clinical application of cell- free dna in...

Clinical application of cell-free DNA in lymphoma

Seok Jin Kim MD, PhDMedicine

Hematology/OncologySamsung Medical Center

I have no personal or financial interests to declare:

I have no financial support from an industry source at the current presentation.

대한혈액학회 Korean Society of Hematology

COI disclosureName of author ： Seok Jin KIM

The Era of Liquid Biopsy

https://www.healio.com/hematology-oncology/

Liquid biopsy for cell-free DNA Accessing tumor DNA with a blood sampling Without the need of an invasive tissue biopsy

Cell-free fragments of DNA Shed into the bloodstream by cells undergoing

apoptosis Circulate at a low concentration in plasma as

double-stranded DNA fragments Predominantly short structure (

Clinical applications of CTC and cfDNA analyses in cancer care

Cancer Discovery 4:650-661

Role of cell-free DNA in cancer Early intervention Diagnose cancer earlier through screening

Localized disease Determine the risk of relapse after treatment

Advanced or disseminated disease Determine treatment selection based on biomarkers

Refractory disease Determine mechanisms of resistance and new targets

Timeline of liquid biopsy development

Oncotarget, 2018, Vol. 9, (No. 2), pp: 2912-2922

Number of publications per year in PubMed


Comparison of methodologies for cell-free DNA analysis


Healthy people vs. lymphoma patients Cell-free DNA in healthy people Primarily from the apoptosis of hematopoietic cells Minimally from other tissues Circulates in concentrations of 1-10 ng/mL of plasma

Cell-free DNA in lymphoma patients Primarily from apoptotic tumor cells Always increased compared with age- and gender-

matched healthy people Mean concentration of 30 ng/mL of plasma

Biochim Biophys Acta. 2007;775(1):181-232; Cell Res. 2014;24(6):766-769. Cancer Res. 2001;61(4):1659-1665. Lancet Oncol. 2015;16(5): 541-549.

Cell-free DNA in lymphoma Levels of cfDNA could be variable Subtypes Higher in aggressive lymphomas than in indolent

lymphomas Tumor volume Higher in advanced stage disease than in limited

stage disease Disease status Higher in progressive disease than in a disease

responding to treatment

Ann Oncol. 2009;20(8):1408-1413. Haematologica 2019; 104(4):648-652

Unmet needs of lymphoma

Haematologica 2019; 104(4)

Clinical relevance of cell-free DNA

• Tumor burden

• Cell of origin

• Genetic alteration

• Detection of resistance mutation

• Relapse prediction

• Transformation prediction

• Relapse prediction

• Transformation prediction

cftDNA: MRD marker in DLBCL IgHTS

Clonotypic Immunoglobulin High-throughput sequencing CAPP-Seq

Cancer personalized profiling by deep sequencing

Scherer et al., Sci. Transl. Med. 8, 364ra155 (2016)

CAPP-Sequencing

Detection of resistance mutation


cfDNA reflects tumor burden 100% of pretreatment samples

Detectable ctDNA 37% of pretreatment samples

Abnormally high LDH Better sensitivity of ctDNA than LDH


ctDNA after Allo-SCT NGS-based detection of cell-free tumor DNA

(cfDNA) after allogeneic SCT 88 patients with B- and T-cell lymphoma Peripheral blood samples: Plasma/PBMC Sequencing of Ig or TCR genes

Detectable cfDNA prior to allo-SCT 9/23 in CR (39%); 14/20 in PR (70%)

Detectable cfDNA after all-SCT 16/19 with disease progression (84%) Median of 3.7 months prior to relapse

BJH, 2016, 175, 841–850

cfDNA after Allo-SCT cfDNA positivity in multivariate analysis

Risk of relapse/progression: HR 10.8, p = 0.0006

BJH, 2016, 175, 841–850

Cell-free DNA in CNS lymphoma

6 patients with CNS lymphoma NGS tumor panel for tumor and reference tissue

(ETDA blood) Digital PCR for cfDNA from plasma and CSF

BMC Cancer (2019) 19:192


BMC Cancer (2019) 19:192


Phase 1b trial Ibrutinib-based therapy in R/R CNS lymphoma

Blood. 2019 Jan 31;133(5):436-445.

Newly diagnosed

Patient

Relapsed/Refractory

Patient

Informed consent

Clinical practice

Follow-up & Data update

Analysis Translational research

Sample: Blood, tissue

Samsung lymphoma cohort

Study Process

Targeted sequencing of tumor sample ( ~ 400 genes)

Newly visited

lymphoma patients

Informed consents for

clinical, pathology, and genomic data

analysis, and blood sampling

Blood sampling, biopsy and

imaging studies

Treatment with curative intent

Serial blood sampling for serum and cell-free tumor DNA

Follow-up and update of survival

status

Targeted sequencing of cell-free tumor DNA (~ 66 genes)

Serum cytokine analysis

• A: At diagnosis or After enrollment

• B: Interim analysis

• C: Final analysis

• D: Relapse or progression

• E: 2nd relapse or progression

LiquidSCAN-Lymphoma Overview

Sample Plasma 3-4ml

Sequencing

Platform Illumina NGS

Library KAPA (Pre-indexed)

Target enrichment Hybrid selection (Agilent)

Target region 66 genes (222 kb)

Detection limit (at 95% confidence) 0.3%

Input cfDNA 10 - 50ng

Raw depth > 20,000X

Unique read depth > 5,000X

Background error 8 Gbp

Goal of cell-free tumor DNA analysis

Lymphoma diagnosis

Tissue

Targeted sequencing

Prospective cohort cell-free tumor DNA

serial collection

1) Correlation of tumor tissue with cell-free tumor DNA

2) Correlation of cell-free tumor

DNA with minimal residual disease

Precision medicine for lymphoma patients

3) Detection of clonal changes with cell-free tumor DNA

Gene list

ACTB BTK CDKN1B GNA13 MAP3K13 PIK3CD TCL1A

AKT1 CARD11 CHEK1 IDH2 MAPK1 PIM1 TNFAIP3

ALK CCND1 CREBBP IRAK1 MAPK3 PRKCB TNFRSF14

B2M CCND3 CTLA4 IRAK4 MEF2B RHOA TP53

BCL2 CD28 CXCR4 IRF4 MTOR SGK1 TP63

BCL6 CD58 DDX3X KMT2D MYC SOCS1 XPO1

BCOR CD79A DUSP22 KRAS MYD88 STAT3

BIRC3 CD79B EZH2 MAP2K1 P2RY8 STAT6

BRAF CDK4 FYN MAP2K4 PAX5 SYK

BTG1 CDK6 GATA3 MAP3K1 PIK3CA TBL1XR1

Allele frequencies

Comparison of mutation between tissue and cell-free

tumor DNA

Comparison of mutation frequency according to treatment and

response

Leuk Lymphoma. 2019 Feb 18:1-10

The allele frequency of ctDNA estimated by targeted deep sequencing was plotted on the x-axis for NHL patients according to (A) NHL subtypes and (B) Ann Arbor stages. Box plot shows ranked normalized allele frequency in the median, 1st and 3rd quartiles, and 1.5 interquartile range (IQR) of 1st and 3rd quartiles.

Allele frequencies


Mutation profiling of tissue and/or plasma samples

Concordance of variants detected in plasma cell-free DNA samples compared with biopsy-based sequencing tests is shown in the chart.


Serial changes in patients


Progressive DLBCL


Prediction of relapse in DLBCL


Cell-free DNA is early outcomePredictors in DLBCL

J Clin Oncol. 2018 Oct 1;36(28):2845-2853

Cell-free DNA in AITL

Newly diagnosed AITL


Cell-free tumor DNA in AITL

Relapsed AITL


Cell-free DNA in ENKTL

Newly diagnosed ENKTL

Follow-up is required


Cell-free DNA in ENKTLRelapsed ENKTL


Comparison with other markers

Unpublished data

Prediction of outcome in T-cell lymphoma

Fold change of VAF at final sample

cfD

NA

amou

nt

(ng/

ml)

Max

imal

VAF

Bas

elin

e sa

mpl

e

CR PR PD

At diagnosis Interim analysis

> 2-fold reduction

< 2-fold reduction

No relapse (Follow-up < 1 year)

No relapse (Follow-up > 1 year)

Relapse within 1 year after treatment

Unpublished data

Clinical application: Cell-free DNA in lymphoma Expected role of cell-free DNA in lymphomas Diagnostic marker Predictive marker for surveillance Detection of minimal residual disease

Detection of clonal changes May help to select appropriate targeted agents Limited coverage of genes due to technological

issues and cost

See you in Busan!

Clinical application of cell-free DNA in lymphoma Slide Number 2The Era of Liquid BiopsyLiquid biopsy for cell-free DNAClinical applications of CTC and cfDNA analyses in cancer careRole of cell-free DNA in cancerTimeline of liquid biopsy developmentNumber of publications per year in PubMedComparison of methodologies for cell-free DNA analysisHealthy people vs. lymphoma patientsCell-free DNA in lymphoma Unmet needs of lymphomaClinical relevance of cell-free DNA cftDNA: MRD marker in DLBCLCAPP-SequencingDetection of resistance mutationcfDNA reflects tumor burdenctDNA after Allo-SCTcfDNA after Allo-SCTCell-free DNA in CNS lymphomaCell-free DNA in CNS lymphomaCell-free DNA in CNS lymphomaSamsung lymphoma cohortStudy ProcessSlide Number 25LiquidSCAN-Lymphoma OverviewGoal of cell-free tumor DNA analysis Gene listAllele frequenciesAllele frequenciesMutation profiling of tissue and/or plasma samples Serial changes in patientsSerial changes in patientsProgressive DLBCLPrediction of relapse in DLBCLCell-free DNA is early outcome�Predictors in DLBCLCell-free DNA is early outcome�Predictors in DLBCLCell-free DNA in AITLCell-free tumor DNA in AITLCell-free DNA in ENKTLCell-free DNA in ENKTLComparison with other markersPrediction of outcome in T-cell lymphoma Clinical application�: Cell-free DNA in lymphoma See you in Busan!

clinical application of cell- free dna in...

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