Current sequencing methods are error-prone, which precludes the identification of low frequency mutations for early cancer detection. Duplex sequencing is a sequencing technology that decreases errors by scoring mutations present only in both strands of DNA. Our aim was to determine whether duplex sequencing could detect extremely rare cancer cells present in peritoneal fluid from women with highgrade serous ovarian carcinomas (HGSOCs). These aggressive cancers are typically diagnosed at a late stage and are characterized by TP53 mutations and peritoneal dissemination. We used duplex sequencing to analyze TP53 mutations in 17 peritoneal fluid samples from women with HGSOC and 20 from women without cancer. The tumor TP53 mutation was detected in 94% (16/17) of peritoneal fluid samples from women with HGSOC (frequency as low as 1 mutant per 24,736 normal genomes). Additionally, we detected extremely low frequency TP53 mutations (median mutant fraction 1/13,139) in peritoneal fluid from nearly all patients with and without cancer (35/37). These mutations were mostly deleterious, clustered in hotspots, increased with age, and were more abundant in women with cancer than in controls. The total burden of TP53 mutations in peritoneal fluid distinguished cancers from controls with 82% sensitivity (14/17) and 90% specificity (18/20). Age-associated, low frequency TP53 mutations were also found in 100% of peripheral blood samples from 15 women with and without ovarian cancer (none with hematologic disorder). Our results demonstrate the ability of duplex sequencing to detect rare cancer cells and provide evidence of widespread, low frequency, age-associated somatic TP53 mutation in noncancerous tissue.TP53 mutations | ultra-deep sequencing | ovarian cancer | clonal hematopoiesis | premalignant mutations T he detection of tumor-specific mutations in clinically accessible samples has enormous potential to transform cancer diagnostics, monitoring, and screening. However, a major limitation is insufficiently accurate sequencing methods. Conventional nextgeneration sequencing (NGS) technologies have a high false positive error rate, which precludes reliable detection of mutations at frequencies <1/100 (1). "Molecular tagging" of single-stranded DNA decreases the rate of false mutations to less than 1 per 10,000 sequenced nucleotides and has been successfully applied to the detection of mutant cancer DNA in a variety of clinical samples (2-6). However, this false positive error rate limits the specificity of this method in challenging situations in which ultra-deep sequencing is needed to detect extremely low frequency mutant molecules (e.g., <1/10,000), as is the case of ovarian cancer DNA in Pap smears (5). Because true mutations are indistinguishable from artifacts, compromised specificity leads to lower sensitivity and overall low diagnostic accuracy. Duplex sequencing is an NGS technology that employs molecular tagging of both strands of DNA independently. True mutations are defined as mutations that are present at the same...

We are interested in developing oncolytic adenoviruses for the treatment of bone metastasis of cancer. A key limitation of systemic delivery of oncolytic adenovirus type 5 (Ad5) is that the majority of the virus is taken up by the liver, causing liver damage and systemic toxicity. Given that Ad5 hexon binding with blood coagulation factor X is a key factor in liver sequestration, and that a rare serotype, Ad48, has a diminished capacity to bind with factor X, we have generated mHAd.luc2, a novel hexon-chimeric oncolytic adenovirus. To create mHAd.luc2, seven hypervariable regions of Ad5 hexon were substituted with the corresponding regions from Ad48. Compared with Ad5-based oncolytic virus Ad.luc2, intravenous injection of mHAd.luc2 into nude mice resulted in significantly reduced liver uptake. A single high dose (1.0×10(11) viral particles/mouse) of Ad.luc2 resulted in 100% animal death by day 3; whereas none of the mice died in the mHAd.luc2 group. Liver enzyme and liver pathology studies indicated that mHAd.luc2 induced significantly less liver toxicity compared with Ad.luc2. Both mHAd.luc2 and Ad.luc2 exhibited similar binding with breast tumor cells, whereas in the presence of factor X, mHAd.luc2 binding was reduced. Both mHAd.luc2 and Ad.luc2 had nearly equal replication potential in breast cancer cells in vitro. Intravenous injection of mHAd.luc2 and Ad.luc2 into nude mice bearing bone metastases resulted in uptake of the viruses into skeletal tumors, and induced significant inhibition of established bone metastases. Thus, liver-detargeted oncolytic adenovirus can be developed for the treatment of breast cancer bone metastasis.

Our lab has developed a new series of self‐immolative MR agents for the rapid detection of enzyme activity in mouse models expressing β‐galactosidase (β‐gal). We investigated two molecular architectures to create agents that detect β‐gal activity by modulating the coordination of water to GdIII. The first is an intermolecular approach, wherein we designed several structural isomers to maximize coordination of endogenous carbonate ions. The second involves an intramolecular mechanism for q modulation. We incorporated a pendant coordinating carboxylate ligand with a 2, 4, 6, or 8 carbon linker to saturate ligand coordination to the GdIII ion. This renders the agent ineffective. We show that one agent in particular (6‐C pendant carboxylate) is an extremely effective MR reporter for the detection of enzyme activity in a mouse model expressing β‐gal.

We have examined the effect of adenoviruses expressing soluble transforming growth factor receptorII-Fc (sTGFβRIIFc) in a 4T1 mouse mammary tumor bone metastasis model using syngeneic BALB/c mice. Infection of 4T1 cells with a non-replicating adenovirus, Ad(E1-).sTβRFc, or with two oncolytic adenoviruses, Ad.sTβRFc and TAd.sTβRFc, expressing sTGFβRIIFc (the human TERT promoter drives viral replication in TAd.sTβRFc) produced sTGFβRIIFc protein. Oncolytic adenoviruses produced viral replication and induced cytotoxicity in 4T1 cells. 4T1 cells were resistant to the cytotoxic effects of TGFβ-1 (up to 10 ng/ml). However, TGFβ-1 induced the phosphorylation of SMAD2 and SMAD3, which were inhibited by co-incubation with sTGFβRIIFc protein. TGFβ-1 also induced IL-11, a well-known osteolytic factor. Intracardiac injection of 4T1-luc2 cells produced bone metastases by day 4. Intravenous injection of Ad.sTβRFc (on days 5 and 7) followed by bioluminescence imaging (BLI) of mice on days 7, 11 and 14 in tumor bearing mice indicated inhibition of bone metastasis progression (p<0.05). X-ray radiography of mice on day 14 showed a significant reduction of the lesion size by Ad.sTβRFc (p<0.01) and TAd.sTβRFc (p<0.05). Replication-deficient virus Ad(E1-).sTβRFc expressing sTGFβRIIFc showed some inhibition of bone metastasis, while Ad(E1-).Null was not effective in inhibiting bone metastases. Thus, systemic administration of Ad.sTβRFc and TAd.sTβRFc can inhibit bone metastasis in the 4T1 mouse mammary tumor model, and can be developed as potential anti-tumor agents for breast cancer.