Cynthia S. Spittle

Proofreading exonuclease activity of human DNA polymerase δ and its effects on lesion-bypass DNA synthesis

Replicative DNA polymerases possess 3′ → 5′ exonuclease activity to reduce misincorporation of incorrect nucleotides by proofreading during replication. To examine if this proofreading activity modulates DNA synthesis of damaged templates, we constructed a series of recombinant human DNA polymerase δ (Pol δ) in which one or two of the three conserved Asp residues in the exonuclease domain are mutated, and compared their properties with that of the wild-type enzyme. While all the mutant enzymes lost more than 95% exonuclease activity and severely decreased the proofreading activity than the wild-type, the bypass efficiency of damaged templates was varied: two mutant enzymes, D515V and D402A/D515A, gave higher bypass efficiencies on templates containing an abasic site, but another mutant, D316N/D515A, showed a lower bypass efficiency than the wild-type. All the enzymes including the wild-type inserted an adenine opposite the abasic site, whereas these enzymes inserted cytosine and adenine opposite an 8-oxoguanine with a ratio of 6:4. These results indicate that the exonuclease activity of human Pol δ modulates its intrinsic bypass efficiency on the damaged template, but does not affect the choice of nucleotide to be inserted.

Generation of a unique strain of multiple intestinal neoplasia (Apc+/Min-FCCC) mice with significantly increased numbers of colorectal adenomas

The relevance of the Apc+/Min mouse model in the study of human colorectal cancer remains uncertain due to the predominance of small intestinal adenomas and few, if any, colorectal adenomas. A new strain of Apc+/Min mice (Apc+/Min‐FCCC) with significantly greater numbers of colorectal adenomas has been generated and characterized. Male C57BL/6J—Apc+/Min mice (the Jackson Laboratory, Bar Harbor, ME) were crossed with wild‐type (Apc+/+) C57BL/6J females from an independent colony at this institution (offspring = Apc+/Min‐FCCC) and 233 animals were evaluated over 20 generations. In order to determine the contribution of genetics to the enhanced colorectal adenoma phenotype, breeding pairs (Apc+/Min male × wild type female C57BL/6J) were purchased from the Jackson Laboratory and offspring (Apc+/Min‐JAX) were maintained in our facility under identical conditions (n = 98). Animals were fed Purina Rodent chow (#5013) diet containing 5% fat. The entire intestinal tract was examined histopathologically in both strains. Both the Apc and Pla2g2a (candidate for Mom1) genes were sequenced and found to be identical for both the Apc+/Min‐FCCC and Apc+/Min‐JAX mouse strains. The multiplicity of colorectal adenomas in the Apc+/Min‐FCCC mice was much higher than reported in the literature and significantly greater than the multiplicity of colorectal adenomas in Apc+/Min‐JAX mice maintained in our facility (P = 0.01). Apc+/Min‐FCCC had a significantly greater incidence of rectal prolapse (P = 0.02) and small intestinal adenocarcinomes (P = 0.001), and multiplicity of small intestinal adenocarcinomas (P = 0.001) compared to Apc+/Min‐JAX mice. Male Apc+/Min‐FCCC mice had significantly greater numbers of colorectal adenomas compared to female Apc+/Min‐FCCC mice (P = 0.0002), as did male Apc+/Min‐JAX mice vs. female Apc+/Min‐JAX mice (P < 0.0001). These results allow us to conclude: (1) Apc+/Min‐FCCC mice are unique in that they develop significantly greater numbers of colorectal adenomas and small intestinal cancers, and a significantly greater incidence of small intestinal cancers and rectal prolapse than Apc+/Min‐JAX mice. (2) This study represents the first report of a significant gender difference in multiplicity of colorectal adenomas. (3) Differences between Apc+/Min‐FCCC and Apc+/Min‐JAX mice in currently undefined genetic modifiers may contribute to the enhanced colorectal phenotype. (4) The Apc+/Min‐FCCC strain is highly suited for the investigation of colorectal neoplastic disease and chemoprevention studies.

Pharmacokinetic analysis of irinotecan plus bevacizumab in patients with advanced solid tumors

PurposeThe purpose of this study was to evaluate the effect of bevacizumab on the pharmacokinetics (PK) of irinotecan and its active metabolite. Exploratory analyses of the impact of variability in uridine diphosphate glucuronosyltransferase 1A (UGT1A) genes on irinotecan metabolism and toxicity were conducted.MethodsThis was an open-labeled, fixed-sequence study of bevacizumab with FOLFIRI (irinotecan, leucovorin, and infusional 5-fluorouracil). Pharmacokinetic assessments were conducted in cycles 1 and 3.ResultsForty-five subjects were enrolled. No difference in dose-normalized AUC0–last for irinotecan and SN-38 between irinotecan administered alone or in combination with bevacizumab was identified. Leukopenia was associated with higher exposure to both irinotecan and SN-38. UGT1A1 polymorphisms were associated with variability in irinotecan PK. Gastrointestinal toxicity was associated with UGT1A6 genotype. No other associations between UGT1A genotypes and toxicity were detected.ConclusionBevacizumab does not affect irinotecan PK when administered concurrently. A variety of pharmacogenetic relationships may influence the pharmacokinetics of irinotecan and its toxicity.

Abstract 3490: Highly sensitive detection of EGFR T790M on Ion Torrent PGM.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Introduction: EGFR T790M mutation leads to treatment resistance in ∼50% of NSCLC patient undergoing TKI treatment. Early detection of the emergence of this resistance mutation allows for tailoring of the treatment regimen. Detection of EGFR T790M requires higher sensitivity techniques than those needed to detect EGFR activating mutations, and quantitative measurement of T790M may also provide value in monitoring disease progression. Here we demonstrate that our proprietary amplicon next-generation sequencing (NGS) on Ion Torrent PGM provides an attractive solution for T790M detection with the advantage of high sensitivity, specificity, and quantification capability. Methods: We developed a proprietary library prep method for amplicon sequencing on the Ion Torrent PGM. Ion Torrent barcode sequences were refined in our validation to minimize barcode cross contamination. This optimization of barcodes allows for accurate mutation quantification, especially critical for variants with a frequency below 1%. This also added the benefit of higher throughput and lower cost. We employ this non-enrichment method to quantitatively measure T790M. DNA from the NCI-H1975 cell line was serially diluted into wild-type NA19240 DNA to determine the limit of detection (LOD), sensitivity, specificity, accuracy and reproducibility of the method. We also used reference FFPE DNA from HorizonDx to validate the accuracy and robustness of the assay. We further validated the test by comparing the performance of our NGS method with ddPCR using the same sample set (serially diluted H1975 into wild-type DNA controls). Results: Our data demonstrated that the LOD for T790M is 0.2%. And the method could reproducibly identify variants at these frequencies. With an input of 10ng of the HorizonDx FFPE DNA, we measured T790M frequency at 6.7%, which is concordant with the 6.5% mutation frequency reported by HorizonDx. We cross-validated our NGS assay with ddPCR, and the mutation frequencies detected by both platforms are nearly identical, and have a regression coefficient of 0.9995, with comparable LOD of 0.2%. Furthermore, our NGS assay allows us to detect other variants located in this amplicon, e.g. SNP Q787Q, with LOD at 1%. The phasing status of compound mutations can also be determined by this assay. To further improve the sensitivity, an enrichment method was developed, and the enriched T790M was sequenced by PGM. This method can semi-quantitatively measure T790M frequency with LOD of 0.03%. Conclusion: Our EGFR T790M NGS assay provides a unique option to reliably quantify T790M down to 0.2% with low DNA input. This highly sensitive and specific detection capability may enable earlier detection of emerging therapeutic resistance, particularly if FFPE results reported here extend to a more accessible specimen type, i.e. plasma circulating DNA, amenable to periodic patient monitoring. Citation Format: Agus Darwanto, Peng Fang, Zhenyu Yan, Weihua Liu, Kim Pelak, Jessica Kristof, Sabita Sankar, Cynthia Spittle, Chad Galderisi, Jin Li. Highly sensitive detection of EGFR T790M on Ion Torrent PGM. [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 3490. doi:10.1158/1538-7445.AM2013-3490

Abstract 3229: Validation of Illumina TruSeq Amplicon Cancer Panel with concordance testing using Ion AmpliSeq Cancer Panel and other methods.

Introduction: The TruSeq Amplicon Cancer Panel (TSACP), a highly multiplexed targeted resequencing assay for use on the Illumina MiSeq platform, is designed for detecting the hotspot mutations in 212 Regions of Interest (ROI) from 48 cancer related genes. Here we report our validation study on the reproducibility, sensitivity and the specificity of detecting single base substitutions and small indels by the TSACP. Methods: We used well characterized cancer cell lines harboring clinically relevant variants as positive controls and HapMap samples NA12878 and NA19240 as wild type control samples. DNA from 8 cancer cell lines was serially diluted into the control DNA NA12878 for validating the Limit of Detection (LOD) of the TSACP assay. A total of 41 FFPE patient specimens representing a variety of cancer types were analyzed in a blinded fashion to evaluate the analytical sensitivity and specificity. DNA quality was assessed using a qPCR assay. With no gold standard available as a reference method to detect mutations with comparable sensitivity, concordance testing was performed using the Ion AmpliSeq Cancer Panel. Variants detected by both panels were considered as true positives. Variants that were only covered by one of the two panels were confirmed by a third method, either Sanger sequencing for variants with frequencies above 10% or a custom Ion TargetSeq Assay for variants with frequencies below 10%. Data were analyzed using MiSeq Reporter software and our proprietary analysis pipeline. In addition to reporting hotspots mutations, we also report “Critical Variants” such as non-synonymous coding mutations and splicing site mutations that fall within the ROI. Results: 95% of our ROIs were sequenced at minimum of 0.2X normalized coverage. A cell line dilution study showed that the LOD of confirmed variants is 5%. DNA extracted from 4 of the 41 FFPE specimens failed the template QC by qPCR and failed in the subsequent sequencing run. A total of 124 unique critical variants, including single base substitution, single- or multi- base (up to 21bp) deletion, one- or two- base insertion, were identified in the cancer cell lines and 37 qualified FFPE samples. The intra-assay and inter-assay reproducibility was ≥96%. Using our proprietary analysis pipeline, the analytical sensitivity and specificity for the FFPE samples were both 99%. One false negative of TSACP was identified by the Ion AmpliSeq Cancer Panel and was further confirmed by Sanger sequencing. This allele drop-off occurred as a result of the capture probe falling on a SNP. Homopolymer indels in KIT and STK11 were accurately identified with the Illumina sequencing chemistry. Conclusions: These studies demonstrate that the TSACP assay is highly specific and sensitive and is suitable for screening patient FFPE tumor specimens for a spectrum of clinically relevant somatic mutations. Citation Format: Peng Fang, Zhenyu Yan, Weihua Liu, Agus Darwanto, Kim Pelak, Kim Anoe, Cynthia Spittle, Sabita Sankar, Chad Galderisi, Jin Li. Validation of Illumina TruSeq Amplicon Cancer Panel with concordance testing using Ion AmpliSeq Cancer Panel and other methods. [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 3229. doi:10.1158/1538-7445.AM2013-3229

Abstract 39: Clinical assessment of PTEN mutation in FFPE tissue: comparison of Sanger sequencing, immunohistochemistry and chromogenic in situ hybridization methods.

Background: PTEN is a tumor suppressor that negatively regulates the PI3K signaling pathway by dephosphorylating PIP3, converting it to PIP2. PTEN loss of function results in activation of Akt and downstream signaling pathways, and has been associated with numerous cancers, most commonly endometrial cancer, glioblastoma, melanoma and prostate cancer. PTEN loss can be a result of multiple different aberrations which include mutations in the coding region leading to frame-shift or stop codons, genomic deletions as well as promoter methylation leading to loss of PTEN protein. Determination of PTEN status, both at the DNA and protein levels, may be important for clinical decision-making because loss of PTEN is associated with resistance to various targeted therapies, while inactivating mutations may confer sensitivity to therapeutics targeting PI3K pathway members. We have developed Sanger sequencing, immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH) assays to assess PTEN mutation status in a set of FFPE patient specimens. Methods: Our Sanger sequencing assay for PTEN was developed to detect known hotspot mutations in exons 5-8, which occur in the majority of PTEN mutations reported in the COSMIC database. The IHC and CISH assays were developed using commercially available reagents. All assays were validated using a combination of plasmids, cell lines and FFPE tissues for specificity, sensitivity, reproducibility and concordance. Results: 22 patient FFPE tissue specimens from a variety of tumor types including colon, lung, pancreas, uterus and prostate were evaluated for PTEN mutation and expression status by both Sanger sequencing and IHC methods. Sequencing results were: 9 wild-type, 12 heterozygous mutant, and 1 hemizygous mutant. 7 of the 9 specimens reported as wild-type by sequencing exhibited normal protein expression levels by IHC, while 2 were PTEN negative by IHC, suggesting that mechanisms other than mutations in exons 5-8 contributed to the loss of expression of PTEN protein. These 2 specimens were further analyzed by CISH to determine copy number. Of the 13 specimens reported as mutant by sequencing, IHC protein expression results indicated that 6 were PTEN positive and 7 were PTEN negative, underscoring the observation that exon 5-8 mutation status alone is not sufficient to predict PTEN expression levels. Data on CISH analysis of the specimen that was hemizygous mutant by sequencing will be reported to confirm the copy loss. Conclusion: The data suggest that additional mutations outside the exon 5-8 region as well as promoter methylation may result in loss of PTEN protein, and that accurate clinical assessment of PTEN mutation and protein expression status in FFPE tissues requires complimentary methods including Sanger sequencing, IHC and CISH methodologies. Citation Format: Kellen Sakrison, Jennifer Wright, Eric Bruening, Stephane Wong, Cynthia Spittle, Sabita Sankar, Chad Galderisi. Clinical assessment of PTEN mutation in FFPE tissue: comparison of Sanger sequencing, immunohistochemistry and chromogenic in situ hybridization methods. [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 39. doi:10.1158/1538-7445.AM2013-39