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“Intrafollicular neoplasia” of nodular lymphocyte predominant Hodgkin lymphoma
Human Pathology (2012) 43, 619–628
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“Intrafollicular neoplasia” of nodular lymphocyte predominant Hodgkin lymphoma Description of a hypothetic early step of the disease Antonino Carbone MD a,⁎, Annunziata Gloghini PhD b a
Division of Pathology, Centro di Riferimento Oncologico Aviano, Istituto Nazionale Tumori, IRCCS, 33081 Aviano, Italy Department of Diagnostic Pathology and Laboratory Medicine, Fondazione IRCCS, Istituto Nazionale Tumori, 20133 Milano, Italy
b
Received 1 July 2011; revised 5 September 2011; accepted 7 September 2011
Keywords: Intrafollicular neoplasia; Nodular NLPHL; NLPHL
Summary The 2008 WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues has addressed the problem of intrafollicular neoplasia/“in situ” lesion for follicular lymphoma. The concept of intrafollicular neoplasia has also been proposed for other lymphomas in which the putative normal counterpart of the tumor cell is located in the germinal center or the mantle zone or the marginal zone of the follicle. However, unlike in situ follicular lymphoma, the precise histologic definition of this early lesion for other lymphomas is still lacking. When applied to nodular lymphocyte predominant Hodgkin lymphoma, another germinal center–derived lymphoma, “intrafollicular neoplasia” may be regarded as a neoplasia at an early stage of development, such as in situ follicular lymphoma. Interestingly, this early lesion can be observed in lymph nodes that otherwise show the most common nodular involvement by nodular lymphocyte predominant Hodgkin lymphoma. The recognition of intrafollicular neoplasia is based on the identification of typical, strongly stained BCL6+, lymphocyte predominant tumor cells located within altered follicles with a recognizable germinal center. Lymphocyte predominant tumor cells, surrounded by rosetting PD1+ T cells, reside in an environment reminiscent of a secondary follicle. Intrafollicular neoplasia in nodular lymphocyte predominant Hodgkin lymphoma is correctly identifiable based on immunohistochemical recognition of the CD23+ meshwork of follicular dendritic cells surrounded by an outer zone containing immunoglobulin D+ B cells. This immunoarchitectural pattern, highlighting the intrafollicular involvement by the neoplasia, is of great utility for diagnosis. An appropriate immunohistochemical characterization for diagnosis should include lymphocyte predominant (BCL6 and CD20) and microenvironmental (CD23, immunoglobulin D, and PD1) cell markers. © 2012 Elsevier Inc. All rights reserved.
The concept of intrafollicular neoplasia has been recently acknowledged by the 2008 WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues [1,2]. The term in situ lymphoma has been provisionally adopted to define a lymphoid neoplasia with an “exclusive intrafollicular” ⁎ Corresponding author. Tel.: +39 0434 659 085; fax: +39 0434 659 370. E-mail address: [email protected] (A. Carbone). 0046-8177/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2011.09.002
growth pattern. Therefore, the concept of intrafollicular neoplasia/in situ lymphoma properly applies when the neoplastic cells are localized in the “place” that is occupied by the normal counterpart of the tumor cell, without invasion of surrounding structures [3]. Among germinal center (GC)–derived lymphomas, a type of follicular lymphoma (FL), originally termed “in situ FL,” has been described, in which the accumulation of
620 neoplastic cells is observed in histologically abnormal follicles within the follicular GC only [4,5]. However, this early lesion has an uncertain clinical behavior and unknown risk to progression to overt lymphoma; therefore, the term of FL-like cells of uncertain significance has recently been used instead of in situ FL. Only a subset of patients with in situ lymphoma was found to have overt lymphoma elsewhere, either before or simultaneously. Few other patients develop overt lymphoma later. Besides “in situ” FL, the concept of intrafollicular neoplasia has been proposed for other lymphomas in which the putative normal counterpart of the tumor cell is located in the GC or the mantle zone or the marginal zone of the follicle [6,7]. These lymphomas include nodular lymphocyte predominant Hodgkin lymphoma (NLPHL), angioimmunoblastic T-cell lymphoma showing limited paracortical involvement, mantle cell lymphoma, and cases of nodal marginal zone lymphoma (reviewed in [6]). In NLPHL, the immunoarchitectural pattern of neoplastic nodules is reminiscent of primary lymphoid follicle [8], although immunohistochemical and molecular studies (reviewed in [9]) indicated that NLPHL derives from GC B cell of secondary lymphoid follicle. Therefore, to recognize morphologically an early phase of the disease, it is reasonable to search for a histologically and immunohistologically identifiable step in which tumor cells are located in a microenvironment still reminiscent of a secondary lymphoid follicle. Intriguingly, authoritative reports from the pertinent literature have described that the large neoplastic nodules that constitute the typical morphological features of NLPHL contain GC or remnants of GC in some cases [8,10-13]. In our experience, neoplastic transformation of secondary follicles could be identified in areas of partially involved lymph nodes that otherwise showed the most common nodular involvement by the tumor [6,14]. In these cases, NLPHL consisted of a mixture of classic tumor nodules and areas of intrafollicular neoplasia where altered follicles with GC remnants contained typical tumor cells, in the same biopsy sample. Importantly, this mixed pattern, having intrafollicular neoplasia as a component, has been observed in lymph nodes of patients with an indolent clinical course [15], suggesting that it may be regarded as an early phase of disease. Therefore, it seems plausible that in the early phases of NLPHL development, a morphological step with intrafollicular neoplasia occurs and may be recognized based on the immunoarchitectural and histologic identification of typical tumor cells within altered follicles with GC remnants. It may be either the dominant or, more frequently, an accompanying component of the tumor. In this review, the concept of intrafollicular neoplasia originally adopted for FL is applied to NLPHL. We assume that this kind of lesion may be regarded as an early involvement and may represent a step potentially leading to frank lymphoma, such as in situ FL. However, unlike in situ FL, the precise definition of this early lesion for NLPHL is still lacking. The relevant impact on the morphology and
A. Carbone, A. Gloghini immunodiagnostic classification of NLPHL of the precise definition of this early lesion for NLPHL will be discussed.
1. Structure, development, and microenvironment of the GC Primary follicles are present in nonstimulated lymphoid tissue and are composed of naive small B cells and an underlying follicular dendritic cell (FDC) meshwork. The histologic appearance of primary follicles characteristically shows small lymphocytes that have a monomorphic appearance; they are positive for B-cell markers including immunoglobulin D (IgD) and BCL2 protein but lack admixed large centroblasts and are negative for the GC markers CD10 and BCL6. Secondary B follicles develop on antigen exposure, forming the GC with a surrounding mantle zone. It is believed that GCs develop, thanks to expansion of a small number of B cells that respond to the antigen presented by follicular-associated dendritic cells (reviewed in [16]). As the GC matures, 2 main compartments become evident. These compartments are termed dark and light zones based on their histologic appearance, in which much of the light zone is occupied by FDC processes, whereas few processes extend into the dark zone where lymphocytes are closely packed. The FDCs of the light zone acquire features that distinguish them from FDCs in the primary follicles, including upregulation of VCAM-1 (vascular cell adhesion protein-1) and FcγRIIB (FC gamma receptor IIB) (reviewed in [17]). The GC reaction produces high-affinity antibodies by random mutation and selective clonal expansion of B cells with high-affinity receptors [18]. Within the GC, B cells undergo affinity maturation involving activation-induced diaminase (AID)–mediated somatic hypermutation (SHM) and class switch recombination (CSR) [19]. Paired box protein 5 (PAX5) and BCL6 are major regulators of B-cell development. PAX5 and BCL6 inhibit plasma cell differentiation, whereas Blimp-1 and XBP-1 are the key regulators promoting plasmacytic development. BCL6 is down-regulated, and Blimp-1, up-regulated due to the loss of PAX5 function [20]. Continued development of the GC is dependent on a specialized subset of T-helper cells termed T-follicular helper (TFH) cells. TFH cells are predominantly composed of antigen-specific activated polyclonal helper T cells (CD4+CD69+CD25−) that are characterized by expression of CD57. The CD57+ TFH cells are predominantly located within the apical light zone, where they mediate costimulatory and CD40-CD40 ligand (CD40-CD154) interactions that promote GC B-cell survival [21]. It has been shown that B-cell division is restricted to the dark zone, with a net vector of B cell movement from the dark zone to the light zone. The decision to return to the dark zone and undergo clonal expansion is controlled by TFH cells in the GC light zone, which discern between light zone B cells based on
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the amount of antigen captured and presented. Thus, TFH cell, and not direct competition for antigen, is the limiting factor in GC selection [18]. TFH cell provide crucial signals to GC B cells undergoing SHM and selection that results in affinity maturation. Tight control of TFH numbers maintains self-tolerance. The microstructure created by B-cell proliferation, SHM, apoptosis, and maturation within the follicles are paralleled by microanatomic complexity in the distribution of T cells and dendritic networks within these environment [19].
The derivation of NLPHL from GC (reviewed in 9) is supported by the expression of the BCL6 gene product and CD40 antigen by neoplastic cells [25,26] as well as by the occurrence of numerous CD4+/CD57+/programmed death 1 (PD1)+ T cells surrounding the lymphocyte predominant (LP) cells, as detectable in normal GC (Fig. 1), and by the presence of a CD23+ FDC meshwork within the nodules. Further evidence indicating that the tumor is derived from GC B cells has been provided by recent molecular studies, based on the global gene expression profile [27].
2. The role of GC in B-cell lymphomagenesis: NLPHL among the GC-derived B-cell lymphomas
3. Intrafollicular neoplasia in NLPHL
GC-derived B-cell lymphomas may share common molecular pathogenetic events that are relevant in B-cell lymphomagenesis. Errors in the processes of CSR and SHM can result in mutation of PAX5 or dysregulated expression of BCL6 through translocation [19]. In particular, NLPHL is targeted by aberrant SHM, as is diffuse large B-cell lymphoma (DLBCL) [22,23]. A recent study, based on microdissected neoplastic cells of NLPHL, reported on the occurrence of aberrant SHM of 4 previously identified aberrant SHM targets (PIM1, PAX5, RhoH/TTF, and c-MYC). Mutations in 1 or more genes were detected in 80% of NLPHLs. The most frequently involved protooncogene was PAX5, mutated in 7 of 9 patients with NLPHL [24].
Previous observations from the pertinent literature (see below) convincingly support the occurrence of a morphologically identifiable phase of NLPHL in which the neoplastic growth involves follicles still reminiscent of secondary follicles, which maintain GC structures. In a study analyzing the morphological and immunophenotypic patterns of NLPHL, Fan et al [28] identified 6 distinct immunoarchitectural patterns (“classic” nodular, serpiginous/interconnected nodular, nodular with prominent extranodular lymphocytic and histiocytic cells, T-cell–rich nodular, diffuse with a T-cell–rich background, and a diffuse B-cell–rich pattern) and 2 variant patterns (presence of small GCs within the nodules and the presence of prominent sclerosis) [28]. Two very recent reports have confirmed the occurrence of these patterns and
Fig. 1 Reactive follicles and intrafollicular neoplasia of NLPHL. Within the GC cell microenvironment (A-C), AID and BCL6 are expressed by the centroblasts of the dark zone, whereas PD1 is expressed by most CD3+/CD4+ T cells. Within the cell microenvironment of intrafollicular neoplasia of NLPHL (D-F), AID and BCL6 are expressed by residual GC B cells. However, intrafollicular neoplasia includes also typical LP cells strongly expressing BCL6 and surrounded by CD4+ PD1+ rosetting T cells.
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Fig. 2 NLPHL: the composite figure shows the immunoarchitectural pattern of intrafollicular neoplasia. In NLPHL, the recognition of intrafollicular neoplasia is based on the localization of morphologically typical and strongly BCL6+, CD20+ LP tumor cells surrounded by rosetting PD1+ T cells, in an environment reminiscent of a secondary follicle (ie, altered follicle with a recognizable GC). The intrafollicular pattern of growth is correctly identifiable based on immunohistochemical recognition of the CD23-positive meshwork of follicular dendritic cells surrounded by a mantle zone containing IgD+ B cells, highlighting the GC involvement by the neoplasia. A, At high magnification, this abnormal follicle contains at the center a CD20-positive LP cell. The inset shows 2 LP cells surrounded by smaller reactive lymphocytes. A reactive background is dominated by smaller nonneoplastic B cells and (B) rosetting PD1+ T cells. This abnormal follicle (C-F) contains a prominent CD23+ FDC meshwork (C) that encompasses CD20 (D) and BCL6-positive cells (E, E inset) surrounded by mantle zone containing IgD+ B-cells (F) highlighting an intrafollicular pattern of growth (E and F). E, inset, The BCL6-positive LP cells (arrows) have a strong intensity of staining and are associated with more weakly stained reactive cells.
Intrafollicular neoplasia/early NLPHL their utility [29,30]. In the variant pattern in which small GCs were identified within the neoplastic nodules, stains directed against CD20, CD21, and MIB1 highlighted these GCs [28]. These small GCs contained, although rarely, LP cells. The histologic pattern described by Fan et al may correspond to early transformed neoplastic follicles, still containing a recognizable GC and reminiscent of secondary follicle. However, this pattern previously not well described in NLPHL did not correlate with any clinical characteristics [28]. Other reports, however, have cited the presence of residual GC and broken up GCs within the nodules of NLPHL [10-12] in accordance with the findings of Fan et al. Mason et al [8], in their article defining NLPHL as a distinct clinicopathologic entity, stated that “the large nodules that constitute the most prominent histologic feature contain broken up remnants of GC in some cases.” The authors added that “the nodules contain an extensive meshwork of FDCs, which are often more prominent than in normal follicles” [8]. We also have come up against some NLPHL cases with an immunoarchitectural pattern revealing that GC or small GC or broken-up GC was present within altered follicles, which contained typical LP tumor cells. This pattern was seen in patients with an indolent clinical course. It was hypothesized that this NLPHL variant pattern could be regarded as an early lesion, which is likely to be an early step of disease [15]. According to these observations, here, we propose that an intrafollicular neoplasia step may occur in the early phases of NLPHL development (Fig. 1). It may be suspected in lymph nodes, where several follicles have a larger size and irregular shape, show a well-preserved mantle zone and contain scattered LP cells with typical morphology. Immunohistologically, these abnormal follicles contain a prominent FDC meshwork that encompasses CD20- and BCL6-positive reactive GC B cells (Fig. 2). This reactive background, dominated by CD23+ FDCs, is surrounded by an outer zone with smaller nonneoplastic IgD+ B cells highlighting an intrafollicular pattern of NLPHL growth. LP cells are strongly positive for BCL6 and CD20 but usually negative for AID (Table 1 and Fig. 1). Furthermore, strongly stained BCL6+ LP cells are associated with more weakly stained reactive cells. The BCL6positive LP cells have an intensity of staining similar to that of the GC B cells of contiguous reactive follicles. LP cells reside in spherical meshworks of FDCs that are filled with nonneoplastic reactive cells, which include also T cells. Most of the LP cells are ringed by CD3+ T cells and, to a lesser extent, by CD57+ T cells. CD3+/CD4+ T cells express markers such as PD1 and interferon regulatory factor 4 (IRF4)/multiple myeloma-1 (MUM1), consistent with a subset of GC T cells. CD4+/CD57+/PD1+ small lymphocytes rosetting around typical CD20+/BCL6+ LP cells are useful for the differential diagnosis with other conditions including progressively transformed GC (PTGC), lympho-
623 Table 1 Nodular lymphocyte predominant Hodgkin lymphoma: histologic and immunoarchitectural pattern of intrafollicular neoplasia Histopathology LP cells in enlarged follicles Immunohistochemistry LP cells Strongly stained for BCL6, CD20 Stained for CD40, OCT2, Ki-67 Variably stained for PAX5, IRF4 [26] Usually unstained for CD30, CD15, IgD, AID GC-reactive (LP cells Strongly stained for PD1 rosetting) T cells Stained for CD4, CD57, IRF4 GC-reactive B cells Stained for BCL6, CD20, CD40 Unstained for IgD FDCs: entrapped LP cells reside in spherical meshwork of FDCs that are expanded and broken up Genetic abnormality BCL6 dysregulation Aberrant somatic hypermutation (PAX5 mutation) AID/aberrant class switch recombination of the Ig genes Differential diagnosis: CD4+, CD57+, PD1+ small lymphocytes rosetting typical CD20+, BCL6+ LP cells are useful for the differential diagnosis with “progressively transformed GC,” “lymphocyte-rich classic HL,” and T/histiocyte cell–rich B-cell lymphoma. Staining for IgD has to be considered in recognizing mantle zone B cells.
cyte-rich classic Hodgkin lymphoma (HL), and T/histiocyte cell–rich B-cell lymphoma (Table 1). A relation between PTGCs and early NLPHL showing intrafollicular neoplasia is suggested by their similar morphological and immunohistochemical appearance (Fig. 3). Further indication for a relation between PTGCs and NLPHL is the occurrence of PTGCs in approximately 15% of patients with NLPHL before, during, or after NLPHL. PTGCs were observed only in 2% of patients with classic HL. The most important difference between PTGCs and NLPHL is the presence of LP cells, representing the tumor B-cell population, only in NLPHL. Moreover, NLPHL usually differs from PTGCs by less well-circumscribed nodules and a broken-up pattern and greater numbers of T cells, which may form aggregates and rings around the LP cells.
4. Neoplastic follicle-like structures in NLPHL Follicle-like structure (Fig. 4) is a well-defined neoplastic nodule reminiscent of a lymphoid follicle for its size, shape, and especially for the presence of a FDC component. In NLPHL with follicle-like structures, the most prominent architectural hallmark is the presence of nodular structures containing LP cells within a reactive cellular background. By conventional histology, the
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Fig. 3 PTGCs and intrafollicular neoplasia of NLPHL. In PTGC (A and B), follicles contain an extensive meshwork of FDCs, which is more prominent than in normal follicles. In intrafollicular neoplasia (C and D), follicles contain spherical meshwork of FDCs that are expanded and broken up. FDCs entrap LP cells. D, The inset highlights a typical LP cell at the margin of FDC meshwork.
nodules simulated expanded primary follicles. GC cells corresponding to centrocytes and centroblasts are usually absent from these nodules. In these cases, the neoplasia shows well-defined follicle-like structures with visible FDC meshworks. CD23 immunodetection discloses an extended meshwork of FDC within the nodules, which engulfed the neoplastic cells and the surrounding T-cell rosettes. LP cells reside exclusively within these nodular structures. In these structures, not only the number of FDCs but also the level of destruction of normal follicle architecture might be observed and might be an important variable for outcome prediction [31].
5. Immunoarchitecture of NLPHL: tumor progression from intrafollicular neoplasia to classic neoplastic nodule In this section, we briefly recapitulate the steps occurring during progression of the disease from the early phases of NLPHL development to the most common and classic histologic features of NLPHL.
1. In NLPHL, a hypothetical first step corresponds to a transformed neoplastic follicle morphologically identifiable as an intrafollicular neoplasia. It still maintains the normal architecture of the secondary follicle including a recognizable GC and a mantle zone (Fig. 2). 2. A subsequent step is a well-defined neoplastic folliclelike structure lacking GC and containing a dense meshwork of FDC (Fig. 4). Probably, it is the result of a fragmentation and collapse of the GC throughout a multistep process that destroys the follicular microarchitecture. Moreover, it is the consequence of a multifocal inward migration of small B cells with a mantle zone phenotype into the follicle center, an event that, in part, resembles the progressive transformation of GC that results, however, in a benign transformed follicle [32]. In intrafollicular neoplasia and the follicle-like neoplastic structures, the neoplastic cells are found to be located only within the follicles. 3. The classic nodules of NLPHL show expression of immunoglobulin M and IgD on the nonneoplastic B-cell component, consistent with a mantle zone
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Fig. 4 NLPHL: the composite figure shows the immunoarchitectural pattern of a follicle-like neoplastic nodule. The term follicle like was used as the neoplastic nodule is reminiscent of a lymphoid follicle for its size, shape, and especially for the presence of a FDC component. A, In this follicle-like neoplastic nodule, most CD20+ B cells within the nodule are IgD+ (B). C, Residual BCL6+ B cells are scarce. The arrows indicate 2 BCL6+ LP cells. D, The FDC CD23+ meshwork is broken up. LP cells are entrapped by FDCs (E) and are surrounded by smaller lymphocytes (arrows) (F).
phenotype. However, evidence has been provided that NLPHL is a GC-derived lymphoma and the normal cell counterpart of the tumor cell is a GC B cell resembling centroblast. The occurrence of GC collapse combined with a migration of small B cells with a mantle zone phenotype into the follicle center
may explain why the immunoarchitectural pattern of the classic neoplastic nodule is reminiscent of the primary follicle. In the classic nodular pattern, described by Fan et al [28], the nodules usually contain a prominent FDC meshwork that encompasses the LP cells. In these cases, the neoplastic LP
626 cells are found to be located predominantly within the nodular structures, but rare LP cells extend outside the nodule (Fig. 5). 4. During the progression of the disease, more LP cells extend outside the nodules and infiltrate the perinodular space. Importantly, the presence of numerous LP cells outside the nodules predicts for progression to a diffuse pattern (Fig. 5) [33]. The presence of many extra-
A. Carbone, A. Gloghini nodular LP cells may characterize the pattern described by Fan et al as “nodular with prominent extranodular LP cells.” It may represent early progression to a diffuse pattern [1,28]. 5. According to this multistep process of tumor progression, an intrafollicular neoplasia in NLPHL is likely to be an in situ neoplasia potentially leading, step by step, to a NLPHL with a diffuse pattern (Fig. 6).
6. Concluding remarks From a pathologic point of view, the definition of early NLPHL should be restricted to cases in which (a) the immunohistologic pattern either consists of intrafollicular neoplasia or (b) it consists of follicle-like neoplastic structures and (c) the LP tumor cells do not expand outside the neoplastic follicles. The scientific bases of the description of intrafollicular neoplasia of NLPHL as an early step of the disease include, at present, histopathologic observations and new findings on the immunohistochemical architecture. The scientific bases of this proposal should be further supported by experimental work and clinicopathologic studies. Regarding genetics, data from a study dealing with microdissected LP cells analyzed for somatic hypermutations [24] were reviewed. Interestingly, we found that, in 1 of the 8 cases in which microdissected LP cells showed PAX5 mutation, intrafollicular neoplasia was the dominant pattern (A. Carbone and A. Liso, unpublished observation). Other experiments studying microdissected single cells for PAX5 mutation and BCL6 translocation need to be done to definitively support the genetic similarity of the LP cells microdissected from intrafollicular neoplasia with those microdissected from neoplastic nodules. As regards the clinical side, we had previously found that patients exhibiting exclusively intrafollicular NLPHL are alive and well 14, 16, 18, and 20 years after the initial diagnosis and are still in follow-up [15]. Clinical outcome studies, comparing intrafollicular NLPHL with NLPHL, need to be done to show the importance of recognizing intrafollicular/early NLPHL. Clinicopathologic correlations will be also useful to define the risk of progression of the disease, the frequency of association with concomitant frank NLPHL, the clinical significance of this association and guidelines for the appropriate management and treatment.
Acknowledgments Fig. 5 Examples of follicle-like NLPHL and nodular NLPHL. A and B, In a follicle-like neoplastic nodule, LP cells expressing OCT2 (A) and CD20 (B) are inside the nodule (arrows) and do not infiltrate its outer zone. C, In a classic nodular NLPHL, several CD20+ LP cells (arrows) are present outside the nodule.
This article is based on the study of more than 100 NLPHL cases diagnosed by the authors during a 20-year period (from 1986 to 2005) at the Centro di Riferimento Oncologico of Aviano, Italy. All cases were diagnosed and studied by combining hematoxylin and eosin and
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Fig. 6 Molecular mechanisms promoting B-cell lymphomagenesis and tumor progression from intrafollicular neoplasia to classic neoplastic nodule in NLPHL. A, Proliferating GC B cells undergo affinity maturation involving AID-mediated SHM and CSR (19). Major transcriptional regulators of B-cell development during the different stages of B-cell activation include PAX5 and BCL6. The repression program of BCL6 is down-regulated by several mechanisms including the transcriptional repression of the BCL6 gene by IRF4 after CD40 stimulation (19). B, Errors in the processes of CSR and SHM can result in mutation of PAX5 or dysregulated expression of BCL6 through translocation (B-C). C, Among these DNA lesions that might keep the tumor precursor cell in the GC stage of development [19], PAX5 mutation, resulting from aberrant SHM, occurs in a significant fraction of NLPHL [24]. In the figure, a typical large LP cell is surrounded by rosetting reactive lymphocytes. D, In early neoplastic lesions (intrafollicular neoplasia) in which altered follicles still contain GC, LP cells reside in spherical meshworks of FDCs that are filled with nonneoplastic inflammatory cells. LP cells are strongly positive for BCL6 and CD20. LP cells are usually positive for PAX5, whereas they are usually negative for AID. Most of the LP cells are ringed by CD3+ T cells and, to a lesser extent, by CD57+ T cells. CD3+/CD4+ T cells express markers such as PD1 and IRF4/MUM1, consistent with a subset of GC T cells. E, In the follicle-like nodules of NLPHL, LP cells do not expand beyond the neoplastic nodule. E, LP cells are within the nodule where they are entrapped by FDCs. E and F, Most reactive CD20+ B cell within the nodules are IgD+ (mantle cell phenotype), whereas residual BCL6+ B cells (GC phenotype) are absent. FDC CD23+ meshwork is broken up. F, LP cells are inside and outside the nodule.
immunohistochemical stainings. Selected subsets of these cases were included in previous studies reporting on the consistent expression of CD40 [34], BCL6 [25], and MUM1/IRF4 [26] on LP tumor cells as well as on the abnormal SHM in NLPHL [24]. The authors thank B. Canal, P. Ceolin, A. Selva, and L. Zannier for the current immunohistochemical reassessment of the whole NLPHL case series. The authors also thank Elettra Gislon (Scientific Direction, Centro di Riferimento Oncologico Aviano, Istituto Nazionale Tumori, IRCCS, Aviano, Italy) for the English revision of the manuscript. This work was supported in parts by grants from the Ministero della Salute, Rome, within the framework of the
“Progetto Integrato Oncologia-Advanced Molecular Diagnostics” project (RFPS-2006-2-339694.1; RFPS-2006-2339723.2).
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A. Carbone, A. Gloghini [21] Carbone A, Gloghini A, Gruss HJ, Pinto A. CD40 ligand is constitutively expressed in a subset of T cell lymphomas and on the microenvironmental reactive T cells of follicular lymphomas and Hodgkin's disease. Am J Pathol 1995;147:912-22. [22] Pasqualucci L, Neumeister P, Goossens T, et al. Hypermutation of multiple proto-oncogenes in B-cell diffuse large-cell lymphomas. Nature 2001;412:341-6. [23] Gaidano G, Pasqualucci L, Capello D, et al. Aberrant somatic hypermutation in multiple subtypes of AIDS-associated non-Hodgkin lymphoma. Blood 2003;102:1833-41. [24] Liso A, Capello D, Marafioti T, et al. Aberrant somatic hypermutation in tumor cells of nodular-lymphocyte–predominant and classic Hodgkin lymphoma. Blood 2006;108:1013-20. [25] Falini B, Bigerna B, Pasqualucci L, et al. Distinctive expression pattern of the BCL-6 protein in nodular lymphocyte predominance Hodgkin's disease. Blood 1996;87:465-71. [26] Carbone A, Gloghini A, Aldinucci D, Gattei V, Dalla-Favera R, Gaidano G. Expression pattern of MUM1/IRF4 in the spectrum of pathology of Hodgkin's disease. Br J Haematol 2002;117: 366-72. [27] Brune V, Tiacci E, Pfeil I, et al. Origin and pathogenesis of nodular lymphocyte–predominant Hodgkin lymphoma as revealed by global gene expression analysis. J Exp Med 2008;205:2251-68. [28] Fan Z, Natkunam Y, Bair E, Tibshirani R, Warnke RA. Characterization of variant patterns of nodular lymphocyte predominant Hodgkin lymphoma with immunohistologic and clinical correlation. Am J Surg Pathol 2003;27:1346-56. [29] Churchill HR, Roncador G, Warnke RA, Natkunam Y. Programmed death 1 expression in variant immunoarchitectural patterns of nodular lymphocyte predominant Hodgkin lymphoma: comparison with CD57 and lymphomas in the differential diagnosis. HUM PATHOL 2010;41: 1726-34. [30] Venkataraman G, Raffeld M, Pittaluga S, Jaffe ES. CD15-expressing nodular lymphocyte–predominant Hodgkin lymphoma. Histopathology 2011;58:803-5. [31] Steidl C, Connors JM, Gascoyne RD. Molecular pathogenesis of Hodgkin's lymphoma: increasing evidence of the importance of the microenvironment. J Clin Oncol 2011;29:1812-26. [32] Jones D. Dismantling the germinal center: comparing the processes of transformation, regression, and fragmentation of the lymphoid follicle. Adv Anat Pathol 2002;9:129-38. [33] Anagnostopoulos I, Hansmann ML, Franssila K, et al. European Task Force on Lymphoma project on lymphocyte predominance Hodgkin disease: histologic and immunohistologic analysis of submitted cases reveals 2 types of Hodgkin disease with a nodular growth pattern and abundant lymphocytes. Blood 2000; 96:1889-99. [34] Carbone A, Gloghini A, Gattei V, et al. Expression of functional CD40 antigen on Reed-Sternberg cells and Hodgkin's disease cell lines. Blood 1995;85:780-9.
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Current topic
“Intrafollicular neoplasia” of nodular lymphocyte predominant Hodgkin lymphoma Description of a hypothetic early step of the disease Antonino Carbone MD a,⁎, Annunziata Gloghini PhD b a
Division of Pathology, Centro di Riferimento Oncologico Aviano, Istituto Nazionale Tumori, IRCCS, 33081 Aviano, Italy Department of Diagnostic Pathology and Laboratory Medicine, Fondazione IRCCS, Istituto Nazionale Tumori, 20133 Milano, Italy
b
Received 1 July 2011; revised 5 September 2011; accepted 7 September 2011
Keywords: Intrafollicular neoplasia; Nodular NLPHL; NLPHL
Summary The 2008 WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues has addressed the problem of intrafollicular neoplasia/“in situ” lesion for follicular lymphoma. The concept of intrafollicular neoplasia has also been proposed for other lymphomas in which the putative normal counterpart of the tumor cell is located in the germinal center or the mantle zone or the marginal zone of the follicle. However, unlike in situ follicular lymphoma, the precise histologic definition of this early lesion for other lymphomas is still lacking. When applied to nodular lymphocyte predominant Hodgkin lymphoma, another germinal center–derived lymphoma, “intrafollicular neoplasia” may be regarded as a neoplasia at an early stage of development, such as in situ follicular lymphoma. Interestingly, this early lesion can be observed in lymph nodes that otherwise show the most common nodular involvement by nodular lymphocyte predominant Hodgkin lymphoma. The recognition of intrafollicular neoplasia is based on the identification of typical, strongly stained BCL6+, lymphocyte predominant tumor cells located within altered follicles with a recognizable germinal center. Lymphocyte predominant tumor cells, surrounded by rosetting PD1+ T cells, reside in an environment reminiscent of a secondary follicle. Intrafollicular neoplasia in nodular lymphocyte predominant Hodgkin lymphoma is correctly identifiable based on immunohistochemical recognition of the CD23+ meshwork of follicular dendritic cells surrounded by an outer zone containing immunoglobulin D+ B cells. This immunoarchitectural pattern, highlighting the intrafollicular involvement by the neoplasia, is of great utility for diagnosis. An appropriate immunohistochemical characterization for diagnosis should include lymphocyte predominant (BCL6 and CD20) and microenvironmental (CD23, immunoglobulin D, and PD1) cell markers. © 2012 Elsevier Inc. All rights reserved.
The concept of intrafollicular neoplasia has been recently acknowledged by the 2008 WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues [1,2]. The term in situ lymphoma has been provisionally adopted to define a lymphoid neoplasia with an “exclusive intrafollicular” ⁎ Corresponding author. Tel.: +39 0434 659 085; fax: +39 0434 659 370. E-mail address: [email protected] (A. Carbone). 0046-8177/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2011.09.002
growth pattern. Therefore, the concept of intrafollicular neoplasia/in situ lymphoma properly applies when the neoplastic cells are localized in the “place” that is occupied by the normal counterpart of the tumor cell, without invasion of surrounding structures [3]. Among germinal center (GC)–derived lymphomas, a type of follicular lymphoma (FL), originally termed “in situ FL,” has been described, in which the accumulation of
620 neoplastic cells is observed in histologically abnormal follicles within the follicular GC only [4,5]. However, this early lesion has an uncertain clinical behavior and unknown risk to progression to overt lymphoma; therefore, the term of FL-like cells of uncertain significance has recently been used instead of in situ FL. Only a subset of patients with in situ lymphoma was found to have overt lymphoma elsewhere, either before or simultaneously. Few other patients develop overt lymphoma later. Besides “in situ” FL, the concept of intrafollicular neoplasia has been proposed for other lymphomas in which the putative normal counterpart of the tumor cell is located in the GC or the mantle zone or the marginal zone of the follicle [6,7]. These lymphomas include nodular lymphocyte predominant Hodgkin lymphoma (NLPHL), angioimmunoblastic T-cell lymphoma showing limited paracortical involvement, mantle cell lymphoma, and cases of nodal marginal zone lymphoma (reviewed in [6]). In NLPHL, the immunoarchitectural pattern of neoplastic nodules is reminiscent of primary lymphoid follicle [8], although immunohistochemical and molecular studies (reviewed in [9]) indicated that NLPHL derives from GC B cell of secondary lymphoid follicle. Therefore, to recognize morphologically an early phase of the disease, it is reasonable to search for a histologically and immunohistologically identifiable step in which tumor cells are located in a microenvironment still reminiscent of a secondary lymphoid follicle. Intriguingly, authoritative reports from the pertinent literature have described that the large neoplastic nodules that constitute the typical morphological features of NLPHL contain GC or remnants of GC in some cases [8,10-13]. In our experience, neoplastic transformation of secondary follicles could be identified in areas of partially involved lymph nodes that otherwise showed the most common nodular involvement by the tumor [6,14]. In these cases, NLPHL consisted of a mixture of classic tumor nodules and areas of intrafollicular neoplasia where altered follicles with GC remnants contained typical tumor cells, in the same biopsy sample. Importantly, this mixed pattern, having intrafollicular neoplasia as a component, has been observed in lymph nodes of patients with an indolent clinical course [15], suggesting that it may be regarded as an early phase of disease. Therefore, it seems plausible that in the early phases of NLPHL development, a morphological step with intrafollicular neoplasia occurs and may be recognized based on the immunoarchitectural and histologic identification of typical tumor cells within altered follicles with GC remnants. It may be either the dominant or, more frequently, an accompanying component of the tumor. In this review, the concept of intrafollicular neoplasia originally adopted for FL is applied to NLPHL. We assume that this kind of lesion may be regarded as an early involvement and may represent a step potentially leading to frank lymphoma, such as in situ FL. However, unlike in situ FL, the precise definition of this early lesion for NLPHL is still lacking. The relevant impact on the morphology and
A. Carbone, A. Gloghini immunodiagnostic classification of NLPHL of the precise definition of this early lesion for NLPHL will be discussed.
1. Structure, development, and microenvironment of the GC Primary follicles are present in nonstimulated lymphoid tissue and are composed of naive small B cells and an underlying follicular dendritic cell (FDC) meshwork. The histologic appearance of primary follicles characteristically shows small lymphocytes that have a monomorphic appearance; they are positive for B-cell markers including immunoglobulin D (IgD) and BCL2 protein but lack admixed large centroblasts and are negative for the GC markers CD10 and BCL6. Secondary B follicles develop on antigen exposure, forming the GC with a surrounding mantle zone. It is believed that GCs develop, thanks to expansion of a small number of B cells that respond to the antigen presented by follicular-associated dendritic cells (reviewed in [16]). As the GC matures, 2 main compartments become evident. These compartments are termed dark and light zones based on their histologic appearance, in which much of the light zone is occupied by FDC processes, whereas few processes extend into the dark zone where lymphocytes are closely packed. The FDCs of the light zone acquire features that distinguish them from FDCs in the primary follicles, including upregulation of VCAM-1 (vascular cell adhesion protein-1) and FcγRIIB (FC gamma receptor IIB) (reviewed in [17]). The GC reaction produces high-affinity antibodies by random mutation and selective clonal expansion of B cells with high-affinity receptors [18]. Within the GC, B cells undergo affinity maturation involving activation-induced diaminase (AID)–mediated somatic hypermutation (SHM) and class switch recombination (CSR) [19]. Paired box protein 5 (PAX5) and BCL6 are major regulators of B-cell development. PAX5 and BCL6 inhibit plasma cell differentiation, whereas Blimp-1 and XBP-1 are the key regulators promoting plasmacytic development. BCL6 is down-regulated, and Blimp-1, up-regulated due to the loss of PAX5 function [20]. Continued development of the GC is dependent on a specialized subset of T-helper cells termed T-follicular helper (TFH) cells. TFH cells are predominantly composed of antigen-specific activated polyclonal helper T cells (CD4+CD69+CD25−) that are characterized by expression of CD57. The CD57+ TFH cells are predominantly located within the apical light zone, where they mediate costimulatory and CD40-CD40 ligand (CD40-CD154) interactions that promote GC B-cell survival [21]. It has been shown that B-cell division is restricted to the dark zone, with a net vector of B cell movement from the dark zone to the light zone. The decision to return to the dark zone and undergo clonal expansion is controlled by TFH cells in the GC light zone, which discern between light zone B cells based on
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the amount of antigen captured and presented. Thus, TFH cell, and not direct competition for antigen, is the limiting factor in GC selection [18]. TFH cell provide crucial signals to GC B cells undergoing SHM and selection that results in affinity maturation. Tight control of TFH numbers maintains self-tolerance. The microstructure created by B-cell proliferation, SHM, apoptosis, and maturation within the follicles are paralleled by microanatomic complexity in the distribution of T cells and dendritic networks within these environment [19].
The derivation of NLPHL from GC (reviewed in 9) is supported by the expression of the BCL6 gene product and CD40 antigen by neoplastic cells [25,26] as well as by the occurrence of numerous CD4+/CD57+/programmed death 1 (PD1)+ T cells surrounding the lymphocyte predominant (LP) cells, as detectable in normal GC (Fig. 1), and by the presence of a CD23+ FDC meshwork within the nodules. Further evidence indicating that the tumor is derived from GC B cells has been provided by recent molecular studies, based on the global gene expression profile [27].
2. The role of GC in B-cell lymphomagenesis: NLPHL among the GC-derived B-cell lymphomas
3. Intrafollicular neoplasia in NLPHL
GC-derived B-cell lymphomas may share common molecular pathogenetic events that are relevant in B-cell lymphomagenesis. Errors in the processes of CSR and SHM can result in mutation of PAX5 or dysregulated expression of BCL6 through translocation [19]. In particular, NLPHL is targeted by aberrant SHM, as is diffuse large B-cell lymphoma (DLBCL) [22,23]. A recent study, based on microdissected neoplastic cells of NLPHL, reported on the occurrence of aberrant SHM of 4 previously identified aberrant SHM targets (PIM1, PAX5, RhoH/TTF, and c-MYC). Mutations in 1 or more genes were detected in 80% of NLPHLs. The most frequently involved protooncogene was PAX5, mutated in 7 of 9 patients with NLPHL [24].
Previous observations from the pertinent literature (see below) convincingly support the occurrence of a morphologically identifiable phase of NLPHL in which the neoplastic growth involves follicles still reminiscent of secondary follicles, which maintain GC structures. In a study analyzing the morphological and immunophenotypic patterns of NLPHL, Fan et al [28] identified 6 distinct immunoarchitectural patterns (“classic” nodular, serpiginous/interconnected nodular, nodular with prominent extranodular lymphocytic and histiocytic cells, T-cell–rich nodular, diffuse with a T-cell–rich background, and a diffuse B-cell–rich pattern) and 2 variant patterns (presence of small GCs within the nodules and the presence of prominent sclerosis) [28]. Two very recent reports have confirmed the occurrence of these patterns and
Fig. 1 Reactive follicles and intrafollicular neoplasia of NLPHL. Within the GC cell microenvironment (A-C), AID and BCL6 are expressed by the centroblasts of the dark zone, whereas PD1 is expressed by most CD3+/CD4+ T cells. Within the cell microenvironment of intrafollicular neoplasia of NLPHL (D-F), AID and BCL6 are expressed by residual GC B cells. However, intrafollicular neoplasia includes also typical LP cells strongly expressing BCL6 and surrounded by CD4+ PD1+ rosetting T cells.
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Fig. 2 NLPHL: the composite figure shows the immunoarchitectural pattern of intrafollicular neoplasia. In NLPHL, the recognition of intrafollicular neoplasia is based on the localization of morphologically typical and strongly BCL6+, CD20+ LP tumor cells surrounded by rosetting PD1+ T cells, in an environment reminiscent of a secondary follicle (ie, altered follicle with a recognizable GC). The intrafollicular pattern of growth is correctly identifiable based on immunohistochemical recognition of the CD23-positive meshwork of follicular dendritic cells surrounded by a mantle zone containing IgD+ B cells, highlighting the GC involvement by the neoplasia. A, At high magnification, this abnormal follicle contains at the center a CD20-positive LP cell. The inset shows 2 LP cells surrounded by smaller reactive lymphocytes. A reactive background is dominated by smaller nonneoplastic B cells and (B) rosetting PD1+ T cells. This abnormal follicle (C-F) contains a prominent CD23+ FDC meshwork (C) that encompasses CD20 (D) and BCL6-positive cells (E, E inset) surrounded by mantle zone containing IgD+ B-cells (F) highlighting an intrafollicular pattern of growth (E and F). E, inset, The BCL6-positive LP cells (arrows) have a strong intensity of staining and are associated with more weakly stained reactive cells.
Intrafollicular neoplasia/early NLPHL their utility [29,30]. In the variant pattern in which small GCs were identified within the neoplastic nodules, stains directed against CD20, CD21, and MIB1 highlighted these GCs [28]. These small GCs contained, although rarely, LP cells. The histologic pattern described by Fan et al may correspond to early transformed neoplastic follicles, still containing a recognizable GC and reminiscent of secondary follicle. However, this pattern previously not well described in NLPHL did not correlate with any clinical characteristics [28]. Other reports, however, have cited the presence of residual GC and broken up GCs within the nodules of NLPHL [10-12] in accordance with the findings of Fan et al. Mason et al [8], in their article defining NLPHL as a distinct clinicopathologic entity, stated that “the large nodules that constitute the most prominent histologic feature contain broken up remnants of GC in some cases.” The authors added that “the nodules contain an extensive meshwork of FDCs, which are often more prominent than in normal follicles” [8]. We also have come up against some NLPHL cases with an immunoarchitectural pattern revealing that GC or small GC or broken-up GC was present within altered follicles, which contained typical LP tumor cells. This pattern was seen in patients with an indolent clinical course. It was hypothesized that this NLPHL variant pattern could be regarded as an early lesion, which is likely to be an early step of disease [15]. According to these observations, here, we propose that an intrafollicular neoplasia step may occur in the early phases of NLPHL development (Fig. 1). It may be suspected in lymph nodes, where several follicles have a larger size and irregular shape, show a well-preserved mantle zone and contain scattered LP cells with typical morphology. Immunohistologically, these abnormal follicles contain a prominent FDC meshwork that encompasses CD20- and BCL6-positive reactive GC B cells (Fig. 2). This reactive background, dominated by CD23+ FDCs, is surrounded by an outer zone with smaller nonneoplastic IgD+ B cells highlighting an intrafollicular pattern of NLPHL growth. LP cells are strongly positive for BCL6 and CD20 but usually negative for AID (Table 1 and Fig. 1). Furthermore, strongly stained BCL6+ LP cells are associated with more weakly stained reactive cells. The BCL6positive LP cells have an intensity of staining similar to that of the GC B cells of contiguous reactive follicles. LP cells reside in spherical meshworks of FDCs that are filled with nonneoplastic reactive cells, which include also T cells. Most of the LP cells are ringed by CD3+ T cells and, to a lesser extent, by CD57+ T cells. CD3+/CD4+ T cells express markers such as PD1 and interferon regulatory factor 4 (IRF4)/multiple myeloma-1 (MUM1), consistent with a subset of GC T cells. CD4+/CD57+/PD1+ small lymphocytes rosetting around typical CD20+/BCL6+ LP cells are useful for the differential diagnosis with other conditions including progressively transformed GC (PTGC), lympho-
623 Table 1 Nodular lymphocyte predominant Hodgkin lymphoma: histologic and immunoarchitectural pattern of intrafollicular neoplasia Histopathology LP cells in enlarged follicles Immunohistochemistry LP cells Strongly stained for BCL6, CD20 Stained for CD40, OCT2, Ki-67 Variably stained for PAX5, IRF4 [26] Usually unstained for CD30, CD15, IgD, AID GC-reactive (LP cells Strongly stained for PD1 rosetting) T cells Stained for CD4, CD57, IRF4 GC-reactive B cells Stained for BCL6, CD20, CD40 Unstained for IgD FDCs: entrapped LP cells reside in spherical meshwork of FDCs that are expanded and broken up Genetic abnormality BCL6 dysregulation Aberrant somatic hypermutation (PAX5 mutation) AID/aberrant class switch recombination of the Ig genes Differential diagnosis: CD4+, CD57+, PD1+ small lymphocytes rosetting typical CD20+, BCL6+ LP cells are useful for the differential diagnosis with “progressively transformed GC,” “lymphocyte-rich classic HL,” and T/histiocyte cell–rich B-cell lymphoma. Staining for IgD has to be considered in recognizing mantle zone B cells.
cyte-rich classic Hodgkin lymphoma (HL), and T/histiocyte cell–rich B-cell lymphoma (Table 1). A relation between PTGCs and early NLPHL showing intrafollicular neoplasia is suggested by their similar morphological and immunohistochemical appearance (Fig. 3). Further indication for a relation between PTGCs and NLPHL is the occurrence of PTGCs in approximately 15% of patients with NLPHL before, during, or after NLPHL. PTGCs were observed only in 2% of patients with classic HL. The most important difference between PTGCs and NLPHL is the presence of LP cells, representing the tumor B-cell population, only in NLPHL. Moreover, NLPHL usually differs from PTGCs by less well-circumscribed nodules and a broken-up pattern and greater numbers of T cells, which may form aggregates and rings around the LP cells.
4. Neoplastic follicle-like structures in NLPHL Follicle-like structure (Fig. 4) is a well-defined neoplastic nodule reminiscent of a lymphoid follicle for its size, shape, and especially for the presence of a FDC component. In NLPHL with follicle-like structures, the most prominent architectural hallmark is the presence of nodular structures containing LP cells within a reactive cellular background. By conventional histology, the
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Fig. 3 PTGCs and intrafollicular neoplasia of NLPHL. In PTGC (A and B), follicles contain an extensive meshwork of FDCs, which is more prominent than in normal follicles. In intrafollicular neoplasia (C and D), follicles contain spherical meshwork of FDCs that are expanded and broken up. FDCs entrap LP cells. D, The inset highlights a typical LP cell at the margin of FDC meshwork.
nodules simulated expanded primary follicles. GC cells corresponding to centrocytes and centroblasts are usually absent from these nodules. In these cases, the neoplasia shows well-defined follicle-like structures with visible FDC meshworks. CD23 immunodetection discloses an extended meshwork of FDC within the nodules, which engulfed the neoplastic cells and the surrounding T-cell rosettes. LP cells reside exclusively within these nodular structures. In these structures, not only the number of FDCs but also the level of destruction of normal follicle architecture might be observed and might be an important variable for outcome prediction [31].
5. Immunoarchitecture of NLPHL: tumor progression from intrafollicular neoplasia to classic neoplastic nodule In this section, we briefly recapitulate the steps occurring during progression of the disease from the early phases of NLPHL development to the most common and classic histologic features of NLPHL.
1. In NLPHL, a hypothetical first step corresponds to a transformed neoplastic follicle morphologically identifiable as an intrafollicular neoplasia. It still maintains the normal architecture of the secondary follicle including a recognizable GC and a mantle zone (Fig. 2). 2. A subsequent step is a well-defined neoplastic folliclelike structure lacking GC and containing a dense meshwork of FDC (Fig. 4). Probably, it is the result of a fragmentation and collapse of the GC throughout a multistep process that destroys the follicular microarchitecture. Moreover, it is the consequence of a multifocal inward migration of small B cells with a mantle zone phenotype into the follicle center, an event that, in part, resembles the progressive transformation of GC that results, however, in a benign transformed follicle [32]. In intrafollicular neoplasia and the follicle-like neoplastic structures, the neoplastic cells are found to be located only within the follicles. 3. The classic nodules of NLPHL show expression of immunoglobulin M and IgD on the nonneoplastic B-cell component, consistent with a mantle zone
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Fig. 4 NLPHL: the composite figure shows the immunoarchitectural pattern of a follicle-like neoplastic nodule. The term follicle like was used as the neoplastic nodule is reminiscent of a lymphoid follicle for its size, shape, and especially for the presence of a FDC component. A, In this follicle-like neoplastic nodule, most CD20+ B cells within the nodule are IgD+ (B). C, Residual BCL6+ B cells are scarce. The arrows indicate 2 BCL6+ LP cells. D, The FDC CD23+ meshwork is broken up. LP cells are entrapped by FDCs (E) and are surrounded by smaller lymphocytes (arrows) (F).
phenotype. However, evidence has been provided that NLPHL is a GC-derived lymphoma and the normal cell counterpart of the tumor cell is a GC B cell resembling centroblast. The occurrence of GC collapse combined with a migration of small B cells with a mantle zone phenotype into the follicle center
may explain why the immunoarchitectural pattern of the classic neoplastic nodule is reminiscent of the primary follicle. In the classic nodular pattern, described by Fan et al [28], the nodules usually contain a prominent FDC meshwork that encompasses the LP cells. In these cases, the neoplastic LP
626 cells are found to be located predominantly within the nodular structures, but rare LP cells extend outside the nodule (Fig. 5). 4. During the progression of the disease, more LP cells extend outside the nodules and infiltrate the perinodular space. Importantly, the presence of numerous LP cells outside the nodules predicts for progression to a diffuse pattern (Fig. 5) [33]. The presence of many extra-
A. Carbone, A. Gloghini nodular LP cells may characterize the pattern described by Fan et al as “nodular with prominent extranodular LP cells.” It may represent early progression to a diffuse pattern [1,28]. 5. According to this multistep process of tumor progression, an intrafollicular neoplasia in NLPHL is likely to be an in situ neoplasia potentially leading, step by step, to a NLPHL with a diffuse pattern (Fig. 6).
6. Concluding remarks From a pathologic point of view, the definition of early NLPHL should be restricted to cases in which (a) the immunohistologic pattern either consists of intrafollicular neoplasia or (b) it consists of follicle-like neoplastic structures and (c) the LP tumor cells do not expand outside the neoplastic follicles. The scientific bases of the description of intrafollicular neoplasia of NLPHL as an early step of the disease include, at present, histopathologic observations and new findings on the immunohistochemical architecture. The scientific bases of this proposal should be further supported by experimental work and clinicopathologic studies. Regarding genetics, data from a study dealing with microdissected LP cells analyzed for somatic hypermutations [24] were reviewed. Interestingly, we found that, in 1 of the 8 cases in which microdissected LP cells showed PAX5 mutation, intrafollicular neoplasia was the dominant pattern (A. Carbone and A. Liso, unpublished observation). Other experiments studying microdissected single cells for PAX5 mutation and BCL6 translocation need to be done to definitively support the genetic similarity of the LP cells microdissected from intrafollicular neoplasia with those microdissected from neoplastic nodules. As regards the clinical side, we had previously found that patients exhibiting exclusively intrafollicular NLPHL are alive and well 14, 16, 18, and 20 years after the initial diagnosis and are still in follow-up [15]. Clinical outcome studies, comparing intrafollicular NLPHL with NLPHL, need to be done to show the importance of recognizing intrafollicular/early NLPHL. Clinicopathologic correlations will be also useful to define the risk of progression of the disease, the frequency of association with concomitant frank NLPHL, the clinical significance of this association and guidelines for the appropriate management and treatment.
Acknowledgments Fig. 5 Examples of follicle-like NLPHL and nodular NLPHL. A and B, In a follicle-like neoplastic nodule, LP cells expressing OCT2 (A) and CD20 (B) are inside the nodule (arrows) and do not infiltrate its outer zone. C, In a classic nodular NLPHL, several CD20+ LP cells (arrows) are present outside the nodule.
This article is based on the study of more than 100 NLPHL cases diagnosed by the authors during a 20-year period (from 1986 to 2005) at the Centro di Riferimento Oncologico of Aviano, Italy. All cases were diagnosed and studied by combining hematoxylin and eosin and
Intrafollicular neoplasia/early NLPHL
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Fig. 6 Molecular mechanisms promoting B-cell lymphomagenesis and tumor progression from intrafollicular neoplasia to classic neoplastic nodule in NLPHL. A, Proliferating GC B cells undergo affinity maturation involving AID-mediated SHM and CSR (19). Major transcriptional regulators of B-cell development during the different stages of B-cell activation include PAX5 and BCL6. The repression program of BCL6 is down-regulated by several mechanisms including the transcriptional repression of the BCL6 gene by IRF4 after CD40 stimulation (19). B, Errors in the processes of CSR and SHM can result in mutation of PAX5 or dysregulated expression of BCL6 through translocation (B-C). C, Among these DNA lesions that might keep the tumor precursor cell in the GC stage of development [19], PAX5 mutation, resulting from aberrant SHM, occurs in a significant fraction of NLPHL [24]. In the figure, a typical large LP cell is surrounded by rosetting reactive lymphocytes. D, In early neoplastic lesions (intrafollicular neoplasia) in which altered follicles still contain GC, LP cells reside in spherical meshworks of FDCs that are filled with nonneoplastic inflammatory cells. LP cells are strongly positive for BCL6 and CD20. LP cells are usually positive for PAX5, whereas they are usually negative for AID. Most of the LP cells are ringed by CD3+ T cells and, to a lesser extent, by CD57+ T cells. CD3+/CD4+ T cells express markers such as PD1 and IRF4/MUM1, consistent with a subset of GC T cells. E, In the follicle-like nodules of NLPHL, LP cells do not expand beyond the neoplastic nodule. E, LP cells are within the nodule where they are entrapped by FDCs. E and F, Most reactive CD20+ B cell within the nodules are IgD+ (mantle cell phenotype), whereas residual BCL6+ B cells (GC phenotype) are absent. FDC CD23+ meshwork is broken up. F, LP cells are inside and outside the nodule.
immunohistochemical stainings. Selected subsets of these cases were included in previous studies reporting on the consistent expression of CD40 [34], BCL6 [25], and MUM1/IRF4 [26] on LP tumor cells as well as on the abnormal SHM in NLPHL [24]. The authors thank B. Canal, P. Ceolin, A. Selva, and L. Zannier for the current immunohistochemical reassessment of the whole NLPHL case series. The authors also thank Elettra Gislon (Scientific Direction, Centro di Riferimento Oncologico Aviano, Istituto Nazionale Tumori, IRCCS, Aviano, Italy) for the English revision of the manuscript. This work was supported in parts by grants from the Ministero della Salute, Rome, within the framework of the
“Progetto Integrato Oncologia-Advanced Molecular Diagnostics” project (RFPS-2006-2-339694.1; RFPS-2006-2339723.2).
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