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Roth, Z., and P. J. Hansen. "324SPHINGOSINE-1-PHOSPHATE PROTECTS CULTURED BOVINE OOCYTES FROM PHYSIOLOGICALLY RELEVANT THERMAL STRESS." Reproduction, Fertility and Development 16, no. 2 (2004): 282. http://dx.doi.org/10.1071/rdv16n1ab324.
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Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that can block the sphingomyelin cell-death pathway by suppressing ceramide-induced apoptosis. The present study was performed to test whether S1P protects oocytes from heat shock during in vitro maturation. Cumulus-oocyte complexes obtained by slicing follicles were placed in maturation medium with or without 50nM S1P and cultured at 38.5°C (CON) or 41°C (41C) for the first 12h of maturation. Incubation during the last 10h of maturation (22-h total maturation time), fertilization, and embryonic development were performed at 38.5°C and 5% (v/v) CO2. Blastocyst development was recorded at 8 days post-insemination (dpi) and activity of group II caspases in 8-day blastocysts was determined using a fluoroprobe, PhiPhiLux-G1D2 (OncoImmunin, Gaithersburg, MD, USA). Data were analysed by least-squares ANOVA with the GLM procedure of SAS. Percentage data were subjected to arcsin transformation before analysis. Exposure of oocytes to thermal stress during the first 12h of maturation reduced cleavage rate (P<0.01) and the number of oocytes developing to the blastocyst stage (P<0.04). There was a temperature x S1P interaction for cleavage rate (P<0.03) because S1P blocked effects of thermal stress on cleavage rate. Without S1P, the percentage of oocytes that cleaved by 3 dpi were 83.6±2.7% and 65.8±2.7% for CON and 41C, respectively. In the presence of S1P, percent cleavage was 86.7±2.7% and 83.9±2.7% for CON and 41C, respectively. There was a trend (P=0.06) for a temperature x S1P interaction for percent oocytes developing to blastocyst stage because S1P blocked effects of heat shock on development. Without S1P, the percentages of oocytes that developed to the blastocyst stage were 28.7±3.0% and 15.2±3.0% for CON and 41C, respectively. In the presence of S1P, percent blastocysts were 24.3±3.4% and 23.9±3.0% for CON and 41C, respectively. When development was expressed as percentage of cleaved embryos, however, there were no effects of temperature, S1P, or temperature x S1P on percent development to the blastocyst stage. Blastocyst caspase activity was not affected by temperature or S1P. In summary, exposure to physiologically relevant thermal stress during the first 12h of maturation has a deleterious effect on oocyte competence and this effect can be reduced by S1P. The fact that heat shock reduced the percentage of oocytes but not the percentage of cleaved embryos that became blastocysts suggests that oocytes that survive effects of heat shock and cleave have normal potential to develop to the blastocyst stage. Moreover, since heat shock did not affect caspase activity, it is likely that blastocysts from heat-shocked oocytes have normal developmental potential, at least as determined by caspase activity. Support: BARD FI-330-2002 and USDA Grants 2002-35203-12664 and 2001-52101-11318.
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Mohankumar, Kumaravel, Gus Wright, Subhashree Kumaravel, et al. "Abstract 236: Nuclear receptor 4A1 ligands target T-cell exhaustion in colorectal cancer." Cancer Research 82, no. 12_Supplement (2022): 236. http://dx.doi.org/10.1158/1538-7445.am2022-236.
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Abstract Colorectal cancer (CRC) is a highly complex disease with multiple risk factors. The orphan nuclear receptor 4A1 (NR4A1) is overexpressed in several cancers and is a negative prognostic factor for cancer patient survival. Previous reports indicate a potential role for overexpression of NR4A1 in T-cell exhaustion and in this study, we aim to investigate the antitumorigenic activity of two bis-indole derived ligands (DIMs) that act as receptor antagonists. Immune competent C57BL/6 mice and mouse MC-38 colon cancer cells were used and tumor Infiltrating Lymphocytes (TILs) were isolated from mice either untreated or treated with CDIM/NR4A1 antagonists. FACS analysis and Real -Time PCR were performed to determine expression of exhaustion markers in these tumor T-cell population. 1,1-Bis(3΄-indolyl)-1-(3-bromo-5-trifluoromethoxyphenyl)methane (DIM-3-Br-5-OCF3) and the 3,5-dichlorophenyl analog (DIM-3,5-Cl2) at doses of 2.5 and 7.5 mg/kg/d inhibited tumor growth and downregulated expression of PD-L1, an NR4A1-regulated gene in MC-38 - derived tumors and cells. The mouse MC-38 colon cancer cell line was used in a syngeneic mouse model of colon cancer and tumor infiltrating lymphocytes (TILs) from MC-38 cell-derived colon tumors exhibited multiple markers of T-cell exhaustion in both CD8+ and CD4+ T-cells. Lymphocytes from an enlarged spleen in these tumor-bearing animals also exhibited markers of CD8+ and CD4+ T-cell exhaustion. Analysis of CD8+ cells from TILs showed that treatment with the NR4A1 antagonists modulated expression of several genes associated with T-cell exhaustion namely a decrease in TOX, TOX2 and NFAT mRNAs, activation of T-Bet. The percentage of CD8+ and CD4+ T-cells from tumors and spleen expressing PD-1, 2B4 and TIGIT was decreased in the treated vs control mice and TIM3 expression was also decreased in CD8+ (tumors and spleen) and CD4+ (tumors) T-cells. These findings suggest that NR4A1 antagonists are highly effective as anticancer agents in this mouse syngeneic colon tumor model by inactivating the pro-oncogenic activities of NR4A1 in tumors and by remediating NR4A1-regulated T-cell exhaustion in tumor and splenic lymphocytes. Citation Format: Kumaravel Mohankumar, Gus Wright, Subhashree Kumaravel, Rupesh Shrestha, Lei Zhang, Maen Abdelrahim, Robert S. Chapkin, Stephen Safe. Nuclear receptor 4A1 ligands target T-cell exhaustion in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 236.
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Mohankumar, Kumaravel, Gus Wright, Subhashree Kumaravel, et al. "732 A novel nuclear receptor 4A1 (NR4A1) antagonists attenuates T-cell exhaustion in colorectal cancer." Journal for ImmunoTherapy of Cancer 9, Suppl 2 (2021): A761—A762. http://dx.doi.org/10.1136/jitc-2021-sitc2021.732.
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BackgroundColorectal cancer (CRC) is a highly complex disease with multiple risk factors and both genetic and environmental components contribute to disease incidence.1 2 Cancer immunotherapy using immune-checkpoint blockades represents a major advance in treatment strategy.3 4 The orphan nuclear receptor 4A1 (NR4A1) is overexpressed in lung, colon, liver and breast cancers and in Rhabdomyosarcoma and is a negative prognostic factor for cancer patient survival.5–8 Previous studies in breast cancer cells showed that PD-L1 was regulated by NR4A1 which activates transcription factor Sp1 bound to the PD-L1 gene promoter. Genome-wide studies have identified NR4A1 as a key mediator of T-cell dysfunction and NR4A1 also plays an important role in regulating genes which are involved in tumor-induced T-cell exhaustion.9 Bis-indole derived NR4A1 ligand that act as receptor antagonists have been developed in this laboratory and these compounds block pro-oncogenic NR4A1-regulated genes/pathways.MethodsImmune competent C57BL/6 mice and mouse MC-38 colon cancer cells were used and tumor Infiltrating Lymphocytes (TILs) were isolated from mice either untreated or treated with CDIM/NR4A1 antagonists. FACS analysis and Real -Time PCR were performed to determine expression of exhaustion markers in these tumor T-cell population.ResultsTwo compounds: 1,1-bis(3′-indolyl)-1-(3-bromo-5-trifluoromethoxyphenyl)methane (DIM-3-Br-5-OCF3) and 3,5-dichlorophenyl analog (DIM-3,5-Cl2) inhibited tumor growth and weight at doses of 2.5 and 7.5mg/kg/day (figure 1). Tumor CD8+ T-cells isolated from mice treated with DIM-3,5-Cl2 and DIM-3-Br-5-OCF3 exhibited decreased mRNA expression of NR4A1 and high mobility group – box transcription factors NFAT, TOX and TOX2 and increased mRNA levels of Interferon-γ (IFNγ), granzyme β (GzB) and perforin compared to control animals (figure 2). As TOX and TOX2 cooperate with NR4A1 to modulate CD8+ T-cell exhaustion, we investigated the expression of several inhibitory receptors of T-cell exhaustion in CD8+ TILs, including PD-1, 2B4, TIGIT and TIM3. Following treatment with DIM-3,5-Cl2 or DIM-3-Br-5-OCF3, there was a significant decrease in the percentage of PD1 and 2B4 cells and a decrease in TIGIT and TIM3 (figure 3). These results indicate that NR4A1 antagonists reverses T-cell exhaustion.ConclusionsNR4A1 plays a critical role in T-cell dysfunction, and this includes T-cell exhaustion.10 11 Our results demonstrate that the NR4A1 antagonists reverse many markers of T-cell exhaustion including activation of cytokines. The combined effects of NR4A1 antagonists in both tumors and T-cells result in inhibition of colon tumorigenesis by targeting pathways/genes in tumor cells and by enhancing immune surveillance via reversal of T-cell exhaustion.ReferencesAhmed M. Colon cancer: a Clinician's perspective in 2019. Gastroenterology Res 2020;13(1):1–10. Epub 2020/02/26. doi: 10.14740/gr1239.Keum N, Giovannucci E. Global burden of colorectal cancer: emerging trends, risk factors and prevention strategies. Nat Rev Gastroenterol Hepatol 2019;16(12):713–32. Epub 2019/08/29. doi: 10.1038/s41575-019-0189-8.Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer 2012;12(4):252–64. Epub 2012/03/23. doi: 10.1038/nrc3239.Ribas A, Wolchok JD. Cancer immunotherapy using checkpoint blockade. Science 2018;359(6382):1350–1355.Yang et al. Distinct epigenetic features of tumor-reactive CD8+ T cells in colorectal cancer patients revealed by genome-wide DNA methylation analysis. Genome Biol 2019;21(1):2. doi: 10.1186/s13059-019-1921-y.Safe S, Karki K. The paradoxical roles of orphan nuclear receptor 4A (NR4A) in cancer. Mol Can Res 2020. Epub 2020/10/28. doi: 10.1158/1541-7786.MCR-20-0707.Lee SO, et al. Diindolylmethane analogs bind NR4A1 and are NR4A1 antagonists in colon cancer cells. Molecular Endocrinology. 2014;28(10):1729–39. doi: 10.1210/me.2014-1102.Hedrick E, et al. The nuclear orphan receptor NR4A1 regulates β1-integrin expression in pancreatic and colon cancer cells and can be targeted by NR4A1 antagonists. Mol Carcinog 2017;56(9):2066–2075.Liu X, et al. Genome-wide analysis identifies NR4A1 as a key mediator of T cell dysfunction. Nature 2019;567(7749):525–9. Epub 2019/03/01. doi: 10.1038/s41586-019-0979-8.Chen J et al. NR4A transcription factors limit CAR T cell function in solid tumours. Nature. 2019;567(7749):530–4. Epub 2019/03/01. doi: 10.1038/s41586-019-0985-x. PubMed PMID: 30814732; PMCID: PMC6546093.Seo H, et al. TOX and TOX2 transcription factors cooperate with NR4A transcription factors to impose CD8(+) T cell exhaustion. Proc Natl Acad Sci USA 2019;116(25):12410–5.Ethics ApprovalAll animal studies were carried out according to the ethical procedures approved by the Texas A&M University Institutional Animal Care and Use Committee. Approval number is 2020-0138.Abstract 732 Figure 1CDIM/NR4A1 antagonists inhibit colon tumor growth. Model for regulation of genes with GC-rich promoters by NR4A1/SP1 (A). C57BL/6 mice bearing MC-38 cells as xenografts were treated for 21 days with corn oil (control), DIM-3-Br-5-OCF3 (2.5 and 7.5 mg/kg/d), DIM-3,5-Cl2 (2.5 and 7.5 mg/kg/d) and effects on tumor volume (B), and tumor weights (C) were determined.Abstract 732 Figure 2CDIM analogs alter transcription factors expression. FACS analysis and CD4 and CD8 – specific antibodies were used to determine T-cell population in tumors of mice treated with corn oil (control), DIM-3-Br-5-OCF3 (2.5 and 7.5 mg/kg/d) and DIM-3,5-Cl2 (2.5 and 7.5 mg/kg/d) (A). Real time PCR was used to determine expression of nuclear factors (B) and cytokine (C) mRNA levels in CD8+ T-cells isolated from tumors. Results are expressed as means ± SD replicates from each treatment group and significant (p<0.05) induction or inhibition is indicated (*)Abstract 732 Figure 3NR4A1 ligands decreases T-cell exhaustion markers. FACS analysis in tumors derived from mice treated with corn oil (control), DIM-3-Br-5-OCF3 (2.5 and 7.5 mg/kg/d) and DIM-3,5-Cl2 (2.5 and 7.5 mg/kg/d) using specific antibodies was carried out to determine percentage of CD8+ T-cells expressing T-cell exhaustion markers - PD1 (A), 2B4 (B), TIGIT (C) and TIM3 (D). Significant (p<0.05) induction or inhibition is indicated (*) and results are expressed as means ± SD for at least 4 separate mice per treatment group.
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D'Angelo, Sandra P., Steven Attia, Jean-Yves Blay, et al. "Identification of response stratification factors from pooled efficacy analyses of afamitresgene autoleucel (“Afami-cel” [Formerly ADP-A2M4]) in metastatic synovial sarcoma and myxoid/round cell liposarcoma phase 1 and phase 2 trials." Journal of Clinical Oncology 40, no. 16_suppl (2022): 11562. http://dx.doi.org/10.1200/jco.2022.40.16_suppl.11562.
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11562 Background: Afami-cel is an autologous, HLA-A*02-restricted, specific peptide enhanced affinity receptor, T-cell therapy engineered to target MAGE-A4+ solid tumors. The pivotal, 2-cohort, single-arm, Phase 2, SPEARHEAD-1 trial (NCT04044768) with afami-cel met its primary endpoint based on Cohort 1 data. As of September 1, 2021, in 47 patients (pts) with metastatic synovial sarcoma (SyS) or myxoid/round cell liposarcoma (MRCLS), the overall response rate (ORR) per independent review was 34% with encouraging durability (Van Tine, et al. Paper 30: CTOS 2021; Virtual). To identify potential stratification factors for response and assess whether response is a proxy for progression-free survival (PFS), we present pooled analyses using data from the prior Phase 1 trial (NCT03132922) and Cohort 1 of the SPEARHEAD-1 trial. Methods: Eligible pts (16–75 years) were HLA-A*02+ with MAGE-A4+ tumors. Pts received afami-cel after lymphodepleting chemotherapy. The pooled analyses evaluated ORR per RECIST v1.1 by investigator review, stratified by 7 factors, and safety. Results: In the pooled data, 69 pts received afami-cel (2.12–9.99×109 transduced T-cells) and were evaluable for response (Phase 1, n = 18; Phase 2, n = 51); all expressed one eligible HLA-A*02 allele. Median (range) for: age was 42 years (19–76), number of prior lines of therapy was 2 (1–12), and tumor MAGE-A4 H-score was 230 (60–300). Median (range) H-score was higher in SyS (256 [60–300]) than in MRCLS (180 [112–230]). The pooled investigator-assessed ORR was 36.2% (40.7% in SyS; 10.0% in MRCLS). Responses occurred across a wide MAGE-A4 H-score range (134–300). Median (range) duration of response was 52 weeks (8.29–75.14). Response rate was higher in the 59 pts with SyS: with ≤2 vs ≥3 prior lines of therapy (55.2% vs 26.7%), baseline target lesion sum of longest diameters <10cm vs ≥10cm (53.1% vs 25.9%), MAGE-A4 H-score ≥200 vs <200 (46.3% vs 27.8%), without vs with bridging therapy (48.6% vs 29.2%), who were female vs male (46.4% vs 35.5%), aged ≥40 vs <40 years (45.7% vs 33.3%), and from North America vs Europe (42.6% vs 33.3%). In responders vs non-responders with SyS, respectively, median PFS was 58.3 vs 11. 0 weeks (log-rank p-value <0.0001); the probability of being progression-free at 24 weeks was 0.8 vs 0.2. The pooled benefit:risk profile of afami-cel was similar to that in the SPEARHEAD-1 trial (Van Tine, et al. Paper 30: CTOS 2021; Virtual.). Conclusions: We show that baseline tumor burden, prior systemic treatment history, and MAGE-A4 tumor expression levels are potential factors associated with response to afami-cel, although their true predictive value for response status awaits confirmation. Our findings will inform the ongoing clinical development of afami-cel in sarcoma, especially for prognostic studies with PFS or overall survival endpoints. Clinical trial information: NCT04044768, NCT03132922.
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D'Angelo, Sandra P., Steven Attia, Jean-Yves Blay, et al. "Identification of response stratification factors from pooled efficacy analyses of afamitresgene autoleucel (“Afami-cel” [Formerly ADP-A2M4]) in metastatic synovial sarcoma and myxoid/round cell liposarcoma phase 1 and phase 2 trials." Journal of Clinical Oncology 40, no. 16_suppl (2022): 11562. http://dx.doi.org/10.1200/jco.2022.40.16_suppl.11562.
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11562 Background: Afami-cel is an autologous, HLA-A*02-restricted, specific peptide enhanced affinity receptor, T-cell therapy engineered to target MAGE-A4+ solid tumors. The pivotal, 2-cohort, single-arm, Phase 2, SPEARHEAD-1 trial (NCT04044768) with afami-cel met its primary endpoint based on Cohort 1 data. As of September 1, 2021, in 47 patients (pts) with metastatic synovial sarcoma (SyS) or myxoid/round cell liposarcoma (MRCLS), the overall response rate (ORR) per independent review was 34% with encouraging durability (Van Tine, et al. Paper 30: CTOS 2021; Virtual). To identify potential stratification factors for response and assess whether response is a proxy for progression-free survival (PFS), we present pooled analyses using data from the prior Phase 1 trial (NCT03132922) and Cohort 1 of the SPEARHEAD-1 trial. Methods: Eligible pts (16–75 years) were HLA-A*02+ with MAGE-A4+ tumors. Pts received afami-cel after lymphodepleting chemotherapy. The pooled analyses evaluated ORR per RECIST v1.1 by investigator review, stratified by 7 factors, and safety. Results: In the pooled data, 69 pts received afami-cel (2.12–9.99×109 transduced T-cells) and were evaluable for response (Phase 1, n = 18; Phase 2, n = 51); all expressed one eligible HLA-A*02 allele. Median (range) for: age was 42 years (19–76), number of prior lines of therapy was 2 (1–12), and tumor MAGE-A4 H-score was 230 (60–300). Median (range) H-score was higher in SyS (256 [60–300]) than in MRCLS (180 [112–230]). The pooled investigator-assessed ORR was 36.2% (40.7% in SyS; 10.0% in MRCLS). Responses occurred across a wide MAGE-A4 H-score range (134–300). Median (range) duration of response was 52 weeks (8.29–75.14). Response rate was higher in the 59 pts with SyS: with ≤2 vs ≥3 prior lines of therapy (55.2% vs 26.7%), baseline target lesion sum of longest diameters <10cm vs ≥10cm (53.1% vs 25.9%), MAGE-A4 H-score ≥200 vs <200 (46.3% vs 27.8%), without vs with bridging therapy (48.6% vs 29.2%), who were female vs male (46.4% vs 35.5%), aged ≥40 vs <40 years (45.7% vs 33.3%), and from North America vs Europe (42.6% vs 33.3%). In responders vs non-responders with SyS, respectively, median PFS was 58.3 vs 11. 0 weeks (log-rank p-value <0.0001); the probability of being progression-free at 24 weeks was 0.8 vs 0.2. The pooled benefit:risk profile of afami-cel was similar to that in the SPEARHEAD-1 trial (Van Tine, et al. Paper 30: CTOS 2021; Virtual.). Conclusions: We show that baseline tumor burden, prior systemic treatment history, and MAGE-A4 tumor expression levels are potential factors associated with response to afami-cel, although their true predictive value for response status awaits confirmation. Our findings will inform the ongoing clinical development of afami-cel in sarcoma, especially for prognostic studies with PFS or overall survival endpoints. Clinical trial information: NCT04044768, NCT03132922.
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Lunt, Colin, Sandra P. D’Angelo, Albiruni Ryan Abdul Razak, et al. "Abstract A038: Enrollment of pediatric and adolescent patients with MAGE-A4+ advanced synovial sarcoma into cohort 2 of SPEARHEAD-1: a phase 2 trial of afamitresgene autoleucel (“afami-cel” [formerly ADP-A2M4])." Clinical Cancer Research 28, no. 18_Supplement (2022): A038. http://dx.doi.org/10.1158/1557-3265.sarcomas22-a038.
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Abstract Background: Afami-cel is an autologous, specific peptide enhanced affinity receptor T-cell therapy genetically engineered to target MAGE-A4+ solid tumors in HLA-A*02+ patients. SPEARHEAD-1 (NCT04044768) is a Phase 2, two-cohort, single-arm, open-label trial evaluating afami-cel in patients with advanced/metastatic synovial sarcoma or myxoid/round cell liposarcoma (MRCLS) and is the largest trial in metastatic synovial sarcoma to date. Preliminary data from Cohort 1 in 47 heavily pre-treated patients aged 16–75 years from 22 centers in North America and Europe, showed an overall response rate (ORR) per independent review of 34.0% (14/39 [35.9%] in synovial sarcoma; 2/8 [25%] in MRCLS) and a favorable benefit–risk profile with mainly low-grade cytokine release syndrome and tolerable/reversible hematologic toxicities.1 The reported ORR in synovial sarcoma in Cohort 1 was higher than reported ORRs for current standard-of-care therapies, such as pazopanib and trabectedin, in the second-line metastatic setting.2 As synovial sarcoma is the most common malignant nonrhabdomyosarcoma soft-tissue sarcoma in children and adolescents with few treatment options, especially for recurrent disease, the trial opened a second cohort to allow enrollment of pediatric patients with MAGE-A4+ synovial sarcoma who experienced disease progression post first-line chemotherapy, and to better understand MAGE-A4 tumor expression in children. Methods: Cohort 2 of the SPEARHEAD-1 trial is enrolling patients with advanced synovial sarcoma who are at least 10 years old and weigh at least 40 kg. The planned enrollment is 45 patients, including up to 13 children, to enable a pooled analysis of ORR in &gt;90 patients across Cohorts 1 and 2. HLA and MAGE-A4+ screening in Cohort 2 is conducted at a central laboratory using the same method as Cohort 1; MAGE-A4 testing is done using a clinical trial assay. All patients enrolled in Cohort 2 undergo apheresis and their isolated T-cells are then transduced with the MAGE-A4c1032 TCR using a lentivirus vector, followed by ex vivo expansion. Prior to afami-cel infusion of 1–10 × 109 transduced T-cells, patients will receive lymphodepleting chemotherapy consisting of fludarabine (30 mg/m2/day for 4 days) and cyclophosphamide (600 mg/m2/day for 3 days). Disease will be assessed by independent review per RECIST v1.1 using computerized tomography or magnetic resonance imaging at weeks 4, 8, 12, 16, 24, and every 2 months thereafter until confirmed disease progression. Patients enter long-term follow-up for 15 years. 1. Van Tine BA, et al. Paper 30: CTOS 2021; Virtual 2. Carroll C, et al. Cancer Res 2021;81(13_Suppl): Abstract nr 2630. Citation Format: Colin Lunt, Sandra P. D’Angelo, Albiruni Ryan Abdul Razak, Michael J. Wagner, Brian A. Van Tine, Kristen Ganjoo, Jean-Yves Blay, Dejka M. Araujo, Mark Agulnik, John W. Glod, Erin Van Winkle, Erica Elefant, Swethajit Biswas, Dennis Williams, Axel Le Cesne. Enrollment of pediatric and adolescent patients with MAGE-A4+ advanced synovial sarcoma into cohort 2 of SPEARHEAD-1: a phase 2 trial of afamitresgene autoleucel (“afami-cel” [formerly ADP-A2M4]) [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A038.
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Labonte, Melissa Janae, Pierre Oliver Bohanes, Dongyun Yang та ін. "Novel colon cancer tumor suppressor gene, β-defensin 1, to predict recurrence in patients with stage II and III colon cancer." Journal of Clinical Oncology 30, № 15_suppl (2012): 3622. http://dx.doi.org/10.1200/jco.2012.30.15_suppl.3622.
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3622 Background: Human β-defensin 1 (hBD-1) encoded by the DEFB1 gene is an antimicrobial peptide involved in the innate immune response and is expressed in epithelial cells, including the colon. hBD-1 has been shown to have tumor suppressor functions in urothelial cancer models. We tested whether 4 germline single nucleotide polymorphisms (SNPs) in DEFB1 could predict time to tumor recurrence (TTR) in stage II and III colon cancer (CC) patients. We then sought to demonstrate if hBD-1 has tumor suppressor functions in CC models. Methods: A total of 234 patients, 105 stage II and 129 stage III, treated with 5-FU-based chemotherapy at the University of Southern California were included. The median follow-up time was 4.4 yrs. SNPs were analyzed from whole blood samples using direct DNA-sequencing. To gain insight into hBD-1’s function, hBD-1 expression in CC cell lines was determined by qRT-PCR and Western blotting. hBD-1 expression was induced ectopically and CC cells and viability, membrane permeability, cell-cycle and apoptosis analyzed. Results: Stage III patients with rs1800972 DEFB1 -44G containing genotypes had longer TTR than patients with C/C genotype (11.3 mo vs 2.7 mo; p=.008). In contrast in stage II, patients with rs1799946 DEFB1 -52A containing genotypes, had significantly longer TTR than patients with G/G genotype (16.8 mo vs 5.9 mo; p=.017). In the multivariate analysis, both DEFB1 -44C/G and DEFB1 -52G/A SNPs remained significant, respectively in stage III (HR=.419; 95%CI .201-.87; p=.020) and in stage II (HR=.360; 95%CI .152-.853; p=.020). Haplotype of all four SNPs was significantly associated with stage-specific TTR (to be presented at the meeting). In CC cell line models, there was a loss of hBD-1 expression, and induction of hBD-1 expression resulted in a loss of cell viability, membrane permeability and the induction of apoptosis. Conclusions: The results demonstrate for the first time evidence of hBD-1’s potential influence on the microenvironment and its role as a tumor suppressor in CC. Further studies need to be conducted to better understand the role of hBD-1 in CC development and progression and evaluate the potential utility of hBD-1 as a therapeutic strategy.
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Kim, Hanjun, Sewoon Kim, Yonghee Song, Wantae Kim, Qi-Long Ying, and Eek-hoon Jho. "Dual Function of Wnt Signaling during Neuronal Differentiation of Mouse Embryonic Stem Cells." Stem Cells International 2015 (2015): 1–10. http://dx.doi.org/10.1155/2015/459301.
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Activation of Wnt signaling enhances self-renewal of mouse embryonic and neural stem/progenitor cells. In contrast, undifferentiated ES cells show a very low level of endogenous Wnt signaling, and ectopic activation of Wnt signaling has been shown to block neuronal differentiation. Therefore, it remains unclear whether or not endogenous Wnt/β-catenin signaling is necessary for self-renewal or neuronal differentiation of ES cells. To investigate this, we examined the expression profiles of Wnt signaling components. Expression levels of Wnts known to induceβ-catenin were very low in undifferentiated ES cells. Stable ES cell lines which can monitor endogenous activity of Wnt/β-catenin signaling suggest that Wnt signaling was very low in undifferentiated ES cells, whereas it increased during embryonic body formation or neuronal differentiation. Interestingly, application of small molecules which can positively (BIO, GSK3βinhibitor) or negatively (IWR-1-endo, Axin stabilizer) control Wnt/β-catenin signaling suggests that activation of that signaling at different time periods had differential effects on neuronal differentiation of 46C ES cells. Further, ChIP analysis suggested thatβ-catenin/TCF1 complex directly regulated the expression ofSox1during neuronal differentiation. Overall, our data suggest that Wnt/β-catenin signaling plays differential roles at different time points of neuronal differentiation.
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Huang, Qian, Jingying Xu, Yanyan Ge, Yue Shi, Fei Wang та Mingli Zhu. "NR4A1 inhibits the epithelial–mesenchymal transition of hepatic stellate cells: Involvement of TGF-β–Smad2/3/4–ZEB signaling". Open Life Sciences 17, № 1 (2022): 447–54. http://dx.doi.org/10.1515/biol-2022-0047.
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Abstract This study aimed to examine whether nuclear receptor 4a1 (NR4A1) is involved in inhibiting hepatic stellate cell (HSC) activation and liver fibrosis through the epithelial–mesenchymal transition (EMT). HSC-T6 cells were divided into the control group, the acetaldehyde (200 μM, an EMT activator) group, and the NR4A1 activation group (Cytosporone B; 1 μM). The expression levels of the epithelial marker E-cadherin, the mesenchymal markers fibronectin (FN), vimentin, smooth muscle alpha-actin (α-SMA), and fibroblast-specific protein 1 (FSP-1), and the components of the transforming growth factor (TGF)-β pathway were detected by real-time polymerase chain reaction and western blotting. Compared with the control group, E-cadherin in the acetaldehyde group was downregulated, whereas FN, FSP-1, vimentin, α-SMA, and COL1A1/COL1A2 were upregulated (P < 0.05). Compared with the acetaldehyde group, NR4A1 agonist upregulated E-cadherin and downregulated FN, FSP-1, vimentin, α-SMA, and COL1A1/COL1A2 (P < 0.05). After acetaldehyde stimulation, TGF-β, Smad2/3/4, and zinc finger E-box-binding homeobox (ZEB) were upregulated, while Smad7 mRNA levels were downregulated (all P < 0.05). Compared with acetaldehyde alone, NR4A1 agonist increased Smad7 mRNA levels and reduced TGF-β, Smad2/3/4, and ZEB mRNA levels (all P < 0.05). NR4A1 activation suppresses acetaldehyde-induced EMT, as shown by epithelial and mesenchymal marker expression. The inhibition of the TGF-β–Smad2/3/4–ZEB signaling during HSC activation might be involved.
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Welsh, James, Danxia Ke, Nahum Puebla Osorio, et al. "376 Radiation sub-study to characterize safety and tolerability of low-dose radiation in combination with afami-cel in patients with advanced cancers (NCT03132922)." Journal for ImmunoTherapy of Cancer 9, Suppl 2 (2021): A407. http://dx.doi.org/10.1136/jitc-2021-sitc2021.376.
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BackgroundAutologous cell therapies with an engineered T-cell receptor targeting MAGE-A4 have shown responses in patients with synovial sarcoma1 with additional responses in myxoid/round cell liposarcoma (MRCLS), head and neck, lung, esophagogastric junction, and melanoma cancers.2 3 Low-dose radiation may control tumor growth locally and modulate stroma of solid tumors,4 potentially facilitating T-cell infiltration into tumors and antitumor activity.MethodsSub-study designed to assess safety, tolerability, and efficacy in up to 10 patients with low-dose radiation in combination with lymphodepleting chemotherapy, followed by afami-cel (an autologous TCR cell T-cell therapy targeting MAGE-A4). Eligible patients are HLA-A*02^+ with MAGE-A4 expressing tumors including urothelial, melanoma, head and neck, ovarian, non-small cell lung, esophageal, gastric, synovial sarcoma, and MRCLS cancers. Patients receive afami-cel by infusion following low-dose radiation and lymphodepleting chemotherapy. Radiation was 4.2–7 Gy per lesion or isocenter (maximum of 5). Lymphodepleting regimen was IV fludarabine 30 mg/m^2/day for 4 days (−7 to −4) and cyclophosphamide 600 mg/m^2/day for 3 days (−7 to −5). Afami-cel doses ranged from 1.2 x 10^9 to 10 x 10^9 transduced cells. Pts receive afami-cel infusion on Day 1.ResultsAs of Dec 27, 2020, a total of 8 patients, including 4 patients (1 male) with melanoma (2), HNSCC (1), or ovarian (1) cancers received low-dose radiation and afami-cel. Most frequently reported AEs (4/4 pts) were leukopenia/decreased white blood cell count, lymphopenia/decreased lymphocyte count, and neutropenia/decreased neutrophil count; all of which were related to the lymphodepletion regimen. The most commonly (>1 patient) reported AEs considered related to T-cell infusion were cytokine release syndrome (2/4 pts) and fatigue (2/4 pts). Two patients had a total of 5 SAEs: adrenal insufficiency, hyperglycemia, neurotoxicity, pneumonia aspiration, and pneumothorax. The only SAE considered to be related to treatment was Grade 3 neurotoxicity. Best overall responses per RECIST 1.1: 1 partial response (melanoma, −42% in target lesions), 2 stable diseases (ovarian cancer, −23%; HNSCC, no change), and 1 patient did not have post-baseline scans yet. Translational analyses showed peripheral persistence and serum cytokine response profiles consistent with that of afami-cel monotherapy, whilst a relatively greater T cell infiltration in tumor biopsies was evident.ConclusionsAfami-cel with low-dose radiation has had an acceptable safety profile. Most AEs were consistent with those typically experienced by cancer patients undergoing lymphodepletion cytotoxic chemotherapy and cellular therapy. Infused T-cells were observed in tumor biopsies at high frequency, and one patient exhibited a clinical partial response.Trial RegistrationNCT03132922ReferencesVan Tine BA, et al. CTOS 2020.Hong DS, et al. ASCO 2020.Hong DS, et al. SITC 2020.De Selm C, et al. Mol Ther 2018;26(11):2542–2552.
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Bianchi, Paola, Klaus Schwarz, Elisa Fermo, et al. "Molecular Analysis of the SEC23B Gene In Patients Affected by Congenital Dyserythropoietic Anemia Type II (CDAII)." Blood 116, no. 21 (2010): 4227. http://dx.doi.org/10.1182/blood.v116.21.4227.4227.
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Abstract Abstract 4227 CDAII, the most frequent type of congenital dyserythropoietic anemia, is an autosomal recessive disease characterized by ineffective erythropoiesis, peripheral hemolysis, erythroblast morphological abnormalities and hypoglycosylation of some RBC membrane proteins. Last year we and others identified SEC23B as the gene responsible for CDAII (Schwarz et al, 2009, Bianchi et al, 2009). SEC23B is a member of the SEC23/SEC24 family, a component of COPII coat protein complex which is involved in protein trafficking through membrane vesicles from the endoplasmic reticulum to the Golgi apparatus. The gene, localized on chromosome 20p11, is split in 20 exons and codifies for a 767 aa protein. The aim of the study was to characterize the molecular defect in a large series of CDAII patients of Caucasian origin. Fifty-one CDAII patients from 49 unrelated families (17 Italians, 20 German or Swiss, 4 Dutch, 2 British, 2 Czech and 1 Greek, Turkish, Bulgarian and Irish origin) were analyzed by direct exon sequencing. We identified 36 different mutations, 25 of which were not described before. Eighteen were disruptive and 18 were missense mutations. The patients' molecular data are summarized in the Figure (novel mutations are reported in bold). All the missense mutations affected highly conserved aminoacids and were not found in 200 normal alleles examined. The c.325G>A mutation was identified in 15 homozygous patients and in two cases in combination with other mutations. The change c.40C>T was detected in 16 unrelated patients as heterozygous mutation and, for the first time, at the homozygous level in one patient only. Considering the entire series of patients (including previously published cases) characterized by our groups (86 CDAII patients from 77 unrelated families) c.325G>A and c.40C>T mutations account for 49% (76/154) of the unrelated mutated alleles and therefore should be firstly screened during molecular diagnosis of CDAII. A c.325G>A mutation usually results in a mild to moderate clinical picture at homozygous level, whereas it may cause a very severe clinical pictures when combined with other mutations (Fermo, et al 2010). Despite the sequencing of all exons and flanking intronic regions, 5 patients displayed only one mutation, suggesting the possibility that mutations could be located in regulatory regions or that a second gene could be involved in the pathogenesis of CDAII. P. Bianchi and K. Schwarz contributed equally to this work. Disclosures: No relevant conflicts of interest to declare.
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Alareeqi, Ola A. A., Yaser H. A. Obady, Mansoor Q. Al-Khulaidi, and Khalid Al-Mureish. "SOME STUDIES ON RED BLOOD CELLS MORPHOLOGY OF HEALTHY AND DIABETIC PATIENTS IN TAIZ, YEMEN." Electronic Journal of University of Aden for Basic and Applied Sciences 2, no. 3 (2021): 109–23. http://dx.doi.org/10.47372/ejua-ba.2021.3.105.
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The aim of this study was to: 1- Identify and quantify the prevalence of RBC abnormalities in healthy and diabetic subjects. 2- Provide supporting evidence about the relation between RBC storage duration at 4oC and alterations to RBC morphology (compare with the morphology at the time of collection). 3- The obtain information about how the number of normal cells in different times of storage declines as a function of the storage period. 4- Estimate the prevalence of red cell morphological changes in diabetic patients.
 One hundred and ninety-six slides of 49 healthy and 49 diabetic patients of different age groups were collected from November 2019 to March 2020.
 Human venous blood samples were taken and anticoagulated with EDTA. samples were divided into 4 groups, group 1 was examined at once, and groups 2-4 were stored at 4oC for 24, 48, and 72 hours respectively.
 During the current study, abnormalities of erythrocyte morphology, prevalence, and histological effects of storage duration on the human blood cells were evaluated. 16 different types of abnormality in shapes of the red blood cells were identified in healthy subjects and 19 different shapes in diabetic subjects, with the difference in the prevalence percentage. Analysis of variance (ANOVA) exhibited statistically significant effects of storage time (24, 48, and 72 hours at 40C) on RBC morphology. The present result also shows that the change in erythrocyte shapes at once beginning and during time storage were statistically significant between healthy and diabetic donors. These results are in line with previous laboratory studies on other parameters.
 In conclusion, our observations indicate that morphological abnormalities of erythrocytes are common in healthy and diabetic subjects, and the slight effects of diabetic Mellitus on the changes observed in erythrocyte compare to healthy subjects over 72 hours of storage.
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Chen, Youhao, Haoming Wang, Nan Li, et al. "A Novel Approach Combined with MIPO Technique for the Treatment of Type C Pilon Fractures." Oxidative Medicine and Cellular Longevity 2022 (June 14, 2022): 1–10. http://dx.doi.org/10.1155/2022/7427255.
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Objective. Type C fracture is a complete intra-articular fracture, and the mainstay of treatment remains open reduction and internal fixation. The purpose of the study is to observe the clinical effect of an anterior ankle C approach (ankle-C) combined with minimal invasive plate osteosystems (MIPO) for tibial pilon fractures (AO/OTA 43C, combined with fibula fractures). Methods. A retrospective comparative analysis was performed on the clinical data of 33 patients with C-type pilon fractures (combined fibula fractures) admitted to our department from July 2018 to July 2021, including 12 cases treated with ankle-C (a-C) approach and 21 cases with conventional approach (including combined approach). All patients were followed up for over 6 months. Visual Analogue Scale (VAS), AOFAS Ankle-Hindfoot Scale (AOFAS-AHS), wound healing time, fracture healing time, and complications were used to evaluate the clinical efficacy. Results. The scores of VAS and AOFAS in the a-C group scored better than the conventional group ( P < 0.05 ), especially in the extent of limited range of motion (LROM) of ankle dorsiflexion-plantarflexion in 1 month after operation and at the last follow-up ( P < 0.01 ). Bone healing was achieved in both groups 6 months after operation, with no implant exposure or infection. Among them, 4 cases in the conventional approach group had wound healing time exceeding 2 weeks. Conclusions. For type C pilon fractures (combined with fibula fractures), ankle-C approach combined with MIPO technique has certain advantages in ankle function recovery and soft tissue repair, which provides an alternative for the treatment of type C pilon fractures.
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D'Angelo, Sandra P., Brian Andrew Van Tine, Steven Attia, et al. "SPEARHEAD-1: A phase 2 trial of afamitresgene autoleucel (Formerly ADP-A2M4) in patients with advanced synovial sarcoma or myxoid/round cell liposarcoma." Journal of Clinical Oncology 39, no. 15_suppl (2021): 11504. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.11504.
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11504 Background: This phase 2, open-label trial (SPEARHEAD-1; NCT04044768) is designed to evaluate the efficacy, safety, and tolerability of afamitresgene autoleucel in 45 patients (pts) with advanced/metastatic synovial sarcoma or Myxoid/Round Cell Liposarcoma (MRCLS). Methods: Eligible pts are HLA-A*02 positive with MAGE-A4-expressing tumors. Pts undergo leukapheresis for collection of autologous T-cells for processing and manufacture into afamitresgene autoleucel cells. Pts were treated with afamitresgene autoleucel doses between 1–10 × 109 transduced T-cells after receiving lymphodepleting chemotherapy. The primary endpoint is overall response rate per RECIST v1.1 by independent review. An independent Data Safety Monitoring Board reviews ongoing safety and benefit: risk during the interventional phase. Results: As of Feb 4, 2021, 32 pts received afamitresgene autoleucel. Of these pts, 59% were male, 87.5% had synovial sarcoma, the median age was 43 yrs (range: 24–73), and they had a median of 3 prior systemic lines of therapy. The MAGE-A4 antigen expression level (histoscore) ranged from 112–300, and the transduced cell dose ranged from 2.7–9.9 x 109. At the data cutoff, 25 pts were evaluable for preliminary efficacy (23 with synovial sarcoma and 2 with MRCLS) and 7 pts (5 with synovial sarcoma and 2 with MRCLS) had insufficient follow-up (<8 weeks follow-up and/or awaiting first scan). Of the 25 evaluable pts, the investigator-assessed responses were: complete response (2 pts), partial response (8 pts), stable disease (11 pts), and progressive disease (4 pts). All responses were confirmed. Nine of the 10 responders had ongoing response at the data cutoff and 3 responders had MAGE-A4 antigen histoscores <200. The most common AEs of any grade (>30% pts) were neutropenia, lymphopenia, nausea, cytokine release syndrome, leukopenia, fatigue, pyrexia, and anemia. Cytokine release syndrome of any grade occurred in 19/32 pts; 95% of those events were ≤Grade 2. No immune effector cell-associated neurotoxicity syndrome (ICANS) has been reported to date. Cytopenia (≥G3) at 4 wks post-infusion was observed in 6 pts (anemia 3 pts, neutropenia 2 pts, and thrombocytopenia 1 pt). Conclusion: These preliminary data demonstrate afamitresgene autoleucel is efficacious and well tolerated in heavily pre-treated pts. Objective responses are reported across a wide range of MAGE-A4 antigen levels and deep responses have been observed. Initial durability data is encouraging. Preliminary response data in SPEARHEAD-1 is comparable to the findings of the prior Phase 1 trial [1]. To date, the safety profile of afamitresgene autoleucel has been favorable, with mainly low-grade cytokine release syndrome and tolerable/reversible hematologic toxicities. [1]. Van Tine BA, et al. CTOS; November 18-21, 2020; Virtual. Clinical trial information: NCT04044768.
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Ramadoss, Jayanth, and Ronald R. Magness. "Multiplexed digital quantification of binge-like alcohol-mediated alterations in maternal uterine angiogenic mRNA transcriptome." Physiological Genomics 44, no. 11 (2012): 622–28. http://dx.doi.org/10.1152/physiolgenomics.00009.2012.
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Genomic studies on fetal alcohol spectrum disorders (FASD) have utilized either genome-wide microarrays/bioinformatics or targeted real-time PCR (RT-PCR). We utilized herein for the first time a novel digital approach with high throughput as well as the capability to focus on one physiological system. The aim of the present study was to investigate alcohol-induced alterations in uterine angiogenesis-related mRNA abundance using digital mRNA technology. Four biological and three technical replicates of uterine arterial endothelial cells from third-trimester ewes were fluorescence-activated cell sorted, validated, and treated without or with binge-like alcohol. A capture probe covalently bound to an oligonucleotide containing biotin and a color-coded reporter probe were designed for 85 angiogenesis-related genes and analyzed with the Nanostring nCounter system. Twenty genes were downregulated (↓) and two upregulated (↑), including angiogenic growth factors/receptors (↓placental growth factor), adhesion molecules (↓angiopoietin-like-3; ↓collagen-18A1; ↓endoglin), proteases/matrix proteins/inhibitors (↓alanyl aminopeptidase; ↓collagen-4A3; ↓heparanase; ↓plasminogen, ↑plasminogen activator urokinase; ↓platelet factor-4; ↓plexin domain containing-1; ↓tissue inhibitor of metalloproteinases-3), transcription/signaling molecules (↓heart and neural crest derivatives-2; ↓DNA-binding protein inhibitor; ↓NOTCH-4; ↓ribosomal protein-L13a1; ↓ribosomal protein large-P1), cytokines/chemokines (↓interleukin-1B), and miscellaneous growth factors (↓leptin; ↓platelet-derived growth factor-α); ↓transforming growth factor (TGF-α; ↑TGF-β receptor-1). These novel data show significant detrimental alcohol effects on genes controlling angiogenesis supporting a mechanistic role for abnormal uteroplacental vascular development in FASD. The tripartite digital gene expression system is therefore a valuable tool to answer many additional questions about FASD from both mechanistic as well as ameliorative perspectives.
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McAlpine, Cheryl, Martin Isabelle, Robyn Broad, et al. "Abstract 892: Afamitresgene autoleucel (afami-cel; formerly ADP-A2M4) demonstrates durable clinical responses by inducing broad immune engagement with anti-tumor activity." Cancer Research 83, no. 7_Supplement (2023): 892. http://dx.doi.org/10.1158/1538-7445.am2023-892.
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Abstract Afami-cel is a mixed CD4+ CD8+ autologous T-cell therapy engineered to target the cancer testis antigen melanoma-associated antigen A4 in HLA-A*02-positive patients with advanced/metastatic synovial sarcoma or myxoid/round cell liposarcoma (MRCLS). Pooled data from the Phase 1 (NCT03132922) and Phase 2 (SPEARHEAD-1, NCT04044768) trials of afami-cel showed an acceptable benefit to risk profile with an overall response rate of 36.2% and a median duration of response of 52.0 weeks.1 To support the continued investigation of potential mechanisms of durable anti-tumor activity, we previously showed that afami-cel induces broad and enduring peripheral cytokine responses2 and that afami-cel tumoral infiltration is associated with increased presence of activated and proliferative cytotoxic T-cells in the tumor microenvironment.3 Here, we report the results of translational analyses exploring the cooperation between afami-cel-induced innate and adaptive immune responses pooled from the Phase 1 and 2 trials. Methods included measurement of 92 biomarkers related to apoptosis, chemotaxis, metabolism, tumor immunity promotion/suppression, and vascular/tissue remodeling in pre- and post-infusion serum samples from 38 patients. We also conducted multiplex immunofluorescence and gene set variation analysis of Reactome immune system pathway categories and microenvironment cell populations in RNA sequencing data from pre- and post-infusion biopsies from ≥15 patients. Serum analyses showed that patients with a clinical benefit as defined by RECIST v1.1 had significantly greater post-infusion levels of chemotactic markers (Kruskal-Wallis; p&lt;0.05 for partial response [PR] compared to progressive disease, p&lt;0.01 for PR compared to stable disease), indicating higher signaling related to immune-cell recruitment towards lesions. Tumor analyses showed increased expression of genes associated with innate and adaptive immunity, and cytokine signaling, in post-infusion biopsies, including T-cell receptor signaling-related expression, which was consistent with relatively greater spatial protein detection of pro-immune infiltrate. This profile was associated with longer progression-free survival. In conclusion, our data suggest that afami-cel induces peripheral and tumoral innate and adaptive immune responses, a hallmark of durable anti-tumor activity. Updated patient sample data will be presented. 1. D'Angelo SP, et al. J Clin Oncol. 2022;40:16_suppl:11562. 2. D’Angelo SP, et al. Poster 146 presented at: CTOS 2021; Virtual. 3. Van Tine, BA et al. Paper 61 presented at: CTOS 2022; Vancouver, BC, Canada. Citation Format: Cheryl McAlpine, Martin Isabelle, Robyn Broad, Revashnee Naidoo, Ashley Liddle, Elizabeth Duperret, Paul Noto, Ruoxi Wang, Dzmitry Batrakou, Sumit Middha, Chris Evans. Afamitresgene autoleucel (afami-cel; formerly ADP-A2M4) demonstrates durable clinical responses by inducing broad immune engagement with anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 892.
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Rowland, Emma, Jordan Walter, Anna Jermakowicz, Robert Suter, Rebecca Riggins, and Nagi Ayad. "Abstract 1747: Targeting metabolic and epigenetic programs to re-sensitize glioblastoma to chemotherapy." Cancer Research 83, no. 7_Supplement (2023): 1747. http://dx.doi.org/10.1158/1538-7445.am2023-1747.
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Abstract Treatment options for glioblastoma (GBM) are limited. Prognosis remains dismal, with an 18 month on average survival rate following diagnosis due to treatment resistance and disease recurrence. The goal of this project is to investigate hallmarks of cancer progression that contribute to temozolomide (TMZ) resistance, a first tine treatment for GBM. Two signaling pathways were investigated in TMZ-sensitive and -resistant GBM cell lines and in primary and recurrent patient-derived xenograft (PDX) tumor cells by genetically and pharmacologically inhibiting methionine adenosyltransferase 2A (MAT2A) and adenosylhomocysteinase (AHCY). Cell growth and survival were assessed by measuring protein expression of proliferation, oxidative stress and cell cycle arrest markers. EPIC array analysis and targeted bisulfite sequencing were conducted to identify changes in genome-wide and specific CpG island methylation. The Seahorse XF Analyzer measured mitochondrial respiratory capacity and oxidative metabolism. Induced pluripotent stem cell organoids were co-cultured with PDX tumor cells to determine if treatments mitigate tumor cell invasiveness. Compared to parental cells (PC), MAT2A gene expression was increased by 1.7-fold in acquired resistant and de novo resistant GBM cells (RC) [(transcript per million): PC, 7386 ± 412; RC, 12941 ± 1023; n=2; p=2.10e-8].Compared to TMZ-sensitive cells (TS), TMZ-resistant cells (TR) demonstrated a 56% increase in baseline oxygen consumption rate [(pmol/min): TS, 179 ± 6.7; TR, 279 ± 13; n=18; p=.0012] and 64% increase in maximal respiratory capacity [(pmol/min): TS, 403 ± 29; TR, 659 ± 35; n=6; p&lt;.0001]. Both primary and recurrent GBM cells were subject to select MAT2A and AHCY inhibitor compounds and EC50s were determined. Recurrent cells demonstrated a vulnerability to TMZ-generated cell death both by genetically knocking down and pharmacologically inhibiting MAT2A and AHCY. MAT2A and AHCY contribute to TMZ resistance and recurrence by dysregulating methylation programs and upregulating antioxidant programs, respectively. These findings provide a foundation for developing novel combinatory therapeutic strategies and inform clinical studies intended to augment the practically negligible rate of remission for GBM and improve treatment outcomes for cancer patients. Citation Format: Emma Rowland, Jordan Walter, Anna Jermakowicz, Robert Suter, Rebecca Riggins, Nagi Ayad. Targeting metabolic and epigenetic programs to re-sensitize glioblastoma to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1747.
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Ligon, John, Woonyoung Choi, Gady Cojocaru, et al. "506 The tumor immune microenvironment of metastatic osteosarcoma is marked by lymphocyte exclusion and impacts patient progression-free survival." Journal for ImmunoTherapy of Cancer 8, Suppl 3 (2020): A541—A542. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0506.
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BackgroundPatients with relapsed metastatic osteosarcoma have no effective treatments available to them,1 and immunotherapy thus far has not succeeded in improving outcomes.2–5 We aim to understand the immune architecture of the tumor microenvironment (TME) of osteosarcoma, with the goal of harnessing the immune system as a major therapeutic strategy for the treatment of patients with osteosarcoma.Methods66 osteosarcoma tissue specimens were stained and analyzed by immunohistochemistry. Tumor-infiltrating lymphocytes (TILs) from 25 specimens were profiled by functional multiparameter flow cytometry (MFC). Distinct regions from 16 pulmonary metastases (PMs) were microdissected, and RNA was extracted to perform comparative transcriptomic studies. Clinical follow-up (median 24 months) was available from resection.ResultsDigital image analysis of immunohistochemistry demonstrated significantly higher infiltrating immune cells in the PMs compared to primary bone tumors, concentrated at the tumor-normal lung ‘PM interface’ region, and elevated expression of multiple immune checkpoint molecules at the PM interface (figure 1). MFC confirmed the increased expression of the immune checkpoint molecules programmed cell death 1 (PD-1, p<0.01) and lymphocyte activation gene 3 (LAG-3, p<0.01), as well as the activation marker IFN-γ (p<0.05) in CD8+ TILs. Gene expression profiling provided further evidence for the presence of TILs with expression of activation markers and inhibitory immune checkpoint molecules at the PM interface compared to the PM interior (figure 2). A strong M2 macrophage signature was present in both regions. Further analysis revealed that genes related to neutrophil and myeloid cell chemotaxis and known to be associated with polymorphonuclear myeloid-derived suppressor cells were highly expressed at the PM interface, along with genes for multiple subsets of dendritic cells (figure 3). Expression of PD-L1, LAG-3, and CSF1R at the PM interface were associated with worse progression-free survival (PFS), while gene sets associated with productive T cell immune response were associated with improved PFS (figure 4).Abstract 506 Figure 1Immunohistochemistry of osteosarcoma pulmonary metastasesA. H&E with demarcation of tumor-normal lung interface (center green line) and area quantified as the ‘PM interface’ (outer green lines). Pulmonary metastases demonstrate a higher concentration of immune cells (CD3 p<0.001, CD8 p<0.001, CD163 p<0.01) and PD-1 (p<0.001)/PD-L1 (p<0.05) at the PM interface.B. H&E with demarcation of PM interface as above. Pulmonary metastases demonstrating increased staining of TIM-3 (p<0.01), LAG-3 (p<0.01) and IDO1 (p<0.0001) at the PM interface (no significant concentration of CSF1R at PM interface).Abstract 506 Figure 2Activated/exhausted lymphocyte signatures at PM interfaceA. Heatmap displaying significant genes that contribute to leading-edge of core enrichment subset via Gene Set Enrichment Analysis (GSEA) demonstrating higher expression of immune regulatory molecules at the PM interface compared to the PM interior. Expression levels were converted into heatmaps and colors quantitatively correspond to fold changes. FDR=GSEA false-discovery rate q-value.B. Heatmap illustrating coefficients of xCell analysis shows higher expression of markers of cytotoxicity and activation, as well as multiple checkpoint molecules, at the PM interface, with evidence that they are being contributed chiefly by T cells. Intensity represents xCell coefficient, which corresponds to the amount that a particular region (PM interior or PM interface) or cell population (T cells, B cells, or myeloid cells) contributes to the expression of a specific gene.Abstract 506 Figure 3Genes related to dendritic cells and MDSCs at PM interfaceA. By GSEA, genes associated with multiple subclasses of antigen-presenting dendritic cells are significantly upregulated at the PM interface (cDC1=conventional type 1 dendritic cell; cDC2=conventional type 2 dendritic cell; pDC=plasmacytoid dendritic cell; moDC=monocyte-derived dendritic cell). FDR=GSEA false-discovery rate q-value.B. Heatmap shows heightened expression of cytokines, chemokines and endothelin transcripts associated with development, recruitment and maintenance of PMNs and granulocytic MDSCs at the PM interface compared to the PM interior.Abstract 506 Figure 4Markers of immune TME at PM interface correlate with PFSA. Hazard ratios for immunohistochemistry markers at the PM interface as they relate to PFS. For absolute count biomarkers (CD3, CD8, Foxp3, PD-1, CD163, and LAG-3) the unit is per 100 cells, and for percentage biomarkers (PD-L1, CSF1R, TIM-3, and IDO1) the unit is per 1%.B. Hazard ratios for gene sets at the PM interface as they relate to PFS. NS=p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001ConclusionsIn contrast to primary bone osteosarcoma ‘immune deserts,’ osteosarcoma PMs represent an ‘immune-excluded’ TME where immune cells are present but are halted at the PM interface. TILs can produce effector cytokines, suggesting their capability of activation and recognition of tumor antigens. Our findings suggest cooperative immunosuppressive mechanisms in osteosarcoma PMs that prevent TILs from penetrating into the PM interior, including immune checkpoint molecule expression and the presence of immunosuppressive myeloid cells. We identify cellular and molecular signatures that are associated with PFS of patients, which could be potentially manipulated for successful immunotherapy.Ethics ApprovalThis study was approved by Johns Hopkins University’s Ethics Board, approval number FWA00005752.ReferencesMirabello L, Troisi RJ, Savage SA. Osteosarcoma incidence and survival rates from 1973 to 2004: Data from the surveillance, epidemiology, and end results program. Cancer 2009;115(7):1531–43.Tawbi HA, Burgess M, Bolejack V, Van Tine BA, Schuetze SM, Hu J, et al. Pembrolizumab in advanced soft-tissue sarcoma and bone sarcoma (SARC028): A multicentre, two-cohort, single-arm, open-label, phase 2 trial. Lancet Oncol 2017;18(11):1493–501.Davis KL, Fox E, Merchant MS, Reid JM, Kudgus RA, Liu X, et al. Nivolumab in children and young adults with relapsed or refractory solid tumours or lymphoma (ADVL1412): A multicentre, open-label, single-arm, phase 1–2 trial. Lancet Oncol 2020;21(4):541–50.D’Angelo SP, Mahoney MR, Van Tine BA, Atkins J, Milhem MM, Jahagirdar BN, et al. Nivolumab with or without ipilimumab treatment for metastatic sarcoma (alliance A091401): Two open-label, non-comparative, randomised, phase 2 trials. Lancet Oncol 2018;19(3):416–26.Paoluzzi L, Cacavio A, Ghesani M, Karambelkar A, Rapkiewicz A, Weber J, et al. Response to anti-PD1 therapy with nivolumab in metastatic sarcomas. Clin Sarcoma Res 2016;6:24.
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Chen, Yongping, Haotian Yang, Tianyuan Yang, et al. "Protective Effects of Low-Dose Alcohol against Acute Stress-Induced Renal Injury in Rats: Involvement of CYP4A/20-HETE and LTB4/BLT1 Pathways." Oxidative Medicine and Cellular Longevity 2021 (October 13, 2021): 1–14. http://dx.doi.org/10.1155/2021/4475968.
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Low-dose alcohol possesses multiple bioactivities. Accordingly, we investigated the protective effect and related molecular mechanism of low-dose alcohol against acute stress- (AS-) induced renal injury. Herein, exhaustive swimming for 15 min combined with restraint stress for 3 h was performed to establish a rat acute stress model, which was verified by an open field test. Evaluation of renal function (blood creatinine and urea nitrogen), urine test (urine leukocyte esterase and urine occult blood), renal histopathology, oxidative stress, inflammation, and apoptosis was performed. The key indicators of the cytochrome P450 (CYP) 4A1/20-hydroxystilbenetetraenoic acid (20-HETE) pathway, cyclooxygenase (COX)/prostaglandin E2 (PGE2) pathway, and leukotriene B4 (LTB4)/leukotriene B4 receptor 1 (BLT1) pathway were measured by real-time PCR and ELISA. We found that low-dose alcohol (0.05 g/kg, i.p.) ameliorated AS-induced renal dysfunction and histological damage. Low-dose alcohol also attenuated AS-induced oxidative stress and inflammation, presenting as reduced malondialdehyde and hydrogen peroxide formation, increased superoxide dismutase and glutathione activity, and decreased myeloperoxidase, interleukin-6, interleukin-1β, and monocyte chemoattractant protein-1 levels ( P < 0.05 ). Moreover, low-dose alcohol alleviated AS-induced apoptosis by downregulating Bax and cleaved caspase 3 protein expression and reduced numbers of terminal deoxynucleotidyl transferase-mediated dUTP nick-end label-positive cells ( P < 0.01 ). Correlation analysis indicated that 20-HETE was strongly correlated with oxidative stress, while LTB4 was strongly correlated with inflammation. Low-dose alcohol inhibited AS-induced increases in CYP4A1, CYP4A2, CYP4A3, CYP4A8, and BLT1 mRNA levels and LTB4 and 20-HETE content ( P < 0.01 ). Interestingly, low-dose alcohol had no effect on COX1 or COX2 mRNA expression or the concentration of PGE2. Furthermore, low-dose alcohol reduced calcium-independent phospholipase A2 mRNA expression, but did not affect secreted phospholipase A2 or cytosolic phospholipase A2 mRNA expression. Together, these results indicate that low-dose alcohol ameliorated AS-induced renal injury by inhibiting CYP4A/20-HETE and LTB4/BLT1 pathways, but not the COX/PGE2 pathway.
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Mardilovich, Katerina, Lilli Wang, Rachel Kenneil, et al. "95 Inhibition of AKT signaling during expansion of TCR-engineered T-cells from patient leukocyte material generates SPEAR T-cells with enhanced functional potential in vitro." Journal for ImmunoTherapy of Cancer 8, Suppl 3 (2020): A106. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0095.
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BackgroundT-cells attributes for adoptive cell therapy of patients with advanced cancer can be optimized during ex vivo expansion culture. Autologous TCR-engineered T-cells targeting the MAGE-A4 antigen with Specific Peptide Enhanced Affinity Receptors (SPEAR T-cells) have shown promise in the clinic.1 The highly variable leukocyte material obtained from individual patients during apheresis can present a manufacturing challenge for autologous T-cell therapies. The degree of ex vivo expansion and the functional attributes of the expanded T-cell product impact therapeutic efficacy and can be suboptimal for some patient apheresis material. Both TCR and cytokine growth factor signals used for ex vivo T-cell expansion promote robust activation of AKT (Protein Kinase B) signaling, which drives T-cell activation, proliferation, and terminal differentiation. It is hypothesized that inhibition of AKT signaling during T-cell expansion may uncouple proliferation and terminal differentiation, leading to the generation of less differentiated T-cells that may have functional benefit in vivo.2 3MethodsWe evaluated use of an AKT inhibitor during SPEAR T-cell manufacturing using leukocytes from healthy donors and patients with advanced solid cancers.ResultsAKT inhibition resulted in the generation of a more consistent expansion and phenotype of the final T-cell product. This was observed using two SPEAR T-cell constructs, ADP-A2M4 and ADP-A2M4CD8. Ex vivo SPEAR T-cell expansion in the presence of an AKT inhibitor generated CD8+ T-cells that maintained a less differentiated phenotype (based on CCR7+CD45RA+ and CD62L+ expression). AKT inhibition was associated with enhanced antigen-specific responses of SPEAR T-cells in vitro, including effector cytokine production, target-cell killing, ability to proliferate in response to prolonged antigen-stimulation and maintenance of cytotoxic activity following antigen re-stimulation.ConclusionsWe plan to introduce AKT inhibition into the GMP manufacturing process, and evaluate the efficacy of the resulting products in ongoing clinical studies.AcknowledgementsWe are extremely grateful to the patients, who were previously enrolled in our clinical trials, and healthy donors for their consent for R&D studies. This was a collaborative cross-functional project, and we are grateful for the contributions of the following Scientists: Garth Hamilton, Adel Toth, Abigail Kay, Sophie Badie, Josh Griffiths, Kaushik Sarkar, Anoop Chandran.Ethics ApprovalThe experimental study was conducted in accordance with the principles of the Declaration of Helsinki and the International Conference on Harmonization Good Clinical Practice guidelines and was approved by local authorities. An independent ethics committee or institutional review board approved the clinical protocol at each participating center. All the patients provided written informed consent before study entry.ReferencesHong DS, Van Tine BA, Olszanski AJ, et al, Phase 1 dose escalation and expansion trial to assess safety and efficacy of ADP-A2M4 in advanced solid tumors. J Clin Oncol 2020;38;A102.Klebanoff C, Crompton J, Leonardi A, et al. Inhibition of AKT signaling uncouples T cell differentiation from expansion for receptor-engineered adoptive immunotherapy. JCI Insight 2017;2:e95103.van der Waart A, van de Weem N, Maas F, et al. Inhibition of Akt signaling promotes the generation of superior tumor-reactive T cells for adoptive immunotherapy. Blood. 2014;124;3490-3500
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Carneiro, Benedito A., Maria Diab, Brian A. Van Tine, et al. "Abstract CT116: First-in-human study of AZD8853, an anti-growth and differentiation factor 15 (GDF15) antibody, in patients (pts) with advanced/metastatic solid tumors." Cancer Research 83, no. 8_Supplement (2023): CT116. http://dx.doi.org/10.1158/1538-7445.am2023-ct116.
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Abstract Background: The cytokine GDF15 is overexpressed in solid malignant tumors such as colorectal, lung and urothelial cancer, where it modulates T cells, dendritic cells (DCs) and myeloid-derived cells, driving the tumor microenvironment toward an immunosuppressive, tumor-promoting state. AZD8853 is a humanized immunoglobulin G1 monoclonal antibody that binds to, and neutralizes, GDF15. Anti-GDF15 treatment increased T cell proliferation and DC activation, leading to an antitumor immune response in preclinical studies of anti-PD-L1 resistant models. In vitro and in vivo preclinical data support the potential antitumor activity of AZD8853 in pts with selected advanced/metastatic cancers. Methods: This Phase I/IIa, first-in-human, open-label study (NCT05397171) assesses the safety, pharmacokinetics (PK), pharmacodynamics (PD) and preliminary efficacy of AZD8853 in pts with histologically or cytologically confirmed locally advanced, unresectable or metastatic mismatch repair-proficient colorectal cancer (pMMR-CRC), non-small-cell lung cancer (NSCLC) and urothelial carcinoma (UC). Up to 165 pts will be enrolled in 3 parts: Part A, dose escalation; Part B, pharmacodynamics expansion; and Part C, efficacy expansion. All pts receive AZD8853 IV. Eligible pts are ≥18 years old with ≥1 measurable target lesion per RECIST v1.1, ECOG PS of 0/1, life expectancy ≥12 weeks and adequate organ and bone marrow function. Pts with NSCLC must have had ≥1 prior line of systemic treatment in the advanced/metastatic setting, and no sensitizing EGFR or ALK aberrations. Pts with pMMR-CRC must have had ≥2 prior treatments in the advanced/metastatic setting. Pts with UC must have had ≥1 prior treatment in the advanced/metastatic setting including platinum-containing therapy and/or a PD-(L)1-inhibitor. Pts with Grade ≥2 unresolved toxicities from prior therapy, symptomatic CNS metastases or leptomeningeal disease, or prespecified active/ongoing infections are excluded. The primary objective is safety, including dose-limiting toxicities, adverse events (AEs), serious AEs and AEs leading to AZD8853 discontinuation. The secondary objectives include assessment of efficacy (objective response rate, disease control rate, duration of response, percentage change from baseline in target lesion size, change from baseline in circulating tumor DNA, and progression-free and overall survival), PK and immunogenicity. Changes in GDF15 serum levels are measured in Parts A and B. Tumoral CD8+ T cell infiltration is measured in a subset of pts from Part B using PET/CT imaging and IHC of paired biopsies. The study is currently recruiting at centers in the USA and Canada with additional sites planned in the UK, France and Spain. Citation Format: Benedito A. Carneiro, Maria Diab, Brian A. Van Tine, Anthony F. Shields, Albiruni Abdul Razak, John F. Hilton, Rafael Santana-Davila, Elhan Sanai, Jorge Zeron-Medina, Veronique Bragulat, Kath Lowery, Arthur Lambert, John Hood, Rakesh Kumar, Duncan Jodrell, Patricia LoRusso. First-in-human study of AZD8853, an anti-growth and differentiation factor 15 (GDF15) antibody, in patients (pts) with advanced/metastatic solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT116.
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Holowiecki, Jerzy, Sebastian Giebel, Malgorzata Krawczyk-Kulis, et al. "Lower Relapse Incidence after Non-Cryopreserved Autologous Bone Marrow Transplantation Compared to Peripheral Blood Stem Cell Transplantation for High-Risk Hodgkin’s Lymphoma." Blood 104, no. 11 (2004): 912. http://dx.doi.org/10.1182/blood.v104.11.912.912.
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Abstract In a number of studies on Hodgkin’s lymphoma (HL), autologous transplantation of peripheral blood hematopoietic stem cells (autoPBSCT) was proved to result in faster hematopoietic recovery compared to bone marrow transplantation (autoBMT), however, no difference regarding long-term outcome has been demonstrated so far. In Katowice transplant centre we developed a new method of autoBMT with bone morrow not-cryopreserved but stored for 3 days in 40C and reinfused 24 hours after completion of CBV conditioning. In this study we analyzed outcome of 40 HL patients treated with this method in comparison with 125 patients given autoPBSCT between 1993–2004. In this setting patients were treated with CBV (n=32), BEAM+/− procarbazine (n=63) or other preparative regimens (n=30). In the autoBMT group patients were transplanted in high-risk CR1 (achieved after >1 line of therapy) (25%), CR>=2 (22.5%), PR1 (35%), PR>=2 (5%), and primary or secondary refractoriness (12.5%). The indications for autoPBSCT were comparable. As well, both groups did not differ in terms of age, histological subtype, disease stage at diagnosis, organ involvement or preceding therapy. At six years, the overall- and progression-free survival for autoBMT and autoPBSCT group equaled 88% vs. 72% (p=0.1) and 69% vs. 54% (p=0.08), respectively. The relapse incidence was significantly lower for patients given autoBMT compared with autoPBSCT (23% vs. 41%, p=0.03). In a univariate analysis, among other analyzed factors (age, disease stage at diagnosis and at transplantation, organ involvement, histological subtype, conditioning regimen, preceding therapy), the only factor influencing the risk of relapse was disease status at transplantation (CR or PR vs. NR - 32% vs. 62%, p=0.001). In a multivariate analysis the impact of both disease status (p=0.001) and the source of stem cells (p=0.04) remained statistically significant. The 100 days incidence of non-relapse mortality equaled 2.5% for autoBMT and 1% for autoPBSCT (p=NS). The neutrophil >0.5 G/L recovery was faster in the autoPBSCT group compared with autoBMT (14.7 vs. 17 d., p=0.006) whereas the difference regarding platelet >50 G/L recovery was not significant (17.5 vs. 19 d., p=NS). Both procedures did not differ in terms of severe adverse events as well as the need for blood products substitution, iv antibiotic therapy, cytokine tharapy or the time of hospital stay. CONCLUSION: AutoBMT without cryopreservation results in lower relapse rate in high-risk HL patients compared with autoPBSCT. Although the neutrophil recovery is longer by 2.5 days, the toxicity of both procedures as well as the need for supportive treatment is comparable.
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Strandgaard, Trine, Iver Nordentoft, Karin Birkenkamp-Demtröder, et al. "Abstract 3340: Bladder field cancerization impacts tumor development and T-cell exhaustion, and is reflected in urinary tumor DNA." Cancer Research 83, no. 7_Supplement (2023): 3340. http://dx.doi.org/10.1158/1538-7445.am2023-3340.
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Abstract Introduction: Patients with high-risk non-muscle invasive bladder cancer (NMIBC) are treated with Bacillus Calmette-Guérin (BCG), which targets altered normal-appearing urothelium (field cancerization). Field cancerization may affect treatment response and outcome. High urinary tumor DNA (utDNA) levels in patients with NMIBC have been associated with worse clinical outcomes, and utDNA may be used for real-time assessment of residual disease. The prognostic and predictive values of field cancerization and utDNA need further investigation. Materials and methods: We analyzed samples procured from 134 patients with NMIBC - including 210 tumors, 751 selected site biopsies (SSBs) and 187 urine samples. The median follow-up time was 7.8 years. Samples were collected before and after BCG-treatment. Sixty-four patients (48%) progressed to muscle-invasive bladder cancer or experienced high-grade NMIBC recurrence within two years after BCG treatment, indicating BCG-failure. Tumor samples were analyzed using whole-exome and RNA-sequencing. DNA from SSBs and urine samples was subjected to tumor-informed deep-targeted sequencing. SSB DNA was sequenced to a mean coverage of 1,357X, and urinary DNA to a mean coverage of 2,153X. Unique molecular identifiers were applied and the deepSNV pipeline was used for error-robust mutation calling. The level of field cancerization in each patient was estimated by the highest number of called mutations adjusted by the number of mutations on the sequencing panel. Results: We detected mutations in 458 out of 751 analyzed SSBs. Mutations in normal-appearing SSBs were detected at a mean frequency of 0.04 (range: 0.002-0.39). The level of field cancerization increased with age (p=0.0027). utDNA levels reflected the bladder disease status, increasing with higher tumor stage and multiplicity and was shed from both bladder field cancerization and tumors. High utDNA levels after BCG-treatment were associated with high recurrence rates (p&lt;0.001), high risk of progression (p=0.0014) and worse high-grade recurrence-free survival (p=0.047). Field cancerization level was not associated with response to BCG treatment. High levels of field cancerization and utDNA were associated with high tumor-associated CD8 T-cell exhaustion. Additionally, a high field cancerization level correlated with a high tumor neoantigen load (p=0.029), indicating a possible link to the observed CD8 T-cell exhaustion. Conclusion: A high level of field cancerization was associated with an immune exhausted phenotype of the tumor. Measurements of utDNA reflected the overall disease status of the bladder, including field cancerization. Collectively, these results suggest that field cancerization may impact the tumor microenvironment and utDNA levels may be used for assessing clinical risk in NMIBC and for guiding surveillance and early treatment interventions. Citation Format: Trine Strandgaard, Iver Nordentoft, Karin Birkenkamp-Demtröder, Liina Salminen, Frederik Prip, Emil Christensen, Philippe Lamy, Tine Ginnerup Andreasen, Sia Viborg Lindskrog, Michael Knudsen, Julie Rasmussen, Torben Steiniche, Jørgen Bjerggaard Jensen, Lars Dyrskjøt. Bladder field cancerization impacts tumor development and T-cell exhaustion, and is reflected in urinary tumor DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3340.
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Van Tine, Brian A. "Abstract IA022: Arginine, serine, xCT and ME, the therapeutic metabolism of different sarcomas." Clinical Cancer Research 28, no. 18_Supplement (2022): IA022. http://dx.doi.org/10.1158/1557-3265.sarcomas22-ia022.
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Abstract The absolute diversity of the biology of the sarcomas makes therapeutic development challenging. As most of tumor metabolism is composed of transporters and enzymes, finding metabolic dependencies should allow for small molecule targeting. Due to the rapid metabolic evolution that tumors undergo in response to the targeting of any one metabolic pathway, a deep understanding of sarcoma metabolism is needed. First, the most common metabolic adaptation that sarcomas undergo is loss of expression of argininosuccinate synthetase 1 (ASS1), which is silenced in ~90% of cases by methylation. This results in sarcomas being arginine auxotrophic and sensitive to arginine starvation therapies, such as with arginine deiminase (ADI-PEG20). Not only does arginine starvation alter the Warburgian biology of sarcomas making them dependent of glutamine, but it can be used to upregulate the expression of cell surface transports such as hENT, which allows to gemcitabine internalization. In addition, mouse modeling has demonstrated that vesicular trafficking is key to overcoming initial arginine starvation in vivo, a process that can be blocked with chloroquine. This arginine dependency is likely related to the mesenchymal origin of sarcomas, which would explain its high recurrence rate of ASS1 silencing across histologies. Next, due to the upregulation of 3-phosphoglycerate dehydrogenase (PHGDH), osteosarcoma preferentially utilizes glucose to make the serine that enters the folate cycle, as opposed to completing lower glycolysis. In addition, as osteosarcoma does not seem to depend on extracellular serine import, inhibition of PHGDH leads to pro-survival compensation by the mTORC pathway. This can be targeted on both the AMP Kinase and the AKT dependent parts of the MTORC pathway that converge at FOXO3. When both are inhibited approach demonstrates unique triple synergy and therapeutic strategy for osteosarcoma due to its unique serine biology. Finally, synovial sarcoma lack the expression of malic enzyme 1 (ME1), also likely due to its cell of origin. This leads to reduced glucose oxidation, enhanced glycolysis, and compensatory increased flux through the pentose phosphate pathway for cytoplasmic NADPH production. Additionally, absence of ME1 in SS results in significant reductions in the GSH/GSSG ratio as well as a reduced glutathione synthesis. Sensitivity to GSH pathway inhibition is also reduced while sensitivity to inhibition of the thioredoxin system is significantly increased. ME1 absence results in increases in the labile iron pool that sensitizes synovial sarcoma to ferroptosis. Therefore, ME1 null SS is exquisitely sensitive to induction of ferroptosis with xCT inhibition. This can be accomplished by erastin analogs in vitro and ACXT-3102 (a tumor targeted erastin) in vivo. As we learn more about each sarcoma, we must understand the underlying metabolism of each subtype and their cell of origin. Given the targetable nature of metabolic enzymes and transporters, an ever deeper understanding of sarcoma metabolism is warranted. Citation Format: Brian A. Van Tine. Arginine, serine, xCT and ME, the therapeutic metabolism of different sarcomas [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr IA022.
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Leonard, Alexis, Aylin Bonifacino, Venina Marcela Dominical, et al. "Bone Marrow Characterization in Sickle Cell Disease: Inflammation and Stress Erythropoiesis Lead to Suboptimal CD34 Recovery Compared to Normal Volunteer Bone Marrow." Blood 130, Suppl_1 (2017): 966. http://dx.doi.org/10.1182/blood.v130.suppl_1.966.966.
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Abstract Introduction Gene therapy for sickle cell disease (SCD) requires modification of a high number of long term engrafting hematopoietic stem cells (LT-HSCs) sufficient to sustain production of the gene of interest at levels capable of overcoming the pathogenic HbSS phenotype. Unlike β-Thalassemia, the inflammatory bone marrow (BM) environment and stress erythropoiesis associated with SCD may have significant impacts on HSC quality and yield necessary for disease amelioration. Important work to optimize gene therapy through improvement in gene transfer efficiency, editing strategies, or transplant conditioning can only improve gene therapy in SCD if enough autologous HSCs are LT-HSCs, thus characterization of SCD BM and CD34+ HSCs is required. Collection type, storage, and delays in processing may further impact CD34+ recovery and should be investigated as a strategy to maximize LT-HSC recovery. Methods Twenty milliliters of BM from subjects with SCD (HbSS genotype) and normal volunteers was collected in different anticoagulants (Heparin, ACD-A) and processed immediately(day 0) or stored at 40C and processed the following day(day 1). After isolation via Ficoll density gradient centrifugation, the mononuclear (MN) layer was stained with antibodies against inflammatory markers (CD36, CD35, CD11b, CD62L, CD62P), non-MN cells (GPA, CD66b, CD41/61), or processed for CD34+ selection using a magnetic microbead CD34+ selection kit and stained for CD34, CD45, and GPA expression. Data were analyzed by conventional and imaging flow cytometry, the latter confirming post-CD34+ selection flow data and demonstrating antibody intensity as a characterization of HSC heterogeneity and progenitor lineage. Complete blood count and hemoglobin (Hb) electrophoresis were obtained at the time of BM collection. Statistical analyses were performed using unpaired t-tests. Results BM was collected from 18 subjects (16 with SCD; 11M; age 21-41 years). Median Hb (8.6 vs. 13.5 gm/dL, p&lt;0.01) and white blood cell count (8.8 vs. 4.2 K/mcL, p&lt;0.05) differed significantly between SCD and non-SCD subjects. Median percent sickle Hb in SCD subjects was 62%. Inflammatory markers and contamination with red cell and platelet markers in the post-Ficoll MN layer were higher in SCD vs. non-SCD BM regardless of anticoagulant (CD35 24% vs. 13%, p&lt;0.05; CD36 22% vs. 11%, p&lt;0.05; CD62P 16% vs. 3%, p&lt;0.05; GPA 16% vs. 4%, p&lt;0.05; CD41/61 19% vs. 3%, p&lt;0.05), and trended higher on day 1 in SCD BM in both anticoagulants, significantly in Heparin (GPA 23% vs. 33% on day 1, p&lt;0.05). Total CD45 expression was lower in SCD vs. non-SCD BM in both anticoagulants (p&lt;0.05) and on day 0 (p&lt;0.05) and 1 (p&lt;0.01), with Amnis data confirming a higher CD34+CD45- population in SCD BM (4 ± 2% vs. 0.5 ± 0.3%, p&lt;0.05). While there was no significant difference in total CD34+ cell count between SCD and non-SCD BM after selection post-Ficoll, there was a trend for lower CD34+ count in SCD in both anticoagulants (2.6x10^5 vs. 4.7x10^5, p=0.1). SCD CD34+ cells were characterized by higher GPA expression (28 ± 5% vs. 13 ± 3% in non-SCD BM, p&lt;0.01) that worsened in Heparin on day 1 (22 ± 6.3% vs. 35 ± 12.4%, p&lt;0.05). Image cytometry confirmed a majority of GPA expression in SCD BM is from single cell CD34+CD45+GPA+ and CD34+CD45-GPA+ HSCs in addition to red cell aggregates, with an increase in CD34+CD45-GPA+ HSCs on day 1 (10 ± 5% vs. 0.6 ± 0.2 % on day 0, p&lt;0.05). Furthermore, the percentage of CD34hi HSCs was lower in SCD vs. non-SCD BM, with &gt;50% SCD HSCs characterized as CD34dim (56% vs. 4% in non-SCD BM, p&lt;0.001). Lastly, the purity of CD34+ selection worsened from day 0 to day 1 in SCD BM in heparin (94% vs. 68 ± 8%, p&lt;0.05) and ACD-A (88% vs. 68 ± 0.7%, p&lt;0.05). Conclusions SCD BM is characterized by increased inflammation and cell contamination in the MN layer regardless of anticoagulant that worsens over time in Heparin more significantly than in ACD-A. Compared to non-SCD BM, CD34+ HSC yield post-Ficoll is lower in SCD subjects, and is characterized by a larger proportion of CD34+CD45+GPA+ and CD34+CD45-GPA+ HSCs that rise with delays in processing. This indication of early differentiation along the erythroid lineage, with more than 50% of HSCs losing CD34+ intensity suggesting they are not LT-HSCs, suggests suppression of inflammation and stress erythropoiesis, combined with early cell processing may be critical for maximal HSC recovery necessary for successful gene therapy in SCD. Disclosures Luo: bluebird bio Inc.: Employment. Pierciey: bluebird bio: Employment.
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Spigel, David R., Eugene Ahn, Herbert L. Duvivier, et al. "Abstract CT152: Phase I study of MT-6402, a novel engineered toxin body (ETB) targeting PD-L1, in patients with PD-L1 expressing advanced solid tumors." Cancer Research 82, no. 12_Supplement (2022): CT152. http://dx.doi.org/10.1158/1538-7445.am2022-ct152.
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Abstract This is the first-in-human study of MT-6402, a unique, first-in-class potent PD-L1-targeted engineered toxin body (ETB) capable of direct killing of PD-L1 expressing cells via rapid PD-L1-mediated internalization of a fused Shiga-like toxin A subunit (SLTA) resulting in permanent ribosomal inactivation. MT-6402 also delivers an HLA-A*02 restricted pp65 cytomegalovirus (CMV) antigen into PD-L1 expressing tumor cells leading to MHC-I presentation to existing CMV-specific cytotoxic T cells (antigen seeding). MT-6402 functions by targeting tumor and inhibitory immune cells directly and altering tumor immunophenotype to re-direct antiviral cytotoxic T cells into the tumor microenvironment. MT-6402 shows picomolar cytotoxic activity across several PD-L1 expressing cancer cell lines and treatment of human PBMCs results in selective depletion of PD-L1 positive cells. MT-6402 was well tolerated in non-human primates at doses above those which induce pharmacodynamic (PD) effect (reduction of CD14+ monocytes) in humans. This first-in-human study in patients with PD-L1-expressing advanced solid tumors will employ a modified toxicity probability interval design to determine MTD and will then enroll additional subjects at the MTD, to further explore safety and efficacy and determine RP2D. Results of the first dose cohort (16 micrograms/kg) are presented. Six patients received MT-6402. A significant and sustained reduction in CD14+ monocytes starting in cycle 2 was observed in patients on therapy beyond one cycle and was maintained with each MT-6402 administration, indicating HLA-independent PD effect consistent with preclinical observations. One HLA-A*02 and CMV+ patient with osseous metastases from NSCLC showed marked CMV-specific T-cell extravasation at day 8 and serum cytokine signatures consistent with antigen dependent responses and T cell mobilizations, suggesting engagement of MT-6402 antigen seeding. This patient has reduced intensity of metastatic bone lesions with 3/4 lesions resolving; the remaining lesion showing reduced uptake. Two patients had cytokine elevations at day 15, manifested by transient (1-12h), grade 2 infusion-related reactions and grade 2 cytokine release syndrome, which were subsequently prevented by steroid premedication. These results describe a novel approach to checkpoint modulation, leveraging direct PD-L1 cell kill and antigen seeding technology by the ETB. The results support further dose escalation and hold promise for development of MT-6402 for solid tumors, including in the R/R setting. Citation Format: David R. Spigel, Eugene Ahn, Herbert L. Duvivier, Drew Rasco, Agnes Rethy, Chris Moore, Amy Yuet, Sandra Hankins, Swati Khanna, Joseph Dekker, Brian Van Tine. Phase I study of MT-6402, a novel engineered toxin body (ETB) targeting PD-L1, in patients with PD-L1 expressing advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT152.
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Cranmer, Lee D., Andrew J. Wagner, Vinod Ravi, et al. "Abstract LB288: Biomarker analysis from AMPECT correlating response to nab-sirolimus with TSC1 and TSC2 inactivating alterations." Cancer Research 83, no. 8_Supplement (2023): LB288. http://dx.doi.org/10.1158/1538-7445.am2023-lb288.
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Abstract nab-Sirolimus is an mTOR inhibitor (mTORi) approved in the US for the treatment of adult patients with locally advanced, unresectable, or metastatic malignant perivascular epithelioid cell tumor (PEComa) based on clinical efficacy and safety data from the phase 2, multicenter, open-label AMPECT trial (NCT02494570). TSC1 and TSC2 are tumor suppressor genes and key upstream regulators of mTOR complex 1 (mTORC1); inactivating alterations (loss-of-function mutations or deletions) in these genes lead to mTORC1 hyperactivation, which may contribute to tumor formation. Phosphorylation of S6 ribosomal protein (pS6) is a reliable surrogate for mTORC1 activity. Here we present the results of an exploratory biomarker analysis performed on samples from patients enrolled in the AMPECT study. Targeted exome next-generation sequencing (NGS) using a 500-gene OncoPanel test (Center for Advanced Molecular Diagnostics, Brigham and Women’s Hospital, Boston, MA) assessed mutations, copy number changes, and translocation events. pS6 was assessed by immunohistochemistry (IHC). Twenty-five patients had tissue sufficient for NGS, 56% (14/25) had either TSC1 (N=5, 20%) or TSC2 (N=9, 36%) mutations: TSC1, 1 frameshift, 2 splice site, 1 missense, and 1 nonsense mutation; TSC2, 1 nonsense, 7 frameshift, and 1 homozygous deletion. In this dataset, TSC1 and TSC2 inactivating alterations were mutually exclusive. Patients with TSC1 or TSC2 inactivating alterations achieved a clinically meaningful benefit: 64.3% (9/14) objective overall responses (including complete and partial responses) and median (95% CI) duration of response (DOR) of 45.7 (5.6, not reached [NR]) months, progression-free survival (PFS) of 41.2 (5.5, NR) months, and overall survival (OS) of NR (31.6, NR) months. Mutations in TP53, RB1, and ATRX were also common (48%, 24%, and 20%, respectively). Of tissue samples evaluable for IHC (N=25), responses occurred in 58.8% (10 of 17) of patients with pS6 positive tumors versus 0% (0 of 8) with pS6 negative tumors; absent pS6 staining was negatively associated with response to nab-sirolimus (P=0.008, Fisher’s exact). Median DOR for pS6 positive responders was 39.7 (5.6, NR) months. In pS6 positive vs pS6 negative patients, the median PFS was 24.4 (2.8, 53.1) vs 5.5 (2.8, NR) months, and OS was 53.1 (18.0, NR) vs 37.0 (16.6, NR) months. A variety of pathogenic inactivating alterations were observed in TSC1 and TSC2 genes, though TSC2 mutations were most commonly frameshift mutations; no recurring mutations were observed. A tumor-agnostic study (PRECISION 1: NCT05103358) is now recruiting patients with pathogenic inactivating TSC1 or TSC2 alterations to further examine these biomarker findings. Citation Format: Lee D. Cranmer, Andrew J. Wagner, Vinod Ravi, Richard F. Riedel, Kristen N. Ganjoo, Brian A. Van Tine, Rashmi Chugh, Erlinda M. Gordon, David J. Kwiatkowski, Jason L. Hornick, Heng Du, Li Ding, Anita N. Schmid, Willis H. Navarro, Loretta M. Itri, Mark A. Dickson. Biomarker analysis from AMPECT correlating response to nab-sirolimus with TSC1 and TSC2 inactivating alterations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB288.
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Okera, Meena, Brian A. Van Tine, Joleen M. Hubbard, et al. "Abstract OT2-11-01: A phase 1 study of the novel immunotoxin MT-5111 in patients with HER2+ tumors: interim results." Cancer Research 83, no. 5_Supplement (2023): OT2–11–01—OT2–11–01. http://dx.doi.org/10.1158/1538-7445.sabcs22-ot2-11-01.
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Abstract MT-5111 is a 55kD engineered toxin body targeting HER2 in solid tumors that binds to an epitope distinct from trastuzumab and pertuzumab, offering potential combination strategies with other HER2-targeting agents. MT-5111 works by internalizing, self-routing through intracellular compartments to the cytosol, and inducing potent cell-kill via the enzymatic and permanent inactivation of ribosomes. This is a phase 1 study in adults with advanced HER2+ solid tumors. MT-5111 is dosed weekly IV over 30 min in every 21-day cycle until disease progression, unacceptable toxicity, death, or withdrawn consent. The study has dose escalation (Part A) cohorts enrolling patients (pts) with any HER2+ cancer (CA) and expansion (Part B) cohorts for HER2+ breast cancer (BC), Gastric or Gastroesophageal junction adenocarcinoma (GEA), or other HER2+ solid CA. As of 30 June 2022, 42 total pts had enrolled (36 in Part A on 0.5-23 µg/kg/dose, 6 pts with BC in Part B1 on 10 µg/kg/dose). Median age 65 years, 28 (66.78%) pts were female, median of 4 prior systemic and 2 prior HER2-targeting treatment (tx). 17 pts with BC, 6 with biliary CA, 9 with GEA, and 10 with other solid CA have enrolled. Of the 17 BC pts, 15 received ≥ 10 µg/kg/dose. Tx emergent adverse events (TEAEs) have been reported in 40 (95%) pts, and tx-related AEs (TRAE) occurred in 23 (55%) pts. No pt experienced G4-G5 TRAE. G1 troponin elevations were noted in 5 pts without clinical signs of cardiotoxicity (1 pt 6.75 µg/kg, 2 pts 10 µg/kg, 1 pt 17 µg/kg, 1 pt 23 µg/kg). Reversible G1/G2 infusion-related reactions were reported in 2 pts. Tx-related G1-G3 rash was observed in 5 pts (4 pts ≥ 10 µg/kg/dose); maculopapular in 2 pts, acneiform in 1 pt and associated with pruritus in 3 pts. The G3 rash developed one wk after first dose of 23 µg/kg, was declared a DLT, improved with systemic steroid therapy and the pt continued tx at the same dose without recurrence. Best overall response per RECIST thus far is stable disease (SD) in 17 pts, non-CR/non-PD in 1 pt, and progressive disease (PD) in 14 pts. 1 pt had non-CR/non-PD for 30 wks (1 μg/kg, BC); 1 pt had SD for 24wks (10 μg/kg, pancreatic); 1 pt is on tx with SD through 8 cycles (10 μg/kg, BC). Of the 10 BC pts who received ≥ 10 μg/kg/dose, the best response was 5 SD. The mean serum concentration of MT-5111 has increased in a dose-proportional manner starting at 6.75µg/kg/dose (Table 1). The soluble HER2 (sHER2) levels at end of tx were higher compared to baseline in cohorts that received ≤ 4.5µg/kg/dose, but similar or lower in cohorts that received ≥ 6.75µg/kg/dose. Higher MT-5111 doses have been well tolerated and may saturate circulating sHER2, leading to more predictable serum concentrations and tumor penetration. The Cmax in humans at doses ≥6.75 µg/kg/dose is above the in vitro IC50 for high HER2+ cell lines (0.029nM) and at 17 µg/kg/dose, above the IC50 for moderate HER2+ cells (1.6nM). In conclusion, the dose proportionate increase in serum concentration with levels above the in vitro IC50 and the leveling off/reduction of sHER2 indicate exposure to MT-5111 is at clinically therapeutic levels. Skin toxicity at higher doses may indicate on-target effect as observed in other EGFR-targeted therapies where it is associated with clinical response and a better prognosis. sHER2 biomarker data is expected for all cohorts with PK correlation and 23µg/kg safety and efficacy data. PK profile of MT-5111, C1D1 values Citation Format: Meena Okera, Brian A. Van Tine, Joleen M. Hubbard, Minal Barve, Erika Hamilton, Monica M. Mita, Frances Valdes-Albini, Daniel Ahn, Admasu Mamuye, Joshua Pelham, Amy Yuet, Diana Yurewicz, Yanning Liu, Andres Machado Sandri, William J. Edenfield, Aki Morikawa, William Gradishar, Rajiv Kumar, Zev A. Wainberg. A phase 1 study of the novel immunotoxin MT-5111 in patients with HER2+ tumors: interim results [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT2-11-01.
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Van Tine, Brian A., Monica Mita, Minal A. Barve, et al. "Abstract P2-13-45: Interim results of a phase 1 study of the novel immunotoxin MT-5111 in patients with HER2+tumors." Cancer Research 82, no. 4_Supplement (2022): P2–13–45—P2–13–45. http://dx.doi.org/10.1158/1538-7445.sabcs21-p2-13-45.
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Abstract Background: Engineered toxin bodies (ETBs) are composed of a de-immunized Shiga-like Toxin A subunit genetically fused to an antibody-like binding domain. ETBs can force receptor internalization, induce potent cell-kill via enzymatic and permanent inactivation of ribosomes, and may not be subject to resistance mechanisms of other therapeutics. MT-5111 is a 55 kD ETB targeting HER2 in solid tumors that binds to an epitope distinct from trastuzumab and pertuzumab, offering potential combination strategies with other HER2-targeting agents. MT-5111 may demonstrate efficacy in patients (pts) resistant to other HER2-targeting agents, as its mechanism of action does not rely on inhibition of kinase signaling or cytoskeletal or DNA damage. Methods: The primary objective is to determine the maximum tolerated dose (MTD) of MT-5111 monotherapy in adult pts with advanced HER2+ solid tumors. Secondary objectives are pharmacokinetics (PK), efficacy, and immunogenicity. Using a modified 3+3 design, the dose-escalation part of the study enrolls pts with HER2+ tumors into 7 cohorts: 0.5, 1, 2, 3, 4.5, 6.75, and 10 µg/kg/dose. Three dose-expansion cohorts will follow for HER2+ breast cancer, gastro-esophageal cancer, and other HER2+ tumors. All pts receive MT-5111 weekly as 30-min IV infusions in each 21-day treatment (tx) cycle (C) until disease progression (PD), unacceptable toxicity, death, or withdrawn consent (NCT04029922). Results: Per data cut in June 2021, 21 pts (mean age 64 years, range 34-78; 38% male) in cohorts 1-6 with breast (n=7), biliary (n=6), gastric (n=3), pancreatic (n=2), lung (n=2), and colon (n=1) cancer were treated (Table 1). Pts had a median of 4 prior lines of systemic therapies (range, 1-8) and 2 prior lines of HER2-targeting treatments (range, 0-6). No Grade (G)4 or 5 tx-emergent (TE) adverse events (AEs) occurred. Tx-related AEs occurred in 11 (52%) pts; the most common was fatigue (n=7, 33%). One pt (4.5 µg/kg) had a possibly (per PI) related G3 serious AE (SAE) of dyspnea and hypoxia, but also lymphangitic carcinomatosis and H. influenzae infection. The other related SAE occurred in a pt (6.75 µg/kg) who had a G1 transient troponin increase without concomitant cardiac symptoms or ECG/ECHO changes. All other related AEs were ≤G2. There were no clinically significant changes in cardiac biomarkers (troponin, ECG, left ventricular ejection fraction) nor were there cases of capillary leak syndrome. Two pts (3 µg/kg and 4.5 µg/kg) had reversible G2 infusion-related reactions. Best response to date has been stable disease. AUClast data matched PK simulations based on non-human primate studies, and Cmax data at 6.75 µg/kg indicated that current in-human exposure was between the IC50 values of high and medium HER2-expressing cell lines (approximately 10-89 ng/mL). Thus, a dose of at least 10 µg/kg may be required to achieve effective exposure. To date, no dose-limiting toxicities have been observed and the 10 µg/kg/dose cohort is now accruing. Conclusions: MT-5111 was well tolerated with no clinically significant immuno- or cardiotoxicity. Dose escalation is ongoing and nearing levels expected to be required for efficacious exposure. Table 1.Metastatic HER2 status and MT-5111 treatment by cohortDose0.5 µg/kg1.0 µg/kg2.0 µg/kg3.0 µg/kg4.5 µg/kg6.75 µg/kgCohort123456Number of patients treated with MT-5111433335Metastatic HER2 2+ by IHC: ALL/BC1/11/11/01/10/04/0Metastatic HER2 3+ by IHC: ALL/BC3/12/02/12/13/01/1BC, breast cancer. Citation Format: Brian A. Van Tine, Monica Mita, Minal A. Barve, Erika P. Hamilton, Andrew J. Brenner, Frances Valdes, Daniel Ahn, Joleen Hubbard, Jason Starr, Angela Georgy, Joshua Pelham, Banmeet S. Anand, Thomas Strack, Andrés Machado Sandri, Zev A. Wainberg. Interim results of a phase 1 study of the novel immunotoxin MT-5111 in patients with HER2+tumors [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-45.
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Kannan, Meganathan, Firdos Ahmad, Birendra K. Yadav, Rajive Kumar, Jawed Fareed, and Renu Saxena. "Glanzmann’s Thrombasthenia Patients with No Mutations in Both the ITGA2B and ITGB3 Genes as Identified by Conformation Sensitive Gel Electrophoresis (CSGE)." Blood 112, no. 11 (2008): 1236. http://dx.doi.org/10.1182/blood.v112.11.1236.1236.
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Abstract Glanzmann’s Thrombasthenia (GT) is an autosomal recessive inherited platelet function disorder that is due to defect platelet aggregation in response to multiple physiologic agonists. The defect may be because of mutations in the genes encoding either ITGA2B or ITGB3 that result in qualitative or quantitative abnormalities of the platelet receptor aIIbb3. A total of 45 unrelated GT patients were analyzed for mutations in all the exons of ITGA2B and ITGB3 genes by a mutation screening technique, Conformation Sensitive Gel Electrophoresis (CSGE). Mutation was identified in 36 out of 45 patients; and in 9 patients no causative gene alterations were identified in both the genes. Only polymorphisms were identified in 5 patients; however, 4 patients did not show any sequence variation in both the genes. Though their mutation status was not identified, their hematological parameters, platelet aggregation, flow cytometry and western blot revealed that they were definite GT (Table 1). Of these 9 patients with no mutations, 5 patients had family history of bleeding; that included death episode, due to prolonged bleeding in all four families and bleeding complication in sibling in one family. The clinical manifestations included epistaxis, Gum bleeding and petechiae in 8 patients and Echymotic spots in 6 patients. The major bleeding complication of gastro- intestinal bleed and eye bleed was seen in 4 patients and 2 patients respectively; one patient had hematuria. Among the 9 patients with no mutation, a blood transfusion was required in 8 patients. Normal values of Prothrombin time, Activated partial thromboplastin time and platelet count in these patients excluded the possibility of other factor deficiencies and thrombocytopenia. Platelet aggregation was absent with all the four agonists in all these patients. Platelet aIIbb3 analysis by flow cytometry classified 5 patients as type I, one as type II and 3 patients as type III GT. Western blot analysis revealed complete absence of both aIIb and b3 in 6 patients. In remaining 3 patients, one had trace amount of aIIb and reduced amount of b3; another had mild amount of b3 with no trace of aIIb, the third patient had abnormal aIIb protein. GT in these 9 patients may be due to defect in a regulatory element affecting the transcription of these two genes or abnormalities in mechanisms that are responsible for post-translational modifications and trafficking of integrin subunits. Moreover, the strategy to detect mutations in these patients was based on CSGE, whose sensitivity is not 100%. Even though the CSGE technique was carefully set up and tested with known mutations, making it unlikely that nucleotide substitutions were missed in these patients, the literature reports that the sensitivity of the CSGE technique is approximately 80%. The above data shows that these patients need to be considered as definite GT and treated based on Clinical, hematological parameters and flow cytometry, even though they did not show mutations. Hematological evaluation protein analysis GT No Age/Sex CG FH Bld Tfn Bleeding Symptoms P/S PC BT (min) CR (%) Platelet aggregation FCM Western blot Polymorphisms ADP ADR AA Collag. Risto. aIIb/b3 aIIb b3 4 15/M N Y Y P, Ep, GB, HU Isolated plt N 8′ 20 Abs Abs Red Abs Red 20.4% AN N IIb c.2188-7C&gt;G IIIa c.1126-30delT IIIa c.1545C&gt;A IIbc.2188-7C&gt;G 5 19/F Y Y Y P, Ep, GB, Mr Isolated plt N &gt;15′ 5 Abs Abs Abs Abs N 0.13% Abs Red IIb c.2187+34_42 del 9bp 6 44/M N Y Y P, ES, Ep, GB, GI Isolated plt N &gt;15′ 0 Abs Abs Abs Abs N 7.72% Red Red Nil 7 12/M N Y Y P, ES, Ep, GB Isolated plt N &gt;15′ 12 Abs Abs Abs Abs N 1.02% Abs Red Nil 8 46/M N Y Y P, ES, Ep, GB, GI Isolated plt N &gt;15′ 0 Abs Abs Abs Abs N 0.27% Abs Abs Nil 13 4/M Y N N P, ES, GB Isolated plt N &gt;15′ 0 Abs Abs Abs Abs N 20% Abs Abs IIbc.2188-7C&gt;G IIbc.2188-47C&gt;T IIb c.1600+100delT IIIa c.2040T&gt;C 21 14/M N N Y ES, Ep, GB, GI, EB Isolated plt N &gt;15′ 0 Abs Abs Abs Abs N 0.2% Abs Abs Nil 22 11m/M Y N Y P, ES, Ep, GI, EB Isolated plt N 9′ 0 Abs Abs Abs Abs N 21.7% Abs Abs IIb c.2188-7C&gt;G IIb c.3063C&gt;T 33 1/F Y N Y P, Ep, GB Isolated N &gt;15′ 0 Abs Abs Abs Abs N 3.5% Abs Abs IIb c.2188-7C&gt;G Table 1: The clinical and hematological parameters along with polymorphisms identified in GT patients with no mutations Note: CG: Consanguinity, Bld Tfn: Blood transfusion, P/S: Peripheral smear, PC: Platelet count, BT: Bleeding time, CR: Clot retraction, FCM: Flow cytometry, ADP: Adenosine 5′-diphosphate, ADR: Adrenaline, AA: Arachidonic acid, Collag: Collagen, Risto: Ristocetin sulphate, P: Purpura, Ep: Epistaxis, GB: Gum bleed, HU: Hematuria, Mr: Menorrhagia, ES: Epistaxis, GI: Gastrointestinal bleed, EB: Eye bleed
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Nakamaki, Tsuyoshi, Kunihiko Fukuchi, Takashi Maeda, et al. "Constitutive Cyclin A1 Expression Impairs Retinoic Acid-Induced Growth Arrest and Differentiation of Myeloid Leukemia Cells." Blood 116, no. 21 (2010): 2894. http://dx.doi.org/10.1182/blood.v116.21.2894.2894.
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Abstract Abstract 2894 Acute promyelocytic leukemia (APL) differentiation syndrome (DS) in all-trans retinoic acid (ATRA) therapy is often associated with increase of leukocyte count (hyperleukocytosis). It suggests deregulated cell proliferation of ATRA-induced APL cells is possibly involved in the development of DS. The molecular mechanism(s) of hyperleukocytosis are unknown. We previously found increased expression of cyclin A1 mRNA were associated with both increase of initial leukocyte count and development of DS in the therapy with ATRA in APL. To clarify role(s) of cyclin A1 in the proliferation and differentiation of myeloid leukemia cells, we generated U937.A1 which is stably transfected with cyclin A1 gene. U937.A1 showed increased expression of cyclin A1 protein compared with U937.C, which carries pCDNA3.1Hisc (2.5 fold, immunoblot). U937.A1 showed increased in vitro colony formation compared with U937.C (1.3 fold). Flow cytometry (FCM) analysis of cells stained with propidium iodide(PI) showed that U937.A1 was characterized with significant decrease in cells in G1 and increase in G2M (P<0.01)(U937.C=G1:70.1±1.1,S:26.5±1.1,G2M:13.8±1.0% and U937.A1=G1:55.5±0.9,S:30.1±2.9,G2M:21.1±0.8%). U937.A1 showed constitutive higher Cdk1 kinase activities compared with U937.C more than 100 times, as measured by ELISA which quantitate enzyme activities by detecting phospholyrated synthetic peptide using monoclonal antibody 4A4. It suggests that inappropriate activation of cyclin A1/Cdk1 complex is associated with altered cell cycle progression of U937.A1 cells. Immunoblot analysis showed that U937.A1 over-expressed checkpoint kinase 1(Chk1) protein compared with U937.C.(1.4 fold)suggesting deregulated G2M checkpoint in U937.A1. Anti-apoptotic protein, bcl2, was increased in U937.A1 cells(1.5 fold). Incubation with 1μ M ATRA for 4 days significantly inhibited cell growth in U937.C (U937.C=44.9±1% and U937.A1=70.8±3%, p<0.01, cell No. with ATRA/without ATRA). ATRA-induced growth inhibition in U937.C was accompanied with significant increase of cells in G1 (U937.C=88.9±0.9% and U937.A1=68.2±0.8%) and decreased in the G2M(U937.C=4.9±0.5% and U937.A1=19.7±1.2%). Expression of both cyclin A2 and cyclin B proteins were markedly down-regulated at days 3 and 4 in U937.C in the culture (U937.C=69.1±1.3%(cyclin A2) and 52.1±2.1%(cyclin B), U937.A1=90.2±1.7%(cyclin A2)and 94.5±2.4%(cyclin B),day4, ATRA/without ATRA) Cdk1 protein expression was also significantly down-regulated in U937.C(U937.C=53.5±2.5% and U937.A1=115±5%, p<0.01, ATRA/without ATRA). In both of U937.C and U937.A1 cells, Cdk1 kinase activities were significantly suppressed by 4 days' culture with 1μ M ATRA,however, the activities in U937.A1 still remained 10 times higher than those in U937.C (U937.C=0.06±0.01/0.25±0.02 and U937.A1=0.19±0.01/1.04±0.04,day4, O.D., ATRA/without ATRA). Incubated with ATRA, expression of Chk1 protein in U937.C cells time-dependently decreased (U937.C=44.3±0.1% and U937.A1=86.1±0.1%, p<0.01, day4. with ATRA/without ATRA) Checkpoint kinase activities, as measured by ELISA using anti-phospho-Cdc25C serine 216 specific antibody, was significantly suppressed by ATRA in U937.C (U937.C=1.25±0.02/2.69±0.09 and U937.A1=3.42±0.06/2.79±0.22,day4, O.D., ATRA/without ATRA). Incubation with Go6976 at 10 μ M, a specific chk1 inhibitor, produced significant cell growth inhibition in U937.A1 in combination with ATRA. The growth inhibition by ATRA plus Go6976 was accompanied with accumulation of cells in G1(79.2±0.9%) and decrease of cells in G2M (7.7%±0.3%)compared with those incubated with ATRA alone, suggesting deregulated chk1 activities are involved in impaired ATRA-induced growth inhibition in U937.A1 cells. ATRA-induced down-regulation of expression of bcl2 protein was significant in U937.C (U937.C=41.8±0.1% and U937.A1=77.8±0.2%, day4, with ATRA/without ATRA). Decreased bcl2 protein was inversely co-related with up-regulation of CD11b induced by ATRA (U937.C=4.3±0.2% and U937.A1=3.9±0.1%, FCM, day4). The present results showed that forced expression of cyclin A1 resulted in impaired ATRA-induced growth suppression and differentiation of U937 cells. It was related with inappropriately activated Chk1-Cdk1 pathway. Deregulated G2M cell cycle checkpoint(S)is a candidate for the therapeutic target in ATRA-induced hyperleukocytois in APL. Disclosures: No relevant conflicts of interest to declare.
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Cui, Bing, Liguang Chen, Laura Z. Rassenti, et al. "High-Level Expression of ROR1 Associates with Early Disease Progression in Patients with Chronic Lymphocytic Leukemia." Blood 126, no. 23 (2015): 1713. http://dx.doi.org/10.1182/blood.v126.23.1713.1713.
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Abstract ROR1 is a type-1 tyrosine kinase-like orphan-receptor that ordinarily is expressed during embryogenesis, but that also is found on leukemia cells of patients (pts) with chronic lymphocytic leukemia (CLL). In prior studies we found ROR1 served as a receptor for Wnt5a, which could promote survival/growth of CLL cells. We found Wnt5a in the plasma is significantly higher in pts with CLL (3.2±1.6 ng/ml (mean ±S.D.), N = 36) than in healthy adults (0.14±0.16 ng/ml, N=14, p<0.01) and also may be elaborated by accessory cells in the CLL microenvironment. On the other hand, reducing expression of ROR1 on CLL cells via siRNA adversely affected leukemia-cell survival. We hypothesized that expression of ROR1 in CLL could play a role in the pathogenesis and/or progression of this disease. Early studies failed to define differences in the expression level of ROR1 on CLL cells of different pts in a small cohort. However, upon further analyses, we observed heterogeneity in the expression levels of ROR1 among samples of different pts in larger cohort. We investigated whether the level of ROR1 expression was associated with disease progression in a cohort of 1,568 CLL pts followed by CLL Research Corium (CRC). CLL samples were uniformly examined for the expression of ROR1 using the same preparation of an Alexa-647-conjugated anti-ROR1 mAb (4A5) and a standardized procedure by the CRC Tissue Core. We used 10 CLL cases, and tested ROR1 expression at 3 different time-points for each case. We found ROR1 expression was stable over time in all CLL cases. Then, we randomly segregated pt samples into either of two cohorts, one a training set of 797, and the other a validation set of 773 cases. For the 797 cases in the training dataset, we used the profile-likelihood method in a Cox regression model of treatment-free survival (TFS) from diagnosis to determine the optimal threshold level of ROR1 expressionthat could segregate pts into 2 subgroups. In the training cohort, 294 pts were stratified into a "ROR1-Low" subgroup and 503 pts were assigned to a "ROR1-High" subgroup, defining a threshold absolute mean fluorescence intensity of CLL cells stained for ROR1 of 29. The subgroup of pts in the ROR1- High subgroup had a median TFS of 6.0 years, whereas those pts in the ROR1-Low subgroup had a median TFS of 11.0 years. We applied this threshold to segregate the 773 samples of the validation cohort into ROR1-High and ROR1-Low subgroups. We found that high-level expression of ROR1retained its strong predictive adverse effect on TFS in this validation set. The subgroup of pts in the ROR1- High subgroup had a median TFS of 5.7 years, whereas those pts in the ROR1-Low subgroup had a median TFS of 11.8 years. The immunoglobulin heavy-chain variable region (IGHV) mutation status was known for 593 of the 797 samples in the training set (74%) and 586 of the 773 samples in the validation set (76%). A significantly lower percentage of the ROR1-Low cases (34%, or 69 of 203) used unmutated IGHV than did the ROR1-High cases (54%, or 209 of 390) (p<0.001). Nonetheless, using a multivariate Cox regression model with either a non-delayed entry or a delayed entry, we observed that high-level expression of ROR1 had predictive value for short TFS (non-delayed entry HR 1.4, p<0.05; delayed entry HR 1.9, p<0.001) even when the IGHV mutation status was taken into consideration (non-delayed entry HR 2.8, p<0.001; delayed entry HR 2.7, p<0.001). Taken together, this study demonstrates that there is heterogeneity in the expression of ROR1 on CLL B cells. Furthermore, we find that a high level expression of ROR1 is associated with early disease progression in pts with CLL. Table 1. Number of cases with high versus low ROR1 and mutated versus unmutated IGHV in the training and validation cohort Training Dataset Validation Dataset ROR1 MFI distribution (Median) 0~475 (35.5) 0~424(34.6) ROR1Low with Mutated IGHV 139 132 ROR1Low with Unmutated IGHV 64 84 ROR1High with Mutated IGHV 181 156 ROR1High with Unmutated IGHV 209 214 Total With Known IGHV Mutation Status 593 586 Disclosures Keating: Celgene Corp.: Consultancy; Glaxo-Smith-Kline Inc.: Other: Advisory board. Kay:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Tolero Pharma: Research Funding; Genentech: Research Funding; Hospira: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding. Byrd:Acerta Pharma BV: Research Funding. Gribben:Celgene: Consultancy, Honoraria; Janssen: Honoraria; Roche/Genentech: Honoraria; Pharmacyclics: Honoraria; Gilead: Honoraria. Kipps:Pharmacyclics Abbvie Celgene Genentech Astra Zeneca Gilead Sciences: Other: Advisor.
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Chaturvedi, Shalini, Anke Weispfenning, David Bauer, et al. "Abstract CT082: Next-generation sequencing (NGS) and cytokine assessment from a phase III study of copanlisib in combination with rituximab in patients with indolent non-Hodgkin lymphoma (iNHL) - associations with survival endpoints." Cancer Research 83, no. 8_Supplement (2023): CT082. http://dx.doi.org/10.1158/1538-7445.am2023-ct082.
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Abstract Introduction: The PI3K inhibitor copanlisib (C) plus the anti-CD20 antibody rituximab (R) was superior to R plus placebo (P) in patients (pts) with relapsed iNHL (Matasar et al. Lancet Oncol 2021). We previously reported that C+R improved median progression-free survival (PFS) in the iNHL, follicular lymphoma (FL), and non-FL groups, with a statistically significant improvement in the PTEN-positive population (Shalini et al., AACR 2022). Here we conducted NGS analysis from pts treated with either C+R or P+R for possible impact on PFS outcomes. We also determined if there was an association between plasma cytokine levels at baseline with survival. Methods: Adult pts with relapsed B-cell iNHL were randomly assigned 2:1 to either C+R or P+R; standard dosing on a 28-day cycle applied. Either fresh or archival tumor tissues were collected for central pathology review and biomarker analysis. NGS was conducted using the TSO500 platform for 476 genes. Analyses included EZH2 and BCL2 mutation status in FL subset, and other genes for FL subsets and overall iNHL, as prognostic and predictive markers. The effect of mutations on PFS and overall survival (OS) outcomes was assessed. Cytokines from baseline plasma were assessed using the multiplex MSD platform (71-plex). Association between cytokine levels and PFS and OS were explored. Results: Baseline tumor samples were evaluable by NGS from 200 pts; 127 C+R and 73 P+R. Variant alleles with frequencies &gt;10% were analyzed. One quarter (25.7%) of FL pts were determined to have EZH2 mutations. C+R treatment significantly improved PFS for FL pts with in mutant EZH2 (p=0.0066) compared to P+R. FL pts with mutant BCL2 had a statistically improved PFS compared to WT BCL2 when receiving C+R treatment. Plasma samples were available for 71-plex assessment in 373 pts; 246 C+R and 127 P+R. Pts were assigned as high or low baseline cytokine groups based on the median values. For majority of cytokines, the low cytokine group correlated with better PFS for all pts, FL and Other iNHL pts in the C+R arm. For OS, there were too few events at the time of primary data cutoff to make any associations. However, with longer follow up (2 years from primary completion) there was a significant improvement in OS in iNHL pts with low IL2 values at baseline receiving C+R compared to pts receiving P+R; HR 0.495 (95%CI: 0.270,0.908), p=0.0230. This association was only seen for pts with low (or undetected) levels of IL2 at baseline. Conclusions: Mutation status was associated with improvements in PFS in FL pts with mutant BCL2 or mutant EZH2 treated with C+R. iNHL pts with low plasma levels of IL2 levels treated with C+R showed a significant improvement in OS. Citation Format: Shalini Chaturvedi, Anke Weispfenning, David Bauer, Rong Du, Tine Descamps, Sara Bellinvia, Teresa Lunt, Lidia Mongay Soler, Barrett H. Childs, Pier Luigi Zinzani. Next-generation sequencing (NGS) and cytokine assessment from a phase III study of copanlisib in combination with rituximab in patients with indolent non-Hodgkin lymphoma (iNHL) - associations with survival endpoints [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT082.
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Yao, Baojin, Chaonan Wang, Zhenwei Zhou, et al. "Comparative transcriptome analysis of the main beam and brow tine of sika deer antler provides insights into the molecular control of rapid antler growth." Cellular & Molecular Biology Letters 25, no. 1 (2020). http://dx.doi.org/10.1186/s11658-020-00234-9.
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Abstract Background Deer antlers have become a valuable model for biomedical research due to the capacities of regeneration and rapid growth. However, the molecular mechanism of rapid antler growth remains to be elucidated. The aim of the present study was to compare and explore the molecular control exerted by the main beam and brow tine during rapid antler growth. Methods The main beams and brow tines of sika deer antlers were collected from Chinese sika deer (Cervus nippon) at the rapid growth stage. Comparative transcriptome analysis was conducted using RNA-Seq technology. Differential expression was assessed using the DEGseq package. Functional Gene Ontology (GO) enrichment analysis was accomplished using a rigorous algorithm according to the GO Term Finder tool, and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis was accomplished with the R function phyper, followed by the hypergeometric test and Bonferroni correction. Quantitative real-time polymerase chain reaction (qRT-PCR) was carried out to verify the RNA levels for differentially expressed mRNAs. Results The expression levels of 16 differentially expressed genes (DEGs) involved in chondrogenesis and cartilage development were identified as significantly upregulated in the main beams, including transcription factor SOX-9 (Sox9), collagen alpha-1(II) chain (Col2a1), aggrecan core protein (Acan), etc. However, the expression levels of 17 DEGs involved in endochondral ossification and bone formation were identified as significantly upregulated in the brow tines, including collagen alpha-1(X) chain (Col10a1), osteopontin (Spp1) and bone sialoprotein 2 (Ibsp), etc. Conclusion These results suggest that the antler main beam has stronger growth capacity involved in chondrogenesis and cartilage development compared to the brow tine during rapid antler growth, which is mainly achieved through regulation of Sox9 and its target genes, whereas the antler brow tine has stronger capacities of endochondral bone formation and resorption compared to the main beam during rapid antler growth, which is mainly achieved through the genes involved in regulating osteoblast and osteoclast activities. Thus, the current research has deeply expanded our understanding of the intrinsic molecular regulation displayed by the main beam and brow tine during rapid antler growth.
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Westbrook, Lindsey, Kevin R. Regner, Ashley C. Johnson, et al. "Abstract 1: Loss of Nuclear Receptor 4a1 on a Hypertensive Genetic Background Promotes Immune-Mediated Renal Injury." Hypertension 60, suppl_1 (2012). http://dx.doi.org/10.1161/hyp.60.suppl_1.a1.
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Nuclear hormone receptors of the NR4A subgroup have been implicated in cancer, atherosclerosis, and most prominently in metabolic disease. However, little is known about the role of NR4A1 in kidney health or disease. Fawn-hooded hypertensive (FHH) Nr4a1 -/- deficient animals were evaluated for blood pressure, proteinuria, renal function and metabolic parameters from 4-24 weeks of age. At week 24, Nr4a1 -/- rats exhibited 4-fold higher proteinuria (81±11.5 mg/24hrs/100gBW) compared to FHH (21±1.0). No difference in telemetry measured blood pressure was observed at any time point, but glomerular filtration rate was significantly decreased in the Nr4a1 -/- (542±78.9 ul/min/gKW) vs FHH (937±75.2). Moderate increases in glomerulosclerosis were seen, but were not different between strains. The severity of tubular atrophy, casts, and fibrosis was significantly increased in Nr4a1 -/- compared to FHH (19±1.86% vs 12±0.8%, respectively). Tubulointerstitial macrophage infiltration was also significantly increased in kidneys from Nr4a1 -/- (245±62 foci per field) compared to FHH (50±13.4) at week 24. Microarray analysis of kidney from week 8-24 found significant up-regulation of important immune mediators, including TGF-β1-2, CCL2 (MCP-1), CCL9 (MIP-1γ), and other chemokines and interleukins. The specific injury (tubulointerstitial), immune cell infiltration, and immune gene expression profile led to the hypothesis that loss of Nr4a1 contributes to the observed injury by an immune-mediated mechanism. To test this hypothesis, bone marrow (bm) cross transplantation studies were performed in 3 groups of irradiated (irr) animals: (1) bmFHH into irrFHH (FHH control); (2) bmFHH into irrNr4a1 -/- ; and (3) bmNr4a1 -/- into irrNr4a1 -/- (Nr4a1 control). The bmFHH into irrNr4a1 -/- animals showed a significant attenuation of proteinuria starting as early as week 12 and continuing through week 24 (35±4.6 mg/24hrs/100gBW) compared to the Nr4a1 -/- control (60±7.5). These data strongly suggest that the mechanism of Nr4a1 -/- renal injury is mediated via an immune mechanism, likely due to the predisposition of the FHH to develop renal injury. Further studies are now underway to better understand the precise immune mechanism that promotes injury in this model.
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Cooper, Elizabeth, Zoe Woolf, Molly E. V. Swanson, et al. "Single-cell image analysis reveals overexpression of organic-anion transporting polypeptides (OATPs) in human glioblastoma tissue." Neuro-Oncology Advances, October 14, 2022. http://dx.doi.org/10.1093/noajnl/vdac166.
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Abstract Background Glioblastoma (GBM) is the most common and aggressive primary brain tumour in adults. Whilst the role of the efflux transporters are well established in GBM, the expression and function of uptake transporters, such as the organic anion-transporting polypeptide (OATP) family, are not well understood. OATPs possess broad substrate specificity that includes anti-cancer agents; therefore, we sought to investigate the expression of four OATP isoforms in human GBM cell types using patient tumour tissue. Methods We used fluorescent immunohistochemical labelling of paraffin-embedded surgically resected tissues and single-cell image analysis methods to explore the expression of the OATP isoforms in different tumour cell types through co-labelling with cell-type-specific markers, such as IBA1 (pan-myeloid), GFAP (tumour cell), PDGFRβ (stromal cell), and UEA-1-lectin (endothelial). Results We found significant over-expression of all the OATP isoforms (OATP1A2, 2B1, 1C1 and 4A1) in GBM tumour sections when compared to non-neoplastic brain. A single-cell image analysis revealed that OATPs were significantly upregulated throughout the tumour parenchyma, with significantly higher expression found on lectin-positive blood vessels and IBA1-positive myeloid cells in GBM compared to non-tumour brain tissue. Qualitative analysis of the four OATP isoforms demonstrated greater expression of OATP4A1 in peri-necrotic regions of GBM tissue, which correlated with hypoxia-related markers within the Ivy GAP RNAseq dataset. Conclusion Here, we demonstrate, for the first time, the protein expression of four OATPs in human GBM tissue, including upregulation within the tumour microenvironment by myeloid cells and tumour vasculature, and isoform-specific upregulation within hypoxic niches.
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Jahan, M. N., M. R. Munir, M. Sohag, M. M. Alam, and M. R. Alam. "Changes of caprine (Capra hircus) blood during prolong storage for transfusion." Bangladesh Journal of Veterinary Medicine 17, no. 2 (2020). http://dx.doi.org/10.33109/bjvmjd19rm4.
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Background: This experiment was performed to investigate the effects of acid citrate dextrose (ACD) and citrate phosphate dextrose adenine-1 (CPDA-1) on the keeping qualities of various haematological and biochemical parameters of caprine blood during long time preservation and storage for transfusion. Methods: Sixteen healthy goats were selected and divided into 2 equal groups (A, n=8 and B, n=8). Fifty ml of blood was collected from each goat and preserved with ACD for group A (n=8) and CPDA-1 for group B (n=8). All the samples were stored at 40C in refrigerator for 28 days. The recorded blood parameters include total erythrocyte count (TEC), total leucocyte count (TLC), haemoglobin (Hb), packed cell volume (PCV), total protein (TP) and pH. The blood parameters were analyzed immediately after collection and thereafter on day-1, day-3, day-7, day-14, day-21 and day-28 for both the groups. Results: In both groups, the TEC, TLC, Hb and PCV values were decreased gradually from day-1 onward. In ACD preserved blood, the control values of TEC (11.27±0.26 million/cumm), TLC (8.85±0.22 thousand/cumm), Hb (8.61±0.13 g/dl) and PCV (30.75±0.59%) were decreased to TEC (9.21±0.38 million/cumm), TLC (7.58±0.10 thousand/cumm), Hb (7.03±0.06 g/dl) and PCV (22.25±0.53%) respectively on day-7 which was statistically significant (p‹0.05). However, the gradual decrease in the parameters was also noticed from day-7 onward. On the other hand, in case of CPDA-1 preserved blood, the control values of TEC (11.88±0.28 million/cumm), TLC (8.91±0.26 thousand/cumm), Hb (8.91±0.42 g/dl) and PCV (32.13±0.79%) were found decreasing slightly with the progression of the preservation period, but the changes were statistically significant (p‹0.05) on day-21 [TEC (8.06±0.22 million/cumm), TLC (6.28±0.34 thousand/cumm), Hb (6.28±0.16 g/dl) and PCV (25.02±0.46%) respectively] and onward. Changes in the TP and pH values were also noticed in both the groups during the experiment but CPDA-1 group showed less alteration than ACD group as compared to the control values. Conclusion: The results of this study revealed that CPDA-1 can be used for storing caprine blood longer period for transfusion in comparison to ACD with greater RBC viability.