Person: MacConaill, Laura
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MacConaill
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Laura
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MacConaill, Laura
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Publication Unique, dual-indexed sequencing adapters with UMIs effectively eliminate index cross-talk and significantly improve sensitivity of massively parallel sequencing(BioMed Central, 2018) MacConaill, Laura; Burns, Robert T.; Nag, Anwesha; Coleman, Haley A.; Slevin, Michael K.; Giorda, Kristina; Light, Madelyn; Lai, Kevin; Jarosz, Mirna; McNeill, Matthew S.; Ducar, Matthew D.; Meyerson, Matthew; Thorner, Aaron R.Background: Sample index cross-talk can result in false positive calls when massively parallel sequencing (MPS) is used for sensitive applications such as low-frequency somatic variant discovery, ancient DNA investigations, microbial detection in human samples, or circulating cell-free tumor DNA (ctDNA) variant detection. Therefore, the limit-of-detection of an MPS assay is directly related to the degree of index cross-talk. Results: Cross-talk rates up to 0.29% were observed when using standard, combinatorial adapters, resulting in 110,180 (0.1% cross-talk rate) or 1,121,074 (0.29% cross-talk rate) misassigned reads per lane in non-patterned and patterned Illumina flow cells, respectively. Here, we demonstrate that using unique, dual-matched indexed adapters dramatically reduces index cross-talk to ≤1 misassigned reads per flow cell lane. While the current study was performed using dual-matched indices, using unique, dual-unrelated indices would also be an effective alternative. Conclusions: For sensitive downstream analyses, the use of combinatorial indices for multiplexed hybrid capture and sequencing is inappropriate, as it results in an unacceptable number of misassigned reads. Cross-talk can be virtually eliminated using dual-matched indexed adapters. These results suggest that use of such adapters is critical to reduce false positive rates in assays that aim to identify low allele frequency events, and strongly indicate that dual-matched adapters be implemented for all sensitive MPS applications. Electronic supplementary material The online version of this article (10.1186/s12864-017-4428-5) contains supplementary material, which is available to authorized users.Publication Colorectal Cancers from Distinct Ancestral Populations Show Variations in BRAF Mutation Frequency(Public Library of Science, 2013) Hanna, Megan C.; Go, Christina; Roden, Christine; Jones, Robert T.; Pochanard, Panisa; Javed, Ahmed Yasir; Javed, Awais; Mondal, Chandrani; Palescandolo, Emanuele; Van Hummelen, Paul; Hatton, Charles; Bass, Adam; Chun, Sung Min; Na, Deuk Chae; Kim, Tae-Im; Jang, Se Jin; Osarogiagbon, Raymond U.; Hahn, William; Meyerson, Matthew; Garraway, Levi; MacConaill, LauraIt has been demonstrated for some cancers that the frequency of somatic oncogenic mutations may vary in ancestral populations. To determine whether key driver alterations might occur at different frequencies in colorectal cancer, we applied a high-throughput genotyping platform (OncoMap) to query 385 mutations across 33 known cancer genes in colorectal cancer DNA from 83 Asian, 149 Black and 195 White patients. We found that Asian patients had fewer canonical oncogenic mutations in the genes tested (60% vs Black 79% (P = 0.011) and White 77% (P = 0.015)), and that BRAF mutations occurred at a higher frequency in White patients (17% vs Asian 4% (P = 0.004) and Black 7% (P = 0.014)). These results suggest that the use of genomic approaches to elucidate the different ancestral determinants harbored by patient populations may help to more precisely and effectively treat colorectal cancer.Publication Mutational Profiling Reveals PIK3CA Mutations in Gallbladder Carcinoma(Springer Science + Business Media, 2011) Deshpande, Vikram; Nduaguba, Afamefuna Maxwell; Zimmerman, Stephanie M; Kehoe, Sarah M; MacConaill, Laura; Lauwers, Gregory Y.; Ferrone, Cristina; Bardeesy, Nabeel; Zhu, Andrew; Hezel, Aram FPublication Genotyping Cancer-Associated Genes in Chordoma Identifies Mutations in Oncogenes and Areas of Chromosomal Loss Involving CDKN2A, PTEN, and SMARCB1(Public Library of Science, 2014) Choy, Edwin; MacConaill, Laura; Cote, Gregory; Le, Long P.; Shen, Jacson K.; Nielsen, Gunnlaugur; Iafrate, Anthony; Garraway, Levi; Hornicek, Francis; Duan, ZhenfengThe molecular mechanisms underlying chordoma pathogenesis are unknown. We therefore sought to identify novel mutations to better understand chordoma biology and to potentially identify therapeutic targets. Given the relatively high costs of whole genome sequencing, we performed a focused genetic analysis using matrix-assisted laser desorption/ionization-time of flight mass spectrometer (Sequenom iPLEX genotyping). We tested 865 hotspot mutations in 111 oncogenes and selected tumor suppressor genes (OncoMap v. 3.0) of 45 human chordoma tumor samples. Of the analyzed samples, seven were identified with at least one mutation. Six of these were from fresh frozen samples, and one was from a paraffin embedded sample. These observations were validated using an independent platform using homogeneous mass extend MALDI-TOF (Sequenom hME Genotyping). These genetic alterations include: ALK (A877S), CTNNB1 (T41A), NRAS (Q61R), PIK3CA (E545K), PTEN (R130), CDKN2A (R58*), and SMARCB1 (R40*). This study reports on the largest comprehensive mutational analysis of chordomas performed to date. To focus on mutations that have the greatest chance of clinical relevance, we tested only oncogenes and tumor suppressor genes that have been previously implicated in the tumorigenesis of more common malignancies. We identified rare genetic changes that may have functional significance to the underlying biology and potential therapeutics for chordomas. Mutations in CDKN2A and PTEN occurred in areas of chromosomal copy loss. When this data is paired with the studies showing 18 of 21 chordoma samples displaying copy loss at the locus for CDKN2A, 17 of 21 chordoma samples displaying copy loss at PTEN, and 3 of 4 chordoma samples displaying deletion at the SMARCB1 locus, we can infer that a loss of heterozygosity at these three loci may play a significant role in chordoma pathogenesis.Publication Whole-exome sequencing and clinical interpretation of FFPE tumor samples to guide precision cancer medicine(2013) Allen, Eliezer M. Van; Wagle, Nikhil; Stojanov, Petar; Perrin, Danielle L.; Cibulskis, Kristian; Marlow, Sara; Jane-Valbuena, Judit; Friedrich, Dennis C.; Kryukov, Gregory; Carter, Scott L.; McKenna, Aaron; Sivachenko, Andrey; Rosenberg, Mara; Kiezun, Adam; Voet, Douglas; Lawrence, Michael; Lichtenstein, Lee T.; Gentry, Jeff G.; Huang, Franklin; Fostel, Jennifer; Farlow, Deborah; Barbie, David; Gandhi, Leena; Lander, Eric; Gray, Stacy; Joffe, Steven; Janne, Pasi; Garber, Judy; MacConaill, Laura; Lindeman, Neal; Rollins, Barrett; Kantoff, Philip; Fisher, Sheila A.; Gabriel, Stacey; Getz, Gad; Garraway, LeviTranslating whole exome sequencing (WES) for prospective clinical use may impact the care of cancer patients; however, multiple innovations are necessary for clinical implementation. These include: (1) rapid and robust WES from formalin-fixed paraffin embedded (FFPE) tumor tissue, (2) analytical output similar to data from frozen samples, and (3) clinical interpretation of WES data for prospective use. Here, we describe a prospective clinical WES platform for archival FFPE tumor samples. The platform employs computational methods for effective clinical analysis and interpretation of WES data. When applied retrospectively to 511 exomes, the interpretative framework revealed a “long tail” of somatic alterations in clinically important genes. Prospective application of this approach identified clinically relevant alterations in 15/16 patients. In one patient, previously undetected findings guided clinical trial enrollment leading to an objective clinical response. Overall, this methodology may inform the widespread implementation of precision cancer medicine.Publication The impact of tumor profiling approaches and genomic data strategies for cancer precision medicine(BioMed Central, 2016) Garofalo, Andrea; Sholl, Lynette; Reardon, Brendan; Taylor-Weiner, Amaro; Amin-Mansour, Ali; Miao, Diana; Liu, David; Oliver, Nelly; MacConaill, Laura; Ducar, Matthew; Rojas-Rudilla, Vanesa; Giannakis, Marios; Ghazani, Arezou; Gray, Stacy; Janne, Pasi; Garber, Judy; Joffe, Steve; Lindeman, Neal; Wagle, Nikhil; Garraway, Levi; Van Allen, EliezerBackground: The diversity of clinical tumor profiling approaches (small panels to whole exomes with matched or unmatched germline analysis) may engender uncertainty about their benefits and liabilities, particularly in light of reported germline false positives in tumor-only profiling and use of global mutational and/or neoantigen data. The goal of this study was to determine the impact of genomic analysis strategies on error rates and data interpretation across contexts and ancestries. Methods: We modeled common tumor profiling modalities—large (n = 300 genes), medium (n = 48 genes), and small (n = 15 genes) panels—using clinical whole exomes (WES) from 157 patients with lung or colon adenocarcinoma. We created a tumor-only analysis algorithm to assess germline false positive rates, the impact of patient ancestry on tumor-only results, and neoantigen detection. Results: After optimizing a germline filtering strategy, the germline false positive rate with tumor-only large panel sequencing was 14 % (144/1012 variants). For patients whose tumor-only results underwent molecular pathologist review (n = 91), 50/54 (93 %) false positives were correctly interpreted as uncertain variants. Increased germline false positives were observed in tumor-only sequencing of non-European compared with European ancestry patients (p < 0.001; Fisher’s exact) when basic germline filtering approaches were used; however, the ExAC database (60,706 germline exomes) mitigated this disparity (p = 0.53). Matched and unmatched large panel mutational load correlated with WES mutational load (r2 = 0.99 and 0.93, respectively; p < 0.001). Neoantigen load also correlated (r2 = 0.80; p < 0.001), though WES identified a broader spectrum of neoantigens. Small panels did not predict mutational or neoantigen load. Conclusions: Large tumor-only targeted panels are sufficient for most somatic variant identification and mutational load prediction if paired with expanded germline analysis strategies and molecular pathologist review. Paired germline sequencing reduced overall false positive mutation calls and WES provided the most neoantigens. Without patient-matched germline data, large germline databases are needed to minimize false positive mutation calling and mitigate ethnic disparities. Electronic supplementary material The online version of this article (doi:10.1186/s13073-016-0333-9) contains supplementary material, which is available to authorized users.Publication BreaKmer: detection of structural variation in targeted massively parallel sequencing data using kmers(Oxford University Press, 2015) Abo, Ryan P.; Ducar, Matthew; Garcia, Elizabeth P.; Thorner, Aaron; Rojas-Rudilla, Vanesa; Lin, Ling; Sholl, Lynette M.; Hahn, William; Meyerson, Matthew; Lindeman, Neal I.; Van Hummelen, Paul; MacConaill, LauraGenomic structural variation (SV), a common hallmark of cancer, has important predictive and therapeutic implications. However, accurately detecting SV using high-throughput sequencing data remains challenging, especially for ‘targeted’ resequencing efforts. This is critically important in the clinical setting where targeted resequencing is frequently being applied to rapidly assess clinically actionable mutations in tumor biopsies in a cost-effective manner. We present BreaKmer, a novel approach that uses a ‘kmer’ strategy to assemble misaligned sequence reads for predicting insertions, deletions, inversions, tandem duplications and translocations at base-pair resolution in targeted resequencing data. Variants are predicted by realigning an assembled consensus sequence created from sequence reads that were abnormally aligned to the reference genome. Using targeted resequencing data from tumor specimens with orthogonally validated SV, non-tumor samples and whole-genome sequencing data, BreaKmer had a 97.4% overall sensitivity for known events and predicted 17 positively validated, novel variants. Relative to four publically available algorithms, BreaKmer detected SV with increased sensitivity and limited calls in non-tumor samples, key features for variant analysis of tumor specimens in both the clinical and research settings.Publication Comparison of Prevalence and Types of Mutations in Lung Cancers Among Black and White Populations(American Medical Association (AMA), 2017) Campbell, Joshua David; Lathan, Christopher; Sholl, Lynette; Ducar, Matthew; Vega, Mikenah; Sunkavalli, Ashwini; Lin, Ling; Hanna, Megan; Schubert, Laura; Thorner, Aaron; Faris, Nicholas; Williams, David; Osarogiagbon, Raymond U.; van Hummelen, Paul; Meyerson, Matthew; MacConaill, LauraImportance Lung cancer is the leading cause of cancer death in the United States in all ethnic and racial groups. The overall death rate from lung cancer is higher in black patients than in white patients. Objective To compare the prevalence and types of somatic alterations between lung cancers from black patients and white patients. Differences in mutational frequencies could illuminate differences in prognosis and lead to the reduction of outcome disparities by more precisely targeting patients’ treatment. Design, Setting, and Participants Tumor specimens were collected from Baptist Cancer Center (Memphis, Tennessee) over the course of 9 years (January 2004-December 2012). Genomic analysis by massively parallel sequencing of 504 cancer genes was performed at Dana-Farber Cancer Institute (Boston, Massachusetts). Overall, 509 lung cancer tumors specimens (319 adenocarcinomas; 142 squamous cell carcinomas) were profiled from 245 black patients and 264 white patients. Main Outcomes and Measures The frequencies of genomic alterations were compared between tumors from black and white populations. Results Overall, 509 lung cancers were collected and analyzed (273 women [129 black patients; 144 white patients] and 236 men [116 black patients; 120 white patients]). Using 313 adenocarcinomas and 138 squamous cell carcinomas with genetically supported ancestry, overall mutational frequencies and copy number changes were not significantly different between black and white populations in either tumor type after correcting for multiple hypothesis testing. Furthermore, specific activating alterations in members of the receptor tyrosine kinase/Ras/Raf pathway including EGFR and KRAS were not significantly different between populations in lung adenocarcinoma. Conclusions and Relevance These results demonstrate that lung cancers from black patients are similar to cancers from white patients with respect to clinically actionable genomic alterations and suggest that clinical trials of targeted therapies could significantly benefit patients in both groups.Publication Somatic mutations in CDH1 and CTNNB1 in primary carcinomas at 13 anatomic sites(Impact Journals LLC, 2017) Busch, Evan; Hornick, Jason; Umeton, Renato; Albayrak, Adem; Lindeman, Neal; MacConaill, Laura; Garcia, Elizabeth P.; Ducar, Matthew; Rebbeck, TimothyMetastases are involved in most cancer deaths. Evidence has suggested that cancer cell detachment from primary tumors might occur largely via the mechanism of epithelial-mesenchymal transition (EMT) activated by epigenetic events, but data addressing other possible triggers of detachment, particularly genetic mutations, have been limited. Using the Profile study of cancer genomics at Dana-Farber Cancer Institute, we examined somatic mutations in the EMT genes CDH1 in 5,106 primary carcinomas and CTNNB1 in 7,578 primary carcinomas across 13 anatomic sites: urinary bladder, breast, colon/rectum, endometrium, esophagus, kidney, lung, ovary, pancreas, prostate, skin (non-melanoma), stomach, and thyroid. For each gene and anatomic site, we calculated the prevalence of primary carcinomas with at least one mutation. Across all anatomic sites, 4% of carcinomas had at least one CDH1 mutation and 4% of carcinomas had at least one CTNNB1 mutation. By anatomic site, the observed prevalence of carcinomas with at least one mutation was less than 5% at 10 sites for CDH1 and 12 sites for CTNNB1. Tumor stage data were available for a subset of breast, colorectal, lung, and prostate tumors. Among patients from this subset who were diagnosed with regional or distant disease, only 4% had a CDH1 mutation and 1% had a CTNNB1 mutation in the primary tumor. The low mutation prevalences, especially among those with diagnoses of regional or distant disease, suggest that somatic mutations in CDH1 and CTNNB1 are unlikely to explain a substantial proportion of cancer cell detachment from primary carcinomas originating at most anatomic sites.Publication Novel Genetic Mutations in a Sporadic Port-Wine Stain(American Medical Association (AMA), 2014) Lian, Christine; Sholl, Lynette; Zakka, Labib; O, Teresa M.; Liu, Cynthia; Xu, Shuyun; Stanek, Ewelina; Garcia, Elizabeth; Jia, Yonghui; MacConaill, Laura; Murphy, George; Waner, Milton; Mihm, MartinImportance Port-wine stains (PWSs) are common congenital cutaneous capillary malformations. A somatic GNAQ mutation was recently identified in patients with sporadic PWSs and Sturge-Weber syndrome. However, subsequent studies to confirm or extend this observation are lacking. Observations We report a long-standing, unilateral facial PWS of a man in his early 70s confirmed by histopathological analysis. Staged surgical excision of the vascular malformation was performed, and genomic DNA was extracted from the vascular malformation specimen and normal skin. Targeted next-generation sequencing of the coding sequence of 275 known cancer genes including GNAQ was performed in both specimens. A single-nucleotide variant (c.548G>A, p.Arg183Gln) in GNAQ was identified in the PWS-affected tissue but not in the normal skin sample. In addition, this sequencing approach uncovered several additional novel somatic mutations in the genes SMARCA4, EPHA3, MYB, PDGFR-β, and PIK3CA. Conclusions and Relevance Our findings confirm the presence of somatic mutations in GNAQ in the affected skin of a patient with congenital PWS, as well as alterations in several other novel genes of possible importance in the pathogenesis of PWS that may also offer substantial therapeutic targets.