Research - International Journal of Medical Research & Health Sciences ( 2020) Volume 9, Issue 10
Hyper-CVAD Protocol versus UKALL Protocol and the Minimal Residual Disease Status in Adult Acute Lymphoblastic Leukemia PatientsAhmed Mjali1*, Bassam Francis Matti2, Yassmin Ali Abdul Kareem2, Dhulfiqar Azeez Hasan3, Anmar Alharganee4, Alaa Fadhil Alwan5, Aladdin Sahham Naji6, Tareq Abdullah Saleh2, Amer Shareef Alhachmi7, Ibrahim Khalil Al Shemmari8, Haider Hasan Jaleel Al- Shammari6, Talib Hussein Kamoona9, Nareen Tawfeeq Abbas10, Saja Khudhair Abbas1 and Ahmed Ibrahim Shukr11
2Baghdad Teaching Hospital, Medical City, Baghdad, Iraq
3Teaching Laboratory, Al Sader Medical City, Najaf, Iraq
4Nottingham University Hospital NHS UK, Baghdad Medical City/Oncology Teaching Hospital, Iraq
5The National Center of Hematology, Mustansiriyah University, Baghdad, Iraq
6Baghdad University, College of Medicine,Baghdad, Iraq
7University of Thi-Qar, College of Medicine, Thi-Qar, Iraq
8Department of Internal Medicine and Hematology, Imamain Khadumain Medical City, Baghdad, Iraq
9Kufa University, College of Medicine, Najaf, Iraq
10Department of Hematology, Hiwa Hematology/Oncology Hospital, Sulaymaniyah, Iraq
11Kirkuk Oncology/Hematology Center, Kirkuk, Iraq
Ahmed Mjali, Department of Hematology/Oncology, Al- Hussein Medical City, Karbala, Iraq, Tel: 962 6 580 4804, Email: [email protected]
Received Date: Sep 14, 2020 / Accepted Date: Oct 23, 2020 / Published Date: Oct 30, 2020
Introduction: In Iraq acute lymphoblastic leukemia (ALL) represents the most common hematological malignancy contributing 41% of all leukemia cases. Hyper-CVAD protocol and UKALL protocol are commonly used in ALL treatment. In this study, we tried to assess minimal residual disease (MRD) post induction therapy for both hyperCVAD protocol and UKALL protocol by using 8 colors flow cytometry.
Material and Method: Data collected retrospectively from 85 adult patients with acute lymphoblastic leukemia (≥ 14 years old) received either hyper-CVAD or UKALL chemotherapy protocol, with MRD level results post induction therapy of each protocol therapy, from January 2017 till October 2019. Fifty patients (58.82%) with B-ALL, while T-ALL were 35 patients (41.18%). Patients treated with hyper-CVAD protocol were 52 patients (61.18%) while 33 patients (38.82%) were treated with UKALL protocol.
Results: Patients with de novo ALL of Philadelphia chromosome negative, male to female ratio 2:1 and the mean age was 23 years. Thirty seven patients (43.53%) achieved MRD negative. Of these MRD negative patients, 14 patients (37.84%) were post UKALL while 23 patients (62.16%) were post hyper-CVAD protocol with (p=0.9). Conclusion: Both hyper-CVAD and UKALL protocol therapy in adult ALL have a good efficacy without significant difference in achieving MRD negativity.
KeywordsMinimal residual disease, Hyper-CVAD protocol, UKALL protocol
Acute lymphoblastic leukemia (ALL), is a clonal expansion of hematopoietic blasts, it is an extremely heterogeneous disease that including different entities for which special therapeutic approaches are needed. In developed countries ALL represents approximately 12% of all childhood and adult leukemia and about 60% of diagnosed patients were under 20 years of age. In Iraq, leukemia represents more than 6% of cancer patients and ALL represents the most common hematological malignancy contributing 41% of all leukemia cases [1-3].
The outcomes of ALL treatment have been a successful story in pediatric oncology, while in adults were inferior. Poor results in adults ALL has been variously due to high prevalence of high-risk leukemia with increase drug resistance, reduced tolerance and treatment compliance, reluctance to accept certain temporary toxic effects and less effective treatment regimens as compared with childhood ALL . The successful response in pediatric protocols leads to inspire these protocols in treatment of adult ALL patients. UKALL 2003 is the most common pediatric protocol used in Iraq which proved to be effective up to 24 years old [5-7]. These protocols use non-myelosuppressive drugs, such as glucocorticoids and L-asparaginase and fewer myelosuppressive drugs, such as anthracycline, but L-asparagines side effects was a great concern .
Current intensive treatment protocols such as hyper-CVAD is widely used in many centers and have improved the outcomes in adults ALL patients, with more than 80% response rate, nonetheless we still have to substitute our strategies to increase the rates of long-term survival which range from 30 to 45% [8,9].
Minimal residual disease (MRD) represents the low level disease that traditional morphology cannot detect. MRD monitoring can be a way to accurately predict early response to treatment and detect persistence disease in ALL patients [10,11]. Nowadays, the most important technique to assess MRD in ALL are Flow cytometric analysis (FCM) and Polymerase chain reaction (PCR) .
The assessment of MRD by flow cytometry needs to identify immune phenotype markers with selected positive expression in leukemia cells comparable to negative expression in normal hematopoietic lineage cells [13,14]. Differences in gating strategies, antibody panels and the applied immune-staining protocols are significantly different according to centers and chemotherapy protocols making MRD as subjective expert procedures . Here in this study, we tried to assess MRD post induction therapy of both hyper-CVAD protocol and UKALL protocol by using 8 colors flow cytometry, supposing the differences in efficacy degree on MRD status of each protocol.
Patients and Methods
This is a retrospective analysis from different Iraqi centers experience with MRD status data in adult ALL patients between January 2017 and October 2019. We included all data for ALL patients ≥ 14 years from both genders. All patients were Philadelphia chromosome negative without significant co-morbidity and good performance status by ECOG scale (less than 3). On other side we excluded patients who previously treated or relapsed, patients with Burkitt lymphoma/leukemia, patients with significant co-morbidities or poor performance status by ECOG scale (≥ 3), patients with Philadelphia chromosome positive and those with CNS involvement at diagnosis to avoid any additional risk.
Data were obtained from the records of each patient. ALL patients in our study were treated either with hyper-CVAD protocol or UKALL protocol, according to hematologists experiences and preferences. Protocols details shown in Tables 1 and 2.
|Phase and Therapy||Dose||Route of Administration||Days Administered|
|Course 1, 3, 5, 7|
|Cyclophosphamide||300 mg/m2||b.i.d IV (2 hours infusion)||D1-3|
|Doxorubicin||50 mg/m2||IV (2 hours infusion)||D 4|
|Dexamethasone||40 mg||I.V. or oral||D1-4 , 11-14|
|Course 2, 4, 6, 8|
|Methotrexate||1000 mg/m2||Iv (24 hours infusion) With folinic acid rescue||D1|
|Cytarabine||2000 mg/m2||b.i.d IV (2 hours infusion)|
|Methotrexate||12 mg||IT||D2 of each course|
|Cytarabine||100 mg||IT||D7 of each course|
*All courses with G-CSF support , repeated every 3 weeks
|Phase and Therapy||Dosage||Route of Administration||Days Administered|
|Phase 1, weeks 1-4|
|Daunorubicin||60 mg/m2||IV||D1, 8, 15, 22|
|Vincristine||1.4 mg/m2||IV||D1, 8, 15, 22|
|l-asparaginase||10000 U||IV or IM||D17-28|
|Phase 2, weeks 5-8|
|Cyclophosphamide||650 mg/m2||IV||D1, 15, 29|
|Cytarabine||75 mg/m2||IV||D1-4, 8-11, 15-18, 22-25|
|Methotrexate||12.5 mg||IT||D1, 8, 15, 22|
|Intensification/CNS prophylaxis (3 cycles)|
|Methotrexate||3000mg/m2||IV (with folinic acid rescue)||D1,8,22|
|Asparagines||10000 IU||IV||D2, 9,23|
*Followed by transplantation or consolidation and maintenance therapy
Response Assessments and MRD
MRD was tested by flow cytometry analysis for the bone marrow aspiration; as the MRD levels in B-cell precursor ALL tend to be 1 to 3 logs lower in peripheral blood than in bone marrow . European clinical guidelines and protocols recommend testing patients who achieve a complete hematological remission (CR) for MRD for the purpose of risk stratification which drives treatment decisions and level ≥ 0.01% or 10-4 cell considered as MRD positive [17,18]. In general, the assessment made at the end of induction therapy when the peripheral blood indices recovery (hemoglobin ≥ 10 g/dl, ANC ≥ 1500 mml and platelets count ≥ 100 × 109 without blasts in peripheral blood) [19,20].
It is important to note that the first sample taken from the bone marrow should be send for MRD analysis, to prevent dilution with peripheral blood (as dilution may lead to underestimation of MRD). The bone marrow sample/peripheral blood collected in either EDTA or heparin. The sample transported at room temperature and the assay was complete within 48 hours. Before starting the staining, it is important to get the cell counts on the sample to be processed. The bone marrow sample can be directly run on an automated cell counter to get the counts. It is optimal to start with 1-2 × 106 cells in 100 μL sample volume .
The samples are all processed by a Stain-lyse-wash approach and the same is followed for MRD analysis by 8 color flow cytometry from BD. Red cells lysis is performed using a fixative containing lysing reagent like buffered NH4Cl containing 0.25% formaldehyde. Since cytoplasmic/nuclear markers are also used, the tube after surface staining is premeabilised using commercial permeabilising reagent as per company specific protocol, cytoplasmic antibodies are added. After the optimal staining time, the cells are washed once, the volume is brought to 500 μL sheath and cells are ready for acquisition . Regarding gating markers, in our lab we use the protocols that shown in Tables 3 and 4.
|Florochromes||V450||v 500||FITC||PE||PerCP Cy 5.5||Pe-Cy7||APC||APC H7|
|TUBE 1||CD Marker||CD20||CD45||CD58||CD66c||CD 34||CD19||CD10||CD38|
|TUBE 2||CD Marker||CD73||CD45||CD123||CD200||CD34||CD19||CD10||CD81|
The results were analyzed statistically in all tables, and Chi square procedure was used to find the significant differences at the level of significance (p ≤ 0.05).
Patients Characteristics and Treatment Protocols
In this study, there were 85 patients with de novo ALL and Philadelphia chromosome negative, of these patients, 57 patients (67.06%) were male and 28 patients (32.94%) were female, with a male to female ratio 2:1. Mean age was 23 years, most of the patients (48%) with age group between 12-19 years old. There were 50 patients (58.82%) with B-ALL, while T-ALL were 35 patients (41.18%). Fifty two patients (61.18%) were treated with hyper-CVAD protocol while 33 patients (38.82%) were treated with UKALL protocol. Most of patients ≥ 25 years treated with hyper-CVAD protocol (22 patients) while only 5 patient ≥ 25 years treated with UKALL protocol.
Minimal Residual Disease Status
From all cases, there were 37 patients achieved MRD negative, 14 patients (37.84%) achieved MRD negativity post induction with UKALL protocol while 23 patients (62.16%) post induction with hyper-CVAD protocol with (p=0.9) as shown in Table 5.
|Negative||14 (37.84%)||23 (62.16%)||37 (100%)||0.9|
|Positive||19 (39.58%)||29 (60.42%)||48 (100%)||0.9|
MRD in Related to Age, Gender, Type of ALL after Induction
By analysis of MRD status according to the age, gender and different types of ALL patients, there was no significant difference between MRD status regarding age and gender. Regardless protocol therapy, T cell type ALL tend to be more MRD positive with 29 patients (82.86%), while only 19 patients (38%) of B-ALL stay with MRD positive (p=0.0001) as shown in Table 6.
|Variable||MRD Negative||MRD Positive||RR (95%CI)||p-value|
|≥ 25||14 (51.85%)||13(48.15%)||0.92||0.9|
|Gender||Female||13 (46.43%)||15 (53.57%)||1.15||0.9|
|Male||24 (42.11%)||33 (57.89%)||1.37||0.9|
|Type of All||B Type||31 (62%)||19 (38%)||0.61||0.1|
|T Type||6 (17.14%)||29 (82.86%)||4.83||0.000*|
|Type of Protocl||Hyper-CVAD||23 (44.23%)||29 (55.77%)||1.26||0.9|
|UKALL||14 (42.42%)||19 (57.58%)||1.35||0.9|
*means the significant differences, Relative Risk (RR) , Confidence Interval (CI)
Types of ALL Regarding Protocol Types and MRD
Nineteen patients (61.29%) with B-ALL post induction therapy with Hyper-CVAD protocol were MRD negative while 12 patients (38.71%) were MRD negative post induction therapy with UKALL protocol (p=0.900). In T-ALL patients 4 patients (66.7%) were MRD negative post induction treatment with Hyper-CVAD while only 2 patients (33.3%) were MRD negative post induction treatment with UKALL protocol and (p=0.900) as shown in Table 7.
|Protocol Type||Negative MRD||Positive MRD|
|B-ALL||Hyper-CVAD||19 (61.29%)||11 (57.89%)|
|UKALL||12 (38.71%)||8 (42.11%)|
|Total||31 (100%)||19 (100%)|
|T-ALL||Hyper-CVAD||4 (66.67%)||18 (62.1%)|
|UKALL||2 (33.33%)||11 (37.9%)|
|Total||6 (100%)||29 (100%)|
Although clinical factors and cytogenetics play an important role in guiding therapy, MRD has become standard practice to evaluate patients for using molecular techniques such as flow cytometry and PCR . The first studies on MRD detection in ALL date back from the 1980s, using immunofluorescence microscopy. Three decades later, monitoring of MRD has become routine clinical practice in frontline treatment of ALL patients and guide treatment decision [15,20,26].
In our study from 85 adult ALL patients, there were 37 patients (43.53%) achieved MRD negative post induction therapy. For those who achieved MRD negative, 37.84% of them were post induction with UKALL protocol and 62.16% were post hyper-CVAD protocol with no significant statistical differences. Siegel et al, accounted six comparisons of disease control with hyper-CVAD and pediatric regimens, two were reports of comparable outcome and four reported inferior outcome with hyper-CVAD . From 52 patients who received hyper-CVAD protocol 23 patients (44.23%) were MRD negative. This was close to study done by Cassaday, et al. where 42% of patients were MRD negative at 1st assessment .
In our study there were 42.4% patients achieved MRD negativity post UKALL protocol without any significant dif ference between MRD status and age. While in other studies for patients who received UKALL protocol showed 52% had MRD negative and those with aged ≥ 16 years old were more MRD high risk compared to younger age groups [7,28].
The T-ALL patients were tend to be more MRD positive with 29 patients (82%), while only 19 patients of B- ALL (38%) stay MRD positive (p=0.000). Our result was consistent with previous studies that suggest those patients with T-ALL had a poorer event free survival and an inferior overall survival than patients with other leukemia subtypes. It is generally associated with high levels of MRD during and at the end of remission induction therapy . In the same time most of the patients (48%) with age group between 12-19 years old and older adolescents were more likely to have T-cell ALL and detectable minimal residual disease during or at the end of remission induction compared with younger patients .
Hyper-CVAD and UKALL protocols in adults ALL are effective as same as regarding achieving MRD negativity without significant difference. Subsequent MRD follow up after the end of treatment is advisable to assess the stability of response status of each protocol therapy.
Conflicts of Interest
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
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