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Immunohistochemical Analysis of Peritoneal Mesothelioma

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					                                                                 Anatomic Pathology / PERITONEAL MESOTHELIOMA AND SEROUS CARCINOMA
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   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                                                                   Malignant mesothelioma of the peritoneum (PMM) is
        The role of immunohistochemical markers in                       less common than its counterpart in the pleural cavity,
   distinguishing peritoneal mesothelioma from primary                   accounting for only around 10% of all mesotheliomas.1
   or metastatic serous papillary carcinoma of the                       However, like pleural mesothelioma, the diagnosis of PMM
   peritoneum was evaluated. We immunostained 20                         can be challenging, particularly in the female peritoneum,
   peritoneal mesotheliomas (from 14 men and 6 women),                   where it may mimic serous carcinoma of the ovary or peri-
   14 primary peritoneal carcinomas, and 14 metastatic                   toneum. Nevertheless, accurate distinction of these tumors is
   serous ovarian carcinomas with a panel of 16                          important for clinical and medicolegal reasons. Malignant
   antibodies. Positive staining for calretinin was                      mesothelioma is a chemotherapy- and radiotherapy-resistant
   identified in 17 (85%) of 20 mesotheliomas, but all                   tumor with a dismal prognosis, whereas these treatment
   carcinomas were negative. Positive staining for Ber-                  modalities can significantly improve patient survival in pri-
   EP4 was identified in 27 (96%) of 28 carcinomas and                   mary and metastatic peritoneal serous carcinoma (PSC).2-5 No
   in 2 (10%) of 20 mesotheliomas. Estrogen receptors                    etiologic relationship has been established between asbestos
   were positive in 26 (93%) of 28 carcinomas, and                       exposure and PSC. PMM, however, is recognized to occur
   progesterone receptors were positive in 8 (29%) of 28                 after asbestos exposure, and this diagnosis, therefore, might
   carcinomas. All mesotheliomas were negative for                       lead to medicolegal compensation.1,6 It should be noted, how-
   estrogen and progesterone receptors. The other                        ever, that in women, the proportion of cases with asbestos
   antibodies evaluated were insufficiently sensitive and/or             exposure is lower than in men and it usually is of the paraoc-
   specific to be diagnostically useful. In conjunction with             cupational (domestic) type.7
   calretinin and Ber-EP4, estrogen and progesterone                          Histologically, epithelioid malignant mesothelioma might
   receptors are useful discriminatory markers for                       have an appearance identical to that of primary or metastatic
   distinguishing peritoneal mesothelioma from primary                   serous carcinoma.8,9 Furthermore, mucin stains are consider-
   or metastatic serous carcinoma.                                       ably less helpful for differentiating between these tumors than
                                                                         for distinguishing epithelioid malignant mesothelioma from
                                                                         metastatic adenocarcinoma in the pleura.10 Immunohisto-
                                                                         chemical 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


   © American Society for Clinical Pathology                                                               Am J Clin Pathol 2006;125:67-76   67
                                                                                                    67    DOI: 10.1309/8FCHQ3VPBWM7B5X9      67
Barnetson et al / PERITONEAL MESOTHELIOMA AND SEROUS CARCINOMA



that can reliably distinguish epithelioid malignant mesothe-       examination subsequently showed the presence of a primary
lioma from primary or metastatic ovarian serous carcinoma of       ovarian adenocarcinoma.
the peritoneum. Our antibody panel included well-recognized             For PPC, the specimens consisted of open biopsy speci-
markers of mesothelioma and adenocarcinoma. Antibodies to          mens or specimens from debulking surgery. The diagnosis of
estrogen receptor (ER) and progesterone receptor (PR) also         PPC was based on appropriate microscopic features and con-
were included.                                                     firmation of lack of ovarian involvement at laparoscopy or by
                                                                   surgical pathology. Similar to PMM, the diagnoses were sup-
                                                                   ported by appropriate immunohistochemical, ultrastructural,
                                                                   or postmortem findings.
Materials and Methods
                                                                        MOCs consisted of open biopsy specimens or peritoneal
                                                                   metastases removed at the time of oophorectomy. All MOCs
Tumor Samples                                                      included in this study were classified as papillary serous ade-
      Retrospective paraffin-embedded tissue samples were          nocarcinomas based on appropriate histopathologic features.
retrieved from the pathology records of the Western Infirmary,
Royal Infirmary, and Southern General Hospital, Glasgow,           Immunohistochemical Analysis
Scotland; the Victoria Infirmary, Kirkaldy, Fife, Scotland; and         Information about the antibodies selected is given in
the Western General, Edinburgh, Scotland. The specimens            ❚Table 1❚. For immunohistochemical analysis, 3-µm sections
included 14 samples from men and 8 samples from women              were immunostained by using a DAKO Autostainer using
initially diagnosed with PMM, 14 samples from women with           EnVision Peroxidase (DAKO, Ely, England). Briefly, sections
metastatic ovarian carcinoma (MOC), and 14 samples from            were washed with tris(hydroxymethyl)aminomethane-
women with primary peritoneal carcinoma (PPC).                     buffered saline containing polysorbate (Tween) buffer
      For PMM, the specimens consisted of needle or open           (TBS/TB) and then blocked with normal goat serum (dilution
biopsy specimens, and the microscopic features were consid-        1:20) for 20 minutes. Sections then were incubated with pri-
ered consistent with those reported in standard texts. These       mary antibody for 30 minutes followed by another wash in
histologic findings were supported by appropriate immuno-          TBS/TB. Following this step, the sections were incubated
histochemical, ultrastructural, or postmortem findings. In each    with DAKO EnVision for 30 minutes. After a final wash in
case, the clinical findings were of a diffuse peritoneal tumor     TBS/TB, color was developed with diaminobenzidine for 10
with no evidence of a primary tumor elsewhere in the body,         minutes and sections counterstained with hematoxylin. For
and, in most cases, there was a well-documented history of         antigen retrieval, slides were microwaved under pressure in 1
asbestos exposure. By using these criteria, 2 cases initially      mmol/L of EDTA, pH 8.0, for 5 minutes or incubated with
diagnosed as PMM occurring in women were excluded. In              0.1% trypsin, pH 7.8, for 25 minutes at 37°C. Appropriate
one case, electron microscopy had been undertaken on the           control material (according to the manufacturer’s instructions)
tumor showing the presence of short microvilli more in keep-       was used for each run. For negative control samples, the pri-
ing with adenocarcinoma. In the other case, a postmortem           mary antibody was omitted.


❚Table 1❚
Antibodies Used for Immunohistochemical Analysis

Antibody                                   Clone                        Source                    Dilution               Pretreatment

Carcinoembryonic antigen                II-7              DAKO, Ely, Cambridge, England            1:400                     Trypsin
Human epithelial antigen                Ber-EP4           DAKO                                     1:200                     Trypsin
LeuM1                                   LeuM1             Becton Dickinson, Oxford, England        1:200                     MWPC
Mesothelin                              5B2               Becton Dickinson                         1:20                      MWPC
CA 125                                  OV185:1           Vector, Peterborough, England            1:400                     MWPC
Epithelial-related antigen              MOC31             DAKO                                     1:20                      Trypsin
Thyroid transcription factor-1          SPT 24            Vector                                   1:50                      MWPC
Estrogen receptor                       6F11              Vector                                   1:50                      MWPC
Progesterone receptor                   PGR636            DAKO                                     1:400                     MWPC
Cytokeratin 5                           XM26              Vector                                   1:200                     MWPC
Cytokeratin 7                           OV-TLR12/30       DAKO                                     1:500                     MWPC
Cytokeratin 20                          Ks20.8            DAKO                                     1:500                     MWPC
Calretinin                              5A5               Vector                                   1:100                     MWPC
Thrombomodulin                          15C8              Vector                                   1:200                     Trypsin
WT1                                     6F-H2             DAKO                                     1:50                      MWPC
CD44H                                   F10-44-2          DAKO                                     1:100                     MWPC

MWPC, pressure cook, microwave.



68    Am J Clin Pathol 2006;125:67-76                                                             © American Society for Clinical Pathology
68    DOI: 10.1309/8FCHQ3VPBWM7B5X9
                                                                                                                        Anatomic Pathology / ORIGINAL ARTICLE


Interpretation of Results                                                               Ber-EP4, Carcinoembryonic Antigen, LeuM1, and
     The slides were evaluated semiquantitatively, and the per-                         MOC31
centage of positive tumor cells was described as follows: 1+,                                Strong, membranous staining for Ber-EP4 was seen in
staining 1% to 49% of tumor cells, or 2+, staining 50% to                               27 (96%) of 28 carcinomas. Of the PMMs, 2 (10%) of 20
100% of tumor cells. All cases showing staining of less than                            showed 1+ staining for Ber-EP4. All PMMs occurring in
1% of tumor cells were regarded as negative. Weak staining or                           both men and women were negative for carcinoembryonic
staining that was difficult to interpret was regarded as equivo-                        antigen (CEA) and LeuM1. Positive staining for CEA was
cal. The pattern of staining was recorded as membranous,                                observed in only 1 (4%) of 28 PSCs (a PPC). Of 28 carci-
cytoplasmic, or nuclear for each antibody. The sections were                            nomas, 14 (50%) were positive for LeuM1, although stain-
assessed independently by 2 observers (R.J.B. and F.R.). For                            ing was focal in the majority of cases. Strong membranous
cases in which there was substantial disagreement, the slides                           staining for MOC31 was seen in all 28 PSCs. Of 20
were assessed by a third observer (R.A.B.), and consensus                               PMMs, 5 (25%) showed 1+ staining for MOC31.
was reached.
                                                                                        CK5, CK7, and CK20
                                                                                             Of 20 PMMs, 18 (90%) stained strongly with CK5. CK5
Results                                                                                 staining was seen in 14 (50%) of 28 PSCs. Strong CK7 stain-
    The results are summarized in ❚Table 2❚, ❚Table 3❚, ❚Table 4❚,                      ing was observed in 17 (85%) of 20 PMMs and in all 28 PSCs
❚Table 5❚, and ❚Table 6❚ and shown in ❚Image 1❚ and ❚Image 2❚.                          (100%). CK20 was negative in all PMMs and PSCs.


❚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)            0        1            0        2          5        0     0    12      14       0          9             12        6        8       10        0
 Female (n = 6)           0        1            0        1          4        0     0     6       3       0          4              5        3        3        5        0
 Total (n = 20)           0        2            0        3          9        0     0    18      17       0         13             17        9       11       15        0
Carcinoma
 MOC (n = 14)             0        13         8         14        13       13      5     8      14       0         13              0        1       10        2        0
 PPC (n = 14)             1        14         6         14        13       13      3     6      14       0         12              0        0       11        2        1
 Total (n = 28)           1        27        14         28        26       26      8    14      28       0         25              0        1       21        4        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❚                                                                               ❚Table 4❚
Immunohistochemical Staining of Peritoneal Mesothelioma in                              Immunohistochemical Staining of Peritoneal Mesothelioma in
14 Men*                                                                                 6 Women*

Antibody                      2+            1+         Equivocal         Negative       Antibody                    2+             1+           Equivocal         Negative

CEA                            0            0                0               14         CEA                         0               0               0                 6
Ber -EP4                       0            1                0               13         Ber -EP4                    0               1               0                 5
LeuM1                          0            0                0               14         LeuM1                       0               0               0                 6
MOC31                          0            4                1               11         MOC31                       0               1               0                 5
CA 125                         2            3                0                9         CA 125                      2               2               0                 2
ER                             0            0                0               14         ER                          0               0               0                 6
PR                             0            0                0               14         PR                          0               0               0                 6
CK5                           10            2                0                2         CK5                         4               2               0                 0
CK7                           14            0                0                0         CK7                         3               0               0                 3
CK20                           0            0                0               14         CK20                        0               0               0                 6
Mesothelin                     6            3                0                5         Mesothelin                  4               0               0                 2
Calretinin                    10            2                0                2         Calretinin                  4               1               0                 1
Thrombomodulin                 1            5                0                8         Thrombomodulin              0               3               0                 3
WT1                            2            6                0                6         WT1                         2               1               1                 2
CD44H                          8            2                1                3         CD44H                       5               0               0                 1
TTF-1                          0            0                0               14         TTF-1                       0               0               0                 6

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

   proprietary information, see Table 1.                                                   proprietary information, see Table 1.



© American Society for Clinical Pathology                                                                                           Am J Clin Pathol 2006;125:67-76        69
                                                                                                                           69     DOI: 10.1309/8FCHQ3VPBWM7B5X9            69
Barnetson et al / PERITONEAL MESOTHELIOMA AND SEROUS CARCINOMA


❚Table 5❚                                                                               ❚Table 6❚
Immunohistochemical Staining of 14 Metastatic Serous                                    Immunohistochemical Staining of 14 Primary Peritoneal
Ovarian Carcinomas*                                                                     Carcinomas*

Antibody                   2+              1+          Equivocal          Negative      Antibody                   2+              1+          Equivocal         Negative

CEA                         0               0               0                14         CEA                          1              0               0                13
Ber -EP4                   12               1               0                 1         Ber -EP4                    12              2               0                 0
LeuM1                       1               7               0                 6         LeuM1                        2              4               0                 8
MOC31                       8               6               0                 0         MOC31                       10              4               0                 0
CA 125                     12               1               1                 0         CA 125                      11              2               1                 0
ER                         10               3               1                 0         ER                          12              1               0                 1
PR                          1               4               0                 9         PR                           1              2               1                10
CK5                         0               8               2                 4         CK5                          1              5               2                 6
CK7                        13               1               0                 0         CK7                         14              0               0                 0
CK20                        0               0               0                14         CK20                         0              0               0                14
Mesothelin                 12               1               1                 0         Mesothelin                  10              2               0                 2
Calretinin                  0               0               0                14         Calretinin                   0              0               0                14
Thrombomodulin              0               1               0                13         Thrombomodulin               0              0               1                13
WT1                        10               0               2                 2         WT1                         10              1               1                 2
CD44H                       0               2               0                12         CD44H                        1              1               0                12
TTF-1                       0               0               0                14         TTF-1                        1              0               0                13

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




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


70     Am J Clin Pathol 2006;125:67-76                                                                                             © American Society for Clinical Pathology
70     DOI: 10.1309/8FCHQ3VPBWM7B5X9
                                                                                           Anatomic Pathology / ORIGINAL ARTICLE


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).
© American Society for Clinical Pathology                                                            Am J Clin Pathol 2006;125:67-76   71
                                                                                              71    DOI: 10.1309/8FCHQ3VPBWM7B5X9      71
Barnetson et al / PERITONEAL MESOTHELIOMA AND SEROUS CARCINOMA


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).

72   Am J Clin Pathol 2006;125:67-76                                                             © American Society for Clinical Pathology
72   DOI: 10.1309/8FCHQ3VPBWM7B5X9
                                                                                                Anatomic Pathology / ORIGINAL ARTICLE



     The present study was planned to ascertain the usefulness                Calretinin is a 29-kd calcium-binding protein, and it is
of commercially available, well-established antibodies in the            expressed in a range of tissues, including normal and neoplastic
distinction of PMM from primary or metastatic serous adeno-              mesothelium.29 It has been studied extensively in malignant
carcinoma. The panel included markers considered relatively              mesothelioma of the pleura with some disparate results, although
specific and sensitive for both mesothelioma and adenocarci-             many authors have reported positive staining in 80% to 100%
noma. In addition, because serous MOCs and PPCs are                      of mesotheliomas with specificities of up to 100%.12-14,16,29
tumors of the primary and secondary müllerian systems,                   Similar levels of positivity for calretinin have been reported in
respectively, ER and PR also were included. These have been              PMM.8,9 Ordóñez13 found that discrepancies with this marker
reported to be negative in PMM.22                                        were due mainly to the type of antibody used. Cury et al17 sug-
     Ber-EP4 is a monoclonal antibody produced from the                  gested that only nuclear staining should be regarded as posi-
MCF-7 breast carcinoma cell line that recognizes epithelial              tive in distinguishing mesothelioma from carcinoma. In our
glycoproteins.23 It has been used in many studies to discrim-            study, calretinin was a good positive marker for mesothe-
inate between pleural mesothelioma and adenocarcino-                     lioma with strong nuclear and cytoplasmic staining in 85% of
ma11-14,16,24 and to a lesser extent in the peritoneal setting.8-10,24   PMMs (in 12/14 men and 5/6 women), whereas all PPCs and
Ordóñez8 found Ber-EP4 positivity in 100% of 45 PSCs,                    MOCs were negative (85% sensitivity and 100% specificity).
whereas only 11% of PMMs (4/35) showed positivity in a lim-              In some cases, the staining was focal, and this, in addition to
ited number of cells. Similarly Attanoos et al9 found positivi-          the slightly lower sensitivity, might reflect the use of a mon-
ty for Ber-EP4 in 95% of papillary serous ovarian cancers                oclonal rather than a polyclonal antibody. The specificity of
(19/20) and 9% of PMMs (3/32). Our study confirmed the                   this antibody is, however, 100% compared with polyclonal
high sensitivity and specificity of Ber-EP4 with strong mem-             antibodies, which have been shown to stain up to 9% of
branous staining in 96% of PSCs (27/28; 13/14 MOCs and                   serous adenocarcinomas.8,29
14/14 PPCs). There was unequivocal positive staining for Ber-                 CA 125 and CEA have been shown to be of little value in
EP4 in 10% of PMMs (2/20; in 1 woman and 1 man).                         differentiating PMMs from serous adenocarcinomas.8,10,15
Although both of these cases also showed focal positivity for            Our results concur with these findings: CA 125 was not spe-
MOC31, there was, in addition, 2+ staining for calretinin and            cific, staining 42% of PMMs (8/19) and 93% of PSCs (26/28),
focal staining for thrombomodulin.                                       whereas CEA was not sensitive, staining only 1 case of PPC.
     ER is a ligand-activated transcription factor that mediates         Similarly, in our study, LeuM1 showed lower sensitivity than
the effect of the steroid hormone 17β-estradiol in men and               Ber-EP4; although staining was negative in all PMMs, it was
women.25 PR is an estrogen-inducible protein activated by                positive in 50% of PSCs (14/28). Furthermore, the staining
progesterone and synthetic progestins, provoking its phospho-            was focal in the majority of cases. Attanoos et al9 also found
rylation and DNA-binding ability and inducing its regulatory             LeuM1 to be of low sensitivity with positive staining of 35%
activities. Its expression is indicative of an intact ER pathway         of MOCs and 0% of PPCs. Other studies have reported high-
and, as such, might identify tumors that are hormonally                  er sensitivity with positive staining between 57% and
responsive to estrogen therapy, improving the overall predic-            80%.8,10,30,31 One of these studies, however, showed focal
tive value of steroid receptor assays in selected tumors.26              staining in 3 PMMs, and 1 study was of autopsy material,
Halperin et al27 found positive staining in PPC for ER and PR            which might show differences in antigen expression. MOC31
in 30.8% and 42%, respectively, whereas MOC showed posi-                 is a monoclonal antibody to a cell surface glycoprotein and
tive staining for ER and PR in 72.7% and 90.9%, respective-              shows membranous staining in the majority of adenocarcino-
ly. Henzen-Logmans et al28 found ER and PR staining in 46%               mas.32-34 It also has been shown to stain between 2.3% and
and 34%, respectively, in MOC. There is scant literature                 20% of mesotheliomas.33,35,36 In our study, MOC31 was pos-
describing the use of these markers in PMM, but Trupiano et              itive in 100% of PSCs with staining of 15% of PMMs (3/20).
al22 found ER and PR staining in 6% and 0%, respectively, in             However, the staining in PMMs tended to be patchy compared
their study of 33 pleural mesotheliomas and PMMs.                        with the diffuse membranous staining seen in PSC. In our
     In our study, none of the 20 cases of PMM stained for               hands, this antibody does not seem to offer advantages over
either ER or PR. In contrast, 93% of PSCs (13/14 PPCs and                Ber-EP4.
13/14 MOCs) showed strong nuclear staining for ER, yielding                   Differential cytokeratins (CK7 and CK20) frequently are
a sensitivity of 93% and specificity of 100%. PR was positive            used when the pathologist is faced with metastatic carcinoma
in 29% of PSCs (3/14 PPCs and 5/14 MOCs), yielding 29%                   from an unknown primary site, eg, in the differentiation of
sensitivity but 100% specificity in our hands. These results             ovarian serous metastases (CK7+; CK20–) from colorectal
suggest that these antibodies, used in an appropriate panel,             adenocarcinoma (CK7–; CK20+).37,38 In tumors involving
offer good specificity, and, in the case of ER, good sensitivity         the pleura, CK7 and CK20 may be useful additions to an anti-
in differentiating PSC from PMM.                                         body panel because expression of CK20 with or without CK7


© American Society for Clinical Pathology                                                                  Am J Clin Pathol 2006;125:67-76   73
                                                                                                    73   DOI: 10.1309/8FCHQ3VPBWM7B5X9       73
Barnetson et al / PERITONEAL MESOTHELIOMA AND SEROUS CARCINOMA



positivity is more in keeping with metastases from the gas-                Initial reports suggested that antibodies to CD44H, a
trointestinal tract.39 However, for distinguishing PMM from          receptor for hyaluronic acid, were useful positive markers for
PSC, these antibodies have poor sensitivity and specificity.         mesothelioma.15 Later reports concluded that this antibody
We found CK20 to be negative in all PMMs and PSCs,                   was of limited specificity.12 We found that 75% of PMMs
whereas CK7 was positive in 85% of PMMs (17/20) and                  (15/20) were positive for CD44H, whereas 14% of PSCs were
100% of 28 PSCs.                                                     positive (4/28), suggesting a limited role for this antibody in
      TTF-1 is a 38-kd homeodomain containing DNA-binding            an appropriate panel.
protein originally identified in follicular cells of the thyroid           We found thrombomodulin to be positive in only 45% of
and subsequently in pneumocytes. Many studies have shown             PMMs (9/20). One of the PSCs was immunoreactive for this
it to be a sensitive and specific marker of lung adenocarcino-       antibody, but this case also showed staining for MOC31, Ber-
ma,40-43 and, therefore, it is considered an important part of the   EP4, and LeuM1. Studies have shown positivity for thrombo-
antibody panel for differentiating pleural mesothelioma from         modulin in 80% to 100% of pleural mesotheliomas and 10%
metastatic adenocarcinoma of lung (or thyroid).13,18 There is        to 15% of adenocarcinomas.45,46 In PMMs, Ordóñez8 and
scant evidence in the literature on positive staining for TTF-1      Attanoos et al9 reported 74% and 56% positivity, respectively.
in serous carcinomas. A study by Hecht et al43 on paraffin-          In the majority of our cases, staining for thrombomodulin was
embedded sections of cell block preparations derived from            focal. This patchy reactivity is well recognized47 and suggests
effusion and fine-needle aspiration specimens showed focal           that staining for thrombomodulin might be affected by tissue
weak reactivity in 1 MOC.                                            fixation in larger specimens. This may be one reason for its
      In our study, all PMMs were negative for TTF-1, but 1          limited usefulness in PMM.
PSC (a PPC) unexpectedly showed strong nuclear staining                    Similarly, mesothelin that is known to be positive in non-
for this antibody. This tumor was positive for CK7, Ber-EP4,         mucinous ovarian carcinomas48 was positive in 65% of PMMs
LeuM1, CA 125, ER, PR, TTF-1, and CD44 and negative for              (13/20) and 89% of PSCs (25/28) and was not considered
calretinin, CK5, CK20, mesothelin, thrombomodulin, CEA,              helpful for distinguishing these tumors. The level of positivity
and WT1. Subsequent staining for thyroglobulin was nega-             for mesothelin in PMM is somewhat lower than that reported
tive. This diffuse peritoneal-based tumor developed 14 years         in studies by Ordóñez48,49 of 100%; however, this antibody
after hysterectomy and bilateral salpingo-oophorectomy for           has not been studied extensively in PMM.
benign disease in the patient, a nonsmoker. She was treated                WT1 has been studied in the context of pleural mesothe-
with systemic chemotherapy (carboplatin) and initially had a         lioma in which positive nuclear staining has been reported in
response. However, her condition slowly deteriorated, and            70% to 93% of pleural mesotheliomas.13,33,50,51 WT1 also has
there was a rising serum CA 125 level. Despite further               been shown to be positive in most ovarian serous carcinomas,
chemotherapy and hormonal therapy, she died 32 months                and staining with this marker is useful for excluding metastases
after diagnosis. The chest radiographic findings were normal         from other sites to the peritoneum.52,53 Studies have reported
until 7 months before death, when multiple pulmonary                 positivity for WT1 in 93% to 100% of ovarian serous carcino-
metastases developed. The behavior of this tumor was entire-         mas.54-57 We found positive staining for WT1 in 55% of PMMs
ly in keeping with PPC, and, therefore, we conclude that this        (11/20) and in 75% of PSCs (21/28). The explanation for this
represents true TTF-1 staining in a PPC.                             lower level of positivity is unclear but might reflect the archival
      Studies have shown that strong expression of CK5/6 is a        nature of the tissue samples used in this study with resultant
sensitive and specific marker for distinguishing mesothe-            degradation of nuclear antigens. Regardless of the low rate of
lioma from metastatic adenocarcinoma in the pleura.44                expression in both PMM and PSC, in the present study, WT1
Ordóñez13 found that 100% of 60 epithelioid pleural                  was not helpful for distinguishing PMM from PSC.
mesotheliomas stained positively for CK5/6, whereas only 1                 As highlighted by previous studies, we have shown that
(2%) of 50 metastatic adenocarcinomas was positive. In               there is no single marker that is diagnostic for PMM or PSC.8-10
addition, Ordóñez,8 in a study of PMMs and peritoneal and            Similarly, immunohistochemical analysis cannot distinguish
ovarian serous carcinomas, found positive staining for               MOC of the serous type from PPC. When faced with the
CK5/6 in 100% of 35 PMMs and 11 (24%) of 45 serous car-              dilemma of diagnosing a mesothelioma in which the differen-
cinomas. In our study, by using CK5, we found positive               tial diagnosis includes metastatic adenocarcinoma, a panel of
staining in 90% of PMMs (18/20) and in 50% of PSCs                   antibodies comprising a series of positive and negative mark-
(14/28). The lack of specificity casts doubt on the usefulness       ers for mesothelioma and adenocarcinoma should be used.
of this antibody in the context of differentiating these peri-       This also applies to the peritoneum in which mesothelioma
toneal tumors. This is in agreement with the findings of             has to be separated from histologically similar tumors such as
Attanoos et al,9 who similarly found this antibody to be of          primary and secondary serous carcinoma. A diagnostic error
limited use in differentiating PMM and PSC.                          in this situation might have therapeutic implications and/or


74   Am J Clin Pathol 2006;125:67-76                                                                  © American Society for Clinical Pathology
74   DOI: 10.1309/8FCHQ3VPBWM7B5X9
                                                                                               Anatomic Pathology / ORIGINAL ARTICLE



medicolegal consequences. This study showed that ER and PR              14. Roberts F, Harper CM, Downie I, et al. Immunohistochemical
are useful additions to a “diffuse peritoneal malignancy”                   analysis still has a limited role in the diagnosis of malignant
                                                                            mesothelioma: a study of thirteen antibodies. Am J Clin Pathol.
workup and, in combination with Ber-EP4 and calretinin,                     2001;116:253-262.
form a sensitive and specific diagnostic panel.                         15. Attanoos RL, Webb R, Gibbs AR. CD44H expression in
                                                                            reactive mesothelium, pleural mesothelioma and pulmonary
From the Departments of Pathology, 1University of Glasgow                   adenocarcinoma. Histopathology. 1997;30:260-263.
Western Infirmary, 2Glasgow Royal Infirmary, and 3Southern              16. Carella R, Deleonardi G, D’Errico A, et al.
General Hospital, Glasgow, Scotland.                                        Immunohistochemical panels for differentiating epithelial
                                                                            malignant mesothelioma from lung adenocarcinoma: a study
     Supported by the June Hancock Mesothelioma Research                    with logistic regression analysis. Am J Surg Pathol. 2001;25:43-
Fund, Sheffield, England.                                                   50.
     Address reprint requests to Dr Roberts: Dept of Pathology,         17. Cury PM, Butcher DN, Fisher C, et al. Value of the
Western Infirmary, Dumbarton Rd, Glasgow, G11 6NT, Scotland.                mesothelium-associated antibodies thrombomodulin,
     Acknowledgments: We are grateful to A. Lessels, FRCPath,               cytokeratin 5/6, calretinin, and CD44H in distinguishing
Edinburgh, Scotland, and I. Nawroz, FRCPath, Kirkaldy, Scotland,            epithelioid pleural mesothelioma from adenocarcinoma
for supplying additional material.                                          metastatic to the pleura. Mod Pathol. 2000;13:107-112.
                                                                        18. Ordóñez NG. Value of thyroid transcription factor-1, E-
                                                                            cadherin, BG8, WT1, and CD44S immunostaining in
                                                                            distinguishing epithelial pleural mesothelioma from pulmonary
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76   Am J Clin Pathol 2006;125:67-76                                                                    © American Society for Clinical Pathology
76   DOI: 10.1309/8FCHQ3VPBWM7B5X9

				
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