<p>Canine primary lung cancer cell line sensitivity to lapatinib. Four canine cell lines (three HER2WT and one HER2V659E) were treated with 14 lapatinib doses ranging from 100 μM to 5.5x10-2 nM for 72 hours with CellTiterGlo viability endpoints were measured and shown as percent survival relative to DMSO vehicle control.</p>
<p>Somatic copy number plots derived from exome sequencing of five primary canine pulmonary adenocarcinomas (cPAC) and matched constitutional DNA. Tumor copy number states determined by tCoNutT analysis of tumors and matched constitutional DNA from five cPAC cases is shown with each canine chromosome plotted on the x-axis (shown in alternating green and black) and log2 fold change shown on the y-axis.</p>
<p>Canine primary lung cancer cell line sensitivity to erlotinib. Five canine cell lines (three HER2WT and two HER2V659E) and one human cell line BT474 (HER2amp) were treated with 10 erlotinib doses ranging from 5x10-8 to 50 μM for 72 hours with CellTiterGlo viability endpoints measured and shown as percent growth inhibition relative to DMSO vehicle control.</p>
<p>HER2 cellular location and function in primary canine lung cancer. (A) Canine papillary adenocarcinoma with intense, complete, circumferential membrane (white arrow) and lateral cytoplasmic membrane (black arrow) anti-HER2 antibody positive staining (brown) in a patient with wild-type HER2. (B) Canine papillary adenocarcinoma with moderate cytoplasmic (black arrow) anti-HER2 antibody positive staining (light brown) in a patient with wild-type HER2. x 40; bar 50 µm. (C) Anti-HER2 immunohistochemistry of a Grade 1 canine papillary adenocarcinoma wild type for HER2. x 20. (D) Segmentation mark-up of the tumor from adjacent normal lung. Tumor is identified by green, whereas red is area within tumor that contains no tissue, and yellow represents areas of non-tumor such as necrosis or tumor stroma. x20.</p>
Abstract Accurate detection of minimal residual disease (MRD) can guide individualized management of early stage cancer patients, but current diagnostic approaches lack adequate sensitivity. Circulating tumor DNA (ctDNA) analysis has shown promise for recurrence monitoring but MRD detection immediately after neoadjuvant therapy or surgical resection has remained challenging. We have developed TARgeted DIgital Sequencing (TARDIS) to simultaneously analyze multiple patient-specific cancer mutations in plasma and improve sensitivity for minute quantities of residual tumor DNA. In 77 reference samples at 0.03%-1% mutant allele fraction (AF), we observed 93.5% sensitivity. Using TARDIS, we analyzed ctDNA in 34 samples from 13 patients with stage II/III breast cancer treated with neoadjuvant therapy. Prior to treatment, we detected ctDNA in 12/12 patients at 0.002%-1.04% AF (0.040% median). After completion of neoadjuvant therapy, we detected ctDNA in 7/8 patients with residual disease observed at surgery and in 1/5 patients with pathological complete response (odds ratio, 18.5, Fisher’s exact p=0.032). These results demonstrate high accuracy for a personalized blood test to detect residual disease after neoadjuvant therapy. With additional clinical validation, TARDIS could identify patients with molecular complete response after neoadjuvant therapy who may be candidates for nonoperative management. One Sentence Summary A personalized ctDNA test achieves high accuracy for residual disease.
e16185 Background: Comprehensive sequencing efforts have identified fibroblast growth factor receptor (FGFR) translocations in numerous cancers. FGFR2 fusions were recently identified as an actionable therapeutic target in ̃17% of cholangiocarcinoma patients, predominantly found in patients with intrahepatic cholangiocarcinoma. Genomic partners for FGFR fusions are variable. In addition, obtaining high yield tissue biopsies for CCA remains challenging and tumor tissue available for molecular analyses is scarce. When combined, these two factors make detection of FGFR fusions in CCA challenging. An accurate molecular assay to detect FGFR fusions in CCA could improve utilization of FGFR-targeted therapies. Methods: The objective of our study was to develop an approach for targeted RNA analysis to improve sensitivity for FGFR fusion detection from limited tumor material. We adapted the sensitive targeted digital sequencing (TARDIS) approach recently developed in our lab for analysis of RNA. This approach utilizes a combination of open-ended targeted amplification followed by ligation, enabling detection of tyrosine kinase fusions without prior knowledge of the precise sequence of the fusion breakpoint or identity of the fusion partner. For evaluation of analytical performance, we analyzed RNA from a CCA organoid model with a known FGFR2 fusion, and Seraseq Fusion RNA Mix v3 Reference Material with two known FGFR3 fusions. Results: Without prior knowledge the partner or coordinates of the fusion breakpoint, we detected an FGFR2-KIF5C fusion in RNA from a CCA organoid model. The fusion breakpoint coordinates predicted by TARDIS-RNA were validated by comparison with RNA-Seq. To assess sensitivity and quantitative performance of the assay, we analyzed a serial dilution of the reference RNA sample from Seraseq with concentration of RNA molecules representing candidate fusions verified by digital PCR. Using TARDIS-RNA to analyze replicates with 5 ng RNA input, we were able to detect candidate fusions represented by as few as 2.7 fusion molecules on average. Concentrations of fusion molecules measured using the two methods were highly correlated (R = 0.96). Conclusions: These results demonstrate sensitivity and quantitative performance of a targeted RNA sequencing assay to detect and quantify FGFR fusions in tumor specimen from intrahepatic cholangiocarcinoma. On-going work is focused on further evaluating assay performance and characterizing FGFR fusions using additional tissue specimen.
<p>Somatic mutation signatures identified by exome sequencing in primary canine lung cancers. (A) The distribution of somatic single nucleotide mutation types in their trinucleotide context from tumor/normal exome sequencing of five cPAC cases. (B) The most common mutation signatures based on trinucleotide context and frequency of somatic single nucleotide mutations from tumor/normal exome sequencing of five cPAC cases. COSMIC Signature 1A (C>T substitutions at NpCpG trinucleotides that are associated with age) was present in four cases. COSMIC Signature 2 (C>T and C>G substitutions at TpCpN, associated with APOBEC cytidine deaminase activity) was present in two cases.</p>
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