Minimal Residual Disease in AML can be Monitored Utilizing
Cell-Free DNA

H029

Lisa Chamberlain, Zhiyi Xie , Andrew Carson, Valerie McClain, Ogeen Kiya, Bradley Patay, Martin Blankfard, Timothy Stenzel*, Jeffrey Miller.
Invivoscribe, Inc., San Diego, CA USA

Introduction
Cell-Free DNA (cfDNA) isolated from plasma can be a source of tumor DNA and has immense potential for use fortracking disease status. Though our MyMRD assay was developed for use with genomic DNA from whole blood, here
we demonstrate its utility using cfDNA as the specimen type. At least one study has demonstrated that measuring themolecular disease burden in B-cell lymphoma patient specimens in plasma is higher compared with circulating cells(1) and that malignant mutations match those observed in bone marrow biopsies (2). Additionally, cfDNA can often
contain mutations not observed in peripheral blood, blasts, or bone marrow (3). Thus, cfDNA has great potential foruse in AML and other malignancies.
Here we demonstrate our ability to store whole blood for up to 72 hours prior to fractionation into plasma, increasing
the feasibility of using cfDNA as a specimen type. Additionally we generate whole genome libraries from cfDNA, while
maintaining the characteristic size range of cfDNA. The pooling and subsequent hybridization of these whole genome
libraries with our MyMRD probes to generate targeted libraries for sequencing also maintains cfDNA size range. The
ability to track mutations in cfDNA to at least 0.1% mutational load is demonstrated, indicating high utility of thismethod with MRD in hematological malignancies.

II: Plasma preservation in two different preservation tube types

A comparison of two plasma preservation tubes was performed (Vendor A and Vendor B), with plasma being
processed at 2 hours and 48 hours. No significant difference in tubes was observed, indicating that either
preservation tube can be used for this timeframe prior to plasma processing (Figure 3, Table 2).
Figure 3: cfDNA from preservation tubes is stable forup to 48 hours post draw in multiple tube types

Materials & Methods
cfDNA collection, plasma processing (fractionation of plasma from blood), and extraction of cfDNA from plasma were
optimized prior to processing samples from patients and healthy donors. All EDTA samples were processed within 2hours of blood draw, while samples stored in plasma preservation tubes were stored for indicated time periods prior
to plasma processing. cfDNA concentrations were assessed using fluorometric methods. DNA fragments with sizes
similar to cfDNA were generated for initial feasibility and linearity studies. Genomic DNA was fragmented bysonication and selected for a mean size of ~160bp. Whole genome libraries, generated from cfDNA and fragmented
DNA, were hybridized with MyMRD® probes. Enriched libraries were sequenced using Illumina platforms. Sequencing
data was analyzed using proprietary Invivoscribe MyInformatics® software.

Results
I: Plasma preservation for up to 72 hours

cfDNA is easily contaminated by cellular DNA, thus typical methods require fractionation of whole blood from EDTA
tubes into plasma within 2 hours of blood draw. A 2 hour time limit on plasma processing is difficult to accomplish in
the real world, thus we worked with plasma preservation tubes from Vendor A to explore their ability to maintain
cfDNA over time. Whole blood from 10 healthy subjects was drawn into 4 tubes, one EDTA, and 3 Vendor A plasmapreservation tubes. Plasma processed from blood stored in Vendor A tubes maintained a cell-plasma interface for theduration of the experiment. However, plasma at late time points was visually observed to have greater hemolysis
compared to plasma from EDTA tubes processed within 2 hours of blood draw (Figure 1). This hemolysis effect (seenas a red tint in plasma at cell-plasma interface) is highly dependent on the subject and is not expected to affect cfDNA
concentrations or variant read frequencies.
cfDNA concentrations from plasma derived from EDTA (2 hour) and Vendor A tubes at multiple time points (2, 24, 72hours) exhibit no significant increase over time, indicating that the tubes are maintaining cellular structures andkeeping cfDNA and genomic DNA separate (Figure 2, Table 1).
Figure 1: Plasma interface for EDTA vsplasma preservation tube
Subject

EDTA < 2 hrs

Vendor A<2 hrsVendor A~24 hrs

Figure 2: cfDNA from preservation tubes is similar
to EDTA for up to 72 hours post draw

Vendor A~72 hrs2346

Table 1: cfDNA from preservation tubes is similar toEDTA for up to 72 hours post draw

78910

TubeEDTA

Vendor Ahrs

222472

Mean Conc.
(ng/ul)

0.58

0.37

0.5

0.54

Std Dev

0.26

0.1

0.18

0.2

%CV

44.17

27.89

35.62

36.97

Table 2: cfDNA from preservation tubes
is stable for up to 48 hours post draw inmultiple tube types
Tubehrs

Vendor A
248

Vendor B
248

N

10101010

Mean Conc.
(ng/ul)

0.23

0.25

0.25

0.32

Std Dev

0.09

0.11

0.12

0.14

%CV37.4043.40

47.06

43.12

V. MyMRD cfDNA Assay can detect VAF as low as 0.1%

Libraries generated from cfDNA of 7 clinical samples were sequenced on MiSeq (Figure 6 blue lines).
Libraries generated from cfDNA of 28 healthy donors were sequenced on NextSeq (Figure 6 red lines). Totalof 25ng cfDNA was used for library preparation with the exception of one sample (see*) with only 10ng
cfDNA available. The read coverage decreased significantly for this sample when comparing with samples
with 25ng DNA input (Figure 6). The statistic read coverage data for cfDNA samples run on the NextSeq issummarized in Table 4. The required read coverage for expected LoDs are labeled as vertical dash lines inthe Figure 6. The MyMRD® cfDNA Assay could detect a VAF of 0.25% over 96% of the targeted region, and a
VAF of 0.1% over 82% of target region (Table 4). If the 10 ng sample is excluded, that number rises to 86%.
The data indicates that, with 25ng cfDNA input, the MyMRD cfDNA Assay could detect VAF as low as 0.1%
in the majority of the targeted region.
Figure 6: Sequencing depth for cfDNA samples using low sequencing vs highsequencing depth

Low sequencing depth, duplicate removalHigh sequencing depth, family formationand duplicate removal*

III: Sequencing Library Generation from cfDNA

cfDNA extracted from plasma displayed a mononucleosomal peak at ~166 bp and following di- and tri- nuclosomal
peaks (Figure 4A). Whole genome libraries were generated using 10-25 ng of DNA input (Figure 4B), these libraries
exhibit similar characteristics to the extracted DNA. Libraries were then hybridized to our MyMRD® panel baits. An
example bioanalyzer trace of a cfDNA final library is shown below in Figure 4C. As seen in whole genome libraries,mononucleosomal, di- and tri- nucleosomal peaks were maintained from cfDNA extraction through final librarygeneration.
Figure 4: cfDNA extracted from plasma, whole genome libraries, and final libraries all exhibit
typical characteristic peaks of cfDNAA) cfDNAB) Whole Genome LibraryC) Final Library

5%

1%

0.5%

0.1%

Table 4: Read coverage for cfDNA samples with high sequencing depth

Expected LoD

Read Depth Required

0.10%

Percentage of Targets Met Required Read Depth
MeanSD%CV

9151

82.97%

16.61%

20.02%

0.25%

3659

96.49%

3.91%

4.05%

0.50%

1829

99.06%

1.48%

1.50%

1%

913

99.78%

0.50%

0.50%

2.50%

364

100.00%

0.02%

0.02%

5%

181

100.00%

0.00%

0.00%

Conclusions

IV. Excellent Linearity of the MyMRD cfDNA Assay

A linearity panel was generated by diluting DNA from 5 cell lines containing known variants into a background ofgenome in a bottle (NA12878) DNA from 20% to 0.1%. Contrived samples were then sheared to generate
fragments of approximately 160 bp to mimic cfDNA. Samples were sequenced to an average depth of 1372.
Representative variants with Expected variant allele frequency (VAF) is plotted against observed variant readfrequency (VRF) in Figure 5. R2 values are listed in Table 3.
Figure 5: Expected variants demonstrate excellent
linearity using cell line DNA sheared to cfDNA sizes

15Results

Results

Table 3: Expected variantsdemonstrate excellent linearity using
cell line DNA sheared to cfDNA sizes
GeneVariant TypeR2

DNMT3A G/A

SNV

0.975

FLT3 C/A

SNV

0.993

FLT3 ins30

Indel

0.991

NPM1 '+TCTG

Indel

0.981

TP53 dupC

Indel

0.998

The MyMRD® cfDNA assay was designed to detect at least one clinically actionable driver variants in ~95%
of all AML patients for purposes for monitoring minimal residual disease. Plasma, containing cfDNA can bepreserved for up to 72 hours, providing the possibility for clinical application. Higher sensitivity, as low as
0.1% VAF, can be obtained through deeper sequencing with error correction utilizing unique molecular
identifiers and family formation. This assay can potentially replace invasive BM sampling and provide analternative accurate test for monitoring of patients receiving targeted therapy.
The MyMRD® assay is currently available for research use only (RUO) and will be available as a CLIA/CAP
registered assay by LabPMM LLC starting in January.

References and Acknowledgements
1) D.M. Kurtz et.al. Noninvasive monitoring of diffuse large B-cell lymphoma by immunoglobulin highthroughput sequencing. Blood, 11 June 2015. Volume 125 #24.
2) O. Kis et.al. Circulating tumor DNA sequence analysis as an alternative to multiple myeloma bonemarrow aspirates. Nature Communications, 22 May 2017. 8:15086.
3) Hocking, et.al. Liquid biopsies for liquid tumors: emerging potential of circulating free nucleic acidevaluation for the management of hematologic malignancies. Cancer Biol Med, 2016 10.20892.
The authors would like to thank Michael Smitherman for assistance with figure generation.

AMP 2018, November 1-3, San Antonio, TX, USA*Present affiliation: Office of In Vitro Diagnostics and Radiological Health, FDA.