Excessive vincristine exposure in a child being treated for acute lymphoblastic leukaemia with underlying Dubin-Johnson syndrome: a case report.

BACKGROUND: Dubin-Johnson syndrome is a rare benign autosomal recessive condition that causes an isolated increase of conjugated bilirubin in the serum. Impaired biliary excretion is due to mutation in the multiple drug-resistance protein 2 gene (MRP2). CASE PRESENTATION: We describe the case of a 4-year-old girl being treated for acute lymphoblastic leukaemia who had a history of conjugated hyperbilirubinaemia and persistently elevated bilirubin levels on initiation of chemotherapy. During treatment for leukaemia, she was diagnosed with Dubin-Johnson syndrome for the underlying condition. Following administration of vincristine at the recommended dose of 1.5 mg/m2, an abnormally high vincristine exposure was observed (AUC > 200 µg/L*h), approximately 3 times higher than previously reported exposures in a comparable clinical setting. Vincristine dose reductions were applied on subsequent cycles of treatment and resulted in markedly reduced drug exposures, within the normal target range. CONCLUSION: This case provided a rare opportunity to assess the impact of MRP2 mutations associated with Dubin-Johnson syndrome on the pharmacokinetics of vincristine and strongly indicates that a marked dose reduction should be recommended. Clinicians should be made aware of the potential for altered drug disposition for agents such as vincristine in patients with this rare genetic condition.

Development and validation of an ELISA method for quantification of the anti-HER3 antibody HMBD-001 in human serum.

Background: HMBD-001 is an IgG1 humanized monoclonal antibody specifically targeting HER3, a receptor highly expressed on cancer cells in certain tumors. A bioanalytical method was required to quantify HMBD-001 in human serum, with high selectivity and without interference from HER3. Methods and results: A bridging ELISA using an anti-idiotypic monoclonal capture and detection was developed and validated for quantitative measurement of HMBD-001 in human serum. The assay is sensitive, with a lower limit of quantification of 250 ng/ml, has a broad dynamic range of 250-7000 ng/ml HMBD-001, and exhibits excellent precision and overall accuracy. Conclusion: We have developed and validated a sensitive and selective method for measuring HMBD-001 in human serum. This assay is now being used in a clinical trial setting.

Pharmacokinetics and Pharmacogenetics of Cyclophosphamide in a Neonate and Infant Childhood Cancer Patient Population.

Infants and young children represent an important but much understudied childhood cancer patient population. The pharmacokinetics and pharmacogenetics of the widely used anticancer prodrug cyclophosphamide were investigated in children <2 years of age. Concentrations of cyclophosphamide and selected metabolites were determined in patients administered cyclophosphamide at doses ranging from 100-1500 mg/m2 (5-75 mg/kg), with various infusion times as determined by the standard treatment regimen that each patient was receiving. Polymorphisms in genes including CYP2B6 and CYP2C19 were investigated. Data generated for cyclophosphamide were analysed using a previously published population pharmacokinetic model. Cyclophosphamide pharmacokinetics was assessed in 111 samples obtained from 25 patients ranging from 4-23 months of age. The average cyclophosphamide clearance for the patients was 46.6 mL/min/m2 (ranging from 9.4-153 mL/min/m2), with marked inter-patient variability observed (CV 41%). No significant differences in cyclophosphamide clearance or exposure (AUC) were observed between patient groups as separated by age or body weight. However, marked differences in drug clearance and metabolism were noted between the current data in children <2 years of age and recently published results from a comparable study conducted by our group in older children, which reported significantly lower cyclophosphamide clearance values and metabolite exposures using the same population pharmacokinetic model for analysis. Whilst this study demonstrates no significant differences in cyclophosphamide clearance in patients <2 years, it highlights large differences in dosing protocols across tumour types. Furthermore, the study suggests marked differences in cyclophosphamide clearance in children less than two years of age as compared to older patients.

Label-Free Leukemia Monitoring by Computer Vision.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. While there are a number of well-recognized prognostic biomarkers at diagnosis, the most powerful independent prognostic factor is the response of the leukemia to induction chemotherapy (Campana and Pui: Blood 129 (2017) 1913-1918). Given the potential for machine learning to improve precision medicine, we tested its capacity to monitor disease in children undergoing ALL treatment. Diagnostic and on-treatment bone marrow samples were labeled with an ALL-discriminating antibody combination and analyzed by imaging flow cytometry. Ignoring the fluorescent markers and using only features extracted from bright-field and dark-field cell images, a deep learning model was able to identify ALL cells at an accuracy of >88%. This antibody-free, single cell method is cheap, quick, and could be adapted to a simple, laser-free cytometer to allow automated, point-of-care testing to detect slow early responders. Adaptation to other types of leukemia is feasible, which would revolutionize residual disease monitoring. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Genetic Polymorphisms Affecting Cardiac Biomarker Concentrations in Children with Cancer: an Analysis from the "European Paediatric Oncology Off-patents Medicines Consortium" (EPOC) Trial.

BACKGROUND AND OBJECTIVES: Doxorubicin plays an essential role in the treatment of paediatric cancers. Defining genotypes with a higher risk for developing anthracycline-induced cardiotoxicity could help to reduce cardiotoxicity. METHODS: Data originated from a phase II study assessing the pharmacokinetics of doxorubicin in 100 children. Studied patients (0-17 years) were treated for solid tumours or leukaemia. Two cycles of doxorubicin were studied. Concentrations of natriuretic peptides proANP, BNP and NT-proBNP and cardiac troponins T and I were measured at five time points before, during and after two cycles of doxorubicin treatment. Genotypes of 17 genetic polymorphisms in genes encoding for anthracycline metabolizing enzymes and drug transporters were determined for each patient. We analysed the influence of genotypes on cardiac biomarker concentrations at different time points by a Kruskal-Wallis test. To perform a pairwise comparison significant genetic polymorphisms with more than two genotypes were analysed by a post hoc test. RESULTS: The Kruskal-Wallis tests and the post hoc-tests showed a significant association for seven genetic polymorphisms (ABCB1-rs1128503, ABCB1-rs1045642, ABCC1-rs4148350, CBR3-rs8133052, NQO2-in/del, SLC22A16-rs714368 and SLC22A16-rs6907567) with the concentration of at least one biomarker at one or more time points. We could not identify any polymorphism with a consistent effect on any biomarker over the whole treatment period. CONCLUSIONS: In this study of patients treated with doxorubicin for different tumour entities, seven genetic polymorphisms possibly influencing the pharmacokinetics and pharmacodynamics of doxorubicin could lead occasionally to differences in the concentration of cardiac biomarkers. Since, the role of cardiac biomarkers for monitoring anthracycline-induced cardiotoxicity has not yet been clarified, further trials with a long follow-up time are required to assess the impact of these genetic polymorphisms on chemotherapy-related cardiotoxicity. TRIAL REGISTRATION: EudraCT number: 2009-011454-17.

Detection of Circulating and Disseminated Neuroblastoma Cells Using the ImageStream Flow Cytometer for Use as Predictive and Pharmacodynamic Biomarkers.

PURPOSE: Circulating tumor cells (CTCs) serve as noninvasive tumor biomarkers in many types of cancer. Our aim was to detect CTCs from patients with neuroblastoma for use as predictive and pharmacodynamic biomarkers. EXPERIMENTAL DESIGN: We collected matched blood and bone marrow samples from 40 patients with neuroblastoma to detect GD2 +/CD45- neuroblastoma CTCs from blood and disseminated tumor cells (DTCs) from bone marrow using the Imagestream Imaging flow cytometer (ISx). In six cases, circulating free DNA (cfDNA) extracted from plasma isolated from the CTC sample was analyzed by high-density single-nucleotide polymorphism (SNP) arrays. RESULTS: CTCs were detected in 26 of 42 blood samples (1-264/mL) and DTCs in 25 of 35 bone marrow samples (57-291,544/mL). Higher numbers of CTCs in patients with newly diagnosed, high-risk neuroblastoma correlated with failure to obtain a complete bone marrow (BM) metastatic response after induction chemotherapy (P < 0.01). Ex vivo Nutlin-3 (MDM2 inhibitor) treatment of blood and BM increased p53 and p21 expression in CTCs and DTCs compared with DMSO controls. In five of six cases, cfDNA analyzed by SNP arrays revealed copy number abnormalities associated with neuroblastoma. CONCLUSIONS: This is the first study to show that CTCs and DTCs are detectable in neuroblastoma using the ISx, with concurrently extracted cfDNA used for copy number profiling, and may be useful as pharmacodynamic biomarkers in early-phase clinical trials. Further investigation is required to determine whether CTC numbers are predictive biomarkers of BM response to first-line induction chemotherapy.

A novel imaging flow cytometry method for the detection of histone H4 acetylation in myeloid cells.

BACKGROUND: The histone deacetylase inhibitor (HDACI) valproic acid has been shown to inhibit the growth of multiple paediatric tumour types and is well tolerated in a childhood cancer setting. The current study was designed to develop a novel imaging flow cytometry method for the detection of histone H4 acetylation in white blood cells obtained from childhood cancer patients treated with valproic acid. MATERIALS AND METHODS: HL-60 cells and whole blood samples from healthy volunteers were incubated with valproic acid (0-8 mM) for 0-24 hours, with additional blood samples collected from ependymoma patients receiving valproic acid on the SIOP Ependymoma II clinical trial. An imaging flow cytometry method was developed using an ImageStream®χ flow cytometer, collecting 100 000 images per sample following excitation of PE tagged acH4 antibody and DAPI. RESULTS: The mean percentage of acH4-positive cells increased to a greater extent than increases in mean and median fluorescence intensity following incubation with valproic acid. Comparable results were observed for in vitro and ex vivo experiments, and the assay was shown to be appropriate for clinical sample analysis. Myeloid cells exhibited a smaller proportion of acH4-positive cells than the lymphoid population, but a greater fold increase above basal levels. CONCLUSIONS: The percentage of acH4-positive myeloid cells has the potential to be used as a robust pharmacodynamic biomarker for the measurement of acH4 for HDACIs. The developed assay is now being utilised in a clinical trial involving the treatment of childhood ependymoma patients with valproic acid.

Inhibition of ATR acutely sensitizes acute myeloid leukemia cells to nucleoside analogs that target ribonucleotide reductase.

The ataxia telangiectasia and Rad3-related (ATR) protein kinase promotes cancer cell survival by signaling stalled replication forks generated by replication stress, a common feature of many cancers including acute myeloid leukemia (AML). Here we show that the antileukemic activity of the chemotherapeutic nucleoside analogs hydroxyurea and gemcitabine was significantly potentiated by ATR inhibition via a mechanism involving ribonucleotide reductase (RNR) abrogation and inhibition of replication fork progression. When administered in combination with gemcitabine, an inhibitor of the M1 RNR subunit, the ATR inhibitor VX-970, eradicated disseminated leukemia in an orthotopic mouse model, eliciting long-term survival and effective cure. These data identify a synergistic interaction between ATR inhibition and RNR loss that will inform the deployment of small molecule inhibitors for the treatment of AML and other hematologic malignancies.

Neutrophils: driving progression and poor prognosis in hepatocellular carcinoma?

BACKGROUND: Irrespective of the underlying aetiology, 90% of hepatocellular carcinomas arise and progress on a background of chronic inflammation. We have explored the independent prognostic value of circulating inflammatory cells. METHODS: Peripheral blood count data sets from 583 consecutive patients presenting to a single UK centre (2000-2010) were analysed for associations with tumour stage, liver function, performance status (PST) and survival. Validation was in an independent Hong Kong cohort (585 patients; 2007-2013). RESULTS: In both UK and Hong Kong cohorts, neutrophils, platelets, lymphocytes, the neutrophil/lymphocyte ratio (NLR) and the Systemic Immune-Inflammation Index (SII) correlated stepwise, either increasing or decreasing (lymphocytes), with tumour node metastasis (TNM) and Childs-Pugh stage, PST and consequently with the combined Barcelona Clinic for Liver Cancer stage. Survival analyses confirmed the NLR and SII as highly significant prognostic biomarkers. Focused on individual cell types, only the neutrophil count was independently associated with both TNM stage and PST, as well as being significantly and independently associated with poorer survival. CONCLUSIONS: In this study of 1168 patients, neutrophils alone, rather than lymphocytes or platelets, were independently associated with outcome. These data support further characterisation of a potentially distinctive role for neutrophils as facilitators of tumour progression and deteriorating performance.

Gemcitabine and docetaxel versus doxorubicin as first-line treatment in previously untreated advanced unresectable or metastatic soft-tissue sarcomas (GeDDiS): a randomised controlled phase 3 trial.

BACKGROUND: For many years, first-line treatment for locally advanced or metastatic soft-tissue sarcoma has been doxorubicin. This study compared gemcitabine and docetaxel versus doxorubicin as first-line treatment for advanced or metastatic soft-tissue sarcoma. METHODS: The GeDDiS trial was a randomised controlled phase 3 trial done in 24 UK hospitals and one Swiss Group for Clinical Cancer Research (SAKK) hospital. Eligible patients had histologically confirmed locally advanced or metastatic soft-tissue sarcoma of Trojani grade 2 or 3, disease progression before enrolment, and no previous chemotherapy for sarcoma or previous doxorubicin for any cancer. Patients were randomly assigned 1:1 to receive six cycles of intravenous doxorubicin 75 mg/m2 on day 1 every 3 weeks, or intravenous gemcitabine 675 mg/m2 on days 1 and 8 and intravenous docetaxel 75 mg/m2 on day 8 every 3 weeks. Treatment was assigned using a minimisation algorithm incorporating a random element. Randomisation was stratified by age (≤18 years vs >18 years) and histological subtype. The primary endpoint was the proportion of patients alive and progression free at 24 weeks in the intention-to-treat population. Adherence to treatment and toxicity were analysed in the safety population, consisting of all patients who received at least one dose of their randomised treatment. The trial was registered with the European Clinical Trials (EudraCT) database (no 2009-014907-29) and with the International Standard Randomised Controlled Trial registry (ISRCTN07742377), and is now closed to patient entry. FINDINGS: Between Dec 3, 2010, and Jan 20, 2014, 257 patients were enrolled and randomly assigned to the two treatment groups (129 to doxorubicin and 128 to gemcitabine and docetaxel). Median follow-up was 22 months (IQR 15·7-29·3). The proportion of patients alive and progression free at 24 weeks did not differ between those who received doxorubicin versus those who received gemcitabine and docetaxel (46·3% [95% CI 37·5-54·6] vs 46·4% [37·5-54·8]); median progression-free survival (23·3 weeks [95% CI 19·6-30·4] vs 23·7 weeks [18·1-20·0]; hazard ratio [HR] for progression-free survival 1·28, 95% CI 0·99-1·65, p=0·06). The most common grade 3 and 4 adverse events were neutropenia (32 [25%] of 128 patients who received doxorubicin and 25 [20%] of 126 patients who received gemcitabine and docetaxel), febrile neutropenia (26 [20%] and 15 [12%]), fatigue (eight [6%] and 17 [14%]), oral mucositis (18 [14%] and two [2%]), and pain (ten [8%] and 13 [10%]). The three most common serious adverse events, representing 111 (39%) of all 285 serious adverse events recorded, were febrile neutropenia (27 [17%] of 155 serious adverse events in patients who received doxorubicin and 15 [12%] of 130 serious adverse events in patients who received gemcitabine and docetaxel, fever (18 [12%] and 19 [15%]), and neutropenia (22 [14%] and ten [8%]). 154 (60%) of 257 patients died in the intention-to-treat population: 74 (57%) of 129 patients in the doxorubicin group and 80 (63%) of 128 in the gemcitabine and docetaxel group. No deaths were related to the treatment, but two deaths were due to a combination of disease progression and treatment. INTERPRETATION: Doxorubicin should remain the standard first-line treatment for most patients with advanced soft-tissue sarcoma. These results provide evidence for clinicians to consider with their patients when selecting first-line treatment for locally advanced or metastatic soft-tissue sarcoma. FUNDING: Cancer Research UK, Sarcoma UK, and Clinical Trial Unit Kantonsspital St Gallen.

Temporary Single-Cell Coating for Bioprocessing with a Cationic Polymer.

Temporary single-cell coating is a useful tool for cell processing, allowing manipulation of cells to prevent cell attachment and agglomeration, before re-establishing normal cell function. In this work, a speckled coating method using a known polycation [poly(l-lysine), PLL] is described to induce cell surface electrostatic charges on three different cell types, namely, two bone cancer cell lines and fibroblasts. The morphology of the PLL speckled coating on the cell surface, internalization and metabolization of the polymer, and prevention of cellular aggregations are reported. Polymer concentration was found to be the key parameter controlling both capsule morphology and cell health. This approach allows a temporary cell coating over the course of 1-2 h, with cells exhibiting phenotypically normal behavior after ingesting and metabolizing the polymer. The process offers a fast and efficient alternative to aid single-cell manipulation for bioprocessing applications. Preliminary work on the application of PLL speckled cell coating in enabling reliable bioprinting is also presented.

Pharmacogenetic association of MBL2 and CD95 polymorphisms with grade 3 infection following adjuvant therapy for breast cancer with doxorubicin and cyclophosphamide.

Life-threatening infection as an adverse reaction to cytotoxic therapy of cancer remains a major problem, potentially limiting efficacy. Administration of colony-stimulation factors benefits only a minority of patients, and improved stratification guidelines are needed to identify those patients likely to benefit. We investigated single nucleotide polymorphisms (SNPs) in two genes related to immune function to identify associations with severe infection following treatment of breast cancer with doxorubicin and cyclophosphamide. CD95 mediates the extrinsic apoptosis pathway in haematopoietic cells and a CD95 promoter SNP (rs2234767) has been shown to result in reduced expression of the receptor. MBL2 activates the classical complement pathway in the presence of pathogens and independently of antibodies. Numerous SNPs have been described including a promoter SNP (rs7096206) which results in decreased expression of the protein. Homozygotes for the CD95 minor allele were more likely to experience a grade 3 infection than heterozygote and homozygote wild-type patients (29%, 3% and 5%, respectively p=0.048). CD95 minor allele homozygotes also had higher basal white blood cell and neutrophil counts compared with wild-type allele carriers, which was sustained throughout therapy. There was an allele-dose association between the MBL2 SNP and grade 3 infection, with 2, 8 and 17% of wild-type homozygotes, heterozygotes and minor allele homozygotes, respectively, experiencing grade 3 infection (p=0.02). These associations demonstrate the utility of a pharmacogenetic approach to identify individuals more likely to acquire a life-threatening infection during chemotherapy. The apparent association with a CD95 SNP and a mild neutrophilia merits further investigation.

Condensin II mutation causes T-cell lymphoma through tissue-specific genome instability.

Chromosomal instability is a hallmark of cancer, but mitotic regulators are rarely mutated in tumors. Mutations in the condensin complexes, which restructure chromosomes to facilitate segregation during mitosis, are significantly enriched in cancer genomes, but experimental evidence implicating condensin dysfunction in tumorigenesis is lacking. We report that mice inheriting missense mutations in a condensin II subunit (Caph2nes) develop T-cell lymphoma. Before tumors develop, we found that the same Caph2 mutation impairs ploidy maintenance to a different extent in different hematopoietic cell types, with ploidy most severely perturbed at the CD4+CD8+ T-cell stage from which tumors initiate. Premalignant CD4+CD8+ T cells show persistent catenations during chromosome segregation, triggering DNA damage in diploid daughter cells and elevated ploidy. Genome sequencing revealed that Caph2 single-mutant tumors are near diploid but carry deletions spanning tumor suppressor genes, whereas P53 inactivation allowed Caph2 mutant cells with whole-chromosome gains and structural rearrangements to form highly aggressive disease. Together, our data challenge the view that mitotic chromosome formation is an invariant process during development and provide evidence that defective mitotic chromosome structure can promote tumorigenesis.

A phase I pharmacokinetic and pharmacodynamic study of the oral mitogen-activated protein kinase kinase (MEK) inhibitor, WX-554, in patients with advanced solid tumours.

PURPOSE: We performed a multi-centre phase I study to assess the safety, pharmacokinetics (PK) and pharmacodynamics (PD) of the orally available small molecule mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor, WX-554, and to determine the optimal biological dose for subsequent trials. EXPERIMENTAL DESIGN: Patients with treatment-refractory, advanced solid tumours, with adequate performance status and organ function were recruited to a dose-escalation study in a standard 3 + 3 design. The starting dose was 25 mg orally once weekly with toxicity, PK and PD guided dose-escalation with potential to explore alternative schedules. RESULTS: Forty-one patients with advanced solid tumours refractory to standard therapies and with adequate organ function were recruited in eight cohorts up to doses of 150 mg once weekly and 75 mg twice weekly. No dose-limiting toxicities were observed during the study, and a maximum tolerated dose (MTD) was not established. The highest dose cohorts demonstrated sustained inhibition of extracellular signal-regulated kinase (ERK) phosphorylation in peripheral blood mononuclear cells following ex-vivo phorbol 12-myristate 13-acetate stimulation. There was a decrease of 70 ± 26% in mean phosphorylated (p)ERK in C1 day 8 tumour biopsies when compared with pre-treatment tumour levels in the 75 mg twice a week cohort. Prolonged stable disease (>6 months) was seen in two patients, one with cervical cancer and one with ampullary carcinoma. CONCLUSIONS: WX-554 was well tolerated, and an optimal biological dose was established for further investigation in either a once or twice weekly regimens. The recommended phase 2 dose is 75 mg twice weekly.

Imagestream detection and characterisation of circulating tumour cells - A liquid biopsy for hepatocellular carcinoma?

BACKGROUND & AIMS: The lack of progress in developing and delivering new therapies for hepatocellular carcinoma (HCC) is in part attributed to the risk related avoidance of tumour biopsy at diagnosis. Circulating tumour cells (CTCs) are a potential source of tumour tissue that could aid biological or biomarker research, treatment stratification and monitoring. METHODS: An imaging flow cytometry method, using immunofluorescence of cytokeratin, EpCAM, AFP, glypican-3 and DNA-PK together with analysis of size, morphology and DNA content, for detection of HCC CTCs was developed and applied to 69 patient and 31 control samples. The presence of CTCs as a prognostic indicator was assessed in multivariate analyses encompassing recognised prognostic parameters. RESULTS: Between 1 and 1642 CTCs were detected in blood samples from 45/69 HCC patients compared to 0/31 controls. CTCs positive for the epithelial markers cytokeratin and EpCAM were detected in 29% and 18% of patients respectively, while an additional 28% of patients had CTCs negative for all markers other than size and evidence of hyperploidy. CTC number correlated significantly with tumour size and portal vein thrombosis (PVT). The median survival of patients with >1 CTC was 7.5months versus >34months for patients with <1 CTC (p<0.001, log-rank), with significance retained in a multivariate analysis (HR 2.34, 95% CI 1.005-5.425, p=0.049) including tumour size and PVT. CONCLUSIONS: The use of multiple parameters enhanced HCC CTC detection sensitivity, revealing biological associations and predictive biomarker potential that may be able to guide stratified medicine decisions and future research. LAY SUMMARY: Characteristics of tumour tissues can be used to predict outcomes for individual patients with cancer, as well as help to choose their best treatment. Biopsy of liver cancers carries risks, however, and is usually avoided. Some cancer cells enter the blood, and although they are very rare, we have developed a method of finding and characterising them in patients with liver cancer, which we hope will provide a low risk means of guiding treatment.

Phase 2 multicentre trial investigating intermittent and continuous dosing schedules of the poly(ADP-ribose) polymerase inhibitor rucaparib in germline BRCA mutation carriers with advanced ovarian and breast cancer.

BACKGROUND: Rucaparib is an orally available potent selective small-molecule inhibitor of poly(ADP-ribose) polymerase (PARP) 1 and 2. Rucaparib induces synthetic lethality in cancer cells defective in the homologous recombination repair pathway including BRCA-1/2. We investigated the efficacy and safety of single-agent rucaparib in germline (g) BRCA mutation carriers with advanced breast and ovarian cancers. METHODS: Phase II, open-label, multicentre trial of rucaparib in proven BRCA-1/2 mutation carriers with advanced breast and or ovarian cancer, WHO PS 0-1 and normal organ function. Intravenous (i.v.) and subsequently oral rucaparib were assessed, using a range of dosing schedules, to determine the safety, tolerability, dose-limiting toxic effects and pharmacodynamic (PD) and pharmacokinetic (PK) profiles. RESULTS: Rucaparib was well tolerated in patients up to doses of 480 mg per day and is a potent inhibitor of PARP, with sustained inhibition ⩾24 h after single doses. The i.v. rucaparib (intermittent dosing schedule) resulted in an objective response rate (ORR) of only 2% but with 41% (18 out of 44) patients achieved stable disease for ⩾12 weeks and 3 patients maintaining disease stabilisation for >52 weeks. The ORR for oral rucaparib (across all six dose levels) was 15%. In the oral cohorts, 81% (22 out of 27) of the patients had ovarian cancer and 12 out of 13, who were dosed continuously, achieved RECIST complete response/partial response (CR/PR) or stable disease (SD) ⩾12 weeks, with a median duration of response of 179 days (range 84-567 days). CONCLUSIONS: Rucaparib is well tolerated and results in high levels of PARP inhibition in surrogate tissues even at the lowest dose levels. Rucaparib is active in gBRCA-mutant ovarian cancer and this activity correlates with platinum-free interval. The key lessons learned from this study is that continuous rucaparib dosing is required for optimal response, the recommended phase 2 dose (RP2D) for continuous oral scheduling has not been established and requires further exploration and, thirdly, the use of a PD biomarker to evaluate dose-response has its limitations.

Genetic variants in the HER2 gene: Influence on HER2 overexpression and loss of heterozygosity in breast cancer.

Human epidermal growth factor receptor 2 (HER2) overexpression in breast cancer is an indicator of poor prognosis and is the pre-requisite for treatment with the agents targeting this member of the epidermal growth factor receptor family. In order to determine the influence of these common single-nucleotide polymorphisms (SNPs) in the HER2 gene, genomic DNA was obtained from 361 patients with breast cancer, aged between 29 and 82 years. Samples of tumour tissue were obtained from 241 (66%) patients and material for extraction of DNA is isolated from surrounding normal tissue by laser capture microdissection. Genotyping was performed using the Taqman fluorogenic 5' nuclease assay. Of the 360 patients with definitive determination of HER2 status, 49% were positive. The Ile655Val SNP had no influence on the frequency of HER2 expression. However, the proline allele of the Ala1170Pro SNP was associated with a higher frequency of HER2 overexpression (56% versus 43%, p = 0.015). Where the germline genotype was homozygous, the tumour genotype was identical in every case and for both SNPs. In HER2-positive tumours, heterozygosity was maintained in only 15% and 18% of the Ile655Val and Ala1170Pro SNPs, respectively. This was lower than in the HER2-negative tumours (46% and 43%, respectively). Normal breast tissue (n = 23) retained the germline genotype in all but one case. The underlying link between the Ala1170Pro SNP and HER2 positivity is not known, nor is the significance of HER2 overexpression and loss of heterozygosity in breast cancer. However, these results illustrate the complexity of HER2 genotype and overexpression in this disease.

Cyclophosphamide pharmacokinetics and pharmacogenetics in children with B-cell non-Hodgkin's lymphoma.

INTRODUCTION: Variation in cyclophosphamide pharmacokinetics and metabolism has been highlighted as a factor that may impact on clinical outcome in various tumour types. The current study in children with B-cell non-Hodgkin's lymphoma (NHL) was designed to corroborate previous findings in a large prospective study incorporating genotype for common polymorphisms known to influence cyclophosphamide pharmacology. METHODS: A total of 644 plasma samples collected over a 5 year period, from 49 B-cell NHL patients ≤ 18 years receiving cyclophosphamide (250 mg/m(2)), were used to characterise a population pharmacokinetic model. Polymorphisms in genes including CYP2B6 and CYP2C19 were analysed. RESULTS: A two-compartment model provided the best fit of the population analysis. The mean cyclophosphamide clearance value following dose 1 was significantly lower than following dose 5 (1.83 ± 1.07 versus 3.68 ± 1.43 L/h/m(2), respectively; mean ± standard deviation from empirical Bayes estimates; P < 0.001). The presence of at least one CYP2B6*6 variant allele was associated with a lower cyclophosphamide clearance following both dose 1 (1.54 ± 0.11 L/h/m(2) versus 2.20 ± 0.31 L/h/m(2), P = 0.033) and dose 5 (3.12 ± 0.17 L/h/m(2) versus 4.35 ± 0.37 L/h/m(2), P = 0.0028), as compared to homozygous wild-type patients. No pharmacokinetic parameters investigated were shown to have a significant influence on progression free survival. CONCLUSION: The results do not support previous findings of a link between cyclophosphamide pharmacokinetics or metabolism and disease recurrence in childhood B-cell NHL. While CYP2B6 genotype was shown to influence pharmacokinetics, there was no clear impact on clinical outcome.

GEANT4 models for the secondary radiation flux in the collimation system of a 300MeV proton microbeam.

In Harbin, we are developing a 300MeV proton microbeam for many applications in space science including upset studies in microelectronic devices, radiation hardness of materials for satellites and radiation effects in human tissues. There are also applications of this facility proposed for proton therapy. The microbeam system will employ a purpose-built proton synchrotron to provide the beam. However there are many challenges to be addressed in the design, construction and operation of this facility. Here we address two important design aspects for which we apply GEANT4 modeling. First, the high energy proton beam interacts strongly with beam line materials, especially the collimation slits, to produce showers of secondary particles which could introduce significant background signals and degrade the resolution of the proton microbeam. Second, the beam transport within the residual vacuum of the beam line may also introduce undesirable background radiation. In both cases mitigation strategies need to be incorporated during the design phase of the new system. We study the use of a dipole magnet following the aperture collimator to reduce the flux of secondary particles incident on the analysis chamber. Monte Carlo simulations are performed using GEANT4 and SRIM. By inserting the dipole magnet, we find as expected a significant reduction in the scattering of protons and other particles, such as neutrons and gamma rays, at the collimation system exit position. Secondary radiation from the residual gas pressure within the beam line vacuum system are also modelled and found to be negligible under the standard operating conditions.