Cytokine/Chemokine Release Patterns and Transcriptomic Profiles of LPS/IFNγ-Activated Human Macrophages Differentiated with Heat-Killed Mycobacterium obuense, M-CSF, or GM-CSF.

Macrophages (Mφs) are instrumental regulators of the immune response whereby they acquire diverse functional phenotypes following their exposure to microenvironmental cues that govern their differentiation from monocytes and their activation. The complexity and diversity of the mycobacterial cell wall have empowered mycobacteria with potent immunomodulatory capacities. A heat-killed (HK) whole-cell preparation of Mycobacterium obuense (M. obuense) has shown promise as an adjunctive immunotherapeutic agent for the treatment of cancer. Moreover, HK M. obuense has been shown to trigger the differentiation of human monocytes into a monocyte-derived macrophage (MDM) type named Mob-MDM. However, the transcriptomic profile and functional properties of Mob-MDMs remain undefined during an activation state. Here, we characterized cytokine/chemokine release patterns and transcriptomic profiles of lipopolysaccharide (LPS)/interferon γ (IFNγ)-activated human MDMs that were differentiated with HK M. obuense (Mob-MDM(LPS/IFNγ)), macrophage colony-stimulating factor M-MDM(LPS/IFNγ)), or granulocyte/macrophage colony-stimulating factor (GM-MDM(LPS/IFNγ)). Mob-MDM(LPS/IFNγ) demonstrated a unique cytokine/chemokine release pattern (interleukin (IL)-10low, IL-12/23p40low, IL-23p19/p40low, chemokine (C-x-C) motif ligand (CXCL)9low) that was distinct from those of M-MDM(LPS/IFNγ) and GM-MDM(LPS/IFNγ). Furthermore, M-MDM(LPS/IFNγ) maintained IL-10 production at significantly higher levels compared to GM-MDM(LPS/IFNγ) and Mob-MDM(LPS/IFNγ) despite being activated with M1-Mφ-activating stimuli. Comparative RNA sequencing analysis pointed to a distinct transcriptome profile for Mob-MDM(LPS/IFNγ) relative to both M-MDM(LPS/IFNγ) and GM-MDM(LPS/IFNγ) that comprised 417 transcripts. Functional gene-set enrichment analysis revealed significant overrepresentation of signaling pathways and biological processes that were uniquely related to Mob-MDM(LPS/IFNγ). Our findings lay a foundation for the potential integration of HK M. obuense in specific cell-based immunotherapeutic modalities such as adoptive transfer of Mφs (Mob-MDM(LPS/IFNγ)) for cancer treatment.

Perturbations in Mitochondrial Dynamics Are Closely Involved in the Progression of Alcoholic Liver Disease.

BACKGROUND: Mitochondria play a fundamental role in the pathogenesis of alcoholic liver disease (ALD). The preservation of functional mitochondria during toxic alcohol insults is essential for cell survival and is maintained by key processes known as mitochondrial dynamics, including fragmentation and fusion, which are regulated by mitochondria-shaping proteins (MSP). We have shown mitochondrial dynamics to be distorted by alcohol in cellular and animal models, but the effect in humans remains unknown. METHODS: Hepatic gene expression of the main MSP involved in the mitochondrial fusion and fragmentation pathways was evaluated in patients with alcoholic hepatitis (AH) by DNA microarray (n = 15) and Reverse Transcription Polymerase Chain Reaction (n = 32). The activation of dynamin-1-like protein (Drp1) was also investigated in mitochondria isolated from liver biopsies of ALD patients (n = 8). The effects of alcohol on mitochondrial dynamics and on MSP protein expression were studied in human precision-cut liver slices (PCLS) exposed for 24 hours to increasing doses of ethanol (EtOH; 50 to 250 mM). RESULTS: A profound hyperactivation of the fragmentation pathway was observed in AH patients, with a significant increase in the expression of Drp1 and its adapters/receptors. The translocation of Drp1 to the mitochondria was also induced in patients with severe ALD and was affected in the PCLS with short-term exposure to EtOH but only mildly. The fusion pathway was not altered in ALD, and this was confirmed in the PCLS model. CONCLUSIONS: The present study reveals the role of mitochondrial dynamics in human ALD, confirming our previous observations in animal and cell culture models of ALD. Taken together, we show that alcohol has a significant impact on the fragmentation pathway, and we confirm Drp1 as a potential therapeutic target in severe ALD.

Enhanced effect of checkpoint inhibitors when given after or together with IMM-101: significant responses in four advanced melanoma patients with no additional major toxicity.

BACKGROUND: The use of checkpoint inhibitors (ipilimumab, pembrolizumab, nivolumab) has revolutionised the treatment of metastatic melanoma. However still more than the half the patients do not respond to single-agent immunotherapy. This has led to the development of combining these agents in an attempt to enhance the anti-cancer activity. More than 300 different studies with 15 different drug doses are currently ongoing. Combining different checkpoint inhibitors (CPIs) does indeed lead to an increase in response rate, but this is associated with significant toxicity. IMM-101 is a heat killed Mycobacterium preparation which induces marked immune modulation and little systemic toxicity. It has been reported as having activity in melanoma as single agent and in pancreatic cancer in combination with gemcitabine, the latter in a randomised study. METHODS: Here we report the effect of adding CPIs to 3 patients who had previously been on IMM-101, either as a trial or a named patient programme and a patient who received the IMM-101 together with nivolumab. RESULTS: All 4 patients had rapid and very good responses, three of them maintained over 18 months with no significant additional toxicity. CONCLUSIONS: The rapid and complete clinical responses seen in these patients may suggest that IMM-101 is activating a complementary pathway which is synergistic with CPI treatment.

Increased carcinoembryonic antigen (CEA) following neoadjuvant chemotherapy predicts poor prognosis in patients that undergo hepatectomy for liver-only colorectal metastases.

BACKGROUND: The importance of preoperative chemotherapy in a multimodality management of patients with colorectal liver metastases (CRLM) has been demonstrated. We analyse the carcinoembryonic antigen (CEA) changes following neoadjuvant chemotherapy in patients with CRLM who underwent liver resection. METHODS: The final cohort included 107 eligible patients. Increased CEA levels following neoadjuvant chemotherapy were defined as the increase of baseline CEA level at diagnosis of CRLM compared with the CEA level after completion of neoadjuvant chemotherapy. Disease-free survival (DFS), post-recurrence survival (PRS) and overall survival (OS) were calculated using both Kaplan-Meier and multivariate Cox-regression methods. RESULTS: CEA increase was associated with decreased PRS and OS (HR 2.69; 95 % CI, 1.28-5.63; p = 0.009, and HR 2.50; 95 % CI, 1.12-5.56; p = 0.025, respectively) in multivariate analysis, but there was no association between CEA changes and DFS. CEA increase was only associated with disease progression during preoperative chemotherapy (p = 0.014). Interestingly, this association was not absolute, as only 5 of the 11 patients with disease progression demonstrated CEA increase. Regarding the remaining 12 patients with CEA increase, according to RECIST criteria, eight patients demonstrated partial response and four patients stable disease. CONCLUSION: In this study, we demonstrated the CEA increase following neoadjuvant chemotherapy as an adverse prognostic factor for PRS, and OS but not for DFS in patients undergoing liver resection for liver-only colorectal metastases.

Randomised, open-label, phase II study of gemcitabine with and without IMM-101 for advanced pancreatic cancer.

BACKGROUND: Immune Modulation and Gemcitabine Evaluation-1, a randomised, open-label, phase II, first-line, proof of concept study (NCT01303172), explored safety and tolerability of IMM-101 (heat-killed Mycobacterium obuense; NCTC 13365) with gemcitabine (GEM) in advanced pancreatic ductal adenocarcinoma. METHODS: Patients were randomised (2 : 1) to IMM-101 (10 mg ml(-l) intradermally)+GEM (1000 mg m(-2) intravenously; n=75), or GEM alone (n=35). Safety was assessed on frequency and incidence of adverse events (AEs). Overall survival (OS), progression-free survival (PFS) and overall response rate (ORR) were collected. RESULTS: IMM-101 was well tolerated with a similar rate of AE and serious adverse event reporting in both groups after allowance for exposure. Median OS in the intent-to-treat population was 6.7 months for IMM-101+GEM v 5.6 months for GEM; while not significant, the hazard ratio (HR) numerically favoured IMM-101+GEM (HR, 0.68 (95% CI, 0.44-1.04, P=0.074). In a pre-defined metastatic subgroup (84%), OS was significantly improved from 4.4 to 7.0 months in favour of IMM-101+GEM (HR, 0.54, 95% CI 0.33-0.87, P=0.01). CONCLUSIONS: IMM-101 with GEM was as safe and well tolerated as GEM alone, and there was a suggestion of a beneficial effect on survival in patients with metastatic disease. This warrants further evaluation in an adequately powered confirmatory study.

Two cases of gallbladder metastasis from renal cell carcinoma and review of literature.

BACKGROUND: Renal cell carcinoma accounts for 90% of renal neoplasms and metastatic disease is common. One third of newly diagnosed cases will have synchronous metastases at diagnosis and further 25-50 % will develop metachronous disease. CASE PRESENTATION: This study presents two new cases of gallbladder metastasis from renal cell carcinoma (RCC) from our institution and reviews the published literature. The final cohort included 52 evaluable patients. M/F ratio was 2:1 and median age was 62.5 years. Most patients were diagnosed incidentally after follow-up or staging imaging for RCC. Of the patients with available histology, all except one were clear cell type (n = 39) and 92% were polypoid. Thirty-six patients demonstrated metachronous gallbladder metastasis with median disease-free interval (DFI) from nephrectomy of 4 years. The most frequent site of metastasis was the contralateral kidney (46.7%) followed by the pancreas and lung. The median disease-free interval (DFS) after cholecystectomy was 37 months. Three- and five-year OS rates were 74 and 62%, respectively. Age younger than 45 years (p = 0.008) and DFI <24 months (p = 0.049) were associated with decreased OS. CONCLUSIONS: RCC metastasis to the gallbladder is associated with an unusual pattern of concomitant metastasis. Symptoms are not common. Simple cholecystectomy is associated with increased OS and nil local or port site recurrence. Young age and short DFI are associated with decreased OS.

The Preoperative Lymphocyte-to-Monocyte Ratio is Prognostic of Clinical Outcomes for Patients with Liver-Only Colorectal Metastases in the Neoadjuvant Setting.

BACKGROUND: Circulating monocyte-derived, tumor-associated macrophages are associated with a poor prognosis for various cancers. Conversely, circulating lymphocytes are the source of tumor-infiltrating lymphocytes, which are associated with an improved prognosis. This study evaluated the prognostic value of the preoperative blood lymphocyte-to-monocyte ratio (LMR) for patients undergoing hepatectomy for liver-only colorectal metastases. METHODS: This retrospective study examined 140 consecutive patients with liver-only colorectal metastases. Disease-free survival (DFS), post-recurrence survival (PRS), cancer-specific survival (CSS), and overall survival (OS) were analyzed in relation to LMR values using both Kaplan-Meier and multivariate Cox-regression methods. RESULTS: In the multivariate analysis, high LMR (>3) was significantly associated with increased OS [hazard ratio (HR), 2.43; 95 % confidence interval (CI), 1.32-4.48; P = 0.004], CSS (HR 2.15; 95 % CI 1.13-4.10; P = 0.020), and PRS (HR 2.15; 95 % CI 1.15-4.01; P = 0.016) but not with DFS. An LMR lower than 3 may have been associated with decreased CSS and PRS by increasing the rate of multifocal recurrence (P = 0.063). In the multivariate analysis comparing LMR, the neutrophil-lymphocyte ratio, and the platelet-lymphocyte ratio, LMR remained the only significant prognostic variable of CSS. CONCLUSION: This study identified preoperative LMR as an independent prognostic factor for PRS, CSS, and OS but not for DFS in patients undergoing hepatectomy for liver-only colorectal metastases. In the future, interventions to augment immune function could improve survival for low-LMR patients.

Neutrophil to lymphocyte ratio predicts pattern of recurrence in patients undergoing liver resection for colorectal liver metastasis and thus the overall survival.

OBJECTIVE: We investigate the neutrophil to lymphocyte ratio (NLR) as a potential prognostic factor for patients undergoing curative liver resection for colorectal liver metastasis (CRLM). METHODS: We identified patients who underwent liver resection, via our prospectively accumulated surgical database at the Royal Marsden Hospital and The London Clinic, by two liver surgeons (SM and AZK) between January 2005 and December 2012 and patients included had liver resection for CRLM and received preoperative chemotherapy with an NLR > 2.5 considered elevated. NLR's role in disease-free, post-recurrence and overall survival was determined by univariate and multivariate Cox regression models. RESULTS: One hundred and sixty nine patients were enrolled. Seventy-one patients (42%) demonstrated NLR > 2.5. Elevated NLR was associated with decreased OS in univariate and multivariate analysis (HR 2.12; 95% CI, 1.18-3.82; P = 0.012) but not with DFS. Analyzed as continuous variable, higher NLR was associated with decreased OS(HR 1.17; 95% CI, 1.03-132; P = 0.011) and associated with increased risk of extrahepatic/multifocal recurrence (P = 0.007), linked in this way with a decreased post-recurrence survival (HR 1.24, 95% CI, 1.02-1.52; P = 0.032). CONCLUSIONS: Elevated NLR in patients who undergo hepatectomy following neoadjuvant chemotherapy for CRLM increases risk of extrahepatic/multifocal recurrence and is an independent predictor of overall survival.

A case of metastatic pancreatic adenocarcinoma with prolonged survival after combination of neoadjuvant FOLFIRINOX therapy and synchronous distal pancreatectomy and hepatectomy.

In this case report we detail the treatment of a patient with pancreatic ductal adenocarcinoma and a solitary liver metastasis who received nine cycles of FOLFIRINOX therapy with favourable response. The patient subsequently underwent synchronous distal pancreatectomy and hepatectomy with an R0 resection followed by three further cycles of FOLFIRINOX. At the last follow-up, 2 years from operation and 28 months from the diagnosis of metastatic pancreatic adenocarcinoma the patient remains disease free.

CALy Prognostic Score (CEA, ALP, Lymphocyte Count) as a predictor of survival after hepatectomy for liver-only colorectal metastases.

PURPOSE: The discovery of prognostic factors for patients who undergo hepatectomy for colorectal liver metastases (CRLM) in the era of neoadjuvant chemotherapy is imperative. This study aimed to establish a simple, cheap and easily available prognostic score for these patients. METHODS: Preoperative carcinoembryonic antigen (CEA), serum alkaline phosphatase (ALP), and lymphocyte count (LC) were used for the establishment of a prognostic score (CALy PS). The cut-off levels of these variables were determined by applying receiver operating curve (ROC) analysis. The final prognostic score assigned one risk point for each variable (CEA>4 µg/L, ALP>93 U/L, and LC≥1.6x10(9)/L). RESULTS: One hundred and thirty-five patients were included. Two risk categories were established with 0-1 and 2-3 points, respectively. CALy 0-1 vs CALy 2-3, and CALy 2-3 were associated with decreased disease free survival (DFS) and overall survival (OS) both in univariate and multivariate analysis (DFS: HR 1.84; 95% CI 1.18-2.86; p=0.007; OS: HR 2.25; 95% CI 1.23-4.11; p=0.008). When four risk categories were established with 0,1,2,and 3 points,CALy was again associated with decreased DFS and OS both in univariate and in multivariate analysis (DFS: HR 1.37; 95% CI 1.083-1.74; p=0.009; OS:HR 1.84; 95% CI 1.31-2.59; p<0.001). Three-year DFS rates for these categories (CALy 0, CALy 1, CALy 2, and CALy 3) were 45, 38, 15 and 7%, respectively, and the 5-year OS rates were 78, 68, 32, and 24%, respectively. CONCLUSION: This simple, cheap, and easily available risk score provides good prognostic accuracy for both DFS and OS for patients undergoing liver resection for liver-only colorectal metastases after neoadjuvant chemotherapy.

Role of the eIF4E binding protein 4E-BP1 in regulation of the sensitivity of human pancreatic cancer cells to TRAIL and celastrol-induced apoptosis.

BACKGROUND INFORMATION: Tumour cells can be induced to undergo apoptosis after treatment with the tumour necrosis factor α-related death-inducing ligand (TRAIL). Although human pancreatic cancer cells show varying degrees of response they can be sensitised to the pro-apoptotic effects of TRAIL in the presence of celastrol, a natural compound extracted from the plant Tripterygium wilfordii Hook F. One important aspect of the cellular response to TRAIL is the control of protein synthesis, a key regulator of which is the eukaryotic initiation factor 4E-binding protein, 4E-BP1. RESULTS: We examined the effects of celastrol and TRAIL in several pancreatic cancer cell lines. In cells that are normally resistant to TRAIL, synergistic effects of TRAIL plus celastrol on commitment to apoptosis and inhibition of protein synthesis were observed. These were associated with a strong up-regulation and dephosphorylation of 4E-BP1. The enhancement of 4E-BP1 expression, which correlated with a threefold increase in the level of the 4E-BP1 transcript, was blocked by inhibitors of reactive oxygen species and the JNK protein kinase. When the expression of 4E-BP1 was reduced by an inducible micro-RNA, TRAIL-mediated apoptosis was inhibited. CONCLUSION: These results suggest that 4E-BP1 plays a critical role in the mechanism by which TRAIL and celastrol together cause apoptotic cell death in human pancreatic tumour cells.

Surgical management of intraabdominal metastases from melanoma: role of the neutrophil to lymphocyte ratio as a potential prognostic factor.

BACKGROUND: The aims of this study were to evaluate the role of surgery in the management of patients with intraabdominal metastases from melanoma and to investigate the neutrophil to lymphocyte ratio (NLR) as prognostic factor in this group of patients. METHODS: Altogether, 44 patients who underwent surgery for Intraabdominal metastases from melanoma with curative, cytoreductive, or palliative intent were analyzed. RESULTS: There were 77 intraabdominal organ resections performed during the 44 operations. R0 resection was achieved in 19 (43 %) cases. Factors associated with R0 resection were an absence of extra-abdominal metastases, low serum lactate dehydrogenase, involvement of fewer than three sites, and the presence of fewer than three metastatic lesions. The 1-, 3-, and 5-year overall survival rates were, respectively, 79, 66, and 44 % in the curative intent group; 36, 18, and 9 % in the cytoreduction group; and 21, 0 and 0 % in the palliation group (curative intent vs. cytoreduction vs. palliation p < 0.001). By Cox's multivariate analysis, the independent prognostic factors were time from excision of primary melanoma to the diagnosis of intraabdominal metastases, NLR, and residual disease after surgery. CONCLUSIONS: Our results confirm the usefulness of major surgical interventions as reported in previous studies. We reviewed recent evidence that immunologic phenomena may explain the unexpectedly good response rate in patients with advanced disease. The simple estimation of the NLR has been advocated as a prognostic marker for several cancers. We show that it is likewise useful in metastatic melanoma. We stress the need for developing additional immunologic markers.

Prognostic factors for primary gastrointestinal stromal tumours: are they the same in the multidisciplinary treatment era?

PURPOSE: Gastrointestinal stromal tumours (GISTs) are the most common mesenchymal tumour of the gastrointestinal tract. The introduction of tyrosine kinase inhibitors (TKIs) has lead to increasing use of combination of medical and surgical therapy. The aim of this study was to look at outcomes from a series of surgically treated GISTs and determine prognostic factors in the context of multimodal therapy. METHODS: We analysed 104 single surgeon's patients with GIST. End points of the study were disease-specific survival (DSS), disease-free survival (DFS) and post-operative complications. RESULTS: Three- and 5-year DSS rates were 96.7 and 94.6 %. On univariate analysis, clear resection margins were predictive of DSS. Patients with R2 resection had a worse prognosis (3-year DSS rate of 83.3 %; 5-year DSS rate of 62.5 %) compared to patients with R0 (3-year DSS rate of 98 %; 5-year DSS rate of 98 %) or R1 resection (3-year DSS rate of 100 %; 5-year DSS rate of 100 %) (R0 vs R1 vs. R2 p = 0.001). Pre-operative factors associated with R2 resection were clinical metastatic disease (p < 0.001), non-gastric tumour site (p = 0.002) and large tumour diameter (p = 0.031). Three- and 5-year DFS rates were 65.5 and 59.8 %. Serosal perforation (p = 0.013) and mitotic rate (p = 0.05) were found to be independently predictive of increased DFS. The presence of serosal perforation was associated with tumour site (p = 0.018), mitotic rate (p = 0.035), tumour diameter (p < 0.001), growth pattern (p = 0.007) and age (p = 0.040). CONCLUSIONS: In the multidisciplinary management of GIST, serosal perforation may represent an additional predictor of recurrence along with mitotic rate. Complete macroscopic surgical resection is the most reliable prognostic factor, and an aggressive surgical approach should be advocated.

Combined ultrasonic aspiration and saline-linked radiofrequency precoagulation: a step toward bloodless liver resection without the need of liver inflow occlusion: analysis of 313 consecutive patients.

BACKGROUND: Hemorrhage is undoubtedly one of the main factors contributing to morbidity and mortality in liver resections. Vascular occlusion techniques are effective in controlling intraoperative bleeding, but they cause liver damage due to ischemia. We evaluated the effectiveness and safety of using a combined technique for hepatic parenchymal transection without liver inflow occlusion. METHODS: Three hundred and thirteen consecutive patients who underwent liver resection in four hepato-pancreato-biliary units. Hepatic parenchymal transection was carried out using a combined technique of saline-linked radiofrequency precoagulation and ultrasonic aspiration without liver inflow occlusion. RESULTS: During the study period 114 minor and 199 major hepatic resections were performed. The mean amount of intraoperative blood loss was 377 ml (SD 335 ml, range 50 to 2,400 ml) and the blood transfusion rate was 10.5%. The median amount of blood loss during parenchymal transection and parenchymal transection time was 222 ml (SD 224 ml, range 40 to 2,100 ml) and 61 minutes (range 12 to 150 minutes) respectively. There were two postoperative deaths (0.6%). Complications occurred in 84 patients (26.8%) and most complications were minor. CONCLUSIONS: Combined technique of saline-linked radiofrequency ablation and ultrasonic aspiration for liver resection is a safe method for both major and minor liver resections. The method is associated with decreased blood loss, reduced postoperative morbidity, and minimal mortality rates. We believe that this combined technique is comparable to other techniques and should be considered as an alternative.

"Chronic" metastatic pancreatic acinar cell carcinoma.

Acinar cell carcinoma (ACC) of the pancreas is a rare exocrine tumour for which there is very limited information about chemotherapy regimens and prognosis. Even though there are clinical guidelines for management of ductal cell carcinoma, a definitive and specific regime has not yet been agreed for this type of pancreatic cancer. We report a case of metastatic ACC of pancreas who has been treated with a multimodal approach, including novel combinations of different targeted drugs with conventional chemotherapy, surgery and radiofrequency ablation since the last 11 years. This degree of long term survival has not been reported so far in such a case of metastatic ACC of the pancreas. This case highlights the importance of a personalised multidisciplinary therapeutic strategy, employing locoregional therapies along with combinations of established and novel systemic therapies to control the disease, and the importance of flexibility when instigating new treatment paradigms for progressive cancer. Also, this case demonstrates that complete tumour eradication may not be the sole purpose of surgical oncology.

Splenectomy for non-haematological metastatic malignant disease.

PURPOSE: Multiple cases and small series of patients who have undergone splenectomy for metastatic malignant disease have been reported. This study examines the outcome of patients with metastatic malignant disease to the spleen treated by splenectomy at a tertiary oncology centre and a review of cases published in the last 10 years. METHODS: The hospital histopathology database was searched over a 25-year period up to 2004 for patients who had undergone splenectomy for non-haematological malignancy. Medical records of these patients were reviewed and clinical course was examined. The literature review was undertaken using a search of PubMed for the terms "splenectomy" and "metastasis" from 2000 to 2010. RESULTS: Twenty-one cases at our institution were identified. The most common primary site of malignancy was ovary (nine cases), followed by malignant melanoma (three) and pancreas (three). There were two cases of metastatic disease from colonic primary and one each from renal, breast, nasopharyngeal and unknown primary disease. There were two cases of long-term disease-free survival (both primary ovarian tumours) and four cases of patients who survived more than 4 years but had disease recurrence (ovarian and colonic primaries). The literature review provided a further 115 cases. CONCLUSIONS: More favorable outcomes were seen in patients with metachronous disease. There was a trend to improved outcome in ovarian and colorectal primaries over malignant melanoma. It is postulated that improved outcome may be seen in patients for whom there were effective adjuvant chemotherapeutic options, low probability of other metastatic disease and less aggressive tumour biology. However, frequently the presentation is indicative of aggressive widespread disease with a poor prognosis.

Clinical utility of circulating tumor cell measurement in the diagnosis of indeterminate lesions of the pancreas.

The quantification of circulating tumor cells has been historically problematic due to the different methods applied to their measurement. Following the development of standardized technology, they are now becoming well-established prognostic and predictive markers in patients with breast, colon and prostate cancer. While they represent a real-time noninvasive test, their use in diagnostics has seldom been reported. We report their use to help diagnose an indeterminate pancreatic mass. The use of an automated circulating tumor cell platform as described is likely to have utility as an aid to differential diagnosis, although larger studies will be required to ascertain its positive or negative predictive value.

Dissecting the stress activating signaling pathways in acute pancreatitis.

BACKGROUND/AIMS: Acute pancreatitis is a severe and frequently a life-threatening disease which can lead to pancreatic necrosis, acute lung injury, SIRS and MODS. The pathophysiologic pattern of acute pancreatitis is being investigated, when guidelines for treatment are being modified often and novel therapeutic strategies have failed to show real benefit in clinical practice. METHODOLOGY: The present paper reviews the role of nuclear factors NF-kappaB and AP-1, TNFalpha, and TLR-4 in acute pancreatitis. CONCLUSION: Understanding the inflammatory mediators expression, regulation of apoptosis and dissecting the stress activating signaling pathways in acute pancreatitis, is of paramount importance in order to achieve in the near future adequate therapeutic interventions.

Synergistic activity of agents targeting growth factor receptors, CDKs and downstream signaling molecules in a panel of pancreatic cancer cell lines and the identification of antagonistic combinations: Implications for future clinical trials in pancreatic cancer.

Pancreatic cancer is one of the most aggressive, heterogeneous and fatal type of human cancers for which more effective therapeutic agents are urgently needed. Here, we investigated the sensitivity of a panel of seven human pancreatic cancer cell lines (HPCCLs) to treatment with various tyrosine kinase inhibitors (TKIs), cyclin­dependent kinase (CDK) inhibitors, an inhibitor of STAT3 stattic, and a cytotoxic agent gemcitabine both as single agents and in combination. The membranous expression of various receptors and the effect of selected agents on cell cycle distribution, cell signaling pathways and migration was determined using flow cytometry, western blot analysis and scratch wound healing assays, respectively. While the expression of both HER­3 and HER­4 was low or negative, the expression of EGFR and HER2 was high or intermediate in all HPCCLs. Of all the agents examined, the CDK1/2/5/9 inhibitor, dinacicilib, was the most potent agent which inhibited the proliferation of all seven HPCCLs with IC50 values of ≤10 nM, followed by SRC targeting TKI dasatinib (IC50 of ≤258 nM), gemcitabine (IC50 of ≤330 nM), stattic (IC50 of ≤2 µM) and the irreversible pan­HER TKI afatinib (IC50 of ≤2.95 µM). Treatment with afatinib and dasatinib inhibited the ligand­induced phosphorylation of EGFR and SRC respectively. Statistically significant associations were found between HER2 expression and response to treatment with the ALK/IGF­IR/InsR inhibitor ceritinib and fibroblast growth factor receptor (FGFR)1/2/3 inhibitor AZD4547, HER3 and IGF­IR expression and their response to treatment with TKIs targeting HER family members (erlotinib and afatinib), and c­MET and ALK7 expression and their response to treatment with stattic. Interestingly, treatment with a combination of afatinib with dasatinib and gemcitabine with dasatinib resulted in synergistic tumor growth inhibition in all HPCCLs examined. In contrast, the combination of afatinib with dinaciclib was found to be antagonistic. Finally, the treatment with afatinib, dasatinib and dinaciclib strongly inhibited the migration of all HPCCLs examined. In conclusion, the CDK1/2/5/9 inhibitor dinaciclib, irreversible pan­HER TKI afatinib and SRC targeting TKI dasatinib were most effective at inhibiting the proliferation and migration of HPCCLs and the combination of afatinib with dasatinib and gemcitabine with dasatinib led to synergistic tumor growth inhibition in all HPCCLs examined. Our results support further investigation on the therapeutic potential of these combinations in future clinical trials in pancreatic cancer.