Fluorescent mRNA HER2 FISH
A Potential Auxiliary Cytogenetic Tool for HER2 Diagnosis in Breast Cancer
- Fig. 1A+B: MDA-MB-231 (A), BT20 (B), ZR.75.1(C), and SK-BR-3 (D) paraffin embedded breast cancer cell lines probed anti-HER2-mRNA for establishment. Low (A), moderate (B and C), and high HER2-mRNA expression levels are observed and scored 1 (A), 2 (B and C) and 3 (D). DNA-FISH signals of 25 nuclei were counted respectively.
- Fig. 1C+D: MDA-MB-231 (A), BT20 (B), ZR.75.1(C), and SK-BR-3 (D) paraffin embedded breast cancer cell lines probed anti-HER2-mRNA for establishment. Low (A), moderate (B and C), and high HER2-mRNA expression levels are observed and scored 1 (A), 2 (B and C) and 3 (D). DNA-FISH signals of 25 nuclei were counted respectively.
- Fig. 2: Breast cancer tissues with her2 gene amplification but different levels of her2 expression (mRNA): low (A), moderate (B), and high (C) HER2-mRNA expression representing score 1, 2, and 3. Inserts a, b, and c: same images as A, B and C but w/o DAPI channel. DNA-FISH signals of 25 nuclei were counted respectively.
- Fig. 3: Breast cancer tissues with diploid (normal) her2 gene copy numbers but different levels of her2 expression (mRNA): low (A), and high (B) HER2-mRNA expression scored 1 and 3. Inserts a, and b: same images as A, and B but w/o DAPI channel. DNA-FISH signals of 25 nuclei were counted respectively.
- Fig. 4: Breast cancer tissues with polysomal chromosome 17 but different levels of her2 expression (mRNA): low (A), and moderate (B) HER2-mRNA expression scored 1 and 2. Inserts a, and b: same images as A, and B but w/o DAPI channel. DNA-FISH signals of 25 nuclei were counted respectively.
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  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 . 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 . 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 . 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).
HER2 mRNA FISH: Examples of Findings
Figure 1 is an example of four cell lines with no (A) moderate (B and C) and high (D) mRNA expression levels. Evaluation of expression density is based on both fluorescence intensity and degree of granularity. Expression levels were scored 1 (no or low expression), 2 (moderate expression), and 3 (high expression) and this scoring was applied to primary breast cancer specimens.
Figure 2 shows three breast cancer tissue samples with strong her2 gene amplification. The examples illustrate that despite of high her2 gene copy numbers (her2 gene CEN 17 ratios) the HER2 mRNA expression varies significantly. Example B represents her2 gene amplification along with only moderate mRNA expression but no detectable receptor protein.
Figure 3 gives an example of two diploid breast cancer specimens with low (A) and moderate to high (B) HER2-mRNA expression respectively. Both samples show nearly now protein expression (IH score 1+).
Two examples of polysomic specimens with a gene/CEN ratio of 1.1 and 1.2 are displayed in figure 4. HER2-mRNA expression is low (A) or moderate (B). We never found high mRNA expression (score 3) in the presence of polysomy.
Conclusions and Perspectives
We demonstrate here that fluorescent mRNA FISH can be integrated into slide based diagnostic procedures in a modular way i. e. it can be combined with DNA-FISH and (fluorescent) IH targeted to different gene loci and antigens respectively. Fluorescence based mRNA FISH should be considered as part of an individually designed multiplexed diagnostic approach. We provide examples of different mRNA expression levels at different levels of gene copy numbers. Missing receptor protein expression in the presence of high mRNA levels, for instance, might represent a transient state of receptor (down-)regulation.
The examples suggest that the integration of slide based mRNA analysis by in-situ-hybridization might augment the diagnostic value of well established approaches. The potential predictive value of integrated mRNA FISH needs to be evaluated by the investigation of larger cohorts of breast cancer specimens. The evaluation of HER2 receptor expression on mRNA level could complement well established diagnostic procedures as IH and DNA-FISH and could bridge discrepancies between findings derived by either method. mRNA FISH however can provide additional information about the HER2 status and its complex regulation. It might furthermore corroborate patient stratification and therapeutic directives for anti-HER2 targeted therapies.
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