ALBERT

Description: Autologous stem cell transplantation (ASCT) leads to induction of molecular remission in mantle cell lymphoma (MCL). A large proportion of the patients, however, relapse after ASCT. Increasing levels of minimal residual disease (MRD) in consecutive BM samples after ASCT have been observed prior to relapse by us and others, whereas patients in continuous clinical remission have remaining low levels of MRD. In the present 2nd Nordic MCL Phase 2 trial we aimed to direct preemptive treatment to patients in clinical remission but with increasing levels of MRD at high risk of relapse after ASCT. We used a combined standard nested and quantitative real-time PCR analysis to estimate MRD levels (Andersen et al, 2002, Exp. Hematol). According to the protocol consecutive BM and PB samples were procured and shipped for central PCR analysis every 3–4 months post-transplant. Preemptive therapy consisted of four weekly doses 375 mg/m2 of Rituximab. Of the 161 MCL cases included in the trial 81 cases underwent ASCT and had PCR markers available. CR-rate after ASCT was 92%. In total 852 post-transplant BM/PB samples were monitored for MRD. 47 of 81 (58%) cases remained standard nested PCR negative after ASCT for a median follow-up time of 2.7 years (range: 0.14–5.7 years). In 4 (8%) of these a clinical relapse was observed without any PCR detectable MRD present in BM or PB after ASCT, including at time of clinical relapse. In 34 (42%) of 81 cases standard nested PCR was positive at least once after ASCT. The majority of the PCR positive cases (26/34 cases, 76%) converted from standard nested PCR negative to positive during post-transplant follow-up, thus, these cases relapsed molecularly. 8 (24%) of 34 cases remained standard nested PCR positive after ASCT. In these, a rising level of MRD was detected by real-time PCR analysis in 4 cases. The remaining 4 cases either had stable low or declining levels of MRD. Of the 30 cases which relapsed molecularly 8 cases simultaneously underwent a clinical relapse leaving no therapeutical window for preemptive treatment. One case refused preemptive treatment. All molecular relapse occurred within 3 years after ASCT, except in 1 case. In total 21 cases have received preemptive treatment. 19/21 (90%) cases became standard nested PCR negative (18 cases) or reduced to low MRD level (1 case). 2/21 cases remained PCR positive and relapsed after 3 and 6 months, respectively. 16/21 cases remain in clinical CR for a median follow-up time of 1.4 years after preemptive treatment (range: 0.25 to 3.8 years) and 5/21 cases have relapsed. Of the latter cases the 3 of the 5 became PCR negative for 6–9 months before relapse. Of note, two cases have received preemptive treatment twice after a second molecular relapse after which they again became PCR negative. Preemptive treatment has not been reported in lymphoma before. Our results in MCL suggest that the large number of cases who remain in molecular remission after intensified ASCT may be followed by MRD monitoring and treated at molecular relapse instead of receiving maintenance therapy. However, 4 of these cases relapsed. Here, more than PCR methods are needed for early stage disease detection. Our results indicate that preemptive treatment using Rituximab can successfully reinduce molecular remission and prolong time to relapse. Finally, more patients may have PCR markers available by applying frozen diagnostic lymph node material.

Description: Background: Outcome for elderly patients with Hodgkin’s lymphoma is not as good as for younger patients, partly due to inferior capability to tolerate appropriate chemotherapy. There is no consensus on standard treatment. In 2000 we introduced CHOP-21 as standard chemotherapy for elderly Hodgkin’s lymphoma patients at our institution with the aim to improve treatment results. CHOP-21 is known to be well tolerated in older patients with non-Hodgkin’s lymphoma and contains active drugs for Hodgkin’s disease. Patients and methods: Twenty-nine consequtive patients admitted to our institution from 2000–2004, 60 years and older with Hodgkin’s lymphoma were included in this retrospective survey. Stage I/IIA patients received 2–4 cycles of CHOP-21 followed by 30–35 Gy involved field radiotherapy. Stage IIB-IV patients were treated with 6–8 cycles of CHOP-21. Selected cases received 30–35 Gy involved field radiotherapy against residual tumors. Results: The median age was 71 years (range 60–91). Sixty-two percent presented in stage IIB-IV and 38% in stage I-IIA. Forty-five percent of the early stage patients had bulky disease and/or elevated SR. For the advanced stage patients IPS score of 2–3 was the most common category (72%). B-symptoms occurred in 52% of the total population. Nodular sclerosis was the most frequent histology subgroup (38%), followed by mixed cellularity (17%). Fifty-five percent had co-morbidity prior to treatment, cardiac conditions were most common. Two cases of treatment-related deaths were seen (7%). The most common toxicity during therapy was febrile neutropenia (31%). Treatment modifications were necessary in 38% of cases. The complete response rate after CHOP +/− radiotherapy was as high as 93%. Median follow-up for the whole population is now three years. Five patients have relapsed, and four have died from Hodgkin’s lymphoma. Total overall survival (OS) at three year was 80%, and better for the stage I-IIA subgroup (90% versus 60%). Total Hodgkin-specific survival (HSS) at three years was 73%, and 80% versus 60% for early and advanced stages, respectively. No relapses have occurred after 2 years. Outcome with CHOP +/− radiotherapy for Hodgkin’s lymphoma patients 60 years and older Response 29 pts (%) NE = not evaluable, patients died after one cycle of CHOP. CR 27 (93) PR 1 (3) ORR 28 (97) NE 1 (3) Relapsed 5 (18) Cause of death Hodgkin’s lymphoma 4 (14) Toxicity 2 (7) Other 1 (3) Conclusion: The data shows that CHOP-21 is a well tolerated and highly effective first line treatment for elderly patients with Hodgkin’s lymphoma. Of particular interest were the findings that advanced stage patients did so well and that no relapses have been seen after two years.

Description: Mantle cell lymphoma (MCL) is considered incurable. Intensive immunochemotherapy with stem cell support has not been tested in large, prospective series. In the 2nd Nordic MCL trial, we treated 160 consecutive, untreated patients younger than 66 years in a phase 2 protocol with dose-intensified induction immunochemotherapy with rituximab (R) + cyclophosphamide, vincristine, doxorubicin, prednisone (maxi-CHOP), alternating with R + high-dose cytarabine. Responders received high-dose chemotherapy with BEAM or BEAC (carmustine, etoposide, cytarabine, and melphalan/cyclophosphamide) with R-in vivo purged autologous stem cell support. Overall and complete response was achieved in 96% and 54%, respectively. The 6-year overall, event-free, and progression-free survival were 70%, 56%, and 66%, respectively, with no relapses occurring after 5 years. Multivariate analysis showed Ki-67 to be the sole independent predictor of event-free survival. The nonrelapse mortality was 5%. The majority of stem cell products and patients assessed with polymerase chain reaction (PCR) after transplantation were negative. Compared with our historical control, the Nordic MCL-1 trial, the event-free, overall, and progression-free survival, the duration of molecular remission, and the proportion of PCR-negative stem cell products were significantly increased (P 〈 .001). Intensive immunochemotherapy with in vivo purged stem cell support can lead to long-term progression-free survival of MCL and perhaps cure. Registered at www.isrctn.org as #ISRCTN 87866680.

Description: CHOP – based chemotherapy for aggressive lymphomas in patients with age-adjusted International Prognostic Index (IPI) score of 2–3 resulted in a historical 3-year progression free survival of approximately 30% in a previous Nordic phase III study. The aim of the present study is to determine whether an intensified regimen with chemoimmunotherapy and CNS prophylaxis improves outcome. Methods. From October 2004-June 2008 patients in Norway, Finland, Sweden and Denmark were included in a phase II study. Inclusion criteria: Age 18–64 years, newly diagnosed de novo DLBCL or FL grade III, no clinical sign of CNS disease and negative CSF cytology/flow cytometry by lumbar puncture, HIV negative, WHO performance status grade 0–3, adequate organ functions. Schedule: Six courses of rituximab and CHOP with the addition of etoposide 100 mg/m2 day 1–3 by i.v. route given every 14 day. Pegfilgrastim 6 mg sc. day 4 of each cycle. One course of cytarabine 12 g/m2 (6 g/m2 for patients 60–64 years). One course of methotrexate 3 g/m2 (1.5 g/m2 for patients 60–64 years). Biopsy and/or 18FDG PET/CT exam. of residual masses after fulfilled therapy was recommended, but not mandatory. Radiotherapy was given to residual masses of uncertain significance. Results. Demographic data: 160 patients were included (99 males). Median age: 54 years (range 20–64). Histology: DLBCL: 148, FL grade 3: 12. Age adjusted IPI score: 2: 120; 3: 40. Stage 3–4: 154 patients. LDH elevated: 154 patients. Performance status 2–3: 53 patients. B-symptoms were registered in 40% of the patients, more than one extranodal site in 23%, and bulky lesions (≥ 10 cm) in 43%. Data on toxicity and response rates were registered for 127 patients by Aug. 1st 2008 after the end of therapy and will be available for all patients by Dec 1st. Toxicity: Two toxic deaths were registered, one after large bowel perforation and one after an acute toxic necrosis of the liver. Hematological toxicity grade 4 was seen in 78% of the patients, infection grade 4 in 8%. Seven patients (5.5%) had major protocol deviations due to toxicity. Response: Two of the patients were non-responders at evaluation after 3 courses and were taken off therapy. Radiotherapy was given to 25 patients (20%). Response rates at end of therapy: CR: 54 (43%), CRu: 38 (30%), PR: 27 (21%), SD: 1 (1%), PD: 7 (5%). The majority (23/27) of the PR patients were considered to have residual masses and not viable tumor tissue and were observed without further treatment. Conclusions: Preliminary data indicate a low rate of toxic deaths despite intensive therapy. Remission rates are highly satisfactory for this subgroup of patients. Data are too premature for survival analysis at this time point.

Description: Mantle cell lymphoma (MCL) is considered incurable, with a median survival of 4 years. Intensive immunochemotherapy and autologous stem-cell (ASC) support has appeared promising in small patient cohorts, but has not been tested in large, consecutive series. Here we report the final results of the 2nd Nordic MCL (MCL2) trial after a median of 3 years follow-up from study entry. Methods: This unrandomized phase-II trial included 159 untreated patients younger than 66 years, 84% stage IV, 128 with classical, 31 with blastoid/pleomorphic cytology. Following 6 cycles of intensive induction immunochemotherapy with alternating cycles of rituximab (R) + maxi-CHOP and R+ high-dose AraC, responders received BEAM/BEAC with in-vivo purged (R) ASC support. Results: 153 patients (96%) responded to induction therapy with CR in 55% and PR in 41%. The 5-year event-free (EFS) and overall survival (OAS) are 63% and 74% respectively on intention-to-treat, and the 144 (91%) responders who completed treatment had 72% 5-year response duration, with plateaus emerging in all three curves at these levels. Figure Figure There were 6 treatment-related deaths (3,8%). Of 77 patients with available primers, 90% had become PCR-negative two months posttransplant; those who remained PCR-negative more than 1 year posttransplant had a significantly longer clinical response duration than patients who did not (P

Description: Abstract 932 The Nordic Lymphoma Group has since 1996 conducted three consecutive phase II trials for front-line treatment of MCL patients ≤ 65 years of age. The first protocol (MCL1) 1996-2000 introduced high-dose chemotherapy with autologous stem cell support (unpurged or ex vivo purged) as consolidation after 4 cycles of intensified CHOP (maxi-CHOP). The results were disappointing, as the majority of patients relapsed. 1 Being in CR pre-transplant was the most important factor for outcome. Hence, in the second trial (MCL2) 2000-2006 induction therapy was intensified by adding high-dose Ara-C and rituximab to the regimen. Compared to MCL1 this led to significant improvement of event-free and overall survival, and the rate of PCR negative stem cell grafts and bone marrow samples.2 Again, responders in less than CR pre-transplant had a significantly poorer outcome. We therefore made a further intensification for the MCL3 study (2006-2009) by adding 90Y-Ibritumomab tiuxetan (Zevalin®) to the high-dose BEAC/BEAM to responders not in CR. Methods: As in the MCL1 and 2 studies newly diagnosed stage II-IV MCL patients ≤ 65 years were included. Induction treatment was identical to that of the MCL2 study with alternating cycles of maxi-CHOP-rituximab (3 cycles) and Ara-C-rituximab (3 cycles). Response evaluation was done after cycle 5. PET/CT was recommended, but could not influence the response evaluation, which was done according to the International Workshop criteria. Responders underwent in vivo purged harvest of stem cells after cycle 6 (Ara-C + 2 doses of rituximab). Patients in CRu or PR received a standard dose 90Y-Ibritumomab tiuxetan (0.4 mCi/kg) one week prior to the BEAM/BEAC, CR patients received BEAM/BEAC alone. Patients are followed by CT-scans, bone marrow and blood samples, including PCR for minimal residual disease or molecular relapse. For molecular relapse preemptive treatment with 4 standard doses of rituximab, as in the MCL2 study3, is given. Results: The planned accrual of 160 patients was reached in June 2009. The patient characteristics are similar to those of the MCL2 trial with a median age of 57 years (28-65), the majority male (80%) and in stage IV (89%) with bone marrow involvement (74%). The response rates pre-transplant so far compare favorably with data from MCL2 with 50% in CR, 18% in CRu, and 28% in PR. Only 4 out of 128 evaluable patients did not respond (3%) and there was one case (1%) of treatment-related mortality during induction therapy. While it is still too early to assess the impact of the 90Y-Ibritumomab tiuxetan on the progression-free survival, the side effects were similar to those of the MCL2 study including a treatment related mortality of 4%. Fifty-five patients in CRu or PR have so far been treated with 90Y-Ibritumomab tiuxetan, with no indication of any added toxicity. Only 12 out of 133 patients (10%) have not undergone transplant, 5 due to stem cell harvest failure, 3 due to toxicity and 4 due to non response to induction treatment. PET-scan prior to transplant was positive in 2% of CR patients, 20% of CRu patients and 54% of PR patients. Patients with a positive PET-scan pre-transplant had a 36% chance of achieving a molecular remission post-transplant, compared to 92% of cases with a negative PET-scan (p

Description: Despite the success of passive immunotherapy with monoclonal antibodies (mAbs), many lymphoma patients eventually relapse. Induction of an adaptive immune response may elicit active and long-lasting antitumor immunity, thereby preventing or delaying recurrence. Immunomodulating mAbs directed against immune cell targets can be used to enhance the immune response to achieve efficient antitumor immunity. Anti-CD137 agonistic mAb has demonstrated antitumor efficacy in various tumor models and has now entered clinical trials for the treatment of solid tumors. Here, we investigate the therapeutic potential of anti-CD137 mAb in lymphoma. We found that human primary lymphoma tumors are infiltrated with CD137+ T cells. We therefore hypothesized that lymphoma would be susceptible to treatment with anti-CD137 agonistic mAb. Using a mouse model, we demonstrate that anti-CD137 therapy has potent antilymphoma activity in vivo. The antitumor effect of anti-CD137 therapy was mediated by both natural killer (NK) and CD8 T cells and induced long-lasting immunity. Moreover, the antitumor activity of anti-CD137 mAb could be further enhanced by depletion of regulatory T cell (Tregs). These results support the evaluation of anti-CD137 therapy in clinical trials for patients with lymphoma.

Description: Background. The short synthetic peptide 302 has been shown to induce rapid membrane disruption of lymphoma cells in vitro and necrosis of local tumors in the A20 lymphoma model in Balb/c mice. In order to stimulate the immune system to generate an anti-tumor response we designed a model where intra-tumor injections of peptide 302 was combined with sc injections of the Toll-like receptor 9 binding synthetic oligonucleotide CpG 1826. Methods. Balb/c mice were inoculated with A20 lymphoma cells sub-cutaneously (s.c.) on the abdomen. When the tumors reached a size of 5–7 mm, 302 peptide was administered directly into the tumors on days 1 and 6. CpG 1826 was injected s.c. on days 1–4 and 6–8. Tumor growth was measured repeatedly during follow-up. A20-specific T-cell responses were detected by culturing peripheral blood lymphocytes from treated animals for 24 hours with A20 lymphoma cells and analyzing for intracellular IFN-γ production by flow cytometry. To dissect the role of T-cell subtypes, the treatment was performed in animals depleted for CD4 or CD8 positive T-cells. In order to show A20 specific immunological memory, cured animals were re-challenged with the A20 lymphoma or the carcinoma cell line CT26. Results. Combined treatment with peptide 302 and CpG 1826 cured 8 out of 10 mice, compared to only 2 out of 10 mice who received peptide 302 alone or CpG 1826 alone. Cured mice were followed for 9 weeks without relapsing. Similar results with the combination of peptide 302 and CpG were observed in a separate experiment. The highest cure rate was achieved when injecting CpG 1826 s.c. in the tumors draining lymph node area as compared to administration of CPG 1826 in a non-draining lymph node region or intra-peritoneal. Animals treated with peptide 302 + CpG 1826 or CpG 1826 alone developed CD8-specific IFN-γ responses against A20 cells. In one separate experiment CD8 knock-out mice did not respond to the treatment, unlike animals depleted for CD4+ cells and normal mice. Only 1 out of 10 cured animals re-challenged with the A20 lymphoma developed a new tumor, a result that was reproduced in a second experiment. Conclusion: Treatment with intra-tumor injections of the anti-lymphoma peptide 302 in combination with CpG 1826 s.c. in the draining lymph node region cured established A20 tumors, induced tumor-specific CD8 positive tumor-reactive T-cells, and induced specific immunological memory. This principle represents a novel therapeutic vaccine approach.

Description: The 2nd. Nordic Lymphoma Group mantle cell lymphoma (MCL2) protocol has demonstrated the importance of Ara-C and Rituximab in the induction chemotherapy and stem-cell mobilisation before high-dose therapy and autologous stem-cell transplant (1). By July 2005, 128 patients (83% stage IV) had completed protocol treatment consisting of 3 series of R-CHOP and 3 series of R-Ara-C, stem-cell harvest and high-dose therapy with BEAM/BEAC with ASCT. The 5-year failure-free and overall survival is 50% and 83% respectively, significantly higher than the historic control group of the Nordic MCL1 protocol with the same treatment without HD-Ara-C and Rituximab (P

Description: Abstract 759 Introduction: Tumor infiltrating T cells present within biopsy specimens of human B cell non-Hodgkin's lymphomas (NHL) provide a valuable opportunity to examine immune system function in the presence of cancer. We recently used flow cytometry to characterize signaling in subpopulations of tumor samples from patients with follicular lymphoma (FL). In FL, we identified a novel lymphoma cell subset with impaired B cell antigen receptor (BCR) signaling, the prevalence of which correlated with adverse clinical outcome. Here, we turned our attention to signaling differences in subsets of the tumor-infiltrating T cells from FL and two other NHLs, diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL). Signaling differences that distinguish the tumor infiltrating T cells from each malignancy might be expected to be a reflection of the specific disease microenvironment, whereas T cell signaling differences distinguishing cases of the same malignancy might be related to the biology of each patient's tumor. Methods: Single cell flow cytometry measurements of signaling were acquired for samples of DLBCL (N=13), MCL (N=20), and FL (N=14). Phosphorylation of 14 signaling proteins was measured under 12 stimulation conditions in every cell, including lymphoma B cells and tumor-infiltrating T cells within the same specimen. Stimulation conditions included those that were B cell specific (BCR crosslinking, CD40 ligand), T cell specific (IL-7), and those that stimulated both B and T cells (IL-4, IL-10, IL-21, PMA + ionomycin, and IFN-γ). Results: Striking differences were observed in the signaling responses of tumor infiltrating T cells. T cells infiltrating FL patient samples showed significantly lower responses to cytokines where signal transduction is mediated by the common γ chain receptor. Specifically, we observed significant lower induction of p-STAT6 after IL-4 stimulation, p-STAT5 after IL-7 stimulation, and p-STAT3 after IL-21 stimulation (p 〈 0.001 for FL vs. MCL in all cases). In contrast, receptor-independent signaling was not significantly different as FL tumor infiltrating T cells responded at a level comparable to MCL and DLBCL tumor infiltrating T cells when stimulated with PMA and ionomycin. The lower response to common γ chain family cytokines could be the result of a partial suppression of all tumor infiltrating T cells or a complete suppression of a distinct subset. To distinguish between these possibilities, we analyzed signaling in tumor infiltrating T cell subsets. This single cell approach showed that tumor infiltrating T cells were a heterogeneous mixture of non-responsive cells and highly responsive T cells in response to cytokines. Specifically, the mean percentage of T cells that did not induce p-STAT3 after IL-21 stimulation was 50.3% in FL samples in contrast to only 26.2% in MCL samples. Phenotypic analysis showed that the vast majority of T cells infiltrating FL patient samples were CD4+CD45RO memory cells, and the single cell signaling approach revealed that the FL nonresponsive T cell subset had this phenotype. Furthermore, FL T cells were composed of a significantly larger fraction of T regulatory cells than MCL T cells, on average 17% FoxP3+CD25+ cells compared to only 9% in MCL (p