ALBERT

Description: INTRODUCTION: Circular RNAs (circRNAs) are covalently closed endogenous RNA molecules with tissue- and disease specific expression patterns, that are emerging as potential diagnostic and prognostic biomarkers. These molecules exert diverse regulatory functions, and several studies have reported prognostic relevance and functional significance of circRNAs in cancer. However, no studies have yet examined the role of circRNAs in Mantle Cell Lymphoma (MCL). We have previously shown that the Nanostring nCounter technology enables specific, sensitive and accurate quantification of circRNAs in formalin-fixed, paraffin-embedded (FFPE) tissue samples from patients (Dahl et al, 2018). Here, we quantified circRNA expression in MCL tumors to address whether certain circRNAs could serve as prognostic markers in MCL. Samples were obtained from patients treated in the two Nordic clinical trials MCL2 and MCL3, all treated with immuno-chemotherapy and autologous stem cell transplant, which due to the improved prognosis observed with this regimen, remains the current standard of care. MATERIALS AND METHODS: We profiled the genome-wide circRNA expression in MCL using high-throughput RNA-sequencing (RNA-seq) of 14 diagnostic MCL samples, from which high-quality RNA from lymph nodes with MCL tumor infiltration was available. Based on these data, we designed assays for analyses of 41 differentially expressed circRNAs and quantified the expression using the NanoString nCounter technology. We examined the prognostic potential of individual circRNAs in a training cohort of 75 patients (MCL2) and confirmed these results in an independent, but similarly treated, validation cohort of 90 patients (MCL3). RESULTS: The total cohort consisted of 165 previously untreated MCL patients

Description: Background: Follicular lymphoma (FL) is the most common subtype of indolent non Hodgkin's lymphoma (NHL). Median survival is long (〉10 years), but current chemo-immunotherapy regimens used for FL are usually not curative. While T cells in the FL tumor microenvironment are considered dysfunctional and associated with disease progression, a better understanding of T-cell signaling may reveal how to productively engage tumor-infiltrating T cells to kill lymphoma B cells. Our previous study showed that expression of the immune checkpoint receptor PD-1 was directly correlated with reduced cytokine signaling in FL T cells (Myklebust et al., Blood 2013). Antibody immunotherapy targeting the PD-1/PD-L1 pathway has shown significant activity in solid tumors, but these benefits have not been as profound in NHLs, including FL. Co-blockade of checkpoint inhibitors may therefore be necessary to generate optimal anti-tumor responses in FL. The hypothesis underlying this study was that characterizing signaling responses in FL tumor-infiltrating T cells will identify new targets for combination of checkpoint blockade. Methods: Surface expression of 9 checkpoint receptors governing T cell function was measured in subsets of CD4 and CD8 T cells from FL lymph node tumors (n = 14) and from healthy donor tonsils (n= 11) and peripheral blood samples (n = 7) using fluorescence flow cytometry. Patterns of checkpoint receptor expression were compared with 1) intracellular phospho-protein signaling response and 2) cytokine production for subsets of T cells infiltrating FL tumors and the corresponding T-cell populations in healthy tonsils. Phospho-specific flow cytometry measured phosphorylation of STATs and T cell receptor (TCR) signaling effectors within minutes following stimulation by IL-4, IL-7, IL-21, or α-CD3+α-CD28 (TCR stimulation) antibodies. Results: CD4 and CD8 T cells infiltrating FL tumors were gated into subsets defined by PD-1 and ICOS protein expression, and compared to cognate T cell subsets in healthy tonsils. FL and tonsil T cells closely matched in their signaling responses to IL-4, IL-7, and IL-21 stimulation, with PD-1 expressing cells (CD4+PD-1hiICOS+ (TFH) and CD8+PD-1int T cells) exhibiting modest phospho-protein signaling responses compared to T cells not expressing PD-1. Furthermore, TCR membrane proximal signaling events (p-CD3ζ, p-SLP76) following TCR stimulation were comparable in FL and tonsil T cells. This contrasted reduced phospho-ERK signaling in all CD4 and CD8 T cell subsets infiltrating FL tumors which distinguished them from tonsillar T cells. IFN-γ production also differed between FL and tonsils, as CD8 T cells infiltrating FL tumors produced less IFN-γ. Reduced IFN-γ production was independent of PD-1 expression, suggesting suppressed function in these T cells which could be due to inhibitory receptors other than PD-1. Of the 9 checkpoint receptors measured, PD-1 and T cell Ig and ITIM domain (TIGIT) were expressed at the highest frequency. In FL, TIGIT was expressed in 58% and 80% of CD8 effector and effector memory cells, respectively, as compared to 43% and 68% of the cognate healthy tonsillar subsets. TIGIT was also frequently expressed in CD4 FL T cells, as 52% and 79% of effector and effector memory cells expressed TIGIT, respectively, as compared to 16% and 59% of the corresponding subsets from healthy tonsils. viSNE analysis demonstrated that TIGIT and PD-1 were frequently co-expressed in FL T cells, and a large fraction of PD-1int T cells had high expression of TIGIT (Figure 1). These results provide a rationale for co-blockade of PD-1 and TIGIT in FL and for investigation of how co-blockade impacts T cell functions. Conclusions: These results reveal specific suppression of cytokine signaling in CD8 effector T cells infiltrating FL tumors and identify TIGIT and PD-1 as strong candidates for co-checkpoint blockade in FL. A deeper understanding of the interplay between checkpoint receptors and key T cell cytokine signaling events in FL will further assist in engineering immuno-therapeutic regiments that improve FL patient clinical outcomes. Disclosures Kolstad: Nordic Nanovector: Other: Membership of Scientific Advisory Board. Levy:Kite Pharma: Consultancy; Five Prime Therapeutics: Consultancy; Innate Pharma: Consultancy; Beigene: Consultancy; Corvus: Consultancy; Dynavax: Research Funding; Pharmacyclics: Research Funding. Irish:Incyte: Research Funding; Janssen: Research Funding; Cytobank, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Description: Eskelund et al examined clonal hematopoiesis (CH) in a cohort of patients with mantle cell lymphoma (MCL) treated with first-line chemotherapy and autologous stem cell transplantation. In young, good-risk MCL patients, CH after first-line therapy arises almost entirely from preexisting clones, stabilizes after a period of expansion posttransplantation, and does not negatively impact survival.

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: Minimal residual disease monitoring has been shown to be of relevance in mantle cell lymphoma (MCL) to evaluate quality of remission and predict clinical relapse. The main objectives of the present study were to determine the value of minimal residual disease (MRD) monitoring to guide pre-emptive treatment with rituximab, and to predict clinical relapse in MCL following autologous stem cell transplantation (ASCT) in two prospective trials (MCL2 and MCL3) with long-term follow-up conducted by the Nordic Lymphoma Group. Methods: Patients treated in the two studies received a total of 6 alternating cycles with R-CHOP and R-Ara-C followed by a peripheral blood stem cell harvest and high-dose therapy with ASCT. Additionally, responding patients not in CR before ASCT in the MCL3 trial received yttium-90 ibritumomab tiuxetan (0.4 mCi/kg) as intensification one week prior to the standard conditioning with BEAM/C. Staging included physical examination, blood tests, computed tomography (CT) scans and bone marrow (BM) aspiration and biopsy. Clinical and molecular response evaluation was repeated 2-3 months and 6 months after transplant, and then every 6 months until relapse or 5 years follow-up. A combined standard nested and quantitative real-time PCR assay was used to estimate MRD involvement in consecutive post-transplant BM/PB samples. Molecular relapse after ASCT was defined as; 1. Conversion from standard nested PCR negative to standard nested PCR positive, or 2. For patients who were MRD positive post-ASCT, a significant (〉5 fold) increase of the real time quantitative PCR detectable MRD level in two consecutive BM samples. Patients in clinical remission who developed a molecular relapse in both studies received 4 weekly doses of rituximab (375 mg/m2). This treatment could be repeated in case of recurrent molecular relapses. Results: An MRD marker for Bcl-1 or IgH rearrangement was obtained for 94 out of 160 patients (59%) recruited in the Nordic MCL2 trial, and for 121 out of 160 (76%) in the consecutive MCL3 trial. 183 patients, who had completed induction therapy and autologous stem cell transplantation (ASCT) in the two studies and where a PCR marker in blood or bone marrow was obtained, were included in the current analysis. Median follow-up from inclusion was 8.5 years among survivors. Patients who were MRD negative post-ASCT had significantly longer relapse-free survival (RFS) and overall survival (OS) compared to those who were MRD positive (P

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: Abstract 1804 Introduction: Advanced stage follicular lymphoma (FL) cannot be cured by current conventional therapies. Treatment with idiotype cancer vaccines has thus far been disappointing, and is expensive and labor intensive. Hence, simpler principles for vaccinations that do not require production of patient-specific tumor antigens are warranted. We designed a novel strategy involving intratumoral injections of low-dose anti-CD20 antibody (Rituximab) and autologous dendritic cells (DC) in irradiated lymph nodes. The aim was to facilitate antigen up-take and presentation by DCs to T cells to achieve systemic anti-tumor responses in patients with FL. Method: Ten untreated patients with stage III/IV follicular lymphoma not in need of conventional therapy underwent standard work-up, including CT-scans, FDG PET/CT and bone marrow samples. Single tumor cells were frozen for later immunoassays. Apheresis was performed for isolation of monocytes subsequently cultured with IL-4 and GM-CSF for 5 days to generate immature dendritic cells (DC). The first 6 patients received a single 8 Gy dose of radiotherapy against an enlarged node on day 1, followed by intratumoral injections of DCs (1 × 108) and GM-CSF (50 μg) on days 3 and 4. The treatment was repeated against a different enlarged node after 6 weeks. Thereafter, the protocol was changed, and patients received intratumoral injections of a small dose of rituximab (5 mg) on days 1 and 3 in order to enhance ADCC, radiotherapy (8 Gy) on day 2 and intratumoral injections of DCs and GM-CSF sc on days 4 and 5. This therapy was then repeated after 2 and 4 weeks, targeting other lymph nodes. Follow-up included repeated PET/CT-scans, bone marrow sampling and preparation of peripheral blood mononuclear cells (PBMC) for later immune studies. Tumor-specific helper and cytotoxic T cell responses were monitored by flow cytometry after a 5-day co-culture of single tumor cells with autologous PBMC sampled before, and 2 and 4 months after, treatment. Proliferation was studied by incorporation of CFSE, whereas degranulation (CD107a/b) and interferon gamma release was measured following re-stimulation with tumor cells for 5h. Result: Ten patients with FL of a total of 20 planned for this study were treated so far. No adverse events were observed. Among the first 6 patients treated by the original strategy, two showed a systemic metabolic response by PET/CT and one of these had a good response by regular CT not qualifying for PR. The protocol was then modified as described above. The first patient treated by the new strategy that included intratumoral rituximab, had a systemic metabolic response by PET/CT after 2 and 4 months and was PET negative with a normal CT by 8 months (Figure 1). Another patient showed a systemic PET response at 2 and 4 months. Analysis of T cell responses against autologous tumor cells has been initiated and we have so far demonstrated a vigorous CD8 dominated T cell response, as assayed by proliferation, degranulation and cytokine production, in the patient who achieved a negative PET-scan at 8 months (Figure 2). In parallel with the metabolic response, the immune response became stronger during follow-up after treatment. One patient without clinical response was also analysed, and no immune response was demonstrated. Further analysis of immune responses in the other patients is on-going. Conclusion: The present strategy is a novel principle to generate T cell responses and clinical anti-tumor responses detected by PET/CT in FL. Disclosures: No relevant conflicts of interest to declare.

Description: Global gene expression profiling of the tumor microenvironment in diffuse large B-cell lymphoma (DLBCL) has revealed broad innate immune signatures that distinguish the heterogeneous disease subtypes and correlate with good treatment outcome. However, we still lack tools to identify the relatively large group of patients that are refractory to initial therapy and have a dismal prognosis. Here, we used mass cytometry and serum profiling in a systems-level approach to analyze immune responses in 36 patients with aggressive B cell lymphoma and age- and sex-matched healthy controls. Stochastic neighbor embedding (t-SNE) analysis of protein profiles divided patients into two distinct clusters, with cluster 2 representing patients with a more severe deviation in their protein expression compared to healthy controls. Patients in cluster 2 showed a more dramatic perturbation of their immune cell repertoires with expansion of myeloid-derived suppressor cells (MDSCs), increased T cell differentiation and significantly higher expression of metabolic markers such as GLUT-1 and activation markers, including Ki67, CD38 and PD-1. An extended analysis of serum protein profiles in two independent cohorts (n=69 and n=80 patients, respectively) revealed that that the identified systemic immune signatures were linked to poor progression free survival (PFS) and inferior overall survival (OS). Immune monitoring during chemo-immunotherapy showed that most patients normalized their serum protein profiles. Notably, non-responding patients retained higher than normal expression of several proteins, including PD-L1, CD70, IL-18, granzyme A and CD83. These studies demonstrate distinct patterns of disease-driven alterations in the systemic immune response of DLBCL patients that are associated with poor survival and persist in patients who are refractory to therapy. Figure 1 System-level immune signatures associated with poor prognosis in DLBCL. A) Altered serum profiles in patients compared to healthy controls. Two clusters of patients were identified based on t-SNE analysis of serum profiles. B) Patients in cluster 2 had bulky disease and B symptoms. C) t-SNE map of all patients (n=36) and controls (n=17). Relative abundance of cells from healthy controls and patients in all areas of the t-SNE clustering, highlighting cell subsets that are larger or smaller in patients compared to healthy donors. Colors indicate the difference in kernel density estimation of the t-SNE data for patients and healthy controls. D) Abundance of monocytic myeloid-derived suppressor cells as percentage of all CD45+ cells in healthy donors and the two patient clusters. White, Healthy controls; Blue, Cluster 1; Red, Cluster 2. E) Major phenotypic differences between patient clusters shown as mean mass intensity (MMI) or percent positive cells for selected markers (CD38 and PD-1) across multiple subsets. White, Healthy controls; Blue, Cluster 1; Red, Cluster 2. F-G) Overall survival in patients with serologically defined immune signatures belonging to cluster 1 or 2. H) Abundance of serum proteins in patients that stayed in remission (n=24) compared to those that did not (n=6). Figure 1 Disclosures Olweus: Gilead Kite: Research Funding; Intellia: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Wahlin:Roche and Gilead: Consultancy. Fehniger:Cyto-Sen Therapeutics: Consultancy; Horizon Pharma PLC: Other: Consultancy (Spouse). Holte:Novartis: Honoraria, Other: Advisory board. Kolstad:Nordic Nanovector: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding. Malmberg:Fate Therapeutics, Inc.: Consultancy, Research Funding; Vycellix: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 144 Introduction: Mantle cell lymphoma (MCL) is an aggressive non-Hodgkin's lymphoma. Although newer treatment regimens including intensive chemo-immunotherapy followed by stem cell transplant have improved survival to a median approaching 6 years, MCL is still incurable in many cases. The use of flow cytometry to investigate stimulation induced signaling at the single cell level represents an opportunity to obtain each patient's signaling profile as well as to discover tumor cell heterogeneity and identify signaling events potentially responsible for poor outcomes. We recently used this method to characterize signaling in subpopulations of tumor samples from patients with follicular lymphoma (FL). In FL, we identified a lymphoma negative prognostic (LNP) cell subset with impaired B cell antigen receptor (BCR) signaling, the prevalence of which correlated with adverse clinical outcome (Irish et al., PNAS 2010). In the present study we used the same approach to identify signaling responses with large variation within MCL patient samples, and tested whether these signaling events might predict patients' clinical outcomes. By understanding the biology based on lymphoma signaling profile, we might identify prognostic significant factors such as the LNP subset observed in FL. Method: Single cell flow cytometry measurements of signaling were acquired for samples of MCL (n=25). Of these, 21 MCL specimens were obtained prior to any treatment. Median age was 61 years, and follow up time ranged from ½ until 15 years. Treatment regimens varied. Samples from diffuse large B cell lymphoma (DLBCL, n=12), FL (n=14), chronic lymphocytic leukemia (CLL, n=14) and tonsils from healthy donors (n=4) were also included for comparison. Phosphorylation of 14 signaling proteins was measured under 12 different stimulation conditions in every cell within the patient specimens, including lymphoma B cells and in tumor-infiltrating T cells. Stimulation conditions included BCR crosslinking, CD40 ligand, CpG oligodeoxynucleotides (CpG ODN) and several cytokines (IL-4, IL-10, IL-21, IFN-γ). Result: The magnitude of most cytokine induced signaling responses in lymphoma cells from MCL patients was higher than in lymphoma cells from FL. This included IL-10 induced phosphorylation of STAT3 (p

Description: Key PointsAddition of lenalidomide to R-B is highly active in patients with untreated MCL, but associated with unexpected high rates of infections and SPMs.