Cancer stem cells (CSC) are supported by the tumor microenvironment, and non-CSCs can regain CSC phenotypes in certain niches, leading to limited clinical benefits of CSC-targeted therapy. A better understanding of the mechanisms governing the orchestration of the CSC niche could help improve the therapeutic targeting of CSCs. Here, we report that Rab13, a small GTPase, is highly expressed in breast CSCs (BCSC). Rab13 depletion suppressed breast cancer cell stemness, tumorigenesis, and chemoresistance by reducing tumor-stroma cross-talk. Accordingly, Rab13 controlled the membrane translocation of C-X-C chemokine receptor type 1/2 (CXCR1/2), allowing tumor cells to interact with tumor-associated macrophages and cancer-associated fibroblasts to establish a supportive BCSC niche. Targeting the Rab13-mediated BCSC niche with bardoxolone-methyl (C-28 methyl ester of 2-cyano-3, 12-dioxoolen-1, 9-dien-28-oic acid; CDDO-Me) prevented BCSC stemness in vitro and in vivo. These findings highlight the novel regulatory mechanism of Rab13 in BCSC, with important implications for the development of therapeutic strategies for disrupting the BCSC niche. SIGNIFICANCE: Targeting Rab13 perturbs formation of the breast cancer stem cell niche by inhibiting cross-talk between cancer cells and the tumor microenvironment, providing a therapeutic opportunity for niche-targeted breast cancer treatment.

A plethora of treatment options exist for cancer therapeutics, but many are limited by side effects and either intrinsic or acquired resistance. The need for more effective targeted cancer treatment has led to the focus on forkhead box (FOX) transcription factors as possible drug targets. Forkhead factors such as FOXA1 and FOXM1 are involved in hormone regulation, immune system modulation, and disease progression through their regulation of the epithelial-mesenchymal transition. Forkhead factors can influence cancer development, progression, metastasis, and drug resistance. In this review, we discuss the various roles of forkhead factors in biological processes that support cancer as well as their function as pioneering factors and their potential as targetable transcription factors in the fight against cancer.

Innate immune cells participate in the detection of tumor cells via complex signaling pathways mediated by pattern-recognition receptors, such as Toll-like receptors and nucleotide-binding and oligomerization domain-like receptors. These pathways are finely tuned via multiple mechanisms, including epigenetic regulation. It is well established that hematopoietic progenitors generate innate immune cells that can regulate cancer cell behavior, and the disruption of normal hematopoiesis in pathologic states may lead to altered immunity and the development of cancer. In this review, we discuss the epigenetic and transcriptional mechanisms that underlie the initiation and amplification of innate immune signaling in cancer. We also discuss new targeting possibilities for cancer control that exploit innate immune cells and signaling molecules, potentially heralding the next generation of immunotherapy.

Erratum in Cancer Res. 2022 Aug 3;82(15):2808.

The FDA has recently approved a high tumor mutational burden (TMB-high) biomarker, defined by ≥10 mutations/Mb, for the treatment of solid tumors with pembrolizumab, an immune checkpoint inhibitor (ICI) that targets PD1. However, recent studies have shown that this TMB-high biomarker is only able to stratify ICI responders in a subset of cancer types, and the mechanisms underlying this observation have remained unknown. The tumor immune microenvironment (TME) may modulate the stratification power of TMB (termed TMB power), determining if it will be predictive of ICI response in a given cancer type. To systematically study this hypothesis, we inferred the levels of 31 immune-related factors characteristic of the TME of different cancer types in The Cancer Genome Atlas. Integration of this information with TMB and response data of 2,277 patients treated with anti-PD1 identified key immune factors that determine TMB power across 14 different cancer types. We find that high levels of M1 macrophages and low resting dendritic cells in the TME characterized cancer types with high TMB power. A model based on these two immune factors strongly predicted TMB power in a given cancer type during cross-validation and testing (Spearman Rho = 0.76 and 1, respectively). Using this model, we predicted the TMB power in nine additional cancer types, including rare cancers, for which TMB and ICI response data are not yet publicly available. Our analysis indicates that TMB-high may be highly predictive of ICI response in cervical squamous cell carcinoma, suggesting that such a study should be prioritized. SIGNIFICANCE: This study uncovers immune-related factors that may modulate the relationship between high tumor mutational burden and ICI response, which can help prioritize cancer types for clinical trials.

Androgen receptor (AR) pathway inhibitors are the mainstay treatment for advanced prostate cancer, but resistance to therapy is common. Here, we used a CRISPR activation screen in metastatic castration-sensitive prostate cancer cells to identify genes that promote resistance to AR inhibitors. Activation of the TGFβ target gene paired-related homeobox2 (PRRX2) promoted enzalutamide resistance. PRRX2 expression was the highest in double-negative prostate cancer (DNPC), which lack AR signaling and neuroendocrine differentiation, and a PRRX2-related gene signature identified a subset of patients with DNPC with reduced overall survival. PRRX2-expressing cells showed alterations in the CDK4/6/Rb/E2F and BCL2 pathways. Accordingly, treatment with CDK4/6 and BCL2 inhibitors sensitized PRRX2-expressing, castration-resistant tumors to enzalutamide. Overall, PRRX2 was identified as a driver of enzalutamide resistance. The PRRX2 signature merits investigation as a biomarker of enzalutamide resistance in prostate cancer that could be reversed with CDK4/6 and BCL2 inhibitors. SIGNIFICANCE: PRRX2 mediates enzalutamide resistance via activation of the E2F and BCL2 pathways, which can be targeted with CDK4/6 and BCL2 inhibitors to reverse resistance.

Targeting immune checkpoints such as programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) has transformed cancer treatment, with durable clinical responses across a wide range of tumor types. However, a high percentage of patients fail to respond to anti-PD-1/PD-L1 treatment. A greater understanding of PD-L1 regulation is critical to improving the clinical response rate of PD-1/PD-L1 blockade. Here, we demonstrate that PD-L1 is phosphorylated and stabilized by casein kinase 2 (CK2) in cancer and dendritic cells (DC). Phosphorylation of PD-L1 at Thr285 and Thr290 by CK2 disrupted PD-L1 binding with speckle-type POZ protein, an adaptor protein of the cullin 3 (CUL3) ubiquitin E3 ligase complex, protecting PD-L1 from CUL3-mediated proteasomal degradation. Inhibition of CK2 decreased PD-L1 protein levels by promoting its degradation and resulted in the release of CD80 from DC to reactivate T-cell function. In a syngeneic mouse model, combined treatment with a CK2 inhibitor and an antibody against T-cell immunoglobulin mucin-3 (Tim-3) suppressed tumor growth and prolonged survival. These findings uncover a mechanism by which PD-L1 is regulated and suggest a potential antitumor treatment option to activate DC function by blocking the CK2-PD-L1 pathway and inhibiting Tim-3. SIGNIFICANCE: This work identifies a role for CK2 in immunosuppression by phosphorylation and stabilization of PD-L1, identifying CK2 inhibition as an immunotherapeutic approach for treating cancer.

Understanding the genetic variation underlying transcript splicing is essential for fully dissecting the molecular mechanisms of common diseases. The available evidence from splicing quantitative trait locus (sQTL) studies using pancreatic ductal adenocarcinoma (PDAC) tissues have been limited to small sample sizes. Here we present a genome-wide sQTL analysis to identify SNP that control mRNA splicing in 176 PDAC samples from TCGA. From this analysis, 16,175 sQTLs were found to be significantly enriched in RNA-binding protein (RBP) binding sites and chromatin regulatory elements and overlapped with known loci from PDAC genome-wide association studies (GWAS). sQTLs and expression quantitative trait loci (eQTL) showed mostly nonoverlapping patterns, suggesting sQTLs provide additional insights into the etiology of disease. Target genes affected by sQTLs were closely related to cancer signaling pathways, high mutational burden, immune infiltration, and pharmaceutical targets, which will be helpful for clinical applications. Integration of a large-scale population consisting of 2,782 patients with PDAC and 7,983 healthy controls identified an sQTL variant rs1785932-T allele that promotes alternative splicing of ELP2 exon 6 and leads to a lower level of the ELP2 full-length isoform (ELP2_V1) and a higher level of a truncated ELP2 isoform (ELP2_V2), resulting in decreased risk of PDAC [OR = 0.83; 95% confidence interval (CI), 0.77-0.89; P = 1.16 × 10-6]. The ELP2_V2 isoform functioned as a potential tumor suppressor gene, inhibiting PDAC cell proliferation by exhibiting stronger binding affinity to JAK1/STAT3 than ELP2_V1 and subsequently blocking the pathologic activation of the phosphorylated STAT3 (pSTAT3) pathway. Collectively, these findings provide an informative sQTL resource and insights into the regulatory mechanisms linking splicing variants to PDAC risk. SIGNIFICANCE: In pancreatic cancer, splicing quantitative trait loci analysis identifies a rs1785932 variant that contributes to decreased risk of disease by influencing ELP2 mRNA splicing and blocking the STAT3 oncogenic pathway.

Inactivating mutations of von Hippel-Lindau (VHL) are highly prevalent in clear cell renal cell carcinoma (ccRCC). Improved understanding of the vulnerabilities of VHL-deficient ccRCC could lead to improved treatment strategies. The activity of DNA dioxygenase ten-eleven translocation (TET)2 is significantly reduced in multiple cancers by different mechanisms, but its role in ccRCC progression remains unclear. Here, we report that increased expression of TET2, but not TET1 and TET3, is negatively associated with tumor metastasis and advanced tumor stage and is positively associated with good prognosis uniquely in ccRCC among all 33 types of cancer in The Cancer Genome Atlas datasets. TET2 restrained glycolysis and pentose phosphate pathway metabolism in a VHL deficiency-dependent manner, thereby suppressing ccRCC progression. Notably, TET2 and VHL mutations tended to cooccur in ccRCC, providing genetic evidence that they cooperate to inhibit the progression of ccRCC. Mechanistically, TET2 was recruited by transcription factor HNF4α to activate FBP1 expression, which antagonized the function of hypoxia-inducible factor-1/2α (HIF1/2α) in metabolic reprogramming to impede ccRCC growth. Stimulating the TET2-FBP1 axis with vitamin C repressed the growth of VHL-deficient ccRCC with wild-type TET2 and increased the sensitivity to glycolysis inhibitors. Moreover, combined expression levels of the HNF4α-TET2-FBP1 axis served as a biomarker of prognosis in patients with ccRCC. This study reveals a unique function of TET2 in the suppression of tumor metabolism and HIF signaling, and it also provides therapeutic targets, potential drugs, and prognostic markers for the management of ccRCC. SIGNIFICANCE: The identification of TET2-mediated inhibition of HIF signaling and tumor metabolic reprogramming provides insights for new therapeutic strategies for VHL-deficient ccRCC.

Comment in Cancer Res. 2022 Jun 6;82(11):2069-2071.

Comment on Cancer Res. 2017 Nov 1;77(21):e104-e107.

Comment on Cancer Res. 2022 Jun 6;82(11):2156-2170.

The protein tyrosine phosphatase SHP2 is crucial for oncogenic transformation of acute myeloid leukemia (AML) cells expressing mutated receptor tyrosine kinases. SHP2 is required for full RAS-ERK activation to promote cell proliferation and survival programs. Allosteric SHP2 inhibitors act by stabilizing SHP2 in its autoinhibited conformation and are currently being tested in clinical trials for tumors with overactivation of the RAS/ERK pathway, alone and in various drug combinations. In this study, we established cells with acquired resistance to the allosteric SHP2 inhibitor SHP099 from two FLT3-ITD (internal tandem duplication)-positive AML cell lines. Label-free and isobaric labeling quantitative mass spectrometry-based phosphoproteomics of these resistant models demonstrated that AML cells can restore phosphorylated ERK (pERK) in the presence of SHP099, thus developing adaptive resistance. Mechanistically, SHP2 inhibition induced tyrosine phosphorylation and feedback-driven activation of the FLT3 receptor, which in turn phosphorylated SHP2 on tyrosine 62. This phosphorylation stabilized SHP2 in its open conformation, preventing SHP099 binding and conferring resistance. Combinatorial inhibition of SHP2 and MEK or FLT3 prevented pERK rebound and resistant cell growth. The same mechanism was observed in a FLT3-mutated B-cell acute lymphoblastic leukemia cell line and in the inv(16)/KitD816Y AML mouse model, but allosteric inhibition of Shp2 did not impair the clonogenic ability of normal bone marrow progenitors. Together, these results support the future use of SHP2 inhibitor combinations for clinical applications. SIGNIFICANCE: These findings suggest that combined inhibition of SHP2 and FLT3 effectively treat FLT3-ITD-positive AML, highlighting the need for development of more potent SHP2 inhibitors and combination therapies for clinical applications.

The molecular characterization of cancer through genomics, data from multiomics technologies, molecular-driven clinical trials, and internet-enabled devices capturing patient context and real-world data are creating an unprecedented big data revolution across the cancer research-care continuum. While big data has translated to benefit for some patients, it has also created new problems. Our intent in this brief communication is to explore some examples of progress and key challenges that remain. The problems are not intractable, but success will require rethinking and rebuilding an information and evidence-based learning system that moves beyond paralysis to shape a better future for patients with cancer.

Circular RNAs (circRNA) containing retained introns are normally sequestered in the nucleus. Dysregulation of cellular homeostasis can drive their nuclear export, which may be involved in cancer metastasis. However, the mechanism underlying circRNA nuclear export and its role in lymph node (LN) metastasis of bladder cancer remain unclear. Here, we identify an intron-retained circRNA, circNCOR1, that is significantly downregulated in LN metastatic bladder cancer and is negatively associated with poor prognosis of patients. Overexpression of circNCOR1 inhibited lymphangiogenesis and LN metastasis of bladder cancer in vitro and in vivo. Nuclear circNCOR1 epigenetically promoted SMAD7 transcription by increasing heterogeneous nuclear ribonucleoprotein L (hnRNPL)-induced H3K9 acetylation in the SMAD7 promoter, leading to inhibition of the TGFβ-SMAD signaling pathway. Nuclear retention of circNCOR1 was regulated by small ubiquitin-like modifier (SUMO)ylation of DDX39B, an essential regulatory factor responsible for circRNA nuclear-cytoplasmic transport. Reduced SUMO2 binding to DDX39B markedly increased circNCOR1 retention in the nucleus to suppress bladder cancer LN metastasis. By contrast, SUMOylated DDX39B activated nuclear export of circNCOR1, impairing the suppressive role of circNCOR1 on TGFβ-SMAD cascade activation and bladder cancer LN metastasis. In patient-derived xenograft (PDX) models, overexpression of circNCOR1 and inhibition of TGFβ signaling significantly repressed tumor growth and LN metastasis. This study highlights SUMOylation-induced nuclear export of circNCOR1 as a key event regulating TGFβ-SMAD signaling and bladder cancer lymphangiogenesis, thus supporting circNCOR1 as a novel therapeutic agent for patients with LN metastatic bladder cancer. SIGNIFICANCE: This study identifies the novel intron-retained circNCOR1 and elucidates a SUMOylation-mediated DDX39B-circNCOR1-SMAD7 axis that regulates lymph node metastasis of bladder cancer.

Immunosuppressive elements within the tumor microenvironment, such as tumor-associated macrophages (TAM), can present a barrier to successful antitumor responses by cytolytic T cells. Here we employed preclinical syngeneic p53 null mouse models of triple-negative breast cancer (TNBC) to develop a treatment regimen that harnessed the immunostimulatory effects of low-dose cyclophosphamide coupled with the pharmacologic inhibition of TAMs using either a small-molecule CSF1R inhibitor or an anti-CSF1R antibody. This therapeutic combination was effective in treating several highly aggressive TNBC murine mammary tumor and lung metastasis models. Single-cell RNA sequencing characterized tumor-infiltrating lymphocytes including Th cells and antigen-presenting B cells that were highly enriched in responders to combination therapy. In one model that exhibited long-term posttreatment tumor regression, high-dimensional imaging techniques identified the close spatial localization of B220+/CD86+-activated B cells and CD4+ T cells in tertiary lymphoid structures that were present up to 6 weeks posttreatment. The transcriptional and metabolic heterogeneity of TAMs was also characterized in two closely related claudin-low/mesenchymal subtype tumor models with differential treatment responses. A murine TAM signature derived from the T12 model was highly conserved in human claudin-low breast cancers, and high expression of the TAM signature correlated with reduced overall survival in patients with breast cancer. This TAM signature may help identify human patients with claudin-low breast cancer that will benefit from the combination of cyclophosphamide and anti-CSF1R therapy. These studies illustrate the complexity of the tumor immune microenvironment and highlight different immune responses that result from rational immunotherapy combinations. SIGNIFICANCE: Immunostimulatory chemotherapy combined with pharmacologic inhibition of TAMs results in durable treatment responses elicited by Th cells and B cells in claudin-low TNBC models.

Identifying colorectal cancer patient populations responsive to chemotherapy or chemoradiation therapy before surgery remains a challenge. Recently validated mouse protocols for organoid irradiation employ the single hit multi-target (SHMT) algorithm, which yields a single value, the D0, as a measure of inherent tissue radiosensitivity. Here, we translate these protocols to human tissue to evaluate radioresponsiveness of patient-derived organoids (PDO) generated from normal human intestines and rectal tumors of patients undergoing neoadjuvant therapy. While PDOs from adenomas with a logarithmically expanded Lgr5+ intestinal stem cell population retain the radioresistant phenotype of normal colorectal PDOs, malignant transformation yields PDOs from a large patient subpopulation displaying marked radiosensitivity due to reduced homologous recombination-mediated DNA repair. A proof-of-principle pilot clinical trial demonstrated that rectal cancer patient responses to neoadjuvant chemoradiation, including complete response, correlate closely with their PDO D0 values. Overall, upon transformation to colorectal adenocarcinoma, broad radiation sensitivity occurs in a large subset of patients that can be identified using SHMT analysis of PDO radiation responses. SIGNIFICANCE: Analysis of inherent tissue radiosensitivity of patient-derived organoids may provide a readout predictive of neoadjuvant therapy response to radiation in rectal cancer, potentially allowing pretreatment stratification of patients likely to benefit from this approach.

Comment in Cancer Res. 2022 Jun 15;82(12):2216-2218.

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease with few effective treatments. Here we show that the mitochondrial calcium uniporter (MCU) promotes PDAC cell migration, invasion, metastasis, and metabolic stress resistance by activating the Keap1-Nrf2 antioxidant program. The cystine transporter SLC7A11 was identified as a druggable target downstream of the MCU-Nrf2 axis. Paradoxically, despite the increased ability to uptake cystine, MCU-overexpressing PDAC demonstrated characteristics typical of cystine-deprived cells and were hypersensitive to cystine deprivation-induced ferroptosis. Pharmacologic inhibitors of SLC7A11 effectively induced tumor regression and abrogated MCU-driven metastasis in PDAC. In patient-derived organoid models in vitro and patient-derived xenograft models in vivo, MCU-high PDAC demonstrated increased sensitivity to SLC7A11 inhibition compared with MCU-low tumors. These data suggest that MCU is able to promote resistance to metabolic stress and to drive PDAC metastasis in a cystine-dependent manner. MCU-mediated cystine addiction could be exploited as a therapeutic vulnerability to inhibit PDAC tumor growth and to prevent metastasis. SIGNIFICANCE: Elevated mitochondrial calcium uptake in PDAC promotes metastasis but exposes cystine addiction and ferroptosis sensitivity that could be targeted to improve pancreatic cancer treatment.

Cancer cells can disseminate during very early and sometimes asymptomatic stages of tumor progression. Though biological barriers to tumorigenesis have been identified and characterized, the mechanisms that limit early dissemination remain largely unknown. We report here that the orphan nuclear receptor nuclear receptor subfamily 2, group F, member 1 (NR2F1)/COUP-TF1 serves as a barrier to early dissemination. NR2F1 expression was decreased in patient ductal carcinoma in situ (DCIS) samples. High-resolution intravital imaging of HER2+ early-stage cancer cells revealed that loss of function of NR2F1 increased in vivo dissemination and was accompanied by decreased E-cadherin expression, activation of wingless-type MMTV integration site family, member 1 (WNT)-dependent β-catenin signaling, disorganized laminin 5 deposition, and increased expression of epithelial-mesenchymal transition (EMT) genes such as twist basic helix-loop-helix transcription factor 1 (TWIST1), zinc finger E-box binding homeobox 1 (ZEB1), and paired related homeobox 1 (PRRX1). Furthermore, downregulation of NR2F1 promoted a hybrid luminal/basal phenotype. NR2F1 expression was positively regulated by p38α signaling and repressed by HER2 and WNT4 pathways. Finally, early cancer cells with NR2F1LOW/PRRX1HIGH staining were observed in DCIS samples. Together, these findings reveal the existence of an inhibitory mechanism of dissemination regulated by NR2F1 in early-stage breast cancer cells. SIGNIFICANCE: During early stages of breast cancer progression, HER2-mediated suppression of NR2F1 promotes dissemination by inducing EMT and a hybrid luminal/basal-like program.

Immunoediting includes three temporally distinct stages, termed elimination, equilibrium, and escape, and has been proposed to explain the interactions between cancer cells and the immune system during the evolution of cancer. However, the status of immunoediting in cancer remains unclear, and the existence of neoantigen depletion in untreated cancer has been debated. Here we developed a distribution pattern-based method for quantifying neoantigen-mediated negative selection in cancer evolution. The method can provide a robust and reliable quantification for immunoediting signal in individual patients with cancer. Moreover, this method demonstrated the prevalence of immunoediting in the immunotherapy-naive cancer genome. The elimination and escape stages of immunoediting can be quantified separately, where tumor types with strong immunoediting-elimination exhibit a weak immunoediting-escape signal, and vice versa. The quantified immunoediting-elimination signal was predictive of clinical response to cancer immunotherapy. Collectively, immunoediting quantification provides an evolutionary perspective for evaluating the antigenicity of neoantigens and reveals a potential biomarker for precision immunotherapy in cancer. SIGNIFICANCE: Quantification of neoantigen-mediated negative selection in cancer progression reveals distinct features of cancer immunoediting and can serve as a potential biomarker to predict immunotherapy response.

The epitranscriptome represents the more than 140 types of chemically varying and reversable RNA modifications affecting RNA fate. Among these, the most relevant for this review are the mRNA modifications N6-methyladenosine and N6,2'-O-dimethyladenosine. Epitranscriptomic mRNA biology involves RNA methyltransferases (so-called "writers"), RNA demethylases ("erasers"), and RNA-binding proteins ("readers") that interact with methylation sites to determine the functional outcome of the modification. In this review, we discuss the role of a specific RNA demethylase encoded by the fat mass and obesity-associated gene (FTO) in cancer. FTO initially became known as the strongest genetic link for human obesity. Only in 2010, 16 years after its discovery, was its enzymatic function as a demethylase clarified, and only recently has its role in the development of cancer been revealed. FTO functions are challenging to study and interpret because of its genome-wide effects on transcript turnover and translation. We review the discovery of FTO and its enzymatic function, the tumor-promoting and suppressive roles of FTO in selected cancer types, and its potential as a therapeutic target.

Comment on Cancer Res. 2022 Jun 15;82(12):2269-2280.

Preclinical studies provide valuable data in the early development of novel drugs for patients with cancer. Many cancer treatment regimens now utilize multiple agents with different targets to delay the emergence of drug-resistant tumor cells, and experimental agents are often evaluated in combination with FDA-approved drugs. The Biological Testing Branch (BTB) of the U.S. NCI has evaluated more than 70 FDA-approved oncology drugs to date in human xenograft models. Here, we report the first release of a publicly available, downloadable spreadsheet, ROADMAPS (Responses to Oncology Agents and Dosing in Models to Aid Preclinical Studies, dtp.cancer.gov/databases_tools/roadmaps.htm), that provides data filterable by agent, dose, dosing schedule, route of administration, tumor models tested, responses, host mouse strain, maximum weight loss, drug-related deaths, and vehicle formulation for preclinical experiments conducted by the BTB. Data from 70 different single targeted and cytotoxic agents and 140 different xenograft models were included. Multiple xenograft models were tested in immunocompromised mice for many cancer histologies, with lung cancer as the most broadly tested (24 models). Many of the dose levels and schedules used in these experiments were comparable with those tolerated in humans. Targeted and cytotoxic single agents were included. The online spreadsheet will be updated periodically as additional agent/dose/model combinations are evaluated. ROADMAPS is intended to serve as a publicly available resource for the research community to inform the design of clinically relevant, tolerable single and combinatorial regimens in preclinical mouse models. SIGNIFICANCE: ROADMAPS includes data that can be used to identify tolerable dosing regimens with activity against a variety of human tumors in different mouse strains, providing a resource for planning preclinical studies.

Forty-five years ago, Cancer Research published the study by Leon and colleagues in which the authors described the observation of increased levels of cell-free DNA (cfDNA) in the serum of patients with cancer as compared with healthy individuals, with the highest serum levels in patients with metastatic cancer. Most interestingly was the correlation between serum DNA concentrations and therapy outcome: Increased serum DNA levels were associated with poor response to treatment, whereas decreases in DNA levels during treatment appeared to be a sign of better prognosis. Since the discovery of the prognostic value of blood DNA, much research has been focused on the characterization of cfDNA to understand its origins and increase the sensitivity and specificity of using cfDNA as a prognostic and predictive marker in the battle against cancer. Tumor-specific cfDNA markers that were discovered include genetic alterations, chromosomal aberrations, epigenetic modifications, and DNA fragmentation size. In recent years, due to the development of highly sensitive molecular technologies, cfDNA-based assays are now being introduced into the clinic as the so called "liquid biopsy." The advantages of a liquid biopsy over traditional biopsy and imaging have led to the implementation in the clinic for early cancer detection, improved cancer staging, early detection of relapse, real-time monitoring of therapeutic efficacy, and detection of therapeutic targets and resistance mechanisms. Despite Leon and colleagues' initial skepticism about the potential diagnostic value of serum DNA, cfDNA-based liquid biopsy has become one of the most important tools for personalized cancer treatment. See related article by Leon and colleagues, Cancer Res 1977;37:646-50.