Clonal Differences Research Articles

Effects of Reduced-Dose Anti-Human T-Lymphocyte Globulin on Overall and Donor-Specific T-Cell Repertoire Reconstitution in Sensitized Kidney Transplant Recipients.

BackgroundPre-sensitized kidney transplant recipients have a higher risk for rejection following kidney transplantation and therefore receive lymphodepletional induction therapy with anti-human T-lymphocyte globulin (ATLG) whereas non-sensitized patients are induced in many centers with basiliximab. The time course of lymphocyte reconstitution with regard to the overall and donor-reactive T-cell receptor (TCR) specificity remains elusive.Methods/DesignFive kidney transplant recipients receiving a 1.5-mg/kg ATLG induction therapy over 7 days and five patients with 2 × 20 mg basiliximab induction therapy were longitudinally monitored. Peripheral mononuclear cells were sampled pre-transplant and within 1, 3, and 12 months after transplantation, and their overall and donor-reactive TCRs were determined by next-generation sequencing of the TCR beta CDR3 region. Overall TCR repertoire diversity, turnover, and donor specificity were assessed at all timepoints.ResultsWe observed an increase in the donor-reactive TCR repertoire after transplantation in patients, independent of lymphocyte counts or induction therapy. Donor-reactive CD4 T-cell frequency in the ATLG group increased from 1.14% + -0.63 to 2.03% + -1.09 and from 0.93% + -0.63 to 1.82% + -1.17 in the basiliximab group in the first month. Diversity measurements of the entire T-cell repertoire and repertoire turnover showed no statistical difference between the two induction therapies. The difference in mean clonality between groups was 0.03 and 0.07 pre-transplant in the CD4 and CD8 fractions, respectively, and was not different over time (CD4: F(1.45, 11.6) = 0.64 p = 0.496; CD8: F(3, 24) = 0.60 p = 0.620). The mean difference in R20, a metric for immune dominance, between groups was -0.006 in CD4 and 0.001 in CD8 T-cells and not statistically different between the groups and subsequent timepoints (CD4: F(3, 24) = 0.85 p = 0.479; CD8: F(1.19, 9.52) = 0.79 p = 0.418).ConclusionReduced-dose ATLG induction therapy led to an initial lymphodepletion followed by an increase in the percentage of donor-reactive T-cells after transplantation similar to basiliximab induction therapy. Furthermore, reduced-dose ATLG did not change the overall TCR repertoire in terms of a narrowed or skewed TCR repertoire after immune reconstitution, comparable to non-depletional induction therapy.

Abstract P1-04-04: Dna barcoding reveals ongoing immunoediting of clonal cancer populations during metastatic progression and in response to immunotherapy

Abstract As cancers develop and spread they must continually evade immune destruction. Understanding mechanisms of immune evasion in cancer is clinically significant as demonstrated with the successes of immune checkpoint inhibitors. Breast cancer is known to be highly immune evasive and responds poorly to the current immunotherapies, indicating alternative immune pathways must be targeted. We hypothesise that there are unidentified genetic mechanisms that enable immune evasion in breast cancer. We aim to uncover and target these mechanisms to sensitise immune evasive breast cancer cells to immune destruction in the context of immunotherapy treatment. DNA barcoding technology offers a new approach to understanding immune evasion. By stably integrating a unique DNA barcode sequence into each cell, we can study clonal immune evasion in vivo. Using this technology, we identified cancer cell clones from the 4T1 murine mammary carcinoma cell line that are highly enriched in lung metastases following treatment with combination immunotherapy (anti-CTLA-4 plus anti-PD-1). We isolated these specific immune evasive clones and established them as clonal cell lines. We have identified stark clonal differences in both PD-L1 and MHC I expression at both the RNA and protein level, and shown that MHC I expression is only partially controlled by epigenetic mechanisms. In addition, immune evasive subclones co-cultured with stimulated T cells resulted in less activated T cells than their less evasive counterparts. Furthermore, RNA sequencing of these clones has identified a gene signature that is strongly associated with decreased survival in both the METABRIC and TCGA cohorts. We have demonstrated ongoing immunoediting in the 4T1 model in vivo, both during metastasis and immunotherapy treatment. We have also identified subclonal populations of cells within a single tumour utilising different mechanisms of immune evasion. RNA sequencing has revealed a gene signature strongly associated with poor survival of basal-like breast cancer in two cohorts. Further pathway-level analysis of the resulting gene signature is required to elucidate the drivers of this aggressive and immune evasive phenotype. By targeting newly identified mechanisms of immune evasion in combination with current immunotherapies, we hope to improve the long-term survival of breast cancer patients. Citation Format: Louise A Baldwin, Nenad Bartonicek, Jessica Yang, Sunny Z Wu, Niantao Deng, Daniel Roden, Chia-Ling Chan, Ghamdan Al-Eryani, Damien J Zanker, Belinda S Parker, Alexander Swarbrick, Simon Junankar. Dna barcoding reveals ongoing immunoediting of clonal cancer populations during metastatic progression and in response to immunotherapy [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-04-04.

The Relationship Between Convergent IGH Signatures and Severity of COVID-19 Patients by Next-Generation Sequencing of B-Cell Repertoire.

ObjectTo reveal convergent IGH signatures and the association with severity of coronavirus disease 2019 (COVID-19) patients.MethodA total of 25 COVID-19 inpatients were classified into three clinical conditions: mild, severe, and critical. We analyzed convergent IGH signatures by ImmuHub® B-cell receptor (BCR) profiling system.ResultsIGH singleton frequency in patients is significantly lower than that of healthy donors (HDs). The clonality index of IGH in patients is significantly higher than that in HDs. Nevertheless, no significant difference was observed among the three groups. The difference in IGH clonality (top five clones) between post- and pretreatment was significant in the improvement and deterioration groups. Three common public motifs were shared by all COVID-19 patients: ARDYGG, RWYFDY, and YYYYGMDV.ConclusionB cells could recognize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and produce clonal expansion. Patients who had better outcomes after treatment had higher IGH clonality. Three common public motifs—ARDYGG, RWYFDY, and YYYYGMDV—might be used for vaccine development (ChiCTR2000029626).

Landscape of Stress Response and Virulence Genes Among Listeria monocytogenes Strains.

The pathogenic microorganism Listeria monocytogenes is ubiquitous and responsible for listeriosis, a disease with a high mortality rate in susceptible people. It can persist in different habitats, including the farm environment, the food production environments, and in foods. This pathogen can grow under challenging conditions, such as low pH, low temperatures, and high salt concentrations. However, L. monocytogenes has a high degree of strain divergence regarding virulence potential, environmental adaption, and stress response. This review seeks to provide the reader with an up-to-date overview of clonal and serotype-specific differences among L. monocytogenes strains. Emphasis on the genes and genomic islands responsible for virulence and resistance to environmental stresses is given to explain the complex adaptation among L. monocytogenes strains. Moreover, we highlight the use of advanced diagnostic technologies, such as whole-genome sequencing, to fine-tune quantitative microbiological risk assessment for better control of listeriosis.

Changes in T-cell subsets and clonal repertoire during chemoimmunotherapy with pembrolizumab and paclitaxel or capecitabine for metastatic triple-negative breast cancer

BackgroundChemoimmunotherapy is a standard treatment for triple-negative breast cancer (TNBC), however, the impacts of different chemotherapies on T-cell populations, which could correlate with clinical activity, are not known. Quantifying T-cell...

Variation in Growth, Biomass Production and its Components in Eucalyptus Clones

Eucalypts are among the most widely cultivated forest trees in the world under a range of different climates for products that include pulp, paper fuel wood and solid wood products such as poles, furniture and construction timber. Large scale plantations have been raised in government owned and private farm lands; the planting is continuing. Productivity and profitability of plantations of Eucalyptus have been revolutionized with the development of genetically improved, fast growing and high yielding clonal planting stock of Eucalypts. Eucalypts clonal planting has been said to have advantages which includes quick provision of benefits associates with fast growth, short rotation for production of pulp wood (of around 70 t ha -1 in 6 years) ready marketing and easy establishment and less maintenance needs. Clonal planting one among the approach for management of water and nutrients compared to the other conventional strategies. Studies relating to clonal difference and evaluation for dry matter production will help to overcome productivity loss due to deficit rainfall and optimum utilization of available natural resources for higher wood production. The present study was carried out to test the hypothesis that there exists a clonal variation in growth, biomass production and components and the present study gives an insight in to clonal variation with reference to growth, biomass production and components.

The Spatial Heterogeneity in Newly Diagnosed Multiple Myeloma Patients - from Sub-Clonal Architecture to the Immune Microenvironment

The Spatial Heterogeneity in Newly Diagnosed Multiple Myeloma Patients - from Sub-Clonal Architecture to the Immune Microenvironment

Stress survival islets contribute to clonal and serotype-specific differences in L. monocytogenes

Listeria monocytogenes is an important opportunistic foodborne pathogen causing listeriosis, an often fatal infection leading to meningitis, sepsis, or infection of the fetus and abortion in susceptible individuals. Diverse ready-to-eat food (RTE) like dairy, meat, fish, vegetables, and complex foods are often linked with listeriosis outbreaks. L. monocytogenes is capable of surviving in stressful environmental conditions and grow in refrigerated foods. Regarding stress-related genes, SSI-1 contributes to the survival of cells under suboptimal conditions, such as high salt content and acidic environment. At the same time, SSI-2 is responsible for persistence under alkaline and oxidative stresses.

Clonal variation in coppiced and uncoppiced growth, root sprout stem formation, and biomass partitioning in Salix interior on two highly disturbed site types

Salix interior Rowlee (INT) is a wide-ranging North American willow from the small taxonomic group Salix section Longifoliae, notable for its ability to form multi-stemmed vegetative stem colonies arising via root sprouts (RS) from a shallow horizontal root network. This study quantifies biomass production for both 1-year-old coppiced plants and the original 4-year-old plants established from dormant, unrooted stem cuttings, as well as for the RS stems associated with each ortet (original mother plant) using eight selected INT clones established on two distinct site types. Significantly greater coppiced and uncoppiced ortet stem dry mass was recorded on coarse-textured, shale rock overburden (SO); possibly due to significantly greater fertility, compared to adjacent gravel outwash deposits (GD), which had greater RS stem mass. The total stem dry mass (ortet + associated RS) was comparable across the two different site types for both the 1-year-old coppiced plants and the 4-year-old uncoppiced plants, showing rapid recovery of biomass growth and yield in the year following coppicing. Significant clonal differences, as well as site type × clone interactions, were found for ortet stem dry mass, especially on SO sites, indicating that clonal selection might be useful to increase biomass production on different site types. When expressed as a fraction of total stem dry mass produced on 2 m × 2 m biomass plots, the RS component represented a significant 57% of total stem dry mass per plot (ortet + associated RS) on GD sites and only 18% on SO sites. The use of colony-forming willows such as INT minimizes the need for periodic replanting, providing a cost advantage over conventional short-rotation, coppice-based woody biomass plantations using species that do not have the ability to produce new stems via root sprouting. This root-sprouting trait enables INT to rapidly colonize and spread on difficult-to-manage sites, making it suitable for use in land reclamation.

Nuaulu Use and Management of Culturally Salient Polymorphisms inCodiaeum variegatum: Explaining the Biocultural Dimensions of Leaf Variegation in a Southeast Asian Ornamental

Codiaeum variegatum has become a well-known ornamental plant in Europe and North America and has long been culturally significant in Southeast Asia and the Pacific, for example as a boundary plant. This paper asks, firstly, how variations in foliage are classified, managed, and valued in one population (Nuaulu on the island of Seram, eastern Indonesia), and how this relates to the range of uses to which these variations are put. Secondly, the paper suggests that this particular case helps shed light on the importance of leaf variegation as an organoleptic quality in the context of biocultural evolution. It is noted that the features that evolved in its area of endemism are those making it attractive as an ornamental globally. While color variations in foliage combining genotypic cultivar differences, clonal differences, and age-dependent differences produce phenotypic instability and are a problem for ornamental plant producers in a commercial context, they are not a problem for Nuaulu.

T-cell receptor pharmacodynamics associated with survival and response to tremelimumab (T) in combination with durvalumab (D) in patients (pts) with unresectable hepatocellular carcinoma (uHCC).

4087 Background: Study 22, a phase 2 clinical study (NCT02519348) evaluating T (anti-CTLA-4) and D (anti-PD-L1) as monotherapies and in combination indicated the best efficacy-safety profile with a novel combination regimen containing a single, priming dose of T (T300+D). Additionally, an expansion of proliferative CD8+ lymphocytes at Day 15 was observed with T300+D that was associated with improved response. Here, an exploratory molecular analysis of peripheral blood T cell receptors is presented. Methods: Immune-checkpoint inhibitor-naïve pts were randomized to 1 of 2 T+D combinations: T300+D (T 300 mg [1 dose] + D 1500 mg, then D every 4 weeks [Q4W]) or T75+D (T 75 mg Q4W + D 1500 mg Q4W [4 doses], then D Q4W); or single agent D (1500 mg Q4W) or T (750 mg Q4W [7 doses] then Q12W). DNA was isolated from PAXgene-preserved whole blood collected at baseline and on Day 29 during the first cycle of Q4W dosing, and then underwent CDR3 sequencing of T-cell receptor β using the immunoSEQ Assay (Adaptive Biotechnologies, Seattle, WA). Associations with objective response rate (ORR) and overall survival (OS) were evaluated. Results: The number of evaluable pts, samples, and overall ORR and OS are provided (Table). Immunosequencing analysis did not reveal significant differences in baseline T-cell clonality across arms. Increased T-cell clonal expansion at Day 29 appeared to be T dose dependent (Table), with no significant difference in the median expansion between the D and T75+D arms. Across all arms, responders had a larger median number of expanded T-cell clones on Day 29 than nonresponders (77.5 vs 40), and this greater expansion trended with longer OS (Table). Further evaluation by arm demonstrated an increase in T-cell clonal expansion in responders vs nonresponders in the T300+D arm. Pts with T-cell expansion above the median in the T300+D and T75+D arms also exhibited longer OS. Both newly expanded and total expanded clones on Day 29 vs Day 1 were associated with improved OS. Conclusions: The observed T dose-dependent increase in T-cell clonal expansion trended with improved ORR and longer OS, with the greatest overall benefit seen with T300+D vs T75+D, D and T. This is consistent with the previously reported observation that T300+D led to the highest median proliferating CD8+ T-cell counts and radiographic response. Further work is needed to differentiate the relative contributions of CD4 and CD8 clonal expansion to increased efficacy. T300+D and D are being evaluated in the phase 3 HIMALAYA study (NCT03298451) in uHCC vs sorafenib. Funding: AstraZeneca. Clinical trial information: NCT02519348. [Table: see text]

Phenotypic Characteristics of the Tumour Microenvironment in Primary and Secondary Hepatocellular Carcinoma.

Simple SummaryHow liver cancer changes as it progresses from where it initially arises, to metastatic sites distant from the liver, is unclear. We obtained a unique set of paired samples from patients, both from the original site and from metastases. We compared the mutation burden, transcriptional profile, and immune cell infiltrate between primary and secondary samples. We found that liver cancer metastases retain the ability to exclude the immune system from the tumour core as they spread.(1) Background: The intra-tumoural heterogeneity (ITH) of hepatocellular carcinoma (HCC) and its microenvironment (TME) across primary and secondary disease is poorly characterised. (2) Methods: Intra-tumoural (IT) and peri-tumoural (PT) staining of matched primary and secondary samples was conducted to evaluate the distribution of CD4+/FOXP3+ and CD8+/PD1+ T-cells. Samples underwent PD-L1/2 immunostaining, tumour mutational burden (TMB) evaluation, and high-resolution T-cell receptor (TCR) sequencing to derive T-cell clonality and targeted transcriptomics. (3) Results: We analysed 24 samples from matched primary (n = 11) and secondary (n = 13; 5 synchronous, 6 metachronous) deposits, 11 being extrahepatic (84.6%). IT CD8+ density was lower than PT in both primary (p = 0.005) and secondary deposits (p = 0.01), consistent with immune exclusion. PD-L1+ tumours displayed higher IT and PT CD8+/PD1+ cell density compared to PD-L1- (p < 0.05), and primary IT infiltrate was enriched in CD4+/FOXP3+ cells, compared to PT regions (p = 0.004). TCR-sequencing demonstrated enrichment of the top T-cell clonotype in secondary versus primary HCC (p = 0.02), without differences in overall productive clonality (p = 0.35). TMB was similar across primary versus secondary HCC (p = 0.95). While directed gene set analysis demonstrated the uniformity of transcriptional signatures of individual immune cell types, secondary deposits demonstrated higher COLEC12 (p = 0.004), CCL26 (p = 0.02), CD1E (p = 0.02) and CD36 (p = 0.03) expression with downregulation of CXCL1 (p = 0.03), suggesting differential regulation of innate immunity. (4) Conclusion: Immune exclusion is a defining feature of the HCC TME. Despite evidence of homogeneity in somatic TMB, secondary HCC is characterised by the expansion of a distinct T-cell clonotype and differential regulation of innate immune pathways.

How clonal differences and within-tree heterogeneity affect pore properties of hybrid aspen wood and biochar?

Production of applicable and homogeneous biochar for soil amendment purposes would benefit from knowledge on how feedstock heterogeneity impacts key biochar pore properties and how the properties are transformed due to pyrolysis. This study aimed (1) to quantify how clonal differences and within-tree heterogeneity of a hybrid aspen feedstock (wood) impact biochar pore properties and (2) to estimate how pore properties of non-pyrolysed wood materials are transformed when pyrolysed into biochar. The study was conducted by collecting samples from a hybrid aspen (Populus tremula L. × Populus tremuloides Michx.) clonal field trial. Key pore properties of non-pyrolysed and pyrolysed wood samples were quantified with 3D X-ray imaging and quantitative image analyses. The results demonstrated how pyrolysis shifted distinctively bi-modal pore size distributions of the wood samples towards smaller pore size regions. The bi-modal wood tissue structure controlled the pore structure also in the biochars. Due to decreasing cell wall thicknesses, the pyrolysis increased the porosity of the materials. While the thermal process homogenized differences in the wall thicknesses, the thicknesses of the feedstock were also shown to control the resulting thicknesses in the biochars. Mechanisms of biochar pore property formation can be considered important when designing applicable biochars for a chosen purpose. Clonal differences and within-tree heterogeneity had a direct impact only on the wall thicknesses and the pore diameters of vessels. These impacts can be of interest when planning feedstock utilization in biochar production. However, the results suggest that relatively homogeneous biochar can be produced from hybrid aspen feedstocks.

Characteristics of T-Cell Receptor Repertoire and Correlation With EGFR Mutations in All Stages of Lung Cancer.

Lung cancer is the leading cause of cancer-related deaths worldwide, and its occurrence is related to the accumulation of gene mutations and immune escape of the tumor. Sequencing of the T-cell receptor (TCR) repertoire can reveal the immunosurveillance status of the tumor microenvironment, which is related to tumor escape and immunotherapy. This study aimed to determine the characteristics and clinical significance of the TCR repertoire in lung cancer. To comprehensively profile the TCR repertoire, results from high-throughput sequencing of samples from 93 Chinese patients with lung cancer were analyzed. We found that the TCR clonality of tissues was related to smoking, with higher clonality in patients who had quit smoking for less than 1 year. As expected, TCR clonality was correlated with stages: patients with stage IV disease showed higher clonality than others. The correlation between TCR repertoire and epidermal growth factor receptor (EGFR) status was also investigated. Patients with EGFR non-L858R mutations showed higher clonality and a lower Shannon index than other groups, including patients with EGFR L858R mutation and wild-type EGFR. Furthermore, we analyzed the TCR similarity metrics—that is, the TCR shared between postoperative peripheral blood and tissue of patients with non-distant metastasis of lung cancer. A similar trend was found, in which patients with EGFR L858R mutations had lower overlap index (OLI) and Morisita index (MOI) scores. Moreover, the OLI showed a positive correlation with several clinical characteristics, including the tumor mutational burden of tissues and the maximum somatic allele frequency of blood; OLI showed a negative correlation with the ratio of CD4+CD28+ in CD4+ cells and the ratio of CD8+CD28+ in CD8+ cells. In conclusion, TCR clonality and TCR similarity metrics correlated with clinical characteristics of patients with lung cancer. Differences in TCR clonality, Shannon index, and OLI across EGFR subtypes provide information to improve understanding about varied responses to immunotherapy in patients with different EGFR mutations.

Comparison of the Initial Growth of Different Poplar Clones on Four Sites in Western Slovakia\u2014Preliminary Results

This study was conducted to evaluate four hybrid poplar comparison tests along a groundwater availability gradient in Western Slovakia. The weather fluctuation during the 3-year study period was described with indices, such as the Forestry Aridity Index (FAI) or the hydrothermal coefficient (HTC). The soil chemical and physical parameters were determined from soil samples from the two upper horizons. The nutrient status and supply of the trees were categorized based on leaf elemental analysis. Altogether, 21 different clones from 6 genomic groups were compared. The survival (SRV), diameter at breast height (DBH), and height of the trees (H) had been measured annually since the plantations were established, and from these measurements, mean annual height increment (MAHI) values were derived. These weather, edaphic, and clonal factors were evaluated and compared. Significant effects of the site (edaphic factors) were found as the primary source of variance and clonal differences as secondary sources of variance among the growth of trees. The interaction of site × clone effects was not significant. The results showed that for short rotation forestry (SRF), the site parameters—especially groundwater availability—are key factors.

English

Growth response of two clones of Manihot esculenta Crantz (Cassava), TMS 30555 and TMS 30572 to Bentex T soil treatment was studied. Mycorrhizal root colonization in relation to growth parameters such as stomata size, plant water content, plant foliation, as well as height and stem circumference was examined. Bentex T, a fungicide which could be used to limit the growth of Arbuscular Mycorrhizal (AM) fungi was added to soil at the concentrations of zero 0 (control), 50, 100, 500 and 1000 µg a.i. /g soil. Growth parameters had minimal variations (pEƒ 0.05) between treatments in both clones of the plant. However, clonal differences at (pE‚ 0.01) occurred in some of the growth parameters. The level of root colonization by the AM fungi affected the growth response of the plant. The untreated soil (control) with the highest AM fungi root colonization (84%) had the least plant foliation (15 and 16) and height (34.1 and 28.5 cm) for TMS 30572 and TMS 30555, respectively. The highest values obtained for stomata size (width and length) were at 50 µg/g bentex concentration; 0.040 and 0.019mm for TMS 30572 and 0.017 and 0.007 for TMS 30555, respectively. The least value obtained for the stomata size was at the zero (0) µg/g bentex concentration. Plants from soil treated with 100 µg/g bentex T concentration had the highest amount of water; 75% for TMS 30572 and 76% for TMS 30555. The untreated soil had plants with the least amount of water. Implications of Bentex T soil treatment of cassava plants was discussed in relation to mycorrhizal colonization rating and some growth parameters of the test plant. Key words: AM fungal colonization rating, growth response, cassava, Bentex T Soil Treatment

BIOM-17. BRAF MUTATION IS AN EARLY EVENT IN THE EVOLUTION OF A SUBSET OF GLIOBLASTOMAS AND IS ASSOCIATED WITH INCREASED PD-L1 EXPRESSION

Abstract INTRODUCTION BRAF is a RAF-family kinase that regulates MAPK/ERK signaling. Activating BRAF mutations, including V600E, are common in circumscribed low-grade gliomas of childhood and young adulthood, but are uncommon in infiltrative astrocytomas, including glioblastoma. Their role in glioblastoma initiation and progression requires analysis of large datasets given the low frequency (1.0%) in TCGA IDH-wild-type glioblastomas. METHODS Molecular profiling was done on 4679 FFPE gliomas by next-generation sequencing at Caris Life Sciences, of which 3170 underwent RNA-sequencing for gene fusion and 4603 DNA-sequencing for mutations. MGMT promoter methylation was tested by pyrosequencing and PD-L1 IHC was performed using the SP142 clone. RESULTS Excluding variants of uncertain significance, BRAF mutations/fusions were most common in pleomorphic xanthoastrocytoma (PXA; N=12/24, 50%), glioneuronal tumors (N=6/13, 46%), pilocytic astrocytoma (PA; N=15/48, 31%), and ganglioglioma (n=5/18, 28%). BRAF fusions were uncommon (N=17), most frequent in PA (N=8/31, 26%) where they were associated with older age at daignosis (P=0.043), and typically involved KIAA1549 as fusion partner (70%). BRAF-mutated/fused glioblastoma patients (N=59/3126, 2%) were younger than BRAF-wild-type glioblastoma patients (54 versus 59 years, P=3.5x10-3); more likely to be MGMT-unmethylated (75% versus 56%, P=5.0x10-3); and 3x more likely to express PD-L1 (55% versus 17%, P=2.1x10-10). In tumors harboring a V600E mutation, the variant allele frequency (VAF) was similar in glioblastoma as in PXA, PA, ganglioglioma, and glioneuronal tumors (median VAF=35%). CONCLUSIONS BRAF-mutated glioblastoma were 3x more likely to express PD-L1 than BRAF-wild-type glioblastoma. We observed no differences in BRAF V600E clonality in BRAF-mutated glioblastoma compared to BRAF-mutated PXA, PA, ganglioglioma, and glioneuronal tumors, suggesting BRAF mutation is an initiating event in the clonal evolution of a subset of glioblastoma. There is rationale to evaluate combined BRAF inhibition with checkpoint inhibition in BRAF-mutated glioblastoma, potentially synergizing the complete response profile of the former with the durable response profile of the latter.

Responses of isoprene emission and photochemical efficiency to severe drought combined with prolonged hot weather in hybrid Populus.

Isoprene emissions have been considered as a protective response of plants to heat stress, but there is limited information of how prolonged heat spells affect isoprene emission capacity, particularly under the drought conditions that often accompany hot weather. Under combined long-term stresses, presence of isoprene emission could contribute to the maintenance of the precursor pool for rapid synthesis of essential isoprenoids to repair damaged components of leaf photosynthetic apparatus. We studied changes in leaf isoprene emission rate, photosynthetic characteristics, and antioxidant enzyme activities in two hybrid Populus clones, Nanlin 1388 (relatively high drought tolerance) and Nanlin 895 (relatively high thermotolerance) that were subjected to long-term (30 d) soil water stress (25% versus 90% soil field capacity) combined with a natural heat spell (day-time temperatures of 35-40 °C) that affected both control and water-stressed plants. Unexpectedly, isoprene emissions from both the clones were similar and the overall effects of drought on the emission characteristics were initially minor; however, treatment effects and clonal differences increased with time. In particular, the isoprene emission rate only increased slightly in the Nanlin 895 control plants after 15 d of treatment, whereas it decreased by more than 5-fold in all treatment × clone combinations after 30 d. The reduction in isoprene emission rate was associated with a decrease in the pool size of the isoprene precursor dimethylallyl diphosphate in all cases at 30 d after the start of treatment. Net assimilation rate, stomatal conductance, the openness of PSII centers, and the effective quantum yield all decreased, and non-photochemical quenching and catalase activity increased in both control and water-stressed plants. Contrary to the hypothesis of protection of leaf photosynthetic apparatus by isoprene, the data collectively indicated that prolonged stress affected isoprene emissions more strongly than leaf photosynthetic characteristics. This primarily reflected the depletion of isoprene precursor pools under long-term severe stress.

Biomass allocation and nutrient content of hybrid aspen clones grown on former agricultural land in Finland

ABSTRACT Vegetatively propagated hybrid aspen (Populus tremula × P. tremuloides) is one of the fastest growing tree species in Finland. To study clonal variation in element concentrations in wood, bark and branches, and the amount of elements bound in above-ground biomass, seven clones were selected from a field trial in Southern Finland, when the trees were 13 years old. Significant clonal differences in the growth and allocation of biomass in stem wood, stem bark and branches were found. On average, the biomass consisted of 56% of stem wood, 10% of stem bark and 34% of branches. The clone with the highest total biomass production also had the highest fraction of biomass allocated to branches (43%). The study also revealed significant clonal differences in the concentrations of most of the studied elements in wood, bark and branches, and in the amounts of elements bound in the leafless above-ground biomass. For most of the elements, the highest concentrations were found in the stem bark followed by branches. Hybrid aspen bark was especially rich in Ca compared to some other broad-leaved tree species. There were also significant clonal differences in the amount of heavy metals (Cd, Cu and Zn) bound in the biomass.

Clonal evaluation of prostate cancer molecular heterogeneity in biopsy samples by dual immunohistochemistry and dual RNA in situ hybridization.

Prostate cancer is frequently multifocal. Although there may be morphological variation, the genetic underpinnings of each tumor are not clearly understood. To assess the inter and intra tumor molecular heterogeneity in prostate biopsy samples, we developed a combined immunohistochemistry and RNA in situ hybridization method for the simultaneous evaluation of ERG, SPINK1, ETV1, and ETV4. Screening of 601 biopsy cores from 120 consecutive patients revealed multiple alterations in a mutually exclusive manner in 37% of patients, suggesting multifocal tumors with considerable genetic differences. Furthermore, the incidence of molecular heterogeneity was higher in African Americans patients compared with Caucasian American patients. About 47% of the biopsy cores with discontinuous tumor foci showed clonal differences with distinct molecular aberrations. ERG positivity occurred in low-grade cancer, whereas ETV4 expression was observed mostly in high-grade cancer. Further studies revealed correlation between the incidence of molecular markers and clinical and pathologic findings, suggesting potential implications for diagnostic pathology practice, such as defining dominant tumor nodules and discriminating juxtaposed but molecularly different tumors of different grade patterns.