by Dr Weijie Li
Cytogenetic and molecular testing has been becoming more and more important in the diagnosis of human diseases. These tests are indispensable for the diagnosis and/or prognostic prediction for some hematopoietic malignancies. This article discusses a group of lymphomas with similar morphology and phenotype but distinct cytogenetic/molecular abnormalities, with emphasis on the importance of proper cytogenetic/molecular testing in the diagnosis of these diseases.

Background

Burkitt lymphoma (BL) is an aggressive high-grade B-cell lymphoma (HGBCL), which typically shows sheets of monomorphic mediumsized tumour cells with round nuclei, finely dispersed chromatin, and multiple basophilic small to intermediate-sized nucleoli (Fig. 1a). The cytoplasm is moderately abundant and highly basophilic with multiple lipid vacuoles better visualized on Wright’s and/or Giemsa stained air-dried smears or imprint slides. There are many mitotic and apoptotic figures, and numerous intermixed tingible body macrophages resulting in a so-called ‘starry sky’ pattern. The immunophenotype of typical BL is that of germinal centre B-cell-type (GCB), positive for immunoglobulin (Ig) M, CD19, CD20, CD22, and CD79a, CD10 and BCL6 (see Table 1 for definitions). BCL2 is usually negative. The proliferative index (as determined by Ki-67 levels) is nearly 100% (Fig. 1a) [1].
There are three clinical variants of BL: endemic, sporadic and immunodeficiency-associated. They differ mainly in their geographic distribution, clinical presentation and anatomic localization of primary tumour. Regardless of the clinical variants, the molecular hallmark of BL is the translocation of the MYC proto-oncogene (MYC) (8q24) to the Ig heavy chain region (14q32), t(8;14) (q24;q32), or less commonly to the Ig light chain kappa locus on 2p12 or the Ig light chain lambda locus on 22q11. There are other molecular cytogenetic changes, which include copy-number gains involving 1q, 7 and 12 and losses involving 6q, 13q32-34 and 17p. Gene expression profiling studies have defined a molecular signature characteristic of BL, which is different from other lymphomas such as diffuse large B-cell lymphoma (DLBCL) [2]. Mutations of the transcription factor 3 (TCF3) gene or the gene for its negative regulator, inhibitor of DNA binding 3 (ID3) have been frequently detected by next-generation sequencing analysis in sporadic BL cases. The resulting mutant proteins can activate B-cell receptor signalling, which sustains BL cell survival by engaging the phosphoinositide- 3-kinase pathway. Other mutations in CCND3, TP53, RHOA, SMARCA4 and ARID1A genes are also detected in certain BLs. Endemic cases show fewer mutations overall and lower proportion of mutations in TCF3 or ID3 [1].
Technically, all BL cases should have MYC rearrangement. It is controversial to make a diagnosis of BL when MYC rearrangement is not detectable. However, routine fluorescence in situ hybridization (FISH) and chromosomal analysis may not be able to detect this translocation owing to complex karyotype or more complicated translocation. Additionally, by gene expression profile study, some MYC-negative cases show a typical BL molecular signature [2]. Therefore, in the presence of classic morphology and phenotype, the diagnosis of BL should be made even without the demonstration of this translocation. Recent studies [3, 4] show that some true MYC-negative cases have characteristic 11q aberration, which has been recognized by the 2016 WHO classification as a provisional new entity [1, 5] and will be discussed more in detail next.
Historically, there has been no consensus on the diagnostic classification of the lymphomas with morphology resembling BL but showing more cytological pleomorphism and/or other atypical morphologic or phenotypic features. These were so-called ‘grey-zone lymphomas’ and classified as atypical BL/Burkitt-like lymphoma (BLL) by the 2001 WHO Classification of tumours of hematopoietic and lymphoid tissues. The name was then changed to B-cell lymphoma, unclassifiable, with features intermediate between BL and DLBCL by 2008 WHO classification [6]. These lymphomas were thought to represent a continuum between BL and DLBCL.

2016 revision of the WHO classification

The 2016 WHO classification of tumours of hematopoietic and lymphoid tissues has shed some light on this field with the modification of the grey-zone lymphoma with features intermediate between BL and DLBCL, and the creation of Burkitt-like lymphoma with 11q aberration (BLL-11q) and large B-cell lymphoma with IRF4 rearrangement (LBL-IRF4) [1, 5].
HGBCL with MYC and BCL2 and/or BCL6 rearrangement and HGBCL, not otherwise specified
The grey-zone lymphoma with features intermediate between BL and DLBCL has been divided into two categories: HGBCL with MYC and BCL2 and/or BCL6 rearrangement (double-hit or triple-hit lymphomas); and HGBCL, not otherwise specified (NOS). These lymphomas usually occur in older adults, with men and women affected equally. They are usually present as a rapidly enlarging mass involving lymph nodes or extranodal sites with advanced stage. These lymphomas have very poor prognosis with no optimal therapeutic strategy [7–9]. Double-hit (DH)/triple-hit (TH) refers to the co-occurrence of MYC and BCL2 and/or BCL6 translocations. MYC rearrangements usually differ from those seen in BL: they often involve non-IG partner genes. These lymphomas usually have complex karyotypes. The majority (60–70%) of them have MYC/BCL2 rearrangements, with a minority (15–30%) harbouring MYC/BCL6 rearrangements and fewer (10%) harbouring rearrangements of all three genes. Rare cases of follicular lymphoma or B-lymphoblastic leukemia/lymphoma can have these DH/TH cytogenetic changes. They should not be included in this category. HGBCLs with DH/TH morphologically can resemble BL, DLBCL or lymphoblastic lymphoma. They are phenotypically mature B-cell lymphomas with the presence of CD19, CD20, CD79a, and Pax-5 and lack of TdT proteins. Most cases also produce CD10 and BCL6. HGBCL, NOS includes cases with atypical BL features or blastoid morphology without DH or TH cytogenetic findings, regardless of MYC status. They are phenotypically mature B-cell lymphomas. For cases with blastoid morphology, cyclin D1 and SOX-11 should be stained for to rule out blastoid variant of mantle cell lymphoma. Approximately 20–35% of HGBCL, NOS cases have a MYC rearrangement [1].
BLL-11q lymphomas
BLL-11q encompasses cases with morphologic, phenotypic, and gene expression resemblance to BL, but lacking MYC translocation and harbouring characteristic proximal 11q gains at 11q23.3 and distal 11q loss at 11q24-qter (Fig. 1b) [1, 3–5]. These 11q changes are likely to be the result in the upregulation of oncogenes PAFAH1B2, USP2, and CBL located in the gained regions of 11q23, and corresponding down-regulation of tumour suppressor candidate genes FLI1, ETS1, TBRG1, and EI24 located in the regions of 11q24-qter loss. Cases of BLL-11q commonly show some degree of cytomorphologic pleomorphism and tend to have a more complex karyotype than BL [1]. The phenotype is that of GCB in nearly all cases [3, 4]. Besides the classic gains and losses within 11q, additional frequently identified abnormalities include del(6q) and trisomy 12 [10]. A recent study has shown that BLL-11q lymphomas have mutational landscape distinct from BL [11], which indicates that BLL-11q is truly a distinct entity. BLL-11q cases occur over a wide age range but are more common in children and young adults. They are more frequently nodal than BL and tend to present as a single dominant mass or conglomerate mass [4, 10]. Patients tend to present with limited disease without involvement of bone marrow or central nervous system, and prognosis appears to be favourable, similar to classical BL [1].
LBL-IRF4
LBL-IRF4 most commonly affects children and young adults [1, 5]. It mainly involves the Waldeyer’s ring and cervical lymph nodes and usually presents as low stage disease. Microscopically, the tumour cells are medium to large with finely clumped chromatin and small basophilic nucleoli. A starry sky pattern is usually absent, though proliferation rate is usually high by Ki-67 stain. These lymphomas may have a diffuse growth pattern, follicular growth pattern, or follicular/diffuse pattern. The tumour cells are positive for B-cell specific markers (CD20, CD79a, Pax-5), and characteristically show high levels of IRF4and BCL6. Over 50% of the cases are also positive for BCL2 and CD10. Despite the high levels of IRF4, these cases have a GCB signature by gene expression profiling. Most cases have a cytogenetically cryptic rearrangement of IRF4 with an Ig heavy chain locus. BCL6 alterations may be detected in some cases, but essentially all cases lack MYC and BCL2 rearrangement. Most cases have shown good response to chemotherapy [12, 13].

Diagnosis of these lymphomas

All the lymphomas described above except BL are uncommon and quite confusing to most general pathologists. They can be easily misdiagnosed in the absence of a proper work-up plan. Based on our practical experience, a step-by-step diagnostic algorithm has been designed, as shown in Figure 2. This diagnostic scheme is not recommended for the practice in BL endemic areas, where morphology and phenotype are likely to be adequate for the diagnosis of most BL cases. When a lymphoid malignancy is encountered with BL or BLL morphology, and mature B-cell phenotype is demonstrated by flow cytometry and/or immunochemical staining, the first check required is for MYC rearrangement. If MYC rearrangement is detected by FISH or other methods, BL will be the diagnosis in the presence of classic BL morphology and phenotype. If there is morphologic and/or phenotypic atypia, further testing for BCL2 and BCL6 rearrangement is necessary. In the presence of BCL2 and/or BCL6 rearrangement, the diagnosis will be HGBCL with DH or TH. Without the rearrangements of BCL2 and BCL6, the diagnosis will be HGBCL, NOS. Based on our experience in a children’s hospital in the USA, sporadic BL cases frequently show a certain degree of atypical morphologic features. And HGBCL with DH or TH is exceedingly rare in children [7, 8, 14]. Therefore, testing for DH/TH is usually not necessary for pediatric cases. These pediatric cases should be diagnosed as BL as long as MYC rearrangement is detected unless morphologic and phenotypic atypia is significant. The HGBCL, NOS category is not applicable for pediatric cases, considering the excellent prognosis of most pediatric HGBCL cases and their distinct molecular features [14–16]. In the pediatric population, MYC+ cases with significant morphologic and/or phenotypic atypia but no DT/TH should be diagnosed as DLBCL, NOS.
Although high Myc levels (in >80% of nuclei) are present in most cases of BL, there is much more variation in the HGBCLs with DH/ TH. Although most studies have concluded that Myc staining is not reliable enough to be used for the selection of cases for cytogenetic or molecular testing, some studies suggest using a cut-off of >30% or >70% Myc positivity for case selection [17, 18].
The rearrangements of MYC, BCL2, and BCL6 should be detected by a cytogenetic/molecular method such as FISH. The presence of only copy-number changes or somatic mutations, without an underlying rearrangement, is not enough to qualify a case for this category. So-called double-expresser DLBCLs show immunohistochemical overexpression of Myc and BCL2 protein and have a relatively poor prognosis [19]. However, overexpression cannot be used as a surrogate marker for DH cytogenetic status, because most double-expressers are not DH lymphomas (although most DH lymphomas are also double-expressers).
If MYC rearrangement is absent (MYC−), the newly proposed entity, BLL with 11q, should be considered and properly tested. The diagnosis of BLL-11q is based on the presence of characteristic gain/loss patterns of 11q, together with BL/BLL morphology, GCB phenotype, and lack of MYC rearrangement. The characteristic 11q aberration is key to making the diagnosis, but its presence alone is neither specific nor diagnostic since it may also be present in MYC+ BL or DLBCL [10, 20]. The most sensitive method for detecting this characteristic cytogenetic finding is DNA microarray. The 11q aberration can be visualized by chromosomal analysis. However, chromosomal analysis relies on tumour cell viability and metaphase morphology, and the finding may not be characteristic for this aberration if the resolution is low. Another potential diagnostic strategy is FISH for chromosome 11 abnormalities. Commercially available FISH probes for chromosome 11 regions may be used to detect gains within 11q and 11q terminal loss. As a result of the variation of gain/loss spots among the cases, depending on the probes used, FISH alone may miss some cases. If MYC rearrangement is absent and there is no typical 11q aberration, the diagnosis should be HGBCL, NOS for adult patients. For pediatric patients, these lymphomas often have a Burkitt or intermediate molecular gene expression profile and show an excellent prognosis. These cases should not be classified as HGBCL, NOS. For the pediatric cases with morphology and phenotype close to BL and relatively simple karyotype, the diagnosis should be BL; otherwise the diagnosis should be DLBCL, NOS. If the case shows diffuse and levels of of IRF4 and BCL6, especially in Waldeyer’s ring and cervical regions, LBL-IRF4 with diffuse growth pattern should be considered. The diagnosis of LBL-IRF4 should be confirmed by FISH analysis for IRF4 rearrangement [21].

Conclusion

With the advances in cytogenetic/molecular studies of lymphomas with BL or BLL morphology, new classification of these lymphomas has been proposed. Accurate diagnosis of these lymphomas needs the combination of cytogenetic/molecular testing results, morphology and phenotype. More specific or targeted treatment for these lymphomas may be on the horizon, and hence accurately diagnosing them is clinically important.

The author

Weijie Li MD, PhD Department of Pathology and Laboratory Medicine,
Children’s Mercy Hospital, University of Missouri-Kansas City School of
Medicine, Kansas City, MO 64108, USA

E-mail: wli@cmh.edu