the phosphoinositol-3-kinase/Akt pathway.Am J Hum Genet. 2003; 73: 404-411Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). PCR-based DGGE and subsequent sequencing revealed 16 intragenic PTEN mutations in 15 (83%) of the 18 samples (table 1). By using LCM, we were able to microdissect affected and adjacent nonaffected tissues in four samples (LDD-G5, LDD-5, LDD-7, and LDD-19). Nonaffected tissue sections were available for another two patients (LDD-9a and LDD-14) and served as sources of germline DNA. Thus, six had matched germline DNA, and all six were found to have germline mutations. In the sample from patient LDD-19, in addition to the germline mutation Y88H, which was detected in DNA from both nonaffected bone tissue and affected cerebellar tissue, a second mutation, H141R, was only detected in DNA from the affected cerebellar tissue, suggesting that the latter is a somatic mutation. Of the 16 mutations found in 15 patients with LDD, 10 were truncating mutations, comprising 4 nonsense mutations and 6 frameshifting microdeletions or -insertions, and 6 were missense mutations. Mutation R130X was detected in two unrelated patients, LDD-G4 and LDD-8c. Similarly, mutation Y88H was detected in the germline of two unrelated patients, LDD-9a and LDD-19 (table 1). The recurrent R130X and Y88H as founder mutations were excluded by genotype analysis (data not shown). No sequence variants in the promoter region were found in the three LDD samples without intragenic PTEN mutations (LDD-G3, LDD-2, and LDD-17) (table 1). Loss-of-heterozygosity (LOH) analysis was performed on four LDD samples using two microsatellite markers flanking PTEN, D10S1765 and D10S541, as previously described (Marsh et al. Marsh et al., 1999Marsh DJ Kum JB Lunetta KL Bennett MJ Gorlin RJ Ahmed SF Bodurtha J Crowe C Curtis MA Dasouki M Dunn T Feit H Geraghty MT Graham Jr, JM Hodgson SV Hunter A Korf BR Manchester D Miesfeldt S Murday VA Nathanson KL Parisi M Pober B Romano C Eng C et al.PTEN mutation spectrum and genotype-phenotype correlations in Bannayan-Riley-Ruvalcaba syndrome suggest a single entity with Cowden syndrome.Hum Mol Genet. 1999; 8: 1461-1472Crossref PubMed Scopus (448) Google Scholar). Of the four samples analyzed, three were informative at a minimum of one marker, and one was not informative for either marker. LOH was scored when at least one of the two polymorphic loci showed evidence of LOH. We found that two LDD samples had LOH at the PTEN locus in the affected cerebellar tissue, thus leaving the remaining germline mutant allele (LDD-G5 and LDD-7) (table 1). No LOH was detected in the third informative LDD sample (LDD-5). We next examined the PTEN expression level and P-Akt level in these LDD samples by immunohistochemistry using antibodies against PTEN (6H2.1) and P-Akt (Ser 473) (Cell Signaling Technology), respectively. Immunohistochemical detection of PTEN and P-Akt was carried out following protocols described elsewhere (Perren et al. Perren et al., 1999Perren A Weng LP Boag AH Ziebold U Thakore K Dahia PL Komminoth P Lees JA Mulligan LM Mutter GL Eng C Immunohistochemical evidence of loss of PTEN expression in primary ductal adenocarcinomas of the breast.Am J Pathol. 1999; 155: 1253-1260Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar; Zhou et al. Zhou et al., 2000aZhou XP Gimm O Hampel H Niemann T Walker MJ Eng C Epigenetic PTEN silencing in malignant melanomas without PTEN mutation.Am J Pathol. 2000; 157: 1123-1128Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). As noted in other studies (Perren et al. Perren et al., 1999Perren A Weng LP Boag AH Ziebold U Thakore K Dahia PL Komminoth P Lees JA Mulligan LM Mutter GL Eng C Immunohistochemical evidence of loss of PTEN expression in primary ductal adenocarcinomas of the breast.Am J Pathol. 1999; 155: 1253-1260Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar; Gimm et al. Gimm et al., 2000Gimm O Perren A Weng LP Marsh DJ Yeh JJ Ziebold U Gil E Hinze R Delbridge L Lees JA Mutter GL Robinson BG Komminoth P Dralle H Eng C Differential nuclear and cytoplasmic expression of PTEN in normal thyroid tissue, and benign and malignant epithelial thyroid tumors.Am J Pathol. 2000; 156: 1693-1700Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar; Zhou et al