Controvers ies in Test ing for HER2

By Giuseppe Viale, MD, FRCPath

Overview: There are still several highly controversial aspectsof HER2 testing in breast cancer despite decades of extensiveexperience with immunohistochemical (IHC) and in situ hy-bridization (ISH) assays, the availability of standardized re-agents and kits, and the publication of guidelines andrecommendations describing how to optimally perform theassays and evaluate and score the results. Indeed, the accu-racy and reproducibility of the test results still are a majorconcern worldwide: the optimal thresholds for a positive IHCor ISH assay are debatable; there is lack of consensus aboutthe clinical implications of intratumoral heterogeneity of HER2status; the existence of polysomy of chromosome 17 hasbeen challenged; and it is unclear whether tumors may changetheir HER2 status during progression, thus implying that it is

uncertain whether a rebiopsy of the metastatic sites is justi-fied to inform a tailored systemic treatment of the patients.To address all these controversial issues, the scientific com-munity should be willing to reconsider at least some of theassumptions on which the current assays and the scoringsystems are based. By tackling the controversies, we mayeventually foster our understanding of the biology, the clinicalcourse, and the responsiveness to targeted therapies ofHER2-positive breast cancers, thus improving the accuracy inthe selection of patients who are candidates for HER2-targeted therapies. This will hopefully bring us closer to theidentification of the responsive patients to different therapeu-tic options.

HER2 HAS most, if not all, of the features of the perfectmarker for breast cancer: it is deregulated in a consid-

erable fraction (approximately 20%) of the tumors, and itsstatus correlates with the clinical outcome of the disease(prognostic factor) and with the response to different sys-temic therapies (predictive factor). More importantly, HER2is the specific target for the new and highly efficaciousbiologic therapies with monoclonal antibodies or small mol-ecules interfering with its intracellular pathway.

By contrast with other tumor markers for breast carci-noma, there is a very strong correlation between the defectat the gene level (gene amplification) and the expression ofthe encoded mRNA and protein. Thus, HER2 status can beassessed using several different approaches, which yieldhighly concordant results when performed properly. IHCand ISH assays with fluorescent (FISH) or chromogenic(CISH) probes are most commonly used, and different U.S.Food and Drug Administration (FDA)-approved reagentsand ready-to-use kits are commercially available.1

Guidelines and recommendations describing how to opti-mally perform the assays and evaluate and score the resultshave been issued and recently updated.2,3 These assays havebeen clinically validated in several studies demonstratingthe high predictive value of an HER2-positive status for theefficacy of HER2-targeted treatments.

It is then paradoxical that—after decades of extensivetesting of HER2 status in breast cancer and several years ofclinical application of the test results to identify patientswho are candidates for HER2-targeted therapies—we stillhave to tackle so many controversial issues in HER2 testing.In particular, accuracy and reproducibility of the test resultsare still highly unsatisfactory; the thresholds for a positivetest and the scoring systems are debatable; the clinicalimplications of intratumoral heterogeneity of HER2 statusare currently unknown; the existence of polysomy of chro-mosome 17 has been challenged; and it is unclear whethertumors may change their HER2 status during progression,thus implying that there is uncertainty about the value of arebiopsy of the metastatic sites to inform a tailored systemictreatment for the patients.

Most of these controversial aspects likely derive fromtechnical limitations or drawbacks, and they could be tack-led by improving accuracy and reproducibility of the assaysor even by developing new and more reliable testing proce-

dures.4 It is also possible, however, that some of thesedebated issues simply reflect a still limited understanding ofthe biologic characteristics of HER2-positive breast cancer.Indeed, we have arbitrarily defined thresholds for HER2positivity and have assumed that scoring systems based onthe quantitative evaluation of protein expression by IHCand gene amplification by ISH would have fully reflectedboth tumor aggressiveness and responsiveness to HER2-targeted therapies. Controversies now arise because tumorscontinuously challenge our assumptions and dogmas byshowing unexpected outcomes and counterintuitive re-sponses to targeted interventions.5 This should prompt arenovated willingness to try and unveil peculiar aspects ofHER2-positive breast cancer that have been apparentlyoverlooked or misinterpreted until now.

Accuracy and Reproducibility of HER2 Testing

First, it is unacceptable that accuracy and interlaboratoryreproducibility of the assessment of HER2 status continue tobe worldwide concerns. The results of several quality controlschemes, as well as central re-evaluation of locally positiveassays on the tumor samples of patients enrolled in clinicaltrials of adjuvant trastuzumab, have consistently shown avery high rate of interlaboratory discordance in the assess-ment of HER2 status both with IHC (with as many as 15%to 20% false-positive results) and FISH assays.6-9

The causes for a discordant result may be manifold andinclude preanalytic, analytic, and interpretative issues, ei-ther alone or in combination. Overfixation in formalin oralcoholic postfixation of samples suboptimally fixed in for-malin may be the most important preanalytic sources ofunderestimation or overestimation of HER2 overexpressionby IHC assays. However, it is most likely that the inappro-priate evaluation and scoring of the staining results bypathologists lacking specific knowledge and expertise is amajor determinant of inaccurate IHC and ISH assays.

From the Department of Pathology, European Institute of Oncology and University ofMilan, Milan, Italy.

Author’s disclosures of potential conflicts of interest are found at the end of this article.Address reprint requests to Giuseppe Viale, MD, Department of Pathology, European

Institute of Oncology, Via Ripamonti 435, 20141 Milan, Italy; e-mail: giuseppe.viale@ieo.it.© 2011 by American Society of Clinical Oncology.1092-9118/10/1-10

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To minimize the risk of false-positive assessments it isrecommended that whenever possible the tissue block se-lected for HER2 testing includes at least a minor componentof non-neoplastic breast parenchyma. This should remainunstained (or very weakly stained) in IHC assays andexhibit the normal number of copies of the HER2 gene whenassayed by ISH techniques. If this is not the case, then therisk of false-positive results in the assessment of HER2status in the tumor cells is very high. Additionally, it mustbe always kept in mind that well-differentiated and low-proliferating ductal and lobular breast carcinomas are veryrarely, if ever, HER2-positive.

The inaccurate assessment of HER2 status not only isdetrimental for an optimal selection of patients who arecandidates for tailored systemic treatments, but also jeop-ardizes our understanding of the biology of breast cancer.This is best exemplified by the controversies surroundingthe actual occurrence of changes of HER2 status betweenthe primary tumor and synchronous or late metastases.10,11

Indeed, the reported prevalence of a discordant HER2 statusinevitably is affected by the error rate of the assay, to thepoint that it may be almost impossible to reliably ascertainthe actual rate of HER2 discordance in the metastases.12

Future studies of discordant HER2 status in metastatic

tumors should require mandatory retesting of HER2 in theprimary and the metastatic lesions, using both IHC and ISHassays run simultaneously on the primary and correspond-ing metastatic samples, thus minimizing the risk of interassayvariability of the results. Only when an accurate estimate ofthe likelihood of HER2—and estrogen receptor—discordancein relapsing tumors is eventually defined, will it be possibleto reach a consensus about the clinical value of a rebiopsy ofthe metastatic sites.

Thresholds for a Positive Assay and Scoring ofthe Results

According to the regulatory agencies worldwide and thetrastuzumab package insert, only patients whose tumorsoverexpress HER2 by IHC in more than 10% of invasivetumor cells or show HER2 gene amplification (four or morecopies of the gene/cell, or a ratio � 2 between the gene copynumber and the chromosome 17 centromeres) are candi-dates for trastuzumab treatment. The recommendationsissued by the American Society of Clinical Oncology(ASCO)/College of American Pathologists (CAP)—raisingthe threshold for a positive IHC assay to more than 30%overexpressing tumor cells and for a positive ISH assay to aratio of 2.2 or greater or a gene/cell copy number of morethan six—were not intended, and actually not entitled, toreplace the original thresholds, but to increase the concor-dance rate between the two assays.3,13 However, clinicalstudies are still needed to assess whether the updatedguidelines are better at predicting response to HER2-targeted therapies. For the time being, adoption of the newASCO/CAP thresholds may keep patients from receiving atargeted therapy, despite the fact that the same patientswould have been eligible for the adjuvant trastuzumab trialsthat led to its approval.

It is worth re-emphasizing, however, that although IHCresults are evaluated and scored at the individual cell level(by assessing the actual percentage of positive cells), ISH isa population-based assay, averaging the number of copies ofthe gene among all the neoplastic cells of one or moremicroscopic fields. Accordingly, a tumor with 20% cellsshowing HER2 overexpression will be scored as positive byIHC (according to the FDA scoring system), but it may endup negative by ISH because of the dilution effect of thenonamplified cells, despite the fact that the same 20% tumorcells actually show gene amplification. Indeed, to reach anHER2:chromosome 17 ratio of 2, each of the amplified cellsshould have at least 12 copies of the gene. As a consequence,the likelihood of such a case to be eventually reported asamplified or not amplified depends on the actual number ofcopies of the gene in the amplified cells.

The different criteria for scoring IHC and ISH resultswould not be an issue at all if the entire neoplastic cellpopulation of every breast cancer were homogeneouslyHER2-positive or -negative, an assumption stemming fromthe concept of monoclonality of the tumors and supported byprevious data derived from the analysis of freshly frozentumor samples.14 These earlier investigations demonstratedrelatively little cell-to-cell heterogeneity within a givencancer, with essentially all neoplastic cells overexpressingthe protein to a similar level in frozen samples of HER2-amplified tumors. However, formalin fixation and paraffinembedding did result in loss of antigenicity and introductionof IHC heterogeneity that was not encountered in the

KEY POINTS

● Accuracy and reproducibility of HER2 testing are lessthan satisfactory worldwide, and they should be im-proved by a more stringent compliance with availableguidelines and recommendations, and by wider par-ticipation in national or international quality assur-ance schemes.

● Overfixation in formalin or alcoholic postfixation ofsamples suboptimally fixed in formalin may be themost important preanalytic sources of underestima-tion or overestimation of HER2 overexpression byimmunohistochemistry (IHC) assays.

● Patients whose tumors overexpress HER2 by IHC inmore than 10% of invasive tumor cells or show HER2gene amplification (four copies of the gene/cell or aratio � 2 between the number of the gene copies andof chromosome 17 centromeres) are candidates fortrastuzumab treatment.

● Breast cancers often show heterogeneity in HER2overexpression and amplification, with only a fractionof tumor cells being HER2-positive (genetic heteroge-neity). Therefore, it is recommended to indicate theoccurrence of genetic heterogeneity in the final re-port, even if any evidence for the responsiveness ofthe disease to HER2-targeted therapies is currentlylacking.

● Polysomy of chromosome 17 is a very rare event inbreast cancer, and all tumors showing an apparentpolysomy at in situ hybridization assays should beclassified according to the mean number of HER2copies/cell as amplified (� six gene copies/cell) or notamplified (� four gene copies/cell).

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corresponding frozen samples. These findings led to theconclusion that FISH should be preferred as the screeningassay for formalin-fixed paraffin-embedded samples becauseDNA is more stable and less affected by the preanalyticprocedures.

Unfortunately, there is now overwhelming evidence thatbreast cancers often show heterogeneity in both HER2overexpression and amplification, with only a minor fractionof invasive tumor cells being HER2-positive. This heteroge-neity may be easily justified when it is encountered inhistologically mixed breast cancers, where one component(most commonly a high-grade ductal carcinoma) is HER2-positive, whereas the associated component(s) is negative.However, in the majority of the cases, HER2 heterogeneityoccurs within tumors that are histologically homogeneous,and it appears as discrete foci of cells with HER2 overex-pression/amplification (Figure 1) or a variable number ofpositive cells scattered among the prevalent negative cellpopulation. In both cases, positive and negative cells aremorphologically indistinguishable, and a plausible justifica-tion of this kind of heterogeneity has not been reached thusfar.

In heterogeneous tumors, especially when there is only aminor component of HER2-positive cells, it is very likelythat the results of IHC and ISH assays are discordant, withthe former assay being positive and the latter negative forthe reasons discussed above. This would militate against theadoption of ISH as the ideal screening test for all breastcancers because it would misclassify as HER2-negativethose showing a more pronounced heterogeneity.

Genetic Heterogeneity of Breast Cancer

That intratumoral heterogeneity is not such an uncom-mon event as previously suggested and cannot be neglectedany longer is also witnessed by the recent guidelines issuedby CAP.15 In these guidelines, the panelists endorsed thedefinition of “genetic heterogeneity” to identify tumors withonly 5% to 50% of the neoplastic cells showing amplificationof the HER2 gene. Most commonly this minor component ofamplified cells is not sufficient enough to attain an overallHER2 to chromosome 17 ratio of 2.2 or higher, and, there-fore, the tumor does not qualify for ISH-positive. The pan-

elists, however, recommended to indicate the occurrence ofgenetic heterogeneity in the final report, even if any evi-dence for the responsiveness of the disease to HER2-targeted therapies is currently lacking. In a recent survey of6,461 patients with breast cancer, the prevalence of casesshowing genetic heterogeneity according to the above defi-nition was as high as 33.5%.16

How could we tackle tumor heterogeneity without incur-ring in any discordance between the results of IHC and ISHassays that would eventually imply a different treatment ofthe patients according to the assay used for screening? Afeasible approach would be to use the same scoring system,based on the actual percentage of positive cells, for bothassays. Accordingly, a tumor would be considered HER2-positive if more than 10% of the neoplastic cells are overex-pressing the protein (3� by IHC) and/or carrying geneamplification.

A final aspect that deserves further consideration is thecurrent consensus that patients with tumors exhibiting 10%or fewer HER2-positive cells are not candidates for theHER2-targeted therapy because of these patients were noteligible for the clinical trials of adjuvant trastuzumab. It isimpossible, however, to justify on a biologic basis why apatient with a tumor overexpressing HER2 in 10% or fewerneoplastic cells would respond differently to a targetedtherapy as compared with a patient harboring a tumor with11% immunoreactive cells. Under the current regulatoryconstraint, not to deny any patient with 10% or fewerimmunoreactive tumor cells the possible benefit of targetedinterventions, it may be wise to test HER2 status in addi-tional tumor blocks, or even in the lymph node metastases (ifpresent) to assess whether the more than 10% thresholdmay be eventually reached in at least one of these samples.

Does Polysomy of Chromosome 17 Occur inBreast Cancer?

The adoption of the HER2:chromosome 17 ratio to assessgene amplification was originally intended to avoid misclas-sification of polysomic tumors as amplified. The rationalebehind this scoring system was that in case of chromosome17 polysomy the increased number of copies of HER2 genewould not be because of an actual amplification at the genelevel, but instead because of repeated duplications of theentire chromosome 17. Polysomy of chromosome 17 did notsubstantially influence the response to trastuzumab in theN9831 clinical trial or to lapatinib in metastatic breastcancer, and it correlates unpredictably with overexpressionof the HER2 protein.17,18 Indeed, patients with tumorsshowing chromosome 17 polysomy were eligible for entry inclinical trials of adjuvant trastuzumab (and are now candi-dates for the targeted treatment) only in case of concurrentoverexpression of the protein as documented by IHC assays.

According to the HER2:chromosome 17 ratio, breast can-cers may be classified into three different categories: ampli-fied, not amplified, and polysomic. However, in the last fewyears, different studies using alternative approaches toassess chromosome 17 status have consistently shown thattrue polysomy of chromosome 17 is an exceedingly rare—ifat all possible—event in breast cancer. Almost all the caseswith an increased number of chromosome 17 signals in ISHassays actually carry gain or amplification of the centromereregion of chromosome 17, and not true polysomy.19-21 Inseveral instances, scoring of the results by the HER2:

Fig. 1. Single focus of HER2-overexpressing invasive tumor cells—representing less than 5% of the neoplastic population—in an other-wise negative breast cancer.

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chromosome 17 ratio led to overlooking an actual HER2gene amplification that was coexistent with a gain or ampli-fication of the CEP17 region.

If polysomy of chromosome 17 is such a rare event inbreast cancer, all tumors showing an apparent polysomy atdual-color ISH assays should be classified according to themean number of HER2 copies/cell as amplified (� six genecopies/cell) or not amplified (� four gene copies/cell).22 Caseswith four to six HER2 copies/cell and three or more CEP17signals/cell would represent an equivocal category, and theprescription of HER2-targeted therapy would be eventuallyinformed by the results of IHC assays.

Conclusion

Many oncologists seem to believe that pathologists arenow infallible in assessing HER2 status of breast cancer,and that a very accurate identification of patients who arecandidates for HER2-targeted therapies may be informed bythe pathologic report. Accordingly, oncologists are now al-ready asking the next clinical question, whether it is possi-ble to identify among the patients who are candidates thosewho will actually respond to the different anti-HER2 in-terventions.23 Preclinical data and initial findings in retro-spective series of patients have already identified a numberof potential predictive markers whose clinical validation iseagerly awaited.24-27

In this scenario, it is frustrating and irritating that somany controversial aspects of basic HER2 testing still need

to be addressed and eventually solved before we can moveforward to try and predict the differential responsiveness ofHER2-positive breast carcinomas to targeted treatments.Ongoing trials in the field, including proposals to study theeffect of HER-2 targeted therapies in patients with lowHER2 status, will hopefully clarify some of these debatedissues.

Until now, we have accepted without major criticisms thearbitrary thresholds for a HER2-positive tumors and thescoring rules dictated by earlier assumptions and dogmas.Therefore, patients with tumors showing 10% or fewerIHC-positive cells, patients with an overall HER2:chromo-some17 ratio less than 2 despite the presence of geneticheterogeneity, or patients diagnosed as polysomic have beendenied access to the clinical trials of HER2-targeted thera-pies and they are not candidates for these treatments in theclinical practice. A few of these patients, however, have beeninadvertently enrolled in the clinical trials of adjuvanttrastuzumab and they actually showed a similar benefitfrom the targeted therapy as the right patient population.5

This should be taken as a strong signal for us to reconsiderat least some of the assumptions and dogmas that haveoriginated many of the current controversies in HER2 test-ing, with the aim of improving the accuracy and reproduc-ibility in the selection of patients who are candidates forHER2-targeted therapies, and of getting closer to the iden-tification of patients who are differentially responsive to theincreasing number of therapeutic options.

Author’s Disclosures of Potential Conflicts of Interest

Author

Employment orLeadershipPositions

Consultant orAdvisory Role

StockOwnership Honoraria

ResearchFunding

ExpertTestimony

OtherRemuneration

Giuseppe Viale DAKO GlaxoSmithKline,Roche

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