Genome-wide cell-free DNA fragmentation in patients with cancer.

Cell-free DNA in the blood provides a non-invasive diagnostic avenue for patients with cancer1. However, characteristics of the origins and molecular features of cell-free DNA are poorly understood. Here we developed an approach to evaluate fragmentation patterns of cell-free DNA across the genome, and found that profiles of healthy individuals reflected nucleosomal patterns of white blood cells, whereas patients with cancer had altered fragmentation profiles. We used this method to analyse the fragmentation profiles of 236 patients with breast, colorectal, lung, ovarian, pancreatic, gastric or bile duct cancer and 245 healthy individuals. A machine learning model that incorporated genome-wide fragmentation features had sensitivities of detection ranging from 57% to more than 99% among the seven cancer types at 98% specificity, with an overall area under the curve value of 0.94. Fragmentation profiles could be used to identify the tissue of origin of the cancers to a limited number of sites in 75% of cases. Combining our approach with mutation-based cell-free DNA analyses detected 91% of patients with cancer. The results of these analyses highlight important properties of cell-free DNA and provide a proof-of-principle approach for the screening, early detection and monitoring of human cancer.

Genome-wide repeat landscapes in cancer and cell-free DNA.

Genetic changes in repetitive sequences are a hallmark of cancer and other diseases, but characterizing these has been challenging using standard sequencing approaches. We developed a de novo kmer finding approach, called ARTEMIS (Analysis of RepeaT EleMents in dISease), to identify repeat elements from whole-genome sequencing. Using this method, we analyzed 1.2 billion kmers in 2837 tissue and plasma samples from 1975 patients, including those with lung, breast, colorectal, ovarian, liver, gastric, head and neck, bladder, cervical, thyroid, or prostate cancer. We identified tumor-specific changes in these patients in 1280 repeat element types from the LINE, SINE, LTR, transposable element, and human satellite families. These included changes to known repeats and 820 elements that were not previously known to be altered in human cancer. Repeat elements were enriched in regions of driver genes, and their representation was altered by structural changes and epigenetic states. Machine learning analyses of genome-wide repeat landscapes and fragmentation profiles in cfDNA detected patients with early-stage lung or liver cancer in cross-validated and externally validated cohorts. In addition, these repeat landscapes could be used to noninvasively identify the tissue of origin of tumors. These analyses reveal widespread changes in repeat landscapes of human cancers and provide an approach for their detection and characterization that could benefit early detection and disease monitoring of patients with cancer.

DNA methylation and gene expression as determinants of genome-wide cell-free DNA fragmentation.

Circulating cell-free DNA (cfDNA) is emerging as an avenue for cancer detection, but the characteristics of cfDNA fragmentation in the blood are poorly understood. We evaluate the effect of DNA methylation and gene expression on genome-wide cfDNA fragmentation through analysis of 969 individuals. cfDNA fragment ends more frequently contained CCs or CGs, and fragments ending with CGs or CCGs are enriched or depleted, respectively, at methylated CpG positions. Higher levels and larger sizes of cfDNA fragments are associated with CpG methylation and reduced gene expression. These effects are validated in mice with isogenic tumors with or without the mutant IDH1, and are associated with genome-wide changes in cfDNA fragmentation in patients with cancer. Tumor-related hypomethylation and increased gene expression are associated with decrease in cfDNA fragment size that may explain smaller cfDNA fragments in human cancers. These results provide a connection between epigenetic changes and cfDNA fragmentation with implications for disease detection.

Early detection of ovarian cancer using cell-free DNA fragmentomes and protein biomarkers.

Ovarian cancer is a leading cause of death for women worldwide in part due to ineffective screening methods. In this study, we used whole-genome cell-free DNA (cfDNA) fragmentome and protein biomarker (CA-125 and HE4) analyses to evaluate 591 women with ovarian cancer, benign adnexal masses, or without ovarian lesions. Using a machine learning model with the combined features, we detected ovarian cancer with specificity >99% and sensitivity of 72%, 69%, 87%, and 100% for stages I-IV, respectively. At the same specificity, CA-125 alone detected 34%, 62%, 63%, and 100% of ovarian cancers for stages I-IV. Our approach differentiated benign masses from ovarian cancers with high accuracy (AUC=0.88, 95% CI=0.83-0.92). These results were validated in an independent population. These findings show that integrated cfDNA fragmentome and protein analyses detect ovarian cancers with high performance, enabling a new accessible approach for noninvasive ovarian cancer screening and diagnostic evaluation.

Cell-Free DNA Fragmentomes in the Diagnostic Evaluation of Patients With Symptoms Suggestive of Lung Cancer.

BACKGROUND: The diagnostic workup of individuals suspected of having lung cancer can be complex and protracted because conventional symptoms of lung cancer have low specificity and sensitivity. RESEARCH QUESTION: Among individuals with symptoms of lung cancer, can a blood-based approach to analyze cell-free DNA (cfDNA) fragmentation (the DNA evaluation of fragments for early interception [DELFI] score) enhance evaluation for the possible presence of lung cancer? STUDY DESIGN AND METHODS: Adults were referred to Bispebjerg Hospital (Copenhagen, Denmark) for diagnostic evaluation of initial imaging anomalies and symptoms consistent with lung cancer. Numbers and types of symptoms were extracted from medical records. cfDNA from plasma samples obtained at the prediagnostic visit was isolated, sequenced, and analyzed for genome-wide cfDNA fragmentation patterns. The relationships among clinical presentation, cancer status, and DELFI score were examined. RESULTS: A total of 296 individuals were analyzed. Median DELFI scores were higher for those with lung cancer (n = 98) than those without cancer (n = 198; 0.94 vs 0.19; P < .001). In a multivariate model adjusted for age, smoking history, and presenting symptoms, the addition of the DELFI score improved the prediction of lung cancer for those who demonstrated symptoms (area under the receiver operating characteristic curve, 0.74-0.94). INTERPRETATION: The DELFI score distinguishes individuals with lung cancer from those without cancer better than suspicious symptoms do. These results represent proof-of-concept support that fragmentation-based biomarker approaches may facilitate diagnostic resolution for patients with concerning symptoms of lung cancer.

Single-molecule genome-wide mutation profiles of cell-free DNA for non-invasive detection of cancer.

Somatic mutations are a hallmark of tumorigenesis and may be useful for non-invasive diagnosis of cancer. We analyzed whole-genome sequencing data from 2,511 individuals in the Pan-Cancer Analysis of Whole Genomes (PCAWG) study as well as 489 individuals from four prospective cohorts and found distinct regional mutation type-specific frequencies in tissue and cell-free DNA from patients with cancer that were associated with replication timing and other chromatin features. A machine-learning model using genome-wide mutational profiles combined with other features and followed by CT imaging detected >90% of patients with lung cancer, including those with stage I and II disease. The fixed model was validated in an independent cohort, detected patients with cancer earlier than standard approaches and could be used to monitor response to therapy. This approach lays the groundwork for non-invasive cancer detection using genome-wide mutation features that may facilitate cancer screening and monitoring.

Detecting Liver Cancer Using Cell-Free DNA Fragmentomes.

Liver cancer is a major cause of cancer mortality worldwide. Screening individuals at high risk, including those with cirrhosis and viral hepatitis, provides an avenue for improved survival, but current screening methods are inadequate. In this study, we used whole-genome cell-free DNA (cfDNA) fragmentome analyses to evaluate 724 individuals from the United States, the European Union, or Hong Kong with hepatocellular carcinoma (HCC) or who were at average or high-risk for HCC. Using a machine learning model that incorporated multifeature fragmentome data, the sensitivity for detecting cancer was 88% in an average-risk population at 98% specificity and 85% among high-risk individuals at 80% specificity. We validated these results in an independent population. cfDNA fragmentation changes reflected genomic and chromatin changes in liver cancer, including from transcription factor binding sites. These findings provide a biological basis for changes in cfDNA fragmentation in patients with liver cancer and provide an accessible approach for noninvasive cancer detection. SIGNIFICANCE: There is a great need for accessible and sensitive screening approaches for HCC worldwide. We have developed an approach for examining genome-wide cfDNA fragmentation features to provide a high-performing and cost-effective approach for liver cancer detection. See related commentary Rolfo and Russo, p. 532. This article is highlighted in the In This Issue feature, p. 517.

Detection and characterization of lung cancer using cell-free DNA fragmentomes.

Non-invasive approaches for cell-free DNA (cfDNA) assessment provide an opportunity for cancer detection and intervention. Here, we use a machine learning model for detecting tumor-derived cfDNA through genome-wide analyses of cfDNA fragmentation in a prospective study of 365 individuals at risk for lung cancer. We validate the cancer detection model using an independent cohort of 385 non-cancer individuals and 46 lung cancer patients. Combining fragmentation features, clinical risk factors, and CEA levels, followed by CT imaging, detected 94% of patients with cancer across stages and subtypes, including 91% of stage I/II and 96% of stage III/IV, at 80% specificity. Genome-wide fragmentation profiles across ~13,000 ASCL1 transcription factor binding sites distinguished individuals with small cell lung cancer from those with non-small cell lung cancer with high accuracy (AUC = 0.98). A higher fragmentation score represented an independent prognostic indicator of survival. This approach provides a facile avenue for non-invasive detection of lung cancer.

Integrative Tumor and Immune Cell Multi-omic Analyses Predict Response to Immune Checkpoint Blockade in Melanoma.

In this study, we incorporate analyses of genome-wide sequence and structural alterations with pre- and on-therapy transcriptomic and T cell repertoire features in immunotherapy-naive melanoma patients treated with immune checkpoint blockade. Although tumor mutation burden is associated with improved treatment response, the mutation frequency in expressed genes is superior in predicting outcome. Increased T cell density in baseline tumors and dynamic changes in regression or expansion of the T cell repertoire during therapy distinguish responders from non-responders. Transcriptome analyses reveal an increased abundance of B cell subsets in tumors from responders and patterns of molecular response related to expressed mutation elimination or retention that reflect clinical outcome. High-dimensional genomic, transcriptomic, and immune repertoire data were integrated into a multi-modal predictor of response. These findings identify genomic and transcriptomic characteristics of tumors and immune cells that predict response to immune checkpoint blockade and highlight the importance of pre-existing T and B cell immunity in therapeutic outcomes.

Multimodal genomic features predict outcome of immune checkpoint blockade in non-small-cell lung cancer.

Despite progress in immunotherapy, identifying patients that respond has remained a challenge. Through analysis of whole-exome and targeted sequence data from 5,449 tumors, we found a significant correlation between tumor mutation burden (TMB) and tumor purity, suggesting that low tumor purity tumors are likely to have inaccurate TMB estimates. We developed a new method to estimate a corrected TMB (cTMB) that was adjusted for tumor purity and more accurately predicted outcome to immune checkpoint blockade (ICB). To identify improved predictive markers together with cTMB, we performed whole-exome sequencing for 104 lung tumors treated with ICB. Through comprehensive analyses of sequence and structural alterations, we discovered a significant enrichment in activating mutations in receptor tyrosine kinase (RTK) genes in nonresponding tumors in three immunotherapy treated cohorts. An integrated multivariable model incorporating cTMB, RTK mutations, smoking-related mutational signature and human leukocyte antigen status provided an improved predictor of response to immunotherapy that was independently validated.

White blood cell and cell-free DNA analyses for detection of residual disease in gastric cancer.

Liquid biopsies are providing new opportunities for detection of residual disease in cell-free DNA (cfDNA) after surgery but may be confounded through identification of alterations arising from clonal hematopoiesis. Here, we identify circulating tumor-derived DNA (ctDNA) alterations through ultrasensitive targeted sequencing analyses of matched cfDNA and white blood cells from the same patient. We apply this approach to analyze samples from patients in the CRITICS trial, a phase III randomized controlled study of perioperative treatment in patients with operable gastric cancer. After filtering alterations from matched white blood cells, the presence of ctDNA predicts recurrence when analyzed within nine weeks after preoperative treatment and after surgery in patients eligible for multimodal treatment. These analyses provide a facile method for distinguishing ctDNA from other cfDNA alterations and highlight the utility of ctDNA as a predictive biomarker of patient outcome to perioperative cancer therapy and surgical resection in patients with gastric cancer.

Early Noninvasive Detection of Response to Targeted Therapy in Non-Small Cell Lung Cancer.

With the advent of precision oncology, there is an urgent need to develop improved methods for rapidly detecting responses to targeted therapies. Here, we have developed an ultrasensitive measure of cell-free tumor load using targeted and whole-genome sequencing approaches to assess responses to tyrosine kinase inhibitors in patients with advanced lung cancer. Analyses of 28 patients treated with anti-EGFR or HER2 therapies revealed a bimodal distribution of cell-free circulating tumor DNA (ctDNA) after therapy initiation, with molecular responders having nearly complete elimination of ctDNA (>98%). Molecular nonresponders displayed limited changes in ctDNA levels posttreatment and experienced significantly shorter progression-free survival (median 1.6 vs. 13.7 months, P < 0.0001; HR = 66.6; 95% confidence interval, 13.0-341.7), which was detected on average 4 weeks earlier than CT imaging. ctDNA analyses of patients with radiographic stable or nonmeasurable disease improved prediction of clinical outcome compared with CT imaging. These analyses provide a rapid approach for evaluating therapeutic response to targeted therapies and have important implications for the management of patients with cancer and the development of new therapeutics.Significance: Cell-free tumor load provides a novel approach for evaluating longitudinal changes in ctDNA during systemic treatment with tyrosine kinase inhibitors and serves an unmet clinical need for real-time, noninvasive detection of tumor response to targeted therapies before radiographic assessment.See related commentary by Zou and Meyerson, p. 1038.

Dynamics of Tumor and Immune Responses during Immune Checkpoint Blockade in Non-Small Cell Lung Cancer.

Despite the initial successes of immunotherapy, there is an urgent clinical need for molecular assays that identify patients more likely to respond. Here, we report that ultrasensitive measures of circulating tumor DNA (ctDNA) and T-cell expansion can be used to assess responses to immune checkpoint blockade in metastatic lung cancer patients (N = 24). Patients with clinical response to therapy had a complete reduction in ctDNA levels after initiation of therapy, whereas nonresponders had no significant changes or an increase in ctDNA levels. Patients with initial response followed by acquired resistance to therapy had an initial drop followed by recrudescence in ctDNA levels. Patients without a molecular response had shorter progression-free and overall survival compared with molecular responders [5.2 vs. 14.5 and 8.4 vs. 18.7 months; HR 5.36; 95% confidence interval (CI), 1.57-18.35; P = 0.007 and HR 6.91; 95% CI, 1.37-34.97; P = 0.02, respectively], which was detected on average 8.7 weeks earlier and was more predictive of clinical benefit than CT imaging. Expansion of T cells, measured through increases of T-cell receptor productive frequencies, mirrored ctDNA reduction in response to therapy. We validated this approach in an independent cohort of patients with early-stage non-small cell lung cancer (N = 14), where the therapeutic effect was measured by pathologic assessment of residual tumor after anti-PD1 therapy. Consistent with our initial findings, early ctDNA dynamics predicted pathologic response to immune checkpoint blockade. These analyses provide an approach for rapid determination of therapeutic outcomes for patients treated with immune checkpoint inhibitors and have important implications for the development of personalized immune targeted strategies.Significance: Rapid and sensitive detection of circulating tumor DNA dynamic changes and T-cell expansion can be used to guide immune targeted therapy for patients with lung cancer.See related commentary by Zou and Meyerson, p. 1038.

Integrated Genomic, Epigenomic, and Expression Analyses of Ovarian Cancer Cell Lines.

To improve our understanding of ovarian cancer, we performed genome-wide analyses of 45 ovarian cancer cell lines. Given the challenges of genomic analyses of tumors without matched normal samples, we developed approaches for detection of somatic sequence and structural changes and integrated these with epigenetic and expression alterations. Alterations not previously implicated in ovarian cancer included amplification or overexpression of ASXL1 and H3F3B, deletion or underexpression of CDC73 and TGF-beta receptor pathway members, and rearrangements of YAP1-MAML2 and IKZF2-ERBB4. Dose-response analyses to targeted therapies revealed unique molecular dependencies, including increased sensitivity of tumors with PIK3CA and PPP2R1A alterations to PI3K inhibitor GNE-493, MYC amplifications to PARP inhibitor BMN673, and SMAD3/4 alterations to MEK inhibitor MEK162. Genome-wide rearrangements provided an improved measure of sensitivity to PARP inhibition. This study provides a comprehensive and broadly accessible resource of molecular information for the development of therapeutic avenues in ovarian cancer.