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Table S3 and Fig. S1-S10

Beyond tertiary lymphoid structures, a significant number of immune-rich areas without germinal center-like structures are observed in non–small cell lung cancer. Here, we integrated transcriptomic data and digital pathology images to study the prognostic implications, spatial locations, and constitution of immune rich areas (immune hotspots) in a cohort of 935 patients with lung cancer from The Cancer Genome Atlas. A high intratumoral immune hotspot score, which measures the proportion of immune hotspots interfacing with tumor islands, was correlated with poor overall survival in lung squamous cell carcinoma but not in lung adenocarcinoma. Lung squamous cell carcinomas with high intratumoral immune hotspot scores were characterized by consistent upregulation of B-cell signatures. Spatial statistical analyses conducted on serial multiplex IHC slides further revealed that only 4.87% of peritumoral immune hotspots and 0.26% of intratumoral immune hotspots were tertiary lymphoid structures. Significantly lower densities of CD20+CXCR5+ and CD79b+ B cells and less diverse immune cell interactions were found in intratumoral immune hotspots compared with peritumoral immune hotspots. Furthermore, there was a negative correlation between the percentages of CD8+ T cells and T regulatory cells in intratumoral but not in peritumoral immune hotspots, with tertiary lymphoid structures excluded. These findings suggest that the intratumoral immune hotspots reflect an immunosuppressive niche compared with peritumoral immune hotspots, independent of the distribution of tertiary lymphoid structures. A balance toward increased intratumoral immune hotspots is indicative of a compromised antitumor immune response and poor outcome in lung squamous cell carcinoma.Significance:Intratumoral immune hotspots beyond tertiary lymphoid structures reflect an immunosuppressive microenvironment, different from peritumoral immune hotspots, warranting further study in the context of immunotherapies.

The relationship among B-cell, T-cell percentages and CD8+/CD4+FOXP3+ ratio in individual intratumoral IH and peritumoral IH without TLS or LAG. A, Percentages of B-cell subsets in individual intratumoral (n = 262) and peritumoral IH (n = 392) classified by CD8+/CD4+FOXP3+ ratio. The high and low groups were determined by the cut-off of median CD8+/CD4+FOXP3+ ratio in intratumoral and peritumoral IH, respectively. B, Percentages of CD8+ T cells and CD4+FOXP3+ Treg cells in individual intratumoral (n = 262) and peritumoral IH (n = 392) classified by the percentage of B cells. This B-cell percentage was calculated as the total count of all B-cell subsets divided by the total count of all 6 immune cell types. C, Percentages of CD8+ T cells in individual intratumoral (n = 262) and peritumoral IH (n = 392) classified by CD4+FOXP3+ Treg-cell percentages. Statistical significance was evaluated by the Wilcoxon rank sum test and adjusted by the Benjamini–Hochberg method.

Diversity of lymphocyte interactions in intratumoral and peritumoral IH without TLS or LAG. A, Examples of lymphocyte interactions in individual peritumoral and intratumoral IH. Lymphocytes identified on the serial IHC slides were connected by the Delaunay triangle graph. Lymphocyte subsets are represented by dots of different colors. Interactions significant in the univariate logistic regression are denoted by red, otherwise blue. B, Shannon diversity of lymphocyte densities and interactions in individual peritumoral (n = 392) and intratumoral IH (n = 262). Statistical significance was evaluated by the Wilcoxon signed-rank test. C, Univariate logistic regression predicting peritumoral and intratumoral IH with individual lymphocyte interactions as variables, n = 654. The coefficients of the significant variables were all negative, suggesting a higher frequency of the interactions in peritumoral IH compared with intratumoral IH. P values were adjusted by the Benjamini–Hochberg method. Regions identified as TLS or LAG were excluded from the above analyses.

Characteristics of the included patients and their association with the Sintra/immune, Sintra/cancer, and Sintra/tissue.

Lymphocyte composition and the presence of TLS at intratumoral and peritumoral IH. A, Example regions showing annotations of TLS at the IH and pathologically defined lymphoid aggregates without a prominent germinal center (LAG) at the intratumoral IH. B, Lymphocyte subset densities at intratumoral (n = 10) and peritumoral IH (n = 10). Cell densities were measured as the cell counts divided by the total area of IHs. C, Pie chart displaying the average percentages of peritumoral IH and intratumoral IH identified as TLS, LAG, and non-TLS/LAG IH. D, Densities of TLS and LAG at intratumoral (n = 10) and peritumoral IH (n = 10). E, Densities of B- and T-cell subsets in TLS (n = 10), LAG (n = 10), and non-TLS/LAG IH (n = 10). Statistical significance was evaluated by the Wilcoxon signed-rank test, followed by the Benjamini–Hochberg method for P value adjustment. *, P < 0.05; **, P < 0.01. The median is marked by the horizontal line; the first and third quartiles are shown as box edges; whiskers indicate the range of error.

Lists of TCGA LUSC and LUAD cases in the discovery and validation cohort

Immune hotspots in LUSC. A, Illustrative examples of automatic cell classification results and hotspot distribution with zoomed-in regions showing areas enriched with immune cells and cancer–immune coclustering. Cancer hotspots (red) and IHs were identified based on the local clustering of cancer and lymphocytes. Intratumoral IH (green) was determined as regions enriched with both immune and cancer cells, while peritumoral IH (blue) were regions enriched with only immune cells. B, Schematic representations of tissue compartments including cancer hotspots, peritumoral IH, intratumoral IH based on automated spatial analysis, and pathologically defined TLSs and lymphoid aggregates without clear separation of T- and B-cell zones (LAG). The three spatial scores Sintra/immune, Sintra/cancer, and Sintra/tissue were determined as the proportion of intratumoral IH in the immune-rich area (intratumoral IH and peritumoral IH), cancer-rich area (intratumoral IH and cancer hotspots), and the entire tissue, respectively. C, Kaplan–Meier curves to illustrate the difference in OS in LUSC patients stratified by Sintra/immune, with the dichotomization optimized for the discovery cohort (n = 231) and assessed in the validation cohort (n = 231). D, Forest plots to show the prognostic value of Sintra/immune in multivariate models adjusted for stage, age, and smoking pack years in the validation cohort. E, Spearman correlation between lymphocyte percentage and Sintra/immune in the TCGA LUSC cohort (n = 462). F, Kaplan–Meier curve to illustrate the difference in OS in LUSC patients stratified by both Sintra/immune and lymphocyte percentage, with the stratification of the entire cohort shown (n = 462).

B-cell dominance of high-Sintra/immune group in LUSC. A, Gene Ontology (GO) biological processes found to be enriched in the DEGs according to patient groups stratified by Sintra/immune. The red line denotes the ratio of the number of DEGs in a pathway to the total number of genes in that pathway. FDR, false discovery rate. B, Volcano plot showing DEGs in the high-Sintra/immune group compared with the low group. Genes with log-fold change (logFC) smaller than 1 were excluded. Genes involved in immune responses are highlighted in yellow. C, Consistently upregulated B-cell signatures in high-Sintra/immune group across seven bioinformatics methods using the TCGA LUSC cohort, n = 459. Statistical significance was evaluated by the Wilcoxon rank sum test and adjusted by the Benjamini–Hochberg method.

Supplementary Figure 2: Localization of endogenous Akt and pAkt (pT308) in SKBR3 cells and FFPE human breast tumor tissues using TSA amplified immunofluorescence.

Beyond tertiary lymphoid structures, a significant number of immune-rich areas without germinal center-like structures are observed in non–small cell lung cancer. Here, we integrated transcriptomic data and digital pathology images to study the prognostic implications, spatial locations, and constitution of immune rich areas (immune hotspots) in a cohort of 935 patients with lung cancer from The Cancer Genome Atlas. A high intratumoral immune hotspot score, which measures the proportion of immune hotspots interfacing with tumor islands, was correlated with poor overall survival in lung squamous cell carcinoma but not in lung adenocarcinoma. Lung squamous cell carcinomas with high intratumoral immune hotspot scores were characterized by consistent upregulation of B-cell signatures. Spatial statistical analyses conducted on serial multiplex IHC slides further revealed that only 4.87% of peritumoral immune hotspots and 0.26% of intratumoral immune hotspots were tertiary lymphoid structures. Significantly lower densities of CD20+CXCR5+ and CD79b+ B cells and less diverse immune cell interactions were found in intratumoral immune hotspots compared with peritumoral immune hotspots. Furthermore, there was a negative correlation between the percentages of CD8+ T cells and T regulatory cells in intratumoral but not in peritumoral immune hotspots, with tertiary lymphoid structures excluded. These findings suggest that the intratumoral immune hotspots reflect an immunosuppressive niche compared with peritumoral immune hotspots, independent of the distribution of tertiary lymphoid structures. A balance toward increased intratumoral immune hotspots is indicative of a compromised antitumor immune response and poor outcome in lung squamous cell carcinoma. Significance: Intratumoral immune hotspots beyond tertiary lymphoid structures reflect an immunosuppressive microenvironment, different from peritumoral immune hotspots, warranting further study in the context of immunotherapies.

Environmental carcinogenic exposures are major contributors to global disease burden yet how they promote cancer is unclear. Over 70 years ago, the concept of tumour promoting agents driving latent clones to expand was first proposed. In support of this model, recent evidence suggests that human tissue contains a patchwork of mutant clones, some of which harbour oncogenic mutations, and many environmental carcinogens lack a clear mutational signature. We hypothesised that the environmental carcinogen

Adaptive immunity recognizes and responds to tumors, although they are part of the immunological "self." T cells, both CD4+ and CD8+, play a key role in the process, and the specific set of receptors which recognize tumor antigens therefore has the potential to provide prognostic biomarkers for tracking tumor growth after cancer therapy, including immunotherapy. Most published data on the T cell repertoire continue to rely on commercial proprietary methods, which often do not allow access to the raw data, and are difficult to validate. We describe an open-source protocol for amplifying, sequencing, and analyzing T cell receptors which is economical, robust, sensitive, and versatile. The key experimental step is the ligation of a single-stranded oligonucleotide to the 3' end of the T cell receptor cDNA, which allows easy amplification of all possible rearrangements using only a single set of primers per locus, while simultaneously introducing a unique molecular identifier to label each starting cDNA molecule. After sequencing, this molecular identifier can be used to correct both sequence errors and the effects of differential PCR amplification efficiency, thus producing a more accurate measure of the true T cell receptor frequency within the sample. Samples are then tagged with unique pairs of indices, facilitating robotic scale-up and significantly reducing cross-sample contamination from index hopping. This method has been applied to the analysis of tumor-infiltrating lymphocytes and matched peripheral blood samples from patients with a variety of solid tumors.

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

Approximately 50% of patients with early-stage non-small-cell lung cancer (NSCLC) who undergo surgery with curative intent will relapse within 5 years1,2. Detection of circulating tumor cells (CTCs) at the time of surgery may represent a tool to identify patients at higher risk of recurrence for whom more frequent monitoring is advised. Here we asked whether CellSearch-detected pulmonary venous CTCs (PV-CTCs) at surgical resection of early-stage NSCLC represent subclones responsible for subsequent disease relapse. PV-CTCs were detected in 48% of 100 patients enrolled into the TRACERx study3, were associated with lung-cancer-specific relapse and remained an independent predictor of relapse in multivariate analysis adjusted for tumor stage. In a case study, genomic profiling of single PV-CTCs collected at surgery revealed higher mutation overlap with metastasis detected 10 months later (91%) than with the primary tumor (79%), suggesting that early-disseminating PV-CTCs were responsible for disease relapse. Together, PV-CTC enumeration and genomic profiling highlight the potential of PV-CTCs as early predictors of NSCLC recurrence after surgery. However, the limited sensitivity of PV-CTCs in predicting relapse suggests that further studies using a larger, independent cohort are warranted to confirm and better define the potential clinical utility of PV-CTCs in early-stage NSCLC.

Highlights • We found a high asymptomatic rate in vulnerable people with epilepsy. • Enhanced surveillance allows to quickly contain outbreaks. • We report a low rate of COVID-19 morbidity and mortality in a long-term care facility. • Preventative measures allow reducing resident-to-resident and -to-caregiver transmission. • Children and young adults appear to have lower infection rates., In this cohort study, we aim to compare outcomes from coronavirus disease 2019 (COVID-19) in people with severe epilepsy and other co-morbidities living in long-term care facilities which all implemented early preventative measures, but different levels of surveillance. During 25-week observation period (16 March–6 September 2020), we included 404 residents (118 children), and 1643 caregivers. We compare strategies for infection prevention, control, and containment, and related outcomes, across four UK long-term care facilities. Strategies included early on-site enhancement of preventative and infection control measures, early identification and isolation of symptomatic cases, contact tracing, mass surveillance of asymptomatic cases and contacts. We measured infection rate among vulnerable people living in the facilities and their caregivers, with asymptomatic and symptomatic cases, including fatality rate. We report 38 individuals (17 residents) who tested severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-positive, with outbreaks amongst residents in two facilities. At Chalfont Centre for Epilepsy (CCE), 10/98 residents tested positive: two symptomatic (one died), eight asymptomatic on weekly enhanced surveillance; 2/275 caregivers tested positive: one symptomatic, one asymptomatic. At St Elizabeth’s (STE), 7/146 residents tested positive: four symptomatic (one died), one positive during hospital admission for symptoms unrelated to COVID-19, two asymptomatic on one-off testing of all 146 residents; 106/601 symptomatic caregivers were tested, 13 positive. In addition, during two cycles of systematically testing all asymptomatic carers, four tested positive. At The Meath (TM), 8/80 residents were symptomatic but none tested; 26/250 caregivers were tested, two positive. At Young Epilepsy (YE), 8/80 children were tested, all negative; 22/517 caregivers were tested, one positive. Infection outbreaks in long-term care facilities for vulnerable people with epilepsy can be quickly contained, but only if asymptomatic individuals are identified through enhanced surveillance at resident and caregiver level. We observed a low rate of morbidity and mortality, which confirmed that preventative measures with isolation of suspected and confirmed COVID-19 residents can reduce resident-to-resident and resident-to-caregiver transmission. Children and young adults appear to have lower infection rates. Even in people with epilepsy and multiple co-morbidities, we observed a high percentage of asymptomatic people suggesting that epilepsy-related factors (anti-seizure medications and seizures) do not necessarily lead to poor outcomes.

Immunotherapy containing regimens have become the standard of care first-line therapy for non-small cell lung cancer (NSCLC) in the metastatic setting and are being explored in the adjuvant and neo-adjuvant setting. However, as intratumor heterogeneity (ITH) is pervasive in cancer we investigated the impact of ITH on established and putative biomarkers predictive of immunotherapy response. We have used whole exome sequencing, RNA sequencing and PDL1 immunohistochemistry (IHC) derived from the multiregion treatment naïve TRACERx cohort to investigate this. Whole exome sequencing data was available for 432 primary tumours from 421 patients with greater than 1500 unique tumour regions. Tumour mutational burden (TMB) was calculated using best practice guidelines from the TMB harmonisation project. Using the FDA approved threshold of ≥ 10 mutations per megabase to define high TMB we found that 41% of patients with high TMB had at least one region with In conclusion we demonstrate significant heterogeneity of immune biomarkers in NSCLC and in particular a lack of robustness of expression-based predictors of response to immunotherapy. This highlights the need for biomarkers that are robust to intra-tumour heterogeneity and sampling bias or the need for more representative tumour sampling techniques. Citation Format: Crispin T. Hiley, Kevin Litchfield, Oriol Pich, David Moore, Cristina Naceur-Lombardelli, Selvaraju Veeriah, Maise Al Bakir, Simranpreet Summan, Kristiana Grigoriadis, Carlos Martinez Ruiz, Clare Puttick, Katey Enfield, Sophia Ward, Alexander Frankell, Dhruva Biswas, Rachel Rosenthal, Nicolai J. Birkbak, Mariam Jamal-Hanjani, Nicholas McGranahan, Charles Swanton, TRACERx Consortium. Heterogeneity of immunotherapy biomarkers in the TRACERx non-small cell lung cancer multi-region lung cancer cohort study [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 645.

Patient-derived xenograft (PDX) models have become important models in cancer biology. They are thought to mimic tumor biology more closely than traditional cell lines as a consequence of their in vivo heterocellularity and cell-matrix interactions, their 3D architecture and their relatively recent derivation. The genomic fidelity of PDXs is integral to their applications in both basic research and in translational medicine. We derived multi-region PDX models from patients enrolled in TRACERx - a study of the evolutionary dynamics of non-small cell lung cancer (NSCLC) that uses a multi-region deep whole-exome sequencing (WES) approach - using more extensive spatial sampling to better understand the histological and genomic fidelity of the PDX approach. We transplanted regional NSCLC tumor tissue subcutaneously into immunocompromised NSG mice. 134 regions were attempted, resulting in 60 passagable xenografts (44.8%). Of these, 44 regional xenografts (32.8% of all regions) were NSCLC-derived, while 16 (11.9% of all regions) were B-cell lymphoproliferative disease. Multi-region sampling revealed heterogeneous success of PDX derivation between regions within individual patient tumors. There was correlation between PDX derivation and tumor purity by WES. Overall, NSCLC PDX models were established for 22 patients (50%; range 1-5 regional models per patient; Table 1). Histologically, broad similarity was observed between PDX models and corresponding patient tumor regions, although in a small number of models, variability was noted between the patient tumor and the corresponding PDX model, or during PDX passaging. PDXs were subjected to WES for comparison with matched patient tumor regions. Overall, our study demonstrates the feasibility of systematic multi-region PDX derivation and suggests that multiple spatial sampling of tumors could improve PDX take rates and generate PDXs that represent the intratumor diversity of heterogenous NSCLCs. Table 1. Engraftment rates of multiple spatial regions from non-small cell lung cancer tumors. Attempted NSCLC PDX B-Lymphoproliferations No Xenograft: Regions No Xenograft: Any Region LUAD 48 (21) 12 (7) 9 (7) 27 (10) LUSC 61 (17) 20 (11) 6 (5) 35 (4) Other 25 (6) 12 (4) 1 (1) 12 (1) TOTAL 134 (44) 44 (22) 16 (13) 74 (15) Citation Format: Robert E. Hynds, David R. Pearce, Ayse U. Akarca, Sophia Ward, Ariana Huebner, Oriol Pich, Gareth A. Wilson, Kate H. Gowers, Rebecca Towns, Assma Ben Aissa, Selvaraju Veeriah, Sergio A. Quezada, Mariam Jamal-Hanjani, Sam M. Janes, Nicholas McGranahan, David A. Moore, Teresa Marafioti, Charles Swanton. Multi-region patient-derived xenograft models from non-small cell lung cancer patients enrolled in lung TRACERx [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 3096.

Murine tissues harbour signature, resident γδ T cell compartments with profound yet differential impacts on carcinogenesis. γδ T cell knockout mice have heightened susceptibility to carcinogenesis. However, it is now clear in the murine setting that IFNγ-producing γδ T cells reject tumours whilst IL-17-producing γδ T cells promote them. Nonetheless, many human γδ T cell compartments are distinct from that in mice and vice versa. The extent to which human tissues and tumours harbour resident γδ T cells, their effector function and their role in cancer is less clear. Although a large scale in silico study of 5000+ patients with cancer found that intratumoural γδ T cells were the most important correlate of survival, smaller studies have found that these cells to be associated with either survival or progression. Many historical studies have however been limited by the availability of technologies to rigorously identify, isolate, and examine tissue-resident γδ T cells. To address these issues, we present data from stage I-III non-small cell lung cancers and paired non-tumour (NT) tissue obtained at primary surgery from 25 patients enrolled in the TRACERx Study. Using flow cytometry and quantitative T cell receptor sequencing, we demonstrate that NT lung tissues harbour a resident population of Vδ1 γδ T cells, entirely distinct to blood. Compared with NT lung tissues, resident-memory and effector-memory Vδ1 T cells are enriched in tumours. RNA sequencing revealed that intratumoural Vδ1 T cells are skewed towards cytolysis and T-helper-1 functions, akin to intratumoural NK and CD8+ T cells. Importantly, we found no evidence of T-helper-17 skew that has been implicated in tumour promotion in murine models. Ongoing remission after surgery was significantly associated with the presence of CD103+ tissue-resident Vδ1 T cells in non-malignant lung tissues and the presence of CD45RA-/CD27+ effector-memory Vδ1 T cells in tumours. Moreover, patients with a greater proportion of intratumoural Vδ1 T cell clones shared with paired NT tissues were more likely to remain in remission, consistent with the cells’ proposed immunosurveillance function in steady state epithelial tissues. Whilst immunotherapies modulating αβ T cells have been successful for some patients, including those with non-small cell lung cancer, clinical trials of γδ T cells have so far demonstrated poor efficacy in solid cancers. These trials have hitherto exclusively utilised Vδ2 T cells, a subset which is found predominantly in peripheral blood and more commonly associated, albeit still rarely, with IL-17 production. The first-in-human clinical trial of Vδ1 T cell immunotherapy has just opened for patients with acute myeloid leukaemia. Thus, our findings have immediate translational relevance and support the utilisation of these as-yet-untapped Vδ1 T cells in solid cancer immunotherapy. Citation Format: Yin Wu, Dhruva Biswas, Ieva Usaite, Angelova Mihaela, Stefan Boeing, Takahiro Karasaki, Selvaraju Veeriah, Justyna Czyzewska-Khan, James Reading, Andrew Georgiou, Maise Al-Bakir, Nicholas McGranahan, Mariam Jamal-Hanjani, Allan Hackshaw, TRACERx Consortium, Sergio Quezada, Adrian Hayday, Charles Swanton. V-delta-1 T cells are resident in the human lung and associate with survival in patients with non-small cell lung cancer in the TRACERx Study [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 5636.

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.

In the version of this article initially published, the list of members and affiliations for The TRACERx Consortium was provided as a Supplementary Note. The list should have been provided in the online version. Also, an incorrect version of Fig. 2b was published. The correct version is provided below. The errors have been corrected in the HTML and PDF versions of the article.

// Angus J.M. Cameron 1 , Selvaraju Veeriah 2,3 , Jacqueline J.T. Marshall 3 , Elizabeth R. Murray 1 , Banafshe Larijani 4 and Peter J. Parker 3,5 1 Kinase Biology Laboratory, Barts Cancer Institute, Queen Mary University of London, John Vane Science Centre, Charterhouse Square, London, UK 2 Translational Cancer Therapeutics Laboratory, Paul O’Gorman Building, University College London Cancer Institute, London, United Kingdom 3 Protein Phosphorylation Laboratory, Francis Crick Institute, London, UK 4 Cell Biophysics Laboratory, Ikerbasque Basque Foundation for Science, Research Centre for Experimental Marine Biology and Biotechnology (PiE) & Biofisika Instituto (UPV/EHU, CSIC), University of the Basque Country, Areatza Hiribidea, Plentzia, Spain 5 Division of Cancer Studies, King’s College London, New Hunts House, Guy’s Campus, London, UK Correspondence to: Angus J.M. Cameron, email: // Peter J. Parker, email: // Keywords : mTORC2, CRIM, Akt, xenograft, Sin1 Received : December 29, 2016 Accepted : June 26, 2017 Published : August 09, 2017 Abstract Mammalian target of rapamycin (mTOR) is a central regulator of growth and metabolism. mTOR resides in two distinct multi-protein complexes – mTORC1 and mTORC2 – with distinct upstream regulators and downstream targets. While it is possible to specifically inhibit mTORC1 with rapamycin, or inhibit both mTOR complexes together with ATP pocket directed mTOR kinase inhibitors, it is not possible to assess the specific roles for mTORC2 pharmacologically. To overcome this, we have developed a novel, inducible, dominant negative system for disrupting substrate recruitment to mTORC2. Previously we identified the mTORC2 specific subunit Sin1 as a direct binding partner for AGC kinases Akt and PKC. Sin1 mutants, which retain the ability to bind Rictor and mTOR, but fail to recruit their AGC client kinases, inhibit AKT and PKC priming and block cell growth. In this study, we demonstrate that uncoupling mTORC2 from AGC kinases in DLD1 colon cancer cells inhibits Akt activation and blocks tumour growth in vivo . Further we demonstrate, using time resolved two-site amplified FRET (A-FRET) analysis of xenograft tumours, that inhibition of tumour growth correlates with the degree of mTORC2 uncoupling from its downstream targets, as demonstrated for Akt. These data add weight to the body of evidence that mTORC2 represents a pharmacological target in cancer independently of mTORC1.

Background: \ud Among patients with non–small-cell lung cancer (NSCLC), data on intratumor heterogeneity and cancer genome evolution have been limited to small retrospective cohorts. We wanted to prospectively investigate intratumor heterogeneity in relation to clinical outcome and to determine the clonal nature of driver events and evolutionary processes in early-stage NSCLC.\ud \ud Methods: \ud In this prospective cohort study, we performed multiregion whole-exome sequencing on 100 early-stage NSCLC tumors that had been resected before systemic therapy. We sequenced and analyzed 327 tumor regions to define evolutionary histories, obtain a census of clonal and subclonal events, and assess the relationship between intratumor heterogeneity and recurrence-free survival.\ud \ud Results: \ud We observed widespread intratumor heterogeneity for both somatic copy-number alterations and mutations. Driver mutations in EGFR, MET, BRAF, and TP53 were almost always clonal. However, heterogeneous driver alterations that occurred later in evolution were found in more than 75% of the tumors and were common in PIK3CA and NF1 and in genes that are involved in chromatin modification and DNA damage response and repair. Genome doubling and ongoing dynamic chromosomal instability were associated with intratumor heterogeneity and resulted in parallel evolution of driver somatic copy-number alterations, including amplifications in CDK4, FOXA1, and BCL11A. Elevated copy-number heterogeneity was associated with an increased risk of recurrence or death (hazard ratio, 4.9; P=4.4×10−4), which remained significant in multivariate analysis.\ud \ud Conclusions: \ud Intratumor heterogeneity mediated through chromosome instability was associated with an increased risk of recurrence or death, a finding that supports the potential value of chromosome instability as a prognostic predictor. (Funded by Cancer Research UK and others; TRACERx ClinicalTrials.gov number, NCT01888601.)

Remarkable progress in molecular analyses has improved our understanding of the evolution of cancer cells towards immune escape(1–5). However, the spatial configurations of immune and stromal cells, which may shed light on the evolution of immune escape across tumor geographical locations, remain unaddressed. We integrated multi-region exome and RNA-seq data with spatial histology mapped by deep learning in 100 non-small cell lung cancer (NSCLC) patients from the TRAcking Cancer Evolution through Therapy (Rx) (TRACERx) cohort(6). Cancer subclones derived from immune cold regions were more closely related in mutation space, diversifying more recently than subclones from immune hot regions. In TRACERx and in an independent multi-sample cohort of 970 lung adenocarcinoma (LUAD) patients, the number of immune cold regions significantly correlated with risk of relapse, independently of tumor size, stage and number of samples per patient. In LUAD, but not lung squamous cell carcinoma (LUSC), geometrical irregularity and complexity of the cancer-stromal cell interface significantly increased in tumor regions without disruption of antigen presentation. Decreased lymphocyte accumulation in adjacent stroma was observed in tumors with low clonal neoantigen burden. Collectively, immune geospatial variability elucidates tumor ecological constraints that may shape the emergence of immune evading subclones and aggressive clinical phenotypes.

Automatic cell detection in histology images is a challenging task due to varying size, shape and features of cells and stain variations across large cohorts. Conventional deep learning methods regress the probability of each pixel belonging to the centre of a cell followed by detection of local maxima. We propose a three stage method (MapDe) to improve cell detection. (a) The dot annotations are convolved with a mapping filter to generate artifical labels. (b) A convolutional neural network (CNN) is modified to convolve its output with the same mapping filter. The mapping filter is fixed during training forcing the network to generate better probability maps. (c) Output of the trained CNN is deconvolved to generate points as cell detection. The results show that (1) local maxima performs better cell detection with probability maps generated using fixed convolution filter, (2) the results can be further improved by deconvolving the output with fewer parameters to tune.

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.

Introduction Minimal residual disease (MRD) detection in solid tumors describes isolation of circulating tumor DNA (ctDNA) molecules in plasma following definitive treatment of a cancer. Detection of MRD following surgical tumor excision categorizes patients as high risk for disease recurrence. Establishing an MRD approach to treating early-stage NSCLC will facilitate escalation of standard of care (SoC) treatment only in patients destined to relapse from their cancer and overcome challenges associated with conventional adjuvant drug-trial design. Here, we present data from the lung TRACERx study where patients with early-stage NSCLC underwent phylogenetic ctDNA profiling following resection. Methods Patient specific anchored-multiplex PCR (AMP) enrichment panels were generated for 78 lung TRACERx patients who underwent surgery for stage I-III NSCLC; 608 plasma samples were analyzed. Extensive patient-specific cfDNA enrichment panels targeted a median of 196 (range 72 to 482) clonal and subclonal variants detected in primary tumor tissue by multi-region exome sequencing. A novel MRD-caller controlled and estimated background sequencing error to maximize ctDNA detection at low mutant allele frequencies (MAFs). Analytical validation experiments benchmarked assay performance. Results Analytical validation of a 50-variant AMP-MRD assay demonstrated a sensitivity of 89% for mutant DNA at a MAF of 0.008% (with 25ng of DNA input into the assay), specificity was 100% experimentally and 99.9% (95% CI: 99.67 to 99.99%) modelled in-silico. 45 patients suffered relapse of their primary NSCLC; ctDNA was detected at or before clinical relapse in 37 of 45 patients. In these 37 patients the median ctDNA lead-time (time from ctDNA detection to clinical relapse) was 151 days (range 0 to 984 days) and the median time to relapse from surgery was 413 days (range 41 to 1242 days). In 10 of 10 patients who developed second primary cancers during follow-up no ctDNA was detected, reflecting specificity of the MRD assay toward the primary tumor. In 23 patients who remained relapse-free during a median of 1184 days of study follow-up, ctDNA was detected in 1 of 199 time-points analyzed. Analysis of SoC adjuvant surveillance imaging (CT, PET-CT or MRI, 220 encounters) revealed examples of MRD positive patients where SoC radiological surveillance was negative for impending relapse. Through application of large cfDNA enrichment panels targeting up to 483 variants per patient we observed dynamic changes in clonal composition and copy-number status prior to NSCLC relapse, categorized relapse as monoclonal or polyclonal and identified distinct subclonal dynamics during systemic intervention for disease recurrence. Conclusions ctDNA is an adjuvant biomarker capable of both detecting MRD following surgery and defining the clonality of relapsing disease. These data pave the way for clinical trials predicated on escalation of adjuvant standard of care in NSCLC patients who exhibit MRD positive status following surgery. Citation Format: Chris Abbosh, Alexander Frankell, Aaron Garnett, Thomas Harrison, Morgan Weichert, Abel Licon, Selvaraju Veeriah, Bob Daber, Mike Moreau, Adrian Chesh, Kevin Litchfield, Emilia Lim, Daniel Cooke, Clare Puttick, Maise Al Bakir, Fabio Gomes, Akshay Patel, Lizi Manzano, Ariana Huebner, Nicolas Carey, Joan Riley, Paula Roberts, Todd Druley, Jacqui A. Shaw, Nicholas McGranahan, Mariam Jamal-Hanjani, Nicolai Birkbak, Josh Stahl, Charles Swanton, Lung TRACERx consortium. Phylogenetic tracking and minimal residual disease detection using ctDNA in early-stage NSCLC: A lung TRACERx study [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT023.

Somatic mutations together with immunoediting drive extensive heterogeneity within non-small-cell lung cancer (NSCLC). Herein we examine heterogeneity of the T cell antigen receptor (TCR) repertoire. The number of TCR sequences selectively expanded in tumors varies within and between tumors and correlates with the number of nonsynonymous mutations. Expanded TCRs can be subdivided into TCRs found in all tumor regions (ubiquitous) and those present in a subset of regions (regional). The number of ubiquitous and regional TCRs correlates with the number of ubiquitous and regional nonsynonymous mutations, respectively. Expanded TCRs form part of clusters of TCRs of similar sequence, suggestive of a spatially constrained antigen-driven process. CD8+ tumor-infiltrating lymphocytes harboring ubiquitous TCRs display a dysfunctional tissue-resident phenotype. Ubiquitous TCRs are preferentially detected in the blood at the time of tumor resection as compared to routine follow-up. These findings highlight a noninvasive method to identify and track relevant tumor-reactive TCRs for use in adoptive T cell immunotherapy.

An aim of molecular biomarkers is to stratify patients with cancer into disease subtypes predictive of outcome, improving diagnostic precision beyond clinical descriptors such as tumor stage1. Transcriptomic intratumor heterogeneity (RNA-ITH) has been shown to confound existing expression-based biomarkers across multiple cancer types2–6. Here, we analyze multi-region whole-exome and RNA sequencing data for 156 tumor regions from 48 patients enrolled in the TRACERx study to explore and control for RNA-ITH in non-small cell lung cancer. We find that chromosomal instability is a major driver of RNA-ITH, and existing prognostic gene expression signatures are vulnerable to tumor sampling bias. To address this, we identify genes expressed homogeneously within individual tumors that encode expression modules of cancer cell proliferation and are often driven by DNA copy-number gains selected early in tumor evolution. Clonal transcriptomic biomarkers overcome tumor sampling bias, associate with survival independent of clinicopathological risk factors, and may provide a general strategy to refine biomarker design across cancer types. TRACERx Lung: Intratumoral transcriptional heterogeneity, which often hinders the development of clinically useful RNA-expression-biased biomarkers for cancer, can now be overcome with an approach for the identification of clonal expression biomarkers.

Immune evasion is a hallmark of cancer. Losing the ability to present neoantigens through human leukocyte antigen (HLA) loss may facilitate immune evasion. However, the polymorphic nature of the locus has precluded accurate HLA copy-number analysis. Here, we present loss of heterozygosity in human leukocyte antigen (LOHHLA), a computational tool to determine HLA allele-specific copy number from sequencing data. Using LOHHLA, we find that HLA LOH occurs in 40% of non-small-cell lung cancers (NSCLCs) and is associated with a high subclonal neoantigen burden, APOBEC-mediated mutagenesis, upregulation of cytolytic activity, and PD-L1 positivity. The focal nature of HLA LOH alterations, their subclonal frequencies, enrichment in metastatic sites, and occurrence as parallel events suggests that HLA LOH is an immune escape mechanism that is subject to strong microenvironmental selection pressures later in tumor evolution. Characterizing HLA LOH with LOHHLA refines neoantigen prediction and may have implications for our understanding of resistance mechanisms and immunotherapeutic approaches targeting neoantigens. VIDEO ABSTRACT.

The fusogenic lipid diacylglycerol is essential for remodeling gamete and zygote nuclear envelopes (NE) during early embryogenesis. It is unclear whether upstream signaling molecules are likewise conserved. Here we demonstrate PLCγ and its activator SFK1, which co-operate during male pronuclear envelope formation, also promote the subsequent male and female pronuclear fusion. PLCγ and SFK1 interact directly at the fusion site leading to PLCγ activation. This is accompanied by a spatially restricted reduction of PtdIns(4,5)P2. Consequently, pronuclear fusion is blocked by PLCγ or SFK1 inhibition. These findings identify new regulators of events in the early embryo and suggest a conserved "toolkit" of fusion machinery drives successive NE fusion events during embryogenesis.

Coordinate control of different classes of cyclins is fundamentally important for cell cycle regulation and tumor suppression, yet the underlying mechanisms are incompletely understood. Here we show that the PARK2 tumor suppressor mediates this coordination. The PARK2 E3 ubiquitin ligase coordinately controls the stability of both cyclin D and cyclin E. Analysis of approximately 5,000 tumor genomes shows that PARK2 is a very frequently deleted gene in human cancer and uncovers a striking pattern of mutual exclusivity between PARK2 deletion and amplification of CCND1, CCNE1 or CDK4-implicating these genes in a common pathway. Inactivation of PARK2 results in the accumulation of cyclin D and acceleration of cell cycle progression. Furthermore, PARK2 is a component of a new class of cullin-RING-containing ubiquitin ligases targeting both cyclin D and cyclin E for degradation. Thus, PARK2 regulates cyclin-CDK complexes, as does the CDK inhibitor p16, but acts as a master regulator of the stability of G1/S cyclins.

At the point of cancer diagnosis, molecular biomarkers aim to stratify patients into precise disease subtypes predictive of outcome independent of standard clinical parameters such as tumour stage. Although prognostic gene expression signatures have been derived for many cancer types, seldom have they been shown to improve therapeutic decision making, limiting their clinical use. While intra-tumour transcriptomic heterogeneity (RNA-ITH) has been shown to bias existing biomarkers, efforts to control for this biological parameter have not been considered in biomarker development. Here, we analyse multi-region RNA-seq and whole-exome data for 156 tumour regions from 48 TRACERx patients to explore RNA-ITH in NSCLC. We show that chromosomal instability is a major driver of RNA-ITH, through the generation of heterogeneous copy number events within tumours, and that existing prognostic gene expression signatures are vulnerable to sampling bias. To address this issue, we develop the Outcome Risk Associated Clonal Lung Expression (ORACLE) assay, comprised of genes expressed homogeneously within individual tumours but heterogeneously between patients. These genes are enriched in modules associated with cell proliferation, such as mitosis and nucleosome assembly, that are often selected for through copy number gain events occurring early in tumour evolution. Our approach to identify “clonal” transcriptomic biomarkers in NSCLC overcomes tumour sampling bias, improves survival risk forecasting over current clinicopathological risk factors, and may be generalised to other cancer types, whilst revealing the early evolutionary selection of high risk DNA copy number events driving poor clinical outcome. Citation Format: Dhruva Biswas, Nicolai J. Birkbak, Rachel Rosenthal, Crispin T. Hiley, Emilia L. Lim, Krisztian Papp, Marcin Krzystanek, Dijana Djureinovic, Yin Wu, David A. Moore, Marcin Skrypski, Christopher Abbosh, Maise Al Bakir, Thomas B. Watkins, Selvaraju Veeriah, Gareth A. Wilson, Mariam Jamal-Hanjani, Arul M. Chinnaiyan, Patrick Micke, Jiri Bartek, Istvan Csabai, Zoltan Szallasi, Javier Herrero, Nicholas McGranahan, Charles Swanton. A clonal expression biomarker improves prognostic accuracy: TRACERx lung [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2678.

In order to avoid immune predation, particularly in response to immunotherapy, tumors must acquire mechanisms to evade immune detection or withstand its activity. Disruption of human leukocyte antigen (HLA), which may lead to reduced neoantigen presentation, has been proposed as an immune escape strategy in many cancers, including lung cancer. A high proportion of cancer types have been found to downregulate HLA expression, potentially leading to the evasion of T cell-mediated destruction (Hicklin, 1999; Garrido, 2017) with reduced HLA expression associating with decreased overall survival and tumor progression (Mehta, 2008). Another irreversible means of HLA disruption is via loss of heterozygosity (LOH) at the HLA locus (Koopman, 2000). However, to date, the diversity of the HLA locus has impeded copy number analysis and determination of HLA loss. To investigate the prevalence, timing, and importance of HLA disruption, we developed LOHHLA (Loss Of Heterozygosity in Human Leukocyte Antigen), a computational tool to determine HLA allele-specific copy number from sequencing data. Supporting the notion that loss of antigen presentation may play an important role in immune evasion during tumor evolution, HLA LOH was identified in 40% of the NSCLC samples analyzed, frequently as a late event in tumor evolution and associated with a high subclonal neoantigen burden, increase in immune infiltration, and PD-L1 positivity. Characterizing HLA haplotype specific copy number with LOHHLA refines neoantigen prediction and may have implications for immunotherapeutic approaches targeting neoantigens. Citation Format: Nicholas McGranahan, Rachel Rosenthal, Crispin T. Hiley, Andrew J. Rowan, Thomas B.K. Watkins, Gareth A. Wilson, Nicolai J. Birkbak, Selvaraju Veeriah, Peter Van Loo, Javier Herrero, Charles Swanton. Allele-specific HLA loss and immune escape in lung cancer evolution [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr IA23.

12009Background: The lung TRACERx study is a prospective study exploring the cancer genome evolution of NSCLC. Data analysis from the first 100 patients enrolled into the study has shown an increas...

Apple juice is considered to be an important component of the healthy diet, with anticancer activities in colon cancer animal models and key ingredients have numerous chemoprotective activities in human colon cells in vitro.Since only little is known on comparable activities in the human colon in vivo, here a pilot study was performed to assess related mechanisms caused by ileostomy samples from volunteers that had consumed apple juice.Ileostomy samples were collected after intervention (0-8 h) with cloudy apple juice (1 l). They were characterized analytically for major apple polyphenols and biologically in HT29 colon cells for their potential to cause genotoxic damage, protect from the genotoxic insult by hydrogen peroxide (H(2)O(2)) and modulate the expression of GSTT2, an enzyme related to antioxidative defence against different peroxides.The analytical determination of polyphenols in the ileostomy samples revealed that the majority of the compounds were recovered in the samples collected 2 h after intervention. The comparison of genotoxic effects of samples before intervention and 2 h after intervention revealed a considerable variation of genotoxic response, but there was a trend for reduced genotoxicity in three of eight persons (P) after intervention. Samples collected at 2 h protected HT29 cells from genotoxic damage by H(2)O(2) (for 4 of 8 persons), resulted in an increased GSTT2 expression (for 2 of 6 persons) and of GSTT2 promotor activity (2 of 6 persons).The intervention with apple juice results in bioavailable concentrations of related polyphenols in the gut lumen, which could contribute to reduced genotoxicity, enhanced antigenotoxicity and favorable modulation of GSTT2 gene expression in some individuals. The pilot study for the first time used this combination of faecal biomarkers which in larger cohorts may either reveal overall significant alterations of chemoprotection or may be used to identify individuals which could particularly benefit from a personalized nutrition.

Flavonoids from fruits and vegetables probably reduce risks of diseases associated with oxidative stress, including cancer. Apples contain significant amounts of flavonoids with antioxidative potential. The objectives of this study were to investigate such compounds for properties associated with reduction of cancer risks. We report herein that apple flavonoids from an apple extract (AE) inhibit colon cancer cell growth and significantly modulate expression of genes related to xenobiotic metabolism. HT29 cells were treated with AE at concentrations delivering 5-50 microM of one of the major ingredients, phloridzin ("phloridzin-equivalents," Ph.E), to the cell culture medium, with a synthetic flavonoid mixture mimicking the composition of the AE or with 5-100 microM individual flavonoids. HT29 cell growth was inhibited by the complex extract and by the mixture. HT29 cells were treated with nontoxic doses of the AE (30 microM, Ph.E) and after 24 h total RNA was isolated to elucidate patterns of gene expression using a human cDNA-microarray (SuperArray) spotted with 96 genes of drug metabolism. Treatment with AE resulted in an upregulation of several genes (GSTP1, GSSTT2, MGST2, CYCP4F3, CHST5, CHST6, and CHST7) and downregulation of EPHX1, in comparison to the medium controls. The enhanced transcriptional activity of GSTP1 and GSTT2 genes was confirmed with real-time qRT-PCR. On the basis of the pattern of differential gene expression found here, we conclude that apple flavonoids modulate toxicological defense against colon cancer risk factors. In addition to the inhibition of tumor cell proliferation, this could be a mechanism of cancer risk reduction.

There is evidence that consumption of certain dietary ingredients may favourably modulate biotransformation of carcinogens. Associated with this is the hypothesis that the risk for developing colorectal cancer could be reduced, since its incidence is related to diet. Two main groups of biotransformation enzymes metabolize carcinogens, namely Phase I enzymes, which convert hydrophobic compounds to more water-soluble moieties, and Phase II enzymes (e.g. glutathione S-transferases [GST]), which primarily catalyze conjugation reactions. The conjugation of electrophilic Phase I intermediates with glutathione, for instance, frequently results in detoxification. Several possible colon carcinogens may serve as substrates for GST isoenzymes that can have marked substrate specificity. The conjugated products could be less toxic/genotoxic if GSTs are induced, thereby reducing exposure. Thus, numerous studies have shown that the induction of GSTs by antioxidants enables experimental animals to tolerate exposure to carcinogens. One important mechanism of GST induction involves an antioxidant-responsive response element (ARE) and the transcription factor nuclear factor E2-related factor 2 (Nrf2), which is bound to the Kelch-like ECH associated protein 1 (Keap1) in the cytoplasm. Antioxidants may disrupt the Keap-Nrf2 complex, allowing Nrf2 to translocate to the nucleus and mediate expression of Phase II genes via interaction with the ARE. GSTs are also induced by butyrate, a product of gut flora-derived fermentation of plant foods, which may act via different mechanisms, e.g. by increasing histone acetylation. GSTs are expressed with high inter-individual variability in human colonocytes, which points to large differences in cellular susceptibility to xenobiotics. Enhancing expression of GSTs in human colon tissue could therefore contribute to reducing cancer risks. However, it has not been demonstrated in humans that this mechanism is associated with cancer prevention. In the future, it will be useful to determine GSTs during dietary intervention studies to enhance our understanding of this protective mechanism.