CAP TODAY Pathology/Laboratory Medicine/Laboratory Management
The study found that aggregate frequency distributions of ER and PgR results were of particular interest. They highlight the relative homogeneity of ER expression (85.2 percent of cases showed strong average intensity of ER staining and 83.9 percent of cases with 91 to 100 percent of cells with nuclear positivity) and the more heterogeneous expression of PgR in contrast (60 percent showed strong average intensity of staining and 30.7 percent moderate expression, and 61 percent of PgR-tested cases showed 91 to 100 percent of cells with nuclear positivity). Says Dr. Brown: “Those of us who perform large numbers of these studies have conversationally noted for some time that there is a basic difference in receptor expression, with ER typically either completely positive, low positive, or negative. There is little in between. In contrast, there is sizable variation in PgR expression from case to case.”
This anecdotal experience is supported by the CAP proficiency testing program for ER and PgR, he says, in which “there is a high level of agreement for ER but, in many of the challenges, sufficient variation in interpretation of the PgR cores to preclude grading by 80 percent consensus of at least one tissue core.”
“The data from this study provide additional evidence of the inherent variation in PgR expression.”
Dr. Mais
The largest percentage of tumors—58.5 percent—were ER/PgR-positive and HER2-negative, an expected rate. “That would be fairly typical,” Dr. Mais says. “That’s how the majority of our cases tend to stain.” Triple-negative tumors represented 8.2 percent of cases and triple-positive tumors represented 3.6 percent of cases. “We all know the triple-positive group exists and that it’s a small number of patients, but it’s an under-studied group. We’ve looked long and hard at triple-negative patients, but not much work has been done on the triple-positive group. And it would be an interesting group to look at.”
Laboratories will want to assess the reason why their rates might differ from those in the Q-Probes study. Differing expression results for these predictive markers could occur because the patient population is unique in some way, Dr. Mais says. “You may have a particular ethnic group or age group overrepresented. Or you may have a grade of tumor overrepresented.”
One can never be certain, Dr. Brown says. “But in general, the results, if deviating from the mean significantly, should be explainable by known associations. For example, one would expect high rates of ER and PR expression if the patient population were predominantly women who are postmenopausal with well-differentiated tumors. A population in which young women with aggressive tumors were overrepresented would be expected to have lower ER and PR expression rates.”
But there could be analytic factors that labs would want to check out. “The major place to look would be your test systems,” Dr. Mais says. “There could be something anomalous with the reagent antibody you are using or the hardware of the testing system. Or you may be overinterpreting those slides. Things that other people would call 2+ for HER2, you are calling 3+, for example. You’d even want to evaluate whether you are picking the right blocks for staining. We find that we get more reliable results when we choose blocks that have an internal control we can use.”
If a laboratory thinks it might have a problem with HER2 staining, the best thing to do may be to retest those cases by FISH or retest in another laboratory and compare the results, Dr. Mais says. “But also reassess the H&E slides in those cases and make sure your grade is appropriate—that you haven’t undergraded cases. In my lab, for example, if I have a HER2-positive tumor and I see that it was graded as one, that would cause me to hesitate before reporting those results.”
Laboratories that lie above the 90th or below the 10th percentiles in this Q-Probes study should examine their procedures to ensure that the level of sensitivity is appropriate, Dr. Brown recommends. “This involves careful evaluation of internal controls—normal breast tissue—and external positive and negative controls, as well as recent results on proficiency testing challenges. In some cases, a revalidation against tissues with known receptor expression may be warranted.”
ER, PgR, and HER2 testing points clinicians to the right way to treat patients, Dr. Guidi notes, and adds, “We don’t want to get that wrong. To make sure we are reporting correctly, we have to participate in proficiency testing, which I think most laboratories do, and also look at benchmark data and follow it over time to make sure nothing drifts.”
He’s a strong believer in using benchmark data to maintain quality and gives an example of the reason based on his laboratory’s experience. “It’s fairly unusual to get patients who are negative for ER and positive for PgR. In the last Q-Probes study of ER and PgR, that combination occurred just over two percent of the time.”
His laboratory saw that combination less than one percent of the time. “But a year or two ago we noticed, because we looked at our data, that this rate was rising in our laboratory. It was approaching the four percent range. This wasn’t picked up through proficiency testing, but by trending data and looking at it.” The laboratory realized it needed to retool its PgR immunostain process, it made modifications, and the rate is once again less than one percent a year. This experience confirms the value of benchmarking, he says. “We need to get it right, and benchmarking is one way, one tool in our tool belt, to do that.”
The key takeaway of this study for Dr. Brown is that ongoing attention to quality is essential.
“We know from clinical trials in which central review is performed that there can be significant variation in the way in which these predictive markers are performed and reported. Recent data from CAP Surveys suggest that in that cohort of participating laboratories, concordance is excellent. However, we must all remain vigilant, as the immunostains currently in use require significant antigen retrieval, which is known to be a cause of both false-positive and false-negative results, depending on the integrity of tissue fixation and processing.” So comparison of positivity rates and patterns of expression against those of other laboratories and against prior performance within the laboratory, he says, should remain an important tool for quality management.
Anne Paxton is a writer and attorney in Seattle. Full results of the study will be submitted for publication in the Archives of Pathology & Laboratory Medicine.