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Personalized Oncology Through Integrative High-Throughput Sequencing:

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Personalized Oncology Through Integrative High-Throughput Sequencing:

  1. 1. Sci Transl Med 3, 111ra121 (2011); DOI: 10.1126/scitranslmed.3003161 Raunak Shrestha 30th October 2012 1
  2. 2. • Pair-end Whole Genome Sequencing (5X – 15 X) • Targeted Exome Sequencing (Tumor and Matched Germline Samples) • Pair-end Transcriptome Sequencing of Tumor 2
  3. 3. TRAININGTEST • 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 3 (mouse-xenografts)
  4. 4. 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 v38 Normal Exome 101 X Transcriptome NA - Patient 2 > 90 % Whole Genome 4.27 X Illumina HiSeq 2000 - Tumor Exome 82 X Agilent v38 Normal Exome 87 X Transcriptome NA - Patient 3 60 - 70 % Whole Genome 4.8 X Illumina HiSeq 2000 - Tumor Exome 126 X Roche V2.0 Normal Exome 128 X Transcriptome NA - Patient 4 75 - 80 % Whole Genome 4.8 X Illumina HiSeq 2000 - Tumor Exome 124 X Roche V2.0 Normal Exome 121 X Transcriptome NA - 4
  5. 5. • low PTEN expression in this patient relative to an existing prostate RNA-Seq cohort • These findings were only evaluated in the xenograft specimen 5
  6. 6. • 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 6
  7. 7. • 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 7
  8. 8. • 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 8
  9. 9. • 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) 9
  10. 10. 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 10
  11. 11. 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 11
  12. 12. 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 12

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