الفهرس 
TitleOnly 14 pages are availabe for public view
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Abstract
B cell lymphoma (BCL) were a group of distinct entities with widely varying clinical features, histology, immunophenotypes, and genetic abnormalities. This cancer began in certain immune system cells, and were classified as either Hodgkin lymphoma or non-Hodgkin lymphoma (NHL). The most common types of NHL were (follicular lymphoma, diffuse large B cell lymphoma, small lymphocytic lymphoma, mucosa-associated lymphoid tissue lymphoma and Mantle Cell Lymphoma (MCL)) they represented about 75% of non-Hodgkin’s lymphoma. Non-Hodgkin’s lymphoma (NHL) had much higher global incidence than HL with wider variations across the world.
In Egypt, lymphoma was known to be the fourth most prevalent adult tumor, accounting for 76.6% non-Hodgkin lymphoma (NHL) and 23.4% Hodgkin lymphoma (HL). The age-standardized incidence rates of the NHLs were (16.3/100 000 person) according to the Middle East Cancer Consortium in Egypt. This very high incidence made NHLs the third most common cancer in Egyptian men and the second most common cancer in women identified by the National Cancer Institute (NCI) representing 10.9% of all cancers diagnosed annually in Egypt.
The aim of this research was to investigate the expression of nuclear factor kappa (NF-κB), signal transduction and activator of transcription 6 (STAT6), forkhead box P3 (FOXP3) in B- cell non-Hodgkin’s and Hodgkin’s lymphoma using immunohistochemistry and image analysis trying to identify the possible molecular pathogenetic mechanisms contributing to their development and correlating the gene expression of these biomarkers with different clinicopathological parameters including stages, international prognostic index, extra nodal metastasis, bone marrow invasion, Deauville score, performance Scales: Karnofsky & ECOG Scores, response to therapy, relapse, progression, and survival outcome.
This study was a cohort comparative non-randomized retrospective and prospective study including 87 patients with pathologically proven B-cell non-Hodgkin and Hodgkin lymphoma with available blocks containing adequate material suitable for immunohistochemical studies, and available clinical data was followed for 2 years during the period from 2012 to 2017.
The patients were divided into 5 groups: The first group included 28 cases of Hodgkin lymphoma (HL), The second group included 24 cases of diffuse large B-cell non Hodgkin lymphoma (DLBCL), The third group included 14 cases of follicular B-cell non Hodgkin lymphoma (FL), the fourth group included 18 cases of small B-cell lymphocytic lymphoma (SLL/CLL) and the fifth group included 3 cases of mucosa associated lymphoid tissue (MALT).
All cases included in our study were subjected to: Histopathologic examination, immunohistochemical streptavidin biotin peroxidase staining procedure was used for (FOXP3, NFKB and STAT6), expression of immunohistochemical staining results was performed through the image analysis technique.
In our study, patients with elevated FOXP3-positive cell infiltration >67 in (DLBC, FL and HL) were associated with a significantly improved prognosis in early-stage patients
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with a good prognostic index 1 low risk, 2.3 low intermediate risk), low performance Scales: Karnofsky & ECOG Scores who achieved prolonged overall survival, responded to therapy with decreased risk of relapse, progression, extra nodal metastasis and bone marrow infiltration on the contrary in SLL/CLL increased expression of FOXP3 was associated with bad prognosis, shortened overall survival, late stages patients with bad prognostic index, high performance Scales: Karnofsky & ECOG Scores refractory to therpy with increased risk of replase, progression, extranodal metastasis and bone marrow infiltration while patients expressed low FOXP3-positive cell infiltration ≤67 were associated with bad prognosis, late stages patients with bad prognostic index (4,5 high intermediate risk, 6 high risk), shortened overall survival, refractory to therapy, increased risk of replase, progression, extra nodal metastasis and bone marrow infiltration. Significant negative correlation was the relationship between FOXP3 and stages (p= 0.028*) in HL, FOXP3 and stages(p= 0.003*)in NHL, FOXP3 and limited /advanced stages (p= 0.023*) in HL, limited /advanced stages and FOXP3 (p= 0.007*) in NHL, ECOG grades and FOXP3 (p= 0.012*) in HL, ECOG grades and FOXP3(p= 0.018*) in NHL, number of lymph nodes and FOXP3(p= 0.018*) in NHL, Deauville score and FOXP3 (p= 0.003*) in NHL but positive correlation between Karnofsky performance status scale and FOXP3(p= 0.010*) in HL,Karnofsky performance status scale and FOXP3(p= 0.005*) in NHL.The increased expression of FOXP3 was predictive of good prognosis and improved overall survival in (DLBC, FL and HL) while in (SLL) was predictive of bad prognosis and shortened overall survival.
In our study, patients with a low NF-κB ≤196 and STAT6 ≤151.5 positive cell infiltration in (DLBC, FL, HL and SLL/CLL) were associated with a substantially better prognosis, early stages,good prognostic index, (1 low risk, 2,3 low intermediate risk), low performance Scales: Karnofsky & ECOG Scores prolonged overall survival (OS), responded to therapy,decresed risk of relapse,progression, extra nodal metastasis and bone marrow infiltration while patients with high NF-κB >196 and STAT6 >151.5 positive cell infiltration were associated with bad prognosis, late stages, bad prognostic index (4,5 high intermediate risk, 6 high risk),high performance Scales: Karnofsky & ECOG Scores achieved no metabolic response, increased risk of replase, progression, extra nodal metastasis and bone marrow infiltration. Significant positive correlation was the relationship between NF-κB and stages (p= 0.047*) in HL, NF-κB and stages(p= <0.001*) in NHL, STAT 6 and stages (p= 0.003*)in HL, stages and STAT6 (p= <0.001*) in NHL,Limited /advanced stages and NF-κB (p= 0.015*) in HL, limited /advanced stages and NF-κB (p= <0.001*) in NHL, limited /advanced stages and STAT6 (p= 0.003*) in HL, limited /advanced stages and STAT6 (p= <0.001*) in NHL, number of lymph nodes and NF-κB (p=0.041*) in HL, number of lymph nodes and STAT6 (p= 0.003*) in HL,ECOG grades and NF-κB (p= 0.008*) in HL, Deauville score and NF-κB (p= 0.014*) in NHL, Deauville score and STAT 6 (p= 0.022*) in NHL, response to therapy and NF-κB (p= 0.024*) in NHL, negative correlation between STAT6 and response to therapy (p= 0.001*) in NHL in addition negative correlation between Karnofsky performance status scale and NF-κB (p= 0.009*) in HL, Karnofsky performance status scale and NF-κB (p= 0.023*) in NHL,Karnofsky performance status scale and STAT6 (p= 0.035*)in HL and Karnofsky performance status scale and STAT6 (p= 0.022 *) in NHL. Increased expression of NF-κB & STAT 6 were predictive of bad prognosis and shortened overall survival in (DLBCL,FL, SLL/CLL, HL).
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Therefore the three (FOXP3, NF-κB, STAT6) biomarkers in tumor microenvironment might represent new strategies for (HL) and (NHL) diagnosis, prognosis and targeted immunotherapy.
6.2 Conclusions.
from the present study we concluded that:
The high expression of FOXP3 in (DLBCL), (FL), (HL) was correlated with favorable prognostic factors such as early stages, good performance status, good international prognostic index, response to therapy, decreased risk of infiltration of the bone marrow and extra nodal metastasis, prolonged overall survival so that predictor of good prognosis while the elevated expression of FOXP3 in small cell lymphoma (SLL/CLL) was correlated with unfavorable prognostic factors such as late stages, low performance status, bad international prognostic index, existence of infiltration of the bone marrow and extra nodal metastasis, no metabolic response to therapy, shortened overall survival therefore predictor of poor prognosis.
High FOXP3 expression emerged as a predictor of superior outcome in (HL), (DLBCL), (FL) in both univariate and multivariate independent statistical analyses and predictor of poor outcome in (SLL).
Elevated expression of FOXP3 genes in patients, further supporting the role of tregs cells in the occurrence, progression and response to therapy.
FOXP3 (Tregs) played a role in changing immune responses in lymphoma patients, and could be useful in immunotherapy and new anti-lymphoma strategies involved tregs depletion.
Elevated expression of NF-κB in (HL) and (NHL) was correlated with unfavorable prognostic factors such as late stages, poor performance status, poor international prognostic index, increased risk of bone marrow infiltration , extra nodal metastasis, no therapeutic response, shortened overall survival , therefore suggested poor prognosis.
NF-κB played a role in the initiation, progression and resistance to chemotherapy in (HL) and (NHL) patients and might be useful in immunotherapy and risk stratification of the patients therby development of new strategies were essential to restrict tumor cell growth and suppressed both intrinsic and therapy-induced chemoresistance.
Elevated expression of STAT6 in (DLBCL), (FL), (HL) and (SLL) was correlated with unfavorable prognostic factors such as late stages, low performance status, poor international prognostic index, presence of infiltration of the bone marrow , extra nodal metastasis, no metabolic response to therapy, shortened overall survival and therefore predicted poor prognosis.
STAT6 played a role in initiation, progression, recurrence and chemoresistence in patients with (HL)& (NHL) therefore could be useful in determining the outcome of the patients and immunotherapy.
A categorical score of FOXP3, NF-κB, STAT6 was able to discriminate patients with particularly poor and good risk in both early stages and advanced stages disease.
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The three immunological biomarkers of (FOXP3, NF-κB, STAT6) in tumor microenvironment represented new strategies for (HL) and (NHL) diagnosis, prognosis and targeted immunotherapy.
There was significant negative correlation between FOXP3 and NF-κB (p=0.031*), and insignificant correlation between STAT6 and NF-κB and between STAT6 and FOXP3 in Hodgkin’s lymphoma. There was significant positive correlation between NF-κB and STAT6 (p=<0.001*), and insignificant correlation between FOXP3 and STAT6 and between NF-κB and FOXP3 in Non-Hodgkin’s lymphoma.
Univariate analysis showed significant impact on overall survival and patients prognostic factors amongst all groups studied including ECOG grades 1&2, 3&4 (p=<0.001*),prognostic index (p=<0.001*), therapy response (p=<0.001*),extra nodal metastasis (p=<0.001*), bone marrow infiltration (p= <0.001*), FOXP3 (Percentage) ≤67 and >67 (p=0.001*), NF-κB (Percentage) ≤ 196 and > 196 (p=0.001*) and STAT6 (Percentage) ≤ 151.5 and > 151.5 (p=<0.001*).
An increased understanding of molecular mechanism of (HL) and (NHL) offered the potential for novel therapeutic intervention, targeting therapy and influenced the course of the disease.
Our data fostered our understanding of (HL) and (NHL) immune biology that could help to innovate effective immunological control strategies, therapeutically targeted a prognostic factor and therefore improved the prognosis of the patients.
6.3 Recommendations.
1. Further research through the role of FOXP3 in pathogenesis, prognosis, survival in (HL) and (NHL) patients will provide a method to potentially hinder the development of FOXP3 in lymphoma, as well as to introduce innovative new immunotherapy approaches.
2. FOXP3(Tregs) were correlated with development and progression of lymphoma so that these findings postulated the potential importance of tregs in modifying immune responses in patients with lymphomas therfore large scale studies will be necessary to confirm these findings, which will provide basis for new anti-lymphoma strategies involving interference in tregs biology or depletion of tregs.
3. Modulating the action of tregs within larger clinical trials will represent one aspect in the prevention of tumor ”immune’ escape, which potentially will enhance immunosurveillance, the effects of other immunotherapeutic modalities and to determine the significance of these cells for clinical effectiveness of cancer immunotherapy.
4. Further subsequent studies will be required to examine the precise function of NF-κB pathogenesis and it’s role in prognosis, recurrence, chemoresistance in (HL) and (NHL) patients that will be essential to understand lymphoma immunobiology, potentially mediate the development of new immunological therapies and predict patients outcomes.
5. Therapeutic agents targeting the NF-κB pathway had been introduced into the therapeutic regimen of patients with HL & NHL therefore it will important to
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identify groups of patients for whom such therapy will be most appropriate or subsets of patients who’ll never benefit.
6. Additional research on the exact role of STAT6 in pathogenesis, prognosis and progression, recurrence, chemoresistence in (HL) and (NHL) will provide clues to hindering, possibly delaying progression, recurrence of the already formed lymphoma, improving prognostic stratification, survival of the patients, and to design newer innovative immunological therapies.
7. Further large cohort retrospective and prospective studies on the mechanism influencing pathogenesis and progression of (HL) and (NHL) will improve the efficacy of treatment, elongate the survival time of patients and enhance their quality of life.
8. It will be essential to search for clinical parameters and biomarkers that will help to better (HL) and (NHL) characterization, risk stratification and promise a better possibility of personalized and targeted therapy.
9. An increased understanding of molecular mechanism of (HL) and (NHL) will offer the potential for novel therapeutic targeted intervention, and influence the course of the disease.
10. Recent therapies targeting cells in the microenvironment or disrupting the microenvironment dependent signaling in malignant cells will be effective in relapsed HL and NHL patients.
11. The complexity of the involved pathways and the overall heterogeneity of the disease,which also involved non-genetic mechanisms will emphasize the need for precise patient stratification and in particular patients with poor prognosis in order to deliver more effective targeted therapies.
12. The development of precision personalized medicine in (HL) and (NHL) will not only require identification of mutations that drive tumorigenesis and disease progression, but will also involve characterization of cooperating mutations that confer chemoresistence to therapy.
13. Adaptive trial designs with enriched patient populations and markers of early response, integrating translational and clinical knowledge, must remain a key focus for future research. Research proposal and stratification of subsets of patients shall be based not only on clinical characteristics but also on tumor biology.