To study early treatment response in peripheral blood on Day 15 in B cell Acute Lymphoblastic Leukemia patients
- Conditions
- B cell Acute Lymphoblastic Leukemia
- Registration Number
- CTRI/2016/08/007180
- Lead Sponsor
- Tata Memorial Hospital
- Brief Summary
**PROTOCOL**
**An observational study to evaluate early treatment response in peripheral blood on Day 15 by flow cytometry in B cell Acute Lymphoblastic Leukemia patients treated with ICiCLe protocol.**
*Principal Investigator*:
**Dr.** **P G Subramanian**
Associate professor,
Hematopathology Laboratory,
Tata Memorial Hospital
*Co-principal Investigators*:
**Dr. S. D. Banavali**
Professor and head,
Department of pediatric oncology,
Tata Memorial Hospital
**Dr. Brijesh Arora**
Professor,
Department of pediatric oncology,
Tata Memorial Hospital
*Co-Investigators*:
**Dr. Prashant Tembhare**
Assistant professor,
Hematopathology Laboratory,
Tata Memorial Hospital
**Dr. Sumeet Gujral**
Professor,
Hematopathology Laboratory,
Tata Memorial Hospital
**Dr, Nikhil Patkar**
Assistant professor,
Hematopathology Laboratory,
Tata Memorial Hospital
**Dr. Gaurav Narula**
Associate professor,
Department of pediatric oncology,
Tata Memorial Hospital
**Dr. Ketan Ingle**
Junior resident,
Department of pathology,
Tata Memorial Hospital
**STUDY SUMMARY**
**1. INTRODUCTION AND RATIONALE:**
Acute lymphoblastic leukemia is the neoplasm of precursor lymphoid cells. It is the most common malignancy affecting children worldwide. The estimated worldwide incidence is 1-4.75/100,000 persons per year(1). Over 75% of children affected by ALL are less than 6 years of age. B cell ALL is most common in ALLs.
The treatment of ALL has improved over the years. General plan of treatment of B-ALL under various protocols consists of remission-Induction phase, intensification or consolidation phase followed by maintenance or continuation phase. The treatment consists of steroids, L-asparginase and multiple (three to four) chemotherapeutic agents along with CNS-directed therapy during Induction phase(2).
Apart from the pretreatment factors such as age, cytogenetics and total leukocyte count at diagnosis, response to treatment has been shown to be independent important prognostic factor(3,4). In particular, early response to treatment can be used to stratify patients according to the risk of relapse, on basis of which further treatment intensification or de-escalation may be done(5,6).
The early response to therapy can be assessed at various time points. First is after completion of prephase, which is a part of remission-induction phase in BFM (Berlin-Frankfurt-Munster) based protocols. This determines the response to corticosteroids. Early corticosteroid response has been shown to be an important independent prognostic factor(7). This is measured usually by cytomorphological assessment of peripheral blood for presence of leukemic blasts. Studies show that Prednisone poor responders (PPR) having peripheral blood blast on Day 7 more than 1000/µL have significantly less event free survival as compared to prednisone good responders (PGR)(8,9). In poor corticosteroid responders, treatment escalation has yielded improved results(10).
Conventionally, the next point of treatment response assessment has been a morphological assessment of end of induction bone marrow. If the blast percentage is less than 5% it is a good response and has better outcome than blast percentage at or above 5%. Morphological assessment has problems in terms of sensitivity and reproducibility. Regenerative normal B cell precursors are morphologically indistinguishable from blasts. To overcome these limitations and to increase the sensitivity to pick up presence of residual disease ancillary techniques like multi-parameter Flow cytometry and molecular methods have been used. The presence of low level disease, detected by ancillary methods has been termed as Minimal Residual Disease.
Minimal residual disease (MRD) has been found to be the major predictor of response irrespective of all other risk factors(11)(12)(13). The evaluation of MRD at specific time points during treatment of B-ALL is useful for risk stratification and subsequent modification of treatment(11)(14).
MRD evaluation of bone marrow aspirate at the end of remission-induction phase is an integral part of all of B-ALL protocols. But various studies have shown that MRD evaluation even at earlier time points can improve detection of high risk cases(15) and identification of subgroup having good prognosis(16,17). This can lead to further improvement in treatment stratification by MRD.
Among the earlier end points, Day 15 bone marrow was the best early MRD time-point to differentiate between patients with high, intermediate and low risk of relapse(18). Basso et al (AIEOP-BFM-ALL 2000 protocol) showed that measurement of MRD by flow cytometry in day 15 bone marrow was the most powerful early predictor of relapse. Even MRD at one single time point in day 15 bone marrow has been shown to have a robust prognostic impact (19) In study by Panzer-Grümayer et al (ALL-BFM 90 and 95 protocol), all patients with negative or low day-15 MRD had excellent prognoses and were in the MRD-based low-risk group. Thus, after only 2 weeks of treatment, it was possible to identify a patient population of 20% who may benefit from the least intensive treatment(17).
In addition, Ratei et al(AIEOP-BFM-ALL 2000 protocol) have shown that Blast count at day 15 bone marrow by flow cytometry is the strongest predictive parameter for the remission status(20).
Thus, early MRD measurements at day 15 post remission-induction in B-ALL can provide additional information for identification of very good responders and a small subgroup of poor responders, with further potential of treatment modification.
Few previous studies have evaluated role of MRD detection in peripheral blood at various time points(13,16,20–23). The studies show that peripheral blood MRD gives additional prognostic information and is also helpful in risk-stratification.
Volejnikova et al (ALL IC-BFM 2002 protocol) concluded that MRD in peripheral blood at day 15 identified a large group of patients with an excellent prognosis and added prognostic information to the risk stratification based on minimal residual disease at day 33 and week 12. This trial did not have MRD based treatment modification after post induction in its protocol. At day 15, a level of minimal residual disease in blood lower than 10-4 as detected by PCR was associated with an excellent 5-year relapse-free survival (100% *versus* 69±7%; *P*=0.0003). Subgroups defined by the level of minimal residual disease in blood at day 15 (high-risk: ≥10-2, intermediate-risk: <10-2 and ≥10-4, standard-risk: <10-4) partially correlated with bone marrow based stratification. Apart from Day 15, no other time point analyses were predictive of outcome in peripheral blood, except for a weak association at day 8.(16).
The study of B-ALL patients by Borowitz et al treated on COG (Children Oncology Group) protocols which involves four drug induction protocol from day 1, showed that Peripheral blood MRD negativity at Day 8 identified a subset of patients with favourable prognosis. Day 8 peripheral blood MRD status has also been found to be well correlated with end induction MRD status in various other studies (13,14,20,21). As ICiCLe protocol is BFM (Berlin–Frankfurt–Münster) based protocol, it involves 7 day Prednisolone only prephase followed by three/four drug induction with chemotherapeutic agents, the evaluation of MRD in peripheral blood on day 15 of treatment will actually reflect the early response to chemotherapeutic agents; equivalent to day 8 in COG/UK-ALL other non-BFM based protocols.
The present study will evaluate the role of MRD evaluation in peripheral blood at Day 15 post induction by flow cytometry in B-ALL patients treated on ICiCLe (Indian Childhood Collaborative Leukaemia Group) protocol- a BFM based protocol, already IRB approved vide project no. 1312, June 2, 2014. Peripheral blood MRD detection is less invasive method than that of bone marrow. Also, MRD levels in peripheral blood do not have the possibility of getting affected by patchy distribution of residual disease. But there is approx one log less blasts in the peripheral blood as compared to bone marrow at Day 15 shown by studies doing simultaneous assessment of peripheral blood and bone marrow.
Minimal Residual Disease detection:
The contemporary methods for detection of MRD include RQ-PCR for Immunoglobulin and TCR gene rearrangementsand Flow cytometric immunophenotyping.
Flow cytometry is a quantitative, widely applicable, faster & comparatively cheaper method and also provides sensitivity up to 0.01% as well as specificity in detection of leukemic blasts(14,24). Besides, there is high level of concordance between PCR and flow cytometry, especially during early induction-MRD(25). Flow cytometry is further more useful in detection of early induction MRD, as residual disease is more evident because of the lower background level of hematogones(26). Recent improvements in flow cytometric methods, such as 8-10 color analysis and new leukemia markers, have further increased sensitivity and allow better differentiation from hematogones in regenerating bone marrows. The major factor limiting the sensitivity of Flow cytometry is the number of cells/ events acquired. The published studies include acquisition of cells/events of up to 1-5 x 105 cells only, thereby reducing the sensitivity of detection. The current study proposes to acquire over a million (106) events to make the peripheral blood blast level sensitivity to nearly reach the sensitivity of bone marrow while avoiding an invasive procedure.
Issues in Flow cytometry techniques:
Early flow cytometry MRD studies relied on expertise of flow cytometrist for reliable interpretation. This results in a human component with its attendant subjectivity. The MRD analysis requires expertise and experience which is still limited in India. In the western world this is done in a centralized laboratory for multicentre trials. The samples are perishable and required to be processed in a short period of time (preferably 24hrs). Hence there is need for expertise in interpretation to be available in all centres. In India the expertise is currently scarce and limited to a handful of centres, where as the patients and treating centres are present widely. There is need for automated analysis with lesser human input in India.
The EuroFlow consortium has introduced new high-throughput concepts in flow MRD, based on multivariate analysis, e.g. principal component analysis. N-dimension (e.g. principal component analysis)-based deviations from normal hematogones is used. This is achieved using novel software (e.g. Infinicyt). Multiple parameters can be assessed in a single plot as opposed to two parameters per plot in conventional analysis. Estimated sensitivity of flow cytometry with these tools is 10-4-10-5(2.5-5.0 x 106 cells analyzed). The software is validated in multicentre studies and is claimed to reduce the skill, complexity and subjectivity of data interpretation of MRD by flow cytometry(27,28).
In the present study, we propose to analyse the MRD status using software tools which will give better sensitivity, objectivity and standardization. As a corollary, we will compare the flow cytometry MRD analysis by automated software with manual (observer based) analysis. If the claim is proven this will enable standardised MRD evaluation for multicentre studies and reduce training and experience need for doing MRD.
**2. AIMS/ OBJECTIVES**:
- To study day 15 peripheral blood residual blasts level as an early indicator of treatment response in B-ALL and assess the predictive value of peripheral blood day 15 treatment response for Post induction (day 35) MRD levels.
- To evaluate role of automated evaluation in detection of MRD using software tools based on principle component analysis(multivariate analysis of flow cytometric marker intensities)
- To study the 4 year relapse rates for B- ALL children on ICiCLe protocol based on Day 15 and post induction MRD. (This aim will not be the part of student thesis)
**3. METHODS:**
This is an observational, non-randomized, prospective study.
**3a.Eligibility criteria**:
**Inclusion Criteria:**
All patients under 15 years of age with newly diagnosed B-ALL enrolled in ICiCLe protocol.
**Exclusion Criteria:**
Patients with following high risk cytogenetics-
(i) MLL rearranged leukaemias, e.g. t(4;11) (q21;q23)
(ii) Low hypodiploidy (<45 chromosomes)
(iii) t(17;19) (q22;p13) (TCF3-HLF)
(iv) Intrachromosomal amplification of chromosome 21 (iAMP21)
(v) t(9;22) (q34;q11) (BCR-ABL)
**3b. Ethics:**
The study will be conducted after clearance from Institutional Ethics Committee. The study will also be conducted in accordance with the guidelines of the Indian Council of Medical Research 2006.
**3c. Study Site:**
Tata Memorial Hospital and ACTREC
**3d. Study Procedure:**
100 newly diagnosed Precursor B-Acute Lymphoblastic leukemia patients, as confirmed by flow cytometric immunophenotyping (done as a part of standard of care) which are undergoing therapy based on ICiCLe protocol, will be enrolled in the study.
As a part of routine investigations, B-ALL patients are assessed for Day 15 automated Complete Blood Count. The remaining blood sample after the Automated Complete Blood Count and peripheral blood smear examination will be utilized for flow cytometric assessment of Minimal Residual Disease (MRD). The results of the flow cytometric analyses will be blinded from the treating physicians. So, there will be no bearing on management of patients.
Also, as per standard of care, these patients will undergo routine flow cytometric examination of bone marrow for assessment of post remission-induction minimal residual disease on day 35.
Meanwhile, patients will continue to receive treatment as per ICiCLe protocol and will be followed up accordingly.
**Flow Cytometric Immunophenotyping:**
Specimens will be stained within 24 hours of collection with a panel of antibodies mentioned below. Erythrocytes in specimen will be lysed by incubating with NH4Cl based lysing solution. Specimens will be then washed with phosphate buffered saline (PBS) to remove cytophilic antibodies before determining cell number. 2 x 106 cells in 50 μL cell suspension in PBS will be stained with a cocktail of antibodies and incubated for 20 min at room temperature (maintained at 21-23°C). The antibody panels will include antibodies against CD3, CD10, CD19, CD20, CD25, CD34, CD38, CD44, CD45, CD58, CD73, CD81, CD86, CD123 and SYTO13.
Antibody Panel:
| | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| **Tube no.**
**BV510**
**BV421/Pacifc Blue**
**FITC**
**PE**
**PE-CF594**
**PerCP-Cy5.5/ PC5.5**
**PC7**
**APC**
[**APC\_AF\_700**](mailto:APC_Af@700)
[**APC-AF\_750**](mailto:APC-AF@750)
| ***1***
**CD20**
**CD123**
**CD58**
**CD86**
**CD25**
**CD19**
**CD10**
**CD34**
**CD45**
**CD38**
| ***2***
**CD20**
**CD73**
**CD44**
**CD81**
**X**
**CD19**
**CD10**
**CD34**
**CD45**
**CD38**
| ***3***
**X**
**CD3**
**SYTO13**
**X**
**X**
**CD19**
**X**
**X**
**CD45**
**X**
Cells will be acquired on 10 color flow cytometer (Navios, BeckmanCoulter). Acquisition will be done to reach at least 1 million events per tube. Flow cytometric data thus generated will be analyzed by using two softwares:
1. Kaluza software from Beckman Coulter (Observer based/Manual analysis)
2. Infinicyt software from Cytognos (Automated analysis)
Analysis with each of the software will be done in separate sets and will be blinded for the patient name or case numbers. Both the analyzers will be blinded from the results of the other analysis software.
**3e. Statistical Analysis:**
Pearson’s chi square test and Fischer’s exact test will be used. Correlation analysis will be done using Pearson’s correlation coefficient.
In the later (long-term) part of the study, survival analysis will be done using Kaplan-Meier analysis and cox proportional hazard model.
The evaluation of automated software MRD levels versus manual analysis will be done using correlation coefficient and bland altman analysis.
**4. References:**
1. Redaelli A, Laskin B l., Stephens J m., Botteman M f., Pashos C l. A systematic literature review of the clinical and epidemiological burden of acute lymphoblastic leukaemia (ALL). Eur J Cancer Care (Engl). 2005 Mar 1;14(1):53–62.
2. Inaba H, Greaves M, Mullighan CG. Acute lymphoblastic leukaemia. Lancet Lond Engl. 2013 Jun 1;381(9881):1943–55.
3. Conter V, Bartram CR, Valsecchi MG, Schrauder A, Panzer-Grümayer R, Möricke A, et al. Molecular response to treatment redefines all prognostic factors in children and adolescents with B-cell precursor acute lymphoblastic leukemia: results in 3184 patients of the AIEOP-BFM ALL 2000 study. Blood. 2010 Apr 22;115(16):3206–14.
4. Pui C-H, Robison LL, Look AT. Acute lymphoblastic leukaemia. The Lancet. 2008;371(9617):1030–43.
5. Vora A, Goulden N, Wade R, Mitchell C, Hancock J, Hough R, et al. Treatment reduction for children and young adults with low-risk acute lymphoblastic leukaemia defined by minimal residual disease (UKALL 2003): a randomised controlled trial. Lancet Oncol. 2013;14(3):199–209.
6. Vora A, Goulden N, Mitchell C, Hancock J, Hough R, Rowntree C, et al. Augmented post-remission therapy for a minimal residual disease-defined high-risk subgroup of children and young people with clinical standard-risk and intermediate-risk acute lymphoblastic leukaemia (UKALL 2003): a randomised controlled trial. Lancet Oncol. 2014 Jul;15(8):809–18.
7. Riehm H, Reiter A, Schrappe M, Berthold F, Dopfer R, Gerein V, et al. [Corticosteroid-dependent reduction of leukocyte count in blood as a prognostic factor in acute lymphoblastic leukemia in childhood (therapy study ALL-BFM 83)]. Klin Pädiatr. 1987 Jun;199(3):151–60.
8. Schrappe M, Reiter A, Ludwig W-D, Harbott J, Zimmermann M, Hiddemann W, et al. Improved outcome in childhood acute lymphoblastic leukemia despite reduced use of anthracyclines and cranial radiotherapy: results of trial ALL-BFM 90. Blood. 2000 Jun 1;95(11):3310–22.
9. Manabe A, Ohara A, Hasegawa D, Koh K, Saito T, Kiyokawa N, et al. Significance of the complete clearance of peripheral blasts after 7 days of prednisolone treatment in children with acute lymphoblastic leukemia: the Tokyo Children’s Cancer Study Group Study L99-15. Haematologica. 2008 Aug 1;93(8):1155–60.
10. Aricò M, Valsecchi MG, Conter V, Rizzari C, Pession A, Messina C, et al. Improved outcome in high-risk childhood acute lymphoblastic leukemia defined by prednisone-poor response treated with double Berlin-Frankfurt-Muenster protocol II. Blood. 2002 Jul 15;100(2):420–6.
11. Campana D. Role of minimal residual disease monitoring in adult and pediatric acute lymphoblastic leukemia. Hematol Oncol Clin North Am. 2009 Oct;23(5):1083–98.
12. Brüggemann M, Raff T, Kneba M. Has MRD monitoring superseded other prognostic factors in adult ALL? Blood. 2012 Nov 29;120(23):4470–81.
13. Borowitz MJ, Devidas M, Hunger SP, Bowman WP, Carroll AJ, Carroll WL, et al. Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia and its relationship to other prognostic factors: a Children’s Oncology Group study. Blood. 2008 Jun 15;111(12):5477–85.
14. Schrappe M. Minimal residual disease: optimal methods, timing, and clinical relevance for an individual patient. ASH Educ Program Book. 2012 Dec 8;2012(1):137–42.
15. Karsa M, Dalla Pozza L, Venn NC, Law T, Shi R, Giles JE, et al. Improving the Identification of High Risk Precursor B Acute Lymphoblastic Leukemia Patients with Earlier Quantification of Minimal Residual Disease. PLoS ONE. 2013 Oct 11;8(10):e76455.
16. Volejnikova J, Mejstrikova E, Valova T, Reznickova L, Hodonska L, Mihal V, et al. Minimal residual disease in peripheral blood at day 15 identifies a subgroup of childhood B-cell precursor acute lymphoblastic leukemia with superior prognosis. Haematologica. 2011 Dec 1;96(12):1815–21.
17. Panzer-Grümayer ER, Schneider M, Panzer S, Fasching K, Gadner H. Rapid molecular response during early induction chemotherapy predicts a good outcome in childhood acute lymphoblastic leukemia. Blood. 2000 Feb 1;95(3):790–4.
18. Sutton R, Venn NC, Tolisano J, Bahar AY, Giles JE, Ashton LJ, et al. Clinical significance of minimal residual disease at day 15 and at the end of therapy in childhood acute lymphoblastic leukaemia. Br J Haematol. 2009 Aug 1;146(3):292–9.
19. Basso G, Veltroni M, Valsecchi MG, Dworzak MN, Ratei R, Silvestri D, et al. Risk of Relapse of Childhood Acute Lymphoblastic Leukemia Is Predicted By Flow Cytometric Measurement of Residual Disease on Day 15 Bone Marrow. J Clin Oncol. 2009 Nov 1;27(31):5168–74.
20. Ratei R, Basso G, Dworzak M, Gaipa G, Veltroni M, Rhein P, et al. Monitoring treatment response of childhood precursor B-cell acute lymphoblastic leukemia in the AIEOP-BFM-ALL 2000 protocol with multiparameter flow cytometry: predictive impact of early blast reduction on the remission status after induction. Leukemia. 2008 Nov 20;23(3):528–34.
21. O’Connor D, Jesson J, Bahey M, Eyre L, Lawson S. Analysis of early disease response in childhood acute lymphoblastic leukaemia: can peripheral blood replace bone marrow analysis? Br J Haematol. 2013 Jun 1;161(5):743–5.
22. Coustan-Smith E, Sancho J, Hancock ML, Razzouk BI, Ribeiro RC, Rivera GK, et al. Use of peripheral blood instead of bone marrow to monitor residual disease in children with acute lymphoblastic leukemia. Blood. 2002 Oct 1;100(7):2399–402.
23. van der Velden VHJ, Jacobs DCH, Wijkhuijs AJM, Comans-Bitter WM, Willemse MJ, Hählen K, et al. Minimal residual disease levels in bone marrow and peripheral blood are comparable in children with T cell acute lymphoblastic leukemia (ALL), but not in precursor-B-ALL. Leukemia. 2002 Aug;16(8):1432–6.
24. Dongen JJM van, Velden VHJ van der, Brüggemann M, Orfao A. Minimal residual disease diagnostics in acute lymphoblastic leukemia: need for sensitive, fast, and standardized technologies. Blood. 2015 Jun 25;125(26):3996–4009.
25. Gaipa G, Cazzaniga G, Valsecchi MG, Panzer-Grümayer R, Buldini B, Silvestri D, et al. Time point-dependent concordance of flow cytometry and real-time quantitative polymerase chain reaction for minimal residual disease detection in childhood acute lymphoblastic leukemia. Haematologica. 2012 Oct 1;97(10):1582–93.
26. Coustan-Smith E, Sancho J, Behm FG, Hancock ML, Razzouk BI, Ribeiro RC, et al. Prognostic importance of measuring early clearance of leukemic cells by flow cytometry in childhood acute lymphoblastic leukemia. Blood. 2002 Jul 1;100(1):52–8.
27. Costa ES, Pedreira CE, Barrena S, Lecrevisse Q, Flores J, Quijano S, et al. Automated pattern-guided principal component analysis vs expert-based immunophenotypic classification of B-cell chronic lymphoproliferative disorders: a step forward in the standardization of clinical immunophenotyping. Leukemia. 2010 Nov;24(11):1927–33.
28. Pedreira CE, Costa ES, Lecrevisse Q, van Dongen JJM, Orfao A, EuroFlow Consortium. Overview of clinical flow cytometry data analysis: recent advances and future challenges. Trends Biotechnol. 2013 Jul;31(7):415–25.
- Detailed Description
Not available
Recruitment & Eligibility
- Status
- Completed
- Sex
- All
- Target Recruitment
- 100
B acute lymphoblastic leukemia patients treated on Indian Childhood Collaborative Leukemia group(ICiCLe)protocol.
Patients with following high risk cytogenetics- (i) MLL rearranged leukaemias, e.g. t(4;11) (q21;q23) (ii) Low hypodiploidy (<45 chromosomes) (iii) t(17;19) (q22;p13) (TCF3-HLF) (iv) Intrachromosomal amplification of chromosome 21 (iAMP21) (v) t(9;22) (q34;q11) (BCR-ABL).
Study & Design
- Study Type
- Observational
- Study Design
- Not specified
- Primary Outcome Measures
Name Time Method Level of MRD by manual analysis and automated analysis on day 15 post induction peripheral blood (%) and categorization into-MRD (Day15 PB) positive/negative Day 15 post induction
- Secondary Outcome Measures
Name Time Method 4 year relapse and survival rates for B- ALL patients based on Day 15 and post induction MRD. (This will not be a part of student thesis) 4 years from start of treatment
Trial Locations
- Locations (1)
Hematopathology Laboratory, Tata Memorial Hospital
🇮🇳Mumbai, MAHARASHTRA, India
Hematopathology Laboratory, Tata Memorial Hospital🇮🇳Mumbai, MAHARASHTRA, IndiaDr Papagudi Subramanian Associate Professor and Officer incharge Hematopathology LaboratoryPrincipal investigator9819135574pgs_mani@yahoo.com