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It is special because it is not a routine stain that is performed on a tissue sample. The routine stain for tissue sections is the H and E (Hematoxylin and Eosin). This stain has stood the test of time for nearly the entire 20th century.  Hematoxylin stains tissue a deep blue while eosin stains a deep red. Depending upon the tissue, all cells are composed of a nearly infinite combination of the various hues and shades of these two stains.  Most cells have a reproducible staining pattern, regardless of the tissue. For example, cell nuclei usually are deeply blue (or basophilic), preferentially staining with hematoxylin.  Squamous cells, such as that comprising the skin, are usually red (or eosinophilic). 

The term usual is used to distinguish it from immunoperoxidase stains.  These stains are not monoclonal antibodies and thus are not as specific for the tissue it stains. Nontheless, for many years, this panel of stains aided pathologists in diagnoses.  A common stain is the mucicarmine stain which highlights mucin a bright pink on a background of yellow.  The presence of mucin within a malignant tumor may indicate glandular differentiation suggesting it is an adenocarcinoma.

These stains became part of the pathologists' armamenterium during the 1980's.  It has revolutionized the practice of diagnostic pathology as well as opened new insights into basic research.  These stains are really monoclonal antibodies.  These are antibodies which are raised against specific proteins and amplified by cloning.  The antibodies are then labelled with a marker which stains with a brown or red counterstain.  Depending upon the antibody used and the tissue which is stained, the pathologist may use these to narrow a differential diagnosis or confirm an initial impression.  For example, a poorly differentiated tumor which has no differentiating features by routine tissue examination may be strongly positive for immunoperoxidase stains S-100 and vimentin, while negative for cytokeratin and leukocyte common antigen.  Based upon these results, a diagnosis of melanoma can be strongly suspected. 

OUTLINE

Quality Assurance
Stains and Antibodies
Commonly Used Terms
Internet Links

QUALITY ASSURANCE	CHARACTERIZATION
GENERAL
Quality assurance in immunohistochemistry: results of an interlaboratory trial involving 172 pathologists. Rudiger T, Hofler H, Kreipe HH, Nizze H, Pfeifer U, Stein H, Dallenbach FE, Fischer HP, Mengel M, von Wasielewski R, Muller-Hermelink HK. Institute of Pathology, Technical University Munchen, Germany.	Am J Surg Pathol 2002 Jul;26(7):873-82 Abstract quote The practicability of quality assurance in immunohistochemistry and its integration into the diagnostic process were both tested in this Germany-wide interlaboratory trial. One hundred seventy-two pathologists received one hematoxylin and eosin and five unstained slides from five cases; all cases were selected by a panel because immunohistochemistry was required for their final diagnosis. Participants rendered a morphologic diagnosis and then substantiated it immunohistochemically. Stained slides and evaluation sheets were reviewed by the panel, and the diagnostic process was analyzed in individual steps: morphologic diagnosis, selection of antibodies, staining quality, interpretation of stained slides, conclusions, and final diagnosis. Diagnosis-independent immunohistochemical performance was tested using a multisample tissue block (30 samples) that was stained and evaluated for six common antigens. For individual cases, corresponding to their difficulty, 21-89% of the final diagnoses (altogether 57% from 828 diagnoses) were correct. In a statistical analysis, the tentative diagnosis, the interpretation of stains and conclusions drawn from immunohistochemistry, were independent factors in reaching the diagnosis. Sensitivity to detect estrogen receptors on the multisample tissue block was only 48%. However, 24% of the stains were interpreted as falsely negative. The low staining sensitivity was not correlated to the number of correct diagnoses. The major problem of applying immunohistochemistry in surgical pathology appears to be its integration into the diagnostic process and not the staining quality. Both future quality control projects and training will have to regard these integrative requirements. Multisample tissue blocks provide a promising tool to standardize quantitative immunohistochemical parameters, such as receptor or proliferation scores.
ANTIGEN RETRIEVAL
Improved 1-h rapid immunostaining method using intermittent microwave irradiation: practicability based on 5 years application in Toyama Medical and Pharmaceutical University Hospital. Kumada T, Tsuneyama K, Hatta H, Ishizawa S, Takano Y. Department of Pathology, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Sugitani, Toyama, Japan.	Mod Pathol. 2004 Sep;17(9):1141-9. Abstract quote   Immunostaining depending on antigen-antibody specificity is the commonest approach for determining the localization of specific antigens in tissue sections. This procedure is applicable not only with frozen or specially fixed samples, but also has proved reliable with formalin-fixed paraffin-embedded tissue sections through improvement of antigen-retrieval. Immunostaining is thus firmly established as a tool for diagnostic pathology and in our institute multiple antibodies are applied for 13-15% of the cases examined, as well as H and E staining. With the standard approach, approximately 3 h is necessary from the beginning of deparaffinization till covering sections with the Envision system. We utilized intermittent microwave irradiation for 10 min during hybridization with primary and secondary antibodies in a special moist-chamber, to achieve all immunostaining steps within 1 h in 178 primary antibodies frequently used for diagnostic pathology. According to our 5 years experience, such microwave irradiation not only obtained significant specific staining for enhancing the specificity of antigen-antibody reactions, but also inhibited nonspecific binding. We present herein the details of the methodology and recommendations for its application with particular primary antibodies. This method can contribute to savings in time and energy, allowing pathologists to rapidly obtain diagnostic information.
A Molecular Mechanism of Formalin Fixation and Antigen Retrieval Seshi R. Sompuram, PhD, Kodela Vani, MS, Elizabeth Messana, and Steven A. Bogen, MD, PhD	Am J Clin Pathol 2004;121:190-199 Abstract quote Despite the popularity of antigen-retrieval techniques, the precise molecular mechanism underlying the process remains enigmatic. We examined the molecular features underlying the loss of immunoreactivity following formalin fixation, with subsequent recovery by antigen retrieval. To do this, we first created a molecular model using short peptides that mimic the antibody-binding site of common clinical protein targets. The advantage of this model is that we know the amino acid sequence in and around the antibody-binding site. We observed that some, not all, of the peptides exhibited the formalin-fixation and antigen-retrieval phenomenon. Other peptides did not lose their ability to be recognized by antibody, even after prolonged incubation in formalin. A third, intermediate group exhibited the formalin-fixation and antigen-retrieval phenomenon only if another irrelevant protein was mixed with the peptide before fixation. Amino acid sequence analysis indicates that fixation and antigen retrieval are associated with a tyrosine in or near the antibody-binding site and with an arginine elsewhere, implicating the Mannich reaction as important in fixation and antigen retrieval.
Effect of Formalin Tissue Fixation and Processing on Immunohistochemistry Martin Werner, M.D.; Andreas Chott, M.D.; Alfredo Fabiano, M.D.; Hector Battifora, M.D. From the Institut für Allgemeine Pathologie und Pathologische Anatomie, Klinikum rechts der Isar, Technische Universität München, Germany (M.W.); Institut für klinische Pathologie, Allgemeines Krankenhaus Wien, Universität Wien, Austria (A.C.); Servizio Anatomia Patologica, Ospedale Fatebenefratelli, Isola Tiberina, Rome, Italy (A.F.); and Immunohistochemistry, Pacific Coast Reference Laboratories, Cypress, California, U.S.A. (H.B.).	Am J Surg Pathol 2000;24:1016-1019 Abstract quote Although immunohistochemistry is routinely performed by many pathology laboratories, its standardization still lags behind. A major cause of variation in the reproducibility of immunohistochemical staining is induced by tissue fixation and, to a lesser degree, tissue processing. This report, stemming from the first meeting of the International Consensus Group on Standardization and Quality Control (ICGSQC) in Nice, France, summarizes the problem and suggests solutions to begin to achieve standardization of fixation and processing. Most laboratories use neutral-buffered formalin (10%) for tissue fixation which introduces cross-links, whereas coagulative fixatives are less popular. Problems with formalin fixation comprise delay of fixation and variations in the duration of the fixation mainly. Solutions to these problems could be to start fixation soon (<30 min) after surgical removal of the tissue and to avoid overfixation (>24–48 hrs). For tissue processing, the most important problem is inadequate tissue dehydration prior to paraffin embedding. This can be prevented by preparing all solutions freshly every week, depending on the volume of tissue processed. If consistently applied, these procedures could eliminate some of the sources of variation in immunohistochemical stains.
TISSUE MICROARRAY
Large-scale molecular and tissue microarray analysis of mesothelin expression in common human carcinomas. Frierson HF Jr, Moskaluk CA, Powell SM, Zhang H, Cerilli LA, Stoler MH, Cathro H, Hampton GM.	Hum Pathol. 2003 Jun;34(6):605-9. Abstract quote The 40-kilodalton processed glycoprotein, mesothelin, is highly expressed in epithelial mesotheliomas and adenocarcinomas of the ovary (serous papillary) and pancreas, but its expression in a large series of other common carcinomas has not been completely explored. In the present study, we used oligonucleotide and tissue microarrays to profile the expression of the mesothelin gene (MSLN) and encoded protein, respectively. Among 150 carcinomas of multiple anatomic sites, we found the highest average expression of MSLN in serous carcinomas of the ovary and adenocarcinomas of the pancreas, consistent with previous reports, as well as measurable but less-striking expression in pulmonary, gastric/esophageal, and colorectal adenocarcinomas. On tissue microarrays containing 621 carcinomas derived from the same and additional sites as those profiled by gene expression, mesothelin immunoreactivity was highest in cancers of the ovary (serous papillary, endometrioid, and undifferentiated) and pancreas, with less frequent staining seen in adenocarcinomas of the endometrium, lung, and stomach/esophagus. Some immunopositivity was observed in 42% of pulmonary adenocarcinomas, including 18% that had >50% of tumor cells that were immunoreactive. Some 14% of breast and 30% of colorectal adenocarcinomas showed immunopositivity, but no case contained >50% tumor cells that were immunoreactive. Mesothelin was either entirely absent or present in <5% of carcinomas of the prostate, bladder/ureter, liver, kidney, and thyroid. Overall, we observed good concordance between the results obtained by oligonucleotide and tissue microarrays. This large study of the MSLN gene and protein expression in common carcinomas provides data for future investigations that evaluate the utility of mesothelin/megakaryocyte potentiating factor as a potential serum tumor marker or target of immunotoxin-based therapy in human cancers.
Tissue Array Technology for Testing Interlaboratory and Interobserver Reproducibility of Immunohistochemical Estrogen Receptor Analysis in a Large Multicenter Trial Reinhard von Wasielewski, MD, Michael Mengel, MD, Birgitt Wiese, Thomas Rüdiger, MD Hans Konrad Müller-Hermelink, MD and Hans Kreipe, MD	Am J Clin Pathol 2002;118:675-682 Abstract quote Semiquantitative immunohistochemical assessment of estrogen receptor (ER) is used to predict the likelihood of response to antiestrogen therapy in breast carcinoma. If semiquantitative immunohistochemical analysis leads to therapeutic decisions, the importance of standardization and quality control increases. ER assessment reproducibility was studied among 172 laboratories using tissue microarray slides with 20 tissue spots negative and 10 tissue spots expressing ER at low, medium, or high levels. More than 80% of the laboratories demonstrated ER positivity in the medium- and high-expressing tissue spots, but only about 43% succeeded with tissue spots with low expression. Poor interlaboratory agreement was based on insufficient retrieval efficacy as shown by additional tests using autoclave pretreatment. The immunohistochemical scores used to quantify therapeutic target molecules remain inconclusive as long as progress toward standardized immunohistochemical procedures and evaluation is not achieved. Tissue microarray technology has proved its suitability for large-scale immunohistochemical trials, giving rise to new dimensions in control assessment.
Tissue Micro-Array: A Cost and Time-Effective Method for Correlative Studies by Regional and National Cancer Study Groups Martha Milanes-Yearsley, M.D., M. Elizabeth H. Hammond, M.D., Thomas F. Pajak, Ph.D., Jay S. Cooper, M.D., Chu Chang, M.D., Thomas Griffin, M.D., Diana Nelson, M.D., George Laramore, M.D. and Milijenko Pilepich, M.D. Forrest D. Hsu, M.Sc., Torsten O. Nielsen, M.D., Abdulmohsen Alkushi, M.D., Bev Dupuis, R.T., David Huntsman, M.D., Chih Long Liu, B.Sc., Matt van de Rijn, M.D. and C. Blake Gilks, M.D. Department of Pathology, Ohio State University (MM-Y), Columbus, Ohio; Department of Pathology, LDS Hospital (MEHH), Salt Lake City, Utah; Radiation Therapy Oncology Group, Statistical Unit (TFP), Philadelphia, Pennsylvania; Department of Radiation Oncology, New York University Medical Center (JSC), New York, New York; Department of Radiation Oncology (CC), Columbia Presbyterian Medical Center, New York, New York; Griffin Corporation (TG), Seattle, Washington; Department of Radiation Oncology, Mayo Clinic (DN), Rochester, Minnesota; Department of Radiation Oncology, University of Washington Medical Center (GL), Seattle, Washington; and Department of Radiation Oncology, St. Joseph Mercy Hospital (MP), Ann Arbor, Michigan	Modern Pathology 2002;15:1366-1373 Abstract quote Tissue micro-arrays have been used for molecular and immunohistochemical studies. We sought to evaluate whether such arrays could substitute for whole sections in correlative studies performed by the Radiation Therapy Oncology Group. Four multitumor 150-sample arrays were built using formalin-fixed, paraffin-embedded, archival prostate, brain, and head/neck tumor blocks from RTOG tissue bank. p53 immunostaining of arrays and whole sections was done. Blind evaluation of each slide was made, and agreement rates between the two techniques were determined in various scenarios. Cost was also evaluated. Results demonstrate excellent agreement for p53 between slides and arrays. Agreement improved when three or four replicate arrays were used. Findings based on one to four arrays agree well with those obtained from analysis of the whole tissue samples. Minimal tissue damage, improved tissue salvage, cost reduction, ease of interpretation, and significant time savings were realized by using the arrays. Tissue micro array technique is a valuable tool for evaluation of patient materials associated with clinical trials.
Tissue Microarrays Are an Effective Quality Assurance Tool for Diagnostic Immunohistochemistry Forrest D. Hsu, M.Sc., Torsten O. Nielsen, M.D., Abdulmohsen Alkushi, M.D., Bev Dupuis, R.T., David Huntsman, M.D., Chih Long Liu, B.Sc., Matt van de Rijn, M.D. and C. Blake Gilks, M.D. Department of Pathology and Genetic Pathology Evaluation Centre, Vancouver General Hospital and the University of British Columbia (FDH, TON, AA, BD, DH), Vancouver, British Columbia, Canada; and the Departments of Biochemistry (CLL) and Pathology (MvdR), Stanford University, Stanford, California	Modern Pathology 15:1374-1380 Abstract quote There has been considerable variability in the reported results of immunohistochemical staining for some diagnostically relevant antigens. Our objectives in this study were to (1) use a multitumor tissue microarray with tissue from 351 cases received in our department, representing 16 normal tissues and 47 different tumor types, to compare immunohistochemical staining results in our laboratory with published data, using a panel of 22 antibodies; (2) assess interlaboratory variability of immunohistochemical staining for S-100 using this microarray; and (3) test the ability of hierarchical clustering analysis to group tumors by primary site, based on their immunostaining profile. Tissue microarrays consisting of duplicate 0.6-mm cores from blocks identified in the hospital archives were constructed and stained according to our usual protocols. Antibodies directed against the following antigens were used: B72.3, bcl-2, carcinoembryonic antigen, c-kit, pankeratin, CD 68, CD 99, CK 5/6, CK 7, CK 8/18, CK19, CK 20, CK 22, epithelial membrane antigen, estrogen receptor, melan-A, p53, placental alkaline phosphatase, S-100, synaptophysin, thyroid transcription factor-1, and vimentin. Staining results on the array cases were compared with published results, and hierarchical clustering analysis was performed based on the immunohistochemical staining results. Unstained slides of the multitumor tissue microarray were sent to five other diagnostic immunohistochemistry laboratories and stained for S-100 protein. The staining results from the different laboratories were compared. Staining results using our current methods and samples from our laboratory were compatible with those described in the literature for most antigens. Placental alkaline phosphatase staining was not specific with our protocol, showing staining of a broad spectrum of different tumors; this finding initiated a review of our recent requests for placental alkaline phosphatase immunostaining and revealed two instances in which placental alkaline phosphatase positivity was incorrectly interpreted as evidence of a germ cell tumor. S-100 staining was less sensitive but more specific for the diagnosis of melanoma or neural tumor in our laboratory, compared to some published reports. Assessment of interlaboratory variability of S-100 immunostaining showed that there was more frequent staining of carcinomas in some laboratories, resulting in decreased specificity of S-100 staining in distinguishing melanoma from carcinoma. Hierarchical clustering analysis showed a strong trend for tumors to cluster by tissue of origin, but there were significant exceptions. We conclude that multiple-tumor microarrays are an efficient method for assessing the sensitivity and specificity of staining with any antibody used diagnostically. As a tool for quality assurance, they offer the advantage of taking into account local differences in tissue fixation, processing, and staining. They also allow cost-effective assessment of interlaboratory variability in immunohistochemical staining. Results of hierarchical clustering analysis show the potential for panels of immunohistochemical stains to identify the primary site of metastatic carcinomas but also confirm the limitations of currently available antibodies in giving unequivocal tissue-specific staining patterns.

 

INDIVIDUAL IMMUNOPEROXIDASE STAINS	CHARACTERIZATION
ALPHA-METHYLACYL -CoA RACEMASE
Alpha-Methylacyl-CoA Racemase: A Novel Tumor Marker Over-expressed in Several Human Cancers and Their Precursor Lesions. Zhou M, Chinnaiyan AM, Kleer CG, Lucas PC, Rubin MA. Departments of Pathology (M.Z., A.M.C., C.G.K., P.C.L., M.A.R.) and Urology (A.M.C., M.A.R.), University of Michigan School of Medicine, Ann Arbor, Michigan, U.S.A.	Am J Surg Pathol 2002 Jul;26(7):926-31 Abstract quote alpha-Methylacyl-CoA racemase (AMACR) is a mitochondrial and peroxisomal enzyme involved in the metabolism of branched-chain fatty acid and bile acid intermediates. Recently, AMACR has been demonstrated to be over-expressed in localized and metastatic prostate cancer, suggesting that it may be an important tumor marker. This study examines AMACR expression in a variety of human cancers and their precursor lesions. A survey of online Expressed Sequence Tags (ESTs) and Serial Analysis of Gene Expression (SAGE) databases revealed that AMACR was over-expressed in multiple cancers. The findings were confirmed by AMACR immunohistochemistry performed on several tissue microarrays containing common human tumors, including prostate, colon, and breast. Based on prior work, AMACR protein expression was divided into two categories: negative (negative to weak staining intensity) and positive (moderate to strong staining intensity). AMACR protein over-expression was found in a number of cancers, including colorectal, prostate, ovarian, breast, bladder, lung, and renal cell carcinomas, lymphoma, and melanoma. Greatest over-expression was seen in colorectal and prostate cancer with positive staining in 92% and 83% cases, respectively. AMACR over-expression was present in 44% of breast cancer cases. AMACR was also over-expressed in precursor lesions. Sixty-four percent of high-grade prostatic intraepithelial neoplasia and 75% colonic adenomas demonstrated positive AMACR protein expression. Reverse transcriptase-polymerase chain reaction for AMACR using laser capture microdissected prostate tissue confirmed gene over-expression at the mRNA level. In conclusion, our study suggests that AMACR is potentially an important tumor marker for several cancers and their precursor lesions, especially those linked to high-fat diets.
CADHERINS
Epithelial-type and neural-type cadherin expression in malignant noncarcinomatous neoplasms with epithelioid features that involve the soft tissues. Laskin WB, Miettinen M. Department of Pathology, Northwestern University Medical School, Chicago, Ill (Dr Laskin); and the Department of Soft Tissue Pathology, Armed Forces Institute of Pathology, Washington, DC (Dr Miettinen).	Arch Pathol Lab Med 2002 Apr;126(4):425-31 Abstract quote Context.-Transmembrane adhesion molecules, epithelial-type cadherin (ECAD) and neural-type cadherin (NCAD), help in regulating transformations between epithelial and mesenchymal cells in the developing embryo and in maintaining the epithelioid phenotype. Consequently, the presence of epithelioid cells in certain malignant noncarcinomatous neoplasms raises speculation that the expression of ECAD and NCAD in these neoplasms may have diagnostic significance. Objective.-To investigate the utility of ECAD and NCAD immunoexpression in distinguishing malignant (noncarcinomatous) neoplasms with epithelioid features that involve the soft tissues. Design.-Membranous immunoreactivity of anti-ECAD and anti-NCAD was evaluated on archived cases selected from the files of the Armed Forces Institute of Pathology. Results.-Epithelial-type cadherin was found in biphasic synovial sarcoma (35 of 35 cases), malignant melanoma (13/21), monophasic fibrous synovial sarcoma (13/26), clear cell sarcoma (4/9), poorly differentiated synovial sarcoma (3/13), diffuse mesothelioma (4/20), malignant epithelioid peripheral nerve sheath tumor (1/6), and epithelioid sarcoma (5/62). Neural-type cadherin was observed in chordoma (11/11), biphasic synovial sarcoma (30/35), diffuse mesothelioma (14/20), malignant melanoma (14/25), epithelioid sarcoma (24/63), epithelioid angiosarcoma (1/4), poorly differentiated synovial sarcoma (2/13), clear cell sarcoma (1/10), and monophasic fibrous synovial sarcoma (1/26). Eighteen cases of primary cutaneous squamous cell carcinomas all tested positive for ECAD, whereas NCAD was focally observed in 5 cases. No expression of either molecule was observed in cases of epithelioid hemangioendothelioma (n = 9), alveolar soft part sarcoma (n = 8), and extraskeletal myxoid chondrosarcoma (n = 7). Conclusions.-Epithelial-type and neural-type cadherins are found in a variety of noncarcinomatous neoplasms with epithelioid features that involve the soft tissues and can be utilized, in association with other immunomarkers, in distinguishing chordoma (100% NCAD) from extraskeletal myxoid chondrosarcoma and conventional chondrosarcoma of bone (0% NCAD), squamous cell carcinoma (100% ECAD) from epithelioid sarcoma (8% ECAD), and biphasic synovial sarcoma (100% ECAD) from diffuse mesothelioma (20% ECAD).
CALRETININ
Calretinin expression in human normal and neoplastic tissues: A tissue microarray analysis on 5233 tissue samples. Lugli A, Forster Y, Haas P, Nocito A, Bucher C, Bissig H, Mirlacher M, Storz M, Mihatsch MJ, Sauter G.	Hum Pathol. 2003 Oct;34(10):994-1000. Abstract quote   Calretinin is a calcium-binding protein expressed in different normal and neoplastic tissues. Early studies suggested that calretinin is a useful marker to differentiate adenocarcinomas from malignant mesotheliomas of the lung, but subsequent work has shown that calretinin can be expressed in several other tumor types. To systematically investigate the epidemiology of calretinin expression in normal and neoplastic tissues, we used tissue microarrays (TMAs) to analyze the immunohistochemically detectable expression of calretinin in 5233 tissue samples from 128 different tumor categories and 76 different normal tissue types. At least 1 case with weak expression could be found in 74 of 128 (58%) different tumor types and 46 entities (36%) had at least 1 tumor with strong positivity. In normal tissues, a particularly strong expression was found in Leydig cells of the testis, neurons of the brain, theca-lutein and theca interna cells of the ovary, and mesothelium. In tumors, strong calretinin expression was most frequently found in malignant mesotheliomas (6 of 7), Leydig cell tumors of the testis (5 of 5), adenomas of adrenal gland (5 of 9), and adenomatoid tumors (4 of 9). In summary, calretinin is frequently expressed in many different tumor types. Metastases of various different origins must be included in the differential diagnosis of calretinin-positive pleura tumors.
CD138
Expression of T/NK-Cell and Plasma Cell Antigens in Nonhematopoietic Epithelioid Neoplasms An Immunohistochemical Study of 447 Cases Peiguo G. Chu, MD, PhD, Daniel A. Arber, MD, and Lawrence M. Weiss, MD	Am J Clin Pathol 2003; 120:64-70 Abstract quote We studied the expression of CD2, CD3, CD4, CD5, CD7, CD8, CD56, and CD138 in 447 cases of common human neoplasms with epithelioid features. CD2, CD3, CD4, and CD8 antigens were detected in none of 447 cases of nonhematopoietic tumors. CD5 and CD7 antigens were expressed in 12.3% and 19.5% of cases of nonhematopoietic tumors, respectively. Their expression was found primarily in adenocarcinomas from the gastrointestinal tract, breast, and female reproductive organs. The high expression of CD5 and CD7 antigen in pancreatic ductal carcinoma and cholangiocarcinoma and high expression of CD7 in epithelioid sarcoma may have diagnostic value. One quarter of cases were positive for CD56. Overexpression of CD56 antigen was detected mainly in neuroendocrine tumors or adenocarcinomas with neuroendocrine differentiation. Its consistent overexpression in adrenal cortical and thyroid tumors may have diagnostic usefulness. Virtually all tumor types studied were CD138+ with a variable positivity rate. The negative staining of CD138 in malignant mesothelioma may be useful for separating mesothelioma from metastatic adenocarcinoma.
CD163 (Hemoglobin Scavenger Receptor)
Expression of CD163 (Hemoglobin Scavenger Receptor) in Normal Tissues, Lymphomas, Carcinomas, and Sarcomas Is Largely Restricted to the Monocyte/Macrophage Lineage. Nguyen TT, Schwartz EJ, West RB, Warnke RA, Arber DA, Natkunam Y. From the Department of Pathology, Stanford University School of Medicine, Stanford, CA.	Am J Surg Pathol. 2005 May;29(5):617-24. Abstract quote   CD163, a hemoglobin scavenger receptor, is expressed in monocytes and macrophages. We tested the expression of the CD163 protein in 1,105 human malignancies and normal tissues using tissue microarrays and conventional paraffin-embedded tissue sections. Besides staining nonneoplastic monocytes and histiocytes (tissue macrophages), membranous/cytoplasmic staining for CD163 was primarily limited to neoplasms with monocytic/histiocytic differentiation. CD163 reactivity was not observed in normal tissues, lymphomas, carcinomas, and in a majority of mesenchymal neoplasms, including follicular dendritic cell tumors (0 of 4), although it stained admixed histiocytes. Staining for CD163 was seen in Rosai-Dorfman disease (5 of 6), histiocytic sarcoma (3 of 4), littoral cell angioma (6 of 6), and Langerhans cell histiocytosis (3 of 5). A subset of atypical fibrous histiocytomas (9 of 16), benign fibrous histiocytomas (6 of 9), and atypical fibroxanthomas (1 of 3) also showed CD163 staining. Our studies also confirm earlier work showing that CD163 is expressed in acute myeloid leukemia with monocytic differentiation (AML, FAB subtype M5) (2 of 6), as well as a majority of giant cell tenosynovial tumors (7 of 8). Its limited range of expression and tissue specificity indicate that CD163 may have significant diagnostic utility in separating specific tumors with monocytic and histiocytic derivation from other entities in their differential diagnosis.
FASCIN
Actin-binding protein fascin expression in skin neoplasia. Goncharuk VN, Ross JS, Carlson JA. Department of Pathology, Albany Medical College, Albany, NY, Divisions of Dermatopathology and Dermatology, Albany Medical College, Albany, NY, USA.	J Cutan Pathol 2002 Aug;29(7):430-8 Abstract quote BACKGROUND: Fascin containing actin bundles provide mechanical support to cellular protrusions and stress fibers. In cancers, some malignant cells (e.g. subsets of breast and ovarian carcinomas) express fascin. In skin cancer, the role of fascin is unknown. METHODS: Cases of 61 keratocytic neoplasms, 35 melanocytic neoplasms, nine extramammary Paget's disease (four with adenocarcinoma) and five sarcomas (angiosarcoma and atypical fibroxanthoma) were examined by immunohistochemistry, using monoclonal antihuman fascin antibody, clone 55 k-2 (Dako Corporation, Carpinteria, CA, USA). RESULTS: Fascin labeled all sarcomas and all keratinocytic neoplasms except for pagetoid pattern Bowen's disease. The regions of most intense fascin labeling were seen in the basal cells of infiltrative tumor margins. A minority of Merkel cell carcinomas exhibited weak or absent immunoreactivity. All melanocytic nevi except for some junctional nests of dysplastic melanocytic nevi expressed fascin. However, pagetoid cells of melanoma in situ and epithelioid cells of invasive melanoma weakly expressed or did not express fascin, whereas melanoma cells exhibiting spindle cell morphologies labeled intensely with fascin. Lastly, all cells of extramammary Paget's disease and most associated adenocarcinomas cells did not or were faintly labeled by fascin antibodies. Decreased or absent fascin expression was significantly associated with skin cancers with a high risk for metastasis (e.g. melanoma) vs. those with a low risk (e.g. basal cell carcinoma) (24% vs. 100% with > 50% immunoreactivity; p = 0.0001, chi-squared test). CONCLUSION: Fascin is expressed by skin tumors that locally infiltrate and replace surrounding tissues indicating a role for fascin in cell adhesion, cell motility and invasiveness. No or weak fascin expression is exhibited by cancers with pagetoid intraepidermal spread and by invasive tumors with a high risk of metastasis. Downregulation or loss of fascin's actin-bundling properties, probably associated with disorganization of cell-cell and cell-matrix contacts, may be a crucial step in the progression from locally invasive to widely disseminating cancers.
MESOTHELIN
Application of mesothelin immunostaining in tumor diagnosis. Ordonez NG. University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.	Am J Surg Pathol. 2003 Nov;27(11):1418-28. Abstract quote   Mesothelin is a differentiation antigen that was first described as the antigenic target of the monoclonal antibody K1. Using this antibody, it was demonstrated that mesothelin is strongly expressed in normal mesothelial cells, mesotheliomas, nonmucinous ovarian carcinomas, and some other malignancies. Immunostaining with the K1 antibody was suggested to be useful in the diagnosis of mesothelioma in the early 1990s. This, however, could not be further explored until recently because of the lack of commercially available anti-mesothelin antibodies. In a recent investigation by this author, all epithelioid mesotheliomas and about 40% of the lung adenocarcinomas reacted with the 5B2 anti-mesothelin antibody, which has only recently become commercially available. It was concluded that immunostaining with this antibody has limited value in discriminating between these conditions. The aim of the current study was to further investigate the potential application of the 5B2 antibody in tumor diagnosis. Mesothelin expression was evaluated in formalin-fixed, paraffin-embedded samples of normal tissues and in 471 tumors of various origins. The carcinomas that most frequently exhibited strong mesothelin reactivity were nonmucinous carcinomas of the ovary (14 of 14 serous, 3 of 3 endometrioid, 6 of 8 clear cell, and 4 of 4 transitional cell carcinoma), and adenocarcinomas of the pancreas (12 of 14), the ampulla of Vater (3 of 3), endometrium (7 of 11), lung (14 of 34), and liver (7 of 19 cholangiocarcinomas). The carcinomas that did not express mesothelin included renal cell carcinomas, hepatomas, carcinomas of the thyroid, adrenal cortical carcinomas, prostatic adenocarcinomas, and carcinoid tumors. All germ cell tumors, with the exception of teratomas, were consistently negative for mesothelin. Because of the strong mesothelin expression in nonmucinous carcinomas of the ovary, but not in a variety of tumors with which these lesions may be confused (eg, clear cell carcinoma of the ovary versus endodermal sinus tumor or renal cell carcinoma, clear cell type; transitional cell carcinoma of the ovary versus TCC of the urinary tract), immunostaining for this marker could be useful in establishing the differential diagnosis. The strong mesothelin expression in the large majority of pancreatic ductal adenocarcinomas (12 of 14), but not in normal pancreas, confirms that this marker may have some diagnostic utility in discriminating between neoplastic and nonneoplastic pancreatic ductal epithelium. The mesothelin expression in about one-third of the cholangiocarcinomas, but not in hepatomas, suggests that this marker may have some utility in distinguishing between these two malignancies when they are poorly differentiated. In the group of small round blue cell tumors, only desmoplastic small round cell tumors exhibited mesothelin positivity (7 of 12). Of the soft tissue tumors, only the epithelial component of biphasic synovial sarcomas (9 of 9) expressed mesothelin. These findings indicate that, in some instances, mesothelin immunostaining can assist in the diagnosis of these tumors. Finally, the strong mesothelin reactivity seen in the adenomatoid tumors (3 of 3) provides further support for a mesothelial derivation for this lesion.
Nup88 (Karyoporin)
Nup88 (karyoporin) in human malignant neoplasms and dysplasias: correlations of immunostaining of tissue sections, cytologic smears, and immunoblot analysis. Gould VE, Orucevic A, Zentgraf H, Gattuso P, Martinez N, Alonso A. Department of Pathology, Rush Medical College, Chicago, IL 60612, USA.	Hum Pathol 2002 May;33(5):536-44 Abstract quote Nuclear pore complexes (NPCs) are elaborate macromolecular structures that regulate the bidirectional nucleocytoplasmic traffic system. In vertebrate cells, NPCs include a family of 50 to 100 proteins termed nucleoporins (Nups). The 88-kD Nup has been found to be linked in a dynamic subcomplex with the oncogenic CAN/Nup214. Applying a polyclonal antiserum to Nup88 on paraffin sections, we found that it immunoreacts with numerous malignant neoplasms. All carcinomas reacted irrespective of site, type, or degree of differentiation; often, high-grade carcinomas stained more strongly and extensively. Some sarcomas (e.g., fibrosarcomas, leiomyosarcomas, liposarcomas, and rhabdomyosarcomas) reacted intensely; melanomas, gliomas, mesotheliomas, and malignant lymphomas also stained. In situ carcinomas of the colon, stomach, breast, and prostate stained convincingly, as did in situ melanomas; some samples of fetal tissues also reacted. Cytologic smears of some of the aforementioned tumors also stained. In selected samples, enhanced immunostaining of tissue sections and cytologic smears correlated strongly and consistently with immunoblot data. Immunoblots of the same tumors with antibodies to 2 other Nups (Nup214 and Nup153) showed no comparable enhancement. Therefore, it seems that in some malignant tumors, Nup88 overexpression is not parallelled by an overexpression of other Nups. Benign tumors, hyperplasias, and normal tissues showed weak and sporadic staining or absence of staining; immunoblots of the same samples yielded weak signals. Occasional highly proliferative hyperplastic-reactive processes showed focal staining. Thus, our correlative histologic, cytologic, and molecular data indicate that Nup88 may be viewed as a potentially useful, broadly based histodiagnostic and molecular marker of many malignancies and premalignant dysplasias, and further suggest that in some malignant tumors, Nup88 may be selectively overexpressed as compared with other Nups. Thus, we propose that Nup88 be designated as karyoporin.
p27
Inadequate formalin fixation decreases reliability of p27 immunohistochemical staining: probing optimal fixation time using high-density tissue microarrays. De Marzo AM, Fedor HH, Gage WR, Rubin MA. Department of Urology, The Johns Hopkins Medical Institutions, Baltimore, MD 21231-1000, USA	Hum Pathol 2002 Jul;33(7):756-60 Abstract quote Immunohistochemical analysis of molecular targets in clinical tissues is increasingly becoming central to our ability to render diagnoses, to predict prognosis, to select patients for appropriate therapies, and to provide surrogate end points for therapeutic monitoring. For example, reduction of immunohistochemical staining for the cyclin-dependent kinase inhibitor p27(Kip1) has been proposed as a potential prognostic biomarker in prostate, breast, and gastrointestinal tumors. We observed that with our standard formalin fixation in rapidly processed (same-day) radical prostatectomy specimens, there is often a gradient of p27(Kip1) staining in normal prostate epithelium, with more staining near the periphery and less staining toward the center of the sample. This raised the hypothesis that the reliability of staining for p27(Kip1) is decreased in inadequately fixed tissues. The implications of this, if true, are that many studies using p27(Kip1) for prognostic purposes may be subject to unpredictable artifacts, and hence unreliable results, if the fixation of the specimens is not well controlled. The objectives of the present study were (1) to formally test the hypothesis that inadequate fixation time is responsible for apparent loss of p27(Kip1) nuclear staining and (2) to test a recently proposed method for improving the uniformity of immmunohistochemical staining using formalin injection. Prostate tissue sections from radical prostatectomy specimens were either processed immediately (zero time fixation) or fixed for 1, 2, 3, or 8 days in 10% neutral buffered formalin before processing into paraffin. To assure identical antigen retrieval and immunohistochemical staining conditions for specimens fixed for different lengths of time, 2 high-density tissue microarrays (TMAs), containing 564 tissue samples (0.6 mm in diameter) were constructed. Based on an estimate of the percentage of nuclei in normal prostatic epithelial secretory cells with strong staining, quality of p27(Kip1) staining was graded in a blinded fashion with respect to fixation time. There was a significant increase in the percentage of cores that were scored as "strong" as fixation time increased from 0 (same-day processing) to 1 or more days (P <.0001). Interestingly, even at 8 days of fixation, there was excellent staining that was superior to the same-day processing. Based on these results, we conclude the following: (1) for large clinical specimens that have been fixed briefly to decrease diagnostic turn-around time, the reliability of interpretation of immunohistochemical staining may be quite limited; (2) for p27(Kip1), decreased antigen staining as a result of the widely held concept of "overfixation" is much less of a problem than "underfixation"; (3) formalin injection produces a marked improvement in staining for several markers, including p27(Kip1); and (4) high-density TMAs, which assure identical test conditions, provide an excellent platform on which to evaluate the effects of tissue fixation on immunohistochemical staining.
p63
Taking Advantage of Basic Research: p63 Is a Reliable Myoepithelial and Stem Cell Marker: On: p63, a p53 homologue, is a selective nuclear marker of myoepithelial cells of the human breast. Barbareschi M, Pecciarini L, Cangi MG, et al. Am J Surg Pathol 2001;25:1054-1060. Reis-Filho JS, Schmitt FC. At the *School of Health Sciences, University of Minho, Braga, Portugal; the dagger Medical Faculty, University of Porto, Portugal; and the * dagger Institute of Molecular Pathology and Immunology, University of Porto, Portugal.	Adv Anat Pathol 2002 Sep;9(5):280-9 Abstract quote p63 is a recently characterized p53-homolog that is consistently expressed by basal/somatic stem cells of stratified epithelia, myoepithelial cells of the breast and salivary glands, and proliferative compartment of gastric mucosa. p63 is located on the long arm of chromosome 3 (3q27) and it encodes six isoforms, three transactivating (TA) and three DeltaN-isoforms. While the first three isoforms may act as tumor suppressor genes, the DeltaN-isoforms may inhibit the p53/TA-p63-driven cell cycle arrest and apoptosis. Recently, the putative applications of p63 in the identification of myoepithelial cells of the breast and basal cells of the prostate acini have been evaluated; however, no critical systematic analysis of its role in surgical pathology practice have hitherto been reported. We review the putative roles of p63 in surgical pathology practice and provide guidelines for future directions on p63 translational research.
PROTEIN GENE PRODUCT 9.5 (PGP 9.5)
Protein gene product 9.5 (PGP 9.5) is not a specific marker of neural and nerve sheath tumors: an immunohistochemical study of 95 mesenchymal neoplasms. Campbell LK, Thomas JR, Lamps LW, Smoller BR, Folpe AL. University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.	Mod Pathol. 2003 Oct;16(10):963-9. Abstract quote In non-neoplastic tissues, the expression of protein gene product 9.5 (PGP 9.5), a member of the ubiquitin hydrolase family of proteins, is confined to neural and neuroendocrine cells. Although it has been claimed that PGP 9.5 is a specific marker of neural and/or nerve sheath differentiation in human tumors, careful review of the literature suggests that relatively few nonneural or nerve sheath tumors have been studied. Prompted by our recent observation of a PGP 9.5-positive malignant fibrous histiocytoma, we undertook a study of PGP 9.5 expression in a large group of well-characterized mesenchymal neoplasms. Sections from 95 mesenchymal tumors were retrieved from our archives and immunostained for PGP 9.5 using standard avidin-biotin complex technique and heat-induced epitope retrieval. Scoring was as follows: negative, 1+ (<10-25% of cells), 2+ (25-50% of cells), and 3+ (>50% of cells). Normal nerves and fibrous tissue were internal positive and negative controls, respectively. Positive immunostaining was seen in 80/95 (84%) of cases. Positive results by tumor subtype were as follows: (1) nerve sheath tumors: malignant peripheral nerve sheath tumor (7/10), neurofibromas (10/10), and perineuriomas (3/3); (2) (Myo) fibroblastic tumors: malignant fibrous histiocytoma (18/20), low-grade fibromyxoid sarcomas (8/9), fibromatoses (7/7), and desmoplastic fibroblastomas (2/2); (3) vascular tumors: angiosarcomas (4/4), hemangioendotheliomas (3/5), and hemangiomas (3/4); and (4) other non-nerve sheath tumors: pleomorphic liposarcoma (4/4), dermatofibrosarcoma protuberans (2/5), rhabdomyosarcomas (2/2), synovial sarcomas (8/8), melanomas (1/2). All positive cases were 2-3+ except 6 malignant peripheral nerve sheath tumor, 1 neurofibroma, 3 malignant fibrous histiocytoma, 2 low-grade fibromyxoid sarcoma, and 1 dermatofibrosarcoma protuberans. Positive staining was seen in normal smooth muscle and germinal centers in addition to nerves. We conclude that in this, the largest study to date of PGP 9.5 expression in mesenchymal neoplasms, we have found strong (2-3+) expression in the vast majority of nonneural or nerve sheath neoplasms studied. Although PGP 9.5 is a sensitive neural/nerve sheath marker, it is essentially totally nonspecific for diagnostic purposes. It is possible that our findings reflect cross-reactivity of the 13C4 clone with epitopes present on other ubiquitin hydrolases. Alternatively, PGP 9.5 expression may be aberrantly up-regulated in a variety of mesenchymal neoplasms.
TTF-1
Incidence and significance of cytoplasmic thyroid transcription factor-1 immunoreactivity. Bejarano PA, Mousavi F. Department of Pathology, University of Miami School of Medicine, Jackson Memorial Hospital, Miami, Fla.	Arch Pathol Lab Med 2003 Feb;127(2):193-5 Abstract quote Context.-The immunohistochemical identification of thyroid transcription factor-1 (TTF-1) is regarded as the presence of a nuclear pattern of staining and is used to identify tumors of thyroid or pulmonary origin. Although there have been reports of cytoplasmic expression of TTF-1, the significance of this pattern has not been studied in detail. Objectives.-To determine the incidence at which cytoplasmic immunostaining for TTF-1 occurs and to analyze the diagnostic value of this pattern of immunoreactivity. Design.-Histologic sections of 361 consecutive cases of neoplasms stained for TTF-1 were reviewed, and those showing cytoplasmic staining in the tumor cells or in nonneoplastic cells were selected. Clinical correlation was obtained on the latter cases regarding the origin of the tumor. Setting.-An immunohistochemistry laboratory in a tertiary-care institution. Results.-The 361 tumors were obtained from 29 organ sites and corresponded to primary and metastatic neoplasms. Twenty-three (6.3%) tumors showed cytoplasmic staining for TTF-1. In 13 of these, the primary site of origin was established with certainty: 7 were lung carcinomas (3 primary lung adenocarcinomas, 1 primary large cell carcinoma, 1 metastatic small cell carcinoma to the liver, 1 metastatic adenocarcinoma to a neck lymph node, and 1 metastatic adenocarcinoma to thigh soft tissue), 3 colonic adenocarcinomas (2 metastases to vertebrae and 1 to lung), 1 metastatic breast ductal adenocarcinoma to femur, 1 metastatic laryngeal squamous cell carcinoma to liver, and 1 meningioma involving the orbit bone. There were 3 lung carcinomas with concomitant nuclear immunostaining. Nonneoplastic liver tissue was the most frequent nontumoral tissue in which the cytoplasm stained with TTF-1 antibody. Conclusion.-Occasional cytoplasmic staining for TTF-1 in tumors is seen, but it is a nonspecific finding; when present, it should be disregarded for diagnostic purposes.

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