Introduction: Myeloproliferative disorders are characterized by proliferation of one or more myeloid lineages cells. In order to assess the burden of these illness for public health planning, it is important to know their frequency. Objectives: A study to determine the clinical, hematological, cytogenetic, and molecular profile in chronic myeloid leukemia (CML) patient in and around Eastern UP, India. Materials and Methods: Newly diagnosed and follow-up adult and pediatric cases of myeloproliferative disorder were taken into study. Detailed history, physical, and systemic examination was done with informed consent. Investigations like complete blood count including hemoglobin level, platelet count, total and differential leucocyte count, general blood picture, and bone marrow aspiration/biopsy were done. Molecular and cytogenetic studies were also done whenever required. Results: In total, 90 patients were enrolled in the study. The median age of presentation of CML was 37 years and the mean age was 38.6 years. M: F ratio of 1.4:1.75 patients (83%) were in CML-chronic phase (CP), 11 patients (12%) in CML-accelerated phase (AP) phase, and 4 patients (5%) were found in CML-blast crisis (BC) phase. The common symptoms of the patients were fullness of the abdomen (66.6%). Among these 69 cases, Philadelphia chromosome was present in 65 (94.2%) cases. Revers transcriptase polymerase chain reaction (RT–PCR) was done in 40 out of 90 cases, breakpoint cluster region (BCR)-Abelson oncogene (ABL) gene came out to be positive in all the 40 cases. Conclusion: Most CML patients in eastern UP (India) are relatively young (31–40 years). In addition, males were more commonly affected.
Keywords: BCR-ABL; CML; cytogenetics
Introduction
Myeloproliferative disorders are characterized by proliferation of one or more myeloid lineages cells. These includes chronic myelogenous leukemia, primary myelofibrosis, polycythemia Vera, and essential thrombocythemia.[[
Objectives
The objectives of this study were to determine the clinical, hematological, cytogenetic, and molecular profile in CML patient in and around Eastern UP, India.
Materials and Methods
This study was conducted between June 2016 and June 2018 in the Department of Pathology, in collaboration with Department of Medicine and Department of Pediatrics at a tertiary care hospital. Newly diagnosed and follow-up adult and pediatric cases of myeloproliferative disorder were taken into study. The study was approved by the Ethical Committee of the Institute. Detailed history, physical and systemic examination was done with informed consent. Investigations such as complete blood count including hemoglobin level, platelet count, total and differential leucocyte count, general blood picture, and bone marrow aspiration/biopsy were done. Molecular studies such as RT–PCR for BCR-ABL fusion and cytogenetic studies for Philadelphia chromosome were also done whenever required.
Statistical analysis
Data were analyzed by using SPSS version 20. The data were presented by mean ± standard deviation for continuous variables and frequencies with their respective percentages were given for categorical variables. Spearman correlation coefficient was used to measure the degree of association between two variables. A P value < 0.05 was considered as statistically significant.
Results
In total, 90 patients presented with chronic myeloproliferative disorders in which all the patient were diagnosed with CML. The median age of presentation of CML was 37 years and the mean age was 38.6, the patients were in the age range of 5–72 years. Overall most common age group affected were between 31 and 40 year. About 52 (57.8%) were male and 38 (42.2%) were female with M:F ratio of 1.4:1. [Figure 1] is showing the geographical distribution of CML in Eastern Uttar Pradesh and adjoining areas of Bihar State. About 75 patients (83%) were in CML-chronic phase (CP), 11 patients (12%) in CML-accelerated phase (AP) phase, and 4 patients (5%) were found in CML-blast crisis (BC) phase. All patients of BC are of myeloblasts [Figure 2]. All patients were symptomatic at the time of diagnosis. The common symptoms of the patients were fullness of the abdomen (66.6%) followed by weakness (63%), fever (59%), and fatigue (55.5%) [Table 1]. Splenomegaly and anemia was the most common sign [Table 2]. About 13 (17.3%) cases had mild splenomegaly, 20 (26.6%) cases had moderate splenomegaly, and 42 (56%) cases had massive splenomegaly. In AP, 1 (10%) case had mild splenomegaly and 10 (90%) cases had massive splenomegaly whereas in blast crises phase, 100% had massive splenomegaly. Among all patients, 56 (62.2%), 20 (22.2%), and 14 (15.6%) patients presented with massive, moderate, and mild splenomegaly, respectively. In CP, 45 (64.2%) cases presented with moderate anemia, 21 (30%) cases presented with mild anemia, whereas only 4 (5.71%) cases presented with severe anemia. In AP, 54.5% presented with severe anemia and in BC phase 75% presented with moderate anemia. Sixty one (72%) and 24 (28%) patients had hemoglobin concentration less and greater than 10 g/dL. Most of the patients of CP (56%) had leucocyte count in the range (100–250 × 103/dL), whereas leucocyte count >250 × 103/dL seen in AP (63.6%) of CML. About 25% of patients in BC showed leucocyte below 100 × 103/dL. Patients of CP (68%) and AP (72.7%) had normal platelet count (100–450 × 103/dL). Increased platelet count (>450 × 103/dL) was seen more in patients of CP (34.8%). Thrombocytopenia was noted more among patients of CP (53.3%) and BC (25%) as compared with other phases of CML.{Figure 1}{Figure 2}{Table 1}{Table 2}
All the patients (100%) of CP had <10% blast in their peripheral blood and 89.3% of these patients had basophils <10%. About 10 patients (90.9%) had blasts between 10%–19% in peripheral blood. Criteria of 10%–19% blast in AP, 1 patients (9.%) had <10% blasts but basophils >20%; thus, on the criteria of basophils, >20% in peripheral blood in AP. Four patients had >20% blasts fulfilling criteria of BC and 75% of these patients had basophil <10%.
Majority of the patients (58.8%) presented with hemoglobin in the range of 7–10 g/dL and severe anemia (<7 g/dL) was seen in 13% of patients. Total leucocyte were seen in the range (100–250) × 103/dL (52.2%). About 68.9% of patients had platelet count in the range of (100–450) × 103/dL. About 24.4% of the patients had thrombocytosis (>450 × 103/dL) [Table 3], There was a significant direct correlation between spleen size and WBC count (Pearson's correlation: 0.488; P, 0.0001), and inverse correlation between spleen size and hemoglobin levels of patients (Pearson correlation: −0.424; P, 0.001) in CP of the disease. Similarly, WBC counts had significant inverse correlation with hemoglobin levels (Pearson correlation: −0.464; P, 0.001). There was insignificant relation between spleen size and platelet counts of patients [Table 4]. In patients with CP of CML, 47/75 (62.6%) had LAP scores between 0 and 13 (normal: 14–100). In AP of CML, 6 (54.5%) patients had LAP scores <14, 2 (18%) patients had LAP scores >100, whereas the remaining had LAP score within normal limits. In patients with myeloid BC, 1/4 (25%) had LAP score <14, whereas 3/4 (75%) patients had LAP score within normal limits. Among 90 patients, cytogenetic study was done only in 69 patients (76.6%) due to financial constrain. Among these 69 cases, Philadelphia chromosome was present in 65 (94.2%) cases and absent in 4 (5.8%) cases. Among the Philadelphia chromosome positive cases, 53 (81.5%) cases were in CP, 8 (12.3%) cases in AP and 4 (6.15%) cases in BC. All the Philadelphia chromosome negative CML patients (4; 5.8%) were of CP. The four cases, which came negative for Philadelphia chromosome by conventional cytogenetic method, were subjected to FISH analysis and all of them were Philadelphia chromosome positive. RT–PCR was done in 40 out of 90 cases as remaining 50 patients were not affordable. BCR-ABL gene came out to be positive in all the 40 cases. About 78 cases of CML had increased LDH value (normal: 105–248 U/L), among them 65 (86.7%) were in CP, 9 (81.8%) in AP, and all (100%) cases of BC had increased LDH level. None of the patients have reduced LDH level. There is no significant correlation between CP, AP, BC with LDH.{Table 3}{Table 4}
In our study, 14 (18.7%) patients in CP, 4 (36.4%) patients in AP, and 2 (50%) patients in BC had hyperuricemia. There is no significant finding.
Discussion
The pattern of this disease in India and other Asian countries is still unknown due to paucity of studies reported from these areas. The present study was undertaken to obtain the clinical, hematological, cytogenetic, and molecular profile in CML patient in and around Eastern UP and adjoining district of other state. There were few publications on the clinical and hematological spectrum of patients in CML from the Indian subcontinent.[[
In this study, the frequency of all three phases of CML at the time of clinical presentation was 83%, 12%, and 5% for CP, AP, and BC, respectively, were observed among 90 patients suffering from CML. Motatalib et al. from Bangladesh reported frequency of all three phases of CML was 81.25%, 14.58%, and 4.17% for CP, AP, and BC, respectively.[[
Hepatomegaly and lymphadenopathy were seen in 58% and 6% cases, which was similar to as compared in Indian study. In international study, hepatomegaly was seen in 2.2% a case only, which was not comparable to our study. The lymphadenopathy seen in Indian scenario could be due to associated infections; one of them may be tuberculosis. In our study, mean value of total leucocyte count was 182.5 × 103/μL. Minimum TLC was 15.4 × 103/μL and maximum was 546.3 × 103/μL. In Tashfeen et al. study found the mean TLC in the CML group was 184.5 × 109/L. Minimum TLC was 17.9 × 109/L and maximum was 552.3 × 109/L.[[
Chronic phase of CML
In our study, 83% of the patients were in CP. Among the patients in CP, 43 (57.3%) were males and 32 (42.7%) were females. The male to female ratio was 1.3:1. Maximum patients were within age range of 31–40 years (36%). Most of the patients of CP had anemia (93.3%). Most of the patients had hemoglobin in the range 7–10 g/dL (64.2%) followed by mild anemia (>10 g/dL) seen in 30% of cases. This is similar to Bhatti et al. study with most patients with hemoglobin in the range 7.1–10 g/dL. The mean hemoglobin is 9.4 g/dL.[[
Accelerated phase of CML
In our study, 12.2% of the patients were in AP. Among the patients in AP, 6 (54.5%) were males and 5 (45.5%) were females. In the AP, majority of the patients were between 41 and 50 years (36.3%). All the patients had anemia (100%). Most of the patients had severe anemia (<7 g/dL; 54.5% cases), followed by mild anemia (>10 g/dL; 27.2% cases). These findings are against to Bhatti et al. study.[[
Most of the patients had leucocyte count >250 × 103/L (63.6%). Majority of the patients had platelet count in the range of (100–450) × 103/μL (72.7.9%). About 18.2% had platelet count >450 × 103/μL. Thrombocytopenia was seen in 9.0% of cases. In this study, thrombocytosis is seen in 18.2% of the patients of AP which is lower as compared with chronic (26.%), but the study of Bhatti et al. showed thrombocytosis seen maximum in patients of AP. One patient (9%) had <10% blasts but basophils >20% in peripheral blood, thus fulfilling criteria of basophils >20% in peripheral blood in AP.[[
Blast crisis phase
About 4.4% of the patients were in BC. Among the patients in BC, 3 (75%) were males and 1 (25%) were females. Majority of the patients were between 51 and 60 years of age. All the patients had anemia (100%). Most of the patients had hemoglobin in the range 7–10 g/dL (75%). Severe anemia (<7 g/dL) is noted in 25% of cases, which was more than seen in patients of chronic (17.6%). These findings are similar to study by Bhatti et al.[[
Among 90 cases, only 69 cases have undergone conventional cytogenetics for Philadelphia chromosome due to financial constraints. By cytogenetics study, 65 cases were Philadelphia chromosome positive, whereas four cases were Philadelphia chromosome negative. These four cases were further subjected for FISH analysis which confirms Philadelphia chromosome positive. Among the 69 cases, 40 cases are subjected for RT–PCR and all these cases showed p210 kDa BCR-ABL fusion protein positive. De Lemos et al. studied 25 CML patients all cases shows p210 kDa BCR-ABL fusion protein positive.[[
In our study, molecular testing was used only in certain cases, because karyotyping and molecular testing are not available at this center and many patients are not affordable. Indeed, in our population, majority of patients are of low socioeconomic status. However, according to the WHO, detection of the Philadelphia (Ph) chromosome and/or BCR-ABL1 fusion gene is essential baseline investigation to confirm the diagnosis of CML. Follow-up label of BCR-ABL fusion gene product is essential to see the effect of therapy. Thus, more resources are needed to facilitate access to cytogenetic and molecular diagnosis of all patients in Eastern UP. Lack of conventional cytogenetic study and molecular testing was the lacunae of our study. Liver function test and renal function test parameters were also evaluated. In patients of chronic myeloid leukemia, median value of total bilirubin, LDH, ALT, AST, total protein, albumin, urea, uric acid, creatinine, sodium, potassium, and chloride were 0.6 mg/dL, 903.5 U/L, 26 U/L, 36 U/L, 7.6 g/dL, 4.1 g/dL, 26.7 mg/dL, 5.75 mg/dL, 1.0 mg/dL, 139 mmol/L, 4.42 mmol/L, and 101.3 mmol/L, respectively. In our study, majority of CML patients had normal creatinine level in all phases of disease progression, whereas 14 (18.6%) cases in CP, 2 (18.2%) cases in AP, and 1 (25%) case in BC had increased serum creatinine level. These findings have no significance. About 78 cases of CML had increased LDH value (normal: 105–248 U/L), among them 65 (86.7%) cases in CP, 9 (81.8%) cases in AP, and all (100%) cases of BC. None of the patients have reduced LDH level. There is no significant correlation between CP, AP, and BC with LDH. In our study, 14 (18.7%) patients in CP, 4 (36.4%) patients in AP, 2 (50%) patients in BC had hyperuricemia. This is also a not significant finding. There were limitations in our study. There are overlapping clinical, laboratory, and morphologic findings in different type of myeloproliferative neoplasm. For example, most MPNs result in increased numbers of granulocytes, RBCs, and/or platelets. Despite being common malignancy in India, literature search does not shows any study from this part of country which few studies seen from areas, such as Gujarat, Haryana, Assam, northern Karnataka, Delhi, Mumbai, Calcutta, etc., but not in this geographical area. Therefore, to conclude, most CML patients in eastern UP are relatively young (31–40 years). In addition, males were more commonly affected.
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Conflicts of interest
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By Sandip Kumar; Vikas Gupta; Anju Bharti; Lalit Meena; Vineeta Gupta and Jyoti Shukla
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