Khakoo, Yasmin

Abstract Intrinsic brainstem tumors arising in pediatric, adolescent, and young adult patients comprise a spectrum of entities, predominantly diffuse midline gliomas (DMG) and IDH mutant astrocytomas. Accurate molecular diagnosis is essential for prognostication and optimal therapy. Therapeutic considerations include inclusion (in IDH mutant) or exclusion (in H3K27M) of adjuvant Temozolomide post radiotherapy and use of molecular targeted therapy (IDH inhibitors). Surgical biopsy, however, is associated with increased risk of permanent neurological deficits and may yield insufficient or non-diagnostic tissue. We hypothesized that minimally-invasive “liquid biopsy” of cerebrospinal fluid (CSF) could represent a superior diagnostic modality in this patient population, employing molecular analysis of cell free DNA (cfDNA). We analyzed 44 CSF samples from 38 unique patients for recurrent driver mutations in brainstem gliomas, including H3K27M, IDH1, and IDH2. MSK-IMPACT, a NY state-authorized hybridization capture-based next-generation panel DNA sequencing assay, was used for analysis. Samples without a detectable driver mutation by MSK-IMPACT testing were further subjected to gene targeted testing by droplet digital PCR. In all, 10/44 (22.7%) samples had mutations detected by MSK-IMPACT using standard calling criteria and 13 of the remaining 34 samples (38.2%) had supporting evidence of a mutation based on manual review. Further testing by ddPCR was performed on a subset of cases (based on DNA availability) which confirmed 7 of the previous low-level mutations and uncovered 4 additional mutations. Overall, 27/44 (61.4%) of cases had detectable evidence of a mutation, and of the 23 patients without a known tissue diagnosis, a driver in the CSF was identified for 47.8% of patients (11/23). Minimally-invasive analysis of CSF cfDNA in patients with intrinsic brainstem tumors has a high diagnostic yield and may obviate the need for tissue biopsy in a majority of patients. Testing with high sensitivity assays is valuable to maximize the rate of detection.

Abstract PURPOSE: A subset of pediatric, adolescent and young adult (AYA) gliomas are located in the brainstem, eloquent locations, or present with diffuse/leptomeningeal disease, and are associated with high risk and low yield of biopsy. At the same time, accurate molecular diagnosis is necessary to direct optimal therapy. In such cases, analysis of cell free DNA (cfDNA) form cerebral spinal fluid (CSF) may represent a diagnostic alternative to biopsy. METHODS: We investigated the utility of CSF cfDNA sequencing through a stepwise approach, using clinically validated, targeted molecular assays. Testing was performed using a broad hybrid capture next generation sequencing assay (MSK-IMPACT) and subsequent targeted digital droplet PCR in a subset of cases. RESULTS: We analyzed 17 CSF samples from 17 pediatric (n=6) and AYA (n=11) glioma patients with primary or recurrent disease. Thirteen had tumors located within the brainstem, and four had leptomeningeal involvement. Somatic alterations were detected in 12/17 samples (71%). In 3/4 patients with leptomeningeal involvement, cfDNA testing revealed a BRAF fusion consistent with the diagnosis of diffuse leptomeningeal glioneuronal tumor (DLGNT). Among the 13 patients with brainstem involvement, four had somatic H3 K27M mutations, three had IDH mutations, and one had TP53 and ATRX mutations; five patients had no detectable mutations. CONCLUSION: In our analysis, we found that established glioma hotspot mutations were able to be detected within the CSF. We propose that in patients for whom tissue biopsy is high risk, not feasible, or tissue was nondiagnostic, CSF cfDNA sequencing has a substantial diagnostic yield and should be considered as a valuable novel diagnostic tool. Ongoing research is aiming to further increase the sensitivity of cfDNA testing, especially in patients with very low levels of CSF cfDNA.

Abstract OBJECTIVE: We hypothesize that patients with neurocutaneous melanocytosis-associated melanoma and ventriculoperitoneal shunts are at risk of developing intraperitoneal spread of melanoma. BACKGROUND: Neurocutaneous melanocytosis, a rare condition characterized by excessive proliferation and deposition of melanocytes in the leptomeninges and brain parenchyma, typically occurs in children with large congenital melanocytic nevi and multiple smaller congenital nevi. These patients are at heightened risk for developing NRAS+ melanomas in the central nervous system, which in turn may lead to symptomatic hydrocephalus requiring cerebrospinal fluid diversion for symptom relief. METHODS: Retrospective single-institution study of patients with histologically or radiographically confirmed NCM evaluated at Memorial Sloan Kettering Cancer Center (MSKCC) from 2001-2022. RESULTS: Of the 47 patients with a diagnosis of NCM, 44 patients had symptomatic neurological complications. Eleven patients developed hydrocephalus, 10 had CNS melanoma, and required ventriculoperitoneal shunt placement. Nine of the 10 patients ultimately died of their disease. Three patients were diagnosed with intraperitoneal melanoma, though data are unavailable for the remaining eight. CONCLUSIONS: All (n=11) patients with NCM-associated CNS melanoma required VP shunts for symptomatic relief. Ten of these patients died within 4.3 years of VP shunt placement, with a range of 1 month to 13.5 years prior to succumbing to their disease. While the intraperitoneal pathology remains unknown for 7 of the cases, 3 had confirmed intraperitoneal melanoma, suggesting that VP shunts provided the conduit to CNS melanoma seeding of the peritoneum. Obtaining baseline abdominal imaging studies prior to VP shunt placement may be helpful in the follow-up of these patients.