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ACCME/DisclosuresThe USCAP requires that anyone in a position to influence or control the content of CME disclose

any relevant financial relationship WITH COMMERCIAL INTERESTS which they or their

spouse/partner have, or have had, within the past 12 months, which relates to the content of

this educational activity and creates a conflict of interest.

Dr. David Wu declares having received research funding for studies with Adaptive Biotechnology,

Seattle, WA.

Clinical Application of NGS for MRD Monitoring in Lymphoid Neoplasms

David Wu, MD, PhDDepartment of Laboratory MedicineUniversity of Washington, Seattle

http://www.uscap.org/meetings/detail/2016‐annual‐meeting/sessions/2333

Agenda

• Background

• Technical considerations• Defining the clone• Ensuring minimal bias in multiplexed PCR• Sensitivity and specificity• Limitations

• Clinical applications• Acute lymphoblastic leukemia• Mature B-cell lymphomas and plasma cell neoplasms• Mature T-cell lymphomas

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Agenda

• Background

• Technical considerations• Defining the clone• Ensuring minimal bias in multiplexed PCR• Sensitivity and specificity• Limitations

• Clinical applications • Acute lymphoblastic leukemia• Mature B-cell lymphomas and plasma cell neoplasms• Mature T-cell lymphomas

Diversity of the immune system

IGHV D J C

D‐J

V‐DJ

Generic structure of IGH or TRB locus after VDJ rearrangement

Warren E H et al. Blood 2013;122:19-22©2013 by American Society of Hematology

NGS provides unprecedented TRB detail

H. Robins et al., Blood, 2009; 114:4099©2009 by American Society of Hematology

TRBV 10

J segments

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Immune profiling

Nature Biotechnology, 31(3):184‐186

• Academic institutions• Adaptive Biotechnologies (Seattle, WA and San 

Francisco, CA)

• Atreca (San Carlos, CA)

• Clontech Laboratories, Inc. (Mountain View, CA)  

• EuroClonality‐NGS Consortium• iRepertoire (Huntsville, AL), www.r10k.org

• ImmuMetrix (Palo Alto, CA)

• Invivoscribe (San Diego, CA)

• “Main objectives of the EuroClonality‐NGS consortium are to develop, standardize, and validate IG/TR NGS assays for (i) clonality assessment; (ii) MRD analysis; and, (iii) repertoire analysis

• Focus on standardization, which not only concerns the analytical phase, but also the pre‐analytical (e.g. sample preparation, target choice) and the post‐analytical phases (e.g. bioinformatics pipeline).

• Finally, a very important aim of the consortium is to validatethe technology via large‐scale testing of clinical samples and in the context of clinical trials.”

http://www.euroclonality.org/

Strategies for MRD assessment

• Multi-parametric flow cytometry (10-4 to 10-5)+ Widely used, reasonably sensitive and specific− Operator/laboratory dependent, difficult to standardize

• Molecular-based methods (10-5 to 10-6)+ More “objective”− Laborious, time-consuming− Patient-specific, requires individualized validation

Agenda

• Background

• Technical considerations• Defining the clone• Ensuring minimal bias in multiplexed PCR• Sensitivity and specificity• Limitations

• Clinical applications • Acute lymphoblastic leukemia• Mature B-cell lymphomas and plasma cell neoplasms• Mature T-cell lymphomas

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Identification of clonal TRB sequences

©2012 by American Society of Hematology

Gawad C et al. Blood 2012;120:4407-4417

• 51 random patients with B‐ALL 

• >145 million reads• Average >750K reads per sample

• 5% threshold in this cohort defined as index clonotypes

Defining a clone

B‐ALL samples      Bone marrow control

10‐1

10‐3

Freq

uency

10‐5

Controlling PCR bias is critical

Standard Multiplex PCR Assay Bias Controlled Assay

15

Courtesy of Harlan Robins, Fred Hutchinson Cancer Research Center, Seattle, WA 

Addressing multiplex PCR bias

C. Carlson et al., Nat. Commun. 2013 Oct 25;4:2680. doi: 10.1038/ncomms3680.

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Addressing PCR bias using a synthetic immune system

• Known versus unknown immune repertoire • Create TRG 56 synthetic templates (14 V x 4 J) • (Separate work with IGH 1,116 templates)

• Empirical primer titration, with computational correction of residual bias

C. Carlson et al., Nat. Commun. 2013 Oct 25;4:2680. doi: 10.1038/ncomms3680. C. Carlson et al., Nat. Commun. 2013 Oct 25;4:2680. doi: 10.1038/ncomms3680.

56 synthetic templates, rank ordered

Amplificatio

n bias, r

elative to m

ean 1st iteration

2nd iteration

3rd iteration

8th iteration

Bi-allelic TRG rearrangements should be counted comparably

C. Carlson et al., Nat. Commun. 2013 Oct 25;4:2680. doi: 10.1038/ncomms3680.

Log10 (Allele 1) 

Log 1

0(Allele 2) 

©2012 by American Society of Hematology

Faham M et al., Blood 2012; 120:5173-5180

NGS can be highly sensitivity

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©2012 by American Society of Hematology

Faham M et al., Blood 2012; 120:5173-5180

…and accurate vs. flow cytometry and ASO‐PCR

Sequences are largely specific

TRB

TRG

Wu et al., Sci Transl Med. 2012 May 16;4(134):134ra63. doi: 10.1126/scitranslmed.3003656.

Specificity: cross-patient comparisons

• TRB coincidences: 3 of 1,344 comparisons (32 x 42) = 0.22%

• TRG coincidences: 17 of 1,512 comparisons (36 x 42) = 1.12%

G. Cazzaniga and A. Biondi, Haematologica 2005; 90:382‐390 

More similarities than expected

• Sequence CDR3 regions from TRB of CD8+ T cells of 7 adults

• The CDR3 repertoire is biased toward specific V-J pairs, primarily by sequences with few inserted nucleotides

• Actual variation is < 0.1% of the estimated 5 x 1011 possible sequences

• Of 3 to 4 million T cells, only 5 TRB should be shared; however, finding is the > 10,000 TRB shared among 2 unrelated persons

Robins et al., Sci Transl Med. 2010 Sep 1;2(47):47ra64. 

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Advantages of NGS for MRD• Broad applicability• Improve standardization • Circumvent laboratory work-flow

challenges • Enhance sensitivity and specificity of

MRD detection

• MRD detection generally requires sequence of index clone

• Sample input defines limit of detection• Analytic precision is dependent on sequencing

coverage (read-depth), but limited by Poisson sampling and sequencing error

• Must understand disease biology• Clonal sequences may not define disease• Clonal sequences may not be stable through therapy

Points to consider…

Agenda

• Background

• Technical considerations• Defining the clone• Ensuring minimal bias in multiplexed PCR• Sensitivity and specificity• Limitations

• Clinical applications• Acute lymphoblastic leukemia• Mature B-cell lymphomas and plasma cell neoplasms• Mature T-cell lymphomas

NGS applied to ALL MRD

• T-ALL cohort (COG AALL0934)• 43 patients samples • Paired pre- and day 29 post-treatment

• B-ALL cohort (COG AALL0932)• 98 patients samples • Paired pre- and day 29 post-treatment

Wu et al., Science Transl. Med. 2012; Wu et al. Clin. Cancer Res. 2014

Can high‐throughput sequencing (HTS) of TCR/IG gene rearrangements detect MRD ?

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D29 Post-treatment T-ALL MRD by HTS

9 cases 10 cases 12 cases

HTS versus Flow

HTS− / Flow− HTS+ / Flow− HTS+ / Flow+

Clon

e Freq

uency

Wu et al., Sci Transl Med. 2012 

HTS+/Flow+23 cases 

HTS+/Flow−28 cases

HTS− / Flow−40 cases  

Wu et al. Clin. Cancer Res. 2014

Similar findings with B‐ALL cohort

Clon

e Freq

uency

~ 92% of unselected B‐ALL cases had clonal IGH suitable for MRD monitoring by sequencing

Questions to consider• What sensitivity is needed?• What are clinical implications of cases that are

HTS+positve and Flow−negative?

Recent papers in B-ALL

• Berlin-Frankfurt-Munster protocol with 210 samples from 76 patients

• 56 patients with B-cell ALL enrolled in Children’s Oncology Group trial ASCT0431.

M. Kotrova et al., Blood 126(8):1045 (2015)Pulsipher et al., Blood, 125(22):3501 (2015) 

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HTS+/Flow+23 cases 

HTS+/Flow−28 cases

HTS− / Flow−40 cases 

Wu et al. Clin. Cancer Res. 2014

Clon

e Freq

uency

Clinical significance of discordant cases requires further study.

What is significance of HTS+ but flow negative cases?

Flow cytometry-sorting of “mature” B cells

Sequencing

Hypothesis:  Treatment‐induced antigenic shift may explain presence of clonal IGH sequences

x 3

“mature” B cells

+

−

detector

Clonal IGH sequence may be found in triple flow-sorted “mature” B cells

Pre

Post

Wu et al. Clin. Cancer Res. 2014

Index clonal IGH sequence at 0.01% is detected in flow‐sorted “mature” B cells

Clonal “evolution” of IGH frequent in B-ALL

• Relapse clones often found retrospectively in pre-treatment samples at very low levels (Li et al., Leuk Res 2001; 25-1033; Li et al., Blood 2003; 102:4520; Choi et al., 2007; 110:632; Mullighan CG et al., Science. 2008;322(5906):1377-80.)

• Burden of relapse clone in pretreatment sample correlates with time to relapse (Choi et al. Blood 2007; 110:632)

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Mechanisms for clonal “evolution” of IGH

1. Incomplete DJ clones: on-going VH rearrangement

1. Complete VDJ clones: 5’ VH replacement modelV DJ

C

5’

5’

5’

3’

3’

3’

VDJ

VDJ

C

C

©2012 by American Society of Hematology

Gawad C et al. Blood 2012;120:4407-4417

Example of “evolved” IGH clones

Gawad C et al. Blood 2012;120:4407-4417©2012 by American Society of Hematology

Example on‐going rearrangement 

Patient 36:No evolution

Patient 50: High level of evolution

IGH‐J IGH‐V

IndexNormalEvolved

Freq

uency

NGS for ALL

• Adequate sensitivity and specificity• May contribute to detection of so-called “evolved”

clones• Further clinical validation needed

• Time point for testing?• Significance of low level MRD?

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Non-Hodgkin B-cell lymphomas and plasma cell neoplasms

• Detecting Hodgkin lymphoma• Detecting clonal IGH as circulating DNA• MRD detection for plasma cell neoplasms

Armand P, et al., Detection of circulating tumour DNA in patients with aggressive B‐cell non‐Hodgkin lymphoma, Br J Haematol. 2013 Oct;163(1):123‐6. PMID: 23795711.

Roschewski M, et al., Circulating tumour DNA and CT monitoring in patients with untreated diffuse large B‐cell lymphoma: a correlative biomarker study, Lancet Oncol. 2015 May;16(5):541‐9.  PMID:25842160.

Oki Y, et al., Detection of classical Hodgkin lymphoma specific sequence in peripheral blood using a next‐generation sequencing approach, Br J Haematol. 2015 Jun;169(5):689‐93. PMID: 25818067.

Martinez‐Lopez J, et al., Prognostic value of deep sequencing method for minimal residual disease detection in multiple myeloma, Blood. 2014 May 15;123(20):3073‐9. PMID: 24646471.

Mature T-cell lymphomas

• Help to define diagnostic boundary of reactive versus neoplastic

• Permit much more sensitive and specific monitoring

Weng WK, et al., Minimal residual disease monitoring with high‐throughput sequencing of T cell receptors in cutaneous T cell lymphoma. Sci Transl Med. 2013 Dec 4;5(214):214ra171. PMID:24307695.

Kirsch IR, et al., TCR sequencing facilitates diagnosis and identifies mature T cells as the cell of origin in CTCL, Sci Transl Med. 2015 Oct 7;7(308):308ra158. PMID: 26446955.

CTCL vs. reactive skin lesions

Kirsch IR, et al., Sci Transl Med. 2015 Oct 7;7(308):308ra158

Summary

• Clonality testing by NGS in lymphoid neoplasms is well underway

• Focus on standardization for quantitation • Ensuring optimal test utilization and assessing

clinical validity

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Acknowledgements

• University of Washington• Brent Wood, faculty colleagues, and staff of UW

Molecular Hematopathology Laboratory• T-ALL and B-ALL work

• COG collaborators: M. Loh, Stuart Winter, Kim Dunsmore (T-ALL), Anne Angiolillo (B-ALL)

• Adaptive Biotechnologies, Seattle, WA: Lanny Kirsch and A. Sherwood

• Fred Hutchinson Cancer Research Center• Harlan Robins, Fred Hutchinson Cancer Research

Center