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News Article | November 16, 2016
Site: www.nature.com

We analysed TCGA data for association between mRNA expression level of 16 candidate immune-related genes (ARG1, IL10, FOXP3, CD68, IL12A, IL12B, IFNG, CD8A, CD4, ITGAM (also known as CD11B), CD14, TNF, IL1A, IL1B, IL6 and CCL5) and 5 year overall survival. Illumina HiSeq RNaseqV2 mRNA expression and clinical data for 520 head and neck squamous cell carcinoma samples were downloaded from the TCGA data portal. Median follow-up from diagnosis was 1.8 years with a range of 0.01 years to 17.6 years. Follow-up time was truncated at 5-years for analysis and 200 deaths occurred in this period. For each of the 16 candidate immune response genes, we scored subjects as above (high) or below (low) the median expression and compared survival using a log-rank test at 5% significance. HPV+ patients were stratified into a favourable immune profile if they had expression above the median for the significant genes IL12A, IL12B, IFNG, CD8A and below the median for IL6. Kaplan–Meier curves were plotted for these two groups. Similar methods were used to examine association of these 16 genes with 720 lung adenocarcinoma and 876 gastric carcinoma samples using the publically available data from KM Plotter29. In lung adenocarcinomas, 12 genes were significantly associated with survival; patients were scored as having a favourable immune profile if 7 or more of the 12 significant genes had expression in the favourable direction. In 876 gastric cancer samples, 8 genes were significantly associated with survival. Patients were scored as having a favourable immune profile if 5 out of the 8 genes had expression in the favourable direction. We investigated 66 immune-related genes in four functional classes, 17 genes related to antigen presentation (HLA class I and II molecules), 24 genes surveying T cell activation, 20 innate immune response genes (IL6, CCL7 and others) and 5 genes related to cancer cell signalling. These genes changed expression in response to PI3Kγ inhibition for association with survival in HPV+ and HPV− TCGA HNSCC and lung adenocarcinoma cohorts. Within each cancer type, we scored subjects as above or below the median expression for each gene and compared survival using a log-rank test, using 10% false discovery rate (FDR) within each class as the significance threshold. HPV+ and HPV− HNSCC survival were investigated separately, as HPV− HNSCC generally has a worse prognosis. Within each cohort, patients were classified as having a favourable PI3Kγ immune response profile if they had expression levels above or below the median in the direction of low PI3Kγ activity for the genes identified as significant. We compared the survival experience of favourable versus less-favourable profiles of patients using Kaplan–Meier curves. Out of the 66 experimentally identified PI3Kγ-regulated genes, 43 showed significant association with overall survival in the HPV+ cohort (FDR < 10% within each functional class). Comparison of these genes between HPV+ and HPV− cohorts showed that HPV− samples generally had significantly (P < 0.05) lower expression of 42 genes in the antigen presentation and T cell activation classes, consistent with a pattern of adaptive immune suppression, and higher expression of genes in the innate immune response and cancer cell signalling classes, which were negatively associated with survival. Only MALT1 was not differentially expressed between the two groups (P = 0.7). Pik3cg−/− and Pik3cg−/−,PyMT mice were generated as previously described13. Cd8−/− and Cd4−/− mice with a C57Bl/6J background were purchased from the Jackson Laboratory and crossed with syngeneic Pik3cg−/− mice. All animal experiments were performed with approval from the Institutional Animal Care and Use Committee of the University of California. Animals were euthanized before the IACUC maximum allowable tumour burden of 2 cm3 per mouse was exceeded. Wild-type or Pik3cg−/− 6–8 week-old female or male syngeneic C57Bl/6J (LLC lung, PyMT breast and MEER HPV+ HNSCC) or C3He/J (SSCVII HPV− HNSCC) mice were implanted with 106 tumour cells by subcutaneous injection (LLC, MEER, SCCVII) or by orthotopic injection (PyMT) (n = 10–15) and tumour growth was monitored for up to 30 days. Tumour dimensions were measured once when tumours were palpable. Tumour volumes were calculated using the equation (l2 × w)/2. In some studies, wild-type and Pik3cg−/− mice with LLC tumours were treated with gemcitabine (150 mg kg−1) or saline by intraperitoneal (i.p.) injection on day 7 and day 14 (n = 10). LLC were acquired from ATCC, PyMT were from L. Ellies (University of California), HPV+ MEER were from J. Lee (Cancer Biology Research Center, Sanford Research/USD) and SCCVII squamous carcinoma cells were from S. Schoenberger (La Jolla Institute for Allergy and Immunology). All cell lines were tested for mycoplasma and mouse pathogens and checked for authenticity against the International Cell Line Authentication Committee (ICLAC; http://iclac.org/databases/cross-contaminations/) list. In some studies, mice bearing LLC, PyMT, HPV+ MEER or HPV− HNSCC tumour cells were treated once daily by oral gavage with vehicle (5% NMP and 95% PEG 400), 15 mg kg−1 per day of the PI3Kγ inhibitor IPI-549 or by i.p. injection with 2.5 mg kg−1 twice per day of TG100-115 (ref. 13) beginning on day 8 post-tumour injection and continuing daily until euthanasia. IPI-549 is an orally bioavailable PI3Kγ inhibitor with a long plasma half-life and a K value of 0.29 nM for PI3Kγ with >58-fold weaker binding affinity for the other class I PI3K isoforms17. Enzymatic and cellular assays confirmed the selectivity of IPI-549 for PI3Kγ (>200-fold in enzymatic assays and >140-fold in cellular assays over other class I PI3K isoforms17). To study the effect of IPI-549 on lung tumour growth, LLC tumour cells were passaged three times in C57BL/6 albino male mice. When tumour volume reached 1,500 mm3, tumours were collected and single-cell suspensions were prepared. This tumour cell suspension was implanted subcutaneously in the hind flank of C57BL/6 albino male mice at 106 cells per mouse. Prior to initiating treatment with once daily IPI-549 (15 mg kg−1 orally), groups were normalized on the basis of tumour volume. In some studies, wild-type- and Pik3cg−/−-tumour-bearing mice were treated with 100 μg of anti-CD8 (clone YTS 169.4) or an isotype-control clone (LTF-2) from Bio X Cell administered by i.p. injections on day 7, 10 and 13 of tumour growth. For all tumour experiments, tumour volumes and weights were recorded at death. C57Bl/6J (wild-type) or Pik3cg−/− 6–8 week-old male or female mice (MEER HPV+ HNSCC) or C3He/J (SCCVII HPV− HNSCC) were implanted with tumour cells by subcutaneous injection (106 MEER or 105 SCCVII). In HPV+ MEER studies, wild-type and Pik3cg−/− mice were treated with four doses of 250 μg of anti-PD-1 antibody (clone RMP-14, Bio X Cell) or rat IgG2a isotype control (clone 2A3, Bio X Cell) every 3 days, starting when tumours became palpable on day 11 (n = 12–14 mice per group). Wild-type mice bearing HPV+ tumours were also treated with the PI3Kγ inhibitor TG100-115 (ref. 13) twice per day by i.p. injection, beginning on day 11. Tumour regressions were calculated as a percentage of the difference in tumour volume between the date treatment was initiated and the first date of death of the control group. For HPV− SCCVII studies, C3He/J mice were treated with PI3Kγ inhibitor (2.5 mg kg−1 TG100-115 i.p.) beginning on day 6 post-tumour inoculation and with six doses of anti-PD-1 antibody (250 μg clone RMP-14, Bio X Cell) or rat IgG2a isotype control (clone 2A3, Bio X Cell) every 3 days beginning on day 3 (n = 12 mice per group) or with a combination of the two. Alternatively, mice were treated with 5 mg kg−1 TG100-115 twice per day ± anti-PD-1 (250 μg every 3 days) beginning on day 1 (Fig. 4). Mice that completely cleared HPV+ MEER tumours were re-injected with HPV+ tumour cells contralateral to the initial tumour injection and tumour growth was monitored. The growth and metastasis of spontaneous mammary tumours in female PyMT+ (n = 13) and Pik3cg−/−,PyMT+ (n = 8) mice was evaluated over the course of 0–15 weeks. Total tumour burden was determined by subtracting the total mammary gland mass in PyMT− mice from the total mammary gland mass in PyMT+ mice. Lung metastases were quantified macroscopically and microscopically in H&E tissue sections at week 15. Septic shock was induced in wild-type and Pik3cg−/− mice via i.p. injection of 25 mg kg−1 LPS (Sigma, B5:005). Survival was monitored every 12 h and liver, bone marrow and serum were collected 24 h after LPS injection. C57Bl/6J female mice were implanted with 106 LLC tumour cells by subcutaneous injection. When the average tumour size was 250 mm3, mice were treated by i.p. injection with 1 mg per mouse clodronate or control liposomes (www.clodronateliposomes.com) every 4 days for 2 weeks in combination with daily administration of vehicle or IPI-549 (15 mg kg−1 per day orally). In other studies, 6-week-old female BALB/c mice were injected subcutaneously with 2.5 × 105 CT26 mouse colon carcinoma cells in 100 μl phosphate buffered saline (PBS) in the right flank. Eight days later, tumour-bearing mice were arranged into four groups (n = 15) with an average tumour volume of 70 mm3. Oral administration of IPI-549 (15 mg kg−1) or vehicle (5% NMP and 95% PEG 400) and anti-CSF-1R antibody (50 mg kg−1 i.p. 3× per week, clone AFS98, Bio X Cell) began on day 8 after tumour injection via oral gavage at a 5 ml kg−1 dose volume and continued daily for a total of 18 doses. Six-week-old female BALC/c mice were injected subcutaneously with 2.5 × 105 CT26 mouse colon carcinoma cells in 100 μl PBS in the right flank. On day 8 after tumour injection, tumour-bearing mice were grouped and treated daily with IPI-549 (15 mg kg−1, orally) or vehicle (5%NMP and 95% PEG 400). In addition, mice were injected i.p. with 50 mg kg−1 anti-CD115 (Bio X Cell clone AFS98) or 50 mg kg−1 rat IgG2a isotype control (Bio X Cell clone 2A3) antibodies as described above for a total of three injections. Two days after the final injection mice were euthanized, tumours were digested in a mixture of 0.5 mg ml−1 collagenase IV and 150 U ml−1 DNase I in RPMI-1640 for 30 min at 37 °C and tumour-infiltrating myeloid cells were analysed by flow cytometry. CD11b+Gr1− cells were isolated from single-cell suspensions of LLC tumours from donor mice by fluorescence-activated cell sorting (FACS) or serial magnetic bead isolation. Additionally, for some experiments, primary bone-marrow-derived macrophages were polarized and collected into a single-cell suspension. Purified cells were admixed 1:1 with LLC tumour cells and 5 × 105 total cells were injected subcutaneously into new host mice. Tumour dimensions were measured three times per week beginning on day 7. In antibody blocking studies, CD11b+Gr1− cells were incubated with 5 μg anti-IL12 (clone RD1-5D9) or isotype (clone LTF-2, Bio X Cell) for 30 min before the addition of tumour cells. Mice were additionally treated intradermally with 5 μg of antibody 3 and 6 days after tumour cell inoculation. In some studies, CD11b+Gr1− cells were pre-incubated with inhibitors of arginase (nor-NOHA, 50 μM, Cayman Chemical), iNOS (1400W dihydrocholoride, 100 μM, Tocris), mTOR (rapamycin, 10 μM Calbiochem), or IκKβ (ML120B, 30 μM, Tocris) for 30 min before the addition of tumour cells. Inoculated mice were further treated by intradermal injection with inhibitors at 3 and 6 days after inoculation. Donor C57Bl/6J (WT) or Pik3cg−/− mice were implanted with 106 LLC tumour cells by subcutaneous injection. On day 14 after tumour implantation, CD90.2+, CD4+ or CD8+ cells were harvested by magnetic bead isolation (Miltenyi Biotec). T cells were mixed 1:1 with viable LLC tumour cells. Cell mixtures containing 5 × 105 total cells were injected into the flanks of naive wild-type or Pik3cg−/− mice (n = 8–10 per group). Tumour growth, intratumoral apoptosis and necrosis were investigated over 0–16 days. In other studies, wild-type T cells were incubated at 37 °C and 5% CO for 6 h with 10 or 100 nM IPI-549 (Infinity Pharmaceuticals) or Cal-101 (Selleck Chem). After 6 h, T cells were washed, admixed 1:1 with LLC tumour cells, and 106 total cells were injected subcutaneously into recipient mice. Tumour growth was monitored for 14 days. Tumours were isolated, minced in a Petri dish on ice and then enzymatically dissociated in Hanks balanced salt solution containing 0.5 mg ml−1 collagenase IV (Sigma), 0.1 mg ml−1 hyaluronidase V (Sigma), 0.6 U ml−1 dispase II (Roche) and 0.005 MU ml−1 DNase I (Sigma) at 37 °C for 5–30 min. The duration of enzymatic treatment was optimized for greatest yield of live CD11b+ cells per tumour type. Cell suspensions were filtered through a 70-μm cell strainer. Red blood cells were solubilized with red cell lysis buffer (Pharm Lyse, BD Biosciences) and the resulting suspension was filtered through a cell strainer to produce a single-cell suspension. Cells were washed once with PBS before use in flow cytometry analysis or magnetic bead purification. Thioglycollate-elicited peritoneal macrophages were collected 96 h after i.p. injection of a 3% thioglycollate solution. Cells were collected from the peritoneal cavity in 10 ml of PBS and macrophage enrichment was performed by plating cells in RPMI with 10% FBS and 1% penicillin/streptomycin for 2 h at 37 °C and 5% CO . After 2 h, non-adherent cells were removed with three PBS washes, and cells were analysed via flow cytometry and qPCR analysis. Single-cell suspensions (106 cells in 100 μl total volume) were incubated with aqua live dead fixable stain (Life Technologies), FcR-blocking reagent (BD Biosciences) and fluorescently labelled antibodies and incubated at 4 °C for 1 h. Primary antibodies to cell surface markers directed against F4/80 (BM8), CD45 (30-F11), CD11b (M1/70), Gr1 (RB6-8C5), CD3 (145-2C11), CD4 (GK1.5), CD8 (53-6.7), CD273 (B7-DC), CD274 (B7-H1) were from eBioscience; Ly6C (AL-21), Ly6G (1A8), CD11c (HL3), and MHC-II (AF6-120.1) from BD Pharmingen, CCR2 (475301) from R&D Systems and CD206 (MR5D3) from AbD Serotech. For intracellular staining, cells were fixed, permeabilized using transcription factor staining buffer set (eBioscience) and then incubated with fluorescently labelled antibodies to FoxP3 (FJK-16 s) from eBioscience. Multicolour FACS analysis was performed on a BD Canto RUO 11 colour analyser. All data analysis was performed using the flow cytometry analysis program FloJo (Treestar). Single-cell preparations from bone marrow or tumours were incubated with FcR-blocking reagent (BD Biosciences) and then with 20 μl magnetic microbeads conjugated to antibodies against CD11b, Gr1, CD90.2, CD4 and CD8 (Miltenyi Biotech MACS Microbeads) per 107 cells for 20 min at 4 °C. Cells bound to magnetic beads were then removed from the cell suspension according to the manufacturer’s instructions. For cell sorting, single-cell suspensions were stained with aqua live dead fixable stain (Life Technologies) to exclude dead cells and anti-CD11b-APC (M1/70, eBioscience) and anti-Gr1-FITC (RB6-8C5, eBioscience) antibodies. FACS sorting was performed on a FACS Aria 11 colour high speed sorter at the Flow Cytometry Core at the UC San Diego Center for AIDS Research. Live cells were sorted into the following populations: CD11b+Gr1−, CD11b+Gr1lo, CD11b+Gr1hi and CD11b−Gr1− cells. CD11b-positive cells were defined by increased staining over the isotype control, and Gr1 levels were defined both by comparison to the isotype control and relative staining to other populations. Bone-marrow-derived cells were aseptically collected from 6–8 week-old female mice by flushing leg bones of euthanized mice with PBS, 0.5% BSA, 2 mM EDTA, incubating in red cell lysis buffer (155 mM NH Cl, 10 mM NaHCO and 0.1 mM EDTA) and centrifuging over Histopaque 1083 to purify the mononuclear cells. Approximately 5 × 107 bone-marrow-derived cells were purified by gradient centrifugation from the femurs and tibias of a single mouse. Purified mononuclear cells were cultured in RPMI + 20% serum + 50 ng ml−1 mCSF (PeproTech). Human leukocytes from apheresis blood products were obtained from the San Diego Blood Bank. Cells were diluted in PBS, 0.5% BSA, 2 mM EDTA, incubated in red cell lysis buffer (155 mM NH Cl, 10 mM NaHCO and 0.1 mM EDTA) and centrifuged over Histopaque 1077 to purify mononuclear cells. Approximately 109 bone-marrow-derived cells were purified by gradient centrifugation from one apheresis sample. Purified mononuclear cells were cultured in RPMI + 20% serum + 50 ng ml−1 Human mCSF (PeproTech). Non-adherent cells were removed after 2 h by washing and adherent cells were cultured for 6 days to differentiate macrophages fully. Bone-marrow-derived macrophages were polarized with IFNγ (20 ng ml−1, Peprotech) + LPS (100 ng ml−1, Sigma) or LPS alone for 24 h, or IL4 (20 ng ml−1, Peprotech) for 24–48 h. For inhibitor studies, PI3Kγ inhibitors (1 μM) (IPI-549, Infinity Pharmaceuticals and TG100-115, Targegen/Sanofi-Aventis), rapamycin (10 μM, Selleck), or ML120B (30 μM) were incubated with macrophages 1 h before the addition of polarizing stimuli. Total RNA was harvested from macrophages using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Freshly isolated mouse bone marrow cells from nine wild-type and nine Pik3cg−/− mice were pooled into three replicates sets of wild-type or Pik3cg−/− cells and differentiated into macrophages for 6 days in RPMI + 20% FBS+ 1% penicillin/streptomycin + 50 ng ml−1 mCSF. Each replicate set of macrophages was then treated with mCSF, IL4 or IFNγ/LPS. Macrophages were removed from dishes, and RNA was collected using Qiagen Allprep kit. In addition, RNA was harvested from day 14 (500 mm3) LLC tumours or purified CD11b+Gr1-F480+ TAMs from wild-type (C57BL/6) and Pik3cg−/− mice. RNA was collected using the Qiagen Allprep kit. RNA libraries were prepared from 1 μg RNA per sample for sequencing using standard Illumina protocols. RNA sequencing was performed by the University of California, San Diego Institute for Genomic Medicine. mRNA profiles were generated by single read deep sequencing, in triplicate, using Illumina HiSeq2000. Sequence analysis was performed as previously described16. Sequence files from Illumina HiSeq that passed quality filters were aligned to the mouse transcriptome (mm9 genome build) using the Bowtie2 aligner4. Gene-level count summaries were analysed for statistically significant changes using DESeq. Individual P values were adjusted for multiple testing by calculating Storey’s q values using fdrtooltrimmer. For each gene, the q value is the smallest false discovery rate at which the gene is found significant. We analysed biological processes as defined by the Gene Ontology Consortium. Each gene ontology term defines a set of genes. The entire list of genes, sorted by the q value in ascending order, was subjected to a non-parametric variant of the gene set enrichment analysis (GSEA), in which the parametric Kolmogorov–Smirnov P value was replaced with the exact rank-order P value. We perform a Bonferroni adjustment of gene set P values for the number of gene sets tested. Heat maps of expression levels were created using in-house hierarchical clustering software that implements Ward clustering. The colours qualitatively correspond to fold changes. cDNA was prepared using 1 μg RNA with the qScript cDNA Synthesis Kit (Quanta Biosciences). Sybr green-based qPCR was performed using human and mouse primers to Arg1, Ifng, Il10, Il12p40, Il1b, Il6, Ccl2, Vegfa, Gapdh, Nos2, Tgfb1, Tnfa and mouse H2-Aa, H2-Ab1, H2-Eb1, and H60a (Qiagen QuantiTect Primer Assay). mRNA levels were normalized to Gapdh and reported as relative mRNA expression or fold change. Freshly isolated bone-marrow-derived CD11b+ myeloid cells or differentiated macrophages were transfected by electroporation using an AMAXA mouse macrophage nucleofection kit with 100 nM of siRNA or 2 μg Pik3cgCAAX or pcDNA control plasmid. Non-silencing (Ctrl_AllStars_1) siRNA and Cebpb (MmCebpb_4 and MmCebpb_6), and Mtor (Mm_Frap1_1 and Mm_Frap1_2) siRNAs were purchased from Qiagen. After transfection, cells were cultured for 36–48 h in RPMI containing 10% serum and 10 ng ml−1 mCSF (PeproTech) or polarized as described above. Whole tumours, CD11b+Gr1− cells, CD90.2+ cells, CD4+ cells and CD8+ cells isolated from LLC tumours were lysed in RIPA buffer and total protein concentrations were determined using a BCA protein assay (Pierce). Macrophage supernatants (100 μl) or 500 μg of total protein lysate from tumours were used in ELISAs to detect CCL2, TGFβ, IL1β, TNFα, IL6, IFNγ, IL10, IL12 and granzyme B (ready set go ELISA, eBioscience). Protein expression was normalized to total volume (supernatants) or mg total protein (tumour lysates). The QuantiChrom arginase assay kit (DARG-200, BioAssay Systems) was used to measure arginase activity in primary mouse bone-marrow-derived macrophages from wild-type and Pik3cg−/− mice according to the manufacturer’s instructions. For all conditions, cells were harvested and lysed in 10 mM Tris (pH 7.4) containing 1 μM pepstatin A, 1 μM leupeptin, and 0.4% (w/v) Triton X-100. Samples were centrifuged at 20,000g at 4 °C for 10 min. To measure NFκB and C/EBPβ activation, TransAM NFκB family and C/EBP transcription factor assay kits (43296 and 44196, Active Motif) were used according to the manufacturer’s protocol. Briefly, wild-type and Pik3cg−/− bone-marrow-derived macrophages were stimulated with LPS (100 ng ml−1) or IL4 (20 ng ml−1) and nuclear extracts were prepared in lysis buffer AM2 (Active Motif). Nuclear extracts were incubated with the immobilized consensus sequences and RelA, cRel or C/EBPβ were detected using specific primary antibodies. Quantification was performed via colourimetric readout of absorbance at 450 nm. IL4 and LPS macrophage cultures were solubilized in RIPA buffer containing protease and phosphatase inhibitors. Thirty micrograms of protein was electrophorezed on Biorad precast gradient gels and electroblotted onto PVDF membranes. Proteins were detected by incubation with 1:1,000 dilutions of primary antibodies, washed and incubated with goat anti-rabbit-HRP antibodies and detected after incubation with a chemiluminescent substrate. Primary antibodies directed against Akt (11E7), p-Akt (244F9), IκBα (L35A5), IκKβ (D30C6), p-IκKα/β (16A6), RelA (D14E12), pRelA (93H1), C/EBPβ (#3087), p-CEBPβ (#3082), IRAK1 (D51G7), TBK1 (D1B4) and PI3Kγ (#4252) were from Cell Signaling Technology and pTBK1 (EPR2867(2)) was from Abcam. CD90.2+ tumour-derived T cells were purified from LLC tumour-bearing wild-type and Pik3cg−/− or TG100-115 and control treated mice and then co-incubated with LLC tumour cells (target cells) at 2.5:1, 5:1 and 10:1 ratios of T cells to tumour cells (2 × 103 LLC tumour cells per well) for 6 h. Target cell killing was assayed by collecting the supernatants from each well for measurement of the lactate dehydrogenase release (Cytotox96 non-radioactive cytotoxicity assay kit, Promega). Tumour samples were collected and cryopreserved in OCT, sections (5 μm) were fixed in 100% cold acetone, blocked with 8% normal goat serum for 2 h, and incubated anti-CD8 (53-6.7, 1:50 BD Biosciences) for 2 h at room temperature. Sections were washed three times with PBS and incubated with Alexa594-conjugated secondary antibodies. Slides were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) to identify nuclei. Immunofluorescence images were collected on a Nikon microscope (Eclipse TE2000-U) and analysed using Metamorph image capture and analysis software (Version 6.3r5, Molecular Devices). The detection of apoptotic cells was performed using a TUNEL-assay (ApopTag fluorescein in situ apoptosis detection kit, Promega) according to the manufacturer’s instructions. Slides were washed and mounted in DAKO fluorescent mounting medium. Immunofluorescence images were collected on a Nikon microscope (Eclipse TE2000-U) and analysed with MetaMorph software (version 6.3r5) or SPOT software (version 4.6). Pixels per field or cell number per field were quantified in five 100× fields from ten biological replicates. Primary tumour samples with mRNA expression data were scored as above or below the median expression level, and tested for association with patient survival using a log-rank test at 5% significance. For studies evaluating the effect of drugs on tumour size, tumour dimensions were measured directly before the start of treatment, tumour volumes were computed and mice were randomly assigned to groups so that the mean volume ± s.e.m. of each group was identical. A sample size of ten mice per group provided 80% power to detect a mean difference of 2.25 standard deviation (s.d.) between two groups (based on a two-sample t-test with two-sided 5% significance level). Sample sizes of 15 mice per group provided 80% power to detect one s.d. difference between two groups. Data were normalized to the standard (control). Analysis for significance was performed by one-way ANOVA with a Tukey’s post-hoc test for multiple pairwise testing with more than two groups and by parametric or nonparametric Student’s t-test when only two groups were compared. We used a two-sample t-test (two groups) and ANOVA (multiple groups) when data were normally distributed and a Wilcoxon rank sum test (two groups) when data were not normally distributed. All mouse studies were randomized and blinded; assignment of mice to treatment groups, tumour measurement and tumour analysis was performed by coding mice with randomly assigned mouse number, with the key unknown to operators until experiments were completed. In tumour studies for which tumour size was the outcome, mice removed from the study owing to health concerns were not included in endpoint analyses. All experiments were performed at least twice; n refers to biological replicates. RNA sequencing data can be accessed using numbers GSE58318 (in vitro macrophage samples) and GSE84535 (in vivo tumour and tumour-associated macrophages samples) at www.ncbi.nlm.nih.gov/geo.


News Article | November 7, 2016
Site: globenewswire.com

BASKING RIDGE, N.J., Nov. 07, 2016 (GLOBE NEWSWIRE) -- Caladrius Biosciences, Inc. (NASDAQ:CLBS) (“Caladrius” or the “Company”), a cell therapy company combining an industry-leading development and manufacturing services provider through its subsidiary PCT, LLC a Caladrius Company™ (“PCT”) with a select therapeutic development pipeline, announces financial results for the three and nine months ended September 30, 2016. Business and financial highlights for the third quarter of 2016 and recent weeks include: “Throughout 2016, we consistently increased revenues at our PCT subsidiary and are poised to achieve our stated goal of 2016 annual revenue in excess of $30 million, which represents annual growth of more than 30%. In addition, year-to-date we secured over $50 million in strategic and/or committed financings, about half of which was non-dilutive with the remainder under favorable terms relative to the market rates for companies like ours. We also paid back to our lender over $9 million of long-term debt, significantly reduced our operating expenses and monetized non-core assets, all while advancing our key immune modulation program’s Phase 2 clinical study of CLBS03 to treat T1D,” stated David J. Mazzo, Ph.D., Chief Executive Officer of Caladrius. “We are particularly pleased with the progress of The Sanford Project: T-Rex Study in T1D.  We completed enrollment of the first cohort of 19 patients and were delighted that the DSMB recommended continuation of the study based on a favorable safety assessment. We have begun enrolling patients into the second cohort of this 111-patient study earlier than originally expected and plan to reach the 50% enrollment mark in mid-2017. Enrollment of the 70th patient, which triggers an $8.4 million capital infusion under the terms of the September private placements, is expected to occur shortly thereafter.  We continue to benefit from the support of Sanford Research. In addition to their equity investment, Sanford has agreed to extend operational support at their clinical sites for the remainder of the study. We continue to be excited by the promise of CLBS03, a novel therapeutic being developed to address the significant unmet medical need in this chronic disease that affects children and young adults. “We have made significant achievements across a number of key areas that we believe position Caladrius for continued growth and success throughout the balance of 2016 and beyond,” concluded Dr. Mazzo. Total revenues for the third quarter of 2016 increased 58% to $9.3 million compared with $5.9 million for the third quarter of 2015 and increased 12% compared with $8.3 million for the second quarter of 2016.  Gross margin on revenues was 8% in the third quarter of 2016 compared with 18% in the third quarter of 2015, directly impacted by the non-payment of approximately $600,000 in billings for work the Company performed and billed to a single customer during the third quarter of 2016, which customer is currently experiencing financial difficulties. Accordingly, the Company has delayed revenue recognition until such time as payment is reasonably assured.  The Company continues to work with this client to obtain payment and will recognize any such receipts as revenue in the periods received. Research and development (R&D) expenses for the third quarter of 2016 decreased 58% to $2.6 million compared with $6.3 million for the third quarter of 2015. The majority of current quarter expenses were dedicated to the Company’s immune modulation platform and, specifically, costs related to the T-Rex Study. The decline in R&D expenses over the prior year quarter was primarily related to the discontinuation of non-core R&D programs announced at the beginning of 2016 and related reductions in R&D staffing and departmental costs. Selling, general and administrative (SG&A) expenses for the three months ended September 30, 2016 were $4.9 million, a small reduction from $5.1 million reported for the same period in 2015. This reflected significantly lower operational and compensation-related costs during the current year quarter compared with the prior year quarter, partially offset by higher equity-based compensation expenses compared with the prior year quarter. The operating loss for the third quarter of 2016 was $6.9 million compared with an operating loss of $10.4 million for the third quarter of 2015, reflecting higher revenues and lower R&D expenses. The Company reported a net loss attributable to Caladrius common stockholders for the third quarter of 2016 of $6.9 million, or $1.09 per share, compared with a net loss attributable to Caladrius common stockholders for the third quarter of 2015 of $11.4 million, or $2.06 per share. Total revenues for the nine months ended September 30, 2016 increased 68% to $25.1 million compared with $14.9 million for the first nine months of 2015.  Gross margin for the first nine months of 2016 was 13% compared with 6% for the first nine months of 2015. R&D expenses for the first nine months of 2016 decreased to $12.5 million compared with $20.7 million for the first nine months of 2015. The decline in R&D expenses over the first nine months of 2015 was primarily related to the discontinuation of non-core R&D programs announced at the beginning of 2016 and related reductions in R&D staffing and departmental costs. SG&A expenses decreased to $16.1 million for the first nine months of 2016 compared with $25.0 million for the same period in 2015.  This reflected operational and compensation-related cost reductions, as well as equity-based compensation expenses that were significantly below the prior year SG&A expense levels. The operating loss for the first nine months of 2016 was $25.4 million compared with an operating loss of $54.1 million for the first nine months of 2015. The net loss attributable to Caladrius common stockholders for the nine months ended September 30, 2016 was $26.7 million, or $4.45 per share, compared with a net loss attributable to Caladrius common stockholders for the nine months ended September 30, 2015 of $47.7 million, or $10.40 per share. As of September 30, 2016, Caladrius had cash and cash equivalents of $18.6 million, which included $10.6 million received from the previously announced equity financing in September 2016 and the payment of $3.0 million to Oxford Finance LLC for repayment of long-term debt. Net cash used in operating activities for the nine months ended September 30, 2016 was $20.3 million, compared with $30.5 million for the nine months ended September 30, 2015. Caladrius also invested $2.3 million in capital expenditures, primarily related to improvements to PCT’s Allendale, N.J. manufacturing facility. The Company updates its previous 2016 guidance as follows: As previously announced, Caladrius management will host a conference call to discuss these results and provide a company update today at 5:00 pm Eastern Time. To participate in the conference call, dial 877-562-4460 (U.S.) or 513-438-4106 (international) and provide conference ID 95709220. To access the live webcast, visit the Investor Relations section of the Company’s website at www.caladrius.com/events.  The webcast will be archived on the website for 90 days. Caladrius Biosciences, Inc. is advancing a proprietary platform technology for immunomodulation by pioneering the use of regulatory T cells as an innovative therapy for recent onset type 1 diabetes.  The product candidate, CLBS03, is the subject of an ongoing Phase 2 clinical trial (The Sanford Project: T-Rex study) in collaboration with Sanford Research, and has been granted Orphan Drug and Fast Track designation by the U.S. Food and Drug Administration and Advanced Therapeutic Medicinal Product classification by the European Medicines Agency.  The Company’s PCT subsidiary is a leading development and manufacturing partner to the cell therapy industry.  PCT works with its clients to overcome the fundamental challenges of cell therapy manufacturing by providing a wide range of innovative services including product and process development, GMP manufacturing, engineering and automation, cell and tissue processing, logistics, storage and distribution, as well as expert consulting and regulatory support. PCT and Hitachi Chemical Co., Ltd. have entered into a strategic global collaboration to accelerate the creation of a global commercial cell therapy development and manufacturing enterprise with deep engineering expertise.  For more information, visit www.caladrius.com. This press release contains forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995. Forward-looking statements reflect management’s current expectations, as of the date of this press release, and involve certain risks and uncertainties. All statements other than statements of historical fact contained in this press release are forward-looking statements. The Company’s actual results could differ materially from those anticipated in these forward-looking statements as a result of various factors. Factors that could cause future results to materially differ from the recent results or those projected in forward-looking statements include the “Risk Factors” described in the Company’s Annual Report on Form 10-K filed with the Securities and Exchange Commission (“SEC”) on March 15, 2016, and in the Company’s other periodic filings with the SEC. The Company’s further development is highly dependent on, among other things, future medical and research developments and market acceptance, which are outside of its control.


BASKING RIDGE, N.J., Nov. 02, 2016 (GLOBE NEWSWIRE) -- Caladrius Biosciences, Inc. (NASDAQ:CLBS) (“Caladrius” or the “Company”), a cell therapy company combining an industry-leading development and manufacturing services provider (PCT) with a unique, promising therapeutic development pipeline, announces today that the Company will release financial results for the third quarter of 2016 after the U.S. financial markets close on Monday, November 7, 2016. Caladrius’ management will host a conference call for the investment community beginning at 5:00 pm ET on Monday, November 7, 2016, to discuss the financial results, provide a company update and answer questions. Shareholders and other interested parties may participate in the conference call by dialing 877-562-4460 (U.S.) or 513-438-4106 (international) and providing conference ID 95709220. The call will also be broadcast live on the Internet via the Company’s website at www.caladrius.com/events. The webcast will be archived on the Company’s website for 90 days. About Caladrius Biosciences Caladrius Biosciences, Inc. is advancing a proprietary platform technology for immunomodulation by pioneering the use of T regulatory cells as an innovative therapy for recent onset type 1 diabetes.  The product candidate, CLBS03, is the subject of an ongoing Phase 2 clinical trial (The Sanford Project: T-Rex study) in collaboration with Sanford Research, and has been granted Orphan Drug and Fast Track designation by the U.S. Food and Drug Administration and Advanced Therapeutic Medicinal Product classification by the European Medicines Agency.  The Company’s PCT subsidiary is a leading development and manufacturing partner to the cell therapy industry.  PCT works with its clients to overcome the fundamental challenges of cell therapy manufacturing by providing a wide range of innovative services including product and process development, GMP manufacturing, engineering and automation, cell and tissue processing, logistics, storage and distribution, as well as expert consulting and regulatory support. PCT and Hitachi Chemical Co., Ltd. have entered into a strategic global collaboration to accelerate the creation of a global commercial cell therapy development and manufacturing enterprise with deep engineering expertise.   For more information, visit www.caladrius.com. Forward Looking Statements This press release contains forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995. Forward-looking statements reflect management’s current expectations, as of the date of this press release, and involve certain risks and uncertainties. All statements other than statements of historical fact contained in this press release are forward-looking statements. The Company’s actual results could differ materially from those anticipated in these forward-looking statements as a result of various factors. Factors that could cause future results to materially differ from the recent results or those projected in forward-looking statements include the “Risk Factors” described in the Company’s Annual Report on Form 10-K filed with the Securities and Exchange Commission (“SEC”) on March 15, 2016, and in the Company’s other periodic filings with the SEC. The Company’s further development is highly dependent on, among other things, future medical and research developments and market acceptance, which are outside of its control.


Sanford Research to Provide Operational Support at its Clinical Sites for Remainder of Trial BASKING RIDGE, N.J., Oct. 31, 2016 (GLOBE NEWSWIRE) -- Caladrius Biosciences, Inc. (NASDAQ:CLBS) (“Caladrius” or the “Company”), a cell therapy company combining a unique, promising therapeutic development pipeline with an industry-leading development and manufacturing services provider through its subsidiary PCT, LLC a Caladrius Company™ (“PCT”), today announced that the independent Data Safety Monitoring Board (“DSMB”) for its Phase 2, The Sanford Project: T-Rex Study recommended that the trial proceed with the enrollment of the prescribed second cohort of subjects in the study to meet the target of 111 total participants. The Company’s decision to resume enrollment ahead of the original timeline represents an acceleration of the program. The decision was made in consultation with external clinical advisors based on a favorable safety profile for the first cohort of 19 subjects who reached their 28-day post-treatment follow-up. Sanford Research, the Company’s collaborator for the study, has agreed to extend operational support at its participating clinical sites for the remainder of the Study. The study is evaluating CLBS03 (the Company’s product candidate consisting of autologous expanded regulatory T cells, or Tregs) as a treatment for recent-onset type 1 diabetes (T1D). Caladrius has modified the T-Rex study protocol to allow for an earlier than planned resumption of enrollment for the remaining patients based on the DSMB recommendation after the 28-day, rather than three-month, evaluation of the first cohort. Based on the earlier than originally expected recommencement of Study enrollment and the high interest from subjects and investigators in the Study experienced from the first cohort, the Company now expects to reach the important milestone of treating 50% of subjects by mid-2017. The results from a pre-specified interim analysis of early therapeutic effect for the Study triggered after approximately 50% of subjects reach the 6-month post-treatment follow-up visit is expected to be announced late 2017/early 2018. The enrollment of the 70th subject in the Study, expected to occur in mid-2017, will trigger an expected $8.4 million infusion of capital pursuant to the terms of the recently announced private placements of Caladrius common stock. CLBS03 has received Fast Track and Orphan Drug designations from the FDA as well as Advanced Therapeutic Medicinal Product (ATMP) classification from the European Medicines Agency. “We are delighted to have identified multiple means by which we can accelerate our timeline for the T-Rex study and to have achieved this positive assessment of clinical safety earlier than originally planned. These results augment the existing body of evidence supporting the notion that CLBS03 has a favorable safety profile,” said David J. Mazzo, Ph.D., Chief Executive Officer of Caladrius. “We look forward to efficiently executing on the remainder of the T-Rex study, driving towards an understanding of CLBS03’s therapeutic effect in adolescents with recent-onset type 1 diabetes, starting with the planned interim analysis based upon a 6-month follow-up of ~50% of the targeted enrollment planned to occur late 2017 or early 2018.” Sanford Research (“Sanford”), has extended the agreement with Caladrius to provide operational support for the T-Rex Study beyond the first cohort to the remainder of the study. Sanford will provide and cover the costs of its clinical performance sites. Sanford’s initial support resulted in the enrollment of most of the first cohort of subjects and its recent agreement to make a $5 million equity investment in Caladrius will contribute to the execution of the remainder of the Study. Sanford Research is a non-profit organization that is part of Sanford Health and supports an emerging translational research center focused on finding a cure for T1D, called the Sanford Project. “Patients with type 1 diabetes benefit from having access to the newest and most innovative treatment options like cell therapy,” said David Pearce, Ph.D., executive vice president for Sanford Research. “Sanford understands how essential collaborations with organizations like Caladrius are to discovering those treatments and making them accessible to patients through clinical trials.” The landmark study, which is being conducted in collaboration with Sanford Research, a Sanford Health subsidiary, is a prospective, randomized, placebo-controlled, double-blind Phase 2 clinical trial to evaluate the safety and efficacy of CLBS03 as a treatment for T1D, in 111 subjects age 12 to 17 with recent-onset T1D. Subjects are randomized into one of three groups to receive, through a single administration, either a high dose of CLBS03, a low dose of CLBS03 or placebo. Enrollment of the first cohort of 19 subjects, designated for a preliminary safety evaluation, was completed in August 2016. The key endpoints for the trial are the standard medical and regulatory endpoints for a T1D trial and include preservation of C-peptide, an accepted measure for pancreatic beta cell function; insulin use; severe hypoglycemic episodes; and glucose and hemoglobin A1c levels. For more information on The Sanford Project: T-Rex Study, please visit https://clinicaltrials.gov/ct2/show/NCT02691247. Sanford Health is an integrated health system headquartered in the Dakotas. It is one of the largest health systems in the nation with 45 hospitals and nearly 300 clinics in nine states and four countries. Sanford Health’s 28,000 employees, including more than 1,300 physicians, make it the largest employer in the Dakotas. Nearly $1 billion in gifts from philanthropist Denny Sanford have allowed for several initiatives, including global children's clinics, genomic medicine and specialized centers researching cures for type 1 diabetes, breast cancer and other diseases. For more information, visit sanfordhealth.org. Caladrius Biosciences, Inc. is advancing a proprietary platform technology for immunomodulation by pioneering the use of T regulatory cells as an innovative therapy for recent onset type 1 diabetes.  The product candidate, CLBS03, is the subject of an ongoing Phase 2 clinical trial (The Sanford Project: T-Rex Study) in collaboration with Sanford Research, and has been granted Orphan Drug and Fast Track designation by the U.S. Food and Drug Administration and Advanced Therapeutic Medicinal Product classification by the European Medicines Agency.  The Company’s PCT subsidiary is a leading development and manufacturing partner to the cell therapy industry.  PCT works with its clients to overcome the fundamental challenges of cell therapy manufacturing by providing a wide range of innovative services including product and process development, GMP manufacturing, engineering and automation, cell and tissue processing, logistics, storage and distribution, as well as expert consulting and regulatory support. PCT and Hitachi Chemical Co., Ltd. have entered into a strategic global collaboration to accelerate the creation of a global commercial cell therapy development and manufacturing enterprise with deep engineering expertise.   For more information, visit www.caladrius.com. This press release contains forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995. Forward-looking statements reflect management’s current expectations, as of the date of this press release, and involve certain risks and uncertainties. All statements other than statements of historical fact contained in this press release are forward-looking statements, including statements regarding the enrollment of the second cohort of subjects in the T-Rex Study, the planned interim analysis based upon a 6-month follow-up of approximately 50% of the targeted enrollment, and the enrollment of the 70th subject in the Study. The Company’s actual results could differ materially from those anticipated in these forward-looking statements as a result of various factors. Factors that could cause future results to materially differ from the recent results or those projected in forward-looking statements include the “Risk Factors” described in the Company’s Annual Report on Form 10-K filed with the Securities and Exchange Commission (“SEC”) on March 15, 2016, and in the Company’s other periodic filings with the SEC. The Company’s further development is highly dependent on, among other things, future medical and research developments and market acceptance, which are outside of its control.


BASKING RIDGE, N.J., Feb. 23, 2017 (GLOBE NEWSWIRE) -- Caladrius Biosciences, Inc. (NASDAQ:CLBS) (“Caladrius” or the “Company”), a cell therapy company combining a select therapeutic development pipeline with an industry-leading development and manufacturing services provider (PCT), announces today that the California Institute for Regenerative Medicine (CIRM) has awarded a grant to Caladrius, providing up to $12.2 million for the development of CLBS03. CLBS03 is the Company’s investigational cell therapy currently being evaluated as a treatment for recent onset type 1 diabetes (T1D) in a Caladrius-sponsored Phase 2 trial, the Sanford Project: T-Rex Study, in collaboration with Sanford Research, a subsidiary of Sanford Health. The grant from CIRM, which was recommended for approval by its distinguished and independent panel of scientific reviewers, is a significant endorsement of the potential for Caladrius’ novel approach for treating T1D with cell therapy by restoring immune balance. The award has important implications as it is expected to fund a significant portion of the remaining cost of the Company’s Phase 2 trial currently underway. The grant will be used to cover expenses including all manufacturing and development based in California and other trial costs dependent upon the proportion of subjects enrolled in California, with consumption of at least $6 million of the award expected. CLBS03 uses the patient’s own regulatory T cells (Tregs) to treat autoimmune disease. Tregs are a natural part of the human immune system that regulate the activity of T effector cells, which are responsible for protecting the body from viruses and other foreign antigens. When Tregs function properly, only harmful foreign materials are attacked by T effector cells. In autoimmune diseases, it is thought that deficient Treg activity permits the T effector cells to attack the body’s own beneficial cells and, in the case of T1D, insulin-producing pancreatic beta cells, thereby reducing and eventually eliminating the body’s ability to produce sufficient amounts of insulin. Caladrius’ novel approach seeks to restore immune balance by augmenting the number and activity of a patient’s own Tregs and using their innate capabilities to modulate multiple facets of the effector arm of the immune system. CLBS03 has received Orphan Drug and Fast Track designations from the U.S. Food and Drug Administration (FDA), and Advanced Therapeutic Medicinal Product classification from the European Medicines Agency. Patients are currently being enrolled in the second cohort of the Phase 2 trial, with an interim analysis of early therapeutic effect expected by the end of 2017. "We are grateful to CIRM and the experts who reviewed and endorsed our application. We firmly believe that this therapy has the potential to improve the lives of people with T1D and this grant helps us advance our Phase 2 clinical study with the goal of determining the potential for CLBS03 to be an effective therapy in this important indication," said David J. Mazzo, PhD, Caladrius’ Chief Executive Officer. "This grant substantiates our approach to identify and secure non-dilutive funding for our development programs and helps position Caladrius as a leader among cell therapy and autoimmune disease therapy developers." About Caladrius Biosciences Caladrius Biosciences, Inc. is a cell therapy development company with cell therapy products in development based on multiple technology platforms and targeting autoimmune and cardiology indications. The company’s subsidiary, PCT, is a leading development and manufacturing partner exclusively focused on the cell therapy industry and has served over 100 clients since 1999. PCT provides a wide range of innovative services including product and process development, GMP manufacturing, engineering and automation, cell and tissue processing, logistics, storage and distribution, as well as expert consulting and regulatory support. For more information on Caladrius please visit www.caladrius.com and for more information on PCT please visit www.pctcaladrius.com. This press release contains forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995. Forward-looking statements reflect management’s current expectations, as of the date of this press release, and involve certain risks and uncertainties. All statements other than statements of historical fact contained in this press release are forward-looking statements. The Company’s actual results could differ materially from those anticipated in these forward-looking statements as a result of various factors. Factors that could cause future results to materially differ from the recent results or those projected in forward-looking statements include the “Risk Factors” described in the Company’s Annual Report on Form 10-K filed with the Securities and Exchange Commission (“SEC”) on March 15, 2016, and in the Company’s other periodic filings with the SEC. The Company’s further development is highly dependent on, among other things, future medical and research developments and market acceptance, which are outside of its control.


News Article | February 15, 2017
Site: www.prweb.com

A national breast cancer research project has chosen Sanford Health to be the repository for the specimens collected as part of the 100,000 woman study. The five-year study is called Women Informed to Screen Depending on Measures of risk, or WISDOM, and is being conducted by the Athena Breast Health Network, a collaboration among five University of California medical centers and Sanford Health’s Edith Sanford Breast Center. Investigators are studying routine annual screenings and personalized screenings based on genetic information to determine which method is more effective in reducing false positives and misdiagnoses of breast cancer. Sanford’s genomic lab will house the specimens collected from consenting women as part of this research. The study will enroll 100,000 women across the United States. Researchers from across the globe will have access to the data collected to use in other breast cancer studies. “Sanford Health has been an outstanding partner in our breast health research and clinical care initiatives here at the University of California,” said Sandy Borowsky, the principal investigator at the University of California, Davis site of Athena. “Sanford demonstrated a commitment to improving care for women through innovative thinking and the hard work of collecting data across multiple sites. Their professionalism and institutional support for a first class biospecimen lab made the choice clear. It’s a true win-win opportunity.” The Athena Breast Health Network in 2015 received a $14.1 million grant from the Patient-Centered Outcomes Research Institute, or PCORI, to fund WISDOM. Edith Sanford Breast Center has been a member of Athena since 2013. Other partners include the University of California campuses in San Francisco, San Diego, Davis, Los Angeles and Irvine. “Sanford Health enjoys beneficial integration of its clinical and research operations, which has provided us the infrastructure to support the WISDOM repository,” said David Pearce, Ph.D., executive vice president for Sanford Research. “The data gathered and stored here have the potential to improve breast cancer screenings for women everywhere.” Women age 40-74 years old who have not had a prior breast cancer diagnosis and receive care at an Athena site are eligible to enroll. Edith Sanford Breast Center in Sioux Falls expects to open enrollment in early 2017. About Sanford Health Sanford Health is an integrated health system headquartered in the Dakotas. It is one of the largest health systems in the nation with 45 hospitals and nearly 300 clinics in nine states and four countries. Sanford Health’s 28,000 employees, including more than 1,300 physicians, make it the largest employer in the Dakotas. Nearly $1 billion in gifts from philanthropist Denny Sanford have allowed for several initiatives, including global children's clinics, genomic medicine and specialized centers researching cures for type 1 diabetes, breast cancer and other diseases. For more information, visit sanfordhealth.org.


News Article | February 28, 2017
Site: www.prweb.com

Cancer Breakthroughs 2020, the world’s most comprehensive cancer collaborative initiative focusing on combination immunotherapy, has selected Sanford Health as one of three sites nationwide to launch the program’s first clinical trial. The study will explore an immunotherapy vaccine for patients with certain types of advanced breast cancer. Cancer Breakthroughs 2020 was created in early 2016 by Dr. Patrick Soon-Shiong, to facilitate collaboration among multinational pharmaceutical, biotechnology companies, academic centers and community oncologists in an effort to test novel immunotherapy protocols in combination with other treatment methods. “Our mission at Cancer Breakthroughs 2020 is to turn cancer into a chronic disease, not a life-threatening disease,” said Soon-Shiong. “Sanford Health is a key partner in helping us achieve that goal. Their drive to bring advanced cancer treatment options to even the most rural areas shows their commitment to fighting cancer.” Amy Sanford, M.D., an oncologist at Edith Sanford Breast Center in Sioux Falls, is the trial’s principal investigator. Her team will study if an immunotherapy vaccine designed to strengthen the body’s immune system can help patients fight breast cancer that cannot be treated with surgery or that has metastasized and is characterized by the HER2 gene. “After years of offering clinical trials and ranking the potential of immunotherapy high on Sanford’s list of priorities, the power of our comprehensive clinical research program is evident by Cancer Breakthroughs 2020’s selection of Sanford to help start the inaugural study of the project,” said David Pearce, Ph.D., executive vice president for Sanford Research. “The next generation standard of care will be the result of teamwork among organizations like Cancer Breakthroughs 2020 and Sanford Health.” Sanford Health has been involved in Cancer Breakthroughs 2020 since its inception early last year. In February, Sanford was announced as a founding member of the Cancer Breakthroughs 2020 Pediatrics Consortium. Sam Milanovich, M.D., is among the national experts using comprehensive cancer molecular diagnostic testing to study pediatric forms of cancer. This summer, Patrick Soon-Shiong, M.D., the founder of Cancer Breakthroughs 2020, finalized a relationship with Sanford Health to work clinical trials for human papillomavirus-, or HPV-, related cancers like head and neck cancer, cervical cancer and anal cancer. The study is open at Edith Sanford Breast Center in Sioux Falls. For information or to enroll, call 1-87-SURVIVAL. About Cancer Breakthroughs 2020 The Cancer Breakthroughs 2020 program is one of the most comprehensive cancer collaborative initiative launched to date, seeking to accelerate the potential of combination immunotherapy as the next generation standard of care in cancer patients. This initiative aims to explore a new paradigm in cancer care by initiating randomized Phase II trials in patients at all stages of disease in 20 tumor types in 20,000 patients within the next 36 months. These findings will inform Phase III trials and the aspirational breakthroughs to develop an effective vaccine-based immunotherapy to combat cancer by 2020. For more information, please visit http://www.cancerbreakthroughs2020.org and follow @Cancer2020 on Twitter. About Sanford Health Sanford Health is an integrated health system headquartered in the Dakotas. It is one of the largest health systems in the nation with 45 hospitals and nearly 300 clinics in nine states and four countries. Sanford Health’s 28,000 employees, including more than 1,300 physicians, make it the largest employer in the Dakotas. Nearly $1 billion in gifts from philanthropist Denny Sanford have allowed for several initiatives, including global children's clinics, genomic medicine and specialized centers researching cures for type 1 diabetes, breast cancer and other diseases. For more information, visit sanfordhealth.org.


Munce T.A.,Sanford Sports Science Institute | Munce T.A.,Sanford Burnham Institute for Medical Research | Munce T.A.,University of South Dakota | Dorman J.C.,Sanford Sports Science Institute | And 8 more authors.
Medicine and Science in Sports and Exercise | Year: 2015

Football players are subjected to repetitive impacts that may lead to brain injury and neurologic dysfunction. Knowledge about head impact exposure (HIE) and consequent neurologic function among youth football players is limited. Purpose: This study aimed to measure and characterize HIE of youth football players throughout one season and explore associations between HIE and changes in selected clinical measures of neurologic function. Methods: Twenty-two youth football players (11-13 yr) wore helmets outfitted with a head impact telemetry (HIT) system to quantify head impact frequency, magnitude, duration, and location. Impact data were collected for each practice (27) and game (9) in a single season. Selected clinical measures of balance, oculomotor performance, reaction time, and self-reported symptoms were assessed before and after the season. Results: The median individual head impacts per practice, per game, and throughout the entire season were 9, 12, and 252, respectively. Approximately 50% of all head impacts (6183) had a linear acceleration between 10g and 20g, but nearly 2% were greater than 80g. Overall, the head impact frequency distributions in this study population were similar in magnitude and location as in high school and collegiate football, but total impact frequency was lower. Individual changes in neurologic function were not associated with cumulative HIE. Conclusion: This study provides a novel examination of HIE and associations with short-term neurologic function in youth football and notably contributes to the limited HIE data currently available for this population. Whereas youth football players can experience remarkably similar head impact forces as high school players, cumulative subconcussive HIE throughout one youth football season may not be detrimental to short-term clinical measures of neurologic function. Copyright © 2015 by the American College of Sports Medicine.


De P.,Sanford Research | Miskimins K.,Cancer Biology Sanford Research | Dey N.,Sanford Research | Leyland-Jones B.,Sanford Research
Cancer Treatment Reviews | Year: 2013

The PI3K-AKT-mTOR network has been the major focus of attention for cancer researchers (both in the clinic and the laboratory) in the last decade. An incomplete knowledge of the molecular biology of this complex network has seen an expansion of first generation allosteric mTOR inhibitors, rapalogues, but also biomarker studies designed to identify the best responders of these agents. Currently, research in this pathway has focused on the dual nature of mTOR that is integrated by mTOR-RAPTOR complex (mTORC1) and mTOR-RICTOR complex (mTORC2). These two complexes are regulated by different upstream proteins and also regulated by multiple different compensatory feedback loops. The related advantage of feedback regulation of signaling systems is that it allows diversification in signal response. This deeper understanding has facilitated the development of a novel second generation of inhibitors that are able to affect both mTORC1 and mTORC2, and their downstream effectors, through inhibition of their catalytic activity (ATP competitive inhibitors, attacking the kinase domain of this protein) than binding to the FKBP12 regulatory proteins as for rapalogues. This article reviews the newest insight in the signaling network of the mTOR pathway, preclinical/clinical status of mTOR inhibitors (including second generation of kinase inhibitors) and then focuses on the development of a new wave of research related to combination therapies in subset specific breast tumors. © 2012 Elsevier Ltd.


PubMed | University of North Dakota, Ohio State University and Sanford Research
Type: Journal Article | Journal: Applied and environmental microbiology | Year: 2016

Neorickettsia spp. are bacterial endosymbionts of parasitic flukes (Digenea) that also have the potential to infect and cause disease (e.g., Sennetsu fever) in the vertebrate hosts of the fluke. One of the largest gaps in our knowledge of Neorickettsia biology is the very limited information available regarding the localization of the bacterial endosymbiont within its digenean host. In this study, we used indirect immunofluorescence microscopy to visualize Neorickettsia sp. within several life cycle stages of the digenean Plagiorchis elegans Individual sporocysts, cercariae, metacercariae, and adults of P. elegans naturally infected with Neorickettsia sp. were obtained from our laboratory-maintained life cycle, embedded, sectioned, and prepared for indirect immunofluorescence microscopy using anti-Neorickettsia risticiihorse serum as the primary antibody. Neorickettsiasp. was found within the tegument of sporocysts, throughout cercarial embryos (germ balls) and fully formed cercariae (within the sporocysts), throughout metacercariae, and within the tegument, parenchyma, vitellaria, uteri, testes, cirrus sacs, and eggs of adults. Interestingly, Neorickettsia sp. was not found within the ovarian tissue. This suggests that vertical transmission of Neorickettsia within adult digeneans occurs via the incorporation of infected vitelline cells into the egg rather than direct infection of the ooplasm of the oocyte, as has been described for other bacterial endosymbionts of invertebrates (e.g.,Rickettsia and Wolbachia).

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