Katrin S. Reiners

Serum autotaxin is increased in pruritus of cholestasis, but not of other origin, and responds to therapeutic interventions†‡

Pruritus is a seriously disabling symptom accompanying many cholestatic liver disorders. Recent experimental evidence implicated the lysophospholipase, autotaxin (ATX), and its product, lysophosphatidic acid (LPA), as potential mediators of cholestatic pruritus. In this study, we highlight that increased serum ATX levels are specific for pruritus of cholestasis, but not pruritus of uremia, Hodgkins disease, or atopic dermatitis. Treatment of patients with cholestasis with the bile salt sequestrant, colesevelam, but not placebo, effectively reduced total serum bile salts and fibroblast growth factor 19 levels, but only marginally altered pruritus intensity and ATX activity. Rifampicin (RMP) significantly reduced itch intensity and ATX activity in patients with pruritus not responding to bile salt sequestrants. In vitro, RMP inhibited ATX expression in human HepG2 hepatoma cells and hepatoma cells overexpressing the pregnane X receptor (PXR), but not in hepatoma cells in which PXR was knocked down. Treatment of severe, refractory pruritus by the molecular adsorbents recirculation system or nasobiliary drainage improved itch intensity, which, again, correlated with the reduction of ATX levels. Upon reoccurrence of pruritus, ATX activity returned to pretreatment values. Conclusion: Serum ATX activity is specifically increased in patients with cholestatic, but not other forms of, systemic pruritus and closely correlates with the effectiveness of therapeutic interventions. The beneficial antipruritic action of RMP may be explained, at least partly, by the PXR‐dependent transcriptional inhibition of ATX expression. Thus, ATX likely represents a novel therapeutic target for pruritus of cholestasis. (HEPATOLOGY 2012)

A genome-wide association study of Hodgkin's lymphoma identifies new susceptibility loci at 2p16.1 ( REL ), 8q24.21 and 10p14 ( GATA3 )

To identify susceptibility loci for classical Hodgkins lymphoma (cHL), we conducted a genome-wide association study of 589 individuals with cHL (cases) and 5,199 controls with validation in four independent samples totaling 2,057 cases and 3,416 controls. We identified three new susceptibility loci at 2p16.1 (rs1432295, REL, odds ratio (OR) = 1.22, combined P = 1.91 × 10−8), 8q24.21 (rs2019960, PVT1, OR = 1.33, combined P = 1.26 × 10−13) and 10p14 (rs501764, GATA3, OR = 1.25, combined P = 7.05 × 10−8). Furthermore, we confirmed the role of the major histocompatibility complex in disease etiology by revealing a strong human leukocyte antigen (HLA) association (rs6903608, OR = 1.70, combined P = 2.84 × 10−50). These data provide new insight into the pathogenesis of cHL.

Dendritic cells release HLA-B-associated transcript-3 positive exosomes to regulate natural killer function.

NKp30, a natural cytotoxicity receptor expressed on NK cells is critically involved in direct cytotoxicity against various tumor cells and directs both maturation and selective killing of dendritic cells. Recently the intracellular protein BAT3, which is involved in DNA damage induced apoptosis, was identified as a ligand for NKp30. However, the mechanisms underlying the exposure of the intracellular ligand BAT3 to surface NKp30 and its role in NK-DC cross talk remained elusive. Electron microscopy and flow cytometry demonstrate that exosomes released from 293T cells and iDCs express BAT3 on the surface and are recognized by NKp30-Ig. Overexpression and depletion of BAT3 in 293T cells directly correlates with the exosomal expression level and the activation of NK cell-mediated cytokine release. Furthermore, the NKp30-mediated NK/DC cross talk resulting either in iDC killing or maturation was BAT3-dependent. Taken together this puts forward a new model for the activation of NK cells through intracellular signals that are released via exosomes from accessory cells. The manipulation of the exosomal regulation may offer a novel strategy to induce tumor immunity or inhibit autoimmune diseases caused by NK cell-activation.

Gonadal Function and Fertility in Survivors After Hodgkin Lymphoma Treatment Within the German Hodgkin Study Group HD13 to HD15 Trials

PURPOSE To optimize fertility advice in patients with Hodgkin lymphoma (HL) before therapy and during survivorship, information on the impact of chemotherapy is needed. Therefore, we analyzed gonadal functions in survivors of HL. PATIENTS AND METHODS Women younger than age 40 and men younger than 50 years at diagnosis in ongoing remission at least 1 year after therapy within the German Hodgkin Study Group HD13 to HD15 trials for early- and advanced-stage HL were included. Hormone parameters, menstrual cycle, symptoms of hypogonadism, and offspring were evaluated. RESULTS A total of 1,323 (55%) of 2,412 contacted female and male survivors were evaluable for the current analysis (mean follow-up, 46 and 48 months, respectively). Follicle-stimulating hormone, anti-Müllerian hormone, and inhibin B levels correlated significantly with therapy intensity (P < .001). Low birth rates were observed in survivors after advanced-stage treatment within the observation time (women, 6.5%; men, 3.3%). Regular menstrual cycle was reported by more than 90% of female survivors of early-stage HL (recovery time mostly ≤ 12 months). After six to eight cycles of bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, and prednisone, menstrual activity was strongly related to age (< v ≥ 30 years: 82% v 45%, respectively; P < .001; prolonged recovery time). Thirty-four percent of women age ≥ 30 years suffered severe menopausal symptoms (three- to four-fold more frequently than expected). In contrast, male survivors had mean levels of testosterone within the normal range and reported no increased symptoms of hypogonadism. CONCLUSION The present analysis in a large group of survivors of HL provides well-grounded information on gonadal toxicity of currently used treatment regimens and allows risk-adapted fertility preservation and comprehensive support during therapy and follow-up.

Dendritic cell-derived exosomes as maintenance immunotherapy after first line chemotherapy in NSCLC

ABSTRACT Dendritic cell-derived exosomes (Dex) are small extracellular vesicles secreted by viable dendritic cells. In the two phase-I trials that we conducted using the first generation of Dex (IFN-γ-free) in end-stage cancer, we reported that Dex exerted natural killer (NK) cell effector functions in patients. A second generation of Dex (IFN-γ-Dex) was manufactured with the aim of boosting NK and T cell immune responses. We carried out a phase II clinical trial testing the clinical benefit of IFN-γ-Dex loaded with MHC class I- and class II-restricted cancer antigens as maintenance immunotherapy after induction chemotherapy in patients bearing inoperable non-small cell lung cancer (NSCLC) without tumor progression. The primary endpoint was to observe at least 50% of patients with progression-free survival (PFS) at 4 mo after chemotherapy cessation. Twenty-two patients received IFN-γ-Dex. One patient exhibited a grade three hepatotoxicity. The median time to progression was 2.2 mo and median overall survival (OS) was 15 mo. Seven patients (32%) experienced stabilization of >4 mo. The primary endpoint was not reached. An increase in NKp30-dependent NK cell functions were evidenced in a fraction of these NSCLC patients presenting with defective NKp30 expression. Importantly, MHC class II expression levels of the final IFN-γ-Dex product correlated with expression levels of the NKp30 ligand BAG6 on Dex, and with NKp30-dependent NK functions, the latter being associated with longer progression-free survival. This phase II trial confirmed the capacity of Dex to boost the NK cell arm of antitumor immunity in patients with advanced NSCLC.

Soluble ligands for NK cell receptors promote evasion of chronic lymphocytic leukemia cells from NK cell anti-tumor activity

Natural killer (NK) cells are a major component of the anti-tumor immune response. NK cell dysfunctions have been reported in various hematologic malignancies, including chronic lymphocytic leukemia (CLL). Here we investigated the role of tumor cell-released soluble and exosomal ligands for NK cell receptors that modulate NK cell activity. Soluble CLL plasma factors suppressed NK cell cytotoxicity and down-regulated the surface receptors CD16 and CD56 on NK cells of healthy donors. The inhibition of NK cell cytotoxicity was attributed to the soluble ligand BAG6/BAT3 that engages the activating receptor NKp30 expressed on NK cells. Soluble BAG6 was detectable in the plasma of CLL patients, with the highest levels at the advanced disease stages. In contrast, NK cells were activated when BAG6 was presented on the surface of exosomes. The latter form was induced in non-CLL cells by cellular stress via an nSmase2-dependent pathway. Such cells were eliminated by lymphocytes in a xenograft tumor model in vivo. Here, exosomal BAG6 was essential for tumor cell killing because BAG6-deficient cells evaded immune detection. Taken together, the findings show that the dysregulated balance of exosomal vs soluble BAG6 expression may cause immune evasion of CLL cells.

Rescue of Impaired NK Cell Activity in Hodgkin Lymphoma With Bispecific Antibodies In Vitro and in Patients

Natural killer (NK) cells represent a key component of the innate immune system against cancer. Nevertheless, malignant diseases arise in immunocompetent individuals despite tumor immunosurveillance. Hodgkin lymphoma (HL) is characterized by CD30+ tumor cells and a massive infiltration of immune effector cells in affected lymph nodes. The latter obviously fail to eliminate the malignant cell population. Here, we tested for functional NK cell defects in HL and suggest an improvement of NK function by therapeutic means. We demonstrate that peripheral NK cells (pNK) from patients with HL fail to eliminate HL cell lines in ex vivo killing assays. Impaired NK cell function correlated with elevated serum levels of soluble ligands for NK cell receptors NKp30 (BAG6/BAT3) and NKG2D (MICA), factors known to constrict NK cell function. In vitro, NK cell cytotoxicity could be restored by an NKG2D/NKp30-independent bispecific antibody construct (CD30xCD16A). It artificially links the tumor receptor CD30 with the cytotoxicity NK cell receptor CD16A. Moreover, we observed that NK cells from patients treated with this construct were generally activated and displayed a restored cytotoxicity against HL target cells. These data suggest that reversible suppression of NK cell activity contributes to immune evasion in HL and can be antagonized therapeutically.

Lenalidomide in patients with refractory or multiple relapsed Hodgkin lymphoma

Most Hodgkin Lymphoma (HL) patients can be cured with chemotherapy, radiotherapy or combined modality treatment. However, current treatment is associated with toxicities, such as infertility, cardiovascular damage and secondary malignancies (Fuchs et al, 2006). Moreover, about 10% of all HL patients have refractory disease despite high dose chemotherapy and autologous or allogeneic stem cell transplantation (Cashen & Bartlett, 2008; Laport, 2008; Sureda et al, 2008). Lenalidomide (Revlimid), a thalidomide-derivate, belongs to a novel class of immunomodulatory drugs (IMIDs) approved for the treatment of Multiple Myeloma and Myelodysplastic Syndrome with deletion (-q5) (ChananKhan & Cheson, 2008). Lenalidomide has multiple modes of action, including direct induction of apoptosis in tumour cells, antiangiogenic effects and the activation of immune cells, such as Natural Killer cells and T-cells (Marriott et al, 2001; Bartlett et al, 2004). Apoptosis resistance, increased neoangiogenesis and impaired immunity critically contribute to HL (Re et al, 2005; Enblad et al, 2007). We therefore hypothesized that single agent lenalidomide might be active in HL patients who have failed conventional chemotherapy. Lenalidomide was provided by Celgene (Munich, Germany) for each individual patient within a named patient program. Between February 2007 and November 2008, 12 patients with relapsed or refractory HL were included in this program in four German centres (Table I). Participants gave written informed consent, had no curative treatment options, an Eastern Cooperative Oncology Group performance status £2 and normal organ functions including peripheral blood counts within the normal range. Patients received oral Lenalidomide 25 mg/d for 21 d of a 28-day cycle. Patients were staged and restaged with computed tomography (CT) of the initially involved sites; the use of PET was optional. Response was defined according to the National Cancer institute Sponsored International Working Group criteria (Cheson et al, 1999) and evaluated after two cycles and every other cycle thereafter. Treatment was continued until disease progression or intolerable toxicity occurred. Concomitant anti-thrombotic prophylaxis with oral acetylsalicylic acid 100 mg/d was mandatory. Patients with a history of thrombosis or thrombembolic events received low molecular heparin. All patients were informed regarding the teratogenic risks of lenalidomide and agreed to use effective contraception at least during treatment and 4 weeks afterwards. Toxicities were assessed at each study visit according to National Cancer Institute Common Terminology Criteria (CTC) for Adverse Events version 3.0 (http://ctep.cancer.gov/protocolDevelopment/electronic_ applications/docs/ctcaev3.pdf). Twelve adult patients (median age 34Æ5 years, range 20– 64 years) with relapsed (Patients 4 and 8) or refractory HL (all other patients) were treated with single-agent lenalidomide. Two patients had lymphocyte predominant HL, all other patients had classical HL; nodular sclerosis was the most frequent histological subtype. All patients had relapsed after at least four chemotherapies (mean 6, range 4–9 previous therapies) and 10 of the 12 patients had advanced-stage disease. All but two patients had previously undergone highdose chemotherapy and autologous stem cell transplantation; one patient had relapsed after allogeneic stem cell transplantation. Most patients (10 of 12) had not responded to the previous treatment. One patient discontinued lenalidomide because of asymptomatic pulmonary embolism, which was detected on a routine CT scan. One patient withdrew consent after two cycles of lenalidomide. All other patients completed a minimum of four treatment cycles. None of the patients received concomitant medication other than anticoagulation. Four patients continued the treatment after the first four cycles and three patients are currently still on treatment. The mean number of cycles applied to date was 7 (range 2–25). Overall toxicity was very low with no toxicity occurring with a CTC grade above 2. Non-haematological toxicities observed were one CTC grade-2 pulmonary embolism in a patient who discontinued the treatment and CTC grade-1 constipation and CTC grade-1 dyspnea in two other patients. One patient experienced an episode of CTC grade-2 diarrhoea with positive testing for Norovirus-RNA, and another patient developed a non-squamous, spontaneously resolving CTC grade-2 rash. Haematological toxicity included CTC grade-2 leucopenia in two patients and CTC grade-2 thrombocytopenia in another patient. All patients continued on the same dose level and recovered without further intervention. For two patients (No 7 and 9) the dose of lenalidomide was increased to 25 mg/d continuously after the sixth cycle due to the decision of the treating physician. None of the 12 patients showed radiological evidence of progression after two cycles of lenalidomide. Overall, six of correspondence

Delayed development of chronic lymphocytic leukemia in the absence of macrophage migration inhibitory factor

UNLABELLED Survival of chronic lymphocytic leukemia (CLL) cells depends on stimuli provided by a suitable microenvironment. The factors and mechanisms providing this growth support for CLL cells are not fully understood. We found that plasma levels of macrophage migration inhibitory factor (MIF), a proinflammatory and immunoregulatory chemokine, were elevated in CLL patients. Therefore, we characterized the functional role of MIF in a CLL mouse model. For this purpose, we crossed Eμ-TCL1 mice with MIF knockout (MIF-/-) mice. The resulting TCL1+/wtMIF/ mice showed a delayed onset of leukemia, reduced splenomegaly and hepatomegaly, and a longer survival than TCL1+/wtMIFwt/wt controls. Immunohistochemical examination of the lymphoid organs showed that the numbers of macrophages were significantly reduced in the spleen and bone marrow of TCL1+/wtMIF/ mice compared with TCL1+/wtMIFwt/wt controls. Mechanistic studies in vitro revealed that the absence of MIF rendered CLL cells more susceptible to apoptosis. Accordingly, incubation with an anti-MIF antibody reduced the survival of CLL cells on a macrophage feeder layer. In addition, the migratory activity of TCL1+/wtMIF/ macrophages was decreased compared with TCL1+/wtMIFwt/wt macrophages. Taken together, our results provide evidence that MIF supports the development of CLL by enhancing the interaction of CLL cells with macrophages. KEY POINTS Targeted deletion of the gene for macrophage migration inhibitory factor (MIF) delays development of chronic lymphocytic leukemia and prolongs survival in mice. MIF recruits leukemia-associated macrophages to spleen or liver.

Galectin-1 serum levels reflect tumor burden and adverse clinical features in classical Hodgkin lymphoma

Galectin-1 (Gal1) is a member of a highly conserved family of carbohydrate-binding proteins. It modulates innate and adaptive immune responses and fosters tumor-immune escape. Hodgkin lymphoma (HL) Reed-Sternberg cells overexpress and secrete Gal1, which selectively kills T helper (Th)1 and Th17 cells and cytotoxic T cells and promotes the immunosuppressive Th2/regulatory T-cell-predominant HL microenvironment. We developed a sandwich enzyme-linked immunosorbent assay and assessed serum Gal1 levels in 293 newly diagnosed, previously untreated patients with classical HL (cHL) enrolled in 3 risk-adapted clinical trials. Serum Gal1 levels were significantly higher in patients with cHL than in normal controls (P < .0001). Gal1 serum levels also increased with Ann Arbor stage (P = .012), areas of nodal involvement (P < .0001), and the International Prognostic Score (2-7, P = .019). We conclude that Gal1 serum levels are significantly associated with tumor burden and related clinical features in newly diagnosed cHL patients.