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Trophogram, an immunohistochemistry-based algorithmic approach, in the differential diagnosis of trophoblastic tumors and tumorlike lesions
Annals of Diagnostic Pathology 11 (2007) 228 – 234
Review Article
Trophogram, an immunohistochemistry-based algorithmic approach, in the differential diagnosis of trophoblastic tumors and tumorlike lesions Ie-Ming Shih Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA
Abstract
Keywords:
Recent histopathologic and molecular studies of trophoblastic cells in the normal placenta and in a variety of trophoblastic diseases have revealed that the latter recapitulate the differentiation of normal trophoblast in the early developing placenta. This new knowledge, especially the identification and characterization of the protein markers expressed in human trophoblast, not only helps elucidate the pathogenesis of trophoblastic lesions but also provides a repertoire of immunohistochemical markers that may facilitate the diagnosis of various trophoblastic diseases. This article reviews the recent advances in the trophoblast-associated markers that have been reported to be useful in the differential diagnosis of trophoblastic tumors and tumorlike lesions. Moreover, a “trophogram,” which is a stepwise and rational immunohistochemistry-based approach, will be introduced. The trophogram may prove to be useful in assisting the differential diagnosis of various trophoblastic diseases in diagnostic pathology. © 2007 Elsevier Inc. All rights reserved. Protein markers; Trophoblast; Trophogram
1. Introduction Gestational trophoblastic disease can be broadly divided into 3 groups: hydatidiform moles, which represent abnormally formed placentas, benign tumorlike lesions, and true trophoblastic neoplasms (Table 1). In recent years, progress has been made in elucidating the biology of human trophoblast. The identification and characterization of the genes expressed in human trophoblast has led to a further understanding of the lineage and the differentiation program of trophoblast and related this to trophoblastic diseases. It has become clear that trophoblastic lesions recapitulate the trophoblast present in the early developing placenta and implantation site. In human placentas, cytotrophoblast is the germinative or “stem cell” component that differentiates into either syncytiotrophoblast, which functions as a hormone and pseudovascular organ, or
This review article was presented in the Surgical Pathology Update at Leipzig, Germany on June 14, 2007. E-mail address: [email protected]. 1092–9134/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.anndiagpath.2007.04.001
intermediate trophoblastic (IT) cells. The IT cells that infiltrate the decidua, myometrium, and spiral arteries of the placental site are termed implantation site IT, and those in the chorion laeve in the fetal membranes are termed chorionic-type IT. The recent research findings suggest that after neoplastic transformation of trophoblastic stem cells, presumably cytotrophoblast, specific differentiation programs determine the type of trophoblastic tumor that will develop [1-4]. Accordingly, choriocarcinoma is composed of variable amounts of neoplastic cytotrophoblast, syncytiotrophoblast, and intermediate trophoblast and resembles the previllous blastocyst, which is composed of a similar mixture of trophoblastic populations. In contrast, the neoplastic cytotrophoblastic cells in placental site trophoblastic tumors (PSTTs) differentiate mainly into implantation site IT cells, whereas the neoplastic trophoblastic cells in epithelioid trophoblastic tumors (ETTs) differentiate into chorionic-type IT cells. Therefore, choriocarcinoma is the most primitive trophoblastic tumor, whereas PSTT and ETT are relatively more differentiated. Clinically, distinguishing trophoblastic tumors from other types of neoplastic disease is important as far as the treatment
I.-M. Shih / Annals of Diagnostic Pathology 11 (2007) 228–234 Table 1 Classification of gestational trophoblastic diseases Hydatidiform moles (abnormally formed placentas) Complete mole Partial mole Invasive mole Trophoblastic tumorlike lesions (benign lesions) Exaggerated placental site/reaction Placental site nodule Trophoblastic tumors (neoplastic diseases) PSTT ETT
plans are concerned [5]. Furthermore, diagnosis of different types of trophoblastic tumors vs tumorlike lesions is critical, as the former may likely involve surgical intervention and/or chemotherapy. However, the differential diagnosis can be difficult based on morphology alone. As a result, application of immunohistochemistry on trophoblast-associated markers may be very useful in difficult cases. Therefore, it is critical to review the trophoblast-associated markers from the literature and to generate an algorithm that allows pathologists to conveniently apply commercially available antibodies in a stepwise fashion to assist differential diagnosis of trophoblastic diseases. This review article briefly highlights the clinicopathologic features of trophoblastic tumors and tumorlike lesions and summarizes the recent advance in studying trophoblast-associated markers. Finally, a “trophogram,” which is a stepwise immunohistochemistry-based approach by combining those markers, will be introduced. It is expected that the trophogram may prove to be useful in assisting the differential diagnosis of various trophoblastic tumors and tumorlike lesions. 2. Clinicopathologic features of trophoblastic tumors and tumorlike lesions 2.1. Choriocarcinoma Gestational choriocarcinoma is a highly malignant epithelial tumor that can be associated with any type of gestational event, most often with a complete hydatidiform mole [5]. Choriocarcinoma may arise from neoplastic transformation of cytotrophoblast and recapitulates the differentiation of cytotrophoblastic cells in early gestation [6]. Neoplastic cytotrophoblast, like its normal counterpart, retains its full capacity to differentiate into syncytiotrophoblast and intermediate trophoblast. As a result, an intimate mixture of cytotrophoblast, intermediate trophoblast, and syncytiotrophoblast characterizes choriocarcinoma. At lower magnification, the cytotrophoblast and IT cells tend to grow in clusters and sheets, separated by syncytiotrophoblast, forming the characteristic dimorphic growth pattern of mononucleate trophoblast and syncytiotrophoblast. The diagnosis of a choriocarcinoma is usually straightforward given its unique histopathologic pattern and the soaring levels of serum β-hCG. However, a choriocarcinoma can be
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confused with other types of carcinoma and trophoblastic neoplasms, especially for those cases with attenuated syncytiotrophoblastic components. 2.2. Placental site trophoblastic tumor Placental site trophoblastic tumor is a relatively uncommon tumor and is composed of neoplastic implantation site IT cells [7-10]. In contrast to the normal implantation site in which invasion of intermediate trophoblast is tightly regulated and is confined to the inner third of the myometrium, the tumor cells of PSTT are highly invasive and infiltrate deeply into the myometrium, occasionally penetrating through the uterine wall. Microscopically, PSTT resembles the trophoblastic infiltration of the endometrium and myometrium of the placental site during early pregnancy but tends to form confluent masses or sheets with enlarged and highly atypical nuclei. Mitotic figures may vary from case to case but generally are very few (b5 mitoses per 10 high-power fields). Although most patients with their tumors confined to the uterus are curable after tumor resection or hysterectomy, the clinical behavior of PSTTs can be unpredictable in some cases, and tumor recurrence and metastasis have been reported several years after initial treatment [11,12]. 2.3. Epithelioid trophoblastic tumor Epithelioid trophoblastic tumor is another unusual type of trophoblastic neoplasm that is distinct from PSTT and choriocarcinoma, with morphological features resembling a carcinoma [4]. The tumors are composed of chorionic-type IT cells based on both morphological and immunohistochemical studies [4]. Microscopically, ETTs can assume several growth patterns including nodules, solid sheets, masses, and infiltrating nests. Geographic tumor cell necrosis and/or focal calcification are frequently observed. The tumor nests in an ETT are frequently accompanied by eosinophilic fibrinoid material, giving the tumor an appearance similar to a keratinizing squamous carcinoma. Because placental site nodules (see section below) are also composed of chorionictype intermediate trophoblast, it has been hypothesized that some placental site nodules may represent an intermediate stage in tumor progression to ETTs. This view is supported by observations that some proliferative placental site nodules with slightly greater cytologic atypia tentatively classified as atypical placental site nodules have features intermediate between typical placental site nodules and ETTs. Moreover, in some cases there is an intimate association of an ETT with a placental site nodule. Further molecular genetic studies are necessary to confirm this hypothesis. The clinical behavior of an ETT is similar to that of a PSTT. Most ETTs behave in a benign fashion, but malignant ETTs with local invasion and distant metastasis can also occur. 2.4. Placental site nodule Placental site nodules are small, well-circumscribed nodular aggregates of chorionic-type IT cells that are
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embedded in a hyalinized stroma. Placental site nodules have been thought to represent a portion of uninvoluted placental site from a remote gestation. However, the constituent cells in placental site nodules are morphologically more closely related to the intermediate trophoblast of the chorion laeve (chorionic-type intermediate trophoblast) than to the intermediate trophoblast of the placental site [3]. In addition, the trophoblastic cells in the placental site nodule exhibit an immunophenotype similar to that of trophoblastic cells in the chorion laeve but distinct from implantation site IT cells. It remains a mystery as to how these cells (from chorion leave) are retained and survive in the uterus after delivery. Placental site nodules are usually incidental findings in cervical biopsy and endocervical or endometrial curettage specimens. Placental site nodules are benign lesions, and no further clinical management is required once the diagnosis is established. 2.5. Exaggerated placental site Exaggerated placental site (EPS) is a benign nonneoplastic lesion characterized by an exuberant number of implantation site IT cells that extensively infiltrate the endometrium and underlying myometrium [2]. The EPS can occur in a normal pregnancy, an abortion from the first trimester, or a molar pregnancy, especially a complete mole. The trophoblastic cells in an EPS display an identical morphological and immunophenotypic profile to the implantation site IT cells in the normal placental site [10]. Despite the extensive infiltration of IT cells in an EPS, the Ki-67 indices of IT cells are near zero, suggesting that the increased number of IT cells in EPS is probably not the result of de novo proliferation of intermediate trophoblast in the implantation site [10,13]. Rather, it has been thought that EPS represents a physiologic presentation in the placental bed, especially from an early gestation. Therefore, there is no clinical significance associated with EPS.
3. Trophoblast-associated markers In the past decades, studies on the trophoblastassociated markers have provided fundamental insights into the biology of human trophoblastic cells during implantation and placentation. Furthermore, several of these proteins, especially for those that can be detected by commercially available antibodies, have emerged as new diagnostic markers for trophoblastic tumors and tumorlike lesions. Some of the markers are expressed in several types of trophoblastic subpopulations, whereas others are specifically expressed in certain trophoblastic subpopulations. The following discussion summarizes those markers and their relevance to diagnostic pathology (Table 2).
Table 2 Markers for trophogram and antibody sources Marker
Application
Antibody source
HSD3B p63 hPL β-hCG Cyclin E Ki-67
Distinguish GTD vs non-GTD Distinguish ETT vs PSTT Distinguish PSTT vs ETT Highlight ST in choriocarcinoma Distinguish ETT vs PSN Distinguish ETT vs PSN; PSTT vs EPS
Abnova (3C11-D4 a) Neomarker (4A4) Dako (polyclonal) Dako (polyclonal) Zymed (HE12) Dako (MIB-1)
GTD indicates gestational trophoblastic disease; PSN, placental site nodule; ST, syncytiotrophoblast. a Clone name for the monoclonal antibody.
3.1. Hydroxyl-δ-5-steroid dehydrogenase— a pan-trophoblastic marker Hydroxyl-δ-5-steroid dehydrogenase (HSD3B1) is an enzyme involved in steroid hormone synthesis by catalyzing the oxidative conversion of δ-5-3 β-hydroxy steroids to the δ-4-3-keto configuration [14]. Using a commercially available anti-HSD3B1 monoclonal antibody on paraffin sections, Mao et al [15] have demonstrated intense HSD3B1 immunoreactivity in implantation site and chorionic-type IT cells as well as in syncytiotrophoblast in all cases of early placentas and complete hydatidiform moles. More importantly, HSD3B1 immunoreactivity was detected in all trophoblastic tumors including PSTTs, ETTs and choriocarcinomas, and in all tumorlike lesions including placental site nodules and EPSs. Hydroxyl-δ-5-steroid dehydrogenase immunoreactivity was always diffuse and intense in most trophoblastic tumors and tumorlike lesions, with an exception in a few choriocarcinomas that are predominantly composed of cytotrophoblastlike tumor cells. In contrast, only 3 (b1%) of 323 nontrophoblastic carcinomas from the uterus, lung, and breast react with the HSD3B1 antibody. Moreover, the immunoreactivity in the positive nontrophoblastic cases is focal and weak and thus is in contrast to the diffuse and intense immunostaining pattern observed in trophoblastic tumors and tumorlike lesions. The specificity of HSD3B1 expression in trophoblastic tissues was further supported by analyzing the gene expression profile of HSD3B1 in silica using the serial analysis of gene expression database deposited in public domain. In that study, HSD3B1 was not expressed in any of 159 libraries of breast, lung, colorectal, pancreatic, ovarian carcinomas, and a wide variety of normal adult and fetal tissues [15]. The results from the above studies strongly suggest that compared with other trophoblastic markers, HSD3B1 could be a specific and sensitive marker being expressed in all types of trophoblastic tumors and tumorlike lesions but rarely in a variety of carcinomas that have been examined. 3.2. p63—a trophoblastic marker for chorionic-type IT differentiation p63 is a transcription factor belonging to the p53 gene family. [16,17] It is localized on chromosome 3q27-29 and
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has a strong homology to p53 in nucleotide sequence and biologic function. p63 isoforms are present as a result of differential promoter usage [18,19]. The transcription activation (TA) isoform uses an upstream (the most 5′) promoter, generating p63 proteins with the TA domain, which binds to p53-consensus sequences and induces p53-target genes. In contrast, transcription from the downstream (3′) promoter results in a smaller p63 isoform that lacks the TA domain and is referred to as ΔN isoform (Np63). The TA and the ΔN isoforms regulate distinct sets of downstream genes and have distinct biologic functions [9,22]. Specifically, the TA isoforms have a p53-like tumor suppressor function, whereas the Np63 isoforms exert an oncogenic effect. A previous study has demonstrated that different trophoblastic subpopulations are characterized by unique expression patterns of p63 isoforms. Cytotrophoblast expresses the ΔN isoforms, whereas the chorionic-type IT cells in fetal membrane (chorion laeve) express the TA isoforms. Implantation site intermediate trophoblast and syncytiotrophoblast do not express either of the p63 isoforms. In trophoblastic lesions, expression of TAp63 is detected in placental site nodules and ETTs but not in EPS or PSTTs. Thus, TAp63 is unique among known trophoblastic markers in that it is diffusely expressed in ETTs but not in PSTTs, serving as a marker for their differential diagnosis. When p63 immunoreactivity is considered for diagnostic purpose, an appropriate antibody should be used to detect the TA isoform rather than the ΔN isoform only. The 4A4 antibody, which reacts with both TA and ΔNp63 isoforms, can be ideal. 3.3. Human placental lactogen—a trophoblastic marker for implantation site IT differentiation Human placental lactogen (hPL) is a pregnancy-related hormone that is normally synthesized by syncytiotrophoblast and IT cells located at the implantation sites. As expected, among the trophoblastic tumors and tumorlike lesions, hPL is expressed in most of the trophoblastic cells of PSTTs and EPSs [20] but in only a very small percentage (b5%) of trophoblastic cells in ETTs and placental site nodules [2,4]. This expression pattern indicates the value by applying immunohistochemistry of this marker in distinguishing lesions that are related to implantation site IT cells (PSTT and EPS) from those that are related to IT cells in chorion laeve (ETT and placental site nodule). Unlike other IT markers such as HSD3B1, HLA-G, and Mel-CAM (CD146), which are expressed in nearly all PSTTs, the percentage of hPL-positive cells in PSTT can vary from case to case. Among neoplastic diseases, hPL appears to be highly specific to PSTT, as other human tumors rarely express this hormone except in some mixed germ cell tumors with trophoblastic differentiation. 3.4. Human chorionic gonadotropin— a syncytiotrophoblastic marker Human chorionic gonadotropin (hCG) is the first and probably the most well-known trophoblastic marker that
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plays a critical role in maintaining the placental function during pregnancy. It is synthesized and secreted predominantly by syncytiotrophoblast in placentas throughout the course of gestation. Further studies have demonstrated that molecular composition of hCG is different between normal pregnancy and gestational trophoblastic tumors. The former produces intact, normally glycosylated hCG, whereas the latter secretes a wide range of hCG variants that may not be detected by conventional assays. Human chorionic gonadotropin is composed of α and β subunits. Because the β subunit is relatively specific to hCG, antibodies that react to this subunit have been routinely used in diagnostic pathology. In fact, the β-hCG has become an ideal marker to demonstrate the syncytiotrophoblastic component in cases suspicious for a choriocarcinoma in which the syncytiotrophoblast within the tumor is inconspicuous based on hematoxylin-eosin staining. In PSTT and ETT, β-hCG– positive cells are extremely rare, and if any, they are limited to the multinucleated IT cells. 3.5. Ki-67—a proliferation marker Ki-67 antigen is the most frequently used immunostaining marker in diagnostic pathology to assess cell proliferation activity in tissues [21]. It was first identified by an antibody approach in the city of Kiel in 1991. The Ki-67 gene encodes a large nuclear protein with 2 isoforms in which their biologic functions remain unclear. Ki-67 immunoreactivity can be found in all phases of cell cycle except in the quiescent G0 phase. During early G1 phase, Ki-67 staining is generally weak and is located at discrete foci in the nuclei, whereas during late G1 phase, Ki-67 immunoreactivity progressively condenses in larger perinucleolar granules. During S and G2 phases, Ki-67 staining is detected largely in the nucleolar region, and during mitosis, Ki-67 demonstrates an intense expression associated with the surface of condensed chromosomes. This intensity rapidly disappears toward the anaphase-telophase phase. In normal early placentas, cytotrophoblastic cells are labeled with Ki-67 staining in 25% to 50% of cells, whereas in complete hydatidiform moles, almost all cytotrophoblastic cells are positive for Ki-67, reflecting the hyperplastic nature of molar placentas. Syncytiotrophoblast and normal implantation site IT cells are negative for Ki-67. Very interestingly, the chorionic-type IT cells are positive for Ki-67 in approximately 3% to 5% of cells. The low proliferation activity may be required to gradually increase the trophoblastic cell population in chorion laeve (fetal membrane) as gestation proceeds. In trophoblastic tumors, the Ki-67 labeling index elevates in all trophoblastic tumors. Choriocarcinomas are diffusely labeled with Ki-67 (N50%), whereas most of PSTTs and ETTs contain approximately 8% to 20% of Ki-67–positive cells, although in malignant cases [22], the labeling index can be much higher. In contrast, the IT cells in an EPS are negative for Ki-67 staining, and the trophoblastic cells in placental site nodules are only positive for Ki-67 in less than 10% of trophoblastic cells [3]. The
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above findings suggest that determining the Ki-67 labeling index can be useful for the differential diagnosis of trophoblastic tumors and tumorlike lesions, especially between PSTT and EPS [22]. 3.6. Cyclin E—a marker that distinguishes ETT vs placental site nodule Cyclin E participates in cell cycle control and has emerged as a critical molecule involved in the development of many types of human cancer [23,24]. In normal tissues, cell cycle progression is tightly regulated by a set of molecules that act in concert to ensure that cell proliferation takes place as scheduled. Deregulated cell cycle is an inherent and characteristic feature in neoplastic diseases, as the checkpoints for cell cycle progression are always defective, allowing uncontrolled cellular proliferation in tumors. It has been well established that there are 2 main cell cycle checkpoints, that is, the G0→G1 transition and the G1→S transition. At the molecular levels, these events are directly mediated by cyclin and cyclin-dependent kinase (Cdk) families. Cyclin, the regulatory unit, interacts with Cdk, the catalytic component, in a specific manner. The cyclin E–Cdk2 complex mediates the G1→S transition through phosphorylation and inactivation of the retinoblastoma protein; an event releases the E2F transcription factor, which plays a critical role in DNA synthesis. Cyclin E expression is deregulated in a variety of epithelial and nonepithelial cancers through mutation or amplification of the gene or by mutations in other components of the related pathways [25,26]. In normal placentas and implantation sites, cyclin E immunoreactivity is detected in trophoblastic columns of anchoring villi and implantation site IT cells [27]. In gestational trophoblastic diseases, it has been demonstrated that cyclin E is expressed in both PSTTs and ETTs but not in placental site nodules except a low level of cyclin E immunoreactivity in so-called atypical placental site nodule that may represent a transition from a conventional placental site nodule to an ETT [28]. This finding suggests that cyclin E immunoreactivity may serve as marker to differentiate an ETT from a benign placental site nodule. 4. The trophogram—a stepwise immunohistochemistry approach for differential diagnosis Although the diagnosis of trophoblastic tumors and tumorlike lesions is usually straightforward based on morphological features, the differential diagnosis between trophoblastic diseases and nontrophoblastic lesions may be difficult in some cases. Furthermore, distinguishing different types of trophoblastic tumors and tumorlike lesions can be problematic. It cannot be overemphasized how a precise diagnosis of a trophoblastic disease can impact clinical management of patients, and 3 such examples are illustrated below. First, choriocarcinomas, especially those with a
monomorphic appearance (attenuated syncytiotrophoblastic component), can be confused with a poorly differentiated neoplasm, PSTT, or ETT. The recognition of a choriocarcinoma rather than the other types of neoplastic diseases is important, as the clinical behavior and management are very different. Choriocarcinoma is highly sensitive to chemotherapy, whereas PSTT and ETT are relatively indolent neoplasms for which surgical resection or total hysterectomy is considered the treatment of choice because they tend to be refractory to conventional chemotherapy. Second, ETTs may be confused with hyalinizing squamous cell carcinomas of the cervix not only because both tumors share similar histopathologic features (epithelioid growth and eosinophilic fibrinoid deposition in ETT that may resemble keratin in squamous carcinoma) but also because many ETTs develop at the cervix or lower uterine segment from reproductive ages of women [4]. Third, the differential diagnosis between ETT and placental site nodule can be at times very difficult because they are composed of chorionic-type IT cells [2-4]. Epithelioid trophoblastic tumors are neoplastic diseases with malignant potential that requires surgical intervention and close follow-up. In contrast, placental site nodules are benign lesions without the need for further clinical management. Given that trophoblast-associated markers can be useful as adjuncts in assisting differential diagnosis, it is likely that development of an algorithmic approach by combining those markers should be desirable in diagnostic pathology. The stepwise immunohistochemical assessment of a lesion suspected of a trophoblastic tumor or a tumorlike lesion not only provides a rational ground of an immunostaining approach but also offers a cost-effective way to reach the diagnosis with only essential antibodies being used. Therefore, an algorithm, termed trophogram, is proposed for this purpose (Fig. 1). Trophogram is a 3-tiered sequential staining procedure that uses the panel of commercially available antibodies as listed in Table 2. The first tier is to discriminate a trophoblastic vs a nontrophoblastic lesion. The second tier is to determine if the lesion is a choriocarcinoma, a lesion related to implantation site IT cells, or a lesion related to chorionic-type IT cells. The third tier is to distinguish a benign tumorlike lesion vs a true trophoblastic neoplasm. The antibodies that specifically detect each trophoblast-associated marker in each tier are all commercially available and have been used in formalin-fixed and paraffinembedded sections. Based on the trophogram, HSD3B1 expression should be first assessed in a suspected trophoblastic lesion. A diffuse and intense HSD3B1 immunoreactivity suggests that the lesion is trophoblast related. Other markers such as lowmolecular-weight cytokeratin 8 or 18 may also be considered to further validate the trophoblastic origin. All trophoblastic tumors and tumorlike lesions are diffusely positive for lowmolecular-weight cytokeratin. However, cytokeratin 8 or 18 is not recommended as the first line marker because it is expressed in a wide variety of carcinomas; therefore, cytokeratin antibodies can only be used to further support
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Fig. 1. Applying trophogram in the differential diagnosis of trophoblastic tumors and tumorlike lesions. An algorithm is proposed by combining a set of trophoblast-associated markers to facilitate an immunohistochemistrybased diagnosis of trophoblastic diseases. The trophogram is a 3-tiered sequential staining procedure that uses the panel of commercially available antibodies as listed in Table 2. The first tier is to discriminate a trophoblastic vs a nontrophoblastic lesion. The second tier is to determine if the lesion is a choriocarcinoma, a lesion related to implantation site intermediate trophoblast, or a lesion related to chorionic-type intermediate trophoblastic cells. The third tier is to distinguish a benign tumorlike lesion vs a true trophoblastic neoplasm. PSN indicates placental site nodule.
the diagnosis in HSD3B1-positive cases. Once a trophoblastic lesion is established, the diagnosis of a choriocarcinoma should be entertained, as it is the most common trophoblastic neoplasm. The presence of β-hCG in syncytiotrophoblast confirms the diagnosis of choriocarcinoma. In some choriocarcinomas where the syncytiotrophoblastic component is attenuated, β-hCG–positive syncytiotrophoblast tends to assume a slender and serpiginous structure that outlines the mononucleate trophoblastic masses. Occasionally, β-hCG–positive multinucleated IT cells are present in PSTT and ETT, and they should not be confused with syncytiotrophoblast. If choriocarcinoma has been ruled out, both hPL and p63 expression should be evaluated to distinguish if the lesion is related to implantation site intermediate trophoblast or chorionic-type intermediate trophoblast (Fig. 1). In PSTT and EPS, hPL is positive and p63 is negative. In contrast, in ETT and placental site nodule, hPL is negative but p63 is positive. The antibody to be used to detect p63 should be the one that recognizes the TAp63 isoform because the p63 antibody that only recognizes the ΔNp63 isoform does not stain ETT or placental site nodule [8]. In further distinguishing an ETT vs a placental site nodule and a PSTT vs an EPS, Ki-67 labeling index has been shown to be valuable in this setting. The Ki-67 indices are near zero (b1%) in EPS, whereas the indices are usually more than 8% in PSTTs. Similarly, the Ki-67 indices in placental site nodules are generally low (b8%), whereas the indices in ETTs are more than 10%. One of the main pitfalls in determining Ki-67 labeling index is that several normal cell types including NK cells and T cells that are resident in the endomyometrium can be proliferating and thus Ki-67
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labeled [22]. Thus, special precaution should be exercised to recognize the enlarged and highly pleomorphic nuclei that are characteristic to trophoblastic cells in an effort to avoid the possibility that inflammatory cells are mistakenly counted as trophoblastic cells. In practical sense, application of Ki-67 labeling index appears much more reliable in diagnosing PSTTs vs EPS rather than ETTs vs placental site nodules because the margin of Ki-67 labeling index between ETT and placental site nodule is relatively narrow in some cases. To improve the performance of the trophogram, Mao et al [28] has recently reported that cyclin E immunoreactivity permits the discrimination between ETTs and placental site nodules. Cyclin E is diffusely expressed in most ETTs but rarely in placental site nodules. When applying the trophogram as an immunostaining tool for differential diagnosis of trophoblastic tumors and tumorlike lesions, it should be emphasized that the sequence in interpreting the staining results as outlined in the Fig. 1 should be followed. For example, p63 immunoreactivity is meaningful only in the context of distinguishing trophoblastic tumors and tumorlike lesions that are related to either implantation site IT cells (PSTT and EPS) or chorionic-type IT cells (ETT and placental site nodule). p63 immunostaining should not be used to diagnose trophoblastic vs nontrophoblastic lesions. Likewise, cyclin E immunoreactivity can help in distinguishing ETTs from placental site nodules but may not be useful in other diagnostic settings in the trophogram.
5. Conclusions Trophoblastic tumors and tumorlike lesions are a group of unique but rare diseases, and correct diagnosis is important because each of these lesions may require different therapeutic approaches. Microscopically, a trophoblastic lesion can be confused with a variety of nontrophoblastic tumors and other trophoblastic lesions. In those difficult cases, application of a stepwise and algorithmic immunostaining approach, that is, the trophogram, may greatly assist the differential diagnosis. The commercially available antibodies that recognize the trophoblast-associated markers in the panel of trophogram should facilitate the introduction of trophogram in routine diagnostic pathology. References [1] Shih IM. Gestational trophoblastic neoplasms–Pathogenesis and target-based therapy. Lancet Oncology 2000 [in press]. [2] Shih IM, Kurman RJ. The pathology of intermediate trophoblastic tumors and tumor-like lesions. Int J Gynecol Pathol 2001;20:31-47. [3] Shih IM, Seidman JD, Kurman RJ. Placental site nodule and characterization of distinctive types of intermediate trophoblast. Hum Pathol 1999;30:687-94. [4] Shih I-M, Kurman RJ. Epithelioid trophoblastic tumor—a neoplasm distinct from choriocarcinoma and placental site trophoblastic tumor simulating carcinoma. Am J Surg Pathol 1998;22:1393-403.
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Review Article
Trophogram, an immunohistochemistry-based algorithmic approach, in the differential diagnosis of trophoblastic tumors and tumorlike lesions Ie-Ming Shih Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA
Abstract
Keywords:
Recent histopathologic and molecular studies of trophoblastic cells in the normal placenta and in a variety of trophoblastic diseases have revealed that the latter recapitulate the differentiation of normal trophoblast in the early developing placenta. This new knowledge, especially the identification and characterization of the protein markers expressed in human trophoblast, not only helps elucidate the pathogenesis of trophoblastic lesions but also provides a repertoire of immunohistochemical markers that may facilitate the diagnosis of various trophoblastic diseases. This article reviews the recent advances in the trophoblast-associated markers that have been reported to be useful in the differential diagnosis of trophoblastic tumors and tumorlike lesions. Moreover, a “trophogram,” which is a stepwise and rational immunohistochemistry-based approach, will be introduced. The trophogram may prove to be useful in assisting the differential diagnosis of various trophoblastic diseases in diagnostic pathology. © 2007 Elsevier Inc. All rights reserved. Protein markers; Trophoblast; Trophogram
1. Introduction Gestational trophoblastic disease can be broadly divided into 3 groups: hydatidiform moles, which represent abnormally formed placentas, benign tumorlike lesions, and true trophoblastic neoplasms (Table 1). In recent years, progress has been made in elucidating the biology of human trophoblast. The identification and characterization of the genes expressed in human trophoblast has led to a further understanding of the lineage and the differentiation program of trophoblast and related this to trophoblastic diseases. It has become clear that trophoblastic lesions recapitulate the trophoblast present in the early developing placenta and implantation site. In human placentas, cytotrophoblast is the germinative or “stem cell” component that differentiates into either syncytiotrophoblast, which functions as a hormone and pseudovascular organ, or
This review article was presented in the Surgical Pathology Update at Leipzig, Germany on June 14, 2007. E-mail address: [email protected]. 1092–9134/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.anndiagpath.2007.04.001
intermediate trophoblastic (IT) cells. The IT cells that infiltrate the decidua, myometrium, and spiral arteries of the placental site are termed implantation site IT, and those in the chorion laeve in the fetal membranes are termed chorionic-type IT. The recent research findings suggest that after neoplastic transformation of trophoblastic stem cells, presumably cytotrophoblast, specific differentiation programs determine the type of trophoblastic tumor that will develop [1-4]. Accordingly, choriocarcinoma is composed of variable amounts of neoplastic cytotrophoblast, syncytiotrophoblast, and intermediate trophoblast and resembles the previllous blastocyst, which is composed of a similar mixture of trophoblastic populations. In contrast, the neoplastic cytotrophoblastic cells in placental site trophoblastic tumors (PSTTs) differentiate mainly into implantation site IT cells, whereas the neoplastic trophoblastic cells in epithelioid trophoblastic tumors (ETTs) differentiate into chorionic-type IT cells. Therefore, choriocarcinoma is the most primitive trophoblastic tumor, whereas PSTT and ETT are relatively more differentiated. Clinically, distinguishing trophoblastic tumors from other types of neoplastic disease is important as far as the treatment
I.-M. Shih / Annals of Diagnostic Pathology 11 (2007) 228–234 Table 1 Classification of gestational trophoblastic diseases Hydatidiform moles (abnormally formed placentas) Complete mole Partial mole Invasive mole Trophoblastic tumorlike lesions (benign lesions) Exaggerated placental site/reaction Placental site nodule Trophoblastic tumors (neoplastic diseases) PSTT ETT
plans are concerned [5]. Furthermore, diagnosis of different types of trophoblastic tumors vs tumorlike lesions is critical, as the former may likely involve surgical intervention and/or chemotherapy. However, the differential diagnosis can be difficult based on morphology alone. As a result, application of immunohistochemistry on trophoblast-associated markers may be very useful in difficult cases. Therefore, it is critical to review the trophoblast-associated markers from the literature and to generate an algorithm that allows pathologists to conveniently apply commercially available antibodies in a stepwise fashion to assist differential diagnosis of trophoblastic diseases. This review article briefly highlights the clinicopathologic features of trophoblastic tumors and tumorlike lesions and summarizes the recent advance in studying trophoblast-associated markers. Finally, a “trophogram,” which is a stepwise immunohistochemistry-based approach by combining those markers, will be introduced. It is expected that the trophogram may prove to be useful in assisting the differential diagnosis of various trophoblastic tumors and tumorlike lesions. 2. Clinicopathologic features of trophoblastic tumors and tumorlike lesions 2.1. Choriocarcinoma Gestational choriocarcinoma is a highly malignant epithelial tumor that can be associated with any type of gestational event, most often with a complete hydatidiform mole [5]. Choriocarcinoma may arise from neoplastic transformation of cytotrophoblast and recapitulates the differentiation of cytotrophoblastic cells in early gestation [6]. Neoplastic cytotrophoblast, like its normal counterpart, retains its full capacity to differentiate into syncytiotrophoblast and intermediate trophoblast. As a result, an intimate mixture of cytotrophoblast, intermediate trophoblast, and syncytiotrophoblast characterizes choriocarcinoma. At lower magnification, the cytotrophoblast and IT cells tend to grow in clusters and sheets, separated by syncytiotrophoblast, forming the characteristic dimorphic growth pattern of mononucleate trophoblast and syncytiotrophoblast. The diagnosis of a choriocarcinoma is usually straightforward given its unique histopathologic pattern and the soaring levels of serum β-hCG. However, a choriocarcinoma can be
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confused with other types of carcinoma and trophoblastic neoplasms, especially for those cases with attenuated syncytiotrophoblastic components. 2.2. Placental site trophoblastic tumor Placental site trophoblastic tumor is a relatively uncommon tumor and is composed of neoplastic implantation site IT cells [7-10]. In contrast to the normal implantation site in which invasion of intermediate trophoblast is tightly regulated and is confined to the inner third of the myometrium, the tumor cells of PSTT are highly invasive and infiltrate deeply into the myometrium, occasionally penetrating through the uterine wall. Microscopically, PSTT resembles the trophoblastic infiltration of the endometrium and myometrium of the placental site during early pregnancy but tends to form confluent masses or sheets with enlarged and highly atypical nuclei. Mitotic figures may vary from case to case but generally are very few (b5 mitoses per 10 high-power fields). Although most patients with their tumors confined to the uterus are curable after tumor resection or hysterectomy, the clinical behavior of PSTTs can be unpredictable in some cases, and tumor recurrence and metastasis have been reported several years after initial treatment [11,12]. 2.3. Epithelioid trophoblastic tumor Epithelioid trophoblastic tumor is another unusual type of trophoblastic neoplasm that is distinct from PSTT and choriocarcinoma, with morphological features resembling a carcinoma [4]. The tumors are composed of chorionic-type IT cells based on both morphological and immunohistochemical studies [4]. Microscopically, ETTs can assume several growth patterns including nodules, solid sheets, masses, and infiltrating nests. Geographic tumor cell necrosis and/or focal calcification are frequently observed. The tumor nests in an ETT are frequently accompanied by eosinophilic fibrinoid material, giving the tumor an appearance similar to a keratinizing squamous carcinoma. Because placental site nodules (see section below) are also composed of chorionictype intermediate trophoblast, it has been hypothesized that some placental site nodules may represent an intermediate stage in tumor progression to ETTs. This view is supported by observations that some proliferative placental site nodules with slightly greater cytologic atypia tentatively classified as atypical placental site nodules have features intermediate between typical placental site nodules and ETTs. Moreover, in some cases there is an intimate association of an ETT with a placental site nodule. Further molecular genetic studies are necessary to confirm this hypothesis. The clinical behavior of an ETT is similar to that of a PSTT. Most ETTs behave in a benign fashion, but malignant ETTs with local invasion and distant metastasis can also occur. 2.4. Placental site nodule Placental site nodules are small, well-circumscribed nodular aggregates of chorionic-type IT cells that are
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embedded in a hyalinized stroma. Placental site nodules have been thought to represent a portion of uninvoluted placental site from a remote gestation. However, the constituent cells in placental site nodules are morphologically more closely related to the intermediate trophoblast of the chorion laeve (chorionic-type intermediate trophoblast) than to the intermediate trophoblast of the placental site [3]. In addition, the trophoblastic cells in the placental site nodule exhibit an immunophenotype similar to that of trophoblastic cells in the chorion laeve but distinct from implantation site IT cells. It remains a mystery as to how these cells (from chorion leave) are retained and survive in the uterus after delivery. Placental site nodules are usually incidental findings in cervical biopsy and endocervical or endometrial curettage specimens. Placental site nodules are benign lesions, and no further clinical management is required once the diagnosis is established. 2.5. Exaggerated placental site Exaggerated placental site (EPS) is a benign nonneoplastic lesion characterized by an exuberant number of implantation site IT cells that extensively infiltrate the endometrium and underlying myometrium [2]. The EPS can occur in a normal pregnancy, an abortion from the first trimester, or a molar pregnancy, especially a complete mole. The trophoblastic cells in an EPS display an identical morphological and immunophenotypic profile to the implantation site IT cells in the normal placental site [10]. Despite the extensive infiltration of IT cells in an EPS, the Ki-67 indices of IT cells are near zero, suggesting that the increased number of IT cells in EPS is probably not the result of de novo proliferation of intermediate trophoblast in the implantation site [10,13]. Rather, it has been thought that EPS represents a physiologic presentation in the placental bed, especially from an early gestation. Therefore, there is no clinical significance associated with EPS.
3. Trophoblast-associated markers In the past decades, studies on the trophoblastassociated markers have provided fundamental insights into the biology of human trophoblastic cells during implantation and placentation. Furthermore, several of these proteins, especially for those that can be detected by commercially available antibodies, have emerged as new diagnostic markers for trophoblastic tumors and tumorlike lesions. Some of the markers are expressed in several types of trophoblastic subpopulations, whereas others are specifically expressed in certain trophoblastic subpopulations. The following discussion summarizes those markers and their relevance to diagnostic pathology (Table 2).
Table 2 Markers for trophogram and antibody sources Marker
Application
Antibody source
HSD3B p63 hPL β-hCG Cyclin E Ki-67
Distinguish GTD vs non-GTD Distinguish ETT vs PSTT Distinguish PSTT vs ETT Highlight ST in choriocarcinoma Distinguish ETT vs PSN Distinguish ETT vs PSN; PSTT vs EPS
Abnova (3C11-D4 a) Neomarker (4A4) Dako (polyclonal) Dako (polyclonal) Zymed (HE12) Dako (MIB-1)
GTD indicates gestational trophoblastic disease; PSN, placental site nodule; ST, syncytiotrophoblast. a Clone name for the monoclonal antibody.
3.1. Hydroxyl-δ-5-steroid dehydrogenase— a pan-trophoblastic marker Hydroxyl-δ-5-steroid dehydrogenase (HSD3B1) is an enzyme involved in steroid hormone synthesis by catalyzing the oxidative conversion of δ-5-3 β-hydroxy steroids to the δ-4-3-keto configuration [14]. Using a commercially available anti-HSD3B1 monoclonal antibody on paraffin sections, Mao et al [15] have demonstrated intense HSD3B1 immunoreactivity in implantation site and chorionic-type IT cells as well as in syncytiotrophoblast in all cases of early placentas and complete hydatidiform moles. More importantly, HSD3B1 immunoreactivity was detected in all trophoblastic tumors including PSTTs, ETTs and choriocarcinomas, and in all tumorlike lesions including placental site nodules and EPSs. Hydroxyl-δ-5-steroid dehydrogenase immunoreactivity was always diffuse and intense in most trophoblastic tumors and tumorlike lesions, with an exception in a few choriocarcinomas that are predominantly composed of cytotrophoblastlike tumor cells. In contrast, only 3 (b1%) of 323 nontrophoblastic carcinomas from the uterus, lung, and breast react with the HSD3B1 antibody. Moreover, the immunoreactivity in the positive nontrophoblastic cases is focal and weak and thus is in contrast to the diffuse and intense immunostaining pattern observed in trophoblastic tumors and tumorlike lesions. The specificity of HSD3B1 expression in trophoblastic tissues was further supported by analyzing the gene expression profile of HSD3B1 in silica using the serial analysis of gene expression database deposited in public domain. In that study, HSD3B1 was not expressed in any of 159 libraries of breast, lung, colorectal, pancreatic, ovarian carcinomas, and a wide variety of normal adult and fetal tissues [15]. The results from the above studies strongly suggest that compared with other trophoblastic markers, HSD3B1 could be a specific and sensitive marker being expressed in all types of trophoblastic tumors and tumorlike lesions but rarely in a variety of carcinomas that have been examined. 3.2. p63—a trophoblastic marker for chorionic-type IT differentiation p63 is a transcription factor belonging to the p53 gene family. [16,17] It is localized on chromosome 3q27-29 and
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has a strong homology to p53 in nucleotide sequence and biologic function. p63 isoforms are present as a result of differential promoter usage [18,19]. The transcription activation (TA) isoform uses an upstream (the most 5′) promoter, generating p63 proteins with the TA domain, which binds to p53-consensus sequences and induces p53-target genes. In contrast, transcription from the downstream (3′) promoter results in a smaller p63 isoform that lacks the TA domain and is referred to as ΔN isoform (Np63). The TA and the ΔN isoforms regulate distinct sets of downstream genes and have distinct biologic functions [9,22]. Specifically, the TA isoforms have a p53-like tumor suppressor function, whereas the Np63 isoforms exert an oncogenic effect. A previous study has demonstrated that different trophoblastic subpopulations are characterized by unique expression patterns of p63 isoforms. Cytotrophoblast expresses the ΔN isoforms, whereas the chorionic-type IT cells in fetal membrane (chorion laeve) express the TA isoforms. Implantation site intermediate trophoblast and syncytiotrophoblast do not express either of the p63 isoforms. In trophoblastic lesions, expression of TAp63 is detected in placental site nodules and ETTs but not in EPS or PSTTs. Thus, TAp63 is unique among known trophoblastic markers in that it is diffusely expressed in ETTs but not in PSTTs, serving as a marker for their differential diagnosis. When p63 immunoreactivity is considered for diagnostic purpose, an appropriate antibody should be used to detect the TA isoform rather than the ΔN isoform only. The 4A4 antibody, which reacts with both TA and ΔNp63 isoforms, can be ideal. 3.3. Human placental lactogen—a trophoblastic marker for implantation site IT differentiation Human placental lactogen (hPL) is a pregnancy-related hormone that is normally synthesized by syncytiotrophoblast and IT cells located at the implantation sites. As expected, among the trophoblastic tumors and tumorlike lesions, hPL is expressed in most of the trophoblastic cells of PSTTs and EPSs [20] but in only a very small percentage (b5%) of trophoblastic cells in ETTs and placental site nodules [2,4]. This expression pattern indicates the value by applying immunohistochemistry of this marker in distinguishing lesions that are related to implantation site IT cells (PSTT and EPS) from those that are related to IT cells in chorion laeve (ETT and placental site nodule). Unlike other IT markers such as HSD3B1, HLA-G, and Mel-CAM (CD146), which are expressed in nearly all PSTTs, the percentage of hPL-positive cells in PSTT can vary from case to case. Among neoplastic diseases, hPL appears to be highly specific to PSTT, as other human tumors rarely express this hormone except in some mixed germ cell tumors with trophoblastic differentiation. 3.4. Human chorionic gonadotropin— a syncytiotrophoblastic marker Human chorionic gonadotropin (hCG) is the first and probably the most well-known trophoblastic marker that
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plays a critical role in maintaining the placental function during pregnancy. It is synthesized and secreted predominantly by syncytiotrophoblast in placentas throughout the course of gestation. Further studies have demonstrated that molecular composition of hCG is different between normal pregnancy and gestational trophoblastic tumors. The former produces intact, normally glycosylated hCG, whereas the latter secretes a wide range of hCG variants that may not be detected by conventional assays. Human chorionic gonadotropin is composed of α and β subunits. Because the β subunit is relatively specific to hCG, antibodies that react to this subunit have been routinely used in diagnostic pathology. In fact, the β-hCG has become an ideal marker to demonstrate the syncytiotrophoblastic component in cases suspicious for a choriocarcinoma in which the syncytiotrophoblast within the tumor is inconspicuous based on hematoxylin-eosin staining. In PSTT and ETT, β-hCG– positive cells are extremely rare, and if any, they are limited to the multinucleated IT cells. 3.5. Ki-67—a proliferation marker Ki-67 antigen is the most frequently used immunostaining marker in diagnostic pathology to assess cell proliferation activity in tissues [21]. It was first identified by an antibody approach in the city of Kiel in 1991. The Ki-67 gene encodes a large nuclear protein with 2 isoforms in which their biologic functions remain unclear. Ki-67 immunoreactivity can be found in all phases of cell cycle except in the quiescent G0 phase. During early G1 phase, Ki-67 staining is generally weak and is located at discrete foci in the nuclei, whereas during late G1 phase, Ki-67 immunoreactivity progressively condenses in larger perinucleolar granules. During S and G2 phases, Ki-67 staining is detected largely in the nucleolar region, and during mitosis, Ki-67 demonstrates an intense expression associated with the surface of condensed chromosomes. This intensity rapidly disappears toward the anaphase-telophase phase. In normal early placentas, cytotrophoblastic cells are labeled with Ki-67 staining in 25% to 50% of cells, whereas in complete hydatidiform moles, almost all cytotrophoblastic cells are positive for Ki-67, reflecting the hyperplastic nature of molar placentas. Syncytiotrophoblast and normal implantation site IT cells are negative for Ki-67. Very interestingly, the chorionic-type IT cells are positive for Ki-67 in approximately 3% to 5% of cells. The low proliferation activity may be required to gradually increase the trophoblastic cell population in chorion laeve (fetal membrane) as gestation proceeds. In trophoblastic tumors, the Ki-67 labeling index elevates in all trophoblastic tumors. Choriocarcinomas are diffusely labeled with Ki-67 (N50%), whereas most of PSTTs and ETTs contain approximately 8% to 20% of Ki-67–positive cells, although in malignant cases [22], the labeling index can be much higher. In contrast, the IT cells in an EPS are negative for Ki-67 staining, and the trophoblastic cells in placental site nodules are only positive for Ki-67 in less than 10% of trophoblastic cells [3]. The
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above findings suggest that determining the Ki-67 labeling index can be useful for the differential diagnosis of trophoblastic tumors and tumorlike lesions, especially between PSTT and EPS [22]. 3.6. Cyclin E—a marker that distinguishes ETT vs placental site nodule Cyclin E participates in cell cycle control and has emerged as a critical molecule involved in the development of many types of human cancer [23,24]. In normal tissues, cell cycle progression is tightly regulated by a set of molecules that act in concert to ensure that cell proliferation takes place as scheduled. Deregulated cell cycle is an inherent and characteristic feature in neoplastic diseases, as the checkpoints for cell cycle progression are always defective, allowing uncontrolled cellular proliferation in tumors. It has been well established that there are 2 main cell cycle checkpoints, that is, the G0→G1 transition and the G1→S transition. At the molecular levels, these events are directly mediated by cyclin and cyclin-dependent kinase (Cdk) families. Cyclin, the regulatory unit, interacts with Cdk, the catalytic component, in a specific manner. The cyclin E–Cdk2 complex mediates the G1→S transition through phosphorylation and inactivation of the retinoblastoma protein; an event releases the E2F transcription factor, which plays a critical role in DNA synthesis. Cyclin E expression is deregulated in a variety of epithelial and nonepithelial cancers through mutation or amplification of the gene or by mutations in other components of the related pathways [25,26]. In normal placentas and implantation sites, cyclin E immunoreactivity is detected in trophoblastic columns of anchoring villi and implantation site IT cells [27]. In gestational trophoblastic diseases, it has been demonstrated that cyclin E is expressed in both PSTTs and ETTs but not in placental site nodules except a low level of cyclin E immunoreactivity in so-called atypical placental site nodule that may represent a transition from a conventional placental site nodule to an ETT [28]. This finding suggests that cyclin E immunoreactivity may serve as marker to differentiate an ETT from a benign placental site nodule. 4. The trophogram—a stepwise immunohistochemistry approach for differential diagnosis Although the diagnosis of trophoblastic tumors and tumorlike lesions is usually straightforward based on morphological features, the differential diagnosis between trophoblastic diseases and nontrophoblastic lesions may be difficult in some cases. Furthermore, distinguishing different types of trophoblastic tumors and tumorlike lesions can be problematic. It cannot be overemphasized how a precise diagnosis of a trophoblastic disease can impact clinical management of patients, and 3 such examples are illustrated below. First, choriocarcinomas, especially those with a
monomorphic appearance (attenuated syncytiotrophoblastic component), can be confused with a poorly differentiated neoplasm, PSTT, or ETT. The recognition of a choriocarcinoma rather than the other types of neoplastic diseases is important, as the clinical behavior and management are very different. Choriocarcinoma is highly sensitive to chemotherapy, whereas PSTT and ETT are relatively indolent neoplasms for which surgical resection or total hysterectomy is considered the treatment of choice because they tend to be refractory to conventional chemotherapy. Second, ETTs may be confused with hyalinizing squamous cell carcinomas of the cervix not only because both tumors share similar histopathologic features (epithelioid growth and eosinophilic fibrinoid deposition in ETT that may resemble keratin in squamous carcinoma) but also because many ETTs develop at the cervix or lower uterine segment from reproductive ages of women [4]. Third, the differential diagnosis between ETT and placental site nodule can be at times very difficult because they are composed of chorionic-type IT cells [2-4]. Epithelioid trophoblastic tumors are neoplastic diseases with malignant potential that requires surgical intervention and close follow-up. In contrast, placental site nodules are benign lesions without the need for further clinical management. Given that trophoblast-associated markers can be useful as adjuncts in assisting differential diagnosis, it is likely that development of an algorithmic approach by combining those markers should be desirable in diagnostic pathology. The stepwise immunohistochemical assessment of a lesion suspected of a trophoblastic tumor or a tumorlike lesion not only provides a rational ground of an immunostaining approach but also offers a cost-effective way to reach the diagnosis with only essential antibodies being used. Therefore, an algorithm, termed trophogram, is proposed for this purpose (Fig. 1). Trophogram is a 3-tiered sequential staining procedure that uses the panel of commercially available antibodies as listed in Table 2. The first tier is to discriminate a trophoblastic vs a nontrophoblastic lesion. The second tier is to determine if the lesion is a choriocarcinoma, a lesion related to implantation site IT cells, or a lesion related to chorionic-type IT cells. The third tier is to distinguish a benign tumorlike lesion vs a true trophoblastic neoplasm. The antibodies that specifically detect each trophoblast-associated marker in each tier are all commercially available and have been used in formalin-fixed and paraffinembedded sections. Based on the trophogram, HSD3B1 expression should be first assessed in a suspected trophoblastic lesion. A diffuse and intense HSD3B1 immunoreactivity suggests that the lesion is trophoblast related. Other markers such as lowmolecular-weight cytokeratin 8 or 18 may also be considered to further validate the trophoblastic origin. All trophoblastic tumors and tumorlike lesions are diffusely positive for lowmolecular-weight cytokeratin. However, cytokeratin 8 or 18 is not recommended as the first line marker because it is expressed in a wide variety of carcinomas; therefore, cytokeratin antibodies can only be used to further support
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Fig. 1. Applying trophogram in the differential diagnosis of trophoblastic tumors and tumorlike lesions. An algorithm is proposed by combining a set of trophoblast-associated markers to facilitate an immunohistochemistrybased diagnosis of trophoblastic diseases. The trophogram is a 3-tiered sequential staining procedure that uses the panel of commercially available antibodies as listed in Table 2. The first tier is to discriminate a trophoblastic vs a nontrophoblastic lesion. The second tier is to determine if the lesion is a choriocarcinoma, a lesion related to implantation site intermediate trophoblast, or a lesion related to chorionic-type intermediate trophoblastic cells. The third tier is to distinguish a benign tumorlike lesion vs a true trophoblastic neoplasm. PSN indicates placental site nodule.
the diagnosis in HSD3B1-positive cases. Once a trophoblastic lesion is established, the diagnosis of a choriocarcinoma should be entertained, as it is the most common trophoblastic neoplasm. The presence of β-hCG in syncytiotrophoblast confirms the diagnosis of choriocarcinoma. In some choriocarcinomas where the syncytiotrophoblastic component is attenuated, β-hCG–positive syncytiotrophoblast tends to assume a slender and serpiginous structure that outlines the mononucleate trophoblastic masses. Occasionally, β-hCG–positive multinucleated IT cells are present in PSTT and ETT, and they should not be confused with syncytiotrophoblast. If choriocarcinoma has been ruled out, both hPL and p63 expression should be evaluated to distinguish if the lesion is related to implantation site intermediate trophoblast or chorionic-type intermediate trophoblast (Fig. 1). In PSTT and EPS, hPL is positive and p63 is negative. In contrast, in ETT and placental site nodule, hPL is negative but p63 is positive. The antibody to be used to detect p63 should be the one that recognizes the TAp63 isoform because the p63 antibody that only recognizes the ΔNp63 isoform does not stain ETT or placental site nodule [8]. In further distinguishing an ETT vs a placental site nodule and a PSTT vs an EPS, Ki-67 labeling index has been shown to be valuable in this setting. The Ki-67 indices are near zero (b1%) in EPS, whereas the indices are usually more than 8% in PSTTs. Similarly, the Ki-67 indices in placental site nodules are generally low (b8%), whereas the indices in ETTs are more than 10%. One of the main pitfalls in determining Ki-67 labeling index is that several normal cell types including NK cells and T cells that are resident in the endomyometrium can be proliferating and thus Ki-67
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labeled [22]. Thus, special precaution should be exercised to recognize the enlarged and highly pleomorphic nuclei that are characteristic to trophoblastic cells in an effort to avoid the possibility that inflammatory cells are mistakenly counted as trophoblastic cells. In practical sense, application of Ki-67 labeling index appears much more reliable in diagnosing PSTTs vs EPS rather than ETTs vs placental site nodules because the margin of Ki-67 labeling index between ETT and placental site nodule is relatively narrow in some cases. To improve the performance of the trophogram, Mao et al [28] has recently reported that cyclin E immunoreactivity permits the discrimination between ETTs and placental site nodules. Cyclin E is diffusely expressed in most ETTs but rarely in placental site nodules. When applying the trophogram as an immunostaining tool for differential diagnosis of trophoblastic tumors and tumorlike lesions, it should be emphasized that the sequence in interpreting the staining results as outlined in the Fig. 1 should be followed. For example, p63 immunoreactivity is meaningful only in the context of distinguishing trophoblastic tumors and tumorlike lesions that are related to either implantation site IT cells (PSTT and EPS) or chorionic-type IT cells (ETT and placental site nodule). p63 immunostaining should not be used to diagnose trophoblastic vs nontrophoblastic lesions. Likewise, cyclin E immunoreactivity can help in distinguishing ETTs from placental site nodules but may not be useful in other diagnostic settings in the trophogram.
5. Conclusions Trophoblastic tumors and tumorlike lesions are a group of unique but rare diseases, and correct diagnosis is important because each of these lesions may require different therapeutic approaches. Microscopically, a trophoblastic lesion can be confused with a variety of nontrophoblastic tumors and other trophoblastic lesions. In those difficult cases, application of a stepwise and algorithmic immunostaining approach, that is, the trophogram, may greatly assist the differential diagnosis. The commercially available antibodies that recognize the trophoblast-associated markers in the panel of trophogram should facilitate the introduction of trophogram in routine diagnostic pathology. References [1] Shih IM. Gestational trophoblastic neoplasms–Pathogenesis and target-based therapy. Lancet Oncology 2000 [in press]. [2] Shih IM, Kurman RJ. The pathology of intermediate trophoblastic tumors and tumor-like lesions. Int J Gynecol Pathol 2001;20:31-47. [3] Shih IM, Seidman JD, Kurman RJ. Placental site nodule and characterization of distinctive types of intermediate trophoblast. Hum Pathol 1999;30:687-94. [4] Shih I-M, Kurman RJ. Epithelioid trophoblastic tumor—a neoplasm distinct from choriocarcinoma and placental site trophoblastic tumor simulating carcinoma. Am J Surg Pathol 1998;22:1393-403.
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