Importance of formalin fixing conditions for HER2 testing in gastric cancer: immunohistochemical staining and fluorescence in situ hybridization

Abstract

BackgroundAccurate and reliable assessment of human epidermal growth factor receptor type 2 (HER2) status is important for selecting patients with gastric cancer who may benefit from trastuzumab treatment. Here we examined the impact of formalin fixing conditions on HER2 immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) in xenografted tumor tissues.MethodsXenografted tumor tissues of the human gastric cancer cell lines NCI-N87, SCH, and SNU-16 were collected and kept at room temperature for 0, 6, or 24 h before being fixed with 10 % neutral buffered formalin (NBF) for 24 h or 5, 7, or 10 days and embedded in paraffin. Use of 10 % NBF, 20 % NBF, or nonbuffered formalin as fixative was investigated.ResultsThe HER2 IHC scores for NCI-N87, SCH, and SNU-16 tumors were 3+, 2+, and 1+, respectively, when specimens were fixed with 10 % NBF for 24 h immediately after resection of the tumors. Specimens left for longer than 6 h before fixation had shrinkage of the tumor periphery and decreased immunostaining intensity in this region in all specimens. In SCH and SNU-16 specimens, starting fixation 24 h after tumor tissue collection induced autolysis and reduction of the number of stained cells, and 10-day-fixation lowered the HER2 score. Prolongation of fixation time did not affect FISH results, but if samples were left for more than 6 h before fixation, the FISH score was strongly reduced in SCH specimens (2.3 to 1.3). Reduced IHC staining intensity was observed with 20 % NBF and nonbuffered formalin compared to 10 % NBF.ConclusionsThe time to and length of fixation of tumor specimens can affect HER2 IHC and FISH scores. The fixative used can affect IHC results.

Expression of P40 and P63 in lung cancers using fine needle aspiration cases. Understanding clinical pitfalls and limitations

“…In general, TMAs are constructed using well-select tumor tissues, however, the FNA cases are represent randomly sampled specimens, thus, the sampling error may have potential effect on the specificity. Second, the period of tumor sample in normal saline and the time of tumor fixation may also have potential effect on the detection of cellular proteins [37]. Taken together, the variable sensitivity and specificity might be related to type of the specimen, tumor heterogeneity, tissue sampling and cross-reaction.…”

Diagnostic utility of phosphorylated signal transducer and activator of transcription 5 immunostaining in the diagnosis of mammary analogue secretory carcinoma of the salivary gland: A comparative study of salivary gland cancers

“…Although the cause for this discrepancy is unclear, it is known that immunostaining can cause instability of protein expression depending on the fixation method, and the use of 10% neutral buffered formalin (eg, human epidermal growth factor receptor 2 testing of breast cancer) is recommended for histological samples. 31 Because the same patient's materials with paired histology and cytology were used for almost all MASCs, we conclude that p-STAT5 is highly sensitive for MASC. In a detailed comparative study, we added MMG by IHC and ICC because MMG is known to be positive in MASC.…”

“…In the current study, we found p‐STAT5–positive staining in all 7 MASCs with cytology (100%) by ICC, whereas this result was discordant with the IHC results (7 of 8 or 87.5%) of histology. Although the cause for this discrepancy is unclear, it is known that immunostaining can cause instability of protein expression depending on the fixation method, and the use of 10% neutral buffered formalin (eg, human epidermal growth factor receptor 2 testing of breast cancer) is recommended for histological samples . Because the same patient's materials with paired histology and cytology were used for almost all MASCs, we conclude that p‐STAT5 is highly sensitive for MASC.…”

“…IHC and ICC were regarded as positively expressing p-STAT5 when the qualitative intensity was 21 or 31 on the basis of the staining strength ([0] negative, [1] weak, [2] moderate, and [3] strong) and more than 20% of the cells were reactive. 19,21 The extent of staining of MMG proteins (21) or strong staining (31) in greater than 25% of the tumor cells (extent, 2-41) were considered positive for MMG. 6 A known case of MASC with p-STAT5 positivity was used as a positive control for histology and cytology.…”

HER2 testing of gastro-oesophageal adenocarcinoma: a commentary and guidance document from the Association of Clinical Pathologists Molecular Pathology and Diagnostics Committee

“…It has been reported that, compared with 10% neutral buffered formalin (NBF), fixation in 20% NBF or non-buffered formalin reduces intensity of HER2 immunostaining 12. Therefore, only 10% NBF is recommended for gastro-oesophageal adenocarcinoma specimens (biopsies and resection specimens) which may potentially undergo HER2 testing.…”