Adam-28 Overexpression in All and Its Relation to CNS Involvement in Adult Egyptian Patients

INTRODUCTION

Acute lymphoblastic leukemia (acute lymphocytic leukemia, ALL) is a malignant (clonal) disease of the bone marrow in which early lymphoid precursors proliferate and replace the normal hematopoietic cells of the marrow. ALL is the most common type of cancer and leukemia in children in the United States. (reference)

The malignant cells of acute lymphoblastic leukemia (ALL) are lymphoid precursor cells (ie, lymphoblasts) that are arrested in an early stage of development. This arrest is caused by an abnormal expression of genes, often as a result of chromosomal translocations or abnormalities of chromosome number. (reference)

A review of the genetics, cell biology, immunology, and epidemiology of childhood leukemia by Greaves concluded that B-cell precursor acute lymphoblastic leukemia (ALL) has a multifactorial etiology. The first step occurs in utero, when fusion gene formation or hyperdiploidy generates a covert, pre-leukemic clone. The second step is the postnatal acquisition of secondary genetic changes that drive conversion to overt leukemia. (reference)

A lot of studies have shown increased risk of ALL in association with polymorphisms of the following: Arylamine N-acetyltransferases 1 and 2, MMP-8 promoter genotypes, HLA alleles, ARID5B, IKZF1, CEBPE, CDKN2A, PIP4K2A, LHPP, ELK3

 ALL cells that had entered the CNS and were infiltrating the meninges were characterized by high expression of vascular endothelial growth factor A (VEGF). Although cellular viability, growth, proliferation, and survival of ALL cells were found to be independent of VEGF, transendothelial migration through CNS microvascular endothelial cells was regulated by VEGF. (reference)

The importance of VEGF produced by ALL cells in mediating leukemia-cell entry into the CNS and leptomeningeal infiltration was further demonstrated by specific reduction of CNS leukemia on in vivo VEGF capture by the anti-VEGF antibody bevacizumab. Thus, we identified a mechanism of ALL-cell entry into the CNS, which by targeting VEGF signaling may serve as a novel strategy to control CNS leukemia in patients replacing conventional CNS-toxic treatment.(3)

ADAM-28 This gene encodes a member of the ADAM (a disintegrin and metalloprotease domain) family. Members of this family are membrane-anchored proteins structurally related to snake venom disintegrins, and have been implicated in a variety of biological processes involving cell-cell and cell-matrix interactions, including fertilization, muscle development, and neurogenesis. The protein encoded by this gene is a lymphocyte-expressed ADAM protein. This gene is present in a gene cluster with other members of the ADAM family on chromosome 8. Alternative splicing results in multiple transcript variants. Biased expression in stomach (RPKM 21.4), gall bladder (RPKM 11.1) and 12 other tissue.(1)

ADAM-28 in Neurological Disorders .Although normally at low levels, ADAM is expressed in multiple cell types in the central nervous system (CNS), in particular in neurons, oligodendrocytes, astrocytes, microglia and endothelial cells. As CNS relevant substrates, ‘close homologue of L1’ (CHL1), a neuronal cell adhesion molecule and amyloid precursor protein (APP) have been described. (reference)

Other substrates of ADAM8 in the CNS are not CNS-specific, but contribute substantially to CNS pathology. In the inflamed or injured CNS, ADAM shows a notable increase in reactive glia or endothelial cells, detected either under conditions of neurodegeneration or spinal cord injury, respectively. (reference)

In neurodegeneration, ADAM has a protective role by shedding of the TNF-α receptor 1 (TNF-R1), slowing down the progression of motor neuron death. The role of ADAM in spinal cord injury remains elusive; however, the co-localization of ADAM with the endothelial marker CD31 (PECAM) implies a function in angiogenesis. (reference)

By shedding of CNS-specific substrates CHL1 and APP abbreviation of what ? , ADAM8 could contribute to regeneration processes in the CNS. Apart from this positive role, the progression of autoimmune diseases in the CNS is negatively affected by ADAM. In an animal model for neuroinflammation, the administration of a recombinant ADAM disintegrin domain had a suppressive effect on disease progression, most likely due to inhibition of ADAM in the CNS, demonstrating a beneficial effect of ADAM inhibition in the inflamed CNS. (reference)

Recent studies in numerous cancer types have revealed upregulation of ADAM which in some cases is correlated with increased malignancy and bad prognosis of cancer patients. According to Genevestigator (‘Microarray data’), upregulation of ADAM is highest in leukemias, in adenocarcinomas of pancreas (PDAC) and lung when compared to transcript levels in normal matching tissues. (reference)

 In addition, brain tumors (glioblastomas), renal cell carcinomas and prostate cancer show high expression levels of ADAM8 (listed in Koller et al.). In lung cancer, ADAM was detected as a soluble form in patient serological samples and was proposed as a marker for early stage diagnosis of lung cancer. (reference)

In PDAC cell lines, increased ADAM levels were shown to mediate invasiveness of tumor cells. Silencing of ADAM by siRNA reduced invasiveness of tumor cells but not proliferation, suggesting that ADAM is an invasion promoting factor rather than a growth promoting one. ADAM was also described as a marker for metastasis: a novel splice form of ADAM contributes to the aggressive bone metastatic phenotype of lung cancer. In addition to invasion-promoting properties of ADAM8 by proteolysis of ECM molecules, effects on cancer cells due to cleavage of TNF-R1 can also be considered. It is likely that TNF-R1 shedding directly inhibits TNF-α stimulated apoptosis of cancer cells, so that tumor cells overexpressing ADAM confer protection against immune-regulated apoptosis, a tumor-induced process mainly regulated by tumor-associated macrophages. (reference)

Several peptidomimetics were constructed tos bind the integrin-binding loop of ADAM and a cyclic peptide mimicking the integrin-binding loop was found to be effective in inhibiting ADAM8 activation in vivo. This peptide shows high specificity towards ADAM and can serve as a ‘gold standard’ inhibitor for the development of ADAM8 inhibiting drugs (‘ADAM patent’). Together with the finding that ADAM is dispensible under physiological conditions, specific inhibition of ADAM in diseases is not expected to have severe side effects.(2) (reference)

AIM OF THE WORK

The aim of the present study is to compare the ALL with CNS infiltration and without CNS infiltration and ADAM-28 overexpression in ALL and its relation to CNS involvement in adult Egyptian patients.

PATIENTS AND METHODS

Type of the study: Prospective study. Study setting: the study will be conducted on patients diagnosed as Acute Lymphocytic Leukemia ALL attending hematology  department of Ain Shams University. Study period: Six months. Study population:50 patient (25  patient with ALL and CNS infiltration , 25  patient with ALL and  had No CNS infiltration)

Inclusion criteria: Patients aged 16 years old and more less than 60 years old Patients with Acute Lymphocytic Leukemia ALL.

Exclusion criteria: Patients aged less than 16 years old and more than 60 years old and Patients with other malignancy.

Sampling method: 25  patient with ALL and CNS infiltration , 25  patient with ALL and  had No CNS infiltration will be recruited from Clinical Hematology and Oncology Division, Internal Medicine Department, Ain Shams University

Sample size: 50 participants. Ethical considerations: Informed written consent will be obtained from all patients participating in the study. The Study is approved by Ethical Committee Board of Ain Shams University and in accordance with Declaration of Helsinki.

Study tools: All patients will be subjected to all of the following at time of recruitment:

Full history taking and Clinical examination, Laboratory investigations including;

Complete blood count with differential, reticulocyte percentage, Kidney and Liver function test, Disease-specific labs:-Bone marrow aspiration, flowcytometry and cytogenetic study and CSF examination

Measurement of ADAM-28 level in serum using ELISA, Radiological finding (MRI, CT Brain, PETSCAN)

Statistical Analysis: Proper statistical analysis that deemed to be necessary will be carried out.

RESULTS

Table 1: Comparison between patients group and control group regarding BMI of the studied patients Immunophenotype, karyo type and risk groups distribution among the studied patients

Table 2: Comparison between patients group and control group regarding laboratory data

P-value >0.05: Non significant (NS); P-value

•: Independent t-test; ‡: Mann Whitney test

Table 3: Comparison between patients group and control group regarding ADAM-28m and ADAM-28s

P-value >0.05: Non significant (NS); P-value

•: Independent t-test

Table 4: Kaplan-Meier analysis for the comparison between patients group and control group regarding overall survival (months)

Table 5: ROC curve of ADAM between patients group and control group

Correlation between ADAM-28m and the other studied parameters

Table 6: Relation between ADAM-28m and the other studied parameters

P-value >0.05: Non significant (NS); P-value

•: Independent t-test; ••: One Way ANOVA test

Table 7: Distribution of cases group according to follow-up outcome

P-value >0.05: Non significant (NS); P-value

*:Chi-square test

Table 8: Correlation between MRD and findings in ADAM-28

DISCUSSION

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

The aim of the present study is to evaluate ADAM-28 expression in adult Egyptian patients with either denovo or relapsed ALL with or without CNS infiltration.

In the present study we found that there was no statistically significant difference between the studied groups as regards age and sex.

In agreement with our results, Reman et al. (2008) showed thatthere was no statistically significant difference between the studied groups as regards age and sex. There were 947 males and 546 females with amedian age of 32 years (range, 15–60 years).

In contrast to our results, Del Principe et al. (2021) showed that there was an equal male/female ratio among studied groups.

In our study we found that there was high statistically significant difference between the studied groups as regard BMI.

In contrast to our findings, Liu et al. (2021) showed thatthere was no statistically significant difference between the studied groups as regard BMI.

In agreement with our results, Atkinson et al. (2015) reported thatthere was statistically significant difference between the studied groups as regard BMI.

In the current study we found that according to Immunophenotype there were 6 (30%) pro-B-ALL, 13 (65%) c-ALL and 1 (5%) pre-B-ALL, according to Karyotype there were 5 (25%)  BCR-ABL, 1 (5%) hypodiploidy, 2 (10%) complex karyotype, 3 (15%) MLL-translocation and 9 (45%) other karyotypes and according to risk group there were 12 (60%) with poor risk and 8 (40%) with standard risk.

Reman et al. (2008) Showed that there was also some difference in the proportion within the different phenotype groups: T-lineage ALL, 41% versus 25%, Blineage ALL, 51% versus 69%. Patients with CNS disease were less likeky to present with Ph chromosome (8% versus 18%).

In the present study we found that there was high statistically significant difference between the studied groups as regard Hb, WBCs, Plts, PT, blast and ESR.

Reman et al. (2008) was in agreement with our results as regard Hb, there was statistically significant difference between the studied groups as regard Hb (P=0.001) and was in contrast to our results as regards WBCs and platelets, there was no statistically significant difference between the studied groups as regards WBCs and platelets.

In contrast to our results, Del Principe et al. (2021) showed that there was no statistically significant difference between the studied groups as regards WBCs. 

Sharma et al. (2016) showed thathematological parameters showed hemoglobin (range 2.3-15.8 g/dl; median 7.5 g/dl), white blood cell count of (range 1.0-436.0 × 109 /L; median 10.3 × 109 /L) and platelet count of (range 2.0-508.0 × 109 /L; median 28.0 × 109 /L).

In our study we found that there was no statistically significant difference between the studied groups as regard Liver and kidney function tests. Also we found that there was no viral infection detected.

In agreement with our results Reman et al. (2008) showed that there was no statistically significant difference between the studied groups as regards hepatomegaly and splenomegaly.

In contrast to our results, Pavlovsky et al. showed that the incidence of CNS leukemia in patients with initial organomegaly (liver, spleen and/or lymph node enlargement present at time of diagnosis) was higher (46%) compared with those without initial organomegaly (25%) at 20 mo, respectively: confrontation with these two curves showed a significant difference (p < 0.05).

In agreement with our results Zhang et al. (2019) reported that level in cerebrospinal fluid (CSF) of patients with central nervous system leukemia (CNSL) was significantly higher than that without CNSL (2.87±0.81 vs 0.50±0.08, p<0.001). These data suggested the up-regulated expression of ADAM28 might be correlated with relapse.

Zhang et al. (2015) showed that the data strongly suggest that in leukemia cells, ADAM28 is involved in the degradation of IGFBP-3 and IGF-I-induced proliferation in the leukemic cells. The overexpression of ADAM28 is implicated in the poor prognosis of many cancers. Our previous study revealed that ADAM28 expression in B-ALL patients was significantly increased. Patients experiencing disease relapse exhibited significantly increased ADAM28 expression compared with those with favorable outcomes. In addition, ADAM28 overexpression was associated with lower probabilities of relapse-free (RFS) and EFS, suggesting that ADAM28 may serve as a prognostic factor in B-ALL.

In our study we found that according to CNS infiltration there were 3 (15%) with CNS infiltration.

Similar results were reported by Reman et al. (2008) who reported that 109 patients (15%) presented a CNS relapse.

Sancho et al. (2006) showed that the main result of the current study consisted of a similar frequency (5.8%) of CNS recurrence (isolated or  combined) compared with other studies in which CNS prophylaxis included cranial radiotherapy.

In the current study we found that in the follow-up there were 15 (75%) were alive and 5 (25%) were dead.

With the limitations of its small sample size, the results of our study demonstrate that in adult patients with ALL, FCM allows to more precisely identify and quantify the number of patients with a CNS involvement at diagnosis and that this impacts significantly on the clinical course and outcome of the disease, thus enabling a further refinement of the current diagnostic risk stratification process. This refined CNS evaluation should become a routine tool for the work-up of ALL patients at presentation. Further and larger prospective studies are needed to further standardize the procedures and permit an optimal clinical application of this technique.

CONCLUSION

We conclude that adult patients with ALL who develop CNS recurrence have a very poor prognosis. ADAM28 expression levels also identified a new subgroup at a higher risk for relapse and with a poor prognosis in the favorable-risk patients, Although the disease in the CSF frequently responds to IT chemotherapy, most patients eventually develop a systemic recurrence and succumb to the disease. Effective CNS prophylaxis remains the best strategy for potential long-term survival. Prospective studies looking at better strategies to manage patients with this complication are needed.