Are Assessment Guidelines for HER2 Status Leading to Many False Positives?

The use of alternative control probes to classify HER2 status in breast cancer could lead to substantial false-positive rates, according to a new study.

The American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) have specified criteria for the assessment of HER2 amplification status in recent years. The 2013 recommendations included both the average HER2 gene number per tumor cell, as well as the ratio of average HER2-to-internal control chromosome 17 centromere (CEP17) for assessment using fluorescence in situ hybridization (FISH). This assessment resulted in five possible ASCO-CAP FISH groups, with one of them (ASCO-CAP FISH group 4) labeled as “HER2-equivocal,” meaning neither amplified nor non-amplified (a 2018 clinical practice update has changed these guidelines, and this name is no longer used).

“According to the guidelines, this ambiguous status may be resolved with alternative controls to replace CEP17 for assessment of HER2 FISH ratios using genes other than CEP17,” wrote study authors led by Michael F. Press, MD, PhD, of the Keck School of Medicine at the University of Southern California in Los Angeles. The 2018 clinical practice update noted that the "indiscriminate" use of alternative probes can in fact lead to false positives, and the expert panel recommended against their routine use and instead recommended immunohistochemistry recounts to resolve certain cases. However, as Press and the study authors noted, "[the] approach was widely adopted by both commercial testing laboratories and academic centers."

The use of alternative control probes could lead to false positives because the genetic loci used for these probes are often heterozygously deleted in breast cancer. The use of these loci could then lead to a HER2-to-control ratio of greater than or equal to 2.0, which is classified as “ISH-positive.”

For the new study, the researchers retrospectively assessed the use of the recommended alternative control genomic sites, including TP53, D17S122, SMS, RARA, and TOP2A, among a cohort of 1,915 patients in the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC). The results were published in JAMA Oncology.

They found that heterozygous deletions were “relatively common,” especially on the p-arm of chromosome 17, in both HER2-amplified and HER2-non-amplified breast cancers. Among cancers considered to have HER2 copy number gain but not amplification (which was most likely to be representative of ISH-equivocal tumors), regional losses occurred across the various targets at rates exceeding 60%.

The researchers also compared results in a group of 100 ASCO-CAP FISH group 4 breast cancers and 100 group 5 cancers. Among the 100 cases deemed to be group 4, the HER2-to-control ratio was at least 2.0 in 61 cases using SMS, 65 cases using TP53, and 30 cases using D17S122. Using RARA or TOP2A resulted in fewer such ratios.

In the 100 cases deemed to be group 5, meaning HER2-not-amplified or ISH-negative, heterozygous deletions were again seen in many cases. Use of the alternative controls in these cases would have resulted in upgrading of HER2 status to ISH-positive in many of these, meaning they would be false-positive results.

“HER2-equivocal breast cancers with these false-positive ratios do not have HER2 protein overexpression and these patients do not have clinical outcomes that differ from either other patients with HER2-equivocal breast cancers not upgraded to positive or from HER2-negative disease,” the authors concluded. “Use of HER2-directed therapies in a population falsely classified as HER2-positive is expected to produce inferior clinical and pharmacoeconomic outcomes.”