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

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.

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.

Abstract Circulating, cell free DNA (cfDNA) is a non-invasive sample source that contains tumor associated DNA. Next generation sequencing (NGS) of cfDNA has shown that tumor specific mutations can be detected, providing an effective means to monitor disease, treatment efficacy, and characterize the genome wide methylation state of cfDNA. While hypermethylation is observed for specific gene promoters, on a genome wide perspective, hypomethylation of cfDNA is observed in cancer, making cfDNA an attractive target to assess cancer burden. The challenges in deep sequencing of cfDNA include: mandatory fast turnaround times due to sample degradation, limited sample material, short DNA fragments, and limit of detection issues caused by the presence of both normal and tumor DNA. This study describes a novel library preparation for the Illumina platforms that is cost-effective, sensitive, and specific to assess the methylation status of cfDNA. To characterize the methylation status of cfDNA, NGS libraries were generated utilizing a chemistry that sequentially ligates the adapters to each end of the DNA molecules. Since the library is generated after bisulfite treatment, a high recovery of DNA library molecules is observed, thus enabling high complexity library preparation from 5 ng of cfDNA. Upon establishment of this technique, we obtained cfDNA from healthy subjects as well as from subjects with a spectrum of cancers. Following bisulfite-conversion and library preparation of these samples, we sequenced each sample on the Illumina MiSeq to a depth of 10 million reads. A minimum threshold of 1.1% was used to determine significant hypomethylation. Upon analysis, preliminary analysis of the hypomethylation status of the cfDNA from 4 of the 5 cancer subjects ranged from 3% to 9% when compared to the healthy controls. The cfDNA sample which was negative for hypomethylation originated from the plasma of a subject with a high grade serous adenocarconima in the fallopian tube. The most hypomethylated cfDNA came from the plasma of subject with metastatic adenocarcinoma of the colon which had metastasized to the liver. Other tumor types which resulted in cfDNA hypomethylation between these two extremes included invasive breast carcinoma as well as pancreatic ductal carcinoma. This method provides the basis for a technique to reliably prepare and characterize cfDNA via NGS deep sequencing. We obtained results 5 days after resection and reliably made high complexity library using only 5 ng of cfDNA. We have demonstrated reproducibility and sensitivity for genome wide methylation status, while also observing that the utility of this assay may vary with cancer type. To further define biologically significant thresholds for methylation status, we will present a longitudinal study examining the cfDNA methylation status of individuals before, during, and after treatment, thereby generating a methylation gradient as a function of time and treatment. Citation Format: Cassie A. Schumacher, Sukhinder Sandhu, Vladimir Makarov. An assay to detect hypomethylation in circulating cell free DNA and monitor cancer burden. [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 1051. doi:10.1158/1538-7445.AM2015-1051

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.

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.

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.

OBJECTIVE: Glioblastoma multiforme is the most frequent primary brain tumor in adults. The major obstacle for successful treatment is the invasive growth pattern. Metabolically, glioblastomas are highly glycolytic, leading to increased levels of lactic acid production. The carbonic anhydrase IX (CAIX) moderates the extrusion of hydrogen ions into the extracellular space, which may enhance tumor invasion by activating proteolytic enzymes. We therefore induced glycolysis in glioblastoma cells and investigated the extracellular pH, cathepsin B expression, and its subcellular distribution and secretion parallel to the invasive behavior of the cells upon CAIX knockdown. METHODS: U251 glioblastoma cells were transfected with a CAIX siRNA construct and cultured in a Biocoat Matrigel invasion chambers with an 8-mm pore size membrane. The chambers were incubated in a humified 5% CO2 modular with either 21% oxygen and 25 mM glucose (control) or 0% oxygen plus 125 mM glucose (glycolysis). Invasion was quantified by counting the cells on the lower membrane surface. Extracellular pH was measured using a pH-meter. Cathepsin B expression and localization was investigated by RT-PCR, Western blot, and immunofluorescence staining, respectively. The cathepsin B secretion into the supernatant was measured using a cathepsin B activity assay. RESULTS: In vitro glycolysis caused a significant increase of cathepsin B expression and secretion combined with massive invasion of glioblastoma cells. In addition, the subcellular distribution of the enzyme was shifted to the cell periphery. The extracellular pH dropped significantly under glycolytic conditions, antagonized by CAIX knockdown. In addition, CAIX knockdown did not influence cathepsin B expression but attenuated the intracellular change of cathepsin B distribution and reduced both the cathepsin B secretion and glioma cell invasion. CONCLUSION: Our data demonstrate that CAIX moderates invasion in glycolytic glioma cells via acidification of the extracellular milieu and enhanced secretion of cathepsin B.

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.