DiagnosticUtility of Interleukin-6 Expression by Immunohistochemistry in Differentiating Castleman Disease Subtypes and Reactive Lymphadenopathies

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1 474 Available online at Annals of Clinical & Laboratory Science, vol. 46, no. 5, 2016 DiagnosticUtility of Interleukin-6 Expression by Immunohistochemistry in Differentiating Castleman Disease Subtypes and Reactive Lymphadenopathies Ginell R. Post, Robert C. Bell*, Anwar Rjoop, Rodolfo Henrich Lobo, Youzhong Yuan, and Steven R. Post Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, AR, USA Abstract. The objective of the study was to evaluate the expression pattern of interleukin-6 (IL-6) to determine its utility in differentiating Castleman Disease subtypes and reactive lymphadenopathies. Paraffinembedded tissue blocks from 20 cases referred for assessment of Castleman Disease (CD) and 4 cases of reactive hyperplasia were selected for immunohistochemical staining with an IL-6 antibody. Six pathologists evaluated the hematoxylin and eosin stained tissue sections and IL-6 expression pattern. Of 20 CD referral cases, the pathologic diagnosis was CD in 14 cases and included 6 hyaline-vascular (HV-CD), 6 plasma cell (PC-CD) and 2 mixed type -CD cases. The remaining 6 referral cases showed morphologic features consistent with reactive lymphadenopathy. Patients with non-cd, reactive lymphadenopathies had clinical and/or laboratory features of systemic lupus erythematosus, Hashimoto s disease, viral infection or chronic cellulitis. The pattern of IL-6 expression differed between CD subtypes and non-cd cases. In PC-CD, IL-6 expression was detected in plasma cells and vascular endothelial cells; whereas IL-6 immunoreactivity was detected primarily in vascular endothelial cells in HV-CD. Interfollicular plasma cells were prominent in PC-CD and reactive lymphadenopathies; however, IL-6 expression was significantly increased in PC-CD compared to reactive lymph nodes. Together with morphologic features, the expression pattern of IL-6 detected by immunohistochemistry is helpful to distinguish CD subtypes and reactive mimics. Key words: Interleukin-6, immunohistochemistry, Castleman Disease. Introduction Castleman Disease (CD) is a rare, reactive lymphoproliferative disorder. Histopathologic findings were first described in 1956 by Dr. Benjamin Castleman in lymph nodes excised from patients with mediastinal lymphadenopathy [1]. In subsequent years, Fendrig and Schillings and Keller et al. described two pathologic variants of CD; termed hyaline vascular (HV-CD) and plasma cell (PC- CD) [2,3]. Current CD classification incorporates histopathology, clinical features, probable etiologic agent(s), and associated lesions. Disease management includes surgical excision, radiotherapy, rituximab/steroid therapy, lymphoma type therapy or anti-interleukin-6 (IL-6) therapy [4,5]. The risk of developing malignancy differs amongst CD *Current affiliation: Department of Pathology, University of Arizona, Tucson, AZ, USA. Address correspondence to Ginell R. Post, MD, PhD, Associate Professor, Department of Pathology, University of Arkansas for Medical Sciences, 4301 W, Markham St., slot 502, Little Rock, AR, 72205, USA; phone: ; fax: ; e mail: gpost@uams.edu subtypes. For example, HV-CD patients are at risk for developing follicular dendritic cell neoplasms and/or vascular/stromal neoplasms. PC-CD is associated with POEMS syndrome (plasma cell neoplasms), and HHV-8 positive CD is associated with aggressive lymphoma and Kaposi sarcoma [6]. Therefore, it is important for a pathologist to recognize CD and distinguish the morphologic subtypes. Morphologically, HV-CD is characterized by small, inactive germinal centers with dysplastic follicular dendritic cells and concentric proliferation of mantle zone lymphocytes without parafollicular plasmacytosis. The germinal centers and parafollicular areas often show vascular proliferation and hyaline fibrosis [5,6]. PC-CD morphology often shows hyperplastic follicles admixed with involuted follicles. In contrast to HV-CD, the interfollicular areas characteristically show aggregates of polytypic plasma cells [5,6]. The presence of hyaline vasculartype follicles in cases with increased interfollicular /16/ by the Association of Clinical Scientists, Inc.

2 Table 1. Comparison of Clinical and Laboratory Features. IL-6 expression by immunohistochemistry in Castleman Disease 475 Pathologic Diagnosis Age Male: % Multi- Associated Diseases Symptoms (average Female centric ±SD) Castleman (14) PC (6) 47±9 5:1 66% POEMS (67%) 66% Plasmacytoma (16%) PC Unicentric (1) PC- Multicentric (5) HV (6) 28±8 1:2 33% None 16% HV-Unicentric (4) HV-Multicentric (2) Mixed (2) 30±6 2:0 100% 50% Reactive LN (10) 56±9 2:3 80% SLE (2) 20% Hashimoto s (1) Renal transplant, CMV (1) EBV (1) Chronic cellulitis (1) IgG4-related adenopathy (1) Chronic Sclerosing Sialoadenitis (1) Sleep apnea (1) plasma cells has led some pathologists to designate these cases as mixed type or CD-not otherwise specified [6]. However, there are no standardized diagnostic criteria, including immunohistochemical staining patterns, to distinguish CD subtypes from reactive conditions, including viral and autoimmune-related lymphadenopathies. Despite heterogeneous morphologic and clinical features, the pathophysiology of CD involves IL-6 [7,8]. IL-6 promotes B- and Th2-lymphocyte and plasma cell proliferation, induces vascular endothelial growth factor (VEGF) secretion resulting in increased lymph node vascularity, and mediates autoimmune and pro-inflammatory responses resulting in systemic symptoms [5,8]. Surgical excision of CD lymph nodes or therapeutic intervention with anti-il6 or anti-il6r monoclonal antibodies abrogates the symptomology and associated lymphadenopathy in CD [4,5]. Although the role of IL-6 in CD pathophysiology is established, the diagnostic utility of examining IL-6 expression in different CD subtypes and other reactive lymph nodes has not been systematically investigated. In this retrospective analysis, we used an immunohistochemical approach to assess IL-6 expression patterns in CD and non-cd lymph node biopsies. Materials and Methods The University of Arkansas for Medical Sciences (UAMS) is an international referral center for Castleman disease. In this IRB-approved, retrospective case analysis, we reviewed 20 tissue biopsies, including 18 excisional biopsies and 2 needle cores (mediastinal lymph nodes), received in consultation for CD from 2005 to Four additional non-cd cases were chosen for comparison. Clinical notes and imaging studies were reviewed to determine the presence or absence of systemic symptoms (fever, weight loss and/or night sweats), the extent of adenopathy (unicentric vs. multicentric disease) and previous treatment. Laboratory tests were evaluated, as available, including complete blood count, serum IL-6 levels, serum VEGF levels, acute phase markers (ESR and/or CRP), serum protein electrophoresis results, serologic/ molecular tests for HIV, HHV-8, CMV, and autoimmune disorders (eg ANA). Formalin-fixed and paraffin-embedded tissue specimens were sectioned at 4 µm and stained with hematoxylin and eosin (H&E) or incubated with a rabbit polyclonal IL-6 antibody (Santa Cruz; sc-7920) at a 1:100 dilution. Tissue sections were then incubated with goat anti-rabbit followed by avidin-biotin-peroxidase complex (Dako) and developed with diaminobenzidine. As a positive control, prostate tissue sections were incubated with the same concentration of IL-6 and secondary antibodies. In prostate tissue, IL-6 immunoreactivity was localized to

3 476 Annals of Clinical & Laboratory Science, vol. 46, no. 5, 2016 Figure 1. Representative photomicrographs and IL-6 expression in PC-CD, HV-CD and reactive lymph node biopsies. A-D: Histopathology and IL-6 expression in PC-CD: A and C: H&E stained sections at 20x and 200x show expanded interfollicular zones with increased plasma cells; B and D: The IL-6 Immunohistochemical stain shows that the plasma cells and blood vessel endothelial cells are positive for IL-6. E-H: Histopathology and IL-6 expression in HV-CD: E and G: H&E stained sections at 20x and 200x show hyalinized germinal centers, prominent interfollicular vessels (arrow; 200x) and a relative paucity of interfollicular plasma cells; F and H: The blood vessel endothelial cells are positive for IL-6 (arrow, 200x). I-L: Histopathology and IL-6 expression in a reactive lymph node. I and J: H&E stained sections at 20x and 200x show hyperplastic and regressed follicles and abundant parafollicular plasma cells. J and L: The corresponding IL-6 IHC shows that the plasma cells are negative for IL-6. the epithelial cells [9]. Using IL-6 immunoreactivity in prostate tissue as a positive control, staining intensity was scored as negative (0 points), dim positive (1 point) or positive (2 points) in germinal center cells, vascular endothelial cells and parafollicular plasma cells by pathologists blinded to the final pathologic and clinical diagnosis. The average score assigned by each pathologist was calculated and compared by 2 way ANOVA followed by a Tukey post-hoc test using GraphPad Prism 6 (GraphPad Software, Inc). Differences were considered significant if p<0.05.

4 IL-6 expression by immunohistochemistry in Castleman Disease 477 Results Morphologic Diagnosis and Patient Characteristics. In this retrospective analysis, 20 biopsies from patients referred to UAMS from 2005 to 2015 for assessment of CD were reviewed. Morphologic features evaluated on H&E stained tissue sections included: 1) Size, number and type of follicles (e.g., primary and/or secondary, hyperplastic, twinning); 2) Germinal center morphology (hyalinized with/without penetrating sclerotic vessels); 3) Mantle zone lymphocyte polarization, and/ or concentric layering; 4) Interfollicular vascular proliferation/ hyalinization, vasculitis, or fibrosis; and 5) Presence/Distribution of plasma cells. Using these morphologic features and available previous immunohistochemical stains, 14 cases were morphologically consistent with CD, and 6 cases considered non-diagnostic for CD were classified as reactive lymphadenopathy. Of the CD cases, the pathologic diagnoses was PC-CD in 6 cases, HV- CD in 6 cases or mixed type CD in 2 cases. Four additional, non-cd cases showing reactive lymphadenopathy were chosen for comparison. Since CD is a clinicopathologic entity, patient demographics, clinical presentation, imaging studies and associated diseases were evaluated. In this cohort, the average age of patients diagnosed with PC-CD was 47±9 years (Table 1). Five of six patients (83%) were male, had a multicentric disease, and were symptomatic (fever, weight loss, and night sweats). Associated diseases included POEMS (4 cases) or plasmacytoma (1 case). Four patients were previously treated. Laboratory abnormalities at the time of morphologic evaluation included anemia (16% of cases), elevated ESR or CRP (83%), elevated serum IL-6 (67%), elevated VEGF (83%), and polyclonal hypergammaglobulinemia (33%). None of the patients were positive for HIV or HHV-8. The average age of patients with HV-CD was 28±8 years with a male:female ratio of 1:2. HV-CD cases were unicentric in 4 cases and multicentric in 2 cases. Patients were asymptomatic in 83% of cases. Laboratory data was available for 4 patients and included anemia (67%), elevated ESR or CRP (20%), and elevated serum IL-6 (25%). None of the patients had been previously treated, exhibited hypergammaglobulinemia or increased serum VEGF. In two cases, the morphologic diagnosis was mixed-type CD. Both patients were male with multicentric involvement. One patient was symptomatic, and both patients had received previous therapy. Associated laboratory abnormalities at the time of referral included anemia, elevated ESR or CRP (1 patient), elevated serum IL-6 and VEGF in one patient. Neither patient had hypergammaglobulinemia or was positive for HHV-8 or HIV. Due to the low number of patients in this diagnostic group, mixed type CD was excluded from further analyses. Ten lymph node biopsies were morphologically consistent with reactive lymphadenopathy. The average age of patients with reactive lymphadenopathy was 56±9 years with the majority of patients being female (60%). Generalized lymphadenopathy was present in 80% of cases. Clinically, associated autoimmune disorders were present in three patients and included systemic lupus erythematosus (SLE), Hashimoto s disease or IgG4-related disease. Viral infection (2 patients) or chronic cellulitis (1 patient) was present in the remaining patients. One patient in this group had a history of previously treated multicentric PC-CD. Associated laboratory abnormalities included anemia (40%), elevated ESR or CRP (67%), elevated serum IL-6 (62%), elevated VEGF (25%) and polyclonal hypergammaglobulinemia (50%). Patient characteristics are presented in Table 1. IL-6 Expression. IL-6 stained tissue sections (Figure 1) of 24 selected cases were reviewed by six pathologists. IL-6 staining intensities in germinal center cells (GC), blood vessel endothelial cells (EC), and plasma cells (PC) were scored as negative (0), dim positive (1) or positive (2). A 2-way ANOVA was used to compare staining intensities, and a significant effect was found between cell type and pathologic diagnosis, and a Tukey s post-hoc test was used to determine significant differences (α 0.05) between individual intensities (Figure 2). In PC-CD, IL-6 reactivity was detected in both endothelial cells and plasma cells. However, in HV- CD cases, IL-6 expression was primarily limited to endothelial cells. IL-6 expression was dim positive or negative in all cell types in the majority of reactive lymph node cases (Figure 2). There were no significant differences in the staining intensity of IL-6 in germinal center cells of the different CD

5 478 Annals of Clinical & Laboratory Science, vol. 46, no. 5, 2016 Figure 2. Intensity of IL-6 Staining in PC-CD, HV-CD and reactive lymph node biopsies. IL-6 immunohistochemistry intensity in germinal center cells (GC), vascular endothelial cells (EC) and plasma cells (PC) in plasma cell CD (PC-CD), hyaline vascular CD (HV-CD) and non-cd disease cases (Reactive) as assessed by six pathologists. Key: 0= negative for IL-6; 1 = dim positive; 2 = positive for IL-6. The scores were averaged and the means + SD are shown. Graph depicts individual values with mean ± 95% CI, with significant differences determined by Tukey s multiple comparisons test shown in the table. subtypes or reactive lymph nodes. Although IL-6 immunoreactivity tended to be higher in the endothelial cells of CD cases compared to reactive lymph nodes, this difference was not statistically significant. In contrast, IL-6 expression was significantly higher in plasma cells in PC-CD compared to HV- PC (p<0.0001) and reactive lymphadenopathies (p<0.0001). Discussion CD is a rare, heterogeneous lymphoproliferative disorder. Although there are no standard diagnostic criteria, CD can be divided based on histopathologic and clinical features. PC-CD, including multicentric or unicentric presentations, is the least common clinicopathologic entity, comprising approximately 10-20% of cases. However, due to the referral nature of our institution, 6 of 20 referral cases were morphologically and clinically consistent with PC-CD (Table 1). Histologically, PC-CD showed altered, but non-effaced lymphoid architecture with hyperplastic and regressed follicles and increased interfollicular plasma cells [4-6]. As in previous studies, the majority of patients were male, older than patients with HV-CD variant, and had elevated VEGF, ESR and CRP [5,6,10]. Six of 20 cases were morphologically and clinically consistent with HV-CD. Lymph node sections in HV-CD showed increased numbers of regressed and hyalinized germinal centers, prominent interfollicular vessels and a relative lack of interfollicular plasma cells [2-6]. Clinically, the majority of patients had unicentric involvement and were asymptomatic (Table 1; [4-6]). Patients with non- CD, reactive lymphadenopathies had clinical and/ or laboratory features of SLE (2), Hashimoto s disease (1), viral infection (2) or chronic cellulitis (1) (Table 1). Hyperplastic follicles, polarized mantle zones, and increased interfollicular plasma cells were common in reactive lymph nodes; however these features were also seen in PC-CD. The majority of non-cd patients had generalized lymphadenopathy, elevated ESR or CRP and elevated serum IL-6. Thus, while CD subtypes have some distinguishing pathologic features, there is substantial clinical and morphologic overlap with reactive conditions.

6 IL-6 expression by immunohistochemistry in Castleman Disease 479 IL-6 is a pro-inflammatory cytokine that plays a central role in the pathophysiology of CD, and blockade of IL-6 and IL-6R signaling by monoclonal antibodies has been shown to have therapeutic benefit [5]. Studies have shown a correlation between serum IL-6 concentration, clinical features and IL-6 expression in CD lymph nodes, particularly in germinal center cells [7,8]. This correlation suggests that deregulated IL-6 expression in the lymph node may contribute to the difference in morphologic features and systemic manifestations of CD vs. reactive lymphadenopathies. In this study, we evaluated IL-6 immunohistochemical reactivity in CD and other reactive lymph node biopsies. Similar to Leger-Ravet et al. [7], our results show that IL-6 expression is more commonly detected in cases of CD disease compared to reactive non-cd lymph nodes, and the pattern of IL-6 expression differs amongst the different CD subtypes. While previous studies using immunohistochemistry and/or in situ hybridization showed increased IL-6 expression in germinal center cells of CD lymph nodes compared to hyperplastic lymph nodes [7,8], we did not detect significant differences in germinal center cell IL-6 immunoreactivity in this cohort. Rather, our results showed differences in the pattern of IL-6 expression outside the germinal centers. In HV-CD, the IL-6 expression is seen primarily in interfollicular blood vessel endothelial cells; whereas, IL-6 reactivity was detected in both blood vessels and plasma cells in the majority of PC-CD cases (Figures 1 and 2). IL-6 immunoreactivity in plasma cells in CD cases has not been previously reported [7,8]. Unlike HV-CD, which shows a relative paucity of interfollicular plasma cells, PC-CD and reactive conditions showed increased interfollicular plasma cells. Importantly, while IL-6 is expressed in interfollicular plasma cells in PC-CD, IL-6 expression in plasma cells is dim or absent in reactive lymph nodes, a feature that helps to distinguish these two entities. The differential expression pattern and intensity of IL-6 in CD subtypes and reactive lymph nodes suggests that inclusion of IL-6 in a panel of immunohistochemical stains increases diagnostic accuracy in CD diagnosis and subclassification. Acknowledgment The authors thank Ms. Jennifer James for performing IL-6 immunohistochemistry. This study was supported by funds from the Department of Pathology. References 1. Castleman, B., L. Iverson, and V.P. Menendez, Localized mediastinal lymphnode hyperplasia resembling thymoma. Cancer, (4): p Keller, A.R., L. Hochholzer, and B. Castleman, Hyalinevascular and plasma-cell types of giant lymph node hyperplasia of the mediastinum and other locations. Cancer, (3): p Flendrig, J. and P. Schillings, Benign giant lymphoma: the clinical signs and symptoms. Folia Medica Neerlandica, : p Casper, C., The aetiology and management of Castleman disease at 50 years: translating pathophysiology to patient care. Br J Haematol, (1): p van Rhee, F., K. Stone, S. Szmania, B. Barlogie, and Z. Singh, Castleman disease in the 21st century: an update on diagnosis, assessment, and therapy. Clin Adv Hematol Oncol, (7): p Cronin, D.M. and R.A. Warnke, Castleman disease: an update on classification and the spectrum of associated lesions. Adv Anat Pathol, (4): p Leger-Ravet, M.B., M. Peuchmaur, O. Devergne, J. Audouin, M. Raphael, J. Van Damme, P. Galanaud, J. Diebold, and D. Emilie, Interleukin-6 gene expression in Castleman's disease. Blood, (11): p Yoshizaki, K., T. Matsuda, N. Nishimoto, T. Kuritani, L. Taeho, K. Aozasa, T. Nakahata, H. Kawai, H. Tagoh, T. Komori, and et al., Pathogenic significance of interleukin-6 (IL- 6/BSF-2) in Castleman's disease. Blood, (4): p Royuela, M., M. Ricote, M.S. Parsons, I. Garcia-Tunon, R. Paniagua, and M.P. de Miguel, Immunohistochemical analysis of the IL-6 family of cytokines and their receptors in benign, hyperplasic, and malignant human prostate. J Pathol, (1): p Frizzera, G., P.M. Banks, G. Massarelli, and J. Rosai, A systemic lymphoproliferative disorder with morphologic features of Castleman's disease. Pathological findings in 15 patients. Am J Surg Pathol, (3): p