Temporal evolution and inter-patient heterogeneity in primary and recurrent head and neck squamous cell carcinoma

Abstract

Background: Head and neck squamous cell carcinomas (HNSCCs) are heterogeneous in terms of origin and aetiology. In addition, there is uncertainty about the genetic evolution from initial diagnosis to recurrence after primary treatments and further disease progression following systemic treatment. Changes in the genetic profile have implications on the selection of appropriate treatments for patients, especially in the era of targeted therapies and immunotherapies.

Methods: We analysed a cohort of nine HNSCC patients with metachronous recurrence. All patients had paired primary and recurrent samples suitable for whole-exome sequencing, while transcriptomic data from seven patients could be analysed (multiple recurrent samples collected at different time points were available for three patients).

Results: At the genomic level, the recurrences shared a fraction of the somatic single nucleotide variants (SNVs) with the index primary tumours, but they also acquired many additional mutations, while losing only a few others. A similar behaviour was also observed when examining the changes of mutational signatures between primary and recurrent samples. Overall, recurrences appeared thus more genetically diverse than the respective primary tumours. The transcriptomic analysis showed that recurrent samples had lower immune cell presence, which was also confirmed by the multiplex immunofluorescence (IF) histology assays performed on the PhenoCycler platform. Several genes related to immune response were significantly downregulated compared to the primary samples.

Conclusions: Our results underline the importance of analysing multiple samples per patient to obtain a more complete picture of the patient's tumour and advocate a re-biopsy in the event of recurrence and treatment failure, in order to select the most appropriate therapeutic strategy.

Fig. 1. Single nucleotide variants (SNVs) detected in the tumour samples.

a Overview of the number of detected SNVs in primary tumours, in recurrences or in both, for each patient. b Same overview is shown for non-synonymous SNVs. *Patients P01 and P07 were alive and well at the time of analysis.

Fig. 2. Mutational signatures in primary and recurrent samples.

“No change” indicates that the same signatures were detected in the primary tumours and in the recurrences. “Gain in R sample(s)” indicates that the recurrence(s) acquired additional signatures, not present in the primary tumours. Associations to known mechanisms are reported next to the Signature legend. MMR = mismatch repair, 5meC = 5-Methylcytosine. *Patients P01 and P07 were alive and well at the time of analysis.

Fig. 3. Results from differential expression analysis.

a Number of significantly up- and downregulated genes in recurrent samples, compared to primary tumours. b Heatmap of the RPKM expression, shown as gene-wise Z-scores, of the genes with the lowest p-values (< 0.005) in all samples of the cohort. Values of -1 and lower are shown with the same light yellow shade, values of 1 and larger with the same dark green shade. Side bar indicates primary tumours (blue) and recurrences (orange). c Gene ontology terms significantly over-represented in the list of downregulated genes. FDR = false discovery rate. * Patients P01 and P07 were alive and well at the time of analysis.

Fig. 4. Immunoprofile of primary and recurrent samples.

a Percentages of immune cells present in the primary and recurrent samples, obtained with quanTIseq. b Phenocycler IF staining of samples T1 and R1 from patient P08 (see Figs. S5–S7 for images of the remaining patients). Whole-slide imaging at single-cell resolution of B cells (CD20) and T cells (CD3, CD8, FoxP3). Insets show representative 20x zoom-in images. Counterstaining: DAPI; scale bar: 250 µm. *Patients P01 and P07 were alive and well at the time of analysis. **Biopsy R2 was taken while patient was receiving treatment and responding to it.