Intra-Tumor Heterogeneity Revealed by Mass Spectrometry Imaging Is Associated with the Prognosis of Breast Cancer

Abstract

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.

Prognostic Value of Molecular Intratumor Heterogeneity in Primary Oral Cancer and Its Lymph Node Metastases Assessed by Mass Spectrometry Imaging

“…We have previously found that a higher level of intratumor heterogeneity addressed by MALDI-MSI was associated with better outcomes in patients with HER2-positive breast cancers. Moreover, increased heterogeneity observed in that study was associated with the presence of tumor-infiltrating lymphocytes, which prompted us to hypothesize that a more favorable prognosis of breast cancer patients who had a higher level of ITH revealed by MALDI-MSI was associated with a higher level of immune cells enhancing the response to treatment [ 21 ]. We found here that continuous/dense pattern of lymphocyte infiltration at the tumor/host interface correlated with a higher level of phenotypic ITH, which suggested similar mechanisms in patients with locally advanced oral cancer.…”

“…The acquired MSI spectra were preprocessed by computationally performed mass channels unification, baseline subtraction, outlying spectra identification, peak alignment, TIC normalization, and peak detection using Gaussian mixture modeling (GMM) of the average spectrum; the procedure of our spectra processing pipeline was described in detail elsewhere [ 21 , 41 ]. Abundance of a particular component was estimated by pairwise convolution of the GMM components and individual spectra.…”

“…Populations of computed similarity values were plotted as cumulative distribution functions to visualize the intra-ROI heterogeneity and inter-ROIs differences with relevance to the NED and PD patient groups. The deglomerative divisive iK-means (DivIK) algorithm with region-driven feature selection was applied for unsupervised molecular image segmentation as described in detail elsewhere [ 18 , 21 , 30 ]. Spectra from (i) all 77 T-ROIs, and spectra from (ii) 37 patients for whom paired specimens of the T-ROI and N-ROI were available, created two distinct datasets that were clustered separately.…”

“…However, one should emphasize that the available papers addressing tumor heterogeneity had a rather methodological character and focused on the development and optimization of tools used for data analysis [ 17 , 18 , 19 ]. Hence, there are only few reports where MSI has been employed in the analysis of the actual biological role of ITH, which showed differences between the primary tumor and metastatic spread in thyroid cancer [ 20 ] or association between ITH and the prognosis of breast cancer [ 21 ].…”

Proteomics-Based Identification of Dysregulated Proteins in Breast Cancer

“…A new method combining MALDI-MSI with IHC, termed MALDI-IHC, has been described, allowing the high-plex MSI of a wide range of biomarkers in various tissues, including BC [ 17 ]. In advanced HER2+ invasive ductal carcinoma, MALDI-MSI emphasized the association between intratumor heterogeneity and the prognosis of BC; the higher heterogeneity of tumors with a better prognosis reflects the presence of infiltrating immune cells that facilitate the treatment response [ 29 ]. An integrated experimental design based on IHC and histology-directed MSI defined the proteome profile of tumor microenvironment (TME), suggesting that the phosphatase and tensin homolog (PTEN) expression may be associated with different collagen types and regulation by PT sites of modification [ 30 ].…”