Clonal Differences Research Articles

Analysis of the clonal origin and differences in the biological behavior of multifocal papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) exhibits a trend of multifocal growth. However, the clonal origin of multiple cancer foci in the thyroid gland remains an issue of ongoing debate. In order to investigate the clonal origin and biological behavior differences of multifocal PTC (MPTC) from a unique perspective, a combination of dual gene and dual protein detection methods was used. The present study included 52 patients with MPTC. Immunohistochemical staining was used to assess the expression of v-raf murine sarcoma viral oncogene homolog B1 (BRAF) and telomerase reverse transcriptase (TERT) proteins, while quantitative PCR and Sanger sequencing were used to identify BRAF and TERT gene mutations. Based on the results, MPTC cases were classified into two clonal origins, namely intraglandular metastatic (71.2%) and independent multicentric origin (28.8%). BRAF protein expression and BRAF gene mutation were significantly higher in the intraglandular metastasis group than in the multicentric cancer group. However, no significant differences in TERT protein expression and TERT gene mutation were observed between the two groups. Sex, central lymph node metastasis rate, Hashimoto's thyroiditis and tumor distribution laterality were not found to differ significantly between the two groups. However, significant differences were detected in age at initial diagnosis, lateral cervical lymph node metastasis rate, tumor capsule invasion rate and maximum tumor diameter. The study found that MPTC predominantly occurs due to intraglandular metastasis, which is associated with stronger tumor invasiveness than cancer foci with multiple independent origins, as it is more likely to exhibit pathogenic gene mutations and abnormal protein expression, cervical lymph node metastasis and capsule invasion. Therefore, it is recommended that the surgical approaches and follow-up strategies for intraglandular metastatic MPTC should be aggressive and individualized.

Evaluation of MDR-specific phage Pɸ-Mi-Pa loaded mucoadhesive electrospun nanofibrous scaffolds against drug-resistant Pseudomonas aeruginosa- induced wound infections in an animal model

Given the escalating prevalence of drug-resistant wounds, there is a justified imperative to explore innovative and more efficacious therapies that diverge from conventional, ineffective wound healing approaches. This research has introduced a strategy to address multi-drug resistant (MDR) Pseudomonas aeruginosa infections in a chronic wound model, employing MDR-specific phage Pɸ-Mi-Pa loaded onto mucoadhesive electrospun scaffolds. A cocktail of three isolates of P. aeruginosa-specific lytic phages, Pɸ-Mi-Pa 51, Pɸ-Mi-Pa 120, and Pɸ-Mi-Pa 133 were incorporated into varying ratios of fabricated PCL-PVP polymer. These formulations were assessed for their therapeutic efficacy in achieving bacterial clearance in P. aeruginosa-induced wound infections. The study encompassed biological characterization through in vivo wound healing assessments, histology, and histomorphometry. Additionally, morphological, mechanical, and chemical analyses were conducted on the fabricated PCL-PVP electrospun nanofibrous scaffolds. Three clonal differences of the MDR P. aeruginosa-specific phages (Pɸ-Mi-Pa 51, Pɸ-Mi-Pa 120, and Pɸ-Mi-Pa 133) produced lytic activity and were seen to produce distinct and clear zones of inhibition against MDR P. aeruginosa strains Pa 051, Pa 120 and Pa 133 respectively. The average porosity of the nanofibrous scaffolds PB 1, PB 2, PB 3, and PB 4 were 12.2 ± 0.3 %, 22.1 ± 0.7 %, 31.1 ± 2.4 %, 28.0 ± 0.8 % respectively. In vitro cumulative release of MDR-specific phage Pɸ-Mi-Pa from the mucoadhesive electrospun nanofibrous scaffolds was found to be 70.91 % ± 1.02 % after 12 h of incubation after an initial release of 42.8 % ± 3.01 % after 1 h. Results from the in vivo wound healing study revealed a substantial reduction in wound size, with formulations PB 2 and PB 3 exhibiting the most significant reduction in wound size, demonstrating statistically significant results on day 5 (100 % ± 31.4 %). These findings underscore the potential of bacteriophage-loaded electrospun PCL-PVP nanofibrous scaffolds for treating drug-resistant wounds, generating tissue substitutes, and overcoming certain limitations associated with conventional wound care matrices.

Identification of Enhanced Vaccine Mimotopes for the p15E Murine Cancer Antigen.

The MHC-I-restricted p15E tumor rejection epitope is expressed in multiple murine cancer lines and is used as a marker of antitumor cellular immunity, but has seen limited success as a vaccine immunogen. An in vivo screening approach based on a positional peptide microlibraries is used to identify enhanced p15E mimotopes bearing amino acid mutations that induce significantly improved functional immunogenicity relative to vaccination with the wild-type epitope.

Clonal differences underlie variable responses to sequential and prolonged treatment

Cancer cells exhibit dramatic differences in gene expression at the single-cell level, which can predict whether they become resistant to treatment. Treatment perpetuates this heterogeneity, resulting in a diversity of cell states among resistant clones. However, it remains unclear whether these differences lead to distinct responses when another treatment is applied or the same treatment is continued. In this study, we combined single-cell RNA sequencing with barcoding to track resistant clones through prolonged and sequential treatments. We found that cells within the same clone have similar gene expression states after multiple rounds of treatment. Moreover, we demonstrated that individual clones have distinct and differing fates, including growth, survival, or death, when subjected to a second treatment or when the first treatment is continued. By identifying gene expression states that predict clone survival, this work provides a foundation for selecting optimal therapies that target the most aggressive resistant clones within a tumor. A record of this paper's transparent peer review process is included in the supplemental information.

Feasibility and performance of cross-clone Raman calibration models in CHO cultivation.

Raman spectroscopy is widely used in monitoring and controlling cell cultivations for biopharmaceutical drug manufacturing. However, its implementation for culture monitoring in the cell line development stage has received little attention. Therefore, the impact of clonal differences, such as productivity and growth, on the prediction accuracy and transferability of Raman calibration models is not yet well described. Raman OPLS models were developed for predicting titer, glucose and lactate using eleven CHO clones from a single cell line. These clones exhibited diverse productivity and growth rates. The calibration models were evaluated for clone-related biases using clone-wise linear regression analysis on cross validated predictions. The results revealed that clonal differences did not affect the prediction of glucose and lactate, but titer models showed a significant clone-related bias, which remained even after applying variable selection methods. The bias was associated with clonal productivity and lead to increased prediction errors when titer models were transferred to cultivations with productivity levels outside the range of their training data. The findings demonstrate the feasibility of Raman-based monitoring of glucose and lactate in cell line development with high accuracy. However, accurate titer prediction requires careful consideration of clonal characteristics during model development.

Single Cell Multi-Omic Correlation of Single Nucleotide Variants, Copy Number Variation and Surface Epitopes for Clonal Profiling of Myeloma

Introduction Multiple myeloma (MM) is a cancer of plasma cells with approximately 200,000 new diagnoses each year and a 54% 5-year overall survival rate. As myeloma cells expand, clonal genetic differences lead to relapse due to acquired resistance in 100% of patients, mandating regular, long-term surveillance. Improvements in precision medicine for MM have come from monoclonal antibodies and antibody-drug conjugates that specifically target the offending cells. However, while existing diagnostic modalities offer exquisite sensitivity, they are unable to correlate subclonal genetic changes and putative antibody targets on refractory myeloma cells, leaving the clinician to use their best judgment on salvage therapy which can lead to unnecessary expense and toxicity to patients due to ineffective treatment. Specifically knowing actual targets on refractory MM cells would better facilitate precision medicine and potentially improve outcomes. Methods Cryopreserved, human MM patient samples were processed on the Mission Bio Tapestri platform, enabling simultaneous single cell quantification of subclones by single nucleotide variants (SNV), copy number variants (CNV), and surface protein analysis. These single time point samples were thawed from frozen bone marrow mononuclear cells previously enriched for CD38, stained with a 45-plex antibody-oligo cocktail to label common heme-specific surface markers for sequencing analysis, and processed with a 733-plex DNA primer panel that combined whole-genome CNV coverage with MM gene hotspots. Sequencing was performed on an Illumina system with the raw data being analyzed using Mission Bio proprietary algorithms. Results We show complex clonal evolution of MM in individual samples as copy gains and losses that were sequentially acquired and correlated with expression changes of MM markers. Across the cases analyzed, complex branching phylogenetic trees were reconstructed with as many as five subclones and clear associated shifts in MM-marker expression. When averaged across subclones, each sample had CNV profiles that matched patient bulk genome-wide array records, ranging from a single gene-level copy gain to arm-level gains and losses across the majority. The progression of protein expression within different samples could be mapped together on a single plot and correlated with generally higher MM-markers as subclonal genetic variants were acquired, though occasional branches saw reversal. The fraction of other cell phenotypes, such as T-cells and those with low viability, decreased within subclones as mutational burden grew. Conclusion This high-resolution, single cell assay offers a potential new modality for the diagnosis and surveillance of patients with suspected MGUS, SGUS or high-risk MM. We have demonstrated: 1) exceptional results from cryopreserved human specimens, 2) the ability to use genetic lesion profiling to positively identify subclonal MM and, most importantly, 3) correlate cell surface protein expression of potential therapeutic targets with each clonal and subclonal population

Drought effects on water status, plant hydraulics and growth in Eucalyptus grandis and clonal hybrids with red gums and E. urophylla

Afforestation with Eucalyptus species occupies over a million hectares in Uruguay, and E. grandis is one of the most cultivated species, mainly for the timber industry. However, the increasing frequency of drought and heat waves in the region raises the need to evaluate hybrids with red gums (E. camaldulensis and E. tereticornis), since they are known to sustain gas exchange and growth even under severe drought conditions. Hybrids with E. urophylla are also evaluated for plantations in subtropical regions. This study aimed to compare the effects of drought on plant water status, hydraulics, and growth of E. grandis (one clone; G), E. grandis × camaldulensis (one clone; GC), E. grandis × tereticornis (one clone; GT), and E. grandis × urophylla (two clones; GU1 and GU2). Two drought-stress cycles were applied to six-month rooted cuttings from mid-spring to early fall under greenhouse environmental conditions. Predawn leaf water potential (?pd), midday leaf water potential (?md), relative leaf water content (RWC), stomatal conductance (gs), and plant growth were measured across each cycle. Daily fluctuation of Ψ was calculated as ΔΨ = Ψpd - Ψmd. Specific (kS) and leaf-specific (kL) hydraulic conductivities and specific leaf area (SLA) were measured at the end of the experiment. Percentage loss of hydraulic conductivity (PLC) was calculated after removing the xylem embolism. Preliminary results suggest that both red-gum hybrids had higher PLC under moderate-to-severe drought, as a consequence of maintaining high water potential gradients to sustain transpiration and carbon fixation throughout the experiment. Under the well-watered condition, no clonal difference was observed. Also, GT showed a three-fold increase in cavitation and had 1.5 times stomatal opening (gs) than the most drought-sensitive clone (E. grandis, G), and exhibited the lowest reduction (17%) in diameter growth under drought. Also, GC attained the highest height at the end of the study, 40% higher than E. grandis, and increased by 25% the SLA under drought. GU clones reached the highest diameter at the end of the study. Correlations among functional variables are explored, and the water supply capacity to the foliage among clones is discussed for a better understanding of drought resistance strategies and growth performance of the different taxa.

Clonal Targeting of DNA Damage Response Pathways Eradicates Myeloproliferative Neoplasms

Clonal diversity of myeloproliferative neoplasms (MPN) plays a key role in poor therapeutic outcomes. MPN cells usually accumulate spontaneous DNA damage including highly toxic DNA double-strand breaks (DSBs) induced by metabolic products and replication stress. To repair numerous DSBs and survive, MPN cells activate the DNA damage response (DDR) involving the pathways that sense (ATM and ATR kinases) and repair (RAD51-mediated homologous recombination = HR, RAD52-mediated transcription associated homologous recombination = TA-HR and single strand annealing = SSA, DNA-PK -mediated non-homologous end-joining = NHEJ, PARP1/Polq-dependent microhomology-mediated end-joining = MMEJ) DSBs. Thus, DDR is a legitimate therapeutic target. Numerous MPN-driving mutations [ JAK2(V617F), TETmut, DNMT3Amut, IDH1mut] regulate DDR and affect the sensitivity of leukemia cells to DDR inhibitors (DDRi). The genetic landscape of malignant clones in a patient may be complicated since individual clones can carry multiple mutations. Thus, individual MPN clones may respond differently to DDRi depending on their mutational profile. In our experimental protocol MPN cells from individual patients were treated with various DDRi followed by single-cell targeted DNA sequencing (sctDNA-seq) to integrate patient's leukemia clonal composition with response to the inhibitors. We developed a sctDNA-seq myeloid platform interrogating up to 1394 genetic variants of 54 known leukemia driver genes, which unraveled the clonal landscape of MPN at a single-cell resolution before and after the treatment. Based on these results we designed a “clonal attack”, a patient-tailored combination of DDRi targeting all MPN clones in a patient sample. Here we show that DDRi simultaneously attacking different clones caused MPN clonal attrition in vitro. For example, phylogenetic tree analysis of MPN patient P349 sample detected linear multi-clonal architecture with 3 Lin-CD34+ clones carrying specific sets of mutations. sctDNA-seq followed by fish plot analysis revealed clonal similarities and differences in response to DDRi. The clone carrying KMT2A(L2373H) + SETBP1(H1100R) was more sensitive to ATRi than RAD52i, conversely clones with KMT2A(L2373H) alone and KMT2A(L2373H) + SETBP1(H1100R) + FLT3(R834L) were more sensitive to RAD52i than ATRi. Based on this observation, we hypothesized that simultaneous treatment with ATRi+RAD52i should result in elimination of all 3 MPN P349 clones. Remarkably, the combination of RAD52i + ATRi was >100x more effective in inhibiting clonogenic growth of Lin-CD34+ P349 cells (all clonogenic cells were eradicated). On the other hand, combination of RAD52i + ATMi, the two DDR inhibitors displaying similar pattern of clonal targeting was only 2x better than individual inhibitors. Phylogenetic tree analysis of Lin-CD34+ cells from another MPN patient P350 sample showed linear multi-clonal architecture with 4 clones. Again, sctDNA-seq followed by fish plot analysis revealed clonal similarities and differences in response to DDRi. All 4 clones displayed similar sensitivity to RAD52i and ATMi, but they responded differently to PARPi and ATRi. Clones carrying TET2(P363L) + NRAS(G12D) were more sensitive to ATRi than PARPi, whereas clones with TET2(P363L) + NRAS(G12D) + DNMT3A(W330C) and TET2(P363L) + NRAS(G12D) + DNMT3A(W330C) + IDH2(R132C) responded better to PARPi than ATRi. Importantly, the combination of PARPi + ATRi was >9x more effective in inhibiting clonogenic growth of Lin-CD34+ P349 cells, whereas RAD52i + ATMi was only 2x better than individual inhibitors. In conclusion, we postulate that sctDNA-seq combined with in vitro DDRi sensitivity testing (sctDNA-seq/DDRi) is a powerful tool to interrogate clonal sensitivity of MPN to these agents. This clonal medicine approach may become a novel therapeutic regimen to overcome clonal complexity of MPN in a cohort of patients. The “clonal attack” by DDR inhibitors shifts the paradigm of genotoxic therapies from those using non-discriminative cytotoxic drugs to those selectively attacking DDR vulnerabilities in MPN clones with minimal harm to normal cells. Since clonal heterogeneity and DNA damage are hallmarks of cancer, the “clonal attack” may be broadly applicable to the quest for cancer cure.

Rooting dynamics of ‘kustee’ honeybush (Cyclopia genistoides) as affected by seasonality and the manipulation of stock plant characteristics

Honeybush (Cyclopia spp.), a legume endemic to the Fynbos biome of South Africa, has gained popularity as a herbal tea, particularly for its unique honey-like flavour, low tannin content and high antioxidant status. However, extensive wild-harvesting practices are a concern as this continued practice is placing pressure on natural stands. To ensure a sustainable supply of high-quality tea, a switch to clonal cultivation is required. In this study, the rooting potential of cuttings from four clonal types of Cyclopia genistoides was evaluated, following rejuvenation of stock plants over various regrowth periods, with cutting material collected over different seasons and phenological phases. The highest rooting success of >90 % was achieved following either six- or nine months of regrowth in winter and spring. Cutting dry weight positively correlated with rooting percentages in summer and autumn rooted cuttings. Extended periods of regrowth highlighted clonal differences in rooting success whilst no clonal differences occurred in shorter regrowth periods of less than a year. Cuttings (clones ‘GK3’ and ‘GK5’) made following regrowth of more than 12 months differed significantly, with ‘GK3’ rooting percentages that were lower in comparison to ‘GK5’ cutting material, when harvested in winter (30 %; 82 %) and the following autumn (37 %; 100 %), but were comparable when harvested in the preceding summer, autumn and spring. Stock plant phenology of C. genistoides at harvesting influenced rooting as low rooting percentages consistently coincided when the harvesting of cutting material coincided with the flowering period. The use of terminal cuttings collected from the current season's growth during a vegetative phase, following a six- or nine-month regrowth period, is recommended for C. genistoides stock plants to achieve acceptable to high rooting success in cutting material.

A Comparative Study between ‘Parson Brown’ and ‘Hamlin’ Sweet Oranges Growing under Endemic Huanglongbing Conditions in Florida

Citrus greening, or huanglongbing (HLB), caused by the phloem-limited bacterium Candidatus Liberibacter asiaticus (CaLas), threatens the global citrus industry. Field observations have demonstrated that some citrus cultivars are more tolerant to the CaLas pathogen than others. ‘Parson Brown’ is an early maturing sweet orange variety that has consistently exhibited minimal leaf and fruit drop in the field compared with the ‘Hamlin’ sweet orange under similar conditions. This study aimed to understand performance of the ‘Parson Brown’ cultivar in several locations across the citrus production regions of Florida. Results indicated that the CaLas bacterial titer in both cultivars were similar with the quantitative polymerase chain reaction cycle threshold values ranging between 24.99 and 28.61 in ‘Hamlin’ and between 25.48 and 30.89 in ‘Parson Brown’. Leaves from the ‘Parson Brown’ trees however demonstrated higher chlorophyll content and total phenolic compounds in most of the locations. We also detected higher relative expression of CsPR1 and CsPR2 transcripts in ‘Parson Brown’ leaves in the first sampling period (March) and the fourth period (November). Additionally, Phloem protein 2 transcripts were downregulated in ‘Parson Brown’ leaves compared with ‘Hamlin’ at all locations. The ‘Hamlin’ juice had higher acidity, whereas ‘Parson Brown’ juice demonstrated a higher Brix to acidity ratio and juice color. The oil content in the juice ranged between 0.020% and 0.042%, and there was variation in the oil content between the locations, which could indicate clonal differences. ‘Parson Brown’ juice however contained higher limonin and nomilin content than ‘Hamlin’ juice in most of the locations. Taken together, the current results confirmed the enhanced tolerance of ‘Parson Brown’ trees to HLB when compared with ‘Hamlin’ in all sampled locations.

P18.04.B SINGLE CELL TRANSCRIPTOMIC ANALYSIS IDENTIFIES DISTINCT INTRATUMORAL SUBCLONES IN MENINGIOMA

Abstract BACKGROUND Meningiomas arise from the leptomeninges of the brain and spinal cord. Despite being the most common intracranial tumor in adults, risk stratification remains challenging. Currently, tumors are graded based on histology as well as molecular markers. In recent years, several epigenomic classification systems with a correlation to patient outcome have been proposed for meningiomas. However, these classification systems do not discriminate between effects of the tumor microenvironment and the tumor itself or consider distinct intratumoral subpopulations with different phenotypes. To address this, we set out to investigate intratumoral differences and correlated them with gene expression differences. MATERIAL AND METHODS We applied single nuclei RNA sequencing (10X Genomics 3’) to 30 meningioma samples to obtain transcriptomic profiles on single cell level. Copy number variations were estimated for single cells based on the transcriptomic profiles to identify clonal differences within a given tumor. RESULTS Based on aberrations in the copy number profile, we were able to identify distinct subclones within the same tumor, which could in many cases be connected to a change in the transcriptomic profile of the respective subclone. Different patterns of subclonal diversity were observed between tumor grades. Further insight into the subpopulations was used to infer a novel model for prognostication which was validated by methylation data. CONCLUSION Altogether, these results demonstrate that diverse subclones co-exist within meningioma, especially within higher-grade tumors. Effects of meningioma subclones on tumor growth and their relevance for clinical risk prediction remain to be elucidated.

Bulk T-cell receptor sequencing confirms clonality in pediatric eosinophilic esophagitis and identifies a food-specific repertoire.

Eosinophilic esophagitis (EoE) involves a chronic immune-mediated response to dietary antigens. Recent work identifies T-cell clonality in children with EoE, however, it is unknown whether this is true in adults or whether there is a restricted food-specific T-cell repertoire. We sought to confirm T-cell receptor (TCR) clonality in EoE and assess for differences with specific food triggers. Bulk TCR sequencing was performed on mRNA isolated from esophageal biopsies obtained from adults and children with EoE (n = 15) who had food triggers confirmed by endoscopic evaluation. Non-EoE adult and pediatric controls (n = 10) were included. Differences in TCR clonality by disease and treatment status were assessed. Shared and similar V-J-CDR3s were assessed based on specific food triggers. Active EoE biopsies from children but not adults displayed decreased unique TCRα/β clonotypes and increased relative abundance of TCRs comprising >1% of the total compared to non-EoE controls and paired inactive EoE samples. Among patients in which baseline, post diet elimination, and food trigger reintroduction samples (n = 6) were obtained, we observed ~1% of TCRs were shared only between pre-diet elimination and trigger reintroduction. Patients with a shared EoE trigger (milk) had a greater degree of shared and similar TCRs compared to patients with differing triggers (seafood, wheat, egg, soy). We confirmed relative clonality in children but not adults with active EoE and identified potential food-specific TCRs, particularly for milk-triggered EoE. Further studies are needed to better identify the broad TCR repertoire relevant to food triggers.

ESBL-Producing and Non-ESBL-Producing Escherichia coli Isolates from Urinary Tract Differ in Clonal Distribution, Virulence Gene Content and Phylogenetic Group.

The objectives of this study are to determine the differences in clonality, virulence gene (VG) content and phylogenetic group between non extended-spectrum beta-lactamase-producing E. coli (non-ESBL-EC) and ESBL-EC isolates from urine. This study characterized a total of 100 clinical E. coli isolates consecutively obtained from the inpatients hospitalized in The First Affiliated Hospital of Ningbo University in China by polymerase-chain reaction (PCR). Phylogenetic group B2 was found to be the most prevalent in both ESBL-EC and non-ESBL-EC group. Among 100 clinical isolates, the count of acquired virulence genes in group B2 was found to be significantly higher than that in group A, B1, and D (p <0.001). Additionally, the presence of content within virulence genes (the total number of virulence genes detected per isolate) in B2 of non-ESBL-EC and ESBL-EC showed a significant difference (p<0.001). ST131 was detected exclusively in ESBL-EC, while ST95 and ST73 were the main sequence types in non-ESBL-EC. Our study demonstrated the different distribution of MLST, phylogenetic group in ESBL-EC and non-ESBL-EC group. The inverse association between beta-lactamase resistance and VG content performed in this study should get a lot more attention. At the same time, we should also be wary of the appearance of non-ESBL-EC isolates of group B2 harboring more virulence genes which will lead to high pathogenicity.

Population dynamics of Zeuzera spp. (Lepidoptera: Cossidae) on Eucalyptus pellita plantation in Central Kalimantan, Indonesia

Abstract. Suheri M, Haneda NF, Anwar R, Jung Y, Sukeno S, Park J. 2022. Population dynamics of Zeuzera spp. (Lepidoptera: Cossidae) on Eucalyptus pellita plantation in Central Kalimantan, Indonesia. Biodiversitas 23: 5782-5789. The red coffee borer, Zeuzera spp. (Lepidoptera: Cossidae) damage to Eucalyptus pellita forests was one of the problems in the forestry sector. The population dynamic of these insects is monitored and surveyed on E. pellita at PT Korintiga Hutani. The present study aimed to analyze the contributing factors of Zeuzera spp. damage to E. pellita plantation and observe the population dynamics of Zeuzera spp. using pheromone traps. The survey results showed that the accumulated damage in planting block 5 was more serious (29%) than in planting block 6 (9%). The impact of the multi-regression analysis indicates that the main factors affecting the damage are the location and age of the plantation. At the same time, the E. pellita clonal difference is minor compared to those two factors. The damage is more severe and occurs at an older age of plantations. The population dynamics of Zeuzera spp. observed in E. pellita plantation showed a peak flight trend in October, followed by a reasonably high population presence from late May to early June. This information could be a primary consideration for these insect pest need to control the significant damage to E. pellita trees and determine the management of Zeuzera spp. based on the timing of larvae infestation in the upcoming after-peak flight.

Abstract PO002: Pretreatment immune cell composition and checkpoint ligand expression define the response to immunotherapy in advanced HCC: a study using single-cell sequencing

Abstract The novel combination of atezolizumab/bevacizumab has demonstrated unprecedented response rates in a subgroup of advanced hepatocellular carcinoma (aHCC) patients. In order to understand why only a subset of aHCC patients respond to immunotherapy targeting the PD-1/PD-L1 axis, we subjected pre-treatment tissue biopsies from 31 aHCC patients, with mainly non-viral etiologies, to simultaneous single-cell transcriptome (scRNAseq) and T-cell receptor (scTCRseq) sequencing, providing an integrative view of the tumor-microenvironment of aHCC (n=91 169 cells). The therapeutic targets, PD-1 and PD-L1 are expressed in tumor-infiltrating T-cells versus myeloid cells and tumor cells, respectively. Myeloid cells in tumors responding to immunotherapy (according to mRECIST) express significantly higher levels of PD-L1 (p=0.014) compared to non-responders and this is driven by pro-inflammatory PD-L1 expressing CXCL10+ macrophages, involved in T-cell recruitment. In contrast, non-responding tumors are infiltrated by immature CX3CR1+ macrophages and CCR2+ ‘monocyte-like’ macrophages (p=0.009 and p=0.01, respectively). Interestingly, the composition of tumor-infiltrating T-cells did not differ significantly between patient groups. However, responding tumors were characterized by a more clonal baseline T-cell receptor repertoire (p=0.007). Single-cell T-cell receptor sequencing revealed that this clonality was present in activated, effector T-cells, while non-responders had a higher proportion of non-clonal T-cells (p=0.01) concentrated within naïve and memory-like T-cell phenotypes. In conclusion, this is the first and largest aHCC cohort to correlate single-cell sequencing data with clinical outcomes. Prior to treatment, differences in abundancy and ligand expression in small clusters of immune cells as well as difference in baseline T-cell clonality drive response to checkpoint inhibition in aHCC. Citation Format: Sarah Cappuyns, Gino Philips, Vincent Vandecaveye, Chris Verslype, Eric Van Cutsem, Diether Lambrechts, Jeroen Derkervel. Pretreatment immune cell composition and checkpoint ligand expression define the response to immunotherapy in advanced HCC: a study using single-cell sequencing [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO002.

Abstract PR03: Pretreatment immune cell composition and checkpoint ligand expression define the response to immunotherapy in advanced HCC: a study using single-cell sequencing

Abstract The novel combination of atezolizumab/bevacizumab has demonstrated unprecedented response rates in a subgroup of advanced hepatocellular carcinoma (aHCC) patients. In order to understand why only a subset of aHCC patients respond to immunotherapy targeting the PD-1/PD-L1 axis, we subjected pre-treatment tissue biopsies from 31 aHCC patients, with mainly non-viral etiologies, to simultaneous single-cell transcriptome (scRNAseq) and T-cell receptor (scTCRseq) sequencing, providing an integrative view of the tumor-microenvironment of aHCC (n=91 169 cells). The therapeutic targets, PD-1 and PD-L1 are expressed in tumor-infiltrating T- cells versus myeloid cells and tumor cells, respectively. Myeloid cells in tumors responding to immunotherapy (according to mRECIST) express significantly higher levels of PD-L1 (p=0.014) compared to non-responders and this is driven by pro-inflammatory PD-L1 expressing CXCL10+ macrophages, involved in T-cell recruitment. In contrast, non-responding tumors are infiltrated by immature CX3CR1+ macrophages and CCR2+ ‘monocyte-like’ macrophages (p=0.009 and p=0.01, respectively). Interestingly, the composition of tumor-infiltrating T-cells did not differ significantly between patient groups. However, responding tumors were characterized by a more clonal baseline T-cell receptor repertoire (p=0.007). Single-cell T-cell receptor sequencing revealed that this clonality was present in activated, effector T-cells, while non-responders had a higher proportion of non-clonal T-cells (p=0.01) concentrated within naïve and memory-like T-cell phenotypes. In conclusion, this is the first and largest aHCC cohort to correlate single-cell sequencing data with clinical outcomes. Prior to treatment, differences in abundancy and ligand expression in small clusters of immune cells as well as difference in baseline T-cell clonality drive response to checkpoint inhibition in aHCC. Citation Format: Sarah Cappuyns, Gino Philips, Vincent Vandecaveye, Chris Verslype, Eric Van Cutsem, Diether Lambrechts, Jeroen Derkervel. Pretreatment immune cell composition and checkpoint ligand expression define the response to immunotherapy in advanced HCC: a study using single-cell sequencing [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PR03.

Peripheral blood T-cell receptor repertoire profiling of advanced non-small cell lung cancer patients receiving PD-1/PD-L1 treatment.

e21004 Background: Whether peripheral blood T cell receptor (TCR) repertoire profiling can serve as a biomarker to predict clinical benefit from anti-PD-1/PD-L1 checkpoint immunotherapy is not well understood. Moreover, it is not known which methods for TCR repertoire analysis are most clinically meaningful. To address this, we have performed TCR repertoire analysis of patients with advanced/metastatic non-small cell lung cancer (NSCLC) receiving PD-1/PD-L1 treatment. Methods: We analyzed 29 patients receiving PD-1/PD-L1 monotherapy or combination therapy in any line of treatment, excluding patients with EGFR mutations or Alk alterations. Genomic DNA was extracted from peripheral blood examples, and CDR3 regions of the rearranged TCR beta genes were amplified and sequenced using the immunoSEQ platform (Adaptive Biotechnologies, Seattle, WA). Libraries were sequenced using Illumina HiSeq 2500. TCR clonality, diversity, evenness, and the percentage of high-frequency clones (&gt; 0.1% of the repertoire) were calculated at pre-treatment and post-treatment (1 – 3 months) timepoints. Morisita’s overlap index was calculated for 25 paired pre- and post-treatment samples. Metrics were compared in patients with and without durable clinical response at 6 months using the Mann-Whitney test. Results: There were no statistically significant differences in TCR repertoire clonality, diversity, evenness, or high-frequency clones between patients with and without durable clinical benefit, either at pre-treatment or post-treatment time points (p &gt; 0.05). Conclusions: TCR repertoire metrics including clonality, diversity, evenness, percentage of high-frequency clones, and Morisita’s overlap index do not predict immunotherapy responses in this cohort of advanced/metastatic NSCLC patients receiving PD-1/PD-L1 monotherapy or combination therapy. Limitations of this study include sample size and heterogeneity of the patient cohort. This highlights the need for advanced TCR repertoire metrics, for example, considering shared TCR specificities and clonal identity. In ongoing work, we are defining TCR specificity groups and TCR clones in NSCLC as biomarkers of immunotherapy responsiveness.

Clonal and Diversity Differences of Extravascular B cells in the Influenza-Experienced Lung

Abstract Memory B cells are a vital arm of the antiviral response. Recent literature has characterized a population of murine lung resident memory B cells after influenza A virus (IAV) infection capable of in situ differentiation into anti-influenza antibody secreting cells. Separately, PDL2+/CD80+ B cells appear to have a “memory-like” phenotype while the double-negative counterparts are “naïve-like” with low mutation frequency and class-switching. Additionally, our group has shown that PDL2+ EBCs impact Streptococcus pneumoniae immunity but their role in anti-viral immunity is understudied. While extravascular B cells (EBCs) may play a critical immune function, it’s unclear if the PDL2+/CD80+ population is clonally distinct from the PDL2-/CD80- one as diversity can impact immune capabilities. We hypothesize PDL2+/CD80+ EBCs have a signature clonal repertoire influenced by infection history and tissue localization. We infected BALB/cJ mice twice with related IAVs separated by four weeks. After a six-week rest period, we isolated PDL2-/CD80- and PDL2+/CD80+ EBCs from the lung and spleen. Immunoglobulin sequencing (IgSeq) was performed on IgM and IgG libraries that were analyzed with in-house clonal partitioning software (Cloanalyst). There was significant increase in the number of EBCs in the lung and the proportion of PDL2+/CD80+ EBCs increased dramatically. Mutation frequency of PDL2+/CD80+ EBCs within the IAV-exposed lung rose, suggesting a role of tissue localization on B cell signature. Furthermore, diversity analysis using Shannon and Simpson estimators suggests differences between tissues and PDL2/CD80 expression. Together, our data highlight a unique clonal niche occupied by PDL2+/CD80+ EBCs. Supported by grants from NIH (R33 HL-137081, T32 AI-007309)

The Effects of Frost Cracks and Large Poplar Borer Damage on Stem Rot in Hybrid Aspen (Populus tremula L. × Populus tremuloides Michx.) Clones

Hybrid aspen (Populus tremula L. × Populus tremuloides Michx.) plantations may produce valuable sawlogs for the growing timber market and contribute to carbon sequestration. However, environmental risks such as stem rot, the spread of which is facilitated by insect or frost damage, may reduce the proportion of valuable timber. It is important to understand the various factors affecting the spread of aspen rot to mitigate negative impacts with tree breeding. This study aimed to assess the impact of frost cracks and large poplar borer on stem rot in hybrid aspen clones in two clonal trials in Latvia. Genetic parameters for the traits were also estimated. The presence of insect passages substantially increased the probability of stem rot without distinct clonal differences. A negative and mainly insignificant correlation was observed between rot and stem cracking. The highest broad-sense heritability (H2 = 0.21) and strong site-site genotypic correlation (0.86) showed that the probability of stem rot is genetically determined in the study material. Significant differences in diameter at breast height, the presence of stem rot, and its severity were found among the clones, albeit without undesirable positive correlation between growth and presence of decay. This indicated its potential to improve both productivity and rot resistance.

Clonal differences in nitrogen use efficiency and macro-nutrient uptake in young clonal cocoa (Theobroma cacao L.) seedlings from Indonesia

Nitrogen (N) is one of the most required nutrients in cocoa production. Unlike in many other crops, there is limited knowledge on genotypic variation in nitrogen use efficiency (NUE) in clonal cocoa. We therefore evaluated the effect of nitrogen application on cocoa NUE in 10 clonal cocoa scions that were top-grafted on 4-month old rootstock seedlings of cocoa clone MCC 02. Grafted seedlings received either 0 or 2.3 g/plant of N at the beginning of the experiment, which lasted for five months and was arranged in a nursery of the Indonesian Coffee and Cocoa Research Institute (ICCRI) in Jember, Indonesia. A factorial randomized block design was used with four replications. We found that clones significantly affected rootstock stem diameter and dry biomass both in roots and shoots. Nitrogen application decreased rootstock stem diameter and root biomass but increased shoot-root ratio. Following the N application, we observed significantly higher N concentration and uptake both in roots and shoots. This higher N concentration and uptake contributed to significantly lower NUE in both roots and shoots. We did not observe clonal differences in N concentration, uptake, NUE in either roots or shoots and ANR (Apparent Nitrogen Recovery). Nitrogen application influenced P, K, Mg, and Ca uptake in both roots and shoots. There was a clonal effect on P, K and Mg uptake and on P and Mg use efficiency in both roots and shoots. This implies that the studied clones vary in their P and Mg nutrient use efficiency but not in NUE.