The Genetic Landscape of Primary Breast Marginal Zone Lymphoma Identifies a Mutational-driven Disease With Similarities to Ocular Adnexal Lymphoma.

Extranodal marginal zone lymphomas (eMZL) can occur in any organ and site of the body. Recent research has shown that they differ from organ to organ in terms of their mutational profile. In this study, we investigated a cohort of primary breast marginal zone lymphomas (PBMZL) to get a better insight into their morphologic and molecular profile. A cohort of 15 cases (14 female and 1 male) was characterized by immunohistochemistry (IHC) for 19 markers, fluorescence in situ hybridization (FISH), and high throughput sequencing (HTS) using a lymphoma panel comprising 172 genes. In addition, PCR for the specific detection of Borrelia spp. and metagenomics whole genome sequencing were performed for infectious agent profiling. Follicular colonization was observed in most cases, while lymphoepithelial lesions, though seen in many cases, were not striking. All 15 cases were negative for CD5, CD11c, and CD21 and positive for BCL2 and pan B-cell markers. There were no cases with BCL2 , BCL10 , IRF4 , MALT1 , or MYC translocation; only 1 had a BCL6 rearrangement. HTS highlighted TNFAIP3 (n=4), KMT2D (n=2), and SPEN (n=2) as the most frequently mutated genes. There were no Borrelia spp. , and no other pathogens detected in our cohort. One patient had a clinical history of erythema chronicum migrans affecting the same breast. PBMZL is a mutation-driven disease rather than fusion-driven. It exhibits mutations in genes encoding components affecting the NF-κB pathway, chromatin modifier-encoding genes, and NOTCH pathway-related genes. Its mutational profile shares similarities with ocular adnexal and nodal MZL.

The role of infectious agents in cancer of the ocular region.

The purpose of the study was to investigate the association between infectious agents and the development of cancer in the ocular adnexa. A comprehensive literary study was carried out, reviewing and summarizing previous reports on the topic. A broad range of malignancies of the ocular adnexa are associated with infectious agents. A strong association and possible causal relationship between the infectious agent and the development of ocular adnexal cancer are seen in Merkel cell carcinoma (Merkel cell polyomavirus), Burkitt lymphoma (Epstein-Barr virus) and Kaposi sarcoma (human herpesvirus 8). Infection with Chlamydia psittaci has been associated with the development of extranodal marginal zone B-cell lymphoma in Italy. Human papillomavirus infection has been associated with the development of squamous cell carcinomas of the ocular adnexa, although with a highly variable reported prevalence. By exploring the role of infectious agents in the ocular adnexa and the mechanism by which they contribute to oncogenesis, the diagnostics, management and prevention of these malignancies may also improve. Antibiotic treatment and vaccines against infectious agents may be valuable in future treatment. Additionally, the presence of infectious agents within the tumours may have a prognostic or predictive value.

Prevalence of Chlamydia psittaci, Chlamydia pneumoniae, and Chlamydia trachomatis Determined by Molecular Testing in Ocular Adnexa Lymphoma Specimens.

OBJECTIVES: To assess the prevalence of Chlamydia psittaci, Chlamydia pneumoniae, and Chlamydia trachomatis in ocular adnexa lymphoma (OAL) determined by molecular testing in different countries and the potential association of Chlamydia infection with mucosa-associated lymphoid tissue (MALT) histotype by performing a systematic review and meta-analysis. METHODS: Electronic databases were searched for studies assessing the presence of Chlamydia in OAL. Pooled prevalence of the three Chlamydia species was calculated in each country. An odds ratio was calculated for the association between Chlamydia and MALT histotype, with a significant P < .05. RESULTS: Thirty-seven studies with 1,188 OALs were included. Pooled prevalence of C psittaci, C pneumoniae, and C trachomatis by country was done. Chlamydia infection was significantly associated with MALT histotype (odds ratio, 2.183; P = .027). CONCLUSIONS: The involvement of C psittaci in OAL is highly variable, with the highest prevalence in Italy and Korea. Chlamydia is associated with MALT histotype.

Ocular adnexal marginal zone lymphoma: Clinical presentation, pathogenesis, diagnosis, prognosis, and treatment.

Ocular adnexal marginal zone lymphoma (OAML) represents 1-2% of all non Hodgkin lymphomas. In the last few years many advances in understanding the pathogenesis and the molecular basis involved in its development have been done. Many potential risk factors have been proposed; a dysregulation of immune response in association with a chronic antigenic stimulation, have been hypothesized as possible pathogenic mechanism. In particular, Chlamydia psittaci infection has been related to OAML arising, and eradicating antibiotic therapy has been addressed as a safe and cost-effective approach. Management of OAML is still heterogeneous and matter of debate. There is no consensus about the best upfront treatment and therapeutic decision should take into account several patient-, lymphoma- and treatment-related factors. Novel agents and chemotherapy-free strategies are being investigated to reduce side effects and improve tumor control. This review is focused in recent knowledge improvements in this lymphoma.

Chlamydia and ocular adnexal lymphomas: An Indian experience.

CHLAMYDIA AND OCULAR ADNEXAL LYMPHOMAS: AN INDIAN EXPERIENCE: Ocular adnexal lymphomas (OALs) are a heterogeneous group of malignancies, majority being extranodal mucosa-associated lymphoid tissue (MALT) type. Different geographical regions have reported association of Chlamydia with OALs (MALT type). In India, role of Chlamydia in OALs remains unexplored. The aim of this study was to detect Chlamydia and to correlate with clinicopathological features of OALs in India. The clinicopathological features of 41 OAL cases were studied prospectively. Chlamydia DNA was detected by genus specific PCR amplifying major outer membrane protein (MOMP) gene followed by DNA sequencing. Chlamydia immunoexpression was evaluated by immunofluorescence and immunohistochemistry. The results were correlated with clinicopathological features including follow-up and survival. Chlamydia genome was detected in 3/41 (7.3%) OAL cases by PCR. Direct sequencing revealed C. trachomatis in 3 positive cases. Immunofluorescence and immunohistochemistry showed Chlamydia antigen in 5/41 and 1/41 cases respectively. Immunofluorescence demonstrated higher sensitivity than immunohistochemistry. A significant association was observed between Chlamydia positivity and orbital location (P=0.05). Follow-up revealed relapse in 2 Chlamydia positive cases (P=0.056). Our results demonstrate for the first time presence of C. trachomatis genome in 7.3% OAL cases in India. As no other reports are documented, more detailed studies from different regions within India are needed to explore status of Chlamydia in OALs.

The spectrum of MALT lymphoma at different sites: biological and therapeutic relevance.

Extranodal marginal zone (MZ) B-cell lymphomas of the mucosa-associated lymphoid tissue (MALT) arise from lymphoid populations that are induced by chronic inflammation in extranodal sites. The best evidence of an etiopathogenetic link is provided by the association between Helicobacter pylori-positive gastritis and gastric MALT lymphoma. Indeed, successful eradication of this microorganism with antibiotics can be followed by gastric MALT lymphoma regression in most cases. Other microbial agents have been implicated in the pathogenesis of MZ lymphoma arising at different sites. Apart from gastric MALT lymphoma, antibiotic therapies have been adequately tested only in ocular adnexal MALT lymphomas where upfront doxycycline may be a reasonable and effective initial treatment of patients with Chlamydophila psittaci-positive lymphoma before considering more aggressive strategies. In all other instances, antibiotic treatment of nongastric lymphomas remains investigational. Indeed, there is no clear consensus for the treatment of patients with gastric MALT lymphoma requiring further treatment beyond H pylori eradication or with extensive disease. Both radiotherapy and systemic treatments with chemotherapy and anti-CD20 antibodies are efficacious and thus the experience of individual centers and each patient's preferences in terms of adverse effects are important parameters in the decision process.

Genome-wide DNA methylation profiles according to Chlamydophila psittaci infection and the response to doxycycline treatment in ocular adnexal lymphoma.

PURPOSE: To compare genome-wide DNA methylation profiles according to Chlamydophila psittaci (Cp) infection status and the response to doxycycline treatment in Korean patients with ocular adnexal extranodal marginal zone B-cell lymphoma (EMZL). METHODS: Twelve ocular adnexal EMZL cases were classified into two groups (six Cp-positive cases and six Cp-negative cases). Among the 12 cases, eight were treated with doxycycline as first-line therapy, and they were divided into two groups according to their response to the treatment (four doxy-responders and four doxy-nonresponders). The differences in the DNA methylation states of 27,578 methylation sites in 14,000 genes were evaluated using Illumina bead assay technology. We also validated the top-ranking differentially methylated genes (DMGs) with bisulfite direct sequencing or pyrosequencing. RESULTS: The Infinium methylation chip assay revealed 180 DMGs in the Cp-positive group (74 hypermethylated genes and 106 hypomethylated genes) compared to the Cp-negative group. Among the 180 DMGs, DUSP22, which had two significantly hypomethylated loci, was validated, and the correlation was significant for one CpG site (Spearman coefficient=0.6478, p=0.0262). Regarding the response to doxycycline treatment, a total of 778 DMGs were revealed (389 hypermethylated genes and 336 hypomethylated genes in the doxy-responder group). In a subsequent replication study for representative hypomethylated (IRAK1) and hypermethylated (CXCL6) genes, the correlation between the bead chip analysis and pyrosequencing was significant (Spearman coefficient=0.8961 and 0.7619, respectively, p<0.05). CONCLUSIONS: Ocular adnexal EMZL showed distinct methylation patterns according to Cp infection and the response to doxycycline treatment in this genome-wide methylation study. Among the candidate genes, DUSP22 has a methylation status that was likely attributable to Cp infection. Our data also suggest that the methylation statuses of IRAK1 and CXCL6 may reflect the response to doxycycline treatment.

Marginal zone lymphomas and infectious agents.

A link with infectious agents, bacteria and viruses in particular, has been reported for many lymphoma entities. Marginal zone lymphomas (extranodal, nodal and splenic forms) are frequently associated with chronic infections, with important clinical, molecular, biological, and therapeutic implications. The well-known correlation between Helicobacter pylori and gastric MALT-lymphoma, the recently reported links between Chlamydophila psittaci and ocular adnexal MALT-lymphoma and Borrelia burgdorferi and cutaneous MALT lymphoma constitute the best studied examples of lymphomagenic activity of bacteria, while the hepatitis C virus represents the most extensively investigated virus associated with marginal zone lymphomas. Biological and clinical features, therapeutic implications and future perspectives of these lymphoma-microbial associations are discussed in this review.

Chlamydophila psittaci eradication with doxycycline as first-line targeted therapy for ocular adnexae lymphoma: final results of an international phase II trial.

PURPOSE: The pathogenic association between Chlamydophila psittaci (Cp) and ocular adnexal marginal zone lymphoma (OAMZL) and the efficacy of doxycycline monotherapy have been investigated in retrospective series with variations in stage, management, and follow-up duration. To our knowledge, this is the first international phase II trial aimed at clarifying Cp prevalence and activity of first-line doxycycline in a homogeneous series of consecutive patients with newly diagnosed stage I OAMZL. PATIENTS AND METHODS: Forty-seven patients were registered. Tumor tissue, conjunctival swabs, and peripheral blood from 44 patients were assessed for seven Chlamydiaceae infections by three polymerase chain reaction protocols. Thirty-four patients with measurable or parametrable disease were treated with doxycycline and assessed for chlamydial eradication and lymphoma response (primary end point). RESULTS: Cp DNA was detected in biopsies of 39 patients (89%); no other Chlamydiaceae were detected. Twenty-nine patients had Cp DNA in baseline swabs and/or blood samples and were evaluable for chlamydial eradication, which was achieved in 14 patients (48%). Lymphoma regression was complete in six patients and partial in 16 (overall response rate, 65%; 95% CI, 49% to 81%); 11 had stable disease, and one had progressive disease. At a median follow-up of 37 months (range, 15 to 62 months), 20 patients remained relapse free (5-year progression-free survival [PFS] ± standard deviation, 55% ± 9%). Cp eradication was associated with improved response rate (86% v 47%; P = .02) and 5-year PFS (68% v 47%; P = .11). CONCLUSION: Upfront doxycycline is a rational and active treatment for patients with stage I Cp-positive OAMZL. Lymphoma regression is consequent to Cp eradication, which can easily be monitored on conjunctival and blood samples. Prospective trials aimed at identifying more effective administration schedules for doxycycline are warranted.

Biased use of the IGHV4 family and evidence for antigen selection in Chlamydophila psittaci-negative ocular adnexal extranodal marginal zone lymphomas.

Extranodal marginal zone lymphomas (EMZL) are the most common lymphomas in the ocular adnexa. The etiology and potential role for antigenic stimulation in these lymphomas are still controversial. We have examined IGHV gene usage and mutations in 67 Chlamydophila psittaci-negative ocular adnexal EMZL. Clonal IGHV gene sequences were identified in 43 tumors originating from the orbit (19), conjunctivae (18) and lacrimal gland (6). Forty four potentially functional clonal IGHV gene sequences were detected with overrepresentation of the IGHV4 family and IGHV4-34 gene. All but 3 sequences were mutated with the average percent homology to the germ line of 93.5±6.1. Multinomial model and Focused binomial test demonstrated evidence for positive and/or negative antigen selection in 59% of the potentially functional IGHV genes. Intraclonal variation was detected in 8 of 11 tumor specimens. Overall our findings demonstrate that C. psittaci-negative ocular adnexal EMZL exhibit biased usage of IGHV families and genes with evidence for intraclonal heterogeneity and antigen selection in multiple tumors, implicating B-cell receptor-mediated antigen stimulation in the pathogenesis of these lymphomas.

Chlamydia psittaci Infection in nongastrointestinal extranodal MALT lymphomas and their precursor lesions.

Extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue (MALT) are associated with various infectious pathogens. We analyzed the presence of Chlamydia psittaci, Chlamydia pneumoniae, and Chlamydia trachomatis DNA in 47 nongastrointestinal and 14 gastrointestinal MALT lymphomas, 37 nonmalignant control samples, and 27 autoimmune precursor lesions by polymerase chain reaction amplification and direct sequencing. In 47 nongastrointestinal MALT lymphomas, 13 (28%) were positive for C psittaci DNA compared with 4 (11%) of 37 nonmalignant control samples (P = .09). C psittaci was detected at variable frequencies in MALT lymphomas of different sites: lung, 100% (5/5; P < .01); thyroid gland, 30% (3/10; P > .05); salivary gland, 13% (2/15; P > .05); ocular adnexa, 15% (2/13); and skin, 25% (1/4). Of 27 autoimmune precursor lesions (11 Hashimoto thyroiditis and 16 Sjögren syndrome), 11 (41%) contained C psittaci DNA. Only 1 (7%) of 14 gastrointestinal MALT lymphomas was positive for C psittaci. All specimens were negative for C trachomatis and C pneumoniae. Besides ocular adnexal lymphomas, C psittaci infection is associated with nongastrointestinal MALT lymphomas and autoimmune precursor lesions, suggesting possible involvement of C psittaci-induced antigenic-driven MALT lymphomagenesis.

Ocular adnexal lymphoma and Helicobacter pylori gastric infection.

There is a causal association between Helicobacter pylori (Hp) gastric infection and the development of gastric MALT lymphoma. In contrast, the link between Hp gastric infection and the development of extragastric lymphoma has not been thoroughly investigated. We, therefore, studied the prevalence of gastric Hp infection at initial diagnosis of ophthalmologic and nonophthalmologic extragastric lymphoma patients. Three cohorts of patients were studied: a first one of 83 patients with OAL, a second one of 101 patients with extraophthalmologic extragastric lymphoma, and a third one of 156 control individuals (control) without malignant lymphoma. Gastric Hp infection was investigated by histopathological analysis and Hp-specific PCR assay on gastric biopsy tissue samples. We found gastric Hp infection in 37 OAL patients (45%), in 25 extraophthalmologic extragastric lymphoma cases (25%), and in 18 controls individuals (12%) (P < 0.0001 OAL/C and P < 0.01 OAL/extra-OAL cases). Gastritis was found in 51% and 9% of Hp-positive and Hp-negative lymphoma patients, respectively (P < 10(-4)). Gastric Hp infection only correlated with MALT/LPL lymphoma (P = 0.03). There is a significant association between gastric Hp infection and MALT/LPL OAL. This suggests a novel mechanism of indirect infection-associated lymphomagenesis whereby chronic local antigen stimulation would lead to the emergence of ectopic B-cell lymphoma.

[Infectious agents in ocular adnexal tumours]. / Erreger bei Tumoren der okulären Adnexe.

In recent years, infectious agents have been increasingly recognised as an important pathogenetic factor for various malignant tumours of the ocular adnexa. Many of these viruses and bacteria affect the cell cycle and physiological apoptosis. Ocular adnexal lymphoma (OAL), especially extranodal marginal cell lymphoma, is associated with Chlamydophila psittaci and Helicobacter pylori in certain geographic regions. Epstein-Barr virus seems to play a role in the natural killer/T-cell lymphoma subtype of the orbit, as has long been described for Burkitt lymphoma. Bacteria seem to induce reactive lymphoid proliferation, while viruses directly infect the lymphoid cells, affecting the cell cycle and suppressing apoptosis, with subsequent malignant transformation. In general, proteins leading to cell cycle progression, like retinoblastoma protein, are elevated, and proteins inhibiting cell cycle progression, like p16 and p21, are absent or unable to function normally. Inactivation of p53 by mutation of its DNA, which leads to elevation of defective p53 protein and inhibition of apoptosis, allows oncogenic by-chance mutations to become effective. Conjunctival intraepithelial neoplasia (CIN) is less strongly associated with HPV infection than is cervical intraepithelial neoplasia. Based on the localisation of CIN, ultraviolet B radiation seems to play a primary role, leading to p53 inactivation and subsequent inhibition of apoptosis. HIV positivity also seems to aid the development of CIN and conjunctival squamous cell carcinoma, with an increasing number of cases during recent years. Kaposi sarcoma rarely occurs at the ocular adnexa in HIV-positive individuals and seems to be associated with Kaposi sarcoma-associated Herpes virus (KSHV) or HHV8. The KSHV-encoded latency associated nuclear antigen (LANA) protein binds to the negative regulator glycogen-synthase kinase-3 (GSK-3), causing a cell cycle-dependent nuclear accumulation of GSK-3, which stabilises beta-catenin and increases its levels. The findings regarding these various infectious agents and cell cycle alterations might aid the development of new therapeutic strategies.

Geographic variation and environmental conditions as cofactors in Chlamydia psittaci association with ocular adnexal lymphomas: a comparison between Italian and African samples.

A particular extra-nodal lymphoma type arises from B cells of the marginal zone (MZ) of mucosa-associated lymphoid tissue (MALT). The aetiology of MZ lymphomas suggests that they are associated with chronic antigenic stimulation by microbial pathogens, among which Helicobacter pylori-associated gastric MALT lymphoma is the best studied. Recently, MALT lymphomas have been described in the context of chronic conjunctivitis, which can be associated with Chlamydia spp. infection. Studies from Italy showed the presence of Chlamydia psittaci in 87% of ocular adnexal lymphomas (OAL), and C. psittaci has been described in a large part of samples from Austria and Korea as well. However, this finding was not always confirmed by other studies, suggesting that the association with C. psittaci may depend on geographic heterogeneity. Interestingly, none of the studies up to now has been carried out in the African population, where a strong association between infectious agents and the occurrence of human neoplasms has been reported. This study was designed to investigate the possible association of Chlamydia psittaci in cases retrieved from Kenya, compared to cases from Italy. Our results showed that there was a marked variation between the two geographical areas in terms of association with C. psittaci, as 17% (5/30) of the samples from Italy were positive for C. psittaci, whereas no association with this pathogen was observed in any of the African samples (0/9), suggesting that other cofactors may determine the OAL occurrence in those areas. OAL cases are often characterized by down-regulation of p16/INK4a expression and promoter hypermethylation of the p16/INK4a gene. Our results showed a partial methylation of p16/INK4a promoter in C. psittaci-negative cases, whereas no hypermethylation of this gene was found in C. psittaci-positive cases, suggesting that mechanisms other than promoter hypermethylation lead to p16/INK4a silencing in C. psittaci-positive cases. We may conclude that the role of epidemiologic, environmental and genetic factors, must be considered in the aetiology of this disease.

[Relationship between primary ocular adnexal mucosa-associated lymphoid tissue lymphoma and eye infection].

OBJECTIVE: To study the role of pathogenic microorganisms commonly associated with chronic eye disease, including Chlamydia psittaci, Chlamydia trachomatis, Chlamydia pneumoniae, herpes simplex virus (HSV) type 1 and type 2, and adenovirus type 8 and type 19, in the development of primary ocular adnexal mucosa-associated lymphoid tissue (MALT) lymphoma in Chinese patients. METHODS: Sixty-eight archival cases of primary ocular adnexal lymphoproliferative lesions, including 38 cases of MALT lymphoma, 3 cases of non-MALT lymphoma and 27 cases of chronic inflammation, were enrolled into the study. DNA was extracted from the paraffin-embedded tissue samples. The presence of DNA of C. psittaci, C. trachomatis, C. pneumoniae, HSV type 1, HSV type 2, adenovirus type 8 and adenovirus type 19 were analyzed by multiplex touchdown enzyme time-release polymerase chain reaction (TETR-PCR). RESULTS: All of the specimens yielded PCR products of over 100 base pairs and were thus suitable for TETR-PCR screening of infectious agents. The prevalence of DNA of C. psittaci, C. trachomatis and adenovirus type 19 were 0 in MALT lymphoma, non-MALT lymphoma and chronic inflammation. There were 2 cases positive for C. pneumoniae DNA, amongst the 38 cases of MALT lymphoma studied (5.3%, 2/38). HSV type 1, HSV type 2 and adenovirus type 8 DNA was found in each of the 3 patients with chronic inflammation. CONCLUSION: The study indicates that C. psittaci, C. trachomatis, C. pneumoniae, HSV type 1, HSV type 2, adenovirus type 8 and adenovirus type 19 probably play little role in the pathogenesis of ocular adnexal MALT lymphoma in Chinese patients.