ABSTRACT
DNA-methylation alterations are common in cancer and display unique characteristics that make them ideal markers for tumor quantification and classification. Here we present MIMESIS, a computational framework exploiting minimal DNA-methylation signatures composed by a few dozen informative DNA-methylation sites to quantify and classify tumor signals in tissue and cell-free DNA samples. Extensive analyses of multiple independent and heterogenous datasets including >7200 samples demonstrate the capability of MIMESIS to provide precise estimations of tumor content and to enable accurate classification of tumor type and molecular subtype. To assess our framework for clinical applications, we designed a MIMESIS-informed assay incorporating the minimal signatures for breast cancer. Using both artificial samples and clinical serial cell-free DNA samples from patients with metastatic breast cancer, we show that our approach provides accurate estimations of tumor content, sensitive detection of tumor signal and the ability to capture clinically relevant molecular subtype in patients' circulation. This study provides evidence that our extremely parsimonious approach can be used to develop cost-effective and highly scalable DNA-methylation assays that could support and facilitate the implementation of precision oncology in clinical practice.
Subject(s)
Breast Neoplasms , Cell-Free Nucleic Acids , Humans , Female , Cell-Free Nucleic Acids/genetics , Precision Medicine , DNA Methylation , Breast Neoplasms/genetics , Biomarkers, Tumor/genetics , DNA, Neoplasm/geneticsABSTRACT
BACKGROUND: Circulating tumor cells (CTCs) are prognostic in patients with advanced breast cancer (ABC). However, no data exist about their use in patients treated with palbociclib. We analyzed the prognostic role of CTC counts in patients enrolled in the cTREnd study, a pre-planned translational sub-study of TREnd (NCT02549430), that randomized patients with ABC to palbociclib alone or palbociclib plus the endocrine therapy received in the prior line of treatment. Moreover, we evaluated RB1 gene expression on CTCs and explored its prognostic role within the cTREnd subpopulation. METHODS: Forty-six patients with ER-positive, HER2-negative ABC were analyzed. Blood samples were collected before starting palbociclib treatment (timepoint T0), after the first cycle of treatment (timepoint T1), and at disease progression (timepoint T2). CTCs were isolated and counted by CellSearch® System using the CellSearch™Epithelial Cell kit. Progression-free survival (PFS), clinical benefit (CB) during study treatment, and time to treatment failure (TTF) after study treatment were correlated with CTC counts. Samples with ≥ 5 CTCs were sorted by DEPArray system® (DA). RB1 and GAPDH gene expression levels were measured by ddPCR. RESULTS: All 46 patients were suitable for CTCs analysis. CTC count at T0 did not show significant prognostic value in terms of PFS and CB. Patients with at least one detectable CTC at T1 (n = 26) had a worse PFS than those with 0 CTCs (n = 16) (p = 0.02). At T1, patients with an increase of at least three CTCs showed reduced PFS compared to those with no increase (mPFS = 3 versus 9 months, (p = 0.004). Finally, patients with ≥ 5 CTCs at T2 (n = 6/23) who received chemotherapy as post-study treatment had a shorter TTF (p = 0.02). Gene expression data for RB1 were obtained from 19 patients. CTCs showed heterogeneous RB1 expression. Patients with detectable expression of RB1 at any timepoint showed better, but not statistically significant, outcomes than those with undetectable levels. CONCLUSIONS: CTC count seems to be a promising modality in monitoring palbociclib response. Moreover, CTC count at the time of progression could predict clinical outcome post-palbociclib. RB1 expression analysis on CTCs is feasible and may provide additional prognostic information. Results should be interpreted with caution given the small studied sample size.
Subject(s)
Breast Neoplasms/drug therapy , Neoplastic Cells, Circulating/pathology , Piperazines/therapeutic use , Protein Kinase Inhibitors/therapeutic use , Pyridines/therapeutic use , Biomarkers, Tumor/blood , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Count , Disease Progression , Female , Humans , Neoplastic Cells, Circulating/drug effects , Neoplastic Cells, Circulating/metabolism , Progression-Free Survival , Receptor, ErbB-2/deficiency , Receptors, Estrogen/metabolism , Receptors, Progesterone/metabolism , Retinoblastoma Binding Proteins/metabolism , Treatment Outcome , Ubiquitin-Protein Ligases/metabolismABSTRACT
BACKGROUND: Within the OMITERC prospective study (OMIcs application from solid to liquid biopsy for a personalised ThERapy of Cancer), we explored the prognostic role of liquid biopsy encompassing cell-free DNA (cfDNA) and circulating tumour cells (CTCs) in KRAS mutated metastatic colorectal cancer (mCRC). METHODS: We defined a workflow including pre-analytical and analytical procedures collecting blood before therapy and every 3 months until disease progression (PD). CTCs were counted by CellSearch® and isolated by DEPArray™. NGS sequencing of CTCs and cfDNA was performed using a panel of cancer/CRC related genes respectively. RESULTS: KRAS mutational status was mostly concordant between tumour tissues and liquid biopsy. The percentage of cfDNA samples with mutations in CRC driver genes was in line with literature. In longitudinal monitoring circulating biomarkers anticipated or overlapped conventional diagnostic tools in predicting PD. The presence of CTCs at baseline was confirmed a negative prognostic marker. CONCLUSIONS: Cell-free DNA and CTCs are readily available candidates for clinical application in mCRC. While CTCs demonstrated a prognostic significance at baseline, cfDNA was confirmed an easily accessible material for monitoring the mutational status of the tumour over time. Moreover, in the longitudinal study, the two markers emerged as complementary in assessing disease progression.
Subject(s)
Biomarkers, Tumor/genetics , Cell-Free Nucleic Acids/genetics , Colorectal Neoplasms/pathology , Neoplastic Cells, Circulating/pathology , Proto-Oncogene Proteins p21(ras)/genetics , Sequence Analysis, DNA/methods , Aged , Aged, 80 and over , Colorectal Neoplasms/genetics , Disease Progression , Female , High-Throughput Nucleotide Sequencing , Humans , Longitudinal Studies , Male , Middle Aged , Mutation , Neoplasm Metastasis , Prognosis , Prospective StudiesABSTRACT
OBJECTIVES: to examine the factors that, in the context of the current pandemic, have influenced the conduct of a randomized clinical trial on hydroxychloroquine in Italy. DESIGN: the trend of enrolment in the PROTECT study, "A randomized study with Hydroxychloroquine versus observational support for prevention or early phase treatment of Coronavirus disease (COVID-19)" (Eudract number: 2020-001501-24, NCT04363827), conducted in the period from May to September 2020, was analysed to evaluate the possible association of the enrolment rate with the amount of information published in the national and local press on hydroxychloroquine. SETTING AND PARTICIPANTS: the PROTECT clinical study is an Italian interventional superiority study, open label, with cluster randomization, aimed at evaluating whether treatment with hydroxychloroquine can reduce the percentage of symptomatic subjects compared to observation only in a population of subjects exposed to SARS-CoV-2 virus consisting of cohabitants/contacts of COVID-19 patients and asymptomatic or paucisymptomatic subjects diagnosed with COVID-19. MAIN OUTCOME MEASURES: the number of asymptomatic or paucisymptomatic COVID-19 patients and the number of contacts/cohabitants of COVID-19 patients enrolled in the Protect study from May to September 2020. RESULTS: from May to September 2020, the number of patients diagnosed with COVID-19 enrolled in the PROTECT clinical trial showed a decrease consistent with the number of news on hydroxychloroquine appearing in the national and local press, starting from the time when the first criticisms of the efficacy of hydroxychloroquine were made known; the number of contacts/cohabitants of COVID-19 patients showed a more marked and more timely decrease. CONCLUSIONS: in the context determined by the current COVID-19 pandemic, conducting a controlled clinical trial is strongly influenced by public opinion on scientific issues. Adherence to a clinical study can become highly problematic and invalidate the possibility of answering a scientific question and the validity of a project. In the current pandemic situation, randomized controlled trials may not always be the optimal tool to reach the expected scientific evidence, due to a number of problems. It is preferable to use a sequential or adaptive design. Furthermore, study protocols should implement innovative approaches that also include the involvement of participants in the decision-making process. In any case, the influence of public information on scientific issues is an extremely important factor to consider in the design of clinical trials in exceptional situations such as a pandemic.
Subject(s)
COVID-19 Drug Treatment , COVID-19 , Hydroxychloroquine , COVID-19/epidemiology , Humans , Hydroxychloroquine/therapeutic use , Italy/epidemiologyABSTRACT
In the era of precision medicine in oncology, pharmacogenomic assessment is a vital step in delivering personalized care. Increasing evidence points towards the importance of assessing molecular features of the advanced disease, rather than relying on the primary tumor sample, in order to appreciate the evolution of the tumor and to target relevant features. Circulating tumor cells (CTCs) represent a novel method of tumor sampling, as they offer a contemporaneous picture of the current disease state without the need for invasive needle biopsy. As they may derive from any number of metastatic sites, the potential to capture the heterogeneity of the disease is increased. Improvements in CTC capture, enrichment and isolation technology now allow sophisticated interrogation of these cells, such that pharmacogenomic assessment of CTCs is now possible, and the clinical potential is being explored. We review current and potential uses for CTCs for pharmacogenomic analysis.
Subject(s)
Neoplasms/genetics , Neoplasms/pathology , Neoplastic Cells, Circulating/metabolism , Neoplastic Cells, Circulating/pathology , Pharmacogenomic Testing/methods , Humans , Precision Medicine/methodsABSTRACT
PURPOSE: To identify prognostic circulating biomarkers to cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6i), we performed a mutational analysis on circulating tumor DNA (ctDNA) samples from patients included in the TREnd trial, which randomly assigned patients to receive the CDK4/6i palbociclib alone or with the endocrine treatment (ET) to which they had progressed. METHODS: Forty-six patients were enrolled in this substudy. Plasma was collected before treatment (T0), after the first cycle of therapy (T1), and at the time of progression (T2). ctDNA hybridization and capture were performed using the Illumina TruSight Tumor 170 Kit. Acquired mutations were confirmed by digital polymerase chain reaction. Progression-free survival analysis was estimated using the Kaplan-Meier method and compared with the log-rank test. RESULTS: The most frequently mutated genes at T0 were ESR1 (23%), PIK3CA (17%), AR, FGFR2, and TP53 (10%). Mutations in ESR1 at T0 conferred higher risk of progression in the entire population (P = .02) and in patients treated with palbociclib + ET (P = .04). ESR1 mutation effect remained significant after correction for clinical variables (P = .03). PIK3CA mutations at T0 were not prognostic, but higher risk of progression was observed when a broader analysis of PI3K pathway was performed (P = .04). At T2, we observed the emergence of nine new mutations in seven genes. CONCLUSION: Mutations in ESR1 and in PI3K pathway genes at T0 were associated with worse prognosis in palbociclib-treated patients. We describe the emergence of newly acquired mutations in palbociclib-treated patients, which might potentially affect subsequent treatment.
Subject(s)
Breast Neoplasms , Circulating Tumor DNA , Piperazines , Pyridines , Receptor, ErbB-2 , Humans , Female , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Circulating Tumor DNA/genetics , Phosphatidylinositol 3-Kinases , Receptors, Estrogen/geneticsABSTRACT
AIM: Anthracyclines are commonly used in breast cancer, although they lack validated predictive biomarkers. We explored the interaction between TOP2A protein by quantitative immunofluorescence (QIF) and anthracycline sensitivity. PATIENTS & METHODS: Patients with estrogen receptor-negative breast cancer received neoadjuvant epirubicin. Pretreatment biopsies were analyzed using AQUA(®). Total, cytoplasmic (C) and nuclear (N) TOP2A protein concentrations were expressed as QIF scores and compared with pathologic complete response (pCR), TOP2A by immunohistochemistry, TOP2A mRNA, TOP2A and HER2 gene status, and Ki-67 level. RESULTS: In total, 76 cases were assessable. C, N, and total scores did not correlate with pCR, or other markers. The N:C ratio differed significantly by HER2 status. No pCRs occurred in patients in the lowest N:C quartile. CONCLUSION: Although no relevant correlation between TOP2A QIF scores and pCR was found, N:C ratio may have a negative predictive role, and may merit further exploration in a multifactorial predictive model that includes tumor and host factors.
Subject(s)
Antigens, Neoplasm/metabolism , Antineoplastic Agents/therapeutic use , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/metabolism , Epirubicin/therapeutic use , Topoisomerase II Inhibitors/therapeutic use , Antigens, Neoplasm/genetics , Breast Neoplasms/mortality , Cell Nucleus/metabolism , Cytoplasm/metabolism , DNA Topoisomerases, Type II/genetics , DNA-Binding Proteins/genetics , Female , Humans , Immunohistochemistry , Neoadjuvant Therapy , Poly-ADP-Ribose Binding Proteins , Prognosis , Protein TransportABSTRACT
Background: This study estimated the economic and organisational impact of using subcutaneous injection (SC) formulations of two monoclonal antibodies (rituximab and trastuzumab) in patients with non-Hodgkin's lymphoma (NHL) and HER2 + breast cancer (BC).Methods: The database of the Unit of Oncology and Haematology of the IRST of Meldola was used. The analysis was structured as follows: i) measurement of the volume of Day-Hospital activity; ii) identification of activities and time to complete the administration; iii) estimate the mean cost of an SC or IV (intravenous infusion); iv) estimate the impact of SC formulations on the volumes of activities provided; v) estimate the social impact of SC or IV formulations.Results: The use of an SC formulation was associated a significant reduction in the time that the patient takes to complete the administration (trastuzumab -1 hr 18 min; rituximab -2 hr 24 min) and in consumption of healthcare resources and related costs for trastuzumab (IV: 1781; SC: 1701) and rituximab (IV: 2116; SC: 1941).Conclusion: The adoption of the SC formulation for rituximab and trastuzumab can generate a greater value for both the national healthcare system and the patient compared to an IV formulation.
Subject(s)
Breast Neoplasms/drug therapy , Lymphoma, Non-Hodgkin/drug therapy , Rituximab/administration & dosage , Trastuzumab/administration & dosage , Antineoplastic Agents, Immunological/administration & dosage , Antineoplastic Agents, Immunological/economics , Breast Neoplasms/economics , Delivery of Health Care/economics , Drug Costs , Female , Humans , Infusions, Intravenous , Injections, Subcutaneous , Italy , Lymphoma, Non-Hodgkin/economics , Pilot Projects , Receptor, ErbB-2/metabolism , Retrospective Studies , Rituximab/economics , Time Factors , Trastuzumab/economicsABSTRACT
BACKGROUND: Metronomic chemotherapy can induce disease control in patients with metastatic breast cancer (MBC) and has better safety profiles than conventional chemotherapy. Evidence suggests that cytotoxics can be anti-angiogenic in pre-clinical models and may have synergistic effects when combined with anti-vascular endothelial growth factor therapies. PATIENTS AND METHODS: Patients pretreated with ≥ 1 prior line of therapy for MBC received oral cyclophosphamide 50 mg daily in combination with oral vinorelbine at escalating doses of 20 mg (V20), 30 mg (V30), and 40 mg (V40) 3 times per week, and intravenous bevacizumab 15 mg/kg every 3 weeks. Patients with human epidermal growth factor receptor 2-positive disease were given the same regimen plus standard trastuzumab. Doses were escalated when 3 patients completed 3 treatment cycles of V20 and V30, without experiencing dose-limiting toxicities. The recommended dose was then tested in a further 6 patients. Circulating tumour cells and circulating endothelial cells (CEC) were measured in 30 mL of whole blood samples at baseline, after cycle 1, and at the disease progression. RESULTS: Fifteen patients were recruited from June 2013 to October 2015. The median age was 61 years (range, 29-72 years); 80% had estrogen receptor-positive and 33% had human epidermal growth factor receptor 2-positive disease. At least 67% had visceral metastases, and 80% had received ≥ 2 lines of prior treatment. No dose-limiting toxicities were observed at the 3 dose-levels, making V40 the recommended dose. Overall 8 (53%) patients developed grade 2 adverse events (arthralgia, n = 3 [20%]; asthenia, n = 2 [13%]; diarrhea, n = 2 [13%]; leukopenia, n = 2 [13%]). Bevacizumab was associated with grade 3 hypertension (n = 3 [20%]). Stable disease as best response was observed in 11 (73.3%) patients. The clinical benefit rate was 66.6% (10/15 patients). The median time to progression was 6.9 months. At baseline, CECs were more commonly detectable than circulating tumor cells; however, no statistical correlation was found between CEC kinetics and response. CONCLUSION: A metronomic vinorelbine dose of 40 mg combined with cyclophosphamide and bevacizumab is a promising treatment regimen in pretreated patients with MBC.
Subject(s)
Antineoplastic Agents/administration & dosage , Bevacizumab/administration & dosage , Breast Neoplasms/drug therapy , Carcinoma/drug therapy , Cyclophosphamide/administration & dosage , Vinorelbine/administration & dosage , Administration, Metronomic , Administration, Oral , Adult , Aged , Breast Neoplasms/pathology , Carcinoma/pathology , Dose-Response Relationship, Drug , Drug Therapy, Combination , Endothelial Cells , Female , Humans , Middle Aged , Neoplastic Cells, Circulating , Pilot Projects , Prospective StudiesABSTRACT
CDK4/6 inhibitors (CDK4/6i) and endocrine therapy are the standard treatment for patients with hormone receptor-positive and HER2 negative (HR+/HER2-) metastatic breast cancer. Patients might show intrinsic and acquired resistance, which leads to treatment failure and progression. Circulating biomarkers have the potential advantages of recognizing patients who might not respond to treatment, monitoring treatment effects and identifying markers of acquired resistance during tumor progression with a simple withdrawal of peripheral blood. Genomic alterations on circulating tumor DNA and serum thymidine kinase activity, but also circulating tumor cells, epigenetic or exosome markers are currently being tested as markers of CDK4/6i treatment response, even though none of these have been integrated into clinical practice. In this review, we discuss the recent advancements in the development of circulating biomarkers of CDK4/6i response in patients with HR+/HER2-breast cancer.
ABSTRACT
Liquid biopsy based on cell-free DNA (cfDNA) enables non-invasive dynamic assessment of disease status in patients with cancer, both in the early and advanced settings. The analysis of DNA-methylation (DNAm) from cfDNA samples holds great promise due to the intrinsic characteristics of DNAm being more prevalent, pervasive, and cell- and tumor-type specific than genomics, for which established cfDNA assays already exist. Herein, we report on recent advances on experimental strategies for the analysis of DNAm in cfDNA samples. We describe the main steps of DNAm-based analysis workflows, including pre-analytics of cfDNA samples, DNA treatment, assays for DNAm evaluation, and methods for data analysis. We report on protocols, biomolecular techniques, and computational strategies enabling DNAm evaluation in the context of cfDNA analysis, along with practical considerations on input sample requirements and costs. We provide an overview on existing studies exploiting cell-free DNAm biomarkers for the detection and monitoring of cancer in early and advanced settings, for the evaluation of drug resistance, and for the identification of the cell-of-origin of tumors. Finally, we report on DNAm-based tests approved for clinical use and summarize their performance in the context of liquid biopsy.
Subject(s)
Cell-Free Nucleic Acids/genetics , DNA Methylation/genetics , Medical Oncology/methods , Animals , Computational Biology , Humans , Liquid BiopsyABSTRACT
PURPOSE: Thymidine kinase 1 (TK1) is downstream to the CDK4/6 pathway, and TK activity (TKa) measured in blood is a dynamic marker of outcome in patients with advanced breast cancer (ABC). This study explores TK1 as a biomarker of palbociclib response, both in vitro and in patients with ABC. EXPERIMENTAL DESIGN: Modulation of TK1 levels and activity by palbociclib were studied in seven estrogen receptor-positive breast cancer cell lines: sensitive (PDS) and with palbociclib acquired resistance (PDR). TKa was assayed in plasma obtained at baseline (T0), after one cycle (T1), and at disease progression on palbociclib (T2) in patients enrolled in the "To Reverse ENDocrine Resistance" (TREnd) trial (n = 46). RESULTS: Among E2F-dependent genes, TK1 was significantly downregulated after short-term palbociclib. Early TKa reduction by palbociclib occurred in PDS but not in PDR cells. In patients, median TKa (mTKa) at T0 was 75 DiviTum units per liter (Du/L), with baseline TKa not proving prognostic. At T1, mTKa decreased to 35 Du/L, with a minority of patients (n = 8) showing an increase-correlating with a worse outcome than those with decreased/stable TKa (n = 33; mPFS 3.0 vs 9.0 months; P = 0.002). At T2, mTKa was 251 Du/L; patients with TKa above the median had worse outcomes on post-study treatment compared with those with lower TKa (2.9 vs 8.7 months; P = 0.05). CONCLUSIONS: TK is a dynamic marker of resistance to palbociclib which may lead to early identification of patients in whom treatment escalation may be feasible. In addition, TKa may stratify prognosis in patients with acquired resistance to palbociclib.
Subject(s)
Breast Neoplasms/drug therapy , Breast Neoplasms/enzymology , Piperazines/therapeutic use , Pyridines/therapeutic use , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/blood , Breast Neoplasms/blood , Breast Neoplasms/pathology , Cell Line, Tumor , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 4/blood , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Cyclin-Dependent Kinase 6/blood , Drug Resistance, Neoplasm , Female , Humans , Middle Aged , Neoplasm Metastasis , Protein Kinase Inhibitors/therapeutic use , Receptors, Estrogen/metabolism , Survival Rate , Thymidine Kinase/blood , Treatment SwitchingABSTRACT
OBJECTIVES: Hydroxychloroquine has shown to have antiviral activity in vitro against coronaviruses, specifically SARS-CoV-2. It is believed to block virus infection by increasing endosomal pH required for virus cell fusion and glycosylation of viral surface proteins. In addition to its antiviral activity, hydroxychloroquine has an immune-modulating activity that may synergistically enhance its antiviral effect in vivo, making it a potentially promising drug for the prevention and the cure of SARS-CoV-19. However, randomized controlled trials are needed to assess whether it can be used safely to treat COVID-19 patients or to prevent infection. The main objective of the present study is to evaluate the efficacy of hydroxychloroquine for (I) the prevention of COVID-19 or related symptoms in SARS-CoV-2-exposed subjects, such as as household members/contacts of COVID-19 patients and (II) the treatment of early-phase asymptomatic or paucisymptomatic COVID-19 patients. TRIAL DESIGN: This is a controlled, open label, cluster-randomized, superiority trial with parallel group design. Subjects will be randomized either to receive hydroxychloroquine or to observation (2:1). PARTICIPANTS: SARS-CoV-2-exposed subjects, including household members and/or contacts of COVID-19 patients and healthcare professionals (Group 1) or patients with COVID-19 (positive PCR test on a rhinopharyngeal or oropharyngeal swab for SARS-CoV-2), asymptomatic or paucisymptomatic in home situations who are not undergoing treatment with any anti COVID-19 medication (Group 2), will be enrolled. Paucisymptomatic patients are defined as patients with a low number of mild symptoms. All subjects must be aged ≥18 years, male or female, must be willing and able to give informed consent and must not have any contraindications to take hydroxychloroquine (intolerance or previous toxicity for hydroxychloroquine/chloroquine, bradycardia or reduction in heart rhythm with arrhythmia, ischemic heart disease, retinopathy, congestive heart failure with use of diuretics, favism or glucose-6-phosphate dehydrogenase (G6PD) deficiency, diabetes type 1, major comorbidities such as advanced chronic kidney disease or dialysis therapy, known history of ventricular arrhythmia, any oncologic/hematologic malignancy, severe neurological and mental illness, current use of medications with known significant drug-drug interactions, and known prolonged QT syndrome or current use of drugs with known QT prolongation). The study is monocentric and will be conducted at Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS. Subjects will be enrolled from a large epidemic region (North-Central Italy). The Public Health Departments of several Italian regions will collaborate by identifying potentially eligible subjects. INTERVENTION AND COMPARATOR: The participants will be randomized (2:1 randomization) to receive either hydroxychloroquine (Arm A) or to Observation (Arm B). Hydroxychloroquine will be administered with the following schedule: Group1: A loading dose hydroxychloroquine 400 mg twice daily on day 1, followed by a weekly dose of hydroxychloroquine 200 mg twice daily on days 8, 15 and 22, for a total of one month of treatment. Group 2: A loading dose hydroxychloroquine 400 mg twice daily on day 1 followed by 200 mg twice daily for a total of 5-7 days. The comparator in this trial is observation given that currently neither treatment is administered to asymptomatic or paucisymptomatic subjects, nor prophylaxis is available for contacts. Hydroxychloroquine will be shipped to subjects within 24 hours of randomization. Given the extraordinary nature of the COVID-19 pandemic, only telephonic interviews will be carried out and electronic Patient Reported Outcomes (ePRO) completed. During treatment, each subject will be contacted every other day for the first week and weekly thereafter (Group 2) or weekly (Group 1) by a study physician to assess early onset of any COVID-19 symptom or any adverse reaction to hydroxychloroquine and to check subject compliance. Furthermore, all subjects will receive periodic ePROs which may be completed through smartphone or tablets to record drug self-administration and onset of any symptom or adverse event. All subjects will be followed up for a total of 6 months by periodic telephonic interviews and ePROs. MAIN OUTCOMES: The primary endpoint/outcome measure for this trial is: for Group 1, the proportion of subjects who become symptomatic and/or swab-positive in each arm within one month of randomization; for Group 2, the proportion of subjects who become swab-negative in each arm within 14 days of randomization. RANDOMIZATION: All household members and/or contacts of each COVID-19 index case, and the COVID-19 patient himself/herself, fulfilling all inclusion criteria will be grouped into a single cluster and this cluster will be randomized (2:1) to either arm A or arm B. Information on each subject will be recorded in specific data records. Randomization lists will be stratified according to the following factors regarding COVID-19 index cases: 1. COVID-19 risk level on the basis of province of residence (high vs. low/intermediate); 2. Index case is a healthcare professional (yes vs.no) 3. Index case with COVID-19 treatment (yes vs. no) An independent statistician not otherwise involved in the trial will generate the allocation sequence, and COVID-19 response teams will be unaware of the allocation of clusters. Randomization will be performed through an interactive web-based electronic data-capturing database. An Independent Data Monitoring Committee has been established. BLINDING (MASKING): This study is open label. NUMBERS TO BE RANDOMIZED (SAMPLE SIZE): For Group 1, a sample size of about 2000 SARS-CoV-2-exposed subjects such as household members and/or contacts of COVID-19 patients will take part in the study. Assuming around 1.5-2.0 asymptomatic household members and/or contacts for each COVID-19 patient, we expect to identify approximately 1000-1300 COVID-19 index cases to be randomized. An interim analysis on efficacy is planned using standard alpha-spending function. For Group 2, sufficient power for primary objective (negative swab within 14 days of randomization) will be reached given a sample size of 300 asymptomatic or paucisymptomatic COVID-19 subjects in home situations not treated for COVID-19 (25%-30% of about 1000-1300 expected index cases). Since up to date reduced evidence about COVID-19 infection epidemiology, the continuous update of diagnostic and therapeutic approaches, the sample size estimation could be updated after a one third of population will be recruited and eventually modified according to a substantial protocol amendment. An interim analysis at 100 enrolled COVID-19 patients is planned. We have planned a Generalized Estimating Equation analysis, which is more efficient than a cluster level analysis, to take advantage of subject-specific covariates. The above reported sample size analysis is therefore to be considered conservative. TRIAL STATUS: The current version of the PROTECT trial protocol is 'Final version, 15 April 2020'. The study started on 9th May 2020. The first patient was enrolled on 14th May 2020. Recruitment is expected to last through September 2020. TRIAL REGISTRATION: The PROTECT trial is registered in the EudraCT database (no. 2020-001501-24) and in ClinicalTrials.gov ( NCT04363827 ), date of registration 24 April 2020. FULL PROTOCOL: The full PROTECT protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interests of expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol (Protocol final version, 15th April 2020). The study protocol has been reported in accordance with Standard Protocol Items: Recommendations for Clinical Interventional Trials (SPIRIT) guidelines (Additional file 2).
Subject(s)
Betacoronavirus , Coronavirus Infections/prevention & control , Hydroxychloroquine/therapeutic use , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Randomized Controlled Trials as Topic , COVID-19 , Cluster Analysis , Female , Humans , Male , Patient Reported Outcome Measures , SARS-CoV-2 , TelemedicineABSTRACT
The key to optimising our approach in early breast cancer is to individualise care. Each patient has a tumour with innate features that dictate their chance of relapse and their responsiveness to treatment. Often patients with similar clinical and pathological tumours will have markedly different outcomes and responses to adjuvant intervention. These differences are encoded in the tumour genetic profile. Effective biomarkers may replace or complement traditional clinical and histopathological markers in assessing tumour behaviour and risk. Development of high-throughput genomic technologies is enabling the study of gene expression profiles of tumours. Genomic fingerprints may refine prediction of the course of disease and response to adjuvant interventions. This review will focus on the role of multiparameter gene expression analyses in early breast cancer, with regards to prognosis and prediction. The prognostic role of genomic signatures, particularly the Mammaprint and Rotterdam signatures, is evolving. With regard to prediction of outcome, the Oncotype Dx multigene assay is in clinical use in tamoxifen treated patients. Extensive research continues on predictive gene identification for specific chemotherapeutic agents, particularly the anthracyclines, taxanes and alkylating agents.
Subject(s)
Biomarkers, Tumor/metabolism , Breast Neoplasms/diagnosis , Antineoplastic Agents/therapeutic use , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Female , Gene Expression Profiling , HumansABSTRACT
Biocharacterization of circulating tumor cells (CTCs) in the peripheral blood of advanced breast cancer (ABC) patients may represent a real-time tumor biopsy. We assessed HER2 status on CTCs from blood samples of ABC patients. CTCs were separated and stained using the CellSearch System((R)). HER2 status was assessed by immunofluorescence and, when technically feasible, by fluorescence in situ hybridization. Blood samples were obtained from 66 ABC patients. Forty patients had a positive CTC sample (61%) and of these, 15 (37%) had HER2 + CTCs. We found non-concordant results in 32% of cases: 29% (8/28) of HER2-negative primary tumors had HER2-positive CTCs and 42% (5/12) of HER2-positive primary tumors had HER2-negative CTCs (k = 0.278). Our study suggests that a subset of patients with HER2-negative primary tumors develops HER2-positive CTCs during disease progression.
Subject(s)
Biomarkers, Tumor/genetics , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Genes, erbB-2/genetics , Neoplastic Cells, Circulating/pathology , Disease Progression , Female , Fluorescent Antibody Technique , Gene Amplification , Humans , Immunomagnetic Separation , Middle Aged , Receptor, ErbB-2/biosynthesis , Receptor, ErbB-2/geneticsABSTRACT
The recent arrival of CDK4/6 inhibitor agents, with an approximate doubling of progression-free survival (PFS) associated with their use in hormone receptor-positive, HER2-negative advanced breast cancer (BC), has radically changed the approach to managing this disease. However, resistance to CDK4/6 inhibitors is considered a near-inevitability in most patients. Mechanisms of resistance to these agents are multifactorial, and research in this field is still evolving. Biomarkers with the ability to identify early resistance, or to predict the likelihood of successful treatment using CDK4/6 inhibitors are yet to be identified, and represent an area of unmet clinical need. Here we present selected mechanisms of resistance to CDK4/6 inhibitors, largely focussing on roles of Rb, cyclin E1, and the PIK3CA pathway, with discussion of associated biomarkers which have been investigated and applied in recent pre-clinical and clinical studies. These biological drivers may furthermore influence clinical treatment strategies adopted beyond CDK4/6 resistance.
ABSTRACT
BACKGROUND: Thymidine kinase 1 (TK1) plays a critical role in DNA synthesis and cell proliferation. Recent studies have shown potential for serum TK1 activity (sTKa) as a prognostic marker and indicator of early response to endocrine therapy in advanced breast cancer. The aim of this study is to assess the correlation between sTKa and patient outcome. PATIENTS AND METHODS: The Evaluation of Faslodex versus Exemestane Clinical Trial (EFECT) was a double-blind, double-dummy, randomised trial of fulvestrant versus exemestane after progression on non-steroidal aromatase inhibitor therapy, in postmenopausal women with advanced breast cancer. Retrospective analyses of serum archived from EFECT were conducted. sTKa was assessed using the DiviTum® assay on samples collected at baseline, after three and six months of endocrine therapy, and at disease progression. RESULTS: The median time to progression (mTTP) for patients with low baseline sTKa levels was 5.03 months (95% confidence interval [CI]: 3.91-5.89) versus 2.57 months (95% CI: 2.04-3.52) in patients with high sTKa baseline levels (P < 0.0001). On treatment, patients whose sTKa increased from baseline had a significantly shorter mTTP (3.39 months, 95% CI: 2.14-4.11) than those without an sTKa increase (5.39 months, 95% CI: 4.01-6.68) (P = 0.0045). Similar results were observed in the separate EFECT treatment arms. After adjusting for major prognostic factors, sTKa remained an independent marker. CONCLUSION: sTKa is a potential circulating prognostic marker in patients with advanced breast cancer treated with endocrine therapy. It may also represent a tool for upfront identification of endocrine therapy resistance and early positive response to therapy. Independent validation of these results is warranted.
Subject(s)
Androstadienes/therapeutic use , Antineoplastic Agents/therapeutic use , Breast Neoplasms/drug therapy , Fulvestrant/therapeutic use , Thymidine Kinase/therapeutic use , Aged , Androstadienes/pharmacology , Antineoplastic Agents/pharmacology , Breast Neoplasms/complications , Female , Fulvestrant/pharmacology , Humans , Neoplasm Metastasis , Prognosis , Thymidine Kinase/pharmacologyABSTRACT
Arsenate tolerance, As accumulation and As-induced phytochelatin accumulation were compared in populations of Silene paradoxa, one from a mine site enriched in As, Cu and Zn, the other from an uncontaminated site. The mine population was significantly more arsenate-tolerant. Arsenate uptake and root-to-shoot transport were slightly but significantly higher in the non-mine plants. The difference in uptake was quantitatively insufficient to explain the difference in tolerance between the populations. As accumulation in the roots was similar in both populations, but the mine plants accumulated much less phytochelatins than the non-mine plants. The mean phytochelatin chain length, however, was higher in the mine population, possibly due to a constitutively lower cellular glutathione level. It is argued that the mine plants must possess an arsenic detoxification mechanism other than arsenate reduction and subsequent phytochelatin-based sequestration. This alternative mechanism might explain at least some part of the superior tolerance in the mine plants.
Subject(s)
Arsenates/toxicity , Industrial Waste , Mining , Phytochelatins/metabolism , Silene/metabolism , Soil Pollutants/toxicity , Arsenic , Copper , Drug Tolerance , Environmental Monitoring/methods , Glutathione/analysis , Glutathione/metabolism , Plant Roots/chemistry , Plant Roots/metabolism , Plant Shoots/chemistry , Plant Shoots/metabolism , ZincABSTRACT
The aim of this study was to investigate if thymidine kinase-1 (TK1), a well-known proliferation marker, could represent a valid circulating biomarker to identify hormone receptor positive (HR+)/HER2 negative (HER2neg) metastatic breast cancer (MBC) patients most likely to benefit from endocrine therapy (ET). We used the DiviTum™ assay to analyze TK1 activity in cell lysates of three HR+/HER2neg BC cell lines and in plasma of 31 HR+/HER2neg MBC patients receiving ET. Blood samples were collected at treatment initiation, after one month and at disease progression. CTCs count and ESR1/PIK3CA mutations in circulating tumor DNA were performed and correlated with TK1 activity. TK1 activity was reduced in the two endocrine-sensitive cell lines after 2 days of treatment. In patients, high baseline TK1 activity correlated with CTCs positivity (p-value=0.014). Patients with low baseline levels of TK1 activity had a significantly better PFS compared to those with high baseline TK1 activity (p-value=0.012). Patients with an early drop of TK1 activity after one month of treatment had a significantly better PFS compared to those who experienced an increase (p-value=0.0026). Our study suggests that TK1 could be a potential prognostic, predictive and monitoring marker of early ET response in HR+/HER2neg MBC patients.
ABSTRACT
Circulating Tumor Cells (CTCs) represent a "liquid biopsy" of the tumor potentially allowing real-time monitoring of cancer biology and therapies in individual patients.The purpose of the study was to explore the applicability of a protocol for the molecular characterization of single CTCs by Next Generation Sequencing (NGS) in order to investigate cell heterogeneity and provide a tool for a personalized medicine approach.CTCs were enriched and enumerated by CellSearch in blood from four metastatic breast cancer patients and singularly isolated by DEPArray. Upon whole genome amplification 3-5 single CTCs per patient were analyzed by NGS for 50 cancer-related genes.We found 51 sequence variants in 25 genes. We observed inter- and intra-patient heterogeneity in the mutational status of CTCs.The highest number of somatic deleterious mutations was found in the gene TP53, whose mutation is associated with adverse prognosis in breast cancer.The discordance between the mutational status of the primary tumor and CTCs observed in 3 patients suggests that, in advanced stages of cancer, CTC characteristics are more closely linked to the dynamic modifications of the disease status.In one patient the mutational profiles of CTCs before and during treatment shared only few sequence variants.This study supports the applicability of a non-invasive approach based on the liquid biopsy in metastatic breast cancer patients which, in perspective, should allow investigating the clonal evolution of the tumor for the development of new therapeutic strategies in precision medicine.