Marilyn Bui, MD, PhD, is a Senior Member in the Department of Pathology and the Scientific Director of the Analytic Microscopy Core at the Moffitt Cancer Center in Tampa, FL. She is also a Professor and the Director of the Cytopathology Fellowship Program at the University of South Florida Morsani College of Medicine. In this article, Dr. Bui, MD, PhD, discusses the advantages and disadvantages of using cytology samples for HER2 IHC testing in breast cancer, particularly focusing on the impact of pre-analytic variables on the reliability of test results.

Cytology specimens, in general, are more highly dispersed cells, such as those that occur in liquid as well as samples from minute tissue fragments, such as in a fine-needle aspiration biopsy.¹ Oftentimes, cytology specimens, such as a fine needle aspirate or exfoliative fluid sample, are submitted for HER2 IHC testing. The cell blocks are typically prepared within 24 hours after the procedure is performed, but there is no standardized method used to generate the cell block pellets before subjecting them to formalin fixation and paraffin embedding.^1,2 In contrast, the handling and processing of core needle biopsies or breast tissue resections are standardized in the 2023 ASCO-CAP clinical practice guidelines.²

In addition, the tumor cells in the cell block may not maintain the architecture of the tumor that is seen in histological tissue samples. Since the main purpose of cytology is to identify metastatic or recurrent breast cancer, the samples received at cytology practices are typically of later or higher stages of the disease.

It is important to keep in mind that metastatic breast cancer samples must be tested for prognostic and predictive biomarkers, such as HER2, as per the ASCO-CAP guidelines.³ During the collection of samples, efforts should be made to generate adequate cell blocks and to use fixatives such as formalin.¹ Though there is no standardized procedure set out in clinical guidelines, a good general practice consensus deems 100 tumor cells to be adequate for evaluation.⁴ Per ASCO-CAP guidelines, the optimal tissue handling requirements include: (1) time from tissue acquisition to fixation should be as short as possible; (2) samples for HER2 testing are fixed in 10% neutral buffered formalin for 6-72 hours; and (3) cytology specimens must be fixed in formalin.²

The cytology samples are freshly collected with dispersed tumor cells that are more accessible to antibody labeling.⁵ Due to its inherent limited cellularity, cell blocks are often inadequate for diagnostic testing and therefore hinder patient management.¹ Some studies have shown that cell blocks from primary or metastatic breast cancer are reliable for HER2 IHC testing, while other studies have questioned the reliability of cytological specimens for HER2 testing due to unstable staining (Figure 1).^4-8 Furthermore, the role of using liquid-based cytology samples for HER2 testing remains undefined.

The 2023 ASCO-CAP guidelines recommend HER2 testing of cytology specimens must be done on specimens that are fixed in formalin.² To ensure accurate and reproducible reporting of HER2 IHC results using cell blocks, it is important to address both the pre-analytic factors affecting proper HER2 testing alongside other relevant issues.⁸ Strict adherence to the 2023 ASCO-CAP HER2 testing and reporting guidelines (the same as those used for histological sections), adequate training, and intradepartmental consensus on HER2-low cases are recommended.

The concordance of HER2 IHC results between cell block and histological specimens is more consistent and robust for HER2-positive tumors when compared to other HER2 expression groups. Precautions should be taken to ensure adequate quality cellularity of the cell block material. Practice standards have yet to be determined for testing on HER2-low using cytological specimens.