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Mutations01:39

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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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Solids in which the atoms, ions, or molecules are arranged in a definite repeating pattern are known as crystalline solids. Metals and ionic compounds typically form ordered, crystalline solids. A crystalline solid has a precise melting temperature because each atom or molecule of the same type is held in place with the same forces or energy. Amorphous solids or non-crystalline solids (or, sometimes, glasses) which lack an ordered internal structure and are randomly arranged. Substances that...
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Salvaging the supernatant: next generation cytopathology for solid tumor mutation profiling.

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Discarded supernatant from fine needle aspiration (FNA) provides valuable DNA for tumor mutation profiling. This study demonstrates its utility for next-generation sequencing and digital PCR, aiding molecular testing without re-biopsy.

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Area of Science:

  • Oncology
  • Pathology
  • Molecular Diagnostics

Background:

  • Targeted therapies require extensive molecular testing of solid tumors.
  • Fine needle aspiration (FNA) yields limited tissue, posing challenges for comprehensive molecular profiling.
  • Supernatant fluid from FNA needle rinses, typically discarded, may contain recoverable DNA.

Purpose of the Study:

  • To evaluate the DNA yield and mutational profiling potential of post-centrifuged supernatant from FNA needle rinses.
  • To assess the utility of supernatant DNA for next-generation sequencing (NGS) and droplet digital PCR (ddPCR).
  • To determine if supernatant DNA analysis can complement or replace tissue from surgical specimens for molecular testing.

Main Methods:

  • Collected and processed supernatant fluid from 25 malignant and 10 benign FNA needle rinses.
  • Quantified DNA yield from supernatant samples.
  • Performed NGS using the Ion AmpliSeq Cancer Hotspot Panel v2 on malignant samples.
  • Confirmed mutations using ddPCR in a subset of cases.
  • Compared NGS results from supernatant with prior or concurrent surgical specimens.

Main Results:

  • Substantial DNA yields were obtained from supernatants (mean 445 ng, median 176.4 ng).
  • NGS detected somatic mutations in all 25 malignant samples; no mutations were found in benign samples.
  • 100% concordance was observed between supernatant DNA mutations and those from surgical specimens.
  • ddPCR successfully confirmed mutations in EGFR, KRAS, BRAF, PIK3CA, and NRAS.

Conclusions:

  • Post-centrifuged FNA supernatant is a robust source of DNA for molecular profiling.
  • Utilizing supernatant DNA enables expanded mutation profiling via NGS and ddPCR.
  • This approach optimizes the use of limited FNA samples, potentially avoiding repeat biopsies.