Genetic evolution of keratinocytes to cutaneous squamous cell carcinoma

Authors

Bishal Tandukar

Delahny Deivendran

Limin Chen

Noel Cruz-Pacheco

Harsh Sharma

Albert Xu

Aravind K. Bandari

Daniel B. Chen

Christopher George

Annika Marty

Raymond J. Cho

Jeffrey Cheng

Drew Saylor

Pedram Gerami

Sarah T. Arron

Boris C. Bastian

A. Hunter Shain

Doi

10.1101/2024.07.23.604673

PMID: 39091884 · DOI: 10.1101/2024.07.23.604673 · Journal: bioRxiv (2024)

TL;DR

This study performed multi-omic profiling (single-cell mutational analysis, targeted DNA sequencing, and spatial transcriptomics) of normal epidermal keratinocytes, precancerous actinic keratoses, and cutaneous squamous cell carcinomas (cSCC) from 15 donors and 16 cSCC patients. The authors found that most normal keratinocytes have remarkably low mutation burdens despite decades of sun exposure, but keratinocytes harboring pathogenic TP53 or NOTCH1 mutations display substantially elevated mutation rates. Phylogenetic reconstruction of matched actinic keratosis-cSCC pairs revealed a stepwise model: TERT promoter and CDKN2A mutations emerge in actinic keratoses, while ARID2 loss-of-function and MAPK-pathway activation mark the transition to invasive carcinoma. Unexpectedly, actinic keratoses were frequently clonally unrelated to their adjacent cSCC.

Cohort & data

  • Normal skin cells: 137 keratinocytes, 131 melanocytes, and 23 fibroblasts from 22 biopsies across 15 donors (ages 35-95), profiled at single-cell resolution via clonal expansion and whole-exome sequencing (~95X coverage) (PMID:39091884).
  • cSCC-AK cohort: 16 archival tissue cases, each with cSCC immediately adjacent to actinic keratosis (AK), sequenced with a cancer gene panel at ~380X depth (PMID:39091884).
  • Spatial transcriptomics: 10X Visium performed on 5 cSCC-AK cases with resolved clonal relationships (PMID:39091884).
  • Validation data: Reanalysis of 160-gene panel data from Kim et al. (2022) covering cSCC and adjacent skin.
  • Dataset: normal_skin_melanocytes_2024; data deposited in dbGaP (phs001979.v1.p1, phs003683.v2.p1, phs003282.v2.p1) and cBioPortal.

Key findings

  • Keratinocyte mutation burdens are low: Median 1.14 mut/Mb for keratinocytes vs. 3.91 mut/Mb for melanocytes and 1.92 mut/Mb for fibroblasts from the same biopsies, despite shared UV exposure in the basal epidermis (PMID:39091884).
  • Pathogenic mutations break UV repair: Every keratinocyte with >3.5 mut/Mb harbored at least one pathogenic mutation. Missense TP53 mutations (dominant-negative) produced the highest mutation burdens (up to 49.71 mut/Mb) (PMID:39091884).
  • Mutational signatures differ by cell type: Keratinocytes showed higher proportions of clock-like signatures (SBS1, SBS5) relative to UV signature SBS7a, whereas melanocytes and fibroblasts were dominated by SBS7a (PMID:39091884).
  • Clonal patches in normal skin: Median keratinocyte clone size was 6.21 mm^2. Clones with pathogenic mutations were not larger than those without, consistent with prior findings by Martincorena et al. (PMID:39091884).
  • AK-to-cSCC progression model: In 5 of 16 matched cases, cSCC clearly evolved from the adjacent AK. TERT promoter and CDKN2A mutations emerged in AK (trunk of phylogenetic trees), while ARID2 loss-of-function and RTK-RAS-MAPK activation marked the cSCC transition (PMID:39091884).
  • Frequent clonal independence: In 6/16 cases, AK and adjacent cSCC shared no somatic alterations, indicating they arose as independent clones (“collisions”) (PMID:39091884).
  • Immune checkpoint at invasive front: Spatial transcriptomics revealed high expression of checkpoint ligands (PVR, NECTIN2, CD274, CD80, CD86) on tumor cells and checkpoint receptors (CTLA4, TIGIT, PDCD1) on lymphocytes at the invasive front of cSCC (PMID:39091884).

Genes & alterations

  • TP53: Missense mutations (dominant-negative) in normal keratinocytes associated with hypermutation; present on trunks of AK-cSCC phylogenies; less common than NOTCH1 in normal skin but more common in cSCC (PMID:39091884).
  • NOTCH1: Loss-of-function mutations frequent in normal keratinocyte clones; typically early/trunk events in cSCC evolution; induce stem/progenitor cell state (PMID:39091884).
  • NOTCH2: Loss-of-function mutations observed in AK; secondary hits to Notch pathway on phylogenetic branches (PMID:39091884).
  • CDKN2A: Loss-of-function mutations rare in normal skin but recurrent in AK; 9p allelic imbalance affecting CDKN2A observed (PMID:39091884).
  • TERT: Promoter gain-of-function mutations emerging in AK (trunk events), upregulating telomerase (PMID:39091884).
  • ARID2: Loss-of-function mutations enriched specifically at the AK-to-cSCC transition; SWI/SNF chromatin remodeling complex disruption, potentially linked to immune evasion (PMID:39091884).
  • CBL: Loss-of-function mutation in example cSCC case, associated with increased phospho-MAPK signaling (PMID:39091884).
  • FAT1: Hippo pathway-activating mutations observed in individual normal keratinocytes, less common than TP53 and NOTCH1 (PMID:39091884).

Clinical implications

  • The stepwise model (TP53/NOTCH1 –> CDKN2A/TERT –> ARID2/MAPK) provides a framework for understanding which actinic keratoses are at highest risk of progressing to cSCC, potentially informing surveillance and early intervention strategies (PMID:39091884).
  • High expression of immune checkpoint molecules (CD274/PD-L1, CTLA4, PDCD1/PD-1) at the invasive front of cSCC supports the rationale for immune checkpoint inhibitor therapy in advanced cSCC (PMID:39091884).
  • Clonal independence of adjacent AK and cSCC lesions means clinical proximity should not be used to assume shared clonal origin; resection margins based on histological boundaries may underestimate tumor extent (PMID:39091884).

Limitations & open questions

  • Clonal expansion bias: Single-cell genotyping required ex vivo clonal expansion, which may bias toward cells that grow well in culture, potentially underestimating mutation burdens (single-cell averages were ~3-fold lower than bulk-cell estimates) (PMID:39091884).
  • Small matched cohort: Only 5 of 16 AK-cSCC pairs showed clear clonal evolution, limiting statistical power for ordering driver events (PMID:39091884).
  • Preprint status: Published on bioRxiv; not yet peer-reviewed at time of extraction.
  • No functional validation: The proposed mutator phenotype of TP53/NOTCH1 mutations is inferred from correlative data; causal experiments were not performed.
  • Limited gene panel for AK-cSCC sequencing: Cancer gene panel at 380X depth prioritized depth over breadth, so some driver events (e.g., non-coding or novel genes) may have been missed.
  • Open question: Whether SWI/SNF complex mutations (ARID2) at the AK-to-cSCC transition directly drive immune evasion, as suggested by emerging evidence from other tumor types.

Citations from this paper used in the wiki

  • “most keratinocytes have remarkably low mutation burdens, despite decades of sun exposure, however keratinocytes with TP53 or NOTCH1 mutations had substantially higher mutation burdens” (PMID:39091884, Abstract).
  • “TERT promoter and CDKN2A mutations emerging in actinic keratoses, whereas additional mutations inactivating ARID2 and activating the MAPK-pathway delineated the transition to squamous cell carcinomas” (PMID:39091884, Abstract).
  • “actinic keratoses were often not related to their neighboring squamous cell carcinoma, indicating that collisions of unrelated neoplasms are common in the skin” (PMID:39091884, Abstract).
  • “We observed higher expression of immune checkpoint ligands (PVR, NECTIN2, CD274, CD80, and CD86) in the tumor cells at the invasive front of the squamous cell carcinomas” (PMID:39091884, Results).
  • “The median mutation burden of keratinocytes was 1.14 mutations per megabase (mut/Mb), which was lower than the mutation burdens of melanocytes (3.91 mut/Mb) and fibroblasts (1.92 mut/Mb)” (PMID:39091884, Results).

This page was processed by crosslinker on 2026-05-04.