Background Immunohistochemistry (IHC) plays a central role in the histopathological classification of diseases, including cancer. More recently, the importance of immunohistochemical staining is increasing. IHC usage in diagnostics is invaluable; however, the genetic and therapeutic significance of biomarker immunostaining has become equally relevant.
Content In this article, we would like to analyse the three distinct roles of IHC and review their individual impacts on modern diagnostic pathology: (1) diagnostic IHC; (2) genetic IHC and (3) therapeutic IHC.
Summary Thus, we will characterise the different analytical processes that are required in the three approaches to IHC usage stated above, as well as the clinical significance and overall importance in patient management. This will allow us to hypothesise on the most appropriate laboratory environment and detection methods for the future.
- Molecular Oncology
- Tumour Biology
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Since the direct method for immunohistochemistry (IHC) described by Coons et al in 19411 and, more importantly, the use of indirect methods of amplification, such as the peroxidase-antiperoxidase complex in 1979,2 and the avidin-biotin complex method soon after, IHC has played a pivotal role in the histopathological taxonomy of diseases. Indeed, the detection of specific ‘patterns of immunohistochemical expression’ and its likely association with a given diagnostic label has become a standard component of a pathologist's diagnostic acumen and, in many ways, dictates much of the ongoing research in diagnostic histopathology. In the last decades, however, new uses have been postulated for some of these immunohistochemical biomarkers. For instance, the presence (or absence) of expression of a protein may be perceived as evidence of an inherited disease. Furthermore, in the era of personalised medicine, immunohistochemical expression may dictate a certain therapeutic intervention. The genetic and therapeutic uses of IHC, in addition to its well-established diagnostic role, may change the paradigm of antibody evaluation and the overall approach to IHC. Here, we would like to review the three main roles of IHC in clinical pathology (diagnostic, genetic and therapeutic) each of which is essential to stratify patients and to tailor effective therapy.
The diagnostic value of IHC
The application of IHC can provide valuable diagnostic information in the initial determination of malignancy. Specific antibodies directed against a range of generic tumour markers determine their presence/absence in disease tissue versus normal, as well as the specific diagnostic label for a given set of histological changes. Although the IHC analysis is often interpretative and, thus, carries a reduced specificity, the identification of immunoreactivity patterns can confirm tumour type. Furthermore, when utilised in combinations (so-called immunopanels, discussed later) and interpreted in the correct clinical context, the value of such markers is greatly enhanced.
The classic example of generic tumour markers is presented by the Cytokeratins (CKs). These are epithelial markers useful for confirming the epithelial nature of tumours and, hence, designation as carcinoma.3 ,4 Normally, the expression of CKs varies with epithelial cell type, extent of differentiation and tissue development;5 however, during malignant transformation, the CK patterns and integrity are maintained, a property that enables their use as tumour markers.6 ,7 Unfortunately, very few CK markers are organ-specific which limits their utility. This non-specificity is a feature of many commonly used antibodies. Indeed, specificity appears to decrease over time as antibodies are applied to and studied in multiple tissue types. S100 protein is highly immunoreactive in benign peripheral nerve sheath tumours,8 ,9 melanoma10 and clear cell sarcoma11 when analysed by IHC. However, widespread expression of S100 limits its diagnostic value beyond that previously stated. Smooth Muscle Actin IHC, which identifies tissue of smooth muscle or myofibroblastic origin, can be positive in many non-muscular/myofibroblastic lesions. Additionally, reactive myofibroblasts are present in several tumour types from various origins.4 CD34 is immunoreactive in many soft tissue tumours, including vascular, solitary fibrous, gastrointestinal stromal, peripheral nerve sheath, epithelioid sarcomas and in a subpopulation of dermal dendritic cells. Due to its non-specificity, this marker is usually complemented by additional markers, such as CD31 for the diagnosis of vascular tumours and CD117 (KIT) or DOG1 for gastrointestinal tumours.4 ,12 Prostate Specific Antigen (PSA) is secreted exclusively from the luminal cells of the glandular epithelial of the prostate gland,13 ,14 and is expressed uniformly in normal prostate in the secretory epithelial cells of prostatic acini when analysed by IHC. The use of PSA to aid diagnosis requires knowledge of the clinical context as well as an appreciation that staining intensity can be weak or absent in poorly differentiated prostatic tumours. This sensitivity varies across monoclonal and polyclonal PSA subtypes but may be increased by additional prostatic markers such as Prostatic acid phosphatase (PSAP). Interestingly, it would appear that in the context of diagnostic IHC, the results always provide a diagnostic likelihood but not a diagnostic certainty, hence, the interpretative approach and the need to consider morphology and wider clinical context.
Due to the mentioned lack of specificity of these single generic tumour markers, diagnostic immunopanels comprising several immunohistochemical stains are employed. Such an approach increases diagnostic accuracy and strengthens single biomarker evaluation. Several examples of diagnostic immunopanels exist. The lymphoma panel is broadly based on morphologic differential diagnosis, and derived from knowledge of lymphocyte development and anatomic compartmentalisation within the lymph node: expression of markers associated with specific stages of lymphocyte development and the immunoarchitectural features facilitate diagnosis. The precise immunopanel varies with the initial morphological analysis and likely differential diagnosis. However, it usually includes analysis of CD20, PAX5 (B cell) and CD3, CD4, CD5, CD8 (T cell) expression. Anatomic architectural alterations are also evaluated: BCL-2, CD10 (follicular patterns) and other markers include CD45, CD23, cyclinD1, CD15 and CD30. Additional markers can identify subgroups of lymphoma.15 ,16 For example, BCL-2 and CD10 characterise the germinal-centre phenotype, typified by follicular lymphoma while post-germinal-centre lymphomas are in the plasma cell pathway and correspondingly express CD138 and MUM1. Although beneficial as a tumour diagnostic, this panel-based approach holds limited genetic or therapeutic value.
The genetic value of IHC
In this particular case, the gain or loss of protein expression detected by an IHC becomes a surrogate of an inherited mutation. Many examples exist whereby the mutational status of certain biomarkers dictates the overexpression/diminished expression of the resultant proteins. Furthermore, IHC analysis of this nature can account for the genetic variability of individuals within the same population, that is, the same cancer type.
Mismatch repair (MMR) gene mutations are the most characterised forms of genetic instability in colorectal cancer (CRC) and hereditary non-polyposis colorectal cancer (HNPCC)/Lynch syndrome. Microsatellite instability (MSI) is a hallmark of HNPCC.17 The persistence of mismatch mutations as a result of defective MMR proteins and enhanced MSI predisposes individuals to HNPCC and CRC.18 HNPCC sufferers inherit one germline mutation in an MMR gene. In this instance, the complete loss or patchy/weak expression of the MMR IHC (MSH2, MSH6 and MLH1, PMS2) has important clinical implications indicating either absent MMR protein, expression of a truncated protein, or loss of the epitope recognised by the antibody due to mutation. It is noteworthy that a small proportion of HNPCC-related tumours do not exhibit abnormal MMR protein expression by IHC, even though the function of the MMR system is defective.19 Indeed, the decision of what should be analysed first (MMR IHC or MSI status) in the overall diagnosis of HNPCC and its cost-effectiveness is an old debate in diagnostic laboratories,20 and the choice is usually dictated by the availability of one of those methods in non-integrated laboratory environments.
The therapeutic value of IHC
The therapeutic information gained from specific biomarker expression tissue studies reaffirms the multifaceted role of IHC in cancer. Assessment of these biomarkers can be directly aligned with specific treatment options for individuals as well as providing important prognostic and predictive information. Relying on near-to-absolute quantification, IHC scores can inform likelihood of response to targeted treatment.
The following examples address the clinical relevance of molecular profiling and immunohistochemical analysis in directing effective tailored targeted therapy. The ultimate goal is translation of research into molecular diagnostics for the clinical use in oncology.
Oestrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 (Her2)
Antibody-defined markers in breast cancer can be employed as prognostic or predictive indicators. The advent of personalised medicine and the inclusion of immunohistochemical molecular profiling to direct specific treatment occurred in the 1970s following the development of the antioestrogen agent, tamoxifen, for breast cancer patients. Oestrogen receptor (ER) and progesterone receptor (PR) are weak prognostic markers of clinical outcome,21 but are strong predictors of therapeutic response. The expression levels of both receptors are reported simultaneously, and the latter is associated with disease-free and overall survival. Patients with ER+ve/PR+ve tumours have a better prognosis than patients possessing ER+ve/PR−ve tumours, who in turn have a more favourable prognosis than patients with ER−ve/PR−ve breast tumours.22 However, ER and PR are strong predictive markers of response to endocrine therapy, such as tamoxifen.23 In particular, the test for ER status has become an important stratification factor to identify patients who would benefit from antioestrogen treatment: ER+ve breast cancer patients are selected for tamoxifen treatment, whereas ER−ve patients do not benefit and require additional therapeutic strategies.24
Determination of Her2 status in breast cancer has emerged as a third important prognostic and predictive marker. Her2 protein overexpression, and/or gene amplification, is an independent prognostic indicator of clinical outcome in breast cancer patients,25 ,26 and Her2 status predicts sensitivity to various anticancer regimens, for example, overexpression of the protein confers resistance to tamoxifen-based therapies in the setting of ER+ve breast cancers.27 Importantly, the humanised monoclonal antibody, trastuzumab (Herceptin), specifically targets Her2. Immunohistochemical analysis of Her2 is an important stratification measure to identify patients who harbour Her2+ve tumours and are, therefore, eligible for trastuzumab treatment. Her2+ve patients receiving trastuzumab have demonstrated improved response rates and survival.28 ,29 Trastuzumab is one of the first successful therapies that has been custom designed alongside a tumour-associated molecule of interest, paving the way for future personalised medicine strategies. Indeed, trastuzumab can now be utilised in the context of gastric cancer too.30 As a whole, Her2 testing has become the best possible paradigm of the strength of therapeutic pathology in tissues, and also a reminder of the controversy of where and by whom should Her2 testing be performed to help our patients better.31
Non-small cell lung cancer
Endothelial growth factor receptor (EGFR) mutations resulting in the downstream activation of proliferative and cell survival signals in tumour cells are central to the biology of specific cancers, including non-small cell lung cancer (NSCLC).32 Somatic mutations within the tyrosine kinase (TK) domain of the EGFR occur in approximately 20% of NSCLC cases33 leading to overexpression of EGFR protein. As opposed to their presence in these well-differentiated adenocarcinomas, EGFR mutations are virtually absent in other lung cancer subtypes (except for adenosquamous carcinoma), forming a distinct clinically favourable biological subset.34 ,35 Importantly, activating mutations of the EGFR are the most reliable predictors of response to EGFR TK inhibitors (TKIs) such as erlotinib and gefitinib.36 NSCLC tumours that harbour mutations in the EGFR gene exhibit enhanced sensitivity to the anticancer effects of TKIs. Molecular testing and stratification according to EGFR mutational status is standard-of-practice in these patient populations. Immunohistochemical analysis of EGFR protein expression has provided useful molecular information for the diagnostic and therapeutic management of NSCLC patients. EGFR IHC using antibodies that react only with the mutant product of the gene alteration can determine the mutational status of the EGFR. Common EGFR mutations include exon 19 deletions and the L858R mutation in exon 21. In IHC companion molecular tests, mutation-specific antibodies exhibit increased immunoreactivity in NSCLC tumours harbouring the corresponding EGFR gene mutation.33 Importantly, molecular mutation detection by IHC predicts the sensitivity of tumour cells to TKIs, and is invaluable for the stratification of patients according to responders (EGFR mutation +ve) and non-responders (EGFR mutation –ve) to these agents. Although detection of the EGFR mutation by PCR-based methods still remains the gold standard, there is a role emerging for IHC-based detections as a preliminary screening tool. Figure 1 depicts one of the ‘rational pathways’ for molecular testing of lung adenocarcinoma, and the place in which IHC EGFR mutation analysis may be of use. Importantly, it is very relevant for these IHC-based approaches to ensure minimal false negative results as they represent a screening approach.
CRC and NSCLC
EGFR IHC for cetuximab treatment selection
The anti-EGFR therapy cetuximab (Erbitux) is approved for use in metastatic CRC and was previously restricted to those patients whose tumours express EGFR, assessed immunohistochemically.37 ,38 Recent studies, however, have shown that cetuximab is efficacious in CRC tumours that are immunohistochemically negative for EGFR. Such studies highlight that routine IHC EGFR testing for the purpose of selecting cetuximab treatment is an unsuitable method for identifying eligible CRC patients. In light of these recent observations, patients who could potentially benefit from cetuximab would be excluded based on traditional criteria.39 The mutational status of KRAS, a downstream effector of the EGFR signalling pathway, has emerged as a superior predictive marker of response to cetuximab therapy in CRC, and might explain why some immunohistochemically EGFR+ve tumours do not respond to cetuximab. KRAS is mutated in approximately 35% of CRCs,40 and the presence of a mutation is associated with an absence of response to cetuximab. Regardless of EGFR expression, mutant KRAS is associated with a downstream activation of the Ras/MAPK pathway. Resultant uncontrolled cell proliferation cannot be significantly inhibited by cetuximab that acts upstream of the K-ras protein.41
Although the therapeutic value of EGFR IHC for cetuximab treatment selection in CRC patients has been refuted, the potential use of IHC to identify EGFR+ve lung tumours that might respond to cetuximab remains to be clarified. In the FLEX trial, post-hoc analysis demonstrates that high EGFR expression, determined immunohistochemically, is predictive of response to cetuximab plus chemotherapy versus chemotherapy alone.42 ,43 An ongoing study evaluating the use of cetuximab (with/without chemotherapy) in patients with metastatic or recurrent NSCLC will examine, prospectively, whether high EGFR protein expression can be used as a predictive indicator of treatment response to cetuximab. In the interim, the potential benefit of EGFR protein expression by IHC remains unclear.44
IHC—different uses demand different approaches
The different types of IHC described demand a different scoring approach. Diagnostic IHC is very much interpretative. Indeed, pathologists can choose to ignore the evidence provided by the expression of an antibody if the overall morphological assessment and/or the evidence of other diagnostic tools points in a different direction. Therapeutic IHC, on the other hand, must be as quantitative as possible, calling for a higher degree of laboratory analytical homogeneity, a certain degree of proficiency in the scoring and, when possible, a degree of automation in the quantitation of the biomarker. This may require, in the long term, a different laboratory setup that may be closer to the new ‘Personalised Medicine’ diagnostic laboratories than to the ‘conventional’ pathology laboratories.
IHC, because of its wide availability and affordability, as well as the possibility of relating the expression of a biomarker to a specific cell type or subcellular localisation, is still the preferred way to introduce single novel biomarkers in the routine diagnostic setting for many, including the pharma industry. Only if this is done quantitatively and with a degree of consistency, will IHC be preferred over other molecular approaches that, on paper, are presumed to be more objective.
In some instances, the true diagnostic or therapeutic value of a biomarker will be assigned to its status at the mRNA level, or to the DNA level, or to the protein level, and the correct assay must then be chosen accordingly. In other instances, however, it may be that the most readily available quantitative method is the one that is adopted. It is, once again, in the hands of the pathologist, to create the rigorous laboratory environment to make IHC the biomarker analysis of choice in genetic and therapeutic diagnostics.
Contributors All authors contributed to the concept, draft reviews, final version of the manuscript.
Competing interests None.
Provenance and peer review Not commissioned; internally peer reviewed.
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