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Recurrent genetic abnormalities detected by FISH in adult B ALL and association with hematological parameters

Abstract Background Recurrent genetic abnormalities influence prognosis in B lymphoblastic leukemia. BCR‐ABL rearrangement is associated with higher leukocyte counts and older age at presentation. Among adults, BCR ‐ABL ‐ is the commonest recurrent abnormality whereas, IgH rearrangements are rare. Aim Aim of this study was to identify common recurrent genetic abnormalities in adult B ALL and study their association with hematological findings. Methods Bone marrow and peripheral blood from patients with B acute lymphoblastic leukemia were analyzed for complete blood counts, bone marrow morphology and cytogenetic abnormalities. The study group was divided into smaller groups based on cytogenetic abnormalities. Hematological parameters and presence of recurrent genetic abnormalities was compared across age groups and gender by non parametric tests. Results BCR‐ABL positive group had a higher leukocyte count than BCR‐ABL negative group. Among groups 1 to 5, group 1 with gains of chromosomes was associated with leucopenia and higher age at presentation. BCR‐ABL is commonest recurrent abnormality followed by IgH rearrangements. Conclusion Patients with gains of chromosomes alone have low total leukocyte counts at presentation.

counts have been associated with prognosis in acute B ALL. 18,19 However, studies associating complete blood counts with genetic abnormalities have been limited to total leukocyte counts.
The aim of this study was to identify common recurrent genetic abnormalities in adult B ALL by fluorescence in situ hybridization (FISH) and study their association with hematological findings.

| MATERIALS AND METHODS
Adults diagnosed with de novo acute B ALL were included in the study. The study included all adults with B ALL whose samples were   IgH rearrangements were seen in ten (9.5%) patients. In four patients, IgH rearrangements coexisted with MYC rearrangement and in three patients it coexisted with BCR-ABL rearrangements.
The study group was divided in to five groups-group 1 through to group 5, based on cytogenetic abnormalities. Group 1 had gains of chromosomes only. There were 10 (9.5%) patients in this group (seven     figure 4. Distribution of haemoglobin and platelets were not found to be significantly different between the five groups {h = 6.46 (4, n = 105) P = .16 for haemoglobin and h = 3.2 (4, n = 105), P = .51 for platelet count, Kruskal Wallis test}. Total leukocyte count and age was found to significantly differ between the groups {h = 12.1 (4, n = 105), P = .01 for total leukocyte count and h = 9.57 (4, n = 105), P = .04 for age distribution, Kruskal Wallis test}.
None of the five age groups (less than 30, 30-40, 41-50, 51-60, and 61 and above) were found to have significant differences in hematological parameters as well as cytogenetic abnormalities (Table 3).
Discussion: In this study which included 105 adults with B ALL, males outnumber females by a ratio of 1.9:1. There was no difference in cytogenetic abnormalities with respect to gender. BCR-ABL rearrangement was seen in 25.7%, followed by IgH rearrangements F I G U R E 8 ALL with IgH rearrangement indicated by one orange signal, one green signal and one fusion signal (arrows) . This is a break apart rearrangement probe Spectrum Green LSI IGHV (IGH-variable) probe covers essentially the entire IGH variable region. Spectrum Orange LSI IGH 3 0 probe lies 3 0 (centromeric) to the IGH locus.
(Magnification 630x). 8.5% were between 18 and 30 years of age, 6.2% between 30 and 40 years, 5.5% between 40 and 50 years, and 5.3% between 50 and 60 years. 2 This study involved 5202 adults and children with both T and B ALL whereas; our study involved only adults with B-ALL, which could be the reason for these differences in frequency among the various age groups. In the same study, TCF3/PBX1 was seen in nearly seven % of patients who were less than 30 years of age. 2 Similar to their study, TCF3/PBX was infrequent and seen in 2.8% of our patients of which, 6% of patients were less than 30 years and 3.8% of patients were between 30 and 40 years. In the present study, BCR-ABL was found to be nearly uniform across the age groups except for a slightly higher frequency (38.4%) in patients aged between 30 and 40 years. In contrast, in the earlier Italian study, frequency of BCR-ABL increased with age-14.4% in 18-25-year age group, 26% in 25 to -30 years, 37.3% in 30 to 40 years, 42.8% at 40 to 50 years and 52.7% at 50 to 60 years. In their study, patients with total leukocyte counts of >50 × 10 9 /L had higher frequency of BCR-ABL positivity (P < .0001) whereas we found total leukocyte counts of >50× 10 9 /L was seen more frequently in the BCR-ABL negative group (P < .0001). This difference could be due to the limited number of patients in the older age groups in our study. This could also be the reason for discrepancy in MLL frequency. MLL was seen in only 2.5% of patients in our study and was limited to two age groups: one less than 30 years and other 30 to 40 years of age. However in the study by the Italian group, it was seen to progressively increase from 3.8% in the 18-25-year age group to 6.4% (25-30 years) to 7.9% (30-40 years) and to 11.7% (40-50 years). They found that MLL rearrangement was associated with higher leukocyte counts across all age groups. We found MLL rearrangement in only two patients in our study group. One of the patients had leukocytosis whereas, the other had leukopenia. In another study, on patients aged 60 and above, it was found that 18% of ALL occurs in elderly. BCR-ABL positivity was observed in 24% of these patients. 3 In our study, BCR-ABL positivity was seen in 18.1% of patients above 61 years. Only 10.4% of our patients were above 61 years. In another study, involving 321 adolescents and young adults aged between 16 and 20 years, BCR-ABL positivity was seen in 4.7% and MLL rearrangement in 2.7%. 4 Similarly, in our study, MLL gene rearrangement was seen in 3% of patients aged less than 30 years. However, in the same age group our BCR-ABL positivity was 24.2%. This discrepancy could be due to the limited number of patients in our study (33 patients were less than 30 years of age). In the GMALL study group, BCR-ABL positivity was seen in 36.2% which is slightly higher than our study (25.7%). In this study, it was also seen that BCR-ABL positivity increased with age from 12.7% (15-24 years), to 30.6% (25-34 years) to 43.7

T A B L E 3 Hematological parameters and frequency of recurrent genetic abnormalities in the different age groups
(35-44 years). 8 We noticed an increase from 24.2% (less than 30 years) to 38.4% (30-40-years). However, a decline was seen in patients aged between 41 and 50 years (18.7%) which remained nearly constant across patients aged between 51 and 61-years and patients aged over 61 years.
In the German multicentre trial, BCR-ABL positivity was prevalent in 37%, with positive patients having a higher white cell count than BCR-ABL negative patients (23.8× 10 9 /L vs 11.5 × 10 9 /L, P = .0001). Patients in the BCR-ABL positive group had a higher median age than negative patients (45 vs 30 years) as well as a marginally higher haemoglobin (102 vs 92 g/ L, P = .004). 12 In our study, median leukocyte counts in the BCR-ABL positive group was significantly higher than the BCR-ABL negative group (P = .02). However, unlike the German study, age and haemoglobin were not seen to vary significantly between the two groups. Among Chinese patients, the mean age of presentation was significantly higher in females than males (19.5 years vs 16.4 years, P = .007). Percentage of patients positive for Philadelphia chromosome did not differ in the two groups (17.7% in males and 17.4% in females, P = .93). 13 However, in our study, age at presentation though marginally higher in females, did not differ significantly among males and females (39.5 years in females and 35 in males, P = .10). Frequency of BCR-ABL positivity also did not differ significantly between males and females (P = 0.24).
In an UK based study, males at presentation were found to be younger than females and a slight male preponderance was observed. 15 Prevalence of BCR-ABL was found to be 15% and like our study, positivity increased with age from 5% (15-19 years) to 11% (20-29 years), to 15% (30-39 years) and 24% (40-49 years) after which, incidence declined and a plateau was reached. 15 In our study, a similar decline and plateau was evident after the fourth decade. The second most common abnormality was MYC rearrangement which was seen in 7%, followed by TCF3/PBX1 in 3% and MLL rearrangement in 4%. 15 In our study too, we found MYC rearrangement in 5.7%, followed by TCF3/PBX in 2.8% and MLL in 1.9%.
Liu et al reported MLL rearrangement in 7.6%, TCF3/PBX rearrangements in 9.8% and BCR-ABL positivity in 26.1%. 16 IgH rearrangements have been reported in association with hyperdiploidy, BCR-ABL positivity, DUX4 overexpression and MLL gene rearrangements. 16,17 In our patients, F I G U R E 9 ALL positive for MYC rearrangement indicated by a normal fusion signal and abnormal separated green and orange signals. Spectrum Orange probe starts centromeric to MYC and is centromeric to the breakpoint region. Spectrum Green begins telomeric to MYC gene and is telomeric to the break point region (Magnification 630 x).
IgH rearrangements were seen in 9.5%, of which 40% were associated with MYC rearrangement and 30% with BCR-ABL rearrangement.
Our study suffers from some limitations. The study group is small and flow cytometry as well as karyotyping findings have not been correlated. The findings must be correlated with prognosis and survival.
In this study, we found that BCR-ABL is the commonest recurrent genetic abnormality in adult B ALL. Positivity for this rearrangement is related to high leukocyte counts. None of the other hematological parameters have any association with cytogenetic abnormalities.
Patients with gains of chromosome only had lower leukocyte counts at presentation and were older at presentation compared to other groups. None of the cytogenetic abnormalities have any predilection for particular age or gender. We need to further investigate this in larger groups to study prognosis and therapeutic response.

ETHICAL STATEMENT
Data were collected from the hospital information system and anonymised. Samples were collected after obtaining informed consent from patients. Ethics approval for the study and publication was obtained retrospectively from the institution ethics committee (Ethics approval number-AMH-C-S-014/07-20).

CONFLICT OF INTEREST
The authors declare no potential conflict of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.