Adjuvant Techniques–Immunohistochemistry, Cytogenetics, and Molecular Genetics

Specimen Handling and Preservation

A prerequisite of successful IHC evaluation in soft tissue and bone pathology is an approach to specimen handling that aims to preserve antigens. Timely and complete formalin fixation is critical to this approach. In samples with incomplete permeation by fixative, a gradient can be observed that reflects more robust IHC detection of antigen expression in the well-fixed, external portions of tissue. In addition to incomplete fixation, necrosis and electrocautery are common causes of lost antigenicity in soft tissue and bone tumors.

Of particular relevance to bony specimens, acidic decalcification solutions, used to render bone amenable to sectioning, can cause the degradation of nucleic acids and, to a lesser extent, proteins. Thus, whenever possible, the palpably soft components of a bone specimen should be dissected free and submitted in a separate paraffin block without exposure to demineralizing agents.

If acid-based decalcification is necessary, the tissue should undergo formalin fixation prior to decalcification, and exposure to decalcifying solutions should be limited to the duration required for histologic sectioning. Ethylenediaminetetraacetic acid (EDTA) is an alternative chelating agent used for decalcification that better preserves biomacromolecules, particularly DNA. The drawback of EDTA is a substantial increase in the required demineralization time compared with that of conventional acid decalcification. Although many antibodies retain functionality for IHC on formalin-fixed paraffin-embedded tissues that have undergone acid-based demineralization, it is sensible to approach these specimens cautiously when interpreting IHC studies, giving particular consideration to the possibility of “false negative” outcomes.

Practical Approach to Immunohistochemistry in Soft Tissue and Bone Pathology

When assessing IHC studies of soft tissue and bone tumors, a methodical approach can help to avoid errors in interpretation. First and foremost, it is essential to be aware of the appropriate internal and external positive and negative controls . With specific regard to the issue of specimen preservation, internal positive controls help to ensure that the diagnostic tissue is adequately preserved. As an example, a vascular component is present in essentially every tissue specimen, providing an internal positive control for endothelial lineage markers CD31 and ERG. In select cases where antigen preservation is of particular concern, it can be useful to evaluate a ubiquitously expressed target, such as SMARCB1 (INI1) or trimethylated H3K27, keeping in mind that background non-neoplastic cells should express these antigens even in tumors characterized by their loss. An inability to detect these antigens, especially in non-neoplastic cells, would indicate a problem with tissue preservation that should prompt caution in the interpretation of all other IHC studies revealing an absence of immunoreactivity.

Attention to the subcellular localization of immunoreactivity is another key to the accurate interpretation of IHC studies. For diagnostic applications in surgical pathology, it is useful to know whether a given epitope is localized to the nucleus, cytoplasm, or cell membrane because immunoreactivity in the wrong subcellular location should not be interpreted as “positive” for expression. Instead, immunoreactivity in an unexpected subcellular site should raise consideration of nonspecific “background” staining. This practical consideration is particularly valuable when assessing nuclear-localized proteins, such as transcription factors. In this context, cytoplasmic or membranous immunoreactivity should not be reported as positive for expression, since the result does not reflect the known characteristics of the protein being interrogated accurately. For some proteins, the subcellular location changes according to a transient state of activation or functional mutation. A notable example is β-catenin , which acts as both a structural protein and transcription factor, translocating to the nucleus in the latter capacity when stimulated by growth factors, or when affected by mutations leading to its constitutive activation. Such activating mutations of β-catenin characterize the vast majority of sporadic desmoid-type fibromatosis cases, for which IHC identification of nuclear-localized β-catenin can be a diagnostically valuable feature. Syndromic cases of desmoid fibromatosis also exhibit aberrantly nuclear-localized β-catenin, albeit due to inactivating mutations of the inhibitory APC gene, rather than mutations of β-catenin itself. Thus, the assessment of subcellular localization is necessary for the accurate diagnostic interpretation of β-catenin IHC.

Just as the subcellular location of antigen expression can influence IHC interpretation, accurate diagnostic assessment requires the appropriate attribution of protein expression to the various cellular components of a tumor. In particular, one should always consider whether immunoreactivity within a tumor reflects the antigen expression by the neoplasm itself or the expression in a non-neoplastic tissue component instead. In tumors extensively infiltrated by immune cells, localizing antigen expression to tumor cells can be particularly challenging. An example encountered with some frequency relates to the issue of CD31 expression by tumor-infiltrating histiocytes, leading to the misattribution of CD31 expression to a neoplastic process and the diagnostic assumption that this reflects a tumor with vascular differentiation. The avoidance of this pitfall requires the diagnostician not only to realize the heterogeneous nature of the cellular component of the tumor, but also to assign immunoreactivity to the morphologic correlate of non-neoplastic histiocytes appropriately. The use of a secondary marker of vascular endothelial differentiation, such as ERG (which is not expressed by histiocytes), can also aid in the accurate interpretation of CD31 expression. A separate example of the potential pitfall of the incorrect attribution of antigen expression occurs in leiomyomas of the gastrointestinal tract, particularly those arising in association with the esophagus. In esophageal leiomyomas, substantial infiltration by the non-neoplastic interstitial cells of Cajal, expressing both KIT and DOG1, can give the appearance that the tumor itself is expressing these GIST markers ( Fig. 2.1 ).

Small, Blue, Round Cell Tumors

The differential diagnosis of “small, blue, round” cell tumors includes both sarcomas and other neoplasms. Among the nonsarcomatous neoplasms that might be included in this differential diagnosis are lymphoma, melanoma, and, in an older patient, small cell carcinoma. The sarcomas that should be included in the differential diagnosis include Ewing sarcoma (ES), rhabdomyosarcoma (RMS), poorly differentiated synovial sarcoma (PDSS), and desmoplastic small round cell tumor, along with so-called Ewing-like sarcomas bearing BCOR-CCNB3 or CIC-DUX translocations. Matrix-producing tumors with small round cell morphology, such as mesenchymal chondrosarcoma, should be considered too, especially in the context of limited biopsy sampling that may fail to capture the matrix component. Particularly in children, additional non-sarcomatous embryonal neoplasms fall within this morphologic differential, including retinoblastoma, Wilms tumor, hepatoblastoma, and neuroblastoma. Table 2.3 presents a screening panel of antibodies and the expected results for these tumors. The results of this panel dictate which additional studies are needed to confirm a specific diagnosis.

Additional IHC workup depending on the suspected diagnosis:

• 1.

  Ewing sarcoma (ES): Diffuse, strong, membranous CD99 expression is an important, but nonspecific, feature of ES ( Fig. 2.2 ). CD99 IHC is susceptible to incomplete fixation and specimen decalcification. NKX2-2 and PAX7 are transcriptional targets of the EWSR1-ETS oncoprotein; detection of these proteins by IHC can aid in classification, particularly in cases in which CD99 staining is difficult to interpret. The common ETS domain-containing 3’ fusion partners of EWSR1, namely FLI1 and ERG, exhibit diffuse nuclear overexpression when present in the fusion. It is noted that both FLI1 and ERG are also markers of the vascular endothelial lineage.

  

• 2.

  Rhabdomyosarcoma (RMS): Small round cell cytomorphology typically coincides with the alveolar subtype of RMS, which is often, but not always, driven by PAX3/7-FOXO1 fusions. Fusion-positive RMS characteristically expresses strong and diffuse myogenin. Desmin and MyoD1 are also expressed. Fusion-negative, often embryonal, RMS cases with round cell morphology are also encountered; these tumors can on occasion show very limited desmin and myogenin expression, requiring an assiduous search for immunoreactivity. Alternative markers of the skeletal muscle lineage, namely MyoD1 and PAX7, may be more diffusely expressed, aiding in the classification ( Fig. 2.3 ).

  

• 3.

  Desmoplastic small round cell tumor (DSRCT): DSRCT distinctively expresses both desmin and keratin in the majority of cases. It is further characterized by diffuse immunoreactivity with antibodies targeting epitopes in the C-terminus of WT1 (present in the EWSR1-WT1 fusion oncoprotein; Fig. 2.4 ), but not the N-terminus (which is absent from the fusion protein). CD99 is also expressed in a subset of DSRCT cases.

  

• 4.

  CIC-DUX sarcoma: Variable CD99 expression can be seen. Diffuse and strong expression of full-length WT1 (N- and C-termini) is a distinctive feature. There is also diffuse immunoreactivity against the c-Myc protein. DUX4 immunohistochemistry can detect those tumors with DUX4 translocations.

• 5.

  BCOR-CCNB3 sarcoma : This tumor expresses both BCOR and CCNB3 on IHC. TLE1 and SATB2 expression are also seen in most cases.

• 6.

  Small cell carcinoma: Although not always present, chromogranin A, synaptophysin, or INSM1 expression supports the diagnosis as markers of neuroendocrine differentiation.

• 7.

  Melanoma: This expresses S-100 protein/SOX10, which is ordinarily strong and diffuse. The diagnosis can be confirmed with additional markers indicative of melanocytic differentiation (HMB45, Melan-A, MITF). A small number of melanomas may be S-100 protein-negative, and occasional melanomas express keratin or desmin. Small cell melanomas of the sinonasal tract are frequently S-100 protein-negative/HMB45-positive ( Fig. 2.5 ).

  

• 8.

  Lymphoma: Lymphoblastic lymphoma may be CD45-negative and CD99/FLI1-positive, which can easily result in misdiagnosis as Ewing sarcoma. Terminal deoxynucleotide transferase (TdT) and/or CD43 may be critical in arriving at the correct diagnosis in CD45-negatifve cases. In adults and children, anaplastic large cell lymphomas, including the small cell variant, may also be CD45-negative. CD30 is useful here ( Fig. 2.6 ).

  

• 9.

  Poorly differentiated synovial sarcoma (PDSS): Keratin expression may be patchy or absent in some cases. Epithelial membrane antigen (EMA) and high-molecular-weight keratins may be positive in such cases. Uniform, strong nuclear expression of TLE1 protein is a highly sensitive, but imperfectly specific, finding in PDSS.

• 10.

  Poorly differentiated osteosarcoma: While SATB2 expression can, in theory, be used to support osteogenic differentiation in the absence of morphological evidence of tumor osteoid, it is also expressed in the majority of BCOR-CCNB3 sarcomas and in some synovial sarcomas. Additionally, SATB2 expression can be seen in essentially any malignant neoplasm showing heterologous osteocartilaginous differentiation (e.g., melanoma, malignant peripheral nerve sheath tumor, others) and should therefore be interpreted with great caution in the absence of clear-cut osteoid production.

• 11.

  Mesenchymal chondrosarcoma: S-100 protein expression is largely within the cartilaginous component, although scattered positivity can be present in undifferentiated round cells. The expression of desmin, MYOD1, and myogenin, usually limited in extent, is observed in a subset of cases, and should not be misinterpreted as evidence of rhabdomyosarcoma. NKX3.1 expression has also emerged as a distinctive feature of mesenchymal chondrosarcoma.

Monomorphic Spindle Cell Tumors

The differential diagnosis of monomorphic spindle cell tumors often includes such entities as fibrosarcoma [usually arising in dermatofibrosarcoma protuberans (DFSP)], monophasic synovial sarcoma, malignant peripheral nerve sheath tumor (MPNST), and solitary fibrous tumor. Particularly in the abdomen, this differential diagnosis may also include a gastrointestinal stromal tumor (GIST), true smooth muscle tumors, and cellular schwannoma. Table 2.4 presents a screening immunohistochemical panel and the expected result for each tumor.

Additional IHC workup depending on the suspected diagnosis:

• 1.

  Synovial sarcoma: Keratin and EMA expression may be focal in monophasic synovial sarcomas. The expression of CD34 is exceptionally rare in synovial sarcoma. TLE1 expression may be helpful ( Fig. 2.7 ) but is nonspecific.

  

• 2.

  MPNST and cellular schwannoma: SOX10 and S-100 protein expression is often weak and focal in MPNST, but diffuse and strong in cellular schwannoma ( Fig. 2.8 ). EMA, claudin-1, and Glut-1 expression may be observed in MPNST with perineurial differentiation. The loss of H3K27me3 expression, if present, would support classification as MPNST versus cellular benign nerve sheath tumor.

  

• 3.

  Fibrosarcomatous DFSP : CD34 expression may be observed only in the DFSP component and lost in the fibrosarcoma component. Smooth muscle actin (SMA) expression, indicative of myofibroblastic differentiation, may be present ( Fig. 2.9 ).

  

• 4.

  Solitary fibrous tumor (SFT): The detection of nuclear STAT6 expression by IHC distinguishes SFT from morphologic mimics. Malignant SFT may have anomalous keratin expression. Dedifferentiated SFTs frequently lose expression of CD34 and can even partially or entirely lose expression of STAT6. Among mesenchymal neoplasms, nuclear STAT6 is also seen in a subset of dedifferentiated liposarcomas owing to STAT6 gene amplification.

• 5.

  Gastrointestinal stromal tumor (GIST): More than 90% of GISTs express KIT (CD117) ( Fig. 2.10 ). The expression of DOG1 may be valuable in cases with weak or absent CD117 expression ( Fig. 2.11 ). It is also useful to note that approximately 70% of GISTs express CD34. GIST may be variably positive for both SMA and S-100 protein, but are typically desmin-negative. KIT expression is not restricted to GIST, and recognition hereof can help to avoid the misdiagnosis of intra-abdominal manifestations of KIT-positive tumors, such as melanoma, Ewing sarcoma, or angiosarcoma.

  
  

• 6.

  Desmoid fibromatosis : Nuclear-localized β-catenin is a manifestation of the constitutive β-catenin signaling activity that characterizes this entity. Importantly, only nuclear-localized immunoreactivity is considered indicative of the transcriptionally active protein representing the underlying oncogenic mutation. Metaplastic carcinoma and low-grade endometrial stromal sarcoma are two notable spindle cell neoplasms that can also exhibit aberrant nuclear-localized β-catenin by IHC. Diffuse nuclear β-catenin expression is also quite characteristic of sinonasal glomangiopericytoma (hemangiopericytoma-like tumor) and intranodal palisaded myofibroblastoma. Desmoid fibromatosis should not express keratin, CD34, or significant desmin.

• 7.

  Perineurioma : Each of EMA, GLUT1, and claudin-1 is expressed by the large majority of perineuriomas. CD34 is expressed in a subset. These are S-100-protein/SOX10, MUC4, and STAT6-negative.

• 8.

  Low-grade fibromyxoid sarcoma (LGFMS) and sclerosing epithelioid fibrosarcoma (SEF) : MUC4 is a fairly sensitive and specific marker of LGFMS/SEF, which also expresses EMA in some cases. The finding of MUC4 by IHC correlates with the presence of an underlying FUS or, less often, EWSR1 rearrangement. EMA expression is seen in up to half of cases. However, keratin, along with S-100 protein, CD34, desmin, and CD45, is not present. DOG1 expression in LGFMS/SEF can be problematic if performed to exclude GIST in the context of an intra-abdominal tumor.

• 9.

  Spindle cell rhabdomyosarcoma : Because a substantial proportion will express only limited desmin and myogenin, it is important to assess MYOD1 expression if spindle cell rhabdomyosarcoma is considered.

• 10.

  Leiomyoma and leiomyosarcoma : High-molecular-weight or “heavy” caldesmon expression appears to be a somewhat more sensitive, and certainly more specific, marker of smooth muscle differentiation than are SMA or desmin. Thus, there may be utility in assessing h-caldesmon in undifferentiated spindle cell sarcomas or myogenic sarcomas lacking rhabdomyoblastic differentiation.

• 11.

  Inflammatory myofibroblastic tumor : In addition to smooth muscle actin, muscle-specific actin (clone HHF35), and calponin, which manifest the myofibroblastic nature of the neoplastic cell, ALK overexpression will be present in slightly more than half of inflammatory myofibroblastic tumors ( Fig. 2.12 ). Keratin and desmin can both be expressed.

  

• 12.

  Kaposi sarcoma : Like other vascular endothelial neoplasms, Kaposi sarcoma will express CD31, ERG, and FLI1. Unique to Kaposi sarcoma among vascular tumors is the strong and diffuse expression of nuclear-localized HHV8 LANA (latency-associated nuclear antigen) ( Fig. 2.13 ).

  

Poorly Differentiated Epithelioid Tumors

The differential diagnosis of poorly differentiated epithelioid tumors includes carcinoma, melanoma, lymphoma (i.e., anaplastic large cell lymphoma), and epithelioid soft tissue tumors, such as epithelioid sarcoma, myoepithelioma, and angiosarcoma. A screening panel for this differential diagnosis is presented in Table 2.5 . This initial screening panel can make a specific diagnosis of melanoma, lymphoma, or anaplastic large cell lymphoma, but generally it is not able to discriminate carcinoma from epithelioid sarcoma or epithelioid angiosarcoma (EAS). In the axial skeleton, this differential diagnosis should also include chordoma. These tumors can be reliably distinguished with the additional panel of antibodies listed in Table 2.5 .

Additional IHC workup depending on the suspected diagnosis:

• 1.

  Carcinoma: With rare exceptions, SMARCB1 (INI1) expression is retained in carcinomas, in contrast to the loss observed in more than 90% of epithelioid sarcomas. Epithelial lineage markers (e.g., TTF-1, CDX-2, p40, GATA3, PAX8, NKX3.1) are also of great value, depending on the clinical scenario.

• 2.

  Melanoma and epithelioid malignant peripheral nerve sheath tumor (E-MPNST): Both are typically diffusely S-100 protein and SOX10 positive. E-MPNST does not express melanocytic markers such as HMB45 or Melan-A. A subset of E-MPNST cases exhibit SMARCB1 loss.

• 3.

  Lymphoma: In addition to CD30, anaplastic lymphoma kinase-1 (ALK-1) protein is expressed by many, but not all, cases of anaplastic large cell lymphoma (ALCL).

• 4.

  Chordoma: Brachyury is a sensitive and specific marker of the notochordal lineage and should be included in the workup of epithelioid tumors of the axial skeleton ( Fig. 2.14 ).

  

• 5.

  Myoepithelioma: Co-expression of keratin, SOX10/ S-100 protein, SMA, p40/p63, and glial fibrillary acidic protein is diagnostic of myoepithelioma, although any individual marker may be negative in a given tumor.

• 6.

  Epithelioid sarcoma: Co-expression of CD34 is seen in 50% of epithelioid sarcomas, but not in carcinomas. SMARCB1 expression is lost in more than 90% of epithelioid sarcomas ( Fig. 2.15 ). Rare proximal-type epithelioid sarcomas exhibit loss of other SWI/SNF proteins instead, such as SMARCA4.

  

• 7.

  Epithelioid angiosarcoma (EAS): Expression of ERG, FLI1, and CD31 may be helpful in the distinction of EAS from carcinoma and epithelioid sarcoma. The diagnosis of EAS requires a high degree of suspicion given that a subset of cases will express keratin, mimicking poorly differentiated carcinoma ( Fig. 2.16 ). Unlike many carcinomas and epithelioid sarcoma, EAS typically does not express high-molecular-weight keratins.

  

• 8.

  Epithelioid hemangioendothelioma (EHE) : More than 80% of EHEs will express diffuse nuclear-localized CAMTA1 as a manifestation of the WWTR1-CAMTA1 fusion oncoprotein ( Fig. 2.17 ). EHE is also positive for vascular endothelial markers CD31, CD34, FLI1, and ERG. Keratin and EMA expression is extremely rare and often limited in extent.

  

Pleomorphic Spindle Cell Tumors

It is critical to realize that histologic findings supersede IHC for many pleomorphic malignant neoplasms in soft tissue and bone. For example, the finding of pleomorphic lipoblasts, osteoid, or low-grade chondrosarcoma can help to establish the diagnoses of pleomorphic liposarcoma, osteosarcoma, or dedifferentiated chondrosarcoma, respectively, regardless of the immunophenotype of the pleomorphic tumor cells. In addition, it can be argued that the most clinically relevant use of IHC in the differential diagnosis of a pleomorphic spindle cell tumor in soft tissue or bone is to exclude the possibility of a non-mesenchymal neoplasm, such as metastatic carcinoma or melanoma. There is, however, evidence that the prognosis for pleomorphic sarcomas showing myogenous differentiation is worse than that of other pleomorphic sarcomas; therefore, an attempt should be made to identify such tumors with careful histologic examination and ancillary IHC for muscle markers. Table 2.6 presents an IHC panel for the evaluation of pleomorphic spindle cell tumors.

Additional IHC workup depending on the suspected diagnosis:

• 1.

  Carcinoma: It is often useful to employ broad-spectrum and high-molecular-weight keratins to fully exclude sarcomatoid carcinoma when clinical suspicion is high. Markers of specific primary epithelial origins (e.g., TTF1, CDX2, GATA3, NKX3.1, PAX8) can also support the diagnosis of carcinoma.

• 2.

  Melanoma: The expression of SOX10/S-100 protein identifies melanoma in an initial IHC panel, but does not per se distinguish melanoma from MPNST. Melanocytic markers (e.g., HMB-45, Melan-A) are expressed in melanoma more specifically. In addition, melanoma more often exhibits diffuse/strong SOX10/S-100 protein expression compared with the heterogeneous, sometimes focal, expression observed in most cases of MPNST.

• 3.

  ALCL: CD30 is included in the primary panel of markers for pleomorphic tumors in order to screen for ALCL. ALK is also a useful marker of the subset of cases bearing ALK translocations. Most cases express CD2, CD4, or CD5. CD45 is commonly expressed as well. The majority of cases are positive for EMA.

• 4.

  Leiomyosarcoma: High-molecular-weight or “heavy” caldesmon is a sensitive and specific marker of smooth muscle differentiation that can be used in addition to desmin. Smooth muscle actin expression alone cannot be used to support classification as leiomyosarcoma, given evidence of expression in many spindle cell tumors. In a desmin-expressing tumor, it is important to exclude rhabdomyosarcoma by examining markers of rhabdomyoblastic differentiation, such as myogenin and MyoD1 ( Fig. 2.18 A–B).

  

• 5.

  Pleomorphic rhabdomyosarcoma: Identification of the expression of myogenic regulatory transcription factors myogenin or MyoD1 is required for the diagnosis of rhabdomyosarcoma in the context of a pleomorphic malignancy. Diffuse expression of desmin is typically seen. The possibility of heterologous differentiation must be considered, as is observed with some frequency in malignant peripheral nerve sheath tumor and dedifferentiated liposarcoma ( Fig. 2.19 ).

  

• 6.

  Dedifferentiated liposarcoma : Diffuse, nuclear-localized MDM2 and CDK4 expression is a useful, but nonspecific feature. While the absence of MDM2/CDK4 expression has excellent negative predictive value, it may be appropriate to confirm MDM2 gene amplification in the context of a pleomorphic spindle cell neoplasm. Intimal sarcoma should also be included in the differential diagnosis of a pleomorphic sarcoma with MDM2 amplification; the distinction of intimal sarcoma from dedifferentiated liposarcoma, in the absence of a well-differentiated liposarcoma component, requires correlation with the clinical and imaging findings of large vessel involvement.

• 7.

  Undifferentiated pleomorphic sarcoma: Limited SMA expression, indicative of myofibroblastic differentiation, may occasionally be observed. Rare cases exhibit focal anomalous keratin expression, while a minority of cases may have focal desmin expression in the absence of SMA, caldesmon, or myogenin/MyoD1 expression; such cases are probably best not considered evidence of myogenous differentiation. The presence or absence of CD68 expression is not helpful in the assessment of sarcomas in particular, because this is a highly nonspecific marker ( Fig. 2.18 C–D); CD163 is a more specific marker of true histiocytic differentiation.