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Influence of decalcification procedures on immunohistochemistry and molecular pathology in breast cancer
Willemijne A M E Schrijver1, Petra van der Groep1,2, Laurien Dc Hoefnagel1
1Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands.
Abstract:
Distant breast cancer metastases are nowadays routinely biopsied to reassess receptor status and to isolate DNA for sequencing of druggable targets. Bone metastases are the most frequent subgroup. Decalcification procedures may negatively affect antigenicity and DNA quality. We therefore evaluated the effect of several decalcification procedures on receptor status and DNA/RNA quality. In 23 prospectively collected breast tumors, we compared ERα, PR and HER2 status by immunohistochemistry in (non-decalcified) tissue routinely processed for diagnostic purposes and in parallel tissue decalcified in Christensen's buffer with and without microwave, EDTA and Formical-4. Furthermore, HER2 fluorescence in situ hybridization and DNA/RNA quantity and quality were assessed. We found that the percentage of ERα-positive cells were on average lower in EDTA (P=0.049) and Formical-4 (P=0.047) treated cases, compared with controls, and PR expression showed decreased antigenicity after Christensen's buffer treatment (P=0.041). Overall, a good concordance (weighted kappa) was seen for ERα, PR and HER2 immunohistochemistry when comparing the non-decalcified control tissues with the decalcified tissues. For two patients (9%), there was a potential influence on therapeutic decision making with regard to hormonal therapy or HER2-targeted therapy. HER2 fluorescence in situ hybridization interpretation was seriously hampered by Christensen's buffer and Formical-4, and DNA/RNA quantity and quality were decreased after all four decalcification procedures. Validation on paired primary breast tumor specimens and EDTA-treated bone metastases showed that immunohistochemistry and fluorescence in situ hybridization were well assessable and DNA and RNA yield and quality were sufficient. With this, we conclude that common decalcification procedures have only a modest negative influence on hormone and HER2 receptor immunohistochemistry in breast cancer. However, they may seriously affect DNA/RNA-based diagnostic procedures. Overall, EDTA-based decalcification is therefore to be preferred as it best allows fluorescence in situ hybridization and DNA/RNA isolation.
Insights
Decalcification of bone metastases in breast cancer has a minor impact on hormone and HER2 receptor immunohistochemistry. However, it significantly degrades DNA/RNA quality, impacting molecular diagnostics. EDTA decalcification is preferred for preserving FISH and nucleic acid integrity.
Area of Science:
- Oncology
- Pathology
- Molecular Diagnostics
Background:
- Distant breast cancer metastases, particularly bone metastases, require biopsy for receptor status reassessment and molecular profiling.
- Standard decalcification procedures may compromise tissue antigenicity and nucleic acid quality, potentially affecting diagnostic accuracy.
- Accurate receptor status and molecular profiling are crucial for guiding targeted therapies in breast cancer patients.
Purpose of the Study:
- To evaluate the impact of different decalcification methods on the accuracy of estrogen receptor alpha (ERα), progesterone receptor (PR), and HER2 assessment by immunohistochemistry (IHC).
- To assess the effect of decalcification on HER2 fluorescence in situ hybridization (FISH) and DNA/RNA quantity and quality.
- To determine the optimal decalcification method for preserving diagnostic information in bone metastases from breast cancer.
Main Methods:
- Twenty-three prospectively collected breast tumors underwent parallel processing: routine diagnostic processing (control) and decalcification using Christensen's buffer (with/without microwave), EDTA, and Formical-4.
- ERα, PR, and HER2 status were evaluated by IHC.
- HER2 FISH, DNA/RNA quantity, and quality were assessed for all methods.
Main Results:
- ERα expression was reduced in EDTA and Formical-4 treated samples; PR antigenicity decreased with Christensen's buffer.
- Overall concordance for IHC was good, but a potential influence on therapeutic decisions was noted in 9% of cases.
- All decalcification methods decreased DNA/RNA quantity and quality; Christensen's buffer and Formical-4 significantly hampered HER2 FISH interpretation.
Conclusions:
- Common decalcification procedures have a modest negative effect on ERα, PR, and HER2 IHC in breast cancer bone metastases.
- Decalcification significantly compromises DNA/RNA quality and quantity, impacting molecular diagnostic procedures.
- EDTA-based decalcification is recommended as it best preserves tissue for FISH analysis and nucleic acid isolation, while maintaining acceptable IHC results.
