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Immunohistochemical Analysis of Peritoneal

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					Anatomic Pathology / PERITONEAL MESOTHELIOMA AND SEROUS CARCINOMA

Immunohistochemical Analysis of Peritoneal Mesothelioma and Primary and Secondary Serous Carcinoma of the Peritoneum Antibodies to Estrogen and Progesterone Receptors Are Useful
Robert J. Barnetson, MBChB,1 Rodney A. Burnett, FRCPath,1 Ian Downie, MSc,2 Christina M. Harper, MRCPath,3 and Fiona Roberts, MRCPath1
Key Words: Peritoneal mesothelioma; Primary peritoneal carcinoma; Metastatic ovarian carcinoma; Estrogen receptor; Progesterone receptor
DOI: 10.1309/8FCHQ3VPBWM7B5X9

Abstract
The role of immunohistochemical markers in distinguishing peritoneal mesothelioma from primary or metastatic serous papillary carcinoma of the peritoneum was evaluated. We immunostained 20 peritoneal mesotheliomas (from 14 men and 6 women), 14 primary peritoneal carcinomas, and 14 metastatic serous ovarian carcinomas with a panel of 16 antibodies. Positive staining for calretinin was identified in 17 (85%) of 20 mesotheliomas, but all carcinomas were negative. Positive staining for BerEP4 was identified in 27 (96%) of 28 carcinomas and in 2 (10%) of 20 mesotheliomas. Estrogen receptors were positive in 26 (93%) of 28 carcinomas, and progesterone receptors were positive in 8 (29%) of 28 carcinomas. All mesotheliomas were negative for estrogen and progesterone receptors. The other antibodies evaluated were insufficiently sensitive and/or specific to be diagnostically useful. In conjunction with calretinin and Ber-EP4, estrogen and progesterone receptors are useful discriminatory markers for distinguishing peritoneal mesothelioma from primary or metastatic serous carcinoma.

Malignant mesothelioma of the peritoneum (PMM) is less common than its counterpart in the pleural cavity, accounting for only around 10% of all mesotheliomas.1 However, like pleural mesothelioma, the diagnosis of PMM can be challenging, particularly in the female peritoneum, where it may mimic serous carcinoma of the ovary or peritoneum. Nevertheless, accurate distinction of these tumors is important for clinical and medicolegal reasons. Malignant mesothelioma is a chemotherapy- and radiotherapy-resistant tumor with a dismal prognosis, whereas these treatment modalities can significantly improve patient survival in primary and metastatic peritoneal serous carcinoma (PSC).2-5 No etiologic relationship has been established between asbestos exposure and PSC. PMM, however, is recognized to occur after asbestos exposure, and this diagnosis, therefore, might lead to medicolegal compensation.1,6 It should be noted, however, that in women, the proportion of cases with asbestos exposure is lower than in men and it usually is of the paraoccupational (domestic) type.7 Histologically, epithelioid malignant mesothelioma might have an appearance identical to that of primary or metastatic serous carcinoma.8,9 Furthermore, mucin stains are considerably less helpful for differentiating between these tumors than for distinguishing epithelioid malignant mesothelioma from metastatic adenocarcinoma in the pleura.10 Immunohistochemical analysis generally is regarded as the most important ancillary technique to distinguish between these tumors.11-14 However, these tumors are uncommon compared with pleural mesothelioma and adenocarcinoma, and there are few studies that have addressed this specific problem. The aim of the present study was to identify the most accurate and clinically useful immunohistochemical panel
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that can reliably distinguish epithelioid malignant mesothelioma from primary or metastatic ovarian serous carcinoma of the peritoneum. Our antibody panel included well-recognized markers of mesothelioma and adenocarcinoma. Antibodies to estrogen receptor (ER) and progesterone receptor (PR) also were included.

Materials and Methods
Tumor Samples Retrospective paraffin-embedded tissue samples were retrieved from the pathology records of the Western Infirmary, Royal Infirmary, and Southern General Hospital, Glasgow, Scotland; the Victoria Infirmary, Kirkaldy, Fife, Scotland; and the Western General, Edinburgh, Scotland. The specimens included 14 samples from men and 8 samples from women initially diagnosed with PMM, 14 samples from women with metastatic ovarian carcinoma (MOC), and 14 samples from women with primary peritoneal carcinoma (PPC). For PMM, the specimens consisted of needle or open biopsy specimens, and the microscopic features were considered consistent with those reported in standard texts. These histologic findings were supported by appropriate immunohistochemical, ultrastructural, or postmortem findings. In each case, the clinical findings were of a diffuse peritoneal tumor with no evidence of a primary tumor elsewhere in the body, and, in most cases, there was a well-documented history of asbestos exposure. By using these criteria, 2 cases initially diagnosed as PMM occurring in women were excluded. In one case, electron microscopy had been undertaken on the tumor showing the presence of short microvilli more in keeping with adenocarcinoma. In the other case, a postmortem
❚Table 1❚ Antibodies Used for Immunohistochemical Analysis
Antibody Carcinoembryonic antigen Human epithelial antigen LeuM1 Mesothelin CA 125 Epithelial-related antigen Thyroid transcription factor-1 Estrogen receptor Progesterone receptor Cytokeratin 5 Cytokeratin 7 Cytokeratin 20 Calretinin Thrombomodulin WT1 CD44H
MWPC, pressure cook, microwave.

examination subsequently showed the presence of a primary ovarian adenocarcinoma. For PPC, the specimens consisted of open biopsy specimens or specimens from debulking surgery. The diagnosis of PPC was based on appropriate microscopic features and confirmation of lack of ovarian involvement at laparoscopy or by surgical pathology. Similar to PMM, the diagnoses were supported by appropriate immunohistochemical, ultrastructural, or postmortem findings. MOCs consisted of open biopsy specimens or peritoneal metastases removed at the time of oophorectomy. All MOCs included in this study were classified as papillary serous adenocarcinomas based on appropriate histopathologic features. Immunohistochemical Analysis Information about the antibodies selected is given in ❚Table 1❚. For immunohistochemical analysis, 3-µm sections were immunostained by using a DAKO Autostainer using EnVision Peroxidase (DAKO, Ely, England). Briefly, sections were washed with tris(hydroxymethyl)aminomethanebuffered saline containing polysorbate (Tween) buffer (TBS/TB) and then blocked with normal goat serum (dilution 1:20) for 20 minutes. Sections then were incubated with primary antibody for 30 minutes followed by another wash in TBS/TB. Following this step, the sections were incubated with DAKO EnVision for 30 minutes. After a final wash in TBS/TB, color was developed with diaminobenzidine for 10 minutes and sections counterstained with hematoxylin. For antigen retrieval, slides were microwaved under pressure in 1 mmol/L of EDTA, pH 8.0, for 5 minutes or incubated with 0.1% trypsin, pH 7.8, for 25 minutes at 37°C. Appropriate control material (according to the manufacturer’s instructions) was used for each run. For negative control samples, the primary antibody was omitted.

Clone II-7 Ber-EP4 LeuM1 5B2 OV185:1 MOC31 SPT 24 6F11 PGR636 XM26 OV-TLR12/30 Ks20.8 5A5 15C8 6F-H2 F10-44-2

Source DAKO, Ely, Cambridge, England DAKO Becton Dickinson, Oxford, England Becton Dickinson Vector, Peterborough, England DAKO Vector Vector DAKO Vector DAKO DAKO Vector Vector DAKO DAKO

Dilution 1:400 1:200 1:200 1:20 1:400 1:20 1:50 1:50 1:400 1:200 1:500 1:500 1:100 1:200 1:50 1:100

Pretreatment Trypsin Trypsin MWPC MWPC MWPC Trypsin MWPC MWPC MWPC MWPC MWPC MWPC MWPC Trypsin MWPC MWPC

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Interpretation of Results The slides were evaluated semiquantitatively, and the percentage of positive tumor cells was described as follows: 1+, staining 1% to 49% of tumor cells, or 2+, staining 50% to 100% of tumor cells. All cases showing staining of less than 1% of tumor cells were regarded as negative. Weak staining or staining that was difficult to interpret was regarded as equivocal. The pattern of staining was recorded as membranous, cytoplasmic, or nuclear for each antibody. The sections were assessed independently by 2 observers (R.J.B. and F.R.). For cases in which there was substantial disagreement, the slides were assessed by a third observer (R.A.B.), and consensus was reached.

Ber-EP4, Carcinoembryonic Antigen, LeuM1, and MOC31 Strong, membranous staining for Ber-EP4 was seen in 27 (96%) of 28 carcinomas. Of the PMMs, 2 (10%) of 20 showed 1+ staining for Ber-EP4. All PMMs occurring in both men and women were negative for carcinoembryonic antigen (CEA) and LeuM1. Positive staining for CEA was observed in only 1 (4%) of 28 PSCs (a PPC). Of 28 carcinomas, 14 (50%) were positive for LeuM1, although staining was focal in the majority of cases. Strong membranous staining for MOC31 was seen in all 28 PSCs. Of 20 PMMs, 5 (25%) showed 1+ staining for MOC31. CK5, CK7, and CK20 Of 20 PMMs, 18 (90%) stained strongly with CK5. CK5 staining was seen in 14 (50%) of 28 PSCs. Strong CK7 staining was observed in 17 (85%) of 20 PMMs and in all 28 PSCs (100%). CK20 was negative in all PMMs and PSCs.

Results
The results are summarized in ❚Table 2❚, ❚Table 3❚, ❚Table 4❚, ❚Table 5❚, and ❚Table 6❚ and shown in ❚Image 1❚ and ❚Image 2❚.

❚Table 2❚ Immunohistochemical Staining of Peritoneal Mesotheliomas and Carcinomas*
Tumor Type CEA Ber-EP4 LeuM1 MOC31 CA 125 ER PR CK5 CK7 CK20 Mesothelin Calretinin TM WT1 CD44H TTF-1

Mesothelioma Male (n = 14) Female (n = 6) Total (n = 20) Carcinoma MOC (n = 14) PPC (n = 14) Total (n = 28)

0 0 0 0 1 1

1 1 2 13 14 27

0 0 0 8 6 14

2 1 3 14 14 28

5 4 9 13 13 26

0 0 0 13 13 26

0 0 0 5 3 8

12 6 18 8 6 14

14 3 17 14 14 28

0 0 0 0 0 0

9 4 13 13 12 25

12 5 17 0 0 0

6 3 9 1 0 1

8 3 11 10 11 21

10 5 15 2 2 4

0 0 0 0 1 1

CEA, carcinoembryonic antigen; CK, cytokeratin; ER, estrogen receptor; MOC, metastatic ovarian carcinoma; PPC, primary peritoneal carcinoma; PR, progesterone receptor; TM, thrombomodulin; TTF-1, thyroid transcription factor-1; WT1, Wilms tumor 1. * For proprietary information, see Table 1.

❚Table 3❚ Immunohistochemical Staining of Peritoneal Mesothelioma in 14 Men*
Antibody CEA Ber -EP4 LeuM1 MOC31 CA 125 ER PR CK5 CK7 CK20 Mesothelin Calretinin Thrombomodulin WT1 CD44H TTF-1 2+ 0 0 0 0 2 0 0 10 14 0 6 10 1 2 8 0 1+ 0 1 0 4 3 0 0 2 0 0 3 2 5 6 2 0 Equivocal 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1 0 Negative 14 13 14 11 9 14 14 2 0 14 5 2 8 6 3 14

❚Table 4❚ Immunohistochemical Staining of Peritoneal Mesothelioma in 6 Women*
Antibody CEA Ber -EP4 LeuM1 MOC31 CA 125 ER PR CK5 CK7 CK20 Mesothelin Calretinin Thrombomodulin WT1 CD44H TTF-1 2+ 0 0 0 0 2 0 0 4 3 0 4 4 0 2 5 0 1+ 0 1 0 1 2 0 0 2 0 0 0 1 3 1 0 0 Equivocal 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 Negative 6 5 6 5 2 6 6 0 3 6 2 1 3 2 1 6

CEA, carcinoembryonic antigen; CK, cytokeratin; ER, estrogen receptor; PR, progesterone receptor; TTF-1, thyroid transcription factor-1; WT1, Wilms tumor 1. * 1+, staining 1% to 49% of tumor cells; 2+, staining 50% to 100% of tumor cells. For proprietary information, see Table 1.

CEA, carcinoembryonic antigen; CK, cytokeratin; ER, estrogen receptor; PR, progesterone receptor; TTF-1, thyroid transcription factor-1; WT1, Wilms tumor 1. * 1+, staining 1% to 49% of tumor cells; 2+, staining 50% to 100% of tumor cells. For proprietary information, see Table 1.

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❚Table 5❚ Immunohistochemical Staining of 14 Metastatic Serous Ovarian Carcinomas*
Antibody CEA Ber -EP4 LeuM1 MOC31 CA 125 ER PR CK5 CK7 CK20 Mesothelin Calretinin Thrombomodulin WT1 CD44H TTF-1 2+ 0 12 1 8 12 10 1 0 13 0 12 0 0 10 0 0 1+ 0 1 7 6 1 3 4 8 1 0 1 0 1 0 2 0 Equivocal 0 0 0 0 1 1 0 2 0 0 1 0 0 2 0 0 Negative 14 1 6 0 0 0 9 4 0 14 0 14 13 2 12 14

❚Table 6❚ Immunohistochemical Staining of 14 Primary Peritoneal Carcinomas*
Antibody CEA Ber -EP4 LeuM1 MOC31 CA 125 ER PR CK5 CK7 CK20 Mesothelin Calretinin Thrombomodulin WT1 CD44H TTF-1 2+ 1 12 2 10 11 12 1 1 14 0 10 0 0 10 1 1 1+ 0 2 4 4 2 1 2 5 0 0 2 0 0 1 1 0 Equivocal 0 0 0 0 1 0 1 2 0 0 0 0 1 1 0 0 Negative 13 0 8 0 0 1 10 6 0 14 2 14 13 2 12 13

CEA, carcinoembryonic antigen; CK, cytokeratin; ER, estrogen receptor; PR, progesterone receptor; TTF-1, thyroid transcription factor-1; WT1, Wilms tumor 1. * 1+, staining 1% to 49% of tumor cells; 2+, staining 50% to 100% of tumor cells. For proprietary information, see Table 1.

CEA, carcinoembryonic antigen; CK, cytokeratin; ER, estrogen receptor; PR, progesterone receptor; TTF-1, thyroid transcription factor-1; WT1, Wilms tumor 1. * 1+, staining 1% to 49% of tumor cells; 2+, staining 50% to 100% of tumor cells. For proprietary information, see Table 1.

Thyroid Transcription Factor-1 All PMMs were negative for thyroid transcription factor1 (TTF-1). Of 28 PSCs, 1 showed strong nuclear staining for TTF-1. This case had been designated as a PPC. CA 125, ER, and PR Strong staining for CA 125 was seen in 9 (45%) of 20 PMMs. Of 28 PSCs, 26 (93%) showed strong positive staining for CA 125. In the remaining 2 cases, staining was considered equivocal. ER and PR were negative in all 20 PMMs. Of 28 PSCs, 26 (93%) were strongly positive for ER. In 1 of the remaining 2 cases (an MOC), staining was considered equivocal. PR was positive in 8 (29%) of 28 PSCs. In 1 of the remaining 20 cases (a PPC), staining was considered equivocal. Calretinin, Mesothelin, Thrombomodulin, CD44H, and WT1 Calretinin was positive in 17 (85%) of 20 PMMs, with nuclear staining in addition to cytoplasmic staining. None of the PSCs was positive for this antibody. Mesothelin was positive in 13 (65%) of 20 PMMs and in 25 (89%) of 28 PSCs. Thrombomodulin was positive in 9 (45%) of 20 PMMs. One of the PSCs was positive for this antibody. CD44H was positive in 15 (75%) of 20 PMMs. One of the remaining 5 cases showed equivocal staining. Of 28 PSCs, 4 (14%) showed positive staining for this antibody. WT1 positivity was seen in 11 (55%) of 20 PMMs. Of 28 PSCs, 21 (75%) showed strong positive staining for this antibody. Another 3 cases showed equivocal staining.
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Discussion
Immunohistochemical analysis has been used extensively to assist in the distinction between malignant mesothelioma of the pleura and metastatic adenocarcinoma, usually of lung origin.11-18 In contrast, the immunohistochemical analysis of PMM has received little attention. The peritoneal serosal lining is subject to a different environment from the pleura, and this may be expressed as a different antigen profile. Furthermore, the majority of biopsy specimens obtained from the peritoneum are larger compared with needle biopsy specimens of the pleura such that differences in tissue fixation might alter antigen expression.19 This is compounded by the apparently decreasing incidence of PMM relative to the dramatic increase in pleural mesotheliomas.1 In women, however, it is likely that the decrease in incidence is due in part to increased recognition of PSC, which previously might have been misdiagnosed as PMM.3,20,21 In the present study we had planned to have equal numbers of cases of PMM from men and women but were able to find only 8 cases in women designated as PMM in our archives. There was, however, no difference in the immunostaining pattern between samples from men and women. Indeed, 2 of the 8 cases originally reported as PMM in women were excluded from the study because review of the cases revealed that postmortem and electron microscopic findings were those of adenocarcinoma. Both of these cases yielded an immunophenotype in keeping with that of a peritoneal serous carcinoma. This highlights the diagnostic difficulties associated with distinguishing between these 2 tumors and supports the case for the use of immunohistochemical analysis.
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A

B

C

D

E

F

❚Image 1❚ A, Malignant mesothelioma from a man showing diffuse infiltration of peritoneal fat by an epithelioid tumor (H&E, ×100). B, Immunohistochemical staining of this mesothelioma for calretinin shows cytoplasmic and nuclear staining of the tumor cells (×100). C, Negative staining for Ber-EP4 in the same mesothelioma as in A (×100). D, Primary peritoneal carcinoma showing papillary areas and occasional psammoma bodies (arrows) (H&E, ×100). E, Negative staining for calretinin in this primary peritoneal carcinoma. Note the positive staining of the mesothelium lining the peritoneum (arrows) (×100). F, Immunohistochemical staining of this primary peritoneal carcinoma for Ber-EP4 shows membranous staining of the tumor cells (×100).
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A

B

C

D

E

F

❚Image 2❚ A, Malignant mesothelioma from a woman showing diffuse infiltration of peritoneal tissues by an epithelioid tumor with pseudoglandular spaces (H&E, ×100). B, Immunohistochemical staining for estrogen receptor (ER) is negative. The brown pigment is hemosiderin from previous hemorrhage (×100). C, Immunohistochemical staining for progesterone receptor (PR) is negative. The brown pigment is hemosiderin from previous hemorrhage (×100). D, Metastatic ovarian serous adenocarcinoma showing papillary and pseudoglandular areas (H&E, ×100). E, There is strong nuclear staining for ER in this metastatic ovarian carcinoma (×100). F, There is focal nuclear positivity for PR in this metastatic ovarian carcinoma (×100).
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The present study was planned to ascertain the usefulness of commercially available, well-established antibodies in the distinction of PMM from primary or metastatic serous adenocarcinoma. The panel included markers considered relatively specific and sensitive for both mesothelioma and adenocarcinoma. In addition, because serous MOCs and PPCs are tumors of the primary and secondary müllerian systems, respectively, ER and PR also were included. These have been reported to be negative in PMM.22 Ber-EP4 is a monoclonal antibody produced from the MCF-7 breast carcinoma cell line that recognizes epithelial glycoproteins.23 It has been used in many studies to discriminate between pleural mesothelioma and adenocarcinoma11-14,16,24 and to a lesser extent in the peritoneal setting.8-10,24 Ordóñez8 found Ber-EP4 positivity in 100% of 45 PSCs, whereas only 11% of PMMs (4/35) showed positivity in a limited number of cells. Similarly Attanoos et al9 found positivity for Ber-EP4 in 95% of papillary serous ovarian cancers (19/20) and 9% of PMMs (3/32). Our study confirmed the high sensitivity and specificity of Ber-EP4 with strong membranous staining in 96% of PSCs (27/28; 13/14 MOCs and 14/14 PPCs). There was unequivocal positive staining for BerEP4 in 10% of PMMs (2/20; in 1 woman and 1 man). Although both of these cases also showed focal positivity for MOC31, there was, in addition, 2+ staining for calretinin and focal staining for thrombomodulin. ER is a ligand-activated transcription factor that mediates the effect of the steroid hormone 17β-estradiol in men and women.25 PR is an estrogen-inducible protein activated by progesterone and synthetic progestins, provoking its phosphorylation and DNA-binding ability and inducing its regulatory activities. Its expression is indicative of an intact ER pathway and, as such, might identify tumors that are hormonally responsive to estrogen therapy, improving the overall predictive value of steroid receptor assays in selected tumors.26 Halperin et al27 found positive staining in PPC for ER and PR in 30.8% and 42%, respectively, whereas MOC showed positive staining for ER and PR in 72.7% and 90.9%, respectively. Henzen-Logmans et al28 found ER and PR staining in 46% and 34%, respectively, in MOC. There is scant literature describing the use of these markers in PMM, but Trupiano et al22 found ER and PR staining in 6% and 0%, respectively, in their study of 33 pleural mesotheliomas and PMMs. In our study, none of the 20 cases of PMM stained for either ER or PR. In contrast, 93% of PSCs (13/14 PPCs and 13/14 MOCs) showed strong nuclear staining for ER, yielding a sensitivity of 93% and specificity of 100%. PR was positive in 29% of PSCs (3/14 PPCs and 5/14 MOCs), yielding 29% sensitivity but 100% specificity in our hands. These results suggest that these antibodies, used in an appropriate panel, offer good specificity, and, in the case of ER, good sensitivity in differentiating PSC from PMM.
© American Society for Clinical Pathology

Calretinin is a 29-kd calcium-binding protein, and it is expressed in a range of tissues, including normal and neoplastic mesothelium.29 It has been studied extensively in malignant mesothelioma of the pleura with some disparate results, although many authors have reported positive staining in 80% to 100% of mesotheliomas with specificities of up to 100%.12-14,16,29 Similar levels of positivity for calretinin have been reported in PMM.8,9 Ordóñez13 found that discrepancies with this marker were due mainly to the type of antibody used. Cury et al17 suggested that only nuclear staining should be regarded as positive in distinguishing mesothelioma from carcinoma. In our study, calretinin was a good positive marker for mesothelioma with strong nuclear and cytoplasmic staining in 85% of PMMs (in 12/14 men and 5/6 women), whereas all PPCs and MOCs were negative (85% sensitivity and 100% specificity). In some cases, the staining was focal, and this, in addition to the slightly lower sensitivity, might reflect the use of a monoclonal rather than a polyclonal antibody. The specificity of this antibody is, however, 100% compared with polyclonal antibodies, which have been shown to stain up to 9% of serous adenocarcinomas.8,29 CA 125 and CEA have been shown to be of little value in differentiating PMMs from serous adenocarcinomas.8,10,15 Our results concur with these findings: CA 125 was not specific, staining 42% of PMMs (8/19) and 93% of PSCs (26/28), whereas CEA was not sensitive, staining only 1 case of PPC. Similarly, in our study, LeuM1 showed lower sensitivity than Ber-EP4; although staining was negative in all PMMs, it was positive in 50% of PSCs (14/28). Furthermore, the staining was focal in the majority of cases. Attanoos et al9 also found LeuM1 to be of low sensitivity with positive staining of 35% of MOCs and 0% of PPCs. Other studies have reported higher sensitivity with positive staining between 57% and 80%.8,10,30,31 One of these studies, however, showed focal staining in 3 PMMs, and 1 study was of autopsy material, which might show differences in antigen expression. MOC31 is a monoclonal antibody to a cell surface glycoprotein and shows membranous staining in the majority of adenocarcinomas.32-34 It also has been shown to stain between 2.3% and 20% of mesotheliomas.33,35,36 In our study, MOC31 was positive in 100% of PSCs with staining of 15% of PMMs (3/20). However, the staining in PMMs tended to be patchy compared with the diffuse membranous staining seen in PSC. In our hands, this antibody does not seem to offer advantages over Ber-EP4. Differential cytokeratins (CK7 and CK20) frequently are used when the pathologist is faced with metastatic carcinoma from an unknown primary site, eg, in the differentiation of ovarian serous metastases (CK7+; CK20–) from colorectal adenocarcinoma (CK7–; CK20+).37,38 In tumors involving the pleura, CK7 and CK20 may be useful additions to an antibody panel because expression of CK20 with or without CK7
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positivity is more in keeping with metastases from the gastrointestinal tract.39 However, for distinguishing PMM from PSC, these antibodies have poor sensitivity and specificity. We found CK20 to be negative in all PMMs and PSCs, whereas CK7 was positive in 85% of PMMs (17/20) and 100% of 28 PSCs. TTF-1 is a 38-kd homeodomain containing DNA-binding protein originally identified in follicular cells of the thyroid and subsequently in pneumocytes. Many studies have shown it to be a sensitive and specific marker of lung adenocarcinoma,40-43 and, therefore, it is considered an important part of the antibody panel for differentiating pleural mesothelioma from metastatic adenocarcinoma of lung (or thyroid).13,18 There is scant evidence in the literature on positive staining for TTF-1 in serous carcinomas. A study by Hecht et al43 on paraffinembedded sections of cell block preparations derived from effusion and fine-needle aspiration specimens showed focal weak reactivity in 1 MOC. In our study, all PMMs were negative for TTF-1, but 1 PSC (a PPC) unexpectedly showed strong nuclear staining for this antibody. This tumor was positive for CK7, Ber-EP4, LeuM1, CA 125, ER, PR, TTF-1, and CD44 and negative for calretinin, CK5, CK20, mesothelin, thrombomodulin, CEA, and WT1. Subsequent staining for thyroglobulin was negative. This diffuse peritoneal-based tumor developed 14 years after hysterectomy and bilateral salpingo-oophorectomy for benign disease in the patient, a nonsmoker. She was treated with systemic chemotherapy (carboplatin) and initially had a response. However, her condition slowly deteriorated, and there was a rising serum CA 125 level. Despite further chemotherapy and hormonal therapy, she died 32 months after diagnosis. The chest radiographic findings were normal until 7 months before death, when multiple pulmonary metastases developed. The behavior of this tumor was entirely in keeping with PPC, and, therefore, we conclude that this represents true TTF-1 staining in a PPC. Studies have shown that strong expression of CK5/6 is a sensitive and specific marker for distinguishing mesothelioma from metastatic adenocarcinoma in the pleura.44 Ordóñez13 found that 100% of 60 epithelioid pleural mesotheliomas stained positively for CK5/6, whereas only 1 (2%) of 50 metastatic adenocarcinomas was positive. In addition, Ordóñez,8 in a study of PMMs and peritoneal and ovarian serous carcinomas, found positive staining for CK5/6 in 100% of 35 PMMs and 11 (24%) of 45 serous carcinomas. In our study, by using CK5, we found positive staining in 90% of PMMs (18/20) and in 50% of PSCs (14/28). The lack of specificity casts doubt on the usefulness of this antibody in the context of differentiating these peritoneal tumors. This is in agreement with the findings of Attanoos et al,9 who similarly found this antibody to be of limited use in differentiating PMM and PSC.
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Initial reports suggested that antibodies to CD44H, a receptor for hyaluronic acid, were useful positive markers for mesothelioma.15 Later reports concluded that this antibody was of limited specificity.12 We found that 75% of PMMs (15/20) were positive for CD44H, whereas 14% of PSCs were positive (4/28), suggesting a limited role for this antibody in an appropriate panel. We found thrombomodulin to be positive in only 45% of PMMs (9/20). One of the PSCs was immunoreactive for this antibody, but this case also showed staining for MOC31, BerEP4, and LeuM1. Studies have shown positivity for thrombomodulin in 80% to 100% of pleural mesotheliomas and 10% to 15% of adenocarcinomas.45,46 In PMMs, Ordóñez8 and Attanoos et al9 reported 74% and 56% positivity, respectively. In the majority of our cases, staining for thrombomodulin was focal. This patchy reactivity is well recognized47 and suggests that staining for thrombomodulin might be affected by tissue fixation in larger specimens. This may be one reason for its limited usefulness in PMM. Similarly, mesothelin that is known to be positive in nonmucinous ovarian carcinomas48 was positive in 65% of PMMs (13/20) and 89% of PSCs (25/28) and was not considered helpful for distinguishing these tumors. The level of positivity for mesothelin in PMM is somewhat lower than that reported in studies by Ordóñez48,49 of 100%; however, this antibody has not been studied extensively in PMM. WT1 has been studied in the context of pleural mesothelioma in which positive nuclear staining has been reported in 70% to 93% of pleural mesotheliomas.13,33,50,51 WT1 also has been shown to be positive in most ovarian serous carcinomas, and staining with this marker is useful for excluding metastases from other sites to the peritoneum.52,53 Studies have reported positivity for WT1 in 93% to 100% of ovarian serous carcinomas.54-57 We found positive staining for WT1 in 55% of PMMs (11/20) and in 75% of PSCs (21/28). The explanation for this lower level of positivity is unclear but might reflect the archival nature of the tissue samples used in this study with resultant degradation of nuclear antigens. Regardless of the low rate of expression in both PMM and PSC, in the present study, WT1 was not helpful for distinguishing PMM from PSC. As highlighted by previous studies, we have shown that there is no single marker that is diagnostic for PMM or PSC.8-10 Similarly, immunohistochemical analysis cannot distinguish MOC of the serous type from PPC. When faced with the dilemma of diagnosing a mesothelioma in which the differential diagnosis includes metastatic adenocarcinoma, a panel of antibodies comprising a series of positive and negative markers for mesothelioma and adenocarcinoma should be used. This also applies to the peritoneum in which mesothelioma has to be separated from histologically similar tumors such as primary and secondary serous carcinoma. A diagnostic error in this situation might have therapeutic implications and/or
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medicolegal consequences. This study showed that ER and PR are useful additions to a “diffuse peritoneal malignancy” workup and, in combination with Ber-EP4 and calretinin, form a sensitive and specific diagnostic panel.
From the Departments of Pathology, 1University of Glasgow Western Infirmary, 2Glasgow Royal Infirmary, and 3Southern General Hospital, Glasgow, Scotland. Supported by the June Hancock Mesothelioma Research Fund, Sheffield, England. Address reprint requests to Dr Roberts: Dept of Pathology, Western Infirmary, Dumbarton Rd, Glasgow, G11 6NT, Scotland. Acknowledgments: We are grateful to A. Lessels, FRCPath, Edinburgh, Scotland, and I. Nawroz, FRCPath, Kirkaldy, Scotland, for supplying additional material.

References
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