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. 2024 Oct 29;12(1):172.
doi: 10.1186/s40478-024-01875-z.

NF1 expression profiling in IDH-wildtype glioblastoma: genomic associations and survival outcomes

Michael Chang  1 , Mohamed Sherief  1 , Maria Ioannou  1 , Viveka Chinnasamy  1 , Lucy Chen  1 , Michael Frost  2 , Michelle Mattson-Hoss  2 , Herb Sarnoff  2 , David O Kamson  1 , Matthias Holdhoff  3 , Debraj Mukherjee  4 , Chetan Bettegowda  4 , Jordina Rincon-Torroella  4 , Victoria Croog  5 , Peng Huang  6 , Fausto J Rodriguez  7 , Calixto-Hope G Lucas  8 , Karisa C Schreck  9   10
Affiliations

NF1 expression profiling in IDH-wildtype glioblastoma: genomic associations and survival outcomes

Michael Chang et al. Acta Neuropathol Commun. .

Abstract

Background: NF1 inactivation is associated with sensitivity to MEK inhibitor targeted therapy in low-grade and some high-grade gliomas. NF1 loss may also be a harbinger of exploitable vulnerabilities in IDH-wildtype glioblastoma (GBM). Accurate and consistent detection of NF1 loss, however, is fraught given the large gene size, challenges with complete coverage and variant calling upon sequencing, and mechanisms of mRNA and protein regulation that result in early degradation in the absence of genomic alterations. Here, we seek to perform a composite analysis for NF1 loss accounting for genomic alterations and protein expression via immunohistochemistry. We also characterize the landscape of NF1 alterations in GBM.

Methods: We assembled a single-institution, retrospective cohort of 542 IDH-wildtype GBM with somatic next generation sequencing to investigate the frequency and nature of detected NF1 alterations. We selected 69 GBMs from which to build a tissue microarray (TMA) of 44 NF1-wildtype and 25 NF1-mutant cases. We performed NF1 immunohistochemistry using two different NF1 antibodies (NFC, Sigma-Aldrich; and iNF-07E, iNFixion Bioscience) and correlated results with clinical, genomic, and other immunohistochemical features.

Results: In our retrospective cohort, we identified 88 IDH-wildtype GBM with NF1 alterations (16%). NF1 alterations were mutually exclusive with EGFR and MDM2 alterations (p-adj < 0.001, 0.05, respectively), but co-occurred with PIK3R1 alterations (Log2(OR) = - 1.6, p-adj = 0.03). Of the 63 scorable sporadic GBMs in the TMA, 14 harbored NF1 inactivating alterations and of those, 12 (86%) demonstrated minimal NF1 immunoreactivity by NFC antibody, compared to 8 (57%) by iNF-07E antibody. Among the 42 scorable NF1-wildtype GBM in the TMA, NF1 immunostaining was minimal in 18 (43%) by NFC antibody compared to 4 (10%) by iNF-07E antibody, potentially reflecting false positives or differential protein regulation. Minimal immunoreactivity by NFC antibody was associated with decreased median overall survival (8.5 vs. 16.4 months, p = 0.011). Cox proportional hazards model correcting for prognostic variables in this subset revealed HR 3.23 (95% CI 1.29-8.06, p = 0.01) associated with decreased NF1 expression by IHC.

Conclusion: NF1 immunostaining may serve as a sensitive surrogate marker of NF1 genomic inactivation and a valuable extension to next-generation sequencing for defining NF1 status. Minimal NF1 immunoreactivity is a poor prognostic marker, even in IDH-wildtype glioblastoma without apparent NF1 genomic alterations, but the underlying molecular mechanism requires further investigation.

Keywords: Biomarker; Glioblastoma; High-grade glioma; NF1; Neurofibromin.

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Conflict of interest statement

CB is a consultant for Depuy-Synthe, Bionaut Labs, Haystack Oncology and Privo Technologies. CB is a co-founder of OrisDx and Belay Diagnostics. iNF-07E monoclonal antibody is owned and supplied by Infixion Bioscience, Inc. (San Diego). KCS has received honoraria from Novartis and institutional research funding from Springworks Therapeutics, Pfizer, Lantern Pharma, and FORE Biotherapeutics. She also sits on a data safety and monitoring board for Advarra.

Figures

Fig. 1
Fig. 1
Genomic and clinical features of NF1 loss in glioblastoma. A Distribution of NF1 alteration types and B ClinVar classification of NF1 alterations detected in JHH GBM cohort (N = 115). C Co-alteration frequencies between NF1 and other selected genes in JHH GBM cohort. Statistical significance was assessed using two-tailed Fisher’s Exact test. D Lollipop plot of NF1 alterations detected in JHH GBM cohort. Box and whisker plots comparing NF1 mRNA expression between NF1 mutant and NF1 wildtype glioblastomas in E TCGA (z-scores, U133 microarray) and F CPTAC (z-scores, log2FPKM) GBM cohorts. Statistical significance was assessed using Welch’s t-test. G Scatter plot comparing NF1 protein abundance ratio (z-scores) and NF1 mRNA expression (z-scores, log2FPKM) in CPTAC GBM cohort. H Kaplan–Meier survival curves displaying overall survival in JHH GBM cohort stratified by NF1 alteration type. VUS = variant of unknown significance, FS = frame shift
Fig. 2
Fig. 2
Oncoprint of 69 glioblastomas included in TMA cohort displaying genetic alterations in selected genes
Fig. 3
Fig. 3
NF1 immunohistochemistry in patient-derived glioblastoma samples. Representative IHC shows loss of NF1 immunostaining in NF1 −/− tumor (patient with neurofibromatosis type 1) with A NFC antibody and C iNF-07E antibody. Representative IHC shows retained NF1 immunostaining in NF1 +/+ tumor with B NFC and D iNF-07E. E Plot of NF1 genomic status and corresponding NF1 IHC scores using two NF1 antibodies. F, G I Bar plots demonstrating no difference in expression of candidate surrogate biomarkers selected a priori—phospho-ERK, phospho-S6, p16, podoplanin (PDPN), ATRX, Ki-67, p53—between tumors with retained and lost NF1 immunostaining
Fig. 4
Fig. 4
Kaplan–Meier curves displaying overall survival in TMA cohort by NF1 immunohistochemistry. Curves are stratified by A NFC immunostaining, B iNF-07E immunostaining among entire TMA cohort
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