Search

Understanding the evolutionary pathways to metastasis and resistance to immune-checkpoint inhibitors (ICI) in melanoma is critical for improving outcomes. Here, we present the most comprehensive intrapatient metastatic melanoma dataset assembled to date as part of the Posthumous Evaluation of Advanced Cancer Environment (PEACE) research autopsy program, including 222 exome sequencing, 493 panel-sequenced, 161 RNA sequencing, and 22 single-cell whole-genome sequencing samples from 14 ICI-treated patients. We observed frequent whole-genome doubling and widespread loss of heterozygosity, often involving antigen-presentation machinery. We found KIT extrachromosomal DNA may have contributed to the lack of response to KIT inhibitors of a KIT-driven melanoma. At the lesion-level, MYC amplifications were enriched in ICI nonresponders. Single-cell sequencing revealed polyclonal seeding of metastases originating from clones with different ploidy in one patient. Finally, we observed that brain metastases that diverged early in molecular evolution emerge late in disease. Overall, our study illustrates the diverse evolutionary landscape of advanced melanoma.Significance:Despite treatment advances, melanoma remains a deadly disease at stage IV. Through research autopsy and dense sampling of metastases combined with extensive multiomic profiling, our study elucidates the many mechanisms that melanomas use to evade treatment and the immune system, whether through mutations, widespread copy-number alterations, or extrachromosomal DNA. more...

Supplementary figure 1: Cohort overview. Number of samples sequenced with whole exome, panel or whole RNA sequencing. Supplementary figure 2: Phylogeny and WGD events in CRUKP1047. Supplementary figure 3: Ploidy and SCNA burden. Supplementary figure 4: Overview of each case. Supplementary figure 5: MEDICC2 copy number sample trees. Supplementary figure 6: MEDICC tree of all exome samples demonstrating that samples cluster together by patient, and not by melanoma subtype. Supplementary figure 7: SCNA frequency of cutaneous (a), acral (b) and melanoma of unknown primary (MUP, c). Supplementary figure 8: Correlation between liver copy number distance to other sites and time of emergence after stage IV diagnosis. Supplementary figure 9: Examination of tumour heterogeneity of alterations to antigen-presentation machinery genes, with site and patient annotation. Supplementary figure 10: Boxplots indicating the proportion of losses in the cohort for each segment. Supplementary figure 11: Balance of expression between nonsynonymous mutations that were not predicted to be neoantigens and clonal predicted neoantigens. Supplementary figure 12: Barplot of TIL infiltration score frequencies, determined by pathologist assessment of histology, across all samples. Supplementary figure 13: Number of samples per patient that are classified as either none-low in terms of TILs or moderate-heavy. Supplementary figure 14: Histogram of purity for samples with RNA-seq data. Supplementary figure 15: TME deconvolution. Supplementary figure 16: The effect of metastatic site on transcriptional profiles. Supplementary figure 17: Association of PHF3 copy number with expression. Supplementary figure 18: Overview of gene fusions identified in RNA-seq data. Supplementary figure 19: Comparison of ploidy estimates from panel sequencing data, exome data and FISH. Supplementary figure 20: Comparison of ploidy estimates in panel, exome, FISH and single cell data. Supplementary figure 21: FACs sort plot for CRUKP2567 diaphragmatic metastasis. Supplementary figure 22: Ploidy and wGII values from single cell sequencing of FACS-sorted tumour cells. Supplementary figure 23: Copy number profiles on chromosome 5 for bulk samples from primary and DI_2_R2, a diaphragmatic metastasis. Supplementary figure 24: Histogram of purity of samples for which RNA-seq was performed faceted by patient. Supplementary figure 25: Histogram of purity of samples for which RNA-seq was performed faceted by tissue site. more...

S. Table 1: List of PEACE Consortium members. S. Table 2: Sample database used for the study for Panel, Exome and RNASeq samples. S. Table 3: Overview of lines of treatment given to each patient. S. Table 4: Lesions and patients included in the lesion-level response to ICI analysis. S. Table 5: Hallmark genesets significantly upregulated in normal tissue vs tumor tissue. Table shows p-values for tissue type, purity; T value for tissue type, purity; q-value for tissue type, purity (FDR corrected). S. Table 6: Hallmark genesets significantly upregulated in tumor tissue vs normal tissue. Table shows p-values for tissue type, purity; T value for tissue type, purity; q-value for tissue type, purity (FDR corrected). S. Table 7: Genes included in the target panel. S. Table 8: Tree topology comparisons between manually constructed trees and pairtree-constructed trees. more...

SummaryAntigen recognition and T-cell mediated cytotoxicity in clear-cell renal cell carcinoma (ccRCC) remains incompletely understood. To address this knowledge gap, we analysed 115 multiregion tumour samples collected from 15 treatment-naïve patients pre- and post-nivolumab therapy, and at autopsy in three patients. We performed whole-exome sequencing, RNAseq, TCRseq, multiplex immunofluorescence and flow cytometry analyses and correlated with clinical response. We observed pre-treatment intratumoural TCR clonal expansions suggesting pre-existing immunity. Nivolumab maintained pre-treatment expanded, clustered TCR clones in responders, suggesting ongoing antigen-driven stimulation of T-cells. T-cells in responders were enriched for expanded TCF7+CD8+T-cells and upregulated GZMK/B upon nivolumab-binding. By contrast, nivolumab promoted accumulation of new TCR clones in non-responders, replacing pre-treatment expanded clonotypes. In this dataset, mutational features did not correlate with response to nivolumab and human endogenous retrovirus expression correlated indirectly. Our data suggests that nivolumab potentiates clinical responses in ccRCC by binding pre-existing expanded CD8+T-cells to enhance cytotoxicity. more...

ADAPTeR is a prospective, phase II study of nivolumab (anti-PD-1) in 15 treatment-naive patients (115 multiregion tumor samples) with metastatic clear cell renal cell carcinoma (ccRCC) aiming to understand the mechanism underpinning therapeutic response. Genomic analyses show no correlation between tumor molecular features and response, whereas ccRCC-specific human endogenous retrovirus expression indirectly correlates with clinical response. T cell receptor (TCR) analysis reveals a significantly higher number of expanded TCR clones pre-treatment in responders suggesting pre-existing immunity. Maintenance of highly similar clusters of TCRs post-treatment predict response, suggesting ongoing antigen engagement and survival of families of T cells likely recognizing the same antigens. In responders, nivolumab-bound CD8+ T cells are expanded and express GZMK/B. Our data suggest nivolumab drives both maintenance and replacement of previously expanded T cell clones, but only maintenance correlates with response. We hypothesize that maintenance and boosting of a pre-existing response is a key element of anti-PD-1 mode of action. more...

This abstract is being presented as a short talk in the scientific program. A full abstract is available in the Proffered Abstracts section (PR002) of the Conference Proceedings. Citation Format: Alexander Coulton, Irene Lobon, Lavinia Spain, Andrew Rowan, Desiree Shnidrig, Scott Shepherd, Ben Shum, Fiona Byrne, Lewis Au, Kim Edmonds, Ellie Carlyle, Alexandra Renn, Christina Messiou, Charlotte Spencer, Andreas M. Schmidt, Zayd Tippu, Aljosja Rogiers, Max Emmerich, Camille Gerard, Husayn Pallikonda, Cristina Naceur-Lombardelli, Floris Foijer, Hilda van den Bos, René Wardenaar, Diana Spierings, Kate Young, Lisa Pickering, Andrew Furness, Elaine Borg, Miriam Mitchison, David Moore, Mary Falzon, Ian Proctor, Ruby Stewart, Ula Mahadeva, Anna Green, James Larkin, Charles Swanton, Mariam Jamal-Hanjani, Kevin Litchfield, Samra Turajlic. Advanced melanoma exhibits a diversity of evolutionary routes to lethality [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A012. more...

AIM To assess clinical outcomes for submucosal (T1b) oesophageal adenocarcinoma (OAC) patients managed with either surgery or endoscopic eradication therapy. METHODS Patients found to have T1b OAC following endoscopic resection between January 2008 to February 2016 at University College London Hospital were retrospectively analysed. Patients were split into low-risk and high-risk groups according to established histopathological criteria and were then further categorised according to whether they underwent surgical resection or conservative management. Study outcomes include the presence of lymph-node metastases, disease-specific mortality and overall survival. RESULTS A total of 60 patients were included; 22 patients were surgically managed (1 low-risk and 21 high-risk patients) whilst 38 patients were treated conservatively (12 low-risk and 26 high-risk). Overall, lymph node metastases (LNM) were detected in 10 patients (17%); six of these patients had undergone conservative management and LNM were detected at a median of 4 mo after endoscopic mucosal resection (EMR). All LNM occurred in patients with high-risk lesions and this represented 21% of the total high-risk lesions. Importantly, there was no statistically significant difference in tumor-related deaths between those treated surgically or conservatively (P = 0.636) and disease-specific survival time was also comparable between the two treatment strategies (P = 0.376). CONCLUSION T1b tumours without histopathological high-risk markers of LNM can be treated endoscopically with good out-comes. In selected patients, endoscopic therapy may be appropriate for high-risk lesions. more...

One of the many routes proposed for the cellular inactivation of endogenous nitric oxide (NO) is by the cytochrome c oxidase of the mitochondrial respiratory chain. We have studied this possibility in human embryonic kidney cells engineered to generate controlled amounts of NO. We have used visible light spectroscopy to monitor continuously the redox state of cytochrome c oxidase in an oxygen-tight chamber, at the same time as which we measure cell respiration and the concentrations of oxygen and NO. Pharmacological manipulation of cytochrome c oxidase indicates that this enzyme, when it is in turnover and in its oxidized state, inactivates physiological amounts of NO, thus regulating its intra- and extracellular concentrations. This inactivation is prevented by blocking the enzyme with inhibitors, including NO. Furthermore, when cells generating low concentrations of NO respire toward hypoxia, the redox state of cytochrome c oxidase changes from oxidized to reduced, leading to a decrease in NO inactivation. The resultant increase in NO concentration could explain hypoxic vasodilation. more...

Background: Patients (pts) with advanced gynecological (GYN) cancers have limited therapeutic options and the prognosis is poor. Early phase trials may be a suitable option for pts with good performance status. Increasingly, molecular characterisation guides pt selection for early phase trials. We sought to determine the outcome of GYN pts treated in a phase 1 unit and examined the role of molecular selection to inform therapeutic decision making. Methods: Medical records of all pts with a GYN malignancy treated within an early phase trial between 2010 and 2016 were reviewed. Data comprising patient and tumor characteristics, prior treatment, trial therapy and outcome were analysed. Results: 81 pts with a median age of 60 years (range 20-75) with a diagnosis of ovarian (OC, 54), endometrial (EC, 15) or cervical/vulval (CC, 12) cancer were identified. The median number of prior therapies for advanced disease was 3 (OC) and 2 (EC and CC) (overall range 1-6). 9 pts (11%) entered a second and 1 pt a third phase 1 study on disease progression. Next Generation Sequencing (NGS) using a targeted panel was performed in 32 pts (40%) with an actionable mutation identified in 9 including; KRAS (3pts), PIK3CA (2pts) and EGFR (2pts). Germline BRCA (gBRCA) testing was performed in 35 OC pts (65%) with 24 gBRCA mutations identified. Pts were allocated, in order of priority, where available, to (1) a trial selected on the basis of NGS or gBRCA (‘genomic’ 35%), (2) a ‘tumor specific’ cohort within an early phase trial (15%) or (3) a ‘generic’ study (51%). For the whole cohort there was an overall response rate (ORR) of 18% with 41% stable disease (SD) and median progression free survival (PFS) and overall survival (OS) of 13 and 46 weeks respectively. Outcomes were best for pts in the genomic group. Both PFS and OS were significantly longer with genomic selection (p < 0.01 for both, Mantel-cox test) with median PFS of 29.7, 14.2, 8.0 weeks and OS of 84.1, 69.7, 33.6 weeks for genomics, tumor specific and generic studies respectively. The ORR was also greatest for the genomic cohort (32%) compared to the tumour specific (7%) and generic (11%) groups. Within the heavily pre-treated EC and CC cohorts there was an OS of 30 and 42 weeks respectively. 24% of EC pts had an ORR with a further 24% with stable disease (SD). There was only 1 response (9%) in the CC cohort, however SD was seen in 64%. The OS for the OC was 55 weeks with an ORR of 20% and 46% SD. Conclusions: Early phase trials represent a good option for pts with advanced GYN malignancies. Whilst applicable to all GYN cancers, this is particularly relevant for EC and CC pts as standard treatment options are limited. For OC patients (median 3 prior lines of chemotherapy in this cohort) where standard treatment options exist, early access to phase 1 genomic trials may result in improved response rates and allow further standard options to be given subsequently. NGS is feasible in real time and may have a positive impact on outcome. Citation Format: Rowan E. Miller, Michael John Devlin, Nicholas Brown, Kin Woo, Tami Grunewald, Arran Speirs, Miriam Mitchison, Michelle Lockley, Mary McCormack, Jonathan Ledermann, Martin Forster, Tim Meyer, Rebecca Kristeleit. Guidance by molecular selection improves the outcome of early phase treatment for gynecological (GYN) cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2726. doi:10.1158/1538-7445.AM2017-2726 more...

Introduction Oesophageal adenocarcinoma (OAC) carries a poor prognosis compared to other gastrointestinal tract tumours. Advances in minimally invasive endoscopic therapy can provide curative intervention of early OAC arising in Barrett’s oesophagus (BE). Historically, patients with OAC and submucosal involvement (T1b) were offered surgery due to risk of lymph node metastases (LNM). Endoscopic resection (ER) is a viable alternative to surgery in T1b tumours with low risk (LR) histopathological features. Aim: To assess the overall risk of LNM and long-term outcomes of patients with LR and high risk (HR) T1b OAC. Method All patients with T1b OAC on endoscopic resection specimens at a single centre during 2008–2015 were included, with no sign of metastatic disease. All specimens were reviewed by two expert histopathologists. LR tumour was defined as completely resected, well or moderately differentiated, with only superficial submucosal involvement ( 500 µm) or lymphovascular invasion. Disease was considered metastatic if there was lymph node involvement in surgical resection specimens or advanced OAC on follow-up. Results 59 patients with T1b OAC on ER specimens were included: 13 (22%) had LR and 46 (78%) HR tumours. In total 37 patients were treated conservatively (12 LR, 25 hour) and 22 patients (37%) underwent oesophagectomy (1 LR, 21 hour). There was no surgery-related mortality. After a median follow-up of 38 months (range 12–87) no patients with LR OAC developed metastatic disease. Ten of the 46 hour patients (22%) developed metastatic disease at latest follow up; 4 of these had LNM in surgical resection specimens and 6 had metastatic disease detected on follow-up. In the HR group that were treated conservatively compared to those treated with surgery, although a trend was observed towards better survival in the surgery group, there was no statistically significant long-term survival benefit (log rank p=0.22). Conclusion Our results show rate of LNM in T1b OAC with LR histopathological features to be very low and endoscopic treatment in these patients is preferred to surgery. HR T1b cancers in our series carry a significant lymph node metastases risk of 22%. In selected HR patients in whom surgery may not be an option, a conservative approach is a valid alternative but rigorous follow-up is required due to the risk of LNM. Disclosure of Interest None Declared more...

Introduction Screening colonoscopy has resulted in increasing identification rates of colorectal polyps which can be endoscopically removed. Almost 10% will histologically prove to be pT1 cancers. Factors associated with cancer recurrence post polypectomy have been described (ie lymphovascular invasion, poor differentiation, R1 Methods Patients with pT1 cancers that had either endotherapy+surgery or endotherapy alone between 2008–2014 were included in the study (n = 61). Endoscopic, histologic and surgery data, including complication rates from both modalities and overall/ cancer-free survival were recorded. Management was compared between two different periods (2008–11, 2012–14) in order to identify changes in practice. Results 38 (62%) patients were treated with endoscopic resection alone. 23 had a completion surgery, 15 (65%) due to R1 resection. Adenomatous tissue was found in 6 surgical specimens but only one had residual cancer. Major complications occurred in 2 (3%) patients post endoscopy versus 6 (26%) postoperatively (p = 0.044), 2 of whom died. There were no cancer relapses at mean follow up of 41±24 months. 17/31 (55%) patients treated prior to 2012 had surgery compared to 6/30 (20%, p = 0.008) treated in 2012–14. More than one risk factors were apparent in 8 (33%) patients treated with endoscopy only within the last 3 years compared to no one previously (p = 0.017). Out of the overall 38 patients with R1 resection, 14 (74%) had completion surgery between 2008–11 compared to 31% recently treated (p = 0.022). Conclusion Endoscopic resection of polyp cancers is a safe and successful therapy with minor complications and recurrence rates similar to surgery. Changing our practice recently in favour of endotherapy alone did not lead to increasing cancer relapses, suggesting that presence of risk factors should not be an absolute indication for surgery, but instead an individualised decision should be made based on combination of factors. Disclosure of Interest None Declared more...

Introduction Colorectal cancer is now thought to arise from more than one molecular pathway. Lately, the serrated pathway has been placed in the limelight as it may account for 20–30% of colorectal cancers. However, sessile serrated lesions are easily missed on colonoscopy and in histopathological analysis despite their neoplastic potential. We conducted an observational study to understand the presentation, characteristics and significance of sessile serrated lesions detected in colonoscopies done for a variety of indications. Methods A retrospective analysis of all sessile serrated lesions identified through the colorectal histopathological database from January 2012 – March 2015 was performed in our tertiary centre. Histology was reported by specialist gastrointestinal pathologists as per national standards. Results 138 colonoscopies were performed in 118 patients who were found to have sessile serrated lesions. The table below shows the breadth of indications for colonoscopy in these cases. A total of 351 serrated lesions were noted, with just over half in the right colon (180/351, 51.3%). 67/351 polyps were >1 cm in size. 42/118 patients did have polyps noted on previous colonoscopies (19 had serrated lesions). The prevalence of co-existing adenomas in this cohort was 50% (59/118). A subgroup analysis identified 13 patients in the entire cohort who met the WHO criteria for serrated polyposis syndrome. 4 of these patients (4/13; 30.8%) had been diagnosed with colorectal cancer in the past compared with 16/105 (15.2%) in the non serrated polyposis syndrome group. Only 8/13 patients underwent genetic screening and of this, 2 had hyperplastic polyposis syndrome and 2 had MYH polyposis. Conclusion This large series of sessile serrated lesions has demonstrated the heterogeneity of circumstances in which they are detected during colonoscopy. Only 16% of patients were found to have serrated lesions on a previous colonoscopy. This may indicate that a large proportion of these polyps were missed or not recognised previously. A small proportion of these patients will have a serrated polyposis syndrome that increases their colorectal cancer risk even further and genetic screening is advocated in all these patients. Disclosure of Interest None Declared more...

A 62-year-old woman presented with abdominal pain and diarrhoea. She had a history of primary biliary cirrhosis and renal transplantation secondary to reflux nephropathy. Cross-sectional imaging and a colonoscopy demonstrated terminal ileal inflammation and ulceration, but histological biopsies were inconclusive and cytomegalovirus staining was negative. The patient subsequently developed spontaneous small bowel perforation due to deep ulceration, necessitating an emergency right hemicolectomy. Her postoperative period was complicated by several episodes of rectal bleeding. After a prolonged admission at her local hospital, the patient was referred to our unit for further management. On arrival, the patient's immunosuppression consisted of tacrolimus and low-dose prednisolone. Mycophenolate mofetil … more...