An Orthotopic Patient-Derived Xenograft (PDX) Model Allows the Analysis of Metastasis-Associated Features in Colorectal Cancer.

Metastasis is the primary cause of death in patients with colorectal cancer (CRC), urging the need for preclinical models that recapitulate the metastatic process at the individual patient level. We used an orthotopic patient-derived xenograft (PDX) obtained through the direct implantation of freshly dissociated CRC cells in the colon of immunocompromised mice to model the metastatic process. Ortho-PDX engraftment was associated to a specific set of molecular features of the parental tumor, such as epithelial-to-mesenchymal transition (EMT), TGF-ß pathway activation, increased expression of stemness-associated factors and higher numbers of circulating tumor cells (CTCs) clusters expressing the metastatic marker CD44v6. A parallel analysis of orthotopic/metastatic xenografts and organoids showed that tumor cells underwent mesenchymal-to-epithelial transition at the metastatic site and that metastasis-derived organoids had increased chemotherapy resistance. These observations support the usefulness of ortho-PDX as a preclinical model to study metastasis-related features and provide preliminary evidence that EMT/stemness properties of primary colorectal tumors may be crucial for orthotopic tumor engraftment.

An organoid model of colorectal circulating tumor cells with stem cell features, hybrid EMT state and distinctive therapy response profile.

BACKGROUND: Circulating tumor cells (CTCs) are responsible for the metastatic dissemination of colorectal cancer (CRC) to the liver, lungs and lymph nodes. CTCs rarity and heterogeneity strongly limit the elucidation of their biological features, as well as preclinical drug sensitivity studies aimed at metastasis prevention. METHODS: We generated organoids from CTCs isolated from an orthotopic CRC xenograft model. CTCs-derived organoids (CTCDOs) were characterized through proteome profiling, immunohistochemistry, immunofluorescence, flow cytometry, tumor-forming capacity and drug screening assays. The expression of intra- and extracellular markers found in CTCDOs was validated on CTCs isolated from the peripheral blood of CRC patients. RESULTS: CTCDOs exhibited a hybrid epithelial-mesenchymal transition (EMT) state and an increased expression of stemness-associated markers including the two homeobox transcription factors Goosecoid and Pancreatic Duodenal Homeobox Gene-1 (PDX1), which were also detected in CTCs from CRC patients. Functionally, CTCDOs showed a higher migratory/invasive ability and a different response to pathway-targeted drugs as compared to xenograft-derived organoids (XDOs). Specifically, CTCDOs were more sensitive than XDOs to drugs affecting the Survivin pathway, which decreased the levels of Survivin and X-Linked Inhibitor of Apoptosis Protein (XIAP) inducing CTCDOs death. CONCLUSIONS: These results indicate that CTCDOs recapitulate several features of colorectal CTCs and may be used to investigate the features of metastatic CRC cells, to identify new prognostic biomarkers and to devise new potential strategies for metastasis prevention.

A pre-existing population of ZEB2+ quiescent cells with stemness and mesenchymal features dictate chemoresistance in colorectal cancer.

BACKGROUND: Quiescent/slow cycling cells have been identified in several tumors and correlated with therapy resistance. However, the features of chemoresistant populations and the molecular factors linking quiescence to chemoresistance are largely unknown. METHODS: A population of chemoresistant quiescent/slow cycling cells was isolated through PKH26 staining (which allows to separate cells on the basis of their proliferation rate) from colorectal cancer (CRC) xenografts and subjected to global gene expression and pathway activation analyses. Factors expressed by the quiescent/slow cycling population were analyzed through lentiviral overexpression approaches for their ability to induce a dormant chemoresistant state both in vitro and in mouse xenografts. The correlation between quiescence-associated factors, CRC consensus molecular subtype and cancer prognosis was analyzed in large patient datasets. RESULTS: Untreated colorectal tumors contain a population of quiescent/slow cycling cells with stem cell features (quiescent cancer stem cells, QCSCs) characterized by a predetermined mesenchymal-like chemoresistant phenotype. QCSCs expressed increased levels of ZEB2, a transcription factor involved in stem cell plasticity and epithelial-mesenchymal transition (EMT), and of antiapototic factors pCRAF and pASK1. ZEB2 overexpression upregulated pCRAF/pASK1 levels resulting in increased chemoresistance, enrichment of cells with stemness/EMT traits and proliferative slowdown of tumor xenografts. In parallel, chemotherapy treatment of tumor xenografts induced the prevalence of QCSCs with a stemness/EMT phenotype and activation of the ZEB2/pCRAF/pASK1 axis, resulting in a chemotherapy-unresponsive state. In CRC patients, increased ZEB2 levels correlated with worse relapse-free survival and were strongly associated to the consensus molecular subtype 4 (CMS4) characterized by dismal prognosis, decreased proliferative rates and upregulation of EMT genes. CONCLUSIONS: These results show that chemotherapy-naive tumors contain a cell population characterized by a coordinated program of chemoresistance, quiescence, stemness and EMT. Such population becomes prevalent upon drug treatment and is responsible for chemotherapy resistance, thus representing a key target for more effective therapeutic approaches.

Colorectal cancer: towards new challenges and concepts of preventive healthcare.

Colorectal cancer (CRC) holds the woeful record of being the most preventable but least prevented type of cancer. Although healthy lifestyles and screening programs associated to early polyp removal greatly reduce CRC incidence and CRC-related death, the overall lack of information and of effective preventive policies is promoting an overwhelming global escalation of this disease. Moreover, new challenges such as the increasing occurrence of aggressive CRC in young adults highlight underlying changes not only in the incidence but also in the nature of this disease. In this scenario, CRC prevention should also undergo a significant transformation, embracing not only individual initiatives but also issues of social and international relevance. The nascent network of countries surrounding the Mediterranean basin (COLOMED, the COLOrectal cancer MEDiterranean network) serves as a paradigm of international cooperation aimed at broadening scientific collaboration and promoting effective health policies in CRC research and prevention.

Laparoscopic liver resection for hepatocellular carcinoma in cirrhotic patients: single center experience of 90 cases.

BACKGROUND: Hepatocellular carcinoma (HCC) with or without underlying liver disease can be treated by surgical resection. The aim of this study was to evaluate the feasibility, morbidity and mortality of a laparoscopic approach in cirrhotic patients with HCC. METHODS: From 2004 to September 2014, 90 patients underwent a laparoscopic liver resection (LLR) for HCC. Data were collected in a prospectively maintained database since 2001. Preoperative patient evaluation was based on a multidisciplinary team meeting assessment. RESULTS: Median age was 63 years; 67 (74.4%) patients were male. Median body mass index (BMI) was 26.7. Underlying liver disease was known in 68 patients: in 46 patients' hepatitis C virus (HCV)-related, in 15 patients to hepatitis B virus (HBV)-related, in 5 patients alcohol-related. Child-Pugh Score was of grade A in 85 patients and of grade B in 5 patients; 63 patients had a Model for End-stage Liver Disease (MELD) <10 and 27 patients MELD >10. A total of 18 left lateral sectionectomies, 1 left hepatectomy and 71 wedge resections or segmentectomies were performed. Conversion to laparotomy was necessary in 7 (7.7%) patients (five cases for bleeding and two cases for oncological reasons). In 90 patients, 98 HCC nodules were resected: 79 patients had one nodule, 8 patients had two nodules and 1 patient had three nodules. HCC nodules medium diameter was 29 mm (range, 4-100 mm) with median value of 25 mm. Tumor margins distance was 16 mm (range, 0-35 mm) with a median of 5 mm. Seventy nodules were located within the anterior sectors and 28 nodules within the posterior sectors. CONCLUSIONS: LLR for HCC can be performed with acceptable morbidity in patients with underlying liver disease. The use of laparoscopic surgery in cirrhotic patients may be proposed as the first-line treatment for HCC or as bridge treatment before liver transplantation.

Laparoscopic first step approach in the two stage hepatectomy.

Resection is the gold standard therapeutic option for patients with colorectal liver metastases. However, only 20-30% of patients are resectable. In patients with a concomitant future liver remnant (FLR) less than 25-30%, a single stage resection is not feasible. The aim of this study is to evaluate the feasibility and the rates of morbidity and mortality of the laparoscopic approach in the first-step of two stage hepatectomy. From 2004 to March 2014, 73 patients underwent a two stage hepatectomy: of these, four underwent a totally laparoscopic first step [wedge left liver resection and right portal vein ligation (PVL)]. All the patients were male. Median age was 55 years. One patient underwent an atypical wedge resection of segment II-III and a laparoscopic PVL (LPVL), one patient had a first wedge resection of segment II and LPVL, and two patients underwent a wedge resection of segment III and LPVL. First step surgical mean time was 189 (range, 160-244) min, mean blood loss was 22 (range, 0-50) cc. No transfusion was required in this series. The results of our study demonstrate that the first step of hepatic resection and PVL is feasible with a laparoscopic approach in patients with bilobar liver metastases.

Laparoscopic liver resections in normal and cirrhotic livers: a retrospective analysis in a tertiary hepato-biliary unit.

BACKGROUND: Liver surgery in patients with underlying liver disease results in higher mortality and morbidity rates compared to patients without underlying liver disease. Laparoscopy seems to have good results in patients with normal liver in terms of postoperative outcomes, but is more challenging in cirrhotic patients. Aim of this study was to evaluate the feasibility of laparoscopic liver resection both in normal and cirrhotic livers, and secondary endpoint was to compare the surgical results. METHODS: We retrospectively evaluated 105 patients who underwent laparoscopic liver resection between November 2001 and January 2012. Candidates for laparoscopic liver resection were divided into two groups according to the presence or absence of an underlying liver disease. RESULTS: 105 patients (52.4% males, median age 56.1 years) were enrolled, and 37.1% had liver cirrhosis. Hepatocellular carcinoma in hepatitis C virus-related cirrhosis (89.7%) and liver metastases (57.6%) were the main indications for surgery in patients with cirrhosis and non-cirrhotic livers, respectively. None of the patients died post-operatively. Cirrhotic patients had greater blood loss (100 vs 50 ml; p<0.012) and longer hospital stays (6 vs 4 days; p<0.031) compared to non-cirrhotics. CONCLUSIONS: Laparoscopic liver resections are safe and feasible procedures in both patients with cirrhotic and non-cirrhotic livers.

Cystic echinococcosis in a single tertiary care center in Rome, Italy.

BACKGROUND: Cystic echinococcosis (CE) is a chronic, clinically complex, and neglected disease. Its prevalence in Italy, a country of medium to high endemicity, remains poorly defined, as notification has long ceased to be mandatory. METHODS: We set up a retrospective cohort study involving all CE patients followed at our institute between January 2005 and December 2012. Demographical and clinical features were recorded and analyzed. RESULTS: CE was found in 28 patients (64.3%), mostly Italians from the central regions (50%), followed by subjects from the islands (33.3%) and Southern Italy (16.7%). Their median age was 45 years (IQR: 38.5-66.5), with Eastern Europeans being significantly younger (28 years, IQR: 19-39) than other patients (P ≤ 0.0001). A total of 149 cysts, mostly with hepatic localization (96%), were described. Based on the WHO classification, the cysts were mainly small (80.5%) and active (CE1 (73.8%); CE2 (7.4%)). Active cysts were more common in Eastern Europeans (85.7%) than Italians (66.7%). CONCLUSION: Our data confirm CE occurrence in Italy. We emphasize the importance to have a national CE registry, opportunely recently introduced. This is essential to assess CE prevalence in this country, implement appropriate control measures, and improve patient management.

Use of cold-stored vein allografts for venous reconstruction during pancreaticoduodenectomy.

INTRODUCTION: Venous resections and reconstructions of portal vein and/or superior mesenteric vein in course of pancreaticoduodenectomy are becoming a common practice and many surgical options have been described, from simple tangential resection and venorrhaphy to large segmental resections followed by interposition grafting. The aim of this study was to report the first experience of using fresh cadaveric vein allografts for venous reconstruction during pancreaticoduodenectomy focusing on technical feasibility and postoperative outcomes. METHODS: From January 2001 to October 2012, out of 151 patients undergoing pancreaticoduodenectomy for pancreatic head tumor, 22 (14.5 %) received a vascular resection of the mesentericoportal axis. In five of these patients, vascular reconstruction was accomplished by using cold-stored venous allografts of iliac and femoral veins from donor cadaver. Patients' data, surgical techniques, and clinical outcomes were analyzed. RESULTS: Five patients undergoing pancreaticoduodenectomy were selected to receive a vascular reconstruction using a fresh venous allograft for patch closure in three cases, conduit interposition in one case and a Y-shaped graft in the last case. No graft thrombosis or stenosis occurred postoperatively and at long-term follow-up. Mortality rate was zero. CONCLUSION: The use of fresh vein allografts is a feasible and effective technique for venous reconstruction during pancreaticoduodenectomy. However, prospective surveys including large cohorts of patients are necessary to confirm these results.

Outcome of chronic pilonidal disease treatment after ambulatory plain midline excision and primary suture.

BACKGROUND: Pilonidal sinus (PS) is a common chronic disorder of the sacrococcygeal region. The optimal treatment for PS remains controversial, and recent reports have advocated different surgical approaches. METHODS: A prospective study was performed on 103 patients with nonrecurrent quiescent chronic discharging sinus. Excision with primary closure was performed on all patients. Patients were subdivided randomly into 2 groups. In group A, the excision was associated with drainage of the wound; in group B, the wound was not drained. RESULTS: Excision with primary closure and drainage was performed in 53 patients (group A). Drainage was omitted in 50 patients (group B). Minor wound complications occurred in 3 patients in group A and in 36 patients in group B. No complete dehiscence of the wound was observed in patients in group A and in 8 patients in group B. Complete healing was fastest in patients in group A. Sinus recurrence occurred in 1 patient in group A and in 2 patients in group B. CONCLUSION: Short- and long-term results suggest that limited midline excision with primary closure and wound drainage is a simple and effective procedure in the surgical treatment of uncomplicated PS. More demanding flap techniques and plasties should be reserved for complicated PS, which requires a wider excision.

Intra-operative ultrasound for detection of liver metastases from colorectal cancer.

OBJECTIVE: The aim of this study was to evaluate the accuracy of intra-operative ultrasound (IOUS) imaging in detecting liver secondaries at the time of primary colorectal surgery and to evaluate the impact of IOUS on patient management. METHODS: Data from 167 patients with primary colorectal cancer who were admitted for elective surgery between January 1995 and December 2003 were prospectively evaluated and analysed. All patients underwent pre-operative abdominal ultrasonography (US) and computed tomography (CT), as well as IOUS. The final diagnosis of liver metastases was made by means of histological examination of either biopsy or surgical specimens. The sensitivities of pre-operative US and CT were compared with the sensitivity of IOUS, referred to histology. Changes in surgical management owing to IOUS findings were noted. RESULTS: IOUS supplied additional information in the case of 31 patients. In 28 of these patients, this information had a major impact on the intra-operative strategy, in that the procedure was altered. CONCLUSIONS: IOUS is safe, simple to perform and more accurate than pre-operative imaging. It reduces the number of patients subjected to superfluous surgery. The use of IOUS is therefore encouraged during colorectal cancer surgery.

Treatment of hydatid bronchobiliary fistulas: 30 years of experience.

BACKGROUND: Bronchobiliary fistula (BBF) is an uncommon but severe complication of hydatid disease of the liver. Operation is considered the treatment of choice but the most appropriate operation is uncertain. The aim of this study was to evaluate the early and long-term outcomes following different surgical procedures. METHODS: A retrospective evaluation of 31 patients with BBF was performed. Surgical access consisted of laparotomy, thoracotomy or a thoracoabdominal (TA) incision. Surgical procedures for the treatment of the cyst were classified as conservative or radical. RESULTS: Radical treatment including lung resection and pericystectomy was performed in all patients in whom the surgical exposure was obtained by either thoracotomy or TA. Of the patients treated by laparotomy, two had a pericystectomy, and four had drainage of the cyst. There were two deaths among the seven thoracotomy patients and one among the 18 TA patients. Pleural effusion was observed in six of the TA, two of the thoracotomy, and three of the laparotomy patients. Biliary fistula occurred in two of the five thoracotomy patients surviving operation and in two laparotomy patients (2/6). Progression of the lung disease was observed in four laparotomy patients and in one thoracotomy patient. CONCLUSIONS: The better outcome achieved in TA patients is the result of the simultaneous radical treatment of all the pathological aspects of BBF.

Surgical treatment of liver metastases from colorectal cancer in elderly patients.

INTRODUCTION: The liver is the most frequent site of liver metastases (LM) from colorectal cancer. Because of short life expectancies and improved nonoperative modalities, the role of liver resection in elderly patients with LM is unclear. METHODS: During a 15-year period, 197 patients underwent liver resection for colorectal metastases. This study was designed to compare morbidity, mortality, and long-term outcome after hepatic resection in patients aged 70 years and older and in patients younger than 70. According to the age at the time of operation, patients were divided into two groups. Group A included patients aged 70 years or older and group B included younger patients. RESULTS: The clinical and pathologic parameters of the two groups were compared and tested as factors affecting early and long-term outcomes after resection. A modified oncologic clinical risk score (CRS) was tested on this series of patients. Overall morbidity was 16.3% (group A 20.7% vs group B 14.6%; P=0.18). Hospital mortality was 3% (5.7% in group A and 2.1% in group B; P=0.19). Actuarial 5 years survival were 30% in group A and 38% in group B (P=ns). DISCUSSION: The presence of more than three Fong's CRS parameters and microscopic involvement of resectional margin directly affected survival. Under meticulous preoperative assessment and postoperative care, liver resection for LM is justified in patients over 70 years of age; age by itself may not be a controindication to surgery.