Wide spectrum screening keratin as a marker of metaplastic spindle cell carcinoma of the breast: an immunohistochemical study of 24 patients

Wide spectrum screening keratin as a marker of metaplastic spindle cell carcinoma of the breast: an immunohistochemical study of 24 patients
Aims : Metaplastic spindle cell carcinomas may be difficult to distinguish histologically from other spindle cell lesions in the breast. Variable staining with cytokeratin immunomarkers has been reported for metaplastic carcinomas. We evaluated the diagnostic utility of anti-cytokeratin polyclonal antibody, wide spectrum screening keratin, to assess spindle cell breast lesions.
Methods and results : Twenty-four patients with spindle cell breast carcinoma and 31 patients with benign or malignant spindle cell tumours were studied using a panel of antibodies directed against multiple cytokeratins (AE1/AE3, CAM5.2, wide spectrum screening keratin), epithelial membrane antigen (EMA), and vimentin. Sites of origin for the 31 controls included breast, bone, and soft tissue. All but one (95.8%) metaplastic carcinomas stained positively with wide spectrum screening keratin. Only rare or focal immunoreactivity was observed with AE1/AE3 in four cases; however, sensitivity of AE1/AE3 was improved in 13 cases using steam EDTA as an antigen retrieval technique. Three cases were immunoreactive with CAM5.2 and eight cases were immunoreactive with EMA. All control cases lacked immunoreactivity with the cytokeratin panel and EMA. The spindle cells in the metaplastic breast tumours (88%) and in the controls (97%) stained with vimentin.
Conclusions : Wide spectrum screening keratin may be the most useful and convenient antibody in differentiating metaplastic spindle cell carcinoma from other spindle cell lesions in the breast.     

Staining for factor VIII related antigen and Ulex europaeus agglutinin I (UEA-I) in 230 tumours. An assessment of their specificity for angiosarcoma and Kaposi''s sarcoma

In this study we examined the staining reactivity of commercially available antisera to factor VIII related antigen (F VIII RAg) and Ulex europaeus agglutinin I (UEA-I) on sections from 230 formalin fixed paraffin embedded tumours. These included 196 sarcomas, 20 carcinomas and 14 angiomas. All angiomas showed positive staining for F VIII RAg; all carcinomas showed negative staining; the vasoformative areas of all angiosarcomas stained positively but only four of six angiosarcomas showed positive staining of their solid areas; of seven Kaposi's sarcomas, all showed positive staining of vessels and six showed positive staining of the spindle cell component. In the remaining 181 non-vascular sarcomas there was a false positive result in four tumours (2.2%), three of which had a history of irradiation. Pre-radiotherapy biopsies of these three tumours stained negatively with anti-F VIII RAg. UEA-I was demonstrated in all the angiomas studied, in all angiosarcomas (including the solid components) and in well-formed vessels of all Kaposi's sarcomas, but only in the spindle cell component of 3/6. However, there was an unacceptably high rate of false positive staining amongst the carcinomas and non-vascular sarcomas. In conclusion, F VIII RAg is a specific but not a sensitive marker of angiosarcomas; UEA-I is a sensitive but not a specific marker of angiosarcomas.     

Vimentin: an evaluation of its role as a tumour marker

In this study we examined 198 sarcomas, 38 carcinomas, 13 'tumours with a spindle cell component' and 22 malignant melanomas with a commercial monoclonal vimentin antibody. All histopathological material was formalin fixed and paraffin embedded. The results show this antibody to be a sensitive and specific marker of mesenchymal derivation or differentiation. It is a useful tool in separating sarcomas from most carcinomas, and in separating malignant melanomas from carcinomas. When used in combination with a cytokeratin antibody it identifies carcinosarcomas and synovial sarcomas.     

Comparison of keratin monoclonal antibodies MAK-6, AE1:AE3, and CAM-5.2

Two routinely used antikeratin monoclonal antibodies, AE1:AE3 (Hybritech Inc., La Jolla, CA) and CAM-5.2 (Becton-Dickinson, Mountain View, CA), were compared with a new antikeratin monoclonal antibody mixture, MAK-6 (Triton Biosciences, Inc., Alameda, CA). Various poorly differentiated epithelial neoplasms, lymphomas, melanomas, and sarcomas were studied with the use of the avidin-biotin-peroxidase technic. All three antibodies had similar staining profiles, however, some differences were noted. The MAK-6 and CAM-5.2 antibodies illustrated stronger staining for some grade III transitional cell carcinomas, whereas the AE1:AE3 was variably positive to negative in all cases. Three renal cell carcinomas were positive with all three antibodies, two were negative with all three, and one had scattered positive cells with MAK-6 only. All lymphomas and plasmacytomas were negative with MAK-6 and CAM-5.2, however, AE1:AE3 faintly stained two of three plasmacytomas and two of the seven large cell lymphomas. Two out of three hepatomas evaluated were strongly positive with CAM-5.2 and MAK-6 and variably positive with AE1:AE3. The third case had both positive and negative cells with all three antibodies. In conclusion, MAK-6 antikeratin antibody is as useful as AE1:AE3 and CAM-5.2 for identification of poorly differentiated epithelial neoplasms.     

Synovial sarcoma: an immunohistochemical study

The immunohistochemical staining pattern of 18 cases of synovial sarcoma with two epithelial-specific monoclonal antibodies is described. This is compared with normal synovium, cases of giant cell tumour of tendon sheath (benign synovioma) and a variety of spindle celled sarcomas. Sixteen cases of synovial sarcoma showed staining of the epithelial component with at least one antibody. No staining was seen in normal synovium or in giant cell tumours of tendon sheath. A small number of malignant schwannomas contained groups of cells which stained positively whilst other spindle cell sarcomas either did not stain or showed 'cross-reaction' type staining only. These results add weight to the proposition that synovial sarcomas do not arise from normal synovium, despite their morphological similarities, but from mesenchymal connective tissue. It is also shown that immunohistochemical staining with anti-epithelial antibodies will emphasize the biphasic pattern of synovial sarcomas allowing their distinction from other sarcomas.     

Anti-mesothelial markers in sarcomatoid mesothelioma and other spindle cell neoplasms

AIMS: To undertake a comparative evaluation of three antimesothelial markers (thrombomodulin, cytokeratin 5/6 and calretinin) with broad spectrum cytokeratin (AE1/AE3) in differentiating between sarcomatoid mesothelioma and a spectrum of spindle cell neoplasms. METHODS AND RESULTS: Thirty-one malignant sarcomatoid mesotheliomas were studied. Calretinin expression was focally identified in 12 (39%) tumours and thrombomodulin and cytokeratin 5/6 immunoreactivity was seen in nine (29%) cases. In comparison there was strong diffuse cytoplasmic reactivity with the broad spectrum cytokeratin (AE1/AE3) in 24 of 31 (77%) tumours. Thirty mixed spindle cells neoplasms were studied. No calretinin expression was identified in any case. Thrombomodulin immunoreactivity was identified in four (16%) cases (two angiosarcomas, two high-grade sarcomas, not otherwise specified). Cytokeratin 5/6 expression was seen in one high-grade pulmonary sarcoma originally termed malignant fibrous histiocytoma. None of the antimesothelial markers was expressed in the four spindle cell carcinomas studied. In contrast, broad spectrum cytokeratin was diffusely expressed in all four spindle cell carcinomas (three pulmonary, one renal), both synovial sarcomas, both malignant mixed Müllerian tumours, one of three pulmonary leiomyosarcomas and two of nine sarcomas, not otherwise specified. CONCLUSIONS: Immunohistochemistry has a more limited role in the diagnosis and distinction of sarcomatoid mesothelioma from other spindle cell neoplasms. The combination of a broad spectrum cytokeratin with calretinin combines both high sensitivity (77% for AE1/AE3) with high specificity (100% for calretinin) for sarcomatoid mesothelioma and can be diagnostically useful. The mesothelial markers, thrombomodulin and cytokeratin 5/6, are not useful alone in the diagnosis of sarcomatoid mesothelioma as each shows insufficient antibody sensitivity, although together they complement calretinin.     

Immunohistochemical examination of 25 cases of Merkel cell carcinoma: a comparison with small cell carcinoma of the lung and oesophagus, and a review of the literature.

Merkel cell carcinomas (MCC) were compared to small cell carcinomas of the lung (SCCL) and oesophagus (SCCO). Most MCC were of the intermediate cell type while SCCL and SCCO were usually of the small cell type. Only MCC of trabecular type could be separated from SCCL and SCCO by means of histopathological examination alone. All MCC (25) stained with cytokeratin CAM 5.2, 20 of which in a "paranuclear globular" or combined "paranuclear globular"/diffuse pattern while 17 MCC stained with cytokeratin AE1/AE3. Cytokeratin CAM 5.2 reacted with 60 percent of the SCCL and 86 percent of the SCCO, and cytokeratin AE1/AE3 with 33 and 28 percent respectively. Neurofilament stained 17 MCC in a "paranuclear globular" pattern but none of the SCCL and SCCO. All MCC with a diffuse staining pattern for cytokeratin CAM 5.2 were negative for neurofilament. The results of this study and review of the literature indicate that in most instances Merkel cell carcinoma can be separated from other SCC, pulmonary as well as extrapulmonary, by means of histopathological and, above all, immunohistochemical examinations.     

Immunohistochemistry of cytokeratin proteins in squamous and transitional cell lesions of the urinary tract.

Expression of low and high molecular weight cytokeratin proteins was investigated immunohistochemically in a variety of transitional and squamous epithelial lesions of the urinary tract with and without schistosomiasis. The monoclonal antibodies used were CAM 5.2 and NCL5D3 for low, PK 63 and 121 for high, and MAK 6 for a broad range of intermediate molecular weight cytokeratins. On staining with CAM 5.2 and NCL5D3, urothelial hyperplasias (n = 12) and grades 1 (n = 5) and 2 (n = 10) papillary transitional cell carcinomas showed labelling patterns quite distinct from carcinoma in situ (n = 4) and non-papillary grades 2 (n = 6) and 3 tumours (n = 3). Among squamous lesions only focal positivity was obtained in 14 of 22 moderate to poorly differentiated squamous cell carcinomas. By contrast, PK 63 and 121 stained squamous lesions exclusively. MAK 6 stained the whole range of urothelial and squamous lesions with the exception of squamous metaplasias. Polyclonal antikeratin adequately labelled spindle cell areas of high grade tumours. The distinctive staining patterns given by these or similar antibodies may help in the identification of squamous metaplasia and in diagnosing tumours of the urothelium.     

Heterogeneity of epithelial marker expression in routinely processed, poorly differentiated carcinomas.

The application of immunohistochemical markers against epithelial antigens has proved useful for studying tumor differentiation and in aiding tumor diagnosis. However, the reactivity of various epithelial markers with poorly differentiated carcinomas (the situation in which they are most often used) has not been well established. As a result, it is unclear how negative results should be interpreted and how often more than one antibody may be needed to document the epithelial nature of poorly differentiated neoplasms. We studied 98 poorly differentiated epithelial tumors with AE1, CAM 5.2, and EMA to assess the use of these markers in their diagnosis. Both CAM 5.2 and EMA provided support for epithelial differentiation in 71% (70/98) of the cases, while AE1 stained 50% (49/98) of the tumors; CAM 5.2 was the single most useful marker in the subset of poorly differentiated neuroendocrine carcinomas, staining 20 (77%) of 26 tumors. Use of these markers in pairs increased the recognition of epithelial differentiation (at least one marker showing positive staining) as follows: AE1/CAM 5.2, 80% (78/98); AE1/EMA, 87% (85/98); and CAM 5.2/EMA, 99% (97/98). Thirty carcinomas stained with all three markers, 34 with two markers, and in 34 cases only one antibody supported epithelial differentiation. Twelve (21%) of 58 tumors showed evidence of S100 reactivity. None of the 71 cases to which PD7 was applied showed staining This study indicates that poorly differentiated carcinomas are heterogeneous in their expression of antigens recognized by AE1, CAM 5.2, and EMA. Moreover, these results quantitate the probability of reactivity with poorly differentiated carcinomas for each marker and support the use of one or more antibodies in a "backup" panel when a negative result is obtained with a single antibody and the diagnosis of carcinoma is still suspected.     

Cytokeratin-specific monoclonal antibodies are reactive with tumours of smooth muscle derivation. An immunocytochemical and biochemical study using antibodies to intermediate filament cytoskeletal proteins

Formalin fixed and paraffin wax embedded tissue from 13 leiomyosarcomas, three leiomyoblastomas, five leiomyomas and fresh tissue from one leiomyosarcoma and one leiomyoma was studied. Tumours were stained by the avidin-biotin-peroxidase technique with antibodies to desmin, vimentin, cytokeratins and S-100 protein. Neoplastic cells in all the cases were positive for desmin and vimentin but were negative for S-100; antibody CAM 5.2 gave strong reactivity with tumour cells of 12 leiomyosarcomas and all leiomyomas, but failed to stain the leiomyoblastomas; LP34 weakly stained two leiomyosarcomas and one leiomyoblastoma. Immunoblot studies using whole cell extracts of one leiomyosarcoma and one leiomyoma respectively, showed 55-58 kd and 35-38 kd bands reactive with monoclonal antibody CAM 5.2. However, no cytokeratin components were demonstrated when cytoskeletal extracts of the same tissue samples were immunoblotted with CAM 5.2. The implications of these findings for the diagnostic histopathologist are discussed.     

Expression of a new mucin-type glycoprotein in select epithelial dysplasias and neoplasms detected immunocytochemically with Mab A-80

We studied by immunocytochemistry 573 tissue and 106 cytologic samples of human tumors, non-neoplastic proliferative lesions and normal tissues with the monoclonal antibody (Mab) A-80 that recognizes a mucinous glycoprotein from the colon carcinoma cell line LS-174T. The spectrum of benign and malignant breast lesions was studied as were epithelial tumors of the colon, stomach, pancreas, lung, salivary glands, thyroid, prostate, kidney, endometrium, skin and mesothelium; non-epithelial tumors included lymphomas, melanomas, gliomas, meningiomas, and sarcomas of soft tissue and bone. With a single exception, breast carcinomas regardless of histologic type were reactive while few fibroadenomas stained weakly and focally. In fibrocystic disease, the presence and intensity of the reactivity paralleled the severity of the epithelial proliferation, e.g. staining was strong in foci of severe or atypical hyperplasia, borderline lesions and carcinomas in situ; apocrine metaplasia stained often but less strongly. Barrett's mucosa, colonic polyps and most gastric and colonic carcinomas stained regardless of glandular features while small cell neuroendocrine carcinomas did not. Adenocarcinomas of the pancreas and lung, and a subset of large cell lung carcinomas reacted whereas neuroendocrine carcinomas of those sites did not. Carcinomas of endometrium, ovary and prostate reacted variably whereas thyroid and renal carcinomas and mesotheliomas were either negative or weakly reactive despite the presence of glands. Lymphomas, skin adnexal tumors, nevi, schwannomas, melanomas, gliomas and sarcomas generally did not react but occasional A-80-positive cells were seen in rare sarcomas and meningiomas. Immunostaining patterns in cytologic specimens were similar to the aforementioned. We conclude that Mab A-80 is an excellent marker for breast carcinomas, and for certain proliferative forms of fibrocystic disease that may precede or be associated with carcinomatous transformation. In colonic, pulmonary and gastric carcinomas, staining with Mab A-80 revealed exocrine features regardless of the absence of glands whereas in renal and thyroid carcinomas and in mesotheliomas staining was focal and weak or absent irrespective of glandular features. We suggest that Mab A-80 is a very promising immunolabel for select exocrine carcinomas, and for some of the dysplasias that may precede their development; its ease of application on tissue sections and cytologic specimens should broaden its usefulness.     

p63 is useful in the diagnosis of mammary metaplastic carcinomas

AIMS: p63 has been recently reported to be expressed in sarcomatoid/metaplastic carcinoma of the breast, in addition to its role as a myoepithelial marker. A large series of 34 metaplastic carcinomas, including cases with pure epithelial component (squamous cell and adenosquamous carcinomas), biphasic tumours with carcinomatous and sarcomatoid components and monophasic tumours with only spindle cell component, were evaluated for p63 expression with respect to the different cellular components. METHODS: All of the metaplastic carcinomas were assessed for p63 and conventional epithelial and mesenchymal markers of AE1/3, CAM5.2 and vimentin by immunohistochemistry. RESULTS: All of the different categories of metaplastic carcinomas showed similar clinico-pathological features (patient age, tumour size, nuclear grade, mitotic activity, lymph node status and hormonal receptor status). For metaplastic carcinoma with epithelial component only, p63 was only expressed in the squamous cell component, but not the adenocarcinoma component. Eight of the 10 tumours were positive for p63. For the tumours with sarcomatoid component, either singly or together with carcinomatous component, p63 was positive in 14 of 24 cases. Pure sarcomas and carcinomas were all negative for p63 staining by immunohistochemistry, thus rendering p63 staining highly specific for diagnosing metaplastic carcinoma. CONCLUSIONS: Using p63 for diagnosis of metaplastic carcinoma gives a sensitivity of 65%, a specificity of 96%, a positive predictive value of 96%, and a negative predictive value of 66% and an accuracy of 78%. p63 may be used as an adjunct marker in the diagnosis of metaplastic carcinoma.     

Mac387: its non-specificity as a tumour marker or marker of histiocytes

A commercially available monoclonal antibody which detects histiocytes in paraffin sections, Mac387, was reacted with 148 soft tissue sarcomas, 29 carcinomas and 10 malignant melanomas. The soft tissue sarcomas had been previously immunophenotyped. All categories of sarcomas, with the exception of angiosarcomas, were positive. Most showed staining in less than 50% of the tumour cells. Six of 10 adenocarcinomas and three of nine basal cell carcinomas were positive, while squamous carcinomas and malignant melanomas were negative. Normal squamous mucosa and epidermis were also positive. The results of this study suggest that Mac387 is of little value in the differential diagnosis of soft tissue tumours, nor is it a specific histiocytic marker.     

KP-1: not a specific marker. Staining of 137 sarcomas, 48 lymphomas, 28 carcinomas, 7 malignant melanomas and 8 cystosarcoma phyllodes

This study documents the reactions of the monoclonal antibody KP-1, which detects histiocytes in paraffin sections, with 137 sarcomas, 48 lymphomas, 28 carcinomas, 7 malignant melanomas and 8 cystosarcoma phyllodes. The soft tissue sarcomas had been previously immunophenotyped. Positive staining was obtained in all categories of sarcoma except clear-cell sarcomas. Most categories of sarcoma showed staining in less than 10% of tumour cells although a minority of leiomyosarcomas showed more extensive staining. Five of 7 malignant melanomas were also positive while all lymphomas and carcinomas were negative. We conclude that KP-1 positivity is not helpful in supporting the histiocytic origin of a tumour and is of limited value in the differential diagnosis of soft tissue sarcomas or their separation from other categories of malignancy.     

Monoclonal antibody to cytokeratin for use in routine histopathology

CAM 5.2 is a murine monoclonal antibody, raised against the colon carcinoma cell line HT29, which recognises lower molecular weight intracellular cytokeratin proteins within secretory epithelia. Extensive indirect immunohistochemical studies have confirmed that this antibody stains formalin fixed (and freshly frozen) normal and malignant human tissue in a consistent manner. Reliable staining of conventionally processed pathological tissues provides more accurate identification and staging of human malignant epithelial diseases.     

A comparative immunohistochemical study of malignant mesothelioma and renal cell carcinoma: the diagnostic utility of Leu-M1, Ber EP4, Tamm-Horsfall protein and thrombomodulin

Metastatic renal cell carcinoma has occasionally been reported to mimic malignant pleural mesothelioma. Morphologically, histochemically and immunohistochemically, similarities in the two tumours exist making their differentiation difficult, particularly in biopsy specimens. The aim of this study was to make a comparative immunohistochemical analysis of the two tumours by use of a panel of four antibodies (Leu M1; Ber EP4; thrombomodulin and Tamm-Horsfall protein). Their suitability in differentiating between the two tumours was assessed. We examined 20 cases of renal cell carcinoma and 20 cases of malignant pleural mesothelioma. On immunostaining with Leu M1, 14 of 20 renal cell carcinomas were positive, yielding 70% sensitivity and 95% specificity and one of 20 mesotheliomas. In comparison, Ber EP4 antibody stained only seven of 20 of the renal cell carcinomas. In addition, it was noted that four tubulopapillary pattern renal cell carcinomas stained positively with both anti-Leu M1 antibody and Ber EP4 antibody. Thrombomodulin immunostaining was present in 11 of 20 mesotheliomas (55% sensitivity and demonstrated 95% specificity) and one of 20 renal cell carcinomas. For epithelial mesotheliomas only, thromobomodulin staining was identified in 10 of 14 cases. In the differentiation of renal cell carcinoma from epithelial mesothelioma we recommend the use of Leu M1 and thrombomodulin as diagnostically useful markers. None of the antibodies used in this study was effective in distinguishing sarcomatoid renal cell carcinoma from sarcomatous mesothelioma. Tamm-Horsfall protein showed little diagnostic utility in differentiating the two tumours.     

An immunoperoxidase study of renal cell carcinomas: correlation with nuclear grade, cell type, and histologic pattern

We applied a panel of antibodies to formalin-fixed, paraffin-embedded sections of 55 renal cell carcinomas using a three-stage immunoperoxidase technique. The antibody panel included two anti-keratins, AE1 and CAM5.2, anti-epithelial membrane antigen (EMA), anti-vimentin, anti-S100 protein, and the anti-leukocyte marker PD7/26. Forty-eight of 55 renal cell carcinomas expressed keratins. CAM5.2 stained 46 tumors (84%) and AE1 stained 37 neoplasms (67%). AE1 reacted with two CAM5.2-negative tumors. EMA was expressed by 35 carcinomas (64%), including three of the CAM5.2-negative neoplasms. Therefore, using all three antibodies, 50 neoplasms (91%) expressed antigens of epithelial differentiation. Anti-EMA and AE1 were complementary to each other; the combination stained 46 of the carcinomas, comparable with CAM5.2 alone. Vimentin was expressed by 26 tumors (47%), and S100 was expressed by one. PD7/26 did not stain any of the cases. Vimentin expression correlated with nuclear grade; low nuclear grade neoplasms infrequently expressed vimentin, while the converse was true for high nuclear grade tumors. Keratin expression was related to tumor cell type and histologic pattern, as fewer neoplasms of clear cell type and with a solid pattern expressed keratins. In contrast, all papillary and eight of nine (89%) spindled carcinomas expressed keratins.     

Cytokeratin 7: a useful adjunct in the diagnosis of chromophobe renal cell carcinoma

AIMS: The histopathological diagnosis of chromophobe renal cell carcinoma can present a diagnostic challenge, as these tumours can resemble either conventional renal cell carcinoma or oncocytoma. The aim of this study was to determine whether cytokeratin 7 expression is of practical use in the distinction of these three entities. METHODS AND RESULTS: A total of 40 cases previously diagnosed as either chromophobe renal cell carcinoma, conventional renal cell carcinoma or oncocytoma were identified. A representative section of each was stained with H&E and cytokeratin 7. Following independent review of the cases by three pathologists, a consensus diagnosis for each case was reached and the pattern of cytokeratin 7 staining was assessed. There were 12 cases of chromophobe renal cell carcinoma in the study, all of which showed a characteristic peripheral membrane pattern of staining for cytokeratin 7. Seventeen of the 18 cases of conventional renal cell carcinoma studied were negative for cytokeratin 7, while one case showed weak focal staining of <5% of the cells. The 10 cases of oncocytoma showed patchy weak to moderate cytoplasmic expression of cytokeratin 7, without the characteristic peripheral membrane accentuation seen in the chromophobe carcinomas. CONCLUSIONS: Immunohistochemical staining for cytokeratin 7 appears to be a useful adjunct in the diagnosis of chromophobe renal cell carcinoma, and in distinguishing this tumour from both oncocytoma and conventional renal cell carcinoma.