Biological characterization of stage I follicular lymphoma according to extranodal or nodal primary origin and t(14;18) status using high-resolution array-based comparative genomic hybridization.

Abstract

Follicular lymphoma (FL) is the most frequent indolent lymphoma and has a variable clinical course. Although generally characterized by an indolent clinical behavior, many cases eventually transform to an aggressive large B-cell lymphoma 1. Although most of the patients have advanced disease at presentation, approximately 10–20% of patients with FL present in limited stages (Stage I–II) 1. In a retrospective analysis, more than half of patients with early stage FL remained untreated at a median of 6 or more years, and survival was comparable to that observed in patients undergoing immediate treatment 1. FLs are generally characterized by the presence of the t(14;18)(q32;q21), which causes a fusion of the BCL2 oncogene with the immunoglobulin heavy chain joining region. The IGH/BCL2 rearrangement is a relatively specific molecular marker of FL, frequently used for diagnosis and monitoring of disease 2. We have recently shown that the incidence of t(14;18) is significantly lower in limited stage FL and the status of t(14;18) appears to be predictive of clinical outcome 2. Little is known of the molecular genetics of t(14;18)-negative FL or of the genetic differences between nodal and extranodal FL. Tagawa et al. has shown that trisomy 3 is a specific genomic aberration of t(14;18)-negative FL 3. Leich et al. did not confirm this finding in their comparison of FLs with and without t(14;18) 4. Katzenberger found that a distinctive subset of t(14;18)-negative nodal FL is characterized by a predominantly diffuse growth pattern and deletions in the chromosomal region 1p36 5. The goal of the current study was to characterize stage I FL using high resolution array-based comparative genomic hybridization (aCGH). Validation of findings in aCGH were performed on 20 stage I FL as well as an additional 28 stage I and II FL cases by both RNA gene expression and DNA copy number. Twenty stage I FL were identified from the pathology database of Stanford University as previously described 2. Agilent 60-mer oligonucleotide microarrays were used to analyze DNA copy number variations. FFPE tissue DNA isolation, labeling, and microarray processing and feature extraction were preformed according to the manufacture instructions (Agilent Techologies, Santa Clara, CA). The obtained data was analyzed using both Agilent DNA Analytics 4.0 Software and CGH-miner (Stanford, CA) software. Only gains and losses identified by both methods were considered significant. Cases were compared both by origin from extranodal versus nodal site and by t(14;18) status. Genomic DNA was isolated using Ambion-Applied Biosystems (Foster City, CA) total nucleic Acid Isolation Kit. Multiplex PCR were performed on an ABI 7900HT PCR systems. RNA were isolated according to Ambion-Applied Biosystems. GAPDH was used as the reference gene. PCR were performed on an ABI 7900HT PCR systems. Data were analyzed using ABI SDS2.3 software. Statistical analysis was performed using student's T test and Mann-Whitney U test. 20 stage 1 FL cases were used for array CGH and were composed of 10 cases with t(14;18) and 10 without this translocation and confirmed with FISH (previously described in 2). All cases showed similar age, distribution of grade (with predominance of Grade 1 and 2) and similar immunohistochemical stainining pattern and follow up time (Supporting Information Table I). Overall array-based comparative genomic hybridization data is shown in Figure 1. Most frequent gains observed in stage I FL are located in chromosomes 1, 6p, 7q, 12p, 15q, 17 while most frequent deletions are located in chromosomes 1q and 17q. The region with the most number of genes with gains was located on 14q12 (16 of 25 genes, P = 0.03). Cases lacking t(14;18) contained significant gains in chromosome 1q42, 3q13, 3p22, 14q12 and a loss in 6q12 as compared to t(14;18)+ cases. Cases that were positive for t(14;18) contained gains in chromosome 3q13 and deletion in 15q12 (genes listed in Supporting Information Table II). Array cGH findings were confirmed on RNA and DNA level in total of 48 FL cases (Supporting Information Table III). Overall array-based comparative genomic hybridization data in 20 stage 1 FL cases. Gains are displayed in green bars and losses are displayed in red bars. FL is one of the most common types of non-Hodgkin B cell lymphoma in the United States and Europe. FL is generally characterized by the presence of the (14;18) chromosomal translocation but prior reports indicate that 15–20% of FL lack this translocation 1. Earlier studies using traditional cytogenetic and FISH analysis found that t(14;18) negative FL had higher frequency of Grade 3 and BCL6 translocations but these studies looked at all stages of FL. To clarify the molecular mechanism of low stage FL, we performed an array CGH analysis and confirmed the results with DNA copy number and RNA gene expression. We found that in low stage FL the most frequent gains are in chromosomes 1, 6p, 7q, 12p, 15q, X, and 17 while the most frequent deletions are in chromosomes 1q, 17q, and X. Comparison of our results with previous study of 127 FL by CGH method shows overall similar results 6. Notable difference present in stage I FL cases includes gains in 16p and deletions in 17q. When comparing by t(14;18) status, we found that cases lacking t(14;18) showed gains in 1q, 3, 14q, and loss in 6q. Similar to Cheung et al., we did not find gains in 18q in our t(14;18) negative cases 6 but we did find gains in chromosome three similar to Tagawa et al. 5. We also found that extranodal FL cases had significant gains 3p, 4q, 7p, and 17p as compared to nodal FL. At the RNA and DNA expression level, we found that RIMS1 and IRF2 genes are more frequently present in the t(14;18) negative cases. When evaluating by extranodal versus nodal status, differences in expression of RIMS1, TGFBR2, MBD4, IRF2, IRF9, and BTLA genes was found. These genes, including RIMS1 and BTLA as well as others have been described in hematologic malignancies. Limited stage (Stage I and II) FL is reported in 10–20% of all FLs and frequently lacks t(14;18). This is the first study reporting on genetic differences within this subgroup of FL. Comparing our results with genome wide profiling of FL in all stages shows unique characteristics in this group. Olga K. Weinberg1*, Lisa Ma2, Richard T. Hoppe3, Daniel A. Arber2 1Department of Pathology Boston Children's Hospital Boston, Massachusetts 2Department of Pathology, Stanford University Medical Center, Stanford, California 3Department of Radiation Oncology, Stanford University Medical Center Stanford, California Additional Supporting Information may be found in the online version of this article. Supplementary Tables Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.