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While tumor genome sequencing has become widely available in clinical and research settings, the interpretation of tumor somatic variants remains an important bottleneck. Here we present the Cancer Genome Interpreter, a versatile platform that automates the interpretation of newly sequenced cancer genomes, annotating the potential of alterations detected in tumors to act as drivers and their possible effect on treatment response. The results are organized in different levels of evidence according to current knowledge, which we envision can support a broad range of oncology use cases. The resource is publicly available at http://www.cancergenomeinterpreter.org .

Circulating free tumor DNA (ctDNA) analysis is gaining popularity in precision oncology, particularly in metastatic breast cancer, as it provides non-invasive, real-time tumor information to complement tissue biopsies, allowing for tailored treatment strategies and improved patient selection in clinical trials. Its use in early breast cancer has been limited so far, due to the relatively low sensitivity of available techniques in a setting characterized by lower levels of ctDNA shedding. However, advances in sequencing and bioinformatics, as well as the use of methylome profiles, have led to an increasing interest in the application of ctDNA analysis in early breast cancer, from screening to curative treatment evaluation and minimal residual disease (MRD) detection. With multiple prospective clinical trials in this setting, ctDNA evaluation may become useful in clinical practice. This article reviews the data regarding the analytical validity of the currently available tests for ctDNA detection and the clinical potential of ctDNA analysis in early breast cancer.

A mutation conferring resistance to novel irreversible EGFR inhibitors is identified in cell-free plasma DNA from lung cancer patients. Here we studied cell-free plasma DNA (cfDNA) collected from subjects with advanced lung cancer whose tumors had developed resistance to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) AZD9291. We first performed next-generation sequencing of cfDNA from seven subjects and detected an acquired EGFR C797S mutation in one; expression of this mutant EGFR construct in a cell line rendered it resistant to AZD9291. We then performed droplet digital PCR on serial cfDNA specimens collected from 15 AZD9291-treated subjects. All were positive for the T790M mutation before treatment, but upon developing AZD9291 resistance three molecular subtypes emerged: six cases acquired the C797S mutation, five cases maintained the T790M mutation but did not acquire the C797S mutation and four cases lost the T790M mutation despite the presence of the underlying EGFR activating mutation. Our findings provide insight into the diversity of mechanisms through which tumors acquire resistance to AZD9291 and highlight the need for therapies that are able to overcome resistance mediated by the EGFR C797S mutation.

Anti-BRAF/EGFR therapy was recently approved for the treatment of metastatic BRAF V600E colorectal cancer (mCRC BRAF-V600E ). However, a large fraction of patients do not respond, underscoring the need to identify molecular determinants of treatment response. Using whole-exome sequencing in a discovery cohort of patients with mCRC BRAF-V600E treated with anti-BRAF/EGFR therapy, we found that inactivating mutations in RNF43 , a negative regulator of WNT, predict improved response rates and survival outcomes in patients with microsatellite-stable (MSS) tumors. Analysis of an independent validation cohort confirmed the relevance of RNF43 mutations to predicting clinical benefit (72.7% versus 30.8%; P  = 0.03), as well as longer progression-free survival (hazard ratio (HR), 0.30; 95% confidence interval (CI), 0.12–0.75; P  = 0.01) and overall survival (HR, 0.26; 95% CI, 0.10–0.71; P  = 0.008), in patients with MSS- RNF43 mutated versus MSS- RNF43 wild-type tumors. Microsatellite-instable tumors invariably carried a wild-type-like RNF43 genotype encoding p.G659fs and presented an intermediate response profile. We found no association of RNF43 mutations with patient outcomes in a control cohort of patients with MSS-mCRC BRAF-V600E tumors not exposed to anti-BRAF targeted therapies. Overall, our findings suggest a cross-talk between the MAPK and WNT pathways that may modulate the antitumor activity of anti-BRAF/EGFR therapy and uncover predictive biomarkers to optimize the clinical management of these patients. The presence of inactivating mutations in RNF43 , a negative regulator of WNT, in tumor cells predicts improved response rates and survival outcomes in patients with metastatic BRAF V600E colorectal cancer treated with anti-BRAF/EGFR therapy.

BackgroundLiquid biopsy offers a minimally invasive alternative to tissue-based evaluation of mutational status in cancer. The goal of the present study was to evaluate the aggregate performance of OncoBEAM RAS mutation analysis in plasma of colorectal cancer (CRC) patients at 10 hospital laboratories in Spain where this technology is routinely implemented.MethodsCirculating cell-free DNA from plasma was examined for RAS mutations using the OncoBEAM platform at each hospital laboratory. Results were then compared to those obtained from DNA extracted from tumour tissue from the same patient.ResultsThe overall percentage agreement between plasma-based and tissue-based RAS mutation testing of the 236 participants was 89% (210/236; kappa, 0.770 (95% CI: 0.689–0.852)). Re-analysis of tissue from all discordant cases by BEAMing revealed two false negative and five false positive tumour tissue RAS results, with a final concordance of 92%. Plasma false negative results were found more frequently in patients with exclusive lung metastatic disease.ConclusionsIn this first prospective real-world RAS mutation performance comparison study, a high overall agreement was observed between results obtained from plasma and tissue samples. Overall, these findings indicate that the plasma-based BEAMing assay is a viable solution for rapid delivery of RAS mutation status to determine mCRC patient eligibility for anti-EGFR therapy.

Patritumab deruxtecan (HER3-DXd) exhibits promising efficacy in breast cancer, with its activity not directly correlated to baseline ERBB3 /HER3 levels. This research investigates the genetic factors affecting HER3-DXd’s response in women with early-stage hormone receptor-positive and HER2-negative (HR+/HER2-) breast cancer. In the SOLTI-1805 TOT-HER3 trial, a single HER3-DXd dose was administered to 98 patients across two parts: 78 patients received 6.4 mg/kg (Part A), and 44 received a lower 5.6 mg/kg dose (Part B). The CelTIL score, measuring tumor cellularity and infiltrating lymphocytes from baseline to day 21, was used to assess drug activity. Part A demonstrated increased CelTIL score after one dose of HER3-DXd. Here we report CelTIL score and safety for Part B. In addition, the exploratory analyses of part A involve a comprehensive study of gene expression, somatic mutations, copy-number segments, and DNA-based subtypes, while Part B focuses on validating gene expression. RNA analyses show significant correlations between CelTIL responses, high proliferation genes (e.g., CCNE1, MKI67 ), and low expression of luminal genes (e.g., NAT1, SLC39A6 ). DNA findings indicate that CelTIL response is significantly associated with TP53 mutations, proliferation, non-luminal signatures, and a distinct DNA-based subtype (DNADX cluster-3). Critically, low HER2DX  ERBB2 mRNA, correlates with increased HER3-DXd activity, which is validated through in vivo patient-derived xenograft  models. This study proposes chemosensitivity determinants, DNA-based subtype classification, and low ERBB2 expression as potential markers for HER3-DXd activity in HER2-negative breast cancer. Patritumab deruxtecan (HER3-DXd) is a promising therapy for breast cancer, targeting HER3. Here, the authors analyse the genomic factors that affect the response to HER3-DXd in patients with early-stage HER2-negative breast cancer as part of the SOLTI-1805 TOT-HER3 clinical trial and report outcomes for Part B of the trial using lower HER3-DXd dose in patients with HER2-negative breast cancer.

Liquid biopsy has proven valuable in identifying individual genetic alterations; however, the ability of plasma ctDNA to capture complex tumor phenotypes with clinical value is unknown. To address this question, we have performed 0.5X shallow whole-genome sequencing in plasma from 459 patients with metastatic breast cancer, including 245 patients treated with endocrine therapy and a CDK4/6 inhibitor (ET + CDK4/6i) from 2 independent cohorts. We demonstrate that machine learning multi-gene signatures, obtained from ctDNA, identify complex biological features, including measures of tumor proliferation and estrogen receptor signaling, similar to what is accomplished using direct tumor tissue DNA or RNA profiling. More importantly, 4 DNA-based subtypes, and a ctDNA-based genomic signature tracking retinoblastoma loss-of-heterozygosity, are significantly associated with poor response and survival outcome following ET + CDK4/6i, independently of plasma tumor fraction. Our approach opens opportunities for the discovery of additional multi-feature genomic predictors coming from ctDNA in breast cancer and other cancer-types. Plasma ctDNA is a promising method to determine patient outcome in multiple cancer types. Here, the authors use shallow WGS to create machine learning signatures to identify tumor phenotypes and predict therapy response in patients with metastatic breast cancer.

Identifying all essential genomic components is critical for the assembly of minimal artificial life. In the genome‐reduced bacterium Mycoplasma pneumoniae , we found that small ORFs (smORFs; < 100 residues), accounting for 10% of all ORFs, are the most frequently essential genomic components (53%), followed by conventional ORFs (49%). Essentiality of smORFs may be explained by their function as members of protein and/or DNA/RNA complexes. In larger proteins, essentiality applied to individual domains and not entire proteins, a notion we could confirm by expression of truncated domains. The fraction of essential non‐coding RNAs (ncRNAs) non‐overlapping with essential genes is 5% higher than of non‐transcribed regions (0.9%), pointing to the important functions of the former. We found that the minimal essential genome is comprised of 33% (269,410 bp) of the M. pneumoniae genome. Our data highlight an unexpected hidden layer of smORFs with essential functions, as well as non‐coding regions, thus changing the focus when aiming to define the minimal essential genome. Synopsis A genome essentiality analysis in the genome‐reduced bacterium Mycoplasma pneumoniae , reveals that protein essentiality should be considered at the domain level and that small proteins (< 100 aa) and ncRNAs are frequently essential genomic elements. A genome essentiality analysis is performed using two mini‐transposon mutant libraries of M. pneumoniae . The results indicate that ORF essentiality should be considered at the protein domain level. Small ORFs are as essential as conventional ORFs and they can interact with DNA. Some essential antisense ncRNAs are involved in the regulation of essential ORF expression. Graphical Abstract A genome essentiality analysis in the genome‐reduced bacterium Mycoplasma pneumoniae , reveals that protein essentiality should be considered at the domain level and that small proteins (< 100 aa) and ncRNAs are frequently essential genomic elements.

Dormant or slow-cycling tumor cells can form a residual chemoresistant reservoir responsible for relapse in patients, years after curative surgery and adjuvant therapy. We have adapted the pulse-chase expression of H2BeGFP for labeling and isolating slow-cycling cancer cells (SCCCs). SCCCs showed cancer initiation potential and enhanced chemoresistance. Cells at this slow-cycling status presented a distinctive nongenetic and cell-autonomous gene expression profile shared across different tumor types. We identified TET2 epigenetic enzyme as a key factor controlling SCCC numbers, survival, and tumor recurrence. 5-Hydroxymethylcytosine (5hmC), generated by TET2 enzymatic activity, labeled the SCCC genome in carcinomas and was a predictive biomarker of relapse and survival in cancer patients. We have shown the enhanced chemoresistance of SCCCs and revealed 5hmC as a biomarker for their clinical identification and TET2 as a potential drug target for SCCC elimination that could extend patients' survival.

The human transcriptome contains thousands of small open reading frames (sORFs) that encode microproteins whose functions remain largely unexplored. Here, we show that TINCR lncRNA encodes pTINCR, an evolutionary conserved ubiquitin-like protein (UBL) expressed in many epithelia and upregulated upon differentiation and under cellular stress. By gain- and loss-of-function studies, we demonstrate that pTINCR is a key inducer of epithelial differentiation in vitro and in vivo. Interestingly, low expression of TINCR associates with worse prognosis in several epithelial cancers, and pTINCR overexpression reduces malignancy in patient-derived xenografts. At the molecular level, pTINCR binds to SUMO through its SUMO interacting motif (SIM) and to CDC42, a Rho-GTPase critical for actin cytoskeleton remodeling and epithelial differentiation. Moreover, pTINCR increases CDC42 SUMOylation and promotes its activation, triggering a pro-differentiation cascade. Our findings suggest that the microproteome is a source of new regulators of cell identity relevant for cancer. Small proteins encoded by previously assumed non-coding RNAs can have cell regulatory functions. Here the authors report that TINCR lncRNA encodes pTINCR, a ubiquitin-like protein (UBL) that promotes epithelial differentiation through the SUMOylation and activation of CDC42, and it has tumour suppressor activity in epithelial cancers.