Circulating tumor DNA as a marker of treatment response in BRAF V600E mutated non-melanoma solid tumors

Purpose: We evaluated longitudinal tracking of BRAF V600E in circulating cellfree DNA (cfDNA) as a marker of treatment response to BRAF inhibitor (BRAFi) combination therapies in non-melanoma solid tumors included in the Copenhagen Prospective Personalized Oncology (CoPPO) program. Experimental design: Patients with BRAF V600E-mutated tumors were treated with combination therapies including BRAFi. Quantification of mutant cfDNA from plasma was determined and correlated to clinical outcomes. Exome sequencing was performed to identify possible resistance mutations. Results: Twenty-three patients had BRAF-mutated tumors out of 455 patients included in CoPPO and 17 started BRAFi combination (EGFRi/MEKi) therapy. Tumor responses were achieved in 8 out of 16 evaluable patients and the median overalland progression-free survival (OS and PFS) was 15 and 4.8 months, respectively. Longitudinal measurements of BRAF V600E-mutant cfDNA indicated disease progression prior to radiological evaluation and a reduction in the mutant fraction of more than 50% after 4 and 12 weeks of therapy was associated with a significantly longer PFS (p=0.003 and p=0.029) and OS (p=0.029 and p=0.017). Furthermore, the baseline mutant fraction and total level of cfDNA positively correlated with tumor burden (p=0.026 and p=0.024). Finally, analysis of cfDNA at progression revealed novel mutations potentially affecting the MAPK pathway. Conclusion: BRAFi combination therapies showed a response rate of 50% in BRAF V600E-mutated non-melanoma tumors. The fraction of BRAF-mutant cfDNA represent a sensitive indicator for clinical outcomes with plasma collected at week 4 and 12 as crucial time points for monitoring response and disease progression.This study was supported by the Danish Cancer Society, The Harboe Foundation, and the Oncological Research Fund, Department of Oncology, Copenhagen University Hospital, Denmark

Tropical summer induces DNA fragmentation in boar spermatozoa: implications for evaluating seasonal infertility

Summer infertility continues to undermine pig productivity, costing the pig industry millions in annual losses. The boar’s inefficient capacity to sweat, non-pendulous scrotum and the extensive use of European breeds in tropical conditions, can make the boar particularly vulnerable to the effects of heat stress; however, the link between summer heat stress and boar sperm DNA damage has not yet been demonstrated. Semen from five Large White boars was collected and evaluated during the early dry, late dry and peak wet seasons to determine the effect of seasonal heat stress on the quality and DNA integrity of boar spermatozoa. DNA damage in spermatozoa during the peak wet was 16-fold greater than during the early dry and nearly 9-fold greater than during the late dry season. Sperm concentration was 1.6-fold lower in the peak wet than early dry whereas no difference was found across several motility parameters as determined by computer-assisted sperm analysis. These results demonstrate that tropical summer (peak wet season) induces DNA damage and reduces concentration without depressing motility in boar spermatozoa, suggesting that traditional methods of evaluating sperm motility may not detect inherently compromised spermatozoa. Boar management strategies (such as antioxidant supplementation) need to be developed to specifically mitigate this problem

Defective germline reprogramming rewires the spermatogonial transcriptome.

Defective germline reprogramming in Piwil4 (Miwi2)- and Dnmt3l-deficient mice results in the failure to reestablish transposon silencing, meiotic arrest and progressive loss of spermatogonia. Here we sought to understand the molecular basis for this spermatogonial dysfunction. Through a combination of imaging, conditional genetics and transcriptome analysis, we demonstrate that germ cell elimination in the respective mutants arises as a result of defective de novo genome methylation during reprogramming rather than because of a function for the respective factors within spermatogonia. In both Miwi2-/- and Dnmt3l-/- spermatogonia, the intracisternal-A particle (IAP) family of endogenous retroviruses is derepressed, but, in contrast to meiotic cells, DNA damage is not observed. Instead, we find that unmethylated IAP promoters rewire the spermatogonial transcriptome by driving expression of neighboring genes. Finally, spermatogonial numbers, proliferation and differentiation are altered in Miwi2-/- and Dnmt3l-/- mice. In summary, defective reprogramming deregulates the spermatogonial transcriptome and may underlie spermatogonial dysfunction

⁶⁴Cu-DOTATATE positron emission tomography (PET) of <i>borrelia burgdorferi</i> infection:In vivo imaging of macrophages in experimental model of lyme arthritis

Macrophages play a key role in the inflammatory response in Lyme arthritis (LA) and could be a target for diagnosing and monitoring active Borrelia burgdorferi sensu lato (Bb) infection. Therefore, we evaluated the potential of macrophage imaging using 64Cu-DOTATATE PET/CT for detection of Bb activity in a murine model of LA. LA was established in C3H/HeNRj mice infected with Bb B31 strain ML23 pBBE22luc. Bioluminescence imaging was performed to detect migration of spirochetes and inflammatory phagocytes to the joints. Three weeks post-infection 64Cu-DOTATATE PET/CT imaging was performed at an early (3 h) and late (48 h) time point. Plasma levels of a systemic macrophage marker in plasma CD163 were measured. 64Cu-DOTATATE uptake in infected joints was increased at the early (p &lt; 0.0001) and late time points (p = 0.0005) compared with uptake in non-infected controls. No significant difference in plasma levels of CD163 was measured. 64Cu-DOTATATE PET allows for in vivo detection and quantification of LA locally in the joints through non-invasive visualization of macrophages. In contrast, measurement of a systemic macrophage marker in plasma, CD163, did not allow to detect disease. We suggest that 64Cu-DOTATATE PET could become a valuable diagnostic tool for in situ detection of Bb infection-related inflammation