Follicular lymphoma and marginal zone lymphoma: how many diseases?

FL is characterized by effacement of normal lymph node architecture by closely packed neoplastic follicles with loss of polarization, absence of body macrophages, and attenuated mantle zones. The neoplastic cells are characteristically a mixture of CC and CB. The 3rd and 4th edition World Health Organization (WHO) classifications of lymphomas [1, 4] recommended FL grading based on the number of CB per high-power field (HPF): grade 1 < 5CB/HPF; grade 2 = 5 to 15 CB/HPF; and grade 3 or high-grade > 15 CB/HPF. Grade 3 is subdivided into grades 3A and 3B; the latter is assigned to FL cases, where neoplastic follicles are essentially composed of sheets of CB. Moreover, because grades 1 and 2 comprise a morphologic continuum and are both clinically indolent, the designation grade 1–2 (low-grade FL) was introduced and widely used [1, 4]. However, FL grading based on strict counting methods of CB and CC (cells that may exhibit variation in their cytological features) lacks reproducibility and its clinical impact, especially between grades 1, 2, and 3A, appears to be less significant in the era of rituximab. Therefore, the 5th edition of the WHO [3] proposes grading to be optional in FL, gathering grades 1, 2, and 3A as classic FL, whereas FL grade 3B was renamed as follicular large B-cell lymphoma (FLBL). Although, the 2022 ICC [2] recognizes the biological relationship between grades 1, 2, and 3A, and acknowledges some difficulties in FL grading, the consensus among clinicians was to retain histologic grading, in part to allow for investigation of the response of new agents. Whether patients with grade 3A have a more adverse prognosis [5, 6] or deserve different management remains debatable and needs to be re-evaluated in the future, given evolving non-cytotoxic therapeutic approaches [5‐9]. Of note, some FLs are composed of neoplastic cells that are not classic CB but rather small-sized blasts/blastoid cells or large cleaved cells/large centrocytes that do not strictly fit the FL3A or FL3B 2017 WHO criteria [10]. Based on the challenges in distinguishing FL 3A and 3B, the 2022 ICC recommends that emphasis should be placed on ancillary studies in conjunction with morphological assessment. The demonstration of BCL2 rearrangement (BCL2-R) and CD10 expression both favor grade 3A [2]. The 5th edition WHO recommends the name of FL with uncommon features (uFL) for these and other cases with variant morphology [3]. FL commonly has a follicular growth pattern sustained by a follicular dendritic cell (FDC) network. Sometimes more and less extended diffuse areas can be observed. Diagnostic reports traditionally identify the growth pattern, with designation as mainly follicular pattern (at least 75%); FL with follicular and diffuse pattern (25–75%); predominantly diffuse (< 25%); and totally diffuse (no follicles). Moreover, t(14;18)-negative CD23⁺ FCL, often having a diffuse growth pattern, should be also distinguished from conventional FL (see below). As recommended in the last WHO 2017 [1] and 2022 ICC [2], the presence of entirely or predominantly diffuse areas, in cases with high-grade cytology, will support a diagnosis of diffuse large B-cell lymphomas (DLBCL). However, treatment decisions in individual patients should not be based on pathology information alone but rather on integration of clinical and pathologic data [3].

In addition, several cytologic variants including FL with marginal zone B-cell differentiation (monocytoid-like cells), floral variant, or sclerosing variant (Castleman-like features) of FL have been described but are not separated from conventional FL. Other cytologic FL variants are less commonly seen.

FL cells express the pan B-lymphocyte antigens (CD19, CD20, CD22, CD79a, PAX-5) and IgM with or without IgD. Similar to normal GC cells, FL cells express CD10 and BCL6, usually stronger in the neoplastic follicles than in interfollicular areas. Moreover, a subset of FL especially grade 3B can be negative for CD10 and/or BCL6. In this context, other GC markers including GCET1, HGAL (GCET2), LMO2, and/or MEF2B should be performed to establish a GC phenotype and rule out other B-cell lymphoma diagnoses.

FL cells typically overexpress BCL2 as a result of the genetic hallmark of FL, t(14;18) IGH::BCL2 translocation. Similar to CD10 staining, the frequency of BCL2 expression is higher in low-grade (85–90%) than in high-grade (50–70%) FL. Overall 10–15% of FL cases, especially grade 3B [10, 11], remain BCL2-negative due to the lack of BCL2-R. The variable expression of BCL2 may also be due to mutations in the BCL2 gene that alter the epitopes recognized by some monoclonal antibodies used for diagnosis. In these “pseudo-negative” cases, other BCL2 antibodies and/or FISH for BCL2-R should be tested [12‐14].

FL is commonly negative for IRF4/MUM1. However, it can be expressed in some cases, especially with grade 3B cytology. In these particular cases of IRF4/MUM1-positive FL grade 3B, NOTCH1/2 mutations have been reported to be associated with poorer prognosis [15]. In addition, all IRF4/MUM1-positive FL with high-grade cytological features, especially grade 3B and uFL [2, 3], should be evaluated for IRF4 alterations, especially in younger patients to rule out LBCL-IRF4 (see below).

The follicular pattern of FL may be assessed by the identification of FDC networks that are positive for CD21, CD23, and/or CD35, usually absent in cases with diffuse areas. FL with grade 1–2 usually shows a lower Ki-67 proliferation index (PI) than grade 3. However, some FL 1–2 can exhibit a high PI within follicles. The significance of an increased proliferative rate within follicles is not established [16‐18]. Assessment of PI using Ki67 staining can be difficult because its distribution is not uniform within follicles and its expression may be highly variable between follicular and interfollicular areas. Altogether, Ki67 staining can be specified in diagnostic reports, but still has uncertain clinical significance in isolation [19], and is not required for grading.

In 2009, Katzenberger et al. [45], described a diffuse variant of FL with unusual clinical and pathological features. Subsequently, it was found that these cases frequently carry STAT6 mutations [31]. Further studies reported a close association of CD23 expression with STAT6 mutation, features which correlated with localized disease. Moreover, it was appreciated that at least 30% of the cases were purely follicular [33]. Due to their characteristic, clinical, morphological, and molecular features, the 2022 ICC proposed recognizing these cases as a provisional entity [2]. The specific category of BCL2-R-negative CD23⁺ FCL is not recognized in the 5th WHO [3], although there is some overlap with the subtype identified as “diffuse variant.” BCL2-R-negative CD23⁺FCL is more frequent in women (M:F = 2:1), and often presents with inguinal involvement, but non-inguinal presentations including cervical, axillary, and even retroperitoneum can occur [31]. Patients usually present with low clinical stage. The characteristic histological features are illustrated in Fig. 1. Diagnostic criteria include the absence of the t(14;18), expression of CD23, and at least one GC marker. BCL2 staining is usually negative or weak positive. The molecular profile includes a high frequency of STAT6 and CREBBP co-mutation as well as 1q gain and a recurrent loss 1p36 loss/TNFRS14 abnormalities. The latter has been described at variable frequencies (30–97%) [33, 46]. As noted, CD23 expression is a helpful surrogate marker for the detection of STAT6 mutations [33]. Interestingly, BCL2-R-negative CD23⁺ FCL without STAT6 mutations carry SOCS1 mutation, which is known to be upstream of STAT6 and thereby contributes to STAT6 activation [33].

PTFL was recognized as a definite entity in the WHO 2017 classification [1]. It occurs in children and adolescents, and has an excellent prognosis with conservative management. PTFL is usually characterized by an expanded and serpiginous follicular proliferation of monotonous CB, often with a starry sky pattern and moderate to high proliferation. Neoplastic cells express GC markers whereas BCL2 is typically negative. BCL2-R, BCL6-R, MYC-R, and IRF4-R are negative. The mutational profile is distinct from that observed in conventional FL with a high frequency of MAP2K1 mutations and 1p36/TNFRS14 alterations (30–70%) [47‐49]. Additional mutations in IRF8, a tumor suppressor gene, are also common in PTFL (15–50%) [49‐51]. Mutations in epigenetic modifiers are uncommon, in contrast to conventional FL. Among them, KMT2D is the most frequent mutated gene, observed in 16% but alterations in other genes such as CREBBP, EP300, MEF2B, and EZH2 are rare. PTFL frequently shows evidence of marginal zone differentiation, and recent studies support the view that pediatric-type nodal marginal zone lymphoma (pNMZL) is part of the spectrum of PTFL, with both disorders having similar molecular profiles, clinical presentations, and outcomes [51]. Importantly, FISH testing and mutational profile are advised to rule out a diagnosis of conventional grade 3B FL as well as LBCL-IRF4, especially in young patients.

PCFL will be discussed elsewhere in this issue with other cutaneous lymphoproliferative disorders.

Testicular follicular lymphoma occurs in children and is only rarely reported in adults. This variant has been recognized as a distinct form in 2022 ICC [2] but still considered as a subtype of FL in the 5th WHO [3]. It shares the same morphological and molecular profile as FL BCL2-R-negative with negative or weak expression of BCL2 and absent BCL2-R. Frequent TNFRSF14 alterations, as seen in PCFL, and MAPK mutations, as observed in PTFL, have been reported [52] but further studies are needed to define the molecular landscape of this specific variant.

LBCL-IRF4 was introduced as a provisional entity in the WHO 2017 classification under the group of FL [1], and now is upgraded to a definite entity in the 2022 ICC. It remains within the group of FL [2]. The 5th WHO classification [3] also recognizes LBCL-IRF4 as a definite entity but grouped this disease with large B-cell cell lymphomas, which might be misleading, giving the impression that LBCL-IRF4 is an aggressive disease. LBCL-IRF4 is most common in children and young adults and preferentially involves the head and neck region, particularly Waldeyer’s ring but also the intestine [53]. However, it can also occur in adults [54, 55]. It usually presents as a localized disease (stages 1–2) with excellent prognosis, regardless of the growth pattern (see below). Some cases presenting with purely follicular growth patterns, in children, have been reported to achieve complete remission without systemic treatment [56, 57], stressing the indolent nature of the disease. LBCL-IRF4 is characterized by large B-cells with the expression of at least one GC marker and constitutive expression of MUM1/IRF4, which correlates with IRF4 translocation (Fig. 2). The growth pattern might be follicular, follicular/diffuse, or diffuse [54]. The cells lack BCL2-R or MYC-R. However, some cases could harbor BCL6-R together with IRF4-R. FISH for IRF4-R is preferred for diagnosis, but cases lacking demonstrable rearrangements should have evidence of either IGH or IGK/IGL breaks. Some cases can display a cryptic IRF4 rearrangement leading to a false IRF4-R-negative by FISH. In these cases, the detection of IRF4 mutation may support the diagnosis. In addition, IRF4-R can occur as a secondary event in other aggressive B-cell lymphomas in adults, and in this context, the rearrangement is not specific for the diagnosis of LBCL-IRF4 [54, 55].