key: cord-0040397-6j1n4778
authors: HAWES, DEBRA; SHI, SHAN-RONG; DABBS, DAVID J.; TAYLOR, CLIVE R.; COTE, RICHARD J.
title: Immunohistochemistry
date: 2009-10-30
journal: Modern Surgical Pathology
DOI: 10.1016/b978-1-4160-3966-2.00016-3
sha: 40f5ac0baa74f427b11a575998426e80d2729158
doc_id: 40397
cord_uid: 6j1n4778

nan

Debra haWeS n ShaN-roNg Shi n DaViD J. DabbS cliVe r. Taylor n richarD J. coTe c h a P T e r 5

Technical Considerations Limitations Antigen Retrieval

Although they do not publicize it, pathologists have long recognized their fallibility. 1 As a result, more objective means of validating morphologic judgments have been sought. Stains using histochemical methods are of value in accentuating morphologic features but do not provide objective evidence of the lineage or biologic potential of a cell. The objective of immunohistochemistry is to use anti bodies to identify antigens, increasing the specificity of the stain for the tissue with which it reacts. In doing so, immuno histology has transformed surgical pathology from a highly subjective discipline into a much more objective science, while still taking advantage of the light microscope and standard morphologic practices. Immunohistochemistry, as the name implies, is the com bination of histology and immunology. The resulting tech nique is a powerful tool that not only enables pathologists to detect whether particular antigens are present within a given cell but also allows the identification of the micro anatomic (cellular) location of the antigen. These abilities permit the lineage of cell populations to be identified, an important consideration when confronted with a poorly differentiated neoplasm of undetermined origin. The tech nique is also useful in defining distinct populations of cells within the same lineage and defining functional differences. In addition, this technique preserves the histologic archi tecture and enables the pathologist to confirm that the positive cells are the cells in question. This confirmation is not possible with molecular methods, such as reverse tran scriptase polymerase chain reaction or standard flow cy tometry methods.

Immunohistochemistry is used by a variety of disciplines to study a wide range of questions. This chapter discusses the application of this technology in surgical pathology, in which immunohistochemistry has had a profound and fun damental impact on the practice of pathology. 1, 2 

Immunohistochemistry has the potential to transform sur gical pathology from a subjective art to an objective science, based on the recognition of cells by microscopic methods. Although this potential has resulted in its almost universal use, immunohistochemistry has not produced uniformly high standards of practice. 3, 4 Therefore, certain technical considerations must be borne in mind to ensure the accu racy of results. It is selfevident that the quality of an immuno histochemical stain depends on the integrity of an antibodyantigen interaction and on the extent to which the relevant antigen has been preserved during tissue fixation and processing. 1 There is a high degree of variability in the way tissues are initially prepared. These variations include differences in the fixative used; the amount, age, and pH of the fixative; how long the tissue sat unfixed before being placed in fixative; the thickness of the tissue when first placed in fixative; and the time the tissue is left in fixative. All these variables, which may not alter the results of routine hematoxylineosin (H&E) staining to a significant degree, can lead to widely discrepant results when it comes to immunohistochemistry. These variables cannot always be predicted or remedied, but they can be mitigated to a great extent by the advent of successful and relatively simple antigen retrieval methods (discussed later). 5, 6 In addition to the variables in tissue fixation, as in all other laboratory tests, the reagents and techniques used must be optimized and thoroughly validated to ensure con sistent, reliable, and clinically meaningful results. When developing an immunohistochemical protocol, it is impor tant for each laboratory performing a test to validate every reagent used. This validation includes a determination of the specificity and the optimal working dilution of each primary antibody, secondary antibody, linking antibody, labeling reagent, and substrate. Repeat validation is required for each new lot of reagents because of variations in origin, composition, concentration, and specificity that can occur among different lots even when supplied by the same company. 1 Also, reputable manufacturers should be used. The higher standards and more rigorous quality control of products from the better manufacturers have been counter balanced by the concurrent proliferation of smaller manu facturers that are able to produce or otherwise acquire and market large numbers of different antibodies through mono clonal antibody technology, molecular engineering, and the like. In addition to the need for highquality antibodies and reagents, proper incubation times and ideal temperatures for each antibody must be determined. The optimal buf fering agent must be determined, as well as the need for any predigestion techniques or antigen enhancement procedures.

Premanufactured, allinclusive kits have been marketed in an attempt to simplify the performance of immunohisto chemistry; for example, these kits obviate the need for each individual clinical laboratory to validate each reagent because preoptimized working dilutions and recommended working protocols are provided. Nevertheless, there are some pitfalls that should be kept in mind when working with premanufactured kits. The protocols and reagents have been formulated to work on the prototype tissue used by the manufacturer and may not be as effective on the actual tissues tested because of laboratories' different fixation and processing protocols, all of which may have adverse effects on the results. Adjustments in the recommended protocol are necessary to optimize the kits in each indi vidual laboratory, which effectively means that the kits must be customized, and any changes made in the manufacturer's protocol require that the entire staining procedure be reval idated by the performing laboratory. 1 This procedure may be complicated by the fact that many of the working dilu tions of the reagents supplied are already at the critical level of sensitivity.

Another method that can potentially enhance consistency and reproducibility is automation. A variety of automated immunostaining systems are now commercially available. The theoretical advantages of these systems over manual staining techniques include improved reproducibility, facil itation of interlaboratory comparisons, reduced reagent expenses, and increased technician productivity. Automa tion does not mitigate the need to thoroughly validate each step of the staining procedure or the need to evaluate every reagent used to ensure highquality, consistent results. The same quality control issues that apply to manual staining apply to automated systems. As with manual staining, it is important that a complete reevaluation be performed if there is any departure from the validated protocol. 1 Auto mation does not guarantee an optimal result. Finally, auto mation cannot replace the pathologist, who must choose the appropriate antibodies and then interpret the final result.

There is a growing need to be able to quantify immunohis tochemical staining results, which is probably best accom plished by automated image analysis. Current systems, including the Automated Cellular Imaging System II (ACIS II Clarient, Aliso Viejo, Calif), have the ability to assess marker positivity in terms of both percentage positive and intensity of staining. The ability to quantify markers more precisely is especially important in the identification of targets of therapy. One example of this is Her2/neu. When Her2/neu protein expression by immunohistochemistry was compared with gene amplification by fluorescence in situ hybridization using routine manual methods and the assistance of a digital microscope, both accuracy and reli ability were improved when the digital microscope was used. 7,7a Precise quantification of hormone receptors may also be important because there is evidence that in patients with high levels of hormone receptors, the addition of cyto toxic chemotherapy has a deleterious effect on outcome. 8, 9 Another exciting development in the field of automated cellular imaging is spectral imaging. This technology allows multiple markers to be assessed on the same slide-even on a single cell. Computer software can isolate a single chromogen from other chromogens present based on its emission spectrum. Automated cellular imaging provides greater objectivity and reproducibility and thus minimizes interobserver discrepancies.

Immunohistochemical tests performed and interpreted in the absence of the appropriate controls are valueless and even dangerous. Minimal controls should include a tissue known to express the particular antigen of interest, pro cessed in a manner analogous to that of the unknown tissue (the positive control), and a second section of the test specimen in which the primary antibody is replaced either by diluent or, better, by an irrelevant antibody of the same isotype, from the same species, and at the same concentra tion (the negative control). In the positive control, only cells expected to express the antigen should show positiv ity; all other cells and structural elements should be nega tive. In the negative control, there should be no specific staining. The "sausage" technique, in which samples of multiple tissues are gathered into a single tissue block, is a useful control method. 10 Controls are performed for a variety of purposes; in addi tion to indicating whether a reaction occurred (or not), they are essential for judging the nature of the reaction. A vast array of immunohistochemical tests are judged not by a positive or negative result but by the intensity and localiza tion of the result (a good example is Her2/neu and hormone receptor analysis in breast cancer). Immunohistochemistry results should never be interpreted in the absence of the known positive results because the assessment of quality and quantity of the reaction is essential.

Interpretation of the results of immunohistochemical stains is the province of the surgical pathologist and is best accom plished by pathologists who have the appropriate level of experience not only in the morphologic aspects of diagnosis but also with regard to immunohistochemical findings. As in any other area of pathology, experience matters: A pathol ogist with little experience with immunohistochemistry, who runs a few different tests each week or month, will obtain very different results from a pathologist who per forms and interprets immunohistochemical tests on a daily basis. As the impact of immunohistochemistry on surgical pathology increases, these differences will become more profound.

As described previously, many factors influence the results. All these factors must be considered by the pathol ogist in interpreting the findings. Negative, weak, or unin terpretable results should lead to a repetition of the test after the use of antigen retrieval. 11, 12 One measure of antigen preservation is to test for expression of the intermediate filament vimentin, a fixationsensitive protein that is typi cally expressed by vascular or connective components; this technique often serves as an internal indicator of the con servation or loss of antigenicity. 11 Test results may also be affected by technical artifacts and by the nature of the tissue under study. For instance, if tumor cells are crushed, false positive or nonspecific staining may be encountered. Non viable areas of tissue from a necrotic tumor may also be a source of falsepositive results, attributable in part to leakage of serum proteins (e.g., immunoglobulins). The subcellular distribution of immunoreactivity is critical to the interpreta tion of immunohistochemical results. For example, Her 2/neu shows membranous staining, whereas antibodies to estrogen and progesterone receptors produce nuclear stain ing. When unexpected staining patterns are observed with an antibody, the results should be discounted. 1 To interpret the results effectively, the pathologist must have extensive knowledge of the staining patterns of the primary antibod ies under consideration, including a detailed knowledge of tissue specificity and subcellular localization of the antigen, and an awareness of technical variables. Each lab oratory performing immunohistochemical staining should have established written criteria for determining and report ing positive and negative findings for each immunohisto chemical stain, with particular reference to stains that are expected to produce cell surface membrane, cytoplasmic nuclear, or extracellular staining. Although it seems obvious, it often is overlooked that staining should be recorded as positive only if it occurs in the expected cellular or tissue location.

The Food and Drug Administration's increased attention to the reagents used in immunohistochemistry has undoubt edly contributed to an improvement in their quality. 13 It is highly recommended that all laboratories performing di a g nostic immunohistochemistry participate in the College of American Pathologists' certification program, which includes a checklist of the essential elements required for a successful immunostaining program. 7a With regard to staff qualifications, the National Society of Histotechnologists has focused its efforts on continuing education and certifi cation programs for technologists performing immunohis tochemical staining.

Federal law requires a high degree of testing and valida tion. In the United States, laboratories performing immu nohistochemistry are required under the Clinical Laboratory Improvement Amendments of 1988 to validate the perfor mance of their test reagents for accuracy, specificity, sensi tivity, and precision. 14 First, the testing procedure is optimized (as described earlier), and performance expecta tions are established. During the validation process of each analyte, multiple slides with known pathology (generally 20 representative cases) are evaluated with the optimized procedure to assess the accuracy of diagnostic staining, sensitivity of signal, and reproducibility. Validations that meet specifications must be signed by qualified individuals, and the documents are maintained in the laboratory. Quality control and proficiency testing must be performed to monitor performance.

Although immunohistochemistry is an extremely valuable technique in experienced hands, its limitations must be recognized for it to be used to its maximum potential.

Although immunohistochemistry is more objective than routine morphologic examination, the experience of the pathologist assessing the slides is critical. A firm under standing of the principles of immunohistochemical staining is necessary because the reporting pathologist must be equipped to deal with the unexpected and conflicting results that inevitably occur. To evaluate the immunohistochemical slides properly, the pathologist must have a firm under standing of the limitations of antibodies in terms of their technical aspects as well as their inherent specificity, sensi tivity, and expected subcellular location.

The advent and refinement of the hybridoma technique for the production of monoclonal antibodies have produced a large number of available antibodies. Often a newly devel oped antibody is hailed as exquisitely specific. In time, however, most are found to be considerably less specific than initially hoped, generally because the antigen the anti body detects has a wider distribution than expected. This fact does not negate the usefulness of the antibody in ques tion, but it may mean that panels of antibodies must be used in conjunction with standard morphologic features and clinical history.

Many studies have shown that a loss of antigenicity can occur on cut paraffin sections that have been stored for varying lengths of time. 1517 Among the antibodies studied, those most adversely affected by storage include p53, 15, 17 MIB1, 16, 17 factor VIII-related antigen, 15 estrogen receptor, 15 bcl2, 15 p27kip1, 16 CD44s, 16 and androgen receptor. 16 In many cases, the use of carefully selected and tested antigen retrieval techniques can compensate for this loss. 17

Formalin is the most widely used fixative in surgical pathol ogy. Crosslinking of proteins is the essential feature of formalin fixation. This crosslinking interferes with the antigen's ability to react with the primary antibody. In 1991 the antigen retrieval technique was developed. 12, 18 This technique is a heatinduced modification of the protein con formation that allows the antigen to be accessible again for chemical reactions, in this case, antibody binding. Hydrolysis of crosslinking resulting from formalin fixation probably plays a major role in this modification process. 1922 The application of antigen retrieval to sections derived from formalinfixed, paraffinembedded blocks produces consistent results of acceptable quality, 6 although a few anti gens remain undetectable even after antigen retrieval has been performed. Antigen retrieval methods have revolution ized immunohistochemistry and have become a standard part of diagnostic immunohistochemistry in surgical pathol ogy. These methods result in higher sensitivity and more consistent antibody reactivity. Antigen retrieval technology has led to a proliferation of protocols that may produce different results in different laboratories. The successful application of these methods allows the detection of some antigens that were previously undetectable in paraffin sec tions, rendering much of the early literature (prior to 1993) obsolete. This fact continues to escape the notice of some practicing pathologists, leading to errors of interpretation (Fig. 51) .

It should be recognized that two major factors influence the effectiveness of antigen retrieval: the conditions under which heating takes place, and the pH value of the buffer solution used during the heating process. 5, 11, 23 The most critical factor is the combination of the temperature and the duration of heating, which have a reverse correlation. Based on these two factors (heating conditions and buffer pH), a test battery approach has been developed to establish optimal antigen retrieval protocols for immunostaining on archival paraffinembedded tissue sections. 1, 6, 23 A typical test battery consists of nine serial sections of a specimen known to express the antigen under study. The sections are evaluated with buffer at three different pH values (e.g., pH 1 to 2, 7 to 8, and 10 to 11) and three heating conditions (e.g., 90°C, 100°C, and 120°C) for various lengths of time (or some other comparable heating versus time schedule). The best result is selected as the optimal retrieval condition for that antigen. In the event that a satisfactory result is not obtained, other variations may be explored, including dif ferent buffer solutions and more or less vigorous heating methods. 19, 23 Protocols for antigen retrieval differ in their effectiveness for retrieving certain antigens, and a single universally effective retrieval method does not exist. Many laboratories use more than one method for different anti body and antigen combinations. Overall, citrate buffer at pH 6.0 has the broadest applicability for the widest range of antigens, although several studies have demonstrated that the use of higher pH retrieval solutions yields satisfac tory results. 21, 24 Retrieval solutions with lower pH values, TRIS (tromethamine) buffer at pH 8.0, and EDTA (ethyl enediaminetetraacetic acid)NaOH solution (pH 8.0), are effective in certain special situations. 2527 The selection of heating method (water bath, steamer, microwave, pres sure cooker, or autoclave) is influenced by custom and availability.

Immunohistochemistry has become an integral and essen tial part of surgical pathology. It is applied to define tumor 

origin, establish prognosis, and determine treatment response. In this textbook, the role of immunohistochem istry in defining the origin, prognosis, and treatment response of tumors is discussed in the chapters devoted to the specific organ systems; therefore, a full discussion is not provided here. Because the evaluation of tumors of unknown origin does not fall under any particular organ system, it is discussed in this chapter.

Tumors are classified most often by their tissue of origin (e.g., breast, colon, prostate) or histogenetically (e.g., tissue of epithelial, mesenchymal, or neural origin). A tumor cannot be staged accurately, and proper therapy cannot be administered, without such classification. Although accepted and fairly reproducible criteria exist for the morphologic diagnosis of most tumors, there is inherent subjectivity in any morphologic evaluation. It is well recognized that morphologic features often overlap among different entities and that one disease can present with myriad histologic pictures. Most tumors can be classified correctly by routine histologic techniques when the clinical situation is clear (e.g., a breast mass); however, an important subset of tumors defies morphologic interpretation. The magnitude of this problem is substantial. The diagnosis of "metastatic cancer of unknown primary site" is the eighth most common cancer diagnosis and may represent up to 15% of cancers at large hospitals. 28 Much more common is the diagnosis of "tumor of uncertain origin." This occurs when (1) the tumor is first identified in a metastatic site, and the primary site is not apparent; (2) the tumor is so poorly differentiated that no specific morphologic features can be identified; (3) the morphologic appearance of the tumor is compatible with more than one distinct tissue (e.g., epithelial versus lymphoid origin); and (4) the histogenesis of a tumor is clear (e.g., adenocarcinoma), but the primary site is in ques tion. This distinction has important consequences to the patient.

Immunohistochemical tests should be performed with a defined objective in mind. The results of a single immuno histochemical procedure can be misleading not only because of variables in the staining procedure but also because of unanticipated patterns of reactivity of certain antibodies. 1 Although myriad antibodies are available, the choice in a particular case should be judicious and designed to address the diagnostic possibilities. The use of too few antibodies rarely provides sufficient information to support a specific diagnosis and can produce misleading information. Anti bodies should be selected on the basis of their ability to affirm or exclude considerations in the differential diagno sis. This socalled problemoriented approach is based on the selection of appropriate panels of antibodies. When selecting antibodies, factors that should be considered include the clinical history, morphologic features of the tumor, and results of other tests that may have been per formed, including serologic and radiographic tests. Pathol ogists can find guidance in the literature and in a few specialized textbooks that address the use of immunohisto chemistry, 2,29 but this is a rapidly evolving field. The limited antibody panels of a few years ago are inadequate to deal with tumors of unknown or uncertain primary sites today. With this in mind, the panels presented here must be con sidered elementary guides.

When evaluating tumors of uncertain origin by immuno histochemistry, certain basic guidelines of interpretation must be kept in mind. A positive staining reaction is gener ally more helpful than a negative one because a lack of immunoreactivity may represent a technical problem with the tissue or the way it was fixed, as discussed earlier.

The more poorly differentiated a tumor is, the less likely it is to express tissue differentiation antigens. There is often staining variation within a tumor; by extension, vari ations in staining patterns may be seen between the primary tumor and the metastatic focus. Most important, the final diagnosis should never depend on immunohistochemistry alone; it must be made using all the clinical, serologic, radiographic, morphologic, and epidemiologic data avail able. Other techniques, such as the assessment of specific DNA alterations, are becoming increasingly important adjuncts to the pathologic evaluation. Although immuno histochemical evaluation is essential, it is only one of many tools that must be used in the evaluation of pathologic processes.

The application of a primary panel of antibodies to characterize tumor histogenesis (epithelial, mesenchymal, neural, or hematopoietic) is often the first step. When this panel has been established, additional antibodies can be used to identify the tumor type more specifically. Included in the first tier would be antibodies against pankeratin, vimentin, S100 protein, neuronspecific enolase (NSE) and CD45 (common leukocyte antigen) to differentiate epithe lial, mesenchymal, melanomatous, neural, and lymphoid malignancies (Table 51 ; Fig. 52 ).

The expression of intermediate filament proteins, which function as the supporting cytoskeleton in normal and neo plastic cells, is extremely useful in the initial assessment of tumors of unknown primary origin. 1 There are five major classes of intermediate filaments, based on protein compo sition and cellular distribution: cytokeratin, vimentin, desmin, neurofilament, and glial fibrillary acidic protein (GFAP). 30 Most neoplasms show the predominant expres sion of one or more of these intermediate filaments. Carci nomas usually express cytokeratin; sarcomas, melanomas, 

Cytokeratins are present in almost all epithelial cells and are highly sensitive markers for carcinomas. In a generic sense, malignant cells expressing keratin positivity indicate an epithelial origin. Antibodies against keratin are also extremely useful as markers for occult metastases (micro metastases) in the peripheral blood, bone marrow, and lymph nodes (discussed later). There are more than 20 different subtypes of cytokeratin found in human epithelial cells. These subtypes are distin guishable by their molecular weight and isoelectric pH. 31 Monoclonal antibodies specific for many of these subtypes have been developed. Carcinomas of different types tend to express characteristic keratin profiles. 32 There is a general correlation between the complexity of the epithelium from which the tumor is derived and the complexity of the keratin subunits expressed. Lowmolecularweight or non squamous keratins appear early in development and pre dominate in tumors derived from simple, nonstratified epithelium (e.g., ductal carcinoma of the breast, gastroin testinal adenocarcinoma). Highmolecularweight or squa mous keratins appear in more complex stratified epithelium and predominate in tumors derived from stratified epithe lium (e.g., squamous cell carcinoma). Some tumors, such as those derived from pseudostratified columnar epithe lium, contain a mixture of high and lowmolecularweight keratins, with a predominance of the latter. In some instances, especially in extremely poorly differentiated tumors, as few as 5% of tumor cells may express keratin reactivity. 1 When a tumor of uncertain primary site has been defined as epithelial by either immunohistochemistry or morphol ogy, it is important to attempt to define its specific origin. This presents a problem when, for example, a patient with a prior history of breast carcinoma presents with a lung mass that, on biopsy, is adenocarcinoma. Determining whether the lung mass represents a primary pulmonary tumor or metastasis from the breast has enormous conse quences in terms of patient outcome and choice of specific treatment. Although the immunohistochemical evaluation of primary epithelial tumors is problematic, advances have been made. Monoclonal antibodies against keratin subtypes may help determine the origin of certain poorly differenti ated neoplasms. Hepatocellular carcinoma (positive for AE3 and CAM 5.2 but negative for AE1) can be distinguished from bile duct carcinoma and adenocarcinoma metastatic to the liver (positive for AE1). 33 In particular, the differential expression of cytokeratins 7 and 20 (CK7, CK20) is extremely useful in the characterization of epithelial neo plasms (Fig. 53) . 32,3436 These patterns are not absolute, but they can be useful guides in establishing origin.

Cytokeratin 5/6 (CK5/6) has received considerable attention recently. CK5/6 has been found to be positive in the majority of squamous cell carcinomas, basal cell carci nomas, thymomas, salivary gland tumors, and biphasic malignant mesotheliomas and in a subset of endometrial adenocarcinomas, transitional cell carcinomas, and pancre atic adenocarcinomas. 37 CK5/6 is rarely positive in adeno carcinoma of the lung and has therefore been used to distinguish malignant mesothelioma from pulmonary ade nocarcinoma. 37,38 In addition, p63 is frequently seen in squamous cell carcinoma and transitional cell carcinoma, whereas mesothelioma is uniformly negative for p63. There fore, positive immunostaining for both p63 and CK5/6 is highly predictive of a primary tumor of squamous epithelial origin. 39,40 Because p63 is also known to immunoreact on the basal cell nuclei in benign prostate glands, this marker can be used to distinguish prostate cancer from benign mimics. 41 There is an increasing array of tissue-specific markers, such as prostatespecific antigen (PSA) and thyroglobulin, as well as tissue-associated markers, such as GCDFP15 and mammaglobin for breast epithelium, 42,43 OC125 for ovary, 44 uroplakins for urothelium, 45,46 and synaptophysin for neuroendocrine lesions (Fig. 54) . 47 Table 52 summarizes many of these tissueassociated antibodies. It is important to keep in mind patterns of antigenic coexpression, which can lead to erroneous assessments. Also, as mentioned earlier, the more poorly differentiated the neoplasm, the less likely it is to express tissuespecific or associated antigens. Nevertheless, the specific origin of an epithelial neoplasm of uncertain primary site can be elucidated in an increasing proportion of cases through careful evaluation of morphol ogy, clinical data, and antigen expression.

Lymphomas often present morphologically as undifferenti ated malignant neoplasms. A large cell lymphoma may be difficult to differentiate from carcinoma or melanoma. Ana plastic large cell lymphomas occasionally react with anti bodies against keratin or epithelial membrane antigen. Similarly, small cell lymphomas often resemble other tumors, such as small cell undifferentiated carcinoma. Immunohistochemistry can be invaluable in classifying a tumor as lymphoid in origin. Immunohistochemistry is also used widely to aid in the subclassification of nonHodgkin's lymphoma (discussed in detail in Chapter 41). Another important application of immunohistochemistry is the phe notyping of a lesion to determine the immunoglobulin lightchain expression. One can distinguish between malig nant and benign lymphoid proliferation by demonstration of lightchain (or heavychain) restriction.

CD45 is an excellent screening marker to determine whether a tumor is lymphoid in origin. Staining is charac teristically membranous. Some neoplasms of lymphoid origin may not express CD45, and rare nonlymphoid neo plasms may show cytoplasmic staining for CD45. More specific lymphoid markers such as CD3, which marks T cells, and CD20, which marks B cells, may further delineate a lesion. Immunohistochemistry can also be used in cases of Hodgkin's disease, in which markers useful in identi fying ReedSternberg cells include CD15, CD30, and BLA36. 48,49 Figure 55 illustrates an algorithmic approach to the immunohistochemical diagnosis of malignant lymphomas.

Melanomas are typically (but not always) negative for cytokeratin 50 and positive for vimentin. S100 protein is a sensitive marker for melanoma and occurs, in almost all cases, in a nuclear pattern (see Fig. 52C ). S100 is not specific for melanoma, however, and is seen in a variety of lesions, including Langerhans histiocytes, many sarcomas, and certain carcinomas. 51,52 Positive immunoreactivity for HMB45, Melan A, or tyrosinase, which are much more specific markers for melanoma and melanocytes, can confirm the diagnosis. 53 This topic is discussed in detail in Chapter 49.

A diagnosis of sarcoma is worth considering when a spindle cell neoplasm expresses vimentin but not keratin, CD45, or HMB45. Immunohistochemical analysis can delineate a specific type of sarcoma such as myogenic and fibrohistio cytic tumors, tumors with neural differentiation, and vas cular sarcomas. The differentiation of soft tissue tumors by immunohistochemistry is discussed in Chapter 46. The major subtypes are described briefly here.

Myogenic sarcomas react with antibodies to muscle specific actin and desmin. Antibodies against myoD1 smooth muscle actin show preferential reactivity with leio myosarcomas. 54 Alveolar soft part sarcomas typically coex press vimentin and desmin. 55 Tumors derived from skeletal muscle often contain myoglobin; positive immunoreactivity for musclespecific actin, desmin, and myoglobin indicates that the tumor in question may be a rhabdomyosarcoma.

Fibrohistiocytic tumors, including malignant fibrous his tiocytoma, are the most common form of soft tissue sarcoma in adults. Malignant fibrous histiocytoma reacts with vimen tin and lacks significant reactivity for keratin. These lesions Neurogenic tumors, including malignant peripheral nerve sheath tumors and schwannomas, are positive for antibody against S100 protein, myelin basic protein, and Leu7. 59, 60 Although most benign neurogenic tumors (schwannomas and neurofibromas) contain S100, less than half of malignant peripheral nerve sheath tumors contain detectable S100 protein. 61 Normal and neoplastic vessels can be identified with endothelial markers such as factor VIII-related antigen and Ulex europaeus lectin. Antibodies to factor VIII-related antigen react only with endothelial cells and megakaryo cytes and are more specific than Ulex europaeus. Other TdT −/+ S100+; CD1a+; CD68−/+ markers of endothelial cells include CD34 and CD31; these are more sensitive but less specific than factor VIII. Other tumors to be considered in the differential diag nosis of anaplastic spindle cell tumors include liposarco mas, chondrosarcomas, osteogenic sarcomas, fibrosarcomas, and synovial sarcomas. Although certain variants of liposar coma may be diagnosed by histologic criteria alone, the diagnosis of pleomorphic liposarcoma is aided by immuno histochemistry. Most liposarcomas react with vimentin and S100 but are nonreactive for HMB45, in contrast to most melanomas. The same pattern is seen for chondrosarco mas. 62 Osteosarcomas react with vimentin. Antibodies to the socalled osteonectin or osteosarcoma antigens also may assist in the diagnosis. 62 Fibrosarcomas are rare neoplasms that express only vimentin. Figure 56 provides the immu nostaining patterns characteristic of anaplastic spindle cell tumors.

Cytogenetic profiling of sarcomas of unknown origin is becoming common. Tumors with known abnormalities include endometrial sarcoma, myeloid sarcoma, synovial sarcoma, Ewing's sarcoma, and visceral clear cell sarcoma, among others. 6370 This topic is discussed in more detail in Chapter 46.

Neural and neuroendocrine tumors may be classified as neural tumors, neuroepithelial tumors, or neural neoplasms of mesenchymal origin. These three categories are based on the different predominant intermediate filaments found in the cytoplasm of these lesions. Neural tumors usually express neurofilament, NSE, chromogranin, and synapto physin. 7173 Examples include neuroblastomas, paraganglio mas, and pheochromocytomas. Neuroepithelial tumors coexpress keratin and neuroendocrine markers. These tumors include carcinoids, Merkel cell carcinomas, and small cell carcinomas. Neural neoplasms of mesenchymal origin, which consist of primitive neuroectodermal tumors, Ewing's sarcomas, and medulloblastomas, are positive for vimentin and may express NSE and Leu7. 74 A good marker for small cell tumors such as Ewing's sarcoma, primitive neuroectodermal tumor, and peripheral neuroepithelial tumor is O13, which identifies the CD99 (p30/32, mic2, HBA71) antigen. 75 NSE, although a sensitive marker for neuroendocrine tumors, lacks specificity and can be seen in a wide variety of tumor types. Chromogranin and synaptophysin are more specific than NSE but lack sensitivity. Chromogranin tends to be positive in betterdifferentiated neuroendocrine tumors but is less often positive in the more poorly differentiated tumors, such as small cell carcinoma. 72, 7678 Table 53 pro vides immunostaining patterns of endocrine tumors, and Figure 57 illustrates an algorithm used to distinguish small round cell tumors.

GFAP is expressed by glial cells and is seen in astrocytomas, ependymomas, medulloblastomas, some oligodendroglio mas, and choroid plexus tumors. 79, 80 It has also been reported in a few extracerebral tumors, including pleomorphic ade nomas of the salivary gland, 81 neurofibromas, and schwanno mas. 82 Intracerebral tumors in which GFAP is not expected to be positive include meningiomas, lymphomas, and 

Cancer results from defects in gene structure, expression, or both. Alterations in chromosome and DNA structureincluding cytogenetic changes, point mutations, deletions, amplifications, translocations, and DNA methylationare being identified at an increasing rate. Characterization of these defects is becoming an important component of tumor evaluation, particularly in terms of prognosis and response to treatment. In addition, genetic defects highly characteristic of specific tumor types are being identified. The identification of DNA alterations is also becoming an increasingly important component in the evaluation of tumors of uncertain primary site. Molecular and genetic evaluation is particularly useful in the evaluation of hema topoietic and soft tissue tumors. 83, 84 Recently, a highly sophisticated evaluation of hundreds of genes using so called microarray chip technology was used to identify gene expression differences in several tumor types 8486 and will no doubt become an important component of tumor evaluation in the future. Transcription factors are proteins involved in the regulation of gene expression that bind to the promoter elements upstream of genes and either facilitate or inhibit transcrip tion. They may be tissue specific, or they may be present in more than one tissue type. Even the socalled tissuespecific transcription factors are, however, usually not restricted to a single tumor type. Examples include thyroid transcription factor1 (TTF1), which is found in the thyroid and lung, and the pituitary transcription factor PIT1, which is found in the placenta as well as the pituitary gland. Nevertheless, they can be useful in determining the primary site of tumors of unknown origin (see Table 52 ).

TTF-1 TTF1 belongs to a family of homeodomain transcription factors and plays a role in regulating genes expressed within the thyroid, lung, and diencephalons. TTF1 is considered a reliable marker for distinguishing primary tumors of the lung, including adenocarcinoma (75%), non-small cell car cinoma (63%), neuroendocrine and small cell carcinoma (>90%), and squamous cell carcinoma (10%), 8790 and thyroid from other tumor types. However, primary adeno carcinoma of the colon is positive in some cases. 36, 91, 92 The pattern of reactivity is nuclear. Hepatocytes and hepatocel lular carcinoma reportedly show cytoplasmic positivity. 93 CDX-2 CDX-2 is a homeobox gene that encodes a transcription factor involved in the development of intestinal epithelium. It is expressed in normal colonic epithelium and in most colorectal adenocarcinomas and is a useful marker to iden tify colorectal metastases. 9496 CDX2 is also useful in extra mammary Paget's disease; the endodermal subtype is positive for CDX2, whereas the cutaneous subtype is negative. 97

Activating transcription factor 3 (ATF3) is a member of the basic leucine zipper/cyclic adenosine monophosphateresponsive element binding protein family of transcription factors. Studies indicate that ATF3 is an androgenregulated gene that stimulates cell proliferation. ATF3 protein detected by immunohistochemistry is present in prostate tumors; it has increased expression in highGleasonscore disease and in tumors refractive to therapy. 98

One of the outstanding achievements of modern medicine is the ability to predict the behavior of tumors based on specific clinical and pathologic criteria. Tumor stage and grade provide only general estimates of outcome for a par ticular patient, however. Current clinical and pathologic staging parameters cannot identify those patients who are destined to experience relapse or those whose disease will be cured by local therapy alone. These considerations have obvious consequences for the patient and enormous eco nomic implications.

Efforts are under way to identify enzymes, oncogenes, or tumor suppressor genes whose presence or absence may predict more accurately the biologic behavior of tumors.

Such studies represent a fundamental shift in the means by which tumor behavior is defined-a change from an outcomebased empirical analysis (i.e., prediction of what a tumor will do based on what it has done) to one focused on tumor biology (i.e., predictions of behavior based on specific genetic alterations). The immunohistochemical analysis of tumors is also undergoing a profound shift in emphasis. Although initial studies focused on defining tumor histogenesis, the goal of much current research is to reveal the biologic potential of tumors, providing a more scientific basis for patient management.

The use of advanced technologies to define tumor prog nosis is described in detail throughout this book. Presented here are some general principles that pertain to the prog nostic evaluation of all tumors.

The most important factor affecting the outcome of patients with invasive cancers is whether the tumor has spread either regionally (to regional lymph nodes) or systemically. A proportion of patients with no evidence of systemic dis semination as evaluated by routine methods (careful patho logic, clinical, biochemical, and radiologic evaluation) develop recurrent disease. In addition, the success of adju vant therapy is assumed to stem from its ability to eradicate occult metastases before they become clinically evident. 99 Immunohistochemistry is commonly used to identify occult metastatic cancer cells in the bone marrow, peripheral blood, and lymph nodes of patients with cancer. Although many of the initial studies focused on breast cancer, 100102 tumors from other organs such as the stomach, 103, 104 colon, 105, 106 prostate, 107,108 lung, 109,110 nervous system, 111 and skin 112 have been investigated. Immunohistochemical methods are based on the ability of monoclonal antibodies to distinguish between cells of different histogenesis (e.g., epithelial cancer cells versus the hematopoietic and stromal cells of the bone marrow and lymph nodes). The results indicate that it is possible to identify occult metastatic cancer cells in lymph nodes and bone marrow before their detection by other methods and that the presence of these cells may be an important risk factor for disease recurrence (Fig. 58) .

The most widely used monoclonal antibodies to detect occult metastatic carcinoma cells are directed toward epi theliumspecific antigens. These antibodies do not react with normal hematopoietic or stromal cells present in the bone marrow or lymph nodes. None of the antibodies used in any study is specific for cancer; all react with normal and malignant epithelial cells. They are useful because they can identify an extrinsic population of epithelial cells in bone marrow or lymph nodes, where there are normally no epi thelial elements. The reported sensitivity of immunohisto chemistry ranges from the detection of 1 epithelial cell in 10,000 to 2 to 5 epithelial cells in 1 million hematopoietic cells. 100, 113 A potentially more sensitive approach for the detection of occult metastasis is the reverse transcriptase polymerase chain reaction (RTPCR) technique, which has been applied to several malignancies using a variety of marker transcripts as targets. Since the first study by Smith and colleagues in 1991, 114 many authors have reported molecular diagnoses in the lymph nodes, blood, and bone marrow in cancer patients. 42,107,115119 Application of RTPCR in regional and sentinel lymph nodes has been described for a number of cancers, including melanoma, colorectal cancer, and cancers of the prostate, breast, and lung. 118, 120124 Many of these compare immunocytochemicalbased detection with RT PCR for sensitivity and conclude that RTPCR may achieve enhanced detection, provided the target markers are suffi ciently specific. Various formats of RTPCR assays 125127 have also been used to detect disseminated tumor cells in the bone marrow of patients with cancers of the breast, colon, and lung, among others. With the exception of some organ specific markers such as maspin or mammaglobin for breast cancer 116, 128 or uroplakins for urothelial tumors, 129 most of the molecular targets used in these RTPCR assays lack the requisite specificity owing to illegitimate expression in nontarget hematopoietic cells. 130132 Unlike immunohisto chemistry, morphologic confirmation of the cells in ques tion to verify tumor origin is not possible with RTPCR. RTPCR has also been used to enhance the sensitivity of the detection of tumor cells in the peripheral blood in a variety of cancers, including prostate, breast, gastrointesti nal tract, colorectal, and head and neck cancers and mela noma. 117, 133135 Concerns about nonspecificity owing to illegitimate transcription of target genes in the nontarget hematopoietic cells also apply to the blood, which has ham pered the use of these assays in routine clinical diagnosis.

In breast cancer, the bone marrow is the single most common site of metastasis, and 80% of patients with recurrent tumors develop bone marrow metastases at some point during the evolution of their disease. 136 Immunohistochemistry can show the presence of occult metastases in the bone marrow in approximately 10% to 45% of patients with lowstage disease. 102, 127, 137143 Several studies have addressed the clini cal significance of these early metastatic cells in the bone marrow, including a pooled analysis of more than 4700 patients. 143 They have found that the presence of such cells is an independent prognostic indicator of diseasefree sur vival and overall survival. 144146 Occult metastases in the bone marrow are also prognostically important in other malignancies, including primary non-small cell lung cancer, 109, 110, 147, 148 esophageal and gastric cancers, 103 colo rectal cancer, 149 and neuroblastoma. 111 The finding of posi tive cells in the bone marrow of patients with colorectal cancer-a tumor that rarely shows overt metastasis to the bone-indicates that this may be a general indicator of tumor dissemination. In addition, the prognostic signifi cance of occult metastatic cells in the blood is under investigation.

Peripheral blood has the advantage of being easier to access than bone marrow. However, detection rates are con siderably lower than with bone marrow, a fact that has hampered studies to date.

In the detection of tumor cells in the bone marrow and blood, epithelial cell adhesion molecule in conjunction with immunomagnetic enrichment has been used to detect circulating tumor cells in breast cancer patients. Circulating tumor cells in the blood have been used to monitor response to therapy in patients with metastatic cancer. 150,151,151a The presence of epithelial adhesion molecule-positive cells before and after the initiation of therapy was found to be an independent prognostic factor. Other markers used in patients with breast cancer include mammaglobin, epi dermal growth factor receptor, and carcinoembryonic antigen. 116, 152, 153 Although none of these markers is entirely specific for the detection of metastatic breast cancer, and although the sensitivity of peripheral blood is less than that of bone marrow, there is growing evidence that the detec tion of occult metastatic cells in the peripheral blood has a negative impact on prognosis. 154 A recent study found that the presence of five or more tumor cells in the periph eral blood from patients with breast cancer examined upon the initiation of therapy was important in predicting outcome. 151 Peripheral blood from patients with colorectal, 106 stomach, 104 prostate, 155 and skin 155, 156 cancer have also been studied. Markers that have been studied in colorectal cancer include cytokeratins, 106 carcinoembryonic antigen, 157, 158 apolipoprotein, 159 and CD44v6. 160 PSA messenger RNA (mRNA) is the most commonly used marker in patients with prostate cancer. 155, 161 Tyrosinase mRNA is the marker 

Studies undertaken to detect occult lymph node metastases by routine histologic methods have generally been per formed by cutting serial sections from all paraffin blocks containing lymph nodes, followed by routine staining and microscopic review. 163 Several studies simply reviewed the original histologic slides. Newer studies involve cytokeratin immunohistochemistry on one or more lymph node sec tions. PSA immunohistochemistry has also been used to confirm the prostatic origin of cytokeratinpositive cells in the lymph nodes of patients with prostate cancer. 108 All these studies have shown that deposits of tumor can be detected using these methods. In previously determined nodenegative cases of breast cancer, 7% to 33% convert to nodepositive status after review. Neville and colleagues 164 found the mean conversion rate to be approximately 13%. Although virtually all studies have shown that lymph node metastases can be overlooked, there has been surprising disagreement about the prognostic importance of these occult tumor deposits. 165167 However, it is now widely accepted that the detection of occult lymph node metastases is an important predictor of outcome in patients with histologically nodenegative cancer. 168170 In a key study (Ludwig Trial V), occult breast cancer metastases were detected by immunohistochemistry in 20% of patients and were associated with significantly poor diseasefree and overall survival in postmenopausal patients but not in pre menopausal patients. 168 Additional studies in patients with breast cancer found occult lymph node metastases to be predictive of a poorer outcome. 169, 170 Studies in patients with lung, 171,172 prostate, 107,108 and colorectal 173, 174 cancer suggest that occult metastases in the lymph nodes in these patients may also predict a worse prognosis. The finding that occult bone marrow and lymph node metastases are prognostically important has motivated several major clinical trials, notably by the American College of Surgeons Oncology Group, in breast cancer (Z0010) and lung cancer (Z0040). The advent of the use of sentinel lymph node biopsy in tumor surgery (for breast cancer and melanoma) has caused physicians to examine these lymph nodes by more sensitive techniques, owing to the limited material available for histologic review. 175 It is likely that the detection of occult metastases will soon be the general stan dard of care; this is true at many institutions that treat large numbers of patients with cancer.

Her2/neu (or cerb B2) is a protooncogene. The gene encodes for a protein (185 kD) that shows homology with epidermal growth factor and displays tyrosine activity. Amplification of the gene coding for Her2/neu has been described in breast, ovarian, prostate, gastric, salivary gland, lung, colon, and squamous cell carcinoma. 176186 When over expressed, the protein accumulates at the cell membrane and is seen as a crisp membrane stain; a cytoplasmic stain ing pattern is not associated with protein or gene overex pression. 176, 183 Although Her2/neu overexpression and amplification have been described in several tumor systems, it has been studied most extensively in the breast. Her2/neu overex pression occurs in 10% to 34% of primary breast carcino mas 187 and is restricted to cancer cells. There is an inverse association between Her2/neu amplification and the expres sion of estrogen and progesterone receptors. Her2/neu overexpression is also associated with highgrade tumors 176, 188 and is considered an adverse prognostic indictor in patients with breast cancer. 187 The presence of Her2/neu overexpres sion is associated with resistance to tamoxifen therapy 189191 and to CMF (cyclophosphamide, methotrexate, 5fluoro uracil) adjuvant chemotherapy but is associated with an increased response to regimens that use highdose doxoru bicin. 192195 Recent studies have linked amplification of the Her2/neu and topoisomerase IIα genes to the effects of anthracyclines. Preliminary data suggest that coamplifica tion of these two genes may identify a subgroup of highrisk breast cancer patients who might benefit from individually tailored and doseescalated adjuvant anthracyclines. 196, 197 Her2/neu can also be assessed through amplification of the gene by fluorescence in situ hybridization.

Epidermal growth factor receptor (EGFR) belongs to a family of growth factor receptors involved in normal growth. The gene is located on chromosome 7p12. It is the receptor for epidermal growth factor and is a member of the receptor tyrosine kinase family. It is closely related to Her2/neu, Her3, and Her4. EGFR is known to be involved in carci nogenic processes such as cell proliferation, apoptosis, angiogenesis, cell motility, and metastasis. The expression of EGFR has been examined in a wide variety of tissues, and in many cases, increased expression of EGFR is predic tive of tumor progression (e.g., cancer of the breast, esoph agus, adrenals, lung, bladder, thyroid, and gastrointestinal tract and glioblastoma multiforme). 198205 In addition to immunohistochemical methods, fluorescence in situ hybrid ization has been used successfully to identify EGFR muta tion or deletions on formalinfixed, paraffinembedded tissue. 206 EGFR is also showing promise as a therapeutic target. Studies are under way in lung and colorectal cancer to determine the usefulness of targeting EGFR for anticancer therapy. 207, 208 

The primary characteristics of tumor suppressor genes are that they encode normal cellular products involved in growth control, and both alleles must be inactivated for loss of function (i.e., loss of tumor suppression) to occur. The most well known are retinoblastoma (Rb) protein, p53, p27, p21, and p16. The two best characterized are the Rb and p53 genes. Both are thought to be involved in growth control through the regulation of transcription.

The Rb gene is located on chromosome 13q14 and is dys functional in a number of types of cancer. Its normal func tion is to prevent the replication of damaged DNA; it does so by preventing cell replication by binding and inhibiting the transcription factor E2F. 209, 210 The retinoblastoma protein (pRb) is activated when it is dephosphorylated and inacti vated when it is phosphorylated. Alterations in this gene have been described in many human tumors, including retinoblastoma, osteosarcoma, other sarcomas, leukemias, lymphomas, and certain carcinomas, including breast, lung, prostate, bladder, kidney, and testicular carcinoma. 29, 211, 212 Gene alterations are associated with advanced tumor grade and stage in a variety of tumors. 211, 213 Alterations in the Rb gene correlate with loss of expression of pRb as determined by immunohistochemistry. 214 Assessment of Rb gene loss by immunohistochemistry is based on the loss of detectable nuclear staining for pRb. There is growing evidence that gene alterations may identify tumors that have a higher risk of developing metastases. 215 Loss of heterozygosity, muta tions, or deletions of the Rb gene usually result in the loss of pRb expression, which has been regarded as an indicator of loss of pRb function in human tumors. In addition to loss of pRb expression, aberrantly high pRb expression indi cates a loss of pRb function in bladder tumors compared with moderate pRb expression. 210, 215 It has been shown that tumors with pRb overexpression demonstrate pRb hyper phosphorylation, mediated in part by the loss of p16 expres sion or overexpression of cyclin D1. 210

The p53 gene is located on chromosome 17p13.1. The p53 protein is expressed by all normal cells, but the halflife of the normal protein is so short (6 to 30 minutes) that it does not accumulate in levels high enough to be detected by standard immunohistochemical techniques. Mutant p53 protein, by contrast, has an extended halflife, accumulates, and is readily detectable in the cell nucleus; mutation is indicated by positive staining. Alterations of the p53 gene are extremely common in human cancer and have been described in bladder, colon, lung, breast, and other carcino mas; astrocytomas; leukemias; sarcomas; and mesothelio mas. 1, 29, 213, 216, 217 Because of the importance of p53 alterations in human cancer and the ease of detecting p53 mutations by molecular or immunohistochemical methods, p53 alter ations have been the focus of intense examination. As with Rb alterations, p53 alterations are associated with tumors of high histologic grade and a high proliferative index. There is growing evidence that, at least for some types of tumors, p53 alterations identify patients with shorter diseasefree intervals and poorer overall survival. 217, 218 

The cyclindependent kinase inhibitors are a family of cell cycle regulators. Their primary function seems to be the formation of stable complexes with cyclindependent kinase proteins and the subsequent inhibition of the cell cycle. These complexes inactivate the catalytically operative units. Among the most well known and clinically relevant are p21, p27, and p16.

A member of the WAF/CIP/KIP family of cyclindependent kinase inhibitors, p21 is probably the best characterized. It acts as a regulator of epithelial carcinogenesis and differen tiation and is thought to play an important role in tumor suppression by regulating cell cycle progression, DNA rep lication, and DNA repair. 29, 219 The protein expression of p21 has been studied in a variety of tumor types, including breast, 220 gastric, 221 ovary, 222 colorectal, 223 and bladder 213, 224 carcinomas. The alteration of protein expression assessed by immunohistochemical methods has been associated with higher tumor grade and worse prognosis in patients with bladder cancer. 123, 213 p27

The p27 inhibitor is involved in the regulation of the cell cycle at the G 1 S transition, ultimately through the inhibi tion of pRb phosphorylation. 225 Mutations in the human p27 gene appear to be rare. 226 Loss of p27 expression is associated with colon, breast, prostate, and gastric cancer progression. 227231 p16 Also known as p16 INK4 and CDKN2A, p16 is a tumor sup pressor protein encoded on the INK4a/ARF locus of chro mosome 9p21, which is one of the most frequent sites of genetic loss in human cancer. 232 Numerous studies have found abnormal p16 protein in a variety of tumor types, including melanomas; gliomas; esophageal, pancreatic, lung, and bladder carcinomas; and certain types of lympho mas. 232240 In addition, p16 is known to regulate Rb, and immunohistochemical expression of pRb and p16 is inversely correlated in a variety of tumors. 241, 242 combined effects of p53, p21, and prb It is known that, individually, p53, p21, and pRb are inde pendent predictors of time to recurrence and overall sur vival in patients with bladder cancer. 215, 217, 224 Efforts have therefore been made to examine these determinants in com bination. 123, 213 In one study, patients were analyzed accord ing to whether none, one, two, or all three markers were positive. The 5year survival rates were 70%, 58%, 33%, and 8%, respectively. These data suggest that alterations in p53, p21, and pRb act in cooperative or synergistic ways to promote bladder cancer progression.

Cyclin D1 plays a key role in the regulation of the G 1 S transition phase of the cell cycle. It has been linked to a number of different cancers, including colorectal, esop h ageal, gastric, lung, head and neck, and pancreatic cancer. 243

Although a major purpose of the molecular assessment of cancer is to better understand the risk for disease progres sion, advanced technologies are also being used to under stand the specific patterns of response and resistance to therapeutic regimens. The traditional means of determining appropriate systemic treatment generally involved histo genic classification. It has long been recognized, however, that response to hormonal therapy can be predicted spe cifically by molecular determinants (e.g., the expression of estrogen and progesterone receptors in breast and other cancers of reproductive organs). 244 Tumors arising from the breast, prostate, endometrium, and ovary are known to be regulated by steroid sex hor mones (estrogens, androgens). It was discovered that removing the source of hormones that control tumor growth (by oophorectomy, orchiectomy, or chemical methods) sometimes resulted in dramatic tumor remission. 244 Growth regulation was found to be associated with the amount of specific hormone receptors: Tumors that expressed high levels of these receptors tended to respond well to hormone ablation, whereas those with few or no receptors tended not to respond to this type of therapy. Accurate methods for determining the presence or absence of hormone recep tors are essential for determining the best method of treatment.

The availability of monoclonal antibodies to estrogen, progesterone, and androgen receptors has made immuno histochemical detection of hormone receptor status the current method of choice. These immunohistochemical methods can be performed on formalinfixed, paraffin embedded tissue and on cytology specimens. Immunohis tochemical antireceptor assays allow one to predict breast cancer's response to hormonal treatment. 245, 246 Tumors that do not express estrogen or progesterone receptors have a low probability of responding to hormonal manipulation, whereas estrogen receptor-and progesterone receptor-pos itive tumors have a high probability of responding to such treatment. Many practitioners believe that the only relevant result for hormone receptors in breast cancer is "positive" or "negative." However, some investigators have shown that the level of hormone receptor is important as well. 8, 9 Although a proportion of patients with low levels of hormone receptor will respond to hormone therapy, most benefit from the addition of systemic cytotoxic chemother apy. In contrast, in patients with high levels of hormone receptor, the addition of cytotoxic chemotherapy has a del eterious effect on outcome. 8, 9 Recently, attention has focused on expression of the estrogen receptor subtypes α and β and on various isoforms of the β subtype. It has been found that estrogen receptor α-negative tumors express significant levels of estrogen receptor β1 and β5 and that their expression levels are no different from levels in estrogen receptor α-positive tumors. 246 Therefore, these two estrogen receptor isoforms may be potential molecular targets for designing chemopre ventive drugs to treat estrogen receptor α-negative breast cancers.

Pglycoprotein is a transmembrane protein of 170 kD that has been associated with intrinsic and acquired resis tance to certain chemotherapeutic agents, particularly anthracyclines and vinca alkaloids. Pglycoprotein also may play a role in tumor progression and has been associated with blood vessel invasion and lymph node metastases. 2 Some tumors inherently express Pglycoprotein, whereas other tumors acquire expression only after exposure to certain chemotherapeutic agents. 2 Overexpression is associ ated with failure of chemotherapy. 2, 247 Other predictors of response to specific forms of chemo therapy are being explored. The prevailing view has been that p53 alterations should result in a chemoresistant phe notype. This view is based on a body of evidence showing that wildtype p53 is required for entrance into the apop totic pathway at the G 1 to Sphase transition. 248, 249 Because chemotherapy works through the induction of apoptosis, p53 alterations may result in resistance to such agents. We are conducting a clinical trial concerning the role of p53 in predicting progression and response in patients with bladder cancer. 250 It is also possible that p53 may promote chemo resistance by other mechanisms, such as through induction of the multidrug resistance (MDR-1) gene. 249, 251 In tumors in which p53 alterations confer increased (selective) che mosensitivity, combining agents that have different actions (e.g., DNA damage versus inhibition of the G 2 M check point) may have synergistic effects on tumor cell killing, a finding that has important implications in the design of new combination chemotherapy regimens. 252 The expression of thymidylate synthase in colorectal tumors predicts resistance to the most common type of systemic chemotherapy used in that disease, 5fluoroura cil. 253, 254 As mentioned earlier, Her2/neu overexpression in breast cancer predicts resistance to hormone therapy in estrogen receptor-positive tumors 189, 190, 255 and resistance to some types of chemotherapy, but increased sensitivity to doxorubicinbased regimens. 193, 194 Her2/neu and EGFR are specific targets of antibodydirected therapy. In the case of Her 2/neu, it seems that only those tumors that overexpress the target are likely to respond to such therapies. 256258 In the case of colorectal cancer, therapy directed against EGFR appears to work only in tumors with wildtype KRAS; tumors with mutant KRAS do not respond to EGFR therapy. 258a Similar findings are seen in lung cancer. 258b The ability to predict the specific response of individual tumors to chemotherapeutic agents can have a profound effect on treatment decisions for patients with cancer. It is not difficult to envision the day when drug selection is based on the resistance patterns of individual tumors to specific agents. Treatment decisions will become less organ based and will better reflect the biology of the tumors.

Traditionally, the stains available to surgical pathologists to identify infectious organisms in tissue sections consisted of Gram stain, variations of the acidfast stain, periodic acid-Schiff, and silver stains. There is now a wide range of immu nohistochemical or in situ hybridization techniques available for the detection of specific types of organisms within fixed paraffin sections. Although culturing tech niques remain the most important method for diagnosing most infections, immunohistochemical methods are as effective as, or even superior to, culture and routine H&E methods for the detection of certain infectious organisms such as cytomegalovirus (Fig. 59) , mycobacteria, Toxoplasma, Pneumocystis carinii, Histoplasma capsulatum, Helicobacter pylori, and human papillomavirus. 259269 Table 54 summarizes some of the infectious agents that can be iden tified by immunohistochemistry. 

Immunomicroscopy: A Diagnostic Tool for the Surgical Pathologist

Immunohistochemistry and related marking techniques

The current role of immunohistochemistry in diagnostic pathology

Quality assurance in immunohistochemistry: A discipline coming of age

Antigen retrieval for immunohistochem istry status and need for greater standardization

Standardization of immuno histochemistry based on antigen retrieval technique for routine formalinfixed tissue sections

Enhanced accuracy and reliability of Her2/ neu immunohistochemical scoring using digital microscopy

of Clinical Oncology/College of American Pathologists guideline rec ommendations for human epidermal growth factor receptor 2 testing in breast cancer

Estrogen receptor status by immunohistochemistry is superior to the ligandbinding assay for predicting response to adjuvant endocrine therapy in breast cancer

Estrogenreceptor status and outcomes of modern chemotherapy for patients with nodepositive breast cancer

The checkerboard tissue block: An improved multitissue control block

Antigen retrieval immunohistochem stry under the influence of pH using monoclonal antibodies

Antigen retrieval in formalinfixed, par affin embedded tissues: An enhancement method for immunohisto chemical staining based on microwave oven heating of tissue sections

FDA issues final rule for classification/reclassification of immunochemistry reagents and kits

Department of Health and Human Services, Health Care Financing Administration, et al: Clinical Laboratory Improvement Amendments of 1988

Loss of tumor markerimmunostaining intensity on stored paraffin slides of breast cancer

Quantitative analysis of the decay of immunoreactivity in stored prostate needle biopsy sec tions

Paraffin section storage and immunohistochemistry: Effects of time, temperature, fixation, and retrieval protocol with emphasis on p53 protein and MIB1 antigen

An enhancement method for immunohistochemical staining of proliferating cell nuclear antigen in archival rodent tissue

Antigen retrieval immunohistochemis try: Past, present, and future

Modeling formalin fixation and antigen retrieval with bovine pancreatic ribonuclease A:I-structural and functional alterations

Mechanisms of heatinduced antigen retrieval: Analysis in vitro employing SDSPAGE and immunohistochemistry

A molecular mecha nism of formalin fixation and antigen retrieval

Antigen retrieval immunohisto chemistry: Practice and development

Role of calcium chelation in high temperature antigen retrieval at different pH values

Microwavestimulated antigen retrieval is pH and temperature dependent

Antigen retrieval techniques in immunohistochemistry: Comparison of different methods

Reversing the effects of formalin fixation with citraconic anhydride and heat: A universal antigen retrieval method

An analysis of 1539 patients with cancer of unknown primary site

Immunohistochemical detection of tumor suppressor genes using antigen retrieval technique

A monoclonal antibody against a smooth muscle actin: A new probe for smooth muscle differentiation

Histiogenesis of alveolar soft part sarcoma: An immu nohistochemical and biochemical study

Demonstration of alpha1antitrypsin and alpha1 antichymotrypsin in fibrous histiocytomas using the immunoper oxidase technique

The value of electron microscopy and immunohistochem istry in the diagnosis of soft tissue sarcoma: A study of 200 cases

Antichymotrypsin staining of 194 sarcomas, 38 carcinomas, and 17 malignant melanomas: Its lack of specificity as a tumor marker

Synaptophysin: A novel marker for neurons, certain neuroendocrine cells and their neoplasms

Malignant peripheral nerve sheath tumor, an immu nohistochemical study of 62 cases

Value of S100 protein in the diagnosis of soft tissue tumors with particular reference to benign and malignant schwann cell tumors

Chondrosarcoma with additional mesenchymal com ponent (dedifferentiated chondrosarcoma) II: An immunohisto chemical and electron microscopic study

Cytogenetic profile of unknown primary tumors: Clues for their pathogenesis and clinical management

Cytogenetic and molecular genetic analyses of endo metrial stromal sarcomas: Nonrandom involvement of chromosome arms 6p and 7p and confirmation of JAZF1/JJAZ1 gene fusion in t(7;17)

Rearrangement of MLL in a patient with congenital acute monoblastic leukemia and granulocytic sarcoma associated with t(1;11)(p36;q23) translocation

Primary myeloid sarcoma of the testicle with t(15;17)

Primary pleuro pulmonary synovial sarcoma diagnosed by fine needle aspiration with cytogenetic confirmation: A case report

Genomic profiling of myeloid sarcoma by array com parative genomic hybridization

Visceral clear cell sarcoma of soft tissue with con firmation by EWSATF1 fusion detection

Advances in the pathologic diagnosis and biology of acute myeloid leukemia

Enolase isoenzymes as markers for dif ferential diagnosis of neuroblastoma, rhabdomyosarcoma, and Wilm's tumor

Detection of chromogranin in neuroendocrine cells with monoclonal antibody

HNKIdefined antigen detected in paraffin embedded neuroectoderm tumors and those derived from cells of the amine precursor uptake and decarboxylation system

Primitive neuroectodermal tumors of the central nervous system: Patterns of expression of neuroendocrine markers, and all classes of intermediate filament proteins

Immunohistochemical analysis of Ewing's sarcoma cell surface antigen p30/32MIC2

Immunohistochemical spectrum of rhabdomyosarcoma and rhabdomyosarcomalike tumors: Expression of cytokeratin and the 68 kD neurofilament protein

Small cell lung cancer, endocrine cells of the fetal bronchus, and other neuroendocrine cells express the Leu7 anti genic determinant present on natural killer cells

Immunoreactive neuronspecific enolase, bombesin, and chromogranin as markers for neuroendocrine lung tumors

Use of monoclonal antibodies to glial fibrillary acidic protein in the cytologic diagnosis of brain tumors

Recent applications of immunoperoxidase histochemistry in human neurooncology: An update

Expression of glial filament protein (GFP) in nerve sheaths and nonneural cells reexamined using monoclonal antibodies, with special emphasis on the coexpression of GFP and cytokeratin in epithelial cells of human salivary gland and pleomor phic adenomas

Expression of glial fibrillary acidic protein (GFAP) in peripheral nerve sheath tumors: A comparative study of immuno reactivity of GFAP, vimentin, S100 protein and neurofilament in 38 schwannomas and 18 neurofibromas

Mechanisms of bcl2 activation in human follicular lymphoma

Contribution of molecular genetic data to the classification of sarcomas

Detection of the SYTSSX fusion transcripts in form aldehydefixed, paraffinembedded tissue: A reverse transcription polymerase chain reaction amplification assay useful in the diagnosis of synovial sarcoma

Recent advances in soft tissue tumor diagnosis

Immunocytochemical expression of tissue specific transcription factor1 in lung carcinoma

TTF1 protein expression in pleural malignant mesotheliomas and adenocarcinomas of the lung

TTF1 gene expression and human lung cancer tumors

Surfactant proteins and thyroid transcription factor 1 in pulmonary and breast carcinomas

Immunoexpression of HBME1, high molecular weight cytokeratin, cytokeratin 19, thyroid transcription factor1, and Ecadherin in thyroid carcinomas

Variable sensitivity and specificity of TTF1 anti bodies in lung metastatic adenocarcinoma of colorectal origin

Recent immunohistochemical markers in the differential diagnosis of primary and metastatic carcinomas of the liver

CDX2 homeobox gene expression is a reliable marker of colorectal adenocarcinoma metastases to the lungs

Immunocytochemical identification of carcino mas of unknown primary in serous effusions

CDX2 expression in pulmonary fineneedle aspira tion specimens: A useful adjunct for the diagnosis of metastatic colorectal adenocarcinoma

Potential diagnostic utility of CDX2 immunophenotyping in extramammary Paget's disease

The expression of transcription factor activating transcription factor 3 in the human prostate and its regulation by androgen in prostate cancer

Rationale for adjuvant chemotherapy

Sensitivity of immunocytochemical detection of breast cancer cells in human bone marrow

Monoclonal antibodies for detec tion of occult carcinoma cells in bone marrow of breast cancer patients

Prediction of early relapse in patients with operable breast cancer by detection of occult bone marrow metastases

Bone marrow micrometastases predict early post operative recurrence following surgical resection of oesophageal and gastric carcinoma

Detection of micrometastasis of gastric carcinoma in peripheral blood circulation

Epithelial tumor cells in bone marrow of patients with colorectal cancer: Immunocytochemical detection, phenotypic characterization, and prognostic significance

Detection of micrometastasis in peripheral blood by multisampling in patients with colorectal cancer

Comparison of immunohistochemistry with reverse transcriptasePCR for the detection of micrometastatic prostate cancer in lymph nodes

Detection of occult lymph node metastases in locally advanced (pT3) nodenegative prostate cancer

Detection of occult bone marrow metastases in patients with operable lung carcinoma

Prognostic significance of micrometastasis in nonsmallcell lung cancer

Detection of micrometastasis of neuroblastoma to bone marrow and tumor dissemination to hematopoietic autographs using flow cytometry and reverse transcriptasepolymerase chain reaction

Clinical relevance of molecular staging for melanoma: Comparison of RTPCR and immunohistochemistry staining in sen tinel lymph nodes of patients with melanoma

Immunohistochemical detection of occult carci noma in bone marrow and blood

Detection of melanoma cells in peripheral blood by means of reverse transcriptase and polymerase chain reaction

Detection of carcinoembryonic antigen messenger RNA in lymph nodes from patients with colorectal cancer

Detection of circulating mammary carcinoma cells in the peripheral blood of breast cancer patients via nested reverse transcriptase polymerase chain reaction assay for mammaglobin mRNA

Prediction of distant metastasis by using reverse transcriptasepolymerase chain reaction for epithelial and variant CD44 mRNA in the peripheral blood of patients with colorec tal cancer

Detection of telomerase expression in mediastinal lymph nodes of patients with lung cancer

Prognostic value of cytokeratin 19 fragment (CYFRA211) and cytokeratin positive cells in bone marrow samples of breast cancer patients

Correlation of cyclooxygenase2 expression with molecular markers, pathological features and clinical outcome of transitional cell carcinoma of the bladder

Clinical relevance of reverse transcriptasepoly merase chain reaction for the detection of axillary lymph node metas tases in breast cancer

Examination of regional lymph nodes by sentinel node biopsy and molecular analysis provides new staging facilities in primary cutaneous melanoma

p53, p21, pRb, and p16 Expression predict clinical outcome in cystectomy with bladder cancer

Maspin and mammaglobin genes are specific markers for RTPCR detection of minimal residual disease in patients with breast cancer

Detection of epithelial cancer cells in peripheral blood by reverse transcriptasepolymerase chain reaction

A novel method for monitoring response to chemo therapy based on the detection of circulating cancer cells: A case report

Detection of isolated tumor cells in bone marrow is an independent prognostic factor in breast cancer

Mammaglobin expression in gynecologic malig nancies and malignant effusions detected by nested reverse transcrip tase polymerase chain reaction

Detection of circulating cancer cells expressing uro plakins and epidermal growth factor receptor in bladder cancer patients

Molecular and immunological detection of circulating tumor cells and micrometastases from solid tumors

Utilization of polymerase chain reaction technology in the detection of solid tumors

Molecular detection of micro metastases and circulating tumor cells in solid tumors

Detection of rare disseminated tumor cells identi fies head and neck cancer patients at risk of treatment failure

Prognostic value of circulating melanoma cells detected by reverse transcriptasepolymerase chain reaction

Detection of circulating uroplakin positive cells in patients with transitional cell carcinoma of the bladder

Metastatic bone disease: Clinical and therapeutic aspects

Detection and management of bone marrow micrometastases in breast cancer

Simultaneous immunohistochemical detection of tumor cells in lymph nodes and bone marrow aspirates in breast cancer and its correlation with other prognostic factors

Epithelial cells in bone marrow of breast cancer patients at the time of primary surgery: Clinical outcome during longterm followup

The fate and prognostic value of occult metastatic cells in the bone marrow of patients with breast carcinoma between primary treatment and recurrence

The prognostic value of isolated tumor cells in bone marrow in breast cancer patients: Evaluation of morphological cat egories and the number of clinically significant cells

Isolated tumor cells in bone marrow three years after diagnosis in diseasefree breast cancer patients predict unfavor able clinical outcome

A pooled analysis of bone marrow micrometastasis in breast cancer

Cytokeratinpositive cells in the bone marrow and survival of patients with stage I, II or III breast cancer

Micrometastatic breast cancer cells in bone marrow at primary surgery: Prognostic value in comparison with nodal status

Outcome of primarybreastcancer patients with micrometastases: A longterm followup study

Frequency and prognostic significance of isolated tumor cells in bone marrow of patients with nonsmall cell lung cancer without overt metastases

Isolated tumor cells in bone marrow predict reduced survival in nodenegative nonsmall cell lung cancer

Prognostic significance of micrometastatic tumor cells in bone marrow of colorectal cancer patients

Circulating tumor cells, disease progression, and survival in metastatic breast cancer

Circulating tumor cells: A novel prognostic factor for newly diagnosed metastatic breast cancer

Potential applications for cir culating tumor cells expressing the insulinlike growth factor1 receptor

Mammaglobin gene expression: A superior marker of breast cancer cells in peripheral blood in comparison to epidermalgrowthfactor receptor and cytokeratin19

Clinical significance of circulating cancer cells in peripheral blood detected by reverse transcriptasepolymerase chain reaction in patients with breast cancer

Molecular detection of cytokeratin19positive cells in the peripheral blood of patients with operable breast cancer: Evaluation of their prognostic significance

The role of the detection of hematogenous microme tastasis in prostate adenocarcinoma and malignant melanoma by RTPCR

Immunomagnetic detection and clinical significance of micrometastatic tumor cells in malignant melanoma patients

Realtime PCR (TaqMan PCR) quantification of car cinoembryonic antigen (CEA) mRNA in the peripheral blood of colorectal cancer patients

Detection of epithelial tumour RNA in the plasma of colon cancer patients is associated with advanced stages and circulat ing tumour cells

Apolipoprotein AI reverse transcriptasepoly merase chain reaction analysis for detection of hematogenous colon cancer dissemination

Limitations of CD44v6 ampli fication for the detection of tumour cells in the blood of colorectal cancer patients

Reverse transcriptase polymerase chain reaction (RTPCR) detection of melanoma related transcripts in peripheral blood and bone marrow of patients with malignant melanoma

Polymerase chain reaction for selection of melanoma in peripheral blood: Too early to assess clinical value

The reliability of histologi cally negative axillary lymph nodes in breast cancer

Axillary lymph node micrometastases and breast cancer

Immunohistochemical detection and significance of axillary lymph node micrometastases in breast cancer-a study of 97 cases

The use of monoclonal antibodies for the histologic detectioon of mammary axillary micrometastases

The immmunohistochemical detection of lymph node micrometastases from infiltrating lobular carcinoma of the breast

Role of immunohistochemistry detection of lymph node metastases in management of breast cancer

The prognostic significance of axillary lymphnode micrometastases in breast cancer patient

The association of cytokeratinonlypositive sen tinel lymph nodes and subsequent metastases in breast cancer

Immunohistochemical assessment of individual tumor cells in lymph nodes of patients with nonsmall cell lung cancer

Micrometastasis to lymph nodes in stage I left lung cancer patients

Identification of occult micrometastases in peri colic lymph nodes of Duke's B colorectal cancer patients using mono clonal antibodies against cytokeratins and CC49

Micrometastasis in regional lymph nodes of extir pated colorectal carcinoma: Immunohistochemical study using anti cytokreratin antibodies AE1/AE3

Sentinel lymphadenectomy in breast cancer

Studies of the Her2/neu protooncogene in human breast and ovarian cancer

Comparative investigation of cerbB2/ neu expression in head and neck and mammary cancer

Expression of the erbB2 protein in benign and malignant salivary gland tumors

Immunohistochemical detection of cerbB2 protein in human benign and neoplastic prostate

cerb B2 Oncoprotein expression in primary human tumors

Enhanced cerbB2neu expression in human ovarian cancer cells correlates with more severe malignancy that can be sup pressed by EIA

Double immunostaining for cerbB2 and p53 in human stomach cancer cells

Her2/neu expression in nodenegative breast cancer: Direct tissue quantitation by computerized image analysis and asso ciation of overexpression with increased risk of recurrent disease

Differential expression of the cerbB2 gene in human small cell lung cancer

Expression of the neu geneencoded protein (p185neu) in human nonsmall cell carcinoma of the lung

Expression of the cerbB2 gene product (p185) at different stages of neoplastic progression in the colon

Her2/neu (cerbB2) gene and protein in breast cancer

Expression of cerbB2 oncoprotein: A prognostic indicator in human breast cancer

Prognostic and predictive value of cerbB2 overex pression in primary breast cancer, alone and in combination with other prognostic markers

Predicting response to adjuvant and radiation therapy in patients with early stage breast carcinoma

Role of Her2/neu in tumor progression and therapy

Prognostic and predictive relevance of CerbB2 and ras expression in node positive and negative breast cancer

cerbB2 Antigen levels and decreased response to hormone therapy of breast cancer

Oncogene amplification and prognosis in breast cancer: Relationship with systemic treatment

cerbB2 Expression and response to adjuvant therapy in women with nodepositive early breast cancer

Her2/neu and topoisomerase IIa gene amplification and protein expression in invasive breast carcinomas

Topoisomerase IIa gene amplification predicts favor able treatment response to tailored and doseescalated anthracycline based adjuvant chemotherapy in Her2/neuamplified breast cancer: Results from the randomized Scandinavian breast group trial 9401

Epidermal growth factor receptor (EGFr); results of a 6 year followup study in operable breast cancer with emphasis on the node negative subgroup

Immunohistologic detection of the epidermal growth factor receptor in human esophageal squamous cell carcinoma

Immunohistochemical expression of epidermal growth factor receptors in human adrenocortical carcinoma

Epidermal growth factor receptors in human bladder cancer: Comparison of invasive and superficial tumours

Epidermal growth factor receptors in plasma membranes of normal and diseased human thyroid glands

Epidermal growth factor receptor and bladder cancer

Epidermal growth factor receptor expression correlates with poor survival in gastric adenocarcinoma from Mexican patients: A multivariate analysis using a standardized immunohisotchemical detection system

Prognostic and pathologic significance of quan titative protein expression profiling in human gliomas

Demonstration of EGFR gene copy loss in colorectal carcinomas by fluorescence in situ hybridization (FISH): A surrogate marker for sensitivity to specific antiEGFR therapy

Growth factor receptors as targets for lung cancer therapy

Epidermal growth factor receptor as a target for chemotherapy

E2FRb complexes regulating transcription of genes important for differentiation and development

Hyperphosphorylation of pRb: A mechanism for RB tumour suppressor pathway inactivation on bladder cancer

Altered expression of the retinoblastoma gene product: Prognostic indicator in bladder cancer

Studies of retinoblastoma gene in sarcomas

Combined effect of p53, p21 and pRb expression in the progression of bladder transitional cell carcinoma

Absence of retinoblastoma protein expression in primary nonsmall cell lung carcinomas

Elevated and absent pRb expression is associated with bladder cancer progression and has cooperative effects with p53

Genetic alterations of the p53 gene are a feature of malignant mesotheliomas

Accumulation of nuclear p53 and tumor progression in bladder cancer

Immunohistochemical study of p53 expression in cancer tissues from patients undergoing radiation therapy

p21CIP1 Acts as a positive regulator of cyclin B through carboxyterminal association with novel protein

p21WAFa/Cip1 Is associated with cyclin D1CCND1 expression and tubular differentiation but is independent of p53 overexpression in human breast carcinoma

Expression of p53 and p21 are independent prog nostic factors in patients with serosal invasion by gastric carcinoma

Increased level of p21 in human ovarian tumors is associated with increased expression of cdk2, cyclin A and PCNA

Loss of p21/WAF1/Cip1 protein expression accompanies progression of sporadic colorectal neoplasms but not hereditary nonpolyposis colorectal cancers

Effect of p21WAF/CIP1 expression on tumor progres sion in bladder cancer

Mutation of cellcycle regulators: Biological and clinical implications for human neoplasia

Mutational analysis of human cyclindependent kinase inhibitor p27/kip1 in primary breast carcinomas

p27Kip1: Chromosomal mapping to 12p1212p13.1 and absence of mutations in human tumors

Association of p27Kip1 levels with recurrence and survival in patients with stage C prostate carcinoma

Increased proteasomedependent degradation of the cyclindependent kinase inhibitor p27 in aggressive colorectal carci nomas

Expression of cellcycle regulators p27Kip1 and cyclin E, alone or in combination, correlate with survival in young breast cancer patients

Expression of cellcycle regulators p27 and cyclin E cor relates with survival in gastric carcinoma patients

INK4a/ARF: A multifunctional tumor suppressor locus

Frequent somatic mutations and homozygous dele tions of the p16 (MTS1) gene in pancreatic adenocarcinoma

Candidate tumorsuppressor genes MTS1 (p16INK4A) and MTS2 (p15INK4B) display frequent homozygous deletions in primary cells from T but not from Bcell lineage acute lymphoblastic leukemias

Deletion of p16 and p15 genes in brain tumors

Frequent somatic mutation of the MTS1/CDK4I (mul tiple tumor suppressor/cyclindependent kinase 4 inhibitor) gene in esophageal squamous cell carcinoma

CDKN2(p16/MTS1) gene deletion or CDK4 ampli fication occurs in the majority of glioblastomas

Inhibitors of mammalian G1 cyclindependent kinases

Effects of exogenous wildtype p16 gene trans fection on the expression of cell cyclerelated proteins in bladder cancer cell lines

p16 Expression in primary malignant mela noma is associated with prognosis and lymph node status

Correlation of abnormal Rb, p16ink4a, and p53 expression with 3p loss of heterozygosity, other genetic abnormali ties, and clinical features in 103 primary nonsmall cell lung cancer

Role of p16/MTS1, cyclin D1 and Rb in primary oral cancer and oral cancer cell lines

Molecular prognostic markers in pancreatic cancer: A systematic review

Receptors reconsidered: A 20 year perspective

Prediction of endocrine response in breast cancer by immunocytochemical detection of oestrogen receptor in fine needle aspirates

The prognostic value of immunohistochemical estrogen receptor analysis in paraffinembedded and frozen sections versus that of steroid binding assays

Chemosensitization and drug accumulation effects of VX710, verapamil, cyclosporin A, MS209 and GF120918 in multidrug resistant HL60/ADR cells expressing multidrug resis tanceassociated protein MRP

Molecular determinants of outcome in bladder cancer

p53 and Treatment of bladder cancer

Modulation of activity of the promoter of the human MDR1 gene by Ras and p53

UCN01: A potent abrogator of G2 checkpoint function in cancer cells with disrupted p53

p53 and Thymidylate synthase expression in untreated stage II colon cancer: Association with recurrence, survival and site

p53 Point mutations and thymidylate synthase mes senger RNA levels in disseminated colorectal cancer: An analysis of response and survival

cerbB2 Overexpression decreases the benefit of adjuvant tamoxifen in earlystage breast cancer without axillary lymph node metastases

Current status of taxanes as adjuvant therapy for early stage breast cancer

NCCN task force report: Adjuvant therapy for breast cancer

Trastumab after primary treatment for early stage Her2positive breast cancer reduces recurrence

Wildtype KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer

Role of KRAS and EGFR as biomarkers of response erlotinib in National Cancer Institute of Canada Clinical Trials Group Study BR.21

Rapid diagnosis of disseminated histoplasmosis in tissues

Immunohistologic analysis of myobacterial anti gens by monoclonal antibodies in tuberculosis and mycobacteriosis

Identification of CMV in formalinfixed, paraffin embedded tissues: Comparison of immunocytochemistry and in situ DNA hybridization

Three sensitive methods for the detection of cyto megalovirus in lung cancer tissue of patients with interstitial pneu monitis

Disseminated Pneumocystis carinii infection causing extrapulmonary organ failure: Clinical, pathologic and immunohis tochemical analysis

Comparative analysis of human papillomavirus detection of polymerase chain reaction and Virapap/Viratype kits

Immunocytochemical identification of Helicobacter pylori in formalinfixed gastric biopsies

Analysis of lower genital tract and lesions suspicious for condyloma by in situ hybridization and consensus sequence PCR for the detection of HPV

Involvement of the pancreas in AIDS: A prospec tive study of 109 postmortems

Human decidual cells activity in women with spontaneous abortions of probable CMV aetiology during the first trimester of gestation: An immunohistochemical study with CMV associated antigen

Detection of Helicobacter pylori in the middle ear fluid of patients with otitis media with effu sion