Liquid biopsy-based circulating tumour (ct)DNA analysis of a spectrum of myeloid and lymphoid malignancies yields clinically actionable results.

AIMS: Liquid biopsy (LBx)-based next-generation sequencing (NGS) of circulating tumour DNA (ctDNA) can facilitate molecular profiling of haematopoietic neoplasms (HNs), particularly when tissue-based NGS is infeasible. METHODS AND RESULTS: We studied HN LBx samples tested with FoundationOne Liquid CDx, FoundationOne Liquid, or FoundationACT between July 2016 and March 2022. We identified 271 samples: 89 non-Hodgkin lymphoma (NHL), 43 plasma-cell neoplasm (PCN), 41 histiocytoses, 27 myelodysplastic syndrome (MDS), 25 diffuse large B-cell lymphoma (DLBCL), 22 myeloproliferative neoplasm (MPN), 14 Hodgkin lymphoma (HL), and 10 acute myeloid leukaemia (AML). Among 73.4% with detectable pathogenic alterations, median maximum somatic allele frequency (MSAF) was 16.6%, with AML (36.2%), MDS (19.7%), and MPN (44.5%) having higher MSAFs than DLBCL (3.9%), NHL (8.4%), HL (1.5%), PCN (2.8%), and histiocytoses (1.8%) (P = 0.001). LBx detected characteristic alterations across HNs, including in TP53, KRAS, MYD88, and BTK in NHLs; TP53, KRAS, NRAS, and BRAF in PCNs; IGH in DLBCL; TP53, ATM, and PDCD1LG2 in HL; BRAF and MAP2K1 in histiocytoses; TP53, SF3B1, DNMT3A, TET2, and ASXL1 in MDS; JAK2 in MPNs; and FLT3, IDH2, and NPM1 in AML. Among 24 samples, the positive percent agreement by LBx was 75.7% for variants present in paired buffy coat, marrow, or tissues. Also, 75.0% of pairs exhibited alterations only present on LBx. These were predominantly subclonal (clonal fraction of 3.8%), reflecting the analytical sensitivity of LBx. CONCLUSION: These data demonstrate that LBx can detect relevant genomic alterations across HNs, including at low clonal fractions, suggesting a potential clinical utility for identifying residual or emerging therapy-resistant clones that may be undetectable in site-specific tissue biopsies.

LambdaPP: Fast and accessible protein-specific phenotype predictions.

The availability of accurate and fast artificial intelligence (AI) solutions predicting aspects of proteins are revolutionizing experimental and computational molecular biology. The webserver LambdaPP aspires to supersede PredictProtein, the first internet server making AI protein predictions available in 1992. Given a protein sequence as input, LambdaPP provides easily accessible visualizations of protein 3D structure, along with predictions at the protein level (GeneOntology, subcellular location), and the residue level (binding to metal ions, small molecules, and nucleotides; conservation; intrinsic disorder; secondary structure; alpha-helical and beta-barrel transmembrane segments; signal-peptides; variant effect) in seconds. The structure prediction provided by LambdaPP-leveraging ColabFold and computed in minutes-is based on MMseqs2 multiple sequence alignments. All other feature prediction methods are based on the pLM ProtT5. Queried by a protein sequence, LambdaPP computes protein and residue predictions almost instantly for various phenotypes, including 3D structure and aspects of protein function. LambdaPP is freely available for everyone to use under embed.predictprotein.org, the interactive results for the case study can be found under https://embed.predictprotein.org/o/Q9NZC2. The frontend of LambdaPP can be found on GitHub (github.com/sacdallago/embed.predictprotein.org), and can be freely used and distributed under the academic free use license (AFL-2). For high-throughput applications, all methods can be executed locally via the bio-embeddings (bioembeddings.com) python package, or docker image at ghcr.io/bioembeddings/bio_embeddings, which also includes the backend of LambdaPP.

Therapeutically Increasing MHC-I Expression Potentiates Immune Checkpoint Blockade.

Immune checkpoint blockade (ICB) therapy revolutionized cancer treatment, but many patients with impaired MHC-I expression remain refractory. Here, we combined FACS-based genome-wide CRISPR screens with a data-mining approach to identify drugs that can upregulate MHC-I without inducing PD-L1. CRISPR screening identified TRAF3, a suppressor of the NFκB pathway, as a negative regulator of MHC-I but not PD-L1. The Traf3-knockout gene expression signature is associated with better survival in ICB-naïve patients with cancer and better ICB response. We then screened for drugs with similar transcriptional effects as this signature and identified Second Mitochondria-derived Activator of Caspase (SMAC) mimetics. We experimentally validated that the SMAC mimetic birinapant upregulates MHC-I, sensitizes cancer cells to T cell-dependent killing, and adds to ICB efficacy. Our findings provide preclinical rationale for treating tumors expressing low MHC-I expression with SMAC mimetics to enhance sensitivity to immunotherapy. The approach used in this study can be generalized to identify other drugs that enhance immunotherapy efficacy. SIGNIFICANCE: MHC-I loss or downregulation in cancer cells is a major mechanism of resistance to T cell-based immunotherapies. Our study reveals that birinapant may be used for patients with low baseline MHC-I to enhance ICB response. This represents promising immunotherapy opportunities given the biosafety profile of birinapant from multiple clinical trials.This article is highlighted in the In This Issue feature, p. 1307.

Mapping the Relative Biological Effectiveness of Proton, Helium and Carbon Ions with High-Throughput Techniques.

Large amounts of high quality biophysical data are needed to improve current biological effects models but such data are lacking and difficult to obtain. The present study aimed to more efficiently measure the spatial distribution of relative biological effectiveness (RBE) of charged particle beams using a novel high-accuracy and high-throughput experimental platform. Clonogenic survival was selected as the biological endpoint for two lung cancer cell lines, H460 and H1437, irradiated with protons, carbon, and helium ions. Ion-specific multi-step microplate holders were fabricated such that each column of a 96-well microplate is spatially situated at a different location along a particle beam path. Dose, dose-averaged linear energy transfer (LETd), and dose-mean lineal energy (yd) were calculated using an experimentally validated Geant4-based Monte Carlo system. Cells were irradiated at the Heidelberg Ion Beam Therapy Center (HIT). The experimental results showed that the clonogenic survival curves of all tested ions were yd-dependent. Both helium and carbon ions achieved maximum RBEs within specific yd ranges before biological efficacy declined, indicating an overkill effect. For protons, no overkill was observed, but RBE increased distal to the Bragg peak. Measured RBE profiles strongly depend on the physical characteristics such as yd and are ion specific.

Clonal tracing reveals diverse patterns of response to immune checkpoint blockade.

BACKGROUND: Immune checkpoint blockade (ICB) therapy has improved patient survival in a variety of cancers, but only a minority of cancer patients respond. Multiple studies have sought to identify general biomarkers of ICB response, but elucidating the molecular and cellular drivers of resistance for individual tumors remains challenging. We sought to determine whether a tumor with defined genetic background exhibits a stereotypic or heterogeneous response to ICB treatment. RESULTS: We establish a unique mouse system that utilizes clonal tracing and mathematical modeling to monitor the growth of each cancer clone, as well as the bulk tumor, in response to ICB. We find that tumors derived from the same clonal populations showed heterogeneous ICB response and diverse response patterns. Primary response is associated with higher immune infiltration and leads to enrichment of pre-existing ICB-resistant cancer clones. We further identify several cancer cell-intrinsic gene expression signatures associated with ICB resistance, including increased interferon response genes and glucocorticoid response genes. These findings are supported by clinical data from ICB treatment cohorts. CONCLUSIONS: Our study demonstrates diverse response patterns from the same ancestor cancer cells in response to ICB. This suggests the value of monitoring clonal constitution and tumor microenvironment over time to optimize ICB response and to design new combination therapies. Furthermore, as ICB response may enrich for cancer cell-intrinsic resistance signatures, this can affect interpretations of tumor RNA-seq data for response-signature association studies.

CCL5 promotes breast cancer recurrence through macrophage recruitment in residual tumors.

Over half of breast-cancer-related deaths are due to recurrence 5 or more years after initial diagnosis and treatment. This latency suggests that a population of residual tumor cells can survive treatment and persist in a dormant state for many years. The role of the microenvironment in regulating the survival and proliferation of residual cells following therapy remains unexplored. Using a conditional mouse model for Her2-driven breast cancer, we identify interactions between residual tumor cells and their microenvironment as critical for promoting tumor recurrence. Her2 downregulation leads to an inflammatory program driven by TNFα/NFκB signaling, which promotes immune cell infiltration in regressing and residual tumors. The cytokine CCL5 is elevated following Her2 downregulation and remains high in residual tumors. CCL5 promotes tumor recurrence by recruiting CCR5-expressing macrophages, which may contribute to collagen deposition in residual tumors. Blocking this TNFα-CCL5-macrophage axis may be efficacious in preventing breast cancer recurrence.

RBE Model-Based Biological Dose Optimization for Proton Radiobiology Studies.

PURPOSE: The purpose of the current study was (1) to develop a straightforward and rapid method to incorporate a dose-averaged linear energy transfer (LET d )-based biological effect model into a dose optimization algorithm for scanned protons; and (2) to apply a novel beam delivery strategy with increased LET d within the target, thereby enhancing the biological effect predicted using the selected relative biological effectiveness (RBE) model. MATERIALS AND METHODS: We first generated pristine dose Bragg curves in water and their corresponding LET d distributions for 94 groups of proton beams, using experimentally validated Geant4 Monte Carlo simulations. Next, we developed 1-dimensional dose optimization algorithms by using the Python programming language. To calculate the RBE of protons for biological dose optimization, we invoked the McNamara RBE model and applied the radiobiological parameters of the lung cancer H460 cell line with 137Cs reference photons. RESULTS: High-accuracy optimization results were obtained. The relative difference between the delivered dose and the prescribed dose was approximately within ±1.0% in the target. In addition, we obtained the RBE enhancement within the target by applying the LET-painting technique. For example, considering a simple case in which 2 opposed downslope dose fields were superimposed to form a uniform dose in the 5- to 10-cm target region, the center RBE was 1.23 ± 0.01, which was greater than the center RBE of 1.16 ± 0.01 found when using the traditional method of delivering 2 opposed flat dose fields. CONCLUSION: We have successfully developed an easy-to-implement method to perform the biological dose optimization procedure by invoking the McNamara RBE model in the iteration process using the Python programming language. According to the RBE model predictions, we conclude that the increased target LET d enhances the RBE. The accuracy of the RBE model predictions needs to be validated in radiobiological experiments.

Foxo-dependent Par-4 Upregulation Prevents Long-term Survival of Residual Cells Following PI3K-Akt Inhibition.

Tumor recurrence is a leading cause of death and is thought to arise from a population of residual cells that survive treatment. These residual cancer cells can persist, locally or at distant sites, for years or decades. Therefore, understanding the pathways that regulate residual cancer cell survival may suggest opportunities for targeting these cells to prevent recurrence. Previously, it was observed that the proapoptotic protein (PAWR/Par-4) negatively regulates residual cell survival and recurrence in mice and humans. However, the mechanistic underpinnings on how Par-4 expression is regulated are unclear. Here, it is demonstrated that Par-4 is transcriptionally upregulated following treatment with multiple drugs targeting the PI3K-Akt-mTOR signaling pathway, and identify the Forkhead family of transcription factors as mediators of this upregulation. Mechanistically, Foxo3a directly binds to the Par-4 promoter and activates its transcription following inhibition of the PI3K-Akt pathway. This Foxo-dependent Par-4 upregulation limits the long-term survival of residual cells following treatment with therapeutics that target the PI3K-Akt pathway. Taken together, these results indicate that residual breast cancer tumor cell survival and recurrence requires circumventing Foxo-driven Par-4 upregulation and suggest that approaches to enforce Par-4 expression may prevent residual cell survival and recurrence. Mol Cancer Res; 16(4); 599-609. ©2018 AACR.