Dominant Subtype Research Articles

H5Nx Viruses Emerged during the Suppression of H5N1 Virus Populations in Poultry.

ABSTRACTHighly pathogenic avian influenza (HPAI) H5 viruses have posed a substantial pandemic threat through repeated human infection since their emergence in China in 1996. Nationwide control measures, including vaccination of poultry, were implemented in 2005, leading to a sharp reduction in H5N1 virus outbreaks. In 2008, novel non-N1 subtype (H5Nx) viruses emerged, gradually replacing the dominant H5N1 subtype and causing global outbreaks. The cause of this major shift in the ecology of HPAI H5 viruses remains unknown. Here, we show that major H5N1 virus lineages underwent population bottlenecks in 2006, followed by a recovery in virus populations between 2007 and 2009. Our analyses indicate that control measures, not competition from H5Nx viruses, were responsible for the H5N1 decline, with an H5N1 lineage capable of infecting poultry and wild birds experiencing a less severe population bottleneck due to circulation in unaffected wild birds. We show that H5Nx viruses emerged during the successful suppression of H5N1 virus populations in poultry, providing an opportunity for antigenically distinct H5Nx viruses to propagate. Avian influenza vaccination programs would benefit from universal vaccines targeting a wider diversity of influenza viruses to prevent the emergence of novel subtypes.IMPORTANCE A major shift in the ecology of highly pathogenic avian influenza (HPAI) H5 viruses occurred from 2008 to 2014, when viruses with non-N1 neuraminidase genes (termed H5Nx viruses) emerged and caused global H5 virus outbreaks. Here, we demonstrate that nationwide control measures, including vaccination in China, successfully suppressed H5N1 populations in poultry, providing an opportunity for antigenically distinct H5Nx viruses to emerge. In particular, we show that the widespread use of H5N1 vaccines likely conferred a fitness advantage to H5Nx viruses due to the antigenic mismatch of the neuraminidase genes. These results indicate that avian influenza vaccination programs would benefit from universal vaccines that target a wider diversity of influenza viruses to prevent potential emergence of novel subtypes.

Clinical characteristics of children infected with different subtypes/genotypes of human respiratory syncytial virus in Beijing from 2009 to 2017

Objective: To explore the different clinical characteristics of children infected with different subtype/genotype of human respiratory syncytial virus (HRSV) in Beijing. Methods: Respiratory specimens for positive HRSV were randomly collected from children with acute respiratory tract infection (ARTI) in the epidemic season of HRSV from November of each year to January of the next year during 2009 and 2017. G genes of HRSV were amplified and sequenced for subtyping and genotyping by bioinformatics analysis. Clinical data were collected and analyzed. Results: Out of 590 children, 376 (63.7%) with subtype A, and 214 (36.3) with subtype B. The annual dominant subtypes of HRSV from 2009 to 2017 were B-A-A-B-AB-A-A-B-A, respectively, whilst a total of 10 genotypes were detected with 95.8% assigned to genotype ON1 and NA1 of subtype A, and genotype BA9 of subtype B. Children infected with subtype B (96 cases, 44.9%) were more likely aged 0-3 month old than those with subtype A (118 cases, 31.4%) (P=0.001), and more likely to be admitted to Intensive Care Unit(ICU) ((124 cases, 57.9%) than those with subtype A (172 cases, 45.7%)) (P=0.005). Statistical significance were shown among children infected with genotype ON1, NA1 or BA9, in the possibility of infection in children aged 0-3 month (P=0.003), proportion of admission into ICU (P=0.007), length of stay in hospital (P=0.001), and clinical outcome (P=0.001), respectively. Conclusion: Children infected with different subtype or genotype of HRSV have different clinical characteristics, which stresses the important role of the monitoring HRSV subtypes and genotypes among children.

New Estimation of Antibiotic Resistance Genes in Sediment Along the Haihe River and Bohai Bay in China: A Comparison Between Single and Successive DNA Extraction Methods.

Sediment is thought to be a vital reservoir for antibiotic resistance genes (ARGs). Often, studies describing and comparing ARGs and their potential hosts in sediment are based on single DNA extractions. To date, however, no study has been conducted to assess the influence of DNA extraction efficiency on ARGs in sediment. To determine whether the abundance of ARGs is underestimated, we performed five successive extraction cycles with a widely used commercial kit in 10 sediment samples collected from the Haihe River and Bohai Bay. Our results showed that accumulated DNA yields after five extractions were 1.8–3.1 times higher than that by single DNA extractions. High-throughput sequencing showed that insufficient DNA extraction could generate PCR bias and skew community structure characterization in sediment. The relative abundances of some pathogenic bacteria, such as Enterobacteriales, Lactobacillales, and Streptomycetales, were significantly different between single and successive DNA extraction samples. In addition, real-time fluorescent quantitative PCR (qPCR) showed that ARGs, intI1, and 16S rRNA gene abundance strongly increased with increasing extraction cycles. Among the measured ARGs, sulfonamide resistance genes and multidrug resistance genes were dominant subtypes in the study region. Nevertheless, different subtypes of ARGs did not respond equally to the additional extraction cycles; some continued to have linear growth trends, and some tended to level off. Additionally, more correlations between ARGs and bacterial communities were observed in the successive DNA extraction samples than in the single DNA extraction samples. It is suggested that 3–4 additional extraction cycles are required in future studies when extracting DNA from sediment samples. Taken together, our results highlight that performing successive DNA extractions on sediment samples optimizes the extractable DNA yield and can lead to a better picture of the abundance of ARGs and their potential hosts in sediments.

Molecular epidemiology of respiratory syncytial virus among children and adults in India 2016 to 2018

Respiratory syncytial virus (RSV) is a common cause of respiratory tract infections among children less than 5 years of age and the elderly. This study intended to determine the circulating genotypes of RSV among severe acute respiratory illness (SARI) cases during the period 2016–2018 in India, among hospitalized acute febrile illness cases of age ranging from 1 to 65 years. Throat/nasopharyngeal swab samples were subjected for testing RSV and subgroups by real-time reverse transcriptase polymerase chain reaction (RT-PCR), further sequencing and phylogenetic analysis were performed for the second hypervariable region of the G gene. RSV-A and B subtypes co-circulated during the years 2016, 2017, and 2018, with RSV-A as the dominant subtype in 2016, and RSV-B as the dominant subgroup in 2017 and 2018. Phylogenetic analysis revealed that the circulating genotypes of RSV were GA2 (16/16), of RSV-A, and GB5 (23/23) of RSV-B in the South, North, and Northeast region of India during the period between 2016 and 2018. Here we report the first study comprising the distribution of RSV-A and B genotypes in the different geographic regions of India among children and adults during the year 2016 to 2018. We also report GA2.3.7 lineage of GA2 genotype for the first time in India to the best of our knowledge.

Molecular Subtypes of Primary SCLC Tumors and Their Associations With Neuroendocrine and Therapeutic Markers

IntroductionA new molecular subtype classification was recently proposed for SCLC. It is necessary to validate it in primary SCLC tumors by immunohistochemical (IHC) staining and define its clinical relevance. MethodsWe used IHC to assess four subtype markers (ASCL1, NEUROD1, POU2F3, and YAP1) in 194 cores from 146 primary SCLC tumors. The profiles of tumor-associated CD3+ and CD8+ T-cells, MYC paralogs, SLFN11, and SYP were compared among different subtypes. Validation was performed using publicly available RNA sequencing data of SCLC. ResultsASCL1, NEUROD1, POU2F3, and YAP1 were the dominant molecular subtypes in 78.2%, 5.6%, 7%, and 2.8% of the tumors, respectively; 6.3% of the tumors were negative for all four subtype markers. Notably, three cases were uniquely positive for YAP1. Substantial intratumoral heterogeneity was observed, with 17.6% and 2.8% of the tumors being positive for two and three subtype markers, respectively. The non-ASCL1/NEUROD1 tumors had more CD8+ T-cells and manifested more frequently an “inflamed” immunophenotype. L-MYC and MYC were more often associated with ASCL1/NEUROD1 subtypes and non-ASCL1/NEUROD1 subtypes, respectively. SLFN11 expression was absent in 40% of the tumors, especially those negative for the four subtype markers. SYP was often expressed in the ASCL1 and NEUROD1 subtypes and was associated with less tumor-associated CD8+ T-cells and a “desert” immunophenotype. ConclusionsWe validated the new molecular subtype classification in primary SCLC tumors by IHC and identified several intriguing associations between subtypes and therapeutic markers. The new subtype classification may potentially assist treatment decisions in SCLC.

Performance of full-scale aerobic composting and anaerobic digestion on the changes of antibiotic resistance genes in dairy manure

Understanding the different performances of full-scale active composting (AC) and anaerobic digestion (AD) on the changes of antibiotic resistance genes (ARGs) in dairy manure is crucial to uncover the dissemination risks of ARGs in post-biotreated manure. In this regard, metagenomic sequencing was deployed to reveal the variations of ARGs in dairy manure in an intensive dairy farm. Results showed that the total abundance of ARGs increased from 150.64 reads/ng DNA to 204.06 reads/ng DNA in dairy manure, and it is mainly attributed to the contributions of AC (85.49%) rather than AD (14.51%). In AC, more ARG subtypes were induced and the dominant ARG subtypes were shifted completely, probably due to the enrichment of Proteobacteria and Actinobacteria which could be the hosts of multiple ARGs. These results inspire us to further evaluate the dissemination risks of ARGs along the route from composted manure to soil and to plants.

Genomic and evolutionary classification of lung cancer in never smokers.

Lung cancer in never smokers (LCINS) is a common cause of cancer mortality but its genomic landscape is poorly characterized. Here high-coverage whole-genome sequencing of 232 LCINS showed 3 subtypes defined by copy number aberrations. The dominant subtype (piano), which is rare in lung cancer in smokers, features somatic UBA1 mutations, germline AR variants and stem cell-like properties, including low mutational burden, high intratumor heterogeneity, long telomeres, frequent KRAS mutations and slow growth, as suggested by the occurrence of cancer drivers' progenitor cells many years before tumor diagnosis. The other subtypes are characterized by specific amplifications and EGFR mutations (mezzo-forte) and whole-genome doubling (forte). No strong tobacco smoking signatures were detected, even in cases with exposure to secondhand tobacco smoke. Genes within the receptor tyrosine kinase-Ras pathway had distinct impacts on survival; five genomic alterations independently doubled mortality. These findings create avenues for personalized treatment in LCINS.

MP44-09 UNDERSTANDING PRUNE BELLY SYNDROME AT SINGLE CELL RESOLUTION

Prune Belly Syndrome (PBS) is characterized by bladder dysmyogenesis, yielding a dysfunctional compliant thick wall with excess collagen deposition. To dissect the cellular heterogeneity and gene expression networks altered in PBS, we report the cell type composition and transcriptional activity of PBS human bladder by using single cell RNA sequencing (scRNA-seq). Using IRB-approved methods, bladder dome from 2 PBS and 6 non-PBS control (CO) males underwent fresh single-cell digestion. scRNA-seq was performed and 5277 and 31828 bladder cells from PBS and CO patients was detected, respectively. Cell type clusters were graphically displayed by Uniform Manifold Approximation and Projection (UMap) plot and differentially expressed genes (DEGs) were generated to assign each cluster identity. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis was performed for PBS affected genes. We identified 17 distinct bladder cell clusters, including 6 fibroblast (1, 2, 3, and 4, immunofibroblast, myofibroblast), 1 smooth muscle (SM), and 2 urothelial (umbrella and basal+intermediate) clusters (Fig 1A-B). Counts of individual cell types were expressed as relative proportions, identifying significant PBS fibroblast enrichment, (67% PBS vs 40% CO). Five of 6 PBS fibroblast sub-types are proportionately fewer in number than in CO. The exception is a dominant fibroblast sub-type we label as fibroblast 4, (61% of all PBS fibroblasts vs <10% CO fibroblast subtypes). SM and urothelial cell populations are dramatically reduced in PBS (SM: 5% PBS vs 11% CO and urothelial: <1% PBS vs 7% CO) (Fig 1C-E). PBS fibroblast DEGs, but not SM cells, are enriched in collagen genes. Fibroblast markers (DCN and PLA2G2A) and SM genes (DES, TPM2 and TAGLN) are reduced (by 4, 13, 2, 4, and 2x respectively) in PBS cells (Fig 1G). KEGG pathways analysis for fibroblasts and SM showed a highly similar enrichment for neurodegenerative disease pathways (Fig 1H-I). Using scRNA-seq, we identified and characterized the disarrayed cell type populations in PBS bladders, generating their unbiased transcriptomic signatures which highlight commonality with neurodegenerative diseases. This PBS transcriptomic map is a step toward potential markers for diagnosis and therapeutic intervention.[Figure: see text]Source of Funding:NIH DK100483, DK127589 PI: Baker, L.

The Effect of The Body Composition to Prognosis in Young Breast Cancer Patients

Objective:In cancer patients, the effect of the body composition on prognosis is a new clinical area of interest. In patients with class 2 or 3 obesity (BMI >35), survival found to be worse control groups in a pooled analysis. BMI category is not truly representing body composition and hard to use to determine the true muscle and fat quantity. Computed tomography (CT) is a frequent method to determine body composition precisely. Methods:Axial CT images, including all abdominal muscles (psoas, erector spinae, quadratus lumborum) external and internal oblique and rectus abdominis) total skeletal muscle area (SMA), was calculated. Besides, axial CT images of the body fat subcutaneous adipose and visceral adipose tissue distribution (VAT, SAT) areas were calculated in cm2 using threshold values of -30 to -190 for adipose tissue. Results:Eighty-four women were included in the study. Most of the patients were normal or over-weighted. Invasive carcinoma NST was the dominant histological subtypes, with 94% of the study population. The count of the sarcopenic and non-sarcopenic patients was 11 and 68 respectively. Although the median OS cannot be reached at the end of the follow-up period for both groups, the difference between groups statistically insignificant. The median OS was not reached for both groups, the difference between low and high TVAT groups was statistically significant. In multivariate analysis, TVAT was found to be an independent risk factor. Conclusion:In this study, we demonstrate sarcopenia may be seen in patients with breast cancer under 40 years old, and it may not have a prognostic effect.

Blood component therapy, demographic and outcome feature of pediatric acute lymphoblastic leukemia

Introduction: Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy, with an annual incidence rate of three to four cases per 100000 children. Most children with ALL frequently receive blood products including packed cells, platelets, fresh frozen plasma (FFP) and whole blood in the course of chemotherapy and these transfusions may affect ALL outcomes. Objectives: This study aimed to evaluate blood component therapy together with demographic and outcome features of pediatric ALL patients. Patients and Methods: Demographic information of 208 patients with pediatric ALL from February 2011 to August 2019 enrolled in this cross-sectional study. Data is gathered and rechecked from archive files and e-files of Motahari hospital. Results: The mean age of patients at diagnosis was 5.48±3.38 years and Pre-B ALL was the most common phenotype (94.3%). 130 Out of 208 patients were treated with the new protocol and 78 patients were treated with the BFM98 protocol. The majority of relapses were in the bone marrow. The average of received packed cell, platelet and FFP were 4.32±2.93, 5.97±7.09 and 5.29±6.6 units, respectively. The mean overall survival of patients was 3.42±2.58 years in 10 years. Conclusion: According to this study, most of the patients were 5 to 15 years old. Dominant subtype of disease was B-cell type. Most of the deaths were one to 6 years after diagnosis. The relapse rate was about 31% and most of them were in the bone marrow.

Single-Cell RNA-Sequencing Portraying Functional Diversity and Clinical Implications of IFI6 in Ovarian Cancer.

Ovarian cancer (OC) is one of the most lethal gynecologic malignancies. Most patients die of metastasis due to a lack of other treatments aimed at improving the prognosis of OC patients. In the present study, we use multiple methods to identify prognostic S1 as the dominant subtype in OC, possessing the most ligand–receptor pairs with other cell types. Based on markers of S1, the consensus clustering algorithm is used to explore the clinical treatment subtype in OC. As a result, we identify two clusters associated with distinct survival and drug response. Notably, IFI6 contributes to the cluster classification and seems to be a vital gene in OC carcinogenesis. Functional enrichment analysis demonstrates that its functions involve G2M and cisplatin resistance, and downregulation of IFI6 suppresses proliferation capabilities and significantly potentiates cisplatin-induced apoptosis of OC cells in vitro. To explore possible mechanisms of IFI6 influencing OC proliferation and cisplatin resistance, GSEA is conducted and shows that IFI6 is positively correlated with the NF-κB pathway, which is validated by RT-qPCR. Significantly, we develop a prognostic model including IFI6, RiskScore, which is an independent prognostic factor and presents encouraging prognostic values. Our findings provide novel insights into elucidating the biology of OC based on single-cell RNA-sequencing. Moreover, this approach is potentially helpful for personalized anti-cancer strategies and predicting outcomes in the setting of OC.

Anticipatory 50-kHz Precontact Ultrasonic Vocalizations and Sexual Motivation: Characteristic Pattern of Ultrasound Subtypes in an Individual Analyzed Profile.

We verified the hypothesis of the existence of forms of individual-specific differences in the emission of anticipatory precontact vocalization (PVs) indicating individualization related to sexual experience and motivation in male rats. Long-Evans males were individually placed in a chamber and 50-kHz ultrasounds were recorded during 5-min periods. In experiment 1, PVs were recorded before the introduction of a female in four consecutive sessions during the acquisition of sexual experience. In experiment 2, PVs were analyzed in three groups of sexually experienced males: with the highest, moderate, and the lowest sexual motivation based on previous copulatory activity. In both experiments, the total number of ultrasounds, as well as 14 different specific subtypes, was measured. The ultrasound profiles for each male were created by analyzing the proportions of specific dominant subtypes of so-called 50-kHz calls. We decided that the dominant ultrasounds were those that represented more than 10% of the total recorded signals in a particular session. The number of PVs was positively correlated with the acquisition of sexual experience and previous copulatory efficiency (measured as the number of sessions with ejaculation). Furthermore, PVs showed domination of the frequency modulated signals (complex and composite) as well as flat and short with upward ramp ultrasounds with some individual differences, regardless of the level of sexual motivation. The results show a characteristic pattern of PVs and confirm the hypothesis that the number of PVs is a parameter reflecting the level of sexual motivation.

Differential expression of retinoic acid alpha and beta receptors in neuronal progenitors generated from human embryonic stem cells in response to TTNPB (a retinoic acid mimetic)

Retinoic acid (RA), an active metabolite of vitamin A, plays a critical role in the morphogenesis and differentiation of various tissues, especially in the central nervous system. RA is the most commonly used morphogen for the differentiation of human embryonic stem cells (hESCs) into neuronal progenitor cells (NPCs), an abundant source of healthy neuronal tissues for regenerative therapy. During the differentiation process, the activity of RA is governed by the involvement of RA receptor subtypes (RAR α, β, and γ) and their isoforms in the nucleus. However, little is known about the involvement of specific RAR subtypes during neuronal differentiation in humans. It is essential to elucidate the dynamic function of different RAR subtypes and their influence on the phenotypic outcome. Here in this study, we used TTNPB, an analog and stabilized form of retinoic acid that potently and selectively activates retinoic acid receptors. Here we determined the optimum concentration of TTNPBfor the efficient generation of early NPCs from hESCs. Using the optimized concentration of -TTNPB, we found that RARα is the functionally dominant subtype and controls the RA-mediated neurogenesis of hESCs. Importantly, we also found that the RARγ subtype also played a role in neuronal differentiation. In contrast, the RARβ subtype negatively correlates with neuronal differentiation. Therefore, pharmacological inhibition of RARβ in the TTNPB-mediated differentiation process could be used as a strategy to generate a large number of NPCs in vitro. In summary, our results show that RARα and RARγ play a vital role in the TTNPB-mediated neuronal differentiation of hESCs, -whereas RARβ acts as a negative regulator.

Nonmotor symptoms and quality of life in Parkinson's disease with different motor subtypes.

To explore the differences in the features and impact on quality of life (QOL) of nonmotor symptoms (NMS) of the tremor dominant (TD) and non-tremor dominant (NTD) subtypes of Parkinson's disease (PD) and elucidate the characteristic NMS and determinants of QOL for the subtypes. This cross-sectional study included 77patients with PD. The NMS questionnaire was used to assess the range and prevalence of NMS. The modified Hoehn and Yahr staging and the unified PD rating scale (UPDRS) were used to assess symptom severity. Cognitive abilities were investigated using the mini-mental status examination (MMSE). Emotional state was assessed using the Hamilton depression scale (HAMD) and the Hamilton anxiety scale (HAMA) and QOL was evaluated using the Parkinson's disease questionnaire-39 (PDQ-39). Overall, 39(50.65%) patients were classified as the TD subtype, and 38(49.35%) were classified as the NTD subtype. Patients with NTD subtype presented a significantly higher number of NMS total scores (P = 0.007) and significantly higher score in the cognition domain of PDQ-39 (P = 0.021) than patients with TD subtype. Patients with NTD subtype had significantly higher NMS prevalence than patients with TD subtype with constipation (P = 0.021), incomplete bowel emptying (P = 0.050), anxiety (P = 0.030), daytime sleepiness (P = 0.017) and intense vivid dreams (P = 0.016). The predictors for the NTD subtype were intense vivid dreams (P = 0.012), anxiety (P = 0.030) and constipation (P = 0.044). The UPDRS, NMS total, and MMSE scores were the determinants of QOL in patients with PD (P < 0.05). The NMS is aprominent manifestation of PD. Patients with the NTD subtype had ahigher prevalence of NMS than those with the TD subtype and intense vivid dreams, anxiety and constipation were characteristic of the NTD subtype. NMS is one of the important factors that determine the QOL of patients with PD especially those with the NTD subtype.

NAADP-induced intracellular calcium ion is mediated by the TPCs (two-pore channels) in hypoxia-induced pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a form of obstructive vascular disease. Chronic hypoxic exposure leads to excessive proliferation of pulmonary arterial smooth muscle cells and pulmonary arterial endothelial cells. This condition can potentially be aggravated by [Ca2+] i mobilization. In the present study, hypoxia exposure of rat's model was established. Two‐pore segment channels (TPCs) silencing was achieved in rats' models by injecting Lsh‐TPC1 or Lsh‐TPC2. The effects of TPC1/2 silencing on PAH were evaluated by H&E staining detecting pulmonary artery wall thickness and ELISA assay kit detecting NAADP concentrations in lung tissues. TPC1/2 silencing was achieved in PASMCs and PAECs, and cell proliferation was detected by MTT and BrdU incorporation assays. As the results shown, NAADP‐activated [Ca2+] i shows to be mediated via two‐pore segment channels (TPCs) in PASMCs, with TPC1 being the dominant subtype. NAADP generation and TPC1/2 mRNA and protein levels were elevated in the hypoxia‐induced rat PAH model; NAADP was positively correlated with TPC1 and TPC2 expression, respectively. In vivo, Lsh‐TPC1 or Lsh‐TPC2 infection significantly improved the mean pulmonary artery pressure and PAH morphology. In vitro, TPC1 silencing inhibited NAADP‐AM‐induced PASMC proliferation and [Ca2+] i in PASMCs, whereas TPC2 silencing had minor effects during this process; TPC2 silencing attenuated NAADP‐AM‐ induced [Ca2+] i and ECM in endothelial cells, whereas TPC1 silencing barely ensued any physiological changes. In conclusion, TPC1/2 might provide a unifying mechanism within pulmonary arterial hypertension, which can potentially be regarded as a therapeutic target.

Reductive soil disinfestation attenuates antibiotic resistance genes in greenhouse vegetable soils

Reductive soil disinfestation (RSD) is an emerging technique that ameliorates soil degradation, but its effects against antibiotic resistance genes (ARGs) were unclear. Here, we examined soil properties, ARG types and numbers, and ARG profiles, and bacterial community compositions following 4 soil treatments: control; straw addition (SA); water flooding (WF); and RSD, both straw addition and water flooding. The results showed that the numbers of ARG types and subtypes decreased by 10.8% and 21.1%, respectively, after RSD, and the numbers of ARGs decreased by 18.6%. The attenuated multidrug, beta-lactam, macrolide, and phenicol resistance genes in the RSD soil corresponded to a decreased relative abundance of ARG subtypes (i.e., adeF, mdtM, TypeB_NfxB, mecA, nalC, OXA-60, and cmlA4). Taxa in phyla Proteobacteria, Actinobacteria, and Deinococcus-Thermus were the main hosts for dominant ARG subtypes and were inhibited by RSD. The selected bacterial genera and soil properties explained 83.4% of the variance in ARG composition, suggesting that the improved soil properties and the reduced potential ARG hosts produced by the interactions of straw addition and water flooding are likely responsible for ARG attenuation by RSD. Therefore, RSD has the potential to mitigate ARG pollution in soils.

Abstract 865: Genomic and evolutionary classification of lung cancer in never smokers from the Sherlock-Lung study

Abstract Lung cancer in never smokers (LCINS) is a common cause of cancer mortality, but is poorly characterized. Here we show, through high coverage whole genome sequencing of 232 tumors/blood pairs, that LCINS can be separated into three subtypes, defined by somatic copy number aberrations. While the dominant subtype (‘piano'), rarely found in lung cancer from smokers, is characterized by quiet copy number profiles, the other subtypes are associated with specific arm-level amplifications and EGFR mutations (‘mezzo-forte'), and whole genome doubling (‘forte'). Piano tumors feature somatic UBA1 mutations, germline AR variants, and stem cell-like properties, including low mutational burden, infrequent TP53 alterations, high intra-tumor heterogeneity, long telomeres, and slow growth as suggested by the occurrence of cancer driver genes decades prior to tumor diagnosis. In contrast, driver mutations in mezzo-forte and forte tumors are generally late clonal events acquired close to tumor diagnosis, thus potentially facilitating target identification with a single biopsy. The mutational signature associated with direct exposure to tobacco smoking (SBS4) was not observed in our study, even in cases with reported exposure to second-hand tobacco smoke. No significant differences of mutational spectra were found between passive and non-passive smokers. While genes within the RTK-RAS pathway have distinct impacts on survival, mutations in TP53, CHEK2, EGFR, or loss of 15q or 22q independently doubled mortality. These genomic alterations in forte and mezzo-forte subtypes, and stem cell-like features in piano tumors create avenues for personalized therapeutic strategies in LCINS. Citation Format: Tongwu Zhang, Ludmil B. Alexandrov, Wei Zhao, Jian Sang, Jianxin Shi, Bin Zhu, Dmitry A. Gordenin, Montserrat Garcia-Closas, Qing Lan, Nathaniel Rothman, Stephen J. Chanock, David C. Wedge, Maria Teresa Landi. Genomic and evolutionary classification of lung cancer in never smokers from the Sherlock-Lung study [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 865.

Oestrogen receptors: A potential therapeutic target in oesophageal adenocarcinoma?

Oesophageal cancer is the seventh most common cancer in the world and adenocarcinoma is the dominant subtype in Western industrialised nations. The global 5-year relative survival rate for oesophageal adenocarcinoma is 12%. Chemotherapy is a standard treatment offered to patients with both resectable and unresectable disease. However, there are only a few established chemotherapeutic drug options and progress in this area is limited. Recent efforts have focused on targeted molecular therapies. Epidemiological evidence points towards hormonal influences on disease development, particularly sex hormones. Several research studies have demonstrated oestrogen receptor (ER) expression in oesophageal adenocarcinoma tissue, making them a possible option for targeting with ER modulating agents. ERs are also present in laboratory models of the disease and experiments in ER-positive cell lines suggest that ER modulator therapy may be effective. A deeper understanding of the roles of ERα and ERβ in this disease would be valuable for future translation into clinical practice. In this review, we discuss the association between oestrogens and the development of oesophageal adenocarcinoma and the potential to modulate ER signalling networks for therapeutic benefit.

Abstract 669: A comprehensive single cell analysis of cancer-associated fibroblasts (CAFs) reveals subtypes with prognostic impact in pancreatic ductal adenocarcinoma

Abstract Introduction The presence of heterogeneity in cancer-associated fibroblasts (CAFs) of pancreatic ductal adenocarcinoma (PDAC) is well-known. The presence of mouse inflammatory CAFs (iCAFs) and myofibroblastic CAFs (myCAFs) were published earlier. Recently, we identified the first patient-derived sub-populations of CAFs (pCAFassigner) with different phenotypic and pathological characteristics. We demonstrated the presence of intra-tumoral heterogeneity in patients, differential sensitivity of CAF subtypes in co-cultures with cancer cells to existing PDAC treatment options and prognostic impact of these subtypes. The presence of antigen-presenting CAFs (apCAFs) in humans and mouse was introduced recently. However, a comprehensive, unbiased analysis is required to reconcile different CAF classifications and understand the prognostic significance of these subtypes for future clinical translation. Methods Mouse and human PDAC single-cell RNA sequencing (scRNA-seq)-based suite of analyses were performed to assign CAFs into pCAFassigner and other subtypes. Enrichment (hypergeometric) and differential gene expression (DGE) analyses were performed to identify sample- and gene-wise similarities between classifications, as well as develop and validate a new pCAFassigner gene signature. For prognostic impact, a published single sample classifier was used to identify a dominant CAF subtype in 1071 whole tumor samples, for which clinical information was available for 618 samples. The overall survival (OS) analysis was performed using Kaplan-Meier survival analysis. Results As expected, our analysis revealed the synonymity between subtype-A and myCAF (91 %), subtypes-B and -C with iCAF (69 %) and subtype D with apCAF (91 %, FDR &amp;lt; 0.001). We revised our pCAFassigner gene signature from a prediction analysis of microarrays (PAM) centroid to an 82 gene signature unique to each subtype. Given the similarities between these separate classifications, we sought to identify association of iCAF (subtype-B/C), apCAF (subtype-D) and myCAF (subtype-A) with survival. We observed a significant difference in OS with apCAF- and myCAF-dominant samples having the poorest prognosis, while patients with dominant iCAF had prolonged survival, questioning the understanding of iCAFs being pro-tumorigenic given its secretory nature and consequently, its presumed association with poor survival. Conclusions Our results indicate that while methodologies to derive each CAF classification is distinct, ultimately, they have proven to be similar. Further, we reveal prognostic impact of iCAFs, apCAFs and myCAFs, supporting the use of these CAF subtypes as independent prognostic indicators for resected PDAC and will aid better management of the disease. Citation Format: Aasia Hussain, Jiahui Ji, Prashant K. Srivastava, Anguraj Sadanandam. A comprehensive single cell analysis of cancer-associated fibroblasts (CAFs) reveals subtypes with prognostic impact in pancreatic ductal adenocarcinoma [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 669.

Abstract 967: Phosphodiesterase 4D depletion enhances anti-tumor effects of tyrosine kinase inhibitor in renal cell carcinoma involving CRAF-ERK pathway

Abstract Clear cell renal cell carcinoma (ccRCC) is the most lethal form of kidney cancer. Small molecule Vascular Endothelial Growth Factor Receptor (VEGFR) inhibitors are widely used but are not curative and various resistant mechanisms have been described. Previous studies have indicated a crosstalk between the cAMP pathway and the MAPK pathway, and the elevation of cAMP levels by Phosphodiesterase 4 (PDE4) inhibition can result in growth arrest and/or cell death. In this study we focused on the effects of PDE4 in ccRCC. We showed for the first time that PDE4D is the dominant subtype of PDE4 in kidney. PDE4D depletion in ccRCC, Caki-1 cells, using CRISPR Knock Out (KO) enhanced sensitivity of Tyrosine Kinase Inhibitor (TKI), sorafenib and increased cytotoxicity. We also showed synergistic effects of PDE4 inhibitor, roflumilast and sorafenib, in increasing cytotoxicity of ccRCC in the absence of PDE4D. Our results further demonstrated that PDE4D deficiency increased intracellular cAMP levels and resulted in downregulation of MAPK but not PI3K/AKT signaling in a CRAF dependent manners. In conclusion, we provide a preclinical rationale for dual PDE4/VEGFR inhibition in patients with ccRCC. While the MAPK signaling pathway is activated in ccRCC, dual inhibitions may improve the anti-tumor effects in patients with proven CRAF signaling activation. Citation Format: Minghua Cao, Amrandra K. Ajay, Martin Gasser, Li-LI Hsiao, Ana Maria Waaga-Gasser. Phosphodiesterase 4D depletion enhances anti-tumor effects of tyrosine kinase inhibitor in renal cell carcinoma involving CRAF-ERK pathway [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 967.