The Journal of Molecular Diagnostics, Vol. 24, No.

6, June 2022

jmdjournal.org

Sensitive Measurement of Minimal Residual

Disease in Blood by HAT-PCR
Sue Latham,* Elizabeth Hughes,* Bradley Budgen,* David Ross,* Matthew Greenwood,yz Kenneth Bradstock,x
Luciano Dalla-Pozza,{ Libby Huang,k Tamara Law,k Louise Doculara,k Nicola Venn,k Shahid Ullah,* Rosemary Sutton,k** and
Alexander A. Morley*

From the College of Medicine and Public Health,* Flinders University, Adelaide; the Royal North Shore Hospital,y Sydney; University of Sydney,z Sydney; the
Westmead Hospital,x Sydney; the Children’s Hospital Westmead,{ Sydney; the Children’s Cancer Institute,k Sydney; and the School of Women’s and
Children’s Health,** University of New South Wales, Sydney, New South Wales, Australia

Accepted for publication

March 3, 2022.
Address correspondence to
Alexander A. Morley, M.D.,
3/28 Springfield Ave., Toorak,
VIC 3142, Australia.
E-mail: alec.morley@flinders.
edu.au.
PCR is widely used to measure minimal residual disease (MRD) in lymphoid neoplasms, but its sensitivity
is limited. High Adenine/Thymine PCR and High Annealing Temperature PCR (HAT-PCR) is a modified
PCR designed to minimize nonspecificity and hence increase sensitivity. It was evaluated in the lab-
oratory and the clinic, using samples from 58 patients. Of these patients, 57 were adolescents or young
adults who were participating in the Australasian Leukemia and Lymphoma Group ALL06 trial in which
MRD was measured in blood principally by HAT-PCR and in marrow by conventional PCR. HAT-PCR
produced significantly less nonspecificity than conventional PCR, and its limit of detection was <10
6
in 90% of patients. In 196 samples, an excellent correlation was found between blood and marrow
MRD. Variable partitioning of leukemic cells between blood and marrow was observed. Measurement of
MRD in blood by HAT-PCR was noninferior to measurement of MRD in marrow by conventional PCR, in
terms of both detecting disease and predicting clinical outcome. At a median follow-up of 3 years and
for MRD levels in blood at the end of consolidation treatment, an MRD level of >104 cells/L signif-
icantly predicted relapse and mortality, whereas undetectable MRD significantly predicted relapse-free
survival and overall survival. HAT-PCR is a simple, quick, cheap and sensitive method for measurement
of MRD, and its adoption for MRD in blood may be clinically useful. (J Mol Diagn 2022, 24: 632e641;
https://doi.org/10.1016/j.jmoldx.2022.03.007)

Following the initial reports1e3 almost 3 decades ago that
the outcomes of patients with acute lymphoblastic leukemia
(ALL) could be predicted by measurement of minimal re-
sidual disease (MRD) using quantitative PCR and an allele-
specific oligonucleotide (ASO) primer, PCR quantification
of MRD has become widely used for patient management,
both to predict outcome and to make decisions on treatment,
and in clinical trials as a surrogate for patient outcome. Two
unresolved issues in the use of PCR quantification of MRD
in ALL are whether the sensitivity of MRD assays can be
improved and whether blood can be used for MRD analysis.
A more sensitive assay system would widen the use of
MRD assay and make it more accurate, whereas use of
blood would have advantages for both patients and clini-
cians. These two issues are linked because in B-cell ALL
(B-ALL), the level of MRD in blood is lower than the level
in marrow4e7 and a more sensitive assay system could
compensate for this lower level and facilitate the use of
blood for monitoring MRD.
Although it is often stated that PCR quantification of
MRD has a sensitivity of 105, the published data indicate
that PCR as currently performed fails to reach this level of
sensitivity in a substantial proportion of patients.8
Supported by Monoquant Pty. Ltd. Grant 30485 (A.A.M. and Flinders
University).
Disclosures: Monoquant Pty. Ltd. provided research support, which
included the salaries of S.L., E.H., and B.B., and it has applied for a related
patent (US application 16999594; August 20, 2020). Monoquant Pty. Ltd.
played no role in the design of the study, collection and analysis of the data,
or preparation of the manuscript. A.A.M., S.L., E.H., and B.B. hold equity
in Monoquant Pty. Ltd. The other authors declare no potential conflict of
interest.

Copyright ª 2022 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.
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Nonspecificity, manifested as a false-positive signal in the
PCR, is often responsible. We previously used three round,
nested real-time quantitative PCRs to avoid nonspecificity
and achieve highly sensitive quantification of MRD in
ALL,9 then investigated two round, nested PCRs and finally
developed High Adenine/Thymine PCR and High
Annealing Temperature PCR (HAT-PCR) as the one-round
technique for assay. The use of high annealing temperature
and the acronym HAT-PCR were originally reported by
Mecklenburg,10 but his work seems to have been largely
forgotten, and this study was performed without knowledge
of his work. During the development of HAT-PCR, an
analysis of MRD levels in blood and marrow was performed
in young adults with ALL. This report details the method of
HAT-PCR, compares the levels of MRD in blood as
measured by HAT-PCR with the levels of MRD in marrow
as measured by conventional PCR, and provides data on the
prediction of outcome afforded by measurement of blood
MRD by HAT-PCR. A preliminary report has been
published in abstract form.11
Materials and Methods
Study Participants
Adolescents and young adults 15 to 40 years of age with
Philadelphia-negative ALL participated in study ALL06 of
the Australasian Leukemia and Lymphoma Group.12 Patients
were treated with a pediatric-type protocol of multiagent
chemotherapy stratification to high-risk treatment, and allo-
geneic transplant in first remission was largely based on
MRD levels in marrow. Outcomes were assessed after me-
dian follow-up of 36 months had been reached. All patients
provided informed consent, and samples of blood for the
current study were obtained from a subset of all patients.
Conventional PCR
The level of MRD in marrow was measured by the Sydney
laboratory using PCR performed and analyzed according to
Berlin-Frankfurt-Münster practice and EuroMRD guidelines,
using an ASO primer, a generic segment-specific primer, a
probe, and 500 ng of remission DNA in triplicate.13e19 The
PCR was a touch-down PCR, which started at 10 C above
the final annealing temperature and decreased by 1 C per
cycle until the final annealing temperature was reached. The
final annealing temperature was usually 61 C, and amplifi-
cation at this temperature was continued for an additional 45
cycles. Nonspecificity was tested for using DNA isolated
from peripheral blood leukocytes (PBLs) and pooled from
five individuals without leukemia. Each primer was evaluated
for nonspecificity using three wells that each contained 0.5
mg of PBL DNA in 25 mL of water, and nonspecificity during
the MRD assay was evaluated using six wells that each
contained 0.5 mg of PBL DNA in 25 mL of water. The
threshold cycle (CT) values resulting from nonspecificity
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Sensitivity of MRD in Blood by HAT-PCR
wells were used at the time in determination of primer
sensitivity and MRD results but were not permanently
recorded, although record was made of the presence or
absence of positive results.
HAT-PCR
The Sydney laboratory provided to the Flinders laboratory
the sequences for rearranged regions of the IGH, TCB, and
TCG genes that had been used. The level of MRD in blood
from diagnosis and treatment samples was measured in the
Flinders laboratory. Unless otherwise stated, all PCRs used
an ASO primer, a J segment specific primer, and a Taqman
probe directed to the J region of IGH or TCR. Early samples
were measured by two round, nested real-time quantitative
PCR, but HAT-PCR was ultimately used for most mea-
surements. For two round, nested PCRs, the primers were
designed and PCR was performed as previously described,9
except that only two rounds of PCR were performed, with
the first comprising 18 cycles and the final 45 cycles.
HAT-PCR is a one-round ASO PCR that aims to mini-
mize nonspecificity and achieve greater sensitivity. The
important features are as follows: an A or T base at each of
the one, two, three, four, five, or six most 30 positions of
both the forward and the reverse primer; annealing
temperature within 3 C of critical annealing temperature
(the highest annealing temperature that still gives efficient
amplification); and a relatively high concentration of Taq
polymerase
Primers were designed in the laboratory and had one to
four A or T bases at the 30 end. The melting temperature
(Tm) was assessed using the OligoAnalyzer tool from IDT
(Coralville, IA) and using values of 5 mmol/L Mgþþ and
0.3 mmol/L dNTPs. For nearly all patients, primers with a
Tm of 69 C to 71 C and a PCR with an annealing temper-
ature of 72 C were used. Occasionally, longer or shorter
primers with a higher or lower Tm were used, but their
performance appeared satisfactory provided the basic primer
design and PCR features were maintained. The reverse J
primers were designed similarly to the forward ASO primers
except that their Tm was slightly higher, between 71.5 C
and 72.5 C. Sequences of J primers used are given in
Table 1.
The constituents of the PCR were as follows: potassium
chloride, 50 mmol/L; magnesium chloride, 5 mmol/L;
dNTPs, 300 mmol/L; primers, 400 nmol/L; probe, 160
nmol/L; Taq, 4 U; Tris hydrochloride, 20 mmol/L (pH 8.4);
and water, 50 mL. The PCR amplification protocol was as
follows: 91 C for 3 minutes; 5 cycles of 97 C for 15 sec-
onds and 72 C for 30 seconds; 5 cycles of 96 C for 15
seconds and 72 C for 30 seconds; and 35 cycles of 94 C for
15 seconds and annealing at 72 C for 30 seconds.
Between one and three candidate ASO primers were
synthesized for most patients, although more were synthe-
sized during the learning phase and for what were assessed
as potentially problem sequences. Using an eight-well
633
Latham et al

Table 1 IGH and TCRb Primers Used Set B Comprised the Most Commonly Used Primers for IGH. JG1.3/2.3 and JG1.1/2.1 Were Used for
TCRg14
Primer Sequence Melting temperature,  C
IGH J
SetA J1 50 -CCTCTGCCCTCCTGCTTCTCCCATACA-30 72.6
SetA J2 50 -GCTAAGTGACAGCAGGGCTCTGGCAT-30 72.4
SetA J3 50 -CGTGGTCCCAAACAGCCGGAGAA-30 71.4
SetA J4 50 -CTGTTGCCTCAGGGCATCCTCCTGA-30 72.1
SetA j5 50 -GAGGACAGGCTGGGTTCCCATTCGAA-30 72
SetA J6 50 -GCCCAGGTCCCCTCGGAACAT-30 71.2
SetB J1 50 -GGCTCCCCGCTATCCCCAGACA-30 72.3
SetB J2 50 -GCCTGGTGCCTGGACAGAGAAGACT-30 71.9
SetB J3 50 -CTGGGCCCAGAGAAAGGAGGCAGAA-30 72.1
SetB J4 50 -CCCTTCAGAGTTAAAGCAGGAGAGAGGTTGTGA-30 71.9
SetB J5 50 -CCCTGGCAAGCTGAGTCTCCCTAAGT-30 71.5
SetB J6 50 -GGCAGTAGCAGAAAACAAAGGCCCTAGAGT-30 71.7
TCRb J
TCR1.1b 50 -TTCCCTGTGACGGATCTGCAAAAGAACCTGA-30 72.8
TCR1.2b 50 -CCCTCCTAGAGACCCCCAGCCTTACCTACAA-30 73.6
TCR1.3b 50 -CAAGTTCCCAGCTGTCCAGCCTTGACTTACT-30 72.7
TCR1.4b 50 -CCAGGAACTCCGACCTTATGATACACTATCCCGAAAGAA-30 72.9
TCR1.5b 50 -ATGGCCATACCACCCTGATTCTGCAACTTACCTA-30 73.3
TCR1.6b 50 -GAGTCAAGAGTGGAGCCCCCATACCTGT-30 72.4
TCR2.1b 50 -CACCTGGAGCCCCCTTCTTACCTAGCA-30 72.6
TCR2.2b 50 -CGGAGCCCCAACCGCCTCCTT-30 73.6
TCR2.3b 50 -GGAGCCCCGCTTACCGAGCACT-30 72.9
TCR2.4b 50 -CCGGCGGCCCCAGCTT-30 71.6
TCR2.5b 50 -GCGCTCACCGAGCACCAGGA-30 71.8
TCR2.5c 50 -CCGCGCTCACCGAGCACCAGGA-30 71.6
TCR2.5d 50 -CCCGCGCTCACCGAGCACCAGGA-30 71.6
TCR2.6b 50 -CGCGAAAACTCACCCAGCACGGTCA-30 73.1
TCR2.7d 50 -GGAAGGTGGGGAGACGCCCGAAT-30 72.6
Set B comprised the most commonly used primers for IGH. JG1.3/2.3 and JG1.1/2.1 were used for TCRg.14

gradient of annealing temperature, typically ranging from MRD Assay

68 C to 75 C for a primer with a Tm between 69 C and
71 C, each primer was assessed to determine its critical
annealing temperature and hence the chosen annealing
temperature and to determine its propensity to produce
nonspecific amplification. The six wells at the higher tem-
peratures contained 400 pg of diagnosis leukemic DNA, and
the two wells at the lower temperatures each contained 1 mg
of DNA isolated from PBLs pooled from five individuals
without leukemia.
Droplet digital PCR (ddPCR) was performed using the
Bio-Rad (Hercules, CA) QX100 instrument and according
to the manufacturer’s instructions. Using ddPCR, a refer-
ence gene, GALT, was used to assess DNA integrity of all
samples, and for diagnostic samples specific primers were
used to determine the number of leukemic sequences per
mass of genomic DNA. For the test samples, a preliminary
two-well HAT-PCR was performed to determine the
approximate level of MRD, and to exclude any inhibition in
the sample, another HAT-PCR was performed that involved
four wells that contained diagnosis DNA to an MRD level
of 103, with two wells containing and two wells not
containing 1 mg of test DNA.
The definitive PCR assay of blood for MRD always
involved 45 cycles. Specificity for each primer was tested
using at least 20 wells, each containing 1 mg of DNA pooled
from five individuals without leukemia (PBL DNA). With
HAT-PCR, single targets amplify to produce a positive
signal before 40 cycles have occurred.20 and positive signals
with PBL DNA were therefore recorded as false-positive
results because of nonspecificity if they developed when
the CT was <40 and were ignored if they developed when
the CT was >40. Patient samples were quantified using 1 mg
of PBL DNA per well, with three wells being assayed if the
CT of the preliminary PCR was <35; otherwise, 30 wells
were used, except for occasional cases when the amount of
DNA available was limited. All wells had a final volume of
50 mL. Samples were analyzed and quantified using a
standard curve of dilutions of diagnosis DNA, but for
samples with a low MRD level and 30 wells being assayed,
it also proved possible to perform a digital analysis based on
the proportion of the assay wells that showed no amplifi-
cation. In these instances, the geometric mean of the
analogue and digital values was calculated. For the

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occasional assays in which nonspecificity was detected, the
result for the test DNA was accepted if it was above the
MRD calculated from the 90% upper confidence limit of the
number of positive PBL wells.21 If below, the result was
considered not detected and less than the value calculated
from the 90% upper confidence limit.
Nonspecificity produced by HAT-PCR in the Flinders
laboratory was compared with that produced by conven-
tional PCR in the Sydney laboratory by determining the
percentage of control wells that had nonspecific amplifica-
tion in MRD assays. Because the PCR protocols in the two
laboratories differed in several respects, for HAT-PCR the
percentage of positive control wells after completion of the
45 cycles was used, and for conventional PCR the results
were recalculated to determine the percentage that would
have resulted if the wells had contained 1 mg rather that 0.5
mg of DNA. An additional study was also performed in the
Flinders laboratory to directly compare the nonspecificity
produced by the two different PCR protocols and primers.
Statistical tests that involved MRD assays were per-
formed using the logarithms of the results. For sensitive
detection of MRD marrow samples for which leukemic cells
had been isolated using Ficoll-Hypaque, log3 was sub-
tracted from the logarithm of the MRD value on the
assumption that there had been threefold enrichment of the
leukemic cells. The final estimates of the ratios between
marrow and blood MRD levels are presented as arithmetic
values.
The variance of the partitioning of leukemic cells between
marrow and blood was obtained by subtracting published
variances of MRD assay and marrow sampling22 from the
total variance for the difference between marrow and blood
observed in the current study. The total variance of parti-
tioning was then separated into interindividual and intra-
individual components by performing analysis of variance.
Patient outcomes were reviewed when the median follow-
up reached 36 months. The primary hypothesis was that the
level of MRD predicted outcome, and the secondary hy-
pothesis was that levels of MRD that predicted a high or low
probability of relapse could be identified. Time to relapse
was measured from the date of registration to the date of
Table 2 Occurrence of False-Positive Results Caused by Nonspecificity Produced by 58 Allele-Specific Oligonucleotide Primers in Relation
to the Characteristics of the Primer (When Level of MRD Is the Level That Would Have Been Obtained if the False-Positive Result Had Been a
True-Positive Result)
Variable Values
N regions, n 2 1
Bases in N region >7
Primers tested, n
Nonspecificity absent 24 14
Nonspecificity present 0 0
Level of MRD NA
MRD, mean residual disease; NA, not applicable.
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Sensitivity of MRD in Blood by HAT-PCR
relapse. Patients were censored at relapse, date of loss to
follow-up, end of follow-up, transplantation, or death. Cox
proportional hazards regression models were applied to
examine the relapses between cut points of each MRD
measure. The models were adjusted by age. The estimates
were calculated using the likelihood ratio method and were
expressed as hazard ratios (HRs). Proportional hazards
assumption was tested by log-log plot of survival and
Schoenfeld residuals. Survival for patients with MRD
values above or below the cut point for each MRD measure
were evaluated by standard Kaplan-Meier survival curves,
and groups were compared by the log-rank test. Goodness
of fit was evaluated by C statistics. The one-sided test was
performed for all analyses.
Results
Primers from 57 patients from the ALL06 study, 48 with B-
ALL and 9 with T-cell ALL (T-ALL), and from 1 child
patient with B-ALL were studied. Table 2 gives the results of
testing for nonspecificity. Results were negative with primers
for 52 patients (90%), indicating that any nonspecificity was
<3.4  107. Nonspecificity was seen with primers for 6
patients, with levels ranging from 1.7  105 to 3.4  107.
In these 6 patients, the rearrangement was a partial DNJ
rearrangement with two to four bases in the N region.
Figure 1A shows accuracy and potential sensitivity of
HAT-PCR using dilutions of leukemic DNA in non-
leukemic DNA. Figure 1B shows the precision of HAT-
PCR as measured by the SD calculated from duplicate
measurements on dilution samples. Assays of the 106 and
5  107 samples involved 30 mg of nonleukemic DNA,
and MRD was detected in 24 of 25 assays at the 106 level
and 6 of 8 of assays at the 5  107 level.
In total, 233 MRD assays were performed on samples of
blood from the 57 patients in ALL06, 64 by two-round
PCR, 161 by HAT-PCR, and 8 by both methods. A total of
49 samples were taken on day 15, 50 at the end of in-
duction, 52 at the end of consolidation, and 74 on various
days after consolidation. A total of 107 HAT-PCRs had
7 6 5 4 3 2
5 4 1 2 1 1
0 0 0 3 2 1
7.0  10-6 1.9  10-6 1.7  10-5
7.1  10-7 3.4  10-7
7.1  10-7
635
 Latham et al

A B 0.4 7
10
-3 0.35

0.3
-4
0.25
Observed MRD

11
11

SD
0.2
y = 1x + 0.0074 6
-5
r = 0.98. n = 14 0.15 14

0.1 10
-6
0.05

-7 0
-7 -6 -5 -4 -3 -2 -7 -6 -5 -4 -3 -2
Expected MRD MRD (log)

Figure 1 Accuracy, sensitivity, and precision of HAT-PCR. Leukemic DNA was diluted in 30 mg of nonleukemic DNA in various proportions to provide
artificial samples, which were then assayed. A: Observed mean residual disease (MRD) values in relation to expected values. The open square symbol indicates a
sample in which MRD was not detected and the value shown is the less than value. B: Results of experiments when different samples were assayed twice, by two
different individuals on two different days, and the SD of the two results was calculated. The mean SDs  1 SE are shown in relation to the levels of MRD, which
are collated into 0.5 log groups. The numbers shown are the number of samples assayed.

positive results, 25 between 104 and 105, 31 between All the T-ALL blood samples were assayed by HAT-
105 and 106, and 7 <106, with the lowest being PCR. Of the B-ALL blood samples, 107 were analyzed by
3.4  107. A total of 191 sets of results were available HAT-PCR, and there were 55 marrow-blood pairs of
from paired blood and marrow samples from 57 patients. quantified samples. These pairs also had significant regres-
Leukemic cells had been isolated from marrow samples by sion between levels in blood and marrow (y Z 1.0849x e
density-gradient separation in 51 instances. The marker 0.9449, r Z 0.89, P < 0.001) and a marrow-blood ratio of
rearrangement involved IGH in 47, TCRb in 6, and TCRg 14.1 (t Z 10.1, P < 0.001). Table 3 gives the frequency
in 4 patients. Figures 2 and 3 show the significant corre- with which MRD was detected, either in blood by HAT-
lation between the levels of MRD in blood and marrow for PCR or in marrow by conventional PCR.
both B-ALL and T-ALL. The marrow-blood ratio was 16.1 The observations of nonspecificity with conventional
for B-ALL samples (71 pairs, t70 Z 12.03, P < 0.001) and PCR and HAT-PCR during MRD assays are shown in
13.5 for T-ALL samples (12 pairs, t11 Z 3.85, P Z 0.003). Figure 4A. Conventional PCR produced significantly more
Variation was analyzed in the log mode and only for B- nonspecificity (P < 0.001), but interpretation was uncertain
ALL samples because there were too few T-ALL samples. owing to differences between the two laboratories in the
For the mean MRD difference between marrow and blood samples of nonleukemic DNA tested and the method of
of 1.21, total variance was 0.72, intraindividual partitioning calculation of the level of nonspecificity. A direct compar-
variance was 0.182, and interindividual partitioning vari- ison of the two methods was therefore performed in the
ance was 0.100. Flinders laboratory. Figure 4B shows the results and

0 Not Detected-
Detected Not Quantified
-1
Figure 2 Mean residual disease (MRD) measured
-2
in marrow and blood for IGH rearrangements. For
IGH Blood MRD (log10)

-3
marrow MRD measured by conventional PCR, the re-
sults were classified in accordance with EuroMRD
-4 guidelines into those that were detected and quanti-
fiable, those that were detected but below the quan-
-5 tifiable range, and those that were not detected. For
y = 1.071x- 1.0424 blood MRD measured by HAT-PCR, the results were
-6 classified into only two categories: those that were
r = 0.86 n = 71 P<0.001
detected and quantified and those that were not
-7
detected. The dotted lines mark the limits of these
-8 53 15
categories.

-9
-7 -6 -5 -4 -3 -2 -1 0 1
IGH Marrow MRD (log10)

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 Sensitivity of MRD in Blood by HAT-PCR

1
Not Detected -
0 Detected Not Quantified

-1

-2 Figure 3 Mean residual disease (MRD) measured

TCR Blood MRD (log10)

in marrow and blood for TCR rearrangements. For

-3
marrow MRD measured by conventional PCR, the re-
sults were classified into those that were detected and
-4
quantifiable, those that were detected but below the
-5 quantifiable range, and those that were not detected,
in accordance with EuroMRD guidelines. For blood
-6 y = 0.7639x - 1.6422 MRD measured by HAT-PCR, the results were classified
r = 0.74 n = 12 P = 0.006 into only two categories: those that were detected
-7 and quantified and those that were not detected. The
dotted lines mark the limits of these categories.
-8 Not
6
Detected
-9
-7 -6 -5 -4 -3 -2 -1 0 1

TCR Marrow MRD (log10)

indicates that conventional PCR produced significantly the variance of the partitioning of leukemic cells between
more nonspecificity (P Z 0.014). blood and marrow. Using this value, the SD of an assay of
The potential for false-positive results caused by non- MRD in a blood sample was estimated to be approximately
specificity was also studied in eight marrow samples for 0.60.
which the MRD level had been scored by conventional PCR Patient outcome was assessed after follow-up for a me-
as detectable but not quantifiable, which is usually between dian duration of 36 months. Occasional samples were
104 and 105. Measurement of MRD by HAT-PCR using missing or limited. Twenty-one patients received trans-
4 to 9 mg of DNA gave four detectable results (4.48  105, plants. Relapse occurred in 10 patients without transplants
1.46  105, 7.23  106, and 5.75  106) and 4 non- and in 2 patients with transplants. The relationships between
detectable results (<1.45  106, 1.09  106, MRD at the end of induction and outcome are shown in
1.02  106, and 7.44  107), suggesting that for the latter Supplemental Figure S1. The findings were not statistically
four samples the conventional PCR results were false pos- significant. Relationships between MRD at the end of
itive because of nonspecificity. These four samples came consolidation and relapse-free survival are shown in
from three patients. Figure 5, AeD. Figure 5A shows the results of MRD in
Analysis of the variance of the difference between the marrow. Figure 5B shows that patients with an undetectable
paired blood and marrow samples gave a value of 0.282 for level of blood MRD had an excellent prognosis. Figure 5C
indicates that patients with a high level of blood MRD had a
high probability of relapsing. Figure 5D shows the rela-
Table 3 Frequency of Detection of MRD for Assays of Blood
tionship between MRD level in blood and outcome after
Performed by HAT-PCR
censoring patients at the time of transplant. Twelve patients
Marrow MRD died, 10 from leukemia, 1 from graft-versus-host disease,
Blood MRD Positive Nonquantified Negative and 1 from sepsis. An undetectable level of marrow or blood
T-ALL MRD at the end of consolidation significantly predicted
Positive 12 4 4 overall survival, and a high level of blood MRD signifi-
Negative 0 0 6 cantly predicted mortality (Supplemental Figure S2, AeC).
B-ALL
Positive 55 11 5
Discussion
Negative 1 9* 26
Positive indicates that MRD was detected and quantifiable, nonquantified Nonspecificity limits sensitivity, and HAT-PCR is a modi-
indicates that MRD was detected and but below the quantifiable range, and fication of conventional PCR that aims to prevent non-
negative indicates that MRD was not detected.
specificity and hence increase sensitivity. Its essential
*Present and previous data suggest that approximately half of these
results are likely to be false positive. features are the use of an annealing temperature that is as
B-ALL, B-cell acute lymphoblastic leukemia; MRD, mean residual disease; close as possible to the maximum and the presence of one or
T-ALL, T-cell acute lymphoblastic leukemia. more A or T bases at the 30 end of the forward and reverse

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Latham et al

A Nonspecificity B -4.0

-4
Conven onal PCR HAT-PCR

-5.0

MRD (log)
-5

MRD (log) -6.0

-6 7 12
-7.0
P < 0.001
Conven onal PCR HAT - PCR
ND = 22 ND = 37 P < 0.014
-7 -8.0

Figure 4 Levels of nonspecificity observed with conventional PCR or HAT-PCR. A: Comparison based on historical data from the two laboratories. The gray
symbols are minimum values and result from all wells being positive. There was a highly significant difference in the results observed with the two methods.
B: Results of a direct comparison in the Flinders laboratory using the different PCR protocols, the separately designed primers for 15 patients, and quantifying
false-positive results in 20 mg of DNA. The boxed numbers refer to the number of primers that did not show nonspecificity, indicating that the level was
<3.4  107. Testing the results in panel A by a one-tailed U test and the results in panel B by a one-tailed Wilcoxon signed rank test showed that HAT-PCR
produced significantly less nonspecificity than conventional PCR. ND, not detected.

primers. Nonspecificity at a relatively low level was experiments using artificial mixtures with MRD at 106
observed in 10% of patients. The association with single and and 107 likewise indicated detection of single targets.
short N regions has been previously reported.8 When MRD levels are low, stochastic Poisson variation in
In 90% of patients, nonspecificity was not observed, the number of targets in an assay predominates, and
and MRD down to the level produced by the presence of probability of detection is 95% when three targets are
a single intact target sequence was able to be detected. present. Therefore, in most patients, the limit of detection
This level corresponds to an MRD level of 3.4  107 if of HAT-PCR is approximately 7  107 when 30 mg of
20 mg of DNA is assayed and 2.2  107 if 30 mg of DNA is assayed and approximately 1  106 when 20
DNA is assayed. The frequency of positive assays in the mg of DNA is assayed.

A MRD in Marrow at End of Consolidaon B MRD in Blood at End of Consolidaon

1.0 1.0 MRD Negave

(n=24)

0.8 MRD Negave 0.8

(n=33)

MRD Posive MRD Posive

RFS

RFS

0.6 0.6
(n=19) (n=20)

0.4 0.4

P = 0.028 P = 0.013

0.2 0.2
0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7
Time, years Time, years

C MRD in Blood at End of Consolidaon

D MRD in Blood at End of Consolidaon

1.0 1.0 MRD Negave

(n=24)
MRD
< 104 / L
(n=36) 0.8
0.8

P = 0.0027 MRD Posive

RFS

RFS

0.6 0.6 (n=20)

MRD
0.4 >104 / L 0.4
(n=13) P = 0.029

0.2 0.2
0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7
Time, years Time, years

Figure 5 Relapse-free survival (RFS) related to mean residual disease (MRD) at the end of consolidation in blood, as measured by HAT-PCR, or in marrow,
as measured by conventional PCR. A: RFS depending on whether MRD was detectable in marrow. B: RFS depending on whether MRD was detectable in blood.
C: RFS depending on whether the MRD level in blood was >104 cells/L; median RFS was 30.5 months in patients with MRD >104 cells/L. D: RFS depending on
whether MRD was detectable in blood, but with patient results being censored at the time of transplantation. The level of MRD corresponding to detection of
1 target per assay was 2.2  107 for assay of blood and 4.5  106 for assay of marrow.

638 jmdjournal.org - The Journal of Molecular Diagnostics

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With HAT-PCR, nonspecificity is regarded as a problem
that can arise frequently and unpredictably, and the PCR is
designed to achieve maximal prevention. A consequence is
that HAT-PCR is characterized by sustained amplification
efficiency of close to 100% and detection of single targets.20
With conventional PCR, nonspecificity is regarded as occa-
sional, and the protocol is modified only when false-positive
results caused by nonspecificity are observed.15,19 However,
this approach has several consequences that prevent sensitive
detection of MRD. First, the quantifiable range does not
reach 104 in approximately one-third of cases8 because of
false positivity in many cases and possibly because of PCR
inhibition in others. Second, the current data and the data of
previous studies5,23 taken in aggregate suggest that approxi-
mately 50% of the positive results that are below the quan-
titative range, between approximately 104 and 105, are
false-positives. Third, the data in Figure 4, A and B indicate
that false-positive results also occur at levels that are below
the detectable range of conventional PCR. The frequency of
occurrence of false -positive results, their occurrence below a
level of 104, and the unpredictability of their occurrence
indicate that PCR as conventionally performed is an unreli-
able method for quantification of low levels of MRD. This
conclusion is supported by additional unpublished finding
that conventional PCR intended to quantify a low level of
MRD may sometimes give a negative result, without pro-
ducing a false-positive signal.
The present results indicate that the levels of MRD in
blood and marrow were significantly correlated and that the
levels of MRD in marrow are significantly higher than those
in blood. The findings agree with previous reports except for
the finding of a high marrow-blood ratio for T-ALL. This
finding was based on only 12 results and may have been a
statistical anomaly. For MRD in T-ALL, although the data
are small, the frequency of detection in blood by HAT-PCR
was greater than the frequency of detection in marrow by
conventional PCR. For MRD in B-ALL, when allowance is
made for the proportion of detected but not quantifiable
assay results likely to be false-positives, the frequency of
detection of MRD in blood by HAT-PCR was comparable
to the frequency of detection by conventional PCR. There-
fore, for monitoring of MRD, for T-ALL the assay of blood
by HAT-PCR may be more informative than the assay of
marrow by conventional PCR, and for B-ALL the two
approaches may be equally informative.
Measurement of MRD in a sample of blood or marrow
aims to provide an indirect estimate of the total number of
leukemic cells in the marrow and thus enable assessment of
response and prediction of outcome. Measurement of MRD
in marrow is subject to two sources of variation, which can
produce an inaccurate result; there is sampling error, which
can be substantial,22 and the result is expressed as leukemic
cells per total cells and hence is affected by variation in the
number of nonleukemic cells. Measurement in blood is not
affected by sampling error because blood flows through all
areas of the marrow, and the assay result can be converted to
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Sensitivity of MRD in Blood by HAT-PCR
an absolute value of cells per unit volume. However, as
leukemic cells partition between the blood and marrow
compartments, variation in the degree of partitioning will
affect the accuracy with which a result for MRD in blood
provides an estimate of the total number of leukemic cells in
marrow. The result for the variance of partitioning, although
approximate, suggests that the magnitude of partitioning error
is similar to the previously reported magnitude of sampling
error of marrow aspiration.22 The results also suggest that
partitioning variation results from variation between within
individuals, the latter possibly attributable to therapy.
Blood has obvious advantages for measurement of MRD,
but the limited sensitivity of current measurement methods
has minimized its investigation and use. Studies using flow
cytometry have suggested that measuring MRD in blood may
be useful in T-ALL because the level of MRD in blood
differs little or not at all from the level of MRD in marrow.
However, although similar studies in B-ALL provided some
prediction of outcome, the degree of prediction appeared
to be inferior to that provided by measuring MRD in
marrow.24,25
The current study found a significant relationship between
outcome and the level of MRD in blood at the end of
consolidation. An undetectable MRD level indicated a high
probability of sustained remission and survival, whereas a
level >104 leukemic cells/L suggested a high probability of
relapse and death (Figure 5, BeD, and Supplemental Figure
S2). These relationships were evident despite patients with
high MRD levels in marrow being treated more intensively,
particularly by transplantation. Comparison of Figure 5, A
and B suggests that outcome is more clearly related to the
level of MRD in blood than to the level of MRD in marrow.
This difference may have been attributable to chance because
the number of relapsing patients was small, to the greater
sensitivity of HAT-PCR outweighing the lower level of
MRD in blood, or to measurement in blood being qualita-
tively superior to measurement in marrow.
The sensitivity of HAT-PCR provides advantages for
measurement of MRD in marrow and blood as well as in
lymphoid neoplasms in general. For measurement in
marrow, MRD not detectable by current conventional PCR
can be detected and quantitated. MRD that is currently
detectable by PCR but at a low level can be measured more
accurately owing to the elimination of Poisson variation,
which, as seen in Figure 1B, is the main source of mea-
surement variation when the MRD level is low. Measuring
MRD in blood has advantages for the patient, physician, and
payer. Measurement in blood by HAT-PCR may be
particularly useful when frequent measurement by a sensi-
tive method is desired, for example, to detect impending
relapse in adult patients with ALL or after transplantation or
chimeric antigen receptor T-cell therapy.
HAT-PCR is simple to perform, inexpensive, and able to
be performed by any institution that has a molecular labo-
ratory. It requires the initial steps of sequencing of the
marker rearrangement followed by primer synthesis and
639
Latham et al
testing. Obtaining sequence information is the most labo-
rious initial step but will in the future be simplified by the
use of next-generation sequencing (NGS). Initial costs can
be amortized if multiple estimations are performed.
MRD can now be measured by three sensitive techniques:
next-generation flow, NGS, and HAT-PCR. The strengths
and weaknesses of each of these three evolving techniques
will become apparent over time as the result of progressive
evaluation of each becomes available and comparisons are
made between them. For a given sample size, both HAT-
PCR and NGS are more sensitive than flow because both
can detect a single target. Compared with NGS, HAT-PCR
is simpler, more inexpensive, and quicker and would be
more widely available, but it cannot detect clonal evolution,
which is an important factor in ALL. Therefore, HAT-PCR
and NGS could be used in a complementary fashion for
monitoring MRD in ALL, with NGS being used at diag-
nosis to obtain sequence information and at occasional in-
tervals to detect clonal evolution and with HAT-PCR being
used for more frequent monitoring. HAT-PCR may have a
greater role in chronic lymphocytic leukemia and myeloma
because clonal evolution is much less common in these
diseases.
Acknowledgments
We thank all members and staff of the Australasian Leu-
kemia and Lymphoma group for their contribution to the
study, the Children’s Cancer Institute Tumor Bank for
sample reception and initial processing, and Prof. Chris
Frampton for statistical advice. The Children’s Cancer
Institute is affiliated with both Sydney Children’s Hospital
Network and the University of New South Wales.
Supplemental Data
Supplemental material for this article can be found at
http://doi.org/10.1016/j.jmoldx.2022.03.007.
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