ORIGINAL ARTICLE
Perivascular Epithelioid Cell Neoplasm (PEComa) of the Gynecologic Tract Clinicopathologic and Immunohistochemical Characterization of 16 Cases John Kenneth Schoolmeester, MD,* Brooke E. Howitt, MD,w Michelle S. Hirsch, MD, PhD,w Paola Dal Cin, PhD,w Bradley J. Quade, MD, PhD,w and Marisa R. Nucci, MDw
Abstract: Perivascular epithelioid cell tumor (PEComa) belongs to a family of tumors characterized by coexpression of melanocytic and muscle markers. Recent studies have shown that sporadic and tuberous sclerosis complex–associated PEComa may respond to mTOR inhibitors underscoring the importance of recognizing this tumor. However, its occurrence in the gynecologic tract continues to be disputed owing to its common misclassification as other types of uterine sarcoma and its controversial relationship with epithelioid smooth muscle tumors. To more fully characterize PEComa of the female genital tract, 16 cases of gynecologic PEComa were identified (1990 to 2012) and formed the basis of this study. Each case was analyzed for conventional morphologic and immunohistochemical characteristics established for PEComa of extrauterine sites; clinical outcome data were obtained for all cases. The 16 patients were aged 28 to 60 (mean 49; median 50) years, and 1 had a history of tuberous sclerosis complex. Thirteen cases were primary of the uterus, 2 of the adnexa, and 1 of the vagina. Tumor size ranged from 0.3 to 25.0 (mean 8.7) cm. Three patients died of disease, 6 were alive with disease, and 7 were alive without evidence of disease at last follow-up (1 mo to 13 y follow-up; mean 26 mo). All patients with an adverse outcome met established criteria for malignancy as proposed for extrauterine sites (ie, 2 or more features present: size Z5 cm, high-grade nuclear features, infiltration, necrosis, lymphovascular invasion, or a mitotic rate Z1/50 high-power fields). Of the melanocytic markers, HMB45 was most commonly expressed (16/16 positive, 100%), followed by microphthalmia transcription factor (11/12 positive, 92%), MelanA (14/16 positive, 88%), and S100 protein (2/10 positive, 20%). Of the smooth muscle markers, desmin was most commonly expressed (15/15 cases, 100%), followed by SMA (14/15 cases, 93%) and h-caldesmon (11/12 cases, 92%). TFE3 im-
From the *Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN; and wDivision of Women’s and Perinatal Pathology, Brigham and Women’s Hospital, Boston, MA. Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article. Correspondence: Marisa R. Nucci, MD, Department of Pathology, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115 (e-mail: [email protected]). Copyright r 2013 by Lippincott Williams & Wilkins
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munopositivity was identified in 5 of 13 cases; however, 3 tested cases were negative for a TFE3 rearrangement by fluorescence in situ hybridization. Current criteria for malignancy appear to be valid in the female genital tract, although modified criteria, as described herein, may be more specific. Awareness of the characteristic features of PEComa is important to help distinguish it from epithelioid smooth muscle tumors and other mimics as PEComa may respond to unique chemotherapeutic regimens. Key Words: PEComa, gynecologic, perivascular epithelioid cell (Am J Surg Pathol 2014;38:176–188)
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erivascular epithelioid cell neoplasm, commonly abbreviated as PEComa, is a rare mesenchymal tumor of perivascular epithelioid cell (PEC) lineage.1 In 1992, the seemingly disparate lesions of angiomyolipoma (AML) and clear cell “sugar” tumor (CCST) of the lung were first noted by Bonetti and colleagues2 to feature an unconventional cell, one that is epithelioid in morphology with clear to eosinophilic cytoplasm, perivascular in orientation, expresses melanocytic and muscle markers, and is what we now regard as the PEC. The inception of the PEC as the cell of origin of PEComa was formally introduced by Zamboni et al3 after characterization of a pancreatic tumor resembling CCST of the lung. Although no normal histologic counterpart has currently been recognized for the PEC,4,5 the unique phenotypic biology of PEComa has led to its identification in soft tissue, viscera, and several other organ sites.6–12 Subsequent studies now include PEComa in a family of tumors linked by its similar morphologic and immunohistochemical (IHC) phenotypes and consist of AML, CCST of the lung, and lymphangioleiomyomatosis (LAM), among others.4,13 Regardless of its name, each member of this family displays a neoplastic proliferation of PECs. PECs are pathologically defined by the World Health Organization (WHO) as peritheliomatous cells of epithelioid or spindled cytology that show myomelanocytic differentiation by IHC.1 Clinically, certain members of the family, largely AML and LAM, are linked to the tuberous sclerosis complex (TSC), with a reduced frequency of disease Am J Surg Pathol
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association in the other familial constituents.4,14–17 The heterogeneity of this family extends beyond their syndromic relationship, with inherent differences in the natural history and histology of each tumor. Perhaps it is because of this pathobiological variation and because Mu¨llerian PEComa appears uncommon with approximately 50 cases reported that its existence in the gynecologic tract is a topic of dispute. Some authors propose that uterine PEComa is not a distinct entity but rather is an epithelioid smooth muscle tumor (ESMT) with melanocytic marker immunopositivity.18–21 This debate stems from the morphologic similarity of PEComa and ESMT coupled with their immunophenotypic overlap. To explore this controversy and better delineate the clinical and pathologic characteristics of PEComa of the female genital tract, this study analyzed the histopathologic features and IHC profile of a series of cases. We applied and evaluated current criteria for malignancy by correlating histopathologic parameters with clinical outcome, as such parameters have yet to be fully established for Mu¨llerian PEComa. In addition, TFE3 rearrangements have been observed in some PEComa and suggest that these lesions may be a separate, if closely related, entity22,23; therefore, in a subset of cases demonstrating notable immunoexpression of TFE3, fluorescence in situ hybridization (FISH) analysis for TFE3 rearrangements was performed. Recent advances in the molecular biology of PEComa are also reviewed, as the consequence in differentiating this tumor from ESMT has significant therapeutic implications. Herein, we present 16 cases of PEComa of gynecologic origin, the largest single series to date.
MATERIALS AND METHODS Case Selection Sixteen cases of gynecologic PEComa were retrieved from the institutional and consultation archives of the Department of Pathology of the Brigham and Women’s Hospital (BWH). A retrospective search for keywords “PEComa” or “Perivascular epithelioid cell tumor” was used to identify material for the study. Each case was rereviewed to confirm its diagnosis according to WHO established definitions of PEComa.1 No cases identified in the search were excluded. Available original 4-mm-thick hematoxylin and eosin (H&E) sections and corresponding IHC performed at BWH were evaluated. When necessary or available, unstained slides or paraffin-embedded tissue blocks were used for additional H&E, IHC, or FISH studies. Clinical history and patient outcomes were obtained from institutional records or the referring pathologist or gynecologist. Gross pathology findings were recorded from institutional and consultation reports. A single consultation case was previously reported.24 Study approval was granted by the Institutional Review Board of BWH.
Morphologic Analysis For each case, H&E slides were assessed for the following histologic features: cytomorphology (epithelioid or spindled, and the estimated percentage of each component), r
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significant nuclear atypia (absence or presence of significant nuclear atypia observable by a 10 objective), degree of cellularity (graded as hypercellular or hypocellular in comparison with a normocellular uterine leiomyoma), tumor border interface (infiltrative, pushing, or permeative/ endometrial stromal sarcoma-like), tumor necrosis (absence or presence with estimated percentage), lymphovascular invasion (absence or presence), stromal hyalinization (absence or presence with estimated percentage), nested growth (absence or presence), tumor cell discohesion (absence or presence), spider cell–like giant cells (absence or presence), multinucleated tumor cells (absence or presence), melanoma-like macronucleoli (absence or presence), hemangiopericytic vessels (absence or presence), intranuclear pseudoinclusions (absence or presence), total mitotic index (counted separately in 10 and 50 high-power fields [HPF]), and atypical mitotic figures (counted separately in 10 and 50 HPF, with respective percentage of total mitotic index).
Immunohistochemistry IHC studies were carried out using an antibody panel directed against the following antigens: HMB45 (clone HMB45; dilution 1:400; no antigen treatment; Dako, Carpinteria, CA), MelanA (clone A103; dilution 1:200; citrate buffer pressure cooker; Dako), microphthalmia transcription factor (MiTF) (clone 34CA5; dilution 1:75; citrate buffer pressure cooker; Leica, Buffalo Grove, IL), S100 protein (polyclonal; dilution 1:500; no antigen treatment; Dako), AE1AE3 (clone AE1AE3; dilution 1:200; 10 min proteinase digestion; Dako), smooth muscle actin (SMA) (clone 1A4; dilution 1:20,000; no antigen treatment; Sigma, St Louis, MO), desmin (clone DEU-10; dilution 1:5000; citrate buffer pressure cooker; Sigma), h-caldesmon (clone h-CD; dilution 1:300; citrate buffer pressure cooker; Dako), PAX8 (polyclonal; dilution 1:400; citrate buffer pressure cooker; Proteintech Group, Chicago, IL), TFE3 (polyclonal; dilution 1:500; citrate buffer pressure cooker; Cell Marque, Rocklin, CA), and cathepsin K (clone 3F9; dilution 1:500; citrate buffer pressure cooker; Abcam, Cambridge, MA). The Envision Plus detection system (Dako) was utilized for all antibodies with positive and negative controls as necessary. Tumor cell immunoreactivity was semiquantitatively graded by percentage of tumor cell staining: 0 (negative); 1+ (1% to 5%); 2+ (6% to 25%); 3+ (26% to 50%); 4+ (>50%). Tumor cell immunoreactivity was also semiqualitatively graded: weak (W), heterogenous (HET), or strong (S). For calculation of IHC totals, a score of 1+ with weak, heterogenous, or strong staining was considered positive for all antibodies except TFE3. A minimum of 3+ was required for TFE3 immunopositivity.
Fluorescence In Situ Hybridization FISH analysis for TFE3 rearrangements was performed in 3 cases demonstrating significant TFE3 immunoreactivity (at least 3+, W or greater). Unstained slides sectioned at 4 mm were subjected to a previously validated FISH assay for TFE3 rearrangement utilizing a break-apart probe strategy.25 Bacterial artificial chromosome www.ajsp.com |
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clone selection was performed using the BACPAC Resources Center at Children’s Hospital Oakland Research Institute (Oakland, CA). The 30 end of TFE3 was detected by RP118A2 (labeled by Spectrum Orange/Red), and the 50 end of TFE3 was detected by RP11-528A24 (labeled by Spectrum Green) (Amersham Inc., Piscataway, NJ). The previously detailed protocol26 was followed for processing, analysis, and interpretation.
Statistics Analysis of morphologic features for statistical significance was made using Fisher exact tests. P values 1/50 HPF (P = 0.011). Infiltration was not statistically significant (P = 1.0).
DISCUSSION The Morphologic and IHC Spectrum of PEComa The term PEComa was first introduced by Zamboni et al in 19963 to describe a family of mesenchymal tumors differentiating toward the PEC, which has distinctive morphologic and IHC features. As originally described, PECs have an epithelioid appearance with clear to eosinophilic and granular cytoplasm and centrally located round to oval nuclei with a nucleolus. The tumor cells typically grow in sheets or nests, and the cells often are intimately associated with a prominent vascular component. It is this characteristic perivascular distribution that led initial observers to speculate that they may originate from the wall of blood vessels. Since this classic descrip-
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tion, histologic variants of PEComa are now known to occur and include tumors with a variable and often prominent spindled tumor cell component (eg, as described in clear cell myelomelanocytic tumor of the falciform ligament) as well as tumors with extensive stromal hyalinization or so-called sclerosing PEComa.27 In comparing the present series to the accepted morphologic spectrum of PEComa, all cases showed typical morphology: clear to granular eosinophilic cytoplasm with a nested growth pattern. The majority of tumors contained a mixture of epithelioid and spindled cells, the ratio of which varied in each case. Three cases had purely epithelioid morphology, but none was composed entirely of spindled cells. The pattern of invasion into surrounding tissues was often multiple, with the majority having destructively infiltrative growth (81%), followed by a pushing border (63%). A significant subset (25%) had a permeative pattern akin to that seen in endometrial stromal sarcoma, a pattern in keeping with what has been previously described r
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FIGURE 2. The cytologic spectrum of gynecologic PEComa. PEComa is typically composed of epithelioid (A) and spindled (B) cells with clear-pink granular cytoplasm and a nested growth pattern accompanied by a delicate vasculature. Other less common features include heavily pigmented cells (C) and clear cells with glycogen-rich–appearing cytoplasm (D). Some PEComas show anaplastic features along with multinucleated tumor giant cells, melanoma-like macronucleoli, and intranuclear inclusions (E). Benign multinucleated tumor cells may also be present (F).
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TABLE 4. IHC Profile of Gynecologic PEComa
Case 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Total (n [%])
HMB45
MelanA
MiTF
S100 Protein
2+, HET 1+, S 1+, S 1+, W 2+, HET 1+, S 4+, HET 2+, S 1+, S 2+, S 2+, HET 1+, W 1+, HET 1+, W 2+, HET 4+, HET 16/16 (100)
1+, HET 1+, S 1+, S 1+, S 0 2+, HET 1+, W 2+, S 0 1+, W 1, HET 1+, W 1+, W 1+, W 1+, HET 4+, S 14/16 (88)
3+, W 2+, W 3+, W 4+, W 4+, W 0 2+, W — 4+, S — — 4+, W 1+, W 4+, HET 3+, HET — 11/12 (92)
0 0 0 0 — 1+, S 0 0 — — — — 1+, W 0 0 — 2/10 (20)
AE1 AE3 0 0 3+, HET 0 — 0 0 — — 0 — — — 0 0 — 1/9 (11)
SMA
Desmin
3+, HET 4+, S 1+, S 3+, S 4+, S 4+, S 2+, S 2+, S 4+, S 4+, S 1+, S 1+, S 4+, S — 0 3+, S 4+, S 4+, S 4+, S 4+, S 1+, S 1+, S — — 4+, S 3+, S 4+, HET 4+, S 1+, H 1+, H 1+, W 1, W 14/15 (93) 15/15 (100)
hCaldesmon
PAX8
1+, W 4+, HET 4+, HET — 3+, S 1+, S 4+, S — 2+, HET 4+ HET 0 — 4+, S 2+, S 1+, W — 11/12 (92)
0 0 0 0 0 0 — — — — — — 0 0 — — 0/9 (0)
TFE3
Cathepsin K
4+, W 2+, HET 2+, W 3+, HET 4+, W 4+, S 0 4+, S 4+, W 4+, HET 0 4+, S 0 4+, S — — 4+, W 4+, S — — 0 — — — 1+, W 4+, HET 3+, HET 2+, S 0 — 0 4+, S 5/13 (38) 11/11 (100)
— indicates not performed. HET indicates heterogenous; S, strong; W, weak.
in PEComa of the uterus.28 Differing amounts of stromal hyalinization were present in two thirds of tumors, and extensive stromal hyalinization, consistent with sclerosing PEComa, was identified in 2. Several tumors also demonstrated features that have been less commonly emphasized in the literature: 2 cases had a prominent hemangiopericytoma-like vascular pattern, and 2 cases contained PECs with glycogenated/lipid-distended cytoplasm1 (Fig. 2D). A single case exhibited heavy melanin pigmentation (Fig. 2C); although published as a case report,24 this consultation case highlights an unusual finding in gynecologic PEComa albeit its morphology resembles recently described melanotic Xp11 neoplasms. The tumor cells often exhibited nuclear features similar to those seen in malignant melanoma, including 12 cases (75%) with multinucleation, 10 (63%) with macronucleoli, and 6 (38%) with nuclear pseudoinclusions. The results of our IHC are in general agreement with published immunophenotypic data. All cases demonstrated expression of both melanocytic and smooth muscle markers. HMB45 was the most sensitive stain and was positive in 100% of cases, followed by MiTF (92%) and MelanA (88%). Cathepsin K has recently been shown to be consistently and strongly positive in PEComa29–32 and was expressed in 100% of tested cases. Cathepsin K also demonstrated more diffuse positivity with staining of >50% of neoplastic cells (8/11 cases; 72% with 4+ positivity), when compared with HMB45 (2/16 cases; 13% 4+ positivity) and MelanA (1/16 cases; 6% 4+ positivity). S100 protein immunopositivity, which has been reported in approximately 30% of PEComa, was less frequent (2 cases; 10%) in our series, was cytoplasmic, and appears to be of limited value.33 For smooth muscle markers, desmin was the most sensitive and was positive in all cases (15/15; 100%); labeling of SMA (14/15; 93%) and h-caldesmon (11/12; 92%) showed nearly similar sensitivity. Our findings are in contrast to those de-
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scribed by Vang and Kempson28; half of their cases (4/8; 50%) showed no desmin labeling, and one third were also negative for SMA (2/6; 33%). In our series, 25% of cases (4/ 16) showed positivity in 50% of cells), which is contrary to the findings of Folpe et al33 in which 5 cases with TFE3 expression showed focal or no MiTF staining, although it is not clear whether these cases corresponded to PEComa of soft tissue or gynecologic origin. Cases in our series with TFE3 labeling also had substantial expression of smooth muscle markers, with all cases showing positivity for at least 1 stain in >50% of cells. This finding diverges from reports of TFE3-rearranged PEComa, which display strong TFE3 immunoreactivity but minimal smooth muscle expression.23 Our data suggest that PEComa with TFE3 immunoexpression that also show substantial reactivity for smooth muscle markers are unlikely to have TFE3 gene rearrangement. r
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PEComa of the Female Genital Tract: Its Controversial Relationship to ESMT and Proposed Criteria for its Recognition as a Distinct Entity The occurrence of PEComa in the gynecologic tract is a dispute that has persisted for many years.18–21,28,34–36 Validation of its existence in the gynecologic tract and which tumors should be classified as PEComa continues to be debated. Some authors propose that gynecologic tumors with morphology and an immunophenotype otherwise diagnostic of PEComa of soft tissue and other sites should be designated within the spectrum of ESMTs as has been done in the past. Their argument for this is threefold. First, changing terminology requires both clinicians and pathologists to revise their diagnostic vernacular at the risk of creating confusion and incorrectly treating patients. However, when a particular diagnosis alters therapeutic options offered to patients, it is our strong belief that the benefit of appropriate treatment far exceeds the unlikely risk of confusing practitioners. Research has elucidated unique therapeutic targets for PEComa and most members of its family as it has a relatively welldefined pathogenesis involving TSC1 or TSC2 mutations and resultant dysregulation of the mTOR signaling pathway.37–41 Several clinical trials aimed at exploring the role of mTOR inhibitors in treatment of members of the PEComa family25,41–44 have shown promise. One trial treated advanced cases of malignant PEComa with sirolimus and found a radiographic response in all patients as assessed by serial imaging studies.45 Another trial evaluated the efficacy of sirolimus in cases of LAM and reported stabilization of pulmonary function as well as reduction of symptoms and improved quality of life.46 The outcomes of these trials underscore the importance of the inclusion of PEComa as a diagnostic entity in gynecologic neoplasms. By distinguishing it from ESMTs, the most effective chemotherapeutic agent is made available to patients. This concept is further supported by the patient in our series (case 15) who showed a dramatic response to mTOR inhibitor therapy after reclassification of her tumor as PEComa. Although this patient recurred, a recent review of mTOR inhibition therapy in PEComa found that patients tended to show either complete resolution or an initially dramatic response, followed by disease progression.43 Second, the PEC’s current lack of a normal histologic counterpart prompts questions of whether it exists independently or is a derivative of a cell of smooth muscle lineage. When Bonetti and colleagues formally introduced the PEC, it was hypothesized to originate from the walls of blood vessels, as it is often seen radiating from vascular structures.2,4,5 Since then, however, additional studies have further characterized the distinctive morphologic, IHC, ultrastructural, and molecular genetic properties of the PEC.1 Morphologically, the PEC is known to demonstrate either an epithelioid or spindled shape, and PEComa may show either pure epithelioid or spindled forms or, more commonly, a mixture of both. In comparison with non-neor
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plastic and neoplastic smooth muscle, the PEC has clear to eosinophilic and granular cytoplasm that differs from the more densely eosinophilic cytoplasm of smooth muscle cells. Moreover, PEComa may also contain cells with glycogen or lipid-distended cytoplasm, a feature that is not characteristic of smooth muscle tumors. Some reports have found HMB45 immunopositivity in leiomyomas and epithelioid leiomyosarcomas.20,47–49 However, there are histologic and IHC features that distinguish PEComa from ESMTs. Multinucleated tumor cells, spider cell–like giant cells, stromal hyalinization, and an arborizing vasculature encasing nests of cells are not typically seen in ESMTs. In our experience, uterine leiomyosarcomas are uncommonly positive for HMB45, and these cases showed only focal expression (4/35 tumors; 11%) but are not positive for MelanA.50 Therefore, although HMB45 expression is classically associated with all types of PEComa, MelanA appears to be a more specific marker for gynecologic PEComa and allows for its distinction from smooth muscle tumors. In addition, ESMTs are often positive for keratins, whereas PEComa rarely shows positivity. The ultrastructure of the PEC comprises premelanosomes, cytoplasmic glycogen, thin filaments, and dense bodies,51–53 which is distinct from smooth muscle tumors. Genetic evidence supporting PEComa as a distinct entity includes cytogenetic findings linking PEComa to TSC. One study found frequent deletions of chromosome 16p in about two thirds of tested tumors.54 Additionally, the authors found the genetic profile of their case of uterine PEComa to be entirely dissimilar to other comparative genomic hybridization studies of uterine smooth muscle tumors. Thus, there is a unique and defined set of histologic, immunophenotypic, ultrastructural and genetic features of PEComa that are sufficient to separate it from smooth muscle tumors. Third, is the issue of definition. The WHO defines PEComa as “mesenchymal tumors composed of distinctive cells that show a focal association with blood vessel walls and usually express melanocytic and smooth muscle markers.”1 However, given the controversy between PEComa and ESMT, we propose that a tumor exhibiting morphologic features of PECs with at least focal IHC expression of 2 melanocytic markers, preferably HMB45 and MelanA, with concurrent expression of at least a single muscle marker such as SMA, desmin, or h-caldesmon should be classified as PEComa. It is noteworthy that 2 cases in this series were positive for HMB45 but negative for MelanA. These cases showed strong and diffuse positivity for cathepsin K, and thus we propose that this strain may be used in cases that lack MelanA to support the diagnosis of PEComa in morphologically compatible cases.
Evaluation of Proposed Criteria for Malignancy Fewer than 50 cases of gynecologic PEComa have been reported in the English-language literature, most as single cases or small series.55–58 As a result, criteria for malignancy have yet to be firmly established. The current classification system33,38 delineates 3 categories for PEComa (extrauterine and uterine): benign, uncertain maligwww.ajsp.com |
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TABLE 5. Classification of PEComa by Clinical Outcome Using Current Criteria
Classification Benign Uncertain malignant potential Malignant
Cases With Known Metastasis Meeting Criteria (n [%])
Definition Tumors showing: 5 cm, infiltrative growth, high-grade nuclear features, necrosis, vascular invasion, or a mitotic index Z1/50 HPF
Cases Without Known Metastasis Meeting Criteria* (n [%])
0/9 (0)
0/7 (0)
0/9 (0)
0/7 (0)
9/9 (100)
4/7 (57)
*3 cases without known metastasis could not be classified using current criteria because of the presence of only a single feature concerning for malignancy that did not qualify it as uncertain for malignant potential (for example: infiltrative growth only or mitotically active only).
nant potential, and malignant. Benign is defined as displaying no concerning gross or histologic features typically associated with malignancy: gross size
Perivascular Epithelioid Cell Neoplasm (PEComa) of the Gynecologic Tract Clinicopathologic and Immunohistochemical Characterization of 16 Cases John Kenneth Schoolmeester, MD,* Brooke E. Howitt, MD,w Michelle S. Hirsch, MD, PhD,w Paola Dal Cin, PhD,w Bradley J. Quade, MD, PhD,w and Marisa R. Nucci, MDw
Abstract: Perivascular epithelioid cell tumor (PEComa) belongs to a family of tumors characterized by coexpression of melanocytic and muscle markers. Recent studies have shown that sporadic and tuberous sclerosis complex–associated PEComa may respond to mTOR inhibitors underscoring the importance of recognizing this tumor. However, its occurrence in the gynecologic tract continues to be disputed owing to its common misclassification as other types of uterine sarcoma and its controversial relationship with epithelioid smooth muscle tumors. To more fully characterize PEComa of the female genital tract, 16 cases of gynecologic PEComa were identified (1990 to 2012) and formed the basis of this study. Each case was analyzed for conventional morphologic and immunohistochemical characteristics established for PEComa of extrauterine sites; clinical outcome data were obtained for all cases. The 16 patients were aged 28 to 60 (mean 49; median 50) years, and 1 had a history of tuberous sclerosis complex. Thirteen cases were primary of the uterus, 2 of the adnexa, and 1 of the vagina. Tumor size ranged from 0.3 to 25.0 (mean 8.7) cm. Three patients died of disease, 6 were alive with disease, and 7 were alive without evidence of disease at last follow-up (1 mo to 13 y follow-up; mean 26 mo). All patients with an adverse outcome met established criteria for malignancy as proposed for extrauterine sites (ie, 2 or more features present: size Z5 cm, high-grade nuclear features, infiltration, necrosis, lymphovascular invasion, or a mitotic rate Z1/50 high-power fields). Of the melanocytic markers, HMB45 was most commonly expressed (16/16 positive, 100%), followed by microphthalmia transcription factor (11/12 positive, 92%), MelanA (14/16 positive, 88%), and S100 protein (2/10 positive, 20%). Of the smooth muscle markers, desmin was most commonly expressed (15/15 cases, 100%), followed by SMA (14/15 cases, 93%) and h-caldesmon (11/12 cases, 92%). TFE3 im-
From the *Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN; and wDivision of Women’s and Perinatal Pathology, Brigham and Women’s Hospital, Boston, MA. Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article. Correspondence: Marisa R. Nucci, MD, Department of Pathology, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115 (e-mail: [email protected]). Copyright r 2013 by Lippincott Williams & Wilkins
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munopositivity was identified in 5 of 13 cases; however, 3 tested cases were negative for a TFE3 rearrangement by fluorescence in situ hybridization. Current criteria for malignancy appear to be valid in the female genital tract, although modified criteria, as described herein, may be more specific. Awareness of the characteristic features of PEComa is important to help distinguish it from epithelioid smooth muscle tumors and other mimics as PEComa may respond to unique chemotherapeutic regimens. Key Words: PEComa, gynecologic, perivascular epithelioid cell (Am J Surg Pathol 2014;38:176–188)
P
erivascular epithelioid cell neoplasm, commonly abbreviated as PEComa, is a rare mesenchymal tumor of perivascular epithelioid cell (PEC) lineage.1 In 1992, the seemingly disparate lesions of angiomyolipoma (AML) and clear cell “sugar” tumor (CCST) of the lung were first noted by Bonetti and colleagues2 to feature an unconventional cell, one that is epithelioid in morphology with clear to eosinophilic cytoplasm, perivascular in orientation, expresses melanocytic and muscle markers, and is what we now regard as the PEC. The inception of the PEC as the cell of origin of PEComa was formally introduced by Zamboni et al3 after characterization of a pancreatic tumor resembling CCST of the lung. Although no normal histologic counterpart has currently been recognized for the PEC,4,5 the unique phenotypic biology of PEComa has led to its identification in soft tissue, viscera, and several other organ sites.6–12 Subsequent studies now include PEComa in a family of tumors linked by its similar morphologic and immunohistochemical (IHC) phenotypes and consist of AML, CCST of the lung, and lymphangioleiomyomatosis (LAM), among others.4,13 Regardless of its name, each member of this family displays a neoplastic proliferation of PECs. PECs are pathologically defined by the World Health Organization (WHO) as peritheliomatous cells of epithelioid or spindled cytology that show myomelanocytic differentiation by IHC.1 Clinically, certain members of the family, largely AML and LAM, are linked to the tuberous sclerosis complex (TSC), with a reduced frequency of disease Am J Surg Pathol
Volume 38, Number 2, February 2014
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Volume 38, Number 2, February 2014
association in the other familial constituents.4,14–17 The heterogeneity of this family extends beyond their syndromic relationship, with inherent differences in the natural history and histology of each tumor. Perhaps it is because of this pathobiological variation and because Mu¨llerian PEComa appears uncommon with approximately 50 cases reported that its existence in the gynecologic tract is a topic of dispute. Some authors propose that uterine PEComa is not a distinct entity but rather is an epithelioid smooth muscle tumor (ESMT) with melanocytic marker immunopositivity.18–21 This debate stems from the morphologic similarity of PEComa and ESMT coupled with their immunophenotypic overlap. To explore this controversy and better delineate the clinical and pathologic characteristics of PEComa of the female genital tract, this study analyzed the histopathologic features and IHC profile of a series of cases. We applied and evaluated current criteria for malignancy by correlating histopathologic parameters with clinical outcome, as such parameters have yet to be fully established for Mu¨llerian PEComa. In addition, TFE3 rearrangements have been observed in some PEComa and suggest that these lesions may be a separate, if closely related, entity22,23; therefore, in a subset of cases demonstrating notable immunoexpression of TFE3, fluorescence in situ hybridization (FISH) analysis for TFE3 rearrangements was performed. Recent advances in the molecular biology of PEComa are also reviewed, as the consequence in differentiating this tumor from ESMT has significant therapeutic implications. Herein, we present 16 cases of PEComa of gynecologic origin, the largest single series to date.
MATERIALS AND METHODS Case Selection Sixteen cases of gynecologic PEComa were retrieved from the institutional and consultation archives of the Department of Pathology of the Brigham and Women’s Hospital (BWH). A retrospective search for keywords “PEComa” or “Perivascular epithelioid cell tumor” was used to identify material for the study. Each case was rereviewed to confirm its diagnosis according to WHO established definitions of PEComa.1 No cases identified in the search were excluded. Available original 4-mm-thick hematoxylin and eosin (H&E) sections and corresponding IHC performed at BWH were evaluated. When necessary or available, unstained slides or paraffin-embedded tissue blocks were used for additional H&E, IHC, or FISH studies. Clinical history and patient outcomes were obtained from institutional records or the referring pathologist or gynecologist. Gross pathology findings were recorded from institutional and consultation reports. A single consultation case was previously reported.24 Study approval was granted by the Institutional Review Board of BWH.
Morphologic Analysis For each case, H&E slides were assessed for the following histologic features: cytomorphology (epithelioid or spindled, and the estimated percentage of each component), r
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significant nuclear atypia (absence or presence of significant nuclear atypia observable by a 10 objective), degree of cellularity (graded as hypercellular or hypocellular in comparison with a normocellular uterine leiomyoma), tumor border interface (infiltrative, pushing, or permeative/ endometrial stromal sarcoma-like), tumor necrosis (absence or presence with estimated percentage), lymphovascular invasion (absence or presence), stromal hyalinization (absence or presence with estimated percentage), nested growth (absence or presence), tumor cell discohesion (absence or presence), spider cell–like giant cells (absence or presence), multinucleated tumor cells (absence or presence), melanoma-like macronucleoli (absence or presence), hemangiopericytic vessels (absence or presence), intranuclear pseudoinclusions (absence or presence), total mitotic index (counted separately in 10 and 50 high-power fields [HPF]), and atypical mitotic figures (counted separately in 10 and 50 HPF, with respective percentage of total mitotic index).
Immunohistochemistry IHC studies were carried out using an antibody panel directed against the following antigens: HMB45 (clone HMB45; dilution 1:400; no antigen treatment; Dako, Carpinteria, CA), MelanA (clone A103; dilution 1:200; citrate buffer pressure cooker; Dako), microphthalmia transcription factor (MiTF) (clone 34CA5; dilution 1:75; citrate buffer pressure cooker; Leica, Buffalo Grove, IL), S100 protein (polyclonal; dilution 1:500; no antigen treatment; Dako), AE1AE3 (clone AE1AE3; dilution 1:200; 10 min proteinase digestion; Dako), smooth muscle actin (SMA) (clone 1A4; dilution 1:20,000; no antigen treatment; Sigma, St Louis, MO), desmin (clone DEU-10; dilution 1:5000; citrate buffer pressure cooker; Sigma), h-caldesmon (clone h-CD; dilution 1:300; citrate buffer pressure cooker; Dako), PAX8 (polyclonal; dilution 1:400; citrate buffer pressure cooker; Proteintech Group, Chicago, IL), TFE3 (polyclonal; dilution 1:500; citrate buffer pressure cooker; Cell Marque, Rocklin, CA), and cathepsin K (clone 3F9; dilution 1:500; citrate buffer pressure cooker; Abcam, Cambridge, MA). The Envision Plus detection system (Dako) was utilized for all antibodies with positive and negative controls as necessary. Tumor cell immunoreactivity was semiquantitatively graded by percentage of tumor cell staining: 0 (negative); 1+ (1% to 5%); 2+ (6% to 25%); 3+ (26% to 50%); 4+ (>50%). Tumor cell immunoreactivity was also semiqualitatively graded: weak (W), heterogenous (HET), or strong (S). For calculation of IHC totals, a score of 1+ with weak, heterogenous, or strong staining was considered positive for all antibodies except TFE3. A minimum of 3+ was required for TFE3 immunopositivity.
Fluorescence In Situ Hybridization FISH analysis for TFE3 rearrangements was performed in 3 cases demonstrating significant TFE3 immunoreactivity (at least 3+, W or greater). Unstained slides sectioned at 4 mm were subjected to a previously validated FISH assay for TFE3 rearrangement utilizing a break-apart probe strategy.25 Bacterial artificial chromosome www.ajsp.com |
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clone selection was performed using the BACPAC Resources Center at Children’s Hospital Oakland Research Institute (Oakland, CA). The 30 end of TFE3 was detected by RP118A2 (labeled by Spectrum Orange/Red), and the 50 end of TFE3 was detected by RP11-528A24 (labeled by Spectrum Green) (Amersham Inc., Piscataway, NJ). The previously detailed protocol26 was followed for processing, analysis, and interpretation.
Statistics Analysis of morphologic features for statistical significance was made using Fisher exact tests. P values 1/50 HPF (P = 0.011). Infiltration was not statistically significant (P = 1.0).
DISCUSSION The Morphologic and IHC Spectrum of PEComa The term PEComa was first introduced by Zamboni et al in 19963 to describe a family of mesenchymal tumors differentiating toward the PEC, which has distinctive morphologic and IHC features. As originally described, PECs have an epithelioid appearance with clear to eosinophilic and granular cytoplasm and centrally located round to oval nuclei with a nucleolus. The tumor cells typically grow in sheets or nests, and the cells often are intimately associated with a prominent vascular component. It is this characteristic perivascular distribution that led initial observers to speculate that they may originate from the wall of blood vessels. Since this classic descrip-
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tion, histologic variants of PEComa are now known to occur and include tumors with a variable and often prominent spindled tumor cell component (eg, as described in clear cell myelomelanocytic tumor of the falciform ligament) as well as tumors with extensive stromal hyalinization or so-called sclerosing PEComa.27 In comparing the present series to the accepted morphologic spectrum of PEComa, all cases showed typical morphology: clear to granular eosinophilic cytoplasm with a nested growth pattern. The majority of tumors contained a mixture of epithelioid and spindled cells, the ratio of which varied in each case. Three cases had purely epithelioid morphology, but none was composed entirely of spindled cells. The pattern of invasion into surrounding tissues was often multiple, with the majority having destructively infiltrative growth (81%), followed by a pushing border (63%). A significant subset (25%) had a permeative pattern akin to that seen in endometrial stromal sarcoma, a pattern in keeping with what has been previously described r
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FIGURE 2. The cytologic spectrum of gynecologic PEComa. PEComa is typically composed of epithelioid (A) and spindled (B) cells with clear-pink granular cytoplasm and a nested growth pattern accompanied by a delicate vasculature. Other less common features include heavily pigmented cells (C) and clear cells with glycogen-rich–appearing cytoplasm (D). Some PEComas show anaplastic features along with multinucleated tumor giant cells, melanoma-like macronucleoli, and intranuclear inclusions (E). Benign multinucleated tumor cells may also be present (F).
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TABLE 4. IHC Profile of Gynecologic PEComa
Case 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Total (n [%])
HMB45
MelanA
MiTF
S100 Protein
2+, HET 1+, S 1+, S 1+, W 2+, HET 1+, S 4+, HET 2+, S 1+, S 2+, S 2+, HET 1+, W 1+, HET 1+, W 2+, HET 4+, HET 16/16 (100)
1+, HET 1+, S 1+, S 1+, S 0 2+, HET 1+, W 2+, S 0 1+, W 1, HET 1+, W 1+, W 1+, W 1+, HET 4+, S 14/16 (88)
3+, W 2+, W 3+, W 4+, W 4+, W 0 2+, W — 4+, S — — 4+, W 1+, W 4+, HET 3+, HET — 11/12 (92)
0 0 0 0 — 1+, S 0 0 — — — — 1+, W 0 0 — 2/10 (20)
AE1 AE3 0 0 3+, HET 0 — 0 0 — — 0 — — — 0 0 — 1/9 (11)
SMA
Desmin
3+, HET 4+, S 1+, S 3+, S 4+, S 4+, S 2+, S 2+, S 4+, S 4+, S 1+, S 1+, S 4+, S — 0 3+, S 4+, S 4+, S 4+, S 4+, S 1+, S 1+, S — — 4+, S 3+, S 4+, HET 4+, S 1+, H 1+, H 1+, W 1, W 14/15 (93) 15/15 (100)
hCaldesmon
PAX8
1+, W 4+, HET 4+, HET — 3+, S 1+, S 4+, S — 2+, HET 4+ HET 0 — 4+, S 2+, S 1+, W — 11/12 (92)
0 0 0 0 0 0 — — — — — — 0 0 — — 0/9 (0)
TFE3
Cathepsin K
4+, W 2+, HET 2+, W 3+, HET 4+, W 4+, S 0 4+, S 4+, W 4+, HET 0 4+, S 0 4+, S — — 4+, W 4+, S — — 0 — — — 1+, W 4+, HET 3+, HET 2+, S 0 — 0 4+, S 5/13 (38) 11/11 (100)
— indicates not performed. HET indicates heterogenous; S, strong; W, weak.
in PEComa of the uterus.28 Differing amounts of stromal hyalinization were present in two thirds of tumors, and extensive stromal hyalinization, consistent with sclerosing PEComa, was identified in 2. Several tumors also demonstrated features that have been less commonly emphasized in the literature: 2 cases had a prominent hemangiopericytoma-like vascular pattern, and 2 cases contained PECs with glycogenated/lipid-distended cytoplasm1 (Fig. 2D). A single case exhibited heavy melanin pigmentation (Fig. 2C); although published as a case report,24 this consultation case highlights an unusual finding in gynecologic PEComa albeit its morphology resembles recently described melanotic Xp11 neoplasms. The tumor cells often exhibited nuclear features similar to those seen in malignant melanoma, including 12 cases (75%) with multinucleation, 10 (63%) with macronucleoli, and 6 (38%) with nuclear pseudoinclusions. The results of our IHC are in general agreement with published immunophenotypic data. All cases demonstrated expression of both melanocytic and smooth muscle markers. HMB45 was the most sensitive stain and was positive in 100% of cases, followed by MiTF (92%) and MelanA (88%). Cathepsin K has recently been shown to be consistently and strongly positive in PEComa29–32 and was expressed in 100% of tested cases. Cathepsin K also demonstrated more diffuse positivity with staining of >50% of neoplastic cells (8/11 cases; 72% with 4+ positivity), when compared with HMB45 (2/16 cases; 13% 4+ positivity) and MelanA (1/16 cases; 6% 4+ positivity). S100 protein immunopositivity, which has been reported in approximately 30% of PEComa, was less frequent (2 cases; 10%) in our series, was cytoplasmic, and appears to be of limited value.33 For smooth muscle markers, desmin was the most sensitive and was positive in all cases (15/15; 100%); labeling of SMA (14/15; 93%) and h-caldesmon (11/12; 92%) showed nearly similar sensitivity. Our findings are in contrast to those de-
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scribed by Vang and Kempson28; half of their cases (4/8; 50%) showed no desmin labeling, and one third were also negative for SMA (2/6; 33%). In our series, 25% of cases (4/ 16) showed positivity in 50% of cells), which is contrary to the findings of Folpe et al33 in which 5 cases with TFE3 expression showed focal or no MiTF staining, although it is not clear whether these cases corresponded to PEComa of soft tissue or gynecologic origin. Cases in our series with TFE3 labeling also had substantial expression of smooth muscle markers, with all cases showing positivity for at least 1 stain in >50% of cells. This finding diverges from reports of TFE3-rearranged PEComa, which display strong TFE3 immunoreactivity but minimal smooth muscle expression.23 Our data suggest that PEComa with TFE3 immunoexpression that also show substantial reactivity for smooth muscle markers are unlikely to have TFE3 gene rearrangement. r
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PEComa of the Female Genital Tract: Its Controversial Relationship to ESMT and Proposed Criteria for its Recognition as a Distinct Entity The occurrence of PEComa in the gynecologic tract is a dispute that has persisted for many years.18–21,28,34–36 Validation of its existence in the gynecologic tract and which tumors should be classified as PEComa continues to be debated. Some authors propose that gynecologic tumors with morphology and an immunophenotype otherwise diagnostic of PEComa of soft tissue and other sites should be designated within the spectrum of ESMTs as has been done in the past. Their argument for this is threefold. First, changing terminology requires both clinicians and pathologists to revise their diagnostic vernacular at the risk of creating confusion and incorrectly treating patients. However, when a particular diagnosis alters therapeutic options offered to patients, it is our strong belief that the benefit of appropriate treatment far exceeds the unlikely risk of confusing practitioners. Research has elucidated unique therapeutic targets for PEComa and most members of its family as it has a relatively welldefined pathogenesis involving TSC1 or TSC2 mutations and resultant dysregulation of the mTOR signaling pathway.37–41 Several clinical trials aimed at exploring the role of mTOR inhibitors in treatment of members of the PEComa family25,41–44 have shown promise. One trial treated advanced cases of malignant PEComa with sirolimus and found a radiographic response in all patients as assessed by serial imaging studies.45 Another trial evaluated the efficacy of sirolimus in cases of LAM and reported stabilization of pulmonary function as well as reduction of symptoms and improved quality of life.46 The outcomes of these trials underscore the importance of the inclusion of PEComa as a diagnostic entity in gynecologic neoplasms. By distinguishing it from ESMTs, the most effective chemotherapeutic agent is made available to patients. This concept is further supported by the patient in our series (case 15) who showed a dramatic response to mTOR inhibitor therapy after reclassification of her tumor as PEComa. Although this patient recurred, a recent review of mTOR inhibition therapy in PEComa found that patients tended to show either complete resolution or an initially dramatic response, followed by disease progression.43 Second, the PEC’s current lack of a normal histologic counterpart prompts questions of whether it exists independently or is a derivative of a cell of smooth muscle lineage. When Bonetti and colleagues formally introduced the PEC, it was hypothesized to originate from the walls of blood vessels, as it is often seen radiating from vascular structures.2,4,5 Since then, however, additional studies have further characterized the distinctive morphologic, IHC, ultrastructural, and molecular genetic properties of the PEC.1 Morphologically, the PEC is known to demonstrate either an epithelioid or spindled shape, and PEComa may show either pure epithelioid or spindled forms or, more commonly, a mixture of both. In comparison with non-neor
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plastic and neoplastic smooth muscle, the PEC has clear to eosinophilic and granular cytoplasm that differs from the more densely eosinophilic cytoplasm of smooth muscle cells. Moreover, PEComa may also contain cells with glycogen or lipid-distended cytoplasm, a feature that is not characteristic of smooth muscle tumors. Some reports have found HMB45 immunopositivity in leiomyomas and epithelioid leiomyosarcomas.20,47–49 However, there are histologic and IHC features that distinguish PEComa from ESMTs. Multinucleated tumor cells, spider cell–like giant cells, stromal hyalinization, and an arborizing vasculature encasing nests of cells are not typically seen in ESMTs. In our experience, uterine leiomyosarcomas are uncommonly positive for HMB45, and these cases showed only focal expression (4/35 tumors; 11%) but are not positive for MelanA.50 Therefore, although HMB45 expression is classically associated with all types of PEComa, MelanA appears to be a more specific marker for gynecologic PEComa and allows for its distinction from smooth muscle tumors. In addition, ESMTs are often positive for keratins, whereas PEComa rarely shows positivity. The ultrastructure of the PEC comprises premelanosomes, cytoplasmic glycogen, thin filaments, and dense bodies,51–53 which is distinct from smooth muscle tumors. Genetic evidence supporting PEComa as a distinct entity includes cytogenetic findings linking PEComa to TSC. One study found frequent deletions of chromosome 16p in about two thirds of tested tumors.54 Additionally, the authors found the genetic profile of their case of uterine PEComa to be entirely dissimilar to other comparative genomic hybridization studies of uterine smooth muscle tumors. Thus, there is a unique and defined set of histologic, immunophenotypic, ultrastructural and genetic features of PEComa that are sufficient to separate it from smooth muscle tumors. Third, is the issue of definition. The WHO defines PEComa as “mesenchymal tumors composed of distinctive cells that show a focal association with blood vessel walls and usually express melanocytic and smooth muscle markers.”1 However, given the controversy between PEComa and ESMT, we propose that a tumor exhibiting morphologic features of PECs with at least focal IHC expression of 2 melanocytic markers, preferably HMB45 and MelanA, with concurrent expression of at least a single muscle marker such as SMA, desmin, or h-caldesmon should be classified as PEComa. It is noteworthy that 2 cases in this series were positive for HMB45 but negative for MelanA. These cases showed strong and diffuse positivity for cathepsin K, and thus we propose that this strain may be used in cases that lack MelanA to support the diagnosis of PEComa in morphologically compatible cases.
Evaluation of Proposed Criteria for Malignancy Fewer than 50 cases of gynecologic PEComa have been reported in the English-language literature, most as single cases or small series.55–58 As a result, criteria for malignancy have yet to be firmly established. The current classification system33,38 delineates 3 categories for PEComa (extrauterine and uterine): benign, uncertain maligwww.ajsp.com |
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TABLE 5. Classification of PEComa by Clinical Outcome Using Current Criteria
Classification Benign Uncertain malignant potential Malignant
Cases With Known Metastasis Meeting Criteria (n [%])
Definition Tumors showing: 5 cm, infiltrative growth, high-grade nuclear features, necrosis, vascular invasion, or a mitotic index Z1/50 HPF
Cases Without Known Metastasis Meeting Criteria* (n [%])
0/9 (0)
0/7 (0)
0/9 (0)
0/7 (0)
9/9 (100)
4/7 (57)
*3 cases without known metastasis could not be classified using current criteria because of the presence of only a single feature concerning for malignancy that did not qualify it as uncertain for malignant potential (for example: infiltrative growth only or mitotically active only).
nant potential, and malignant. Benign is defined as displaying no concerning gross or histologic features typically associated with malignancy: gross size
