STEP 2:
Exclusionary requirements

Expert guidance - IgG4-related disease (IgG4-RD)

Among AI/AI-Ds, IgG4-RD is one of the most frequently biopsied, because of its common mass-like presentation, either nodal or extra nodal, which raises the clinical suspicion of a lymphoma.

IgG4-RD is defined by clinical and histologic parameters, the former including increased serum IgG4 levels >135 mg/dL, and the latter including an increase in IgG4 positive plasma cells at immunohistochemistry (10 IgG4-positive plasma cells per high-power/x400 field and a IgG4/IgG ratio ≥40%), but a wide variation of IgG4 positive plasma cells cut-offs and combination of the primary histologic lesions is described in different tissues.¹ In particular, among the 5 described lymph node histologic patterns one resembling a PC- or mixed type iMCD is listed. Since iMCD (particularly PC-type) can present with positive autoantibodies, increase in IgG4 serum levels (up to 50% patients) and up to one fourth of iMCD patients have IgG4/IgG+ ratio >40% and meet diagnostic criteria of IgG4-RD, the distinction may be very challenging.²

IgG4-RD can be favoured if there is eosinophilia and/or tissue eosinophils, uncommon tissues are involved (like pancreas, eyes, salivary glands), the IgG4 positive plasma cell increase also occurs within the GCs of the lymphoid follicles, horse-shoe like non necrotizing granulomas are observed around follicles, steroids are clinically effective.^2-5 iMCD would be more likely in cases of significant elevation of serum IL-6, CRP, LDH, IgA, tissue IgA increase among plasma cells, no/minimal eosinophilia or tissue eosinophils, systemic symptoms, no response to steroids, and/or no extra nodal sites of involvement.

Expert guidance - general considerations for autoimmune/autoinflammatory diseases (AI/AI-Ds)

AI/AI-Ds are more commonly defined on clinic-laboratory basis for which diagnostic criteria are set. However iMCD can associate with serum autoantibodies and partially or totally meet criteria for AI/AI-Ds.

Among clinical presentation, multiple – usually small – lymphadenopathies are common in AI/AI-Ds, though rarely biopsied unless of clinical significance. Most AI/AI-Ds do not show specific histologic features and specific diseases cannot be reliably diagnosed on this basis, since similar features can be observed in other reactive conditions, such as viral lymphadenopathies. The most common features include cortical B-zone expansion with follicular lymphoid hyperplasia (with florid polarised GCs in most cases) or prevalent T-cell paracortical hyperplasia with possible increase in vessels and sometimes vague reminiscence of angioimmunoblastic type lymphadenopathy. Eosinophils can be fairly represented as well as plasma cells. The kappa/lambda as well as the CD4/CD8 ratios have to be preserved (roughly 3 to 1 for both). A CD4/CD8 double negativity in the T-cell compartment can raise suspicion of an autoimmune lymphoproliferative syndrome that needs to be checked for mutations in genes affecting the extrinsic apoptotic pathway (FAS, FASL,CASP10). An increase in plasmacytoid mature dendritic cells can be observed in AI/AI-Ds. These appear as variably sized aggregates of lymphoid-like cells in the paracortical area, close to high endothelial venules, composed of small to medium-size elements with thin cytoplasm and dispersed chromatin without evident nucleoli, positive for CD303, CD123, TCL1 and CD4 molecules, and Bcl2 negative.

Epithelioid histiocytes or non necrotizing microgranulomas may occur. A significant increase in plasma cells with lymph node capsule involvement should prompt syphilis investigation (including Warthin-Starry stain), while a predominance of CD8+ T-cells would speak in favour of a virus – associated lymph adenopathy.

T- and B-cell clonality are not usually necessary to be performed, but they should show polyclonal peaks. In case of monoclonality, wider panels of antigens must be applied as well as additional genetic investigations to exclude T- or B-cell lymphomas.

Occasionally almost all AI/AI-Ds may show CD-like features in the lymph nodes, as the result of the active cyto/chemokine secretion in these diseases, possibly raising the suspicion of iMCD. In most instances, the strict application and grading of the 5 diagnostic criteria defined for iMCD allow its exclusion for the absence of a consistent spectrum of significantly represented features, but it may not always be the case. That is why a multidisciplinary approach is recommended.

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Abbreviations

AI, autoimmune disease; AI-Ds, autoinflammatory diseases; CD, Castleman Disease; GCs, germinal centres; iMCD, idiopathic Multicentric Castleman Disease.

Expert guidance - multiple myeloma/plasma cell neoplasms

Plasma cell neoplasms must also be considered in the very rare event of iMCD-PC with prominent and/or predominant interfollicular proliferation of monotypic plasma cells.

Lymph nodes with plasma cell neoplasms may in fact also show a variable number of dispersed lymphoid follicles, more commonly with hyperplastic GCs. This can lead to a possible suspicion of monotypic CD-PC or B-cell lymphoma with plasma cell differentiation (see expert guidance on lymphomas). Tests for immunoglobulin gene rearrangements are mandatory to confirm monoclonality in addition to light chain immunohistochemical restriction.

Using the patient’s history and the clinic-laboratory and instrumental data is critical in the interpretation of these challenging conditions in order to exclude a nodal involvement by a systemic multiple myeloma or a POEMS syndrome. A bone marrow biopsy is mandatory to detect the clonal (mostly lambda in POEMS) plasma cell proliferation. However, a monoclonal gammopathy can rarely occur in iMCD without clear evidence of a concurrent plasma cell neoplasm.

In case of a localised/unicentric lymph-adenopathy with CD-PC type and monotypic plasma cells, the possibility of a primary extramedullary (lymph node) plasmacytoma has to be considered. These are very rare events, for which clonality tests and EBV integration should be investigated, but UCD cases are very rarely of the PC-type and in this case a suspicion of a oligocentric CD could be raised.

Additional stainings for Cyclin D1/Bcl1, CD56, PAX5, CD117 and ISH technique for EBER 1-2 probe should be performed as any positivity would suggest a plasma cell neoplasm. Multiple myeloma-associated recurrent gene rearrangements should also be analysed with FISH.

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Abbreviations

CD, Castleman Disease; EBV, Epstein-Barr virus; FISH, fluorescence in situ hybridization; GCs, germinal centres; iMCD, idiopathic Multicentric Castleman Disease; PC, plasmacytic; POEMS, polyneuropathy, organomegaly, endocrinopathy, monoclonal paraprotein, skin changes; UCD, Unicentric Castleman Disease.

Expert guidance - follicular dendritic cell sarcoma (FDCS)¹

The development of a FDCS (previously termed ‘tumour’) usually relates to UCD and/or iMCD-HyperV. The lymph node structure is partially or totally effaced by a cohesive proliferation of large cells, with fascicular, or storiform or whorled diffuse growth pattern, with atypical vescicular nuclei with evident nucleoli. iMCD-HyperV type features can be observed in the uninvolved lymph node areas, as well as aspects of FDC dysplasia within the regressed GC. On rare occasions, large clusters of dysplastic FDC within the GC exiting the GC and bridging GC to the interfollicular areas can be seen as possible steps of progression from FDC dysplasia to FDCS.

The staining for CD21, which is part of the basic immunohistochemical panel of markers necessary for a diagnosis of CD, would clearly highlight the exuberant and atypical FDC proliferation in most cases. However, multiple FDC-associated markers have to be investigated due to possible antigen loss in FDCS. FDC-associated markers are CD21, CD23, CD35, clusterin, CXCL13 and podoplanin. Epithelial Growth Factor Receptor is reported positive, though with low specificity; EMA, S100, smooth muscle actin and CD68 have occasionally reported, as well as CD30. EBV search is mandatory (for a diagnosis of EBV-positive inflammatory follicular dendritic cell/fibroblastic reticular cell tumour or EBV-positive inflammatory FDCS now designated as a separate entity from EBV negative FDCS).

Expert guidance – lymphomas that may more commonly pose overlap with iMCD

A) SMALL B-CELL LYMPHOMAS WITH PLASMA CELL DIFFERENTIATION – LYMPHOPLASMACYTIC LYMPHOMA (LPL) AND NODAL MARGINAL ZONE LYMPHOMA WITH PLASMACYTIC DIFFERENTIATION (NMZL-PC)

This would apply in those rare (~10%) MCD cases of PC- or mixed types with monotypic light chain restriction at immunohistochemistry (mostly lambda) and/or monoclonal immunoglobulin gene rearrangement.

LPL commonly presents with IgM monoclonal gammopathy. The diffuse proliferation shows a more intimate mixture of small B-lymphocytes, lymphoplasmactyoid cells and plasma cells, rather than the clear-cut separation of lymphoid follicles and interfollicular plasma cells sheets observed in CD. Mast cells are usually increased and regressed or hyperplastic residual GCs are uncommon in LPL. Therefore, this differentiation is rarely posed. There are no specific immunophenotypic markers but most LPL cases have an L265P mutation in the MYD88 gene that defines LPL’s neoplastic origin.

NMZL can occasionally present with significant plasma cell differentiation (NMZL-PC) with side sheets of mature bland looking monotypic plasma cells (either kappa or lambda light chain restricted) that occupy the interfollicular areas with sporadic lymphoid follicles. An expansion of the marginal zone area of the lymphoid follicles (possible evidenced by MNDA and/or IRTA-1 antigens) as well as spilling of marginal zone B-lymphocytes over the plasma cell sheets can be observed, whereas in CD a presence of increase in IgA and/or IgG4 positive plasma cells is more common. The GCs in NMZL-PC should look or normal appearing or colonised rather than atretic or frankly hyperplastic as seen in CD, and FDC meshwork should be disarranged rather than prominent or expanded as observed in CD. Neither immunophenotype nor genetics provide specific and/or recurrent findings, although the detection of genetic aberrations at next-generations-sequencing analysis would speak in favour of a neoplastic nature.

It is recommended that a diagnosis of iMCD-PC with monotypic plasma cells can be favoured when obvious CD-type follicles are present, whereas in their absence a diagnosis of HN may be more appropriate.¹ iMCD and NMZL-PC may clinically overlap with advanced stages disease with multiple lymph-adenopathies and usually no serum monoclonal gammopathy; no specific alterations of serum protein levels are detected in NMZL-PC, whereas hypergammaglobulinemia would favour CD.

B) FOLLICULAR LYMPHOMA (FL) WITH CD-LIKE FEATURES

FL may occasionally show depleted GCs, lack the typical back-to-back follicular growth pattern, show twinning GCs and sclerosis. In these conditions a suspicion of iMCD with a hypervascular subtype (iMCD-HyperV) may be raised. Immunohistochemistry can highlight a follicular and often interfollicular B-cell proliferation expressing GC-associated markers (BCL6, CD10) in FL; most cases would also show BCL2 protein expression and/or the (14;18)(q32;q21) translocation at FISH analysis. A subset of FL lacks the BCL2 rearrangement and BCL2 protein expression, and this is relatively more frequent in cases arising at inguinal sites where lymph node vascularity is commonly and physiologically increased: these aspects may lead to the differential diagnosis with or a misdiagnosis of iMCD-HyperV. CD23 staining can be of aid in some BCL2 negative FL being positive in the neoplastic follicular and interfollicular proliferation, in addition to FDC. The latter may look expanded in FL with CD-like features but are not as dense as in the regressed GCs of CD.^2-5

In the case series of FL with CD-like features reported by Pina-Oviedo et al. the majority of the studied lymph nodes did show areas fully consistent with a morphologic and phenotypic diagnosis of FL.⁴

In addition to FISH analysis, clonality and genetic sequencing tests provide useful support for a diagnosis of FL.

On clinical grounds, both FL and iMCD can present with multiple adenopathies and systemic symptoms. However, it is uncommon for alterations of inflammatory indices and cytopenia to be observed in FL if the bone marrow is involved.

C) T-CELL LYMPHOMA - MOST COMMONLY T-CELL LYMPHOMAS OF T-FOLLICULAR HELPER CELL (TFH) PHENOTYPE AND SPECIFICALLY ANGIOIMMUNOBLASTIC (AITL) SUBTYPE, IN THE EARLY PHASES OF DEVELOPMENT

In the early stages of development, the differentiation of T-cell lymphomas of TFH phenotype from iMCD-HyperV is posed due to an only moderately expanded interfollicular area, with an increased in high endothelial venules and increase in polytypic plasma cells. Eosinophils are also numerous and this is rarely observed in CD. The neoplastic lymphoid cells show minimal to overt cytologic atypia and often represent a minor subset of the whole T-cell population, hard to identify at the sole histology. Suggestive hints are medium size cells, with relatively wide and clear cytoplasm, with a trend to aggregate in small clusters in the outer part of the B-cell follicle or in intimate association with interfollicular vessels. AITL typically shows FDC hyperplasia outside the lymphoid follicle which does not occur in CD. The B-cell compartment can still be widely represented in the early stages of the disease, and either hyperplastic or regressed/depleted GCs are observed. Immunohistochemistry is diagnostic, and shows the neoplastic T-cell subset (positive for CD3 and CD4, with complete or variably defective expression of the remaining T-cell markers) with expression of at least 2, preferably 3, widely used TFH-associated markers (PD1, ICOS, Bc16, CD10, CXCL13) and frequent high proliferation index. Assessment of T-cell clonality of both (γ and β chains) is confirmatory in most cases. If available, recurrent mutations in regulatory genes and histone methylation (TET2 and DNMT3A), in IDH2 and RHOA genes may provide support to the diagnosis.

AITL and iMCD share many clinical features, multiple lymphadenopathies, systemic symptoms, increase of inflammatory indices, hypergammaglobulinemia, autoimmune manifestiations, and cytopenia.

D) MANTLE CELL LYMPHOMA (MCL)

Few cases of association of MCL and iMCD have been reported. MCL detection can be an incidental finding as an in situ mantle cell neoplasm (with a thin rim of neoplastic B- lymphocytes surrounding regressed GC) or suspected in cases with wide mantle zone expansions. Since the latter feature is frequently observed in CD and given that normal mantle-zone B-lymphocytes can express CD51 in CD, the suspicion of iMCD can be arisen.⁶ The mantle zone expansion in iMCD frequently assumes an “onion-skin” appearance and the negativity for cyclin D1 and SOX11 antigens as well as the t(11;14)(q13;132) are key factors in ruling out MCL.

E) CLASSIC HODGKIN LYMPHOMA (CHL)

Hodgkin lymphoma is the most common type of lymphoma associated with CD or CD-like features.⁷ This is likely due to the pronounced inflammatory environment that characterises this lymphoma which can produce CD-like morphology (of either PC or HyperV sub types). The latter can be histologically misleading and conceal the neoplastic Hodgkin-Reed-Sternberg (HRS) cells, particularly in minimally involved lymph nodes or in cases with few neoplastic cells. For this reason it is suggested to include CD30 in the standard panel applied in the suspicion of CD and in case HRS cells are reliably detected, to add staining for CD15 and Pax5 as well as EBV tests (LMP1 by immunohistochemistry; EBER 1-2 by ISH technique). The HL can be EBV positive or negative, and the most frequent subtype is the mixed cellular, with interfollicular spread. Serum IL-6 can also be increased in CHL.

F) INDOLENT LYMPHOBLASTIC PROLIFERATION (IT-LBP)

This is a rare condition characterised by a TdT positive T-lymphoblastic cell increase in tissues other than the thymus, and lymph nodes seem to be one of the most common sites. Recent publications reported a relatively high occurrence of IT-LBP in lymph nodes with CD/CD-like features.⁸ IT-LBP bears good prognosis, the proliferative index is low, and the disease does not show progression or genetic features associated with T-ALL. Surgical excision without any treatment is indicated. The pathogenesis remains unclear but an IL-6 or cytokine mediated release of immature thymic T-lymphoblasts from the thymus towards lymph nodes is suggested during the course of autoimmune disorders, CD or tumours.^8-9

Expert guidance – general considerations for malignancies

Haematologic neoplasms (HNs) must be excluded to diagnose iMCD. However, HNs can be observed alongside is 1 word histopathologic CD-features, either concurrently or sequentially, in the same or different lymph node. HNs can induce CD-like features or mimic CD, and in rare cases, HNs can truely associate with CD. Therefore, determining which feature developed first is critical. If a HN is diagnosed before, or concurrently (either in the same or a different lymph node) or shortly after iMCD, its likely that the CD-like features are caused by the production of CD-like cytokines and chemokines from the neoplastic or environmental cells. Therefore, iMCD should not be diagnosed. If a HN is diagnosed a year or more after iMCD and no evidence of HN is detected in the original diagnostic lymph node following review, the initial diagnosis of iMCD should not be overturned.

FOR THE DIAGNOSTIC CRITERIA OF THE ACKNOWLEDGED LYMPHOMA CATEGORIES REFER TO:

• The International Consensus Classification of Mature Lymphoid Neoplasms: A Report from the Clinical Advisory
• The 5th edition of the World Health Organization Classification of Haematolymphoid Tumours: Lymphoid Neoplasms.

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Abbreviations

CD, Castleman Disease; HNs, haematologic neoplasms; iMCD, idiopathic Multicentric Castleman Disease.

Expert guidance

Excisional lymph node biopsies should be first evaluated morphologically (H&E; optionally Giemsa). The following workflow is suggested to perform a complete complementary analysis (IHC, FCM, ISH for EBV, clonality analysis if required) in addition to morphological evaluation of the lymph node biopsy.

IMMUNOHISTOCHEMICAL FIRST-STEP PANEL:

CD20, CD3, CD138, Kappa, Lambda, CD21, HHV-8/LANA1, IgG, IgG4, CD30, BCL2

FCM PANEL:

CD19, CD20, CD5, sKappa, sLambda, CD38, CD3, CD4, CD8, TCR GD, CD56, CD10, CD22, CD23, CD200, CD79b, CD43, CD45

IN-SITU HYBRIDIZATION (ISH):

EBV/EBER

CLONALITY ANALYSIS (IN SELECTED CASES OF SUSPECTED B/T CELL LYMPHOMA):

Immunoglobulin and/or T-cell receptor gene rearrangements

Excisional lymph node biopsies should be evaluated for the presence of HHV-8 infection by IHC (which would define the HHV8-positive variant of CD) and integration of the Epstein-Barr virus (EBV) by ISH (to detect EBV driven lymphoproliferations including infectious mononucleosis or chronic-active EBV infection).

Evaluations of Kappa and Lambda light chains either by IHC and/or FCM would allow for the exclusion of plasma cell neoplasia with CD type changes and B-cell lymphoma with plasmacytic differentiation, although sporadic cases of CD with monotypic light chain restriction may occur.

Some lymphoma types (Hodgkin lymphoma, follicular lymphoma, marginal zone lymphoma, angioimmunoblastic T-cell lymphoma) may show CD like histopathological changes. If a lymphoma is suspected, adequate IHC/FCM panels are required.

Clonality analysis must be used to confirm the presence of a clonal B or T cell population although iMCD cases may rarely show monoclonal immunoglubulin gene rearrangement (in such instances, a monotypic/monoclonal CD should be considered only if typical morphologic features are clearly present). The presence of lymphoma-specific genetic aberrations may be helpful.

The bone marrow biopsy in iMCD-NOS (not otherwise specified) may show polytypic plasmacytosis, reactive interstitial or aggregated lymphocytes and increased megakaryocytes, while bone marrow biopsy in iMCD-TAFRO are hypercellular with megakaryocytic hyperplasia (with frequent myelodysplastic- or myeloproliferayive-like features ) and increased reticulin deposition. In addition, bone marrow biopsies should be analysed to rule out plasma cell neoplasm in the work-up of a patients with a diagnosis of CD.

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Abbreviations

CD, Castleman Disease; EBV, Epstein-Barr virus; FHC, flow cytometry; HHV-8, human herpesvirus-8; IgG, immunoglobulin G; IgG4, immunoglobulin G4; IHC, immunohistochemistry; iMCD, idiopathic Multicentric Castleman Disease; NOS, not otherwise specified; ISH, in situ hybridization; POEMS, polyneuropathy, organomegaly, endocrinopathy, monoclonal paraprotein, skin changes; TAFRO, thrombocytopenia, anasarca, fever, reticulin myelofibrosis, organomegaly.