Dolores C. García-Olmo

Autologous expanded adipose-derived stem cells for the treatment of complex cryptoglandular perianal fistulas: a phase III randomized clinical trial (FATT 1: fistula Advanced Therapy Trial 1) and long-term evaluation.

Background: Autologous adipose-derived stem cells may represent a novel approach for the management of complex fistula-in-ano. After successful phase I and II clinical trials, a phase III trial was performed to investigate the safety and efficacy. Design: In this multicenter, randomized, single-blind, add-on clinical trial, 200 adult patients from 19 centers were randomly assigned to receive 20 million stem cells (group A, 64 patients), 20 million adipose-derived stem cells plus fibrin glue (group B, 60 patients), or fibrin glue (group C, 59 patients) after closure of the internal opening. Fistula healing was defined as reepithelization of the external opening and absence of collection >2 cm by MRI. If the fistula had not healed at 12 weeks, a second dose (40 million stem cells in groups A and B) was administered. Patients were evaluated at 24 to 26 weeks (primary end point) and at 1 year (long-term follow-up). Results: All results are according to the “blinded evaluator” assessment. After 24 to 26 weeks, the healing rate was 39.1%, 43.3%, 37.3% in groups A, B, and C (p = 0.79). At 1 year, the healing rates were 57.1%, 52.4%, and 37.3 % (p = 0.13). On analysis of the subpopulation treated at the technique’s pioneer center, healing rates were 54.55%, 83.33%, and 18.18%, at 24 to 26 weeks (p < 0.001). No SAEs were reported. Conclusions: In treatment of complex fistula-in-ano, a dose of 20 or 60 million adipose-derived stem cells alone or in combination with fibrin glue was considered a safe treatment, achieving healing rates of approximately 40% at 6 months and of more than 50% at 1-year follow-up. It was equivalent to fibrin glue alone. No statistically significant differences were found when the 3 groups where compared. Clinical trials registration: www.clinicaltrials.gov, identifier NCT00475410; Sponsor, Cellerix SA.

Functionality of Circulating DNA

Abstract: The detection of free, circulating tumor DNA in the plasma of cancer patients opens up new possibilities for the diagnosis and prognostication of cancer. Whatever might be the mechanism for the presence of such DNA, it is now clear that oncogenes can circulate in the plasma fraction of blood and we can now ask whether this phenomenon has potentially important implications in cancer patients. The results of our experiments, together with previous observations of other authors, have led us to propose the “Hypothesis of Genometastasis”, which suggests that metastases might develop as a result of transfection of susceptible cells in distant target organs with dominant oncogenes that circulate in the plasma and are derived from the primary tumor.

Adipose-derived stem cells in Crohn's rectovaginal fistula.

Therapeutic options for recto-vaginal fistula in the setting of Crohns 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 Crohns 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 Crohns disease environment.

Circulating nucleic acids in plasma and serum (CNAPS): applications in oncology

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.

Experimental evidence does not support use of the "no-touch" isolation technique in colorectal cancer.

PURPOSE: The benefits of the “no-touch” isolation technique usually performed to prevent the circulation of tumor cells are not evident. The aim of this study was to determine whether conventional surgical procedures for treatment of colon cancer could provoke the circulation of tumor cells detected by a genetic technology. METHODS: Sixteen patients undergoing resection for colorectal cancer and two patients with irresectable tumors were studied. No patient showed liver or lung metastasis. With specific primers for carcinoembryonic antigen, we used reverse transcriptasepolymerase chain reaction to analyze tumor biopsy specimens and blood samples obtained from the antecubital vein before and after surgery and from the main drainage vein of the tumor when the tumor had been extracted. Peritoneal fluid was also collected in irrecsectable cases. RESULTS: Amplification of cDNA with carcinoembryonic antigen-specific primers was achieved with all tumor biopsies and samples of peritoneal fluid. In two patients carcinoembryonic antigen reverse transcriptase-polymerase chain reaction products were detected in antecubital vein blood before surgery and in one of them also after surgery. Only in one patient (Dukes C) were carcinoembryonic antigen reverse transcriptase-polymerase chain reaction products detected from the main drainage vein of the tumor. In serial dilution experiments we determined that the limit of detection of this method was ten tumor cells in 2 ml of blood. CONCLUSION: Our data suggest that the use of no-touch isolation techniques in colorectal cancer is not justified, based on lack of evidence indicating the detachment of cells from the tumor at surgery.

Quantitation of cell-free DNA and RNA in plasma during tumor progression in rats

BackgroundTo 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.MethodsTo 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.ResultsThe 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.ConclusionsThe 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.

In vitro activation of macrophages by a novel proteoglycan isolated from corms of Crocus sativus L

Saffron corms contain a proteoglycan that is highly cytotoxic on human tumor cells. The present work was undertaken to study the possible immunomodulatory and anti-invasive properties of this compound. Non-cytotoxic concentrations of this glycoconjugate promoted significant macrophage activation, detected by the release of nitric oxide. A rapid activation of protein kinase C and NF-kappaB was obtained after proteoglycan treatment, which could explain the induction of nitric oxide synthase. Proteoglycan concentrations ranging from 10-1000 ng/ml specifically promoted apoptosis of macrophages, probably triggered by their activation. This molecule did not inhibit in vitro migration or invasion of human tumor cells. Altogether these results support a plausible immuno-modulating activity for this saffron Crocus compound.

Release of cell-free DNA into the bloodstream leads to high levels of non-tumor plasma DNA during tumor progression in rats

To examine the implications of cell-free DNA in the plasma in neoplastic disease, it is necessary to clarify various features of this DNA, such as the contribution of DNA from the hosts normal cells and the kinetics of the release of this latter DNA. To quantify non-tumor DNA in the plasma of tumor-bearing rats and to correlate levels of this DNA with tumor progression, we injected DHD/K12-PROb colon cancer cells subcutaneously into BD-IX rats and recorded tumor diameters weekly. After euthanasia, we collected plasma from each rat and quantified non-mutated and mutated DNA in the plasma. Overall, levels of non-mutated (non-tumor) DNA in plasma of tumor-bearing animals were significantly higher than those in healthy animals measured by real-time PCR (p=0.001). However, 5 weeks after inoculation, levels were similar to those in healthy animals. As a whole, levels of non-mutated DNA were not significantly related to tumor size or to metastasis. However, when we excluded animals that were analyzed earliest, we found a positive and statistically significant correlation between levels of non-mutated DNA and tumor diameter (p=0.002). Release of cell-free DNA into the plasma during tumor progression appears to follow a predictable time course in a homogeneous population. In addition, large amounts of non-tumor DNA are released during tumor progression and, in particular, at early stages. Our findings support the hypothesis that interactions between tumor cells and host cells result in release of cell-free DNA.

The concentration of deoxyribonucleic acid in plasma from 73 patients with colorectal cancer and apparent clinical correlations

BACKGROUND Detection of cell-free plasma DNA has considerable potential as a tool for the diagnosis and assessment of the prognosis of many types of cancer. The aim of the present study was to quantify, by spectrophotometry, the cell-free DNA in plasma samples from patients with colorectal cancer at different stages of the disease and to attempt to correlate the resultant values with the clinical picture. METHODS We reviewed the medical reports of 73 patients, who had undergone resection of primary colorectal cancer. Samples of blood had been taken from each patient immediately prior to surgery. DNA was extracted from samples of plasma and quantified, by spectrophotometry, after a storage period of no longer than 2 years in 89% of the cases examined. RESULTS The mean(+/-S.D.) concentration of DNA in plasma samples was 108+/-156 ng/microl. We found a statistically significant correlation between the concentration of DNA and the presence of metastases (mainly liver metastases). CONCLUSION The detection and quantitation of cell-free DNA in plasma, using this simple technique, might be of clinical value for the surveillance of colon cancer patients and the detection of metastases.

Surgery and Hematogenous Dissemination: Comparison Between the Detection of Circulating Tumor Cells and of Tumor DNA in Plasma Before and After Tumor Resection in Rats

BackgroundTo examine the effects of the surgical manipulation of tumors on the hematogenous dissemination of tumors, we compared rates of detection of tumor-derived DNA in the buffy coat and in plasma from tumor-bearing rats before and after tumor resection.MethodsWe injected DHD/K12-PROb cells subcutaneously into BD-IX rats. Three weeks later, we removed the tumors surgically. Group PERI was sacrificed 3 hours after surgery, group POST-2 was sacrificed 2 weeks later, group POST-4 was sacrificed another 2 weeks later, and group POST-LONG was sacrificed when rats were close to death. In group PERI, four perioperative blood samples were taken. In the other groups, only one blood sample was taken per rat, immediately before euthanasia. We used polymerase chain reaction to detect tumor-derived DNA in buffy-coat, plasma, and lung samples.ResultsIn group PERI, tumor DNA in plasma was more frequent than circulating tumor cells at all perioperative time points. The difference was statistically significant 3 hours after surgery (P = .035). In group POST-2, there was no detectable metastasis or tumor DNA in blood samples. There were lymphatic and lung metastases in most animals in group POST-4 and in all animals in group POST-LONG. In the last two groups, the frequencies of tumor DNA in the buffy coat and in plasma were similar.ConclusionsIn our animal model, the hematogenous dissemination of tumors due to surgery seemed to be more closely related to tumor-derived cell-free DNA than to circulating tumor cells. In addition, the surgical resection of primary tumors did not inhibit the development of metastases.