Diagnostic Immunohistochemistry Dabbs Pdf Free

Posted on 12/25/2017by admin

Adobe Flash Player is required to view this feature. If you are using an operating system that does not support Flash, we are working to bring you alternative formats. Original Article HER2 and Response to Paclitaxel in Node-Positive Breast Cancer Daniel F. Hayes, M.D., Ann D. Thor, M.D., Lynn G. Dressler, Dr.Ph., Donald Weaver, M.D., Susan Edgerton, M.A., David Cowan, B.A., Gloria Broadwater, M.S., Lori J.

By David J Dabbs MD PDF. Read Best Book Online Diagnostic Immunohistochemistry: Theranostic and Genomic Applications, Expert Consult: Online and Print, 4e, ebook download Diagnostic Immunohistochemistry: Theranostic and Genomic Applications, Expert Consult: Online and Print, 4e, pdf epub free download. Unlimited access to ALL articles including Application Notes; News, interviews & opinions from leading industry experts; Receive print (and PDF) copies of The. Because of their diversity, they're one of the most problematic areas in diagnostic breast pathology – so we routinely use IHC analysis to assist with diagnosis (3).

Goldstein, M.D., Silvana Martino, D.O., James N. Ingle, M.D., I. Craig Henderson, M.D., Larry Norton, M.D., Eric P. Winer, M.D., Clifford A. The Da Vinci Code Full Movie Free Download In English here. Hudis, M.D., Matthew J.

Ellis, M.B., Ph.D., and Donald A. Berry, Ph.D., for the Cancer and Leukemia Group B (CALGB) Investigators N Engl J Med 2007; 357:1496-1506 DOI: 10.1056/NEJMoa071167. Methods We randomly selected 1500 women from 3121 women with node-positive breast cancer who had been randomly assigned to receive doxorubicin (60, 75, or 90 mg per square meter of body-surface area) plus cyclophosphamide (600 mg per square meter) for four cycles, followed by four cycles of paclitaxel (175 mg per square meter) or observation. Tissue blocks from 1322 of these 1500 women were available. Immunohistochemical analyses of these tissue specimens for HER2 with the CB11 monoclonal antibody against HER2 or with a polyclonal-antibody assay kit and fluorescence in situ hybridization for HER2 amplification were performed.

Results No interaction was observed between HER2 positivity and doxorubicin doses above 60 mg per square meter. HER2 positivity was, however, associated with a significant benefit from paclitaxel. The interaction between HER2 positivity and the addition of paclitaxel to the treatment was associated with a hazard ratio for recurrence of 0.59 (P=0.01). Patients with a HER2-positive breast cancer benefited from paclitaxel, regardless of estrogen-receptor status, but paclitaxel did not benefit patients with HER2-negative, estrogen-receptor–positive cancers. Conclusions The expression or amplification, or both, of HER2 by a breast cancer is associated with a benefit from the addition of paclitaxel after adjuvant treatment with doxorubicin (. Figure 1 Clinical Outcomes in Patients Treated with or without Paclitaxel, According to HER2 Status. Patients were randomly assigned to receive four cycles of paclitaxel (175 mg per square meter) or no further chemotherapy with paclitaxel after completion of four cycles of doxorubicin plus cyclophosphamide.

Disease-free survival for group 1 (Panels A and B), overall survival for group 1 (Panels C and D), disease-free survival for group 2 (Panels E and F), and overall survival for group 2 (Panels G and H) were assessed according to negative or positive HER2 expression, as determined by means of immunohistochemical analysis with the CB11 monoclonal antibody. Figure 2 Disease-free Survival among Patients Treated with or without Paclitaxel According to Estrogen-Receptor Status and HER2 Expression. Patients were randomly assigned to receive four cycles of paclitaxel (175 mg per square meter) or no further chemotherapy with paclitaxel after completion of four cycles of doxorubicin and cyclophosphamide. Disease-free survival for patients in groups 1 and 2 combined was determined according to negative HER2 expression (Panels A and B) or positive HER2 expression (Panels C and D), as determined by immunohistochemical analysis with the CB11 monoclonal antibody, or according to negative estrogen-receptor (Panels A and C) or positive estrogen-receptor (Panels B and D) expression, as determined at the local institutions.

The Cancer and Leukemia Group B (CALGB) 8541 trial showed that increasing the dose of a doxorubicin (Adriamycin)–based regimen from a relatively low dose (30 mg per square meter of body-surface area) to what is now considered to be a standard dose (60 mg per square meter) is highly beneficial. Subsequently, a randomized trial (CALGB 9344/INT0148) examined the effects of increased doses of doxorubicin, above 60 mg per square meter, when combined with cyclophosphamide (Cytoxan) at a dose of 600 mg per square meter, plus four subsequent cycles of paclitaxel (Taxol). Overall, there was no benefit from doses of doxorubicin above 60 mg per square meter, but the addition of paclitaxel improved both disease-free survival and overall survival.

Adjuvant chemotherapy causes substantial morbidity and occasional life-threatening toxic effects, and it is costly. No biomarkers have been identified that can reliably predict a clinical benefit from paclitaxel or escalating doses of doxorubicin in women with breast cancer. HER2, a member of the epidermal growth factor receptor family, is amplified, overexpressed, or both in 15 to 20% of breast cancers.

HER2 overexpression and amplification in breast-cancer cells are strong predictors of a benefit from treatment with trastuzumab, and the status of HER2 in the tumor might predict the results of other treatments of breast cancer. In a correlative study of breast-cancer tissues from trial 8541, HER2 amplification and overexpression were associated with benefits from standard doses of doxorubicin but not from low doses of doxorubicin. However, no studies have examined whether the overexpression or amplification of HER2 can predict a benefit of increasing the dose of doxorubicin above 60 mg per square meter. Results of preclinical studies and preliminary clinical studies regarding an interaction between HER2 status and paclitaxel are inconsistent. For these reasons, we investigated whether HER2 expression in the tumor identifies patients with breast cancer who are likely to benefit from doses of doxorubicin above 60 mg per square meter, the addition of paclitaxel after adjuvant treatment with doxorubicin plus cyclophosphamide, or both.

Tissue Collection, Processing, Storage, and Distribution Approximately 90% of women enrolled in this trial gave written informed consent for the collection, storage, and analysis of formalin-fixed paraffin-embedded primary breast-cancer tissue. A tissue block or unstained tissue sections were requested from each patient's primary institution by the pathology coordinating office of each of the participating cooperative groups (CALGB, Southwest Oncology Group, Eastern Cooperative Oncology Group, and North Central Cancer Treatment Group) and were collected at the CALGB pathology coordinating office. Coded tissue sections were placed on glass slides without any patient identifiers and were distributed to the various investigators' laboratories for analysis in a blinded fashion. HER2 data were submitted by each investigator to the CALGB Statistical Center for correlations with clinical outcomes. Estrogen-receptor status was determined by the local institutions by means of standard procedures, and positive or negative status was designated according to each institution's policy as recorded on the case-report form submitted to CALGB. Estrogen-receptor data were available for 99.4% of patients enrolled in the clinical trial. Immunohistochemical Analysis for HER2 Expression Immunohistochemical analysis with the CB11 monoclonal antibody was performed as previously described.

Tissue specimens were considered to be positive for HER2 if 50% or more of breast-cancer cells stained with CB11. Immunohistochemical analysis with a polyclonal-antibody kit (Herceptest, Dako) was performed by the clinical histology laboratory of the Fletcher Allen Health Care Center according to the instructions of the manufacturer (Dako).

The standard scoring method was used, and only 3+ staining was considered to be positive for HER2 overexpression. Statistical Analysis The statistical analyses were specified in advance in a written correlative protocol that was approved by the Breast Cancer Intergroup of North America Correlative Science Committee and subsequently by the institutional review board of each of the laboratory investigators' institutions. The immunohistochemical assay with the CB11 monoclonal antibody and FISH for HER2 amplification constituted the principal objectives of this study; results of the polyclonal-antibody test were also evaluated. To preserve tissue and resources, a sampling scheme was developed prospectively.

Two distinct and randomly selected groups of tissue samples from 750 patients were identified from the full sample of nearly 2800 available specimens. The two groups of patients were similar with respect to the number of positive lymph nodes, estrogen-receptor status, age, and treatment assignment. Results of the two principal assays in the combined groups were analyzed, whereas results of the polyclonal-antibody test were analyzed in only one group. We requested tissue specimens from 1500 women who participated in the clinical trial, and we obtained 643 and 679 tissue specimens from groups 1 and 2, respectively (a total of 1322 specimens). The primary end point was disease-free survival, defined in the parent study as the interval from study entry until the first local or distant recurrence or death due to any cause. The interaction effect was defined as the ratio of the hazard ratios for recurrence or death with paclitaxel treatment in women with HER2-positive tumors and in those with HER2-negative tumors. The principal analysis was based on a multivariate Cox proportional-hazards model for disease-free survival with the following covariates: paclitaxel therapy, dose of doxorubicin, square root of the number of positive nodes, tumor size, menopausal status (premenopausal vs.

Perimenopausal or postmenopausal), estrogen-receptor status, HER2 status, and the presence or absence of an interaction between HER2 positivity and paclitaxel. Adjustments for multiple comparisons were not performed. Disease-free survival for the paclitaxel and no-paclitaxel groups was estimated with Kaplan–Meier curves. Third-order interactions of HER2 positivity or negativity with estrogen-receptor status and receipt or nonreceipt of paclitaxel were analyzed for hypothesis generation, and no significance levels are given for such interactions. The two methods of assessing each of the biomarkers were compared by calculating their level of agreement with the use of the kappa statistic. Results of this study are presented in accordance with reporting recommendations for tumor-marker prognostic studies (REMARK) criteria.

Outcomes and Characteristics of Selected Subgroups The overall results of the CALGB 9344 trial with approximately 5 years of follow-up have been reported. With approximately 10 years of follow-up, the results have remained qualitatively similar. No significant differences in either disease-free survival or overall survival were observed for doxorubicin doses above 60 mg per square meter. The hazard ratios for recurrence and death with the use of doxorubicin doses of 60 mg per square meter as compared with 90 mg per square meter were 0.97 (95% confidence interval [CI], 0.86 to 1.13; P=0.73) and 1.03 (95% CI, 0.89 to 1.23; P=0.56), respectively.

The addition of paclitaxel resulted in significant improvements in disease-free and overall survival. The hazard ratios for recurrence and death with the addition of paclitaxel after doxorubicin plus cyclophosphamide were 0.81 (95% CI, 0.73 to 0.91; P. Doxorubicin Dose and HER2 Status No significant association between HER2 overexpression, according to immunohistochemical analysis with the CB11 monoclonal antibody, and escalation of the doxorubicin dose to 75 mg per square meter or 90 mg per square meter was observed in group 1 or 2 or in the combined groups.

The rate of disease-free survival at 5 years in the combined groups for patients with a HER2-positive tumor who received 60 mg per square meter or for those who received 90 mg per square meter was 63% (95% CI, 52 to 76) and 63% (95% CI, 53 to 73), respectively, whereas the rate of disease-free survival at 5 years for patients with a HER2-negative tumor who received 60 mg per square meter and for those who received 90 mg per square meter was 72% (95% CI, 67 to 77) and 69% (95% CI, 64 to 74), respectively. Paclitaxel and HER2 Status Disease-free survival and overall survival among patients who did or did not receive paclitaxel were analyzed according to HER2 status as established by immunohistochemical analysis with the CB11 monoclonal antibody ( Figure 1 Clinical Outcomes in Patients Treated with or without Paclitaxel, According to HER2 Status. Patients were randomly assigned to receive four cycles of paclitaxel (175 mg per square meter) or no further chemotherapy with paclitaxel after completion of four cycles of doxorubicin plus cyclophosphamide. Disease-free survival for group 1 (Panels A and B), overall survival for group 1 (Panels C and D), disease-free survival for group 2 (Panels E and F), and overall survival for group 2 (Panels G and H) were assessed according to negative or positive HER2 expression, as determined by means of immunohistochemical analysis with the CB11 monoclonal antibody.

The apparent interaction between HER2 positivity and the addition of paclitaxel in group 1 was not significant (hazard ratio for recurrence in the interaction between HER2 positivity and the addition of paclitaxel, 0.63; P=0.15) ( Table 2 Results of Multivariate Models of the Interaction of HER2 Positivity and Paclitaxel Treatment. We further examined this interaction in group 2.

In group 2, the hazard ratio for recurrence in the interaction between HER2 positivity and the addition of paclitaxel was 0.52 (P=0.03) ( ) ( ). When the two groups were combined, in multivariate analyses the hazard ratio for recurrence in the interaction between HER2 positivity and the addition of paclitaxel to the treatment was 0.59 (P=0.01) ( Table 3 Multivariate Analysis of Disease-free Survival for Groups 1 and 2 Combined. The hazard ratio for death in the combined groups for the HER2–paclitaxel interaction was 0.57 (P=0.01) ( ). Paclitaxel and HER2 and Estrogen-Receptor Status Since an estrogen-receptor–positive tumor can be a negative predictive factor for the response to chemotherapy in breast cancer, we performed an exploratory analysis of the benefit of paclitaxel based on HER2 positivity and estrogen-receptor status ( Figure 2 Disease-free Survival among Patients Treated with or without Paclitaxel According to Estrogen-Receptor Status and HER2 Expression.

Patients were randomly assigned to receive four cycles of paclitaxel (175 mg per square meter) or no further chemotherapy with paclitaxel after completion of four cycles of doxorubicin and cyclophosphamide. Disease-free survival for patients in groups 1 and 2 combined was determined according to negative HER2 expression (Panels A and B) or positive HER2 expression (Panels C and D), as determined by immunohistochemical analysis with the CB11 monoclonal antibody, or according to negative estrogen-receptor (Panels A and C) or positive estrogen-receptor (Panels B and D) expression, as determined at the local institutions.

The log-rank P value in each panel is for the comparison of Kaplan–Meier disease-free survival curves in the paclitaxel and no-paclitaxel groups and does not represent the three-way interaction among HER2 positivity, estrogen-receptor negativity, and a benefit from paclitaxel. For this analysis, we examined disease-free survival in the combined groups, and HER2 was evaluated with the CB11 antibody. Shows Kaplan–Meier curves for disease-free survival in the paclitaxel and no-paclitaxel groups for each estrogen-receptor–HER2 combination. Log-rank P values are provided as a measure of discordance and should be viewed as descriptive, not inferential. In this analysis, paclitaxel was associated with improved disease-free survival among patients with HER2-positive tumors, an effect that was independent of estrogen-receptor status ( ). In the small subgroup of patients with cancers that were estrogen-receptor–positive and HER2-positive, paclitaxel appeared to be beneficial ( ). However, paclitaxel did not benefit patients with estrogen-receptor–positive, HER2-negative cancers ( ).

This subgroup included more than 50% of patients in this study. Paclitaxel and Method of HER2 Analysis We compared the results of immunohistochemical analysis with the CB11-antibody test, with the polyclonal-antibody test, and with FISH. The qualitative results of all three assays were similar ( Figure 3 Disease-free Survival among Patients Treated with or without Paclitaxel According to Different Methods of HER2 Analysis. Patients were randomly assigned to receive four cycles of paclitaxel (175 mg per square meter) or no further chemotherapy after completion of four cycles of doxorubicin and cyclophosphamide. Disease-free survival for patients in group 1 was determined according to negative HER2 expression (Panels A and C) or positive HER2 expression (Panels B and D), as determined by immunohistochemical analysis with the CB11 monoclonal antibody (Panels A and B) or the polyclonal-antibody test (Panels C and D), or according to HER2 amplification (Panel F) or no amplification (Panel E), as determined by fluorescence in situ hybridization (FISH).

The interaction term between HER2 expression and a benefit from paclitaxel (within group 1 only) reached significance in only one of the three comparisons (with the polyclonal-antibody test, P=0.04; P=0.06 with FISH). The mean (±SD) kappa statistic was 85±2.6% for the level of agreement between immunohistochemical analysis with the CB11-antibody test and the polyclonal-antibody test, 79±3.2% for the level of agreement between immunohistochemical analysis with the CB11-antibody test and FISH, and 80±3.0% for the level of agreement between the polyclonal-antibody test and FISH. Discussion We observed a significant interaction between the HER2 status of the tumor and the benefit of adjuvant paclitaxel in patients with node-positive breast cancer who received four cycles of doxorubicin plus cyclophosphamide. Our results indicate that HER2 positivity can predict improvement in disease-free survival and overall survival by the addition of paclitaxel to doxorubicin plus cyclophosphamide. In contrast, no interaction was observed between HER2 status and doses of doxorubicin above 60 mg per square meter. The HER2–paclitaxel interaction was observed regardless of whether HER2 positivity was determined by means of either of two immunohistochemical methods or whether HER2 gene amplification was determined by means of FISH.

Our data do not suggest that one assay is more robust than any other of the three. In this study, we used dichotomous cutoffs to designate whether a tumor was positive or negative for expression or amplification of HER2.

A panel convened by the American Society of Clinical Oncology and the College of American Pathologists recently issued guidelines for HER2 testing by means of immunohistochemical analysis or FISH in which they proposed categories of HER2 results that should be considered to be equivocal. In our study, these categories applied to only 13 and 16 patients, respectively, precluding meaningful analyses. In an exploratory analysis, we observed an apparent three-way interaction among HER2 positivity, estrogen-receptor negativity, and a benefit from paclitaxel. We found no benefit of paclitaxel in patients with HER2-negative, estrogen-receptor–positive breast cancer ( ).

This subgroup represents more than half the patients with node-positive breast cancer who participated in the CALGB 9344 trial and who would, under most current circumstances, receive a taxane with or after cyclophosphamide plus an anthracycline. Our studies suggest that such patients could avoid the toxic effects associated with adjuvant paclitaxel when given after doxorubicin plus cyclophosphamide. Our results require validation before adoption into clinical practice, however. CALGB trials that enrolled patients with node-positive breast cancer from 1985 to 1997, including the CALGB 9344 trial, showed incremental benefits in disease-free survival and overall survival. These studies compared what would now be considered insufficient doses of cyclophosphamide, doxorubicin, and fluorouracil with higher doses of the same regimen (CALGB 8541), the addition of paclitaxel to standard doses of doxorubicin and cyclophosphamide (CALGB 9344), and more recently, the administration of doxorubicin, cyclophosphamide, and paclitaxel every 2 weeks instead of every 3 weeks (CALGB 9741). In each case, the additional benefit of the investigational strategy as compared with the standard treatment was substantially greater in patients with estrogen-receptor–negative breast cancer than in patients with estrogen-receptor–positive breast cancer.

However, this differential benefit was not limited to the estrogen-receptor–negative subgroup in any of these studies, suggesting that estrogen-receptor status is not an absolute predictor of a benefit from additional or dose-dense chemotherapy. The results of the present study suggest that HER2 assessment can refine predictions of a benefit from chemotherapy. Previous studies have shown that the response to regimens containing doxorubicin at a dose of up to 60 mg per square meter is strongly correlated with HER2 amplification, overexpression, or both. Other investigators showed that any benefit from an anthracycline is associated with HER2 status and that HER2 positivity may be a surrogate for abnormalities in the topoisomerase II gene, which is present on the same amplicon as HER2. Our data, however, indicate that there is no detectable HER2–doxorubicin effect when the dose of doxorubicin is higher than 60 mg per square meter. Preclinical data regarding HER2 status and the response to taxanes are contradictory. However, in one trial, patients with HER2-positive metastatic breast cancer were more likely to benefit from a paclitaxel-containing regimen than patients with HER2-negative disease; this finding is consistent with our results.

Trastuzumab, a humanized monoclonal antibody against HER2, decreases the risks of recurrence and death among women with HER2-positive breast cancer by approximately one half and one third, respectively. We cannot speculate on how the addition of trastuzumab might affect our results. However, the benefits of trastuzumab would not affect our observation that paclitaxel appeared to have little, if any, benefit in patients with HER2-negative, estrogen-receptor–positive tumors. We found a significant association between HER2 positivity and a benefit from the addition of paclitaxel after adjuvant treatment with doxorubicin plus cyclophosphamide in women with node-positive, stage II breast cancer. Our data raise the possibility of a three-way interaction among HER2 negativity, estrogen-receptor positivity, and a lack of benefit from paclitaxel. Supported by grants from the National Institutes of Health (CA092461, to Dr. Hayes; and CA33601, to Dr.

Berry) and from the Breast Cancer Research Foundation and the Fashion Footwear Charitable Foundation of New York/QVC Presents Shoes on Sale (to Dr. Dako provided the Herceptest kits for HER2 immunohistochemical analysis.

The research for the CALGB 9344 trial was supported in part by grants from the National Cancer Institute (CA31946, to Cancer and Leukemia Group B) and to the CALGB Statistical Center (CA33601). The views expressed in this article are solely those of the authors and do not necessarily represent the official views of the National Cancer Institute. Norton, Goldstein, and Berry report receiving consulting fees from Bristol-Myers Squibb; Dr. Berry, consulting fees from Abbott; and Drs. Winer and Goldstein, clinical research support from Bristol-Myers Squibb. No other potential conflict of interest relevant to this article was reported.

We thank the pathology coordinating offices of the respective participating cooperative groups (CALGB, Eastern Cooperative Oncology Group, North Central Cancer Treatment Group, and Southwest Oncology Group), and in particular Laura Monovich and Scott Jewell for their tireless efforts in cataloguing and preparing tissues for this study; Jeannette Mitchell and the immunohistochemistry laboratory of Fletcher Allen Health Care, who performed the Herceptest assays; and Dako for providing the Herceptest assay kits and special technical training. Appendix The following investigators participated in the CALGB trial: Fox Chase Cancer Center, Philadelphia — L. Goldstein; Mayo Clinic, Rochester, MN — J.N. Ingle; CALGB Statistical Center, Duke University Medical Center, Durham, NC — S. Crawford; Dana–Farber Cancer Institute, Partners HealthCare, Boston — E.P. Winer; Dartmouth Medical School, Norris Cotton Cancer Center, Lebanon, NH — M.S.

Ernstoff; Long Island Jewish Medical Center, Lake Success, NY — M. Citron; Massachusetts General Hospital, Boston — M.L. Grossbard; Medical University of South Carolina, Charleston — M.

Green; Memorial Sloan-Kettering Cancer Center, New York — C. Hudis; Mount Sinai School of Medicine, New York — L.R. Silverman; North Shore University Hospital, Manhasset, NY — D.R. Budman; Rhode Island Hospital, Providence, RI — W. Sikov; Roswell Park Cancer Institute, Buffalo, NY — E. Levine; State University of New York Upstate Medical University, Syracuse — S.L.

Graziano; University of Alabama at Birmingham, Birmingham — R. Diasio; University of California at San Diego, La Jolla — J. Mortimer; University of California at San Francisco, San Francisco — A.P. Venook; University of Chicago, Chicago — G. Fleming; University of Illinois, Chicago — L.E. Feldman; University of Iowa, Iowa City — G. Clamon; University of Maryland Greenebaum Cancer Center, Baltimore — M.

Edelman; University of Massachusetts Medical School, Worcester — W.V. Walsh; University of Minnesota, Minneapolis — B.A Peterson; University of Missouri, Ellis Fischel Cancer Center, Columbia — M.C. Perry; University of Nebraska Medical Center, Omaha — A. Kessinger; University of North Carolina at Chapel Hill, Chapel Hill — T.C. Shea; University of Tennessee, Memphis — H.B. Niell; Vermont Cancer Center, Burlington — H.B.

Muss; Wake Forest University School of Medicine, Winston-Salem, NC — D.D. Hurd; Walter Reed Army Medical Center, Washington, DC — T. Reid; Washington University School of Medicine, St. Bartlett; Weill Medical College of Cornell University, New York — S.

References • 1 Early Breast Cancer Trialists' Collaborative Group (EBCTCG). Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials.

Lancet 2005;365:1687-1717 • 2 Smith I, Chua S. Medical treatment of early breast cancer. Adjuvant treatment.

BMJ 2006;332:34-37 • 3 Wood WC, Budman DR, Korzun AH, et al. Dose and dose intensity of adjuvant chemotherapy for stage II, node-positive breast carcinoma. N Engl J Med 1994;330:1253-1259[Erratum, N Engl J Med 1994;331:139.] • 4 Budman DR, Berry DA, Cirrincione CT, et al. Dose and dose intensity as determinants of outcome in the adjuvant treatment of breast cancer.

J Natl Cancer Inst 19-1211 • 5 Henderson IC, Berry DA, Demetri GD, et al. Improved outcomes from adding sequential paclitaxel but not from escalating doxorubicin dose in an adjuvant chemotherapy regimen for patients with node-positive primary breast cancer. J Clin Oncol 2003;21:976-983 • 6 Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL.

Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 1987;235:177-182 • 7 Mass RD, Press MF, Anderson S, et al. Evaluation of clinical outcomes according to HER2 detection by fluorescence in situ hybridization in women with metastatic breast cancer treated with trastuzumab. Clin Breast Cancer 2005;6:240-246 • 8 Yamauchi H, Stearns V, Hayes DF.

When is a tumor marker ready for prime time? A case study of c-erbB-2 as a predictive factor in breast cancer. J Clin Oncol 20-2356 • 9 Muss HB, Thor AD, Berry DA, et al. C-erbB-2 expression and response to adjuvant therapy in women with node-positive early breast cancer. N Engl J Med 1994;330:1260-1266[Erratum, N Engl J Med 1994;331:211.] • 10 Thor AD, Berry DA, Budman DR, et al. ErbB2, p53, and efficacy of adjuvant therapy in lymph node-positive breast cancer.

J Natl Cancer Inst 19-1360 • 11 Dressler LG, Berry DA, Broadwater G, et al. Comparison of HER2 status by fluorescence in situ hybridization and immunohistochemistry to predict benefit from dose escalation of adjuvant doxorubicin-based therapy in node-positive breast cancer patients. J Clin Oncol 20-4297 • 12 Yu D. Mechanisms of ErbB2-mediated paclitaxel resistance and trastuzumab-mediated paclitaxel sensitization in ErbB2-overexpressing breast cancers. Semin Oncol 2001;28:Suppl 16:12-17 • 13 Formenti SC, Spicer D, Skinner K, et al. Low HER2/neu gene expression is associated with pathological response to concurrent paclitaxel and radiation therapy in locally advanced breast cancer.

Int J Radiat Oncol Biol Phys 2002;52:397-405 • 14 Sjostrom J, Collan J, von Boguslawski K, et al. C-erbB-2 expression does not predict response to docetaxel or sequential methotrexate and 5-fluorouracil in advanced breast cancer. Eur J Cancer 2002;38:535-542.

Adobe Flash Player is required to view this feature. If you are using an operating system that does not support Flash, we are working to bring you alternative formats. Original Article Effect of Occult Metastases on Survival in Node-Negative Breast Cancer Donald L. Weaver, M.D., Takamaru Ashikaga, Ph.D., David N. Krag, M.D., Joan M. Skelly, M.S., Stewart J. Anderson, Ph.D., Seth P.

Harlow, M.D., Thomas B. Julian, M.D., Eleftherios P.

Mamounas, M.D., and Norman Wolmark, M.D. N Engl J Med 2011; 364:412-421 DOI: 10.1056/NEJMoa1008108. Methods We randomly assigned women with breast cancer to sentinel-lymph-node biopsy plus axillary dissection or sentinel-lymph-node biopsy alone. Paraffin-embedded tissue blocks of sentinel lymph nodes obtained from patients with pathologically negative sentinel lymph nodes were centrally evaluated for occult metastases deeper in the blocks. Both routine staining and immunohistochemical staining for cytokeratin were used at two widely spaced additional tissue levels.

Treating physicians were unaware of the findings, which were not used for clinical treatment decisions. The initial evaluation at participating sites was designed to detect all macrometastases larger than 2 mm in the greatest dimension.

Results Occult metastases were detected in 15.9% (95% confidence interval [CI], 14.7 to 17.1) of 3887 patients. Log-rank tests indicated a significant difference between patients in whom occult metastases were detected and those in whom no occult metastases were detected with respect to overall survival (P=0.03), disease-free survival (P=0.02), and distant-disease–free interval (P=0.04). The corresponding adjusted hazard ratios for death, any outcome event, and distant disease were 1.40 (95% CI, 1.05 to 1.86), 1.31 (95% CI, 1.07 to 1.60), and 1.30 (95% CI, 1.02 to 1.66), respectively. Five-year Kaplan-Meier estimates of overall survival among patients in whom occult metastases were detected and those without detectable metastases were 94.6% and 95.8%, respectively.

Figure 2 Kaplan-Meier Survival Estimates According to the Presence or Absence of Occult Metastases Detected in Initially Negative Sentinel Lymph Nodes. Panel A shows the probability of overall survival. The Kaplan–Meier estimate of overall survival at 60 months among patients in whom occult metastases were not detected was 95.8%; among patients in whom occult metastases were detected, it was 94.6%. Panel B shows the probability of disease-free survival. The Kaplan–Meier estimate of disease-free survival at 60 months among patients in whom occult metastases were not detected was 89.2%; among patients in whom occult metastases were detected, it was 86.4%. Panel C shows the probability of distant-disease–free survival.

The Kaplan–Meier estimate of distant-disease–free survival at 60 months among patients in whom occult metastases were not detected was 92.5%; among patients in whom occult metastases were detected, it was 89.7%. A landmark 1948 article by Saph and Amromin showed that the routine analysis of lymph nodes in breast cancer was insufficient to detect all metastases present. Although the practice of additional pathological analysis was not adopted, the concept of occult metastases (metastases that are not detected initially but are detected with further evaluation) was introduced and has been the subject of considerable research and controversy over the ensuing decades. The National Surgical Adjuvant Breast and Bowel Project (NSABP) trial B-32 was designed to evaluate whether sentinel-lymph-node biopsy alone was equivalent to complete axillary dissection with respect to overall survival and local and regional control. This trial was an opportunity to investigate the clinical significance of occult metastatic disease in selected axillary lymph nodes — namely, the sentinel nodes that had already been shown to be 4.3 times as likely to contain overt metastases and 12.3 times as likely to contain occult metastases as nonsentinel nodes.

Retrospective studies of occult metastases have important limitations: they have not used a standardized analysis of nodes and have lacked a concerted effort to exclude women with macrometastases (deposits >2.0 mm in the greatest dimension) from the study population. B-32, a prospective trial designed with a standard pathological approach to sentinel-lymph-node evaluation, excluded patients with macrometastases from the population evaluated for occult metastases. In addition, the results of the central analysis of occult metastases were blinded; thus, this cohort analysis was a global outcome evaluation within a randomized, phase 3 trial in which the effect that micrometastases and isolated tumor-cell clusters exerted on disease-free survival and overall survival were assessed without the influence of treatment bias. Trial Design We randomly assigned women to sentinel-lymph-node biopsy with immediate axillary dissection or to sentinel-lymph-node biopsy alone, as previously described.

The randomization process stratified patients according to age (≤49 years or ≥50 years), clinical tumor size (≤2.0 cm, 2.1 to 4. Serial Key For Net Protector 2014. 0 cm, or ≥4.1 cm in the greatest dimension), and planned surgical treatment (lumpectomy or mastectomy) within each participating institution ( Figure 1 Randomization and Results of Evaluation for Occult Metastases. The patients who underwent sentinel-lymph-node (SLN) biopsy plus axillary dissection and the patients who underwent SLN biopsy alone were combined into two analytic cohorts: patients in whom occult metastases were detected and patients in whom occult metastases were not detected.

The categories for metastasis size (isolated tumor-cell clusters, micrometastases, and macrometastases) were used for subgroup analysis. Patients who underwent sentinel-lymph-node biopsy alone in whom one or more positive sentinel lymph nodes were detected on either intraoperative cytologic assessment or subsequent assessment of a permanent section also underwent complete axillary dissection. The primary outcomes of the trial included overall survival and disease-free survival among all randomly assigned patients with pathologically negative sentinel lymph nodes. Overall survival was defined as the time from randomization to death from any cause. Disease-free survival was defined as the time from randomization to any local, regional, or distant disease; diagnosis of a second cancer other than breast cancer; or death from any cause. Secondary outcomes were breast-cancer–related death and distant disease.

The distant-disease–free interval was defined as the interval without any distant cancer, but data on death without evidence of distant disease were censored. All outcome results were reported as of December 31, 2009.

Procedures for Detecting Occult Metastases Participating sites were instructed to slice sentinel lymph nodes at approximately 2.0-mm intervals, embed all slices in paraffin tissue blocks, and examine one slide, routinely stained with hematoxylin and eosin, from each block. Findings that were suggestive of metastases on initial sections could be confirmed or refuted with immunohistochemical staining, but the routine use of immunohistochemical analysis or analysis of deeper tissue levels was prohibited. These results, which were documented on study-specific data forms and in the pathology report, were used for clinical treatment decisions. Tissue blocks of sentinel lymph nodes obtained from all patients in whom metastases were not detected by the participating site were sent to the University of Vermont for further evaluation. Additional sections that were approximately 0.5 mm and 1.0 mm deeper in the block relative to the original surface were evaluated for occult metastases with the use of hematoxylin and eosin and immunohistochemical staining at each level.

This evaluation protocol involving two widely spaced levels was designed to detect virtually all occult metastases larger than 1.0 mm in the greatest dimension and to randomly detect a proportion of occult metastases smaller than 1.0 mm that were present in the initially negative sentinel-lymph-node blocks. For our blinded analysis, the patients who underwent sentinel-lymph-node biopsy plus axillary dissection and the patients who underwent sentinel-lymph-node biopsy alone were combined into one group, and each patient was classified according to whether occult metastases were detected or not detected. A subgroup analysis according to metastasis size was also undertaken, with the use of American Joint Committee on Cancer definitions of isolated tumor-cell clusters (≤0.2 mm in the greatest dimension), micrometastases (>0.2 mm and ≤2.0 mm), and macrometastases (>2.0 mm).

Statistical Analysis The primary outcomes — overall survival, disease-free survival, and distant-disease-free interval — were characterized with the use of Kaplan-Meier plots, and log-rank tests were used to compare the outcomes between the group of patients with no detectable occult metastases and the group with detectable occult metastases. Cox proportional-hazards models were developed to estimate the hazard ratio for occult metastases with and without adjustments for stratification measures, the use or nonuse of systemic and radiation therapy, and study group (sentinel-lymph-node biopsy plus axillary dissection or sentinel-lymph-node biopsy alone).

Interaction effects for the size of occult metastases were analyzed for each stratification and therapy variable. The size distribution of occult metastases with stratification variables (patient age, ≤49 years or ≥50 years; tumor size, ≤2.0 cm, 2.1 to 4.0 cm, or ≥4.1 cm; and surgical treatment plan, lumpectomy or mastectomy), systemic therapy (chemotherapy, endocrine therapy, or other therapy), use or nonuse of radiation therapy, and study group was examined with the use of the Kruskal-Wallis rank-sum test. Event-free survival rates were compared between patients without occult metastasis and patients with occult metastasis by means of Fisher's exact test. Prevalence estimates for occult metastases and event-free outcomes are reported with 95% confidence intervals.

Reported P values are two-tailed. The NSABP B-32 trial was undertaken after approval from local institutional review boards and in accordance with an assurance filed with and approved by the Department of Health and Human Services. Written informed consent was obtained from each participant.

The pathological-outcome study of occult metastases was also approved by the institutional review board of the University of Vermont. The third author initiated the trial; the first author designed the pathological-outcome study.

Patient recruitment and randomization and collection of outcome data were conducted by the NSABP. Participating sites sent sentinel-lymph-node blocks to the University of Vermont for an evaluation that was funded by the National Cancer Institute. These data were linked with trial outcome data by the NSABP, were transferred to the University of Vermont in a format that eliminated identifying characteristics of the patients, and were analyzed by the second author. Characteristics of the Patients A total of 5611 women with operable, clinically node-negative, invasive breast cancer were randomly assigned to either sentinel-lymph-node biopsy plus axillary dissection or sentinel-lymph-node biopsy alone. In 3989 of these 5611 patients (71.1%), no metastases were detected in initial sentinel-lymph-node sections evaluated at participating sites.

Pathological material was available from 3887 of these patients (97.4%), and they agreed to participate in planned pathological studies: 1927 in the group of patients who underwent sentinel-lymph-node biopsy plus axillary dissection and 1960 in the group of patients who underwent sentinel-lymph-node biopsy alone. Of these 3887 patients, follow-up information was available for 3884 (99.9%): 637 had outcome events, 302 died, and 120 died from breast cancer. The median time in the study was 95.3 months. Among the reported adverse events associated with the trial, 46 (0.8%) were allergic reactions and 26 (0.6%) were surgical events. Prevalence of Occult Metastases Occult metastases were detected in 15.9% (95% confidence interval [CI], 14.7 to 17.1) of the 3887 patients: 11.1% with isolated tumor-cell clusters, 4.4% with micrometastases, and 0.4% with macrometastases. Significant differences in size distributions of occult metastases were observed according to age group, clinical tumor size, type of planned surgical treatment, and type of systemic therapy ( Table 1 Prevalence of Occult Metastases According to Demographic and Clinical Characteristics. Trial Outcomes and Occult Metastases Log-rank tests indicated a significant decrease in overall survival (P=0.03), disease-free survival (P=0.02), and distant-disease–free interval (P=0.04) between patients in whom occult metastases were detected and patients in whom occult metastases were not detected.

The unadjusted hazard ratios were 1.37 (95% CI, 1.03 to 1.81) for death, 1.27 (95% CI, 1.04 to 1.55) for any outcome event, and 1.29 (95% CI, 1.02 to 1.63) for distant disease, and the corresponding adjusted hazard ratios were 1.40 (95% CI, 1.05 to 1.86), 1.31 (95% CI, 1.07 to 1.60), and 1.30 (95% CI, 1.02 to 1.66) ( Table 2 Multivariable Hazard Ratios for Death, Any Outcome Event, and Distant Disease. No interaction effects for occult metastases were detected. The 5-year Kaplan-Meier survival estimates for patients in whom occult metastases were detected were 94.6% for overall survival, 86.4% for disease-free survival, and 89.7% for distant-disease–free interval; the survival estimates for patients in whom occult metastases were not detected were 95.8%, 89.2%, and 92.5%, respectively ( Figure 2 Kaplan-Meier Survival Estimates According to the Presence or Absence of Occult Metastases Detected in Initially Negative Sentinel Lymph Nodes. Panel A shows the probability of overall survival. The Kaplan–Meier estimate of overall survival at 60 months among patients in whom occult metastases were not detected was 95.8%; among patients in whom occult metastases were detected, it was 94.6%.

Panel B shows the probability of disease-free survival. The Kaplan–Meier estimate of disease-free survival at 60 months among patients in whom occult metastases were not detected was 89.2%; among patients in whom occult metastases were detected, it was 86.4%. Panel C shows the probability of distant-disease–free survival.

The Kaplan–Meier estimate of distant-disease–free survival at 60 months among patients in whom occult metastases were not detected was 92.5%; among patients in whom occult metastases were detected, it was 89.7%. Subgroup analysis of outcomes according to the size of occult metastases indicated that smaller metastases had less of an effect on outcomes than larger metastases. The adjusted hazard ratios for detection of isolated tumor-cell clusters (vs. No detection) were 1.27 (95% CI, 1.04 to 1.54) for death, 1.18 (95% CI, 1.02 to 1.33) for any outcome event, and 1.19 (95% CI, 1.00 to 1.41) for distant disease; the corresponding adjusted hazard ratios for detection of micrometastases or macrometastases (vs.

No detection) were 1.60 (95% CI, 1.32 to 1.96), 1.38 (95% CI, 1.15 to 1.60), and 1.41 (95% CI, 1.19 to 1.68) ( ). Exclusion of the 14 patients with occult macrometastases from the Cox regression model resulted in only minimal changes: the hazard ratios in the subgroup analyses of isolated tumor-cell clusters and micrometastases were 1.29 and 1.66 for death, 1.19 and 1.41 for any outcome event, and 1.19 and 1.42 for distant disease, respectively. A subgroup analysis of size of metastases and death from breast cancer also indicated that smaller metastases had a smaller effect; the hazard ratio for death was 1.38 (95% CI, 1.02 to 1.87) among patients with isolated tumor-cell clusters and 1.91 (95% CI, 1.41 to 2.59) among patients with micrometastases or macrometastases, as compared with patients in whom metastases were not detected.

The 5-year Kaplan-Meier estimates of the proportions of patients who did not die from breast cancer were 98.4%, 97.8%, and 96.0% among patients without detectable occult metastases, those with isolated tumor-cell clusters, and those with micrometastases or macrometastases, respectively; however, confidence in these estimates is limited by the small number of outcome events. Occult Metastases and Treatment Failure The sites of first treatment failure are summarized in Table 3 First Treatment Failure, According to Status with Respect to Occult Metastases and Site of Recurrence.. Among patients in whom no occult metastases were detected, there were 14 regional recurrences (0.4%) and 94 distant recurrences (2.9%). Among patients in whom occult metastases were detected, there were 7 regional recurrences (1.1%) and 23 distant recurrences (3.7%).

The overall disease-free survival was significantly higher among patients in whom no occult metastases were detected (2751 of 3268 patients [84.2%]; 95% CI, 82.9 to 85.4) than among those in whom occult metastases were detected (496 of 616 patients [80.5%]; 95% CI, 77.2 to 83.6; P=0.03). Discussion This cohort analysis within a randomized, prospective trial examined the effect of occult metastases in sentinel lymph nodes on disease-free survival and overall survival. Clinical treatment was based on a standard evaluation of sentinel lymph nodes without routine immunohistochemical analysis or analysis of deeper tissue levels. This initial analysis was followed by a blinded analysis of the sentinel nodes for residual occult metastases.

Our findings are consistent with the hypothesis that nodal tumor burden is a continuous variable and indicate that occult metastases are an independent prognostic factor, with unadjusted and adjusted hazard ratios greater than 1.00 for death, any outcome event, and distant disease in patients in whom occult metastases in sentinel lymph nodes were detected as compared with patients in whom no occult metastases were detected. Furthermore, the subgroup analysis of the size of occult metastases indicates that the risk associated with isolated tumor-cell clusters is lower than the risk associated with micrometastases; this finding provides support for the current prognostic segregation of these two categories. The differences observed between patients in whom occult metastases were detected and those in whom occult metastases were not detected with respect to 5-year Kaplan-Meier estimates of overall survival (between-group difference, 1.2 percentage points), disease-free survival (2.8 percentage points), and distant-disease-free interval (2.8 percentage points) were statistically significant but relatively small. Additional follow-up, particularly for hormone-receptor–positive tumors, will be required to determine whether these estimates will converge or continue to diverge. Occult metastases were not discriminatory predictors of cancer recurrence. A total of 138 of 3884 patients (3.6%) had regional or distant recurrences as first events and only 30 of these events (21.7%) (in 0.8% of all the patients) occurred in patients with occult metastases. Conversely, 496 of 616 patients with occult metastases (80.5%) were alive and free of disease.

Identification of occult metastases does not appear to be clinically useful for patients with newly diagnosed disease in whom systemic therapy can be recommended on the basis of the characteristics of the primary tumor. Among women who underwent sentinel-lymph-node biopsy alone, the outcome differences between women with and those without occult metastases were also small (between-group difference, 0.5 percentage points for event-free outcome and 1.3 percentage points for combined rates of regional and distant recurrence).

These minimal differences do not justify changes in clinical management. The outcomes in this group of women are highly relevant; sentinel-lymph-node biopsy alone has been widely adopted and endorsed as an alternative to axillary dissection, and the overall outcome in this trial shows no significant disadvantage for women who underwent sentinel-lymph-node biopsy alone as compared with women who underwent sentinel-lymph-node biopsy plus axillary dissection ( ). In general, the overall rate of regional or distant recurrence (3.5%) was low. The 15.9% prevalence of occult metastases in the current study is within the range reported for axillary nodes (9 to 33%) and is similar to the prevalence in our preliminary study leading to this trial (11.5%). The unfavorable outcome associated with occult metastases in our sentinel-lymph-node study is similar to the results of recent pooled analyses examining the effect of occult metastases in axillary nodes, although our hazard ratios were generally lower. In the pooled analyses, “conclusions could not be drawn from sentinel-lymph-node biopsy studies because studies were limited by small patient groups and short follow-up.” Our analysis, which involved 3884 patients (616 in whom occult metastases were detected) followed for a median of more than 95 months, is limited neither by small size nor short follow-up. The prevalence of occult metastases was significantly associated with an age of less than 50 years, a clinical tumor size of more than 2.0 cm in the greatest dimension, and planned mastectomy.

The higher prevalence in these subgroups is not surprising: planned mastectomy is an indicator of larger tumor size; younger women are more likely than older women to have large, poorly differentiated tumors; and tumor size is highly correlated with the presence or absence of lymph-node metastases. Occult metastases may be more important in the case of larger tumors (combined hazard ratio, 1.32×1.40=1.85) ( ).

This observation was also noted in an analysis of the National Cancer Institute's Surveillance, Epidemiology, and End Results data with respect to the clinical significance of micrometastases. Occult metastases were more likely to be present in patients receiving adjuvant therapy; however, therapy was not based on the presence of occult metastases because their presence was not known to the treating physicians. Thus, other factors known at the initiation of therapy most likely correlate with the presence of occult metastases. Perhaps the most interesting interaction was with endocrine therapy, indicating that occult metastases are associated with estrogen-receptor-positive tumors, a favorable prognostic factor, and that endocrine therapy markedly reduces the risk of a poor outcome; for example, the overall hazard ratio for death among patients with occult metastases was reduced to 0.74 when endocrine therapy was administered (combined hazard ratio, 1.40×0.53=0.74) ( ).

Thus, occult metastases were observed in tumors with favorable prognostic features as well as in tumors with unfavorable prognostic features, underscoring the complex and unpredictable relationship among prevalence, treatment, and outcome. The protocol for sentinel-lymph-node examination at participating sites was designed to identify all macrometastases (deposits >2.0 mm in the greatest dimension). Less than 0.4% of patients had detectable occult macrometastases; this indicates that slicing sentinel lymph nodes at approximately 2.0-mm intervals and examining a single section stained with hematoxylin and eosin from each slice is an effective method for identifying macrometastases.

Furthermore, the analysis of occult metastases in this trial can be considered a study of residual isolated tumor-cell clusters and micrometastases. Isolated tumor-cell clusters had a smaller effect on outcome than micrometastases for every outcome evaluated, including overall survival, disease-free survival, distant-disease–free interval, and death from breast cancer, regardless of whether occult macrometastases were included or excluded. The magnitude of the difference in 5-year Kaplan-Meier estimates for death from breast cancer was small for detection of isolated tumor-cell clusters versus no detection (0.6 percentage points) and for detection of micrometastases versus no detection (2.4 percentage points). A subgroup analysis according to metastasis size was not part of our original planned survival analysis because there is generally less statistical power in smaller samples. Despite this limitation, this trial will probably remain the largest controlled cohort study within a randomized trial to examine this issue. Our findings argue against analysis of additional tissue levels or routine immunohistochemical analysis for sentinel-lymph-node evaluation.

This conclusion is similar to that of the American College of Surgeons Oncology Group Z0010 investigators. Their observed difference in 5-year survival (0.7 percentage points) between patients in whom occult metastases were detected and patients in whom occult metastases were not detected by means of immunohistochemical analysis in initially negative sentinel lymph nodes was not significant (P=0.53). The prevalence of occult metastases in the Z0010 study (10.5%) was lower than the prevalence in this trial (15.9%).

An important limitation of our analysis is not unique to our study: no examination detects all occult metastases present. In fact, our quality-assurance studies show that both micrometastases and isolated tumor-cell clusters are present in unexamined tissue between the levels examined and in the tissue remaining in the paraffin blocks. Although isolated tumor-cell clusters may indicate micrometastases deeper in the sentinel-lymph-node blocks, this misclassification error occurs in a minority of cases (22%).

With extrapolation from our data, it is reasonable to conclude that, regardless of whether they are detected in initial sections or in additional deeper levels, isolated tumor-cell clusters and micrometastases have less prognostic significance than macrometastases and should be classified separately. The strength of our study is that it was specifically designed to exclude macrometastases from the study population and to include virtually all residual metastases larger than 1.0 mm in the greatest dimension, and a significant proportion of occult metastases smaller than 1.0 mm by statistical chance, providing a robust outcome analysis linked to a specific standardized sentinel-lymph-node evaluation. Any analysis following our standardized approach would be expected to have similar results with respect to prevalence and outcome. In summary, we found that small occult metastases in sentinel nodes are an independent predictor of overall survival, disease-free survival, and distant-disease–free interval.

Multivariate analysis suggests that multiple factors (e.g., age and tumor size) influence the prevalence of occult metastases and the outcome and that local radiation therapy and adjuvant systemic therapy, particularly endocrine therapy, attenuate the unfavorable effect of occult metastases. The magnitude of the differences in outcome between patients with and those without occult metastases was small (1 to 3 percentage points) at 5 years but warrants continued follow-up and analysis. The views expressed in this article are solely those of the authors and do not necessarily represent the official views of the National Cancer Institute or the U.S.

Supported by grants (UO1-CA65121, P30-CA22435, U10CA-12027, U10CA-69974, U10CA-37377, and U10CA-69651) from the Public Health Service of the National Cancer Institute. Provided by the authors are available with the full text of this article at NEJM.org. This article (10.1056/NEJMoa1008108) was published on January 19, 2011, at NEJM.org. We thank the NSABP study participants, participating pathologists, members of the Histology Department at Fletcher Allen Health Care, and the pathology review team at the University of Vermont, including Drs. Abiy Ambaye, Scott Anderson, Thuy Tran, and Brenda Waters, for their dedication to this effort; and Sarah Howe for assistance in preparation and submission of an earlier version of the manuscript. Source Information From the University of Vermont College of Medicine and Vermont Cancer Center, Departments of Pathology, Surgery, and Medical Biostatistics, Burlington (D.L.W., T.A., D.N.K., J.M.S., S.P.H.); the National Surgical Adjuvant Breast and Bowel Project (NSABP) (D.L.W., T.A., D.N.K., S.J.A., S.P.H., T.B.J., E.P.M., N.W.), the NSABP Biostatistical Center and the University of Pittsburgh Graduate School of Public Health, Department of Biostatistics (S.J.A.), and Allegheny General Hospital (T.B.J., N.W.) — all in Pittsburgh; and the Aultman Cancer Center, Canton, OH (E.P.M.).

Address reprint requests to Dr. Weaver at the Department of Pathology, Given Courtyard, University of Vermont College of Medicine, 89 Beaumont Ave., Burlington, VT,. References • 1 Saphir O, Amromin GD. Obscure axillary lymph node metastases in carcinoma of the breast.

Cancer 1948;1:238-241 • 2 Dowlatshahi K, Fan M, Snider HC, Habib FA. Lymph node micrometastases from breast carcinoma: reviewing the dilemma. Cancer 19-1197 • 3 de Boer M, van Dijck JA, Bult P, Borm GF, Tjan-Heijnen VC. Breast cancer prognosis and occult lymph node metastases, isolated tumor cells, and micrometastases.

J Natl Cancer Inst 2010;102:410-425 • 4 Weaver DL. Sentinel lymph nodes and breast carcinoma: which micrometastases are clinically significant? Am J Surg Pathol 2003;27:842-845 • 5 Krag DN, Anderson SJ, Julian TB, et al.

Technical outcomes of sentinel-lymph-node resection and conventional axillary-lymph-node dissection in patients with clinically node-negative breast cancer: results from the NSABP B-32 randomised phase III trial. Lancet Oncol 2007;8:881-888 • 6 Weaver DL, Krag DN, Ashikaga T, Harlow SP, O'Connell MJ. Pathologic analysis of sentinel and nonsentinel lymph nodes in breast carcinoma: a multicenter study.

Cancer 20-1107 • 7 Weaver DL, Le UP, Dupuis SL, et al. Metastasis detection in sentinel lymph nodes: comparison of a limited widely spaced (NSABP protocol B-32) and a comprehensive narrowly spaced paraffin block sectioning strategy. Am J Surg Pathol 20-1589 • 8 Weaver DL.

Pathology evaluation of sentinel lymph nodes in breast cancer: protocol recommendations and rationale. Mod Pathol 2010;23:Suppl:S26-S32 • 9 Greene FL, Page DL, Fleming ID, et al., eds.

AJCC cancer staging manual. New York: Springer, 2002. • 10 Kaplan EL, Meier P. Nonparametric estimation from incomplete observations. J Am Stat Assoc 1958;53:457-481 • 11 Mantel N, Haenszel W. Statistical aspects of the analysis of data from retrospective studies of disease. J Natl Cancer Inst 1959;22:719-748 • 12 Cox DR.

Regression models and life-tables. J R Stat Soc [B] 1972;34:187-220 • 13 Gibbons JD. Nonparametric statistical inference.

New York: McGraw-Hill, 1971. • 14 Krag DN, Anderson SJ, Julian TB, et al. Sentinel-lymph-node resection compared with conventional axillary-lymph-node dissection in clinically node-negative patients with breast cancer: overall survival findings from the NSABP B-32 randomised phase 3 trial.

Lancet Oncol 2010;11:927-933 • 15 Lyman GH, Giuliano AE, Somerfield MR, et al. American Society of Clinical Oncology guideline recommendations for sentinel lymph node biopsy in early-stage breast cancer. J Clin Oncol 20-7720 • 16 Carter CL, Allen C, Henson DE. Relation of tumor size, lymph node status and survival in 24,740 breast cancer cases. Cancer 1989;63:181-187 • 17 Chen SL, Hoehne FM, Giuliano AE. The prognostic significance of micrometastases in breast cancer: a SEER population-based analysis. Ann Surg Oncol 20-3384 • 18 Cote R, Giuliano AE, Hawes D, et al.

ACOSOG Z0010: a multicenter prognostic study of sentinel node (SN) and bone marrow (BM) micrometastases in women with clinical T1/T2 N0 M0 breast cancer. J Clin Oncol 2010;28:Suppl:18-18.