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1.
Oncoimmunology ; 11(1): 2052410, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35371621

RESUMO

Major immunotherapy challenges include a limited number of predictive biomarkers and the unusual imaging features post-therapy, such as pseudo-progression, which denote immune infiltrate-mediated tumor enlargement. Such phenomena confound clinical decision-making, since the cancer may eventually regress, and the patient should stay on treatment. We prospectively evaluated serial, blood-derived cell-free DNA (cfDNA) (baseline and 2-3 weeks post-immune checkpoint inhibitors [ICIs]) for variant allele frequency (VAF) and blood tumor mutation burden (bTMB) changes (next-generation sequencing) (N = 84 evaluable patients, diverse cancers). Low vs. high cfDNA-derived average adjusted ΔVAF (calculated by a machine-learning model) was an independent predictor of higher clinical benefit rate (stable disease ≥6 months/complete/partial response) (69.2% vs. 22.5%), and longer median progression-free (10.1 vs. 2.25 months) and overall survival (not reached vs. 6.1 months) (all P < .001, multivariate). bTMB changes did not correlate with outcomes. Therefore, early dynamic changes in cfDNA-derived VAF were a powerful predictor of pan-cancer immunotherapy outcomes.


Assuntos
Inibidores de Checkpoint Imunológico , Neoplasias , Frequência do Gene , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Biópsia Líquida , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia
2.
Adv Biochem Eng Biotechnol ; 110: 47-66, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18528666

RESUMO

Interaction defective alleles (IDAs) are alleles that contain mutations affecting their ability to interact with their wild type binding partners. The locations of the mutations may lead to the identification of protein interaction domains and interaction interfaces. IDAs may also distinguish different binding interfaces of multidomain proteins that are part of large complexes, thus shedding light on large protein structures that have yet to be determined. IDAs may also be used in conjunction with RNAi to dissect protein interaction networks. Here, the wild type allele is knocked down and replaced with an IDA that has lost the ability to interact with a specific binding partner. As a result, interactions are disrupted rather than knocking out the entire gene. Thus, IDAs have the potential to be extremely valuable tools in protein interaction network analysis. IDAs can be isolated by reverse two-hybrid analysis, which was demonstrated over a decade ago, but high background levels caused by truncated IDAs have prevented its widespread adoption. We recently described a novel method for full-length allele library generation that eliminates this background and increases the efficiency of the reverse two-hybrid protocol (and IDA isolation) significantly. Here we discuss our strategy for allele library generation, the potential uses of IDAs as outlined above, and additional applications of allele libraries.


Assuntos
Alelos , Mapeamento de Interação de Proteínas/métodos , Proteínas/genética , Proteínas/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Substituição de Aminoácidos , Biblioteca Gênica , Mutação
3.
Oncotarget ; 9(29): 20304-20322, 2018 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-29755653

RESUMO

The current algorithm for Lynch syndrome diagnosis is highly complex with multiple steps which can result in an extended time to diagnosis while depleting precious tumor specimens. Here we describe the analytical validation of a custom probe-based NGS tumor panel, TumorNext-Lynch-MMR, which generates a comprehensive genetic profile of both germline and somatic mutations that can accelerate and streamline the time to diagnosis and preserve specimen. TumorNext-Lynch-MMR can detect single nucleotide variants, small insertions and deletions in 39 genes that are frequently mutated in Lynch syndrome and colorectal cancer. Moreover, the panel provides microsatellite instability status and detects loss of heterozygosity in the five Lynch genes; MSH2, MSH6, MLH1, PMS2 and EPCAM. Clinical cases are described that highlight the assays ability to differentiate between somatic and germline mutations, precisely classify variants and resolve discordant cases.

4.
Oncotarget ; 7(42): 68206-68228, 2016 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-27626691

RESUMO

The development of targeted therapies for both germline and somatic DNA mutations has increased the need for molecular profiling assays to determine the mutational status of specific genes. Moreover, the potential of off-label prescription of targeted therapies favors classifying tumors based on DNA alterations rather than traditional tissue pathology. Here we describe the analytical validation of a custom probe-based NGS tumor panel, TumorNext, which can detect single nucleotide variants, small insertions and deletions in 142 genes that are frequently mutated in somatic and/or germline cancers. TumorNext also detects gene fusions and structural variants, such as tandem duplications and inversions, in 15 frequently disrupted oncogenes and tumor suppressors. The assay uses a matched control and custom bioinformatics pipeline to differentiate between somatic and germline mutations, allowing precise variant classification. We tested 170 previously characterized samples, of which > 95% were formalin-fixed paraffin embedded tissue from 8 different cancer types, and highlight examples where lack of germline status may have led to the inappropriate prescription of therapy. We also describe the validation of the Affymetrix OncoScan platform, an array technology for high resolution copy number variant detection for use in parallel with the NGS panel that can detect single copy amplifications and hemizygous deletions. We analyzed 80 previously characterized formalin-fixed paraffin-embedded specimens and provide examples of hemizygous deletion detection in samples with known pathogenic germline mutations. Thus, the TumorNext combined approach of NGS and OncoScan potentially allows for the identification of the "second hit" in hereditary cancer patients.


Assuntos
Biologia Computacional/métodos , Variações do Número de Cópias de DNA , Regulação Neoplásica da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias/genética , Análise Mutacional de DNA/métodos , Frequência do Gene , Predisposição Genética para Doença/genética , Humanos , Mutação , Neoplasias/patologia , Inclusão em Parafina , Polimorfismo de Nucleotídeo Único , Reprodutibilidade dos Testes , Fixação de Tecidos
5.
Cancers (Basel) ; 7(3): 1313-32, 2015 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-26193321

RESUMO

The molecular characterization of tumors using next generation sequencing (NGS) is an emerging diagnostic tool that is quickly becoming an integral part of clinical decision making. Cancer genomic profiling involves significant challenges including DNA quality and quantity, tumor heterogeneity, and the need to detect a wide variety of complex genetic mutations. Most available comprehensive diagnostic tests rely on primer based amplification or probe based capture methods coupled with NGS to detect hotspot mutation sites or whole regions implicated in disease. These tumor panels utilize highly customized bioinformatics pipelines to perform the difficult task of accurately calling cancer relevant alterations such as single nucleotide variations, small indels or large genomic alterations from the NGS data. In this review, we will discuss the challenges of solid tumor assay design/analysis and report a case study that highlights the need to include complementary technologies (i.e., arrays) and germline analysis in tumor testing to reliably identify copy number alterations and actionable variants.

6.
Mol Cell Proteomics ; 6(3): 514-26, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17151022

RESUMO

The yeast reverse two-hybrid method was developed to identify mutations disrupting protein-protein interactions. Adoption of the method has been slow, in large part, due to the high frequency of truncation and frameshift mutants typically observed with current protocols. We have developed a new strategy, based on in vitro recombinational cloning and full-length selection in Escherichia coli, to eliminate this background and dramatically increase the efficiency of the reverse two-hybrid protocol. The method was tested by generating an allele library of MyoD1 and selecting for alleles with defective interaction with Id1. Our results confirm that most of the interaction-defective alleles contain a single point mutation in the known interaction domain, the basic helix-loop-helix region. Moreover analysis of the crystal structure of MyoD reveals that the majority of these mutations occurred at the interaction interface. The results obtained using this novel approach for allele library generation demonstrate a significant advancement in the application of yeast reverse two-hybrid screens. Furthermore this method is applicable to any loss-of-function mutant screen where truncated proteins are a source of high background.


Assuntos
Alelos , Técnicas do Sistema de Duplo-Híbrido , Animais , Escherichia coli/genética , Biblioteca Gênica , Proteína 1 Inibidora de Diferenciação/genética , Proteína 1 Inibidora de Diferenciação/metabolismo , Camundongos , Mutação , Proteína MyoD/genética , Proteína MyoD/metabolismo , Fases de Leitura Aberta/genética , Saccharomyces cerevisiae/genética
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