Development of the prognostic model
ypN0
A forward stepwise approach was used to build the prognostic model. In particular, starting from a null Cox model for EFS, variables selected from a pool were added following the AIC method [function “steps” from the “stats” R package (v4.0.5)Before calculating the signature scores, genes with a total read count across all samples < 20 were excluded, and normalization was performed by dividing gene counts by the sample mean gene count followed by logWith regards to BCR and TCR measures, the same procedure followed for uni- and multi-variate analysis was applied in NeoALTTO (i.e., transformation according to the Shapiro-Francia test, then centering and scaling).The model was developed on a cohort of 221 patients from the NeoALTTO trial with all data available. Subsequent analysis regarding the prognostic score in NeoALTTO were then performed in a cohort of 233 patients with all the variables selected for the model available, except for the analyses including pCR in breast + axilla (ypT0/is ypN0), which were performed on 224 patients.A prognostic score (HER2-EveNT) was computed multiplying the coefficient obtained for each variable in the final Cox model (Supplementary data The score was therefore calculated as:BCR evenness (standardized value) × 0.300436587 + TILs (%) × −0.024107504 + pCR (0 or 1) × −0.568111234 + HR status (0 or 1) × −0.525011259 + nodal status (0 or 1) × −0.504914127.As the coefficients are the natural logarithm of the HRs for each variable, the score represents a measure of the relative risk of relapse calculated based on the values of each variable in the single patient, with a lower score being associated with a better prognosis. Variables with a negative coefficient were associated with improved EFS, while a positive coefficient was associated with worse prognosis.This score was then divided into tertiles. As EFS > 90% was reached for the first tertile in NeoALTTO, the cutoff between the first and the intermediate tertiles was used to define the two final prognostic groups. An external independent validation was performed testing the model in 230 patients from the CALGB 40601 trial with pCR, hormone receptor, clinical nodal status, TILs, and BCR evenness data available. As BCR evenness was not log transformed in NeoALTTO, the BCR evenness values in CALGB 40601 were only centered by removing the mean of NeoALTTO BCR evenness (0.7917893) and scaled dividing the obtained values by the NeoALTTO BCR evenness standard deviation (0.1004482) before computing the HER2-EveNT score.The concordance index (Harrel’s C-index) was assessed to estimate the predictive performance of the model. Cutoffs derived from NeoALTTO were applied on the score calculated in CALGB 40601 to identify correspondent prognostic groups.