Double-hit lymphomas: clinical, morphological, immunohistochemical and cytogenetic study in a series of Brazilian patients with high-grade non-Hodgkin lymphoma

Double-hit lymphomas (DHL) are aggressive neoplasms presenting translocation phenomenon involving the MYC gene combined with another event involving translocation of the BCL2 or BCL6 gene [1‐6]. The combination of MYC translocation with translocations of BCL2 and BCL6 can also occur, defining the triple-hit lymphomas (THL) [1, 4, 7‐9]. A diagnosis of DHL is only determined following the results of a cytogenetic test, such as fluorescence in situ hybridization (FISH) [10‐12]. It represents approximately 15% of the provisional entity called B-cell lymphoma unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma (B-CLU/DLBCL/LB), Classification of Tumors of Haematopoietic and Lymphoid Tissues of the World Health Organization (WHO) in 2008 [1‐4].

In 2016, Swerdlow et al. [13] published a proposal of revision in the WHO classification of 2008. This group of authors recognized DHL and THL as clinical and pathological categories, designated high-grade B-cell lymphomas (HGBL) with MYC and BCL2 and/or BCL6 rearrangements. Cases with morphological aspects of HGBL without MYC and BCL2 and/or BCL6 rearrangements were placed in the HGBL, NOS. However, there is not a specific guideline to select which cases should have FISH studies [13]. The morphological and/or immunophenotypic parameters do not seem to effectively predict the detection of cytogenetic translocations involving MYC, BCL2 and BCL6 [4, 14]. Molecular cytogenetics, like FISH, is not a readily available technique, mainly due to its high costs. Thus, determining which group of patients must have their biopsies submitted for this kind of test is a subject of controversy [4, 14, 15]. Despite the difficulties in interpreting the immunohistochemical profiles of Myc, Bcl2 and Bcl6, several studies have been published on how this technique can be used as a method of screening for molecular research, since it is being incorporated into the clinical routine as this cytogenetic information becomes increasingly relevant in therapeutic practice and prognosis [16, 17].

This study identified cases of DHL in a series of high-grade non-Hodgkin lymphomas in Brazil, characterizing them according to their clinical, morphological, immunophenotypic and molecular features. In addition, the associations between the expression of Myc, Bcl2 and Bcl6 proteins assessed by immunohistochemistry and the detection of translocations involving these genes were investigated in order to contribute to the discussions about how to select the cases of lymphoma for further molecular studies.

The 120 patients were submitted for clinical review, morphological analysis, immunohistochemistry and molecular evaluation. However, in 30 cases, flaws were verified in molecular testing in one or more of the three markers, related to issues of material fixation. FISH analysis detected three cases of DHL. The three groups defined by the cytogenetics investigation were BL (n = 15), DHL (n = 3) and DLBCL (n = 72); the sociodemographic, clinical, morphological, immunohistochemical and cytogenetic data are shown in Table 1.

DHL are rare, aggressive neoplasms that account for less than 10% of B-cell lymphomas and about 4% of high-grade lymphomas [4, 12]. This first Brazilian study found three cases of DHL, two with translocations involving MYC and BCL2, and one with translocations involving MYC and BCL6. The combination that is most often reported is the double translocation involving the MYC gene and the BCL2 gene (62%) [4, 20].

The most important clinical and prognostic factors of DHL patients are the advanced stage at diagnosis, the presence of extranodal masses, the bone marrow or CNS infiltration and the age of over 60 years old [4, 10, 12, 16, 21]. The three patients identified in this study were elderly; however, they did not present high Ann-Arbour stages or bone marrow/CNS infiltration, though two of the patients exhibited extranodal masses.

Survival is lower in patients with DHL. In this study, all three patients with DHL died, an average of 4 months after diagnosis. According to the literature [16], the overall median survival rate is 8.2 months in this group of patients versus 56.8 months in patients with other high-grade lymphomas. Snurdel et al. (2010) [10] reported 70% of deaths within eight months in patients with DHL. The aggressiveness of DHL is possibly explained by the synergistic action of MYC and BCL2: proliferative activity with inhibition of apoptosis in the context of a complex karyotype [4, 20, 22, 23].

In this study, the best agreements between immunohistochemical and FISH results occurred with respect to the MYC gene and, mainly, the BCL2 gene, with high NPV. Evaluations of the MYC gene and the BCL2 gene shows good results, however, there is a fair agreement by Kappa coefficient, indicating the need for caution in assessing the markers with the immunohistochemical method.

Immunohistochemical evaluation of Myc, Bcl2 and Bcl6 is a problem discussed by several hematopathology groups [12, 22‐24]. The cutoff for determining positivity for MYC varies between 30 and 50% in the literature, while for Bcl2, the variation is between 30 and 70% [22, 23, 27].

Perry et al. [24] studied the immunohistochemical expression of Myc and Bcl2 of 106 patients diagnosed with DLBCL, correlating their data with prognosis. Positivity for these markers, especially when both were positive, was related to worse prognosis, increasing the chances of death nine-fold. However, Perry et al. (2014) did not correlate their results with cytogenetic data [24].

Green et al. [25] studied the immunohistochemical and cytogenetic profiles of 193 patients diagnosed with DLBCL, of which 6% were DHL identified by FISH, and showed poor prognosis. Regarding immunohistochemistry, 29% of patients were positive for Myc and Bcl2, and 54% of these patients had translocations involving one of these genes. These patients, regardless of the cytogenetic test results, showed worse survival rates compared with other patients [25].

Yan et al. [26] evaluated 336 patients with DLBCL by immunohistochemistry and FISH, and found a high specificity (approximately 90%) of positivity for Myc and Bcl2 in the immunohistochemistry in relation to FISH positivity for MYC and BCL2 translocations, favoring its potential use for stratification in cases of DLBCL with aggressive behavior [26]. Kawamoto et al. [15] studied only DLBCL patients, by FISH and immunohistochemistry. They found that MYC translocation and Bcl2 immunohistochemical expression were independent prognostic factors and must be investigated at the initial diagnosis [15].

Kalaw et al. [28] studied the cell proliferation rate, measured by the percentage of staining for Ki67, as a potential selection parameter of patients for cytogenetic tests. The cutoff values used were 75% or 90% and both were not associated with detection of MYC translocations, as was seen in this study.

Mahmound et al. (2015) [27] studied the agreement in the evaluation of Myc expression by immunohistochemistry and noted great heterogeneity in the results, reflecting the subjective interference and the lack of a clear standard regarding the interpretation of this marker [27].

In this study, the “starry sky” morphological pattern, in the DLBCL context, positive immunohistochemistry for Myc in over 50% of cells, and positive immunohistochemistry for Bcl2 above 70% are indicated as potential selector parameters for patients eligible for cytogenetic study. However, further studies with larger case samples are required, for more detailed analysis of these associations, in special morphological pattern studies because it is a conventional and accessible method of evaluation in the pathology routine.

Swerdlow (2014) [23] emphasizes the importance of identifying cases of aggressive large B cell lymphomas, mainly due to the absence of a specific therapeutic protocol [23]. In this context, there is great interest in studies of agreement between immunohistochemical and cytogenetic results, mainly in relation to selector elements for molecular screening. In fact, difficulties related to the standardization of defining criteria of positivity for Myc and Bcl2 in immunohistochemical reactions has been reported.

This study presents the first Brazilian series of patients with double-hit lymphoma identified by cytogenetic study performed by FISH on 120 cases of high-grade lymphomas. For this study, high grade B-cell lymphomas, particularly when there is a “starry sky” pattern, must be submitted to immunohistochemistry for, at least, Myc andBcl2. When Myc and/or Bcl2 are positive, if it is possible, the investigation must continue with FISH test for MYC, BCL2 and BCL6. These cases are high grade lymphomas and the molecular markers must be reported. In the situation that molecular approach is not possible, the immunohistochemistry results must be reported too, because they may represent a prognostic factor.