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4.2.1. Immunohistochemistry of Infectious Disease

Infectious organisms sometimes induce cytopathic or histologic changes that are easily recognizable on hematoxylin and eosin (H&E)-stained sections or that can be highlighted by various histochemical stains such as Ziehl-Neelsen (for acid-fast bacilli [AFB]) or Grocott's methenamine silver (GMS) stain (for fungal organisms). In the case of GMS, the interpretation of positive versus negative is usually not difficult, as the organisms of interest are fairly large and visible at scanning magnification. On the other hand, searching for mycobacteria on an AFB stain can be an arduous task given the small size and scarcity of the organisms in the typical case. In addition to the ability to highlight the infectious agent, the specific identity of the organism can be elucidated by IHC (30). There are antibodies available that recognize numerous infectious agents, of which antibodies against viral antigens such as CMV and HSV and bacterial antigens associated with Helicobacter pylori are among the most commonly used. With the appropriate specimen preparation and control material, these tools can increase the sensitivity and specificity of tissue examination for infectious agents. Although the degree of antibody specificity varies depending on the organism, the turnaround time on IHC is often far faster than

Membranous And Cytoplasmic Staining
Fig. 2. Membranous and cytoplasmic staining for EBV-LMP1 in neoplastic cells from a case of Hodgkin's lymphoma.

cultures or molecular tests. Markers for certain fungal organisms, such as Cryptococcus neoformans, are quite specific, showing virtually no crossreactivity with other fungi. On the other hand, the current iterations of antibodies that recognize Mycobacterium tuberculosis (MTB) show crossreactivity with other mycobacterial species such as Mycobacterium-avium intracellular complex. Likewise, IHC for H. pylori shows cross reactivity with the closely related H. heilmannii, although the morphology of these two organisms is sufficiently distinct to allow differentiation. Molecular studies available for mycobac-teria and for H. pylori do not have these issues of crossreactiv-ity, but are more time-consuming and expensive than IHC. A culture of these micro-organisms can take weeks to months, whereas IHC or molecular studies for MTB can provide results in a matter of days.

4.2.2. Detection of Oncogenic Viruses Epstein-Barr virus (EBV) is one member of the herpesvirus family that has oncogenic potential. It has been linked to the development of a variety of lymphoid and epithelial malignancies with varying frequencies (31,32). It is virtually always associated with nasopharyngeal carcinoma, commonly seen in posttransplant lymphoproliferative disorder (PTLD) and less frequently in Burkitt lymphoma and Hodgkin lymphoma. More recently, an association has been suggested with gastric and breast carcinomas (32,33) although the latter is still a matter of debate (33). Although the exact mechanisms of malignant transformation are yet to be elucidated, the detection of viral nucleic acids or proteins can serve as a useful marker for diagnosis in some cases. EBV can persist as a latent infection in non-neoplastic B-cells and is also present in the latent form in the EBV-associated tumors. The latently infected cells express a limited number of viral genes, coding for six EBV nuclear antigens (EBNAs), three latent membrane proteins (LMPs), and early RNAs (EBERs). Interestingly, different tumors show consistent patterns of EBV gene expression and can be categorized as latency types I, II, and III based on the pattern of expression of these genes (34).

There are two main methods of detecting EBV in tissue: EBER in situ hybridization and immunohistochemical detection of EBV latent proteins (35). Currently, detection of EBER by in situ hybridization is regarded as the "gold standard" for detection and localization of EBV in tissues. EBERs are consistently expressed at high levels in all EBV-associated tumors, as well as in the lymphoid tissues of patients with acute infection (infectious mononucleosis). Development of antibodies against viral proteins has permitted IHC detection of EBV-infected cells. Antibodies against LMP1 are currently used to detect some EBV-associated tumors (Fig. 2), but LMP1 expression is seen only in latency type II (Hodgkin lymphoma and nasopharyngeal carcinoma) and type III (PTLD) tumors, not in latency type I (Burkitt lymphoma) tumors. The development of antibodies against other EBV proteins such as EBNA1, EBNA2, and LMP2 might help further characterize these tumors. For instance, all EBNAs and LMPs are expressed in latency type III, whereas latency type II tumors express EBNA1 and the three LMPs, and Burkitt lymphoma expresses only EBNA1.

Other viruses implicated in the development of human neoplasms include human papilloma viruses (HPVs), which are associated with squamous cell carcinoma of the cervix, and human herpesvirus 8 (HHV-8 or KSV), which is associated with Kaposi sarcoma (KS), body cavity lymphomas, as well as multicentric Castleman disease. At least one study has demonstrated that antibodies against HHV-8 latent nuclear antigen detect the virus in cases of KS with good correlation with poly-merase chain reaction (PCR) methods. (36). On the other hand, detection of HPV in Pap smears by IHC has focused on looking at p16, a cyclin-dependent kinase inhibitor whose expression is upregulated by HPV in squamous and glandular cervical neoplasms (37,38).

4.3. APPLICATIONS OF IMMUNOHISTOCHEMISTRY TO TUMOR DIAGNOSIS Before the era of modern laboratory tests such as molecular pathology and IHC, pathology diagnoses were made on the basis of H&E morphology, a few histochemical stains, and clinical data. It is well recognized that morphology can be misleading, as there is considerable overlap in the appearance of various disease entities. In tumor pathology, the examples often given are renal cell carcinoma and melanoma as the great morphologic mimics. With a panel of immunohistochemical markers, these tumors can now usually be diagnosed with confidence. IHC has greatly enhanced the accuracy of tumor diagnosis (39), particularly in the diagnosis of poorly differentiated tumors and lymphomas, defining the origins of metastatic tumors of unknown primary and spindle cell neoplasms. The contributions of IHC to resolving these problems with some specific examples are illustrated in the following subsections.

4.3.1. Diagnosis of an Undifferentiated Malignant Neoplasm It is not uncommon to encounter undifferentiated malignant neoplasms whose H&E appearance gives few clues as to its origin. In the past, such tumors might have been signed out as "poorly differentiated malignant neoplasm," which is a frustrating diagnosis for the clinician, pathologist, and patient alike. Immunohistochemical analysis using a limited panel of antibodies such as a "cytokeratin cocktail" (mixture of various high- and low-molecular-weight cytokeratins), S-100 and/or human melanoma black (HMB)-45, and the leukocyte common antigen (LCA or CD45) can help establish the lineage of the tumor as epithelial, melanocytic, or hematopoietic. The ability to differentiate melanoma, carcinoma, and hematologic malignancies is essential to appropriate patient management. Additional studies might further subclassify these entities to help the clinician more precisely tailor therapy.

4.3.2. Establish the Site of Origin in Metastatic Carcinoma of Unknown Primary In other instances, metastatic disease is the first or only manifestation of a carcinoma. If a primary is not detected clinically, it is up to the pathologist to suggest a potential site of origin for the neoplas-tic process. Again, IHC can help detect the site of origin in cases without a clinically recognizable primary (40). There are a few markers that suggest a particular primary site such as prostate-specific antigen (PSA) in prostate, thyroglobulin in thyroid neoplasms, gross cystic disease fluid protein (GCDFP-15) and ER/PR in breast cancer, HepPar-1 in hepatocellular carcinoma, and thyroid transcription factor (TTF)-1 for lung or thyroid primaries. Even though these markers give clues to the origin of the tumor, none is diagnostic of a particular primary site. In some cases, when an antibody is introduced as a site- or type-specific marker, the passage of time and experience with more tumor types shows some immunohistochemical overlap with unrelated tumors. For this reason, interpretation of these markers should be done with foreknowledge of possible pitfalls. Because most tumors do not have a pathognomonic immunohistochemical marker, one way to help narrow the field of possibilities is to use combinations of antibodies to create an antigen expression profile that will increase or decrease the likelihood of certain primary sites (40,41). Such a panel might include various low- and high-molecular-weight cytokeratins, CK7 and CK20, carcinoembryonic antigen (CEA), and possibly TTF-1 (Table 1).

4.3.3. Lymphoma Diagnosis In some fields, notably hematopathology, immunophenotype is an essential component

Table 1

IHC Panel in Carcinoma of Unknown Origin

Table 1

IHC Panel in Carcinoma of Unknown Origin


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