Expression of BCL2L12and LIPOCALIN 2 in Adult Patient with Acute Myeloid Leukemia and Correlation with Clinical Response

Background: Acute myeloid leukemia (AML) is a malignancy arising within the bone marrow (BM), in which leukemic cells proliferate uncontrollably in association with a disruption of normal hematopoiesis.Aim of the work to evaluate the expression of LCN and BCL2L2, in newly diagnosed bone marrow samples from adult with AML and to correlate their expression levels with clinical and Laboratory data of the patients especially that known to have a prognostic feature. Methods: This study was carried out on 87 consecutive newly diagnosed adult AML patients of which 75 are evaluated for both LCN, BCL2L12 (All 87 are evaluated for LCN). In addition, 20 donors of matched age and sex healthy individuals from donors for bone marrow transplantation were included as a control group. Results: No statistical significant correlation was found between LCN over-expression and the control group , there was no statistical significance between its expression and age, sex, hepatomegally, splenomegally and lymphadenopathy, also there was no statistical significance regarding peripheral blood and bone marrow findings, immunophenotyping, cytogenetics or molecular findings and MRD at day 15.No statistical significance was found between BCL2L12 expression and the control group again there was no statistical significance between its expression and age, sex, hepatomegally, splenomegally and lymphadenopathy, also there was no statistical significance regarding peripheral blood and bone marrow findings, immunophenotyping, cytogenetics or molecular findings and MRD at day 15. Conclusion: LCN and BCL2L12 were found to be expressed with non significant difference in AML as in normal subjects; however studies on large number of cases are needed to confirm our finding. The role of LCN and BCL2L12 need to be verified by further large-scale sample and further studies.


Expression of BCL2L12and LIPOCALIN 2 in Adult Patient with Acute Myeloid Leukemia and Correlation with Clinical Response
of leukemia. NGAL does not appear to influence the balance between survival and death of bone marrow stem and progenitor CD34+ cells, mature granulocytes, and T and B lymphocytes. Whereas it blocks the maturation of lineage-committed myeloid cells into mature erythrocytes and monocytes (Wang et al., 2019).
BCL2L12 Bcl-2, like protein 12, is a protein that in humans is encoded by the BCL2L12 gene. The protein encoded by this gene belongs to the Bcl-2 protein family. BCL2L12 expression is upregulated in most human glioblastomas. Expression of BCL2L12 results in resistance to apoptosis. BCL2L12 directly neutralizes caspase-7 (CASP7) and indirectly neutralizes caspase-3 (CASP3) by an indirect mechanism. Both caspase enzymes are known to play essential roles in the execution phase of apoptosis (Chia-Hua, 2012). In our study we aimed at evaluating the expression of LCN and BCL2L2, in newly diagnosed bone marrow samples from adult with AML and to correlate their expression levels with clinical and Department of Clinical and Chemical Pathology, National Cancer Institute, Cairo University, Egypt. *For Correspondence: luciekhozam@hotmail.com

Lucie G khozam*, Reham A.A Elshimy, Roxan E Shafik
Laboratory data of the patients, especially those known to have a prognostic feature.

Patients and Methods
This study was carried out on 87 consecutive newly diagnosed adult AML patients, who presented to adult medical oncology department outpatient clinic at the National Cancer Institute, Cairo University over the period of two years. In addition, 20 healthy donors of matched age and sex from donors for bone marrow transplantation were included as a control group. De novo AML was defined if the patient had no history of prior treatment with chemotherapy and had no prior diagnosis of myelodysplastic syndrome or chronic myeloid leukemia. An informed consent was obtained from all the participants prior to the study accompanied by detailed explanation of the procedure and its outcome. This study was carried out in accordance to the guidelines approved by the Ethics Committee, National Cancer Institute, Cairo University; Local institutional research board approval was also acquired prior to the study. The data were collected from patients' files after permission.

Exclusion criteria
Pediatric, Non-Egyptians or Treated acute myeloid leukemia patients and Acute promyelocytic leukemia patients.
Patients were analyzed for the following: A: Demographic characteristics including, B: Full history taking including, C: Full clinical examination, D: Laboratory investigations.
• Bone marrow aspiration and examination, supplemented with cytochemical stains such as Myelo-Peroxidase (MPO) or Sudan Black Stain (SBB), Esterase, Acid Phosphatase and Periodic Acid Schiff (PAS) when indicated.
• Quantitative analysis of LCN, BCL2L12 genes expression by real time reverse transcriptase polymerase chain reaction (RT-PCR).

Assessment of LCN, BCL2L12 expression by real-time (RT-PCR)
Total RNA was extracted from bone marrow cells RT-PCR was performed using a total volume of 20 μl, and the thermal reaction conditions were as follows: 95°C for 10 min (polymerase activation), followed by 40 cycles of 95°C for 30s (Denaturation), 60°C for 60s (annealing and extension), in which fluorescence was acquired and detected by Step One Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Relative expression of LCN, BCL2L12 gene were analyzed by the comparative Ct method (2 −ΔΔCt ), in which data were expressed as the fold changes in LCN, BCL2L12 gene expressions in the patients normalized to the expression levels of the endogenous control gene (B Actin, GAPDH) respectively and relative to the healthy controls.

Statistical Methods
Data management and analysis were performed using SPSS, (version 20 for Windows; SPSS Inc., Chicago, IL, USA). Categorical data were summarized as percentages; numerical data were summarized using means and standard deviation or medians and ranges. Relation between LCN, BCL2L12 and other variables were assessed using Chi-square test. All tests of hypotheses were conducted at the alpha level of 0.05, with 95% confidence interval. A p-value < 0.05 was considered significant.

Results
The current study included 87 newly diagnosed adult AML patients with age ranged from 20 to 70 years, out of which,75 patients were evaluated for both LCN and BCL2L12, and all 87 patients were evaluated for LCN, together with twenty age and sex matched healthy controls. Expression levels of LCN, BCL2L12 were determined by real time quantitative reverse transcriptase polymerase chain reaction (RT-q PCR). (Demographic and laboratory characteristics of patients are summarized in Table 1).
The median value of gene expression in control group was taken as a cut off (1.5±0.32 for LCN gene). Below this value the patients are considered as low expressers and above it as high expressers, Seven AML patients (8.2%) had elevated LCN expression and seventy-eight patients (91.3%) had low expression. No statistical significant difference was detected between AML group and control group as regard LCN gene expression levels (p value: 0.48) ( Table 2) regarding relapse risk (RR) (P value: 0.27) and regarding survival (P value: 0.54, Figure 1).
Regarding BCL2L12 gene expression, we examined 75 patients only (due to inadequate samples). For BCL2L12 gene expression there was no statistical significant difference was detected between AML group and control group as regard BCL2L12 gene expression levels (p value:0.48) (Table3).
There was statistical significant relation between high and low BCL2L12 expressers AML patients regarding the sex (P value: 0.01).
Comparison revealed no statistical significant relation between high and low BCL2L12 gene expression in AML patients regarding demographic and clinical data: age (P value:0.45), hepatomegaly (P value: 0.66) and splenomegaly (P value: 0.73) and lymphadenopathy (P value: 0.65).
Regarding FLT3 there was no statistical significant relation (P value: 0.41) with BCL2L12 gene expressions. There was no statistical significant difference between There was statistical significant relation between high and low LCN gene expression in AML patients regarding the sex (P value: 0.01). Comparison revealed no statistical significant relation between high and low LCN gene expression in AML patients regarding demographic and clinical data: age (P value:0.77), hepatomegaly (P value: 0.66), splenomegaly (P value: 0.73) and lymphadenopathy (P value:0.65). Regarding FLT3 there was no statistical significant relation (P value: 0.41) with LCN gene expressers. There was no statistical significant difference between high and low LCN gene expressers AML patients regarding outcome of treatment on day 15 (P value: 0.41),

Discussion
AML has a great variability in the pathogenesis. It requires the cooperation between at least two classes of gene mutations (Godley and Shimamura, 2017).
Thus, in this study we evaluated the expression of Lipocalin 2 (LCN) and BCL2L12, in newly diagnosed bone marrow samples taken from adult with Acute Myeloid Leukemia and to correlate their expression levels with clinical, laboratory data of the patients and prognosis and patient survival.
In our study LCN was under expressed in 78 (91.7 %) patients out of 85 adult AML patients, and there were 7 (8.3%) patients have LCN overexpression. There was no significant statistical difference between over expression and under expression of Lipocalin2 in the studied adult AML bone marrow samples compared to the control BM samples as regard outcome of treatment on day 15 (P value: 0.41), or relapse risk (RR) (P value: 0.27) and overall survival (P value :0.54).
In our study, no statistical significant difference was found between the expression level of LCN2 and age, clinical parameters and hematological parameters (WBC count, hemoglobin, platelets, peripheral blood and bone marrow blasts), immunophenotyping and karyotyping.
On the contrary to our study, Yang et al 2013 also investigated the expression of LCN2 in a patient's group and found LCN2 high expression was associated with better prognosis, and FLT3 status had an adjuvant effect on overall survival.
On the other hand, (Bouch and Bauvois 2014) noticed that NGAL expression in the bone marrow is lower in AML patients than in normal controls. Likewise, NGAL expression increased in patients achieving complete remission and falls in patients with refractory disease. In addition, a combination of FLT3-ID mutation status and high NGAL levels is predictive of the best survival rates in patients with AML. These data suggest that MMP-9 and NGAL might be surrogate markers of disease status in patients with AML. Also, study conducted by Bauvois 2018 revealed that the levels of bone marrow NGAL transcript are found to be lower in AML patients than in healthy individuals, and these levels recover to normal values following complete remission and then decline again at relapse.
In patients with AML, higher bone marrow NGAL mRNA expression is observed in individuals with a good prognosis than in individuals with a poor prognosis and independently of the French-American-British (FAB) classification.
Up to our knowledge, there were no other studies done on LCN2 expression in AML patients and our study is the first study in Egypt.
However, some studies have reported differentially altered neutrophil gelatinase-associated lipocalin (NGAL) levels in several solid malignancies. They evaluated NGAL measured in plasma or urine as both prognostic and diagnostic marker for different types of human tumor. Roli et al., (2017) noticed that positive NGAL expression was associated with a decrease of DFS in patients affected by breast and colorectal cancers, advancing a predictive role of malignancy recurrence.
Likewise, this study revealed a significant association between NGAL overexpression and OS for colorectal and endometrial cancers, suggesting that it could be a useful marker to predict patient survival in these kinds of disease. This finding is also coherent with the positive association reported in single studies on liver, lung, esophageal, oral and pancreatic and kidney cancers. In contrast, the study did not find any meaningful association between overexpression of NGAL and OS for gastric cancer.
In our study there were 65 (86.6 %) patients who have BCL2L12 under expression out of 75 adult AML patients, and 10 (13.4 %) patients have BCL2L12 overexpression. There was no significant statistical difference between over expression and Under expression of BCL2L12 in the studied adult AML bone marrow samples compared to the control BM samples as regard outcome of treatment on day 15 (P value:1), or relapse risk (RR) (P value: 0.1) and overall survival (P value:0.94) In our study, no statistical significant difference was found between the expression level of BCL2 and age, clinical parameters and hematological parameters (WBC count, hemoglobin, platelets, peripheral blood and bone marrow blasts), immunophenotyping and karyotyping.
This result is in agreement with the study done by Zhou et al., (2019), which revealed that BCL2 over expression identified specific FAB subtypes of AML, but it did not affect prognosis.
Also, with subsequent studies done by Kulsoom et al., (2018) which showed that Expression of Bax and Bcl-2 does not differ significantly among AML patients in terms of remission, relapse, resistance, overall survival, and disease-free survival suggest that there is no significant association between the expression of Bax and Bcl-2 and their ratio with clinical response, or with 1-year DFS and OS.
Although both Bax and Bcl-2 had higher expression in those with persistent remission (good response group), a higher Bax/Bcl-2 ratio was more common among those who were resistant or had relapse (poor response group).
In contrast to our study Thomadaki et al., (2012). noticed that Leukemia patients expressing high level of BCL2L12 were 3 times more likely to relapse (p=0.004) or die (p=0.007) than patients with low level of BCL2L12 expression.
Additionally, statistical significant relationships were found between BCL2L12 expression level and CD117 expression, the presence of splenomegaly and chemotherapy response.
Also, on the contrary to our study, Campos et al., (1993) showed that high expression of BCL2 was associated with a low complete remission rate after intensive chemotherapy, and with a significantly shorter survival.
In multivariate analysis, the percentage of bcl-2+ cells (or the blast survival in culture), age, and the percentage of CD34+ cells were independently associated with poor survival.
Up to our knowledge, it is to be noted that our study is the only study to detect both LCN and BCL2L12 expression in AML patients. The discrepancy between our results and other studies may be due to the small sample size and difference in ethnicity, geography, gender and other factors.
In conclusion in our study LCN, BCL2 show no significant difference in the studied adult AML bone marrow samples compared to the control BM samples, but needed to be studied on a large scale of patients.

NOTE!!!
The contributions of all authors must be described in the following manner: The authors confirm contribution to the paper as follows: study conception and design: X. Author, Y. Author; data collection: Y. Author; analysis and interpretation of results: X. Author, Y. Author. Z. Author; draft manuscript preparation: Y. Author. Z. Author. All authors reviewed the results and approved the final version of the manuscript.