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Gene expression profiling of FL subtypes
ber of centroblasts), FL3B only harbors centroblasts and centrocytes are not present.1 Although criteria for the his- tological grading of FL are well-defined,1,2 its precise assessment is challenging even for expert hematopatholo- gists, in some cases resulting in interobserver variability in daily routine diagnostics.3,4 On the genetic level, approxi- mately 85% of FL are characterized by the hallmark translocation t(14;18)(q32;q21), resulting in the juxtaposi- tion of BCL2 to the IGH gene locus and, subsequently, to constitutive overexpression of BCL2 and inhibition of apoptosis. The evolution of novel cell clones with modi- fied growth potential, morphologically often character- ized by a higher number of centroblasts and/or by an increased proliferation index, is characteristic of progres- sion of FL.2 Approximately 30% of FL transform to a more aggressive lymphoma, usually diffuse large B-cell lym- phoma (DLBCL), which is typically associated with inferi- or clinical outcome.5 However, the genetic mechanisms underlying the progression and transformation of FL are poorly understood.
Since FL grade 3A often co-exist with an FL1/2 compo- nent, and harbor the t(14;18) in approximately 60% of cases, a linear progression model of FL1, FL2 and FL3A has been developed, although FL3A does not necessarily evolve from FL1/2.3 FL3B, on the other hand, is presumed to be more closely related to DLBCL, and both FL3B and DLBCL are often simultaneously present in a lymph node.2,6,7 Although a molecular characterization of FL3A and FL3B versus FL1/2 has been attempted in the past, many reports have only addressed either immunohisto- chemical and/or genetic differences.6,8,9 The main goal of the present study, therefore, was the comprehensive genetic analysis of a well-defined set of FL3A and FL3B and their comparison with related entities such as GCB- type DLBCL and DLBCL with an FL3B component by gene expression profiling, immunohistochemistry, and genetic analysis by fluorescence in situ hybridization (FISH).
Methods
Sample selection and histological grading
All samples were collected by the Molecular Mechanisms in Malignant Lymphomas (MMML) network project, for which cen- tral and local ethical approval had been obtained. Due to the ret- rospective nature of the study, patients had been treated with var- ious chemotherapy regimens, including (although only in a few cases) rituximab.
Altogether, 98 tumor samples were included: 12 FL1, 10 FL2, 16 FL3A, 6 FL3B with a purely follicular growth pattern, 9 DLBCL with an additional FL3B component (DLBCL/FL3B), and 45 DLBCL of GCB-type, as determined by gene expression profil- ing.10 All tumor samples were classified and graded on the basis of routine hematoxylin and eosin (H&E), Giemsa and Perjodic acid Schiff (PAS) stainings according to the criteria of the World Health Organization (WHO) classification of tumors of hematopoetic and lymphoid tissues within a panel review process conducted by expert reference hematopathologists of the MMML.1,10
Immunohistochemical staining, fluorescence in situ hybridization and gene expression profiling
Paraffin sections were immunostained with antibodies against CD20, CD10, BCL2, BCL6, IRF4/MUM1 and Ki67 as previously described.10,11 For the detection of BCL2-, BCL6- and
MYC-translocations, FISH was performed as described in the Online Supplementary Appendix.12 Gene expression profiles were generated as described in the Online Supplementary Appendix.10
Statistical analysis
Differential gene expression analysis, ANOVA, determination of gene expression indices, classification analyses, and evaluation of the clinical outcome were assessed as described in the Online Supplementary Appendix.
Results
Immunohistochemical and FISH analyses delineate FL1-3A from FL3B and DLBCL/FL3B
According to FISH analyses, BCL2 breaks were the pre- dominant genetic feature of FL1/2 (18 of 22, 82%). The number of cases with BCL2 alterations was lower in FL3A (12 of 16, 75%), FL3B (3 of 6, 50%) and in GCB-DLBCL (13 of 43, 30%), and BCL2 breaks were only infrequently detected in DLBCL/FL3B (1 of 9, 11%) (Table 1 and Online Supplementary Figure S1A). The substantial difference in the incidence of BCL2 breaks between FL3B and DLBCL/FL3B in comparison with FL1/2 or FL3A did not achieve statistical significance due to the small number of cases. Rearrangements of the BCL6 gene locus were most frequently encountered in DLBCL/FL3B (4 of 9, 44%) and in GCB-DLBCL (15 of 44, 34%), but were also detected in FL1/2 (2 of 22, 9%), FL3A (5 of 16, 31%), and FL3B (1 of 6, 17%) (Table 1 and Online Supplementary Figure S1A).
Signal constellations indicative of an MYC break were most frequently observed in DLBCL/FL3B (2 of 9, 22%) and, to a lesser extent, also in FL3B (1 of 6, 17%) and GCB- DLBCL (4 of 44, 9%). Occasional MYC alterations were also detected in FL1/2 (1 of 22, 5%) and FL3A (1 of 16, 6%) (Table 1 and Online Supplementary Figure S1A). According to immunohistochemistry, CD10 positive samples (>25% positive cells) were equally distributed within FL1/2 (13 of 21, 62%), FL3A (8 of 12, 67%), and FL3B/DLBCL (4 of 7, 57%), and, to a lesser extent, in GCB-DLBCL (14 of 42, 33%). CD10 was also expressed in 2 of 2 FL3B tested (Table 1 and Online Supplementary Figure S1B). While none of the FL1/2 were IRF4/MUM1 positive (≥26%), reactivity for this protein was significantly increased in FL3A (4 of 8, 50%; P<0.001), FL3B (2 of 3, 67%; P<0.001), and DLBCL/FL3B (2 of 6, 33%; P<0.05) (Table 1). With increas- ing grade, the number of cases with high Ki67 indices (≥70%) rose. While 3 of 20 FL1/2 (15%) showed reactivity for Ki67 of 70% or over, such a staining pattern was observed in 4 of 15 FL3A (27%) and in 3 of 5 FL3B (60%; P<0.05). A higher proportion of DLBCL/FL3B (7 of 8, 88%) were Ki67-high, although the difference did not reach significance when compared with FL3B or DLBCL (20 of 39, 51%) (Table 1 and Online Supplementary Figure S1B).
Tumor samples with high BCL2 (>50%) expression were equally distributed among the different subtypes. FL3B and GCB-DLBCL showed the lowest numbers of BCL2 expressing cases (2 of 4, 50% and 24 of 43, 56%, respectively, vs. a mean frequency of 84% in the other lymphoma subtypes) (Table 1). All samples showed high numbers of BCL6-expressing cells ranging from 71% to 100% (Table 1).
To summarize, FISH and immunohistochemical profiles pointed to a profound biological difference between FL1/2
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