Is a Novel Marker for Follicular Dendritic Cell Tumors

Anatomic Pathology / D2-40 IN FOLLICULAR DENDRITIC CELL TUMORS

Podoplanin (D2-40) Is a Novel Marker for Follicular Dendritic Cell Tumors Hongbo Yu, MD, PhD, Joanna A. Gibson, MD, PhD, Geraldine S. Pinkus, MD, and Jason L. Hornick, MD, PhD Key Words: Immunohistochemistry; D2-40; Podoplanin; Follicular dendritic cell sarcoma; Soft tissue tumors DOI: 10.1309/7P8U659JBJCV6EEU

Podoplanin, recognized by monoclonal antibody D2-40, may be a useful marker for follicular dendritic cell (FDC) tumors. Paraffin sections of 125 dendritic cell, histiocytic, and spindle cell lesions were studied, including 11 FDC tumors, 5 interdigitating dendritic cell tumors, 10 histiocytic sarcomas, 5 Langerhans cell histiocytosis, 5 sinus histiocytosis with massive lymphadenopathy, 5 inflammatory pseudotumors of lymph node or spleen, 9 nodal Kaposi sarcomas, 6 inflammatory myofibroblastic tumors (IMTs), 29 gastrointestinal stromal tumors (GISTs), and 10 cases each of malignant peripheral nerve sheath tumor, leiomyosarcoma, monophasic synovial sarcoma (SS), and solitary fibrous tumor. All FDC tumors and Kaposi sarcomas showed strong immunoreactivity for podoplanin (predominantly membranous). Podoplanin expression was only occasionally observed in the other tumor types, including 7 GISTs (24%), 2 IMTs (33%), and 3 SS (30%), and was generally weak and cytoplasmic. Reactivity for podoplanin was more common in spindle cell GISTs (5/13 [38%]) than in epithelioid or mixed-type GISTs (2/16 [13%]). Podoplanin is a highly sensitive marker for FDC tumors and may be useful to help confirm the diagnosis in conjunction with conventional FDC markers, particularly in the differential diagnosis of dendritic cell and histiocytic lesions.

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Am J Clin Pathol 2007;128:776-782 DOI: 10.1309/7P8U659JBJCV6EEU

Podoplanin is a transmembrane glycoprotein expressed in a variety of human cell types, including glomerular epithelium, lymphatic endothelium, mesothelium, type I alveolar cells, and epithelial cells of the choroid plexus.1-3 Although the biologic function of podoplanin is not well understood, it has been implicated in the development of lymphatic vessels.4 Podoplanin has also been shown to promote platelet aggregation and may be involved in cancer cell invasion, metastasis, and malignant progression.5-7 Investigators have shown that podoplanin is homologous to T1a (also known as human Aggrus) of type I alveolar cells8,9 and the M2A antigen expressed in testicular germ cell tumors,10 and it is recognized by the recently commercially available monoclonal antibody D2-40.10 In the course of evaluating clinical cases, we have noticed that follicular dendritic cells (FDCs) within lymphoid follicles are strongly positive for podoplanin when using the D2-40 monoclonal antibody. However, podoplanin expression in FDC tumors has not previously been examined. The purpose of this study was to evaluate the expression and specificity of podoplanin in FDC tumors compared with other nodal and extranodal dendritic cell, histiocytic, and spindle cell neoplasms.

Materials and Methods Cases were retrieved from the surgical pathology files of Brigham and Women’s Hospital, Boston, MA, and the consultation files of Christopher D.M. Fletcher, MD, FRCPath (see acknowledgment). In total, 125 tumors were studied: 11 FDC tumors; 5 interdigitating dendritic cell tumors; 10 histiocytic sarcomas; 5 cases of Langerhans cell histiocytosis; 5 cases of © American Society for Clinical Pathology

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Abstract

Anatomic Pathology / ORIGINAL ARTICLE

Sections were counterstained with hematoxylin, dehydrated, and mounted. Isotype control studies (IgG1) were also performed using the same immunoglobulin concentration as the primary antibody. Immunoreactivity was graded semiquantitatively according to the percentage of positive tumor cells: 0, fewer than 5% of tumor cells reactive; 1+, 5% to 25% of tumor cells reactive; 2+, more than 25% to 50% of tumor cells reactive; 3+, more than 50% to 75% of tumor cells reactive; and 4+, more than 75% of tumor cells reactive. The intensity of staining was graded as weak, moderate, or strong, and the predominant staining pattern (cytoplasmic or membranous) was noted. This study was approved by the Brigham and Women’s Hospital Institutional Review Board.

Results The results of this study are summarized in ❚Table 1❚. Cases with more than 5% tumor cells reactive were considered positive. For internal controls, endothelial cells of lymphatic vessels were always strongly positive for podoplanin, as were FDCs within the germinal centers of lymph nodes ❚Image 1❚. IgG1 isotype control studies were negative. All FDC tumors (100%) showed strong immunoreactivity for podoplanin ❚Image 2❚. The extent of immunoreactivity varied among cases, with 4 cases showing 2+ staining, 2 showing 3+ staining, and 5 showing 4+ staining. The staining pattern in FDC tumor cells was largely membranous, with some cases showing cytoplasmic immunoreactivity as well. Similarly, all 9 cases of Kaposi sarcoma of lymph

❚Table 1❚ Podoplanin (D2-40) Immunoreactivity in Dendritic Cell, Histiocytic, and Other Spindle Cell Tumors Extent of Reactivity* Tumor Type Follicular dendritic cell sarcoma/tumor Interdigitating dendritic cell sarcoma/tumor Histiocytic sarcoma Langerhans cell histiocytosis Sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease) Inflammatory pseudotumor of lymph node/spleen Kaposi sarcoma of lymph node Inflammatory myofibroblastic tumor Malignant peripheral nerve sheath tumor Leiomyosarcoma Monophasic synovial sarcoma Solitary fibrous tumor Gastrointestinal stromal tumor Epithelioid Spindle cell Mixed *

No. of Cases

No. (%) Positive

0

1+

2+

3+

4+

Predominant Staining Pattern

11 5 10 5 5

11 (100) 0 (0) 2 (20) 0 (0) 0 (0)

0 5 8 5 5

0 0 0 0 0

4 0 1 0 0

2 0 0 0 0

5 0 1 0 0

Membranous — Cytoplasmic — —

5

1 (20)

4

0

1

0

0

Cytoplasmic

9 6 10 10 10 10 29 7 13 9

9 (100) 2 (33) 1 (10) 1 (10) 3 (30) 0 (0) 7 (24) 1 (14) 5 (38) 1 (11)

0 4 9 9 7 10 22 6 8 8

0 0 1 0 1 0 2 0 2 0

0 0 0 0 1 0 1 0 1 0

0 1 0 1 1 0 3 0 2 1

9 1 0 0 0 0 1 1 0 0

Membranous Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic — Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic

0, 25%-50% tumor cells reactive; 3+, >50%-75% tumor cells reactive; 4+, >75% tumor cells reactive.

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DOI: 10.1309/7P8U659JBJCV6EEU

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sinus histiocytosis with massive lymphadenopathy (RosaiDorfman disease); 5 inflammatory pseudotumors of lymph node (n = 4) or spleen (n = 1); 9 Kaposi sarcomas of lymph node; 6 inflammatory myofibroblastic tumors (IMTs); 29 gastrointestinal stromal tumors (GISTs), including 13 spindle cell, 7 epithelioid, and 9 mixed type; and 10 cases each of malignant peripheral nerve sheath tumor, leiomyosarcoma, monophasic synovial sarcoma, and solitary fibrous tumor. Representative H&E-stained slides and previously performed immunostains were reviewed by two of us (H.Y. and J.L.H.) in all cases to confirm the diagnoses, based on standard and widely accepted criteria.11,12 All FDC tumors evaluated were positive for CD21 and CD35, and 3 (27%) were positive for epithelial membrane antigen; all tumors were negative for S100 protein and keratin proteins. Immunohistochemical studies were performed on 4-µmthick, formalin-fixed, paraffin-embedded tissue sections using mouse monoclonal antibody clone D2-40 (Signet Laboratories, Dedham, MA). Following deparaffinization, slides were treated with 3% hydrogen peroxide to eliminate endogenous peroxidase activity. Antigen retrieval was performed using 0.01 mol/L of citrate buffer, pH 6.0 (Zymed Laboratories, South San Francisco, CA) in a pressure cooker. Slides were then incubated for 1 hour at room temperature with D2-40 antibody (1:100 dilution), then with peroxidase-labeled polymer conjugated to goat antimouse immunoglobulin antibody (EnVision+, DAKO, Carpinteria, CA; 30 minutes). Antibody localization was effected by using a peroxidase reaction with 3,3'-diaminobenzidine tetrahydrochloride (DAB+, DAKO) as the chromogen.

Yu et al / D2-40 IN FOLLICULAR DENDRITIC CELL TUMORS

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❚Image 1❚❚ Follicular dendritic cells within a germinal center in a reactive lymph node show strong immunoreactivity for podoplanin (D2-40) (×200).

Discussion FDCs reside in the germinal centers of lymphoid follicles and participate in the immune system by presenting and retaining antigens for B cells and stimulating B-cell proliferation and differentiation. Most investigators believe that FDCs are mesenchymal in origin, in contrast with interdigitating dendritic cells, which instead are derived from hematopoietic stem cells through the monocyte-macrophage lineage.11 Proliferation of FDCs occurs in various reactive

B

❚Image 2❚❚ A, Follicular dendritic cell tumor composed of spindle cells with fibrillary, eosinophilic cytoplasm showing a fascicular to storiform growth pattern and frequent admixed lymphocytes (H&E, ×400). B, Neoplastic cells exhibit strong, diffuse immunoreactivity for podoplanin (D2-40) in a membranous and cytoplasmic staining pattern (×400).

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nodes (100%) showed 4+ strong membranous staining for podoplanin ❚Image 3❚. Podoplanin expression was only occasionally observed in the other tumor types examined and, in contrast with the predominantly membranous staining pattern seen in FDC tumors and Kaposi sarcoma, reactivity was usually restricted to the cytoplasm. For example, 7 (24%) of 29 GISTs showed immunoreactivity for podoplanin, more commonly in GISTs of spindle cell type (5/13 [38%]) ❚Image 4❚ than in epithelioid GISTs (1/7 [14%]) or those of mixed type (1/9 [11%]). Moreover, the staining intensity was weak in epithelioid and mixed-type GISTs and variable in spindle cell GISTs, with a granular cytoplasmic staining pattern. Of the other histiocytic and dendritic cell neoplasms, only 2 (20%) of 10 histiocytic sarcomas were positive for podoplanin, 1 with 2+ weak cytoplasmic staining and 1 with 4+ moderate to strong staining in a variably cytoplasmic, dotlike, and focally membranous distribution. All IDC tumors, Langerhans cell histiocytosis, and sinus histiocytosis with massive lymphadenopathy cases were negative. Of the other spindle cell tumors examined, 3 (30%) of 10 monophasic synovial sarcomas ❚Image 5❚, 2 (33%) of 6 IMTs, 1 (10%) of 10 malignant peripheral nerve sheath tumors, and 1 (10%) of 10 leiomyosarcomas showed immunoreactivity for podoplanin. Of 5 inflammatory pseudotumors of lymph node, 1 (20%) showed 2+ staining of stromal cells. All solitary fibrous tumors were negative. In contrast with FDC tumors and Kaposi sarcoma, the intensity of staining for podoplanin was weak in all but 7 of the other tumors that were positive. Most of the positive cases showed only focal cytoplasmic staining.


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and neoplastic conditions, such as follicular hyperplasia, nodular lymphocyte predominant Hodgkin lymphoma, and angioimmunoblastic T-cell lymphoma. FDC tumors are rare neoplasms that most commonly involve lymph nodes, but may also arise at a variety of extranodal sites, most often in the gastrointestinal tract and mesentery, but also in the liver, spleen, tonsil, and a variety of other anatomic sites.11,13 FDC tumors occur in association with Castleman disease in 10% to 20% of cases, typically the hyaline vascular type.11 The behavior of FDC tumors is usually indolent, with local recurrence in 40% to 50% of cases and metastases in 25%, often following local recurrence.11 Extranodal location, large tumor size, and high mitotic rate have been associated with more aggressive clinical behavior.14 Cytologically and/or clinically malignant cases are classified as FDC sarcoma. Histologically, FDC tumors are composed of ovoid to spindled cells with syncytial, eosinophilic cytoplasm arranged in sheets, fascicles, and whorls, at times with a storiform growth pattern. The tumor cells are characteristically admixed with small lymphocytes, usually B cells. Given the rarity of these lesions, the diagnosis of FDC tumors is often challenging and requires immunophenotypic studies. By immunohistochemical analysis, the tumor cells are positive for the complement receptors CD21 and CD35 and for the IgE receptor CD23.11,14 However, these antigens are not entirely specific for FDC tumors and may also be expressed in various B-cell lymphomas.15,16 In addition, FDC tumors are usually positive for vimentin, desmoplakin,14 and clusterin,17,18 whereas epithelial membrane antigen is positive in 40% of cases19 and S-100 protein in a smaller subset.19 The latter 2 markers can raise problematic differential diagnostic considerations (ie, metastatic carcinoma and melanoma).

Staining for CD1a, lysozyme, myeloperoxidase, CD34, CD79a, CD3, CD30, HMB-45, and cytokeratins is consistently negative.11,14 The most distinctive feature on electron microscopy is the presence of numerous long, slender cytoplasmic processes, often connected by desmosomes.20 Prompted by the observation that FDCs within lymphoid follicles are strongly positive for podoplanin, we evaluated podoplanin immunoreactivity with the monoclonal antibody D240 in FDC tumors in comparison with other nodal and extranodal dendritic cell, histiocytic, and spindle cell tumors. Although our present study is the first to examine the expression of podoplanin in FDC tumors, a recent study by Schacht et al5 using normal human tissue microarrays also found that podoplanin is expressed by normal FDCs of lymphoid organs, including thymus, tonsils, and lymph nodes, using monoclonal antibody D2-40. In their study, staining of serial sections for the FDC marker CD21 and D2-40 confirmed that CD21+ FDCs express podoplanin. We found that all FDC tumors (100%) showed strong immunoreactivity for podoplanin, with a predominantly membranous staining pattern. Similar results were seen for Kaposi sarcoma of lymph nodes. Of the other histiocytic and dendritic cell tumors examined, only 2 histiocytic sarcomas (20%) were positive for podoplanin. In addition, podoplanin expression was only occasionally observed in the other spindle cell tumor types examined. In the other tumors, only cytoplasmic staining was seen. Moreover, the intensity of staining for podoplanin was weak in all but 7 of the other tumors that were positive. Thus, podoplanin (D2-40) is a novel marker for FDC tumors that may be clinically useful in diagnosis. Podoplanin is a protein that is strongly and selectively expressed in lymphatic endothelium. In normal tissues, it stains the endothelium of lymphatic channels but not of blood Am J Clin Pathol 2007;128:776-782



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❚Image 3❚❚ A, Kaposi sarcoma involving lymph node (H&E, ×200). B, Strong membranous reactivity for podoplanin (D2-40) is observed in lesional cells (×200).


B

C

D

❚Image 4❚❚ A, Gastrointestinal stromal tumor (GIST) composed of spindle cells with paranuclear vacuoles (H&E, ×400) and B, showing granular cytoplasmic staining for podoplanin (D2-40) (×400). Nearly 40% of spindle cell GISTs were at least focally positive for podoplanin. In contrast, nearly all epithelioid GISTs (C, H&E, ×400) were completely negative for podoplanin (D, ×400).

vessels. Previous studies have shown that D2-40 is positive in lymphangiomas, benign tumors of undisputed lymphatic origin, but not in most benign neoplasms and tumor-like lesions of putative blood vessel origin.21,22 In addition, strong podoplanin expression has also been reported in Kaposi sarcoma (of skin and gastrointestinal tract),2,22,23 kaposiform hemangioendothelioma,24 hobnail hemangioma,25 and a subset of angiosarcomas.2,22,26 To our knowledge, our study is the first to examine podoplanin expression in nodal Kaposi sarcoma. As expected, we also found diffuse podoplanin expression in all cases of Kaposi sarcoma involving lymph nodes. Although the extensive expression of podoplanin in both FDC tumors and Kaposi sarcoma could potentially pose problems in differential diagnosis, the use of a panel of markers (eg, 780 780


CD21 and CD35 for FDC tumors; human herpesvirus-8 and endothelial markers CD34 and CD31 for Kaposi sarcoma) can aid in distinguishing these entities. Recent studies have also shown that D2-40 may be a useful marker in demonstrating lymphatic invasion by tumors.27,28 Previous studies have shown that podoplanin is strongly expressed in seminomas5,10,29,30 and epithelioid mesotheliomas.3,26,31-34 In general, seminomas exhibit uniform, strong membranous staining for podoplanin, whereas the staining in other germ cell components is at most focal.30 Furthermore, podoplanin is highly specific and sensitive for epithelioid mesothelioma (compared with pulmonary adenocarcinoma). D2-40 usually shows a continuous membranous staining pattern in these tumor types.26 © American Society for Clinical Pathology


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Few previous studies have examined the expression of podoplanin in nonvascular spindle cell sarcomas. For example, Ordonez26 reported podoplanin expression in the epithelioid component of 4 of 6 biphasic synovial sarcomas. However, the spindle cell component in these tumors, as well as the 2 monophasic synovial sarcomas studied, did not react with this marker.26 This contrasts somewhat with our finding that a subset of monophasic synovial sarcomas shows limited staining. In another study, Kimura and Kimura35 examined podoplanin expression using D2-40 in 6 GISTs. In their study, all 6 cases evaluated (5 from stomach and 1 from small intestine) were reportedly negative for podoplanin. In contrast, in our present study, we examined a much larger series of GISTs and found that nearly 25% showed at least focal immunoreactivity for podoplanin, usually with a granular, cytoplasmic staining pattern. Interestingly, podoplanin positivity was more common in spindle cell GISTs (38%) than in epithelioid (14%) or mixed-type GISTs (11%). The basis for this discrepancy is uncertain but may reflect differences in immunohistochemical technique. We have shown that podoplanin (using monoclonal antibody D2-40) is a highly sensitive marker for FDC tumors. However, nodal Kaposi sarcomas also show strong membranous expression of podoplanin. Of other dendritic cell and histiocytic neoplasms, only occasional cases of histiocytic sarcoma are positive for podoplanin. Among extranodal spindle cell neoplasms, a subset of GISTs, IMTs, and monophasic synovial sarcomas show immunoreactivity for podoplanin, albeit with a cytoplasmic staining pattern. Although its expression in spindle cell neoplasms is not entirely specific for FDC tumors, podoplanin may be useful to help confirm the diagnosis in

conjunction with conventional FDC markers, particularly in the differential diagnosis of dendritic cell and histiocytic lesions. From the Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA. Presented in part at the 96th Annual Meeting of the United States and Canadian Academy of Pathology; March 24-30, 2007; San Diego, CA. Address reprint requests to Dr Hornick: Dept of Pathology, Brigham and Women’s Hospital, 75 Francis St, Boston, MA 02115. Acknowledgment: We are grateful to Christopher D.M. Fletcher, MD, FRCPath, for providing some of the cases used for this study.

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❚Image 5❚❚ A, Monophasic synovial sarcoma composed of highly cellular short fascicles of spindle cells with overlapping nuclei and prominent stromal collagen (H&E, ×400). B, Patchy cytoplasmic staining for podoplanin (D2-40) is observed in tumor cells (×400).


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