Emerging treatments for complex perianal fistula in Crohn's disease

Complex perianal fistulas have a negative impact on the quality of life of sufferers and should be treated.Correct diagnosis,characterization and classification of the fistulas are essential to optimize treatment.Nevertheless, in the case of patients whose fistulas are associated with Crohn's disease,complete closure is particularly difficult to achieve.Systemic medical treatments(antibiotics, thiopurines and other immunomodulatory agents,and, more recently,anti-tumor necrosis factor-αagents such as infliximab)have been tried with varying degrees of success.Combined medical(including infliximab)and less aggressive surgical therapy(drainage and seton placement)offer the best outcomes in complex Crohn's fistulas while more aggressive surgical procedures such as fistulotomy or fistulectomy may increase the risk of incontinence.This review will focus on emerging novel treatments for perianal disease in Crohn's patients. These include locally applied infliximab or tacrolimus, fistula plugs,instillation of fibrin glue and the use of adult expanded adipose-derived stem cell injection.More welldesigned controlled studies are required to confirm the effectiveness of these emerging treatments.

Stem cell therapy for faecal incontinence: Current state and future perspectives

Faecal continence is a complex function involving different organs and systems. Faecal incontinence is a common disorder with different pathogeneses, disabling consequences and high repercussions for quality of life. Current management modalities are not ideal, and the development of new treatments is needed. Since 2008, stem cell therapies have been validated, 36 publications have appeared(29 in preclinical models and seven in clinical settings), and six registered clinical trials are currently ongoing. Some publications have combined stem cells with bioengineering technologies. The aim of this review is to identify and summarise the existing published knowledge of stem cell utilization as a treatment for faecal incontinence. A narrative or descriptive review is presented. Preclinical studies have demonstrated that cellular therapy, mainly in the form of local injections of muscle-derived(muscle derived stem cells or myoblasts derived from them) or mesenchymal(bone-marrow-or adipose-derived) stem cells, is safe. Cellular therapy has also been shown to stimulate the repair of both acute and subacute anal sphincter injuries, and some encouraging functional results have been obtained. Stem cells combined with normal cells on bioengineered scaffolds have achieved the successful creation and implantation of intrinsically-innervated anal sphincter constructs. The clinical evidence, based on adipose-derived stem cells and myoblasts, is extremely limited yet has yielded some promising results, and appears to be safe. Further investigation in both animal models and clinical settings is necessary to drawing conclusions. Nevertheless, if the preliminary results are confirmed, stem cell therapy for faecal incontinence may well become a clinical reality in the near future.

Further the liquid biopsy:Gathering pieces of the puzzle of genometastasis theory

Metastasis is the major cause of mortality in cancer disease and still constitutes one of the most controversial mechanism,not yet fully understood.What is almost beyond doubt is that circulatory system is crucial for cancer propagation.Regarding this system,much attention has been recently paid to liquid biopsy.This technique is aimed to detect circulating tumor cells (CTCs) and circulating nucleic acids so it can be used as a tool for diagnostic,prognostic and follow-up of patients.Whereas CTCs tend to be scarce in serum and plasma from cancer patient,abundant circulating nucleic acids can be detected in the same location.This fact,together with the genetic origin of cancer,stands out the relevance of circulating nucleic acids and shed light into the role of nucleic acids as drivers of metastasis,a recently discovered phenomenon called Genometastasis.This innovative theory supports the transfer of oncogenes from cancer cells to normal and susceptible cells located in distant target organs through circulatory system.What is more,many biological processes haven been described to deliver and secrete circulating nucleic acids into the circulation which can allow such horizontal transfer of oncogenes.In this review,we focus not only on these mechanisms but also we demonstrate its putative role in cancer propagation and give insights about possible therapeutic strategies based on this theory.Our objective is to demonstrate how findings about cell-to-cell communications and previous results can agree with this unprecedented theory.

Rat model of anal sphincter injury and two approaches for stem cell administration

AIM To establish a rat model of anal sphincter injury and test different systems to provide stem cells to injured area.METHODS Adipose-derived stem cells(ASCs) were isolated from BDIX rats and were transfected with green fluorescent protein(GFP) for cell tracking. Biosutures(sutures covered with ASCs) were prepared with 1.5 × 10~6 GFPASCs, and solutions of 10~6 GFP-ASCs in normal saline were prepared for injection. Anorectal normal anatomy was studied on Wistar and BDIX female rats. Then, we designed an anal sphincter injury model consisting of a 1-cm extra-mucosal miotomy beginning at the anal verge in the anterior middle line. The sphincter lesion was confirmed with conventional histology(hematoxylin and eosin) and immunofluorescence with 4', 6-diamidino-2-phenylindole(commonly known as DAPI), GFP and α-actin. Functional effect was assessed with basal anal manometry, prior to and after injury. After sphincter damage, 36 BDIX rats were randomized to three groups for:(1) Cell injection without repair;(2) biosuture repair; and(3) conventional suture repair and cell injection. Functional and safety studies were conducted on all the animals. Rats were sacrificed after 1, 4 or 7 d. Then, histological and immunofluorescence studies were performed on the surgical area.RESULTS With the described protocol, biosutures had been covered with at least 820000-860000 ASCs, with 100% viability. Our studies demonstrated that some ASCs remained adhered after suture passage through the muscle. Morphological assessment showed that the rat anal anatomy is comparable with human anatomy; two sphincters are present, but the external sphincter is poorly developed. Anal sphincter pressure data showed spontaneous, consistent, rhythmic anal contractions, taking the form of "plateaus" with multiple twitches(peaks) in each pressure wave. These basal contractions were very heterogeneous; their frequency was 0.91-4.17 per min(mean 1.6980, SD 0.57698), their mean duration was 26.67 s and mean number of peaks was 12.53. Our morphological assessment revealed that with the aforementioned surgical procedure, both sphincters were completely sectioned. In manometry, the described activity disappeared and was replaced by a gentle oscillation of basal line, without a recognizable pattern. Surprisingly, these findings appeared irrespective of injury repair or not. ASCs survived in this potentially septic area for 7 d, at least. We were able to identify them in 84% of animals, mainly in the muscular section area or in the tissue between the muscular endings. ASCs formed a kind of "conglomerate" in rats treated with injections, while in the biosuture group, they wrapped the suture. ASCs were also able to migrate to the damaged zone. No relevant adverse events or mortality could be related to the stem cells in our study. We also did not find unexpected tissue growths. CONCLUSION The proposed procedure produces a consistent sphincter lesion. Biosutures and injections are suitable for cell delivery. ASCs survive and are completely safe in this clinical setting.

Detection of KRAS G12D in colorectal cancer stool by droplet digital PCR

AIMTo assess KRAS G12D mutation detection by droplet digital PCR (ddPCR) in stool-derived DNA from colorectal cancer (CRC) patients.METHODSIn this study, tumor tissue and stool samples were collected from 70 patients with stage I-IV CRC diagnosed by preoperative biopsy. KRAS mutational status was determined by pyrosequencing analysis of DNA obtained from formalin-fixed paraffin-embedded (FFPE) tumor tissues. The KRAS G12D mutation was then analyzed by ddPCR in FFPE tumors and stool-derived DNA from patients with this point mutation. Wild-type (WT) tumors, as determined by pyrosequencing, were included as controls; analysis of FFPE tissue and stool-derived DNA by ddPCR was performed for these patients as well.RESULTSAmong the total 70 patients included, KRAS mutations were detected by pyrosequencing in 32 (45.71%), whereas 38 (54.29%) had WT tumors. The frequency of KRAS mutations was higher in left-sided tumors (11 located in the right colon, 15 in the left, and 6 in the rectum). The predominant point mutation was KRAS G12D (14.29%, n = 10), which was more frequent in early-stage tumors (I-IIA, n = 7). In agreement with pyrosequencing results, the KRAS G12D mutation was detected by ddPCR in FFPE tumor-derived DNA, and only a residual number of mutated copies was found in WT controls. The KRAS G12D mutation was also detected in stool-derived DNA in 80% of all fecal samples from CRC patients with this point mutation.CONCLUSIONddPCR is a reliable and sensitive method to analyze KRAS G12D mutation in stool-derived DNA from CRC patients, especially at early stages. This non-invasive approach is potentially applicable to other relevant biomarkers for CRC management.