Current Murine Models and New Developments in H3K27M Diffuse Midline Gliomas

Diffuse Midline Gliomas with Histone 3-Lysine-27-Methionine (H3K27M) mutation constitute the majority of Diffuse Intrinsic Pontine Glioma (DIPG), which is the most aggressive form of pediatric glioma with a dire prognosis. DIPG are lethal tumors found in younger children with a median survival <1 year from diagnosis. Discovery of the characteristic H3K27M mutations offers opportunity and hope for development of targeted therapies for this deadly disease. The H3K27M mutation, likely through epigenetic alterations in specific H3 lysine trimethylation levels and subsequent gene expression, plays a significant role in pathogenesis of DIPG. Animal models accurately depicting molecular characteristics of H3K27M DIPG are important to elucidate underlying pathologic events and for preclinical drug evaluation. Here we review the past and present DIPG models and describe our efforts developing patient derived cell lines and xenografts from pretreated surgical specimens. Pre-treated surgical samples retain the characteristic genomic and phenotypic hallmarks of DIPG and establish orthotopic tumors in the mouse brainstem that recapitulate radiographic and morphological features of the original human DIPG tumor. These models that contain the H3K27M mutation constitute a valuable tool to further study this devastating disease and ultimately may uncover novel therapeutic vulnerabilities

Prospective Evaluation of TMVR for Failed Surgical Annuloplasty Rings: MITRAL Trial Valve-in-Ring Arm 1-Year Outcomes

OBJECTIVES: The authors report 1-year outcomes of high-risk patients with failed surgical annuloplasty rings undergoing transseptal mitral valve-in-ring (MViR) with the SAPIEN 3 aortic transcatheter heart valve (THV). BACKGROUND: The MITRAL (Mitral Implantation of Transcatheter Valves) trial is the first prospective study evaluating transseptal MViR with the SAPIEN 3 aortic THV in high-risk patients with failed surgical annuloplasty rings. METHODS: Prospective enrollment of high-risk patients with symptomatic moderate to severe or severe mitral regurgitation (MR) or severe mitral stenosis and failed annuloplasty rings at 13 U.S. sites. The primary safety endpoint was technical success. The primary THV performance endpoint was absence of MR grade ≥2+ or mean mitral valve gradient ≥10 mm Hg (30 days and 1 year). Secondary endpoints included procedural success and all-cause mortality (30 days and 1 year). RESULTS: Thirty patients were enrolled between January 2016 and October 2017 (median age 71.5 years [interquartile range: 67.0 to 76.8 years], 36.7% women, median Society of Thoracic Surgeons score 7.6% [interquartile range: 5.1% to 11.8%], 76.7% in New York Heart Association functional class III or IV). Technical success was 66.7% (driven primarily by need for a second valve in 6 patients). There was no intraprocedural mortality or conversion to surgery. The primary performance endpoint was achieved in 85.7% of survivors at 30 days (24 of 28) and 89.5% of patients alive at 1 year with echocardiographic data available (17 of 19). All-cause mortality at 30 days was 6.7% and at 1 year was 23.3%. Among survivors at 1-year follow-up, 84.2% were in New York Heart Association functional class I or II, the median mean mitral valve gradient was 6.0 mm Hg (interquartile range: 4.7 to 7.3 mm Hg), and all had ≤1+ MR. CONCLUSIONS: Transseptal MViR was associated with a 30-day mortality rate lower than predicted by the Society of Thoracic Surgeons score. At 1 year, transseptal MViR was associated with symptom improvement and stable THV performance

Prospective Study of TMVR Using Balloon-Expandable Aortic Transcatheter Valves in MAC: MITRAL Trial 1-Year Outcomes

OBJECTIVES: The aim of this study was to evaluate 1-year outcomes of valve-in-mitral annular calcification (ViMAC) in the MITRAL (Mitral Implantation of Transcatheter Valves) trial. BACKGROUND: The MITRAL trial is the first prospective study evaluating the feasibility of ViMAC using balloon-expandable aortic transcatheter heart valves. METHODS: A multicenter prospective study was conducted, enrolling high-risk surgical patients with severe mitral annular calcification and symptomatic severe mitral valve dysfunction at 13 U.S. sites. RESULTS: Between February 2015 and December 2017, 31 patients were enrolled (median age 74.5 years [interquartile range (IQR): 71.3 to 81.0 years], 71% women, median Society of Thoracic Surgeons score 6.3% [IQR: 5.0% to 8.8%], 87.1% in New York Heart Association functional class III or IV). Access was transatrial (48.4%), transseptal (48.4%), or transapical (3.2%). Technical success was 74.2%. Left ventricular outflow tract obstruction (LVOTO) with hemodynamic compromise occurred in 3 patients (transatrial, n = 1; transseptal, n = 1; transapical, n = 1). After LVOTO occurred in the first 2 patients, pre-emptive alcohol septal ablation was implemented to decrease risk in high-risk patients. No intraprocedural deaths or conversions to open heart surgery occurred during the index procedures. All-cause mortality at 30 days was 16.7% (transatrial, 21.4%; transseptal, 6.7%; transapical, 100% [n = 1]; p = 0.33) and at 1 year was 34.5% (transatrial, 38.5%; transseptal, 26.7%; p = 0.69). At 1-year follow-up, 83.3% of patients were in New York Heart Association functional class I or II, the median mean mitral valve gradient was 6.1 mm Hg (IQR: 5.6 to 7.1 mm Hg), and all patients had ≤1+ mitral regurgitation. CONCLUSIONS: At 1 year, ViMAC was associated with symptom improvement and stable transcatheter heart valve performance. Pre-emptive alcohol septal ablation may prevent transcatheter mitral valve replacement-induced LVOTO in patients at risk. Thirty-day mortality of patients treated via transseptal access was lower than predicted by the Society of Thoracic Surgeons score. Further studies are needed to evaluate safety and efficacy of ViMAC

Prospective Evaluation of Transseptal TMVR for Failed Surgical Bioprostheses: MITRAL Trial Valve-in-Valve Arm 1-Year Outcomes

OBJECTIVES: The aim of this study was to assess 1-year clinical outcomes among high-risk patients with failed surgical mitral bioprostheses who underwent transseptal mitral valve-in-valve (MViV) with the SAPIEN 3 aortic transcatheter heart valve (THV) in the MITRAL (Mitral Implantation of Transcatheter Valves) trial. BACKGROUND: The MITRAL trial is the first prospective study evaluating transseptal MViV with the SAPIEN 3 aortic THV in high-risk patients with failed surgical mitral bioprostheses. METHODS: High-risk patients with symptomatic moderate to severe or severe mitral regurgitation (MR) or severe mitral stenosis due to failed surgical mitral bioprostheses were prospectively enrolled. The primary safety endpoint was technical success. The primary THV performance endpoint was absence of MR grade ≥2+ or mean mitral valve gradient ≥10 mm Hg (30 days and 1 year). Secondary endpoints included procedural success and all-cause mortality (30 days and 1 year). RESULTS: Thirty patients were enrolled between July 2016 and October 2017 (median age 77.5 years [interquartile range (IQR): 70.3 to 82.8 years], 63.3% women, median Society of Thoracic Surgeons score 9.4% [IQR: 5.8% to 12.0%], 80% in New York Heart Association functional class III or IV). The technical success rate was 100%. The primary performance endpoint in survivors was achieved in 96.6% (28 of 29) at 30 days and 82.8% (24 of 29) at 1 year. Thirty-day all-cause mortality was 3.3% and was unchanged at 1 year. The only death was due to airway obstruction after swallowing several pills simultaneously 29 days post-MViV. At 1-year follow-up, 89.3% of patients were in New York Heart Association functional class I or II, the median mean mitral valve gradient was 6.6 mm Hg (interquartile range: 5.5 to 8.9 mm Hg), and all patients had MR grade ≤1+. CONCLUSIONS: Transseptal MViV in high-risk patients was associated with 100% technical success, low procedural complication rates, and very low mortality at 1 year. The vast majority of patients experienced significant symptom alleviation, and THV performance remained stable at 1 year

Modulating glioma-mediated myeloid-derived suppressor cell development with sulforaphane.

Glioblastoma is the most common primary tumor of the brain and has few long-term survivors. The local and systemic immunosuppressive environment created by glioblastoma allows it to evade immunosurveillance. Myeloid-derived suppressor cells (MDSCs) are a critical component of this immunosuppression. Understanding mechanisms of MDSC formation and function are key to developing effective immunotherapies. In this study, we developed a novel model to reliably generate human MDSCs from healthy-donor CD14+ monocytes by culture in human glioma-conditioned media. Monocytic MDSC frequency was assessed by flow cytometry and confocal microscopy. The resulting MDSCs robustly inhibited T cell proliferation. A cytokine array identified multiple components of the GCM potentially contributing to MDSC generation, including Monocyte Chemoattractive Protein-1, interleukin-6, interleukin-8, and Macrophage Migration Inhibitory Factor (MIF). Of these, Macrophage Migration Inhibitory Factor is a particularly attractive therapeutic target as sulforaphane, a naturally occurring MIF inhibitor derived from broccoli sprouts, has excellent oral bioavailability. Sulforaphane inhibits the transformation of normal monocytes to MDSCs by glioma-conditioned media in vitro at pharmacologically relevant concentrations that are non-toxic to normal leukocytes. This is associated with a corresponding increase in mature dendritic cells. Interestingly, sulforaphane treatment had similar pro-inflammatory effects on normal monocytes in fresh media but specifically increased immature dendritic cells. Thus, we have used a simple in vitro model system to identify a novel contributor to glioblastoma immunosuppression for which a natural inhibitor exists that increases mature dendritic cell development at the expense of myeloid-derived suppressor cells when normal monocytes are exposed to glioma conditioned media

Age-adjusted antitumoral therapy based on the demonstration of increased apoptosis as a mechanism underlying the reduced malignancy of tumors in the aged

AbstractIn view of the constant increase in the aged population, age-adjusted cancer therapy becomes an urgent target. Although cancer incidence rises with age, paradoxically, growth rate and metastasis often proceed at a slower rate in the aged. Determining the mechanism(s) underlying this reduced tumor progression in the old might have implications for a rational design of age-adjusted therapy. Thus far, decreased cell proliferation or immune response modifications were suggested as possible mechanisms. We show here that an increased tendency to apoptotic tumor cell death in the aged could constitute an additional mechanism. Based on this mechanism, we compared the therapeutic efficacy of two apoptosis inducers, hydrocortisone and adriamycin, on AKR lymphoma and B16 melanoma growth in young and old mice. Treatment with hydrocortisone acetate inhibited tumor growth practically only in old mice in the two tumor systems. Similar effects were obtained with adriamycin treatment of AKR lymphoma but opposite results were seen with B16 melanoma. We thus demonstrated, in three of the four tumor-therapeutic modality systems examined, an age-related antitumoral efficacy of two apoptosis-inducing agents, with tendency for a remarkably more pronounced effect in aged mice

SFN decreases immunosuppressive MDSC formation in response to GCM but increases CD14-/HLA-DR+ cells.

<p>All experiments performed under hypoxic (1% O₂) conditions. A) Representative dot plots showing CD14/HLA-DR expression in CD14+ monocytes cultured alone or in the presence of BT114 GCM +/- SFN. Monocytes cultured alone are largely CD14+/HLA-DR+ with relatively few mMDSC’s (CD14+/HLA-DR-; 4.75%) (top left). Exposure to BT114 GCM causes a marked increase in mMDSC’s (CD14+/HLA-DR-; 71.3%) (top right). Addition of SFN is associated with a dose-dendent reduction in mMDSC’s (5uM SFN = 42.5% mMDSC’s; 10uM SFN = 5.2% mMDSC’s) (bottom left and right; percent shown in red). Interestingly, this reduction in mMDSC’s is associated with an increase in CD14-/HLA-DR+ cells (percent shown in blue). B) SFN dose-dependent reductions in CD14+/HLA-DR- MDSCs in response to three different GCM’s were seen across monocytes from two unique healthy donors. C) Representative histograms showing immunosuppressive PD-L1 expression in monocytes exposed to BT116 GCM that decreases in a dose-dependent fashion with addition of SFN. D) Bar graph showing median fluorescence intensity of PD-L1 expression in monocytes from three donors exposed to BT116 GCM +/- SFN. E) Bar graph showing increasing population of CD14- / HLA-DR+ cells after SFN exposure among CD14+ cells cultured in GCM (from same experiments as 5A-B). * P < 0.05, ** = P< 0.01,**** = P<0.0001.</p

SFN promotes dendritic cell development from monocytes in both fresh media and GCM.

<p>All experiments performed under hypoxic (1% O₂) conditions. A) Representative dot plots showing CD14 and HLA-DR expression in CD14+ monocytes cultured in fresh media (DMEM) or BT116 GCM +/- SFN. Note that in addition to reductions in CD14+/HLA-DR- mMDSC’s (percent shown in red), there is a SFN-dependent increase in CD14- / HLA-DR+ cells suggestive of dendritic cells (percent shown in blue) in both fresh media and to a lesser extent in GCM. B) Representative dot plots showing CD14, HLA-DR, CD80, and CD83 expression in CD14+ monocytes cultured in DMEM +/- SFN. C) Representative dot plots showing CD14, HLA-DR, CD80, and CD83 epxression in CD14+ monocytes cultured in BT116 GCM +/- SFN. D) Bar graphs showing mean frequency of immature dendritic cells (CD14-/HLA-DR+/CD80+/CD83-) after culturing CD14+ monocytes from three donors in fresh media (DMEM) or BT116 GCM +/- SFN. Note that there is a SFN dose-dependent increase in immature dendritic cells with fresh media. E) Bar graphs showing mean frequency of mature dendritic cells (CD14-/HLA-DR+/CD80+/CD83+) after culturing CD14+ monocytes from three donors in fresh media (DMEM) or BT116 GCM +/- SFN. Note that there is a SFN dose-dependent increase in mature dendritic cells with GCM. n.s. = not significant, ** = P<0.01, *** = P<0.001.</p

Macrophage Migration Inhibitory Factor (MIF) is a critical component of GCM promoting MDSC formation.

<p>A) ELISA results confirming the presence of MIF in glioma-conditioned media. An MIF neutralizing antibody blocks the formation of CD14+/HLA-DR- cells when cultured in hypoxic (1% O₂) GCM. B) Representative dot plots showing CD 14 and HLA-DR expression in monocytes in serum free DMEM (top left), GCM from BT 116 (top right), GCM + 20 ug/ml anti-MIF Ab (bottom left), and GCM + 40 ug/ml anti-MIF Ab (bottom right). There is a reduction in the amount of CD14+/HLA-DR− cells (mMDSC’s) in the anti-MIF treated monocytes (percent shown in red). C) Bar graphs showing a significant reduction in the mean frequency of CD14+/HLA-DR- cells generated from monocytes cultured in GCM in the presence of anti-MIF antibodies. **** = P < 0.0001.</p

SFN treated monocytes cultured in both fresh media and GCM promote T-cell proliferation.

<p>All experiments performed under hypoxic (1% O₂) conditions. A) An experiment schematic with representative histograms showing proliferation of CFSE stained T-cells stimulated with anti-CD3/anti-CD28 antibodies with or without prior exposure to autologous monocytes cultured in fresh media (DMEM) or BT116 GCM +/- addition of SFN. B) Bar graphs showing mean T cell proliferation (from three donors) in response to anti-CD3 / anti-CD28 antibodies in the conditions outlined. Exposure to monocytes pre-cultured in DMEM caused a modest but statistically significant reduction in proliferation compared to T cells alone. This could be increased to proliferation greater than baseline by additional exposure to SFN. Exposure to monocytes pre-cultured in GCM (i.e. MDSC-enriched) caused a much greater inhibition of T cell proliferation which could be at least partly reversed in a SFN dose-dependent fashion. * P < 0.05, ** P < 0.01, *** P < .001.</p