Fanconi anemia (FA) is a heritable malformation, bone marrow failure and cancer predisposition syndrome that confers an exceptionally high risk of squamous carcinomas. These carcinomas originate in ...epithelia lining the mouth, proximal esophagus, vulva and anus: their origins are not understood, and no effective ways have been identified to prevent or delay their appearance. Many FA‐associated carcinomas are also therapeutically challenging: they may be multi‐focal and stage‐advanced at diagnosis, and most individuals with FA cannot tolerate standard‐of‐care systemic therapies such as DNA cross‐linking drugs or ionizing radiation due to constitutional DNA damage hypersensitivity. We developed the Fanconi Anemia Cancer Cell Line Resource (FA‐CCLR) to foster new work on the origins, treatment and prevention of FA‐associated carcinomas. The FA‐CCLR consists of Fanconi‐isogenic head and neck squamous cell carcinoma (HNSCC) cell line pairs generated from five individuals with FA‐associated HNSCC, and five individuals with sporadic HNSCC. Sporadic, isogenic HNSCC cell line pairs were generated in parallel with FA patient‐derived isogenic cell line pairs to provide comparable experimental material to use to identify cell and molecular phenotypes driven by germline or somatic loss of Fanconi pathway function, and the subset of these FA‐dependent phenotypes that can be modified, complemented or suppressed. All 10 FANC‐isogenic cell line pairs are available to academic, non‐profit and industry investigators via the “Fanconi Anemia Research Materials” Resource and Repository at Oregon Health & Sciences University, Portland OR.
What's new?
People with Fanconi anemia are at high risk of squamous cell carcinomas (SCC), but it is not well understood how these cancers originate and there is no effective prevention strategy. Here, the authors describe a new tool for studying the origin, treatment and prevention of these carcinomas. The Fanconi Anemia Cancer Cell Line Resource consists of 10 new FANC‐isogenic cell line pairs generated from 5 people with FA‐associated head and neck SCC and 5 with sporadic HNSCC. These cell lines are available for distribution to investigators studying FA or sporadic HNSCC.
Panorama is a web application for storing, sharing, analyzing, and reusing targeted assays created and refined with Skyline, an increasingly popular Windows client software tool for targeted ...proteomics experiments. Panorama allows laboratories to store and organize curated results contained in Skyline documents with fine-grained permissions, which facilitates distributed collaboration and secure sharing of published and unpublished data via a web-browser interface. It is fully integrated with the Skyline workflow and supports publishing a document directly to a Panorama server from the Skyline user interface. Panorama captures the complete Skyline document information content in a relational database schema. Curated results published to Panorama can be aggregated and exported as chromatogram libraries. These libraries can be used in Skyline to pick optimal targets in new experiments and to validate peak identification of target peptides. Panorama is open-source and freely available. It is distributed as part of LabKey Server, an open source biomedical research data management system. Laboratories and organizations can set up Panorama locally by downloading and installing the software on their own servers. They can also request freely hosted projects on https://panoramaweb.org, a Panorama server maintained by the Department of Genome Sciences at the University of Washington.
Early detection of Primary Immunodeficiencies Disorders (PIDDs) is of paramount importance for effective treatment and disease management. Many PIDDs would be strong candidates for newborn screening ...(NBS) if robust screening methods could identify patients from dried blood spots (DBS) during the neonatal period. As majority of congenital PIDDs result in the reduction or absence of specific proteins, direct quantification of these target proteins represents an attractive potential screening tool. Unfortunately, detection is often limited by the extremely low protein concentrations in blood cells and limited blood volume present in DBS. We have recently developed a robust novel method for quantification of low abundance proteins in DBS for PIDDs using peptide immunoaffinity enrichment coupled to selected reaction monitoring (immuno-SRM). Here, we further generated a multiplexed Immuno-SRM panel for simultaneous screening of eight signature peptides representing five PIDD-specific and two cell-type specific proteins from DBS. In samples from 28 PIDD patients including two carriers, representing X-Linked Agammaglobulinemia (XLA), Wiskott-Aldrich Syndrome (WAS), X-Linked Chronic Granulomatous Disease (XL-CGD), DOCK8 Deficiency and ADA deficiency, peptides representing each disease are significantly reduced relative to normal controls and patient identification had excellent agreement with clinical and molecular diagnosis. Also included in the multiplex panel are cell specific markers for platelets (CD42) and Natural Killer Cells (CD56). In patients with WAS, CD42 levels were found to be significantly reduced consistent with characteristic thrombocytopenia. A patient with WAS analyzed before and after bone marrow transplant showed normalized WAS protein and platelet CD42 after treatment highlighting the ability of immuno-SRM to monitor the effects of PIDD treatment. The assay was readily reproduced in two separate laboratories with similar analytical performance and complete agreement in patient diagnosis demonstrating the effective standardized methods. A high-throughput Immuno-SRM method screens PIDD-specific peptides in a 2.5-min runtime meeting high volume NBS workflow requirements was also demonstrated in this report. This high-throughput method returned identical results to the standard Immuno-SRM PIDD panel. Immuno-SRM peptide analysis represents a robust potential clinical diagnostic for identifying and studying PIDD patients from easily collected and shipped DBS and supports a significant potential for early PIDD diagnosis through newborn screening.
Among the pleotropic roles of transforming growth factor-β (TGFβ) signaling in cancer, its impact on genomic stability is least understood. Inhibition of TGFβ signaling increases use of alternative ...end joining (alt-EJ), an error-prone DNA repair process that typically functions as a "backup" pathway if double-strand break repair by homologous recombination or nonhomologous end joining is compromised. However, the consequences of this functional relationship on therapeutic vulnerability in human cancer remain unknown. Here, we show that TGFβ broadly controls the DNA damage response and suppresses alt-EJ genes that are associated with genomic instability. Mechanistically based TGFβ and alt-EJ gene expression signatures were anticorrelated in glioblastoma, squamous cell lung cancer, and serous ovarian cancer. Consistent with error-prone repair, more of the genome was altered in tumors classified as low TGFβ and high alt-EJ, and the corresponding patients had better outcomes. Pan-cancer analysis of solid neoplasms revealed that alt-EJ genes were coordinately expressed and anticorrelated with TGFβ competency in 16 of 17 cancer types tested. Moreover, regardless of cancer type, tumors classified as low TGFβ and high alt-EJ were characterized by an insertion-deletion mutation signature containing short microhomologies and were more sensitive to genotoxic therapy. Collectively, experimental studies revealed that loss or inhibition of TGFβ signaling compromises the DNA damage response, resulting in ineffective repair by alt-EJ. Translation of this mechanistic relationship into gene expression signatures identified a robust anticorrelation that predicts response to genotoxic therapies, thereby expanding the potential therapeutic scope of TGFβ biology.
Stable isotope standards and capture by antipeptide antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution and detection by multiple reaction monitoring mass ...spectrometry to provide quantitative measurement of peptides as surrogates for their respective proteins. In this report, we describe a feasibility study to determine the success rate for production of suitable antibodies for SISCAPA assays in order to inform strategies for large-scale assay development. A workflow was designed that included a multiplex immunization strategy in which up to five proteotypic peptides from a single protein target were used to immunize individual rabbits. A total of 403 proteotypic tryptic peptides representing 89 protein targets were used as immunogens. Antipeptide antibody titers were measured by ELISA and 220 antipeptide antibodies representing 89 proteins were chosen for affinity purification. These antibodies were characterized with respect to their performance in SISCAPA-multiple reaction monitoring assays using trypsin-digested human plasma matrix. More than half of the assays generated were capable of detecting the target peptide at concentrations of less than 0.5 fmol/μl in human plasma, corresponding to protein concentrations of less than 100 ng/ml. The strategy of multiplexing five peptide immunogens was successful in generating a working assay for 100% of the targeted proteins in this evaluation study. These results indicate it is feasible for a single laboratory to develop hundreds of assays per year and allow planning for cost-effective generation of SISCAPA assays.
Primary immunodeficiency disorders (PIDD) comprise a group of life-threatening congenital diseases characterized by absent or impaired immune responses. Despite the fact that effective, curative ...treatments are available with optimal clinical outcomes when diagnosed early, newborn screening does not exist for the majority of these diseases due to the lack of detectable, specific biomarkers or validated methods for population-based screening. Peptide immunoaffinity enrichment coupled with selected reaction monitoring mass spectrometry (immuno-SRM) is a sensitive proteomic assay, involving antibody-mediated peptide capture, that allows for concurrent quantification of multiple analytes. This assay has promise for use in potential newborn screening of PIDDs that lead to diminished or absent target proteins in the majority of cases.
To determine and evaluate if a multiplex assay based on immuno-SRM is able to reliably and precisely distinguish affected patients with X-linked agammaglobulinemia (XLA), Wiskott-Aldrich Syndrome (WAS), and CD3ϵ-associated severe combined immunodeficiency (SCID) from one another and from unaffected normal control dried blood spot (DBS) samples.
We performed a blinded, multiplexed analysis of proteolytically-generated peptides from WASp, BTK, and CD3ϵ (for WAS, XLA, and SCID, respectively) in DBS samples from 42 PIDD patients, 40 normal adult controls, and 62 normal newborns. The peptide ATPase copper transporting protein (ATP7B) 1056 was simultaneously monitored for quality assurance purposes.
The immuno-SRM assays reliably quantified the target peptides in DBS and accurately distinguished affected patients from normal controls. Analysis of signature peptides found statistically significant reduction or absence of peptide levels in affected patients compared to control groups in each case (WASp and BTK:
= 0.0001, SCID:
= 0.05). Intra and inter-assay precision ranged from 11 to 22% and 11 to 43% respectively; linearity (1.39-2000 fmol peptide), and stability (≤ 0.09% difference in 72 h) showed high precision for the multiplexed assay. Inter-laboratory assay comparison showed high concordance for measured peptide concentrations, with R
linearity ≥ 0.97 for the WASp 274, CD3ϵ 197, BTK 407, and ATP7B 1056 peptides.
Immuno-SRM-based quantification of proteotypic peptides from WASp, BTK, and CD3ϵ in DBS distinguishes relevant PIDD cases from one another and from controls, raising the possibility of employing this approach for large-scale multiplexed newborn screening of selective PIDDs.
A major goal in cell signaling research is the quantification of phosphorylation pharmacodynamics following perturbations. Traditional methods of studying cellular phospho-signaling measure one ...analyte at a time with poor standardization, rendering them inadequate for interrogating network biology and contributing to the irreproducibility of preclinical research. In this study, we test the feasibility of circumventing these issues by coupling immobilized metal affinity chromatography (IMAC)-based enrichment of phosphopeptides with targeted, multiple reaction monitoring (MRM) mass spectrometry to achieve precise, specific, standardized, multiplex quantification of phospho-signaling responses. A multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay targeting phospho-analytes responsive to DNA damage was configured, analytically characterized, and deployed to generate phospho-pharmacodynamic curves from primary and immortalized human cells experiencing genotoxic stress. The multiplexed assays demonstrated linear ranges of ≥3 orders of magnitude, median lower limit of quantification of 0.64 fmol on column, median intra-assay variability of 9.3%, median inter-assay variability of 12.7%, and median total CV of 16.0%. The multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay enabled robust quantification of 107 DNA damage-responsive phosphosites from human cells following DNA damage. The assays have been made publicly available as a resource to the community. The approach is generally applicable, enabling wide interrogation of signaling networks.
The application of “omics” technologies to biological samples generates hundreds to thousands of biomarker candidates; however, a discouragingly small number make it through the pipeline to clinical ...use. This is in large part due to the incredible mismatch between the large numbers of biomarker candidates and the paucity of reliable assays and methods for validation studies. We desperately need a pipeline that relieves this bottleneck between biomarker discovery and validation. This paper reviews the requirements for technologies to adequately credential biomarker candidates for costly clinical validation and proposes methods and systems to verify biomarker candidates. Models involving pooling of clinical samples, where appropriate, are discussed. We conclude that current proteomic technologies are on the cusp of significantly affecting translation of molecular diagnostics into the clinic.
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