Prospective Study of Frontline Antibiotics Therapy in the Treatment of Early-stage HP-negative Gastric MALT Lymphoma

Gastric mucosa-associated lymphoid tissue (MALT) lymphoma (marginal zone B-cell lymphoma) is the most common extranodal MALT lymphoma. Since the association between Helicobacter pylori (HP) infection and gastric and duodenal ulcers, the administration of antibiotics plus proton pump inhibitors (PPIs) eradicating HP has been reported to cure the disease of ulcers and decrease the incidence of subsequent gastric cancer. The prevalence of HP-positive gastric MALT lymphoma has decreased over the last two decades, whereas that of HP-negative gastric MALT lymphoma has increased. For example, Raderer et al. showed an increased prevalence of HP-negative infection in patients with gastric MALT lymphoma diagnosed after 2004 compared with those diagnosed before 2004 (31.2% versus 18%; a total of 97 cases). Mendes et al. also found that the prevalence of HP-negative infection in gastric MALT lymphoma diagnosed between 2005 and 2013 was 67.6% (25/37).

In contrast to most HP-positive gastric MALT lymphomas that respond to frontline HP eradication therapy (HPE), the efficacy of frontline antibiotics in the treatment of HP-negative gastric MALT lymphomas remains ambiguous. Several case series reported that a proportion of patients with localized HP-negative gastric MALT lymphoma can be cured by a frontline HPE-like regimen. Between January 1, 2005, and June 30, 2014, we reviewed 25 patients with newly diagnosed stage IE/IIE1 primary HP-negative gastric MALT lymphoma who underwent frontline HPE-like regimen, which consist of 500 mg of amoxicillin administered four times a day (or 1000 mg of amoxicillin administered twice a day), 500 mg of clarithromycin administered twice a day, and 20 mg of omeprazole or 30 mg lansoprazole administered twice a day for 2 weeks as frontline treatment. An HP-negative status was defined as total negative results on histology (including HP, atrophic gastritis, and intestinal metaplasia), a rapid urease test, a 13C urea breath test, and serology. Based on the criteria of Groupe d'Etude des Lymphomes de l'Adult (GELA) for tumor response, we observed antibiotic responses in 9 (36.0%; 95% confidence interval \[CI\], 17.2-54.8%; complete remission \[CR\], n = 8; partial remission \[PR\], n = 1) out of 25 patients, and the median time to a CR was 7 months (95% CI, 0.1-13.9 months). Of 20 patients receiving serum immunofixation electrophoresis (IFE) assessments, immunoglobulin M lambda monoclonal gammopathy was detected in 3 (25.0%) of the 12 antibiotic-unresponsive tumors, but not in the 8 antibiotic-responsive tumors (p = 0.242). At a median follow-up of 51.0 months (95% CI, 34.7-67.3 months), all patients with responsive tumors after HPE therapy were alive and free of lymphomas and progression.

In addition to eradicating HP and HP-like bacteria, clarithromycin has been reported to have an effect in the cure of a proportion of extragastric MALT lymphoma. Ferreri et al. reported that high-dose clarithromycin (2 g a day for 14 days for each course) resulted in a CR rate of 26.9% in patients with relapsed or refractory extranodal MALT lymphoma. In a B-cell lymphoma cell line derived from a BALB/c mouse model, O'Hara et al. showed that clarithromycin inhibited cell viability and induced apoptosis through down-regulating Bcl-2 expression. Mizunoe et al. showed that macrolides, either clarithromycin (100 ug/ml) or azithromycin (100 ug/ml), caused apoptosis of activated lymphocytes via Annexin-V analyses, and the effect of augmentation of apoptosis is through attenuation of Bcl-xL expression. Another macrolide, erythromycin, was found to have an inhibitory effect on the proliferation of human Jurkat T cells, and a possible mechanism is the down-regulation of NF-kB expression. In CD4+ T-cells, azithromycin effectively inhibited cell proliferation and cytokine secretion through down-regulation of the activity of mammalian target of rapamycin (mTOR). The aforementioned immunosuppressive effect on CD4+ T-cells was also observed at a higher concentration of clarithromycin (40 mg/L). At 40 mg/L of azithromycin and clarithromycin, secretion of cytokines, such as IL-2, IL-10, IL-13, IL-17, and IFN-gamma, was reduced in their study.

For patients with antibiotic-unresponsive MALT lymphoma, several new drugs, including bendamustine, bortezomib, and new compounds of alkylating agents, have been demonstrated to provide approximately 50% ORR but result in grade III/IV myelosuppression. Therefore, it is important to seek biological agents that provide excellent efficacy and result in fewer adverse effects for these patients (since most patients are older). Considering that most patients with MALT lymphoma are newly diagnosed at an older age, our pilot study evaluates the efficacy of low-dose single chlorambucil or low-dose single cyclophosphamide in patients with antibiotic-unresponsive MALT lymphoma. We reported that a single low-dose cyclophosphamide (50 mg daily for 21 days, every 28 days) alone resulted in the ORR of 44.4 % in 9 patients with antibiotic-unresponsive MALT lymphoma. In addition to killing lymphoma cells, single low-dose cyclophosphamide (i.e., 25-100 mg in adults), is an option for restoring immune response in patients with advanced cancer. The main mechanism of cyclophosphamide in counteracting immunosuppression in cancer is the result of selective suppression of regulatory T cells (Tregs) and restoration of T and natural killer (NK) effector functions.

Considering that clarithromycin and low-dose cyclophosphamide are found to have anti-tumor effects and immune-modulatory effects in MALT lymphoma in our and other investigators' results, we speculated that combined clarithromycin and low-dose cyclophosphamide can increase the ORR rate as well as durable tumor remission and decrease aggressive chemotherapy-related adverse effects in antibiotic-unresponsive HP-negative gastric MALT lymphoma. Therefore, in this proposal, we will design a prospective phase II study to evaluate the treatment efficacy of frontline bismuth-based quadruple therapy of HPE regimens followed by 4 weeks of clarithromycin in patients with HP-negative gastric MALT lymphoma. In addition, we investigate whether the combined clarithromycin and low-dose cyclophosphamide (combined regimen: CAMC) is an effective regimen for targeting patients with antibiotic-unresponsive HP-negative gastric MALT lymphoma. We will explore the potential biological markers to predict the antibiotic's responsiveness and the responses of CAMC in this project.

We hypothesized that a proportion of patients with localized HP-negative gastric MALT lymphoma may well respond to frontline HPE and remain lymphoma-free for the long term. In addition, we hypothesized that for patients with antibiotics-unresponsive HP-negative gastric MALT lymphoma, the administration of a combination of clarithromycin and low-dose cyclophosphamide (CAMC) could result in tumor regression through the direct anti-neoplastic effects and the indirect immune modulatory effect.

Therefore, in this proposal, we will design a prospective phase II study to evaluate the treatment efficacy of frontline HPE, consisting of two weeks of quadruple HPE regimen, including esomeprazole 40 mg 1# bid PO Days 1-14, bismuth tripotassium dicitrate 300 mg 1# qid PO Days 1-14, tetracycline 500 mg 1# qid PO Days 1-14, and metronidazole 500 mg 1# tid PO Days 1-14, followed by four weeks of clarithromycin in patients with stage IE/IIE1 HP-negative gastric MALT lymphoma. In addition, we will treat patients who did not respond to HPE using combination regimens of CAMC.

• The clinical outcomes of HP-negative gastric MALT lymphoma after antibiotics treatment and low-dose cyclophosphamide and clarithromycin [Time frame: 3 years]