1

Early DiagnosisPrecision TherapyDynamic Monitoring

LIQUID BIOPSY FOR THE STUDY OF CTDNA IN PERIPHERAL BLOOD

IS THE GENOMA GROUP DIVISIONDEDICATED TO ONCOGENOMICS,

AND INCLUDES THREE TYPES OF ANALYSIS

ONCONEXT RISK

Test for the detection of germinalmutations predisposing to

tumors development.

TEST FOR THE DETECTION OF CIRCULATINGTUMOR DNA IN PERIPHERAL BLOOD

SAMPLES

ONCONEXT TISSUE

Test for the detection of somatic mutations in samples of freshor paraffin embedded tumor

tissue

3

IS A DIAGNOSTIC TEST THAT DETECTS THE PRESENCE OF SOMATIC MUTATIONS BY ANALYZING

CIRCULATING TUMOR DNA (CTDNA)

THE CTDNA HAS A GREAT CLINICAL UTILITY:

• Identification of genetic determinants for targeted therapies

• Real time evaluation of resistance acquisition.• Evaluation of the tumor burden• Evaluation of the response to radiation

therapy• Monitoring of the post-surgical minimal

residual disease• Early diagnosis of the tumor progression • Individual biomarker

THE NON-INVASIVE STUDY OF THE MUTATIONAL HETEROGENEITY CANBE PERFORMED AS AN ALTERNATIVE TO TISSUE GENOTYPING WHEN THE SAMPLE IS NOT SUFFICIENT, OR IN CONJUNCTION TOIT WITH THE PURPOSE OF EXPANDING THE INFORMATION.

Apoptosis

ctDNANormal cfDNA

Necrosis

TISSUE BIOPSY

Invasive and expensive Non-invasive with a good benefit/cost ratio

Strongly influenced by tumor position Independent from tumor position

Representativeness limited by genomic heterogeneity

Good representativeness of genomic heterogeneity

Sometimes diff icult or impossible to perfom

Simple and always accessible blood sample

Non-functional if the primary tumor has been removed

Can be used after surgery for early evaluation of residual disease or recovery

It often need preparation No preparation needed

The repetition of tissue sampling issubject to limitations

The repetition of the blood sample is always possible

Often it needs adequate structures with specialised surgery area

Sampling always performable in any sampling point

It often needs long waiting times Rapid waiting times both for the sampling and for the report receiving

Does not allow the dynamic monitoring of the therapeutic response or the development of resistance

Useful tool for dynamic monitoring of the therapeutic response and development and resistance

LIQUID BIOPSY THROUGH THE CTDNA ANALYSIS: A NON-INVASIVE EXAMTHAT PRESENTS NUMEROUS BENEFITS COMPARED TO TRADITIONAL BIOPSY

5

STUDIES THE MUTATIONAL HETEROGENEITY OF TUMOR AND ALLOWS IDENTIFYING ACTIONABLE MUTATIONS

TUMOR TISSUE IS CHARACTERIZED BY AREAS WITH VARIOUS GENETIC PROFILES. TISSUE GENOTYPING ON SAMPLES OBTAINED BY TISSUE BIOPSY MAY NOT DETECT ALL THE ACTIONABLE MUTATIONS PRESENT IN THE TUMOR, DUE TO:

• Limited representativeness of the tissue sample, which expresses information circumscribed to neoplasm area in which tissue sampling was performed.

• Inability to collect the sample from all the possible metastasis.

Most cancer patients are carriers of actionable mutations, therefore if a complete genetic profile is available at the time of diagnosis, it is possible to ensure a better therapeutic appropriateness both in terms of treatment success and in the optimization of health resources.

1) Schwaederle et al. “Detection Rate of Actionable Mutations in Diverse Cancers Using a Biopsy-Free (blood) Circula-ting Tumor Cell DNA Assay.” Oncotarget 7.9 (2016): 9707–9717.

0

10

20

30

40

50

60

70

80

90

100

Perc

enta

ge

Number of alterations 0 1 >2

OVERALL (N

=171

)

Lung (N

=40)

Breast

(N=4

0)

Glioblas

tom

a (N=3

3)

Genito

urinar

y (N=1

0)

Gastro

intestin

al (N

=6)

Other

(N=4

2)

32 %

26%

42%

20%

20%

60%

30%

25%

45%

20%

40%

17%

83%

40%

73%

24%

3%

62%

31%

7%

% Actionable Mutation (tot 171)

% Non-Actionable Mutation (tot 171)

0

9

18

27

36

45

54

63

72

81

90

Overall

Lung

Breast

Glioblastoma

Genitourinary

Gastrointestinal

Other

Table. 1. Mutations detected on 171 patients un-derwent to liquid biopsy.1

Table. 2. Percentage of mutations detected: actionable vs not actionable.1

ANTI-TUMOR THERAPIES CANREDUCE TUMOR BURDEN, BUT ALSO INCREASE THE MUTATIONAL COMPLEXITY DUE TO THE THERAPEUTIC SELECTIVE PRESSURE.2

CTDNA ANALYSIS ALLOWS STUDYING THETUMOR DYNAMIC DURING TREATMENT AND EARLY DETECTING ACQUIRED RESISTANCE.3

A study shows that the CTDNA monitoring allows detecting acquired resistance mutations (T790M not present at the pre-treatment tissue genotyping) up to 2,2 months before clinical progression, in almost 50% of patients treated with TKI.4

2) Diaz and Bardelli. “Liquid Biopsies: Genotyping Circulating Tumor DNA.” Journal of clinical oncology : official journalof the American Society of Clinical Oncology 32.6 (2014): 579–586.

3) Murtaza et al., Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA. Natu-re. 2013;497:108–112.

4) Zheng et al., Plasma EGFR T790M ctDNA status is associated with clinical outcome in advanced NSCLC patientswith acquired EGFR-TKI resistance. Sci. Rep. 2016;6:20913.

ALLOWS REAL TIME MONITORING OF RESISTANCE ACQUISITION TO THERAPEUTIC TREATMENT

7

5) Yang, Meng et al. “Circulating Mutational Portrait of Cancer: Manifestation of Aggressive Clonal Events in Both Earlyand Late Stages.” Journal of Hematology & Oncology 10 (2017): 100.

IS USEFUL TO EVALUATE THE QUANTITATIVE AND QUALITATIVE TUMOR BURDEN

A RECENT STUDY CONDUCTED ON ONCOLOGIC PATIENTS, SHOWS THE MANTEINANCE OF THE CORRELATION BETWEEN TUMOR BURDEN AND LOWER LIFE EXPECTANCY, REGARDLESS OF THE TYPE OF TUMOR.5

Fig. 1. n = number of mutations; n. pts = number of patients

IS USEFUL FOR MONITORING THE EFFECTIVENESS OF RADIATION THERAPIES

FOLLOWING RADIATION TREATMENTS, THE CTDNA LEVELS INCREASE DUE TO CELL DEATH, AND SUBSEQUENTLY DECREASE UNTIL THEY ARE NO MORE DETECTABLE IF THE TREATMENTS HAD SUCCESS.6

The employment of liquid biopsy for monitoring ctDNA levelsin the patient (before, during and as follow-up of radiotherapy)to evaluate its effectiveness, is to be considered as the mostpromising option today. 6

6) Chaudhuri et al. “Predicting Radiotherapy Responses and Treatment Outcomes through Analysis of Circulating Tumor DNA.” Seminars in radiation oncology 25.4 (2015): 305–312.

Surveillance by imaging (CT and/or PET-CT) of the patient treated with radiotherapy may have an unfavorable risk/benefit relationship.6

TIME (H)

ctD

NA

RADIOTHERAPY

9

The test for the study of ctDNA should be performed 6-8 weeks after surgery, and before the adjuvant treatment. 2

2) Diaz and Bardelli. “Liquid Biopsies: Genotyping Circulating Tumor DNA.” Journal of clinical oncology : official journal of the American Society of Clinical Oncology 32.6 (2014): 579–586.

7) Diehl, Frank et al. “Circulating Mutant DNA to Assess Tumor Dynamics.” Nature medicine 14.9 (2008): 985–990.

8) Garcia-Murillas et al. Mutation tracking in circulating tumor DNA predicts relapse in early breast cancer.Sci. Transl. Med. 2015, 7, 302ra133.

ALLOWS MONITORING THE RESPONSE TO SURGERY

A STUDY7 CONDUCTED ON COLORECTAL CANCER PATIENTS SHOWS THAT THE CTDNA PRECENSE, IF STILL DETECTABLE AFTER TUMOR SURGERY REMOVAL, CONSIDERABLY INCREASES THE RELAPSE RISK

Another study8 conducted on women with non-metastatic breast cancer, who underwent to pre- and post- surgical ctDNA test, shows that ctDNA levels that are still significant after surgery suggest the presence of residual disease.

The > 90% of ctDNA+ patients had a relapse.

The ctDNA monitoring test was performed every six months as follow-up after surgery.

90% ctDNA+

10% ctDNA-

PATIENTS WITH RESIDUAL DISEASE AND POST-SURGICAL RELAPSE

ALLOWS THE EARLY DETECTING OF THE PROGRESSIVE DEVELOPMENT(UP TO 16 WEEKS BEFORE THE IMAGING) 9

THE CTDNA IS ALREADY DETECTABLE FOR MASSES OF JUST 50 MILLION CELLS, SIZE LOWER THAN THE LIMITS OF RESOLUTION OF RADIATION TECHNIQUES (7-10 MM, 1 BILLION CELLS). 10

A clinical study11 suggests that the serial evaluation of ctDNA can predict the progression about 100 days prior to the objective clinical progression

9) Oellerich et al., Using circulating cell-free DNA to monitor personalized cancer therapy, Crit Rev Clin Lab Sci. 2017 May;54(3):205-218.

10) Glenn Francis e Sandra Stein. Circulating Cell-Free Tumour DNA in the management of Cancer. Int. J. Mol. Sci. 2015, 16, 14122-14142

11) Kato et al. “Numerical Indices Based on Circulating Tumor DNA for the Evaluation of Therapeutic Response and Disease Progression in Lung Cancer Patients.” Scientific Reports 6 (2016).

20X

50 MILLION CELLSNot detectable by imaging

1 BILLION CELLS (7-10 MM)Detectable by imaging

11

The ctDNA can also be used as biomarker for all tumors for which there are no alternative protein biomarkers.

2) Diaz and Bardelli. “Liquid Biopsies: Genotyping Circulating Tumor DNA.” Journal of clinical oncology :official journal of the American Society of Clinical Oncology 32.6 (2014): 579–586.

12) Pereira E. et al.(2015) Personalized Circulating Tumor DNA Biomarkers Dynamically Predict Treatment Responseand Survival In Gynecologic Cancers. PLoS ONE 10 (12): e0145754. doi:10.1371/journal.pone.0145754.

ALLOWS THE EMPLOYMENT OF CTDNA AS BIOMARKER

THESE CHARACTERISTICS FEATURES CONVENIENTCOMPARED TO OTHER BIOMARKERS SUBJECT TO FALSE POSITIVES (E.G. PSA, CA19-9 AND CA125). 2

THE CTDNA IS AN INDIVIDUAL BIOMARKER:• Specific for disease and patient• Short half-life (from a few minutes to a few hours)• Fast increase/decrease proportionally to tumor burden

In a 2015 study 12 , on patients with ovarian cancer, the ctDNA provides information about the tumor growth,

which will implicate the bowel resection of the intestine, about 6 months earlier compared to CA-125 and 4

months before compared to CT-PET.

Ct P

ET: N

egat

ive

4 MONTHSctDNA v CT (false negative)

PT 137

7 MONTHSctDNA v Surgery

6 MONTHSctDNA v CA-125

30

25

20

15

10

5

0

600

500

400

300

200

100

Surg

ery:

Tum

or -

Bow

el R

esec

tion

25M 26M 27M 28M 29M 30M 31M 32M 33M

ctD

NA

(mut

ant

copi

es p

er 1

mL

seru

m)

CA-125 (U

/mL)

ENSURES THE HIGHESTQUALITATIVE STANDARDS

RELIABILITYTARGET COVERAGE - MINIMUM COVERAGE OF 25 000 READS

ON OVER THE 99% OF THE VARIATIONS STUDIED

SENSITIVENESS AND SPECIFICITYVERY HIGH RESOLUTION - DETECTION OF TARGETED

MUTATIONS UP TO 0.1% (MUTANT ALLELE FREQUENCY - MAF)

HIGH TECH POTENTIAL15 NGS SEQUENCERS (ILLUMINA AND THERMOFISHER)

FAST TAT15 DAYS

HIGHLY QUALIFIED STAFF80 PROFESSIONALS (MOLECULAR BIOLOGISTS AND

LABORATORY TECHNICIANS)

13

CLEAR AND COMPLETE REPORTING

TECHNICAL REPORTTHE REPORT IS FURNISHED WITH A DETAILED TECHNICAL

REPORT, WHICH INCLUDES INFORMATION ABOUT THE

PURPOSE OF THE TEST, THE INTERPRETATION OF RESULTS

AND THE PARAMETERS OF THE ANALYSIS.

THERAPIES AND CLINICAL TRIALS REPORTADDITIONAL REPORT THAT CONTAINS THE INTERPRETATION

OF THE VARIATIONS WITH FDA, NCCN, ESMO AND EMA

INDICATIONS FOR THE TARGETED TREATMENT OF THE

PATHOLOGY ON THE BASIS OF THE IDENTIFIED MUTATION

AND THE ELIGIBILITY OF THE PATIENT TO CLINICAL STUDIES

IN PROGRESS.

THE AVAILABILITY OF 6 DIFFERENT MULTIPLE GENETIC PANELS ALLOWS SATISFYING ANY DIAGNOSTIC NEED

AKT1 ALK APC AR CTNNB1ATM EGFRBRAF CDH1 CDKN2A

GNA11 GNAQ GNAS HNF1A

KRASHRAS MAP2K1IDH1 KDR KIT

ERBB2 ERBB3

ERBB4 ESR1 FGFR3EZH2 FOXL2FBXW7 FGFR1 FGFR2

MET MLH1 MPL NOTCH1 NRAS PDGFRA

PTPN11 RB1PIK3CA PTEN RET ROS1 SF3B1 SMAD4

TP53

SMARCB1

VHL

SMO SRC STK11

50

AKT1 ALK AR BRAF CTNNB1 EGFR ERBB2 ESR1 FOXL2 GNA11

ROS1 SMAD4 TP53

KIT

KRAS MEK1 (MAP2K1) MET

GNAQ

NRAS PDGFRA PIK3CA PTEN RET

23

15AKT1 BRAF EGFR ERBB2 FOXL2 GNA11 GNAQ KIT KRAS MET PDGFRA

PIK3CA RET TP53

NRAS

COLONAKT1 APC BRAF CTNNB1 EGFR ERBB2 FBXW7 GNAS KRAS MAP2K1 PIK3CA

SMAD4 TP53

NRAS

BREASTAKT1 EGFR ERBB2 ERBB3 ESR1 FBXW7 KRAS PIK3CA SF3B1 TP53

LUNGALK BRAF EGFR ERBB2 KRAS MAP2K1 MET NRAS PIK3CA ROS1 TP53

15

TABLE OF THE GENES INCLUDED IN THE ONCONEXT LIQUID PANELS AND MAIN REFERENCE TISSUES

Gene Lung Gastrointestinal Breast Gynecological Prostate/Urinary Other

AKT1

ALK

APC

AR

ATM

BRAF

CDH1

CDKN2A

CTNNB1

EGFR

ERBB2

ERBB3

ERBB4

ESR1

EZH2

FBXW7

FGFR1

FGFR2

FGFR3

FOXL2

GNA11

GNAS

GNAQ

HNF1A

HRAS

IDH1

KDR

KIT

KRAS

MEK1

MET

MLH1

MPL

NOTCH1

NRAS

PDGFRA

PIK3CA

PTEN

PTPN11

RB1

RET

ROS1

SF3B1

SMAD4

SMARCB1

SMO

SRC

STK11

TP53

VHL

desi

gn b

y ev

erm

ind.

it

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WHY CHOOSE ONCONEXT TM

BY

Test performed in Italy(Rome or Milan)

20 years experience inmolecular diagnostics

Laboratories ISO 17025accredited withgroundbreakingtecnologies

Over 200.000 genetictests / year

Fast TAT: 15 days

Medical geneticists team

Test availableworldwide

Dedicated R&D teamNumerous peer reviewedpaper published in renownedinternational joutnals