Recommendations for the collection and annotation of biosamples for analysis of biomarkers in neurofibromatosis and schwannomatosis clinical trials.

INTRODUCTION: Neurofibromatosis 1 and schwannomatosis are characterized by potential lifelong morbidity and life-threatening complications. To date, however, diagnostic and predictive biomarkers are an unmet need in this patient population. The inclusion of biomarker discovery correlatives in neurofibromatosis 1/schwannomatosis clinical trials enables study of low-incidence disease. The implementation of a common data model would further enhance biomarker discovery by enabling effective concatenation of data from multiple studies. METHODS: The Response Evaluation in Neurofibromatosis and Schwannomatosis biomarker working group reviewed published data on emerging trends in neurofibromatosis 1 and schwannomatosis biomarker research and developed recommendations in a series of consensus meetings. RESULTS: Liquid biopsy has emerged as a promising assay for neurofibromatosis 1/schwannomatosis biomarker discovery and validation. In addition, we review recommendations for a range of biomarkers in clinical trials, neurofibromatosis 1/schwannomatosis-specific data annotations, and common data models for data integration. CONCLUSION: These Response Evaluation in Neurofibromatosis and Schwannomatosis consensus guidelines are intended to provide best practices for the inclusion of biomarker studies in neurofibromatosis 1/schwannomatosis clinical trials, data, and sample annotation and to lay a framework for data harmonization and concatenation between trials.

Liquid biopsies in pediatric oncology: opportunities and obstacles.

PURPOSE OF REVIEW: Liquid biopsies have emerged as a noninvasive alternative to tissue biopsy with potential applications during all stages of pediatric oncology care. The purpose of this review is to provide a survey of pediatric cell-free DNA (cfDNA) studies, illustrate their potential applications in pediatric oncology, and to discuss technological challenges and approaches to overcome these hurdles. RECENT FINDINGS: Recent literature has demonstrated liquid biopsies' ability to inform treatment selection at diagnosis, monitor clonal evolution during treatment, sensitively detect minimum residual disease following local control, and provide sensitive posttherapy surveillance. Advantages include reduced procedural anesthesia, molecular profiling unbiased by tissue heterogeneity, and ability to track clonal evolution. Challenges to wider implementation in pediatric oncology, however, include blood volume restrictions and relatively low mutational burden in childhood cancers. Multiomic approaches address challenges presented by low-mutational burden, and novel bioinformatic analyses allow a single assay to yield increasing amounts of information, reducing blood volume requirements. SUMMARY: Liquid biopsies hold tremendous promise in pediatric oncology, enabling noninvasive serial surveillance with adaptive care. Already integrated into adult care, recent advances in technologies and bioinformatics have improved applicability to the pediatric cancer landscape.

Cell-free DNA ultra-low-pass whole genome sequencing to distinguish malignant peripheral nerve sheath tumor (MPNST) from its benign precursor lesion: A cross-sectional study.

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.

Urine cell-free DNA multi-omics to detect MRD and predict survival in bladder cancer patients.

Circulating tumor DNA (ctDNA) sensitivity remains subpar for molecular residual disease (MRD) detection in bladder cancer patients. To remedy this problem, we focused on the biofluid most proximal to the disease, urine, and analyzed urine tumor DNA in 74 localized bladder cancer patients. We integrated ultra-low-pass whole genome sequencing (ULP-WGS) with urine cancer personalized profiling by deep sequencing (uCAPP-Seq) to achieve sensitive MRD detection and predict overall survival. Variant allele frequency, inferred tumor mutational burden, and copy number-derived tumor fraction levels in urine cell-free DNA (cfDNA) significantly predicted pathologic complete response status, far better than plasma ctDNA was able to. A random forest model incorporating these urine cfDNA-derived factors with leave-one-out cross-validation was 87% sensitive for predicting residual disease in reference to gold-standard surgical pathology. Both progression-free survival (HR = 3.00, p = 0.01) and overall survival (HR = 4.81, p = 0.009) were dramatically worse by Kaplan-Meier analysis for patients predicted by the model to have MRD, which was corroborated by Cox regression analysis. Additional survival analyses performed on muscle-invasive, neoadjuvant chemotherapy, and held-out validation subgroups corroborated these findings. In summary, we profiled urine samples from 74 patients with localized bladder cancer and used urine cfDNA multi-omics to detect MRD sensitively and predict survival accurately.

Phase I trial of Ganitumab plus Dasatinib to Cotarget the Insulin-Like Growth Factor 1 Receptor and Src Family Kinase YES in Rhabdomyosarcoma.

PURPOSE: Antibodies against insulin-like growth factor (IGF) type 1 receptor have shown meaningful but transient tumor responses in patients with rhabdomyosarcoma (RMS). The SRC family member YES has been shown to mediate IGF type 1 receptor (IGF-1R) antibody acquired resistance, and cotargeting IGF-1R and YES resulted in sustained responses in murine RMS models. We conducted a phase I trial of the anti-IGF-1R antibody ganitumab combined with dasatinib, a multi-kinase inhibitor targeting YES, in patients with RMS (NCT03041701). PATIENTS AND METHODS: Patients with relapsed/refractory alveolar or embryonal RMS and measurable disease were eligible. All patients received ganitumab 18 mg/kg intravenously every 2 weeks. Dasatinib dose was 60 mg/m2/dose (max 100 mg) oral once daily [dose level (DL)1] or 60 mg/m2/dose (max 70 mg) twice daily (DL2). A 3+3 dose escalation design was used, and maximum tolerated dose (MTD) was determined on the basis of cycle 1 dose-limiting toxicities (DLT). RESULTS: Thirteen eligible patients, median age 18 years (range 8-29) enrolled. Median number of prior systemic therapies was 3; all had received prior radiation. Of 11 toxicity-evaluable patients, 1/6 had a DLT at DL1 (diarrhea) and 2/5 had a DLT at DL2 (pneumonitis, hematuria) confirming DL1 as MTD. Of nine response-evaluable patients, one had a confirmed partial response for four cycles, and one had stable disease for six cycles. Genomic studies from cell-free DNA correlated with disease response. CONCLUSIONS: The combination of dasatinib 60 mg/m2/dose daily and ganitumab 18 mg/kg every 2 weeks was safe and tolerable. This combination had a disease control rate of 22% at 5 months.

Estimated Prevalence, Tumor Spectrum, and Neurofibromatosis Type 1-Like Phenotype of CDKN2A-Related Melanoma-Astrocytoma Syndrome.

Importance: Knowledge about the prevalence and tumor types of CDKN2A-related melanoma-astrocytoma syndrome (MAS) is limited and could improve disease recognition. Objective: To estimate the prevalence and describe the tumor types of MAS. Design, Setting, and Participants: This retrospective cohort study analyzed all available MAS cases from medical centers in the US (2 sites) and Europe (2 sites) and from biomedical population genomic databases (UK Biobank [United Kingdom], Geisinger MyCode [US]) between January 1, 1976, and December 31, 2020. Patients with MAS with CDKN2A germline pathogenic variants and 1 or more neural tumors were included. Data were analyzed from June 1, 2022, to January 31, 2023. Main Outcomes and Measures: Disease prevalence and tumor frequency. Results: Prevalence of MAS ranged from 1 in 170 503 (n = 1 case; 95% CI, 1:30 098-1:965 887) in Geisinger MyCode (n = 170 503; mean [SD] age, 58.9 [19.1] years; 60.6% women; 96.2% White) to 1 in 39 149 (n = 12 cases; 95% CI, 1:22 396-1:68 434) in UK Biobank (n = 469 789; mean [SD] age, 70.0 [8.0] years; 54.2% women; 94.8% White). Among UK Biobank patients with MAS (n = 12) identified using an unbiased genomic ascertainment approach, brain neoplasms (4 of 12, 33%; 1 glioblastoma, 1 gliosarcoma, 1 astrocytoma, 1 unspecified type) and schwannomas (3 of 12, 25%) were the most common malignant and benign neural tumors, while cutaneous melanoma (2 of 12, 17%) and head and neck squamous cell carcinoma (2 of 12, 17%) were the most common nonneural malignant neoplasms. In a separate case series of 14 patients with MAS from the US and Europe, brain neoplasms (4 of 14, 29%; 2 glioblastomas, 2 unspecified type) and malignant peripheral nerve sheath tumor (2 of 14, 14%) were the most common neural cancers, while cutaneous melanoma (4 of 14, 29%) and sarcomas (2 of 14, 14%; 1 liposarcoma, 1 unspecified type) were the most common nonneural cancers. Cutaneous neurofibromas (7 of 14, 50%) and schwannomas (2 of 14, 14%) were also common. In 1 US family, a father and son with MAS had clinical diagnoses of neurofibromatosis type 1 (NF1). Genetic testing of the son detected a pathogenic CDKN2A splicing variant (c.151-1G>C) and was negative for NF1 genetic alterations. In UK Biobank, 2 in 150 (1.3%) individuals with clinical NF1 diagnoses had likely pathogenic variants in CDKN2A, including 1 individual with no detected variants in the NF1 gene. Conclusions and Relevance: This cohort study estimates the prevalence and describes the tumors of MAS. Additional studies are needed in genetically diverse populations to further define population prevalence and disease phenotypes.

A Phase II Trial of Guadecitabine in Children and Adults with SDH-Deficient GIST, Pheochromocytoma, Paraganglioma, and HLRCC-Associated Renal Cell Carcinoma.

PURPOSE: Succinate dehydrogenase (dSDH)-deficient tumors, including pheochromocytoma/paraganglioma, hereditary leiomyomatosis and renal cell cancer-associated renal cell carcinoma (HLRCC-RCC), and gastrointestinal stromal tumors (GIST) without KIT or platelet-derived growth factor receptor alpha mutations are often resistant to cytotoxic chemotherapy, radiotherapy, and many targeted therapies. We evaluated guadecitabine, a dinucleotide containing the DNA methyltransferase inhibitor decitabine, in these patient populations. PATIENTS AND METHODS: Phase II study of guadecitabine (subcutaneously, 45 mg/m2/day for 5 consecutive days, planned 28-day cycle) to assess clinical activity (according to RECISTv.1.1) across three strata of patients with dSDH GIST, pheochromocytoma/paraganglioma, or HLRCC-RCC. A Simon optimal two-stage design (target response rate 30% rule out 5%) was used. Biologic correlates (methylation and metabolites) from peripheral blood mononuclear cells (PBMC), serum, and urine were analyzed. RESULTS: Nine patients (7 with dSDH GIST, 1 each with paraganglioma and HLRCC-RCC, 6 females and 3 males, age range 18-57 years) were enrolled. Two patients developed treatment-limiting neutropenia. No partial or complete responses were observed (range 1-17 cycles of therapy). Biologic activity assessed as global demethylation in PBMCs was observed. No clear changes in metabolite concentrations were observed. CONCLUSIONS: Guadecitabine was tolerated in patients with dSDH tumors with manageable toxicity. Although 4 of 9 patients had prolonged stable disease, there were no objective responses. Thus, guadecitabine did not meet the target of 30% response rate across dSDH tumors at this dose, although signs of biologic activity were noted.

Genomic Patterns of Malignant Peripheral Nerve Sheath Tumor (MPNST) Evolution Correlate with Clinical Outcome and Are Detectable in Cell-Free DNA.

Malignant peripheral nerve sheath tumor (MPNST), an aggressive soft-tissue sarcoma, occurs in people with neurofibromatosis type 1 (NF1) and sporadically. Whole-genome and multiregional exome sequencing, transcriptomic, and methylation profiling of 95 tumor samples revealed the order of genomic events in tumor evolution. Following biallelic inactivation of NF1, loss of CDKN2A or TP53 with or without inactivation of polycomb repressive complex 2 (PRC2) leads to extensive somatic copy-number aberrations (SCNA). Distinct pathways of tumor evolution are associated with inactivation of PRC2 genes and H3K27 trimethylation (H3K27me3) status. Tumors with H3K27me3 loss evolve through extensive chromosomal losses followed by whole-genome doubling and chromosome 8 amplification, and show lower levels of immune cell infiltration. Retention of H3K27me3 leads to extensive genomic instability, but an immune cell-rich phenotype. Specific SCNAs detected in both tumor samples and cell-free DNA (cfDNA) act as a surrogate for H3K27me3 loss and immune infiltration, and predict prognosis. SIGNIFICANCE: MPNST is the most common cause of death and morbidity for individuals with NF1, a relatively common tumor predisposition syndrome. Our results suggest that somatic copy-number and methylation profiling of tumor or cfDNA could serve as a biomarker for early diagnosis and to stratify patients into prognostic and treatment-related subgroups. This article is highlighted in the In This Issue feature, p. 517.