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Background: The ability to undertake molecular analysis to inform on prognosis and predictors of response to therapy is limited by accessibility of tissue. Measurement of total circulating free DNA (cfDNA) or circulating tumor DNA (ctDNA) in peripheral blood may allow easier access to tumor material and help to predict clinical outcomes.Methods: A systematic review of electronic databases identified publications exploring the association between cfDNA or ctDNA and overall survival (OS) in solid tumors. HRs for OS were extracted from multivariable analyses and included in a meta-analysis. Pooled HRs were computed and weighted using generic inverse variance and random-effect modeling. For studies not reporting multivariable analyses, univariable ORs were estimated from Kaplan–Meier curves for OS at 1 and 3 years.Results: Thirty-nine studies comprising 4,052 patients were included in the analysis. Detection of ctDNA was associated with a significantly worse OS in multivariable analyses [HR, 2.70; 95% confidence interval (CI), 2.02–3.61; P < 0.001). Similar results were observed in the univariable analyses at 3 and 1 year (OR, 4.83; 95% CI, 3.20–7.28; P < 0.001).There was also a statistically significant association between high total cfDNA and worse OS for studies reporting multivariable and univariate data at 3 years (HR, 1.91; 95% CI, 1.59–2.29; P < 0.001 and OR, 2.82; 95% CI, 1.93–4.13; P < 0.001, respectively).Conclusions: High levels of total cfDNA and presence of ctDNA are associated with worse survival in solid tumors.Impact: Circulating DNA is associated with worse outcome in solid tumors. Cancer Epidemiol Biomarkers Prev; 25(2); 399–406. ©2015 AACR.

Supplementary Figure 1: Forest plot showing the pooled odds ratio (OR) for overall survival at 1 year for circulating tumor DNA. Supplementary Figure 2: Forest plot showing the pooled odds ratio (OR) for overall survival at 1 year for total circulating DNA. Supplementary Figure 3: Forest plot showing A: pooled hazard ratio (HR) for overall survival for total tumor circulating DNA based on method of collection (plasma versus serum) and B: pooled hazard ratio (HR) for overall survival for t total circulating DNA based on method of collection (plasma versus serum).

Detailed description of the included studies.

It has been proposed that cell-free nucleic acids in the plasma participate in tumorigenesis and the development of metastases via transfection-like uptake of such nucleic acids by susceptible cells. This putative phenomenon is tentatively referred to as “genometastasis.” In the present study, we examined the effects on cultured cells of plasma from healthy individuals and from patients with colon cancer. Cultures of NIH-3T3 cells and human adipose-derived stem cells (hASC) were supplemented with samples of plasma from patients with K-ras–mutated colorectal tumors or from healthy subjects using two different protocols: direct addition of plasma to cultures in standard plates and addition in the absence of contact between plasma and cells, which were separated by a membrane with 0.4-μm pores. In plasma-treated hASCs, no K-ras–mutated sequences were detected by real-time PCR. In contrast, in most cultures of plasma-treated NIH-3T3 cells (murine cells), the transfer of human DNA occurred, as verified by the detection of human K-ras sequences, p53 sequences, and β-globin–encoding sequences. Moreover, NIH-3T3 cells that had been cultured with plasma from patients with colon cancer were oncogenically transformed, as shown by the development of carcinomas in nonobese diabetic–severe combined immunodeficient mice after the injection of such cells. Microscopic analysis of membranes that had separated plasma from cultured cells confirmed the complete absence of cells in the plasma. We only observed noncell particles, having diameters of

Supplementary Methods from Cell-Free Nucleic Acids Circulating in the Plasma of Colorectal Cancer Patients Induce the Oncogenic Transformation of Susceptible Cultured Cells

Background Traumatic brain injury (TBI) is one of the most frequent and severe neurological diseases. In the last few decades, significant advances have been made in TBI pathophysiology and monitoring, however new treatments have not emerged. Although the central nervous system (CNS) has been historically defined as an immunologically privileged organ, recent studies show the increasingly predominant role of inflammatory and apoptotic phenomena in the pathogenesis of TBI. Inflammatory response mediators can be eliminated with continuous renal replacement therapies (CRRT). Our aim was to investigate whether hemofiltration protects the brain after head trauma in an experimental study in animals. Methods and results A model of TBI and CVVH was performed in anesthetized New Zealand white rabbits without acute renal failure. The experimental group TBI ( +)-CVVH ( +) was compared with a TBI ( +)-CVVH (−) and a TBI (−)-CVVH ( +) control groups. Rabbits were assessed immediately (NES1) and 24 h hours after (NES2) TBI and/or CVVH using a functional Neurological Evaluation Score (NES) and histology of the brains after sacrifice. There was evidence to support a difference of NES1 comparing with the TBI (−)-CVVH ( +), but not with TBI ( +)-CVVH (−) with only 15% of the rabbits treated with CVVH and TBI showing a favorable neurological course. The final neurological outcome (mortality at 24 h) was 0%, 22% and 53% in the TBI(−) + CVVH( +), TBI( +)-CVVH(−) and TBI( +)-CVVH( +) groups respectively. The use of hemofiltration before or after TBI did not make a difference in regards the outcome of the rabbits. There was evidence in the histology to support an increase of mild ischemia, hemorrhage and edema in the experimental group compared with the other two groups. Conclusions CVVH in rabbits without renal failure used with the intention to protect the brain may worsen the prognosis in TBI.

Differential presence of exons (DPE) is a method of interpretation of exome sequencing, which has been proposed to design a predictive algorithm with clinical value in patients with colorectal cancer (CRC). The goal of the present study was to examine the reproducibility in a rat model of metastatic colon cancer. DHD/K12-TRb cells were injected in syngenic immunocompetent BD-IX rats. Cells were from two stocks with low and normal metastatic potential, and injected into two separate groups of rats. Five to ten weeks after injection, blood samples were taken prior euthanasia and whole exome sequencing performed. Through DPE analysis, we identified a set of exons whose differential presence in plasma allowed us to compare both groups of tumor-bearing animals. A verification test was performed to confirm that the algorithm was able to classify extracted samples into their corresponding groups of origin. The highest mean probability was 0.8954. In conclusion, the DPE analysis in tumor-bearing animals was able to discriminate between different disease status, which fully supports previous results in CRC patients., This studywas funded by two grants from"Instituto de Salud Carlos III", Spain (FIS; refs. PS09/01815 and PI13/01924)

Most colorectal cancer (CRC)-related deaths are due to liver metastases. PKCζ is a tumor suppressor in CRC with reduced expression in metastasis. Given the importance of microRNAs (miRNAs) in regulating cellular plasticity, we performed an unbiased screening and identified the miR-200 family as the most relevant miRNAs downregulated by PKCζ deficiency. The regulation of the intracellular levels of miR-200 by PKCζ is post-transcriptional and involves their secretion in extracellular vesicles. Here, we identified ADAR2 as a direct substrate of PKCζ in CRC cells. Phosphorylation of ADAR2 regulates its editing activity, which is required to maintain miR-200 steady-state levels, suggesting that the PKCζ/ADAR2 axis regulates miR-200 secretion through RNA editing. Loss of this axis results in epithelial-to-mesenchymal transition (EMT) and increased liver metastases, which can be inhibited in vivo by blocking miR-200 release. Therefore, the PKCζ/ADAR2 axis is a critical regulator of CRC metastases through modulation of miR-200 levels. Shelton et al. demonstrate that the loss of the tumor suppressor PKCζ in colorectal cancer cells results in the downregulation of miR-200, leading to increased epithelial-to-mesenchymal transition, cell invasion, and liver metastasis. This is mediated by an increase in miR-200 secretion through the inactivation of the RNA editing enzyme ADAR2, identifying a key vulnerability in liver metastasis, Research was supported by grants from the NIH (R01DK108743, R01CA172025, and R01CA207177 to J.M.; R01CA192642 and R01CA218254 to M.T.D.-M.)

Increasingly effective therapies targeting the androgen receptor have paradoxically promoted the incidence of neuroendocrine prostate cancer (NEPC), the most lethal subtype of castration-resistant prostate cancer (PCa), for which there is no effective therapy. Here we report that protein kinase C (PKC)λ/ι is downregulated in de novo and during therapy-induced NEPC, which results in the upregulation of serine biosynthesis through an mTORC1/ATF4-driven pathway. This metabolic reprogramming supports cell proliferation and increases intracellular S-adenosyl methionine (SAM) levels to feed epigenetic changes that favor the development of NEPC characteristics. Altogether, we have uncovered a metabolic vulnerability triggered by PKCλ/ι deficiency in NEPC, which offers potentially actionable targets to prevent therapy resistance in PCa.

Highlights • Plasma levels of GAPDH cfDNA might be useful as complementary methods for diagnosis and prognosis of renal cell carcinoma. • Pre and postoperative levels of GAPDH cfDNA were independently associated with OS in renal cell carcinoma. • hTERT cfRNA was not a marker useful for diagnosis nor prognosis of renal cell cancer., Introduction The current challenge on renal cell carcinoma (RCC) is to finding a non-invasive biomarker for improving their diagnostic and therapeutic management. In the present study, we analyzed the clinical value of plasma levels of cell-free DNA (cfDNA) and RNA (cfRNA) of two genes: glyceraldehyde 3-phosphate-dehydrogenase (GAPDH) and human telomerase reverse transcriptase (hTERT). Materials and methods We recruited 82 patients with RCC, and 20 healthy subjects. Using RT-PCR techniques, plasma levels of cfDNA and cfRNA from hTERT and GAPDH genes were quantified pre- and post-operatively, and one year after surgery. Relationships between such plasma levels and clinicopathological features and evolution of disease were analyzed. Results Levels of GAPDH cfDNA and cfRNA were significantly higher in patients than in healthy subjects. hTERT cfDNA was detected in plasma from 35% of RCC patients and in none healthy subject. At diagnosis, plasma levels of GAPDH cfDNA were higher in advanced pT and TNM stages, and hTERT cfDNA in patients with 3–4 Fuhrman grade and affected lymph nodes. Levels of cfNAs were not related to the presence of metastasis. Following nephrectomy, GAPDH cfDNA levels dropped, and patients with higher levels before and after nephrectomy, showed lower overall survival (OS). However, Cox's multivariate model did not prove any association of the cfNA levels with progression. Conclusion Plasma levels of cfDNA from GADPH and hTERT genes were correlated to tumor diagnosis and progression and, thus, such analyses might help to diagnosis and prognosis of RCC patients.

Background: To examine the usefulness of green fluorescent protein (GFP) mice for studying the interactions between normal cells and tumor cells in a host, we used a melanoma model in such “green” mice [C57BL/6-Tg(CAG-EGFP)1Osb mice]. Mice were given a subcutaneous injection of B16-F10 cells, and the resultant primary tumors were removed. Then cells from individual tumors were cultured. Results: The proportion of EFGP+ cells was determined by fluorescence-activated cell sorting (FACS) and was 6.8% ± 3.2% (mean ± s.d.) on day 1 of culture, 0.6% ± 0.3% on day 2, and 0.02% ± 0.01% at day 7. In all cases, isolated cells grew at a constant rate, but fluorescence decreased over time and became undetectable on day 14. Cells were tested using PCR for the presence of an EGFP-specific sequence, and results were negative in all cases, thus indicating that the cells did not harbor the host's reporter gene. Cells were also tested for the presence of EGFP mRNA, which was consistently detected for 22 days after the start of culture. The tumorogenicity of the cultured cells was confirmed in GFP mice injected with cells from a selection of cultures. Conclusions: In a melanoma model in GFP mice, the detection of “green” cells in tumors was not equivalent to the detection of host-derived cells. Such “masking” was caused by a transient, but lasting, transfer of EGFP mRNA from the host's normal cells to tumor cells. Thus, an analysis of tumors postmortem by techniques that yield only a single snapshot can lead to incorrect interpretations and erroneous conclusions.How to cite: Garcia-Olmo D, Picazo MG, García-Olmo D. Melanoma transplants in “green” mice: fluorescent cells in tumors are not equivalent to host-derived cells. Electron J Biotechnol 2018;34. https://doi.org/10.1016/j.ejbt.2018.04.007. Keywords: Cancer models, Development of tumors, EGFP, GFP mice, Green-fluorescent cells, Melanoma, Mouse, Oncogenic transformation

The E1a gene from adenovirus has become a major tool in cancer research. Since the discovery of E1a, it has been proposed to be an oncogene, becoming a key element in the model of cooperation between oncogenes. However, E1a's in vivo behaviour is consistent with a tumour suppressor gene, due to the block/delay observed in different xenograft models. To clarify this interesting controversy, we have evaluated the effect of the E1a 13s isoform from adenovirus 5 in vivo. Initially, a conventional xenograft approach was performed using previously unreported HCT116 and B16-F10 cells, showing a clear anti-tumour effect regardless of the mouse's immunological background (immunosuppressed/immunocompetent). Next, we engineered a transgenic mouse model in which inducible E1a 13s expression was under the control of cytokeratin 5 to avoid side effects during embryonic development. Our results show that E1a is able to block chemical skin carcinogenesis, showing an anti-tumour effect. The present report demonstrates the in vivo anti-tumour effect of E1a, showing that the in vitro oncogenic role of E1a cannot be extrapolated in vivo, supporting its future use in gene therapy approaches., This work was supported by grants from Fundación Leticia Castillejo Castillo and MINECO (SAF 2015 64215-R) and from JCCM (PPII10-0141-0404) to RSP. F. Cimas was supported by Fundación Leticia Castillejo Castillo and Cátedra Enresa. RSP research institution received support from the European Community through the regional development funding programme (FEDER).

KRAS mutations are responsible for resistance to anti-epidermal growth factor receptor (EGFR) therapy in colorectal cancer patients. These mutations sometimes appear once treatment has started. Detection of KRAS mutations in circulating cell-free DNA in plasma (“liquid biopsy”) by droplet digital PCR (ddPCR) has emerged as a very sensitive and promising alternative to serial biopsies for disease monitoring. In this study, KRAS G12V mutation was analyzed by ddPCR in plasma DNA from 10 colorectal cancer patients and compared to six healthy donors. The percentage of KRAS G12V mutation relative to wild-type sequences in tumor-derived DNA was also determined. KRAS G12V mutation circulating in plasma was detected in 9 of 10 colorectal cancer patients whose tumors were also mutated. Colorectal cancer patients had 35.62 copies of mutated KRAS/mL plasma, whereas in healthy controls only residual copies were found (0.62 copies/mL, p = 0.0066). Interestingly, patients with metastatic disease showed a significantly higher number of mutant copies than M0 patients (126.25 versus 9.37 copies/mL, p = 0.0286). Wild-type KRAS was also significantly elevated in colorectal cancer patients compared to healthy controls (7718.8 versus 481.25 copies/mL, p = 0.0002). In conclusion, KRAS G12V mutation is detectable in plasma of colorectal cancer patients by ddPCR and could be used as a non-invasive biomarker., This study was funded by a grant from “Fondo de Investigaciones Sanitarias-FEDER”, Ministry of Health, Spain (FIS; PI13/01924) and the Spanish Ministry of Health and Consumer Affairs (via a cooperative network-FEDER [TerCel RD12- 0019-0035])

[Background]: The ability to undertake molecular analysis to inform on prognosis and predictors of response to therapy is limited by accessibility of tissue. Measurement of total circulating free DNA (cfDNA) or circulating tumor DNA (ctDNA) in peripheral blood may allow easier access to tumor material and help to predict clinical outcomes. [Methods]: A systematic review of electronic databases identified publications exploring the association between cfDNA or ctDNA and overall survival (OS) in solid tumors. HRs for OS were extracted from multivariable analyses and included in a meta-analysis. Pooled HRs were computed and weighted using generic inverse variance and random-effect modeling. For studies not reporting multivariable analyses, univariable ORs were estimated from Kaplan-Meier curves for OS at 1 and 3 years. [Results]: Thirty-nine studies comprising 4,052 patients were included in the analysis. Detection of ctDNA was associated with a significantly worse OS in multivariable analyses [HR, 2.70; 95% confidence interval (CI), 2.02-3.61; P < 0.001). Similar results were observed in the univariable analyses at 3 and 1 year (OR, 4.83; 95% CI, 3.20-7.28; P < 0.001).There was also a statistically significant association between high total cfDNA and worse OS for studies reporting multivariable and univariate data at 3 years (HR, 1.91; 95% CI, 1.59-2.29; P < 0.001 and OR, 2.82; 95% CI, 1.93-4.13; P < 0.001, respectively). [Conclusions]: High levels of total cfDNA and presence of ctDNA are associated with worse survival in solid tumors. [Impact]: Circulating DNA is associated with worse outcome in solid tumors., This work was supported by grants from CRIS cancer foundation (to A. Ocaña and A. Pandiella).

Some experimental procedures are associated with placement of animals in wire-bottom cages. The goal of this study was to evaluate stress-related physiological parameters (heart rate [HR], body temperature [BT], locomotor activity [LA], body weight [BW] and food consumption) in rats under two housing conditions, namely in wire-bottom cages and in bedding-bottom cages. Telemetry devices were surgically implanted in male Sprague-Dawley rats. HR, BT and LA were recorded at 5 min intervals. Analysis under each housing condition was performed from 16:00 to 08:00 h of the following day (4 h light, 12 h dark). During almost all of the light phase, the HR of rats housed in wire-bottom cages remained high (371 ± 35 bpm; mean ± SD; n = 6) and was significantly different from that of rats housed in bedding-bottom cages (340 ± 29 bpm; n = 6; P < 0.001; Student's t-test). In general, BT was similar under the two housing conditions. However, when rats were in wire-bottom cages, BT tended to fluctuate more widely during the dark phase. LA decreased when animals were housed in wire-bottom cages, in particular during the dark phase. Moreover, there was a significant difference with respect to the gain in BW: BW of rats housed in bedding-bottom cages increased 12 ± 2 g, whereas that of rats in wire-bottom cages decreased by 2 ± 3 g (P < 0.001). Our results demonstrate that housing rats in wire-bottom cages overnight leads to immediate alterations of HR, BW and LA, which might be related to a stress response.

The authors evaluated the efficiency and safety of a technique for drawing blood from the cranial vena cava of the hamster (Mesocricetus auratus). They collected blood from Golden Syrian hamsters (n = 15) and from epileptic hamsters (GASH/Sal strain; n = 10) that were anesthetized with isoflurane. Four epileptic hamsters constituted a control group that underwent anesthesia but not blood collection. For blood sampling, a needle connected to a syringe was inserted at a site between 0.2 and 0.6 cm from the manubrium; the depth of insertion was 0.3-1.0 cm. Blood collection was successful in all hamsters except one, and the volume of collected blood ranged from 0.2 ml to 1.5 ml. All hamsters recovered quickly from the procedure, and none showed obvious signs of pain or stress. At necropsy (24 h after the procedure), six subjects showed small local hematomas, and eight showed vascular lacerations of limited severity. Lungs and hearts did not show any damage related to vein puncture. This relatively simple blood collection technique seems to be efficient and safe in hamsters.

Background: To clarify the implications of cell-free nucleic acids (cfNA) in the plasma in neoplastic disease, it is necessary to determine the kinetics of their release into the circulation.Methods: To quantify non-tumor and tumor DNA and RNA in the plasma of tumor-bearing rats and to correlate such levels with tumor progression, we injected DHD/K12-PROb colon cancer cells subcutaneously into syngenic BD-IX rats. Rats were sacrificed and their plasma was analyzed from the first to the eleventh week after inoculation.Results: The release of large amounts of non-tumor DNA into plasma was related to tumor development from its early stages. Tumor-specific DNA was detected in 33% of tumor-bearing rats, starting from the first week after inoculation and at an increasing frequency thereafter. Animals that were positive for tumor DNA in the plasma had larger tumors than those that were negative (p = 0.0006). However, the appearance of both mutated and non-mutated DNA fluctuated with time and levels of both were scattered among individuals in each group. The release of non-tumor mRNA was unaffected by tumor progression and we did not detect mutated RNA sequences in any animals.Conclusions: The release of normal and tumor cfDNA into plasma appeared to be related to individual-specific factors. The contribution of tumor DNA to the elevated levels of plasma DNA was intermittent. The release of RNA into plasma during cancer progression appeared to be an even more selective and elusive phenomenon than that of DNA, This work was financed in part by grants from Fondo Europeo de Desarrollo Regional and Fondo de Investigación Sanitaria (nos. PI031540 and PS09/00874) and from Junta de Comunidades de Castilla La Mancha (no. 03047), Spain

It has been proposed that cell-free nucleic acids in the plasma participate in tumorigenesis and the development of metastases via transfection-like uptake of such nucleic acids by susceptible cells. This putative phenomenon is tentatively referred to as “genometastasis.” In the present study, we examined the effects on cultured cells of plasma from healthy individuals and from patients with colon cancer. Cultures of NIH-3T3 cells and human adipose-derived stem cells (hASC) were supplemented with samples of plasma from patients with K-ras–mutated colorectal tumors or from healthy subjects using two different protocols: direct addition of plasma to cultures in standard plates and addition in the absence of contact between plasma and cells, which were separated by a membrane with 0.4-μm pores. In plasma-treated hASCs, no K-ras–mutated sequences were detected by real-time PCR. In contrast, in most cultures of plasma-treated NIH-3T3 cells (murine cells), the transfer of human DNA occurred, as verified by the detection of human K-ras sequences, p53 sequences, and β-globin–encoding sequences. Moreover, NIH-3T3 cells that had been cultured with plasma from patients with colon cancer were oncogenically transformed, as shown by the development of carcinomas in nonobese diabetic–severe combined immunodeficient mice after the injection of such cells. Microscopic analysis of membranes that had separated plasma from cultured cells confirmed the complete absence of cells in the plasma. We only observed noncell particles, having diameters of

Therapeutic options for recto-vaginal fistula in the setting of Crohn's disease are limited and many data are available in the literature. The manuscript describes the history of a patient who has been the pioneer of our Clinical Trials in treating this disease in fistulizing Crohn's disease environment. We believe it is the first time that a patient with this disease has been treated by adipose-derived stem cells in allogeneic form. The conclusion of our study with Mary is that the use of mesenchymal stem cells derived from adipose tissue is secure, either in autologous or allogeneic form. Furthermore, we have proved that if we use multi-dose and multiple applications on a patient, it does not produce any adverse effect, which confirms us the safety of using these cells in patients at least in the fistulizing Crohn's disease environment.

Tracheal fistulas represent a challenging management problem. The standard treatment in most cases is based on surgical procedures. Various endoscopic treatment modalities have been proposed in patients at high surgical risk but the results are often unsatisfactory. The fistulas frequently recur with additional complications and sometimes death. The case history is reported of a patient with a tracheomediastinal fistula resulting from laser treatment of tracheal cancer. The fistula was treated by bronchoscopic injection of autologous adipose-derived stem cells (ASC) suspended in fibrin glue. The patient's stem cells were isolated from lipoaspirate according to a standard-ised procedure. No complications due to treatment with ASC occurred during the follow-up period and no fistula-related symptoms were detected. Postoperative bronchoscopic examination of the wall and base of the fistula revealed re-epithelialisation and neovascularisation that led to successful closure. To our knowledge, this is the first report of treatment of tracheal fistula by autologous cell therapy. Further studies are needed to confirm whether this new method should become routine practice.

The presence of small amounts of circulating nucleic acids in plasma and serum (CNAPS) is not a new finding. The verification that such amounts are significantly increased in cancer patients, and that CNAPS might carry a variety of genetic and epigenetic alterations related to cancer development and progression, has aroused great interest in the scientific community in the last decades. Such alterations potentially reflect changes that occur during carcinogenesis, and include DNA mutations, loss of heterozygosity, viral genomic integration, disruption of microRNA, hypermethylation of tumor suppressor genes, and changes in the mitochondrial DNA. These findings have led to many efforts toward the implementation of new clinical biomarkers based on CNAPS analysis. In the present article, we review the main findings related to the utility of CNAPS analysis for early diagnosis, prognosis, and monitoring of cancer, most of which appear promising. However, due to the lack of harmonization of laboratory techniques, the heterogeneity of disease progression, and the small number of recruited patients in most of those studies, there has been a poor translation of basic research into clinical practice. In addition, many aspects remain unknown, such as the release mechanisms of cell-free nucleic acids, their biological function, and the way by which they circulate in the bloodstream. It is therefore expected that in the coming years, an improved understanding of the relationship between CNAPS and the molecular biology of cancer will lead to better diagnosis, management, and treatment., Video abstract

To the editor: Horizontal transfer of DNA and the “genometastasis hypothesis” In a recent issue of Blood, Holmgren et al[1][1] raised the question of whether DNA can be transferred from one cell to another via the phagocytosis of apoptotic bodies. Their data show conclusively that DNA can be