Cancer Therapy: Clinical

Pathologic Complete Response to Trastuzumab-Based Neoadjuvant Therapy Is Related to the Level of HER-2 Amplification Laurent Arnould,1Patrick Arveux,1Jerome Couturier,2 Marion Gelly-Marty,1Catherine Loustalot,1 Francette Ettore,4 Christine Sagan,5 Martine Antoine,3 Frederique Penault-Llorca,6 BerangereVasseur,7 Pierre Fumoleau,1and Bruno P. Coudert1

Abstract Purpose: Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) are used to determine human epidermal growth factor receptor-2 (HER-2) status and patient eligibility for trastuzumab therapy. Using FISH and IHC, we analyzed the relationship between pathologic complete response to trastuzumab-based neoadjuvant therapy and level of HER-2 amplification in locally advanced breast cancer. Experimental Design: Breastbiopsiesfrom93HER-2^ positivepatients treatedwithtrastuzumab- based neoadjuvant therapy were centrally collected and analyzed retrospectively for HER-2 amplification using FISH and HER-2 overexpression using IHC. Tumors were classified by FISH as no, low, or high amplification. Biopsies were reassessed centrally by IHC and graded 0, 1+, 2+, or 3+. Results: HER-2 status of tumor samples as assessed by FISH and IHC correlated: 16 no amplifi- cation (11IHC 1+ and 5 IHC 2+), 27 low amplification (26 IHC 3+ and 1IHC 2+), and 50 high amplification (all IHC 3+). Trastuzumab-based neoadjuvant therapy achieved pathologic complete response in 35 of 93 (37.6%) tumors. Pathologic complete response rate in low- and high-amplification tumors was significantly higher than in no-amplification tumors (44% versus 6%; P < 0.004). Pathologic complete response rate in high-amplification tumors was significantly higher compared with low-amplification tumors (56% versus 22%; P < 0.005). In the subgroup of low- plus high-amplification tumors, no correlation was found between pathologic complete response rate and IHC score, treatment regimen,Tor N stage, tumor grade, or hormonal receptors. Conclusions: This is the first study to show positive correlation between level of HER-2 amplifi- cation assessed by FISH and rate of pathologic complete response to trastuzumab-based neoadjuvant treatment.

The human epidermal growth factor receptor-2 (HER-2) gene, to 8.5 months when given as first-line treatment in combina- which plays an important role in tumor formation and growth tion with a taxane (3, 4) in women with HER-2–positive processes, is amplified in approximately 20% to 30% of all metastatic breast cancer. In five major adjuvant clinical trials breast cancers (1, 2). Patients whose tumors overexpress HER-2 involving >13,000 women with HER-2–positive early breast are more likely to experience a shorter time to relapse and a cancer, trastuzumab significantly reduced the risk of recurrence significantly lower overall survival rate (1, 2). Treatment and improved overall survival by one third (5–8). with trastuzumab (Herceptin), a recombinant monoclonal In the neoadjuvant setting, primary systemic therapy with antibody against HER-2, results in significant clinical benefits trastuzumab-based combination chemotherapy has also shown in patients diagnosed with HER-2–positive disease. In phase clinical benefit in terms of both overall response and II/III trials, trastuzumab significantly improved survival by up 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 Authors’Affiliations: 1CLCCG-FLeclerc and1FR100, Dijon, France; 2CLCC Institut Curie; 3Ho“ pital Tenon, Paris, France; 4CLCC A Lacassagne, Nice, France; 5Hopital prove particularly beneficial for women with HER-2–positive Lae« nnec, Nantes, France; 6CLCC Jean Perrin, Clermont-Ferrand, France; and locally advanced breast cancer and, as such, accurate and 7Laboratoire Roche-Pharma, Neuilly sur Seine, France efficient HER-2 status testing should be done at the earliest Received 12/20/06; revised 6/26/07; accepted 7/10/07. opportunity after diagnosis (10, 15, 16). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance Currently, both immunohistochemistry (IHC) and fluores- with 18 U.S.C. Section 1734solely to indicate this fact. cence in situ hybridization (FISH) are the established HER-2 Note: Presented in part at the 29th San Antonio Breast Cancer Symposium, testing methods. IHC detects the amount of HER-2 protein San Antonio,TX, December14-17, 2006. expressed on the surface of cells and relies on a qualitative Requests for reprints: Laurent Arnould, Department of Pathology, Centre G-F scoring system from 0 to 3+, with 0 to 1+ being negative for Leclerc, 1 Rue Pr Marion, 21000 Dijon Cedex, France. Phone: 33-380-737-723; Fax: 33-380-737-717; E-mail: [email protected]. HER-2 overexpression, 2+ being borderline, and 3+ indicating F 2007 American Association for Cancer Research. positive HER-2 status. FISH detects amplification of the HER-2 doi:10.1158/1078-0432.CCR-06-3022 gene and thus tumors are interpreted as HER-2 negative or

Clin Cancer Res 2007;13(21) November 1, 2007 6404 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2007 American Association for Cancer Research. Trastuzumab-Based Neoadjuvant Therapy positive by counting the number of HER-2 gene copies. IHC is The pathologist who did FISH analyses was blinded to all other patient relatively simple and cost effective; however, results are reliant data, including the original and central IHC test results. For each tumor, on several variables and can be associated with interpretation the mean of HER-2 signals was calculated by counting signals in z60 errors (17–19). In the North American clinical trials of tumor cell nuclei. The degree of HER-2 amplification in tumors, as adjuvant trastuzumab, a moderately high level of false-positive assessed by single-color FISH, was classified as follows: no amplifica- tion (mean, <6 signals/nuclei); low amplification (mean, 6-10 signals HER-2 status cases were initially reported in patients tested per nuclei); or high amplification (mean, >10 signals/nuclei or locally with IHC (20, 21). However, if IHC procedures are uncountable due to clusters of signals). Several studies have proposed standardized and calibrated with FISH, a high degree of the cutoff value of six copies between no amplification and concordance between IHC and FISH exists (22, 23). Other amplification as appropriate for single-color FISH (29–31) and this is studies have also reported relatively good concordance the cutoff recommended by the American Society of Cinical Oncology/ between IHC 3+ and FISH-positive as well as IHC 0/1+ and College of American Pathologists (32). The cutoff of 10 gene copies FISH-negative scores but differences between the two assays number per nuclei between low and high amplification is somewhere for cases surrounding the IHC 2+ score (18, 24, 25). The best arbitrary but it was chosen because it is the same are those proposed way to assess HER-2 status for trastuzumab therapy is still in with chromogenic in situ hybridization (33–35). It was also chosen debate (26). because over this cutoff, due to clusters and small aggregates, signals are Trastuzumab-based neoadjuvant studies that enrolled both almost always not possible to be precisely counted. IHC 2+ and 3+ patients showed that patients who scored IHC Borderline tumors (mean approaching six signals per nuclei) were analyzed by double-color FISH using a HER-2 gene–specific probe and 3+ were more responsive to trastuzumab treatment than those a centromeric probe for chromosome 17 (PathVysion HER-2 DNA who scored IHC 2+ (11, 27). In clinical practice, most IHC 2+ Probe kit, Vysis-Abbott) to determine HER-2 amplification. In these scores are followed by additional FISH testing to determine cases, HER-2 amplification was defined by a ratio of HER-2 over HER-2 status accurately. As pathologic complete response is chromosome 17 centromeric signals of z2.2 (32). often predictive of postsurgical disease-free survival and overall The scores obtained by FISH for each tumor were subsequently survival, we sought to ascertain whether a relationship between compared with multiple patient and tumor variables (including level of HER-2 amplification, as assessed by FISH, and treatment regimen, patient age, tumor-node-metastasis staging, and pathologic complete response existed in women diagnosed IHC score) and pathologic complete response rates to determine if FISH with HER-2–positive locally advanced breast cancer who were testing could predict more accurately patient response to neoadjuvant treated preoperatively with a combination of trastuzumab plus treatment with trastuzumab plus chemotherapy. The subpopulation of patients eligible for trastuzumab treatment (IHC 3+ and/or FISH chemotherapy. positive) was also analyzed for variables predictive of pathologic complete response.

Materials and Methods

Patients. Breast biopsies from 93 patients who had received Table 1. Baseline patient characteristics trastuzumab in combination with chemotherapy as primary systemic therapy for operable, HER-2–positive, stage II/III breast cancer were Characteristic Patients (n = 93), n (%) collected from 19 centers in France. All patients provided written, Mean age (range), y 46 (27-67) informed consent for their tissue material and clinical data to be Tumor stage centrally collected and used for research purposes. T2 66 (71) Patients were aged 27 to 67 years and had unilateral, histologically T3 21 (22.6) confirmed, T2 or T3,N0-1, nonmetastatic, noninflammatory, HER-2– T4 5 (5.4) positive breast cancer requiring mastectomy. Most patients were treated Unknown 1 (1.1) preoperatively in two open-label, phase II clinical trials: TAXHER01 Nodal status (n = 21) and GETNA01 (n = 61; refs. 11, 13). An additional 11 patients N0 46 (49.5) who had been treated with the same preoperative regimen as in the N1 43 (46.2) N 4 (4.3) TAXHER01 trial were also included in the analyses. 2 Tumor grade All patients had received weekly neoadjuvant trastuzumab (4 mg/kg SBR1 3 (3.2) loading dose followed by 2 mg/kg once weekly) in combination with SBR2 44 (47.3) 2 either docetaxel (100 mg/m every 3 weeks for six cycles; n = 32) or SBR3 41 (44.1) 2 docetaxel (75 mg/m every 3 weeks for six cycles) plus carboplatin (area Unknown 5 (5.4) under the curve of 6 every 3 weeks for six cycles; n = 61). Three weeks Hormone receptor status after the last course of trastuzumab-based neoadjuvant treatment, Positive 51 (54.8) pathologic complete response was assessed according to Chevallier’s Negative 36 (38.7) classification (28); no carcinoma evidence either in the breast or in the Unknown 6 (6.5) Treatment lymph nodes, with or without in situ carcinoma, was considered as TH 32 (34.4) pathologic complete response. TCH 61 (65.6) HER-2 testing. All patients had initially tested IHC 3+ for HER-2 Pathologic response status, as determined by local participating centers. For this study, pCR 35 (37.6) HER-2 status was analyzed retrospectively and centrally assessed using Non-pCR 58 (62.4) both IHC and FISH. Central IHC analyses for HER-2 overexpression were done with A485 Abbreviations: SBR, Scarff-Bloom-Richardson; TH, trastuzumab + polyclonal antibody (dilution 1:1,800) on the BenchMark XT system docetaxel; TCH, trastuzumab + docetaxel + carboplatin; pCR, (Ventana Medical Systems, Inc.). Biopsies were graded according to the complete pathologic response; non-pCR, absence of complete HercepTest (DAKO) scoring system (0, 1+, 2+, or 3+). FISH analyses pathologic response. were done using the HER-2 Probe (Oncor) and BenchMark XT system.

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Table 2. Patient and tumor characteristics according to HER-2 gene amplification

Patients P NA (n = 16), n (%) LA (n = 27), n (%) HA (n = 50), n (%)

Mean age (range), y 46.4 (32-62) 47.6 (29-62) 45.6 (27-67) Treatment TCH 11 (69) 18 (67) 32 (64) TH 5 (31) 9 (33) 18 (36) 0.932 Tumor stage

T2 12 (75) 17 (63) 37 (74) T3 3 (19) 7 (26) 11 (22) T4 0 3 (11) 2 (4) 0.610 Unknown 1 (6) 0 0 Nodal status

N0 9 (56) 9 (34) 28 (56) N1 or N2 7 (44) 18 (66) 22 (44) 0.138 Tumor grade SBR1 1 (6.5) 0 2 (4) SBR2 8 (53.5) 19 (70) 17 (34) SBR3 6 (40.0) 7 (26) 28 (56) 0.021 Unknown 1 (6.0) 1 (4) 3 (6) Hormone receptor status Positive 13 (81) 16 (59) 22 (44) Negative 0 9 (33) 27 (54) 0.001 Unknown 3 (19) 2 (7) 1 (2) Central IHC score 1+ 11 (69) 0 0 2+ 5 (31) 1 (4) 0 <0.0001 3+ 0 26 (96) 50 (100) Pathologic response pCR 1 (6) 6 (22) 28 (56) Non-pCR 15 (94) 21 (78) 22 (44) <0.0001

Abbreviations: NA, no amplification; LA, low amplification; HA, high amplification.

Statistics. All analyses were done with Stata software. Univariate mean of 8.6 for the 27 tumors. For two tumors with the lowest 2 analyses were done using Pearson’s m or Fisher’s exact test with a mean (6.9 and 7.2), amplification was confirmed by double- bilateral 5% type I error. Initial analyses involved the whole studied color FISH, with the ratio HER-2/chromosome 17 being >2.2. population. Analyses were then done on the subpopulation of patients Sixteen (17.2%) tumors were categorized as no amplifica- eligible for trastuzumab treatment [central IHC 3+ (n = 76) and central tion, with 11 (69%) rated 1+ on central IHC review and 5 IHC 2+ with FISH amplification (n = 1)] to study the prognostic factors of pathologic complete response. Logistic regression analysis was used (31%) rated 2+. Mean signals per tumor nuclei was 2.6 (range, to estimate odds ratios and two-sided 95% confidence intervals. In the 1.1-5.2) for the 16 tumors in this category. The absence of model of logistic regression, we included variables with a P < 0.05 in amplification for the tumor with the highest mean (5.2) was the univariate analysis; treatment and Scarff-Bloom-Richardson grade confirmed by double-color FISH, with the ratio HER-2/ were also included because of their clinical relevance. chromosome 17 being <1.8. Analysis of tumor characteristics according to FISH results. There Results were no significant differences between the three groups of FISH amplification (no amplification, low amplification, and Patients. Baseline patient demographics are summarized in high amplification) in terms of treatment given, age, or T or N Table 1. All patients had invasive breast cancer, with most stage (Table 2). As expected, amplification of the HER-2 gene as patients having T2,N0-1, and grade 2/3 disease. No patients assessed by FISH correlated with tumor grade, with significantly showed evidence of metastatic disease. Mean age was 46 years more grade 3 tumors displaying high HER-2 amplification (range, 27-67 years). According to Chevalier’s classification, 35 [28 of 49 (56%); P = 0.021]. High amplification of HER-2 of 93 (37.6%) patients had a pathologic complete response, also correlated with hormone receptor status: a significant whereas 57 of 93 (61.3%) patients experienced pathologic number of hormone receptor–negative tumors [27 of 36 partial or no response. (75%)] were classified as high amplification and no hormone HER-2 testing: comparison of FISH and IHC results. FISH receptor–negative tumors were classified as no amplification analysis rated 50 (53.8%) tumors as high amplification, all of (P = 0.001). which scored 3+ on central IHC review, with >80% of labeled Central IHC testing revealed no HER-2 1+ tumors displaying cells in all cases. Of the 27 (29%) tumors categorized as low FISH amplification, 1 of 6 HER-2 2+ tumors displaying low amplification, 1 (4%) was 2+ on central IHC review and the amplification, and all HER-2 3+ tumors displaying low remaining tumors (96%) were 3+, with >80% of labeled cells in amplification or high amplification (P < 0.0001). Complete all cases. In this category of low-amplification tumors, the pathologic response was significantly related to presence of mean signals per tumor nuclei ranged from 6.9 to 9.8, with a HER-2 amplification, with pathologic complete response

Clin Cancer Res 2007;13(21) November 1, 2007 6406 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2007 American Association for Cancer Research. Trastuzumab-Based Neoadjuvant Therapy observed in 1 of 16 (6%) no-amplification tumors, 6 of 27 Table 4. (22%) low-amplification tumors, and 28 of 50 (56%) high- Analysis of factors that can predict pathologic complete response in the population amplification tumors (P < 0.001). eligible for trastuzumab treatment after centrally Analysis of variables that can predict pathologic complete confirmed HER-2 status response. Tumor characteristics according to pathologic complete response are summarized in Table 3. There was Patients, n (%) P no relationship between pathologic complete response and pCR Non-pCR treatment regimen, patient age, T or N stage, or Scarff-Bloom- Treatment Richardson grade. Pathologic complete response was signifi- TCH (n = 50) 21 (42) 29 (58) cantly more frequent in FISH-amplified tumors (44%) than in TH (n = 27) 13 (48) 14 (52) 0.604 nonamplified tumors (6%; P = 0.004). As expected, pathologic Tumor stage n complete response also occurred significantly more frequently T2 ( = 54) 25 (54) 29 (46) T (n = 18) 7 (39) 11 (61) in HER-2 3+ tumors (P = 0.009). There was also a trend toward 3 T4 (n = 5) 2 (40) 3 (60) 0.845 pathologic complete response in hormone receptor–negative Nodal status tumors (P = 0.051). N0 (n = 37) 17 (46) 20 (54) n Analysis of variables predictive of pathologic complete response N1 or N2 ( = 40) 17 (42) 23 (58) 0.761 in patients eligible for trastuzumab treatment after central HER-2 Tumor grade SBR1 or 2 (n = 38) 13 (34) 25 (66) testing. Table 4 summarizes the analysis of factors that might SBR3 (n = 35) 19 (54) 16 (46) 0.084 predict pathologic complete response in the subpopulation of Hormone receptor status tumors that were IHC 3+ and/or FISH amplified (n = 77) and Positive (n = 38) 15 (40) 23 (60) thus eligible for treatment with trastuzumab. In this population Negative (n = 36) 18 (50) 18 (50) 0.363 IHC central review of tumors, the only variable related to pathologic complete 2+ (n = 1) 0 1 (100) response was the level of HER-2 amplification as assessed by 3+ (n = 76) 34 (44) 42 (56) 0.371 FISH, with 28 of 50 (56%) high-amplification tumors showing HER-2 amplification pathologic complete response compared with 6 of 27 (22%) HA (n = 50) 28 (56) 22 (44) n 0.004 low-amplification tumors (P = 0.004). Treatment regimen, LA ( = 27) 6 (22) 21 (78) patient age, T or N stage, Scarff-Bloom-Richardson grade, hormone receptor status, and IHC score were not significantly related to pathologic complete response. determine which variables were related to pathologic complete Multivariate analysis. Results of the multivariate analysis response. Results show that only level of amplification as done with logistic regression are summarized in Table 5. In this assessed by FISH was related to pathologic complete response model, we combined treatment regimen (to exclude treatment (P = 0.01). effect), tumor grade, and level of HER-2 amplification to

Discussion Table 3. Analysis of factors that can predict pathologic complete response To our knowledge, this is the first study to show a positive correlation between level of HER-2 amplification, as assessed Patients, n (%) P by FISH, and rate of pathologic complete response to pCR Non-pCR trastuzumab-based neoadjuvant treatment in locally advanced

Treatment HER-2–positive breast tumors. TCH (n = 61) 22 (36) 39 (64) Several phase II studies of trastuzumab plus chemotherapy as TH (n = 32) 13 (41) 19 (59) 0.66 primary systemic therapy have shown substantially improved Tumor stage response rates in this population of patients at high risk for n T2 ( = 66) 26 (39) 40 (61) relapse, with most overall response rates z75% (for detailed T (n = 21) 7 (33) 14 (67) 3 review, see ref. 13). However, pathologic complete response T4 (n = 5) 2 (40) 3 (60) 0.879 Nodal status rates, which are often considered more indicative of disease-free n N0 ( = 46) 18 (39) 28 (61) and overall survival, have been more variable, ranging from 7% n N1 or N2 ( = 47) 17 (36) 30 (64) 0.76 in one small scale study (n = 14) of trastuzumab plus docetaxel Tumor grade SBR1 (n = 3) 1 (33) 2 (67) and epirubicin to >40% in several larger studies (for detailed SBR2 (n = 44) 12 (27) 32 (73) review, see ref. 13). Thus, any baseline factor that could be used SBR3 (n = 41) 20 (49) 21 (51) 0.122 to help in predicting which patients would benefit most from Hormone receptor status trastuzumab-based neoadjuvant regimens would be invaluable n Positive ( = 51) 15 (29) 36 (71) to clinicians. Negative (n = 36) 18 (50) 18 (50) 0.051 IHC score In our study, the only variable related to pathologic complete 1+ (n = 11) 0 11 (100) response in the subpopulation of patients eligible for treatment 2+ (n = 6) 1 (17) 5 (83) with trastuzumab (IHC 2+/3+ and/or FISH positive) was the 3+ (n = 76) 34 (45) 42 (55) 0.009 level of HER-2 amplification as assessed by FISH. Two FISH analysis LA + HA (n = 77) 34 (44) 43 (56) neoadjuvant studies of trastuzumab plus a taxane, which NA (n = 16) 1 (6) 15 (94) 0.004 enrolled both IHC 2+ and 3+ patients, have shown that patients who scored IHC 3+ were more likely to respond to

www.aacrjournals.org 6407 Clin Cancer Res 2007;13(21) November 1, 2007 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2007 American Association for Cancer Research. Cancer Therapy: Clinical trastuzumab treatment than those patients who scored IHC 2+ no significant difference in pathologic complete response rates for HER-2 overexpression (13, 27). Although all FISH-positive according to FISH status (FISH positive versus FISH negative) tumors were centrally confirmed as IHC 2+ or 3+ in this study, was found. Unexpectedly, FISH status also did not affect rates of our classification of HER-2 amplification by FISH was more either progression-free or overall survival in this study. precise in identifying those patients who would have benefited Although our results seem to contradict this study, the most from trastuzumab. Indeed, pathologic complete response population analyzed may be different. Indeed, IHC testing, was seen significantly more frequently in high-amplification used for the patient screening, seemed very sensitive in the FISH tumors compared with low-amplification tumors in Hurley et al. study, as only 60% of IHC 3+ tumors were FISH patients who were eligible to receive trastuzumab treatment. positive, whereas reported rates in the literature are f90% (18, This degree of subclassification with regard to HER-2 status 24, 25, 39). Although our study did not evaluate survival, these would not have been possible using IHC. Therefore, FISH may differing results about the relationship between FISH positivity be a more accurate HER-2 testing method to predict pathologic and pathologic complete response highlight the need to complete response in the neoadjuvant setting. standardize both IHC and FISH procedures for determination Interestingly, our results are in line with retrospective of HER-2 gene status. analyses done on patients enrolled in the pivotal trials of Currently, there is variability with respect to HER-2 testing in trastuzumab in HER-2–positive metastatic breast cancer. terms of a particular commercial kit, antibody, probe, or cutoff Whereas patients with IHC scores of 2+ and 3+ were initially value, and discrepancies due to variability between kits may occur eligible for the phase III trials, subset analysis by Slamon et al. (40). In terms of cutoff values with FISH amplification, our results, (4) revealed that the clinical benefits of trastuzumab plus which were obtained on clinicopathologic data, are in line with paclitaxel were greatest in patients whose tumors were graded those obtained with biological data in a recently published study IHC 3+. Furthermore, retrospective analyses revealed that by Dal Lago et al. (29) and those proposed recently by the patients who tested HER-2 positive by FISH experienced American Society of Clinical Oncology and the College of significantly better response rates and improved survival with American Pathologists (32). In the Dal Lago et al. study, cutoff trastuzumab than those patients whose tumors were FISH values of 4 and 6 copies for no amplification and amplification negative. This suggests that FISH may predict more accurately were evaluated with single-color FISH. Good correlation only which patients will obtain the most clinical benefit from existed between the cutoff value of 6 and their mRNA assay. trastuzumab therapy in the metastatic setting (36). The results of our study also highlight that accurate HER-2 Although the number of cases included in this study is not testing is essential for optimal patient management with large and despite the size of the confidence intervals, our trastuzumab. In the neoadjuvant setting, initial IHC screening analysis shows statistically significant differences between the (which would be negative for the majority for tumors) could two groups of amplification in term of pCR. These results have be used, followed by FISH testing for IHC 2+ or 3+ tumors. certainly to be confirmed in the future in a larger series. This supplementary FISH testing would exclude false-positive Moreover, our results are in concordance with those published tumors by confirming HER-2 amplification and precisely recently by Giuliani et al. (37) in metastatic HER-2–positive determine the level of HER-2 amplification. This need of breast cancer treated with trastuzumab alone or combined with accuracy was particularly emphasized in two large trials that chemotherapy (mainly paclitaxel). In the group of patient have shown discordance between local and central HER-2 treated with trastuzumab and chemotherapy, they have shown testing for IHC (20) or for IHC and FISH (41). a significantly positive correlation between level of HER-2 gene In conclusion, our study showed a significantly positive amplification and the clinical objective response. correlation between pathologic complete response rate and Nevertheless, in a recent neoadjuvant study by Hurley et al. level of HER-2 amplification as assessed by FISH in stage II/III (38) evaluating trastuzumab plus docetaxel and cisplatin as breast tumors. These findings may have clinical implications for primary systemic therapy for patients with HER-2–positive the management of patients with HER-2–positive locally (IHC 2+/3+ or FISH positive) locally advanced breast cancer, 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 Table 5. Multivariate statistical analysis of factors that can predict pathologic complete response in trastuzumab-based chemotherapy. However, it remains un- the population of patients eligible for trastuzumab known whether a relationship exists between pathologic treatment (IHC 2+ or 3+ centrally confirmed with complete response, HER-2 level of amplification as assessed FISH amplification) by FISH, and postoperative survival rates in women with HER- 2–positive locally advanced breast cancer who are treated No. patients Odds 95% P (n = 73) ratio confidence interval preoperatively with trastuzumab-based regimens. Further stud- ies on the effect of level of HER-2 amplification on the behavior HER-2 amplification of tumors treated with trastuzumab in both the metastatic and LA 26 1 adjuvant breast cancer settings are also needed. HA 47 4.75 1.5-15.4 0.01 Treatment TCH 46 1 TH 27 1.54 0.5-4.6 0.44 Acknowledgments Tumor grade SBR1 or 2 38 1 We thank the Ligue de Cote d’Or Contre le Cancer, C. Harris, and D. James SBR3 35 1.76 for assistance in manuscript preparation; Drs. J. Garnier and F. Campana (Roche Laboratories);Prof.N.NamerandP.Atalli(GETNA);andG.Milla(OSMO).

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