Silent theatre 1 – Monday 4 November

12:40 pm-2:00 pm

Room: Hall 4


Programme session type(s): Silent theatre

A potent synergy between FOXG1 overexpression and Wnt signaling drives cell cycle re-entry in quiescent glioblastoma stem cells
Speaker: Faye Robertson
Affiliation: University of Edinburgh, Edinburgh, UK

Abstract:

Glioblastoma is a malignant brain tumour which is universally fatal. Stem cells within the tumour exist in a quiescent state, evade destruction and reactivate, causing relapse. These stem cells are known to overexpress the transcription factor FOXG1.

 

 

 

Method

We use an in vitro model of quiescence in mouse neural stem cells, incorporating a doxycycline inducible human FOXG1 overexpression cassette, to identify, through high content pharmacological screening, a synergistic relationship between high FOXG1 expression and inhibition of glycogen synthase kinase 3 (GSK3) in driving cells into an active, proliferative state.

We quantify this effect using EdU incorporation assays and demonstrate the high efficiency of cell cycle re-entry with colony forming assays. We use pharmacological and genetic approaches to demonstrate that the synergy is effected by canonical Wnt signaling and that it is relevant in human glioblastoma stem cells (GSCs).

Results

The effect of GSK3 inhibition can be phenocopied both by Wnt3a and by inducible constitutively active beta-catenin, suggesting that the synergy is effected through beta-catenin, the key downstream effector of canonical Wnt signaling. Furthermore, the combined effect of FOXG1 overexpression and GSK3 inhibition on exit from quiescence can be abrogated by Wnt inhibitors.

This effect is present in patient-derived human GSCs and it is abolished by excision of FOXG1. Additionally, we show that the groucho-binding domain of the FOXG1 protein is necessary for the response of human GSCs to GSK3 inhibition, consistent with a putative mechanism whereby FOXG1 may sequester TLE1/groucho, a co-repressor at Wnt target genes.

 

Finally, we have developed a mouse model of glioblastoma by excision of NF-1 and Pten and overexpression of EGFRvIII in cells with inducible active beta-catenin and FOXG1.

Conclusion

Targeting the synergistic relationship between FOXG1 and beta-catenin may provide an exciting therapeutic opportunity in preventing relapse and improving the prognosis of glioblastoma.

Overcoming sorafenib resistance in hepatocellular carcinoma by fasting
Speaker: Jelena Krstic
Affiliation: Medical University of Graz, Graz, Austria, Gottfried Schatz Research Center for Cell Signaling, Metabolism and Aging, Graz, Austria

Abstract:

Fasting is suggested as adjuvant for cancer treatment, since it increases cancer cell sensitivity to chemo- and targeted therapy in various cancer models. The mechanisms of this pleiotropic effect of fasting (i.e. starvation) are not fully elucidated, especially in therapy-resistant cancers. We investigated whether starvation can sensitize therapy-resistant hepatocellular carcinoma (HCC) cells to sorafenib, the only first-line treatment currently in use.

Method

HepG2 cells were grown in growth or starvation medium and treated with sorafenib for 24h. Cell viability was analyzed by colorimetric assay and by flow cytometry. Energy metabolism was assayed using XF96 Seahorse analyzer. Components of growth pathways (mTOR and Ras/Raf/MEK/ERK) were analyzed by western blot. p53 knock-out cells were generated using Crispr/Cas9 system. For xenograft assays, cells were injected subcutaneously into the flanks of NMRI-Foxn1nu mice, which were then treated with vehicle or sorafenib and fed ad libitum or intermittently fasted during four weeks. 

Results

Sorafenib-resistant HepG2 cells were sensitized by starvation, with viability decreasing below 15% after 24h of combined treatment. Similar effect was observed in xenografts: tumors grew at a slower rate only when sorafenib-treated mice were exposed to an intermittent fasting regimen. Mechanistically, bioenergetic profiling suggested abrogated oxidative phosphorylation in HepG2 cells, as an early event in sensitization. Starvation-supported sorafenib also blocked the two major growth pathways, mTOR and Ras/Raf/MEK/ERK. Furthermore, the sensitizing effect of starvation was blunted in p53-deficient cells indicating dependency on p53-mediated cell death.

Conclusion

We tested whether starvation can augment the effects of sorafenib in therapy-resistant HCC. Combined treatment could sensitize resistant cancer cells in vitro as well as in xenografts and resulted in complete inhibition of mitochondrial respiration and ATP production, multiple growth pathway blockage, and subsequent p53-mediated cell death. Thus, our study suggests fasting and sorafenib treatment as potential polytherapy for HCC.

Integrating Germline and Somatic Genetic Test Result Data into the English National Cancer Registration and Analysis Service
Speaker: Steven Hardy
Affiliation: National Disease Registration, Public Health England, London, UK

Abstract:

The National Cancer Registration and Analysis Service, run by Public Health England, collects, collates and quality assures data on every cancer diagnosed or treated within English NHS hospitals. We are now linking this population-level clinical data to germline and somatic genetic test results collected from NHS laboratories.

Method

We have established regular, high quality, somatic test result feeds from eleven NHS genetics and pathology laboratories, covering approximately 80% of solid tumour molecular diagnostic testing within England. We also have germline data submissions from ten genetics laboratories; we have developed a novel method to pseudonymise germline demographics upon upload, thus protecting the identity of healthy people undergoing predictive genetic testing.

We have developed the NCRAS registration system to capture source laboratory data in diverse formats; records are standardised by a combination of computational methods and manual registration by a core group of staff specially trained in interpreting molecular test results.

Results

Our pilot work covering 12 months’ worth of somatic testing data has recorded and linked >60,000 genetic test results on >26,000 tumours, covering >1,100 distinct combinations of gene and tumour site. In addition, we have collated data on germline tests on the BRCA1 and BRCA2 genes and released summary variant counts on >22,000 individuals back to the NHS clinical genetics community. The released aggregate data are used to calculate variant frequencies and assist in national consensus reclassification of BRCA1/2 variants of uncertain clinical significance.

Conclusion

This work will enable audit of the scope, availability and usage of molecular diagnostics within NHS cancer pathways and services, and will support epidemiological research and clinical trials. Patient-level and tumour-level linkage of molecular result data embedded within NCRAS is already changing clinical management for high risk BRCA1/2 families, and future work will allow new discoveries about associations between individual DNA aberrations, treatments, and overall outcomes and survival.

Anti-oestrogen medication use in breast cancer patients and subsequent risk of gastrointestinal cancers: a two country pooled analysis
Speaker: Úna McMenamin
Affiliation: Queen’s University Belfast, Belfast, UK

Abstract:

There is a strong male predominance in the incidence of oesophageal and stomach cancer, and to a lesser extent, colorectal cancer, suggesting that oestrogens may be protective against the development of these cancers. In a pooled analysis of two large European breast cancer cohorts, we aimed to evaluate associations between anti-oestrogen medication use and risk of upper GI (oesophageal or stomach) and colorectal cancer.

Method

Breast cancer patients were identified from the Belgian Cancer Registry (2004-2014) and the Scottish Cancer Registry (2009-2017). Linkages to national health insurance records (Belgium) and the Prescribing Information System (Scotland) provided detailed information on anti-oestrogen medication use, including tamoxifen and aromatase inhibitors. The primary outcome was GI cancer (UGI or colorectal). Time-dependent Cox regression was used to calculate hazard ratios (HRs) and 95% confidence intervals (CIs) for hormone therapy use and GI cancer risk, adjusting for potential confounders. Fixed effects meta-analysis was used to pool results between the two cohorts.

Results

A total of 113,771 breast cancer patients (Belgium: 87,166, Scotland: 26,605), were included. During follow-up, 701 GI cancers occurred including 151 UGI and 550 colorectal. Overall, no association was shown for anti-oestrogen medication use and risk of GI cancer in pooled analysis (adjusted HR: 0.92, 95% CI 0.74, 1.15). In analysis by cancer site, findings were similar for colorectal cancer (pooled adjusted HR: 0.84, 95% CI 0.66, 1.07) and although HRs were raised for UGI cancer, results were non-significant (pooled adjusted HR: 1.47, 95% CI 0.85, 2.53). Similar results were obtained in dose-response analysis.

Conclusion

In a two-country study of breast cancer patients, we found little evidence of an association between anti-oestrogen medications and risk of GI cancers of the UGI or colorectum. Given the relatively small number of UGI cancer cases, further studies, preferably through pooled analyses of multiple cohorts, are needed to validate our findings.

The landscape of hepatocellular carcinoma in the UK in the past 20 years: the HCCUK/NCRAS partnership.
Speaker: Anya Burton
Affiliation: Public Health England, London, UK

Abstract:

The HCC-UK/NCRAS partnership was created in 2017 to facilitate a wide programme of research relating to hepatocellular carcinoma (HCC) using data available within the National Cancer Registration and Analysis Service (NCRAS). NCRAS data includes tumour- and patient-specific variables, diagnosis and treatment information. These individual-level data are linked to multiple datasets including Hospital Episodes Statistics (HES).

Aim: The partnership programme is examining the epidemiology of HCC in England, including regional variation in incidence, routes to diagnosis, treatment and survival, as well as the economic burden.

Method

HCC cases were identified using ICD10-O-2 code C22.0 and morphology code M8170.  Demographic characteristics were explored and European age-standardised incidence and mortality rates per 100,000 person years calculated. Linked HES codes were used to identify the presence and severity of cirrhosis.

Results

62,135 primary liver cancer cases were diagnosed in England between 1997 and 2016. 29,906 of these were HCC.  For HCC the mean age at diagnosis was 68.4 years and the male to female ratio was 3.4. Overall 25% of all HCC cases were from the most deprived population quintile.  58% of HCC cases were identified as having cirrhosis and, of these, 42% had decompensated cirrhosis, through linked HES data.  The majority of HCC patients did not receive specific anticancer treatment. The two most common Routes to Diagnosis were emergency presentation (35%) and GP referral (30%).

Conclusion

HCC incidence and mortality have tripled over the last 20 years; the most deprived individuals are most at risk. HCC is often associated with cirrhosis and more than one in five individuals diagnosed with HCC has advanced cirrhosis such that treatment options for HCC are severely limited. These trends highlight the urgent need to address prevention strategies for both liver disease and hepatocellular carcinoma specifically at regional and population level.

A reference guide for patient and public involvement contributors. How the ECMC Network PPI group developed a resource for patients and the public
Speaker: Nikki Hayward
Affiliation: Oxford University Hospitals NHS Trust, Oxford, UK

Abstract:

Patient and public involvement is now embedded into many aspects of cancer research, including gaining thoughts and opinions on the earliest design phases, through to the provision of lay perspectives on ethics committees, trial steering committees and service provision.

When the ECMC network patient and public involvement group was originally established, they identified a gap in training for lay people involved in early phase cancer research. To address this gap, the group decided to produce a handbook which would support people affected by cancer who were taking part in PPI activities. 

Method

The handbook evolved over a period of two years, through face to face meetings of the working group, teleconferences and virtual review of content by the wider group. The handbook was produced by a combination of lay members and professionals. Reviews of content by PPI committees across the UK were sought to provide the widest possible perspective prior to deciding on the final version.

Results

To collate a condensed version of many aspects of early phase research, and ensure the information could be understood by lay people was challenging and relied on the input and knowledge of a combination of professionals and people affected by cancer.

Feedback from the wider PPI groups gave us clearer direction for the final version, especially with regards to layout. The results of the combined efforts of the ECMC PPI working group, the handbook will be published later this year.

Conclusion

Patients and public who get involved with activities to support cancer research are expected to give feedback on complicated proposals which use scientific language, with no previous background in the topic. Providing a handbook for those involved in PPI activity will aid understanding of the projects being reviewed and enable lay people to give feedback with confidence.

Clinical utility of circulating microRNAs in malignant germ cell tumours
Speaker: Matthew Murray
Affiliation: University of Cambridge, Cambridge, UK

Abstract:

The protein biomarkers AFP/HCG have limited sensitivity/specificity for diagnosing malignant germ-cell-tumours (GCTs). We previously showed that microRNAs from the miR-371-373 and miR-302/367 clusters are universally overexpressed in all malignant GCT tissues, regardless of patient age, tumour site or histological subtype, but are not co-ordinately over-expressed in any other cancer type or disease state. Here we provide an overview of our research studying these microRNAs as GCT biomarkers and consider prospects for future clinical applications.

Method

We have developed a highly sensitive pre-amplified qRT-PCR technique for robust detection of microRNAs from the miR-371-373 and miR-302/367 clusters in biospecimens. Our method has been adopted by multiple other groups internationally and validated in over 2,000 patients. Our pipeline includes quality control checks, use of an exogenous spike-in control and normalisation against the endogenous microRNA miR-30b-5p, prior to data analysis. Cost analysis and patient/public involvement initiatives regarding acceptability of circulating microRNA testing are underway.

Results

Results from our group and others show that a four-serum miRNA panel (miR-371a-3p, miR-372-3p, miR-373-3p and miR-367-3p) has high sensitivity/specificity for diagnosing malignant GCTs. Individually, miR-371a-3p shows the most utility as it is most dynamic and most accurately reflects disease activity. Levels of these microRNAs are useful for disease-monitoring and early detection of relapse.

Conclusion

Circulating microRNAs should improve future clinical management of patients with malignant GCTs. Potential benefits include: (i) reducing CT scans in follow-up, lowering cumulative radiation exposure to patients and offering cost savings to healthcare systems; (ii) identifying patients with apparent clinical stage I (CSI) seminoma who have persistently elevated serum microRNA levels post-orchidectomy, suggesting micrometastatic disease. Such patients may benefit from adjuvant chemotherapy to prevent subsequent recurrence. This approach will also prevent overtreatment of the majority of CSI patients who are not destined to relapse. Importantly, circulating microRNA testing appears highly acceptable to patients.

Investigating COX isoform dependency in intestinal tumourigenesis
Speaker: Noha-Ehssan Mohamed
Affiliation: Cancer Research UK Beatson Institute, Glasgow, UK

Abstract:

Inflammation is known to play important roles in sporadic colorectal cancer as well as in colitis-associated cancer. Cyclooxygenase-2 (COX-2), the inducible COX isoform responsible for prostaglandin production, is highly expressed in colorectal adenomas and adenocarcinomas. Targeting COX-2 using non-selective (e.g. aspirin) as well as selective (e.g. celecoxib) COX-2 inhibitors reduces the incidence of colorectal carcinoma and prevents adenoma recurrence. Recently, it has been shown that COX-1 expression is elevated in response to COX-2 deletion in melanoma cell lines and in fibroblasts, resulting in production of prostaglandins. Whether a similar compensation happens in intestinal tumours is not known.

Method

To fully investigate COX isoform dependency, a genetically-engineered mouse (GEM) with a knockout of Ptgs2 (gene coding for COX-2) was crossed to the small intestinal adenoma ApcMin/+ mouse, to investigate tumour initiation and progression. To investigate possible compensation by COX-1, another GEM where Ptgs1 (gene coding for COX-1) is knocked into the Ptgs2 locus, characterized by a full loss of COX-2 protein and overexpression of COX-1 protein (the COX-1KI/KI mouse), was used. The COX-1KI/KI mouse was crossed to the ApcMin/+ mouse.

Results

In the ApcMin/+ adenoma mouse, COX-2 knock out delayed tumour initiation, prolonged survival, and decreased intestinal tumour formation. Similarly, in the ApcMin/+ COX-1K1/KI mouse, tumour initiation in the small intestine (SI) was delayed as reflected by a decrease in the total number of tumours developed as well as the size of the tumours in SI collected from 120 days old mice. Survival benefit was evident upon ageing ApcMin/+ COX-1KI/KI mice, and this was associated with a decrease in SI tumour numbers. These tumours were characterized by being less proliferative and highly infiltrated by T-cells.

Conclusion

COX-2 is the isoform controlling intestinal tumour initiation and progression. COX-1 does not compensate for COX-2 in intestinal tumourigenesis.

Improving the effectiveness of breast cancer multidisciplinary team (MDT) meetings: Implementation of a pre-MDT triage meeting
Speaker: Hannah Winter
Affiliation: St Bartholomew’s Hospital

Abstract:

Multidisciplinary team (MDT) working is considered gold standard for cancer care. With increasing workload and patient complexity, the concept of streamlining MDT meetings (MDMs) has been suggested to improve MDT efficiency and facilitate more in-depth discussion of complex patients.

Method

Across 3 satellite breast units, a pre-MDT triage meeting was initiated in September 2018, involving an oncologist, surgeon, radiologist, breast care nurse and MDM co-ordinator.

All preoperative patients were discussed and those who fitted predefined treatment protocols were not then discussed in the main MDM. Established benign and metastatic MDMs were used to full potential. Patients with pending investigations and those where an appropriate plan could be made were also removed.

Outcomes were recorded on electronic patient records and documented on the main MDM list.

Triage outcome data was collected prospectively and changes to suggested outcomes audited within April 2019. 62 day targets were reviewed.

Results

594 patients were discussed in triage MDM between September 2018 and June 2019. Plans were established in 417 (70%), with these patients removed from main MDM discussion. Of these, 77 (18.4%) fulfilled predefined treatment protocols, 67 (16%) were awaiting tests, issues were addressed via email in 33 (8%), a clear plan could be formed in 141 (33.8%) and reports were being double checked in 82 (19.7%).

Within April 2019, a change in proposed plan was seen in 3/74 (4%), with patient choice accountable for 2. 62 day targets improved following the introduction of triage MDM.

Conclusion

The initiation of a pre-MDT triage meeting has dramatically reduced the number of cases discussed within the main MDM facilitating time for discussion of more complex patients and reducing preparation time for MDT clinicians. No detrimental effect on patient safety has been seen and the triage has been widely accepted within the unit as a safe method of improving the effectiveness of the main breast MDM.

Genome-wide CRISPR screen in 3D intestinal organoids to define novel mechanisms critical for colorectal tumorigenesis
Speaker: Patrizia Cammareri
Affiliation: ECRC

Abstract:

The rapid expansion of cancer genome sequencing projects has led to the identification of numerous somatic mutations with undefined function. In the context of colorectal cancer (CRC), mutations commonly occur in a small number of well-studied genes such as APC, KRAS, TP53, which are currently used to guide the treatment selection. However, many mutations occur in genes, which are still yet to be functionally characterized. Although these genes are mutated at moderate/low frequency in CRC, they could be relevant for CRC formation and therefore pose as potential new druggable targets. 

Method

To identify genes that cooperate with loss of Apc to promote intestinal tumorigenesis, we performed a genome-wide CRISPR knockout screen in Apc heterozygous murine intestinal 3D organoids and we used R-spondin withdrawal from the culture medium as selection for tumour initiation. This provides a strong selection assay for wnt independent growth, which is a property of intestinal tumour initiation.

Results

By next generation sequencing, we identified around 700 hits that are potentially involved in CRC initiation. As expected, we found an over representation of a number of well-established wnt regulators genes (Apc or β-catenin) along with genes with poorly characterized role in CRC. We are currently generating a sub-library containing gRNAs targeting the identified hits for further validation. In parallel, we are carrying out characterization of a number of selected candidates such as Fbxw8 and Lzts2. Interestingly, loss of Fbxw8 induces increased clonogenic potential of Apc heterozygous organoids, without altering the expression of stem cell markers, resembling a fetal-like/repair phenotype.

Conclusion

Our genome-wide approach provides a workframe for understanding the relevance of the entire spectrum of mutations in CRC and suggests that moderate/low frequency mutated genes, rather than a “mutational noise”, can contribute to CRC initiation.

Rapid Analysis of Disease State in Liquid Human Serum combining Infrared Spectroscopy and “Digital Drying”
Speaker: Alexandra Sala
Affiliation: University of Strathclyde

Abstract:

In recent years, the use of infrared (IR) spectroscopy to analyse disease state in biofluids has been largely employed with promising results. Diagnosis of brain tumours has been previously investigated with attenuated total reflectance – Fourier transform infrared (ATR-FTIR) spectroscopy on dried human serum samples to eliminate spectral interferences of the water component. Rapid analysis of liquid samples would represent a promising approach for clinical translation.

Method

This research evaluates ATR-FTIR on both liquid and dried samples to investigate “digital drying” as a novel approach for the analysis of spectra obtained from liquid samples. Quantum cascade laser infrared (QCL-IR) based spectroscopic imaging was also utilized on liquid samples to assess the implications of this novel light source on disease classification.

Results

Random Forest (RF) classifications showed better results for ATR-FTIR dried spectra compared to ATR-FTIR liquid spectra, giving a sensitivity of 91.8% versus 89.9% and a specificity of 83.2% versus 81.2% respectively. Both digital drying approaches of water subtraction and least-squares method demonstrated a greater classification performance compared with the dried spectra, reaching sensitivity values higher than 93.0% and specificity values higher than 83.0%. However, the RF classification of QCL-IR data provided lower sensitivity and specificity when compared to ATR-FTIR results, with sensitivity and sensitivity amounting to 85.1% and 75.3% respectively.

Conclusion

We present and discuss, for the first time, analyses of the potential use of computational drying approaches combined with ATR-FTIR spectroscopy for analysis of liquid human serum samples in clinical translation with promising preliminary results.