The advent of Immunohistochemistry as a diagnostic tool in characterizing malignant tumours could be traced to the work of Coons and colleagues in 1942. They introducted immunofluorescence for the detection of antigens in frozen tissues.
In the early 1980’s , monoclonal antibodies became commercially available. This led to the science of antigen detection via visibly tagged antibodies through research which popularized immuno-histochemical applications in tissues though initial staining was weak due to the horseradish peroxidase enzyme detection system used then. The system was soon to be replaced by the more sensitive avidin-biotin system.
The visualization system with diaminobenzidine remain useful till today with some improvements with tyramide amplification. Tyramide is a highly sensitive polymer based labeling system.
In the early days, antibodies were only applied to frozen sections. No staining with formalin fixation was advisable.
It is pertinent to note that this revolutionary tumour diagnoses technique was widespread in Europe and America. Most countries in Africa was left behind including Nigeria. Very few research centers and Tertiary health centers knew about and used this technique.
In 1991, Shi etal discovered heat induced epitope retrieval (HIER) using microwave or pressure cooker along with a metal salt buffered solution. Modern histochemical staining depend largely on this work as more antigens could be detected on routine formalin fixed archive tissues. Staining time for most antigens was reduced. The need for frozen sections for lymphomas was eliminated with superior morphology.
The introduction of automation has now made it possible to standardize staining for quantitation though manual staining still subsists in most laboratories especially in Nigeria.. Automation has also made it possible to record the effect of fixation time on preservation of tissue antigen.
Specific examples of the impact of IHC in tumour diagnoses was summarized in a recent work by Jargidir etal. as follows:
‘The availability of immunostains for myoepithelium has helped delete breast diagnoses such as "highly suspicious for microinvasion" from our lexicon to the more definitive "microinvasion identified/not identified." In addition, the use of immunohistochemical markers in breast cancer has transformed pathology from simple diagnoses to predictive and prognostic necessities. Immunohistochemistry has assisted in guiding adjuvant therapy decisions and sentinel node staging; subtyping a carcinoma as ductal or lobular, basal (cytokeratin [CK] 5/6) or luminal; distinguishing invasive carcinoma from mimics; and establishing that a metastatic carcinoma of unknown primary site has originated in the breast or elsewhere. In the thyroid, CK19 has been considered a useful ancillary to diagnosing papillary carcinoma of thyroid especially in cytology specimens with a high sensitivity and specificity with the caveat that proper sample and controls need to be used when applying IHC to cytology. Another marker that is useful, although not sensitive and not entirely specific, in detection of thyroid malignancies, is HBME-1. HBME-1 detects an unknown antigen on micro-villi of mesothelioma cells and elsewhere.
Very few markers are specific to the lung and lung tumors. The best marker that we have now is thyroid transcription factor 1 (TTF-1), which is widely used in differentiating a lung primary from other neoplasms, including mesothelioma. However, it is not without its shortcomings and is close to desired specificity but lacks sensitivity, being absent or sparsely positive in poorly differentiated lung cancers and primary squamous cell carcinomas of the lung. Another promising marker on the horizon that appears to complement TTF-1 in detecting a lung primary is napsin A. In our study of more than 1000 carcinomas of diverse origin, we found that napsin A was complementary to TTF-1 in the detection of lung primary and more sensitive than TTF-1 (I. Sainz, P. T. Cagle, MD, J. Jagirdar, MD, unpublished data, September 2007). Napsin A is a functional aspartic proteinase that is expressed in the normal lung parenchyma in type II pneumocytes and in the proximal and convoluted tubules of the kidney. It is weakly expressed in pancreatic tumors and is well expressed in some renal tumors and thyroid carcinomas. Until now there were no good positive mesothelial markers, and a diagnosis of mesothelioma was one of exclusion and depended on having several negative epithelial markers. Now we have calretinin, which is the single best sensitive marker for mesothelial differentiation. The source of calretinin is important, with Zymed antibodies (Zymed Laboratories, South San Francisco, Calif) outperforming the others. Cytokeratin 5/6 is another positive marker for mesothelioma. The issue of separating benign mesothelial proliferation from mesothelioma still relies on morphology. However, it is aided by desmin, which is most frequently positive in benign mesothelial proliferations, whereas epithelial membrane antigen and p53 are positive in malignant ones.
Immunohistochemistry has proven to be a useful tool in the diagnosis of infectious diseases in tissue samples. It is especially useful in the identification of microorganisms that are present in low numbers, stain poorly, are fastidious to grow, are noncultivable, or exhibit an atypical morphology. A caveat to remember when staining pathogens is that there may be widespread occurrence of common antigens among bacteria and pathogenic fungi and both monoclonal and polyclonal antibodies must be tested for possible cross-reactivity with other organisms. Examples of some newer pathogens that can be immunostained now are Hantavirus, parvovirus B19, Rocky Mountain spotted fever, Candida, Aspergillus, and mycobacteria.
By using IHC in selected cases, the rate of false-negative and false-positive diagnoses can be reduced in the genitourinary tract, with some patients getting more specific or effective therapy. Prostate-specific antigen remains one of the best organ-specific markers with sensitivity available for the prostate. Uroplakin III is specific for terminally differentiated urothelial cells and is present in 60% of bladder cancers. α-Methylacyl CoA racemase (clone P504S) was identified using expression profiling and was found preferentially in prostatic carcinoma as compared with normal tissue. It is also positive in prostatic intraepithelial neoplasia and less commonly in nodular hyper-plasia, atrophic glands, and nephrogenic adenoma. Cocktails of high-molecular-weight keratin, keratin 903, p63 (a basal cell marker), and α-methylacyl CoA racemase is used in confirming a morphologic diagnosis of minimal prostatic carcinoma. In testicular tumors, hematopoietic marker CD30 is used to distinguish seminoma (except spermatocytic seminoma and intratubular germ cell tu-mor) and embryonal carcinoma from other germ cell tumors. In seminomas, the staining is focal. CD117, a trans-membrane tyrosine kinase receptor protein, is also strongly positive in seminoma as opposed to other nonsemino-matous germ cell tumors. Conversely, CAM 5.2 is positive in nonseminomatous germ cell tumors. Eighty-five percent of clear cell renal cell carcinomas are positive for CD10, another hematopoietic marker. Inhibin is a highly specific marker for ovarian and testicular sex cord tumors and a sensitive marker for adrenal cortical neoplasms.
By using IHC in selected cases, the rate of false-negative and false-positive diagnoses can be reduced in the genitourinary tract, with some patients getting more specific or effective therapy. Prostate-specific antigen remains one of the best organ-specific markers with sensitivity available for the prostate. Uroplakin III is specific for terminally differentiated urothelial cells and is present in 60% of bladder cancers. α-Methylacyl CoA racemase (clone P504S) was identified using expression profiling and was found preferentially in prostatic carcinoma as compared with normal tissue. It is also positive in prostatic intraepithelial neoplasia and less commonly in nodular hyper-plasia, atrophic glands, and nephrogenic adenoma. Cocktails of high-molecular-weight keratin, keratin 903, p63 (a basal cell marker), and α-methylacyl CoA racemase is used in confirming a morphologic diagnosis of minimal prostatic carcinoma. In testicular tumors, hematopoietic marker CD30 is used to distinguish seminoma (except spermatocytic seminoma and intratubular germ cell tu-mor) and embryonal carcinoma from other germ cell tumors. In seminomas, the staining is focal. CD117, a trans-membrane tyrosine kinase receptor protein, is also strongly positive in seminoma as opposed to other nonsemino-matous germ cell tumors. Conversely, CAM 5.2 is positive in nonseminomatous germ cell tumors. Eighty-five percent of clear cell renal cell carcinomas are positive for CD10, another hematopoietic marker. Inhibin is a highly specific marker for ovarian and testicular sex cord tumors and a sensitive marker for adrenal cortical neoplasms.
In soft tissue tumors, the characteristic translocation in Ewing sarcoma/primitive neuroectodermal tumor, t(11; 22)(q24;q12) involving Ewing sarcoma (EWS) gene on chromosome 22 and the FLI-1 gene on chromosome 11 results in overexpression of FLI-1 protein, which can be detected immunohistochemically in nuclei of ~70% of Ewing sarcoma/primitive neuroectodermal tumors. Another new abnormally expressed protein product of chromosomal aberration 2p23, ALK1(p80), is present in approximately 50% of pediatric inflammatory myofibroblastic tumors and in some other malignant soft tissue tumors. The staining is usually cytoplasmic in inflammatory myofi-broblastic tumor as opposed to nuclear and cytoplasmic in anaplastic large cell lymphoma. Alveolar soft part sarcomas are now theorized to be related to muscle tumors because of their desmin positivity. However, staining with newer muscle markers such has MyoD1 and myogenin are negative. Recently, a characteristic X:17 translocation has been identified in alveolar soft part sarcomas resulting in an ASPL-TFE3 fusion gene. Antibody TFE3 detects the presence of overexpressed gene product in alveolar soft part sarcomas. The same protein is also overexpressed in certain pediatric renal cell carcinomas.
Some liver lesions can be problematic. Differentiation between hepatic adenoma and focal nodular hyperplasia can be problematic and can now be aided by β-catenin, which is found in hepatic adenomas. Hepatocellular carcinomas can be distinguished from cirrhotic nodules and benign liver tumors with the aid of a new marker glypican 3. Hepar-1 is a liver-specific antigen that can be used in the context of hepatocellular carcinoma look-alikes such as renal cell carcinoma and adrenal carcinoma. However, it is not specific for the liver and must be used with a panel of markers. Distinguishing an appendiceal adeno-carcinoma from a colonic carcinoma is now possible via MUC5AC, which is positive in appendiceal carcinoma and not in colonic carcinoma, whereas β-catenin is negative in appendiceal lesions and almost always positive in colonic cancers. β-Catenin and villin are both highly unusual in ovarian mucinous carcinomas as compared with colonic mucinous carcinomas.
In summary, we will continue to make significant progress as more organ-specific markers emerge as a result of proteomics and genomics in conjunction with microar-rays’.
Outlook for the future tends toward evidence based meta analysis in conjuction with tissue microarrays. This is expected to facilitate rapid evaluation of antibody profiles on thousands of cases. It is also expected that the art of immunohistochemistry will develop into a full blown ‘science’..
References s
1. Coons AH, Creech HJ, Jones RN. Immunological properties of an antibody containing a fluorescent group. Proc Soc Exp Biol Med. 1941;47:200.
2. Shi SR, Key ME, Kalra KL. Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem. 1991;39:741-748.
3.Immunohistochemistry: Then and Now
Archives of Pathology & Laboratory Medicine , Mar 2008 by Jagirdar, Jaishree
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