We developed a novel phasing approach, based solely on molecules and genotype frequency, that does not rely on inference of new alleles. We initiated the project because of errors that were detected ...in the phased 1000 Genomes Project data. The algorithm first combined identical genotypes into clusters and ranked them by descending frequency. Using alleles defined in homozygotes, it combined them to produce expected genotypes that were dismissed and subtracted them from remaining genotypes to define additional new putative alleles. Putative alleles had to be confirmed by identifying them in independent genotypes, and the process was iterated until all alleles were identified. The new approach was validated using single-molecule sequencing of eight loci, 145 (8 to 35 per locus) alleles were identified, and an average 98.2% (range, 95.0% to 99.9%) of 1000 genome individuals at these loci were explained. The accuracy of the new method was compared with that from PHASE and SHAPEIT2 to the experimentally determined genotypes based on single-molecule sequencing. Our method was comparable to PHASE and SHAPEIT2 in accuracy but was, on average, 14.6- and 10.8-fold faster, respectively.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Manganese superoxide dismutase-2 (SOD2) plays a crucial role in cells' protection against mitochondrial oxidative damage. A genetic polymorphism in the mitochondrial targeting sequence of the SOD2 ...gene has been implicated in various diseases, including prostate cancer. Paller et al. have shown an increase in prostate-specific antigen (PSA) doubling time in patients with the Ala/Ala (wildtype) genotype when treated with pomegranate/grape extract antioxidants. We developed and validated a pyrosequencing assay that detects the common germline SOD2 SNP (rs_4880) with the aim of identifying men with castrate-resistant prostate cancer eligible for an antioxidant therapy clinical trial. We first selected 37 samples from the 1000 genomes study with known genotypes determined using Illumina-based sequencing and confirmed them by Sanger sequencing. In a blinded design, we then performed the new pyrosequencing assay on these samples and assigned genotypes. Genotypes for all 37 samples (13 homozygous Ala, 12 heterozygous Ala/Val, and 12 homozygous Val) were all concordant by pyrosequencing. The pyrosequencing assay has been live since May 2018 and has proven to be robust and accurate.
Suicide gene therapy using gene-directed enzyme prodrug therapy (GDEPT) is based on delivering a gene-encoded enzyme to cells that converts a nontoxic prodrug into its toxic metabolite. The bystander ...effect is thought to compensate for inefficiencies in delivery and expression because the produced toxic metabolite can spread to adjacent non-expressing cells.
The purpose of this study was to assess the significance of bystander effect in GDEPT over the long term in vivo.
We performed experiments using mixtures of yeast cytosine deaminase (yCD) expressing and empty vector (EV) containing cells. First, the bystander effect was assessed in various ratios of colon cancer cell lines RKO with yCD/EV in 2D and 3D culture. Next, tumors raised from RKO with yCD/EV in mice were treated with the prodrug 5-fluorocytosine (5-FC) for 42 days to assess bystander effect in vivo. Cell types constituting relapsed tumors were determined by 5-FC treatment and PCR.
We were able to demonstrate bystander effect in both 2D and 3D. In mice, tumors initially regressed, but they all eventually recurred including those produced from 80% yCD expressing cells. Cells explanted from the recurrent tumors demonstrated that suicide gene expressing cells had been selected against during in vivo treatment with 5-FC.
We conclude that gene therapy of malignant tumors in patients using the yCD/5-FC system will require targeting well over 80% of the malignant cells, and therefore will likely require improved bystander effect or repeated treatment.
McCune-Albright Syndrome (MAS) is a rare sporadic syndrome caused by post-zygotic mutations in the
GNAS
oncogene, leading to constitutional mosaicism for these alterations. Somatic activating
GNAS
...mutations also commonly occur in several gastrointestinal and pancreatic neoplasms, but the spectrum of abnormalities in these organs in patients with MAS has yet to be systematically described. We report comprehensive characterization of the upper gastrointestinal tract in seven patients with MAS and identify several different types of polyps, including gastric heterotopia/metaplasia (7/7), gastric hyperplastic polyps (5/7), fundic gland polyps (2/7), and a hamartomatous polyp (1/7). In addition, one patient had an unusual adenomatous lesion at the gastroesophageal junction with high-grade dysplasia. In the pancreas, all patients had endoscopic ultrasound findings suggestive of intraductal papillary mucinous neoplasm (IPMN), but only two patients met the criteria for surgical intervention. Both of these patients had IPMNs at resection, one with low-grade dysplasia and one with high-grade dysplasia.
GNAS
mutations were identified in the majority of lesions analyzed, including both IPMNs and the adenomatous lesion from the gastroesophageal junction. These studies suggest that there is a broad spectrum of abnormalities in the gastrointestinal tract and pancreas in patients with MAS and that patients with MAS should be evaluated for gastrointestinal pathology, some of which may warrant clinical intervention due to advanced dysplasia.
Abstract
Introduction: Ultrasensitive point mutation detection can facilitate minimal residual disease detection, early detection of cancer, and therapeutic monitoring of cancer patients. However, ...nearly identical mutant and wildtype molecules exhibit crosstalk with most technologies, thus producing high background levels. To evaluate technologies against each other for accurate ultrasensitive point mutation detection it is crucial to supply laboratories with reference samples of various dilutions.
Methods: We tested a series of sample mixes with digital-droplet PCR and next generation sequencing. Using linearity and accuracy as the gold standard, we empirically introduced two corrections to generate improved quality control reagents. Baseline variant allele frequency (VAF) in the parental cell line was used to correct for copy number variation of variant, while haplotype counting was used to correct technical errors (cell counting and pipetting).
Results: Correlation between the level of dilution and the measured VAF was relatively good (R2 = 0.80) when measured using ddPCR. However, correcting for the parental VAFs substantially improved the correlation (R2 = 0.97). In contrast, correlation of the cell count dilution and the measured VAF, as determined by next generation sequencing (AmpliSeq), was initially relatively poor (R2 = 0.63). However, substantial improvement in correlation was achieved by correcting for the parental VAFs (R2 = 0.94). In addition, correcting by haplotype counting alone only slightly improved accuracy in both technologies (ddPCR, from R2 = 0.80 to R2 = 0.81; AmpliSeq, from R2 = 0.63 to R2 = 0.64). Furthermore, correcting for both factors resulted in the best correlation for ddPCR-generated data (R2 = 0.99); however, introducing both corrections for AmpliSeq data did not prove more beneficial than correcting for parental VAFs alone (both corrections, R2 = 0.94 compared to parental VAF correction only, R2 = 0.94).
Conclusions: Validation of ultrasensitive variant detection requires standardized reference samples. These will also permit assessing various ultrasensitive detection technologies against one another.
Citation Format: Marija Debeljak, Lisa Haley, Derek A. Anderson, Emily M. Adams, Masaya Suenaga, Katie Beierl, Ming-Tseh Lin, Michael Goggins, Christopher D. Gocke, James R. Eshleman. Generating quality control reference standards to evaluate ultrasensitive variant detection. 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 1378.
Next-generation sequencing shows great promise by allowing rapid mutational analysis of multiple genes in human cancers. Recently, we implemented the multiplex PCR-based Ion AmpliSeq Cancer Hotspot ...Panel (>200 amplicons in 50 genes) to evaluate EGFR, KRAS , and BRAF in lung and colorectal adenocarcinomas. In 10% of samples, automated analysis identified a novel G873R substitution mutation in EGFR . By examining reads individually, we found this mutation in >5% of reads in 50 of 291 samples and also found similar events in 18 additional amplicons. These apparent mutations are present only in short reads and within 10 bases of either end of the read. We therefore hypothesized that these were from panel primers promiscuously binding to nearly complementary sequences of nontargeted amplicons. Sequences around the mutations matched primer binding sites in the panel in 18 of 19 cases, thus likely corresponding to panel primers. Furthermore, because most primers did not show this effect, we demonstrated that next-generation sequencing may be used to better design multiplex PCR primers through iterative elimination of offending primers to minimize mispriming. Our results indicate the need for careful sequence analysis to avoid false-positive mutations that can arise in multiplex PCR panels. The AmpliSeq Cancer panel is a valuable tool for clinical diagnostics, provided awareness of potential artifacts.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
Introduction: Our current understanding of the molecular changes predisposing Ductal carcinoma in situ (DCIS) to progress to invasive breast cancer (IBC) is limited. The aim of this study is ...to characterize the molecular changes of DCIS to improve DCIS risk assessment.
Methods: We obtained paraffin-embedded tissue of 210 DCIS samples with no concurrent or antecedent IBC from 5 cancer institutions, and extracted DNA and RNA. Transcriptome analysis: Illumina’s TruSeq RNA Exome kit was used for library construction, followed by sequencing.
Methylome analysis: Bisulfite treated genomic DNA was restored and arrayed using the Illumina 450K methylation chips. DNA Copy number (CNV) analysis: CNV was estimated from the methylation data set using the EpiCopy R package.
Results: Samples passing Q/C metrics: Transcriptome: 59 cases, 63 controls. Methylome & CNV: 93 cases, 98 controls. We classified samples into their intrinsic PAM50 subsets. Cases showed an increase in the Her2 subtype, and the controls were enriched in LuminalA (LumA) samples, while Basal and LuminalB (LumB) subtypes were evenly distributed. Compared to the TCGA IBC dataset, proportions of Her2 and LumB subtypes in DCIS were increased while the LumA cohort was decreased. Unsupervised clustering of the transcriptome data resulted in 3 clusters with key differences between PAM50 subtypes: one cluster predominated in Basal and Her2 subtypes, and a second was enriched in hormone-positive samples (LumA and LumB). For the methylome data, the optimal number of clusters was 6. Again, several clusters showed correlation with PAM50 subtype, e.g., one was enriched for hormone-negative subtypes (Basal and Her2), while another was enriched for hormone-positive subtypes (LumA and LumB). For the CNV data, the optimal number of clusters was 4. Here, one CNV cluster appeared predominantly in the Basal subtype, and there were multiple regions showing significant subtype-specific differences with the TCGA IBC data set. While the well-known heterogeneity of DCIS prevents the above broad molecular categories from strongly stratifying DCIS by outcome, we are continuing to analyze our data sets for predictive molecular signatures. Since the long-term outcome of all DCIS in this cohort is known, we are in the unique position to pursue additional questions such as PAM50 subtype differences in times-to-events or lineage fidelity (i.e., whether the subtype of the subsequent IBC matches the preceding DCIS), and it appears that the time to IBC diagnosis is shortest in Basal DCIS, while at the same time, lineage fidelity is lowest in Basal DCIS, as has been previously reported.
Conclusions: Our subtype-stratified analyses identified multiple molecular differences both between intrinsic subtypes as well as between DCIS and IBC that suggest subtype-specific characteristics that may be exploitable for risk stratification of DCIS.
Citation Format: Marija Debeljak, Soonweng Cho, Bradley Downs, Michael Considine, Brittany Avin-McKelvey, Yongchun Wang, William Grizzle, Katherine Hoadley, Charles Lynch, Brenda Hernandez, Paul van Diest, Wendy Cozen, Ann Hamilton, Debra Hawes, Edward Gabrielson, Ashley Cimino-Mathews, Liliana Florea, Leslie Cope, Christopher B. Umbricht. Multimodal genome-wide survey of progressing and non-progressing DCIS abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3126.
Fields of forensics, transplantation, and paternity rely on human identity testing. Currently, this is accomplished through amplification of microsatellites followed by capillary electrophoresis. An ...alternative and theoretically better approach uses multiple single-nucleotide polymorphisms located within a small region of DNA, a method we initially developed using HLA-A and called haplotype counting. Herein, we validated seven additional polymorphic loci, sequenced a total of 45 individuals from three of the 1000 Genomes populations (15 from each), and determined the number of haplotypes, heterozygosity, and polymorphic information content for each locus. In addition, we developed a multiplex PCR that amplifies five of these loci simultaneously. Using this strategy with a small cohort of leukemic patients who underwent allogeneic bone marrow transplantation, we first attempted to define a threshold (0.26% recipient) by examining seven patients who tested all donor and did not relapse. Although this initial threshold will need to be confirmed in a larger cohort, we detected increased recipient DNA above this threshold 90 to 145 days earlier than microsatellite positivity, and 127 to 142 days before clinical relapse in four of eight patients (50%). Haplotype counting using these novel loci may be useful for ultrasensitive detection in fields such as bone marrow transplantation, solid organ transplant rejection, patient identification, and forensics.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
Introduction: Fields of forensics, paternity, and hematopoietic stem cell transplantation (HSCT) require human identity testing. The polymorphic nature of short tandem repeats (STRs) makes ...them most frequently used but they are relatively insensitive. In 2000, we demonstrated proof-of-principle for the use of SNPs for bone marrow engraftment testing; however, due to the high error rate of NGS platforms, use of a single SNP for this purpose is not possible. Here, we hypothesized that using multiple SNPs on a short amplicon (microhaplotype) could overcome the inherently high error rate of NGS, and use it for ultrasensitive detection of human DNA mixes.
Methods: As proof-of-principle, we identified a 119 bp region on HLA-A Exon 3 that contains a cluster of 18 SNPs. Using cell lines with known HLA-A haplotypes, we made serial dilutions from 1% to 0.001%. By sequencing each dilution at least twice we were able to determine accuracy and limit of detection. Samples collected from patients after BMT that tested as all donor were analyzed using this approach. Sample preparation and sequencing was done per manufacturer's protocol using the Ion Torrent Personal Genome Machine and 200 base chemistry. Additionally, we analyzed the 1000 Genomes database, using 300 bp windows, and identified many other loci that contained clusters of at least 9 SNPs.
Results: Alignment and analysis of common European HLA-A haplotypes of this region show mean number of differences of ∼6 SNPs. Homozygous samples with known HLA-A genotypes that varied by multiple SNPs were analyzed and demonstrated no cross-talk between them. Analyzing serially diluted samples we were able to easily achieve a limit of detection of 1 in 10,000 molecules (0.01%). In BMT samples that tested as all donor by STR analysis, we were able to detect patient DNA of up to 1.5%. From 1000 Genomes data, we identified 4,349 loci that contained at least 9 SNPs within a 300 bp window. Informativity of identified loci was determined for 3 major ethnic groups using 1000 Genomes phased haplotype data.
Conclusions: Ultrasensitive detection of human DNA mixes can be achieved using multiple SNPs within a short amplicon despite the high error rates associated with NGS. This assay may be useful to detect early relapse following bone marrow transplantation and other clinical applications.
Citation Format: Marija Debeljak, Donald N. Freed, Jane A. Welch, Lisa Haley, Katie Beierl, Brian S. Iglehart, Aparna Pallavajjala, Christopher D. Gocke, Mary S. Leffell, Ming-Tseh Lin, Laura D. Wood, Jonathan Pevsner, Sarah J. Wheelan, James R. Eshleman. NGS based microhaplotype counting for ultrasensitive human DNA detection. abstract. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4270. doi:10.1158/1538-7445.AM2015-4270
Fields of forensics, transplantation, and paternity rely on human identity testing. Currently, this is accomplished through amplification of microsatellites followed by capillary electrophoresis. An ...alternative and theoretically better approach uses multiple single-nucleotide polymorphisms located within a small region of DNA, a method we initially developed using HLA-A and called haplotype counting. Herein, we validated seven additional polymorphic loci, sequenced a total of 45 individuals from three of the 1000 Genomes populations (15 from each), and determined the number of haplotypes, heterozygosity, and polymorphic information content for each locus. In addition, we developed a multiplex PCR that amplifies five of these loci simultaneously. Using this strategy with a small cohort of leukemic patients who underwent allogeneic bone marrow transplantation, we first attempted to define a threshold (0.26% recipient) by examining seven patients who tested all donor and did not relapse. Although this initial threshold will need to be confirmed in a larger cohort, we detected increased recipient DNA above this threshold 90 to 145 days earlier than microsatellite positivity, and 127 to 142 days before clinical relapse in four of eight patients (50%). Haplotype counting using these novel loci may be useful for ultrasensitive detection in fields such as bone marrow transplantation, solid organ transplant rejection, patient identification, and forensics.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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