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Bortezomib is a proteasome inhibitor with pleotropic antitumor effects, including putative activity targeting NF-κB. Elevated levels of NF-κB DNA binding have been documented in HL and NF-κB activity is thought to mediate resistance to standard therapeutic approaches. Despite this rationale, and significant activity in non-Hodgkin lymphoma (NHL), the experience to date with bortezomib in HL has been disappointing. Studies in solid tumor lines have suggested that bortezomib markedly enhances cytotoxicity of gemcitabine, a nucleoside analog with significant activity against HL. Thus, we combined bortezomib with gemcitabine in a prospective trial for patients (pts) with relapsed HL. Eligible pts had confirmed relapsed classical HL and normal organ function. Based upon phase I data in solid tumors (Cancer107:2482), bortezomib (1mg/m2 days 1, 4, 8, 11) was administered prior to gemcitabine (800 mg/m2 days 1 and 8) on a 21 day cycle. Response was assessed following two cycles of therapy; patients could then proceed to stem cell collection and ASCT or continue with gemcitabine and bortezomib for up to 6 cycles. 18 pts (10 female) were enrolled. Median age was 36 (range 19–62). 10 pts had received more than one prior chemotherapy regimen. 4 patients had toxicity requiring gemcitabine to be held during the first two cycles of therapy: 1 for thrombocytopenia, and 3 for grade 3 transaminase elevation. 1 additional pt had grade II transaminase elevation; LFTs returned to baseline after cessation of therapy. Other grade 3 toxicities included neutropenia (n=4), thrombocytopenia (n=2), and 5 single events likely unrelated to therapy. 4 pts experienced transient rash. Therapy administered was 5 cycles (n=1); all other pts 2 cycles. 4 pts (22%; 95% CI 3–42%) responded (1CR; 3PR) after 2 cycles of therapy. 7 pts had PET/CT imaging performed before and after therapy; in 4, standardized uptake values (SUV) following treatment were all ≤ 3.0 and 1 additional pt had significantly decreased SUV following treatment suggesting metabolic response. All 12 pts planned for ASCT eventually received it after salvage; 1 pt required harvest due to inadequate mobilization. Biochemical assays were performed to measure activity of the 20S proteasome pre/post treatment on day 1 and day 8 in all patients. Following the first dose of bortezomib, 16 pts demonstrated significant proteasome inhibition in whole blood compared to baseline (median 50%, range −39% to 77%). Proteasome inhibition on day 8 before and after bortezomib was less, with a median inhibition of 36%. Additionally, EMSA analysis for NF-κB activity in peripheral blood mononuclear cells was performed before and after treatment; there was no correlation between results from this assay and clinical response. In this study, there was no obvious improvement in clinical outcome with the gemcitabine/bortezomib combination over what would be expected with gemcitabine alone (39% RR; JCO18:2615). Moreover, unexpected, severe but reversible transaminase elevations requiring modification of prescribed therapy were noted in 16% of pts. Despite this lack of clinical response, we demonstrated proteasome inhibition occurred in nearly all patients following bortezomib, and there was no correlation between degree of proteasome inhibition in blood and clinical response to therapy. We conclude the combination of gemcitabine and bortezomib should not be pursued in HL, and caution should be exercised when combining these agents in other diseases, including NHL, due to potential for severe liver toxicity.

The marginal zone lymphomas are a recently defined group of related diseases likely arising from a common cell of origin, the marginal zone B cell. The clinical presentation varies; data on therapy for subtypes other than gastric MALT has been largely limited to retrospective case series. We therefore undertook this prospective phase 2 study of fludarabine 25 mg/m2 for 5 days with rituximab 375 mg/m2 on day 1 for the treatment of marginal zone lymphomas. To be eligible, patients were required to have newly diagnosed or relapsed, histologically confirmed MALT, marginal zone lymphoma, or a CD5/CD10 negative low-grade B cell lymphoproliferative disorder. They could not be candidates for curative local therapy. From 2004 to 2007, 26 patients were enrolled with a median age of 64 (31–84) and a median time from diagnosis to treatment of 1.6 months. This was the initial therapy for 21 of 26 patients (81%). Seven were diagnosed with MALT lymphomas (27%), 12 with nodal marginal zone lymphomas (46%), 3 with splenic marginal zone lymphoma (12%) and 4 with CD5/10 negative low-grade lymphoproliferative disorders (15%). FISH for BCL-6, trisomy 3, MALT1 and chromosome 1 rearrangements was attempted on 18 available tissue biopsies. Of these, four were normal, three showed BCL-6 rearrangement with other abnormalities, four had chromosome 3 abnormalities, two MALT1 rearrangements and one chromosome 1 abnormality. The majority of patients had stage IV disease (18; 69%), with 5 stage 3, 2 stage 2 and 1 stage 1E disease. Of the 23 patients who have completed therapy, 18 completed at least 4 cycles (78%), with 12 patients completing the planned 6 cycles (52%). Nine patients discontinued therapy due to unacceptable toxicity (39%), six for hematologic toxicity, two for grade 3 rash and one for a delayed grade 3 reaction to rituximab. Of 26 patients evaluable for toxicity, 46% developed grade 4 toxicity (solely hematologic), and 35% grade 3 toxicity. Grade 3–4 toxicities included: neutropenia 14 (54%), thrombocytopenia 5 (19%), febrile neutropenia 2 (8%), rash 3 (11%), myositis 1 (4%), allergic reaction 1 (4%). Two delayed opportunistic pneumonias were observed, one Nocardia and one P. jiroveci. The ORR in the 23 patients who have completed therapy and are evaluable for response is 83% (95% CI 61–95%), with 12 patients achieving CR/CRu (52%). Three patients have relapsed. Two patients have died, one due to small cell lung cancer diagnosed after study enrollment, and the other due to urosepsis with bone marrow aplasia. At the median follow-up of 1.8 years, the PFS is 84% (95% CI 68–99%), and OS 94% (95% CI 82–99%). Concurrent fludarabine and rituximab is therefore a highly effective regimen in the treatment of marginal zone lymphoma but one which is complicated by significant hematologic toxicity and allergic hypersensitivity. These toxicities prevented half the patients from completing the planned therapy and were more severe than usually seen in other low-grade lymphomas, emphasizing the need to study marginal zone lymphomas as a separate entity.

The malignant microenvironment of FL, including T cells, macrophages and follicular dendritic cells, contributes to disease pathogenesis and represents a rational therapeutic target. Gene expression profiling and immunohistochemistry studies have confirmed that degree and type of immune response in the malignant FL lymph node impacts clinical outcome. CpG oligonucleotides have pleotropic effects on both the innate and adaptive immune systems, including increased antigen expression, enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) and shifting the immune response in a TH-1 direction. These agents therefore have significant potential to manipulate the immune response to FL, and alter the malignant microenvironment in a favorable way. We combined a CpG oligonucleotide (1018 ISS, 0.2 mg/kg sc weekly x 4 beginning day 8) with standard R in patients (pts) with relapsed/refractory, histologically confirmed FL, and comprehensively evaluated immunological changes following the combination. 22 pts (10 female; median age 55) with FL sensitive or naive to R were enrolled. FcγRIIIA polymorphism status was FF n=9, VF n=12, VV n=1. Treatment was well-tolerated with no significant adverse events attributable to therapy. Baseline and post therapy evaluations were performed on blood and the CpG injection sites in all pts. Compared to baseline, 7 pts demonstrated significant increases in ADCC after therapy using a conventional chromium-release assay. Circulating CD3-positive cells increased in 11 pts. Increased IFN-γrelease by Elispot occurred in 5 pts. 10 pts had significant increases in neutrophils, T cells, and macrophages in skin biopsies of CpG injection sites performed 24 hours post therapy compared with baseline. Pre- and post-biopsies of tumor tissue (including lymph nodes and bone marrow) were performed in 5 pts, and in all cases a significant infiltration of T cells (CD8 > CD4) and macrophages were observed following treatment. Clinical responses per Cheson criteria were observed in 45% of pts; median PFS was 18 months in responding pts, and 9 months for entire group; 12 pts have not yet required further therapy for FL. In conclusion, this group of pts with relapsed FL had favorable clinical outcome despite adverse prognostic factors. The addition of CpG to R had measurable and reproducible local and systemic immune effects in a substantial subset of pts. Unlike other cytokine/antibody combinations, this study is the first to histologically confirm perturbation of the local immune malignant microenvironment following systemic biological therapy of FL.

Immunostimulatory oligonucleotides containing nonmethylated CpG motifs (ISS) have multiple actions on adaptive and innate immune responses, including enhanced antigen presentation and costimulatory molecule expression, stimulation of dendritic cell maturation, and induction of cytokines that result in enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) and Th1 immune responses. Through these mechanisms, we hypothesized ISS might be able to overcome R resistance. Our completed phase I study combining an ISS (1018 ISS, Dynavax Technologies) with R in patients (pts) with relapsed or refractory, advanced stage NHL, demonstrated that the combination is safe, and causes a dose-related increase in the expression of several interferon (IFN)-inducible genes.(Blood 105:489). We have subsequently enrolled 16 pts (median age 60; 9 female) with R-naïve or sensitive FL in a phase II trial to further investigate the efficacy and biological activity of this rational combination. Pts received 4 injections of 1018 ISS (0.2 mg/kg sc) at weekly intervals, beginning at the time of the second of 4 weekly standard R infusions. No grade III/IV toxicities occurred. Genotyping of 13 evaluable pts for the FcγRIII-A 158 polymorphism revealed VF (n=6) and FF (n=7). Flow cytometry of peripheral blood demonstrated a significant expansion of CD3+ cells in 9 pts following 4 weeks of 1018 ISS. When tested with allogeneic stimulation using an EBV transformed B cell line (Mann) in ELISPOT assay, T cell secretion of IFN-γ was increased following 1018 ISS (mean count pre 1018 ISS: 362; mean count post 1018 ISS: 565; p = 0.05), whereas T cell secretion of IL-2 did not change, both consistent with an emerging Th1 immune response. Using a standard chromium release assay, 7/13 pts (54%) demonstrated increases in ADCC of peripheral blood mononuclear cells following ISS compared to baseline (effector:target ratio of 25:1); including 5 pts with FCγRIIIA genotype 158 FF. Skin biopsies were obtained in all patients at baseline, and following ISS therapy at the injection site and a distant site. Following 1018 ISS, 5 pts (42%) had significant skin infiltration with CD8+ lymphocytes; and 5 pts had increased numbers of CD68+ cells at injection sites. Biopsies of tumor were obtained at baseline, and post 1018 ISS treatment when possible, and evidence of CD8+ T cell infiltration within residual FL nodules was observed following R and 1018 ISS therapy. As of 08/05, at a median follow-up of 9 months, only 3 pts have experienced disease progression, one of whom subsequently converted to CRu without further therapeutic intervention. We conclude that the combination of 1018 ISS and rituximab is safe, and demonstrates significant local and systemic biological activity. Moreover, the demonstration of enhanced ADCC in pts with an FCγRIIIA 158 FF genotype (expected poor prognosis following R in pts with FL), the associated development of a Th1 response, and measurable alteration of the tumor microenvironment all suggest ISS could overcome both innate and acquired R resistance mechanisms in pts with FL.