Affiliations of authors: G. L. Mutter, J. T. Fitzgerald, Department of Pathology, Brigham and Women's Hospital, Boston, MA; M.-C. Lin, Department of Pathology, Brigham and Women's Hospital, and Department of Pathology, Taiwan National University Hospital, Taipei; J. B. Kum, Clinical Cancer Genetics and Human Cancer Genetics Programs, Ohio State University Comprehensive Cancer Center, Columbus, and Charles A. Dana Human Cancer Genetics Unit, Dana-Farber Cancer Institute, Boston; J. P. A. Baak, Department of Pathology, Medisch Centrum Alkmaar, and Amsterdam Free University Hospital, The Netherlands; J. A. Lees, Massachusetts Institute of Technology, Cambridge; L.-P. Weng, Clinical Cancer Genetics and Human Cancer Genetics Programs, Ohio State University Comprehensive Cancer Center; C. Eng, Clinical Cancer Genetics and Human Cancer Genetics Programs, Ohio State University Comprehensive Cancer Center, and Cancer Research Campaign Human Cancer Genetics Research Group, University of Cambridge, U.K.
Correspondence to: George L. Mutter, M.D., Department of Pathology, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115 (e-mail: gmutter{at}rics.bwh.harvard.edu).
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ABSTRACT |
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INTRODUCTION |
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Patients with endometrioid endometrial adenocarcinoma (1,2) account for 80% of endometrial cancer patients in the United States (9,10). Among all histologic subtypes of endometrial adenocarcinomas, the endometrioid subtype appears to have the highest rate of somatic PTEN mutations (1,2). Routine histopathology readily discriminates endometrioid endometrial adenocarcinomas from nonendometrioid tumors, such as the papillary serous and clear-cell adenocarcinomas that also occur in the endometrium. Risk for endometrioid endometrial adenocarcinomas is increased in patients with high estrogen levels that are unopposed by progestins (11) and in patients with a physically distinctive precancerous lesion (12). Interaction between genetic and hormonal events during the premalignant phases of endometrial tumorigenesis has been hypothesized, yet it has never been precisely elucidated.
The inaccessibility of premalignant tissues, the controversy concerning their interpretation, and the paucity of high-yield candidate genes are long-standingbut now fast-disappearingbarriers to productive exploration of the biology of endometrial precancers. Polymerase chain reaction (PCR)-based methods, including detailed mutational (13), clonal (14), and even lineage reconstruction (15) analyses, have improved the analytic repertoire suited to physically small precancers. Accurate diagnosis of the precancers themselves, typically termed "hyperplasias" in the widely used World Health Organization nomenclature (16), has been difficult to standardize (17). Even when criteria are agreed upon, reproducibility (18) is suboptimal. Previous reports of PTEN mutations in putative endometrial precancers (1921) have used subjective diagnostic criteria. Objective computerized morphometry (12,22,23), which uses image analysis algorithms that have excellent ability to predict concurrent (23) or future (12) carcinoma, has been shown to improve the reproducibility of histopathologic diagnoses. We have previously validated computerized morphometric analysis as an accurate means of precancer identification by showing that most of the lesions that it classifies as precancers are, in fact, monoclonal neoplasms (24), albeit benign ones prone to malignant transformation.
We have performed PTEN mutation analysis and protein expression studies in a spectrum of precisely classified endometrial tissues to test our hypothesis that changes in PTEN structure and function are among the earliest events in the pathway to endometrioid endometrial cancer. A series of unopposed estrogen-exposed endometria was included to determine if altered PTEN function might precede the appearance of endometrial intraepithelial neoplasia (EIN), which we define as a precancer diagnosed by morphometry.
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MATERIALS AND METHODS |
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Histologic Classification by Use of Computerized Morphometric Analysis
Diagnostic classification was accomplished by a combination of review by a pathologist (G. L. Mutter) and computerized morphometry. First, carcinomas were distinguished from premalignant lesions by the presence of at least one of three diagnostic features: 1) myometrial invasion, 2) solid areas of neoplastic epithelium, or 3) extensively meandering, interconnected glandular structures. Endometrial tissues that were judged not to constitute carcinomas were circumscribed with ink on the glass slide. Computerized morphometric analysis of corresponding delineated regions on hematoxylineosin-stained sections was performed (by J. P. A. Baak) by use of the QProdit 6.1 system (Leica, Cambridge, U.K.) as described previously (12,23,25). For each lesion, the D score was calculated from the volume percentage stroma (VPS), standard deviation of shortest nuclear axis (SDSNA), and gland outer surface density (OUTSD) [D = 0.6229 + (0.0439 x VPS) - (3.9934 x ln [SDSNA]) - (0.1592 x OUTSD) (12,23)] and was then classified as precancerous (EIN) (D<0), indeterminate (0D
1), or benign (D>1) based on the previously developed (12,23) outcome-predictive formula. Endometrial areas scored as benign were subclassified by pathologist (G. L. Mutter) review. Atrophic, cycling, or reactive endometrium was identified and grouped as "normal." Unopposed estrogen-exposed endometria were diagnosed by the appearance of occasional glandular cysts in a disordered proliferative field without sufficient glandular crowding or atypia to qualify as a precancer. The source of unopposed estrogen was either endogenous (anovulatory cycles) or exogenous (pharmacologic estrogens).
DNA Isolation and Amplification Genomic DNA from endometrial tissues (normal, precancer, or cancer) obtained at hysterectomy was isolated by selective UV irradiation (14) of areas of the paraffin sections that were typically 3 mm in diameter and contained dozens of individual glands. PTEN-coding sequences were amplified by PCR by use of target-specific oligodeoxynucleotide primers. Intron-based PCR primers were used to minimize coamplification of the processed (intronless) PTEN pseudogene on chromosome 9 (26). In the following list of the primers that we used, each like-numbered pair comprises the forward ("FGC") and reverse ("RGC") primers for the correspondingly numbered PTEN exon: 1FGC (5'-CGT CTG CCA TCT CTC TCC TCC T-3'), 1RGC (5'-CGC CCG CCG CGC CCC GCG CCC GGC CCG CCG CCC CCG CCC GAA ATA ATA AAT CCG TCT ACT CCC ACG TTC T-3'), 2FGC (5'-CGT CCC GCG TTT GAT TGC TGC ATA TTT CAG-3'), 2RGC (5'-CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCC GTC TAA ATG AAA ACA CAA CAT G-3'), 3FGC (5'-CGC CCG CCG CGC CCC GCG CCC GGC CCG CCG CCC CCG CCC GTA AAT GGT ATT TGA GAT TAG-3'), 3RGC (5'-GCG CGA AGA TAT TTG CAA GCA TAC A-3'), 4FGC (5'-CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCC GAA ATA ATA AAC ATT ATA AAG ATT CAG GCA ATG-3'), 4RGC (5'-GAC AGT AAG ATA CAG TCT ATC-3'), 5.1FGC (5'-CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCC GTT TTT TCT TAT TCT GAG GTT ATC-3'), 5.1RGC (5'-TCA TTA CAC CAG TTC GTC C-3'), 5.2FGC (5'-TCA TGT TGC AGC AAT TCA C-3'), 5.2RGC (5'-CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCC GGAA GAG GAA AGG AAA AAC ATC-3'), 6FGC (5'-GCG CGT TTC AAT TTG GCT TCT CTT T-3'), 6RGC (5'-CGC CCG CCG CGC CCC GCG CCC GGC CCG CCG CCC CCG CCC GAA ATA ATA AAT AAG AAA ACT GTT CCA ATA C-3'), 7FGC (5'-CGT CCC GCA ATA CTG GTA TGT ATT TAA C-3'), 7RGC (5'-CGC CCG CCG CGC CCC GCG CCC GGC CCG CCG CCC CCG CCC GGA TAT TTC TCC CAA TGA AAG-3'), 8FGC (5'-CGG TTT CAC TTT TGG GTA AAT A-3'), 8RGC (5'-CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCC GAC CCC CAC AAA ATG TTT AAT-3'), 9FGC (5'-CGC CCG CCG CGC CCC GCG CCC GGC CCG CCG CCC CCG CCC GTC ACT AAA TAG TTT AAG ATG-3'), and 9RGC (5'-TTC ATT CTC TGG ATC AGA GT-3'). Since each sense primer had a 30- to 45-base-pair GC clamp at its 5' end (e.g., primer 1RGC), each amplicon (PCR product) included a domain with a lower melting temperature (the sequence to be analyzed for mutations) and a domain with a higher melting temperature (the GC clamp).
Detection and Sequence Analysis of PTEN Mutations Denaturing gradient gel electrophoresis (DGGE) separates amplicons on the basis of melting temperature, which varies with nucleotide composition. We used a 10% polyacrylamide gel containing 5% glycerol and a linear 15%50% ureaformamide gradient, which simulates a temperature gradient, and subjected our samples to electrophoresis at 100 V for 16 hours at 60°C (27). Under these conditions, a double-stranded PCR product moves through the gel until it reaches the level corresponding to the melting temperature of its lower melting domain, whereupon that domain melts instantly. Since the high-melting GC clamp holds the amplicon together, migration ceases. PCR products of mutant DNAs exhibit altered migration during DGGE and usually appear as doublets of mutant PTEN products admixed with wild-type DNA that was contributed by contaminating normal tissues or the companion allele.
PCR products were visualized by UV transillumination of ethidium bromide-stained gels. DNA was isolated from bands identified as aberrant and was further amplified, and a nested sequencing primer was used to generate fluorescence-labeled sequencing products that were analyzed on a semiautomated DNA sequencer (ABI377; Perkin-Elmer Corp., Norwalk, CT) as described previously (6). DGGE gels and sequencing chromatograms were independently read by J. B. Kum, C. Eng, and J. T. Fitzgerald or G. L. Mutter.
Analysis of Loss of Heterozygosity
DNA from carcinoma and adjacent normal myometrium was amplified in the presence of [-32P]thymidine 5'-triphosphate with primers that define D10S541 and D10S215 (MapPairs; Research Genetics, Huntsville, AL), which are polymorphic microsatellite loci at the 3' and 5' ends, respectively, of PTEN. PCR products of these polymorphic microsatellites were separated on nondenaturing polyacrylamide gels (28,29). The intensities of bands representing PCR products of tumor alleles were visually compared with those on a reference set of calibrated autoradiographs (30) of normal myometrium and scored as positive for loss of heterozygosity (LOH) when there was at least a 50% reduction in the intensity of the band corresponding to one allele.
Immunohistochemistry Monoclonal antibody 6H2.1, raised against a 100-amino acid oligopeptide identical to the C-terminal end of human PTEN protein (31), was used in all of the immunocytochemical analyses. Specificity has been demonstrated previously by western blot analysis of wild-type and PTEN-null cell lines (31). Furthermore, when the PTEN-specific antibody was incubated with competing synthetic PTEN peptide (the native antigen) and used to immunostain paraffin-embedded sections of known PTEN-expressing tissues, no immunostaining was observed (31).
Since PTEN immunohistochemistry by use of the 6H2.1 antibody requires freshly cut paraffin sections from recently embedded (within 612 months) tissues to maximize the signal, we used our second series of endometrial tissue samples, which met this requirement, for immunohistochemistry. Formalin-fixed tissue samples were embedded in paraffin by standard histologic procedures. Immunostaining was performed by use of a microwave antigen-retrieval protocol as described previously (31). Sections were incubated with monoclonal antibody 6H2.1 (dilution 1:100 in phosphate buffer) for 1 hour at room temperature, washed, and incubated with a secondary biotinylated horse anti-mouse immunoglobulin G (Vecstatin ABC kit; Vector Laboratories, Inc., Burlingame, CA). PTEN expression, as reflected by immunostaining, was detected by sequential addition of avidin peroxidase (Vector Laboratories, Inc.) and 3,3'-diaminobenzidine (Sigma Chemical Co., St. Louis, MO), which gives a brown reaction product. The intensity of the epithelial staining was scored (by G. L. Mutter and J. T. Fitzgerald) in methyl green-counterstained slides from 0 (absent) to 3 (intense). Endometrial stroma and/or normal endometrial epithelium provided an internal positive control, and negative controls without addition of primary antibody showed low background staining in all cases.
Statistical Analysis Fisher's exact tests were performed by use of SYSTAT v. 9.0 (Statistical Package for Social Sciences, Chicago, IL). All P values are two-sided.
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RESULTS |
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PCR-based analysis to determine LOH of markers within or flanking PTEN was performed on the series of 30 endometrial carcinoma samples shown in Table 1. Overall, the LOH frequency was 23% (seven of 30) (data not shown), and all samples with LOH had PTEN mutations in the remaining allele, indicating inactivation of both PTEN alleles. Attempts to perform LOH analysis on precancers were confounded by the presence of contaminating normal stromal tissue.
The number of genetically altered PTEN alleles within individual endometrial adenocarcinomas can be estimated by combining deletion (LOH) and mutation (DGGE, Table 1) data. Ten (33%) of 30 endometrioid carcinomas had homozygous PTEN inactivation (seven with LOH of one allele and mutation of the second allele and three with mutations in two or more PTEN exons), and another 50% (15 of 30) had hemizygous PTEN genomic lesions (DGGE-detected mutation in one allele only, without LOH of second allele).
Probable biallelic inactivation of PTEN is reflected in lack of PTEN protein expression, which can be assessed by immunohistochemistry. Fig. 2 shows immunohistochemical detection of PTEN protein (brown precipitate) by antibody 6H2.1 in areas of endometrial adenocarcinoma, endometrial precancer, and benign endometrium. Although all of the tissue samples shown in Fig. 2
are from one patient, it illustrates the salient PTEN immunohistochemical findings that are typical of malignant, premalignant, and estrogen-driven endometria from the 81 (excluding nonendometrioid cancers) endometrial tissue samples that we have examined. The carcinoma in Fig. 2, A
, is devoid of PTEN staining, but adjacent endometrial stromal cells and vascular endothelium contain cytoplasmic and nuclear PTEN protein. A zone of precancerous glands devoid of PTEN protein (Fig. 2, B
; upper left) contrasts with abundant stromal staining and an adjacent region of normal endometrial glands (Fig. 2, B
; lower right) that show both nuclear and cytoplasmic PTEN staining. The high-magnification views (Fig. 2, C and E
) of the upper-right corner of Fig. 2, B
, show the interface between PTEN-negative (precancerous) and PTEN-positive (benign) glands, including one transition within an individual gland (Fig. 2, E
). Companion hematoxylineosin-stained sections (Fig. 2, D and F
) are the equivalent of those immunostained with anti-PTEN antibody and show the histologic structure of the tissues.
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Of nine computerized morphometrydefined indeterminate endometrial samples, five (56%) showed no PTEN protein expression. Of 20 morphometrically diagnosed normal tissues, only one had no PTEN expression. This was an atrophic endometrium.
Both adjacent endometrial stroma and endothelial cells of blood vessels in immediate proximity to the tumor were moderately PTEN protein positive. (Fig. 2, A, is a typical example.) Precancerous (EIN) lesions had no discernible PTEN protein expression in 75% (nine of 12) of the examples, most commonly in closely packed expanses of PTEN-negative glands offset by dispersed benign glands having a different cytology. (Panels B and C in Fig. 2
are examples.) A less frequent pattern of heterogeneous PTEN staining was seen in some "transitional" benignprecancer examples without cytologic changes (Fig. 3, AC
). No statistically significant difference in PTEN protein expression (absent versus present at any intensity) as a function of diagnosis (cancer versus EIN) was observed (P = .491), although the small sample size (33 cancers and 12 EIN lesions) limits the power of this comparison.
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DISCUSSION |
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Loss of PTEN protein in nests of crowded endometrial glands follows the predictions of monoclonal growth (14,34), namely, that all cells in the lesion share the same PTEN status. Precancer diagnosis by use of computerized morphometric analysis requires histologic sections with crowded groups of endometrial glands over a field of several square millimeters. It is these clusters of crowded glands that correspond to premalignant lesions usually diagnosed by pathologists as atypical endometrial hyperplasias and which we have designated as EIN by use of morphometry. In practice, diagnosis of endometrial precancers by use of hyperplasia (16) terminology is only modestly reproducible (17,18,35), especially for nonatypical hyperplasias, a category containing monoclonal putative precancers and polyclonal benign tissues. Computerized morphometry, however, appropriately classifies "nonatypical" hyperplasias into high- and low-risk subgroups (24). Fig. 1 shows that mutations in the computerized morphometry-defined high-risk group (D<0, precancerous) are evenly distributed across a rather broad range of D scores and are not clustered at some extreme negative D score distant from the threshold of 0. This validates the idea that all endometrial specimens with a D score below 0 have a high likelihood of having a PTEN mutation.
More troublesome to diagnose are lesions with histologic features intermediate between benign and precancerous, as represented by the "indeterminate" category in Table 1. It is this group of patients in whom PTEN immunohistochemistry may elucidate clonal loss of PTEN expression among a strongly PTEN-positive background of convoluted and "busy" glands. Immunohistochemical identification of individual isolated PTEN-negative endometrial glands in endometria with unopposed estrogen exposure pushes the limits of detection of precancers to an earlier stage of development than was previously possible. The natural history of individual PTEN-negative glands has not yet been determined experimentally, but a rich epidemiologic literature (32,33) showing a 3.1- to 7.3-fold increased risk of endometrial adenocarcinoma in women exposed to unopposed estrogens is consistent with the notion that, in some women, these single glands may progressively expand into histologically recognizable premalignant, and ultimately malignant, processes.
Suppression of PTEN expression in a mitotically active estrogenic environment (unopposed by progestins) may compromise growth control more than loss of PTEN protein in mitotically quiescent cells. Highly mitotic cells, such as normal estrogen-stimulated proliferative endometrial glands, contain abundant PTEN protein. Progesterone, which is known to prevent many of the tumorigenic effects of estrogens, diminishes in vivo endometrial epithelial PTEN protein expression over a period of 45 days, to a point where it is no longer detected in the glands of midsecretory endometrium (data not shown). If these fluctuations in PTEN protein are indeed driven by changing physiologic requirements for the protein, then it is reasonable to predict that the reduced ability to make PTEN protein has a greater effect under estrogenic than under progestenic conditions.
PTEN inactivation (loss of protein) in endometrioid adenocarcinomas and in several other tumor types cannot be explained solely on the basis of observed mutations. This observation suggests that expression of PTEN is repressed at the transcriptional and translational levels by other mechanisms. Fewer than 30% of hematologic malignancies have a structural PTEN alteration, but 70% are PTEN negative as judged by western blot analysis (36). Forty percent of breast cancers are PTEN genetic hemizygotes, and fewer than 5% of cases have biallelic PTEN genomic lesions, yet 15% are devoid of protein that is detectable by immunohistochemistry (31). In this study, inactivation of both PTEN alleles, as a result of either a mutation or a deletion (LOH), was observed in only 33% of endometrial cancers, but 61% of those cancers did not express PTEN protein. More frequent is a hemizygous PTEN genotype in 50% of endometrial cancer cases. Candidate mechanisms for inactivation of the second allele include (undetected) mutation in regulatory regions, epigenetic modification of flanking DNA sequences (e.g., by methylation), or decreased translation. Increased protein degradation in a hemizygous state could also give negative PTEN immunohistochemistry results that would be indistinguishable from biallelic inactivation.
The PTEN mutation rate of 83% that we observed in our series of endometrial adenocarcinomas is about double that of most previous reports (1,2,19,20), probably because of the combined effects of our mutation-detection and sample-selection methods. The DGGE-screening method is very sensitive in PTEN mutation detection compared with the single-strand conformational polymorphism analysis, and its specificity has been confirmed by direct sequencing of DGGE-identified mutations (27). DGGE can detect variants, even at mutant-to-normal allele ratios of 1:100, while sequencing requires this ratio to be at least 35:100 [reviewed in (37)].
The tissue samples that we used for mutational analysis were selected for the presence of both malignant and premalignant endometrial tissues at the time of hysterectomy, thereby enriching the study population for those tumors that develop through a hierarchy of progressive events. Endometrioid adenocarcinoma and its precursors have severalfold higher PTEN mutation rates than those malignant tumors (nonendometrioid, including papillary serous type) that arise abruptly without displaying an intermediate premalignant phase. It is unlikely that the balance of microsatellite-stable and microsatellite-unstable tumors can explain the high PTEN mutation rate that we observed, since both had similar PTEN mutation rates in our series.
The mechanism of diminished PTEN protein expression was indirectly addressed in our study, in which, for technical reasons, genomic and protein expression PTEN analyses were carried out on independent tissue series. Our ability to relate changes in PTEN expression to causal genomic events is thus inferential and limited by our sample size. Simultaneous scoring of PTEN mutation and deletion against expression in individual tissues would determine whether these mechanisms alone can explain the majority of lost PTEN expression. One advantage of using this particular series of tissue samples for PTEN mutational analysis is that it was subjected previously to a number of specialized analyses. Of the 30 cancers analyzed, 10 were microsatellite unstable and 20 were microsatellite stable (14,15,24), with PTEN mutation rates of 90% (nine of 10) and 80% (16 of 20), respectively. In 29 precancers, 57% (12 of 21) of microsatellite-stable and 50% (four of eight) of microsatellite-unstable (14,15,24) lesions had at least one PTEN mutation. KRAS mutations (13) were observed in 21% (six of 29) of the cancers, and all (six of six) of these also had PTEN mutations in at least one exon.
Our observations have demonstrated that complete inactivation of PTEN occurs in the great majority of endometrial carcinomas, especially those of the endometrioid subtype, and even in half of all precancers (EIN). Nearly all (97%) of the endometrial cancer tissue samples that we tested had either complete absence of PTEN protein expression or reduced expression of PTEN protein (Table 1). Inactivation could be a result of structural changes (mutation or LOH) or epigenetic modification of the PTEN gene itself or its regulatory elements. Although relatively few endometrial carcinomas had biallelic structural alterations (either two or more PTEN mutations affecting both alleles or PTEN mutation in one allele and LOH of the other), we found complete loss of PTEN protein expression in 61% (20 of 33) (Table 1
). Although the distribution of multiple exonic hits between one or two alleles is unknown, the number of PTEN exons affected by mutation provides some indication of that fraction of cases that are candidates for biallelic mutational inactivation.
Morphometrically defined precancers are usually diagnosed as atypical endometrial hyperplasias. It is, therefore, of interest to note that, among computerized morphometrically diagnosed precancers in which approximately half had PTEN mutations, three quarters displayed complete absence of PTEN protein (Table 1). Although only nine computerized morphometry-defined indeterminate endometrial samples were available for analysis, more than half (56%) showed no PTEN protein expression. In contrast, only one of 20 morphometrically diagnosed normal tissues did not express PTEN protein.
PTEN is a major gene involved in the pathogenesis of endometrioid endometrial adenocarcinoma. Our data suggest that altered PTEN function is partly responsible for the etiology of the majority of endometrial cancers with a premalignant phase and participates in their progression to carcinoma. Thus, decreased PTEN expression or function is a marker of the earliest endometrial precancers, and we propose that use of PTEN immunostaining in a clinical setting may be informative in identifying premalignant lesions that are likely to progress to carcinoma.
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Manuscript received October 21, 1999; revised March 16, 2000; accepted March 29, 2000.
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