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Originally published as JCO Early Release 10.1200/JCO.2005.07.083 on June 20 2005 © 2005 American Society of Clinical Oncology. Regression of Ocular Adnexal Lymphoma After Chlamydia Psittaci–Eradicating Antibiotic TherapyFrom the Departments of Radiochemotherapy, Pathology, Ophthalmology, and Hematology, San Raffaele H Scientific Institute, Milan; Immunovirology and Biotherapy Unit, and Division of Experimental Oncology 1, Department of Pre-Clinical and Epidemiological Research, Centro di Riferimento Oncologico, Istituto di Ricovero e Cura a Carattere Scientifico National Cancer Institute, Aviano, Italy; and First Department of Medicine, Semmelweis University, Budapest, Hungary Address reprint requests 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
PURPOSE: Some infectious agents contributing to lymphomagenesis have been considered targets for new therapeutic strategies. Chlamydia psittaci DNA has been detected in 80% of ocular adnexal lymphomas. The present pilot study was carried out to assess whether C psittaci–eradicating antibiotic therapy is associated with tumor regression in ocular adnexal lymphomas. PATIENTS AND METHODS: Nine patients with C psittaci–positive marginal-zone B-cell lymphoma of the ocular adnexa at diagnosis or relapse were treated with doxycycline 100 mg, bid orally, for 3 weeks. The presence of C psittaci DNA in peripheral-blood mononuclear cells (PBMCs) was also assessed before and after treatment in seven patients. Objective lymphoma regression was assessed 1, 3, and 6 months after therapy conclusion and every 6 months during follow-up. RESULTS: All patients completed antibiotic therapy with excellent tolerability. At 1 month from doxycycline assumption, chlamydial DNA was no longer detectable in PBMCs of all four positive patients. Objective response was complete in two patients, partial response (> 50%) was observed in two patients, and minimal response (< 50%) was observed in three patients. Duration of response in the seven responders was 12+, 29+, 31+, 8+, 7+, 2+, and 1+ months, respectively. CONCLUSION: C psittaci–eradicating antibiotic therapy with doxycycline is followed by objective response in patients with ocular adnexal lymphoma, even after multiple relapses of the disease. A confirmatory, large, phase II trial is warranted to confirm whether this fast, cheap, and well-tolerated therapy could replace other more aggressive strategies as first-line treatment against ocular adnexal lymphomas.
A pathogenic link between some infectious agents, mainly bacteria and viruses, and non-Hodgkin's lymphomas has been clearly established.1-3 In particular, in marginal zone lymphomas of mucosa-associated lymphoid tissue (MALT) type, the chronic antigenic stimulation provided by these agents has been suggested to elicit host immune responses able to promote and sustain clonal B-cell expansion.4 These findings provided the rationale for new therapeutic modalities aimed at eliminating the source of chronic antigenic stimulation using specific antimicrobial drugs.5 This approach was successfully associated with objective tumor regression in a variable proportion of different lymphoma entities.5 The largest experience in this context is represented by Helicobacter pylori eradication in patients with stage I marginal-zone lymphomas of MALT type of the stomach, which is followed by objective tumor response in 70% to 80% of patients.5-7 Moreover, some cases of tumor regression after antimicrobial therapy have been reported in Borrelia burgdorferi–associated cutaneous MALT lymphomas,8,9 hepatitis C virus–associated splenic lymphomas with villous lymphocytes,10 and, more recently, Campylobacter jejuni–associated immunoproliferative small intestinal disease.11 Chlamydia psittaci DNA has been recently detected in tumor biopsies from 80% of patients with ocular adnexal lymphomas (ie, conjunctiva, lachrymal gland, and orbital soft tissues) and in the peripheral-blood mononuclear cells (PBMCs) of 40% of these patients.12 C psittaci is the etiologic agent of psittacosis, a human infection caused by exposure to infected birds, cats, and other pets.13-15 This microorganism is an obligate intracellular bacterium growing in eukariotic cells and has a tendency to cause persistent infections.16 In particular, C psittaci–related follicular conjunctivitis16,17 may favor the development of ocular adnexal lymphomas through a chronic antigenic stimulation.12 Similar to what was adopted for gastric lymphomas, for which H pylori eradication is a well-established therapeutic strategy,7 eradication of C psittaci infection with a specific antibiotic therapy may constitute an attractive, novel therapeutic approach for ocular adnexal lymphomas. In this article, we report the results, in terms of C psittaci eradication rate and objective lymphoma response, of a pilot study carried out in nine patients with C psittaci–positive, marginal-zone, B-cell lymphoma of MALT type of the ocular adnexa treated with the antibiotic doxycycline.
Study Group Nine immunocompetent adults with C psittaci–associated, marginal-zone, B-cell lymphoma of MALT type of the ocular adnexa (lachrymal gland, conjunctiva, and orbital soft tissue) and measurable disease at diagnosis (n = 4) or relapse (n = 5) were included in this pilot experience. Preliminary therapeutic data of patients 1 through 4 have been previously included in our article describing the association between C psittaci and ocular adnexal lymphoma.12 All patients but one were diagnosed and treated at the San Raffaele H Scientific Institute of Milan, Italy; the remaining patient (patient 8) was diagnosed and treated at the Semmelweis University of Budapest, Hungary. In all these patients, C psittaci DNA was detected in formalin-fixed, paraffin-embedded tumor biopsies (diagnostic samples) by using a multiplex touchdown enzyme time-release polymerase chain reaction that was designed to simultaneously detect Chlamydia trachomatis, pneumoniae, and psittaci DNA at bacterial loads less than 1 infection-forming unit and by following a previously reported protocol.18 Staging work-up consisted of physical examination, ultrasonography of the neck, total-body computed tomography scan, gastroscopy with multiple gastric biopsies, and bilateral bone marrow biopsy. Stage of disease was defined according to the Ann Arbor staging system.19
Trial Design The primary end points of this study were eradicating activity and objective lymphoma response after a 3-week doxycycline regimen. The presence of C psittaci DNA in patients' PBMC was the parameter for eradicating activity assessment. PBMC analysis was performed before the start of antibiotic therapy in seven patients (in three patients treated at diagnosis and in four patients treated at relapse); in relapsed patients, the period between the diagnosis of lymphoma and PBMC sampling was longer than 5 years. C psittaci eradication was monitored by assessing patients' PBMC obtained at 1 month after completion of antibiotic therapy and at 1 year of follow-up; all molecular analyses were performed by blinded investigators. All nine patients were assessable for antilymphoma activity. Objective response was assessed 1, 3, and 6 months after conclusion of antibiotic therapy and every 6 months during follow-up using contrasted computed tomography scan or magnetic resonance imaging of the orbits and any other radiologic procedure that was positive before antibiotic therapy. Objective response was defined according to the WHO criteria,20 and duration of response was calculated from therapy conclusion to date of lymphoma progression. This pilot study conformed to the tenets of the Declaration of Helsinki and was approved by the institutional review boards of the participating institutions; the nine enrolled patients provided signed informed consent.
Study Group The clinical characteristics of the nine patients entered onto this study are listed in Table 1. Sites of disease at diagnosis and relapse and previous therapeutic management of the nine patients are listed in Table 2. Experimental treatment with doxycycline was indicated at lymphoma diagnosis in four patients (patients 1, 7, 8, and 9) and at lymphoma relapse in five patients. The median age of the patients at the time of antibiotic therapy was 72 years (range, 52 to 87 years); six patients were females. All patients had an Eastern Cooperative Oncology Group performance status of  1, both at diagnosis and at the time of enrollment. No patient had increased serum levels of lactate dehydrogenase or B symptoms. Five patients reported prolonged contact with household animals, and seven had a history of chronic conjunctivitis. Patients 4, 5, and 6 had H pylori–associated chronic gastritis detected at the gastroscopy performed during staging immediately after lymphoma diagnosis. They had been treated with a triple antibiotic combination, achieving early bacterial eradication, and none experienced H pylori reinfection. The duration of the period between H pylori eradication and doxycycline assumption was 67, 32, and 65 months for patients 4, 5, and 6, respectively.
Tolerability and Eradication Rate All patients completed the antibiotic therapy, but one elderly patient received doxycycline irregularly (patient 2); no symptomatic toxicity or changes in biochemical parameters were observed. Four of the six assessed patients (patients 2, 3, 4, and 7) had C psittaci DNA in PBMCs before antibiotic therapy (Table 3). At 1 month after doxycycline therapy, Chlamydiae DNA was no longer detectable in the PBMCs of the patients investigated (Fig 1). Eradication persisted over time, as confirmed by PBMC samples collected at 1 year of follow-up in patients 2, 3, and 4.
Objective Lymphoma Response After a median follow-up from doxycycline assumption of 12 months (range, 1 to 31 months), complete lymphoma remission was observed in patients 4 (Figs 2A and 2B) and 5, whereas patients 1 and 6 had a partial response (> 50% tumor reduction), and patients 7 and 8 had a response of less than 50% tumor reduction (Table 3). Patient 9 showed minimal reduction in tumor size at 1 month from doxycycline assumption. Time to best response was variable, ranging from 3 (patient 4) to 31 months (patient 1). In the latter patient, a single lymphomatous lesion of the soft tissues of the left orbit (Fig 2C) displayed a continuing regression, which exceeded a 50% volume reduction only 2 years after doxycycline therapy (Fig 2D). In patient 5, complete response was achieved at 6 months from antibiotic therapy; an ultrasonography performed at 1 month from therapy showed a complete disappearance of multiple cervical lymphadenopathies, which constituted the major part of measurable disease (Figs 2E and 2F), followed by gradual regression of a minimal enhanced area in previously irradiated right lachrymal gland (magnetic resonance imaging). In patient 6, doxycycline therapy was associated with partial regression of both lymphomatous lesions of the left lachrymal gland and conjunctiva. In patient 7, clinical and radiologic examination performed at 6 months of antibiotic therapy showed a gradual regression (< 50%) of both lesions of the left lachrymal gland and right conjunctiva. No cases of tumor progression after response were observed; duration of response was 31+, 29+, 12+, 8+, 7+, 2+, and 1+ months in the seven responding patients. Patients with unresponsive lymphomas received further conventional treatment; patient 2 received four other lines of chemotherapy, and patient 3 was treated with intralesional injections of interferon. Both of these patients are alive with partial lymphoma regression.
Lymphoma regression was observed when doxycycline was used either as first-line treatment or as salvage therapy; in particular, tumor regression was observed in all of the four patients with a follow-up longer than 3 months who received doxycycline as first- or second-line treatment (patients 1, 4, 6, and 7) but only in one (patient 5) of the three patients treated with doxycycline as fourth-line treatment. Interestingly, lymphoma regression was observed both in previously irradiated (patient 5) and nonirradiated (patients 4 and 6) areas, and it was evident both in primary sites of disease (orbit, conjunctiva, and lachrymal gland; Figs 2B and 2D) and in regional lymphadenopathies (patient 5; Fig 2F).
The present pilot experience confirms our preliminary observations12 and expands our knowledge on C psittaci–eradicating antibiotic therapy in MALT lymphomas of the ocular adnexa, providing further evidence for doxycycline as a novel active strategy against these malignancies. In fact, the use of a 3-week regimen of doxycycline was associated with eradication of C psittaci DNA in all of the four patients with positive PBMC and with objective lymphoma response in five of the seven patients with a follow-up longer than 3 months. Tumor response was observed even in a patient who previously received up to three lines of treatment, and duration of response was longer than 2 years in two patients. Despite the limited number of treated patients, lymphoma regression observed after eradication with doxycycline further supports a causal role of C psittaci infection in the development of ocular adnexal lymphomas and in sustaining the disease over time. Available evidence suggests that, like other MALT-type lymphomas, ocular adnexal lymphomas also may be antigen-driven disorders, at least in the earliest phases of their development. In fact, clonally rearranged immunoglobulin heavy-chain variable genes showed, in most cases, a mutation pattern consistent with the likely derivation from postgerminal-center memory B cells.21 Moreover, the mutation rates observed in the immunoglobulin variable genes of these lymphomas are similar to those reported for gastrointestinal marginal-zone lymphomas,22 which have a well-established association with chronic antigenic stimulation by H pylori.23 In this scenario, the recent detection of C psittaci DNA in 80% of ocular adnexal lymphomas12 and the induction of objective responses in a proportion of patients by specific antibiotic therapy is consistent with the possibility that C psittaci–derived antigens may play a pathogenic role in these lymphomas. Interestingly, five of our patients reported prolonged contacts with household animals, and seven patients had a history of chronic conjunctivitis. In four of these patients, C psittaci DNA was detected in PBMC samples collected, in some cases, more than 5 years after lymphoma diagnosis, with a concurrent relapse of the disease. These findings suggest that the natural history of ocular adnexal lymphomas is associated with the persistence of the C psittaci infection over time, further supporting an etiologic role of this bacterium. In this light and similar to what reported for H pylori–associated gastric MALT lymphoma, objective responses induced by doxycycline in patients with C psittaci–positive ocular adnexal lymphomas may be a result of the elimination of a pathogenically relevant chronic antigenic stimulation provided by the microorganism. Further studies are required to formally prove this conclusion. The oral treatment with doxycycline was well tolerated in these aged and heavily pretreated patients and resulted in a high eradication rate, with the disappearance of C psittaci DNA from patients' PBMC at 1 month after the completion of antibiotic therapy, which was confirmed at least 1 year later. Periodic assessment of C psittaci eradication in PBMC may constitute a useful tool to monitor the treatment in these lymphomas. This pilot experience provides, for the first time, details regarding the objective regression of ocular adnexal lymphoma in patients treated with doxycycline, which closely recapitulates the objective response induced in gastric MALT lymphomas by H pylori–eradicating therapy. As reported in gastric MALT lymphomas,24 tumor regression in our patients was slow (Table 3); in particular, patient 1 achieved a 50% volume reduction only after 24 months of follow-up, and a further regression in lymphoma lesion was observed in the following months. These observations seem to suggest that ocular adnexal lymphoma patients with residual disease after eradicating therapy should be followed for a longer period before starting a salvage treatment. Accordingly, one could hypothesize that, with a longer follow-up, patients 6 through 9 may achieve a better objective lymphoma regression. However, patients 2 and 3 did not experience lymphoma response despite successful C psittaci eradication; this recapitulates previous observations of gastric MALT lymphomas that were not responsive to bacterium eradication (20% to 30% of patients).5,24 In patient 2, unresponsiveness could be a result of the irregular doxycycline assumption, although the rapid progression of the disease rather suggests a transformation to high-grade lymphoma, which was probably no longer dependent on microenvironmental growth-promoting stimuli, such as putative C psittaci–derived antigens. Again, in analogy with gastric MALT lymphomas,25 these features point to the need to define which genetic alterations may allow the identification of potentially unresponsive patients at the time of therapeutic decision. An important, original observation in this pilot experience suggests that, at a variance that is reported for gastric MALT lymphomas, eradicating antibiotic therapy is able to induce an objective regression even of regional lymphadenopathies in patients with relapsed lymphoma of the ocular adnexa (patient 5; Figs 2E and 2F). This finding carries two major implications. First, it suggests that a systemic chlamydial infection, confirmed by the presence of C psittaci DNA in patients' PBMC, furnishes a continuous antigenic stimulation sustaining the disseminated disease; and, second, it provides the rationale to assess the role of C psittaci–eradicating antibiotic therapy also in patients with disseminated lymphoma in future trials. Here, we also describe for the first time a case of C psittaci–related ocular adnexal lymphoma (patient 8) diagnosed and treated in a country different from Italy (Hungary). This observation, even if still limited, suggests that this bacteria-lymphoma relationship is not restricted to Northern Italy and stimulates further studies aimed at assessing the prevalence of this association on a large series of patients diagnosed in different geographical areas. In conclusion, our pilot experience reveals that a 3-week regimen of doxycycline may be a fast, cheap, well-tolerated, and active approach to ocular adnexal lymphomas, even for aged and heavily pretreated patients. In addition, the periodic assessment of the presence of C psittaci DNA in patient's PBMC may constitute a useful tool to evaluate eradication or detect reinfection or reactivation. As original contributions, our experience suggests that achieving an objective response in ocular adnexal lymphoma patients treated with doxycycline is slow, requiring a long observation period, and that regression of lymphomatous lesions in previously irradiated areas and of relapsed disease involving regional lymphadenopathies may be achieved with this strategy. These observations deserve to be confirmed in a large, prospective, phase II trial in patients with newly diagnosed or relapsed lymphoma of the ocular adnexa. If confirmed, these results will support the use of C psittaci–eradicating therapy as a new strategy against ocular adnexal lymphomas, avoiding the risk of undesirable and sometimes severe side effects linked to current therapeutic approaches.
The authors indicated no potential conflicts of interest.
We thank Adam Meijer, MD (Research Laboratory for Infectious Disease, National Institute of Public Health and the Environment, Bilthoven, the Netherlands) for the generous gift of molecular biology reagents. We thank Jeanette Toth, MD (1st Department of Ophthalmology, Semmelweis University, Budapest, Hungary), Erika Toth, MD (National Institute of Oncology, Budapest, Hungary), and Laszlo Krenacs, MD (Laboratory of Tumor Pathology and Molecular Diagnostics, Institute of Biotechnology, Bay Zoltan Foundation for Applied Research, Szeged, Hungary). We appreciate the helpful suggestions of Franco Cavalli, MD (Istituto Oncologico della Svizzera Italiana, Ospedale San Giovanni, Bellinzona, Svizzera) and Letterio Politi, MD (Department of Neuroradiology, San Raffaele H Scientific Institute, Milan, Italy). We also thank Antonio Cassone, MD (Dipartimento di Malattie Infettive, Istituto Superiore di Sanità, Rome, Italy) for his sustained scientific collaboration.
Supported in part by a grant from the Italian Association for Cancer Research (R.D.). Presented in part as an oral presentation at the Presidential Symposium of the 9th Annual Meeting of the European Hematology Association, Geneva, Switzerland, June 12, 2004; and published in abstract form as: Ferreri AJM, Guidoboni M, Ponzoni M, et al: The high prevalence of Chlamydia psittaci infection in ocular adnexal lymphomas (OAL) provides the rationale for the use of antibiotics as a potential therapeutic strategy in these patients. Hematol J 5:S111, 2004 (suppl 2, abstr 321). A.J.M.F., M.P., and M.G. contributed equally to this work. Authors' disclosures of potential conflicts of interest are found at the end of this article.
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Copyright © 2005 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
