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Department of Pathology, Section of Cytology, The University of Texas MD Anderson Cancer Center, Houston, Texas
Corresponding author: Nour Sneige, MD, Department of Pathology, Unit 53, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030; Fax: (713) 792-2499; email@example.com
To better define the cytomorphologic spectrum of endosalpingiosis in peritoneal washings (PWs) and thereby facilitate their distinction from well differentiated serous carcinoma, the authors examined PWs from women who underwent surgery and pathologic staging of lesions other than Mullerian malignancies and correlated the findings with surgical specimens.
This was a retrospective review of medical records and PW specimens from 100 consecutive patients who had PWs coded as both “endosalpingiosis” and “negative for carcinoma” between 2002 and 2012. Thirty-eight of these patients had no gynecologic malignancies. Specimens had been prepared using cytocentrifugation and were stained using the Papanicolaou method. The cytologic findings evaluated were cell arrangement, number of cell groups per case, cellular atypia, and psammoma bodies. Smears also were assessed for paired box-8 (PAX8) immunostaining. The authors compared patients' staging biopsy findings with the findings from a review of the PWs.
PW specimens from 35 of 38 patients (92%) exhibited classic endosalpingiosis features: tubular or small branching papillary structures, some with psammoma bodies. Specimens from the 3 remaining patients displayed nonclassic features consistent with dislodged fallopian tube epithelium or endometriosis. From 2 to 20 clusters per slide and from 4 to 50 groups per case were identified. In a few cases, some cell clusters exhibited up to moderate cytologic atypia. Surgical findings included endometriosis, endosalpingiosis, both endometriosis and endosalpingiosis (12 patients; 31.6%), and a variety of unrelated pelvic lesions. All cases were PAX8-positive, confirming their Mullerian origin.
Since the 1950s, peritoneal washing (PW) cytology has been used to evaluate gynecologic cancers; and, in 1975, the International Federation of Gynecology and Obstetrics included PW cytology in the staging process for ovarian carcinomas. The presence of malignant cells in PW cytologic specimens raises the International Federation of Gynecology and Obstetrics stage from stage IA/IB or IIA/IIB to stage IC or IIC, respectively, for ovarian carcinoma. However, the cytologic evaluation of PWs is not always straightforward, and some series have reported up to a 4.5% false-positive rate.[2-4] Diagnostic difficulties have been attributed to reactive mesothelial hyperplasia, pelvic inflammatory disease, endometriosis, psammoma bodies, and endosalpingiosis.[3-9]
In the majority of cases, reactive mesothelial cells can be accurately recognized based on certain cell features10; in some cases, however, reactive changes may result in tight clusters of mesothelial cells with a high nuclear:cytoplasmic ratio that mimic low-grade adenocarcinoma or serous carcinoma. In such cases, the use of immunohistochemical panels that include 2 markers of epithelial cells and 2 markers of mesothelial cells can resolve the diagnosis.[4, 11] Assessment of paired box-8 (PAX8) expression, a highly sensitive and specific transcription factor for tumors of Mullerian origin as well as for normal or non-neoplastic Mullerian epithelium, complements the epithelial markers when a Mullerian origin is suspected.[12, 13]
Endometriosis is recognized through the presence of endometrial glandular and stromal cells together with hemosiderophages.[10, 14] However, the absence of these key cytologic features in many cases of endometriosis, coupled with the presence of reactive changes, may lead to a false-positive diagnosis. And, in contrast to the case of reactive mesothelial cells versus Mullerian epithelium, no cell marker is available to distinguish endometriosis from low-grade tumors of Mullerian origin, because both epithelia share similar immunophenotypic features.[12, 13]
Endosalpingiosis usually presents as cohesive papillary/tubular structures, often forming a single layer of epithelium surrounding psammoma bodies.[5, 7, 8] The epithelial cells range from cuboidal to low columnar and are arranged in an orderly fashion. In some cases, however, atypical/reactive changes in the Mullerian epithelium may result in cytologic features that are indistinguishable from serous tumors of low malignant potential or well differentiated serous carcinoma. No specific marker distinguishes endosalpingiosis from low-grade serous carcinoma, as in the previous example, and the final interpretation of PWs must rely on cytomorphology.
Authors of previous studies have suggested various approaches for accurately assessing PWs. Some have suggested that the challenges to interpreting PWs can only be resolved by evaluating histologic material from the resected corresponding lesions. Others have stressed the use of an algorithm that analyzes the cytomorphologic and/or immunohistochemical characteristics of these specimens and correlates them with the histology of the surgical material to enhance the accuracy of PW cytology.[3, 4, 8, 10] Although those studies were significant in highlighting diagnostic pitfalls and providing guidelines for accurately assessing PWs, they were limited by the small number of cases studied,[3, 8, 15-18] and their interpretations were based purely on morphology without the support of marker studies to confirm the origin of the cells in question.
To further assess the cytomorphologic spectrum of benign Mullerian epithelium as it presents in PWs and, thus, to facilitate the distinction of benign Mullerian epithelium from its mimickers, we undertook this study of “endosalpingiosis”/Mullerian epithelium in PWs from women who had benign gynecologic conditions. We also confirmed these findings as Mullerian through an immunohistochemical analysis of PAX8 expression, thus providing the foundation necessary for an accurate assessment of PWs.
MATERIALS AND METHODS
From a search of our electronic database, we obtained the records of 100 consecutive patients who had PW specimens coded as “endosalpingiosis” and/or “negative for carcinoma” between 2002 and 2012 at The University of Texas MD Anderson Cancer Center. Of the 100 patients, 38 met inclusion criteria (no gynecologic malignancies).
Of the 38 patients who were included in this study, 13 underwent laparoscopic surgery, and the remaining 25 patients underwent laparotomy. PWs had been obtained in the following manner: as soon as the peritoneal cavity was visible or opened, any free fluid was aspirated, and the peritoneal surfaces were lavaged with warm physiologic saline. The fluid was then aspirated and sent immediately to the cytopathology laboratory. There, it was centrifuged, and the Shandon Cytospin centrifuge (ThermoFisher Scientific, Waltham, Mass) was used to prepare slides from the resuspended cell pellet after the supernatant was decanted. The slides were fixed in modified Carnoy solution and stained using the Papanicolaou method.
Slides of 42 PWs from 38 patients (2 patients had 2 and 3 PW specimens each) were available for review. Two to 4 cytospin smears were available per patient. The presence of endosalpingiosis and psammomatous microcalcifications was noted using previously published criteria.[5, 7, 8] Briefly, endosalpingiosis (or Mullerian metaplasia) is recognized by the presence of a second, distinct cell population consisting of cuboidal to columnar or cylindrical cells with scant basophilic cytoplasm, sometimes with cilia, and eccentric oval nuclei with fine granular chromatin and round small nucleoli. The cells are often arranged in simple, smoothly contoured groups with occasional nonbranching papillary formation lacking intercellular windows and surrounding 1 or more psammoma bodies.
The cytologic findings that we evaluated were cell arrangement, the number of cell groups per case, cellular atypia, and associated psammoma bodies. Cellular atypia was defined as epithelial cells with increased nuclear:cytoplasmic ratio, nuclear hyperchromasia with or without nuclear membrane irregularities, and nuclear pleomorphism; the degree of each of these features was graded as mild, moderate, or high. The smears also were assessed for PAX8 immunostaining (a Mullerian marker).
For PAX8 immunostaining, the polymeric biotin-free horseradish peroxide method for the Bond Max stainer (Leica Microsystems, Wetzlar, Germany) was used. In each case, the coverslip was removed by immersing the smears in xylene for a varied length of time until the coverslip became loose and could easily be removed. The slides were rinsed in alcohol 3 times and then placed in deionized water. After heat-induced epitope retrieval with citrate buffer for 10 minutes at 100°C, the slides were incubated with antibody PAX8 (clone ab53490; Protein Tech Group, Chicago, Ill) at 1:100 dilution. The Refine polymer detection kit was used to detect bound antibody, with 3,3′-diaminobenzidine serving as the chromogen (Leica Microsystems). The slides were counterstained with Mayer hematoxylin. Known positive and negative tissue controls were run with the immunostains.
Positive nuclear staining of the epithelioid cell groups and/or of individual epithelioid cells was considered as PAX8-positive, and the number of positive cell groups was recorded. Mesothelial cells served as a built-in negative control. The results of immunostaining were correlated with those noted on the Papanicolaou-stained smears. We compared patients' staging biopsy findings with the findings from our reviews of the PWs. Follow-up data were obtained when available. This study was approved by the MD Anderson Cancer Center Institutional Review Board (protocol lab PA11-0964).
Patients ranged in age from 17 to 74 years (mean, 44 years). We identified 2 to 20 epithelial clusters per slide (mean, 6 clusters) and 4 to 50 groups per patient. In 35 of 38 patients (92%), the PW specimens exhibited typical endosalpingiosis features: small clusters or branching tubular structures, often forming a single layer of epithelium surrounding psammoma bodies. The epithelial cells were cuboidal to tall columnar cells and, for the most part, displayed uniform nuclei with smooth nuclear membranes and a fine chromatin pattern. In a few specimens, however, some of the cell clusters exhibited up to a moderate degree of cytologic atypia characterized by increased nuclear:cytoplasmic ratio, nuclear hyperchromasia, and/or nuclear pleomorphism (Fig. 1). Specimens from the 3 remaining patients displayed nonclassic features consistent with displaced fallopian tube epithelium with papillary hyperplasia, normal fallopian tube epithelium, or endometriosis (1 patient each). The displaced fallopian tube epithelium presented as large strips and sheets of ciliated, columnar cells with uniform oval-to-round nuclei and a fine chromatin pattern (Fig. 2). In addition, in the patient with associated hyperplasia, the cells exhibited a moderate degree of nuclear hyperchromasia with nuclear crowding and formed small papillae (Fig. 3). In the patient with endometriosis, there were numerous small clusters or loose sheets of moderately hyperchromatic and pleomorphic epithelial cells with micropapillary arrangement (almost 50 clusters) in a background of florid histiocytoid and chronic inflammatory cells. However, stromal cells and hemosiderphages characteristic of endometriosis were absent (Fig. 4). Histologic findings of the surgical biopsy specimens are summarized in Table 1.
Table 1. Histologic Findings in Surgical Biopsy Specimens of 38 Patients
No. of Patients (%)
One patient from each of these groups exhibited nonclassic features of endosalpingiosis (total, 3 patients). The remaining patients exhibited classic features of endosalpingiosis.
Other associated lesions were mature teratoma (1 patient) and mucinous cystadenoma (1 patient).
Other associated lesions were serous cystadenoma (1 patient), granulosa cell tumor (1 patient), and fibroma (1 patient).
These patients included 1 with serous cystadenoma.
These patients included 1 with fallopian tube hyperplasia.
Peritoneal washings were part of staging for appendiceal goblet cell adenocarcinoma (2 patients), cervical carcinoma in situ (1 patient), and risk-reducing salpingo-oophorectomy (4 patients).
Endometriosis and/or endosalpingiosis accounted for only 12 of 38 patients (31.6%); however, surgical staging was limited or was not performed in the majority of patients (30 of 38), because intraoperative findings were believed to be benign. Sampling of resected specimens, especially those from the fallopian tube, also was limited. It is noteworthy that 2 of the 3 specimens with nonclassic endosalpingiosis (shown in Figs. 3, 4) were from patients who underwent laparoscopic surgery, which is believed to have contributed to mechanical displacement of fallopian tube epithelium.
Immunostaining for PAX8 was positive in all patients, confirming the Mullerian origin of the clusters. The PAX8-positive cells paralleled those noted on the Papanicolaou stains, both quantitatively and qualitatively (Figs. 1-4).
Our findings demonstrate that Mullerian epithelium can be observed in PW specimens from women with benign gynecologic conditions and in patients who have no detectable or apparent histologic peritoneal lesions (ie, endosalpingiosis or endometriosis). Therefore, awareness of this finding has paramount importance when assessing PWs during the cancer staging process. In the current study, only 33% (12 of 38 patients) of PW findings could be explained on the basis of their corresponding surgical specimens, whereas the majority of the PWs (26 of 38 patients) had no apparent histologic lesions of the peritoneal surface. Although this may be partly attributed to the limited surgical staging in some of the patients and to the finding that cytology may be more representative of the peritoneal surfaces than tissue biopsy sampling, it also may be attributed to the shedding of Mullerian epithelium into the peritoneal cavity, hence the detection of Mullerian epithelium in PWs without its being necessarily implanted on the peritoneal surface.
The presence of Mullerian epithelium in PWs from women with benign gynecologic conditions or with no associated pelvic lesion has been noted previously.[3, 8, 9, 15, 16, 19] In a study by Zuna and Mitchell, such findings (which were referred to as “atypical”) accounted for 18 of 149 benign cases(12.1%). Among their patients, those with non-neoplastic cysts represented the largest percentage of cases (23%). Other conditions included pelvic inflammatory reactions (22.2%) and benign ovarian tumors (12.2%). Although the exact cell origin could not be documented at the time of the study, their illustrated patients point to a Mullerian-type epithelium. Other reports that included associated benign conditions consisted of a single patient with endosalpingiosis[7, 8] or endometriosis. Endosalpingiosis also was detected in 7 of 32 patients undergoing risk-reduction surgery. However, to our knowledge, the current study is the first to document the Mullerian nature of these benign structures in PWs, as documented by PAX8 immunoreactivity.
PAX8 is a nuclear transcription factor that has limited expression in normal and neoplastic tissues in a cell lineage-dependent manner. Recent studies have demonstrated constant, strong, and diffuse PAX8 expression in epithelial cells of the endocervix and endometrium. Benign lesions related to the Mullerian epithelium, such as endometriosis, endosalpingiosis, adenomyosis, parovarian Mullerian embryonic rests, paratubal cysts, and ovarian epithelial inclusions, constantly express PAX8, whereas stromal cells at these sites are uniformly negative for PAX8 expression. In the fallopian tube, only basal and secretory cells, but not ciliated cells, stain positive for PAX8 expression. In the current study, all cell clusters that were thought to morphologically represent Mullerian epithelium had strong and uniformly positive staining for PAX8 expression, thereby confirming their Mullerian origin. Furthermore, the morphologic features and the number of cell clusters paralleled those observed on PAX8 immunostaining; individual cells with positive nuclear staining were rare. Because the staining pattern of PAX8 is maintained in serous, endometrioid, and clear cell carcinoma,[12, 13] it is important not to mistake PAX8-positive benign epithelial cells in PWs for PAX8-positive metastatic tumor cells. Familiarity with the full spectrum of the cytomorphologic features of benign Mullerian epithelium, including metaplasia and hyperplasia, and correlation with the tumor histology are necessary for a correct diagnosis.
The origin of these structures in PWs has been debated for the past half century. Recent studies, however, point to the fallopian tube as an important site of origin of many pelvic serous carcinomas and other epithelial abnormalities.[20-23] In a report by Kurman and colleagues, papillary tubal hyperplasia was frequently associated with atypical proliferative serous tumors in the ovary with implants and also closely resembled not only primary ovarian atypical proliferative serous tumors but also noninvasive epithelial implants and endosalpingiosis. On the basis of their findings, those authors proposed a model for the development of ovarian and extraovarian, low-grade serous proliferations (atypical proliferative serous tumors, noninvasive epithelial implants, and endosalpingiosis) postulating that all of these lesions derive from papillary tubal hyperplasia, which appears to be induced by chronic inflammation. According to the authors, papillary tubal hyperplasia, which is characterized by small, rounded clusters of tubal epithelial cells and small papillae, with or without associated psammoma bodies, can shed and implant tubal epithelium on ovarian and peritoneal surfaces, thereby resulting in a variety of low-grade serous proliferations. On the ovary, the lesion is termed “cortical inclusion cyst,” and, when it involves extraovarian sites, it is termed “endosalpingiosis.” If mutation of KRAS or BRAF occurs in any of these lesions, then an atypical proliferative serous tumor develops.
In a recent review by Mehrad et al, those authors noted that a wide spectrum of epithelial changes in the fallopian tube epithelium, ranging from metaplasia to hyperplasia with or without atypia to carcinoma, is the source of the cells that are shed or implanted on ovarian and peritoneal surfaces and, thus, the reason for the different pathology of proliferative lesions. In general, 4 histologic categories of epithelial changes have been described: 1) metaplasias (including focally accentuated secretory vacuoles, eosinophilic changes, mucinous metaplasia, and endometrioid metaplasia); 2) nonmalignant atypias, including reactive atypia, pregnancy-related change, salpingolysis, and complex epithelial alterations (epithelial pseudoneoplasia associated with salpingitis, adenofibroma, and papillary lesions of the tube); 3) potential precursors, including secretory cell outgrowths and p53 signature, that are recognized morphologically by the presence of linear outgrowths of secretory cells, often appearing more pseudostratified, and are distinct from the surrounding heterogeneous cells; and 4) tubal intraepithelial carcinomas, which are recognized by the presence of conspicuous epithelial stratification and a loss of polarity, a high nuclear:cytoplasmic ratio, and loss of ciliated cells.
Considering the wide spectrum of the aforementioned changes that can be encountered in the fallopian tube epithelium and their accessibility to the peritoneal cavity, at least some of these changes can be anticipated in PWs. A review of the literature, however, indicates that the cytologic counterparts of the aforementioned changes have been described only rarely and mostly as case reports.[3, 9, 19, 24] For the majority of cases (92%) in our current series of 38 patients with benign gynecologic conditions, the cytologic findings in PWs were characteristic of endosalpingiosis and consisted of cohesive cell groups with micropapillary and/or tubular arrangement. The cells were small cuboidal to low columnar with dense cytoplasm and mild-to-moderate atypia. Only 3 cases also exhibited nonclassic features of endosalpingiosis, consistent with displaced fallopian tube epithelium in 2 cases (with associated fallopian tube hyperplasia in 1 case) and endometriosis in the third case.
In addition to the fallopian tube, metaplasia of the coelomic epithelium has been suggested as a source of Mullerian epithelium in PWs,[21, 25] which may explain the PW findings in some of the cases noted here. Again, the limited surgical staging procedure did not allow for further documentation of Mullerian metaplasia of the peritoneal surfaces as a source of these findings.
Recent studies also have demonstrated that current techniques, such as the placement of an intrauterine manipulator for laparoscopic surgery, may cause retrograde dissemination of the lining Mullerian epithelium (endometrial or fallopian), thus contributing to the finding of Mullerian cells in PWs. In 1 study, the incidence of positive PWs in patients with low-risk endometrial cancer who underwent laparoscope-assisted vaginal hysterectomy was higher than that in the control population (10.3% vs 2.8%), suggesting that an intrauterine manipulator may cause retrograde dissemination of cancer cells. In the current study, 13 of 38 patients underwent laparoscopic surgery before PW. The PW findings in these patients were similar to those in other patients who had specimens obtained after laparotomy, consisting of tubular or papillary clusters of small cuboidal cells with psammoma bodies. However, in 2 patients, there were also large epithelial cell groups not typical of endosalpingiosis but, rather, more suggestive of dislodged/displaced fallopian tube epithelium with associated hyperplasia in 1 of the 2 patients. These findings indicate that intrauterine mechanical manipulation during laparoscopy may contribute to the finding of Mullerian epithelium; therefore, careful attention to the surgical method is necessary when evaluating PW specimens.
The rate of detecting benign Mullerian epithelium in PWs in women is not known. In the study by Zuna and Mitchell, “atypical cytologic findings” in PWs from women with benign gynecologic conditions were encountered in 18 of 149 patients (12.1%), and patients with non-neoplastic cysts (including endosalpingiosis) comprised the largest percentage (23%). Other associated benign conditions included benign epithelial-stromal tumors of the ovary (cystadenoma and teratoma), pelvic inflammatory disease, endometriosis, and uterine leiomyoma. In the study by Colgan and colleagues, endosalpingiosis was noted in 7 of 32 PW specimens (21.9%). Such findings in PWs are likely common but probably go undetected unless they become reactive or atypical to the extent that they raise the possibility of an atypical or suspicious diagnosis.
In conclusion, benign Mullerian epithelium may be detected in the PW specimens from women with benign gynecologic conditions. The cytomorphologic features, in the majority of women, are characteristic and, in conjunction with PAX8 positivity, should allow its recognition with a high degree of accuracy. However, in hypercellular specimens and/or in cases with atypical cytologic features (complex architecture or nuclear atypia and nuclear overlap), correlation with the surgical biopsy findings and attention to the intraoperative surgical techniques at the time of PW specimen procurement are needed to ensure accurate diagnosis and, thus, optimal management.
This research is supported in part by The University of Texas MD Anderson Cancer Center Support Grant CA016672.