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Background: The optimal timing of surgery in relation to chemoradiation is still controversial. Retrospective analysis has demonstrated in the recent decades that the regression of adenocarcinoma can be slow and not complete until after several months. More recently, increasing pathologic Complete Response rates have been demonstrated to be correlated with longer time interval. The purpose of the trial is to demonstrate if delayed timing of surgery after neoadjuvant chemoradiotherapy actually affects pathologic Complete Response and reflects on disease-free survival and overall survival rather than standard timing., Methods: The trial is a multicenter, prospective, randomized controlled, unblinded, parallel-group trial comparing standard and delayed surgery after neoadjuvant chemoradiotherapy for the curative treatment of rectal cancer. Three-hundred and forty patients will be randomized on an equal basis to either robotic-assisted/standard laparoscopic rectal cancer surgery after 8 weeks or robotic-assisted/standard laparoscopic rectal cancer surgery after 12 weeks., Discussion: To date, it is well-know that pathologic Complete Response is associated with excellent prognosis and an overall survival of 90%. In the Lyon trial the rate of pCR or near pathologic Complete Response increased from 10.3 to 26% and in retrospective studies the increase rate was about 23-30%. These results may be explained on the relationship between radiation therapy and tumor regression: DNA damage occurs during irradiation, but cellular lysis occurs within the next weeks. Study results, whether confirmed that performing surgery after 12 weeks from neoadjuvant treatment is advantageous from a technical and oncological point of view, may change the current pathway of the treatment in those patient suffering from rectal cancer., Trial Registration: ClinicalTrials.gov NCT3465982.

Three-dimensional (3D) cancer models are overlooking the scientific landscape with the primary goal of bridging the gaps between two-dimensional (2D) cell lines, animal models and clinical research. Here, we describe an innovative tissue engineering approach applied to colorectal cancer (CRC) starting from decellularized human biopsies in order to generate an organotypic 3D-bioactive model. This in vitro 3D system recapitulates the ultrastructural environment of native tissue as demonstrated by histology, immunohistochemistry, immunofluorescence and scanning electron microscopy analyses. Mass spectrometry of proteome and secretome confirmed a different stromal composition between decellularized healthy mucosa and CRC in terms of structural and secreted proteins. Importantly, we proved that our 3D acellular matrices retained their biological properties: using CAM assay, we observed a decreased angiogenic potential in decellularized CRC compared with healthy tissue, caused by direct effect of DEFA3. We demonstrated that following a 5 days of recellularization with HT-29 cell line, the 3D tumor matrices induced an over-expression of IL-8, a DEFA3-mediated pathway and a mandatory chemokine in cancer growth and proliferation. Given the biological activity maintained by the scaffolds after decellularization, we believe this approach is a powerful tool for future pre-clinical research and screenings., (© 2017 Wiley Periodicals, Inc.)

MiR-182 expression was evaluated by qRT-PCR and in situ hybridization in 20 tubular adenomas, 50 colorectal carcinoma (CRC), and 40 CRC liver metastases. Control samples obtained from patients with irritable bowel syndrome, or tumor-matched normal colon mucosa were analyzed (n=50). MiR-182 expression increased progressively and significantly along with the colorectal carcinogenesis cascade, and in CRC liver metastases. The inverse relation between miR-182 and the expression of its target gene ENTPD5 was investigated by immunohistochemical analysis. We observed that normal colocytes featured a strong ENTPD5 cytoplasmic expression whereas a significantly and progressively lower expression was present along with dedifferentiation of the histologic phenotype. Plasma samples from 51 CRC patients and controls were tested for miR-182 expression. Plasma miR-182 concentrations were significantly higher in CRC patients than in healthy controls or patients with colon polyps at endoscopy. Moreover, miR-182 plasma levels were significantly reduced in post-operative samples after radical hepatic metastasectomy compared to preoperative samples. Our results strengthen the hypothesis of a central role of miR-182 dysregulation in colon mucosa transformation, demonstrate the concomitant progressive down-regulation of ENTPD5 levels during colon carcinogenesis, and indicate the potential of circulating miR-182 as blood based biomarker for screening and monitoring CRC during the follow-up.

The mechanism by which tissue microecology influences invasion and metastasis is largely unknown. Recent studies have indicated differences in the molecular architecture of the metastatic lesion compared to the primary tumor, however, systemic analysis of the alterations within the activated protein signaling network has not been described. Using laser capture microdissection, protein microarray technology, and a unique specimen collection of 34 matched primary colorectal cancers (CRC) and synchronous hepatic metastasis, the quantitative measurement of the total and activated/phosphorylated levels of 86 key signaling proteins was performed. Activation of the EGFR-PDGFR-cKIT network, in addition to PI3K/AKT pathway, was found uniquely activated in the hepatic metastatic lesions compared to the matched primary tumors. If validated in larger study sets, these findings may have potential clinical relevance since many of these activated signaling proteins are current targets for molecularly targeted therapeutics. Thus, these findings could lead to liver metastasis specific molecular therapies for CRC.

Background: Currently there is no reliable technique for predicting clinical or pathologic complete tumor response after radiochemotherapy (RCT) in patients with rectal cancer. We applied reverse phase protein microarray (RPMA) technology to find a signal pathway that may predict the response to preoperative treatment., Patients and Methods: Fifteen rectal cancer samples were collected during preoperative RCT. Seven patients had a good response to preoperative therapy (Mandard grade I-II) and 8 patients had a poor response (Mandard grade III-V). Using laser capture microdissection (LCM) and RPMA analysis, we measured the phosphorylation level of nearly 80 end points and analyzed the signaling pathways., Results: We identified 4 signaling proteins whose phosphorylation levels were significantly different (P < .05) between the good vs. poor responders; CHK2 and β-catenin were more highly phosphorylated in poor responders, whereas PDK1 and glycogen synthase kinase (GSK)-3α/β had lower phosphorylation levels in poor responders. Interestingly GSK-3α/β, β-catenin, and PDK1 are all present in the phosphatidylinositol-3-kinase (PI3K)-AKT signaling pathway., Conclusions: Based on our results, we hypothesize that the activating state of the PI3K-AKT pathway can stratify patients who could benefit most from neoadjuvant treatment. Moreover, identification of theranostic targets has the potential to pinpoint new therapeutic strategies for the nonresponsive population., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Although surgery is the gold standard treatment of hepatic metastasis from colorectal cancer (CRC), many patients ultimately die of their disease. We tested the hypothesis that the detection of circulating tumor cells (CTC) might identify patients at high risk of dying of disease recurrence after apparently radical liver surgery., Methods: We considered 50 patients undergoing radical surgery for liver-confined hepatic metastasis from CRC. The expression of a panel of cancer-related genes, as assessed by quantitative real-time PCR, was used to detect CTC in the peripheral blood of these patients immediately before surgery. Survival analysis was performed by the Cox regression model., Results: Univariate analysis of the expression levels of CD133 (a marker of colon cancer stem cells) and survivin (an antiapoptotic factor) resulted in statistically significant association with patient survival [hazard ratio (HR) 2.7, 95% confidence interval (CI) 1.9-3.7, P < 0.0001; and hazard ratio 2.1, 95% CI 1.4-3.2, P < 0.0001, respectively]. Remarkably, multivariate analysis found that only the transcriptional amount of CD133 resulted in statistical significance (HR 2.6, 95% CI 1.9-3.6, P < 0.0001), indicating that this biomarker can independently predict the survival of these patients., Conclusions: CD133-positive CTC may represent a suitable prognostic marker to stratify the risk of patients who undergo liver resection for CRC metastasis, which opens the avenue to identifying and potentially monitoring the patients who are most likely to benefit from adjuvant treatments.

Tissues are complex structures composed of different cell types, each of which present specific functions and characteristics. To better understand and measure the effect of tumor cell enrichment on protein pathway profiling and drug target activation measurements, the signaling activation portraits of laser capture microdissected (LCM) cancer epithelium and tumor stroma were compared with patient-matched whole-tissue specimens from 53 primary colorectal cancer samples. Microdissected material and whole-tissue lysate from contiguous cryostat sections were subjected to reverse-phase protein microarray analysis to determine the level of phopshorylation and expression of 75 different proteins known to be involved in cancer progression. The results revealed distinct differences in the protein activation portraits of cancer epithelium and stroma. Moreover, we found that the signaling activation profiles of the undissected whole-tissue specimens are profoundly different from the matched LCM material. Attempts to rescale the undissected pathway information based on percent endogenous tumor epithelium content were unsuccessful in recapitulating the LCM tumor epithelial signatures. Analysis of epidermal growth factor receptor phosphorylation and COX2 expression in these same sample sets revealed wholesale differences in the rank ordering of patient determination when LCM was compared with undissected samples. On the basis of these data, we conclude that accurate protein pathway activation status, which is under evaluation as a basis for patient selection and stratification for personalized therapy, must include upfront cellular-enrichment techniques such as LCM to generate accurate drug target activation status.

Background: The hypothesis was tested that systemic chemotherapy might contribute to improving overall survival (OS) of patients with unresectable colorectal liver metastases treated with hepatic arterial infusion (HAI)., Patients and Methods: We considered 153 consecutive patients retrospectively divided into group A (n=72) treated with HAI alone (floxuridine [FUDR] + leucovorin [LV]), and group B (n=81) treated with HAI combined with systemic chemotherapy (5-fluorouracil [5FU] + LV)., Results: No significant difference in OS was observed between the two groups. Median OS was better in patients with <50% of liver involvement (21.3 vs. 13.2 months; p<0.0001) and in responders vs. non-responders (24.4 vs. 13.4 months; p<0.0001). The combination of low tumor load with good tumor response to HAI was the only variable retained on multivariate survival analysis, associated with a better clinical outcome (median OS: 34.2 months)., Conclusion: Our study does not support the use of FUDR-based HAI combined or not with 5FU-based systemic chemotherapy as the first-line therapeutic approach to unresectable colorectal cancer liver metastases. The identification of responsive patients would improve the therapeutic index of this HAI regimen.

The identification of prognostic determinants of colorectal cancer (CRC), including prediction of occult metastasis, is of urgent consideration, based on the tremendous differences in outcome and survival between patients who present with metastasis or develop metastasis versus those patients with organ-confined or nonrecurrent disease. Currently, a great deal of attention has been focused on using gene expression profiles of tumor specimens as a launch point for prognostic biomarker discovery. In our study, we chose to focus on functional protein-based pathway biomarkers as a new information archive because it is these proteins that form the functional signaling networks that control cell growth, motility, apoptosis, survival, and differentiation. We used reverse-phase protein microarray analysis of laser capture microdissected CRC tumor specimens to profile broad cell signaling pathways from patients who presented with liver metastasis versus patients who remained recurrence free after follow-up. Our results indicate that members of the EGFR and COX2 signaling pathways appear differentially activated in the primary tumors of patients with synchronous metastatic disease. If validated in larger study sets, this pathway defect might be useful as a prognostic clinical tool as well as a guide to potential therapeutic intervention strategies that target occult disease and/or preventative measure.

A celiomesenteric trunk (CMT) is an extremely rare anatomic variant that consists of celiac and superior mesenteric arteries having a common origin from the aorta. CMT accounts for less than 1% of all splanchnic artery anomalies. Aneurysm involving a CMT is an even rarer vascular abnormality, and, to our knowledge, only eight cases of CMT aneurysm have been reported in literature. We describe a case of the incidental finding of CMT aneurysm in an asymptomatic patient. It was found after dorsolumbar column radiography and successive computed tomography and arteriography confirmed the diagnosis. Even if asymptomatic, we decided to repair it surgically with aneurysmectomy and suture of the neck due to risk of rupture.

Phosphoprotein driven cellular signaling events represent most of the new molecular targets for cancer treatment. Application of reverse-phase protein microarray technology for the study of ongoing signaling activity within breast tumor specimens holds great potential for elucidating and profiling signaling activity in real-time for patient-tailored therapy. Analysis of laser capture microdissection primary human breast tumors and metastatic lesions reveals pathway specific profiles and a new way to classify cancer based on functional signaling portraits. Moreover, the data demonstrate the requirement of laser capture microdissection for analysis and reveal the metastasis-specific changes that occur within a new microenvironment. Analysis of biopsy material from clinical trials for targeted therapeutics demonstrates the feasibility and utility of comprehensive signal pathway activation profiling for molecular analysis.

Leiomyosarcomas rarely arise in primary veins, especially the great saphenous vein. We have found only 20 case reports of leiomyosarcoma arising in the great saphenous vein, most of which manifested as nonspecific symptoms of advanced disease, such as a palpable mass, swelling, and back or abdominal pain. We report the case of greater saphenous vein leiomyosarcoma diagnosed in a 48-year-old man with a 4-month history of an inguinal mass. Ultrasonography and computed tomography showed a 6-cm mass attached to the right superficial femoral vein. Fine-needle aspiration biopsy confirmed that it was a vascular sarcoma. At the time of surgery there was no evidence of distant metastasis; therefore, we removed the tumor en bloc along with the sartorius muscle, inguinal lymph nodes, and 10 cm of the common femoral vein, and replaced the femoral vein with a polytetrafluoroethylene graft. A pathological examination revealed poorly differentiated leiomyosarcoma of the great saphenous vein, involving the deep femoral vein, without lymph node involvement. During follow-up, a thrombosis of the prosthesis developed, followed by proximal stenosis, which was treated successfully with percutaneous transluminal angioplasty. The patient was found to have lung metastases 25 months after surgery and he died about 5 months later.

Background: Mass spectrometry (MS)-based profiling was used to determine whether ion fingerprints could distinguish women with stage 1 breast cancer from women without breast cancer., Methods: The initial study population consisted of 310 subjects: 155 women with yearly negative breast examination and negative mammography findings for at least 4 years, and 155 women undergoing surgery for pathology-proven stage 1 invasive ductal carcinoma. High-resolution SELDI-TOF (surface-enhanced laser desorption ionization-time of flight) analysis was performed on serum obtained from blood samples collected before mammography in controls, and before surgery in patients with breast cancer. Samples were divided into a training (109 controls and 109 cancers) and blinded (46 controls and 46 cancers) testing set; each group had similar age demographics. In addition, an independent study set of 46 serum samples was analyzed 14 months after the initial study to validate the robustness of the classifier., Results: A discriminatory profile consisting of seven ion peaks found in the training set, when applied to the blinded test set, achieved a sensitivity and specificity of 95.6% and 86.5%, respectively. This same seven-peak profile achieved a 96.5% sensitivity and 85.7% specificity, with correct identification of all of 17 T1a tumors when applied to the validation study set., Conclusions: Mass spectrometry profiling of human serum generated a robust classifier composed of seven low-molecular-weight ions that yielded a highly sensitive and specific diagnostic procedure for the discrimination of women with stage 1 breast cancer compared with women without breast cancer in this research study set.

Background: The identification of gastric tumors associated with a higher risk of lymph node metastasis could help surgeons select patients who may benefit from extended lymph node dissection. The aim of this study was to screen the genome in the search of primary gastric cancer gene expression profiles that might predict lymph node status., Methods: The gene expression profile was evaluated in frozen tumor samples obtained from 32 patients with primary gastric adenocarcinomas. The array consisted of a duplicated spot panel of 5,541 human genes. To classify node-positive (N+) and node-negative (N-) cases, a logistic regression model was fitted optimizing the Akaike Information Criteria after a stepwise gene selection. The accuracy was evaluated by means of leave-one-out cross validation., Results: All patients underwent radical gastrectomy and extended lymphadenectomy. Of all the cases, 21 were N+ and 11 demonstrated no lymph node involvement (N-). After quality filtering, the analysis of variance selected a set of 136 genes potentially correlated with nodal involvement (P value <.05). Of these 136 genes, 5 were differentially expressed (adjusted P value <.05). After a stepwise gene selection, only three genes (Bik, aurora kinase B, eIF5A2) were retained in the logistic model, which could correctly predict lymph node status in 30 of 32 cases., Conclusions: If our findings were confirmed, the identified gene pattern might be used to tailor the extent of lymph node dissection on a single patient basis.

Background: Since specific epidermal growth factor receptor (EGFR) somatic mutations have been demonstrated to influence the response to anti-EGFR therapy in non-small cell lung cancer, EGFR gene mutational analysis of different types of neoplasm is under investigation., Patients and Methods: EGFR protein expression and gene mutations in exons 18, 19 and 21 were investigated in 49 gastric adenocarcinomas., Results: no specific EGFR gene mutations were detected, while EGFR positive staining was detected in 6% of the cases., Conclusion: Our findings indicate that in gastric adenocarcinoma, specific EGFR gene mutations are very rare or absent and the rate of EGFR protein expression is low.

Background: Liver metastases represent the major determinant of survival in patients with colorectal cancer (CRC). In cases with unresectable liver disease, more effective agents are needed, since chemotherapy achieves median survival of only 15 months. Protein kinases coordinate complex functions that are often disregulated in cancer and are therefore considered important targets for molecular therapeutics. In this study, we investigated the phosphoproteomic status of different protein kinases in primary CRC and in liver metastases., Methods: The status of 29 key endpoints was evaluated using reverse phase protein array on laser capture microdissected neoplastic cells from five primary CRCs without metastases, three patient-matched primary CRCs and synchronous liver metastases and five CRC metachronous liver metastases., Results: Unsupervised hierarchical two-way clustering analysis showed an entirely different phosphoproteomic profile in primary CRCs compared to liver metastases. This difference was observed also in primary and metastatic patient-matched lesions., Conclusions: Our findings of different signaling pathways between primary and metastatic CRC suggest a possible microenvironment effect, and emphasize the need to perform molecular network analysis of metastatic tissue when molecular targeting is considered.

Metastatic spread to the liver is the major contributor to mortality in patients with colorectal carcinoma (CRC). In order to seek for gene expression patterns associated with metastatic potential in primary CRC, we compared the transcriptional profiles of 10 radically resected primary CRCs from patients who did not develop distant metastases within a 5-year follow-up period with those of 10 primary/metastatic tumor pairs from patients with synchronous liver metastases. To focus selectively on neoplastic cells, the study was conducted on laser-microdissected bioptic tissues. Arrays of 7,864 human cDNAs were utilized. While a striking transcriptional similarity was observed between the primary tumors and their distant metastases, the nonmetastasizing primary tumors were clearly distinct from the primary/metastatic tumor pairs. Of 37 gene expression differences found between the 2 groups of primary tumors, 29 also distinguished nonmetastasizing tumors from metastases. The gene encoding for mannosyl (alpha-1,3-)-glycoprotein beta-1,4-N-acetyl-glucosaminyl-transferase (GnT-IV) became significantly upregulated in primary/metastatic tumor pairs (p < 0.001). GnT-IV upregulation was confirmed by RT-PCR. These data support the existence of a specific transcriptional signature distinguishing primary colon adenocarcinomas with different metastatic potential, the further pursuit of which may lead to relevant clinical and therapeutic applications., (Copyright 2005 Wiley-Liss, Inc)

Background and Objectives: Low tumor expression of the p27(Kip1) protein, which is involved in cell cycle control and apoptosis, is considered a negative prognostic factor in different types of cancer. The aim of this study was to evaluate the clinical and pathological significance of low p27(Kip1) protein expression in patients who had undergone resection for gastric adenocarcinoma., Methods: p27(Kip1) protein was studied by immunohistochemistry in formalin-fixed tumor sections from 95 patients who underwent resection for gastric adenocarcinoma between 1991 and 1996. Based on the median value of protein expression, p27(Kip1) protein expression was classified as low or high., Results: Low p27(Kip1) protein expression was significantly associated with tumor de-differentiation, increased penetration through the gastric wall, lymph node metastasis, and advanced tumor stage. In the group of 84 patients who underwent curative surgery, 5-year survival was 74% in cases with high p27(Kip1) protein expression and 38% in those with low p27(Kip1) protein expression (P < 0.001). At multivariate analysis, low p27(Kip1) protein expression was an independent negative prognostic factor for survival (RR = 3.671; P = 0.004)., Conclusions: In gastric adenocarcinoma, low p27(Kip1) protein expression is associated with poorly differentiated and advanced tumors and is a negative prognostic factor of potential clinical value., (Copyright 2002 Wiley-Liss. Inc.)