Despite increasing recognition of the importance of immune checkpoint inhibitor-associated AKI, data on this complication of immunotherapy are sparse.
We conducted a multicenter study of 138 patients ...with immune checkpoint inhibitor-associated AKI, defined as a ≥2-fold increase in serum creatinine or new dialysis requirement directly attributed to an immune checkpoint inhibitor. We also collected data on 276 control patients who received these drugs but did not develop AKI.
Lower baseline eGFR, proton pump inhibitor use, and combination immune checkpoint inhibitor therapy were each independently associated with an increased risk of immune checkpoint inhibitor-associated AKI. Median (interquartile range) time from immune checkpoint inhibitor initiation to AKI was 14 (6-37) weeks. Most patients had subnephrotic proteinuria, and approximately half had pyuria. Extrarenal immune-related adverse events occurred in 43% of patients; 69% were concurrently receiving a potential tubulointerstitial nephritis-causing medication. Tubulointerstitial nephritis was the dominant lesion in 93% of the 60 patients biopsied. Most patients (86%) were treated with steroids. Complete, partial, or no kidney recovery occurred in 40%, 45%, and 15% of patients, respectively. Concomitant extrarenal immune-related adverse events were associated with worse renal prognosis, whereas concomitant tubulointerstitial nephritis-causing medications and treatment with steroids were each associated with improved renal prognosis. Failure to achieve kidney recovery after immune checkpoint inhibitor-associated AKI was independently associated with higher mortality. Immune checkpoint inhibitor rechallenge occurred in 22% of patients, of whom 23% developed recurrent associated AKI.
This multicenter study identifies insights into the risk factors, clinical features, histopathologic findings, and renal and overall outcomes in patients with immune checkpoint inhibitor-associated AKI.
Tumor responses to programmed cell death protein 1 (PD-1) blockade therapy are mediated by T cells, which we characterized in 102 tumor biopsies obtained from 53 patients treated with pembrolizumab, ...an antibody to PD-1. Biopsies were dissociated, and single-cell infiltrates were analyzed by multicolor flow cytometry using two computational approaches to resolve the leukocyte phenotypes at the single-cell level. There was a statistically significant increase in the frequency of T cells in patients who responded to therapy. The frequency of intratumoral B cells and monocytic myeloid-derived suppressor cells significantly increased in patients' biopsies taken on treatment. The percentage of cells with a regulatory T-cell phenotype, monocytes, and natural killer cells did not change while on PD-1 blockade therapy. CD8(+) memory T cells were the most prominent phenotype that expanded intratumorally on therapy. However, the frequency of CD4(+) effector memory T cells significantly decreased on treatment, whereas CD4(+) effector T cells significantly increased in nonresponding tumors on therapy. In peripheral blood, an unusual population of blood cells expressing CD56 was detected in two patients with regressing melanoma. In conclusion, PD-1 blockade increases the frequency of T cells, B cells, and myeloid-derived suppressor cells in tumors, with the CD8(+) effector memory T-cell subset being the major T-cell phenotype expanded in patients with a response to therapy.
Receptor tyrosine kinases (RTKs) are cell surface receptors that bind growth factor ligands and initiate cellular signaling. Of the 20 classes of RTKs, 7 classes, I-V, VIII, and X, are linked to head ...and neck cancers (HNCs). We focus on the first class of RTK, epidermal growth factor receptor (EGFR), as it is the most thoroughly studied class. EGFR overexpression is observed in 20% of tumors, and expression of EGFR variant III is seen in 15% of aggressive chemoradiotherapy resistant HNCs. Currently, the EGFR monoclonal antibody (mAb) cetuximab is the only FDA approved RTK-targeting drug for the treatment of HNCs. Clinical trials have also included EGFR mAbs, with tyrosine kinase inhibitors, and small molecule inhibitors targeting the EGFR, MAPK, and mTOR pathways. Additionally, Immunotherapy has been found to be effective in 15 to 20% of patients with recurrent or metastatic HNC as a monotherapy. Thus, attempts are underway for the combinatorial treatment of immunotherapy and EGFR mAbs to determine if the recruitment of immune cells in the tumor microenvironment can overcome EGFR resistance.
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Background: Natural botanical drugs containing flavonoids are under clinical investigation for the treatment of malignancies. A phase I clinical study for patients with locally advanced head and ...neck cancer with a new class of botanical drug APG-157 showed excellent tolerability, systemic absorption via sublingual administration, and enhanced immune cell infiltration into the tumor microenvironment and suppression of T cell inhibitory inflammatory cytokine production. We sought to evaluate the efficacy of tumor control for the combination of APG-157 and checkpoint blockade immunotherapy using a head and neck cancer mouse model. Methods: SCCVII (HPV-) and MEER (HPV+) head and neck cancer murine cell line models were utilized in in vivo tumor growth study. After optimization for route and cell number, we measured tumor growth after treating mice with following groups: i) control, ii) APG-157, iii) anti-PD-1, iv) anti-CTLA-4, v) APG-157 + anti-PD-1, vi) APG-157 + anti-CTLA-4, vii) anti-PD-1 and anti-CTLA-4, viii) APG-157 + anti-PD-1 + anti-CTLA-4. Tumors were collected after 2 injections of immune checkpoint inhibitors and characterized for immunophenotype by multi-color flow cytometry, immunohistochemistry, and RNA-sequence analysis. Fecal microbiome was analyzed by 16S rRNA sequence for SCCVII model to assess the impact of APG-157 on the microbiome as we observed the shift of oral microbiome species in the human trial. Results: We confirmed by serum concentration of APG-157 metabolites that the best route of administration is via oral route by mixing with APG-157 with mouse chow. Tumor growth optimization experiments determined that 100,000 cells of SCCVII is the ideal number of cells to inject and day 5-6 is the time to initiate treatment. Among 8 treatment groups, APG-157 + anti-CTLA-4 demonstrated best tumor control (p = 0.0065 compared to control), followed by anti-PD-1 + anti-CTLA-4 treatment group (p = 0.48 compared to control). The immunophenotype assessment by flow cytometry showed over 30% of CD8+ T cell in APG-157 + anti-CTLA-4 group compared to 4-5% of CD8+ T cell for the control group. GSEA analysis from the SCCVII tumors revealed that APG-157 + anti-CTLA-4 group showed an enriched set of genes for inflammatory response, apoptotic signaling pathways. The fecal microbiome analysis showed a substantial difference of lactobacillus genus among groups, highest in the group of APG-157 + anti-CTLA-4 treatment group. The MEER model (HPV positive) tested with same immunotherapy as single or in combination with APG-157 did not grow the tumor, thus we were unable to collect the tumor for immunophenotyping and other studies. Conclusions: These study results indicate that APG-157 is able to modulate the immune system and fecal microbial species that could potentially lead to improved anti-tumor immunity and warrants further studies to define mechanism and the potential for human clinical trial.
Abstract only
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Background: Natural botanical drugs, such as curcumin, resveratrol and related flavonoids, are under clinical studies. Previous pilot study of curcumin, a polyphenol, for normal ...and patients with oral squamous cell carcinoma (OSCC) showed significant inhibition of inflammatory cytokines in saliva. Phase I investigation was performed on APG-157 to evaluate the potential utility an as oral drug for the treatment of OSCC. Methods: A double-blind, randomized, placebo-controlled phase I clinical trial was conducted with a botanical preparation containing a combination of curcumin related polyphenol molecules (pharmaceutical name APG-157). 12 Subjects with oral cancer and 13 normal control subjects were recruited. Two doses of the drug, 3x100 mg and 3x200 mg, were tested. The drug was administered orally each hour for 3 consecutive hours. Blood and saliva were collected pre-treatment and 1, 2, 3, and 24 hours post-treatment. Salivary cells and supernatants were analyzed for the expression of cytokines by multiplex ELISA and microbial content by 16S RNA sequence. Pre- and post-treatment tumor biopsies of one subject were studied for expression using the RNA seq and immunofluorescence (IF). Results: This study did not reveal any toxicity and there was a dose dependent inhibition of inflammatory cytokines, IL-1β, TNF-alpha and IL-8 in the salivary supernatant of cancer subjects treated with the drug. Tumor RNA-seq revealed down regulation of gene ontologies of cell adhesion, cell cycle and cell division and up regulation of generation of precursor metabolite/energy in the post-treatment tumor sample. Microbiome study showed significant decrease in Bacterioides after 24 hours of treatment. There was also a trend of decreasing Bacteroides among other cancer subjects treated with APG-157. IF showed a marked increase in the number of CD4, CD8 T cells in post-treatment tumor. PD-L1 expression was up-regulated in the post-treatment tumor sample. Conclusions: APG-157 is found to be safe and toxicity was not observed. The drug has shown a decrease in inflammatory cytokines. Moreover, there was a markedly increased CD4, CD8 T cells infiltration on a subject and decreased Bacteriodes microbial population after APG-157 treatment suggesting that it might have potential synergistic effect with immune checkpoint blockade immunotherapy. Decreased expression of cell growth related genes and increased expression of growth inhibitory genes pointed to a potential anti-tumor activity of APG-157.
Background
Although curcumin's effect on head and neck cancer has been studied in vitro and in vivo, to the authors' knowledge its efficacy is limited by poor systemic absorption from oral ...administration. APG‐157 is a botanical drug containing multiple polyphenols, including curcumin, developed under the US Food and Drug Administration's Botanical Drug Development, that delivers the active components to oromucosal tissues near the tumor target.
Methods
A double‐blind, randomized, placebo‐controlled, phase 1 clinical trial was conducted with APG‐157 in 13 normal subjects and 12 patients with oral cancer. Two doses, 100 mg or 200 mg, were delivered transorally every hour for 3 hours. Blood and saliva were collected before and 1 hour, 2 hours, 3 hours, and 24 hours after treatment. Electrocardiograms and blood tests did not demonstrate any toxicity.
Results
Treatment with APG‐157 resulted in circulating concentrations of curcumin and analogs peaking at 3 hours with reduced IL‐1β, IL‐6, and IL‐8 concentrations in the salivary supernatant fluid of patients with cancer. Salivary microbial flora analysis showed a reduction in Bacteroidetes species in cancer subjects. RNA and immunofluorescence analyses of tumor tissues of a subject demonstrated increased expression of genes associated with differentiation and T‐cell recruitment to the tumor microenvironment.
Conclusions
The results of the current study suggested that APG‐157 could serve as a therapeutic drug in combination with immunotherapy.
Lay Summary
Curcumin has been shown to suppress tumor cells because of its antioxidant and anti‐inflammatory properties. However, its effectiveness has been limited by poor absorption when delivered orally.
Subjects with oral cancer were given oral APG‐157, a botanical drug containing multiple polyphenols, including curcumin. Curcumin was found in the blood and in tumor tissues. Inflammatory markers and Bacteroides species were found to be decreased in the saliva, and immune T cells were increased in the tumor tissue.
APG‐157 is absorbed well, reduces inflammation, and attracts T cells to the tumor, suggesting its potential use in combination with immunotherapy drugs.
When delivered transorally, curcumin and its analogues in APG‐157 are absorbed into the blood and tumor tissue, with an inhibitory effect noted on salivary Bacteroides species, possibly through decreases in NF‐κB–driven inflammatory cytokines. Immune cell recruitment to the tumor cell microenvironment indicates that APG‐157 could serve as a neoadjuvant therapeutic drug.
We have observed overexpression of PACS-1, a cytosolic sorting protein in primary cervical tumors. Absence of exonic mutations and overexpression at the RNA level suggested a transcriptional and/or ...posttranscriptional regulation. University of California Santa Cruz genome browser analysis of PACS-1 micro RNAs (miR), revealed two 8-base target sequences at the 3′ terminus for hsa-miR-34a and hsa-miR-449a. Quantitative RT-PCR and Northern blotting studies showed reduced or loss of expression of the two microRNAs in cervical cancer cell lines and primary tumors, indicating dysregulation of these two microRNAs in cervical cancer. Loss of PACS-1 with siRNA or exogenous expression of hsa-miR-34a or hsa-miR-449a in HeLa and SiHa cervical cancer cell lines resulted in DNA damage response, S-phase cell cycle arrest, and reduction in cell growth. Furthermore, the siRNA studies showed that loss of PACS-1 expression was accompanied by increased nuclear γH2AX expression, Lys382-p53 acetylation, and genomic instability. PACS-1 re-expression through LNA-hsa-anti-miR-34a or -449a or through PACS-1 cDNA transfection led to the reversal of DNA damage response and restoration of cell growth. Release of cells post 24-h serum starvation showed PACS-1 nuclear localization at G1-S phase of the cell cycle. Our results therefore indicate that the loss of hsa-miR-34a and hsa-miR-449a expression in cervical cancer leads to overexpression of PACS-1 and suppression of DNA damage response, resulting in the development of chemo-resistant tumors.
Abstract
Introduction: A small subset of T cells from patients treated with MART-1 engineered adoptive T cell transfer (ACT) were found to co-express Yamanaka transcription factors SOX2, OCT3/4, and ...NANOG (TSON). Identifying these T cells may have potential use in future T cell based immunotherapy.
Methods: Flow (FC) and mass cytometry (MC) were used to evaluate T cells with induced pluripotent stem cell (iPS)-like markers. Peripheral mononuclear cells (PBMC) were collected from 3 melanoma patients (MP) treated with ACT and 1 healthy donor (HD0). Bone marrow (BM) samples were collected from patients with Non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM). T cell populations were then screened for stem cell markers: SOX2 OCT3/4 NANOG TRA-1-81 SSEA4 TRA-1-60 CD3+ CD34-. Naïve T cells (CD45RO- CD62L+) from HD0 were exposed to IL-7/IL-15/IL-21 with or without pan-AKT inhibitor (AKTi). These cells were analyzed after 7 days for TSON expression and the following surface markers: CD27 CD28 CD45RO CD45RA CD57 CD62L CD95 CD122 CD127 CCR4 CCR6 CCR7. PBMC collections from 5 healthy donors (HD) were expanded with or without IL-7/IL-15/IL-21 exposure in G-Rex bioreactors for 7 days. TSON expression was analyzed using MC and characterized for the already mentioned markers as well as CXCR3 and Ki67. Lastly, expanded CD95+ cells were single-cell sorted for TSON and their RNA was extracted using the FRISCR method for single-cell RNA sequencing (scRNA-seq). The iPS cell line H1 was included as a positive control.
Results: T cells across all 3 MP had a small percentage of TSON expression (0.0002%, 0.0001%, 0.0001%, respectively). This small cohort of T cells co-expressed TRA-1-81, SSEA4, and TRA-1-60 (0.05%, 0.05%, 0.8%, respectively). TSON expression for HD0 was 0.0000013%, while co-expression of TRA-1-81, SSEA4, and TRA-1-60 was undetectable. BM samples NHL and MM also showed TSON expression (0.18% vs 0.06%). Naïve T cells treated with IL-7/IL-15/IL-21 had a significantly higher percentage of TSON after 7 days of exposure compared to T cells treated with IL-7/IL-15/IL-21/AKTi (0.008% vs 0.001%, respectively). Furthermore TSON cell subsets in both treatment populations displayed a phenotype similar to T memory stem cells (TMSC) and central memory T cells (TCM) (CD45RA+ CD27+ CD28+ CD62L+ CCR7+ CD95+ CD57- CD45RO+). Concerning PBMCs from the 5 HD, the proliferation marker Ki67 was displayed on 3 non-stimulated HD and all stimulated HD with a range of median intensities 10, 67. The presence of CD62L CD27 CD95 CCR7 CD28 CD45RO in both non-stimulated and stimulated cell populations was similar to TCM phenotype. Validation of scRNA-seq is still in process. Early results show that OCT3, NANOG, and cMYC are detected in both TSON and H1 while SOX2 could only be detected in H1.
Conclusion: Our data show evidence of T cells with natural TSON expression. However, additional studies are needed to further understand the role of TSON in immunotherapy.
Citation Format: Michael Cerniglia, Helena Escuin-Ordinas, Caroline Porter, Maria Alexandrovna Aleshin, Gardenia Cheung-Lau, Zoran Galic, Inbal Abraham-Davidi, Daniel Sanghoon Shin, Orit Rosenblatt-Rosen, Antoni Ribas, Begoña Comin-Anduix. Phenotypic characteristics of T cells co-expressing SOX2, OCT3/4, and NANOG abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1551.
Abstract
Gigaxonin, the protein product of the Giant axonal neuropathy (GAN) gene, is a E3 ubiquitin ligase involved in neural cell and fibroblast intermediate filament processing. We have previously ...shown that Gigaxonin interacts with p16 in cisplatin-mediated ubiquitination of NF-κB. We analyzed cervical and head and neck cancer cell lines and primary tumors and have found exon 8 c.1293C>T single nucleotide polymorphism (SNP) to occur in both HPV positive and negative tumor cells lines and primary tumors. The prevalence of this SNP in the tumor samples was double that of the normal population (47.25% vs. 22%) and there was no relationship to HPV status. Cell lines containing the SNP showed higher expression of gigaxonin, and an inverse relationship to in vitro tumor cell line growth and cisplatin sensitivity. There was a direct relationship between the expression of gigaxonin and e-cadherin and inverse relationship to the expression of twist, snail and n-cadherin. CRISPR-Cas9 mediated conversion of T to C allele in gigaxonin overexpressing cervical cancer cell line ME 180 resulted in decreased gigaxonin expression accompanied by increased expression of twist, and snail and increased in vitro cell growth. Re-expression of gigaxonin in the CRISPR-Cas9 clone with a plasmid gigaxonin cDNA construct resulted in decreased expression of twist pointing to gigaxonin mediated downregulation of the EMT marker. We therefore hypothesize that GAN, associated with the neurodegenerative disease, is also a human tumor cell growth suppressor gene.
Citation Format: Mysore S. Veena, Daniel Sanghoon Shin, Chan Jeong, Jenna R. Chatoff, Natarajan Venkatesan, Saroj K. Basak, David Gray, Sharon P. Wilczynski, Sharon P. Wilczynski, Steven J. Gray, Donald B. Kohn, Marilene B. Wang, Eri S. Srivatsan. Inverse relationship between Giant Axonal Neuropathy gene expression and EMT in human tumors abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5986.
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