Background
Plasmablastic lymphoma (PBL) is a distinct and rare subtype of B-cell lymphoma that exhibits a plasmablastic morphology but shows a plasma cell-like immunophenotype [
1,
2]. Previous studies have reported that PBL predominantly occurs in immunocompromised individuals, such as those with human immunodeficiency virus (HIV) infection, organ transplantation, and autoimmune diseases [
1,
3]. Frequent Epstein-Barr virus (EBV) infection and
MYC gene aberrations present in PBL patients are reported as adverse prognostic factors and may contribute to lymphomagenesis in these patients [
4]. However, the exact pathogenesis of PBL remains largely unknown and requires further investigation.
Diagnosing PBL is sometimes challenging, as it shares some similar clinicopathological features with myeloma and diffuse large B-cell lymphoma (DLBCL). Although comparative genomic hybridization analysis has revealed that the genomic aberration profile of PBL seems to be more similar to that of DLBCL than plasma cell myeloma (PCM) [
5], the prognosis of PBL was reported to be significantly worse than to that of DLBCL [
6], with an estimated 2-year overall survival (OS) of < 50% [
7,
8]. Therefore, a better understanding of the biology and pathophysiology of PBL may help in improving the survival outcomes.
As most studies on PBL were case reports and small case series, the pathogenesis, standard treatment approaches, and prognostic factors remain largely unknown. Furthermore, clinicopathological features and survival outcomes may vary across populations. Therefore, this study aimed to analyze the clinicopathologic characteristics, therapeutic approaches, and clinical outcomes of PBL patients in a Chinese population. In addition, RNA-sequencing was performed to identify the differences between PBL and DLBCL.
Methods
Patient group
All the patients diagnosed with PBL between January 2008 and October 2019 at the Fudan University Shanghai Cancer Center, Hunan Cancer Hospital, and the Second Xiangya Hospital were examined in this study. Most patients were referred to our institution for consultation after a biopsy was performed. Clinical data such as patients’ age, sex, HIV status, medical history, primary tumor site, Ann Arbor stage, therapies, and clinical outcomes were obtained from medical records. This study was performed in accordance with the Declaration of Helsinki and approved by the ethics committee of each participating medical center. All participants provided written informed consent.
The main immunohistochemical markers (CD20, CD79a, PAX-5, CD30, CD38, CD138, MUM1, and Ki67) were reviewed by two pathologists (Wan and Yu).
Statistical analysis
Progression-free survival (PFS) was calculated from the date of diagnosis to the date of disease progression, relapse, or death by any cause. OS was defined as the time from the date of initial diagnosis to the date of death or last contact. Using univariate analysis, the prognostic role of patient age, sex, disease stage, B symptoms, increased serum lactate dehydrogenase (LDH) level, Ki67, EBER, and patient complete remission (CR) status was evaluated. Prognostic factors (p < 0.05) were further examined using multivariate analysis with Cox regression. PFS and OS were estimated using the Kaplan–Meier method. Survival curves were compared using the log-rank test. Chi-square tests or Fisher’s precision probability tests was used to analyze the clinical differences in different patient groups. Statistical analysis was performed using GraphPad Prism (version 5, GraphPad Software) and R (version 3.5.1, R Foundation for Statistical Computing, Vienna, Austria). Statistical significance was set at P < 0.05.
Immunohistochemistry (IHC) and in situ hybridization for EBV-encoded RNA
Hematoxylin and eosin staining and IHC studies were performed on formalin-fixed and paraffin-embedded tissue sections using standard methods. Primary antibodies against CD20, CD79a, PAX-5, CD38, CD138, and MUM1 (Ventana Medical Systems, USA) were applied on a BenchMark XT automated immunostainer (Ventana Medical Systems) with Cell Conditioning heat retrieval solution (Ventana Medical Systems). Appropriate internal controls (lymphocytes) and external controls (tonsils) were also included in each section. The morphology and IHC results were reviewed by two pathologists (Wan and Yu).
Detection of EBV-encoded small RNA (EBER)-1/2 was performed with proper controls using an ISH kit (Triplex International Bioscience, China) following the manufacturer’s instructions.
RNA-sequencing
RNA was extracted using the AllPrep Kit (Qiagen). Sequencing libraries for RNA-sequencing were prepared using TruSeq RNA Library Prep Kit V2 (Illumina). Paired-end 100 bp read sequencing was performed on a HiSeq 2500 system using Illumina TruSeq V3 chemistry. Paired-end reads were mapped to the human genome (NCBI build 37) by the gapped aligner STAR 2.4.117, using the two-pass method and parameters recommended by the NCI Genomic Data Commons (GDC) 18. The alignment file was used to calculate the raw digital gene expression values by HTseqcount software 0.7.219, using the intersection-nonempty model, which were further analyzed to provide digital gene expression values. The alignment file was also used for variant calling by VarScan2 with selection based on variant read count ≥ 3 and variant read frequency ≥ 0.1.
Reverse-transcriptase quantitative PCR (RT-qPCR)
RT-qPCR was performed as previously described [
9,
10]. RNA was extracted from paraffin-embedded tissue using the AllPrep Kit, according to the manufacturer’s instructions (Qiagen, San Diego, CA, USA). cDNA was synthesized with the Reverse Transcriptase Kit (Takara, # RR036A). Then, qPCR was performed using SYBR Green according to a standard protocol (Takara, #RR420A). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as internal control. The primer sequences are provided in Additional file
1: Table S1.
Discussion
PBL is a rare type of non-Hodgkin lymphoma with overlapping features of B-cell lymphoma and plasma cell neoplasms [
11]. It is characterized by male predominance, frequent involvement of the oral cavity, and an aggressive disease course, with a high rate of HIV and EBV infection [
1,
3]. The exact pathogenesis, standard treatment strategies, and prognostic factors have not yet been defined. This study examined the clinicopathological characteristics, molecular features, and clinical outcomes of PBL in Chinese population.
We found that the clinicopathologic features, such as patient immune status, lesion location, and clinical outcome, were different between Chinese and Western populations. Many studies have confirmed that PBL mainly affects immunocompromised individuals, such as those with HIV infection or other autoimmune diseases [
1‐
3,
8,
11]. Patients in our study group were HIV-negative, and most cases were immunocompetent, consistent with the findings of a review of 60 Chinese PBL patients [
12]. In addition, the most involved sites in our study were the lymph nodes and gastrointestinal tract, followed by the oral cavity, which differed from the findings of previous research that reported that PBL commonly involved the oral cavity and other extra nodal sites in patients with immunodeficiency in Europe [
1,
3,
8]. Similar to previous findings [
1,
3,
7], plasma cell markers (CD38, CD138, and MUM1) were commonly expressed in our patients.
There is no optimal therapeutic approach for PBL. Although CHOP was the most commonly used regimen and patients achieved an overall response rate of approximately 60–70% after treatment, the NCCN guidelines demonstrated that it is inadequate therapy and recommends a more intensive regimen, including hyper-CVAD, CODOX-M/IVAC, and DA-EPOCH [
13]. Previous studies have shown no apparent survival benefit of intensive chemotherapy over CHOP regimens in PBL patients [
14,
15]. The prognosis of PBL remains poor, with a median OS of 6–32 months [
1,
7,
8]. However, the survival outcome of our cohort was relatively longer, with 2-year PFS and OS of 62.7% and 65.2%, respectively. More than half of the patients in our cohort were at an early stage of disease and this may partly explain the prognosis.
Some important prognostic factors, such as disease stage, serum LDH level, EBV status, and CR status, have been identified in PBL [
1,
2]. In our study, clinical stage, LDH level, EBV status, patient CR status, and IPI were significant prognostic factors in the univariate analysis, consistent with the findings of other studies [
1,
2]. However, only CR status remained a significant independent factor in the multivariate analysis [
1]. Many studies have reported that CR after treatment is associated with better outcomes in PBL patients, suggesting that CR is one of the strongest prognostic factors.
The chronic activation of the B cell receptor (BCR) and various downstream signals has been reported to be important for the survival of B cell lymphoma [
16,
17]. In recent years, BCR signaling has emerged as an established target in lymphoma, and BCR inhibitors have achieved clinical effects in B-cell lymphoma [
18,
19]. In our study, BCR signal was downregulated in PBL compared to DLBCL, which was consistent with the findings of a previous study [
20], indicating that the BCR signal was not central to the pathogenesis of PBL and that PBL was distinct from DLBCL.
The tumor suppressor gene
TP53, which encodes the p53 transcription factor and then regulates many target genes in various cancers, was reported to be a barrier to tumor development [
21‐
25]. Mutations in
TP53 occurred in around 20% of DLBCLs and loss of P53 function could contribute to lymphomagenesis [
25,
26]. In ABC-DLBCL, loss of P53 function could facilitate tumor progression by suppressing the pathogenic cooperation of IKK2ca-enforced canonical NF-kB [
24]. Our results showed that P53 signaling pathway was significantly downregulated in PBL compared to GCB-DLBCL, indicating that inactivated P53 may contribute to lymphomagenesis in PBL and serve as a potential therapeutic target in the future.
The tumor microenvironment can interact with cancer cells and play a critical role in tumor development and drug resistance [
27,
28]. Cell adhesion molecules (CAMs) can mediate interactions between tumor cells and stromal cells. Recent studies have reported that high expression of CAMs contributes to the activation of multiple signaling pathways and promotes the development of cancer in plasma cell neoplasms and lymphoma [
27‐
29]. Targeting CAMs such as CD38 and CD138/SDC1 with monoclonal antibodies have achieved promising results in multiple myeloma [
30,
31]. Daratumumab, a monoclonal antibody directed against CD38, was reported to be effective in advanced-stage large B-cell lymphoma (LBCL) with plasmablastic features [
32]. In this study, four PBL patients achieved durable response (12–31 months and ongoing) after the treatment of daratumumab combined with DA-EPOCH [
32]. Our results showed that the expression of CAMs was significantly higher in PBL than in DLBCL, suggesting that CAMs may play important roles in the development of PBL. In addition, CAMs may be potential therapeutic targets in PBL and require further investigation in the future.
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