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Supplementary Table S1. List of genes with an absolute copy number of h >=5 for >=25% of TNBCs.

PDF file - 1575K, Supplementary Figure 1. Set-up of the two bone marrow metastatic niche models - Inhibitory Niche (Bone Marrow Cell lines, BMCL) and Supportive Niche (Human Bone Marrow Stromal Cells, BMSC). Supplementary Figure 2. Set-up of bone marrow metastatic niche models, including coculture with mouse BMSC and the cell lines 293t, fetal kidney, U2OS and MG63, osteosarcoma and organ-specific metastatic sub-lines of MDA-MB-231, 1833 BoM and 4175 LM2. Supplementary Figure 3. Control experiments for set-up of bone marrow metastatic niche models. Supplementary Figure 4 Breast cancer cells in IN co-culture did not undergo apoptosis were viable and capable to proliferate. Supplementary Figure 5. Conditioned medium from IN culture induced cell cycle arrest in breast cancer cells. Supplementary Figure 6. Effects of small molecule inhibitors SB431542 (Alk5), SB203580 (p38) and S1042 (RTK) in 3D co-cultures of breast cancer cell lines. Supplementary Figure 7. Treatment with small molecule inhibitors induced growth of BCC in the subcutaneous implantation model. Supplementary Figure 8 Breast cancer cell lines proliferation in orthotopic and metastasis mouse models (intratibial and intracardiac injection).

Metastatic non-small cell lung cancer (NSCLC) results from a complex evolutionary process in which cancer cells migrate from a primary tumour to a new anatomical site. Immunotherapy is frequently used to treat NSCLC, but drug resistance is frequent (>50%) and metastasis remains the most common cause of death. Recent studies of primary and metastatic tumours suggest that anti-cancer treatments can impact cancer evolutionary dynamics by applying selective pressures that promote the development of treatment resistance in genetically distinct subpopulations of cancer cells, or tumour clones. However, the relationship between metastatic progression and the development of resistance to immunotherapy has not been fully explored. In particular, whether metastases become resistant to immunotherapy as a result of the migration of resistant cells or whether distinct metastatic sites converge in parallel toward an immunotherapy resistance phenotype irrespective of cell migration, is not known. In this work, we investigate the role of metastatic cell migration in the development of immunotherapy resistance using DNA sequencing data from primary and metastatic tumour samples from twelve immunotherapy-treated patients with NSCLC co-recruited to the national TRACERx and PEACE studies. Using computational approaches, we identified distinct tumour clones in primary and metastatic tumours and used these to reconstruct tumour phylogenies for each patient. By evaluating tumour clonal dynamics in relation to treatment, we classified tumour clones as either sensitive or resistant to immunotherapy. We reconstructed the metastatic migration histories of these clones using existing computational methods based on somatic single nucleotide variants. Our results suggest that heterogeneous mechanisms of immune evasion develop in parallel at distinct metastatic sites. The ongoing analysis of the genetic changes that distinguish resistant from sensitive clones, and migrating from non-migrating clones, will provide further detail regarding the impact of metastatic migrations on immunotherapy resistance. Using this approach to map the evolutionary history of metastatic NSCLC, we provide insight into the mechanism by which immunotherapy resistance develops at distinct metastatic sites. Citation Format: Sonya Hessey, Ariana Huebner, Oriol Pich, Abi Bunkum, Wing Liu, David A. Moore, Cristina Naceur-Lombardelli, Selvaraju Veeriah, Sophie Ward, Roberto Salgado, Nicholas McGranahan, Simone Zaccaria, Charles Swanton, Mariam Jamal-Hanjani. Tracking the emergence of immunotherapy resistance in lung cancer metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2037.

Murine tissues harbor signature γδ T cell compartments with profound yet differential impacts on carcinogenesis. Conversely, human tissue-resident γδ cells are less well defined. In the present study, we show that human lung tissues harbor a resident Vδ1 γδ T cell population. Moreover, we demonstrate that Vδ1 T cells with resident memory and effector memory phenotypes were enriched in lung tumors compared with nontumor lung tissues. Intratumoral Vδ1 T cells possessed stem-like features and were skewed toward cytolysis and helper T cell type 1 function, akin to intratumoral natural killer and CD8

Background: The adaptive immune system plays an important role in tumor evolution. A key source of cytotoxic T cell response in cancer is neoantigens, cancer cell specific mutations that result in mutant peptides that elicit a T cell mediated immune response. However, a mutation can only engender a neoantigen if the associated mutant peptide is presented to T cells by HLA molecules, and, as such, transcriptional repression or loss of the HLA genes can have important implications for immune evasion. Methods: We elucidate allele specific genomic and transcriptomic disruption to class I and II HLA genes. In whole exome sequencing (WES) data, our new tool (LOHHLA2.0) assesses loss of heterozygosity (LOH) status and somatic mutations. In RNA sequencing (RNAseq) data, LOHHLA2.0 quantifies allele specific expression and transcriptional repression referencing matched tumor adjacent normal samples. We applied LOHHLA2.0 to the TRACERx421 dataset, including 1554 WES tumor regions from 421 patients and 941 RNAseq regions from 357 tumor patients, 96 of which also had RNAseq from tumor adjacent normal samples. Results: We find that our method is more accurate than existing tools (RSEM) at calling gene level expression, e.g. in HLA-DPB1, RSEM under-calls HLA expression by a factor of two. 36% of TRACERx421 primary tumors harbored HLA LOH of at least one class I HLA gene, validating our previous findings in the TRACERx100 cohort. Strikingly, we found that 74% (71/96) of primary tumors with matched tumor adjacent normal tissue exhibited transcriptional repression of one or more class I HLA alleles, and 81% (78/96) exhibited class II allele transcriptional repression. Class I HLA transcriptional repression was more likely to occur subclonally than LOH. In a subset of tumors, we observed convergence upon disruption of the same allele through alternative mechanisms; with genomic loss in one tumor region and transcriptional repression in another region of the same tumor. Across the tumor regions, we found a concordance between HLA expression and immune infiltrate levels, with immune hot tumors exhibiting higher HLA class I expression. Conclusions: In this study, we find significant disruption to class I and II HLA expression adding to the diversity of immune evasion processes evident in early stage treatment naive NSCLC. Citation Format: Clare Puttick, Oriol Pich, Michelle Leung, Carlos Martinez-Ruiz, Robert Bentham, Rachel Rosenthal, Sonya Hessey, James R. Black, Emilia L. Lim, Katey Enfield, Emma Colliver, Krijn Dijkstra, Crispin T. Hiley, Takahiro Karasaki, Ariana Huebner, Maise Al Bakir, Thomas B. Watkins, Alexander M. Frankell, Simone Zaccaria, Mariam Jamal-Hanjani, Nicholas McGranahan, Charles Swanton. Pervasive allele specific transcriptional repression of the class I and II HLA genes in TRACERx non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1394.

To provide timetabled opportunities for core medical trainees (CMTs) at the Royal Free Hospital to practise essential procedural skills under direct supervision by expert clinicians, with a view to gain the required competences for Annual Review of Competence Progression (ARCP). An online survey using SurveyMonkey was created for CMTs to determine their pre-existing experience in essential procedural skills, how confident they felt performing these procedures, and how …

Introduction: Studies of cancer evolution have relied on archival tissue surplus to diagnostic requirements from living patients obtained in early stage disease and less commonly relapsed/metastatic disease. PEACE (Posthumous Evaluation of Advanced Cancer Environment) is a national research autopsy study aiming to understand the biological processes driving metastatic disease and cancer evolution. Experimental procedures: Patients recruited into the national TRACERx (TRAcking Cancer Evolution through therapy (Rx)) lung study who subsequently developed metastatic disease were enrolled into PEACE. Here we present a cohort of 15 TRACERx/PEACE patients in whom multi-region tumor sampling was performed at diagnosis +/- relapse and at autopsy. Fresh frozen tissue was subjected to whole-exome sequencing (mean depth 390) and germline DNA from blood was used for reference. Single nucleotide variants (SNVs) were identified using VarScan2 and the subclonal composition of each tumor was inferred using PyClone and used to reconstruct phylogenetic trees. Summary of data: Disease progression was associated with increased genomic complexity. Different patterns of progression occurred; monoclonal/monophyletic, polyclonal/monophyletic and polyclonal/polyphyletic dissemination. Both early and late divergence relative to the last clonal sweep were observed. Putative drivers were found to occur both clonally and subclonally. SNVs and copy number aberrations were used to reconstruct migration patterns. Conclusions: Preliminary analysis in this cohort demonstrates increasing genomic complexity with disease progression and variation in patterns of metastatic dissemination. We are yet to establish the impact of the tumor and immune microenvironment on such patterns as well as clinical presentation and outcome. Ongoing PEACE analysis includes study of the somatic copy number landscape and the characterisation of subclones that seed metastases. Patient & sample characteristicsN (%)Total patients15Median age (IQR)73 (65 - 80)Female sex (%)6 (40%)Histological subtypeAdenocarcinoma7 (47%)Squamous cell carcinoma5 (33%)Large cell carcinoma2 (13%)Adenosquamous carcinoma1 (7%)Treatment receivedRadiotherapy8 (53%)Chemotherapy6 (40%)Immunotherapy5 (33%)Targeted therapy4 (27%)Smoking statusCurrent smoker2 (13%)Ex-smoker12 (80%)Never smoker1 (7%)Total metastases sampled289Median samples per patient (range)19 (5 - 41)Number of samples per patient5-104 (27%)11-205 (33%)>206 (40%)Number of tissue types sampled17 Citation Format: Ariana Huebner, Sonya Hessey, Cristina Naceur-Lombardelli, Mita Akther, Selvaraju Veeriah, Maise Al Bakir, David Moore, Simone Zaccaria, Nicholas McGranahan, Charles Swanton, Mariam Jamal-Hanjani, TRACERx and PEACE consortium. Lung cancer evolutionary trajectories in PEACE [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3123.

Mortality of patients with breast cancer is due overwhelmingly to metastatic spread of the disease. Although dissemination is an early event in breast cancer, extended periods of cancer cell dormancy can result in long latency of metastasis development. Deciphering the mechanisms underlying cancer cell dormancy and subsequent growth at the metastatic site would facilitate development of strategies to interfere with these processes. A challenge in this undertaking has been the lack of models for cancer cell dormancy. We have established novel experimental systems that model the bone microenvironment of the breast cancer metastatic niche. These systems are based on 3D cocultures of breast cancer cells with cell types predominant in bone marrow. We identified conditions in which cancer cells are dormant and conditions in which they proliferate. Dormant cancer cells were able to proliferate upon transfer into supportive microenvironment or upon manipulation of signaling pathways that control dormancy. These experimental systems will be instrumental for metastasis studies, particularly the study of cellular dormancy. Cancer Res; 73(23); 6886–99. ©2013 AACR.

Background Palliative care is increasingly incorporated into undergraduate curriculums in UK medical schools. Training needs of junior doctors who frequently provide care for dying patients, however, are less well elucidated. Aim We set out to assess junior doctors’ confidence in various areas of End of Life Care in order to identify specific training needs. Method We adapted the University College London Hospital Care of the Dying Confidence Questionnaire to focus on areas of End of Life Care relevant to junior doctors. We distributed a paper and online version of this questionnaire to trainees in acute, surgical and medical specialties at the Royal Free NHS Foundation Trust. The statistical significance of the difference between Likert scale medians was determined by Kruskal-Wallis tests. Results We received 95 survey responses from doctors in foundation (71%), core (19%) and specialty registrar (10%) training years. Unsurprisingly, higher training grade was associated with increased confidence in providing End of Life Care, in particular in discussing dying and ceiling of care with patients and their family members. All trainees were confident (Likert median=6) in verifying death and writing death certificates. Trainees reported low confidence (Likert median=4) in discussing the discontinuation of nutrition and hydration as well as in caring for a dying patient who becomes unconscious. Conclusion Junior doctors are confident in skills routinely taught and tested during medical school. There remains a need for additional End of Life Care training, in particular for foundation trainees. To optimise the End of Life Care provided by junior doctors this training should focus on areas of low confidence such as discussing nutrition and hydration.