Evaluation of ADAM28 expression in adult Egyptian Acute Myeloid Leukemia patients and its impact on outcome

Azzazi, Mohamed Ossman; El Kourashy, Shaza Abd El Wahab; Nabih, Nermeen Adel; Abdou Hussein, Aliaa Nabil

doi:10.21608/ejhbmt.2021.193653

Evaluation of ADAM28 expression in adult Egyptian Acute Myeloid Leukemia patients and its impact on outcome

Document Type : Original Article

Authors

Internal Medicine and Clinical Hematology Department, Faculty of Medicine, Ain Shams University

10.21608/ejhbmt.2021.193653

Abstract

Background: Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults and is fatal as a result of primary refractoriness, relapse, and treatment-related mortality. Although the majority of patients with AML enter remission upon induction chemotherapy, the risk of relapse is considerable.
Aim of the Work: To evaluate ADAM28 expression in newly diagnosed adult Egyptian Acute Myeloid Leukemia patients and to assess its impact on outcome
Patients and Methods: This was a prospective case control study was conducted at Ain Shams university hospitals. Clinical hematology division of internal medicine department including 60 participants: 30 adult patients with newly diagnosed AML and 30 healthy age and sex matched control. The duration of the study was 6 months.
Results: The main findings of the study revealed that: There was no statistically significant difference between the studied groups as regard demographic data, according to FAB score there were 8 (26.7%) M1, 10 (33.3%) M2, 8 (26.7%) M3 and 4 (13.3%) M4, there was high statistically significant difference between the studied groups as regard WBCs, Plts and ESR and statistically significant difference as regard Hb, there was no statistically significant difference between the studied groups as regard Liver and kidney function tests, according to Cytogenetic risk category there were 4 (13.3%) favorable, 18 (60%) intermediate and 8 (26.7%) unfavorable, there was high statistically significant difference between the studied groups as regard ADAM-28m and ADAM-28s, according to response to chemotherapy in the induction phase there were 13 patients in remission and 11 patients did not respond to chemotherapy and there were 6 who died .
Conclusion: There was high statistically significant difference between the studied groups as regard ADAM-28m and ADAM-28s. There was significant correlation between ADAM-28 and response to chemotherapy. ADAM28 improved the growth and dissemination of AML.

Keywords

Main Subjects

Acute myeloid leukemias

Full Text

INTRODUCTION

Acute myeloid leukemia (AML) is the most common type of acute leukemia in adultsand is fatal as a result of primary refractoriness, relapse, or treatment-related mortality (Wouters and Delwel, 2016). Although the majority of patients with AML enter remission upon induction chemotherapy, the risk of relapse is considerable (Bower et al., 2016). Transplantation regimens can be curative, but it remains challenging to identify high risk patients suitable for early transplantation (Cornelissen and Blaise, 2016).

A disintegrin and metalloproteinases (ADAMs) are a new gene family of proteins with sequence similarity to the reprolysin family of snake venomases that share the metalloproteinase domain with matrix metalloproteinases (MMPs). They are structurally classified into two groups: the membrane-anchored ADAM and ADAM with thrombospondin motifs (ADAMTS) (Zhang et al., 2016).

These molecules are involved in various biological events such as cell adhesion, cell fusion, cell migration, membrane protein shedding and proteolysis. Studies on the biochemical characteristics and biological functions of ADAMs are in progress, and accumulated lines of evidence have shown that some ADAMs are expressed in malignant tumors and participate in the pathology of cancers. The activities of ADAMs are regulated by gene expression, intracytoplasmic and pericellular regulation, activation of the zymogens and inhibition of activities by inhibitors (Dong et al., 2015).

Many ADAM species, including ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17, ADAM19, ADAM28, ADAMTS1, ADAMTS4 and ADAMTS5, are expressed in human malignant tumors. Many of them are involved in the regulation of growth factor activities and integrin functions, leading to promotion of cell growth and invasion, although the precise mechanisms of these are not clear at the present time (Reiss and Saftig, 2009).

ADAM28, a member of the ADAM family, cleaves various substrates including von Willebrand factor. Two isoforms of ADAM28: the membrane associated (ADAM28m) and the secreted (ADAM28s) were identified. Both were overexpressed in solid tumors including breast carcinoma, non-small cell lung cancer, and bladder transitional cell carcinoma (Hubeau et al., 2020).

ADAM28 has been shown to relate with tumor proliferation and prognosis However, little is known about expression and potential role of ADAM28 in hematological malignancies. Limited studies suggested that the expression of ADAM28 is up-regulated in acute myeloid leukemia (Zhang et al., 2019). However, the mechanism by which ADAM28 regulates the leukemic cell and the prognostic relevance with AML remain unknown.

AIM OF THE WORK

To evaluate ADAM28 expression in newly diagnosed adult Egyptian Acute Myeloid Leukemia patients and to assess its impact on outcome.

PATIENTS AND METHODS

Study design: This study is a prospective case control study.

Setting: This study was carried out Ain Shams university hospitals. Clinical hematology division of internal medicine department.

Time of the study: from December 2020 till June 2021.

Sample size: 60 participants: 30 adult patients with newly diagnosed AML and 30 healthy age and sex matched control.

Methods:

Clinical assessment: complete history taking such as age, sex, residency, occupation, smoker or ex-smoker, current complaint and presence of comorbidities were evaluated and clinical examination.

Laboratory assessment: (routine and general evaluation tests): Complete blood count with peripheral blood film, ESR, LDH, uric acid, kidney and Liver function functions, coagulation profile, Bone marrow aspiration, flowcytometry, cytogenetics and study measurement of ADAM-28 level in the control & the cases.

ADAM-28 level test: The kit uses a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) to assay the level of ADAM-28 level in samples. Add ADAM-28 level to monoclonal antibody Enzyme well which is pre-coated with Human ADAM-28 level monoclonal antibody, incubation; then, add ADAM-28 level antibodies labeled with biotin, and combined with Strept avidin-HRP to form immune complex; then carry out incubation and washing again to remove the uncombined enzyme. Then add Chromogen Solution A, B, the color of the liquid changes into the blue, and at the effect of acid, the color finally becomes yellow. The chroma of color and the concentration of the Human Substance ADAM-28 level of sample were positively correlated.

Statistical analysis: Analysis of data was done using Statistical Program for Social Science version 20. Quantitative variables were described in the form of mean and standard deviation. Qualitative variables were described as number and percent. In order to compare parametric quantitative variables between two groups, Student t test was performed. Qualitative variables were compared using chi-square (X2) test or Fisher’s exact test when frequencies were below five. Pearson correlation coefficients were used to assess the association between two normally distributed variables. When a variable was not normally distributed, A P value < 0.05 is considered significant.

RESULTS

Table 1: Descriptive data of the studied groups:

		Cases (AML) (n=30)	Control (Healthy) (n=30)	P	Sig.
Age (years)	Mean±SD	37 ± 9.78	41.33 ± 10.84	0.109^*	NS
	Range	23 – 58	23 – 58
Sex	Males	18 (60.0%)	16 (53.3%)	0.602•	NS
	Females	12 (40.0%)	14 (46.7%)
Hb (g/dL)	Mean±SD	9.13 ± 3.02	11.1 ± 1.17	0.002^*	S
	Range	4.4 – 14.2	9.4 – 13
WBCs (x10⁹/L)	Median (IQR)	14.2 (8.7 – 22.6)	5.8 (4.9 – 6.6)	<0.001^≠	HS
	Range	2.2 – 220.1	4.1 – 7.4
Plts (x109/L)	Median (IQR)	44 (21.5 – 84.3)	197.5 (175.8 – 239.3)	<0.001^≠	HS
	Range	5 – 102	155 – 248
Peripheral blasts %	Median (IQR)	24.5 (11 – 63.8)
	Range	1 – 97
ESR (mm/hr)	Median (IQR)	87.5 (69.5 – 92)	11 (7.3 – 14)	<0.001^≠	HS
	Range	60 – 98	1 – 20
ALT (IU/L)	Median (IQR)	27.5 (22 – 34.8)	22 (15 – 29)	0.078^≠	NS
	Range	14 – 40	12 – 39
AST (IU/L)	Median (IQR)	26 (21 – 32.8)	25 (17 – 30.8)	0.283^≠	NS
	Range	14 – 40	12 – 40
Albumin (g/dL)	Mean±SD	3.46 ± 0.28	3.53 ± 0.28	0.331^*	NS
	Range	3 – 3.9	3 – 4
Creatinine (mg/dL)	Mean±SD	0.94 ± 0.18	0.92 ± 0.16	0.711^*	NS
	Range	0.7 – 1.2	0.7 – 1.2
Urea (mg/dL)	Median (IQR)	18 (11 – 24.8)	18.5 (10.3 – 21.8)	0.390^≠	NS
	Range	8 – 30	7 – 30

Table 2: Distribution of cases group according to FAB score:

FAB score	Cases (n=30)
	No.	%
M1	8	26.7
M2	10	33.3
M3	8	26.7
M4	4	13.3

Table (3): Distribution of cases group according to Cytogenetic risk category

Cytogenetic risk category	Cases (n=30)
	No.	%
Favorable	4	13.3
Intermediate	18	60.0
Unfavorable	8	26.7

Table (4): Comparison between studied groups as regard ADAM-28

	Cases (n=30)	Control (n=30)	p	Sig.
ADAM-28m
Range	1.83 – 2.38	0.3 – 0.49	<0.001^*	HS
Mean ± SD	2.08 ± 0.18	0.4 ± 0.06	<0.001^*	HS
ADAM-28s
Range	259.5 – 330.2	101.7 – 139.1	<0.001^*	HS
Mean ± SD	290.94 ± 19.36	119.52 ± 12.49	<0.001^*	HS

Table (5): Comparing ADAM-28 level between responders and non-responders

to induction chemo therapy of cases

	non-responders (n=11)	responders (n=13)	P	Sig.
ADAM-28m
Range	1.84 – 2.38	1.83 – 2.37	0.040^*	S
Mean ± SD	2.15 ± 0.18	2.02 ± 0.15	0.040^*	S
ADAM-28s
Range	260.5 – 330.2	259.5 – 322.6	0.049^*	S
Mean ± SD	298.79 ± 20.75	284.94 ± 16.37	0.049^*	S

Table (6): Comparing ADAM-28 level between dead and alive

	Dead (n=6)	Alive (n=24)	P	Sig.
ADAM-28m
Range	1.93 – 2.38	1.83 – 2.37	0.006^*	S
Mean ± SD	2.25 ± 0.16	2.03 ± 0.16	0.006^*	S
ADAM-28s
Range	277.7 – 330.2	259.5 – 322.6	0.008^*	S
Mean ± SD	309.12 ± 18.35	286.40 ± 17.08	0.008^*	S

Table (7): Correlation between Age and finding in ADAM-28

Age
	r	p	Sig.
ADAM-28m	-0.197	0.132	NS
ADAM-28s	-0.227	0.081	NS

Table (8): Relation between FAB classification and finding in ADAM-28

		M1	M2	M3	M4	Test value	P-value	Sig.
		No. = 8	No. = 10	No. = 8	No. = 4
ADAM-28m	Mean±SD	2.08 ± 0.23	2.07 ± 0.15	2.07 ± 0.17	2.09 ± 0.20	0.021^●	0.996	NS
	Range	1.83 – 2.38	1.85 – 2.34	1.86 – 2.29	1.84 – 2.29
ADAM-28s	Mean±SD	290.70 ± 26.63	290.87 ± 16.20	290.54 ± 16.14	292.43 ± 23.86	0.008^●	0.999	NS
	Range	259.5 – 330.2	266.4 – 321.8	268 – 316	260.5 – 316.4

Table (9): Relation between cytogenetics and ADAM-28

		Unfavorable	Intermediate	Favorable	Test value	P-value	Sig.
		No. = 8	No. = 18	No. = 4
ADAM-28m	Mean±SD	2.01 ± 0.20	2.11 ± 0.16	2.07 ± 0.24	0.813^●	0.454	NS
	Range	1.83 – 2.37	1.86 – 2.34	1.85 – 2.38
ADAM-28s	Mean±SD	282.63 ± 22.42	294.36 ± 15.82	292.20 ± 27.84	1.030^●	0.371	NS
	Range	259.5 – 322.6	268 – 321.8	266.4 – 330.2

DISCUSSION

A prospective case control study was conducted to evaluate ADAM28 expression in the newly diagnosed adult Egyptian Acute Myeloid Leukemia patients and to assess its impact on outcome.

The patients received induction chemotherapy according to the disease subtype and the response was assessed by bone marrow examination and as a result, there were 13 patients in remission and 11 patients who remains with active disease after exclusion of 6 mortality cases.

The results were in agreement with the study of Zou et al. (2017), Also Østgård et al. (2018) revealed that there was no significant difference between cases of AML and control group as regard age, sex, liver and kidney functions and demographic data.

Our study shows that as regard CBC; there was significant difference between the studied groups as regard leucocytic count, Platelets, and ESR and hemoglobin, which was also described in the study of Sayad et al. (2017) and Zhang et al. (2019).

Our study showed that according to FAB score there were 8 (26.7%) M1, 10 (33.3%) M2, 8 (26.7%) M3 and 4 (13.3%) M4 and there was no statistically significant relation between ADAM-28 and FAB classification.

According to Cytogenetic risk category in cases group, there were 4 (13.3%) favorable, 18 (60%) intermediate and 8 (26.7%) unfavorable and there was no statistically significant relation between ADAM-28 and cytogenetic risk.

In the study of Zhang et al. (2019), it demonstrated that patients with high ADAM28 expression levels had a significantly higher cumulative incidence of relapse in the favorable and intermediate risk group which might be useful in identifying high risk of relapse in favorable risk group.

Our study shows that there was high statistically significant difference between the studied groups as regard ADAM-28m and ADAM-28s as it demonstrates that AML cases group has mean of ADAM-28m= 2.08 (±0.18 SD) and mean of ADAM-28s =290.94 (±19.36 SD). While in control healthy group has mean of ADAM-28m= 0.4 (± 0.06 SD) and mean of ADAM-28s =119.52 (±12.49 SD).

Our study shows that the mean serum level of ADAM28 in the patients who didn’t respond to chemotherapy was higher than that of the patients that responded to chemotherapy as the study shows that in non-responder group, mean of ADAM28m 2.15 (± 0.18 SD) and mean of ADAM-28s 298.79 (±20.75 SD) While in responder group, mean of ADAM-28m 2.02 (± 0.15 SD) and mean of ADAM-28s 284.94 (±16.37 SD)..

The study of Zhang et al. (2019) found that the expression levels of ADAM28 were upregulated in de novo AML patients and differed significantly between patients suffering a relapse and those remaining in CR. These data suggested that ADAM28 levels might be related to the incidence of relapse in patients with AML.

In addition, ADAM28 overexpression was associated with lower probabilities of relapse-free (RFS) suggesting that ADAM28 may serve as a prognostic factor in B-ALL (Zhang et al., 2015).

Our study shows that there was statistically significant relation between mortality and ADAM28 m and ADAM-28s as in mortality cases, mean of ADAM-28m 2.25 (± 0.16SD) and mean of ADAM-28s 309.12 (±18.35 SD).While in Alive patients, mean of ADAM-28m 2.03 (± 0.16 SD) and mean

of ADAM-28s 286.40 (±17.08 SD)..

CONCLUSION

There was high statistically significant difference between the studied groups as regard ADAM-28m and ADAM-28s. There was significant correlation between ADAM-28 and the response to chemotherapy in the induction phase. ADAM28 improved the growth and dissemination of AML.

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