H053
Validate an NGS Based Measurable Residual Disease (MRD) Assay for B-Cell Lymphoproliferative Diseases

P. Shah1, L. Hebert1, O. Diaz II1, A.A. Street1, J. Li1, Ekaterina Rudenko1, A.M. Zlotnicki1, C. Morrison2, S. Glenn2, R. Bomben3, A. Medina Herrera4, A. Wong5, and Y. Huang1 | 1Invivoscribe, Inc., San Diego, United States; 2Roswell Park Comprehensive Cancer Center, New-York, UnitedStates; 3Reference Oncologic Center, National Cancer Institute, Aviano, Italy; 4Salamanca University Hospital, Salamanca, Spain; 5Lahey Hospital & Medical Center, Burlington, United States.

BackgroundResults: Carryover and Crossover Contamination

Results: LOB, LOD, and LOQ

MRD assessment in B-cell lymphoproliferative diseases has proven utility in assessing responseto therapy, refining treatment and predicting clinical outcome in patients previously treated witha combination of treatments (Garcia-Marco et al. Haematologica 2019; Pui et al. Leukemia 2017).
Next-generation sequencing (NGS) based approaches for MRD detection have provided highsensitivity and specificity to identify and track measurable residual disease using clonal
immunoglobulin heavy chain (IGH) gene rearrangements (Rawstron, Leukemia, 2016).
Here, we report a sensitive NGS-based B-cell MRD Assay to detect MRD in B-cell malignancies
such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), multiple
myeloma (MM) and mantle cell lymphoma (MCL) with IGH FR1 and FR3.

Methods
Sample PrepØ LOB Results: No clonal sequence was detected in 10 healthy bone marrow samples tested
at 0.5 µg and 20 µg (N= 180 per master mix lot) with both IGH FR1 and FR3 gene targets.
Thus, LOB was zero.

DNA Input(µg)

Clonal cell estimates

Clonal MRD Frequency

Clonal cell estimates

LOD [95% CI]

LOD [95% CI]

LOQ [95% CI]

Calculated (Clonal) MRD
Frequency
LOQ [95% CI]

0.5

7.0 [4.9 - 12.1]

9.1E-05 [6.4E-05 - 1.6E-04]

5.4 [3.8 - 8.5]

7.0E-05 [4.9E-05 - 1.1E-04]

2

4.7 [3.5 - 7.7]

1.5E-05 [1.1E-05 - 2.5E-05]

4.6 [2.6 - 7.9]

1.5E-05 [8.5E-06 - 2.6E-05]

20

5.4 [4.0 - 8.5]

1.8E-06 [1.3E-06 - 2.7E-06]

7.3 [5.4 - 9.2]

2.4E-06 [1.7E-06 - 3.0E-06]

IGH FR3 - LOD/LOQ Results

Assay Workflow

DNA Input(µg)

Clonal cell estimates

Clonal MRD Frequency

Clonal cell estimates

LOD [95% CI]

LOD [95% CI]

LOQ [95% CI]

Calculated (Clonal) MRD
Frequency
LOQ [95% CI]

0.5

3.9 [3.0-7.2]

5.11E-05 [3.9E-05 – 9.4E-05]

6.4 [5.1-8.0]

8.3E-05[6.6E-05-1.0E-04]

2

4.7 [3.7-7.4]

1.54E-05[1.2E-05 - 2.4E-05]

12.1 [5.2-28.2]

3.9E-05[1.7E-05-9.2E-05]

20

5.7 [4.2-10.1]

1.84E-06[1.4-06 – 3.3E-06]

51.4*[6.7-396.9]

1.7E-05[2.2E-06-1.3E-04]

Crossover≥ 2 reads≥165,160

1.21E-05

1.11E-06

Carryover≥ 2 reads≥ 19,730

1.01E-04

7.91E-06

Sadler’s Precision Profile
IGH FR1%CV per Malignant Cell LevelResults: Linearity
Ø Sample DNA Input: 0.5 µg to 20 µg
Ø MRD Positive & Negative Controls: Run controls
Ø MRD Internal Controls: Quantitative Controls (Spiked into samples and Positive Control)Ø B-cell MRD Software: Designed to track clonal sequences and analyze data in follow-up samples tested
with the IGH FR1/FR3 MRD assay on an Illumina NextSeqDx® instrument, and generate reports for use by
the clinical lab to report on the assessed MRD signal of each sample.

IGH FR1

Results: Cell vs DNA Contriving
R2: 0.87

IGH FR3

Log-transformed Fit

DNA Input(µg)

SlopeIntercept

R2

0.5
220

1.06
1.06
1.01

-0.0050.021-0.542

0.91
0.88
0.85

Log-transformed FitClonal
FR1 Clonality
Assay
Non-Clonal

2000200216

216

216

216

216

216

216

Malignant Cell Input

615.4

215.4

61.5

21.5

8.0

6.2

4.0

2.2

1.5

0.6

DNA Input

12.9

10.0

14.3

10.4

0.0

5.3

13.3

0.0

22.6

24.1

Operator

3.4

0.0

0.0

0.0

5.1

0.0

0.0

0.0

0.0

3.8

MM Lot/Instrument/Run/Day

0.0

0.5

0.0

0.0

7.7

2.0

9.4

0.0

4.6

0.0

Random Error

35.4

34.3

37.2

52.9

59.0

63.6

71.4

97.2 102.3 173.8

Precision(Total Variation)

37.8

35.8

39.8

53.9

59.2

63.8

72.7

97.2 104.7 175.5

IGH FR3%CV per Malignant Cell LevelN (Total data points)

216

216

216

216

216

216

216

216

216

216

Malignant Cell Input

615.4 215.4

61.5

21.5

8.0

6.2

4.0

2.2

1.5

0.6

DNA Input

25.8

30.5

30.4

19.5

30.8

26.5

26.4

24.6

25.9

30.2

0.5
2
20

1.05
1.06
1.05

-0.017-0.020-0.588

0.80
0.82
0.74*

Operator

13.3

14.7

0.0

7.0

13.1

16.3

0.0

0.0

6.5

0.0

MM Lot/Instrument/Run/Day

10.7

11.1

9.4

8.6

14.6

0.0

0.0

0.0

0.0

35.1

Random Error

41.9

44.2

46.2

60.3

64.0

72.5

78.3100.6

110.4 178.4

Precision(Total Variation)

52.1

56.8

56.1

64.3

73.6

78.9

82.6

103.6

113.6 184.4

N =220

Not Detected

216

R2

Ø 10 Healthy bone marrow and 20 contrived MRD clinical samples (CLL, MM, and MCL) were tested
with B-Cell MRD Assay for the IGH FR1 and FR3.
Ø The clonal sequences detected during baseline testing with the reference method (clonality assay)
were used to track MRD in contrived clinical samples.
Ø Positive Percent agreement (PPA) and Negative Percent Agreement (NPA) was 100% for both IGH
FR1 and FR3 gene targets.
Detected

216

Intercept

Results: Clinical Accuracy

N =220216

Slope

Ø MRD Detection was linear across the entire detection range of the B-cell MRD Assay for both IGH FR1 and FR3 (8.0E-03 to 2.6E-06)

IGH FR1

N (Total data points)

DNA Input(µg)*R2 = 0.85 when removing data for one sample that was an outlier

Intercept- 0.32

Ø Study was conducted with two Multiple Myeloma cell lines – U-266 and LP-1
contrived in healthy bone marrow background that covered dynamic range from2.0E-01 to 3.0E-07.
Ø MRD detection of cell and DNA contrived samples was observed to be equivalent
with R2 of 0.87 and slope of 1.09 in regards to calculated (calc.) MRD Frequency(i.e Clonal MRD Frequency)

Average RateØ The clonal sequence of a sample is searched in a previous run for carryover analysis andwithin the same run for crossover contamination. No two samples should have the sameclonal sequence in a previous or within the same run.
Ø If target reads of a sample are greater than minimum threshold reads , a Z-score iscalculated to determine the probability (p-value) for a sequence to be detected.
• If p-value < 0.05 then the sample is considered detected, it’s a true positive• If p-value ≥ 0.05 then the sample is considered not detected, and signal is a falsepositive.

*LOQ is 14.4 cells which is equivalent 4.7E-6 when removing data for one sample that was an outlier

Slope
1.09

Rate
95% CI ± 2 sd

Results: Precision

Ø LOD is highest value at 20 µg : 5.7 cells which is equivalent to 1.8E-6
Ø LOQ is highest value at 20 µg: 51.4 cells which is equivalent to 1.7E-5

Transformed Fit to Log

Minimum
Threshold Reads

IGH FR1- LOD/LOQ Results

Ø LOD is highest value at 20 µg : 7.0 cells which is equivalent to 2.3E-6
Ø LOQ is highest value at 20 µg: 7.3 cells which is equivalent to 2.4E-6

Ø Sample Input volume: ~1-2 mL
Ø Specimen type: Bone Marrow Aspirates
Ø Two methods for DNA extraction•
Manual using QIAmp DNA Blood Midi Kit•
Automated using the Kingfisher Flex

Study

Read Cutoff(MRDDetection)

ClonalFR3 Clonality
AssayNon-Clonal

IGH FR3Detected Not Detected
2000200

Association For Molecular Pathology (AMP), Nov 1-5, 2022 Phoenix, Arizona – Invivoscribe, Inc.

Ø Study was conducted with two master mix lots, 2 operators, 2 instruments, 3 DNA
inputs with 9 clinical samples (CLL, MM and MCL).
Ø CV% decreases with increase in Clonal Malignant cell levelConclusions
Ø IGH FR1 exhibited a LoB at 0, LoD at 2.3E-06, LoQ at 2.4E-06, good linearity with R2>0.88 across 4.5 logs andprecision with %CV ranging from 27.6% to 63.5% at the LoD.
Ø IGH FR3 exhibited a LoB at 0, LoD at 1.8E-06, LoQ at 1.7E-05, good linearity with R2>0.74 across 4.5 logs andprecision with %CV ranging from 46.6% to 89.4% at the LoD.
Ø The B-cell MRD Assay demonstrated excellent clinical performance; yielding 100% agreement in an accuracy
study that included 20 clinical specimens (CLL, MM and MCL) and 10 healthy donors.