Rapid Autopsy Research Articles

Molecular Profiling of Coronavirus Disease 2019 (COVID-19) Autopsies Uncovers Novel Disease Mechanisms

Current understanding of coronavirus disease 2019 (COVID-19) pathophysiology is limited by disease heterogeneity, complexity, and a paucity of studies assessing patient tissues with advanced molecular tools. Rapid autopsy tissues were evaluated using multiscale, next-generation RNA-sequencing methods (bulk, single-nuclei, and spatial transcriptomics) to provide unprecedented molecular resolution of COVID-19-induced damage. Comparison of infected/uninfected tissues revealed four major regulatory pathways. Effectors within these pathways could constitute novel therapeutic targets, including the complement receptor C3AR1, calcitonin receptor–like receptor, or decorin. Single-nuclei RNA sequencing of olfactory bulb and prefrontal cortex highlighted remarkable diversity of coronavirus receptors. Angiotensin-converting enzyme 2 was rarely expressed, whereas basigin showed diffuse expression, and alanyl aminopeptidase, membrane, was associated with vascular/mesenchymal cell types. Comparison of lung and lymph node tissues from patients with different symptoms (one had died after a month-long hospitalization with multiorgan involvement, and the other had died after a few days of respiratory symptoms) with digital spatial profiling resulted in distinct molecular phenotypes. Evaluation of COVID-19 rapid autopsy tissues with advanced molecular techniques can identify pathways and effectors, map diverse receptors at the single-cell level, and help dissect differences driving diverging clinical courses among individual patients. Extension of this approach to larger data sets will substantially advance the understanding of the mechanisms behind COVID-19 pathophysiology.

MP33-01 METASTATIC CASTRATION-RESISTANT PROSTATE CANCER REMAINS DEPENDENT ON ONCOGENIC DRIVERS IN PRIMARY TUMORS

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology I (MP33)1 Sep 2021MP33-01 METASTATIC CASTRATION-RESISTANT PROSTATE CANCER REMAINS DEPENDENT ON ONCOGENIC DRIVERS IN PRIMARY TUMORS Seiji Arai, David Einstein, Kazuhiro Suzuki, Steven Balk, Adam Sowalsky, and Joshua Russo Seiji AraiSeiji Arai More articles by this author , David EinsteinDavid Einstein More articles by this author , Kazuhiro SuzukiKazuhiro Suzuki More articles by this author , Steven BalkSteven Balk More articles by this author , Adam SowalskyAdam Sowalsky More articles by this author , and Joshua RussoJoshua Russo More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002042.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Localized prostate cancer (PC) can be curable, but metastatic recurrence is unfortunately common. Metastatic PC is initially sensitive to androgen receptor inhibition, but eventually becomes castration-resistant (mCRPC). Early use of more intensive therapies targeting androgen receptor and other oncogenic drivers may be more effective than in advanced mCRPC. However, as oncogenic alterations found in mCRPC are less frequent in treatment-naïve primary PC, analysis of primary tumors may not reveal targetable metastatic drivers that are subclonal in the primary tumor or acquired at metastatic sites. METHODS: We analyzed PC samples spanning one patient’s clinical course: diagnostic biopsies, pre-/post-enzalutamide metastatic biopsies, and rapid autopsy samples including a patient-derived xenograft (PDX). RESULTS: Consistent with mCRPC being driven by non-truncal alterations, we identified RB1 loss throughout mCRPC samples, but not in the primary tumor. However, we identified a truncal EGFR-activating mutation in the primary that was maintained in mCRPC samples and the PDX, and showed that the PDX responded to the EGFR inhibitor. CONCLUSIONS: Truncal alterations can drive advanced mCRPC, and suggest that earlier detection and targeting of these truncal alterations may be effective at halting disease progression. Source of Funding: Congressionally Directed Medical Research Program (CDMRP), Prostate Cancer Research Program (Physician Research Award W81XWH-17-1-0350, Early Investigator Award W81XWH-18-1-0531, Idea Development Awards W81XWH-18-1-0379 and W81XWH-20-1-0925), the Prostate Cancer Foundation (Young Investigator Award 18YOUN24), the Intramural Research Program of the NIH, National Cancer Institute (NIH P01 - CA163227, SPORE in Prostate Cancer P50 – CA090281, and NIH P20 – CA233255) © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e606-e606 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Seiji Arai More articles by this author David Einstein More articles by this author Kazuhiro Suzuki More articles by this author Steven Balk More articles by this author Adam Sowalsky More articles by this author Joshua Russo More articles by this author Expand All Advertisement Loading ...

Multiregional genetic evolution of metastatic uveal melanoma

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults and leads to deadly metastases for which there is no approved treatment. Genetic events driving early tumor development are well-described, but those occurring later during metastatic progression remain poorly understood. We performed multiregional genomic sequencing on 22 tumors collected from two patients with widely metastatic UM who underwent rapid autopsy. We observed multiple seeding events from the primary tumors, metastasis-to-metastasis seeding, polyclonal seeding, and late driver variants in ATM, KRAS, and other genes previously unreported in UM. These findings reveal previously unrecognized temporal and anatomic complexity in the genetic evolution of metastatic uveal melanoma, and they highlight the distinction between early and late phases of UM genetic evolution with implications for novel therapeutic approaches.

LMD-07. In Vitro AND In Vivo Culture of Patient Derived-Cerebral Spinal Fluid-Circulating Tumor Cells (PD-CSF-CTCs) in Leptomeningeal Disease (LMD) From Melanoma to Identify Novel Treatment Strategies

Abstract Background Approximately 5% of melanoma patients (pts) will develop LMD. Currently there is no effective treatments for this disease. A significant barrier to the development of effective therapies has been the inability to culture CSF-CTCs for functional analysis. For the first time, we were able to successfully expand CSF-CTCs in vitro and in vivo. We assessed gene signatures of PD-CSF-CTCs to determine novel targets for therapy. As a proof of concept, we tested the efficacy of combining ceritinib (cer), an IGF-1R inhibitor and trametinib (tra), a MEK inhibitor, against LMD. Methods CSF from 11 pts were collected from various sources (ie: LPs, Ommayas, rapid autopsies). PD-CSF-CTCs were expanded in vitro in conditioned media and in vivo using cell line-derived xenograft model. Single-cell RNA-sequencing (scRNAseq) analysis was performed to assess transcriptional profiles of PD-CSF-CTCs. Results Of the total 61 PD-CSF-CTCs collected from 11 pts (avg: 4.07 CSF collections/patient), we successfully cultured PD-CSF-CTCs from 3 pts (20%) and were able to grow them in vivo from 2 pts (18%). scRNAseq identified IGF-1R, Sox9, ErbB3 and MLANA were among the enriched genes for PD-CSF-CTCs. IGF-1R inhibition by cer and depletion by CRISPR suppressed cell growth. We evaluated the responses of cer + tra treatment in vitro and found that combining these agents produced drug synergy against PD-CSF-CTCs and resensitized BRAF inhibitor-resistant melanoma cell line, WM164R. In vivo LMD xenograft model showed cer + tra treatment significantly prolonged median survival of PD-CSF-CTCs LMD (control: 27 days vs treatment: 38.5 days; P value < 0.032) and WM164R LMD (control: 35 days vs treatment: MS not reached; P value < 0.047). Conclusions Though the sample size is small, this is the first report of the successful in vitro and in vivo culture of CSF-CTCs from pts with LMD.

Combined Longitudinal Clinical and Autopsy Phenomic Assessment in Lethal Metastatic Prostate Cancer: Recommendations for Advancing Precision Medicine

BackgroundSystematic identification of data essential for outcome prediction in metastatic prostate cancer (mPC) would accelerate development of precision oncology. ObjectiveTo identify novel phenotypes and features associated with mPC outcome, and to identify biomarker and data requirements to be tested in future precision oncology trials. Design, setting, and participantsWe analyzed deep longitudinal clinical, neuroendocrine expression, and autopsy data of 33 men who died from mPC between 1995 and 2004 (PELICAN33), and related findings to mPC biomarkers reported in the literature. InterventionThirty-three men prospectively consented to participate in an integrated clinical-molecular rapid autopsy study of mPC. Outcome measurements and statistical analysisData exploration with correction for multiple testing and survival analysis from the time of diagnosis to time to death and time to first occurrence of severe pain as outcomes were carried out. The effect of seven complications on the modeled probability of dying within 2 yr after presenting with the complication was evaluated using logistic regression. Results and limitationsFeature exploration revealed novel phenotypes related to mPC outcome. Four complications (pleural effusion, severe anemia, severe or controlled pain, and bone fracture) predict the likelihood of death within 2 yr. Men with Gleason grade group 5 cancers developed severe pain sooner than those with lower-grade tumors. Surprisingly, neuroendocrine (NE) differentiation was frequently observed in the setting of high serum prostate-specific antigen (PSA) levels (≥30 ng/ml). In 4/33 patients, no controlled (requiring analgesics) or severe pain was detected, and strikingly, 14/15 metastatic sites studied in these men did not express NE markers, suggesting an inverse relationship between NE differentiation and pain in mPC. Intracranial subdural metastasis is common (36%) and is usually clinically undetected. Categorization of “skeletal-related events” complications used in recent studies likely obscures the understanding of spinal cord compression and fracture. Early death from prostate cancer was identified in a subgroup of men with a low longitudinal PSA bandwidth. Cachexia is common (body mass index <0.89 in 24/31 patients) but limited to the last year of life. Biomarker review identified 30 categories of mPC biomarkers in need of winnowing in future trials. All findings require validation in larger cohorts, preferably alongside data from this study. ConclusionsThe study identified novel outcome subgroups for future validation and provides “vision for mPC precision oncology 2020–2050” draft recommendations for future data collection and biomarker studies. Patient summaryTo better understand variation in metastatic prostate cancer behavior, we assembled and analyzed longitudinal clinical and autopsy records in 33 men. We identified novel outcomes, phenotypes, and aspects of disease burden to be tested and refined in future trials.

Abstract 3139: Study of tumor heterogeneity and subclonality in primary pancreatic and metastatic sites from rapid autopsy patients in PDMR

Abstract Background: A major set of preclinical models derived from specimens acquired from rapid autopsy patients in the National Cancer Institute Patient-Derived Models Repository (NCI PDMR, https://pdmr.cancer.gov) were from pancreatic adenocarcinoma (PAAD) patients, with metastatic specimens originating from liver, colon, omentum, and lung. Genomic characterization of these preclinical models provides a unique opportunity to study tumor heterogeneity and subclonality associated with the metastatic process and potential treatment resistance. Methods: To date, 30 rapid autopsy patient-derived xenograft (PDX)/patient-derived cell (PDC)/patient-derived organoid (PDOrg ) models derived from pancreatic adenocarcinoma patients (n = 9) have been sequenced using whole-exome sequencing (WES) and RNASeq. Tumor heterogeneity between primary and metastatic sites was studied based on somatic mutation, copy number alteration (CNA) and gene expression data. A bioinformatics workflow was developed to stably infer and visualize the tumor subclonality by integrating the tools of PyClone, SCHISM, and TIMESCAPE, using somatic mutations and site-specific copy number data of multiple samples generated from PDX models in primary and metastatic sites. Results: Among 30 rapid autopsy preclinical models from primary and metastatic sites, liver is the most common metastatic site in PAAD (9/19=47%) compared to other sites. Driver mutations are conserved in all preclinical model specimens derived from a given patient. KRAS p.G12D is present in 28 PDX/PDC/PDOrg models as well as the corresponding patient specimens, and BRAF p.V600E is present in other preclinical models. The fraction of the genome affected by CNA remains stable within a PDX model across passages (n=18, mean=7.63%, sd=5.90%). However, we found that this increased when comparing PDX models derived from metastatic sites versus the primary site (n=16, mean=19.47%, sd=9.69%). This indicates the presence of tumor heterogeneity between metastatic and primary sites. Site-specific subclones were identified in PDX models from two patients (521955 and 485368) and a phylogenetic tree of primary and metastasis sites indicates that one liver metastasis had a unique seeding event compared to the other metastatic sites for both patients. Conclusion: Tumor heterogeneity and subclonality was observed in preclinical models generated from PAAD patients in the NCI PDMR. These models provide a unique resource for preclinical studies in tumor evolution, metastatic spread mediators, and drug resistance. Citation Format: Li Chen, Biswajit Das, Yvonne A. Evrard, Chris A. Karlovich, Tomas Vilimas, Amanda Peach, Chapman Alyssa, Nikitha Nair, Anna L. Fong, Luis Romero, Ting-Chia Chang, Shahanawaz Jiwani, Lindsay Dutko, Kelly Benauer, Marianne Morton, Kelly Dougherty, Michelle A. Eugeni, Dianne Newton, Melinda G. Hollingshead, P. Mickey Williams, James H. Doroshow. Study of tumor heterogeneity and subclonality in primary pancreatic and metastatic sites from rapid autopsy patients in PDMR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3139.

Abstract 37: Convergence of resistance patterns in breast cancer after multiple lines of treatment through analysis of rapid autopsy samples

Abstract Determining patterns and mechanisms of drug resistance is fundamentally required for improving clinical outcome of cancer treatments. The ability to study multiple samples from different metastatic sites of the same patient is a clinically challenging task, which has become possible with the advent of “rapid” autopsy procedures (&amp;lt;6 hours from death) conducted on the patients deceased from cancer. We have obtained and analyzed whole-exome, whole genome and transcriptome sequencing data from advanced breast cancer samples derived from multiple cancer lesions of the same patient post-treatment through the Massachusetts General Hospital Rapid Autopsy program. We have integrated genomic and transcriptomic data through advanced clonal reconstruction methods (PhylogicNDT) to investigate how the patients developed resistance to multiple lines of anti-cancer therapy. Additionally, we have used previously collected cell-free DNA samples to establish both a spatial and temporal picture of cancer drug resistance and progression. We have examined clonal heterogeneity and resistance to targeted therapies in 10 metastatic ER+ breast cancer patients over 120 distinct metastatic sites. The majority of patients in this cohort were treated with CDK4/6 cell cycle inhibitors in addition to hormone therapy. We discovered that multiple metastatic sites often share similar clonal structure, but overall progeny of several sibling clones spreads throughout the body, creating “families” of more closely related lesions. These distinct lesion populations often develop independent resistance mechanisms to the many lines of treatment that the patents have received. At least 8 out of 10 patients showed multiple known and potentially novel resistance mechanisms in separate branches of the phylogenetic tree, often converging on distinct mutations in the same resistance genes. Notably we have identified ESR1, KRAS and chromatin modifier recurrent and convergent mutations across the cohort. Temporal analysis using cfDNA taken over the course of treatment allows us to identify clones that expanded in response to a given treatment. Using RNA expression and pathway analysis, we identify unique transcriptional programs, differentially expressed genes and ER-signaling changes between distinct clones within one patient, as well as compare genetically similar clones across patents. Citation Format: Ignaty Leshchiner, Elizabeth E. Martin, Christopher T. Chen, Elizaveta Leshchiner, Thomas Zhang, Christopher Pinto, Filippo Utro, Kahn Rhrissorrakrai, Chaya Levovitz, Brian P. Danysh, Kara Slowik, Raquel A. Jacobs, Laxmi Parida, Gad Getz, Dejan Juric. Convergence of resistance patterns in breast cancer after multiple lines of treatment through analysis of rapid autopsy samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 37.

Identifying Phenotypically Distinct Fibroblast Subsets in Type 2 Diabetes-Associated Iatrogenic Laryngotracheal Stenosis.

Iatrogenic laryngotracheal stenosis (iLTS) is the pathologic narrowing of the glottis, subglottis, and/or trachea secondary to intubation or tracheostomy related injury. Patients with type 2 diabetes mellitus (T2DM) are more likely to develop iLTS. To date, the metabolomics and phenotypic expression of cell markers in fibroblasts derived from patients with T2DM and iLTS are largely unknown. Controlled in vitro cohort study. Tertiary referral center (2017-2020). This in vitro study assessed samples from 6 patients with iLTS who underwent surgery at a single institution. Fibroblasts were isolated from biopsy specimens of laryngotracheal scar and normal-appearing trachea and compared with controls obtained from the trachea of rapid autopsy specimens. Patients with iLTS were subcategorized into those with and without T2DM. Metabolic substrates were identified by mass spectrometry, and cell protein expression was measured by flow cytometry. T2DM iLTS-scar fibroblasts had a metabolically distinct profile and clustered tightly on a Pearson correlation heat map as compared with non-T2DM iLTS-scar fibroblasts. Levels of itaconate were elevated in T2DM iLTS-scar fibroblasts. Flow cytometry demonstrated that T2DM iLTS-scar fibroblasts were associated with higher CD90 expression (Thy-1; mean, 95%) when compared with non-T2DM iLTS-scar (mean, 83.6%; P = .0109) or normal tracheal fibroblasts (mean, 81.1%; P = .0042). Scar-derived fibroblasts from patients with T2DM and iLTS have a metabolically distinct profile. These fibroblasts are characterized by an increase in itaconate, a metabolite related to immune-induced scar remodeling, and can be identified by elevated expression of CD90 (Thy-1) in vitro.

Cell-free DNA captures tumor heterogeneity and driver alterations in rapid autopsies with pre-treated metastatic cancer

In patients with metastatic cancer, spatial heterogeneity of somatic alterations may lead to incomplete assessment of a cancer’s mutational profile when analyzing a single tumor biopsy. In this study, we perform sequencing of cell-free DNA (cfDNA) and distinct metastatic tissue samples from ten rapid autopsy cases with pre-treated metastatic cancer. We show that levels of heterogeneity in genetic biomarkers vary between patients but that gene expression signatures representative of the tumor microenvironment are more consistent. Across nine patients with plasma samples available, we are able to detect 62/62 truncal and 47/121 non-truncal point mutations in cfDNA. We observe that mutation clonality in cfDNA is correlated with the number of metastatic lesions in which the mutation is detected and use this result to derive a clonality threshold to classify truncal and non-truncal driver alterations with reasonable specificity. In contrast, mutation truncality is more often incorrectly assigned when studying single tissue samples. Our results demonstrate the utility of a single cfDNA sample relative to that of single tissue samples when treating patients with metastatic cancer.

Mechanisms and Stages of Covid-19 Immunopathology Revealed by Tissue-Specific Proteomes

Background: Tissue inflammation in fatal COVID-19 is concentrated in the lung and spleen. Anti-inflammatory therapy reduces mortality but knowledge on the host response at the level of inflamed tissues is incomplete. Methods: We performed targeted proteomic analysis of pulmonary and splenic tissues from 13 fatal cases of COVID-19 that underwent rapid autopsy, and compared to control tissues from cancer resection (lung) and deceased organ donors (spleen). Viral RNA presence was determined by multiplex PCR, and protein was isolated from tissue by phenol extraction. Targeted multiplex immunoassay panels were used for protein detection and quantification. Findings: Pulmonary proteins with increased abundance in COVID-19 included the monocyte/macrophage chemoattractant MCP-3, antiviral TRIM21 and pro-thrombotic TYMP. The lung injury markers OSM and EN-RAGE/S100A12 were highly correlated and associated with tissue inflammation severity. Unsupervised clustering of lung proteomes clearly defined two COVID-19 clusters; these differed by viral presence, tissue inflammation severity and illness duration and were annotated ‘early viral’ and ‘late inflammatory’ groups. In the spleen, lymphocyte chemotactic factors and CD8A were decreased in COVID-19, with pro-apoptotic factors, B-cell signalling components and macrophage colony stimulating factor (CSF-1) all increased. To contextualise our findings, we cross-referenced an existing meta-analysis of host factors in COVID-19 (MAIC). Overlap with a substantial sub-set of factors (including DDX58, OSM, TYMP, IL-18, MCP-3 and CSF-1) was found, with numerous additional proteins also identified by our study. Interpretation: Tissue proteomes from fatal COVID-19 identify disease subsets and dissect host immunopathologic signatures. In doing so, this may afford unique opportunities for therapeutic intervention.Funding Information: This work was funded by UK Research and Innovation (UKRI) (Coronavirus Disease [COVID-19] Rapid Response Initiative; MR/V028790/1 to C.D.L., D.A.D., and J.A.H.), LifeArc (through the University of Edinburgh STOPCOVID funding award, to K.D, D.A.D, C.D.L), The Chief Scientist Office (RARC-19 Funding Call, ‘Inflammation in Covid-19: Exploration of Critical Aspects of Pathogenesis; COV/EDI/20/10’ to D.A.D, C.D.L, C.D.R, J.K.B and D.J.H), and Medical Research Scotland (CVG-1722-2020 to DAD, CDL, CDR, JKB, and DJH). C.D.L is funded by a Wellcome Trust Clinical Career Development Fellowship (206566/Z/17/Z). J.K.B. and C.D.R. are supported by the Medical Research Council (grant MC_PC_19059) as part of the ISARIC Coronavirus Clinical Characterisation Consortium (ISARIC-4C). C.D.R. is supported by an Edinburgh Clinical Academic Track (ECAT)/Wellcome Trust PhD Training Fellowship for Clinicians award (214178/Z/18/Z). J.A.H. is supported by the U.S. Food and Drug Administration (contract 75F40120C00085, Characterization of severe coronavirus infection in humans and model systems for medical countermeasure development and evaluation’). G.C.O is funded by an NRS Clinician award. N.N.G. is funded by a Pathological Society Award. A.R.A. is supported by a Cancer Research UK Clinician Scientist Fellowship award (A24867).Declaration of Interests: All authors have declared that no competing interests exist.Ethics Approval Statement: Written informed consent to undertake postmortem examinations was obtained from next-of-kin. Ethical approval was granted by the East of Scotland Research Ethics Service (16/ES/0084).

Concordance of DNA damage repair (DDR) gene mutations in paired primary and metastatic prostate cancer (PC) samples.

5020 Background: Mutations in DDR genes represent actionable alterations that can be used to guide precision medicine strategies in men with advanced PC. However, acquisition of contemporary tissue samples for advanced molecular testing can be a barrier to deploying precision medicine approaches. We hypothesized that most DDR alterations represent early truncal events in PC and that archival primary tissue would faithfully reflect mutations found in cell-free circulating tumor (ctDNA) and/or metastatic tissue. Methods: Patients were included in this study if a DDR pathway mutation was detected in metastatic tissue or ctDNA and primary tissue sequencing was available for comparison. Sequencing data from three cohorts were analyzed: 1) FoundationOne, 2) University of Washington (UW-OncoPlex or SU2C/PCF International Dream Team sequencing pipelines) and 3) University of Washington rapid autopsy series. Only pathogenic somatic mutations were included and we required ≥30 days between primary tumor tissue and ctDNA/tumor tissue acquisition. Clonal hematopoiesis of indeterminant potential (CHIP) and germline events were adjudicated by an expert molecular pathologist and excluded. Variants detected only in plasma were considered likely to be CHIP or low subclones if the variant fraction was &lt;1% and/or &gt;5-fold less than the estimate tumor content in plasma. Results: Paired primary and ctDNA/metastatic samples were sequenced from 72 individuals with known DDR alterations. After excluding ctDNA cases where only CHIP (N=13) and/or germline events (N=7) were observed, 51 subjects remained and were included in the final analysis. The median time from acquisition of primary tissue to acquisition of ctDNA or tumor tissue was 52 mos (range: 1 – 193 mos). Concordance in DDR genes across samples was 86% (95% CI: 74-93%). Rates of concordance between metastatic-primary and ctDNA-primary pairs were similar when CHIP cases were excluded (87% and 85%, respectively). BRCA2 reversion mutations associated with resistance to PARP inhibitors and platinum chemotherapy were detected in ctDNA from two subjects. Conclusions: These data provide evidence that primary prostate tissue accurately reflect the mutational status of actionable DDR genes in men with metastatic PC, supporting the hypothesis that DDR alterations are early truncal events. After excluding likely CHIP events, ctDNA profiling accurately captured these truncal DDR mutations, while also detecting reversion alterations that may suggest potential resistance mechanisms.[Table: see text]

Multi-omic profiling of lung and liver tumor microenvironments of metastatic pancreatic cancer reveals site-specific immune regulatory pathways

BackgroundThe majority of pancreatic ductal adenocarcinomas (PDAC) are diagnosed at the metastatic stage, and standard therapies have limited activity with a dismal 5-year survival rate of only 8%. The liver and lung are the most common sites of PDAC metastasis, and each have been differentially associated with prognoses and responses to systemic therapies. A deeper understanding of the molecular and cellular landscape within the tumor microenvironment (TME) metastasis at these different sites is critical to informing future therapeutic strategies against metastatic PDAC.ResultsBy leveraging combined mass cytometry, immunohistochemistry, and RNA sequencing, we identify key regulatory pathways that distinguish the liver and lung TMEs in a preclinical mouse model of metastatic PDAC. We demonstrate that the lung TME generally exhibits higher levels of immune infiltration, immune activation, and pro-immune signaling pathways, whereas multiple immune-suppressive pathways are emphasized in the liver TME. We then perform further validation of these preclinical findings in paired human lung and liver metastatic samples using immunohistochemistry from PDAC rapid autopsy specimens. Finally, in silico validation with transfer learning between our mouse model and TCGA datasets further demonstrates that many of the site-associated features are detectable even in the context of different primary tumors.ConclusionsDetermining the distinctive immune-suppressive features in multiple liver and lung TME datasets provides further insight into the tissue specificity of molecular and cellular pathways, suggesting a potential mechanism underlying the discordant clinical responses that are often observed in metastatic diseases.

Evolution of delayed resistance to immunotherapy in a melanoma responder.

Despite initial responses1-3, most melanoma patients develop resistance4 to immune checkpoint blockade (ICB). To understand the evolution of resistance, we studied 37 tumor samples over 9 years from a patient with metastatic melanoma with complete clinical response to ICB followed by delayed recurrence and death. Phylogenetic analysis revealed co-evolution of seven lineages with multiple convergent, but independent resistance-associated alterations. All recurrent tumors emerged from a lineage characterized by loss of chromosome 15q, with post-treatment clones acquiring additional genomic driver events. Deconvolution of bulk RNA sequencing and highly multiplexed immunofluorescence (t-CyCIF) revealed differences in immune composition among different lineages. Imaging revealed a vasculogenic mimicry phenotype in NGFRhi tumor cells with high PD-L1 expression in close proximity to immune cells. Rapid autopsy demonstrated two distinct NGFR spatial patterns with high polarity and proximity to immune cells in subcutaneous tumors versus a diffuse spatial pattern in lung tumors, suggesting different roles of this neural-crest-like program in different tumor microenvironments. Broadly, this study establishes a high-resolution map of the evolutionary dynamics of resistance to ICB, characterizes a de-differentiated neural-crest tumor population in melanoma immunotherapy resistance and describes site-specific differences in tumor-immune interactions via longitudinal analysis of a patient with melanoma with an unusual clinical course.

Pulmonary stromal expansion and intra-alveolar coagulation are primary causes of COVID-19 death

Most COVID-19 victims are old and die from unrelated causes. Here we present twelve complete autopsies, including two rapid autopsies of young patients where the cause of death was COVID-19 ARDS. The main virus induced pathology was in the lung parenchyma and not in the airways. Most coagulation events occurred in the intra-alveolar and not in the intra-vascular space and the few thrombi were mainly composed of aggregated thrombocytes. The dominant inflammatory response was the massive accumulation of CD163 + macrophages and the disappearance of T killer, NK and B-cells. The virus was replicating in the pneumocytes and macrophages but not in bronchial epithelium, endothelium, pericytes or stromal cells. The lung consolidations were produced by a massive regenerative response, stromal and epithelial proliferation and neovascularization. We suggest that thrombocyte aggregation inhibition, angiogenesis inhibition and general proliferation inhibition may have a roll in the treatment of advanced COVID-19 ARDS.

A molecular single-cell lung atlas of lethal COVID-19.

Respiratory failure is the leading cause of death in patients with severe SARS-CoV-2 infection1,2, but the host response at the lung tissue level is poorly understood. Here we performed single-nucleus RNA sequencing of about 116,000 nuclei from the lungs of nineteen individuals who died of COVID-19 and underwent rapid autopsy and seven control individuals. Integrated analyses identified substantial alterations in cellular composition, transcriptional cell states, and cell-to-cell interactions, thereby providing insight into the biology of lethal COVID-19. The lungs from individuals with COVID-19 were highly inflamed, with dense infiltration of aberrantly activated monocyte-derived macrophages and alveolar macrophages, but had impaired T cell responses. Monocyte/macrophage-derived interleukin-1β and epithelial cell-derived interleukin-6 were unique features of SARS-CoV-2 infection compared to other viral and bacterial causes of pneumonia. Alveolar type 2 cells adopted an inflammation-associated transient progenitor cell state and failed to undergo full transition into alveolar type 1 cells, resulting in impaired lung regeneration. Furthermore, we identified expansion of recently described CTHRC1+ pathological fibroblasts3 contributing to rapidly ensuing pulmonary fibrosis in COVID-19. Inference of protein activity and ligand-receptor interactions identified putative drug targets to disrupt deleterious circuits. This atlas enables the dissection of lethal COVID-19, may inform our understanding of long-term complications of COVID-19 survivors, and provides an important resource for therapeutic development.

Rapid Autopsy Programs and Research Support: The Pre– and Post–COVID-19 Environments

Each rapid autopsy is a powerful opportunity to supply multiple researchers with many valuable tissue specimens at the same time. Since the beginning of the development of rapid autopsy, the overriding organizing principle for all rapid autopsy programs has been that the samples or organs must be removed and processed as rapidly as possible. To accomplish this, some rapid autopsy programs are focused on only 1 tumor type, whereas others accept patients demonstrating all tumor types and sometimes other diseases as well. Rapid autopsy programs are logistically complicated and labor-intensive structures; therefore, the key to their success is program flexibility and maintaining a multidisciplinary focus. The necessary collaborations in the complex relationships between clinicians and researchers can be broken down into a series of thought and action steps that must be understood, accepted, and practiced by all participants. A crucial part of the precase steps (prior to death) for a rapid autopsy is the study consenting process. It is extremely important that this individualized consent is obtained for postmortem specimens and that it is written in terms general enough to be used for patients with all types of diseases and for an appropriate range of future research uses. The advent of SARS-CoV-2/COVID-19 (severe acute respiratory syndrome coronavirus 2/coronavirus disease 2019) has presented new challenges and opportunities to the field of autopsy pathology. Guidelines and practice had to be created and adapted to protect physicians and staff while maximizing diagnostic yield. However, any autopsy performed on a patient dying of or with COVID-19 represents a unique opportunity to contribute to understanding the disease mechanisms and to improve death certification, thus assisting in both clinical care and the development of health public policy.

Molecular subtype variation within metastasis of urothelial carcinoma.

471 Background: Molecular subtyping of cancer based on gene expression is a new prognostic tool with potential to guide treatment in the future. Urothelial carcinoma is one such cancer for which numerous molecular subtyping systems have been developed, but the diversity of these systems has hindered their clinical application. Recently, a consensus classification system was derived from six independent systems, defining six molecular classes with distinct oncogenic mechanisms and mutations (Kamoun A, et al. 2020). Considering the high heterogeneity in urothelial carcinoma, we hypothesized that molecular subtype variation may occur between primary and metastatic samples. We further evaluated whether variation in subtype was associated with any unique patient characteristics. Methods: As part of the University of Washington Bladder Cancer Rapid Autopsy Program (BCRAP), primary and metastatic tumor tissue samples were acquired from 14 deceased patients with urothelial carcinoma within 6 hours of death. Patient history was collected and deidentified for analysis. RNA exome sequencing was used for assigning molecular subtype for each of the 61 tumor samples, using the consensus and six comprising systems. Results: Molecular subtype variation within metastatic tumors according to any classification system was detected in 8 out of 14 patients, independent of histologic morphologies. Amongst the patients with variation, on average 2.1 out of 7 classification systems identified a major difference in subtype between sites. Patients with variation (mean age 70 years (SD 7 years)) were older than those without variation (mean age 59 years (SD 11 years), P = 0.04). Furthermore, patients with variation tended to have decreased survival from diagnosis and received less chemotherapy, although these were not statistically significant (p &gt; 0.05). Conclusions: Molecular subtype variation within metastasis is relatively common amongst BCRAP patients with urothelial carcinoma. Older patients are more likely to have variation, possibly due to a higher tumor mutation burden. Potential variation must be taken into account when considering prognosis and developing a recommended drug regimen specific to molecular subtypes.

Abstract PS19-16: Utility of rapid autopsy in cancer research: Unexpected findings and lessons learned from warm autopsies of metastatic breast cancer patients

Abstract Metastatic disease is understudied largely because of inaccessibility to quality specimens for research use. The Legacy Project, a rapid or “warm”, autopsy program at City of Hope, seeks to overcome this challenge by collecting tissue from metastatic patients immediately (within 6 hours) after their death. This paradigm serves as a specimen resource to address many clinically relevant questions, such as disease heterogeneity and mechanisms driving disease progression. Using this model, we uncovered clinically relevant disease information that is normally unavailable while a subject is alive. In this study, 9 metastatic breast cancer patients and their families were approached and consented prior to death. The cohort includes a diversity of clinical presentations in terms of disease subtype, progression history, organ involvement, and final cause of death. A total of 533 specimens were collected across 9 subjects. The average time from death to specimen acquisition was 6.1 hours (range: 4.03 - 7.66 hours; median: 5.71 hours). Total number of specimens collected from each participant ranged from 38-75, with an average of 60 across all patients; the mean number of tumor-positive specimens collected was 29 (range 12-46); the mean number of non-cancer specimens collected was 31 (range 25-45). In patients with primary estrogen receptor (ER) positive disease, we observed variable heterogeneity in estrogen, progesterone, and ki67 status across metastatic lesions. Furthermore, we observed a profound shift in disease phenotype towards end of life, trending towards complete loss of hormone receptor expression and stark increase of Ki67 levels. At the time of procurement, one third of subjects exhibited clinically unidentified diseased sites in organs not commonly associated with breast cancer metastases a, including ovary, kidney, and pancreas. In two other instances, “resolved” bone specimens (as measured by absence of FTG uptake in PET/CT imaging) were later determined to be &amp;gt;30% tumor positive when assessed by H&amp;E. While these preliminary findings generate more questions than answers regarding mechanisms of metastatic progression and resistance to therapy, they highlight the utility of rapid autopsy in a research setting. We suggest that many unanswered clinical questions can be addressed through interrogation of post-mortem tissues and we urge research institutions to thoughtfully consider adoption of the “rapid autopsy” model. Citation Format: Eliza R Bacon, Kena Ihle, Colt Egelston, Weihua Guo, Diana Simons, Peter P Lee, James Waisman. Utility of rapid autopsy in cancer research: Unexpected findings and lessons learned from warm autopsies of metastatic breast cancer patients [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-16.

Abstract PS17-45: Metastatic tissues display organ specific immune infiltration archetypes; lessons from a rapid autopsy tissue collection study

Abstract Immune composition in the tumor microenvironment (TME) of patient tumors has proven to play a central role in the development of metastases and response to therapy. Evidence has suggested that the metastatic TME is immune aberrant, however difficulty in obtaining biopsies of metastatic tumors has made assessment of the immune TME difficult. Here we utilize a rapid autopsy tissue collection protocol to assess the infiltration and composition of the immune TME in numerous metastatic tissue sites, paired disease-free tissue sites, and the associated tissue draining lymph nodes. Post-mortem tissues were collected from nine metastatic breast cancer patients shortly after death through City of Hope’s “Legacy Project for Rapid Tissue Donation” Program. The average post-mortem interval (PMI) for tissue collection was 6 hours. Collected specimens include metastatic lesions and paired non-cancer samples from every cancer-involved organ, disease-free specimens from non-involved major organs, distant and tumor-draining lymph nodes (both cancer-infiltrated and disease free), as well as blood and spleens. Immediately following collection, specimens were processed into single cell suspension for flow cytometry. Over 80 immune cell phenotypes were assessed, including CD8+ and CD4+ T cell subsets, B cell subsets, natural killer (NK) cells, tumor associated macrophages (TAMs), dendritic cell subsets, and other immune cells. Tumor infiltrated tissues were found to have comparable immune cell densities and composition compared to paired disease-free tissues of the same organ type. However, immune cell densities in metastatic tissues and disease-free tissues were significantly different between organ types, with lung immune infiltration consistently being greater than liver, brain, and skin tissues. Differences in immune composition between tissue sites were also observed. Notably, liver tissues favored the presence of IL-2 producing central memory CD8+ T cells, while lung tissues favored the presence of CD8+ tissue resident memory T cells and CD16+ NK cells. Relative to disease-free lung tissues, tumor infiltrated lungs contained diminished frequencies of CD8+ tissue resident memory T cells and altered B cell and monocyte phenotypes. Increased levels of targetable immune pathways were observed, including increased PD-L1 and CTLA-4 expressing cells in skin metastases, and increased GARP+ B cells in bone marrow metastases. These data suggest that immune monitoring and trafficking of metastatic tissues site is dictated by organ type, which can be altered in composition by tumor infiltration. Further studies such as these may reveal organ-specific mechanisms of response to therapeutic interventions. Identity of organ location for tumor metastases may guide choices for immunotherapeutic interventions. Citation Format: Colt Egelston, Weihua Guo, Eliza R. Bacon, Kena Ihle, Diana Simons, Christian Avalos, JIayi Tan, Jian Ye, Lei Wang, Minhui Lim, James R. Waisman, Peter P Lee. Metastatic tissues display organ specific immune infiltration archetypes; lessons from a rapid autopsy tissue collection study [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-45.

Abstract PD15-01: Snord67 promotes lymph node metastasis through U6-mediated alternative splicing in breast cancer

Abstract Background: While the presence of lymph node (LN) metastases in solid tumor malignancies is clinically significant and associated with increased risk for distant metastases, there is little evidence that lymphatic metastasis is distinct from hematogenous dissemination. Recent studies showed that cancer cells in LNs can directly give rise to distant metastases, but the mechanisms behind how cancer cells exit lymph nodes to establish distant metastases is still unknown. Materials and Methods: We developed an immunocompetent mouse model of lymph node metastasis using the 4T1 murine breast cancer cell line. In this model, 4T1 cells micro-surgically injected into axillary lymph nodes (AxLN) are capable of spontaneously establishing distant metastases. We then performed microarray analyses of subclones derived from MFP tumors, AxLN-injected tumors (AxLN), and lung metastases that arose from AxLN-injected tumors (AxLN-LuM) to evaluate for non-coding RNAs (ncRNAs) that were differentially expressed in AxLN tumors. Results: Microarray analyses of ncRNAs expressed in MFP, AxLN, and AxLN-LuM subclones showed that a class of ncRNAs, small nucleolar RNAs (snoRNAs), were enriched in AxLN tumors compared to MFP and AxLN-LuM. We identified the snoRNA SNORD67 as a key regulator of LN metastasis. Knockout of SNORD67 by both CRISPR and antisense oligonucleotide (ASO) resulted in significantly decreased LN tumor growth and subsequent development of distant metastases. This was at least in-part due to loss of targeted 2’-O-methylation of the small nuclear RNA U6, a component of the spliceosome. RNA-Seq analyses revealed distinct alternative splicing in SNORD67 knockouts. Using rapid autopsy breast cancer cases, we found that matched primary tumor and LN metastases revealed similar alternatively spliced genes, including several genes with previously described cancer-promoting splice variants. Conclusions: Small nucleolar RNAs (snoRNAs) have long been regarded as “housekeeping genes”, important for ribosomal biogenesis and protein synthesis. However, there is increasing evidence that this largely ignored class of non-coding RNAs (ncRNAs) also have wide-ranging, non-canonical functions in diseases, including cancer. SnoRNAs have been shown to have both oncogenic and tumor suppressor roles, yet whether snoRNAs regulate metastasis is unknown. Here we show that expression of certain snoRNAs are enriched in lymph node (LN) metastases in a micro-surgical, immune-competent mouse model of breast cancer. Moreover, our results show that SNORD67 is critical for growth of LN metastases and subsequent spread to distant metastases, and suggest that snoRNA-guided modifications of the spliceosome represent a previously unappreciated, yet targetable pathway in cancer. Citation Format: Yvonne Chao, Yinzhou Zhu, Hannah J Wiedner, Yi-Hsuan Tsai, Lily Wilkinson, Alessandro Porrello, Amanda ED Van Swearingen, Lisa Carey, Jimena Giudice, Christopher L Holley, Chad V Pecot. Snord67 promotes lymph node metastasis through U6-mediated alternative splicing in breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD15-01.