Pre-Analytical Evaluation of Streck Cell-Free DNA Blood Collection Tubes for Liquid Profiling in Oncology.

Excellent pre-analytical stability is an essential precondition for reliable molecular profiling of circulating tumor DNA (ctDNA) in oncological diagnostics. Therefore, in vitro degradation of ctDNA and the additional release of contaminating genomic DNA from lysed blood cells must be prevented. Streck Cell-Free DNA blood collection tubes (cfDNA BCTs) have proposed advantages over standard K2EDTA tubes, but mainly have been tested in healthy individuals. Blood was collected from cancer patients (n = 53) suffering from colorectal (n = 21), pancreatic (n = 11), and non-small-cell lung cancer (n = 21) using cfDNA BCT tubes and K2EDTA tubes that were processed immediately or after 3 days (BCTs) or 6 hours (K2EDTA) at room temperature. The cfDNA isolated from these samples was characterized in terms of yield using LINE-1 qPCR; the level of gDNA contamination; and the mutation status of KRAS, NRAS, and EGFR genes using BEAMing ddPCR. CfDNA yield and gDNA levels were comparable in both tube types and were not affected by prolonged storage of blood samples for at least 3 days in cfDNA BCTs or 6 hours in K2EDTA tubes. In addition, biospecimens collected in K2EDTA tubes and cfDNA BCTs stored for up to 3 days demonstrated highly comparable levels of mutational load across all respective cancer patient cohorts and a wide range of concentrations. Our data support the applicability of clinical oncology specimens collected and stored in cfDNA BCTs for up to 3 days for reliable cfDNA and mutation analyses.

Prospective study evaluating dynamic changes of cell-free HPV DNA in locoregional viral-associated oropharyngeal cancer treated with induction chemotherapy and response-adaptive treatment.

BACKGROUND: Human papillomavirus (HPV)-associated oropharyngeal cancer (OPC) has a favorable prognosis which has led to efforts to de-intensify treatment. Response-adaptive de-escalated treatment is promising, however improved biomarkers are needed. Quantitative cell-free HPV-DNA (cfHPV-DNA) in plasma represents an attractive non-invasive biomarker for grading treatment response and post-treatment surveillance. This prospective study evaluates dynamic changes in cfHPV-DNA during induction therapy, definitive (chemo)radiotherapy, and post-treatment surveillance in the context of risk and response-adaptive treatment for HPV + OPC. METHODS: Patients with locoregional HPV + OPC are stratified into two cohorts: High risk (HR) (T4, N3, [Formula: see text] 20 pack-year smoking history (PYH), or non-HPV16 subtype); Low risk (LR) (all other patients). All patients receive induction chemotherapy with three cycles of carboplatin and paclitaxel. LR with ≥ 50% response receive treatment on the single-modality arm (minimally-invasive surgery or radiation alone to 50 Gy). HR with ≥ 50% response or LR with ≥ 30% and < 50% response receive treatment on the intermediate de-escalation arm (chemoradiation to 50 Gy with cisplatin). All other patients receive treatment on the regular dose arm with chemoradiation to 70 Gy with concurrent cisplatin. Plasma cfHPV-DNA is assessed during induction, (chemo)radiation, and post-treatment surveillance. The primary endpoint is correlation of quantitative cfHPV-DNA with radiographic response. DISCUSSION: A de-escalation treatment paradigm that reduces toxicity without compromising survival outcomes is urgently needed for HPV + OPC. Response to induction chemotherapy is predictive and prognostic and can select candidates for de-escalated definitive therapy. Assessment of quantitative cfHPV-DNA in the context of response-adaptive treatment of represents a promising reliable and convenient biomarker-driven strategy to guide personalized treatment in HPV + OPC. TRIAL REGISTRATION: This trial is registered with ClinicalTrials.gov on October 1st, 2020 with Identifier: NCT04572100 .

Routine Molecular Screening of Patients with Advanced Non-SmallCell Lung Cancer in Circulating Cell-Free DNA at Diagnosis and During Progression Using OncoBEAMTM EGFR V2 and NGS Technologies.

BACKGROUND AND OBJECTIVES: The use of ultra-sensitive diagnostic tests to detect clinically actionable somatic alterations within the gene encoding the epidermal growth factor receptor (EGFR) within circulating cell-free DNA is an important first step in determining the eligibility of patients with non-small cell lung cancer to receive tyrosine kinase inhibitors. METHODS: We present the clinical validation (accuracy, sensitivity, and specificity) of a highly sensitive OncoBEAMTM EGFR V2 test, which we compare to a custom next-generation sequencing assay, for the treatment of patients with non-small cell lung cancer with EGFR tyrosine kinase inhibitor therapies. The OncoBEAMTM digital-polymerase chain reaction method detects 36 different EGFR alterations in circulating cell-free DNA, whereas the next-generation sequencing assay covers major solid tumor oncodrivers. Of the 540 samples analyzed with the OncoBEAMTM EGFR V2 test, 42.4% of patients had undergone molecular testing at diagnosis (N = 229/540) and 57.7% of patients during disease progression (N = 311/540). RESULTS: The sensitivity and specificity were measured for this BEAMing assay. The number of mutant beads and mutant allelic fraction were measured for each EGFR alteration and the level of detection was established at 0.1% for a median of 2861 genome equivalent (GE) in each reaction using HD780 horizon control DNA, as well as by an internal quality reference standard. Approximately 10%, 27%, and 63% of the 540 samples contained < 1500 GE, a range of 1500-3000 GE, and > 3000 GE, which corresponded to a maximal assay sensitivity of 2.0%, 0.5-0.1%, and 0.1-0.05% mutant allelic fraction, respectively. In a routine hospital setting, 11.4% of non-small cell lung cancer tumors were positive at diagnosis for EGFR alterations, while 43.7% samples harbored EGFR mutations at progression, among which 40.3% expressed EGFR resistance mutations after first-line tyrosine kinase inhibitor treatment with first- and second-generation drugs. CONCLUSIONS: The OncoBEAMTM EGFR V2 is a sensitive, robust, and accurate assay that delivers reproducible results. Next-generation sequencing and BEAMing technologies act complementarily in the routine molecular screening. We show that using a next-generation sequencing assay, despite its lower sensitivity, enables the identification of rare EGFR alterations or resistance mechanisms (mutation, deletion, insertion, and copy number variation) to orient first- and second-line treatments.

Expanded Low Allele Frequency RAS and BRAF V600E Testing in Metastatic Colorectal Cancer as Predictive Biomarkers for Cetuximab in the Randomized CO.17 Trial.

PURPOSE: Expanded RAS/BRAF mutations have not been assessed as predictive for single-agent cetuximab in metastatic colorectal cancer (mCRC), and low mutant allele frequency (MAF) mutations are of unclear significance. We aimed to establish cetuximab efficacy in optimally selected patients using highly sensitive beads, emulsion, amplification, and magnetics (BEAMing) analysis, capable of detecting alterations below standard clinical assays. PATIENTS AND METHODS: CO.17 trial compared cetuximab versus best supportive care (BSC) in RAS/BRAF-unselected mCRC. We performed RAS/BRAF analysis on microdissected tissue of 242 patients in CO.17 trial using BEAMing for KRAS/NRAS (codons 12/13/59/61/117/146) and BRAF V600E. Patients without BEAMing but with previous Sanger sequencing-detected mutations were included. RESULTS: KRAS, NRAS, and BRAF mutations were present in 53%, 4%, and 3% of tumors, respectively. Cetuximab improved overall survival [OS; HR, 0.51; 95% confidence interval (CI), 0.32-0.81; P = 0.004] and progression-free survival (PFS; HR, 0.25; 95% CI, 0.15-0.41; P < 0.0001) compared with BSC in RAS/BRAF wild-type patients. Cetuximab did not improve OS/PFS for KRAS-, NRAS-, or BRAF-mutated tumors, and tests of interaction confirmed expanded KRAS (P = 0.0002) and NRAS (P = 0.006) as predictive, while BRAF mutations were not (P = 0.089). BEAMing identified 14% more tumors as RAS mutant than Sanger sequencing, and cetuximab lacked activity in these patients. Mutations at MAF < 5% were noted in 6 of 242 patients (2%). One patient with a KRAS A59T mutation (MAF = 2%) responded to cetuximab. More NRAS than KRAS mutations were low MAF (OR, 20.50; 95% CI, 3.88-96.85; P = 0.0038). CONCLUSIONS: We establish single-agent cetuximab efficacy in optimally selected patients and show that subclonal RAS/BRAF alterations are uncommon and remain of indeterminate significance.

Detection of TP53 and PIK3CA Mutations in Circulating Tumor DNA Using Next-Generation Sequencing in the Screening Process for Early Breast Cancer Diagnosis.

Circulating tumor DNA (ctDNA) has emerged as a non-invasive "liquid biopsy" for early breast cancer diagnosis. We evaluated the suitability of ctDNA analysis in the diagnosis of early breast cancer after mammography findings, comparing PIK3CA and TP53 mutations between tumor biopsies and pre-biopsy circulating DNA. Matched plasma and frozen fresh tissue biopsies from patients with Breast Imaging-Reporting and Data System (BIRADS) 4c/5 mammography findings and subsequent diagnosis of primary breast cancer were analyzed using NGS TruSeq Custom Amplicon Low Input Panel (Illumina) and plasma SafeSEQ (Sysmex Inostics). The same plasma and tumor mutations were observed in eight of 29 patients (27.6%) with four in TP53 and five in PIK3CA mutations. Sequencing analysis also revealed four additional ctDNA mutations (three in TP53 and one in PIK3CA) previously not identified in three patients tissue biopsy. One of these patients had mutations in both genes. Age, tumor grade and size, immunohistochemical (IHC) subtype, BIRADS category, and lymph node positivity were significantly associated with the detectability of these blood tumor-derived mutations. In conclusion, ctDNA analysis could be used in early breast cancer diagnosis, providing critical clinical information to improve patient diagnosis.

Performance of Streck cfDNA Blood Collection Tubes for Liquid Biopsy Testing.

OBJECTIVES: Making liquid biopsy testing widely available requires a concept to ship whole blood at ambient temperatures while retaining the integrity of the cell-free DNA (cfDNA) population and stability of blood cells to prevent dilution of circulating tumor DNA (ctDNA) with wild-type genomic DNA. The cell- and DNA-stabilizing properties of Streck Cell-Free DNA BCT blood collection tubes (cfDNA BCTs) were evaluated to determine if they can be utilized in combination with highly sensitive mutation detection technologies. METHODS: Venous blood from healthy donors or patients with advanced colorectal cancer (CRC) was collected in cfDNA BCTs and standard K2EDTA tubes. Tubes were stored at different temperatures for various times before plasma preparation and DNA extraction. The isolated cfDNA was analyzed for overall DNA yield of short and long DNA fragments using qPCR as well as for mutational changes using BEAMing and Plasma Safe-Sequencing (Safe-SeqS). RESULTS: Collection of whole blood from healthy individuals in cfDNA BCTs and storage for up to 5 days at room temperature did not affect the DNA yield and mutation background levels (n = 60). Low-frequency mutant DNA spiked into normal blood samples as well as mutant circulating tumor DNA in blood samples from CRC patients collected in cfDNA BCTs were reliably detected after 3 days of storage at room temperature. However, blood samples stored at ≤ 10°C and at 40°C for an extended period of time showed elevated normal genomic DNA levels and an abnormally large cellular plasma interface as well as lower plasma volumes. CONCLUSION: Whole blood shipped in cfDNA BCTs over several days can be used for downstream liquid biopsy testing using BEAMing and Safe-SeqS. Since the shipping temperature is a critical factor, special care has to be taken to maintain a defined room temperature range to obtain reliable mutation testing results.

Detection and Quantification of KIT Mutations in ctDNA by Plasma Safe-SeqS.

We have designed a highly sensitive assay based on the Safe-SeqS technology to detect de novo mutations in the KIT gene and tested its performance. This assay was applied to plasma samples of GIST patients before and after treatment with regorafenib (GRID III trial) and mutations at known and novel sites of potential secondary resistance were identified.

In vivo activity and pharmacokinetics of nemorosone on pancreatic cancer xenografts.

Pancreatic cancer is one of the leading cancer-related causes of death in the western world with an urgent need for new treatment strategies. Recently, hyperforin and nemorosone have been described as promising anti-cancer lead compounds. While hyperforin has been thoroughly investigated in vitro and in vivo, in vivo data for nemorosone are still missing. Thus, we investigated the growth-inhibitory potential of nemorosone on pancreatic cancer xenografts in NMRI nu/nu mice and determined basic pharmacokinetic parameters. Xenograft tumors were treated with nemorosone and gemcitabine, the current standard of care. Tumor sections were subjected to H&E as well as caspase 3 and Ki-67 staining. Nemorosone plasma kinetics were determined by HPLC and mass spectrometry. Induction of CYP3A4 and other metabolizing enzymes by nemorosone and hyperforin was tested on primary hepatocytes using qRT-PCR. At a dose of 50 mg/kg nemorosone per day, a significant growth-inhibitory effect was observed in pancreatic cancer xenografts. The compound was well tolerated and rapidly absorbed into the bloodstream with a half-life of approximately 30 min. Different metabolites were detected, possibly resembling CYP3A4-independent oxidation products. It is concluded that nemorosone is a potential anti-cancer lead compound with good bioavailability, little side-effects and promising growth-inhibitory effects, thus representing a valuable compound for a combination therapy approach.

Comparison of the cytotoxic effects of enantiopure PPAPs, including nemorosone and clusianone.

The synthesis of an unnatural polyprenylated acylphloroglucinol (PPAP), regioisomeric with nemorosone and clusianone, has been accomplished. The separated enantiomers of this new PPAP, along with those of nemorosone and clusianone, have been screened for activity against HeLa (cervix carcinoma), MIA-PaCa-2 (pancreatic carcinoma), and MCF7 (mamma carcinoma) cancer cell lines. All of the isomers examined gave surprisingly similar results in the screens.

Replication and virus-induced transcriptome of HAdV-5 in normal host cells versus cancer cells--differences of relevance for adenoviral oncolysis.

Adenoviruses (Ads), especially HAdV-5, have been genetically equipped with tumor-restricted replication potential to enable applications in oncolytic cancer therapy. Such oncolytic adenoviruses have been well tolerated in cancer patients, but their anti-tumor efficacy needs to be enhanced. In this regard, it should be considered that cancer cells, dependent on their tissue of origin, can differ substantially from the normal host cells to which Ads are adapted by complex virus-host interactions. Consequently, viral replication efficiency, a key determinant of oncolytic activity, might be suboptimal in cancer cells. Therefore, we have analyzed both the replication kinetics of HAdV-5 and the virus-induced transcriptome in human bronchial epithelial cells (HBEC) in comparison to cancer cells. This is the first report on genome-wide expression profiling of Ads in their native host cells. We found that E1A expression and onset of viral genome replication are most rapid in HBEC and considerably delayed in melanoma cells. In squamous cell lung carcinoma cells, we observed intermediate HAdV-5 replication kinetics. Infectious particle production, viral spread and lytic activity of HAdV-5 were attenuated in melanoma cells versus HBEC. Expression profiling at the onset of viral genome replication revealed that HAdV-5 induced the strongest changes in the cellular transcriptome in HBEC, followed by lung cancer and melanoma cells. We identified prominent regulation of genes involved in cell cycle and DNA metabolism, replication and packaging in HBEC, which is in accord with the necessity to induce S phase for viral replication. Strikingly, in melanoma cells HAdV-5 triggered opposing regulation of said genes and, in contrast to lung cancer cells, no weak S phase induction was detected when using the E2F promoter as reporter. Our results provide a rationale for improving oncolytic adenoviruses either by adaptation of viral infection to target tumor cells or by modulating tumor cell functions to better support viral replication.

Microarray analysis of nemorosone-induced cytotoxic effects on pancreatic cancer cells reveals activation of the unfolded protein response (UPR).

BACKGROUND AND PURPOSE: Pancreatic cancer is one of the leading cancer-related causes of death due to high chemo-resistance and fast metastasation. Nemorosone, a polycyclic polyprenylated acylphloroglucinol, has recently been identified as a promising anticancer agent. Here, we examine its growth-inhibitory effects on pancreatic cancer cells. Based on transcription profiling, a molecular mode of action is proposed. EXPERIMENTAL APPROACH: Nemorosone cytotoxicity was assessed by the resazurin proliferation assay on pancreatic cancer cells and fibroblasts. Apoptosis was determined by Annexin V/propidium iodide staining as well as cytochrome c and caspase activation assays. Staining with the voltage-dependent dye JC-1 and fluorescence microscopy were used to detect effects on mitochondrial membrane potential. Total RNA was isolated from treated cell lines and subjected to microarray analysis, subsequent pathway identification and modelling. Gene expression data were validated by quantitative polymerase chain reaction and siRNA-mediated gene knock-down. KEY RESULTS: Nemorosone significantly inhibited cancer cell growth, induced cytochrome c release and subsequent caspase-dependent apoptosis, rapidly abolished mitochondrial membrane potential and elevated cytosolic calcium levels, while fibroblasts were largely unaffected. Expression profiling revealed 336 genes to be affected by nemorosone. A total of 75 genes were altered in all three cell lines, many of which were within the unfolded protein response (UPR) network. DNA damage inducible transcript 3 was identified as a key regulator in UPR-mediated cell death. CONCLUSIONS AND IMPLICATIONS: Nemorosone could be a lead compound for the development of novel anticancer drugs amplifying the already elevated UPR level in solid tumours, thus driving them into apoptosis. This study forms the basis for further investigations identifying nemorosone's direct molecular target(s).