Intra-tumor heterogeneity (ITH) results from the coexistence of genetically distinct cancer cell (sub)populations, their phenotypic plasticity, and the presence of heterotypic components of the tumor microenvironment (TME). Here we addressed the potential association between phenotypic ITH revealed by mass spectrometry imaging (MSI) and the prognosis of breast cancer. Tissue specimens resected from 59 patients treated radically due to the locally advanced HER2-positive invasive ductal carcinoma were included in the study. After the on-tissue trypsin digestion of cellular proteins, peptide maps of all cancer regions (about 380,000 spectra in total) were segmented by an unsupervised approach to reveal their intrinsic heterogeneity. A high degree of similarity between spectra was observed, which indicated the relative homogeneity of cancer regions. However, when the number and diversity of the detected clusters of spectra were analyzed, differences between patient groups were observed. It is noteworthy that a higher degree of heterogeneity was found in tumors from patients who remained disease-free during a 5-year follow-up (n = 38) compared to tumors from patients with progressive disease (distant metastases detected during the follow-up, n = 21). Interestingly, such differences were not observed between patients with a different status of regional lymph nodes, cancer grade, or expression of estrogen receptor at the time of the primary treatment. Subsequently, spectral components with different abundance in cancer regions were detected in patients with different outcomes, and their hypothetical identity was established by assignment to measured masses of tryptic peptides identified in corresponding tissue lysates. Such differentiating components were associated with proteins involved in immune regulation and hemostasis. Further, a positive correlation between the level of tumor-infiltrating lymphocytes and heterogeneity revealed by MSI was observed. We postulate that a higher heterogeneity of tumors with a better prognosis could reflect the presence of heterotypic components including infiltrating immune cells, that facilitated the response to treatment.

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Objectives This study evaluated the usefulness of artificial intelligence (AI) algorithms as tools in improving the accuracy of histologic classification of breast tissue. Methods Overall, 100 microscopic photographs (test A) and 152 regions of interest in whole-slide images (test B) of breast tissue were classified into 4 classes: normal, benign, carcinoma in situ (CIS), and invasive carcinoma. The accuracy of 4 pathologists and 3 pathology residents were evaluated without and with the assistance of algorithms. Results In test A, algorithm A had accuracy of 0.87, with the lowest accuracy in the benign class (0.72). The observers had average accuracy of 0.80, and most clinically relevant discordances occurred in distinguishing benign from CIS (7.1% of classifications). With the assistance of algorithm A, the observers significantly increased their average accuracy to 0.88. In test B, algorithm B had accuracy of 0.49, with the lowest accuracy in the CIS class (0.06). The observers had average accuracy of 0.86, and most clinically relevant discordances occurred in distinguishing benign from CIS (6.3% of classifications). With the assistance of algorithm B, the observers maintained their average accuracy. Conclusions AI tools can increase the classification accuracy of pathologists in the setting of breast lesions.