Download Gene Expression Analysis Platform Evaluation for FFPE Specimen

013 - Gene Expression Analysis Platform Evaluation
for FFPE Specimen Material-Based Studies
014 - Evaluation of Immunosignature Profile in
Medulloblastoma
Mateusz Koptyra, PhD
Center for Data-Driven Discovery in Biomedicine (D3b)
Children’s Hospital of Philadelphia
Gene Expression Analysis Platform Evaluation for FFPE
Specimen Material-Based Studies for data generation
The overwhelming majority of archival samples of cancer tissue are formalin-fixed,
paraffin-embedded (FFPE)
Archived FFPE specimens present potential for transcriptomic/genomic data
generation especially for rare cancers
Fixative selection as well as storage conditions can compromise nucleic acid quality.
We identified the need to evaluate and establish the system for FFPE material derived
transcriptomic/genomic data generation
The Evaluation
Demonstrate directional correlation between mRNA analysis form RNAseq and HTG,
and NanoString pipelines.
Cases:
5 high-grade gliomas (HGG) and primitive neuro-ectodermal tumors (PNET) FFPE
specimens
Frozen tissue total RNA
RNAseq - BGI@CHOP (data obtained from CBTTC)
FFPE
HTG EdgeSeq analysis – mRNA oncology biomarker panel (OBP) for mRNA gene
expression including 2560 genes.
NanoString nCounter® PanCancer Pathways panel for mRNA gene expression
including 770 genes.
HTG EdgeSeq technology overview
Technology Overview:
-
Bind target RNA to protection probe
-
Eliminate excess protection probes and
RNA with S1 nuclease
-
Eliminate target RNA with heat and
base
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Add sequencing adaptors and tags
-
Quantitate, pool, and sequence
NanoString nCounter technology overview
Technology Overview:
-
Individual reporter and capture probe
for each analyzed transcript
-
Solution based mRNA hybridization
-
Reporter and capture probe complexes
surface binding
-
Surface imaging and reporter probes
decoding
Fortina, P. & Surrey, S., 2008 Nature Biotechnology
Distribution of correlation coefficient
The correlation coefficient calculated for each gene using the normalized data from all
5 samples measured by two different platforms.
Average expression distribution
The distribution of gene expression measurements from 3 platforms.
Average expression correlation
Comparison of the average expression measurements from 5 samples between 3
platforms. The data was adjusted, the medians of the means of all 3 data sets are the
same. Highlighted (red) genes present the lowest correlation coefficient (< 0.2).
Currently ongoing and future plans:
- Establishing within platform correlations
- Single target gene expression validation studies
- miRNA whole transcriptome analysis for all analyzed platforms
- Additional platforms considered – (TaqMan® Gene Expression array /
RT2 Profiler PCR Array)
- Bouin fixative specimen transcriptomic analysis
ImmunoSignature Assay
Johnson, Stephen A., TRILOGY STEM Group 2012
ImmunoSignature Assay recognizes brain tumor immunopattern in serum
The heat-map presents peptides that differentiated glioblastoma patients from healthy
persons obtained from two independent cohorts. The colored bars on the right indicate
clusters that define groups of peptides.
Hughes A. 2012, PLOS One
Immunosignature assay for Medulloblastoma Subtyping
- 10 plasma specimens from medulloblastoma tumor cases
- 28 whole blood specimen from medulloblastoma tumor cases
- Specimen type based evaluation (frozen whole blood vs plasma) analysis
- Clinical medulloblastoma subtyping
- RNAseq medulloblastoma subtyping
Phillip B. Storm, MD
Adam Resnick, PhD
Jena Lilly, MS
Angela Waanders, MD, MPH
Zhe (Jim) Zhang, PhD
Namrata Choudhari, MS
Yaunkun Zhu,BS
Mariarita Santi, MD, PhD
Bo Zhang, BS
Elizabeth Ampert, BS
Robert Wechsler-Reya, PhD
Dustin Hatefi, MD, MPH
Phillip Stafford, PhD
Stephen Albert Johnston, PhD