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Related Concept Videos

Cancers Originate from Somatic Mutations in a Single Cell02:21

Cancers Originate from Somatic Mutations in a Single Cell

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Cancer arises from mutations in the critical genes that allow healthy cells to escape cell cycle regulation and acquire the ability to proliferate indefinitely. Though originating from a single mutation event in one of the originator cells, cancer progresses when the mutant cell lines continue to gain more and more mutations, and finally, become malignant. For example, chronic myelogenous leukemia (CML) develops initially as a non-lethal increase in white blood cells, which progressively...
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Mutations01:39

Mutations

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Mutations01:35

Mutations

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Mutations are changes in the sequence of DNA. These changes can occur spontaneously or they can be induced by exposure to environmental factors. Mutations can be characterized in a number of different ways: whether and how they alter the amino acid sequence of the protein, whether they occur over a small or large area of DNA, and whether they occur in somatic cells or germline cells.
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The first human genome sequencing project cost $2.7 billion and was declared complete in 2003, after 15 years of international cooperation and collaboration between several research teams and funding agencies. Today, with the advent of next-generation sequencing technologies, the cost and time of sequencing a human genome have dropped over 100 fold.
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A mutation is a change in the sequence of bases of DNA or RNA in a genome. Some mutations occur during replication of the genome due to errors made by the polymerase enzymes that replicate DNA or RNA. Unlike DNA polymerase, RNA polymerase is prone to errors because it is not capable of “proofreading” its work. Viruses with RNA-based genomes, like HIV, therefore accrue mutations faster than viruses with DNA-based genomes. Because mutation and recombination provide the raw material...
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Mutation, Gene Flow, and Genetic Drift

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In a population that is not at Hardy-Weinberg equilibrium, the frequency of alleles changes over time. Therefore, any deviations from the five conditions of Hardy-Weinberg equilibrium can alter the genetic variation of a given population. Conditions that change the genetic variability of a population include mutations, natural selection, non-random mating, gene flow, and genetic drift (small population size).
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Detection of Rare Mutations in CtDNA Using Next Generation Sequencing
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Implementation of next generation sequencing technology for somatic mutation detection in routine laboratory

Tindaro Giardina1, Cleo Robinson2, Fabienne Grieu-Iacopetta1

  • 1Anatomical Pathology, PathWest Laboratory Medicine, QEII Medical Centre, Nedlands, WA, Australia.

Pathology
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Summary

Targeted next-generation sequencing (NGS) provides a reliable method for detecting multiple genetic mutations in cancer specimens, showing high concordance with existing platforms. This approach is suitable for various sample types, including those with limited material or low tumor cell content.

Keywords:
Next generation sequencinggene panelsmolecular diagnostic testing laboratorysomatic mutation

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Area of Science:

  • Oncology
  • Genetics
  • Molecular Diagnostics

Background:

  • Routine diagnostic use of next-generation sequencing (NGS) is developing, facing challenges with complexity.
  • Targeted NGS offers an efficient alternative to single-target assays for identifying multiple cancer-related genetic aberrations.
  • Clinical demand for comprehensive genetic testing in cancer specimens is increasing.

Purpose of the Study:

  • To validate targeted NGS performance against established mutation detection platforms in a diagnostic laboratory setting.
  • To assess the concordance of targeted NGS results with Sanger sequencing, pyrosequencing, CAST PCR, and Cobas assays.
  • To evaluate the suitability of targeted NGS for various formalin-fixed, paraffin-embedded (FFPE) cancer sample types.

Main Methods:

  • A blinded validation study was conducted on 113 FFPE tumor samples (core biopsies, resections, cytology) from non-small cell lung cancer (NSCLC), colorectal cancer (CRC), malignant melanoma (MM), and gastrointestinal stromal tumor (GIST).
  • DNA was extracted, and libraries were prepared using the TruSight Tumor 26 gene panel for targeted NGS on a MiSeq instrument.
  • NGS results were compared with those obtained from Sanger sequencing, pyrosequencing, CAST PCR, and Cobas assays.

Main Results:

  • Targeted NGS demonstrated high concordance (94.7%) with conventional methods, with 107 out of 113 cases showing agreement.
  • False negatives were attributed to sequencing quality failures or mutations falling outside the NGS panel's target range.
  • NGS identified 113 additional mutations, including 26 with known clinical importance and 37 with potential clinical significance, using low DNA input (10-20 ng) and samples with <50% tumor cell content.

Conclusions:

  • Targeted NGS is highly concordant with established mutation testing platforms and is suitable for routine diagnostic use.
  • The method performs well across diverse FFPE sample types, including those with limited material or low tumor cell content.
  • This study established quality parameter settings for robust mutation data generation via NGS in a diagnostic laboratory.