Cassie A. Schumacher

Lrp5 and Lrp6 redundantly control skeletal development in the mouse embryo.

The role of Wnt signaling in osteoblastogenesis in the embryo remains to be fully established. Although β-catenin, a multifunctional protein also mediating canonical Wnt signaling, is indispensable for embryonic osteoblast differentiation, the roles of the key Wnt co-receptors Lrp5 and Lrp6 are unclear. Indeed, global deletion of either Lrp5 or Lrp6 did not overtly affect osteoblast differentiation in the mouse embryo. Here, we generated mice lacking both receptors specifically in the embryonic mesenchyme and observed an absence of osteoblasts in the embryo. In addition, the double-deficient embryos developed supernumerary cartilage elements in the zeugopod, revealing an important role for mesenchymal Lrp5/6 signaling in limb patterning. Importantly, the phenotypes of the Lrp5/6 mutant closely resembled those of the β-catenin-deficient embryos. These phenotypes are likely independent of any effect on the adherens junction, as deletion of α-catenin, another component of the complex, did not cause similar defects. Thus, Lrp5 and 6 redundantly control embryonic skeletal development, likely through β-catenin signaling.

Wntless functions in mature osteoblasts to regulate bone mass

Recent genome-wide association studies of individuals of Asian and European descent have found that SNPs located within the genomic region (1p31.3) encoding the Wntless (Wls)/Gpr177 protein are associated significantly with reduced bone mineral density. Wls/Gpr177 is a newly identified chaperone protein that specifically escorts Wnt ligands for secretion. Given the strong functional association between the Wnt signaling pathways and bone development and homeostasis, we generated osteoblast-specific Wls-deficient (Ocn-Cre;Wls-flox) mice. Homozygous conditional knockout animals were born at a normal Mendelian frequency. Whole-body dual-energy X-ray absorptiometry scanning revealed that bone-mass accrual was significantly inhibited in homozygotes as early as 20 d of age. These homozygotes had spontaneous fractures and a high frequency of premature lethality at around 2 mo of age. Microcomputed tomography analysis and histomorphometric data revealed a dramatic reduction of both trabecular and cortical bone mass in homozygous mutants. Bone formation in homozygotes was severely impaired, but no obvious phenotypic change was observed in mice heterozygous for the conditional deletion. In vitro studies showed that Wls-deficient osteoblasts had a defect in differentiation and mineralization, with significant reductions in the expression of key osteoblast differentiation regulators. In summary, these results reveal a surprising and crucial role of osteoblast-secreted Wnt ligands in bone-mass accrual.

Cdc42 and the Guanine Nucleotide Exchange Factors Ect2 and Trio Mediate Fn14-Induced Migration and Invasion of Glioblastoma Cells

Malignant glioblastomas are characterized by their ability to infiltrate into normal brain. We previously reported that binding of the multifunctional cytokine TNF-like weak inducer of apoptosis (TWEAK) to its receptor fibroblast growth factor–inducible 14 (Fn14) induces glioblastoma cell invasion via Rac1 activation. Here, we show that Cdc42 plays an essential role in Fn14-mediated activation of Rac1. TWEAK-treated glioma cells display an increased activation of Cdc42, and depletion of Cdc42 using siRNA abolishes TWEAK-induced Rac1 activation and abrogates glioma cell migration and invasion. In contrast, Rac1 depletion does not affect Cdc42 activation by Fn14, showing that Cdc42 mediates TWEAK-stimulated Rac1 activation. Furthermore, we identified two guanine nucleotide exchange factors (GEF), Ect2 and Trio, involved in TWEAK-induced activation of Cdc42 and Rac1, respectively. Depletion of Ect2 abrogates both TWEAK-induced Cdc42 and Rac1 activation, as well as subsequent TWEAK-Fn14–directed glioma cell migration and invasion. In contrast, Trio depletion inhibits TWEAK-induced Rac1 activation but not TWEAK-induced Cdc42 activation. Finally, inappropriate expression of Fn14 or Ect2 in mouse astrocytes in vivo using an RCAS vector system for glial-specific gene transfer in G-tva transgenic mice induces astrocyte migration within the brain, corroborating the in vitro importance of the TWEAK-Fn14 signaling cascade in glioblastoma invasion. Our results suggest that the TWEAK-Fn14 signaling axis stimulates glioma cell migration and invasion through two GEF-GTPase signaling units, Ect2-Cdc42 and Trio-Rac1. Components of the Fn14-Rho GEF-Rho GTPase signaling pathway present innovative drug targets for glioma therapy. Mol Cancer Res; 10(7); 958–68. ©2012 AACR.

TROY (TNFRSF19) Promotes Glioblastoma Survival Signaling and Therapeutic Resistance

Of the features that characterize glioblastoma, arguably none is more clinically relevant than the propensity of malignant glioma cells to aggressively invade into the surrounding normal brain tissue. These invasive cells render complete resection impossible, confer significant resistance to chemo- and radiation-therapy, and virtually assure tumor recurrence. Expression of TROY (TNFRSF19), a member of the TNF receptor superfamily, inversely correlates with patient survival and stimulates glioblastoma cell migration and invasion in vitro. In this study, we report that TROY is overexpressed in glioblastoma tumor specimens and TROY mRNA expression is increased in the invasive cell population in vivo. In addition, inappropriate expression of TROY in mouse astrocytes in vivo using glial-specific gene transfer in transgenic mice induces astrocyte migration within the brain, validating the importance of the TROY signaling cascade in glioblastoma cell migration and invasion. Knockdown of TROY expression in primary glioblastoma xenografts significantly prolonged survival in vivo. Moreover, TROY expression significantly increased resistance of glioblastoma cells to both IR- and TMZ-induced apoptosis via activation of Akt and NF-κB. Inhibition of either Akt or NF-κB activity suppressed the survival benefits of TROY signaling in response to TMZ treatment. These findings position aberrant expression and/or signaling by TROY as a contributor to the dispersion of glioblastoma cells and therapeutic resistance. Implications: Targeting of TROY may increase tumor vulnerability and improve therapeutic response in glioblastoma. Mol Cancer Res; 11(8); 865–74. ©2013 AACR.

Genomic Analysis of Uterine Lavage Fluid Detects Early Endometrial Cancers and Reveals a Prevalent Landscape of Driver Mutations in Women without Histopathologic Evidence of Cancer: A Prospective Cross-Sectional Study

Background Endometrial cancer is the most common gynecologic malignancy, and its incidence and associated mortality are increasing. Despite the immediate need to detect these cancers at an earlier stage, there is no effective screening methodology or protocol for endometrial cancer. The comprehensive, genomics-based analysis of endometrial cancer by The Cancer Genome Atlas (TCGA) revealed many of the molecular defects that define this cancer. Based on these cancer genome results, and in a prospective study, we hypothesized that the use of ultra-deep, targeted gene sequencing could detect somatic mutations in uterine lavage fluid obtained from women undergoing hysteroscopy as a means of molecular screening and diagnosis. Methods and Findings Uterine lavage and paired blood samples were collected and analyzed from 107 consecutive patients who were undergoing hysteroscopy and curettage for diagnostic evaluation from this single-institution study. The lavage fluid was separated into cellular and acellular fractions by centrifugation. Cellular and cell-free DNA (cfDNA) were isolated from each lavage. Two targeted next-generation sequencing (NGS) gene panels, one composed of 56 genes and the other of 12 genes, were used for ultra-deep sequencing. To rule out potential NGS-based errors, orthogonal mutation validation was performed using digital PCR and Sanger sequencing. Seven patients were diagnosed with endometrial cancer based on classic histopathologic analysis. Six of these patients had stage IA cancer, and one of these cancers was only detectable as a microscopic focus within a polyp. All seven patients were found to have significant cancer-associated gene mutations in both cell pellet and cfDNA fractions. In the four patients in whom adequate tumor sample was available, all tumor mutations above a specific allele fraction were present in the uterine lavage DNA samples. Mutations originally only detected in lavage fluid fractions were later confirmed to be present in tumor but at allele fractions significantly less than 1%. Of the remaining 95 patients diagnosed with benign or non-cancer pathology, 44 had no significant cancer mutations detected. Intriguingly, 51 patients without histopathologic evidence of cancer had relatively high allele fraction (1.0%–30.4%), cancer-associated mutations. Participants with detected driver and potential driver mutations were significantly older (mean age mutated = 57.96, 95% confidence interval [CI]: 3.30–∞, mean age no mutations = 50.35; p-value = 0.002; Benjamini-Hochberg [BH] adjusted p-value = 0.015) and more likely to be post-menopausal (p-value = 0.004; BH-adjusted p-value = 0.015) than those without these mutations. No associations were detected between mutation status and race/ethnicity, body mass index, diabetes, parity, and smoking status. Long-term follow-up was not presently available in this prospective study for those women without histopathologic evidence of cancer. Conclusions Using ultra-deep NGS, we identified somatic mutations in DNA extracted both from cell pellets and a never previously reported cfDNA fraction from the uterine lavage. Using our targeted sequencing approach, endometrial driver mutations were identified in all seven women who received a cancer diagnosis based on classic histopathology of tissue curettage obtained at the time of hysteroscopy. In addition, relatively high allele fraction driver mutations were identified in the lavage fluid of approximately half of the women without a cancer diagnosis. Increasing age and post-menopausal status were associated with the presence of these cancer-associated mutations, suggesting the prevalent existence of a premalignant landscape in women without clinical evidence of cancer. Given that a uterine lavage can be easily and quickly performed even outside of the operating room and in a physician’s office-based setting, our findings suggest the future possibility of this approach for screening women for the earliest stages of endometrial cancer. However, our findings suggest that further insight into development of cancer or its interruption are needed before translation to the clinic.

Characterization of genetically engineered mouse models carrying Col2a1-cre-induced deletions of Lrp5 and/or Lrp6.

Mice carrying Collagen2a1-cre-mediated deletions of Lrp5 and/or Lrp6 were created and characterized. Mice lacking either gene alone were viable and fertile with normal knee morphology. Mice in which both Lrp5 and Lrp6 were conditionally ablated via Collagen2a1-cre-mediated deletion displayed severe defects in skeletal development during embryogenesis. In addition, adult mice carrying Collagen2a1-cre-mediated deletions of Lrp5 and/or Lrp6 displayed low bone mass suggesting that the Collagen2a1-cre transgene was active in cells that subsequently differentiated into osteoblasts. In both embryonic skeletal development and establishment of adult bone mass, Lrp5 and Lrp6 carry out redundant functions.

Abstract 5391: Low frequency variant detection and tissue-of-origin exploration using liquid biopsies

The promise of liquid biopsy assays lies in the non-invasive monitoring of diseases, such as cancer, through circulating, cell-free DNA (cfDNA) or circulating tumor cell DNA. This may assist in advancing early-stage diagnosis while simultaneously monitoring treatment response over time. Since these materials are often limited, most liquid biopsy assays incorporate targeted sequencing to enable cost-effective deep coverage of target loci for detection of low frequency pathogenic variants. Yet a critical aspect in attaining the necessary sensitivity is an assay that produces uniform, comprehensive coverage from low DNA input quantities. We have developed a liquid biopsy workflow to enable low frequency variant detection from a 10 mL blood draw using the Promega Maxwell RSC combined with Swift Biosciences Accel-NGS 2S® library preparation methodologies. In addition, we explore whether methylation patterns of the extracted cfDNA possess information of the tissue-of-origin. Whole blood samples were collected in Streck cell-free DNA BCT vials from patients with late stage cancer and cfDNA was extracted with the Promega Maxwell RSC. This instrument yielded DNA outputs ranging from 8-32 ng, with a size profile defined by a predominant peak of ~170bp and a mean Alu repeat qPCR integrity score of 0.22, characteristic of high quality cfDNA lacking cellular DNA content. A total of 20 ng cfDNA was used to make an Accel-NGS 2S Hyb library followed by hybridization capture using Agilent SureSelect Human All Exon probes. The Accel-NGS 2S Hyb Kit exhibits a 90% library conversion rate with cfDNA and provides high complexity libraries with uniform target coverage. In addition, molecular barcodes were incorporated to label each library molecule uniquely prior to PCR amplification. These molecular barcodes were utilized for accurate removal of PCR duplicates while simultaneously preserving naturally occurring fragmentation and strand duplicates to maximize data recovery. Secondly, these barcoded molecules were grouped to generate consensus sequences after removal of false positives originating from PCR and sequencing errors. Variant calling was performed using Vardict and Lofreq enabling highly sensitive and precise detection of variants down to a 0.5% allele frequency. In parallel, we have developed a workflow to determine if the epigenetic status of cfDNA can identify tissue-of-origin. This workflow utilizes the Accel-NGS Methyl-Seq DNA Library Kit to enable unbiased characterization from low (5 ng) cfDNA inputs. Through whole genome bisulfite sequencing, using a priori knowledge of differentially methylated regions characteristic of different human tissues, we can identify the predominant tissue source of cfDNA in blood. Citation Format: Justin S. Lenhart, Ashley Wood, Sukhinder Sandhu, Cassie Schumacher, Laurie Kurihara, Vladimir Makarov, Tim Harkins. Low frequency variant detection and tissue-of-origin exploration using liquid biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5391. doi:10.1158/1538-7445.AM2017-5391

Abstract 5392: Targeted next-generation sequencing of cell-free tumor DNA to longitudinally monitor cancer burden and progression

Targeted sequencing of cell-free (cfDNA) and circulating tumor DNA (ctcDNA) from blood enables detection of cancer-related mutations using minimally-invasive sample collection methods, and may make early detection of cancer possible, as well as improve monitoring of disease burden in translational research studies. We have developed a series of targeted panels for detection of multiple cancer-related mutations. The panels are designed to efficiently amplify damaged or short fragments of DNA derived from FFPE and cfDNA/ctcDNA, where hundreds of primer pairs can be amplified in a single tube from overlapping targets using only 10 ng input material, making these panels ideal for limiting liquid biopsy samples. A panel which covers known “hotspot” mutations in 56 oncology-related genes has been used in a pilot research study to monitor gynecological cancer in 11 women in a longitudinal study, which found a correlation between the presence of cancer mutations and morbidity and mortality. In 2 of 11 women, the initial absence of mutations above 1% allele-frequency was followed by the appearance of mutations in 1-3 genes at allele frequencies of 5-78% in the later time point. These 2 patients experienced increased morbidity or mortality. In 9 of the 11 women, no mutations were observed, and 6 remain in remission, while 3 are living with cancer. In an effort to further improve both workflow and performance, we are developing two technologies to incorporate into the panel design for future studies. The first will normalize library yield during PCR amplification for simple library pooling, which eliminates the requirement for library quantification and minimizes the time from sample to sequence. The second technology is a molecular ID (MID) system to tag each amplicon uniquely to allow data tracking to individual DNA fragments from the sample, and to increase confidence in variant calling by filtering PCR and sequencing errors. By incorporating technologies that reduce steps in the workflow, the likelihood of error is minimized, and combined with methods that increase confidence in low frequency variant calling, an ideal workflow for liquid biopsy samples is created. Citation Format: Jonathan C. Irish, Cassie A. Schumacher, Navya Nair, Olga Camacho-Vanegas, Jordan Rose- Figura, Ashley Wood, Sukhinder Sandhu, Sushma Chaluvadi, Sergey Chupreta, Laurie Kurihara, Timothy Harkins, John A. Martignetti, Vladimir Makarov. Targeted next-generation sequencing of cell-free tumor DNA to longitudinally monitor cancer burden and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5392. doi:10.1158/1538-7445.AM2017-5392

Abstract 5345: Complementary NGS approaches on digitally sorted pure tumor cells reveal hidden molecular characteristics in low tumor content FFPE specimens

Introduction: Precise characterization of tumor cell populations is an essential requirement for guiding the cancer care, allowing patients to receive personalized therapies. Poor biopsies with low-tumor content represent a significant barrier for sample enrollment in clinical trials. Here we describe a multi-level approach for precisely characterizing the genetic mutation landscape of pure tumor cell populations sorted by the DEPArray™ technology from low-cellularity FFPE samples. Methods: 50μm sections of FFPE from breast infiltrating ductal adenocarcinoma with 10% tumor content were processed by DEPArray sorting protocol. Illumina-compatible libraries were prepared from sorted stromal (n=497) and tumor (n=419) cell pools, and from the unsorted sample. An aliquot of these libraries was processed using SeqCap EZ MedExome enrichment kit (Roche) for whole-exome sequencing (WES) with a HiSeq 2500, reaching a mean coverage of 27x for tumor and 25x for stromal libraries. A second aliquot was used for low-pass (≈1M fragments per sample) whole-genome sequencing (WGS) on a MiSeq to analyze copy-number alterations (CNA). Other cell lysates from stromal (n=104, n=112) and pure tumor (n=75) populations were used in DEPArray OncoSeek amplicon-based assay for focused analysis of clinically relevant somatic variants and copy-number alterations. Results: In sorted pure tumor populations, WES analysis of B-allele frequencies of germline heterozygous SNPs clearly outlined an aberrant profile, precisely revealing several Loss-of-Heterozygosity (LOH) and copy-altered regions. Conversely, unsorted gDNA showed a flat profile non-distinguishable from sorted stromal cells, as expected for a low-cellularity tumor sample. Similar results were obtained by low-pass WGS, where the huge number of copy number aberrations (≈1.2 Gbp) in tumor is contrasted by lack of gains and losses in stromal cells and unsorted gDNA. Noteworthy, WES, low-pass and targeted sequencing by OncoSeek panel highlighted a focal amplification of ERBB2 gene (>13 copies), which was just barely detectable as a 1-copy gain in bulk gDNA. Genetic analyses showed a high concordance between WES and targeted panel data, with two non-synonymous homozygous somatic mutations found in TP53 (p.L111R) and ERBB2 (p.D769Y). In the unsorted sample, the TP53 mutation was missed because allelic frequency was below the limit of detection due to normal-cell dilution, while the ERBB2 mutation was still detectable only because of the high-level amplification. Conclusions: DEPArray sorting technology is an indispensable tool for accurately investigating cancer genomes, enabling multi-level applications for obtaining a fine-grain characterization of copy-numbers, LOH, and tumor-specific variants, independent of original tumor content. Citation Format: Claudio Forcato, Alberto Ferrarini, Genny Buson, Cassie Schumacher, Chiara Bolognesi, Paola Tononi, Valentina del Monaco, Chiara Mangano, Francesca Fontana, Gianni Medoro, Timothy Harkins, Nicolo Manaresi. Complementary NGS approaches on digitally sorted pure tumor cells reveal hidden molecular characteristics in low tumor content FFPE specimens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5345. doi:10.1158/1538-7445.AM2017-5345

Abstract 3637: Targeted exome and panel analysis from low input and FFPE DNA using hybridization capture for cancer genome studies

Introduction Hybridization-based target enrichment techniques coupled with Next Generation Sequencing (NGS) provide a useful and cost-efficient means to study disease specific target regions including whole exomes and gene panels. More than 500 whole exome analyses are behind The Cancer Genome Atlas (TCGA), which is the largest knowledge base for cancer studies including research, prevention, treatment, and care. Clinical samples may be limited in input and of compromised quality due to formalin fixation, however most current NGS library preparation methods require 50-100 ng high quality DNA for such studies. Here we present an efficient method which enables high quality target enrichment and variant calling from inputs as low as 1-25 ng. Method NGS libraries for hybridization capture were made from 1 to 100 ng of Coriell Hapmap samples (NA12878, etc), clinical Formalin Fixed Paraffin Embedded (FFPE) and Horizon Discovery (HDx) reference DNA (HDx 701) using the Accel-NGS® 2S Hyb DNA Library Kit. Amplified libraries were then enriched using specific targeted panels (xGen® Pan-Cancer and xGen® AML) or SeqCap™ EZ MedExome using manufacturer9s specifications (IDT™ and Roche NimbleGen™). Enriched libraries were captured using streptavidin beads. Captured libraries were then amplified according to the manufacturer9s specifications. Targeted panels were sequenced on an Illumina® MiSeq® using V2 chemistry and MedExome on a HiSeq® using V4 chemistry. Sequence analysis was performed with custom pipelines using BWA for alignment and GATK, Samtools, and Freebayes for variant calling. Results Sequencing analyses yielded a minimum average coverage of 30x and more than thirty fold enrichment. Enriched libraries exhibited significant sequence complexity with minimal duplicates and without any base-composition bias. The percent on-target varied with type of input and ranged from 50-80%. Germline variant calling results had > 99% concordance with the NIST GIAB truth list with sensitivity and precision of > 98%, even from inputs as low as 1 ng of DNA. Somatic variant calling down to 1-5% allele frequency was also evaluated at various DNA input quantities and greater depth of sequencing; results for FFPE and DNA standards will be presented. Conclusions Accel-NGS 2S Hyb technology can be used for whole exome and targeted enrichment studies from low quantity and low quality FFPE clinical samples. High complexity libraries with minimal bias yield high quality sequence data which enables discovery and detection of somatic variants in tumor samples to identify molecular drivers associated with different cancer types. The technique facilitates better understanding of complex cancer genomes and guide precision medicine. Citation Format: Sukhinder K. Sandhu, Cassie Schumacher, Laurie Kurihara, Tim Harkins, Vladimir Makarov. Targeted exome and panel analysis from low input and FFPE DNA using hybridization capture for cancer genome studies. [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 3637.