1. Sci Transl Med 3, 111ra121 (2011); DOI: 10.1126/scitranslmed.3003161
Raunak Shrestha
30th October 2012 1

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. 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. 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. • 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. • 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. • 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. • 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. • 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. 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. 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. 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