personalized oncology through integrative high-throughput sequencing:
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Sci Transl Med 3, 111ra121 (2011); DOI: 10.1126/scitranslmed.3003161
Raunak Shrestha
30th October 2012
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• Pair-end Whole Genome Sequencing (5X – 15 X)
• Targeted Exome Sequencing (Tumor and Matched Germline Samples)
• Pair-end Transcriptome Sequencing of Tumor
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TRAI
NIN
GTE
ST• Tumor mouse-xenografts
from two living patients
• No mention of how xenografts were established
• Assumes that genomic landscape/events in xenograft and patient are similar
• Findings in training set were only evaluated in the xenograft specimen and not intended to deliver therapy
(mou
se-x
enog
raft
s)
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Patient Tumor Content
Sequencing Type
Estimated Coverage
Sequencing Platform Exome Capture Kit
Patient 1 > 90 %
Whole Genome 3.76 X
Illumina HiSeq 2000
-Tumor Exome 86 X
Agilent v38Normal Exome 101 XTranscriptome NA -
Patient 2 > 90 %
Whole Genome 4.27 X
Illumina HiSeq 2000
-Tumor Exome 82 X
Agilent v38Normal Exome 87 XTranscriptome NA -
Patient 3 60 - 70 %
Whole Genome 4.8 X
Illumina HiSeq 2000
-Tumor Exome 126 X
Roche V2.0Normal Exome 128 XTranscriptome NA -
Patient 4 75 - 80 %
Whole Genome 4.8 X
Illumina HiSeq 2000
-Tumor Exome 124 X
Roche V2.0Normal Exome 121 XTranscriptome NA -
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• low PTEN expression in this patient relative to an existing prostate RNA-Seq cohort
• These findings were only evaluated in the xenograft specimen
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• CPNE4-NEK11 gene fusion has unknown clinical significance but warrants further biological validation
• Polo-like kinases regulate the transition from G2 to M phase and are being targeted as a class due to their ubiquitous expression in cancer
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• homozygous inactivation of TP53 (via point mutation and copy number loss)
• dual copy number gain and point mutation in Aurora kinase A (AURKA)
• point mutations in smooth muscle myosin heavy chain (MYH11) and FAS death receptor
• copy number gains of EGFR• a large region of chromosome 13 containing
CDK8 was prominently amplified• CDK8 was also overexpressed in the RNA-
Seq outlier analysis
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• Most of the findings were biologically interesting but not clinically significant– tumor had a point mutation in MYH11, which is rearranged in AML and
reported in intestinal cancer – functional role of mutation in FAS death receptor not known (though it is
known that FAS intracellular mutation can protect against apoptosis)– Role of ASMTL-AS1/PPP2R3B gene fusion unknown though it has been
reported in colon and lung tumors
• Patient potentially matched to clinical trials with MEK, PI3K or CDK inhibitors
• Current clinical testing often disregards NRAS because of its low frequency (2%) in CRC
• but activating mutations in NRAS are biologically similar to KRAS (35 to 40% of CRC), which predict resistance to antibody therapies against EGFR
• trials may include CRC patients with KRAS or BRAF mutations for Raf inhibitors, but fail to include patients with NRAS mutations
• amplification of CDK8 has been implicated in 15 to 20% of CRC as a positive regulator of catenin signaling and is a viable target for CDK inhibitors in clinical trials
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• point mutation in the ETS transcription factor family member ELK1 (R74C)
• could potentially qualify for an upcoming trial of combined treatment with PI3K and MEK inhibitors for specified solid tumor malignancies with KRAS, NRAS, and BRAF mutations (NCT01363232)
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Clinical Validation
• The pilot study was implemented in a research setting• Any results that affect clinical decision-making must be
validated using a Clinical Laboratory Improvement Amendments (CLIA)-certified test
• CLIA validation of results through Sanger sequencing, qPCR, or FISH will be performed
• The author’s lab was in a process of getting CLIA-certified so clinical validations were not performed till the publication date
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Conclusions
• Integrative sequencing/analysis helpful to find potential informative aberrations – Can provide orthogonal support for some key
findings
• Effect of low tumor content can be compensated by high depth of sequencing
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Conclusions
• Both patients 3 and 4 had potentially informative aberrations, but these patients did not fit into available trials.
• Highlighted the need to restructure the eligibility criteria for trials of molecularly targeted therapies
• Highlighted the issue that most clinical trials or pharmaceutical research interested in high frequency mutation– Low frequency mutation can be critical from the
perspective of personalized oncology
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