@article{01HZH190Y2S4RYSCC3HEY20KWA,
  abstract     = {{Currently, we cannot provide a conclusive diagnosis for 3 to 5% of people who are confronted with cancer. These patients have Cancer of Unknown Primary (CUP), i.e. a metastasized cancer for which the tissue-of-origin cannot be determined. Studies have shown that the DNA methylation profile is a unique ‘fingerprint’ that can be used to classify tumors. Here we use cfRRBS (cell-free reduced representation bisulfite sequencing), a technique that allows us to identify the methylation profile starting from minimal amounts of highly fragmented DNA, for CUP diagnosis on FFPE tissue and liquid biopsies. We collected 80 primary tumor FFPE samples covering 16 tumor entities together with 15 healthy plasma samples to use as custom cfRRBS reference dataset. Entity specific methylation regions (ESRs) are defined for each entity to build a classifier based on non-negative least squares (NNLS) deconvolution. This classification framework was tested on 30 FFPE, 19 plasma and 40 pleural and peritoneal effusion samples of both known metastatic tumors and clinical CUPs for which pathological investigation finally resulted in a cancer diagnosis. Using this framework, 27/30 FFPE (all CUPs) and 16/19 plasma samples (10/13 CUPs) obtained an accurate diagnosis, with a minimal DNA input of 400 pg. Of the 40 pleural and peritoneal effusion samples, diagnosis is possible in 9/27 samples with negative/inconclusive cytology (6/13 CUPs), showing that cfDNA methylation profiling could complement routine cytological analysis. However, a low “cfDNA – high molecular weight DNA ratio” has a considerable impact on the prediction accuracy. Moreover, the accuracy improves significantly if the predicted tumor percentage is higher than 7%. This proof-of-concept study shows the feasibility of using DNA methylation profiling on FFPE and liquid biopsy samples such as blood, ascites and pleural effusions in a fast and affordable way. Our novel RRBS-based technique requires minimal DNA input, can be performed in less than one week and is highly adaptable to specific diagnostic problems as we only use 5 FFPE references per tumor entity. We believe that cfRRBS methylation profiling could be a valuable addition to the pathologist’s toolbox in the diagnosis of CUPs.}},
  articleno    = {{102091}},
  author       = {{De Wilde, Jilke and Van Paemel, Ruben and De Koker, Andries and Roelandt, Sofie and Van de Velde, Sofie and Callewaert, Nico and Van Dorpe, Jo and Creytens, David and De Wilde, Bram and De Preter, Katleen}},
  issn         = {{0023-6837}},
  journal      = {{LABORATORY INVESTIGATION}},
  keywords     = {{cancer of unknown primary,circulating cell-free DNA,DNA methylation,computational deconvolution,formalin-fixed paraffin embedded tissue,liquid biopsies,CELL-FREE DNA,PROGNOSTIC-FACTORS,MALIGNANT ASCITES,LIQUID BIOPSY,TUMOR-TISSUE,CLASSIFICATION,MULTICENTER,IMMUNOHISTOCHEMISTRY,IDENTIFICATION,VALIDATION}},
  language     = {{eng}},
  number       = {{8}},
  pages        = {{16}},
  title        = {{A fast, affordable and minimally-invasive diagnostic test for Cancer of Unknown Primary (CUP) using DNA methylation profiling}},
  url          = {{http://doi.org/10.1016/j.labinv.2024.102091}},
  volume       = {{104}},
  year         = {{2024}},
}

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