| Abstract|| |
Context: This study was done to assess the Serial peripheral blood and bone marrow changes in patients of Acute Lymphoblastic Leukemia on chemotherapy. Aims: To assess the therapy related serial bone marrow changes in patients of Acute Lymphoblastic Leukemia. Settings and Design: Prospective study, carried out in Lymphoma- Leukemia Lab, Department of Pathology, K.G.M.U from March 2011 to March 2012. A total of 60 cases were studied Materials and Methods: History, complete hemogram, bone marrow examination at pretherapy (Day-0), intratherapy (Day-14), and end of induction chemotherapy (Day-28) were done. Peripheral blood smears were evaluated at regular interval to assess clearance of blast cells. Statistical analysis used: The statistical analysis was done using SPSS (Statistical Package for Social Sciences) Version 15.0 statistical Analysis Software. The values were represented in Number (%) and Mean ± SD. The following Statistical formulas were used: Mean, standard deviation, Chi square test, Paired "t" test, Student 't' test, Level of significance P Results: Incidence of ALL-L1 (46.7%) and ALL-L2 (53.3%) was equal. ALL-L2 patients had poor survival.Day 0 (D-0) bone marrow was hypercellular with flooding of marrow by leukemic cells. High levels of tumor load at D'0' were associated with poor survival. 14 th day of Induction phase showed significant decrease in hemoglobin and TLC as compared to D '0' parameters. D28 showed marrow regeneration. Cellularity, Blast%, and Leukemic Index showed significant drop from day '0' to day 14 due to myelosupression, whereas regeneration reflected by increased cellularity as per day 28 marrow. Lymphocytosis (>20%) at end of induction chemotherapy had better survival and longer remission.Risk of mortality was directly proportional to blast clearance and was a major independent prognostic factor for achievement of complete remission. Conclusions: A bone marrow examination at the end of induction chemotherapy provides information whether patient has achieved remission with regeneration of cells or still has residual leukemia. If the patient is in remission, maintenance treatment is started and if not more intensive chemotherapy or bone marrow transplantation may be embarked upon.
Keywords: Acute lymphoblastic leukemia, bone marrow changes, childhood, pediatric, prognostic factors
|How to cite this article:|
Kushwaha R, Kumar A, Aggrawal K, Nigam N, Kumar A. Post chemotherapy blood and bone marrow regenerative changes in childhood acute lymphoblastic leukemia a prospective study. Indian J Pathol Microbiol 2014;57:72-7
|How to cite this URL:|
Kushwaha R, Kumar A, Aggrawal K, Nigam N, Kumar A. Post chemotherapy blood and bone marrow regenerative changes in childhood acute lymphoblastic leukemia a prospective study. Indian J Pathol Microbiol [serial online] 2014 [cited 2020 Jul 15];57:72-7. Available from: http://www.ijpmonline.org/text.asp?2014/57/1/72/130903
| Introduction|| |
Acute Lymphoblastic Leukemia (ALL) is the most common malignancy of childhood, and dramatic advances in its treatment over the past three decades have changed what was essentially a universally fatal disease to one that is now cured nearly 80% of the time.
In different population-based cancer registries, leukemias constitute 27 to 52% of childhood cancers in males and 19 to 52% in females. It is estimated that within a population of 882 million, six thousand children develop acute lymphoblastic leukemia each year in India. 
For diagnosis of Acute Leukemia, peripheral blood smears and bone marrow examination are indispensable tools. Pre-therapy bone marrow examination is essential for evaluation of cellularity and type of leukemia. Cellularity in bone marrow is essential for estimating the efficacy of treatment by comparing the change in cellularity during and after treatment.
A bone marrow examination done subsequently at the end of induction chemotherapy provides information whether patient has achieved remission with regeneration of cells or still has Residual Leukemia. If the patient is in remission, maintenance treatment is started and if not more intensive chemotherapy or bone marrow transplantation may be embarked upon. ,,
To assess the therapy-related serial bone marrow changes viz aplasia, hypoplasia, and regeneration in patients of Acute Lymphoblastic Leukemia.
| Materials and Methods|| |
This prospective study was carried out from March 2011 to March 2012. A total of 60 cases were studied.
A record of detailed history and examination of the patient was made. After obtaining informed consent, collection of blood samples and bone marrow aspiration was done.
Following parameters were done for each patient as under.
- Complete Hemogram
- Peripheral blood smears- Every alternate day,i.e., on day 0, 2, 4, 6, 8, 10, 12, 14 after starting induction chemotherapy, differential leukocyte count (DLC) was carried out and the rate of disappearance of blast cells were noted.
- Bone Marrow Aspiration and clot sectioning: Evaluated for adequacy, cellularity, morphology and maturation of hemopoeitic precursor cells and for the presence of blast cells. Bone Marrow Aspiration and Bone Marrow Clot Sectioning were done.
- Before chemotherapy was started (DAY 0)
- Intratherapy (DAY 14)
- End of induction chemotherapy (DAY 28 / DAY 42)
The statistical analysis was done using SPSS (Statistical Package for Social Sciences) Version 15.0 statistical Analysis Software. The values were represented in Number (%) and Mean ± SD.
The following Statistical formulas were used:
Mean, standard deviation, Chi square test, Paired "t" test, Student 't' test, Level of significance P
| Results|| |
A total of 60 patients undergoing treatment for ALL were included in the study. Majority of cases were of ALL-L2 type (n = 32; 53.3%). A total of 28 (46.7%) were of ALL-L1 type.
Age of patients ranged from 8 months to 12 years. Mean age of patients was 7.0 years. There were 7 females, all aged between 1-10 years. Among males too, majority (n = 44; 83%) were aged between 1-10 years. There were 2 (3.8%) males of less than 1 year of age and 7 (13.2%) were above 10 years.
Immunophenotyping was done in 50 cases, 24 of ALL-L1 and 26 of ALL-L2. Majority of patients with B-ALL were ALL-L1 (56.8%), whereas majority of patients with T-ALL were ALL-L2 types (75%). Statistically, the association was significant (P = 0.037).
Fever, pallor and weakness were most common clinical presentations followed by lymphadenopathy (n = 38; 63.3%), hepatomegaly (n = 34; 56.7%), and splenomegaly (n = 56.7%). Abdominal distension (n = 8; 13.3%) and gum hypertrophy (n = 1; 1.7%) were some of the less common clinical features [Figure 1].
On analyzing the biochemical parameters, a significant association was found between Serum uric acid levels and TLC (P = 0.003). However, no significant association with blast percentage was seen.
With increasing TLC, a significant increase in serum LDH levels were observed (P = 0.005); however, no statistically significant association was observed between blast cell and S. LDH levels (P = 0.906).
We could follow only 59 cases in induction phase as one patient expired on 11 th day of induction chemotherapy.
A decrease in mean value of Hemoglobin and TLC was observed at day 14 as compared to baseline, whereas as compared to baseline an increase in platelet count was noted at day 14. Statistically, the change was significant for hemoglobin and TLC levels only (P < 0.05). Change in platelet count was not significant statistically (P > 0.05). An increase in hemoglobin levels and platelet count and a decrease in mean TLC were observed between baseline and day 28. Statistically, the change was significant for all the three parameters (P < 0.001). An increase in levels of all the three parameters was observed between day 14 and day 28. Statistically, this change was significant (P < 0.001) [Table 1].
|Table 1: Combined pattern of change in hematological parameters in all cases at day 0, 14, and 28|
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A decrease in mean % blast cell, cellularity, and leukemic index was observed between day 0 and day 14 of treatment. For all the parameters, the decrease was statistically significant. Similarly, a significant (P < 0.001) decrease was observed in mean % blast cell, cellularity, and leukemic index between day 0 and day 28/42 of treatment [Table 2].
|Table 2: Combined pattern of change in blast cell %, cellularity % and leukemic index of patients of all cases at day 0, 14, and 28 (n = 59)|
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At day 14, majority of cases had moderate to marked degree of megakaryocytic regeneration (n = 37; 62.7%). A slightly less than majority (n = 28; 47.5%) had erythroid regeneration. However, only 19 (32.2%) showed marked myeloid regeneration. Similarly, at day 28 too, 79.7% cases showed marked megakaryocytic regeneration, followed by erythroid regeneration in 39 cases [Table 3].
Majority of cases (n = 41; 68.3%) had disappearance of blast cells from peripheral blood before 10 days. There were 18 (31.7%) cases in whom disappearance of blast cells from peripheral blood took equal to or more than10 days after start of treatment.
It was observed that patients who took equal to or more than 10 days in disappearance of blast cells from peripheral blood had higher mortality (64.3%) as compared to cases undergoing remission (3.1%). Statistically, this association was significant (P < 0.001).
ALC count was <1000 cells/μl in 3 (5.1%) cases at day 14 and 24 (40.7%) cases at day 28. A significant change in ALC count status was observed between two intervals (P < 0.001).
It was observed that the patients who had ALC count <1000 cells/μl on day 28 expired, whereas majority of patients who had ALC count >1000 cells/ μl survived. Statistically, the association between ALC count and outcome was significant (P < 0.001).
Among those who expired or relapsed, a total of 10 of 27 (37%) had day 28 bone marrow lymphocyte percentage <20 as compared to majority of those who had a long survival (65.6%). Majority of patients in remission had day 28 bone marrow lymphocytosis, association was statistically significant (P = 0.028).
| Discussion|| |
The present study was done to evaluate serial peripheral blood and bone marrow changes in childhood ALL patients on therapy. Our study group comprised of a total of 60 cases of ALL proven by bone marrow examination.
In our study, of 60 cases of ALL slight, prevalence of ALL-L2 subtype was seen as compared to ALL-L1.
Among patients enrolled, majority were males and were in age group of 1-10 years with mean age of 7 years. All the patients below 1 year and above 10 years were males and this correlation is significant as all three are the poor prognostic criteria. Various studies correlate with our findings.
Perentesis JP observed that despite the use of similar intensive chemotherapy regimens, adults with ALL exhibit a strikingly inferior outcome when compared to children. They conclude that lymphoblast of childhood ALL appear to arise in a developmental compartment that is "poised" for apoptotic death and are particularly sensitive to glucocorticoides, antimetabolites, and other cytotoxic agents. In contrast, adult ALL commonly possesses the poor-prognosis Philadelphia chromosome and is often drug-resistant. 
Similarly, Hoelzer et al. stated that age is an important but complex risk factor in ALL. Adults and infants with ALL have a poorer prognosis than age group of 2-10 years.  Moncke A et al. concluded infancy as the most unfavorable outcome and best results were achieved at toddler and preschool age. The proportion of T-ALL as compared to precursor B-cell ALL was lower in younger children.  Alison M. Friedmann et al. suggested that the prognostic features in ALL are age and WBC at diagnosis; infants (less than one year), adolescents (greater than nine years), and children with WBC above 50,000/μl being at higher risk. 
A.D. Leiper et al. assessed that the children of less than 1 year of age, presented with a higher prevalence of ALL, TLC >100 × 10  /l, hepatosplenomegaly, higher CNS relapse rate, and short duration of remission. 
Miller et al. noted male sex as an adverse effect on continuous complete remission in ALL patients. 
On comparing morphological and immunophenotypic classification, it has been noticed that B-cell ALL were more common than T cell ALL. Tcell ALL and ALL L2 morphological subtype both of them correlated with the adverse prognosis. It was found that patients with T-ALL had adverse prognosis and lesser chances to achieve remission as compared to B-ALL. Tcell ALL accounts for 15 % of ALL in children according to a study done by Pullen DJ et al. and Crist WM et al. , It is rare in infants less than 1 year of age. Tcell ALL occur more frequently in males and is frequently associated with high WBC count at diagnosis. T-cell ALL is associated with poor prognostic factors such as high initial WBC count, age more than 15 years, massive Splenomegaly, FAB L2 morphology, and abnormal karyotype. Our findings correlated with the study of Pui CH et al. and Ludwig WD et al. ,
In ALL, symptoms generally reflect bone marrow failure and involvement of extramedullary sites by leukemic cells. On clinical examination, it was well evident that fever, pallor, and weakness were the most common clinical presentation among ALL patients. Next common features were hepatomegaly, splenomegaly, and lymphadenopathy due to leukemic infiltration, palpitations, and signs of bleeding due to thrombocytopenia. Similar findings were reported by Biswas S et al. 
On analyzing the biochemical parameters, we found that the serum LDH levels and serum uric acid levels were elevated in most patients and showed significant relation with high TLC. High S. LDH levels correlates well with the size of leukemic infiltrates and prognosis. Our study correlated with the studies done by Pui CH et al.
Kornberg A et al. concluded that in cases of acute leukemias, markedly elevated serum LDH levels are suggestive of ALL and in individual patients, the LDH levels correlate with the number of blast during remission and relapse.  Similarly, Hafiz MG et al. concluded that significant decrease of peripheral and bone marrow blast cell percentage at day 29 of induction chemotherapy is associated with significant decrease in levels of serum LDH. So, measurement of S. LDH levels can be accepted as an enzymatic tool for presumption of childhood ALL and response to chemotherapy during induction of remission. 
Jones et al.described that large leukemic cell burden with a high rate of cell turnover may produce tumor lysis syndrome and multiple metabolic disturbances , the most prominent of which is elevation of serum uric acid level. 
Assessment of changes in hematological parameters during induction chemotherapy
On examining hematological parameters on 14 th day of induction phase, we found that Hb and TLC showed significant decrease in comparison to day '0' parameters. However, on day 28 of induction phase, we noted bone marrow regeneration in the form of statistically significant increase in hemoglobin, platelet count, and total leukocyte count. [Table 3].
Assessment of bone marrow changes during induction therapy
Bone marrow parameters, i.e., cellularity, blast %, and leukemic index showed statistically significant drop from day 0 to day 28 due to cytotoxic and myelosuppressive effect of chemotherapeutic agents [Table 2]. Day 14 marrow was aplastic and had only fat.
Pretherapy marrow of all cases were hyperceullar with virtual replacement of fat by leukemic cells. Overall mean bone marrow cellularity was 93.8%. Various workers Frisch et al., Krause et al., and Wittles et al. had described that bone marrow in ALL is hypercellular with minimal residual hemopoeisis. Glick et al. observed that marrow in ALL is very cellular and of homogenous appearance in 75% of cases. High percentage of blast cells and leukemic index at day 0 is associated with poor prognosis. 
However, on comparing day 14 and day 28 bone marrow, there was a significant increase in cellularity due to regeneration of marrow but blast percentage was decreased.
Bone marrow regeneration
On successive analysis of bone marrow cellularity and pattern of cell regrowth/regeneration between day '0' to day 14 to day 28, we found that in our series of cases, Megakaryocytes were first to regenerate followed by erythroid precursors and myeloid precursors as depicted in [Table 3].
In contrast to our findings, in a study done by Islam, S et al sequence of regeneration at one week after end of chemotherapy was erythroid precursors followed by myeloid and megakarocytes. 
Disappearance of blast from peripheral blood and survival
In follow up of patients, we did repeated peripheral blood smear examination after start of induction chemotherapy and the day of disappearance of blast was observed. Median value of blast clearance was 6 days with a range from 3 to 34 days. In our study group, we found that patients who showed complete disappearance of blast from peripheral blood within 10 days had better prognosis. We could demonstrate that patients with a slow response and associated poor survival can be identified by simple differential peripheral cell counts during early induction phase well before or even instead of performing bone marrow aspiration.
Our results are supported by observations of Rautonen et al. which indicate that rate of blast clearance can be used as a criteria in identifying subgroup of patient with poor prognosis, as risk of death or relapse is directly proportional to blast clearance.  Arnar Gajjar et al. supported above findings as persistence of circulating blasts after one week of multi agent chemotherapy confers poor prognosis in childhood ALL.
Absolute lymphocyte count (ALC) and prognosis
ALC is a significant, independent prognostic indicator in ALL. ALC was studied at days 15 (ALC-15) and 28 (ALC-28) of treatment. 1000 cell/μl was the median ALC value for patients who died. ALC ≤1000 cells/μm during induction predicts poor prognosis in children with ALL. In our study, ALC-28 showed statistically significant association, whereas ALC-15 did not show any such association.
Findings were in congruence with study done by Anoceto Martinez A et al. in 2011, which suggested that patients who have higher ALC at day 15 or day 28 (>1000 cells/μl) predicts excellent outcome, whereas ALC-15/ALC-28 ≤1000cells/μl show poor outcome. 
Two studies done by Guillermo De Angulo et al. in 2008 and Sun Di et al. in 2012 observed similar findings in support to our findings. They both took cut off value of ALC as 350 cells/μl. ,
Bone marrow lymphocytosis and prognosis
Bone Marrow Lymphocytosis (BML) occurring during remission of ALL was commonly believed to a poor prognostic sign that may presage early subsequent relapse. Inclusion in the excellent response category required that a total number of lymphocyte be less than 20% of marrow nucleated cells in children.
Bone marrow lymphocytosis on day 28 showed two subgroups, Group I containing 29 patients with <20% bone marrow lymphocytes and group II containing ≥20% bone marrow lymphocytes. We followed the patients in remission for 3 months. Median duration of remission of group I was >3 month while is group II was >3 months; on further follow up 10 of 31 patients of group I expired whereas 17/27 patients of group II had poor survival. Association of bone marrow lymphocytosis with long term outcome was significant.
Correlation of pancytopenia at diagnosis and outcome of patient
There were 9 cases of 60 who presented with pancytopenia at the time of diagnosis (Hb <6gm%; TLC<4000cells/cu.mm.; PC < 1.0 lacs/mm). Majority of the patients had remission with marrow hypoplasia as outcome at the end of induction chemotherapy. Subject matter has not been studied so far as per literature.
| Conclusion|| |
This study was taken up to study the sequential peripheral blood and bone marrow changes in patients of ALL who are on chemotherapy. Changes in cellularity and blast percentage as compared to the pretherapy marrow indicate response to chemotherapy. A bone marrow examination done subsequently at the end of induction chemotherapy provides information whether patient has achieved remission with regeneration of cells or still has residual leukemia. If the patient is in remission, maintenance treatment is started and if not more intensive chemotherapy or bone marrow transplantation may be embarked upon.
| References|| |
|1.||Bhutani M, Kochupillai V, Bakshi S. childhood acute lymphoblastic leukemia: Indian experience. Indian J Med Pediatr Oncol 2003;24:3-8. |
|2.||Nachman JB, Sather HN, Sensel MG, Trigg ME, Cherlow JM, Lukens JN, et al. Augmented post induction therapy for children with high risk acute lymphoblastic leukemia and a slow response to initial therapy. N Engl J Med 1998;338:1663-71. |
|3.||Schrappe M, Reiter A, Ludwig WD, 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. German-Austrian-Swiss ALL-BFM Study Group. Blood 2000;95:3310-22. |
|4.||Pui CH, Evans WE. Treatment of acute lymphoblastic leukemia. N Engl J Med 2006;354:166-78. |
|5.||Perentesis JP. Why is age such an important independent prognostic factor in acute lymphoblastic leukemia. Leukemia1997;11 Suppl 4:54-7. |
|6.||Hoelzer D, Gale RP. Acute lymphoblastic leukemia in adults: Recent progress, future directions. Semin Hematol 1987;24-39. |
|7.||Möncke A, Zimmermann M, Reiter A, Gardner H, Odenwald E, Harbott J et al. Prognostic impact of age in children and adolescents with acute lymphoblastic leukemia: Data from the trials ALL-BFM 86, 90, and 95. Klin Pediatr 2005;217:310-20. |
|8.||Alison MF, Howard JW. The role of prognostic features in the treatment of childhood acute lymphoblastic leukemia. Oncologist 2000;5:321-8. |
|9.||Leiper AD, Chessells J. Acute Lymphoblastic Leukemia under 2 years. Arch Dis Child 1986;61:1007-12. |
|10.||Miller DR, Leikin S, Albo V, Sather H, Karon M, Hammond D. Prognostic factors and therapy in Acute lymphoblastic leukemias of childhood (CG 14). Cancer 1983;5:1041-9. |
|11.||Pullen DJ, Boyett JM, Crist WM, Falletta JM, Roper M, Dowell B, et al. Pediatric oncology group utilization of immunologic markers in the designation of acute lymphoblastic leukemia (ALL) subgroups. Influence on treatment response. Ann N Y Acad Sci 1984;428:26-48. |
|12.||Crist WM, Grossi CE, Pullen DJ, Cooper MD. Immunologic markers in childhood acute lymphoblastic leukemia. Semin Oncol 1985;12:105-21. |
|13.||Pui CH, Behm FG, Singh B, Schell MJ, Williams DL, Rivera GK, et al. Heterogeneity of presenting features and their relation to treatment outcome in 120 childrens with T-cell acute lymphoblastic leukemia. Blood1990;75:174-9. |
|14.||Ludwig WD, Teichmann JV, Sperling C, Komischke B, Ritter J, Reiter A, et al. Incidence, clinical markers and prognostic significance of immunological subtypes of acute lymphoblastic leukemia (ALL) in children: Experiences of the ALL-BFM 83 and 86 studies. Klin Padiatr 1990;202:243-52. |
|15.||Biswas S, Chakrabarti S, Chakraborty J, Paul PC, Konar A, Das S. Childhhod acute leukemia in West Bengal, India. Asian Pac J Cancer Prev2009;10:903-6. |
|16.||Pui CH, Williams DL, Kalwinsky DK, Look AT, Melvin SL, Dodge RK, et al. Cytogenetic features and serum lactate dehydrogenase level predicts a poor treatment outcome for children with pre B cell leukemia. Blood 1986;67:1688-92. |
|17.||Kornberg A, Polliack A. serum lactate dehydrogenase (LDH) levels in acute leukemia: Marked elevations in lymphoblastic leukemia. Blood1980;56:351-5. |
|18.||Hafiz MG, Mannan MA. Serum lactate dehydrogenase level in childhood acute lymphoblastic leukemia. Bangladesh Med Res Counc Bull2007;33:88-91. |
|19.||Jones DP, Stapleton FB, Kalwinsky D, McKay CP, Kellie SJ, Pui CH. Renal dysfunction and hyperuricemia at presentation and relapse of acute lymphoblastic leukemia. Med Pediatr Oncol1990;18;283-6. |
|20.||Roberts MM, Juttner CA, To LB, Kimber RJ. Bone marrow biopsy during induction chemotherapy for Acute myeloid leukemia identifies only 50% of patients with resistant disease. LeukRes1988;12:817-21. |
|21.||Islam A, Catovsky D, Galton DA. Histological Study of Bone marrow regeneration following chemotherapy for Acute myeloid Leukemia and chronic granulocytic leukemia in Blast transformation. Br J Haematel1980;45:535-40. |
|22.||Rautonen J, Hovi L, Siimes MA. Slow disappearance of peripheral blast cells: An independent risk factor indicating poor prognosis in children with acute lymphoblastic leukemia. Blood 1988;71:989-91. |
|23.||Gajjar A, Ribeiro R, Hancock ML, Rivera GK, Mahmoud H, Sandlund JT, et al. Persistence of Circulating Blasts After 1 Week of Multiagent Chemotherapy Confers a Poor Prognosis in Childhood Acute Lymphoblastic Leukemia. Blood1995;86:1292-5. |
|24.||Anoceto Martínez A, González Otero A, Guerchicoff de Svarch E, Arencibia Nuñez A, Jaime JC, Dorticos E, et al. Absolute Lymphocyte Count as a prognostic factor in children with acute lymphoblastic leukemia. An Pediatr (Barc)2012;76:10. |
|25.||De Angulo G, Yuen C, Palla SL, Anderson PM, Zweidler-McKay PA. Absolute Lymphocyte count is a novel prognostic indicator in ALL and AML. Cancer 2008;112:407-15. |
|26.||Sun D, Elson P, Liedtke M, Medeiros BC, Earl M, Alizaden A, et al. Absolute lymphocyte count at day 28 independently predicts event free and overall survival in adults with newly diagnosed acute lymphoblastic leukemia. AmJHematol2012;87:957-60. |
Flat 504, T.G.Hostel, Khadra, Lucknow, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3]