Early Detection Programme 7th Annual Symposium: Poster Area

Welcome to the 2022 Symposium Poster Area

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Oncogene dose heterogeneity in senescence and immune surveillance of premalignant cells

Authors: Adelyne Chan, Haoran Zhu, Liam Cassidy, Andrew Young, Shwethaa Raghunathan, Matthew Hoare, Tim Halim, Masako Narita and Masashi Narita

Affiliation: Cancer Research UK Cambridge Institute, University of Cambridge, United Kingdom

Poster abstract: Oncogene activation leads to diverse phenotypes, including senescence (a state of stable proliferative arrest) and malignant transformation. Both these phenotypes have been shown to require critically high doses of oncogenic RAS, however the mechanisms governing this decision remains unknown. Initially, oncogenic RAS exists as somatic mutations, with inevitably low expression insufficient for both senescence and transformation. Incidental upregulation of RAS activity confers survival benefits, thus is selected for during early tumorigenesis.

Over time, RAS expression may elevate to levels sufficient to trigger senescence, or cancer when coupled with senescence escape/bypass. Additionally, senescent cells provoke an immune reaction, mediating its removal from the tissue (‘senescence surveillance’), further reinforcing its tumour suppressive role.

We have developed in vitro and in vivo systems for modelling this RAS dose-dependency. In cultured human diploid cells, only the highest RAS-expressing cells senesce, with modest-RAS instead conferring competitive advantage. NFKB-driven inflammatory senescence-associated secretory phenotype (SASP) was activated exclusively in a subset of senescent cells, suggesting i) a SASP threshold along the RAS dosage axis and ii) substantial heterogeneity among senescent cells.

These observations were recapitulated in vivo in a liver senescence mouse model, which further demonstrates that senescence surveillance requires critical RAS dose. Remarkably, high-RAS-driven immune responses appear sufficient for eliminating regional modest-RAS hepatocytes, indicating bystander effect. Thus, the nature of oncogene dose heterogeneity determines collective phenotype within largely normal cellular microenvironments and we propose a ‘fish net’ model of senescence surveillance whereby recruited immune cells also act on RAS-expressing but non-senescent cells in the immediate microenvironment.


Single cell profiling of healthy pleura - a path towards better understanding of early mesothelioma biology

Authors: Joanna Obacz1, Taylor S. Adams2, Jonas C. Schupp2, Naftali Kaminski2, Giuseppe Aresu3, Aman S. Coonar3, Adam Peryt3, Doris M. Rassl4, Robert C. Rintoul5, Stefan J. Marciniak6

Affiliations: 1British Lung Foundation-Victor Dahdaleh Foundation MesobanK Fellow, Cambridge Institute for Medical Research (CIMR), University of Cambridge, Cambridge, UK; 2Section of Pulmonary, Critical Care, and Sleep Medicine, Yale School of Medicine, New Haven, USA; 3Thoracic Surgery, Royal Papworth Hospital NHS Foundation Trust, Cambridge, UK; 4Department of Histopathology, Royal Papworth Hospital NHS Foundation Trust, Cambridge, UK; 5Department of Thoracic Oncology, Royal Papworth Hospital NHS Foundation Trust, Cambridge, UK; Department of Oncology, University of Cambridge, Cambridge, UK; 6Cambridge Institute for Medical Research (CIMR), University of Cambridge, Cambridge, UK; Department of Medicine, University of Cambridge, Addenbrooke’s Hospital and Royal Papworth Hospital NHS Foundation Trust, Cambridge, UK

Poster abstract: Pleural mesothelioma is an aggressive malignancy of the thoracic cavity lining that primarily develops as a result of previous exposure to asbestos.

Owing to its insidious presentation, only a minority of patients are diagnosed with early stage disease that is amenable to surgery. For inoperable cases, palliative chemotherapy with pemetrexed and cisplatin is used, but this increases survival by only 3 months. Recently, immunotherapy and VEGF inhibitors have been shown to confer further modest survival benefits in selected patients, but there remains a huge unmet need for effective therapies.

A key challenge in the development of safe and effective treatments for mesothelioma is an inadequate understanding of the biology of healthy pleura. To address this, we have established a reproducible protocol for the isolation and culture of primary mesothelial cells from human pleural tissue. By comparing the transcriptomes of primary healthy mesothelial cells with those of primary mesothelioma cell lines obtained from MesobanK UK, we aim to identify biomarkers for early detection of mesothelioma. This may also provide novel therapeutic targets.

Additionally, using single-cell RNA profiling we explored the cellular heterogeneity of human healthy pleura in 8 patients undergoing surgery for prior pneumothorax. This provides a comprehensive parietal pleural atlas comprising mesothelial, stromal and immune cells. These novel datasets fill significant gaps in our current knowledge and should be of value to those studying many aspects of pleural biology.


Detecting tumour-specific methylated markers from circulating free DNA using a mass sensitive low-cost platform

Authors: Ermira Lleshi, Radoslaw Lach, Gahee Park, Anne Babbage, Sara Pita, Vincent Gnanapragasam, Anne George, CUTRACT Team, Andrew Flewitt and Charlie Massie.

Abstract: Cell free DNA (cfDNA) methylation analysis relies on DNA sequencing, a costly and labour-intensive method involving long processes which can be a barrier to clinical application.

Inspired by the performance of cfDNA methylation markers in recent studies and the need of implementing clinically a rapid, inexpensive, cancer-specific ultrasensitive blood-based test, here I show the principle of an assay design that allows for enrichment of cancer-specific methylated targets and their detection from a low-cost Thin Film Bulk Acoustic Resonator (TFBAR).

Low abundance of circulating tumour DNA requires this assay to be sufficiently sensitive. To address this criterion, I demonstrate enrichment data that show methylation-specific binding protein 2 (MBD2-seq) outperforming antibody immunoprecipitation (MeDIP-seq) in the enrichments of pre-defined methylation targets in cfDNA from a prostate cancer cell line model (PC3).

We show that MBD2-seq performs well even at very low input cfDNA (5ng). From differential analysis, using low input MBD2-seq conducted on a training cohort of 38 patients (metastatic and benign cfDNA prostate samples), we will determine a selected panel of cancer - specific region (pre-defined and novel) to help design specific probes to be utilised for label-free detection using TFBAR sensing device.

Early detection of T-cell lymphoma with T follicular helper phenotype by RHOA mutation analysis

Authors: Rachel Dobson, Peter Y Du, Lívia Rásó-Barnett, Wen-Qing Yao, Zi Chen, Calogero Casa, Hesham Ei-Daly, Lorant Farkas, Elizabeth Soilleux, Penny Wright, John W Grant, Manuel Rodriguez-Justo, George A Follows, Hala Rashed, Margarete Fabre, E Joanna Baxter, George Vassiliou, Andrew Wotherspoon, Ayoma D Attygalle, Hongxiang Liu, Ming-Qing Du.

Abstract: Angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphoma with T follicular helper phenotype (PTCL-TFH) are a group of complex clinicopathological entities that originate from TFH cells and share a similar mutation profile. Their diagnosis is often a challenge, particularly at an early stage, due to a lack of specific histological and immunophenotypic features, paucity of neoplastic T cells and prominent polymorphous infiltrate.

The lymphoma specific RHOA Gly17Val (c.50G>T) mutation is detected in 60-70% AITL/PTCL-TFH cases. We have investigated whether the mutation is present in the early ‘reactive’ lesions for cases which develop AITL/PTCL-TFH.

The RHOA Gly17Val mutation was detected by quantitative PCR using a LNA probe specific to the mutation, and a PNA clamp to suppress wild-type allele amplification. The assay was highly sensitive and specific, detecting Gly17Val mutation at 0.03% allele frequency.

Among 37 cases of AITL/PTCL-TFH investigated, RHOA Gly17Val was detected in 62.2% (23/37) of which 19 had multiple biopsies available, including preceding biopsies in 10 and follow up biopsies in 11 cases. RHOA Gly17Val was detected in each of these preceding or follow up biopsies, including 18 specimens that showed no evidence of lymphoma by combined histological, immunophenotypic and clonality analyses. Interestingly, the mutation was detected in preceding biopsies 0-26.5 months (mean=7.87 months) prior to lymphoma diagnosis. Meanwhile, the assay did not detect the mutation in 61 control samples.

Our results show that the RHOA Gly17Val mutation is valuable in the early detection of AITL/PTCL-TFH, since it is detected in the early ‘reactive’ lesions of AITL/PTCL-TFH cases.

Estimating the contribution of somatic variants in TP53 to germline cells from blood samples

Authors: H MacGregor, D Easton, J Blundell

Abstract: Deleterious germline variants in TP53 are strongly linked to Li-Fraumeni syndrome, a condition which predisposes people to the development of multiple tumours in early life. However, there is some uncertainty as to the true prevalence of TP53 germline variants and it is suspected that somatic TP53 variants in blood may be being confused for true germline variants in large-scale blood sequencing studies e.g. in the UKBiobank.

To determine to what extent somatic variants might be contributing to the prevalence of observed TP53 variants, we developed a model which captures the dynamics of known somatic TP53 variants in the blood of 4,160 pre-treatment cancer patients. We apply this model to predict the prevalence of somatic TP53 variants in the UK Biobank 200k exomes cohort. We predict that there should be 2-22 variants above 20% VAF and 0-2 variants above 30% VAF, suggesting that it is unlikely that a substantial proportion of apparently germline TP53 SNVs in UK Biobank are actually somatic.

Risks of Developing a Second Primary Cancer in Male Breast Cancer Survivors: A Systematic Review and Meta-Analysis

Authors: Isaac Allen, Eleni Sofianopoulou, Hend Hassan, Marc Tischkowitz, Paul Pharoah, Antonis Antoniou

Affiliation: Centre for Cancer Genetic Epidemiology, Department of Public Health and Primary Care, University of Cambridge

Abstract:

Objective:
Cancer risk stratification is critical for optimum targeting of early detection strategies. We undertook a systematic review and meta-analysis of second primary non-breast cancer risks in male survivors of a first invasive primary breast cancer (BC).

Methods:
PubMed, Embase, and Web of Science were searched for studies published up to January 2021. Studies meeting a nominal sample size which allowed extraction of standardized incidence ratios (SIRs) with 95% confidence intervals were accepted. SIRs were synthesised assuming a random-effects model using the generic inverse variance method. Meta-analyses were stratified by continent of study location, as well as age at and time elapsed since first BC onset. Syntheses of SIRs for the risk of site-specific second primaries were also performed.

Results:
9 papers fulfilled the selection criteria. All were population-based retrospective cohort studies. Most SIRs reported for the development of any second non-breast primary ranged from 1.05 to 1.34, with the summary SIR being estimated as 1.15 (95%CI: 1.03 – 1.29). The summary SIR for men diagnosed with a first BC at under age 50 was estimated as 1.50 (95%CI: 1.22 – 1.84), which was significantly higher in comparison to the older age group (p = 0.035). When examining site-specific cancer risks, male BC survivors were found to be at increased risk of colorectal (SIR of 1.30 (95%CI: 1.01 – 1.66)), pancreatic (SIR of 1.67 (95%CI: 1.13 – 2.47), and thyroid (SIR of 5.58 (95%CI: 1.04 – 30.05)) cancers. There was also a suggestion for increased risks of prostate and stomach cancers, but the associations were not significant.

Conclusion:
The higher risk of developing second primaries among male BC survivors may inform clinical management decisions, particularly for those whose first BC developed at a younger age. Our work shows how meta-analyses of risks can identify populations that would benefit from targeted early detection strategies.