Collection of cell-free DNA (cfDNA) from the blood of individuals with cancer has permitted noninvasive tumor genome analysis. Detection and characterization of cfDNA in ascites and pleural effusions have not yet been reported. Herein, we analyzed cfDNA in the ascites and pleural effusions from six individuals with metastatic cancer. In all cases, cfDNA copy number variations (CNV) were discovered within the effusate. One individual had a relevant alteration with a high copy amplification in EGFR in a never smoker with lung cancer, who showed only MDM2 and CDK4 amplification in a prior tissue biopsy. Another subject with metastatic breast cancer had cytology-positive ascites and an activating PIK3CA mutation identified in the tissue, blood, and ascites collectively. This individual had tumor regression after the administration of the mTOR inhibitor everolimus and had evidence of chromotripsis from chromosomal rearrangements noted in the cell-free ascitic fluid. These results indicate that cfDNA from ascites and pleural effusions may provide additional information not detected with tumor and plasma cell-free DNA molecular characterization, and a context for important insights into tumor biology and clonal dynamic change within primary tumor and metastatic deposits. Mol Cancer Ther; 16(5); 948-55. Ó2017 AACR.

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The interaction of an animal with its environment during a critical period in early postnatal life has lifelong effects on the structure and function of sensory and motor systems. To gain insight into the molecular mechanisms of experience-dependent development, we challenged young rats to adapt to a new environment that engenders novel motor behavior. Rats born in the gravitational field (1G) of the earth subsequently were reared for 2 weeks either in the absence of gravity (microgravity) or at 1G. A comparison of gene expression using microarrays led to the identification of a panel of differentially regulated transcripts. We report here that the abundance of serum-and glucocorticoid-inducible kinase (SGK) is increased in spinal cord tissue from animals reared in microgravity in comparison with 1G-reared controls. The induction of SGK expression also can be achieved by administration of glucocorticoids to animals at 1G or neurons in vitro. Expression of constitutively active SGK in neurons leads to the elaboration of neuronal dendrites and their branching. Glucocorticoids also lead to dendrite elaboration, and this effect can be abrogated by inhibiting SGK activity. Changes in the level of expression of SGK could be part of the mechanism for experience-dependent acquisition of mature neuronal properties.

Background: There is increased interest in molecular analysis of cell-free DNA isolated from body fluids for the evaluation of tumor progression. These “liquid” biopsies are usually obtained from blood. Their advantages include the fact that they can be repeatedly accessed, are less invasive, and may be sensitive to changes in tumor profile from multiple metastatic sites. Materials and Methods: Eleven ascites samples from patients with metastatic epithelial neoplasms (gastric, N = 3; pancreas, N = 3; ovarian, N = 2; breast, N = 2; and lung cancer, N = 1) were investigated. Cell-free DNA was isolated from supernatant of ascites fluid (50 ml) after centrifugation using commercially available DNA purification kits (Norgen Bioteck Corp and Qiagen), and analyzed using the OncoScan FFPE Assay kit. Results: Cell-free DNA yields ranged from 1.7 ng to 230 ng per mL of ascites fluid, indicating wide variability in DNA content. Of the 11 patients, all had detectable aberrations, including copy number alterations affecting from <1%- 37% of the genome. A sample from a patient with metastatic breast cancer had apparent chromosome 12 chromothripsis (chromosome breakage with numerous clustered chromosomal rearrangements). Another sample (metastatic lung cancer) had high overall genomic stability (T); the lung tumor tissue did not show these alterations though the patient has never smoked. Other aberrations were seen in genes including: p53 (N = 4 patients), EGFR (N = 3), ERBB2 (N = 2), PI3KCA (n = 2). Conclusions. Combined copy number and somatic analysis of ascites cell-free DNA revealed clinically relevant aberrations, including ones not found in primary tumor tissue. Comparison to circulating cells in ascites, cell-free DNA in blood, as well as tumor molecular profile is ongoing. Citation Format: Hatim Husain, Sumathi Venkatapathy, German Gomez, Brian Woodward, Suzanna Lee, Lubena Khambaty, Lily Chen, Radha Duttagupta, Eric T. Fung, Razelle Kurzrock. Cell-free DNA derived from ascites: Detection of copy number and somatic mutations using OncoScan FFPE® Assay. [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 2410. doi:10.1158/1538-7445.AM2015-2410

Background Copy number (CN) and somatic mutation studies of cancer are powerful tools for discovering reliable biomarkers that can predict clinical outcomes. We have shown that the Molecular Inversion Probe (MIP) assay used in the OncoScan™ platform works well with thousands of degraded FFPE samples.1 In collaboration with leading cancer researchers, the coverage of the existing platform was further enhanced by supplementing the probe panel with new probes to cover an additional 200 cancer-relevant genes. The OncoScan™ FFPE Express 2.0 platform is able to deliver three types of high-quality data (SNP, CN, and somatic mutation calls) from just 75 ng non-amplified genomic DNA. Experimental design New MIP probes were designed to target the 200 cancer relevant genes and provide: (a) greater marker density; (b) high MAF (minor allele frequency) for SNP analysis; (c) good quantitative performance for reproducibility and dynamic range. Our platform employs a measurement comparing adjacent markers across the genome: this median of absolute pairwise distribution, or MAPD, is a reliable metric for assessing data quality.2 Results The median resolution increased from 1500 to 500 bp/probe in tumor suppressor genes and from 3500 to 1500 bp/probe in oncogenes. The performance of additional probes were assessed in the following categories: (1) For genotyping - the average call rate for 48 HapMap samples with 335K passing SNPs is 99.82%; for normal FFPE, 99.78%. The average accuracy for 48 HapMap is 99.38%. (2) For copy number - probe reproducibility was measured at CN = 0, 1 and 2. The average reproducibility of copy number changes is >95% as measured at the probe level in the SNPs that reside in the 200 genes. Furthermore, >97% of the newly designed probes passed this criteria. (3) For somatic mutation - the new assay was tested in cell lines with known mutations, including EGFR_pE746_A750del, KRAS_pG12D/V, PIK3CA_pE545K and PIK3CA_pH1047. Concordance of 100% was observed from six repeats of each mutation; testing was performed in two different laboratories. The false positive rate is 0.63% when assessing performance with normal samples. Conclusion We have developed a powerful new version of the OncoScan™ platform that works well on both frozen and archival FFPE samples. All three types of mutation analysis (SNP, CN, and somatic mutations) can be interrogated by the same assay, offering an unprecedented opportunity for both retrospective studies where only FFPE samples are available and for ongoing clinical research where only small amounts of biopsy sample are accessible References: 1 Thompson P.A et al. Selective genomic copy number imbalances and probability of recurrence in early-stage breast cancer. PLoS One. 6(8):e23543 (2011). 2 Wang Y., et al. High Quality Copy Number and Genotype Data from FFPE Samples Using Molecular Inversion Probe (MIP) Microarrays. BMC Med Genomics. 19(2):8 (2009). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1259. doi:1538-7445.AM2012-1259