Defining best practices for tissue procurement in immuno-oncology clinical trials: consensus statement from the Society for Immunotherapy of Cancer Surgery Committee.

Immunotherapy is now a cornerstone for cancer treatment, and much attention has been placed on the identification of prognostic and predictive biomarkers. The success of biomarker development is dependent on accurate and timely collection of biospecimens and high-quality processing, storage and shipping. Tumors are also increasingly used as source material for the generation of therapeutic T cells. There have been few guidelines or consensus statements on how to optimally collect and manage biospecimens and source material being used for immunotherapy and related research. The Society for Immunotherapy of Cancer Surgery Committee has brought together surgical experts from multiple subspecialty disciplines to identify best practices and to provide consensus on how best to access and manage specific tissues for immuno-oncology treatments and clinical investigation. In addition, the committee recommends early integration of surgeons and other interventional physicians with expertise in biospecimen collection, especially in clinical trials, to optimize the quality of tissue and the validity of correlative clinical studies in cancer immunotherapy.

Mechanistic Justifications of Systemic Therapeutic Oxygenation of Tumors to Weaken the Hypoxia Inducible Factor 1α-Mediated Immunosuppression.

Long-term studies of anti-pathogen and anti-tumor immunity have provided complementary genetic and pharmacological evidence for the immunosuppressive and immunomodulatory effects of Hypoxia-HIF-1α and adenosine-mediated suppression via the A2A adenosine receptor signaling pathway (Hypoxia-A2A-adenosinergic). This pathway is life saving when it protects inflamed tissues of vital organs from collateral damage by overactive anti-pathogen immune cells or enables the differentiation of cells of adaptive immunity. However, the Hypoxia-A2A-adenosinergic immunosuppression can also prevent tumor rejection by inhibiting the anti-tumor effects of T and NK cells. In addition, this suppressive pathway has been shown to mask tumors due to the hypoxia-HIF-α-mediated loss of MHC Class I molecules on tumor cells. It is suggested that it will be impossible to realize the full anti-tumor capacities of current cancer immunotherapies without simultaneous administration of anti-Hypoxia-A2A-Adenosinergic drugs that inactivate this tumor-protecting mechanism in hypoxic and adenosine-rich tumors.Here, we overview the supporting evidence for the conceptually novel immunotherapeutic motivation to breathe supplemental oxygen (40-60%) or to repurpose already available oxygenation agents in combination with current immunotherapies. Preclinical studies provide strong support for oxygen immunotherapy to enable much stronger tumor regression by weakening immunosuppression by A2A adenosine receptors and by the Hypoxia➔HIF-1α axis. The results of these studies emphasize the value of systemic oxygenation as clinically feasible, promising, and as a valuable tool for mechanistic investigations of tumor biology and cancer immunology. Perhaps the most effective and feasible among individual members of this novel class of anti-tumor drugs are oxygenation agents.

Suppression of stress induction of the 78-kilodalton glucose regulated protein (GRP78) in cancer by IT-139, an anti-tumor ruthenium small molecule inhibitor.

In many cancers, combination therapy regimens are successfully improving response and survival rates, but the challenges of toxicity remain. GRP78, the master regulator of the unfolded protein response, is emerging as a target that is upregulated in tumors, specifically following treatment, and one that impacts tumor cell survival and disease recurrence. Here, we show IT-139, an antitumor small molecule inhibitor, suppresses induction of GRP78 from different types of endoplasmic reticulum (ER) stress in a variety of cancer cell lines, including those that have acquired therapeutic resistance, but not in the non-cancer cells being tested. We further determined that IT-139 treatment exacerbates ER stress while at the same time suppresses GRP78 induction at the transcriptional level. Our studies revealed a differential effect of IT-139 on chaperone protein family expression at multiple levels in different cancer cell lines. In xenograft studies, IT-139 decreased BRAF inhibitor upregulation of GRP78 expression in the tumor, while having minimal effect on GRP78 expression in the adjacent normal cells. The preferential decrease in GRP78 levels in tumor cells over normal cells, supported by the manageable safety profile seen in the Phase 1 clinical trial, reinforce the value IT-139 brings to combination therapies as it continues its clinical development.

Imaging the delivery of drug-loaded, iron-stabilized micelles.

Nanoparticle drug carriers hold potential to improve current cancer therapy by delivering payload to the tumor environment and decreasing toxic side effects. Challenges in nanotechnology drug delivery include plasma instability, site-specific delivery, and relevant biomarkers. We have developed a triblock polymer comprising a hydroxamic acid functionalized center block that chelates iron to form a stabilized micelle that physically entraps chemotherapeutic drugs in the hydrophobic core. The iron-imparted stability significantly improves the integrity of the micelle and extends circulation pharmacokinetics in plasma over that of free drug. Furthermore, the paramagnetic properties of the iron-crosslinking exhibits contrast in the tumors for imaging by magnetic resonance. Three separate nanoparticle formulations demonstrate improved anti-tumor efficacy in xenograft models and decreased toxicity. We report a stabilized polymer micelle that improves the tolerability and efficacy of chemotherapeutic drugs, and holds potential for non-invasive MRI to image drug delivery and deposition in the tumor.

PCR-based bioprospecting for homing endonucleases in fungal mitochondrial rRNA genes.

Fungal mitochondrial genomes act as "reservoirs" for homing endonucleases. These enzymes with their DNA site-specific cleavage activities are attractive tools for genome editing and gene therapy applications. Bioprospecting and characterization of naturally occurring homing endonucleases offers an alternative to synthesizing artificial endonucleases. Here, we describe methods for PCR-based screening of fungal mitochondrial rRNA genes for homing endonuclease encoding sequences, and we also provide protocols for the purification and biochemical characterization of putative native homing endonucleases.

Evolutionary dynamics of introns and their open reading frames in the U7 region of the mitochondrial rnl gene in species of Ceratocystis.

The mtDNA rnl-U7 region has been examined for the presence of introns in selected species of the genus Ceratocystis. Comparative sequence analysis identified group I and group II introns encoding single and double motif LAGLIDADG open reading frames (ORFs) at the following positions L1671, L1787, and L1923. In addition downstream of the rnl-U7 region group I introns were detected at positions L1971 and L2231, and a group II intron at L2059. A GIY-YIG type ORF was located within one mL1923 LAGLIDADG type ORF and a degenerated GIY-YIG ORF fused to a nad2 gene fragment was found in association with the mL1971 group I intron. The diversity of composite elements that appear to be sporadically distributed among closely related species of Ceratocystis illustrates the potential for homing endonucleases and their associated introns to invade new sites. Phylogenetic analysis showed that single motif LADGLIDADG ORFs related to the mL1923 ORFs have invaded the L1787 group II intron and the L1671 group I intron. Phylogenetic analysis of intron encoded single and double motif LAGLIDADG ORFs also showed that these ORFs transferred four times from group I into group II B1 type introns.

The mtDNA rns gene landscape in the Ophiostomatales and other fungal taxa: twintrons, introns, and intron-encoded proteins.

Comparative sequence analysis of the mitochondrial small subunit ribosomal RNA (rns) gene among species of Ophiostoma, Grosmannia, Ceratocystiopsis and related taxa provides an overview of the types of introns that have invaded this gene within the ophiostomatoid fungi. The rns gene appears to be a reservoir for a number of group I and group II introns along with intron-associated open reading frames such as homing endonucleases and reverse transcriptases. This study uncovered two twintrons, one at position mS917 where a group ID intron encoding a LAGLIDADG ORF invaded another ORF-less group ID intron. Another twintron complex was detected at position mS1247 here a group IIA1 intron invaded the open reading frame embedded within a group IC2 intron. Overall the distribution of the introns does not appear to follow evolutionary lineages suggesting the possibility of rare horizontal gains and frequent losses. Results of this study will make a significant contribution to the understanding of the complexity of the mitochondrial intron landscape, and offer a resource to those annotating mitochondrial genomes. It will also serve as a resource to those that bioprospect for ribozymes and homing endonucleases.

Identification of group I introns within the SSU rDNA gene in species of Ceratocystiopsis and related taxa.

During a recent phylogenetic study, group I introns were noted that interrupt the nuclear small subunit ribosomal RNA (SSU rDNA) gene in species of Ceratocystiopsis. Group I introns were found to be inserted at the following rDNA positions: S943, S989, and S1199. The introns have been characterized and phylogenetic analysis of the host gene and the corresponding intron data suggest that for S943 vertical transfer and frequent loss appear to be the most parsimonious explanation for the distribution of nuclear SSU rDNA introns among species of Ceratocystiopsis. The SSU rDNA data do suggest that a recent proposal of segregating the genus Ophiostoma sensu lato into Ophiostoma sensu stricto, Grosmannia, and Ceratocystiopsis has some merit but may need further amendments, as the SSU rDNA suggests that Ophiostoma s. str. may now represent a paraphyletic grouping.

Molecular evolution of the mtDNA encoded rps3 gene among filamentous ascomycetes fungi with an emphasis on the Ophiostomatoid fungi.

The mitochondrial ribosomal protein S3 (rps3) gene within the fungi is extremely diverse in location and organization, some versions of this gene have been incorporated into a group I intron, others appear to have gained large insertions, microsatellite expansions, or have been invaded by homing endonucleases. Among the ascomycetes fungi the group I intron encoded version of rps3 appears to have a rather complex evolutionary history including first the acquisition of rps3 by a group I intron (mL2449), the loss of the mL2499 intron and the establishment of rps3 as a free-standing gene, and the eventual loss of the intron derived version of rps3.

The sporadic occurrence of a group I intron-like element in the mtDNA rnl gene of Ophiostoma novo-ulmi subsp. americana.

The presence of group I intron-like elements within the U7 region of the mtDNA large ribosomal subunit RNA gene (rnl) was investigated in strains of Ophiostoma novo-ulmi subsp. americana from Canada, Europe and Eurasia, and in selected strains of O. ips, O. minus, O. piceae, O. ulmi, and O. himal-ulmi. This insertion is of interest as it has been linked previously to the generation of plasmid-like mtDNA elements in diseased strains of O. novo-ulmi. Among 197 O. novo-ulmi subsp. americana strains tested, 61 contained a 1.6kb insertion within the rnl-U7 region and DNA sequence analysis suggests the presence of a group I intron (IA1 type) that encodes a potential double motif LAGLIDADG homing endonuclease-like gene (HEG). Phylogenetic analysis of rnl-U7 intron encoded HEG-like elements supports the view that double motif HEGs originated from a duplication event of a single-motif HEG followed by a fusion event that combined the two copies into one open reading frame (ORF). The data also show that rnl-U7 intron encoded ORFs belong to a clade that includes ORFs inserted into different types of group I introns, e.g. IB, ID, IC3, IA1, present within a variety of different mtDNA genes, such as the small ribosomal subunit RNA gene (rns), apo-cytochrome b gene (cob), NADH dehydrogenase subunit 5 (nad5), cytochrome oxidase subunit 1 gene (coxI), and ATPase subunit 9 gene (atp9). We also compared the occurrence of the rnl-U7 intron in our collection of 227 strains with the presence of the rnl-U11 group I intron and concluded that the U7 intron appears to be an optional element and the U11 intron is probably essential among the strains tested.