Minimal Residual Disease in Adult Acute Lymphoblastic Leukemia: Egyptian Experience

Background: Acute lymphoblastic leukemia (ALL) is a clonal disease that affects early lymphoid progenitors in the bone marrow. Minimal residual disease (MRD) is assessed by different methods to monitor disease kinetics after treatment. Aim: to Assess MRD post-induction, at 6 and 12 months after intensive chemotherapy in adult patients with ALL. Patients and Methods: Seventy adult newly diagnosed acute lymphoblastic leukaemia patients were enrolled between July 2018 and July 2019 at the Clinical Hematology Unit, Ain Shams University hospitals, Egypt. MRD was assessed on the bone marrow samples using multi-parameter four color flow cytometry with 0.01% cut-off; below which cases are deemed MRD negative. Results: After the end of induction period, 13 out of 46 patients (28%) had positive MRD. However, MRD positivity is demonstrable in 14/32(43.8%), and 10/28(35.7%) patients at 6 and 12 months; respectively. MRD positivity was significantly associated with older age group (more than 39 years) and high NCCN risk stratum with p-values <0.05. Moreover, most of MRD positive patients at 12 months of therapy were of T-ALL immunophenotype (P value 0.002). Patients with complete remission and negative MRD exhibited significantly higher overall survival when compared to patients having MRD positivity (P value 0.027). Conclusion: MRD is a powerful predictor of outcome in ALL and its positivity at different time points is associated with poor prognostic factors as well as survival outcomes.


Introduction
Acute lymphoblastic leukaemia (ALL) is a type of haematologic cancer where lymphoid blasts exhibit arrested maturation and differentiation. This in turn results into the accumulation of early lymphoid progenitors in the bone marrow, peripheral blood as well as extramedullary sites; leading to perturbed haematopoiesis and extramedullary infiltrations (Della Starza et al., 2019).
Minimal residual disease (MRD) assessment in ALL patients has been routinely adopted in paediatric ALL. followed up for one year.
Patients included in the study were newly diagnosed de-novo adult cases of acute lymphoblastic leukaemia that were deemed fit to intensive chemotherapy. Secondary cases on top of chronic myeloid leukaemia, and those who were too frail to receive chemotherapy of curative intent were excluded.
Patients were risk stratified according to the National Comprehensive Cancer Network (NCCN) risk stratification criteria of 2015 into standard risk and high risk (Alvarnas et al., 2015). High risk patients exhibited high risk features including Philadelphia chromosome positivity, complex karyotype, age>35 years, the presence of extramedullary disease, high WBC count at diagnosis (>30 x10 9 /L for B-ALL; > 100 x 10 9 /L for T-ALL), or 11q23 positivity. Patients who did not show any of the high-risk criteria were considered standard risk.

Treatment protocols
A l l p a t i e n t s r e c e i v e d h y p e r f r a c t i o n a t e d cyclophosphamide, vincristine, doxorubicin, dexamethasone (Hyper-CVAD) cycles alternating with high dose methotrexate and cytarabine cycles that are detailed elsewhere (Kantarjian et al., 2004) Patients with Philadelphia chromosome positive ALL received imatinib 400 mg PO OD in conjunction with the chemotherapy cycles. After the achievement of complete remission, repetition of the cycles was done till a maximum of 4 cycles. After that, maintenance treatment using prednisone, vincristine, methotrexate and 6-mercaptopurine (POMP) was given as mentioned elsewhere (Kantarjian et al., 2004). Patients were followed up for one year.

Response criteria
Response was assessed according to the NCCN guidelines published in 2015. Complete remission(CR) is considered when bone marrow blasts <5%, absolute neutrophil count >1*10 9 /L, and platelet count >100*10 9 /L, with the disappearance of any extramedullary disease. This should persist for at least four weeks. Failure to achieve these targets define chemoresistance, whereas reemergence of bone marrow blasts or de-novo extramedullary disease after the initial response defines relapse (Alvarnas et al.,2015). Minimal residual disease is considered negative at a cut-off 0.01% (Campana, 2010).

Statistical analysis
Recorded data were analyzed using the statistical package for social sciences, version 23.0 (SPSS Inc., Chicago, Illinois, USA). Quantitative data were reported as mean±SD, while qualitative data were reported as frequencies and percentages. Student t test was used to compare quantitative data between two independent groups. Chi square test was used to compare qualitative data between different groups. Kaplan Meier survival analysis with Log rank test were used to compare time to event between different groups. Level of confidence was set to 95% and margin of error was accepted at 5%. Differences were considered to be statistically significant when P<0.05.

Table 1, exhibits the clinical and laboratory profile of the patients
The study included 70 patients. They had an average age of around 30.8±11 years (range 21-56). About 2/3 of them belonged to adolescents young adults (AYA) group. Most of them (62.9%) were males. Expectedly, B-ALL patients were the majority (68.6%, n=48).
About one third of them fitted in the standard risk stratum, whereas the remainder had high risk features. Philadelphia chromosome was there in 14 patients (20%). By the end of induction, analysis of the remission status was performed in conjunction with MRD assessment for those who developed CR. Fourteen patients died during induction and 10 patients were chemo-resistant (14.3%). Forty-six patients went into CR. MRD was negative in 33/46 (72%) and positive in 13/46 (28%).
Further follow-up of these patients showed that MRD positivity was achieved in 14/32 (43.8%), and 10/28 (35.7%) patients at 6 and 12 months; respectively.By the end of one year follow up, 40% of cases enrolled were alive (n=28) with a median survival of 85 days. Deaths were mainly due to sepsis during nadir either after induction or in the following cycles.
After dissecting patients by their MRD status at different timepoints, comparative analysis of their different demographic and clinico-pathologic features of patients and disease has been made as shown in Table 2.
In regard to post-induction MRD, it is demonstrable that the majority of patients exhibiting post-induction measurable residual disease were belonging to the older age category (age>39 years) whereas adolescent young adult group had statistically significant association to those having extramedullary disease, this did not yield any statistical value (P>0.05).
Minimal residual disease status at 6 and 12 months timepoints have been plotted against the same aforementioned variables used with post induction MRD reproducing the same results. However; MRD positivity MRD negativity with P-value 0.023. Moreover, high NCCN risk was remarkably related to positive postinduction MRD (P-value 0.034).
Despite the fact that MRD positivity was much more common among males, T-ALL immunophenotype, and    at 12 months was significantly associated with T-ALL (P 0.002). This is well-depicted in tables (3) and (4).
There is significant association between CR and negative MRD with OS (P value 0.027 in Figure 1.

Discussion
Minimal residual disease in acute lymphoblastic leukaemia is the persistence of residual leukemic cells in the bone marrow and /or peripheral blood of the patients after receiving cytotoxic chemotherapy and radiotherapy. It is used as a way to foretell the probability of relapse and the survival rate. Moreover, it guides the clinician decision in regard to escalating or de-escalating therapeutic intensity (Kruse et al.,2020).
Many laboratory methods are utilized to assess MRD; of them multi-parameter 3 or 4 colour flowcytometry and polymerase chain reaction (PCR) were the most extensively used techniques. Multi-parameter colour flowcytometry depends upon the detection of peculiar leukemia associated immunophenotype (Abou Dalle et al., 2020) This includes either aberrant expression of myeloid associated antigens or reduced or enhanced expression of lymphoid antigens normally expressed on benign B or T-cell progeny. However; PCR relies on the amplification of immunoglobulin or T-cell receptor gene rearrangements or recurrent genetic abnormalities (Abou Dalle et al., 2020).
In this current study, we assessed MRD in adult ALL patients after completing the induction chemotherapy, at 6 and 12 months timepoints and plotted these results against different patients as well as disease prognostic variables.
In pediatric ALL, MRD post induction range between 25-30% (Borowitz et al., 2008;Patkar et al., 2017), in our study 13 out of 46 patients (28%) had positive MRD post induction, furthermore MRD positivity is demonstrable in 14 out of 32 patients (43.8%), and 10 out of 28 patients (35.7%) at 6 and 12 months; respectively. It is noticeable that older age category was associated with MRD positivity at different timepoints in our study. This is concordant with the results obtained by Kikuchi and coworkers who showed that age >55 years was associated with measurable residual disease on day 100 by univariate analysis. However; multivariate analysis failed to prove age as an independent predictor of positive MRD in that study (Kikuchi et al., 2010) In our study, minimal residual disease positivity at 12 months was associated with T-ALL immunophenotype. Yet; this is not reproducible at post-induction or 6 months timepoints. No other studies have studied such an   association which may warrant further studies as it may explain the higher incidence of relapse among T-ALL cases. In addition, it may be explained by the slower pace of T-ALL disease in responding to therapy when compared to B-cell ALL (Raetz and Teachey, 2016) In our study, minimal residual disease status was consistently associated with cytogenetic risk; with MRD positivity being linked to poor risk cytogenetics. This is concordant with the study held by Borowitz and colleagues in 2008 on childhood ALL cases. They observed that MRD positivity in patients of standard risk with poor risk cytogenetics was about double that encountered in patients with favourable cytogenetics like TEL-AML1 (Borowitz et al., 2008).
We failed to prove an association between MRD status in our cohort and extramedullary disease. This may be explained by the need of larger sample size to study such a relationship.
In our study, MRD negative cases enjoyed significantly longer overall survival in comparison to MRD positive counterparts. This mirrors the results obtained by Gökbuget et al who evaluated molecular response among Philadelphia negative ALL cases using PCR and identified molecular failure as a predictor of lower survival rates (Gökbuget et al., 2014) This is also in line with several other studies (Kikuchi et al., 2010;Berry et al., 2017;Bassan et al., 2019).
In conclusion, MRD positivity was significantly associated with older age group, high NCCN risk stratum and T-ALL immunophenotype. Patients with complete remission and negative MRD exhibited significantly higher overall survival. minimal residual disease assessment in adult ALL patients should be incorporated as an essential component of patient's follow up. This helps us to tailor therapy as per the patient's needs to achieve better disease control without undue toxicities.

Author Contribution Statement
Rasha Ibrahim did Study Design and Statistical Analysis, Alia Saeed did Data Collection and Literature Search, all authors contributed to Data Interpretation, Manuscript Preparation.