Microscaled proteogenomic methods for precision oncology

Shankha Satpathy,Mark A Watson,Cynthia Ma,Anna Malovannaya,Eric J Jaehnig,Alexander B Saltzman,Meenakshi Anurag,Beom Jun Kim ,Suhas Vasaikar ,Bing Zhang,Ari Gao,Bo Wen,Purba Singh,Jeremy Hoog,Kimberly Holloway ,Foluso O Ademuyiwa,Terry Hyslop,Steven A Carr,Chen Huang,George Miles,Doug W Chan,Yongchao Dou,Karl R Clauser,Mothaffar F Rimawi,D R Mani,Charles M Perou,David G Mutch ,Rachel Schiff,Michael A Gillette,Noel Namai,Karsten Krug,Samuel A Jacobs ,Matthew J Ellis

doi:10.1038/s41467-020-14381-2

Abstract

Cancer proteogenomics promises new insights into cancer biology and treatment efficacy by integrating genomics, transcriptomics and protein profiling including modifications by mass spectrometry (MS). A critical limitation is sample input requirements that exceed many sources of clinically important material. Here we report a proteogenomics approach for core biopsies using tissue-sparing specimen processing and microscaled proteomics. As a demonstration, we analyze core needle biopsies from ERBB2 positive breast cancers before and 48–72 h after initiating neoadjuvant trastuzumab-based chemotherapy. We show greater suppression of ERBB2 protein and both ERBB2 and mTOR target phosphosite levels in cases associated with pathological complete response, and identify potential causes of treatment resistance including the absence of ERBB2 amplification, insufficient ERBB2 activity for therapeutic sensitivity despite ERBB2 amplification, and candidate resistance mechanisms including androgen receptor signaling, mucin overexpression and an inactive immune microenvironment. The clinical utility and discovery potential of proteogenomics at biopsy-scale warrants further investigation.

Highlights

Cancer proteogenomics promises new insights into cancer biology and treatment efficacy by integrating genomics, transcriptomics and protein profiling including modifications by mass spectrometry (MS)
To microscale specimen processing an optimal cutting temperature (OCT)-embedded core biopsy is serially sectioned with alternating 50um sections transferred into 3 different 1.5 ml tubes (Fig. 1a)
We illustrate the potential microscaled proteogenomics in a proof-of-principle breast cancer clinical study designed to detect the immediate effects of inhibiting the ERBB2 pathway

Summary

Introduction

Cancer proteogenomics promises new insights into cancer biology and treatment efficacy by integrating genomics, transcriptomics and protein profiling including modifications by mass spectrometry (MS). Cancer proteogenomics integrates data from cancer genomics and transcriptomics with cancer proteomics to provide deeper insights into cancer biology and therapeutic vulnerabilities Both by improving the functional annotation of genomic aberrations and through insights into pathway activation, this multi-dimensional approach to the characterization of human tumors shows promise for application to precision oncology[1,2,3,4,5,6,7]. For clinical diagnostics a single snap-frozen tumor-rich core needle biopsy (∼ < 20 mg) must provide sufficient DNA, RNA and protein for deep-scale proteogenomic profiling To reduce these tissue requirements, we describe methods to generate highquality DNA, RNA and protein for deep-scale DNA and RNA sequencing and proteome and phosphoproteome analysis from a single 14 G core needle biopsy (Biopsy Trifecta Extraction, (BioTExt)) and a microscaled liquid chromatography-mass spectrometry (LC-MS/MS)-based proteome and phosphoproteome analysis pipeline (MiProt) that requires only 25 μg peptide per sample. We choose trastuzumab-based treatment for ERBB2 + breast cancer as an example of an oncogenic kinasedriven tumor where proteogenomic analyses and pharmacodynamic studies should provide significant insights into variability in treatment outcomes[9,10]

Methods

Results

Conclusion