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As availability of precision therapies expands, a well-validated circulating cell-free DNA (cfDNA)-based comprehensive genomic profiling assay has the potential to provide considerable value as a complement to tissue-based testing to ensure potentially life-extending therapies are administered to patients most likely to benefit. Additional data supporting the clinical validity of cfDNA-based testing is necessary to inform optimal use of these assays in the clinic. The FoundationOne.sup.® Liquid CDx assay is a pan-cancer cfDNA-based comprehensive genomic profiling assay that was recently approved by FDA. Validation studies included >7,500 tests and >30,000 unique variants across >300 genes and >30 cancer types. Clinical validity results across multiple tumor types are presented. Additionally, results demonstrated a 95% limit of detection of 0.40% variant allele fraction for select substitutions and insertions/deletions, 0.37% variant allele fraction for select rearrangements, 21.7% tumor fraction for copy number amplifications, and 30.4% TF for copy number losses. The limit of detection for microsatellite instability and blood tumor mutational burden were also determined. The false positive variant rate was 0.013% (approximately 1 in 8,000). Reproducibility of variant calling was 99.59%. In comparison with an orthogonal method, an overall positive percent agreement of 96.3% and negative percent agreement of >99.9% was observed. These study results demonstrate that FoundationOne Liquid CDx accurately and reproducibly detects the major types of genomic alterations in addition to complex biomarkers such as microsatellite instability, blood tumor mutational burden, and tumor fraction. Critically, clinical validity data is presented across multiple cancer types.

BackgroundAssays of the abundance of immune cell populations in the tumor microenvironment promise to inform immune oncology research and the choice of immunotherapy for individual patients. We propose to measure the intratumoral abundance of various immune cell populations with gene expression. In contrast to IHC and flow cytometry, gene expression assays yield high information content from a clinically practical workflow. Previous studies of gene expression in purified immune cells have reported hundreds of genes showing enrichment in a single cell type, but the utility of these genes in tumor samples is unknown. We use co-expression patterns in large tumor gene expression datasets to evaluate previously reported candidate cell type marker genes lists, eliminate numerous false positives and identify a subset of high confidence marker genes.MethodsUsing a novel statistical tool, we use co-expression patterns in 9986 samples from The Cancer Genome Atlas (TCGA) to evaluate previously reported cell type marker genes. We compare immune cell scores derived from these genes to measurements from flow cytometry and immunohistochemistry. We characterize the reproducibility of our cell scores in replicate runs of RNA extracted from FFPE tumor tissue.ResultsWe identify a list of 60 marker genes whose expression levels measure 14 immune cell populations. Cell type scores calculated from these genes are concordant with flow cytometry and IHC readings, show high reproducibility in replicate RNA samples from FFPE tissue and enable detailed analyses of the anti-tumor immune response in TCGA. In an immunotherapy dataset, they separate responders and non-responders early on therapy and provide an intricate picture of the effects of checkpoint inhibition. Most genes previously reported to be enriched in a single cell type have co-expression patterns inconsistent with cell type specificity.ConclusionsDue to their concise gene set, computational simplicity and utility in tumor samples, these cell type gene signatures may be useful in future discovery research and clinical trials to understand how tumors and therapeutic intervention shape the immune response.

Homologous recombination repair (HRR) is a cellular pathway for high-fidelity double strand DNA break repair that uses the sister chromatid as a guide to ensure chromosomal integrity and cell viability. Deficiency in the HRR pathway (HRD) can sensitize tumors to poly (ADP-ribose) polymerase inhibitors (PARPi) and platinum-based chemotherapy, offering an avenue to identify patients who may benefit from targeted therapies. HRD signature (HRDsig) is a pan-solid-tumor biomarker on the FoundationOne®CDx (F1CDx®) assay that employs a DNA scar-based approach to calculate a score based on copy number features (e.g., segment size, oscillation patterns, and breakpoints per chromosome arm) and does not rely on HRR gene alterations, enabling detection of genomic and epigenetic mechanisms of HRD. After finalizing the HRDsig algorithm, analytical validation was conducted in a CAP-accredited, CLIA-certified laboratory on 278 solid tumor and normal tissue specimens. HRDsig results were compared with an independent HRD biomarker, defined by the presence of a reversion mutation restoring HRR gene function. In this evaluation, 100 HRD-positive and 126 HRD-negative samples showed a positive percent agreement of 90.00% and a negative percent agreement of 94.44%. The limit of detection (LoD) was estimated at 23.04% tumor purity, with the limit of blank (LoB) confirmed as zero in 60 normal tissue replicates. Reproducibility testing on 11 positive and 11 negative samples across multiple labs, reagent lots, and sequencers yielded agreement in 99.49% of positive and 99.73% of negative replicates. HRDsig status remained consistent in the presence of interfering substances, demonstrating 100% concordance in spiked samples. These validation results underscore the high analytical concordance, low false-positive rate, and overall robustness of HRDsig for reliable assessment of homologous recombination deficiency.

12 microRNA expression profiling platforms are compared for their reproducibility, sensitivity, accuracy and specificity, and the strengths and weaknesses of each platform are discussed. MicroRNAs are important negative regulators of protein-coding gene expression and have been studied intensively over the past years. Several measurement platforms have been developed to determine relative miRNA abundance in biological samples using different technologies such as small RNA sequencing, reverse transcription–quantitative PCR (RT-qPCR) and (microarray) hybridization. In this study, we systematically compared 12 commercially available platforms for analysis of microRNA expression. We measured an identical set of 20 standardized positive and negative control samples, including human universal reference RNA, human brain RNA and titrations thereof, human serum samples and synthetic spikes from microRNA family members with varying homology. We developed robust quality metrics to objectively assess platform performance in terms of reproducibility, sensitivity, accuracy, specificity and concordance of differential expression. The results indicate that each method has its strengths and weaknesses, which help to guide informed selection of a quantitative microRNA gene expression platform for particular study goals.

Emerging technologies focused on the detection and quantification of circulating tumor DNA (ctDNA) in blood show extensive potential for managing patient treatment decisions, informing risk of recurrence, and predicting response to therapy. Currently available tissue-informed approaches are often limited by the need for additional sequencing of normal tissue or peripheral mononuclear cells to identify non-tumor-derived alterations while tissue-naïve approaches are often limited in sensitivity. Here we present the analytical validation for a novel ctDNA monitoring assay, FoundationOne®Tracker. The assay utilizes somatic alterations from comprehensive genomic profiling (CGP) of tumor tissue. A novel algorithm identifies monitorable alterations with a high probability of being somatic and computationally filters non-tumor-derived alterations such as germline or clonal hematopoiesis variants without the need for sequencing of additional samples. Monitorable alterations identified from tissue CGP are then quantified in blood using a multiplex polymerase chain reaction assay based on the validated Signatera TM assay. The analytical specificity of the plasma workflow is shown to be 99.6% at the sample level. Analytical sensitivity is shown to be >97.3% at ≥5 mean tumor molecules per mL of plasma (MTM/mL) when tested with the most conservative configuration using only two monitorable alterations. The assay also demonstrates high analytical accuracy when compared to liquid biopsy-based CGP as well as high qualitative (measured 100% PPA) and quantitative precision (<11.2% coefficient of variation).

Pathological and genomic profiling have transformed breast cancer care by matching patients to targeted treatments. However, tumors evolve and evade therapeutic interventions often through the acquisition of genomic mutations. Here we examine patients profiled with tissue (TBx) and liquid biopsy (LBx) as part of routine clinical care, to characterize the tumor evolutionary landscape and identify potential vulnerabilities in the relapsed setting. Real-world evidence demonstrates that LBx is utilized later in care and identifies associations with intervening therapy. While driver events are frequently shared, acquired LBx alterations are detected in a majority of patients, with the highest frequency in ER+ disease and in patients with longer biopsy intervals. Acquired mutations are often polyclonal and present at lower allelic fractions, suggesting multi-clonal convergent evolution. In addition to well-characterized resistance mutations (e.g., ESR1 , NF1 , RB1 , ERBB2) , we observe a diversity of rarer but potentially targetable mutations (e.g., PIK3CA, HRAS / NRAS / KRAS , FGFR1/2/3 , BRAF ) and fusions (e.g., FGFR1/2 , ERBB2 , RET ), as well as BRCA1/2 reversions through a variety of mechanisms, including splice alterations and structural deletions. This study provides insights on treatment and selection-driven tumor evolution and identifies potential combinatorial treatment options in advanced breast cancer. Liquid biopsies could be valuable tools to monitor breast cancer progression and evolution. Here, the authors investigate genomic profiling of tissue and liquid biopsies in a large cohort of patients with breast cancer during the course of therapy to characterise tumour evolution and acquired mutations.

Mir-290 through mir-295 (mir-290–295) is a mammalian-specific microRNA (miRNA) cluster that, in mice, is expressed specifically in early embryos and embryonic germ cells. Here, we show that mir-290–295 plays important roles in embryonic development as indicated by the partially penetrant lethality of mutant embryos. In addition, we show that in surviving mir-290–295-deficient embryos, female but not male fertility is compromised. This impairment in fertility arises from a defect in migrating primordial germ cells and occurs equally in male and female mutant animals. Male mir-290–295–/– mice, due to the extended proliferative lifespan of their germ cells, are able to recover from this initial germ cell loss and are fertile. Female mir-290–295–/– mice are unable to recover and are sterile, due to premature ovarian failure.

MicroRNAs (miRNAs) post-transcriptionally regulate the expression of thousands of distinct mRNAs. While some regulatory interactions help to maintain basal cellular functions, others are likely relevant in more specific settings, such as response to stress. Here we describe such a role for the mir-290-295 cluster, the dominant miRNA cluster in mouse embryonic stem cells (mESCs). Examination of a target list generated from bioinformatic prediction, as well as expression data following miRNA loss, revealed strong enrichment for apoptotic regulators, two of which we validated directly: Caspase 2, the most highly conserved mammalian caspase, and Ei24, a p53 transcriptional target. Consistent with these predictions, mESCs lacking miRNAs were more likely to initiate apoptosis following genotoxic exposure to gamma irradiation or doxorubicin. Knockdown of either candidate partially rescued this pro-apoptotic phenotype, as did transfection of members of the mir-290-295 cluster. These findings were recapitulated in a specific mir-290-295 deletion line, confirming that they reflect miRNA functions at physiological levels. In contrast to the basal regulatory roles previously identified, the pro-survival phenotype shown here may be most relevant to stressful gestations, where pro-oxidant metabolic states induce DNA damage. Similarly, this cluster may mediate chemotherapeutic resistance in a neoplastic context, making it a useful clinical target.

Genomic tumour profiling informs targeted treatment options. Entrectinib is a tyrosine kinase inhibitor with efficacy in NTRK fusion‐positive (‐fp) solid tumours and ROS1‐fp non‐small cell lung cancer. FoundationOne® Liquid CDx (F1L CDx), a non‐invasive in vitro next‐generation sequencing (NGS)‐based diagnostic, detects genomic alterations in plasma circulating tumour DNA (ctDNA). We evaluated the clinical validity of F1L CDx as an aid in identifying patients with NTRK‐fp or ROS1‐fp tumours and assessed the genomic landscape pre‐ and post‐entrectinib treatment. Among evaluable pre‐treatment clinical samples (N = 85), positive percentage agreements between F1L CDx and clinical trial assays (CTAs) were 47.4% (NTRK fusions) and 64.5% (ROS1 fusions); positive predictive value was 100% for both. The objective response rate for CTA+ F1L CDx+ patients was 72.2% in both cohorts. The median duration of response significantly differed between F1L CDx+ and F1L CDx− samples in ROS1‐fp (5.6 vs. 17.3 months) but not NTRK‐fp (9.2 vs. 12.9 months) patients. Fifteen acquired resistance mutations were detected. We conclude that F1L CDx is a clinically valid complement to tissue‐based testing to identify patients who may benefit from entrectinib and those with acquired resistance mutations associated with disease progression. Entrectinib is a kinase inhibitor that targets both the NTRK and ROS1 oncogenes. We show that NTRK and ROS1 fusions are present in 46% and 64%, respectively, of blood samples from patients enrolled on a clinical trial, with a response rate of 72%. Profiling blood at progression showed that resistance occurs via mutations in NTRK/ROS1 or via downstream MAP‐kinase reactivation.

The amounts of circulating cell-free DNA (cfDNA) and circulating-tumor DNA (ctDNA) present in peripheral blood liquid biopsies can vary due to preanalytic/analytic variables. In this study, we examined the impact of patient age, sex, stage, and tumor type on cfDNA yield, ctDNA fraction, and estimated ctDNA quantity from a large cohort of clinical liquid biopsy samples. We performed a retrospective analysis of 12 139 consecutive samples received for liquid biopsy (FoundationOne® Liquid) clinical testing. Significant differences in both cfDNA yield and estimated ctDNA quantity were observed based on the underlying tumor type that initiated the liquid biopsy analysis and the stage of the patient (P < 0.001). In addition, significant differences in ctDNA quantity were present based in both the patient age and sex (P < 0.001). Importantly, we saw a significantly higher success rate of issuing a clinically useful report in patients with higher levels of cfDNA yield and ctDNA quantity (P < 0.001). In this study, we show that ctDNA quantity varied significantly based on patient age, sex, stage, and tumor type, which could offer an explanation as to why certain liquid biopsy specimens are more likely to fail sequencing or provide clinically meaningful results. In addition, this could affect future clinical decisions on the blood sample volumes required to allow successful liquid biopsy testing.