Leiomyosarcoma and liposarcoma are common subtypes of soft tissue sarcoma (STS). Patients with metastatic leiomyosarcoma or dedifferentiated liposarcoma (DDLPS) typically have worse outcomes compared ...with localized leiomyosarcoma or well-differentiated liposarcoma (WDLPS). A better understanding of genetic changes between primary/metastatic leiomyosarcoma and between WDLPS/DDLPS may provide insight into their genetic evolution.
We interrogated whole-exome sequencing (WES) from "trios" of normal tissue, primary tumor, and metastatic tumor from individual patients with leiomyosarcoma (
= 9), and trios of normal tissue, well-differentiated tumor, and dedifferentiated tumor from individual patients with liposarcoma (
= 19). Specifically, we performed mutational, copy number, and tumor evolution analyses on these cohorts and compared patterns among leiomyosarcoma and liposarcoma trios.
Leiomyosarcoma cases harbored shared drivers through a typical parent/child relationship where the metastatic tumor was derived from the primary tumor. In contrast, while all liposarcoma cases shared the characteristic focal chromosome 12 amplicon, most paired liposarcoma cases did not share additional mutations, suggesting a divergent evolutionary pattern from a common precursor. No highly recurrent genomic alterations from WES were identified that could be implicated as driving the progression of disease in either sarcoma subtype.
From a genomic perspective, leiomyosarcoma metastases contain genetic alterations that are also found in primary tumors. WDLPS and DDLPS, however, appear to divergently evolve from a common precursor harboring 12q amplification, rather than as a transformation to a higher-grade tumor. Further efforts to identify specific drivers of these distinct evolutionary patterns may inform future translational and clinical research in STS.
'Introduction': The objective of this cross-sectional, descriptive study was to identify unpaid caregiver differences in demographics, competing work responsibilities, support, health, caregiver ...burden, and interaction with healthcare professionals in US metropolitan and non-metropolitan settings.
'Methods': This study leveraged the nationally representative survey of older adults and their unpaid caregivers residing in the USA: the 2017 National Health and Aging Trends Study and National Study of Caregiving. Participants were unpaid caregivers for community-dwelling older adults.
'Results': A total of 2278 unique unpaid caregivers corresponding to 1431 care recipients were investigated for this study. Non-metropolitan caregivers had significantly lower income than their metropolitan counterparts, were more likely to be married or have a partner, missed less work, and, when traveling to provide care, traveled on average fewer minutes than metropolitan caregivers. However, there were no significant differences in relationship to caregiver, impact on primary work responsibilities, financial assistance, resource utilization or access, caregiver burden, relationship with primary care providers, or self-rated caregiver health.
'Conclusion': Non-metropolitan caregivers experience lower income with possible greater familial support, but despite the financial disparities do not have higher caregiver burden, poorer self-rated health, or differences in other important measures. Additional studies that further divide the non-metropolitan cohort into more refined categories by population and with larger sample sizes are essential for designing policy and programs to learn from rural caregivers and build resilience among all care providers.
Primary and metastatic prostate cancers have low mutation rates and recurrent alterations in a small set of genes, enabling targeted sequencing of prostate cancer-associated genes as an efficient ...approach to characterizing patient samples (compared to whole-exome and whole-genome sequencing). For example, targeted sequencing provides a flexible, rapid, and cost-effective method for genomic assessment of patient-derived cell lines to evaluate fidelity to initial patient tumor samples.
We developed a prostate cancer-specific targeted next-generation sequencing (NGS) panel to detect alterations in 62 prostate cancer-associated genes as well as recurring gene fusions with ETS family members, representing the majority of common alterations in prostate cancer. We tested this panel on primary prostate cancer tissues and blood biopsies from patients with metastatic prostate cancer. We generated patient-derived cell lines from primary prostate cancers using conditional reprogramming methods and applied targeted sequencing to evaluate the fidelity of these cell lines to the original patient tumors.
The prostate cancer-specific panel identified biologically and clinically relevant alterations, including point mutations in driver oncogenes and ETS family fusion genes, in tumor tissues from 29 radical prostatectomy samples. The targeted panel also identified genomic alterations in cell-free DNA and circulating tumor cells (CTCs) from patients with metastatic prostate cancer, and in standard prostate cancer cell lines. We used the targeted panel to sequence our set of patient-derived cell lines; however, no prostate cancer-specific mutations were identified in the tumor-derived cell lines, suggesting preferential outgrowth of normal prostate epithelial cells.
We evaluated a prostate cancer-specific targeted NGS panel to detect common and clinically relevant alterations (including ETS family gene fusions) in prostate cancer. The panel detected driver mutations in a diverse set of clinical samples of prostate cancer, including fresh-frozen tumors, cell-free DNA, CTCs, and cell lines. Targeted sequencing of patient-derived cell lines highlights the challenge of deriving cell lines from primary prostate cancers and the importance of genomic characterization to credential candidate cell lines. Our study supports that a prostate cancer-specific targeted sequencing panel provides an efficient, clinically feasible approach to identify genetic alterations across a spectrum of prostate cancer samples and cell lines.
Advances in next generation sequencing technologies provide approaches to comprehensively determine genomic alterations within a tumor that occur as a cause or consequence of neoplastic growth. ...Though providers offering various cancer genomics assays have multiplied, the level of reproducibility in terms of the technical sensitivity and the conclusions resulting from the data analyses have not been assessed.We sought to determine the reproducibility of ascertaining tumor genome aberrations using whole exome sequencing (WES) and RNAseq. Samples of the same metastatic tumors were independently processed and subjected to WES of tumor and constitutional DNA, and RNAseq of RNA, at two sequencing centers. Overall, the sequencing results were highly comparable. Concordant mutation calls ranged from 88% to 93% of all variants including 100% agreement across 154 cancer-associated genes. Regions of copy losses and gains were uniformly identified and called by each sequencing center and chromosomal plots showed nearly identical patterns. Transcript abundance levels also exhibited a high degree of concordance (r2 ≥ 0.78;Pearson). Biologically-relevant gene fusion events were concordantly called. Exome sequencing of germline DNA samples provided a minimum of 30X coverage depth across 56 genes where incidental findings are recommended to be reported. One possible pathogenic variant in the APC gene was identified by both sequencing centers.The findings from this study demonstrate that results of somatic and germline sequencing are highly concordant across sequencing centers that have substantial experience in the technological requirements for preparing, sequencing and annotating DNA and RNA from human biospecimens.
Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease that largely arises from antigen-exposed B-cells that transit through the germinal center (GC). DLBCL is further classified into ...transcriptional subtypes - activated B-cell (ABC) and GC B-cell (GCB). ABC DLBCLs exhibit genetic alterations in NF-kB modifiers and proximal components of the B-cell receptor (BCR) pathway and perturbed terminal B-cell differentiation. GCB DLBCLs have reported alterations in chromatin-modifying enzymes, PI3K signaling and Gα-migration pathway components and frequent translocations of BCL2 .
Despite the recognized molecular heterogeneity in DLBCL, previously published genomic analyses have largely focused on single types of alterations - mutations, copy number alterations (CNAs) or structural variants (SVs) - in smaller data sets with more limited clinical annotation. An unbiased comprehensive analysis of all three alteration types is needed to define discrete, clinically annotated subtypes of DLBCL.
We performed whole exome sequencing (WES) of 304 newly diagnosed DLBCLs, using an expanded bait set that captures recurrent SVs; 85% of study patients were uniformly treated with R-CHOP and had long-term follow-up. Somatic alterations (mutations, CNAs and SVs) and their clonality were determined with analytical pipelines developed at the Broad Institute. Notably, half of our DLBCL cohort lacked patient-matched normal samples, prompting the successful development of new methods to analyze tumor-only WES data. Significantly mutated genes (SMGs) were identified with MutSig2CV andrecurrent CNAs were defined with GISTIC2.0 .
With increased sample size and improved methodology, we identified 158 SMGs, CNAs and SVs; the 98 SMGs included ones previously unreported in DLBCL but described in other lymphoid malignancies or cancer. Additional insights into the putative biological function of newly identified alterations were obtained by overlaying the predicted protein changes onto their 3-dimensional protein structures.
We analyzed the mutational signatures in these DLBCLs and found that the majority of exome mutations were caused by spontaneous deamination at CpGs, a clock-like genetic signature associated with aging; only a minority of mutations were attributed to activation-induced cytidine deaminase (AID), an enzyme required for physiologic immunoglobulin receptor editing and aberrant somatic hypermutation.
IGH, BCL2, BCL6 and MYC were the most frequently rearranged genes (40%, 21%, 19% and 8%, respectively). There were 18 arm-level and 18 focal regions of copy gain and 2 arm-level and 32 focal regions of copy loss with frequencies ranging between 5-32%.
SMGs were significantly more likely to reside within focal CNAs (p=1x10-44), suggesting that these driver genes were perturbed by multiple mechanisms. Individual DLBCLs had a median of 17 genetic drivers, highlighting the need for a more comprehensive analysis. Therefore, we applied an integrated clustering approach to the recurrent mutations, CNAs and SVs and delineated 5 genetically distinct DLBCL subtypes and determined the likely temporal order of alterations within each cluster.
This unbiased approach identified a previously unappreciated favorable risk ABC subset with genetic features of an extrafollicular, possibly marginal zone origin; a more tightly defined poor risk group of ABC DLBCLs with frequent BCL2 gain and concordant MYD88L265P / CD79B mutations; a distinct poor risk subset of GCB tumors with BCL2 SVs and alterations of PTEN and epigenetic enzymes and a discrete group of good risk GCB DLBCLs with specific alterations in BCR/PI3K, JAK/STAT and BRAF pathway components and multiple histones; and a Cell-of-Origin-independent subset with biallelic inactivation of TP53, 9p21.3/CDKN2A and associated genomic instability. These findings likely explain the variability in outcome predictions with the binary ABC- vs. GCB-DLBCL transcriptional classification and the challenges of therapeutically targeting less well defined DLBCLs.
The genetically distinct DLBCL subsets provide a framework for assessing previously unrecognized heterogeneity in this disease, characterizing combinations of genetic alterations that drive DLBCL biology and guiding the development of rational single-agent and combination therapies in patients with the greatest need.
Cerhan:Janssen: Other: Scientific Advisory Board (REMICADELYM4001); Janssen: Other: Multiple Myeloma Registry Steering. Rodig:Bristol-Myers Squibb: Honoraria, Research Funding. Neuberg:Synta Pharmaceuticals: Other: Stock shares. Shipp:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Other: Scientific Advisory Board; Cell Signaling: Honoraria; Bayer: Research Funding; Takeda: Other: Scientific Advisory Board; Merck: Other: Scientific Advisory Board; AstraZeneca: Honoraria.
Abstract
Lung adenocarcinoma is the leading cause of cancer-related death worldwide. Recent molecular characterization of this disease through large-scale sequencing efforts has identified distinct ...subsets driven by mutant oncogenes or kinase fusion proteins, many of which can be inhibited with targeted therapies. Despite these advances, almost half of all lung cancers still lack an identifiable driver. Here, we describe the genomic profiling of 230 normal-paired lung adenocarcinoma samples included as part of The Cancer Genome Atlas (TCGA) effort. All samples were subjected to whole exome analysis, copy number profiling and a subset were also subjected to whole genome sequencing. Mutation calling was performed with the MuTect algorithm. To identify significantly mutated genes, we used the MutSig CV algorithm, a statistically rigorous analysis that takes into account nucleotide context, gene-expression, replication time, and somatic background mutation rate. Mutation rate in lung adenocarcinoma was quite high with an average of 242 mutations/tumor observed (median: 161, range: 11-1328). In total, we identified mutations in over 13,500 genes of which 10 genes reached statistical significance (q<0.1). One significant gene was excluded from further analyses as it was not expressed in RNA-seq data. In addition to mutant genes with established roles in lung adenocarcinoma (e.g. TP53, KRAS, STK11, EGFR, RB1, KEAP1, and BRAF), we also identified other statistically significant mutant genes whose role in lung tumorigenesis is presently unclear. These included mutations in the RNA-binding protein RBM10, and the integrin protein ITGAL. Although statistically insignificant by a small degree, we also identified mutations in the splicing factor U2AF1, and the SWI/SNF complex proteins SMARCA4 and ARID1A. We are currently analyzing whole genome sequences to confirm these events, and identify known and novel fusion events that may be contributing to tumorigenesis. In conclusion, we have analyzed the exomes of 230 lung adenocarcinoma samples and identified known and unknown mutations in this disease. Ultimately, these data will be integrated with ongoing expression, methylation, pathway, miRNA, and proteomic analyses. At its conclusion, this effort will represent the most comprehensive profiling of lung adenocarcinoma samples to date, and will provide a detailed integrative picture of the molecular mechanisms contributing to this disease.
Citation Format: Juliann Chmielecki, Mara Rosenberg, Marcin Imielinski, Bryan Hernandez, Michael Lawrence, Andrey Sivachenko, Kristian Cibulskis, Douglas Voet, Carrie Sougnez, Stacey Gabriel, Gad Getz, Matthew Meyerson, The Cancer Genome Atlas Research Network. Whole exome and whole genome sequence analysis of lung adenocarcinoma. abstract. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1112. doi:10.1158/1538-7445.AM2013-1112
Abstract
Background: Circulating cell-free DNA (cfDNA) has largely been used to monitor blood for specific tumor mutations, but genome-wide discovery from cfDNA has not been well established. Here, ...we establish a scalable approach for whole-exome sequencing (WES) of cfDNA, making it possible to perform comprehensive genomic characterization of metastatic cancer in a routine and minimally-invasive manner.
Comprehensive genomic characterization of metastatic cancer stands to uncover novel alterations of clinical significance. A major challenge is that metastatic tumors are infrequently biopsied. Cell-free DNA is shed abundantly into the bloodstream from metastatic tumors, presenting an opportunity for genomic discovery in advanced cancers that are rarely biopsied in routine clinical care. We report an efficient process to qualify and sequence whole-exomes from cfDNA at scale and systematically compare the somatic mutations, indels, and copy number alterations detected in WES of cfDNA to WES of matched tumor biopsies.
Methods: We consented 86 patients with metastatic breast or prostate cancers for blood collection. We isolated cfDNA and germline DNA from blood and performed low coverage sequencing to estimate tumor content based on genome-wide copy number. We screened patient blood samples and prioritized those with higher tumor fractions for WES. In parallel, we analyzed cfDNA and germline DNA from healthy donors to calibrate our methods and assess false positive rate for genomic alterations.
Results: We found the vast majority of patients with metastatic prostate or breast cancer to have detectable tumor-derived cfDNA. WES of cfDNA from healthy donors revealed very low false positive rates for somatic mutations, indels and copy number alterations (SCNAs). By analyzing WES of cfDNA and tumor biopsies from dozens of patients with metastatic breast or prostate cancers, we established guidelines for the coverage and tumor fraction required for mutation discovery in WES of cfDNA. We found WES of cfDNA to uncover 91% of the clonal mutations, 59% of the subclonal mutations, and 75% of the SCNAs detected in WES of matched tumor biopsies. In several cases, we observed mutations exclusive to cfDNA that were confirmed in later blood draws, suggesting that cfDNA-exclusive mutations may be derived from unsampled metastases. In some cases, cfDNA revealed clinically actionable mutations that were not detected in matched tumor biopsies.
Conclusions: WES of cfDNA uncovers the majority of somatic mutations, indels, and SCNAs found in matched tumor biopsies of metastatic cancer. The high degree of concordance suggests that comprehensive sequencing of cfDNA can be leveraged for genomic discovery in settings where conventional biopsies are difficult to access. Furthermore, the detection of mutations in cfDNA that are not detected in concurrent biopsies suggests that cfDNA may be complementary to tumor biopsies for both translational studies and precision cancer medicine.
Citation Format: Viktor A. Adalsteinsson, Gavin Ha, Sam Freeman, Atish D. Choudhury, Daniel G. Stover, Heather A. Parsons, Gregory Gydush, Sarah Reed, Denis Loginov, Dimitri Livitz, Daniel Rosebrock, Ignat Leshchiner, Ofir Cohen, Coyin Oh, Jaegil Kim, Chip Stewart, Mara Rosenberg, Huiming Ding, Maxwell R. Lloyd, Sairah Mahmud, Karla E. Helvie, Margaret S. Merrill, Rebecca A. Santiago, Edward P. O’Connor, Seong H. Jeong, Joseph F. Kramkowski, Jens G. Lohr, Laura Polacek, Nelly Oliver, Lori Marini, Joshua Francis, Lauren C. Harshman, Eliezer M. Van Allen, Eric P. Winer, Nancy U. Lin, Mari Nakabayashi, Mary-Ellen Taplin, Levi A. Garraway, Todd R. Golub, Jesse S. Boehm, Nikhil Wagle, Gad Getz, Matthew Meyerson, Christopher J. Love. High concordance of whole-exome sequencing of cell-free DNA and matched biopsies enables genomic discovery in metastatic cancer. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-136.
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