Fig 1: GPRC5A, TPBG, NEU1, and SP140L protein levels are associated with lack of pCR to neadjuvant anti-HER2 therapy in three proteomics datasets(A) Analogous limma models to those described in Figure S3A were applied to the DP1 study after excluding potential false-positive protein outliers. The Venn diagrams show the overlap between upregulated (top) and downregulated (bottom) proteins from the comparison of non-pCR to pCR for the THL + TH arms of the current study and the DP1 study (limma moderated t test, p < 0.05). Numbers included for each study in parentheses indicate numbers of differential proteins among the 8,488 proteins evaluated by both studies. p values for overlap are from hypergeometric tests evaluating the probability that the overlapping sets contain as many proteins as observed here by chance given the numbers of differential proteins for each study and the background of 8,488 proteins.(B) Heatmap showing protein levels in biopsies (duplicate biopsies from same tumor are included as separate samples) from the THL + TH arms in the current study and from the DP1 study for candidates identified by the workflow described in (A). Upper panel shows 15 proteins that had significantly higher expression in non-pCR than in pCR, and lower panel shows the nine proteins that were higher in pCR (limma moderated t test, p < 0.05). Asterisks indicate genes that were also significant in the current RNA dataset from CALGB 40601.(C) Comparison of non-pCR to pCR samples from a recently published proteomics dataset for tumors from patients treated with dual antibody therapy28 revealed that GPRC5A (top), TPBG (middle), and NEU1 (bottom) proteins were also significantly elevated in and the SP140L protein was significantly lower in non-pCR in that study as well. Boxplots show the median (center) and first (upper bound) and third (lower bound) quartiles of respective levels of each protein in pCR and non-pCR tumors. p values are from using Wilcoxon rank-sum tests comparing the two groups.(D) TPBG IHC of control sections. Positive controls include placenta and renal cell carcinoma (RCC) tissue and MCF7 cells, while the negative control is the RAJI cell line (a human lymphoblastoid cell line). 100 micron scale bar in the upper left image applies to all images in the panel.(E) TPBG IHC of representative sections from tumors from the CALGB 40601 trial for each ordinal score category (0, 1 ,2, 3). 100 micron scale bar in the upper left image applies to all images in the panel.(F) Comparison of mass spectroscopy data for TPBG protein (y axis) to IHC scores. Boxplots show the median (center) and first (upper bound) and third (lower bound) quartiles of TPBG protein log2 TMT ratios for each group (tumors scoring as 2 or 3 vs. those scoring as 0 and 1), and whiskers extend to 1.5× interquartile range (IQR). For tumors with duplicate biopsies, the mean TPGB log2 TMT ratio was used. p value is from Wilcoxon rank-sum test comparing these groups.See also Tables S2 and S3.