International Journal of Gynecological Pathology 34:3–8, Lippincott Williams & Wilkins, Baltimore r 2014 International Society of Gynecological Pathologists
Original Article
Two Types of Ovarian Cortical Inclusion Cysts: Proposed Origin and Possible Role in Ovarian Serous Carcinogenesis Natalie Banet,
M.D.
and Robert J. Kurman,
M.D.
Summary: Ovarian cortical inclusion cysts (CICs) have been long regarded as a possible site of origin of epithelial ovarian carcinoma. It has been proposed that they develop from invagination of ovarian surface epithelium (OSE) which then undergoes metaplasia to form mullerian-type tissue and then undergoes neoplastic transformation. Recent studies have challenged this view, at least for high-grade serous carcinoma, proposing that the latter arise from occult carcinomas in the fallopian tube. Although there is compelling evidence supporting this view, it does not account for the origin of all high-grade serous carcinomas. We have postulated that a subset of high-grade serous carcinoma may develop from CICs, but that they are derived from implantation of tubal epithelium when the OSE is disrupted at ovulation. If true, it would be expected that the number of CICs would increase with age and that CICs would not be present before menarche. To test this hypothesis we examined ovaries removed at autopsy for the presence of CICs and correlated their presence with age. In addition, we used immunohistochemistry for PAX8 (mullerian marker) and calretinin (mesothelial marker). CICs were defined as either ciliated (tubal-type, PAX8-positive) or flat (OSE-type, calretinin-positive). As it has been argued that steroid hormones convert mesothelial-derived OSE to mullerian-type tissue, we performed immunohistochemistry for estrogen and progesterone receptors. CICs lined by tubal-type epithelium were found only in postmenarchial women and 20/21 (95%) were PAX8-positive; none of the 5 flat cysts expressed PAX8 but 4/5 (80%) expressed calretinin. Estrogen receptor was expressed in 1 of 21 (5%) ciliated CICs, whereas it was negative in all 5 flat CICs. Progesterone receptor was expressed in 14 of 21 (66%) ciliated CICs, and in none of the 5 flat cysts. The findings suggest that there are 2 types of CICs, 1 from OSE and 1 from tubal epithelium that probably develop at the time of ovulation. Key Words: Cortical inclusion cysts—Ovarian carcinoma origin—Serous carcinoma.
Ovarian cortical inclusion cysts (CICs) have been long regarded as the site of origin of epithelial ovarian carcinoma and it has been maintained that
they develop from invagination of the ovarian surface epithelium (OSE) (1). The latter is a flat, nondescript, single layer of epithelium which, in fact, is mesothelium continuous with that lining the peritoneal cavity. Skeptics of this theory have, therefore, questioned why epithelial ovarian tumors, if they are derived from OSE, are carcinomas with a mu¨llerian phenotype rather than mesotheliomas. To explain this contradiction it was proposed that the invaginated OSE undergoes metaplasia, possibly from hormonal stimulation, resulting in the development of ciliated (tubal-type) epithelium before undergoing malignant transformation (2).
From the Department of Pathology (N.B., R.J.K.), Division of Gynecologic Pathology; Departments of Gynecology and Obstetrics (R.J.K.); and Oncology (R.J.K.), The Johns Hopkins University School of Medicine, Baltimore, Maryland. The authors declare no conflict of interest. Address correspondence and reprint requests to Natalie Banet, MD, Department of Pathology, Division of Gynecologic Pathology, The Johns Hopkins University School of Medicine, 410 North Broadway, Weinberg 2242, Baltimore, MD 21213. E-mails: [email protected]; [email protected].
3
DOI: 10.1097/PGP.0000000000000120
4
N. BANET AND R.J. KURMAN
This series of events has been recently challenged, at least for high-grade serous carcinomas (HGSCs), by studies in which the ovaries and fallopian tubes of women at high risk of developing ovarian cancer have been completely processed and found to contain occult carcinomas in the fimbria of the fallopian tubes that closely resemble ovarian HGSC (3–9). Subsequent studies that thoroughly evaluated the fallopian tubes of women presenting with sporadic tubal, ovarian, and primary peritoneal HGSC revealed STICs in 50% to 60% of these cases (mostly in the fimbria) leading some investigators to suggest that the majority of ovarian HGSCs are derived from the fallopian tube (10–13). The source of the remaining 40% to 50% of HGSCs in which an associated tubal carcinoma was not detected remained a mystery. In some instances it is likely that the small tubal carcinoma was overgrown and obliterated by the invasive cancer but we have also entertained the thought that some HGSCs may be derived from CICs. A recent study describing aneuploidy in some CICs but not in OSE supports this proposal (14). In contrast to the view that the CICs are derived from OSE, we have postulated that some result from implantation of tubal epithelium directly into the ovary when the OSE is disrupted at ovulation. If true it would be expected that the number of CICs would increase as a function of age and that they would not be present before menarche. The present study was undertaken to test this hypothesis by examining ovaries removed at autopsy for the presence of CICs and to correlate their presence and number with age. In addition, we attempted to characterize CICs as being of either tubal or OSE origin by performing analysis with a panel of immunostains. PAX8 is a transcription factor that plays an important role in the development of a number of organs, including those derived from the mu¨llerian duct. It is positive in a variety of gynecologic tissues, including the fallopian tube epithelium (15,16), and has been shown to be negative in a majority of benign mesothelial lesions (16,17). Calretinin, which is occasionally expressed in gynecologic tissues, is a reliable marker for cells of mesothelial origin, including ovarian surface epitheium (18–20). Because of the possible role of hormones in the development of CICs, we also examined them for the presence of estrogen receptor (ER) and progesterone receptor (PR). Finally, in our experience, intraepithelial leukocytes are always present in fallopian tube epithelium (Fig. 3) but rarely in OSE. Accordingly, we performed immunohistochemistry for CD45 to help determine whether CICs were related to tubal epithelium or OSE. Int J Gynecol Pathol Vol. 34, No. 1, January 2015
MATERIALS AND METHODS The autopsy files from The Johns Hopkins Hospital over the last 12 yr were searched for cases in which ovaries were available. These cases were reviewed and the presence or absence of ovarian CICs was recorded, as was the extent of ovarian sampling. In addition, the type of epithelial lining the cysts (flat or ciliated) was noted. Immunohistochemical stains for calretinin, PAX8, CD45, ER, and PR were performed on all of the specimens with cysts. Immunohistochemical stains for PAX8, ER, and PR were considered positive if at least 10% of the cells showed nuclear labeling; calretinin was interpreted as positive if there was nuclear or cytoplasmic labeling in at least 10% of the cells.
RESULTS Our search identified 123 cases in which ovarian tissue had been submitted and could be analyzed for CICs. The ages of the entire cohort ranged from 0 (in utero death) to 94 yr (mean, 45 yr; median, 48 yr). Of the 123 cases, the ovaries of 35 (28%) patients contained CICs, some of which had more than one type of cyst (Table 1). An average of 1.3 blocks of ovary was sampled per patient, both in the entire cohort and in the subpopulation with cysts. Cysts were primarily located in a subcortical location. Ciliated columnar epithelium with interspersed secretory cells lined 21 cysts (60%), 11 (31%) contained cysts with both ciliated and flat epithelium, and 5 (14%) contained cysts lined only by flat epithelium devoid of cilia. The ages of the women with CICs TABLE 1. Cysts characteristics by patient age Number Mean Median Range 0–10 11–20 21–30 31–40 41–50 51–60 61–70 71+
No cysts
Cyst cohort
Flat cysts
Cysts with cilia*
87 38 39 0–94 18 7 10 10 12 10 9 11
35 61 58 38–94 0 0 0 3 6 10 7 9
5 63 65 52–76 0 0 0 0 0 2 2 1
32 62 58 38–94 0 0 0 4 5 9 4 10
Total number of cases = 122. *Cysts with cilia includes ciliated cysts and mixed cysts, for the purpose of this table. Some cases contained ciliated and flat cysts. Cysts from all cases did not persist on further sectioning for immunohistochemistry, which accounts for the discordance between the total cyst cohort and the subcategories.
TWO TYPES OF OVARIAN CORTICAL INCLUSION CYSTS ranged from 38 to 94 yr (mean, 61 yr; median, 58 y) and a correlation of cyst type with age is shown in Figure 1. The ages of the women without CICs ranged from 0 to 94 (mean, 38; median, 39). Of the 35 cases with CICs, some had >1 block available in which cysts were present, whereas in others the cysts were exhausted on deeper levels resulting in 29 cases in which immunohistochemical staining could be performed. Ciliated cysts were present in 21 (72%) of these 29 cases and expressed PAX8 in 20/21 (95%) and calretinin in 4/21 (19%). None of the 5 flat, nonciliated cysts expressed PAX8, whereas 4/5 (80%) expressed calretinin. All of the 11 cysts lined by both flat and ciliated columnar epithelium (hybrid cysts) stained for PAX8, whereas only 4 (36%) stained for calretinin. ER was expressed in 1 of 21 (5%) ciliated CICs, whereas it was negative in all 5 flat CICs. It was positive in 1 of 11 (9%) hybrid cysts. PR was expressed in 14 of 21 (67%) ciliated CICs, and in none of the 5 flat cysts. It was positive in 5 of 11 (45%) of the hybrid cysts. OSE is easily denuded during removal of the ovaries at autopsy and was therefore detected in only 21 (72%) of 29 cases. Among the cases in which OSE was present, 2/20 (10%) were positive for PAX8, whereas 19/21 (90%) were positive for calretinin. ER was expressed in OSE in 1 of 20 (5%) cases and PR in 3/20 (15%) (Table 2, Fig. 2). Lymphocytes were present in only 2 of 18 (11%) cases in OSE. One of these cases was PAX8-negative and the other could not be further evaluated because the OSE was lost during the process of sectioning for the additional immunohistochemistry studies. Lymphocytes were present in all 20 ciliated CICs as well as in all 9 hybrid cysts (in 2 cases the cysts were lost during preparation of additional sections for immu-
FIG. 1. Breakdown of cysts type by patient age. The ciliated cysts and mixed cysts (those with both flat and ciliated epithelium) have been combined for the purpose of this figure.
5
TABLE 2. Immunohistochemistry results in cysts OSE Ciliated Flat Mixed
Present
PAX8
CRT
ER
PR
21/29 21/29 5/29 11/29
2/20 20/21 0/5 11/11
19/21 4/21 4/5 4/11
1/20 1/21 0/5 1/11
3/20 14/21 0/5 5/11
Ciliated indicates ciliated cortical inclusion cysts; CRT, calretinin; ER, estrogen receptor; Flat, flat cortical inclusion cysts; Mixed, flat and ciliated cortical inclusion cysts; OSE, ovarian surface epithelium; PR, progesterone receptor.
nohistochemistry). Lymphocytes were found in 1 of 3 flat cysts. The CD45 findings are summarized in Table 3 and Figure 3.
DISCUSSION In this study we observed that most CICs are lined by ciliated, tubal-type epithelium, whereas substantially fewer are lined by flattened epithelium resembling OSE, a finding similar to that reported by Li et al (21). There were no CICs in girls before the age of 12 which is the mean age of menarche in the United States (22). In contrast, in a study by Blaustein et al. (2), cysts lined by flattened epithelium were reported in premenarchal girls but CICs lined by ciliated epithelium were not found in this age group. In the present study we found that vast majority of ciliated CICs expressed PAX8 but not calretinin and that CICs lined by flattened epithelium expressed calretinin but not PAX8. Interestingly, cysts lined by ciliated and flattened epithelium were almost all PAX8-positive and rarely calretinin-positive, suggesting that these ‘‘hybrid’’ cysts were actually ciliatedtype CICs in which part of the lining epithelium became flattened. We speculate that expansion of these cysts resulted in compression and flattening of the ciliated epithelium along one margin. Thus, there appear to be 2 types of CICs, 1 containing ciliatedtype epithelium and 1 containing flattened type epithelium. Li et al. (21) came to a similar conclusion. Our findings and those reported by Blaustein and colleagues showing that ciliated CICs are not found in premenarchal girls supports our hypothesis that they develop from tubal epithelium at the time of ovulation when the fimbria come into direct contact with the rupture site on the ovary. The trend in increasing numbers of ciliated-type CICs with age that we observed indirectly supports this proposal. The relative absence of ER and PR in the flat CICs and OSE suggests that it is unlikely that OSE gets transformed to tubal-type epithelium under the Int J Gynecol Pathol Vol. 34, No. 1, January 2015
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N. BANET AND R.J. KURMAN
FIG. 2. PAX8 and calretinin in fallopian tube, ovarian surface epithelium, and cysts. Normal fallopian tube (A), which marks diffusely for PAX8 and not for calretinin (CRT) (B and C). Ovarian surface epithelium (D), which does not stain for PAX8 (E), but does stain for CRT (F). Ciliated and mixed cysts (G and J, respectively) are diffusely and strongly positive for PAX8 (H and K), but not for CRT (I and L). The flat cyst (M) does not mark for PAX8 (N), but is positive for CRT (O). H&E indicates hematoxylin and eosin; OSE, ovarian surface epithelium.
influence of steroid hormones. In contrast, the presence of CICs lined by flattened epithelium in premenarchal girls as reported by Blaustein and colleagues suggest that they develop from invagination of OSE as the ovaries develop. The occasional Int J Gynecol Pathol Vol. 34, No. 1, January 2015
finding of PAX8-positive cells on the OSE and also the occasional finding of ciliated cells in that location probably result from implanted tubal epithelium, a finding that has also been recently reported (21). It is conceivable that this results from implantation of
TWO TYPES OF OVARIAN CORTICAL INCLUSION CYSTS
7
FIG. 3. Fallopian tube epithelium (A), which labels diffusely for PAX8 (B), but not for calretinin (CRT) (C). A CD45 immunostain highlights scattered positive lymphocytes (D).
tubal epithelium on the OSE and if the epithelium does not invaginate it is sloughed off. In summary, the findings in this, and other studies, showing that CICs lined by ciliated epithelium occur only in postmenarchal women are consistent with our hypothesis that this type of cyst is derived from implanted fallopian tube epithelium, possibly occurring at the time of ovulation, rather than as a result of TABLE 3. Presence of CD45+ lymphocytes CD45+ lymphocytes present n (%) OSE Ciliated Flat Mixed
2/18 20/20 1/3 9/9
(11) (100) (33) (100)
The results of CD45 immunostaining in OSE, ciliated, flat, and mixed cysts. Ciliated indicates ciliated cortical inclusion cysts; Flat, flat cortical inclusion cysts; Mixed, flat and ciliated cortical inclusion cysts; OSE, ovarian surface epithelium.
metaplasia occurring in cysts that are derived from invagination of the OSE. Even the most meticulous sampling of the fallopian tubes in studies of pelvic HGSC reveals STICs in only 60% of cases (13). In some instances this is likely due to overgrowth of STICs by the invasive carcinoma but we believe this probably does not account for all of the remaining 40%. Our findings that a large proportion of CICs are lined by tubal-type epithelium in conjunction with the report of aneuploidy in CICs suggest the tubaltype CIC may be another source of HGSC.
REFERENCES 1. Scully R, Young R, Clement P, eds. Tumors of the Ovary, Maldeveloped Gonads, Fallopian Tube, and Broad Ligament. Washington, DC: Armed Forces Institution of PathologyChapter 1 title; 1998:1–21. 2. Blaustein A, Kantius M, Kaganowicz A, et al. Inclusions in ovaries of females aged day 1-30 years. Int J Gynecol Pathol 1982;1:145–53.
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3. Callahan MJ, Crum CP, Medeiros F, et al. Primary fallopian tube malignancies in BRCA-positive women undergoing surgery for ovarian cancer risk reduction. J Clin Oncol 2007;25:3985–90. 4. Carcangiu ML, Radice P, Manoukian S, et al. Atypical epithelial proliferation in fallopian tubes in prophylactic salpingo-oophorectomy specimens from BRCA1 and BRCA2 germline mutation carriers. Int J Gynecol Pathol 2004;23:35–40. 5. Colgan TJ, Murphy J, Cole DE, et al. Occult carcinoma in prophylactic oophorectomy specimens: prevalence and association with BRCA germline mutation status. Am J Surg Pathol 2001;25:1283–9. 6. Finch A, Shaw P, Rosen B, et al. Clinical and pathologic findings of prophylactic salpingo-oophorectomies in 159 BRCA1 and BRCA2 carriers. Gynecol Oncol 2006;100:58–64. 7. Medeiros F, Muto MG, Lee Y, et al. The tubal fimbria is a preferred site for early adenocarcinoma in women with familial ovarian cancer syndrome. Am J Surg Pathol 2006;30:230–6. 8. Paley PJ, Swisher EM, Garcia RL, et al. Occult cancer of the fallopian tube in BRCA-1 germline mutation carriers at prophylactic oophorectomy: a case for recommending hysterectomy at surgical prophylaxis. Gynecol Oncol 2001; 80:176–80. 9. Shaw PA, Rouzbahman M, Pizer ES, et al. Candidate serous cancer precursors in fallopian tube epithelium of BRCA1/2 mutation carriers. Mod Pathol 2009;22:1133–8. 10. Carlson JW, Miron A, Jarboe EA, et al. Serous tubal intraepithelial carcinoma: its potential role in primary peritoneal serous carcinoma and serous cancer prevention. J Clin Oncol 2008;26:4160–5. 11. Roh MH, Kindelberger D, Crum CP. Serous tubal intraepithelial carcinoma and the dominant ovarian mass: clues to serous tumor origin? Am J Surg Pathol 2009;33:376–83. 12. Seidman JD, Zhao P, Yemelyanova A. ‘‘Primary peritoneal’’ high-grade serous carcinoma is very likely metastatic from serous tubal intraepithelial carcinoma: assessing the new paradigm of ovarian and pelvic serous carcinogenesis and its
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13. 14.
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implications for screening for ovarian cancer. Gynecol Oncol 2011;120:470–3. Przybycin CG, Kurman RJ, Ronnett BM, et al. Are all pelvic (nonuterine) serous carcinomas of tubal origin? Am J Surg Pathol 2010;34:1407–16. Ko¨rner M, Burckhardt E, Mazzucchelli L. Different proportions of aneusomic cells in ovarian inclusion cysts associated with serous borderline tumours and serous high-grade carcinomas support different pathogenetic pathways. J Pathol 2005;207:20–6. Ozcan A, Liles N, Coffey D, et al. PAX2 and PAX8 expression in primary and metastatic mu¨llerian epithelial tumors: a comprehensive comparison. Am J Surg Pathol 2011;35: 1837–47. Ozcan A, Shen SS, Hamilton C, et al. PAX 8 expression in non-neoplastic tissues, primary tumors, and metastatic tumors: a comprehensive immunohistochemical study. Mod Pathol 2011;24:751–64. Xiang L, Zheng W, Kong B. Detection of PAX8 and p53 is beneficial in recognizing metastatic carcinomas in pelvic washings, especially in cases with suspicious cytology. Gynecol Oncol 2012;127:595–600. Okamoto S, Ito K, Sasano H, et al. Ber-EP4 and anti-calretinin antibodies: a useful combination for differential diagnosis of various histological types of ovarian cancer cells and mesothelial cells. Tohoku J Exp Med 2005;206:31–40. Ordo´n˜ez NG. Value of calretinin immunostaining in diagnostic pathology: a review and update. Appl Immunohistochem Mol Morphol 2013. [Epub ahead of print]. Cao QJ, Jones JG, Li M. Expression of calretinin in human ovary, testis, and ovarian sex cord-stromal tumors. Int J Gynecol Pathol 2001;20:346–52. Li J, Abushahin N, Pang S, et al. Tubal origin of ‘‘ovarian’’ low-grade serous carcinoma. Mod Pathol 2011;24:1488–99. Diaz A, Laufer MR, Breech LL. Menstruation in girls and adolescents: using the menstrual cycle as a vital sign. Pediatrics 2006;118:2245–50.
Original Article
Two Types of Ovarian Cortical Inclusion Cysts: Proposed Origin and Possible Role in Ovarian Serous Carcinogenesis Natalie Banet,
M.D.
and Robert J. Kurman,
M.D.
Summary: Ovarian cortical inclusion cysts (CICs) have been long regarded as a possible site of origin of epithelial ovarian carcinoma. It has been proposed that they develop from invagination of ovarian surface epithelium (OSE) which then undergoes metaplasia to form mullerian-type tissue and then undergoes neoplastic transformation. Recent studies have challenged this view, at least for high-grade serous carcinoma, proposing that the latter arise from occult carcinomas in the fallopian tube. Although there is compelling evidence supporting this view, it does not account for the origin of all high-grade serous carcinomas. We have postulated that a subset of high-grade serous carcinoma may develop from CICs, but that they are derived from implantation of tubal epithelium when the OSE is disrupted at ovulation. If true, it would be expected that the number of CICs would increase with age and that CICs would not be present before menarche. To test this hypothesis we examined ovaries removed at autopsy for the presence of CICs and correlated their presence with age. In addition, we used immunohistochemistry for PAX8 (mullerian marker) and calretinin (mesothelial marker). CICs were defined as either ciliated (tubal-type, PAX8-positive) or flat (OSE-type, calretinin-positive). As it has been argued that steroid hormones convert mesothelial-derived OSE to mullerian-type tissue, we performed immunohistochemistry for estrogen and progesterone receptors. CICs lined by tubal-type epithelium were found only in postmenarchial women and 20/21 (95%) were PAX8-positive; none of the 5 flat cysts expressed PAX8 but 4/5 (80%) expressed calretinin. Estrogen receptor was expressed in 1 of 21 (5%) ciliated CICs, whereas it was negative in all 5 flat CICs. Progesterone receptor was expressed in 14 of 21 (66%) ciliated CICs, and in none of the 5 flat cysts. The findings suggest that there are 2 types of CICs, 1 from OSE and 1 from tubal epithelium that probably develop at the time of ovulation. Key Words: Cortical inclusion cysts—Ovarian carcinoma origin—Serous carcinoma.
Ovarian cortical inclusion cysts (CICs) have been long regarded as the site of origin of epithelial ovarian carcinoma and it has been maintained that
they develop from invagination of the ovarian surface epithelium (OSE) (1). The latter is a flat, nondescript, single layer of epithelium which, in fact, is mesothelium continuous with that lining the peritoneal cavity. Skeptics of this theory have, therefore, questioned why epithelial ovarian tumors, if they are derived from OSE, are carcinomas with a mu¨llerian phenotype rather than mesotheliomas. To explain this contradiction it was proposed that the invaginated OSE undergoes metaplasia, possibly from hormonal stimulation, resulting in the development of ciliated (tubal-type) epithelium before undergoing malignant transformation (2).
From the Department of Pathology (N.B., R.J.K.), Division of Gynecologic Pathology; Departments of Gynecology and Obstetrics (R.J.K.); and Oncology (R.J.K.), The Johns Hopkins University School of Medicine, Baltimore, Maryland. The authors declare no conflict of interest. Address correspondence and reprint requests to Natalie Banet, MD, Department of Pathology, Division of Gynecologic Pathology, The Johns Hopkins University School of Medicine, 410 North Broadway, Weinberg 2242, Baltimore, MD 21213. E-mails: [email protected]; [email protected].
3
DOI: 10.1097/PGP.0000000000000120
4
N. BANET AND R.J. KURMAN
This series of events has been recently challenged, at least for high-grade serous carcinomas (HGSCs), by studies in which the ovaries and fallopian tubes of women at high risk of developing ovarian cancer have been completely processed and found to contain occult carcinomas in the fimbria of the fallopian tubes that closely resemble ovarian HGSC (3–9). Subsequent studies that thoroughly evaluated the fallopian tubes of women presenting with sporadic tubal, ovarian, and primary peritoneal HGSC revealed STICs in 50% to 60% of these cases (mostly in the fimbria) leading some investigators to suggest that the majority of ovarian HGSCs are derived from the fallopian tube (10–13). The source of the remaining 40% to 50% of HGSCs in which an associated tubal carcinoma was not detected remained a mystery. In some instances it is likely that the small tubal carcinoma was overgrown and obliterated by the invasive cancer but we have also entertained the thought that some HGSCs may be derived from CICs. A recent study describing aneuploidy in some CICs but not in OSE supports this proposal (14). In contrast to the view that the CICs are derived from OSE, we have postulated that some result from implantation of tubal epithelium directly into the ovary when the OSE is disrupted at ovulation. If true it would be expected that the number of CICs would increase as a function of age and that they would not be present before menarche. The present study was undertaken to test this hypothesis by examining ovaries removed at autopsy for the presence of CICs and to correlate their presence and number with age. In addition, we attempted to characterize CICs as being of either tubal or OSE origin by performing analysis with a panel of immunostains. PAX8 is a transcription factor that plays an important role in the development of a number of organs, including those derived from the mu¨llerian duct. It is positive in a variety of gynecologic tissues, including the fallopian tube epithelium (15,16), and has been shown to be negative in a majority of benign mesothelial lesions (16,17). Calretinin, which is occasionally expressed in gynecologic tissues, is a reliable marker for cells of mesothelial origin, including ovarian surface epitheium (18–20). Because of the possible role of hormones in the development of CICs, we also examined them for the presence of estrogen receptor (ER) and progesterone receptor (PR). Finally, in our experience, intraepithelial leukocytes are always present in fallopian tube epithelium (Fig. 3) but rarely in OSE. Accordingly, we performed immunohistochemistry for CD45 to help determine whether CICs were related to tubal epithelium or OSE. Int J Gynecol Pathol Vol. 34, No. 1, January 2015
MATERIALS AND METHODS The autopsy files from The Johns Hopkins Hospital over the last 12 yr were searched for cases in which ovaries were available. These cases were reviewed and the presence or absence of ovarian CICs was recorded, as was the extent of ovarian sampling. In addition, the type of epithelial lining the cysts (flat or ciliated) was noted. Immunohistochemical stains for calretinin, PAX8, CD45, ER, and PR were performed on all of the specimens with cysts. Immunohistochemical stains for PAX8, ER, and PR were considered positive if at least 10% of the cells showed nuclear labeling; calretinin was interpreted as positive if there was nuclear or cytoplasmic labeling in at least 10% of the cells.
RESULTS Our search identified 123 cases in which ovarian tissue had been submitted and could be analyzed for CICs. The ages of the entire cohort ranged from 0 (in utero death) to 94 yr (mean, 45 yr; median, 48 yr). Of the 123 cases, the ovaries of 35 (28%) patients contained CICs, some of which had more than one type of cyst (Table 1). An average of 1.3 blocks of ovary was sampled per patient, both in the entire cohort and in the subpopulation with cysts. Cysts were primarily located in a subcortical location. Ciliated columnar epithelium with interspersed secretory cells lined 21 cysts (60%), 11 (31%) contained cysts with both ciliated and flat epithelium, and 5 (14%) contained cysts lined only by flat epithelium devoid of cilia. The ages of the women with CICs TABLE 1. Cysts characteristics by patient age Number Mean Median Range 0–10 11–20 21–30 31–40 41–50 51–60 61–70 71+
No cysts
Cyst cohort
Flat cysts
Cysts with cilia*
87 38 39 0–94 18 7 10 10 12 10 9 11
35 61 58 38–94 0 0 0 3 6 10 7 9
5 63 65 52–76 0 0 0 0 0 2 2 1
32 62 58 38–94 0 0 0 4 5 9 4 10
Total number of cases = 122. *Cysts with cilia includes ciliated cysts and mixed cysts, for the purpose of this table. Some cases contained ciliated and flat cysts. Cysts from all cases did not persist on further sectioning for immunohistochemistry, which accounts for the discordance between the total cyst cohort and the subcategories.
TWO TYPES OF OVARIAN CORTICAL INCLUSION CYSTS ranged from 38 to 94 yr (mean, 61 yr; median, 58 y) and a correlation of cyst type with age is shown in Figure 1. The ages of the women without CICs ranged from 0 to 94 (mean, 38; median, 39). Of the 35 cases with CICs, some had >1 block available in which cysts were present, whereas in others the cysts were exhausted on deeper levels resulting in 29 cases in which immunohistochemical staining could be performed. Ciliated cysts were present in 21 (72%) of these 29 cases and expressed PAX8 in 20/21 (95%) and calretinin in 4/21 (19%). None of the 5 flat, nonciliated cysts expressed PAX8, whereas 4/5 (80%) expressed calretinin. All of the 11 cysts lined by both flat and ciliated columnar epithelium (hybrid cysts) stained for PAX8, whereas only 4 (36%) stained for calretinin. ER was expressed in 1 of 21 (5%) ciliated CICs, whereas it was negative in all 5 flat CICs. It was positive in 1 of 11 (9%) hybrid cysts. PR was expressed in 14 of 21 (67%) ciliated CICs, and in none of the 5 flat cysts. It was positive in 5 of 11 (45%) of the hybrid cysts. OSE is easily denuded during removal of the ovaries at autopsy and was therefore detected in only 21 (72%) of 29 cases. Among the cases in which OSE was present, 2/20 (10%) were positive for PAX8, whereas 19/21 (90%) were positive for calretinin. ER was expressed in OSE in 1 of 20 (5%) cases and PR in 3/20 (15%) (Table 2, Fig. 2). Lymphocytes were present in only 2 of 18 (11%) cases in OSE. One of these cases was PAX8-negative and the other could not be further evaluated because the OSE was lost during the process of sectioning for the additional immunohistochemistry studies. Lymphocytes were present in all 20 ciliated CICs as well as in all 9 hybrid cysts (in 2 cases the cysts were lost during preparation of additional sections for immu-
FIG. 1. Breakdown of cysts type by patient age. The ciliated cysts and mixed cysts (those with both flat and ciliated epithelium) have been combined for the purpose of this figure.
5
TABLE 2. Immunohistochemistry results in cysts OSE Ciliated Flat Mixed
Present
PAX8
CRT
ER
PR
21/29 21/29 5/29 11/29
2/20 20/21 0/5 11/11
19/21 4/21 4/5 4/11
1/20 1/21 0/5 1/11
3/20 14/21 0/5 5/11
Ciliated indicates ciliated cortical inclusion cysts; CRT, calretinin; ER, estrogen receptor; Flat, flat cortical inclusion cysts; Mixed, flat and ciliated cortical inclusion cysts; OSE, ovarian surface epithelium; PR, progesterone receptor.
nohistochemistry). Lymphocytes were found in 1 of 3 flat cysts. The CD45 findings are summarized in Table 3 and Figure 3.
DISCUSSION In this study we observed that most CICs are lined by ciliated, tubal-type epithelium, whereas substantially fewer are lined by flattened epithelium resembling OSE, a finding similar to that reported by Li et al (21). There were no CICs in girls before the age of 12 which is the mean age of menarche in the United States (22). In contrast, in a study by Blaustein et al. (2), cysts lined by flattened epithelium were reported in premenarchal girls but CICs lined by ciliated epithelium were not found in this age group. In the present study we found that vast majority of ciliated CICs expressed PAX8 but not calretinin and that CICs lined by flattened epithelium expressed calretinin but not PAX8. Interestingly, cysts lined by ciliated and flattened epithelium were almost all PAX8-positive and rarely calretinin-positive, suggesting that these ‘‘hybrid’’ cysts were actually ciliatedtype CICs in which part of the lining epithelium became flattened. We speculate that expansion of these cysts resulted in compression and flattening of the ciliated epithelium along one margin. Thus, there appear to be 2 types of CICs, 1 containing ciliatedtype epithelium and 1 containing flattened type epithelium. Li et al. (21) came to a similar conclusion. Our findings and those reported by Blaustein and colleagues showing that ciliated CICs are not found in premenarchal girls supports our hypothesis that they develop from tubal epithelium at the time of ovulation when the fimbria come into direct contact with the rupture site on the ovary. The trend in increasing numbers of ciliated-type CICs with age that we observed indirectly supports this proposal. The relative absence of ER and PR in the flat CICs and OSE suggests that it is unlikely that OSE gets transformed to tubal-type epithelium under the Int J Gynecol Pathol Vol. 34, No. 1, January 2015
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FIG. 2. PAX8 and calretinin in fallopian tube, ovarian surface epithelium, and cysts. Normal fallopian tube (A), which marks diffusely for PAX8 and not for calretinin (CRT) (B and C). Ovarian surface epithelium (D), which does not stain for PAX8 (E), but does stain for CRT (F). Ciliated and mixed cysts (G and J, respectively) are diffusely and strongly positive for PAX8 (H and K), but not for CRT (I and L). The flat cyst (M) does not mark for PAX8 (N), but is positive for CRT (O). H&E indicates hematoxylin and eosin; OSE, ovarian surface epithelium.
influence of steroid hormones. In contrast, the presence of CICs lined by flattened epithelium in premenarchal girls as reported by Blaustein and colleagues suggest that they develop from invagination of OSE as the ovaries develop. The occasional Int J Gynecol Pathol Vol. 34, No. 1, January 2015
finding of PAX8-positive cells on the OSE and also the occasional finding of ciliated cells in that location probably result from implanted tubal epithelium, a finding that has also been recently reported (21). It is conceivable that this results from implantation of
TWO TYPES OF OVARIAN CORTICAL INCLUSION CYSTS
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FIG. 3. Fallopian tube epithelium (A), which labels diffusely for PAX8 (B), but not for calretinin (CRT) (C). A CD45 immunostain highlights scattered positive lymphocytes (D).
tubal epithelium on the OSE and if the epithelium does not invaginate it is sloughed off. In summary, the findings in this, and other studies, showing that CICs lined by ciliated epithelium occur only in postmenarchal women are consistent with our hypothesis that this type of cyst is derived from implanted fallopian tube epithelium, possibly occurring at the time of ovulation, rather than as a result of TABLE 3. Presence of CD45+ lymphocytes CD45+ lymphocytes present n (%) OSE Ciliated Flat Mixed
2/18 20/20 1/3 9/9
(11) (100) (33) (100)
The results of CD45 immunostaining in OSE, ciliated, flat, and mixed cysts. Ciliated indicates ciliated cortical inclusion cysts; Flat, flat cortical inclusion cysts; Mixed, flat and ciliated cortical inclusion cysts; OSE, ovarian surface epithelium.
metaplasia occurring in cysts that are derived from invagination of the OSE. Even the most meticulous sampling of the fallopian tubes in studies of pelvic HGSC reveals STICs in only 60% of cases (13). In some instances this is likely due to overgrowth of STICs by the invasive carcinoma but we believe this probably does not account for all of the remaining 40%. Our findings that a large proportion of CICs are lined by tubal-type epithelium in conjunction with the report of aneuploidy in CICs suggest the tubaltype CIC may be another source of HGSC.
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