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High-Throughput MicroRNA (miRNAs) Arrays Unravel the Prognostic Role of MiR-211 in Pancreatic Cancer

Background Only a subset of radically resected pancreatic ductal adenocarcinoma (PDAC) patients benefit from chemotherapy, and identification of prognostic factors is warranted. Recently miRNAs emerged as diagnostic biomarkers and innovative therapeutic targets, while high-throughput arrays are opening new opportunities to evaluate whether they can predict clinical outcome. The present study evaluated whether comprehensive miRNA expression profiling correlated with overall survival (OS) in resected PDAC patients. Methodology/Principal Findings High-resolution miRNA profiles were obtained with the Torays 3D-Gene™-miRNA-chip, detecting more than 1200 human miRNAs. RNA was successfully isolated from paraffin-embedded primary tumors of 19 out of 26 stage-pT3N1 homogeneously treated patients (adjuvant gemcitabine 1000 mg/m2/day, days-1/8/15, every 28days), carefully selected according to their outcome (OS<12 (N = 13) vs. OS>30 months (N = 6), i.e. short/long-OS). Highly stringent statistics included t-test, distance matrix with Spearman-ranked correlation, and iterative approaches. Unsupervised hierarchical analysis revealed that PDACs clustered according to their short/long-OS classification, while the feature selection algorithm RELIEF identified the top 4 discriminating miRNAs between the two groups. These miRNAs target more than 1500 transcripts, including 169 targeted by two or more. MiR-211 emerged as the best discriminating miRNA, with significantly higher expression in long- vs. short-OS patients. The expression of this miRNA was subsequently assessed by quantitative-PCR in an independent cohort of laser-microdissected PDACs from 60 resected patients treated with the same gemcitabine regimen. Patients with low miR-211 expression according to median value had a significantly shorter median OS (14.8, 95%CI = 13.1–16.5, vs. 25.7 months, 95%CI = 16.2–35.1, log-rank-P = 0.004). Multivariate analysis demonstrated that low miR-211 expression was an independent factor of poor prognosis (hazard ratio 2.3, P = 0.03) after adjusting for all the factors influencing outcome. Conclusions/Significance Through comprehensive microarray analysis and PCR validation we identified miR-211 as a prognostic factor in resected PDAC. These results prompt further prospective studies and research on the biological role of miR-211 in PDAC.

Incidence, Diagnosis, and Treatment of Chylous Leakage After Laparoscopic Live Donor Nephrectomy

Background. Chylous leakage (CL) is a rare complication of laparoscopic live donor nephrectomy (LLDN). It may lead to malnutrition and immunological deficits because of protein and lymphocyte depletion. Methods. Data from 208 consecutive LLDN performed at two institutions, between April 2000 and September 2010, were reviewed to identify the anatomical basis behind CL along with its diagnostic and therapeutic options. Results. CL developed in eight donors (3.8%), as determined by high-volume drainage (range 540–800 mL/24 hr) of triglyceride-rich fluid. All donors were managed conservatively. Seven were put on total parenteral nutrition plus octreotide. One received low-fat diet, medium-chain triglyceride supplementation, and octreotide. Chylous fistulas resolved in 5 to 16 days (mean time 12.3 days). Drains were removed before hospital discharge, and no donor was readmitted and/or needed outpatient care. Conclusions. CL is a potentially insidious and perhaps misdiagnosed complication after LLDN. It occurs in nearly 4% of LLDN and it seems to be uniquely associated to left-sided kidney recovery because of distinctive lymphatics distribution around the periaortic area of dissection. Conservative therapy is effective in most donors and should be initially attempted. Surgical ligatures or fibrin sealants may be indicated in case of refractory CL before the arising of malnutrition and/or relevant immunodeficiency.

The odd case of a small and mucinous-like acinar cell cystoadenocarcinoma of the pancreas

Improvement of imaging has made the incidental diagnosis of pancreatic cysts very common. Interpretation of diagnostic information, however, is not always straightforward. We report on one of such patients, initially thought to have a mucinous cystic tumor of the pancreas, and eventually diagnosed with a higher grade acinar cell cystoadenocarcinoma (ACC). In April 2009, a 63-year old woman underwent total gastrectomy because of gastric cancer (undetermined tumor type, T2bN0M0–G1; 48 examined lymph nodes). During preoperative work-up contrast enhanced computed tomography (CT) showed a uniloculated cystic lesion in the head of the pancreas. During gastrectomy the cyst was aspirated. Cytology revealed protein rich fluid without evidence of epithelial cells. The patient underwent a 4 month course of multi-agent adjuvant chemotherapy, based on FOLFIRI (irinotecan, 5-fluoracil, and folinic acid), platinum and taxotere. During the follow-up CT showed progressive increase of the size of the cyst up to 30 mm, and the patient was referred to our center. Magnetic resonance cholangiopancreatography and CT (Fig. 1) demonstrated no communication between the cyst and the main pancreatic duct (MPD). Although the more likely preoperative diagnosis was mucinous cystadenoma, we preferred to proceed with pancreaticoduodenectomy mostly because of the growth of the cystic lesion, despite the patient was receiving multi-agent chemotherapy. We also considered that the digestive changes associated with total gastrectomy could have blurred the interpretation of developing complaints. Finally, concerns on pursuing major surgery in a patient recently operated because of gastric cancer were, at least in part, addressed by favorable tumor staging. Frozen section histology suggested side branch intraductal papillary mucinous neoplasm with normal MPD. On gross examination, the cyst measured 3 2 cm, communicated with the MPD, and was filled with milky content. Final pathology disclosed ACC (Fig. 2). Resection margins were negative and no metastasis was discovered in 30 regional lymph nodes. Post-operative course was uneventful and the patient was discharged on post-operative day 9. She received gemcitabine-based adjuvant chemotherapy for 5 months. One year after pancreaticoduodenectomy the patient is alive and disease-free. Acinar cell carcinoma of the pancreas is a rare solid epithelial exocrine tumor (1–2% of all exocrine pancreatic tumors) [1–5]. It is defined as a carcinoma exhibiting exocrine differentiation and lacking significant (<25%) ductal or endocrine differentiation [2,3].

Total Robotic Pancreaticoduodenectomy

Context The “da Vinci” surgical system reintroduces much of the operative dexterity lost during laparoscopic operations and offers the unique opportunity to verify if pancreaticoduodenectomy (PD) can be safely performed through a minimally invasive approach. Objective We report our technique for total robotic PD employed in 39 consecutive patients. This experience was earned at a high-volume center of pancreatic surgery, having extensive experience in advanced laparoscopy and robotic surgery. Methods Our technique for total robotic PD is unique in several respects: pure laparoscopy is not used at any stage; only the right colonic flexure is mobilized; a total of five ports are used; the camera port is placed along the right pararectal line to allow optimal view of the uncinate process (UP); the third robotic arm is placed on the patient’s left side and it is used to “hang” the duodenum during dissection of the UP; the gallbladder is used to retract the liver; the first jejunal loop is fully mobilized but it is not sectioned until the specimen is ready for removal, to facilitate jejunal rotation behind the mesenteric vessels. Results No PD was converted to open surgery or laparoscopy, despite 3 patients required segmental resection of the mesenteric vein and reconstruction by a jump graft. Mean operative time was 597 minutes (range: 420-960 minutes). Thirty-day operative mortality was nil. No pseudoaneurysm of the gastroduodenal artery was noted. Only 4 patients developed grade B pancreatic fistulas and none grade C fistulas. Mean hospital-stay was 23 days (range: 10-86 days). Malignant tumors were diagnosed in 51% of the patients. Overall, the mean number of lymph nodes retrieved was 32 (range 15-76). None of the margins was positive. Conclusions In selected patients total robotic PD is feasible. As compared to hybrid techniques, coupling laparoscopic dissection with robotic reconstruction, a total robotic procedure spares unnecessary dissections and allows optimal control of large peripancreatic vessels permitting segmental vein resection and tailored reconstruction. Technology refinements and improvement of surgical technique could make robotic PD an appealing alternative to open PD in selected patients.

Analysis of Instrument Traffic During Laparoscopic Robot-Assisted Pancreaticoduodenectomy

Context Robotic surgery entails specific issues that are not present, or are not equally relevant, in open surgery or conventional laparoscopy. Instrument traffic (IT) is one of such issues. IT is the time during which surgery is paused because the surgeon at the console is waiting for the action of the surgeon the table (e.g., instrument change, camera cleaning, introduction/withdrawal of needles). Objective We provide the first objective evaluation of IT during robotic pancreaticoduodenectomy (PD). Methods The operative videos of 12 robot-assisted PDs were reviewed to define IT. The analysis included: crude IT time (CITT), relative IT time (RITT) (defined as the percentage of operative time spent for IT), number of robotic instruments changes (RIC), time spent for RIC (TRIC), number of pure laparoscopic actions (PLA), and time spent for PLA (TPLA). Figures were estimated for the entire operation as well as for dissection and reconstruction phases. Details on pancreaticojejunostomy (PJ) or hepaticojejunostomy (HJ) were related to IT to define their relative impact on operative time. Results Mean operative time was 517 min (range 420-600 min). Mean CITT was 3,681.6 sec (RITT 11.89%). Mean RIC or PLA was 315.7. Each RIC or PLA paused surgery for 11.8 sec. Mean RIC was 184.4 (TRIC 2,633.8 sec). Mean PLA was 131.4 (TPLA 1,039.5 sec). Mean dissection time was 326.9 min. Mean CITT was 2,095.1 sec (RITT 10.68%). Mean RIC was 105.8 (TRIC 1,645.2 sec). Mean PLA was 35.7 (TPLA 382.5 sec). Each RIC or PLA paused surgery for 14.4 sec. PJ was made by invaginating technique or duct-to-mucosa. The last one required fewer stitches, but did not reduced CITT or RIC. HJ was performed using either 4 half running sutures or interrupted external stitches plus inner half running sutures. Despite similar CITT the former technique was associated with fewer RIC. Conclusions Some 12% of operative time of laparoscopic robot-assisted PD is wasted because of IT. Since in this series operative time of robotic PD averaged 517 minutes, IT prolonged surgery of more than one hour. Technology improvements and/or refinements in surgical technique are expected to reduce IT during robotic PD.

The New Automated Algorithm to Evaluate Ki67 in pNET. The Five “W” law: Who, When, Where, What, and Why

Context Ki67 index (Ki67-I), is the percentage Ki67 immunoreactive cells, expressing tumor proliferation, with important clinical relevance in pancreatic neuroendocrine tumors (pNET) and to standardize its evaluation is extremely important. The pNET guideline indicate to evaluate at list 2 mm 2 of tissue or 2,000 tumor cells. However, this type of evaluation is currently done by subjective opinion of pathologist concerning the area of interest (AI). Objective We elaborated a new algorithm of analysis able to catch all tumor cells present in the selected area according to the pathologist’s criteria. Methods The program (D-Sight, 2.0, Menarini, Florence, Italy) catch all color intensity on the tissue surface, to understand whether which type of sensitivity to immunohistochemistry could match with the Ki67-I. The system returns automatically the number of both total and stained cells. Results The first attempts made on the samples previously evaluated (15 cases with the oldest algorithm) showed an improvement of two important parameters: 1) a better evaluation of total tumor cells present in AI; and 2) a good evaluation of nuclei aggregation. These two data showed a better Ki67-I in pNET, very close to the pathologist’s interpretation. Conclusion The possibility to standardize a fully automated methodology to evaluate Ki67 value can improve both pathological evaluation (i.e., grading of the tumor) and clinical management of pancreatic neuroendocrine patients. This application could open also the new analytical evaluations of other protein markers involved in clinical outcome of oncologic patients.

PTEN and MIR-21 Expression in IPMN and PDAC

Context The prognosis of invasive IPMN is better than for PDAC and many authors believe that IPMNs have distinct genetic and biological characteristics underlying this different clinical behavior. Objective Since previous studies correlated miR-21 expression with PTEN levels and worse prognosis in PDAC, we compared PTEN and miR-21 expression in invasive IPMNs and PDACs. Methods Ten invasive IPMN and 16 PDAC were evaluated for both PTEN expressions, with a validated immunohistochemistry method: 4 degrees of score (0 absent, 1 weak, 2 moderate, 3 strong). The miR-21 expression, as assessed by PCR in mRNA isolated from laser-microdissected samples. According to the miR21 quantification, all samples were identified as follow: (IPMN-L, IPMN-H, PDAC-L and PDAC-H). Statistical analysis was performed using ANOVA tests. Results IPMNs with high mi-R21 expression presented a negative/weak PTEN cytoplasmatic staining, with only few scattered positive cells, while IPMNs characterized by low mi-R21, had a moderate or strong cytoplasmic PTEN expression. This inverse correlation of miR-21 and PTEN expression was also observed in PDAC. However, we observed a significant difference comparing PTEN IPMN-L vs . PDAC-L (P=0.021), IPMN-L vs . IPMN-H (P=0.041) and all groups (P=0.037). Conclusion PTEN expression correlated with miR-21 in both invasive IPMNs and PDACs. Moreover, PDAC had significantly higher levels of miR-21 and lower levels of PTEN than IPMNs, suggesting that these biological characteristics might underline the better clinical outcome of IPMN compared to PDAC.

Pancreatic Metastasis from Colorectal Cancer

Context Pancreatic metastases are rare (2% of all pancreatic carcinomas). Very few cases about surgical treatment of colorectal cancer metastases to the pancreas are reported. Case report We report a case of single colorectal cancer metastasis to the pancreas managed by distal splenopancreatectomy in a patient undergone to left hemicolectomy for the primary tumor eight years before and to middle lung lobectomy for metastasis one year before. A 61-year-old asymptomatic woman with a history of colorectal cancer was admitted to our department after that during the oncological imaging follow-up a thoracic-abdominal contrast-enhanced computed tomography (CT) demonstrated a single 25 mm hypodense lesion in the pancreatic tail. She also presented high levels of CEA (61.7 ng/mL) and CA 19-9 (82.9 U/mL) before the admission. Eight years before the patient underwent to left hemicolectomy for a B2-Dukes classification colorectal cancer. The resected margins were free of tumor and no regional lymph nodes were positive. One year before the patient underwent to a lung lobectomy for a single 30 mm pulmonary metastases. Considering history and imaging findings, the pancreatic lesion was suspected a colorectal cancer metastasis. A distal splenopancreatectomy was performed. The patient was discharged in healthy conditions. Final pathology disclosed the pancreatic lesion was a colorectal cancer metastasis (CD20+, CK7-) with infiltration of the peri-pancreatic adipose tissue. The resected margins were free of tumor and no lymph nodes were metastasized. The patient is still alive. Conclusion Metastases to the pancreas are commonly considered rare, especially those from colorectal cancer. The improvement of imaging techniques has led to an increase of diagnoses and surgical procedures for metastases to the pancreas. Secondary tumors may be considered in the differential diagnosis of primitive pancreatic lesions. The diagnosis may be facilitated by clinical history and serum markers assessment. Metastatic colorectal cancer to the pancreas is an indication for pancreatic resection to increase the overall survival and, as palliative procedure, to treat symptoms like jaundice and pain.

Gemcitabine-Ukrain Combination Affects MMP9 Expression in Primary Pancreatic Adenocarcinoma Cell Cultures (PPCCs)

Context Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal tumors mostly because of its invasive behavior and resistance to most chemotherapy regimens. Our previous results suggested that NSC-631570 (ukrain) modulates extracellular matrix remodeling of PDAC cell lines [1]. This study investigates the modulation of key determinants of invasive behavior such as MMP9 protein by gemcitabine-ukrain, using appropriate preclinical models. Objective This study investigates the modulation of key determinants of invasive behavior such as MMP9 protein by gemcitabine-ukrain, using appropriate preclinical models. Methods Two PPCCs and two cell lines of PDAC were seeded in multi-well chamber slides (8,000 each/well) and exposed to gemcitabine (10 nM), ukrain (1 µM) and their combination. After 48-h treatment the cells were stained with the polyclonal antibody (CST-Euroclone) for MMP9. Untreated cells were used to evaluate the basal level of MMP9, and while non-stained cells were employed as negative control. Protein expression levels were evaluated with novel software for image analysis, checking both nuclei and cytoplasm staining intensity. Differences in expression values were compared by t-test/ANOVA analyses. Results We observed a significant reduction of MMP9 expression in both PPCCs treated with gemcitabine-ukrain combination with respect to their controls and to cells treated with gemcitabine or ukrain alone (P<0.01). Moreover, drug combination significantly reduced the number cells, and modified the structure of most nuclei with respect to untreated cells. Conclusion New approaches to reduce the metastatic behavior of PDAC are warranted, and gemcitabine-ukrain showed promising results in our preclinical studies. The new computerized approach to evaluate MMP9 staining at ICC is an ease-to-use and rapid method that should be further developed both in preclinical models and for IHC analyses of PDAC tissues.

Robotic Pancreatectomy for Pancreatic and Periampullary Cancer

Context Minimally invasive surgery, when feasible, should accept no oncologic compromise in the setting of pancreatic and periampullary cancer since local radicality is key for all these tumor types. Objective We herein report on 50 patients undergoing robotic pancreatic resection because of pancreatic or periampullary cancer. Methods Fifty patients diagnosed with malignant tumors were selected for laparoscopic robot-assisted pancreatectomy between October 2008 to June 2012. There were 28 males (56%) and 22 females (44%), with a mean age of 60 years (range 24-78 years). Twenty-five patients underwent pancreaticoduodenectomy (PD) (50%), 16 distal pancreatectomy (DP) (32%), 7 total pancreatectomy (TP) (14%), and 2 to central pancreatectomy (CP) (4%). Results Final pathology disclosed neuroendocrine carcinoma (NEC) in 7 patients (14%), cancer arising on IPMN in 9 cases (18%), ductal adenocarcinoma (DA) in 19 cases (38%), cholangiocarcinoma (CHC) in 5 patients (10%), carcinoma of the papilla of Vater in 5 cases (10%) (4 PD), solid pseudopapillary tumor in 2 (4%) and adenosquamous carcinoma in 1 case (2%). Resection margins were all negative. A mean number of 30 lymph nodes (range 5-74) was retrieved en-bloc with the specimen. 22 patients had lymph node metastasis (44%) including 11 diagnosed with DA (60%), 4 with CHC (80%) and 4 with NEC (5.7%). After a mean follow-up period of 14.1 months (range 1-42 months) all but 2 patients are disease-free (96%). Conclusions After a learning curve, best completed on patients with benign pancreatic diseases, laparoscopic robot-assisted pancreatic resection seems to offer the potential for radical tumor clearance in selected patients without locally advanced pancreatic and periampullary cancer. Further experience and longer follow-up are both needed before any final conclusion can be drawn.