Is endometrial carcinoma in situ a precursor lesion of endometrial carcinoma?

Original Contribution Kitasato Med J 2018; 48: 1-8 Is endometrial carcinoma in situ a precursor lesion of endometrial carcinoma? Hisako Inoue, Makoto Saegusa Department of Pathology, Kitasato University School of Medicine Objective: To clarify the significance of the endometrial intraepithelial carcinoma in situ(ecis) to neoplasia in the uterus. Method: A total of 130 endometrial carcinomas including 121endometrioid and 9 serous lesions were immunohistochemically investigated, using antibodies for bcl-2, p53 and estrogen and progesterone receptors. A polymerase chain reaction (PCR)/loss of heterozygosity (LOH) analysis for p53 and deleted in colorectal cancer (DCC) genes was also performed. Results: An endometrial carcinoma in situ (ECIS) was found in 15/130 (11.5%) cases. Comparative immunohistochemical assessment of the primary tumor and the ECIS in each case revealed 4 (25%) of 16 combinations (1 had 2 ECIS lesions) that had identical immunophenotypes. PCR-LOH analysis also demonstrated identical allelotypes for the p53 and DCC genes in all informative cases. These findings indicate that while a proportion of ECIS lesions are directly associated with tumors, suggestive of secondary growth they may also be precursors because some cases showed different immunophenotypes, and one case had multiple ECISs. Conclusion: The present study demonstrated that ECIS may be either due to intraepithelial spread from primary tumors or multicentric development of endometrial carcinomas. Key words: endometrial intraepithelial carcinoma, ECIS, p53, DCC, bcl-2 Introduction wo different pathways, one estrogen-dependent and T the other estrogen-independent, leading respectively to type I and type II tumors, are generally considered to be involved in endometrial carcinogenesis. 1,2 It has been demonstrated that atypical endometrial hyperplasia closely linked with hyperestrogenism is a precursor for type I endometrial carcinomas because a long-term follow-up study revealed 25% of untreated atypical hyperplasias progress to endometrioid carcinomas, although the majority of the tumor appears to develop from non-hyperplastic endometrium. 3 Carcinomas in the type II category show a higher histological grade, more aggressive growth and a less favourable prognosis; and while they frequently develop from atrophic endometrium in older women, there is no known precursor lesion. Recently, the concept of endometrial carcinoma in situ (ECIS) 4 or endometrial intraepithelial carcinoma (EIC) 5 characterized by spreading or replacement of endometrial surface epithelium and glands by cytologically malignant cells, has been proposed. Ambros et al. 5 demonstrated that a frequent and specific association of ECIS with uterine tumors displayed serous differentiation. Spiegel 4 also stressed that in many postmenopausal women, either surface epithelium or individual glands can undergo malignant transformation and progress to an invasive state without any preliminary atypical hyperplasia phase. However, considering the finding that ECIS is occasionally in direct histological continuity with primary invasive carcinomas, 6 the possibility it may be due to intraepithelial spread from primary tumors cannot be precluded. In the present study, to clarify the significance of ECIS to the development or progression of endometrial carcinomas, we immunohistochemically investigated the endometrium in patients who underwent a hysterectomy for malignant tumors, using bcl-2, p53, estrogen receptor, and progesterone receptor antibodies. A polymerase chain reaction (PCR)/-loss of heterozygosity (LOH) analysis Received 31 October 2017, accepted 13 November 2017 Correspondence to: Hisako Inoue, Department of Pathology, Kitasato University School of Medicine 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0374, Japan E-mail: dm13008y@st.kitasato-u.ac.jp 1

Inoue H. et al. for p53 and deleted in colorectal cancer (DCC) genes was also performed to determine the degree of identity between pairs of lesions. atrophic or weakly proliferative, and in 21 it was hyperplastic. In contrast all serous carcinoma cases had a background of atrophic endometrium. Materials and Methods Cases A total of 130 endometrial carcinoma cases surgically resected at the Kitasato University Hospital from 1990 to 1995 were investigated. Cases in which the endometrium was completely replaced by invasive carcinomas were excluded. To obtain samples from the entire endometrium, at least 40 sections for each case were systematically prepared by making slides through the uterus at 5-mm intervals, which were conventionally 10% formalin fixed and paraffin embedded. Histopathologic diagnoses were made according to the criteria of the International Federation of Gynecology and Obstetrics. 7 The cases investigated comprised 121 endometrioid carcinomas [69 grade (G) 1, 31 G2, and 21 G3] and 9 serous carcinomas. Histopathological examination Classification of ECIS was performed using the criteria described by Spiegel, 4 with a minor modification. Briefly, surface carcinoma in situ (SCIS) was defined as an area of replacement of the surface epithelium with cells showing feature of malignancy without papillary formation, and glandular carcinoma in situ (GCIS) was defined as the presence of one or two glands lined with such cells. A group of several altered glands was designated as glandular thin carcinoma (CA), and SCIS with papillary growth was defined as papillary thin CA. Thin CA with slight disruption of the basement membrane and astromal reaction including fibrotic change and inflammatory cell infiltration was definded as microinvasive. The majority of findings for ECIS were obtained using sections from areas free of primary tumors. When both ECIS and primary tumor lesions were present in the same slide, assessment was only performed when they were separated by at least 5 mm of non-cancerous endometrium. ECIS lesions adjacent to invasive carcinomas were excluded since they frequently demonstrated inter connections. Non-cancerous endometrium was histologically divided into three categories: normal tissue including the secretory phase; atrophic or proliferative endometrium; and hyperplasia including simple, complex, and atypical types. In the endometrioid carcinoma cases, 23 demonstrated normal background tissue, in 77 it was 2 Immunohistochemistry In 94 endometrioid carcinomas (53 G1, 26 G2 and 15 G3) randomly selected from 121 cases, and 9 serous carcinomas, immunohistochemistry was performed using a combination of the microwave-oven heating and the standard streptavidin-biotin-peroxidase complex (Histofine SAB-PO [M] kit, Nichirei Co., Tokyo) methods. Briefly, the slides were heated in 10-mM citrate buffer (ph 6.0) for three 5-minute cycles using a microwave oven and were then incubated overnight at 4 with optimum dilutions of primary antibodies. The antibodies used were anti-human bcl-2 mouse monoclonal antibody ( 100 dilution, Dako, Copenhagen, Denmark), CM 1 ( 1,000 dilution, Novocastra Lab, Ltd., Newcastle, UK) for p53 protein, anti-estrogen receptor mouse monoclonal antibody ( 80 dilution, Novocastra Lab) and anti-progesterone receptor mouse monoclonal antibody ( 80 dilution, Novocastra Lab). As positive internal controls, infiltrating lymphocytes for bcl-2, and stromal and myometrial cells for estrogen and progesterone receptor (ER and PR) were present in each slide. As a p53-positive control a gastric carcinoma was applied. For negative controls, to confirm the immunospecificity of each antibody, 0.01 M phosphatebuffered saline or either normal mouse or rabbit serum ( 500 dilution) were supplied instead of primary antibodies. The positivity for bcl-2, p53, ER, and PR was classified into four levels: negative, 5% positive cells; positive, 1+, 5% but 30% positive cells; 2+, 30% but 50% positive cells; 3+, 50% positive cells. DNA extraction and PCR-LOH assay DNA samples were obtained by scraping off tumor cells under a dissecting microscope, carefully avoiding contamination with areas of more than 30% normal cells, using serial 10μm-thick paraffin sections stained with 1% methyl green solution. DNA extraction was performed with a conventional phenol/chloroform treatment method. DNA samples from non-tumor areas were also extracted in the same manner. For detection of LOH at the p53 locus, two oligonucleotide primer sets were used: TP53 8 (sense: 5'-AGGGATACTATTCAGCCCGAGGTG-'3 and antisense: 5'-ACTGCCACTCCTTGCCCCATTC-3') and a variable number of tandem repeat (VNTR) locus in p53 intron 1 9 (sense: 5'-ACTCCAGCCTGGGCAATAAGAGCT-3' and

ECIS a precursor of endometrial carcinoma antisense: 5'-ACAAAACATCCCCTACCAAACAGC-3'). The total reaction volume of 10μl contained 50 ng DNA, 0.1μM of each primer and 0.5 U Taq DNA polymerase (Takara, Shiga). The PCR procedure featured 45 cycles of 0.5 minutes at 94, 1 minute at 60 and 1 minute at 72. For detection of LOH at the DCC locus, a primer set recognizing a VNTR within the DCC gene 10 (sense: 5'-GATGACATTTTCCCTCTAG-3' and antisense: 5'-GTGGTTATTGCCTTGAAAAG-3') was used and the PCR was performed with 45 cycles of 0.5 minutes at 94, 2 minutes at 58, and 2 minutes at 72. The PCR products were electrophoresed on 8% polyacrylamide or 3% agarose gels and detected by ethidium bromide staining. LOH was defined as a more than 50% decrease in the relative intensity of alleles between normal and tumor DNA, as described previously. 11 As a negative control, water was applied instead of DNA. 0.0001, P = 0.0077, respectively). Eight of nine serous carcinomas, demonstrated p53 immunoreactivity (88.9%) while only three each were immunopositive for bcl-2, ER, and PR (33.3%) (Table 1). Microscopical findings for ECIS SCIS lesions demonstrated a single layer or stratified cell layers of columnar or polygonal shaped cells with large nuclei and distinct nucleoli, occasionally replacing Statistics Statistical analysis of the relations among antibody immunopositivity, ECIS foci and histological grading of endometrioid carcinomas was performed using the χ 2 test. Results Bcl-2, p53, ER, and PR immunoreactivity in primary tumors Bcl-2 immunoreactivity was found in cytoplasmic and perinuclear sites, with a heterogenous distribution in positive tumor cells, while immunoreactivity for p53, ER, and PR was revealed as distinct dense nuclear staining. Marked heterogeneity of ER and/or PR immunopositive cells was observed in invasive carcinomas. A variety of distributions of p53 positive cells was found in endometrioid carcinomas, while serous carcinomas occasionally showed a diffuse immunostaining pattern. Of 94 endometrioid carcinoma cases examined, 58 (61.7%) were positive for bcl-2, 38 (40.4%) for ER, 74 (78.8%) for PR, and 15 (16.0%) for p53 (Table 1). The rates for bcl-2, ER, PR, and p53 immunopositivity were closely correlated with the histological grade in endometrioid carcinomas (P = 0.0232, P = 0.0002, P = Table 1. Expression of bcl-2, ER, PR, and p53 in endometrial carcinomas Tumor type bcl-2 ER PR p53 Endometrioid (n = 94) 58 (61.7) 38 (40.4) 74 (78.7) 15 (16.0) Serous (n = 9) 3 (33.3) 3 (33.3) 3 (33.3) 8 (88.9) 3 Figure 1. A. SCIS. Note replacement of the surface epithelium by malignant cells without papillary formation (case 84, E-26). H&E staining, 400. B. Glandular thin carcinoma. A group of several glands composed of cells with malignant features is present in the atrophic endometrium (case 89, E-10). An atrophic endometrial gland is indicated by the arrow. H&E staining, 200. C. Papillary thin carcinoma. An SCIS demonstrating papillary growth is present on the surface of an atrophic endometrium (cases 41, E-62). H&E staining, 100.

Inoue H. et al. Table 2. Summary of endometrial carcinoma cases with ECIS lesions Case Age Non-cancer Section No. (year) lesion* No.** Histology*** Immunohistochemistry PCR-LOH assay bcl-2 ER PR p53 TP53 p53 VNTR DCC VNTR 2 46 W. Prolif. 7 45 Prolif. 18 44 Com. Hyper. 23 60 Atrophy 28 48 W. Prolif. 40 42 Sim. Hyper. 41 55 Atrophy 42 55 Atrophy 79 51 Atrophy 80 77 Atrophy P-6 Em Ca-G3 - - 1+ - N N P E-9 GCIS W+ + + - N NA P P-8 Em Ca-G1 3+ - 3+ - N N H E-10 GCIS W+ F+ F+ - N NA H P-33 Em Ca-G1 - - 2+ - NA H H E-18 GCIS - W+ F+ - NE NE H P-7 Em Ca-G2 - - 2+ 2+ P H NE E-28 GCIS - W+ F+ + P H NE P-31 Em Ca-G2 3+ 1+ 3+ 2+ N N NE E-10 Microinvasivethin CA + + + - N N NE P-21 Em Ca-G2 2+ - - - N NA H E-7 Papillary thin CA W+ + - - NE NE H P-22 Em Ca-G3 1+ - - - N H H E-62 Papillary thin CA - - - + NE NE H P-5 Em Ca-G2 3+ - - - N N H E-12 GCIS W+ F+ + - N NA H P-10 Em-Ca-G2 - - - - NA NA H E-4 SCIS - - - - NE NE H P-19 Serous - 1+ - 3+ N H H E-9 Papillary thin CA - - - + N NE H P-8 Serous 3+ - - - N N H 84 70 Atrophy E-26 SCIS - - - - N N H E-28 Microinvasivethin CA - + + - N N H 87 51 A. Hyper. 89 72 Atrophy 104 52 W. Prolif. 108 49 Secretory P-14 Em Ca-G1 3+ - 3+ - P N H E-22 GCIS - + + - NE NE H P-9 Em Ca-G1 3+ 2+ 3+ - NE NE H E-10 Gland thin CA W+ + + - NE NE H P-13 Em Ca-G3 2+ - - - N N H E-14 Microinvasivethin CA F+ - - - N NA H P-10 Em Ca-G1 3+ 2+ 3+ - N N N E-9 GCIS + + + - N N N *W. Prolif., weakly proliferativeendometrium; **P, section number including primary lesion; ***Em Ca, endometrioidcarcinoma Com. Hyper., complex hyperplasia; Sim. Hyper., simple hyperplasia; A. Hyper., atypical hyperplasia; E, section number involving ECIS foci; G1, grade 1; G2, grade 2; G3, grade 3; Gland thin CA, glandular thincarcinoma; GCIS, glandular carcinoma in situ; Microinvasive Thin CA, microinvasive thin carcinoma; Papillary thin CA, papillary thin carcinoma, SCIS; surface carcinoma in situ. Immunopositivity of ECIS: W, weakly; F, focal. PCR-LOH assay results: N, negative, P; positive, NE; not examined NA; not amplified, H; homozygous 4

ECIS a precursor of endometrial carcinoma the surface epithelium of areas of atrophic endometrium (Figure 1A). Change to non-cancerous epithelium was abrupt. Sometimes SCIS foci were observed in connection with GCIS lesions. Both glandular and papillary thin CA foci were occasionally located in the superficial endometrial layers (Figure 1B,C). Heterogeneity of immunoreactivity for bcl-2, ER, and PR was observed in ECIS foci, with p53 frequently showing a diffuse immunopositivity (Figure 2). Figure 2. Semiserial sections through a papillary thin carcinoma (case 80, E-9). A. H&E staining, 100. B. Note the diffuse p53 immunoreactivity, 100. Relation between primary tumors and ECIS foci ECIS lesions, including SCIS, GCIS, and thin CA types, were found in 13 (10.7%) of 121 cases of endometrioid carcinomas. Although intraepithelial carcinoma foci with or without microinvasive features were identified in 6 (66.7%) of 9 serous carcinoma cases, 4 were excluded from this study because physical connections between the primary tumors and the foci were revealed by careful observation of semi-serial sections. The results for Table 3. Relation between primary tumor grade and the type of ECIS Endometrioid carcinoma (n = 13) Serous carcinoma Grade 1 Grade 2 Grade 3 (n = 4) (n = 6) (n = 3) (n = 2)* GCIS 4 3 0 0 SCIS 0 1 0 1 Thin CA 0 2 3 P = 0.063 2 *Case 84 had 2 ECIS lesions GCIS; glandular carcinoma in situ, SCIS; surface carcinoma in situ, Thin CA; thin carcinoma Table 4. Identity of immunoreactive markers between primary tumors and ECIS Identical markers Combinations between primary tumor and ECIS n = 16* (%) 4 4 (25.0%) 3 7 (43.8%) 2 4 (25.0%) 1 1 (6.2%) 0 0 Table 5. Combination of LOH of p53 and DCC genes in primary tumor and ECIS LOH in p53-loh primary tumor/ecis (n = 10) DCC-LOH Positive/Positive 1 1 Negative/Negative 9 1 Negative/Positive 0 0 Positive/Negative 0 0 *One case had 2 ECIS lesions. 5

Inoue H. et al. primary tumors and ECIS lesions for the p53 gene were gained for 10 of 16 (62.5%) lesions, but in only 2 cases for DCC. Identical patterns for the presence or absence of LOH of either p53 or DCC genes were found in all informative cases (Figure 3 and Table 5). Figure 3. Results of PCR-LOH assays of p53 and DCC gene loci. A. Allelic deletion of the TP53 locus is found not only in the primary tumor (P) but also the ECIS (E) lesion (case 23) (N, normal). B. The VNTR locus in the DCC gene. LOH is detected in both lesions (P, primary tumor; E, ECIS; N, normal) in case 2 but not in case 108. M, molecular markers (DNA molecular weight markers V, Boehringer Mannheim/Yamanouchi). endometrial carcinoma cases with ECIS are detailed in Table 2. Two ECIS lesions were found in case 84. Comparative analysis of primary tumors and ECIS demonstrated GCIS to be frequently found in associated with G1 endometrioid carcinomas, while thin CA lesions were predominantly detected along with G3 endometrioid and serous carcinomas (Table 3). As shown in Table 4, identical immunoreactive phenotypes for all four antibodies used were found for 4 (25%) of the 16 combinations between primary and ECIS lesions, and there were no cases in which the two lesions showed completely different immunophenotypes. Comparison of the LOH patterns of p53 and DCC genes between primary and ECIS lesions As shown in Table 2, informative combinations between 6 Discussion Multifocal development of lesions has been described for a variety of human malignant tumors, such as hepatocellular, 12,13 breast 14 and esophageal carcinomas, 15 so that a clear distinction between metastasis from a single primary tumor and synchronous appearance of multiple primary neoplasms must be made. In endometrial carcinoma cases, it has been noted that the absence of any background of hyperplasia in more aggressive uterine carcinomas may be due to rapid overgrowth and replacement of antecedent hyperplasia. 16 In the present study, the fact that intraepithelial tumor spreading lesions showing ECIS-like features were found in some endometrial carcinoma cases indicates that diagnosis should only be made after careful observation of many sections obtained from the entire endometrium. High incidences or levels of expression of bcl-2, ER, and PR have been demonstrated not only during normal menstrual cycles but also in endometrial carcinomas. 17,18 Ito et al. 19 have proposed that p53 immunohistochemical evaluation of endometrial carcinomas may be useful for identifying aggressive cases, especially in the early stages. Sherman et al. 20 also demonstrated a high incidence of p53 immunopositivity in serous carcinomas, in contrast to a low positivity in endometrioid carcinomas. In the present study, relatively higher bcl-2, ER, and PR immunoreactivity was found in endometrioid carcinomas, while p53 accumulation was predominantly detected in serous carcinomas. Therefore, it seems likely that these immunohistochemical markers reflect processes of importance to endometrial carcinoma development. Intratumoral antigenetic heterogeneity raises questions as to whether assessment of immunohistochemical phenotypes can serve to discriminate between tumor foci. Esteban et al. 21 demonstrated that phenotypic diversity was minimal between primary breast and gastric carcinomas and metastatic foci when the presence or absence of a reaction to a particular antibody was the criterion used as opposed to the degree of expression or the distribution. Dawson et al. 13 also concluded that in most breast carcinomas the immunocytochemical phenotype is a relatively stable property of tumor cells that may be used as a basis for comparison between

ECIS a precursor of endometrial carcinoma different tumor foci. Moreover, stability of both K- rasmutations and p53 gene abnormalities (mutations and allelic loss) has also been observed in primary endometrial and ovarian carcinomas and metastatic tumor lesions. 22 It is widely accepted that allelic losses are common chromosomal events in human malignant tumors. 23 An earlier allelotype analysis of endometrial carcinomas demonstrated that although LOH may be found for loci on almost all chromosomes, those involving loci on chromosomes 18q (DCC) and 17p (p53) were significantly more frequent than at other sites, indicating an association with tumor progression. 24 A close link between p53 gene changes including either mutations or LOH and survival has also been documented, indicating the possibility that acquisition of such alterations leads to rapid development of endometrial carcinomas without the necessity of passing through a phase of hyperplasia. 25 The significance of ECIS is related to whether endometrial carcinomas are of multi- or unicentric origin. Ambros et al. 5 proposed that ECIS is the most likely precursor of serous carcinomas. However, this cannot be considered to always be true, because as shown in the present study, ECIS is often present in endometrium along with typical endometrioid carcinomas. The present study demonstrated identical immunohistochemical phenotypes in 4 (25%) of 16 combinations of primary tumors and ECIS lesions, as well as an apparent association between the histological grade of primary tumors and the degree of progression of ECIS (Table 3). Moreover, the LOH analysis of p53 and DCC genes demonstrated identical allelic patterns in all informative cases. These findings, therefore, suggest that a proportion of ECIS-like lesions may develop monocentrically from primary tumors. However, these results do not preclude a precursor role, given the cases with varying immunophenotypes and the one case (case 84) with non-identical multifocal lesions. In conclusion, the present study indicates that ECIS may be either due to intraepithelial spread from single primary tumors or constitute an early event in multicentric tumor development of endometrial carcinomas. References 1. Bokhman KV. Two pathogenetic types of endometrial carcinoma. Gynecol Oncol 1983; 15: 10-7. 2. Deligdisch L, Cohen CJ. Histologic correlates and virulence implications of endometrial carcinoma associated with adenomatous hyperplasia. Cancer 1985; 56: 1452-5. 3. Kurman RJ, Kaminski PF, Norris HJ. The behavior of endometrial hyperplasia: A long-term study of "untreated" hyperplasia in 170 patients. Cancer 1985; 56: 403-12. 4. Spiegel GW. Endometrial carcinomas in situ in postmenopausal women. Am J Surg Pathol 1995; 19: 417-32. 5. Ambros RA, Sherman ME, Zahn CM, et al. Endometrial intraepithelial carcinoma: a distinctive lesion specifically associated withtumors displaying serous differentiation. Hum Pathol 1995; 26: 1260-7. 6. Sherman ME, Bitterman P, Rosenshein NB, et al. Uterine serous carcinoma: A morphologically diverse neoplasm with unifying clinicopathologic features. Am J Surg Pathol 1992; 16: 600-10. 7. Creasman WT. Announcement, FIGO stage: 1988 revisions. Gynecol Oncol 1989: 35: 125-7. 8. Jones MH, Nakamura Y. Detection of loss of heterozygosity at the human TP53 locus using a dinucleotide repeat polymorphism. Genes Chromosomes Cancer 1992; 5: 89-90. 9. Hahn M, Serth J, Fislage R, et al. Polymerase chain reaction detection of a highly polymorphic VNTR segment in intron 1 of the human p53 gene. Clin Chem 1993; 39: 549-50. 10. Hung Y, Boynton RF, Bloount PL, et al. Loss of heterozygosity involves multiple tumor suppressor genes in human esophageal cancers. Cancer Res 1992; 52: 6525-30. 11. Saegusa M, Takano Y, Kamata Y, et al. Bcl-2 expression and allelic loss of the p53 gene in gastric carcinomas. J Cancer Res Clin Oncol 1996; 7: 427-32. 12. Tsuda H, Oda T, Sakamoto M, et al. Different pattern of chromosomal allele loss in multiple hepatocellular carcinomas as evidence of their multifocal origin. Cancer Res 1992; 52: 1504-9. 13. Terada T, Nakanuma Y. Multiple occurrence of borderline hepatocellular nodules in human cirrhotic livers: possible multicentric origin of hepatocellular carcinoma. Virchows Arch 1995; 427: 379-83. 14. Dawson PJ, Baekey PA, Clark RA. Mechanisms of multifocal breast cancer: an immunocytochemical study. Hum Pathol 1995; 26: 965-9. 15. Wang LD, Zhou Q, Hong J-Y, et al. p53 protein accumulationand gene mutations in multifocal esophageal precancerous lesions from symptom free subjects in a high incidence area for esophageal carcinoma in Henan, China. Cancer 1996; 77: 1244-9. 16. Beckner ME, Mori T, Silverberg SG. Endometrial carcinoma: nontumor factors in prognosis. Int J Gynecol Pathol 1985; 4: 131-45. 7

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