A SENSITIVE NGS ASSAY TO DETECT MEASURABLE RESIDUAL DISEASE (MRD) IN 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, United States; 3Reference Oncologic Center, National Cancer
45Institute, Aviano, Italy; Salamanca University Hospital, Salamanca, Spain; Lahey Hospital & Medical Center, Burlington, United States.
Results: Cell vs DNA Contriving

BackgroundResults: LOB, LOD, and LOQ

MRD assessment in B-cell lymphoproliferative diseases has proven utility in assessing
response to therapy, refining treatment and predicting clinical outcome in patientspreviously treated with a 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 high sensitivity and specificity to identify and track
measurable residual disease using clonal immunoglobulin heavy chain (IGH) generearrangements (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.

 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.

Methods
Sample Prep

IGH FR1- LOD/LOQ Results
DNA Input Clonal cell estimates(µg)
LOD [95% CI]

Clonal MRD Frequency

Clonal cell estimates

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]

 LOD is highest value at 20 µg : 7.0 cells which is equivalent to 2.3 x10-6
 LOQ is highest value at 20 µg: 7.3 cells which is equivalent to 2.4 x10-6

DNA Input Clonal cell estimates(µg)
LOD [95% CI]

Clonal MRD Frequency

Clonal cell estimates

LOD [95% CI]

LOQ [95% CI]

Calculated (Clonal) MRDFrequency
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]

Transformed Fit to Log
Slope
1.09

Results: Precision
Sadler’s Precision Profile LOD is highest value at 20 µg : 7.0 cells which is equivalent to 2.3 x10-6
 LOQ is highest value at 20 µg: 51.4 cells which is equivalent to 1.7 x10-5

Assay Workflow%CV per Malignant Cell Level*LOQ is 14.4 cells which is equivalent 4.7 x10-6 when removing data for one sample that was an outlier

IGH FR1

Results: Linearity

 Sample DNA Input: 0.5 µg to 20 µg
 MRD Positive & Negative Controls: Run controls
 MRD Internal Controls: Quantitative Controls (Spiked into samples and Positive Control)

IGH FR1

Results: Carryover and Crossover Contamination
 The clonal sequence of a sample is searched in a previous run for carryover analysis and within thesame run for crossover contamination. No two samples should have the same clonal sequence in aprevious or within the same run.
 If target reads of a sample is greater than minimum threshold reads , a Z-score is calculated based ontarget reads, average rate and standard deviation to calculate the probability (p-value) for asequence 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 false positive.
Study

CrossoverCarryoverRead Cutoff(MRDDetection)≥ 2 reads≥ 2 reads

Minimum
Threshold Reads≥165,160≥ 19,730

Rate
95% CL ± 2 sd
1.21E-05
1.01E-04

Average Rate

1.11E-06
7.91E-06

Slope

Intercept

R2

0.5

1.06

-0.005

0.91

21.06

0.021

0.88

20

1.01

-0.542

0.85

0.5
220

IGH FR3

Log-transformed Fit

DNA Input(µg)

DNA Input
(µg)

Slope

Intercept

1.05
1.06
1.05

R2-0.017-0.020-0.588

0.80
0.82
0.74*

 MRD Detection is Linear for spanning the whole detection range of the IGH FR1 and FR3 MRD Assay (8.0E-03 to 2.6E-06)

Results: Clinical Accuracy 10 Healthy bone marrow and 20 contrived MRD clinical samples (CLL, MM, and MCL) were tested
with IGH FR1 and FR3 MRD Assay.
 The clonal sequence detected at baseline level with clonality assay (reference method) was used totrack in contrived MRD clinical samples.
 Positive Percent agreement (PPA) and Negative Percent Agreement (NPA) was 100% for both IGH
FR1 and FR3 gene targets.
N =220Clonal
FR1 Clonality
AssayNon-Clonal

DetectedNot Detected2000200

N =220Clonal
FR3 Clonality
Assay
Non-Clonal

IGH FR3
Detected Not Detected
2000200N (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

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

71.497.2 102.3 173.8

Precision(Total Variation)

37.8

35.8

39.8

53.9

59.2

63.8

72.7

97.2 104.7 175.5%CV per Malignant Cell Level

Log-transformed Fit*R2 = 0.85 when removing data for one sample that was an outlier

IGH FR1

Intercept- 0.32

 Study was conducted with two Multiple Myeloma cell lines – U-266 and LP-1
contrived in healthy bone marrow background that covered dynamic range from
2.0E-01 to 3.0E-07.
 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 (akaClonal MRD Frequency)

IGH FR3 - LOD/LOQ Results Sample Input volume: ~1-2 mL
 Specimen type: Bone Marrow Aspirates
 Two methods for DNA extraction•
Manual using QIAmp DNA Blood Midi Kit•
Automated using the Kingfisher Flex

R2: 0.87

IGH FR3

N (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

30.2Operator

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

78.3 100.6 110.4 178.4

Precision(Total Variation)

52.1

56.8

56.1

64.3

73.6

78.9

82.6 103.6 113.6 184.4

 Study was conducted with two master mix lot, 2 operators, 2 instruments, 3 DNA
inputs with 9 clinical samples (CLL, MM and MCL).
 CV% decreases with increase in Clonal Malignant cell levelConclusions
 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.
 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.
 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.

EUROPEAN HEMATOLOGY ASSOCIATION, JUNE 9-12, 2022 VIENA, AUSTRIA – INVIVOSCRIBE, Inc.