INTRODUCTION

Gamma secretases are intramembranous multisubunit protease complexes that are composed of four core components (presenilin, nicastnin, presenilin enhancer 2[Pen2] and anterior pharynx-defective 1 (Aph1), and several associated proteins(De Strooper and Annaert,2010).

Many of physiological and pathological activities of gamma secretaseis derived from its activity as a protease against NOTCH, a central molecule in the control of growth and differentiation (De strooper et al.,1999).

NOTCH activation plays an important role in the genesis of T cell acute lymphoblastic leukemia (Weng et al.,2004).

In contrast, the role ofNOTCH activation in B cell malignancies is not clear (Chiaramnonte et al.,2003).

Due to the central role of gamma secretase in these malignancies, considerable efforts have been made to characterize this unique protease,however, to the best of our knowledge, all the studies were directed to an exo cell assay of gamma secretase expression and activity and even in vitro inhibition of gamma secretase activity in some cell lines of B cell malignancies (Shelton et al.,2009; Ramakrishnanet al.,2012;and Secchiero et al.,2017).

So, modulation or inhibition of gamma secretase activity and hence altered signal pathways, could be a light path as a line of therapy against various types of tumor cells (Selkoe and Walfe,2007;Shih and Wang,2007;and Groth and Fortini,2012).

chronic lymphocytic leukaemia (CLL), the most frequent type of leukaemia in adults, is a lymphoproliferative disorder that is characterized by the expansion of monoclonal, mature CD5⁺CD23⁺ B cells in the peripheral blood, secondary lymphoid tissues and bone marrow. CLL is an incurable disease with a heterogeneous clinical course, for which the treatment decision still relies on conventional parameters (such as clinical stage and lymphocyte doubling time)(Bosch and Dalla-Favera, 2019).

METHODOLOGY:

• Type of Study : cross sectional study
• Study Setting: Internal medicine department, clinical hematology inpatient unit and outpatient department (OPD),Ain Shams University Hospitals.
• Study Time: between 2020-2021
• Study Population 30 patients with chronic lymphocytic leukemia
• Inclusion criteria:

Patients with chronic lymphocytic leukemia at different status (new,relapsed and resistant cases),above 50 years old.

• Exclusion criteria

Patients with other associated malignancies

• Sample Size :30 cases, according to recommendations of community department

An explaoratory study will be carried out upon at least 30 patients with chronic lymphocytic leukemia to evaluate the in vivo expression of gamma secretase catalytic subunit (presenilin) in among them.

• Ethical Considerations :

Written informed consent will be obtained from all patients before participation. Blood samples will be collected once (for real time PCR).

• Study Procedures :

All patients was subjected to following:

1)     Full history

2)     Clinical examination

3)     Laboratory investigations :

-        CBC with differential count

-        Corrected reticulocyte count

-        Coomb's test direct and indirect

-        Viral markers : hepatitis b and c

-        Complete renal functions

-        Complete liver functions, LDH

-        Bone Marrow Aspirate (if possible)

-        Immunophenotyping on either bone marrowor peripheral blood by flowcytometry

-        Convensional cytogenetic study (when available )

-        Fish for 17p deletion and immunological heavy chain mutation (if possible)

4)     Imaging for patients with:

Ultrasound or CT scan

5)     Real Time PCR for Presenilin subunit of gamma secretase enzyme on either Bone marrow or peripheral blood samples.

A control group consisting of 12 age and sex matched patients were also included in the study for comparison.

Operational design: The researcher introduced himself to all participants included in this study and asked them to participate after illustrating the goal of the study.

All selected participants received comprehensive information regarding objective and the expected benefit of the study. All ethical considerations were taken throughout the whole work.

A Pilot study was carried out on (10% of study sample) to test feasibility, applicability and clarity of methods.

Statistical analysis:

Analysis of data was done usingStatistical Package for Social Science (IBM SPSS) version 23. The quantitative data were presented as mean, standard deviations and ranges when parametric and median, inter-quartile range (IQR) when data found non-parametric. Also qualitative variables were presented as number and percentages. The comparison between groups with qualitative data were done by using Chi-square test. The comparison between two groups with quantitative data and parametric distribution were done by using Independent t-test. ; While the comparison between two groups with quantitative data and non parametric distribution was done by using Mann-Whitney test. The comparison between more than two groups with quantitative data and parametric distribution were done by using One Way ANOVA test. ; While the comparison between more than two groups with quantitative data and non parametric distribution was done by using Kruskall Wallis test.Spearman correlation coefficients were used to assess the correlation between two quantitative parameters in the same group. Receiver operating characteristic curve (ROC) was used in the quantitative form to determine best cut of point, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and Area under curve (AUC.P ≤ 0.05:is consideredSignificant.

Approvales:An informed verbal consent from all participants was taken and confidentiality of information was assured, an official written administrative permission letter was obtained from dean of faculty of medicine, Ain Shams university hospital. The title and objectives of the study were explained to them to ensure their cooperation,

Ethical committee:Permission from the faculty of medicine ethical committee was also obtained.

Cost of the study: The study was totally afforded by the researcher.

Limitations:no limitations.

RESULTS:

Table(1): Comparison between the patient group and the control group using Chi -Square test as regard Age and sex:

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

*:Chi-square test; •: Independent t-test

There was no stastically significance between control group and patient group as regard age and sex.

Table(2):The clinical characteristics of CLL patients included in the study:

Table(3): The laboratory characteristics of CLL patients included in the study:

Table(4):The Qualitative data of CLL patients included in the study :

Table(5):Comparison between the patient and the control groups as regard the expression of gamma secretase catalytic subunit (presenilin) using Mann-Whitney test:

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

•: Mann-Whitney test.

Figure(1):Showingthat expression of presenilin was higher in cll patients in than control group

There was high statistically significant difference between the patient and control groups as regard the gamma secretase gene catalytic subunit presenilinexpression.

Table(6):Correlation between laboratory characteristics of Cll patients and gamma secretase catalytic subunit presenilingene expression

Spearman correlation coefficients

There was no statistically significance correlationbetween ageof Cll patients and gamma secretase catalytic subunit presenilingene expression.

There was no statistically significance correlationbetween CBC parameters or LDH of Cll patients and gamma secretase catalytic subunit presenilingene expression.

Table(7): Correlation between expression of gamma secretase catalytic subunit presenilin withthe major characteristics of CLL patients included in the study:

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

‡: Mann Whitney test; ‡‡: Kruskal Wallis test

There was no statistically significant correlation between the gamma secretase catalytic subunit presenilinexpression with sex, stage of the disease, bone marrow cellularity,presence of atypical flow pattern.

There was high statistically significant correlation between the gamma secretase catalytic subunit presenilin expression with type of patients being higher in relapsed and resistant cases than newly diagnosed.

Figure(2):Showing that gamma secretase catalytic subunit presenilin gene expression was higher in relapsed and resistant group in comparision to newly diagnosed group

Table(8): Comparison between control and different types of CLL patients included in the study as regard the gamma secretase catalytic subunit presenilin expression usingKruskal Wallis test:

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

‡‡: Kruskal Wallis test

There was high statistically significant correlation of the Normalized target gene expression being higher in resistant - relapsed group > newly diagnosed> control group

Figure(3):Showing that the gamma secretase catalytic subunit presenilin expression was highest in resistant relapsed group then newly diagnosed cll patients then control group

DISCUSSION:

Gamma secretases are intramembranous multisubunit protease complexes that are composed of four core components (presenilin, nicastnin, presenilin enhancer 2[Pen2] and anterior pharynx-defective 1 (Aph1), and several associated proteins (De Strooper and Annaert,2010).

Gamma secretase was primarily identified in Alzehimer's disease.as it cleaves amyloid precursor protein (APP)(Laguarta and Pera, 2010).

Gamma secretase may influence on tumorigensis also via its role in angiogenesis as many of substrates are shown to regulate the formation and development of new blood vessels (Boulton et al.,2008).

Many of physiological and pathological activities of gamma secretaseis derived from its activity as a protease against NOTCH, a central molecule in the control of growth and differentiation (De strooper et al.,1999).

NOTCH activation plays an important role in the genesis of T cell acute lymphoblastic leukemia (Weng et al.,2004).

In contrast, the role ofNOTCH activation in B cell malignancies is not clear (Chiaramnonte et al.,2003).

There are several reports of NOTCH activation in Hodgkin's lymphoma, multiple myeloma and chronic B cell lymphocytic leukemia (Jundt et al.,2002;Nefedova et al.;2008and Rosati et al.,2009).

Due to the central role of gamma secretase in these malignancies, considerable efforts have been made to characterize this unique protease,however, to the best of our knowledge, all the studies were directed to an exo cell assay of gamma secretase expression and activity and even in vitro inhibition of gamma secretase activity in some cell lines of B cell malignancies (Shelton et al.,2009; Ramakrishnanet al.,2012;and Secchiero et al.,2017).

Gamma secretase inhibitors (GSIs) make up a new class of drugs targeting the Notch signal transduction pathway that are currently in clinical trials for a variety of malignancies and Alzheimer’s disease. The target of GSIs, .-secretase, is a large multi-protein transmembrane complex composed of presenilin, Aph1, Pen-2 and nicastrin(De Strooperet al., 1999).

Chronic lymphocytic leukaemia (CLL), the most frequent type of leukaemia in adults, is a lymphoproliferative disorder that is characterized by the expansion of monoclonal, mature CD5+CD23+ B cells in the peripheral blood, secondary lymphoid tissues and bone marrow. CLL is an incurable disease with a heterogeneous clinical course, for which the treatment decision still relies on conventional parameters (such as clinical stage and lymphocyte doubling time) (Bosch and Dalla-Favera, 2019).

Current studies, showed thatexpression of Notch2, Jagged1, and Jagged2 protein by Western blot in B-CLL cells that B-CLL cells but not normal B cells constitutively express Notch1 and Notch2 proteins as well as their ligands Jagged1 and Jagged2, suggesting that the expression of these proteins is a peculiar property of these malignant cells(Rosati et al., 2009).

To the best of our knowledge, this is the first report to evaluate theinvivo expression of presenilin gene in CLL patients usingrealtimepcr.

In our study, there was high statistically significant difference between the patient group and the control as regard presenilin gene expression. That was consistent with Shen and Yin(2020) who compared The expression of presenilin 1 (PS1) by Western blot in HCC cells and healthy liver cell lines showing that Expression of PS1 was increased in HCC tissue and several HCC cell lines.

In comparison toXinet al. (2011)who investigated the gene expression levels of Notch signal pathway members (Notch1, Jagged1 and Delta1) in bone marrow mononuclear cells by realtime quantitative PCR) expression was elevated in the bone marrow from patients with newly diagnosed AML, compared with normal controls.

There was high statistically significant difference between different types of Cll patients included in the study being higher in resistant- relapsed group in comparsion to newly diagnosed and that may be an important prognostic factor in Cll. That was consistent with Yun et al.(2019)experienced Expression of PSEN and other gamma secretase subunits in pancreatic cancer patients found that the survival rate of patients with pancreatic cancer with high PSEN1, APH1A, and PSENEN expression was low.

That was consistent withPaul et al. (2018)who experienced co-administration of the Notch inhibitor GSI-XII with the chemotherapeutic agent Ara-C In NOG-mouse-based xenograft models of B-ALL lowered bone marrow leukemic burden compared with DMSO or Ara-C alone, thus prolonging mouse survival.

In our study,there was no statistically significant correlation between the sex of the Cll patients and the presenilingene expression.

There was no statistically significant correlation between the patient Rai,Binet stage and the presenilin gene expression.

There was no statistically significant difference between the patients initial WBCs as regard the presenilin gene expression.

CONCLUSION:

Expression of presenilin gene in Cll patients is significantly elevated compared to healthy individuals.It has a prognostic value being higher in resistant –relapsed compared to newly diagnosed cases.