CLINICOHEMATOLOGICAL, IMMUNOPHENOTYPING, MOLECULAR PROFILE, AND OVERALL SURVIVAL IMPACT IN ACUTE LYMPHOID LEUKEMIA PATIENTS FROM NORTH INDIA

Objective: Cytogenetic plays an inevitable role in predicting the diagnosis of acute leukemia. The recurrent chromosomal aberrations in acute leukemia have provided critical insights into the pathophysiological mechanism of leukemogenesis. Cytogenetics findings at diagnostics provide important information for decision-making in both childhood and adult acute lymphoblastic leukemia (ALL). The cure rate for ALL is >80% in children and 35% in adults. Despite the therapeutic advances in ALL, several important biological and pathophysiological questions remain to be answered to achieve an accurate diagnosis, timely prognosis, and maximum therapeutic benefit. 
Methods: The present study was carried out at tertiary care hospital, New Delhi, India. A total of 144 newly diagnosed ALL patients were analyzed for clinicohematological profile, immunophenotyping, conventional, and molecular cytogenetics. 
Results: The study population was found to have normal karyotypes in most of the cases; however, abnormalities also reported. Our study clearly indicates that the application of fluorescence in situ hybridization has increased sensitivity and accuracy for detecting various chromosomal abnormalities, more so with the cryptic rearrangements. 
Conclusion: We observed that the prevalence of the molecular subgroup of leukemia with a potential for a favorable clinical outcome (ETV6-RUNX1 and hyperdiploidy) in precursor B-ALL is higher in the North India.


INTRODUCTION
Acute leukemia is characterized by the uncontrolled multiplication of undifferentiated hematopoietic precursors in blood, bone marrow, and lymphoid organs. It can originate from myeloid (acute myeloid leukemia [AML]) or lymphoid (acute lymphoid leukemia [ALL]) progenitor cells, with the latter having either T-cell or B-cell lineage origins (T-ALL or B-ALL). ALL is a neoplastic disease that results from a somatic mutation in a single lymphoid progenitor cell at one of the several discrete stages of development. In leukemia, an inframe fusion gene is formed which results in a protein with altered properties. ALL is the most common malignancy in children [1]. It accounts for 25% of all childhood cancers and approximately 75% of all cases of childhood leukemia [2].
The cure rate for ALL is >80% in children and approximately 35% in adults. Remarkable advances have been made over the past 15 years in the treatment of ALL, and the understanding of its pathophysiology and several important biomarkers have been established.
Pediatric ALL is often cited as one of the true success stories of modern medicine. The cure rates have improved from virtually zero, before the advent of modern chemotherapy and radiotherapy (in the 1950s), to the current overall event-free survival rates of approximately 80% [2]. This success and exemplary progress is largely due to the identification of various biomarkers responsible for good and poor prognosis, diagnosis, and improvement in the understanding of how to combine and use the chemotherapeutic agents effectively with minimum toxicity in supportive care.
Despite advances in the treatment, 20-30% of children with ALL in whom remission is achieved after initial induction chemotherapy, subsequently relapse. An array of clinical and lymphoblastic biological features have been identified as prognostically significant in childhood ALL, including age, presenting leukocyte count, immunophenotype, chromosomal abnormalities, the presence of overt central nervous system leukemia, and the rapidity with which the patients demonstrate a response to initial induction chemotherapy [3].

Study design
The study was conducted at the Department of Molecular Biology and Transplant Immunology, Indraprastha Apollo Hospitals, New Delhi. A total of 144 newly diagnosed ALL patients were included in the study with their written informed consent. The clinics and other details were taken (Table 1). These patients were further classified on the basis of risk stratification, i.e., low-risk and high-risk patients on the basis of age, sex, platelets, and WBC count at the time of diagnosis as per the criteria of the National Cancer Institute, USA (Table 2). Immunophenotyping was done to further categorize these patients. Karyotyping was done as a routine investigation in the patients. Fluorescence in situ hybridization (FISH) was performed on Pre B-ALL patients wherever possible. Five years survival analysis was also done in follow-up patient (Fig. 1a).

Kumar et al.
added (GIBCO cat. no. 10270), COLCEMID (0.05 µg/ml) (Biological Industries 10 μg/ml cat. no. 12004-1D) was added for the past 60 min of culture, followed by hypotonic treatment with a 0.075-KCl solution and a final fixation in methanol/acetic acid (3:1). Chromosomes were G-banded for identification whenever possible, at least, 20 metaphases were analyzed according to the International System for Cytogenetic Human Nomenclature, 2016.

FISH
FISH is a technique that involves the precise annealing of a singlestranded fluorescently labeled DNA probe to the complementary target sequences. The hybridization of the probe with the cellular DNA site is visible by direct detection using fluorescence microscopy. The interphase cells obtained from bone marrow specimens are processed with a hypotonic treatment with 0.075M KCl followed by fixation in Carnoy's fixative (3 methanol:1 acetic acid). FISH was carried out by standard protocols and hybridization procedure was modified according to the probe manufacturer (Vysis-Abbott Molecular Abbott Park, Illinois, USA). Following hybridization excess and unbound probe were removed by the series of washes as recommended in the protocol. Finally, chromosome and nuclei were counterstained with DNA specific stain DAPI (4,6 diamidino-2-phenylindole) that fluoresces blue.
The following probes were used for FISH analysis a. Dual-Color, Single-Fusion, Extra Signal Probes: ETV6/RUNX1 gene: The FISH assay for the cryptic 12; 21 translocations (ETV6/RUNX1 or, historically, TEL/AML1) has applications in the diagnosis and monitoring of ALL. The 12; 21 cryptic translocation cannot be seen on G-banded metaphases; therefore, FISH and other molecular methods are needed to detect this rearrangement. In a normal nucleus, the expected pattern for a cell hybridized with the LSI TEL/AML1 ES Dual Color Translocation probe is the two orange (AML1), two green (TEL) (2O2G) signal pattern. An abnormal cell with the translocation would show the ETV6/RUNX1 fusion as yellow (red + green on the derivative chromosome 21), one green signal (uninvolved chromosome 12), and one large red signal (uninvolved chromosome 21). b. Dual-Color, Dual-Fusion Probes: BCR/ABL1 gene: The dual-color, dual-fusion BCR (green)/ABL1 (red) probe set allows for detection of all forms of the BCR/ABL1 fusion (yellow), i.e., t(9;22), variant translocations, and cryptic translocations or insertions. In a normal nucleus, the expected pattern is two orange, two green (2O2G) signal pattern. In a nucleus containing a balanced t(9;22), one orange, one green signal, and two orange/green (yellow) fusion signals are observed (1O1G2F). c. Dual-Color, Break-Apart Probe: MLL gene: Structural rearrangements of the mixed-lineage leukemia (MLL) gene at 11q23 are welldocumented recurring abnormalities and are observed in ALL. The majority of MLL rearrangements occur as a result of an established chromosomal translocation involving 11q23. The probe is a dualcolor break-apart probe made of differentially labeled (red and green) DNA segments located on either side of the MLL breakpoint cluster region. The separation of red and green signals indicates MLL break for the 3' and 5' regions of the gene. The advantage of this kind of FISH assessment is that it can detect all recurrent and possibly novel MLL rearrangements in a single experiment.

Survival analysis
Analysis of disease outcome was examined as overall survival (OS). The OS was measured from the date of initial diagnosis of ALL to the date of death from any cause or date of the last contact using the Kaplan-Meier method which is a nonparametric (actuarial) technique for estimating time-related events (the survivorship function) (Fig. 1a).

Survival analysis
Out of 144 patients, five-year survival data available for 70 patients were analyzed using Prism 7 software. A patient diagnosed with hyperploid had better overall survival than patient with hypoploidy ( Fig. 1a).

DISCUSSION
Acute leukemia is characterized by the uncontrolled clonal proliferation of hematopoietic precursor cells coupled with aberrant or arrested differentiation. ALL is the most common cancer diagnosed in children and represents 23% of cancer diagnoses among children younger than 15 years.
Numerous clinical and laboratory findings, including prognostic factors such as age, gender, cell count, pathophysiological, and cytogenetics at the time of diagnosis helps in determining the intensity and severity of disease and to predicts the best clinical outcome.

Demographic and clinical findings
In the present study, the age of the study subjects varied between 1.5 and 15.8 years with a mean and the median age of 7.2 (±4.1) and 6.5 years, respectively. Advani et al. [4] reported a median age of 8.8 years in their study. The majority of patients (74.6%) in our study population were below 10 years of age indicating a younger study population. More so, a larger number of patients (38.6%) were <5 years of age. Our results are concurrent with Advani et al. [4]. The authors reported 60% and 57% patients, respectively, aged between 2 and 9 years [5,6]. Further reports by Wessels et al. and Silverman et al. have also supported our findings [7,8].
The gender distribution of the study subjects revealed that there were 112 male (78.1%) and 32 female (21.9%) with a male to female ratio of 3.5:1. A male to female ratio of 2.9:1, 2.6:1, and 2.14:1 was reported previously [4]. This distorted sex ratio is not uncommon from studies in India [4][5][6].
In the laboratory findings, the TLC ranged between 600 and 7 lakhs/L. The mean and the median TLC were 39,000/cumm and 8300/cumm, respectively. A mean TLC of 38.8 and 62.7×109/L in two different population from South Africa, respectively [8]. In contrast, Silverman et al. reported a mean WBC count of 9.8×10 9 /L in 1255 patients [7]. In our study, the TLC was >50.0×10 9 /L in 28 (23.7%) patients. In three separate studies, it was observed that 26-30.9% of their patients had TLC >50.0×10 9 /L [4][5][6]. These findings are in concordance with our observation. However, Shanta et al. had reported TLC >60.0×10 9 /L in 60% of patients in their series previously [9].
In the present study, hyperleukocytosis (defined as TLC >100.0×10 9 /L) was found in 8 (7.8%) patients. Studies from other Indian centers reported hyperleukocytosis in 15.3-23.2% cases [6,10]. Silverman et al. reported hyperleukocytosis in 10.8% of the ALL cases [7]. In our experience, the incidence of hyperleukocytosis was lower than the figures reported in other Indian centers and was similar to the prevalence reported from other nations. Children with TLC >50,000 were considered in the high-risk group.

Cytogenetic analysis
Mrozek et al. have described that the standard cytogenetic analysis can be obtained in most of the patients with ALL [11]. In large studies of adult ALL, between 70% and 75% of samples analyzed cytogenetically were deemed successful. Higher success rates, 83 and 91%, were reported by two large studies of childhood ALL [12,13]. Among successfully analyzed patients, one or more clonal aberration has been detected in 57-82% of children with ALL [12,13].
In our study, out of the 144 patients, successful karyotype results were available in 131 (91%) patients and poor morphology in the remaining 13 (9%) patients. Waghray et al. in their study reported successful karyotyping in 52% of the cases [14]. Similarly, in a study by Yang, karyotyping was possible in 86% of the patients [15]. In another study by Forestier et al., cytogenetic analyses were carried out in 1372 (66%) patients. Among these, 787 (57%) displayed clonal chromosomal abnormalities [16]. Perez-Vera et al. in a study including 150 Mexican children aged from 5 months to 16 years with ALL reported successful karyotyping in 131 (87%) children [17]. These studies show that the successful karyotyping rate in our study was either comparable or better than most of the other studies. This could be due to stringent quality control and aseptic measures followed in our laboratory. Besides, most of the bone marrow samples received in the lab are from within the hospital and are transported in ambient temperature to the laboratory without any delay in time.
Whitlock and Gaynon have already explained in their study that both chromosome number (ploidy) and structural alterations have independent prognostic significance in childhood ALL [18].  -65) generally occurs in cases with clinically favorable prognostic factors (patients aged 1-9 years with a low WBC count) and is itself an independent favorable prognostic factor [22].
A very useful explanation about the mechanism of gain or loss of chromosome has been described by Pederson-Biergaard and Rowley in 1994 [23]. They described that non-disjunction at mitosis may be the possible mechanism of gain or loss of a whole chromosome. The other mechanism which leads to extensive chromosome loss in the hypodiploid may be the development of a haploid karyotype with a   [26]. Among the translocations, we observed that the most common translocation was t(9;22) in 5 (14.28%) patients. Padhi et al. in their study carried out on 31 subjects observed that translocations as their major structural abnormality [27]. The t(9;22) was observed in approximately 10% of their patients.

Fish analysis
FISH can be easily performed on specimens prepared for cytogenetic studies, is more sensitive, able to diagnose cryptic rearrangements, and provides rapid results. Application of FISH, therefore, represents an efficient and effective strategy to maximize the information obtained from clinical specimens. In our setting, as a routine, we usually perform karyotyping on all ALL samples and FISH for ETV6-RUNX1 fusion, BCR-ABL and MLL rearrangements for pre B-ALL patients.
The t(12;21) (p13;q22) involving the RUNX1 (AML1 and CBFA2) gene is the most frequent translocation in children with ALL. This translocation is present in 25% of childhood precursor-BALL and 2% of adult precursor-B-ALL and is correlated with a moderate to favorable prognosis [28][29][30]. Although this translocation can be detected by both conventional karyotype and FISH, according to "Acute Lymphoblastic Leukemia Best Practice Guidelines (2011) V1. 00'6," it is mandatory to perform FISH testing for ETV6/RUNX1 in all infants and pediatric cases due to its cryptic nature and its prognostic significance. ETV6/RUNX1 probe, in addition to detecting cryptic t(12;21) (p13;q22) translocation also detects: (i) Amplification of RUNX1 which signifies intrachromosomal amplification of chromosome 21 (iAMP21) and (ii) extra signals of RUNX1 which suggests hyperdiploid karyotype.
Interphase FISH testing for confirmation for high hyperdiploidy should be performed by including probes for chromosomes X, 4, 6, 10, 14, 17, and 18 especially in cases when a normal karyotype is obtained, and interphase FISH identifies extra signals for RUNX1 or where chromosome analysis is unsuccessful. The t(12;21) (p13;q22) (cryptic on karyotyping) in the production of a fusion protein that acts in a dominant-negative pattern and inhibits the transcription of RUNX1 gene. After the first detection of the ETV6-RUNX1 fusion by FISH, a large number of studies demonstrated that the t(12;21) is rarely the only abnormality present. Additional abnormalities include del(6q), Del(11q), rearrangements of 12p, and del(16q), and often these abnormalities provide a clue that a t(12;21) might be present. The t(12;21) has a favorable prognosis with cure span in ≥90%, especially if other favorable factors are present. The frequency of this translocation has been found in a range from 14% to 25% by molecular techniques [31]. In our study, we detected t(12;21) (p13;q22) with a frequency of 43.3% which included detection by either of the techniques. Therefore, we reported a higher incidence of ETV6/ RUNX1 involving B lineage ALL than previously reported studies in this region [32]. It is worth noting that the lower frequency of this fusion gene has also been observed in other studies from within India (6%), Mexico (9.6%), Argentina (11.6%), Thailand (12%), China (17.9%), and Taiwan (19%) [33][34][35][36][37][38]. The higher frequency of ETV6/RUNX1 gene along with hyperdiploidy provides a clue to the higher frequency of molecular subgroup of leukemia with a potential for favorable clinical outcome in precursor B-ALL from the North India. This is in complete contrast with the study done by Inamdar et al. from Bombay, and Padhi et al. from Southern India who found a very low frequency of B-ALL with favorable clinical outcome [27,33]. These geographical variations within India can be explained on the basis that the prevalence of this gene in the North Indian population per se could be more as compared to other parts of India. Furthermore, India is a big country has diverse social and cultural population, thereby bringing heterogeneity in the patient population as well. The difference in the patients' inclusion criteria could also be a reason for this high incidence.
For 30 patients, it was possible to perform both karyotype and FISH panel for BCR-ABL gene in our study. The Philadelphia (Ph) chromosome is derived from the t(9;22) (q34;q11.2). The incidence of t(9;22) in childhood ALL varies from 3% to 5%, but various authors have reported its incidence ranging from 2% to 50% [39][40][41][42][43]. At the molecular level, the breakpoints in B-ALL and CML differ, and this variation leads to the production of p190 and p210 fusion proteins, respectively. In our study, both karyotype and FISH analysis were positive for BCR-ABL gene in 5 (16.66%) out of 30 patients which is in concordance with the other studies [39][40][41][42][43].
Russo et al. found partial or complete monosomy seven in approximately 25% of 57 children with Ph+ ALL [44]. In our study, although the positivity rate of this fusion was low (5 patients, 16.66%), we did observe monosomy seven in two out of 5 patients (40%). This observation is notable because the loss of one copy of chromosome seven generally characterizes myeloproliferative disorders that progress to acute myeloid leukemia. This subgroup of children with Ph+/−7 ALL comprised mainly older males with early B-lineage ALL, whose induction failure rate (3.1%) was much higher than that among Ph-cases. These findings suggest that leukemic transformation in such patients is a multistep process involving the interaction of a dominant oncogene (Ph; BCR-ABL) with a tumor suppressor gene (−7). In both children and adults, t(9;22) ALL has the worst prognosis among patients with ALL. Oyekunle et al. have described that the deletions of the IKZF1 gene confer an adverse risk profile in Ph-positive ALL [45].
The IKZF1 gene has a coding function for a transcription regulator involved in T-and B-cell differentiation.
In our study, it was not possible to perform both karyotype and FISH panel for MLL gene in 30 patients due to some reasons. The detection of MLL rearrangements by karyotyping is although sensitive but sometimes problematic, especially, when the MLL-rearrangement is a subtle anomaly, and chromosome preparations are of poor quality. The duplications and deletion involving the MLL genes are even more difficult to detect by karyotyping. For translocations detection, irrespective of the translocation partner, FISH is the method of choice [46]. In our study, out of the four patients positive for MLL rearrangements, two patients (50%) were detected only by FISH analysis. We, therefore, highly recommend the use of FISH for the detection of MLL rearrangements to overcome the shortcomings associated with the karyotyping.
We found that the frequency of MLL rearrangements was up to 13.3%. This was quite high as compared to study carried out by Safaei et al. attributed their low frequency of 1.5% to the fact that the occurrence of this abnormality is more frequently seen in infants under 1 year

Kumar et al.
and secondary ALL [47]. Because our positive sample size was too low, we cannot say for sure whether this was a chance occurrence or not. MLL translocations in ALL are associated with the pre-B-ALL immunophenotype (CD19+/CD10-) and are characterized by a poor prognosis, particularly in infants [48]. Leukemia is a disease with heterogeneous causes and with well-defined cytogenetic molecular abnormalities inducing clinical manifestations [53]. Leukemic cells have a very challenging heterogeneous environment with different receptivity to prescribed drug or chemotherapeutic agents [54]. Therefore, accurate diagnosis using molecular cytogenetics and other advanced techniques plays a crucial role in the overall survival of the patient.

Survival analysis
This study has some limitations. First, not all children were tested with all diagnostic techniques and therefore, the sample size for comparison between the two techniques, i.e., karyotyping and FISH was quite low.
Further studies with increased sample size would be worthwhile to analyze the two techniques. Second, advanced molecular methods to detect subtle abnormalities, including chromosomal microarray, were not available to substantiate our observations. Finally, many patients were lost to follow-up, and therefore, a complete picture of the disease course cannot be said with confidence.

CONCLUSION
We found that normal karyotypes in our study population were more frequent. Our study indicated that employing FISH technique with increased sensitivity helps in detecting various chromosomal abnormalities, more so with the cryptic rearrangements. Observation of monosomy seven in two out of BCR-ABL positive 5 patients (40%) is notable, because the loss of one copy of chromosome seven generally characterizes myeloproliferative disorders that progress to acute myeloid leukemia and suggest that leukemic transformation in such patients is a multistep process involving the interaction of a dominant oncogene (Ph; BCR-ABL) with a tumor suppressor gene (-7). Unlike other studies both in India and in other countries, higher frequency of molecular subgroup of leukemia with a potential for the favorable clinical outcome (ETV6-RUNX1, hyperdiploidy) in precursor B-ALL was observed from the North India. With the availability of NGS and other techniques, we understand the human genome variability and its impact on disease susceptibility and drug response. With the availability of multiple genomic panels for investigation, the whole scenario of diagnosis and decision-making is changing. However, cytogenetics analysis including karyotyping and FISH with clinical details are gold standard, and we recommend their use to provide a more accurate and reliable characterization of ALL for better prognosis and best possible clinical outcome with improved cure rates and decreased drug toxicity.

ACKNOWLEDGMENT
We greatly acknowledge all the patients who kindly consented to be a part of the study. The authors also thank the whole-hearted support and encouragement from the management of Indraprastha Apollo Hospitals, and Dr. Ashok K Chauhan, Founder President, Amity Group of Institutions.

AUTHORS' CONTRIBUTIONS
Dr. Mohit Chowdhry, Dr. RN Makroo, and Dr. Pankaj Sharma participated in the conception, design, and coordination of the study; Manoj Kumar, Deepika Rani, and Vandana Sharma performed the study; Manoj Kumar, and Dr. Mohit Chowdhry drafted the manuscript.