EDITORIAL      277

Molecular Profiling of Tumours by Immunohistochemistry

In recent decades, molecular techniques have defined the genetic profiles of many tumours and contributed significantly to the advancement of clinical management and therapeutic approaches. But, due to lack of facilities, only a few molecular techniques have reached from the bench to the bedside. Immunohistochemistry (IHC) has emerged as a major ‘doable’ tool for pathologists. The application of IHC to detect various antigenic components using specific antibodies has revolutionized diagnostic pathology.1 IHC can be used to define molecular alterations in cancers, which not only confirms the diagnosis of certain tumours, but also helps in their prognostication and therapeutic management, especially in this era of targeted molecular therapy. The greatest benefit of this technique seems to be in countries such as India where molecular laboratories are not viable everywhere. Understanding the concept of ‘molecular histochemistry’ can thus modernize the practice of oncology. Another advantage of IHC is that it can be easily applied to formalin-fixed, paraffin-embedded tissues, which form the backbone of tissue pathology. On the other hand, most molecular techniques require fresh cryopreserved tissue for extraction of good quality DNA, RNA or protein, which is often difficult to obtain. However, the results of IHC need to be interpreted with caution because the level of protein expression may not exactly correlate with the corresponding genetic alterations.
   In the traditional practice of diagnostic pathology, the role of IHC is mainly to localize specific epitopes in tissue sections by either monoclonal or polyclonal antibodies. However, it is indispensable in the diagnosis of poorly differentiated carcinomas (cytokeratin-positive), sarcomas (vimentin-positive) and lymphomas (CD 45-positive).1 In addition, IHC has a specific role in detecting tumour differentiation by identifying specific intermediate filaments. It also helps to identify the source of a tumour in case of metastasis from an unknown primary, e.g. if the origin is from the breast (oestrogen/progesterone receptor-positive), thyroid/lung (thyroid transcription factor-1-positive) or from the gastrointestinal tract (by analysing the panel of CK 7/CK 20 antibodies).1
   Genomic alterations, which are molecular signatures of specific malignancies, can be identified using ‘molecular histochemistry’. Some common genetic alterations in cancer are deletion, mutation and silencing of tumour suppressor genes, tumourspecific translocations and proto-oncogene amplifications. These alterations manifest as loss, gain or abnormal subcellular localization of proteins, which can be studied using IHC and available antibodies.1 This information may contribute immensely to the diagnosis, prognosis and treatment of cancers.

Markers for diagnosis
E-cadherin is a transmembrane glycoprotein that mediates calcium-dependent intercellular adhesion and is specifically involved in epithelial cell-to-cell adhesion in the breast. Loss of E-cadherin expression is the molecular signature of lobular carcinomas of the breast and helps to differentiate them from infiltrating duct carcinomas. E-cadherin alterations can occur either by somatic mutation, allelic loss or methylation of the gene. Molecular techniques to detect these alterations can be costly and cumbersome. Whatever the mechanism of the genetic alteration, the end result is the loss of expression of E-cadherin, which can be easily detected by IHC.2,3
   Rhabdoid tumours are highly malignant neoplasms which occur in children in the kidney, brain and extrarenal soft tissues. Histologically, rhabdoid tumours need to be differentiated from primitive neuroectodermal/embryonal tumours (PNET) because they have a very poor prognosis and are non-responsive to chemotherapeutic regimens used for the management of other malignant small round cell tumours. The genetic hallmark of atypical teratoid/rhabdoid tumours (AT/RTs) is mutation or loss of the INI 1 (hSNF5/SMARCB1) gene locus at chromosome 22q11.2.4 This results in the loss of INI 1 protein expression in almost all AT/RTs. In normal tissue and in most tumours, INI 1 is expressed as a nuclear protein. However, in AT/RTs, there is loss of nuclear expression of INI 1 in tumour cells. Immunohistochemical staining for expression of INI 1 protein is a sensitive and specific marker for the diagnosis of AT/RTs. Hence, it has become mandatory to identify the INI 1 protein as a molecular signature of hSNF5/ INI 1 gene mutation in AT/RTs of the brain or in renal/extrarenal rhabdoid tumours.4
   IHC can distinguish a particular tumour type from a subset of tumours with similar histomorphology by identifying the unique tumour-specific fusion protein formed out of a specific chromosomal translocation. A characteristic recurrent chromosomal translocation—t(11;22)— between EWSR1/FLI-1 genes in Ewing sarcoma (ES) leads to the formation of a fusion protein, where the usual RNA-binding function is replaced by DNA-binding activity of FLI-1, resulting in prolonged transcription factor activity in this tumour.1 Immunohistochemical detection of FLI-1 protein in tumours (normally expressed in endothelial and haematopoietic cells) confirms the diagnosis of ES/PNET.1
   Similarly, IHC for ALK-1 protein in anaplastic large cell lymphoma is an indicator of the translocation t(2;5) (NPH/ALK-1) and helps in diagnosis.5 Also, the characteristic IHC expression of bcl-2 due to t(14;18) seen in synovial sarcoma helps to differentiate it from other soft tissue sarcomas.1
   Molecular histochemistry is essential in classifying lymphomas according to the current WHO lymphoma classification.5 For example, t(11;14) is seen in mantle cell, hairy cell and in some splenic marginal zone lymphomas. This translocation is reflected by IHC expression of bcl-1 in these tumours. Similarly, bcl-2 expression represents t(14;18) in follicular lymphomas and helps to differentiate these from reactive follicles.5
   The tumour suppressor gene p53 is mutated in a large number of cancers. The normal p53 gene codes for the wild-type protein, which has a short half-life, and hence cannot be detected by IHC. However, mutation of the p53 gene leads to expression of a mutant protein which, by virtue of its stability and longer half-life, can be easily detected by IHC in tissue sections. Hence, IHC for p53 protein can be used as a surrogate marker for detection of p53 mutation in tumours.6 Astrocytic tumours of grades II to IV show an immunopositivity for p53, while grade I pilocytic astrocytomas are immunonegative. This can be used to differentiate grade I from grade II tumours, which differ in biological behaviour and management; radiotherapy is a must for grade II tumours following surgical resection.7–9 The 2 types of glioblastomas differ in their molecular pathways of genesis—primary glioblastomas are associated with epidermal growth factor receptor (EGFR) amplification and secondary glioblastomas with p53 mutations. Clinically, the differentiation is important because secondary glioblastomas occur more commonly in young patients and have a better prognosis while primary glioblastomas arise in the elderly and have a poor outcome. IHC can detect p53 and EGFR protein expression and aid in this differentiation.7–9

Markers of prognosis
IHC can identify mutations or alterations in methylation of the promoter sequence of mismatch repair genes in colorectal adenocarcinomas in Lynch syndrome and in 10%–15% of sporadic adenocarcinomas. These alterations result in loss of expression of the specific mismatch repair genes (MMRs)—hMSH1, hMSH2, hMLH1, hPSM1 or hPSM2. The absence of a properly functioning mismatch repair mechanism may result in microsatellite instability (MSI). Loss of MMR activity corresponds to the loss of protein expression (as detected by IHC). Hence, this phenotype can be detected (in 93% of cases) without resorting to complicated molecular techniques.10 Even though molecular MSI testing is the gold standard for assessing tumour DNA mismatch repair competency, studies show that IHC for detecting hMLH1 and hMSH2 is a sensitive, rapid and cost-effective method. Adjuvant chemotherapy improves overall 5-year survival in patients with colorectal microsatellite-stable tumours or tumours exhibiting low-frequency microsatellite instability. However, there is no benefit of adjuvant chemotherapy in those with high-frequency microsatellite instability.11 The detection of loss of MMRs is being increasingly used in routine clinical practice.
   Overexpression of p53 has been detected in 91% of colorectal carcinoma patients in northern India. Routine assessment of this marker is helpful as it is indicative of a poor postoperative course in colorectal carcinomas.6
   Adenoma polyposis coli (APC) gene mutation has been shown to be one of the earliest events in the progression of familial adenomatous polyposis and Gardner syndrome. The APC gene acts through interaction with β-catenin.9 Mutation of the APC gene can be identified early in colorectal carcinogenesis by using monoclonal antibodies against β-catenin in colon biopsies. Increased and ectopic expression of β-catenin in colorectal carcinoma has been associated with a reduction of E-cadherin
  TABLE I. Immunohistochemistry as a window to molecular alterations

CHITRA SARKAR
PRASENJIT DAS
RANAJOY GHOSH
Department of Pathology
All India Institute of Medical Sciences
New Delhi
sarkar.chitra@gmail.com