European Journal of Cardiovascular Medicine

Cancer has surpassed heart disease, as the top cause of mortality for people under 85years of age(1). Chronic Myeloid Leukemia (CML) is a myeloproliferative neoplasm with an incidence of 1-2 cases per 1,00,000 adults (2) and the average age at diagnosis is 65 years (3). Since age is a major factor related to the incidence of the disease, cancer remains to be a major source of morbidity, mortality and financial burden in the population. Therefore adequate cure of the disease with knowledge about the pathogenesis, molecular and genetic alterations involved and thereby developing effective therapy becomes important.

CML differs from other myeloproliferative neoplasms due to the existence of a chimeric BCR-ABL gene, which is made up of fragments of the BCR gene on chromosome 22 and the ABL gene on chromosome 9. (4,5) The discovery that BCR-ABL plays a critical role in the pathogenesis of CML led naturally to the hypothesis that inhibiting BCR-ABL could be an effective treatment strategy. The development of tailored treatment has significantly changed how the disease progresses. Tyrosine kinase inhibitor therapy, particularly in individuals with early disease, produces long-lasting remissions with tolerable side effects and postpones the development of blast crisis, possibly by inhibiting the proliferative drive that leads to the acquisition of additional mutations.(4).

Patients receiving treatment with tyrosine kinase inhibitors frequently have cancers that contain mutations in the BCR-ABL kinase domain that prevent the drugs from binding after relapses. The significant anticancer effects of BCR-ABL inhibitors explain the selective expansion of these cells, which implies that these resistant tumours are nonetheless "reliant" on the progrowth signals supplied by BCR-ABL.

The only recognised curative therapy for CML is bone marrow transplant, which carries a high risk of morbidity and mortality in a large number of patients do not react to all TKIs. BCL- 2 is an anti-apoptotic molecule. Through a variety of ways, overexpression of BCL-2 is a frequent occurrence that protects tumour cells against apoptosis. Tyrosine kinase inhibitor resistance and CML development are both influenced by genes in the BCL-2 family.

p53 is a tumor suppressor gene. Loss of prodeath BCL-2 family transcription and activation is caused by p53 deletion, which is likely the most frequent genetic abnormality in cancer. Tp53 mutations are typically not sufficiently specific to diagnose cancer on their own, and their absence does not rule it out either. p53 activation might be an optional strategy for treating various cancers. MDM2 normally inactivates the p53 gene, however in chronic myeloid leukaemia, it becomes active (5)

Haematopoietic stem cells in the bone marrow which remains quiescent are responsible for relapse of the disease and they are found to upregulate the anti apoptotic BCL-2 gene. Studies have shown that p53 regulates the quiescent state of haematopoietic stem cells (6). The majority of treatments are ineffective against the cancer stem cells. Eliminating cancer stem cells offers a novel approach to cancer treatment by totally eradicating the source of cancerous cells. This study is intented to examine the expression of BCL2 and p53 in the survival of CML cells.

This is a Cross sectional study.Patients diagnosed with Chronic Myeloid Leukemia identified from the records available in the Department Of Pathology, GMC Kozhikode. Data To Be Collected Over A Period Of 4 Years From January 1st 2017 To December 31st 2020 and to be followed up for one year subsequently.

STUDY VARIABLES

1. Diagnosed cases based on final histopathology report from Department of Pathology, Government medical college,
2. Expression of BCL2 and p53
3. Haematologic parameters such as Haemoglobin, total count and platelet count
4. Age and gender

DATA COLLECTION TOOLS

1. Bowins fluid fixed paraffin embedded bone marrow trephine biopsy blocks
2. Glass slides and coverslips for
3. Haematoxylin and Eosin
4.  
5. IHC Marker: BCL2 and p53
6. Secondary antibodies to use in
7. Hospital
8. A structural proforma to collect

STUDY POPULATION

Haematologically confirmed cases of Chronic myeloid leukemia received in Department Of Pathology, GMC Kozhikode.

INCLUSION CRITERIA

• Who are diagnosed with Chronic Myeloid Leukemia and whose case records are available at government medical college
• Who are willing to co-operate for the study by sharing their course of the disease for the purpose of

EXCLUSION CRITERIA

• Whose case records are not available at GMC kozhikode
• Who do not give consent for followup studies
• Bone marrow biopsies of poor quality
  • Myeloproliferative Disorders other than Chronic Myeloid Leukemia

DATA COLLECTION TECHNIQUES AND PROCEDURE

After ethical clearance was obtained, the case records of all patients diagnosed as chronic myeloid leukemia were studied from 1st January 2017 to 31st December 2020. The details of patients with variables like age, sex, Hb, total count, platelet count and imaging was collected from in patient medical records and from records in the Department of Pathology. Bouins solution fixed decalcified bone marrow biopsies were retrieved and stained with hematoxylin and eosin and assessed for leukemic cells. WHO criteria for diagnosis of chronic myeloid leukemia was followed. CML was diagnosed on the basis of a CBC as well as subsequent bone marrow examination. 4 microns thick deparaffinized adequate bone marrow biopsies were subjected to BCL2 and p53 IHC markers. The percent of p53 immunoreactive tumor cells was scored as 0 to 4+ in p53 positive regions. The percent of BCL-2 immunoreactive tumor cells was scored as 0 to 3+. In cases with BCL2 and p53 immunoreactivity, prognostic parameters and their treatment response was looked into. Patients were followed up over phone or directly when the patient came to hospital for review in OPD.

BCL2 and p53 marker used for this study is from Pathinsitu.

Procedure for immunohistochemistry

1. 5 micrometre sections made from the paraffin blocks are incubated at room temperature overnight and then at 600 Celsius for 1
2. Deparaffinise the section by dipping thrice in xylene solution for 5 minutes
3. Hydration is done by immersing the section in descending concentrations of isopropanol of 100 %, 95 %, 70 % and 50 % for 5 minutes
4. Wash the sections with distilled water, two changes of 2 minutes
5. Antigen retrieval is done in citrate buffer, at a pH of 6.5 for 15-20 minutes by using MERS technique by heating at a sub boiling temperature of 900
6. Wash the sections again in distilled water, two changes of 2 minutes
7. Wash in TBST (Tris buffered saline tween) for 2 minutes. (TBST contains Tris base 42g, Sodium chloride 8g in 1 litre of water. The pH is adjusted to 7.6 with concentrated HCl and stored at 40 Celsius. At the time of use Tween 20 is added).
8. Endogenous peroxidase blocking is done (20 minutes) by using 3% hydrogen peroxide solution in methanol/the solution received with the antibody
9. Rinse it with distilled water and TBST
10. Primary antibody is added to the prepared section and kept in room temperature for 45
11. Rinse with distilled water and TBST
12. Poly Excel Target Binder is added and kept for 20-30 minutes at room temperature (to enhance the reaction)
13. Rinse with distilled water and TBST
14. DAB (Diamino benzidine) working solution is added and kept for 5 minutes at room temperature in a dark room
15. Rinse with TBST
16. Counterstain with haematoxylin for 30 seconds and wash with water
17. Dehydration is done by using ascending concentrations of isopropanol 50%, 75%, 95% and 100%.
18. Dip the section in Xylene for 5 minutes, two changes and mount with

Further the immunohistochemical expression of BCL-2 and p53 was compared with status of remission upon treatment.

Statistical analysis

Data entered in MS Excel; statistical analysis was done using SPSS statistical software version 23. Categorical variables were expressed as frequency and percentage. Continuous variables were expressed as mean and standard deviation. Comparison of categorical variable between two group was analysed by chi square test. Comparison of continuous variable between two group was analysed by independent sample t test. A p value less than 0.05 was considered as statistically significant.

Ethical considerations

Institutional Ethics Committee clearance was obtained prior to commencement of study. Data collection was started only after getting ethics committee approval for study. Confidentiality have been ensured and maintained.

12 patients out of the 21 patients (57%) who were not in remission were elderly in the age  group above 65 years whereas, 94% of patients in remission were <65 years of age.

Table 1 : Distribution of age according to status of remission

The greater proportion of patients among the study population, were  Males constituting 74.5% and females constituted 25.5%.

Table 2: Characteristics of gender

Table 3 : Distribution of cases according to haemoglobin

Greater proportion of patients in remission(64.7%) and not in remission(52.4%) had haemoglobin in the range between 7-10 mg%.

Table 4: Distribution of Heamoglobin according to status of remission

Table 5: Distribution of cases according to total count

Majority of the population under study had a total count above 25,000 per microlitre irrespective of the status of remission.

Table 6: Distribution of total count according to the status of remission

Table 7: Distribution of cases according to platelet count

The study population irrespective of the status of remission had platelet count ranging between 1 lakh and 10 lakh.

Table 8: Distribution of Platelet according to status of remission

Table 9: Organomegaly among the cases

90.9% of the population under study presented with massive splenomegaly at initial presentation. 9% of the cases showed no organomegaly at presentation. 69% of the patients were in chronic phase. 25% were in blast crisis and 5% were in accelerated phase of CML.

Table 10: Distribution of cases according to the phase of the disease

On followup of the population over a period of 1 year, 61.8% of patients were responding well to therapy and 38.2% patients showed worsening of symptoms on TKI therapy.

Table 11: Distribution of cases according to the status of remission

97% of patients in remission were in chronic phase and 3% were in accelerated phase. Blast crisis was not seen in patients under remission. 67% of patients who were not in remission were in blast crisis phase and the rest 24% of patients were in chronic phase and the relation was found to be statistically significant.

Table 12 : Status of remission of patients in different phases of CML

Table 13:Comparison of phases of CML with status of remission

Among the study population, 71% showed loss of p53 and 29% showed a nuclear positivity of score 1 + (i.e in <50% of tumour cells).

Table 14: Expression of p53 in the study population

Among the 55 patients, 34 patients were in remission of which 67.6 % showed loss of p53 tumor suppressor gene. Of the 21 patients who were not in remission (worsened on Imatinib therapy) 76.2% showed loss of p53 expression.

Table 15: Correlation of expression of p53 and status of remission

Majority (71%) of the patients in chronic phase showed negative expression of p53. All three patients in accelerated phase showed negative p53 expression. 86% of patients in blast crisis also showed negative p53 expression

Table 16: Distribution of p53 in different phases of CML

Association between p53 immunoreactivity and the phases of CML was done using Chi- square test/ Fisher’s exact test. It was found that there is no association between p53 and the different phases of CML (p>0.05)

Table 17: Correlation of p53 with different phases of CML

Majority of the study population (61.8%) showed absence of over expression of BCL-2.

Table 18: Expression of BCL2 in the study population

Among the 34 patients in remission, 85.3 % showed negative expression for BCL2 and rest showed a score of 2+(overexpression). 76.2% of the cases who were not in remission showed BCL2 overexpression.

Table 19: Correlation of expression of BCL-2 and status of remission

93% of patients in BC phase showed positive immunoreactivity for BCL2. 66% of cases in AP also showed similar positivity. 84% of patients in CP didn’t show any expression of BCL2 protein.

Table 20: Distribution of BCL-2 immunoreactivity in different phases of CML

Association between BCL2 immunoreactivity and the phases of CML was done using Chi- square test/ Fisher’s exact test. It was found that there is an association between BCL2 and the different phases of CML (p<0.05)

Table 21: Association between BCL-2 and different phases of CML

BMT 7931/20

Fig 1: CML chronic phase showing p53 immunoreactivity of 1+

BMT 6985/18

In the present study 55 cases of CML were selected. The paraffin embedded BMT biopsy blocks were retrieved and clinical details of the patients were collected from medical records. BCL-2 and p53 immunohistochemical staining was done in all cases. All the data collected were entered in Microsoft excel sheet & was analysed using SPSS statistical software. Qualitative variables were expressed as frequency and percentages. Association between P53, BCL2, haemoglobin, total count, platelet and age with NIR/IR was done using Chi-square test/ Fisher’s exact test. Comparison of haemoglobin, total count, platelet and age among patients in remission and not in remission was done using independent sample t test. A p value of <0.05 was considered statistically significant.

91% of the population under study presented with massive splenomegaly at initial presentation. 9% of the cases showed no organomegaly at presentation. In a study by Dhruv et al. splenomegaly is the most common clinical finding present in >50% of the patients.

The haemoglobin of the patients in the current study ranged from 5 mg% to 14.2 mg% and mean haemoglobin in the study population was 9.12 mg%. Majority of the patients had haemoglobin in the range of 7-10 mg%. This was close to the study conducted by Kumar et al. where the mean haemoglobin was 9.41 mg% and the range was similar to current study between 7-10 mg%(58)

The total count of the patients ranged from 12,500 to 2,75,700 per microlitre and mean count was 1,11,067 per microlitre.

In a study conducted by kumar et al. the mean total count was 1,82,000 per microlitre (58).

The Platelet count of the patients ranged from 7,000 to 10,40,000 and mean platelet count was 4,82,218 per microlitre. The maximum (87%) number of patients had a platelet count in the range between 1,00,000 to 10,00,000 per microlitre.

In a study conducted by kumar et al. the platelet count ranged between 1,50,000 to 4,00,50,000 per microlitre similar to our study(58).

In the present study, among the study population, 71% of patients with CML showed loss of p53 immunoreactivity. In a study conducted by Lanza et al. it was found that p53 acts as a negative regulator of myeloid proliferation both of mature cells and CD34+ progenitor cells(5) similar to our study. Negative gene regulation means the type of gene regulation that prevents gene expression.

In another study done by Peterson et al, it was shown that inhibition of HDM2 (human homolog double minute 2) and consequently p53 stabilization induce CML cells to undergo apoptosis regardless of the presence of mutation in the BCR–ABL kinase domain.(51)

Similar study by Luke et al. showed that HDM2 inhibition and increasing p53 activity induced apoptosis in CML stem cells, and its effect was enhanced by the tyrosine kinase inhibitor imatinib(50). In the present study, a large proportion of patients irrespective of the remission status showed loss of p53 immunoreactivity implicating that overall loss of p53 expression is a bad prognostic factor in cases of CML. However, no significant association between p53 and status of remission was found out in our study (P value <0.05).

In the present study it was found that, majority of the study population (61.8%) showed absence of over expression of BCL-2. 76.2% of the cases who were not in remission showed BCL2 overexpression (Score 2+). In a study conducted by Goff et al, it was found that BCL2 genes maintains a protective microenvironment for the malignant stem cells and suggested that inhibition of BCL2 family proteins can eliminate quiescent, TKI-resistant LSC in CML. In a similar study done by carter et al. (48) it was suggested that BCL-2 is a key survival factor for CML stem/progenitor cells.

97% of patients in remission were in chronic phase and 3% were in accelerated phase. Blast crisis was not seen in patients under remission. 67% of patients who were not in remission were in blast crisis phase and the rest 24% of patients were in chronic phase. Greater proportion of cases who were not in remission had loss of nuclear p53 compared with those in remission. Compared to the cases in remission, the population of patients who were not in remission showed an overexpression of BCL2 (72%). A study by Lanza et al suggests that alterations in the p53 gene are frequently related with the blast crisis of chronic myeloid leukaemia (CML), but infrequently with chronic phase of the disease. (5)

In the present study it was found that there is an association between BCL2 and the status of remission (p value<0.05). Hence Overexpression of BCL2 is associated with a bad prognosis in CML. In our study, 72% of patients who were not in remission showed positive immunoreactivity for BCL2. Also there is a statistically significant association between BCL-2 immunoreactivity and the phases of CML. Strong BCL-2 expression was associated with Blast Crisis in our study. p53 acted as a negative regulator for gene expression in cases of CML in our study. The association between p53 expression and status of remission was not statistically significant in our study. But majority of the cases in our study showed loss of nuclear immunoreactivity for p53 irrespective of the status of remission or phase of the disease. Majority of the patients who were not in remission were in blast crisis phase of the disease and greater proportion of them showed loss of p53 staining.

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