Significance: Development of methods for rapid intraoperative assessment of breast pathologies is important for decreasing the rate of surgical reexcisions during breast-conserving therapy. Frozen-section analysis is the most widely used method for intraoperative margin assessment but is time consuming, has limited diagnostic accuracy, and produces freezing artifacts. This manuscript demonstrates nonlinear microscopy for rapid assessment of surgical breast specimens without need for fixation, embedding, and sectioning required for conventional histology. Blinded reading of nonlinear microscopy images by three pathologists achieved 95.4% sensitivity, 93.3% specificity, and 94.1% overall accuracy compared with histology for identifying invasive cancer and ductal carcinoma in situ versus benign breast tissue. The results suggest that nonlinear microscopy is a promising method for intraoperative assessment of breast surgical excision specimens.

Purpose: Emerging data suggest immune checkpoint inhibitors have reduced efficacy in heavily pretreated triple-negative breast cancers (TNBC), but underlying mechanisms are poorly understood. To better understand the phenotypic evolution of TNBCs, we studied the genomic and transcriptomic profiles of paired tumors from patients with TNBC. Experimental Design: We collected paired primary and metastatic TNBC specimens from 43 patients and performed targeted exome sequencing and whole-transcriptome sequencing. From these efforts, we ascertained somatic mutation profiles, tumor mutational burden (TMB), TNBC molecular subtypes, and immune-related gene expression patterns. Stromal tumor-infiltrating lymphocytes (stromal TIL), recurrence-free survival, and overall survival were also analyzed. Results: We observed a typical TNBC mutational landscape with minimal shifts in copy number or TMB over time. However, there were notable TNBC molecular subtype shifts, including increases in the Lehmann/Pietenpol-defined basal-like 1 (BL1, 11.4%–22.6%) and mesenchymal (M, 11.4%–22.6%) phenotypes, and a decrease in the immunomodulatory phenotype (IM, 31.4%–3.2%). The Burstein-defined basal-like immune-activated phenotype was also decreased (BLIA, 42.2%–17.2%). Among downregulated genes from metastases, we saw enrichment of immune-related Kyoto Encyclopedia of Genes and Genomes pathways and gene ontology (GO) terms, and decreased expression of immunomodulatory gene signatures (P < 0.03) and percent stromal TILs (P = 0.03). There was no clear association between stromal TILs and survival. Conclusions: We observed few mutational shifts, but largely consistent transcriptomic shifts in longitudinally paired TNBCs. Transcriptomic and IHC analyses revealed significantly reduced immune-activating gene expression signatures and TILs in recurrent TNBCs. These data may explain the observed lack of efficacy of immunotherapeutic agents in heavily pretreated TNBCs. Further studies are ongoing to better understand these initial observations. See related commentary by Savas and Loi, p. 526

Failure to precisely distinguish malignant from healthy tissue has severe implications for breast cancer surgical outcomes. Clinical prognoses depend on precisely distinguishing healthy from malignant tissue during surgery. Laser Raman spectroscopy (LRS) has been previously shown to differentiate benign from malignant tissue in real time. However, the cost, assembly effort, and technical expertise needed for construction and implementation of the technique have prohibited widespread adoption. Recently, Raman spectrometers have been developed for non-medical uses and have become commercially available and affordable. Here we demonstrate that this current generation of Raman spectrometers can readily identify cancer in breast surgical specimens. We evaluated two commercially available, portable, near-infrared Raman systems operating at excitation wavelengths of either 785 nm or 1064 nm, collecting a total of 164 Raman spectra from cancerous, benign, and transitional regions of resected breast tissue from six patients undergoing mastectomy. The spectra were classified using standard multivariate statistical techniques. We identified a minimal set of spectral bands sufficient to reliably distinguish between healthy and malignant tissue using either the 1064 nm or 785 nm system. Our results indicate that current generation Raman spectrometers can be used as a rapid diagnostic technique distinguishing benign from malignant tissue during surgery.

Motivation: A major goal of biomedical research is to identify molecular features associated with a biological or clinical class of interest. Differential expression analysis has long been used for this purpose; however, conventional methods perform poorly when applied to data with high within class heterogeneity. Results: To address this challenge, we developed EMDomics, a new method that uses the Earth mover’s distance to measure the overall difference between the distributions of a gene’s expression in two classes of samples and uses permutations to obtain q-values for each gene. We applied EMDomics to the challenging problem of identifying genes associated with drug resistance in ovarian cancer. We also used simulated data to evaluate the performance of EMDomics, in terms of sensitivity and specificity for identifying differentially expressed gene in classes with high within class heterogeneity. In both the simulated and real biological data, EMDomics outperformed competing approaches for the identification of differentially expressed genes, and EMDomics was significantly more powerful than conventional methods for the identification of drug resistance-associated gene sets. EMDomics represents a new approach for the identification of genes differentially expressed between heterogeneous classes and has utility in a wide range of complex biomedical conditions in which sample classes show within class heterogeneity. Availability and implementation: The R package is available at http://www.bioconductor.org/packages/release/bioc/html/EMDomics.html Contact:abeck2@bidmc.harvard.edu Supplementary information: supplementary data are available at Bioinformatics online.

CD8 ⁺ tumor-infiltrating lymphocytes (TILs) are associated with improved survival in triple-negative breast cancer (TNBC) yet have no association with survival in estrogen receptor–positive (ER ⁺ ) BC. The basis for these contrasting findings remains elusive. We identified subsets of BC tumors infiltrated by CD8 ⁺ T cells with characteristic features of exhausted T cells (T _EX ). Tumors with abundant CD8 ⁺ T _EX exhibited a distinct tumor microenvironment marked by amplified interferon-γ signaling–related pathways and higher programmed death ligand 1 expression. Paradoxically, higher levels of tumor-infiltrating CD8 ⁺ T _EX associated with decreased overall survival of patients with ER ⁺ BC but not patients with TNBC. Moreover, high tumor expression of a CD8 ⁺ T _EX signature identified dramatically reduced survival in premenopausal, but not postmenopausal, patients with ER ⁺ BC. Finally, we demonstrated the value of a tumor T _EX signature score in identifying high-risk premenopausal ER ⁺ BC patients among those with intermediate Oncotype DX Breast Recurrence Scores. Our data highlight the complex relationship between CD8 ⁺ TILs, interferon-γ signaling, and ER status in BC patient survival. This work identifies tumor-infiltrating CD8 ⁺ T _EX as a key feature of reduced survival outcomes in premenopausal patients with early-stage ER ⁺ BC.