EVALUATION OF YKL-40 AS A PROGNOSTIC MARKER IN ADULT ACUTE MYELOID LEUKEMIA PATIENTS

Mohamed Eissa 1,2 and Amal Ahmad Zidan 1 . 1. Clinical Pathology Department, Faculty of Medicine, Zagazig University 2. Pathology Department College of Medicine, King Khalid University ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Sample preparation:-It was diluted in 1:10 with the sample diluent buffer and mixed thoroughly. Twenty five μl of sample was added to 225 μl of sample diluent buffer.

Reagent preparation:-
Reconstitution of the human Chitinase 3-like 1 standard: Chitinase 3-like 1 standard solution was prepared no more than 2 hours prior to the experiment. Two tubes of Chitinase 3-like 1 standard (10 ng per tube) were included in each kit. 1. 10,000 pg/ml of human Chitinase 3-like 1 standard solution: 1 ml sample diluent buffer was added into one tube; the tube was kept at room temperature for 10 min and mixed thoroughly. 2.
4000pg/ml of human Chitinase 3-like 1 standard solution: 0.4 ml of the above 10,000 pg/ml Chitinase 3-like 1 standard solution was added into 0.6 ml sample diluent buffer and mixed thoroughly. 3. 2000 pg/ml→62.5 pg/ml of human Chitinase 3-like 1 standard solutions: Six eppendorf tubes were labeled with 2000 pg/ml, 1000pg/ml, 500 pg/ml, 250 pg/ml, 125 pg/ml, 62.5 pg/ml; respectively. Three hundred µl of the sample diluent buffer was added into each tube. 300 µl of the above 4000 pg/ml Chitinase 3-like 1 standard solution was added into 1st tube and mixed. 300 µl from 1st tube was transferred to 2nd tube and mixed. 300 µl from 2nd tube was transferred to 3rd tube and mixed, and so on.
Preparation of biotinylated anti-human Chitinase 3-like 1 antibody working solution: The solution was prepared no more than 2 hours prior to the experiment. 1. The total volume was: 0.1 ml/well x (the number of wells). 2. Biotinylated anti-human Chitinase 3-like 1 antibody was diluted in 1:100 with the antibody diluent buffer and mixed thoroughly.

Preparation of Avidin-Biotin-Peroxidase Complex (ABC) working solution:-
The solution was prepared no more than 1 hour prior to the experiment. 1. The total volume was: 0.1 ml/well x (the number of wells). 2. Avidin-Biotin-Peroxidase Complex (ABC) was diluted in 1:100 with the ABC dilution buffer and mixed thoroughly.
Procedure:-1. One hundred µl of the 10,000 pg/ml, 4000pg/ml, 2000 pg/ml, 1000 pg/ml, 500 pg/ml, 250 pg/ml, 125 pg/ml and 62.5 pg/ml human Chitinase 3-like 1 standard solutions were added into the precoated wells, one hundred µl of the sample diluent buffer was added to the control (Zero well). 2. One hundred µl of each properly diluted sample of serum was added to the well. 3. The plate was sealed with the cover and incubated at 37°C for 90 min. 4. The cover was removed, plate content was discarded, and the plate was blotted onto paper towels. 5. One hundred µl of biotinylated anti-human Chitinase 3-like1 antibody working solution was added to each well and the plate was incubated at 37°C for 60 min. 6. The plate was washed 3 times with PBS, and each time washing buffer stayed in the wells for 1-2 min. The washing buffer was discarded and the plate was blotted onto paper towels. 7. One hundred µl of prepared ABC working solution was added to each well and the plate was incubated at 37°C for 30 min. 8. The plate was washed 5 times with PBS as in step 6. 9. Ninety hundred μl of prepared TMB color developing agent was added into each well. The plate was incubated at 37°C in dark for 30 min. 10. One hundred μl of prepared TMB stop solution was added into each well. The color changes into yellow immediately. 11. The plate was read at 450 nm in a microplate reader within 30 min. after the stop solution was added.    (1) show comparison between YKL-40 levels in two studied groups. There was a significant difference between levels of YKL-40 in control and patient groups before the treatment (P=0.002). Moreover, there was a highly significant difference between patient groups before and after treatment (P< 0.001).             (7) show that there was a significant difference between CR and NR groups as regard YKL-40 level (P=0.004).

Discussion:-
Acute myeloid leukemia (AML) is a clonal malignant proliferation of myeloid blast cells in the marrow with impaired normal hematopoiesis. (20) It is the most common acute leukemia among adults and its incidence increases with age. (21) Despite the advancement in treatment options for AML, its prognosis is very variable, ranging from survival of few days to cure. Many patients die either from complications of intensive chemotherapy, resistance to the current treatment options or they experience relapse after initial response to traditional chemotherapy. (22) Moreover, there is still a need for simple reliable and easy measured factors which has an impact on the prognosis especially in area where modern technology is not usually available. So, in this study we measured the level of YKL-40 in serum of do novo AML patients to evaluate its role in prognosis and response to treatment. It was estimated at the onset of the disease and after 1 month of induction therapy. This cohort study was carried out in Clinical pathology and Medical Oncology & Hematology Departments, Faculty of Medicine, Zagazig University Hospitals from February 2014 to February 2015 on 48 subjects classified into two groups: control group (24 normal individuals) and patient group (24 adult newly diagnosed AML patients).
In this study there was no significant difference between the control and patients as regards age and sex. This is in agreement with a study done by El Hefni et al., 2015 as both studies were done on adult AML patients. There was a slight female predominance in both studies but still with no significant difference with the control group. (23) Among the newly diagnosed AML patients, fever was the most common clinical manifestation. It was found in 66.7% of patients, followed by organomegaly in 54.2%, gum bleeding in 37.5%, purpura in 29.2%, Lymphadenopathy in 25% and CNS manifestation in 8.4%of patients. This finding is in agreement with Weinblatt et al., 2004 who stated that fever is the most common initial clinical presentation. (24) In the current study, there was a highly significant difference between the patients and control groups as regards TLC, hemoglobin concentration, platelets count and LDH. This was expected as these are directly attributable to the leukemic infiltration of bone marrow with resultant cytopenia (El Hefni et al., 2015). (23) ng/ml 1901 Our studied patients were subdivided according to FAB classification into M1 (3 patients), M2 (12 patients), M3 (0 patient), M4 (5 patients) and M5 (4 patients). The majority of our patients were classified as M2, while no M3 subgroup was included in the study. According to national cancer institute, Brunning et al., 2001 stated that M2 is the most common morphologic type. (25) As regard cytogenetic risk classification, the majority of the patients were included in the intermediate subgroup where the patients were subdivided into bad (16.7%), intermediate (58.3%), good (25%) prognosis subgroups.
In this study, the levels of YKL-40 before induction therapy in patient group were significantly higher than those in control group. After induction therapy, it was noted that the level of YKL-40 was significantly decreased in patient group when compared with its level before treatment (98.5 versus 139ng/ml; respectively).
These were similar to the results of the studies performed by Bergmann et al., 2005 and El Hefni et al., 2015 who mentioned that YKL-40 plays a role in the malignant phenotype as a cellular survival factor. Furthermore, YKL-40 modulates vascular endothelial cell morphology by promoting the formation of branching tubules, indicating that YKL-40 has a role in angiogenesis by stimulating the migration and reorganization of vascular endothelial cells. YKL-40 also acts as a chemoattractant for endothelial cells, stimulates their migration and promotes the migration and adhesion of vascular smooth muscle cells. (26,23) In this study, YKL-40 could predict response to treatment among AML patients at a cut-off ≥137 ng/ml with sensitivity 83.3% and specificity 77.8%.On the other hand, in the study done by Anil et al., 2013 the cut-off was 112 ng/ml where they used the 90 th percentile of YKL-40 values for the normal control group. (17) In this study, when comparing patient groups with cut-off ≥ 137 ng/ml with that < 137 ng/ml, we found that there was a significant difference between the two groups as regard platelets count and LDH. In agreement with our results Bergmann et al. 2005 found that there was a significant difference between high and low YKL-40 groups as regard LDH but no difference was observed in platelet count. This might be attributed to different sample size in both studies. (26) Of note, no significant difference was found between the two groups as regard cytogenetic risk categories and FAB classification types. This goes hand in hand with the study done by Bergmann et al. 2005 who reported that the influence of cytogenetic was underestimated due to the small number of patients in both good and bad prognosis subgroups. (26) After induction therapy, it was noted that CR rate was significantly lower in the patient group with YKL-40 level ≥ 137 ng/ml (54.5%) than that in those with YKL-40 level < 137 ng/ml (92.3%), indicating that high level of YKL-40 confers a poor outcome. This assumption can be further supported by our finding of high level of YKL-40 in NR group when compared with CR group (365ng/ml versus 127.5ng/ml; respectively).  (27,28) In this study, there was significant difference between the two groups (CR and NR) as regard CNS and lymphadenopathy. As their incidence was significantly more in those patients who did not achieve complete remission or got into relapse.
In addition, we found a significant correlation between YKL-40 and TLC, platelets count and LDH, which are considered prognostic factors. In a study done by Schmidt et al., 2005 on metastatic melanoma, they found that combination of YKL-40 and LDH as prognostic factors was able to divide patients into 3 prognostic groups that affect survival of patients and their elevation together was with the poorest prognosis group. (29) Moreover, there was a significant relation between both CR and NR groups and cytogenetic risk categories which is considered the most prognostic factor identified at the time of diagnosis. On the other hand, Bergmann et al., (2005) and 1902 EL Hefni et al., (2015) did not find any relation with the cytogenetic risk categories. This might be attributed to selection criteria of studied groups. (26,23) Taking together, high serum level of YKL-40 seems to add prognostic information in patients with AML. However, its predictive value is still unclear.
In the current study, DFS was shortened in patients with serum level of YKL-40 ≥ 137ng/ml compared to the ones with level < 137ng/ml. Our results go hand in hand with that reported by Bergmann et al., (2005) who stated that high serum YKL-40 was related to short recurrence free interval and short overall survival. Moreover, Jensen et al., (2003) in breast cancer patients found that high serum YKL-40 was related to recurrence and less response to treatment. (26,30) Our study shows that the high serum YKL-40 is a new independent prognostic biomarker in patients with AML. It may hold promise for developing novel therapeutic agent targeting YKL-40 that is over expressed in a broad range of human cancers.

Conclusion:-
It can be concluded that high serum level of YKL-40 was associated with bad response to treatment and confers poor outcome. Moreover, it could serve as valuable prognostic marker.