Medulloblastoma Circulating Tumor Cell Clusters as a Novel Tumor Biomarker and Mechanism for Hematogenous Spread Public

Abstract

Medulloblastoma is an embryonal tumor of the cerebellum and accounts for most pediatric malignant disease of the central nervous system (CNS). RNA sequencing and DNA methylation studies reveal four major subgroups of disease: SHH-activated, WNT-activated, Group 3 and Group 4 [22, 23]. While grouping carries prognostic sig- nificance, metastatic spread remains the single most important indicator of outcomes [25].

Previously, medulloblastoma spread was thought to occur only by direct tumor shedding. However, there is a growing recognition for a hematogenous route for metastasis [13].

Literature in several adult cancers recognize the role of circulating tumor cells (CTCs) as a mechanism of seeding disease at remote sites [2], with CTC clusters (CTCCs) carrying even greater potential for metastasis [6]. Traditional methods of CTC detection rely on immunolabeling. This requires a universal and tumor-specific surface marker, whereas no similar markers are known in medulloblastoma cells. A novel microfluidic chip (Cluster-Chip) was developed to capture CTCCs from unpro- cessed blood using a label-free approach, achieving 99% efficiency on cluster sizes of 4 or more cells [24]. Using the Cluster-Chip technology, we describe the presence of CTCCs in patients with medulloblastoma. CTCCs therefore may represent a novel biomarker for tumor and present an exciting new direction to study hematogenous disease spread in medulloblastoma.

We enrolled 44 pediatric patients in a longitudinal study. CTCCs are quantified at enrollment and additionally at routine 3-month intervals in medulloblastoma pa- tients. From this study cohort, we report the presence of CTCCs in all patients with medulloblastoma and at all phases of therapy, while none are detected in patients without malignancy.

Identifying CTCCs in blood from patients with medulloblastoma is promising, but their role in metastasis pathophysiology is not currently understood. We use multiple change point detection to identify significant changes in CTCC quantity from sequential peripheral whole blood data, and next-generation high-throughput RNA sequencing to describe CTCC transcriptome compared to whole blood through gene set enrichment analysis and immune cell deconvolution. 

Table of Contents

1 Introduction and Background 1

1.1 Medulloblastoma ............................. 1 1.1.1 MedulloblastomaGenomics ................... 2 1.1.2 MetastasisandSurvivalOutcomes ............... 2 1.2 CirculatingTumorCellsandCellClusters . . . . . . . . . . . . . . . 3 1.2.1 CirculatingTumorCellCapture................. 4

2 Methods 5

2.1 Patient Enrollment and Human Subjects Research . . . . . . . . . . . 5 2.2 ChangePointDetection ......................... 6 2.3 CTCCEnrichmentandBulk-RNASeq ................. 7 2.3.1 Enrichment ............................ 7 2.3.2 Sequencing ............................ 7 2.3.3 DifferentialGeneExpressionAnalysis . . . . . . . . . . . . . . 9 2.3.4 BatchEffect............................ 9 2.3.5 Stemness.............................. 10 2.3.6 GeneSetEnrichmentAnalysis.................. 10 2.3.7 ImmuneDeconvolution...................... 11 2.4 ContributionsfromCollaborators .................... 12 2.5 Funding .................................. 12

3 Results 13

3.1 PatientEnrollmentandCellCapture .................. 13 3.2 CTCC Quantification and Change Point Detection . . . . . . . . . . 15 3.3 BulkRNA-SeqQualityControl ..................... 17 3.4 BulkRNASequencing .......................... 20 3.5 GeneSetEnrichmentAnalysis...................... 23 3.5.1 ImmuneDeconvolution...................... 25

4 Discussion 27

4.1 Circulating Tumor Cell Cluster Quantification . . . . . . . . . . . . . 27

4.2 TranscriptomicAnalysisofCapturedCTCCs. . . . . . . . . . . . . . 28

4.3 CTCCGeneSetEnrichmentAnalysis.................. 29

4.4 Immunedeconvolution .......................... 30

4.5 BatchEffect................................ 31

4.6 FutureStudies............................... 31

5 Conclusions 33 

About this Master's Thesis

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School	Laney Graduate School
Department	Computer Science and Informatics
Degree	M.S.
Submission	Master's Thesis
Language	English
Research Field	Computer Science Biology, Bioinformatics Biology, Cell
Mot-clé	Bioinformatics Medulloblastoma Transcriptomics Circulating Tumor Cells RNA-sequencing
Committee Chair / Thesis Advisor	Manoj Bhasin, PhD, Emory University
Committee Members	Zhaohui Qin, PhD, Emory University Matthew Reyna, PhD, Emory University Swati Bhasin, PhD, Emory University

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