Response to Rituximab in Non-Hodgkin Lymphoma Patients at National Oncology Center, Aden, Yemen, 2017-2020:  Retrospective Cohort Study

<jats:title>Abstract</jats:title>         <jats:p><jats:bold>Background:</jats:bold> Rituximab is currently approved for the treatment of relapsed and refractory indolent lymphomas, which was added to the protocol in Yemen although it was highly costly. The study aimed to determine the long-term response of rituximab on Non-Hodgkin Lymphomas (NHL) patients. <jats:bold>Methods:</jats:bold> A retrospective cohort study was conducted in the National Oncology Center (NOC) in Aden by review of medical registries for NHL patients from 2017-2020. Sociodemographic and clinical data were extracted from patients’ files by a well-trained team. SPSS version 23 was used for the analysis of data, and Kaplan Meier survival curve was used to measure overall survival (OS) and progression-free survival (PFS) between two groups CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisolone) and rituximab-CHOP group in two follow up periods (12-months and 4-years).<jats:bold> </jats:bold>Cox regression was used to evaluate the association between the risk factors. A P-value of &lt; 0.05 at a confidence interval of 95% is considered statistically significant.<jats:bold> </jats:bold><jats:bold>Results:</jats:bold> Of 100 patients, 50 for each group of CHOP and R-CHOP. The under 60 years represented 70%. Advanced Stage represented 64% and diffuse large B cell lymphoma represented 47% of all subtypes of NHL. Significant association difference in OS in two periods of follow-up (12-month and 4-years) for CHOP vs. R-CHOP was 63% vs. 86%, and 10% vs. 33%, respectively (<jats:italic>P=0.01 and P=0.04</jats:italic>). In contrast, the difference was non-statically significant in the PFS in two periods 39% vs. 66%, and 12% vs. 27% for CHOP vs. R-CHOP, respectively (<jats:italic>P=0.10 and P=0.31</jats:italic>). The age group &gt; 60 years, ≥ 6 cycles of chemotherapy received, abnormality of LDH, and married patients were the significantly hazard proportion that was associated with poor prognostic factors (<jats:italic>P=</jats:italic> 0.009, 0.001,0.010, and 0.011, respectively).<jats:bold> </jats:bold><jats:bold>Conclusion</jats:bold>: The addition of rituximab to CHOP chemotherapy had a statistical difference in overall survival and there is no difference in PFS. Elderly, married patients, more than 6 cycles of chemotherapy received, and abnormality of LDH were the most important prognostic factors. Therefore, providing the oncology center with Rituximab for ongoing use of NHL patients especially those under 60 years, further assessment for hospital-related risk factors that lead to decreased survival are recommended.</jats:p>

section_content: Non-Hodgkin lymphoma (NHL) is an aggressive chronic lymphoproliferative disorder of the immune system that comprises many subtypes, each with distinct epidemiology, etiology, histology, immunophenotypic, genetic, clinical features; and responses to therapy, which can make diagnosis di cult. B-cell NHLs represented almost all of the cases 85%, while T/Natural killer cells represented 15% [1]. 

Anaplastic large cell, follicular, B cell lymphoma, diffuse large cell, lymphoblastic, mantle cell, mycosis fungoides, primary CNS lymphoma, and cutaneous Tcell lymphoma are the subtypes of NHL. Furthermore, it may result from chromosomal translocations, infections, environmental factors, immunode ciency states, and chronic in ammation [2]. 

In 1982, the NHL was classi ed by morphology and clinical behavior (i. e., low, intermediate, or high grade). Then in1990s, the Revised European-American Lymphoma (REAL) classi cation attempted to apply immunophenotypic and genetic features in identifying distinct clinicopathologic NHL entities [3]. The World Health Organization (WHO) classi cation divided NHL into those of B-cell origin and those of T-cell and natural killer (NK)-cell origin according to the REAL approach [4]. 

The treatment of non-Hodgkin lymphoma (NHL) varies greatly, depending on many factors: Tumor stage, Phenotype (B-cell, T-cell or Natural Killer cell/nullcell), Histology (i. e., low, intermediate, or high grade), Symptoms, Performance status, Patient age, Comorbidities [5]. 

Rituximab is a monoclonal antibody that targets the CD20 antigen, which is expressed on the surface of pre-B and mature B-lymphocytes. Rituximab binds to CD20 after mediates B-cell lysis (or breakdown). The possible mechanisms of cell lysis include complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity [6]. 

NHL is the most prevalent hematopoietic neoplasm, representing approximately 4. 3% of all cancer diagnoses and ranking seventh in frequency among all cancers. The incidence of NHL has increased 168% since 1975 (while survival has improved by 158%) [7]. 

Globally, the latest GLOBOCAN data in 2018 estimated 509,600 new cases of NHL were diagnosed; comprising 2. 8% of worldwide cancer diagnoses and the age-standardized risk of NHL was 6. 7 among men and 4. 7 among women. In addition, an estimated 248,700 deaths were attributable to NHL, and the NHL's 5year survival from 2010 to 2016 in the US was 72. 7% [7]. 

In the Eastern Mediterranean region EMRO, 2020, according to GLOBOCAN, the NHL represented as the eighth rank of all cancer in the region, incidence rate of 4. 2%, and case mortality rate of 3. 4%, the prevalence of 5-years 10. 68/100,000 of the population [8]. 

In Yemen, Non-Hodgkin Lymphoma was a major health problem that represents the second most common type of cancer (10. 1%) in males, and the third common type in females (6. 6%) in 2011 [9]. In Aden governorate, the NHL represented the rst common hematological malignancy in 2010 [10]. In 2013, Lymphoma represented 9. 8% of all cancer patients who attended the national oncology center, and the NHL represented 65% of all lymphomas patients [11]. 

Rituximab entered into the protocol of NHL in NOC, although of its higher cost, there is no previous study published in Yemen and neighboring countries to measure the impact and response of it on lymphomas patients. The present study aims to determine the long-term response of rituximab on NHL patients and measure the survival rate among patients when treated with R-CHOP or CHOP.
section_name: Background

section_name: Methods

section_content: A retrospective cohort study was conducted during the period of (2017-2020).
section_name: Study design

section_content: The study was conducted in the National Oncology Center (NOC), Aden governorate. NOC was established in 2013 at Alsadaqa hospital. The majority of attendees to the center are from Aden governorate and many neighboring Governorates such as Lahj, Abian, Aldhale, Shabowh, and other governorates. The number of tumor cases until October 2021 reached 8960 cases for the center, where the number of males reached 3157 cases, females 4303, and children 1550, with an increase of cases in the last years. In addition, the center receives approximately 1200 cases annually.
section_name: Study area and Setting

section_content: All the Non-Hodgkin lymphoma patients that registered in NOC from 2017-2020 and received at least four cycles of R-CHOP& CHOP protocol were included in this study.
section_name: Study Population

section_content: Exposed cases were de ned as all lymphoma patients > 16 years in NOC received at least four cycles of R-CHOP protocol [1] and lymphoma patients > 16 years in NOC received at least four cycles of CHOP protocol were de ned as non-exposed.
section_name: De nitions and selection criteria

section_content: Patients with NHL received at least four cycles of R-CHOP protocol or CHOP, the age more than 16 years and diagnosed between 2017-2020 years.
section_name: Inclusion criteria:

section_content: The patient received less than four cycles of chemotherapy (R-CHOP and CHOP) or received other protocols than R-CHOP or CHOP protocol or both protocols of R-CHOP and CHOP. In addition, patients with age less than 16 years or diagnosed before 2017 and after 2020.
section_name: Exclusion criteria:

section_content: We reviewed patients' registries and les in NOC by a trained team (principal investigator & data collectors). Abstracting form used to collect all variables of the patient les regarding patients' demographics (e. g., sex, age, job, place of birth, marital status) and clinical features such as stage of disease, a subtype of NHL, B symptoms, and outcomes. In addition, the abstracting form included data about date of diagnosis, start chemotherapy, follow up, lack of follow up, death, negative scan, and relapse for each patient as well as laboratory investigations
section_name: Data collection tools and procedures

section_content: The principal investigator monitored all the processes of data collection from patient registries and each sample of the abstracting forms was evaluated to be sure that collection is done perfectly as instructed in training. Data collected from all collectors and frequencies were used to check data before data entry.
section_name: Quality assurance and control

section_content: Data entered in the excel format of the abstracting form and exported into SPSS version 23 for analysis. Univariate and multivariate cox regression analysis was conducted with the nal model. Secondary clinical indicators assessed at the end of chemotherapy protocols were overall survival (OS) is the percentage age of people who can still live for a de nite time after they were clinically diagnosed with a certain disease like cancer or started therapy for that disease. 

Progression-free survival (PFS), The PFS is the period during treatment and after the management of a malignant disease like cancer, patient lives with the disease but it does not get worse. That measured from date of diagnosis to date of relapse or progression of the lesion or death. 

Kaplan Meier survival curve analyses were employed to measure OS and PFS for the two groups of cases (CHOP/ R-CHOP) to nd any difference in response due to treatment protocols. A P-value of less than 0. 05 at a con dence interval of 95% is considered statistically signi cant.
section_name: Data entry and management

section_name: Results

section_content: A total of 200 cases of NHL were admitted at NOC-Aden in the last four years, 100 cases of them were well-matched to inclusion and exclusion criteria of this study, of whom 50 cases were treated according to CHOP and 50 cases with R-CHOP. The overall mean age of patients was 48. 8 years (± 16). Table 1 shows the characteristics of NHL patients, aged less than 60 years, male, non-employed, and Married patients formed the majority of cases, 70%, 57%, 61%and 81%, The abnormality of laboratory investigations of LDH 62%, B2M 61%, while normality of WBC 84% and bone marrow 95%. Positive cases for CD20 represent 67%. 

Advanced stages of disease and mortality were found to be signi cantly associated with patients who were treated with CHOP. (p=0. 03 and p =0. 02), respectively.
section_name: Descriptive analysis

section_content: Two periods to follow up with the patients in our study were determined. First, the patients of NHL during four years were followed up every 12 months; from the date of diagnosis to the end period for each group (CHOP & R-CHOP). The overall survival (OS) for 12-month for CHOP and R-CHOP was 63% and 86%, respectively with a p-value of (P=0. 01). While the progression-free survival (PFS) for the same period was 39% and 66% for CHOP and R-CHOP respectively with a p-value (P= 0. 10) (Figure 1 ). 

Second, the patients were followed up for four years, the overall survival (OS) for CHOP and R-CHOP was with little difference 10% and 33%, respectively with P-value (P=0. 04). While progression-free survival (PFS) during four years follow-up was 12% and 27% for CHOP and R-CHOP respectively with P-value (P= 0. 31) (Figure 2 )

Table 2 illustrates the hazard proportion in many characteristics of patients by using cox regression. The age group more than 60 years, more than 6 cycles of chemotherapy, abnormality of LDH, and married patients were the signi cantly hazard proportion that was associated with poor prognostic factors with Pvalue of 0. 009, 0. 001,0. 010, and 0. 011, respectively.
section_name: Survival analysis

section_content: The burden of the NHL has increased distinctly in the last years in Yemen. In patients with NHL, the addition of rituximab to standard treatment signi cantly enhanced response to therapy and overall outcomes. Rituximab is currently approved for the treatment of relapsed and refractory indolent lymphomas as single-agent therapy and as initial therapy in combination with standard chemotherapy regimens [12]. 

NHL is most often diagnosed in an elderly patient that is aged >60 years, but there is an exception for some types of NHL that are diagnosed in an adult patient [13]. Our study showed the mean age of the patient was 48 years, which is similar to the previous study conducted in Aden, Hadramoot governorates [10, 14], and other studies in China and Mexico [1, 15]. This is due to our patients being contained in all subtypes of NHL. 

Regarding gender, our study showed no great difference between males and females (57%). This result is consistent with previous studies in Southeastern governorates of Yemen, Saudia Arabia, and Mexico [1, [16] [17] [18]. In contrast, studies conducted in Yemen, and among Chinese patients showed that females more than males, [14, 15] this attributed to the male developing disease more likely than females [19]. 

Regarding pathological characteristics, advanced stages (, ) represented the majority of cases; these ndings were in agreement with studies reported in Saudia Arabia, Mexico, and France [1, 18, 20, 21]. On other hand, a study in China revealed that most cases were in stages, [15]. This difference may be due to the lack of early screening of disease in our country. 

About 30-40% of NHL patients complain of B-symptoms at the time of diagnosis [5]. our nding revealed that two-thirds of patients did not complain of Bsymptoms (67%), which is nearly similar to studies conducted in Mexico and Korea that were (65%, 68%) respectively, and higher than reported from Saudia Arabia (52%) [1, 18, 22]. 

Cervical lesions represent 28% of all cases in the rst of other lesion sites, agrees with a study conducted in Hadramoot [14], and different with a study conducted in Aden which revealed the common lesion was in the abdomen [17]. 

Diffuse large B cell lymphoma was the most frequent subtype of NHL in our study; this result is similar to a study conducted in Hadramoot Yemen, Aseer Saudia Arabia, and a previous study Middle East region [14, 18, 23]. This is due to DLBCL being the most common form of lymphoma that represents about 30% of NHL [24]. 

Regarding laboratory investigations, our study showed the abnormality of LDH and B2M in most cases, this nding was agreed with a study conducted in Korea [22], and disagreed with the ndings of a study conducted in China. [15] In addition, most cases were a normal count of WBC; a similar result was reported in Saudia Arabia [18]. and CD20 was positive in 67% of patients in our study, which is lower than ndings from Saudia Arabia 82% [18]. This difference may be due to variances in sample size, and the percentage of B cell lymphoma that contains about 98% of positive CD20. Furthermore, Only 5% of cases had bone marrow in ltration, which is consistent with ndings reported in a previous study [22]. 

The incidence rate of new cases of non-Hodgkin lymphoma was 19. 6 /100,000 per year and the death rate was 5. 3 /100,000 per year globally, furthermore, the mortality was about fourth of diagnosed cases of NHL [25]. Our study found that the overall fatality rate was 20% and 30% & 10% for CHOP and R-CHOP, respectively, which is close to the global rate. This result is slightly lower than a study conducted in Korea that found the overall fatality rate was 31% and 57%& 14% for each group, and another study in France showed 35% and (41% & 29%) [20, 22]. 

In this study, the survival outcome of two groups (CHOP& R-CHOP) was estimated in two follow-up periods in 12 months and 4-year, according to previously evaluated cancer survival analysis [26]. Our ndings revealed that the overall survival (OS) in 12-month was 63% and 86%, for CHOP and R-CHOP respectively.
section_name: Discussion

section_name: Acknowledgments

section_content: The authors would like to acknowledge the Training Programs in Epidemiology & Public Health Interventions Network (TEPHINET) and Yemen Field Epidemiology Training Program for their technical support. In addition, the National Oncology Center, Aden facilitate all process of data collection.
section_name: Acknowledgments

section_name: Funding

section_content: This work was supported by TEPHNET; the funder had a role only in data collection
section_name: Funding

section_name: Availability of data and materials

section_content: All relevant data are presented in this paper, and more information can be provided upon reasonable request from the corresponding author.
section_name: Availability of data and materials

section_name: Declarations

section_content: respectively. 25 % of cases were diagnosed in 2017. Pathological characteristics of NHL, Ann-Arbor advance stage, represented 64, and with an absence of B-symptoms (fever, drenching night sweats, and loss of more than 10 percent of body weight over 6 months) in 67% of cases. Nodal lesion represented 54% and diffuse large B cell lymphoma (DLBCL) represent 47% of all NHL subtypes, (66% in R-CHOP group). 

This was nearly similar to a study conducted in China for the same period 12-month (75%& 93. 8%) [27]. In addition, the progression-free period (PFP) in our study was 39% and 66% for CHOP and R-CHOP, respectively, lower than studies conducted in China (52. 8%& 81. 2%) [27]. This could be due to many causes such as the low quality of medical services in Yemen compared with other countries, decrease patient outcomes that lead to poor regular follow-up, and most patients coming in advanced stages. 

The survival rate has been improving since 1997, thanks to treatment advances. From 2009 to 2018, the death rate decreased by 2% annually. The overall 5year survival rate for people with NHL is 73% [28]. The 4-years survival rate for patients in our study was 10% and 33% for overall survival of CHOP and R-CHOP groups, respectively. This result was much lower than 5-years survival in the United States (73%), a study conducted in Mexico (61. 8% & 65%), and in Korea (94. 7% &84. 7%) [1, 28, 29]. on the other hand, the progression-free survival (PFS) was 12% and 27% for CHOP and R-CHOP, respectively. This is also much lower than other studies conducted in Mexico (63. 8 and 51. 2%), Korea (50. 0% vs. 79. 0%), and Germany (59% & 79%) [1, 22, 30]. 

Our nding revealed signi cant overall survival (OS) of two groups and not signi cant progression-free survival (PFS), this was consistent with the previous study conducted in China and France that found signi cant overall survival [15, 20]. In contrast, previous studies conducted in Korea found signi cant progression-free survival and not signi cant overall survival, and another study conducted in Mexico found no signi cance for both overall and progressionfree survival [1, 22]. This difference may be due to the most patients were in advanced stages. 

The prognosis for non-Hodgkin lymphoma (NHL) varies with the histology, the stage of disease at diagnosis, and the response of the disease to therapy. Furthermore, age ≥60 years, elevated lactate dehydrogenase (LDH) level, Stage III or IV disease, Eastern Cooperative Oncology Group (ECOG) performance status ≥2, and two or more extra-nodal sites [13]. In our study, the signi cant prognostic factors were the age > 60 years, >6 cycles chemotherapy, abnormality of LDH, and married patient with P-value <0. 05. This conforms to studies conducted in Korea and China that found age > 60 years were poor prognostic factors [15, 22]. In contrast, many studies conducted in different countries found these factors not signi cantly associated with protocol treatment [1, 20, 29]. The main limitation in our study was the poor documentation for some processes of treatment of patients and the follow-up results. Lack of a computerized system in the treatment center leads to a lack of the outcome of many patients. 

In conclusion, the addition of rituximab to CHOP chemotherapy had a statistical difference in overall survival and there is no difference in PFS. Elderly, married patients, more than 6 cycles of chemotherapy received, and abnormality of LDH were the most important prognostic factors. Therefore, providing the oncology center with Rituximab for ongoing use of NHL patients especially those under 60 years, further assessment for hospital-related risk factors that lead to decreased survival are recommended.

section_content: Authors' contributions MA the principal investigator, collect data, analyzed it, and wrote the manuscript. GA eld supervisor. MA revised data analysis. YG general supervisor of the study, all authors approved the nal draft of the study.
section_name: Declarations

section_content: Study submitted to the Ethical Committee at the Ministry of Public Health& Population (MoPHP) for approval (at 18. 8. 2021, accepted at 31. 8. 2021, reference number 5146). Authorization for conducting the study in NOC was obtained from Aden Health O ce and the director of the NOC. As well as patient's con dentiality secured using a unique ID. The study did not involve experiments on a human subject or human participants under the age of 18 years.
section_name: Ethics approval and consent to participate

section_content: This study does not include any identi able human images or data thus does not require consent to publish.
section_name: Consent for publication

section_content: The author(s) declared no potential con icts of interest concerning the research, authorship, and/or publication of this article. 

Author Details
section_name: Competing interests