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Immunotherapy with checkpoint blockade induces rapid and durable immune control of cancer in some patients and has driven a monumental shift in cancer treatment. Neoantigen-specific CD8+ T cells are at the forefront of current immunotherapy strategies, and the majority of drug discovery and clinical trials revolve around further harnessing these immune effectors. Yet the immune system contains a diverse range of antitumour effector cells, and these must function in a coordinated and synergistic manner to overcome the immune-evasion mechanisms used by tumours and achieve complete control with tumour eradication. A key antitumour effector is the natural killer (NK) cells, cytotoxic innate lymphocytes present at high frequency in the circulatory system and identified by their exquisite ability to spontaneously detect and lyse transformed or stressed cells. Emerging data show a role for intratumoural NK cells in driving immunotherapy response and, accordingly, there have been renewed efforts to further elucidate and target the pathways controlling NK cell antitumour function. In this Review, we discuss recent clinical evidence that NK cells are a key immune constituent in the protective antitumour immune response and highlight the major stages of the cancer–NK cell immunity cycle. We also perform a new analysis of publicly available transcriptomic data to provide an overview of the prognostic value of NK cell gene expression in 25 tumour types. Furthermore, we discuss how the role of NK cells evolves with tumour progression, presenting new opportunities to target NK cell function to enhance cancer immunotherapy response rates across a more diverse range of cancers.This Review discusses the key role that natural killer (NK) cells play in driving an antitumour immune response throughout the progression of cancer from its initial development to its metastatic spread and eventual treatment, defined herein as the cancer–NK cell immunity cycle.

Metastatic colorectal cancer (CRC) with a mismatch repair-deficiency (MMR-D)/microsatellite instability-high (MSI-H) phenotype carries unique characteristics such as increased tumour mutational burden and tumour-infiltrating lymphocytes. Studies have shown a sustained clinical response to immune checkpoint inhibitors with dramatic clinical improvement in patients with MSI-H/MMR-D CRC. However, the observed response rates range between 30% and 50% suggesting the existence of intrinsic resistance mechanisms. Moreover, disease progression after an initial positive response to immune checkpoint inhibitor treatment points to acquired resistance mechanisms. In this review article, we discuss the clinical trials that established the efficacy of immune checkpoint inhibitors in patients with MSI-H/MMR-D CRC, consider biomarkers of the immune response and elaborate on potential mechanisms related to intrinsic and acquired resistance. We also provide a perspective on possible future therapeutic approaches that might improve clinical outcomes, particularly in patients with actionable resistance mechanisms.

Assays that measure steroid hormones in patient care, public health, and research need to be both accurate and precise, as these criteria help to ensure comparability across all clinical and research applications. This review addresses major issues relevant to assay variability and describes recent activities by the US Centers for Disease Control and Prevention (CDC) to improve assay performance. Currently, high degrees of accuracy and precision are not always met for testosterone and estradiol measurements; although technologies for steroid hormone measurement have advanced significantly, measurement variability within and across laboratories has not improved accordingly. Differences in calibration and specificity are discussed as sources of variability in measurement accuracy. Ultimately, a combination of factors appears to cause inaccuracy of steroid hormone measurements, with nonuniform assay calibration and lack of specificity being two major contributors to assay variability. Within-assay variability for current assays is generally high, especially at low analyte concentrations. The CDC Hormone Standardization (HoSt) Program is improving clinical assays, as evidenced by a 50% decline in mean absolute bias between mass spectrometry assays and the CDC reference method from 2007 to 2011. This program provides the measurement traceability to CDC reference methods and helps to minimize factors affecting measurement variability.

Gastric-type cervical adenocarcinoma (GCA) is an aggressive type of endocervical adenocarcinoma characterized by mucinous morphology, gastric-type mucin, lack of association with human papillomavirus (HPV) and resistance to chemo/radiotherapy. We characterized the landscape of genetic alterations in a large cohort of GCAs, and compared it with that of usual-type HPV-associated endocervical adenocarcinomas (UEAs), pancreatic adenocarcinomas (PAs) and intestinal-type gastric adenocarcinomas (IGAs). GCAs (n = 68) were subjected to massively parallel sequencing targeting 410–468 cancer-related genes. Somatic mutations and copy number alterations (CNAs) were determined using validated bioinformatics methods. Mutational data for UEAs (n = 21), PAs (n = 178), and IGAs (n = 148) from The Cancer Genome Atlas (TCGA) were obtained from cBioPortal. GCAs most frequently harbored somatic mutations in TP53 (41%), CDKN2A (18%), KRAS (18%), and STK11 (10%). Potentially targetable mutations were identified in ERBB3 (10%), ERBB2 (8%), and BRAF (4%). GCAs displayed low levels of CNAs with no recurrent amplifications or homozygous deletions. In contrast to UEAs, GCAs harbored more frequent mutations affecting cell cycle-related genes including TP53 (41% vs 5%, p < 0.01) and CDKN2A (18% vs 0%, p = 0.01), and fewer PIK3CA mutations (7% vs 33%, p = 0.01). TP53 mutations were less prevalent in GCAs compared to PAs (41% vs 56%, p < 0.05) and IGAs (41% vs 57%, p < 0.05). GCAs showed a higher frequency of STK11 mutations than PAs (10% vs 2%, p < 0.05) and IGAs (10% vs 1%, p < 0.05). GCAs harbored more frequent mutations in ERBB2 and ERBB3 (9% vs 1%, and 10% vs 0.5%, both p < 0.01) compared to PAs, and in CDKN2A (18% vs 1%, p < 0.05) and KRAS (18% vs 6%, p < 0.05) compared to IGAs. GCAs harbor recurrent somatic mutations in cell cycle-related genes and in potentially targetable genes, including ERBB2/3. Mutations in genes such as STK11 may be used as supportive evidence to help distinguish GCAs from other adenocarcinomas with similar morphology in metastatic sites.

Breast cancer remains a leading cause of cancer mortality worldwide, underscoring the urgent need for novel and effective therapeutic strategies. Eph receptor tyrosine kinases, particularly EphB4, exhibit diverse roles in cancer biology, acting as either tumor promoters or suppressors depending on the cellular environment and ligand engagement. EphB4 is frequently overexpressed in breast cancer and contributes to dysregulated signaling and tumor progression through the abnormal interaction with its ligand Ephrin‐B2. We herein developed an improved anti‐EphB4 monoclonal antibody, Eb4Mab‐7‐mG2a, which can be characterized as a subclass‐switched IgG2a variant designed to enhance immune effector function, specifically antibody‐dependent cellular cytotoxicity (ADCC) and complement‐dependent cytotoxicity (CDC). Our findings showed that Eb4Mab‐7‐mG2a effectively blocked Ephrin‐B2‐induced ERK phosphorylation and proliferation in EphB4‐positive MCF‐7 breast cancer cells but had no effect on EphB4‐knockout (KO) MCF‐7 cells. Flow cytometry confirmed high‐affinity binding between Eb4Mab‐7‐mG2a and EphB4‐expressing cells, whereas in vitro assays demonstrated potent and selective ADCC and CDC activities against EphB4‐positive tumor cells. In vivo experiments showed that Eb4Mab‐7‐mG2a significantly suppressed xenograft growth in models bearing EphB4‐overexpressing CHO‐K1 and EphB4‐positive MCF‐7, but showed no therapeutic effect in EphB4‐negative CHO‐K1 and EphB4‐KO MCF‐7 xenografts. Immunohistochemical analysis revealed reduced Ki‐67 proliferation indices in treated tumors, supporting the antiproliferative effects of the developed antibody. Overall, these findings demonstrate that Eb4Mab‐7‐mG2a exerts dual‐action antitumor activity through ligand blockade and immune effector engagement. Further evaluations in other EphB4‐overexpressing cancers and in combination with immune checkpoint inhibitors are warranted. Humanization and tumor‐selective engineering may enhance its clinical potential for precision oncology. Eb4Mab‐7‐mG2a, an anti‐EphB4 monoclonal antibody, significantly suppressed tumor growth in the MCF‐7 xenograft model. Tumor volume and weight were markedly reduced in treated mice, demonstrating its antitumor efficacy against EphB4‐positive breast cancer.

The expression profiles and spatial distributions of RNAs regulate many cellular functions. Image-based transcriptomic approaches provide powerful means to measure both expression and spatial information of RNAs in individual cells within their native environment. Among these approaches, multiplexed error-robust fluorescence in situ hybridization (MERFISH) has achieved spatially resolved RNA quantification at transcriptome scale by massively multiplexing single-molecule FISH measurements. Here, we increased the gene throughput of MERFISH and demonstrated simultaneous measurements of RNA transcripts from ∼10,000 genes in individual cells with ∼80% detection efficiency and ∼4% misidentification rate. We combined MERFISH with cellular structure imaging to determine subcellular compartmentalization of RNAs. We validated this approach by showing enrichment of secretome transcripts at the endoplasmic reticulum, and further revealed enrichment of long noncoding RNAs, RNAs with retained introns, and a subgroup of protein-coding mRNAs in the cell nucleus. Leveraging spatially resolved RNA profiling, we developed an approach to determine RNA velocity in situ using the balance of nuclear versus cytoplasmic RNA counts. We applied this approach to infer pseudotime ordering of cells and identified cells at different cell-cycle states, revealing ∼1,600 genes with putative cell cycle-dependent expression and a gradual transcription profile change as cells progress through cell-cycle stages. Our analysis further revealed cell cycle-dependent and cell cycle-independent spatial heterogeneity of transcriptionally distinct cells. We envision that the ability to perform spatially resolved, genome-wide RNA profiling with high detection efficiency and accuracy by MERFISH could help address a wide array of questions ranging from the regulation of gene expression in cells to the development of cell fate and organization in tissues.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified as the etiologic agent associated with coronavirus disease, which emerged in late 2019. In response, we developed a diagnostic panel consisting of 3 real-time reverse transcription PCR assays targeting the nucleocapsid gene and evaluated use of these assays for detecting SARS-CoV-2 infection. All assays demonstrated a linear dynamic range of 8 orders of magnitude and an analytical limit of detection of 5 copies/reaction of quantified RNA transcripts and 1 x 10 50% tissue culture infectious dose/mL of cell-cultured SARS-CoV-2. All assays performed comparably with nasopharyngeal and oropharyngeal secretions, serum, and fecal specimens spiked with cultured virus. We obtained no false-positive amplifications with other human coronaviruses or common respiratory pathogens. Results from all 3 assays were highly correlated during clinical specimen testing. On February 4, 2020, the Food and Drug Administration issued an Emergency Use Authorization to enable emergency use of this panel.

The focus of tumour-specific antigen analyses has been on single nucleotide variants (SNVs), with the contribution of small insertions and deletions (indels) less well characterised. We investigated whether the frameshift nature of indel mutations, which create novel open reading frames and a large quantity of mutagenic peptides highly distinct from self, might contribute to the immunogenic phenotype. We analysed whole-exome sequencing data from 5777 solid tumours, spanning 19 cancer types from The Cancer Genome Atlas. We compared the proportion and number of indels across the cohort, with a subset of results replicated in two independent datasets. We assessed in-silico tumour-specific neoantigen predictions by mutation type with pan-cancer analysis, together with RNAseq profiling in renal clear cell carcinoma cases (n=392), to compare immune gene expression across patient subgroups. Associations between indel burden and treatment response were assessed across four checkpoint inhibitor datasets. We observed renal cell carcinomas to have the highest proportion (0·12) and number of indel mutations across the pan-cancer cohort (p<2·2 × 10−16), more than double the median proportion of indel mutations in all other cancer types examined. Analysis of tumour-specific neoantigens showed that enrichment of indel mutations for high-affinity binders was three times that of non-synonymous SNV mutations. Furthermore, neoantigens derived from indel mutations were nine times enriched for mutant specific binding, as compared with non-synonymous SNV derived neoantigens. Immune gene expression analysis in the renal clear cell carcinoma cohort showed that the presence of mutant-specific neoantigens was associated with upregulation of antigen presentation genes, which correlated (r=0·78) with T-cell activation as measured by CD8-positive expression. Finally, analysis of checkpoint inhibitor response data revealed frameshift indel count to be significantly associated with checkpoint inhibitor response across three separate melanoma cohorts (p=4·7 × 10−4). Renal cell carcinomas have the highest pan-cancer proportion and number of indel mutations. Evidence suggests indels are a highly immunogenic mutational class, which can trigger an increased abundance of neoantigens and greater mutant-binding specificity. Cancer Research UK, UK National Institute for Health Research (NIHR) at the Royal Marsden Hospital National Health Service Foundation Trust, Institute of Cancer Research and University College London Hospitals Biomedical Research Centres, the UK Medical Research Council, the Rosetrees Trust, Novo Nordisk Foundation, the Prostate Cancer Foundation, the Breast Cancer Research Foundation, the European Research Council.

The Cyclin-dependent kinase 1, as a serine/threonine protein kinase, is more than a cell cycle regulator as it was originally identified. During the last decade, it has been shown to carry out versatile functions during the last decade. From cell cycle control to gene expression regulation and apoptosis, CDK1 is intimately involved in many cellular events that are vital for cell survival. Here, we provide a comprehensive catalogue of the CDK1 upstream regulators and substrates, describing how this kinase is implicated in the control of key ‘cell cycle-unrelated’ biological processes. Finally, we describe how deregulation of CDK1 expression and activation has been closely associated with cancer progression and drug resistance.