Chlamydia Psittaci negative MALT Lymphomas of the Ocular Adnexa Are Characterized by Biased Use of the VH4 Family and Evidence for Antigen Selection

Abstract 2644

Mucosa-associated lymphoid tissue (MALT) lymphomas are the most common lymphomas in the ocular adnexa. Similar to extranodular MALT lymphomas in other tissues, ocular adnexa MALT lymphomas (OAMALTL) are frequently characterized by an indolent clinical course and often remain localized for many years. While chronic infection (e. g. Helicobacter pylori) or autoimmune diseases (e.g. Hashimoto's thyroiditis and Sjogren's syndrome) frequently precede and predispose patients to develop MALT lymphomas in the stomach, thyroid and salivary glands, respectively, the etiology and pathogenesis of OAMALTL are still controversial. An Italian group and investigators from several other geographic regions demonstrated Chlamydia psittaci (C. psittaci) DNA present in OAMALTL, suggesting that this pathogen may be implicated in the development of these lymphomas. However, similar studies performed by us and other investigators on US-based patients have failed to corroborate this finding. Furthermore, DNA from other bacteria was also not detected by us, thus not supporting a bacterial etiology in US-based patients. Since non-bacterial antigens may predispose patients to OAMALTL, we have examined immunoglubulin (Ig) heavy chain variable region (VH) usage and mutations in the largest to date cohort of C psittaci negative OAMALTL originating from a single institution. DNA was extracted by standard techniques from 68 fresh OAMALTL tissues and used for direct PCR or PCR followed by cloning and sequencing using family specific VH leader and JH primers. To control for potential PCR error, all patient samples were evaluated by 2 independent PCR reactions and sequencing and only cases with identical CDR3 regions in the independent PCR runs were defined as clonal. Using this definition, clonal rearranged V_HDJ_H sequences were identified in 44 (64.7%) tumors originating from the orbit (19), conjunctivae (19) and lacrimal gland (6). Forty seven clonal V_H gene sequences were detected (3 of the patients had 2 clonal sequences) with 46 potentially functional and 1 harboring an out-of-frame junction with a stop codon. In 14 (31.8%) cases the PCR product could be sequenced directly, whereas in 30 (68.2%) cases PCR amplicons had to be subcloned to identify the V_H gene. The 46 potentially functional V_H were derived from 4 of the 7 human V_H gene families with the following distribution: VH1, 13.1%; VH2, 2.2%; VH3, 39.1%; VH4, 45.6%, demonstrating a biased overrepresentation of the VH4 gene family (p=0.001). The most frequently encountered genes were VH4-34 (n=8), VH3-30 (n=6), VH3-23 (n=5) and VH4-30 (n=4). VH4-34 represented 17% of all the potentially functional V_H genes identified in this study in contrast to its usage in 4–7% in adult peripheral B lymphocytes. Only 3 V_H gene sequences were unmutated. The average percent homology to the germ line sequence in the 43 functional mutated sequences was 93.2% (range 71.5–99.6). Multinomial algorithm for antigen selection revealed evidence for positive selection in the CDR in 15 sequences, negative selection in the FR in 17 sequences, and selection in both CDR and FR (in the same sequence) in 7 sequences. Selection analysis using the focused binomial test demonstrated evidence of selection in the FR in 12 sequences and in the CDR in only 4 sequences. Selection was detected in 5 of the 8 functional VH4-34 sequences. Analysis of the tumor-derived CDR3 sequences revealed low similarity and an absence of stereotyped sequences with no homology to antibacterial and other previously published antibodies. The average CDR3 isoelectric point was 5.91±1.89 (SD). The average CDR3 length was 15.73±13.62 (SD) amino acids, with 19 sequences harboring 15–19 amino acids and 7 CDR3 sequences longer than 19 amino acids. Intraclonal variation was assessed by extensive molecular cloning in 8 potentially functional V_H gene isolates from 8 randomly selected tumor specimens. Confirmed ongoing mutations were detected in the 6 of the 8 analyzed sequences. Overall our findings demonstrate that C. psittaci negative OAMALTL exhibit biased usage of VH families and genes with evidence for intraclonal heterogeneity and antigen selection in multiple tumors, implicating immunological stimulation in the pathogenesis of these lymphomas. The nature of the antigens potentially playing role in these processes is currently unknown and requires further studies.