A Multiplex Assay for Fast PIK3CA Hotspot Mutation Characterization in a Single Specimen by 3-Color Digital PCR Analysis.

BACKGROUND: Activating mutations in the phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) gene have been detected often in solid tumors. Targeted therapy for mutant PIK3CA is now available in the clinic, making molecular diagnostics pivotal. Our aim was to design a multiplex digital PCR (dPCR) assay to evaluate the 4 most common PIK3CA hotspot mutations simultaneously to characterize and quantify these in liquid biopsies. METHODS: A multiplex assay was developed to detect exon 9 p.E542K and p.E545K mutations, and exon 20 p.H1047L and p.H1047R mutations using the Stilla 3-color dPCR Naica system. The assay was evaluated on stock and pre-amplified DNA from cell lines with the above mutations as single and pooled samples, and on cell-free DNA (cfDNA) from healthy blood donors (HBDs) and breast cancer patients, to determine detection thresholds and diagnostic accuracy. RESULTS: The assay distinguished all 4 PIK3CA mutations in (cf)DNA, and also when dual mutations were present. Detection thresholds of stock and pre-amplified cfDNA samples were 0.11 and 0.40 copies/uL (cp/uL) for mutant copies concentration, and 0.003% and 0.68% for variant allele frequencies (VAFs), respectively. The assay confirmed the PIK3CA (mutation) status as defined by targeted next-generation sequencing (NGS) in 82 out of 96 patients that were mutant for PIK3CA, and in 11 out of 12 patients with wild-type PIK3CA. CONCLUSIONS: Our designed multiplex dPCR assay detected PIK3CA mutations with high accuracy in stock and pre-amplified cfDNA. Furthermore, it is affordable and demands less cfDNA input when compared to available uniplex dPCR assays and NGS analyses.

Epigenetic and Genomic Hallmarks of PARP-Inhibitor Resistance in Ovarian Cancer Patients.

BACKGROUND: Patients with advanced-stage epithelial ovarian cancer (EOC) receive treatment with a poly-ADP ribose-polymerase (PARP) inhibitor (PARPi) as maintenance therapy after surgery and chemotherapy. Unfortunately, many patients experience disease progression because of acquired therapy resistance. This study aims to characterize epigenetic and genomic changes in cell-free DNA (cfDNA) associated with PARPi resistance. MATERIALS AND METHODS: Blood was taken from 31 EOC patients receiving PARPi therapy before treatment and at disease progression during/after treatment. Resistance was defined as disease progression within 6 months after starting PARPi and was seen in fifteen patients, while sixteen patients responded for 6 to 42 months. Blood cfDNA was evaluated via Modified Fast Aneuploidy Screening Test-Sequencing System (mFast-SeqS to detect aneuploidy, via Methylated DNA Sequencing (MeD-seq) to find differentially methylated regions (DMRs), and via shallow whole-genome and -exome sequencing (shWGS, exome-seq) to define tumor fractions and mutational signatures. RESULTS: Aneuploid cfDNA was undetectable pre-treatment but observed in six patients post-treatment, in five resistant and one responding patient. Post-treatment ichorCNA analyses demonstrated in shWGS and exome-seq higher median tumor fractions in resistant (7% and 9%) than in sensitive patients (7% and 5%). SigMiner analyses detected predominantly mutational signatures linked to mismatch repair and chemotherapy. DeSeq2 analyses of MeD-seq data revealed three methylation signatures and more tumor-specific DMRs in resistant than in responding patients in both pre- and post-treatment samples (274 vs. 30 DMRs, 190 vs. 57 DMRs, Χ2-test p < 0.001). CONCLUSION: Our genome-wide Next-Generation Sequencing (NGS) analyses in PARPi-resistant patients identified epigenetic differences in blood before treatment, whereas genomic alterations were more frequently observed after progression. The epigenetic differences at baseline are especially interesting for further exploration as putative predictive biomarkers for PARPi resistance.

Comprehensive characterization of circulating tumor cells and cell-free DNA in patients with metastatic melanoma.

Advances in therapeutic approaches for melanoma urge the need for biomarkers that can identify patients at risk for recurrence and to guide treatment. The potential use of liquid biopsies in identifying biomarkers is increasingly being recognized. Here, we present a head-to-head comparison of several techniques to analyze circulating tumor cells (CTCs) and cell-free DNA (cfDNA) in 20 patients with metastatic melanoma. In this study, we investigated whether diagnostic leukapheresis (DLA) combined with multimarker flow cytometry (FCM) increased the detection of CTCs in blood compared to the CellSearch platform. Additionally, we characterized cfDNA at the level of somatic mutations, extent of aneuploidy and genome-wide DNA methylation. Both CTCs and cfDNA measures were compared to tumor markers and extracranial tumor burden on radiological imaging. Compared to the CellSearch method applied on peripheral blood, DLA combined with FCM increased the proportion of patients with detectable CTCs from 35% to 70% (P = 0.06). However, the median percentage of cells that could be recovered by the DLA procedure was 29%. Alternatively, cfDNA mutation and methylation analysis detected tumor load in the majority of patients (90% and 93% of samples successfully analyzed, respectively). The aneuploidy score was positive in 35% of all patients. From all tumor measurements in blood, lactate dehydrogenase (LDH) levels were significantly correlated to variant allele frequency (P = 0.004). Furthermore, the presence of CTCs in DLA was associated with tumor burden (P < 0.001), whereas the presence of CTCs in peripheral blood was associated with number of lesions on radiological imaging (P < 0.001). In conclusion, DLA tended to increase the proportion of patients with detectable CTCs but was also associated with low recovery. Both cfDNA and CTCs were correlated with clinical parameters such as LDH levels and extracranial tumor burden.

Machine learning-based somatic variant calling in cell-free DNA of metastatic breast cancer patients using large NGS panels.

Next generation sequencing of cell-free DNA (cfDNA) is a promising method for treatment monitoring and therapy selection in metastatic breast cancer (MBC). However, distinguishing tumor-specific variants from sequencing artefacts and germline variation with low false discovery rate is challenging when using large targeted sequencing panels covering many tumor suppressor genes. To address this, we built a machine learning model to remove false positive variant calls and augmented it with additional filters to ensure selection of tumor-derived variants. We used cfDNA of 70 MBC patients profiled with both the small targeted Oncomine breast panel (Thermofisher) and the much larger Qiaseq Human Breast Cancer Panel (Qiagen). The model was trained on the panels' common regions using Oncomine hotspot mutations as ground truth. Applied to Qiaseq data, it achieved 35% sensitivity and 36% precision, outperforming basic filtering. For 20 patients we used germline DNA to filter for somatic variants and obtained 245 variants in total, while our model found seven variants, of which six were also detected using the germline strategy. In ten tumor-free individuals, our method detected in total one (potentially germline) variant, in contrast to 521 variants detected without our model. These results indicate that our model largely detects somatic variants.

Detection of circulating tumour DNA after neoadjuvant chemoradiotherapy in patients with locally advanced oesophageal cancer.

Active surveillance instead of standard surgery after neoadjuvant chemoradiotherapy (nCRT) has been proposed for patients with oesophageal cancer. Circulating tumour DNA (ctDNA) may be used to facilitate selection of patients for surgery. We show that detection of ctDNA after nCRT seems highly suggestive of major residual disease. Tumour biopsies and blood samples were taken before, and 6 and 12 weeks after, nCRT. Biopsies were analysed with regular targeted next-generation sequencing (NGS). Circulating cell-free DNA (cfDNA) was analysed using targeted NGS with unique molecular identifiers and digital polymerase chain reaction. cfDNA mutations matching pre-treatment biopsy mutations confirmed the presence of ctDNA. In total, 31 patients were included, of whom 24 had a biopsy mutation that was potentially detectable in cfDNA (77%). Pre-treatment ctDNA was detected in nine of 24 patients (38%), four of whom had incurable disease progression before surgery. Pre-treatment ctDNA detection had a sensitivity of 47% (95% CI 24-71) (8/17), specificity of 85% (95% CI 42-99) (6/7), positive predictive value (PPV) of 89% (95% CI 51-99) (8/9), and negative predictive value (NPV) of 40% (95% CI 17-67) (6/15) for detecting major residual disease (>10% residue in the resection specimen or progression before surgery). After nCRT, ctDNA was detected in three patients, two of whom had disease progression. Post-nCRT ctDNA detection had a sensitivity of 21% (95% CI 6-51) (3/14), specificity of 100% (95% CI 56-100) (7/7), PPV of 100% (95% CI 31-100) (3/3), and NPV of 39% (95% CI 18-64) (7/18) for detecting major residual disease. The addition of ctDNA to the current set of diagnostics did not lead to more patients being clinically identified with residual disease. These results indicate that pre-treatment and post-nCRT ctDNA detection may be useful in identifying patients at high risk of disease progression. The addition of ctDNA analysis to the current set of diagnostic modalities may not improve detection of residual disease after nCRT. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

CABA-V7: a prospective biomarker selected trial of cabazitaxel treatment in AR-V7 positive prostate cancer patients.

BACKGROUND: Metastatic castration-resistant prostate cancer (mCRPC) patients with positive AR-V7 expression in their circulating tumour cells (CTCs) rarely derive benefit from abiraterone and enzalutamide. DESIGN: We performed a prospective, multicenter, single arm phase II clinical trial (CABA-V7) in mCRPC patients previously treated with docetaxel and androgen deprivation therapy. OBJECTIVE: In this trial, we investigated whether cabazitaxel treatment resulted in clinically meaningful PSA response rates in patients with positive CTC-based AR-V7 expression and collected liquid biopsies for genomic profiling. RESULTS: Cabazitaxel was found to be modestly effective, with only 12% of these patients obtaining a PSA response. Genomic profiling revealed that CTC-based AR-V7 expression was not associated with other known mCRPC-associated alterations. CTC-based AR-V7 status and dichotomised CTC counts were observed as independent prognostic markers at baseline. CONCLUSIONS: AR-V7 positivity predicted poor overall survival (OS). However, cabazitaxel-treated AR-V7 positive patients and those lacking AR-V7 positivity, who received cabazitaxel as standard of care, appeared to have similar OS. Therefore, despite the low response rate, cabazitaxel may still be an effective treatment in this poor prognosis, AR-V7 positive patient population.

Liquid biopsy in esophageal cancer: a case report of false-positive circulating tumor DNA detection due to clonal hematopoiesis.

Circulating tumor DNA (ctDNA) analysis is a promising non-invasive technique for active surveillance after chemoradiotherapy for locally advanced resectable esophageal carcinoma. In other malignancies false-positive results in ctDNA analysis have been reported due to clonal hematopoiesis. In this case, we present a 66-year-old male who had adenocarcinoma of the gastroesophageal junction for which he received neoadjuvant chemoradiotherapy and underwent a transhiatal esophagectomy. Postoperatively our patient received follow-up with ctDNA analysis using next generation sequencing (NGS) and droplet digital PCR (ddPCR). This case report illustrates a number of the current challenges in ctDNA diagnostics in esophageal carcinoma. Firstly, the TP53 c.524G>A; p.R175H mutation that was found in preoperative tumor biopsies became detectable in ctDNA only after distant metastases had already been confirmed by clinical symptoms and standard imaging- and biopsy techniques. Secondly our patient repeatedly had false-positive outcomes of ctDNA analysis. Genomic analysis of white blood cells revealed that the origin of these discordant mutations lies in clonal hematopoiesis. Failure to detect TP53 c.524G>A; p.R175H in cell-free DNA (cfDNA) is most likely due to the amount of ctDNA in the cfDNA fraction being below the limit of detection for NGS and ddPCR analyses. Clinicians should be aware of the possibility of finding mutations originating from clonal hematopoiesis when using ctDNA analysis during active surveillance for esophageal carcinoma. We recommend correlation of mutations in cfDNA with mutations in tumor biopsies.

Optimizing Nanopore sequencing-based detection of structural variants enables individualized circulating tumor DNA-based disease monitoring in cancer patients.

Here, we describe a novel approach for rapid discovery of a set of tumor-specific genomic structural variants (SVs), based on a combination of low coverage cancer genome sequencing using Oxford Nanopore with an SV calling and filtering pipeline. We applied the method to tumor samples of high-grade ovarian and prostate cancer patients and validated on average ten somatic SVs per patient with breakpoint-spanning PCR mini-amplicons. These SVs could be quantified in ctDNA samples of patients with metastatic prostate cancer using a digital PCR assay. The results suggest that SV dynamics correlate with and may improve existing treatment-response biomarkers such as PSA. https://github.com/UMCUGenetics/SHARC .

Detection of oncogenic mutations in paired circulating tumor DNA and circulating tumor cells in patients with hepatocellular carcinoma.

BACKGROUND AND AIMS: Circulating tumor cells (CTCs) or circulating tumor DNA (ctDNA) may be used for diagnostic or prognostic purposes in patients with hepatocellular carcinoma (HCC). We aim to determine whether CTCs or ctDNA are suitable to determine oncogenic mutations in HCC patients. METHODS: Twenty-six mostly advanced HCC patients were enrolled. 30 mL peripheral blood from each patient was obtained. CellSearch system was used for CTC detection. A sequencing panel covering 14 cancer-relevant genes was used to identify oncogenic mutations. TERT promoter C228T and C250T mutations were determined by droplet digital PCR. RESULTS: CTCs were detected in 27% (7/26) of subjects but at low numbers (median: 2 cells, range: 1-15 cells) and ctDNA in 77% (20/26) of patients. Mutations in ctDNA were identified in several genes: TERT promoter C228T (77%, 20/26), TP53 (23%, 6/26), CTNNB1 (12%, 3/26), PIK3CA (12%, 3/26) and NRAS (4%, 1/26). The TERT C228T mutation was present in all patients with one or more ctDNA mutations, or detectable CTCs. The TERT C228T and TP53 mutations detected in ctDNA were present at higher levels in matched primary HCC tumor tissue. The maximal variant allele frequency (VAF) of ctDNA was linearly correlated with largest tumor size and AFP level (Log10). CtDNA (or TERT C228T) positivity was associated with macrovascular invasion, and positivity of ctDNA (or TERT C228T) or CTCs (≥ 2) correlated with poor patient survival. CONCLUSIONS: Oncogenic mutations could be detected in ctDNA from advanced HCC patients. CtDNA analysis may serve as a promising liquid biopsy to identify druggable mutations.

Detection of tumor-derived extracellular vesicles in plasma from patients with solid cancer.

BACKGROUND: Extracellular vesicles (EVs) are actively secreted by cells into body fluids and contain nucleic acids of the cells they originate from. The goal of this study was to detect circulating tumor-derived EVs (ctEVs) by mutant mRNA transcripts (EV-RNA) in plasma of patients with solid cancers and compare the occurrence of ctEVs with circulating tumor DNA (ctDNA) in cell-free DNA (cfDNA). METHODS: For this purpose, blood from 20 patients and 15 healthy blood donors (HBDs) was collected in different preservation tubes (EDTA, BCT, CellSave) and processed into plasma within 24 h from venipuncture. EVs were isolated with the ExoEasy protocol from this plasma and from conditioned medium of 6 cancer cell lines and characterized according to MISEV2018-guidelines. RNA from EVs was isolated with the ExoRNeasy protocol and evaluated for transcript expression levels of 96 genes by RT-qPCR and genotyped by digital PCR. RESULTS: Our workflow applied on cell lines revealed a high concordance between cellular mRNA and EV-RNA in expression levels as well as variant allele frequencies for PIK3CA, KRAS and BRAF. Plasma CD9-positive EV and GAPDH EV-RNA levels were significantly different between the preservation tubes. The workflow detected only ctEVs with mutant transcripts in plasma of patients with high amounts (> 20%) of circulating tumor DNA (ctDNA). Expression profiling showed that the EVs from patients resemble healthy donors more than tumor cell lines supporting that most EVs are derived from healthy tissue. CONCLUSIONS: We provide a workflow for ctEV detection by spin column-based generic isolation of EVs and PCR-based measurement of gene expression and mutant transcripts in EV-RNA derived from cancer patients' blood plasma. This workflow, however, detected tumor-specific mutations in blood less often in EV-RNA than in cfDNA.

TP53 Mutations in Serum Circulating Cell-Free Tumor DNA As Longitudinal Biomarker for High-Grade Serous Ovarian Cancer.

The aim of this study was to determine an optimal workflow to detect TP53 mutations in baseline and longitudinal serum cell free DNA (cfDNA) from high-grade serous ovarian carcinomas (HGSOC) patients and to define whether TP53 mutations are suitable as biomarker for disease. TP53 was investigated in tissue and archived serum from 20 HGSOC patients by a next-generation sequencing (NGS) workflow alone or combined with digital PCR (dPCR). AmpliSeq™-focused NGS panels and customized dPCR assays were used for tissue DNA and longitudinal cfDNAs, and Oncomine NGS panel with molecular barcoding was used for baseline cfDNAs. TP53 missense mutations were observed in 17 tissue specimens and in baseline cfDNA for 4/8 patients by AmpliSeq, 6/9 patients by Oncomine, and 4/6 patients by dPCR. Mutations in cfDNA were detected in 4/6 patients with residual disease and 3/4 patients with disease progression within six months, compared to 5/11 patients with no residual disease and 6/13 patients with progression after six months. Finally, mutations were detected at progression in 5/6 patients, but not during chemotherapy. NGS with molecular barcoding and dPCR were most optimal workflows to detect TP53 mutations in baseline and longitudinal serum cfDNA, respectively. TP53 mutations were undetectable in cfDNA during treatment but re-appeared at disease progression, illustrating its promise as a biomarker for disease monitoring.

High-throughput isolation of circulating tumor DNA: a comparison of automated platforms.

The emerging interest in circulating tumor DNA (ctDNA) analyses for clinical trials has necessitated the development of a high-throughput method for fast, reproducible, and efficient isolation of ctDNA. Currently, the majority of ctDNA studies use the manual QIAamp (QA) platform to isolate DNA from blood. The purpose of this study was to compare two competing automated DNA isolation platforms [Maxwell (MX) and QIAsymphony (QS)] to the current 'gold standard' QA to facilitate high-throughput processing of samples in prospective trials. We obtained blood samples from healthy blood donors and metastatic cancer patients for plasma isolation. Total cell-free DNA (cfDNA) quantity was assessed by TERT quantitative PCR. Recovery efficiency was investigated by quantitative PCR analysis of spiked-in synthetic plant DNA. In addition, a ß-actin fragmentation assay was performed to determine the amount of contamination by genomic DNA from lysed leukocytes. ctDNA quality was assessed by digital PCR for somatic variant detection. cfDNA quantity and recovery efficiency were lowest using the MX platform, whereas QA and QS showed a comparable performance. All platforms preferentially isolated small (136 bp) DNA fragments over large (420 and 2000 bp) DNA fragments. Detection of the number variant and wild-type molecules was most comparable between QA and QS. However, there was no significant difference in variant allele frequency comparing QS and MX to QA. In summary, we show that the QS platform has comparable performance to QA, the 'gold standard', and outperformed the MX platform depending on the readout used. We conclude that the QS can replace the more laborious QA platform, especially when high-throughput cfDNA isolation is needed.

IGF1R signaling drives antiestrogen resistance through PAK2/PIX activation in luminal breast cancer.

Antiestrogen resistance in estrogen receptor positive (ER+) breast cancer is associated with increased expression and activity of insulin-like growth factor 1 receptor (IGF1R). Here, a kinome siRNA screen has identified 10 regulators of IGF1R-mediated antiestrogen with clinical significance. These include the tamoxifen resistance suppressors BMPR1B, CDK10, CDK5, EIF2AK1, and MAP2K5, and the tamoxifen resistance inducers CHEK1, PAK2, RPS6KC1, TTK, and TXK. The p21-activated kinase 2, PAK2, is the strongest resistance inducer. Silencing of the tamoxifen resistance inducing genes, particularly PAK2, attenuates IGF1R-mediated resistance to tamoxifen and fulvestrant. High expression of PAK2 in ER+ metastatic breast cancer patients is correlated with unfavorable outcome after first-line tamoxifen monotherapy. Phospho-proteomics has defined PAK2 and the PAK-interacting exchange factors PIXα/ß as downstream targets of IGF1R signaling, which are independent from PI3K/ATK and MAPK/ERK pathways. PAK2 and PIXα/ß modulate IGF1R signaling-driven cell scattering. Targeting PIXα/ß entirely mimics the effect of PAK2 silencing on antiestrogen re-sensitization. These data indicate PAK2/PIX as an effector pathway in IGF1R-mediated antiestrogen resistance.

Increased MAPK1/3 Phosphorylation in Luminal Breast Cancer Related with PIK3CA Hotspot Mutations and Prognosis.

INTRODUCTION: While mutations in PIK3CA are most frequently (45%) detected in luminal breast cancer, downstream PI3K/AKT/mTOR pathway activation is predominantly observed in the basal subtype. The aim was to identify proteins activated in PIK3CA mutated luminal breast cancer and the clinical relevance of such a protein in breast cancer patients. MATERIALS AND METHODS: Expression levels of 171 signaling pathway (phospho-)proteins established by The Cancer Genome Atlas (TCGA) using reverse phase protein arrays (RPPA) were in silico examined in 361 breast cancers for their relation with PIK3CA status. MAPK1/3 phosphorylation was evaluated with immunohistochemistry on tissue microarrays (TMA) containing 721 primary breast cancer core biopsies to explore the relationship with metastasis-free survival. RESULTS: In silico analyses revealed increased phosphorylation of MAPK1/3, p38 and YAP, and decreased expression of p70S6K and 4E-BP1 in PIK3CA mutated compared to wild-type luminal breast cancer. Augmented MAPK1/3 phosphorylation was most significant, i.e. in luminal A for both PIK3CA exon 9 and 20 mutations and in luminal B for exon 9 mutations. In 290 adjuvant systemic therapy naïve lymph node negative (LNN) breast cancer patients with luminal cancer, high MAPK phosphorylation in nuclei (HR=0.49; 95% CI, 0.25-0.95; P=.036) and in tumor cells (HR=0.37; 95% CI, 0.18-0.79; P=.010) was related with favorable metastasis-free survival in multivariate analyses including traditional prognostic factors. CONCLUSION: Enhanced MAPK1/3 phosphorylation in luminal breast cancer is related to PIK3CA exon-specific mutations and correlated with favorable prognosis especially when located in the nuclei of tumor cells.

Somatic Tumor Mutations Detected by Targeted Next Generation Sequencing in Minute Amounts of Serum-Derived Cell-Free DNA.

The use of blood-circulating cell-free DNA (cfDNA) as 'liquid-biopsy' is explored worldwide, with hopes for its potential in providing prognostic or predictive information in cancer treatment. In exploring cfDNA, valuable repositories are biobanks containing material collected over time, however these retrospective cohorts have restrictive resources. In this study, we aimed to detect tumor-specific mutations in only minute amounts of serum-derived cfDNA by using a targeted next generation sequencing (NGS) approach. In a retrospective cohort of ten metastatic breast cancer patients, we profiled DNA from primary tumor tissue (frozen), tumor-adjacent normal tissue (formalin-fixed paraffin embedded), and three consecutive serum samples (frozen). Our presented workflow includes comparisons with matched normal DNA or in silico reference DNA to discriminate germline from somatic variants, validation of variants through the detection in at least two DNA samples of an individual, and the use of public databases on variants. By our workflow, we were able to detect a total of four variants traceable as circulating tumor DNA (ctDNA) in the sera of three of the ten patients.

Application of circulating tumor DNA in prospective clinical oncology trials - standardization of preanalytical conditions.

Circulating tumor DNA (ctDNA) has emerged as a potential new biomarker with diagnostic, predictive, and prognostic applications for various solid tumor types. Before beginning large prospective clinical trials to prove the added value of utilizing ctDNA in clinical practice, it is essential to investigate the effects of various preanalytical conditions on the quality of cell-free DNA (cfDNA) in general and of ctDNA in particular in order to optimize and standardize these conditions. Whole blood samples were collected from patients with metastatic cancer bearing a known somatic variant. The following preanalytical conditions were investigated: (a) different time intervals to plasma isolation (1, 24, and 96 h) and (b) different preservatives in blood collection tubes (EDTA, CellSave, and BCT). The quality of cfDNA/ctDNA was assessed by DNA quantification, digital polymerase chain reaction (dPCR) for somatic variant detection and a ß-actin fragmentation assay for DNA contamination from lysed leukocytes. In 11 (69%) of our 16 patients, we were able to detect the known somatic variant in ctDNA. We observed a time-dependent increase in cfDNA concentrations in EDTA tubes, which was positively correlated with an increase in wild-type copy numbers and large DNA fragments (> 420 bp). Using different preservatives did not affect somatic variant detection ability, but did stabilize cfDNA concentrations over time. Variant allele frequency was affected by fluctuations in cfDNA concentration only in EDTA tubes at 96 h. Both CellSave and BCT tubes ensured optimal ctDNA quality in plasma processed within 96 h after blood collection for downstream somatic variant detection by dPCR.

LRG1 mRNA expression in breast cancer associates with PIK3CA genotype and with aromatase inhibitor therapy outcome.

BACKGROUND: PIK3CA is the most frequent somatic mutated oncogene in estrogen receptor (ER) positive breast cancer. We previously observed an association between PIK3CA genotype and aromatase inhibitors (AI) treatment outcome. This study now evaluates whether expression of mRNAs and miRs are linked to PIK3CA genotype and are independently related to AI therapy response in order to define potential expressed biomarkers for treatment outcome. MATERIALS AND METHODS: The miR and mRNA expression levels were evaluated for their relationship with the PIK3CA genotype in two breast tumor datasets, i.e. 286 luminal cancers from the TCGA consortium and our set of 84 ER positive primary tumors of metastatic breast cancer patients who received first line AI. BRB Array tools class comparison was performed to define miRs and mRNAs whose expression associate with PIK3CA exon 9 and 20 status. Spearman correlations established miR-mRNA pairs and mRNAs with related expression. Next, a third dataset of 25 breast cancer patients receiving neo-adjuvant letrozole was evaluated, to compare expression levels of identified miRs and mRNAs in biopsies before and after treatment. Finally, to identify potential biomarkers miR and mRNA levels were related with overall survival (OS) and progression free survival (PFS) after first-line AI therapy. RESULTS: Expression of 3 miRs (miR-449a, miR-205-5p, miR-301a-3p) and 9 mRNAs (CCNO, FAM81B, LRG1, NEK10, PLCL1, PGR, SERPINA3, SORBS2, VTCN1) was related to the PIK3CA status in both datasets. All except miR-301a-3p had an increased expression in tumors with PIK3CA mutations. Validation in a publicly available dataset showed that LRG1, PGR, and SERPINA3 levels were decreased after neo-adjuvant AI-treatment. Six miR-mRNA pairs correlated significantly and stepdown analysis of all 12 factors revealed 3 mRNAs (PLCL1, LRG1, FAM81B) related to PFS. Further analyses showed LRG1 and PLCL1 expression to be unrelated with luminal subtype and to associate with OS and with PFS, the latter independent from traditional predictive factors. CONCLUSION: We showed in two datasets of ER positive and luminal breast tumors that the expression of 3 miRs and 9 mRNAs associate with the PIK3CA status. Expression of LRG1 is independent of luminal (A or B) subtype, decreased after neo-adjuvant AI-treatment, and is proposed as potential biomarker for AI therapy outcome.

Cell-free DNA mutations as biomarkers in breast cancer patients receiving tamoxifen.

The aim was to identify mutations in serum cell-free DNA (cfDNA) associated with disease progression on tamoxifen treatment in metastatic breast cancer (MBC). Sera available at start of therapy, during therapy and at disease progression were selected from 10 estrogen receptor (ER)-positive breast cancer patients. DNA from primary tumor and normal tissue and cfDNA from minute amounts of sera were analyzed by targeted next generation sequencing (NGS) of 45 genes (1,242 exons). At disease progression, stop-gain single nucleotide variants (SNVs) for CREBBP (1 patient) and SMAD4 (1 patient) and non-synonymous SNVs for AKAP9 (1 patient), PIK3CA (2 patients) and TP53 (2 patients) were found. Mutations in CREBBP and SMAD4 have only been occasionally reported in breast cancer. All mutations, except for AKAP9, were also present in the primary tumor but not detected in all blood specimens preceding progression. More sensitive detection by deeper re-sequencing and digital PCR confirmed the occurrence of circulating tumor DNA (ctDNA) and these biomarkers in blood specimens.

Somatic mutation detection using various targeted detection assays in paired samples of circulating tumor DNA, primary tumor and metastases from patients undergoing resection of colorectal liver metastases.

Assessing circulating tumor DNA (ctDNA) is a promising method to evaluate somatic mutations from solid tumors in a minimally-invasive way. In a group of twelve metastatic colorectal cancer (mCRC) patients undergoing liver metastasectomy, from each patient DNA from cell-free DNA (cfDNA), the primary tumor, metastatic liver tissue, normal tumor-adjacent colon or liver tissue, and whole blood were obtained. Investigated was the feasibility of a targeted NGS approach to identify somatic mutations in ctDNA. This targeted NGS approach was also compared with NGS preceded by mutant allele enrichment using synchronous coefficient of drag alteration technology embodied in the OnTarget assay, and for selected mutations with digital PCR (dPCR). All tissue and cfDNA samples underwent IonPGM sequencing for a CRC-specific 21-gene panel, which was analyzed using a standard and a modified calling pipeline. In addition, cfDNA, whole blood and normal tissue DNA were analyzed with the OnTarget assay and with dPCR for specific mutations in cfDNA as detected in the corresponding primary and/or metastatic tumor tissue. NGS with modified calling was superior to standard calling and detected ctDNA in the cfDNA of 10 patients harboring mutations in APC, ATM, CREBBP, FBXW7, KRAS, KMT2D, PIK3CA and TP53. Using this approach, variant allele frequencies in plasma ranged predominantly from 1 to 10%, resulting in limited concordance between ctDNA and the primary tumor (39%) and the metastases (55%). Concordance between ctDNA and tissue markedly improved when ctDNA was evaluated for KRAS, PIK3CA and TP53 mutations by the OnTarget assay (80%) and digital PCR (93%). Additionally, using these techniques mutations were observed in tumor-adjacent tissue with normal morphology in the majority of patients, which were not observed in whole blood. In conclusion, in these mCRC patients with oligometastatic disease NGS on cfDNA was feasible, but had limited sensitivity to detect all somatic mutations present in tissue. Digital PCR and mutant allele enrichment before NGS appeared to be more sensitive to detect somatic mutations.

Ovarian cancer cell line panel (OCCP): clinical importance of in vitro morphological subtypes.

Epithelial ovarian cancer is a highly heterogeneous disease and remains the most lethal gynaecological malignancy in the Western world. Therapeutic approaches need to account for inter-patient and intra-tumoural heterogeneity and detailed characterization of in vitro models representing the different histological and molecular ovarian cancer subtypes is critical to enable reliable preclinical testing. There are approximately 100 publicly available ovarian cancer cell lines but their cellular and molecular characteristics are largely undescribed. We have characterized 39 ovarian cancer cell lines under uniform conditions for growth characteristics, mRNA/microRNA expression, exon sequencing, drug response for clinically-relevant therapeutics and collated all available information on the original clinical features and site of origin. We tested for statistical associations between the cellular and molecular features of the lines and clinical features. Of the 39 ovarian cancer cell lines, 14 were assigned as high-grade serous, four serous-type, one low-grade serous and 20 non-serous type. Three morphological subtypes: Epithelial (n = 21), Round (n = 7) and Spindle (n = 12) were identified that showed distinct biological and molecular characteristics, including overexpression of cell movement and migration-associated genes in the Spindle subtype. Comparison with the original clinical data showed association of the spindle-like tumours with metastasis, advanced stage, suboptimal debulking and poor prognosis. In addition, the expression profiles of Spindle, Round and Epithelial morphologies clustered with the previously described C1-stromal, C5-mesenchymal and C4 ovarian subtype expression profiles respectively. Comprehensive profiling of 39 ovarian cancer cell lines under controlled, uniform conditions demonstrates clinically relevant cellular and genomic characteristics. This data provides a rational basis for selecting models to develop specific treatment approaches for histological and molecular subtypes of ovarian cancer.