Decalcification in breast cancer pathology
INTRODUCTION Multiple studies have shown that the expression of predictive tissue markers, such as estrogen receptor alpha (ERÎą), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2) may differ between the primary tumor and distant metastases (“receptor conversionâ€?) in a significant proportion of breast cancer patients 1-3. Therefore, several guidelines nowadays advice to biopsy distant metastases to reassess hormone and HER2 receptor status by immunohistochemistry whenever possible 4,5. However, in bone metastases this could potentially lead to inappropriate systemic treatment, since antigenicity may be altered by decalcifying agents that enable sectioning of bone 6,7. On the other hand, some studies report that decalcifying methods can be applied without significant loss of immunoreactivity 8,9. The same contradictory results about influence of decalcifying buffers have been seen when RNA or DNA integrity and interpretation of in situ hybridization were taken into account 10-14. These inconsistencies may be explained by usage of different decalcifying agents. Strong acids such as hydrochloric and nitric acid are traditionally widely used for their rapid decalcifying properties, but they are known to have a detrimental influence on immunoreactivity and DNA integrity 15. Therefore, weak(er) acidic buffers, containing formic or trichloracetic acid, are now more popular. EDTA, a chelating agent with neutral pH, requires longer time periods for the complete removal of calcium salts, but produces the best morphological results 8. Singh et al. recently tested ten commercially available decalcification agents and concluded that the best preservation of nucleic acids is achieved with decalcifying agents that contain either EDTA or formic acid or a combination of both11. Since bone is a frequent metastatic site among breast cancer patients 16,17, we set out to evaluate the influence of three routinely used decalcifying agents (containing formic acid and/or EDTA) on assessment of hormone and HER2 receptor status and DNA/RNA quality in breast cancer.
MATERIALS AND METHODS Material Prospectively, tissue from 23 breast tumors was collected and processed according to routine procedures at the department of Pathology of the University Medical Center Utrecht, The Netherlands. Original diagnoses were made between August 2012 and July 2015. Clinicopathological characteristics are shown in Table 1 (test cohort). Four tumor biopsies (0.4-0.8 cm3 in size) were removed from each breast tumor (Figure 1). All tissue samples were fixed in 4% buffered formaldehyde for 24-48 hours. After fixation, breast cancer tissue collected for diagnostic purposes was processed according to a standard 211
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