Randomised trial of radiotherapy with weekly cisplatin or cetuximab in low risk HPV associated oropharyngeal cancer (TROG 12.01)- a Trans-Tasman Radiation Oncology Group study

International journal of radiation oncology, biology, physics

2021 Apr 24

Rischin, D;King, M;Kenny, L;Porceddu, S;Wratten, C;Macann, A;Jackson, JE;Bressel, M;Herschtal, A;Fisher, R;Fua, T;Lin, C;Liu, C;Hughes, BGM;McGrath, M;McDowell, L;Corry, J;
PMID: 34098030 | DOI: 10.1016/j.ijrobp.2021.04.015

The excellent prognosis of patients with low risk HPV associated oropharyngeal squamous cell carcinoma has led to concerns about overtreatment and excessive toxicity with radiotherapy and cisplatin, leading to interest in de-intensification trials. We investigated whether cetuximab, an EGFR targeting antibody, when combined with radiotherapy would result in a decrease in symptom burden and toxicity with similar efficacy when compared to weekly cisplatin.XXXX, a randomised, multicentre trial involving 15 sites in XXXX enrolled patients with HPV associated oropharyngeal squamous cell carcinoma, AJCC 7th edition Stage III (excluding T1-2N1) or stage IV (excluding T4 and/or N3 and/or N2b-c if smoking history >10 pack years and/or distant metastases). Patients were randomised (1:1) to receive radiotherapy (70Gy in 35 fractions) with either weekly cisplatin, 7 doses of 40mg/m2 or cetuximab, loading dose of 400mg/m2 followed by 7 weekly doses of 250 mg/m2. The primary outcome was symptom severity assessed by the MD Anderson Symptom Inventory Head and Neck Symptom Severity Scale from baseline to 13 weeks post completion of radiotherapy using the area under the curve (AUC). Trial was registered on ClinicalTrials.gov: XXXX RESULTS: Between 17th June 2013 and 7th June 2018, 189 patients were enrolled, with 92 on cisplatin arm and 90 on cetuximab included in the main analysis. There was no difference in the primary endpoint of symptom severity; difference in AUC cetuximab - cisplatin was 0.05 (95%CI: -0.19, 0.30), p= 0.66. The T-score (mean number of ≥ grade 3 acute adverse events) was 4.35 (SD 2.48) in the cisplatin arm and 3.82 (SD 1.8) in the cetuximab arm, p= 0.108. The 3 -year failure-free survival rates were 93% (95% CI: 86-97%) in the cisplatin arm and 80% (95% CI: 70-87%) in the cetuximab arm (hazard ratio = 3.0 (95% CI: 1.2-7.7); p=0.015.For patients with low risk HPV associated oropharyngeal cancer, radiotherapy and cetuximab had inferior failure-free survival without improvement in symptom burden or toxicity compared to radiotherapy and weekly cisplatin. Radiotherapy and cisplatin remains the standard of care.

Evaluating a New Class of AKT/mTOR Activators for HIV Latency Reversing Activity Ex Vivo and In Vivo

Journal of virology

2021 Feb 03

Gramatica, A;Schwarzer, R;Brantley, W;Varco-Merth, B;Sperber, HS;Hull, PA;Montano, M;Migueles, SA;Rosenthal, D;Hogan, LE;Johnson, JR;Packard, TA;Grimmett, ZW;Herzig, E;Besnard, E;Nekorchuk, M;Hsiao, F;Deeks, SG;Snape, M;Kiernan, B;Roan, NR;Lifson, JD;Estes, JD;Picker, LJ;Verdin, E;Krogan, NJ;Henrich, TJ;Connors, M;Ott, M;Pillai, SK;Okoye, AA;Greene, WC;
PMID: 33536176 | DOI: 10.1128/JVI.02393-20

An ability to activate latent HIV-1 expression could benefit many HIV cure strategies, but the first generation of latency reversing agents (LRAs) has proven disappointing. We evaluated AKT/mTOR activators as a potential new class of LRAs. Two glycogen synthase kinase-3 inhibitors (GSK-3i's), SB-216763 and tideglusib (the latter already in phase II clinical trials) that activate AKT/mTOR signaling were tested. These GSK-3i's reactivated latent HIV-1 present in blood samples from aviremic individuals on antiretroviral therapy (ART) in the absence of T cell activation, release of inflammatory cytokines, cell toxicity, or impaired effector function of cytotoxic T lymphocytes or NK cells. However, when administered in vivo to SIV-infected rhesus macaques on suppressive ART, tideglusib exhibited poor pharmacodynamic properties and resulted in no clear evidence of significant SIV latency reversal. Whether alternative pharmacological formulations or combinations of this drug with other classes of LRAs will lead to an effective in vivo latency-reversing strategy remains to be determined.IMPORTANCE If combined with immune therapeutics, latency reversing agents (LRAs) have the potential to reduce the size of the reservoir sufficiently that an engineered immune response can control the virus in the absence of antiretroviral therapy. We have identified a new class of LRAs that do not induce T-cell activation and that are able to potentiate, rather than inhibit, CD8+ T and NK cell cytotoxic effector functions. This new class of LRAs corresponds to inhibitors of glycogen synthase kinase-3. In this work, we have also studied the effects of one member of this drug class, tideglusib, in SIV-infected rhesus monkeys. When tested in vivo, however, tideglusib showed unfavorable pharmacokinetic properties, which resulted in lack of SIV latency reversal. The disconnect between our ex vivo and in vivo results highlights the importance of developing next generation LRAs with pharmacological properties that allow systemic drug delivery in relevant anatomical compartments harboring latent reservoirs.

Expression of NR5A2, NUP153, HNF4A, USP15 and FNDC3B is consistent with their use as novel biomarkers for bovine mammary stem/progenitor cells

Journal of molecular histology

2021 Jan 05

Choudhary, RK;Capuco, AV;
PMID: 33400051 | DOI: 10.1007/s10735-020-09948-8

Mammary stem cells (MaSC) are essential for growth and maintenance of mammary epithelium. Previous studies have utilized morphological characteristics or retention of bromodeoxyuridine (BrdU) label to identify MaSC and progenitor cells, these approaches may not be feasible or may not identify all resident stem cells. Alternatively, these special cells may be identified by assessing protein and mRNA expression of appropriate markers. The focus of this study was to assess the staining patterns and in situ quantification of novel candidate markers for bovine MaSC/progenitor cells. The candidate markers for MaSC/progenitor cells for immunohistochemical analysis were: NR5A2, NUP153, HNF4A, USP15 and FNDC3B and for in situ transcripts quantification were HNF4A and NUP153. We also evaluated protein expression pattern of presumptive MaSC markers known from the literature namely, ALDH1, MSI1 and Notch3. We found that NR5A2, NUP153, HNF4A and USP15-labeled cells represented 2.5-6% of epithelial cells prepubertally and were distributed in a fashion consistent with the location and abundance of MaSC/progenitor cells. A transient increase (10-37%) in expression of these markers was observed at peak lactation. FNDC3B was localized mainly in the nucleus prepubertally and in the cytoplasm of myoepithelial cells and nuclei of a limited number of alveolar cells during lactation. Abundant expression (~ 48%) and luminal localization of ALDH1 precludes its use as a bovine MaSC marker but may include transamplifying progenitor cells. MSI1 staining was consistent with MaSC localization. Onset of lumen formation in mammary ducts of prepubertal gland was associated with Notch 3 expression in the apical surface of luminal cells. RNAscope analysis of HNF4A and NUP153 transcripts in calf mammary gland showed very low copy numbers in a few epithelial cells, supporting the idea that these markers are expressed by fewer cells of epithelial origin. This study suggests that NR5A2, NUP153, HNF4A, USP15 and FNDC3B are likely markers for bovine MaSC/progenitor cells. Quantification of RNA transcripts of HNF4A and NUP153 in bovine MEC as potential MaSC markers are novel. Further studies to correlate protein expression of these markers with their transcripts level using single cell analysis in larger samples in lactating cow at different physiological stages are warranted.

Detection of SARS-CoV-2 RNA by In Situ Hybridization in Lung-Cancer Cells Metastatic to Brain and in Adjacent Brain Parenchyma

Pathogens (Basel, Switzerland)

2023 May 29

Valyi-Nagy, T;Fredericks, B;Wilson, J;Shukla, SD;Setty, S;Slavin, KV;Valyi-Nagy, K;
PMID: 37375462 | DOI: 10.3390/pathogens12060772

The mechanisms by which severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may spread to the human brain are poorly understood, and the infection of cancer cells in the brain by SARS-CoV-2 in Coronavirus disease 2019 (COVID-19) patients has been the subject of only one previous case report. Here, we report the detection of SARS-CoV-2 RNA by in situ hybridization in lung-cancer cells metastatic to the brain and adjacent brain parenchyma in a 63-year-old male patient with COVID-19. These findings suggest that metastatic tumors may transport the virus from other parts of the body to the brain or may break down the blood-brain barrier to allow for the virus to spread to the brain. These findings confirm and extend previous observations that cancer cells in the brain can become infected by SARS-CoV-2 in patients with COVID-19 and raise the possibility that SARS-CoV-2 can have a direct effect on cancer growth and outcome.

Improved in situ sequencing for high-resolution targeted spatial transcriptomic analysis in tissue sections

Journal of genetics and genomics = Yi chuan xue bao

2023 Feb 14

Tang, X;Chen, J;Zhang, X;Liu, X;Xie, Z;Wei, K;Qiu, J;Ma, W;Lin, C;Ke, R;
PMID: 36796537 | DOI: 10.1016/j.jgg.2023.02.004

Spatial transcriptomics enables the study of localization-indexed gene expression activity in tissues, providing the transcriptional landscape that in turn indicates the potential regulatory networks of gene expression. In situ sequencing (ISS) is a targeted spatial transcriptomic technique, based on padlock probe and rolling circle amplification combined with next-generation sequencing chemistry, for highly multiplexed in situ gene expression profiling. Here, we present improved in situ sequencing (IISS) that exploits a new probing and barcoding approach, combined with advanced image analysis pipelines for high-resolution targeted spatial gene expression profiling. We develop an improved combinatorial probe anchor ligation chemistry using a 2-base encoding strategy for barcode interrogation. The new encoding strategy results in higher signal intensity as well as improved specificity for in situ sequencing, while maintaining a streamlined analysis pipeline for targeted spatial transcriptomics. We show that IISS can be applied to both fresh frozen tissue and formalin-fixed paraffin-embedded tissue sections for single-cell level spatial gene expression analysis, based on which the developmental trajectory and cell-cell communication networks can also be constructed.

Evaluation of Human Kidney Injury Molecule 1 (hKIM-1) Expression in Tumors From Various Organs by Messenger RNA In Situ Hybridization

American journal of clinical pathology

2021 Feb 20

Sarami, I;Shi, J;Lin, B;Liu, H;Monroe, R;Lin, F;
PMID: 33608720 | DOI: 10.1093/ajcp/aqaa236

Human kidney injury molecule 1 (hKIM-1) is a sensitive and specific marker for detection of clear cell renal cell carcinoma (CRCC), papillary renal cell carcinoma (PRCC), and ovarian clear cell carcinoma (OCCC). Its use was limited to a few surgical pathology laboratories because this specific antibody to hKIM-1 was not commercially available. We investigated the diagnostic utility of RNA in situ hybridization/RNAscope in the detection of hKIM-1 in tumors from various organs. RNAscope for hKIM-1 was performed on 1,252 cases on tissue microarray sections, including CRCC (n = 185), PRCC (n = 59), chromophobe renal cell carcinoma (n = 18), oncocytoma (n = 12), OCCC (n = 27), and metastatic CRCC (n = 46). Fifty-nine (100%) of 59 PRCCs, 94 (95%) of 99 low-grade CRCCs, 83 (96%) of 86 high-grade CRCCs, and 24 (89%) of 27 OCCCs, and 44 (96%) of 46 metastatic CRCCs were positive for hKIM-1. In contrast, hKIM-1 expression was not seen in normal renal tubules or in most nonrenal tumors. Low-level expression could be seen in a small percentage of urothelial, hepatocellular, and colon carcinomas. hKIM-1 is a sensitive and relatively specific marker (1) for diagnosing PRCC, CRCC, and OCCC when working on a tumor of unknown origin and (2) for differentiating CRCC from chromophobe renal cell carcinoma and oncocytoma.

Intratumoral dendritic cell-CD4+ T helper cell niches enable CD8+ T cell differentiation following PD-1 blockade in hepatocellular carcinoma

Nature medicine

2023 Jun 01

Magen, A;Hamon, P;Fiaschi, N;Soong, BY;Park, MD;Mattiuz, R;Humblin, E;Troncoso, L;D'souza, D;Dawson, T;Kim, J;Hamel, S;Buckup, M;Chang, C;Tabachnikova, A;Schwartz, H;Malissen, N;Lavin, Y;Soares-Schanoski, A;Giotti, B;Hegde, S;Ioannou, G;Gonzalez-Kozlova, E;Hennequin, C;Le Berichel, J;Zhao, Z;Ward, SC;Fiel, I;Kou, B;Dobosz, M;Li, L;Adler, C;Ni, M;Wei, Y;Wang, W;Atwal, GS;Kundu, K;Cygan, KJ;Tsankov, AM;Rahman, A;Price, C;Fernandez, N;He, J;Gupta, NT;Kim-Schulze, S;Gnjatic, S;Kenigsberg, E;Deering, RP;Schwartz, M;Marron, TU;Thurston, G;Kamphorst, AO;Merad, M;
PMID: 37322116 | DOI: 10.1038/s41591-023-02345-0

Despite no apparent defects in T cell priming and recruitment to tumors, a large subset of T cell rich tumors fail to respond to immune checkpoint blockade (ICB). We leveraged a neoadjuvant anti-PD-1 trial in patients with hepatocellular carcinoma (HCC), as well as additional samples collected from patients treated off-label, to explore correlates of response to ICB within T cell-rich tumors. We show that ICB response correlated with the clonal expansion of intratumoral CXCL13+CH25H+IL-21+PD-1+CD4+ T helper cells ("CXCL13+ TH") and Granzyme K+ PD-1+ effector-like CD8+ T cells, whereas terminally exhausted CD39hiTOXhiPD-1hiCD8+ T cells dominated in nonresponders. CD4+ and CD8+ T cell clones that expanded post-treatment were found in pretreatment biopsies. Notably, PD-1+TCF-1+ (Progenitor-exhausted) CD8+ T cells shared clones mainly with effector-like cells in responders or terminally exhausted cells in nonresponders, suggesting that local CD8+ T cell differentiation occurs upon ICB. We found that these Progenitor CD8+ T cells interact with CXCL13+ TH within cellular triads around dendritic cells enriched in maturation and regulatory molecules, or "mregDC". These results suggest that discrete intratumoral niches that include mregDC and CXCL13+ TH control the differentiation of tumor-specific Progenitor exhasuted CD8+ T cells following ICB.

SARS-CoV-2 detection by digital polymerase chain reaction and immunohistochemistry in skin biopsies from 52 patients with different COVID-19-associated cutaneous phenotypes

Dermatology (Basel, Switzerland)

2023 Apr 19

Marzano, AV;Moltrasio, C;Genovese, G;De Andrea, M;Caneparo, V;Vezzoli, P;Morotti, D;Sena, P;Venturini, M;Battocchio, S;Caputo, V;Rizzo, N;Maronese, CA;Venegoni, L;Boggio, FL;Rongioletti, F;Calzavara-Pinton, P;Berti, E;
PMID: 37075721 | DOI: 10.1159/000530746

COronaVIrus Disease 19 (COVID-19) is associated with a wide spectrum of skin manifestations, but SARS-CoV-2 RNA in lesional skin has been demonstrated only in few cases.To demonstrate SARS-CoV-2 presence in skin samples from patients with different COVID-19-related cutaneous phenotypes.Demographic and clinical data from 52 patients with COVID-19-associated cutaneous manifestations were collected. Immunohistochemistry and digital PCR (dPCR) were performed in all skin samples. RNA in situ hybridization (ISH) was used to confirm the presence of SARS-CoV-2 RNA.Twenty out of 52 (38%) patients presented SARS-CoV-2 positivity in the skin. Among these, 10/52 (19%) patients tested positive for spike protein on immunohistochemistry, five of whom had also positive testing on dPCR. Of the latter, one tested positive both for ISH and ACE-2 on immunohistochemistry while another one tested positive for nucleocapsid protein. Twelve patients showed positivity only for nucleocapsid protein on immunohistochemistry.SARS-CoV-2 was detected only in 38% of patients, without any association with a specific cutaneous phenotype, suggesting that the pathophysiology of cutaneous lesions mostly depends on the activation of the immune system. The combination of spike and nucleocapsid immunohistochemistry has higher diagnostic yield than dPCR. Skin persistence of SARS-CoV-2 may depend on timing of skin lesions, viral load and immune response.S. Karger AG, Basel.

Spatial Transcriptomics: Emerging Technologies in Tissue Gene Expression Profiling

ChemRxiv Analytical Chemistry

2023 Apr 28

Robles-Remacho*, A;Sanchez-Martin, R;Diaz-Mochon*, J;
| DOI: 10.26434/chemrxiv-2023-n20f0

Spatial transcriptomics technologies are providing new insights to study gene expression, allowing researchers to investigate the spatial organization of transcriptomes in cells and tissues. This approach enables the creation of high-resolution maps of gene expression patterns within their native spatial context, adding an extra layer of information to bulk sequencing data. Spatial transcriptomics has expanded significantly in recent years and is making a notable impact on a range of fields, including tissue architecture, developmental biology, cancer, neurodegenerative and infectious diseases. The latest advancements in spatial transcriptomics have resulted in the development of highly multiplexed methods, transcriptomic-wide analysis, and single-cell resolution, utilizing diverse technological approaches. In this perspective, we provide a detailed analysis of the molecular foundations behind the main spatial transcriptomics technologies, including methods based on microdissection, in situ sequencing, single-molecule FISH, spatial capturing, selection of regions of interest and single-cell or nuclei dissociation. We contextualize the detection and capturing efficiency, strengths, limitations, tissue compatibility, and applications of these techniques, as well as provide information on data analysis. In addition, this perspective discusses future directions and potential applications of spatial transcriptomics, highlighting the importance of the continued development to promote widespread adoption of these techniques within the research community.

Simultaneous detection and quantification of spike mRNA and protein in SARS-CoV-2 infected airway epithelium

MethodsX

2023 Feb 03

Jerome, K;Sattar, S;Mehedi, M;
PMID: 36779029 | DOI: 10.1016/j.mex.2023.102050

Visualizing and quantifying mRNA and its corresponding protein provides a unique perspective of gene expression at a single-molecule level. Here, we describe a method for differentiating primary cells for making airway epithelium and detecting SARS-CoV-2 Spike (S) mRNA and S protein in the paraformaldehyde-fixed paraffin-embedded severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected airway epithelium. For simultaneous detection of mRNA and protein in the same cell, we combined two protocols: 1. RNA fluorescence-based in situ hybridization (RNA-FISH) based mRNA detection and 2. fluorescence-based immunohistochemistry (IHC) based protein detection. The detection of mRNA and proteins in the same cell also allows for quantifying them using the open-source software QuPath, which provides an accurate and more straightforward fluorescent-based quantification of mRNA and protein in the microscopic images of the infected cells. Additionally, we can achieve the subcellular distribution of both S mRNA and S protein. This method identifies SARS-CoV-2 S gene products' (mRNA and protein) degree of expression and their subcellular localization in the infected airway epithelium. Advantages of this method include: •Simultaneous detection and quantification of mRNA and protein in the same cell.•Universal use due to the ability to use mRNA-specific primer-probe and protein-specific antibodies.•An open-source software QuPath provides a straightforward fluorescent-based quantification.

Considerations for the Use of Viral Vectors in Nonhuman Primates

Vectorology for Optogenetics and Chemogenetics

2023 Feb 07

Bohlen, M;Tremblay, S;
| DOI: 10.1007/978-1-0716-2918-5_16

The development of new genetic tools has revolutionized our ability to study the functional role of specific neuronal populations and circuits generating behavior. Although this revolution has already taken place in small animal models such as mice, adoption of these techniques has been relatively slow for animals more closely related to humans, such as nonhuman primates. Current challenges include effective delivery to much larger structural targets in the primate brain, cell-type specific transduction, and immunological responses. In this chapter, we will review some of the challenges and considerations that are specific to using these viral technologies in the nonhuman primate brain. Ultimately, these challenges can be met with new advances in surgical technique and gene therapy that will spin out new viral vectors with enhanced features able to compensate for the limitations of current vectors. As the existing challenges are circumvented, this will lead to a revolution in primate neuroscientific research and a greater understanding of the functional role circuits play in complex behaviors relevant to human neurological and psychiatric diseases.

HIV integration in the human brain is linked to microglial activation and 3D genome remodeling

Molecular cell

2022 Dec 15

Plaza-Jennings, AL;Valada, A;O'Shea, C;Iskhakova, M;Hu, B;Javidfar, B;Ben Hutta, G;Lambert, TY;Murray, J;Kassim, B;Chandrasekaran, S;Chen, BK;Morgello, S;Won, H;Akbarian, S;
PMID: 36525955 | DOI: 10.1016/j.molcel.2022.11.016

To explore genome organization and function in the HIV-infected brain, we applied single-nuclei transcriptomics, cell-type-specific chromosomal conformation mapping, and viral integration site sequencing (IS-seq) to frontal cortex from individuals with encephalitis (HIVE) and without (HIV+). Derepressive changes in 3D genomic compartment structures in HIVE microglia were linked to the transcriptional activation of interferon (IFN) signaling and cell migratory pathways, while transcriptional downregulation and repressive compartmentalization of neuronal health and signaling genes occurred in both HIVE and HIV+ microglia. IS-seq recovered 1,221 brain integration sites showing distinct genomic patterns compared with peripheral lymphocytes, with enrichment for sequences newly mobilized into a permissive chromatin environment after infection. Viral transcription occurred in a subset of highly activated microglia comprising 0.33% of all nuclei in HIVE brain. Our findings point to disrupted microglia-neuronal interactions in HIV and link retroviral integration to remodeling of the microglial 3D genome during infection.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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