Integrated Histomolecular Diagnosis in Mediastinal Gray Zone Lymphomas

Abstract

Mediastinal gray zone lymphoma is now recognized as a distinct entity in the newly updated classification of hematolymphoid malignancies. In recent years, several clinicopathological and molecular studies have helped to clarify the spectrum of this entity, which is intermediate between Classical Hodgkin Lymphoma and Primary Mediastinal B-cell Lymphoma. The mediastinal location remains an important feature of the disease and it needs to be cautious to do this diagnosis in extramediastinal location. In addition, cases with EBV association should be better classified in polymorphic EBV+ B-cell lymphomas until a better understanding of this entity. As in PMBCL and CHL, MGZ exhibits a high rate of immune escape with CIITA break and PDL1/PDL2 abnormalities.

1. Introduction

Mediastinal Gray Zone Lymphoma (MGZL) was first described in 1998 [1] and recognized as a provisional entity in the 2008 WHO classification [2, 3], referred to as “B-cell lymphoma, unclassifiable, with features intermediate between Diffuse Large B-Cell Lymphoma (DLBCL) and Classic Hodgkin Lymphoma (CHL)”. It is now a definitive entity of the 2022 WHO (World Health classification of hematologic malignancies) and the ICC (International Consensus Conferences), so-called “Mediastinal gray zone lymphoma” [4].

It is indeed a B-cell lymphoma with overlapping clinical, morphological, phenotypic, and molecular features between Primary Mediastinal (thymic) B-Cell Lymphoma (PMBL) and Classical Hodgkin Lymphoma (CHL), more particularly Nodular Sclerosis CHL (NSCHL). The definition of this entity has evolved over the years, in particular thanks to the development of molecular techniques which made it possible to dismember this entity. MGZL is classified into two subtypes, CHL-like-GZL and PMBCL-like-GZL [5]. CHL-like-GZL is identified based on its morphological similarity to CHL, but with a PMBCL-like immunophenotype characterized by strong and homogeneous expression of B-cell markers on all tumor cells. The second, so-called PMBL- or LBCL-like-GZL corresponds to cases that were morphologically more closely related to LBCL, in particular PMBL, but harbored a CHL immunophenotype. The introduction of Gene Expression Profiling (GEP) [6] and Next Generation Sequencing [7] has led to the reclassification of LBCL-like GZL into two distinct categories. True PMBL-type GZL, which is related to lymphomas of thymic origin (such as CHL and PMBL), and DLBCL-like GZL, which seems to have a different cell of origin and will therefore be excluded from the spectrum of MGZ in the future. In addition, EBV-associated gray zone lesions can be more accurately classified as polymorphic-EBV-Large B-cell lymphoma. In the new 2022 WHO classification, as well as in the ICC, the term inclassable B-cell lymphoma with features intermediate between CHL and Large B-cell lymphoma has been replaced by MGZ. This new nomenclature is used to specifically identify this entity as a primary mediastinal disease.

All studies suggest a common cell of origin for lymphomas of the thymic-anatomic niche, including CHL, PMBL, and MGZL, which may be derived from the same cell of origin, a thymic B lymphocyte [6, 8], although the etiology is unknown. This may explain overlapping disease evolution of these entities [9].

2. Mediastinal gray zone lymphoma

2.1 Epidemiology and clinical presentation

The epidemiology of MGZ is not well known.

Mediastinal Gray Zone Lymphoma commonly affects people between the ages of 20 and 40, with a median age of 32 years (13–83 years) [10] and it is slightly more common in men than in women [10]. The age of onset is thus common with CHL and PMBL, but the latter is more frequent in young female [11]. Most cases have been reported from North America. Like NSCHL, the disease appears to be less common in black people and Asians.

Clinical presentation of GZL includes general symptoms of lymphomas, such as alteration of the general condition (tiredness, loss of appetite, loss of weight), B-symptoms (night sweats, fever), itchy skin or cytopenia (anemia, lymphopenia…). There are signs associated with the presence of a mediastinal mass, which is classically a large anterior prevascular mass. These symptoms include vena cava syndrome, and which sometimes causes noisy symptoms such as tracheal compression and respiratory distress in cases of bulky disease (one third of cases) [10]. Disease is more often disseminated at diagnosis, with predominance of Ann Arbor stages III–IV [8, 10]. In these cases, supraclavicular lymph nodes involvement is frequent, but other peripheral lymph node groups are less commonly involved. Pleural or pericardial effusion/extension is common, and there is a possibility of direct spread to the lung. However, involvement in subdiaphragmatic locations and/or bone marrow extension is uncommon (less than 10% of cases) [10], except in the rare cases of disseminated disease [6].

Rare cases without anterior mediastinal involvement, referred to as non-mediastinal GZL with primary extra-mediastinal presentation (PEMGZL), have been previously reported. These cases generally occur in older patients (median age 65 years), and there is no gender predisposition associated with this condition. The definition of PEMGZL is a topic of ongoing discussion. It is important to note that this diagnosis should be made with caution and restricted primarily to (clinical) research studies.

2.2 Diagnosis

2.2.1 Histology

Most cases of MGZL are composed of a diffuse proliferation of pleomorphic tumor cells in a diffusely fibrotic stroma, sometimes giving the impression of a vaguely nodular architecture in cases with fibrous band. The cells are medium or large, round or oval, and may even appear spindle-shaped due to cell crush artifacts, similar to what can be observed in PMBL. However, the proliferation is more pleomorphic than in the typical case of PMBL, due to the association with lacunar cells and/or Hodgkin cells. The neoplastic lymphocytes show indeed a broad spectrum of cytological appearances, resembling typical Hodgkin and Reed-Sternberg cells, lacunar cells, centroblasts and immunoblasts. This broad spectrum of cytological appearance, with different areas of the tumor showing variations in cytological appearance, is a characteristic feature of MGZL. The background inflammatory infiltrate may contain eosinophils, lymphocytes, plasma cells, and histiocytes, although eosinophils are less abundant than in NSCHL. Sheets of necrosis are usually present, classically without neutrophilic infiltrate.

As seen before, the morphological spectrum of MGZL is broad, extending from CHL-like-GZL to PMBCL-like-GZL. These subtypes may coexist within the same tumor specimen [6].

The majority of MGZL resemble to CHL, particularly the nodular sclerosis CHL (NSCHL) (60%) [5] and are called CHL-like-GZL. Typical cases (group 0) have a morphology closely related to NSCHL and often show confluent, sheet-like growth of pleomorphic cells within a variable abundant microenvironment (including eosinophils and plasma cells) and dense fibrotic stroma. Some cases are less typical (group 1), including some CHL features but associated with intermediate cells and/or diffuse areas, with a background less rich in eosinophils and plasma cells. In this second case, the differential diagnosis with PMBL can be difficult based on morphologic criteria alone.

A minority of MGZL mimic PMBL and are called PMBL-like-GZL. Typical cases (group 3) have a morphology closely related to large B-cell lymphomas, in particular PMBL. These cases present with a rather monomorphic appearance, with sheets of medium to large atypical cells in a variable, dense fibrotic stroma with paucicellular inflammatory infiltrate. Marked pleomorphism and some Hodgkin cells may be encountered. Some cases are also less typical (group 2), with predominance of medium to large atypical and pleomorphic tumor cells, with clear cells, Hodgkin cells, and/or Reed-Sternberg (RS) cells.

2.2.2 Immunohistochemistry

The spectrum of MGZL ranges from cases with a morphology more closely related to CHL but showing a strong B-cell phenotype to cases in which morphology is more closely related to DLBCL but with a CHL-like immunophenotype. This challenging diagnosis should not be overdone and must be considered after exclusion of CHL and PMBL.

The diagnosis of CHL-like-GZL, especially in its classical form (group 0), is based on the discrepancy between morphological appearance and immunophenotype. Indeed, the diagnosis of MGZL should be evoked in case of conservation of the B-cell program on Hodgkin cells, with expression of membranous B-cell marker (CD20, CD19, and CD79a) and of nuclear transcription factor (PAX5, OCT2, BOB1). Both CD30 and IRF4 (MUM1) are usually diffusely positive, with variable intensity, but CD15 may be inconstant [5, 10]. CD23 expression is not classical but may be seen in about 30% of CHL-like-GZL [5]. MAL expression is seen in 49% of cases [5, 12]. Expression of BCL6 is variable. CD10 and ALK are negative, such as surface and cytoplasmic immunoglobulins [5]. The background lymphocytes are predominantly CD3+/CD4+ T-cells, as seen in CHL. MGZL is infrequently positive for EBV, and EBV positivity should prompt suspicion for EBV-positive CHL [5, 13].

PMBL-like-GZL are, at the contrary, characterized by loss of the B-cell program, including loss of CD20 expression (22% of cases) [5] or loss of another B-cell marker. Most cases show intense and diffuse expression of CD30 and CD15. In cases where CD30 intensity is medium to low, CD15 must be diffuse and intense, and vice versa. MAL expression is reported in 61% of cases, and CD23 expression in 49% of cases [5, 12]. MGZL is infrequently positive for EBV, and EBV positivity should prompt suspicion for EBV-positive DLBCL [5, 13].

Morphological and phenotypical data are resumed in Table 1 and illustrated in Figure 1. As these complex histological features are often not reliably identifiable in core needle biopsies, an excisional biopsy is required to arrive at the diagnosis of MGZL. Moreover, this diagnosis rests on the mismatch between morphological and immunohistochemical findings and require therefore a large panel of antibodies.

	CHL-like-GZL (group 0)	CHL-like-GZL (group 1)	PMBL-type GZL (group 2)	PMBL-type GZL (group 3)
Morphology	≈ CHL; Pleomorphic cells with lacunar and Hodgkin/RS cells.; Inflammatory background.	CHL features associated with intermediate cells and/or diffuse areas.; Less inflammatory background.	Predominance of medium to large atypical and pleomorphic tumor cells, with clear cells, Hodgkin cells, or RS cells.	≈ PMBL; Monomorphic appearance.; Paucicellular inflammatory infiltrate.
B-cell markers	CD20 +; PAX5 +; and another B-cell marker + (CD19, CD79a, BOB1, OCT2).		Partial or complete loss of B-cell marker.
CD30; CD15	+++ (variable intensity); +/−		Strong and uniform positive expression of CD30 and/or CD15.

Table 1.

Summary of morphological and immunohistochemical features to differentiate mediastinal gray zone lymphoma (MGZL) subtypes.

Figure 1.

Illustration of morphological and immunohistochemical features to differentiate mediastinal gray zone lymphoma (MGZL) subtypes.

Moreover, patients may present with an unambiguous diagnosis of CHL and PMBL at different time points during the course of the disease, in the same or different anatomic sites, with some composite or sequential/metachronous cases, suggesting a certain degree of plasticity between these entities. These cases should not be classified as MGZL, instead, it is recommended to name and classify the individual components separately. The same holds true for “composite” lymphomas diagnosed at any localization composed of DLBCL and CHL, which are generally not biologically related [2, 14, 15]. For these reasons, this diagnosis is very difficult on core needle biopsy, and an excision biopsy is recommended, and if necessary an expert pathological review and a multidisciplinary integration of clinical and pathological.

2.3 Molecular pathology

Using fluorescence in situ hybridization, cases with PMBL-like morphology harbor more frequent PDL1/PDL2 or CIITA rearrangements than CHL-like-GZL (63 vs. 32% using a CIITA break-apart probe, and 32 vs. 6% using a PDL1/PDL2 break apart probe) [5]. MYC rearrangement is rare, but some studies demonstrated gains of 8q24 (MYC) in among 27% of cases [5]. BCL2 and BCL6 rearrangements are rare (respectively 6 and 0%) [7] in MGZL and their detection should lead to a re-evaluation of the diagnosis of DLBCL with secondary mediastinal location. In cytogenetics, gains at 2p16.1 (REL/BCL11A locus) and alterations at 9p24.1 (JAK2/PDL2 locus) are observed in 33 and 55% of cases respectively [14]. Cytogenetic abnormalities involving the JAK2/PDL2 may be responsible of an increased expression of PDL1 (CD274).

Studies using gene expression profiling (GEP) have further dismembered the CHL/MGZL/PMBL spectrum [6, 16]. Expression signatures revealed differences between CHL and PMBL, and confirmed the presence of different subtypes of MGZL, some clustering more with CHL, corresponding to CHL-like GZL from a morphological and immunohistochemical point of view, and others clustering with PMBL, corresponding to PMBL-like GZL. They also highlighted some characteristics of CHL, shared with the CHL-like GZL, as the lower expression of germinal center genes (GCB) and IFN regulatory factor 4 (IRF4) genes in MGZL and CHL, indicating downregulation of the B-cell program in these entities compared with PMBL and PMBL-like GZL. Conversely, infiltrating T-cells genes (IL6ST, CTLA4, CD28, and ICOS), immune regulation genes (IL1R2, IL32, IL7R, and TNIP3), and macrophage signature were higher in CHL and CHL-like GZL consistent with the presence of an inflammatory background [10, 16].

Studies on the mutational landscape of MGZL have further clarified its characteristics and have allowed distinguishing it from other large B-cell lymphomas that do not have a mediastinal origin/location. The genetical landscape of MGZL shows many similarities to PMBL and NSCHL, reinforcing the hypothesis that it can be the missing link between these two entities. MGZL cases with thymic niche involvement exhibit a pattern of mutation which was very similar to CHL and PMBL, including SOCS1 (45%), B2M (45%), TNFAIP3 (35%), GNA13 (35%), LRRN3 (32%), and NFKBIA (29%) mutations, involving the JAK/STAT and NF-κB pathways. In contrast, cases without thymic niche involvement, so-called DLBCL-like GZL, have a significantly distinct pattern, enriched in mutations related to apoptosis defects (TP53 [39%], BCL2 [28%], BIRC6 [22%]) and depleted in GNA13, XPO1, or NF-κB signaling pathway mutations [7].

2.4 Differential diagnosis

As described earlier, the diagnosis of MGZL should only be considered after careful exclusion of other entities, in particular CHL and PMBL. In daily practice, although the exact number of B-cell markers required to be expressed for MGZL diagnosis remains controversial, the diagnosis of CHL-like-GZL should be reserved for cases with intense and diffuse expression of CD20, associated with expression of PAX5 and with the expression of another B-cell marker on Hodgkin cells [10]. If additional B-cell markers are negative (solely CD20 + and PAX5 +), the diagnosis of CHL will be favored.

Concerning PMBL-like forms, the diagnosis of MGZL should not be based on CD30 expression alone but may be made only in cases of loss of the B-cell program, or, if B-markers are retained, in cases of intense and diffuse co-expression of CD30 and CD15. Phenotypes that should favor the diagnosis of PMBL-like-GZL rather than PMBL in front of a lymphocytic infiltrate made up of large, fairly pleomorphic cells are: (i) CD20 +++, CD30 + intense and diffuse, CD15 +/−; (ii) CD20 +++, CD15 + intense and diffuse, CD30 +/−; or (iii) CD20 -, CD79A +, CD30 +, and CD15 + .

EBV+ MGZL is extremely rare and alternative differential diagnoses as mentioned should be excluded in these cases. In particular, EBV positivity must argue for the diagnosis of EBV+ DLBCL or B-cell lymphoproliferative disorder in immunodeficiency settings, as well as T-cell lymphomas with EBV-positive cells, in particular angio-immunoblastic T-cell lymphoma.

A monoclonal B population is classically present and may help the pathologist to differentiate CHL-like GZL from T-cell lymphomas with Hodgkin or RS-cells, in particular follicular subtype of T-follicular helper (TFH) lymphoma.

As discussed earlier, molecular data can help in the differential diagnosis of MGZ. Thus, the demonstration of a CIITA rearrangement can lead to the diagnosis of PMBL-like-GZL, whereas in case of MYC, BCL2 or BCL6 rearrangements, the diagnosis of DLBCL should be discussed. Similarly, the demonstration of a mutational profile similar to that observed in CHL or PMBL will reinforce the diagnosis of MGZL instead of DLBCL. Finally, techniques using RNA expression such as the nCounter NanoString Assay, developed after the model of GEP studies, can also lead the diagnosis toward CHL or PMBL instead of DLBCL. The demonstration of transcriptional enrichment in genes of the tumor microenvironment, notably those involved in immune escape, may also be an argument favoring the diagnosis of MGZL. These data illustrate the importance of a comprehensive integration of clinical, histological, and molecular data in the differential diagnosis of MGZL, PMBL, CHL, or DLBCL.

2.5 Prognosis and treatment

In retrospective series, the outcome seems to be worse when compared to PMBCL or CHL with a lower response rate to regimens effective in CHL, such as ABVD. Any consensus at this time is described for a consensus treatment.

3. Conclusions

The diagnosis of MGZL should be made with caution especially because no therapeutic consensus exists at this time. A diagnosis of CHL with few B-cell markers and PMBCL with loss of B-cell markers should be considered to adapt the therapeutic options. Not mediastinal cases and EBV cases must be included with careful integration of all data.

Conflict of interest

The authors declare no conflict of interest.