Therapeutic PD-1 pathway blockade augments with other modalities of immunotherapy T-cell function to prevent immune decline in ovarian cancer.

The tumor microenvironment mediates induction of the immunosuppressive programmed cell death-1 (PD-1) pathway, and targeted interventions against this pathway can help restore antitumor immunity. To gain insight into these responses, we studied the interaction between PD-1 expressed on T cells and its ligands (PD-1:PD-L1, PD-1:PD-L2, and PD-L1:B7.1), expressed on other cells in the tumor microenvironment, using a syngeneic orthotopic mouse model of epithelial ovarian cancer (ID8). Exhaustion of tumor-infiltrating lymphocytes (TIL) correlated with expression of PD-1 ligands by tumor cells and tumor-derived myeloid cells, including tumor-associated macrophages (TAM), dendritic cells, and myeloid-derived suppressor cells (MDSC). When combined with GVAX or FVAX vaccination (consisting of irradiated ID8 cells expressing granulocyte macrophage colony-stimulating factor or FLT3 ligand) and costimulation by agonistic α-4-1BB or TLR 9 ligand, antibody-mediated blockade of PD-1 or PD-L1 triggered rejection of ID8 tumors in 75% of tumor-bearing mice. This therapeutic effect was associated with increased proliferation and function of tumor antigen-specific effector CD8(+) T cells, inhibition of suppressive regulatory T cells (Treg) and MDSC, upregulation of effector T-cell signaling molecules, and generation of T memory precursor cells. Overall, PD-1/PD-L1 blockade enhanced the amplitude of tumor immunity by reprogramming suppressive and stimulatory signals that yielded more powerful cancer control.

Maintaining dendritic cell viability in culture.

When mouse dendritic cells (DCs) are isolated from tissues, purified and placed in a nutritive culture they die more rapidly than would be expected from their normal turnover in vivo. This can distort culture assays of DC function. We therefore tested several approaches to prolonging DC survival in culture. Of several cytokines tested granulocyte-macrophage colony stimulating factor was most effective at preserving the viability of conventional DCs (cDCs) but was ineffective for plasmacytoid DCs (pDCs). Surprisingly, Fms-like tyrosine kinase 3 ligand, crucial for DC development, produced only a marginal improvement in DC survival in culture, and interleukin-3, reported to prevent apoptosis of human pDCs, produced only a minor improvement in survival of mouse DCs. Genetic manipulation of cell death pathways was also tested, to avoid activation effects exerted by cytokine signalling. The isolation of DCs from mice overexpressing Bcl-2 was especially effective in maintaining pDC viability but gave a lesser improvement in cDC viability. DCs isolated from Bim(-/-)Noxa(-/-) mice also showed improved culture survival, but in this case with pDCs showing the least improvement.

Adaptive sequence evolution in a color gene involved in the formation of the characteristic egg-dummies of male haplochromine cichlid fishes.

BACKGROUND: The exceptionally diverse species flocks of cichlid fishes in East Africa are prime examples of parallel adaptive radiations. About 80% of East Africa's more than 1 800 endemic cichlid species, and all species of the flocks of Lakes Victoria and Malawi, belong to a particularly rapidly evolving lineage, the haplochromines. One characteristic feature of the haplochromines is their possession of egg-dummies on the males' anal fins. These egg-spots mimic real eggs and play an important role in the mating system of these maternal mouthbrooding fish. RESULTS: Here, we show that the egg-spots of haplochromines are made up of yellow pigment cells, xanthophores, and that a gene coding for a type III receptor tyrosine kinase, colony-stimulating factor 1 receptor a (csf1ra), is expressed in egg-spot tissue. Molecular evolutionary analyses reveal that the extracellular ligand-binding and receptor-interacting domain of csf1ra underwent adaptive sequence evolution in the ancestral lineage of the haplochromines, coinciding with the emergence of egg-dummies. We also find that csf1ra is expressed in the egg-dummies of a distantly related cichlid species, the ectodine cichlid Ophthalmotilapia ventralis, in which markings with similar functions evolved on the pelvic fin in convergence to those of the haplochromines. CONCLUSION: We conclude that modifications of existing signal transduction mechanisms might have evolved in the haplochromine lineage in association with the origination of anal fin egg-dummies. That positive selection has acted during the evolution of a color gene that seems to be involved in the morphogenesis of a sexually selected trait, the egg-dummies, highlights the importance of further investigations of the comparative genomic basis of the phenotypic diversification of cichlid fishes.

Saturable metabolism of continuous high-dose ifosfamide with mesna and GM-CSF: a pharmacokinetic study in advanced sarcoma patients. Swiss Group for Clinical Cancer Research (SAKK).

BACKGROUND: The aim of this study was to assess the pharmacology, toxicity and activity of high-dose ifosfamide mesna +/- GM-CSF administered by a five-day continuous infusion at a total ifosfamide dose of 12-18 g/m2 in adult patients with advanced sarcomas. PATIENTS AND METHODS: Between January 1991 and October 1992 32 patients with advanced or metastatic sarcoma were entered the study. Twenty-seven patients were pretreated including twenty-three with prior ifosfamide at less than 8 g/m2 total dose/cycle. In 25 patients (27 cycles) extensive pharmacokinetic analyses were performed. RESULTS: The area under the plasma concentration-time curve (AUC) for ifosfamide increased linearly with dose while the AUC's of the metabolites measured in plasma by thin-layer chromatography did not increase with dose, particularly that of the active metabolite isophosphoramide mustard. Furthermore the AUC of the inactive carboxymetabolite did not increase with dose. Interpatient variability of pharmacokinetic parameters was high. Dose-limiting toxicity was myelosuppression at 18 g/m2 total dose with grade 4 neutropenia in five of six patients and grade 4 thrombocytopenia in four of six patients. Therefore the maximum tolerated dose was considered to be 18 g/m2 total dose. There was one CR and eleven PR in twenty-nine evaluable patients (overall response rate 41%). CONCLUSION: Both the activation and inactivation pathways of ifosfamide are non-linear and saturable at high-doses although the pharmacokinetics of the parent drug itself are dose linear. Ifosfamide doses greater than 14-16 g/m2 per cycle appear to result in a relative decrease of the active metabolite isophosphoramide mustard. These data suggest a dose-dependent saturation or even inhibition of ifosfamide metabolism by increasing high dose ifosfamide and suggest the need for further metabolic studies.

Hereditary diffuse leukoencephalopathy with axonal spheroids: More than just a rare disease.

Hereditary diffuse leukoencephalopathy with spheroids (HDLS) is a progressive white matter disease with a wide range of clinical symptoms including dementia, behavioral changes, seizures, pyramidal signs, ataxia, and parkinsonism.(1-3) Affected individuals develop symptoms in their early 40s with an average survival time of 10 years. HDLS is inherited as an autosomal dominant trait. Recently, mutations in the colony-stimulating factor 1 receptor gene (CSF-1R) were identified as the genetic cause of HDLS.(4) White matter lesions, easily demonstrated on MRI studies, involve predominantly the frontal lobes and corpus callosum with subsequent cortical atrophy. MRI abnormalities are present prior to symptom onset.(5,6) Histopathology shows widespread myelin and axon destruction with axonal dilations termed spheroids, as well as pigmented macrophages.

Dual Blockade of PD-1 and CTLA-4 Combined with Tumor Vaccine Effectively Restores T-Cell Rejection Function in Tumors.

Tumor progression is facilitated by regulatory T cells (Treg) and restricted by effector T cells. In this study, we document parallel regulation of CD8(+) T cells and Foxp3(+) Tregs by programmed death-1 (PD-1, PDCD1). In addition, we identify an additional role of CTL antigen-4 (CTLA-4) inhibitory receptor in further promoting dysfunction of CD8(+) T effector cells in tumor models (CT26 colon carcinoma and ID8-VEGF ovarian carcinoma). Two thirds of CD8(+) tumor-infiltrating lymphocytes (TIL) expressed PD-1, whereas one third to half of CD8(+) TIL coexpressed PD-1 and CTLA-4. Double-positive (PD-1(+)CTLA-4(+)) CD8(+) TIL had characteristics of more severe dysfunction than single-positive (PD-1(+) or CTLA-4(+)) TIL, including an inability to proliferate and secrete effector cytokines. Blockade of both PD-1 and CTLA-4 resulted in reversal of CD8(+) TIL dysfunction and led to tumor rejection in two thirds of mice. Double blockade was associated with increased proliferation of antigen-specific effector CD8(+) and CD4(+) T cells, antigen-specific cytokine release, inhibition of suppressive functions of Tregs, and upregulation of key signaling molecules critical for T-cell function. When used in combination with GVAX vaccination (consisting of granulocyte macrophage colony-stimulating factor-expressing irradiated tumor cells), inhibitory pathway blockade induced rejection of CT26 tumors in 100% of mice and ID8-VEGF tumors in 75% of mice. Our study indicates that PD-1 signaling in tumors is required for both suppressing effector T cells and maintaining tumor Tregs, and that PD-1/PD-L1 pathway (CD274) blockade augments tumor inhibition by increasing effector T-cell activity, thereby attenuating Treg suppression. Cancer Res; 73(12); 3591-603. ©2013 AACR.

CSF1R mutations link POLD and HDLS as a single disease entity.

OBJECTIVE: Pigmented orthochromatic leukodystrophy (POLD) and hereditary diffuse leukoencephalopathy with axonal spheroids (HDLS) are rare neurodegenerative disorders characterized by cerebral white matter abnormalities, myelin loss, and axonal swellings. The striking overlap of clinical and pathologic features of these disorders suggested a common pathogenesis; however, no genetic or mechanistic link between POLD and HDLS has been established. Recently, we reported that mutations in the colony-stimulating factor 1 receptor (CSF1R) gene cause HDLS. In this study, we determined whether CSF1R mutations are also a cause of POLD. METHODS: We performed sequencing of CSF1R in 2 pathologically confirmed POLD families. For the largest family (FTD368), a detailed case report was provided and brain samples from 2 affected family members previously diagnosed with POLD were re-evaluated to determine whether they had HDLS features. In vitro functional characterization of wild-type and mutant CSF1R was also performed. RESULTS: We identified CSF1R mutations in both POLD families: in family 5901, we found c.2297T>C (p.M766T), previously reported by us in HDLS family CA1, and in family FTD368, we identified c.2345G>A (p.R782H), recently reported in a biopsy-proven HDLS case. Immunohistochemical examination in family FTD368 showed the typical neuronal and glial findings of HDLS. Functional analyses of CSF1R mutant p.R782H (identified in this study) and p.M875T (previously observed in HDLS), showed a similar loss of CSF1R autophosphorylation of selected tyrosine residues in the kinase domain for both mutations when compared with wild-type CSF1R. CONCLUSIONS: We provide the first genetic and mechanistic evidence that POLD and HDLS are a single clinicopathologic entity.

Atopic dermatitis-like disease and associated lethal myeloproliferative disorder arise from loss of Notch signaling in the murine skin.

BACKGROUND: The Notch pathway is essential for proper epidermal differentiation during embryonic skin development. Moreover, skin specific loss of Notch signaling in the embryo results in skin barrier defects accompanied by a B-lymphoproliferative disease. However, much less is known about the consequences of loss of Notch signaling after birth. METHODOLOGY AND PRINCIPAL FINDINGS: To study the function of Notch signaling in the skin of adult mice, we made use of a series of conditional gene targeted mice that allow inactivation of several components of the Notch signaling pathway specifically in the skin. We demonstrate that skin-specific inactivation of Notch1 and Notch2 simultaneously, or RBP-J, induces the development of a severe form of atopic dermatitis (AD), characterized by acanthosis, spongiosis and hyperkeratosis, as well as a massive dermal infiltration of eosinophils and mast cells. Likewise, patients suffering from AD, but not psoriasis or lichen planus, have a marked reduction of Notch receptor expression in the skin. Loss of Notch in keratinocytes induces the production of thymic stromal lymphopoietin (TSLP), a cytokine deeply implicated in the pathogenesis of AD. The AD-like associated inflammation is accompanied by a myeloproliferative disorder (MPD) characterized by an increase in immature myeloid populations in the bone marrow and spleen. Transplantation studies revealed that the MPD is cell non-autonomous and caused by dramatic microenvironmental alterations. Genetic studies demontrated that G-CSF mediates the MPD as well as changes in the bone marrow microenvironment leading to osteopenia. SIGNIFICANCE: Our data demonstrate a critical role for Notch in repressing TSLP production in keratinocytes, thereby maintaining integrity of the skin and the hematopoietic system.

The effect of endocrine responsiveness on high-risk breast cancer treated with dose-intensive chemotherapy: results of International Breast Cancer Study Group Trial 15-95 after prolonged follow-up.

BACKGROUND: The role of adjuvant dose-intensive chemotherapy and its efficacy according to baseline features has not yet been established. PATIENTS AND METHODS: Three hundred and forty-four patients were randomized to receive seven courses of standard-dose chemotherapy (SD-CT) or three cycles of dose-intensive epirubicin and cyclophosphamide (epirubicin 200 mg/m(2) plus cyclophosphamide 4 mg/m(2) with filgrastim and progenitor cell support). All patients were assigned tamoxifen at the completion of chemotherapy. The primary end point was disease-free survival (DFS). This paper updates the results and explores patterns of recurrence according to predicting baseline features. RESULTS: At 8.3-years median follow-up, patients assigned DI-EC had a significantly better DFS compared with those assigned SD-CT [8-year DFS percent 47% and 37%, respectively, hazard ratio (HR) 0.76; 95% confidence interval 0.58-1.00; P = 0.05]. Only patients with estrogen receptor (ER)-positive disease benefited from the DI-EC (HR 0.61; 95% confidence interval 0.39, 0.95; P = 0.03). CONCLUSIONS: After prolonged follow-up, DI-EC significantly improved DFS, but the effect was observed only in patients with ER-positive disease, leading to the hypothesis that efficacy of DI-EC may relate to its endocrine effects. Further studies designed to confirm the importance of endocrine responsiveness in patients treated with dose-intensive chemotherapy are encouraged.

Long-term results of a multicenter SAKK trial on high-dose ifosfamide and doxorubicin in advanced or metastatic gynecologic sarcomas.

BACKGROUND: Dose intensive chemotherapy has not been tested prospectively for the treatment of gynecologic sarcomas. We investigated the antitumor activity and toxicity of high-dose ifosfamide and doxorubicin, in the context of a multidisciplinary strategy for the treatment of advanced and metastatic, not pretreated, gynecologic sarcomas. PATIENTS AND METHODS: Thirty-nine patients were enrolled onto a phase I-II multicenter trial of ifosfamide, 10 g/m2 as a continuous infusion over 5 days, plus doxorubicin intravenously, 25 mg/m2/day for 3 days with Mesna and granulocyte-colony-stimulating factor every 21 days. Salvage therapy was allowed after chemotherapy. RESULTS: Among the 37 evaluable patients, the tumor was locally advanced (n = 11), with concomitant distant metastases (n = 5) or with distant metastases only (n = 21). After a median of three (range 1-7) chemotherapy cycles, six patients experienced a complete response and 12 a partial response for an overall response rate of 49% (95% CI 32% to 66%). The response rate was higher in poorly differentiated tumors (62%) compared with moderately well differentiated ones (18%), but was not different according to histology subtypes. Eleven patients had salvage therapy, either immediately following chemotherapy (n = 7) or at time of progression (n = 4). With a median follow-up time of 5 years, the median overall survival was 30.5 months. Hematological toxicity was as expected neutropenia, thrombopenia and anemia > or = grade 3 at 50%, 34% and 33% of cycles respectively. No toxic death occurred. CONCLUSIONS: High-dose ifosfamide plus doxorubicin is an active regimen for all subtypes of gynecological sarcomas. Its toxicity was manageable in a multicentric setting. The prolonged survival might be due to the multidisciplinary strategy that was possible in one-third of the patients.

Awakening dormant haematopoietic stem cells.

Haematopoietic stem cells (HSCs) in mouse bone marrow are located in specialized niches as single cells. During homeostasis, signals from this environment keep some HSCs dormant, which preserves long-term self-renewal potential, while other HSCs actively self renew to maintain haematopoiesis. In response to haematopoietic stress, dormant HSCs become activated and rapidly replenish the haematopoietic system. Interestingly, three factors - granulocyte colony-stimulating factor, interferon-alpha and arsenic trioxide - have been shown to efficiently activate dormant stem cells and thereby could break their resistance to anti-proliferative chemotherapeutics. Thus, we propose that two-step strategies could target resistant leukaemic stem cells by priming tumours with activators of dormancy followed by chemotherapy or targeted therapies.

G-CSF-induced remission in two cases of acute myeloid leukemia.

We report on two elderly patients with newly diagnosed acute myeloid leukemia (AML) who were treated in palliative intention because of comorbidities and intermediate or poor risk cytogenetics. Both received G-CSF to reduce the risk of infection related to neutropenia. Interestingly, one patient achieved a full hematological remission and the other a peripheral remission with dramatic reduction of the bone marrow blast count. Although a direct therapeutic effect of myeloid growth factors seems to be unusual in AML, the use of G-CSF or GM-CSF may be recommended in patients such as elderly patients who are not suited for intensive chemotherapy.

Innate Immune Sensing of Fusarium culmorum by Mouse Dendritic Cells.

Chronic inhalation of grain dust is associated with asthma and chronic bronchitis in grain worker populations. Exposure to fungal particles was postulated to be an important etiologic agent of these pathologies. Fusarium species frequently colonize grain and straw and produce a wide array of mycotoxins that impact human health, necessitating an evaluation of risk exposure by inhalation of Fusarium and its consequences on immune responses. Data showed that Fusarium culmorum is a frequent constituent of aerosols sampled during wheat harvesting in the Vaud region of Switzerland. The aim of this study was to examine cytokine/chemokine responses and innate immune sensing of F. culmorum in bone-marrow-derived dendritic cells and macrophages. Overall, dendritic cells and macrophages responded to F. culmorum spores but not to its secreted components (i.e., mycotoxins) by releasing large amounts of macrophage inflammatory protein (MIP)-1α, MIP-1β, MIP-2, monocyte chemoattractant protein (MCP)-1, RANTES, and interleukin (IL)-12p40, intermediate amounts of tumor necrosis factor (TNF), IL-6, IL-12p70, IL-33, granulocyte colony-stimulating factor (G-CSF), and interferon gamma-induced protein (IP-10), but no detectable amounts of IL-4 and IL-10, a pattern of mediators compatible with generation of Th1 or Th17 antifungal protective immune responses rather than with Th2-dependent allergic responses. The sensing of F. culmorum spores by dendritic cells required dectin-1, the main pattern recognition receptor involved in β-glucans detection, but likely not MyD88 and TRIF-dependent Toll-like receptors. Taken together, our results indicate that F. culmorum stimulates potently innate immune cells in a dectin-1-dependent manner, suggesting that inhalation of F. culmorum from grain dust may promote immune-related airway diseases in exposed worker populations.

Proteasome inhibition reduces proliferation, collagen expression, and inflammatory cytokine production in nasal mucosa and polyp fibroblasts

Proteasome inhibitors, used in cancer treatment for their proapoptotic effects, have anti-inflammatory and antifibrotic effects on animal models of various inflammatory and fibrotic diseases. Their effects in cells from patients affected by either inflammatory or fibrotic diseases have been poorly investigated. Nasal polyposis is a chronic inflammatory disease of the sinus mucosa characterized by tissue inflammation and remodeling. We tested the hypothesis that proteasome inhibition of nasal polyp fibroblasts might reduce their proliferation and inflammatory and fibrotic response. Accordingly, we investigated the effect of the proteasome inhibitor Z-Leu-Leu-Leu-B(OH)2 (MG262) on cell viability and proliferation and on the production of collagen and inflammatory cytokines in nasal polyp and nasal mucosa fibroblasts obtained from surgery specimens. MG262 reduced the viability of nasal mucosa and polyp fibroblasts concentration- and time-dependently, with marked effects after 48 h of treatment. The proteasome inhibitor bortezomib provoked a similar effect. MG262-induced cell death involved loss of mitochondrial membrane potential, caspase-3 and poly(ADP-ribose) polymerase activation, induction of c-Jun phosphorylation, and mitogen-activated protein kinase phosphatase-1 expression. Low concentrations of MG262 provoked growth arrest, inhibited DNA replication and retinoblastoma phosphorylation, and increased expression of the cell cycle inhibitors p21 and p27. MG262 concentration-dependently inhibited basal and transforming growth factor-β-induced collagen mRNA expression and interleukin (IL)-1β-induced production of IL-6, IL-8, monocyte chemoattractant protein-1, regulated on activation normal T cell expressed and secreted, and granulocyte/macrophage colony-stimulating factor in both fibroblast types. MG262 inhibited IL-1β/tumor necrosis factor-α-induced activation of nuclear factor-κB. We conclude that noncytotoxic treatment with MG262 reduces the proliferative, fibrotic, and inflammatory response of nasal fibroblasts, whereas high MG262 concentrations induce apoptosis.

Proteasome inhibition reduces proliferation, collagen expression, and inflammatory cytokine production in nasal mucosa and polyp fibroblasts

Proteasome inhibitors, used in cancer treatment for their proapoptotic effects, have anti-inflammatory and antifibrotic effects on animal models of various inflammatory and fibrotic diseases. Their effects in cells from patients affected by either inflammatory or fibrotic diseases have been poorly investigated. Nasal polyposis is a chronic inflammatory disease of the sinus mucosa characterized by tissue inflammation and remodeling. We tested the hypothesis that proteasome inhibition of nasal polyp fibroblasts might reduce their proliferation and inflammatory and fibrotic response. Accordingly, we investigated the effect of the proteasome inhibitor Z-Leu-Leu-Leu-B(OH)2 (MG262) on cell viability and proliferation and on the production of collagen and inflammatory cytokines in nasal polyp and nasal mucosa fibroblasts obtained from surgery specimens. MG262 reduced the viability of nasal mucosa and polyp fibroblasts concentration- and time-dependently, with marked effects after 48 h of treatment. The proteasome inhibitor bortezomib provoked a similar effect. MG262-induced cell death involved loss of mitochondrial membrane potential, caspase-3 and poly(ADP-ribose) polymerase activation, induction of c-Jun phosphorylation, and mitogen-activated protein kinase phosphatase-1 expression. Low concentrations of MG262 provoked growth arrest, inhibited DNA replication and retinoblastoma phosphorylation, and increased expression of the cell cycle inhibitors p21 and p27. MG262 concentration-dependently inhibited basal and transforming growth factor-β-induced collagen mRNA expression and interleukin (IL)-1β-induced production of IL-6, IL-8, monocyte chemoattractant protein-1, regulated on activation normal T cell expressed and secreted, and granulocyte/macrophage colony-stimulating factor in both fibroblast types. MG262 inhibited IL-1β/tumor necrosis factor-α-induced activation of nuclear factor-κB. We conclude that noncytotoxic treatment with MG262 reduces the proliferative, fibrotic, and inflammatory response of nasal fibroblasts, whereas high MG262 concentrations induce apoptosis.