A 56-year-old female was initially diagnosed with HR-positive, HER2-negative (IHC 1+), Grade 1 invasive ductal carcinoma. Three years after initial treatment with mastectomy and adjuvant hormone therapy, the cancer metastasized to liver and bone. At this stage, a core needle biopsy was performed on the metastatic site in the liver and the sample was processed for a repeat HER2 IHC testing.1
Why is it important to control pre-analytic variables for this case?
Pre-analytic variables may influence HER2 IHC results and should be considered in all specimen types from both the primary and metastatic sites.2 Deviations from standard pre-analytic variables may result in false-negative results.2-5 This is especially important when assessing HER2 IHC at the low end of the spectrum as a slight change in staining may impact treatment decisions.2,6
Pre-analytic variables should be carefully controlled, including:
Cold ischemic time and fixation
The following optimal tissue handling requirements are outlined in ASCO-CAP guidelines6:
- Time from tissue acquisition to fixation should be as short as possible
- Fix samples in 10% neutral buffered formalin for 6-72 hours
Cold ischemic time should ideally be under 1 hour, as prolonged cold ischemic time may change HER2 IHC status.4,5,7 Prompt fixation preserves tissue quality and avoids compromising sample integrity due to rapid protein degradation after the interruption of the blood flow.2 Over- or under-fixation may affect the degree of protein cross-linking and antigen retrieval necessary for proper assay performance.2 This effect may also lead to incorrect hybridization in ISH testing and increase the chance of false-negative or false-positive results.2
Tissue processing
The following optimal tissue handling requirements are outlined in ASCO-CAP guidelines6:
- Section samples at 5-10 mm intervals
- Process samples within 6 weeks of sectioning
Surgical resection specimens should be ideally sliced at 5-10 mm intervals to allow for optimal formalin penetration into the tissue; the chemical fixation should be uniform, with consistent fixative and fixation time.6
Optimally, HER2 staining should be performed on sections freshly cut from tissue blocks; however, if this is not feasible, the stored unstained sections should be stained within 6 weeks to ensure optimal preservation of antigenicity.6
Why was HER2 IHC not performed on the bone specimen in this case?
In this case, a bone biopsy was not performed for HER2 IHC testing as the access to the metastatic site on the liver was more easily achieved. If processed appropriately, bone biopsy specimens are optimal for HER2 IHC testing: however, standardization of protocols and decalcification methods is necessary to avoid altering results.
Strong acids (e.g., hydrochloric acid) may rapidly macerate tissue and cause a loss of staining.8-10 Weak acids (e.g., formic acid) are slower in action and are more widely used for decalcification since they are less likely to interfere with immunostaining.8-10 Decalcification using chelating agents (e.g., EDTA) is best for preserving histomorphology and can be reliably used for IHC or ISH.8-10 However, they may be less suitable for urgent specimens due to longer processing time.9 Time for decalcification may also vary depending on the size or type of bone specimen (e.g., core biopsy, curettage, or resection).9
It is essential to validate the duration and the method of declassification, if decalcification of a bone specimen is deemed necessary, to ensure that they do not result in clinically relevant discordance.8 Prior to placing a specimen in the decalcification solution, consider removing any soft tissue within and/or adherent to bone and submit for histology processing without declassification. Often the soft tumoral tissues removed are sufficient for biomarker testing. Moreover, small amounts of bone spicules in the bone biopsies also can be processed normally without decalcification, and do not result in difficulty in the interpretation of histology or IHC.*
What does the HER2 IHC staining indicate?
The HER2 IHC slide at 40X power of magnification shows faint/barely perceptible, incomplete membrane staining, which is found in around 5% of tumor cells.1 Based on the latest ASCO-CAP guideline update, this case is scored as IHC 0 with membrane staining that is incomplete and faint/barely perceptible in ≤10% of tumor cells.6
