|Year : 2017 | Volume
| Issue : 3 | Page : 99-107
Prognostic role of tissue expression and serum level of YKL-40 in patients with diffuse large B-cell lymphoma
Maaly M Mabrouk1, Omnia AbdElfattah2, Tamer A Elbedewy3, Dareen A Aziz4, Asmaa E Bedeer4
1 Department of Clinical Pathology, Faculty of Medicine, Tanta University, Tanta, Egypt
2 Department of Clinical Oncology, Faculty of Medicine, Tanta University, Tanta, Egypt
3 Department of Internal Medicine, Faculty of Medicine, Tanta University, Tanta, Egypt
4 Department of Pathology, Faculty of Medicine, Tanta University, Tanta, Egypt
|Date of Submission||26-Jun-2017|
|Date of Acceptance||08-Aug-2017|
|Date of Web Publication||9-Nov-2017|
Tamer A Elbedewy
Department of Internal Medicine, Tanta University, 51719
Source of Support: None, Conflict of Interest: None
Background Serum YKL-40 levels are increased in various inflammatory disorders and a wide range of malignancies. Moreover, these elevated levels correlate with poor prognosis of patients with cancer, suggestive of YKL-40 as a prognostic biomarker. The effect of YKL-40 on non-Hodgkin lymphoma prognosis has not been fully explained.
Aim The aim of this article was to study the serum levels and expression of YKL-40 in tissue specimens of patients with diffuse large B-cell lymphoma (DLBCL) for assessing its prognostic value and shedding light on their effect on survival.
Patients and methods The study included 60 patients with DLBCL. Enzyme-linked immunosorbent assay was used to assess the serum YKL-40 levels. Immunohistochemical staining was used to detect YKL-40 protein expression in lymphoma specimens.
Results YKL-40 serum levels were significantly higher in patients with DLBCL when compared with the control group. YKL-40 protein was expressed in 66.67% of examined specimens. Receiver–operator curve analysis showed serum YKL-40 at a cutoff value of greater than or equal to 95.5 ng/ml had a sensitivity of 70% and a specificity of 95% for DLBCL diagnosis. In patients with DLBCL, progression-free and overall survival rates significantly decreased with increased serum levels of YKL-40 above the cutoff level as well as in YKL-40 positive expressed patients.
Conclusion Serum YKL-40 and its tissue expression could be a valuable prognostic marker in patients with DLBCL.
Keywords: diffuse large B-cell lymphoma, overall survival, progression-free survival, YKL-40
|How to cite this article:|
Mabrouk MM, AbdElfattah O, Elbedewy TA, Aziz DA, Bedeer AE. Prognostic role of tissue expression and serum level of YKL-40 in patients with diffuse large B-cell lymphoma. Egypt J Haematol 2017;42:99-107
|How to cite this URL:|
Mabrouk MM, AbdElfattah O, Elbedewy TA, Aziz DA, Bedeer AE. Prognostic role of tissue expression and serum level of YKL-40 in patients with diffuse large B-cell lymphoma. Egypt J Haematol [serial online] 2017 [cited 2018 Sep 23];42:99-107. Available from: http://www.ehj.eg.net/text.asp?2017/42/3/99/217879
| Introduction|| |
Diffuse large B-Cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma (NHL), accounting for nearly 30% of all newly diagnosed cases and more than 80% of aggressive lymphomas . DLBCL is a heterogeneous disease, but it is readily curable, even in the most advanced cases. However, its treatment represents a great challenging as DLBCL is not a single entity ,.
Angiogenesis is a requirement of malignant tumor progression and metastatic spread, as observed by both experimental and clinical studies ,. Several studies have suggested that angiogenesis is highly relevant to lymphoma subtypes by assessment of the angiogenic characters of the tumor cells and the vascular microenvironment ,.
YKL-40 (chitinase 3-like protein 1) is a 40-kDa glycoprotein, classified in the chitinase gene family ,. However, YKL-40 is deficient in chitinase/hydrolase activity owing to substitution of an essential glutamic acid to leucine in the chitinase 3-like catalytic domain . YKL-40 has been documented as a growth factor for fibroblasts and connective tissue cells. This proposes a role of YKL-40 in tissue remodeling and as an antiapoptotic protein ,. YKL-40 acts as a chemoattractant factor for endothelial cells . It also stimulates migration and vascular smooth muscle cells adhesion  and has an angiogenetic role, although in-vivo proof of this is yet to be obtained .
Increased serum levels of YKL-40 have been described in many benign diseases, illustrated by inflammation and tissue remodeling, for example, rheumatoid arthritis .
In carcinogenesis, the exact role of YKL-40 is not established yet. Although, it is observed that YKL-40 may participate in cancer cells proliferation and differentiation. It has been declared to promote an antiapoptotic effect on cancer cells, promotion of angiogenesis, renewal of extracellular tissue, and the stimulation of fibroblasts near the tumor .
YKL-40 is expressed in many types of malignancies (breast, colon, lung, kidney, ovarian, glioblastoma, germ cell tumors, prostate, uterine, osteosarcoma, and oligodendroglioma) ,. Elevated serum YKL-40 levels have been associated with recurrence of tumor and decreased survival rate in many patients with cancer .
| Aim|| |
The aim was to study the serum levels and expression of YKL-40 in tissues specimens of patients with DLBCL for assessing its prognostic value and shedding light on its effect on survival.
| Patients and methods|| |
The present study was conducted on a group of 60 histologically confirmed newly diagnosed patients with DLBCL (37 males and 23 females; aged 29–73 years, median 55), treated and followed up at Tanta University Hospitals, Egypt, between July 2013 and April 2017. The study was conducted in accordance with the local ethical committee, and all the participants gave an informed consent. Overall, 30 healthy individuals (age and sex matched) served as controls.
Patients were selected in our study after fulfilling the following criteria: age greater than 18 years, not received any pervious chemotherapy or radiotherapy with normal cardiac functions, and adequate liver and kidney functions without comorbidity. Patients experiencing other malignancies were excluded from the study.
All patients were subjected to the following:
- Before starting of the DLBCL treatment, every patient underwent baseline full history taking, complete physical examination including B symptoms, and routine laboratory studies including complete blood count, ESR, serum lactate dehydrogenase level, bone marrow examination, and kidney and liver function tests.
- SerumYKL-40 estimation.
- Review and confirmation of diagnostic pathology. Immunohistochemistry was done using a panel of monoclonal antibodies including: CD3, CD20, CD30, and CD15.
- Immunohistochemistry for YKL-40.
- Radiological assessment, including computed tomography (CT) scan of the neck, thorax, abdomen, and pelvis according to the site of involvement and echocardiography.
Diagnosis and staging
DLBCL was diagnosed based on histopathological examination of lymph nodes and/or extranodal tissue biopsy specimen according to the 2016 revision of the WHO . Patients were staged according to the Ann Arbor staging system with Cotswolds modifications . Patients’ performance status was assessed using Eastern Cooperative Oncology Group performance status . International Prognostic Index (IPI) was used for determining the prognosis of DLBCL .
Serum YKL-40 determination
Peripheral venous blood samples were collected at the time of the diagnosis and before the start of treatment in a sterile test tube. The samples were centrifuged at centrifugation for 15 min at 1000g. The aliquots were stored at less than or equal to −20°C. Serum YKL-40 concentrations were measured using a commercially available, quantitative sandwich enzyme immunoassay technique (Quantikine ELISA, Human Chitinase 3-like 1 Immunoassay, Catalog Number DC3L10; R&D systems, Minneapolis, Minnesota, USA) in accordance with the manufacturer’s instructions. The sensitivity ranged from 1.25 to 8.15 pg/ml. Intra-assay coefficient of variation (CV)% and interassay CV% are 4.3–4.7% and 5.3–6.9%, respectively.
Paraffin-embedded lymphoma specimens were cut to 6-mm thickness and processed for the staining of YKL-40. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide for 20 min. For antigen retrieval, slides were heated in a microwave oven for 10 min in 10 mmol/l citrate buffer, pH 6.0. Nonspecific binding was prevented by incubation with blocking buffer at room temperature for 30 min The sections were then incubated at 4°C overnight with primary antibodies against YKL-40 (1 : 150 dilution, rabbit polyclonal IgG; Quidel, San Diego, CA, USA). After a concise wash with phosphate-buffered saline, the sections were incubated for 30 min with a peroxidase-labeled polymer conjugated to anti-rabbit immunoglobulins (DAKO EnVision System/HRP K4010; DAKO Produktionsvej, Glostrup, Denmark). The final detection was performed using 3,3′-diaminobenzidine as the chromogen and 3% hematoxylin as the counter stain. Breast cancer tissue was used as positive control (immunereactivity was noted in the tumor cells). A negative control without a primary antibody was used.
YKL-40 staining was based on cytoplasmic analysis and evaluated as sum of two scores based on the percent and intensity of positive stained cells. The scores for percentage of cells stained is as follows: no staining cells is 0 points, 10% of cells stained is one point, 11–50% of cells stained is two points, and 50% of cells stained is three points. The intensity of staining is scored as follow: no staining is zero points, weak staining is one point, moderate staining is two points, and strong staining is three points. Thus, YKL-40 scores were further classified into three groups: negative/low (0–2 points), medium (3–4 points), and high (5–6 points) of YKL-40 staining. For statistical analysis, we consider medium and high score as positive and low or negative score as negative.
All patients were treated with six cycles of chemoimmunotherapy R-CHOP protocol . The protocol recycled every 21 days. Before every cycle of chemotherapy, complete clinical examination and complete hematological workup were done, and toxicity was evaluated according to National Cancer Institute (NCI) toxicity criteria .
A total of 18 (30%) patients received radiotherapy 2 weeks after the end of chemotherapy and with the assurance of bone marrow recovery. Radiotherapy was delivered for sites of initial bulky disease and extranodal sites whenever feasible. Radiation dose was 36 Gy delivered in 1.8 Gy daily fractions, 5 days a week. Radiotherapy fields were limited to regions of initial involvement.
Evaluation of tumor response
Cheson’s criteria were used to define the response to treatment . Patients’ response to initial therapy was assessed at midtreatment unless response was clinically apparent (three to four cycles) by CT scan to identify nonresponse or progression despite therapy. Six courses were completed if greater than or equal to 50% partial remission or complete remission has been achieved. Progressive-free survival (PFS) is described as the time from entry into a study until lymphoma progression or death as a result of any cause. Overall survival (OS) is identified as the time from entry into the clinical trial until death as a result of any cause or last follow up .
Patients had been reviewed clinical and by routine investigations every 3 months and by CT scan every 6 months for 36 months after treatment . The follow-up period ranged between 3 and 36 months, with a median of 22 months.
The collected data were analyzed using SPSS (version 17 software; SPSS Inc., Chicago, Illinois, USA). Comparison of continuous data between two groups was made by Mann–Whitney test, and Kruskal–Wallis were used for comparison between three groups for nonparametric data. Fisher’s exact and χ2-tests were used for comparison between categorical data. The sensitivity, specificity, and accuracy were calculated by using receiver-operating characteristic analysis. Survival analysis was done using Kaplan–Meier method, and comparison between two survival curves was done using log-rank test. The accepted level of significance in this work was stated at 0.05 (P≤0.05 was considered significant).
| Results|| |
The characteristics of the study population are presented in [Table 1].
The expression of YKL-40 in 60 DLBCL specimens was investigated by immunohistochemistry. YKL-40 was positive in 40 (66.67%) of 60 DLBCL specimens. YKL-40 positive expression was detected significantly in patients greater than 60 years (P<0.0001), advanced stage (P=0.0212), and advanced IPI (P=0.0003) ([Table 2] and [Figure 1]A–[Figure 1]D).
|Table 2 Relationship between YKL-40 expression and different clinicopathological parameters|
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|Figure 1 Cases of DLBCL showed. A: Negative expression of YKL-40 immunostaining (X400). B: Low expression of YKL-40 immunostaining (X400). C: Medium expression of YKL40 immunostaining (x400). D: Strong expression of YKL-40 immunostaining (x400).|
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Serum YKL-40 levels were significantly higher in patients with DLBCL, patients who died, and YKL-40 positive expressed patients when compared with the controls, alive patients, and negative expression patients (P=0.0000). Moreover, an increase in serum YKL-40 was observed with increase in the patients staging (P=0.0098) ([Table 3]).
For diagnosis of DLBCL, receiver-operating characteristic curve analysis showed that serum YKL-40 at a cutoff value of greater than or equal to 95.5 ng/ml had a sensitivity of 70%, a specificity of 95%, a positive predictive value of 97.67%, and a negative predictive value of 51.35%, and AUC was 0.897 ([Table 4] and [Figure 2]).
|Table 4 Sensitivity, specificity, positive prediction value, and negative prediction value of serum YKL-40 level among the studied patients|
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In patients with DLBCL, PFS and OS rates significantly decreased with increased serum levels of YKL-40 above the cutoff level as well as in YKL-40 positive expressed patients. Progression-free and OS rates significantly decreased with increase in the patients staging ([Table 5], [Table 6] and [Figure 3] and [Figure 4]).
|Table 5 Progression-free survival probability for different groups of patients|
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|Figure 3 Kaplan–Meier analysis of the progression free survival probability.|
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| Discussion|| |
B-cell lymphomas represent most NHL cases, and of these, DLBCL is the major subtype . Nearly half of patients with aggressive NHL will not be cured by standard treatment; consequently it is critical to recognize substitute markers for treatment response and outcome .
Angiogenesis is a multistep process having a vital function in the progression and metastasis of various tumors, including hematolymphoid malignancies . YKL-40 is a potent angiogenic factor capable of promoting tumor vascularization controlled by endothelial cells and maintenance of vascular integrity continued by smooth muscle cells, both of which are associated with a more aggressive cancer type . YKL-40 has been found to be a potential serum tumor marker for the evaluation of genesis and progression and prognosis of many kinds of human malignant tumors, such as lymphomas .
In this study, YKL-40 protein was found to be expressed in 66.67% of DLBCL specimens. YKL-40 positive expression was detected significantly in patients greater than 60 years, advanced stage, and advanced IPI, implying a close relation of YKL-40 positive expression with poor prognosis of patients with DLBCL. Therefore, YKL-40 expression emerges as a predictable marker for DLBCL.
In this study, serum YKL-40 levels were significantly higher in patients with DLBCL, patients who died, and YKL-40 positive expressed patients when compared with the controls, alive patients, and negative expression patients. Moreover, an increase in serum YKL-40 was observed with increase in the patients’ staging.
Biggar et al.  found that serum levels of YKL-40 are increased in pretreatment patients with Hodgkin lymphoma and in patients with more advanced stages of the disease. El-Galaly et al.  found in their study on patients with NHL that YKL-40 levels increased significantly in patients obtaining complete remission than in patients not achieving remission.
Shao et al.  showed a close connection between tissue expression and serum levels of YKL-40 in the same malignant patients. YKL-40 expression in malignant tissue could reveal the levels of serum concentration in the blood. So, the measurement of serum levels of YKL-40 as a cancer biomarker might have a promising role value in the diagnosis and prognosis of malignancies. The role of YKL-40 in tumor angiogenesis may suggest a novel therapeutic agent for targeting YKL-40.
Kazakova et al.  indicated the YKL-40 mRNA level and protein expression in the tumor site and in the serum of patients with glioma equivalent with the malignant stage of the tumor, signifying a possible unfavorable prognostic effect of YKL-40 in tumor pathogenesis.
In this study, PFS and OS rates significantly decrease in patients with increased serum levels of YKL-40 above the cutoff level and YKL-40 positive expressed patients. Progression-free and OS rates significantly decrease with increase in the patients staging. Thus, the positive tissue expression or elevated serum levels of YKL-40 are suggestive of an unfavorable response in DLBCL patients.
Johansen et al.  observed in their study on gastrointestinal cancer that participants with increased plasma YKL-40 had shorter survival than participants with low-plasma YKL-40. They hypothesized that cancer cells with elevated production of YKL-40 have a more aggressive phenotype with increased proliferation and differentiation rate and a high metastatic potential, all factors linked with short survival.
Shao et al.  used a multidisciplinary approach to identify YKL-40 mechanisms promoting tumor growth and proliferation. They explained that elevated serum levels of YKL-40 are associated with poorer prognosis and shorter disease-free survival in patients with broad types of cancers.
Pelloski et al.  found that YKL-40 expression is associated with poorer response to radiation and shorter OS in glioblastoma.
YKL-40 stimulates the phosphoinositide 3-kinase (PI3K)/AKT pathway and the Ras signaling pathway as well as the mitogen-activated protein kinase, both of which are critical pathways connected with mitogenesis and cell survival . Neutralizing antibody against YKL-40 suppresses the angiogenic activity . YKL-40 controls cellular and tissue responses through IL-13 receptor α2 . Moreover, it is proposed that YKL-40 contributes in inflammatory processes which control vital features of tumor proliferation, cell death, progression and matrix remodeling and then encourages tumor growth and metastasis .
In lymphoma malignancies, He et al. and Karin and Greten  have postulated that YKL-40 is a growth and differentiation factor for malignant cells, protects them from apoptosis, and has a role in angiogenesis and tissue repair. YKL-40 is regulated by IL-6, a B-cell growth and differentiation factor with a main role in lymphoma. Plasma IL-6 levels are increased in patients with NHL, and high levels are probably related with an unfavorable prognosis ,.
| Conclusion|| |
The serum level or tissue expression of YKL-40 could be a valuable prognostic marker and may provide insight into creating a new therapeutic modality.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Swerdlow S, Campo E, Harris NL, editors. International agency for research on cancer. WHO classification of tumours of haematopoietic and lymphoid tissue
. Geneva, Switzerland: World Health Organization 2008.
Campo E, Swerdlow SH, Harris NL, Pileri S, Stein H, Jaffe ES. The 2008 WHO classification of lymphoid neoplasms and beyond: evolving concepts and practical applications. Blood
Ninan MJ, Wadhwa PD, Gupta P. Prognostication of diffuse large B-cell lymphoma in the rituximab era. Leuk Lymphoma
Folkman J. Role of angiogenesis in tumor growth and metastasis. Semin Oncol
Griffioen AW, Molema G. Angiogenesis: potentials for pharmacologic intervention in the treatment of cancer, cardiovascular diseases, and chronic inflammation. Pharmacol Rev
Raica M, Cimpean AM, Ribatti D. Angiogenesis in premalignant conditions. Eur J Cancer
Ruan J, Hajjar K, Rafii S, Leonard JP. Angiogenesis and antiangiogenic therapy in non-Hodgkin’s lymphoma. Ann Oncol
Fusetti F, Pijning T, Kalk KH, Bos E, Dijkstra BW. Crystal structure and carbohydrate-binding properties of the human cartilage glycoprotein-39. J Biol Chem
Hu B, Trinh K, Figueira WF, Price PA. Isolation and sequence of a novel human chondrocyte protein related to mammalian members of the chitinase protein family. J Biol Chem
Renkema GH, Boot RG, Au FL, Donker-Koopman WE, Strijland A, Muijsers AO et al.
Chitotriosidase, a chitinase, and the 39-kDa human cartilage glycoprotein, a chitin-binding lectin, are homologues of family 18 glycosyl hydrolases secreted by human macrophages. Eur J Biochem
Recklies AD, White C, Ling H. The chitinase 3-like rote in human cartilage glycoprotein 39(HC-gp39) stimulates proliferation of human connective-tissue cells and activates both extra cellular signal-regulated kinase-and protein kinaseB-mediated signaling pathways. Biochem J
Scully S, Yan W, Bentley B, Cao QJ, Shao R. Inhibitory activity of YKL-40 in mammary epithelial cell differentiation and polarization induced by lactogenic hormones: a role in mammary tissue involution. PloS One
Malinda KM, Ponce L, Kleinman HK, Shackelton LM, Millis AJ. Gp38k, a protein synthesized by vascular smooth muscle cells, stimulates directional migration of human umbilical vein endothelial cells. Exp Cell Res
Nishikawa KC, Millis AJ. gp38k (CHI3L1) is a novel adhesion and migration factor for vascular cells. Exp Cell Res
Shao R, Hamel K, Petersen L, Cao QJ, Arenas RB, Bigelow C et al.
YKL-40, a secreted glycoprotein, promotes tumor angiogenesis. Oncogene
Nielsen KR, Steffensen R, Boegsted M, Baech J, Lundbye-Christensen S, Hetland ML. Promoter polymorphisms in the chitinase 3-like1gene influence the serum concentration of YKL-40 in Danish patients with rheumatoid arthritis and in healthy subjects. Arthritis Res Ther
Francescone RA, Scully S, Faibish M, Taylor SL, Oh D, Moral L et al.
Role of YKL-40 in the angiogenesis, radio resistance, and progression of glioblastoma. J Biol Chem
Horbinski C, Wang G, Wiley CA. YKL-40 is directly produced by tumor cells and is inversely linked to EGFR in glioblastomas. Int J Clin Exp Pathol
Thom I, Andritzky B, Schuch G, Burkholder I, Edler L, Johansen JS et al.
Elevated pretreatment serum concentration of YKL-40 An independent prognostic biomarker for poor survival in patients with metastatic non small cell lung cancer. Cancer
Johansen JS, Schultz NA, Jensen BV. Plasma YKL-40: a potential new cancer biomarker? Future Oncol
Swerdlow SH, Campo E, Pileri SA, Harris NL, Stein H, Siebert R et al.
The 2016 revision of the World Health Organization classification of lymphoid neoplasms. Blood
Lister TA, Crowther D, Sutcliffe SB, Glatstein E, Canellos GP, Young RC et al.
Report of a committee convened to discuss the evaluation and staging of patients with Hodgkin’s disease: cotswolds meeting. J Clin Oncol
Oken MM, Creech RH, Tormey DC, Horton J, Davis TE, McFadden ET, Carbone PP. Toxicity and response criteria of the Eastern Cooperative Oncology Group. Am J Clin Oncol
The International Non-Hodgkin’s Lymphoma Prognostic Factors Project. A predictive model for aggressive non-Hodgkin’s lymphoma. N Engl J Med
Cheson BD, Horning SJ, Coiffier B, Shipp MA, Fisher RI, Connors JM et al.
Report of an international workshop to standardize response criteria for non-Hodgkin’s lymphomas. J Clin Oncol
Cheson BD, Pfistner B, Juweid ME, Gascoyne RD, Specht L, Horning SJ et al.
International Harmonization Project on Lymphoma. Revised response criteria for malignant lymphoma. J Clin Oncol
Pedersen LM, Klausen TW, Davidsen UH, Johnsen HE. Early changes in serum IL-6 and VEGF levels predict clinical outcome following first line therapy in aggressive non-Hodgkin ’s lymphoma. Ann Hematol
Shao R. YKL-40 acts as an angiogenic factor to promote tumor angiogenesis. Front Physiol
El-Galaly TC, Bilgrau AE, Gaarsdal E, Klausen TW, Pedersen LM, Nielsen KR et al.
Circulating tumor necrosis factor-α and YKL-40 level is associated with remission status following salvage therapy in relapsed non-Hodgkin lymphoma. Leuk Lymphoma
Biggar RJ, Johansen JS, Smedby KE, Rostgaard K, Chang ET, Adami HO et al.
Serum YKL-40 and interleukin 6 levels in Hodgkin lymphoma. Clin Cancer Res
Kazakova MH, Staneva DN, Koev IG, Staikov DG, Mateva N, Timonov PT et al.
Protein and mRNA levels of YKL-40 in high-grade glioma. Folia Biol (Praha)
Johansen JS, Bojesen SE, Mylin AK, Frikke-Schmidt R, Price PA, Nordestgaard BG. Elevated plasma YKL-40 predicts increased risk of gastro-intestinal cancer and decreased survival after any cancer diagnosis in the general population. J Clin Oncol
Pelloski CE, Mahajan A, Maor M, Chang EL, Woo S, Gilbert M et al.
YKL-40 expression is associated with poorer response to radiation and shorter overall survival in glioblastoma. Clin Cancer Res
He CH, Lee CG, Dela Cruz CS, Lee CM, Zhou Y, Ahangari F et al.
Chitinase 3-like 1 regulates cellular and tissue responses via IL-13 receptor α 2. Cell Rep
Johansen JS. Studies on serum YKL-40 as a biomarker in diseases with inflammation, tissue remodeling, fibrosis and cancer. Dan Med Bull
Karin M, Greten FR. NF-kappa β: linking inflammation and immunity to cancer development and progression. Nat Rev Immunol
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]