Abstract With the emergence of checkpoint blockade and other immunotherapeutic drugs, and the growing adoption of smaller, more flexible adaptive clinical trial designs, there is an unmet need to develop diagnostics that can rapidly immunophenotype patient tumors. The ability to longitudinally profile the tumor immune infiltrate in response to immunotherapy also presents a window of opportunity to illuminate mechanisms of resistance. We have developed a fine needle aspirate biopsy (FNA) platform to perform immune profiling on thoracic malignancies. Matching peripheral blood, bulk resected tumor, and FNA were analyzed from 13 mesothelioma patients. FNA samples yielded greater numbers of viable cells when compared to core needle biopsies. Cell numbers were adequate to perform flow cytometric analyses on T cell lineage, T cell activation and inhibitory receptor expression, and myeloid immunosuppressive checkpoint markers. FNA samples were representative of the tumor as a whole as assessed by head-to-head comparison to single cell suspensions of dissociated whole tumor. Parallel analysis of matched patient blood enabled us to establish quality assurance criteria to determine the accuracy of FNA procedures to sample tumor tissue. FNA biopsies provide a diagnostic to rapidly phenotype the tumor immune microenvironment that may be of great relevance to clinical trials.
<p>Supplemental Figure S6 shows clone family UMAPs, proportion change in response to drug, and gene signature set expression at untreated and acute drug treatment.</p>
Abstract Objectives To identify molecular characteristics of keratosis of unknown significance and to nominate pathways of molecular progression to oral cancer. Our work could provide a rationale for monitoring and treating these lesions definitively. Methods Patients with oral leukoplakia were eligible for our prospective observational study. We correlated alterations in cancer‐associated genes with clinical and histopathologic variables (keratosis of unknown significance vs. moderate‐to‐severe dysplasia) and compared these alterations to a previously molecularly characterized oral cancer population. Results Of 20 enrolled patients, 13 (65%) had evidence of keratosis of unknown significance, while seven (35%) had dysplasia. Nine patients (45%) developed oral cancer (4/13 with keratosis of unknown significance, 5/7 with dysplasia). At a median follow‐up of 67 (range 22–144) months, median overall survival was significantly shorter for patients with dysplasia (hazard ratio 0.11, p = .02). KMT2C and TP53 alterations were most frequent (75% and 35%, respectively). There were molecular similarities between keratosis of unknown significance and dysplasia patients, with no significant differences in mutational frequency among genes with ≥15% rate of alteration. Conclusions Among patients with leukoplakia, both patients with keratosis of unknown significance and patients with dysplasia developed oral cancer. Molecular alterations between these two groups were similar at this sample size.
<p>PDF file, 109K, Clonogenic survival of lung SqCC after radiation (S1); High-throughput profiling of lung SqCC after radiation (S2); Mutations in NFE2L2 and KEAP1 in lung SqCC cell lines (S3); MSigDB Gene Sets from Fig S5 (S4); TP53 alterations, LOH, and mRNA levels in lung SqCC cell lines (S5).</p>
<p>Table S5. Variants, copy number alterations and rearrangements identified in 24 samples by targeted next generation sequencing (NGS). In the copy number alterations column samples were classified based on gene copy number in two: (A) = amplified (> 2 copies of the gene) and (L) = Low (< 2 copies of the gene). Sample 2 is reported in the main text as CG-42.</p>
Abstract For a majority of patients with non–small cell lung cancer with EGFR mutations, treatment with EGFR inhibitors (EGFRi) induces a clinical response. Despite this initial reduction in tumor size, residual disease persists that leads to disease relapse. Elucidating the preexisting biological differences between sensitive cells and surviving drug-tolerant persister cells and deciphering how drug-tolerant cells evolve in response to treatment could help identify strategies to improve the efficacy of EGFRi. In this study, we tracked the origins and clonal evolution of drug-tolerant cells at a high resolution by using an expressed barcoding system coupled with single-cell RNA sequencing. This platform enabled longitudinal profiling of gene expression and drug sensitivity in response to EGFRi across a large number of clones. Drug-tolerant cells had higher expression of key survival pathways such as YAP and EMT at baseline and could also differentially adapt their gene expression following EGFRi treatment compared with sensitive cells. In addition, drug combinations targeting common downstream components (MAPK) or orthogonal factors (chemotherapy) showed greater efficacy than EGFRi alone, which is attributable to broader targeting of the heterogeneous EGFRi-tolerance mechanisms present in tumors. Overall, this approach facilitates thorough examination of clonal evolution in response to therapy that could inform the development of improved diagnostic approaches and treatment strategies for targeting drug-tolerant cells. Significance: The evolution and heterogeneity of EGFR inhibitor tolerance are identified in a large number of clones at enhanced cellular and temporal resolution using an expressed barcode technology coupled with single-cell RNA sequencing.
<p>Supplementary Figure 1. Shown in (A) are the IC50 values for the inhibition of proliferation for BRAF ( ), KRAS ( ), and NRAS ( ) mutant as well as "WT" ( ) cell lines. Below the graph Fischer's exact test using 1, 2, and 2.5ï�M sensitivity cutoffs are provided. (B) Comparison of cell line sensitivities for LXH254 and RAF709. LXH254 values are provided in this manuscript (Sup. Table 3) and RAF709 values obtained from (25). (C) mRNA expression (RNASeq, https://portals.broadinstitute.org/ccle/data) for cell lines presented in (A) with the 2.0ï�M LXH254 sensitivity threshold delineated. (D) ARAF mRNA expression in KRAS mutant and wild type cells from TCGA Pan-cancer Atlas Project (47), using the cBIO cancer genomics portal (https://www.cbioportal.org/, (1,2))</p>
