Page 150 - Haematologica Vol. 107 - September 2022

ARTICLE - FL3B and simultaneous DLBCL
K. Koch et al.
tern of the germinal center microenvironment, such as follicular dendritic cells (CD21), follicular T helper cells (ICOS, STAT4), T cells (CD3, TCF7, TXK) and CXCL12, which has previously been described to be upregulated in FL stromal cells.20 Furthermore, genes more highly expressed in follicular areas reflected a greater abundance of cells of innate immunity (C1QB, SIGLEC1, TPSAB1) including natural killer cells (KLRB1, KLRG1) compared to the DLBCL component. In line with a higher content of immune cells, genes involved in trafficking e.g. by adhesion and motility, were more highly expressed in follicular areas (CCR7 and its ligand CCL21, CDH1, ITGA6, ITGB6). Alterations of the extracellular matrix between follicular and diffuse areas are reflected by differential expression of collagen III a and fibronectin which were more highly expressed in the DLBCL component. Of note, two of 12 genes more highly expressed in the diffuse part are known to be involved in T-cell activation (CD70 and TNFSF4/OXL40). Only a few of the differentially expressed genes could be assigned to B- cell differentiation (e.g. CD27) suggesting that the gene ex- pression pattern of the neoplastic B cells does not differ substantially between follicular (FL3B) and diffuse (DLBCL) areas. No differences were observed in genes de- scribed to be associated with transformation or an ag- gressive phenotype of FL (MYC, NOTCH, NFkB signaling pathway genes, data not shown).
Molecular genetic features are stable between follicular lymphoma grade 3B and diffuse large B-cell lymphoma Genes recurrently mutated in mature B-cell lymphoma were analyzed by targeted sequencing in pure FL3B (n=11) and FL3B+DLBCL (n=16). The FL3B+DLBCL group consisted of two cases containing follicular and diffuse components, ten cases with a confluent and a diffuse component and four cases with all growth patterns in the same specimen. Areas representing growth patterns were separated by macrodissection and analyzed individually (yielding 36 se- quencing results). Sequencing retrieved a mean read depth of 4,258 (min 276, max 10,131). Overall, 81 genetic variants were detected (Online Supplementary Table S2). The genetic variants were mostly missense variants (n=56), followed by nonsense (n=9), and splice site or splice region variants (n=8), frameshift (n=4) and inframe variants (n=4). The pure FL3B group consisted of nine cases with a confluent growth pattern, one case with a follicular growth pattern and one case with a follicular and confluent growth pattern of which only the confluent component was analyzed. Se- quencing retrieved a mean read depth of 5,146 (min 967, max 9,769). Overall, 22 genetic variants were detected (On- line Supplementary Table S2). The genetic variants were missense in most cases (n=14), followed by nonsense (n=7) and splice site or splice region variants (n=1). Since germline DNA was not available, variant effect prediction using EN- SEMBLE was performed to estimate the functional rel-
evance of the genetic alterations. In the FL3B+DLBCL group, variant effect prediction identified 16/81 (20%) variants with high impact, 17/81 (21%) with moderate/deleterious effects, 29/81 (36%) with moderate/mixed effects, 13/81 (16%) with moderate/tolerated or unclear effects and 6/81 (7%) with low impact. In the pure FL3B group, 9/22 (41%) variants were predicted to have high impact, 6/22 (27%) with mod- erate/deleterious effects, 4/22 (18%) with moderate/mixed effects and 3/22 (14%) with moderate/tolerated effects. The number of variants detected per case was significantly dif- ferent between pure FL3B (0 to 6; mean 2) and FL3B+DLBCL (1 to 11; mean 5.1; P=0.0037, unpaired t-test). Overall, 62/81 (77%) variants in FL3B+DLBCL and 19/22 (86%) variants in pure FL3B were predicted to affect protein function negatively. Considering only variants with poten- tially negative effects on protein function, significant dif- ferences between FL3B and FL3B+DLBCL could still be observed (FL3B: 0 to 6 variants per case, mean 1.7 and FL3B+DLBCL: 1 to 8 variants per case, mean 3.9; P=0.0091, unpaired t-test). However, as a targeted sequencing ap- proach was applied, this does not allow conclusions on the whole load of genetic variants in individual tumors. The most frequently altered genes with potentially negative ef- fects on protein function were KMT2D (6/16 cases, 38%), CREBBP (6/16 cases, 38%) and KMT2C (5/16 cases, 31%) in FL3B+DLBCL and TP53 (3/11 cases, 27%), MYD88 (2/11 cases, 18%) and KMT2D (2/11 cases, 18%) in pure FL3B. No signifi- cant differences were found between FL3B and FL3B+DLBCL considering variants in specific genes (Online Supplementary Table S4).
In 14/16 (88%) cases of FL3B+DLBCL all variants were shared between the different specimens/areas of the cor- responding sample. Thus, overall 77/81 (95%) variants did not differ between FL3B and DLBCL occurring in the same patient (Figure 2). In only two cases, four variants were de- tected that differed between FL and DLBCL within the same patient. In one sample (case 35) a variant in the CREBBP gene was detected in the follicular component but no sequencing result was obtained for the DLBCL com- ponent due to low coverage. In the other sample (case 28), three variants were discrepant between the FL3B (con- fluent pattern) and the DLBCL component. Two variants affecting NOTCH1 and SOCS1 were identified in the con- fluent and the DLBCL component, respectively; both had a very low frequency (<10% of reads) in the other com- ponent and were therefore initially not considered. In contrast, a KMT2D mutation was present in the FL3B and completely absent in the DLBCL component (Figure 2). This variant was predicted to have a moderate/mixed effect.
The clinical features of follicular lymphoma grade 3B alone and with diffuse large B-cell lymphoma
do not differ
To understand whether the co-occurrence of DLBCL with
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