Gestational trophoblastic diseases: update on new immunohistochemical findings

Current Diagnostic Pathology (2003) 9, 1^10

c 2003 Elsevier Science Ltd. All rights reserved. doi:10.1054/cdip.2003.0150, available online at http://www.sciencedirect.com

MINI-SYMPOSIUM: IMMUNOHISTOLOGY IN GYNAECOLOGICAL PATHOLOGY

Gestational trophoblastic diseases: update on new immunohistochemical ¢ndings J. M. Lage, S. Minamiguchi and M. S. Richardson Departments of Pathology and Laboratory Medicine, Medical University of South Carolina,165 Ashley Avenue, P.O. Box 250908 Charleston, SC 29425, USA; and Laboratory of Anatomic Pathology, Kyoto University Hospital, Kyoto, Japan

KEYWORDS trophoblast, choriocarcinoma, hydatidiform mole, placental site trophoblastic tumour, epithelioid trophoblastic tumour, gestational trophoblastic disease, immunohistochemistry

Summary Recent advances in the immunohistochemical analysis of gestational trophoblastic diseases allow pathologists to classify more accurately speci¢c types ofgestational trophoblastic lesions. The morphologic features of some forms of gestational trophoblastic tumours are dependent on the parental origin of the genetic contributions.Genomic imprinting results in the production of certain protein products based on the parental genetic origin. By analysing the protein product of an imprinted gene, such as p57kip2, a hydatidiform mole may be classi¢ed as complete or partial.In addition, we review CD 34, p53, bcl-2,Bax, p21waf1/cip1 and PCNA immunohistochemical expression in complete hydatidiform moles. A similar immunohistochemical review is performed for partial hydatidiform moles. We discuss the implications of various immunohistochemical ¢ndings in the nonvillous trophoblastic tumours: placental site trophoblastic tumour, epithelioid trophoblastic tumour and choriocarcinoma. Speci¢c antibodies reviewedin detail for these lesionsinclude inhibin-alpha,CK18,MIB-1 (Ki-67), Mel-CAM, cyclins A, B, D1 and E, cdk 2 and 4, and p53. In summary, advances in immunohistochemistry aid the histopathologist in correctly classifying gestational trophoblastic tumours.

c 2003 Elsevier Science Ltd. All rights reserved.

INTRODUCTION Gestational trophoblastic diseases encompass a wide range of clinical presentations, pathological features and genetic compositions. Accurate prognostication and successful treatment in gestational trophoblastic disease hinges on the pathologist’s ability to classify the tumour type correctly. The most frequent form of gestational trophoblastic disease, the complete hydatidiform mole, results from an abnormal conception event. Since most pregnancies, whether normal or abnormal, are diagnosed very soon after conception, it follows that the pathologist will be required to diagnose an early complete hydatidiform mole before the full histological features are in evidence. Furthermore, adding to these di⁄culties is a new entity in gestational trophoblastic disease, the epithelioid trophoblastic tumour which has been described recently.1,2 While this tumour has unique histological features, results of immunohistochemical Correspondence to: JML.Tel.: +1 843 792 3121; Fax: +1 843 792 0555; E-mail: [email protected]

studies may be most helpful in elucidating the correct diagnosis.1^3 This brings us to even more fundamental issues: the generic category of trophoblast. In learning to recognize trophoblast, one cannot underestimate the value of a simple pancytokeratin stain (Fig.1).This report provides an update on the immunohistochemical armamentarium available to the general diagnostic pathologist for use in arriving at a correct diagnosis in gestational trophoblastic disease.

COMPLETE HYDATIDIFORM MOLE The hydatidiform moles are divided into two main types: (1) complete hydatidiform mole; and (2) partial hydatidiform mole.The complete hydatidiform mole is an androgenetic conceptus derived from the fertilization of an oocyte which has extruded its female pronucleus. This ‘empty egg’ that is fertilized by either one sperm which then duplicates its own DNA, or by two spermatozoa, results in a 46, XX or 46, XYconceptus. As it contains only paternal (androgenetic) nuclear DNA, it forms a

2

Figure 1 Gestational endometrium with normal implantation site trophoblast stained with cytokeratin AE1/AE3 (Dako). Individual implantation site trophoblasts are highlighted along with super¢cial endometrial epithelium (immunoperoxidase stain, original magni¢cation,10x).

very exuberant placenta with trophoblastic hyperplasia called a complete hydatidiform mole. The maternal serum b human chorionic gonadotropin (b-HCG) levels are markedly elevated for the gestational age. b-HCG values of100 000 mIU/ml or greater are not uncommon. The complete hydatidiform mole conveys a signi¢cant risk of developing persistent gestational trophoblastic disease. While various studies have cited di¡ering rates of persistent gestational trophoblastic disease following complete hydatidiform mole, many large studies in the USA, the UK and Japan have found persistent disease rates of approximately 17^33%.4 ^ 6 Women with persistent gestational trophoblastic disease following complete hydatidiform mole require chemotherapy and, occasionally, radiotherapy to eradicate the proliferating trophoblast. Following treatment, the vast majority of these women will achieve complete gonadotropin remission and be cured. Awoman who has had a complete hydatidiform mole has an increased risk of having another subsequent hydatidiform mole. All subsequent pregnancies in such women need to be monitored carefully. Choriocarcinoma follows complete hydatidiform mole in approximately 2^5% of cases.7,8 Its occurrence rate is even higher in Asian women. A more aggressive chemotherapeutic approach is required to achieve gonadotropin remission in choriocarcinoma. There appears to be no increased risk of anomalies occurring in the children of subsequent pregnancies in women treated for gestational trophoblastic diseases. Menopause is reported to be hastened by 3 years in women given chemotherapy to treat gestational trophoblastic disease.9 Complete hydatidiform mole is characterized by a uniform enlargement of the placental villi that, in many instances, is so striking that the ultrasonographer

CURRENT DIAGNOSTIC PATHOLOGY

suggests the diagnosis at the ¢rst obstetrical ultrasonographic examination. These enlarged villi contain proliferating, multilayered cytotrophoblast and mounds of syncytiotrophoblast. This hyperplastic trophoblast proliferates in a circumferential fashion on the villi.The abundance of the syncytiotrophoblast causes a markedly elevated maternal serum b-HCG in complete hydatidiform moles. The villous stroma is cellular and contains karyorrhectic debris. This villous stromal hypercellularity is in striking contrast with early normal villi or an early partial hydatidiform mole. A cistern, or central cavity, forms in the centre of the molar villi. As the cistern enlarges, its edges become sharply demarcated from the remaining villous stroma. The issue of villous blood vessels in complete hydatidiform mole has caused some controversy.The very young complete hydatidiform mole often has quite well-formed villous blood vessels which may even contain wellformed fetal nucleated red blood cells, some of which appear megaloblastic. The blood vessels disintegrate some time after their formation so that by mid-second trimester, they are not apparent.The issue of embryonic tissue is even more controversial. Some have argued quite cogently that a severely abnormal, stunted embryo may form very early in the complete hydatidiform mole. It is not often identi¢ed because it may be tiny and inapparent, or it may have dissolved by the time of uterine evacuation. A few stunted embryos have been reported in complete hydatidiform moles in the literature. Most commonly, no embryonic tissue remains identi¢able by the time the pathologist encounters the specimen. It would be extremely uncommon for embryonic tissues to be selected at random for microscopy from the voluminous tissues of a molar pregnancy. Many senior trophoblastic disease pathologists have learned to recognize the complete hydatidiform mole by the striking atypicality of the implantation site trophoblast, termed the ‘molar implantation site’.This atypicality is apparent in the very early complete hydatidiform moles and increases in severity in the more established forms.

Immunohistochemical studies (Table 1) Immunohistochemical studies of complete hydatidiform mole have revolved around: (1) identifying the normal components of a complete hydatidiform mole; and (2) distinguishing a complete hydatidiform mole from a partial hydatidiform mole. The early complete hydatidiform mole has well-formed fetal blood vessels, often containing nucleated red blood cells. Most commonly, immunohistochemistry for factor-VIII-related antigen is negative in these endothelial cells. Recent immunohistochemical studies using the monoclonal antibody QBEND/10 have demonstrated CD34 expression in villous stromal endothelial cells.10 As the complete hydatidiform mole

GESTATIONALTROPHOBLASTIC DISEASES

3

Table 1 Immunohistochemical studies in partial and complete hydatidiform moles

Inhibin-a (cytoplasmic) Ki-67 (nuclear) PCNA (P10) (nuclear) Mel-CAM (CD146) (membranous)c Cytokeratin AE1/AE3 (cytoplasmic) p57kip2 (nuclear) p53 (nuclear)

Bax (nuclear membrane or cytoplasm) bcl-2 (nuclear membrane or cytoplasm) P21waf1/cip1 (nuclear) a

Complete hydatidiform mole

Partial hydatidiform mole

Positive in SCT Positive in16^32%b; 84.6+/ 10.3% in ITand CTa 89.3+/ 11.1% in ITand CTa

Positive in SCT 65.3+/ 17.6% in CTa

Positive Absent or very low in CT and villous stromal cells, present in intervillous cell islands Wild-type p53 expression in10 ^ 50% in IT and CT, negative wild-type p53 in SCT; absence of mutant p53 Positive in CT, negative in ST Varying positivity in ST; negative CT, IT and villous stroma Positive in 20+/ 5.0% of ST

Positive Strong expression in CT and villous stromal cells

24 b

79.9+/ 12.3% in CT a

Positive in 9.8+/ 9.3 % of ST

c

Ostrzega et al. ; seen in villous and extravillous trophoblast; Mel-CAM stains intermediate trophoblast of implantation site origin; dpregnancy-associated major basic protein stains intermediate trophoblast; SCT, syncytiotrophoblast;IT, intermediate trophoblast; CT, cytotrophoblast.Cellular location of positive staining, whether nuclear, membranous or cytoplasmic, is indicated in parentheses for the newer antibodies. See text for explanation and references. Mel-CAM, melanoma cell adhesion molecule; PCNA, proliferating cell nuclear antigen.

matures, the villous stromal blood vessels degenerate and the endothelial cells fragment. It is interesting that although the endothelial cells disintegrate, in the more mature complete hydatidiform mole, CD34 linear staining in the villous stroma will highlight their previous location.11 Due to its lack of maternal nuclear DNA, only paternal DNA and its gene products should be present in the complete hydatidiform mole. p57kip2 (CDKNIC) is an imprinted gene. Due to some ambiguity of terminology in the literature regarding genetic imprinting, p57kip2 may be described as a paternally imprinted or maternally imprinted gene.12^14 This gene resides on chromosome 11p15.15 p57kip2 is the protein product of this gene which inhibits cyclin-dependent kinases (CDK) thus serving to inhibit cell proliferation and suppress tumour growth. Altered cell di¡erentiation and proliferation have been described in mice lacking p57kip2.16 This alteration, as well as the protein expression, may be tissue speci¢c.17 11p15.5 has been implicated in the development of Beckwith ^Wiedemann syndrome.18 One gene involved with Beckwith ^Wiedemann syndrome has been localized to 11p15.5 and is associated with loss or point mutation of the maternal p57kip2.18 When this region is duplicated on the paternally inherited chromosome, or when there is uniparental disomy for two paternally inherited chromosome 11s, the phenotype is expressed. Rarely, Beckwith ^Wiedemann syndrome is identi¢ed when this region is deleted on the maternal chromosome 11.18 Some time ago, it was suggested that there were similarities between the gross and histological ¢ndings of

Figure 2 P57kip2 immunohistochemical stain of diploid complete hydatidiform mole,9 weeks’gestational age.The villous cytotrophoblast, syncytiotrophoblast and stroma (mesenchyme) are negative for p57kip2. Photograph courtesy of Dr Masaharu Fukunaga,Tokyo, Japan (immunoperoxidase stain, original magni¢cation, 200x).

the placental hydrops of Beckwith ^Wiedemann syndrome and those of a hydatidiform mole.19 The recent genetic ¢ndings augment the notion that there may be a common genetic aetiology for part of the placental phenotypic expressions of these two disorders.19 p57kip2 is absent or expressed at a low level in the villous cytotrophoblast and villous stromal cells in complete hydatidiform mole (Fig. 2).12^14 Intervillous trophoblastic cell islands and decidualized stromal cells serve as positive internal controls for p57kip2 in most

4 complete hydatidiform moles.12,13 In one study, absence of p57kip2 in villous mesenchymal cells and cytotrophoblast was found in 58 of 59 complete hydatidiform moles, regardless of gestational age.13 Given that this protein product inhibits cell proliferation, the lack of gene expression in the villous stroma may account for the increased villous stromal cellularity previously recognized morphologically20 in early complete hydatidiform moles.14 It is not clear why the intervillous trophoblast in complete hydatidiform mole should show normal expression of p57kip2. Considerations include relaxation of gene imprinting or incomplete imprinting. The wild-type form of p53 is a tumour suppressor gene which appears to downregulate cell proliferation. Mutated p53 gene is seen in many malignancies and appears to signal cell transformation. In complete hydatidiform moles, p53 expression is found in 10 ^50% of cytotrophoblast and intermediate trophoblast while absent in the syncytiotrophoblast.21,22 bcl-2 prolongs cell survival and is involved in the regulation of programmed cell death.21 In the complete hydatidiform mole, bcl-2 is expressed with variable intensity as nuclear membrane or cytoplasmic immunopositivity in the syncytiotrophoblast alone. All proliferating cells such as cytotrophoblast, intermediate trophoblast and villous stroma are negative.21 Overexpression of the protein Bax allows for apoptosis. It is the counterbalance to bcl-2 which allows for cell survival. Expression of Bax in complete hydatidiform moles is con¢ned to the nuclear membrane or cytoplasm of cytotrophoblast. It is negative in the syncytiotrophoblast and villous stromal cells.21 p21WAF1/CIP1 is a protein that, like p57kip2, inhibits CDK.23 This inhibition halts progression through the cell cycle and thus inhibits cell division. Wild-type p53 induces expression of p21WAF1/CIP1 whereas the mutated forms of p53 inhibit its expression.22 Cheung et al. found this protein expressed in approximately 20% of syncytiotrophoblastic nuclei of complete hydatidiform mole.22 Unfortunately, its expression in complete hydatidiform moles failed to predict subsequent development of persistent gestational trophoblastic disease.22 Looking at two nuclear antigens expressed only in cycling cells, Ki- 67 and proliferating cell nuclear antigen (PCNA), Ostrzega et al. demonstrated elevated rates for both antigens in complete hydatidiform moles.23 Using MIB-1 (Westbrook, ME, USA) to analyse Ki- 67 expression in cytotrophoblast and intermediate trophoblast, Ostrzega et al. found 84.6 +/ 10.3% positivity in complete moles compared with 8.7+/ 10.0% in hydropic abortusesFa 10 -fold di¡erence.23 Similar results were identi¢ed for PCNA using PCNA P10 (Dako): 89.2+/ 11.1% nuclear staining in complete hydatidiform moles compared with 23.1 +/ 19.8% for hydropic abortuses.23 This elegant work con¢rms the longheld histological

CURRENT DIAGNOSTIC PATHOLOGY

impression of trophoblastic hyperplasia in complete hydatidiform moles.

PARTIAL HYDATIDIFORM MOLE The partial hydatidiform mole is an abnormal conceptus resulting from an extra haploid dose of paternal DNA. The fertilized conceptus contains 69 chromosomes with a variety of sex chromosomal combinations, apart from YYY which is lethal. In contrast to the complete hydatidiform mole which is devoid of maternal nuclear DNA, the partial hydatidiform mole has preserved the maternal nuclear DNA contribution. This biparental DNA allows for embryonic/fetal development, albeit morphologically abnormal. The majority of women with partial hydatidiform mole undergo spontaneous gonadotropin remission after evacuation. Partial hydatidiform mole has a persistent gestational trophoblastic disease rate far lower than that for the complete hydatidiform mole, being in the order of 0 ^12%.4 ^ 6 Treatment of persistent disease that develops in the setting of partial molar pregnancy requires far less chemotherapy to achieve gonadotropin remission compared with that required following complete molar pregnancy. Rare instances of choriocarcinoma have followed partial hydatidiform mole. This should not be surprising since approximately 22.5% of choriocarcinomas follow a normal pregnancy.24 The histology of partial hydatidiform mole was described over 20 years ago. It consists of two populations of villi with scalloped villous outlines, stromal trophoblastic inclusions, and focal syncytiotrophoblastic hyperplasia. Fetal/embryonic tissues are often found. The villous stromal blood vessels are usually preserved and often contain fetal nucleated red blood cells. Occasional examples of Beckwith ^Wiedemann syndrome may be confused with partial hydatidiform mole as massive hydrops of isolated villi are common in Beckwith ^ Wiedemann syndrome.19 The studied morphological pathologist should be able to distinguish the placenta of Beckwith ^Wiedemann syndrome from a partial hydatidiform mole since the histological features of scalloped villous outlines, stromal trophoblastic inclusions and syncytiotrophoblastic hyperplasia seen in the latter are lacking in the former.

Immunohistochemical studies (Table 1) The concept of trophoblastic hyperplasia in partial hydatidiform mole is a somewhat di⁄cult issue. Generally, it consists of knots or knuckles of syncytiotrophoblast akin to the accelerated villous maturation with increased syncytial knot formation seen in a pre-eclamptic placenta. Immunohistochemical attempts to evaluate this parameter have used antibodies to proteins expressed in the proliferative phases of the cell cycle. Suresh et al.

GESTATIONALTROPHOBLASTIC DISEASES

examined PCNA expression in partial hydatidiform moles and hydropic abortions, and concluded that PCNA was of no value in distinguishing between these entities as there was no signi¢cant di¡erence in the percentage of cells expressing PCNA in either category.25 This is in contrast to the work of Ostrzega et al.23 wherein the partial hydatidiform moles had a PCNA expression rate of 79.9 +/ 12.4% in the cytotrophoblast and intermediate trophoblast compared with 23.1+/ 19.8% for the hydropic abortuses. Similar proliferation values were found using Ki- 67 (MIB-1): 65.3+/ 17.6% for partial hydatidiform moles compared with 8.7+/ 10.0% for hydropic abortuses.23 The studies of Ostrzega et al.23 con¢rm the standard criteria of trophoblastic hyperplasia in partial molar pregnancy.23 When access to immunohistochemistry is limited, one is left with the eyes and the brainFadequate equipment in most instances to discern the di¡erence between partial hydatidiform mole and hydropic abortus. Although not the topic for this commentary, there is no shame in resorting to £ow or image cytometry, cytogenetic studies or £uorescence in situ hybridization (FISH) for chromosome content to distinguish triploid from diploid DNA content in di⁄cult cases.4 ^ 6 As described above, immunohistochemical studies for the di¡erentially imprinted gene products such as p57kip2 show normal expression of this protein in the villous mesenchyme and villous cytotrophoblast in partial moles and normal placentas (Fig. 3). This is compared with its absence in the cytotrophoblast and mesenchyme of complete moles (vide supra).12 Another CDK inhibitor, p21WAF1/CIP1 is found in approximately10% of syncytiotrophoblastic nuclei in a partial hydatidiform mole compared with 20% in a complete hydatidiform mole.22 In

5

these terminally di¡erentiated cells, it is postulated that p21 maintains growth arrest.22

PLACENTAL SITE TROPHOBLASTIC TUMOUR There are three potentially malignant gestational trophoblastic tumours which do not form villi.They are the placental site trophoblastic tumour, the epithelioid trophoblastic tumour1 and choriocarcinoma. The placental site trophoblastic tumour tends to follow a normal pregnancy or, much less commonly, a complete hydatidiform mole.26 In an interesting study, the antecedent gestation was found to be far more commonly female, suggesting that a paternally imprinted X chromosome was required for tumourigenesis.27 In contrast to complete hydatidiform mole and choriocarcinoma, this tumour often spreads by direct local extension and contiguous nodal involvement before widespread dissemination occurs. Maternal serum b HCG remains in a low-positive range in placental site trophoblastic tumour. The majority of women, approximately 85%, are cured of this disease. The mainstay of treatment is surgical resection, with chemotherapy added as clinically indicated. The placental site trophoblastic tumour consists of a proliferation of intermediate trophoblast mimicking the normally invasive intermediate trophoblast of an implantation site.This intermediate trophoblast tends to be uninucleate, although binucleate and multinucleate forms may exist. Occasionally, admixed syncytiotrophoblast may be present, but ¢nding alternating syncytiotrophoblast and cytotrophoblast with abundant haemorrhagic necrosis warrants a diagnosis of choriocarcinoma. A characteristic feature of placental site trophoblastic tumour is the perivascular accumulation of large, eosinophilic tumour cells and striking endovascular growth wherein tumour cells cling to the endothelial lining, focally replacing endothelium, and/or lying freely in the vascular lumen.

Immunohistochemical features (Tables 2 and 3)

Figure 3 p57kip2 immunohistochemical stain of triploid partial hydatidiform mole,11 weeks’gestational age. Positive (brown nuclear) staining is found in villous cytotrophoblast and villous stroma (mesenchyme) indicating presence of maternal DNA. Photograph courtesy of Dr Masaharu Fukunaga, Tokyo, Japan (immunoperoxidase stain, original magni¢cation, 200x).

The placental site trophoblastic tumour stains positively for pancytokeratin cocktails, cytokeratin 18, epithelial membrane antigen, human placental lactogen (HPL), and placental alkaline phosphatase (PLAP). A rare cell shows focal staining for HCG, generally con¢ned to a few syncytiotrophoblasts or to an occasional uninucleate trophoblast within the tumour. The tumour is strongly positive for inhibin-a, a protein which serves to inhibit follicle stimulating hormone and to regulate HCG production in the placenta, among other functions.3,28 MelCAM, a marker originally identi¢ed in a melanoma cell line, is found in intermediate trophoblast, cerebellar

6

CURRENT DIAGNOSTIC PATHOLOGY

Table 2 Immunohistochemical studiesin exaggerated placental site reaction, placental site trophoblastic tumour, epithelioid trophoblastic tumour and choriocarcinoma Exaggerated placental site Inhibin-a (cytoplasmic) Ki-67 (nuclear)

o1%

Mel-CAM (CD146) (membranous)b Cytokeratin AE1/AE3 Cytokeratin18 p53 (wild-type and mutated p53) Epithelial membrane antigen E-cadherin HPL HCG

Di¡use positive Positive Positive

Placental site trophoblastic tumor

Epithelioid trophoblastic tumor25

Choriocarcinoma

Positive in IT 14+/ 6.9%, or 410%; 21.1+/ 12.5% (range = 8.3^44.6%) Di¡use positive

1^3+, di¡use 17.7+/ 4.5%, range10^25%

Positive in SCT

1+, focal in 71%; di¡use in two cases 4+, di¡use Positive

1^3+

Positive Positive Positive in o50% of cells Positive

EGF-R pMBP (intracellular and extracellular)c

Positive Positive

1^3+, di¡use

3+,100% Positive Negative

3+,96% positive Negative to occasional positive cells

100% positive

77% positive, including positivity for eosinophilic hyaline material

2^4+, di¡use 1+, focal, in 64% 1+, focal, in 43%

1^3+ 2^3+

3^4+, di¡use

a

seen in villous and extravillous trophoblast; bMel-CAM stains intermediate trophoblast of implantation site origin; cPregnancyassociated major basic protein stains intermediate trophoblast; by immuno£uorescence or immunohistochemistry; SCT, syncytiotrophoblast; IT intermediate trophoblast; CT, cytotrophoblast; 1+, 6^25% cells positive; 2+, 26^50% cells positive; 3+, 51^75% cells positive; 4+,475% cells positive.Cellular location of positive staining, whether nuclear, membranous or cytoplasmic, is indicated in parentheses for the newer antibodies. See text for explanation and references.HPL, human placental lactogen; HCG, human chorionic gonadotropin; EGF-R, epithelial growth factor receptor; Mel-CAM, melanoma cell adhesion molecule.

Table 3 Proliferative activity of the nonvillous trophoblastic lesions Normal implantation site

Exaggerated placental site

Molar implantation site

Epithelioid trophoblastic tumour

Placental site trophoblastic tumour

Choriocarcinoma

Ki-67 (MIB-1)

0%

0%

5.2+/ 4.0%

17.7+/ 4.5%, range10^25%

69+/ 20%

Mitotic indexa

0

0

14+/ 6.9%; 21.1+/ 12.5 (range = 8.3^44.6%) 0.2 ^ 6.8, average = 1.85 Variable, 0^6

Mitotic count/10 hpf

Variable, 0^10, average = 2

2^22.7, mean = 9.3 High, 2^22

*Number of mitoses/10 high power ¢elds in in Mel-CAM-positive cells (intermediate trophoblast). See text for explanation and references.

cortex, hair follicles, endothelium and smooth muscle. It seems to be a sensitive, although not speci¢c, marker of intermediate trophoblast. It is expressed in a membranous (cell-surface) distribution in this tumour.29 The nuclear cell proliferation antigen, Ki- 67, is expressed in cells that have entered the cell cycle and are committed to cell division. An antibody to Ki- 67, MIB-1,

is positive in more than 14%+/ 6.9% of Mel-CAM-positive cells in placental site trophoblastic tumour.30 This helps distinguish the placental site trophoblastic tumour from an exaggerated placental site which has nearly zero MIB-1-positive cells in the Mel-CAM-positive intermediate trophoblast. In using this immunohistochemical stain without Mel-CAM identi¢cation of intermediate

GESTATIONALTROPHOBLASTIC DISEASES

trophoblast, care must be taken to avoid counting the MIB-1-positive T-cell lymphocyte nuclei and the large granular lymphocytes. An example of the usefulness of proliferation markers in the di¡erentiation between placental site trophoblastic tumour and exaggerated placental site reaction is seen in Fig. 4. A uterine curettage specimen revealed multiple, classic, placental site nodules with a few clusters of admixed atypical intermediate trophoblast. The latter was examined immunohistochemically for Ki- 67 expression. Despite the lack of dual Mel-CAM staining, the large nuclei and the abundance of cytoplasm in these cells con¢rmed their intermediate trophoblastic nature. Positivity for Ki- 67 demonstrated a brisk proliferative rate, and a diagnosis of placental site trophoblastic tumour was made (Fig. 4). Subsequent hysterectomy con¢rmed placental site trophoblastic tumour. Ichikawa et al.31 studied the immunohistochemical pro¢le of placental site trophoblastic tumours.Their extensive evaluation included many cyclins, CDKs, Ki- 67 and p53. These investigators found cyclin A, cyclin B, cyclin D1, cyclin E,CDK 2,CDK4,CDC2 and p53 expressed in placental site trophoblastic tumours. Interestingly, in some tumours, over 50% of the tumour cells were positive for cyclin E.31 All pregnant women have pregnancy-associated eosinophil granule major basic protein in their sera. In placental site trophoblastic tumour, it is localized by immuno£uorescence and immunohistochemistry to the intermediate trophoblast and multinucleated placental

Figure 4 Ki-67 expression using MIB-1 antibody (Immunotech) in uterine currettings of a placental site trophoblastic tumour. Positive cells indicated by nuclear staining in intermediate trophoblast which exceed thatexpected in exaggerated implantation site and placental site nodule. This case had admixed benign placental site nodules as well. Diagnosis of placental site trophoblastic tumour was con¢rmed on subsequent hysterectomy. (Note few immunopositive lymphocytes as well.) (immunoperoxidase stain, original magni¢cation,40x).

7 site giant cells. It is not found in other trophoblasts.32 As such, like Mel-CAM, it serves as a sensitive marker for intermediate trophoblast and, as the name implies, eosinophils. It is present in 78% of all placental site trophoblastic tumours compared with 96% positivity for HPL in the same tumours.32

EPITHELIOID TROPHOBLASTIC TUMOUR The epithelioid trophoblastic tumour is a very rare trophoblastic tumour. Shih and Kurman described 14 cases in their initial report in 1998.1 According to the authors, this intermediate trophoblastic tumour is derived from the intermediate trophoblast of the chorion laeve (of the placental membranes) and consequently has a di¡ering natural history from the placental site trophoblastic tumour which arises from implantation site trophoblast. The epithelioid trophoblastic tumour seems to follow normal pregnancy in most instances. These are most commonly found in the uterus, but sites of extra-uterine involvement have been reported.1,2 We have seen a case clinically followed by ultrasonography as a lower uterine segment leiomyoma for a number of years, only to prove to be an epithelioid trophoblastic tumour. Fortunately, these tumours appear to be amenable to surgical intervention. Combining similarly reported cases shows that although the majority of women with epithelioid trophoblastic tumours are alive and well, 25% develop recurrence, and 10% have died.1 This tumour is characterized by a striking geographical appearance with a few cellular islands of uniform, viable tumour, inconspicuous stroma and central blood vessels, and a surrounding sea of eosinophilic, necrotic debris admixed with ¢brillar, hyaline-like material (Fig. 5). The entire tumour assumes a geographical appearance, which is undoubtedly due to large bands of ischaemic necrosis which have accreted over time as the tumour continued to outpace its blood supply. The tumour cells are mainly uninucleate, with a round, uniform nucleus, ¢nely dispersed chromatin with inconspicuous nucleoli, and clear to eosinophilic cytoplasm. Scattered cells have larger, cleaved nuclei with eosinophilic cytoplasm (Fig. 6). Scattered lymphocytes and plasma cells are admixed within, and in the periphery of, tumour islands. Interestingly, in the study of Shih and Kurman, nearly half of the cases had associated foci of placental site nodule, placental site trophoblastic tumour or choriocarcinoma.1 Is it not possible that some of these tumours are a dedi¡erentiated form of the neoplasm of origin, akin to the unusual variants of metastatic gestational choriocarcinoma reported by Mazur33 and Jones et al.,34 and the nodules of intermediate trophoblast of Silva et al.35 which followed hydatidiform moles? Shih and Kurman noted that some epithelioid trophoblastic tumours even have transitional features between placental site nodule

8

CURRENT DIAGNOSTIC PATHOLOGY

determine whether this is a unique lesion, or simply a histological variant of another. Only then could one discern whether these tumours will follow the expected course of the incident tumour, or, quite possibly, take a divergent path. Such musings fall far short of the challenge, controversy and animus currently inherent in diagnosing a malignant ¢brous histiocytoma.

Immunohistochemical features (Tables 2 and 3)

Figure 5 Epithelioid trophoblastic tumour. Note central island of viable tissue with nests and islands of tumour cells surrounded by pale, acellular eosinophilic debris.Tumour cells have open chromatin, ample eosinophilic cytoplasm and moderate mitotic activity. Admixed lymphocytes are present (H&E, original magni¢cation,10x).

The epithelioid trophoblastic tumour is, like all gestational trophoblastic tumours, strongly positive for pancytokeratins and CK18. The tumour also expresses epithelial membrane antigen, epidermal growth factor receptor, inhibin-a and E-cadherin.1 Inhibin-a distribution is variable. In addition, very focal positive immunostaining for HPL is found in 64% of cases, HCG in 43%, placental alkaline phosphatase in 57% and Mel-CAM in 71%.1 Most of the cells are negative for HPL and HCG. Ki- 67 proliferation rates are relatively low for a trophoblastic tumour, being between 10 and 25%.1

CHORIOCARCINOMA

Figure 6 Epithelioid trophoblastic tumour. Viable tumour cells are embedded in eosinophilic hyaline-like material.The cells have abundant eosinophilic to clear cytoplasm. Each cell has a large nucleus with a prominent single, large nucleolus, or has dispersed chromatin withtiny chromocentres.There are two mitotic ¢gures, scattered lymphocytes and plasma cells (H&E, original magni¢cation,40x).

and epithelioid trophoblastic tumour.1 Thus, they propose that this lesion represents the ‘... neoplastic counterpart of the placental site nodule.’1 Hamazaki et al. suggest that epithelioid trophoblastic tumour may represent dedi¡erentiation of a choriocarcinoma which has lost some level of trophoblastic di¡erentiation.2 Since the known natural histories of the incident tumours (normal placenta, hydatidiform mole, placental site nodule, placental site trophoblastic tumour and choriocarcinoma) are so disparate, it will be interesting to identify additional examples of this tumour in order to

Choriocarcinoma is the most aggressive form of gestational trophoblastic disease. It may develop on its own, termed ‘ab initio’, or occur subsequent to another form of gestation, most commonly, a complete hydatidiform mole. Approximately 25% of cases follow apparently normal pregnancy.24 Some instances have been identi¢ed in situ in term placentas. Women with choriocarcinoma generally have a very high serum b-HCG value. Metastasis at the time of initial diagnosis is not uncommon. Choriocarcinoma was the ¢rst tumour to be eradicated by chemotherapy. Even in advanced, widely disseminated disease, cure rates in excess of 90% are reported with multi-agent chemotherapy, occasionally combined with radiotherapy and surgery as indicated. Choriocarcinoma is generally easily recognized by the pathologist when adequate tissue is provided for examination. Since this tumour tends to be highly necrotic, uterine curettage specimens often contain only necrotic tissues, debris and a few viable cells.The tumour consists of sheets of mononuclear cytotrophoblast and intermediate trophoblast with scattered syncytiotrophoblast. Viable tumour cells exist on the fringe of necrotic debris. Destructive tissue invasion is a hallmark.Vascular space invasion is often seen, with a unique featureFthe tumour often appears to be heading out of the vessels into the surrounding tissues rather than vice versa.

Immunohistochemistry (Tables 2 and 3) Although rarely needed, immunohistochemical studies of choriocarcinoma reveal strong, di¡use pancytokeratin and HCG staining, and focal HPL staining.

GESTATIONALTROPHOBLASTIC DISEASES

SUMMARY Immunohistochemical studies of gestational trophoblastic tumours have advanced our ability to determine the cell of origin of some of these tumours. Being epithelial tumours, all trophoblastic tumours are positive for pancytokeratin (AE1/AE3) and CK18.The addition of antibodies to Mel-CAM and inhibin-a allows us to further subclassify such tumours by suggesting a possible trophoblastic origin. While both of the latter markers are sensitive for trophoblastic tumours, they are not speci¢c as Mel-CAM, originally identifed from a melanoma cell line, is expressed in other tissues such as cerebellar cortex, hair follicles, endothelium and smooth muscle, and inhibin-a is expressed in virtually all tissues of adrenal cortical origin.3,29 Due to vagaries of imprinting, early hydatidiform moles may be further subclassi¢ed immunohistochemically as partial or complete depending on stromal and cytotrophoblastic staining of p57kip2. No expression is found in these cellular constituents in complete hydatidiform moles in contrast to normal positive expression in partial hydatidiform moles.Of note, the syncytiotrophoblastic nuclei of complete hydatidiform moles show greater expression of P21WAF1/CIP1 than partial moles. This is interesting because this gene product inhibits the action of CDKs and thus shuts down cell division.This comports with the notion that these cells are synthesizing bHCG, not preparing to divideFwhich they are not able to do anyway, perhaps in part because of p21WAF1/CIP1. We hope that these very informative immunohistochemical studies may guide us in formulating strategies for identifying trophoblastic-speci¢c gene products. These will allow for the development of chemotherapeutic agents speci¢cally targeted to eradicate these tumours.

PRACTICE POINTS Complete hydatidiform mole Di¡usedly hydropic villous tissue K Trophoblastic hyperplasia, moderate to marked K Embryo generally lacking kip2 absent in villous mesenchyme and villous K p57 cytotrophoblast K High MIB-1 (Ki-67) and PCNA expression in cytotrophoblast and intermediate trophoblast K

Partial hydatidiform mole Two populations of villi K Focal trophoblastic hyperplasia K Embryonic tissues common kip2 staining present in villous mesenchyme and K p57 villous cytotrophoblast K Moderately elevated MIB-1 (Ki-67) and PCNA in cytotrophoblast and intermediate trophoblast K

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Placental site trophoblastic tumour K Mononuclear cells, predominantly uninucleate K Non-destructive myometrial invasion K Perivascular and intravascular growth K Pancytokeratin, HPL, inhibin-alpha and Mel-CAM positive K MIB-1 (Ki-67) in14% of tumour cells Epithelioid trophoblastic tumour Geographic necrosis, extensive K Central blood vessels with surrounding viable tumour cells K Pancytokeratin,CK-18, inhibin-alpha positive; mostly negative for HCG and HPL, except for an occasional, strongly positive cell K MIB-1 (Ki-67) in10^25% of cells K

Choriocarcinoma K Biphasic tumour with syncytiotrophoblast, intermediate trophoblast and cytotrophoblast K Extensive, di¡use, haemorrhagic necrosis K Destructive stromal invasion K Intravascular growth with tumour invading surrounding tissues K Pancytokeratin and HCG positive; focal HPL staining

ACKNOWLEDGEMENT The authors sincerely thank Dr Masaharu Fukunaga,The Jikei Daisan Hospital, Tokyo, Japan for his kind assistance in providing original photomicrographs of p57 expression in partial and complete hydatidiform moles (Figs 2 and 3).

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CURRENT DIAGNOSTIC PATHOLOGY

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