Background The leading cause of mortality for patients with the neurofibromatosis type 1 (NF1) cancer predisposition syndrome is the development of malignant peripheral nerve sheath tumor (MPNST), an aggressive soft tissue sarcoma. In the setting of NF1, this cancer type frequently arises from within its common and benign precursor, plexiform neurofibroma (PN). Transformation from PN to MPNST is challenging to diagnose due to difficulties in distinguishing cross-sectional imaging results and intralesional heterogeneity resulting in biopsy sampling errors. Methods and findings This multi-institutional study from the National Cancer Institute and Washington University in St. Louis used fragment size analysis and ultra-low-pass whole genome sequencing (ULP-WGS) of plasma cell-free DNA (cfDNA) to distinguish between MPNST and PN in patients with NF1. Following in silico enrichment for short cfDNA fragments and copy number analysis to estimate the fraction of plasma cfDNA originating from tumor (tumor fraction), we developed a noninvasive classifier that differentiates MPNST from PN with 86% pretreatment accuracy (91% specificity, 75% sensitivity) and 89% accuracy on serial analysis (91% specificity, 83% sensitivity). Healthy controls without NF1 (participants = 16, plasma samples = 16), PN (participants = 23, plasma samples = 23), and MPNST (participants = 14, plasma samples = 46) cohorts showed significant differences in tumor fraction in plasma (P = 0.001) as well as cfDNA fragment length (P < 0.001) with MPNST samples harboring shorter fragments and being enriched for tumor-derived cfDNA relative to PN and healthy controls. No other covariates were significant on multivariate logistic regression. Mutational analysis demonstrated focal NF1 copy number loss in PN and MPNST patient plasma but not in healthy controls. Greater genomic instability including alterations associated with malignant transformation (focal copy number gains in chromosome arms 1q, 7p, 8q, 9q, and 17q; focal copy number losses in SUZ12, SMARCA2, CDKN2A/B, and chromosome arms 6p and 9p) was more prominently observed in MPNST plasma. Furthermore, the sum of longest tumor diameters (SLD) visualized by cross-sectional imaging correlated significantly with paired tumor fractions in plasma from MPNST patients (r = 0.39, P = 0.024). On serial analysis, tumor fraction levels in plasma dynamically correlated with treatment response to therapy and minimal residual disease (MRD) detection before relapse. Study limitations include a modest MPNST sample size despite accrual from 2 major referral centers for this rare malignancy, and lack of uniform treatment and imaging protocols representing a real-world cohort. Conclusions Tumor fraction levels derived from cfDNA fragment size and copy number alteration analysis of plasma cfDNA using ULP-WGS significantly correlated with MPNST tumor burden, accurately distinguished MPNST from its benign PN precursor, and dynamically correlated with treatment response. In the future, our findings could form the basis for improved early cancer detection and monitoring in high-risk cancer-predisposed populations.

One of the most common malignancies affecting adults with Neurofibromatosis type 1 (NF1) is the malignant peripheral nerve sheath tumor (MPNST), an aggressive and often fatal sarcoma that commonly arises from benign plexiform neurofibromas. Despite advances in our understanding of MPNST pathobiology, there are few effective therapeutic options, and no investigational agents have proven successful in clinical trials. To further understand the genomic heterogeneity of MPNST, and to generate a preclinical platform that encompasses this heterogeneity, we developed a collection of NF1-MPNST patient-derived xenografts (PDX). These PDX were compared with the primary tumors from which they were derived using copy number analysis, whole exome sequencing, and RNA sequencing. We identified chromosome 8 gain as a recurrent genomic event in MPNST and validated its occurrence by FISH in the PDX and parental tumors, in a validation cohort, and by single-cell sequencing in the PDX. Finally, we show that chromosome 8 gain is associated with inferior overall survival in soft-tissue sarcomas. These data suggest that chromosome 8 gain is a critical event in MPNST pathogenesis and may account for the aggressive nature and poor outcomes in this sarcoma subtype.

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One of the most common malignancies affecting adults with Neurofibromatosis type 1 (NF1) is the malignant peripheral nerve sheath tumor (MPNST), an aggressive and often fatal sarcoma which commonly arises from benign plexiform neurofibromas. Despite advances in our understanding of MPNST pathobiology, there are few effective therapeutic options, and no investigational agents have proven success in clinical trials. To further understand the genomic heterogeneity of MPNST, and to generate a preclinical platform that encompasses this heterogeneity, we developed a collection of NF1-MPNST patient derived xenografts (PDX). These PDX were compared to the primary tumors from which they were derived using copy number analysis, whole exome and RNA sequencing. We identified chromosome 8 gain as a recurrent genomic event in MPNST and validated its occurrence by FISH in the PDX and parental tumors, in a validation cohort, and by single cell sequencing in the PDX. Finally, we show that chromosome 8 gain is associated with inferior overall survival in soft tissue sarcomas. Taken together, these data suggest that chromosome 8 gain is a critical event in MPNST pathogenesis, and may account for the aggressive nature and poor outcomes in this sarcoma subtype.

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BackgroundThe leading cause of mortality for patients with the Neurofibromatosis type 1 (NF1) cancer predisposition syndrome is development of Malignant Peripheral Nerve Sheath Tumor (MPNST), an aggressive soft tissue sarcoma. In the setting of NF1, this cancer type frequently arises from within its common and benign precursor, plexiform neurofibroma (PN). Transformation from the benign PN to MPNST is challenging to diagnose due to difficulties in distinguishing cross-sectional imaging results and intralesional heterogeneity resulting in biopsy sampling errors.Methods and FindingsThis multi-institutional study used ultra-low-pass whole genome sequencing (ULP-WGS) of plasma cell-free DNA (cfDNA) to distinguish between MPNST and PN in patients with NF1. Using copy number to estimate the fraction of plasma cfDNA originating from tumor (tumor fraction), we developed a noninvasive classifier which differentiates MPNST from PN with 91% specificity. Healthy controls without NF1 (subjects = 14, plasma samples= 14), PN (subjects = 45, plasma samples = 45), and MPNST (subjects = 14, plasma samples = 48) cohorts showed significant differences in tumor fraction in plasma (P = 0.006) as well as cfDNA fragment length (P < 0.001) with MPNST samples harboring shorter fragments and being enriched for tumor-derived cfDNA relative to PN and healthy controls. Mutational analysis demonstrated focal NF1 copy number loss in PN and MPNST patient plasma but not in healthy controls. Genomic alterations associated with malignant transformation (focal copy number gains in chromosome 8 and copy number losses in SUZ12, SMARCA2, CDKN2A/B, and chromosome arm 9p) were more prominently observed in MPNST plasma. Furthermore, the sum of longest tumor diameters (SLD) visualized by cross-sectional imaging correlated significantly with paired tumor fractions in plasma from MPNST patients (ρ = 0.50, P = 0.0007). On case series analysis, tumor fraction levels in plasma correlated with treatment response to therapy and minimal residual disease (MRD) detection before relapse.ConclusionsTumor fraction levels derived from copy number alteration analysis of plasma cfDNA using ULP-WGS significantly correlated with MPNST tumor burden, distinguished MPNST from its benign PN precursor, and correlated with treatment response. In the future, our findings could form the basis for improved early cancer detection and monitoring in high-risk cancer-predisposed populations.

Background: Malignant peripheral nerve sheath tumors (MPNST) are aggressive soft tissue sarcomas that, in the setting of neurofibromatosis type 1 (NF1), arise within pre-malignant atypical neurofibroma (AN) and benign plexiform neurofibroma (PN). Early surgical resection improves prognosis, however, early detection by imaging and tissue biopsies is challenging due to tissue heterogeneity. In this multi-institutional study we analyze fragmentomic profiles of plasma cell free DNA (cfDNA) to non-invasively distinguish between NF1 associated PN, AN and MPNST. Accurate classification would inform clinical care: standards of care for PN is observation, AN is narrow-margin resection, and MPNST is wide-margin resection. Methods: We performed whole genome sequencing of plasma cfDNA samples from healthy controls (n = 21), patients with PN (n = 113), AN (n = 39) and MPNST (n = 71). cfDNA fragment profiles were analyzed using two complementary approaches. First, we used unsupervised non-negative matrix factorization (NMF) to obtain global fragment length signatures to infer tumor fragment length distributions. The optimal cutpoint was determined after receiver operating characteristic analysis by Youden’s index in one-versus-one (OVO) disease state comparisons. Additionally, we implemented a bin-wise fragmentomic analysis based on DELFI, training a classifier on the ratios of short (100-150bp) and long (151-220bp) fragments in 5 megabase regions across the genome and arm-level features. Results: NMF accurately distinguished disease states on OVO comparisons: MPNST v AN (acc 0.71), MPNST v PN (acc 0.75), MPNST v healthy (acc 0.84), AN v PN (acc 0.70), AN v healthy (acc 0.80) and PN v healthy (acc 0.87). Accuracies were moderately improved in nearly all conditions with bin-wise fragmentomics: MPNST v AN (acc 0.62), MPNST v PN (acc 0.83), MPNST vs healthy (acc 0.86), AN v PN (acc 0.87), AN vs healthy (acc 0.72) and PN vs healthy (acc 0.88). Strikingly, the two AN with the DELFI scores most closely resembling MPNST were separately identified by independent clinical care teams to have very high-risk features and recent history warranting short-interval follow up. Conclusions: This study demonstrates that the spectrum of benign, pre-malignant and malignant peripheral nerve sheath tumors have distinct, disease state specific fragmentomic signatures. Fragmentomics alone outperformed our previously published copy number based cfDNA classifier in all conditions, most notably in low mutational burden healthy, PN and AN states. Finally, preliminary clinical vignettes suggest that this approach may be able to inform surveillance intervals by identifying higher risk premalignant lesions. Together, this work has the potential to enable earlier detection of clinically actionable AN and early-stage MPNST, thereby improving survival outcomes. Citation Format: Alex C. Pan, Russell Taylor Sundby, Jeffrey J. Szymanski, Paul A. Jones, Peter K. Harris, Aadel A. Chaudhuri, Angela C. Hirbe, Jack F. Shern. Cell-free DNA fragmentomics distinguish between benign, pre-malignant and malignant peripheral nerve sheath tumors in neurofibromatosis type 1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 997.

INTRODUCTION: Metastasis is a complex aspect of malignancy that is constantly being studied and monitored in advanced cases. In most cases, different types of cancers can be predictable in how they metastasize, and this can affect management and treatment.