Anchored multiplex PCR for targeted next-generation sequencing

Context: In 2013, an evidence-based guideline was published by the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology to set standards for the molecular analysis of lung cancers to guide treatment decisions with targeted inhibitors. New evidence has prompted an evaluation of additional laboratory technologies, targetable genes, patient populations, and tumor types for testing. Objective: To systematically review and update the 2013 guideline to affirm its validity; to assess the evidence of new genetic discoveries, technologies, and therapies; and to issue an evidence-based update. Design: The College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology convened an expert panel to develop an evidence-based guideline to help define the key questions and literature search terms, review abstracts and full articles, and draft recommendations. Results: Eighteen new recommendations were drafted. The panel also updated 3 recommendations from the 2013 guideline. Conclusions: The 2013 guideline was largely reaffirmed with updated recommendations to allow testing of cytology samples, require improved assay sensitivity, and recommend against the use of

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“…A capture-based sequencing approach, involving DNA or RNA, may be more sensitive and more readily integrated into a large multigene panel. 76 …”

Section: Expert Consensus Opinionmentioning

confidence: 99%

Analytical Validation of BRAF Mutation Testing from Circulating Free DNA Using the Amplification Refractory Mutation Testing System

Aung

Donald

Ellison

et al. 2014

The Journal of Molecular Diagnostics

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“…Focusing on relevant fusions in NSCLC, molecular events involving ALK, ROS1, RET and NTRK1-3 genes would hopefully, in the next future, routinely been simultaneously sounded out (60), allowing the exploitation of small amounts of material. Moreover, deep sequencing analysis can be performed on FFPE samples and not necessarily on frozen tissues (61). With special regard to ALK rearrangement, NGS feasibility and reliability have been already proven (46).…”

Section: Next-generation Techniquesmentioning

confidence: 99%

Tackling ALK in non-small cell lung cancer: the role of novel inhibitors

Facchinetti¹,

Tiseo²,

Maïo³

et al. 2016

Transl. Lung Cancer Res.

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Abstract:Crizotinib is an oral inhibitor of anaplastic lymphoma kinase (ALK) with remarkable clinical activity in patients suffering from ALK-rearranged non-small cell lung cancer (NSCLC), accounting to its superiority compared to chemotherapy. Unfortunately, virtually all ALK-rearranged tumors acquire resistance to crizotinib, frequently within one year since the treatment initiation. To date, therapeutic strategies to overcome crizotinib resistance have focused on the use of more potent and structurally different compounds. Second-generation ALK inhibitors such as ceritinib (LDK378), alectinib (CH5424802/ RO5424802) and brigatinib (AP26113) have shown relevant clinical activity, consequently fostering their rapid clinical development and their approval by health agencies. The third-generation inhibitor lorlatinib (PF-06463922), selectively active against ALK and ROS1, harbors impressive biological potency; its efficacy in reversing resistance to crizotinib and to other ALK inhibitors is being proven by early clinical trials. The NTRK1-3 and ROS1 inhibitor entrectinib (RXDX-101) has been reported to act against NSCLC harboring ALK fusion proteins too. Despite the quick development of these novel agents, several issues remain to be discussed in the treatment of patients suffering from ALK-rearranged NSCLC. This position paper will discuss the development, the current evidence and approvals, as long as the future perspectives of new ALK inhibitors beyond crizotinib. Clinical behaviors of ALK-rearranged NSCLC vary significantly among patients and differential molecular events responsible of crizotinib resistance account for the most important quote of this heterogeneity. The precious availability of a wide range of active anti-ALK compounds should be approached in a critical and careful perspective, in order to develop treatment strategies tailored on the disease evolution of every single patient.

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“…21 Briefly, genomic DNA was isolated from blood or bone marrow aspirates (QIAcube; Qiagen, Valencia, CA). The genomic DNA was sheared with the Covaris (Woburn, MA) M220 instrument followed by end-repair, adenylation, and ligation with an adapter.…”

Section: Targeted Dna-seq Using Anchored Multiplex Pcrmentioning

confidence: 99%

“…A sequencing library that targeted hotspots and exons in 39 genes (Supplemental Table S1) was generated with two hemi-nested PCR reactions with the use of one primer specific to a sequence in the gene of interest and one specific to a universal sequence in the adapter, for each PCR reaction. 21 Illumina (San Diego, CA) MiSeq 2 Â 151 bp paired-end sequencing results were aligned to the hg19 human genome reference with the use of BWA-MEM. 22 PCR/optical duplicates were removed on the basis of unique start sites of sequenced molecules.…”

Section: Targeted Dna-seq Using Anchored Multiplex Pcrmentioning

confidence: 99%

Detection of Dual IDH1 and IDH2 Mutations by Targeted Next-Generation Sequencing in Acute Myeloid Leukemia and Myelodysplastic Syndromes

Platt

Fathi

Borger

et al. 2015

The Journal of Molecular Diagnostics

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Studies in myeloid neoplasms have described recurrent IDH1 and IDH2 mutations as primarily mutually exclusive. Over a 6-month period of clinical testing with a targeted next-generation sequencing assay, we evaluated 92 patients with acute myeloid leukemia, myelodysplastic syndrome, and chronic myelomonocytic leukemia and identified a subset of 21 patients (23%) who harbored mutations in either IDH1 or IDH2. Of the 21 patients with IDH mutations, 4 (19%) were found to have single nucleotide variants in both IDH1 and IDH2. An additional patient included in the study was found to have two different IDH2 mutations. The mutations were typically present at different variant allelic frequencies, with one predominating over the other, consistent with the presence of multiple subclones in a single patient. In one case, the variant allelic frequencies in both IDH1 and IDH2 were equally low in the setting of a high percentage of blasts, suggesting that the IDH mutations were unlikely to be present in the founding clone. Given these data, we conclude that dual IDH1/2 mutations likely were previously underestimated, a finding that may carry important treatment implications. (J Mol Diagn 2015, 17: 661e668; http://dx.doi.org/10.1016/j.jmoldx.2015 In recent years, whole-genome sequencing of acute myeloid leukemias (AMLs) led to the identification of frequent heterozygous mutations in isocitrate dehydrogenase 1 gene (IDH1).1,2 Subsequent exome sequencing and targeted resequencing studies in AML also identified frequent IDH2 mutations. 3,4 Overall, approximately 15% to 30% of AML have mutations in IDH1 or IDH2, almost exclusively at codon Arg132 for IDH1 and at codons Arg140 and Arg172 for IDH2.1,3e6 Patients with cytogenetically normal AML are enriched for these mutations. The mutations confer neomorphic enzyme activity through the NADPH-dependent reduction of the normal end-product a-ketoglutarate to the putative oncometabolite 2-hydroxyglutarate. The accumulation of high levels of 2-hydroxyglutarate in the IDH1/2-mutant tumor provides an important mechanism of cellular transformation through the targeting of epigenetic regulators. 7IDH mutations were also identified in preleukemic clonal malignancies, including myelodysplastic syndromes (MDSs) and myeloproliferative neoplasms (approximately 5% of MDS/myeloproliferative neoplasm and approximately 20% of AML arising from MDS or myeloproliferative neoplasm).

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Anchored multiplex PCR for targeted next-generation sequencing

Cited by 731 publications

References 31 publications

Analytical Validation of BRAF Mutation Testing from Circulating Free DNA Using the Amplification Refractory Mutation Testing System

Analytical Validation of BRAF Mutation Testing from Circulating Free DNA Using the Amplification Refractory Mutation Testing System

Tackling ALK in non-small cell lung cancer: the role of novel inhibitors

Detection of Dual IDH1 and IDH2 Mutations by Targeted Next-Generation Sequencing in Acute Myeloid Leukemia and Myelodysplastic Syndromes

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