HAT-PCR is More Sensitive Than Flow for Quantifying MRD in Chronic Lymphocytic Leukemia

Background:
Sensitive quantification of minimal residual disease (MRD) is becoming important in chronic lymphocytic leukaemia (CLL) owing to the development of highly effective forms of treatment. Flow cytometry is widely used for quantification of MRD. HAT-PCR (High Annealing Temperature or High A/T)-PCR is a recently developed method which markedly decreases nonspecificity and thus enables measurement of MRD usually down to 5 x 10–7. Its simplicity and low cost make it particularly valuable for repeated measurement of MRD.

Aims:
To compare flow cytometry and HAT-PCR for measurement of MRD in CLL.

Methods:
We compared measurement of MRD by HAT-PCR with measurement of MRD by flow cytometry using samples obtained at various times during treatment from patients participating in Study CLL6 of the Australasian Leukaemia and Lymphoma Group.

Quantification of MRD by flow cytometry was performed on whole peripheral blood or bone marrow using a bulk lysis technique with ammonium chloride. Multiparameter flow cytometry using 10 colours in a single tube was performed to assess MRD with a sensitivity of 10–4 – 10–5. This method was validated against the international standardised flow cytometric approach for MRD detection in CLL (ERIC) treated patients.

Quantification of MRD by HAT-PCR involved sequencing of the IGH rearrangement by next-generation sequencing (NGS); design and synthesis of 1–3 candidate patient-specific primers; and testing of these primers for amplification efficiency and lack of nonspecificity. The final assay was a single round quantitative PCR which used DNA from Ficoll-Hypaque separated cells and which measured up to 30 μg of patient DNA for maximum sensitivity and 20 μg of pooled non-leukemic DNA to detect any nonspecificity.

Results:
A total of 127 samples, either blood (100) or marrow (27), were obtained from 36 patients at various .times during treatment. The number of samples / patient ranged from 1 to 9, with a median of 3. Nonspecificity was only seen with primers from one patient, at a level equivalent to an MRD value of 3.5 x 10–7. A total of 107 samples were analysed by both flow cytometry and HAT-PCR and the results are shown in the Figure. Flow measured down to 10–5 whereas HAT PCR measured down to 3.5 x 10–7. There was excellent correlation (r = 0.86) between HAT-PCR and flow when MRD could be measured in a sample by both methods. There were 28 samples in which MRD was not detected by flow but was quantified by HAT-PCR; we suspect that poor sample quality may have been responsible for failure of flow to detect some samples for which a high level of MRD was detected by HAT-PCR. There were 3 samples, all from the same patient, in which MRD was detected by flow at a very low level but was not detected by HAT-PCR; we suspect that flow was detecting a low level of nonspecificity. There were 5 samples from one patient, the results shown as open circles in the figure, in which high values for MRD were determined by flow and low values were determined by HAT-PCR; we suspect there was an anomalous immunophenotype in this patient. MRD was not detected by either method in 5 samples.


Summary/Conclusion:
HAT-PCR is substantially more sensitive and possibly more reliable than flow cytometry for measurement of MRD. The clinical significance of CLL detection at levels below 10–4 remains uncertain but HAT-PCR may be of value in assessment in future in patients with very low levels or undetectable disease in whom cessation of therapy is being considered.