Keywords
Acute lymphoblastic leukemia - Berlin–Frankfurt–Munster - flow cytometry - L-asparaginase
- minimal residual disease
Introduction
Acute lymphoblastic leukemia (ALL) is a heterogeneous hematologic disease characterized
by the proliferation of immature lymphoid cells in the bone marrow, peripheral blood,
and other organs with peak incidence in between 2 and 5 years of age. Survival in
pediatric ALL has improved to roughly 90% in trials with risk stratification by biological
features of leukemic cells and response to treatment, treatment modification based
on patients’ pharmacodynamics and pharmacogenomics, and improved supportive care.[1]
Minimal residual disease (MRD) in ALL refers to the presence of leukemic cells below
the threshold of detection using conventional morphologic methods. Patients who experienced
a Complete Remission (CR) according to morphologic assessment alone can potentially
harbor a large number of leukemic cells in the bone marrow: up to 1010 malignant cells.
The most frequently used methods for MRD assessment include multicolor flow cytometry
(FCM) to detect abnormal immune phenotypes and polymerase chain reaction (PCR) assays
to detect clonal rearrangements in immunoglobulin heavy chain genes and/or T-cell
receptor genes. Current FCM or PCR methods can detect leukemic cells at a sensitivity
threshold of fewer than 1 × 10−4 (<0.01%) bone marrow mononuclear cells. The concordance
rate for detecting MRD between these methods is high.
The clinical significance of MRD has been conclusively demonstrated in both childhood
and adult ALLs. In most studies, MRD positivity is defined by the presence of 0.01%
or more ALL cells; the risk of relapse is generally proportional to the level of MRD,
particularly when measured during or at the end of remission induction therapy. The
prevalence of MRD during early therapy differs among genetic and biologic ALL subtypes.
However, being a measurement of drug resistance in vivo and reflecting multiple cellular, host, and treatment variables, MRD is typically
an independent prognostic factor. MRD is now used in several clinical trials for risk
assignment and to guide clinical management overall.[2]
[3]
Owing to the unique anticancer mechanism of action, L-asparaginase has been introduced
to the multidrug chemotherapy in children and adults with ALL, which has contributed
to significant improvement of therapy outcomes and to achieve complete remission in
about 90% of patients. L-asparaginase acts by reducing the exogenous supply of asparagine
by catalyzing its hydrolysis, inducing a relative deficiency of asparagine which leads
to apoptosis and impaired protein synthesis in leukemic blasts. Notwithstanding its
high therapeutic efficacy, L-asparaginase can increase the risk of thrombosis and
other complications.[4] Previous studies have proposed that enzyme activity >100U/L ensures complete asparagine
depletion.[5]
In Berlin–Frankfurt–Munster (BFM) 95/2000 protocols, Escherichia coli-derived L-asparaginase
was given twice weekly for initial 4 weeks of induction therapy. Within the ALL BFM
2000 trial, therapeutic drug monitoring of L-asparaginase done on day + 3 after administering
5000 IU/m2 of E. coli-derived L-asparaginase was compared with MRD on day + 33. The authors
found no significant relationship between MRD positivity and differences in L-asparaginase
levels. On the contrary, L-asparaginase activity values measured in patients with
severe toxicities significantly exceeded those measured in samples from patients without
toxicity.[6] In light of above and the severe toxicities encountered with L-asparaginase, especially
when co-administered with steroids during induction phase, we planned to study the
effect of the standard dose of E. coli-derived L-asparaginase on early MRD disease by phasing the same total dose to be
given once a week over 8 weeks and study its impact on early MRD at the completion
of 4 weeks and then again after the completion of 8 weeks of induction chemotherapy.
Methods
This study was carried out from February 1, 2014, to April 30, 2016. This study was
approved by the Institutional Scientific and Ethical Committee. Informed and written
consent was taken from the patients and/or their parents/guardians; in case, the patients
were <18 years. It was a prospective, observational, pilot study. Forty-five ALL children
and young adults up to the age of 40 years were enrolled.
Fourteen patients defaulted during induction treatment and were not eligible for further
analysis. Thus, 31 cases were eligible for final analysis. Partially or previously
treated, HIV-positive patients allergic to L-asparaginase and those with serum bilirubin
>2 mg/dl, serum glutamic-pyruvic transaminase >3 times ULN, creatinine clearance <50
ml/min, pancreatitis, and serum fibrinogen <100 mg/dl were excluded from the study.
MRD was assessed using Navios flow cytometer from Beckman Coulter. Six-color FCM analysis
was done using fluorescein isothiocyanate, phycoerythrin (PE), allophycocyanin, PE-Cy5,
PE-Cy7, and electron-coupled dye.
Patients were subjected to complete systemic examination, assessment of performance
status, body surface area and central nervous system (CNS). Investigations done included
complete blood counts, liver & renal function tests, tumor lysis profile, serum fibrinogen
levels, bone marrow aspiration and biopsy, FCM from aspiration sample, cerebrospinal
fluid analysis and 2D Echocardiography. After diagnosing the patients, the patients
were started on preinduction with prednisone along with the first dose of intrathecal
methotrexate. Patients were treated according to modified BFM 95 protocol [Table 1a] and [b].
Table 1a
Modified Berlin-Frankfurt-Munster 95 dose schedule in Phase A induction chemotherapy
|
Phase A
|
Dose
|
Days
|
|
Prednisone
|
60 mg/m2
|
D1-D28
|
|
Vincristine
|
1.5 mg/m2
|
D8, D15, D22, D29
|
|
Daunorubicin
|
30 mg/m2
|
D8, D15, D22, D29
|
|
L-asparaginase
|
5000 IU/m2
|
D8, D15, D22, D29
|
|
Methotrexate Intrathecal (IT)
|
12 mg
|
D1, D8, D36
|
Table 1b
Modified Berlin-Frankfurt-Munster 95 dose schedule in Phase B induction chemotherapy
|
Phase B (INJ)
|
Dose
|
Days
|
|
D – Day; INJ – Injection; IT – Intrathecal; IU – International Units
|
|
Cyclophosphamide
|
1000 mg/m2
|
D1 and D22
|
|
Cytarabine
|
75 mg/m2
|
D1, D8, D15, D22
|
|
L-asparaginase
|
5000 IU/m2
|
D1, D8, D15, D22
|
|
Methotrexate IT
|
12 mg
|
D1, D22
|
MRD1 assessment was done at the end of 4 weeks, i.e., day 36 and MRD2 at the end of
Phase B, i.e., day 72. Up to 1 million events were studied. CD45 blast gating method
was used to discriminate leukemic blasts from the cells of various lineages and to
facilitate the analysis of leukemic blasts present at low frequencies. MRD positive
was defined as residual blasts ≥0.01% whereas MRD negative was labeled as <0.01%.[2] Patients were evaluated with liver function test, amylase, and fibrinogen thrice
a week during Phase A and once a week during Phase B.
For statistical analysis of MRD at the end of 4 weeks and 8 weeks, we applied student’s
t-test (Levene’s test of equality of variance) for continuous variables. For binary
variables, we applied Chi-square test (Fisher’s exact test).
Results
The patient characteristics at baseline are shown in [Table 2] and [Figure 1]. The median age of the patients diagnosed with ALL was 14 years (range, 2–40), both
females and males were diagnosed, and males were found to be more in number than females.
In this study, 22 cases were B-cell immune phenotype and the remaining 9 cases belonged
to T-cell immunophenotype. No case of testicular involvement was seen. The BFM 95
risk group stratified 7, 16, and 8 patients as having standard risk, medium risk,
and high risk (HR), respectively. Good prednisolone response was observed on day 8
in 23 participants as compared to poor prednisolone response which was seen in 8 cases.
Table 2
Characteristics of the study population
|
n
|
|
ALL – Acute lymphoblastic leukemia; SR – Standard risk; MR – Medium risk; HR – High
risk; GPR – Good prednisolone response; PPR – Poor prednisolone response; MRD – Minimal
residual disease; CNS – Central nervous system
|
|
Age (years)
|
|
|
1-10
|
13
|
|
>10-40
|
18
|
|
Gender
|
|
|
Female
|
8
|
|
Male
|
23
|
|
Diagnosis
|
|
|
B-cell ALL
|
22
|
|
T-cell ALL
|
9
|
|
Risk group
|
|
|
SR
|
7
|
|
MR
|
16
|
|
HR
|
8
|
|
Prednisolone response
|
|
|
GPR
|
23
|
|
PPR
|
8
|
|
CNS status
|
|
|
CNS 1
|
29
|
|
CNS 2
|
1
|
|
CNS 3
|
1
|
|
Cytogenetics
|
|
|
High hyperdiploidy
|
2
|
|
Normal karyotype
|
24
|
|
HR karyotype
|
5
|
|
L-asparaginase dose
|
|
|
<8 doses
|
22
|
|
8 doses
|
9
|
|
MRD1 values
|
|
|
MRD negative
|
8
|
|
MRD positive
|
23
|
|
MRD2 values
|
|
|
MRD negative
|
13
|
|
MRD positive
|
18
|
Figure 1: Study population distribution
The CNS status evaluation as per the BFM 95 protocol at diagnosis categorized 29 cases
with CNS Status 1 whereas CNS Status 2 and 3 was diagnosed in one patient each. The
cytogenetic evaluation revealed two patients as high hyperdiploidy and 5 participants
as having HR cytogenetics. The remaining 24 participants had normal karyotype [Table 2].
The L-asparaginase dose was dichotomized into those who received 8 doses and the others
receiving less than eight doses are shown in [Table 3].
Table 3
Results as per the L asparaginase dose intensity
|
Variables
|
Patients receiving 8 doses (n=9)
|
Patients receiving <8 doses (n=22)
|
Statistical significance
|
|
SR – Standard risk; MR – Medium risk; HR – High risk; GPR – Good prednisolone response;
PPR – Poor prednisolone response; MRD – Minimum residual disease; BFM – Berlin-Frankfurt-Munster;
CNS – Central nervous system
|
|
Gender
|
|
|
|
|
Male
|
7
|
16
|
Fisher’s exact test
|
|
Female
|
2
|
6
|
(two-sided) 1.00
|
|
Diagnosis
|
|
|
|
|
B cell
|
7
|
15
|
Fisher’s exact test
|
|
T cell
|
2
|
7
|
(two-sided) 0.689
|
|
Prednisolone response
|
|
|
|
|
GPR
|
7
|
16
|
Fisher’s exact test
|
|
PPR
|
2
|
6
|
(two-sided) 1.00
|
|
Cytogenetics
|
|
|
|
|
High hyperdiploidy
|
1
|
1
|
Fisher’s exact test
|
|
Normal karyotype
|
17
|
6
|
(two-sided) 0.635
|
|
High risk karyotype
|
3
|
2
|
|
|
CNS
|
|
|
|
|
1
|
9
|
20
|
Fisher’s exact test
|
|
2
|
0
|
1
|
two-sided) 1.00
|
|
3
|
0
|
1
|
|
|
BFM risk group
|
|
|
|
|
SR
|
4
|
3
|
Fisher’s exact test
|
|
MR
|
4
|
12
|
(two-sided) 0.189
|
|
HR
|
1
|
7
|
|
|
MRD 2
|
|
|
|
|
Positive
|
7
|
11
|
Fisher’s exact test
|
|
Negative
|
2
|
11
|
(two-sided) 0.237
|
The median dose of L-asparaginase received during Phase A and B was 4 (range, 1–4).
However, the total median dose was 7 (range, 2–8). The main reason behind patients
receiving less than the scheduled eight doses was low serum fibrinogen levels and
liver dysfunction.
There were no cases of pancreatitis, thrombosis, or hypersensitivity. Our results
showed that L-asparaginase dose was not statistically significant with respect to
MRD2 (P = 0.237). The median L-asparaginase dose received in this study was 7, which was
also not statistically significant with respect to MRD2.
Discussion
Owing to the unique anticancer mechanism of action, L-asparaginase has been introduced
to the multidrug chemotherapy in children and adults with ALL, which has contributed
to significant improvement of therapy outcomes and to achieve complete remission in
about 90% of patients.[4]
[7]
[8] BFM-inspired protocols continue to be the most commonly followed treatment for patients
with ALL and L-asparaginase remains a crucial block in the treatment protocols. However,
most of the patients were unable to receive the prescribed doses of L-asparaginase
within a short span either due to derangements in the metabolic parameters such as
low serum fibrinogen levels, deranged liver function tests, thrombotic events, or
due to severe allergy/hypersensitivity.
There were no cases of drug hypersensitivity to L-asparaginase, pancreatitis, or bleeding
in this study. There were no cases of thrombosis in this study whereas coagulopathy
was reported among 2.1–15% and up to 30.2% of patients in the comparable studies.[4]
[9]
Our results clearly demonstrate that there is no correlation between L-asparaginase
dose intensity and MRD levels obtained at the end of induction treatment. These results
are in concordance with the results derived from the therapeutic drug monitoring study
in BFM 2000 trial.[6]
The results of this study suggest that the scheduled eight doses of L-asparaginase
can be safely given over 8 weeks instead of giving all the doses during Phase A as
per the BFM 95 protocol. In this study, 13 patients were MRD2 negative as compared
to only eight patients who were MRD1 negative, and none of these MRD-negative patients
relapsed. This happened probably due to the additional doses of L-asparaginase received
during Phase B induction therapy. It has been well documented in the previous studies
that achievement of MRD-negative status at the end of induction chemotherapy correlates
with long-term outcome in ALL. The detection of MRD is a major prognostic factor for
treatment in ALL of childhood. Several groups showed the predictive value of MRD at
the end of induction therapy with low MRD values correlating with better long-term
outcome.[10]
[11]
This study demonstrates the feasibility of administering 8 doses of L-asparaginase
over two phases rather than over the 4-week period as per the original BFM protocols.
This was associated with reduced toxicity and did not compromise the efficacy of the
therapy.
Conclusion
L-asparaginase is the backbone of induction therapy in ALL for decades. The dosing
schedules vary across all the standard protocols. L-asparaginase dose intensity does
not affect early MRD (neither MRD1 nor MRD2). Phasing L-asparaginase over 8 weeks
could lead to the achievement of more MRD2-negative patients and thereby positively
impacting their long-term outcome. Phasing of L asparaginase over 8 weeks may result
in reduced incidence of adverse drug events.
