Comprehensive proteogenomic characterization of rare kidney tumors.

Non-clear cell renal cell carcinomas (non-ccRCCs) encompass diverse malignant and benign tumors. Refinement of differential diagnosis biomarkers, markers for early prognosis of aggressive disease, and therapeutic targets to complement immunotherapy are current clinical needs. Multi-omics analyses of 48 non-ccRCCs compared with 103 ccRCCs reveal proteogenomic, phosphorylation, glycosylation, and metabolic aberrations in RCC subtypes. RCCs with high genome instability display overexpression of IGF2BP3 and PYCR1. Integration of single-cell and bulk transcriptome data predicts diverse cell-of-origin and clarifies RCC subtype-specific proteogenomic signatures. Expression of biomarkers MAPRE3, ADGRF5, and GPNMB differentiates renal oncocytoma from chromophobe RCC, and PIGR and SOSTDC1 distinguish papillary RCC from MTSCC. This study expands our knowledge of proteogenomic signatures, biomarkers, and potential therapeutic targets in non-ccRCC.

Frozen tissue coring and layered histological analysis improves cell type-specific proteogenomic characterization of pancreatic adenocarcinoma.

BACKGROUND: Omics characterization of pancreatic adenocarcinoma tissue is complicated by the highly heterogeneous and mixed populations of cells. We evaluate the feasibility and potential benefit of using a coring method to enrich specific regions from bulk tissue and then perform proteogenomic analyses. METHODS: We used the Biopsy Trifecta Extraction (BioTExt) technique to isolate cores of epithelial-enriched and stroma-enriched tissue from pancreatic tumor and adjacent tissue blocks. Histology was assessed at multiple depths throughout each core. DNA sequencing, RNA sequencing, and proteomics were performed on the cored and bulk tissue samples. Supervised and unsupervised analyses were performed based on integrated molecular and histology data. RESULTS: Tissue cores had mixed cell composition at varying depths throughout. Average cell type percentages assessed by histology throughout the core were better associated with KRAS variant allele frequencies than standard histology assessment of the cut surface. Clustering based on serial histology data separated the cores into three groups with enrichment of neoplastic epithelium, stroma, and acinar cells, respectively. Using this classification, tumor overexpressed proteins identified in bulk tissue analysis were assigned into epithelial- or stroma-specific categories, which revealed novel epithelial-specific tumor overexpressed proteins. CONCLUSIONS: Our study demonstrates the feasibility of multi-omics data generation from tissue cores, the necessity of interval H&E stains in serial histology sections, and the utility of coring to improve analysis over bulk tissue data.

Proteogenomic insights suggest druggable pathways in endometrial carcinoma.

We characterized a prospective endometrial carcinoma (EC) cohort containing 138 tumors and 20 enriched normal tissues using 10 different omics platforms. Targeted quantitation of two peptides can predict antigen processing and presentation machinery activity, and may inform patient selection for immunotherapy. Association analysis between MYC activity and metformin treatment in both patients and cell lines suggests a potential role for metformin treatment in non-diabetic patients with elevated MYC activity. PIK3R1 in-frame indels are associated with elevated AKT phosphorylation and increased sensitivity to AKT inhibitors. CTNNB1 hotspot mutations are concentrated near phosphorylation sites mediating pS45-induced degradation of ß-catenin, which may render Wnt-FZD antagonists ineffective. Deep learning accurately predicts EC subtypes and mutations from histopathology images, which may be useful for rapid diagnosis. Overall, this study identified molecular and imaging markers that can be further investigated to guide patient stratification for more precise treatment of EC.

Epigenetic and transcriptomic characterization reveals progression markers and essential pathways in clear cell renal cell carcinoma.

Identifying tumor-cell-specific markers and elucidating their epigenetic regulation and spatial heterogeneity provides mechanistic insights into cancer etiology. Here, we perform snRNA-seq and snATAC-seq in 34 and 28 human clear cell renal cell carcinoma (ccRCC) specimens, respectively, with matched bulk proteogenomics data. By identifying 20 tumor-specific markers through a multi-omics tiered approach, we reveal an association between higher ceruloplasmin (CP) expression and reduced survival. CP knockdown, combined with spatial transcriptomics, suggests a role for CP in regulating hyalinized stroma and tumor-stroma interactions in ccRCC. Intratumoral heterogeneity analysis portrays tumor cell-intrinsic inflammation and epithelial-mesenchymal transition (EMT) as two distinguishing features of tumor subpopulations. Finally, BAP1 mutations are associated with widespread reduction of chromatin accessibility, while PBRM1 mutations generally increase accessibility, with the former affecting five times more accessible peaks than the latter. These integrated analyses reveal the cellular architecture of ccRCC, providing insights into key markers and pathways in ccRCC tumorigenesis.

Neoplastic cell enrichment of tumor tissues using coring and laser microdissection for proteomic and genomic analyses of pancreatic ductal adenocarcinoma.

BACKGROUND: The identification of differentially expressed tumor-associated proteins and genomic alterations driving neoplasia is critical in the development of clinical assays to detect cancers and forms the foundation for understanding cancer biology. One of the challenges in the analysis of pancreatic ductal adenocarcinoma (PDAC) is the low neoplastic cellularity and heterogeneous composition of bulk tumors. To enrich neoplastic cells from bulk tumor tissue, coring, and laser microdissection (LMD) sampling techniques have been employed. In this study, we assessed the protein and KRAS mutation changes associated with samples obtained by these enrichment techniques and evaluated the fraction of neoplastic cells in PDAC for proteomic and genomic analyses. METHODS: Three fresh frozen PDAC tumors and their tumor-matched normal adjacent tissues (NATs) were obtained from three sampling techniques using bulk, coring, and LMD; and analyzed by TMT-based quantitative proteomics. The protein profiles and characterizations of differentially expressed proteins in three sampling groups were determined. These three PDACs and samples of five additional PDACs obtained by the same three sampling techniques were also subjected to genomic analysis to characterize KRAS mutations. RESULTS: The neoplastic cellularity of eight PDACs ranged from less than 10% to over 80% based on morphological review. Distinctive proteomic patterns and abundances of certain tumor-associated proteins were revealed when comparing the tumors and NATs by different sampling techniques. Coring and bulk tissues had comparable proteome profiles, while LMD samples had the most distinct proteome composition compared to bulk tissues. Further genomic analysis of bulk, cored, or LMD samples demonstrated that KRAS mutations were significantly enriched in LMD samples while coring was less effective in enriching for KRAS mutations when bulk tissues contained a relatively low neoplastic cellularity. CONCLUSIONS: In addition to bulk tissues, samples from LMD and coring techniques can be used for proteogenomic studies. The greatest enrichment of neoplastic cellularity is obtained with the LMD technique.

Rare cases of medulloblastoma with hypermutation.

BACKGROUND: Medulloblastoma is the most common malignant brain tumor of childhood and is considered a tumor with low mutational burden (~1 Mut/Mb). Therefore, though the medulloblastoma genomes have been extensively characterized in literature, reports on potential hypermutations and underlying mutagenic processes in medulloblastomas are limited. AIM: In this report, we studied the landscape of mutational burden in primary and recurrent medulloblastoma. Furthermore, we wanted to understand the differences in underlying mutagenic mechanisms in medulloblastoma with low and high mutational burdens. METHODS: Fifty-three primary and recurrent medulloblastoma genomic sequence were downloaded from the European Genome Archive as BAM files. Thirty-three cases were obtained from formalin-fixed paraffin-embedded tissues from pathology diagnostic archives of Spectrum Health and Cooperative Human Tissue Network. Somatic mutations were called using Mutect2, following best practices guidelines for Genome Analysis Toolkit V4. Mutational signatures were analyzed using deconstructSigs. RESULTS: We identified nine medulloblastoma cases with high mutational burden (>5 Mut/Mb). Of them, five cases met the criteria of hypermutation (>10Mut/Mb), two of the five tumors had canonical mutations in the POLE proof-reading domain, where a large proportion of mutations in these tumor genomes contributed to signature 10. The hypermutated cases also demonstrated mutational signatures 14, 15, and 21, indicating the role of mis match repair deficiency in their mutagenesis. Of the four known molecular subgroups in medulloblastoma-SHH, WNT, Group 3, and Group 4-both the POLE-mutated cases belonged to the SHH subgroup. This report identifies rare cases of hypermutation in medulloblastoma driven by defects in DNA repair mechanisms. CONCLUSION: Hypermutation in medulloblastoma can impact therapeutic decisions, especially at recurrence in otherwise fatal high risk SHH-medulloblastomas. A defect in DNA repair leading to SHH -medulloblastoma is yet another important mechanism that should be further investigated in the genesis of these tumors. Therefore, this report provides important scientific and clinical rationale for future research looking for incidence of hypermutation in large cohorts of medulloblastoma patients.

Proteogenomic characterization of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with poor patient survival. Toward understanding the underlying molecular alterations that drive PDAC oncogenesis, we conducted comprehensive proteogenomic analysis of 140 pancreatic cancers, 67 normal adjacent tissues, and 9 normal pancreatic ductal tissues. Proteomic, phosphoproteomic, and glycoproteomic analyses were used to characterize proteins and their modifications. In addition, whole-genome sequencing, whole-exome sequencing, methylation, RNA sequencing (RNA-seq), and microRNA sequencing (miRNA-seq) were performed on the same tissues to facilitate an integrated proteogenomic analysis and determine the impact of genomic alterations on protein expression, signaling pathways, and post-translational modifications. To ensure robust downstream analyses, tumor neoplastic cellularity was assessed via multiple orthogonal strategies using molecular features and verified via pathological estimation of tumor cellularity based on histological review. This integrated proteogenomic characterization of PDAC will serve as a valuable resource for the community, paving the way for early detection and identification of novel therapeutic targets.

Impact of Preanalytical Factors on the Measurement of Tumor Tissue Biomarkers Using Immunohistochemistry.

Analysis of formalin-fixed paraffin-embedded (FFPE) tissue by immunohistochemistry (IHC) is commonplace in clinical and research laboratories. However, reports suggest that IHC results can be compromised by biospecimen preanalytical factors. The National Cancer Institute's Biospecimen Preanalytical Variables Program conducted a systematic study to examine the potential effects of delay to fixation (DTF) and time in fixative (TIF) on IHC using 24 cancer biomarkers. Differences in IHC staining, relative to controls with a DTF of 1 hr, were observed in FFPE kidney tumor specimens after a DTF of ≥2 hr. Reductions in H-score and/or staining intensity were observed for c-MET, p53, PAX2, PAX8, pAKT, and survivin, whereas increases were observed for RCC1, EGFR, and CD10. Prolonged TIF of 72 hr resulted in significantly reduced H-scores of CD44 and c-Met in kidney tumor specimens, compared with controls with 12-hr TIF. An elevated probability of altered staining intensity due to DTF was observed for nine antigens, whereas for prolonged TIF an elevated probability was observed for one antigen. Results reported here and elsewhere across tumor types and antigens support limiting DTF to ≤1 hr when possible and fixing tissues in formalin for 12-24 hr to avoid confounding effects of these preanalytical factors on IHC.

The Biospecimen Preanalytical Variables Program: A Multiassay Comparison of Effects of Delay to Fixation and Fixation Duration on Nucleic Acid Quality.

CONTEXT.­: Despite widespread use of formalin-fixed, paraffin-embedded (FFPE) tissue in clinical and research settings, potential effects of variable tissue processing remain largely unknown. OBJECTIVE.­: To elucidate molecular effects associated with clinically relevant preanalytical variability, the National Cancer Institute initiated the Biospecimen Preanalytical Variables (BPV) program. DESIGN.­: The BPV program, a well-controlled series of systematic, blind and randomized studies, investigated whether a delay to fixation (DTF) or time in fixative (TIF) affects the quantity and quality of DNA and RNA isolated from FFPE colon, kidney, and ovarian tumors in comparison to case-matched snap-frozen controls. RESULTS.­: DNA and RNA yields were comparable among FFPE biospecimens subjected to different DTF and TIF time points. DNA and RNA quality metrics revealed assay- and time point-specific effects of DTF and TIF. A quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was superior when assessing RNA quality, consistently detecting differences between FFPE and snap-frozen biospecimens and among DTF and TIF time points. RNA Integrity Number and DV200 (representing the percentage of RNA fragments longer than 200 nucleotides) displayed more limited sensitivity. Differences in DNA quality (Q-ratio) between FFPE and snap-frozen biospecimens and among DTF and TIF time points were detected with a qPCR-based assay. CONCLUSIONS.­: DNA and RNA quality may be adversely affected in some tumor types by a 12-hour DTF or a TIF of 72 hours. Results presented here as well as those of additional BPV molecular analyses underway will aid in the identification of acceptable delays and optimal fixation times, and quality assays that are suitable predictors of an FFPE biospecimen's fit-for-purpose.

Biobanks and the Moral Concerns of Donors: A Democratic Deliberation.

Do members of the public believe that biobanks should accommodate the moral concerns of donors about the types of research done with their biospecimens? The answer to this question is critical to the future of genomic and precision medicine, endeavors that rely on a public willing to share their biospecimens and medical data. To explore public attitudes regarding the requirements of consent for biobank donations, we organized three democratic deliberations involving 180 participants. The deliberative sessions involved small group discussions informed by presentations given by experts in both biobank research and ethics. We found that participants had a sophisticated understanding of the ethical problems of biobank consent and the complexity of balancing donor concerns while promoting research important to the future of health care. Our research shows how deliberative methods can offer policy makers creative ideas for accommodating the moral concerns of donors in the biobank consent process.

Cold Ischemia Score: An mRNA Assay for the Detection of Extended Cold Ischemia in Formalin-Fixed, Paraffin-Embedded Tissue.

Although there are thousands of formalin-fixed paraffin-embedded (FFPE) tissue blocks potentially available for scientific research, many are of questionable quality, partly due to unknown preanalytical variables. We analyzed FFPE tissue biospecimens as part of the National Cancer Institute (NCI) Biospecimen Preanalytical Variables program to identify mRNA markers denoting cold ischemic time. The mRNA was extracted from colon, kidney, and ovary cancer FFPE blocks (40 patients, 10-12 hr fixation time) with 1, 2, 3, and 12 hr cold ischemic times, then analyzed using qRT-PCR for 23 genes selected following a literature search. No genes tested could determine short ischemic times (1-3 hr). However, a combination of three unstable genes normalized to a more stable gene could generate a "Cold Ischemia Score" that could distinguish 1 to 3 hr cold ischemia from 12 hr cold ischemia with 62% sensitivity and 84% specificity.

Small Nucleolar RNA Score: An Assay to Detect Formalin-Overfixed Tissue.

Although there are millions of formalin-fixed paraffin-embedded (FFPE) tissue blocks potentially available for scientific research, many are of questionable quality, partly due to unknown fixation conditions. We analyzed FFPE tissue biospecimens as part of the NCI Biospecimen Preanalytical Variables (BPV) program to identify microRNA (miRNA) markers for fixation time. miRNA was extracted from kidney and ovary tumor FFPE blocks (19 patients, cold ischemia ≤2 hours) with 6, 12, 24, and 72 hours fixation times, then analyzed using the WaferGen SmartChip platform (miRNA chip with 1036 miRNA targets). For fixation time, principal component analysis of miRNA chip expression data separated 72 hours fixed samples from 6 to 24 hours fixed samples. A set of small nuclear RNA (snRNA) targets was identified that best determines fixation time and was validated using a second independent cohort of seven different tissue types. A customized assay was then developed, based on a set of 24 miRNA and snRNA targets, and a simple "snoRNA score" defined. This score detects FFPE tissue samples with fixation for 72 hours or more, with 79% sensitivity and 80% specificity. It can therefore be used to assess the fitness-for-purpose of FFPE samples for DNA or RNA-based research or clinical assays, which are known to be of limited robustness to formalin overfixation.

The College of American Pathologists Biorepository Accreditation Program: Results from the First 5 Years.

The College of American Pathologists (CAP) developed the Biorepository Accreditation Program (BAP) in 2012. This program integrates best practices from the International Society for Biological and Environmental Biorepositories, the National Cancer Institute, the Organisation for Economic Cooperation and Development, the Center for Medicare and Medicaid Services, and the CAP Laboratory Accreditation Program. The goal of this elective program is to provide requirements for standardization in biorepository processes that will result in high-quality specimens that can be used to support research, drug discovery, and personalized medicine. CAP uses a peer inspection model to ensure the inspectors have proper expertise and to promote educational efforts through information sharing. Lead inspectors are comprised of pathologists, PhDs, and managers of biorepositories and they are often supported by CAP staff inspectors. Accreditation is a 3-year continuous cycle of quality with a peer inspection occurring at the start of year 1 and a self-inspection and CAP desk assessment at the start of year 2 and 3. At this time 53 biorepositories are fully CAP BAP accredited and 13 are in the process of obtaining accreditation. There are currently 273 established standards with requirement lists customized based on the scope of activities performed by a biorepository. A total of 90 inspections were completed between May 2012 and December 2016. Sixty-one were initial inspections and 29 were reinspections. A total of 527 deficiencies were identified in the areas of Equipment/Instrumentation (22%), Information Technology (18%), Specimen Handling and QC (15%), Quality Management (16%), Personnel (11%), Safety (10%), Facilities (6%), and Regulatory (2%). Assessment of common deficiencies identifies areas of focus for continuous improvement and educational opportunities. Overall success of the program is high based on the current enrollment of 66 biorepositories, anecdotal participant feedback and increasing national recognition of the BAP in federal documents.

Effect of deliberation on the public's attitudes toward consent policies for biobank research.

In this study, we evaluate the effect of education and deliberation on the willingness of members of the public to donate tissue to biobank research and on their attitudes regarding various biobank consent policies. Participants were randomly assigned to a democratic deliberation (DD) group, an education group that received only written materials, and a control group. Participants completed a survey before the deliberation and two surveys post-deliberation: one on (or just after) the deliberation day, and one 4 weeks later. Subjects were asked to rate 5 biobank consent policies as acceptable (or not) and to identify the best and worst policies. Analyses compared acceptability of different policy options and changes in attitudes across the three groups. After deliberation, subjects in the DD group were less likely to find broad consent (defined here as consent for the use of donations in an unspecified range of future research studies, subject to content and process restrictions) and study-by-study consent acceptable. The DD group was also significantly less likely to endorse broad consent as the best policy (OR = 0.34), and more likely to prefer alternative consent options. These results raise ethical challenges to the current widespread reliance on broad consent in biobank research, but do not support study-by-study consent.

Comprehensive molecular profiling of 718 Multiple Myelomas reveals significant differences in mutation frequencies between African and European descent cases.

Multiple Myeloma (MM) is a plasma cell malignancy with significantly greater incidence and mortality rates among African Americans (AA) compared to Caucasians (CA). The overall goal of this study is to elucidate differences in molecular alterations in MM as a function of self-reported race and genetic ancestry. Our study utilized somatic whole exome, RNA-sequencing, and correlated clinical data from 718 MM patients from the Multiple Myeloma Research Foundation CoMMpass study Interim Analysis 9. Somatic mutational analyses based upon self-reported race corrected for ancestry revealed significant differences in mutation frequency between groups. Of interest, BCL7A, BRWD3, and AUTS2 demonstrate significantly higher mutation frequencies among AA cases. These genes are all involved in translocations in B-cell malignancies. Moreover, we detected a significant difference in mutation frequency of TP53 and IRF4 with frequencies higher among CA cases. Our study provides rationale for interrogating diverse tumor cohorts to best understand tumor genomics across populations.

A call to standardize preanalytic data elements for biospecimens.

CONTEXT: Biospecimens must have appropriate clinical annotation (data) to ensure optimal quality for both patient care and research. Clinical preanalytic variables are the focus of this study. OBJECTIVE: To define the essential preanalytic variables (data fields) that should be attached to every collected biospecimen and to provide a complete list of such variables, along with their relative importance, which can vary, depending on downstream use, institutional needs, and information technology capabilities. DESIGN: The College of American Pathologists Diagnostic Intelligence and Health Information Technology Committee sponsored a Biorepository Working Group to develop a ranked list of the preanalytic variables for annotating biospecimens. Members of the working group were experts in anatomic, clinical, and molecular pathology; biobanking; medical informatics; and accreditation. Several members had experience with federal government programs, such as the National Cancer Institute's Biospecimens and Biorepository Branch and the National Cancer Institute's Community Cancer Center Program. Potential preanalytic variables were identified and ranked along with available supporting evidence, definitions, and potential negative effects if the variable was not attached to the biospecimen. Additional national and international stakeholders reviewed the draft manuscript. RESULTS: The ranked listing of 170 preanalytic variables produced can be used as a guide for site-specific implementation into patient care and/or research biorepository processes. Conclusions.-In our collective experience, it is often difficult to choose which of the many preanalytic variables to attach to any specific set of biospecimens used for patient care and/or research. The provided ranked list should aid in the selection of preanalytic variables for a given biospecimen collection.

Evaluation of sex-specific gene expression in archived dried blood spots (DBS).

Screening newborns for treatable serious conditions is mandated in all US states and many other countries. After screening, Guthrie cards with residual blood (whole spots or portions of spots) are typically stored at ambient temperature in many facilities. The potential of archived dried blood spots (DBS) for at-birth molecular studies in epidemiological and clinical research is substantial. However, it is also challenging as analytes from DBS may be degraded due to preparation and storage conditions. We previously reported an improved assay for obtaining global RNA gene expression from blood spots. Here, we evaluated sex-specific gene expression and its preservation in DBS using oligonucleotide microarray technology. We found X inactivation-specific transcript (XIST), lysine-specific demethylase 5D (KDM5D) (also known as selected cDNA on Y, homolog of mouse (SMCY)), uncharacterized LOC729444 (LOC729444), and testis-specific transcript, Y-linked 21 (TTTY21) to be differentially-expressed by sex of the newborn. Our finding that trait-specific RNA gene expression is preserved in unfrozen DBS, demonstrates the technical feasibility of performing molecular genetic profiling using such samples. With millions of DBS potentially available for research, we see new opportunities in using newborn molecular gene expression to better understand molecular pathogenesis of perinatal diseases.

Basal cell carcinoma: stressful life events and the tumor environment.

CONTEXT: Child emotional maltreatment can result in lasting immune dysregulation that may be heightened in the context of more recent life stress. Basal cell carcinoma (BCC) is the most common skin cancer, and the immune system plays a prominent role in tumor appearance and progression. OBJECTIVE: To address associations among recent severe life events, childhood parental emotional maltreatment, depression, and messenger RNA (mRNA) coding for immune markers associated with BCC tumor progression and regression. DESIGN: We collected information about early parent-child experiences, severe life events in the past year as assessed by the Life Events and Difficulties Schedule, depression, and mRNA for immune markers associated with BCC tumor progression and regression from patients with BCC tumors. SETTING: University medical center. PARTICIPANTS: Ninety-one patients with BCC (ages, 23-92 years) who had a previous BCC tumor. MAIN OUTCOME MEASURES: The expression of 4 BCC tumor mRNA markers (CD25, CD3ε, intercellular adhesion molecule 1, and CD68) that have been linked to BCC tumor progression and regression were assessed in BCC tumor biopsy specimens. RESULTS: Both maternal and paternal emotional maltreatment interacted with the occurrence of severe life events to predict the local immune response to the tumor (adjusted P = .009 and P = .03, respectively). Among BCC patients who had experienced a severe life event within the past year, those who were emotionally maltreated by their mothers (P = .007) or fathers (P = .02) as children had a poorer immune response to the BCC tumor. Emotional maltreatment was unrelated to BCC immune responses among those who did not experience a severe life event. Depressive symptoms were not associated with the local tumor immune response. CONCLUSIONS: Troubled early parent-child relationships, in combination with a severe life event in the past year, predicted immune responses to a BCC tumor. The immunoreactivity observed in BCCs and the surrounding stroma reflects an anti-tumor-specific immune response that can be altered by stress.