Prognostically Significant Fusion Oncogenes in Pakistani Patients with Adult Acute Lymphoblastic Leukemia and their Association with Disease Biology and Outcome

Background and objectives: Chromosomal abnormalities play an important role in genesis of acute lymphoblastic leukemia (ALL) and have prognostic implications. Five major risk stratifying fusion genes in ALL are BCR-ABL, MLL-AF4, ETV6-RUNX11, E2A-PBX1 and SIL-TAL1. This work aimed to detect common chromosomal translocations and associated fusion oncogenes in adult ALL patients and study their relationship with clinical features and treatment outcome. Methods: We studied fusion oncogenes in 104 adult ALL patients using RT-PCR and interphase-FISH at diagnosis and their association with clinical characteristics and treatment outcome. Results: Five most common fusion genes i.e. BCR-ABL (t 9; 22), TCF3-PBX1 (t 1; 19), ETV6-RUNX1 (t 12; 21), MLL-AF4 (t 4; 11) and SIL-TAL1 (Del 1p32) were found in 82/104 (79%) patients. TCF3-PBX1 fusion gene was associated with lymphadenopathy, SIL-TAL1 positive patients had frequent organomegaly and usually presented with a platelets count of less than 50 x109/l. Survival of patients with fusion gene ETV6-RUNX1 was better when compared to patients harboring other genes. MLL-AF4 and BCR-ABL positivity characterized a subset of adult ALL patients with aggressive clinical behaviour and a poor outcome. Conclusions: This is the

80%, treatment results remain unsatisfactory in adults, with a poor overall prognosis and a long term probability of survival less than 40 % (Pui et al., 2004;Bassan., 2005). Biologic differences in the leukemogenesis between adult and childhood ALL is the most likely explanation between the childhood and adult ALL as indicated by the prevalence of various genetic abnormalities and other factors. Genetic abnormalities play a major role in the prognosis and treatment outcome of ALL; however, to induce leukemogenesis (Andreasson et al., 2001;Moorman et al., 2007). Although the impact of genetic is well characterized in childhood ALL, the prognostic It is likely that the distribution of molecular genetic subtypes of ALL may not be uniform in different parts of the world because of the racial and genetic variations that exist among different populations. Previous studies indicate that ALL in adults is a heterogeneous disease with differences in the clinical characteristics and molecular abnormalities in various ethnic groups (Iacobucci et al., 2012). Therefore, it is important to study the disease biology and underlying molecular pathways in different populations in order to develop effective treatment strategies. Acquired recurrent chromosomal abnormalities in the malignant cells are the hallmarks of acute leukemia and define different subsets of the disease in ALL patients (Harrison & Foroni, 2002). The discovery and characterization of these genetic lesions has increased our understanding of the biology of the disease, has shaped direct therapy. The development of treatment strategies the pathogenesis of the disease. In order to improve the outcome in adult ALL, it is essential that targeted Knowledge about the genetic abnormalities obtained by molecular cytogenetics is essential to develop targeted therapies, and to incorporate them in treatment protocols. This work aimed to detect the chromosomal translocations and underlying fusion oncogenes in adult ALL patients and study their association with clinical features and treatment outcome.

Materials and Methods
From March 2009 to February 2012 a total of 128 patients from 4 different centers in Lahore, Pakistan, participated in the study. According to the inclusion criteria, untreated patients between the age of 16 and 70 years with the morphological and cytochemically available. Patients were excluded if they had a prior malignancy, severe systemic illness, or a psychiatric disorder. All patients had adequate renal and hepatic Newly diagnosed without treatment function. Informed consent was obtained from all the patients and the study was approved by the ethical committees of the participating centers.
Immunophenotypic data from the local institutions were used after central review, cytometric analysis and panels of monoclonal antibodies were used for surface marker positivity was at least 20% expression of the leukemia blast cell population. B-lineage antigen Expression of the common ALL antigens was assessed by blood specimens be submitted for molecular cytogenetic analysis for the presence of the fusion genes to the HOPES Group. Fusion genes were studied in 104 adult ALL hybridization (FISH), and their association with clinical features and treatment outcome was analyzed. reagent according to the manufacturer's instruction.
template in PCR reaction. The reverse transcriptase (RT) reaction is catalyzed by enzyme 'Reverse Transcriptase' in the presence of random hexamer primers which are the and other reaction conditions were adopted from van Reaction was carried out by incubating mixture of the 70ºC for 10 min. Then rest of the reagents were added and incubated at 42°C for 60 min, 70°C for 10 min and held at PCR primers and nested-PCR protocols for the Excellence in Molecular Biology, Lahore, Pakistan. For the 1 st was performed containing 5x PCR buffer with KCl, 25mM MgCl 2 along with the forward and reverse primers. Thermal cycling conditions for PCR were initial denaturation at 95°C for 3 min followed by 35 cycles of denaturation  RT-PCR analysis of fusion oncogenes. All probes and kits were purchased from Abbot Laboratories (Illinois USA) and FISH procedures were carried out according to the manufacturer's instructions. Stained slides were analyzed using a FISH analyzer system (Leica microscope; Induction therapy was given over a four-week period in two phases. Phase-I included dexamethasone, vincristine and danuorubicin. In phase-II cyclophosphamide and cytarabine were given. Consolidation therapy was administered every four to six weeks with six alternating cycles of methotrexate, L-asparaginase, high dose cytarabine (recommended dose reduction for patients >70 years of age). Patients also received prophylactic Maintenance protocol included pulses of dextamethasone, daily mercaptopurine, weekly methotrexate, and monthly vincristine.
Patients were considered to be in CR when the results of BM examination were normal (including <5% blasts and >25% cellularity), the neutrophil counts were greater than 1.5×10 9 /l, the platelet count was greater than 100×10 9 /l, and all extramedullary disease had resolved.

Statistical Analysis
We used convenient sampling technique in this study to collect the data; therefore, we used non-parametric tests to analyze the data. Chi Square test was used to study the association between different oncogenotypes and clinical and laboratory parameters of leukemia patients. Kaplan and Meier method was used to calculate the median survival times, while Breslow's test was used to study the survival differences between various patient groups.
basis of statistical criteria when response was analyzed . Seventy-nine of 104 (76%) eligible patients achieved a CR (Table 2). Three patients died before hematopoietic recovery after induction chemotherapy. Thirty-six (46%) treatment. Forty-three (54%) patients required more than 30 days to achieve CR either because of slow recovery of blood counts or marrow cellularity. Seventy-six percent of remissions were achieved within 42 days. Sixteen patients died in remission. Treatment response was strongly patients less than 30 years old achieved a CR, as compared to 23 (62.2%) of 37 patients between the ages of 30-59 years, while only one (12.5%) out of 8 patients 60 years or older achieved CR (P< .001) (Figure 2). : fusion gene was expressed in 10 (9.7 %) patients and the clinical characteristics of the patients are shown in Table 3. At diagnosis these patients frequently had an elevated WBC involvement. The presence of t (4;11) (q21; q23) with the expression of fusion gene characterizes a subset of adult ALL patients with aggressive clinical features and a poor outcome. SIL-TAL1: The fusion gene was expressed in 29 (27.9 %) patients (Table 1). Clinical characteristics of the patients are shown in Table 3. SIL-TAL1+ patients frequently presented with lymphadenopathy, organomegaly and low platelet counts (<50×10 9 /l). The immunophenotyping data available in 24 of patients indicated this fusion gene was associated with with low platelet's count and lymphadenopathy (p < 0.001) and patients in this subgroup had poor survival (Figure 3). BCR-ABL: Clinical characteristics of the patients are shown in Table 3. BCR-ABL fusion gene was expressed in 21 (20.3%) patients (Table 1). The prevalence of BCR-ABL positivity increased with age and was more common between the 30-59 year age group. patients frequently had high leukocyte count (p <0.001) and splenomegaly (p <0.001).
ALL was associated with a 10% lower chance of CR as compared to BCR-ABL negative disease and a poor overall prognosis, with a median survival of 8 months (Figure 3). positive patients are given in Table 3. The fusion gene was expressed in 5 (4.8%) cases (Table 1). There were 4 males and one female and all our positive patients were young below the age of 46 years. Immunophenotyping data was available in two patients and they had B-ALL lineage. One of the patients presented with massive organomegaly and most of the patients had total leukocytic counts below 30×10 9 /l. In most of the cases remission was achieved in less than four weeks and -positive patients had a These clinical features suggest that positive patients have a relatively good prognosis (Figure 3)  (86) 1 (20) 5 (50) 8 (28) 15 (88) Platelet count <50,000 20 (95) 1 (20) 9 (90) 6 (21) 5 (29) >50,000 1 (5) 4 (80) 1 (10) 23 (79) 12 (71)

A B
although their long-term survival was negatively affected by late relapses. TCF3-PBX1: Clinical characteristics of positive patients are shown in Table 3. The fusion gene was expressed in 17 (16.3 %) cases (Table  1). Majority of the patients with were males (15) and only 2 females with ages between 16 to 29 years. Hepato-splenomegaly was uncommon but Most of the positive cases had a total leukocyte counts below 30×10 9 /l along with anemia and thrombocytopenic at presentation. Immunophenotyping data were available in nine patients and all cases belonged to common ALL + + ) lineage. In 11 cases remission was achieved in less than four weeks and 3 patients achieved late remission.

Discussion
Although some of the cytogenetic abnormalities have shown to be independent prognostic factors in pediatric abnormalities other than BCR-ABL (t 9; 22) and abnormalities being reported as both good or poor risk in different studies (Faderl et al., 1998;Thomas et al., 2004;Mancini et al., 2005). The most likely reason for this is the genetic heterogeneity of the disease. The most common fusion oncogenes in adult ALL other than the BCR-ABL (t 9; 22) include (t 1; 19), (t 12; 21), We found that 82 (79%) of the adult ALL patients harbored one of the five fusion oncogenes with the distribution of in 17 (16.3%) patients, in 10 (9.7%), BCR-ABL in 21 (20.3%), in 5 (4.8%) and SIL-TAL1 in 29 (27.9%) patients. some of these rearrangements when compared to reports from other parts of the world. For example Mancini et al found only one patient with fusion gene in a much bigger series, and hardly any patients with this abnormality in one of the largest studies in ALL, MRC UKALLXII/ECOG 2993 trial, while it was present in of this rearrangement in our population (Mancini et al., 2005;Moorman et al;2007). Similarly rearrangement was found in 17 (16.3 %) of our patients, again much more common than previously reported (Thomas et al., 2004;Mancini et al., 2005;Burmeister et al., 2010). The frequency of rearrangement also appears to be higher in our patients (Mancini et al., 2005;Moorman et al;2007). As expected, the CR rates of adult ALL varied among our patients harboring different fusion genes.
Among the various gene rearrangements, the t(4;11) (q21; q23)/ positive ALL is rarely observed in adult patients. In our adult ALL patients, frequency was 9.7%. It was associated with FAB L1 or L2 morphology, immature immunophenotype, B-cell lineage, high leukocyte counts and poor outcome. The t(4;11) [ ] associated disease is generally considered to be a high risk ALL subtype, characterized by a poor accordance with our results. The frequency of BCR-ABL rearrangement in our patients is similar to other studies i.e., around 20-25%. Faiz et al. (2010) reported a higher frequency of BCR-ABL rearrangement in adult ALL patients from Pakistan. However, they only used RT-PCR for their fusion gene any validated techniques like cytogenetic analysis or FISH (Faiz et al., 2011). Iqbal and Akhtar used RT-PCR and cytogenetics interphase FISH techniques and reported the frequency of BCR-ABL to be around 50% in adult ALL patients (Iqbal and Akhtar., 2006). However, they (now Khyber Pakhtunkhwa) and Federal Area of Pakistan geographic area of Pakistan, which further strengthens our hypothesis of ethnic and geographic differences in another study by the same group using interphase FISH techniques (Iqbal et al., 2009).
BCR-ABL fusion gene was also associated with a poor prognosis in our study which was in accordance with tyrosine kinase inhibitors (TKIs) in combination with chemotherapy has produced promising results in this subgroup of patients, although problems have emerged with drug resistance, and relapses are common without a stem cell transplant (Moorman et al., 2007;Soverini et al., 2007;Xing et al., 2012). Unfortunately TKIs were not available for our BCR/ABL+ patients which resulted in lower CR rates and relapse of most of the patients. Identification of this genetic entity at diagnosis and incorporation of TKIs in the treatment of BCR/ABL+ ALL is crucial for an optimal outcome (Fielding., 2010). The t(12;21)[ ] is uncommon and detectable by FISH or PCR analysis in about 3 % of adults with B-ALL. Patients generally have a favorable prognosis (Burmeister et al., 2010). Survival of our patients with was better than the patients with other fusion genes.
(t 1;19) fusion gene may be found in up to 5% of adult ALL patients. We found a much higher frequency of this rearrangement (17/ genetic abnormalities include age of the patient, WBC count and response to initial therapy. We also found that age was an important prognostic factor in this cohort. The CR rate decreased from 90% for patients below the age of 30 years, to 14% for patients older than 50. There was a corresponding increasing risk of resistant disease count, although the 7 patients with WBC count more than 100×10 9 /l had a CR rate of only 52% but it was not accordance with the previously reported literature (The Pui et al., 2008;Burmeister et al., 2010). This study shows the prevalence of different fusion genes and their association with clinical parameters as well as treatment outcome. It also indicates that based upon molecular cytogenetic evaluation at diagnosis and its integration with clinical characteristics, patients can studies have shown that treatment can be improved by the ALL patient subgroups with poor outcome although high relapses may remain a problem (The Group Français Burmeister et al., 2010). Our data and its comparison differences in biology and treatment of adult ALL, and indicates a strong interplay of genetic and environmental factors affecting the outcome of the disease. More comprehensive and large scale studies using advanced genetic techniques like microarray and whole genome sequencing are required to further explore the genetic heterogeneity of adult ALL and its correlation with disease biology and treatment outcome. This will further help treatment outcome.
which investigated the frequency of 5 fusion oncogenes in adult ALL patients, and their association with clinical features, treatment response and outcome. Frequency of some of the oncogenes was different from reported elsewhere and they appear to be associated with distinct clinical characteristics and treatment outcome. This risk adapted management of adult ALL patients.