Longitudinal Assessment of Tumor-Infiltrating Lymphocytes in Primary Breast Cancer Following Neoadjuvant Radiation Therapy.

PURPOSE: Tumor-infiltrating lymphocytes (TILs) have prognostic significance in several cancers, including breast cancer. Despite interest in combining radiation therapy with immunotherapy, little is known about the effect of radiation therapy itself on the tumor-immune microenvironment, including TILs. Here, we interrogated longitudinal dynamics of TILs and systemic lymphocytes in patient samples taken before, during, and after neoadjuvant radiation therapy (NART) from PRADA and Neo-RT breast clinical trials. METHODS AND MATERIALS: We manually scored stromal TILs (sTILs) from longitudinal tumor samples using standardized guidelines as well as deep learning-based scores at cell-level (cTIL) and cell- and tissue-level combination analyses (SuperTIL). In parallel, we interrogated absolute lymphocyte counts from routine blood tests at corresponding time points during treatment. Exploratory analyses studied the relationship between TILs and pathologic complete response (pCR) and long-term outcomes. RESULTS: Patients receiving NART experienced a significant and uniform decrease in sTILs that did not recover at the time of surgery (P < .0001). This lymphodepletive effect was also mirrored in peripheral blood. Our SuperTIL deep learning score showed good concordance with manual sTILs and importantly performed comparably to manual scores in predicting pCR from diagnostic biopsies. The analysis suggested an association between baseline sTILs and pCR, as well as sTILs at surgery and relapse, in patients receiving NART. CONCLUSIONS: This study provides novel insights into TIL dynamics in the context of NART in breast cancer and demonstrates the potential for artificial intelligence to assist routine pathology. We have identified trends that warrant further interrogation and have a bearing on future radioimmunotherapy trials.

The artificial intelligence-based model ANORAK improves histopathological grading of lung adenocarcinoma.

The introduction of the International Association for the Study of Lung Cancer grading system has furthered interest in histopathological grading for risk stratification in lung adenocarcinoma. Complex morphology and high intratumoral heterogeneity present challenges to pathologists, prompting the development of artificial intelligence (AI) methods. Here we developed ANORAK (pyrAmid pooliNg crOss stReam Attention networK), encoding multiresolution inputs with an attention mechanism, to delineate growth patterns from hematoxylin and eosin-stained slides. In 1,372 lung adenocarcinomas across four independent cohorts, AI-based grading was prognostic of disease-free survival, and further assisted pathologists by consistently improving prognostication in stage I tumors. Tumors with discrepant patterns between AI and pathologists had notably higher intratumoral heterogeneity. Furthermore, ANORAK facilitates the morphological and spatial assessment of the acinar pattern, capturing acinus variations with pattern transition. Collectively, our AI method enabled the precision quantification and morphology investigation of growth patterns, reflecting intratumoral histological transitions in lung adenocarcinoma.

Deep Learning Enables Spatial Mapping of the Mosaic Microenvironment of Myeloma Bone Marrow Trephine Biopsies.

Bone marrow trephine biopsy is crucial for the diagnosis of multiple myeloma. However, the complexity of bone marrow cellular, morphologic, and spatial architecture preserved in trephine samples hinders comprehensive evaluation. To dissect the diverse cellular communities and mosaic tissue habitats, we developed a superpixel-inspired deep learning method (MoSaicNet) that adapts to complex tissue architectures and a cell imbalance aware deep learning pipeline (AwareNet) to enable accurate detection and classification of rare cell types in multiplex immunohistochemistry images. MoSaicNet and AwareNet achieved an AUC of >0.98 for tissue and cellular classification on separate test datasets. Application of MoSaicNet and AwareNet enabled investigation of bone heterogeneity and thickness as well as spatial histology analysis of bone marrow trephine samples from monoclonal gammopathies of undetermined significance (MGUS) and from paired newly diagnosed and posttreatment multiple myeloma. The most significant difference between MGUS and newly diagnosed multiple myeloma (NDMM) samples was not related to cell density but to spatial heterogeneity, with reduced spatial proximity of BLIMP1+ tumor cells to CD8+ cells in MGUS compared with NDMM samples. Following treatment of patients with multiple myeloma, there was a reduction in the density of BLIMP1+ tumor cells, effector CD8+ T cells, and regulatory T cells, indicative of an altered immune microenvironment. Finally, bone heterogeneity decreased following treatment of patients with multiple myeloma. In summary, deep learning-based spatial mapping of bone marrow trephine biopsies can provide insights into the cellular topography of the myeloma marrow microenvironment and complement aspirate-based techniques. SIGNIFICANCE: Spatial analysis of bone marrow trephine biopsies using histology, deep learning, and tailored algorithms reveals the bone marrow architectural heterogeneity and evolution during myeloma progression and treatment.

Self-supervised deep learning for highly efficient spatial immunophenotyping.

BACKGROUND: Efficient biomarker discovery and clinical translation depend on the fast and accurate analytical output from crucial technologies such as multiplex imaging. However, reliable cell classification often requires extensive annotations. Label-efficient strategies are urgently needed to reveal diverse cell distribution and spatial interactions in large-scale multiplex datasets. METHODS: This study proposed Self-supervised Learning for Antigen Detection (SANDI) for accurate cell phenotyping while mitigating the annotation burden. The model first learns intrinsic pairwise similarities in unlabelled cell images, followed by a classification step to map learnt features to cell labels using a small set of annotated references. We acquired four multiplex immunohistochemistry datasets and one imaging mass cytometry dataset, comprising 2825 to 15,258 single-cell images to train and test the model. FINDINGS: With 1% annotations (18-114 cells), SANDI achieved weighted F1-scores ranging from 0.82 to 0.98 across the five datasets, which was comparable to the fully supervised classifier trained on 1828-11,459 annotated cells (-0.002 to -0.053 of averaged weighted F1-score, Wilcoxon rank-sum test, P = 0.31). Leveraging the immune checkpoint markers stained in ovarian cancer slides, SANDI-based cell identification reveals spatial expulsion between PD1-expressing T helper cells and T regulatory cells, suggesting an interplay between PD1 expression and T regulatory cell-mediated immunosuppression. INTERPRETATION: By striking a fine balance between minimal expert guidance and the power of deep learning to learn similarity within abundant data, SANDI presents new opportunities for efficient, large-scale learning for histology multiplex imaging data. FUNDING: This study was funded by the Royal Marsden/ICR National Institute of Health Research Biomedical Research Centre.

Glioblastoma cell fate is differentially regulated by the microenvironments of the tumor bulk and infiltrative margin.

Glioblastoma (GBM) recurrence originates from invasive margin cells that escape surgical debulking, but to what extent these cells resemble their bulk counterparts remains unclear. Here, we generated three immunocompetent somatic GBM mouse models, driven by subtype-associated mutations, to compare matched bulk and margin cells. We find that, regardless of mutations, tumors converge on common sets of neural-like cellular states. However, bulk and margin have distinct biology. Injury-like programs associated with immune infiltration dominate in the bulk, leading to the generation of lowly proliferative injured neural progenitor-like cells (iNPCs). iNPCs account for a significant proportion of dormant GBM cells and are induced by interferon signaling within T cell niches. In contrast, developmental-like trajectories are favored within the immune-cold margin microenvironment resulting in differentiation toward invasive astrocyte-like cells. These findings suggest that the regional tumor microenvironment dominantly controls GBM cell fate and biological vulnerabilities identified in the bulk may not extend to the margin residuum.

Bridging clinic and wildlife care with AI-powered pan-species computational pathology.

Cancers occur across species. Understanding what is consistent and varies across species can provide new insights into cancer initiation and evolution, with significant implications for animal welfare and wildlife conservation. We build a pan-species cancer digital pathology atlas (panspecies.ai) and conduct a pan-species study of computational comparative pathology using a supervised convolutional neural network algorithm trained on human samples. The artificial intelligence algorithm achieves high accuracy in measuring immune response through single-cell classification for two transmissible cancers (canine transmissible venereal tumour, 0.94; Tasmanian devil facial tumour disease, 0.88). In 18 other vertebrate species (mammalia = 11, reptilia = 4, aves = 2, and amphibia = 1), accuracy (range 0.57-0.94) is influenced by cell morphological similarity preserved across different taxonomic groups, tumour sites, and variations in the immune compartment. Furthermore, a spatial immune score based on artificial intelligence and spatial statistics is associated with prognosis in canine melanoma and prostate tumours. A metric, named morphospace overlap, is developed to guide veterinary pathologists towards rational deployment of this technology on new samples. This study provides the foundation and guidelines for transferring artificial intelligence technologies to veterinary pathology based on understanding of morphological conservation, which could vastly accelerate developments in veterinary medicine and comparative oncology.

The tumour ecology of quiescence: Niches across scales of complexity.

Quiescence is a state of cell cycle arrest, allowing cancer cells to evade anti-proliferative cancer therapies. Quiescent cancer stem cells are thought to be responsible for treatment resistance in glioblastoma, an aggressive brain cancer with poor patient outcomes. However, the regulation of quiescence in glioblastoma cells involves a myriad of intrinsic and extrinsic mechanisms that are not fully understood. In this review, we synthesise the literature on quiescence regulatory mechanisms in the context of glioblastoma and propose an ecological perspective to stemness-like phenotypes anchored to the contemporary concepts of niche theory. From this perspective, the cell cycle regulation is multiscale and multidimensional, where the niche dimensions extend to extrinsic variables in the tumour microenvironment that shape cell fate. Within this conceptual framework and powered by ecological niche modelling, the discovery of microenvironmental variables related to hypoxia and mechanosignalling that modulate proliferative plasticity and intratumor immune activity may open new avenues for therapeutic targeting of emerging biological vulnerabilities in glioblastoma.

Spatial Positioning of Immune Hotspots Reflects the Interplay between B and T Cells in Lung Squamous Cell Carcinoma.

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.

Artificial intelligence-based morphometric signature to identify ductal carcinoma in situ with low risk of progression to invasive breast cancer.

Ductal carcinoma in situ (DCIS) may progress to ipsilateral invasive breast cancer (iIBC), but often never will. Because DCIS is treated as early breast cancer, many women with harmless DCIS face overtreatment. To identify these women that may forego treatment, we hypothesized that DCIS morphometric features relate to the risk of subsequent iIBC. We developed an artificial intelligence-based DCIS morphometric analysis pipeline (AIDmap) to detect DCIS as a pathologist and measure morphological structures in hematoxylin-eosin-stained (H&E) tissue sections. These were from a case-control study of patients diagnosed with primary DCIS, treated by breast-conserving surgery without radiotherapy. We analyzed 689 WSIs of DCIS of which 226 were diagnosed with subsequent iIBC (cases) and 463 were not (controls). The distribution of 15 duct morphological measurements in each H&E was summarized in 55 morphometric variables. A ridge regression classifier with cross validation predicted 5-years-free of iIBC with an area-under the curve of 0.65 (95% CI 0.55-0.76). A morphometric signature based on the 30 variables most associated with outcome, identified lesions containing small-sized ducts, low number of cells and low DCIS/stroma area ratio. This signature was associated with lower iIBC risk in a multivariate regression model including grade, ER, HER2 and COX-2 expression (HR = 0.56; 95% CI 0.28-0.78). AIDmap has potential to identify harmless DCIS that may not need treatment.

Comparative Evaluation of Tumor-Infiltrating Lymphocytes in Companion Animals: Immuno-Oncology as a Relevant Translational Model for Cancer Therapy.

Despite the important role of preclinical experiments to characterize tumor biology and molecular pathways, there are ongoing challenges to model the tumor microenvironment, specifically the dynamic interactions between tumor cells and immune infiltrates. Comprehensive models of host-tumor immune interactions will enhance the development of emerging treatment strategies, such as immunotherapies. Although in vitro and murine models are important for the early modelling of cancer and treatment-response mechanisms, comparative research studies involving veterinary oncology may bridge the translational pathway to human studies. The natural progression of several malignancies in animals exhibits similar pathogenesis to human cancers, and previous studies have shown a relevant and evaluable immune system. Veterinary oncologists working alongside oncologists and cancer researchers have the potential to advance discovery. Understanding the host-tumor-immune interactions can accelerate drug and biomarker discovery in a clinically relevant setting. This review presents discoveries in comparative immuno-oncology and implications to cancer therapy.

Spatial interplay of tissue hypoxia and T-cell regulation in ductal carcinoma in situ.

Hypoxia promotes aggressive tumor phenotypes and mediates the recruitment of suppressive T cells in invasive breast carcinomas. We investigated the role of hypoxia in relation to T-cell regulation in ductal carcinoma in situ (DCIS). We designed a deep learning system tailored for the tissue architecture complexity of DCIS, and compared pure DCIS cases with the synchronous DCIS and invasive components within invasive ductal carcinoma cases. Single-cell classification was applied in tandem with a new method for DCIS ductal segmentation in dual-stained CA9 and FOXP3, whole-tumor section digital pathology images. Pure DCIS typically has an intermediate level of colocalization of FOXP3+ and CA9+ cells, but in invasive carcinoma cases, the FOXP3+ (T-regulatory) cells may have relocated from the DCIS and into the invasive parts of the tumor, leading to high levels of colocalization in the invasive parts but low levels in the synchronous DCIS component. This may be due to invasive, hypoxic tumors evolving to recruit T-regulatory cells in order to evade immune predation. Our data support the notion that hypoxia promotes immune tolerance through recruitment of T-regulatory cells, and furthermore indicate a spatial pattern of relocalization of T-regulatory cells from DCIS to hypoxic tumor cells. Spatial colocalization of hypoxic and T-regulatory cells may be a key event and useful marker of DCIS progression.

High inter-follicular spatial co-localization of CD8+FOXP3+ with CD4+CD8+ cells predicts favorable outcome in follicular lymphoma.

The spatial architecture of the lymphoid tissue in follicular lymphoma (FL) presents unique challenges to studying its immune microenvironment. We investigated the spatial interplay of T cells, macrophages, myeloid cells and natural killer T cells using multispectral immunofluorescence images of diagnostic biopsies of 32 patients. A deep learning-based image analysis pipeline was tailored to the needs of follicular lymphoma spatial histology research, enabling the identification of different immune cells within and outside neoplastic follicles. We analyzed the density and spatial co-localization of immune cells in the inter-follicular and intra-follicular regions of follicular lymphoma. Low inter-follicular density of CD8+FOXP3+ cells and co-localization of CD8+FOXP3+ with CD4+CD8+ cells were significantly associated with relapse (p = 0.0057 and p = 0.0019, respectively) and shorter time to progression after first-line treatment (Logrank p = 0.0097 and log-rank p = 0.0093, respectively). A low inter-follicular density of CD8+FOXP3+ cells is associated with increased risk of relapse independent of follicular lymphoma international prognostic index (FLIPI) (p = 0.038, Hazard ratio (HR) = 0.42 [0.19, 0.95], but not independent of co-localization of CD8+FOXP3+ with CD4+CD8+ cells (p = 0.43). Co-localization of CD8+FOXP3+ with CD4+CD8+ cells is predictors of time to relapse independent of the FLIPI score and density of CD8+FOXP3+ cells (p = 0.027, HR = 0.0019 [7.19 × 10-6 , 0.49], This suggests a potential role of inter-follicular CD8+FOXP3+ and CD4+CD8+ cells in the disease progression of FL, warranting further validation on larger patient cohorts.

Emerging technologies and their impact on regulatory science.

There is an evolution and increasing need for the utilization of emerging cellular, molecular and in silico technologies and novel approaches for safety assessment of food, drugs, and personal care products. Convergence of these emerging technologies is also enabling rapid advances and approaches that may impact regulatory decisions and approvals. Although the development of emerging technologies may allow rapid advances in regulatory decision making, there is concern that these new technologies have not been thoroughly evaluated to determine if they are ready for regulatory application, singularly or in combinations. The magnitude of these combined technical advances may outpace the ability to assess fit for purpose and to allow routine application of these new methods for regulatory purposes. There is a need to develop strategies to evaluate the new technologies to determine which ones are ready for regulatory use. The opportunity to apply these potentially faster, more accurate, and cost-effective approaches remains an important goal to facilitate their incorporation into regulatory use. However, without a clear strategy to evaluate emerging technologies rapidly and appropriately, the value of these efforts may go unrecognized or may take longer. It is important for the regulatory science field to keep up with the research in these technically advanced areas and to understand the science behind these new approaches. The regulatory field must understand the critical quality attributes of these novel approaches and learn from each other's experience so that workforces can be trained to prepare for emerging global regulatory challenges. Moreover, it is essential that the regulatory community must work with the technology developers to harness collective capabilities towards developing a strategy for evaluation of these new and novel assessment tools.

Unmasking the immune microecology of ductal carcinoma in situ with deep learning.

Despite increasing evidence supporting the clinical relevance of tumour infiltrating lymphocytes (TILs) in invasive breast cancer, TIL spatial variability within ductal carcinoma in situ (DCIS) samples and its association with progression are not well understood. To characterise tissue spatial architecture and the microenvironment of DCIS, we designed and validated a new deep learning pipeline, UNMaSk. Following automated detection of individual DCIS ducts using a new method IM-Net, we applied spatial tessellation to create virtual boundaries for each duct. To study local TIL infiltration for each duct, DRDIN was developed for mapping the distribution of TILs. In a dataset comprising grade 2-3 pure DCIS and DCIS adjacent to invasive cancer (adjacent DCIS), we found that pure DCIS cases had more TILs compared to adjacent DCIS. However, the colocalisation of TILs with DCIS ducts was significantly lower in pure DCIS compared to adjacent DCIS, which may suggest a more inflamed tissue ecology local to DCIS ducts in adjacent DCIS cases. Our study demonstrates that technological developments in deep convolutional neural networks and digital pathology can enable an automated morphological and microenvironmental analysis of DCIS, providing a new way to study differential immune ecology for individual ducts and identify new markers of progression.

Artificial intelligence and digital pathology: Opportunities and implications for immuno-oncology.

The field of immuno-oncology has expanded rapidly over the past decade, but key questions remain. How does tumour-immune interaction regulate disease progression? How can we prospectively identify patients who will benefit from immunotherapy? Identifying measurable features of the tumour immune-microenvironment which have prognostic or predictive value will be key to making meaningful gains in these areas. Recent developments in deep learning enable big-data analysis of pathological samples. Digital approaches allow data to be acquired, integrated and analysed far beyond what is possible with conventional techniques, and to do so efficiently and at scale. This has the potential to reshape what can be achieved in terms of volume, precision and reliability of output, enabling data for large cohorts to be summarised and compared. This review examines applications of artificial intelligence (AI) to important questions in immuno-oncology (IO). We discuss general considerations that need to be taken into account before AI can be applied in any clinical setting. We describe AI methods that have been applied to the field of IO to date and present several examples of their use.

Biomarkers for site-specific response to neoadjuvant chemotherapy in epithelial ovarian cancer: relating MRI changes to tumour cell load and necrosis.

BACKGROUND: Diffusion-weighted magnetic resonance imaging (DW-MRI) potentially interrogates site-specific response to neoadjuvant chemotherapy (NAC) in epithelial ovarian cancer (EOC). METHODS: Participants with newly diagnosed EOC due for platinum-based chemotherapy and interval debulking surgery were recruited prospectively in a multicentre study (n = 47 participants). Apparent diffusion coefficient (ADC) and solid tumour volume (up to 10 lesions per participant) were obtained from DW-MRI before and after NAC (including double-baseline for repeatability assessment in n = 19). Anatomically matched lesions were analysed after surgical excision (65 lesions obtained from 25 participants). A trained algorithm determined tumour cell fraction, percentage tumour and percentage necrosis on histology. Whole-lesion post-NAC ADC and pre/post-NAC ADC changes were compared with histological metrics (residual tumour/necrosis) for each tumour site (ovarian, omental, peritoneal, lymph node). RESULTS: Tumour volume reduced at all sites after NAC. ADC increased between pre- and post-NAC measurements. Post-NAC ADC correlated negatively with tumour cell fraction. Pre/post-NAC changes in ADC correlated positively with percentage necrosis. Significant correlations were driven by peritoneal lesions. CONCLUSIONS: Following NAC in EOC, the ADC (measured using DW-MRI) increases differentially at disease sites despite similar tumour shrinkage, making its utility site-specific. After NAC, ADC correlates negatively with tumour cell fraction; change in ADC correlates positively with percentage necrosis. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov NCT01505829.

The T cell differentiation landscape is shaped by tumour mutations in lung cancer.

Tumour mutational burden (TMB) predicts immunotherapy outcome in non-small cell lung cancer (NSCLC), consistent with immune recognition of tumour neoantigens. However, persistent antigen exposure is detrimental for T cell function. How TMB affects CD4 and CD8 T cell differentiation in untreated tumours, and whether this affects patient outcomes is unknown. Here we paired high-dimensional flow cytometry, exome, single-cell and bulk RNA sequencing from patients with resected, untreated NSCLC to examine these relationships. TMB was associated with compartment-wide T cell differentiation skewing, characterized by loss of TCF7-expressing progenitor-like CD4 T cells, and an increased abundance of dysfunctional CD8 and CD4 T cell subsets, with significant phenotypic and transcriptional similarity to neoantigen-reactive CD8 T cells. A gene signature of redistribution from progenitor-like to dysfunctional states associated with poor survival in lung and other cancer cohorts. Single-cell characterization of these populations informs potential strategies for therapeutic manipulation in NSCLC.

Immune Surveillance in Clinical Regression of Preinvasive Squamous Cell Lung Cancer.

Before squamous cell lung cancer develops, precancerous lesions can be found in the airways. From longitudinal monitoring, we know that only half of such lesions become cancer, whereas a third spontaneously regress. Although recent studies have described the presence of an active immune response in high-grade lesions, the mechanisms underpinning clinical regression of precancerous lesions remain unknown. Here, we show that host immune surveillance is strongly implicated in lesion regression. Using bronchoscopic biopsies from human subjects, we find that regressive carcinoma in situ lesions harbor more infiltrating immune cells than those that progress to cancer. Moreover, molecular profiling of these lesions identifies potential immune escape mechanisms specifically in those that progress to cancer: antigen presentation is impaired by genomic and epigenetic changes, CCL27-CCR10 signaling is upregulated, and the immunomodulator TNFSF9 is downregulated. Changes appear intrinsic to the carcinoma in situ lesions, as the adjacent stroma of progressive and regressive lesions are transcriptomically similar. SIGNIFICANCE: Immune evasion is a hallmark of cancer. For the first time, this study identifies mechanisms by which precancerous lesions evade immune detection during the earliest stages of carcinogenesis and forms a basis for new therapeutic strategies that treat or prevent early-stage lung cancer.See related commentary by Krysan et al., p. 1442.This article is highlighted in the In This Issue feature, p. 1426.

Noninvasive MRI Native T1 Mapping Detects Response to MYCN-targeted Therapies in the Th-MYCN Model of Neuroblastoma.

Noninvasive early indicators of treatment response are crucial to the successful delivery of precision medicine in children with cancer. Neuroblastoma is a common solid tumor of young children that arises from anomalies in neural crest development. Therapeutic approaches aiming to destabilize MYCN protein, such as small-molecule inhibitors of Aurora A and mTOR, are currently being evaluated in early phase clinical trials in children with high-risk MYCN-driven disease, with limited ability to evaluate conventional pharmacodynamic biomarkers of response. T1 mapping is an MRI scan that measures the proton spin-lattice relaxation time T1. Using a multiparametric MRI-pathologic cross-correlative approach and computational pathology methodologies including a machine learning-based algorithm for the automatic detection and classification of neuroblasts, we show here that T1 mapping is sensitive to the rich histopathologic heterogeneity of neuroblastoma in the Th-MYCN transgenic model. Regions with high native T1 corresponded to regions dense in proliferative undifferentiated neuroblasts, whereas regions characterized by low T1 were rich in apoptotic or differentiating neuroblasts. Reductions in tumor-native T1 represented a sensitive biomarker of response to treatment-induced apoptosis with two MYCN-targeted small-molecule inhibitors, Aurora A kinase inhibitor alisertib (MLN8237) and mTOR inhibitor vistusertib (AZD2014). Overall, we demonstrate the potential of T1 mapping, a scan readily available on most clinical MRI scanners, to assess response to therapy and guide clinical trials for children with neuroblastoma. The study reinforces the potential role of MRI-based functional imaging in delivering precision medicine to children with neuroblastoma. SIGNIFICANCE: This study shows that MRI-based functional imaging can detect apoptotic responses to MYCN-targeted small-molecule inhibitors in a genetically engineered murine model of MYCN-driven neuroblastoma.

Geospatial immune variability illuminates differential evolution of lung adenocarcinoma.

Remarkable progress in molecular analyses has improved our understanding of the evolution of cancer cells toward immune escape1-5. 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 multiregion exome and RNA-sequencing (RNA-seq) data with spatial histology mapped by deep learning in 100 patients with non-small cell lung cancer from the TRACERx cohort6. 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 multisample cohort of 970 patients with lung adenocarcinoma, tumors with more than one immune cold region had a higher risk of relapse, independently of tumor size, stage and number of samples per patient. In lung adenocarcinoma, but not lung squamous cell carcinoma, 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.