Persistence of TEL-AML1 fusion gene as minimal residual disease has no additive prognostic value in CD 10 positive B-acute lymphoblastic leukemia: a FISH study

Acute lymphoblastic leukemia (ALL) is the most common malignancy of childhood. Cure of many of these children is difficult to predict and is considered an individual response of the patient to chemotherapy. It is likely that this clinical heterogeneity reflects a diverse pathogenesis of leukemia. The molecular basis of childhood ALL is largely unknown. Furthermore, it is likely that significant advance in the treatment of childhood ALL will be dependent on a better understanding of the molecular events that cause the disease [1, 2].

A recurrent t(12;21)(p13:q22) has been described in several human ALLs. In this translocation the TEL gene fuses to AML1; a gene previously cloned from translocation breakpoints in acute myeloid leukemia. These abnormalities consist of both translocations and deletions. The frequency of t(12;21) was estimated as to be 15–35% in childhood ALL. This translocation has been recognized as the most common chromosomal aberration in childhood ALL [2‐4]. All (95–100%) of TEL-AML1 positive ALL patients found to has a consistent cell surface immunophenotype. (B lineage ALL based on the expression of HLA-DR, CD 10 and CD 19) [2, 4]. Thus, we raised a question if the opposite is true meaning that if CD 10 positive B-ALL immunophenotype will have a similarly high incidence of positive TEL-AML1 fusion gene?. Accordingly can we use this fusion gene as a minimal residual disease (MRD) in this specific subgroup of B-ALL.

It was also reported that patients with the TEL-AML1 fusion have a high sensitivity to chemotherapy [4‐6]. Other investigators have reported that almost 10–28% of relapsed pediatric ALL patients express the TEL-AML1 fusion, but the relapse of patients with the TEL-AML1 fusion is not always associated with a poor prognosis [7‐9]. However, some patients with the TEL-AML1 transcripts and additional molecular lesions had poor outcomes [10]. So, the prognostic significance of TEL-AML1 transcript remains controversial.

Patients with a poor treatment response by morphologic criteria have a high risk of relapse [11, 12]. But morphologic studies will only identify a minority of those children with ALL who eventually fail. Minimal residual disease (MRD) has been of prognostic value in children with ALL. Several studies have shown that children with a high leukemic cell burden at the end of induction therapy have an inferior outcome compared to children with a lower leukemic cell burden [13‐18]. The investigation of MRD using TEL-AML1 fusion gene as a marker has been carried out on a limited number of patients to date although it is a minor examination. The relation between relapse and the persistence of detectable MRD show heterogeneity [19, 20]. As this translocation is often difficult to detect by conventional G-banding analysis, in addition many patients with ALL were diagnosed as normal karyotype or could not examined for karyotype by classic cytogenetic analysis. In particular fluorescence in situ hybridization (FISH) analysis has been applied to hematopoietic malignancies with subtle or complex chromosomal aberrations which are difficult or impossible to detect by standard cytogenetic analysis [3]. Therefore, we conducted a retrospective study to determine the frequency and prognostic significance of TEL-AML1 fusion in CD 10 positive B-ALL, and to clarify whether the persistence of the TEL-AML1 fusion gene as a MRD has an additive value.

Eighty ALL patients were enrolled in the study at our Institute between 2002 and 2006. They males were (n = 56; 70%) and females were (n = 24; 30%), mean age 7.4 ± 2 years they were 44 patients L1 (55%) and 36 L2 (45%). They were all B-lineage ALL positive for CD 10 and CD 19 by immunophenotyping (common and pre B-ALL). Most of our patients were in the standard risk (n = 64; 80%), while (n = 16; 20%) were in the high risk category. The karyotypes: Seven metaphases were available for cytogenetic analysis and they were normal. A precise karyotype was not obtained from other patients because of poor morphology of metaphases.

TEL-AML1 fusion gene was evaluated by FISH which showed a fused yellow signal (Figure 1) on the der (21) chromosome in the metaphase and on the interphase nuclei of leukemic cells. It was measured in newly diagnosed cases (Table 1) and it was positive in 30/80 (37.5%) determining its frequency in B-lineage ALL positive for CD 10 and CD 19. The mean percent of TEL-AML1 fusion gene was 50 ± 22% estimated in 300 interphase cells. A control was performed using, five normal bone marrow samples and the cut-off level in this method was estimated to be 1.2%. There was a favorable significant correlation between TEL-AML1 fusion gene and disease course (r = 0.5, P = 0.003). Of particular interest was the observation that 10/50 (20%) of patients lacking the TEL-AML1 fusion had a very bad course (eight children did not achieve a complete remission after induction chemotherapy (resistant) and two achieved CNS relapse and died). No significant correlation was detected between the presence of TEL-AML1 fusion gene at diagnosis and peripheral WBC count, age, sex, organs, FAB classification, central nervous system disease, and risk category. We analyzed the patients who were positive for the presence of TEL-AML1 fusion at diagnosis (n = 30) to detect its persistence as a MRD in patients who entered in complete remission morphologically (Table 2). It was positive in (n = 15/30; 50%) patients. The mean percent of TEL-AML1 fusion gene was 7 ± 2% estimated in 300 interphase cells. The persistence of TEL-AML1 fusion gene as a MRD, was correlated with a favorable course (r = 0.4, P = 0.01). To be noticed that (n = 12/15; 80%) of MRD positivity were in CRI.

Kaplan-Meier curve for the presence of TEL-AML1 fusion at the diagnosis was associated with a better probability of OS (Figure 2); mean survival time was 47 ± 1 month, in contrast to 28 ± 5 month in its absence (P = 0.006). Also, the persistence at TEL-AML1 fusion as a MRD was not significantly associated with a better probability of OS (Figure 3); the mean survival time was 42 ± 2 months in the presence of MRD and it was 40 ± 1 months in its absence. So, persistence of TEL-AML1 fusion as a MRD had no additive prognostic value over its measurement at diagnosis in terms of predicting the probability of OS.

The TEL gene encodes a member of the ETS family of transcription factors and is rearranged in a wide variety of hematological malignancies. In particular, TEL is fused to the platelet-derived growth factor receptor β in CMML, to the ABL tyrosine kinase in acute myeloid leukemia and ALL, and to the product of the MNI gene in myeloproliferative disorders. AML-I is the DNA-binding subunit of the transcription factor complex core binding factor (CBF-β). It is frequently rearranged in myeloid malignancy either through fusion to ETO as a result of t(8;21)(q22:q22) or to EVII, MDS1, or EAP as a result of t(3;21)(q26:q22).[2, 22] The frequent involvement of TEL and AML-I in chromosomal translocations suggests that these genes play important roles in the pathogenesis of human leukemia. In t(12;21) a high level of expression of the hybrid protein that contains the functional domains of AML-I under the transcriptional control of the TEL promoter may be involved in oncogenic transformation [2, 22]. In this study we demonstrated that the frequency of TEL-AML1 fusion in B-Lineage CD10 positive ALL was 37.5%. versus 30% in a previous study included multicentres and larger number of patients.23 In our data (22%)of patients with t(12;21) were CD34-positive, indicating that the leukemic cells originated from primitive hematopoietic cell similar to those of ALL patients with t(9;22) or 11q23 abnormalities [2]. We also, found that 67% of the t(12;21) positive patients were in (CR1), indicating a favorable course, as previously reported [1, 2].

The relationship between the TEL-AML1 fusion and a favorable prognosis represented by survival has already been described [2, 9]. Rubnitz et al [9] reported that the survival at five years follow-up of a group with the TEL-AML1 fusion was 91 ± 5%. These patients with positive TEL-AML1 fusion who achieved a favorable prognosis were found to be younger, without hyperleukocyosis, with the CD 10 positive B precursor ALL immunophenotyping and chemosensitive [2]. Also, a recent report studying the prognosis of relapsed patients showed an outcome consistent with ours. The median duration of remission of relapsed TEL-AML1-positive patients was reported to be 42.5 versus 27 months in those lacking the gene; P = 0.0001 [24]. Our study was consistent with the pervious studies as TEL-AML1 fusion at the diagnosis was associated with a better probability of overall survival [2, 8, 9]. On the other hand, other studies have reported that some patients with TEL-AML1 transcript had a poor outcome [25]. In many cases, TEL-AML1 transcripts detected by RT-PCR and Southern blotting in childhood ALL disappeared soon after the start of chemotherapy [6, 26]. Others reported that a patient with additional molecular lesions with p16 homozygous deletion in addition to TEL-AML1 transcript relapsed usually late, and the survival was ultimately favorable [8, 9]. An analysis of late or off-treatment relapse of TEL-AML1 positive ALL suggested that leukemic cells in relapse were not derived from the dominant clone at diagnosis. It represents a transformation of cells belonging to a persistent preleukemic clone that was generated by TEL-AML1 fusion in utero and survived chemotherapy [27].

The progress in treatment of ALL patients without conventional risk factors has been hampered by the inability to predict relapse after patients achieved a complete remission [19]. Whereas in large prospective studies on childhood ALL, residual disease is a powerful indicator of treatment outcome [20, 28]. In this study, MRD was detectable in 50% of patients of CD 10 positive B-ALL after 4 to 6 weeks of induction therapy. Whereas, in a prospective study on childhood ALL, MRD was detectable in 25% to 58% of patients after the same period of induction therapy [20]. It was reported that the frequency of MRD positivity is high after induction and decreases gradually during consolidation and maintenance phase being in some genes 88% during early induction to 13% at week 52 [19]. If MRD as a marker was detected, the general opinion is that it could become a risk factor for relapse [20, 28]. In contrast, MRD lasts among some patients in long-term remission in other forms of childhood acute leukemia like t(15;17) and t(8;21). [32,33] Cayuela et al [26], reported that one out of seven patients with the TEL-AML1 transcript, serially evaluated, exhibited persistence of detectable MRD over eight months, and that all the patients were in continuous complete remission. This study was consistent with that reported by others [26, 29, 30] that several patients were found to be positive for TEL-AML1 fusion, but the persistence of detectable MRD was not associated with a better probability of OS. Therefore, the relationship of the MRD level of TEL-AML1 fusion and prognosis shows heterogeneity and further investigation is required to evaluate their association and to design risk adapted therapeutic approaches.

TEL-AML1 fusion gene detected by FISH in newly diagnosed cases of CD 10 positive B-ALL is considered a favorable prognostic marker with a better course. The persistence of TEL-AML1 fusion gene as a MRD has no additive prognostic value. Considering the cost-benefit ratio TEL-AML1 fusion gene done once at diagnosis gives sufficient prognostic information. However, much research about the biologic and clinical significance of TEL-AML1 as MRD in CD 10 positive ALL is needed to determine how to best integrate TEL-AML1 testing into routine patient care.