• TP53 loss initiates chromosomal instability in fallopian tube epithelial cells

      Bronder, Daniel; Tighe, Anthony; Wangsa, D.; Zong, D.; Meyer, T. J.; Wardenaar, R.; Minshall, Paul; Hirsch, D.; Heselmeyer-Haddad, K.; Nelson, Louisa; et al. (2021)
      High-grade serous ovarian cancer (HGSOC) originates in the fallopian tube epithelium and is characterized by ubiquitous TP53 mutation and extensive chromosomal instability (CIN). However, direct causes of CIN, such as mutations in DNA replication and mitosis genes, are rare in HGSOC. We therefore asked whether oncogenic mutations that are common in HGSOC can indirectly drive CIN in non-transformed human fallopian tube epithelial cells. To model homologous recombination deficient HGSOC, we sequentially mutated TP53 and BRCA1 then overexpressed MYC. Loss of p53 function alone was sufficient to drive the emergence of sub-clonal karyotype alterations. TP53 mutation also led to global gene expression changes, influencing modules involved in cell cycle commitment, DNA replication, G2/M checkpoint control, and mitotic spindle function. Both transcriptional deregulation and karyotype diversity were exacerbated by loss of BRCA1 function, with whole-genome doubling events observed in independent p53/BRCA1-deficient lineages. Thus, our observations indicate that loss of the key tumour suppressor TP53 is sufficient to deregulate multiple cell cycle control networks and thereby initiate CIN in pre-malignant fallopian tube epithelial cells.
    • The impact of COVID-19 lockdowns on the genetic integrity of your mouse colonies

      Moncaut, Natalia; Hart-Johnson, S.; Genome Editing and Mouse Models, Cancer Research UK Manchester Institute, University of Manchester, Manchester, UK (2021)
    • Upregulation of human endogenous retroviruses in bronchoalveolar lavage fluid of COVID-19 patients

      Kitsou, K.; Kotanidou, A.; Paraskevis, D.; Karamitros, T.; Katzourakis, A.; Tedder, R.; Hurst, T.; Sapounas, S.; Kotsinas, A.; Gorgoulis, Vassilis G; et al. (2021)
      Severe COVID-19 pneumonia has been associated with the development of intense inflammatory responses during the course of infections with SARS-CoV-2. Given that human endogenous retroviruses (HERVs) are known to be activated during and participate in inflammatory processes, we examined whether HERV dysregulation signatures are present in COVID-19 patients. By comparing transcriptomes of bronchoalveolar lavage fluid (BALF) of COVID-19 patients and healthy controls, and peripheral blood monocytes (PBMCs) from patients and controls, we have shown that HERVs are intensely dysregulated in BALF of COVID-19 patients compared to those in BALF of healthy control patients but not in PBMCs. In particular, upregulation in the expression of specific HERV families was detected in BALF samples of COVID-19 patients, with HERV-FRD being the most highly upregulated family among the families analyzed. In addition, we compared the expression of HERVs in human bronchial epithelial cells (HBECs) without and after senescence induction in an oncogene-induced senescence model in order to quantitatively measure changes in the expression of HERVs in bronchial cells during the process of cellular senescence. This apparent difference of HERV dysregulation between PBMCs and BALF warrants further studies in the involvement of HERVs in inflammatory pathogenetic mechanisms as well as exploration of HERVs as potential biomarkers for disease progression. Furthermore, the increase in the expression of HERVs in senescent HBECs in comparison to that in noninduced HBECs provides a potential link for increased COVID-19 severity and mortality in aged populations. IMPORTANCE SARS-CoV-2 emerged in late 2019 in China, causing a global pandemic. Severe COVID-19 is characterized by intensive inflammatory responses, and older age is an important risk factor for unfavorable outcomes. HERVs are remnants of ancient infections whose expression is upregulated in multiple conditions, including cancer and inflammation, and their expression is increased with increasing age. The significance of this work is that we were able to recognize dysregulated expression of endogenous retroviral elements in BALF samples but not in PBMCs of COVID-19 patients. At the same time, we were able to identify upregulated expression of multiple HERV families in senescence-induced HBECs in comparison to that in noninduced HBECs, a fact that could possibly explain the differences in disease severity among age groups. These results indicate that HERV expression might play a pathophysiological role in local inflammatory pathways in lungs afflicted by SARS-CoV-2 and their expression could be a potential therapeutic target.
    • Enhancer recruitment of transcription repressors RUNX1 and TLE3 by mis-expressed FOXC1 blocks differentiation in acute myeloid leukemia

      Simeoni, Fabrizio; Romero-Camarero, Isabel; Camera, Francesco; Amaral, Fabio M R; Sinclair, Oliver J; Papachristou, E. K.; Spencer, Gary J; Lie-A-Ling, Michael; Lacaud, Georges; Wiseman, Daniel H; et al. (2021)
      Despite absent expression in normal hematopoiesis, the Forkhead factor FOXC1, a critical mesenchymal differentiation regulator, is highly expressed in ?30% of HOXAhigh acute myeloid leukemia (AML) cases to confer blocked monocyte/macrophage differentiation. Through integrated proteomics and bioinformatics, we find that FOXC1 and RUNX1 interact through Forkhead and Runt domains, respectively, and co-occupy primed and active enhancers distributed close to differentiation genes. FOXC1 stabilizes association of RUNX1, HDAC1, and Groucho repressor TLE3 to limit enhancer activity: FOXC1 knockdown induces loss of repressor proteins, gain of CEBPA binding, enhancer acetylation, and upregulation of nearby genes, including KLF2. Furthermore, it triggers genome-wide redistribution of RUNX1, TLE3, and HDAC1 from enhancers to promoters, leading to repression of self-renewal genes, including MYC and MYB. Our studies highlight RUNX1 and CEBPA transcription factor swapping as a feature of leukemia cell differentiation and reveal that FOXC1 prevents this by stabilizing enhancer binding of a RUNX1/HDAC1/TLE3 transcription repressor complex to oncogenic effect.
    • Risk-based decision-making in the treatment of HER2-positive early breast cancer: Recommendations based on the current state of knowledge

      Jackisch, C.; Cortazar, P.; Geyer, C. E.; Gianni, L.; Gligorov, J.; Machackova, Z.; Perez, E. A.; Schneeweiss, A.; Tolaney, S. M.; Untch, M.; et al. (2021)
      Treatment of HER2-positive early breast cancer (EBC) continues to evolve with neoadjuvant (pre-operative) and adjuvant (post-operative) HER2-targeted therapies as standard of care. There are two important decision points. The first involves deciding between neoadjuvant therapy or proceeding directly to surgery. Neoadjuvant chemotherapy (NACT) plus pertuzumab-trastuzumab is appropriate for patients with high-risk HER2-positive EBC (tumour diameter ≥2 cm, and/or node-positive disease). Patients with node-negative disease and tumour diameter <2 cm are candidates for upfront surgery followed by paclitaxel for 12 weeks plus 18 cycles of trastuzumab, with the option to add pertuzumab (if pN+). The second decision point involves the pathohistological result at surgery after neoadjuvant therapy. Total pathological complete response (tpCR: ypT0/is, ypN0) is associated with improved survival endpoints. Patients with tumours ≥2 cm and/or node-positive disease at diagnosis who have a tpCR after dual blockade should continue pertuzumab-trastuzumab in the adjuvant setting to complete 1 year (18cycles) of treatment. For patients with invasive residual disease, 14cycles of post-neoadjuvant trastuzumab emtansine (T-DM1) therapy significantly increases invasive-DFS compared with trastuzumab. Extended adjuvant therapy with neratinib is an option in selected patients (HER2-positive and oestrogen receptor [ER]-positive) who have completed adjuvant trastuzumab-based therapy. Less aggressive chemotherapy regimens are recommended in populations with a lower risk of recurrence (patients with small tumours without axillary involvement; patients unlikely to tolerate anthracycline-taxane or taxane-carboplatin regimens). Ultimately, treatment recommendations should be consistent with local and international guidelines. Further studies will guide optimisation of treatment for patients with HER2-positive EBC according to the risk of disease recurrence.
    • The Magnolia Trial: Zanubrutinib, a next-generation bruton tyrosine kinase inhibitor, demonstrates safety and efficacy in relapsed/refractory marginal zone lymphoma

      Opat, S.; Tedeschi, A.; Linton, Kim M; McKay, P.; Hu, B.; Chan, H.; Jin, J.; Sobieraj-Teague, M.; Zinzani, P. L.; Coleman, M.; et al. (2021)
      Purpose: Marginal zone lymphoma (MZL) is an uncommon non-Hodgkin lymphoma with malignant cells that exhibit a consistent dependency on B-cell receptor signaling. We evaluated the efficacy and safety of zanubrutinib, a next-generation selective Bruton tyrosine kinase inhibitor, in patients with relapsed/refractory (R/R) MZL. Experimental design: Patients with R/R MZL were enrolled in the phase 2 MAGNOLIA (BGB-3111-214) study. The primary endpoint was overall response rate (ORR) as determined by an independent review committee (IRC) based on the Lugano 2014 classification. Results: Sixty-eight patients were enrolled. After a median follow-up of 15.7 months (range, 1.6 to 21.9 months), the IRC-assessed ORR was 68.2% and complete response (CR) was 25.8%. The ORR by investigator assessment was 74.2%, and the CR rate was 25.8%. The median duration of response (DOR) and median progression-free survival (PFS) by independent review was not reached. The IRC-assessed DOR rate at 12 months was 93.0%, and IRC-assessed PFS rate was 82.5% at both 12 and 15 months.with the majority of adverse events (AEs) being grade 1 or 2. The most common AEs were diarrhea (22.1%), contusion (20.6%), and constipation (14.7%). Atrial fibrillation/flutter was reported in two patients; one patient had grade 3 hypertension. No patient experienced major hemorrhage. In total, four patients discontinued treatment due to AEs, of which was considered treatment-related by the investigators. Conclusions: Zanubrutinib demonstrated high ORR and CR rate with durable disease control and a favorable safety profile in patients with R/R MZL.
    • International variation in childhood cancer mortality rates from 2001-2015: comparison of trends in the International Cancer Benchmarking Partnership countries

      Smith, L.; Stiller, C. A.; Aitken, J. F.; Hjalgrim, L. L.; Johannesen, T.; Lahteenmaki, P.; McCabe, Martin G; Phillips, R.; Pritchard-Jones, K.; Steliarova-Foucher, E.; et al. (2021)
      Despite improved survival rates, cancer remains one of the most common causes of childhood death. The International Cancer Benchmarking Partnership (ICBP) showed variation in cancer survival for adults. We aimed to assess and compare trends over time in cancer mortality between children, adolescents and young adults (AYAs) and adults in the six countries involved in the ICBP: UK, Denmark, Australia, Canada, Norway and Sweden. Trends in mortality between 2001 and 2015 in the six original ICBP countries were examined. Age standardized mortality rates (ASR per million) were calculated for all cancers, leukaemia, malignant and benign CNS tumours, and non-CNS solid tumours. ASRs were reported for children (age 0-14 years), AYAs aged 15-39 years, and adults aged 40 years and above. Average annual percentage change (AAPC) in mortality rates per country were estimated using Joinpoint regression. For all cancers combined, significant temporal reductions were observed in all countries and all age groups. However, the overall AAPC was greater for children (-2.9; 95% CI -4.0 to -1.7) compared to AYAs (-1.8; -2.1 to -1.5) and adults aged>40 years (-1.5; -1.6 to -1.4). This pattern was mirrored for leukaemia, CNS tumours and non-CNS solid tumours, with the difference being most pronounced for leukaemia: AAPC for children -4.6 (-6.1 to -3.1) vs AYAs -3.2 (-4.2 to -2.1) and over 40s -1.1 (-1.3 to -0.8). AAPCs varied between countries in children for all cancers except leukaemia, and in adults over 40 for all cancers combined, but not in subgroups. Improvements in cancer mortality rates in ICBP countries have been most marked among children aged 0-14s in comparison to 15-39 and over 40 year olds. This may reflect better care, including centralised service provision, treatment protocols and higher trial recruitment rates in children compared to older patients.
    • Reply to: Comment on: Unsatisfactory quality of E. coli asparaginase biogenerics in India-Implications for clinical outcomes in acute lymphoblastic leukaemia

      Sidhu, J.; Saha, Vaskar; Krishnan, Shekhar; Department of Paediatric Haematology and Oncology, Tata Medical Center, Kolkata, India (2021)
    • Subpathologies and genomic classifier for treatment individualization of post-prostatectomy radiotherapy

      Ramotar, M.; Chua, M. L. K.; Truong, H.; Hosni, A.; Pintilie, M.; Davicioni, E.; Fleshner, N. E.; Dicker, A. P.; Bristow, Robert G; He, H. H.; et al. (2021)
      Purpose/objective: Risk-stratification for post-prostatectomy radiotherapy (PORT) using conventional clinicopathologic indexes leads to substantial over- and under-treatment. Better patient selection could spare unnecessary toxicities and improve outcomes. We investigated the prognostic utility of unfavorable subpathologies intraductal carcinoma and cribriform architecture (IDC/CA), and a 22-gene Decipher genomic classifier (GC) in prostate cancer (PCa) patients receiving PORT. Material/methods: A cohort of 302 men who received PORT at 2 academic institutions was pooled. PORT was predominately delivered as salvage (62% of cases); 20% received HT+PORT. Specimens were centrally reviewed for IDC/CA presence. In 104 cases, GC scores were determined. Endpoints were biochemical relapse-free (bRFR) and metastasis-free (mFR) rates. Results: After a median follow-up of 6.49-years, 135 (45%) and 40 (13%) men experienced biochemical relapse and metastasis, respectively. IDC/CA were identified in 160 (53%) of cases. Men harboring IDC/CA experienced inferior bRFR (HR 2.6, 95%CI 1.8-3.2, P<0.001) and mFR (HR 3.1, 95%CI 1.5-6.4, P = 0.0014). Patients with GC scores, 22 (21%) were stratified low-, 30 (29%) intermediate-, and 52 (50%) high-risk. GC low-risk was associated with superior bRFR (HR 0.25, 95%CI 0.1-0.5, P<0.001) and mFR (HR 0.15, 95%CI 0.03-0.8, P = 0.025). On multivariable analyses, IDC/CA and GC independently predicted for bRFR, corresponding to improved discrimination (C-index = 0.737 (95%CI 0.662-0.813)). Conclusions: IDC/CA subpathologies and GC predict for biochemical relapse and metastasis beyond conventional clinicopathologic indexes in the PORT setting. Patients harboring IDC/CA are at higher risk of relapse after maximal local therapies, thus warranting consideration for treatment intensification strategies. Conversely, for men with absence of IDC/CA and low GC scores, de-intensification strategies could be explored.
    • Genomic and evolutionary classification of lung cancer in never smokers

      Zhang, T.; Joubert, P.; Ansari-Pour, N.; Zhao, W.; Hoang, P. H.; Lokanga, R.; Moye, A. L.; Rosenbaum, J.; Gonzalez-Perez, A.; Martínez-Jiménez, F.; et al. (2021)
      Lung cancer in never smokers (LCINS) is a common cause of cancer mortality but its genomic landscape is poorly characterized. Here high-coverage whole-genome sequencing of 232 LCINS showed 3 subtypes defined by copy number aberrations. The dominant subtype (piano), which is rare in lung cancer in smokers, features somatic UBA1 mutations, germline AR variants and stem cell-like properties, including low mutational burden, high intratumor heterogeneity, long telomeres, frequent KRAS mutations and slow growth, as suggested by the occurrence of cancer drivers' progenitor cells many years before tumor diagnosis. The other subtypes are characterized by specific amplifications and EGFR mutations (mezzo-forte) and whole-genome doubling (forte). No strong tobacco smoking signatures were detected, even in cases with exposure to secondhand tobacco smoke. Genes within the receptor tyrosine kinase-Ras pathway had distinct impacts on survival; five genomic alterations independently doubled mortality. These findings create avenues for personalized treatment in LCINS.
    • Evaluation of senescent cells in intervertebral discs by lipofuscin staining

      Veroutis, D.; Kouroumalis, A.; Lagopati, N.; Polyzou, A.; Chamilos, C.; Papadodima, S.; Evangelou, K.; Gorgoulis, Vassilis G; Kletsas, D.; Molecular Carcinogenesis Group, Department of Histology and Embryology, Medical School, National and Kapodistrian University of Athens, Athens, Greece (2021)
      Intervertebral disc (IVD) degeneration is considered an important contributor of low back pain, a major age-related disease. Interestingly, an unprecedented high number of senescent cells has been reported in aged and degenerated IVDs, most probably affecting tissue homeostasis. In previous studies classical markers of cellular senescence have been used, such as SA-β-gal staining or p16Ink4a expression. Aim of the presented study was a re-evaluation of the number of senescent IVD cells by using a newly established staining procedure for lipofuscin, based on a Sudan Black-B analogue (GL13), which can be used in fresh, as well as in fixed and embedded tissues. In cultures of senescent rat and human IVD cells both SA-β-gal and GL13 gave similar percentages of senescent cells. Similarly, in fresh tissues from old rats the ratios of senescent cells were high with both detection procedures. Finally, in formalin-fixed and paraffin-embedded tissues from humans, a significant increased number of GL13-positive cells was found in herniated tissues, as compared to apparently normal ones, while similar numbers of p16Ink4a-positive cells were observed. These data confirm the significantly enhanced number of senescent cells in aged and degenerated IVDs, most probably contributing to the degeneration of this tissue.
    • Machine learning approaches on high throughput NGS data to unveil mechanisms of function in biology and disease

      Pezoulas, V. C.; Hazapis, O.; Lagopati, N.; Exarchos, T. P.; Goules, A. V.; Tzioufas, A. G.; Fotiadis, D. I.; Stratis, I. G.; Yannacopoulos, A. N.; Gorgoulis, Vassilis G; et al. (2021)
      In this review, the fundamental basis of machine learning (ML) and data mining (DM) are summarized together with the techniques for distilling knowledge from state-of-the-art omics experiments. This includes an introduction to the basic mathematical principles of unsupervised/supervised learning methods, dimensionality reduction techniques, deep neural networks architectures and the applications of these in bioinformatics. Several case studies under evaluation mainly involve next generation sequencing (NGS) experiments, like deciphering gene expression from total and single cell (scRNA-seq) analysis; for the latter, a description of all recent artificial intelligence (AI) methods for the investigation of cell sub-types, biomarkers and imputation techniques are described. Other areas of interest where various ML schemes have been investigated are for providing information regarding transcription factors (TF) binding sites, chromatin organization patterns and RNA binding proteins (RBPs), while analyses on RNA sequence and structure as well as 3D dimensional protein structure predictions with the use of ML are described. Furthermore, we summarize the recent methods of using ML in clinical oncology, when taking into consideration the current omics data with pharmacogenomics to determine personalized treatments. With this review we wish to provide the scientific community with a thorough investigation of main novel ML applications which take into consideration the latest achievements in genomics, thus, unraveling the fundamental mechanisms of biology towards the understanding and cure of diseases.
    • Clinical benefit of pelabresib (Cpi-0610) in combination with ruxolitinib in JAK inhibitor treatment-naive myelofibrosis patients: interim efficacy subgroup analysis from Arm 3 of the MANIFEST Phase 2 Study

      Gupta, V.; Kremyanskaya, M.; Mascarenhas, J.; Palandri, F.; Patriarca, A.; Devos, T.; Harrison, C.; Passamonti, F.; Rampal, R.; Mead, A.; et al. (2021)
      Context: Pelabresib (CPI-0610), a fi rst-in-class, oral, small molecule inhibitor of BET proteins (BETi), has potential to promote disease-modifying activity through altered gene regulation of key oncogenic, fi brotic, and infl ammatory factors in MF. Treatment with JAKi ruxolitinib (rux) or fedratinib in the frontline setting is associated with approximately 30–40% splenic response (spleen volume reduction 35% [SVR35]) at 6 months. BETi pelabresib monotherapy demonstrated clinical activity in heavily pre-treated MF pts. Therefore, combination of pelabresib with rux is expected to achieve higher SVR35 response rate in JAKi treatment-naïve MF pts. Objective: Evaluation of pelabresib in combination with rux in JAKi treatment-naïve MF pts. Design: Pts with MF who were not previously treated with JAKi were enrolled in Arm 3 of MANIFEST. The primary endpoint is SVR35 response at wk 24. Pts were treated with pelabresib 125 mg daily on days 1–14 in a 21-day cycle in combination with rux, which was dosed based on the baseline platelet count. Results: As of 29 September 2020, 78 pts were treated, and 66 pts were ongoing. Baseline characteristics were mean age: 67 years; 72% male; primary MF: 54% pts; DIPSS Int-2: 76% pts; IPSS Int-2: 83%; Hgb <10g/dL: 65%; median platelet: 294 x 109 /L (range: 100, 1849); median spleen volume: 1719 cc (range: 451, 4782); median TSS: 16 (range: 0, 38); high molecular-risk mutations: 55%, JAK2 mutation: 72%. At wk 24, 67% (42/63) pts achieved SVR35 (median % change from baseline: -50%; range: -84.4%, 23.7%). Subgroup analysis showed that mean percentage change in spleen volume at wk 24 was consistent across subgroups based on gender, age, risk score, MF subtype, baseline platelet count, and baseline spleen size. Importantly, subgroup analysis based on molecular fi ndings showed a signifi cant benefi t regardless of the mutational status, including the presence of ASXL1 mutation, which generally carries a poor prognosis. Pelabresib was generally well tolerated. Conclusions: Pelabresib treatment in combination with rux in JAKi treatment-naïve MF pts resulted in spleen volume reduction that was greater than what would be expected with rux alone, regardless of baseline patient disease and demographic characteristics
    • Pelabresib (CPI-0610) improved anemia associated with myelofibrosis: interim results from the ongoing MANIFEST phase 2 study

      Bose, P.; Verstovsek, S.; Kremyanskaya, M.; Mascarenhas, J.; Talpaz, M.; Harrison, C.; Rampal, R.; Patriarca, A.; Gupta, V.; Granacher, N.; et al. (2021)
      Pelabresib (CPI-0610), a first-in-class, oral, small molecule inhibitor of BET proteins, has the potential to promote disease-modifying activity through altered gene regulation of key oncogenic, fi brotic, and infl ammatory factors in MF. Many patients (pts) with MF treated with ruxolitinib (rux) develop worsening anemia and may become RBC transfusion-dependent (TD). Objective: Evaluation of pelabresib monotherapy or as add on to rux in advanced MF pts. Design: In Arm 1, pts refractory, intolerant, or ineligible for JAKi were treated with pelabresib monotherapy. In Arm 2, pts receiving rux but not deriving adequate benefi t were treated with add-on pelabresib. The primary endpoint was achievement of transfusion independence (TI) 12 wks in TD cohorts and 35% spleen volume reduction at wk 24 in non-TD cohorts. Results: As of 29 Sept. 2020, 19 TD pts and 27 non-TD pts were treated in Arm 1. Seventy-six percent of pts had baseline Hgb <10g/dL (median: 9, range: 6–15). In the TD cohort, 21% (3/14) of pts achieved TI (median duration: 44 wk, range: 32–50). In the non-TD cohort, a mean Hgb increase 1.5 g/dL sustained over a 12-wk transfusion-free period was achieved by 59.1% (13/22) of pts. In Arm 2, 52 TD pts and 26 non-TD pts were treated with pelabresib as add-on to rux. Seventy-six percent of pts had baseline Hgb <10g/dL (median: 9, range: 6–13). In the TD cohort, 36% (13/36) pts achieved TI (median duration: 39 wk, range: 18–148); 17.4% (4/23) of non-TD pts had mean Hgb increase 1.5 g/dL sustained over a 12-wk transfusion-free period. Hgb improvement/ achievement of TI has been associated with increased reticulocyte count and/or CD71+ progenitor cells in bone marrow, suggesting a positive effect on erythroid differentiation. Pelabresib was generally well tolerated. Conclusions: Pelabresib monotherapy was associated with a mean increase in Hgb 1.5 g/dL in majority of non-TD pts and conversion of one-fifth of TD pts to TI in Arm 1. Pelabresib add-on to rux in Arm 2 resulted in mean increase in Hgb 1.5 g/dL in 17% of non-TD pts and conversion to TI in more than one-third of TD pts.
    • Nanonets for multiomics blood analysis and cancer biomarker discovery

      Gardner, L.; Rothwell, Dominic G; Dive, Caroline; Kostarelos, K.; Hadjidemetriou, M.; University of Manchester, Manchester (2021)
      Despite the tremendous potential of liquid biopsies to revolutionise cancer care, there has been limited success translating blood-circulating proteomic and genomic biomarkers into the clinic. This is fundamentally due to the extremely low concentration of tumour-derived biomolecules in blood circulation, particularly at an early disease stage, which makes the discovery phase of the biomarker pipeline extremely challenging. Nanotechnology offers a promising solution, with a nanoparticle-biomolecule enrichment tool recently developed to enrich low-abundant, low molecular weight proteins in the blood of ovarian cancer patients.[1] Proteomic analysis followed by immunoassay-based validation of selected proteins demonstrated the potential of the nanoparticle-platform proposed to discover novel biomarkers with greater specificity and sensitivity than the clinically used biomarkers. In addition, we recently confirmed the presence of cell-free DNA (cfDNA) captured onto the surface lipid nanoparticles incubated ex vivo with human plasma.[2] A significantly higher abundance of cfDNA was detected in the nanoparticle-enriched plasma samples of late-stage ovarian cancer patients compared to age-matched female controls. Proteomic analysis of the same samples also revealed tumour-specific elevations in histone proteins, which are commonly found in circulation complexed with cfDNA. These findings have highlighted the opportunity for the development of a nano-proteogenomics platform able to simultaneously purify both proteins and cell-free nucleic acids from human plasma, an important step in the discovery of novel multi-omic biomarker panels. Utilising the above patented nanotechnology, we have compared proteomic and genomic profiles derived from nanoparticle-biomolecule samples of cancer patients with age- and sex-matched controls to uncover new potential blood-based biomarkers in a proof-of-principle study. In brief, ex-vivo plasma samples were incubated with lipid-based nanoparticles and purified using a two-step size-based purification protocol. The purified samples were then analysed by label-free proteomics (LC-MS/MS) and next-generation sequencing to uncover both proteomic and genomic tumour-specific signatures, including differentially abundant proteins, genomic copy number alterations and tumour-specific mutations. This work highlights the potential of our nanotechnology-based enrichment platform to enhance the discovery of cancer-specific proteogenomic biomarker panels, a vital step in developing sensitive and specific liquid biopsies for the early detection of cancer.
    • A phase I/II study of thiotepa-based immunochemotherapy in relapsed/refractory primary CNS lymphoma: the TIER trial

      Fox, C. P.; Ali, A. S.; McIlroy, G. W.; Thust, S. C.; Martinez-Calle, N.; Jackson, A. E.; Hopkins, L.; Thomas, C. M.; Kassam, S.; Wright, J.; et al. (2021)
      Relapsed or refractory primary central nervous system lymphoma (rrPCNSL) confers a poor prognosis with no accepted standard of care. Very few prospective studies have been conducted in this patient group. This multicenter, phase I/II study investigated thiotepa in combination with ifosfamide, etoposide and rituximab (TIER), for the treatment of PCNSL relapsed or refractory to high-dose methotrexate-based chemotherapy. A 3+3 design investigated the recommended phase II dose of thiotepa for a single-stage phase II cohort, assessing activity of two cycles of TIER against rrPCNSL. The primary outcome was overall response rate. The dose-finding study demonstrated 50mg/m2 of thiotepa could be safely delivered within the TIER regimen. No dose-limiting toxicities were encountered in phase I, and TIER was well-tolerated by the 27 patients treated in phase II. The most common Grade 3/4 toxicities were neutropenia (56% of patients) and thrombocytopenia (39%). An overall response was confirmed in 14 (52%) patients, meeting the pre-specified threshold for clinically relevant activity. The median progression-free survival was 3 months (95% confidence interval 2 to 6 months) and overall survival 5 months (3 to 9 months). Exploratory analyses suggest a greater benefit for thiotepa-naïve patients. Six patients successfully completed autologous stem cell transplant consolidation (ASCT), with 4 experiencing durable remissions after median follow-up of 50 months. The TIER regimen can be safely delivered and is active against rrPCNSL, and followed by ASCT can provide durable remission and long-term survival. However, for the majority of patients, prognosis remains poor and novel treatment strategies are urgently needed.
    • Study of innate immune responses during small cell lung cancer (SCLC) development

      Chudziak, Jakub; Galvin, Melanie; Banyard, Antonia; Simpson, Kathryn L; Frese, Kristopher K; Kilgour, Elaine; Hussell, T.; Dive, Caroline; Cancer Research UK Manchester Institute, Alderley Park, (2021)
      SCLC is a highly aggressive neuroendocrine tumor with dismal prognosis. Inability to detect SCLC early renders surgical resections a rare event, limiting investigation of the early stages of tumor development. We sought to address this challenge using a genetically engineered mouse model (GEMM) to investigate how the innate immune system responds to SCLC development. The RPM (Rb1fl/fl, p53fl/fl, MycLSL/LSL) SCLC GEMM was selected to characterize the local immune microenvironment throughout the process of tumor development. Following tumor induction using an intranasally installed Cre bearing adenovirus, lung tissue was collected and analyzed using multi-parametric flow cytometry to assess changes in innate immune cell populations. Induction of SCLC resulted in late stage disease being present in animals approximately 7 weeks after virus exposure. Flow cytometric analysis of immune cell populations of the whole lung showed no significant differences in the overall levels of immune cell types, although an upwards trend in the levels of neutrophils in tumor-bearing mice was observed. The most significant finding was a decrease in the expression levels of major histocompatibility complex class II (MHCII) in both alveolar and interstitial macrophage populations. Further characterization, including separate analysis of the two interstitial macrophage subpopulations described by Chakarov et al. (Science 2019), and Schyns et al. (Nature Communications 2019), showed significant drops in levels of MHCII in all populations as measured by median fluorescent intensity (MFI) (p=0.004 in alveolar macrophages, p=0.0148 in vascular supporting CD206+ve interstitial macrophages, and p=0.0027 in CD206-ve interstitial macrophages). The striking difference between the changes seen in these populations was that alveolar and CD206+ve interstitial macrophages both showed a downward shift in MHCII expression as a whole, while CD206-ve macrophages acquired a novel population completely negative for MHCII accounting for approx. half of the CD206-ve macrophage subpopulation. The development of SCLC has been found to result in all macrophage subtypes expressing lower levels of MHCII, pointing towards a more immunosuppressive immune environment. The population of CD206-ve MHCII-ve interstitial macrophages is of particular interest, as it is contrary to the currently described interstitial macrophage phenotypes. A more in-depth characterization of all macrophage populations in the mouse lung, including their precise phenotype and localization is currently underway using CyTOF and IHC approaches. The precise timings of the observed changes are being elucidated through analysis of a disease time-course ranging from very early stage to late stage disease, in order to better understand the process of SCLC development.
    • A microenvironment-inspired synthetic three-dimensional model for pancreatic ductal adenocarcinoma organoids

      Below, Christopher R; Kelly, Joanna; Brown, A.; Humphries, J. D.; Hutton, Colin; Xu, Jingshu; Lee, Brian Y; Cintas, Celia; Zhang, Xiaohong; Hernandez-Gordillo, V.; et al. (2021)
      Experimental in vitro models that capture pathophysiological characteristics of human tumours are essential for basic and translational cancer biology. Here, we describe a fully synthetic hydrogel extracellular matrix designed to elicit key phenotypic traits of the pancreatic environment in culture. To enable the growth of normal and cancerous pancreatic organoids from genetically engineered murine models and human patients, essential adhesive cues were empirically defined and replicated in the hydrogel scaffold, revealing a functional role of laminin-integrin α3/α6 signalling in establishment and survival of pancreatic organoids. Altered tissue stiffness-a hallmark of pancreatic cancer-was recapitulated in culture by adjusting the hydrogel properties to engage mechano-sensing pathways and alter organoid growth. Pancreatic stromal cells were readily incorporated into the hydrogels and replicated phenotypic traits characteristic of the tumour environment in vivo. This model therefore recapitulates a pathologically remodelled tumour microenvironment for studies of normal and pancreatic cancer cells in vitro.
    • Enhanced target-specific delivery of docetaxel-loaded nanoparticles using engineered T cell receptors

      McDaid, William J; Lissin, N.; Pollheimer, E.; Greene, M.; Leach, A.; Smyth, P.; Bossi, G.; Longley, D.; Cole, D. K.; Scott, C. J.; et al. (2021)
      For effective targeted therapy of cancer with chemotherapy-loaded nanoparticles (NPs), antigens that are selective for cancer cells should be targeted to minimise off-tumour toxicity. Human leukocyte antigens (HLAs) are attractive cancer targets as they can present peptides from tumour-selective proteins on the cell surface, which can be recognised by T cells via T cell receptors (TCRs). In this study, docetaxel-loaded polymeric NPs were conjugated to recombinant affinity-enhanced TCRs to target breast cancer cells presenting a tumour-selective peptide-HLA complex. The TCR-conjugated nanoparticles enabled enhanced delivery of docetaxel and induced cell death through tumour-specific peptide-HLA targeting. These in vitro data demonstrate the potential of targeting tumour-restricted peptide-HLA epitopes using high affinity TCR-conjugated nanoparticles, representing a novel treatment strategy to deliver therapeutic drugs specifically to cancer cells.
    • Murine AGM single-cell profiling identifies a continuum of hemogenic endothelium differentiation marked by ACE

      Fadlullah, Muhammad Z H; Neo, Wen H; Lie-A-Ling, Michael; Thambyrajah, R.; Patel, R; Mevel, Renaud; Aksoy, I.; Do Khoa, N.; Savatier, P.; Fontenille, L.; et al. (2021)
      In vitro generation and expansion of hematopoietic stem cells (HSCs) holds great promise for the treatment of any ailment that relies on bone marrow or blood transplantation. To achieve this, it is essential to resolve the molecular and cellular pathways that govern HSC formation in the embryo. HSCs first emerge in the aorta-gonad-mesonephros region (AGM) where a rare subset of endothelial cells, hemogenic endothelium (HE), undergoes an endothelial-to-hematopoietic transition (EHT). Here, we present full-length single-cell-RNA-sequencing of the EHT process with a focus on HE and dorsal aorta niche cells. By using Runx1b and Gfi1/1b transgenic reporter mouse models to isolate HE, we uncovered that the pre-HE to HE continuum is specifically marked by Angiotensin-I converting enzyme (ACE) expression. We established that HE cells begin to enter the cell cycle near the time of EHT initiation when their morphology still resembles endothelial cells. We further demonstrated that RUNX1 AGM niche cells consist of vascular smooth muscle cells and PDGFRa+ mesenchymal cells and can functionally support hematopoiesis. Overall, our study provides new insights into HE differentiation towards HSC and the role of AGM RUNX1+ niche cells in this process. Our expansive scRNA-seq datasets represents a powerful resource to investigate these processes further.