Recurrent loss of heterozygosity in 1p36 associated with TNFRSF14 mutations in IRF4 translocation negative pediatric follicular lymphomas

Abstract

Pediatric follicular lymphoma is a rare disease that differs genetically and clinically from its adult counterpart. With the exception of pediatric follicular lymphoma with IRF4-translocation, the genetic events associated with these lymphomas have not yet been defined. We applied array-comparative genomic hybridization and molecular inversion probe assay analyses to formalin-fixed paraffin-embedded tissues from 18 patients aged 18 years and under with IRF4 translocation negative follicular lymphoma. All evaluable cases lacked t(14;18). Only 6 of 16 evaluable cases displayed chromosomal imbalances with gains or amplifications of 6pter-p24.3 (including IRF4) and deletion and copy number neutral-loss of heterozygosity in 1p36 (including TNFRSF14) being most frequent. Sequencing of TNFRSF14 located in the minimal region of loss in 1p36.32 showed nine mutations in 7 cases from our series. Two subsets of pediatric follicular lymphoma were delineated according to the presence of molecular alterations, one with genomic aberrations associated with higher grade and/or diffuse large B-cell lymphoma component and more widespread disease, and another one lacking genetic alterations associated with more limited disease.

Figure 1.

(A) Copy number (CN) profiles of 16 pediatric FL. In the x-axis the chromosomes are represented horizontally from 1 to 22, in the y-axis the percentage of cases showing the CN alterations. Gains are represented in the positive y-axis and colored in yellow, whereas losses are represented in the negative y-axis in blue. The most frequent CN alteration was gain/amplification of 6pter-p24.3. (B) Molecular Inverse Probe (MIP)-assay profiles showing CN neutral loss of heterozygosity (CNN-LOH) at 1p36 in the pFL7, pFL10 and pFL14 cases. Allelic events are displayed along the x axis (from pter to qter). Germline homozygosity (e.g. AA, BB alleles) at a given SNP results in calls at the 0 and 1 levels, respectively, germline heterozygosity (AB-alleles) in calls around 0.5 (y-Axis: 0–1). CNN-LOH in the tumor leads to loss of calls around 0.5 and to the presence of allelic imbalance calls derived from a sum of heterozygous normal cell (AB) and homozygous tumor cell (AA or BB) calls for a given locus resulting in values between 0–0.5 or 0.5–1 depending on the amount of cells carrying the aberration. Thus, in the areas with only contribution from one parent (LOH/CNN-LOH), two bands should we expected (0% and 100%->0 and 1.0. y Axis). In the 3 cases (pFL7, pFL10, pFL14), the probes did not reach such thresholds because alterations are detected to be in mosaicism (not germ-line alterations). (C) Summary data of the FISH, CN, CNN-LOH, 1p36, TNFRSF14, EZH2 mutation, IGH clonality, BCL2 expression analyses and DLBCL component for the 18 pediatric FL patients. FISH was considered positive when splits in the BCL2, BCL6, MYC, IRF4 and IGH genes were detected; CN, when chromosomal imbalances were found by MIP-assay or aCGH; 1p36, when CNN-LOH (detected by MIP-assay) or deletions (by MIP-assay and/or aCGH) were observed at that region; TNFRSF14 and EZH2, when mutations were found by direct sequencing; IGH clonality was positive when clonal IGH chain gene rearrangement was detected by PCR; BCL2 expression was positive when more than 25% of the cells were positive. In pFL2 only the region FR3 was evaluable in the IGH PCR reaction. In pFL18 IGH monoclonality was based on an analysis performed in an outside laboratory. CNN-LOH could not be determined in cases 1, 2, 12, 16, 17, and 18.

Figure 2.

(A-H) Histomorphological features of case pFL11 (FL3a) (A-D) without aberrant genotype and case pFL14 (FL3a) (E-H) with aberrant genotype: in both cases an infiltration of the lymph node by enlarged abnormal follicles as well as remnants of normal reactive follicles (indicated by arrows) are seen in the low magnification (A,E, H&E x 25). The neoplastic follicles lack a clear “starry sky” pattern as well as the demarcation by a mantle zone (B,F, H&E x 100). The follicles consist of centroblasts and centrocytes (C,G, H&E x 1000). The lack of zonation is seen with immunohistochemistry for the proliferation marker Ki67 (D, H, Ki67 x 100), nevertheless a hot spot of proliferation can be observed in H.