C-MYC Protein Expression and High Ki-67 Proliferative Index are Predictives of Disease Relapse in Diffuse Large B Cell Lymphoma

C-MYC is one of the most important transcriptional proto-oncogene involved in human carcinogenesis and is the most common mutated gene in lymphomas. This proto-oncogene is regulating a wide range of cellular functions including proliferation, growth and apoptosis [1]. C-MYC oncogene functions as a universal enhancer of already expressed genes in cells rather than directly activating silent genes [2]. Rearrangement of C-MYC gene is crucial in the pathogenesis of B cell Abstract


Introduction
intact C-MYC protein that transforms cells through three main mechanisms such as insertional mutagenesis, gene amplification and chromosomal translocation [4]. Secondary C-MYC gene rearrangement in low grade B-cell lymphomas usually results in transformation into highly aggressive lymphomas with decreased survival rates. The gene changes in DLBCL most often confer an aggressive clinical behavior and chemotherapy resistance [1][2][3][4][5][6]. The adverse prognosis associated with C-MYC gene rearrangement is largely derived from concurrent BCL2 or BCL6 gene rearrangement [7]. DLBCL with C-MYC and either BCL2 or BCL6 rearrangements are subsequently termed double-hit lymphomas, while those with all three rearrangements are referred to as triple-hit lymphomas. Double-hit lymphoma is currently classified as high grade B cell lymphoma [8]. The detection of C-MYC gene translocation and C-MYC protein expression has become essential in the clinical diagnosis and prognosis of aggressive B cell lymphoma. Conventional karyotyping and fluorescence in situ hybridization (FISH) are the two techniques clinically available for the detection of C-MYC gene abnormalities [9]. Immunohistochemical (IHC) detection of C-MYC protein expression does not correspond to C-MY C gene rearrangement. However, there is a correlation between C-MYC protein expression immunohistochemically and its gene translocation in aggressive B cell lymphoma, as over expression of C-MYC protein suggests its gene translocation [10][11]. DLBCL with high level of C-MYC protein expression are likely double-hit lymphomas [8]. IHC staining for detection of C-MYC protein expression is important for prognosis in DLBCL. High level C-MYC protein expression is associated with inferior overall survival irrespective of BCL2 expression [6]. Double expressor lymphoma is a DLBCL that exhibits over expression of both C-MYC and BCL2 proteins. It is associated with worse clinical outcome than other DLBCL [12][13]. Unregulated C-MYC expression promotes cell proliferation, but also induces apoptosis in p53-dependent and p53-independent pathways [14]. Ki-67 is a nuclear non-histone protein that is synthesized at the beginning of cell proliferation [15]. Ki-67 expression has been widely used in clinical practice as an index to evaluate the proliferative activity of lymphoma cells. High Ki-67 proliferative index (PI) is highly associated with worse survival for Non Hodgkin lymphoma [16]. In DLBCL, Ki-67 can distinguish patients with a good and bad prognosis when combined with other prognostic factors [17].
This study aimed to evaluate the status of C-MYC protein expression and Ki-67 PI and clarify their role in predicting relapse of DLBCL using immunohistochemical study.

Materials and Methods
This study was carried out on 50 cases of DLBL diagnosed in the time period from January 2014 till December 2016 in the Pathology Departments of the National Cancer Institute (NCI) Cairo University-Cairo-Egypt, Misr University for Science and Technology (MUST) -Giza-Egypt and private labs of authors, with available follow-up data for the following 3 years from 2015 till the end of year 2017. Approval for this study was obtained from the Medical Ethics Committee of NCI and MUST University. Tissue specimens were preserved in 10% neutral buffered formalin, embedded in paraffin, cut in 4 μm thick tissue sections and stained with H&E stain for conventional histopathologic examination. The diagnosis of DLBL for all cases was confirmed by histologic examination and immunophenotyping using pan B (CD20) and pan T (CD3) markers. All cases revealed diffuse positivity for CD20, while CD3 highlighted only small reactive T lymphocytes. Immunohistochemical analysis was performed using automated Ventana GX immunostainer. The used antibody is C-MYC (EP121) rabbit monoclonal antibody of Cell Marque Tissue Diagnostics, predilute 7 ml vial. C-MYC protein expression was assessed as dense nuclear staining of tumor cells and was judged as positive if the number of stained nuclei is equal to or more than 40% [11]. The used clone for Ki-67 was anti-Ki-67 (30-9) rabbit monoclonal primary antibody from ROCHE Diagnostics. The estimation of Ki-67 PI was done by calculating the percentage of positive tumor nuclei in 1000 tumor cells. The cut-off value of high Ki-67 positivity was set to ≥ 70% [17]. Clinical data as age and gender of the patients were recorded. Excision and core biopsies were obtained from various nodal and extranodal sites, including cervical (n=21), axillary (n=7), inguinal (n=5), spleen (n=4), liver (n=3), colon (n=2), retroperitoneal (n=2), lacrimal gland (n=1), mediastinum (n=1), breast (n=1), supraclavicular (n=1), nasopharyngeal (n=1) and oropharyngeal (n=1) regions. Follow up for all patients included in the study were conducted for the following 3 years and relapsed cases were recorded. The relation between the results of IHC expression of C-MYC protein, Ki-67 PI, clinical data and relapse status during the follow up period were analyzed.

Statistical Analysis
All statistical analyses were performed using Statistical Package for the Social Sciences (SPSS version 25.0; IBM Corporation, Armonk, NY, USA). Demographic and clinical characteristics of the studied patients were presented as frequencies and percentages (%) or mean ± standard deviation (SD). Association between categorical variables were tested for statistical analysis by Chi-square test or Fisher's exact test (if >20% of expected values were less than 5). Shapiro-Wilk's test was used to test for data normality. Difference in mean age (a continuous variable) between binary categorical variables was tested for statistical significance by independent-samples t-test. Multivariate analysis with logistic regression was performed to calculate the age-and sex-adjusted odds ratio of combined C-MYC and Ki-67 for predicting relapses. A p-value <0.05 was considered statistically significant. expression and high Ki-67 PI (Table 3). Multivariate analysis revealed that patients with combined positive C-MYC protein expression and high Ki-67 PI were 19.6 times more likely to have disease relapse when compared to patients with combined negative C-MYC protein expression and low Ki-67 PI; and 16.2 times when compared to patients with either positive C-MYC protein expression alone or high Ki-67 PI alone. In contrast, patients with either positive C-MYC protein expression or high Ki-67 PI alone were only 1.17 times more likely to have relapses when compared to patients with combined negative C-MYC protein expression and low Ki-67 PI.

Results
This study included a total of 50 cases of DLBCL with available follow-up data. Nodal and extra-nodal biopsies were taken from 25 male and 25 female patients. The age of the patients ranged from 4 to 80 years, with a mean age of 56.7± 15 years. The most common biopsy sites were cervical, axillary, and inguinal lymph nodes (42%, 14%, and 10%, respectively). Submitted tissue materials were either core biopsies (56%) or excision biopsies (44%). The Ki-67 PI ranged from 25 to 99 with a mean of 62.40 ± 25.93. Twenty-three cases (46%) had positive C-MYC protein expression, and 29 cases (58%) had high Ki-67 PI (Figure 1). Twenty-two cases (44%) showed disease relapse during the three years follow-up period (

Discussion
DLBCL is a mature B-cell neoplasm with diffuse growth pattern and various immune phenotypes and clinical prognosis. DLBCL accounts for approximately 25-30% of non-Hodgkin lymphoma in Western countries [18]. In Egypt, lymphoma is considered the most common hematologic malignancy and accounts for about 8.4% of all new cancer cases diagnosed annually [19]. The role of C-MYC oncogene in the subtypes of B-cell lymphomas was clarified by many previous studies. In Burkitt's lymphoma, the translocation of C-MYC at 14q32 is the most common, and occurring in approximately 80% of cases [20]. In plasmablastic lymphoma C-MYC rearrangements, most commonly t (8; 14) (q24·1; q32) are found in approximately 49% of cases [21]. On the other hand Kawasaki et al., identified C-MYC gene rearrangement in 5-15% of cases in DLBCL [22]. Previous researches reported that there is a correlation between C-MYC protein expression and its gene translocation in aggressive B cell lymphoma and that over expression of C-MYC protein suggests its gene translocation [10][11].
In this study, C-MYC protein expression and Ki-67 PI were not significantly associated with specific patient's age and sex. These results were in concordance of those reported by Yun et al., who examined 26 males and 16 females with an average age of 58.9 ± 12.3. Specimens were from various sources such as superficial lymph nodes, subcutaneous soft tissue, gastrointestinal tract, mesentery, spleen, and tonsil. Statistical analysis showed that, there was no significant difference in the expression of C-MYC protein related to age or gender of patients [11].
In this study, the cut-off value of Ki-67 positivity was set to be ≥ 70% .The Ki-67 PI ranged from 25 to 99 with a mean of 62.40 ± 25.93. Broyde et al., reported that the mean Ki-67 PI ranges from 26.6% in indolent lymphomas to 97.6% in very aggressive lymphomas. In patients with DLBCL, a Ki-67 PI of 70% was found to significantly discriminate patients with good or bad prognosis [17].
In this study 44% of patients relapsed on the three years follow-up period. Relapses were not significantly   [24]. Moreover, higher rates of relapse were observed in the high Ki-67 expression group compared to the low Ki-67 expression group. In multivariate analysis, Ki-67 expression was a significant prognostic factor for event free survival and borderline significance for overall survival. Elevated Ki-67 expression seems to be associated with higher relapse and inferior free survival in patients with DLBCL [25]. We concluded that C-MYC protein expression and high Ki-67 PI are independently associated with relapses in DLBCL patients. Furthermore, the combined positive C-MYC protein expression and high Ki67 PI test is better than a single positive test in predicting relapses among DLBCL patients.