Cryptic MYC insertions in Burkitt lymphoma: New data and a review of the literature

Abstract

The occurrence of MYC-negative Burkitt lymphoma (BL) has been discussed for many years. The real frequency of the MYC insertion in MYC-negative BL is still unknown. Fine-needle aspiration biopsies of 108 consecutive patients with clinicopathologically suspected BL (suspBL) were evaluated by flow cytometry, classical cytogenetics, and fluorescence in situ hybridization (FISH). We found 12 cases (11%) without the MYC rearrangement by FISH with a MYC breakapart probe: two patients (1.9%) with cryptic MYC/IGH fusion (finally diagnosed as BL) and 10 patients (9.3%) with 11q gain/loss (finally diagnosed as Burkitt-like lymphoma with 11q aberration). The exact breakpoints of the cryptic MYC/IGH were investigated by next-generation sequencing. The MYC insertions' breakpoints were identified in PVT1 in the first case, and 42 kb upstream of 5'MYC in the second case. To date, a molecular characterization of the MYC insertion in BL has only been reported in one case. Detailed descriptions of our MYC insertions in a routinely and consecutively diagnosed suspBL cohort will contribute to resolving the issue of MYC negativity in BL. In our opinion, the presence of the MYC insertions in BL and other lymphomas might be underestimated, because routine genetic diagnostics are usually based on FISH only, without karyotyping.

Fig 1. Pathomorphological features of Case 1.

A: Pathomorphological features of cecal lymph node with partial involvement by Burkitt lymphoma (BL), obtained by surgery of the cecum. This image revealed BL with a reduced number of apoptotic bodies but with starry sky appearance without phagocytosis in histopathology as compared to the cecum tumor with the reduced number of apoptotic bodies and starry sky appearance. Diffuse growth is seen in terms of monomorphic medium-sized lymphoid cells showing a jigsaw puzzle effect of cytoplasmic borders. The round nuclei are similar in size and shape, showing open chromatin without clear nucleoli and with scanty amounts of cytoplasm (paraffin section stained with HE, original magnification, 40×). B-D: The other images show the immunophenotypic (IHC) features of BL in comparison to the part of the uninvolved lymph node (the asterisk indicates the unchanged germinal center (GC) of the lymph node). IHC showed classic MYC-positive BL immunostaining, CD38+^strong (B)/MYC+ ^strong,100% (C)/LMO2− (D) (original magnification 40×). The IHC test shows differences in CD38 staining between plasma cells (the strongest)(green arrows), BL cells (strong)(blue arrows), and T lymphocytes (the weakest, partially negative)(red arrows). On immunohistochemical staining, GC cells have weaker expression of CD38, with CD38+^higher on plasma cells, no MYC, and a strong expression of LMO2 in most cells.

Fig 2. Flow cytometry analysis of CD38 expression of Case 1.

Flow cytometry-based analysis of median fluorescence intensity (MFI) of CD38 expression on BL (698) in R1 was higher (CD38(+)^higher) than that for normal T-lymphocytes (37) in R2 and apoptotic bodies (135) (in a circle).

Fig 3. Flow cytometry immunophenotyping of Case 1.

Fine-needle aspiration biopsy/flow cytometry analysis of BL. A: Forward scatter/side scatter dot plots present both small normal T lymphocytes (red cells) and usually larger lymphoma cells (green cells) with apoptotic bodies (marked by circles). BL expresses CD20/CD19/CD22 (MFI CD20 > CD19 > CD22) (B–D) as well as CD45+^weaker/HLADR+. E–I: BL expresses a homogeneous phenotype of germinal center origin (CD81+^higher/CD10+/BCL6+^higher/CD38+^higher/CD44±^{weaker (very low expression on a small subpopulation of cells)}. J–P: BL expresses CD54±^weaker/CD305±^weaker/CD62L±^{weaker (very low expression on a small subpopulation of cells)} but is negative for BCL2/lambda, with a restricted expression of IgM+ heavy/kappa+ light immunoglobulin chain. In addition, CD71+++ expression was detected in 100% of BL cells. Antigen expression of few macrophages and normal T-lymphocytes is marked with a pink asterisk and boxes, respectively. Dot-plots.

Fig 4. Genetic findings in Case 1.

The thick black arrow indicates chromosome 8 with insertion of IGH and with MYC/IGH fusion. A: Karyotype 46,XY [20]. B: The same metaphase, FISH with IGH BAP probe: two non-rearranged IGH (yellow) signals on chromosomes 14 and one 3′IGH (red) signal on normal chromosome 8 indicating insertion of IGH into MYC. C: The same metaphase, FISH with IGH/MYC:CEP8 dual fusion probe: two centromere 8 (blue) signals on chromosomes 8, two IGH (green) signals on chromosomes 14, one MYC (red) signal on chromosome 8, and one MYC/IGH (yellow) signal on normal chromosome 8, indicating MYC/IGH fusion. D: Detailed breakpoints identified by PCR and Sanger sequencing: the break on chromosome 8 maps to the PVT1 region; the break on chromosome 14 is located 1.6 kb upstream of the Sμ switch region.

Fig 5. Schematic view of the MYC insertion breakpoints in our data and previous literature.

In Case 1, the breakpoint on chromosome 8 was 158 kb downstream of 3′MYC, in the PVT1 region. In Case 2, the breakpoint was 42 kb upstream of the 5′MYC. Green arrows indicate the MYC insertion breakpoints in lymphomas reported in the literature [–19]. Visualization based on Ensembl 101: Aug 2020 [29]. BL control, BL without insertion, but with typical translocation t(8;14)(q24;q32) and with MYC/IGH fusion.

Fig 6. Flow cytometry immunophenotyping including analysis of CD38 expression of Case 2.

FCM analysis of BL cells from the peritoneal fluid. A: Forward scatter/side scatter dot plots present both small normal T lymphocytes (red cells) and larger lymphoma cells (green cells) with a reduce number of apoptotic bodies (marked by circles). B-D: BL expresses: CD20/CD19/CD22 (with median fluorescence intensity (MFI) of CD20> CD19> CD22), as shown by monoclonal antibodies conjugated with the same fluorochrome, APC-A as well as CD45+weaker/HLADR+. E-I: BL expresses a homogeneous phenotype of germinal center origin (CD81+higher/CD10±/CD38+higher/CD44– but BCL6 negative. H (enlarged dot plot): FCM-based analysis of MFI of CD38 expression in BL. MFI of CD38 expression on BL (873) in R1 was higher (CD38(+)higher) compared to normal T-lymphocytes (36) in R2 and apoptotic bodies (599) (in a circle). J-P: BL expresses CD54+higher/CD305+higher/ BCL2±weaker (very low expression on a small subpopulation of cells) but is negative for CD62L/kappa/lambda with a restricted expression of IgM±/IgD±heavy immunoglobulin chain. In addition, CD71+++ expression is detected in 100% of BL cells. Antigen expression of few macrophages was marked with a pink asterisk (CD10/CD38/CD44/CD54/CD71/CD305). Antigen expression of BL cells is compared to the expression on a subpopulation of normal T-lymphocytes (most antigens) (i.e. CD38/CD43/CD44/CD45/CD54/CD81/BCL2) and on macrophages (i.e. CD54/ CD305) of the tumor and described as + higher for an antigen with a higher expression in BL cells compared to normal lymphocytes/ macrophages in 100% of cells; +, positive in 100% of BL cells; + weaker, for an antigen with a weaker expression than in lymphocytes/macrophages in 100% of cells; ± weaker, for an antigen with a weaker expression in BL cells compared to normal lymphocytes/macrophages in >20% to <100% of BL cells;–, no expression (i.e. expression in <20% BL cells). Dot-plots.

Fig 7. Pathomorphological features of Case 2.

A: A monomorphic population of BL cells in the absence of apoptotic bodies in the background is visible in the cytological smear obtained from the FNAB of the liver tumor. Cytologic features with relatively uniform round nuclei, more cells with single, central nucleoli, and thin rims of cytoplasm—“small immunoblast” (cytological smear stained with HE, original magnification, 800×). B: A trephine biopsy showing heavy infiltration with BL. C: Gastric tissue biopsy showing heavy infiltration with BL. B-C: Both these images revealed BL with the reduced number of apoptotic bodies and starry sky appearance in HP. High magnification showing a “squaring off” of the cytoplasm. Also note the slight nuclear irregularity and more cells with single, central nucleoli (B-C: paraffin section stained with HE, original magnification, 800×). D: MYC protein immunostaining is strongly expressed by all BL cells. C-D: The images show stomach wall glands, which are also MYC positive (D) (D: original magnification 800×).

Fig 8. Genetic findings in Case 2.

The thick black arrow indicates chromosome 14 with insertion of the MYC and with the MYC/IGH fusion. A: Karyotype 46,XX,dup(1)(q21q42) [7]/46,idem,del(11)(q23) [6]. B: Metaphase, FISH with IGH/MYC:CEP8 dual fusion probe: two centromere 8 (blue) signals on chromosomes 8, two MYC (red) signals on chromosomes 8, one IGH (green) signal on chromosome 14, and one MYC/IGH (yellow) signal on chromosome 14, indicating MYC/IGH fusion. C: Detailed breakpoints identified by PCR and Sanger sequencing: the break on chromosome 8 maps 43 kb upstream of the 5′MYC; the break on chromosome 14 is 2 kb downstream of 3′IGHD2-2.