Chlamydia Infection and Lymphomas: Association Beyond Ocular Adnexal Lymphomas Highlighted by Multiple Detection Methods

The pathogenic and therapeutic implications of the relationship between bacterial agents and non–Hodgkin's lymphomas are an emerging and highly debated issue. Helicobacter pylori–associated marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT)-type of the stomach is paradigmatic in this context (1). Among these potentially pathogenic associations, we have shown the presence of Chlamydia psittaci [now reclassified as Chlamydophila psittaci (Cp) ref. 2] infection in 80% of patients with ocular adnexal lymphomas (OAL), mostly of MALT type (3). Our data were supported by results from other groups (4, 5), although some series did not report similar figures (4, 6–11). Within this complex scenario, either different geographic Cp infection prevalence rates or differences in sensibility and specificity of PCR-based detection methods could be considered (12, 13). Importantly, Cp eradication with the antibiotic doxycycline induced objective clinical response in a substantial fraction of patients, further supporting a likely causal role for this infection, and resulted in a safe, inexpensive, and effective therapeutic strategy for OAL patients (14).

Nevertheless, several issues about the relationship between Cp infection and lymphomas still remain to be clarified (15): to validate additional Cp detection tools corroborating PCR data on Cp presence; to identify the cellular target of Cp infection within lymphomatous tissue, to better define the Cp role in lymphomagenesis; to investigate whether Cp infection is restricted to OAL or is detectable among the other major types of lymphoma. This last issue is clinically relevant, also in the light of the reported response to the antibiotic doxycycline obtained in a patient with Cp-associated diffuse large B-cell lymphoma (DLBCL) of the bronchus (16).

To address these questions, we investigated chlamydial infection with multiple detection methods, complementary to Cp-DNA based approach and searched for the cell type(s) that could constitute the main chlamydial reservoir in lymphomatous tissue. The prevalence of Chlamydiae infection in human lymphomas was assessed through a screening on 205 cases of nodal and extranodal lymphomas, and in a variety of nonneoplastic lymphoid tissues. We report the morphologic evidence suggesting that Cp within lymphoma-related monocytes/macrophages is a viable microorganism. Cp turned out to be present not only in OAL, but also in an unexpected proportion of nodal and extranodal lymphomas, thus raising important clinical and therapeutic considerations.

Patient population and controls. The patient population was constituted by 35 OAL patients (21 of whom not previously reported; ref. 3) in which we did both molecular studies and immunohistochemistry (IHC) for Chlamydia. Immunofluorescence (IFS), electron microscopy, and laser-capture microdissection were done in 4, 2, and 2 cases, respectively (Table 1).

Moreover, to define the Cp DNA prevalence among different lymphoma categories, we did PCR on 205 nonconsecutive cases of immunocompetent patients cases with nodal or extranodal lymphomas different from OAL, retrieved from the Pathology Unit files at the San Raffaele H Scientific Institute of Milan. Fifty-eight cases were nodal lymphomas (including 27 cases of Hodgkin's lymphoma) and 147 cases occurred at different extranodal sites, including 12 cases with a T-cell immunophenotype (Table 2). Age-matched controls were similarly selected and represented by 31 patients with nonspecific tonsillitis, 36 with nonspecific reactive lymphadenopathies, 7 nonneoplastic spleens, and 61 nonneoplastic skin biopsies.

PCR and sequencing. DNA was isolated from formalin-fixed, paraffin-embedded tissues according to standard procedures (3). Cp DNA analysis was done by PCR amplification of three different genomic regions: portion of the 16S rRNA gene and the 16S-23S spacer region (TETR-PCR; refs. 3, 17), outer membrane protein (ompA; ref. 18), and heat shock protein 60 (19). All PCR products were analyzed and sequenced as previously reported (3, 20). Amplification of the β-globin gene was carried out as a control (3).

IHC and IFS. IHC was done in 54 cases encompassing lymphomatous and nonneoplastic disorders previously evaluated by TETR-PCR (Table 3). The comparison between results from PCR and IHC for Chlamydia detection in this subgroup was done to investigate the value of IHC as a surrogate alternative/complementary tool to PCR techniques in the detection of Chlamydiae. Antigen retrieval was carried out with 0.1 mol/L citrate buffer, at pH 6.0. For Chlamydia IHC, a mouse monoclonal antibody specific for Chlamydia lipopolysaccaride (C-LPS), clone 6J12 contained in the “Imagen Chlamydia test” (Argene), at a working dilution 1:150, and the Novolink detection kit (Novocastra) with 3,3′-diaminobenzidine as chromogen were used. A section of parrot's lung infected by Cp was used as positive control; omission of primary antibody was used as negative control. Results were blindly evaluated by M.P and C.D.

Chlamydia detection by IFS was done with the Imagen Chlamydia test directly in four OAL cases where frozen tissue was available. In two C-LPS–positive cases at IHC, double IFS was done to identify the lineage of LPS positive cells, using the following directly conjugated Zenon Alexa 555 antibodies (Molecular Probes/InVitrogen): monoclonal mouse anti-human CD3, CD20, CD34 antibodies (Novocastra), and CD68R (PGM-1; DAKO), used at conventional working dilutions. Sections were counterstained with 4′,6-diamidino-2-phenylindole. Microscopic observation was done using a Nikon 90 instrument and images were captured by Nikon DS-2MBW digital camera and NIS software (Nikon Instruments Italia S.p.a.).

Laser capture microdissection and molecular analysis on micro dissected cells. Laser capture microdissection was done by Pixcell II LCM system (Molecular Devices Sunnyvale) microdissection microscope, according to manufacturer's recommendations. Three samples known to be positive for Cp DNA and two samples known to be negative for Cp DNA PCR were micro dissected (Table 4). Three distinct types of cell populations were dissected from each slide: (a) a pool of 200 C-LPS–positive cells, microdissected at single-cell level, which showed invariably monocyte/macrophage-like morphology; (b) a pool of 200 C-LPS–negative cells, microdissected at single-cell level, including different cell types (i.e., neoplastic lymphocytes, endothelial cells, and stromal cells); (c) the whole residual tissue (few C-LPS–positive cells were still present in this tissue). These three samples per each case were assessed by TETR-PCR analysis, which was done after DNA preamplification (I-PEP; ref. 21).

Electron microscopy. Two cases of Cp-positive OAL were investigated to visualize, at the ultrastructural level, chlamydial elementary bodies within lymphomatous tissue. Samples were fixed and treated as previously described (22) and examined under a Zeiss EM900 transmission electron microscope.

Statistical analysis. The comparison of the results obtained with PCR and IHC techniques and distribution of Cp infection among different lymphoma categories were analyzed using the χ² or Fisher exact test for categorical variables. All the probability values were two sided, with an overall significance level of 0.05. Analyses were carried out using the Statistica 4.0 statistical package for Windows (Statsoft, Inc.).

Cp detection in OAL. Among the 35 OAL patients evaluated, Cp was detected by TETR-PCR in 26 patients (74%; Table 1). The presence of Cp DNA was further confirmed with a PCR for the detection of heat shock protein 60 gene because the DNA sequence obtained by TETR-PCR amplification may not discriminate Cp from the closely related Chlamydophila abortus (19). This approach was applied, as a proof of principle, to nine cases of TETR-positive Cp-related lymphomas from this series, confirming the specificity of the amplified fragments. Chlamydophila abortus DNA was not detected (Table 1). Three (9%) of 32 assessed OAL patients had concomitant hepatitis C virus infection (seropositivity plus seric hepatitis C virus DNA); 2 hepatitis C virus–positive patients had Cp infection and 1 of them had multifocal extranodal involvement.

Monocytes/macrophages are carriers of Cp. Cells immunoreactive for antichlamydial LPS in IHC were identified in 28 of the 34 PCR-positive cases investigated (82%; Table 3). These cells were rare and irregularly scattered throughout the section, never formed aggregates, and displayed a monocyte/macrophage-like morphology. Immunoreactivity occurred in form of cytoplasmic coarse granules with variable size (Fig. 1A-D). No additional cells were stained with anti–C-LPS antibody. The same specific distribution was confirmed in the two lymphomatous cases studied by IFS (Fig. 1E). In addition, double IFS staining confirmed that Chlamydia signal was detectable within CD68-positive monocytes/macrophages (Fig. 1F) but not in B-lymphocytes, T-lymphocytes, and endothelial cells.

Laser-capture microdissection confirms immunohistochemical results. The presence of Cp DNA in the pool of microdissected C-LPS–positive cells was confirmed by TETR-PCR and by direct sequencing of these PCR products in all cases (Table 4). C-LPS–negative microdissected cells were negative for Cp DNA, excluding the presence of Cp in neoplastic, endothelial, and stromal cells. In the scraped tissues of positive cases, Cp DNA was detected, consistently with the presence, after microdissection, of few LPS-positive cells in the residual tissues (data not shown). Microdissected cells obtained from samples not immunoreactive for anti–C-LPS antibody, used as negative controls, did not contain Cp DNA (Table 4); in these cases, amplification of β-globin gene confirmed the presence of DNA.

IHC is a “surrogate marker” for Cp detection. Concordance between Chlamydial C-LPS–positive and TETR-PCR–positive cases for Cp DNA was high, suggesting that IHC could be used as a surrogate marker for Cp infection (Table 3). In fact, of the 54 cases of lymphomas and controls investigated for PCR/IHC correlation, 23 (43%) cases were PCR+/IHC+ and 15 (28%) were PCR−/IHC−, with a concordance rate of 70% (P = 0.001). Eleven (20%) and 5 (10%) cases were “false negative” and “false positive” cases, respectively. Concordance rate was higher among lymphomas (34 of 45, 76%) in comparison with benign lesions (4 of 9, 44%; P = 0.06). Within lymphomas, discordances were present in 9 (25%) of 26 marginal zone B-cell lymphomas and two (22%) of the 9 cases with other histological subtypes (P = 0.86).

Cp is viable within monocytes/macrophages. The transmission electron microscopy examination confirmed the presence of Chlamydia within mononuclear cells with relatively abundant cytoplasm, which were consistent with monocytes/macrophages. Clusters of intact chlamydial elementary bodies were seen mainly in the host cell cytoplasm but also in the extracellular space (Fig. 2A-B).

Cp prevalence in extra-OAL. By TETR-PCR, chlamydial DNA was detected in 23 of 205 (11%) cases of lymphomas (Table 2). Cp (18 cases, 9%) was significantly more common than Chlamydophila pneumoniae (4 cases) and Chlamydia trachomatis (1 case) in lymphoma samples (P < 0.00001). The prevalence of Cp was similar both in lymphomas (18 cases, 9%) and reactive skin biopsies and lymphoid tissues used as controls (8 cases, 6%; P = 0.33).

In the lymphoma group (Table 2), no significant difference in Cp prevalence was observed between extranodal (10 cases, 7%) and nodal B-cell lymphomas (6 cases, 10%; P = 0.49). In extranodal B-cell lymphomas, Cp DNA was present in one (2%) of the 62 MALT lymphomas and in 9 (15%) of the 73 DLBCL investigated, respectively (P = 0.01). The presence of Cp DNA in extranodal lymphomas was not randomly distributed, with the highest prevalence in skin (3 of 9 cases, 33%) and Waldeyer's ring (3 of 17 cases, 18%; Table 2). Positivity in cutaneous DLBCL was significantly higher than those observed in nonneoplastic skin biopsies (33% versus 5%; P = 0.02), whereas Cp positivity in DLBCL of Waldeyer's ring was not significantly higher from those observed in reactive tonsillitis (18% versus 6%; P = 0.33).

The nested “touchdown” PCR specific for Cp major outer membrane protein gene (ompA; ref. 18) corroborated the TETR-PCR specificity results, showing similar figures in terms of Chlamydophila pneumoniae and Cp detection rates.

In this study, we provide, with multiple detection methods, new insights on the link between chlamydial infection and lymphomas. We showed, for the first time, by electron microscopy, the presence of intact chlamydial elementary bodies, the viable forms of this bacterium, in monocytes/macrophages associated to lymphomatous tissue. Using two additional discriminative tests directed to different and independent regions of Cp genome, (ompA and heat shock protein 60), we corroborated the diagnostic value of TETR-PCR in Cp DNA detection in OAL as well as in nodal or extranodal lymphomas different from OAL. Furthermore, with the aid of laser-capture microdissection and subsequent molecular analysis, we showed that IHC is a reliable, additional tool for Chlamydia detection and may be regarded as a complementary diagnostic test within this scenario. Moreover, electron microscopy, IHC, and IFS allowed the identification of the main cell reservoir of Chlamydia infection in monocytes/macrophages, both in nonneoplastic and lymphomatous tissues. Taking advantage from these complementary approaches, we evaluated a large series of lymphomas, showing that Cp DNA is detectable in a considerable fraction of lymphomas arising in organ different from ocular adnexa; in particular, DLBCL from skin and Waldeyer's ring were the entities most frequently associated with this infection.

The direct visualization of intact Chlamydia elementary bodies by electron microscopy is consistent with the presence of viable Cp within monocytes/macrophages associated with lymphomatous tissue (Fig. 2). The viability of Cp is further substantiated by response to doxycycline (3, 23) and its successful isolation and in vitro growth from both conjunctival swabs and peripheral blood mononuclear cells of patients with Cp-associated OAL (24). These associations seem not to reflect simply a high prevalence of Cp infection in the Italian population, considering that both our previous retrospective analysis (3) and a more recent, prospective case-control study (24) revealed the virtual absence of Cp infection in two large groups of blood healthy donors used as controls.

In humans, Cp infection is mainly known as the causative agent of psittacosis, an acute respiratory disease. In lymphoma patients, Cp can be detected both in tumor tissue and peripheral blood mononuclear cell, either at diagnosis, at relapse, and after a long follow-up (3, 23), thus confirming that this microorganism may be associated with a chronic, although clinically unapparent, infection. The detection of the DNA of such bacteria in peripheral blood mononuclear cell of 40% of patients with Cp-positive OAL indicates that this is often a systemic infection (3, 23), and provides the rationale for the search of possible alternative sites where Cp could contribute to lymphoma development. Our figures show that a 10% prevalence rate of Cp infection was similar among the nodal lymphoma entities investigated (i.e., follicular and DLBCL; Table 2). The most important differences in prevalence rates were appreciable within the extranodal B-cell lymphoma subgroup. In fact, one third of cutaneous DLBCL and about one fifth of Waldeyer's ring DLBCL contained Cp DNA in tumor tissue. Although further confirmations in larger series are needed, these observations are intriguing because they suggest a preferential distribution of this microorganism in lymphomas occurring at extranodal organs considered as “first barriers” to air-transported antigen exposure. It is worth considering that Chlamydiae are commonly spread by aerosol or by contact (25). Intriguingly, in these sites, Cp infection was more common among DLBCL with respect to marginal zone lymphoma (MZL), at a variance with the higher prevalence of this infection in OAL of MALT type. At present, we do not know whether Cp infection occurring at extranodal sites different from ocular adnexae may favor the rapid evolution of indolent lymphoproliferations to large cell lymphomas. Despite this uncertainty, our results may have important therapeutic implications, in the light of the long-lasting remission of a case of Cp-associated DLBCL of the bronchus after doxycycline treatment alone (16). This therapeutic success may parallel the clinical remission reported in Helicobacter pylori–associated high-grade gastric lymphomas treated with antibiotics (26) and may provide the rationale to extend to additional anatomic regions and histotypes the encouraging data on the clinical response induced by doxycycline as first-line treatment in patients with Cp-positive OAL (14). Nevertheless, the use of doxycycline as exclusive treatment for Cp-positive lymphomas of a histotype diverse of MALT-type one remains an investigational approach, which must be carefully assessed by properly designed trials.

From a technical standpoint, TETR-PCR proved to be the best method currently available for the molecular detection of Cp DNA. In fact, the specificity of this approach was further validated by the use of a PCR for ompA, which can discriminate between Cp and Chlamydiophila pneumoniae. Since our original report (3), sequence database have been continuously updated and, at the present time, the fragment amplified by our TETR-PCR could be identical to a portion of Chlamydophila abortus DNA. However, all our cases tested for heat shock protein 60 protein showed with no exception a sequence unambiguously belonging to Cp, thus confirming the specificity of our results. Moreover, the adjunctive diagnostic role of IHC also emerges from the present study. In fact, a significant 70% concordance rate between this technique and TETR-PCR was found, mostly in lymphoma cases, which suggests that IHC could be routinely considered a complementary, fast, and practical method in the detection of chlamydial infection in lymphomas. Notwithstanding, positive results in IHC techniques do not represent a formal proof of Cp infection considering that available monoclonal antibodies detect genus-specific chlamydial LPS, requiring confirmation by PCR and direct sequencing, which remain critical for bacterial strain definition. As well, attention should be paid when evaluating these immunostains due to both the small number and the scattered distribution of C-LPS–positive macrophages within lymphomatous tissues (Fig. 1A-D).

In conclusion, Cp is present as a viable microorganism within lymphomas and, in particular, in extranodal B-cell lymphomas preferentially arising in external-antigen exposed organs, where monocytes/macrophages act as Cp main reservoirs. Together with PCR, IHC may be an important additional diagnostic tool for the evaluation of the presence of chlamydial infection in lymphomas, particularly in view of potential therapeutic implications.

No potential conflicts of interest were disclosed.

We thank Michele Reni, M.D. and Michele Spina, M.D. for critically reading the manuscript, Professor Emilio Berti for providing non-neoplastic skin biopsies, and Mosè Barbaro for photographic assistance. This paper is dedicated to the memory of Sergio Vigo, whose technical skillness has been greatly appreciated.