Authors
Tristan Bergmann, Marianne Hölbling, Monica Sudarsanam, Christiane Driemel, Maximilian E Daniels, Joanna Swierz, Sandra Jaschinski, Maximilian Seidl, Norbert R Kübler, Wolfram T Knoefel, Rui Pl Neves, Karl C Sproll, Nikolas H Stoecklein
Published in
The Journal of pathology. Sep 21, 2026. Epub Sep 21, 2026.
Abstract
Accurate detection of lymph node (LN) metastases is critical for prognosis and surgical decision-making in head and neck squamous cell carcinoma (HNSCC) and other epithelial cancers. Standard histopathology examines only a small fraction of LN tissue, limiting sensitivity for micrometastases and isolated tumour cells. Scalable approaches are needed to improve staging accuracy and reduce overtreatment. We adapted the FDA-approved CELLSEARCH System, originally developed for circulating tumour cell detection, for use on dissociated 50 μm formalin-fixed paraffin-embedded (FFPE) LN sections. Tumour cells were enriched via EpCAM-based immunomagnetic capture, stained with cytokeratin (CK)/DAPI and quantified using the ACCEPT (Automated CTC Classification, Enumeration and Phenotyping Technology) image analysis tool. Specificity and reproducibility were tested in 50 metastasis-positive and 50 metastasis-free LNs from patients with HNSCC. Enriched cells underwent single-cell and pooled whole-genome amplification (WGA), quality-controlled by a genomic integrity index and profiled for copy number alterations (CNA) by low-pass whole-genome sequencing. Tumour cells were detected in 100% of LN metastases (range: 37-12,479), with counts strongly correlating with histological tumour area (p < 0.0001). Rare background events in control LNs were excluded by manual CELLSEARCH review. Based on their frequency, a threshold of > 12 cells was defined to classify samples as positive, yielding an assay specificity and sensitivity of 100%. Reproducibility across adjacent sections was high. Despite FFPE preservation, WGA succeeded in 27% of single cells and > 75% of pooled fractions. CNA profiling yielded 50 high-quality tumour genomes, revealing typical HNSCC alterations and marked inter-/intra-patient heterogeneity. This adapted CELLSEARCH workflow enables sensitive detection and genomic profiling of tumour cells from FFPE LN tissue. Demonstrated in HNSCC as a proof of concept, the approach is broadly applicable to epithelial cancers where LN staging is prognostically decisive. © 2026 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
PMID:
42768301
Bibliographic data and abstract were imported from PubMed on 22 Sep 2026.
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