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PATIENT INFORMATION
Patient Name: Mr Sajjan Raj B Jain Date of Receipt: 23-07-2014
Age/Date of Birth: 58 years Date of Report: 08-08-2014
Gender: Male Referring Hospital: HCG, Bangalore
Specimen type: Saliva (Normal) & FFPE (Tumor) Referred by: Dr. Shekar Patil
Block ID: Not Known Ref/MRN ID: Not Known
Specimen Site : Not Known Laboratory ID: STRAN-000000347

INDICATION FOR TEST: Esophageal carcinoma

TEST DETAILS

Strand Somatic 48-gene Test - detects somatic alterations in hot spot regions of 48 genes and interprets
those with possible therapeutic, clinical or prognostic implications. The test is based on Illumina TruSeq
Amplicon Cancer Panel (see supplementary information for full list of genes). Gene coverage limitations are
listed on page 4.










SUMMARY OF TEST RESULTS

Gene Mutation
*
Therapy
(for tumor type)
*
Therapy
(for another tumor
type)
Relevant drugs
/ Clinical trials
TP53

RefSeq id:
NM_000546
chr17:7577538 C>T
c.743G>A
p.Arg248Gln

May indicate chemoresistance None None
Amplification
EGFR


RefSeq
id:NM_005228
Amplification None May indicate
possible response
to anti-EGFR
therapy
NCT01107639,
NCT01243398,
NCT00686114
(for details, see
clinical trials
section, pg 4)
Quality: The coverage was 500X with a base quality greater than Q20, variants with Phred score greater than 50 were considered for SNV calling.
Some portions of the hot spot regions of these 48 genes were not covered (<500X coverage) by the test; these are listed in the appendix. CDKN2A
gene is poorly covered in all runs.
1) Genes were considered as probably amplified when majority of the amplicons showed >4x reads over normal at 99% confidence level.
2) Next Generation sequencing (NGS): Large insertions, deletions, duplications, inversions and complex rearrangements cannot be characterized
using short-read sequencing data. They have a much higher false-positive and false-negative rates than seen for SNVs (single nucleotide variant).
3) The identified SNVs are NOT validated by Sanger sequencing.
4) For the deletion/duplication nomenclature, the most 3' position possible in the transcript sequence is arbitrarily assigned to have been changed
(Human Genome Variation Society guidelines).
*Approved by Food and Drug Administration (FDA), U.S.A.


RESULTS: A somatic mutation was detected in the TP53 gene. Amplification of the EGFR gene was
detected.
Mutation in TP53 may indicate poor prognosis and resistance to chemotherapy and
radiotherapy.
Amplification of EGFR may indicate response to gefitinib, erlotinib and cetuximab. Clinical
trials are available with all three drugs in esophageal carcinoma (NCT01243398, NCT00686114,
NCT01107639).

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INTERPRETATION

Gene Mutation Mutation Status Exon
TP53 p.Arg248Gln
Known (COSM10662) 7

Gene alteration description

A missense mutation was detected in the TP53 gene.

TP53 is a well known tumor suppressor gene and encodes the p53 protein. It is located on chromosome 17p13.1 and
is known to be inactivated in multiple cancer types. The p53 protein plays a central role in response to DNA damage
by regulating genes involved in cell cycle arrest and apoptosis (Amundson et al., 1998).

The identified mutation causes a missense substitution in exon 7 and the resulting protein change is p.Arg248Gln. This
mutation was identified in 18% of the specimen tumor DNA. This mutation has been reported in cancers of large
intestine, breast, haematopoietic and lymphoid tissue, central nervous system and stomach. While p.Arg248Gln has
not been reported in esophageal cancer, a tryptophan substitution at the same position has been noted (COSMIC
database). The residue Arg248 is located in one of the four highly conserved regions (region IV) in the DNA binding
domain of p53 and is a mutation hot-spot (Reles et al., 2001). p53 protein with the Gln248 residue is known to retain
the wild-type conformation but lacks DNA-binding ability and is hence impaired for sequence-specific transactivation
(Ory et al., 1994). This residue is one of the contact residues involved in p53-Bcl-XL interaction which is crucial for the
apoptotic functions of the protein (Xu et al., 2014). Moreover, this mutant has been shown to result in an oncogenic
gain of function in mice (Hanel et al., 2013).

Prognostic relevance

A recent large scale study using clinical data obtained from The Cancer Genome Atlas (TCGA), analyzed prognostic
associations of TP53 mutations at the hotspot residues, Arg175, Tyr220,.Gly245, Arg248, Arg249, Arg273 and Arg282.
A statistical analysis of patient survival for various cancers (breast, ovary, lung, bladder, colorectal, gliobastoma) in
relation to nonsense and substitution mutations at the residues mentioned above, revealed significantly shorter
overall survival and two fold higher hazard ratios for p.Arg248 and p.Arg282 than even the nonsense mutations (Xu et
al., 2014). Additionally, clinical evidence in esophageal cancer patients with TP53 mutations in exons 5, 6, 7 and 8
exhibited significantly shorter survival, than those without mutations, when treated with chemotherapy (5-
fluorouracil, cisplatin, and alpha-interferon) and concurrent external beam radiotherapy (Ribeiro et al., 1998).

Gene
Mutation
Mutation Status
Exon
EGFR Amplification
Known -

Gene alteration description

Gene amplification was detected in the EGFR gene
EGFR (epidermal growth factor receptor) gene is located on chromosome 7p12 and encodes a membrane glycoprotein
belonging to the receptor tyrosine kinase family. Activation of the EGFR receptor results from binding of growth factor
ligands, leading to activation of several downstream signalling pathways controlling an array of vital cellular processes
(Wells, 1999).

EGFR gene showed amplified signal of >4.7 fold in tumor sample compared to paired normal at the positions, chr7:
55221818_55221937, chr7: 55241675_55241800, chr7: 55248905_55249026, chr7: 55249026_55249152, chr7:
55259513_55259635.
EGFR amplification has been frequently reported in esophageal squamous cell carcinoma (SCC) and is associated with

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overexpression of the EGFR protein (Hanawa et al., 2006). In Barretts adenocarcinoma and esophageal
adenocarcinoma patients, the rate of EGFR amplification has been reported to be 8-11% (Xu et al., 2013).

Therapeutic relevance

The EGFR tyrosine kinase inhibitor (TKI), gefitinib is FDA approved for treatment of non small cell lung cancer (NSCLC)
patients. Although clinical evidence for this drug in esophageal cancer patients does not indicate clear benefits with
respect to EGFR amplification, a trend towards high response in SCC with EGFR overexpression has been noted (Xu et
al., 2013, Janmaat et al, 2006). Inspired by the promising results from gefitinib therapy of EGFR amplified NSCLC
patients, esophageal cancer cell lines were tested for gefitinib efficacy. Such preclinical data revealed significant
response upon gefitinib treatment in this study (Drenckhan et al., 2014). Clinical trials are available for gefitinib
therapy in esophageal cancer (NCT01243398).

Another EGFR tyrosine kinase inhibitor, erlotinib is an FDA approved drug for NSCLC and pancreatic cancer with EGFR
activating mutations. A phase II trial with erlotinib therapy for esophageal cancer indicated partial response in
patients with metastatic esophageal cancer overexpressing EGFR, although no statistically correlated results were
reported (Illson et al., 2011). Taken together, clinical studies with erlotinib suggest responsiveness of SCC with EGFR
overexpression (Xu et al., 2013). An ongoing phase III trial is available for erlotinib therapy in esophageal cancer
(NCT00686114).

A monoclonal antibody, cetuximab, specific to the extracellular domain of EGFR, is approved by the FDA for treatment
of metastatic colorectal cancer and head and neck cancer. Encouraged by the promising results in cetuximab-treated,
EGFR-amplified colorectal and head and neck cancer, multiple clinical trials have been initiated to check the efficacy of
this drug in esophageal cancer (Tew et al., 2005). A study, including esophageal SCC patients with EGFR
overexpression, investigated the efficacy of inclusion of cetuximab in a chemotherapy regimen and reported a
favourable response (Lorenzen et al., 2009). In a pre-clinical set up, cetuximab was shown to induce cytotoxicity in
esophageal SCC cell lines overexpressing EGFR (Kawaguchi et al., 2007). An ongoing phase III clinical trial, comparing
the efficacy of chemotherapy with or without cetuximab has been included here (NCT01107639).
Prognostic relevance

Amplification of EGFR has been frequently associated with invasive disease and poor prognosis for esophageal cancer
(Hanawa et al., 2006, Kitagawa et al., 1996, Marx et al., 2010).

RELEVANT DRUGS:

THERAPY RATIONALE
Cetuximab

Cetuximab is a monoclonal antibody against EGFR, with anti-neoplastic activity. It is an FDA
approved drug for metastatic colorectal cancer and head and neck cancer. It specifically binds to
the extracellular domain of EGFR inhibiting its activation and dimerization. This results in inhibition
of the signalling cascade activated by EGFR. Solid tumors overexpressing EGFR, may benefit from
treatment with this drug.
Gefitinib

Gefitinib is an anilinoquinazoline that inhibits the tyrosine kinase activity of EGFR resulting in
inhibition of EGFR dependent tumor growth. It specifically competes with the ATP binding site in
the receptor, thus blocking its autophosphorylation. It is an FDA approved drug for NSCLC.
Erlotinib

Erlotinib is a hydrochloride salt of a quinazoline derivative which has anti-cancer activity. It binds to
the intracellular catalytic domain of EGFR and reversibly inhibits EGFR phosphorylation. This in turn
results in inhibition of the signalling events downstream. Erlotinib is particularly efficient in
inhibiting EGFR mutants which are constitutively activated. It is an FDA approved drug for NSCLC
and pancreatic cancer.


CLINICAL TRIALS:

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Drug Trial_ID Phase Details
Cetuximab
(Erbitux)
NCT01107639 III
Radiation Therapy and Chemotherapy, With or Without Cetuximab,
Followed by Surgery in Treating Patients With Locally Advanced
Esophageal Cancer That Can Be Removed by Surgery

This phase III trial is studying the effect of inclusion of cetuximab in a
radiation and chemotherapy based therapy regimen in locally
advanced esophageal cancer patients.
Gefitinib
(Iressa)
NCT01243398 III
Gefitinib in Treating Patients With Esophageal Cancer That is
Progressing After Chemotherapy

This phase III trial is a placebo controlled that was designed to check
efficacy of gefitinib therapy on esophageal cancer patients.
Erlotinib
(Tarceva)
NCT00686114 III
Concurrent Chemoradiotherapy Containing Paclitaxel&Cisplatin
With/Without Tarceva in Locally Advanced Esophageal Cancer

This is a phase III trial is investigating the efficacy of chemotherapy
with or without erlotinib in esophageal cancer.
Note: Clinical trials are continuously updated and hence the above may not be a complete list. Kindly visit www.clinicaltrials.gov for details on the
current status by using the clinical trial id provided above.

GENE COVERAGE LIMITATIONS

Summary of regions with reported COSMIC mutations showing low coverage (<500X reads in tumor) and (<20X reads in normal).
The percentage of the gene target regions and COSMIC mutations NOT covered in the test are provided below:

Gene
Tumor Normal
Number of COSMIC
mutation not
covered (<500X)
% Gene with low
coverage (<500X)
Number of COSMIC
mutations not
covered
% Gene with low
coverage (<20X)
ERBB2 31/52 35.66 0/52 0.00
GNA11 0/16 0.96 0/16 0.96
SMO 0/21 1.82 0/21 0.00
SRC 2/2 9.63 0/2 0.00
STK11 9/119 1.17 9/119 1.17
VHL 33/626 4.31 0/626 0.00


REFERENCES

Amundson et al. (1998) Roles for p53 in growth arrest and apoptosis: putting on the brakes after genotoxic stress. Oncogene
17:3287-99. [PMID: 9916991]
Drenckhan et al (2014) Esophageal carcinoma cell line with high EGFR polysomy is responsive to gefitinib. Langenbecks Arch Surg
[PMID: 25070024]

Hanawa et al (2006) EGFR protein overexpression and gene amplification in squamous cell carcinomas of the esophagus. Int J
Cancer 118(5):1173-80. [PMID: 16161046]

Hanel et al (2013) Two hot spot mutant p53 mouse models display differential gain of function in tumorigenesis. Cell Death Differ
20(7):898-909. [PMID: 23538418]

Illson et al (2013) A phase 2 trial of erlotinib in patients with previously treated squamous cell and adenocarcinoma of the
esophagus. Cancer 117(7):1409-14. [PMID: 21425140]


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Janmaat et al (2006) Predictive factors for outcome in a phase II study of gefitinib in second-line treatment of advanced esophageal
cancer patients. J Clin Oncol 24(10):1612-9. [PMID: 16575012]

Kawaguchi et al (2007) Cetuximab induce antibody-dependent cellular cytotoxicity against EGFR-expressing esophageal squamous
cell carcinoma. Int J Cancer 120(4):781-7. [PMID: 17096332]

Kitagawa et al (1996) Further evidence for prognostic significance of epidermal growth factor receptor gene amplification in
patients with esophageal squamous cell. Clin Cancer Res 2(5):909-14. [PMID: 9816249]

Lorenzen et al (2009) Cetuximab plus cisplatin-5-fluorouracil versus cisplatin-5-fluorouracil alone in first-line metastatic
squamous cellcarcinoma of the esophagus: a randomized phase II study of the Arbeitsgemeinschaft Internistische
Onkologie. Ann Oncol 20(10):1667-73. [PMID: 19549707]

Marx et al (2010) Homogeneous EGFR amplification defines a subset of aggressive Barrett's adenocarcinomas with poor prognosis.
Histopathology 57(3):418-26. [PMID: 20840671]

Ory et al (1994) Analysis of the most representative tumour-derived p53 mutants reveals that changesin protein conformation are
not correlated with loss of transactivation or inhibition ofcell proliferation. EMBO J 13(15):3496-504. [PMID: 8062826]

Reles A et al. (2001) Correlation of p53 mutations with resistance to platinum-based chemotherapy and shortened survival in
ovarian cancer. Clin Cancer Res. 7:2984-97. [PMID: 11595686]
Ribeiro et al (1998) p53 sequence analysis predicts treatment response and outcome of patients with esophageal carcinoma. Cancer
83(1):7-18. [PMID:9655287]

Tew et al (2005) Targeted therapies for esophageal cancer. Oncologist 10(8):590-601.[PMID: 16177283]

Wells (1999) EGF receptor. Int J Biochem Cell Biol 31(6):637-43. [PMID: 10404636]

Xu et al (2013) Role of epidermal growth factor receptor tyrosine kinase inhibitors in the treatment of esophageal carcinoma and
the suggested mechanisms of action. Oncol Lett 5(1):19-24. Epub 2012 Oct 24. [PMID: 23255886]

Xu et al (2014) Unequal prognostic potentials of p53 gain-of-function mutations in human cancers associate with drug-metabolizing
activity. Cell Death Dis 5:e1108. [PMID: 24603336]
















Urvashi Bahadur, Ph.D
Associate Director
Medical Genetics and Genomics

Mithua Ghosh, PhD
Director-Research and Development

Preveen Ramamoorthy, MS, PhD
Laboratory Director
Adjoint faculty, National Jewish Health and
University of Colorado School of Medicine,
Denver, USA.
Some supplementary information is listed below. For additional information or queries regarding these results, please call 080
23095252 ; 80-40787263 or E-mail-support.triesta@strandls.com
Digitally signed
by Urvashi
Bahadur, PhD
Date: 2014.08.08
18:29:02 +05'30'
Digitally signed by Preveen
Ramamoorthy
DN: cn=Preveen Ramamoorthy,
o=Strand LIfe Sciences, ou,
email=preveen@strandls.com, c=US
Date: 2014.08.08 20:32:55 +05'30'

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SUPPLEMENTARY INFORMATION

Malignant transformation of cells is driven largely by somatic DNA mutations. While current modes of treatment are successful in limiting
the growth of a large number of tumors, there is considerable variability in the degree of effectiveness of conventional therapies. It is often
difficult to predict the sensitivity of a given therapy for a particular patient. Identification of changes in the genome would enable
personalized treatment. Increasing evidence links somatic mutations in key genes involved in cancer to overall outcome and/or
responsiveness to particular types of therapy. It may thus be useful to determine which of these hot spot mutations is present in a given
patients tumor type.

About the Test: Strand Somatic 48-gene Test is a Next Generation Sequencing based test which profiles a patients tumor genotype in
order to detect genetic alterations in the hot spot regions in a comprehensive panel of well curated 48 tumor genes. Some of the
alterations detected may have bearing on prognosis and/or therapeutic options and may provide relevant information that allows a doctor
to consider various lines of targeted treatment for the patient.

The panel included mutation hot spots in 48 genes: ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2,
ERBB4, FBXW7, FGFR1,FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR, KIT, KRAS, MET, MLH1, MPL,
NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, VHL.

Methodology
Genomic DNA was extracted from FFPE tissue using a commercial kit (Qiagen). DNA from the paired normal sample (saliva) was isolated
using another commercial kit (DNA Genotek). The FFPE DNA was qualified using the illumina Infinium assay. Amplicon based library was
prepared using FFPE and saliva DNA. Libraries were pooled and loaded on Illumina MiSeq platform to yield multitude of reads for each
region of interest.

Analyses
Paired end NGS reads obtained from the IlluminaMiSeq sequencer were aligned against the hg19 reference genome using the MiSeq
Reporter software from Illumina. The aligned files were imported into Avadis NGS 1.5 for QC and downstream analysis. Reads with average
quality below Q20 were filtered. The Avadis NGS variant caller was used to identify single base variants (SBV) and multi-base variants (MBV)
at all locations with coverage of at least 10X. Variants with Phred confidence above 50 were annotated using the dbSNP 137 database. The
SNP effect analysis feature of Avadis NGS was used to identify the functional effects of the variants on RefSeq transcripts of the genes.

Primary Tools
Avadis NGS: Avadis NGS 1.6 (http://www.avadis-ngs.com) is an NGS analysis platform from Strand Life Sciences. It comprises algorithms
for alignment, variant calling, exon deletion/duplication analysis, and structural variant calling. A builtin genome browser enables
inspection of read level data. Several QC steps enable inspection of read quality. Avadis NGS has been cited in at least 50 publications.

Literature review: Strand has developed its text mining technology which is integrated with Strand's proprietary data mining platform -
Avadis. Avadis Text Mining engine can extract key concepts, sentiments, and relationships from textual or "unstructured" data and
convert them to a structured format that can be used to create predictive models. In the first pass, Pubmed abstracts are processed using
this tool to find mutations in key genes. All extracted information is then manually curated for validation.


LIMITATIONS AND DISLAIMER
The test was performed on an experimental protocol using Illumina 48 gene chip for targeted hot spots. Genomic DNA extracted
from FFPE tissue can sometimes lead to DNA degradation or poor quality DNA sequencing due to improper fixation with formalin.
As with any laboratory test, there is a small chance that this result may be inaccurate for a procedural reason, such as an error
during specimen collection and labelling (incorrect patient identification), an error in processing, data collection, or interpretation.
The Strand Somatic 48-gene test analyzed the following types of mutations: nucleotide substitutions, small deletions, small
insertions (including small repeat expansion) and small indels. It is not intended to analyze any of the following types of mutations:
gross deletion/duplications or rearrangements, deep intronic mutations, and other gross chromosomal abnormalities.
Furthermore, we report only those mutations, which are clinically relevant. The pattern and frequencies of mutation types vary
with the genes tested and from tumor to tumor. A negative result from the analysis does not rule out the possibility that the tumor
sample carries mutations at a low frequency. The Strand Somatic 48-gene test analysis has been designed to identify mutations
that occur at >10% frequency in the tumor DNA sample compared to the individuals paired normal DNA. The minimum amount of
tumor content in which Strand Somatic 48-gene test can detect somatic mutations with optimal sensitivity is 30% tumor content
in the specimen. If a tumor specimen has less than 30% tumor content, we cannot guarantee that our Strand Somatic 48-gene test
will be able to detect ANY somatic mutations in such sample. Furthermore even if a tumor has >30% tumor content it is impossible
to estimate the number of mutation specific population of tumor cells hence the sensitivity of the test can vary from sample to
sample. Sample mix up of either tumor or normal samples can result in errors. If the mutations are in regions of low or no coverage,
they may be missed. The Strand Somatic 48-gene test detects a high but variable percentage of known and unknown mutants of
the classes stated above. A negative result from the analysis cannot rule out the possibility that the tested individuals tumor
sample carries at an undetectable frequency one or more the known mutation or rare, novel mutations or mutations in genes not
previously associated with cancer and hence not included in the panel. The quality of sequencing and coverage varies between
regions. Many factors such as homopolymers, GC-rich regions etc. influence the quality of sequencing and coverage. This may

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result in an occasional error in sequence reads or lack of detection of a particular genetic lesion. Tumor cells are highly proliferative
and are prone to genomic instability. During each S-phase (DNA replication); it is likely that several additional mutations will be
incorporated in tumor cells therefore the mutational profile of a particular tumor may differ at different sampling points. Accurate
interpretation of this report is dependent on detailed clinical history of the patient. In the event of unavailability of detailed clinical
history, the lab cannot guarantee the accuracy of the interpretation. Not all mutations detected may be listed in the report.
Inclusion of mutations is dependent upon our assessment of their significance. The therapeutic implications listed in the report are
based on the interpretation derived as a result of extensive analysis of up-to-date scientific literature from various sources and
clinical trials available and are likely to present studied therapeutic options. The report should be carefully assessed by the treating
physician and further interpreted along with clinical, histopathological findings, contradictions and guidelines before deciding the
course of therapy.

COMPLIANCE STATEMENT
This assay is used for clinical purposes and was developed, and its performance characteristics determined by Strand Center for
Genomics and Personalized Medicine at Strand Life Sciences. It has not been cleared or approved by the US Food and Drug
Administration. The FDA has determined that such clearance or approval is not necessary. This laboratory is following the regulatory
requirements and guidelines of Clinical Laboratory Improvement Amendments of 1988 (CLIA-88) and College of American
Pathologists (CAP), NABL and ISO15189. Genetic counselling is recommended for all patients undergoing genetic testing. We follow
the American Society of Medical Genetics (ACMG) guidelines regarding guidelines for test validation, variant classification and
clinical reporting



****************END OF REPORT****************