ALBERT

Description: According to WHO’s current criteria, follicular lymphoma is morphologically divided into grades 1, 2, and 3, and grade 3 is further subdivided into 3a and 3b, depending on the presence of centrocytes and solid sheets of centroblasts. The clinical relevance of this grading system is unclear. Although there is consensus that grades 1 and 2 share clinical characteristics of indolent lymphoma, it is debated whether grade 3 should be treated intensively with anthracycline-containing regimens. Furthermore, the 3a/3b subdivision is not universally implemented. We wanted to investigate the clinical relevance of the grading system with respect to clinical characteristics, treatment and survival. Totally, 186 cases were diagnosed between January 1994 and January 2004 in South Stockholm County with de novo follicular lymphoma and without concomitant transformation. The biopsies were re-graded according to the current WHO criteria by two experienced hematopathologists. 129 cases were grade 1 or 2, 44 grade 3a, and 13 grade 3b. Clinical characteristics in the patients with follicular lymphoma grade 1–2 and 3a are shown in Table 1. The local policy has been to treat all patients with grade 3 disease aggressively. In grade 3a disease, front-line anthracyclines did not seem to improve overall survival (Figure 1). Grades 1–2 and 3a show similar overall survival curves of indolent and incurable lymphoma, while the 13 patients with grade 3b disease (of whom 12 received aggressive front-line therapy) seem to reach a plateau after four years (Figure 2). We conclude that follicular lymphoma grade 3a is not clinically different from grade 1 and 2 disease, and that patients with grade 3a do not seem to benefit from front-line anthracyclines. Although our findings are limited by the few cases of grade 3b, it seems that grade 3b is another disease entity and, when treated aggressively, it is maybe curable with chemotherapy. The histopathological distinction between grade 3a and 3b is clinically of great importance. Table 1 Grade 1–2 Grade 3a P Clinical characteristics in patients with follicular lymphoma grade 1-2 and 3a. Age 〉60 years 50% 61% NS Elevated LDH 32% 49% 0.046 Stage III or IV 68% 80% NS Front-line anthracyclines 41% 73% 0.0004 Subsequent transformation 21% 19% NS Figure Figure Figure Figure

Description: Mantle cell lymphoma is a non-Hodgkin lymphoma with, in general, a poor prognosis. A minor subset of patients with an indolent disease course has however been recognized (1,2). The various growth patterns of MCL, i.e. mantle zone (MZ), nodular (N) or diffuse (D) is assumed to correlate to stage and to disease course. The genetic aberrations underlying the pathogenesis are well defined and correlate to high tumour cell proliferation and poor prognosis. However, the effect of the lymphoma microenvironment in disease development and sustainability is largely unknown. We have used flow cytometry to investigate the non-malignant cell composition of the lymph node microenvironment in a population-based cohort of 154 MCL cases diagnosed from January 1, 1998 to December 31, 2012. Flow cytometry analyses of lymph nodes, performed as part of the diagnostic process, were used to evaluate percentages of tumour cells, remaining non-malignant B-cells and T-cell subsets (CD3+, CD3+CD4+, CD3+CD8+). As lymph node T-cell numbers reflect a high tumor load in the lymph node we also investigated the CD4/CD8 ratio, which is not dependent on T-cell percentage. Data from 26 non-malignant lymph nodes were used for comparison. T-cell percentages are shown in Table 1. Clinical and other pathological parameters of the MCL cases, including MIPI, cell morphology, tumor growth pattern and cell proliferation were also evaluated. Indolent disease (n=15), defined here as requirement of treatment 〉 two years from diagnosis, was associated with higher amount of CD3, CD3+CD4+ and higher CD4/CD8 ratio (p=0.0429, p=0.0211 and p= 0.0032 respectively). Higher tumor cell proliferation correlated negatively with the CD4/CD8 ratio (p= 0.0007). There was a significant difference in CD3 percentages between reactive lymph nodes and MCL irrespective of growth pattern (all p

Description: Background: Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma with a high rate of relapses after therapy. Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with varied outcome. For both diseases there is a need for new therapies. Cannabinoid receptors (CBs), which are overexpressed in most cases of MCL and CLL compared to normal B cells (Islam et al., 2003; Gustafsson et al., 2008; Freund et al., 2016) are promising novel therapeutic targets. CBs are membrane-bound receptors that convey signals from the microenvironment to the cells. There are two types of CBs: CB1 and CB2. CB1 is suggested to be involved in retention and/or egress of MCL cells from the tissue to the blood circulation (Wasik et al., 2014). CB2 is expressed by normal B-cells where it regulates positioning and retention of cells in tissue (Pereira et al., 2009; Basu et al., 2011; Muppidi et al., 2011) and in pre-B-cell acute lymphoblastic leukemia, involved in the energy metabolism (Chan et al., 2017). The retention/egress of the B-cell lymphoma cells is mainly regulated by chemokine receptors and adhesion molecules. The chemokine receptor CXCR4 is one of the most highly expressed chemokine receptors in MCL and CLL. 2-arachidonoylglycerol (2-AG, CB1/CB2 endogenous ligand) and CXCL12 (CXCR4 ligand) are synthetized and secreted by stromal cells in the bone marrow (Kose et al., 2018; Burger and Gribben, 2014). The endocannabinoids levels in cancer are suggested to have a role in cancer progression (Sailler et al., 2014) while CXCL12 is already a candidate target for therapy using a CXCR4 inhibitor AMD3100. Aim: To investigate a possible crosstalk between CBs and CXCR4 in MCL and CLL cells. Methods: Patients with newly diagnosed MCL (n=8) or CLL (n=25) gave informed consent to participate in the study. Lymphoma cells were enriched by negative selection. Fifteen primary lymphoma samples and the JeKo MCL cell line were subjected to chemotaxis towards CXCL12 and/or 2-AG. CXCR4 membrane expression was assessed by flow cytometry. Selective CB1 and CB2 antagonists were used to investigate the underlying mechanisms. CB1, CB2 and CXCR4 encoding genes levels were measured by qPCR and normalized to B cells from tonsil. Results and Conclusion: 2-AG induced chemotaxis in 11/15 MCL and CLL samples. In JeKo, 2-AG-induced migration was blocked by a CB2 antagonist, suggesting that signaling via CB2 is involved. When the primary cells were subjected to migration towards CXCL12, two patterns of chemotaxis were observed. The first pattern was seen in 7/15 samples that migrated towards CXCL12. In these samples, the migration was inhibited when 2-AG was combined with CXCL12. The second type of response was observed in 8/15 samples, those samples did not migrate towards CXCL12 but chemotaxis was enhanced by combining 2-AG and CXCL12. MCL and CLL samples expressed variable mRNA levels of CB1 (RFI range: 0.0-204) and CB2 (RFI range: 0.8-14.3) and all expressed CXCR4 at mRNA (RFI range: 0.1-215.8) and protein (MFI range: 1278-19301) levels that did not differ neither between the two diseases nor between the two migratory groups. When all 15 samples were combined, CB1 mRNA levels, but not CB2 mRNA, correlated to the chemotaxis towards CXCL12 (Spearman correlation coefficient = 0.626; p=0.01). In contrast, CB2 mRNA levels, but not CB1, correlated to chemotaxis towards 2-AG (Spearman correlation coefficient = 0.532; p=0.04), which is in agreement with the effects observed in JeKo. Furthermore, CB1 and CB2 mRNA levels correlated to chemotaxis towards the combination of CXCL12 and 2-AG both (for CB1 mRNA: Spearman correlation coefficient= 0.588; p=0.02 and for CB2 mRNA: 0.589; p=0.02). Neither CXCL12-induced CXCR4 receptor internalization, nor recycling was influenced by 2-AG incubation. Our findings indicate a novel pathway regulating chemotaxis of MCL and CLL implicating a cross-talk between CBs and CXCR4. The fact that the capacity to internalize CXCR4 remained intact after incubation with 2-AG suggests that the reduced CXCL12-mediated migration when 2-AG was combined could be due to an impaired downstream signaling in lymphoma cells. Importance: Lymphoma cells residing in the tissue receive pro-survival stimuli and are protected from chemotherapy by signals from the microenvironment. A better understanding of how lymphoma cell migration and tissue retention are regulated can be a step towards more efficient therapies. Disclosures Wahlin: Gilead: Consultancy, Honoraria, Research Funding; Roche: Research Funding.

Description: The prognostic role of the transcription factor SOX11 in mantle cell lymphoma (MCL) is controversial. We investigated prognostic markers in a population-based cohort of 186 MCL cases. Seventeen patients (9%) did not require any therapy within the first 2 years after diagnosis and were retrospectively defined as having an indolent disease. As expected, indolent MCL had less frequent B symptoms and extensive nodal involvement and 88% of these cases expressed SOX11. In our cohort 13 cases (7.5%) lacked nuclear SOX11 at diagnosis. SOX11− MCL had a higher frequency of lymphocytosis, elevated level of lactate dehydrogenase (LDH), and p53 positivity. The overall survival in the whole cohort, excluding 37 patients receiving autologous stem cell transplantation, was 3.1 year and in patients with indolent or nonindolent disease, 5.9 and 2.8 years, respectively (P = .004). SOX11− cases had a shorter overall survival, compared with SOX11+ cases, 1.5 and 3.2 years, respectively (P = .014). In multivariate analysis of overall survival, age 〉 65 (P = .001), Eastern Cooperative Oncology Group score ≥ 2 (P = .022), elevated LDH level (P = .001), and p53 expression (P = .001) remained significant, and SOX11 lost significance. We conclude that most indolent MCLs are SOX11+ and that SOX11 cannot be used for predicting an indolent disease course.

Description: Introduction Mantle cell lymphoma (MCL) is an incurable disease with a median survival of 3-5 years. Most cases express SOX11, a transcription factor associated with MCL pathobiology, which serves as a diagnostic marker. Current standard first-line therapy for younger MCL patients is rituximab with cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) and cytarabine (Ara-C), consolidated with high dose chemotherapy with autologous stem cell transplantation (ASCT), which is associated with prolonged survival (Eskelund et al. Br J Haematol 2016). Ara-C metabolizes into its active triphosphate form Ara-CTP which inhibits proliferation of the malignant cells. Recent reports suggest that the expression of the mammalian dNTPs hydrolase SAMHD1 determines response to Ara-C in acute myeloid leukemia (AML) by hydrolyzing Ara-CTP and thus by diminishing the anti-proliferative properties of Ara-C. Consequently, in vitro downregulation of SAMHD1 resulted in sensitization of AML cells to Ara-C (Herold et al., Nat Med 2017). SAMHD1 also exhibits anti-tumor properties via regulation of dNTP pool and is recurrently mutated in CLL (Clifford et al. Blood 2014) and T-PLL (Johansson et al. Blood Cancer J. 2018). In CLL a role in DNA-repair has been suggested (Clifford et al. Blood 2014). Thus, SAMHD1 may function as a tumor suppressor. In this study, we investigated for the first time the expression patterns of SAMHD1 in MCL and its association to clinical outcome, especially in patients receiving Ara-C as part of induction for ASCT. Methods SAMHD1 and SOX11 expression was investigated by qPCR, WB and IHC on whole tissue sections or tissue microarrays. MCL cell lines were treated with gene-specific siRNA for SAMHD1 or SOX11. Data on overall survival (OS) was retrieved from patient records. For patients included in the Nordic MCL2 and MCL3 trials data on progression free survival (PFS) and OS were retrieved. Results Initial IHC showed that expression of SAMHD1 is high in normal T-cells and macrophages and low in cells in the mantle zones of reactive tonsils. Co-staining with CD20 in a heterogeneously treated cohort of primary MCL (n=104) showed a large variation between cases (median 73.15%, range: 0.4%-99.6%) and the staining intensity was lower than in T-cells. Sixty two/104 samples were also evaluated for SOX11 expression and the percentage of SOX11 positive cells moderately correlated with SAMHD1 expression (Spearman correlation coefficient 0.27, p=0.036). Analysis of mRNA expression (normalized to mRNA levels of respective genes in Granta519 cell line) of SAMHD1 (median RFI: 2.09, range: 0.18-10.69) and SOX11 (median RFI: 2.00, range: 0.00-7.11) in flow cytometry sorted primary MCL cells (n=19) showed a trend for correlation (Spearman correlation coefficient 0.45, p=0.053). However, downregulation of SOX11 by siRNA did not alter the expression of SAMHD1 and neither did downregulation of SAMHD1 by siRNA change the expression of SOX11 suggesting that the observed correlation is not due to a mutual regulation. We investigated the relation of SAMHD1 expression to clinical and pathological parameters in this cohort and found no correlation to OS, blastoid morphology, proliferation (% Ki67+ tumor cells) nor p53 positivity (〉20% positively stained cells). Further, a cohort of patients treated within the Nordic MCL2 and MCL3 protocols (n=51) were investigated for SAMHD1 expression by IHC. Interestingly the distribution of SAMHD1 expression was different in this cohort (median 34.9%, range: 2.2%-97.1%; Mann-Whitney p=0.0019). Survival analysis showed a trend for better PFS and OS in patients with high (〉75% SAMHD1 positive cells) and low (