Abstract Background The androgen receptor splice variant 7 (AR-V7) is associated with resistance to hormonal therapy in castration-resistant prostate cancer (CRPC). Due to limitations of the methods ...available for AR-V7 analysis, the identification of a reliable detection method may facilitate the use of this biomarker in clinical practice. Objective To confirm AR-V7 as a predictor of resistance to hormonal therapy and develop a new approach to assess AR-V7 by highly sensitive digital droplet polymerase chain reaction (ddPCR) in plasma-derived exosomal RNA. Design, setting, and participants Plasma samples were collected from 36 CRPC patients before they began second-line hormonal treatment. Exosomes were isolated and RNA extracted for analysis of AR-V7 by ddPCR. Outcome measurements and statistical analysis The absolute target gene concentration as copies per milliliter (copies/ml) was determined by ddPCR. Statistical analyses were performed with SPSS software (IBM Corp., Armonk, NY, USA). Results and limitations A total of 26 patients received abiraterone and 10 enzalutamide; 39% of patients were found to be AR-V7 positive (AR-V7+ ). Median progression-free survival was significantly longer in AR-V7 negative (AR-V7− ) versus AR-V7+ patients (20 vs 3 mo; p < 0.001). Overall survival was significantly shorter in AR-V7+ participants at baseline compared with AR-V7− participants (8 mo vs not reached; p < 0.001). Conclusions This study demonstrates that plasma-derived exosomal RNA is a reliable source of AR-V7 that can be detected sensitively by ddPCR assay. We also showed that resistance to hormonal therapy may be predicted by AR-V7, making it a clinically relevant biomarker. Patient summary We report a first study on a method for androgen receptor splice variant 7 (AR-V7) detection in RNA extracted from cancer cell vesicles released in blood. Results confirmed the role of AR-V7 as a predictive biomarker of resistance to hormonal therapy. Our assay showed that vesicles are a reliable source of AR-V7 RNA and that the method is fast, highly sensitive, and affordable.
Liquid biopsy has emerged as an alternative source of nucleic acids for the management of Epidermal Growth Factor Receptor (
)-mutant non-Small Cell Lung Cancer (NSCLC). The use of circulating ...cell-free DNA (cfDNA) has been recently introduced in clinical practice, resulting in the improvement of the identification of druggable
mutations for the diagnosis and monitoring of response to targeted therapy.
-dependent (T790M and C797S mutations) and independent (Mesenchymal Epithelial Transition
gene amplification, Kirsten Rat Sarcoma
, Phosphatidyl-Inositol 4,5-bisphosphate 3-Kinase Catalytic subunit Alpha isoform
, and RAF murine sarcoma viral oncogene homolog B1
gene mutations) mechanisms of resistance to EGFR tyrosine kinase inhibitors (TKIs) have been evaluated in plasma samples from NSCLC patients using highly sensitive methods (i.e., digital droplet PCR, Next Generation Sequencing), allowing for the switch to other therapies. Therefore, liquid biopsy is a non-invasive method able to detect the molecular dynamic changes that occur under the pressure of treatment, and to capture tumor heterogeneity more efficiently than is allowed by tissue biopsy. This review addresses how liquid biopsy may be used to guide the choice of treatment strategy in
-mutant NSCLC.
Modified FOLFIRINOX (mFOLFIRINOX) and gemcitabine + nab-paclitaxel (GemNab) regimens represent a standard treatment in advanced pancreatic cancer (aPC). DPYD and UGT1A1 variants are relevant ...predictors of fluoropyrimidine and irinotecan-associated adverse events (AEs). Furthermore, data about the associations between polymorphisms in ABCB and CDA genes and GemNab-related toxicities are still controversial. The present study analyzes the association between DPYD, UGT, ABCB1, CDA variants, and AEs in aPC patients (pts) treated with mFOLFIRINOX or GemNab. Blood samples collected from 104 aPC pts treated with mFOLFIRINOX and 63 with GemNab were tested for DPYD c.1679T>G, IVS14+1G>A, c.2194G>A, c.2846A>T, UGT1A1*28, CDA c.79A>C, and ABCB1 c.1236C>T, c.2677G>T/A, c.3435C>T by real-time PCR and automatic sequencing. In mFOLFIRINOX cohort, DPYD IVS14+1GA genotype was associated with G4 hematological AEs, while the UGT1A1*28 significantly correlated with the risk of thrombocytopenia (p = 0.006). In the GemNab cohort, a significant association between CDA c.79CC and high-grade nausea was observed (p = 0.002). Moreover, the presence of at least a mutant allele in ABCB1 increased the risk of overall hematological AEs (p = 0.01), both further strengthened by the presence of CDA c.79CC (p = 0.0002). DPYD IVS14+1A allele is confirmed to be associated with fluoropyrimidine life-threatening toxicities, and UGT1A1*28 is related with a higher risk of hematologic AEs following irinotecan treatment. CDA c.79C and ABCB1 c.1236T, c.2677T/A, and c.3435T mutant alleles are predictive biomarkers of GemNab-related AEs. All these variants should be considered in aPC pts candidate to mFOLFIRINOX or GemNab treatments.
Background
It is still unclear how to combine biomarkers to identify patients who will truly benefit from anti-PD-1 agents in NSCLC. This study investigates exosomal mRNA expression of PD-L1 and ...IFN-γ, PD-L1 polymorphisms, tumor mutational load (TML) in circulating cell-free DNA (cfDNA) and radiomic features as possible predictive markers of response to nivolumab and pembrolizumab in metastatic NSCLC patients.
Methods
Patients were enrolled and blood (12 ml) was collected at baseline before receiving anti-PD-1 therapy. Exosome-derived mRNA and cfDNA were extracted to analyse PD-L1 and IFN-γ expression and tumor mutational load (TML) by digital droplet PCR (ddPCR) and next-generation sequencing (NGS), respectively. The PD-L1 single nucleotide polymorphisms (SNPs) c.-14-368 T > C and c.*395G > C, were analysed on genomic DNA by Real-Time PCR. A radiomic analysis was performed on the QUIBIM Precision
®
V3.0 platform.
Results
Thirty-eight patients were enrolled. High baseline IFN-γ was independently associated with shorter median PFS (5.6 months vs. not reached
p
= 0.0057), and levels of PD-L1 showed an increase at 3 months vs. baseline in patients who progressed (
p
= 0.01). PD-L1 baseline levels showed significant direct and inverse relationships with radiomic features. Radiomic features also inversely correlated with PD-L1 expression in tumor tissue. In subjects receiving nivolumab, median PFS was shorter in carriers of c.*395GG vs. c.*395GC/CC genotype (2.3 months vs. not reached,
p
= 0.041). Lastly, responders had higher non-synonymous mutations and more links between co-occurring genetic somatic mutations and ARID1A alterations as well.
Conclusions
A combined multiparametric approach may provide a better understanding of the molecular determinants of response to immunotherapy.
In the "precision oncology" era the characterization of tumor genetic features is a pivotal step in cancer patients' management. Liquid biopsy approaches, such as analysis of cell-free DNA from ...plasma, represent a powerful and noninvasive strategy to obtain information about the genomic status of the tumor. Sequencing-based analyses of cell-free DNA, currently performed with second generation sequencers, are extremely powerful but poorly scalable and not always accessible also due to instrumentation costs. Third generation sequencing platforms, such as Nanopore sequencers, aim at overcoming these obstacles but, unfortunately, are not designed for cell-free DNA analysis.Here we present a customized workflow to exploit low-coverage Nanopore sequencing for the detection of copy number variations from plasma of cancer patients. Whole genome molecular karyotypes of 6 lung cancer patients and 4 healthy subjects were successfully produced with as few as 2 million reads, and common lung-related copy number alterations were readily detected.This is the first successful use of Nanopore sequencing for copy number profiling from plasma DNA. In this context, Nanopore represents a reliable alternative to Illumina sequencing, with the advantages of minute instrumentation costs and extremely short analysis time.The availability of protocols for Nanopore-based cell-free DNA analysis will make this analysis finally accessible, exploiting the full potential of liquid biopsy both for research and clinical purposes.
Circulating cell-free DNA (cfDNA) may help understand the molecular response to pharmacologic treatment and provide information on dynamics of clonal heterogeneity. Therefore, this study evaluated ...the correlation between treatment outcome and activating EGFR mutations (act-EGFR) and T790M in cfDNA in patients with advanced NSCLC given osimertinib.
Thirty-four NSCLC patients resistant to first/second-generation EGFR-TKIs, positive for both act-EGFR and T790M in cfDNA at the time of progression were enrolled in this study. Plasma samples were obtained at osimertinib baseline and after 3 months of therapy; cfDNA was analyzed by droplet digital PCR and results were expressed as mutant allele frequency (MAF).
At baseline, act-EGFR MAF was significantly higher than T790M (p < 0.0001). act-EGFR MAF and T790M/act-EGFR MAF ratio were significantly correlated with disease response (p = 0.02). Cut-off values of act-EGFR MAF and T790M/act-EGFR ratio of 2.6% and 0.22 were found, respectively. The PFS of patients with act-EGFR MAF of > 2.6% and < 2.6%, were 10 months vs. not reached, respectively (p = 0.03), whereas patients with T790M/act-EGFR ≤ 0.22 had poorer PFS than patients with a value of > 0.22 (6 months vs. not reached, respectively, p = 0.01).
act-EGFR MAF and T790M/act-EGFR MAF ratio are potential markers of outcome in patients treated with osimertinib.
KRAS is the most common and, simultaneously, the most ambiguous oncogene implicated in human cancer. Despite KRAS mutations were identified in Non Small Cell Lung Cancers (NSCLCs) more than 20 years ...ago, selective and specific inhibitors aimed at directly abrogating KRAS activity are not yet available. Nevertheless, many therapeutic approaches have been developed potentially useful to treat NSCLC patients mutated for KRAS and refractory to both standard chemotherapy and targeted therapies.The focus of this review will be to provide an overview of the network related to the intricate molecular KRAS pathways, stressing on preclinical and clinical studies that investigate the predictive value of KRAS mutations in NSCLC patients.
A bibliographic search of the Medline database was conducted for articles published in English, with the keywords KRAS, KRAS mutations in non-small cell lung cancer, KRAS and tumorigenesis, KRAS and TKIs, KRAS and chemotherapy, KRAS and monoclonal antibody, KRAS and immunotherapy, KRAS and drugs, KRAS and drug resistance.
Circulating cell-free DNA (cfDNA, liquid biopsy) is a powerful tool to detect molecular alterations. However, depending on tumor characteristics, biology and anatomic localization, cfDNA detection ...and analysis may be challenging. Gliomas are enclosed into an anatomic sanctuary, which obstacles the release of cfDNA into the peripheral blood. Therefore, the advantages of using liquid biopsy for brain tumors is still to be confirmed. The present study evaluates the ability of liquid biopsy to detect IDH1 mutations and its correlation with survival and clinical characteristics of glioma patients.
Blood samples obtained from glioma patients were collected after surgery prior to the adjuvant therapy. cfDNA was extracted from plasma and IDH1 p.R132H mutation analysis was performed on a digital droplet PCR. χ2-test and Cohen k were used to assess the correlation between plasma and tissue IDH1 status, while Kaplan Meier curve and Cox regression analysis were applied to survival analysis. Statistical calculations were performed by MedCalc and GraphPad Prism software.
A total of 67 samples were collected. A concordance between IDH1 status in tissue and in plasma was found (p = 0.0024), and the presence of the IDH1 mutation both in tissue (138.8 months vs 24.4, p < 0.0001) and cfDNA (116.3 months vs 35.8, p = 0.016) was associated with longer median OS. A significant association between IDH1 mutation both in tissue and cfDNA, age, tumor grade and OS was demonstrated by univariate Cox regression analysis. No statistically significant association between IDH1 mutation and tumor grade was found (p = 0.10).
The present study demonstrates that liquid biopsy may be used in brain tumors to detect IDH1 mutation which represents an important prognostic biomarker in patients with different types of gliomas, being associated to OS.
Metronomic chemotherapy (MC) is the frequent, regular administration of drug doses designed to maintain a low, but active, range of concentrations of chemotherapeutic drugs, during prolonged periods ...of time without inducing excessive toxicities. To date, more than 400,000 children and adolescents under the age of 20 are diagnosed with cancer, per year, with 80% survival in most high-income countries, but less than 30% in low- and middle-income ones. In this review, we summarized the principal preclinical and clinical studies involving the use of MC in the most common pediatric tumors, with an overview of efficacy, toxicity, pharmacokinetic profile, and biomarkers. The best advantages of MC are low toxicity, oral administration and, thus, the feasibility of a more comfortable, home-based treatment, therefore improving the quality of life of the children themselves and of their parents and caregivers. Moreover, MC could represent a valid method to reduce the economic burden of anticancer therapy in the pediatric setting.
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