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  • Wound healing inflammatory markers predict prognosis and survival in early breast cancer

    Singh, U; Castle, John; Greenhalgh, S.; Hussain, U.; Descamps, Tine; Nash, S.; Wilson, M.; Hunt, R; Kirwan, Cliona C; Division of Cancer Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, University of Manchester (2021)
  • Patterns of practice for adaptive and real-time radiation therapy: part I intra-fraction motion

    Distefano, G.; Bertholet, J.; Poulsen, P.; Roggen, T.; Garibaldi, C.; Tilly, N.; Booth, J.; Oelfke, U.; Heijmen, B; Aznar, Marianne Camille; et al. (2020)
    Purpose or Objective The patterns of practice for adaptive and real-time radiation therapy (POP-ART RT) study aims to determine to which extent and how these methods are used in clinical practice and to understand the barriers to implementation. Here we report on part I: real-time respiratory motion management (RRMM). Material and Methods An institution-specific questionnaire developed during the 2nd ESTRO physics workshop was distributed worldwide. The focus was both on current practice and wishes for implementation. Therefore, centres not (yet) doing RRMM were encouraged to participate. RRMM was defined as the use of gating in free-breathing (FB) or breath-hold (BH), or tracking if the beam is continuously realigned with the target in real-time (via robotic or gimbal guidance, MLC or couch tracking). Respondents were asked if they used RRMM for selected tumor sites, the percentage of patients treated with RRMM, eligibility criteria and the monitoring signal used to guide gating or tracking. Respondents were also asked if they wished 1) to change or expand their use of RRMM for a tumor site already treated with RRMM and 2) to implement RRMM for a new tumor site and to rank the barriers to implementation in order of importance. Results The questionnaire was filled out by 200 centres from 41 countries. 68% of respondents used RRMM in at least one tumor site (“users”). Inspiration BH was the dominant technique for breast and lymphoma, whereas the spread in technique was greater for other sites (Table 1). Within any given tumor site, users only applied RRMM in a subset of patients. The most frequently selected percentage range of patients treated using RRMM was <25% for lung, pancreas and lymphoma, 25-50% for breast and >75% for liver. However, for liver and pancreas, >50% of users applied RRMM in >50% of patients. The main selection criteria was “left breast” (76%) for breast and SBRT (~50%) for lung, liver and pancreas. Across all tumor sites, external marker was the main RRMM signal used by >60% of respondents. For breast and lymphoma this was followed by surface imaging and breathing volume. KV/MV imaging was frequently used for liver and pancreas (with markers) and for lung (with or without markers) (Fig 1a). Tracking was mainly done on robotic linacs with hybrid monitoring. For breast and lung, 36% and 49% of the centres respectively wish to expand or implement RRMM (Fig 1b). In contrast, for liver and pancreas >55% of centres do not use RRMM and do not wish to implement it. Overall 71% of centres wish to implement RRMM for any new treatment site (Fig 1c) but human/financial resources and capacity on machine were the main barriers Conclusion Thirty-two percent of respondents do not use any form of RRMM. Although RRMM was common in the thorax, it was generally applied for less than half of the patients. There is an unmet need for RRMM solutions, particularly in lung cancer. The main barriers to implement RRMM are human/financial resources and capacity on the machine.
  • Real world interpretation of GEC-ESTRO image-guided brachytherapy recommendations for cervix cancer

    Diez, P.; Bourner, J.; Sharp, A.; Hoskin, Peter J; McCormack, M.; National Radiotherapy Trials Quality Assurance Group, Mount Vernon Cancer Centre, Northwood, (2020)
    Purpose or Objective INTERLACE (NCT01566240) is an international phase III multicentre trial of weekly induction chemotherapy followed by standard chemoradiation versus standard chemoradiation alone in patients with locally advanced cervical cancer. As well as a trial protocol, detailed radiotherapy guidelines were produced with a quality assurance (QA) programme to ensure compliance at the 37 participating centres. The guidelines mandated GECESTRO recommendations for image-guided adaptive brachytherapy (IGABT) and prescribing to the high-risk CTV (HRCTV). This study assesses adherence to these recommendations. Material and Methods As part of the trial QA programme brachytherapy centres were required to submit a brachytherapy dummy run (a patient already treated at their centre) for central review prior to trial participation. Both contouring and planning were reviewed to assess their standard brachytherapy technique. The following parameters have been evaluated: use of MR and whether scanned with implant in situ; HRCTV and intermediate-risk CTV (IRCTV) definition; and dose conformity (HRCTV D90, HRCTV V100, and Vref). Results Of 34 institutions providing a brachytherapy service, 23 stated they performed IGABT, prescribing and optimising to HRCTV D90. However, 3 of these centres did not use MR in this process and 1 performed MR without the implant in situ. 8 centres did not follow GEC-ESTRO HRCTV delineation recommendations (see Figure 1). In 2 of the 11 centres defining an IRCTV this was not a direct expansion of the HRCTV and in a further 2 the vagina was not edited out of the expansion. The plan was assessed for dose conformity around the HRCTV. Only 9 centres did this adequately. Most of the uterus was included within the prescription isodose in 14 centres and a large section of the vagina in 7 centres, instead of conforming to the HRCTV definition Conclusion Moving from a 2D procedure or a standard plan in 3D to IGABT can be a steep learning curve, in particular in defining the target. Use of MRI with the implant in situ, and its careful assessment in all 3 imaging planes, is fundamental for accurate target delineation. Approximately a third of centres under-contoured the HRCTV, therefore increasing the risk of local recurrence. 11 centres over-contoured the target, having the potential to either increase the risk of toxicity or, having to reduce the prescription to remain within OAR tolerance, compromising the chance of tumour control. The same is the case where the prescription dose does not conform to the HRCTV. Only 5 centres adhered to all aspects of the GEC-ESTRO cervical IGABT recommendations that were evaluated. Although feedback was provided to all centres within INTERLACE, ongoing education may be necessary. Prospective central review was therefore carried out on the first 2 patients at each centre, with further cases assessed where required. Adoption of GEC-ESTRO recommendations in their entirety is critical to accurate delivery of IGABT. Partial compliance may prove unsafe.
  • An ESTRO-ACROP Delphi consensus on salvage SBRT for intraprostatic relapse after PCa radiotherapy

    Jereczek-Fossa, B. A.; Marvaso, G.; Pepa, M.; Gugliandolo, S. G.; Zerini, D.; Gandini, S.; Vavassori, A.; De Bari, B.; Alongi, F.; Fonteyne, V.; et al. (2020)
    None
  • Identification and characterization of novel functional markers during the hematopoietic stem cell specification process

    Buchler-Schaff, M.; Kaschutnig, P.; Thambyrajah, Roshana; Nadler, W.; Hanke, S.; Pfaffenholz, S.; Block, M.; Grasel, J.; Kremer, J.; Bayindir-Buchhalter, I.; et al. (2020)
    Endothelial to hematopoietic transition (EHT) is a crucial step in the formation of definitive hematopoietic stem cells during embryonic development. Holding such a critical developmental role not many details regarding molecular changes and cell surface marker (CSM) expression on these transitional cells are known. We think that sub-segregating the process of EHT holds great therapeutic potential by giving new indications to understand the hematopoietic stem cell maturation. We generated embryonic stem (ES) cell lines from Hoxb4-YFP reporter mice (Hills, 2011), in which YFP expression marks functional HSCs in adult and embryonic mice. When these cell lines were subject to embryoid body differentiation assays, we observed a transient Hoxb4/YFP+ cell population, which corresponded to the emergence of hemogenic endothelial cells in culture. We then analyzed the gene expression profile of the Hoxb4+ cells and compared it to immediate precursor (Hoxb4-Flk1+) and daughter (Hoxb4-CD41+) cell populations. As well as documenting the expression of numerous molecular markers previously associated with EHT, we also observed an elevated inflammatory gene expression signature that has previously been characterized as a mediator of hematopoietic specification in vivo. We were additionally able to identify 45 novel cell surface markers that could potentially be used to prospectively isolate and sub segregate cells undergoing EHT. 26 of these targets were subsequently verified using MRM mass spectrometry. To establish if any of these markers were functionally relevant, we generated knockout (KO) cell lines using CRISPR/Cas9. Upon EB differentiation, we observed a profound block in hematopoietic differentiation for ES cells that were KO for the membrane proteins Evi2a and Lyve1. This block was manifest at the EHT stage, as verified by time-lapse video imaging, and was equivalent in magnitude to Runx1 KO, suggesting a crucial role of Evi2a and Lyve1 during the EHT stage of hematopoietic development. Notably, this defect could be rescued by genetic replacement of the deleted gene. When comparing our data to human fetal liver data sets Evi2a is highly expressed in fetal liver, as well as in HSCs isolated from murine AGM and fetal liver.
  • Targeting the procoagulant tumour microenvironment in breast cancer

    Blower, Emma; Castle, J.; Clarke, Robert B; Kirwan, C. C.; Manchester Cancer and Thrombosis Team, Manchester (2021)
    Introduction: Coagulation factors, in particular tissue factor (TF), are produced by breast tumour cells and cancer-associated fi broblasts (CAFs). These signal via protease-activated receptors 1 and 2 (PAR1/2) on the cell membrane and induce invasion, angiogenesis and metastasis. Direct oral anticoagulants (DOACs), including rivaroxaban and Dabigatran, inhibit the TF–factor VIIa–factor Xa complex and thrombin respectively, and could therefore be repurposed as anticancer drugs. Aim: We aim to demonstrate that creating a procoagulant tumour micro environment will increase breast cancer cell proliferation and migration in vitro, with this eff ect being abrogated by DOACs. The mechanism by which this occurs, in terms of downstream phosphorylated proteins will also be examined. Materials and Methods: Breast cancer cell lines (BT474, MCF-7 and MDA-MB-231) were co-cultured with recombinant clotting factors (thrombin, TF, factor Xa (FXa), factor VIIa (FVIIa)) in the presence or absence of the anti-TF antibody 10H10, the direct FXa inhibitor rivaroxaban and the direct thrombin inhibitor dabigatran. Proliferation and migration were assessed in vitro using sulforhodamine B assays and wound closure assays visualised using the Incucyte respectively. The eff ect on downstream phosphorylated proteins was assessed by Western blotting. Results: 10 and 40 units/ml of thrombin increases proliferation in BT474 cells (p<0.0001). TF, FVIIa and FXa in combination increases proliferation in MCF-7 cells (p=0.05). Conversely, FXa at 0.1 and 10 units/ml reduces proliferation in MDA-MB-231 cells (p=0.33, p=0.001). 10H10 decreases proliferation in MCF-7 cells (p=0.02), with a trend for decrease in BT474 cells. 10 units/ml of FXa and TF, FVIIa and FXa in combination increases migration of MDA-MB-231 cells (p=0.05 and p=0.007 respectively), whilst there is a trend towards increased migration with 40 units/ml of thrombin in MDA-MB-231 and MCF-7 cells. DOACs had no eff ect on proliferation or migration in vitro. In BT474 cells, thrombin and TF, FVIIa and FXa in combination appear to cause a relative increase in pERK within 1 minute and pAkt within 15 and 5 minutes respectively. Rivaroxaban and dabigatran both appear to cause a decrease in pAkt within 1 minute. Conclusions: Breast cancer cell line in vitro experiments have shown that thrombin and the TF–FVIIa–FXa complex increase proliferation, whilst 10H10 reduces proliferation. FXa independently and in combination with TF and FVIIa promotes migration. These aff ects appear to be occurring via the PAR-1/2 pathway. Targeting the procoagulant tumour microenvironment is a promising strategy in reducing breast cancer cell proliferation and migration.
  • Long-Term quality of life after (chemo)radiotherapy for high-risk endometrial cancer in PORTEC-3

    Post, C.; De Boer, S. M.; Powell, M. E.; Mileshkin, L.; Katsaros, D.; Bessette, P.; Haie-Meder, C.; Ottevanger, P. B.; Ledermann, J. A.; Khaw, P.; et al. (2020)
    Purpose or Objective The survival results of the PORTEC-3 trial showed a significant improvement in both overall and failure-free survival with chemoradiotherapy versus radiotherapy alone. The present analysis was performed to establish long-term adverse events and health related quality of life (HRQOL). Material and Methods The PORTEC-3 trial is an international randomized phase 3 trial. Women with high-risk endometrial cancer (stage I grade 3 with deep myometrial invasion or lymph-vascular space invasion; stage II/III endometrioid cancer; or stage I-III serous or clear cell cancer) were randomly assigned to receive pelvic radiotherapy alone (RT) or chemoradiotherapy (CTRT, concurrent 2 cycles of cisplatin followed by 4 cycles of carboplatin/paclitaxel). Adverse events (AE) were graded using CTCAE v3.0. HRQOL was measured using the EORTC QLQ-C30 and CX24 and OV28 symptom scales at baseline, after radiotherapy, and at 6, 12, 18, 24, 36 and 60 months. Symptoms rated as “quite a bit” or “very much” were considered as severe. Toxicity and HRQOL were analyzed according to treatment received. HRQOL scores were compared to an agematched European norm-population. Clinical trial information: NCT00411138. Results Between 2006 and 2013, 660 women were randomized, of whom 579 (88%) responded for HRQOL assessment at baseline, 355 at 3 years and 237 at 5 years. Median follow up was 74.6 months. At 5 years, AE grade ≥2 were scored for 80 (39%) patients who had received CTRT vs 50 (26%) who had received RT (p=0.007). Grade 3 AE did not differ significantly between the two groups (8% vs 5%, p=0.24) at 5 years and only one grade 4 AE was reported (ileus/obstruction after CTRT). Sensory neuropathy AE persisted at long-term after CTRT in 7% (vs 0% after RT, p<0.001 at 3 and 5 years). At 3 and 5 years, more women who had CTRT reported severe tingling or numbness at HRQOL (27% vs 8%, p<0.001 at 3 years; 24% vs 9%, p=0.002 at 5 years). At 3 years, more women reported severe weakness of arms/legs (21% vs 5%, p<0.001) and lower physical (79.4 vs 86.6, p<0.001, Fig 1) and role functioning (78.3 vs 88.0, p<0.001) scores. Additionally, a trend towards more reported severe muscle or joint pain was seen (28% vs 16%, p=0.037). At 5 years, no significant differences in these HRQOL symptoms or functioning scales were seen, with scores within range of the norm population scores; however, trends towards a lower global health/QOL score (74.4 vs 79.3, p=0.045), a higher fatigue score (24.1 vs 18.7, p=0.036) and more severe hearing problems (12% vs 4%, p=0.044) were observed after CTRT. Conclusion Chemoradiotherapy causes significantly higher rates of grade >2 AE, worse physical functioning and higher symptom scores as compared with RT, but with clear recovery within 2 years and stable scores from then onwards. The most important long-term AE and QOL impairment after CTRT was sensory neuropathy. These long-term data are essential for patient information and shared decision-making regarding adjuvant chemotherapy for high-risk endometrial cancer.
  • External validation of survival of lung cancer patients due to setup uncertainties towards the heart

    Brink, C.; Bernchou, U.; Bertelsen, A.; Hansen, O.; Schytte, T.; Holloway, L.; Van Herk, Marcel; Johnson-Hart, Corinne; Price, Gareth J; Aznar, Marianne Camille; et al. (2020)
    Purpose or Objective The impact on survival due to heart toxicity for lung cancer patients treated with radiotherapy (RT) has been difficult to quantify, due to correlations between dose to heart and lung with tumor burden. A possible measure that indicates enhanced dose to heart but does not correlate with the other risk factors is the deviation between actual and planned daily distance between isocenter and heart. The average of this daily distance (DeltaD), which can be obtained from CBCT scans, will for positive values indicate larger separation of target and heart and thus reduced heart dose during delivery. DeltaD has previously been reported to impact survival [Johnson-Hart et al IJROBP 2018]. The aim of the current study is to undertake an external validation of this finding in another institution Material and Methods All patients treated in the validation department from April 2010 to end 2015 with daily CBCT, planned dose of 60-66 Gy in 2 Gy fractions and for which a heart delineation was available were collected for analysis. Standard clinical IGRT procedure utilized two registrations for each CBCT 1) tumor region (mask) and 2) overall anatomy (clipbox). The first was used for patient positioning while the latter was used for overall validation of the anatomy. The latter is representative of the heart position, while the first represents the tumor position hence the treatment isocenter. The difference between the registrations can thus be used to estimate the daily shift of the heart relative to the isocenter. Based on clinical data (performance status, GTV volume, Age, T and N stage, histology, tumor stage and dose) a base Cox model predicting survival was created using Lasso for parameter selection. Impact of continuous variable DeltaD was tested by adding DeltaD to the base Cox model. All analyses were performed in R v. 3.6.1. Results 300 patients were included. DeltaD did not correlate statistically significant with any of the clinical factors. Range of DeltaD was -0.6 cm to 0.7 cm. Figure 1 shows Kaplan-Meier plots of patients with positive versus negative DeltaD. It is seen that a split of the curves occurs around 16 months after the start of RT. The base Cox model included performance status, ln(GTV volume) and Age. DeltaD was included in the base model using time dependent Cox regression for which DeltaD effectively was zero until 16 months after RT. This resulted in a statistically significant regression constant of DeltaD of - 1.63 per cm (HR=0.195) with p-value of 0.017 Conclusion As in the original study this validation study demonstrates significant impact on survival due to estimated setup errors in the direction of the heart, even with daily online IGRT corrections. This indicates that dose to heart impacts survival for these patients. However, in contrast to the original study the effect of DeltaD only starts 16 months after RT. This difference in the two studies might reflect differences in treatment or patient cohorts at the two centers, and calls for additional external validation.
  • The relationship between the coagulation and inflammatory phases of wound healing in early breast cancer

    Singh, U; Castle, John; Shaker, H.; Greenhalgh, S.; Hussain, U.; Descamps, Tine; Nash, S.; Wilson, M.; Hunt, R; Kirwan, Cliona C; et al. (2021)
    Introduction: Cancer is likened to a non-healing wound. Markers of coagulation (TF, thrombin, PAR1 and PAR2), the fi rst phase of wound healing, have increased stromal fi broblast expression in poor prognosis breast cancer (estrogen receptor (ER) negative, Her2 positive, high Ki67/grade). We hypothesised that markers of infl ammation, the second phase of wound healing, (CD68, macrophage marker; Heme oxygenase 1 (HO-1), macrophage and cancer cell marker; fi broblast activation protein (FAP), fi broblast marker) would correlate with coagulation markers and predict poor prognosis in early breast cancer. Aim: To assess the relationship between coagulation and infl ammatory markers in early breast cancer. To assess the prognostic value of CD68, HO-1 and FAP infl ammatory markers using clinicopathological variables. Materials and Methods: The prospective cohort study CHAMPion (Cancer induced Hypercoagulability as a Marker of Prognosis) recruited patients with ductal carcinoma in situ(DCIS) and invasive breast cancer. In tissue microarrays of 201 invasive tumours, 58 DCIS tumours and matched normal breast tissue distant from disease, CD68+ tumour associated macrophage(TAM)/CD68+ normal macrophage (normal tissue), epithelial HO-1 and fi broblast FAP expression, quantifi ed by immunohistochemistry(dichotomised: high/present vs low/absent), was correlated with tumour factors (grade, proliferation (Ki67), ER, HER2); demographics, behavioural factors (smoking, alcohol) and survival (disease-free survival (DFS), overall survival (OS)). Correlation between coagulation and infl ammatory markers was assessed using Cramer’s V correlation test (p<0.05). Results: High CD68+ macrophage expression was more commonly seen in invasive breast cancer, compared to DCIS, and normal tissue (59%, 41% and 6%, respectively; p<0.001). In invasive cancer, CD68+ TAM expression was increased with increasing tumour grade (Grade 1: 42%, Grade 2: 58%, Grade 3: 72%; p=0.006), high Ki67 (71% vs 47%; p=0.004), ER negativity (79.4% vs 55.4%; p=0.01) and HER2 (HER2 positive 81.8% vs HER2 negative 56.3%; p=0.03). CD68+ TAM expression was higher in high grade compared to low/ intermediate grade DCIS (44% vs 31%, p=0.52). CD68+ TAM expression was increased patients who self-reported alcohol intake (non-drinker 43% vs drinker 62%; p=0.01). Positive HO-1 and FAP expression was not associated with poor prognosis subgroups however cancer cell expression of HO-1 was associated with shorter DFS (HR 3.22, p=0.027) and OS (HR 2.86, p=0.029). Cancer-associated fi broblast (CAF) and tissue factor (TF) expression weakly correlated with high CD68+ TAM expression (CV 0.12, p=0.09), cancer cell HO-1 expression (CV 0.14, p=0.04) and CAF FAP expression (CV 0.15, p=0.03). In CAFs, PAR2 and FAP expression correlated (CV 0.14, p=0.04). Conclusions: Tumour infl ammation, as assessed by high CD68+ TAM expression is increased in poor prognosis breast cancer sub-types. The association reported here between CD68 and alcohol is suggestive of a mechanism for alcohol as a breast cancer risk factor. The correlation between stromal coagulation (fi broblast TF) and infl ammation highlights wound healing as potentially mechanistic in breast cancer growth.
  • Prostate radiotherapy with carbogen and nicotinamide. Final results of the phase 1b/II PROCON trial

    Yip, K.; Hoskin, Peter J; Thiruthaneeswaran, Nijula; Alonzi, R.; Marie Curie Research Wing, Mount Vernon Cancer Centre, Northwood Middlesex, (2020)
    Purpose or Objective Prostate tumour hypoxia is associated with resistance to radiotherapy (RT) and increased likelihood of relapse post treatment. The concurrent administration of carbogen and nicotinamide (CON) with RT improves overall survival in patients with bladder cancer and regional control rates following laryngeal RT. We evaluated the safety, toxicity of this approach for patients with high risk prostate cancer. Material and Methods 50 patients (with 1 of: T3, Gleason ≥8, PSA ≥20) were recruited into the single arm phase 1b/II PROCON trial between Dec 2011 and Sept 2013. They breathed carbogen via a tight-fitting face mask during RT and took 60mg/kg of nicotinamide daily prior to the delivery of RT (74Gy/37# to the prostate and 60Gy/37# to the pelvic nodes). Twenty men also underwent functional MRI imaging; correction of tumour hypoxia was evaluated by comparing tumour R2* values before and after carbogen administration. Patients were assessed during treatment and 2, 4 and 12 weeks after they had completed RT. The primary endpoints were prevalence of grade 3 (G3) or worse lower gastrointestinal (GI) or genitourinary (GU) toxicities during this period. Secondary end points include PSA progression free survival (PFS) and overall survival (OS) rates at five years as calculated by the Kaplan Meier method. Results The median duration of follow up was 60 months. All patients completed the prescribed 37 fraction schedule of RT over 50 to 56 days (median duration: 52 days). 56% patients experienced grade 1 nausea (CTCAE v 4.0), and 52% patients received anti-emetic treatment. 22% patients had a nicotinamide dose adjustment, 20% patients had a break from nicotinamide and 6% patients discontinued nicotinamide. 96% patients received carbogen with every RT fraction. One patient had an interruption of treatment and received carbogen for 33 of 37 fractions. One patient discontinued carbogen treatment after 7 fractions. No one developed G3 or worse lower GI or GU toxicities; prevalence of G1/G2 toxicities are shown in table 1. The 5 year OS and PSA-PFS rates were 92% and 87% respectively. Six patients experienced biochemical progression. Five patients have died but None had biochemical relapse prior to their death. The functional MRI analysis showed a mean decrease of 5.8% in tumour R2* after the application of carbogen, measured in 72 pairs of pre and post carbogen measurements, indicating that the carbogen exposure resulted in an immediate reduction in tumour hypoxia. Conclusion The prevalence of acute lower GI and GU toxicities among our cohort, together with their 5 year PSA PFS and OS rates, are comparable, or superior to, those reported for patients with high risk prostate cancer receiving RT in other contemporary trials. The concurrent administration of CON with RT is safe and effective. It should be further explored in a phase III setting.
  • Dynamic of broad H3K4me3 domains uncover an epigenetic switch between cell identity and cancer-related genes

    Belhocine, M.; Simonin, M.; Abad Flores, J. D.; Cieslak, A.; Manosalva, I.; Pradel, L.; Smith, C.; Mathieu, E. L.; Charbonnier, G.; Martens, J. H. A.; et al. (2021)
    Broad domains of H3K4 methylation have been associated with consistent expression of tissue-specific, cell identity, and tumor suppressor genes. Here, we identified broad domain-associated genes in healthy human thymic T cell populations and a collection of T-Acute Lymphoblastic Leukemia (T-ALL) primary samples and cell lines. We found that broad domains are highly dynamic throughout T cell differentiation and their varying breadth allows the distinction between normal and neoplastic cells. While broad domains preferentially associate with cell identity and tumor suppressor genes in normal thymocytes, they flag key oncogenes in T-ALL samples. Moreover, the expression of broad domain-associated genes, both coding and noncoding, is frequently deregulated in T-ALL. Using two distinct leukemic models, we demonstrated that the ectopic expression of T-ALL oncogenic transcription factor preferentially impacts the expression of broad domain-associated genes in pre-leukemic cells. Finally, an H3K4me3 demethylase inhibitor differentially targets T-ALL cell lines depending on the extent and number of broad domains. Our results show that the regulation of broad H3K4me3 domains is associated with leukemogenesis and suggest that the presence of these structures might be used as epigenetic prioritization of cancer-relevant genes, including long non-coding RNAs.
  • Highly synchronous mitotic progression in schizosaccharomyces pombe upon relief of transient Cdc2-asM17 inhibition

    Singh, Pawan; Halova, Lenka; Hagan, Iain M; Cell Division Group, CRUK Manchester Institute, The University of Manchester, Alderley Park, UK. (2021)
    Synchronized progression of a cell population through the cell division cycle supports the biochemical and functional dissection of cell cycle controls and execution. The concerted behaviour of the population reflects the attributes of each cell within that population. The reversible imposition of a block to cell cycle progression at the G2-M boundary through transient inactivation of the Cdk1-Cyclin B activating phosphatase, Cdc25, with the temperature sensitive cdc25-22 mutant, has been widely used to study fission yeast mitosis and DNA replication. However, the biology of the compromised Cdc25-22 phosphatase generates significant division abnormalities upon release from mitotic arrest. We show how reversible inhibition of Cdc2-asM17, with the ATP analog 3-BrB-PP1, generates higher levels of synchrony with timing and morphology much more reminiscent of a normal division. We also describe a version of the H1 kinase assay of Cdk1-Cyclin B activity that is widely used to monitor mitotic progression which does not require radiolabeled ATP.
  • Host genetic polymorphisms associated with beta human papillomavirus seropositivity

    Antonsson, A.; Rodriguez-Acevedo, A. J.; Liyanage, U. E.; Hughes, M. C. B.; van der Pols, J. C; Green, Adèle C; QIMR Berghofer Medical Research Institute, Brisbane, Australia. (2021)
    Human papillomaviruses (HPVs) cause superficial epidermal infections and are only cleared if they trigger an immunological response. We analysed SNPs that had previously been investigated for association with HPV infection to determine whether they play a role in the serological response to cutaneous beta-HPVs in an Australian population. Serum samples from 1,142 participants were analysed for seropositivity against the L1 protein of 21 beta-HPV types. Associations between seropositivity to beta-HPV types and the SNPs rs9264942 (HLA-C; HPV-9, p = 0.022, HPV-15, p = 0.043 and HPV-17, p = 0.004), rs12449858 (EVER1; HPV-23, p = 0.029), and rs2981451 (FGFR2; HPV-22, p = 0.049) were identified. We found that certain SNPs could be involved in the serological response to beta-HPVs.
  • Wound healing inflammatory markers predict prognosis and survival in early breast cancer

    Singh, U; Castle, John; Greenhalgh, S.; Hussain, U.; Descamps, Tine; Nash, S.; Wilson, M.; Hunt, R; Kirwan, Cliona C; Division of Cancer Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, University of Manchester (2021)
  • Real world interpretation of GEC-ESTRO image-guided brachytherapy recommendations for cervix cancer

    Diez, P.; Bourner, J.; Sharp, A.; Hoskin, Peter J; McCormack, M.; National Radiotherapy Trials Quality Assurance Group, Mount Vernon Cancer Centre, Northwood, (2020)
    Purpose or Objective INTERLACE (NCT01566240) is an international phase III multicentre trial of weekly induction chemotherapy followed by standard chemoradiation versus standard chemoradiation alone in patients with locally advanced cervical cancer. As well as a trial protocol, detailed radiotherapy guidelines were produced with a quality assurance (QA) programme to ensure compliance at the 37 participating centres. The guidelines mandated GECESTRO recommendations for image-guided adaptive brachytherapy (IGABT) and prescribing to the high-risk CTV (HRCTV). This study assesses adherence to these recommendations. Material and Methods As part of the trial QA programme brachytherapy centres were required to submit a brachytherapy dummy run (a patient already treated at their centre) for central review prior to trial participation. Both contouring and planning were reviewed to assess their standard brachytherapy technique. The following parameters have been evaluated: use of MR and whether scanned with implant in situ; HRCTV and intermediate-risk CTV (IRCTV) definition; and dose conformity (HRCTV D90, HRCTV V100, and Vref). Results Of 34 institutions providing a brachytherapy service, 23 stated they performed IGABT, prescribing and optimising to HRCTV D90. However, 3 of these centres did not use MR in this process and 1 performed MR without the implant in situ. 8 centres did not follow GEC-ESTRO HRCTV delineation recommendations (see Figure 1). In 2 of the 11 centres defining an IRCTV this was not a direct expansion of the HRCTV and in a further 2 the vagina was not edited out of the expansion. The plan was assessed for dose conformity around the HRCTV. Only 9 centres did this adequately. Most of the uterus was included within the prescription isodose in 14 centres and a large section of the vagina in 7 centres, instead of conforming to the HRCTV definition Conclusion Moving from a 2D procedure or a standard plan in 3D to IGABT can be a steep learning curve, in particular in defining the target. Use of MRI with the implant in situ, and its careful assessment in all 3 imaging planes, is fundamental for accurate target delineation. Approximately a third of centres under-contoured the HRCTV, therefore increasing the risk of local recurrence. 11 centres over-contoured the target, having the potential to either increase the risk of toxicity or, having to reduce the prescription to remain within OAR tolerance, compromising the chance of tumour control. The same is the case where the prescription dose does not conform to the HRCTV. Only 5 centres adhered to all aspects of the GEC-ESTRO cervical IGABT recommendations that were evaluated. Although feedback was provided to all centres within INTERLACE, ongoing education may be necessary. Prospective central review was therefore carried out on the first 2 patients at each centre, with further cases assessed where required. Adoption of GEC-ESTRO recommendations in their entirety is critical to accurate delivery of IGABT. Partial compliance may prove unsafe.
  • Identification and characterization of novel functional markers during the hematopoietic stem cell specification process

    Buchler-Schaff, M.; Kaschutnig, P.; Thambyrajah, Roshana; Nadler, W.; Hanke, S.; Pfaffenholz, S.; Block, M.; Grasel, J.; Kremer, J.; Bayindir-Buchhalter, I.; et al. (2020)
    Endothelial to hematopoietic transition (EHT) is a crucial step in the formation of definitive hematopoietic stem cells during embryonic development. Holding such a critical developmental role not many details regarding molecular changes and cell surface marker (CSM) expression on these transitional cells are known. We think that sub-segregating the process of EHT holds great therapeutic potential by giving new indications to understand the hematopoietic stem cell maturation. We generated embryonic stem (ES) cell lines from Hoxb4-YFP reporter mice (Hills, 2011), in which YFP expression marks functional HSCs in adult and embryonic mice. When these cell lines were subject to embryoid body differentiation assays, we observed a transient Hoxb4/YFP+ cell population, which corresponded to the emergence of hemogenic endothelial cells in culture. We then analyzed the gene expression profile of the Hoxb4+ cells and compared it to immediate precursor (Hoxb4-Flk1+) and daughter (Hoxb4-CD41+) cell populations. As well as documenting the expression of numerous molecular markers previously associated with EHT, we also observed an elevated inflammatory gene expression signature that has previously been characterized as a mediator of hematopoietic specification in vivo. We were additionally able to identify 45 novel cell surface markers that could potentially be used to prospectively isolate and sub segregate cells undergoing EHT. 26 of these targets were subsequently verified using MRM mass spectrometry. To establish if any of these markers were functionally relevant, we generated knockout (KO) cell lines using CRISPR/Cas9. Upon EB differentiation, we observed a profound block in hematopoietic differentiation for ES cells that were KO for the membrane proteins Evi2a and Lyve1. This block was manifest at the EHT stage, as verified by time-lapse video imaging, and was equivalent in magnitude to Runx1 KO, suggesting a crucial role of Evi2a and Lyve1 during the EHT stage of hematopoietic development. Notably, this defect could be rescued by genetic replacement of the deleted gene. When comparing our data to human fetal liver data sets Evi2a is highly expressed in fetal liver, as well as in HSCs isolated from murine AGM and fetal liver.
  • Host genetic polymorphisms associated with beta human papillomavirus seropositivity

    Antonsson, A.; Rodriguez-Acevedo, A. J.; Liyanage, U. E.; Hughes, M. C. B.; van der Pols, J. C; Green, Adèle C; QIMR Berghofer Medical Research Institute, Brisbane, Australia. (2021)
    Human papillomaviruses (HPVs) cause superficial epidermal infections and are only cleared if they trigger an immunological response. We analysed SNPs that had previously been investigated for association with HPV infection to determine whether they play a role in the serological response to cutaneous beta-HPVs in an Australian population. Serum samples from 1,142 participants were analysed for seropositivity against the L1 protein of 21 beta-HPV types. Associations between seropositivity to beta-HPV types and the SNPs rs9264942 (HLA-C; HPV-9, p = 0.022, HPV-15, p = 0.043 and HPV-17, p = 0.004), rs12449858 (EVER1; HPV-23, p = 0.029), and rs2981451 (FGFR2; HPV-22, p = 0.049) were identified. We found that certain SNPs could be involved in the serological response to beta-HPVs.
  • Highly synchronous mitotic progression in schizosaccharomyces pombe upon relief of transient Cdc2-asM17 inhibition

    Singh, Pawan; Halova, Lenka; Hagan, Iain M; Cell Division Group, CRUK Manchester Institute, The University of Manchester, Alderley Park, UK. (2021)
    Synchronized progression of a cell population through the cell division cycle supports the biochemical and functional dissection of cell cycle controls and execution. The concerted behaviour of the population reflects the attributes of each cell within that population. The reversible imposition of a block to cell cycle progression at the G2-M boundary through transient inactivation of the Cdk1-Cyclin B activating phosphatase, Cdc25, with the temperature sensitive cdc25-22 mutant, has been widely used to study fission yeast mitosis and DNA replication. However, the biology of the compromised Cdc25-22 phosphatase generates significant division abnormalities upon release from mitotic arrest. We show how reversible inhibition of Cdc2-asM17, with the ATP analog 3-BrB-PP1, generates higher levels of synchrony with timing and morphology much more reminiscent of a normal division. We also describe a version of the H1 kinase assay of Cdk1-Cyclin B activity that is widely used to monitor mitotic progression which does not require radiolabeled ATP.
  • Dynamic of broad H3K4me3 domains uncover an epigenetic switch between cell identity and cancer-related genes

    Belhocine, M.; Simonin, M.; Abad Flores, J. D.; Cieslak, A.; Manosalva, I.; Pradel, L.; Smith, C.; Mathieu, E. L.; Charbonnier, G.; Martens, J. H. A.; et al. (2021)
    Broad domains of H3K4 methylation have been associated with consistent expression of tissue-specific, cell identity, and tumor suppressor genes. Here, we identified broad domain-associated genes in healthy human thymic T cell populations and a collection of T-Acute Lymphoblastic Leukemia (T-ALL) primary samples and cell lines. We found that broad domains are highly dynamic throughout T cell differentiation and their varying breadth allows the distinction between normal and neoplastic cells. While broad domains preferentially associate with cell identity and tumor suppressor genes in normal thymocytes, they flag key oncogenes in T-ALL samples. Moreover, the expression of broad domain-associated genes, both coding and noncoding, is frequently deregulated in T-ALL. Using two distinct leukemic models, we demonstrated that the ectopic expression of T-ALL oncogenic transcription factor preferentially impacts the expression of broad domain-associated genes in pre-leukemic cells. Finally, an H3K4me3 demethylase inhibitor differentially targets T-ALL cell lines depending on the extent and number of broad domains. Our results show that the regulation of broad H3K4me3 domains is associated with leukemogenesis and suggest that the presence of these structures might be used as epigenetic prioritization of cancer-relevant genes, including long non-coding RNAs.
  • Polygenic risk scores stratify keratinocyte cancer risk among solid organ transplant recipients with chronic immunosuppression in a high ultraviolet radiation environment.

    Seviiri, M.; Law, M. H.; Ong, J. S.; Gharahkhani, P.; Nyholt, D. R.; Hopkins, P.; Chambers, D.; Campbell, S.; Isbel, N. M.; Soyer, H. P.; et al. (2021)
    Solid organ transplant recipients (SOTRs) have elevated risks for basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), especially in high UVR environments. We assessed whether polygenic risk scores can improve the prediction of BCC and SCC risks and multiplicity over and above the traditional risk factors in SOTRs in a high UV setting. We built polygenic risk scores for BCC (n = 594,881) and SCC (n = 581,431) using UK Biobank and 23andMe datasets, validated them in the Australian QSkin Sun and Health Study cohort (n > 6,300), and applied them in SOTRs in the skin tumor in allograft recipients cohort from Queensland, Australia, a high UV environment. About half of the SOTRs with a high genetic risk developed BCC (absolute risk = 45.45%, 95% confidence interval = 33.14-58.19%) and SCC (absolute risk = 44.12%, 95% confidence interval = 32.08-56.68%). For both cancers, SOTRs in the top quintile were at >3-fold increased risk relative to those in the bottom quintile. The respective polygenic risk scores improved risk predictions by 2% for BCC (area under the curve = 0.77 vs. 0.75, P = 0.0691) and SCC (area under the curve = 0.84 vs. 0.82, P = 0.0260), over and above the established risk factors, and 19.03% (for BCC) and 18.10% (for SCC) of the SOTRs were reclassified in a high/medium/low risk scenario. The polygenic risk scores also added predictive accuracy for tumor multiplicity (BCC R2 = 0.21 vs. 0.19, P = 3.2 × 10-3; SCC R2 = 0.30 vs. 0.27, P = 4.6 × 10-4).

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