Association of Cell-Free DNA Tumor Fraction and Somatic Copy Number Alterations With Survival in Metastatic Triple-Negative Breast Cancer.
ABSTRACT: Purpose Cell-free DNA (cfDNA) offers the potential for minimally invasive genome-wide profiling of tumor alterations without tumor biopsy and may be associated with patient prognosis. Triple-negative breast cancer (TNBC) is characterized by few mutations but extensive somatic copy number alterations (SCNAs), yet little is known regarding SCNAs in metastatic TNBC. We sought to evaluate SCNAs in metastatic TNBC exclusively via cfDNA and determine if cfDNA tumor fraction is associated with overall survival in metastatic TNBC. Patients and Methods In this retrospective cohort study, we identified 164 patients with biopsy-proven metastatic TNBC at a single tertiary care institution who received prior chemotherapy in the (neo)adjuvant or metastatic setting. We performed low-coverage genome-wide sequencing of cfDNA from plasma. Results Without prior knowledge of tumor mutations, we determined tumor fraction of cfDNA for 96.3% of patients and SCNAs for 63.9% of patients. Copy number profiles and percent genome altered were remarkably similar between metastatic and primary TNBCs. Certain SCNAs were more frequent in metastatic TNBCs relative to paired primary tumors and primary TNBCs in publicly available data sets The Cancer Genome Atlas and METABRIC, including chromosomal gains in drivers NOTCH2, AKT2, and AKT3. Prespecified cfDNA tumor fraction threshold of ≥ 10% was associated with significantly worse metastatic survival (median, 6.4 v 15.9 months) and remained significant independent of clinicopathologic factors (hazard ratio, 2.14; 95% CI, 1.4 to 3.8; P < .001). Conclusion We present the largest genomic characterization of metastatic TNBC to our knowledge, exclusively from cfDNA. Evaluation of cfDNA tumor fraction was feasible for nearly all patients, and tumor fraction ≥ 10% is associated with significantly worse survival in this large metastatic TNBC cohort. Specific SCNAs are enriched and prognostic in metastatic TNBC, with implications for metastasis, resistance, and novel therapeutic approaches.
Project description:Loss of androgen receptor (AR) signaling dependence occurs in approximately 15%-20% of advanced treatment-resistant prostate cancers, and this may manifest clinically as transformation from a prostate adenocarcinoma histology to a castration-resistant neuroendocrine prostate cancer (CRPC-NE). The diagnosis of CRPC-NE currently relies on a metastatic tumor biopsy, which is invasive for patients and sometimes challenging to diagnose due to morphologic heterogeneity. By studying whole-exome sequencing and whole-genome bisulfite sequencing of cell free DNA (cfDNA) and of matched metastatic tumor biopsies from patients with metastatic prostate adenocarcinoma and CRPC-NE, we identified CRPC-NE features detectable in the circulation. Overall, there was markedly higher concordance between cfDNA and biopsy tissue genomic alterations in patients with CRPC-NE compared with castration-resistant adenocarcinoma, supporting greater intraindividual genomic consistency across metastases. Allele-specific copy number and serial sampling analyses allowed for the detection and tracking of clonal and subclonal tumor cell populations. cfDNA methylation was indicative of circulating tumor content fraction, reflective of methylation patterns observed in biopsy tissues, and was capable of detecting CRPC-NE-associated epigenetic changes (e.g., hypermethylation of ASXL3 and SPDEF; hypomethylation of INSM1 and CDH2). A targeted set combining genomic (TP53, RB1, CYLD, AR) and epigenomic (hypo- and hypermethylation of 20 differential sites) alterations applied to ctDNA was capable of identifying patients with CRPC-NE.
Project description:Purpose The aqueous humor (AH) liquid biopsy enables in vivo evaluation of tumor-derived cell-free DNA (cfDNA) from retinoblastoma (RB) eyes. Herein, we test our hypothesis that longitudinal dynamics of AH cfDNA—including tumor fraction (TFx) and somatic copy number alteration (SCNA) amplitude—correspond to therapeutic response. Methods Eyes with ?3 AH extractions during intravitreal chemotherapy (IVM) or at secondary enucleation between 2015 to 2019 were included. AH cfDNA was sequenced to assess RB SCNA amplitude; ichorCNA software was used to estimate TFx. Eyes without SCNAs or with TFx < 0.10 across all samples were excluded. Therapeutic responses for each eye were determined from clinical records. Statistical analyses included Mann-Whitney U and Pearson correlation tests. Results Twenty eyes of 20 patients underwent ?3 AH extractions; 6 eyes lacked SCNAs or had TFx < 0.10 throughout sampling and were excluded. Clinical progression was associated with significantly higher SCNA amplitudes and TFx values than regression (P ? 0.04). Relative increases in TFx (?TFx 1.86 ± 2.22) were associated with disease progression, whereas relative decreases in TFx (?TFx 0.53 ± 0.36) were associated with disease regression (P < 0.00001). A ?15% increase in TFx relative to baseline during treatment was associated with an over 90-fold increased likelihood of clinical progression (odds ratio = 90.67, 95% confidence interval = 8.30–990.16, P = 0.0002). TFx and SCNA amplitude were significantly positively correlated throughout sampling (P ? 0.002). Conclusions Longitudinal changes in AH-derived cfDNA TFx and SCNA amplitude are concordant with clinical responses of intraocular RB during active therapy. Translational Relevance Longitudinal evaluation of AH cfDNA may provide an objective, quantitative way to monitor therapeutic response and disease burden in RB patients.
Project description:The evolutionary changes in immune profiles of triple negative breast cancer (TNBC) are not well understood, although it is known that immune checkpoint inhibitors have diminished activity in heavily pre-treated TNBC patients. This study was designed to characterize immune profile changes of longitudinal tumor specimens by studying immune subsets of tumor infiltrating lymphocytes (TILs) in paired primary and metastatic TNBC in a cohort of "poor outcome" (relapsed within 5 years) patients. Immune profiles of TNBCs in a cohort of "good outcome" (no relapse within 5 years) patients were also analyzed. Immune subsets were characterized for CD4, CD8, FOXP3, CD20, CD33, and PD1 using immuno-fluorescence staining in stroma, tumor, and combined stroma and tumor tissue. TIL subsets in "good outcome" versus "poor outcome" patients were also analyzed. Compared with primary, metastatic TNBCs had significantly lower TILs by hematoxylin and eosin (H&E) staining. Stromal TILs (sTILs), but not tumoral TILs (tTILs) had significantly reduced cytotoxic CD8+ T cells (CTLs), PD1+ CTLs, and total PD1+ TILs in metastatic compared with matched primary TNBCs. Higher PD1+ CTLs, PD1+CD4+ helper T cells (PD1+TCONV) and all PD1+ T cells in sTILs, tTILs and total stromal and tumor TILS (s+tTIL) were all associated with better prognosis. In summary, TIL subsets decrease significantly in metastatic TNBCs compared with matched primary. Higher PD1+ TILs are associated with better prognosis in early stage TNBCs. This finding supports the application of immune checkpoint inhibitors early in the treatment of TNBCs.
Project description:Background: Detection of tumor-specific alterations in cell-free DNA (cfDNA) has proven valuable as a liquid biopsy for several types of cancer. So far, use of cfDNA remains unexplored for pancreatic neuroendocrine tumor (PNET) patients. Methods: From 10 PNET patients, fresh frozen tumor tissue, buffy coat and plasma samples were collected. Whole-exome sequencing of primary tumor and germline DNA was performed to identify tumor-specific variants and copy number variations (CNVs). Subsequently, tumor-specific variants were quantified in plasma cfDNA with droplet digital PCR. In addition, CNV analysis of cfDNA was performed using shallow whole-genome sequencing. Results: Tumor-specific variants were detected in perioperative plasma samples of two PNET patients, at variant allele fractions (VAFs) of respectively 19 and 21%. Both patients had metastatic disease at time of surgery, while the other patients presented with localized disease. In the metastatic patients, CNV profiles of tumor tissue and cfDNA were significantly correlated. A follow-up plasma sample of a metastatic patient demonstrated an increased VAF (57%) and an increased chromosomal instability, in parallel with an increase in tumor burden. Conclusions: We are the first to report the presence of tumor-specific genetic alterations in cfDNA of metastatic PNET patients and their evolution during disease progression. Additionally, CNV analysis in cfDNA shows potential as a liquid biopsy.
Project description:Purpose:Access to genomic tumor material is required to select patients for targeted therapies. However, tissue biopsies are not always feasible and therefore circulating cell-free DNA (cfDNA) has emerged as an alternative. Here we investigate the utility of cfDNA for genomic tumor profiling in the phase I setting. Study design:Peripheral blood was collected from patients with advanced solid cancers eligible for phase I treatment. Patients failing the initial tissue biopsy due to inaccessible lesions or insufficient tumor cellularity (<10%) were included in the study. Genomic profiling of cfDNA including whole exome sequencing (WES) and somatic copy number alterations (SCNAs) analysis (OncoScan). Results:Plasma cfDNA was pro- and retrospectively profiled from 24 and 20 patients, respectively. The median turnaround time was 29 days (N= 24, range 13-87 days) compared to tissue re-analyses of median 60 days (N= 6, range 29-98). Selected cancer-associated alterations (SCAAs) were identified in 70% (31/44) of patients, predominantly by WES due to the low sensitivity of OncoScan on cfDNA. Primarily, inaccessible cases of prostate and lung cancers could benefit from cfDNA profiling. In contrast, breast cancer patients showed a low level of tumor-specific cfDNA which might be due to cancer type and/or active treatment at the time of plasma collection. Conclusion:Plasma cfDNA profiling using WES is feasible within a clinically relevant timeframe and represents an alternative to invasive tissue biopsies to identify possible treatment targets. Especially, difficult-to-biopsy cancers can benefit from cfDNA profiling, but tumor tissue remains the gold standard for molecular analyses.
Project description:Sequencing of the mutant allele fraction of circulating cell-free DNA (cfDNA) derived from tumors is increasingly utilized to detect actionable genomic alterations in cancer. We conducted a prospective blinded study of a comprehensive cfDNA sequencing panel with 54 cancer genes. To evaluate the concordance between cfDNA and tumor DNA (tDNA), sequencing results were compared between cfDNA from plasma and genomic tumor DNA (tDNA). Utilizing next generation digital sequencing technology (DST), we profiled approximately 78,000 bases encoding 512 complete exons in the targeted genes in cfDNA from plasma. Seventy-five patients were prospectively enrolled between February 2013 and March 2014, including 61 metastatic cancer patients and 14 clinical stage II CRC patients with matched plasma and tissue samples. Using the 54-gene panel, we detected at least one somatic mutation in 44 of 61 tDNA (72.1%) and 29 of 44 (65.9%) cfDNA. The overall concordance rate of cfDNA to tDNA was 85.9%, when all detected mutations were considered. We collected serial cfDNAs during cetuximab-based treatment in 2 metastatic KRAS wild-type CRC patients, one with acquired resistance and one with primary resistance. We demonstrate newly emerged KRAS mutation in cfDNA 1.5 months before radiologic progression. Another patient had a newly emerged PIK3CA H1047R mutation on cfDNA analysis at progression during cetuximab/irinotecan chemotherapy with gradual increase in allele frequency from 0.8 to 2.1%. This blinded, prospective study of a cfDNA sequencing showed high concordance to tDNA suggesting that the DST approach may be used as a non-invasive biopsy-free alternative to conventional sequencing using tumor biopsy.
Project description:Metastatic breast cancer is a leading cause of cancer-related deaths in women worldwide. Patients with triple negative breast cancer (TNBCs), a highly aggressive tumor subtype, have a particularly poor prognosis. Multiple reports demonstrate that altered content of the multicopy mitochondrial genome (mtDNA) in primary breast tumors correlates with poor prognosis. We earlier reported that mtDNA copy number reduction in breast cancer cell lines induces an epithelial-mesenchymal transition associated with metastasis. However, it is unknown whether the breast tumor subtypes (TNBC, Luminal and HER2+) differ in the nature and amount of mitochondrial defects and if mitochondrial defects can be used as a marker to identify tumors at risk for metastasis. By analyzing human primary tumors, cell lines and the TCGA dataset, we demonstrate a high degree of variability in mitochondrial defects among the tumor subtypes and TNBCs, in particular, exhibit higher frequency of mitochondrial defects, including reduced mtDNA content, mtDNA sequence imbalance (mtRNR1:ND4), impaired mitochondrial respiration and metabolic switch to glycolysis which is associated with tumorigenicity. We identified that genes involved in maintenance of mitochondrial structural and functional integrity are differentially expressed in TNBCs compared to non-TNBC tumors. Furthermore, we identified a subset of TNBC tumors that contain lower expression of epithelial splicing regulatory protein (ESRP)-1, typical of metastasizing cells. The overall impact of our findings reported here is that mitochondrial heterogeneity among TNBCs can be used to identify TNBC patients at risk of metastasis and the altered metabolism and metabolic genes can be targeted to improve chemotherapeutic response.
Project description:Aqueous humor contains tumor-derived cell-free DNA (cfDNA) and can serve as a liquid biopsy for retinoblastoma. We previously associated somatic copy-number alteration (SCNA) 6p gain with a 10-fold increased risk of enucleation. Here we provide a 2-year update to further explore 6p gain as a prognostic biomarker for ocular survival. Patients diagnosed with retinoblastoma from December 2014 to July 2019 from whom aqueous humor was sampled were included. cfDNA was extracted and shallow whole-genome sequencing performed to identify highly recurrent retinoblastoma SCNAs (gain of 1q, 2p, 6p, loss of 13q, 16q). 116 aqueous humor samples from 50 eyes of 46 patients were included: 27 eyes were salvaged, 23 were enucleated. Highly recurrent retinoblastoma SCNAs were found in 66% eyes. 6p gain was the most prevalent SCNA (50% eyes). It was particularly more prevalent in enucleated eyes (73.9%) than in salvaged eyes (29.6%; P = 0.004). 6p gain in aqueous humor cfDNA portended nearly 10-fold increased odds of enucleation (OR = 9.87; 95% confidence interval = 1.75-55.65; P = 0.009). In the enucleated eyes, 6p gain was associated with aggressive histopathologic features, including necrosis, higher degrees of anaplasia, and focal invasion of ocular structures. With extended follow-up and nearly double the aqueous humor samples, we continue to demonstrate 6p gain as a potential prognostic biomarker for retinoblastoma. IMPLICATIONS: Aqueous humor is a high-yield source of tumor-derived DNA in retinoblastoma eyes. Detection of 6p gain in the aqueous humor allows for targeted, patient-centered therapies based on this molecular prognostic marker. Prospective, multicenter studies with aqueous humor sampled from all eyes at diagnosis are warranted to validate these findings.
Project description:Background:Real-time knowledge of the somatic genome can influence management of patients with metastatic castration-resistant prostate cancer (mCRPC). While routine metastatic tissue biopsy is challenging in mCRPC, plasma circulating tumor DNA (ctDNA) has emerged as a minimally invasive tool to sample the tumor genome. However, no systematic comparisons of matched "liquid" and "solid" biopsies have been performed that would enable ctDNA profiling to replace the need for direct tissue sampling. Methods:We performed targeted sequencing across 72 clinically relevant genes in 45 plasma cell-free DNA (cfDNA) samples collected at time of metastatic tissue biopsy. We compared ctDNA alterations with exome sequencing data generated from matched tissue and quantified the concordance of mutations and copy number alterations using the Fisher exact test and Pearson correlations. Results:Seventy-five point six percent of cfDNA samples had a ctDNA proportion greater than 2% of total cfDNA. In these patients, all somatic mutations identified in matched metastatic tissue biopsies were concurrently present in ctDNA. Furthermore, the hierarchy of variant allele fractions for shared mutations was remarkably similar between ctDNA and tissue. Copy number profiles between matched liquid and solid biopsy were highly correlated, and individual copy number calls in clinically actionable genes were 88.9% concordant. Detected alterations included AR amplifications in 22 (64.7%) samples, SPOP mutations in three (8.8%) samples, and inactivating alterations in tumor suppressors TP53 , PTEN , RB1 , APC , CDKN1B , BRCA2 , and PIK3R1 . In several patients, ctDNA sequencing revealed robust changes not present in paired solid biopsy, including clinically relevant alterations in the AR, WNT, and PI3K pathways. Conclusions:Our study shows that, in the majority of patients, a ctDNA assay is sufficient to identify all driver DNA alterations present in matched metastatic tissue and supports development of DNA biomarkers to guide mCRPC patient management based on ctDNA alone.
Project description:The identification of the molecular drivers of cancer by sequencing is the backbone of precision medicine and the basis of personalized therapy; however, biopsies of primary tumors provide only a snapshot of the evolution of the disease and may miss potential therapeutic targets, especially in the metastatic setting. A liquid biopsy, in the form of cell-free DNA (cfDNA) sequencing, has the potential to capture the inter- and intra-tumoral heterogeneity present in metastatic disease, and, through serial blood draws, track the evolution of the tumor genome. In order to determine the clinical utility of cfDNA sequencing we performed whole-exome sequencing on cfDNA and tumor DNA from two patients with metastatic disease; only minor modifications to our sequencing and analysis pipelines were required for sequencing and mutation calling of cfDNA. The first patient had metastatic sarcoma and 47 of 48 mutations present in the primary tumor were also found in the cell-free DNA. The second patient had metastatic breast cancer and sequencing identified an ESR1 mutation in the cfDNA and metastatic site, but not in the primary tumor. This likely explains tumor progression on Anastrozole. Significant heterogeneity between the primary and metastatic tumors, with cfDNA reflecting the metastases, suggested separation from the primary lesion early in tumor evolution. This is best illustrated by an activating PIK3CA mutation (H1047R) which was clonal in the primary tumor, but completely absent from either the metastasis or cfDNA. Here we show that cfDNA sequencing supplies clinically actionable information with minimal risks compared to metastatic biopsies. This study demonstrates the utility of whole-exome sequencing of cell-free DNA from patients with metastatic disease. cfDNA sequencing identified an ESR1 mutation, potentially explaining a patient's resistance to aromatase inhibition, and gave insight into how metastatic lesions differ from the primary tumor.