The genomic landscape of lung cancer in never-smokers from the Women's Health Initiative.

Over 200,000 individuals are diagnosed with lung cancer in the U.S. every year, with a growing proportion of cases, especially lung adenocarcinoma, occurring in individuals who have never smoked. Women over the age of 50 comprise the largest affected demographic. To understand the genomic drivers of lung adenocarcinoma and therapeutic response in this population, we performed whole genome and/or whole exome sequencing on 73 matched lung tumor/normal pairs from post-menopausal women who participated in the Women's Health Initiative. Somatic copy number alterations showed little variation by smoking status, suggesting that aneuploidy may be a general characteristic of lung cancer regardless of smoke exposure. Similarly, clock-like and APOBEC mutation signatures were prevalent but did not differ in tumors from smokers and never-smokers. However, mutations in both EGFR and KRAS showed unique allelic differences determined by smoking status that are known to alter tumor response to targeted therapy. Mutations in the MYC-network member MGA were more prevalent in tumors from smokers. Fusion events in ALK, RET, and ROS1 were absent, likely due to age-related differences in fusion prevalence. Our work underscores the profound impact of smoking status, age, and sex on the tumor mutational landscape and identifies areas of unmet medical need.

Specification of human regional brain lineages using orthogonal gradients of WNT and SHH in organoids.

The repertory of neurons generated by progenitor cells depends on their location along antero-posterior and dorso-ventral axes of the neural tube. To understand if recreating those axes was sufficient to specify human brain neuronal diversity, we designed a mesofluidic device termed Duo-MAPS to expose induced pluripotent stem cells (iPSC) to concomitant orthogonal gradients of a posteriorizing and a ventralizing morphogen, activating WNT and SHH signaling, respectively. Comparison of single cell transcriptomes with fetal human brain revealed that Duo-MAPS-patterned organoids generated the major neuronal lineages of the forebrain, midbrain, and hindbrain. Morphogens crosstalk translated into early patterns of gene expression programs predicting the generation of specific brain lineages. Human iPSC lines from six different genetic backgrounds showed substantial differences in response to morphogens, suggesting that interindividual genomic and epigenomic variations could impact brain lineages formation. Morphogen gradients promise to be a key approach to model the brain in its entirety.

Evaluating performance and applications of sample-wise cell deconvolution methods on human brain transcriptomic data.

Sample-wise deconvolution methods estimate cell-type proportions and gene expressions in bulk tissue samples, yet their performance and biological applications remain unexplored, particularly in human brain transcriptomic data. Here, nine deconvolution methods were evaluated with sample-matched data from bulk tissue RNA sequencing (RNA-seq), single-cell/nuclei (sc/sn) RNA-seq, and immunohistochemistry. A total of 1,130,767 nuclei per cells from 149 adult postmortem brains and 72 organoid samples were used. The results showed the best performance of dtangle for estimating cell proportions and bMIND for estimating sample-wise cell-type gene expressions. For eight brain cell types, 25,273 cell-type eQTLs were identified with deconvoluted expressions (decon-eQTLs). The results showed that decon-eQTLs explained more schizophrenia GWAS heritability than bulk tissue or single-cell eQTLs did alone. Differential gene expressions associated with Alzheimer's disease, schizophrenia, and brain development were also examined using the deconvoluted data. Our findings, which were replicated in bulk tissue and single-cell data, provided insights into the biological applications of deconvoluted data in multiple brain disorders.

Molecular phenotyping of small cell lung cancer using targeted cfDNA profiling of transcriptional regulatory regions.

We report an approach for cancer phenotyping based on targeted sequencing of cell-free DNA (cfDNA) for small cell lung cancer (SCLC). In SCLC, differential activation of transcription factors (TFs), such as ASCL1, NEUROD1, POU2F3, and REST defines molecular subtypes. We designed a targeted capture panel that identifies chromatin organization signatures at 1535 TF binding sites and 13,240 gene transcription start sites and detects exonic mutations in 842 genes. Sequencing of cfDNA from SCLC patient-derived xenograft models captured TF activity and gene expression and revealed individual highly informative loci. Prediction models of ASCL1 and NEUROD1 activity using informative loci achieved areas under the receiver operating characteristic curve (AUCs) from 0.84 to 0.88 in patients with SCLC. As non-SCLC (NSCLC) often transforms to SCLC following targeted therapy, we applied our framework to distinguish NSCLC from SCLC and achieved an AUC of 0.99. Our approach shows promising utility for SCLC subtyping and transformation monitoring, with potential applicability to diverse tumor types.

Interneuron loss and microglia activation by transcriptome analyses in the basal ganglia of Tourette syndrome.

Tourette syndrome (TS) is a disorder of high-order integration of sensory, motor, and cognitive functions afflicting as many as 1 in 150 children and characterized by motor hyperactivity and tics. Despite high familial recurrence rates, a few risk genes and no biomarkers have emerged as causative or predisposing factors. The syndrome is believed to originate in basal ganglia, where patterns of motor programs are encoded. Postmortem immunocytochemical analyses of brains with severe TS revealed decreases in cholinergic, fast-spiking parvalbumin, and somatostatin interneurons within the striatum (caudate and putamen nuclei). Here, we performed single cell transcriptomic and chromatin accessibility analyses of the caudate nucleus from 6 adult TS and 6 control post-mortem brains. The data reproduced the known cellular composition of the adult human striatum, including a majority of medium spiny neurons (MSN) and small populations of GABAergic and cholinergic interneurons. Comparative analysis revealed that interneurons were decreased by roughly 50% in TS brains, while no difference was observed for other cell types. Differential gene expression analysis suggested that mitochondrial function, and specifically oxidative metabolism, in MSN and synaptic function in interneurons are both impaired in TS subjects. Furthermore, such an impairment was coupled with activation of immune response pathways in microglia. Also, our data explicitly link gene expression changes to changes in cis-regulatory activity in the corresponding cell types, suggesting de-regulation as a factor for the etiology of TS. These findings expand on previous research and suggest that impaired modulation of striatal function by interneurons may be the origin of TS symptoms.

Characterization of enhancer activity in early human neurodevelopment using Massively Parallel Reporter Assay (MPRA) and forebrain organoids.

Regulation of gene expression through enhancers is one of the major processes shaping the structure and function of the human brain during development. High-throughput assays have predicted thousands of enhancers involved in neurodevelopment, and confirming their activity through orthogonal functional assays is crucial. Here, we utilized Massively Parallel Reporter Assays (MPRAs) in stem cells and forebrain organoids to evaluate the activity of ~ 7000 gene-linked enhancers previously identified in human fetal tissues and brain organoids. We used a Gaussian mixture model to evaluate the contribution of background noise in the measured activity signal to confirm the activity of ~ 35% of the tested enhancers, with most showing temporal-specific activity, suggesting their evolving role in neurodevelopment. The temporal specificity was further supported by the correlation of activity with gene expression. Our findings provide a valuable gene regulatory resource to the scientific community.

CD8+ chimeric antigen receptor T cells manufactured in absence of CD4+ cells exhibit hypofunctional phenotype.

BACKGROUND: Cell culture conditions during manufacturing can impact the clinical efficacy of chimeric antigen receptor (CAR) T cell products. Production methods have not been standardized because the optimal approach remains unknown. Separate CD4+ and CD8+ cultures offer a potential advantage but complicate manufacturing and may affect cell expansion and function. In a phase 1/2 clinical trial, we observed poor expansion of separate CD8+ cell cultures and hypothesized that coculture of CD4+ cells and CD8+ cells at a defined ratio at culture initiation would enhance CD8+ cell expansion and simplify manufacturing. METHODS: We generated CAR T cells either as separate CD4+ and CD8+ cells, or as combined cultures mixed in defined CD4:CD8 ratios at culture initiation. We assessed CAR T cell expansion, phenotype, function, gene expression, and in vivo activity of CAR T cells and compared these between separately expanded or mixed CAR T cell cultures. RESULTS: We found that the coculture of CD8+ CAR T cells with CD4+ cells markedly improves CD8+ cell expansion, and further discovered that CD8+ cells cultured in isolation exhibit a hypofunctional phenotype and transcriptional signature compared with those in mixed cultures with CD4+ cells. Cocultured CAR T cells also confer superior antitumor activity in vivo compared with separately expanded cells. The positive impact of CD4+ cells on CD8+ cells was mediated through both cytokines and direct cell contact, including CD40L-CD40 and CD70-CD27 interactions. CONCLUSIONS: Our data indicate that CD4+ cell help during cell culture maintains robust CD8+ CAR T cell function, with implications for clinical cell manufacturing.