Unlocking the Potential of Purinergic Signaling in Transplantation.

Purinergic signaling has been recognized as playing an important role in inflammation, angiogenesis, malignancy, diabetes and neural transmission. Activation of signaling pathways downstream from purinergic receptors may also be implicated in transplantation and related vascular injury. Following transplantation, the proinflammatory "danger signal" adenosine triphosphate (ATP) is released from damaged cells and promotes proliferation and activation of a variety of immune cells. Targeting purinergic signaling pathways may promote immunosuppression and ameliorate inflammation. Under pathophysiological conditions, nucleotide-scavenging ectonucleotidases CD39 and CD73 hydrolyze ATP, ultimately, to the anti-inflammatory mediator adenosine. Adenosine suppresses proinflammatory cytokine production and is associated with improved graft survival and decreased severity of graft-versus-host disease. Furthermore, purinergic signaling is involved both directly and indirectly in the mechanism of action of several existing immunosuppressive drugs, such as calcineurin inhibitors and mammalian target of rapamycin inhibitors. Targeting of purinergic receptor pathways, particularly in the setting of combination therapies, could become a valuable immunosuppressive strategy in transplantation. This review focuses on the role of the purinergic signaling pathway in transplantation and immunosuppression and explores possible future applications in clinical practice.

Insights into the pathogenesis of GvHD: what mice can teach us about man.

Acute graft-vs-host disease (GvHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT). Most of the knowledge about the biology of GvHD is derived from mouse models of this disease and therefore a critical analysis of potential advantages and disadvantages of the murine GvHD models is important to classify and understand the findings made in these models. The central events leading up to GvHD were characterized in three phases which includes the tissue damage-phase, the T cell priming-phase and the effector-phase, when the disease becomes clinically overt. The role of individual cytokines, chemokines, transcription factor or receptors was studied in these models by using gene deficient or transgenic mice in the donor or recipient compartments. Besides, numerous studies have been performed in these models to prevent or treat GvHD. Several recent clinical trials were all based on previously reported findings from the mouse model of GvHD such as the trials on CCR5-blockade, donor statin treatment, vorinostat treatment or adoptive transfer of regulatory T cells for GvHD prevention. The different mouse models for GvHD and graft-vs-leukemia effects are critically reviewed and their impact on current clinical practice is discussed.

Vismodegib.

Vismodegib (GDC-0449, Erivedge®) is a novel small molecule antagonist of the hedgehog (Hh) pathway that binds to smoothened (SMO) and leads to inhibition of an aberrant activation of the Hh pathway. Dysregulated Hh signaling results in uncontrolled proliferation in basal cell carcinoma (BCC) and has also been found present in medulloblastoma, and many other cancers such as those of gastrointestinal tract, brain, lung, breast, and prostate. In January 2012, vismodegib became the first agent to target the Hh pathway to receive approval by the United States Food and Drug Administration (FDA) and in July 2013 approval by the European Medicines Agency (EMA) followed for the treatment of adult patients with symptomatic metastatic BCC, or locally advanced BCC inappropriate for surgery or radiotherapy. At the moment, many trials are ongoing to further investigate the role of vismodegib in other malignancies than BCC.

Prognostic factors for survival after allogeneic transplantation in acute lymphoblastic leukemia.

Allogeneic stem cell transplantation (allo-SCT) offers a curative option in adult patients with acute lymphoblastic leukemia (ALL). Prognostic factors for survival after allo-SCT have not been sufficiently defined: pheno-/genotype, patients´ age, conditioning regimens and remission at allo-SCT are under discussion. We analyzed the outcome of 180 consecutive adult ALL-patients undergoing allo-SCT at our center between 1995 and 2018 to identify specific prognostic factors. In our cohort 19% were older than 55 years, 28% had Philadelphia-positive B-ALL, 24% T-ALL. 54% were transplanted in first complete remission (CR1), 13% in CR2 after salvage therapy, 31% reached no remission (8% within first-line, 23% within salvage therapy). In 66% conditioning contained total body irradiation (TBI). With a median follow-up of 10 years, we observed an overall survival of 33% at 10 years, and a progression free survival of 31%. The cumulative incidence of relapse was 41% at 10 years, the cumulative incidence of non-relapse mortality 28%. Acute graft-versus-host disease (GvHD) II°-IV° occurred in 31%, moderate/severe chronic GvHD in 27%. Survival was better in patients reaching CR before allo-SCT and in those receiving TBI. No difference between patients younger/older than 55 years and between different phenotypes was observed. Survival after allo-SCT improved considerably over the last decades.

High-dose chemotherapy and autologous peripheral blood stem cell transplantation in adult patients with high-risk or advanced Ewing and soft tissue sarcoma.

PURPOSE: Despite the availability of combined-modality treatment for Ewing sarcoma (ES) and soft tissue sarcomas (STS), results from independent groups still indicate a poor prognosis for high-risk and metastasized patients. The benefit of high-dose chemotherapy (HDCT) with autologous peripheral blood stem cell transplantation (ASCT) as compared to standard treatment is not defined. METHODS: Here, we report of HDCT in 35 consecutive adult patients with poor-risk ES or rhabdomyosarcoma (n = 11) and STS (n = 24) undergoing ASCT between July 1992 and March 2003. At a median follow-up of 100.6 months after ASCT, 11 patients are alive, with nine in sustained complete remission (CR) and each one in partial remission (PR) and stable disease. Median overall survival (OS) from ASCT was 17.1 months. Response to pretreatment, Karnofsky index > 80%, R (0) resection and first-line ASCT were associated with long-term OS (p < 0.05). CONCLUSION: These data indicate that (1) patients achieving a CR or PR following induction, with preserved performance status and R (0) resection may benefit from ASCT and (2) that this can be an useful therapeutic modality in a subset of patients, in some achieving remarkable responses.

MicroRNA-146a reduces MHC-II expression via targeting JAK/STAT signaling in dendritic cells after stem cell transplantation.

Acute Graft-versus-host disease (GVHD) is a major immunological complication after allogeneic hematopoietic cell transplantation and a better understanding of the molecular regulation of the disease could help to develop novel targeted therapies. Here we found that a G/C polymorphism within the human microRNA-146a (miR-146a) gene of transplant recipients, which causes reduced miR-146a levels, was strongly associated with the risk of developing severe acute GVHD (n=289). In mice, deficiency of miR-146a in the hematopoietic system or transfer of recipient-type miR-146a-/- dendritic cells (DCs) enhanced GVHD, while miR-146a mimic-transfected DCs ameliorated disease. Mechanistically, lack of miR-146a enhanced JAK2-STAT1 pathway activity, which led to higher expression of class II-transactivator (CIITA) and consecutively increased MHCII-levels on DCs. Inhibition of JAK1/2 or CIITA knockdown in DCs prevented miR-146a-/- DC-induced GVHD exacerbation. Consistent with our findings in mice, patients with the miR-146a polymorphism rs2910164 in hematopoietic cells displayed higher MHCII levels on monocytes, which could be targeted by JAK1/2 inhibition. Our findings indicate that the miR-146a polymorphism rs2910164 identifies patients at high risk for GVHD before allo-HCT. Functionally we show that miR-146a acts as a central regulator of recipient-type DC activation during GVHD by dampening the pro-inflammatory JAK-STAT/CIITA/MHCII axis, which provides a scientific rationale for early JAK1/2 inhibition in selected patients.

Evaluation of immunomodulatory treatment based on conventional and lineage-specific chimerism analysis in patients with myeloid malignancies after myeloablative allogeneic hematopoietic cell transplantation.

Both conventional chimerism analysis (CCA) and lineage-specific chimerism analysis (LCA) have potential pitfalls as diagnostic means for the detection of minimal residual disease after allogeneic hematopoietic cell transplantation (aHCT). Therefore, the present study examines the results of both methods in order to determine how predictive consecutive evaluations were, with respect to the risk that the patient would relapse during post-transplant follow-up and with respect to responsiveness to immunomodulatory treatment. A total of 168 individuals with acute myeloid leukemia (AML) (n = 137) and myelo dysplastic syndrome (n = 31) were investigated with CCA and LCA at mean intervals of 24 days (range: 11-116). The median follow-up after myeloablative aHCT was 22 months (range: 4-49). Of 168 patients, 65 experienced a clinical relapse after aHCT. CCA and LCA were comparatively sensitive and specific for relapse at the intervals of chimerism testing employed in this study. Of 32 patients, 10 who were offered donor lymphocyte infusions (DLI) treatment for increasing (n = 29) or stable (n = 3) mixed chimerism (MC) achieved at least transitory CC. The observation that all patients with increasing MC relapsed despite DLI treatment (54%) or withdrawal of immune suppression (24%) indicates that novel strategies to deal with rapidly evolving relapse in AML patients, such as shortening of chimerism monitoring intervals, need to be evaluated.

PO-33 - Heparin suppresses progression of small cell lung cancer (SCLC) in an orthotopic mouse model.

INTRODUCTION: Lung cancer is the leading cause of cancer-related death worldwide. Small cell lung cancer (SCLC) is thereby a highly aggressive neuroendocrine carcinoma representing about 15% of all lung cancer cases. Due to the highly metastatic behavior and multidrug resistance, the long-term survival of patients is very low. AIM: Current clinical studies revealed an increased survival of SCLC patients treated with heparin. Thus, the role of heparin in SCLC progression was analyzed with the focus on cell adhesion, cell survival and metastasis formation. MATERIALS AND METHODS: Heparins were tested for their capacities to alter migration, adhesion and viability of SCLC cells in vitro as well as tumor growth and metastasis formation in vivo. RESULTS: Unfractionated heparin (UFH) and low molecular weight heparin (LMWH) both strongly inhibited migration as well as adhesion of SCLC cells to fibronectin and stromal cells. In addition, Heparin induced cellular apoptosis and also increased apoptotic effects of conventional chemotherapeutics in vitro. To investigate the role of LMWH on metastasis formation in vivo, an orthotopic xenograft mouse model with spontaneous metastasis formation has been established. The primary tumors in this mouse model show a marked capacity to metastasize to characteristic distant organs, reflecting advanced steps of malignant progression. Treatment of tumor-bearing mice with LMWH suppressed progression of SCLC. CONCLUSIONS: Administration of LMWH in addition to the conventional treatment might reduce metastasis formation and development of chemoresistance, leading to an improved survival rate of patients suffering from SCLC.

Long-term follow-up of therapy-related myelodysplasia and AML patients treated with allogeneic hematopoietic cell transplantation.

The outcome of patients with therapy-related myelodysplasia (t-MDS) or t-AML is very poor. The only curative treatment option implements allogeneic hematopoietic cell transplantation (aHCT); however, long-term follow-up data beyond 5 years are scarce. Here we report on a cohort of 79 consecutive patients with a median age of 58 years (range (r): 20-76) at transplantation and a median follow-up of 7.5 years (r: 0.07-19.0). Only 19 (24.1%) patients were in CR before aHCT. Non-relapse mortality and relapse rates were 23% (95% confidence interval, 15-35%) and 42% (32-55%) at 5 years, and 32% (22-46%) and 44% (34-57%) at 10 years, respectively. Disease-free survival (DFS) and overall survival (OS) rates were 35% (24-46%) and 38% (27-49%) at 5 years, and 24% (14-36%) and 24% (13-36%) at 10 years, respectively. Although cytogenetic aberrations were associated with shorter DFS and higher relapse risk, persistent disease at the time of transplantation, an unrelated donor and patient age were not associated with shorter OS. In conclusion, long-term survival beyond 10 years of t-MDS/t-AML patients after aHCT is possible, even for refractory patients. Therefore, early donor search and rapid transplantation are warranted, also to decrease the risk of disease-related deterioration of patients' performance status.

High grade cardiac lymphoma vitality monitoring by gadolinium-enhanced magnetic resonance imaging (MRI).

Primary cardiac lymphoma (PCL) is a rare disorder with a poor prognosis and response monitoring is often difficult. Delay in the diagnosis and infiltration of cardiac structures contribute to the unfavorable prognosis. We report on a 76-year-old woman who was diagnosed as having an immunoblastic B-cell PCL according to a histology attained by catheter-guided biopsy. Systemic chemotherapy with six cycles of CHOP (Cyclophosphamide, Doxorubicine, Vincristine = Oncovine, Prednisone), combined with the monoclonal anti-CD20 antibody Rituximab induced only a partial remission, based solely on monitoring of tumor size. However, cardiac gadolinium-enhanced magnetic resonance imaging (CMR) disclosed a reduced lymphoma perfusion and, therefore, indicated decreased tumor vitality. Nine months after the final treatment, the cardiac tumor further decreased to 10% of the initial size, and the patient is in sustained remission as monitored by CMR and validated by florine-18 fluorodeoxyglucose positron emission tomography (PET). Determination of PCL perfusion was, in our case, beneficial for clinical decision making on additional therapy.

5-Azacytidine and DLI can induce long-term remissions in AML patients relapsed after allograft.

UNLABELLED: DNA-hypomethylating agents are a viable treatment option for AML/myelodysplastic syndrome (MDS) relapse after allograft by upregulating Ags on blasts before DLI. Seventy-two patients with relapsed AML (n=62), MDS (n=8) and other myeloid neoplasms (n=2) after allograft were treated with low-dose 5-azacytidine and, if feasible, DLI. PATIENT CHARACTERISTICS: median age 62 years (range 20-75), 42% with adverse cytogenetics, 82% not in remission at transplant and 83% received fludarabine-based reduced-toxicity conditioning. Median duration from transplant to 5-azacytidine was 289 days (range 59-2133). Response criteria: CR, temporary disease control or treatment failure. A median of 2.7 courses (range 1-10) were administered; 65 out of 72 patients also received DLI (41 already before 5-azacytidine). Ten patients developed acute GVHD and two succumbed to treatment-related sepsis. CR rate was 9.7% (in two patients lasting >5 years), 44% had temporary disease control (median duration 71 days, range 31-380). Median survival from 5-azacytidine was 108 days, 21 patients proceeded to subsequent transplant. In multivariate analysis, peripheral blood blasts <1% were predictive of longer OS (P=0.03). Taken together, long-term remissions can be induced by this well-tolerated outpatient treatment, particularly in patients without peripheral blood blasts.

Ruxolitinib in corticosteroid-refractory graft-versus-host disease after allogeneic stem cell transplantation: a multicenter survey.

Despite major improvements in allogeneic hematopoietic cell transplantation over the past decades, corticosteroid-refractory (SR) acute (a) and chronic (c) graft-versus-host disease (GVHD) cause high mortality. Preclinical evidence indicates the potent anti-inflammatory properties of the JAK1/2 inhibitor ruxolitinib. In this retrospective survey, 19 stem cell transplant centers in Europe and the United States reported outcome data from 95 patients who had received ruxolitinib as salvage therapy for SR-GVHD. Patients were classified as having SR-aGVHD (n=54, all grades III or IV) or SR-cGVHD (n=41, all moderate or severe). The median number of previous GVHD-therapies was 3 for both SR-aGVHD (1-7) and SR-cGVHD (1-10). The overall response rate was 81.5% (44/54) in SR-aGVHD including 25 complete responses (46.3%), while for SR-cGVHD the ORR was 85.4% (35/41). Of those patients responding to ruxolitinib, the rate of GVHD-relapse was 6.8% (3/44) and 5.7% (2/35) for SR-aGVHD and SR-cGVHD, respectively. The 6-month-survival was 79% (67.3-90.7%, 95% confidence interval (CI)) and 97.4% (92.3-100%, 95% CI) for SR-aGVHD and SR-cGVHD, respectively. Cytopenia and cytomegalovirus-reactivation were observed during ruxolitinib treatment in both SR-aGVHD (30/54, 55.6% and 18/54, 33.3%) and SR-cGVHD (7/41, 17.1% and 6/41, 14.6%) patients. Ruxolitinib may constitute a promising new treatment option for SR-aGVHD and SR-cGVHD that should be validated in a prospective trial.

Donor lymphocyte infusions for multiple myeloma: clinical results and novel perspectives.

Donor lymphocyte infusions (DLIs) provide effective therapy for patients with various hematological malignancies who have relapsed after allogeneic hematopoietic stem cell transplantation (HSCT). In patients with multiple myeloma (MM), DLIs can induce response rates of 40-52%. DLIs were employed as treatment for MM relapse or as prophylaxis for relapse in MM patients undergoing allo-HSCT. The clinically most relevant treatment-related morbidity with DLIs is the occurrence of graft-versus-host disease (GVHD). Secondly, graft failure and the immune escape of extramedullary plasmocytoma have been reported. The fact that previous clinical reports have documented graft-versus-myeloma (GVM) activity without GVHD suggests that at least two distinct immunocompetent cell populations mediating GVHD and/or GVM may exist. Further characterization of the effector cells such as T cells and/or NK cells and their targets may help to clarify the immune response that mediates the GVM effect. This review considers the results of clinical approaches with DLI for MM, with emphasis on strategies to prevent GVHD while preserving the GVM effect. Furthermore, currently investigated molecular antigenic targets for the GVM effect such as MM-specific idiotypic determinant of immunoglobulin variable regions, several PRAME epitopes and antigenic structures encoded by cancer germline-specific genes as candidates for immunotherapy trials are discussed.

Extramedullary vs medullary relapse after autologous or allogeneic hematopoietic stem cell transplantation (HSCT) in multiple myeloma (MM) and its correlation to clinical outcome.

Risk-adapted treatment of multiple myeloma (MM) includes autologous (auto-) and allogeneic (allo-) hematopoietic stem cell transplantation (HSCT). Case reports on extramedullary (EM) compared to bone marrow (BM) relapse after HSCT suggest a dismal prognosis. We compared the outcome of 78 MM patients relapsing after auto- (group A: n = 53) or allo- (group B: n = 25) HSCT, stratified into BM (64 patients) vs EM (14 patients) relapse. The relapse-specific groups were also compared with respect to risk factors, including age, beta2-microglobulin, pretreatment, cytogenetics and stage. EM relapse sites were lungs (5), soft tissue (4), pericardium (2), bone (1), skin (1) and CNS (1). As of May 2004, the overall (OS) and progression-free (PFS) survival after HSCT in patients relapsing from EM sites was not significantly different from BM relapse patients, both after auto- and allo-HSCT. Although MM patients relapsing from EM sites after allo-HSCT used to be regarded as having few therapeutic options, we observed encouraging responses to donor lymphocyte infusions (DLI). Treatment responses to DLIs were observed in 5/9 (56%) BM relapse patients, and in 3/4 (75%) EM relapse patients. These observations suggest that EM relapse after HSCT is common and needs an individualized diagnostic and therapeutic approach in MM during clinical follow-up after HSCT.

Late cytomegalovirus polyradiculopathy following haploidentical CD34+-selected hematopoietic stem cell transplantation.

A 55-year-old man with acute myeloid leukemia in second relapse presented 4 months after haploidentical CD34+-selected hematopoietic stem cell transplantation (HSCT) with symmetric, progressive neurological deficits of the lower extremities. Although there was no molecular evidence for drug resistance in the cerebral-spinal fluid, antiviral combination therapy failed to control the rapidly progressing CMV polyradiculopathy (PRP) and encephalitis, which were confirmed by autopsy studies. Late CMV PRP as an unusual manifestation of CMV disease should be kept in mind in patients with suggestive neurological symptoms after HSCT.

Evidence for a graft-versus-mast-cell effect after allogeneic bone marrow transplantation.

Mast cell leukemia (MCL) is a rare form of aggressive mastocytosis with a reported median survival below 6 months. Casuistic reports suggest the effectiveness of allogeneic bone marrow transplantation (BMT) for MCL. However, these reports lack clear evidence for a graft-versus-mast-cell (GvMC) effect. We prospectively investigated the GvMC at different time points after allogeneic BMT and donor-lymphocyte infusions (DLI). Samples were gathered from a patient with MCL treated with allogeneic BMT from an unrelated HLA identical donor. Parameters for detection of a GvMC effect included flow cytometrical analysis of mast cell (MC) populations in peripheral blood and BM, BM smear and histology, chimerism analysis of flow cytometrically sorted BM CD117+/CD34- MC and testing for anti-mast cell reactivity of donor lymphocytes by interferon (IFN)-gamma ELISPOT. DLIs reduced MC from 5 to 0.5%. MC chimerism analysis demonstrated a complete recipient genotype after BMT, suggesting that the persistent mastocytosis was part of residual neoplastic disease. At 3.7 years after BMT, there is some evidence for relapse. In summary, BMT and DLIs attenuated the mastocytosis from an aggressive to an indolent form and may have improved the patients' prognosis. The in vitro data of our study indicate for the first time the existence of a GvMC effect.

Age-dependent leiomuscular atrophy in vertebral arteries of individuals under low fat diet.

The volumetric density of vascular smooth muscle cells (VSMC) in the proximal vertebral artery was investigated. In order to identify VSMC, paraffin-embedded sections of the proximal vertebral artery, obtained from autopsy specimens, were immunostained for smooth muscle alpha-actin by a modified ABC-technique. The 63 autopsy specimens, including 35 males and 28 females, covered the entire range from 2 months to 85 years. The volumetric density of alpha-actin positive VSMC in the tunica media was morphometrically assayed by the point-counting method. It is important to note that the morphometrical evaluation was performed on arteries obtained from autopsy specimens of the years 1953/54, a post-war time characterized in Germany by low fat diet as compared to the present-day nutrition of most industrial nations. Probably due to their origin, the vertebral arteries showed almost no atheroma. As the main purpose of this study was to find out about the atherosclerosis-independent process of aging, these arteries seemed particularly suitable. The evaluation showed a strictly age-dependent leiomuscular atrophy which became morphometrically evident in early adulthood. The average degree of regression was measured at 0.62% per year. These results may justify the conclusion that the leiomuscular atrophy of the media represents a primary age-related process and does not in any way result as a secondary event from an atheromatous transformation of the intima.

Thrombectomy discloses intravascular growth of chondroid liposarcoma mimicking a long distance vena cava thrombosis.

A 22-year-old woman with a newly detected chondroid liposarcoma located in the iliac muscle was diagnosed as having bilateral pulmonary embolism. Gadolinium-enhanced MRI further revealed a long distance thrombus reaching from the iliac vein to the right atrium. The thrombus was attributed to a hypercoagulability state which has been described for chondroid liposarcoma. High-dose chemotherapy with autologous stem cell support reduced the tumor burden and led to a symptom-free interval of 6 months. Despite therapeutic anticoagulation, repeated imaging showed no reduction or remodeling of the thrombus. However, when the thrombus progressed again, the patient underwent cardiac surgery and histology revealed the intravascular growth of the known chondroid liposarcoma. We conclude that in sarcoma patients intravascular tumor growth must be kept in mind when imaging is suggestive for thrombosis.

[The role of microRNAs for immunoregulation after allogeneic hematopoietic cell transplantation]. / Mikro-RNA: Rolle bei der Immunregulation nach allogener hämatopoetischer Stammzelltransplantation.

Immunoregulation following allogeneic hematopoietic stem cell transplantation (allo-HSCT) involves the delicate balance between the desirable graft-versus-leukemia (GvL) effect and the prevention of the undesirable graft-versus-host disease (GvHD). Emerging evidence has shown that microRNAs (miRNAs) play a role in the pathogenesis of different inflammatory and malignant diseases. Especially in autoimmune diseases, allergy and GvHD numerous dysregulated miRNAs have been identified. In this review, we provide an overview of current knowledge about the role of miRNAs in the immunoregulation after allo-HSCT. Moreover, we give an outlook on potential new diagnostic and therapeutic approaches, including the use of miRNAs as clinical biomarkers and the manipulation of immune responses using miRNA mimetics.