Cytogenetic and pathologic characterization of MYC-rearranged B-cell lymphomas in pediatric and young adult patients

Available hematoxylin and eosin (H&E)-stained sections of diagnostic biopsy tissue were reviewed and classified according to morphologic and immunophenotypic criteria of the International Consensus Classification [1]. Large cell size was defined as tumor nuclei on average being larger than those of background histiocytes. Burkitt-like morphology was defined as having a monomorphic population of medium-sized cells with basophilic cytoplasm, round nuclei, and finely clumped nuclear chromatin, typically with a starry-sky background of tingible body macrophages. A designation of high-grade morphology was used for cases with intermediate-sized cells, typically with fine chromatin, but without definitive features of BL either because of increased nuclear pleomorphism, lack of tingible body macrophages in a well-sampled biopsy, or for morphology resembling lymphoblastic lymphoma (blastoid morphology). Immunophenotypically, a BL-like phenotype was defined as cases expressing CD10, CD20, and BCL6 without or with only very dim/subset expression of BCL2.

FISH analysis was performed as part of diagnostic evaluation using laboratory-defined algorithms or based on a pathologist’s request of specific probes. MYC FISH was performed using concurrent MYC break-apart (BAP) and IGH/MYC dual color, dual fusion (DF) probe strategies (Abbott Laboratories, Des Plaines, IL) in the setting of DLBCL, HGBCL, or BL. IGK/MYC and IGL/MYC DF probe sets were performed using laboratory developed tests. These were performed either at pathologist discretion up front, or as a reflex in the setting of a MYC-R without an IGH partner per laboratory protocol. Cases with a positive MYC-R with any probe strategy were considered as MYC-R and underwent testing with BCL2 and BCL6 BAP probes (Abbott Laboratories) if these were not already performed up front. Where material was available, additional FISH probes were performed retrospectively for these probe sets if not performed at diagnosis.

All FISH analysis was performed on formalin-fixed, paraffin-embedded (FFPE) or fresh tissue using standard FISH pretreatment, hybridization, and fluorescence microscopy in accordance with specimen-specific laboratory protocols [7]. FFPE samples were conserved at room temperature. Fresh blood/bone marrow and tissue were respectively kept in anticoagulant and balanced salt solution at ambient or refrigerated temperature. A minimum of one hundred interphase nuclei for BAP probe strategies and two hundred nuclei for DF strategies were scored using previously described cutoffs [8, 9]. FISH analysis was performed by at least two qualified clinical cytogenetic technologists and interpreted by a board-certified (American Board of Medical Genetics and Genomics) clinical cytogeneticist.

Categorical variables are presented as frequencies and percentages and continuous variables as means and standard deviations or medians and ranges. Chi-square tests or Fisher exact probability tests were used to assess between-group differences in categorical variables and non-parametric tests such as Kruskal–Wallis H and Wicoxon rank sum test were used for non-Gaussian variables. Logistic regression analyses were used to assess the frequency of DH-cytogenetics adjusting for age. All statistical tests were two-sided with a significance level of 0.05. Analyses were performed with R [10] version 4.2.1 using the dplyr [11], ggplot2 [12], and gtsummary [13] packages.

The study cohort comprised 258 pediatric/YA cases, including 129 (50%) pediatric patients and 129 (50%) YA patients (Table 1). Median age in the study population was 17.5 years (range 1–30) and 194 (75%) were male. When considering patients from the pediatric group, median age was 9 years (interquartile range (IQR) 5–13) and 105 (81%) were male. Among YA patients, median age was 25 (IQR 21–28) and 89 (69%) were male.

A MYC-R was detected in 242/256 (94.5%) cases subjected to the MYC BAP (the MYC BAP probe was not performed in 2 cases). In the remaining 14/256 cases, the MYC BAP probe rendered equivocal results in 7 (3%) cases and normal results in 7 (3%) yet exhibited an IGH/MYC fusion by DF FISH. This indicates a 3% false negative (FN) rate of the MYC BAP probe in our cohort and a 5.5% FN rate if equivocals are considered negative. Among the 244 (95%) cases with complete assessment of IG rearrangement partners, an IGH partner was identified in 201 cases (82%). IGK and IGL partners were identified in 7 (3%) cases and 20 (8%) cases, respectively. No IG partner was identified in 16 (6%) cases. The remaining 14 cases did not have complete assessment of IG partners by DF FISH, precluding definitive characterization of MYC-R partners (MYC/IGK DF probe set not performed in 1 case, MYC/IGL DF probe set not performed in 1 case, both MYC/IGK and MYC/IGL DF probe sets not performed in 12 cases). In addition to the MYC-R, a rearrangement of BCL2 was identified in 3/258 cases (1%) while 16 (6%) cases harbored a rearrangement of BCL6. No case displayed triple-hit cytogenetics (concurrent MYC, BCL2, and BCL6 rearrangements) in our cohort.

The distribution of MYC-R partners as identified by FISH was then compared between pediatric and YA patients in whom all three IG probe sets were performed (128 pediatric patients and 116 YA patients). An increased frequency of IG partners was found in the pediatric group in comparison with the YA group (126/128, 98% vs 102/116, 88% respectively, p = 0.001). Specifically, IGH rearrangements were more common in the pediatric group (112/128 (88%) vs 89/116 (77%), p = 0.03)). IGK frequency did not differ between the two groups (3/128 (2%) and 4/116 (3%) respectively, p = 0.6), nor did IGL frequency (11/128 (9%) vs 9/116 (8%), p = 0.8). The absence of an IG partner occurred less often in the pediatric group compared with the YA group (2/128 (2%) and 14/116 (12%), p = 0.001) in keeping with the higher rate of IGH partners in the pediatric group (Fig. 1A).

The frequency of HGBCL-DH-BCL2 and HGBCL-DH-BCL6 was also assessed in the pediatric and YA groups. HGBCL-DH-BCL2 was rare in both pediatric and YA patients (1/129 (1%) and 2/129 (2%), respectively, p > 0.9). HGBCL-DH-BCL2 occurred in a single pediatric patient (11-year-old male) and in two YA patients (27-year-old male and 30-year-old female). The concurrent MYC-R in cases with a BCL2 rearrangement involved an IGL partner in the pediatric case and an IGH and non-IG partner in the YA cases. HGBCL-DH-BCL6 was rare in the pediatric group (1/129, 1%) versus the YA group (15/129, 12%) (p < 0.001). HGBCL-DH-BCL6 was identified in a 14-year-old individual in the pediatric group and in individuals aged 19 to 29 in the YA group. The MYC-R in cases with a BCL6 rearrangement involved an IGL partner in the pediatric case and IGH (5/15), IGL (1/15) and non-IG partner (5/15) in the YA cases (IGK/MYC and IGL/MYC DF probe sets not performed in 4/15 YA cases). In total, the frequency of DH cytogenetics was significantly lower in pediatric vs YA cases (respectively 2/129 (2%) and 17/129 (13%), p < 0.001). Furthermore, the frequency of DH cytogenetics increased statistically significantly with increasing age (coefficient estimate 0.14, p < 0.001) (Fig. 1B).

In our study cohort, 138 cases had slides available for retrospective pathology review. Taking into account morphologic, phenotypic and genetic features, the following diagnoses were rendered: BL (N = 107; 78%), DLBCL (N = 20; 14%), and HGBCL with MYC and BCL2 rearrangements or HGBCL with MYC and BCL6 rearrangements (N = 9; 7%) (Table 2, Fig. 2). Examples of morphologic and genetic correlation and final diagnoses are illustrated in Fig. 3. While BL was the most common diagnosis in both pediatric (89%) and YA (66%) age groups, a significantly higher proportion of pediatric MYC-R B-cell lymphomas were BL (p = 0.002). Most BL cases (49/52; 94%) had a typical phenotype (CD10 + , BCL6 + , BCL2-). The rearrangement partner was usually IGH (94/104, 90%), while IGL (7/104, 7%), IGK (2/104, 2%) and no IG partners (1/104, 1%) were found less frequently. Most DLBCL cases had a germinal center B-cell phenotype per the Hans algorithm (12/13, 92%). In 7/20 (35%) DLBCLs, an immunophenotypic profile compatible with BL was present, yet morphologic attributes precluded a diagnosis of BL (3 pediatric and 4 YA patients). Of these 7 DLBCL with a BL immunophenotype, 4 had MYC::IGH, one IGL::MYC, one had a non-IG partner, and one case was incompletely assessed (no IGL and IGK BAP probes). MYC rearrangement partners among cases with a diagnosis of DLBCL and complete assessment of IG partners by FISH were IGH (8/16; 50%), IGL (2/16; 13%), and IGK (1/16, 6%). No IG partner was detected in 5/16 (31%). The mediastinum was the biopsy site in 3/20 (15%) DLBCL, and all three occurred in YA. These may represent primary mediastinal large B-cell lymphoma, but due to lack of further clinical, imaging, or gene expression profiling information this remains speculative. One of these three had an IGL partner, and the MYC partner was not determined in two cases.

Nine cases had DHL cytogenetics, all of which occurred in YA patients (2 HGBCL-DH-BCL2 and 7 HGBCL-DH-BCL6). Of the HGBCL-DH-BCL2, one had high grade and one had large cell morphology and the one with evaluable stains was GCB phenotype. Of the HGBCL-DH-BCL6, one had high grade (14%) and 6 (86%) had large cell morphology; Hans algorithm on these 7 showed 3 GCB, 2 non-GC, and 2 not available. A BL-like immunophenotype was observed in 1/7 (14%) HGBCL-DH-BCL6 with available information. Of the 4 DHL cases with complete assessment for IG rearrangement partners, 3 (75%) had an IGH partner and 1 (25%) had an IGL partner. No IGK partner was observed.

EBV in situ hybridization (EBER) was performed on 29/138 (21%) cases with available biopsy tissue slides for pathologic review. EBER was positive in 6/19 (32%) cases of BL, 0/4 (0%) of HGBCL and 1/6 (17%) of DLBCL. Among EBV-positive BL, four occurred in the pediatric, and two in the YA population, and all showed IGH partners.

Pathologic review of the biopsy material revealed two unusual cases of MYC-R B-cell lymphoma: a case of a 6-year-old boy with BL-like morphology, isolated MYC::IGH rearrangement, CD10 positivity and a high Ki67 proliferative index (> 95%) showed TdT positivity. Cases like this are now best considered B lymphoblastic leukemia/lymphoma based on recent genetic studies [1, 14]. Another case (27-year-old male) with IGH::MYC rearrangement showed HGBCL morphology without Burkitt-like features and with expression of CD10 and strong BCL2, but without BCL2 or BCL6 rearrangements; it was felt this was best categorized as HGBCL, NOS.