Affiliations of authors: Departments of Radiochemotherapy (AJMF, SD, EV), Pathology (MP, EDC, MF), Ophthalmology (CDC), and Hematology (KF) and Laboratorio di Diagnostica e Ricerca (RI), San Raffaele H Scientific Institute, Milan, Italy; Immunovirology and Biotherapy Unit (MG, LC, AAL, RD) and Division of Experimental Oncology 1 (MB), Department of Pre-Clinical and Epidemiological Research, Centro di Riferimento Oncologico, IRCCS National Cancer Institute, Aviano, Italy
Correspondence to: Andrés J.M. Ferreri, MD, Department of Radiochemotherapy, San Raffaele H Scientific Institute, Via Olgettina 60, 20132 Milan, Italy (e-mail: andres.ferreri{at}hsr.it)
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ABSTRACT |
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INTRODUCTION |
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Chlamydia psittaci is the etiologic agent of psittacosis, a human lung infection caused by exposure to infected birds (3). The occurrence of high rates of chlamydial infection in household cats (8,9) and of asymptomatic carriage of C. psittaci in cats from breeding catteries (10) raises the possibility that human C. psittaci infections deriving from pets other than birds may be underdiagnosed. For example, chronic conjunctivitis by C. psittaci is a rare condition, but there is some indication that this condition may be more common than previously thought (11,12). Hence, a putative association between a persistent chlamydial infection (and chronic antigenic stimulation) and development of ocular adnexal lymphomas (i.e., conjunctiva, lachrymal gland, and orbital soft tissues) represents an attractive issue, laying the basis for new potentially therapeutic strategies for these disorders.
It is noteworthy that ocular adnexal lymphomas share some clinico-pathologic features with gastric lymphomas, for which a pathogenic link with Helicobacter pylorirelated follicular gastritis has been established (13). Indeed, both ocular adnexal and gastric lymphomas are characterized by an indolent course, a large prevalence of marginal zone B-cell histologic type, and a high degree of infiltrating reactive T-cells (13,14). Moreover, similar to what was observed in gastric lymphomas, the presence of somatic mutations within immunoglobulin genes in ocular adnexal lymphomas indicates that the development of these disorders is probably favored by chronic stimulation provided by unknown antigen or antigens (15).
This study was designed to investigate a possible association between chlamydial infection and lymphomas of the ocular adnexa. We investigated the presence of three Chlamydiae speciespneumoniae, trachomatis, and psittaciin both tumor tissue and blood samples from case patients with ocular adnexal lymphoma and from control subjects. The possible pathogenic role of C. psittaci infection in the development of ocular adnexa lymphomas is discussed, providing a rational background for new therapeutic modalities for the treatment of ocular adnexal lymphomas.
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METHODS |
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Adults with histologically proven non-Hodgkins lymphomas of the ocular adnexa (i.e., lachrymal gland, conjunctiva, and orbital soft tissue) diagnosed at the San Raffaele H Scientific Institute (Milan, Italy) from 1988 to 2002 were included in this study. Data regarding age; sex; performance status [according to the Eastern Cooperative Oncology Group criteria (16)]; concomitant morbidity; history of neoplastic, infective, allergic, and ocular diseases; risk factors for C. psittaci infection (e.g., prolonged exposition with household animals and workers of breeding catteries); tumor stage; treatment regimes; and relapse and survival rates were collected. This study conformed to the tenets of the Declaration of Helsinki, and all patients provided oral informed consent.
Tissue and Peripheral Blood Mononuclear Cell (PBMC) Samples
Chlamydiae DNA detection was performed in formalin-fixed, paraffin-embedded 10-µm-thick sections obtained from ocular adnexa lymphoma lesions. Histologic material was available for molecular analysis in 40 patients. Tissue samples from 20 patients who had been referred to the Department of Ophthalmology (San Raffaele H Scientific Institute) for nonneoplastic orbital biopsies (e.g., ectropion, entropion, palpebral ptosis) and from 26 patients who had reactive lymphadenopathies were used as control subjects. The control subjects had similar sociodemographic characteristics (median age = 64 years [range = 4490 years] and male/female ratio = 0.6 for ophthalmologic control subjects; and median age = 58 years [range = 1976 years] and male/female ratio = 0.8 for the reactive lymphadenopathy case patients) to those of the lymphoma case patients. Histologic specimens of ocular adnexal lymphomas, nonneoplastic orbital biopsies, and reactive lymphadenopathies were collected and processed during the same period of time and in the same laboratory.
Chlamydiae DNA detection was also investigated in PBMCs from 21 of the lymphoma patients in our series. The time from lymphoma diagnosis to PBMC collection ranged from 1 to 152 months (median = 12 months). PBMC samples were collected at diagnosis or within 12 months of diagnosis in 11 patients and at random in the other 10 lymphoma patients. In three patients, PBMC collection was concomitant with a relapse of the disease. PBMC samples from 38 healthy donors were used as control samples. Similar to the tissue sample groups described above, the control subjects (healthy donors) had similar sociodemographic characteristics (median age = 48 years [range = 2362 years] and male/female ratio = 0.8) to the case patients (median age = 61 years [range = 3474 years] and male/female ratio = 0.62).
DNA Extraction and Purification Procedures
Two to three 5-µm sections were cut from formalin-fixed, paraffin-embedded samples from lymphoma and control tissue. The sections were deparaffinized with two xylene washes (10 minutes each) and rehydrated through decreasing concentrations of ethanol (100%, 96%, 80%, 50%, and distilled water) at room temperature. After rehydration, tissue sections were then placed in 100 µL of digestion buffer (150 mM NaCl, 50 mM TRIS [pH 7.5], 50 mM EDTA, and 1% Triton X100) containing 2 mg/mL proteinase K to digest at 50°C for 3 days. The proteinase Kdigested mixture was then purified by silica gel spin cartridge (Talent S.R.L, Trieste, Italy) according to the manufacturers instructions.
DNA extraction from PBMC samples was carried out following a standard phenol/chloroform extraction technique after proteinase K digestion. DNA from the PBMC samples was extracted 1 year later than was the DNA from the lymphoma tissue samples, and DNA purification from lymphoma and PBMC samples was carried out in two different institutions: Centro di Riferimento Oncologico, Aviano, Italy, and San Raffaele H Scientific Institute, Milan, Italy, respectively.
Detection of Chlamydiae DNA
A multiplex touchdown, enzyme time-release polymerase chain reaction (PCR) assay, designed to simultaneously detect C. trachomatis, pneumoniae, and psittaci DNA at bacterial loads lower than 1 infection-forming unit was performed according to a previously published protocol (17). Briefly, 10 µL of template DNA was added to a PCR tube containing 40 µL of PCR mixture overlaid with 1 drop of mineral oil. Blank reactions filled with 50 µL of PCR mixture were interspersed every three to four samples to monitor possible contamination of PCR reagents by Chlamydiae DNA and to rule out any false-positive results. DNA equivalent to less than 1 infection-forming unit/PCR tube of C. trachomatis L2, C. pneumoniae TW-183, and C. psittaci ORNI (a gift from Dr. Adam Meijer, Research Laboratory for Infectious Disease, National Institute of Public Health and the Environment, Bilthoven, The Netherlands) were included as positive controls. Amplified DNA was from the end of the 16S rRNA gene and the beginning of the 16S-23S spacer region in the ribosomal genes (17). The primer pairs used specifically for C. trachomatis and C. pneumoniae were located entirely in the 16S rRNA gene; for C. psittaci, one primer was located in the 16S rRNA gene and one primer was located in the 16S-23S spacer region.
PCR products were analyzed by electrophoresis in 2% agarose gels with ethidium bromide staining. DNA fragment size was quantified by image analysis (Image Station 440CF, NEN Life Science Products, Boston, MA). Three amplifications were performed per tissue and PBMC sample, both for case patients and for control subjects. Case-patient samples were considered positive for chlamydial infection when Chlamydiae DNA was amplified in at least two independent experiments.
To further rule out the possibility of a false-positive result, all the molecular procedures were repeated twice for both positive and negative case-patient samples by starting from new pathologic material from the same patients and confirming the results obtained in the first round of experiments. Specificity of the amplified fragments was confirmed by direct sequencing of both sense and antisense strands of purified PCR products using an ABI PRISM 310 Genetic Analyzer (Perkin Elmer, Foster City, CA). Sequence specificity was assessed by BLAST search (http://www.ncbi.nlm.nih.gov/blast), and heterogeneity of Chlamydiae sequences across the different samples was investigated by aligning them with the MultAlin software [http://prodes.toulouse.inra.fr/multalin/multalin.html (18)]. In lymphoma samples that were negative to time-release PCR, amplification of -globin was carried out as previously described (19).
Immunocytochemical Techniques
Formalin-fixed, paraffin-embedded 4-µm thick tissue sections of chlamydia-positive lymphomas from seven case patients were immunostained by the Avidin Biotin-Complex method (20), with a monoclonal antibody specific for chlamydia lipopolysaccharide (antibody was included in the Imagen Chlamydia test, working dilution 1:150, DAKO, Glostrup, Denmark). Antigen retrieval was performed with 0.01 M citrate buffer [pH 8] and with six cycles of 5 minutes each at 750 W in a microwave oven. Specificity control experiments were performed using an autoptic lung of a parrot known to be infected by C. psittaci (provided by Dr. Alfredo Seijo) as a positive control and by either substituting the primary monoclonal antibodies (monoclonal antibody from the Imagen Chlamydia test) with nonimmune horse serum (100 µL, working dilution 1:20; Vectastain, Burlingame, CA) or using a histologic section obtained from a case of human prostatic nodular hyperplasia as negative controls.
Chlamydia psittaciEradicating Antibiotic Therapy
Because of the lack of therapeutic guidelines for treating C. psittaciasymptomatic carriers, antibiotic therapy was proposed to the nine patients who were positive for C. psittaci in both their lymphoma and PBMC samples. Two patients refused treatment, and seven patients were treated with doxycycline 100 mg, twice a day, for 3 weeks. The patients who underwent treatment did not receive any concomitant antiblastic, antibiotic, or steroid treatment. The presence of chlamydial DNA in the patients PBMCs was assessed (using the same molecular approach described above) 1 month after the conclusion of antibiotic treatment.
Before treatment, four of the seven patients had measurable lymphoma lesions in the orbit or conjunctiva (one at diagnosis and three with relapsed disease), whereas the remaining three patients were lymphoma-free with a follow-up of more than 1 year. A contrast computed tomography scan of the orbits was performed every 3 months in the four case patients with measurable disease. Objective response (i.e., to antibiotic treatment) was defined according to World Health Organization criteria (21). Briefly, complete remission was defined as the disappearance of all clinical evidence of the disease, partial response was defined as more than a 50% reduction of all measurable lesions, stable disease was defined as regression of any measurable lesion by less than or equal to 50% (minimal response) or no change for the measurable lesions, and progressive disease was defined as appearance of any new lesion or increase in the size of the previously involved sites of at least 25%. The seven patients who underwent treatment and who were eligible for analysis provided signed informed consent.
Statistical Analysis
Differences in Chlamydiae DNA detection between case patients and control subjects and associations between Chlamydiae DNA expression and clinico-pathologic variables were analyzed using the Fisher exact test for categorical variables (Statistica 4.0 statistical package for Windows, Statsoft, Tulsa, OK). All statistical tests were two-sided, with an overall statistical significance level of .05.
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RESULTS |
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The clinico-pathologic characteristics of the 40 patients who participated in the study are reported in Table 1. Chlamydia psittaci DNA was found in lymphoma samples from 32 of the 40 (80%) case patients analyzed, whereas none of the samples carried DNA sequences for either C. trachomatis or C. pneumoniae (Fig. 1). Sequence analysis of PCR products from 13 case patients positive for C. psittaci confirmed the specificity of the amplified fragments (by BLAST search) and showed a discrete degree of sequence heterogeneity among the DNA sequences from the lymphoma case patients (Fig. 2), indicating that these lymphoma samples carried unrelated variants of C. psittaci and, most important, ruling out possible sample contamination from a common source. The finding that about half of the C. psittacipositive lymphoma samples were not positive for the DNA of this microorganism in all of the three PCR amplifications performed per sample (data not shown) indicates that the bacterial load of C. psittaci was generally low in these tissue samples. Notably, all 20 nonneoplastic conjunctival or orbital biopsy samples were negative for all three of the Chlamydiae species investigated, whereas three of the 26 (12%) reactive lymphadenopathy samples were positive for C. psittaci DNA. Hence, C. psittaci DNA was statistically significantly more common in tissue samples among lymphoma patients than among nonneoplastic biopsy patients (80% versus 0%, respectively; P<.001) and reactive lymphadenopathy patients (80% versus 12%; P<.001).
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The presence of Chlamydiae DNA was also assessed in PBMC samples from 21 patients with C. psittacipositive lymphomas to verify whether a systemic chlamydial infection was present at the time of diagnosis and whether the infection persisted over time (Table 2). Chlamydia psittaci DNA was detected in PBMC samples from nine (43%) of the 21 patients. Five of the nine C. psittacipositive PBMC samples were collected at diagnosis, thus indicating an underlying systemic C. psittaci infection at the onset of the disease. The other four C. psittacipositive PBMC samples were collected more than 5 years after the diagnosis of lymphoma, concomitant with a relapse of the disease in three of the patients. Remarkably, DNA sequences from C. pneumoniae and trachomatis were not detected in PBMC samples from lymphoma case patients, and none of the 38 PBMC samples from healthy donors was positive for Chlamydiae species DNA (Fig. 1) (43% for the case patients versus 0% for the healthy donors; P<.001).
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In the parrots lung that carried C. psittaci infection, anti-chlamydial lipopolysaccharide antibodies stained a limited number of scattered interstitial cells with macrophage-like morphology (data not shown). Chlamydia psittacipositive cells displayed a granular and predominantly intracytoplasmatic immunoreactivity. Interestingly, similar findings were observed in four of the seven ocular adnexal lymphoma samples that were positive for C. psittaci DNA (Fig. 4). In particular, immunoreactive cells were characterized by macrophage-like morphology with a relatively abundant cytoplasm, a granular reactivity, and an overall cell size larger than the surrounding neoplastic lymphocytes; these immunoreactive cells were not found within blood vessel lumina.
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Characteristics of the seven patients treated with C. psittacieradicating antibiotic treatment are summarized in Table 3. All patients completed the antibiotic treatment, and there were no cases of symptomatic toxicity or changes in biochemical parameters observed. One month after doxycycline treatment was concluded, Chlamydiae DNA was no longer detectable in the PBMC samples of the seven treated patients. Objective response among the four case patients who had measurable lymphoma lesions before treatment was progressive disease, stable disease (6 months follow-up), minimal response (reduction of 45% of the tumor mass; 20+ months follow-up), and complete remission (18+ months follow-up).
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DISCUSSION |
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The strength of the association between C. psittaci infection and ocular adnexal lymphoma is also supported by the statistically significantly higher prevalence of C. psittaci infection detected in ocular adnexal lymphoma patients than in the general population (22). Unlike C. pneumoniae, which has a broad diffusion worldwide (3), C. psittaci infection is a rare condition in European countries (23). Although data for the prevalence of C. psittaci DNA detection in blood samples from the general population are lacking, serology data indicate that only 3% of elderly individuals in Northern Europe are C. psittaci IgG-seropositive (22). Serology assays, however, are not an adequate method to detect C. psittaci infection, because they are severely limited by high cross-reactivity among different Chlamydiae species and between Chlamydiae and other microorganisms (24). In fact, lipopolysaccharide, the antigen used in available serologic tests, is a common molecule found among several microorganisms. Moreover, in some geographic areas, the seroprevalence of C. pneumoniae has been reported to range from 40% to 80% (22), limiting the value of serologic screening for C. psittaci. Thus, it is important to emphasize that DNA detection by PCR analysis is the current gold standard for chlamydial infection recognition (25,26).
Chlamydial infections have also been associated with neoplastic diseases (46). In fact, Chlamydiae species are known to establish persistent infections, to be mitogenic in vitro (27), to induce polyclonal cell proliferations in vivo (28), and to cause resistance to apoptosis in infected cells (29). Nevertheless, any association between C. psittaci and cancer has not been convincingly demonstrated thus far. The possibility that C. psittaci infections may be underdiagnosed (8), however, highlights the need for research on the potential contribution of this microorganism to tumor development in humans. Indeed, chronic ocular infections by C. psittaci could be more common than previously recognized (9,11,12). If this is the case, then the parallelism between the association of gastric lymphomas with H. pylorirelated chronic gastritis (13) and the potential pathogenic link between chronic conjunctivitis related to chlamydial infection and ocular adnexal lymphomas appears to be worthy of analysis. Interestingly, the incidence of ocular adnexal lymphomas is growing (30). Notably, this increase in incidence is substantially higher than that reported for systemic lymphomas (31) and does not seem to be biased by demographic or reporting artifacts or by variations in diagnostic criteria (30). Our experience confirms this trend, with a 71% increase in the number of ocular adnexal lymphoma cases diagnosed at our institution (San Raffaele H Scientific Institute) between 19941997 and 19982001. These epidemiologic trends may be related to unrecognized pathogenic factors, among which infectious agents could be included.
Chronic persistence of infectious agents causes prolonged direct antigen stimulation, which is thought to play an important role in the clonal expansion of some lymphomas (e.g., follicular and marginal zone B-cell lymphomas), as indicated by the presence of somatically hypermutated immunoglobulin genes with a pattern of ongoing mutations (32). The presence of somatic hypermutations in clonally rearranged immunoglobulin heavy-chain variable genes in marginal zone B-cell lymphomas of the ocular adnexa shows a molecular similarity with the normal properties of B cells positively selected by a cognate antigen within the germinal center (33). In particular, the mutation rates observed in immunoglobulin-variable genes of ocular adnexal lymphomas are similar to those reported for gastro-intestinal marginal zone lymphomas (15), whose association with chronic antigenic stimulation by H. pylori is well established (13). The possibility that ocular adnexal lymphomas arise in the context of chronic inflammation has also been recently suggested (15); however, the source of the putative antigen or antigens presumably involved in this process is still unknown. Chlamydia psittaci may, indeed, provide such an antigenic stimulus, thus contributing to the pathogenesis of ocular adnexal lymphomas. As reported in Table 1, C. psittaci infection was more common among patients with marginal zone B-cell lymphomas and other low-grade lymphomas (88%) than among those with diffuse large B-cell lymphomas (60%), indicating that there may be different pathways leading toward lymphomagenesis, similar to what has been reported for gastric mucosa-associated lymphoid tissue lymphomas (34). However, the fact that diffuse large B-cell lymphomas were more often negative for C. psittaci DNA than were marginal zone B-cell lymphomas is consistent with the possibility that a proportion of C. psittacipositive marginal zone lymphomas may evolve toward a more aggressive histotype (i.e., diffuse large B-cell lymphomas), which is no longer responsive to (and dependent on) the antigenic stimulation provided by the microorganism. Of note, 38% of the patients in this study who had C. psittacipositive lymphomas had a prior history of chronic conjunctivitis (Table 1). Nevertheless, a more thorough characterization of the association between ocular adnexal lymphoma and C. psittaci is presently limited, most importantly by the facts that sequencing of the C. psittaci genome has not been completed and that reagents suitable to specifically investigate the expression of bacterial proteins in lymphoma tissues are lacking. These limitations need to be overcome to identify candidate bacterial peptides to be used in ex vivo functional assays aimed at demonstrating whether C. psittaci is able to provide stimuli that promote lymphoma cell growth.
This is the first study, to our knowledge, to demonstrate the presence of C. psittaci DNA in PBMCs of humans. In fact, we observed C. psittaci DNA in the PBMCs of half of our patients with chlamydia-positive lymphomas at diagnosis. Thus, our data suggest that infection of PBMCs by C. psittaci persists over time in a high proportion of case patients. In fact, three of the four chlamydia-positive PBMC samples collected more than 5 years after the lymphoma diagnosis were collected concomitantly with a disease recurrence, further supporting the possible involvement of C. psittaci in sustaining long-term lymphoma cell growth. Our findings are similar to recent observations (35), which demonstrated the presence of C. pneumoniae DNA in circulating cells of humans, and support the previously reported (36) hypothesis that a prolonged C. psittaci infection in PBMCs could favor continuous reinfections that could, in turn, chronically trigger antigenic stimulation. Immunohistochemical data from the present study suggest that monocyte/macrophage system components may play a critical role in such a scenario. Although we did not find mononuclear cells immunoreactive for anti-chlamydia antibodies within blood vessel lumina in the ocular adnexal lymphoma samples we studied, available immunohistochemical data do not allow the distinction between these cells being resident or circulating elements.
In conclusion, although only a limited number of lymphoma patients were treated with antibiotics specific for chlamydia infection, our observation of lymphoma regression after C. psittaci eradication with doxycycline treatment provides additional evidence for a role of C. psittaci infection in the development of ocular adnexal lymphomas. Similar to findings observed in gastric marginal zone lymphomas (2), in which H. pylori eradication was followed by an objective response in 70%80% of case patients, two out of four of our case patients with ocular adnexal lymphoma and measurable disease showed objective regression after antibiotic treatment. These data should, however, be interpreted with caution, given the small number of treated patients. Nevertheless, the findings deserve to be confirmed in a phase I/II prospective trial to conclusively assess whether C. psittacieradicating antibiotics may constitute a new therapeutic strategy against ocular adnexal lymphomas.
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NOTES |
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A.J.M. Ferreri, M. Guidoboni, and M. Ponzoni contributed equally to this work.
We thank Dr. Adam Meijer (Research Laboratory for Infectious Disease, National Institute of Public Health and the Environment, Bilthoven, The Netherlands), Dr. Alfredo Seijo (Servicio de Zoonosis, Hospital Francisco J. Muñiz, Buenos Aires, Argentina), and Dr. Ricardo Abello (Facultad de Medicina, Universidad de Buenos Aires, Argentina) for the generous gift of molecular biology reagents and histologic material for immunocytochemical studies. We appreciate the excellent technical assistance of Mrs. Benedetta Mazzi, Mrs. Anna Mauri, and Mr. Roberto Cairella (Department of Pathology, San Raffaele H Scientific Institute, Milan, Italy) and the helpful suggestions of Dr. Federico Gury Dohmen (Instituto de Zoonosis, Instituto Luis Pasteur, Buenos Aires, Argentina). We also acknowledge Dr. José Menezés (Laboratory of Immunovirology, University of Montreal and Sainte-Justine Hospital Research Center, Montreal, Canada) and Professor Antonio Cassone and Dr. Laura Ciervo (Dipartimento di Malattie Infettive, Istituto Superiore di Sanità, Rome, Italy) for their sustained scientific collaboration.
Preliminary results were presented at the 39th Annual Meeting of the American Society of Clinical Oncology, Chicago, Illinois, 2003, and were published in abstract form as Ferreri A.J.M., Guidoboni M., Ponzoni M., De Conciliis C., Fleischhauer K., Caggiari L., Freschi M., Villa E., Boiocchi M., Dolcetti R. Evidence for association between Chlamydia psittaci infection and ocular adnexal lymphoma (OAL). Proc Am Soc Clin Oncol 2003; 22:2273.
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Manuscript received August 13, 2003; revised February 19, 2004; accepted February 27, 2004.
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