Atypical neurofibromas reveal distinct epigenetic features with proximity to benign peripheral nerve sheath tumor entities

Abstract

Background: Plexiform neurofibromas can transform into atypical neurofibromas (ANF) and then further progress to aggressive malignant peripheral nerve sheath tumors (MPNST). ANF have been described to harbor distinct histological features and frequent loss of CDKN2A/B. However, histological evaluation may be rater-dependent, and detailed knowledge about the molecular mechanisms of malignant transformation is scarce. In general, malignant transformation can be accompanied by significant epigenetic changes, and global DNA methylation profiling is able to differentiate relevant tumor subgroups. Therefore, epigenetic profiling might provide a valuable tool to distinguish and characterize ANF with differing extent of histopathological atypia from neurofibromas and MPNST.

Methods: We investigated 40 tumors histologically diagnosed as ANF and compared their global methylation profile to other peripheral nerve sheath tumors.

Results: Unsupervised class discovery and t-SNE analysis indicated that 36/40 ANF cluster with benign peripheral nerve sheath tumors with clear separation from MPNST. 21 ANF formed a molecularly distinct cluster in proximity to schwannomas. Tumors in this cluster had a frequent heterozygous or homozygous loss of CDKN2A/B and significantly more lymphocyte infiltration than MPNST, schwannomas, and NF. Few ANF clustered closely with neurofibromas, schwannomas, or MPNST, raising the question, whether diagnosis based on histological features alone might pose a risk to both over- and underestimate the aggressiveness of these lesions.

Conclusions: Our data suggest that ANF with varying histological morphology show distinct epigenetic similarities and cluster in proximity to benign peripheral nerve sheath tumor entities. Future investigations should pay special respect to correlating this methylation pattern to clinical outcomes.

Keywords: DNA methylation; atypical neurofibroma; classification; diagnostic marker; peripheral nerve sheath tumors.

Figure 1.

Global DNA methylation analysis of 40 ANF. (A) T-SNE showing the proximity of ANF to benign PNST and suggesting a distinct cluster of ANF. (B) The cumulative distribution function (CDF) of consensus matrices related to DNA methylation data reveals an optimal number of 10 clusters. (C) Heatmap representation of unsupervised consensus hierarchical clustering of DNA methylation profiles. For each case, histological diagnosis, Heidelberg brain tumor, and Sarcoma Classifier score over 0.84, immunohistochemical positivity for H3K27-trimethylation, CDKN2A/B status, clinical syndrome, sex, and age of the patients are indicated as well as array type and tumor material. The dashed line indicates 18 years of age. (D) Tracking plot, visualizing item cluster membership across different k confirms that the ANF cluster (yellow) is stable even at higher k values. Color code for clusters corresponds to C. (E) Cumulative copy number plots show loss of chromosome 9 (arrow) in approximately 25% of ANF. MPNST show a wide range of chromosomal gains and losses. Abbreviations: ANF, atypical neurofibromatous lesion; CDKN2A/B, cyclin-dependent kinase inhibitor 2A/B; FF, fresh frozen; FFPE, formalin-fixed paraffin-embedded; H3K27me3, trimethylation of lysine 27 on histone H3 protein subunit; n.a., not available; t-SNE, t-distributed stochastic neighbor embedding; V12.5, Heidelberg brain tumor classifier version 12.5. V12.2: Sarcoma Classifier version 12.2.

Figure 2.

Histological features of ANF and reclassification according to WHO 2021 criteria. ANF show nuclear atypia, variable loss of neurofibroma architecture (A–H), and low to moderate Ki67 indices (I–L). S100 is retained (M–P). 40 ANF were reclassified according to the WHO 2021 criteria (Q). Cases are sorted by result from hierarchical clustering of methylation data. Initial and reevaluated diagnoses show relevant differences. Histological features relevant for reclassification were cytological atypia, hypercellularity, mitotic count, and loss of NF architecture. CDKN2A/B status is depicted as inferred from methylation data. Abbreviations: CDKN2A/B, cyclin-dependent kinase inhibitor 2A/B; lg MPNST, low-grade MPNST; MPNST, malignant peripheral nerve sheath tumor; n.a., not available; WHO 2021, World Health Organization Classification of CNS Tumors from 2021.

Figure 3.

Immune cell infiltration in peripheral nerve sheath tumors inferred from global methylation data and immunohistochemistry. (A–C) DIMEimmune scores calculated from global methylation data for ANF from the ANF cluster show the highest scores for tumor-infiltrating lymphocytes, CD4⁺ and CD8⁺ lymphocytes compared to MPNST, neurofibromas, and schwannomas. (D) Representative immunohistochemical stainings for CD3. (E) Quantification of the percentage of CD3-immunostaining (ratio of DAB-positive area/total tissue area) confirms the highest immune cell infiltration in ANF. Abbreviations: DAB, 3, 3ʹ-diaminobenzidine; DIME, Differential Methylation Analysis for Immune Cell Estimation; TIL, tumor-infiltrating lymphocytes.

Figure 4.

Clinical data and imaging characteristics. (A) Violin plot showing age at diagnosis. (B) Approximate localization of ANF shows no predilection site. (C–E) Sex, type of symptoms, and occurrence of MPNST for tumors of the ANF cluster. (F–I) Imaging characteristics of ANF in three different patients. (F) Case 2 with an ANF of the superior thoracic aperture (arrowheads). Tumor had a polylobulated shape, a heterogeneous signal intensity in T2w, and corresponding metabolic activity (SUV_max = 6.9). Tumor volume was 107 ml. (G) Case 3 with a cervical ANF (arrowheads). Tumor showed heterogeneous signal intensity in T2w and corresponding metabolic activity (SUV_max = 5.8). Tumor volume was 105 ml. (H) Case 7 with an ANF of the pelvis and growth along the great sciatic foramen. Tumor showed heterogeneous signal intensity in T2w and corresponding metabolic activity (SUV_max = 7.7). Tumor volume was 99 ml. Left column: Fat suppressed T2w MRI at 3T in coronal or transverse orientation; middle column: MRI-based segmentation for volumetric assessment; right column: ¹⁸FDG PET/CT in coronal or transverse orientation. (I) Table of radiologic measurements including SUV_max and volumetry of 15 ANF. Abbreviations: SD, standard deviation; SUV, standardized uptake value.