Regression of Ocular Adnexal Lymphoma After Chlamydia Psittaci每Eradicating Antibiotic Therapy
http://www.100md.com
▲還散笫雖悝◎
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
First Department of Medicine, Semmelweis University, Budapest, Hungary
ABSTRACT
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每eradicating antibiotic therapy is associated with tumor regression in ocular adnexal lymphomas.
PATIENTS AND METHODS: Nine patients with C psittaci每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每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.
INTRODUCTION
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每associated cutaneous MALT lymphomas,8,9 hepatitis C virus每associated splenic lymphomas with villous lymphocytes,10 and, more recently, Campylobacter jejuni每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每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每positive, marginal-zone, B-cell lymphoma of MALT type of the ocular adnexa treated with the antibiotic doxycycline.
PATIENTS AND METHODS
Study Group
Nine immunocompetent adults with C psittaci每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
Enrolled patients were treated with doxycycline 100 mg, bid orally, for 3 weeks; they did not receive any concomitant antiblastic, antibiotic, or corticosteroid therapy in the period from the start of antibiotic therapy to the maximum response assessment. Doxycycline (100-mg tablets, Bassado; Pharmacia, Rome, Italy) is a tetracycline derivative with uses similar to those of tetracycline. It was preferred to other tetracyclines because of its fairly reliable absorption from the gastrointestinal tract, its long half-life (12 to 24 hours), its wide distribution in body tissues and fluids, and the fact that it can be administered to patients with renal insufficiency.
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.
RESULTS
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每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).
DISCUSSION
The present pilot experience confirms our preliminary observations12 and expands our knowledge on C psittaci每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每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每associated gastric MALT lymphoma, objective responses induced by doxycycline in patients with C psittaci每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每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每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每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每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每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.
Authors' Disclosures of Potential Conflicts of Interest
The authors indicated no potential conflicts of interest.
Acknowledgment
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 Sanit角, Rome, Italy) for his sustained scientific collaboration.
NOTES
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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Groves FD, Linet MS, Travis LB, et al: Cancer surveillance series: Non-Hodgkin's lymphoma incidence by histologic subtype in the United States from 1978 through 1995. J Natl Cancer Inst 92:1240-1251, 2000
Slater DN: Borrelia burgdorferi每associated primary cutaneous B-cell lymphoma. Histopathology 38:73-77, 2001
Mele A, Pulsoni A, Bianco E, et al: Hepatitis C virus and B-cell non-Hodgkin lymphomas: An Italian multicenter case-control study. Blood 102:996-999, 2003
Wyatt JI, Rathbone BJ: Immune response of the gastric mucosa to Campylobacter pylori. Scand J Gastroenterol Suppl 142:44-49, 1988
Bayerdorffer E, Neubauer A, Rudolph B, et al: Regression of primary gastric lymphoma of mucosa-associated lymphoid tissue type after cure of Helicobacter pylori infection. MALT Lymphoma Study Group. Lancet 345:1591-1594, 1995
Isaacson PG: Gastric MALT lymphoma: From concept to cure. Ann Oncol 10:637-645, 1999
Fischbach W, Goebeler-Kolve ME, Dragosics B, et al: Long term outcome of patients with gastric marginal zone B cell lymphoma of mucosa associated lymphoid tissue (MALT) following exclusive Helicobacter pylori eradication therapy: Experience from a large prospective series. Gut 53:34-37, 2004
Roggero E, Zucca E, Mainetti C, et al: Eradication of Borrelia burgdorferi infection in primary marginal zone B-cell lymphoma of the skin. Hum Pathol 31:263-268, 2000
Kutting B, Bonsmann G, Metze D, et al: Borrelia burgdorferi-associated primary cutaneous B cell lymphoma: Complete clearing of skin lesions after antibiotic pulse therapy or intralesional injection of interferon alfa-2a. J Am Acad Dermatol 36:311-314, 1997
Hermine O, Lefrere F, Bronowicki JP, et al: Regression of splenic lymphoma with villous lymphocytes after treatment of hepatitis C virus infection. N Engl J Med 347:89-94, 2002
Lecuit M, Abachin E, Martin A, et al: Immunoproliferative small intestinal disease associated with Campylobacter jejuni. N Engl J Med 350:239-248, 2004
Ferreri AJ, Guidoboni M, Ponzoni M, et al: Evidence for an association between Chlamydia psittaci and ocular adnexal lymphomas. J Natl Cancer Inst 96:586-594, 2004
Nasisse MP, Guy JS, Stevens JB, et al: Clinical and laboratory findings in chronic conjunctivitis in cats: 91 cases (1983-1991). J Am Vet Med Assoc 203:834-837, 1993
Pudjiatmoko Fukushi H, Ochiai Y, et al: Seroepidemiology of feline chlamydiosis by microimmunofluorescence assay with multiple strains as antigens. Microbiol Immunol 40:755-759, 1996
Pointon AM, Nicholls JM, Neville S, et al: Chlamydia infection among breeding catteries in South Australia. Australian Veterinary Practitioner 21:58-63, 1991
Lietman T, Brooks D, Moncada J, et al: Chronic follicular conjunctivitis associated with Chlamydia psittaci or Chlamydia pneumoniae. Clin Infect Dis 26:1335-1340, 1998
Lietman T, Dawson C, Osaki S: Ocular chlamydial infections. Int Ophthalmol Clin 38:125-135, 1998
Madico G, Quinn TC, Boman J, et al: Touchdown enzyme time release-PCR for detection and identification of Chlamydia trachomatis, C. pneumoniae, and C. psittaci using the 16S and 16S每23S spacer rRNA genes. J Clin Microbiol 38:1085-1093, 2000
Carbone PP, Kaplan HS, Musshoff K, et al: Report of the Committee on Hodgkin's Disease Staging Classification. Cancer Res 31:1860-1861, 1971
Zubrod CG, Scheidermann MA, Frei E: Appraisal of methods for the study of chemotherapy in man: Comparative therapeutic trial of nitrogen mustard and triethylene thiophosphoramide. J Chronic Dis 11:7-33, 1960
Coupland SE, Foss HD, Anagnostopoulos I, et al: Immunoglobulin VH gene expression among extranodal marginal zone B-cell lymphomas of the ocular adnexa. Invest Ophthalmol Vis Sci 40:555-562, 1999
Hara Y, Nakamura N, Kuze T, et al: Immunoglobulin heavy chain gene analysis of ocular adnexal extranodal marginal zone B-cell lymphoma. Invest Ophthalmol Vis Sci 42:2450-2457, 2001
Wotherspoon AC, Ortiz-Hidalgo C, Falzon MR, et al: Helicobacter pylori-associated gastritis and primary B-cell gastric lymphoma. Lancet 338:1175-1176, 1991
Zucca E, Roggero E, Delchier JC, et al: Interim evaluation of gastric MALT lymphoma response to antibiotics in the ongoing LY03 randomised cooperative trial of observation vs. chlorambucil after anti-Helicobacter therapy. Proc Am Soc Clin Oncol 19:5a, 2000 (abstr 12)
Liu H, Ruskon-Fourmestraux A, Lavergne-Slove A, et al: Resistance of t(11;18) positive gastric mucosa-associated lymphoid tissue lymphoma to Helicobacter pylori eradication therapy. Lancet 357:39-40, 2001(Andr谷s J.M. Ferreri, Maur)
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
First Department of Medicine, Semmelweis University, Budapest, Hungary
ABSTRACT
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每eradicating antibiotic therapy is associated with tumor regression in ocular adnexal lymphomas.
PATIENTS AND METHODS: Nine patients with C psittaci每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每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.
INTRODUCTION
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每associated cutaneous MALT lymphomas,8,9 hepatitis C virus每associated splenic lymphomas with villous lymphocytes,10 and, more recently, Campylobacter jejuni每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每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每positive, marginal-zone, B-cell lymphoma of MALT type of the ocular adnexa treated with the antibiotic doxycycline.
PATIENTS AND METHODS
Study Group
Nine immunocompetent adults with C psittaci每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
Enrolled patients were treated with doxycycline 100 mg, bid orally, for 3 weeks; they did not receive any concomitant antiblastic, antibiotic, or corticosteroid therapy in the period from the start of antibiotic therapy to the maximum response assessment. Doxycycline (100-mg tablets, Bassado; Pharmacia, Rome, Italy) is a tetracycline derivative with uses similar to those of tetracycline. It was preferred to other tetracyclines because of its fairly reliable absorption from the gastrointestinal tract, its long half-life (12 to 24 hours), its wide distribution in body tissues and fluids, and the fact that it can be administered to patients with renal insufficiency.
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.
RESULTS
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每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).
DISCUSSION
The present pilot experience confirms our preliminary observations12 and expands our knowledge on C psittaci每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每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每associated gastric MALT lymphoma, objective responses induced by doxycycline in patients with C psittaci每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每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每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每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每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每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.
Authors' Disclosures of Potential Conflicts of Interest
The authors indicated no potential conflicts of interest.
Acknowledgment
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 Sanit角, Rome, Italy) for his sustained scientific collaboration.
NOTES
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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