Showing papers by "Bruce E. Johnson published in 2020"

A single-cell and single-nucleus RNA-Seq toolbox for fresh and frozen human tumors

Abstract: Single-cell genomics is essential to chart tumor ecosystems. Although single-cell RNA-Seq (scRNA-Seq) profiles RNA from cells dissociated from fresh tumors, single-nucleus RNA-Seq (snRNA-Seq) is needed to profile frozen or hard-to-dissociate tumors. Each requires customization to different tissue and tumor types, posing a barrier to adoption. Here, we have developed a systematic toolbox for profiling fresh and frozen clinical tumor samples using scRNA-Seq and snRNA-Seq, respectively. We analyzed 216,490 cells and nuclei from 40 samples across 23 specimens spanning eight tumor types of varying tissue and sample characteristics. We evaluated protocols by cell and nucleus quality, recovery rate and cellular composition. scRNA-Seq and snRNA-Seq from matched samples recovered the same cell types, but at different proportions. Our work provides guidance for studies in a broad range of tumors, including criteria for testing and selecting methods from the toolbox for other tumors, thus paving the way for charting tumor atlases.

A single-cell landscape of high-grade serous ovarian cancer.

Abstract: Malignant abdominal fluid (ascites) frequently develops in women with advanced high-grade serous ovarian cancer (HGSOC) and is associated with drug resistance and a poor prognosis1 To comprehensively characterize the HGSOC ascites ecosystem, we used single-cell RNA sequencing to profile ~11,000 cells from 22 ascites specimens from 11 patients with HGSOC We found significant inter-patient variability in the composition and functional programs of ascites cells, including immunomodulatory fibroblast sub-populations and dichotomous macrophage populations We found that the previously described immunoreactive and mesenchymal subtypes of HGSOC, which have prognostic implications, reflect the abundance of immune infiltrates and fibroblasts rather than distinct subsets of malignant cells2 Malignant cell variability was partly explained by heterogeneous copy number alteration patterns or expression of a stemness program Malignant cells shared expression of inflammatory programs that were largely recapitulated in single-cell RNA sequencing of ~35,000 cells from additionally collected samples, including three ascites, two primary HGSOC tumors and three patient ascites-derived xenograft models Inhibition of the JAK/STAT pathway, which was expressed in both malignant cells and cancer-associated fibroblasts, had potent anti-tumor activity in primary short-term cultures and patient-derived xenograft models Our work contributes to resolving the HSGOC landscape3-5 and provides a resource for the development of novel therapeutic approaches

Genomic characterization of human brain metastases identifies drivers of metastatic lung adenocarcinoma

Abstract: Brain metastases from lung adenocarcinoma (BM-LUAD) frequently cause patient mortality. To identify genomic alterations that promote brain metastases, we performed whole-exome sequencing of 73 BM-LUAD cases. Using case-control analyses, we discovered candidate drivers of brain metastasis by identifying genes with more frequent copy-number aberrations in BM-LUAD compared to 503 primary LUADs. We identified three regions with significantly higher amplification frequencies in BM-LUAD, including MYC (12 versus 6%), YAP1 (7 versus 0.8%) and MMP13 (10 versus 0.6%), and significantly more frequent deletions in CDKN2A/B (27 versus 13%). We confirmed that the amplification frequencies of MYC, YAP1 and MMP13 were elevated in an independent cohort of 105 patients with BM-LUAD. Functional assessment in patient-derived xenograft mouse models validated the notion that MYC, YAP1 or MMP13 overexpression increased the incidence of brain metastasis. These results demonstrate that somatic alterations contribute to brain metastases and that genomic sequencing of a sufficient number of metastatic tumors can reveal previously unknown metastatic drivers.

Expansion Sequencing: Spatially Precise In Situ Transcriptomics in Intact Biological Systems

Abstract: Methods for highly multiplexed RNA imaging are limited in spatial resolution, and thus in their ability to localize transcripts to nanoscale and subcellular compartments. We adapt expansion microscopy, which physically expands biological specimens, for long-read untargeted and targeted in situ RNA sequencing. We applied untargeted expansion sequencing (ExSeq) to mouse brain, yielding readout of thousands of genes, including splice variants and novel transcripts. Targeted ExSeq yielded nanoscale-resolution maps of RNAs throughout dendrites and spines in neurons of the mouse hippocampus, revealing patterns across multiple cell types; layer-specific cell types across mouse visual cortex; and the organization and position-dependent states of tumor and immune cells in a human metastatic breast cancer biopsy. Thus ExSeq enables highly multiplexed mapping of RNAs, from nanoscale to system scale. One Sentence Summary In situ sequencing of physically expanded specimens enables multiplexed mapping of RNAs at nanoscale, subcellular resolution.

Natural Language Processing to Ascertain Cancer Outcomes From Medical Oncologist Notes.

Abstract: PURPOSECancer research using electronic health records and genomic data sets requires clinical outcomes data, which may be recorded only in unstructured text by treating oncologists. Natural langua...

Concurrent osimertinib plus gefitinib for first-line treatment of EGFR-mutated non-small cell lung cancer (NSCLC).

Abstract: 9507Background: First-line treatment with an EGFR tyrosine kinase inhibitor (TKI) is standard of care for patients (pts) with EGFR-mutated NSCLC. The EGFR TKI osimertinib is active against the acqu...

Updated overall survival (OS) and genomic analysis from a single-arm phase II study of dabrafenib (D) plus trametinib (T) in patients (pts) with BRAF V600E mutant (Mut) metastatic non-small cell lung cancer (NSCLC).

Abstract: 9593Background: The phase II multicenter, open label study, which evaluated efficacy and safety of D+T in pretreated (cohort B) and treatment (tx)-naive (cohort C) pts with BRAF V600E mut metastati...

Transcriptional subtype-specific microenvironmental crosstalk and tumor cell plasticity in metastatic pancreatic cancer

Abstract: In pancreatic ductal adenocarcinoma (PDAC), the basal-like and classical transcriptional subtypes are associated with differential chemotherapy sensitivity and patient survival. These phenotypes have been defined using bulk transcriptional profiling, which can mask underlying cellular heterogeneity and the biologic mechanisms that distinguish these subtypes. Furthermore, few studies have interrogated metastases, which are the cause of mortality in most patients with this highly lethal disease. Using single-cell RNA-sequencing of metastatic needle biopsies and matched organoid models, we demonstrate intra-tumoral subtype heterogeneity at the single-cell level and define a continuum for the basal-like and classical phenotypes that includes hybrid cells that co-express features of both states. Basal-like tumors show enrichment of mesenchymal and stem-like programs, and demonstrate immune exclusion and tumor cell crosstalk with specific macrophage subsets. Conversely, classical tumors harbor greater immune infiltration and a relatively pro-angiogenic microenvironment. Matched organoid models exhibit a strong bias against the growth of basal-like cells in standard organoid media, but modification of culture conditions can rescue the basal-like phenotype. This study reframes the transcriptional taxonomy of PDAC, demonstrates how divergent transcriptional subtypes associate with unique tumor microenvironments, and highlights the importance of evaluating both genotype and transcriptional phenotype to establish high-fidelity patient-derived cancer models.

Author Correction: A single-cell and single-nucleus RNA-Seq toolbox for fresh and frozen human tumors.

Abstract: An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Multi-modal pooled Perturb-CITE-Seq screens in patient models define novel mechanisms of cancer immune evasion

Abstract: Resistance to immune checkpoint inhibitors (ICI) that activate T cell mediated anti-tumor immunity is a key challenge in cancer therapy, yet the underlying mechanisms remain poorly understood. To further elucidate those, we developed a new approach, Perturb-CITE-seq, for pooled CRISPR perturbation screens with multi-modal RNA and protein single-cell profiling readout and applied it to screen patient-derived autologous melanoma and tumor infiltrating lymphocyte (TIL) co-cultures. We profiled RNA and 20 surface proteins in over 218,000 cells under ~750 perturbations, chosen by their membership in an immune evasion program that is associated with immunotherapy resistance in patients. Our screen recovered clinically-relevant resistance mechanisms concordantly reflected in RNA, protein and perturbation effects on susceptibility to T cell mediated killing. These were organized in eight co-functional modules whose perturbation distinctly affect four co-regulated programs associated with immune evasion. Among these were defects in the IFNγ-JAK/STAT pathway and in antigen presentation, and several novel mechanisms, including loss or downregulation of CD58, a surface protein without known mouse homolog. Leveraging the rich profiles in our screen, we found that loss of CD58 did not compromise MHC protein expression and that CD58 was not transcriptionally induced by the IFNγ pathway, allowing us to distinguish it as a novel mechanism of immune resistance. We further show that loss of CD58 on cancer cells conferred immune evasion across multiple T cell and Natural Killer cell patient co-culture models. Notably, CD58 is downregulated in tumors with resistance to immunotherapy in melanoma patients. Our work identifies novel mechanisms at the nexus of immune evasion and drug resistance and provides a general framework for deciphering complex mechanisms by large-scale perturbation screens with multi-modal singlecell profiles, including in systems consisting of multiple cell types.

Clinical Pan-Cancer Assessment of Mismatch Repair Deficiency Using Tumor-Only, Targeted Next-Generation Sequencing

Abstract: PURPOSEGiven regulatory approval of immune checkpoint inhibitors in patients with mismatch repair–deficient (MMR-D) cancers agnostic to tumor type, it has become important to characterize occurrenc...

From clinical specimens to human cancer preclinical models-a journey the NCI-cell line database-25 years later

Abstract: The intramural the National Cancer Institute (NCI) and more recently the University of Texas Southwestern Medical Center with many different collaborators comprised a complex, multi-disciplinary team that collaborated to generated large, comprehensively annotated, cell-line related research resources which includes associated clinical, and molecular characterization data. This material has been shared in an anonymized fashion to accelerate progress in overcoming lung cancer, the leading cause of cancer death across the world. However, this cell line collection also includes a range of other cancers derived from patient-donated specimens that have been remarkably valuable for other types of cancer and disease research. A comprehensive analysis conducted by the NCI Center for Research Strategy of the 278 cell lines reported in the original Journal of Cellular Biochemistry Supplement, documents that these cell lines and related products have since been used in more than 14 000 grants, and 33 207 published scientific reports. This has resulted in over 1.2 million citations using at least one cell line. Many publications involve the use of more than one cell line, to understand the value of the resource collectively rather than individually; this method has resulted in 2.9 million citations. In addition, these cell lines have been linked to 422 clinical trials and cited by 4700 patents through publications. For lung cancer alone, the cell lines have been used in the research cited in the development of over 70 National Comprehensive Cancer Network clinical guidelines. Finally, it must be underscored again, that patient altruism enabled the availability of this invaluable research resource.

The impact of quantitative CT-based tumor volumetric features on the outcomes of patients with limited stage small cell lung cancer.

Abstract: Limited stage small cell lung cancer (LS-SCLC) has a poor prognosis. Additional prognostic markers are needed for risk-stratification and treatment intensification. This study compares quantitative CT-based volumetric tumor measurements versus International Association for the Study of Lung Cancer (IASLC) TNM staging to predict outcomes. A cohort of 105 patients diagnosed with LS-SCLC and treated with chemoradiation (CRT) from 2000 to 2013 were analyzed retrospectively. Patients were staged by the Union for International Cancer Control (UICC) TNM Classification, 8th edition. Tumor volumes and diameters were extracted from radiation planning CT imaging. Univariable and multivariable models were used to analyze relationships between CT features and overall survival (OS), locoregional recurrence (LRR), in-field LRR, any progression, and distant metastasis (DM). Median follow-up was 21.3 months. Two-year outcomes were as follows: 38% LRR, 31% in-field LRR, 52% DM, 62% any progression, and 47% OS (median survival 16.5 months). On univariable analysis, UICC T-stage and N-stage were not associated with any clinical outcome. UICC overall stage was only statistically associated with in-field LRR. One imaging feature (3D maximum tumor diameter) was found to be significantly associated with LRR (HR 1.10, p = 0.003), in-field LRR (HR 1.10, p = 0.007), DM (HR 1.10, p = 0.02), any progression (HR 1.10, p = 0.008), and OS (HR 1.10, p = 0.03). On multivariable analysis, this feature remained significantly associated with all outcomes. For LS-SCLC, quantitative CT-based volumetric tumor measurements were significantly associated with outcomes after CRT and may be better predictors of outcome than TNM stage.

Tumor Volume Analysis as a Predictive Marker for Prolonged Survival in Anaplastic Lymphoma Kinase-rearranged Advanced Non-Small Cell Lung Cancer Patients Treated With Crizotinib.

Abstract: Purpose Targeted inhibition of anaplastic lymphoma kinase (ALK) has been widely used for the treatment of advanced non-small cell lung cancer (NSCLC) with ALK rearrangements. We performed tumor volume analysis of ALK-rearranged advanced NSCLC treated with crizotinib to identify an early predictive marker for prolonged survival. Materials and methods Cases of 42 patients with ALK-rearranged advanced NSCLC (16 men, 26 women; median age: 55.7 y) treated with crizotinib as their first ALK-directed therapy were retrospectively studied. Tumor volume measurements of dominant lung lesions were performed on baseline computed tomography and follow-up computed tomography at 8 weeks of therapy. The relationships between the 8-week volume change (%) and overall survival (OS) were investigated. Results The 8-week tumor volume change ranged from -99.3% to 117.5% (median: -57.7%). Using the 25th percentile of the 8-week volume change of -74%, 11 patients with >74% volume decrease at 8 weeks had a significantly longer OS compared with 31 patients with ≤74% decrease (median OS: 92.0 vs. 22.8 mo; P=0.0048). In multivariable analyses using Cox proportional hazards models, the 8-week volume decrease of >74% was significantly associated with longer OS (hazard ratio=0.14, 95% confidence interval: 0.03-0.59; Cox P=0.008) after adjusting for tumor stage (stage IV vs. recurrent NSCLC, hazard ratio=5.6, 95% confidence interval: 1.29-24.3; P=0.02). Conclusions The 8-week tumor volume decrease of >74% is significantly associated with longer OS in patients with ALK-rearranged NSCLC treated with crizotinib.

Abstract CT286: CANOPY program clinical trials: Canakinumab (Cana) in patients (pts) with non-small cell lung cancer (NSCLC)

Abstract: Background: In the CANTOS study, treatment (tx) with Cana (selective IL-1β inhibitor) was associated with reduced incidence and mortality of NSCLC in stable post-myocardial infarction pts with elevated high-sensitivity C-reactive protein levels. The results provided a rationale to investigate the therapeutic role of Cana in NSCLC. Methods: The phase (ph) 2, open-label CANOPY-N study (NCT03968419) is evaluating Cana or pembrolizumab (pembro) alone or in combination as neoadjuvant tx in stage IB-IIIA, tx-naive NSCLC pts eligible for primary resection (except N2 and T4 tumors) with planned surgery in 4-6 weeks (wks) from the 1st dose of study tx. Pts (~110) are randomized 2:2:1 to Cana (2 doses 200 mg SC Q3W), Cana + pembro, or pembro (2 doses 200 mg iv Q3W) for 2 three-wk cycles. Randomization (R) stratification: histology (squamous [sq] vs non-sq). Primary endpoint: major pathological response rate at time of surgery.CANOPY-A (NCT03447769), CANOPY-1 (NCT03631199), and CANOPY-2 (NCT03626545) are ph 3, multicenter, double-blind studies. In CANOPY-A (Cana in adjuvant setting), pts (~1500) with stages IIA-IIIA and IIIB (T>5 cm N2), any histology, completely resected (R0) NSCLC post cisplatin-based chemotherapy (CTx) and radiation therapy (if applicable) are randomized 1:1 to Cana (200 mg SC Q3W)/placebo (PBO; SC Q3W) for 18 cycles. R stratification: AJCC/UICC v.8 stage (IIA vs IIB vs IIIA vs IIIB with T>5 cm, N2 disease), histology (sq vs non-sq), region (western Europe and North America vs eastern Asia vs rest of the world). Primary endpoint: disease-free survival. CANOPY-1 and CANOPY-2 consist of Part 1 (open-label, safety run-in; enrollment complete) and Part 2 (randomized 1:1, PBO-controlled, efficacy & safety; ongoing). CANOPY-1 eligibility: pts with previously untreated stages IIIB/IIIC or IV NSCLC and known PD-L1 status (for Part 2), without EGFR sensitizing mutations and/or ALK rearrangements. Part 1 (3 cohorts of ~9 pts each, based on different platinum-CTx): to confirm the recommended phase 3 regimen (RP3R) for Cana. In Part 2, pts (~600) are randomized to Cana (200 mg SC Q3W)/PBO + pembro + CTx for 4 cycles, followed by maintenance tx (Cana/PBO + pembro ± pemetrexed) until progressive disease (PD). R stratification: PD-L1 status (tumor proportion score Citation Format: Luis Paz-Ares, Edward B. Garon, Tony Mok, Andrea Ardizzoni, Fabrice Barlesi, Byoung Chul Cho, Gilberto de Castro, Pedro De Marchi, Enriqueta Felip, Yasushi Goto, Alastair Greystoke, Shun Lu, Darren Wan-Teck Lim, Martin Reck, Benjamin J. Solomon, David R. Spigel, Daniel SW Tan, Michael Thomas, James Chih-Hsin Yang, Jay M. Lee, Pilar Garrido, Edward Kim, Bruce E. Johnson. CANOPY program clinical trials: Canakinumab (Cana) in patients (pts) with non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT286.

Abstract CT214: CANOPY-1: Safety run-in results from phase (ph) 3 study of canakinumab (CAN) or placebo (PBO) in combination (comb) with pembrolizumab (PEM) plus platinum-based doublet chemotherapy (Ctx) as 1st line therapy in patients (pts) with advanced or metastatic NSCLC

Abstract: Cytokine interleukin-1β (IL-1β) has multiple pro-tumorogenic effects on tumor microenvironment, thereby promoting carcinogenesis, tumor invasiveness, and immunosuppression. CAN is a selective IL-1β inhibitor that aims to target tumor-promoting inflammation to reduce immune suppression, thereby potentiating effects of immunotherapy with PD-1 inhibitors such as PEM. Ph 3 CANTOS study has shown IL-1β inhibition with CAN was associated with reduced incidence of lung cancer (LC) and LC mortality in pts with atherosclerosis, providing a rationale to investigate therapeutic role of CAN in LC. CANOPY-1 (NCT03631199) is a PBO-controlled, double-blind, randomized, ph 3 trial designed to evaluate efficacy and safety of PEM + Ctx ± CAN in tx naive pts with stage IIIB/IIIC (not eligible for definitive chemo-radiation curative tx) or stage IV squamous and nonsquamous NSCLC. The study was divided into 2 parts: part 1 is open labelled, safety run-in part where pts received CAN 200 mg s.c Q3W + PEM 200 mg i.v Q3W + platinum-based Ctx (as induction during first 4 cycles only); Cohort A (A, non-squamous), carboplatin + pemetrexed; Cohort B (B, non-squamous), cisplatin + pemetrexed; Cohort C (C, squamous or non-squamous), carboplatin + paclitaxel. Part 2 is randomized and evaluates efficacy and safety of CAN comb regimen vs PBO comb regimen. Primary objective of safety run-in part: recommended ph 3 dose regimen (RP3R) of CAN comb. Secondary objectives: ORR, DCR, DOR, safety, PK, and immunogenicity. As of 14 May 2019 (follow-up of ≥42 days from C1D1 unless pt discontinued earlier), 10 pts in A, 11 pts in B, and 9 pts in C were treated, of which 73% were male, median age was 63 yrs. In total, 24/30 (80%) pts enrolled were still receiving tx; primary reason for tx discontinuation was progressive disease (3 pts in A and 1 pt each in B and C) and 1 pt died due to study indication. 1 pt reported DLT during first 42 days of study tx (C: grade 3 hepatitis, not related to CAN). RP3R of CAN in comb with standard dose PEM + Ctx was 200 mg SC Q3W based on Bayesian logistic regression model (BLRM). Serious AEs regardless of causality were reported in 8 (27%) pts (2 pts in A and 3 pts each in B and C), none of which were considered to be related to CAN. Most common AEs (≥20%, any grade) across all cohorts (n=30) were nausea (37%), vomiting (30%), constipation and fatigue (each 23%), and neutrophil count decrease (20%). 14 pts (47%) experienced grade 3 AEs and 1 pt experienced grade 4 AE (cardiac tamponade [unrelated]). No fatal serious AEs were reported. AEs leading to discontinuation of one of the study drugs were reported in 3 (10%) pts (hepatitis, peripheral neuropathy, and polyneuropathy) but none were CAN related. AEs leading to dose reduction and dose interruption of one of study drugs were reported in 3 (10%) pts and 5 (17%) pts, respectively. Only 1 DLT was reported with CAN + PEM + Ctx. Based on BLRM and all relevant clinical data, the RP3R of CAN as 200 mg SC Q3W comb was considered safe and well tolerated. Enrollment for the randomized part is completed. Citation Format: Bruce E. Johnson, Tae Min Kim, T. Jeroen N. Hiltermann, Fabrice Barlesi, Christian Grohe, Yasushi Goto, Orvar Gunnarsson, Tobias Overbeck, Noemi Reguart, Martin Wermke, Gilberto Castro Castro, Enriqueta Felip, Alastair Greystoke, Benjamin J. Solomon, Stephanie Deudon, Anne-Laure Louveau, Vanessa Passos, Daniel SW Tan. CANOPY-1: Safety run-in results from phase (ph) 3 study of canakinumab (CAN) or placebo (PBO) in combination (comb) with pembrolizumab (PEM) plus platinum-based doublet chemotherapy (Ctx) as 1st line therapy in patients (pts) with advanced or metastatic NSCLC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT214.

Development and validation of a novel EHR-based tumor progression outcome to support biomarker discovery.

Abstract: e19297Background: Obtaining clinical outcomes for analysis has historically been a critical barrier to cancer genomics research. EHRs could constitute an important data source to bridge this gap, b...

Effects of a Low Saturated Fat, Low Cholesterol Fish Oil Supplement in Hypertriglyceridemic Patients

Abstract: Study Objective:To determine the effects of fish oil supplements low in saturated fat and cholesterol on plasma lipid and lipoprotein levels in hypertriglyceridemic patients. Design:Single...