Immunohistochemistry and Immunofluorescence

The development of antigen-retrieval techniques and the increasing range of monoclonal and polyclonal antibodies have been accompaniedby widespread application of immunohistochemistry to routinely fixed paraffin wax-embedded material. Adequate, controlled fixation is still crucial in obtaining the best results from immunohistochemistry, but there is little indication for the use of fixatives other than formalin outside a research setting.

Heat-mediated antigen-retrieval techniques (HMAR) are particularly important for the detection of nuclear antigens such as ER (oestrogen receptor) and Ki67, low-density surface antigens, e.g., CD5 and other surface antigens such as CD20. Both microwaving and pressure cooking have been used for HMAR, with citrate or EDTA buffer. HMAR avoids the risk of over digestion and loss of morphology that may accompany pretreatments with proteolytic enzymes but can lead to loss of adherence of the section to the slide in a significant fraction of cases. This technical problem may be circumvented by the use of slides pretreated with an adhesive material such as APES.

Other technical advances in immunohistochemistry include the development of highly sensitive detection systems and the increasing use of automated immunostainers. New, polymer-based detection systems (e.g., Envision, Dako) may give superior sensitivity to existing methods but many laboratories will continue to use more established ABC techniques (e.g., Duet, Dako) which appear adequate for routine use. The use of immunohistochemistry has been reported to have risen 600% over the last few years in some laboratories and automation is helpful in managing this rising workload. It may also offer more reproducible staining with less batch-to-batch variation, particularly important with stains for predictive markers such as ER or HER2, which are assessed in a semi-quantitative fashion.

The use of immunohistochemistry continues to increase rapidly. It plays a crucial role in tumour diagnosis in particular and especially in the differential diagnosis of tumour types with similar morphological appearances. For example, in the diagnosis of small round blue cell tumours and soft-tissue lesions and in the differentiation of malignant mesothelioma and metastatic adenocarcinoma. The accurate classification of lymphoma subtypes is crucially dependent on the use of appropriate antibody panels and interpretation of the results; indeed, the latest classifications of lymphoma fully incorporate both immunophenotyping and genotyping results. Panels of immunohistochemical stains may also be used to help determine the likely origin of metastatic carcinoma where the primary remains occult (Table 45.1).

The increasing use of diagnostic immunohistochemistry can cause problems. Anomalous or unexpected patterns of staining may be potentially confusing or indeed misleading and result in an erroneous diagnosis, unless the pathologist is aware of such a possibility. It is generally better to use panels of markers rather than to rely on one or two isolated stains. The use of very extended antibody panels with little consideration given to a differential morphological diagnosis is likely to confuse and be very expensive. Standardised panels are easier to order and to organise on automated immunostainers and may assist in the calculation of costs. Technical pitfalls may also trap the unwary and the results of immunohistochemistry should always be considered critically. Judicious use of positive and negative controls, and correlation with morphological, clinical and radiological findings are essential.

Immunohistochemistry has also been used to detect prognostic and predictive biomarkers in malignant tumours and in pre-malignant conditions. The use of prognostic factors is mainly a research activity with the exception of ER/PR receptors in breast cancer and proliferation indices (Ki67) in lesions such as non-Hodgkin's lymphoma, gastrointestinal stromal tumours and haemangiopericytoma. In multivariate analysis, many new "prognostic biomarkers" do not show effects on prognosis independent of histological grade or tumour stage.

Table 45.1. Immunoprofile of cancer types

System

Tumour/Condition

Marker Panel

Head and Neck

Salivary Gland Tumours

Calponin, S100, SmActin, AE1/AE3

Thyroid Tumours

Thyroglobulin, Calcitonin, CEA, TTF1

Gastrointestinal

Oesophageal Tumours

AE1/AE3, CAM5.2, CK7, CK20

Barrett's Oesophagus

Villin, Ki67, cyclinDI, p53

Gastric and Small bowel

CK7, CK20, CEA

Colorectal Tumours

CK7, CK20, CEA

Hepatocellular Carcinoma

aFetoprotein, HEPAR1, CEA polyclonal

Pancreatico-biliary Carcinoma

CK7, CK20, CEA, CA19.9, CA125

Gastrointestinal Stromal Tumours

CD117, CD34, Sm Actin, Desmin, S100, Ki67, vimentin

Neuroendocrine Carcinoids

Chromogranin, CD56, gastrin, insulin, glucagon

Respiratory

Small Cell Carcinoma

CD45, CAM5.2, CD56, chromogranin

Non-small Cell Carcinoma

TTF1, PE10, EP4

Malignant Mesothelioma

Calretinin, Thrombomodulin, HBME1, CK5/6, WT1, EMA, EP4, CEA

Salivary Gland-type Tumours

Calponin, S100, SmActin, AE1/AE3

Gynaecological

Ovarian Carcinoma

CK7, CK20, CEA, CA125, WT1

Sex Cord Stromal

Calretinin, Vimentin, AE1/AE3, Inhibin, EMA

Uterus, Mesenchymal

CD10, Desmin, Sm actin

Endometrial Carcinoma

CK7, CK20, CD10, ER, p53

Cervix-GGIN

BCL-2, P16, Ki67

Cervical Adenocarcinoma

CEA, vimentin and ER (both - )

Genitourinary

Renal Carcinoma

CK7,CK20, EMA, vimentin

Prostate

PSA, PSAP, 34ßE12

Transitional Carcinoma

CK7, CK20, p53

Testicular Tumours

PLAP, «Fetoprotein, CAM5.2, EMA, CD30

Breast

Breast Carcinoma

ER, PR, HER2/neu, SmActin, CK5/6, CK14, CK8/18, E-cadherin

Soft Tissue

Spindle Cell Sarcomas

Vimentin, CD34, SmActin, desmin, CAM5.2, AE1/AE3, S100

Small round blue cell tumours

CD 45, S100, CD99, desmin, WT1, NB84, vimentin, SmActin, CD56

Skin

Melanoma

S100, MelanA, HMB45

Haematopoetic

Lymphomas

CD45, CD43, CD5, CD10, CD20, CD21, CD23, CD3, CD15, CD30, CD56, CD57, ALK, cyclinDI, MIB1, k&\, BCL-2, BCL-6, BCL-10, LMP-1, EBER, granzyme B, myeloperoxidase

These panels can be adapted and modified to suit individual cases and preferences. It is not really possible to summarise this rapidly expanding and complex area with a simple table. Queries about immunohistochemical staining may be answered by logging onto www.immunoquery.com. Alternatively, a vade mecum, in the form of a help file, may be downloaded from http://ourworld.compuserve.com/homepages/paul.bish/

These panels can be adapted and modified to suit individual cases and preferences. It is not really possible to summarise this rapidly expanding and complex area with a simple table. Queries about immunohistochemical staining may be answered by logging onto www.immunoquery.com. Alternatively, a vade mecum, in the form of a help file, may be downloaded from http://ourworld.compuserve.com/homepages/paul.bish/

A tissue microarray consists of an array of small-calibre core biopsies of tumours (or other tissues) prepared either prospectively from resected tumour specimens or retrospectively from paraffin-embedded tumour tissue. High-density arrays can have hundreds of cores on a single glass slide. This innovation represents an "industrial revolution" for such biomarker investigations and should greatly facilitate large-scale immunohistochemical investigations.

Predictive biomarkers are used to predict the response of a malignancy to either conventional treatments such as chemotherapy and radiotherapy or novel targeted therapies. Examples include CD 20 and Rituximab in certain B cell lymphomas, CD 117 (c-kit) and STI 571 in gastrointestinal stromal tumours and HER2/neu and Trastuzumab in breast cancer. ER/PR and HER2/neu are good examples of biomarkers that are both prognostic and predictive and that have clinical utility in the choice of treatments for patients with breast cancer. The expression levels of such markers in tumours vary both within and between tumours and both technical issues and inter-observer variation may affect the validity and reproducibility of the results.

The increasingly important role of immunohistochemistry and the need for standardisation of assays for predictive markers such as ER has prompted the development of external quality-assurance schemes to ensure acceptable technical standards. Methods have also been developed to improve the reproducibility of scoring, the best examples being the "Histo" and "Quick Score" methods used to score ER expression in breast cancer.

Immunofluorescence continues to be used in many laboratories for the evaluation of renal biopsies and skin biopsies in conditions characterised by the deposition of immune complexes or auto-antibody binding, fluorescence providing high resolution and precise localisation. Immunofluorescence ideally requires frozen sections and specialised fluorescence microscopy equipment. As fluorescent preparations fade, photomicroscopy is needed to provide a permanent record and some laboratories have abandoned this technique for conventional immunoperoxidase.

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