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Supplementary Tables S1-S12

Supplementary tables 1-5

Supplementary Figures S1-S17

Chronic inflammation, including that driven by autoimmunity, is associated with the development of B-cell lymphomas. IL1R8 is a regulatory receptor belonging to the IL1R family, which negatively regulates NF-κB activation following stimulation of IL1R or Toll-like receptor family members. IL1R8 deficiency is associated with the development of severe autoimmune lupus-like disease in lpr mice. We herein investigated whether concomitant exacerbated inflammation and autoimmunity caused by the deficiency of IL1R8 could recapitulate autoimmunity-associated lymphomagenesis. We thus monitored B-cell lymphoma development during the aging of IL1R8-deficient lpr mice, observing an increased lymphoid cell expansion that evolved to diffuse large B-cell lymphoma (DLBCL). Molecular and gene-expression analyses showed that the NF-κB pathway was constitutively activated in Il1r8−/−/lpr B splenocytes. In human DLBCL, IL1R8 had reduced expression compared with normal B cells, and higher IL1R8 expression was associated with a better outcome. Thus, IL1R8 silencing is associated with increased lymphoproliferation and transformation in the pathogenesis of B-cell lymphomas associated with autoimmunity.

PDF file - 89K, Supplementary Table 1 complete list of genes Supplementary Table 2 The array consists of a panel of 96 primer sets used for the amplification of 84 genes relevant to the apoptosis pathway

PDF file - 1992K, Relative viability of CLL cells

Legend for Supplementary Figures S1-S5.

Description of additional methods and procedures used in the study.

Angiogenesis has been postulated to be critical for the pathogenesis of multiple myeloma, a neoplastic disease characterized by abnormal proliferation of malignant plasma cells in the bone marrow (BM). Cleavage of the N- and C-terminal regions of circulating chromogranin A (CgA, CHGA), classically an antiangiogenic protein, can activate latent antiangiogenic and proangiogenic sites, respectively. In this study, we investigated the distribution of CgA-derived polypeptides in multiple myeloma patients and the subsequent implications for disease progression. We show that the ratio of pro/antiangiogenic forms of CgA is altered in multiple myeloma patients compared with healthy subjects and that this ratio is higher in BM plasma compared with peripheral plasma, suggesting enhanced local cleavage of the CgA C-terminal region. Enhanced cleavage correlated with increased VEGF and FGF2 BM plasma levels and BM microvascular density. Using the Vk*MYC mouse model of multiple myeloma, we further demonstrate that exogenously administered CgA was cleaved in favor of the proangiogenic form and was associated with increased microvessel density. Mechanistic studies revealed that multiple myeloma and proliferating endothelial cells can promote CgA C-terminal cleavage by activating the plasminogen activator/plasmin system. Moreover, cleaved and full-length forms could also counter balance the pro/antiangiogenic activity of each other in in vitro angiogenesis assays. These findings suggest that the CgA-angiogenic switch is activated in the BM of multiple myeloma patients and prompt further investigation of this CgA imbalance as a prognostic or therapeutic target. Cancer Res; 76(7); 1781–91. ©2016 AACR.

Characterization of the new α-373 antibody and *373-ELISA (S1); Levels of different CgA-related analytes in peripheral-blood and bone marrow plasma samples obtained from normal subjects and MM patients stratified according to renal failure and treatment with proton pump inhibitors (S2); BM plasma levels of different CgA forms in MM patients at diagnosis in different disease stages (S3); MM and endothelial cells produce the urinary-type plasminogen activator (S4); Alignment of CgA sequence from different species (S5).

PDF file - 2581K, Stratification according to the underlying genetics

Demographic and clinical data regarding the study population (S1); ELISAs used for the characterization of CgA and its fragments in MM plasma samples (S2); Correlation between BM plasma levels of different CgA analytes and clinical variables in MM patients at diagnosis (S3); Correlation between BM plasma levels of different CgA analytes and FGF2 or VEGF in MM patients at diagnosis (S4).

PDF file - 1885K, LT-alpha/TNF-beta protects CLL cells from drug-induced apoptosis

Chimeric antigen receptors (CAR)-modified T cells are an emerging therapeutic tool for chronic lymphocytic leukemia (CLL). However, in patients with CLL, well-known T-cell defects and the inhibitory properties of the tumor microenvironment (TME) hinder the efficacy of CAR T cells. We explored a novel approach combining CARs with lenalidomide, an immunomodulatory drug that tempers the immunosuppressive activity of the CLL TME. T cells from patients with CLL were engineered to express a CAR specific for CD23, a promising target antigen. Lenalidomide maintained the in vitro effector functions of CD23.CAR+ T cells effector functions in terms of antigen-specific cytotoxicity, cytokine release and proliferation. Overall, lenalidomide preserved functional CAR T-CLL cell immune synapses. In a Rag2−/−γc−/−-based xenograft model of CLL, we demonstrated that, when combined with low-dose lenalidomide, CD23.CAR+ T cells efficiently migrated to leukemic sites and delayed disease progression when compared to CD23.CAR+ T cells given with rhIL-2. These observations underline the therapeutic potential of this novel CAR-based combination strategy in CLL.

The previous edition of the consensus guidelines of the International Workshop on Chronic Lymphocytic Leukemia (iwCLL), published in 2008, has found broad acceptance by physicians and investigators caring for patients with CLL. Recent advances including the discovery of the genomic landscape of the disease, the development of genetic tests with prognostic relevance, and the detection of minimal residual disease (MRD), coupled with the increased availability of novel targeted agents with impressive efficacy, prompted an international panel to provide updated evidence- and expert opinion–based recommendations. These recommendations include a revised version of the iwCLL response criteria, an update on the use of MRD status for clinical evaluation, and recommendations regarding the assessment and prophylaxis of viral diseases during management of CLL.

In the phase 3 RESONATE study, ibrutinib demonstrated superior progression-free survival (PFS), overall survival (OS) and overall response rate (ORR) compared with ofatumumab in relapsed/refractory CLL patients with high-risk prognostic factors. We report updated results from RESONATE in these traditionally chemotherapy resistant high-risk genomic subgroups at a median follow-up of 19 months. Mutations were detected by Foundation One Heme Panel. Baseline mutations in the ibrutinib arm included TP53 (51%), SF3B1 (31%), NOTCH1 (28%), ATM (19%) and BIRC3 (14%). Median PFS was not reached, with 74% of patients randomized to ibrutinib alive and progression-free at 24 months. The improved efficacy of ibrutinib vs ofatumumab continues in all prognostic subgroups including del17p and del11q. No significant difference within the ibrutinib arm was observed for PFS across most genomic subtypes, although a subset carrying both TP53 mutation and del17p had reduced PFS compared with patients with neither abnormality. Reduced PFS or OS was not evident in patients with only del17p. PFS was significantly better for ibrutinib-treated patients in second-line vs later lines of therapy. The robust clinical activity of ibrutinib continues to show ongoing efficacy and acceptable safety consistent with prior reports, independent of various known high-risk mutations.

Chronic lymphocytic leukemia (CLL) is a B-cell neoplasia characterized by protumor immune dysregulation involving nonmalignant cells of the microenvironment, including T lymphocytes and tumor-associated myeloid cells. Although therapeutic agents have improved treatment options for CLL, many patients still fail to respond. Some patients also show immunosuppression. We have investigated trabectedin, a marine-derived compound with cytotoxic activity on macrophages in solid tumors. Here, we demonstrate that trabectedin induces apoptosis of human primary leukemic cells and also selected myeloid and lymphoid immunosuppressive cells, mainly through the TRAIL/TNF pathway. Trabectedin modulates transcription and translation of IL6, CCL2, and IFNα in myeloid cells and FOXP3 in regulatory T cells. Human memory CD8+ T cells downregulate PD-1 and, along with monocytes, exert in vivo antitumor function. In xenograft and immunocompetent CLL mouse models, trabectedin has antileukemic effects and antitumor impact on the myeloid and lymphoid cells compartment. It depletes myeloid-derived suppressor cells and tumor-associated macrophages and increases memory T cells. Trabectedin also blocks the PD-1/PD-L1 axis by targeting PD-L1+ CLL cells, PD-L1+ monocytes/macrophages, and PD-1+ T cells. Thus, trabectedin behaves as an immunomodulatory drug with potentially attractive therapeutic value in the subversion of the protumor microenvironment and in overcoming chemoimmune resistance.

Olaptesed pegol (NOX-A12) is a pegylated structured L-oligoribonucleotide that binds and neutralizes CXCL12, a chemokine tightly regulating the life cycle of chronic lymphocytic leukemia cells. The resulting inhibition of CXCR4 and CXCR7 signaling reduces the protective activity of the bone marrow and lymph node microenvironment. CXCL12 inhibition mobilizes chronic lymphocytic leukemia cells into the circulation and prevents their homing into the protective niches. In this phase I/II study, 28 patients with relapsed/refractory chronic lymphocytic leukemia were treated with olaptesed pegol in combination with bendamustine and rituximab. Combination treatment was preceded by single escalating pilot doses of olaptesed pegol in the first ten patients for evaluation of safety and pharmacokinetics. Peak concentrations and systemic exposure of olaptesed pegol were dose-linear; plasma elimination was monophasic with a 53.2 h half-life. A rapid increase in circulating chronic lymphocytic leukemia cells was observed already 1 h after administration of olaptesed pegol and lasted for at least 72 h. Single-agent treatment was well tolerated and no dose-limiting toxicity was observed. The combination regimen yielded an overall response rate of 86%, with 11% of patients achieving a complete response and 75% a partial response. Notably, all ten high-risk patients, including four with a 17p deletion, responded to treatment. The median progression-free survival was 15.4 (95% confidence interval: 12.2, 26.2) months while the median overall survival was not reached with >80% of patients alive after a median follow-up of 28 months. Olaptesed pegol was well tolerated and did not result in additional toxicity when combined with bendamustine and rituximab (ClinicalTrials.gov identifier: NCT01486797). Further clinical development of this novel CXCL12 inhibitor is thus warranted. ispartof: HAEMATOLOGICA vol:104 issue:10 pages:2053-2060 ispartof: location:Italy status: published

// Mimma Bianco 1 , Anna Gasparri 1 , Luca Generoso 1 , Emma Assi 1 , Barbara Colombo 1 , Lydia Scarfo 2, 3, 4 , Maria T.S. Bertilaccio 5 , Cristina Scielzo 5 , Pamela Ranghetti 5 , Eleonora Dondossola 1 , Maurilio Ponzoni 3 , Federico Caligaris-Cappio 3, 4, 5 , Paolo Ghia 2, 3, 4 , Angelo Corti 1, 4 1 Tumor Biology and Vascular Targeting Unit, Division of Experimental Oncology, IRCCS San Raffaele Scientific Institute, Milan 20132, Italy 2 B Cell Neoplasia Unit, Division of Experimental Oncology, IRCCS San Raffaele Scientific Institute, Milan 20132, Italy 3 Clinical Lymphoma Unit, Department of Onco-Hematology, San Raffaele Hospital, Milan 20132, Italy 4 San Raffaele Vita-Salute University, Milan 20132, Italy 5 Lymphoid Malignancies Unit, Division of Experimental Oncology, IRCCS San Raffaele Scientific Institute, Milan 20132, Italy Correspondence to: Angelo Corti, email: corti.angelo@hsr.it Keywords: chromogranin A, vasostatin-1, chronic lymphocytic leukemia, tumor cell trafficking, endothelial barrier function Received: February 16, 2016 Accepted: April 26, 2016 Published: May 17, 2016 ABSTRACT Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of leukemic B cells in peripheral blood, bone marrow (BM) and lymphoid tissues, and by their recirculation between these compartments. We observed that circulating chromogranin A (CgA) and its N-terminal fragment (called vasostatin-1, CgA 1-76 ), two neuroendocrine secretory polypeptides that enhance the endothelial barrier function, are present in variable amounts in the blood of CLL patients. Studies in animal models showed that daily administration of full-length human CgA 1-439 (0.3 μg, i.v., or 1.5 μg/mouse, i.p.) can reduce the BM/blood ratio of leukemic cells in Eμ-TCL1 mice, a transgenic model, and decrease BM, lung and kidney infiltration in Rag2 −/− γc −/− mice engrafted with human MEC1 CLL cells, a xenograft model. This treatment also reduced the loss of body weight and improved animal motility. In vitro , CgA enhanced the endothelial barrier integrity and the trans-endothelial migration of MEC1 cells, with a bimodal dose-response curve. Vasostatin-1, but not a larger fragment consisting of N-terminal and central regions of CgA (CgA 1-373 ), inhibited CLL progression in the xenograft model, suggesting that the C-terminal region is crucial for CgA activity and that the N-terminal domain contains a site that is activated by proteolytic cleavage. These findings suggest that circulating full-length CgA and its fragments may contribute to regulate leukemic cell trafficking and reduce tissue infiltration in CLL.

The role of monocytes/macrophages in the development and progression of chronic lymphocytic leukemia (CLL) is poorly understood. Transcriptomic analyses show that monocytes/macrophages and leukemic cells cross talk during CLL progression. Macrophage depletion impairs CLL engraftment, drastically reduces leukemic growth, and favorably impacts mouse survival. Targeting of macrophages by either CSF1R signaling blockade or clodrolip-mediated cell killing has marked inhibitory effects on established leukemia also. Macrophage killing induces leukemic cell death mainly via the TNF pathway and reprograms the tumor microenvironment toward an antitumoral phenotype. CSF1R inhibition reduces leukemic cell load, especially in the bone marrow, and increases circulating CD20(+) leukemic cells. Accordingly, co-targeting TAMs and CD20-expressing leukemic cells provides a survivalbenefit in the mice. These results establish the important role of macrophages in CLL and suggest therapeutic strategies based on interfering with leukemia-macrophage interactions.

Chronic inflammation, including that driven by autoimmunity, is associated with the development of B-cell lymphomas. IL1R8 is a regulatory receptor belonging to the IL1R family, which negatively regulates NF-κB activation following stimulation of IL1R or Toll-like receptor family members. IL1R8 deficiency is associated with the development of severe autoimmune lupus-like disease in lpr mice. We herein investigated whether concomitant exacerbated inflammation and autoimmunity caused by the deficiency of IL1R8 could recapitulate autoimmunity-associated lymphomagenesis. We thus monitored B-cell lymphoma development during the aging of IL1R8-deficient lpr mice, observing an increased lymphoid cell expansion that evolved to diffuse large B-cell lymphoma (DLBCL). Molecular and gene-expression analyses showed that the NF-κB pathway was constitutively activated in Il1r8−/−/lpr B splenocytes. In human DLBCL, IL1R8 had reduced expression compared with normal B cells, and higher IL1R8 expression was associated with a better outcome. Thus, IL1R8 silencing is associated with increased lymphoproliferation and transformation in the pathogenesis of B-cell lymphomas associated with autoimmunity.

In Chronic Lymphocytic Leukemia (CLL), mature CD5+ B cells accumulate in lymphoid organs such as bone marrow and lymph nodes where they proliferate and expand within localized proliferation centers. In vitro and in vivo data suggest that survival and proliferation of CLL cells within proliferation centers may be also dependent on microenvironmental interactions originating from the surrounding cellular elements (e.g. monocyte-derived nurse-like cells, mesenchymal stromal cells, or CD4+ T lymphocytes), that deliver both membrane-bound and soluble signals to CLL cells. In particular, the role of CD4+ T cells in vivo is less defined and data in animal models are conflicting as they appear to sustain CLL clone expansion and survival through CD40L-CD40 interactions, though in approximately 40% of patients with CLL, aggressive leukemic clones appear to be independent of CD40 stimulation. We aimed at clarifying the role of CD4+ T lymphocytes taking advantage of the Eμ-TCL1 mouse model, which develops a disease that mimics aggressive, human CLL. To this aim, we generated genetically modified Eμ-TCL1 mice lacking either CD4+ T cells (TCL1+/+AB0), CD40 (TCL1+/+CD40-/-), or CD8+ T cells (TCL1+/+TAP-/-). In these mice, disease appearance and progression were monitored in lymphoid organs and blood by flow cytometry and immunohistochemistry analyses. Findings were confirmed by adoptive transfer of leukemic clones into mice either lacking CD4+ T cells, or CD40L, or treated with monoclonal antibodies depleting selected T cell populations, or blocking CD40L-CD40 interactions. Interestingly, we observed that CLL clones did not expand in mice either lacking or depleted of CD4+ T cells, thus confirming that CD4+ T cells are essential for CLL development in Eμ-TCL1 mice. On the contrary, in TCL1+/+TAP-/- mice, lacking CD8+ T cells, disease progression was accelerated, suggesting an anti-tumor activity exerted by this subset of T cells. Specificity of CD4+ T cells was marginal for CLL development, as leukemic clones developed regularly in transgenic mice whose CD4+ T cells had TCR with CLL-unrelated specificities. Similarly, TCL1+/+CD40-/- mice developed frank CLL with no differences compared to controls, as well as leukemic clones expanded when transferred into wild type mice treated with monoclonal antibodies blocking CD40, or into CD40L-/- mice, suggesting a dispensable role for CD40/CD40L stimulation in the development of murine CLL. Analysis of peritoneal fluid, spleen, lymph nodes and bone marrow showed similar CLL development in Eμ-TCL1 and TCL1+/+CD40-/- mice. In conclusion, our data demonstrates that CD8+ and CD4+ T cells exert opposite roles in CLL: CD8+ T cells restrain CLL progression, whereas CD4+ T cells support the expansion of CLL clones in Eμ-TCL1 mice through CD40-indipendent, and apparently non-cognate mechanisms. Further studies are warranted to dissect the nature of the molecules, either soluble or membrane-bound, responsible for the interactions occurring between CD4+ T cells and CLL B cells fueling the onset and expansion of CLL cells. Disclosures Ghia: AbbVie: Consultancy, Honoraria, Research Funding; Acerta/AstraZeneca: Consultancy, Honoraria; ArQule: Consultancy, Honoraria; BeiGene: Consultancy, Honoraria; Dynamo: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Juno/Celgene: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Novartis: Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy.

In chronic lymphocytic leukemia (CLL), the non-hematopoietic stromal microenvironment plays a critical role in promoting tumor cell recruitment, activation, survival, and expansion. However, the nature of the stromal cells and molecular pathways involved remain largely unknown. Here, we demonstrate that leukemic B lymphocytes induce the activation of retinoid acid synthesis and signaling in the microenvironment. Inhibition of RA-signaling in stromal cells causes deregulation of genes associated with adhesion, tissue organization and chemokine secretion including the B-cell chemokine CXCL13. Notably, reducing retinoic acid precursors from the diet or inhibiting RA-signaling through retinoid-antagonist therapy prolong survival by preventing dissemination of leukemia cells into lymphoid tissues. Furthermore, mouse and human leukemia cells could be distinguished from normal B-cells by their increased expression of Rarγ2 and RXRα, respectively. These findings establish a role for retinoids in murine CLL pathogenesis, and provide new therapeutic strategies to target the microenvironment and to control disease progression., The stromal microenvironment plays a key role in the expansion of chronic lymphocytic leukemia. Here, the authors use the Eµ-TCL1 mouse model to show that leukemic B-cells induce the activation of retinoic acid synthesis in stromal cells of the lymphoid microenvironment, and that impacting on retinoic acid signalling via diet or chemical inhibition prolonged survival by preventing leukemia dissemination and accumulation in lymphoid tissues.

Multiple myeloma develops primarily inside the bone marrow microenvironment, that confers pro-survival signals and drug resistance. 3D cultures that reproduce multiple myeloma-bone marrow interactions are needed to fully investigate multiple myeloma pathogenesis and response to drugs. To this purpose, we exploited the 3D Rotary Cell Culture System bioreactor technology for myeloma-bone marrow co-cultures in gelatin scaffolds. The model was validated with myeloma cell lines that, as assessed by histochemical and electron-microscopic analyses, engaged contacts with stromal cells and endothelial cells. Consistently, pro-survival signaling and also cell adhesion-mediated drug resistance were significantly higher in 3D than in 2D parallel co-cultures. The contribution of the VLA-4/VCAM1 pathway to resistance to bortezomib was modeled by the use of VCAM1 transfectants. Soluble factor-mediated drug resistance could be also demonstrated in both 2D and 3D co-cultures. The system was then successfully applied to co-cultures of primary myeloma cells-primary myeloma bone marrow stromal cells from patients and endothelial cells, allowing the development of functional myeloma-stroma interactions and MM cell long-term survival. Significantly, genomic analysis performed in a high-risk myeloma patient demonstrated that culture in bioreactor paralleled the expansion of the clone that ultimately dominated in vivo. Finally, the impact of bortezomib on myeloma cells and on specialized functions of the microenvironment could be evaluated. Our findings indicate that 3D dynamic culture of reconstructed human multiple myeloma microenvironments in bioreactor may represent a useful platform for drug testing and for studying tumor-stroma molecular interactions.

Recent studies on splenic marginal zone lymphoma identified distinct mutations in genes belonging to the B-cell receptor and Toll-like receptor signaling pathways, thus pointing to their potential implication in the biology of the disease. However, limited data is available regarding the exact role of TLRs. We aimed at characterizing the expression pattern of TLRs in splenic marginal zone lymphoma cells and their functional impact on the activation, proliferation and viability of malignant cells in vitro. Cells expressed significant levels of TLR1, TLR6, TLR7, TLR8, TLR9 and TLR10 mRNA; TLR2 and TLR4 showed a low, variable pattern of expression among patients whereas TLR3 and TLR5 mRNAs were undetectable; mRNA specific for TLR signaling molecules and adapters was also expressed. At the protein level, TLR1, TLR6, TLR7, TLR9 and TLR10 were detected. Stimulation of TLR1/2, TLR2/6 and TLR9 with their respective ligands triggered the activation of IRAK kinases, MAPK and NF-κB signaling pathways, and the induction of CD86 and CD25 activation molecules, although in a heterogeneous manner among different patient samples. TLR-induced activation and cell viability were also inhibited by a specific IRAK1/4 inhibitor, thus strongly supporting the specific role of TLR signaling in these processes. Furthermore, TLR2/6 and TLR9 stimulation also significantly increased cell proliferation. In conclusion, we demonstrate that splenic marginal zone lymphoma cells are equipped with functional TLR and signaling molecules and that the stimulation of TLR1/2, TLR2/6 and TLR9 may play a role in regulating disease pathobiology, likely promoting the expansion of the neoplastic clone.

Several lines of evidence indirectly suggest that antigenic stimulation through the B‐cell receptor (BCR) supports chronic lymphocytic leukemia (CLL) development. In addition to self‐antigens, a number of microbial antigens have been proposed to contribute to the selection of the immunoglobulins expressed in CLL. How pathogen‐specific BCRs drive CLL development remains, however, largely unexplored. Here, we utilized mouse models of CLL pathogenesis to equip B cells with virus‐specific BCRs and study the effect of antigen recognition on leukemia growth. Our results show that BCR engagement is absolutely required for CLL development. Unexpectedly, however, neither acute nor chronic exposure to virus‐derived antigens influenced leukemia progression. Rather, CLL clones preferentially selected light chains that, when paired with virus‐specific heavy chains, conferred B cells the ability to recognize a broad range of autoantigens. Taken together, our results suggest that pathogens may drive CLL pathogenesis by selecting and expanding pathogen‐specific B cells that cross‐react with one or more self‐antigens.

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignantCD51 B lymphocytes in blood, bone marrow, and lymphoid organs. CD1d-restricted invariant natural killer T (iNKT) cells are innate-like T lymphocytes strongly implicated in tumor surveillance.Weinvestigated the impact of iNKT cells in the natural history of the disease in the Em-Tcl1 (Tcl1) CLL mouse model and 68 CLL patients.Wefound that Tcl1-CLL cells express CD1d and that iNKT cells critically delay disease onset but become functionally impaired upon disease progression. In patients, disease progression correlates with high CD1d expression on CLL cells and impaired iNKT cells. Conversely, disease stability correlates with negative or low CD1d expression on CLL cells and normal iNKT cells, suggesting indirect leukemia control. iNKT cells indeed hinder CLL survival in vitro by restraining CD1d-expressing nurse-like cells, a relevant proleukemia macrophage population. Multivariable analysis identified iNKT cell frequency as an independent predictor of disease progression. Together, these results support the contribution of iNKT cells to CLL immune surveillance and highlight iNKT cell frequency as a prognostic marker for disease progression. © 2017 by The American Society of Hematology.

Toll-like receptors (TLR) are key players in host defence from infection. They recognize a specific set of molecular patterns of microbial origin, immediately trigger an innate immune response, and bridge innate and adaptive immunity. TLR have also been shown to play a role in tumor development. In this context, chronic B-cell malignancies are an interesting example as clonal B lymphocytes remain responsive to and dependent on stimuli originating from the microenvironment which then become crucial for maintaining and propagating the disease. Emerging evidences suggest that, among other microenvironmental elements, TLR ligands may play a role in the pathogenesis of chronic B-cell lymphoid malignancies. Conceivably, their manipulation may find a place in specific settings of treatment of these tumors.

Purpose: The emerging role of Toll-like receptors (TLR) in the pathogenesis of chronic lymphocytic leukemia (CLL) led us to ask whether TLR stimulation may protect CLL cells from drug-induced apoptosis. Experimental Design: We cultured in vitro malignant B cells freshly isolated from 44 patients with CLLs in the presence or the absence of different concentrations of fludarabine before or after 24-hour TLR stimulation with specific ligands and evaluated cell viability, apoptosis, and molecular pathways involved. Results: Heterogeneity was observed among samples. In leukemic cells from patients bearing adverse prognostic factors, TLR stimulation caused a significant increase of protection to fludarabine treatment, whereas this did not occur in the cells from patients with good prognosis. To identify novel molecular mechanisms accounting for the dichotomy of response between the two groups of patients, we conducted an apoptosis gene expression profile on leukemic cells either unstimulated or stimulated with TLR9 ligand. Strikingly, TLR9 stimulation specifically upregulated the expression of lymphotoxin-α in cells where an increased protection to fludarabine treatment was observed. Also, the expression of miR-155-3p was significantly increased after stimulation of distinct TLR in cells where fludarabine treatment was less effective. Conclusions: These results suggest that at least in a proportion of patients, in vitro sensitivity to fludarabine may be modulated by the stimulation of TLR, likely mimicking microenvironmental signals occurring in vivo. Clin Cancer Res; 19(2); 367–79. ©2012 AACR.

Despite a high proportion of patients with primary CNS lymphoma (PCNSL) experiences failure after/during first-line treatment, a few studies focused on salvage therapy are available, often with disappointing results. Herein, we report feasibility and activity of a combination of rituximab, ifosfamide and etoposide (R-IE regimen) in a multicentre series of patients with PCNSL relapsed or refractory to high-dose methotrexate-based chemotherapy. We considered consecutive HIV-negative patients ≤75 years old with failed PCNSL treated with R-IE regimen (rituximab 375 mg/m2, day 0; ifosfamide 2 g/m2/day, days1–3; etoposide 250 mg/m2, day 1; four courses). Twenty-two patients (median age 60 years; range 39–72; male/female ratio: 1:4) received R-IE as second-line (n = 18) or third-line (n = 4) treatment. Eleven patients had refractory PCNSL, and 11 had relapsing disease. Twelve patients had been previously irradiated. Sixty (68%) of the 88 planned courses were actually delivered; only one patient interrupted R-IE because of toxicity. Grade 4 hematological toxicity was manageable; a single case of grade 4 non-hematological toxicity (transient hepatotoxicity) was recorded. Response was complete in six patients and partial in three (overall response rate = 41%; 95%CI: 21–61%). Seven patients were successfully referred to autologous peripheral blood stem cell collection; four responders were consolidated with high-dose chemotherapy supported by autologous stem cell transplant. At a median follow-up of 24 months, eight responders did not experience relapse, two of them died of neurological impairment while in remission. Six patients are alive, with a 2-year survival after relapse of 25 ± 9%. We concluded that R-IE is a feasible and active combination for patients with relapsed/refractory PCNSL. This regimen allows stem cell collection and successful consolidation with high-dose chemotherapy and autologous transplant. Copyright © 2012 John Wiley & Sons, Ltd.

The online version of this article contains supplemental material. BCR signaling is a central pathogenetic pathway in chronic lymphocytic leukemia (CLL). Most CLL cells express BCRs of IgM and IgD isotypes, but the contribution of these isotypes to functional responses remains incompletely defined.We therefore investigated differences between IgM and IgD signaling in freshly isolated peripheral blood CLL cells and in CLL cells cultured with nurselike cells, a model that mimics the lymph node microenvironment. IgM signaling induced prolonged activation of ERK kinases and promoted CLL cell survival, CCL3 and CCL4 chemokine secretion, and downregulation of BCL6, the transcriptional repressor of CCL3. In contrast, IgD signaling induced activation of the cytoskeletal protein HS1, along with F-actin polymerization, which resulted in rapid receptor internalization and failure to support downstream responses, including CLL cell survival and chemokine secretion. IgM and IgD receptor downmodulation, HS1 and ERK activation, chemokine secretion, and BCL6 downregulation were also observed when CLL cells were cocultured with nurselike cells. The Bruton's tyrosine kinase inhibitor ibrutinib effectively inhibited both IgM and IgD isotype signaling. In conclusion, through a variety of functional readouts, we demonstrate very distinct outcomes of IgM and IgD isotype activation in CLL cells, providing novel insight into the regulation of BCR signaling in CLL.

Hypoxia-inducible transcription factors (HIFs) regulate a wide array of adaptive responses to hypoxia and are often activated in solid tumors and hematologic malignancies due to intratumoral hypoxia and emerging new layers of regulation. We found that in chronic lymphocytic leukemia (CLL), HIF-1α is a novel regulator of the interaction of CLL cells with protective leukemia microenvironments and, in turn, is regulated by this interaction in a positive feedback loop that promotes leukemia survival and propagation. Through unbiased microarray analysis, we found that in CLL cells, HIF-1α regulates the expression of important chemokine receptors and cell adhesion molecules that control the interaction of leukemic cells with bone marrow and spleen microenvironments. Inactivation of HIF-1α impairs chemotaxis and cell adhesion to stroma, reduces bone marrow and spleen colonization in xenograft and allograft CLL mouse models, and prolongs survival in mice. Of interest, we found that in CLL cells, HIF-1α is transcriptionally regulated after coculture with stromal cells. Furthermore, HIF-1α messenger RNA levels vary significantly within CLL patients and correlate with the expression of HIF-1α target genes, including CXCR4, thus further emphasizing the relevance of HIF-1α expression to CLL pathogenesis.

Monoclonal B-cell lymphocytosis (MBL), a newly recognized entity found in approximately 3% of normal persons, precedes chronic lymphocytic leukemia. However, MBLs progress into overt malignancy only in a very minor portion of cases, thus raising the clinical concern of whether and how we can discriminate at diagnosis which rare cases will evolve into a fully fledged tumor. Understanding the molecular/biologic features underlying the risk of progression may significantly modify our strategies for correctly managing B-cell premalignant states. MBL cells bear the same chromosomal abnormalities of chronic lymphocytic leukemia. Genome-wide sequencing and animal models indicate that genetic abnormalities disrupting the control of cell growth and survival cooperate with microenvironment-triggered events, mainly represented by antigen-mediated B-cell receptor and coreceptor stimulation, to trigger and fuel clonal expansion. The initial functional activation of survival/proliferation pathways may later become subsidized by autonomous genetic abnormalities (eg, a single mutation) affecting the same or parallel critical signaling pathway(s).

Subgroups of patients with chronic lymphocytic leukemia (CLL) have distinct expression profiles of Toll-like receptor (TLR) pathway–associated genes. To test the hypothesis that signaling through innate immunity receptors may influence the behavior of the malignant clone, we investigated the functional response triggered by the stimulation of TLRs and NOD2 in 67 CLL cases assigned to different subgroups on the basis of immunoglobulin heavy variable (IGHV ) gene usage, IGHV gene mutational status or B-cell receptor (BcR) stereotypy. Differences in the induction of costimulatory molecules and/or apoptosis were observed in mutated versus unmutated CLL. Different responses were also identified in subsets with stereotyped BcRs, underscoring the idea that “subset-biased” innate immunity responses may occur independently of mutational status. Additionally, differential modulation of kinase activities was induced by TLR stimulation of different CLL subgroups, revealing a TLR7-tolerant state for cases belonging to stereotyped subset #4. The distinct patterns of TLR/NOD2 functional activity in cells from CLL subgroups defined by the molecular features of the clonotypic BcRs might prove relevant for elucidating the immune mechanisms underlying CLL natural history and for defining subgroups of patients who might benefit from treatment with specific TLR ligands.

Monoclonal B-cell lymphocytosis (MBL) is classified as chronic lymphocytic leukemia (CLL)–like, atypical CLL, and CD5− MBL. The number of B cells per microliter divides CLL-like MBL into MBL associated with lymphocytosis (usually detected in a clinical setting) and low-count MBL detected in the general population (usually identified during population screening). After a median follow-up of 34 months we reevaluated 76 low-count MBLs with 5-color flow cytometry: 90% of CLL-like MBL but only 44.4% atypical CLL and 66.7% CD5− MBL persisted over time. Population-screening CLL-like MBL had no relevant cell count change, and none developed an overt leukemia. In 50% of the cases FISH showed CLL-related chromosomal abnormalities, including monoallelic or biallelic 13q deletions (43.8%), trisomy 12 (1 case), and 17p deletions (2 cases). The analysis of the T-cell receptor β (TRBV) chains repertoire showed the presence of monoclonal T-cell clones, especially among CD4highCD8low, CD8highCD4low T cells. TRBV2 and TRBV8 were the most frequently expressed genes. This study indicates that (1) the risk of progression into CLL for low-count population-screening CLL-like MBL is exceedingly rare and definitely lower than that of clinical MBL and (2) chromosomal abnormalities occur early in the natural history and are possibly associated with the appearance of the typical phenotype.

Autoimmune cytopenia is a noninfrequent complication of chronic lymphocytic leukemia (CLL) classified into simple and complex autoimmunity, on the basis of the absence or the concomitance of disease progression. Simple-refractory and complex autoimmunity indicate the need of treatment. Here, we review the epidemiology and the biological mechanisms that account for CLL-associated autoimmune disorders and discuss their prognostic relevance and the indication of treatment.Autoimmune disorders can complicate CLL at any stage and even occur in the preleukemic monoclonal B lymphocytosis. CLL cells can act as antigen-presenting cells, possibly inducing the formation of autoreactive T helper cells (through the production of B-cell activator factor and a proliferation-inducing ligand) and nonfunctional T regulatory cells (via CD27-CD70 interaction). Further, nonmalignant lymphocytes may stimulate via CD154-mediated mechanism both tumor growth and the development of autoimmunity, especially after fludarabine-based regimens of therapy. CLL cells tend to produce monoclonal polyreactive autoantibodies suggesting that autoantigen stimulation via B-cell receptor signaling may affect the natural history of CLL. Though the results of multivariate analysis do not allow to separate autoimmune disorders developing in CLL from conventional prognostic factors, the pathogenetic intertwining between autoimmunity and malignant transformation indicates the importance of defining whether the occurrence of autoimmunity in CLL might be considered an autonomous prognostic indicator that influences treatment decisions.Simple-refractory and complex autoimmunity are independent indicators of therapy for CLL. Further, epidemiological and biological studies will help clarifying the prognostic and possibly also the pathogenetic significance of simple autoimmunity.

Chronic lymphocytic leukemia (CLL), an indolent B-cell malignancy frequently diagnosed in the elderly, is characterized by the relentless accumulation of CD5(+) monoclonal B cells that proliferate in the appropriate tissue microenvironments. Despite many advances achieved by molecular and functional studies, our knowledge of the reciprocal relationship between the CLL cell and its microenvironment at the tissue level is still largely incomplete. In this review we present the relevant current information on the tissue microenvironmental features of CLL, focusing on the events that appear to occur in the lymph node. Special attention is devoted to analyzing the properties of both neoplastic and nonneoplastic bystander cells within proliferation centers, the mysterious structures that likely represent the actual proliferative compartment. (C) 2011 Elsevier Inc. All rights reserved. Chronic lymphocytic leukemia (CLL), an indolent B-cell malignancy frequently diagnosed in the elderly, is characterized by the relentless accumulation of CD5(+) monoclonal B cells that proliferate in the appropriate tissue microenvironments. Despite many advances achieved by molecular and functional studies, our knowledge of the reciprocal relationship between the CLL cell and its microenvironment at the tissue level is still largely incomplete. In this review we present the relevant current information on the tissue microenvironmental features of CLL, focusing on the events that appear to occur in the lymph node. Special attention is devoted to analyzing the properties of both neoplastic and nonneoplastic bystander cells within proliferation centers, the mysterious structures that likely represent the actual proliferative compartment. (C) 2011 Elsevier Inc. All rights reserved. Z8 0 ZR 0 ZS 0 ZB 10 "Chronic lymphocytic leukemia (CLL), an indolent B-cell malignancy frequently diagnosed in the elderly, is characterized by the relentless accumulation of CD5+ monoclonal B cells that proliferate in the appropriate tissue microenvironments. Despite many advances achieved by molecular and functional studies, our knowledge of the reciprocal relationship between the CLL cell and its microenvironment at the tissue level is still largely incomplete. In this review we present the relevant current information on the tissue microenvironmental features of CLL, focusing on the events that appear to occur in the lymph node. Special attention is devoted to analyzing the properties of both neoplastic and nonneoplastic bystander cells within proliferation centers, the mysterious structures that likely represent the actual proliferative compartment."

Purpose: NGR-hTNF exploits the tumor-homing peptide asparagine-glycine-arginine (NGR) for selectively targeting TNF-α to an aminopeptidase N overexpressed on cancer endothelial cells. Preclinical synergism with cisplatin was displayed even at low doses. This study primarily aimed to explore the safety of low-dose NGR-hTNF combined with cisplatin in resistant/refractory malignancies. Secondary aims included pharmacokinetics (PKs), pharmacodynamics, and activity. Experimental Design: NGR-hTNF was escalated using a doubling-dose scheme (0.2–0.4–0.8–1.6 μg/m2) in combination with fixed-dose of cisplatin (80 mg/m2), both given intravenously once every three weeks. PKs and circulating TNF-receptors (sTNF-Rs) were assessed over the first three cycles. Results: Globally, 22 patients (12 pretreated with platinum) received a range of one to ten cycles. Consistently with the low-dose range tested, maximum-tolerated dose was not reached. No dose-limiting toxicities (DLTs) were observed at 0.2 (n = 4) and 0.4 μg/m2 (n = 3). One DLT (grade 3 infusion-related reaction) was observed at 0.8 μg/m2. This dose cohort was expanded to six patients without further DLTs. No DLTs were noted also at 1.6 μg/m2 (n = 3). NGR-hTNF exposure increased dose-proportionally without apparent PK interactions with cisplatin. No shedding of sTNF-Rs was detected up to 0.8 μg/m2. At the dose level of 0.8 μg/m2, expanded to 12 patients for activity assessment, a platinum-pretreated lung cancer patient achieved a partial response lasting more than six months and five patients maintained stable disease for a median time of 5.9 months. Conclusions: The combination of NGR-hTNF 0.8 μg/m2 with cisplatin 80 mg/m2 showed favorable toxicity profile and promising antitumor activity. Clin Cancer Res; 17(7); 1964–72. ©2011 AACR.