Figure 1. QuantStudio™ Digital PCR System A) B) C)

During leukemia treatment mixed chimerism occurs in which both recipient and donor cells are present in the bone marrow or peripheral blood after transplantation. Chimerism analysis is performed to monitor peripheral blood or bone marrow in the recipient after allogenic stem cell transplantation to monitor for leukemic relapse. Observation of increasing mixed chimerism after transplantation is associated with a higher risk of relapse in acute leukemia. Previously, a quantitative PCR (qPCR) technique, using INDEL polymorphisms, was found to predict relapse in 88.2% vs. 44.4% of individuals analyzed by VNTR markers with a median anticipation period of 58 days and a sensitivity of 0.01% vs. 3%. Here we present results from research experiments performed to determine if a digital PCR (dPCR) method is able to predict relapse earlier and with greater accuracy than the qPCR method using retrospective leukemia samples. Research results showed that dPCR using data generated by the QuantStudio™ 3D Digital PCR System and the qPCR method yielded similar percent recipient chimerism values when recipient DNA was present above the 1% level. Furthermore, dPCR using the system was found to be more sensitive than the qPCR method based on the ability to detect the recipient DNA in a relapsed individual about 2 months earlier where the percent recipient chimerism was 0.2% or less. The false positive rate was close to the complete chimerism value of 0.01% for peripheral blood samples.

Quantification of donor/recipient chimerism in leukemia samples by digital PCR

Kathleen C. Hayashibara1, Antonio Jiménez-Velasco2

1Life Technologies, 180 Oyster Point Boulevard, South San Francisco, CA, 94080, USA. 2Hematology Department, Carlos Haya Hospital, Málaga, SPAIN.

ABSTRACT

INTRODUCTION

RESULTS

CONCLUSIONS

Digital PCR is an analytical technique for quantification of nucleic acid samples based on PCR amplification of single template molecules, without a standard curve. In these experiments the number of negative PCR reactions is used to estimate the number of target molecules based on Poisson distribution statistics.

Mixed chimerism is a state in which both recipient and donor cells are present in bone marrow or peripheral blood after transplantation. Chimerism analysis is performed to monitor peripheral blood or bone marrow in the recipient after allogenic stem cell transplantation to avoid leukemic relapse. Increasing mixed chimerism after transplantation is associated with a high risk of relapse in acute leukemia. A qPCR technique was found to predict relapse in 88.2% vs. 44.4% of individuals analyzed by VNTR markers with a median anticipation period of 58 days and a sensitivity of 0.01% vs. 3%. The goal of this project is to compare results from the QuantStudio™ 3D Digital PCR System (Figure 1) to results from qPCR to determine if relapse can be predicted earlier and with greater accuracy by digital PCR.

Pre-Transplantation

Chemotherapy or Radiation Therapy

Stem Cells Post-

Transplantation

Mixed Chimera

No Relapse

Potential Relapse

Example 1

Example 2

Complete Chimera

Sealed System

Amplify Load Read Mix

We thank Patricia Hegerich, Paco Cifuentes, Maria Jesus Garcia-Ortiz and Juan-Antonio Barba for project coordination and helpful discussions.

REFERENCES

ACKNOWLEDGEMENTS

TRADEMARKS/LICENSING The QuantStudio™ 3D Digital PCR System is For Research Use Only. Not for use in diagnostic procedures.

1 mm

DNA Samples Genomic DNA was purified from fresh whole peripheral blood (PB), bone marrow (BM), or umbilical cord blood using standard procedures from 48 DNA samples isolated from 7 donor/recipient pairs. Recipient DNA was analyzed before and after stem cell transplantaion (SCT) at various intervals. Some were known to have relapsed while others had not. Assays Identical nucleotide sequences for were used for both qPCR (5′ Exonuclease-Based Real-Time PCR Assay) and dPCR experiments using Custom TaqMan® Assays. Polymorphisms were selected where donors possessed the deletion (null allele) and recipients possessed the insertion. qPCR For all runs 100 ng of gDNA was used in 20 ml reactions using standard procedures. The b-Globin reference was analyzed in the same run in a separate reaction. The normalized ratio of target vs. reference was used to calculate the fraction of DNA from the recipient(1). dPCR The concentration of purified DNA was sufficiently low (<20ng/ml) so that dilution was not necessary. Each chip was loaded and run on a GeneAmp® 9700 PCR System for 40 cycles using standard procedures. Target assays were labeled with FAM as indicated in blue (above) and the b-Globin reference assay was labeled with VIC and the target and reference assays were run in duplex on each chip. The b-Globin gene is not located on the same chromosome as the target loci. Some reactions therefore contained target only (detected by FAM), some contained the b-Globin gene only (detected by VIC), and some contained both (FAM + VIC) producing 3 clusters of data points. The number of reactions containing each target was calculated in external software using Poisson distribution estimates using the number of non-target containing reactions relative to the total number of reactions. The recipient chimerism was calculated as shown below using the copies/ml calculated by the QuantStudio™ 3D Digital PCR System software for the target and reference.

Recipient Chimerism = (Target/Reference)Sample /(Target/Reference)Pre-SCT

(If the recipient was heterozygous for the insertion then the target/reference ratio was normalized by multiplying by 2.) The recipient chimerism values were compared with those determined by qPCR. The software was also used to calculate 95% confidence intervals of the copies/ml of target and reference measurements on each chip.

1. Jiménez-Velasco et al., (Leukemia (2005) 19, 336–343).

• The QuantStudio™ 3D Digital PCR System produced similar percent recipient chimerism values to qPCR when recipient DNA was present above the 1% level.

• The QuantStudio™ 3D Digital PCR System is more sensitive than qPCR because it is able to detect the presence of recipient DNA earlier than qPCR in samples where percent recipient chimerism is 0.2% or less.

• The false positive rate based on analysis of donor samples was close to the complete chimerism value of 0.01% for peripheral blood samples.

• Increasing mixed chimerism was detected in recipients 1-3 and 5-7 and not in recipient 4 consistent with known outcomes.

A) The QuantStudio™ 3D dPCR Chip. B) Magnified chip surface. C) Workflow.

MATERIALS AND METHODS

Target Chromosome

MID-1039 5

MID-2113 10

MID-R271 22

GSTT1 22

MID-2113 10

SRY Y

GSTM1 1

MID-1732 10

b-Globin Reference 11

Insertion target only (from recipient)

Both insertion (from recipient) and b-Globin (from donor or recipient)

in one reaction

Neither target present b-Globin target only (from donor or recipient)

Digital PCR was able to detect increasing amounts of recipient chimerism in all cases where increasing amounts were also detected by qPCR. However, dPCR was also able to detect increasing recipient chimerism in samples that failed to yield signal in qPCR experiments.

Recipient Pre-SCT Donor +62 Days

+147 Days +174 Days +192 Days

Reactions containing recipient DNA appear in blue (reactions with target alone) or green (reactions with both target and reference DNA molecules). At day 192 the presence of recipient DNA (blue and green cluster) is readily apparent. Recipient DNA was detected as early as day 62 post-SCT.

dP

CR

% R

ecip

ien

t C

him

eris

m

0.0%

0.1%

1.0%

10.0%

100.0%Pre-SCT Donor 62 147 174 192

Samples are from a recipient known to have undergone relapse. Digital PCR detected recipient DNA in all post-SCT samples consistent with relapse. Relapse was also confirmed by qPCR. However, no recipient DNA was detected until day 192 and an additional sample beyond 192 days was required for confirmation by qPCR.

Recipient/ Donor Pair

(Target) Description % Target/

Reference+

dPCR % Recipient Chimerism+

dPCR 95% Confidence

Interval Upper

dPCR 95%

Confidence Interval Lower

qPCR % Recipient Chimerism

1* (MID-1039)

Pre-SCT 55.34 100.00 100.07 99.87

Donor 0.01 0.01 0.07 0.00

60 0.48 0.87 1.06 0.72 0.59

83 2.97 5.37 5.71 5.05 4.51

94 5.19 9.38 9.83 8.95 8.21

125 5.64 10.19 11.07 9.39 9.28

151 0.07 0.13 0.49 0.03 0.07

2 (MID-2113)

Pre-SCT 48.57 100.00 101.98 98.07

Donor 0.00 0.00 0.00 0.00

55 0.02 0.05 0.10 0.02 0.10

61 0.05 0.10 0.16 0.06 0.08

91 1.98 4.08 8.66 1.92 0.76

127 43.71 90.00 107.50 75.41 72.94

3 ** (R271)

Pre-SCT 55.33 100.00 100.00 100.00

Donor 0.02 0.03 0.06 0.01

62 0.11 0.20 0.37 0.10 0.00

147 0.04 0.08 0.17 0.04 0.00

174 0.09 0.16 0.21 0.12 0.00

192 2.80 5.06 5.32 4.82 11.45

4 *** (GSTT1)

Pre-SCT 53.73 100.00 100.54 99.51

Donor 0.01 0.01 0.03 0.00

14 0.10 0.18 0.28 0.12 0.10

21 0.03 0.05 0.09 0.03 0.02

34 0.04 0.07 0.12 0.05 0.04

69 0.01 0.02 0.06 0.01 0.02

5 (SRY)

Pre-SCT 51.46 100.00 100.24 99.77

Donor 0.00 0.00 0.00 0.00

317 0.04 0.07 0.10 0.05 0.05

331 0.01 0.03 0.05 0.02 0.03

376 0.02 0.04 0.07 0.03 0.02

417 0.06 0.11 0.15 0.09 0.06

477 2.14 4.17 4.31 4.02 4.09

6 (GSTM1)

Pre-SCT 51.65 100.00 100.38 99.54

Donor 0.01 0.03 0.18 0.00

26 0.01 0.02 0.04 0.01 0.01

40 0.01 0.02 0.05 0.01 0.04

59 0.14 0.27 0.43 0.17 0.13

61 0.12 0.23 0.37 0.14 0.29

73 1.85 3.58 3.78 3.38

7 (MID-1732)

Pre-SCT 97.79 100.00 100.01 99.99

Donor 0.00 0.00 0.01 0.00

23 6.72 6.87 7.27 6.50 6.79

48 1.40 1.43 1.49 1.38 1.88

108 0.83 0.85 0.90 0.79 0.95

140 1.29 1.32 1.39 1.25 1.14

154 1.74 1.78 1.90 1.67 1.12

184 1.22 1.25 1.44 1.08 1.81

+ These percentages were calculated in external software. *This recipient was treated with inmunosupprants to avoid Graft-Versus-Host-Disease (GVHD) complications. This therapy decreases the Graft-Versus-Leukemia (GVL) effect. The immunosupression therapy continued through day 125. **dPCR was able to detect recipient DNA earlier than qPCR. ***This receipient did not relapse. Both dPCR and qPCR show lack of significant increase in recipient chimerism.

Comparison of % Recipient Chimerism from dPCR and qPCR

Leukemia cells in red, non-leukemia in blue