CD30 Expression and Its Correlation with Clinicopathologic Features in Indonesian Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of Non-Hodgkin Lymphoma (NHL) comprising about 30% 40% cases globally with a high percentage in developing countries [1]. DLBCL is the most common histological subtype (68.2%) of B cells NHL in Indonesia [2]. DLBCL is an aggressive tumor and many cases are diagnosed at an advanced stage. In general, patients show very rapid tumor growth either in single Abstract

Introduction factors for DLBCL [4]. Histopathological examination showed various morphologies of DLBCL including centroblastic, immunoblastic and anaplastic subtypes in which centroblastic subtypes were the most common subtypes and have a better prognosis and survival than other subtypes [5].
CD30 antigens are members of the transmembrane cytokine receptor superfamily 120-kd tumor necrosis factor (TNF) that are identified as cell surface antigens in Hodgkin Lymphoma (Reed-Sternberg cells) [6]. CD30 can also be expressed in various types of T cells and B cells NHL. CD30 is a marker of activation induced in vitro by mitogenic signals and under normal conditions expressed in several B cell and T cell immunoblasts in parafollicular regions and peripheral germinal centers in healthy individuals, as well as being involved in regulation of cell proliferation, activation, differentiation, including controlling cell survival or death by apoptosis or cytotoxicity [7]. The limited distribution of CD30 expression makes CD30 an ideal target for monoclonal antibody therapy in lymphoma patients with positive CD30.
Brentuximab vedotin or SGN-35, monoclonal anti-CD30 which has been approved by U.S. The Food and Drug Administration (FDA) responds to relapsed Hodgkin lymphoma (HL) and Anaplastic Large Cell Lymphoma (ALCL) [8][9]. SGN-35 as a new therapy target continues to be developed in various types of lymphomas with CD30 positive [6-10-11]. Recent studies have shown that CD30 immunohistochemical (IHC) staining also plays an important role in determining the therapy and prognosis of DLBCL disease.
Dr. Sardjito Hospital, Special Region of Yogyakarta, is a referral hospital for patients all over Indonesia. Studies and data related to CD30 expression and clinicopathological characteristics (age, sex, Ann Arbor stage, extranodal involvement and morphological variants) are considerably limited in Indonesia. Therefore, it is important to conduct a study to investigate the association of CD30 immunoreactivity with clinicopathological characteristics including age, sex, Ann Arbor stage, extranodal involvement and morphological variants in Indonesian DLBCL patients.

Study Subjects
This study used a retrospective, non-experimental, observational analytic design with a cross-sectional approach to detect CD30 expression with IHC examination methods and analyzed the correlations between positive CD30 expression with clinicopathological characteristics including age, sex, Ann Arbor stage, extranodal involvement and morphological variants in Indonesian DLBCL patients.
The subjects of this study were patients diagnosed with large cell type NHL at Dr. Sardjito Hospital, Special Region of Yogyakarta, Indonesia and the diagnosis was confirmed by histopathological examination and positive CD20 IHC between January 2015 to December 2018.
All glass slides were retrieved, reviewed and reclassified as DLBCL by 2 independent pathologists, then classified according to their morphology. Eventually, there were 104 samples obtained.
CD30 IHC examination was performed on all samples. The study was conducted in the Anatomical Pathology Laboratory, Sardjito hospital, Special Region of Yogyakarta, Indonesia. Clinical data of the patients were obtained from medical records at the Medical Records Installation, Dr. Sardjito Hospital, Special Region of Yogyakarta, Indonesia.

Immunohistochemistry (IHC)
Detection of CD30 expression in this study was performed using the IHC method. This examination aimed to determine the expression of CD30 antigen/ protein. The formalin-fixed paraffin embedded (FFPE) blocks that met inclusion criteria were then cut into a 4 micron sections. The sections were then incubated, deparaffinized and rehydrated. Antigen retrieval was performed using Starr Track detection kit protocol. Sample sections were stained using monoclonal anti-CD30 antibodies (Mouse monoclonal antibody MoAb CD30 cell marque Ber-H2) to identify CD30 immunoreactivity. Immunoreactive cells were visualized using diaminobenzidine (DAB) chromogen. Lymph node tissue from a confirmed Hodgkin lymphoma case was used as positive control. All glass slides were examined by a pathologist.
CD30 expression assessment was performed using 0% and 20% cutoffs which had been used in several previous studies [12][13]. CD30 positivity was expressed in cytoplasmic membrane and Golgi (paranuclear dot like). CD30 positive percentage was calculated utilizing a total of 200 cells in 5 well-preserved areas (positively stained tumor cells) with various staining intensities calculated in percent units (%) [14].

Statistical Analysis
Correlations between CD30 immunoreactivity and clinicopathological characteristics including age, sex, Ann Arbor stage, extranodal involvement and morphological variants in DLBCL patients were statistically analyzed using Chi-square tests. Statistical associations were considered significant if the p-value < 0.05. Statistical analyses were performed using IBM SPSS version 20 (IBM Corp., Chicago).

Patients' Characteristics
Based on the inclusion and exclusion criteria, 104 tissue samples of patients with DLBCL were included in this study. Male patients were 63 (60.6%) and female patients were 41 (39.4%). Based on age category, there were 45 (43.3%) patients ≥ 60 years and 59 (56.7%) patients < 60 years. Data concerning Ann Arbor stage showed 83 (79.8%) patients were in the early stage (stage I-II) and 21 (20.2%) patients were in the advanced stage (stage III-IV). Based on extranodal involvement,
Slides classified as DLBCL based on the WHO classification system and positive CD20 examination were then reviewed by two independent pathologists to determine morphological variants as centroblastic, immunoblastic or anaplastic. Centroblastic morphological variants were identified in 93 patients (89.4%), immunoblastic variants were identified in 10 patients (9.6%) and anaplastic variants were identified in 1 patient (1%).  In this study, there were 2 cases with > 20% positive CD30 expression. The first case, a 57-year-old male with a lump in the right axillary region and left inguinal lymph nodes enlargement (Ann Arbor stage III) with centroblastic morphological variants. CD10 expression was negative, BCL6 and MUM1 were positive (Figure 2, A-D). Based on Hans algorithm, the case is compatible with activated B-cell like (ABC) subtype. This patient had never started chemotherapy with 7 months overall survival (OS). The second case, a 58-year-old male with a lump in bilateral inguinal lymph nodes (Ann Arbor stage II) and immunoblastic morphological variants. CD10 and BCL 6 expressions were negative. Based on Hans algorithm, this case is compatible with ABC subtype. This patient received R-CHOP therapy with 3 months OS after completion of treatment.

Discussion
This study involved a case by case review of 104 patients with DLBCL. The study revealed that Indonesian DLBCL cases are more common in men than women (60.6%) with an average age of 57 years. According to previous epidemiology study of NHL cases from academic medical centers in Indonesia involving 164 samples, a similar result also revealed that NHL B cells with the most common histological subtype is DLBCL (68.2%) and are more common in men (55%) with an average patient age of 51 years-old [2]. The incidence of lymphoma is more common in men and this is explained in the phenomenon of the immune system in women. Other mechanisms show that the hormone estrogen has an anti-proliferation effect on lymphoid cells via β estrogen receptor signaling [15].
In recent years, CD30 expression has been detected in DLBCL cases [16]. Several previous studies have shown that CD30 is expressed in DLBCL for 9.5-40% and depending on the cutoff used, difficulty in standardizing IHC staining methods, as well as regional and ethnic differences [12][13][14][15][16][17], in 14-25% [18]. CD30 is reported to be a useful predictor of good clinical outcome in DLBCL. Patients with CD 30 positive DLBCL show a better development of survival compared to patients with CD30 negative [16].
The International DLBCL Rituximab-CHOP Consortium Study Program (IDCP) study reported a prevalence of positive CD30 expression of 14% of 903 de novo DLBCL cases using a 20% cutoff [8]. Campuzano-Zaluaga (2014) [20]. Gong (2015) obtained 12% of cases of de novo DLBCL with positive CD30 expression > 0% and 9.5% with positive CD30 expression ≥ 20% [16]. In this study, there were 14 (13.5%) cases from 104 tissue samples of patients with DLBCL with positive CD30 expression > 0% and 2 (1.9%) cases with positive CD30 expression > 20%. The relatively low percentage of positive CD30 expression in this study may be attributed to differences in demographic factors and variability in sampling handling strategies. This lower result might reflect differences in underlying tumor biology from a diverse ethnic population [4-13-16]. CD30 expression assessment is closely related to pre-analytical phase including sample handling and IHC staining processes [21][22][23]. The pre-analytic process is most likely to give false positive or negative results [24][25].
The present study also analyzed the correlations between positive CD30 expression and clinicopathological characteristics (sex, age, Ann Arbor stage, extranodal involvement and DLBCL morphological variants). No significant statistical associations were found between positive CD30 expression (using either a 0% or 20% cutoff) and clinicopathological characteristics. Slack (2014) also examined CD30 expression in 308 cases of de novo DLBCL using a positive threshold of CD30 expression > 0% and > 20% and there was also no statistically significant relationship between positive CD30 expression and clinicopathological characteristics [13].
In our study, there were 2 cases with > 20% positive CD30 expression and based on the Hans algorithm, both of them were compatible with ABC subtype. Salas (2016) also obtained 70% positive CD30 expression in ABC subtype from 40 cases positive CD30 DLBCL [30]. Gandhi (2013) obtained 23% positive CD30 expression in ABC DLBCL higher than 9.5% in GCB DLBCL [31]. CD30 expression by IHC is associated with higher expression levels of CD30 mRNA [32]. ABC DLBCL has   [14-32-33]. Positive CD30 expression status in DLBCL patients may aid management strategies using brentuximab vedotin as an alternative therapy and is associated with improved therapy response and prolonged patient survival [13]. Positive CD30 expression indicates better outcomes in both GCB and non-GCB subtypes because there is increased gene encoding that blocks NF-ĸB activation and lymphocyte cell survival, decreases B cell receptor signal regulation and B cell proliferation with cytokine and stromal signals [8].
In our present study, it was concluded that the prevalence of DLBCL with positive CD30 expression > 0% was 13.5% and positive CD30 expression > 20% was 1.9%. There was no statistically significant association between CD30 immunoreactivity (either with 0% or 20% cutoff) and clinicopathological characteristics (age, sex, Ann Arbor stage, extranodal involvement and morphological variants).