Catherine Shannon

EGF inhibits constitutive internalization and palmitoylation-dependent degradation of membrane-spanning procancer CDCP1 promoting its availability on the cell surface.

Many cancers are dependent on inappropriate activation of epidermal growth factor receptor (EGFR), and drugs targeting this receptor can improve patient survival, although benefits are generally short-lived. We reveal a novel mechanism linking EGFR and the membrane-spanning, cancer-promoting protein CDCP1 (CUB domain-containing protein 1). Under basal conditions, cell surface CDCP1 constitutively internalizes and undergoes palmitoylation-dependent degradation by a mechanism in which it is palmitoylated in at least one of its four cytoplasmic cysteines. This mechanism is functional in vivo as CDCP1 is elevated and palmitoylated in high-grade serous ovarian tumors. Interestingly, activation of the EGFR system with EGF inhibits proteasome-mediated, palmitoylation-dependent degradation of CDCP1, promoting recycling of CDCP1 to the cell surface where it is available to mediate its procancer effects. We also show that mechanisms inducing relocalization of CDCP1 to the cell surface, including disruption of its palmitoylation and EGF treatment, promote cell migration. Our data provide the first evidence that the EGFR system can function to increase the lifespan of a protein and also promote its recycling to the cell surface. This information may be useful for understanding mechanisms of resistance to EGFR therapies and assist in the design of treatments for EGFR-dependent cancers.

Towards a test to predict 5-fluorouracil toxicity: Pharmacokinetic data for thymine and two sequential metabolites following oral thymine administration to healthy adult males.

The fluoropyrimidine drugs 5-fluorouracil and its oral prodrug capecitabine remain first line therapy for solid tumours of the neck, breast and colon. However, significant and unpredictable toxicity affects about 10-25% of patients depending upon the mode of 5-fluorouracil delivery. The pharmacokinetics of thymine (5-methyluracil) may provide an approach for screening for 5-fluorouracil toxicity, based on the rationale that thymine is a close structural analogue of 5-fluorouracil and is catabolized by the same enzymatic pathway. Oral thymine loading tests were performed on 12 healthy volunteers. Each subject was given a single oral dose of 250mg thymine in capsule form. Blood, urine and saliva samples were collected pre-dose and up to 5h post-dose. Concentrations of thymine, and its catabolites dihydrothymine and ß-ureidoisobutyrate were analysed by HPLC-tandem mass spectrometry in plasma, urine and saliva. The pharmacokinetic data of healthy volunteers were analysed assuming a non-compartmental model. Thymine peaked quickly (30-45min) in plasma to a maximum concentration of 170±185μg/L (mean±SD). Clearance was high (mean 57.9L/h/kg) exceeding normal human liver blood flow, suggesting low systemic bioavailability; urinary recovery of the thymine dose was low (<1%). Apparent formation rate-limited kinetics were observed for dihydrothymine, and the plasma concentration of dihydrothymine was consistently 10-fold higher than that of thymine. Plasma ß-ureidoisobutyrate concentrations, on the other hand, were similar to that of thymine. Genotyping confirmed that pathological mutations of the DPYD gene were absent. The urinary excretion ratio of thymine/dihydrothymine was informative of the maximum concentration. Saliva thymine was highly variable. These data are potentially useful as a basis for developing of a screening procedure to prospectively identify patients who are at risk of toxicity from fluoropyrimidine drugs.

Cell line and patient-derived xenograft models reveal elevated CDCP1 as a target in high-grade serous ovarian cancer

Background:Development of targeted therapies for high-grade serous ovarian cancer (HGSC) remains challenging, as contributing molecular pathways are poorly defined or expressed heterogeneously. CUB-domain containing protein 1 (CDCP1) is a cell-surface protein elevated in lung, colorectal, pancreas, renal and clear cell ovarian cancer.Methods:CUB-domain containing protein 1 was examined by immunohistochemistry in HGSC and fallopian tube. The impact of targeting CDCP1 on cell growth and migration in vitro, and intraperitoneal xenograft growth in mice was examined. Three patient-derived xenograft (PDX) mouse models were developed and characterised for CDCP1 expression. The effect of a monoclonal anti-CDCP1 antibody on PDX growth was examined. Src activation was assessed by western blot analysis.Results:Elevated CDCP1 was observed in 77% of HGSC cases. Silencing of CDCP1 reduced migration and non-adherent cell growth in vitro and tumour burden in vivo. Expression of CDCP1 in patient samples was maintained in PDX models. Antibody blockade of CDCP1 significantly reduced growth of an HGSC PDX. The CDCP1-mediated activation of Src was observed in cultured cells and mouse xenografts.Conclusions:CUB-domain containing protein 1 is over-expressed by the majority of HGSCs. In vitro and mouse model data indicate that CDCP1 has a role in HGSC and that it can be targeted to inhibit progression of this cancer.

Practical guidance on the use of olaparib capsules as maintenance therapy for women with BRCA mutations and platinum-sensitive recurrent ovarian cancer

Olaparib is the first oral poly(ADP‐ribose) polymerase inhibitor to be approved as maintenance monotherapy for treatment of patients with platinum‐sensitive relapsed BRCA‐mutated (BRCAm) serous ovarian cancer. This review provides practical guidance on the use of olaparib (capsule formulation) in the maintenance setting. The article focuses on the key toxicities that can arise with olaparib therapy and recommendations for their management. Nausea, vomiting, fatigue and anemia are the most commonly reported adverse events in olaparib clinical trials and are generally mild to moderate and transient in nature in most patients. Implementation of an effective and timely management plan can control many of the side effects. It is vital that health care providers effectively communicate the potential side effects of olaparib, as well as educate patients on management strategies to combat these symptoms. To this end, realistic expectations regarding the potential side effects need to be set, with an understanding that dose interruptions and modifications may be required to allow patients to continue receiving treatment.

Simultaneous determination of thymine and its sequential catabolites dihydrothymine and β-ureidoisobutyrate in human plasma and urine using liquid chromatography-tandem mass spectrometry with pharmacokinetic application

To study in vivo activities of dihydropyrimidine dehydrogenase, dihydropyrimidinase, and ureidoproprionase, a sensitive, accurate, selective and precise method for the determination of the endogenous pyrimidine thymine (THY) and its successive metabolites dihydrothymine (DHT) and β-ureidoisobutyric (UIB) acid in human plasma and urine has been developed and validated. Plasma or diluted urine (200 μL) was mixed with 1 mL of deuterated-THY (internal standard) in acetonitrile, then centrifuged, the supernatant evaporated, and the residue reconstituted in 150 μL 0.1% (w/w) formic acid in water. Separation was performed on a Waters Symmetry C₈ column (150 mm × 3.9 mm; 5 μm particle size), with gradient elution using a mobile phase of 0.1% (w/w) formic acid in water (phase A), and 15% (v/v) methanol in acetonitrile (phase B). The detection system was an Applied Biosystems model 3200 tandem mass spectrometer with atmospheric pressure chemical ionisation, and multiple reaction monitoring mode using the transitions: THY (m/z: 127.1-110.0), DHT (m/z: 129.1-68.9), UIB (m/z: 147.1-86.0), and deuterated-THY (m/z: 131.1-114.0). THY, DHT, and UIB eluted at 5.12 min, 5.17 min and 5.00 min, respectively. Linearity of the calibrations was established from 2 to 500 μg/L. The lower limit of quantification was 5 μg/L in plasma, and 10 μg/L in urine for THY, DHT and UIB. Ion-suppression had negligible effect. A pilot pharmacokinetic study analysed plasmas and urines from 2 healthy male subjects who each received an oral 250 mg THY dose. THY was rapidly absorbed and eliminated with an apparent biphasic log-linear profile. DHT and UIB demonstrated apparent formation-rate limited kinetics.

Phase II, open-label trial of lapatinib and vinorelbine in women with previously treated HER2-positive metastatic breast cancer

To evaluate the efficacy and tolerability of lapatinib (L) and intravenous vinorelbine (V) in patients with metastatic HER2‐positive breast cancer who have previously received two lines of anti‐HER2 therapy (i.e. trastuzumab [T] with chemotherapy and lapatinib with capecitabine [LC]).

Vulvar yolk sac tumor diagnosed during pregnancy, with recurrence during subsequent second pregnancy

Highlights • Vulvar yolk sac tumor (YST) is an exceptionally rare and aggressive extra-gonadal germ cell tumor, previously only described in 15 cases in the literature.• We present a unique case of vulvar YST diagnosed during pregnancy, treated with systemic chemotherapy, who subsequently recurred during a second pregnancy.• Despite salvage chemotherapy and autologous stem cell transplantation at recurrence, our patient succumbed to her disease, 31 months post diagnosis.

A pilot study of intraperitoneal bevacizumab for the palliation of malignant ascites.

To the Editor Malignant ascites is a frequent complication of advanced cancer and has a significant impact on quality of life. Patients are usually managed with repeated paracentesis, but this provides only short-term relief. Preclinical studies have demonstrated the efficacy and safety of intraperitoneal inhibition of vascular endothelial growth factor (VEGF) as therapy to prevent accumulation of ascites. Bevacizumab is a recombinant humanized monoclonal antibody that inhibits the biological activity of VEGF. A number of small studies have reported on the safety and efficacy of intraperitoneal (IP) administration of bevacizumab (BVZ) for the treatment of malignant ascites to date with mixed results. We report on a pilot study in which five patients with refractory malignant ascites were treated with intraperitoneal bevacizumab. Patients with intraabdominal malignant disease of any etiology and no current systemic treatment were eligible for the study. Those with contraindications to bevacizumab treatment (e.g. uncontrolled hypertension or coagulopathy), a history of bowel perforation, symptoms of bowel obstruction or a life expectancy of less than 2 weeks were excluded. The clinical need (as determined by patient and/or doctor), to redrain at 14 days, was used as our primary response measure. All patients had ascites drained to dryness using a standard paracentesis catheter inserted at the site of maximal fluid collection as identified by abdominal ultrasound. The catheter was removed following the intraperitoneal infusion of a single dose of 200-mg bevacizumab in 250 mL 0.9% saline. Blood and ascitic fluid were collected for analysis of pretreatment VEGF levels with citrate, theophylline, adenosine, dipyridamole mix (CTAD) suggest specific anticoagulant use to prepare plasma from blood to minimize processing-induced VEGF release from platelets. All patients met the definition of response in that none required repeat drainage within 14 days. Side effects were

Absent progesterone receptor expression in the lymph node metastases of ER-positive, HER2-negative breast cancer is associated with relapse on tamoxifen

Aims Progesterone receptor (PR) expression is prognostic in early stage breast cancer. There are several reports of discordant expression between primary tumour and axillary lymph node (ALN) metastasis expression of oestrogen receptor (ER) and PR. We sought to determine whether expression of these biomarkers in the synchronous ALN metastases of ER positive (+), HER2 negative (−) breast cancer could provide more accurate prognostic information. Methods The retrospective cohort included 229 patients from a single institution with ER+, HER2− breast cancer who had synchronous ALN metastatic disease (2005–2014). PR expression was correlated with relapse-free survival, and subset analysis was performed for patients who received adjuvant tamoxifen or an aromatase inhibitor. Results One patient had an ER+ primary tumour, which was ER− in the ALN metastasis. 27 (11.3%) were PR− in the primary tumour and 56 (23.6%) in the ALN metastasis. The predominant change was from PR+ in the primary tumour to PR− in the lymph node. Absence of PR expression in the ALN was significantly associated with relapse; however, this was not the case in the primary tumour. In a subset analysis of patients taking adjuvant endocrine therapy, poorer prognosis was limited to those with PR− metastases on tamoxifen (HR=5.203, 95% CI 1.649 to 16.416, p=0.005). No significant prognostic effect of PR− metastases in patients taking aromatase inhibitors was seen (HR=1.519, 95% CI 0.675 to 3.418, p=0.312). Conclusions Evaluation of PR expression in ALN metastasis may enable prediction of patients who are less likely to benefit from adjuvant tamoxifen. This study should be replicated in other cohorts.

EGF-induced recycling of the cancer promoting protein CDCP1

15th International Biennial Congress of the METASTASIS RESEARCH SOCIETY Heidelberg, Germany, June 28th–July 1st, 2014 Springer Science+Business Media Dordrecht 2015