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Differential Nuclear and Cytoplasmic Expression of PTEN in Normal Thyroid Tissue, and Benign and Malignant Epithelial Thyroid Tumors

2000; Elsevier BV; Volume: 156; Issue: 5 Linguagem: Inglês

10.1016/s0002-9440(10)65040-7

1525-2191

Oliver Gimm, Aurel Perren, Liang‐Ping Weng, Deborah J. Marsh, Jen Jen Yeh, Ulrike Ziebold, Elad B. Gil, Raoul Hinze, Leigh Delbridge, Jacqueline A. Lees, George L. Mutter, Bruce G. Robinson, Paul Komminoth, Henning Dralle, Charis Eng,

Coagulation, Bradykinin, Polyphosphates, and Angioedema

Germline mutations in PTEN (MMAC1/TEP1) are found in patients with Cowden syndrome, a familial cancer syndrome which is characterized by a high risk of breast and thyroid neoplasia. Although somatic intragenic PTEN mutations have rarely been found in benign and malignant sporadic thyroid tumors, loss of heterozygosity (LOH) has been reported in up to one fourth of follicular thyroid adenomas (FAs) and carcinomas. In this study, we examined PTEN expression in 139 sporadic nonmedullary thyroid tumors (55 FA, 27 follicular thyroid carcinomas, 35 papillary thyroid carcinomas, and 22 undifferentiated thyroid carcinomas) using immunohistochemistry and correlated this to the results of LOH studies. Normal follicular thyroid cells showed a strong to moderate nuclear or nuclear membrane signal although the cytoplasmic staining was less strong. In FAs the neoplastic nuclei had less intense PTEN staining, although the cytoplasmic PTEN-staining intensity did not differ significantly from that observed in normal follicular cells. In thyroid carcinomas as a group, nuclear PTEN immunostaining was mostly weak in comparison with normal thyroid follicular cells and FAs. The cytoplasmic staining was more intense than the nuclear staining in 35 to 49% of carcinomas, depending on the histological type. Among 81 informative tumors assessed for LOH, there seemed to be an associative trend between decreased nuclear and cytoplasmic staining and 10q23 LOH (P = 0.003, P = 0.008, respectively). These data support a role for PTEN in the pathogenesis of follicular thyroid tumors. Germline mutations in PTEN (MMAC1/TEP1) are found in patients with Cowden syndrome, a familial cancer syndrome which is characterized by a high risk of breast and thyroid neoplasia. Although somatic intragenic PTEN mutations have rarely been found in benign and malignant sporadic thyroid tumors, loss of heterozygosity (LOH) has been reported in up to one fourth of follicular thyroid adenomas (FAs) and carcinomas. In this study, we examined PTEN expression in 139 sporadic nonmedullary thyroid tumors (55 FA, 27 follicular thyroid carcinomas, 35 papillary thyroid carcinomas, and 22 undifferentiated thyroid carcinomas) using immunohistochemistry and correlated this to the results of LOH studies. Normal follicular thyroid cells showed a strong to moderate nuclear or nuclear membrane signal although the cytoplasmic staining was less strong. In FAs the neoplastic nuclei had less intense PTEN staining, although the cytoplasmic PTEN-staining intensity did not differ significantly from that observed in normal follicular cells. In thyroid carcinomas as a group, nuclear PTEN immunostaining was mostly weak in comparison with normal thyroid follicular cells and FAs. The cytoplasmic staining was more intense than the nuclear staining in 35 to 49% of carcinomas, depending on the histological type. Among 81 informative tumors assessed for LOH, there seemed to be an associative trend between decreased nuclear and cytoplasmic staining and 10q23 LOH (P = 0.003, P = 0.008, respectively). These data support a role for PTEN in the pathogenesis of follicular thyroid tumors. The tumor suppressor PTEN, also known as MMAC1 and TEP1,1Li 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): 277Crossref Scopus (4363) Google Scholar, 2Steck PA Pershouse MA Jasser SA Yung WK Lin H Ligon AH Langford LA Baumgard ML Hattier T Davis T Frye C Hu R Swedlund B Teng DH Tavtigian SV Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers.Nat Genet. 1997; 15: 356-362Crossref PubMed Scopus (2552) Google Scholar, 3Li 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 has recently been shown to play an important role in the pathogenesis of a variety of human cancers.1Li 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): 277Crossref Scopus (4363) Google Scholar, 2Steck PA Pershouse MA Jasser SA Yung WK Lin H Ligon AH Langford LA Baumgard ML Hattier T Davis T Frye C Hu R Swedlund B Teng DH Tavtigian SV Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers.Nat Genet. 1997; 15: 356-362Crossref PubMed Scopus (2552) Google Scholar, 4Teng DH Hu R Lin H Davis T Iliev D Frye C Swedlund B Hansen KL Vinson VL Gumpper KL Ellis L El-Naggar A Frazier M Jasser S Langford LA Lee J Mills GB Pershouse MA Pollack RE Tornos C Troncoso P Yung WK Fujii G Berson A Bookstein R Bolen JB Tavtigian SV Steck PA MMAC1/PTEN mutations in primary tumor specimens and tumor cell lines.Cancer Res. 1997; 57: 5221-5225PubMed Google Scholar PTEN is located on chromosome subband 10q23.32Steck PA Pershouse MA Jasser SA Yung WK Lin H Ligon AH Langford LA Baumgard ML Hattier T Davis T Frye C Hu R Swedlund B Teng DH Tavtigian SV Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers.Nat Genet. 1997; 15: 356-362Crossref PubMed Scopus (2552) Google Scholar, 5Myers MP Stolarov JP Eng C Li J Wang SI Wigler MH Parsons R Tonks NK P-TEN, the tumor suppressor from human chromosome 10q23, is a dual-specificity phosphatase.Proc Natl Acad Sci USA. 1997; 94: 9052-9057Crossref PubMed Scopus (738) Google Scholar and encodes a dual-specificity phosphatase with lipid and protein phosphatase activity. The major substrate for PTEN is phosphatidylinositol-3,4,5-trisphosphate, a direct product of phosphoinositol-3-kinase activity.6Maehama T Dixon JE The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate.J Biol Chem. 1998; 273: 13375-13378Crossref PubMed Scopus (2648) Google Scholar, 7Myers MP Pass I Batty IH Van der Kaay J Stolarov JP Hemmings BA Wigler MH Downes CP Tonks NK The lipid phosphatase activity of PTEN is critical for its tumor suppressor function.Proc Natl Acad Sci USA. 1998; 95: 13513-13518Crossref PubMed Scopus (1023) Google Scholar, 8Stambolic V Suzuki A de la Pompa JL Brothers GM Mirtsos C Sasaki T Ruland J Penninger JM Siderovski DP Mak TW Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN.Cell. 1998; 95: 29-39Abstract Full Text Full Text PDF PubMed Scopus (2151) Google Scholar Phosphatidylinositol-3,4,5-trisphosphate mediates growth factor-induced activation of intracellular signaling, in particular through the serine-threonine kinase Akt (also referred to as Akt1, RAC1, or PKB), which is known to promote cell survival and cell proliferation. High levels of PTEN are associated with low levels of phosphorylated Akt which leads to the induction of apoptosis; hence, loss of PTEN function leads to increased activity of Akt and subsequently cell survival.7Myers MP Pass I Batty IH Van der Kaay J Stolarov JP Hemmings BA Wigler MH Downes CP Tonks NK The lipid phosphatase activity of PTEN is critical for its tumor suppressor function.Proc Natl Acad Sci USA. 1998; 95: 13513-13518Crossref PubMed Scopus (1023) Google Scholar, 8Stambolic V Suzuki A de la Pompa JL Brothers GM Mirtsos C Sasaki T Ruland J Penninger JM Siderovski DP Mak TW Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN.Cell. 1998; 95: 29-39Abstract Full Text Full Text PDF PubMed Scopus (2151) Google Scholar, 9Haas-Kogan D Shalev N Wong M Mills G Yount G Stokoe D Protein kinase B (PKB/Akt) activity is elevated in glioblastoma cells due to mutation of the tumor suppressor PTEN/MMAC.Curr Biol. 1998; 8: 1195-1198Abstract Full Text Full Text PDF PubMed Google Scholar, 10Dahia PL Aguiar RC Alberta J Kum JB Caron S Sill H Marsh DJ Ritz J Freedman A Stiles C Eng C PTEN is inversely correlated with the cell survival factor Akt/PKB and is inactivated via multiple mechanisms in haematological malignancies.Hum Mol Genet. 1999; 8: 185-193Crossref PubMed Scopus (270) Google Scholar PTEN may also affect other pathways, such as the focal adhesion kinase and mitogen activated protein kinase pathways.11Tamura M Gu J Matsumoto K Aota S Parsons R Yamada KM Inhibition of cell migration, spreading, and focal adhesions by tumor suppressor PTEN.Science. 1998; 280: 1614-1617Crossref PubMed Scopus (1094) Google Scholar, 12Gu J Tamura M Yamada KM Tumor suppressor PTEN inhibits integrin- and growth factor-mediated mitogen-activated protein (MAP) kinase signaling pathways.J Cell Biol. 1998; 143: 1375-1383Crossref PubMed Scopus (304) Google Scholar In other words, PTEN has been shown to mediate G1 cell-cycle arrest and/or apoptosis via the phosphoinositol-3-kinase-Akt pathway in several cell lines such as glioma, breast, and prostate cell lines.13Furnari FB Huang HJ Cavenee WK The phosphoinositol phosphatase activity of PTEN mediates a serum-sensitive G1 growth arrest in glioma cells.Cancer Res. 1998; 58: 5002-5008PubMed Google Scholar, 14Li DM Sun H PTEN/MMAC1/TEP1 suppresses the tumorigenicity and induces G1 cell cycle arrest in human glioblastoma cells.Proc Natl Acad Sci USA. 1998; 95: 15406-15411Crossref PubMed Scopus (442) Google Scholar, 15Weng LP Smith WM Dahia PL Ziebold U Gil E Lees JA Eng C PTEN suppresses breast cancer cell growth by phosphatase activity-dependent G1 arrest followed by cell death.Cancer Res. 1999; 59: 5808-5814PubMed Google Scholar PTEN therefore seems to play an important role in cell cycle growth, migration, and death. Germline PTEN mutations have been identified in the autosomal dominant hamartoma Cowden syndrome (CS) and Bannayan-Riley-Ruvalcaba syndrome.16Liaw 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 (1730) Google Scholar, 17Marsh DJ Dahia PL Zheng Z Liaw D Parsons R Gorlin RJ Eng C Germline mutations in PTEN are present in Bannayan-Zonana syndrome.Nat Genet. 1997; 16: 333-334Crossref PubMed Scopus (577) Google Scholar Benign thyroid disease is characteristic of both CS and Bannayan-Riley-Ruvalcaba syndrome.18Eng C Genetics of Cowden syndrome: through the looking glass of oncology.Int J Oncol. 1998; 12: 701-710PubMed Google Scholar The risk of nonmedullary thyroid carcinomas is increased in CS.19Wade TR Kopf AW Cowden's disease: a case report and review of the literature.J Dermatol Surg Oncol. 1978; 4: 459-464Crossref PubMed Scopus (28) Google Scholar, 20Thyresson HN Doyle JA Cowden's disease (multiple hamartoma syndrome).Mayo Clin Proc. 1981; 56: 179-184PubMed Google Scholar, 21Starink TM Cowden's disease: analysis of fourteen new cases.J Am Acad Dermatol. 1984; 11: 1127-1141Abstract Full Text PDF PubMed Scopus (128) Google Scholar In humans, these thyroid cancers are most often follicular and very rarely papillary thyroid carcinoma (PTC). In contrast, histopathological analyses of Pten ± chimeric and heterozygous mice showed lesions consistent with well-differentiated PTC22Di Cristofano A Pesce B Cordon-Cardo C Pandolfi PP Pten is essential for embryonic development and tumour suppression.Nat Genet. 1998; 19: 348-355Crossref PubMed Scopus (1321) Google Scholar as well as follicular or papillary thyroid neoplasia.23Podsypanina K Ellenson LH Nemes A Gu J Tamura M Yamada KM Cordon-Cardo C Catoretti G Fisher PE Parsons R Mutation of Pten/Mmac1 in mice causes neoplasia in multiple organ systems.Proc Natl Acad Sci U S A. 1999; 96: 1563-1568Crossref PubMed Scopus (844) Google Scholar Although LOH (loss of heterozygosity) on chromosome band 10q23 has been identified in ∼26. of follicular thyroid adenomas (FAs)24Marsh DJ Zheng Z Zedenius J Kremer H Padberg GW Larsson C Longy M Eng C Differential loss of heterozygosity in the region of the Cowden locus within 10q22–23 in follicular thyroid adenomas and carcinomas.Cancer Res. 1997; 57: 500-503PubMed Google Scholar, 25Dahia PL Marsh DJ Zheng Z Zedenius J Komminoth P Frisk T Wallin G Parsons R Longy M Larsson C Eng C Somatic deletions and mutations in the Cowden disease gene, PTEN, in sporadic thyroid tumors.Cancer Res. 1997; 57: 4710-4713PubMed Google Scholar, 26Yeh JJ Marsh DJ Zedenius J Dwight T Robinson BG Eng C Fine structure deletion analysis of 10q22–24 demonstrates regions of loss and suggests that sporadic follicular adenomas and follicular thyroid adenomas develop along distinct parallel neoplastic pathways.Genes Chromosom Cancer. 1999; 26: 322-328Crossref PubMed Scopus (41) Google Scholar and up to 27. of FTCs,27Halachmi N Halachmi S Evron E Cairns P Okami K Saji M Westra WH Zeiger MA Jen J Sidransky D Somatic mutations of the PTEN tumor suppressor gene in sporadic follicular thyroid tumors.Genes Chromosom Cancer. 1998; 23: 239-243Crossref PubMed Scopus (117) Google Scholar somatic intragenic mutations of PTEN are rare.25Dahia PL Marsh DJ Zheng Z Zedenius J Komminoth P Frisk T Wallin G Parsons R Longy M Larsson C Eng C Somatic deletions and mutations in the Cowden disease gene, PTEN, in sporadic thyroid tumors.Cancer Res. 1997; 57: 4710-4713PubMed Google Scholar, 27Halachmi N Halachmi S Evron E Cairns P Okami K Saji M Westra WH Zeiger MA Jen J Sidransky D Somatic mutations of the PTEN tumor suppressor gene in sporadic follicular thyroid tumors.Genes Chromosom Cancer. 1998; 23: 239-243Crossref PubMed Scopus (117) Google Scholar The human syndromic and murine data for PTEN involvement in thyroid neoplasia is strong. In accordance with the Knudson “two hit” hypothesis of carcinogenesis, if a somatic mutation is found on one allele of PTEN and LOH or a deletion is found on the opposite allele, then biallelic inactivation of PTEN at the structural level is said to occur. However, to date, all genetic studies of PTEN in human primary thyroid tumors have only demonstrated monoallelic structural mutation (either a heterozygous deletion or a single-hit somatic intragenic mutation). Whether PTEN inactivation at the protein level or via other mechanisms apart from structural alteration applies to thyroid tumorigenesis is unknown. Thus, we sought to determine whether functional biallelic inactivation of PTEN occurs in sporadic nonmedullary thyroid adenomas and carcinomas by examining them for PTEN expression using immunohistochemistry in conjunction with LOH analysis. Paraffin blocks from 139 unselected benign and malignant nonmedullary thyroid tumors were ascertained from Germany, Australia, and Switzerland. Histological classification of the thyroid tumors was in accordance with the World Health Organization.28Hedinger C Williams ED Sobin LH The WHO histological classification of thyroid tumors: a commentary on the ed 2.Cancer. 1989; 63: 908-911Crossref PubMed Scopus (521) Google Scholar Of note, five papillary tumors were classified as follicular type (Lindsay tumor), and 13 tumors (seven FAs, five FTCs, one PTC) had a prominent granular eosinophilic-appearing cytoplasm (also known as oxyphilic or Hürthle cell). The monoclonal antibody 6H2.1 raised against the last 100 C-terminal amino acids of PTEN29Perren 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 (339) Google Scholar was used in all immunohistochemical analyses. As biochemical proof of antibody specificity for PTEN, total protein lysates were obtained10Dahia PL Aguiar RC Alberta J Kum JB Caron S Sill H Marsh DJ Ritz J Freedman A Stiles C Eng C PTEN is inversely correlated with the cell survival factor Akt/PKB and is inactivated via multiple mechanisms in haematological malignancies.Hum Mol Genet. 1999; 8: 185-193Crossref PubMed Scopus (270) Google Scholar, 15Weng LP Smith WM Dahia PL Ziebold U Gil E Lees JA Eng C PTEN suppresses breast cancer cell growth by phosphatase activity-dependent G1 arrest followed by cell death.Cancer Res. 1999; 59: 5808-5814PubMed Google Scholar from a series of thyroid cell lines for which PTEN status is known: NPA-87, K-1, FTC-133, and WRO-82–1 (gifts from D. V. Canlapan and D. Wynford-Thomas). Further, as an additional positive control, the wild-type full-length human PTEN cDNA sequence was cloned into the expression vector pcDNA3 and transfected into the PTEN null line FTC-133. Western blot analysis was performed as previously described10Dahia PL Aguiar RC Alberta J Kum JB Caron S Sill H Marsh DJ Ritz J Freedman A Stiles C Eng C PTEN is inversely correlated with the cell survival factor Akt/PKB and is inactivated via multiple mechanisms in haematological malignancies.Hum Mol Genet. 1999; 8: 185-193Crossref PubMed Scopus (270) Google Scholar except that 6H2.1 was used at a 1:250 dilution. Thyroid lines with endogenously expressing or exogenously introduced PTEN all demonstrated a single band at 55 kd, the molecular weight predicted for PTEN, although the PTEN null lines did not cross-react with 6H2.1. No other nonspecific bands were noted, thus proving antibody-specificity (Figure 1). Control antibody against α-tubulin (Sigma, St. Louis, MO), used at 1:10,000 dilution, immunoreacted evenly across protein lysates from all cell lines (Figure 1). The specificity of the antibody 6H2.1 and its suitability for immunohistochemistry in paraffin-embedded tissue has been demonstrated previously.29Perren 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 (339) Google Scholar In brief, we used the antibody against embedded PTEN-transfected U2OS cells, BALBc/3T3, Nalm6, and DU145 as positive controls; MDA-MB-468 with hemizygous deletion of PTEN and a truncation of the remaining allele; A172 which has loss of one PTEN allele, and a truncating mutation in exon 2 of the remaining allele; and PC3, which is null for PTEN.29Perren 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 (339) Google Scholar Further, commercially available peptide corresponding to PTEN has been used to successfully compete away 6H2.1 immunostaining in paraffin-embedded tissue (GL M, unpublished data). Recently, it has been shown that a processed PTEN pseudogene (psiPTEN) can be transcribed in a number of cell lines and tissue types.30Fujii GH Morimoto AM Berson AE Bolen JB Transcriptional analysis of the PTEN/MMAC1 pseudogene, psiPTEN.Oncogene. 1999; 18: 1765-1769Crossref PubMed Scopus (79) Google Scholar For this reason, RNA in situ hybridization is not reliable. PsiPTEN, however, does not seem to be translated, at least in thyroid tumors, and therefore it would not be expected to complicate the analysis in this study. The tissue samples were fixed by immersion in 10% buffered formalin and subsequently embedded in paraffin according to standard protocols. Four-mm sections were cut, mounted on Superfrost plus slides, and baked for 2 hours at 60°C. Subsequently, the sections were deparaffinized and rehydrated by passing through xylene and a graded series of ethanol solutions. Antigen retrieval was performed by boiling at 98°C in 0.01 mol/L sodium citrate buffer, pH 6.4, in a microwave oven for 20 minutes. To block endogenous peroxidase activity, the sections were incubated with 0.3% hydrogen peroxide in methanol for 30 minutes after cooling to room temperature. After blocking for 30 minutes in 0.75% horse serum, the sections were incubated with a PTEN monoclonal antibody 6H2.1 (dilution 1:100) for 1 hour at room temperature. Primary antibody binding was localized by using an avidin-biotin-peroxidase kit (Vector Laboratories, Burlingame, CA. according to the manufacturer's instruction. The chromogenic reaction was carried out with 0.05% 3,3′diaminobenzidine (Sigma, St. Louis, MO. using nickel cobalt amplification which gives a black product.31Werner M Von Wasielewski R Komminoth P Antigen retrieval, signal amplification and intensification in immunohistochemistry.Histochem Cell Biol. 1996; 105: 253-260Crossref PubMed Scopus (145) Google Scholar After counterstaining with Nuclear Fast Red (Rowley Biochemical Institute, Danvers, MA) and mounting, the slides were independently evaluated under a light microscope by two investigators (OG and AP) and randomly spot evaluated by a third investigator (CE). Intensity of staining was classified separately for the nucleus/nuclear membrane and the cytoplasm and graded strong (+++), moderate (++), weak (+), or absent (−). These independent assessments did not differ by more than one grading level. In 95 samples, tumor tissue and blood or corresponding normal tissue (either normal thyroid tissue or adjacent muscle tissue) were available for extraction of paired somatic and germline DNA to study LOH. DNA extraction after microdissection was performed using standard protocols.32Eng C Thomas GA Neuberg DS Mulligan LM Healey CS Houghton C Frilling A Raue F Williams ED Ponder BA Mutation of the RET proto-oncogene is correlated with RET immunostaining in subpopulations of cells in sporadic medullary thyroid carcinoma.J Clin Endocrinol Metab. 1998; 83: 4310-4313PubMed Google Scholar All subsequent polymerase chain reactions were carried out using 0.6 μM each of forward and reverse primer in 1× polymerase chain reaction buffer (Qiagen, Valencia, CA), 4.5 mmol/L MgCl2 (Qiagen), 1× Q-buffer (Qiagen), 2.5 U HotStarTaq polymerase (Qiagen), and 200 μmol/L dNTP (Gibco, Gaithersburg, MD) in a final volume of 50 μl. Reactions were subjected to 35 cycles of 94°C for 1 minute, 55°C to 60°C for 1 minute, and 72°C for 1 minute followed by 10 minutes at 72°C. Potential hemizygosity at the PTEN locus was assessed by screening for a T/G polymorphism within PTEN intron 8 (IVS8 + 32G/T) detected by differential digestion with the restriction endonuclease HincII as previously described25Dahia PL Marsh DJ Zheng Z Zedenius J Komminoth P Frisk T Wallin G Parsons R Longy M Larsson C Eng C Somatic deletions and mutations in the Cowden disease gene, PTEN, in sporadic thyroid tumors.Cancer Res. 1997; 57: 4710-4713PubMed Google Scholar except for using the primers PTEN-E8-F (5′-GCGTGCAGATAATGACAAGG-3′) and PTEN-I8-R (5′-TGTCAAGCAAGTTCTTCATCG-3′). If the result of the digestion was not informative, LOH analysis was performed using markers flanking PTEN, D10S541 (telomeric) and D10S579 (centromeric)1Li 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): 277Crossref Scopus (4363) Google Scholar, 16Liaw 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 (1730) Google Scholar as well as the marker D10S2491 that lies within PTEN.33Cairns P Okami K Halachmi S Halachmi N Esteller M Herman JG Jen J Isaacs WB Bova GS Sidransky D Frequent inactivation of PTEN/MMAC1 in primary prostate cancer.Cancer Res. 1997; 57: 4997-5000PubMed Google Scholar All forward primers were 5′-labeled with either HEX or 6-FAM fluorescent dye (Research Genetics, Huntsville, AL). Polymerase chain reactions were carried out as described above and separated by electrophoresis through 6% denaturing polyacrylamide gels using an Applied Biosystems model 377 automated DNA sequencer (Applied Biosystems, Perkin-Elmer Corp., Norwalk, CT). The results were analyzed by automated fluorescence detection using the GeneScan collection and analysis software (GeneScan, Applied Biosystems). Scoring of LOH was performed by inspection of the GeneScan analysis output. A double peak, observed in the microsatellite marker that was amplified from DNA extracted from the germline sample, indicated heterozygosity. A single peak in DNA extracted and amplified from the corresponding tumor sample indicated a loss of one allele. If normal cells were admixed with tumor cells, a ratio of 1.5:1 or greater of germline DNA peak to tumor DNA peak was also considered LOH.24Marsh DJ Zheng Z Zedenius J Kremer H Padberg GW Larsson C Longy M Eng C Differential loss of heterozygosity in the region of the Cowden locus within 10q22–23 in follicular thyroid adenomas and carcinomas.Cancer Res. 1997; 57: 500-503PubMed Google Scholar To examine the correlative trend between PTEN staining intensity and LOH, we performed a Mantel-Haenszel test34Mantel N Haenszel W Statistical aspects for the analysis from retrospective studies of disease.J Natl Cancer Inst. 1959; 22: 719-748PubMed Google Scholar for trend in the association between the row and column variables. A P < 0.05 was considered statistically significant. Of the total 139 thyroid tumor samples examined for PTEN expression using the monoclonal antibody 6H2.1, 50 had accompanying normal thyroid tissue. Normal follicular thyroid cells showed a uniform strong (+++) to moderate (++) nuclear or nuclear membrane (hereafter referred to as nuclear) signal whereas the cytoplasmic staining was less strong, + to ++ (Figure 2A). Endothelial cells showed strong (+++) to moderate (++) PTEN expression with a nuclear predominance and were useful as internal positive controls (Figure 2, B and E). In contrast, nuclear and cytoplasmic staining intensity of fibrocytes was very heterogeneous and varied from weak (Figure 2A) to strong (Figure 2F). The quality and intensity of PTEN immunostaining in the nucleus and cytoplasm in 132 of 139 thyroid tumors was relatively uniform throughout each specimen. However, in seven carcinomas, PTEN expression differed significantly within different regions of each tumor (see below). Because PTEN expression in each of these seven tumors could not be classified into a single category, these different regions were classified separately as if they were two separate tumors, ie, PTEN expression was classified in 139 + 7 = 146 thyroid tumors. Hence, the intensity of PTEN staining in the nucleus and cytoplasm were assessed for 146 thyroid tumors (Figure 3) for purposes of this study. In FA, the neoplastic nuclei had less intense PTEN immunostaining (+ to ++) compared to normal follicular epithelium whereas the cytoplasmic PTEN staining intensity did not differ significantly from that observed in normal follicular cells (Figure 2, Figure 3). In thyroid carcinomas (FTC, PTC, and undifferentiated thyroid carcinoma [UTC]) as a group, nuclear PTEN immunostaining was mostly weak in comparison with normal thyroid follicular cells and FAs. Among the three classes of carcinomas, FTCs had the strongest immunostaining in both the nucleus and cytoplasm and UTCs the weakest (Figure 2, Figure 3). A few carcinomas, in particular UTCs, showed no PTEN staining in the nucleus (FTC, 3 of 28; PTC, 3 of 37; UTC, 8 of 26) or in the cytoplasm (one FTC, two PTCs, five UTCs; Figure 2E); eight carcinomas (one FTC, two PTCs, five UTCs) had no immunoreactivity in the nucleus or in the cytoplasm. In almost half of all thyroid carcinomas (FTC, 46%; PTC, 49%; UTC, 35%), the cytoplasmic staining was more intense than the nuclear staining (Figure 2, Figure 3). In contrast, the more intense cytoplasmic over nuclear staining was observed in only 7% of FAs. In other words, the stepwise decrease in PTEN immunoreactivity in the nucleus seemed to precede that in the cytoplasm from normal thyroid tissue to FA to carcinomas and finally to UTC (Figure 3). There was no obvious difference in PTEN staining pattern and intensity in Hürthle cell tumors compared to non-Hürthle cell tumors. Fibrocytes seemed to stain a little more intensely in tumor stroma (Figure 2F) compared to normal thyroid stroma (Figure 2A). Seven carcinomas (four UTCs, two PTCs, one FTC), but no adenomas, showed dichotomous regional PTEN staining within each sample. These were characterized either by islands of strongly immunopositive cells among sheets of cells staining more weakly (Figure 2, H and I) or by single cells staining weakly randomly distributed among cells staining strongly. In one UTC, the positive cells (graded +) were small and more regular whereas the PTEN-negative cells were larger, pleiomorphic, and had a more undifferentiated appearance. This correlation, however, was not seen in the other three UTCs. This pattern of immunostaining in the former UTC was replicated several times, thus indicating that this was not an artifact. In 95 tumors, normal and tumor tissue samples were available for LOH analysis. Similar to the immunohistochemical analysis, the four available carcinomas with dichotomous staining intensity within each sample, which had paired normal and tumor tissue, were considered as eight samples. Therefore, 99 total samples were considered. For purposes of this study, which was to compare the immunohistochemical data to the LOH data, one copy of PTEN was considered to be physically deleted only when one or more of the ma