RESUMO
Age-associated changes in the T cell compartment are well described. However, limitations of current single-modal or bimodal single-cell assays, including flow cytometry, RNA-seq (RNA sequencing) and CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing), have restricted our ability to deconvolve more complex cellular and molecular changes. Here, we profile >300,000 single T cells from healthy children (aged 11-13 years) and older adults (aged 55-65 years) by using the trimodal assay TEA-seq (single-cell analysis of mRNA transcripts, surface protein epitopes and chromatin accessibility), which revealed that molecular programming of T cell subsets shifts toward a more activated basal state with age. Naive CD4+ T cells, considered relatively resistant to aging, exhibited pronounced transcriptional and epigenetic reprogramming. Moreover, we discovered a novel CD8αα+ T cell subset lost with age that is epigenetically poised for rapid effector responses and has distinct inhibitory, costimulatory and tissue-homing properties. Together, these data reveal new insights into age-associated changes in the T cell compartment that may contribute to differential immune responses.
Assuntos
Subpopulações de Linfócitos T , Transcriptoma , Criança , Humanos , Idoso , Envelhecimento/genética , Epitopos/metabolismo , Análise de Célula ÚnicaRESUMO
Long COVID or post-acute sequelae of SARS-CoV-2 (PASC) is a clinical syndrome featuring diverse symptoms that can persist for months following acute SARS-CoV-2 infection. The aetiologies may include persistent inflammation, unresolved tissue damage or delayed clearance of viral protein or RNA, but the biological differences they represent are not fully understood. Here we evaluate the serum proteome in samples, longitudinally collected from 55 PASC individuals with symptoms lasting ≥60 days after onset of acute infection, in comparison to samples from symptomatically recovered SARS-CoV-2 infected and uninfected individuals. Our analysis indicates heterogeneity in PASC and identified subsets with distinct signatures of persistent inflammation. Type II interferon signaling and canonical NF-κB signaling (particularly associated with TNF), appear to be the most differentially enriched signaling pathways, distinguishing a group of patients characterized also by a persistent neutrophil activation signature. These findings help to clarify biological diversity within PASC, identify participants with molecular evidence of persistent inflammation, and highlight dominant pathways that may have diagnostic or therapeutic relevance, including a protein panel that we propose as having diagnostic utility for differentiating inflammatory and non-inflammatory PASC.
Assuntos
COVID-19 , Síndrome de COVID-19 Pós-Aguda , Humanos , SARS-CoV-2 , Proteínas Sanguíneas , Progressão da Doença , InflamaçãoRESUMO
Longitudinal bulk and single-cell omics data is increasingly generated for biological and clinical research but is challenging to analyze due to its many intrinsic types of variations. We present PALMO ( https://github.com/aifimmunology/PALMO ), a platform that contains five analytical modules to examine longitudinal bulk and single-cell multi-omics data from multiple perspectives, including decomposition of sources of variations within the data, collection of stable or variable features across timepoints and participants, identification of up- or down-regulated markers across timepoints of individual participants, and investigation on samples of same participants for possible outlier events. We have tested PALMO performance on a complex longitudinal multi-omics dataset of five data modalities on the same samples and six external datasets of diverse background. Both PALMO and our longitudinal multi-omics dataset can be valuable resources to the scientific community.
Assuntos
Multiômica , Humanos , SoftwareRESUMO
Although systemic chemotherapy remains the standard of care for TNBC, even combination chemotherapy is often ineffective. The identification of biomarkers for differential chemotherapy response would allow for the selection of responsive patients, thus maximizing efficacy and minimizing toxicities. Here, we leverage TNBC PDXs to identify biomarkers of response. To demonstrate their ability to function as a preclinical cohort, PDXs were characterized using DNA sequencing, transcriptomics, and proteomics to show consistency with clinical samples. We then developed a network-based approach (CTD/WGCNA) to identify biomarkers of response to carboplatin (MSI1, TMSB15A, ARHGDIB, GGT1, SV2A, SEC14L2, SERPINI1, ADAMTS20, DGKQ) and docetaxel (c, MAGED4, CERS1, ST8SIA2, KIF24, PARPBP). CTD/WGCNA multigene biomarkers are predictive in PDX datasets (RNAseq and Affymetrix) for both taxane- (docetaxel or paclitaxel) and platinum-based (carboplatin or cisplatin) response, thereby demonstrating cross-expression platform and cross-drug class robustness. These biomarkers were also predictive in clinical datasets, thus demonstrating translational potential.
RESUMO
Cancer stem cells (CSCs) are a small subpopulation of self-renewing cancer cells that are present within tumors. CSCs possess tumor initiation potential as well as the ability to resist toxic compounds and chemotherapeutic agents through the upregulation of drug efflux transporters, DNA repair pathways, and survival cascades. Accumulating evidence suggests that CSCs are responsible for tumor relapse and resistance to chemotherapeutic agents and that targeting CSCs is critical to inhibition of cancer progression. Therefore, isolation and characterization of CSCs is important in studying tumor initiation and progression. In this chapter, we provide a detailed method for the identification and isolation of CSCs.
Assuntos
Antineoplásicos , Recidiva Local de Neoplasia , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Humanos , Recidiva Local de Neoplasia/patologia , Células-Tronco Neoplásicas/metabolismoRESUMO
Cancer stem cells (CSCs) are a small subpopulation of self-renewing cancer cells that are present within tumors. In this chapter, we provide a detailed method for the quantification of CSCs in vitro through mammosphere formation.
Assuntos
Neoplasias da Mama , Células-Tronco Neoplásicas , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Células-Tronco Neoplásicas/patologiaRESUMO
Cancer stem cells (CSCs) are a small subpopulation of self-renewing cancer cells that are present within tumors. Calculating the frequency of tumor-initiating cells is important in the assessment of the number of CSCs present in a cell population. In this chapter, we present a protocol developed for quantification of CSCs from breast cancer tumors that can be adapted to CSCs from other types of tumors.
Assuntos
Neoplasias da Mama , Células-Tronco Neoplásicas , Neoplasias da Mama/patologia , Transformação Celular Neoplásica/metabolismo , Feminino , Humanos , Células-Tronco Neoplásicas/metabolismoRESUMO
G Protein-Coupled Receptors (GPCRs) represent the largest superfamily of cell-surface proteins. However, the expression and function of majority of GPCRs remain unexplored in breast cancer (BC). We interrogated the expression and phosphorylation status of 398 non-sensory GPCRs using the landmark BC proteogenomics and phosphoproteomic dataset from The Cancer Genome Atlas. Neuropeptide Y Receptor Y1 (NPY1R) gene and protein expression were significantly higher in Luminal A tumors versus other BC subtypes. The trend of NPY1R gene, protein, and phosphosite (NPY1R-S368s) expression was decreasing in the order of Luminal A, Luminal B, Basal, and human epidermal growth factor receptor 2 (HER2) subtypes. NPY1R gene expression increased in response to estrogen and reduced with endocrine therapy in estrogen receptor-positive (ER+) BC cells and xenograft models. Conversely, NPY1R expression decreased in ER+ BC cells resistant to endocrine therapies (estrogen deprivation, tamoxifen, and fulvestrant) in vitro and in vivo. NPY treatment reduced estradiol-stimulated cell growth, which was reversed by NPY1R antagonist (BIBP-3226) in ER+ BC cells. Higher NPY1R gene expression predicted better relapse-free survival and overall survival in ER+ BC. Our study demonstrates that NPY1R mediates the inhibitory action of NPY on estradiol-stimulated growth of ER+ BC cells, and its expression serves as a biomarker to predict endocrine sensitivity and survival in ER+ BC patients.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias das Glândulas Endócrinas/tratamento farmacológico , Receptor alfa de Estrogênio/genética , Receptores de Neuropeptídeo Y/genética , Animais , Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias das Glândulas Endócrinas/genética , Neoplasias das Glândulas Endócrinas/patologia , Estradiol/farmacologia , Estrogênios/genética , Feminino , Fulvestranto/farmacologia , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Camundongos , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Receptor ErbB-2/genética , Receptores Acoplados a Proteínas G/genética , Tamoxifeno/farmacologiaRESUMO
Lung squamous cell carcinoma (LSCC) remains a leading cause of cancer death with few therapeutic options. We characterized the proteogenomic landscape of LSCC, providing a deeper exposition of LSCC biology with potential therapeutic implications. We identify NSD3 as an alternative driver in FGFR1-amplified tumors and low-p63 tumors overexpressing the therapeutic target survivin. SOX2 is considered undruggable, but our analyses provide rationale for exploring chromatin modifiers such as LSD1 and EZH2 to target SOX2-overexpressing tumors. Our data support complex regulation of metabolic pathways by crosstalk between post-translational modifications including ubiquitylation. Numerous immune-related proteogenomic observations suggest directions for further investigation. Proteogenomic dissection of CDKN2A mutations argue for more nuanced assessment of RB1 protein expression and phosphorylation before declaring CDK4/6 inhibition unsuccessful. Finally, triangulation between LSCC, LUAD, and HNSCC identified both unique and common therapeutic vulnerabilities. These observations and proteogenomics data resources may guide research into the biology and treatment of LSCC.
Assuntos
Carcinoma de Células Escamosas/genética , Neoplasias Pulmonares/genética , Proteogenômica , Acetilação , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise por Conglomerados , Quinase 4 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , Proteínas de Neoplasias/metabolismo , Fosforilação , Ligação Proteica , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , UbiquitinaçãoRESUMO
SARS-CoV-2 has infected over 200 million and caused more than 4 million deaths to date. Most individuals (>80%) have mild symptoms and recover in the outpatient setting, but detailed studies of immune responses have focused primarily on moderate to severe COVID-19. We deeply profiled the longitudinal immune response in individuals with mild COVID-19 beginning with early time points post-infection (1-15 days) and proceeding through convalescence to >100 days after symptom onset. We correlated data from single cell analyses of peripheral blood cells, serum proteomics, virus-specific cellular and humoral immune responses, and clinical metadata. Acute infection was characterized by vigorous coordinated innate and adaptive immune activation that differed in character by age (young vs. old). We then characterized signals associated with recovery and convalescence to define and validate a new signature of inflammatory cytokines, gene expression, and chromatin accessibility that persists in individuals with post-acute sequelae of SARS-CoV-2 infection (PASC).
RESUMO
While G protein-coupled receptors (GPCRs) are known to be excellent drug targets, the second largest family of adhesion-GPCRs is less explored for their role in health and disease. ADGRF1 (GPR110) is an adhesion-GPCR and has an important function in neurodevelopment and cancer. Despite serving as a poor predictor of survival, ADGRF1's coupling to G proteins and downstream pathways remain unknown in cancer. We evaluated the effects of ADGRF1 overexpression on tumorigenesis and signaling pathways using two human epidermal growth factor receptor-2-positive (HER2+) breast cancer (BC) cell-line models. We also interrogated publicly available clinical datasets to determine the expression of ADGRF1 in various BC subtypes and its impact on BC-specific survival (BCSS) and overall survival (OS) in patients. ADGRF1 overexpression in HER2+ BC cells increased secondary mammosphere formation, soft agar colony formation, and % of Aldefluor-positive tumorigenic population in vitro and promoted tumor growth in vivo. ADGRF1 co-immunoprecipitated with both Gαs and Gαq proteins and increased cAMP and IP1 when overexpressed. However, inhibition of only the Gαs pathway by SQ22536 reversed the pro-tumorigenic effects of ADGRF1 overexpression. RNA-sequencing and RPPA analysis revealed inhibition of cell cycle pathways with ADGRF1 overexpression, suggesting cellular quiescence, as also evidenced by cell cycle arrest at the G0/1 phase and resistance to chemotherapy in HER2+ BC. ADGRF1 was significantly overexpressed in the HER2-enriched BC compared to luminal A and B subtypes and predicted worse BCSS and OS in these patients. Therefore, ADGRF1 represents a novel drug target in HER2+ BC, warranting discovery of novel ADGRF1 antagonists.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Proteínas Oncogênicas/genética , Receptor ErbB-2/genética , Receptores Acoplados a Proteínas G/genética , Animais , Neoplasias da Mama/genética , Carcinogênese/genética , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Fase G1/genética , Humanos , Camundongos , Camundongos Nus , Fase de Repouso do Ciclo Celular/genética , Transdução de Sinais/genéticaRESUMO
The epithelial-to-mesenchymal transition (EMT) plays a critical role during normal development and in cancer progression. EMT is induced by various signaling pathways, including TGF-ß, BMP, Wnt-ß-catenin, NOTCH, Shh, and receptor tyrosine kinases. In this study, we performed single-cell RNA sequencing on MCF10A cells undergoing EMT by TGF-ß1 stimulation. Our comprehensive analysis revealed that cells progress through EMT at different paces. Using pseudotime clustering reconstruction of gene-expression profiles during EMT, we found sequential and parallel activation of EMT signaling pathways. We also observed various transitional cellular states during EMT. We identified regulatory signaling nodes that drive EMT with the expression of important microRNAs and transcription factors. Using a random circuit perturbation methodology, we demonstrate that the NOTCH signaling pathway acts as a key driver of TGF-ß-induced EMT. Furthermore, we demonstrate that the gene signatures of pseudotime clusters corresponding to the intermediate hybrid EMT state are associated with poor patient outcome. Overall, this study provides insight into context-specific drivers of cancer progression and highlights the complexities of the EMT process.
Assuntos
Transição Epitelial-Mesenquimal/genética , Redes Reguladoras de Genes , RNA-Seq/métodos , Transdução de Sinais/genética , Análise de Célula Única/métodos , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Perfilação da Expressão Gênica/métodos , Perfilação da Expressão Gênica/estatística & dados numéricos , Humanos , Estimativa de Kaplan-Meier , MicroRNAs/genética , Neoplasias/classificação , Neoplasias/genética , Prognóstico , Modelos de Riscos Proporcionais , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
BACKGROUND: The mechanism by which immune cells regulate metastasis is unclear. Understanding the role of immune cells in metastasis will guide the development of treatments improving patient survival. METHODS: We used syngeneic orthotopic mouse tumour models (wild-type, NOD/scid and Nude), employed knockout (CD8 and CD4) models and administered CXCL4. Tumours and lungs were analysed for cancer cells by bioluminescence, and circulating tumour cells were isolated from blood. Immunohistochemistry on the mouse tumours was performed to confirm cell type, and on a tissue microarray with 180 TNBCs for human relevance. TCGA data from over 10,000 patients were analysed as well. RESULTS: We reveal that intratumoral immune infiltration differs between metastatic and non-metastatic tumours. The non-metastatic tumours harbour high levels of CD8+ T cells and low levels of platelets, which is reverse in metastatic tumours. During tumour progression, platelets and CXCL4 induce differentiation of monocytes into myeloid-derived suppressor cells (MDSCs), which inhibit CD8+ T-cell function. TCGA pan-cancer data confirmed that CD8lowPlatelethigh patients have a significantly lower survival probability compared to CD8highPlateletlow. CONCLUSIONS: CD8+ T cells inhibit metastasis. When the balance between CD8+ T cells and platelets is disrupted, platelets produce CXCL4, which induces MDSCs thereby inhibiting the CD8+ T-cell function.
Assuntos
Neoplasias da Mama/imunologia , Antígenos CD4/genética , Antígenos CD8/genética , Linfócitos T CD8-Positivos/transplante , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Fator Plaquetário 4/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Feminino , Técnicas de Inativação de Genes , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Células Supressoras Mieloides/imunologia , Células Neoplásicas Circulantes/imunologia , Fator Plaquetário 4/administração & dosagem , Fator Plaquetário 4/farmacologia , Análise de Sobrevida , Transplante Isogênico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: The epithelial-mesenchymal transition (EMT) enables dissociation of tumour cells from the primary tumour mass, invasion through the extracellular matrix, intravasation into blood vessels and colonisation of distant organs. Cells that revert to the epithelial state via the mesenchymal-epithelial transition cause metastases, the primary cause of death in cancer patients. EMT also empowers cancer cells with stem-cell properties and induces resistance to chemotherapeutic drugs. Understanding the driving factors of EMT is critical for the development of effective therapeutic interventions. METHODS: This manuscript describes the generation of a database containing EMT gene signatures derived from cell lines, patient-derived xenografts and patient studies across cancer types and multiomics data and the creation of a web-based portal to provide a comprehensive analysis resource. RESULTS: EMTome incorporates (i) EMT gene signatures; (ii) EMT-related genes with multiomics features across different cancer types; (iii) interactomes of EMT-related genes (miRNAs, transcription factors, and proteins); (iv) immune profiles identified from The Cancer Genome Atlas (TCGA) cohorts by exploring transcriptomics, epigenomics, and proteomics, and drug sensitivity and (iv) clinical outcomes of cancer cohorts linked to EMT gene signatures. CONCLUSION: The web-based EMTome portal is a resource for primary and metastatic tumour research publicly available at www.emtome.org .
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Bases de Dados Genéticas , Transição Epitelial-Mesenquimal/genética , Neoplasias/genética , Transcriptoma/genética , Humanos , Internet , Neoplasias/patologiaRESUMO
To explore the biology of lung adenocarcinoma (LUAD) and identify new therapeutic opportunities, we performed comprehensive proteogenomic characterization of 110 tumors and 101 matched normal adjacent tissues (NATs) incorporating genomics, epigenomics, deep-scale proteomics, phosphoproteomics, and acetylproteomics. Multi-omics clustering revealed four subgroups defined by key driver mutations, country, and gender. Proteomic and phosphoproteomic data illuminated biology downstream of copy number aberrations, somatic mutations, and fusions and identified therapeutic vulnerabilities associated with driver events involving KRAS, EGFR, and ALK. Immune subtyping revealed a complex landscape, reinforced the association of STK11 with immune-cold behavior, and underscored a potential immunosuppressive role of neutrophil degranulation. Smoking-associated LUADs showed correlation with other environmental exposure signatures and a field effect in NATs. Matched NATs allowed identification of differentially expressed proteins with potential diagnostic and therapeutic utility. This proteogenomics dataset represents a unique public resource for researchers and clinicians seeking to better understand and treat lung adenocarcinomas.
Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Proteogenômica , Adenocarcinoma de Pulmão/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Carcinogênese/genética , Carcinogênese/patologia , Variações do Número de Cópias de DNA/genética , Metilação de DNA/genética , Feminino , Humanos , Neoplasias Pulmonares/imunologia , Masculino , Pessoa de Meia-Idade , Mutação/genética , Proteínas de Fusão Oncogênica , Fenótipo , Fosfoproteínas/metabolismo , Proteoma/metabolismoRESUMO
We report that neurofibromin, a tumor suppressor and Ras-GAP (GTPase-activating protein), is also an estrogen receptor-α (ER) transcriptional co-repressor through leucine/isoleucine-rich motifs that are functionally independent of GAP activity. GAP activity, in turn, does not affect ER binding. Consequently, neurofibromin depletion causes estradiol hypersensitivity and tamoxifen agonism, explaining the poor prognosis associated with neurofibromin loss in endocrine therapy-treated ER+ breast cancer. Neurofibromin-deficient ER+ breast cancer cells initially retain sensitivity to selective ER degraders (SERDs). However, Ras activation does play a role in acquired SERD resistance, which can be reversed upon MEK inhibitor addition, and SERD/MEK inhibitor combinations induce tumor regression. Thus, neurofibromin is a dual repressor for both Ras and ER signaling, and co-targeting may treat neurofibromin-deficient ER+ breast tumors.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Receptor alfa de Estrogênio/genética , Neurofibromina 1/genética , Motivos de Aminoácidos , Animais , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proteínas Correpressoras , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Células MCF-7 , Camundongos Nus , Camundongos SCID , Mutação , Neurofibromina 1/química , Neurofibromina 1/metabolismo , Transdução de Sinais , Tamoxifeno/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras/metabolismoRESUMO
We undertook a comprehensive proteogenomic characterization of 95 prospectively collected endometrial carcinomas, comprising 83 endometrioid and 12 serous tumors. This analysis revealed possible new consequences of perturbations to the p53 and Wnt/ß-catenin pathways, identified a potential role for circRNAs in the epithelial-mesenchymal transition, and provided new information about proteomic markers of clinical and genomic tumor subgroups, including relationships to known druggable pathways. An extensive genome-wide acetylation survey yielded insights into regulatory mechanisms linking Wnt signaling and histone acetylation. We also characterized aspects of the tumor immune landscape, including immunogenic alterations, neoantigens, common cancer/testis antigens, and the immune microenvironment, all of which can inform immunotherapy decisions. Collectively, our multi-omic analyses provide a valuable resource for researchers and clinicians, identify new molecular associations of potential mechanistic significance in the development of endometrial cancers, and suggest novel approaches for identifying potential therapeutic targets.
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Carcinoma/genética , Neoplasias do Endométrio/genética , Regulação Neoplásica da Expressão Gênica , Proteoma/genética , Transcriptoma , Acetilação , Animais , Antígenos de Neoplasias/genética , Carcinoma/imunologia , Carcinoma/patologia , Neoplasias do Endométrio/imunologia , Neoplasias do Endométrio/patologia , Transição Epitelial-Mesenquimal/genética , Retroalimentação Fisiológica , Feminino , Instabilidade Genômica , Humanos , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Repetições de Microssatélites , Fosforilação , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , Transdução de SinaisRESUMO
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.