3735 results for "*oma"

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• Metagenomic Analysis to Identify Novel Infectious Agents in Systemic Anaplastic Large Cell Lymphoma

  Anaplastic large cell lymphoma (ALCL) is a rare T-cell non-Hodgkin lymphoma (NHL). Risk of systemic ALCL is markedly increased among immunosuppressed people, such as those with human immunodeficiency virus (HIV) infection or solid organ transplant recipients. This increased risk in immunosuppressed populations suggests a viral etiology, given similar elevations for diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma, and Hodgkin lymphoma (caused by Epstein-Barr virus [EBV]) and other virus-related cancers in immunosuppressed individuals. EBV might be a plausible candidate, but most reported ALCL tumors are EBV-negative. Therefore, we conducted a metagenomic analysis of microbial associations with ALCL to determine whether a known or novel pathogen is associated with the tumor. We obtained archived formal fixed paraffin embedded (FFPE) ALCL tumor tissue from the Iowa cancer registry and an additional case from an immunosuppressed individual infected with human immunodeficiency virus. Controls included 3 diffuse large B-cell lymphomas (DLBCLs) in transplant recipients (shown to be related to Epstein-Barr virus [EBV] by in situ hybridization) and 3 breast cancers (no known viral etiology). RNA was extracted from FFPE specimens, and total RNA libraries were prepared using the KAPA RNA HyperPrep Kit (Roche). Five samples (IA01, IA53, IA57, IA07, and IA17) were sequenced on a NextSeq 500 (Illumina) and the remainder on a NovSeq 6000 (Illumina). Using novel computational approaches, we eliminated human reads and aligned residual non-human reads to known microbial references using GATK-PathSeq or conducted de novo assembly of unassigned reads using virID pipeline. Although EBV was detected in the 3 DLBCL controls, we did not detect EBV or any novel pathogen in ALCL tumors or in the breast cancers. Our study had a modest sample size and only one case arising in HIV-infected individual. Therefore, further studies are required to characterize the metagenome of systemic ALCLs, especially cases arising in immunosuppressed people.

  Study phs002064

• Multimodal Mapping of the Immune Landscape in Human Pancreatic Cancer

  Pancreatic ductal adenocarcinoma (PDA) is characterized by an immune-suppressive tumor microenvironment that has rendered it largely refractory to immunotherapy. We used single-cell RNA sequencing on patient tumors and matched blood, and we uncovered a previously unappreciated, complex environment of immune-suppressive cellular interactions. These experiments reveal immune checkpoint expression heterogeneity on individual patient T cells, with corresponding ligands on neoplastic and myeloid cells. TIGIT was upregulated on tumor infiltrating CD8+ T cells and was the only checkpoint receptor consistently expressed by exhausted CD8+ T cells. Clinical annotation of the samples revealed that the exhausted signature is most prominent in patients with advanced stages of disease. Our findings suggest individualized combination therapy, involving anti-TIGIT therapies, may be necessary to extend the benefits of immunotherapy to pancreatic cancer patients.

  Study phs002071

• Role of Tobacco Smoke in Clear Cell Renal Cell Carcinoma

  Background. Clear cell renal cell carcinoma (ccRCC) is the most common histologically defined renal cancer. However, it is not a uniform disease and includes several genetic subtypes with different prognoses. ccRCC is also characterized by distinguished metabolic reprogramming. Tobacco smoking (TS) is an established risk factor for ccRCC with unknown effects on tumor pathobiology.Methods. We investigated the landscape of ccRCCs and paired normal kidney tissues (NKTs) using integrated transcriptomic, metabolomic and metallomic approaches in a cohort of never smokers (NS) and long-term current smokers (LTS) Caucasian males. Results. All three Omics domains consistently identified a distinct metabolic subtype of ccRCCs in LTS, characterized by activation of oxidative phosphorylation (OxPhos) coupled with reprogramming of the malate-aspartate shuttle and metabolism of aspartate, glutamate, glutamine and histidine. Cadmium, copper and inorganic arsenic accumulated in LTS tumors showing redistribution among intracellular pools, including relocation of copper into the cytochrome c oxidase complex. Gene expression signature based on the LTS metabolic subtype provided prognostic stratification of The Cancer Genome Atlas (TCGA) ccRCC tumors that was independent from genomic alterations.Conclusions. The work identified the TS related metabolic subtype of ccRCC with vulnerabilities that can be exploited for precision medicine approaches targeting metabolic pathways. The results provided rationale for the development of metabolic biomarkers with diagnostic and prognostic applications using evaluation of OxPhos status. The metallomic analysis revealed the role of disrupted metal homeostasis in ccRCC highlighting the importance of studying effects of metals from e-cigarettes and environmental exposures.

  Study phs002083

• Demographically Diverse Substance Use Disorder Cohorts of Dr. Stanley H. Weiss

  The Demographically Diverse Substance Use Disorder Cohorts of Dr. Stanley H. Weiss, which constitute the Epidemiology of the Weiss Cohort Projects, consist of a series of inter-connected projects, building upon a set of cohort projects of various groups, mainly drug users from medication-assisted treatment programs, that Dr. Stanley H. Weiss first developed in the 1980’s plus several newer initiatives, each with an array of collaborators. Beginning in the 1980’s, Dr. Stanley H. Weiss started several long-term studies of persons who inject drugs (PWID) across the United States, ultimately enrolling over 10,000 participants through the early 1990’s with an average age then in their 30’s. About a quarter were enrolled from sites in New Jersey (NJ). These studies included the first testing of PWID for the human immunodeficiency virus (HIV) and the human T-cell lymphotropic viruses (HTLV I and HTLV II). Cumulative past support (initiation thru ~ 1999) for these cohort studies included ~ $20 million from intramural resources from the National Cancer Institute (NCI) and the National Institute on Drug Abuse (NIDA), plus multiple grants and in-kind support from the New Jersey Department of Health (NJDOH) totaling ~ $1 million. The Weiss Cohort Projects include the first large AIDS-era cohorts to include women at high risk for HIV. A high percentage of subjects in these studies are black or Latino. Thus, this is an ethnically diverse US cohort, with a high proportion of women included. These subjects are at high risk of parenteral and sexual infection from both drug use and sexual practices. Samples from other studies conducted by Dr. Weiss, in which detailed interviews were conducted, are included as controls (persons documented by us not to have a history of opioid drug use). As one of our groups of subjects have many persons of Haitian ancestry, we specifically included some Haitians who had never used opioids as controls. Our documentation includes such ancestry. These cohorts demonstrated high rates of HIV and HTLV-II infection in PWIDs, including one study initiated in 1981 with confirmation in the later cohorts. In the first two decades of these studies, among numerous publications was the first study showing a very high rate of hepatitis C infection among PWIDs. An example of how the studies’ long-time horizon proved essential was that it first became possible to test whether a person had ever been infected with hepatitis C virus (HCV), as well as how much HCV was in each person’s blood, many years after the specimens were collected. This allowed HCV amounts in blood to be compared for subjects who had died of liver disease early in the study versus those who survived. Then a sequence of published papers culminated in demonstrating, using a nested case-control design, that a high baseline HCV titer was predictive of early progression to death from end-stage liver failure. Outcomes related to HCV (end stage liver disease and hepatocellular carcinoma) remain under study. In the original cohort studies, the mean age at enrollment was ~ 33 years old, so that those still alive in 2022 are mainly now ~ 60 - 75 years old. Many participants have already died. The tincture of time has led to subjects reaching ages when many more are dying from a wide array of outcomes, including from many chronic diseases (including cancer) as well as from infectious agents (especially HIV, HCV) or drug overdose. Renewed collaboration with local drug treatment programs has led to new field-based studies, including examination of some currently evolving problems among drug users. Dr. Weiss joined the National Institute on Drug Abuse (NIDA) Genetics Consortium (NGC) in 2017, and through the NIDA project officer has had access to NGC contract resources (see below). NIH Certificate of Confidentiality, CC-DA-16-214 (attached) protects these studies. Past arrangements related to data on our subjects leads to restrictions on the use of data emanating from our study, such as potential commercialization and restrictions on whom may access and use these data. NIDA Genetics Consortium (NGC) resources further support these endeavors and will be used as part of the NGC analyses studying the genetics of substance use. Study participants signed informed consent for the information collected from them to be used with no time limit and for biologic specimens collected from them to be used without restriction in future research. Serum samples were collected from participants, and from many also plasma, white blood cells and/or urine samples. About 100,000 vials were stored. All specimens have been continuously preserved at sufficiently cold temperatures to prevent deterioration, and many subjects separated white blood cells were processed and frozen in such a way as to maintain viability. Detailed data from the participants has been accumulated over time, and in general, linkage has been retained in each sub-study in accordance with the consent forms and protocols. For some participants, specimens were collected at multiple times (that is, sequential specimens). Multiple specimens from a single person exist in this database, and efforts at de-duplication remain ongoing. Dr. Weiss should be contacted if an investigator requires unique individuals since: • Multiple phases of enrollment occurred, and as our prospective follow-up continues; Dr. Weiss may identify new instances of multiple enrollment. • Some persons are related to each other. • In general, in this dataset for dbGaP, only a single specimen/record form a given person is included. Advances in laboratory testing techniques now permit innovative new uses for our linked research biospecimen repository. The ongoing focus of an interdisciplinary research program based on these cohorts relates subjects’ diseases, behaviors, medical history, and outcomes with biological and exposure markers. Participants’ use of various substances was ascertained on study enrollments, many serially over time. Quantitative frequency of use data, also sometimes sequential over time, were ascertained. Active ascertainment of outcomes is being conducted, including matching to mortality and cancer databases. Investigators interested in collaborations on specific outcomes (which is not part of this dbGaP dataset) or in the use of our stored specimens are encouraged to contact the principal investigator, Dr. Weiss. The processing of the genomic data was done in conjunction with NIDA, and in accordance with some longstanding data cleaning steps used by NIDA in the NIDA Genetics Consortium (NGC), a group to which we shall be contributing these data for collaborative analyses. Since there is the potential for these steps to introduce certain types of potential biases, we summarize these here. Under contract from NIDA, cryopreserved sera or plasma (-80 C) or cells (in liquid nitrogen) were used, with most stored having been stored for 30 to 40 years in our biorepository. In the case of serum or plasma, in which only (largely) cell-free DNA fragments were available, DNA was extracted and restored prior to amplification. Industry standard DNA amplification techniques were done on all samples prior to genotyping in accord with established protocols of the NIDA Genetics Consortium. Our genotype data were run and processed on the Illumina Infinium OmniExpress_v_1.3 array. This array has 714,238 SNPs, and was designed many years ago. There were 628 SNPs on the array that do not correspond to any chromosome position, and these were removed. Genotype data were submitted by NIDA’s contracted genotyping laboratory in six batches over time to NIDA’s contracted dbGaP data management group, which conducted quality control (QC) analyses. QC analysis included an assessment of batch effects on for five of the six batches. (One of the batches, with only 12 samples, was too small for QC analysis of batch effects.) Standard NIDA Genetic Consortium cleaning was performed. Samples with a call rate <.85 were removed. Only one sample per person was retained. When more than one specimen was genotyped from one subject, only the sample with the higher call rate was retained (provided, of course, that that call rate was ≥ 0.85). We have retained some people we know are related, including some found to have been related through genotyping; the pedigree file describes those relationships. In summary, key cleaning steps include: 1. Using PLINK to check gender discrepancy. 2. Using PREST-PLUS and KING (Kinship-based Inference for GWAS) to check relatedness. 3. Using PEDCHECK and PLINK to check/zero-out Mendelian error. 4. Using PLINK to perform sample QC, SNP QC, along with KING to perform chromosome X and chromosome Y QC. 5. SNP-QC: Batch-effect: 5 Batches were compared (one batch, with few samples, was not). These five batches were compared to each other in all ten possible pairs, one batch vs. another batch, examining SNP allele frequency discrepancies by population (from GRAF), Fisher Exact Allelic test, with the criterion of p<5e-8 for removal. 6. SNP-QC: discordant SNPs in QC duplicates. Compared 25 QC duplicated samples with call rate > 0.95, removed SNPs with 3+ discordance. 7. There were 1,056 SNPs that were monomorphic; these have been retained so they can be included in analyses in which our dbGaP data are combined with those from other cohorts (in the latter of which those SNPs may not be monomorphic). The final cleaned dataset submitted has 8,898 samples and 606,793 SNPs.

  Study phs002140

• Small RNA Sequencing across Diverse Biofluids Identifies Optimal Methods for exRNA Isolation

  We tested the efficiency and reproducibility of 10 RNA isolation methods on 5 different biofluids. Pooled human plasma and serum, bile and urine were used along with cell culture supernatants from human embryonic stem cells (hESC), primary neonatal rat ventricular myocytes (NRVM) and human cholangiocarcinoma cells (KMBC). 4 RNA isolation methods designed to isolate total RNA from bio fluids without enrichment of exRNA carriers were used. 6 methods that preferentially enriched for exRNA carriers were also compared. Small RNA libraries were constructed using the NEBNext small-RNA library prep. The efficiency and reproducibility of the different RNA isolation methods was compared using small-RNA seq.

  Study phs002143

• Translating Gene-Calcium Interactions to Precision Medicine for Colorectal Cancer

  Identify functional and rare SNPs and their interactions with Ca intake levels in relation to risk of incident adenoma using whole-exome resequencing of 2468 colorectal adenoma cases and controls from PLCO. We will genotype promising rare and functional SNPs associated with incident adenoma in 570 incident cancer cases, 1,710 healthy controls, 710 adenoma recurrence cases and 966 recurrent adenoma controls from the intervention arm of PLCO to both confirm the associations with adenoma, and to identify when, in the natural history of CRC, chemoprevention by Ca-gene interactions occurs. We hypothesize that Ca-gene interactions prevent early-stage (incident adenoma) and mid-stage (adenoma recurrence) carcinogenesis as well as span the entire CRC carcinogenesis spectrum (i.e. incident CRC).

  Study phs002164

• Serrated Colorectal Cancer: An Emerging Disease Subtype

  This dataset represents two combined study populations. Serrated Colorectal Cancer: An Emerging Disease Subtype (called the Advanced Colorectal Cancer of Serrated Subtype Study or ACCESS Study) was a grant awarded to investigate a newly-recognized, biologically-distinct subtype of colorectal cancer (CRC) called “serrated CRC.” The objective of this project was to characterize factors related to the genetic predisposition, clinical presentation, and prognosis of serrated CRC. The study recruited incident invasive CRC cases diagnosed between April 2016 and December 2018, aged 20-74 years at diagnosis. Cases were identified through the Surveillance, Epidemiology and End Results (SEER) cancer registry serving 13 counties in western Washington State. Eligibility for all individuals was limited to those who were English-speaking and could consent. Participation included completing a baseline epidemiologic questionnaire shortly after diagnosis, optional donation of a saliva sample for genetic analysis, and optional consent to release of medical records and tissue specimens related to their diagnosis. Tumor specimens were tested for serrated CRC-defining molecular characteristics. Further, we have vital status on all participants and cause of death on those that have died since enrollment. Hormones and Colon Cancer: Epigenetic Subtypes, Risks, and Survival (called the Post-Menopausal Hormones Study or PMH Study) was a grant awarded to investigate the impact of post-menopausal hormone use on colon cancer risk, tumor molecular characteristics, and outcomes. Eligible cases were females, newly diagnosed with invasive colorectal adenocarcinoma between October 1998 and February 2002, aged 50 to 74 years. Cases were residents of 10 out of the 13 counties in western Washington State served by the Surveillance, Epidemiology and End Results (SEER) cancer registry. Eligibility for all individuals was limited to those who were English-speaking with available telephone numbers, in which they could be contacted. Unrelated population-based controls were randomly selected according to age distribution (in 5-year age intervals) of the eligible cases by using lists of licensed drivers from the Washington State Department of Licensing (for individuals aged 50 to 64 years) and rosters from the Health Care Financing Administration (now the Centers for Medicare and Medicaid, for individuals older than 64 years). Participation included completing a baseline epidemiologic questionnaire, optional donation of a saliva sample for genetic analysis, and (for cases only) optional consent to release of medical records and tissue specimens related to their diagnosis. Tumor specimens were tested for epigenetic and other molecular characteristics. The ACCESS study was supported by funding from the National Cancer Institute of the National Institutes of Health (NCI/NIH) (R01CA196337, PI: Newcomb, PA), as was the PMH Study (R01CA076366, PI: Newcomb, PA). Additional support for the PMH Study came from the Seattle site of the Colon Cancer Family Registry (SCCFR) (U01CA167551, PI: Jenkins, M, and U01/U24CA074794, PI: Newcomb, PA). Additional support for case ascertainment was provided by the Cancer Surveillance System of the Fred Hutchinson Cancer Center, which is funded by Contract Number HHSN261201300012I; NCI Control Number: N01 PC-2013-00012; Contract Number HHSN261201800004I; and NCI Control Number: N01 PC-2018-00004 from the Surveillance, Epidemiology and End Results (SEER) Program of the National Cancer Institute with additional support from the Fred Hutchinson Cancer Center and the State of Washington. This research was also supported by the Genomics and Bioinformatics, Comparative Medicine, Specialized Pathology, Collaborative Data Services, and Experimental Histopathology Shared Resources of the Fred Hutch/University of Washington Cancer Consortium (P30 CA015704).Tumor marker testing was performed using formalin-fixed paraffin-embedded diagnostic tumor tissue specimens, and DNA extracted from those specimens. Testing for microsatellite instability (MSI) was based on either a 10-gene panel (BAT25, BAT26, BAT40, MYCL, D5S346, D17S250, ACTC, D18S55, D10S197, BAT34C4) or a 4-marker immunohistochemistry panel of DNA mismatch repair proteins (MLH1, MSH2, MSH6, PMS2). CpG island methylator phenotype (CIMP) testing was based on a validated quantitative DNA methylation assay using a five-gene panel (CACNA1G, IGF2, NEUROG1, RUNX3, SOCS1) or eight-gene panel (CACNA1G, IGF2, NEUROG1, RUNX3, SOCS1, MLH1, CRABP1, CDKN2A). Somatic p.V600E BRAF mutation status was tested for using a fluorescent allele-specific PCR assay. KRAS mutations in codons 12 and 13 were also assessed through forward and reverse sequencing of amplified tumor DNA. DNA was extracted from blood/saliva samples using conventional methods. The genotyping panel completed was the Build37 OncoArray500K-C, including 1%-6% blinded duplicates to monitor the quality of the genotyping. Quality control procedures were performed to 1) make sure that there were no patterns of missing data by batch, study, or plate, 2) check for gender discrepancies and kinship, 3) complete Principal Component Analysis, and 4) test for Hardy-Weinberg equilibrium (HWE). Samples were excluded based on call rate, heterozygosity, unexpected duplicates, gender discrepancy, and unexpectedly high identity-by-descent or unexpected genotypic concordance (>65%) with another individual. In addition, variants were excluded based on call rate (98%), lack of HWE in controls (P

  Study phs002171

• Merkel Cell Carcinoma Tissue and Data Repository

  Merkel cell carcinoma (MCC) is a rare, aggressive neuroendocrine skin cancer with increasing incidence in the United States. In this study we performed the first whole genome sequencing of three Merkel cell carcinomas with their matched normal tissues for somatic variant calling. From these data we describe the likely mechanism of Merkel cell polyomavirus integration and focal host genomic amplifications in two tumors and the dominance of UV-mediated mutagenesis in a virus-negative tumor. We also performed poly-A RNA sequencing on a total of six Merkel cell carcinomas to address expression differences between virus-positive and virus-negative MCC.

  Study phs002189

• Genomic and Transcriptomic Profiling of Patients with Malignant Pleural and Peritoneal Mesothelioma: The NCI Cohort

  Malignant mesothelioma is an aggressive cancer with limited treatment options and poor prognosis. Better understanding of mesothelioma genomics and transcriptomics could advance novel therapies. We performed whole-exome sequencing (WXS) of germline and tumors of 122 patients, and RNA-sequencing (RNA-Seq) of tumor samples of 100 patients with pleural, peritoneal, and tunica-vaginalis mesothelioma.The biospecimens used in this study were collected as a part of the Mesothelioma Natural History Protocol (https://clinicaltrials.gov/ct2/show/NCT01950572), wherein, samples were collected from subjects with malignant mesothelioma as listed in the eligibility criteria below and written consent from each participant was obtained before sample acquisition.

  Study phs002207

• HTAN MCL Pre-Cancer Atlas Pilot Project - Targeted Sequencing Development Study

  The HTAN-MCL Pre-Cancer Atlas Pilot Project (PCAPP) is the result of a collaboration between the seven members of the MCL consortium. Across four organ types, PCAPP's goal is to collect and profile pre-malignant lesions for gene expression, DNA mutations, single-cell gene expression and immune-environment. Most PML are small in size and only available come from formalin fixed paraffin embedded archived tissue. The primary goal of PCAPP is to 1) understand the logistical challenges of PML specimen collection, 2) document technical limitations of the assays that are specific to the PML and 3) overcome them to support the generation of a more comprehensive Pre-Cancer Atlas in the future. The current upload provides RNA and DNA sequencing from participants with DCIS who were studied at the University of San Diego and the University of Vermont. Description of the overall study: A. Background/Significance One of the critical barriers to developing new approaches for cancer detection and prevention is the lack of understanding of the key molecular and cellular changes that cause cancer initiation and progression. Unlike the extensive work that has been done profiling advanced stage tumors, few studies have comprehensively profiled the molecular alterations found in precancerous tissues. Premalignant lesions are currently characterized by histologic changes that precede the development of invasive carcinoma1,2.These lesions can often be identified in regions surrounding an invasive tumor or in biopsies taken from patients undergoing diagnostic evaluation for suspicion of cancer. Currently, limited metrics exist to identify lesions that will likely progress to carcinoma and require intervention from those that will naturally regress or remain stable3,4. Characterization of the molecular alterations in premalignant lesions and the corresponding changes in the microenvironment would hasten the development of biomarkers for early detection and risk stratification as well as suggest preventive interventions to reverse or delay the development of cancer. Our pilot study will establish the feasibility of transcriptomic, genomic and immune profiling of FFPE premalignant lesions from multiple organ sites, collected and profiled with uniform SOPs across multiple institutions within the MCL consortium. We will characterize the molecular alterations in precancerous lesions and the corresponding microenvironment in four major organ sites, in order to uncover the molecular and cellular determinants of premalignancy, and establish standardized sequencing and immunohistochemistry protocols on FFPE precancerous tissue. We will also evaluate the technical feasibility of single nuclei sequencing of small FFPE pre-cancer lesions. Successful completion of the proposed pilot study will set the stage for expansion and development of a comprehensive Pre-Cancer Atlas (PCA) as part of the NCI's moonshot.B. Specific Aims Aim 1: Collect premalignant lesions (PML) and their associated microenvironment via LCM from FFPE tissue across four organ sites (breast, lung, pancreas & prostate). Aim 2: Perform bulk RNA and DNA seq on premalignant FFPE samples (and flash frozen tissue where available) and compare the genomic/transcriptomic alterations within and across organ sites. C. ApproachAim 1: Collect premalignant lesions (PML) and their associated microenvironment via LCM from FFPE tissue across four organ sites (breast, lung, pancreas & prostate). MethodsI. Patient Population/Sample Collection: Overview of the sites collecting PML tissue from the respective organs is provided in Table 1 and a full description of the biospecimens to be obtained is described in detail for each organ type below. Table 1. Breakdown of cohort by tissue type and collection site.Organ siteBreastLungPancreasProstateType of PMLDCISAAH, Squamous Dysplasia/CISIPMNsPINCollection of PatientsUCSF/UCSDUVMBU*/UCLAVanderbilt/MoffittMDACC*JHUStanford*# of Patients201920 (10 of each type)20 (10 of each type)242020Total patients per Organ39402440Note: single nuclei/cell RNA-Seq will be performed on 4-5 FFPE samples from each of the organ types 1. DCIS lesions from breast tissue: DCIS lesions will be collected from 39 patients (20 from UCSF/UCSD & 19 from UVM) with primary low or high-grade DCIS diagnosed from a breast core biopsy. Subsequent resected lumpectomy or mastectomy tissues will be prospectively sampled in the vicinity of the prior biopsy site using multiple approaches: 1) Live cells (heterogeneous mix) will be obtained as a cell scrape slurry from the lesion surface or by fine needle aspirate (FNA); 2) For a subset of specimens where size is sufficient, a block of breast tissue with DCIS will be fresh-frozen; 3) The remainder of the specimen will be taken for routine formalin-fixation and paraffin-embedding (FFPE). The FFPE sample will be annotated to identify the matched FFPE tissue block adjacent to the fresh-frozen sample and will be sectioned for use in bulk and single nuclei sequencing . We will dissect DCIS, adjacent normal and when available, associated carcinoma. In addition, when possible, normal tissue will be collected from a tissue block lacking lesions as well as collection of blood. A subset of patients (n = 5 | FFPE, flash frozen and fresh) will be sent to the Broad Institute for single nuclei/cell sequencing.2. AAH and squamous dysplastic/CIS lesions from airway and lung tissue: For squamous cell lung cancer, we will collect endobronchial biopsies from abnormal airway regions identified on autofluroscence bronchoscopy or identify PMLs in the margins of resected lung tissue. We will study 20 patients (5 each from BU/UCLA/Vanderbilt/Moffitt) with pre-invasive squamous lesions (moderate-severe dysplasia or carcinoma in situ (CIS)) identified on pathologic examination. LCM of the premalignant region and adjacent normal epithelium will be performed as well as the invasive tumor for those collected from the resection margin (n=5 from UCLA). On a subset of lesions collected at bronchoscopy (n=5), we will collect additional biopsies that will be flash frozen and fresh for single nuclei and cell sequencing, respectively, performed at the Broad Institute. In parallel to the work at the Broad, BU will perform single cell RNA-seq on these freshly cell sorted tissues (n = 5). Blood will be collected on all patients for genomic studies. For lung adenocarcinoma, we will collect resected FFPE lung tissues from 20 patients (10 from UCLA and 10 from Vanderbilt/Moffitt) with early stage lung adenocarcinoma that harbor atypical adenomatous (AAH) premalignant lesions in the resection margin. We will LCM multiple AAH regions (3-5 per patient) as well as adjacent regions of normal epithelium and invasive adenocarcinoma. In addition, blood will be collected on all patients for genomic studies. 3. IPMNs from pancreatic tissue: For pancreatic cancer PML, we will collect low and high grade lesions from 24 patients representing macroscopic Intraductal Papillary Mucinous Neoplasms (IPMN) (n=24) from surgically resected specimens along with blood samples. Archival FFPE specimens of microscopic PanIN lesions, occurring multi-focally adjacent to invasive PDAC, and archival IPMN lesions (with or without associated invasive cancer), along with the adjacent normal tissue, will undergo LCM and utilized for bulk DNA and RNA sequencing. If matched frozen tissues are available for a subset of these FFPE samples, we will bank for comparison of profiles. Because IPMNs are macroscopic lesions, they provide an opportunity for obtaining the samples fresh and therefore can be used for single cell sequencing (in contrast to PanINs). Therefore, 5 freshly obtained IPMNs will be used for the single cell RNA sequencing studies performed at both the Broad Institute and MDACC, and the matched FFPE and/or frozen sections from these lesions (obtained from the adjacent PML) will be sent to Broad Institute as a pilot to assess "single nuclei" RNA sequencing.4. PINs from prostate tissue: For prostate cancer PML, there will be 40 samples of Prostatic Intraepithelial Neoplasia (PIN) collected between the Stanford and JHU sites (20 cases per site). At the Stanford site, 20 prostate specimens detected by PSA screening who have/will undergo surgery (radical prostatectomy) for clinically localized disease will make up the final cohort. The age range of the participants would be 40-75, and we anticipate that 18 will be Caucasian, 1 Asian and 1 Latino or African American based on the practice demographics practice at Stanford. Clinical and MRI data will also be collected for these samples. We will collect low grade (e.g. Gleason score of 6/Grade group 1; n=10) and high grade (Gleason score 4+3=7 or higher/Grade group 3 or higher; n=10) PINs from FFPE samples that have prostate carcinoma. In addition to obtaining LCM archival samples of low and high grade PIN, we will also obtain normal prostatic epithelial from the peripheral, central and transition zones as well as multiple samples of prostate carcinoma in order to obtain the spectrum of Gleason grades in the carcinoma as needed. LCM samples will be used for bulk DNA and RNA sequencing. In addition, single cells will be dissected from FFPE samples to prepare single cell RNA seq libraries using techniques developed at Stanford, and FFPE tissue will be sent to the Broad for single nuclei sequencing. When available, flash frozen and fresh samples from these prostates will be archived and prepared for single nuclei and cell sequencing, respectively, at the Broad Institute and at Stanford (single cell only). JHU will also capture 10 cases (5 grade group 1 and 5 grade group 2) of high grade PIN, normal and invasive adenocarcinoma using frozen sections from fresh frozen tissues. When possible these will be from the same patients as the FFPE samples. Since frozen sections can be quite challenging to morphologically determine high grade PIN from normal epithelium, for these samples we will perform a number of additional tissue-based characterizations. These will include a multicolor combined basal cells (p63 and CK903) and PIN/carcinoma markers (AMACR) referred to in the cocktail as "PIN4", c-MYC (referred to as MYC) protein5, by IHC and mRNA by in situ hybridization (AM De Marzo, Q Zheng unpublished observations), telomere length by in situ hybridization6 and the 5'ETS/45S rRNA7. For these slides, the whole slides will be scanned with a Hammamatsu Nanozoomer with a 40x objective and regions of interest will be annotated as a guide for LCM.II. Laser-capture microdissection (LCM): FFPE tissue blocks will be sectioned at 7μm thickness and serial sections will be stained with H&E. LCM will be performed utilizing standard LCM systems, such as Leica LMD7000 and ArcturusXT at each site. Regions of premalignancy will be dissected and RNA/DNA will be extracted from microdissected cells using the Qiagen All Prep DNA/RNA FFPE Kit. Aim 2: Perform bulk RNA and DNA seq on premalignant FFPE samples and compare the genomic/transcriptomic alterations within and across organ sites. Rationale: There have been limited studies characterizing the genomic and transcriptomic landscape of premalignant lesions associated with breast, pancreatic, lung or prostate cancers. Characterizing the molecular determinants of premalignant disease that are unique and shared across multiple organs will enable new candidate biomarkers for early detection and novel therapeutic strategies for early intervention. MethodsBulk RNA-seq of LCM FFPE tissue: All participating sites will perform bulk RNA-seq in accordance with SOPs developed at BU. In brief, total RNA will be isolated from LCM'd lesion and associated microenvironment tissue using the Qiagen All Prep DNA/RNA FFPE Kit and quality will be assessed with the Agilent Bioanalyzer. Libraries will be generated with the Illumina TruSeq Access kit (for FFPE samples). They will be sequenced on the Illumina HiSeq2500 with 75base-pair paired-end reads. Quality of FASTQ files will be assessed with FastQC. Reads will be aligned to the human genome with STAR and gene-level and isoform-level expression will be quantified with RSEM. Splice junction saturation, transcript integrity, and biotype distributions will be calculated for each sample with RSeQC. DESeq2 and EdgeR will be used to identify associations between gene expression profiles and clinical variables while controlling for confounding covariates. BU will serve as an RNA-seq Core to assess reproducibility of FFPE RNA-seq methods across sites. We will perform RNA-seq according to the SOP listed above on a subset of samples for each organ type (total n ~ 20). Bulk seq of DNA from FFPE tissue: All participating sites will perform targeted or whole exome-seq (WES) in accordance with SOPs. In brief, DNA from laser captured material will be isolated using the Qiagen All Prep DNA/RNA FFPE Kit and undergo stringent quality control to ensure high quality input material for genomic profiling. Purified DNA (ideally 100-200 ng) will be used for library preparation and amplification, followed by next generation sequencing using standard protocols distributed by CDMG. Exome-seq methods are considered standardized, thus we will not need a DNA-seq Core to assess reproducibility across sites. We anticipate local centers will use Illumina paired end reads, following the following general approach. 1) DNA library preparation: Paired-end libraries will be prepared following the manufacturer's protocols (Illumina and Agilent), fragmented to 150-200 bp 2) Capture of targeted exome: Whole exome capture will be carried out using the protocol for Agilent's SureSelect Human All Exon kit. Purified capture products will be amplified using the SureSelect GA PCR primers (Agilent) for 12 cycles. 3) Sequencing will be carried out for the captured libraries using at least 100 bp paired-end reads. To achieve high level sensitivity and accuracy for detecting all the mutations in the whole exome, each sample will be sequenced at 200X mean depth. 4) Read mapping and alignment and variant analysis: Sequence short reads will be aligned to a reference genome (NCBI human genome assembly build 38) using BWA-MEM. Local realignment of aligned reads will be performed using Genome Analysis Toolkit (GATK).Data QC: To ensure scientific rigor and consistency among sites in RNA and DNA processing we will include a preliminary analysis of steps in processing and analysis. Protocols for extraction of high quality RNA and DNA from formalin fixed paraffin embedded (FFPE) tissues, which will be used extensively in these studies continue to improve and may have variable implementation among the sites participating in this study. To evaluate consistency of preliminary steps in processing and downstream analyses, we will initially distribute slides from one large FFPE fixed cancer of origin from prostate, breast, lung and pancreatic cancer. Analysis of these samples will allow us to review the DNA and RNA characteristics (yield, purity and strand length) among sites. Downstream analysis of these same samples will also allow us to compare among sites the consistency of variant calls among centers. We will be able to identify if there are some times of calls (such as small insertion deletions) that are more variable among centers versus other types of calls (such as relative gene expression or single base pair substitutions) that we expect to be less variable and to characterize the reliability of findings across sites. We are also including a 5% blind duplicate analysis of RNA sequencing. Samples will be analysed by the participating genomics cores without knowledge of the phenotype. RNA seq and CNA analyses are normalized for batch effects. We will also compare the observed sex to the self-reported sex as based on RNA profiles and exome sequencing of X chromosome genes as another check for processing accuracy and sample management. D. References 1. Wacholder, S. Precursors in Cancer Epidemiology: Aligning Definition and Function. Cancer Epidemiol. Prev. Biomark. 22, 521-527 (2013). PMID: 23549395.2. Berman, J. J. Precancer: The Beginning and the End of Cancer. (Jones & Bartlett Learning, 2011).3. Nasiell, K., Nasiell, M. & Vaćlavinková, V. Behavior of moderate cervical dysplasia during long-term follow-up. Obstet. Gynecol. 61, 609-614 (1983). PMID: 6835614.4. Merrick, D. T. et al. Persistence of Bronchial Dysplasia Is Associated with Development of Invasive Squamous Cell Carcinoma. Cancer Prev. Res. (Phila. Pa.) 9, 96-104 (2016). PMID: 26542061.5. Gurel, B. et al. Nuclear MYC protein overexpression is an early alteration in human prostate carcinogenesis. Mod. Pathol. Off. J. U. S. Can. Acad. Pathol. Inc 21, 1156-1167 (2008). PMID: 18567993.6. Meeker, A. K. et al. Telomere shortening is an early somatic DNA alteration in human prostate tumorigenesis. Cancer Res. 62, 6405-6409 (2002). PMID: 12438224.7. Guner, G. et al. Novel Assay to Detect RNA Polymerase I Activity In Vivo. Mol. Cancer Res. MCR 15, 577-584 (2017). PMID: 28119429.

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