A double‐hit High‐grade B‐cell lymphoma with three‐way translocation t(3;8;14)(q27;q24;q32) involving BCL6, MYC, and IGH

Key Clinical Message We describe an High‐grade B‐cell lymphoma case, in which a complex translocation t(3;8;14) with effects on the genes BCL6, MYC, and IGH, was detected. This case could be the first double‐hit lymphoma with a single chromosome rearrangement causing the double effect with three genes involved.


| INTRODUCTION
The World Health Organization (WHO) updated Classification of tumors of Haematopoietic and Lymphoid Tissues (2016) introduced a new subset of mature B-cell neoplasms, Highgrade B-cell lymphoma (HGBL) with MYC and BCL2 and/ or BCL6 rearrangements.
This new subtype is now commonly referred to as doublehit lymphoma (DHL) or Triple-Hit Lymphoma (THL). 1 DHL and THL are associated with an aggressive clinical course and resistance to conventional chemotherapy regimens. The main genetic feature is the occurrence of a translocation involving MYC (8q24) together with rearrangements involving BCL2 (18q21) and/or BCL6 (3q27) genes. The involvement of BCL6 is less frequent compared to BCL2 (10% and 73%, respectively). 2 It has been hypothesized that this different rate can be due to either MYC + /BCL6 + DHL being less frequent or because of an underestimation of BCL6 rearrangements as recorded in the Mitelman's database. 3 Indeed BCL6 breakpoints are located in the subtelomeric region of the long arm of chromosome 3 and may be missed by standard chromosomes analysis. 4 Another hallmark of DHL is the greater degree of cytogenetic complexity compared to Burkitt lymphoma (BL) with which it often shares morphologic features. In particular, MYC + /BCL2 + DHL and THL are cytogenetically as complex as DLBCL, instead of MYC + /BCL6 + DHL that can show a simple karyotype in half cases. 5 MYC + /BCL6 + DHL shares many morphological features with MYC + /BCL2 + DHL, but immunohistochemistry reveals some differences because MYC + /BCL6 + DHL is mostly BCL2 negative, with decreased CD10 expression but apparently increased IRF4/MUM-1 immunoreaction. 5 Furthermore, it is reported that MYC + /BCL6 + DHL is aggressive, with a recurrent extra-nodal localization, poor prognosis, and a germinal center (GC) type phenotype. 5,6 Here, we report a patient presenting with clinical conditions suggesting acute leukemia, then concluded as a case of DHL associated with a three-way chromosomal translocation t(3;8;14)(q27;q24;q32) involving BCL6, MYC, and IGH genes, respectively, confirmed by fluorescence in situ hybridization (FISH) performed on metaphase cells.

| Flow cytometry, cytogenetic analysis, and FISH
Peripheral blood (PB) and bone marrow (BM) aspirate smear, cytometric examinations, and BM biopsy were performed and processed according to standard procedures.

| Clinical presentation
A 66-year-old woman was admitted to our hospital due to dizziness, hearing loss, facial hypoesthesia, and muco-cutaneous bleeding. A physical examination revealed bruises, hematomas, and petecchiae all over the body and in the mouth. A complete neurological examination highlighted left periocular and perioral hypoesthesia, postural instability and left hearing loss. A brain computed tomography (CT) scan was performed, showing a left cerebral subdural bleeding without mass effect. A pathological meningeal contrast enhancement in the left fronto-temporal hemisphere was demonstrated by magnetic resonance imaging (MRI), together with an infiltrative mass of cochlea, semicircular canals, and vestibulocochlear nerve, compatible with disease localization. Positron emission tomography and a CT scan of neck/chest/abdomen were negative except for homogeneous splenomegaly (15 cm longitudinal diameter). Informed consent about authorization for instrumental examinations and genetic analysis on biological sample, processing of personal data, and sample storage was obtained. All clinical findings together with BM morphologic, immunophenotypical, cytogenetic, and FISH evaluation led to a diagnosis of DHL with central nervous system and BM involvement, clinical stage IV.
The patient received a first cycle of R-HyperCVAD B (high doses of Methotrexate and Cytarabine). The chemotherapy was well tolerated with resolution of all the symptoms. However, during hospitalization a clinical relapse arose, characterized by headache, confusion, disorientation and recurrence of dizziness, ipoesthesia, and hearing loss was also observed. A brain MRI confirmed the progression of the disease. After the sixth cycle of R-ICE (Rituximab -Ifosfamide -Carboplatin AUC 5-Etoposide) complete remission (CR) was recorded by brain MRI and BM morphologic, immunophenotypical, cytogenetic, and FISH evaluation. An autologous PB stem cell transplantation was subsequently and successfully performed. The patient is now alive and in CR
MYC and IGH status were at first investigated using Break apart FISH probe. MYC rearrangement was observed in 30/100 interphase nuclei and FISH analysis on DAPI counterstained metaphases showed one yellow fusion signal (1Y) on normal chromosome 8, one red signal (1R) on derivative chromosome 8 and one green signal (1G) on derivative chromosome 14 ( Figure 2B). IGH rearrangement was observed in 24/100 nuclei and on metaphases we found that 1G, corresponding to the 5'IGH end, localized on the telomeric region of the long arm of a chromosome 3 ( Figure 2C). So we tested the BCL6 Break Apart probe to confirm the gene's involvement: rearrangement was observed in 24/100 nuclei and on metaphases we observed 1G, corresponding to the 5'BCL6 end, localized on the derivative chromosome 8 ( Figure 2D). Therefore, the two "hits" MYC and BCL6 were not independent events but joined the same single mechanism itself, more exactly a translocation involving three chromosomes t(3;8;14)(q27;q24;q32). We also performed FISH probes to characterize additional chromosome anomalies observed in the karyotype: we confirmed a duplication of 1q25 including the ABL2 gene and an interstitial deletion in 13q14, which is supposed to be duplicated at a later time ( Figure   2E-F). FISH results allowed to better define the karyotype as follows: 47,XX,dup(1)(q25q32),?del(8)(q24),+13,del(13) (q13q21)x2,add (14) involvement in addition to BCL2 and/or BCL6 rearrangements. 9 These multiple-hit lymphomas are associated with a very aggressive clinical course and resistance to conventional chemotherapy. [10][11][12] Because of their biological, genetic and clinical features, DHL and THL are listed together as a specific category in the 2016 update of WHO classification. 1 This revision highlights the clinical relevance of the characterization of molecular features of these disorders, therefore a specific genetic classification is always required.
In this report, we describe a patient presenting seemingly as acute leukemia at first, then revealed as a DHL associated with an uncommon rearrangement, a three-way chromosomal translocation t ( Double-hit lymphoma is well described in literature but reported cases with complex rearrangements are few, although the records are increasing. [13][14][15] We believe that this lack of data is due to the method used for the analysis. Usually, MYC, BCL2 and BCL6 rearrangements are investigated by FISH performed on formalin-fixed paraffin-embedded lymph node tissue sections, due to disease's localization, using a locusspecific probe. This method is very useful to investigate gene's rearrangement directly on tissues processed for histopathological diagnostics, but the analysis is therefore performed on the interphase nuclei, thus it does not allow to identify either the partner of the rearrangement or the effective underlying mechanism, as on the contrary FISH on metaphases enables. Hence we suppose that the amount of multiple-way translocation in DHL could be underestimate because misunderstood as independent events. MYC + /BCL6 + DHL is aggressive in any case, whatever the molecular mechanism underlying is. It is well known that conventional chromosome analysis is often unsuitable to detect subtle rearrangements, mostly when they are part of a complex karyotype and the quality of the chromosomes is poor, as it is often observed in metaphases derived from BM cultures. Nevertheless, standard cytogenetic analysis should be carried out whenever possible, because it can disclose the presence of additional chromosome anomalies, not identified by FISH performed with locus-specific probes, that could involve important genes and consequently change the response to therapy.