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Decalcified bone specimen: Processing procedures

Dr. Wei, MD, PhD, discusses the use of bone specimens as samples for HER2 IHC in metastatic breast cancer.

The importance of reporting discrete HER2 IHC scores

Shi Wei

MD, PhD

Shi Wei, MD, PhD, is the Professor with Tenure and the Barbara F. Atkinson Endowed Professor, Director of Translational Research at the Department of Pathology and Laboratory Medicine, University of Kansas Medical Center. In this article, Dr. Wei, MD, PhD, discusses the use of bone specimens as samples for HER2 IHC in metastatic breast cancer. He considers the impact of the decalcification procedure on the reliability of test results, establishing the advantages and disadvantages of the different procedures and reagents.

How often do you perform HER2 IHC on bone specimens? How does this differ from other specimen types?

It is quite common to receive bone biopsy specimens from patients with a known history of breast cancer for confirmation of metastatic disease, as well as biomarker studies for systemic treatment decision making.¹ Moreover, it is not uncommon to encounter a bone biopsy from patients without a prior history of malignancy, in whom metastatic disease is the initial presentation of breast cancer.¹ Therefore, IHC testing for HER2 on bone specimens is frequently performed in routine pathology practice.

Bone specimens require decalcification. What is this and why is it important?

Decalcification is a technique for removing minerals from bone or other calcified tissue to obtain high-quality paraffin sections.² It is carried out after thorough fixation and prior to processing in paraffin.²

“It is important to validate the decalcification methods to ensure that they do not result in clinically relevant discordance.”

Which methods can be used for decalcification? Is this standardized across the pathology community?

There are different types of decalcifying agents including strong mineral acids, weaker organic acids, and chelating agents.³ Decalcification using strong acids (such as hydrochloric acid) is not recommended, as it can lead to rapid tissue degradation and loss of nuclear staining, as such their use should be limited.³ Weak acids (such as formic acid) are less likely to interfere with immunostaining as they are slower acting and therefore are used more widely for decalcification.³ Decalcification using chelating agents (such as EDTA) best preserves histomorphology and generally can be more reliably used for IHC, FISH, or PCR (Figure 1).^3,4 However, decalcification using chelating agents is more time-consuming and therefore may not be appropriate for processing urgent specimens.³ The decalcification method of bone specimens is not standardized across the pathology community, therefore it is of great importance to validate decalcifications internally.²

Figure 1. Immunohistochemical stainings for ERα, PR, and HER2 on tumor tissue from patient #5 after decalcification in EDTA. 20X magnification.⁴

How can decalcification of bone samples affect interpretation of HER2 IHC results?

“It is recognized that hydrochloric acid-based and long-term formic acid-based decalcification may induce false-negative results on IHC.”^2,3

In one analysis it was reported that when compared to acetic and hydrochloric/formic acid, EDTA decalcification minimally affects ER, PR, and HER2 results. Although, in this particular case the comparison was between the primary tumor and bone metastasis, not the same tissue with and without decalcification.² In contrast, another study demonstrated that mean IHC scores for several nuclear and membranous/cytoplasmic antigens (including HER2) were similar with formic acid, EDTA, and non-decalcified samples.³ Note that the duration of decalcification was different for various decalcifying agents and across each of the studies.³

What does the 2023 ASCO-CAP guideline update recommend for HER2 testing on bone specimens?

Within CAP reporting templates for biomarker reporting there is additional guidance wherein cases where pre-analytical variables may have an impact on test results, the variables must be reported. This includes decalcification of bone specimens as this could potentially alter immunoreactivity.⁶ In all cases, if tests undergo technical issues that prevent results being reported as positive, negative, or equivocal, results should be reported as indeterminate, and another sample should be requested for testing. This includes cases with inadequate specimen handling, where artifacts make interpretation difficult, or analytic testing failure.⁵

What are your key takeaways for the pathology community to optimize HER2 testing on bone specimens?

“Validation of IHC assays is of great importance, as it creates objective evidence of “fit-for-purpose” for clinical practice.”⁷

The validation should be properly documented and informed to fulfill the requirements of clinical practice guidelines in the era of precision medicine. While the 2023 ASCO-CAP guideline update indicates no absolute reasons for assay exclusions,⁶ it is critical to perform a validation study in the practicing pathologists’ laboratories for receptor HER2 testing to fulfil the ASCO-CAP requirements and provide reliable results for patient care.⁷ In all cases where specimens have undergone decalcification and HER2 testing a disclaimer should be included reporting the decalcification method used and the potential impact it may have had on test results.

ASCO, American Society of Clinical Oncology; CAP, College of American Pathologists; EDTA, ethylenediaminetetraacetic acid; ER, estrogen receptor; FISH, fluorescence in situ hybridization; HER2, human epidermal growth factor receptor 2; IHC, immunohistochemistry; ISH, in situ hybridization; PCR, polymerase chain reaction; PR, progesterone receptor.

1. Soni A, et al. Am J Clin Pathol. 2015; 143(4):471-8. 2. van Es SC, et al. Am J Surg Pathol. 2019; 43(10):1355-1360. 3. Miquelestorena-Standley, et al. Mod Pathol. 2020; 33:1505-1517. 4. Schrijver WA, et al. Mod Pathol. 2016; 29(12):1460-1470. 5. Wolff AC, et al. Arch Pathol Lab Med 2023;147(9):993-1000. 6. Fitzgibbons, et al. Breast. 2018; 1:1. 7. Torlakovic EE, et al. Appl Immunohistochem Mol Morphol. 2017; 25(3):151-159.

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Decalcified bone specimen: Processing procedures

Shi Wei

MD, PhD

How often do you perform HER2 IHC on bone specimens? How does this differ from other specimen types?

Bone specimens require decalcification. What is this and why is it important?

Which methods can be used for decalcification? Is this standardized across the pathology community?

Figure 1. Immunohistochemical stainings for ERα, PR, and HER2 on tumor tissue from patient #5 after decalcification in EDTA. 20X magnification.⁴

How can decalcification of bone samples affect interpretation of HER2 IHC results?

What does the 2023 ASCO-CAP guideline update recommend for HER2 testing on bone specimens?

What are your key takeaways for the pathology community to optimize HER2 testing on bone specimens?

Explore more Spotlight Series

Unusual staining pattern: Basolateral staining in micropapillary carcinoma

In this article, Dr. Jaffer, MD, details the interpretation of biomarker testing in invasive micropapillary carcinomas.

Impact of pre-analytic variables on HER2 IHC results

In this article, Dr. Khoury, MD, reviews several different pre-analytic variables and their impact on HER2 IHC testing.

Recommendations for testing of cytology specimens

In this article, Dr. Bui, MD, PhD, discusses the advantages and disadvantages of using cytology samples for HER2 IHC testing in breast cancer.

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Decalcified bone specimen: Processing procedures

Shi Wei

MD, PhD

How often do you perform HER2 IHC on bone specimens? How does this differ from other specimen types?

Bone specimens require decalcification. What is this and why is it important?

Which methods can be used for decalcification? Is this standardized across the pathology community?

Figure 1. Immunohistochemical stainings for ERα, PR, and HER2 on tumor tissue from patient #5 after decalcification in EDTA. 20X magnification.4

How can decalcification of bone samples affect interpretation of HER2 IHC results?

What does the 2023 ASCO-CAP guideline update recommend for HER2 testing on bone specimens?

What are your key takeaways for the pathology community to optimize HER2 testing on bone specimens?

Explore more Spotlight Series

Unusual staining pattern: Basolateral staining in micropapillary carcinoma

In this article, Dr. Jaffer, MD, details the interpretation of biomarker testing in invasive micropapillary carcinomas.

Impact of pre-analytic variables on HER2 IHC results

In this article, Dr. Khoury, MD, reviews several different pre-analytic variables and their impact on HER2 IHC testing.

Recommendations for testing of cytology specimens

In this article, Dr. Bui, MD, PhD, discusses the advantages and disadvantages of using cytology samples for HER2 IHC testing in breast cancer.

Quick Links

Breast Cancer Links

Gastric Cancer Links

About Us

Legal

What is a Daiichi Sankyo Company or AstraZeneca authorized person?

Figure 1. Immunohistochemical stainings for ERα, PR, and HER2 on tumor tissue from patient #5 after decalcification in EDTA. 20X magnification.⁴