Page 147 - Haematologica Vol. 107 - September 2022

ARTICLE - FL3B and simultaneous DLBCL
K. Koch et al.
steps to enrich the target regions no interpretation of the variant allele fractions was possible. Furthermore, no germline material was available from those samples in order to be able to differentiate between somatic and germline variants. To overcome this limitation, variants were categorized based on the variant effect prediction by SIFT, PolyPhen, FATHMM and Condel into six different groups: (i) high impact; (ii) moderate impact and deleteri- ous or damaging effect; (iii) moderate impact but mixed effect prediction (deleterious/damaging or tolerated based on the different tools used); (iv) moderate impact and tolerated or benign; (v) moderate impact but no ad- ditional information; and (vi) low impact. Variants with high impact, moderate/deleterious effect prediction and moderate/mixed effect prediction were considered to af- fect protein function negatively.
Gene expression analysis
Gene expression was analyzed as previously described ap- plying NanoString technology and the PanCancer Immune Profiling Panel.17 Background thresholding and normaliza- tion were performed by the NSolver software (version 4.0; NanoString Technologies). Twenty housekeeping genes were chosen for normalization in a two-step process: (i) housekeeping genes with average counts <100 were ex- cluded; and (ii) 20 housekeeping genes with the lowest count variability were chosen for normalization. Endoge- nous genes with normalized expression levels below the calculated background threshold in >20% of the analyzed specimens were excluded from further analysis. Fold changes between follicular and diffuse growth patterns were calculated for each individual case. Genes with a fold change of >1.2 or <-1.2 in at least 50% of cases were chosen and mean fold changes of these genes were cal- culated as fold change of the geometric means of the re- spective single case gene expression levels. Genes with a mean fold change of >1.5 or <-1.5 were considered to be differentially expressed.
Clinical analysis
The features of patients with FL3B (n=17) and FL3B+DLBCL (n=16) were analyzed in patients treated in the prospective randomized ‘Positron Emission Tomography-Guided Ther- apy of Aggressive Non-Hodgkin Lymphomas’ trial (PETAL: clinicaltrials.gov NCT00554164 and EudraCT 2006-001641- 33).18 Patients received two cycles of standard R-CHOP fol- lowed by interim [18F]fluorodeoxy-glucose-positron emission tomography (PET). Interim PET-negative patients continued R-CHOP whereas interim PET-positive patients were randomized between continued R-CHOP and an in- tensive methotrexate- and cytarabine-based Burkitt lym- phoma protocol. According to current treatment guidelines19 all patients were treated at the timepoint of first diagnosis.
Statistical analysis
Statistical analysis was performed using GraphPadPrism (version 7.00 for Windows, GraphPad software, La Jolla, CA, USA) applying the c2 test, Fisher exact test and unpaired t- test as indicated in the Results section. Kaplan-Meier curves were generated using SPSS statistics (version 26.0, IBM, Armonk, NY, USA).
Results
Growth pattern of follicular lymphoma grade 3B with
a continuum to diffuse large B-cell lymphoma
Forty-nine lymphomas with a diagnosis FL3B were reana- lyzed for growth pattern. All cases fulfilled the criteria of FL3B and 27/49 (55%) additionally harbored areas with dif- fuse growth representing DLBCL by definition (FL3B+DLBCL). None of the cases included any low-grade FL component. None of the cases showed the typical con- stellation of pediatric type FL according to the current WHO classification.1 Relapse biopsies were available for two pa- tients both of whom had FL3B+DLBCL at primary diagnosis and DLBCL at relapse. The proportion of DLBCL ranged be- tween 10% and 95% in individual cases (mean: 49%). The cytomorphology of the DLBCL component was centro- blastic in 25/27 cases and immunoblastic in 2/27 cases. In all cases, the morphology of the DLBCL component matched the morphology of the FL3B component. Within the areas of follicular growth two different growth patterns were noticed: (i) clearly demarcated, roundish follicles that were separated from each other (follicular pattern) (Figure 1) and (ii) follicles localized close to each other appearing to merge (confluent pattern) (Figure 1). The confluent pat- tern was clearly distinguishable from a diffuse pattern/DLBCL in hematoxylin and eosin staining and could be further highlighted by staining for T cells and follicular dendritic cells (Figure 1). Of the 22 pure FL3B lacking an ad- ditional DLBCL component, only four cases showed a pure follicular pattern (4/22, 18%), whereas 11/22 (50%) cases solely displayed the confluent pattern. A combination of both patterns was detectable in 7/22 (32%) of pure FL3B. The distribution of these growth patterns within the FL3B component was different in FL3B+DLBCL cases (confluent pattern 14/27 [52%], follicular pattern 7/27 [26%] and a com- bination of both patterns 6/27 [22%], P=0.0421, c2 test) al- though the differences must be interpreted with caution becauseofthesmallnumberofcases.Allgrowthpatterns and their combinations are indicated in Table 1.
Homogeneous immunophenotype in follicular lymphoma grade 3B and diffuse large B-cell lymphoma CD20washomogeneouslyexpressedinall49cases.CD10 expression was more variable with 22/49 cases (45%) being completely negative, 4/49 cases (8%) with ex-
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