Extracellular matrix composition defines an ultra-high-risk group of neuroblastoma within the high-risk patient cohort

Abstract

Background: Although survival for neuroblastoma patients has dramatically improved in recent years, a substantial number of children in the high-risk subgroup still die.

Methods: We aimed to define a subgroup of ultra-high-risk patients from within the high-risk cohort. We used advanced morphometric approaches to quantify and characterise blood vessels, reticulin fibre networks, collagen type I bundles, elastic fibres and glycosaminoglycans in 102 high-risk neuroblastomas specimens. The Kaplan-Meier method was used to correlate the analysed elements with survival.

Results: The organisation of blood vessels and reticulin fibres in neuroblastic tumours defined an ultra-high-risk patient subgroup with 5-year survival rate <15%. Specifically, tumours with irregularly shaped blood vessels, large sinusoid-like vessels, smaller and tortuous venules and arterioles and with large areas of reticulin fibres forming large, crosslinking, branching and haphazardly arranged networks were linked to the ultra-high-risk phenotype.

Conclusions: We demonstrate that quantification of tumour stroma components by morphometric techniques has the potential to improve risk stratification of neuroblastoma patients.

Figure 1

Examples and schematic representations showing how blood vessels and Ret Fs behave in the ECM of non-high-risk, high-risk and ultra-high-risk patients. Ret Fs and blood-vessel binarized images are shown in small squares. (A) Non-high-risk sample with predominant capillaries and scant presence and irregularity of sinusoids. (B) High-risk sample with predominantly large sinusoids. (C) Ultra-high-risk sample with irregularly shaped blood vessels, small and abundant sinusoids and small and very irregular venules and arterioles (arterioles in this specific sample). (D) Non-high-risk sample with reduced %SA, low crosslinking and smooth outlines. (E) High-risk sample with curvy, crosslinked and non-ordered Ret Fs network. (F) Ultra-high-risk sample with much more haphazardly arranged and cross-linked Ret Fs networks, forming a less porous and stiffer ECM. Data corresponding to non-high-risk patients are not shown.

Figure 2

Kaplan–Meier graphs showing the different accumulated EFS or OS depending on different variables. In all cases, the straight line corresponds to the group under the data used for dichotomization, and the discontinuous line corresponds to the group over the data used of dichotomization. These data are shown in Table 3. P-values and survival rates are shown. (A–D) Blood vessels morphometric variables. (A) Shape factor of total blood vascularisation. (B) Area of sinusoids. (C) Area of venules and arterioles. (D) Roundness of venules and arterioles. (E–J) Ret Fs morphometric variables. (E) SA. (F) Width. (G) Roundness. (H) Perimeter ratio. (I) Fractal dimension. (J) Branching. (K) Patients grouped depending of the amount of morphometric variables related to ultra-high-risk of progression. (L) Definition of ultra-high-risk patients according to clinical and biological factors (definition currently under debate).