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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