Nuclear Grade Plus Proliferation Grading System for Invasive Ductal Carcinoma of the Breast

Nuclear Grade Plus Proliferation Grading System for Invasive Ductal Carcinoma of the Breast Validation in a Tertiary Referral Hospital Cohort Maria Papadimitriou, MD, 1 Sarantos Kaptanis, MD, JD, 2 Evangelos Polymeropoulos, MD, 3 Grigorios Mitsopoulos, MD, FRCPath, 4 Dimitris Stogiannis, PhD, 5 Chrys Caroni, PhD, 6 George Vaiopoulos, MD, PhD, 7 Joannis G. Panayiotides, MD, PhD, 8 and Petros Karakitsos, MD, PhD 8 From the 1 Laikon Hospital, 1st Internal Medicine Department, University of Athens Medical School, Athens, Greece; 2 Barts Cancer Institute, Queen Mary, University of London, London, England; 3 Athens Red Cross Hospital, Athens, Greece; 4 Ealing Hospital NHS Trust, London, England; 5 Department of Epidemiology & Public Health, University College London, London, England; 6 Department of Mathematics, National Technical University of Athens, Athens, Greece; 7 University of Athens Medical School, Athens, Greece; and 8 Department of Cytopathology, University of Athens Medical School, Attikon University Hospital, Athens, Greece. Key Words: Invasive breast carcinoma grading; MIB-1 (Ki-67) immunohistochemistry; Automation; Survival; Nottingham Prognostic Index Am J Clin Pathol December 2015;144:837-843 ABSTRACT Objectives: For patients with invasive breast cancer, management decisions are informed by tumor grade according to the Nottingham Grading System (NGS), either on its own or as part of the Nottingham Prognostic Index (NPI). A system retaining the nuclear grade element but substituting the two subjective components, mitosis count and tubule formation, of the NGS with a proliferation index based on Ki-67 (MIB-1) has been proposed (nuclear grade plus proliferation [N+P] grading). Methods: We validated the prognostic value of this grading system on a population of 322 women. Results: N+P grading resulted in more grade I tumors (47.9% vs 4.5%) and fewer grade II (32% vs 51.5%) and grade III (20.1% vs 44%) tumors compared with NGS. The NPI calculated based on N+P grade had a similar association with survival (P <.001; odds ratio, 1.729) as the NPI calculated on the basis of the NGS grade (P <.001; odds ratio, 1.668). Conclusions: The N+P system seems equivalent to the NGS system. Therapeutic decisions in patients with breast cancer largely depend on their classification in a manner that predicts their biology: clinical staging (American Joint Committee on Cancer TNM stages I-IV 1 ), histologic grading (1-3 2 ), and molecular biomarker-based classifications (estrogen receptor [ER], 3 progesterone receptor [PR], 4 human epidermal growth factor receptor 2 [HER2] gene, 5 and recently multigene prognostic indices 6,7 ). Histologic grading of breast cancer is commonly done according to the Nottingham (Elston-Ellis) modification of the Scarff, Bloom, and Richardson grading system, 8 also known as the Nottingham Grading System (NGS). In the past, concerns over reproducibility of grading systems have led to reluctance to use grades to aid therapeutic decisions. 9 In 2007, Tawfik et al 10 introduced the nuclear grade plus proliferation (N+P) grading system, a more objective grading system that is claimed to better represent breast cancer biology. The N+P grade substitutes MIB-1 count for tubule formation and mitotic count, with the percentage of the total number of breast cancer cells with nuclear staining classified into three categories: 9% or less, 10% to 25%, and more than 25%, respectively. N+P grade I is defined as a tumor having nuclear grade 1 or 2 and MIB-1 25% or less. N+P grade II refers to a tumor with nuclear grade 3 and MIB-1 25% or less, or nuclear grade 1 or 2 with a MIB-1 more than 25%. N+P grade III describes a tumor having a nuclear grade 3 and MIB-1 more than 25%. The initial results with the N+P grading system showed that it is precise, reliable, and easy to use. MIB-1 assessment Am J Clin Pathol 2015;144:837-843 837

Papadimitriou et al / N+P Grading for Breast Cancer has been shown to outperform mitotic count in interobserver agreement in other cancers. 11 Nonetheless, the inventors of the system did not test whether the N+P grading system might confer additional prognostic information on patient survival when tumor size or lymph node status is taken into account parameters used to determine prognosis in the Nottingham Prognostic Index (NPI). N+P also has not been evaluated with regard to the prognostic information it confers on disease-free survival. The present study sought to compare the additional prognostic information that the N+P grading system may confer over the NGS when other relevant clinical parameters are taken into account. It also sought to establish whether the N+P grading system is valid in predicting disease-free survival, as well as overall survival. Materials and Methods Patient Cohort This was a retrospective study. After obtaining institutional board ethics approval (13/22-10-2010), we reviewed medical records of 322 patients treated surgically for breast cancer, from 2003 to 2007, in the Greek tertiary hospital Elena. These included pathology reports and complete follow-up records. Completion date for the follow-up of all patients was February 28, 2011. Complete data were available for 319 patients, and these were analyzed further. Median follow-up was 65 months (minimum: 7 months; maximum: 97 months). Collection of pathology specimens was according to institutional protocol: briefly, mastectomy and wide local excision specimens were formalin fixed in 10% formalin after the surgical procedure. Following paraffin embedding, the tumor specimens were examined by H&E staining. All specimens were evaluated by two consultant pathologists (J.G.P. and P.K.) who reached consensus on tumor grading according to the Nottingham modification of the Scarff- Bloom-Richardson grading system. Immunohistochemistry Tissue blocks containing representative tumor areas were selected for immunohistochemistry (IHC). IHC was performed on tissue fixed with 10% neutral buffered formalin. Paraffin-embedded tissue blocks were cut to 5-μm sections, deparaffinized in xylene, treated with 100% ethanol, and heat treated in a microwave (0.01 M citrate buffer, ph 6.0 for 15 minutes) for antigen retrieval. Hormone receptors (estrogen, progesterone), Her-2/neu, and Ki-67 (MIB-1) were examined on all specimens using a DAKO Autostainer (DAKO, Carpinteria, CA) and commercially available monoclonal antibodies Table 1. ER and PR were graded Table 1 Protocols for Immunohistochemistry Antibody Vendor Clone Titer ER DAKO, Glostrup, Denmark 1D5 1:300 PR DAKO, Glostrup, Denmark PgR 636 1:400 HER2/neu DAKO, Glostrup, Denmark A0485 1:400 Ki-67 DAKO, Glostrup, Denmark MIB-1 1:60 ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; PR, progesterone receptor. as positive if more than 10% of the tumor cells exhibited nuclear overexpression. HER2/neu was evaluated based on the scoring guidelines of DAKO; scores of 0 and 1+ were considered negative, and 2+ and 3+ were considered positive. At least three sections per tumor were examined. Ki-67 proliferation index was measured using the CAS-200 image analyzer (Becton-Dickinson, San Jose, CA). An average score of 10 areas with the highest staining intensity from each specific lesion was calculated quantitatively using the PI program of the image analyzer. Subtypes We evaluated tumor subtypes using an accepted histologic classification, 12 classifying ER+ or PR+, HER2 as luminal A; ER+ or PR+, HER2+ as luminal B; ER, PR, HER2+ as HER2 overexpressing; and ER, PR, HER2 as triple negative. Prognostic Indices NPI was calculated using the original formula 13 : NPI = [0.2 tumor size in cm] + [number of lymph nodes involved: 0 = 1, 1-3 = 2, >3 = 3] + [grade of tumor], where grade was either the NGS or N+P grade. We used the NPI to stratify our patients into three groups: good prognosis (NPI 3.4), moderate prognosis (NPI 3.41-5.4), or poor prognosis (NPI >5.4). Statistical Analysis We constructed a database wherein the following parameters were included: age, type of surgery (mastectomy or wide local excision), date of operation, ER and PR status, ErbB-2 (HER2) status, tumor size (measured on gross pathologic examination), number of lymph nodes, histologic grade, nuclear grade, Ki-67 staining (percent and classification), history of familial breast cancer, adjuvant chemotherapy, adjuvant hormonal therapy, relapse, chemotherapy treatment on relapse, death, and total length of follow-up. We incorporated the three variables: tumor size, number of lymph nodes, and histologic grade in the NPI. All statistical analysis was performed on SPSS version 22.0 (SPSS, Chicago, IL). We performed univariate Cox analysis of all variables to find which of the parameters in our database could be included in our model and, following 838 Am J Clin Pathol 2015;144:837-843

Table 2 s of Patients (n = 319) a that, built multivariate Cox analysis models both for overall survival and disease-free survival. Our analysis compares the NPI using the NGS grading system and the NPI using the N+P grading system in a unique patient cohort. Results Value Age, mean (SD), y 61.1 (11.6) Female sex 319 (100) Family history Yes 49 (15.4) No 270 (84.6) Operation type Wide local excision 167 (52.4) Mastectomy 148 (46.4) Unknown 4 (1.3) Tumor size, median (IQR), mm 22 (16-30) Lymph nodes 0 138 (43.3) 1-3 107 (33.5) >3 74 (23.2) Ki-67 (MIB-1) 9% 78 (24.5) 10%-25% 155 (48.6) >25% 86 (27.0) ER Positive 237 (74.3) Negative 82 (25.7) PR Positive 213 (66.8) Negative 106 (33.2) HER2 Positive 108 (33.9) Negative 211 (66.1) Tumor subtypes Luminal A (ER+ or PR+, HER2 ) 177 (55.5) Luminal B (ER+ or PR+, HER2+) 79 (24.8) HER2 overexpressing (ER, PR, HER2+) 29 (9.1) Triple negative (ER, PR, HER2 ) 34 (10.7) Adjuvant treatment Chemotherapy 302 (94.7) Hormone therapy 265 (83.1) ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; IQR, interquartile range; PR, progesterone receptor. a Values are presented as number (%) unless otherwise indicated. After reviewing the database, 319 patients, all female, were included in the analysis due to missing data on three patients. Patient demographics are presented in Table 2. Importantly, more patients in our cohort underwent breastconserving surgery than mastectomy (52.4%), and most (68.7%) were placed in the moderate prognosis group according to the NPI. Of the patients, 74.3% were ER positive, 66.8% were PR positive, and 33.9% were HER2 positive. Most patients had adjuvant chemotherapy (94.7%) and hormone therapy (83.1%). The anti-her2 antibody Table 3 N+P vs NGS Grade and NPI Scores (n = 319) a NGS N+P Tumor grade I 13 (4.1) 149 (46.7) II 163 (51.1) 108 (33.9) III 143 (44.8) 62 (19.4) NPI, mean (SD) 4.72 (1.19) 4.04 (1.28) NPI prognosis Good 6 (1.9) 40 (12.5) Moderate 219 (68.7) 222 (69.6) Poor 94 (29.5) 57 (17.9) NGS, Nottingham Grading System; N+P, nuclear grade + proliferation grading system; NPI, Nottingham Prognostic Index. a Values are presented as number (%) unless otherwise indicated. Table 4 Outcomes (n = 319) a Value Disease-free survival at end of follow-up 236 (74.0) Overall survival at end of follow-up 284 (89.0) Total follow-up, mean (SD), mo 65.7 (17.2) Disease-free survival follow-up, mean (SD), mo 60.7 (21.6) Recurrence Local 6 (1.9) Contralateral breast 7 (2.2) Regional lymph nodes 5 (1.6) Distant metastases 64 (20.1) a Values are presented as number (%) unless otherwise indicated. trastuzumab (Herceptin) was not available for treatment of primary breast cancer in Greece during that period. The Ki-67 (MIB-1) proliferation index split patients into three groups: 9% or less (24.5%), 10% to 25% (48.6%), and more than 25% (27.0%). We used these data and nuclear grade to calculate the N+P score for all patients in our cohort and the equivalent NPI, and we compare our findings with the standard NGS grading in Table 3. Although the two systems gave similar results for grade and prognostic index, more patients were placed in the good prognosis group using the N+P score (12.5% vs 1.9%), corresponding to more grade I tumors (47.9% vs 4.5%) and less grade II (32% vs 51.5%) and grade III (20.1% vs 44%) tumors. The importance of prognostic indices is their correlation with patient survival and recurrence (ie, prediction of outcomes) Table 4. At the end of follow-up (median, 65 months), 284 (89.0%) patients were still alive, and 236 (74.0%) patients were disease free. There were six cases of local recurrence, seven recurrences in the contralateral breast, five in the regional lymph nodes, and 64 instances of metastatic disease. We performed univariate Cox regression to evaluate the effect of patient-specific, disease-specific, and treatmentspecific factors on survival and recurrence Table 5. Age (better prognosis with older age, P =.018), ER (better prognosis with positive ER, P =.003), PR (better prognosis Am J Clin Pathol 2015;144:837-843 839

Papadimitriou et al / N+P Grading for Breast Cancer Table 5 Univariate Cox Regression for Survival and Recurrence with positive PR, P =.004), tumor size (better prognosis with smaller tumor size, P =.036), number of positive lymph nodes (better prognosis with less involved lymph nodes, P =.015), lymph node stage (P =.012), tumor grade calculated with the NGS system (better prognosis with lower grade, P =.039), Ki-67 (MIB-1 classes) (P =.017), tumor grade calculated with the N+P system (better prognosis with lower grade, P =.006), NPI calculated with the NGS grade (P <.001), NPI calculated with the N+P grade (P <.001), prognostic groups defined by the NPI calculated with the NGS grade (P =.002), prognostic groups defined by the NPI calculated with the N+P grade (better prognosis with smaller scores, P <.001), IHCdefined subtypes (from better to worse prognosis: luminal B, Overall Survival Disease-Free Survival P Value OR (95% CI) P Value OR (95% CI) Age, y.018 0.965 (0.936-0.994).261 0.989 (0.970-1.008) Family history.725 1.185 (0.459-3.057).162 1.642 (0.820-3.287) Type of surgery.154 0.614 (0.314-1.200).667 0.908 (0.584-1.411) ER status.003 2.697 (1.389-5.238).053 1.580 (0.993-2.512) PR status.004 2.697 (1.381-5.270).191 1.350 (0.861-2.118) HER2 status.780 0.905 (0.450-1.823).529 1.166 (0.723-1.882) Tumor size, mm.036 1.018 (1.001-1.036).137 1.010 (0.997-1.024) No. of positive lymph nodes.015 1.049 (1.009-1.091).011 1.038 (1.009-1.068) Lymph node stage 1.012 1.014 1 2 2.175 (0.902-5.249) 0.884 (0.508-1.538) 3 3.579 (1.501-8.534) 1.908 (1.149-3.166) Grade (NGS) 1.039 0.004 0.223 (0.031-1.616) 2 0.506 (0.255-1.004) 0.506 (0.323-0.794) Nuclear grade 1.082 0.006 0.420 (0.102-1.728) 2 0.528 (0.266-1.048) 0.496 (0.313-0.785) Ki-67, %.525 1.772 (0.302-10.402).505 1.505 (0.451-5.020) Ki-67 (MIB-1) 1.017 0.646 (0.285-1.465).073 0.679 (0.380-1.212) 2 0.331 (0.150-0.729) 0.563 (0.341-0.931) Grade (N+P) 1.006 0.314 (0.143-0.692).002 0.391 (0.227-0.674) 2 0.401 (0.178-0.903) 0.595 (0.347-1.020) NPI (NGS) <.001 1.668 (1.264-2.200) <.001 1.385 (1.155-1.662) NPI (NGS) groups.002 0.374 (0.193-0.725).006 0.541 (0.346-0.844) NPI (N+P) <.001 1.729 (1.326-2.256) <.001 1.399 (1.177-1.662) NPI (N+P) groups <.001 0.285 (0.145-0.562).001 0.447 (0.275-0.725) Subtype classification.006.037 Luminal A 0.367 (0.156-0.867) 0.829 (0.418-1.643) Luminal B 0.293 (0.096-0.898) 0.484 (0.209-1.121) HER2+ 1.155 (0.418-3.192) 1.507 (0.651-3.492) TN 1 1 Adjuvant chemotherapy.474 0.483 (0.066-3.533).211 0.408 (0.100-1.661) Adjuvant hormone therapy.007 2.597 (1.291-5.220).061 1.638 (0.978-2.742) CI, confidence interval; ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; NGS, Nottingham Grading System; N+P, nuclear grade + proliferation; NPI, Nottingham Prognostic Index; OR, odds ratio; PR, progesterone receptor; TN, triple negative. luminal A, triple negative, HER2 overexpressing, P =.011), and adjuvant hormone treatment (better prognosis for those who received hormone treatment, P =.007) were statistically significant factors affecting survival. Number of positive lymph nodes (P =.011), lymph node stage (P =.014), tumor grade (NGS) (P =.004), nuclear grade (P =.006), tumor grade (N+P) (P =.002), NPI (NGS) (P <.001), NPI (NGS) groups (P =.006), NPI (N+P) (P <.001), NPI (N+P) groups (P =.001), and IHC-defined subtype classification (P =.037) were statistically significant factors affecting recurrence. We found that the N+P-based NPI is not inferior to the NGS-based NPI in defining prognostic groups. 840 Am J Clin Pathol 2015;144:837-843

Table 6 Multivariate Cox Regression for OS and DFS Models Multivariate Cox regression was performed using statistically significant variables and removing variables that derive from others and create problems of multicollinearity. Thus, we selected age, ER status, PR status, and the relevant NPI for comparisons Table 6. The prognostic indices retain their statistical significance in the multivariate models. The value of prognostic indices in the clinic is in guiding management decisions for adjuvant chemotherapy and frequency of outpatient follow-up; in this respect, the N+P NPI model proves slightly superior to the NGS NPI model, clearly separating good and moderate prognosis patients from poor prognosis ones both for overall and disease-free survival Figure 1 and Figure 2. Discussion The NGS is endorsed by the World Health Organization, the College of American Pathologists, the American NGS Model N+P Models P Value OR (95% CI ) P Value OR (95% CI) OS Age, y.028 0.966 (0.936-0.996).035 0.967 (0.937-0.998) ER status.425 1.387 (0.621-3.100).521 1.304 (0.579-2.939) PR status.025 2.500 (1.119-5.584).040 2.306 (1.038-5.127) NPI (categ).008 0.401 (0.205-0.786).005 0.365 (0.181-0.737) DFS Subtype.035.058 Luminal A 0.829 (0.418-1.641) 0.911 (0.459-1.809) Luminal B 0.463 (0.200-1.073) 0.497 (0.214-1.153) HER2+ 1.501 (0.648-3.477) 1.504 (0.649-3.485) TN 1 1 NPI (categ).006 0.536 (0.344-0.835).002 0.458 (0.281-0.746) CI, confidence interval; DFS, disease-free survival; ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; NGS, Nottingham Grading System; N+P, nuclear grade + proliferation; NPI (categ), Nottingham Prognostic Index categories: good/moderate vs poor; OR, odds ratio; OS, overall survival; PR, progesterone receptor; TN, triple negative. Cumulative Survival (%) 100 95 90 85 80 0 20 40 Good or moderate Poor 60 80 Overall Survival (mo) Figure 1 Cox regression. Overall survival according to Nottingham Prognostic Index (nuclear grade plus proliferation) prognostic category. Cumulative Survival (%) 100 90 80 70 60 50 Poor Good or moderate 0 20 40 60 80 100 Disease-Free Survival (mo) Figure 2 Cox regression. Disease-free survival according to Nottingham Prognostic Index (nuclear grade plus proliferation) prognostic category. Joint Committee on Cancer Staging manual, the European Union, and the Royal College of Pathologists. It assigns a score of 1 to 3 each to tubule formation (>75% = 1, 10%- 75% = 2, <10% = 3, commonly abbreviated as T), nuclear pleomorphism (an indicator of cellular differentiation; small regular uniform cells = 1, moderate nuclear size and variation = 2, marked nuclear variation = 3, commonly abbreviated as N), and mitotic activity (an indicator of proliferation; with 0-9 mitoses/high-power field [hpf] = 1, 10-19 mitoses/ hpf = 2, >20 mitoses/hpf = 3, commonly abbreviated as M). A tumor with a total score of 3 to 5 is categorized as grade 1, one with a score of 6 or 7 as grade 2, and one with a score of 8 or 9 as grade 3. This system is reported to have significant interobserver agreement and correlates well with both disease-free and overall survival. At 10-year follow-up, grade 1 cancers are expected to have 85% survival, grade 2 about 60%, and grade 3 only 45%. The NGS has a modest degree of reproducibility, 14 although continuous standardization efforts have led to Am J Clin Pathol 2015;144:837-843 841

Papadimitriou et al / N+P Grading for Breast Cancer better results over time. Nonetheless, histologic grade is and remains a partially subjective parameter. Thus arises the need for a more objective grading system that is easier to use at the same time: the N+P system. Antigen Ki-67 is a cellular marker for proliferation. Ki-67, a nonhistone protein, is expressed in proliferating cells through all active phases of the cell cycle (G1, G2, S, M) but is absent from resting cells (G0). Several antibodies against Ki-67 have been assessed, the most commonly used being MIB-1 (DAKO), which, unlike the original Ki-67 antibody, can be used on formalin-fixed, paraffin-embedded tissue. 15 Measurement of Ki-67 and receptor status by image analysis is widely reported in the literature 16,17 and has been shown to closely correlate with visual analysis. 11,18 The advantage of automated, quantitative methods such as the CAS-200 is their objectivity and speed. 19 Assessment of Ki-67 and other immunohistochemical markers by automated systems has known weaknesses. Interobserver agreement as low as 56% has been reported, 20 and there is controversy over Ki-67 assessment by both image analysis and visual inspection. 21 Even though CAS-200 is an image analysis system developed in the 1980s and there are newer, possibly more accurate, methods available or under development today, 22 it remains in use in a variety of institutions. Ki-67 has been assessed as a prognostic factor in a number of studies, 23-27 but it is not part of routine clinical evaluation in breast cancer. 28 There is evidence that wellestablished immunohistochemical markers (ER, PR, HER2, Ki-67) combining hormone receptors with a proliferation index (the IHC4 score) could confer similar prognostic information to the 21-gene Genomic Health recurrence score (GHI-RS, also known as Oncotype DX) at a fraction of the cost. 29 The cutoff points for Ki-67 used in our study and previously suggested by Tawfik et al 10 are far from universal, and more research is needed to evaluate these as prognostic markers. To date, this is the largest patient series from Greece evaluating Ki-67 and derived indexes in breast cancer prognosis. Strengths of our study include population homogeneity since our cohort comes from a single tertiary hospital in a relatively short period of 4 years, no losses to follow-up, and robust, consistent laboratory methods in the assessment of biomarkers from a single university laboratory. Our study has several weaknesses. It is a cohort study and is subject to inherent bias since patient management is not rigorously controlled as in a clinical trial. Apparent indicators of this are that most (95%) of our patients had adjuvant chemotherapy. There is controversy over Ki-67 assessment by both image analysis and visual inspection 21 ; our study did not include a comparison of the automated evaluation with visual assessment. Data collection, although prospective, did not include factors that may have influenced outcomes namely, type of chemotherapy regimens and adjuvant radiotherapy. Our follow-up period, although greater than 5 years on average, may be considered short for breast cancer, a disease with an excellent outcome with current treatment modalities; in fact, our series has 89% overall survival and 74% disease-free survival. We encourage additional prospective study of a more controlled patient group. In conclusion, we have demonstrated the N+P system to be equivalent to the traditional NGS system in the grading of breast cancer in a Greek patient cohort, both as an individual predictor and as part of the NPI. At the same time, the N+P system is simpler and easier to use. Corresponding author: Sarantos Kaptanis, MD, JD, Centre for Tumour Biology, Barts Cancer Institute, Queen Mary, University of London, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ, UK; sarandos@gmail.com. This research received institutional board ethics approval from the University of Athens (13/22-10-2010) and was supported by Greek tertiary hospital Elena. References 1. Walters S, Maringe C, Butler J, et al. Breast cancer survival and stage at diagnosis in Australia, Canada, Denmark, Norway, Sweden and the UK, 2000-2007: a population-based study. Br J Cancer. 2013;108:1195-1208. 2. Rakha EA, Reis-Filho JS, Baehner F, et al. Breast cancer prognostic classification in the molecular era: the role of histological grade. Breast Cancer Res. 2010;12:207. 3. Yu KD, Wu J, Shen ZZ, et al. Hazard of breast cancer specific mortality among women with estrogen receptor positive breast cancer after five years from diagnosis: implication for extended endocrine therapy. J Clin Endocrinol Metab. 2012;97:E2201-E2209. 4. Chapman JA, Nielsen TO, Ellis MJ, et al. Effect of continuous statistically standardized measures of estrogen and progesterone receptors on disease-free survival in NCIC CTG MA.12 trial and British Columbia cohort. Breast Cancer Res. 2013;15:R71. 5. Gudlaugsson E, Klos J, Skaland I, et al. Prognostic comparison of the proliferation markers (mitotic activity index, phosphohistone H3, Ki67), steroid receptors, HER2, high molecular weight cytokeratins and classical prognostic factors in T(1)( )(2)N(0)M(0) breast cancer. Polish J Pathol. 2013;64:1-8. 6. Bastien RR, Rodriguez-Lescure A, Ebbert MT, et al. PAM50 breast cancer subtyping by RT-qPCR and concordance with standard clinical molecular markers. BMC Med Genomics. 2012;5:44. 7. Gokmen-Polar Y, Badve S. Molecular profiling assays in breast cancer: are we ready for prime time? Oncology. 2012;26:350-357, 361. 8. Elston CW, Ellis IO. Pathological prognostic factors in breast cancer, I: the value of histological grade in breast cancer: experience from a large study with long-term follow-up. Histopathology. 1991;19:403-410. 9. Dalton LW, Pinder SE, Elston CE, et al. Histologic grading of breast cancer: linkage of patient outcome with level of pathologist agreement. Mod Pathol. 2000;13:730-735. 842 Am J Clin Pathol 2015;144:837-843

10. Tawfik O, Kimler BF, Davis M, et al. Grading invasive ductal carcinoma of the breast: advantages of using automated proliferation index instead of mitotic count. Virchows Arch. 2007;450:627-636. 11. Warth A, Fink L, Fisseler-Eckhoff A, et al. Interobserver agreement of proliferation index (Ki-67) outperforms mitotic count in pulmonary carcinoids. Virchows Arch. 2013;462:507-513. 12. Onitilo AA, Engel JM, Greenlee RT, et al. Breast cancer subtypes based on ER/PR and Her2 expression: comparison of clinicopathologic features and survival. Clin Med Res. 2009;7:4-13. 13. Galea MH, Blamey RW, Elston CE, et al. The Nottingham Prognostic Index in primary breast cancer. Breast Cancer Res Treat. 1992;22:207-219. 14. Meyer JS, Alvarez C, Milikowski C, et al. Breast carcinoma malignancy grading by Bloom-Richardson system vs proliferation index: reproducibility of grade and advantages of proliferation index. Mod Pathol. 2005;18:1067-1078. 15. Cattoretti G, Becker MH, Key G, et al. Monoclonal antibodies against recombinant parts of the Ki-67 antigen (MIB 1 and MIB 3) detect proliferating cells in microwaveprocessed formalin-fixed paraffin sections. J Pathol. 1992;168:357-363. 16. Pinder SE, Wencyk P, Sibbering DM, et al. Assessment of the new proliferation marker MIB1 in breast carcinoma using image analysis: associations with other prognostic factors and survival. Br J Cancer. 1995;71:146-149. 17. Querzoli P, Albonico G, Ferretti S, et al. MIB-1 proliferative activity in invasive breast cancer measured by image analysis. J Clin Pathol. 1996;49:926-930. 18. Coleman KE, Brat DJ, Cotsonis GA, et al. Proliferation (MIB-1 expression) in oligodendrogliomas: assessment of quantitative methods and prognostic significance. Appl Immunohistochem Mol Morphol. 2006;14:109-114. 19. Laitakari J, Harrison D, Stenback F. Automated image analysis of proliferating cells in carcinoma of the larynx. Acta Otolaryngol. 2003;123:759-766. 20. Makkink-Nombrado SV, Baak JP, Schuurmans L, et al. Quantitative immunohistochemistry using the CAS 200/486 image analysis system in invasive breast carcinoma: a reproducibility study. Anal Cell Pathol. 1995;8:227-245. 21. Varga Z, Diebold J, Dommann-Scherrer C, et al. How reliable is Ki-67 immunohistochemistry in grade 2 breast carcinomas? a QA study of the Swiss Working Group of Breast- and Gynecopathologists. PLoS One. 2012;7:e37379. 22. Suciu C, Muresan A, Cornea R, et al. Semi-automated evaluation of Ki-67 index in invasive ductal carcinoma of the breast. Oncol Lett. 2014;7:107-114. 23. Liu Y, Yin W, Yan T, et al. The clinical significance of Ki-67 as a marker of prognostic value and chemosensitivity prediction in hormone-receptor-positive breast cancer: a meta-analysis of the published literature. Curr Med Res Opin. 2013;29:1453-1461. 24. Wiesner FG, Magener A, Fasching PA, et al. Ki-67 as a prognostic molecular marker in routine clinical use in breast cancer patients. Breast. 2009;18:135-141. 25. Jung SY, Han W, Lee JW, et al. Ki-67 expression gives additional prognostic information on St. Gallen 2007 and Adjuvant! Online risk categories in early breast cancer. Ann Surg Oncol. 2009;16:1112-1121. 26. Ferguson NL, Bell J, Heidel R, et al. Prognostic value of breast cancer subtypes, Ki-67 proliferation index, age, and pathologic tumor characteristics on breast cancer survival in Caucasian women. Breast J. 2013;19:22-30. 27. Urruticoechea A, Smith IE, Dowsett M. Proliferation marker Ki-67 in early breast cancer. J Clin Oncol. 2005;23:7212-7220. 28. Jonat W, Arnold N. Is the Ki-67 labelling index ready for clinical use? Ann Oncol. 2011;22:500-502. 29. Cuzick J, Dowsett M, Pineda S, et al. Prognostic value of a combined estrogen receptor, progesterone receptor, Ki-67, and human epidermal growth factor receptor 2 immunohistochemical score and comparison with the Genomic Health recurrence score in early breast cancer. J Clin Oncol. 2011;29:4273-4278. Am J Clin Pathol 2015;144:837-843 843