Introduction
Nodular lymphocyte predominant Hodgkin lymphoma, alternatively termed nodular lymphocyte predominant B-cell lymphoma in the International Consensus Classification (ICC), is uncommon. Some cases can present challenges in diagnosis and may be difficult to differentiate from other entities with similar histologic features.1,2 The objectives of this article were to review the histopathologic features of this disease, abbreviated as NLP, and to provide some clues in its differentiation from de novo T-cell/histiocyte-rich large B-cell lymphoma (THRLBL) and lymphocyte-rich classic Hodgkin lymphoma (LRCHL).
History and terminology
Early on, NLP was identified as a B-cell neoplasm and later shown to be of germinal center derivation.3,4 It is characterized by either nodular or nodular and diffuse proliferation, containing scattered large mononucleated or multilobated neoplastic cells known as lymphocyte-predominant (LP) cells or popcorn cells.5,6 This entity was previously categorized as one of the variants of Hodgkin lymphoma, most likely due to the histologic features, including sparse neoplastic cells and abundant non-neoplastic cells in the background, similar to classic Hodgkin lymphoma (CHL). A proposal from the International Lymphoma Study Group first suggested a more formal separation of NLP from CHL in 1994.7 Subsequently, based on data indicating greater differences in clinical behavior and treatment approaches, the ICC took this a step further by recommending the removal of the eponymic term “Hodgkin”, from the disease, proposing the term “nodular lymphocyte predominant B-cell lymphoma” to emphasize emerging differences in clinical management for these two lymphomas of B-cell lineage.8 Currently, the term “nodular lymphocyte predominant Hodgkin lymphoma” remains unchanged in the 5th Edition of the World Health Organization classification. In the following discussion, NLP will be the abbreviation for both nodular lymphocyte predominant Hodgkin lymphoma and nodular lymphocyte predominant B-cell lymphoma.
Epidemiology and clinical features
NLP is more common in young patients (aged 30–50 years) with a male predominance and comprises 10% of all Hodgkin lymphomas.9 Patients usually present with localized lymphadenopathy initially, most commonly involving cervical, axillary, and inguinal lymph nodes or rarely mesenteric lymph nodes. Mediastinal and extranodal involvements are uncommon. Overall, the disease demonstrates an indolent clinical course and an excellent outcome with standard therapy.10,11 Systemic B symptoms are uncommon in the absence of advanced disease. Approximately 20% of patients can present with advanced-stage disease at diagnosis with involvement of the spleen, liver, or more rarely bone marrow and bone.12 Transformation of NLP into diffuse large B-cell lymphoma occurs in 5–10% of cases.11,13
Epstein-Barr virus (EBV) has been thought to play a significant role in the pathogenesis of some CHL subtypes. However, it is only rarely positive in NLP (< 5%).14,15 LP cells can be positive for IgD in a subgroup of patients, particularly young male patients with isolated neck or axillary lymphadenopathy.16–18 A subset of IgD+ cases were found to be associated with Moraxella catarrhalis and Rothia mucilaginosa in recent studies,19,20 suggesting a potential role for bacterial antigens in the stimulation of the B-cells that ultimately give rise to the LP cells of NLP.
Microscopy
NLP has a nodular growth pattern in most cases and a diffuse pattern in fewer cases (Figs. 1 and 2a). The initial lesion appears to arise within pre-existing reactive follicles. The follicular structures are usually larger than normal follicles and are associated with expanded follicular dendritic cell (FDC) meshworks. LP cells show a rim of pale cytoplasm, large twisted or multilobated nuclei with thin nuclear membranes, vesicular chromatin, and basophilic nucleoli, which are usually smaller than those in Hodgkin/Reed-Sternberg (HRS) cells (Fig. 2a). Multilobated LP cells have been likened to a “popped kernel of corn”, termed “popcorn cells”. Multinucleated cells may be observed. LP cells reside in an immune niche in a background of predominantly small lymphocytes with a variable number of histiocytes. Occasionally, significantly increased histiocytes, clusters of epithelioid histiocytes, or granulomas can be appreciated.
Various growth patterns of LP cells and the associated lymphocytic background were initially described by Fan et al. (Fig. 1).21 The most common growth patterns in NLP are Fan patterns A and B, comprising about 75% of cases. LP cells are scattered singly but commonly show vague clustering within the follicular structures/nodules of small B-cells with a few reactive T-cells. As the disease progresses, normal small B-cells decrease, while admixed T-cells become more abundant. In addition, Fan pattern C shows LP cells extending beyond the follicular compartment. Further loss of normal B-cells is evident in Fan pattern D. Evidence of follicular remnants disappears in Fan pattern E, which may be entirely diffuse. This THRLBL-like transformation is difficult to differentiate from de novo THRLBL. Fan pattern F also shows loss of follicular structures with a vaguely nodular background containing both B-cells and T-cells, lacking FDC meshworks (Fig. 1).
Immunohistochemical (IHC) stains are crucial to identify the various growth patterns, and more than one pattern can be observed in a single lymph node biopsy. The significance of the various growth patterns was recognized in ICC grading, with Fan patterns A, B, and C being grade 1, while Fan patterns D, E, and F considered grade 2.8,21 Patients with grade 2 disease may warrant treatment as diffuse large B-cell lymphoma, especially with advanced clinical stage.22
Reactive follicular hyperplasia, often with foci of progressive transformation of germinal centers (PTGC), may be seen in adjacent uninvolved nodal tissue. PTGC was reported to precede, follow, or occur concurrently with NLP. However, isolated PTGC is not a significant risk for subsequent NLP.23,24 The follicular structures in PTGC are markedly enlarged, largely as a consequence of the mantle zone expansion. The resultant follicle contains clusters of germinal center cells surrounded by IgD-positive cells. However, typical LP cells are not present. In addition, PTGC follicles are usually enriched for T follicular helper (TFH) cells stained by programmed cell death protein 1 (PD-1) and CD57. Therefore, an increase in TFH cells should not raise significant concern for involvement by NLP.
Immunophenotype
LP cells typically demonstrate strong expression of transcription factors Octamer-binding protein 2 (Oct-2) and B cell Oct binding protein 1 (Bob1), which are critical for functional immunoglobulin (IG) expression. They also maintain other B-cell associated markers such as CD20, CD79a, paired box protein 5 (Pax5), PU-1, and B-cell lymphoma 6 (Bcl-6) but are usually negative for multiple myeloma 1/interferon regulatory factor 4 (MUM-1/IRF4) and CD10 (Fig. 2b). In our experience, Oct-2 facilitates the detection of the neoplastic cells, especially when there are abundant small background B-cells. The background non-neoplastic B-cells show weak staining for Oct-2, as they are typically derived from the residual mantle cuffs (Fig. 2c). Pax5 is positive, but more weakly expressed in tumor cells than background B-cells. In addition, myocyte enhancer binding factor 2B (MEF2B) is expressed in LP cells.25–27 IgD was reported to be positive in 27% of cases, particularly affecting young male patients (Fig. 2d).16–18 LP cells are generally negative for EBV-encoded small RNA (EBER), with EBER rarely positive in 3–5% of cases.14,15,28–30 In some cases LP cells show downregulation or absence of some B-cell markers, such as CD20, Pax5, or CD79a,31 particularly in EBV+ cases.14
FDC markers such as CD21 and CD23 highlight the expanded and often fragmented FDC meshworks in the atypical nodules (Fig. 2e). NLP arises in preexistent follicles. Over time, the background normal B-cells disappear and are replaced by infiltrating T-cells. Most of the T-cells associated with the neoplastic LP cells exhibit a TFH phenotype and are strongly positive for PD-1/CD279. They form tight rosettes around the LP cells, which can help facilitate their detection (Fig. 2f–g). Prolonged close contact between LP cells and the surrounding PD-1+ T-cells was observed and may play a role in tumorigenesis.32,33 However, rosette formation with PD-1+ T-cells is not specific for NLP; it can be found in CHL but is usually less conspicuous.34,35 CD57+ cells are also common in the background of NLP.
Genetic findings
The LP cells possess functional IG rearrangements and express IG transcripts, which are more easily detected in DNA from isolated tumor cells or in tumor-rich samples. However, IG rearrangements are challenging to detect in whole tissue sections with sparse neoplastic cells and rich non-neoplastic cells in the microenvironment. LP cells typically exhibit a high load of somatic hypermutation in IGH variant regions with ongoing mutations, indicating the derivation from germinal center B-cells.4 FISH (fluorescence in situ hybridization) analysis may reveal gene rearrangement of BCL6 with partners including IG genes, IKAROS family genes, ABR, and others.36–38 Next generation sequencing study found mutations in PAX5, PIM1, RHOH (TTF), and MYC,39 as well as somatic mutations in SGK1, DUSP2, and JUNB in some cases of NLP.40 LP cells share mutations in JUNB, DUSP2, SGK1, and SOCS1 with THRLBL which suggests a close relationship between these two entities.41 Aberrations in TNFAIP3 or NFkBIA are rare in LP cells,42 unlike in HRS cells of CHL. Instead, the active NFkB signaling gene signature in LP cells results from different mechanisms.43–45 Familial NLP has been reported in patients with Hermansky-Pudlak syndrome type 2 and autoimmune lymphoproliferative syndrome with mutations in FAS.46,47
Challenging cases of NLP and differential diagnosis
Genetically, the tumor cells of NLP and THRLBL exhibit marked similarity, suggesting they are part of a single entity. The differential diagnosis between LRCHL and NLP can also be challenging in some cases. Herein, we discuss some helpful tips for accurate diagnosis in challenging cases of NLP.
NLP, Fan pattern E vs. THRLBL
NLP Fan pattern E shows sparse tumor cells dispersed in a rich inflammatory background composed largely of T-cells and histiocytes. Background FDC meshworks and nodular areas may be absent or only focally seen. The background T-cells are predominantly CD4+ T-cells, while CD8+ T-cells have been reported to be more frequent in THRLBL (Figs. 1 and 3).
THRLBL is considered an aggressive B-cell lymphoma with sparse large tumor cells scattered in a background of abundant T-cells and/or histiocytes. It most frequently affects middle-aged or older male patients, whereas NLP often affects relatively younger patients, including children. Patients with THRLBL typically present with advanced disease with B symptoms, bulky lymphadenopathy, and/or hepatosplenomegaly. Systemic involvement is much more common than in NLP and frequently involves the spleen, liver, bone marrow, and bone, in addition to lymph nodes.
Tumor cells in THRLBL carry clonal IG gene rearrangement and high levels of somatic mutations. The genomic profile is similar to that of NLP, further supporting a close relationship.48,49
Histologically, tumor cells in THRLBL are evenly dispersed in a background of diffuse T-cells with a variable number of histiocytes. The tumor cells may exhibit variations in size or pleomorphism, and lobated nuclei can be present in some cases (Fig. 3a). A nodular growth pattern is not appreciated on H&E-stained sections. Focal residual nodules, if present, confirm NLP Fan pattern E and argue against de novo THRLBL. Other subtle differences may be helpful. In THRLBL, the background usually contains more histiocytes and fine fibrosis, whereas fibrosis is less often encountered in lymph nodes involved by NLP. Necrosis is also more commonly found in THRLBL.
The immunophenotype of the tumor cells in THRLBL is similar to that of NLP (Fig. 3b). They are negative for CD15, CD30, and EBER in most cases. By flow cytometry, the phenotype of the neoplastic cells in THRLBL and NLP is nearly identical, limiting the utility of flow cytometry for differential diagnosis.50,51 Nevertheless, in THRLBL, the background lymphocytes are more often CD8+ T-cells with an absence of small B-cells (Fig. 3b–c).52 These T-cells show strong activation by 3D measurement.53 PD-1+ T-cell rosettes surrounding tumor cells are generally absent. FDC meshworks are absent as well. In contrast, LP cells in NLP Fan pattern E may still show vague clusters, best highlighted by CD20 and Oct-2 (Fig. 3d–e). In addition, a few residual scattered small B-cells, traces of FDC meshworks, and T-cell rosettes all favor the diagnosis of NLP Fan pattern E (Fig. 3e–f).
In some cases, a clear distinction between THRLBL and NLP Fan pattern E may not be possible. However, if NLP is observed in another site or at the time of recurrence, the diagnosis of NLP may be confirmed.
Morphologic and IHC features of the two entities are summarized in Table 1.
Table 1Differential diagnosis of NLP: Summary of histopathologic and IHC features
| THRLBL | NLP | LRCHL |
|---|
| Neoplastic cells | Morphologic features | Sparse, widely dispersed, variable degrees of pleomorphism. May resemble centroblasts or immunoblasts | Sparse, multilobated nuclei, thin nuclear membranes, basophilic nucleoli. Tend to form loose clusters. May be intrafollicular or interfollicular. | Sparse; may resemble lacunar cells or classic HRS cells. Most common in the marginal zone surrounding regressed follicles. |
| CD30 | −/+ | Negative to weakly + | Strongly + |
| CD15 | – | −/rare + | +/− |
| EBER | – | Rarely EBV+ | EBV + (15–20%) |
| EMA | variable | −/+ | – |
| Pan-B cell markers | + | + | −/weak and variable + |
| MUM-1 | −/+ | −/+ | +, strong |
| Oct-2 | +, strong | +, strong | −/+ |
| Pax5 | + | + (may be weak) | +, weak |
| Others | N/A | Positive for MEF2B;
may be positive for STAT6 in rare cases | Often positive for Fascin, Gata-3, and STAT6 |
| Non-neoplastic Background | Morphologic features | Diffuse lymphocytic background with increased histiocytes and fine fibrosis | Nodular or diffuse lymphocytic background. Variable histiocytes. | Mainly nodular pattern. Regressed follicles present. Rare to absence of eosinophils, neutrophils and plasma cells. Diffuse pattern less common |
| CD20 | Absence of small B-cells | Background B-cells nearly always present | Intact follicles |
| CD4/CD8 | Often CD8> CD4 | Mainly CD4+ | Mainly CD4+ |
| T-cell (PD-1+) rosettes | Absent | Present | Sometimes seen |
| CD21/CD23 | Absent | Expanded FDC meshworks | Compact tight intact FDC meshworks |
NLP vs. LRCHL
LRCHL is a subtype of CHL with scattered HRS cells in a background rich in small lymphocytes and usually lacking other inflammatory cells, such as plasma cells and eosinophils. It is less common than NLP and usually affects older patients compared to NLP and other CHL subtypes, showing a male predominance. Patients with LRCHL show an indolent clinical course, and B symptoms are rare. The disease mainly affects peripheral lymph nodes. Mediastinal involvement is less common than in nodular sclerosis CHL but can be present, while mediastinal disease is rare in NLP. The prognosis of LRCHL is somewhat better than other CHL subtypes.12
Histologically, the most common form of LRCHL exhibits a nodular growth pattern (Fig. 4a–f).1,54 The follicles contain regressed germinal centers and intact mantle zones. CD21 or CD23 highlights small, dense, compact, and intact FDC meshworks associated with the eccentrically located regressed germinal centers (Fig. 4b). The HRS cells are best observed in the marginal zone, surrounding the regressed follicles. This pattern contrasts with what is seen in NLP, where the LP cells are scattered within the altered follicles (Fig. 4g–l). LRCHL with a diffuse pattern is rare and probably closer to the mixed cellularity subtype of CHL.
The HRS cells in LRCHL may resemble “lacunar” type HRS cells with ample cytoplasm and sometimes multilobated nuclei (Fig. 4a). They can also resemble LP cells. The HRS cells exhibit the typical immunophenotype of CHL with strong staining for CD30, CD15 (subset), MUM-1, and dim expression of Pax5 (Fig. 4c–e). CD20 and CD79a are most often negative but can be variably expressed. Occasionally, HRS cells in this subtype can express variable B-cell transcription factors such as Oct-2, Bob-1, and Bcl-6. EBER and latent membrane protein 1 (LMP1) can be positive or negative (Fig. 4f). It should be noted that LP cells in NLP can be rarely positive for CD30 or CD15.21,55–57 However, if positive, CD30 staining tends to be weak in LP cells (Fig. 4i). EBV-positive NLP is another rare finding (Fig. 4j).14,15 In reported cases, Oct-2 usually remains strongly expressed. (Fig. 4k)
Other markers reported in the differential diagnosis of LRCHL and NLP include fascin, GATA-binding protein 3 (Gata-3), and STAT6. These antigens are more likely to be positive in the HRS cells of CHL than in LP cells.58–61 The specificity of STAT6 expression in HRS cells is controversial.61,62 In addition, HRS cells also show strong staining with MUM-1, generally negative in LP cells. While epithelial membrane antigen has been reported to be positive in LP cells, the incidence of truly positive cases is low. A recent study showed that light chain restriction can be demonstrated by ultrasensitive in situ hybridization for kappa and lambda in LP cells but not in HRS cells.63 PD-1+/CD57+ T-cell rosettes around HRS cells can be present but are usually less common than in NLP (Fig. 3k, inset).
Specific studies focusing on the genomic features of HRS cells in LRCHL have not been published. In other work, the failure of HRS cells to express IG transcripts has been partially attributed to a deficiency in the transcription factors Oct-2 and Bob-1. It is of interest that these transcription factors are more often positive in LRCHL.55 Whether this impacts Ig expression in the neoplastic cells is unknown.
The genomic profile of HRS cells differs from that of LP cells in NLP and may provide clues for their distinction if available. HRS cells show alterations in both the NF-kappa B and JAK/STAT pathways.64–66 These include mutations of SOCS1.67–70 The other frequent genetic abnormalities include gains of 2p, 9p, 16p, 17q, 19q, and 20q, losses of 6q, 11q, and 13q, as well as aberrations of 9p24.1 leading to amplification of JAK2 and increased expression of PD-1 ligands.71,72 Inactivating B2M mutations, as well as driver mutations in BCL7A, GNA13, and PTPN1, are also detected in CHL.73,74
Morphologic and IHC features of the two entities are summarized in Table 1.
Conclusions
NLP is an uncommon form of lymphoma. In recent years, there has been a greater appreciation of its distinctive clinical and biological features, with emphasis on major differences from CHL. This information has led to changes in the clinical management of NLP. For these reasons, an accurate diagnosis is of major importance to the practicing pathologist. The lymph node biopsy should always be examined with knowledge of the clinical history. Key diagnostic features include the nature of the neoplastic cells, as well as the characteristic microenvironment. In addition, ancillary techniques, including immunohistochemistry, can lead to the correct interpretation in most cases.
Declarations
Funding
This work was supported by the Intramural Research Program of the Center for Cancer Research, National Cancer Institute (ZIA SC000550).
Conflict of interest
The authors declare no competing financial interest.
Authors’ contributions
Manuscript writing and editing (YD, ESJ). Both authors have made a significant contribution to this study and have approved the final manuscript.