Introduction c-kit 1 2 4 5 6 7 8 9 10 11 12 13 14 15 16 P = 11 Materials and methods Patient selection 17 TMA construction 18 Immunohistochemistry (IHC) 1 Table 1 Details of antibodies used for immunohistochemistry Antibody Isotype  Company Antigen retrieval Concentration c-kit Rabbit polyclonal Dako Ventana 1:100 Her2 SP3 Rabbit monoclonal Lab vision Steam 30 min, 0.05 M TRIS buffer (pH 10) 1:100 ER SP1 Rabbit monoclonal Lab vision Citrate buffer (pH 6) 1:250 Immunohistochemical image processing and scoring http://www.gpecimage.ubc.ca/tma/web/viewer.php. 11 18 ® Statistical analysis 19 2 Table 2 Summary of clinical-pathological characteristics of the 4,444 breast cancer patients  Test set Validation set n % within group % within known values n % within group % within known values Total 2,222   2,222   Age at diagnosis (years) Median (range) 60 (25–95)   60 (23–91)   <40 170 7.7 7.7 157 7.1 7.1 40–54 457 20.6 20.6 468 21.1 21.1 55–69 702 31.6 31.6 809 36.4 36.4 ≥70 893 40.2 40.2 788 35.5 35.5 Gender Female 2,211 99.5 99.5 2,210 99.5 99.5 Male 11 0.5 0.5 10 0.5 0.5 Nodal status Negative 1,272 57.2 57.4 1,256 56.5 56.8 Positive 943 42.4 42.6 957 43.1 43.2 Unknown 7 0.3  9 0.4  Number of positive nodes Median (range) 2 (1–24)   2 (1–28)   1–3 586 62.1 64.3 614 64.2 66.2 4–9 243 25.8 26.7 229 23.9 24.7 ≥10 82 8.7 9.0 84 8.8 9.1 Unknown 32 3.4  30 3.1  ER Status at diagnosis Negative 472 21.2 21.8 472 21.2 21.9 Positive 1,689 76.0 78.2 1,687 75.9 78.1 Unknown 61 2.7  63 2.8  Tumor size (cm) Median (range) 2.0 (0.1–9.9)   2.0 (0.1–9.9)   0.1–1.0 265 11.9 12.1 285 12.8 13.0 1.1–2.0 864 38.9 39.4 864 38.9 39.5 2.1–5.0 949 42.7 43.2 915 41.2 41.8 >5.0 117 5.3 5.3 126 5.7 5.8 Unknown 27 1.2  32 1.4  Tumor grade 1 125 5.6 6.0 103 4.6 4.9 2 876 39.4 42.0 844 38.0 40.2 3 1,083 48.7 52.0 1,153 51.9 54.9 Unknown 138 6.2  122 5.5  n n X-tile analysis 20 P 2 2 Results 2 1 Fig. 1 TMA core showing stromal mast cells stained with c-kit (CD-117). Magnification, 20×. MCs are seen as brown, granular stained oval, spindle or polygonal cells  Training set results Survival analysis 2 P = Fig. 2 a b  Correlation with other biomarkers b P = 2 b P = b P = b P = b P = 3 Table 3 Correlations between mast cells and other biomarkers Training set Mast cells ER Kendall’s tau-b 0.034 Significance (2-tailed) 0.148 N 1,788 EGFR Kendall’s tau-b −0.029 Significance (2-tailed) 0.228 N 1,646 Her2 Kendall’s tau-b 0.049 Significance (2-tailed) 0.052 N 1,746 CK5/6 Kendall’s tau-b −0.003 Significance (2-tailed) 0.906 N 1,624 Bcl2 Kendall’s tau-b 0.077 Significance (2-tailed) 0.002 N 1,616 N All scores binarized as detailed in the text Nodal status  P = P = Multivariate analysis 4 P = Table 4 P-  Significance HR BCSS 95% CI for HR Lower Upper (a) Training set Mast cells 0.041 0.804 0.653 0.991 ER 0.018 0.777 0.631 0.957 Her2 0.003 1.439 1.129 1.834 Size of the lesion     2–5 cm vs. ≤2 cm 0.000 1.884 1.547 2.295     >5 cm vs. ≤2 cm 0.001 1.931 1.330 2.803 Grade*     Grade 1, 2 vs. Grade 3  0.001 1.390 1.139 1.696 Nodal status 0.000 2.380 1.965 2.882 Age     40–49 vs. <40 0.000 0.567 0.420 0.765     50–65 vs. <40 0.013 0.689 0.513 0.924     >65 vs. <40 0.019 0.677 0.489 0.938 (b) Validation set Mast cells 0.128 0.846 0.683 1.049 ER 0.0289 0.793 0 .644 0 .976 Her2 0.0018 1.463 1.152 1.859 Size of the lesion     2−5 cm vs. ≤2 cm −4 1.439 1.181 1.753     >5 cm vs. ≤2 cm −6 2.258 1.618 3.151 Grade*     Grade 1, 2 vs. Grade 3  −6 1.658 1.346 2.042 Nodal status −19 2.410 1.984 2.927 Age     40–49 vs. <40 0.432 1.158 0.803 1.671     50–65 vs. <40 0.634 1.090 0.765 1.552     >65 vs. <40 0.07 1.394 0.973 1.998 * Grade 1—well differentiated Grade 2—moderately differentiated Grade 3—poorly differentiated HR = adjusted hazard ratio, CI = confidence interval and BCSS = Breast Cancer Specific Survival Validation set results Survival analysis 2 P = Multivariate analysis 4 P = X-tile analysis results We assessed the association between patient outcome and number of positively stained MCs in the stroma of tumors using X-tile software. This software allowed us to define an optimal cut-off point that defined the number of MCs needed to predict good prognosis in the cancer patients. The program divided the population into low and high-risk groups based on the number of MCs detected. It converted continuous data into ordinal classes for statistical analysis. The X-tile plot showed that breast cancer patients with any number of stromal MCs had better prognosis compared to those who have no MC infiltration in their stroma. MCs ranging between 1 and 22 in the tumor stroma were present in 507 (27.61%) patients and were grouped together in the low population group of the training set. The high population group comprised of 1,329 (72.39%) patients in the training set. It reiterated the findings obtained by KM survival analysis that the presence of any number of stromal MCs predicts good prognosis in invasive breast cancer patients. Discussion 21 22 23 3 24 13 15 9 25 21 26 27 28 29 30 31 32 33 34 35 13 11 11 P = P = 36 37 38 39 40 41 42 In conclusion, we confirm our earlier pilot study findings and confirm that stromal MCs correlate with a good prognosis in a large cohort of 4,444 invasive breast cancer patients with long-term follow-up. It highlights the critical role that the host stromal reaction, in particular the inflammatory cell infiltrate, plays in modulating cancer progression. MCs can be used as markers for risk stratification in invasive breast cancers.