Double-hit lymphoma (DHL), triple-hit lymphoma (THL), diffuse large B-cell lymphoma (DLBCL), high grade B-cell lymphoma (HGBL) with MYC, BCL2 and/or BCL6 rearrangements

The DH lymphoma most commonly affects elderly patients with a median age of 62-74 years (range 18-96 years), and both sexes are affected with a slight male predominance. The incidence of DH genotype has been difficult to assess because fluorescence in situ hybridization (FISH) studies have not been used widely on unselected series, and the published data may be biased toward a specific type of lymphoma. Nevertheless, DH lymphoma has been reported in 5-15% of patients with DLBCL and aggressive B-cell lymphoma. Comprehensive analysis of Mitelman Database of Chromosome Aberrations in Cancer (Aukema et al, 2011, Oliveira et al 2017), DH lymphomas with breaks at MYC, BCL2 and/or BCL6 regions were identified in 14% of DLBCL cases; 62% of those involved MYC and BCL2 sites, 18% involved MYC and BCL6 while the remaining cases had triple translocations. Using FISH, Barrans et al (2010) identified DH and TH genotypes in 12% cases with de novo DLBCL; the majority of these cases had BCL2 and MYC translocations (66%), BCL6 and MYC breaks in 10% and breaks in all three sites in 24% of cases. MYC and BCL2 translocations are also found in a small proportion of follicular lymphoma. We identified MYC breaks in 7% of t(14;18)-bearing follicular lymphoma cases; all described to have a high grade morphology with Burkitt-like or blastic morphology replacing residual follicles (Mohamed et al, 2001). Pedersen et al have reported that in transformed lymphoma, DH genotype was very frequent found in 21% of cases; all had follicular lymphomas (Pedersen et al 2012).

Patients with DH lymphomas commonly have an aggressive clinical course, characterized by advanced stage disease, extranodal involvement, and high serum lactate dehydrogenase levels. The extranodal sites include the bone marrow, central nervous system, gastrointestinal tract, liver, spleen, lung, pleural fluid, testis, ovaries, skin, and thyroid gland. Bone marrow is involved in about 50% of patients and many have a leukemic disease. The International Prognostic Index score (IPI score) is often intermediate-high or high (Barrans et al 2010, Petrich et al 2014). Most patients have a de novo disease while a minority (10%) present with a history of a low-grade follicular lymphoma or other B-cell lymphoma, then develop DHL presumably by the acquisition of MYC rearrangement.
Double Expresser lymphoma
Immunohistochemistry (IHC) has identified a fraction of DLBCL cases with high expression of MYC and BCL2 proteins (Perry et al 2014, Mahmoud et al 2015). These double expresser lymphomas (DE) are lacking MYC and BCL2 translocations and are usually of non-GC types. DE lymphomas are much more common than DH lymphomas, with approximately 30% of DLBCL being reported as DE lymphomas. Most studies use a cutoff of 40% for MYC IHC and a cutoff of 70% for BCL2 to define DE lymphoma (Green et al 2012 A). Clinically, the DE lymphomas have more aggressive clinical course than those lacking MYC and BCL2 protein expression.

Because DHL is genotypically defined; it is not restricted to a specific histology subtype. Most tumors are medium to large in size, with atypical morphology and very high proliferation rate. Previously, DH lymphomas were diagnosed as diffuse large B-cell lymphoma (DLBCL), aggressive B-cell lymphoma not otherwise specified (NOS) or Burkitt-like lymphoma. Rare cases were classified as lymphoblastic lymphoma. The increase recognition of these lymphomas led the 2008 WHO classification of hematopoietic and lymphoid neoplasms to introduce a provisional category of "B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL", (BCLU) to recognize a subset of very aggressive lymphoma in which the distinction between DLBCL and BL is difficult (Campo et al 2011, Swerdlow et al 2014). The 2008 WHO criteria were somewhat subjective and vaguely defined; as a result, recent studies have not been consistent with survival and disease outcome. Therefore, separation of these cases has become necessary to better define these clinically aggressive tumors. In the revised 2016 WHO classification of lymphoid neoplasms, all LBCL with "double hit" and "triple hit" genotype are included in a single category of "high-grade B-cell lymphomas (HGBL) with MYC and BCL2 and/or BCL6 rearrangements" (Swerdlow et al, 2016).
Immunophenotype
Most double hit lymphomas have a germinal center (GC) phenotype with expression of CD10 and BCL6 and lack of MUM1/IRF4. Expression of BCL2 protein is detected in almost 95% of cases supporting the observation that the BCL2 translocation is mainly found in GC type of DLBCL, and also MYC translocations are associated with a GC molecular profile in DLBCL. The proliferation index as measured by Ki67 immunohistochemical staining is often high (>90%). Therefore, although not very specific, co-expression of CD10, BCL6, BCL2, and a high Ki67 may predict double hit genotype in tumors morphologically diagnosed as DLBCL (Aukema et al 2011, Swerdlow et al 2014).

Standard therapies such as R-CHOP developed for patients with DLBCL are less effective in DHL, with a median overall survival of 12 months or less, as the majority of patients experience disease progression. Currently, more, intensive chemotherapy such as dose adjusted R-EPOCH therapy, is being evaluated which appears to be effective in many DH lymphoma patients. Consideration for frontline stem cell transplant in responding patients are also suggested (Oki et al 2014, Petrich et al 2014, Dunleavy 2015).

Patients with classic, FISH-defined DH lymphomas have a very poor prognosis, usually present with high clinical risk factors, such as high IPI scores and bone marrow involvement. The outcome is dismal with rare long term survival after standard therapy (Perry et al 2014, Petrich et al 2014, Oki et al 2014). Several recent studies have indicated that the outcome of DE lymphomas, as defined by IHC is inferior to that of DLBCL but better than that of DH lymphomas. Therefore, the co-expression of MYC and BCL2 proteins in DLBCL is considered a prognostic biomarker of an inferior outcome (Green et al, 2012 B).

t(8;14)(q24;q32), t(14;18)(q32;q21), t(3;14)(q27;q32) and variants.

Double-hit lymphomas are usually seen in the context of a complex karyotype containing multiple numerical and structural aberrations (Figure 1). Over 2/3 of cases demonstrate dual translocations involving 8q24/MYC and t(14;18)/BCL2 breakpoints. Less commonly the translocations implicate 8q24/MYC and 3q27/BCL6 sites and rare cases have breaks in all three sites, referred to a "triple hit" lymphoma. The partner gene(s) involved in MYC/8q24 translocations are the IG genes in about of 60% of DHL cases; more frequently affecting the IG light chain loci 22q12/IG lambda and 2p12/IG kappa loci than the heavy chain 14q32/IGH (Aukema et al 2011, Swerdlow et al 2014). Non-IG partners are found in 40%, recurrently involved 19p13, 1q24, 9p13, or non-identified partner regions (Figure 1). Deletions of chromosome 17p13/ TP53 are present in 20-30% of cases, which can further induce genomic instability, and contribute to the aggressive course of the disease (Figure 1).

Conventional cytogenetics is not always feasible since fresh tissue is required, and small translocations can be overlooked in a complex karyotype. Therefore, most cases currently are detected by FISH using labeled probes targeting genes of interest. FISH studies can be successfully performed with high reproducibility in almost all cases using formalin-fixed paraffin-embedded tissue sections, although smears, touch imprints, or pellet of metaphase and interphase cells can also be used. Today, most clinical cytogenetic laboratories are using a panel of FISH probes for detection of double hit lymphomas including LSI MYC break-apart (BA), LSI BCL6 BA, and LSI IGH-BCL2 dual fusion translocation probes. The IGH-MYC dual fusion translocation probe is useful in picking up occasional cases missed by the MYC BA probe and also in identifying the MYC partner in a subset of the cases (Figure 2 B). Collectively, FISH is considered the "Gold-Standard" test in identifying DH and TH lymphomas. At present it is a subject of debate, if all DLBCL should have FISH testing to detect MYC, BCL2, and BCL6 rearrangements or should FISH only performed on selected cases with high grade morphology and CG phenotypes. Guidelines are warranted to avoid high cost and at the same time not miss those aggressive lymphomas (Pedersen et al 2012, Sesques and Johnson 2017).

MYC translocations, a biological hallmark of BL, can also be detected in other B-cell lymphomas DLBCL, BCLU, and FL but at lower frequencies (mohamed et al 2001). However, there are some fundamental differences between MYC translocation in BL and in other mature B-cell lymphomas. In BL the MYC translocation always involves one of the IG genes, (IGH, IGL or IGK) and is considered a disease-initiating event which occurs in the context of a rather simple karyotype (Dave et al 2006). In contrast, MYC translocations in other mature B-cell lymphomas frequently involve non-IG partners and are mostly found in complex karyotypes, often in addition to well-known primary aberrations such as t(14;18) or t(3;14) translocation. In these lymphomas, MYC translocation is considered as a secondary event occurs during disease progression. Dave et al reported that the gene expression of MYC translocations in BL tend to be simple and control several genes involved in proliferation. In contrast, MYC aberrations in DLBCL-targeted genes are associated with nuclear factor kB (NF-kB) signaling and antiapoptotic cascades. Other mechanisms of MYC deregulation in aggressive B-cell lymphomas are mutations, increase copy number and transcriptional upregulation, mainly by B-cell receptor and NF-kB signaling. Recent studies show that microRNAs may upregulate MYC expression. In clinical setting, MYC aberrations can be detected by conventional cytogenetics, FISH, and recently by IHC.
In t(14;18) follicular lymphoma, the transcription of BCL2 is "constitutively" upregulated that leads to a survival advantage of the involved B cells. BCL2 overexpression in B cells also leads to impaired DNA repair by blocking nonhomologous end-joining activities of Ku proteins essential for repair of both RAG1 / RAG2 - and AICDA -mediated breakpoints. The t(14;18) is thought to occur early in B-cell development however, it is insufficient to cause follicular lymphoma because t(14;18) is rarely observed as a sole karyotypic abnormality (Mohamed et al 2001) and also can be found in mature B cells in healthy individuals. The over-expression of BCL2 makes the cells susceptible for additional genomic alterations such as MYC or BCL6 mutation and rearrangement. BCL2 protein is detected in over 50% of DLBCL and in 75% of HGBL, but is not expressed in BL.

The "double-hit" lymphoma is a genotype feature of lymphoma that exhibits translocations of both the MYC /8q24 gene and a second site, most often the BCL2 /18q21 or less commonly the BCL6 /3q27 gene. A rare "triple-hit" lymphoma harboring MYC, BCL2, and BCL6 translocations has also been identified. Cytogenetics is necessary in establishing the diagnosis of these lymphomas as they tend to manifest an aggressive behavior, and poor outcome when treated with standard therapy.