Cases in group two (the Sheldons of the world might be irritated that these are addressed in clinical question No. 3) have low HER2 copy number (<4.0 signals per cell) but a HER2/CEP17 ratio ≥2.0 These cases tend to have loss of the centromeric control signal. They also would have been considered eligible for the original clinical trials for HER2-targeted therapy, given their ratio-positive status, so the 2013 guideline considered them positive as well. “We initially didn’t want to exclude them from the therapy option,” Dr. Allison says.
The authors of the new guideline looked at recent data on the rates of concordance between IHC positivity versus negativity. “The findings were that these were largely IHC-negative cases,” Dr. Allison says. They’re frequently ER positive, and they typically don’t have biologic features that would suggest they behave more aggressively than an ER-positive, HER2-negative cancer.
“You would expect a HER2-positive truly amplified, truly protein-overexpressing cancer to behave in a more aggressive way than a HER2-negative cancer that’s ER positive,” she says. But the (admittedly limited) data available from the first generation of adjuvant trastuzumab trials suggest patients in group two didn’t receive much benefit from HER2-targeted therapy. Hence the new guideline’s recommendation: For group-two ISH cases, do a concurrent IHC test. “Some labs might just currently do FISH without looking at protein expression in cases like this,” Dr. Allison explains. IHC is a useful guide for scoring ISH/FISH, she says, particularly because it helps recognize if there is regional heterogeneity, but it also can flag discordant results with unusual ISH result categories.
If the IHC result is 3+ positive, “you can go ahead and result the ISH test as HER2 positive,” Dr. Allison says. If it’s IHC negative (zero or 1+), then it should be called HER2 negative. The guideline provides a suggested comment that can be modified and included in the report to note the aforementioned limitations of the early trials, including the enrollment of only small subsets of group-two cases.
And if the blurriness continues, in the form of a 2+ result? “It’s recommended to double-check the initial in situ hybridization result by having a second observer count at least 20 cells that are in that area of 2+ IHC staining,” she says. The second reviewer should be blinded to the first reviewer’s counts. If the result remains in the group-two category, with a 2+ IHC result, “it’s going to be negative overall result for group-two cases,” she says, and should be noted as such with comment. If the new count moves the result into a different ISH category, the final category should be resolved by internal procedures, the guideline notes.
Dr. Allison characterizes these steps as “a little additional workup.” Labs can choose to adopt the additional ISH counting workup for all group-two cases, if that’s easier for the testing parameters. But at minimum, group-two cases that are IHC 2+ should get a second count. Essentially, these cases—again, ratio-positive, signals-per-cell-negative—are considered positive only if additional IHC testing is 3+ positive.Clinical question No. 4 addresses cases in group three (cue coughing noises from the overfastidious). These are the opposite of group-two cases, in that they are signals-per-call high (≥6.0) but with a HER2/CEP17 ratio <2.0. Should these be considered ISH positive?
Only a few rare cases like this were included in one of the original trials, and only if very high HER2 copy number, Dr. Allison notes, since they were ratio negative. But in the 2013 update these cases were considered positive, based on high copy number alone; the ratio, she says, was originally designed to control for polysomy. That places the picture slightly askew. “So instead of true gene amplification causing protein overexpression, is it a cancer that just has multiple copies of chromosome 17? That might not be a true HER2-positive case was the original thinking during the initial trials.” But more recent data, including studies that looked at scrutinizing other areas of the chromosome, showed that the majority of these cases have co-amplification of large regions of chromosome 17, rather than true polysomy, Dr. Allison says.
“This group was a little more controversial than group two,” she says, given that this is likely a more heterogeneous result category. Some data groups indicated the majority of such cases are IHC negative, with a handful of IHC positives that appeared to have very high-level HER2 amplification. Other groups showed a large number of IHC 3+ cases and high levels of amplification.
“Again, it’s a rare group,” says Dr. Allison, which means these cases will be very sample dependent. “It seems like it’s a mixed group. And that includes some more aggressive features and more frequent ER-negativity. Some of these cases have really high-level HER2 amplification, even though they’re also ratio negative, and they have the positive protein overexpression by IHC to support true HER2-positive phenotype.” Again, the recommendation is for concurrent IHC in the workup of an initial group-three ISH result. A negative IHC means pathologists can call the case negative, although, Dr. Allison adds, “I would also double-check a negative result,” to ensure there were no problems with fixation, decalcification, and so on.
Positive IHC results mean the case can be resulted as positive. “That’s a nice concordant result,” she says.