Pathologic Complete Response to Trastuzumab-Based Neoadjuvant Therapy Is Related to the Level of HER-2 Amplification

The human epidermal growth factor receptor-2 (HER-2) gene, which plays an important role in tumor formation and growth processes, is amplified in approximately 20% to 30% of all breast cancers (1, 2). Patients whose tumors overexpress HER-2 are more likely to experience a shorter time to relapse and a significantly lower overall survival rate (1, 2). Treatment with trastuzumab (Herceptin), a recombinant monoclonal antibody against HER-2, results in significant clinical benefits in patients diagnosed with HER-2–positive disease. In phase II/III trials, trastuzumab significantly improved survival by up to 8.5 months when given as first-line treatment in combination with a taxane (3, 4) in women with HER-2–positive metastatic breast cancer. In five major adjuvant clinical trials involving >13,000 women with HER-2–positive early breast cancer, trastuzumab significantly reduced the risk of recurrence and improved overall survival by one third (5–8).

In the neoadjuvant setting, primary systemic therapy with trastuzumab-based combination chemotherapy has also shown clinical benefit in terms of both overall response and pathologic complete response rates (9–13). The goals of primary systemic therapy are to treat occult systemic disease and decrease tumor size, thus allowing for breast-conserving surgery (14). Primary systemic therapy with trastuzumab may prove particularly beneficial for women with HER-2–positive locally advanced breast cancer and, as such, accurate and efficient HER-2 status testing should be done at the earliest opportunity after diagnosis (10, 15, 16).

Currently, both immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are the established HER-2 testing methods. IHC detects the amount of HER-2 protein expressed on the surface of cells and relies on a qualitative scoring system from 0 to 3+, with 0 to 1+ being negative for HER-2 overexpression, 2+ being borderline, and 3+ indicating positive HER-2 status. FISH detects amplification of the HER-2 gene and thus tumors are interpreted as HER-2 negative or positive by counting the number of HER-2 gene copies. IHC is relatively simple and cost effective; however, results are reliant on several variables and can be associated with interpretation errors (17–19). In the North American clinical trials of adjuvant trastuzumab, a moderately high level of false-positive HER-2 status cases were initially reported in patients tested locally with IHC (20, 21). However, if IHC procedures are standardized and calibrated with FISH, a high degree of concordance between IHC and FISH exists (22, 23). Other studies have also reported relatively good concordance between IHC 3+ and FISH-positive as well as IHC 0/1+ and FISH-negative scores but differences between the two assays for cases surrounding the IHC 2+ score (18, 24, 25). The best way to assess HER-2 status for trastuzumab therapy is still in debate (26).

Trastuzumab-based neoadjuvant studies that enrolled both IHC 2+ and 3+ patients showed that patients who scored IHC 3+ were more responsive to trastuzumab treatment than those who scored IHC 2+ (11, 27). In clinical practice, most IHC 2+ scores are followed by additional FISH testing to determine HER-2 status accurately. As pathologic complete response is often predictive of postsurgical disease-free survival and overall survival, we sought to ascertain whether a relationship between level of HER-2 amplification, as assessed by FISH, and pathologic complete response existed in women diagnosed with HER-2–positive locally advanced breast cancer who were treated preoperatively with a combination of trastuzumab plus chemotherapy.

Patients. Breast biopsies from 93 patients who had received trastuzumab in combination with chemotherapy as primary systemic therapy for operable, HER-2–positive, stage II/III breast cancer were collected from 19 centers in France. All patients provided written, informed consent for their tissue material and clinical data to be centrally collected and used for research purposes.

Patients were aged 27 to 67 years and had unilateral, histologically confirmed, T₂ or T₃, N_0-1, nonmetastatic, noninflammatory, HER-2–positive breast cancer requiring mastectomy. Most patients were treated preoperatively in two open-label, phase II clinical trials: TAXHER01 (n = 21) and GETNA01 (n = 61; refs. 11, 13). An additional 11 patients who had been treated with the same preoperative regimen as in the TAXHER01 trial were also included in the analyses.

All patients had received weekly neoadjuvant trastuzumab (4 mg/kg loading dose followed by 2 mg/kg once weekly) in combination with either docetaxel (100 mg/m² every 3 weeks for six cycles; n = 32) or docetaxel (75 mg/m² every 3 weeks for six cycles) plus carboplatin (area under the curve of 6 every 3 weeks for six cycles; n = 61). Three weeks after the last course of trastuzumab-based neoadjuvant treatment, pathologic complete response was assessed according to Chevallier's classification (28); no carcinoma evidence either in the breast or in the lymph nodes, with or without in situ carcinoma, was considered as pathologic complete response.

HER-2 testing. All patients had initially tested IHC 3+ for HER-2 status, as determined by local participating centers. For this study, HER-2 status was analyzed retrospectively and centrally assessed using both IHC and FISH.

Central IHC analyses for HER-2 overexpression were done with A485 polyclonal antibody (dilution 1:1,800) on the BenchMark XT system (Ventana Medical Systems, Inc.). Biopsies were graded according to the HercepTest (DAKO) scoring system (0, 1+, 2+, or 3+). FISH analyses were done using the HER-2 Probe (Oncor) and BenchMark XT system. The pathologist who did FISH analyses was blinded to all other patient data, including the original and central IHC test results. For each tumor, the mean of HER-2 signals was calculated by counting signals in ≥60 tumor cell nuclei. The degree of HER-2 amplification in tumors, as assessed by single-color FISH, was classified as follows: no amplification (mean, <6 signals/nuclei); low amplification (mean, 6-10 signals per nuclei); or high amplification (mean, >10 signals/nuclei or uncountable due to clusters of signals). Several studies have proposed the cutoff value of six copies between no amplification and amplification as appropriate for single-color FISH (29–31) and this is the cutoff recommended by the American Society of Cinical Oncology/College of American Pathologists (32). The cutoff of 10 gene copies number per nuclei between low and high amplification is somewhere arbitrary but it was chosen because it is the same are those proposed with chromogenic in situ hybridization (33–35). It was also chosen because over this cutoff, due to clusters and small aggregates, signals are almost always not possible to be precisely counted.

Borderline tumors (mean approaching six signals per nuclei) were analyzed by double-color FISH using a HER-2 gene–specific probe and a centromeric probe for chromosome 17 (PathVysion HER-2 DNA Probe kit, Vysis-Abbott) to determine HER-2 amplification. In these cases, HER-2 amplification was defined by a ratio of HER-2 over chromosome 17 centromeric signals of ≥2.2 (32).

The scores obtained by FISH for each tumor were subsequently compared with multiple patient and tumor variables (including treatment regimen, patient age, tumor-node-metastasis staging, and IHC score) and pathologic complete response rates to determine if FISH testing could predict more accurately patient response to neoadjuvant treatment with trastuzumab plus chemotherapy. The subpopulation of patients eligible for trastuzumab treatment (IHC 3+ and/or FISH positive) was also analyzed for variables predictive of pathologic complete response.

Statistics. All analyses were done with Stata software. Univariate analyses were done using Pearson's χ² or Fisher's exact test with a bilateral 5% type I error. Initial analyses involved the whole studied population. Analyses were then done on the subpopulation of patients eligible for trastuzumab treatment [central IHC 3+ (n = 76) and central IHC 2+ with FISH amplification (n = 1)] to study the prognostic factors of pathologic complete response. Logistic regression analysis was used to estimate odds ratios and two-sided 95% confidence intervals. In the model of logistic regression, we included variables with a P < 0.05 in the univariate analysis; treatment and Scarff-Bloom-Richardson grade were also included because of their clinical relevance.

Patients. Baseline patient demographics are summarized in Table 1. All patients had invasive breast cancer, with most patients having T₂, N_0-1, and grade 2/3 disease. No patients showed evidence of metastatic disease. Mean age was 46 years (range, 27-67 years). According to Chevalier's classification, 35 of 93 (37.6%) patients had a pathologic complete response, whereas 57 of 93 (61.3%) patients experienced pathologic partial or no response.

HER-2 testing: comparison of FISH and IHC results. FISH analysis rated 50 (53.8%) tumors as high amplification, all of which scored 3+ on central IHC review, with >80% of labeled cells in all cases. Of the 27 (29%) tumors categorized as low amplification, 1 (4%) was 2+ on central IHC review and the remaining tumors (96%) were 3+, with >80% of labeled cells in all cases. In this category of low-amplification tumors, the mean signals per tumor nuclei ranged from 6.9 to 9.8, with a mean of 8.6 for the 27 tumors. For two tumors with the lowest mean (6.9 and 7.2), amplification was confirmed by double-color FISH, with the ratio HER-2/chromosome 17 being >2.2.

Sixteen (17.2%) tumors were categorized as no amplification, with 11 (69%) rated 1+ on central IHC review and 5 (31%) rated 2+. Mean signals per tumor nuclei was 2.6 (range, 1.1-5.2) for the 16 tumors in this category. The absence of amplification for the tumor with the highest mean (5.2) was confirmed by double-color FISH, with the ratio HER-2/chromosome 17 being <1.8.

Analysis of tumor characteristics according to FISH results. There were no significant differences between the three groups of FISH amplification (no amplification, low amplification, and high amplification) in terms of treatment given, age, or T or N stage (Table 2). As expected, amplification of the HER-2 gene as assessed by FISH correlated with tumor grade, with significantly more grade 3 tumors displaying high HER-2 amplification [28 of 49 (56%); P = 0.021]. High amplification of HER-2 also correlated with hormone receptor status: a significant number of hormone receptor–negative tumors [27 of 36 (75%)] were classified as high amplification and no hormone receptor–negative tumors were classified as no amplification (P = 0.001).

Central IHC testing revealed no HER-2 1+ tumors displaying FISH amplification, 1 of 6 HER-2 2+ tumors displaying low amplification, and all HER-2 3+ tumors displaying low amplification or high amplification (P < 0.0001). Complete pathologic response was significantly related to presence of HER-2 amplification, with pathologic complete response observed in 1 of 16 (6%) no-amplification tumors, 6 of 27 (22%) low-amplification tumors, and 28 of 50 (56%) high-amplification tumors (P < 0.001).

Analysis of variables that can predict pathologic complete response. Tumor characteristics according to pathologic complete response are summarized in Table 3. There was no relationship between pathologic complete response and treatment regimen, patient age, T or N stage, or Scarff-Bloom-Richardson grade. Pathologic complete response was significantly more frequent in FISH-amplified tumors (44%) than in nonamplified tumors (6%; P = 0.004). As expected, pathologic complete response also occurred significantly more frequently in HER-2 3+ tumors (P = 0.009). There was also a trend toward pathologic complete response in hormone receptor–negative tumors (P = 0.051).

Analysis of variables predictive of pathologic complete response in patients eligible for trastuzumab treatment after central HER-2 testing.Table 4 summarizes the analysis of factors that might predict pathologic complete response in the subpopulation of tumors that were IHC 3+ and/or FISH amplified (n = 77) and thus eligible for treatment with trastuzumab. In this population of tumors, the only variable related to pathologic complete response was the level of HER-2 amplification as assessed by FISH, with 28 of 50 (56%) high-amplification tumors showing pathologic complete response compared with 6 of 27 (22%) low-amplification tumors (P = 0.004). Treatment regimen, patient age, T or N stage, Scarff-Bloom-Richardson grade, hormone receptor status, and IHC score were not significantly related to pathologic complete response.

Multivariate analysis. Results of the multivariate analysis done with logistic regression are summarized in Table 5. In this model, we combined treatment regimen (to exclude treatment effect), tumor grade, and level of HER-2 amplification to determine which variables were related to pathologic complete response. Results show that only level of amplification as assessed by FISH was related to pathologic complete response (P = 0.01).

To our knowledge, this is the first study to show a positive correlation between level of HER-2 amplification, as assessed by FISH, and rate of pathologic complete response to trastuzumab-based neoadjuvant treatment in locally advanced HER-2–positive breast tumors.

Several phase II studies of trastuzumab plus chemotherapy as primary systemic therapy have shown substantially improved response rates in this population of patients at high risk for relapse, with most overall response rates ≥75% (for detailed review, see ref. 13). However, pathologic complete response rates, which are often considered more indicative of disease-free and overall survival, have been more variable, ranging from 7% in one small scale study (n = 14) of trastuzumab plus docetaxel and epirubicin to >40% in several larger studies (for detailed review, see ref. 13). Thus, any baseline factor that could be used to help in predicting which patients would benefit most from trastuzumab-based neoadjuvant regimens would be invaluable to clinicians.

In our study, the only variable related to pathologic complete response in the subpopulation of patients eligible for treatment with trastuzumab (IHC 2+/3+ and/or FISH positive) was the level of HER-2 amplification as assessed by FISH. Two neoadjuvant studies of trastuzumab plus a taxane, which enrolled both IHC 2+ and 3+ patients, have shown that patients who scored IHC 3+ were more likely to respond to trastuzumab treatment than those patients who scored IHC 2+ for HER-2 overexpression (13, 27). Although all FISH-positive tumors were centrally confirmed as IHC 2+ or 3+ in this study, our classification of HER-2 amplification by FISH was more precise in identifying those patients who would have benefited most from trastuzumab. Indeed, pathologic complete response was seen significantly more frequently in high-amplification FISH tumors compared with low-amplification tumors in patients who were eligible to receive trastuzumab treatment. This degree of subclassification with regard to HER-2 status would not have been possible using IHC. Therefore, FISH may be a more accurate HER-2 testing method to predict pathologic complete response in the neoadjuvant setting.

Interestingly, our results are in line with retrospective analyses done on patients enrolled in the pivotal trials of trastuzumab in HER-2–positive metastatic breast cancer. Whereas patients with IHC scores of 2+ and 3+ were initially eligible for the phase III trials, subset analysis by Slamon et al. (4) revealed that the clinical benefits of trastuzumab plus paclitaxel were greatest in patients whose tumors were graded IHC 3+. Furthermore, retrospective analyses revealed that patients who tested HER-2 positive by FISH experienced significantly better response rates and improved survival with trastuzumab than those patients whose tumors were FISH negative. This suggests that FISH may predict more accurately which patients will obtain the most clinical benefit from trastuzumab therapy in the metastatic setting (36).

Although the number of cases included in this study is not large and despite the size of the confidence intervals, our analysis shows statistically significant differences between the two groups of amplification in term of pCR. These results have certainly to be confirmed in the future in a larger series. Moreover, our results are in concordance with those published recently by Giuliani et al. (37) in metastatic HER-2–positive breast cancer treated with trastuzumab alone or combined with chemotherapy (mainly paclitaxel). In the group of patient treated with trastuzumab and chemotherapy, they have shown a significantly positive correlation between level of HER-2 gene amplification and the clinical objective response.

Nevertheless, in a recent neoadjuvant study by Hurley et al. (38) evaluating trastuzumab plus docetaxel and cisplatin as primary systemic therapy for patients with HER-2–positive (IHC 2+/3+ or FISH positive) locally advanced breast cancer, no significant difference in pathologic complete response rates according to FISH status (FISH positive versus FISH negative) was found. Unexpectedly, FISH status also did not affect rates of either progression-free or overall survival in this study. Although our results seem to contradict this study, the population analyzed may be different. Indeed, IHC testing, used for the patient screening, seemed very sensitive in the Hurley et al. study, as only 60% of IHC 3+ tumors were FISH positive, whereas reported rates in the literature are ∼90% (18, 24, 25, 39). Although our study did not evaluate survival, these differing results about the relationship between FISH positivity and pathologic complete response highlight the need to standardize both IHC and FISH procedures for determination of HER-2 gene status.

Currently, there is variability with respect to HER-2 testing in terms of a particular commercial kit, antibody, probe, or cutoff value, and discrepancies due to variability between kits may occur (40). In terms of cutoff values with FISH amplification, our results, which were obtained on clinicopathologic data, are in line with those obtained with biological data in a recently published study by Dal Lago et al. (29) and those proposed recently by the American Society of Clinical Oncology and the College of American Pathologists (32). In the Dal Lago et al. study, cutoff values of 4 and 6 copies for no amplification and amplification were evaluated with single-color FISH. Good correlation only existed between the cutoff value of 6 and their mRNA assay.

The results of our study also highlight that accurate HER-2 testing is essential for optimal patient management with trastuzumab. In the neoadjuvant setting, initial IHC screening (which would be negative for the majority for tumors) could be used, followed by FISH testing for IHC 2+ or 3+ tumors. This supplementary FISH testing would exclude false-positive tumors by confirming HER-2 amplification and precisely determine the level of HER-2 amplification. This need of accuracy was particularly emphasized in two large trials that have shown discordance between local and central HER-2 testing for IHC (20) or for IHC and FISH (41).

In conclusion, our study showed a significantly positive correlation between pathologic complete response rate and level of HER-2 amplification as assessed by FISH in stage II/III breast tumors. These findings may have clinical implications for the management of patients with HER-2–positive locally advanced breast cancer. The degree of amplification as assessed by FISH may be an additional source of information for physicians wishing to clarify individual risk-benefit assessments for critical patients considering preoperative treatment with trastuzumab-based chemotherapy. However, it remains unknown whether a relationship exists between pathologic complete response, HER-2 level of amplification as assessed by FISH, and postoperative survival rates in women with HER-2–positive locally advanced breast cancer who are treated preoperatively with trastuzumab-based regimens. Further studies on the effect of level of HER-2 amplification on the behavior of tumors treated with trastuzumab in both the metastatic and adjuvant breast cancer settings are also needed.

We thank the Ligue de Cote d'Or Contre le Cancer, C. Harris, and D. James for assistance in manuscript preparation; Drs. J. Garnier and F. Campana (Roche Laboratories); Prof. N. Namer and P. Atalli (GETNA); and G. Milla (OSMO).