Three main groups constitute the cytoskeleton of human cells: thin, intermediate, and thick filaments. Thin filaments (5 to 6 nm) are composed of α-, β-, and γ-actins; the former are exclusively found in muscle cells and can be distinguished by antibodies such as muscle-specific actin (MSA; HHF35), smooth muscle actin (SMA), and smooth muscle myosin heavy chain (SMMS-1). For example, the IHC staining pattern differentiates between nonmuscle cells and tumors (MSA- SMA- SMMS-1-), skeletal muscle myocytes and rhabdomyosarcomas (MSA+ SMA- SMMS-1-), and smooth muscle myocytes and leiomyosarcomas (MSA+ SMA+ SMMS-1+). Myoepithelial cells and myofibroblasts also stain positively for all three markers, although to varying degrees.
Diagnostically speaking, the most useful cytoskeletal proteins are the intermediate filaments (10 nm). The relative composition of intermediate filaments varies by cell type and allows distinction by IHC. The major intermediate filaments include vimentin, desmin, glial fibrillary acidic protein (GFAP), and cytokeratins.
Vimentin can be found in all mesenchyme-derived cells— fibroblasts, myocytes, osteocytes, chondrocytes, Schwann cells, endothelial cells, and hematopoietic elements—often leading to its dismissal as “nonspecific.” Nonetheless, a vimentin stain serves well to distinguish sarcomas and lymphomas from carcinomas. Even with the most poorly differentiated neoplasms, this distinction can usually be made. Vimentin IHC is also of great utility in confirming that tissue antigenicity has been preserved; most sections have at least focal areas of vimentin-positive cells. Necrotic tissue can be surprisingly informative, as it often maintains some degree of reactivity, often in the original pattern of distribution. In these cases, careful comparison with control tissue and nontumoral tissue in the section is necessary to ensure accurate interpretation.
Desmin shares sequence homology with vimentin and is likewise restricted to mesenchymal cells. However, unlike vimentin, desmin is only expressed at significant levels in smooth, skeletal, and cardiac myocytes. Thus, in a sense, desmin is a marker of myogenic differentiation; although the aforementioned cells contain desmin, primitive mesenchymal cells and neoplasms do not. Desmin-positive tumors include leiomyomas, leiomyosarcomas, and rhabdomyosarcomas. Of special note, desmin expression in most cardiac myocytes is limited to the intercalated discs, whereas the Purkinje fibers show diffuse cytoplasmic staining.
Glial fibrillary acidic protein
(GFAP) is relatively specific for astrocytes and their corresponding neoplasms—astrocytomas, glioblastomas, and other gliomas (32
). Reactive astrocytes are markedly positive and care must be taken to ensure that such a population of cells is not mistaken for the actual neoplasm (32
). Ependymal cells and their derivative neoplasms show variable reactivity for GFAP. Neurofilament
actually a set of three related proteins that form fibers within the cell bodies and processes of neurons; the main diagnostic utility of a neurofilament IHC stain is to highlight neurons within tissue or tumor.
Epithelial cells are easily distinguishable by the presence of distinct cytokeratin
profiles. Carcinomas are positive when using broad-spectrum cytokeratin antibody “cocktails” such as AE1/AE3 or CK7/CK20, which can rule out most lymphomas and sarcomas. Important exceptions include the epithelial component of synovial sarcoma (Figure 1C-5, panel C
), epithelioid sarcoma, and the characteristic cytoplasmic inclusions of malignant rhabdoid tumors; the latter stain for cytokeratin, not muscle markers. More specific antibodies can help highlight organ-specific epithelium, for example, CK19 in breast or biliary tract or TTF1 in the lung and thyroid.
FIGURE 1C-5 • Biphasic synovial sarcoma. The H&E-stained sections (A, B) demonstrate a spindle cell sarcoma with areas of glandular differentiation; the latter are immunohistochemically positive for mixed cytokeratins (C), and the majority of the tumor cells are positive for bcl-2 (D). Conventional cytogenetic analysis (E) demonstrated t(X;18) pathognomonic for the SYT-SSX fusion gene of synovial sarcoma (arrows). Breakapart FISH probes (one red, one green from opposite ends [5′ and 3′, respectively] of the SYT gene) are seen separately instead of together as a single intact yellow signal (as seen in the surrounding normal cells) (F). (Karyotype and FISH analysis courtesy of Dr. Peter vanTuinen, Dynacare Clinical Cytogenetics Laboratory, Medical College of Wisconsin.)
Thick filaments (20 to 25 nm) are composed of β-tubulin and are ubiquitous to all cell types. Thus, their diagnostic utility is limited.
Cell Surface Markers
Cell surface antigens have proven utility not only in IHC but also in flow cytometry and cell sorting. However, while flow cytometry requires fresh tissue or cell-rich fluid, the same
markers can be evaluated on FFPE tissue by IHC. These antigens are indispensable in the diagnosis of hematopoietic neoplasms, and such use is detailed elsewhere in this book (see Chapters 22
Many of these cell surface molecules are numerically designated as a “cluster of differentiation,” or “CD.” For example, the normal constituent cells of the mature immune system can be roughly grouped by their expression of these proteins: B lymphocytes (positive for CD19, CD20 [L26], CD79a; Figure 1C-3
), T lymphocytes (positive for CD3, CD4, CD8), and natural killer cells (positive for CD56). Myeloblasts stain for CD34 and CD117 (c-kit), two markers also associated with other neoplasms; a CD34 stain is positive in synovial sarcoma and some vascular tumors, and CD117 positivity is an important finding in gastrointestinal stromal tumors (35
Macrophages and histiocytes exhibit granular staining in their cytoplasm for CD68, a component of lysosomal membranes (more accurately, a lysosomal
surface marker); CD1a is specific for Langerhans cells and T lymphoblasts (36
). Mast cells have membrane positivity for CD138, as well as cytoplasmic positivity for a characteristic enzyme, tryptase (40
). This enzyme can also be detected by histochemical methods that require frozen tissue.
FIGURE 1C-6 • Classic Hodgkin lymphoma. The H&E-stained section (A) reveals scattered large cells with atypical, convoluted nuclei in a mixed inflammatory background. At lower power, fibrous bands were seen entrapping nodules of tumor. By IHC, the Hodgkin cells are positive for CD15 (B) and CD30 (C). In situ hybridization for EBV-encoded RNA (EBER) is positive in many of these cells (D, red staining).
Endothelial cells, both nascent and tumoral, can be marked with CD31 and CD34, although these antibodies mark some other cells, as well. GLUT-1 is a useful marker for the endothelial cells of infantile hemangiomas (41
), and D2-40 (podoplanin) marks lymphatic endothelium (along with mesothelium and some other tissues) (42
A wide variety of viruses can be detected by antibodies specific for well-conserved antigens, including adenovirus, cytomegalovirus (CMV), herpes simplex virus (HSV) types I and II, parvovirus B19, human herpesvirus 8 (HHV8), human papilloma virus, BK virus (via large T antigen), hepatitis B virus (via surface antigen), and Epstein-Barr virus (EBV, via the latent membrane protein [LMP]). Typically, clinical suspicion, positive serologic testing, or viral cytopathic change seen on routine H&E-stained slides serves as a trigger for further workup by IHC. Companion in situ
hybridization tests are available for both HPV and EBV (Figure 1C-6
panel D); these tests can identify the former as high- or low-risk subtypes. Whenever possible, viral IHC should be performed in tandem with culture and serologic testing (43
IHC is less commonly used to identify bacteria, both because of these organisms’ patchy and often sparse distribution in tissue and the greater detection sensitivity of microbiologic culture methods (43
). However, antibodies against Helicobacter pylori
have supplanted more traditional Steiner, Giemsa, or Alcian Yellow stains at some institutions (45
). Also, Pneumocystis jiroveci
, once thought to be protozoal but now formally classified as a fungus, can be easily highlighted by appropriate antibodies in IHC (47
IHC can assist in confirming the diagnosis and hormone secretion profile of many endocrine tumors. For example, pancreatic endocrine neoplasms (islet cell tumors) can be categorized as derived from alpha, beta, delta, or G cells based on immunohistochemically verifiable expression of glucagon, insulin, somatostatin, or gastrin, respectively. Likewise, VIP-producing tumors and serotonin-secreting carcinoids can be demonstrated by IHC.
In conjunction with clinical presentation, pituitary adenomas can be easily classified by IHC profiling for prolactin, adrenocorticotrophin hormone (ACTH), thyroid-stimulating hormone, growth hormone, follicle-stimulating hormone, and luteinizing hormone (49
). This approach is especially useful with silent adenomas, which have detectable hormone(s) in the tumor cells’ cytoplasm, but not in the patient’s serum.
Medullary thyroid carcinoma stains positively for calcitonin, as do normal C-cells and the hyperplastic foci of multiple endocrine neoplasia syndrome. More generally, thyroid epithelial cells can be highlighted by antibodies to thyroglobulin or thyroid transcription factor-1 (TTF-1). Parathyroid hormone stains are useful in identifying parathyroid tissue, although normal, hyperplastic, and neoplastic tissues react identically.
Although less often useful in the pediatric realm, IHC for estrogen and progesterone receptors has become standard of care in the evaluation of breast cancer, serving to guide the choice of chemotherapy. Germ cell and sex cord tumors can express α-inhibin and β-human chorionic gonadotrophin (β-hCG), and the serum levels of these markers are sometimes used to monitor patients for recurrence.
Embryonal and Cancer Markers
Fetal tissues and neoplasms share expression of primitive traits, including a subset of proteins normally restricted to developmental periods. For example, α-fetoprotein can be found in fetal liver as well as hepatoblastomas, hepatomas, and endodermal sinus tumors. Placental alkaline phosphatase (PLAP) stains germ cell tumors and some carcinomas. The stem cell marker OCT4 is now replacing PLAP as a more sensitive and specific marker of germ cell neoplasms, specifically seminoma/germinoma, embryonal carcinoma, and intratubular germ cell neoplasia. Among the so-called cancer markers, CA-125 is more specific for genitourinary neoplasms, whereas CA19-9 is preferentially expressed in gastrointestinal cancers. Both these markers are also detectable in patient serum.
INI-1 (hSNF, SMARCB1, BAF47) is a particularly helpful antibody in the diagnosis of malignant rhabdoid tumor (and atypical teratoid/rhabdoid tumor), epithelioid sarcoma, and renal medullary carcinoma. Loss of nuclear INI-1 staining is predictive of INI-1 mutation or deletion that typifies this group of tumors, whereas the gene is invariably expressed in normal cells, providing a useful internal control.
Also of note is the WT1 protein, a zinc-finger transcription factor that is expressed in the course of normal urogenital development and which functions as an inactivated tumor suppressor in nephroblastoma (Wilms tumor) and other neoplasms. Wilms tumors show nuclear immunore-activity for WT1 with most antibodies, whereas desmoplastic small round cell tumors, with canonical EWS-WT1
gene rearrangements, are often only positive when using antibodies directed against C-terminal epitopes (51
Anaplastic lymphoma kinase-1 (ALK-1) is the fusion product of a characteristic t
(2;5) translocation found in most anaplastic large cell lymphomas and inflammatory myofibroblastic tumors; its expression in the former is thought to portend favorable prognosis. ALK-1 is also mutated or amplified in about 20% of neuroblastomas, especially in familial variants, and may identify patients eligible for crizotinib therapy (53
). Tyrosine kinases, such as c-kit
(CD117), can help diagnose tumors such as gastrointestinal stromal tumors, as well as predict which lesions might respond to monoclonal antibody therapy (in this case, imatinib).
BRAF, a serine/threonine protein kinase, is often mutated in human cancers. In children, a specific V600E mutation is frequently seen in papillary thyroid carcinoma, aggressive Langerhans cell histiocytosis, and brain tumors (glioblastoma multiforme, pleomorphic xanthoastrocytomas, and gangliogliomas) (56
). The presence of this mutation identifies tumors which might be amenable to targeted therapies.
Cell Cycle and Apoptotic Markers
The most commonly used antibody in this category is MIB-1 (Ki67), a proliferation marker frequently used to assess the proliferative activity of lesions. Although a high MIB-1 index does not define something as neoplastic, it can be used as a corroborating piece of evidence in making such a decision, or in determining the histologic grade of a tumor (Figure 1C-3, panel C
). Some apoptotic markers, such as Bcl-2 and Bcl-6, have utility in identifying the lineage of hematopoietic neoplasms and other tumors (Figure 1C-5, panel D
). β-catenin, a molecule involved in the Wnt signaling pathway, shows nuclear staining in desmoid tumors, Wnt pathway-related
medulloblastomas, and colorectal lesions with aberrations of the APC pathway.
P53 IHC correlates with genetic TP53
status in many pediatric tumors, including gliomas, rhabdomyosarcomas, adrenal cortical neoplasms, Wilms tumors, and pleuropul-monary blastomas (27
). Aberrant p53 expression in the nucleus is usually predictive of a gene mutation; the most common deleterious mutations often interfere with protein turnover. Absent p53 expression by IHC doesn’t always imply an intact p53 pathway. Nonsense mutations, deletions and chromosome 17 loss are other mechanisms by which TP53
function can be lost.