Pediatric Hematology and Oncology, 31:638–646, 2014 C Informa Healthcare USA, Inc. Copyright ISSN: 0888-0018 print / 1521-0669 online DOI: 10.3109/08880018.2014.935837
ORIGINAL ARTICLE Acute Lymphoblastic Leukemia
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Impact of TCF3 Rearrangement on CNS Relapse in Egyptian Pediatric Acute Lymphoblastic Leukemia Hoda M. ElGendi,1 Abeer A. Abdelmaksoud,2 Doaa G. Eissa,1 and Samy A. Abusikkien3 1
Department of Clinical Pathology, Ain Shams University, Cairo, Egypt; 2 Department of Pediatrics, Ain Shams University, Cairo, Egypt; 3 Department of Anatomy, Ain Shams University, Cairo, Egypt
Background: TCF3 rearrangement mostly t(1;19) (q23;p13)/ TCF3-PBX1 gene is associated with favorable outcome in acute lymphoblastic leukemia (ALL) upon treatment with intensification protocols; however, it is associated with higher incidence of central nervous system (CNS) relapse which may affect outcome of patients. Objectives: We aimed to assess TCF3 rearrangement in newly diagnosed pediatric ALL patients in relation to clinical and laboratory parameters, CNS relapse, and clinical outcome. Patients and Methods: Eighty newly diagnosed pediatric ALL patients following at Pediatric Hematology Oncology Clinic, Ain Shams University Hospitals were included in this study. Their ages ranged from 0.75 to 16 years. Seventy six (95%) patients had B-lineage ALL and four (5%) had T-lineage ALL. Data recorded included; age, sex, extramedullary manifestations, CNS, and testes infiltrations, risk stratification, response to treatment, and CBC and BM findings. TCF3 rearrangement was assessed by FISH technique using dual color break-apart probe. Results: TCF3 rearrangement [t(1;19) (q23;p13)] was detected in 16 (20%) out of the 80 studied patients, and it was significantly associated with splenomegaly, lymphadenopathy, CNS infiltration at presentation, high total leucocytic count, low platelet count, high-risk group, and isolated CNS relapse. These results identify a group of high-risk ALL patients with high incidence of CNS relapse and poor response to standard therapeutic regimen. Conclusion: Analysis of TCF3 rearrangement [t(1;19) (q23;p13)] at diagnosis may provide a valuable target for modified and intensified CNSdirected chemotherapeutic protocol aiming to improve the patients’ outcome. Keywords ALL, CNS relapse, t(1;19), TCF3 rearrangement, TCF3-PBX1
INTRODUCTION Cytogenetic and molecular analyses of leukemic cells have identified therapeutically important subgroups of childhood acute lymphoblastic leukemia (ALL). One of the most frequently observed translocations is t(1;19)(q23;p13), which occurs in 2–5% of children with B-lineage ALL[1]. Two forms of translocations are found, one balanced t(1;19)(q23;p13) and one unbalanced der(19) t(1;19)(q23;p13), both giving rise to the TCF3/PBX1 fusion gene [2].
Received 9 March 2014; accepted 13 June 2014. Address correspondence to Abeer A Abdelmaksoud, Assistant Professor of Pediatrics, Ain Shams University, 109, area 3/4, 3rd neighborhood, 5th district, Cairo, Egypt. E-mail: dr abeer ahmed [email protected]
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TCF3, ALL, and CNS Relapse
The N-terminal transactivation domain of TCF3(E2A), a helix–loop–helix protein-coding gene on chromosome 19, is fused to the C-terminal DNA-binding homedomain of PBX1, the pre-B-cell transforming gene on chromosome 1. The resulting aberrant protein product appears to have oncogenic potential by affecting cell differentiation [3]. The presence of t(1;19) has been associated with unfavorable presenting features, such as high white blood cell (WBC) count and absence of high hyperdiploidy (>51 chromosomes), and early reports indicated that patients with t(1;19) had a poor outcome with the use of standard treatment compared with patients lacking this translocation [1]. However, some studies reported that intensified chemotherapy improved outcome of patients with the t(1;19) [4]. Other reports showed that although patients with the t(1;19) have an overall favorable outcome when treated with intensive therapy, they have a significantly higher incidence of central nervous system (CNS) relapse [5]. So, identification of this genotype at diagnosis is important for intensifying CNSdirected therapy to improve the outcome in this group of patients. In order to achieve this issue, our work aimed to measure the incidence of TCF3 rearrangement [t(1;19)] among a sample of Egyptian pediatric ALL patients and evaluate its impact on CNS relapse as well as its correlation to standard prognostic factors. PATIENTS AND METHODS The present study included 80 newly diagnosed pediatric ALL patients. They were followed up regularly at Pediatric Hematology Oncology unit of Ain Shams University Hospitals during the period from June 2011 to February 2013. Informed consent for inclusion in this study was provided by the patients’ guardians. All studied patients were subjected to a detailed history and clinical examination, with an emphasis on the presence of hepatomegaly, splenomegaly, lymphadenopathy, fever, CNS, and testes infiltrations. Laboratory investigations were carried out, including complete blood count (CBC) with an examination of Leishman-stained peripheral blood (PB) films, Bone marrow (BM) aspiration, and examination of Leishman-stained BM smears, cytochemical studies using myeloperoxidase stain, and immunophenotyping of BM or PB samples using Beckman Coulter Epics XL (Flowcytometer, Brea, California, USA) to determine the WHO category using the acute leukemia panel. Conventional cytogenetic analysis using G-banding was performed on PB or BM samples [6]. Detection of TCF3 rearrangement by FISH technique used a dual-color Break apart probe (Cytocell aquarius). Diagnosis of ALL was made by cytomorphological and immunological examination of blood and bone marrow smears. For the diagnosis of ALL; 25% blasts or more in the bone marrow was mandatory. Immunological markers were judged positive if expressed in 20% or more of the malignant cells [7]. Risk stratification was based on clinical data, morphological and immunological studies, day 14 bone marrow response, as well as conventional cytogenetics. Standard-risk ALL group involved patients with age 1–9.99 years, WBC count 5%. Response to chemotherapy was assessed by BM performed on day 28 of induction. Patients were considered in complete remission (CR) if BM blasts were less than 5%. All the studied patients received chemotherapy according to CCG 1991 protocol for standard-risk [8] and CCG 1961 for high-risk patients [9]. Risk classification for CNS disease was proposed: CNS1, denoting the absence of identifiable leukemic cells in CSF; CNS2, denoting the presence of blast cells in a sample that contains 6% (Figure 2). Re-assessment of positive results for TCF3 rearrangement was done using FITC-labeled whole chromosome paint FISH probe for chromosome 1 and TRITClabeled probe for chromosome 19 for verification of positive t(1;19)(q23;p13). Statistical Analysis Statistical analysis of the data was performed by using SPSS 15 software package under Windows 7 operating system. Qualitative data parameters were presented in the form of frequency and percentage; and were analyzed for group differences by using Chi-square test (Chi-square value) or Fisher exact test. Quantitative data parameters were presented in the form of mean and median; and measure of spread was presented as standard deviation, 25th and 75th percentile. Comparative analysis for quantitative data was performed by using Mann–Whitney U test (Z value) for comparison between two independent samples with nonparametric distribution or Student t-test (t value) Pediatric Hematology and Oncology
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TCF3, ALL, and CNS Relapse
FIGURE 2 Interphase cells positive for TCF3-PBX1 rearrangement.
for comparison between two independent samples with normal distribution. Survival analysis was done using the Kaplan Meir curve. Assessment of OS and EFS was done by using Median and standard error of the mean (SEM). Log rank test was used to test the difference between survival curves. Probability or P value of .05). There was a significant statistical association between TCF3-PBX1 positivity and isolated CNS relapse, as 6 out of 16 patients (37.5%) had isolated CNS relapse and two patients had both BM and CNS relapse. On the other hand, none of the 64 TCF3-PBX1-negative patients developed isolated CNS relapse (P = .005) (Table 2). Comparing patients with positive and negative TCF3-PBX1 as regards overall survival (OS); it revealed 16±1.8 and 17±2.1 months, respectively, and for event-free survival (EFS); it was 14±2.1 and 16±2.2 months, respectively, but this was statistically insignificant (P > .05) (Figure 3). DISCUSSION The t(1;19) (q23;p13) was identified in (20%) of our 80 newly diagnosed pediatric ALL patients, which is a higher incidence than that recorded by other studies. Kager et al., [4] identified this fusion gene in 31 patients (3.6%) out of 859 Austarian children with ALL, while Jeha et al., [5] reported that out of 735 children with B-cell precursor ALL, 41(5.6%) patients showed TCF3-PBX1 fusion gene. Moreover, Andersen et al., [3] reported the lowest incidence among all previous studies, as t(1;19) was identified in 47 (1.8%)patients out of 2640 children diagnosed with B- cell precursor ALL in Nordic countries. Pediatric Hematology and Oncology
TCF3, ALL, and CNS Relapse
TABLE 2 Comparison Between t(1;19) Negative and t(1;19) Positive Patients
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Parameter
t(1;19) Negative (n = 64)
t(1;19) Positive (n = 16)
6.0 5.0–12.5
4.0 2.0–12.8
1.102∗
.278
38 (59.38%) 26 (40.63%)
6 (37.50%) 10 (62.50%)
1.237†
.430
30 (46.88%) 34 (53.13%)
0 (0.00%) 16 (100.00%)
6.000†
.016
62 (96.88%) 2 (3.12%)
8 (50.00%) 8 (50.00%)
9.608†
.010
38 (59.38%) 26 (40.63%)
6 (37.50%) 10 (62.50%)
1.237†
.430
32 (50.00%) 32 (50.00%1)
0 (0.00%) 8 (100.00%)
6.667†
.013
60 (93.75%) 4 (6.25%)
16 (100.00%) 0 (0.00%)
.526†
1.000
50 (78.12%) 6 (9.38%) 8 (12.50%)
2 (12.50%) 12 (75.00%) 2 (12.50%)
13.159†
.001
12.90 4.83–77.75
127.50 114.75–144.0
3.365∗
.05).
This discrepancy in incidence of t(1;19) may be explained by racial variability which was reported by Jeha et al., [5] mentioning that TCF3-PBX1 genotype is significantly associated with the Black race. Moreover, a number of t(1;19)-positive cases may be missed during diagnosis as there is discrepancy in results between karyotyping and molecular analysis. Also, the presence of an alternative TCF3-PBX1 fusion transcript that is undetectable by RT-PCR using standard primers for TCF3 and PBX1 may play a role, as reported by Paulsson et al.[12]. In our study, t(1;19)(TCF3-PBX1) was detected in 6/16(37.5%) patients by karyotyping and FISH, while in the remaining 10 cases (62.5%) it was detected only by FISH technique. Kager et al.[4] succeeded to detect it in 17(54.8%) patients out of 31 patients positive for t(1;19) by karyotyping and the remaining cases detected only by FISH. This may argue the need for performing FISH at diagnosis to detect this type of translocation, as frequencies may be decreased when only G-banding is performed. As regards presenting features and clinical data of the 16 t(1;19) positive patients, there were 6(37.5%) males and 10(62.5%) females. Their median age at diagnosis was 4 years, Kager et al., [4] presented 17(54.8%) males and 14 (45.2%) females among 31 Austarian children positive for TCF3-PBX1, with a median age at diagnosis equalling 6.9 years. Considering laboratory data, the median diagnostic WBCs count of our t(1;19) positive patients was 127.5×109 /L which is significantly higher than negative ones. In contrast, Kager et al.,[4] reported significantly lower median diagnostic WBCs count among TCF3-PBX1 positive patients which was 20.7×109 /L. Similar to our results, Jeha et al., [5] found that TCF3-PBX1-positive patients were clinically associated with higher presenting leukocyte count and DNA index
ORIGINAL ARTICLE Acute Lymphoblastic Leukemia
Pediatr Hematol Oncol Downloaded from informahealthcare.com by Nyu Medical Center on 06/14/15 For personal use only.
Impact of TCF3 Rearrangement on CNS Relapse in Egyptian Pediatric Acute Lymphoblastic Leukemia Hoda M. ElGendi,1 Abeer A. Abdelmaksoud,2 Doaa G. Eissa,1 and Samy A. Abusikkien3 1
Department of Clinical Pathology, Ain Shams University, Cairo, Egypt; 2 Department of Pediatrics, Ain Shams University, Cairo, Egypt; 3 Department of Anatomy, Ain Shams University, Cairo, Egypt
Background: TCF3 rearrangement mostly t(1;19) (q23;p13)/ TCF3-PBX1 gene is associated with favorable outcome in acute lymphoblastic leukemia (ALL) upon treatment with intensification protocols; however, it is associated with higher incidence of central nervous system (CNS) relapse which may affect outcome of patients. Objectives: We aimed to assess TCF3 rearrangement in newly diagnosed pediatric ALL patients in relation to clinical and laboratory parameters, CNS relapse, and clinical outcome. Patients and Methods: Eighty newly diagnosed pediatric ALL patients following at Pediatric Hematology Oncology Clinic, Ain Shams University Hospitals were included in this study. Their ages ranged from 0.75 to 16 years. Seventy six (95%) patients had B-lineage ALL and four (5%) had T-lineage ALL. Data recorded included; age, sex, extramedullary manifestations, CNS, and testes infiltrations, risk stratification, response to treatment, and CBC and BM findings. TCF3 rearrangement was assessed by FISH technique using dual color break-apart probe. Results: TCF3 rearrangement [t(1;19) (q23;p13)] was detected in 16 (20%) out of the 80 studied patients, and it was significantly associated with splenomegaly, lymphadenopathy, CNS infiltration at presentation, high total leucocytic count, low platelet count, high-risk group, and isolated CNS relapse. These results identify a group of high-risk ALL patients with high incidence of CNS relapse and poor response to standard therapeutic regimen. Conclusion: Analysis of TCF3 rearrangement [t(1;19) (q23;p13)] at diagnosis may provide a valuable target for modified and intensified CNSdirected chemotherapeutic protocol aiming to improve the patients’ outcome. Keywords ALL, CNS relapse, t(1;19), TCF3 rearrangement, TCF3-PBX1
INTRODUCTION Cytogenetic and molecular analyses of leukemic cells have identified therapeutically important subgroups of childhood acute lymphoblastic leukemia (ALL). One of the most frequently observed translocations is t(1;19)(q23;p13), which occurs in 2–5% of children with B-lineage ALL[1]. Two forms of translocations are found, one balanced t(1;19)(q23;p13) and one unbalanced der(19) t(1;19)(q23;p13), both giving rise to the TCF3/PBX1 fusion gene [2].
Received 9 March 2014; accepted 13 June 2014. Address correspondence to Abeer A Abdelmaksoud, Assistant Professor of Pediatrics, Ain Shams University, 109, area 3/4, 3rd neighborhood, 5th district, Cairo, Egypt. E-mail: dr abeer ahmed [email protected]
Pediatr Hematol Oncol Downloaded from informahealthcare.com by Nyu Medical Center on 06/14/15 For personal use only.
TCF3, ALL, and CNS Relapse
The N-terminal transactivation domain of TCF3(E2A), a helix–loop–helix protein-coding gene on chromosome 19, is fused to the C-terminal DNA-binding homedomain of PBX1, the pre-B-cell transforming gene on chromosome 1. The resulting aberrant protein product appears to have oncogenic potential by affecting cell differentiation [3]. The presence of t(1;19) has been associated with unfavorable presenting features, such as high white blood cell (WBC) count and absence of high hyperdiploidy (>51 chromosomes), and early reports indicated that patients with t(1;19) had a poor outcome with the use of standard treatment compared with patients lacking this translocation [1]. However, some studies reported that intensified chemotherapy improved outcome of patients with the t(1;19) [4]. Other reports showed that although patients with the t(1;19) have an overall favorable outcome when treated with intensive therapy, they have a significantly higher incidence of central nervous system (CNS) relapse [5]. So, identification of this genotype at diagnosis is important for intensifying CNSdirected therapy to improve the outcome in this group of patients. In order to achieve this issue, our work aimed to measure the incidence of TCF3 rearrangement [t(1;19)] among a sample of Egyptian pediatric ALL patients and evaluate its impact on CNS relapse as well as its correlation to standard prognostic factors. PATIENTS AND METHODS The present study included 80 newly diagnosed pediatric ALL patients. They were followed up regularly at Pediatric Hematology Oncology unit of Ain Shams University Hospitals during the period from June 2011 to February 2013. Informed consent for inclusion in this study was provided by the patients’ guardians. All studied patients were subjected to a detailed history and clinical examination, with an emphasis on the presence of hepatomegaly, splenomegaly, lymphadenopathy, fever, CNS, and testes infiltrations. Laboratory investigations were carried out, including complete blood count (CBC) with an examination of Leishman-stained peripheral blood (PB) films, Bone marrow (BM) aspiration, and examination of Leishman-stained BM smears, cytochemical studies using myeloperoxidase stain, and immunophenotyping of BM or PB samples using Beckman Coulter Epics XL (Flowcytometer, Brea, California, USA) to determine the WHO category using the acute leukemia panel. Conventional cytogenetic analysis using G-banding was performed on PB or BM samples [6]. Detection of TCF3 rearrangement by FISH technique used a dual-color Break apart probe (Cytocell aquarius). Diagnosis of ALL was made by cytomorphological and immunological examination of blood and bone marrow smears. For the diagnosis of ALL; 25% blasts or more in the bone marrow was mandatory. Immunological markers were judged positive if expressed in 20% or more of the malignant cells [7]. Risk stratification was based on clinical data, morphological and immunological studies, day 14 bone marrow response, as well as conventional cytogenetics. Standard-risk ALL group involved patients with age 1–9.99 years, WBC count 5%. Response to chemotherapy was assessed by BM performed on day 28 of induction. Patients were considered in complete remission (CR) if BM blasts were less than 5%. All the studied patients received chemotherapy according to CCG 1991 protocol for standard-risk [8] and CCG 1961 for high-risk patients [9]. Risk classification for CNS disease was proposed: CNS1, denoting the absence of identifiable leukemic cells in CSF; CNS2, denoting the presence of blast cells in a sample that contains 6% (Figure 2). Re-assessment of positive results for TCF3 rearrangement was done using FITC-labeled whole chromosome paint FISH probe for chromosome 1 and TRITClabeled probe for chromosome 19 for verification of positive t(1;19)(q23;p13). Statistical Analysis Statistical analysis of the data was performed by using SPSS 15 software package under Windows 7 operating system. Qualitative data parameters were presented in the form of frequency and percentage; and were analyzed for group differences by using Chi-square test (Chi-square value) or Fisher exact test. Quantitative data parameters were presented in the form of mean and median; and measure of spread was presented as standard deviation, 25th and 75th percentile. Comparative analysis for quantitative data was performed by using Mann–Whitney U test (Z value) for comparison between two independent samples with nonparametric distribution or Student t-test (t value) Pediatric Hematology and Oncology
Pediatr Hematol Oncol Downloaded from informahealthcare.com by Nyu Medical Center on 06/14/15 For personal use only.
TCF3, ALL, and CNS Relapse
FIGURE 2 Interphase cells positive for TCF3-PBX1 rearrangement.
for comparison between two independent samples with normal distribution. Survival analysis was done using the Kaplan Meir curve. Assessment of OS and EFS was done by using Median and standard error of the mean (SEM). Log rank test was used to test the difference between survival curves. Probability or P value of .05). There was a significant statistical association between TCF3-PBX1 positivity and isolated CNS relapse, as 6 out of 16 patients (37.5%) had isolated CNS relapse and two patients had both BM and CNS relapse. On the other hand, none of the 64 TCF3-PBX1-negative patients developed isolated CNS relapse (P = .005) (Table 2). Comparing patients with positive and negative TCF3-PBX1 as regards overall survival (OS); it revealed 16±1.8 and 17±2.1 months, respectively, and for event-free survival (EFS); it was 14±2.1 and 16±2.2 months, respectively, but this was statistically insignificant (P > .05) (Figure 3). DISCUSSION The t(1;19) (q23;p13) was identified in (20%) of our 80 newly diagnosed pediatric ALL patients, which is a higher incidence than that recorded by other studies. Kager et al., [4] identified this fusion gene in 31 patients (3.6%) out of 859 Austarian children with ALL, while Jeha et al., [5] reported that out of 735 children with B-cell precursor ALL, 41(5.6%) patients showed TCF3-PBX1 fusion gene. Moreover, Andersen et al., [3] reported the lowest incidence among all previous studies, as t(1;19) was identified in 47 (1.8%)patients out of 2640 children diagnosed with B- cell precursor ALL in Nordic countries. Pediatric Hematology and Oncology
TCF3, ALL, and CNS Relapse
TABLE 2 Comparison Between t(1;19) Negative and t(1;19) Positive Patients
Pediatr Hematol Oncol Downloaded from informahealthcare.com by Nyu Medical Center on 06/14/15 For personal use only.
Parameter
t(1;19) Negative (n = 64)
t(1;19) Positive (n = 16)
6.0 5.0–12.5
4.0 2.0–12.8
1.102∗
.278
38 (59.38%) 26 (40.63%)
6 (37.50%) 10 (62.50%)
1.237†
.430
30 (46.88%) 34 (53.13%)
0 (0.00%) 16 (100.00%)
6.000†
.016
62 (96.88%) 2 (3.12%)
8 (50.00%) 8 (50.00%)
9.608†
.010
38 (59.38%) 26 (40.63%)
6 (37.50%) 10 (62.50%)
1.237†
.430
32 (50.00%) 32 (50.00%1)
0 (0.00%) 8 (100.00%)
6.667†
.013
60 (93.75%) 4 (6.25%)
16 (100.00%) 0 (0.00%)
.526†
1.000
50 (78.12%) 6 (9.38%) 8 (12.50%)
2 (12.50%) 12 (75.00%) 2 (12.50%)
13.159†
.001
12.90 4.83–77.75
127.50 114.75–144.0
3.365∗
.05).
This discrepancy in incidence of t(1;19) may be explained by racial variability which was reported by Jeha et al., [5] mentioning that TCF3-PBX1 genotype is significantly associated with the Black race. Moreover, a number of t(1;19)-positive cases may be missed during diagnosis as there is discrepancy in results between karyotyping and molecular analysis. Also, the presence of an alternative TCF3-PBX1 fusion transcript that is undetectable by RT-PCR using standard primers for TCF3 and PBX1 may play a role, as reported by Paulsson et al.[12]. In our study, t(1;19)(TCF3-PBX1) was detected in 6/16(37.5%) patients by karyotyping and FISH, while in the remaining 10 cases (62.5%) it was detected only by FISH technique. Kager et al.[4] succeeded to detect it in 17(54.8%) patients out of 31 patients positive for t(1;19) by karyotyping and the remaining cases detected only by FISH. This may argue the need for performing FISH at diagnosis to detect this type of translocation, as frequencies may be decreased when only G-banding is performed. As regards presenting features and clinical data of the 16 t(1;19) positive patients, there were 6(37.5%) males and 10(62.5%) females. Their median age at diagnosis was 4 years, Kager et al., [4] presented 17(54.8%) males and 14 (45.2%) females among 31 Austarian children positive for TCF3-PBX1, with a median age at diagnosis equalling 6.9 years. Considering laboratory data, the median diagnostic WBCs count of our t(1;19) positive patients was 127.5×109 /L which is significantly higher than negative ones. In contrast, Kager et al.,[4] reported significantly lower median diagnostic WBCs count among TCF3-PBX1 positive patients which was 20.7×109 /L. Similar to our results, Jeha et al., [5] found that TCF3-PBX1-positive patients were clinically associated with higher presenting leukocyte count and DNA index
