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2.  Proteogenomics connects somatic mutations to signaling in breast cancer 
Nature  2016;534(7605):55-62.
Summary
Somatic mutations have been extensively characterized in breast cancer, but the effects of these genetic alterations on the proteomic landscape remain poorly understood. We describe quantitative mass spectrometry-based proteomic and phosphoproteomic analyses of 105 genomically annotated breast cancers of which 77 provided high-quality data. Integrated analyses allowed insights into the somatic cancer genome including the consequences of chromosomal loss, such as the 5q deletion characteristic of basal-like breast cancer. The 5q trans effects were interrogated against the Library of Integrated Network-based Cellular Signatures, thereby connecting CETN3 and SKP1 loss to elevated expression of EGFR, and SKP1 loss also to increased SRC. Global proteomic data confirmed a stromal-enriched group in addition to basal and luminal clusters and pathway analysis of the phosphoproteome identified a G Protein-coupled receptor cluster that was not readily identified at the mRNA level. Besides ERBB2, other amplicon-associated, highly phosphorylated kinases were identified, including CDK12, PAK1, PTK2, RIPK2 and TLK2. We demonstrate that proteogenomic analysis of breast cancer elucidates functional consequences of somatic mutations, narrows candidate nominations for driver genes within large deletions and amplified regions, and identifies therapeutic targets.
doi:10.1038/nature18003
PMCID: PMC5102256  PMID: 27251275
3.  Using the CPTAC Assay Portal to identify and implement highly characterized targeted proteomics assays 
Summary
The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as an open-source repository of well-characterized targeted proteomic assays. The portal is designed to curate and disseminate highly characterized, targeted mass spectrometry (MS)-based assays by providing detailed assay performance characterization data, standard operating procedures, and access to reagents. Assay content is accessed via the portal through queries to find assays targeting proteins associated with specific cellular pathways, protein complexes, or specific chromosomal regions. The position of the peptide analytes for which there are available assays are mapped relative to other features of interest in the protein, such as sequence domains, isoforms, single nucleotide polymorphisms, and post-translational modifications. The overarching goals are to enable robust quantification of all human proteins and to standardize the quantification of targeted MS-based assays to ultimately enable harmonization of results over time and across laboratories.
doi:10.1007/978-1-4939-3524-6_13
PMCID: PMC5017244  PMID: 26867747
multiple reaction monitoring; selected reaction monitoring; MRM; SRM; PRM; quantitative proteomics; targeted mass spectrometry; quantitative assay database; harmonization; standardization
4.  Aromatase inhibition remodels the clonal architecture of estrogen-receptor-positive breast cancers 
Nature Communications  2016;7:12498.
Resistance to oestrogen-deprivation therapy is common in oestrogen-receptor-positive (ER+) breast cancer. To better understand the contributions of tumour heterogeneity and evolution to resistance, here we perform comprehensive genomic characterization of 22 primary tumours sampled before and after 4 months of neoadjuvant aromatase inhibitor (NAI) treatment. Comparing whole-genome sequencing of tumour/normal pairs from the two time points, with coincident tumour RNA sequencing, reveals widespread spatial and temporal heterogeneity, with marked remodelling of the clonal landscape in response to NAI. Two cases have genomic evidence of two independent tumours, most obviously an ER− ‘collision tumour', which was only detected after NAI treatment of baseline ER+ disease. Many mutations are newly detected or enriched post treatment, including two ligand-binding domain mutations in ESR1. The observed clonal complexity of the ER+ breast cancer genome suggests that precision medicine approaches based on genomic analysis of a single specimen are likely insufficient to capture all clinically significant information.
Aromatase inhibitors are used to treat oestrogen-receptor-positive breast cancer. Here, the authors use genomic approaches to analyse tumours before and after neo-adjuvant treatment and find that treatment alters the clonal landscape of the tumours.
doi:10.1038/ncomms12498
PMCID: PMC4980485  PMID: 27502118
5.  Reproducibility of Differential Proteomic Technologies in CPTAC Fractionated Xenografts 
Journal of Proteome Research  2015;15(3):691-706.
The NCI Clinical Proteomic Tumor Analysis Consortium (CPTAC) employed a pair of reference xenograft proteomes for initial platform validation and ongoing quality control of its data collection for The Cancer Genome Atlas (TCGA) tumors. These two xenografts, representing basal and luminal-B human breast cancer, were fractionated and analyzed on six mass spectrometers in a total of 46 replicates divided between iTRAQ and label-free technologies, spanning a total of 1095 LC–MS/MS experiments. These data represent a unique opportunity to evaluate the stability of proteomic differentiation by mass spectrometry over many months of time for individual instruments or across instruments running dissimilar workflows. We evaluated iTRAQ reporter ions, label-free spectral counts, and label-free extracted ion chromatograms as strategies for data interpretation (source code is available from http://homepages.uc.edu/~wang2x7/Research.htm). From these assessments, we found that differential genes from a single replicate were confirmed by other replicates on the same instrument from 61 to 93% of the time. When comparing across different instruments and quantitative technologies, using multiple replicates, differential genes were reproduced by other data sets from 67 to 99% of the time. Projecting gene differences to biological pathways and networks increased the degree of similarity. These overlaps send an encouraging message about the maturity of technologies for proteomic differentiation.
doi:10.1021/acs.jproteome.5b00859
PMCID: PMC4779376  PMID: 26653538
Differential proteomics; label-free; iTRAQ; quality control; xenografts; technology assessment; CPTAC
6.  Comprehensive Quantitative Analysis of Ovarian and Breast Cancer Tumor Peptidomes 
Journal of Proteome Research  2014;14(1):422-433.
Aberrant degradation of proteins is associated with many pathological states, including cancers. Mass spectrometric analysis of tumor peptidomes, the intracellular and intercellular products of protein degradation, has the potential to provide biological insights on proteolytic processing in cancer. However, attempts to use the information on these smaller protein degradation products from tumors for biomarker discovery and cancer biology studies have been fairly limited to date, largely due to the lack of effective approaches for robust peptidomics identification and quantification and the prevalence of confounding factors and biases associated with sample handling and processing. Herein, we have developed an effective and robust analytical platform for comprehensive analyses of tissue peptidomes, which is suitable for high-throughput quantitative studies. The reproducibility and coverage of the platform, as well as the suitability of clinical ovarian tumor and patient-derived breast tumor xenograft samples with postexcision delay of up to 60 min before freezing for peptidomics analysis, have been demonstrated. Moreover, our data also show that the peptidomics profiles can effectively separate breast cancer subtypes, reflecting tumor-associated protease activities. Peptidomics complements results obtainable from conventional bottom-up proteomics and provides insights not readily obtainable from such approaches.
doi:10.1021/pr500840w
PMCID: PMC4286152  PMID: 25350482
protein degradation; peptidomics; proteases; tumor; ovarian cancer; breast cancer; ischemia
7.  Integrated Bottom-Up and Top-Down Proteomics of Patient-Derived Breast Tumor Xenografts*  
Bottom-up proteomics relies on the use of proteases and is the method of choice for identifying thousands of protein groups in complex samples. Top-down proteomics has been shown to be robust for direct analysis of small proteins and offers a solution to the “peptide-to-protein” inference problem inherent with bottom-up approaches. Here, we describe the first large-scale integration of genomic, bottom-up and top-down proteomic data for the comparative analysis of patient-derived mouse xenograft models of basal and luminal B human breast cancer, WHIM2 and WHIM16, respectively. Using these well-characterized xenograft models established by the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium, we compared and contrasted the performance of bottom-up and top-down proteomics to detect cancer-specific aberrations at the peptide and proteoform levels and to measure differential expression of proteins and proteoforms. Bottom-up proteomic analysis of the tumor xenografts detected almost 10 times as many coding nucleotide polymorphisms and peptides resulting from novel splice junctions than top-down. For proteins in the range of 0–30 kDa, where quantitation was performed using both approaches, bottom-up proteomics quantified 3,519 protein groups from 49,185 peptides, while top-down proteomics quantified 982 proteoforms mapping to 358 proteins. Examples of both concordant and discordant quantitation were found in a ∼60:40 ratio, providing a unique opportunity for top-down to fill in missing information. The two techniques showed complementary performance, with bottom-up yielding eight times more identifications of 0–30 kDa proteins in xenograft proteomes, but failing to detect differences in certain posttranslational modifications (PTMs), such as phosphorylation pattern changes of alpha-endosulfine. This work illustrates the potency of a combined bottom-up and top-down proteomics approach to deepen our knowledge of cancer biology, especially when genomic data are available.
doi:10.1074/mcp.M114.047480
PMCID: PMC4762530  PMID: 26503891
8.  Development and verification of the PAM50-based Prosigna breast cancer gene signature assay 
BMC Medical Genomics  2015;8:54.
Background
The four intrinsic subtypes of breast cancer, defined by differential expression of 50 genes (PAM50), have been shown to be predictive of risk of recurrence and benefit of hormonal therapy and chemotherapy. Here we describe the development of Prosigna™, a PAM50-based subtype classifier and risk model on the NanoString nCounter Dx Analysis System intended for decentralized testing in clinical laboratories.
Methods
514 formalin-fixed, paraffin-embedded (FFPE) breast cancer patient samples were used to train prototypical centroids for each of the intrinsic subtypes of breast cancer on the NanoString platform. Hierarchical cluster analysis of gene expression data was used to identify the prototypical centroids defined in previous PAM50 algorithm training exercises. 304 FFPE patient samples from a well annotated clinical cohort in the absence of adjuvant systemic therapy were then used to train a subtype-based risk model (i.e. Prosigna ROR score). 232 samples from a tamoxifen-treated patient cohort were used to verify the prognostic accuracy of the algorithm prior to initiating clinical validation studies.
Results
The gene expression profiles of each of the four Prosigna subtype centroids were consistent with those previously published using the PCR-based PAM50 method. Similar to previously published classifiers, tumor samples classified as Luminal A by Prosigna had the best prognosis compared to samples classified as one of the three higher-risk tumor subtypes. The Prosigna Risk of Recurrence (ROR) score model was verified to be significantly associated with prognosis as a continuous variable and to add significant information over both commonly available IHC markers and Adjuvant! Online.
Conclusions
The results from the training and verification data sets show that the FDA-cleared and CE marked Prosigna test provides an accurate estimate of the risk of distant recurrence in hormone receptor positive breast cancer and is also capable of identifying a tumor's intrinsic subtype that is consistent with the previously published PCR-based PAM50 assay. Subsequent analytical and clinical validation studies confirm the clinical accuracy and technical precision of the Prosigna PAM50 assay in a decentralized setting.
Electronic supplementary material
The online version of this article (doi:10.1186/s12920-015-0129-6) contains supplementary material, which is available to authorized users.
doi:10.1186/s12920-015-0129-6
PMCID: PMC4546262  PMID: 26297356
9.  Proteogenomic characterization of human colon and rectal cancer 
Nature  2014;513(7518):382-387.
Summary
We analyzed proteomes of colon and rectal tumors previously characterized by the Cancer Genome Atlas (TCGA) and performed integrated proteogenomic analyses. Somatic variants displayed reduced protein abundance compared to germline variants. mRNA transcript abundance did not reliably predict protein abundance differences between tumors. Proteomics identified five proteomic subtypes in the TCGA cohort, two of which overlapped with the TCGA “MSI/CIMP” transcriptomic subtype, but had distinct mutation, methylation, and protein expression patterns associated with different clinical outcomes. Although copy number alterations showed strong cis- and trans-effects on mRNA abundance, relatively few of these extend to the protein level. Thus, proteomics data enabled prioritization of candidate driver genes. The chromosome 20q amplicon was associated with the largest global changes at both mRNA and protein levels; proteomics data highlighted potential 20q candidates including HNF4A, TOMM34 and SRC. Integrated proteogenomic analysis provides functional context to interpret genomic abnormalities and affords a new paradigm for understanding cancer biology.
doi:10.1038/nature13438
PMCID: PMC4249766  PMID: 25043054
10.  Estrogen Receptor Expression Is High But Is of Lower Intensity in Tubular Carcinoma Than in Well-Differentiated Invasive Ductal Carcinoma 
Context
Tubular carcinoma (TC) is a rare, luminal A subtype of breast carcinoma with excellent prognosis, for which adjuvant chemotherapy is usually contraindicated.
Objective
To examine the levels of estrogen receptor (ER) and progesterone receptor expression in cases of TC and well-differentiated invasive ductal carcinoma as compared to normal breast glands and to determine if any significant differences could be detected via molecular testing.
Design
We examined ER and progesterone receptor via immunohistochemistry in tubular (N = 27), mixed ductal/tubular (N = 16), and well-differentiated ductal (N = 27) carcinomas with comparison to surrounding normal breast tissue. We additionally performed molecular subtyping of 10 TCs and 10 ductal carcinomas via the PAM50 assay.
Results
Although ER expression was high for all groups, TC had statistically significantly lower ER staining percentage (ER%) (P = .003) and difference in ER expression between tumor and accompanying normal tissue (P = .02) than well-differentiated ductal carcinomas, with mixed ductal/tubular carcinomas falling between these 2 groups. Mean ER% was 79%, 87%, and 94%, and mean tumor-normal ER% differences were 13.6%, 25.9%, and 32.6% in tubular, mixed, and ductal carcinomas, respectively. Most tumors that had molecular subtyping were luminal A (9 of 10 tubular and 8 of 10 ductal), and no significant differences in specific gene expression between the 2 groups were identified.
Conclusions
Tubular carcinoma exhibited decreased intensity in ER expression, closer to that of normal breast parenchyma, likely as a consequence of a high degree of differentiation. Lower ER% expression by TC may represent a potential pitfall when performing commercially available breast carcinoma prognostic assays that rely heavily on ER-related gene expression.
doi:10.5858/arpa.2013-0621-OA
PMCID: PMC4327939  PMID: 25357113
11.  Endocrine-Therapy-Resistant ESR1 Variants Revealed by Genomic Characterization of Breast-Cancer-Derived Xenografts 
Cell reports  2013;4(6):10.1016/j.celrep.2013.08.022.
SUMMARY
To characterize patient-derived xenografts (PDXs) for functional studies, we made whole-genome comparisons with originating breast cancers representative of the major intrinsic subtypes. Structural and copy number aberrations were found to be retained with high fidelity. However, at the single-nucleotide level, variable numbers of PDX-specific somatic events were documented, although they were only rarely functionally significant. Variant allele frequencies were often preserved in the PDXs, demonstrating that clonal representation can be transplantable. Estrogen-receptor-positive PDXs were associated with ESR1 ligand-binding-domain mutations, gene amplification, or an ESR1/YAP1 translocation. These events produced different endocrine-therapy-response phenotypes in human, cell line, and PDX endocrine-response studies. Hence, deeply sequenced PDX models are an important resource for the search for genome-forward treatment options and capture endocrine-drug-resistance etiologies that are not observed in standard cell lines. The originating tumor genome provides a benchmark for assessing genetic drift and clonal representation after transplantation.
doi:10.1016/j.celrep.2013.08.022
PMCID: PMC3881975  PMID: 24055055
12.  A comparison of PAM50 intrinsic subtyping with immunohistochemistry and clinical prognostic factors in tamoxifen-treated estrogen receptor positive breast cancer 
Purpose
To compare clinical, immunohistochemical and gene expression models of prognosis applicable to formalin-fixed, paraffin-embedded blocks in a large series of estrogen receptor positive breast cancers, from patients uniformly treated with adjuvant tamoxifen.
Methods
qRT-PCR assays for 50 genes identifying intrinsic breast cancer subtypes were completed on 786 specimens linked to clinical (median followup 11.7 years) and immunohistochemical (ER, PR, HER2, Ki67) data. Performance of predefined intrinsic subtype and Risk-Of-Relapse scores was assessed using multivariable Cox models and Kaplan-Meier analysis. Harrell’s C index was used to compare fixed models trained in independent data sets, including proliferation signatures.
Results
Despite clinical ER positivity, 10% of cases were assigned to non-Luminal subtypes. qRT-PCR signatures for proliferation genes gave more prognostic information than clinical assays for hormone receptors or Ki67. In Cox models incorporating standard prognostic variables, hazard ratios for breast cancer disease specific survival over the first 5 years of followup, relative to the most common Luminal A subtype, are 1.99 (95% CI: 1.09–3.64) for Luminal B, 3.65 (1.64–8.16) for HER2-enriched and 17.71 (1.71–183.33) for the basal like subtype. For node-negative disease, PAM50 qRT-PCR based risk assignment weighted for tumor size and proliferation identifies a group with >95% 10 yr survival without chemotherapy. In node positive disease, PAM50-based prognostic models were also superior.
Conclusion
The PAM50 gene expression test for intrinsic biological subtype can be applied to large series of formalin-fixed paraffin-embedded breast cancers, and gives more prognostic information than clinical factors and immunohistochemistry using standard cutpoints.
doi:10.1158/1078-0432.CCR-10-1282
PMCID: PMC2970720  PMID: 20837693
13.  PIK3CA and PIK3CB inhibition produce synthetic lethality when combined with estrogen deprivation in estrogen receptor positive breast cancer 
Cancer research  2009;69(9):3955-3962.
Several phosphoinositide-3-kinase (PI3K) catalytic subunit inhibitors are currently in clinical trial. We therefore sought to examine relationships between pharmacological inhibition and somatic mutations in PI3K catalytic subunits in ER+ breast cancer, where these mutations are particularly common. RNA interference (RNAi) was used to determine the effect of selective inhibition of PI3K catalytic subunits, p110α and p110β, in ER+ breast cancer cells harboring either mutation (PIK3CA) or gene amplification (PIK3CB). p110α RNAi inhibited growth and promoted apoptosis in all tested ER+ breast cancer cells under estrogen deprived-conditions, whereas p110β RNAi only affected cells harboring PIK3CB amplification. Moreover, dual p110α/p110β inhibition potentiated these effects. In addition, treatment with the clinical grade PI3K catalytic subunit inhibitor BEZ235 also promoted apoptosis in ER+ breast cancer cells. Importantly, estradiol suppressed apoptosis induced by both gene knockdowns and by BEZ235 treatment. Our results suggest that PI3K inhibitors should target both p110α and p110β catalytic subunits, whether wild-type or mutant, and be combined with endocrine therapy for maximal efficacy when treating ER+ breast cancer.
doi:10.1158/0008-5472.CAN-08-4450
PMCID: PMC2811393  PMID: 19366795
breast cancer; estrogen receptor; PI3 kinase; endocrine therapy; synthetic lethality

Results 1-13 (13)