Breast cancer classification based on proteotypes obtained by SWATH mass spectrometry

Abstract

Accurate breast cancer classification is vital for patient management decisions, and better tumour classification is expected to enable more precise and eventually personalized treatment to improve patient outcomes. Here, we present a novel quantitative proteotyping approach based on SWATH mass spectrometry and establish key proteins for breast tumour classification derived from proteotype data. The study was based on 96 tissue samples representing five breast cancer subtypes according to conventional classification. Correlation of SWATH proteotype patterns indicated groups that largely recapitulate these subtypes. However, 2 the proteotype-based classification also revealed varying degrees of heterogeneity within the conventional subtypes, with triple negative tumours being the most heterogeneous. Proteins that contributed most strongly to the proteotype-based classification include INPP4B, CDK1, and ERBB2, which are associated with oestrogen receptor status, tumour grade, and HER2 status, respectively. While these three key proteins exhibited high levels of correlation between protein and transcript levels (R>0.67), general correlation did not exceed R=0.29, indicating the value of protein-level measurements of biomarkers and disease-regulated genes. Overall, our data shows how large-scale protein-level measurements by next-generation proteomics can lead to improved patient stratification for precision medicine.

Proteomics Identification and Validation of Desmocollin‐1 and Catechol‐O‐Methyltransferase as Proteins Associated with Breast Cancer Cell Migration and Metastasis

“…Half of them was lymph node negative and half lymph node positive. From the panel of the key proteins presented in Table , SWATH‐MS assays were available for COMT, ERP29, RPN1, NB5R1, ADT2, and KTN1 in an assay library containing 4562 proteins (FDR < 0.01) . We compared levels of these proteins in 8 lymph node positive versus 8 lymph node negative primary tumors and confirmed higher level of COMT in lymph node positive TNBC tissues compared to their lymph node negative counterparts (FC = 2.34, p = 0.028, Table A).…”

“…To validate the changes of DSC1 using human tumor samples, we performed an IHC verification of DSC1 levels in the set of 96 breast cancer tissue samples . After a successful antibody test on specificity (Figure S4, Supporting Information), we showed that DSC1 level was significantly increased in lymph node positive versus negative luminal A grade 1 tumors ( p = 0.004 based on histoscore, Figure E), significantly increased in grade 3 versus grade 1 tumors ( p = 0.002) specifically in lymph node negative tumors ( p = 4e −5 ) but not in lymph node positive ones ( p = 0.828), and also increased in HER2 + versus HER2 − tumors ( p = 0.045, Figure E), see also Figures S5–S7, Supporting Information, for detailed data.…”

“…To validate additional key proteins from the discovery SILAC experiment on MDA‐MB‐231 TNBC cell line using tissue samples, we used the SWATH‐MS dataset of 96 breast cancer tissues recently published by Bouchal and co‐workers and extracted data from 16 tumors of TNBC subtype biologically relevant to previously used TNBC MDA‐MB‐231 cell line model. Half of them was lymph node negative and half lymph node positive.…”

“…A detailed description of the sample set is available in Supplemental file 1, Supporting Information, of a previous publication . Tissue sample processing, spectral library preparation, and SWATH measurements were described recently by Bouchal and co‐workers …”

“…Data for top six transitions per precursor selected in the SWATH‐MS assay library were extracted and up to six peptides per protein were manually selected for protein level quantification using group comparison function of MSStats implemented in Skyline 4.1 with data normalization on two actin representing peptides set as global internal standard. The data for comparison of COMT levels between breast cancer subtypes were extracted from Data file S3, Supporting Information, of the previous study …”