Trastuzumab As Adjuvant Therapy for Early Breast Cancer

Written By: Sam Savage

Published Date: June 14, 2008 Last Edited: June 14, 2008

By Hicks, David G Kulkarni, Swati

* Context.-Overexpression of human epidermal growth factor receptor 2 (HER2) is an adverse prognostic parameter for patients with breast cancer. Patients with HER2- positive tumors are, however, likely to gain significant benefit from targeted therapy with trastuzumab. Four largescale trials have assessed the efficacy and safety of adjuvant trastuzumab. In all 4 trials, disease-free survival and overall survival were significantly improved with adjuvant trastuzumab (36%-52% and 33%-41%, respectively), and improvements were observed in all patient subgroups. Objective.-To describe, in detail, the clinical benefits of using trastuzumab in the adjuvant setting, and to emphasize the importance this places on the pathologist to correctly identify all patients with HER2- positive tumors with a high degree of accuracy.

Data Sources.-Published literature on both HER2 testing and the adjuvant use of trastuzumab.

Conclusions.-Immunohistochemistry and/or fluorescence in situ hybridization are routinely used to determine HER2 status. Maintaining quality assurance throughout a standardized testing process is essential to achieve accurate and reproducible assay results. Adherence to the new American Society of Clinical Oncology/ College of American Pathologists HER2 testing guidelines will help to ensure correct identification of all patients who may benefit from adjuvant trastuzumab and has significant implications for patient outcomes.

(Arch Pathol Lab Med. 2008;132:1008-1015)

Success in the clinical setting has led to trastuzumab (Herceptin; Genentech, Inc, South San Francisco, Calif) becoming an important part of the adjuvant treatment algorithm for patients with human epidermal growth factor receptor 2 (HER2)-positive disease.1 Given the significant clinical benefits available to patients with HER2-positive breast cancer, it is of paramount importance to accurately identify all patients eligible for this therapy. Input from pathologists early in the treatment decision process is therefore vital to determine tumor HER2 status. Here we provide an overview of trastuzumab clinical data in the adjuvant setting and discuss the issues that pathologists may face when identifying patients who may benefit from this therapy.

Breast cancer is the most common form of cancer in women, and nearly half a million new cases are expected to be diagnosed every year in the United States and Europe alone.2 Although in recent years there has been a downward trend in the incidence of breast cancer, it will still account for 25% to 30% of new cancer cases and mu16% of cancer-related deaths in women.2,3 Of these patients, 25% to 30% will have HER2 gene amplification or protein overexpression.4 This has an important bearing on disease course and prognosis, as HER2-positive breast cancer is aggressive and associated with poor clinical outcomes.4-7 Although normal levels of HER2 are important for physiologic cellular function, overexpression of HER2 promotes receptor activation, increased signaling, and excessive cellular division.8 HER2-positive tumor cells therefore have increased proliferation and survival characteristics that typically result in aggressive tumors.4,9-12 As adverse pathologic prognostic factors (higher grade, larger size, lymph node involvement, increased proliferative index) are also associated with HER2 status, this further emphasizes HER2 status as a key molecular marker, which drives both the biology of the tumor as well as the clinical course for the disease.

TRASTUZUMAB

Trastuzumab is an anti-HER2 monoclonal antibody that has been used for the treatment of HER2-positive breast cancer in 420 000 patients worldwide during the last decade (Genentech, data on file). Although the mechanisms of action for trastuzumab are not yet fully defined, key mechanisms include effects on both the extracellular and intracellular domains of the HER2 receptor. Action via the extracellular domain results in antibody-dependent cellmediated cytotoxicity and constant HER2-receptor inhibition. Subsequent actions via the intracellular domain lead to downstream signal transduction inhibition, cellcycle arrest, reduction in angiogenesis, and inhibition of extracellular domain cleavage resulting in HER2-positive cell stasis and death.13-23

Trastuzumab has an antiproliferative effect, and patients with tumors that overexpress the HER2 protein, most commonly resulting from amplification of the HER2 gene, gain the most clinical benefit from this treatment.24-27 Trastuzumab is indicated for use with paclitaxel as firstline treatment or as monotherapy for patients with metastatic breast cancer who have previously received chemotherapy. 28

More recently, 4 large trials have assessed the benefits of adjuvant trastuzumab either in combination with or following chemotherapy for early-stage breast cancer. Results obtained from the 2 randomized, controlled clinical trials in the United States have led to the US Food and Drug Administration (FDA) approving trastuzumab for use in the adjuvant setting. Trastuzumab can now be included as part of a treatment regimen containing doxorubicin, cyclophosphamide, and paclitaxel for patients with HER2- positive, node-positive breast cancer in the United States.28

ADJUVANT TRASTUZUMAB

Trial Design

Four large-scale trials have assessed the efficacy and safety of trastuzumab use in the adjuvant setting. These are the National Surgical Adjuvant Breast and Bowel Project (NSABP) B-31 trial (US; N = 2043); the North Central Cancer Treatment Group (NCCTG) N9831 trial (US; N = 1633); the Herceptin Adjuvant (HERA) trial (non-US; N = 5102); and the Breast Cancer International Research Group (BCIRG) 006 trial (worldwide; N = 3222).

The primary endpoint was disease-free survival (DFS) in all of these multicenter, randomized trials. The adjuvant trial designs are summarized in Figure 1. In general, patients with node-positive and HER2-positive breast cancer were enrolled. Definitions of HER2 positivity are discussed in a later section.

The NSABP B-31 trial compared the outcomes of anthracycline- cyclophosphamide (AC; doxorubicin and cyclophosphamide) with or without trastuzumab.29 Patients were randomized to 1 of 2 arms. Arm 1 received doxorubicin (60 mg/m^sup 2^) and cyclophosphamide (600 mg/ m^sup 2^) for four 3-week cycles, followed by paclitaxel (175 mg/ m^sup 2^) for four 3-week cycles. Arm 2 received the same chemotherapy regimen plus concomitant trastuzumab, administered as a loading dose (4 mg/kg) with the first dose of paclitaxel, then as weekly doses of 2 mg/kg for 51 weeks.

In the NCCTG N9831 trial, patients were randomized among 3 arms.29 Arm A received AC therapy (as in NSABP B-31 arm 1) followed by 12 weekly doses of paclitaxel (80 mg/m^sup 2^). Arm B received the same chemotherapy regimen as arm A, followed by a course of post- pac litaxel trastuzumab therapy. Trastuzumab was given as a loading dose (4 mg/kg) followed by weekly doses of 2 mg/kg for 51 weeks. Arm C received the same chemotherapy regimen as arm A with the addition of concomitant trastuzumab (dosed as described for arm A).29

In the NSABP B-31 and NCCTG N9831 trials, the control arms (arms 1 and A, respectively) and treatment arms 2 and C, respectively, are identical; therefore, the FDA approved a joint analysis of the comparable arms.29

In the HERA study, patients who had previously received a predefined neoadjuvant or adjuvant chemotherapy regimen (for a minimum of 4 cycles or 4 months) with or without radiation therapy were randomized to 1 of 3 arms.30 Arm A received no trastuzumab (observation only control arm). Treatment arms B and C received a loading dose of trastuzumab (8 mg/kg) followed by 6 mg/kg in 3-week cycles for 1 year (arm B) or 2 years (arm C).30,31

In the BCIRG 006 study, patients were randomized among 3 treatment arms.32 As with the NSABP B-31 and NCCTG N9831 trials, the BCIRG 006 study design was based on four 3-week cycles of AC therapy, followed by four 3-week cycles of taxane therapy. Doxorubicin (60 mg/m^sup 2^) was used in combination with cyclophosphamide (600 mg/m^sup 2^), and docetaxel (100 mg/m^sup 2^) replaced paclitaxel in the taxane phase of chemotherapy for arm A and arm B. After completion of docetaxel therapy, arm B also received weekly trastuzumab for up to 1 year (2 mg/kg). Arm C assessed a nonanthracycline chemotherapy combination, as anthracyclines have been associated with an increased risk of cardiac dysfunction in patients receiving concurrent trastuzumab.26,33,34 Arm C was based on the synergy among the 3 agents and efficacy in treating metastatic disease9 and comprised six 3-week cycles of docetaxel (T) (75 mg/m^sup 2^) and carboplatin (C) (AUC6) with a weekly standard dose of trastuzumab (H) during chemotherapy (2 mg/kg), switching to 3-week cycles for 1 year of follow-up. This arm is referred to as the TCH arm.

Efficacy Data

In all 4 adjuvant trials, DFS was significantly higher in the trastuzumab-containing arms compared with control/ observation arms (Figure 2). In the updated joint analysis of NSABP B-31 and NCCTG N9831 (median follow-up of 2.9 years), the addition of trastuzumab to adjuvant chemotherapy significantly improved DFS by 52% compared with chemotherapy alone (hazard ratio [HR] = 0.48; 95% confidence interval [CI], 0.41-0.57; P < .001).35 This improvement in DFS is consistent with an earlier interim analysis (52% HR = 0.48; P < .001).29 In a 2-year follow-up of the HERA trial, DFS was significantly improved by 36% (P < .001) with trastuzumab versus observation only.31 In the BCIRG 006 trial, addition of trastuzumab to adjuvant chemotherapy reduced the risk of recurrence by 33% (P < .001) and 39% (P < .001) for nonanthracycline- and anthracycline- containing adjuvant chemotherapy, respectively.32 Differences in the trastuzumab- containing arms were not statistically signifi- cant.

In subset analysis, improvements in DFS were observed in all groups assessed. DFS was similar for patients with hormone receptor- positive or -negative tumors, in all patient age groups ranging from younger than 40 years to older than 60 years, regardless of tumor size.29,32,36

Overall survival (OS) was measured as a secondary endpoint in these trials. In the NSABP B-31 and NCCTG N9831 trials, OS was significantly increased by 35% at 4 years (P < .001; HR = 0.65; 95% CI, 0.51-0.84).35 Patients enrolled in the HERA (34%; P = .01) trial also achieved significant OS benefit in the trastuzumab-containing arms.31 In the BCIRG 006 trial, improvement in OS was 41% (P < .001) in the anthracycline-containing arm and 34% (P = .02) in the nonanthracycline arm.32

Additional data are also available for the smaller FinHer (Finland Herceptin) trial. A total of 232 patients with HER2- positive early breast cancer were randomized to receive either docetaxel or vinorelbine with or without a short course (9 weeks) of trastuzumab therapy followed by combination chemotherapy.37 Relapse- free survival was significantly higher (58% improvement) in those patients receiving trastuzumab at 3 years (89% vs 78%; HR = 0.42; P = .01). Although these data are interesting, this trial has limitations. The number of patients enrolled was small, the responses were inconsistent (indicated by the confidence interval range), and the follow-up time was short.

The results from the large adjuvant trials demonstrated that trastuzumab significantly improves DFS and OS compared with adjuvant chemotherapy alone in patients with HER2-positive early breast cancer. The National Comprehensive Cancer Network guidelines currently recommend adding 1 year of trastuzumab to standard adjuvant chemotherapy for HER2-positive breast cancer.1

CARDIAC SAFETY

Overall, there was little imbalance in adverse events among all treatment arms. Trastuzumab was generally well tolerated in all 4 trials, and no unexpected adverse events were reported.29,31,32,38

In the 4 large trastuzumab adjuvant trials, the difference in cumulative incidence of congestive heart failure or death due to cardiac causes was less than the Independent Data Monitoring Committee safety cutoff of 4%. This was assessed in patients receiving trastuzumab-containing treatment compared with non- trastuzumab-containing treatment, indicating acceptable cardiac safety.31,36,39,40 The incidence of congestive heart failure was higher in tras tuzumab-treated patients, but this was generally responsive to standard medical treatment.29,31,32,38 Direct comparisons between trials are difficult due to differences in analysis time points, eligibility criteria, and definition of cardiac events. However, the difference in cumulative incidence of congestive heart failure is typically 1.5% to 2.7% (trastuzumab vs nontrastuzumab arms).31,32,35,40 Potential risk factors for cardiac events have been identified and include age (>/=50 years), the need for hypertension medication, and pretrastuzumab left ventricular ejection fraction levels of 50% to 54%.33,38,41 Furthermore, an NCCTG N9831 substudy of cardiac biomarkers indicated that brain natriuretic peptide and troponin I may be promising as predictors of cardiac toxicity.42

IDENTIFYING PATIENTS ELIGIBLE FOR ADJUVANT TRASTUZUMAB THERAPY

Although each trial had specific eligibility criteria (node status, tumor size, hormone receptor status, prior therapy), all patients must have had confirmed HER2-positive breast cancer. HER2 status was assessed by immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH). Each assay is discussed in detail below. A score of IHC 3+ (more than moderate staining in >> 10% of tumor cells) and a FISH HER2/centromere of chromosome 17 (CEP17) ratio greater than 2.0 were considered positive for trial entry. The criteria used to define HER2 status have recently been reevaluated and incorporated into updated testing guidelines and are discussed below.43 With the increasing utilization of needle core biopsies for the primary diagnosis of breast cancer, it has become common practice to perform HER2 testing on these samples. While there is an advantage to having information on the HER2 status at this earlier time in terms of treatment planning, it remains debatable whether doing predictive marker testing on needle biopsies is clearly preferable to testing the excision specimen.

An Immunologic Approach to HER2 Testing

The research assay developed for patient enrollment into the early trastuzumab clinical trials served as the developmental prototype for 2 FDA-approved commercially available HER2 IHC-based assays, the HercepTest assay (Dako North America, Inc, Carpinteria, Calif) and the Ventana PATHWAY assay (Ventana Medical Systems, Tucson, Ariz). In order to semiquantitively assess HER2 expression, a 4-point scale was developed to record both the pattern and intensity of the staining reaction. Staining is recorded as negative (0 and 1+), equivocal for HER2 overexpression (2+), or strongly positive (3+). Only the degree and completeness of membrane staining are regarded as significant in the evaluation of a HER2 IHC assay. An IHC 3+ score was required for entry into the adjuvant clinical trials. This was defined as 10% of tumor cells showing a thick, complete membrane pattern of staining, based on previously established guidelines. The new American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines have updated the IHC definitions. An IHC result of 3+ is defined as uniform complete cell membrane staining in greater than 30% of invasive cancer cells. A score of IHC 2+ most typically demonstrates a nonuniform or weak intensity membrane staining in greater than or equal to 10% of cells or more rarely, intense membrane staining of less than 30% of cells.43

Although some tumors are readily defined as HER2 positive by demonstrating a diffuse and intense staining reaction, a score of IHC 2+ should be considered an equivocal result. These tumors typically have poor correlation with HER2 gene status, and this result should be considered as inconclusive. Tumors with a HER2 IHC 2+ score should be reflex tested for HER2 gene amplification using FISH, as recommended in the updated guidelines.43

Detecting HER2 Gene Amplification by FISH Analysis

In contrast to quantifying the amount of HER2 protein, FISH analysis is designed to assess HER2 gene copy number and detect gene amplification using fluorescence microscopy. The HER2 gene is located on chromosome 17. Interpretation of FISH assays depends on scoring the exact number of HER2 hybridization signals per tumor cell nucleus using a fluorescent-labeled nucleic acid probe specific for the HER2 gene. In the FDA-approved PathVysion assay (Vysis Inc, Downers Grove, Ill), the ratio of the average number of copies of HER2 to the average number of copies of CEP17 helps to distinguish true gene ampli- fication (HER2/CEP17 >> 2) from pseudo- amplification due to multiple copies of chromosome 17 (polysomy; HER2/CEP17 ratio of <2). The INFORM HER2 assay has a different approach and uses the absolute level of HER2 gene signal. This approach could be considered a nuclear criterion of gene amplification (average number of HER2 gene signals per tumor cell nucleus) as opposed to the chromosomal criterion for gene amplification (HER2/ CEP17 ratio) utilized by the PathVysion assay. Both FISHbased assays use defined cutoffs to identify gene ampli- fication. For entry into the adjuvant trials, FISH positivity was defined as a HER2/CEP17 ratio greater than or equal to 2.

Current guidelines now define HER2-positive tumors by FISH as having an average HER2 gene copy number of more than 6 gene copies per nucleus or as a HER2/ CEP17 ratio of more than 2.2.43 The equivocal range for FISH assays is defined as HER2/CEP17 ratio of 1.8 to 2.2 or an average gene copy number between 4.0 and 6.0.

Approximately 3% of tumors will have a HER2/CEP17 ratio very close to the previous 2.0 boundary.43 Although a FISH ratio of 1.8 to 2.2 has now defined a new subgroup, the clinical significance for the HER2 status for tumors in this range remains unclear. The potential benefit from HER2-directed therapy for patients with tumors that fall within this borderline (equivocal) category has not yet been fully evaluated and requires further study.43 Treatment of these women depends on the overall clinical picture and can be facilitated by a discussion between the pathologist and oncologist.

The incidence of polysomy for chromosome 17 is an additional consideration when performing FISH assays. As this is not fully defined in terms of HER2 status and response to trastuzumab, the updated ASCO/CAP guidelines do not address this issue.43,44

ADDRESSING HER2 TESTING ISSUES: APPLYING GUIDELINES TO CLINICAL PRACTICE

Early analysis of the adjuvant trials with trastuzumab highlighted the importance of accurate HER2 testing. Preliminary data from patients enrolled in adjuvant trastuzumab trials revealed poor concordance between local and central laboratory assessments of HER2 status.45,46 To ensure the enrollment of patients with the highest probability of benefiting from trastuzumab, eligibility criteria for these trials were subsequently modified to require central laboratory confirmation of HER2 status. The potential reasons for this discordance and the importance of accurate HER2 testing are discussed below. Central Versus Local Testing

One of the aims of the NSABP B-31 trial was to assess concordance between local and central laboratories.45 Initially, a local HercepTest IHC 3+ score or gene amplifi- cation by FISH allowed participation in the trial. Central review of the first enrolled patients (n = 104) demonstrated that 79% (82/104) of tumors were IHC 3+ by HercepTest with 21% being discordant. A total of 79% (82/104) of tumors were FISH positive by PathVysion assay, with a 21% discordance rate.45

Importantly, there was less discrepancy identified between HER2 tests when a high-volume laboratory (mean tumors assessed per month, >>100) performed the assay, compared with laboratories with lower throughput. In response to this finding, the eligibility criteria for the NSABP B-31 trial were amended to include only those patients whose tumors were IHC 3+ according to an NSABP-approved reference laboratory or tumors demonstrating gene amplification by FISH from any laboratory. The rate of IHC discordance (via central review) subsequently reduced to ~3% (n = 240). A similar analysis has reported comparable findings for the NCCTG N9831 trial (n = 119) with a discordance rate of 26% (local vs central testing).46 In an additional study comparing local and central laboratory testing, there was a marked difference in rates of false-positive results (local vs central IHC, 14%; local IHC vs central FISH, 16%) and false-negative results (local vs central IHC, 18%; local IHC vs central FISH, 23%).47 The question arises as to why these differences occur. Two key factors are likely to be of prime importance: the standardization of testing protocols and differences in test interpretation based on individual pathologist experience.48

Persons and colleagues49 recently reported results from HER2- related FISH surveys. Interlaboratory reproducibility of HER2 analysis was high for tumors with no ampli- fication or high amplification of the HER2 gene. But, as with IHC analysis, the challenge of assessing tumors with low-level or equivocal/ borderline amplification was highlighted as problematic. Testing with FISH found 92% concordance between local and central laboratories versus 77.5% with local versus central IHC. Fluorescence in situ hybridization analysis may therefore be more accurate when central laboratory testing is unavailable.50

Discordance Between Methodologies

Immunohistochemistry 3+ staining and gene amplifi- cation by FISH can both be used to identify those patients who are most likely to benefit from trastuzumab therapy. In theory, IHC and FISH assays should give complementary results with the advantages of each providing additional supportive interpretative data. This is especially important for those patients with equivocal test results.

In practice, however, discordant results (IHC 3+/FISH negative or IHC <3+/FISH positive) between the 2 methodologies have been reported. Discussion is ongoing as to whether this is due to real biologic differences in protein (HER2 receptor) versus gene expression or an artifact of the assay methodology. A number of plausible explanations have been put forth in the literature to try to explain the reported discrepancies encountered between HER2 IHC and FISH. For example, variation in HER2 testing could occur due to adverse effects of variable fixation.51,52 This would tend to have a more detrimental effect on IHC assays given the stability of DNA versus HER2 protein. The potential role of polysomy for chromosome 1753-56 and its effect on HER2 gene expression remains to be fully defined and is a likely explanation for some cases of discrepancy between FISH and IHC. Finally, the inherent subjectivity and reproducibility of IHC scoring criteria, 51,57,58 along with differences in sensitivity and specificity between antibodies used for IHC assays,7,51,59 have been put forth as another explanation for some cases of discordant results.

Despite the lack of concordance that can be encountered between the 2 methodologies, when properly performed and interpreted, patients with tumors characterized as either IHC 3+ (independent of FISH status) or FISH positive (independent of IHC status) had comparable times to progression and overall survival with a trastuzumab-containing therapy.

Of interest is the fact that despite the underlying biology, concordance rates similar to those seen in frozen tumors have not been reported when using IHC for HER2 evaluation in formalin-fixed tissue samples. Good correlation has been reported for HER2 protein/ gene results in formalin-fixed, paraffin-embedded tumor samples by many investigators. Others, however, have reported significant discordant results between FISH and IHC and have questioned the accuracy of IHC HER2 testing in formalin- fixed tissues.7,60-66 The FDA approval for both the IHC and FISH assay methodologies requires that all tissues should be fixed in 10% neutral buffered formalin. Non-formalin-based fixatives or alternative fixation methodologies for breast specimens are discouraged as performance data are limited and extrapolation from formalin- fixed data can be unreliable.43 Current guidelines suggest that tissue fixation should occur as soon after acquisition as possible and that fixation for 6 to 48 hours is optimal. Furthermore, tissue should be transported from the operating room to the pathology department within 30 minutes and an immediate gross examination should be performed to facilitate rapid fixation of the tissue sample. How do these findings and subsequent recommendations impact on pathology laboratory practice? To identify all patients who may benefit from trastuzumab, rigorous quality control together with standardization of reagents, assay performance, and test interpretation are needed to improve agreement between HER2 IHC and FISH results in the clinic.

Adherence to strict laboratory quality control, improvements in tissue fixation and embedding procedures, standardization of IHC methodologies, and the use of automated image analysis to remove some of the subjective nature of assay interpretation may all increase the accuracy of IHC testing for HER2 status.60,67 Computerized automated cellular imaging systems are now commercially available and can improve the precision and reliability of analysis. The high degree of correlation between IHC analysis using automated cellular imaging systems and results obtained using FISH highlights that mistakes in manual IHC interpretation may contribute to discordant results.68 These findings underline the need for standard ization of all aspects of the HER2 testing process from tissue acquisition to accurate assay interpretation and reporting.

The Future of HER2 Testing: Toward Greater Accuracy

In addition to the need to reappraise the HER2 algorithm and assay result definitions, issues surrounding test variation and accuracy were a key driver for the development of the recent ASCO/ CAP task force guidelines. It is important that all pathology laboratories establish a high degree of quality assurance by maintaining high concordance rates (>/=95%) between the 2 assay methodologies and offer only fully validated IHC and FISH tests for clinical decision making. Assay validation must include using comparable and established fixation and tissue handling protocols (in at least 25-100 tumors), demonstrating a high degree of concordance against a previously validated assay methodology. Laboratories must also routinely record a standardized set of reporting elements (methods, dates, controls, etc) for both test methodologies. Pathologists will also undergo ongoing assessments to ensure correct and accurate interpretation of assays. Furthermore, from 2007 onward, external assessment of HER2 testing accuracy in CAP-accredited laboratories will demand performance above 90% (via proficiency testing). Standard laboratory inspection will assess all aspects of the HER2 testing protocol from tissue handling through test validation to assay interpretation as a part of laboratory accreditation.

A key conclusion of the ASCO/CAP expert consensus panel was that the 2 methodologies for assessing HER2 status are both acceptable for identifying patients who are likely candidates for response to trastuzumab therapy. The important point is that the tests must be properly performed and interpreted to identify the eligible patients. Both methodologies have performance and interpretive challenges, and they should be viewed as complementary rather than one versus the other.

FUTURE DIRECTIONS

Early preliminary data from the NSABP B-31 trial suggest that benefit from adjuvant trastuzumab may not be confined to those patients with HER2-positive tumors (IHC 3+ and FISH positive [HER2/ CEP17 ratio >> 2.2]) using our current definitions for HER2 status.69 In this analysis, as expected, those patients with HER2- positive tumors gained benefit from trastuzumab. In addition, there was a limited subset of patients who had tumors that were FISH negative and graded less than IHC 3+ who also achieved significant benefit (P = .03) from adjuvant trastuzumab. Identifying potential differences will help explain why these patients responded to trastuzumab despite testing negative for HER2. These data suggest that the criteria pathologists use to identify HER2- positive tumors may need to be reevaluated and/or modified in the adjuvant setting, but clearly more research is needed. These data are intriguing but preliminary, and caution should be exercised in interpreting the results until further data on more patients can be analyzed.

Current HER2 testing algorithms may need to be modified to account for low-level amplification and other genotypic abnormalities that can be seen in some tumors. Kaufman and colleagues70 demonstrated that tumors with increased chromosome 17 polysomy (low-level amplification; see below), but conventionally defined as HER2 negative for amplification by the HER2/CEP17 ratio, may responded favorably to trastuzumab-based therapy. These data raise the possibility that the HER2/CEP17 ratio may not be the best way to evaluate the HER2 status in all cases and that the absolute HER2 gene copy number (whether increased through amplification or polysomy) may be the more important determinant for trastuzumab response for some patients. Together, these studies suggest the intriguing possibility that even when a patient has a FISH-negative tumor (and

SUMMARY

The significant gains in DFS and OS from adjuvant trastuzumab have been demonstrated in 4 large trials. It is vital that all patients eligible for treatment are accurately identified. Pathologists play a central role in this process.

Immunohistochemistry and FISH are both valid methodologies for assessing HER2 status. Studies showing a lack of concordance between HER2 testing results from different laboratories emphasize the need for standardization in methodology and rigorous quality control. The ASCO/CAP guidelines provide useful recommendations for optimizing HER2 testing, and strict adherence to these guidelines, combined with new testing technologies, should ensure improved accuracy and reproducibility of HER2 testing and ultimately lead to improvements in patient outcomes.

Support for third-party writing assistance for this article was provided by Genentech, Inc.

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David G. Hicks, MD; Swati Kulkarni, MD

Accepted for publication January 8, 2008.

From the Department of Pathology and Laboratory Medicine, University of Rochester, Rochester, NY (Dr Hicks); and Department of Surgical Oncology, Roswell Park Cancer Institute, Buffalo, NY (Dr Kulkarni).

The authors have no relevant financial interest in the products or companies described in this article.

Reprints: David G. Hicks, MD, Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, 601 Elmwood Ave, Box 626, Rochester, NY 14642 (e-mail: David=Hicks@urmc. rochester.edu).

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