Gemcitabine diphosphate choline is a major metabolite linked to the Kennedy pathway in pancreatic cancer models in vivo

Background:

The modest benefits of gemcitabine (dFdC) therapy in patients with pancreatic ductal adenocarcinoma (PDAC) are well documented, with drug delivery and metabolic lability cited as important contributing factors. We have used a mouse model of PDAC: KRAS^G12D; p53^R172H; pdx-Cre (KPC) that recapitulates the human disease to study dFdC intra-tumoural metabolism.

Methods:

LC-MS/MS and NMR were used to measure drug and physiological analytes. Cytotoxicity was assessed by the Sulphorhodamine B assay.

Results:

In KPC tumour tissue, we identified a new, Kennedy pathway-linked dFdC metabolite (gemcitabine diphosphate choline (GdPC)) present at equimolar amounts to its precursor, the accepted active metabolite gemcitabine triphosphate (dFdCTP). Utilising additional subcutaneous PDAC tumour models, we demonstrated an inverse correlation between GdPC/dFdCTP ratios and cytidine triphosphate (CTP). In tumour homogenates in vitro, CTP inhibited GdPC formation from dFdCTP, indicating competition between CTP and dFdCTP for CTP:phosphocholine cytidylyltransferase (CCT). As the structure of GdPC precludes entry into cells, potential cytotoxicity was assessed by stimulating CCT activity using linoleate in KPC cells in vitro, leading to increased GdPC concentration and synergistic growth inhibition after dFdC addition.

Conclusions:

GdPC is an important element of the intra-tumoural dFdC metabolic pathway in vivo.     

Ascites-derived pancreatic ductal adenocarcinoma primary cell cultures as a platform for personalised medicine

Background:

Challenges in developing drugs for pancreatic ductal adenocarcinoma (PDAC) include obtaining metastatic cancer tissue for research and validating biomarkers predicative for personalised therapeutic decisions. We have recently developed a novel therapeutic model for PDAC to address these challenges based on the isolation of viable PDAC cells derived from ascites fluid.

Methods:

Ascites fluid was obtained from PDAC patients undergoing palliative paracentesis. Ascites-derived PDAC primary cells were isolated, cultured and characterised in ovo and in vitro.

Results:

We successfully established ascites-derived primary cell cultures within 2–7 days from 92% (93 out of 101) of the ascites fluid samples obtained (from 36 different patients). Homogeneous epithelial PDAC-enriched cell cultures were identified and characterised. We observed a wide range in doubling times and migration properties among the different patient-derived cell cultures. The diverse nature of each individual patient''s cell cultures was further demonstrated by differences in therapeutic susceptibility and resistance. The tumorigenicity and invasiveness of the cells were demonstrated in vivo using chicken chorioallantoic membrane grafts.

Conclusions:

We have developed a unique ascites-derived PDAC primary cell culture model. This model has the potential to study signalling pathways in PDAC progression and to evaluate targeted therapies for the individual patient expeditiously, thereby supporting personalised treatment decisions.     

hERG1 channels drive tumour malignancy and may serve as prognostic factor in pancreatic ductal adenocarcinoma

Background:

hERG1 channels are aberrantly expressed in human cancers. The expression, functional role and clinical significance of hERG1 channels in pancreatic ductal adenocarcinoma (PDAC) is lacking.

Methods:

hERG1 expression was tested in PDAC primary samples assembled as tissue microarray by immunohistochemistry using an anti-hERG1 monoclonal antibody (α-hERG1-MoAb). The functional role of hERG1 was studied in PDAC cell lines and primary cultures. ERG1 expression during PDAC progression was studied in Pdx-1-Cre,LSL-Kras^G12D/+,LSL-Trp53^R175H/+ transgenic (KPC) mice. ERG1 expression in vivo was determined by optical imaging using Alexa-680-labelled α-hERG1-MoAb.

Results:

(i) hERG1 was expressed at high levels in 59% of primary PDAC; (ii) hERG1 blockade decreased PDAC cell growth and migration; (iii) hERG1 was physically and functionally linked to the Epidermal Growth Factor-Receptor pathway; (iv) in transgenic mice, ERG1 was expressed in PanIN lesions, reaching high expression levels in PDAC; (v) PDAC patients whose primary tumour showed high hERG1 expression had a worse prognosis; (vi) the α-hERG1-MoAb could detect PDAC in vivo.

Conclusions:

hERG1 regulates PDAC malignancy and its expression, once validated in a larger cohort also comprising of late-stage, non-surgically resected cases, may be exploited for diagnostic and prognostic purposes in PDAC either ex vivo or in vivo.     

Desmogleins as prognostic biomarkers in resected pancreatic ductal adenocarcinoma

Background:

Frequent disease relapse and a lack of effective therapies result in a very poor outcome in pancreatic ductal adenocarcinoma (PDAC) patients. Thus, identification of prognostic biomarkers and possible therapeutic targets is essential. Besides their function in cell–cell adhesion, desmogleins may play a role in tumour progression and invasion that has not been investigated in PDAC to date. This study evaluated desmoglein expression as a biomarker in PDAC.

Methods:

Using immunohistochemistry, we examined desmoglein 1 (DSG1), desmoglein 2 (DSG2) and desmoglein 3 (DSG3) expression in the tumour tissue of 165 resected PDAC cases. Expression levels were correlated to the patients'' clinicopathological parameters and postoperative survival times. We confirmed these results in two independent gene expression data sets.

Results:

A total of 36% of the tumours showed high DSG3 expression that correlated significantly with shorter patient survival (P=0.011) and poor tumour differentiation (P<0.001), whereas no such association was detected for DSG1 or DSG2. In RNA-Seq data and in microarray expression data, high DSG3 expression correlated significantly with poor survival (P=0.000356 and P=0.00499).

Conclusions:

We identify DSG3 as a negative prognostic biomarker in resected PDAC, as high DSG3 expression is associated with poor overall survival and poor tumour-specific survival. These findings suggest DSG3 and its downstream signalling pathways as possible therapeutic targets in DSG3-expressing PDAC.     

Early expression of the fractalkine receptor CX3CR1 in pancreatic carcinogenesis

Background:

In pancreatic ductal adenocarcinoma (PDAC), fractalkine receptor CX3CR1 contributes to perineural invasion (PNI). We investigated whether CX3CR1 expression occurs early in PDAC and correlates with tumour features other than PNI.

Methods:

We studied CX3CR1 and CX3CL1 expression by immunohistochemistry in 104 human PDAC and coexisting Pancreatic Intraepithelial Neoplasia (PanIN), and in PdxCre/LSL-Kras^G12D mouse model of PDAC. CX3CR1 expression in vitro was studied by a spheroid model, and in vivo by syngenic mouse graft of tumour cells.

Results:

In total, 56 (53.9%) PDAC expressed CX3CR1, 70 (67.3%) CX3CL1, and 45 (43.3%) both. CX3CR1 expression was independently associated with tumour glandular differentiation (P=0.005) and PNI (P=0.01). Pancreatic Intraepithelial Neoplasias were more frequently CX3CR1+ (80.3%, P<0.001) and CX3CL1+ (86.8%, P=0.002) than matched cancers. The survival of PDAC patients was better in those with CX3CR1+ tumour (P=0.05). Mouse PanINs were also CX3CR1⁺ and -CL1⁺. In vitro, cytokines significantly increased CX3CL1 but not CX3CR1 expression. Differently, CX3CR1 was upregulated in tumour spheroids, and in vivo only in well-differentiated tumours.

Conclusion:

Tumour differentiation, rather than inflammatory signalling, modulates CX3CR1 expression in PanINs and PDAC. CX3CR1 expression pattern suggests its early involvement in PDAC progression, outlining a potential target for interfering with the PanIN transition to invasive cancer.     

Phase I study and preclinical efficacy evaluation of the mTOR inhibitor sirolimus plus gemcitabine in patients with advanced solid tumours

Background:

We conducted a phase I study in patients with advanced solid tumours to identify the recommended dose, assess pharmacokinetics (PK), pharmacodynamic activity and preclinical antitumour efficacy of the combination of sirolimus and gemcitabine.

Methods:

Nineteen patients were treated with sirolimus 2 or 5 mg daily and gemcitabine 800 or 1000 mg m⁻² on days 1 and 8. Dose escalation depended on dose-limiting toxicity (DLT) rate during the first 3-week period. Paired skin biopsies were evaluated for phosphorylated S6 (pS6) as marker of mTOR (mammalian target of rapamycin) inhibition. Pharmacokinetics and preclinical evaluation of efficacy using two different sarcoma cell lines and leiomyosarcoma xenografts were also conducted.

Results:

Three DLTs were observed: grade 3 transaminitis, grade 3 thrombocytopenia and grade 4 thrombocytopenia. Common treatment-related adverse events included anaemia, neutropenia, thrombocytopenia and transaminitis. Pharmacodynamic analyses demonstrated mTOR inhibition with sirolimus 5 mg and PK showed no influence of sirolimus concentrations on gemcitabine clearance. In vitro and in vivo studies suggested mTOR pathway hyperactivation by gemcitabine that was reversed by sirolimus. Tumour growth in leiomyosarcoma xenografts was dramatically inhibited by the treatment.

Conclusions:

Recommended dose was sirolimus 5 mg per 24 h plus gemcitabine 800 mg m⁻². Antitumour activity in preclinical sarcoma models and mTOR signalling inhibition were observed. A phase II study is currently ongoing.     

Phase II study of gemcitabine and bexarotene (GEMBEX) in the treatment of cutaneous T-cell lymphoma

Background:

Both gemcitabine and bexarotene are established single agents for the treatment of cutaneous T-cell lymphoma (CTCL). We investigated the feasibility and efficacy of combining these drugs in a single-arm phase II study.

Methods:

Cutaneous T-cell lymphoma patients who had failed standard skin-directed therapy and at least one prior systemic therapy were given four cycles of gemcitabine and concurrent bexarotene for 12 weeks. Responders were continued on bexarotene maintenance until disease progression or unacceptable toxicity.

Results:

The median age was 65 years, stage IB (n=5), stage IIA (n=2), stage IIB (n=8), stage III (n=8) and stage IVA (n=12), 17 patients were erythrodermic, 17 patients were B1, and 10 patients were both erythrodermic and B1. Thirty (86%) patients completed four cycles of gemcitabine. In all, 80.0% of patients demonstrated a reduction in modified Severity-Weighted Assessment Tool (mSWAT) score although the objective disease response rate at 12 weeks was 31% (partial response (PR) 31%) and at 24 weeks 14% (PR 14%, stable disease (SD) 23%, progressive disease (PD) 54%, not evaluable 9%). Median progression-free survival was 5.3 months and median overall survival was 21.2 months.

Conclusion:

The overall response rate of the combination did not reach the specified target to proceed further and is lower than that previously reported for gemcitabine as a single agent.     

Phase I trial of axitinib combined with platinum doublets in patients with advanced non-small cell lung cancer and other solid tumours

Background:

This phase I dose-finding trial evaluated safety, efficacy and pharmacokinetics of axitinib, a potent and selective second-generation inhibitor of vascular endothelial growth factor receptors, combined with platinum doublets in patients with advanced non-small cell lung cancer (NSCLC) and other solid tumours.

Methods:

In all, 49 patients received axitinib 5 mg twice daily (b.i.d.) with paclitaxel/carboplatin or gemcitabine/cisplatin in 3-week cycles. Following determination of the maximum tolerated dose, a squamous cell NSCLC expansion cohort was enroled and received axitinib 5 mg b.i.d. with paclitaxel/carboplatin.

Results:

Two patients experienced dose-limiting toxicities: febrile neutropenia (n=1) in the paclitaxel/carboplatin cohort and fatigue (n=1) in the gemcitabine/cisplatin cohort. Common nonhaematologic treatment-related adverse events were hypertension (36.7%), diarrhoea (34.7%) and fatigue (28.6%). No grade⩾3 haemoptysis occurred among 12 patients with squamous cell NSCLC. The objective response rate was 37.0% for patients receiving axitinib/paclitaxel/carboplatin (n=27) and 23.8% for patients receiving axitinib/gemcitabine/cisplatin (n=21). Pharmacokinetics of axitinib and chemotherapeutic agents were similar when administered alone or in combination.

Conclusion:

Axitinib 5 mg b.i.d. may be combined with standard paclitaxel/carboplatin or gemcitabine/cisplatin regimens without evidence of overt drug–drug interactions. Both combinations demonstrated clinical efficacy and were well tolerated.     

Metronomic gemcitabine suppresses tumour growth,improves perfusion,and reduces hypoxia in human pancreatic ductal adenocarcinoma

Background:

The current standard of care for pancreatic cancer is weekly gemcitabine administered for 3 of 4 weeks with a 1-week break between treatment cycles. Maximum tolerated dose (MTD)-driven regimens as such are often associated with toxicities. Recent studies demonstrated that frequent dosing of chemotherapeutic drugs at relatively lower doses in metronomic regimens also confers anti-tumour activity but with fewer side effects.

Methods:

Herein, we evaluated the anti-tumour efficacy of metronomic vs MTD gemcitabine, and investigated their effects on the tumour microenvironment in two human pancreatic cancer xenografts established from two different patients.

Results:

Metronomic and MTD gemcitabine significantly reduced tumour volume in both xenografts. However, K^trans values were higher in metronomic gemcitabine-treated tumours than in their MTD-treated counterparts, suggesting better tissue perfusion in the former. These data were further supported by tumour-mapping studies showing prominent decreases in hypoxia after metronomic gemcitabine treatment. Metronomic gemcitabine also significantly increased apoptosis in cancer-associated fibroblasts and induced greater reductions in the tumour levels of multiple pro-angiogenic factors, including EGF, IL-1α, IL-8, ICAM-1, and VCAM-1.

Conclusion:

Metronomic dosing of gemcitabine is active in pancreatic cancer and is accompanied by pronounced changes in the tumour microenvironment.     

Urinary TIMP-1 and MMP-2 levels detect the presence of pancreatic malignancies

Background:

A majority of patients with pancreatic malignancies, including both pancreatic ductal adenocarcinoma (PDAC) and pancreatic neuroendocrine tumours (pNETs), present with advanced disease due to a lack of specific symptoms and current diagnostic limitations, making this disease extremely difficult to detect. Our goal was to determine whether urinary matrix metalloproteases (uMMPs) and/or their endogenous inhibitors, urinary tissue inhibitor of metalloproteases (uTIMPs), could be detected in the urine of patients with pancreatic malignancies and whether they may serve as independent predictors of disease status.

Methods:

Retrospective analyses of urine samples (n=139) from PDAC and pNET patients as well as age- and sex-matched controls were conducted. Urinary MMP-2 and uTIMP-1 levels were determined using ELISA and zymography. Biomarker expression in tumour and normal pancreatic tissues was analysed via immunohistochemistry (IHC).

Results:

Multivariable logistic regression analyses indicated that, when controlling for age and sex, uMMP-2 (P<0.0001) and uTIMP-1 (P<0.0001) but not uMMP-9, were significant independent predictors for distinguishing between PDAC patients and healthy controls. Our data also indicated that uMMP-2 was an independent predictor of the presence of pNET. In addition, uTIMP-1 levels could differentiate the two cancer groups, PDAC and pNET, respectively. Immunohistochemistry analysis confirmed that MMP-2 and TIMP-1 protein expression is significantly upregulated in PDAC tissue compared with the normal pancreas.

Conclusions:

Taken together, our results suggest that the detection of uMMP-2 and uTIMP-1 may have diagnostic value in the detection of pancreatic malignancies and that uTIMP-1 may be useful in distinguishing between pancreatic adenocarcinoma and neuroendocrine tumours.     

MiR-23a/-24-induced gene silencing results in mesothelial cell integration of pancreatic cancer

Background:

Invasion of the surrounding tissue is part of the metastatic cascade. Here, we examined the invasion of pancreatic ductal adenocarcinoma (PDAC) cells into the mesothelial barrier and identified the related microRNA (miRNA) expression profiles.

Methods:

The interactions between PDAC cells and mesothelial monolayers were characterised and quantified using a specific time-lapse videomicroscopy assay. Pancreatic ductal adenocarcinoma cells were further evaluated using the adhesion assay, and miRNA, mRNA and protein expressions were determined using microarray, q-RT–PCR and western blots, respectively. These data were correlated with in vivo dissemination scores.

Results:

Two groups of PDAC cell lines were distinguished by their integration capacity into the mesothelial monolayer using mean elongation factors (MEFs). Adhesion assays showed a concordant relation between adhesive properties and integration capacity. The distant metastases scores were reverse correlated with MEFs. Microarray analysis of these groups revealed that miR-23a and/or miR-24 target for FZD5, HNF1B and/or TMEM92, respectively, and that they are significantly deregulated.

Conclusions:

MiR-23a and/or miR-24 overexpression leads to gene silencing of FZD5, TMEM92 and/or HNF1B. Their downregulation induces deregulated expression and degradation of E-cadherin and β-catenin causing destabilisation of the cadherin/catenin complex, and altered the expression of Wnt-related genes. We propose a molecular (epi)genetic mechanism by which increased EMT-like cell shape transformation and integration into mesothelial monolayers of PDAC cells can be observed.     

Dose escalation to rash for erlotinib plus gemcitabine for metastatic pancreatic cancer: the phase II RACHEL study

Background:

This phase II, open-label, randomised study evaluated whether patients with metastatic pancreatic cancer receiving erlotinib/gemcitabine derived survival benefits from increasing the erlotinib dose.

Methods:

After a 4-week run-in period (gemcitabine 1000 mg m⁻² once weekly plus erlotinib 100 mg per day), patients with metastatic pancreatic cancer who developed grade 0/1 rash were randomised to receive gemcitabine plus erlotinib dose escalation (150 mg, increasing by 50 mg every 2 weeks (maximum 250 mg); n=71) or gemcitabine plus standard-dose erlotinib (100 mg per day; n=75). The primary end point was to determine whether overall survival (OS) was improved by increasing the erlotinib dose. Secondary end points included progression-free survival (PFS), incidence of grade ⩾2 rash, and safety.

Results:

Erlotinib dose escalation induced grade ⩾2 rash in 29 out of 71 (41.4%) patients compared with 7 out of 75 (9.3%) patients on standard dose. Efficacy was not significantly different in the dose-escalation arm compared with the standard-dose arm (OS: median 7.0 vs 8.4 months, respectively, hazard ratio (HR), 1.26, 95% confidence interval (CI): 0.88–1.80; P=0.2026; PFS: median 3.5 vs 4.5 months, respectively, HR, 1.09, 95% CI: 0.77–1.54; P=0.6298). Incidence of adverse events was comparable between randomised arms.

Conclusion:

The erlotinib dose-escalation strategy induced rash in some patients; there was no evidence that the higher dose translated into increased benefit.     

Gemcitabine alone or in combination with cisplatin in patients with biliary tract cancer: a comparative multicentre study in Japan

Background:

A British randomised study of gemcitabine plus cisplatin (GC) combination showed promising results in biliary tract cancer (BTC) patients. In our study, we evaluated the efficacy and safety of this combination compared with gemcitabine alone (G) in Japanese BTC patients.

Methods:

Overall, 84 advanced BTC patients were randomised to either cisplatin 25 mg m⁻² plus gemcitabine 1000 mg m⁻² on days 1, 8 of a 21-day cycle (GC-arm), or single-agent gemcitabine 1000 mg m⁻² on days 1, 8 and 15 of a 28-day cycle (G-arm). Treatments were repeated for at least 12 weeks until disease progression or unacceptable toxicity occurred, up to a maximum of 48 weeks.

Results:

A total of 83 patients were included in the analysis. For the GC and G-arms, respectively, the 1-year survival rate was 39.0 vs 31.0%, median survival time 11.2 vs 7.7 months, median progression-free survival time 5.8 vs 3.7 months and overall response rate 19.5 vs 11.9%. The most common grade 3 or 4 toxicities (GC-arm/G-arm) were neutropenia (56.1%/38.1%), thrombocytopenia (39.0%/7.1%), leukopenia (29.3%/19.0%), haemoglobin decrease (36.6%/16.7%) and γ-GTP increase (29.3%/35.7%).

Conclusions:

Gemcitabine plus cisplatin combination therapy was found to be effective and well tolerated, suggesting that it could also be a standard regimen for Japanese patients.     

Serum levels of IL-6 and IL-1β can predict the efficacy of gemcitabine in patients with advanced pancreatic cancer

Background:

With this study, we sought to characterise the impact of pro-inflammatory cytokines on the outcomes of gemcitabine monotherapy (GEM) in patients with pancreatic cancer (PC).

Methods:

Treatment-naive patients with advanced PC and no obvious infections were eligible for enrolment. All of the patients were scheduled to undergo systemic chemotherapy. Serum pro-inflammatory cytokines were measured using an electro-chemiluminescence assay method before chemotherapy. High cytokine levels were defined as values greater than the median. Clinical data were collected prospectively.

Results:

Sixty patients who received GEM were included in the analysis. High IL-6 and IL-1β levels were poor prognostic factors for overall survival in a multivariate analysis (P=0.011 and P=0.048, respectively). Patients with both a high IL-6 level and a high IL-1β level exhibited shortened overall and progression-free survival, a reduction in the tumour control rate, and a high dose intensity of GEM compared with patients with low levels of both IL-6 and IL-1β.

Conclusion:

The serum levels of IL-6 and IL-1β predict the efficacy of GEM in patients with advanced PC.     

Assessing the benefit–risk of new treatments using generalised pairwise comparisons: the case of erlotinib in pancreatic cancer

Background:

Efficacy and safety are the two considerations when characterising the effects of a new therapy. We sought to apply an innovative method of assessing the benefit–risk balance using data from a completed randomised controlled trial that compared erlotinib vs placebo added to gemcitabine in patients with advanced pancreatic cancer (NCIC CTG PA.3).

Methods:

We applied generalised pairwise comparisons with several prioritised outcome measures (e.g., one or more benefit outcomes and one or more risk outcomes). Here, the first priority outcome was overall survival (OS) time. Differences in OS that exceeded 2 months were considered clinically meaningful. The second priority outcome was toxicity. The overall treatment effect was quantified using the proportion in favour of erlotinib, which can be interpreted as the net proportion of patients who have a better overall outcome with erlotinib as compared with placebo. Sensitivity analyses were performed.

Results:

In this trial 569 patients were randomly assigned in a 1 : 1 ratio to receive gemcitabine plus either erlotinib or a matched placebo. Overall, the method indicated no statistically significant overall treatment effect in favour of erlotinib; if anything, the point estimate of the net proportion leaned in favour of the placebo group (overall proportion in favour of erlotinib=−3.6%, 95% CI, −14.2– 7.1% P=0.51). The net proportion was never in favour of the erlotinib group throughout all sensitivity analyses.

Conclusions:

Generalised pairwise comparisons make it possible to assess the benefit–risk balance of new treatments using a single statistical test for any number of prioritised outcomes. The benefit–risk assessment was not in favour of adding erlotinib to gemcitabine for the treatment of patients with advanced pancreatic cancer.     

A phase I combination dose-escalation study of eribulin mesylate and gemcitabine in patients with advanced solid tumours: a study of the Princess Margaret Consortium

Background:

Eribulin mesylate is a synthetic microtubule inhibitor that showed cytotoxic synergy in combination with gemcitabine preclinically. This combination was assessed in a Phase I dose-finding trial in patients diagnosed with advanced solid tumours who had received up to two prior chemotherapy regimens for metastatic disease (CP cohort).

Methods:

Dose escalation was performed in a 3+3 design to identify the recommended phase II dose (RP2D). Two additional expansion cohorts in women with gynaecologic cancers at the RP2D (G), and further dose escalation of metastatic chemotherapy-naive patients (CN), were evaluated.

Results:

45 patients were treated: 21 (CP), 10 (G) and 14 (CN). The initial combination of eribulin and gemcitabine was administered on days 1, 8, and 15 of a 28-day cycle; however, due to 2 out of 6 dose-limiting haematological toxicities at the first dose level, a reduced dose-intense schedule was assessed. The RP2D was defined at 1.0 mg m⁻² eribulin and 1000 mg m⁻² gemcitabine day 1 and 8 q3 weeks. No other significant toxicities were observed in the G expansion cohort. Neutropenia prevented further dose escalation in the CN cohort. Objective responses were seen in all three cohorts – 2/21 (CP), 1/10 (G) and 2/14 (CN).

Conclusions:

The combination of eribulin and gemcitabine was well tolerated at the RP2D.