Protracted haemangioblastic proliferation and differentiation in von Hippel-Lindau disease

Abstract

Von Hippel-Lindau (VHL) disease is caused by germline mutation of the VHL tumour suppressor gene. Patients frequently develop multiple nervous system tumours, denominated haemangioblastomas. Analysis of affected autopsy tissues suggests that tumourigenesis propagates from developmentally arrested, embryonic cells and progresses with consistent architectural, cytological, and molecular sequences similar to haemangioblastic formation and differentiation in the embryo. In this study, we analysed 156 nervous system tumours, 139 of which had been surgically resected from 83 VHL patients. We demonstrate that large tumours consistently contain epithelioid components characteristic of haemangioblastic differentiation in comparison to small tumours that solely display a poorly differentiated, mesenchymal structure. We further show exclusive activation of HIF2alpha in both small mesenchymal tumours and the mesenchymal component of large tumours, whereas activation of HIF1alpha is associated with epithelioid structure. We also show that the MIB1 proliferative index is variably increased in the epithelioid component of large tumours, with extramedullary haematopoiesis foci within the epithelioid component at 100%. These data provide compelling evidence that nervous system tumourigenesis in VHL disease represents a protracted process of haemangioblastic proliferation and differentiation that parallels haemangioblastic formation and differentiation in the embryo.

Figure 1

Two distinct structural presentations of nervous system neoplasia in VHL disease. Tumour tissues may present with mesenchymal (A–E) and epithelioid structures (F–J). All 156 tumours show areas of mesenchymal structure that are composed of small, loosely scattered, individual tumour cells (A) separated by extensive angiogenic vascularisation. (B) Immunohistochemistry for vascular antigen CD34 reveals prominent reactive vascularization, and neoplastic cells are negative for CD34 (vascular antigen CD31 data are not shown). Scattered mesenchymal tumour cells show activation of HIF2α (C), but not HIF1α (D). Immunohistochemistry using MIB1 reveals a low proliferation index with rare, scattered positive cells (E). Ninety-eight of 156 tumours also reveal areas with epithelioid structure (F) that are composed of large tumour cell clusters encompassed by angiogenic vascularisation. (G) Immunohistochemistry for vascular antigen CD34 reveals encompassing reactive vascularization, and neoplastic cells are negative for CD34 (vascular antigen CD31 data are not shown). In epithelioid areas, there is activation of both HIF2α (H) and HIF1α (I). The MIB1 proliferation index is markedly increased, but highly variable in different cell clusters (J). Only in epithelioid tumour areas is there occasional differentiation of haemangioblastic cells into red blood cell precursors forming extramedullary haematopoiesis foci (K, M); all cells within the extramedullary haematopoiesis focus show positive immunoreactivity for the proliferation marker MIB1 on deeper sections (L, N)

Figure 2

Structural and molecular sequences of nervous system tumour progression in VHL disease, demonstrated in a specimen surgically resected from a cervical nerve root. An intraradicular tumourlet precursor with exclusive mesenchymal structure (box 1) has progressed into a haemangioblastic tumour mass (haemangioblastoma). Swollen and distended axons are shown at lower (A, 10× by objective lens) and higher magnifications (B, 40× by objective lens) by immunohistochemistry for neurofilament triplet protein (NFTP). Adjacent tissue sections show intraradicular mesenchymal neoplastic cells and reactive vascularization (C) with low MIB1 index (D, showing a rare MIB1+ cell). The intraradicular mesenchymal neoplastic cells are positive for HIF2α (E), but negative for HIF1α (F) by immunohistochemistry. The tumour tissue reveals progression into an epithelioid structure characterized by clusters of large tumour cells (box 2, G). The MIB1 proliferation index in individual cell clusters is highly variable (H), but increased compared with the mesenchymal tumour component (box 1, D). Epithelioid neoplastic cells show not only activation of HIF2α (I), but also activation of HIF1α (J). Within epithelioid tumour tissue, red blood cell precursors of the extramedullary haematopoiesis foci (box 3, K) show 100% positive MIB1 immunoreactivity (L)

Figure 3

Correlation between nervous system tumour size and histopathological structures provides evidence for structural sequence of tumour progression. Tumours smaller than 8 mm³ show only mesenchymal structure. Tumours greater than 637 mm³ always show additional epithelioid structure. Tumours between 8 and 637 mm³ are either exclusively mesenchymal or mesenchymal and epithelioid. Asterisks indicate tumours with foci of extramedullary haematopoiesis, which are consistently confined to areas with epithelioid differentiation

Figure 4

Structural sequence of haemangiomesenchymal differentiation in the embryo (A) and of tumourigenesis in the nervous system of VHL patients (B). Haemangiomesenchymal cells undergo architectural and cytological changes during differentiation to haemangioblasts. Haemangioblasts then cluster to form vessels, plasma, and blood islands comprised of red blood cell precursors (modelled after Sabin [17,23]) (A). Haemangiomesenchymal, VHL-deficient cells undergo similar structural changes, yet differ in the intense reactive angiogenesis beginning in the early stages of tumourigenesis (B)