EML4-ALK fusion in a non-small

cell lung cancer

patient detected
high sensitivity
circulating
Comprehensive
non-invasive
tumor by
sequencing:
High
fidelity tumor
sequencing
of tumor-derived
circulating
cell-free DNA across 300
DNA
assay empowers
targeted therapy
decisions
Tracking Cancer in Real-Time

cancer patients

EML4-ALK fusion in a non-small cell lung cancer

patient
detected
by
high
sensitivity
circulating
tumor
Results
Methods
DNA assay empowers targeted therapy decisions

#### Here?Nisha A.

2 Stefanie
2 Richard
2 Helmy
Mohindra, MD1 Becky Nagy
MSMortimer,
LGC,2Dragan
Bahram
Kermani,
PhD,Mei,
PhD,Siew,
MD,Eltoukhy,
Eltoukhy,
Stefanie
Sebisanovic,
Gangwu
Benjamin Mortimer,
J. Schiller, LaiMun
Aubrey Lanman,
Zapanta, Helmy
AmirAli
Talasaz PhD,2
Guardant Health Inc., Redwood
City, CA PhD,2 Jyoti D. Patel, MD1
AmirAli Talasaz,
1 Northwestern University, Chicago IL, 2 Guardant Health, Inc, Redwood City, CA
Results
Introduction

We have shown that our Digital Sequencing Technology (DST) enables ultra-sensitive and ultra-specific detection of rare genomic abnormalities at the single molecule level. Standard
next-gen workflows are plagued by extremely high noise and distortion in sample-prep and sequencing. DST is able to eliminate the error and distortion created by these processes and
produce near-perfect representation of all rare variants. Moreover, our Digital Sequencing workflow enables unprecedented sensitivity as the vast majority of input DNA molecules are
converted to sequencing libraries regardless of length, content or input amount, enabling high-quality, high-diversity sequencing. We compared the sensitivity-specificity of conventional
Illumina SBS sequencing versus DST. We have shown that in sequencing a comprehensive cancer panel of 80kbps in 0.1% cancer cell line titration samples, standard SBS generates a
plethora of high-quality false positives (i.e. overlapped paired-end read
TMbases with Qscore>30) with MAFs of 0.05-5%, while DST results in completely error-free detection of every true
positive without a single false-positive (Figure 2).
We confirmed our ability for ultra-sensitive and specific detection of genomic
Limit 1
of Detection based on LNCap titrations
2
2 DNA from a cancer cell line (LNCaP) into a disease2
abnormalities by titrating cell-free
free cell-free DNA sample at ranges from 30%-0.1%. Our data shows the impressive
ability of DST to monitor 2single-nucleotide variations reliably down
2
2
1 to below 0.1% without
any false positives (Figure 3).

100

cfDNA was collected from a blood sample (2 mL tubes) and analyzed

June 2015
March 2015
using a comprehensive 68-gene Digital Sequencing
panel
1
(Guardant360) to identify
potential
genomicMD
alterations
(Figures
and 3)
Nisha
A. Mohindra,
Becky Nagy,
MS 2LGC,
Bahram Kermani, PhD, Stefanie Mortimer, PhD,
Northwestern University, Chicago IL,
2
Tissue-based approaches for tumor genetic status are limited by
Richard Lanman, MD, Helmy Eltoukhy, PhD, AmirAli Talasaz, PhD, Jyoti D. Patel, MD
Guardant Health, Inc, Redwood City, CA
Guardant360 has high sensitivity (detection of single nucleotide variants, Concordance of tumor biopsy vs. cell-free tumor
patient intolerance to repeat biopsy, insufficient tissue, risks of the
Methods
focal gene amplifications in 16 genes, EGFR indels and fusions in ALK, DST was applied to analysis of tumor-derived cell-free DNA of 61 cancer patient plasma
procedure, and cost
Results
samples. We investigated the concordance of tumor mutation profiles derived from
We have developed a differentiated sequencing assay, DigitalMethods
Sequencing
Digital Sequencing Technology
ROS1,
RET
and
NTRK1
in
87%+
of
advanced
cancer
patients)
and
ultrasequencing cell-free DNA with those derived from sequencing matched tumor biopsy
Technology, which enables true-high quality sequencing of rare variants in
cfDNA
was analyzed using a comprehensive 68-Gene Digital Sequencing
Crizotinib
therapy and
wasmelanoma
initiatedpatients
on theatbasis
the EML4-ALK
with rapid clinical improvement Pre-Crizotinib Imaging Post-Crizotinib
samples in colorectal
variousofdisease
stages. The fusion
high
circulating cell-free DNA.
Our blood-based liquid biopsy approach
analyzes
testingcells
of tumor
tissuetumor
is recommended
cell
high
(>99.9999%)
Genomic
concordance achieved in samples with overlap (90%) is summarized in Figure 4.
circulating
DNA
(ctDNA)specificity
and for
offersnon-small
a simple and
comprehensive
tool for(Guardant360)
realPanel
to identify potential genomic alterations. (Figure 1)
Imaging
Small amounts of cell-free nucleic acids originating from
cancer
tumor genetic targeted
profiling. therapy
lung cancer to determinetime
appropriate
can be found in serum and plasma and can provideTissue-based
an alternative
Guardant360 Panel 2015
approaches for tumor genetic
status are limited by

Collect 10 mL Blood from Patient

Figure 3. Quantification of mutant allele fraction of EML4-ALK fusion breakpoint
source for tumor genetic material for biomarker testingpatient intolerance to repeat biopsy, insufficient tissue, risks of the
Complete* or
Critical
Exon
Coverage
in
68
Genes
De novo detection of rare variants in cfDNA
Genomic testing of tumor tissue is recommended for non-small cell
lung (NSCLC) cancer to determine appropriate targeted therapy

Introduction

Next-Generation Sequencing

TM

can be found in serum and plasma and can provide an alternative

source for tumor genetic material for biomarker testing

AKT1

is summarized
in the
table below.
ALKclinical performance
APC
AR
AFAR
ARID1A ATM

BRCA1

BRCA2

CCND1

CDKN2A

CDKN2B

CTNNB1 EGFR

Figure 1: Workflow for deep sequencing of cell-free DNA using Digital Sequencing technology. This
technology enables true-high quality sequencing through Guardant Healths single-molecule library
preparation, digital sequencing and bioinformatics.

Diagnosis
of lung
adenocarcinoma

Multiple
QNS Biopsies

new lesions in brain.

Biopsy Bone

3/2015
Patient started on
Crizotinib

Patient declined rebiopsy

Initial Response
followed with
2 cycles of
Pemetrexed

9/2013-2/2014
Carboplatin/
pemetrexed x4;
maintenance therapy

KRAS wild type; Quantity

not sufcient for additional
biomarker testing

Carboplatin/
Pemetrexed
4 Cycles

6/2014

2/2015
Blood Sample
Guardant360

EML4-ALK fusion 0.06%

August 2015
Dramatic Response to
Crizotinib (Figure 4)

6/2014

Upon
progression,
gamma knife
surgery and
radiation

6/2014

EML4 ALK

CCND2

BRAF

CCNE1

CDH1

CDK4

CDK6

ERBB2

ESR1

EZH2

FBXW7

Base Position (bp)

(>99.9999%)

Analysis of the patients second tube of blood found an additional DNA

fragment with the identical EML4-ALK fusion breakpoint. Total mutant
allele fraction was 0.06%

Figure 6. Titration of cfDNA from a cancer patient with CNVs in EGFR and ERBB2 into disease-free cfDNA at 100%,
50%,25%,12.5% (absolute levels of 15%,7.5%,3.75%,1.875%). Red regions indicate less than 95% confidence. Black
and grey bars are the mean copy number for a given gene with errors bars indicating 95% confidence intervals. CNVs
are indicated by alternating black or grey bars outside the red region with errors bars that do not overlap with zero.

Crizotinib therapy was initiated on the basis of the EML4-ALK fusion with
rapid clinical improvement (Figure 3)
Repeat Guardant360 analysis revealed that the EML4-ALK fusion is no
longer detected (Figure 4)

TM

100

10

0.1

#"

Tumor Response Map

Somatic
Alteration
Burden

25.2%

0.01

0.3%

15.0%

Figure
4. Pre- and post-Crizotinib lung images. Image A. CT scan of lungs shows persistent disease in left
Figure 4. Concordance of SNVs detected in tumor biopsy vs cfDNA. The pie chart (left) illustrates
the total number of samples in which no overlap was observed (blue), with overlap (red), and the
lower
lobe. Image B. Repeat CT Chest shows near complete resolution of Base
leftPosition
lower
(bp) lobe masses.
samples where no SNVs were detected in the tumor. The bar graph (right) shows the average
concordance seen in all samples (left column) or only in the samples that had at least one
matching SNV between tumor and cfDNA (right column).

Clinical Utility of ctDNA analysis

CT Scan A

CT Scan B

A 62-year old male patient underwent primary tumor excision for melanoma and inguinal
LN dissection 4 years ago (stage III). The patient developed liver, spleen, axillary and
inguinal LN metastases 4 years later. The patient was refractory to two prior regimens and
had no further treatment options. Sequencing of the FFPE tumor block was negative for
BRAF alterations. After obtaining informed consent form, the patients blood was drawn for
cell free DNA sequencing which showed a BRAF V600E mutation. The patient was put on
a BRAF inhibitor for two months and the patient is currently in partial remission with >80%
shrinkage in tumor mass. After four months the patient continues to respond to treatment
with even greater shrinkage in tumor mass (Figure 5).
A

Baseline (0 months)

1st treatment (2 months)

2nd treatment (4 months)

Conclusions
B

Figure 2: Detected minor allele frequencies across an entire 80kb panel in 0.1% LNCaP cfDNA
06.11.14
10.01.14
02.19.15
titration using off-the-shelf next-generation sequencing (top) and Digital SequencingTM (bottom)
workflows. Red points are true positives and black points are false positives. It is clear from the
Whole body
cancertechnologies
tracking are too noisy to
number of false positives that current next-generation
sequencing
at below
multiple
time Digital
pointsSequencing
with TM produces no
accurately detect mutations at concentration
1%, while
Guardant Health
Technology
false positives even down to below 0.01% concentrations
and enables
high-fidelity sequencing of
single molecules (>99.999% accuracy).

Conclusions
The present work shows the strong potential clinical impact of Digital
Sequencing in analysis of circulating cell-free tumor-derived nucleic acids,
thereby allowing researchers and clinicians to comprehensively and noninvasively monitor the genetic dimension of cancer throughout the body.

Related Publications

*NCCN NSCLC biomarkers in blue

1/2015

Cri Zontinib
Therapy,
Rapid Clinical
Improvement

!"

We applied Digital Sequencing to the analysis of 320 cancer patient plasma samples to
Point Mutations
evaluate our clinical performance for the detection of SNVs in ctDNA. The results of our

58 year old female diagnosed with advanced NSCLC in July 2013 after
FGFR1
FGFR2
FGFR3
GATA3
GNA11
GNAQ
GNAS
HNF1A
Single-Molecule Library
Case
she presented with acute right hip pain. Imaging revealed a right
Preparation
HRAS
IDH1
IDH2
JAK2
JAK3
KIT
KRAS
MAP2KI
58 year-old woman, never smoker, was diagnosed with lung
proximal femur lesion, a left lower lobe lung mass, lytic
disease in
herwild type) with bone predominant metastatic
MAP2K2 MET
MLH1
MPL
MYC
NF1
NFE2L2 NOTCH1
adenocarcinoma
(KRAS
NPM1
NRAS
NTRK1
PDGFRA PIK3CA PTEN
PTPN11 RAF1
disease
February
2013. Multiple attempts to Digital
obtainSequencing
tissue for
lumbar spine, and brain metastases (Figure 1). Biopsy of
the inright
femur
additional genotyping were done but yielded insufficient tissue. She
Digital Sequencing
been evaluated
for the SMO
detection of CNVs
RET
RHEB
RHOA Technology
RIT1 has also
ROS1
SMAD4
SRC in 31 out
of 54 genes in our panel to identify clinical relevant gene amplification and deletions in
lesion was consistent with lung adenocarcinoma, KRAS
wild
type,
was treated
with carboplatin/pemetrexed
for 4 cycles with initial
STK11
TERT**
TP53
VHL (Figure 6).
cancer patient
plasma samples
Bioinformatics
response, followed by 2 cycles of maintenance Digital
pemetrexed.
She declined
insufficient tissue for additional molecular testing. She further
began
treatment
Detection of EGFR and ERBB2 CNVs in cfDNA
maintence
therapy and was observed. Subsequently, she
Amplifications
Fusions
Indels
symptomatic
progression
of
her
developed
new
brain
lesions
and
with carboplatin and pemetrexed in September 2013 osseous
and disease.
received
4
ALK
RET
AR
BRAF
CCNE1
CDK4
EGFR exon 19 deletions
She underwent gamma knife surgery and attempted
Generate Report
palliative
radiation to her spine but stopped due Cell-Free
to sideTumor
effects.
ROS1
NTRK1
CDK8
EGFR
ERBB2
FGFR1
EGFR exon 20 insertions
cycles of therapy, followed by 2 cycles of maintenance
pemetrexed.
Burden, She
Principal
Profile, ...sample
decided to undergo cell free DNA (cfDNA) testing
fromTumor
a blood
FGFR2
KRAS
MET
MYC
Maintenance therapy was stopped in February 2014to determine
due toif genomic
patient
*Complete exon coverage for genes in bold
information could be obtained on her tumor.
Figure 1
PDGFRA KIK3CA
RAF1
** Includes TERT promoter region
Figure 2. Work flow for
preference. By October 2014, she had new brain metastases
and
Patient 57 year old Female
Figure 3. Guardant360 68 gene panel
Diagnosis NSCLC
Guardant360
testing
Digital Sequencing
Case
Study
1
progressive bone disease. A repeat biopsy for molecular
assessment
4/2015Major
Genomic Driver EGFR For clinical applications of sequencing ctDNA, we developed a 54 gene panel of
Guardant360 has high
Digital sequencing
80,000 bases consisting of all exonic bases of 18 actionable cancer-related
genes
Guardant360
was recommended however she declined. She was agreeable toand coverage of the hot exons of an additional 36 onco-/tumor-suppressor
Collect 2 tubes of blood
sensitivity (detection of single
technology
uses input from
genes
(e.g., exons containing at least one or more reported somaticcell
mutations
in toResults
nucleotide variants, focal gene
free DNA
create
Guardant360 blood testing in February 2015 to evaluate for tumor cellCOSMIC or separate publications).
Sequence a patients cancer
amplifications in 16 genes,
perfect sequencing and
across
dozens
of
genes
and
EGFR indels and fusions in ALK,
Complete exon and partial intron
Exons covered with reported somaticeliminates
alterations noise created by
identify genomic alterations
free DNA (cfDNA).
EML4-ALK fusion wasstandard
detected
from a single mutant molecule
inNTRK1)
a 10
mL
ROS1, RET and
in 87%+
next generation
Capture the constantly
of advanced cancer patients)
sequencing platforms.
evolving
adaptive
response
of
7/2013
10/2014
tube of blood
and ultra-high specificity
(Figure 2)
Figure 1.
the tumor at any point in time
Initial Presentation
Progression in bones,
6/2014

0.01

Germline

from cancer cells

Small amounts of cell-free nucleic acids originating
Cell-free DNA extraction

4/2014

0.1

True

procedure, and cost

2/2013

False

Figure 3: Titration of LNCap cfDNA in disease-free cfDNA at five different concentrations: 30%, 10%, 1%,
0.3% and 0.1%. At levels below 0.1% in this study, many mutations are harbored by <1-2 molecules and
hence fundamental stochastic capture limits come into play.

Case

10

SNVs

Minor Allele Frequency (MAF, %)

Single Molecule Sequencing Via Digital Sequencing Technology

Minor Allele Frequency (MAF, %)

Introduction

Current approaches based on invasive biopsy genetic analysis can fail to capture
an accurate picture of the real-time cancer profile due to limited spatial window of
biopsy into residual disease throughout the body. Moreover, tumor re-biopsy has
significant challenges to be widely used in practice for serial monitoring of disease
progression or acquired resistance. Analysis of circulating tumor nucleic acids
(ctDNA) by massively parallel DNA sequencing has created an exciting new tool for
capturing an accurate, more complete and real-time picture of a patients tumor
profile throughout the body and over the course of disease. However, the
widespread use of this biomarker has been limited by the high-quality false
positives and non-systematic distortion present in current NGS assays, especially
where the tumor-derived fraction is low (<1%). These limitations of therefore
impeded routine clinical use of circulating DNA in oncology.

21x17mm

14x12mm

1. Lee J, Mortimer S, Mei G, Sebisanovic D, Siew LM, Eltoukhy H, Talasaz A. Ultra-high-quality sequencing assay for
comprehensive genetic panel analysis of tumor-derived circulating cell-free DNA in colorectal cancer patients, J Clin Oncol 32,
2014 (suppl 3; abstract 504)
2. Talasaz A, Sebisanovic D, Mei G, Siew LM, Eltoukhy H. Non-Invasive Solid Tumor Sequencing:True-high quality sequencing
assay for comprehensive panel anaylsis of rare tumor-derived circulating cell-free DNA, 2013 Annual Meeting of The American
Society of Human Genetics, Boston.

10x10mm

3. Mei G, Sebisanovic D, Mir A, Gulzar Z, Brooks J, Jeffrey S, Talasaz A. "Liquid biopsy-based assays to monitor residual disease
in cancer," J Clin Oncol 31, 2013 (suppl; abstract 11096)
4. Schwarz AK, Stanulla M, Cario G, Flohr T, Sutton R, Mricke A, Anker P, Stroun M, Welte K, Bartram CR, Schrappe M,
Schrauder A., "Quantification of free total plasma DNA and minimal residual disease detection in the plasma of children with acute
lymphoblastic leukemia," Ann Hematol. 2009 Sep;88(9):897-905. doi: 10.1007/s00277-009-0698-6

To our knowledge, this is the first case report of EML4-ALK fusion identified
free
circulating
tumor
DNA
analysis
5. Anker P,from
Mulcahy H,biopsyStroun M., "Circulating
nucleic
acids in plasma and serum
as a noninvasive
investigation
for cancer: time
for large-scale clinical studies?," Int J Cancer. 2003 Jan 10;103(2):149-52..

27x16mm
19x8mm
Cancer Metastasis Rev.
1999;18(1):65-73.
With single molecule
sensitivity, this
blood test was 14x8mm
able to identify a therapeutic
option
in a patient who was unable to undergo tissueFigure
5.
Tumor
Response
Map
EML4-ALK clone disappears
fromS, Lyautey
circulation
onE, Stroun
crizotinib
therapy.
7. Chen X, Bonnefoi H, Diebold-Berger
J, Lederrey C, Faltin-Traub
M, Anker P, "Detecting
tumor-related
Figure
5. Patient
AP (top)
andbone
chest (bottom)
CT scans at 0, 2, and
4 months after treatment with a BRAF
NGS
and
had
predominant
disease
based
alterations in plasma or serum DNA of patients diagnosed with breast cancer," Clin Cancer Res. 1999 Sep;5(9):2297-303.
6. Anker P, Mulcahy H, Chen XQ, Stroun M., "Detection of circulating tumour DNA in the blood (plasma/serum) of cancer patients,"

inhibitor for (a) right hip subcutaneous lesion and (b) left axillary metastatic LN. Sites of metastases are
indicated with a yellow arrow and tumor dimensions are indicated under each scan.

8. Mulcahy HE, Lyautey J, Lederrey C, qi Chen X, Anker P, Alstead EM, Ballinger A, Farthing MJ, Stroun M., "A prospective study
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Conclusion

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of small cell lung cancer patients," Nat Med. 1996 Sep;2(9):1033-5.

For more information visit www.guardanthealth.com

To our knowledge, this is the first clinical case report of EML4-ALK fusion identified
from biopsy-free circulating tumor DNA analysis
Based on detection of two molecules harboring an EML4-ALK fusion, cell-free DNA
NGS enabled a successful therapeutic intervention in a patient who was unable to
undergo tissue-based NGS