• Chronic myelomonocytic leukaemia stem cell transcriptomes anticipate disease morphology and outcome

      Wiseman, Daniel H; Baker, S. M.; Dongre, Arundhati V|Gurashi, Kristian; Storer, Joanna A; Somervaille, Tim CP; Batta, Kiran; Epigenetics of Haematopoiesis Laboratory, Division of Cancer Sciences, The University of Manchester, Manchester M20 4GJ, UK. (2020)
      Background: Chronic myelomonocytic leukaemia (CMML) is a clinically heterogeneous stem cell malignancy with overlapping features of myelodysplasia and myeloproliferation. Over 90% of patients carry mutations in epigenetic and/or splicing genes, typically detectable in the Lin-CD34+CD38- immunophenotypic stem cell compartment in which the leukaemia-initiating cells reside. Transcriptional dysregulation at the stem cell level is likely fundamental to disease onset and progression. Methods: We performed single-cell RNA sequencing on 6826 Lin-CD34+CD38-stem cells from CMML patients and healthy controls using the droplet-based, ultra-high-throughput 10x platform. Findings: We found substantial inter- and intra-patient heterogeneity, with CMML stem cells displaying distinctive transcriptional programs. Compared with normal controls, CMML stem cells exhibited transcriptomes characterized by increased expression of myeloid-lineage and cell cycle genes, and lower expression of genes selectively expressed by normal haematopoietic stem cells. Neutrophil-primed progenitor genes and a MYC transcription factor regulome were prominent in stem cells from CMML-1 patients, whereas CMML-2 stem cells exhibited strong expression of interferon-regulatory factor regulomes, including those associated with IRF1, IRF7 and IRF8. CMML-1 and CMML-2 stem cells (stages distinguished by proportion of downstream blasts and promonocytes) differed substantially in both transcriptome and pseudotime, indicating fundamentally different biology underpinning these disease states. Gene expression and pathway analyses highlighted potentially tractable therapeutic vulnerabilities for downstream investigation. Importantly, CMML patients harboured variably-sized subpopulations of transcriptionally normal stem cells, indicating a potential reservoir to restore functional haematopoiesis. Interpretation: Our findings provide novel insights into the CMML stem cell compartment, revealing an unexpected degree of heterogeneity and demonstrating that CMML stem cell transcriptomes anticipate disease morphology, and therefore outcome. Funding: Project funding was supported by Oglesby Charitable Trust, Cancer Research UK, Blood Cancer UK, and UK Medical Research Council. Keywords: CMML; Leukaemia; Stem cells; sc-RNA Seq.
    • Investigating the importance of B cells and antibodies during Trichuris muris infection using the IgMi mouse

      Sahputra, R.; Murphy, E. A.; Forman, R.; Mair, I.; Fadlullah, Muhammad ZH; Waisman, A.; Muller, W.; Else, K. J.; Division of Infection, Immunity and Respiratory Medicine, Lydia Becker Institute for Immunology, The University of Manchester, Manchester, UK. (2020)
      The IgMi mouse has normal B cell development; its B cells express an IgM B cell receptor but cannot class switch or secrete antibody. Thus, the IgMi mouse offers a model system by which to dissect out antibody-dependent and antibody-independent B cell function. Here, we provide the first detailed characterisation of the IgMi mouse post-Trichuris muris (T. muris) infection, describing expulsion phenotype, cytokine production, gut pathology and changes in T regulatory cells, T follicular helper cells and germinal centre B cells, in addition to RNA sequencing (RNA seq) analyses of wild-type littermates (WT) and mutant B cells prior to and post infection. IgMi mice were susceptible to a high-dose infection, with reduced Th2 cytokines and elevated B cell-derived IL-10 in mesenteric lymph nodes (MLN) compared to controls. A low-dose infection regime revealed IgMi mice to have significantly more apoptotic cells in the gut compared to WT mice, but no change in intestinal inflammation. IL-10 levels were again elevated. Collectively, this study showcases the potential of the IgMi mouse as a tool for understanding B cell biology and suggests that the B cell plays both antibody-dependent and antibody-independent roles post high- and low-dose T. muris infection. KEY MESSAGES: During a high-dose T. muris infection, B cells are important in maintaining the Th1/Th2 balance in the MLN through an antibody-independent mechanism. High levels of IL-10 in the MLN early post-infection, and the presence of IL-10-producing B cells, correlates with susceptibility to T. muris infection. B cells maintain gut homeostasis during chronic T. muris infection via an antibody-dependent mechanism. Keywords: B cells; IgMi mouse; Interleukin-10; Intestinal pathology; Th1/Th2; Trichuris muris.
    • Prostate cancer heterogeneity assessment with multi-regional sampling and alignment-free methods

      Murphy, R. G.; Roddy, A. C.; Srivastava, S.; Baena, Esther; Waugh, D. J.; J, M. O. S.; McArt, D. G.; Jain, S.; LaBonte, M. J.; Movember FASTMAN Centre of Excellence, Patrick G Johnston Centre for Cancer Research, School of Medicine, Dentistry and Biomedical Sciences, Queen's University Belfast, Belfast BT9 7AE, UK. (2020)
      Combining alignment-free methods for phylogenetic analysis with multi-regional sampling using next-generation sequencing can provide an assessment of intra-patient tumour heterogeneity. From multi-regional sampling divergent branching, we validated two different lesions within a patient's prostate. Where multi-regional sampling has not been used, a single sample from one of these areas could misguide as to which drugs or therapies would best benefit this patient, due to the fact these tumours appear to be genetically different. This application has the power to render, in a fraction of the time used by other approaches, intra-patient heterogeneity and decipher aberrant biomarkers. Another alignment-free method for calling single-nucleotide variants from raw next-generation sequencing samples has determined possible variants and genomic locations that may be able to characterize the differences between the two main branching patterns. Alignment-free approaches have been applied to relevant clinical multi-regional samples and may be considered as a valuable option for comparing and determining heterogeneity to help deliver personalized medicine through more robust efforts in identifying targetable pathways and therapeutic strategies. Our study highlights the application these tools could have on patient-aligned treatment indications.
    • Mechanisms of drug resistance mediated by long non-coding RNAs in non-small-cell lung cancer

      La Montagna, Manuela; Ginn, Lucy; Garofalo, Michela; Transcriptional Networks in Lung Cancer Group, Cancer Research UK Manchester Institute, University of Manchester, Alderley Park, Manchester, SK10 4TG, UK. (2020)
      Non-small-cell lung cancer (NSCLC) is the most prevalent form of lung cancer and has a poor five-year survival rate of 15%. Chemotherapy and targeted therapies have significantly improved patients' prognosis. Nevertheless, after a successful initial response, some patients relapse when cancer cells become resistant to drug treatments, representing an important clinical limitation. Therefore, investigating the mechanisms of drug resistance is of significant importance. Recently, considerable attention has been given to long non-coding RNAs (lncRNAs), a heterogeneous class of regulatory molecules that play essential roles in tumorigenesis by modulating genes and signalling pathways involved in cell growth, metastasis and drug response. In this article, we review recent research findings on the role of lncRNAs in drug resistance in NSCLC, highlighting their mechanisms of action.
    • Alternative enhancer usage and targeted polycomb marking hallmark promoter choice during t cell differentiation

      Maqbool, M. A.; Pioger, L.; El Aabidine, A. Z.; Karasu, N.; Molitor, A. M.; Dao, L. T. M.; Charbonnier, G.; van Laethem, F.; Fenouil, R.; Koch, F.; et al. (2020)
      During thymic development and upon peripheral activation, T cells undergo extensive phenotypic and functional changes coordinated by lineage-specific developmental programs. To characterize the regulatory landscape controlling T cell identity, we perform a wide epigenomic and transcriptional analysis of mouse thymocytes and naive CD4 differentiated T helper cells. Our investigations reveal a dynamic putative enhancer landscape, and we could validate many of the enhancers using the high-throughput CapStarr sequencing (CapStarr-seq) approach. We find that genes using multiple promoters display increased enhancer usage, suggesting that apparent "enhancer redundancy" might relate to isoform selection. Furthermore, we can show that two Runx3 promoters display long-range interactions with specific enhancers. Finally, our analyses suggest a novel function for the PRC2 complex in the control of alternative promoter usage. Altogether, our study has allowed for the mapping of an exhaustive set of active enhancers and provides new insights into their function and that of PRC2 in controlling promoter choice during T cell differentiation. Keywords: CapSTARR-seq; T cell enhancerome; enhancer and promoter usage; enhancer redundancy; long-distance enhancer-promoter interactions.
    • Mitochondrial spongiotic brain disease: astrocytic stress and harmful rapamycin and ketosis effect

      Ignatenko, O.; Nikkanen, J.; Kononov, Alexander; Zamboni, N.; Ince-Dunn, G.; Suomalainen, A.; Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland. (2020)
      Mitochondrial DNA (mtDNA) depletion syndrome (MDS) is a group of severe, tissue-specific diseases of childhood with unknown pathogenesis. Brain-specific MDS manifests as devastating spongiotic encephalopathy with no curative therapy. Here, we report cell type-specific stress responses and effects of rapamycin treatment and ketogenic diet (KD) in mice with spongiotic encephalopathy mimicking human MDS, as these interventions were reported to improve some mitochondrial disease signs or symptoms. These mice with astrocyte-specific knockout of Twnk gene encoding replicative mtDNA helicase Twinkle (TwKOastro) show wide-spread cell-autonomous astrocyte activation and mitochondrial integrated stress response (ISRmt) induction with major metabolic remodeling of the brain. Mice with neuronal-specific TwKO show no ISRmt Both KD and rapamycin lead to rapid deterioration and weight loss of TwKOastro and premature trial termination. Although rapamycin had no robust effects on TwKOastro brain pathology, KD exacerbated spongiosis, gliosis, and ISRmt Our evidence emphasizes that mitochondrial disease treatments and stress responses are tissue- and disease specific. Furthermore, rapamycin and KD are deleterious in MDS-linked spongiotic encephalopathy, pointing to a crucial role of diet and metabolism for mitochondrial disease progression.
    • Evaluation of drugs for potential repurposing against COVID-19 using a tier-based scoring system

      Jarvis, M. A.; Hansen, F. A.; Rosenke, K.; Haddock, E.; Rollinson, C.; Rule, S.; Sewell, G.; Hughes, Andrew; Feldmann, H.; University of Plymouth, Plymouth, Devon, UK. (2020)
      Background: As the Coronavirus Disease 2019 (COVID-19) pandemic grows daily, we remain with no prophylactic and only minimal therapeutic interventions to prevent or ameliorate severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2). Prior to SARS-CoV-2 emergence, high throughput screens utilizing clinically developed drugs identified compounds with in vitro inhibitory effect on human coronaviruses that may have potential for repurposing as treatment options for COVID-19. However, caution should be applied to repurposing of these drugs when they are taken out of context of human pharmacokinetic parameters associated with normal therapeutic use. Methods: Our aim was to provide a tier-based scoring system to interrogate this data set and match each drug with its human pharmacokinetic criteria, such as route of administration, therapeutic plasma levels and half-life, tissue distribution, and safety. Results: Our analysis excluded most previously identified drugs but identified members of 4 drug classes (antimalarial amino-quinolones, selective estrogen receptor modulators; SERMs, low potency tricyclic antipsychotics and tricyclic antidepressants) as potential drug candidates for COVID-19. Two of them, the tricyclic antipsychotics and tricyclic antidepressants were further excluded based on a high adverse event profile. Conclusions: In summary, our findings using a new pharmacokinetic-based scoring system supports efficacy testing of only a minority of candidates against SARS-CoV-2 infection.
    • Melanoma risk in patients treated with biologic therapy for common inflammatory diseases a systematic review and meta-analysis

      Esse, S.; Mason, K. J.; Green, Ad�le C; Warren, R. B.; Division of Musculoskeletal and Dermatological Sciences, University of Manchester, Manchester, United Kingdom. (2020)
      Importance: Biologic therapies are widely prescribed immunomodulatory agents. There are concerns that compared with treatment with conventional systemic therapy, long-term biologic treatment for common immune-mediated inflammatory diseases, namely inflammatory bowel disease (IBD), rheumatoid arthritis (RA), and psoriasis, may be associated with increased risk of melanoma. Objective: To examine whether biologic treatment of IBD, RA, or psoriasis is associated with an increased risk of melanoma compared with conventional systemic therapy. Data sources: Embase, MEDLINE, and the Cochrane Central Register of Controlled Trials (CENTRAL) were searched for articles published from January 1, 1995, to February 7, 2019, for eligible studies. Study selection: Randomized clinical trials, cohort studies, and nested case-control studies quantifying the risk of melanoma in biologic-treated patients with IBD, RA, and psoriasis compared with patients treated with conventional systemic therapy were included. Data extraction and synthesis: Two reviewers independently extracted key study characteristics and outcomes. Study-specific risk estimates were pooled, and random- and fixed-effects model meta-analyses were conducted. Heterogeneity was assessed using the I2 statistic. The Meta-analysis of Observational Studies in Epidemiology (MOOSE) reporting guidelines were followed. Main outcomes and measures: The pooled relative risk (pRR) of melanoma in biologic-treated patients with IBD, RA, and psoriasis compared with biologic-naive patients treated with conventional systemic therapy. Results: Seven cohort studies comprising 34 029 biologic-treated patients and 135 370 biologic-naive patients treated with conventional systemic therapy were eligible for inclusion. Biologic treatment was positively associated with melanoma in patients with IBD (pRR, 1.20; 95% CI, 0.60-2.40), RA (pRR, 1.20; 95% CI, 0.83-1.74), or psoriasis (hazard ratio, 1.57; 95% CI, 0.61-4.09) compared with those who received conventional systemic therapy, but the differences were not statistically significant. Adjustment for other risk factors was absent from most studies. Conclusions and relevance: The findings suggest that clinically important increases in melanoma risk in patients treated with biologic therapy for common inflammatory diseases cannot be ruled out based on current evidence. However, further studies with large patient numbers that adjust for key risk factors are needed to resolve the issue of long-term safety of biologic therapy.
    • Invivo binding of recombination proteins to non-dsb DNA lesions and to replication forks

      Gonz�lez-Prieto, R.; Cabello-Lobato, Maria J; Prado, F.; Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, The Netherlands. (2021)
      Homologous recombination (HR) has been extensively studied in response to DNA double-strand breaks (DSBs). In contrast, much less is known about how HR deals with DNA lesions other than DSBs (e.g., at single-stranded DNA) and replication forks, despite the fact that these DNA structures are associated with most spontaneous recombination events. A major handicap for studying the role of HR at non-DSB DNA lesions and replication forks is the difficulty of discriminating whether a recombination protein is associated with the non-DSB lesion per se or rather with a DSB generated during their processing. Here, we describe a method to follow the in vivo binding of recombination proteins to non-DSB DNA lesions and replication forks. This approach is based on the cleavage and subsequent electrophoretic analysis of the target DNA by the recombination protein fused to the micrococcal nuclease.
    • The B-MaP-C study: Breast cancer management pathways during the COVID-19 pandemic. Study protocol

      Courtney, A.; O'Connell, R.; Rattay, T.; Kim, B.; Cutress, R. I.; Kirwan, C. C.; Gandhi, Ashu; Fairbrother, P.; Sharma, N.; Cartlidge, C. W. J.; et al. (2020)
      Introduction: Approximately 55,000 women in the United Kingdom are diagnosed with new breast cancer annually. Since emerging in December 2019, SARS-CoV-2 (coronavirus disease 2019, COVID-19) has become a global pandemic, affecting healthcare delivery worldwide. In response to the pandemic, multiple guidelines were issued to assist with rationalising breast cancer care. The primary aim of the B-MaP-C study is to audit and describe breast cancer management of patients newly diagnosed with breast cancer during the COVID-19 pandemic against pre-COVID-19 management practice in the UK. The implications of changes to management will be determined and the impact of a COVID-19 diagnosis on the patient's breast cancer management will be determined. Methods and analysis: This is a multi-centre collaborative audit of consecutive breast cancer patients undergoing treatment decisions during the acute and recovery phases of the COVID-19 pandemic. All patients with newly diagnosed primary breast cancer, whose treatment was decided in a multidisciplinary meeting from the 16th March 2020, are eligible for inclusion. Ethics and dissemination: As this is an audit ethical approval is not required. Each participating centre is required to register the study locally and obtain local governance approvals prior to commencement of data collection. Local audit data will be available to individual participating units for governance purposes. The results of the data analysis will be submitted for publication, as well as disseminated via the ABS newsletter and a webinar. All data will be presented at national and international conferences, circumstances permitting. Registration details: Each participating centre received local governance audit registration.
    • Rivaroxaban compared to no treatment in ER-negative stage I-III early breast cancer patients (the TIP Trial): study protocol for a phase II preoperative window-of-opportunity study design randomised controlled trial

      Castle, John; Blower, Emma; Bundred, Nigel J; Harvey, J. R.; Thachil, J.; Marshall, A.; Cox, K.; Cicconi, S.; Holcombe, C.; Palmieri, C.; et al. (2020)
      Background: Breast cancer patients are at a four-fold increased risk of developing a venous thromboembolism (VTE), a major cause of death in this group. Conversely, coagulation factors promote tumour growth and metastasis. This has been evidenced in preclinical models, with an inhibitory effect of anticoagulants on cancer growth through proliferative, angiogenic, apoptotic, cancer stem cell and metastatic processes. The extrinsic clotting pathway is also more upregulated in patients in the relatively poorer prognosis oestrogen receptor (ER)-negative breast cancer subgroup, with increased tumour stromal expression of the coagulation factors Tissue Factor and thrombin. Rivaroxaban (Xarelto�, Bayer AG, Leverkusen, Germany) is a direct oral anticoagulant (DOAC). It is a Factor Xa inhibitor that is routinely prescribed for the prevention of stroke in non-valvular atrial fibrillation and for both VTE prophylaxis and treatment. This trial will assess the anti-proliferative and other anti-cancer progression mechanisms of Rivaroxaban in ER-negative early breast cancer patients. Methods: This UK-based preoperative window-of-opportunity phase II randomised control trial will randomise 88 treatment-na�ve early breast cancer patients to receive 20 mg OD Rivaroxaban treatment for 11 to 17 days or no treatment. Treatment will be stopped 24 h (range 18-36 h) prior to surgery or repeat core biopsy. All patients will be followed up for 2 weeks following surgery or repeat core biopsy. The primary endpoint is change in tumour Ki67. Secondary outcome measures include tumour markers of apoptosis and angiogenesis, extrinsic clotting pathway activation and systemic markers of metastasis, tumour load and coagulation. Discussion: Laboratory evidence supports an anti-cancer role for anticoagulants; however, this has failed to translate into survival benefit when trialled in patients with metastatic disease or poor prognosis cancers, such as lung cancer. Subgroup analysis supported a potential survival benefit in better prognosis advanced disease patients. This is the first study to investigate the anti-cancer effects of anticoagulants in early breast cancer. Trial registration: UK National Research Ethics Service (NRES) approval 15/NW/0406, MHRA Clinical Trials Authorisation 48380/0003/001-0001. The sponsor is Manchester University NHS Foundation Trust, and the trial is co-ordinated by Cancer Research UK Liverpool Cancer Trials Unit (LCTU). EudraCT 2014-004909-33 , registered 27 July 2015. ISRCTN14785273 .
    • Towards a cancer mission in Horizon Europe: recommendations

      Berns, A.; Ringborg, U.; Celis, J. E.; Heitor, M.; Aaronson, N. K.; Abou-Zeid, N.; Adami, H. O.; Apostolidis, K.; Baumann, M.; Bardelli, A.; et al. (2020)
      A comprehensive translational cancer research approach focused on personalized and precision medicine, and covering the entire cancer research-care-prevention continuum has the potential to achieve in 2030 a 10-year cancer-specific survival for 75% of patients diagnosed in European Union (EU) member states with a well-developed healthcare system. Concerted actions across this continuum that spans from basic and preclinical research through clinical and prevention research to outcomes research, along with the establishment of interconnected high-quality infrastructures for translational research, clinical and prevention trials and outcomes research, will ensure that science-driven and social innovations benefit patients and individuals at risk across the EU. European infrastructures involving comprehensive cancer centres (CCCs) and CCC-like entities will provide researchers with access to the required critical mass of patients, biological materials and technological resources and can bridge research with healthcare systems. Here, we prioritize research areas to ensure a balanced research portfolio and provide recommendations for achieving key targets. Meeting these targets will require harmonization of EU and national priorities and policies, improved research coordination at the national, regional and EU level and increasingly efficient and flexible funding mechanisms. Long-term support by the EU and commitment of Member States to specialized schemes are also needed for the establishment and sustainability of trans-border infrastructures and networks. In addition to effectively engaging policymakers, all relevant stakeholders within the entire continuum should consensually inform policy through evidence-based advice.
    • Clinical and functional characterization of CXCR1/CXCR2 biology in the relapse and radiotherapy resistance of primary PTEN-deficient prostate carcinoma

      Armstrong, C. W. D.; Coulter, J. A.; Ong, C. W.; Maxwell, P. J.; Walker, S.; Butterworth, K. T.; Lyubomska, O.; Berlingeri, S.; Gallagher, R.; O'Sullivan, J. M.; et al. (2020)
      Functional impairment of the tumour suppressor PTEN is common in primary prostate cancer and has been linked to relapse post-radiotherapy (post-RT). Pre-clinical modelling supports elevated CXC chemokine signalling as a critical mediator of PTEN-depleted disease progression and therapeutic resistance. We assessed the correlation of PTEN deficiency with CXC chemokine signalling and its association with clinical outcomes. Gene expression analysis characterized a PTEN LOW/CXCR1HIGH/CXCR2HIGH cluster of tumours that associates with earlier time to biochemical recurrence [hazard ratio (HR) 5.87 and 2.65, respectively] and development of systemic metastasis (HR 3.51). In vitro, CXCL signalling was further amplified following exposure of PTEN-deficient prostate cancer cell lines to ionizing radiation (IR). Inhibition of CXCR1/2 signalling in PTEN-depleted cell-based models increased IR sensitivity. In vivo, administration of a CXCR1/2-targeted pepducin (x1/2pal-i3), or CXCR2-specific antagonist (AZD5069), in combination with IR to PTEN-deficient xenografts attenuated tumour growth and progression compared to control or IR alone. Post-mortem analysis confirmed that x1/2pal-i3 administration attenuated IR-induced CXCL signalling and anti-apoptotic protein expression. Interventions targeting CXC chemokine signalling may provide an effective strategy to combine with RT in locally advanced prostate cancer patients with known presence of PTEN-deficient foci.
    • The genomic and epigenomic evolutionary history of papillary renal cell carcinomas

      Zhu, B; Poeta, ML; Costantini, M; Zhang, T; Shi, J; Sentinelli, S; Zhao, W; Pompeo, V; Cardelli, M; Alexandrov, BS; et al. (2020)
      Intratumor heterogeneity (ITH) and tumor evolution have been well described for clear cell renal cell carcinomas (ccRCC), but they are less studied for other kidney cancer subtypes. Here we investigate ITH and clonal evolution of papillary renal cell carcinoma (pRCC) and rarer kidney cancer subtypes, integrating whole-genome sequencing and DNA methylation data. In 29 tumors, up to 10 samples from the center to the periphery of each tumor, and metastatic samples in 2 cases, enable phylogenetic analysis of spatial features of clonal expansion, which shows congruent patterns of genomic and epigenomic evolution. In contrast to previous studies of ccRCC, in pRCC, driver gene mutations and most arm-level somatic copy number alterations (SCNAs) are clonal. These findings suggest that a single biopsy would be sufficient to identify the important genetic drivers and that targeting large-scale SCNAs may improve pRCC treatment, which is currently poor. While type 1 pRCC displays near absence of structural variants (SVs), the more aggressive type 2 pRCC and the rarer subtypes have numerous SVs, which should be pursued for prognostic significance.
    • Chronic expression of p16(INK4a) in the epidermis induces Wnt-mediated hyperplasia and promotes tumor initiation

      Azazmeh, N; Assouline, B; Winter, E; Ruppo, S; Nevo, Y; Maly, A; Meir, K; Witkiewicz, AK; Cohen, J; Rizou, SV; et al. (2020)
      p16INK4a (CDKN2A) is a central tumor suppressor, which induces cell-cycle arrest and senescence. Cells expressing p16INK4a accumulate in aging tissues and appear in premalignant lesions, yet their physiologic effects are poorly understood. We found that prolonged expression of transgenic p16INK4a in the mouse epidermis induces hyperplasia and dysplasia, involving high proliferation rates of keratinocytes not expressing the transgene. Continuous p16INK4a expression increases the number of epidermal papillomas formed after carcinogen treatment. Wnt-pathway ligands and targets are activated upon prolonged p16INK4a expression, and Wnt inhibition suppresses p16INK4a-induced hyperplasia. Senolytic treatment reduces p16INK4a-expressing cell numbers, and inhibits Wnt activation and hyperplasia. In human actinic keratosis, a precursor of squamous cell carcinoma, p16INK4a-expressing cells are found adjacent to dividing cells, consistent with paracrine interaction. These findings reveal that chronic p16INK4a expression is sufficient to induce hyperplasia through Wnt-mediated paracrine stimulation, and suggest that this tumor suppressor can promote early premalignant epidermal lesion formation.
    • Accurate MR image registration to anatomical reference space for diffuse glioma

      Visser, M; Petr, J; Muller, DMJ; Eijgelaar, RS; Hendriks, EJ; Witte, M; Barkhof, F; van Herk, Marcel; Mutsaerts, H; Vrenken, H; et al. (2020)
      To summarize the distribution of glioma location within a patient population, registration of individual MR images to anatomical reference space is required. In this study, we quantified the accuracy of MR image registration to anatomical reference space with linear and non-linear transformations using estimated tumor targets of glioblastoma and lower-grade glioma, and anatomical landmarks at pre- and post-operative time-points using six commonly used registration packages (FSL, SPM5, DARTEL, ANTs, Elastix, and NiftyReg). Routine clinical pre- and post-operative, post-contrast T1-weighted images of 20 patients with glioblastoma and 20 with lower-grade glioma were collected. The 2009a Montreal Neurological Institute brain template was used as anatomical reference space. Tumors were manually segmented in the patient space and corresponding healthy tissue was delineated as a target volume in the anatomical reference space. Accuracy of the tumor alignment was quantified using the Dice score and the Hausdorff distance. To measure the accuracy of general brain alignment, anatomical landmarks were placed in patient and in anatomical reference space, and the landmark distance after registration was quantified. Lower-grade gliomas were registered more accurately than glioblastoma. Registration accuracy for pre- and post-operative MR images did not differ. SPM5 and DARTEL registered tumors most accurate, and FSL least accurate. Non-linear transformations resulted in more accurate general brain alignment than linear transformations, but tumor alignment was similar between linear and non-linear transformation. We conclude that linear transformation suffices to summarize glioma locations in anatomical reference space. Keywords: computer-assisted; glioma; image processing; linear registration; magnetic resonance imaging; non-linear registration.
    • The automated bone scan index as a predictor of response to prostate radiotherapy in men with newly diagnosed metastatic prostate cancer: an exploratory analysis of STAMPEDE's 'M1|RT comparison'

      Ali, Adnan; Hoyle, Alex P; Parker, CC; Brawley, CD; Cook, A; Amos, C; Calvert, J; Douis, H; Mason, MD; Attard, G; et al. (2020)
      Background: Prostate radiotherapy (RT) is a first-line option for newly diagnosed men with low-burden metastatic prostate cancer. The current criterion to define this clinical state is based on manual bone metastasis counts, but enumeration of bone metastases is limited by interobserver variations, and it does not account for metastasis volume or lesional coalescence. The automated bone scan index (aBSI) is a quantitative method of evaluating bone metastatic burden in a standardised and reproducible manner. Objective: To evaluate whether aBSI has utility as a predictive imaging biomarker to define a newly diagnosed metastatic prostate cancer population that might benefit from the addition of prostate RT to standard of care (SOC) systemic therapy. Design, setting, and participants: This is an exploratory analysis of men with newly diagnosed metastatic prostate cancer randomised in a 1:1 ratio to either SOC or SOC + prostate RT within the STAMPEDE 'M1|RT comparison'. Intervention: The SOC was lifelong androgen deprivation therapy, with up-front docetaxel permitted from December 2015. Men allocated RT received either a daily or a weekly schedule that was nominated before randomisation. Outcome measurements and statistical analysis: Baseline bone scans were evaluated retrospectively to calculate aBSI. We used overall (OS) and failure-free (FFS) survival as the end points. Treatment-aBSI interaction was evaluated using the multivariable fractional polynomial interaction (MFPI) and subpopulation treatment effect pattern plot. Further analysis was done in aBSI quartiles using Cox regression models adjusted for stratification factors. Results and limitations: Baseline bone scans for 660 (SOC: 323 and SOC + RT: 337) of 2061 men randomised within the 'M1|RT comparison' met the software requirements for aBSI calculation. The median age was 68 yr, median PSA was 100 ng/mL, median aBSI was 0.9, and median follow-up was 39 mo. Baseline patient characteristics including aBSI were balanced between the treatment groups. Using the MFPI procedure, there was evidence of aBSI-treatment interaction for OS (p = 0.04, MFPI procedure) and FFS (p < 0.01, MFPI procedure). Graphical evaluation of estimated treatment effect plots showed that the OS and FFS benefit from prostate RT was greatest in patients with a low aBSI. Further analysis in quartiles based on aBSI supported this finding. Conclusions: A low automated bone scan index is predictive of survival benefit associated with prostate RT in men with newly diagnosed metastatic prostate cancer. Patient summary: The widely used bone scan can be evaluated using an automated technique to potentially select men with newly diagnosed metastatic prostate cancer who might benefit from prostate radiotherapy. Keywords: Hormone naïve; Imaging; Metastatic; Radiotherapy.
    • Multiple uses of circulating tumor cells in lung cancer?

      Dive, Caroline; CRUK Manchester Institute Biomarker Sciences Centre, Manchester (2020)
      Liquid biopsies are increasingly being used in clinical studies as prognostic, predictive, and pharmacodynamics biomarkers, as surrogates of tumor response, to detect minimal residual disease after treatment with curative intent and to inform on mechanisms of treatment resistance and treatment switching. There is also steady progress in the development of liquid biopsies for early detection of cancers. The advantages of liquid biopsies are that they are minimally invasive and readily repeatable. The liquid biopsy that has been most widely adopted is circulating tumor DNA (ctDNA) as it is readily measured without specialist equipment in most molecular biology laboratories. Circulating tumor cells (CTCs) are technically more challenging and have yet to realize their full potential as biomarkers in clinic. However, CTC technology is constantly improving, and CTCs have several exciting applications beyond ctDNA that, once optimized, will assist personalized cancer medicine, including their use as cultures for real-time therapy testing and their utility, in some cancer types, to derive mouse models. I will discuss the use of CTCs from patients with lung cancer in this presentation. CTCs in both non-small cell lung cancer (NSCLC) and SCLC enumerated by CellSearch (EpCAM and cytokerin positive, CD45 negative) hold prognostic information. However, while CellSearch CTCs are scarce in NSCLC, most likely due to epithelial-to-mesenchymal transition (EMT) that leads to downrgulation of the EpCAM surface marker used to capture them for enumeration, they are prevalent in SCLC. Single CTC copy number analysis has led to the development of classifier that, with further validation studies, could predict response to chemotherapy alongside ctDNA analysis for therapy monitoring. We have exploited SCLC CTC prevalence to derive mouse models in immune-incompetent mice (termed CDX) that allow us to explore biology and test new therapeutics. I will also update on our research using our biobank of 46 CDX models, exploring inter and intratumoral heterogeneity. The most important aspect of this approach is the ability to generate pre- and post-therapy CDX models allowing interrogation of therapy resistance mechanisms and the biology of progressing disease in a tumor type where tumor evolution is rapid and where serial tumor biopsies are rarely obtained. I will also describe our approaches to study mechanisms of tumor dissemination, including vasculogenic mimicry and new models of brain metastasis. More recently, we are developing direct CTC cultures that may allow real-time therapy testing with reporting to the clinic. I will report on our NSCLC CTC studies within the UK TRACERx consortium to study tumor evolution. We have explored the potential of CTCs found in the pulmonary draining vein of stage I-IIIa patients at surgery with curative intent to predict risk of disease recurrence and shown in a case study that a lethal subclone that gives rise to metastasis 10 months later was identified in the pulmonary vein at surgery. In summary, this presentation will exemplify utility of CTCs in lung cancer that are distinct from but complement the implementation of ctDNA.
    • In vitro and in vivo induction of fetal hemoglobin with a reversible and selective DNMT1 inhibitor

      Gilmartin, AG; Groy, A; Gore, ER; Atkins, C; Long, ER, 3rd; Montoute, MN; Wu, Z; Halsey, W; McNulty, DE; Ennulat, D; et al. (2020)
      Pharmacological induction of fetal hemoglobin (HbF) expression is an effective therapeutic strategy for the management of beta-hemoglobinopathies such as sickle cell disease. DNA methyltransferase (DNMT) inhibitors 5-azacytidine (5-aza) and 5-aza-2'-deoxycytidine (decitabine) have been shown to induce fetal hemoglobin expression in both preclinical models and clinical studies, but are not currently approved for the management of hemoglobinopathies. We report here the discovery of a novel class of orally bioavailable DNMT1-selective inhibitors as exemplified by GSK3482364. This molecule potently inhibits the methyltransferase activity of DNMT1, but not DNMT family members DNMT3A or DNMT3B. In contrast with cytidine analog DNMT inhibitors, the DNMT1 inhibitory mechanism of GSK3482364 does not require DNA incorporation and is reversible. In cultured human erythroid progenitor cells (EPCs), GSK3482364 decreased overall DNA methylation resulting in de-repression of the gamma globin genes HBG1 and HBG2 and increased HbF expression. In a transgenic mouse model of sickle cell disease, orally administered GSK3482364 caused significant increases in both HbF levels and in the percentage HbF-expressing erythrocytes, with good overall tolerability. We conclude that in these preclinical models, selective, reversible inhibition of DNMT1 is sufficient for the induction of HbF, and is well-tolerated. We anticipate that GSK3482364 will be a useful tool molecule for the further study of selective DNMT1 inhibition both in vitro and in vivo. Keywords: DNA methyltransferase; Fetal Hemoglobin; Hemoglobinopathies; Molecular Pharmacology; Sickle Cell Disease.
    • Hypofractionated breast radiotherapy for 1 week versus 3 weeks (FAST-Forward): 5-year efficacy and late normal tissue effects results from a multicentre, non-inferiority, randomised, phase 3 trial

      Murray Brunt, A; Haviland, JS; Wheatley, DA; Sydenham, MA; Alhasso, A; Bloomfield, DJ; Chan, C; Churn, M; Cleator, S; Coles, CE; et al. (2020)
      Background: We aimed to identify a five-fraction schedule of adjuvant radiotherapy (radiation therapy) delivered in 1 week that is non-inferior in terms of local cancer control and is as safe as an international standard 15-fraction regimen after primary surgery for early breast cancer. Here, we present 5-year results of the FAST-Forward trial. Methods: FAST-Forward is a multicentre, phase 3, randomised, non-inferiority trial done at 97 hospitals (47 radiotherapy centres and 50 referring hospitals) in the UK. Patients aged at least 18 years with invasive carcinoma of the breast (pT1-3, pN0-1, M0) after breast conservation surgery or mastectomy were eligible. We randomly allocated patients to either 40 Gy in 15 fractions (over 3 weeks), 27 Gy in five fractions (over 1 week), or 26 Gy in five fractions (over 1 week) to the whole breast or chest wall. Allocation was not masked because of the nature of the intervention. The primary endpoint was ipsilateral breast tumour relapse; assuming a 2% 5-year incidence for 40 Gy, non-inferiority was predefined as ?1·6% excess for five-fraction schedules (critical hazard ratio [HR] of 1·81). Normal tissue effects were assessed by clinicians, patients, and from photographs. This trial is registered at isrctn.com, ISRCTN19906132. Findings: Between Nov 24, 2011, and June 19, 2014, we recruited and obtained consent from 4096 patients from 97 UK centres, of whom 1361 were assigned to the 40 Gy schedule, 1367 to the 27 Gy schedule, and 1368 to the 26 Gy schedule. At a median follow-up of 71·5 months (IQR 71·3 to 71·7), the primary endpoint event occurred in 79 patients (31 in the 40 Gy group, 27 in the 27 Gy group, and 21 in the 26 Gy group); HRs versus 40 Gy in 15 fractions were 0·86 (95% CI 0·51 to 1·44) for 27 Gy in five fractions and 0·67 (0·38 to 1·16) for 26 Gy in five fractions. 5-year incidence of ipsilateral breast tumour relapse after 40 Gy was 2·1% (1·4 to 3·1); estimated absolute differences versus 40 Gy in 15 fractions were -0·3% (-1·0 to 0·9) for 27 Gy in five fractions (probability of incorrectly accepting an inferior five-fraction schedule: p=0·0022 vs 40 Gy in 15 fractions) and -0·7% (-1·3 to 0·3) for 26 Gy in five fractions (p=0·00019 vs 40 Gy in 15 fractions). At 5 years, any moderate or marked clinician-assessed normal tissue effects in the breast or chest wall was reported for 98 of 986 (9·9%) 40 Gy patients, 155 (15·4%) of 1005 27 Gy patients, and 121 of 1020 (11·9%) 26 Gy patients. Across all clinician assessments from 1-5 years, odds ratios versus 40 Gy in 15 fractions were 1·55 (95% CI 1·32 to 1·83, p<0·0001) for 27 Gy in five fractions and 1·12 (0·94 to 1·34, p=0·20) for 26 Gy in five fractions. Patient and photographic assessments showed higher normal tissue effect risk for 27 Gy versus 40 Gy but not for 26 Gy versus 40 Gy. Interpretation: 26 Gy in five fractions over 1 week is non-inferior to the standard of 40 Gy in 15 fractions over 3 weeks for local tumour control, and is as safe in terms of normal tissue effects up to 5 years for patients prescribed adjuvant local radiotherapy after primary surgery for early-stage breast cancer.