Marginal zone lymphoma expression of histidine‐rich glycoprotein correlates with improved survival

Abstract Purpose The abundant hepatocyte‐expressed plasma protein histidine‐rich glycoprotein (HRG) enhances antitumor immunity by polarizing inflammatory and immune cells in several mouse models, however, the clinical relevance of HRG in human cancer is poorly explored. The expression and role of HRG in human B‐cell lymphomas was investigated in order to find new tools for prognosis and treatment. Findings Immunohistochemical (IHC) analysis and RNA hybridization of tissue microarrays showed that (i) HRG was expressed by tumor cells in marginal zone lymphoma (MZL), in 36% of 59 cases. Expression was also detected in follicular lymphoma (22%), mantle cell lymphoma (19%), and indiffuse large B‐cell lymphoma (DLBCL;5%) while primary CNS lymphoma (PCNSL) lacked expression of HRG. (ii) MZL patients positive for HRG showed a superior overall survival outcome (HR = 0.086, 95% CI = 0.014‐0.518, P‐value = .007), indicating a protective role for HRG independent of stage, age and sex. (iii) HRG‐expressing MZL displayed significantly increased transcript and protein levels of the host defense peptide alpha defensin 1. In addition, global transcript analyses showed significant changes in gene ontology terms relating to immunity and inflammation, however, infiltration of immune and inflammatory cells detected by IHC was unaffected by HRG expression. Conclusion HRG expression by MZL tumor cells correlates with an altered transcription profile and improved overall survival.

age and is particularly common in patients older than 80 years [5,6].
Although there are common genetic and clinicopathological features among the MZL subtypes, both prognosis and treatment protocols differ between the subtypes. Most SMZL patients have a slow disease progression, and the median patient survival is 8-10 years. However, SMZL has a heterogeneous clinical course with approximately 25% of the patients surviving only 3-4 years and 5-10% of patients undergoing transformation to diffuse large B-cell lymphoma (DLBCL) [7]. Studies are therefore warranted to identify patients who will present with an aggressive disease course and benefit from chemotherapy. Also in other indolent B-cell lymphomas, such as follicular lymphoma (FL) and mantle cell lymphoma (MCL), the disease course is variable and tools to identify different prognostic subgroups are urgently needed [8].
In the healthy individual, histidine-rich glycoprotein (HRG; 75 kDa) is exclusively produced by hepatocytes and is present in plasma at a concentration of 100-150 ug/mL [9]. HRG consists of two N-terminal cystatin-like domains, followed by a histidine/proline-rich domain organized in 12 pentapeptide repeats. HRG binds divalent metal ions including Zn 2+ as well as heparan sulfate/heparin and heme through its histidine-rich region [10]. In several mouse tumor models HRG exerts gene regulatory effects in tumor-associated macrophages resulting in a switch from an M2 to M1 polarity profile, correlating with enhanced antitumor immunity, decreased tumor growth, and reduced metastatic spread [11]. Treatment of mouse glioma by HRG-adenovirus gene therapy results in reduced infiltration of regulatory T cells and suppressed tumor growth [12]. In humans, HRG serum levels are increased in breast cancer patients as compared with healthy controls [13]. In contrast, in ovarian cancer, HRG serum levels decrease in advanced cancer, compared to lower stage cancer and healthy controls [14]. Whether this decrease is due to impaired liver function, or direct effects on HRG transcript and protein production/turnover remain to be addressed. In sepsis, HRG plasma levels decrease and therefore, monitoring plasma HRG can serve as a sepsis biomarker [15].
The immune modulatory effects of HRG prompted the question of a potential involvement of HRG in lymphoma progression. Here, we show that HRG is expressed in several mature B-cell lymphomas. In MZL, HRG is produced by the tumor cells and the expression correlates with an altered transcription profile and with improved overall survival.

RNA in situ hybridization
In situ hybridization was performed on the MZL TMAs using the

Gene microarray analysis
RNA was isolated from paraffin-embedded slides using RNeasy FFPE kit (Qiagen). RNA concentration was measured by NanoDrop (Thermo Fisher Scientific), and RNA integrity number (RIN) was assessed by Bioanalyzer (Agilent). Microarray assay was performed using Clariom™ DPico Assays, Human (Thermo Fisher Scientific) with RNA from five HRG-negative and four HRG-positive patient samples. The data were deposited at the Gene Expression Omnibus (GEO) database repository; accession number: GSE151360.

HRG protein expression in mature B-cell lymphomas
Although there is ample evidence from mouse models that HRG's impact on inflammation and antitumor immunity can steer tumor progression, HRG's clinical relevance and the molecular mechanisms for its potential role in human cancer remain to be fully explored.
We therefore examined the expression and potential role for HRG in human lymphomas. Mature B-cell lymphoma TMAs including MZL (n = 59, see Table I

HRG is expressed by lymphoma tumor cells
HRG is predominantly produced by liver hepatocytes, distributed throughout the circulation, and deposited in the perivascular stroma in healthy tissues [11]. To determine if HRG protein detected in lymphomas is produced in the liver or expressed locally by lymphoma tumor cells, the presence of HRG transcripts in the tumor samples was investigated using RNAscope. First, healthy liver tissue was used to evaluate the accuracy of the HRG RNAscope probe in parallel with antibody staining on consecutive tissue sections ( Figure 2A). As expected, HRG mRNA and protein were highly expressed by hepatocytes, thereby validating the RNAscope probe. Next, RNA-scope was performed on a subset of the MZL samples ( Figure 2B). Expression of HRG mRNA was found in 8 of 19 samples and HRG mRNA was significantly associated with HRG protein expression (P-value = .04; Table   S1).
These data show that although the HRG protein found in 36% of the MZL cases may be produced in the liver and brought to the tumor by the circulation, it is also endogenously produced by the cancer cells in the lymphoma biopsies.

HRG is an independent marker for improved overall survival in MZL
To a variable degree, we found HRG expression in MZL, FL, MCL, and DLBCL samples. The relatively low prevalence in FL and DLBCL combined with the small patient cohort in MCL made it infeasible to further

HRG expression does not correlate with inflammatory/immune cell status but is associated with increased expression of alpha defensin1
To characterize the inflammatory/immune cell status, IHC stainings were performed on the MZL samples against CD3 (T cell lineage), CD4 (T helper cells), CD8 (T killer cells), and CD68 (monocytic lineage). MLZ samples were scored as either low or high for these markers and the scores were compared to the presence of HRG-positive cells. Results showed no significant association between the presence of HRG and infiltration of macrophages or T-lymphocytes (Table S3)  These particular tumors may belong to the group with a more indolent clinical behavior, although the number of cases was low [19]. These findings prompt future studies to explore whether the HRG-positive patients in the different diagnoses of B-cell lymphomas belong to a specific subgroup with common features. However, such analyses would require larger, well-annotated cohorts, which currently are unavailable.
For indolent B-cell lymphoma patients, the clinical course is very variable; a wait-and-watch approach is often considered for patients with slow progressive disease but biomarkers to identify patient groups that eventually need treatment are urgently needed [8]. Since the median age at diagnosis is usually high, many patients also suffer from comorbidities. A tailored treatment approach to minimize aggressive therapy for indolent cases is therefore important [20]. T-cell attracting cytokines [11]. In the MZL tumor samples in contrast, HRG expression did not correlate with an increased presence of CD4 + or CD8 + T cells, or CD68 + macrophages (Table S2) associated with adverse prognosis in leukemias and lymphomas [28].
We failed however to validate the expression of MZB1 by immunostaining. Another gene, the antimicrobial peptide alpha defensin 1, DEFA1, was significantly increased in HRG-positive MZL patients, which was validated on the protein level by immunofluorescent staining. DEFA1 is produced by neutrophil precursors, and is stored in azurophilic granules of mature neutrophils [29]. It is also expressed at low levels by cancer cells in certain cancer forms including colorectal cancer and renal cell carcinoma [30]. Both HRG and DEFA1 have chemoattractant properties, and both can enhance the antitumoral effects of immune cells and increase macrophage phagocytosis [11,29,31,32]. Thus, HRG expression by MZL tumor cells is accompanied by changes in gene expression, including DEFA1, characteristic of an antitumoral response. Future studies remain to establish whether gain of HRG expression is an early or late event in disease progression, and if the HRG-associated GO profile, including DEFA1, is a cause or consequence of HRG expression. Finally, it remains to be shown whether HRG can be exploited not only as a diagnostic tool but also in therapy design.