The analysis of the HER2 status is a prerequisite for antibody (and kinase-inhibitor) based, target specific therapy in breast cancer. The diagnosis is usually done on protein level by immunohistochemistry (IH) and/or on DNA level by (Fluorescence)-in-situ-Hybridization (ISH, FISH). Strong HER2 overexpression scored 3+ (on a scale ranging from 0 over 1+, 2+ to 3+) by IH is most often based on HER2 gene amplification and either diagnostic finding is basically indicative for Trastuzumab therapy of breast cancer patients. Neither of those diagnostic findings however is in fact predictive for a therapeutic response.

A Meaningful Diagnosis Is the Basic Prerequisite for an Efficient Therapy

Even though (F)ISH based molecular diagnosis has been demonstrated by a number of studies as the more robust procedure over IH [1] it does not warrant therapeutic responsiveness. The lack of any predictive value of HER2 diagnosis has been attributed to a variety of molecular (dis-)functions, albeit non-responsiveness is not necessarily based on the development of molecular/cellular resistance. Insufficient or incomplete molecular diagnosis however can cause uncertain or even wrong therapy indication. Considering the analysis of HER2 a diagnostic approach needs to comply with at least two essential requirements:

• a technical performance characterized by highest accuracy and objectivity
• most relevant mechanisms of HER2 regulation should be addressed.

Against this background it has to keep in mind that not the gene but the HER2 receptor protein represents the therapeutic target and that the HER2 expression density is probably not exclusively determined by the gene copy number. Moreover the cell surface located receptor expression is extensively regulated by posttranslational mechanisms and on the transcriptional level as well [2]. Hence diagnostic uncertainties are supposedly not only due to technical aspects or variabilities produced by individual observers, but in addition are presumed to be subject to cellular/molecular mechansims regulating receptor expression.

Addressing the Regulation of HER2 Receptor Expression by mRNA FISH as a Diagnostic "Add-on"

Extensive HER2 receptor expression on (breast) cancer cells is the conditio-sine-qua-non for a successful Trastuzumab therapy.

A number of molecular mechanisms however dynamically affect the receptor content expressed at a certain time [3]. Posttranslational receptor-downregulation, -degradation, -recycling and -stabilization have been described to modulate HER2 (and other erbB receptor) expression and receptor activation/deactivation (and other mechanisms) are known to affect receptor gene transcription as well [4, 5].
In order to extend well established approaches for HER2 diagnosis (IH, FISH) we previously developed a fluorescence based protein detection in combination with DNA FISH analysis [6]. We demonstrated that intermediate HER2 protein expression (scored 2+) occurs with and w/o her2 gene amplification (and with and w/o polysomy) and all three parameters (i) the protein expression density, (ii) the gene (iii) and centromer copy numbers can be simultaneously visualized (on the same tissue section) using appropriate probes and fluorochromes.
Driven by the aim to improve accuracy and reliability of cytogenetic tissue analysis we later introduced and applied a special software tool that facilitates user independent and objective enumeration of FISH signals in multicolored and 3D-imaged tissue specimens [7, 8]. Our extended application of probes targeted to HER2 related receptor genes revealed HER2 relatives with additional prognostic and potential predictive value within the HER-receptor family [9, 10].
Following previous developments we present here HER2-mRNA FISH as a potential auxiliary cytogenetic tool for HER2 diagnosis in breast cancer. We first established fluorescent mRNA FISH using cell lines with known her2 gene copy numbers and known HER2 receptor expression. Afterwards we probed breast cancer tissue specimens with different her2 gene copy numbers and varying HER2 receptor content.

HER2 mRNA Probe Design and Hybridization

A full-length cDNA clone of the human HER2-mRNA was identified in public data bases and grown according to the manufacturer's recommendations. Plasmid DNA was isolated and verified by sequencing. The DNA was directly labeled with ZyOrange (equivalent to rhodamine from ZytoVision GmbH) using the proprietary ZytoLight Direct Label System (ZytoVision GmbH). Labeled DNA was fragmentized to 300-500 bp in length and mixed with formamide and other proprietary reagents to obtain a ready-to-use FISH probe.
FISH was performed using 10 mM citric acid/ as heat pretreatment solution for 30 min at 98°C. After washing slides in water they were air dried. A ready to use pepsin solution (ZytoVision GmbH) was applied for 7 min at 37°C. Slides were treated with 2 x SSC for 5 min, dehydrated in an ethanol series, air dried and 5 µl of HER2-mRNA FISH probe + 5 µl HER2-CEN-17 DNA-FISH probe (both ZytoVision GmbH) was applied. Co-denaturation of probe DNA and secondary mRNA structures was performed at 73°C for 10 min. After hybridization over night at 58°C, slides were washed at 58°C in 2 x SSC, 1 x SSC, 0.2 x SSC, supplemented with 0.3% Igepal (ICN Biomedicals), 5 min each, afterwareds air dried and counterstained with DAPI/Antifade solution (ZytoVision GmbH).