Prognostic significance of progranulin and B7-H4 in patients with diffuse large B-cell lymphoma

Rasha A Elkholy; Alzahraa A Allam; Shimaa H Elattar; Asmaa M Elkady

doi:10.4103/ejh.ejh_16_19

ORIGINAL ARTICLE

Year : 2019 | Volume : 44 | Issue : 2 | Page : 82-90

Prognostic significance of progranulin and B7-H4 in patients with diffuse large B-cell lymphoma

Rasha A Elkholy MD ¹, Alzahraa A Allam², Shimaa H Elattar², Asmaa M Elkady³
¹ Department of Clinical Pathology, Faculty of Medicine, Tanta University, Tanta, Egypt
² Department of Internal Medicine, Faculty of Medicine, Tanta University, Tanta, Egypt
³ Department of Clinical Oncology and Radiotherapy, Faculty of Medicine, Tanta University, Tanta, Egypt

Date of Submission	08-May-2019
Date of Acceptance	16-Jun-2019
Date of Web Publication	15-Nov-2019

Correspondence Address:
Rasha A Elkholy
Department of Clinical Pathology, Faculty of Medicine, Tanta University, Tanta, 31511
Egypt

Source of Support: None, Conflict of Interest: None

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Read associated Erratum: Erratum: Prognostic significance of progranulin and B7-H4 in patients with diffuse large B-cell lymphoma with this article

DOI: 10.4103/ejh.ejh_16_19

Abstract

Background Progranulin (PGRN) plays a substantial role in tumorigenesis, and its serum level is increased in many types of human cancers. Moreover, B7-H4 plays a remarkable role in regulation of the tumor microenvironment. It is overexpressed in different types of human cancers, and dysregulation of its expression has been associated with tumor progression.
Aim This study aimed to estimate serum levels of PGRN and B7-H4 in patients with newly diagnosed diffuse large B-cell lymphoma (DLBCL) and their relation to patient prognosis and survival.
Patients and methods This case–control study was performed on 50 patients with newly diagnosed DLBCL and 30 apparently healthy individuals as a control group. Enzyme-linked immunosorbent assay was used to measure serum levels of PGRN and B7-H4.
Results This study revealed that serum levels of PGRN and B7-H4 were higher in patients with DLBCL compared with the control group. Receiver operating curve analysis showed that serum PGRN at a cutoff value of greater than 98 ng/ml had a sensitivity of 80% and a specificity of 93.33%, and serum B7-H4 at a cutoff value of greater than 49 ng/ml had a sensitivity of 70% and a specificity of 96.67% for DLBCL diagnosis. In patients with DLBCL, progression-free survival and overall survival significantly decreased with increased serum levels of PGRN and B7-H4 above the cutoff levels.
Conclusion PGRN and B7-H4 can be useful in determining prognosis of patients with DLBCL.

Keywords: B7-H4, diffuse large B-cell lymphoma, progranulin

How to cite this article:
Elkholy RA, Allam AA, Elattar SH, Elkady AM. Prognostic significance of progranulin and B7-H4 in patients with diffuse large B-cell lymphoma. Egypt J Haematol 2019;44:82-90

How to cite this URL:
Elkholy RA, Allam AA, Elattar SH, Elkady AM. Prognostic significance of progranulin and B7-H4 in patients with diffuse large B-cell lymphoma. Egypt J Haematol [serial online] 2019 [cited 2023 Mar 30];44:82-90. Available from: http://www.ehj.eg.net/text.asp?2019/44/2/82/271077

Introduction

Diffuse large B-cell lymphoma (DLBCL) is assorted as one of the most common malignancies worldwide [1]. It comprises ∼30% of adult cases of non‐Hodgkin’s lymphoma [2]. It is characterized by a great variability in tumor type, clinical features, genetic abnormality, response to treatment, and outcome [3].

The immunochemotherapeutic regimen rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) (Rituximab 375/m² day 1, Cyclophosphamide 750 mg/m² day 1, Doxorubicin 50 mg/m² day 1, Vincristine 1.4 mg/m² (maximum 2 mg) day 1 and Prednisolone 100 mg day 1–5) is considered the standard regimen therapy for primary diagnosed DLBCL. However, approximately one-third of patients with DLBCL eventually die from either relapse or resistance to chemotherapy [4],[5],[6].

So, recognizing predictors of poor outcomes has gained special awareness in recent research in DLBCL, in which many biological, clinical, and sociodemographic factors have been specified as correspondents to the dissimilarity in DLBCL outcomes [7],[8],[9].

Progranulin (PGRN) is a protein also known as acrogranin, granulin/epithelin precursor proepithelin, PC cell-derived growth factor, and 88-kDa glycoprotein. It is an autocrine growth and survival factor that was first isolated from molded tissue culture media of exceedingly malignant cells [10],[11].

PGRN is encoded by gene termed granulin, which is located on chromosome 17q21.31 and consists of tandem repeats of a 12-cysteine module termed granulin [12],[13]. It is expressed in a many different tissues such as stromal cells of the bone marrow and cells of the innate and acquired immune system [14].

The growth factor PGRN plays a leading role in tumorigenesis, as it motivates cell division, immigration, infestation, angiogenesis, malignant transformation, resistance to chemotherapy, and also, immune invasion [15].

Elevated PGRN level have been observed in various types of human malignancies such as breast, ovary, liver, kidney, prostate, and brain cancer [16],[17],[18],[19],[20],[21].

Besides, high levels of PGRN either in the tumor tissue or in the peripheral blood have been associated with offensive phenotype and poor outcome in breast cancer, glioblastoma, and ovarian cancer [21],[22],[23]. In addition, in normal tissues, it plays a role in wound repairing, inflammation, and neuronal development [24],[25].

Co-stimulatory and co-inhibitory molecules are fundamental for the regulation of adaptive immune responses [26]. Proteins in this family are present on the surface of antigen-presenting cells and communicate with ligands attached to receptors on the surface of T lymphocytes [27]. B7-H4 is one of the members of B7 co-stimulatory molecules family. It plays a remarkable role in regulation of the tumor microenvironment, and dysregulation of its expression has been associated with tumor progress [28].

It has been demonstrated that B7-H4 suppresses the T-cell response by diminished expansion, cell cycle arrest, diminished cytokine secretion, and induced apoptosis of activated T cells. Moreover, B7-H4 suppresses the function of antigen-presenting cells and encourages the evolution and propagation of regulatory T cells [29].

Several research studies have documented that B7-H4 is frequently overexpressed in malignant tumors such as cancers of ovary, breast, and lung [30],[31],[32].

The expression of B7-H4 in the tumor microenvironment can prohibit the propagation and activity of T cells, and diminished the excretion of immune cytokines such as IL-2 [33],[34].

This study aimed to estimate serum levels of PGRN and B7-H4 in patients with newly diagnosed DLBCL and its relation to patient prognosis and survival.

Patients and methods

This study was conducted on 50 patients newly diagnosed with DLBCL and 30 apparently healthy age-matched and sex-matched individuals, who served as a control group.

Informed consent was obtained from both patient and control groups. This study was carried according to the principles of the local ethical committee.

Inclusion criteria were patients newly diagnosed with DLBCL referred to our hospital.

Exclusion criteria were patients having any malignant diseases other than DLBCL or those who received previous treatment for lymphoma.

Patients were subjected to the following:

Careful history taking, with attention to B symptoms and full physical examination, including lymph node examination and spleen.
Routine laboratory studies, including complete blood count using fully automated cell counter (PCE210N; Erma Inc., Tokyo, Japan), examination of Giemsa-stained peripheral blood films, erythrocyte sedimentation rate, serum lactate dehydrogenase level (LDH), and kidney and liver function tests, which were done on fully automated chemistry analyzer (Konelab Prime 60i, Konelab; Thermo-scientific, Vantaa, Finland).
Bone marrow examination (aspirate and biopsy).
Pathological examination of biopsy specimens with immunohistochemistry using a panel of monoclonal antibodies, including CD3, CD20, CD30, and CD15.
Immunophenotyping on peripheral blood or bone marrow if needed, using the following monoclonal antibodies CD19, CD20, CD2, CD79b, CD23, CD5, FMC-7, kappa, and lambda. Flow cytometric analysis was performed on BD FACS Calibur flow cytometer (BD Biosciences, San Diego, California, USA).
Radiological investigations, including pelvi-abdominal ultrasonography, computed tomography (CT) scan (neck, chest, abdomen, and pelvis), PET scan if available, and echocardiography.
Determination of serum PGRN and B7-H4 levels by enzyme-linked immunosorbent assay (ELISA).

Diagnosis and staging

DLBCL was diagnosed upon histopathological examination of affected lymph nodes and/or extranodal tissue biopsy specimens according to the 2016 revision of the WHO [35].

After diagnosis, patients were staged according to the Ann Arbor staging system with Cotswolds modifications [36].

International prognostic index (IPI) was applied for evaluating the prognosis of DLBCL cases [37].

Patients’ performance status (PS) was determined using Eastern Cooperative Oncology Group PS [38].

Treatment

All patients were treated with six cycles of chemoimmunotherapy R-CHOP protocol [39]. The protocol was recycled every 21 days. Before every cycle of chemoimmunotherapy, complete clinical examination and complete hematological workup were done, and toxicity was evaluated according to National Cancer Institute toxicity criteria [40]. After the first 3 cycles, CT scan (neck, chest, abdomen, and pelvis) was used to assess the effectiveness of chemoimmunotherapy and continue on the same regimen or change the protocol of treatment if the tumor shows progressive course using Cheson’s criteria [41].

Evaluation of tumor response was done in the course of the chemoimmunotherapy and after the end of the six cycles by PET scan [42].

Cheson’s criteria were applied to define the response to treatment [43]. Patients’ response to initial therapy was assessed at mid treatment unless response was clinically apparent (3–4 cycles) by CT scan to identify nonresponse or progression despite therapy[44].

Six courses were completed if more than or equal to 50% partial remission or complete remission has been achieved [45].

Patients were observed for a period of 18 months or until death of the patients, which was considered the end point of the study.

Serum progranulin determination

Peripheral venous blood samples were obtained at the time of the diagnosis and before the start of treatment in a sterile test tube. The samples were centrifuged for 15 min at 1000g. The aliquots were stocked at −20°C until time of estimation. Serum PGRN level was measured using a commercially available, quantitative sandwich enzyme immunoassay technique (human progranulin Quantikine ELISA kit, catalog number DPGRN0; R&D Systems, Minneapolis, Minnesota, USA) in accordance with the manufacturer’s instructions. The sensitivity is 0.54 ng/ml. Intra-assay coefficient of variation (CV)% and interassay CV% are 4.4–5.8 and 7.4–8.3%, respectively.

Serum B7-H4 determination

Peripheral venous blood samples were obtained at the time of the diagnosis and before the start of treatment in a sterile test tube. The samples were centrifuged for 20 min at 1000g. The aliquots were stocked at −20°C until time of estimation. Serum B7-H4 level was measured using a commercially available, quantitative sandwich enzyme immunoassay technique (human VTCN1/B7-H4, ELISA kit, catalog number LS-F51510, Lifespan Biosciences, California, USA) in accordance with the manufacturer’s instructions. The sensitivity is 1.95 ng/ml. Intra-assay CV% and interassay CV% are less than 8 and less than 10%, respectively.

Statistical analysis of the data

Data were fed to the computer and analyzed using IBM SPSS software package version 20.0. (IBM Corp., Armonk, New York, USA).The Kolmogorov–Smirnov, Shapiro, and D’agstino tests were used to verify the normality of distribution of variables, Comparisons between groups for categorical variables were assessed using χ²-test (Fisher or Monte Carlo). Mann–Whitney test was used to compare between two groups for abnormally distributed quantitative variables, whereas analysis of variance was used for comparing the four studied groups. Kruskal–Wallis test was used to compare different groups for abnormally distributed quantitative variables. Spearman coefficient was used to correlate between quantitative variables. Receiver operating characteristic curve (ROC) was used to determine the diagnostic performance of the markers. Area more than 50% gives acceptable performance, and area of ∼100% is the best performance for the test. Kaplan–Meier survival curve was used, and Cox regression analysis was done for the significant relation with progression-free survival (PFS) and overall survival (OS). Significance of the obtained results was judged at the P value up to 0.05 [46].

Results

A total of 80 patients were included in this study, comprising 50 patients newly diagnosed with DLBCL and 30 healthy participants matched in age and sex, who served as a control group. The characteristics of patients group are mentioned in [Table 1].

Table 1 The characteristics of patient group

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Serum PGRN and B7-H4 levels were higher in patients with DLBCL than control group, and the difference between the two groups was statistically significant ([Table 2]).

Table 2 Comparison between serum progranulin and B7-H4 levels in patients with diffuse large B-cell lymphoma and control group

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Serum levels of PGRN and B7-H4 were observed to be increased with patients staging. Comparison between the different stages according to different parameter is shown in [Table 3].

Table 3 Comparison between the different stages according to different parameters

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For diagnosis of DLBCL, ROC curve analysis showed that serum PGRN at a cutoff value of greater than 98 ng/ml had a sensitivity of 80%, a specificity of 93.33%, a positive predictive value of 95.2%, and a negative predictive value of 73.7%, and area under the curve was 0.879, and serum B7-H4 at a cutoff value of greater than 49 ng/ml had a sensitivity of 70%, a specificity of 96.67%, a positive predictive value of 97.2%, and a negative predictive value of 65.9%, and area under the curve was 0.891 ([Table 4] and [Figure 1]).

Table 4 Agreement (sensitivity and specificity) for progranulin and B7-H4 to predict cases versus control

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Figure 1 Receiver operating characteristic curve for progranulin (PGRN) and B7-H4 to predict cases versus control.

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Serum level of PGRN above the cutoff value was founded to be higher in advanced IPI and poor PS, whereas no statistical difference was observed regarding age and staging.

Serum level of B7-H4 above the cutoff value was founded to be higher in advanced stages, advanced IPI, and poor PS, whereas no statistical difference was observed regarding age.

Relation between both PGRN and B7-H4 and age, staging, IPI, and PS is shown in [Table 5].

Table 5 Relation between progranulin, B7-H4 levels and different parameters in cases group

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Correlation study showed that a statistical significant positive correlation was present between both markers and age, LDH, and PS, as shown in [Table 6].

Table 6 Correlation between progranulin and B7-H4 with age, lactate dehydrogenase, and performance status

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In patients with DLBCL, PFS and OS rates are significantly decreased with increased serum levels of PGRN and B7-H4 above the cutoff level ([Table 7] and [Table 8] and [Figure 2] and [Figure 3]).

Table 7 Overall survival probability for different patient groups

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Table 8 Progression-free survival probability for different patient groups

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Figure 2 Kaplan–Meier survival curve of the progression-free survival probability for (a) progranulin, (b) B7-H4, and (c) international prognostic index.

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Figure 3 Kaplan–Meier survival curve of the overall survival probability for (a) progranulin, (b) B7-H4, and (c) international prognostic index.

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Discussion

DLBCL is widely identified as a heterogeneous disease in morphology, genetics, and biologic behavior, so patients with DLBCL display great variability in clinical presentations and outcomes [47].

Although the development of rituximab-CHOP chemotherapy has improved the outcome of patients with DLBCL [42], patients who do not respond well to this regimen require high-dose chemotherapy followed by autologous stem cell transplantation [48].

Therefore, identification of recent prognostic markers has a great importance to recognize high-risk group of patients who required a special regimen from the start.

PGRN (GP-88/PGRN) is a multifunctional protein known to be involved in diverse biological processes, including tumorigenesis, anti-inflammation, and anti-infection [49]. In particular, biological studies have established that PGRN plays a considerable role in stimulating survival, angiogenesis, drug resistance, migration, and invasion, all hallmarks of tumor aggressiveness and poor outcome [15],[50].

B7-H4 is a transmembrane molecule that triggers immunosuppressive signaling in T cells and adjusts T-cell immunity [51]. This protein is widely expressed in many types of cancers, and its expression level is correlated with tumor stage, metastasis, and patient outcome [52].

Therefore, expression of B7-H4 by tumors may be a novel mechanism by which the tumor cells can evade the immune system [53].

In this study, we tried to throw light on the value of serum PGRN and serum B7-H4 estimation in patients with newly diagnosed DLBCL, at different stages, searching for a possible value for their estimation.

Accordingly, 50 untreated patients with DLBCL were studied, and their serum levels of PGRN and B7-H4 were estimated and compared with those of 30 apparently normal healthy individuals comparable in age and sex.

Serum PGRN was significantly higher in patients with DLBCL compared with control group. This was in agreement with Yamamoto et al. [54], who reported that PGRN serum concentrations and lymphoid expression were significantly higher in patients with lymphoid malignancies than in healthy controls. However, this was in contrast with Yamamoto et al. [54], who found no statistical difference between serum level of PGRN and patients staging. This study showed that serum PGRN was founded to be increased with increase in patients’ staging, and the difference among the four stages was statistical significant.

Serum B7-H4 was significantly higher in patients with DLBCL compared with control group. This was in agreement with Xie et al. [55], who reported that B7-H4 concentrations were significantly higher in the serum of patients with non-Hodgkin lymphoma than in healthy controls, and Wu et al. [56], who reported that lymphoma tissues express higher level of B7-H4 than those of adjacent tissues. Moreover, serum levels of B7-H4 were founded to be increased with increased patient’s staging. This is in concordance with Xie et al. [55] who reported that serum levels of B7-H4 tend to be higher in advanced lymphomas than in early one, and Wu et al. [56], who reported that B7-H4 tissue expression was significant correlated with tumor stage.

Based on ROC curve analysis, serum PGRN and serum B7-H4 cutoff concentrations higher than 98 and 49 ng/ml, respectively, distinguished patients with DLBCL from healthy controls.

Regarding PGRN, there was no statistically significant difference in age (<60 or >60 years) or patients staging and PGRN serum level above and below the cutoff value, whereas increased serum PGRN above the cutoff value was related to advanced IPI and poor PS, with statistically significant difference between the two groups. This was in agreement with Yamamoto et al. [54], who reported that serum PGRN above the cutoff value was associated with high IPI and poor PS.

Regarding B7-H4, based on cutoff value, there was no statistically significant difference regarding age (<60 and >60 years), whereas increased serum B7-H4 above cutoff value was linked to advanced stages, advanced IPI, and poor performance, status with statistical significant difference between the two groups.

Regarding correlation study, a statistically significant positive correlation was present between both markers and age, LDH, and PS.

Survival analysis showed that OS and PFS rates were significantly reduced in patients whose serum PGRN levels were above the cutoff level. This is in agreement with Yamamoto et al. [54] who reported that high serum PGRN was associated with poor patient survival.

Moreover, OS and PFS rates were significantly reduced in patients with serum B7-H4 levels above the cutoff value. This is in accordance with Wu et al. [56], who reported that B7-H4 levels were significantly related to poor patient outcome.

Thus, elevated serum levels of PGRN and B7-H4 are suggestive of an unfavorable outcome in patients with DLBCL.

Conclusion

PGRN and B7-H4 serum levels were higher in patients with DLBCL than in control group, and their elevation was associated with advanced stages, poor PS, high international prognostic index, and high LDH. Moreover, their elevation above the cutoff values were also associated with shorter OS and PFS. Therefore, serum levels of PGRN and B7-H4 could be used as markers for unfavorable prognosis in patients with DLBCL.

Acknowledgements

The authors thank the patients and the nurses of Internal Medicine, Clinical Oncology and Radiotherapy, and Clinical Pathology Departments for their assistance in conducting the study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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