BackgroundThe immunohistochemical test (IHC) of HER2 and HR can provide prognostic information and treatment guidance for invasive breast cancer patients. We aimed to develop noninvasive image signatures ISHER2and ISHRof HER2 and HR, respectively. We independently evaluate their repeatability, reproducibility, and association with pathological complete response (pCR) to neoadjuvant chemotherapy.MethodsPre-treatment DWI, IHC receptor status HER2/HR, and pCR to neoadjuvant chemotherapy of 222 patients from the multi-institutional ACRIN 6698 trial were retrospectively collected. They were pre-separated for development, independent validation, and test–retest. 1316 image features were extracted from DWI-derived ADC maps within manual tumor segmentations. ISHER2and ISHRwere developed by RIDGE logistic regression using non-redundant and test–retest reproducible features relevant to IHC receptor status. We evaluated their association with pCR using area under receiver operating curve (AUC) and odds ratio (OR) after binarization. Their reproducibility was further evaluated using the test–retest set with intra-class coefficient of correlation (ICC).ResultsA 5-feature ISHER2targeting HER2 was developed (AUC = 0.70, 95% CI 0.59 to 0.82) and validated (AUC = 0.72, 95% CI 0.58 to 0.86) with high perturbation repeatability (ICC = 0.92) and test–retest reproducibility (ICC = 0.83). ISHRwas developed using 5 features with higher association with HR during development (AUC = 0.75, 95% CI 0.66 to 0.84) and validation (AUC = 0.74, 95% CI 0.61 to 0.86) and similar repeatability (ICC = 0.91) and reproducibility (ICC = 0.82). Both image signatures showed significant associations with pCR with AUC of 0.65 (95% CI 0.50 to 0.80) for ISHER2and 0.64 (95% CI 0.50 to 0.78) for ISHER2in the validation cohort. Patients with high ISHER2were more likely to achieve pCR to neoadjuvant chemotherapy with validation OR of 4.73 (95% CI 1.64 to 13.65,Pvalue = 0.006). Low ISHRpatients had higher pCR with OR = 0.29 (95% CI 0.10 to 0.81,Pvalue = 0.021). Molecular subtypes derived from the image signatures showed comparable pCR prediction values to IHC-based molecular subtypes (Pvalue > 0.05).ConclusionRobust ADC-based image signatures were developed and validated for noninvasive evaluation of IHC receptors HER2 and HR. We also confirmed their value in predicting treatment response to neoadjuvant chemotherapy. Further evaluations in treatment guidance are warranted to fully validate their potential as IHC surrogates.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13058-023-01674-9.