Binding Mode Exploration of B1 Receptor Antagonists' by the Use of Molecular Dynamics and Docking Simulation-How Different Target Engagement Can Determine Different Biological Effects.

The kinin B1 receptor plays a critical role in the chronic phase of pain and inflammation. The development of B1 antagonists peaked in recent years but almost all promising molecules failed in clinical trials. Little is known about these molecules' mechanisms of action and additional information will be necessary to exploit the potential of the B1 receptor. With the aim of contributing to the available knowledge of the pharmacology of B1 receptors, we designed and characterized a novel class of allosteric non-peptidic inhibitors with peculiar binding characteristics. Here, we report the binding mode analysis and pharmacological characterization of a new allosteric B1 antagonist, DFL20656. We analyzed the binding of DFL20656 by single point mutagenesis and radioligand binding assays and we further characterized its pharmacology in terms of IC50, B1 receptor internalization and in vivo activity in comparison with different known B1 antagonists. We highlighted how different binding modes of DFL20656 and a Merck compound (compound 14) within the same molecular pocket can affect the biological and pharmacological properties of B1 inhibitors. DFL20656, by its peculiar binding mode, involving tight interactions with N114, efficiently induced B1 receptor internalization and evoked a long-lasting effect in an in vivo model of neuropathic pain. The pharmacological characterization of different B1 antagonists highlighted the effects of their binding modes on activity, receptor occupancy and internalization. Our results suggest that part of the failure of most B1 inhibitors could be ascribed to a lack of knowledge about target function and engagement.

ADPredict: ADP-ribosylation site prediction based on physicochemical and structural descriptors.

Motivation: ADP-ribosylation is a post-translational modification (PTM) implicated in several crucial cellular processes, ranging from regulation of DNA repair and chromatin structure to cell metabolism and stress responses. To date, a complete understanding of ADP-ribosylation targets and their modification sites in different tissues and disease states is still lacking. Identification of ADP-ribosylation sites is required to discern the molecular mechanisms regulated by this modification. This motivated us to develop a computational tool for the prediction of ADP-ribosylated sites. Results: Here, we present ADPredict, the first dedicated computational tool for the prediction of ADP-ribosylated aspartic and glutamic acids. This predictive algorithm is based on (i) physicochemical properties, (ii) in-house designed secondary structure-related descriptors and (iii) three-dimensional features of a set of human ADP-ribosylated proteins that have been reported in the literature. ADPredict was developed using principal component analysis and machine learning techniques; its performance was evaluated both internally via intensive bootstrapping and in predicting two external experimental datasets. It outperformed the only other available ADP-ribosylation prediction tool, ModPred. Moreover, a novel secondary structure descriptor, HM-ratio, was introduced and successfully contributed to the model development, thus representing a promising tool for bioinformatics studies, such as PTM prediction. Availability and implementation: ADPredict is freely available at www.ADPredict.net. Supplementary information: Supplementary data are available at Bioinformatics online.

Hematopoietic stem/progenitor cells express myoglobin and neuroglobin: adaptation to hypoxia or prevention from oxidative stress?

Oxidative metabolism and redox signaling prove to play a decisional role in controlling adult hematopoietic stem/progenitor cells (HSPCs) biology. However, HSPCs reside in a hypoxic bone marrow microenvironment raising the question of how oxygen metabolism might be ensued. In this study, we provide for the first time novel functional and molecular evidences that human HSPCs express myoglobin (Mb) at level comparable with that of a muscle-derived cell line. Optical spectroscopy and oxymetry enabled to estimate an O2-sensitive heme-containing protein content of approximately 180 ng globin per 10(6) HSPC and a P50 of approximately 3 µM O2. Noticeably, expression of Mb mainly occurs through a HIF-1-induced alternative transcript (Mb-V/Mb-N = 35 ± 15, p < .01). A search for other Mb-related globins unveiled significant expression of neuroglobin (Ngb) but not of cytoglobin. Confocal microscopy immune detection of Mb in HSPCs strikingly revealed nuclear localization in cell subsets expressing high level of CD34 (nuclear/cytoplasmic Mb ratios 1.40 ± 0.02 vs. 0.85 ± 0.05, p < .01) whereas Ngb was homogeneously distributed in all the HSPC population. Dual-color fluorescence flow cytometry indicated that while the Mb content was homogeneously distributed in all the HSPC subsets that of Ngb was twofold higher in more immature HSPC. Moreover, we show that HSPCs exhibit a hypoxic nitrite reductase activity releasing NO consistent with described noncanonical functions of globins. Our finding extends the notion that Mb and Ngb can be expressed in nonmuscle and non-neural contexts, respectively, and is suggestive of a differential role of Mb in HSPC in controlling oxidative metabolism at different stages of commitment.

Surface proteomic analysis of differentiated versus stem-like osteosarcoma human cells.

Cancer stem cell characterization represents a breakthrough in cancer research. Despite evidence showing the existence and the role of cancer stem cells in osteosarcoma (OS) onset and progression, little is known about their specific surface phenotype. To address this issue, we carried out a cytometric analysis with an antibody-array comprising 245 membrane proteins comparing the stem and differentiated OS cells. As experimental model, we chose the stem-like cell line 3aminobenzamide-OS and its parental, differentiated, cell line MG63. We identified 50 differentially expressed, 23 homogeneously expressed, and 172 not expressed proteins in the two cell line models, thus defining a surface protein signature specific for each of them. Furthermore, we selected ERK1/2 (p44/42 mitogen-activated protein kinases) as a potential pathway correlated with processes that characterize tumorigenic potential and stemness of 3aminobenzamide-OS cells.

CD66c is a novel marker for colorectal cancer stem cell isolation, and its silencing halts tumor growth in vivo.

BACKGROUND: Despite the well recognized expression of the cell surface markers cluster of differentiation 44 (homing cell adhesion molecule) and CD133 (Prominin 1) on human colorectal cancer stem cells (CCSCs), these molecules do not appear to be effective targets for stem cell-directed therapies. Because the surface marker CD66c (also known as carcinoembryonic antigen-related cell adhesion molecule 6) has demonstrated promise as a therapeutic target in pancreatic malignancy, the authors evaluated its potential as a target for stem cell-directed treatment of colorectal cancer. METHODS: First, the authors characterized CD66c expression by flow cytometry and immunohistochemistry in colon cancer samples and in normal colon tissues. Then, the coexpression of CD66c and CD133 was evaluated on putative CCSCs. CD66c expression also was measured in stem cell-enriched colon spheres. Finally, the effects of small-interfering RNA-mediated CD66c silencing on the in vitro and in vivo growth of Caco2 colon cancer cells were evaluated. RESULTS: CD66c expression was significantly higher in colon cancers than in contiguous normal colon tissues and paralleled cancer stage. CD66c was absent in CD133-positive cells that were isolated from normal colon, whereas its expression was brightest (CD66c(bright) ) in CD133-positive cells from colon cancer samples. In vitro experiments demonstrated that colon spheres were considerably enriched in a CD66c(bright) population in a fashion comparable to the enrichment observed in fresh liver metastases. In vitro proliferation and clonogenic potential were hampered when CD66c was silenced in Caco2 cells. Finally, in vivo xenograft experiments demonstrated that CD66c silencing almost completely abrogated the tumorigenic potential of Caco2 cells. CONCLUSIONS: CD66c(bright) expression was associated with colon cancer stem cells and CD66c silencing blocked tumor growth, thereby opening the way to a potential new treatment for colon cancer.

Protein cross-talk in CD133+ colon cancer cells indicates activation of the Wnt pathway and upregulation of SRp20 that is potentially involved in tumorigenicity.

The cancer stem cell (CSC) theory represents a breakthrough in cancer research. We characterized the protein pattern of CSCs to identify specific intracellular pathways in this subpopulation of tumor cells. We studied colon CSCs using two different colon cancer cell lines: CaCo-2 and HCT-116. Putative CSCs were separated from non-CSCs by flow cytometry using CD133 as stemness marker. Total protein extracts of CD133+ cells were then compared to protein extracts of CD133- cells by 2D DIGE. The protein spots differentially expressed in the two subpopulations of cells were analyzed by mass spectrometry. Bioinformatics analysis of the identified proteins indicated alteration of two main processes: energy metabolism and the Wnt pathway. Interestingly, we observed upregulation of the splicing factor SRp20, a newly identified target gene of the Wnt/ß-catenin pathway, and we demonstrated a direct cause-effect relationship between Wnt pathway activation and the increased SRp20 expression. Our results also show that SRp20 influences cell proliferation, which suggests it plays a role in the tumorigenicity of CD133+ cells. In conclusion, activation of the Wnt pathway in CD133+ cells and upregulation of SRp20, which is implicated in tumorigenesis, raises the possibility of a sequential series of molecular events occurring in connection with this process.

Preferential nuclear accumulation of JAK2V617F in CD34+ but not in granulocytic, megakaryocytic, or erythroid cells of patients with Philadelphia-negative myeloproliferative neoplasia.

Recently, Dawson et al identified a previously unrecognized nuclear role of JAK2 in the phosphorylation of histone H3 in hematopoietic cell lines. We searched nuclear JAK2 in total bone marrow (BM) cells and in 4 sorted BM cell populations (CD34(+), CD15(+), CD41(+), and CD71(+)) of 10 myeloproliferative neoplasia (MPN) patients with JAK2V617F mutation and 5 patients with wild-type JAK2 MPN. Confocal immunofluorescent images and Western blot analyses of nuclear and cytoplasmic fractions found nuclear JAK2 in CD34(+) cells of 10 of 10 JAK2-mutated patients but not in patients with wild-type JAK2. JAK2 was predominantly in the cytoplasmic fraction of differentiated granulocytic, megakaryocytic, or erythroid cells obtained from all patients. JAK2V617F up-regulates LMO2 in K562 and in JAK2V617F-positive CD34(+) cells. The selective JAK2 inhibitor AG490 normalizes the LMO2 levels in V617F-positive K562 and restores the cyto-plasmic localization of JAK2.

CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice.

Acute myeloid leukemia (AML) with mutated NPM1 shows distinctive biologic and clinical features, including absent/low CD34 expression, the significance of which remains unclear. Therefore, we analyzed CD34(+) cells from 41 NPM1-mutated AML. At flow cytometry, 31 of 41 samples contained less than 10% cells showing low intensity CD34 positivity and variable expression of CD38. Mutational analysis and/or Western blotting of purified CD34(+) cells from 17 patients revealed NPM1-mutated gene and/or protein in all. Immunohistochemistry of trephine bone marrow biopsies and/or flow cytometry proved CD34(+) leukemia cells from NPM1-mutated AML had aberrant nucleophosmin expression in cytoplasm. NPM1-mutated gene and/or protein was also confirmed in a CD34(+) subfraction exhibiting the phenotype (CD34(+)/CD38(-)/CD123(+)/CD33(+)/CD90(-)) of leukemic stem cells. When transplanted into immunocompromised mice, CD34(+) cells generated a leukemia recapitulating, both morphologically and immunohistochemically (aberrant cytoplasmic nucleophosmin, CD34 negativity), the original patient's disease. These results indicate that the CD34(+) fraction in NPM1-mutated AML belongs to the leukemic clone and contains NPM1-mutated cells exhibiting properties typical of leukemia-initiating cells. CD34(-) cells from few cases (2/15) also showed significant leukemia-initiating cell potential in immunocompromised mice. This study provides further evidence that NPM1 mutation is a founder genetic lesion and has potential implications for the cell-of-origin and targeted therapy of NPM1-mutated AML.

Detection of erbB2 copy number variations in plasma of patients with esophageal carcinoma.

BACKGROUND: Mortality is high in patients with esophageal carcinoma as tumors are rarely detected before the disease has progressed to an advanced stage. Here, we sought to isolate cell-free DNA released into the plasma of patients with esophageal carcinoma, to analyze copy number variations of marker genes in the search for early detection of tumor progression. METHODS: Plasma of 41 patients with esophageal carcinoma was prospectively collected before tumor resection and chemotherapy. Our dataset resulted heterogeneous for clinical data, resembling the characteristics of the tumor. DNA from the plasma was extracted to analyze copy number variations of the erbB2 gene using real-time PCR assays. RESULTS: The real-time PCR assays for erbB2 gene showed significant (P = 0.001) copy number variations in the plasma of patients with esophageal carcinoma, as compared to healthy controls with high sensitivity (80%) and specificity (95%). These variations in erbB2 were negatively correlated to the progression free survival of these patients (P = 0.03), and revealed a further risk category stratification of patients with low VEGF expression levels. CONCLUSION: The copy number variation of erbB2 gene from plasma can be used as prognostic marker for early detection of patients at risk of worse clinical outcome in esophageal cancer.

"Molecular Anatomy": a new multi-dimensional hierarchical scaffold analysis tool.

The scaffold representation is widely employed to classify bioactive compounds on the basis of common core structures or correlate compound classes with specific biological activities. In this paper, we present a novel approach called "Molecular Anatomy" as a flexible and unbiased molecular scaffold-based metrics to cluster large set of compounds. We introduce a set of nine molecular representations at different abstraction levels, combined with fragmentation rules, to define a multi-dimensional network of hierarchically interconnected molecular frameworks. We demonstrate that the introduction of a flexible scaffold definition and multiple pruning rules is an effective method to identify relevant chemical moieties. This approach allows to cluster together active molecules belonging to different molecular classes, capturing most of the structure activity information, in particular when libraries containing a huge number of singletons are analyzed. We also propose a procedure to derive a network visualization that allows a full graphical representation of compounds dataset, permitting an efficient navigation in the scaffold's space and significantly contributing to perform high quality SAR analysis. The protocol is freely available as a web interface at https://ma.exscalate.eu .

Androgen receptor signaling induced by supraphysiological doses of dihydrotestosterone in human peripheral blood lymphocytes.

Anabolic androgenic steroids, a class of steroid hormones related to testosterone, are natural ligands of androgen receptor (AR), a member of the nuclear receptor superfamily of ligand-activated transcription factors. AR binds specific DNA elements, known as androgen-response elements. Testosterone, the main male sexual hormone, binds AR directly and indirectly, through conversion into dihydrotestosterone (DHT), its more active metabolite. Anabolic androgenic steroids are frequently detected in the urine of doped athletes; their consumption is also growing among sport amateurs and adolescents. The effects of androgens can differ depending on the target cells and/or tissues. To gain insight into transcription activation mechanisms of AR, we investigated AR protein signaling in human peripheral blood lymphocytes treated with supraphysiological doses of DHT. We performed a comparative proteomic analysis and we identified about 30 differentially expressed proteins. At least five species contained a consensus androgen-response elements sequence in the promoter region of related coding genes. The analysis also revealed that high doses of DHT activate the drug detoxification process, could stimulate an increase in cell motility and exert a prosurvival effect rather than an apoptotic one.

The percentage of CD133+ cells in human colorectal cancer cell lines is influenced by Mycoplasma hyorhinis infection.

BACKGROUND: Mollicutes contamination is recognized to be a critical issue for the cultivation of continuous cell lines. In this work we characterized the effect of Mycoplasma hyorhinis contamination on CD133 expression in human colon cancer cell lines. METHODS: MycoAlert and mycoplasma agar culture were used to detect mycoplasma contamination on GEO, SW480 and HT-29 cell lines. Restriction fragment length polymorphism assay was used to determine mycoplasma species. All cellular models were decontaminated by the use of a specific antibiotic panel (Enrofloxacin, Ciprofloxacin, BM Cyclin 1 and 2, Mycoplasma Removal Agent and MycoZap). The percentage of CD133 positive cells was analyzed by flow cytometry on GEO, SW480 and HT-29 cell lines, before and after Mycoplasma hyorhinis eradication. RESULTS: Mycoplasma hyorhinis infected colon cancer cell lines showed an increased percentage of CD133+ cells as compared to the same cell lines rendered mycoplasma-free by effective exposure to antibiotic treatment. The percentage of CD133 positive cells increased again when mycoplasma negative cells were re-infected by Mycoplasma hyorhinis. CONCLUSIONS: Mycoplasma hyorhinis infection has an important role on the quality of cultured human colon cancer cell lines giving a false positive increase of cancer stem cells fraction characterized by CD133 expression. Possible explanations are (i) the direct involvement of Mycoplasma on CD133 expression or (ii) the selective pressure on a subpopulation of cells characterized by constitutive CD133 expression.In keeping with United Kingdom Coordinating Committee on Cancer Research (UKCCCR) guidelines, the present data indicate the mandatory prerequisite, for investigators involved in human colon cancer research area, of employing mycoplasma-free cell lines in order to avoid the production of non-reproducible or even false data.

Extended flow cytometry characterization of normal bone marrow progenitor cells by simultaneous detection of aldehyde dehydrogenase and early hematopoietic antigens: implication for erythroid differentiation studies.

BACKGROUND: Aldehyde dehydrogenase (ALDH) is a cytosolic enzyme highly expressed in hematopoietic precursors from cord blood and granulocyte-colony stimulating factor mobilized peripheral blood, as well as in bone marrow from patients with acute myeloblastic leukemia. As regards human normal bone marrow, detailed characterization of ALDH+ cells has been addressed by one single study (Gentry et al, 2007). The goal of our work was to provide new information about the dissection of normal bone marrow progenitor cells based upon the simultaneous detection by flow cytometry of ALDH and early hematopoietic antigens, with particular attention to the expression of ALDH on erythroid precursors. To this aim, we used three kinds of approach: i) multidimensional analytical flow cytometry, detecting ALDH and early hematopoietic antigens in normal bone marrow; ii) fluorescence activated cell sorting of distinct subpopulations of progenitor cells, followed by in vitro induction of erythroid differentiation; iii) detection of ALDH+ cellular subsets in bone marrow from pure red cell aplasia patients. RESULTS: In normal bone marrow, we identified three populations of cells, namely ALDH+CD34+, ALDH-CD34+ and ALDH+CD34- (median percentages were 0.52, 0.53 and 0.57, respectively). As compared to ALDH-CD34+ cells, ALDH+CD34+ cells expressed the phenotypic profile of primitive hematopoietic progenitor cells, with brighter expression of CD117 and CD133, accompanied by lower display of CD38 and CD45RA. Of interest, ALDH+CD34- population disclosed a straightforward erythroid commitment, on the basis of three orders of evidences. First of all, ALDH+CD34- cells showed a CD71bright, CD105+, CD45- phenotype. Secondly, induction of differentiation experiments evidenced a clear-cut expression of glycophorin A (CD235a). Finally, ALDH+CD34- precursors were not detectable in patients with pure red cell aplasia (PRCA). CONCLUSION: Our study, comparing surface antigen expression of ALDH+/CD34+, ALDH-/CD34+ and ALDH+/CD34- progenitor cell subsets in human bone marrow, clearly indicated that ALDH+CD34- cells are mainly committed towards erythropoiesis. To the best of our knowledge this finding is new and could be useful for basic studies about normal erythropoietic differentiation as well as for enabling the employment of ALDH as a red cell marker in polychromatic flow cytometry characterization of bone marrow from patients with aplastic anemia and myelodysplasia.

Publisher Correction: Novel selective, potent naphthyl TRPM8 antagonists identified through a combined ligand- and structure-based virtual screening approach.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Novel selective, potent naphthyl TRPM8 antagonists identified through a combined ligand- and structure-based virtual screening approach.

Transient receptor potential melastatin 8 (TRPM8), a nonselective cation channel, is the predominant mammalian cold temperature thermosensor and it is activated by cold temperatures and cooling compounds, such as menthol and icilin. Because of its role in cold allodynia, cold hyperalgesia and painful syndromes TRPM8 antagonists are currently being pursued as potential therapeutic agents for the treatment of pain hypersensitivity. Recently TRPM8 has been found in subsets of bladder sensory nerve fibres, providing an opportunity to understand and treat chronic hypersensitivity. However, most of the known TRPM8 inhibitors lack selectivity, and only three selective compounds have reached clinical trials to date. Here, we applied two virtual screening strategies to find new, clinics suitable, TRPM8 inhibitors. This strategy enabled us to identify naphthyl derivatives as a novel class of potent and selective TRPM8 inhibitors. Further characterization of the pharmacologic properties of the most potent compound identified, compound 1, confirmed that it is a selective, competitive antagonist inhibitor of TRPM8. Compound 1 also proved itself active in a overreactive bladder model in vivo. Thus, the novel naphthyl derivative compound identified here could be optimized for clinical treatment of pain hypersensitivity in bladder disorders but also in different other pathologies.

TRAP1 regulates stemness through Wnt/ß-catenin pathway in human colorectal carcinoma.

Colorectal carcinoma (CRC) is a common cause of cancer-related death worldwide. Indeed, treatment failures are triggered by cancer stem cells (CSCs) that give rise to tumor repopulation upon initial remission. Thus, the role of the heat shock protein TRAP1 in stemness was investigated in CRC cell lines and human specimens, based on its involvement in colorectal carcinogenesis, through regulation of apoptosis, protein homeostasis and bioenergetics. Strikingly, co-expression between TRAP1 and stem cell markers was observed in stem cells located at the bottom of intestinal crypts and in CSCs sorted from CRC cell lines. Noteworthy, TRAP1 knockdown reduced the expression of stem cell markers and impaired colony formation, being the CSC phenotype and the anchorage-independent growth conserved in TRAP1-rich cancer cells. Consistently, the gene expression profiling of HCT116 cells showed that TRAP1 silencing results in the loss of the stem-like signature with acquisition of a more-differentiated phenotype and the downregulation of genes encoding for activating ligands and target proteins of Wnt/ß-catenin pathway. Mechanistically, TRAP1 maintenance of stemness is mediated by the regulation of Wnt/ß-catenin signaling, through the modulation of the expression of frizzled receptor ligands and the control of ß-catenin ubiquitination/phosphorylation. Remarkably, TRAP1 is associated with higher expression of ß-catenin and several Wnt/ß-catenin target genes in human CRCs, thus supporting the relevance of TRAP1 regulation of ß-catenin in human pathology. This study is the first demonstration that TRAP1 regulates stemness and Wnt/ß-catenin pathway in CRC and provides novel landmarks in cancer biology and therapeutics.

HMGA1 silencing reduces stemness and temozolomide resistance in glioblastoma stem cells.

OBJECTIVE: Glioblastoma multiforme (GBM) develops from a small subpopulation of stem-like cells, which are endowed with the ability to self-renew, proliferate and give rise to progeny of multiple neuroepithelial lineages. These cells are resistant to conventional chemo- and radiotherapy and are hence also responsible for tumor recurrence. HMGA1 overexpression has been shown to correlate with proliferation, invasion, and angiogenesis of GBMs and to affect self-renewal of cancer stem cells from colon cancer. The role of HMGA1 in GBM tumor stem cells is not completely understood. RESEARCH DESIGN AND METHODS: We have investigated the role of HMGA1 in brain tumor stem cell (BTSC) self-renewal, stemness and resistance to temozolomide by shRNA- mediated HMGA1 silencing. RESULTS: We first report that HMGA1 is overexpressed in a subset of BTSC lines from human GBMs. Then, we show that HMGA1 knockdown reduces self-renewal, sphere forming efficiency and stemness, and sensitizes BTSCs to temozolomide. Interestingly, HMGA1 silencing also leads to reduced tumor initiation ability in vivo. CONCLUSIONS: These results demonstrate a pivotal role of HMGA1 in cancer stem cell gliomagenesis and endorse HMGA1 as a suitable target for CSC-specific GBM therapy.

CD26-positive/CD326-negative circulating cancer cells as prognostic markers for colorectal cancer recurrence.

The present study evaluated the presence and clinical relevance of a cluster of differentiation (CD)26+/CD326- subset of circulating tumor cells (CTCs) in pre- and post-operative blood samples of colorectal cancer patients, who had undergone curative or palliative intervention, in order to find a novel prognostic factor for patient management and follow-up. In total, 80 colorectal cancer patients, along with 25 healthy volunteers were included. The easily transferable methodology of flow cytometry, along with multiparametric antibody staining were used to selectively evaluate CD26+/CD326- CTCs in the peripheral blood samples of colorectal cancer patients. The multiparametric selection allowed any enrichment methods to be avoided thus rendering the whole procedure suitable for clinical routine. The presence of CD26+/CD326- cells was higher in advanced Dukes' stages and was significantly associated with poor survival and high recurrence rates. Relapsing and non-surviving patients showed the highest number of CD26+/CD326- CTCs. High pre-operative levels of CD26+/CD326- CTCs correctly predicted tumor relapse in 44.4% of the cases, while 69% of post-operative CD26+/CD326- CTC-positive patients experienced cancer recurrence, with a test accuracy of 88.8%. By contrast, post-operative CD26+/CD326- CTC-negative patients showed an increase in the three-year progression-free survival rate of 86%, along with a reduced risk of tumor relapse of >90%. In conclusion, CD26+/CD326- CTCs are an independent prognostic factor for tumor recurrence rate in multivariate analysis, suggesting that their evaluation could be an additional factor for colorectal cancer recurrence risk evaluation in patient management.