Analysis of cell free DNA to predict outcome to bevacizumab therapy in colorectal cancer patients.

To predict outcome to combination bevacizumab (BVZ) therapy, we employed cell-free DNA (cfDNA) to determine chromosomal instability (CIN), nucleosome footprints (NF) and methylation profiles in metastatic colorectal cancer (mCRC) patients. Low-coverage whole-genome sequencing (LC-WGS) was performed on matched tumor and plasma samples, collected from 74 mCRC patients from the AC-ANGIOPREDICT Phase II trial (NCT01822444), and analysed for CIN and NFs. A validation cohort of plasma samples from the University Medical Center Mannheim (UMM) was similarly profiled. 61 AC-ANGIOPREDICT plasma samples collected before and following BVZ treatment were selected for targeted methylation sequencing. Using cfDNA CIN profiles, AC-ANGIOPREDICT samples were subtyped with 92.3% accuracy into low and high CIN clusters, with good concordance observed between matched plasma and tumor. Improved survival was observed in CIN-high patients. Plasma-based CIN clustering was validated in the UMM cohort. Methylation profiling identified differences in CIN-low vs. CIN high (AUC = 0.87). Moreover, significant methylation score decreases following BVZ was associated with improved outcome (p = 0.013). Analysis of CIN, NFs and methylation profiles from cfDNA in plasma samples facilitates stratification into CIN clusters which inform patient response to treatment.

The Impact of a National Cyberattack Affecting Clinical Trials: The Cancer Trials Ireland Experience.

PURPOSE: Cyberattacks are increasing in health care and cause immediate disruption to patient care, have a lasting impact, and compromise scientific integrity of affected clinical trials. On the May 14, 2021, the Irish health service was the victim of a nationwide ransomware attack. Patient care was disrupted across 4,000 locations, including 18 cancer clinical trials units associated with Cancer Trials Ireland (CTI). This report analyses the impact of the cyberattack on the organization and proposes steps to mitigate the impact of future cyberattacks. METHODS: A questionnaire was distributed to the units within the CTI group; this examined key performance indicators for a period of 4 weeks before, during, and after the attack, and was supplemented by minutes of weekly conference call with CTI units to facilitate information sharing, accelerate mitigation, and support affected units. A total of 10 responses were returned, from three private and seven public hospitals. RESULTS: The effect of the attack on referrals and enrollment to trials was marked, resulting in a drop of 85% in referrals and 55% in recruitment before recovery. Radiology, radiotherapy, and laboratory systems are heavily reliant on information technology systems. Access to all was affected. Lack of preparedness was highlighted as a significant issue. Of the sites surveyed, two had a preparedness plan in place before the attack, both of these being private institutions. Of the eight institutions where no plan was in place, three now have or are putting a plan in place, whereas no plan is in place at the five remaining sites. CONCLUSION: The cyberattack had a dramatic and sustained impact on trial conduct and accrual. Increased cybermaturity needs to be embedded in clinical trial logistics and the units conducting them.

ExPeCT: a randomised trial examining the impact of exercise on quality of life in men with metastatic prostate cancer.

PURPOSE: All patients living with cancer, including those with metastatic cancer, are encouraged to be physically active. This paper examines the secondary endpoints of an aerobic exercise intervention for men with metastatic prostate cancer. METHODS: ExPeCT (Exercise, Prostate Cancer and Circulating Tumour Cells), was a multi-centre randomised control trial with a 6-month aerobic exercise intervention arm or a standard care control arm. Exercise adherence data was collected via heart rate monitors. Quality of life (FACT-P) and physical activity (self-administered questionnaire) assessments were completed at baseline, at 3 months and at 6 months. RESULTS: A total of 61 patients were included (69.4 ± 7.3 yr, body mass index 29.2 ± 5.8 kg/m2). The median time since diagnosis was 34 months (IQR 7-54). A total of 35 (55%) of participants had > 1 region affected by metastatic disease. No adverse events were reported by participants. There was no effect of exercise on quality of life (Cohen's d = - 0.082). Overall adherence to the supervised sessions was 83% (329 out of 396 possible sessions attended by participants). Overall adherence to the non-supervised home exercise sessions was 72% (months 1-3) and 67% (months 3-6). Modelling results for overall physical activity scores showed no significant main effect for the group (p-value = 0.25) or for time (p-value = 0.24). CONCLUSION: In a group of patients with a high burden of metastatic prostate cancer, a 6-month aerobic exercise intervention did not lead to change in quality of life. Further exercise studies examining the role of exercise for people living with metastatic prostate cancer are needed. TRIAL REGISTRATION: The trial was registered at clinicaltrials.gov (NCT02453139) on May 25th 2015.

Impact of the COVID-19 pandemic on cancer care in Ireland - Perspectives from a COVID-19 and Cancer Working Group.

Upon the COVID-19 pandemic onset in Ireland, cancer service disruptions occurred due to prioritisation of COVID-19 related care, redeployment of staff, initial pausing of screening, diagnostic, medical and surgical oncology procedures, staff shortages due to COVID-19 infection and impacts on the physical and mental health of cancer healthcare workers. This was coupled with reluctance among people with symptoms suspicious for cancer to attend for clinical evaluation, due to concerns of contracting the virus. This was further compounded by a cyber-attack on national health service IT systems on May 14th 2021. The Irish Cancer Society, a national cancer charity with a role in advocacy, research and patient supports, convened a multi-disciplinary stakeholder group (COVID-19 and Cancer Working Group) to reflect on and understand the impact of the pandemic on cancer patients and services in Ireland, and discuss potential mitigation strategies. Perspectives on experiences were gathered across domains including timeliness of data acquisition and its conversion into intelligence, and the resourcing of cancer care to address cancer service impacts. The group highlighted aspects for future research to understand the long-term pandemic impact on cancer outcomes, while also highlighting potential strategies to support cancer services, build resilience and address delayed diagnosis. Additional measures include the need for cancer workforce recruitment and retention, increased mental health supports for both patients and oncology professionals, improvements to public health messaging, a near real-time multimodal national cancer database, and robust digital and physical infrastructure to mitigate impacts of the current pandemic and future challenges to cancer care systems.

The SARS-CoV-2 Pandemic and Cancer Trials Ireland: Impact, Resolution and Legacy.

BACKGROUND: Cancer Trials Ireland (CTI) is the national cooperative group in Ireland. The SARS-CoV-2 pandemic led to significant ongoing disruptive change in healthcare from March 2020 to the present day. Its impact and legacy on a national clinical trials organisation was assessed. METHODS: A review was conducted of prospectively acquired communications, team logs and time sheets, trial activation, closure and accrual, for the period 2019 to September 2021. An online survey of the impact of the pandemic on clinical investigators and of clinical trials units was performed. A National Cancer Retreat was organised on 21 May 2021 to identify and address pandemic related disruption and develop adaptive strategies. RESULTS: In the weeks after the pandemic was declared, remote working was initiated by all central office staff. Nationally, clinical trial accrual fell by 54% compared to the same period in 2019, radiotherapy trial accrual by 90%, and translational studies by 36%. Staff reassignment of research nurse staff occurred in 60% of units, trial monitoring was reduced in 42%, and trial initiations fell by 67%. Extreme fluctuations in monitoring hours were noted paralleling lockdown measures. Significant impact on all clinical trials units was noted including staff reassignments, reduced access to diagnostic imaging and reduced institutional supports. Remote clinic visits and remote monitoring was widely adopted. The National Cancer Retreat identified flexibility in trial conduct, staff recruitment and retention, the need for harmonisation of processes, and research staff support in the context of remote working as priorities. CONCLUSION: The pandemic has had a significant ongoing negative impact on cancer clinical trial activity in Ireland. Adaptive strategies including trial flexibility, expanded telehealth and remote monitoring, harmonisation of processes and staff support have been identified as priorities to ameliorate this impact, and develop a more sustainable clinical trial ecosystem.

Circulating Tumour Cell Numbers Correlate with Platelet Count and Circulating Lymphocyte Subsets in Men with Advanced Prostate Cancer: Data from the ExPeCT Clinical Trial (CTRIAL-IE 15-21).

Interactions between circulating tumour cells (CTCs) and platelets are thought to inhibit natural killer(NK)-cell-induced lysis. We attempted to correlate CTC numbers in men with advanced prostate cancer with platelet counts and circulating lymphocyte numbers. Sixty-one ExPeCT trial participants, divided into overweight/obese and normal weight groups on the basis of a BMI ≥ 25 or <25, were randomized to participate or not in a six-month exercise programme. Blood samples at randomization, and at three and six months, were subjected to ScreenCell filtration, circulating platelet counts were obtained, and flow cytometry was performed on a subset of samples (n = 29). CTC count positively correlated with absolute total lymphocyte count (r2 = 0.1709, p = 0.0258) and NK-cell count (r2 = 0.49, p < 0.0001). There was also a positive correlation between platelet count and CTC count (r2 = 0.094, p = 0.0001). Correlation was also demonstrated within the overweight/obese group (n = 123, p < 0.0001), the non-exercise group (n = 79, p = 0.001) and blood draw samples lacking platelet cloaking (n = 128, p < 0.0001). By flow cytometry, blood samples from the exercise group (n = 15) had a higher proportion of CD3+ T-lymphocytes (p = 0.0003) and lower proportions of B-lymphocytes (p = 0.0264) and NK-cells (p = 0.015) than the non-exercise group (n = 14). These findings suggest that CTCs engage in complex interactions with the coagulation cascade and innate immune system during intravascular transit, and they present an attractive target for directed therapy at a vulnerable stage in metastasis.

Prognostic value of the 6-gene OncoMasTR test in hormone receptor-positive HER2-negative early-stage breast cancer: Comparative analysis with standard clinicopathological factors.

AIM: The aim of the study was to assess the prognostic performance of a 6-gene molecular score (OncoMasTR Molecular Score [OMm]) and a composite risk score (OncoMasTR Risk Score [OM]) and to conduct a within-patient comparison against four routinely used molecular and clinicopathological risk assessment tools: Oncotype DX Recurrence Score, Ki67, Nottingham Prognostic Index and Clinical Risk Category, based on the modified Adjuvant! Online definition and three risk factors: patient age, tumour size and grade. METHODS: Biospecimens and clinicopathological information for 404 Irish women also previously enrolled in the Trial Assigning Individualized Options for Treatment [Rx] were provided by 11 participating hospitals, as the primary objective of an independent translational study. Gene expression measured via RT-qPCR was used to calculate OMm and OM. The prognostic value for distant recurrence-free survival (DRFS) and invasive disease-free survival (IDFS) was assessed using Cox proportional hazards models and Kaplan-Meier analysis. All statistical tests were two-sided ones. RESULTS: OMm and OM (both with likelihood ratio statistic [LRS] P < 0.001; C indexes = 0.84 and 0.85, respectively) were more prognostic for DRFS and provided significant additional prognostic information to all other assessment tools/factors assessed (all LRS P ≤ 0.002). In addition, the OM correctly classified more patients with distant recurrences (DRs) into the high-risk category than other risk classification tools. Similar results were observed for IDFS. DISCUSSION: Both OncoMasTR scores were significantly prognostic for DRFS and IDFS and provided additional prognostic information to the molecular and clinicopathological risk factors/tools assessed. OM was also the most accurate risk classification tool for identifying DR. A concise 6-gene signature with superior risk stratification was shown to increase prognosis reliability, which may help clinicians optimise treatment decisions.

Harmonising the human biobanking consent process: an Irish experience.

Biobanks are repositories of human biological samples and data. They are an important component of clinical research in many disease areas and often represent the first step toward innovative treatments. For biobanks to operate, researchers need human participants to give their samples and associated health data. In Ireland, research participants must provide their freely given informed consent for their samples and data to be taken and used for research purposes. Biobank staff are responsible for communicating the relevant information to participants prior to obtaining their consent, and this communication process is supported by documentation in the form of Participant Information Leaflets and Informed Consent Forms (PILs/ICFs). PILs/ICFs should be concise, intelligible, and contain relevant information. While not a substitute for layperson and research staff discussions, PILs and ICFs ensure that a layperson has enough information to make an informed choice to participate or not. However, PILs/ICFs are often lengthy, contain technical language and can be complicated and onerous for a layperson to read. The introduction of the General Data Protection Regulation (GDPR) and the related Irish Health Research Regulation (HRR) presented additional challenges to the Irish biobank community. In May 2019, the National Biobanking Working Group (NBWG) was established in Ireland. It consists of members from diverse research backgrounds located in universities, hospitals and research centres across Ireland and a public/patient partner. The NBWG aimed to develop a suite of resources for health research biobanks via robust and meaningful patient engagement, which are accessible, GDPR/HRR-compliant and could be used nationally, including a PIL/ICF template. This open letter describes the process whereby this national biobank PIL/ICF template was produced. The development of this template included review by the Patient Voice in Cancer Research, led by Professor Amanda McCann at University College Dublin and the Health Research Data Protection Network.

Platelet cloaking of circulating tumour cells in patients with metastatic prostate cancer: Results from ExPeCT, a randomised controlled trial.

BACKGROUND: Circulating tumour cells (CTCs) represent a morphologically distinct subset of cancer cells, which aid the metastatic spread. The ExPeCT trial aimed to examine the effectiveness of a structured exercise programme in modulating levels of CTCs and platelet cloaking in patients with metastatic prostate cancer. METHODS: Participants (n = 61) were randomised into either standard care (control) or exercise arms. Whole blood was collected for all participants at baseline (T0), three months (T3) and six months (T6), and analysed for the presence of CTCs, CTC clusters and platelet cloaking. CTC data was correlated with clinico-pathological information. RESULTS: Changes in CTC number were observed within group over time, however no significant difference in CTC number was observed between groups over time. Platelet cloaking was identified in 29.5% of participants. A positive correlation between CTC number and white cell count (WCC) was observed (p = 0.0001), in addition to a positive relationship between CTC clusters and PSA levels (p = 0.0393). CONCLUSION: The presence of platelet cloaking has been observed in this patient population for the first time, in addition to a significant correlation between CTC number and WCC. TRIAL REGISTRATION: ClincalTrials.gov identifier NCT02453139.

Understanding and Attitudes toward Cancer Clinical Trials among Patients with a Cancer Diagnosis: National Study through Cancer Trials Ireland.

Cancer clinical trials (CCTs) are critical to translation and development of better therapies to improve outcomes. CCTs require adequate patient involvement but accrual rates are low globally. Several known barriers impede participation and knowing how subpopulations differ in understanding of CCTs can foster targeted approaches to aid accrual and advance cancer treatments. We conducted the first nationwide survey of 1089 patients attending 14 Irish cancer centres, assessing understanding of fundamental concepts in CCT methodology and factors that influence participation, to help tailor patient support for accrual to CCTs. Two-thirds (66%) of patients reported never having been offered a CCT and only 5% of those not offered asked to participate. Misunderstanding of clinical equipoise was prevalent. There were differences in understanding of randomisation of treatment by age (p < 0.0001), ethnicity (p = 0.035) and marital status (p = 0.013), and 58% of patients and 61% previous CCT participants thought that their doctor would ensure better treatment in CCTs. Females were slightly more risk averse. Males indicated a greater willingness to participate in novel drug trials (p = 0.001, p = 0.003). The study identified disparities in several demographics; older, widowed, living in provincial small towns and fewer years-educated patients had generally poorer understanding of CCTs, highlighting requirements for targeted support in these groups.

Combination of variations in inflammation- and endoplasmic reticulum-associated genes as putative biomarker for bevacizumab response in KRAS wild-type colorectal cancer.

Chemotherapy combined with the angiogenesis inhibitor bevacizumab (BVZ) is approved as a first-line treatment in metastatic colorectal cancer (mCRC). Limited clinical benefit underpins the need for improved understanding of resistance mechanisms and the elucidation of novel predictive biomarkers. We assessed germline single-nucleotide polymorphisms (SNPs) in 180 mCRC patients (Angiopredict [APD] cohort) treated with combined BVZ + chemotherapy and investigated previously reported predictive SNPs. We further employed a machine learning approach to identify novel associations. In the APD cohort IL8 rs4073 any A carriers, compared to TT carriers, were associated with worse progression-free survival (PFS) (HR = 1.51, 95% CI:1.03-2.22, p-value = 0.037) and TBK1 rs7486100 TT carriers, compared to any A carriers, were associated with worse PFS in KRAS wild-type (wt) patients (HR = 1.94, 95% CI:1.04-3.61, p-value = 0.037), replicating previous findings. Machine learning identified novel associations in genes encoding the inflammasome protein NLRP1 and the ER protein Sarcalumenin (SRL). A negative association between PFS and carriers of any A at NLRP1 rs12150220 and AA for SRL rs13334970 in APD KRAS wild-type patients (HR = 4.44, 95% CI:1.23-16.13, p-value = 0.005), which validated in two independent clinical cohorts involving BVZ, MAVERICC and TRIBE. Our findings highlight a key role for inflammation and ER signalling underpinning BVZ + chemotherapy responsiveness.

Pilot study of bevacizumab in combination with docetaxel and cyclophosphamide as adjuvant treatment for patients with early stage HER-2 negative breast cancer, including analysis of candidate circulating markers of cardiac toxicity: ICORG 08-10 trial.

BACKGROUND: Combining bevacizumab and chemotherapy produced superior response rates compared with chemotherapy alone in metastatic breast cancer. As bevacizumab may cause hypertension (HTN) and increase the risk of cardiac failure, we performed a pilot study to evaluate the feasibility and toxicity of a non-anthracycline-containing combination of docetaxel with cyclophosphamide and bevacizumab in early stage breast cancer patients. METHODS: Treatment consisted of four 3-weekly cycles of docetaxel and cyclophosphamide (75/600 mg/m2). Bevacizumab was administered 15 mg/kg intravenously on day 1, and then every 3 weeks to a total of 18 cycles of treatment. Serum biomarker concentrations of vascular endothelial growth factor (VEGF), cardiac troponin-I (cTnI), myeloperoxidase (MPO), and placental growth factor (PlGF) were quantified using enzyme-linked immunosorbent assay (ELISA) in 62 patients at baseline and whilst on treatment to determine their utility as biomarkers of cardiotoxicity, indicated by left ventricular ejection fraction (LVEF). RESULTS: A total of 106 patients were accrued in nine sites. Median follow up was 65 months (1-72 months). Seventeen protocol-defined relapse events were observed, accounting for an overall disease-free survival (DFS) rate of 84%. The DFS rates for hormone receptor positive (HR+) and triple-negative (TN) patients were 95% versus 43%, respectively. The median time to relapse was 25 (12-54) months in TN patients versus 38 (22-71) months in HR+ patients. There have been 13 deaths related to breast cancer . The overall survival (OS) rate was 88%. The 5-year OS rate in HR+ versus TN was 95% versus 57%. None of the measured biomarkers predicted the development of cardiotoxicity. CONCLUSIONS: We observed a low relapse rate in node-positive, HR+ patients; however, results in TN breast cancer were less encouraging. Given the negative results of three large phase III trials, it is unlikely that this approach will be investigated further. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT00911716.

Copy number load predicts outcome of metastatic colorectal cancer patients receiving bevacizumab combination therapy.

Increased copy number alterations (CNAs) indicative of chromosomal instability (CIN) have been associated with poor cancer outcome. Here, we study CNAs as potential biomarkers of bevacizumab (BVZ) response in metastatic colorectal cancer (mCRC). We cluster 409 mCRCs in three subclusters characterized by different degrees of CIN. Tumors belonging to intermediate-to-high instability clusters have improved outcome following chemotherapy plus BVZ versus chemotherapy alone. In contrast, low instability tumors, which amongst others consist of POLE-mutated and microsatellite-instable tumors, derive no further benefit from BVZ. This is confirmed in 81 mCRC tumors from the phase 2 MoMa study involving BVZ. CNA clusters overlap with CRC consensus molecular subtypes (CMS); CMS2/4 xenografts correspond to intermediate-to-high instability clusters and respond to FOLFOX chemotherapy plus mouse avastin (B20), while CMS1/3 xenografts match with low instability clusters and fail to respond. Overall, we identify copy number load as a novel potential predictive biomarker of BVZ combination therapy.

Loss of Chromosome 18q11.2-q12.1 Is Predictive for Survival in Patients With Metastatic Colorectal Cancer Treated With Bevacizumab.

Purpose Patients with metastatic colorectal cancer (mCRC) have limited benefit from the addition of bevacizumab to standard chemotherapy. However, a subset probably benefits substantially, highlighting an unmet clinical need for a biomarker of response to bevacizumab. Previously, we demonstrated that losses of chromosomes 5q34, 17q12, and 18q11.2-q12.1 had a significant correlation with progression-free survival (PFS) in patients with mCRC treated with bevacizumab in the CAIRO2 clinical trial but not in patients who did not receive bevacizumab in the CAIRO trial. This study was designed to validate these findings. Materials and Methods Primary mCRC samples were analyzed from two cohorts of patients who received bevacizumab as first-line treatment; 96 samples from the European multicenter study Angiopredict (APD) and 81 samples from the Italian multicenter study, MOMA. A third cohort of 90 samples from patients with mCRC who did not receive bevacizumab was analyzed. Copy number aberrations of tumor biopsy specimens were measured by shallow whole-genome sequencing and were correlated with PFS, overall survival (OS), and response. Results Loss of chromosome 18q11.2-q12.1 was associated with prolonged PFS most significantly in both the cohorts that received bevacizumab (APD: hazard ratio, 0.54; P = .01; PFS difference, 65 days; MOMA: hazard ratio, 0.55; P = .019; PFS difference, 49 days). A similar association was found for OS and overall response rate in these two cohorts, which became significant when combined with the CAIRO2 cohort. Median PFS in the cohort of patients with mCRC who did not receive bevacizumab and in the CAIRO cohort was similar to that of the APD, MOMA, and CAIRO2 patients without an 18q11.2-q12.1 loss. Conclusion We conclude that the loss of chromosome 18q11.2-q12.1 is consistently predictive for prolonged PFS in patients receiving bevacizumab. The predictive value of this loss is substantiated by a significant gain in OS and overall response rate.

Assessment of concordance between fresh-frozen and formalin-fixed paraffin embedded tumor DNA methylation using a targeted sequencing approach.

DNA methylation is altered in many types of disease, including metastatic colorectal cancer. However, the methylome has not yet been fully described in archival formalin-fixed paraffin embedded (FFPE) samples in the context of matched fresh-frozen (FF) tumor material at base-pair resolution using a targeted approach. Using next-generation sequencing, we investigated three pairs of matched FFPE and FF samples to determine the extent of their similarity. We identified a 'bowing' pattern specific to FFPE samples categorized by a lower CG proportion at the start of sequence reads. We have found no evidence that this affected methylation calling, nor concordance of results. We also found no significant increase in deamination, measured by C>T transitions, previously considered a result of crosslinking DNA by formalin fixation and a barrier to the use of FFPE in methylation studies. The methods used in this study have shown sensitivity of between 60-70% based on positions also methylated in colorectal cancer cell lines. We demonstrate that FFPE material is a useful source of tumor material for methylation studies using targeted sequencing.

Outcome of Colorectal Cancer Patients Treated with Combination Bevacizumab Therapy: A Pooled Retrospective Analysis of Three European Cohorts from the Angiopredict Initiative.

BACKGROUND/AIMS: This study is aimed at analyzing the survival rates and prognostic factors of stage IV colorectal cancer patients from 3 European cohorts undergoing combination chemotherapy with bevacizumab. METHODS: Progression free-survival (PFS) and overall survival (OS) were analyzed in 172 patients using the Kaplan-Meier method and uni- and multivariable Cox proportional hazards regression models. RESULTS: The median PFS was 9.7 and the median OS 27.4 months. Patients treated at centers in Germany (n = 97), Ireland (n = 32), and The Netherlands (n = 43) showed a median PFS of 9.9, 9.2, and 9.7 months, OS of 34.0, 20.5, and 25.1 months, respectively. Patients >65 years had a significantly shorter PFS (9.5 vs. 9.8 months) but not OS (27.4 vs. 27.5 months) than younger patients. High tumor grade (G3/4) was associated with a shorter PFS, T4 classification with both shorter PFS and OS. Fluoropyrimidine (FP) chemotherapy backbones (doublets and single) had comparable outcomes, while patients not receiving FP backbones had a shorter PFS. In multivariable analysis, age and non-FP backbone were associated with inferior PFS, T4 classification and therapy line >2nd were significantly associated with poor PFS and OS. CONCLUSION: The observed survival rates confirm previous studies and demonstrate reproducible benefits of combination bevacizumab regimens. Classification T4, non-FP chemotherapy backbone, and age >65 were associated with inferior outcome.

Characterization and response of newly developed high-grade glioma cultures to the tyrosine kinase inhibitors, erlotinib, gefitinib and imatinib.

High-grade gliomas (HGG), are the most common aggressive brain tumours in adults. Inhibitors targeting growth factor signalling pathways in glioma have shown a low clinical response rate. To accurately evaluate response to targeted therapies further in vitro studies are necessary. Growth factor pathway expression using epidermal growth factor receptor (EGFR), mutant EGFR (EGFRvIII), platelet derived growth factor receptor (PDGFR), C-Kit and C-Abl together with phosphatase and tensin homolog (PTEN) expression and downstream activation of AKT and phosphorylated ribosomal protein S6 (P70S6K) was analysed in 26 primary glioma cultures treated with the tyrosine kinase inhibitors (TKIs) erlotinib, gefitinib and imatinib. Response to TKIs was assessed using 50% inhibitory concentrations (IC(50)). Response for each culture was compared with the EGFR/PDGFR immunocytochemical pathway profile using hierarchical cluster analysis (HCA) and principal component analysis (PCA). Erlotinib response was not strongly associated with high expression of the growth factor pathway components. PTEN expression did not correlate with response to any of the three TKIs. Increased EGFR expression was associated with gefitinib response; increased PDGFR-α expression was associated with imatinib response. The results of this in vitro study suggest gefitinib and imatinib may have therapeutic potential in HGG tumours with a corresponding growth factor receptor expression profile.

The effect of tricyclic antidepressants on cutaneous melanoma cell lines and primary cell cultures.

The tricyclic antidepressants have previously been shown to exert activity against glioma cells in vitro. Initial studies in cell lines suggested that this might extend to melanoma cells. We have therefore conducted a study in primary cell cultures from metastatic cutaneous melanoma deposits using a well established ATP-based tumour chemosensitivity assay to confirm and extend these findings. Two cell lines and eight primary cell cultures from metastatic melanoma deposits were exposed to three tricyclic drugs, amitriptyline, nortriptyline and clomipramine, at concentrations ranging from 200 to 6.25 µmol/l in the ATP-based tumour chemosensitivity assay. All three drugs showed activity, although nortriptyline was more active than clomipramine or amitriptyline in both cell lines and primary cell cultures, with an IC50 of 9, 27 and 33 µmol/l, respectively. Tricyclic agents show activity against melanoma in vitro. This could be related to the lysosomal effects based on their cationic amphiphilic properties, or effects at the mitochondrial membrane.

Imatinib and docetaxel in combination can effectively inhibit glioma invasion in an in vitro 3D invasion assay.

The main problem in the treatment of malignant astrocytomas is their invasive behaviour. Successful resection of the main tumour mass cannot prevent recurrence due to single cells invading the surrounding brain parenchyma at the time of diagnosis. The classical combination therapy, PCV (Procarbazine, CCNU and Vincristine) used for over 30 years; has shown its clinical effectiveness in the treatment of malignant astrocytomas and glioblastomas is still doubtful. Using an in vitro three dimensional invasion model, we tested the effect of the tyrosine kinase inhibitor imatinib and the microtubule inhibitor docetaxel on the invasion activity of a panel of astrocytic tumour cell lines, including two established glioma cell lines, IPSB-18 and SNB-19, and two primary cell lines, originating from glioblastomas, CLOM002 and UPHHJA, and in normal astrocytes. A dose response curve for each drug alone and in combination was determined. The half maximal inhibitory concentration (IC(50)) concentration of imatinib was between 15.7 and 18.7 µM, which did not affect invasion activity of the cell lines. The IC(50) concentration of docetaxel was between 0.7 and 19.8 nM, and at 14.9 nM docetaxel had a slight transient inhibitory effect on invasion activity of all tested cells. The combination of imatinib at 13.5 µM and docetaxel at 14.9 nM, however, synergistically inhibited cell growth and invasion activity and could not be reversed by drug removal. A combination treatment with tyrosine kinase inhibitors and cytotoxic drugs shows promise in tackling both glioma proliferation and invasion, and could present a new treatment regimen for malignant astrocytomas.

Hypoxia helps glioma to fight therapy.

Despite major improvements in the surgical management the prognosis for patients bearing malignant gliomas is still dismal. Malignant gliomas are notoriously resistant to treatment and the survival time of patients is between 3-8 years for low-grade and anaplastic gliomas and 6 - 12 month for glioblastoma. Increasing malignancy of gliomas correlates with an increase in cellularity and a poorly organized tumor vasculature leading to insufficient blood supply, hypoxic areas and ultimately to the formation of necrosis, a characteristic of glioblastoma. Hypoxic/necrotic tumors are more resistant to chemotherapy and radiation. Hypoxia induces either directly or indirectly (through the activation of transcription factors) changes in the biology of a tumor and its microenvironment leading to increased aggressiveness and tumor resistance to chemotherapy and radiation. This review is focused on hypoxia-induced molecular changes affecting glioma biology and therapy.