The Possible Association of Some Long-Non Coding RNAs & ABO Blood Groups with Acute Lymphoblastic Leukaemia in Egyptian Children/ Safeya Abdelfattah Abdelrahman ; Supervisors: Dr. Hanan Muhammad Abdelgawa, Dr. Noha Hussein Sayed, Dr. Mahmoud Hammad Mahmoud.

Thesis (M.Sc.) -Cairo University, 2023.

Bibliography: pages 79-108.

Leukemia is the most frequent form of childhood malignancy, accounting for
30% of cases of childhood cancer. Although there are some associations with host
and environmental factors, most pediatric leukemia cases are idiopathic.
In children, the most common type of leukemia is acute lymphoblastic
leukemia (ALL). ALL affects the immature forms of white blood cells, called
lymphoblasts. B-lymphocytes and T-lymphocytes are the two basic types of
lymphocytes, and their immature forms are the source of the two corresponding
subsets of ALL, T-ALL and B- or pre-B ALL.
Long non-coding RNAs (LncRNAs) regulate several biological processes
including differentiation, development and biogenesis. Many human illnesses,
including some cancers, are associated with lncRNA dysregulation.
LncRNA ANRIL, located in the human chromosome 9p21 region, has been
reported to be involved in tumor progression. ANRIL has been found to regulate
gene expression via recruiting polycomb repressor complex (PRC) 1 and 2 or
titrating miRNA; it also participates in signaling pathways. ANRIL was reported to
be overexpressed in many cancer types and is capable of promoting cell
proliferation and cell cycle progression, and inhibiting apoptosis and senescence.
Furthermore, ANRIL has the potential to serve as a biomarker for diagnosis and
prognosis of several cancers.
CDKN2A, located on the same locus as does ANRIL, is frequently
inactivated or deleted in multiple hematological malignancies and this is a frequent
event in childhood B- and T-lineage ALL.
LncRNA LINC-PINT plays a critical role in many diseases, especially
cancer. In most human tumors, LINC-PINT is down-regulated where it serves as a
tumor suppressor.
HMOX1 is a LINC-PINT associated gene. Its activity prevents the oxidative
stress and inflammation of cells. The cytoprotective effects of HMOX1 are exerted
by itself or mediated by its products.
Different studies have reported conflicting results on the differential
distribution of blood groups in acute leukemia patients.
The current study aimed to investigate the possible association between the
two lncRNAs; ANRIL and LINC-PINT and their downstream targets - CDKN2A
and HMOX1, respectively - with ALL in children. The differential distribution of
ABO blood groups among ALL patients was also studied. This work was further
extended to elucidate the possible correlation of ANRIL, LINC-PINT, CDKN2A,
HMOX1 and ABO blood groups with the patients‟ clinicopathological data. The
Summary and Conclusion
76
novelty of this study was investigating the impact of initial treatment till the end of
remission induction phase on the studied parameters.
This case-control study was carried out on 66 Egyptian pediatric patients
newly diagnosed with ALL with range of age 1-17 years. A group of 39 age- and
sex-matched children was recruited as a control group.
Patients were treated at the National Cancer Institute-Cairo University and
Dar El-Salam hospital. Patients who died before end of remission induction, had
relapsing ALL, had other types of leukemia, were under steroid for any other
disease, or those with inadequate samples were excluded. All children were
recruited during the period from December 2020 to March 2022.
Each participant‟s guardian provided written consent at enrollment after
discussing the experimental procedure and the beneficial expectancies. The study
protocol and all procedures performed were approved by the Research Ethics
Committee for experimental and clinical studies at the Faculty of Pharmacy, Cairo
University, Cairo, Egypt (approval number: BC 2458) and conformed to the 1975
Helsinki declaration, revised in 2008.
Peripheral blood samples were collected from all patients at their routine time
of sampling at the initial investigation, another sample was collected at the end of
remission induction phase (43-46 days from diagnosis). Similarly, blood samples
were taken from controls. Each blood sample was divided into 3 aliquots. One
aliquot was used for blood group typing, the second one was used for total RNA
extraction for measuring of ANRIL, CDKN2A and LINC-PINT expression levels
using quantitative real time PCR (RT-qPCR), and a third aliquot was used for
serum separation to measure protein level of HMOX1 by Human HMOX1 ELISA
Kit.
Results of the current study showed a significant upregulation of lncRNA
ANRIL and CDKN2A plasma expression levels in the newly diagnosed patients
compared to controls. However, both demonstrated a downturn at the end of
remission induction that did not reach significant levels as compared to their levels
at diagnosis.
Moreover, newly diagnosed ALL patients showed a dramatic downregulation
of LINC-PINT expression and HMOX1 protein level compared to control levels. In
addition, both showed significant increase at the end of remission induction as
compared to admission levels.
Furthermore, significant positive correlations were found between ANRIL
and CDKN2A and between LINC-PINT and HMOX1.
Summary and Conclusion
77
There was a significant positive association between ANRIL with both risk
stratification and MRD at the end of remission induction phase. Surprisingly,
LINC-PINT showed a positive significant association with CNS status as well as
MRD at the end of remission induction, while HMOX1 was positively associated
with risk stratification and IPT. Of note, no significant association was found
between ANRIL, LINC-PINT or HMOX1 with other data including age, sex or
TLC. Regarding CDKN2A, no significant association was observed with any of the
ALL clinicopathological features. It was only associated with sex, showing higher
expression levels among boys.
ROC curve analysis showed good diagnostic performance for LINC-PINT
followed by CDKN2A then ANRIL, while an excellent diagnostic performance
was observed for HMOX1.
Logistic regression analysis indicated that LINC-PINT and HMOX1 were
significant predictors in the univariate analysis. In a stepwise forward multivariate
analysis, HMOX1 turned out to be significant independent variable
Concerning blood group distribution in ALL patients, notably, blood group A
was the most observed blood group (36.36%). However, no significant difference
was found between controls and ALL patients regarding the distribution of any of
the ABO blood groups. Similarly, there was no significant association between
ABO blood groups and the patients‟ clinicopathological data.
Finally, we can conclude that:
1. ANRIL, CDKN2A, LINC-PINT and HMOX1 might be associated with the
susceptibility of ALL in pediatric patients.
2. ANRIL and LINC-PINT were associated with poor response of patients to
treatment.
3. There was a significant positive association between ANRIL and HMOX1
with risk stratification. The later was also linked to IPT, while LINC-PINT
showed a positive significant association with CNS status.
4. The initial treatment brought ANRIL to a level approaching control‟s level
and moderated the levels of both LINC-PINT and HMOX1.
5. The study parameters had remarkable diagnostic potential in discriminating
ALL patients, where HMOX1 had superior diagnostic power and it was the
only one demonstrating as significant independent predictor of ALL.
Summary and Conclusion
78
6. There was no considerable difference in ABO blood group distribution
between patients and controls, nor were there any associations between
blood groups and patients‟ clinicopathological data.
Lastly, this study has some limitations. To begin with, patients in the current
study were only recruited from National Cancer Institute-Cairo University and Dar
El-Salam hospital, which necessitates comparable multi-centered studies to obtain
more data and compare outcomes. Furthermore, the impact of COVID-19
pandemic affected our ability to collect more samples as it was the timeframe of the
study. Future studies with larger sample size need to be conducted to replicate the
current results.

يعد سرطان الدم الشكل الأكثر شيوعًا للأورام الخبيثة في مرحلة الطفولة، حيث يمثل۳۰ ٪ من حالات سرطان الأطفال. على الرغم من وجود بعض الارتباط بين العوامل الذاتية والبيئية مع المرض، إلا أن معظم حالات سرطان الدم لدى الأطفال مجهولة السبب.
يعتبر سرطان الدم الليمفاوي الحاد هو النوع الأكثر انتشارًا لسرطان الدم في الأطفال. يؤثر سرطان الدم الليمفاوي الحاد على التكوينات غير الناضجة لخلايا الدم البيضاء التي تسمى الخلايا الليمفاوية. وثمة نوعان من الخلايا الليمفاوية: الخلايا الليمفاوية البائية والخلايا الليمفاوية التائية.وقد ينشأ سرطان الدم الليمفاوي الحاد من أي من نوعي الخلايا الليمفاوية، ولذلك فإن حالات سرطان الدم الليمفاوي الحاد تعرف بأنها سرطان الدم الليمفاوي الحاد للخلية باء (B-ALL) أو الخلية تاء(T-ALL) .
تنظم الأحماض النووية الريبوزية الطويلة الغير مشفرة العديد من العمليات البيولوجية بما في ذلك تميز وانقسام الخلايا والتكوين البيولوجي لها. ترتبط العديد من الأمراض البشرية، بما في ذلك بعض أنواع السرطان، بخلل في مستويات الأحماض النووية الريبوزية الطويلة الغير مشفرة.
ومن الأحماض التي تم دراسة دورها في هذا البحثANRIL والموجود في منطقة الكروموسوم البشري 9p21، وقد تم اثبات صلته بتطور الأورام حيث يستطيع ANRILتنظيم التعبير الجيني عن طريق ارتباطه بوحدتي بروتين الـpolycomb أو عن طريق التأثير على الأحماض النووية الريبوزية متناهية الصغر(miRNA) كما يشارك في مسارات الاشارات. وقد وجد أن مستوى ANRILيزيد في العديد من أنواع السرطان، وانه قادر على تعزيز تكاثر وتطور دورة الخلايا، وتثبيط موت الخلايا المبرمج والشيخوخة. علاوة على ذلك، فإن ANRIL لديه القدرة على العمل كمؤشر حيوي لتشخيص العديد من أنواع السرطان.
يقع CDKN2A في نفس الموقع الجيني للـANRIL و غالبا ما يتم تثبيطه أو حذفه في العديد من أورام الدم الخبيثة، كما يشيع ذلك في سرطان الدم الليمفاوي الحاد بنوعيه لدى الاطفال.
يلعب الحمض الريبوزي النووي الطويل الغير مشفرLINC-PINT دورًا حاسمًا في العديد من الأمراض، وخاصة السرطان. إذ يقل مستوى LINC-PINT في معظم الأورام حيث يعمل كمثبط للورم.
يرتبط جين HMOX1 بـ LINC-PINT، و يمنع الاجهاد التأكسدي والتهاب الخلايا عندما يكون نشطًا. يرجع تأثير HMOX1 في حمايته للخلايا إلى تأثيره المباشر أو بواسطة منتجاته.
أثبتت الدراسات المختلفة نتائج متضاربة حول التباين في توزيع فصائل الدم لدى مرضى سرطان الدم الحاد.

Issues also as CD.

Text in English and abstract in Arabic & English.