Victor Velculescu

Introduction: Liquid biopsies are promising noninvasive tools for cancer detection. Variation in cell-free DNA concentrations (cfDNA-conc) has been observed between individuals with and without cancer. We assessed cfDNA-conc in 2287 treatment-naive individuals including those without cancer, with benign or high-risk conditions, or with one of eight cancer types. Methods: Plasma (0.3 to 9.8 mL) was separated from whole blood collected in Streck or EDTA tubes from individuals with bile duct, breast, colorectal, gastric, ovarian, liver, lung, pancreatic, or metastatic cancer (n=23, 54, 28, 26, 264, 75, 180, 31, 22, respectively) as well as benign or high-risk conditions (n=550, 133, respectively) such as cirrhosis or hepatitis B or C virus infection (HBV/HCV), and without cancer (n=901). cfDNA was extracted using the Qiagen QIAamp Circulating Nucleic Acid Kit. Quality and quantity were assessed using the High …

Liquid biopsies are emerging as powerful minimally invasive approaches that have the potential to solve several long-standing problems spanning the continuum of cancer care: early detection of cancer, minimal residual disease tracking, and refinement of the heterogeneity of clinical responses together with therapeutic response monitoring in the metastatic setting. Existing challenges driven by technical limitations and establishment of the clinical value of liquid biopsies represent fields of active research that call for convergence science approaches to bridge scientific discovery with clinical care.

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 …

Introduction: Pancreatic cancer has a poor prognosis especially when identified at advanced stages. Globally in 2020, >450,000 people died from the disease. For those with locally advanced or metastatic cancer, the standard of care treatment is chemotherapy. Preliminary studies show that some patients with advanced-stage disease respond to immune checkpoint blockade treatment. Determining response to therapy using imaging techniques can be challenging. There is a clinical unmet need for noninvasive approaches that can provide a real-time assessment of response to therapy in these patients. We evaluated two methods for assessing response to therapy using circulating cell-free DNA (cfDNA) in patients with metastatic pancreatic cancer treated with immune checkpoint inhibition and radiation as part of the CheckPAC study (NCT02866383). Methods: For all patients with available samples (n=40), we …

Introduction: Patients with stage II colon cancer not classified as high risk (pT4 microsatellite stable) do not receive adjuvant chemotherapy (ACT) according to Dutch guidelines. However, 15-20% of patients with stage II colon cancer experience a disease recurrence, indicating that there is an unmet clinical need to identify patients who could benefit from adjuvant treatment. Observational studies demonstrate that postoperative circulating tumor DNA (ctDNA) is indicative of minimal residual disease (MRD) and a strong prognostic biomarker for disease recurrence. Aim: The MEDOCC-CrEATE trial aims to assess 1) the proportion of stage II colon cancer patients with detectable postoperative ctDNA accepting ACT and 2) whether ctDNA-guided ACT reduces 2-year recurrence rate (RR). Methods: MEDOCC-CrEATE is an interventional trial following the ‘trial within cohorts’ design. Participants of the Prospective Dutch …

Introduction: Immunotherapy has demonstrated modest effectiveness for patients with platinum-resistant ovarian cancer, which is in part related to the immunosuppressive tumor microenvironment (TME) of ovarian cancers. Epigenetic modulators in combination with immune checkpoint inhibitors (ICIs) may represent a potential way to sensitize ovarian cancer to immunotherapy by TME reprogramming. Methods: We evaluated the effect of epigenetic modulation in combination with ICI by comprehensive transcriptomic analyses of serially biopsied platinum-resistant/refractory ovarian cancer. Leveraging serial tumor samples from 30 patients that received oral azacitidine and pembrolizumab in the TRIO026 phase II clinical trial (NCT02900560), we performed bulk RNA sequencing and direct digital counting of 770 target genes using molecular barcodes (nCounter, NanoString) for 72 serial tumor samples, prior to …

2023-08-11 Assigned to THE JOHNS HOPKINS UNIVERSITY reassignment THE JOHNS HOPKINS UNIVERSITY ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: PHALLEN, Jillian A., VELCULESCU, VICTOR E., CRISTIANO, Stephen, BRUHM, Daniel, MATHIOS, Dimitrios, SCHARPF, Robert B.

Background: Accurate monitoring of treatment response in patients with metastatic colorectal cancer (mCRC) is important to decide when to adjust treatment regimen or to proceed to local therapy of metastases. At present, clinical response is determined by computed tomography (CT) imaging-based assessment of changes in tumor size. However, this monitoring approach is constrained by limited sensitivity in detecting lymph node and peritoneal metastases, as well as inter-reader variability. Detection of circulating tumor DNA (ctDNA) is indicative of the amount of neoplastic cells and may have added clinical value to CT imaging for assessment of treatment response and guidance in treatment decision making in mCRC patients. We recently developed a tumor mutation-independent ctDNA test that utilizes low-coverage whole genome sequencing to analyze the plasma cell-free DNA (cfDNA) fragmentome to …

Cell-free DNA (cfDNA) in the bloodstream is increasingly gaining attention as a diagnostic tool for the early detection of cancer. Nevertheless, the characteristics and sources of cfDNA fragmentation in the blood remain poorly understood. In this study, we sought to unravel the impact of DNA methylation and gene expression on the naturally occurring genome-wide fragmentation of cfDNA. We performed a comprehensive analysis of plasma samples from 969 individuals, including 182 individuals diagnosed with cancer (pancreatic, colorectal, ovarian, lung and breast cancer). In healthy individual cfDNA fragmentation patterns like DNA motifs at cfDNA fraqgments ends, recurrent cfDNA fragment start sites, cfDNA coverage and cfDNA fragment size were compared to publicly available data on cfDNA methylation and gene expression from myeloid cells. We discovered that the ends of cfDNA fragments, particularly …

Background: Liquid biopsies can inform treatment strategies by rapidly and accurately assessing tumor burden; with emerging data supporting the clinical utility of circulating cell-free tumor DNA (ctDNA) as an early endpoint of immunotherapy response. Methods: We performed targeted error-correction sequencing of ctDNA (n=330) and matched white blood cell (WBC) DNA (n=109) from 109 patients with advanced non-small cell lung cancer (NSCLC) who received immune checkpoint inhibitor monotherapy (n=79) or immunotherapy-containing combination regimens (n=30). Variant cellular origin was determined using a tumor-independent WBC DNA-informed approach. The maximum mutant allele fraction (maxMAF) of tumor-derived mutations was tracked longitudinally to assess changes in total cell-free tumor load (cfTL). Results: Among 3,418 variants with resolved cellular origin, 2,452 (72%) were tumor …

Introduction: Primary resistance to immune checkpoint inhibition (ICI) is in part driven by a “cold” tumor microenvironment in the context of low PD-L1 expression, low tumor mutation burden or oncogenic mutations that drive tumor immune exclusion. Radiation has the potential to sensitize tumors to ICI through extra-tumoral T cell activation in response to tumor antigens released from irradiated cancer cells (abscopal effect). Here, we investigate tumor microenvironment and T cell repertoire reshaping leveraging serial biospecimens from a phase 2 randomized clinical trial of stereotactic body radiation therapy followed by pembrolizumab (SBRT arm) vs. pembrolizumab alone (control arm) in non-small cell lung cancer (NSCLC; NCT02492568). Methods: We employed whole exome sequencing, RNA sequencing and T cell receptor (TCR) sequencing to analyze 293 serial tumor and peripheral blood samples collected …

Introduction: Surgery followed by adjuvant chemotherapy (ACT) is standard of care in stage III colon cancer. However, only 15-20% of patients benefit from ACT, as half of patients are cured by surgery, and 25-30% still experience a recurrence. Detection of circulating tumor DNA (ctDNA) after resection of the primary tumor is a strong prognostic biomarker in non-metastatic colon cancer and offers a promising approach to better stratify stage III colon cancer patients for ACT treatment decisions. Aim: The PROVENC3 study aims to determine the clinical validity of post-surgery ctDNA detection in patients with stage III colon cancer treated with ACT. Methods: Blood was collected pre-surgery, post-surgery and post-ACT for stage III colon cancer patients treated with ACT. Tumor-informed detection of ctDNA was performed through integrated whole genome sequencing (WGS) analyses of formalin-fixed paraffin-embedded …

Background The fecal immunochemical test (FIT) is widely used in population based colorectal cancer (CRC) screening programs. Recently, circulating cell-free DNA (cfDNA) analyses have emerged as a new avenue for early cancer detection. The performance of cfDNA methods in comparison with FIT is currently unknown. The present study compared pre-operative cfDNA analyses to FIT for detection of patients with CRC who participated in a population-based screening program. Methods In the Dutch national CRC screening program, individuals aged 55-75 years are biennially invited to perform a single FIT. The database of the Dutch CRC screening program was queried to identify individuals with FIT data who also participated in two cfDNA studies, PLCRC-MEDOCC and PLCRC-PROVENC3 (AACR abstract C. Rubio Alarcón). Sensitivities with 95% confidence intervals (CI) for detecting individuals with CRC …

Noninvasive approaches for detection of tumor-specific mutations in cell-free DNA (cfDNA) have the potential to track a patient’s response to treatment, enabling effective and timely decisions on therapy. However, mutations in cfDNA arising from clonal hematopoeisis (CH) are common and tumor biopsies for definitive identification of the origin of these mutations are not always available. Sequencing of matched cells from buffy coat and the absence of mutations in these cells has been used to rule-out white blood cell (WBC) mutations, but uneven sequencing depths between matched cfDNA and buffy coat and the fraction of mutant alleles are generally ignored by rule-based tests. A probabilistic approach that quantifies the evidence of tumor-derived mutations in cfDNA is needed. We developed a Bayesian framework to estimate the probability that a mutation identified in cfDNA is tumor specific. Our approach …

Introduction: Emergence of resistance to immune checkpoint blockade (ICB) mandates the development of strategies for ICB sensitization. Here we employed a multi-omic approach to understand the effects of epigenetic priming in re-shaping the tumor microenvironment, together with genomic drivers of therapeutic response of epigenetic therapy followed by ICB in non-small cell lung cancer (NSCLC). Methods: We performed whole exome sequencing (WES) on 39 baseline tumors and bulk RNA sequencing (RNAseq) on 18 serial tumors across 42 patients with metastatic immunotherapy-naïve NSCLC, who received azacitidine and entinostat followed by nivolumab (NCT01928576). WES was utilized to assess co-mutations, mutation signatures and genome-wide structural changes. RNAseq was utilized for gene and gene set enrichment analysis (GSEA) and repeat element analysis. Response after nivolumab …

Introduction: The stepwise progression of colorectal cancer from healthy epithelium, to premalignant adenoma, to cancer is accompanied by genome-wide epigenetic reprogramming. However, current analyses of these processes have been hampered by complex mixtures of cells in normal, adenoma and cancer tissue samples, as well as absence of suitable adenoma model systems. Cultured colorectal organoids, comprised of pure epithelial cells, could enable insights on the epigenetic changes leading to progression from healthy tissue to cancer. Methods: Using 43 patient-derived colorectal organoids derived from healthy, adenoma, and cancer tissues, we examined the chromatin landscape of tumor progression using transposase accessible chromatin analyses with next generation sequencing. We defined a consensus set of nucleosome depleted regions for each disease stage and used principal …

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BACKGROUND: The rapid detection of disease progression in patients receiving immune checkpoint inhibitors (ICIs) is challenging since there are no reliable biomarkers of clinical response. Liquid biopsy assays may address this unmet need by using cell-free DNA (cfDNA) to monitor treatment responses. Current targeted next-generation sequencing cfDNA assays are costly and have limited assay sensitivity for detection of cancer-specific mutations at low mutant allele frequency (MAF). Here, we demonstrate the utility of DELFI Tumor Fraction (DELFI-TF), a tumor- and mutation-independent cfDNA fragmentome approach to monitor treatment response in patients with metastatic non-small cell lung cancer (mNSCLC). METHODS: Overall, 422 longitudinal blood samples were collected from 141 mNSCLC patients of which 109 were treated with immunotherapy and 32 with targeted therapy (TT). Plasma-derived …

INTRODUCTION: Clonal hematopoiesis (CH) is the result of the clonal expansion of hematopoietic stem cells which acquire and pass on somatically gained mutations, including SNVs as well as arm level losses and gains, defined here as mosaic deletions and amplifications (mDAs). The proportion of blood cells in circulation containing CH alterations increases with age and environmental exposures such as smoking, leading to a higher prevalence in many high-risk cancer screening populations. CH remains a major confounder for mutation-based liquid biopsy tests evaluating circulating cell-free DNA (cfDNA), since these assays are unable to discriminate between mutations originating from the tumor as opposed to CH. While the impact of CH mutations on fragmentation-based methods of detecting ctDNA appears to be limited, the effect of mDAs has not yet been explored. METHODS: To assess the impact of …

Responders to checkpoint blockade in Non Small Cell Lung Cancer (NSCLC) often feature an inflamed microenvironment prior to therapy. However, the complete set of molecular drivers connecting this histologic observation to enhanced tumor clearance remain enigmatic. In updated analysis of the Stand Up 2 Cancer-Mark Foundation (SU2C-MARK) Cohort - a collection of 393 patients with whole exome and/or RNA sequencing along with matched checkpoint blockade response annotation - we identify a prominent predictive role for inducible components of the immunoproteasome, a non-canonical peptide processing complex upstream of antigen presentation. Notably, these subunits are enriched as predictors relative to interferon-inducible genes as well as proteasome components in general, and are consistently associated with objective response, progression-free survival and overall survival. Expression of …