Bacterobilia in pancreatic surgery-conclusions for perioperative antibiotic prophylaxis.

BACKGROUND: Jaundice or preoperative cholestasis (PC) are typical symptoms of pancreatic masses. Approximately 50% of patients undergo preoperative biliary drainage (PBD) placement. PBD is a common cause of bacterobilia (BB) and is a known surgical site infection risk factor. An adjustment of preoperative antibiotic prophylaxis (PAP) may be reasonable according to the profile of BB. For this, we examined the microbiological findings in routine series of patients. AIM: To investigate the incidence and profile of biliary bacterial colonization in patients undergoing pancreatic head resections. METHODS: In the period from January 2009 to December 2015, 285 consecutive pancreatic head resections were performed. Indications for surgery were malignancy (71%), chronic pancreatitis (18%), and others (11%). A PBD was in 51% and PC was in 42%. The standard PAP was ampicillin/sulbactam. Intraoperatively, a smear was taken from the hepatic duct. An analysis of the isolated species and resistograms was performed. Patients were categorized according to the presence or absence of PC (PC+/PC-) and PBD (PBD+/PBD-) into four groups. Antibiotic efficiency was analyzed for standard PAP and possible alternatives. RESULTS: BB was present in 150 patients (53%). BB was significantly more frequent in PBD+ (n =120) than in PBD- (n = 30), P < 0.01. BB was present both in patients with PC and without PC: (PBD-/PC-: 18%, PBD-/PC+: 30%, PBD+/PC-: 88%, PBD+/PC+: 80%). BB was more frequent in malignancy (56%) than in chronic pancreatitis (45%). PBD, however, was the only independent risk factor in multivariate analysis. In total, 357 pathogens (342 bacteria and 15 fungi) were detected. The five most common groups (n = 256, 74.8%) were Enterococcus spp. (28.4%), Streptococcus spp. (16.9%), Klebsiella spp. (12.6%), Escherichia coli (10.5%), and Enterobacter spp. (6.4%). A polymicrobial BB (PBD+: 77% vs PBD-: 40%, P < 0.01) and a more frequent detection of Enterococcus (P < 0.05) was significantly associated with PBD+. In PBD+, the efficiency of imipenem and piperacillin/tazobactam was significantly higher than that of the standard PAP (P < 0.01). CONCLUSION: PBD-/PC- and PBD-/PC+ were associated with a low rate of BB, while PBD+ was always associated with a high rate of BB. In PBD+ patients, BB was polymicrobial and more often associated with Enterococcus. In PBD+, the spectrum of potential bacteria may not be covered by standard PAP. A more potent alternative for prophylactic application, however, was not found.

The impact of immunosuppression on postoperative graft function after graft-unrelated surgery: a retrospective controlled cohort study.

BACKGROUND: Physicians are faced with a growing number of patients after renal transplantation undergoing graft-unrelated surgery. So far, little is known about the postoperative restitution of graft function and the risk factors for a poor outcome. METHODS: One hundred one kidney transplant recipients undergoing graft-unrelated surgery between 2005 and 2015 were reviewed retrospectively. A risk analysis was performed and differences in creatinine, GFR and immunosuppressive treatment were evaluated. Additional, a comparison with a case-matched non-transplanted control group were performed. RESULTS: Preoperative creatinine averaged 1.88 mg / dl [0.62-5.22 mg / dl] and increased to 2.49 mg / dl [0.69-8.30 mg / dl] postoperatively. Acute kidney failure occurred in 18 patients and 14 patients had a permanent renal failure. Significant risk factors for the development of postoperative renal dysfunction were female gender, a preoperative creatinine above 2.0 mg / dl as well as a GFR below 40 ml / min and emergency surgery. Patients with tacrolimus and mycophenolate mofetil treatment showed a significant lower risk of renal dysfunction than patients with other immunosuppressants postoperatively. Contrary to that, the risk of patients with cyclosporine treatment was significantly increased. Transplanted patients showed a significantly increased rate of postoperative renal dysfunction. CONCLUSIONS: The choice of immunosuppressant might have an impact on graft function and survival of kidney transplant recipients after graft-unrelated surgery. Further investigations are needed.

Prognostic relevance of preoperative bilirubin-adjusted serum carbohydrate antigen 19-9 in a multicenter subset analysis of 179 patients with distal cholangiocarcinoma.

BACKGROUND: Distal cholangiocarcinoma (DCC) is a rare malignancy and validated prognostic markers remain scarce. We aimed to evaluate the role of serum CA19-9 as a potential biomarker in DCC. METHODS: Patients operated for DCC at 6 high-volume surgical centers from 1994 to 2015 were identified from prospectively maintained databases. Patient baseline characteristics, surgical and histopathological parameters, as well as overall survival after resection were assessed for correlation with preoperative bilirubin-adjusted serum carbohydrate antigen 19-9 (CA19-9). Preoperative CA19-9 to bilirubin ratio (CA19-9/BR) was classified as elevated (≥ 25 U/ml/mg/dl) according to the upper serum normal values of CA19-9 (37 U/ml) and bilirubin (1.5 mg/dl) giving a cut-off at ≥ 25 U/ml/mg/dl. RESULTS: In total 179 patients underwent resection for DCC during the study period. High preoperative CA19-9/BR was associated with advanced age and regional lymph node metastases. Median overall survival after resection was 27 months. Elevated preoperative serum CA19-9/bilirubin ratio (HR 1.6, p = 0.025), T3/4 stage (HR 1.8, p = 0.022), distant metastasis (HR 2.5, p = 0.007), tumor grade (HR 1.9, p = 0.001) and R status (HR 1.7, p = 0.023) were identified as independent negative prognostic factors following multivariable analysis. CONCLUSION: Elevated preoperative bilirubin-adjusted serum CA19-9 correlates with regional lymph node metastases and constitutes a negative independent prognostic factor after resection of DCC.

Surgical outcomes of renal transplant recipients after abdominal surgery not connected with transplantation. A retrospective case-control study.

BACKGROUND: Due to the increasing number of patients after kidney transplantation, elective and emergency surgery of transplanted patients is becoming a relevant challenge in clinical routine. The current data on complication rate of patients after kidney transplantation, which must undergo another elective or emergency abdominal surgery, is inhomogeneous. Therefore, the aim of our study was to evaluate the outcome of renal transplant patients undergoing abdominal and abdominal wall surgery. MATERIAL AND METHODS: We performed an observational study of patients after kidney transplantation undergoing graft-unrelated abdominal surgery between 2005 and 2015. We randomly created a non-transplanted control for a case-matched controlled analysis. Primary endpoint was the comparison of complication rate. Secondary, a risk analysis of all patients was performed and differences in mortality, length of hospital stay and reoperation rates were calculated. RESULTS: Overall 101 kidney transplanted patients were eligible for inclusion. 20 (19.8%) died after graft-unrelated surgery and 60 (59.4%) suffered from postoperative complications. Case-matched analysis could be performed for 84 out of these 101 patients. We found no significant difference in morbidity rate (58.3% vs. 45.2%, p = 0.090). Transplanted patients had, however, a significantly higher mortality (19% vs. 2.4%, p = 0.001), a longer hospital stay (28.2 vs. 16.9 days, p = 0.020) and a higher rate of re-operations (38.1% vs. 20.2%, p = 0.017). . CONCLUSIONS: Patients after renal transplantation undergoing graft-unrelated abdominal surgery have a significantly increased mortality risk, are more frequently re-operated and have to stay significantly longer in hospital than non-transplanted patients.

Outcome after hepatic resection for isolated non-colorectal, non-neuroendocrine liver metastases in 100 patients - the role of the embryologic origin of the primary tumor.

BACKGROUND: The indication for hepatic resection (HR) in patients suffering from liver metastases (LM) other than colorectal and neuroendocrine tumors is one focus of current multidisciplinary, oncologic considerations. This study retrospectively analyzes outcome after HR for non-colorectal, non-neuroendocrine (NCNNE) LM in the absence of distant or extrahepatic metastases. METHODS: We included 100 consecutive patients undergoing HR for isolated NCNNE LM from a prospective database in our institution, including postoperative follow-up. Primary tumors were of mesodermal origin in 44%, of ectodermal origin in 29% and of entodermal origin in 27%. Survival analysis was performed by univariate and multivariable methods. Mean follow-up after hepatic surgery was 3.6 years (0.25-16). RESULTS: Median age at the time of HR was 59.5 years. Kaplan-Meier-estimated survival after liver resection was 56.8%, 34.3% and 24.5% after 5, 10 and 15 years, respectively. Univariate analysis after HR revealed residual disease (hepatic or primary; p = 0.02), female gender (p = 0.013), entodermal origin (p = 0.009) and early onset of metastatic disease (≤24 months, p = 0.002), as negative prognostic factors. Multivariable survival analysis confirmed residual disease, female gender, entodermal embryologic origin and early onset of metastatic disease (≤24 months) as independent negative prognostic factors. CONCLUSION: Overall outcome after HR of NCNNE LM results in acceptable long-term outcome. Although individual decision-making today mostly relies on clinical experience for this type of disease, risk factors derived from the embryologic origin of the tumor might help in patient selection.

Does the site of primary colorectal cancer influence the outcome after resection of isolated liver metastases?

INTRODUCTION: In unresectable patients with metastatic colorectal cancer (CRC), the site of the primary is a strong prognostic factor warranting major adjustments in palliative medical treatment. Initial results suggested that the site of CRC influences prognosis after curative resection of colorectal liver metastases (CLM). In this study, we evaluated outcome after resection of isolated CLM with regard to the location of the primary. METHODS: 221 patients with macroscopically complete resection of CLM and no known extrahepatic disease were identified. 63 patients had right-sided and 158 had left-sided CRC. Tumors of the transverse colon and rectum were excluded. Survival was evaluated using the Kaplan-Meier method. RESULTS: Characteristics of CLM, primary tumor stage and chemotherapeutic regimens were not significantly different between the two groups. Kaplan-Meier five-year survival was comparable (41%) in patients with right- or left-sided CRC (p = 0.64). Microscopic resection margin, number of liver metastases, age and nodal status but not the site of the primary tumor significantly influenced survival. CONCLUSION: The site of the colorectal primary in this well-defined group of patients after resection of isolated CLM did not prove to be of significant prognostic value. Whether the primary tumor in CLM is located on the left side or the right should not preclude patients from surgery.

Oncological outcome of laparoscopically assisted pancreatoduodenectomy for ductal adenocarcinoma in a retrospective cohort study.

INTRODUCTION: Laparoscopic resections of the pancreatic head are increasingly performed. Several studies show that they are comparable to open operations in terms of postoperative morbidity. However, since a substantial proportion of pancreatic head resections are necessary for pancreatic adenocarcinoma the oncologic safety and outcome of minimally invasive operations is of interest. In this study we evaluated oncologic outcome and survival after laparoscopically assisted pancreatic head resection for ductal adenocarcinoma. METHODS: Perioperative and oncological outcome of sixty-two laparoscopically assisted pancreatic head resections for pancreatic ductal adenocarcinoma performed between 2010 and 2016 was compared to outcome of 278 open resections between 2001 and 2016 in a retrospective study. Data was continuously collected in a prospectively maintained database. RESULTS: Operation time was significantly longer in the laparoscopic group (477 vs. 428 min. p < 0.001). Tumor size, lymph node yield and lymph node state and need of portal vein resection were comparable. There was a higher rate of free resection margins in the laparoscopic group (87% vs. 71%, p < 0.01). There was no difference in postoperative mortality and morbidity. Patients with laparoscopic resection stayed in hospital significantly shorter (median 14 vs. 16 days, p < 0.003). Postoperative survival after 5 years was not different in both groups. CONCLUSION: Laparoscopically assisted resection of adenocarcinoma of the pancreatic head is equal to open resection concerning oncologic outcome and actuarial survival. However, minimally invasive resection shortened the hospital stay. However, further evaluations with a longer follow up time are needed.

Prognostic influence of hepatic margin after resection of colorectal liver metastasis: role of modern preoperative chemotherapy.

PURPOSE: Modern chemotherapy (CTX) increases survival in stage IV colorectal cancer. In colorectal liver metastases (CLM), neoadjuvant (neo) CTX may increase resectability and improve survival. Due to widespread use of CTX in CLM, recent studies assessed the role of the hepatic margin after CTX, with conflicting results. We evaluated the outcome after resection of CLM in relation to CTX and hepatic resection status. METHODS: Since 2000, 334 patients with first hepatic resection for isolated CLM were analyzed. Thirty-two percent had neoadjuvant chemotherapy (targeted therapy in 42%). Sixty-eight percent never had CTX before hepatectomy or longer than 6 months before resection. The results were gained by analysis of our prospective database. RESULTS: Positive hepatic margins occurred in 8% (independent of neoCTx). Patients after neoCTX had higher numbers of CLM (p < 0.01) and a longer duration of surgery (p < 0.03). After hepatectomy, 5-year survival was 45% and correlated strongly with the margin status (47% in R-0 and 21% in R-1; p < 0.001). Survival also correlated with margin status in the subgroups with neoCTX (p < 0.01) or without neoCTx (p < 0.01). In multivariate analysis of the entire group, hepatic margin status (RR 3.2; p < 0.001) and age > 65 years (RR 1.6; p < 0.01) were associated with poorer survival. In the subgroup of patients after neoCTX (n = 106), only the resection margin was an independent predictor of survival (p < 0.001). CONCLUSION: In patients with isolated colorectal liver metastases undergoing resection, the hepatic margin status was the strongest independent prognostic factor. This effect was also present after neoadjuvant chemotherapy for CLM.

Clinical Characteristics and Outcomes of Surgical Patients with Intensive Care Unit Lengths of Stay of 90 Days and Greater.

BACKGROUND: The aim of this study was to evaluate the influence of prolonged length of stay in an intensive care unit (ICU) on the mortality and morbidity of surgical patients. METHODS: We performed a monocentric and retrospective observational study in the surgical critical care unit of the department of surgery at the Medical Center of the University of Freiburg, Germany. Clinical data was collected from patients assigned to the ICU with a length of stay (LOS) of 90 days and greater. RESULTS: From the total of the 19 patients with ICU LOS over 90 days, ten patients died in the ICU whereas nine patients were discharged to the normal ward. The ICU mortality rate was 52%. The overall survival one year after ICU discharge was 32%. Regarding factors affecting mortality of the patients, significantly higher mortality was associated with age of the patients at the time point of the ICU admission and with postoperative need of renal replacement therapy. CONCLUSIONS: We found a high but in our opinion acceptable mortality rate in surgical patients with ICU LOS of 90 days and greater. We identified age and the need of renal replacement therapy as risk factors for mortality.

Survival outcome and prognostic factors after pancreatoduodenectomy for distal bile duct carcinoma: a retrospective multicenter study.

PURPOSE: Pancreatoduodenectomy is the most common operative procedure performed for distal bile duct carcinoma. Data on outcome after surgery for this rare malignancy is scarce, especially from western countries. The purpose of this study is to explore the prognostic factors and outcome after pancreatoduodenectomy for distal bile duct carcinoma. METHODS: Patients receiving pancreatoduodenectomy for distal bile duct carcinoma were identified from institutional databases of five German and one Russian academic centers for pancreatic surgery. Univariable and multivariable general linear model, Kaplan-Meier method, and Cox regression were used to identify prognostic factors for postoperative mortality and overall survival. RESULTS: N = 228 patients operated from 1994 to 2015 were included. Reoperation (OR 5.38, 95%CI 1.51-19.22, p = 0.010), grade B/C postpancreatectomy hemorrhage (OR 3.73, 95%CI 1.13-12.35, p = 0.031), grade B/C postoperative pancreatic fistula (OR 4.29, 95%CI 1.25-14.72, p = 0.038), and advanced age (OR 4.00, 95%CI 1.12-14.03, p = 0.033) were independent risk factors for in-hospital mortality in multivariable analysis. Median survival was 29 months, 5-year survival 27%. Positive resection margin (HR 2.07, 95%CI 1.29-3.33, p = 0.003), high tumor grade (HR 1.71, 95%CI 1.13-2.58, p = 0.010), lymph node (HR 1.68, 95%CI 1.13-2.51, p = 0.011), and distant metastases (HR 2.70, 95%CI 1.21-5.58, p = 0.014), as well as severe non-fatal postoperative complications (HR 1.64, 95%CI 1.04-2.58, p = 0.033) were independent negative prognostic factors for survival in multivariable analysis. CONCLUSION: Distant metastases and positive resection margin are the strongest negative prognostic factors for survival after pancreatoduodenectomy for distal bile duct carcinoma; thus, surgery with curative intent is only warranted in patients with local disease, where R0 resection is feasible.

The Prognostic Impact of the Carcinoembryonic Antigen in Ampullary Cancer - A Retrospective Single Center Study.

Background: Carcinoembryonic antigen cell adhesion molecule (CEA) is a commonly immunohistochemically used antibody in pathological routine diagnostics with an overexpression in different cancers. We aimed to examine the immunohistochemically detectable CEA level in ampullary cancer and to correlate it with clinico-pathological data. Methods: Shot-gun proteomics revealed CEA in undifferentiated ampullary cancer cell lines. Next, tumor tissue of 40 ampullary cancers of a retrospective single center cohort of 40 patients was stained immunohistochemically for CEA; CEA expression was determined and correlated with clinico-pathological data. Results: Thirty-six patient specimens were included in statistical analysis. CEA expression and lymph node ratio (LNR) were the only independent predictors of overall survival in multivariate analysis. Conclusion: To our knowledge, cell line and patient cohorts are the largest and characterized cohorts examined for CEA so far. Hereby, CEA expression in ampullary cancer cells permits an estimation of outcome and suggests an opportunity for individualized CEA-directed therapy. Further trials with larger cohorts are needed to verify our results and to integrate CEA immunohistochemistry into clinical routine.

Complications after pancreaticoduodenectomy are associated with higher amounts of intra- and postoperative fluid therapy: A single center retrospective cohort study.

BACKGROUND: Perioperative mortality after pancreaticoduodenectomy has decreased significantly in high-volume centers, but morbidity remains high. Restrictive perioperative fluid management may contribute to reduced complication rates after various surgical procedures. The aim of this study was to determine whether there is a correlation between the amount of fluid administered and postoperative complications. We hypothesized that higher amounts of intra- and total fluid is associated with greater postoperative morbidity. MATERIALS AND METHODS: We retrospectively examined data of 553 patients who underwent pancreaticoduodenectomy at University of Freiburg Medical Center between 2001 and 2013. Data on intra - and postoperative fluid administration (until postoperative day 5) were obtained from anesthesiological and surgical records. Data on complications were retrieved from our institutional pancreatic database. RESULTS: The median values for intra- and total fluid administered were 6000 ml (range 400-15,000 ml) and 13,600 ml (range 5000-57,700 ml), respectively. The overall in-hospital mortality was 1.9% (no correlation with fluid administration). Patients who received more than 6000 ml intraoperative fluid had more wound infections (P = 0.049), intra-abdominal abscesses (P = 0.020) and postoperative interventions (P = 0.007). In patients who received more than 14000 ml fluid until postoperative day 5 all evaluated types of postoperative complications (infectious, fistula, delayed gastric emptying, bleeding) and re-interventions occurred significantly more frequently than in patients who received less than 14,000 ml (P < 0.05-0.001). CONCLUSIONS: Higher amounts of fluids may contribute to postoperative complications. More studies are needed to adequately assess the use of intra/postop fluid therapy.

ATM mutations and E-cadherin expression define sensitivity to EGFR-targeted therapy in colorectal cancer.

EGFR-targeted therapy is a key treatment approach in patients with RAS wildtype metastatic colorectal cancers (CRC). Still, also RAS wildtype CRC may be resistant to EGFR-targeted therapy, with few predictive markers available for improved stratification of patients. Here, we investigated response of 7 CRC cell lines (Caco-2, DLD1, HCT116, HT29, LS174T, RKO, SW480) to Cetuximab and correlated this to NGS-based mutation profiles, EGFR promoter methylation and EGFR expression status as well as to E-cadherin expression. Moreover, tissue specimens of primary and/or recurrent tumors as well as liver and/or lung metastases of 25 CRC patients having received Cetuximab and/or Panitumumab were examined for the same molecular markers. In vitro and in situ analyses showed that EGFR promoter methylation and EGFR expression as well as the MSI and or CIMP-type status did not guide treatment responses. In fact, EGFR-targeted treatment responses were also observed in RAS exon 2 p.G13 mutated CRC cell lines or CRC cases and were further linked to PIK3CA exon 9 mutations. In contrast, non-response to EGFR-targeted treatment was associated with ATM mutations and low E-cadherin expression. Moreover, down-regulation of E-cadherin by siRNA in otherwise Cetuximab responding E-cadherin positive cells abrogated their response. Hence, we here identify ATM and E-cadherin expression as potential novel supportive predictive markers for EGFR-targeted therapy.

Postoperative fluid overload is a risk factor for adverse surgical outcome in patients undergoing esophagectomy for esophageal cancer: a retrospective study in 335 patients.

BACKGROUND: Restrictive intraoperative fluid management is increasingly recommended for patients undergoing esophagectomy. Controversy still exists about the impact of postoperative fluid management on perioperative outcome. METHODS: We retrospectively examined 335 patients who had undergone esophagectomy for esophageal cancer at the University Hospital Freiburg between 1996 and 2014 to investigate the relation between intra- and postoperative fluid management and postoperative morbidity after esophagectomy. RESULTS: Perioperative morbidity was 75%, the in-hospital mortality 8%. A fluid balance above average on the operation day was strongly associated with a higher rate of postoperative mortality (21% vs 3%, p < 0.001) and morbidity (83% vs 66%, p = 0.001). Univariate analysis for risk factors for adverse surgical outcome (Clavien ≥ III) identified ASA-score (p = 0.002), smoking (p = 0.036), reconstruction by colonic interposition (p = 0.036), cervical anastomosis (p = 0.017), blood transfusion (p = 0.038) and total fluid balance on the operation day and on POD 4 (p = 0.001) as risk factors. Multivariate analysis confirmed only ASA-score (p = 0.001) and total fluid balance (p = 0.001) as independent predictors of adverse surgical outcome. CONCLUSION: Intra- and postoperative fluid overload is strongly associated with increased postoperative morbidity. Our results suggest restrictive intra- and especially postoperative fluid management to optimize the outcome after esophagectomy.

Significant benefits in survival by the use of surgery combined with radiotherapy for retroperitoneal soft tissue sarcoma.

BACKGROUND: To report the effect of intraoperative electron beam radiotherapy (IOERT) and external beam radiotherapy (EBRT) in addition to surgery as well as to evaluate the role of resectable local recurrence for long-term prognosis. METHODS: In 53 patients who underwent surgery for retroperitoneal soft tissue sarcoma (RSTS) from 2001 to 2014 prognostic and epidemiologic factors were reviewed retrospectively to analyze their impact on survival and recurrence. RESULTS: Twenty three patients (50%) had surgery plus radiotherapy, 23 (50%) had surgery only. Histology showed 73.9% liposarcoma, 15.2% leiomyosarcoma and 6.5% pleomorphic undifferentiated sarcoma respectively. Low grade sarcoma were observed in 52.2%, high grade sarcoma in 47.8%. The latter showed a trend towards a decreased 5-year survival rate (p = 0.125). Margin status was: R0: 60.9%, R1: 23.9%, R2: 15.2%; leading to significant changes in 5-year survival rate (R0: 77.6%; R1: 70.0%; R2: 42.9%; p = 0.03). Age younger than 55 years significantly improved 5-year survival rate (p = 0.039). Patients receiving resection of multiple sarcoma recurrence showed an almost identical improved 5-year survival rate compared to patients without recurrence (no recurrence: 100.0%; single recurrence: 35.0%; multiple recurrence: 91.7%; p = 0.001). Surgery plus radiotherapy led to significantly improved survival (p = 0.04). CONCLUSIONS: There is a significant benefit in terms of 5-year survival after surgery plus some form of radiotherapy and a good prognosis for patients when the recurrence from RSTS was resected. Age older than 55 years and incomplete resection lowered 5-year survival rate significantly.

Histopathological tumor invasion of the mesenterico-portal vein is characterized by aggressive biology and stromal fibroblast activation.

BACKGROUND: Mesenterico-portal vein resection (PVR) during pancreatoduodenectomy for pancreatic head cancer was established in the 1990s and can be considered a routine procedure in specialized centers today. True histopathologic portal vein invasion is predictive of poor prognosis. The aim of this study was to examine the relationship between mesenterico-portal venous tumor infiltration (PVI) and features of aggressive tumor biology. METHODS: Patients receiving PVR for pancreatic ductal adenocarcinoma of the pancreatic head were identified from a prospectively maintained database. Immunohistochemical staining of tumor tissue was performed for the markers of epithelial-mesenchymal transition (EMT) E-Cadherin, Vimentin and beta-Catenin. Morphology of cancer-associated fibroblasts (CAFs) was assessed as inactive or activated. Statistical calculations were performed with MedCalc software. RESULTS: In total, 41 patients could be included. Median overall survival was 25 months. PVI was found in 17 patients (41%) and was significantly associated with loss of membranous E-Cadherin in tumor buds (p = 0.020), increased Vimentin expression (p = 0.03), activated CAF morphology (p = 0.046) and margin positive resection (p = 0.005). CONCLUSION: Our findings suggest that PVI is associated with aggressive tumor biology and disseminated growth less amenable to margin-negative resection.

Spatio-temporal mutation profiles of case-matched colorectal carcinomas and their metastases reveal unique de novo mutations in metachronous lung metastases by targeted next generation sequencing.

BACKGROUND: Targeted next generation sequencing (tNGS) has become part of molecular pathology diagnostics for determining RAS mutation status in colorectal cancer (CRC) patients as predictive tool for decision on EGFR-targeted therapy. Here, we investigated mutation profiles of case-matched tissue specimens throughout the disease course of CRC, to further specify RAS-status dynamics and to identify de novo mutations associated with distant metastases. METHODS: Case-matched formalin-fixed and paraffin-embedded (FFPE) resection specimens (n = 70; primary tumours, synchronous and/or metachronous liver and/or lung metastases) of 14 CRC cases were subjected to microdissection of normal colonic epithelial, primary and metastatic tumour cells, their DNA extraction and an adapted library protocol for limited DNA using the 48 gene TruSeq Amplicon Cancer PanelTM, MiSeq sequencing and data analyses (Illumina). RESULTS: By tNGS primary tumours were RAS wildtype in 5/14 and mutated in 9/14 (8/9 KRAS exon 2; 1/9 NRAS Exon 3) of cases. RAS mutation status was maintained in case-matched metastases throughout the disease course, albeit with altered allele frequencies. Case-matched analyses further identified a maximum of three sequence variants (mainly in APC, KRAS, NRAS, TP53) shared by all tumour specimens throughout the disease course per individual case. In addition, further case-matched de novo mutations were detected in synchronous and/or metachronous liver and/or lung metastases (e.g. in APC, ATM, FBXW7, FGFR3, GNAQ, KIT, PIK3CA, PTEN, SMAD4, SMO, STK11, TP53, VHL). Moreover, several de novo mutations were more frequent in synchronous (e.g. ATM, KIT, PIK3CA, SMAD4) or metachronous (e.g. FBXW7, SMO, STK11) lung metastases. Finally, some de novo mutations occurred only in metachronous lung metastases (CDKN2A, FGFR2, GNAS, JAK3, SRC). CONCLUSION: Together, this study employs an adapted FFPE-based tNGS approach to confirm conservation of RAS mutation status in primary and metastatic tissue specimens of CRC patients. Moreover, it identifies genes preferentially mutated de novo in late disease stages of metachronous CRC lung metastases, several of which might be actionable by targeted therapies.

Prognostic Role of Log Odds of Lymph Nodes After Resection of Pancreatic Head Cancer.

INTRODUCTION: Nodal status is a strong prognostic factor after resection of pancreatic cancer. The lymph node ratio (LNR) has been shown to be superior to the pN status in several studies. The role of log odds of the ratio between positive and negative nodes (LODDS) as a suggested new indicator of prognosis, however, has been hardly evaluated in pancreatic cancer. METHODS: Prognostic factors for overall survival after resection for cancer of the pancreatic head were evaluated in 409 patients from two institutions (prospectively maintained databases). The lymph node status, LNR, and LODDS were separately analyzed and independently compared in multivariate survival analysis. RESULTS: The median numbers of examined and positive lymph nodes were 16 and 2, respectively. Actuarial 3- and 5-year survival rates were 29 and 16 %. All three classifications of nodal disease significantly predicted survival in the entire group (n = 409), in patients with free resection margins (n = 297), and in patients with <12 examined nodes. In multivariate analysis, however, both LNR and LODDS were equally superior to the nodal status. In node-negative patients (n = 110), LODDS could not identify subgroups with different prognosis. CONCLUSION: Both LNR and LODDS are superior to the classical nodal status in predicting prognosis in resected pancreatic cancer. However, LODDS has not shown any advantage over LNR in our series, neither in the entire patient group nor in the subgroups with free margins, negative nodes or a low number of examined nodes. Therefore, the use of LODDS to predict the outcome after resection of pancreatic head cancer cannot be recommended.

Histone modifiers and marks define heterogeneous groups of colorectal carcinomas and affect responses to HDAC inhibitors in vitro.

Little is known about histone modifiers and histone marks in colorectal cancers (CRC). The present study therefore addressed the role of histone acetylation and histone deacetylases (HDAC) in CRCs in situ and in vitro. Immunohistochemistry of primary CRCs (n=47) revealed that selected histone marks were frequently present (H3K4me3: 100%; H3K9me3: 77%; H3K9ac: 75%), partially displayed intratumoral heterogeneity (H3K9me3; H3K9ac) and were significantly linked to higher pT category (H3K9me3: p=0.023; H3K9ac: p=0.028). Furthermore, also HDAC1 (62%), HDAC2 (100%) and HDAC3 (72%) expression was frequent, revealing four CRC types: cases expressing 1) HDAC1, HDAC2 and HDAC3 (49%), 2) HDAC2 and HDAC3 (30%), 3) HDAC1 and HDAC2 (10.5%) and 4) exclusively HDAC2 (10.5%). Correlation to clinico-pathological parameters (pT, pN, G, MSI status) revealed that heterogeneous HDAC1 expression correlated with lymph node status (p=0.012). HDAC expression in situ was partially reflected by six CRC cell lines, with similar expression of all three HDACs (DLD1, LS174T), preferential HDAC2 and HDAC3 expression (SW480, Caco2) or lower HDAC2 and HDAC3 expression (HCT116, HT29). HDAC activity was variably higher in HCT116, HT29, DLD1 and SW480 compared to LS174T and Caco2 cells. Treatment with broad (SAHA) and specific (MS-275; FK228) HDAC inhibitors (HDACi) caused loss of cell viability in predominantly MSIpositive CRC cells (HCT116, LS174T, DLD1; SAHA, MS-275 and in part FK228). In contrast, MSI-negative CRC cells (Caco2, HT29, SW480) were resistant, except for high doses of FK228 (Caco2, HT29). Cell viability patterns were not linked to different efficacies of HDACi on reduction of HDAC activity or histone acetylation, p21 expression and/or induction of DNA damage (γH2A-X levels). In summary, this study reveals inter- and intra-tumoral heterogeneity of histone marks and HDAC expression in CRCs. This is reflected by diverse HDACi responses in vitro, which do not follow known modes of action. Together, this implies further exploitation of histone alterations in CRC for molecular classification and/or novel treatment options.

Characterization of various cell lines from different ampullary cancer subtypes and cancer associated fibroblast-mediated responses.

BACKGROUND: Ampullary cancer is a relatively rare form of cancer and usually treated by pancreatoduodenectomy, followed by adjuvant therapy. The intestinal subtype is associated with markedly improved prognosis after resection. At present, only few cell lines are available for in vitro studies of ampullary cancer and they have not been collectively characterized. METHODS: We characterize five ampullary cancer cell lines by subtype maker expression, epithelial-mesenchymal transition (EMT) features, growth and invasion, drug sensitivity and response to cancer-associated fibroblast conditioned medium (CAF-CM). RESULTS: On the basis of EMT features, subtype marker expression, growth, invasion and drug sensitivity three types of cell lines could be distinguished: mesenchymal-like, pancreatobiliary-like and intestinal-like. Heterogeneous effects from the cell lines in response to CAF-CM, such as different growth rates, induction of EMT markers as well as suppression of intestinal differentiation markers were observed. In addition, proteomic analysis showed a clear difference in intestinal-like cell line from other cell lines. CONCLUSION: Most of the available AMPAC cell lines seem to reflect a poorly differentiated pancreatobiliary or mesenchymal-like phenotype, which is consistent to their origin. We suggest that the most appropriate cell line model for intestinal-like AMPAC is the SNU869, while others seem to reflect aggressive AMPAC subtypes.