HER-2

Characteristics

HER-2 (also called HER-2/neu, c-erbB2, ERBB2 or neu) is a transmembrane receptor tyrosine kinase, which was originally discovered from a rat neuroblastoma cell line (named “neu”) more than 20 years ago. At the same time another research group identified the same gene and found its high homology with human epidermal growth factor receptor (therefore named human epidermal growth factor receptor 2, “HER-2”). The consensus name HER-2 is widely used, as well as the acronym of its gene, ERBB2.

HER-2 is a proto-oncogene, i.e. its activation causes malignant transformation and increases the malignant potential (cell proliferation, invasiveness etc.) of the cells. In human cancers HER-2 is activated via gene amplification, which is a genomic mutation where a small fragment at chromosome band 17q12-q21 is multiplied in a cell up to 50-100 folds. HER-2 is considered as the target oncogene driving the amplification, although neighbouring genes (such as topoisomerase II alpha) may occasionally be co-amplified together with HER-2.

Amplification of HER-2 gene invariably leads to over-expression of its protein product. The magnitude of over- expression is usually 10-100 folds or even more. Over-expressed HER-2 protein disturbs the HER-receptor family signalling networks, i.e. signalling mediated via EGFR receptor, HER-3 and HER-4. HER-2 has no ligand of its own and it acts by forming heterodimers with EGFR, HER-3 and HER-4 upon binding of their ligands. In tumours with gene amplification, vastly over-expressed HER-2 forms auto active HER2-HER2 homodimers, which is considered as the major oncogenic activation mechanism.

A breakthrough finding in cancer biology was made in early 1990’s, when antibodies to the extracellular domain of HER-2 were found to inhibit growth of HER-2 positive cell lines and tumour xenografts. These findings gave rise to test the most promising growth inhibitory antibody as an anti-cancer drug in humans. The clinical efficacy of the monoclonal antibody drug named trastuzumab (“Herceptin”) has now been shown in several randomised clinical studies of metastatic breast cancer. Thus, Herceptin is today standard therapy in HER-2 positive metastatic breast cancer. Most recent clinical studies show promising interim results also when used as adjuvant therapy given after primary breast cancer operation (ASCO 2005 meeting reports).

For these reasons, testing for HER-2 amplification and/or over-expression is currently considered as routine practise in clinical pathology laboratories.

Neoplasms

In tumours, HER-2 is over-expressed in 15-25% of primary breast cancers. Metastases usually have the same amplification status as the primary tumours. HER-2 amplification and over-expression are typical features of hormone receptor negative, rapidly growing histologic grade 2-3 tumours. Of the histologic types, Paget’s disease is almost invariably HER-2 positive, whereas only a small minority of lobular and tubular carcinomas shows HER-2 amplification.

HER-2 amplification and over-expression can also be found in intestinal type gastric and gastroesophageal carcinomas, ovarian carcinomas, high grade endometrial carcinomas and some salivary duct tumours. Low-level copy number increases have been found also in rare cases of lung tumours.

Application

As mentioned above, demonstration of HER-2 amplification and over-expression in tumour tissue is required to define eligibility of the patient to trastuzumab (Herceptin) therapy. In most studies, immunohistochemical staining of HER-2 protein is scored qualitatively as 0/1+/2+/3+ (Fig. 1; see table below). Provided that the system is carefully calibrated, scores 0 or 1+ are almost never (i.e., <1-2%) associated with HER-2 amplification and/or therapeutic response, while score 3+ is associated with gene amplification in 90-100% of cases and with the highest likelihood of Herceptin response. The problematic category is IHC score 2+, with less than one third displaying gene amplification. True responses to Herceptin occur in this category of 2+ positive tumours. Thus, IHC score 2+ is considered to be an indication for a confirmatory gene based test (FISH or CISH).

Because of its central importance in breast cancer therapy selection, standardization of HER-2 IHC assays and slide interpretation are of outmost clinical and economical importance. As shown in the context of clinical trials, analytical variability in Her-2 testing is considerable. It can be minimised by the use of standardised tests, and by interlaboratory quality control assessments.

Visualization

The trastuzumab antibody itself (and its original mAb clone 4D5) are not suitable for use in IHC. Currently the polyclonal antibody used in the HercepTest™ (Dako), and the PATHWAY® / CONFIRM™ rmAb clone 4B5 (Ventana) are most frequently used, and the systems approved by FDA. They are used in paraffin sections with high temperature antigen retrieval. Among Abs used in-house systems, mAb clones CB11 and particularly SP3 are promising. Although the HER-2 protein is known to be localised in the cell membrane, cytoplasmic staining also occurs, especially in tumours with high level protein over-expression.

According to the NordiQC experience, the kits Herceptest (Dako) and Pathway (Ventana) approved by FDA in general performs much better than in house systems. However, a major problem is the correct interpretation of the staining result.

Recently it has been shown that excessive heating of paraffin sections prior to deparaffination and epitope retrieval reduces the staining signal. Thus, baking of slides should not exceed 60 C for one hour.

Scoring of HER-2 immunohistochemistry:
The most widely used scoring method for HER-2 IHC in breast cancer is semiquantitative and based of four classes (0/1+/2+/3+, Fig. 1). The scoring can be verbalised as follows:

Control tissue: Positive and negative controls are essential and should be included in every staining batch. The widely used commercially available HER-2 staining kit “Herceptest” includes control slides, which contain formalin-fixed and paraffin-embedded cells from HER-2 positive and negative breast cancer cell cultures. 

The most suitable human tumour control is Paget’s disease, which almost invariably shows gene amplification and 3+ protein over-expression. Ductal tumours tested positive for gene amplification by FISH or CISH can be used as well.

Assessments

Selected references

Ellis CM, Dyson MJ, Stephenson TJ, Maltby EL. HER2 amplification status in breast cancer: a comparison between immunohistochemical staining and fluorescence in situ hybridisation using manual and automated quantitative image analysis scoring techniques. J Clin Pathol. 2005 Jul;58(7):710-4.

Gancberg D, Jarvinen T, di Leo A, Rouas G, Cardoso F, Paesmans M, Verhest A, Piccart MJ, Isola J, Larsimont D. Evaluation of HER-2/NEU protein expression in breast cancer by immunohistochemistry: an interlaboratory study assessing the reproducibility of HER-2/NEU testing. Breast Cancer Res Treat. 2002 Jul;74(2):113-20.

Isola J, Tanner M, Forsyth A, Cooke TG, Watters AD, Bartlett JM. Interlaboratory comparison of HER-2 oncogene amplification as detected by chromogenic and fluorescence in situ hybridization. Clin Cancer Res. 2004 Jul 15;10(14):4793-8.

Joensuu H, Isola J, Lundin M, Salminen T, Holli K, Kataja V, Pylkkanen L, Turpeenniemi-Hujanen T, von Smitten K, Lundin J. Amplification of erbB2 and erbB2 expression are superior to estrogen receptor status as risk factors for distant recurrence in pT1N0M0 breast cancer: a nationwide population-based study. Clin Cancer Res. 2003 Mar;9(3):923-30.

Todorovic-Rakovic N, Jovanovic D, Neskovic-Konstantinovic Z, Nikolic-Vukosavljevic D. Comparison between immunohistochemistry and chromogenic in situ hybridization in assessing HER-2 status in breast cancer. Pathol Int. 2005 Jun;55(6):318-23.