الفهرس 
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Abstract
Breast cancer is a heterogeneous disease that represents the most common cancer and the primary cause
of mortality due to cancer in females around the World. It is either early localized breast cancer or metastatic
cancer that can commonly transfer to distant organs such as the bone, liver, lung, and brain, which mainly
accounts for its incurability. BC usually starts from ductal hyperproliferation and then develops into benign
tumors or even malignant carcinomas after constant stimulation by various modifiable and non-modifiable risk
factors. Primary and metastatic BC tumors are complex ecosystems composed of neoplastic cells, extracellular
matrix (ECM), and “accessory” nonneoplastic cells. The crosstalk between cancer cells and accessory cells fuels
and shapes tumor development. During tumor formation, the tissue architecture evolves into a highly specialized
microenvironment characterized by a corrupted ECM and chronic inflammation.
The treatment of BC has two categories. The first is local therapy, which is usually composed mainly of
surgical treatment (mastectomy, BCS, SLND) and radiation treatment. The second is systemic BC therapy,
represented by chemotherapy that is divided into adjuvant and neo-adjuvant therapy, as well as targeted therapy
like Her2-directed therapy intended for HER2-positive BC, in addition to endocrine therapy for HR-positive
patients and immunotherapy such as immune checkpoint inhibitors.
Immunotherapy has firmly established itself as a novel pillar of cancer care, from the metastatic stage to
the adjuvant and neoadjuvant settings in numerous cancer types. Its applications include monoclonal antibodies,
small-molecule drugs, adoptive cellular immunotherapy, and cancer vaccines. In some branches of adoptive
cellular immunotherapy, such as the TILs technique, IL-2 is used with lymphocytes, which has an unwanted
effect since IL-2 can inhibit T cell responses, maintaining T regulatory cells, in addition to vascular capillary
leak induction.
Under these circumstances, interleukin-15 (IL-15), which is a 14–15 kDa glycoprotein pleiotropic
cytokine with structural similarities to IL-2, has good immune surveillance against solid cancers. IL-15 promotes
the development, expansion, and homeostasis of several lymphocyte populations, including NK, NKT, and
CD8+ T cells, without IL-2 side effects and with much fewer quantities and great effectiveness. The IL-15 in
vitro activates stable telomerase expression, compensates telomere loss in memory phenotype CD8+ T cells, and
supports its homeostasis. IL-15Rα forms a high-affinity trimeric receptor with IL-2R β and γ c chains, allowing
the cells to respond to low concentrations of the IL-15 cytokine and increased the cellular growth of NK, NKT,
and CD8 T cells more effectively than IL-2 at low doses. And according to these characteristic’s the IL-15 has
the potential to applied in adoptive immune cell therapy for cancer.
Telomeres are DNA–protein structures located at the ends of eukaryotic chromosomes that protect
chromosome ends from degradation and fusion. The telomerase enzyme complex is a specialized reverse
transcriptase that extends the 3’ end of chromosomes by adding TTAGGG repeats that provide the stability of
chromosomes and genome integrity during replication. In the absence of telomerase, gradual shortening of
telomeres occurs with each cell division due to the end-replication problem that leads to replicative senescence
and then cell apoptosis. The two telomerase majority components that carry out the function of telomere repeat
addition are the telomerase reverse transcriptase (TERT) domain and Telomerase RNA component (TERC)
which complexes with TERT provides the template for telomeric sequence synthesis. The IL-15 potently stimulates TERT expression in NK and NKT cells in addition to CD8 T cells and is therefore a valuable tool for
adoptive cell therapies.
Effector NK cells, CD8 T cells and NKT-like cells have been shown to effectively kill tumor cells in
vivo when adoptively transferred. Both the systemic levels of effector NK, NKT-like and CD8 T cells and their
infiltration and activity in tumors correlates with patient survival in a number of cancer types. IL-2 and IL-15
have been identified as agents that can expand these cells in protocols where they are either injected systemically
or used to treat effector cells that are then adoptively transferred. Identifying the optimal doses and
understanding the mechanisms by which these two agents enhance effector cell proliferation is critical to their
successful use in patients.
Breast cancer (BC) cells employ telomerase activity induction and telomere length reduction to evade
usual senescence and apoptosis. A short relative telomere length is associated with an increased risk of breast
cancer, and the nuclear and cytoplasmic presence of hTERT in cancer cells may indicate a poor prognosis. On
the contrary, telomeres in BC patient leukocytes are elongated, serving as a protective factor, and the shortening
of these telomeres is linked to an increased risk of death. Leukocytes exhibit hTR/hTERT expression at a high
pace in BC patients, playing a role in the development, differentiation, and activation of lymphocytes, while
maintaining a healthy balance of expression in non-cancerous states.
In this research, our objective is to explore the impact of IL-15 on in-vitro cultured peripheral blood
mononuclear cells (PBMCs) in non-metastatic breast cancer (BC) patients. We aim to analyze hTERT outcomes
both prior to and following chemotherapy, comparing these hTERT levels across neoadjuvant, adjuvant, and
healthy control groups, and correspond to specific demographic, clinical, and pathological variables.
The present study was conducted on a total of 40 Egyptian non-metastatic breast cancer females
recruited from those referred to the Cancer Management and Research Department, Medical Research Institute,
as well as the Alexandria Clinical Oncology Department (ACOD), Main University Hospital, Faculty of
Medicine, Alexandria University. After thorough examination and following clinicopathological criteria, patients
were classified into 2 groups based on the designed (proposed, adopted) therapeutic guidelines: 20 adjuvant and
20 neoadjuvant chemotherapies. Blood sampling was performed for all patients in the heparin tube twice before
starting as well as 3 weeks after the first cycle of AC chemotherapy was received. Eight healthy females matched
for age were included in the study as negative controls. Then the isolation of human PBMCs by Ficoll
Histopaque for in vitro culture of human PBMCs with the recombinant human IL-15 on cell culture at 50 ng/ml
concentration for 1 week of culture 25 ng/mL was added on the first day, and 25 ng/mL was added on day four.
After that, for quantitative detection of hTERT in cell culture supernatant, an ELISA kit for hTERT detection
was used.
According to the distribution of BC patients receiving adjuvant and neoadjuvant chemotherapy have a
significant difference according to Diabetes Mellitus, since most (%95) of neoadjuvant patients are without DM
(P= 0.02), which also return on comorbidity (%85) in the neoadjuvant patients (P= 0.018). According to
clinicopathological data, (%90) of the adjuvant patients’ group have positive LVI (P<0.001), and (%80) of the
adjuvant patients’ group have positive PR (P= 0.022). Furthermore, there are no significant differences in the
distribution of patients between neoadjuvant and adjuvant breast cancer (BC) groups according to variables such
as age, menopausal status, family history, hypertension, obstetric history, and clinicopathological variables (stage,
diagnosis, T, N, M, type of tumor, grade, ER, HER-2). Additionally, there is no substantial difference in the
number of neoadjuvant and adjuvant patients because each of these groups represents 50% of all BC patients.
In the comparison between neoadjuvant and adjuvant groups, it is observed that adjuvant patients exhibit
a higher hTERT reading and, additionally, experience a substantial DROP in hTERT levels due to chemotherapy
(P=0.018). Conversely, IL-15 induces a non-significant increase in hTERT readings in the neoadjuvant group
(P=0.065). For all breast cancer (BC) patients, pre-chemotherapy spontaneous hTERT results are significantly
higher in those with lymphovascular invasion (LVI) (P=0.02), invasive lobular carcinoma (ILC) (P=0.025), and
progesterone receptor-positive (PR) tumors (P=0.022). Post-chemotherapy, hTERT results are significantly
higher for patients with two gravidities (P=0.023). Moreover, In vitro IL-15-induced hTERT results postchemotherapy show a significantly higher hTERT level for patients with twice or more abortions (P=0.04) and
without hypertension (HTN) (P=0.012).
The comparison of hTERT levels in all BC patients and healthy controls reveals that hTERT is
significantly higher in total BC patient lymphocytes under four conditions (Before chemotherapy without IL15,
Before chemotherapy with IL15, After chemotherapy with IL15, and After chemotherapy with IL15), with a
significant linear positive correlation between all hTERT values of these conditions (P<0.01). Also, the samples
of BC patients after chemotherapy with IL15 show that the hTERT amount correlates positively with abortion
numbers (P=0.046) and negatively with age (P=0.037).
The cultured samples of adjuvant patients have a higher hTERT reading than neoadjuvant patient’s
samples, with a significant difference between the medians of hTERT level among four sample conditions for
those adjuvant patients aged over 50 (P=0.017), in a postmenopausal state (P=0.017), and with comorbidities
(P=0.033) – specifically, diabetes (DM) (P=0.041) and hypertension (HTN) (P=0.042) – as well as those with
grade II tumors (G II) (P=0.019), exhibit the highest hTERT levels ”before chemotherapy/with IL-15” and the
lowest ”after chemotherapy/with IL-15.” Additionally, multiparous individuals (having more than one
pregnancy) show significant differences (P=0.028), as do those with positive lymph nodes (P=0.022), especially
those classified as cN1-cN2 (P=0.027), and those with lymphovascular invasion (LVI) (P=0.011), with the
highest hTERT readings occurring for the sample ”before chemotherapy and with IL-15,” while the lowest
readings manifest ”after chemotherapy without IL-15.” Furthermore, tumors at stage III (P=0.009) register the
highest hTERT levels ”before chemotherapy and without IL-15” and the lowest ”after chemotherapy without IL15.”
In contrast, the neoadjuvant group does not show any significant difference between the medians of
hTERT levels among the four sample conditions for any variable. in both the neoadjuvant and adjuvant groups,
there is no relation between the impact of chemotherapy and the influence of IL-15 on each other’s effects.
We propose a novel breast cancer immunotherapeutic strategy mediated by IL-15 through its application
in adoptive cell therapy. Furthermore, we have elucidated a significant impact of adjuvant chemotherapy and
specific patient characteristics on the activation of PBMCs with IL-15.