Stromal cell diversity associated with immune evasion in human triple-negative breast cancer.

The tumour stroma regulates nearly all stages of carcinogenesis. Stromal heterogeneity in human triple-negative breast cancers (TNBCs) remains poorly understood, limiting the development of stromal-targeted therapies. Single-cell RNA sequencing of five TNBCs revealed two cancer-associated fibroblast (CAF) and two perivascular-like (PVL) subpopulations. CAFs clustered into two states: the first with features of myofibroblasts and the second characterised by high expression of growth factors and immunomodulatory molecules. PVL cells clustered into two states consistent with a differentiated and immature phenotype. We showed that these stromal states have distinct morphologies, spatial relationships and functional properties in regulating the extracellular matrix. Using cell signalling predictions, we provide evidence that stromal-immune crosstalk acts via a diverse array of immunoregulatory molecules. Importantly, the investigation of gene signatures from inflammatory-CAFs and differentiated-PVL cells in independent TNBC patient cohorts revealed strong associations with cytotoxic T-cell dysfunction and exclusion, respectively. Such insights present promising candidates to further investigate for new therapeutic strategies in the treatment of TNBCs.

A single-cell tumor immune atlas for precision oncology.

The tumor immune microenvironment is a main contributor to cancer progression and a promising therapeutic target for oncology. However, immune microenvironments vary profoundly between patients, and biomarkers for prognosis and treatment response lack precision. A comprehensive compendium of tumor immune cells is required to pinpoint predictive cellular states and their spatial localization. We generated a single-cell tumor immune atlas, jointly analyzing published data sets of >500,000 cells from 217 patients and 13 cancer types, providing the basis for a patient stratification based on immune cell compositions. Projecting immune cells from external tumors onto the atlas facilitated an automated cell annotation system. To enable in situ mapping of immune populations for digital pathology, we applied SPOTlight, combining single-cell and spatial transcriptomics data and identifying colocalization patterns of immune, stromal, and cancer cells in tumor sections. We expect the tumor immune cell atlas, together with our versatile toolbox for precision oncology, to advance currently applied stratification approaches for prognosis and immunotherapy.

Prostate cancer cell-intrinsic interferon signaling regulates dormancy and metastatic outgrowth in bone.

The latency associated with bone metastasis emergence in castrate-resistant prostate cancer is attributed to dormancy, a state in which cancer cells persist prior to overt lesion formation. Using single-cell transcriptomics and ex vivo profiling, we have uncovered the critical role of tumor-intrinsic immune signaling in the retention of cancer cell dormancy. We demonstrate that loss of tumor-intrinsic type I IFN occurs in proliferating prostate cancer cells in bone. This loss suppresses tumor immunogenicity and therapeutic response and promotes bone cell activation to drive cancer progression. Restoration of tumor-intrinsic IFN signaling by HDAC inhibition increased tumor cell visibility, promoted long-term antitumor immunity, and blocked cancer growth in bone. Key findings were validated in patients, including loss of tumor-intrinsic IFN signaling and immunogenicity in bone metastases compared to primary tumors. Data herein provide a rationale as to why current immunotherapeutics fail in bone-metastatic prostate cancer, and provide a new therapeutic strategy to overcome the inefficacy of immune-based therapies in solid cancers.

Fluorescence microscopy colocalization of lipid-nucleic acid nanoparticles with wildtype and mutant Rab5-GFP: A platform for investigating early endosomal events.

Endosomal entrapment is known to be a major bottleneck to successful cytoplasmic delivery of nucleic acids (NAs) using cationic liposome-NA nanoparticles (NPs). Quantitative measurements of distributions of NPs within early endosomes (EEs) have proven difficult due to the sub-resolution size and short lifetime of wildtype EEs. In this study we used Rab5-GFP, a member of the large family of GTPases which cycles between the plasma membrane and early endosomes, to fluorescently label early endosomes. Using fluorescence microscopy and quantitative image analysis of cells expressing Rab5-GFP, we found that at early time points (t<1h), only a fraction (≈35%) of RGD-tagged NPs (which target cell surface integrins) colocalize with wildtype EEs, independent of the NP's membrane charge density. In comparison, a GTP-hydrolysis deficient mutant, Rab5-Q79L, which extends the size and lifetime of EEs yielding giant early endosomes (GEEs), enabled us to resolve and localize individual NPs found within the GEE lumen. Remarkably, nearly all intracellular NPs are found to be trapped within GEEs implying little or no escape at early time points. The observed small degree of colocalization of NPs and wildtype Rab5 is consistent with recycling of Rab5-GDP to the plasma membrane and not indicative of NP escape from EEs. Taken together, our results show that endosomal escape of PEGylated nanoparticles occurs downstream of EEs i.e., from late endosomes/lysosomes. Our studies also suggest that Rab5-Q79L could be used in a robust imaging assay which allows for direct visualization of NP interactions with the luminal membrane of early endosomes.

PEGylated cationic liposome-DNA complexation in brine is pathway-dependent.

Cationic liposome-DNA (CL-DNA) complexes, are regarded as promising materials for safe and efficient delivery of genes for therapeutical applications. In order to be used in vivo, these complexes may be coated with a hydrophilic polymer (e.g. polyethylene-glycol, PEG) that provides steric stabilization towards adhesion of proteins and removal by the immune system. In this work we study the influence of the initial salt concentration (Cs) - which modulates the electrostatic interaction between oppositely charged vesicles and DNA - on the structure and stability of PEGylated CL-DNA particles. Previous small-angle X-ray scattering has shown that if non-PEGylated or PEGylated CL-DNA lamellar complexes are prepared in water, their structure is well defined with a high number of lipid membrane-DNA layers (larger than 20). Here we show that if these complexes are transferred to saline media (150mM NaCl or DMEM, both near physiological conditions), this structure remains nearly unchanged. Conversely, if PEGylated complexes are prepared in saline media, their lamellar structure is much looser, with fewer number of layers. This pathway dependent behavior of PEGylated complex formation in brine is modulated by the liposome membrane charge density and the mole fraction of PEG 2000 in the membranes, with the average number of layers decreasing with increasing Cs and in going from 5mol% to 10mol% PEG-lipid. Each of these structures (high and low number of layers) is stable with time, suggesting that despite complex formation being thermodynamically favored, the complexation process in PEGylated membranes, which determines the number of layers per particle, is kinetically controlled. In the extreme case (when polymer repulsions from 10mol% PEG-lipid are maximized and electrostatic attraction between PEGylated CLs and DNA are minimized at low membrane charge density) complex formation is suppressed at high Cs=150mM.

Optimizing cationic and neutral lipids for efficient gene delivery at high serum content.

BACKGROUND: Cationic liposome (CL)-DNA complexes are promising gene delivery vectors with potential application in gene therapy. A key challenge in creating CL-DNA complexes for application is that their transfection efficiency (TE) is adversely affected by serum. In particular, little is known about the effects of a high serum content on TE, even though this may provide design guidelines for application in vivo. METHODS: We prepared CL-DNA complexes in which we varied the neutral lipid [1,2-dioleoyl-sn-glycerophosphatidylcholine, glycerol-monooleate (GMO), cholesterol], the headgroup charge and chemical structure of the cationic lipid, and the ratio of neutral to cationic lipid; we then measured the TE of these complexes as a function of serum content and assessed their cytotoxicity. We tested selected formulations in two human cancer cell lines (M21/melanoma and PC-3/prostate cancer). RESULTS: In the absence of serum, all CL-DNA complexes of custom-synthesized multivalent lipids show high TE. Certain combinations of multivalent lipids and neutral lipids, such as MVL5(5+)/GMO-DNA complexes or complexes based on the dendritic-headgroup lipid TMVLG3(8+) exhibited high TE both in the absence and presence of serum. Although their TE still dropped to a small extent in the presence of serum, it reached or surpassed that of benchmark commercial transfection reagents, particularly at a high serum content. CONCLUSIONS: Two-component vectors (one multivalent cationic lipid and one neutral lipid) can rival or surpass benchmark reagents at low and high serum contents (up to 50%, v/v). We propose guidelines for optimizing the serum resistance of CL-DNA complexes based on a given cationic lipid.

A village in a dish model system for population-scale hiPSC studies.

The mechanisms by which DNA alleles contribute to disease risk, drug response, and other human phenotypes are highly context-specific, varying across cell types and different conditions. Human induced pluripotent stem cells are uniquely suited to study these context-dependent effects but cell lines from hundreds or thousands of individuals are required. Village cultures, where multiple induced pluripotent stem lines are cultured and differentiated in a single dish, provide an elegant solution for scaling induced pluripotent stem experiments to the necessary sample sizes required for population-scale studies. Here, we show the utility of village models, demonstrating how cells can be assigned to an induced pluripotent stem line using single-cell sequencing and illustrating that the genetic, epigenetic or induced pluripotent stem line-specific effects explain a large percentage of gene expression variation for many genes. We demonstrate that village methods can effectively detect induced pluripotent stem line-specific effects, including sensitive dynamics of cell states.

TNFAIP3 Reduction-of-Function Drives Female Infertility and CNS Inflammation.

Women with autoimmune and inflammatory aetiologies can exhibit reduced fecundity. TNFAIP3 is a master negative regulator of inflammation, and has been linked to many inflammatory conditions by genome wide associations studies, however its role in fertility remains unknown. Here we show that mice harbouring a mild Tnfaip3 reduction-of-function coding variant (Tnfaip3I325N) that reduces the threshold for inflammatory NF-κB activation, exhibit reduced fecundity. Sub-fertility in Tnfaip3I325N mice is associated with irregular estrous cycling, low numbers of ovarian secondary follicles, impaired mammary gland development and insulin resistance. These pathological features are associated with infertility in human subjects. Transplantation of Tnfaip3I325N ovaries, mammary glands or pancreatic islets into wild-type recipients rescued estrous cycling, mammary branching and hyperinsulinemia respectively, pointing towards a cell-extrinsic hormonal mechanism. Examination of hypothalamic brain sections revealed increased levels of microglial activation with reduced levels of luteinizing hormone. TNFAIP3 coding variants may offer one contributing mechanism for the cause of sub-fertility observed across otherwise healthy populations as well as for the wide variety of auto-inflammatory conditions to which TNFAIP3 is associated. Further, TNFAIP3 represents a molecular mechanism that links heightened immunity with neuronal inflammatory homeostasis. These data also highlight that tuning-up immunity with TNFAIP3 comes with the potentially evolutionary significant trade-off of reduced fertility.

DNA barcoding reveals ongoing immunoediting of clonal cancer populations during metastatic progression and immunotherapy response.

Cancers evade the immune system through the process of cancer immunoediting. While immune checkpoint inhibitors are effective for reactivating tumour immunity in some cancer types, many other solid cancers, including breast cancer, remain largely non-responsive. Understanding how non-responsive cancers evade immunity and whether this occurs at the clonal level will improve immunotherapeutic design. Here we use DNA barcoding to track murine mammary cancer cell clones during immunoediting and determine clonal transcriptional profiles that allow immune evasion following anti-PD1 plus anti-CTLA4 immunotherapy. Clonal diversity is significantly restricted by immunotherapy treatment in both primary tumours and metastases, demonstrating selection for pre-existing breast cancer cell populations and ongoing immunoediting during metastasis and treatment. Immunotherapy resistant clones express a common gene signature associated with poor survival of basal-like breast cancer patient cohorts. At least one of these genes has an existing small molecule that can potentially be used to improve immunotherapy response.

Transcriptomic and proteomic retinal pigment epithelium signatures of age-related macular degeneration.

There are currently no treatments for geographic atrophy, the advanced form of age-related macular degeneration. Hence, innovative studies are needed to model this condition and prevent or delay its progression. Induced pluripotent stem cells generated from patients with geographic atrophy and healthy individuals were differentiated to retinal pigment epithelium. Integrating transcriptional profiles of 127,659 retinal pigment epithelium cells generated from 43 individuals with geographic atrophy and 36 controls with genotype data, we identify 445 expression quantitative trait loci in cis that are asssociated with disease status and specific to retinal pigment epithelium subpopulations. Transcriptomics and proteomics approaches identify molecular pathways significantly upregulated in geographic atrophy, including in mitochondrial functions, metabolic pathways and extracellular cellular matrix reorganization. Five significant protein quantitative trait loci that regulate protein expression in the retinal pigment epithelium and in geographic atrophy are identified - two of which share variants with cis- expression quantitative trait loci, including proteins involved in mitochondrial biology and neurodegeneration. Investigation of mitochondrial metabolism confirms mitochondrial dysfunction as a core constitutive difference of the retinal pigment epithelium from patients with geographic atrophy. This study uncovers important differences in retinal pigment epithelium homeostasis associated with geographic atrophy.

Retinal ganglion cell-specific genetic regulation in primary open-angle glaucoma.

To assess the transcriptomic profile of disease-specific cell populations, fibroblasts from patients with primary open-angle glaucoma (POAG) were reprogrammed into induced pluripotent stem cells (iPSCs) before being differentiated into retinal organoids and compared with those from healthy individuals. We performed single-cell RNA sequencing of a total of 247,520 cells and identified cluster-specific molecular signatures. Comparing the gene expression profile between cases and controls, we identified novel genetic associations for this blinding disease. Expression quantitative trait mapping identified a total of 4,443 significant loci across all cell types, 312 of which are specific to the retinal ganglion cell subpopulations, which ultimately degenerate in POAG. Transcriptome-wide association analysis identified genes at loci previously associated with POAG, and analysis, conditional on disease status, implicated 97 statistically significant retinal ganglion cell-specific expression quantitative trait loci. This work highlights the power of large-scale iPSC studies to uncover context-specific profiles for a genetically complex disease.

Inhibitor of Differentiation 4 (ID4) represses mammary myoepithelial differentiation via inhibition of HEB.

Inhibitor of differentiation (ID) proteins dimerize with basic HLH (bHLH) transcription factors, repressing transcription of lineage-specification genes across diverse cellular lineages. ID4 is a key regulator of mammary stem cells; however, the mechanism by which it achieves this is unclear. Here, we show that ID4 has a cell autonomous role in preventing myoepithelial differentiation of basal cells in mammary organoids and in vivo. ID4 positively regulates proliferative genes and negatively regulates genes involved in myoepithelial function. Mass spectrometry reveals that ID4 interacts with the bHLH protein HEB, which binds to E-box motifs in regulatory elements of basal developmental genes involved in extracellular matrix and the contractile cytoskeleton. We conclude that high ID4 expression in mammary basal stem cells antagonizes HEB transcriptional activity, preventing myoepithelial differentiation and allowing for appropriate tissue morphogenesis. Downregulation of ID4 during pregnancy modulates gene regulated by HEB, promoting specialization of basal cells into myoepithelial cells.

DNA methylation is required to maintain both DNA replication timing precision and 3D genome organization integrity.

DNA replication timing and three-dimensional (3D) genome organization are associated with distinct epigenome patterns across large domains. However, whether alterations in the epigenome, in particular cancer-related DNA hypomethylation, affects higher-order levels of genome architecture is still unclear. Here, using Repli-Seq, single-cell Repli-Seq, and Hi-C, we show that genome-wide methylation loss is associated with both concordant loss of replication timing precision and deregulation of 3D genome organization. Notably, we find distinct disruption in 3D genome compartmentalization, striking gains in cell-to-cell replication timing heterogeneity and loss of allelic replication timing in cancer hypomethylation models, potentially through the gene deregulation of DNA replication and genome organization pathways. Finally, we identify ectopic H3K4me3-H3K9me3 domains from across large hypomethylated domains, where late replication is maintained, which we purport serves to protect against catastrophic genome reorganization and aberrant gene transcription. Our results highlight a potential role for the methylome in the maintenance of 3D genome regulation.

Cryopreservation of human cancers conserves tumour heterogeneity for single-cell multi-omics analysis.

BACKGROUND: High throughput single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for exploring cellular heterogeneity among complex human cancers. scRNA-Seq studies using fresh human surgical tissue are logistically difficult, preclude histopathological triage of samples, and limit the ability to perform batch processing. This hindrance can often introduce technical biases when integrating patient datasets and increase experimental costs. Although tissue preservation methods have been previously explored to address such issues, it is yet to be examined on complex human tissues, such as solid cancers and on high throughput scRNA-Seq platforms. METHODS: Using the Chromium 10X platform, we sequenced a total of ~ 120,000 cells from fresh and cryopreserved replicates across three primary breast cancers, two primary prostate cancers and a cutaneous melanoma. We performed detailed analyses between cells from each condition to assess the effects of cryopreservation on cellular heterogeneity, cell quality, clustering and the identification of gene ontologies. In addition, we performed single-cell immunophenotyping using CITE-Seq on a single breast cancer sample cryopreserved as solid tissue fragments. RESULTS: Tumour heterogeneity identified from fresh tissues was largely conserved in cryopreserved replicates. We show that sequencing of single cells prepared from cryopreserved tissue fragments or from cryopreserved cell suspensions is comparable to sequenced cells prepared from fresh tissue, with cryopreserved cell suspensions displaying higher correlations with fresh tissue in gene expression. We showed that cryopreservation had minimal impacts on the results of downstream analyses such as biological pathway enrichment. For some tumours, cryopreservation modestly increased cell stress signatures compared to freshly analysed tissue. Further, we demonstrate the advantage of cryopreserving whole-cells for detecting cell-surface proteins using CITE-Seq, which is impossible using other preservation methods such as single nuclei-sequencing. CONCLUSIONS: We show that the viable cryopreservation of human cancers provides high-quality single-cells for multi-omics analysis. Our study guides new experimental designs for tissue biobanking for future clinical single-cell RNA sequencing studies.

A single-cell and spatially resolved atlas of human breast cancers.

Breast cancers are complex cellular ecosystems where heterotypic interactions play central roles in disease progression and response to therapy. However, our knowledge of their cellular composition and organization is limited. Here we present a single-cell and spatially resolved transcriptomics analysis of human breast cancers. We developed a single-cell method of intrinsic subtype classification (SCSubtype) to reveal recurrent neoplastic cell heterogeneity. Immunophenotyping using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) provides high-resolution immune profiles, including new PD-L1/PD-L2+ macrophage populations associated with clinical outcome. Mesenchymal cells displayed diverse functions and cell-surface protein expression through differentiation within three major lineages. Stromal-immune niches were spatially organized in tumors, offering insights into antitumor immune regulation. Using single-cell signatures, we deconvoluted large breast cancer cohorts to stratify them into nine clusters, termed 'ecotypes', with unique cellular compositions and clinical outcomes. This study provides a comprehensive transcriptional atlas of the cellular architecture of breast cancer.

A novel strategy for avian species and gender identification using the CHD gene.

We report on a novel and rapid strategy for the simultaneous identification of both avian species and gender by analyzing a section of the CHD gene. The CHD gene is carried by the avian sex determining chromosomes where a female bird carries both a W and Z chromosome but a cock bird carries two copies of the Z chromosome. Two primer pairs, CHD1F/CHD1R and P2/P8, were used to amplify a part of the CHD gene from 144 samples corresponding to 58 avian species. For all species tested, two fragments were observed at least in one amplification for female samples. All tested species produced species specific size fragments allowing both sex determination and species identification using these primer pairs. However, special care is still warranted as so few samples have been characterised. This novel strategy for avian species and gender identification using the CHD gene was developed for a number of applications from ecology to forensic science.

Id Proteins Promote a Cancer Stem Cell Phenotype in Mouse Models of Triple Negative Breast Cancer via Negative Regulation of Robo1.

Breast cancers display phenotypic and functional heterogeneity and several lines of evidence support the existence of cancer stem cells (CSCs) in certain breast cancers, a minor population of cells capable of tumor initiation and metastatic dissemination. Identifying factors that regulate the CSC phenotype is therefore important for developing strategies to treat metastatic disease. The Inhibitor of Differentiation Protein 1 (Id1) and its closely related family member Inhibitor of Differentiation 3 (Id3) (collectively termed Id) are expressed by a diversity of stem cells and are required for metastatic dissemination in experimental models of breast cancer. In this study, we show that ID1 is expressed in rare neoplastic cells within ER-negative breast cancers. To address the function of Id1 expressing cells within tumors, we developed independent murine models of Triple Negative Breast Cancer (TNBC) in which a genetic reporter permitted the prospective isolation of Id1+ cells. Id1+ cells are enriched for self-renewal in tumorsphere assays in vitro and for tumor initiation in vivo. Conversely, depletion of Id1 and Id3 in the 4T1 murine model of TNBC demonstrates that Id1/3 are required for cell proliferation and self-renewal in vitro, as well as primary tumor growth and metastatic colonization of the lung in vivo. Using combined bioinformatic analysis, we have defined a novel mechanism of Id protein function via negative regulation of the Roundabout Axon Guidance Receptor Homolog 1 (Robo1) leading to activation of a Myc transcriptional programme.

Development and validation of a targeted gene sequencing panel for application to disparate cancers.

Next generation sequencing has revolutionised genomic studies of cancer, having facilitated the development of precision oncology treatments based on a tumour's molecular profile. We aimed to develop a targeted gene sequencing panel for application to disparate cancer types with particular focus on tumours of the head and neck, plus test for utility in liquid biopsy. The final panel designed through Roche/Nimblegen combined 451 cancer-associated genes (2.01 Mb target region). 136 patient DNA samples were collected for performance and application testing. Panel sensitivity and precision were measured using well-characterised DNA controls (n = 47), and specificity by Sanger sequencing of the Aryl Hydrocarbon Receptor Interacting Protein (AIP) gene in 89 patients. Assessment of liquid biopsy application employed a pool of synthetic circulating tumour DNA (ctDNA). Library preparation and sequencing were conducted on Illumina-based platforms prior to analysis with our accredited (ISO15189) bioinformatics pipeline. We achieved a mean coverage of 395x, with sensitivity and specificity of >99% and precision of >97%. Liquid biopsy revealed detection to 1.25% variant allele frequency. Application to head and neck tumours/cancers resulted in detection of mutations aligned to published databases. In conclusion, we have developed an analytically-validated panel for application to cancers of disparate types with utility in liquid biopsy.

Tumor inherent interferon regulators as biomarkers of long-term chemotherapeutic response in TNBC.

Patients diagnosed with triple negative breast cancer (TNBC) have an increased risk of rapid metastasis compared to other subtypes. Predicting long-term survival post-chemotherapy in patients with TNBC is difficult, yet enhanced infiltration of tumor infiltrating lymphocytes (TILs) has been associated with therapeutic response and reduced risk of metastatic relapse. Immune biomarkers that predict the immune state of a tumor and risk of metastatic relapse pre- or mid-neoadjuvant chemotherapy are urgently needed to allow earlier implementation of alternate therapies that may reduce TNBC patient mortality. Utilizing a neoadjuvant chemotherapy trial where TNBC patients had sequential biopsies taken, we demonstrate that measurement of T-cell subsets and effector function, specifically CD45RO expression, throughout chemotherapy predicts risk of metastatic relapse. Furthermore, we identified the tumor inherent interferon regulatory factor IRF9 as a marker of active intratumoral type I and II interferon (IFN) signaling and reduced risk of distant relapse. Functional implications of tumor intrinsic IFN signaling were demonstrated using an immunocompetent mouse model of TNBC, where enhanced type I IFN signaling increased anti-tumor immunity and metastasis-free survival post-chemotherapy. Using two independent adjuvant cohorts we were able to validate loss of IRF9 as a poor prognostic biomarker pre-chemotherapy. Thus, IRF9 expression may offer early insight into TNBC patient prognosis and tumor heat, allowing for identification of patients that are unlikely to respond to chemotherapy alone and could benefit from further immune-based therapeutic intervention.

Genomic stratification and liquid biopsy in a rare adrenocortical carcinoma (ACC) case, with dual lung metastases.

Adrenocortical carcinoma is a rare malignancy with a poor prognosis and few treatment options. Molecular characterization of this cancer remains limited. We present a case of an adrenocortical carcinoma (ACC) in a 37-yr-old female, with dual lung metastases identified 1 yr following commencement of adjuvant mitotane therapy. As standard therapeutic regimens are often unsuccessful in ACC, we undertook a comprehensive genomic study into this case to identify treatment options and monitor disease progress. We performed targeted and whole-genome sequencing of germline, primary tumor, and both metastatic tumors from this patient and monitored recurrence over 2 years using liquid biopsy for ctDNA and steroid hormone measurements. Sequencing revealed the primary and metastatic tumors were hyperhaploid, with extensive loss of heterozygosity but few structural rearrangements. Loss-of-function mutations were identified in MSH2, TP53, RB1, and PTEN, resulting in tumors with mismatch repair signatures and microsatellite instability. At the cellular level, tumors were populated by mitochondria-rich oncocytes. Longitudinal ctDNA mutation and hormone profiles were unable to detect micrometastatic disease, consistent with clinical indicators of disease remission. The molecular signatures in our ACC case suggested immunotherapy in the event of disease progression; however, the patient remains free of cancer. The extensive molecular analysis presented here could be applied to other rare and/or poorly stratified cancers to identify novel or repurpose existing therapeutic options, thereby broadly improving diagnoses, treatments, and prognoses.