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Diagnosis and monitoring of Leptomeningeal Disease using Circula5ng free DNA in the cerebrospinal fluid (CSF cfDNA) R. H. Shah1, E. I. Pentsova2, J. Tang5, A. Boire2, D. You5, S. Briggs2, A. Omuro2, X. Lin2, M. Fleisher3, C. Grommes2, F. Meng5, S. D. Selcuklu5, S. Ogilvie4, N. Distefano4, L.Shagabayeva2, M.Rosenblum2, L. M. DeAngelis2, A. Viale5, I. K. Mellinghoff2,, M. F. Berger1,5, 1Department of Pathology, Memorial Sloan KeTering Cancer Center, New York, NY 10065, USA.2Department of Neurology, Memorial Sloan KeTering Cancer Center, New York, NY 10065, USA. 3Department of Laboratory Medicine, Memorial Sloan KeTering Cancer Center, New York, NY 10065, USA.4Department of Neurosurgery; Memorial Sloan KeTering Cancer Center, New York, NY 10065, USA. 5Center for Molecular Oncology, Memorial Sloan KeTering Cancer Center, New York, NY 10065, USA
Background
Conclusion
Leptomeningeal metastases (LM) in solid tumors (ST) represent a devasta]ng complica]on of cancer with a median survival of only 12-‐14 weeks a_er diagnosis [1]; however, establishing the diagnosis of LM can be difficult, par]cularly at early stages before the pa]ent is disabled. The diagnosis is based on CSF cytologic analysis and/or MRI findings.[2-‐4] Brain and spine MRIs have been increasingly preferred for the ini]al evalua]on of LM because of their non-‐invasive nature and convenience to pa]ents. However, MRI findings are nega]ve in 25%-‐50% of pa]ents [3, 4], and unequivocal findings may only appear in late-‐stage disease when the pa]ent is already debilitated. CSF cytologic analysis provides diagnos]c confirma]on of LM but is associated with a rela]vely low sensi]vity (approximately 50% on the first lumbar puncture) and is highly examiner-‐dependent. Improved diagnos]c tools are required to facilitate early diagnosis. To this end, we explored whether sufficient quan]ty and quality of DNA can be isolated from CSF for genomic study and whether the CSF pellet or CSF supernatant, would be more suitable for detec]ng cfDNA. We used an in-‐house sequencing assay, MSK-‐IMPACT [5], to interrogate 341 clinically relevant cancer genes in tumor-‐derived cfDNA from 53 pa]ents. Results of CSF cfDNA were compared to standard CSF cytopathologic analysis from that same CSF sample and with MRI findings performed at the same ]me. When possible, we compared CSF cfDNA with DNA from tumor ]ssue (primary tumor and non-‐CNS sites) to determine similari]es and differences in gene]c altera]ons between these different compartments.
Acknowledgements
Introduc5on Methods
References Our study demonstrates that genomic analysis of CSF, using a sufficiently sensi]ve and comprehensive plaiorm, may be useful to facilitate diagnosis of tumor in the CNS, monitor the evolu]on of the cancer genome during treatment of CNS cancers, guide the choice of second-‐line agents, and perhaps iden]fy pathways that are uniquely associated with cancer spread to the central nervous system.
Center of Molecular Oncology, Department of Pathology & Department of Neurology
Targeted Capture, Sequencing & Genomic Analysis Captures all protein-‐coding exons of 341 cancer-‐associated genes
Sequence pair-‐end reads (2x100) on HiSeq 2500
Analyse genomic data using methods described previously [5].
Extrac]on of cfDNA.
Centrifuged at 10,000 g for 30 min at 4℃ to remove residual precipitated cellular components QIAamp Circula]ng Nucleic Acid Kit
Cerebrospinal Fluid Collec]on and Prepara]on.
Lumbar Puncture Centrifuged at 1,000 x g, 4°C for 5 min to separate supernatent & pellet
Image 1: Comparison of tumor-‐derived DNA from CSF cell pellet and supernatant. (A) Schema]c of separa]on of CSF pellet and supernatant. Cellular DNA is isolated from the pellet, and cfDNA is isolated from the supernatant. (B) Variant allele frequencies for known muta]ons in CSF cfDNA and pellet DNA. (C) Log2 ra]os of normalized sequence coverage for target exons in CSF-‐cfDNA and pellet DNA for pa]ent 8 . Greater than 10-‐fold amplifica]on of HER2 was observed in CSF-‐cfDNA, whereas HER2 amplifica]on was barely detectable in pellet DNA. (D) Evidence of EML4-‐ALK gene fusion in CSF cfDNA and pellet DNA for pa]ent 6. Read-‐pairs suppor]ng the fusion (red) are visualized using the Integra]ve Genomics Viewer.
Results Muta5ons detected in most pa5ents with LM disease
Image 2: (A) Schema]c showing paTerns of CNS involvement in pa]ents with solid tumors. (B) Percentage of pa]ents for which high-‐confidence soma]c altera]ons were detected by MSK-‐IMPACT. Pa]ents are grouped according to the presence or absence of intraparenchymal brain metastases and leptomeningeal metastases (LM)
Drug-‐resistance mechanisms in CSF in pa5ents with CNS relapse
Image 3: Summary of genomic profiling results from CSF and other tumor sites in pa]ents who developed progressive CNS disease during treatment with the indicated kinase inhibitors. Tumor evolu5on in pa5ents with primary brain tumors.
Image 4: Tumor evolu5on in pa5ents with primary brain tumors. (A) Spa]al and temporal heterogeneity between samples obtained at diagnosis, at recurrence and from CSF in pa]ent 42 with recurrent glioblastoma. CSF cfDNA harbors a PTEN R130* muta]on (VAF=0.25), while resec]on #2 harbors a PIK3CA H1047R muta]on (VAF=0.441). (B) CSF molecular profile for a pa]ent 45 with anaplas]c oligodendroglioma contains the IDH1 R132H muta]on and 1p/19q dele]on found in ]ssue resec]on #2, as well as 454 non-‐silent soma]c muta]ons. 448 SNVs represent C>T/G>A muta]ons demonstra]ng temozolomide-‐induced mutagenesis.
A B
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Pilot: CSF cell pellet vs. CSF cfDNA