Tumour budding-based grading as independent prognostic biomarker in HPV-positive and HPV-negative head and neck cancer

Abstract

Background: The prognostic significance of tumour budding (TB) and minimal cell nest size (MCNS) was shown in human papillomavirus (HPV)-negative head and neck squamous cell carcinomas (HNSCC). However, the optimisation of cutpoints, the prognostic impact in HPV-positive HNSCC, and the comparison with other histopathological grading systems are insufficiently investigated.

Methods: TB and MCNS were analysed digitally in 1 and 10 high-power fields (HPF) of 331 HPV-positive and HPV-negative cases from TCGA. Optimising the cutpoints a new cellular dissociation grading (CDG) system was defined and compared to the WHO grading and the Brandwein-Gensler (BG) risk model.

Results: The two-tiered CDG system based solely on TB yielded optimal prognostic stratification with shortened overall survival for CDG-high cases. Optimal cut-offs were two buds (1 HPF) and six buds (10 HPF), respectively. Analysing MCNS did not add prognostic significance to quantifying TB. CDG was a significant prognostic marker in HPV-negative and HPV-positive tumours and prognostically superior to the WHO and BG systems. High CDG was associated with clinically occult lymph-node metastases.

Conclusions: The most comprehensive study of TB in HNSCC so far confirmed its prognostic impact in HPV-negative tumours and for the first time in HPV-positive tumours. Further studies are warranted to evaluate its applicability for therapy guidance in HNSCC.

Fig. 1. Evaluation of tumour budding (TB) in H&E-stained tissue sections of HNSCC.

While tumour budding (TB) was absent in the TCGA cases a and b, strong TB was observed in the TCGA cases c and d. Exemplary budding foci are highlighted by arrows.

Fig. 2. Optimisation of the cellular dissociation grading (CDG) system.

Two four-tiered grading systems were investigated, both based on tumour classification as CDG1, CDG2 and CDG3 and an additional split of the CDG2 class. a, b The set of CDG2 tumours was split based on the number of budding foci. Hazard ratios compared to CDG1 tumours increased and reached 1.5, 2.1, and 2.5 for CDG2/weak TB, CDG2/moderate TB and CDG3 tumours. c, d The set of CDG2 tumours was split based on the presence or absence of single-cell invasion (SCI). Counterintuitively, CDG2 tumours without SCI showed a (non-significant) trend to unfavourable prognosis compared to CDG2 tumours with SCI. e Relation between the number of budding foci, the classification of TB, and the CDG systems.

Fig. 3. Association of overall survival (OS) with cellular dissociation grading (CDG).

Performance of the 3-tiered CDG system and of the 2-tiered CDG system in the study cohort (a, b), in the subcohort of HPV-negative tumours (c, d) and in the subcohort of HPV-positive tumours (e, f).

Fig. 4. Subgroup analysis of the prognosticity of cellular dissociation grading (CDG).

Comparison of CDG high with CGD low tumours with respect to PFI (a) and OS (b). N, number of patients, E, number of events, HR, hazard ratio, CI, 95% confidence interval.

Fig. 5. Comparison of the 1-HPF and the 10-HPF method for the evaluation of TB.

Evaluation of TB in 10 high-power fields (HPF) and in a single HPF. Linear regression revealed that the detected number of budding foci differed by a factor of about three.

Fig. 6. Analysis of clinically nodal-negative (cN0) patients.

A Higher numbers of budding foci in pathologically nodal-positive (pN+) compared to pN0 patients. B Increasing percentages of pN+ patients with increasing TB.