Circulating tumour DNA analysis to direct therapy in advanced breast cancer (plasmaMATCH): a multicentre, multicohort, phase 2a, platform trial.

BACKGROUND: Circulating tumour DNA (ctDNA) testing might provide a current assessment of the genomic profile of advanced cancer, without the need to repeat tumour biopsy. We aimed to assess the accuracy of ctDNA testing in advanced breast cancer and the ability of ctDNA testing to select patients for mutation-directed therapy. METHODS: We did an open-label, multicohort, phase 2a, platform trial of ctDNA testing in 18 UK hospitals. Participants were women (aged ≥18 years) with histologically confirmed advanced breast cancer and an Eastern Cooperative Oncology Group performance status 0-2. Patients had completed at least one previous line of treatment for advanced breast cancer or relapsed within 12 months of neoadjuvant or adjuvant chemotherapy. Patients were recruited into four parallel treatment cohorts matched to mutations identified in ctDNA: cohort A comprised patients with ESR1 mutations (treated with intramuscular extended-dose fulvestrant 500 mg); cohort B comprised patients with HER2 mutations (treated with oral neratinib 240 mg, and if oestrogen receptor-positive with intramuscular standard-dose fulvestrant); cohort C comprised patients with AKT1 mutations and oestrogen receptor-positive cancer (treated with oral capivasertib 400 mg plus intramuscular standard-dose fulvestrant); and cohort D comprised patients with AKT1 mutations and oestrogen receptor-negative cancer or PTEN mutation (treated with oral capivasertib 480 mg). Each cohort had a primary endpoint of confirmed objective response rate. For cohort A, 13 or more responses among 78 evaluable patients were required to infer activity and three or more among 16 were required for cohorts B, C, and D. Recruitment to all cohorts is complete and long-term follow-up is ongoing. This trial is registered with ClinicalTrials.gov, NCT03182634; the European Clinical Trials database, EudraCT2015-003735-36; and the ISRCTN registry, ISRCTN16945804. FINDINGS: Between Dec 21, 2016, and April 26, 2019, 1051 patients registered for the study, with ctDNA results available for 1034 patients. Agreement between ctDNA digital PCR and targeted sequencing was 96-99% (n=800, kappa 0·89-0·93). Sensitivity of digital PCR ctDNA testing for mutations identified in tissue sequencing was 93% (95% CI 83-98) overall and 98% (87-100) with contemporaneous biopsies. In all cohorts, combined median follow-up was 14·4 months (IQR 7·0-23·7). Cohorts B and C met or exceeded the target number of responses, with five (25% [95% CI 9-49]) of 20 patients in cohort B and four (22% [6-48]) of 18 patients in cohort C having a response. Cohorts A and D did not reach the target number of responses, with six (8% [95% CI 3-17]) of 74 in cohort A and two (11% [1-33]) of 19 patients in cohort D having a response. The most common grade 3-4 adverse events were raised gamma-glutamyltransferase (13 [16%] of 80 patients; cohort A); diarrhoea (four [25%] of 20; cohort B); fatigue (four [22%] of 18; cohort C); and rash (five [26%] of 19; cohort D). 17 serious adverse reactions occurred in 11 patients, and there was one treatment-related death caused by grade 4 dyspnoea (in cohort C). INTERPRETATION: ctDNA testing offers accurate, rapid genotyping that enables the selection of mutation-directed therapies for patients with breast cancer, with sufficient clinical validity for adoption into routine clinical practice. Our results demonstrate clinically relevant activity of targeted therapies against rare HER2 and AKT1 mutations, confirming these mutations could be targetable for breast cancer treatment. FUNDING: Cancer Research UK, AstraZeneca, and Puma Biotechnology

Free-Circulating Methylated DNA in Blood for Diagnosis, Staging, Prognosis, and Monitoring of Head and Neck Squamous Cell Carcinoma Patients: An Observational Prospective Cohort Study

Abstract BACKGROUND Circulating cell-free DNA methylation testing in blood has recently received regulatory approval for screening of colorectal cancer. Its application in other clinical settings, including staging, prognosis, prediction, and recurrence monitoring is highly promising, and of particular interest in head and neck squamous cell carcinomas (HNSCCs) that represent a heterogeneous group of cancers with unsatisfactory treatment guidelines. METHODS Short stature homeobox 2 (SHOX2) and septin 9 (SEPT9) DNA methylation in plasma from 649 prospectively enrolled patients (training study: 284 HNSCC/122 control patients; testing study: 141 HNSCC/102 control patients) was quantified before treatment and longitudinally during surveillance. RESULTS In the training study, 59% of HNSCC patients were methylation-positive at 96% specificity. Methylation levels correlated with tumor and nodal category (P &amp;lt; 0.001). Initially increased methylation levels were associated with a higher risk of death [SEPT9: hazard ratio (HR) = 5.27, P = 0.001; SHOX2: HR = 2.32, P = 0.024]. Disease recurrence/metastases were detected in 47% of patients up to 377 days earlier compared to current clinical practice. The onset of second cancers was detected up to 343 days earlier. In the testing study, sensitivity (52%), specificity (95%), prediction of overall survival (SEPT9: HR = 2.78, P = 0.022; SHOX2: HR = 2.50, P = 0.026), and correlation with tumor and nodal category (P &amp;lt;0.001) were successfully validated. CONCLUSIONS Methylation testing in plasma is a powerful diagnostic tool for molecular disease staging, risk stratification, and disease monitoring. Patients with initially high biomarker levels might benefit from intensified treatment and posttherapeutic surveillance. The early detection of a recurrent/metastatic disease or a second malignancy could lead to an earlier consecutive treatment, thereby improving patients' outcomes. </jats:sec

Triplet therapy with palbociclib, taselisib and fulvestrant in PIK3CA mutant breast cancer and doublet palbociclib and taselisib in pathway mutant solid cancers

Cyclin-dependent kinase-4/6 (CDK4/6) and phosphatidylinositol 3-kinase (PI3K) inhibitors synergise in PIK3CA mutant ER-positive HER2-negative breast cancer models. We conducted a phase Ib trial investigating safety and efficacy of doublet CDK4/6 inhibitor palbociclib plus selective PI3K inhibitor taselisib in advanced solid tumors, and triplet palbociclib plus taselisib plus fulvestrant in 25 patients with PIK3CA mutant, ER-positive HER2-negative advanced breast cancer. The triplet therapy response rate in PIK3CA mutant, ER-positive HER2-negative was 37.5% (95% CI 18.8-59.4). Durable disease control was observed in PIK3CA mutant ER-negative breast cancer and other solid tumors, with doublet therapy. Both combinations were well tolerated at pharmacodynamically active doses. In the triplet group, high baseline cyclin E1 expression associated with shorter progression-free survival (PFS) (HR 4.2, 95% CI 1.3-13.1, p=0.02). Early ctDNA dynamics demonstrated high on-treatment ctDNA association with shorter PFS (HR 5.2, 95% CI 1.4-19.4, p=0.04). Longitudinal plasma ctDNA sequencing provided genomic evolution evidence during triplet therapy

Common breast cancer susceptibility alleles are associated with tumor subtypes in BRCA1 and BRCA2 mutation carriers : results from the Consortium of Investigators of Modifiers of BRCA1/2.

Abstract Introduction Previous studies have demonstrated that common breast cancer susceptibility alleles are differentially associated with breast cancer risk for BRCA1 and/or BRCA2 mutation carriers. It is currently unknown how these alleles are associated with different breast cancer subtypes in BRCA1 and BRCA2 mutation carriers defined by estrogen (ER) or progesterone receptor (PR) status of the tumour. Methods We used genotype data on up to 11,421 BRCA1 and 7,080 BRCA2 carriers, of whom 4,310 had been affected with breast cancer and had information on either ER or PR status of the tumour, to assess the associations of 12 loci with breast cancer tumour characteristics. Associations were evaluated using a retrospective cohort approach. Results The results suggested stronger associations with ER-positive breast cancer than ER-negative for 11 loci in both BRCA1 and BRCA2 carriers. Among BRCA1 carriers, single nucleotide polymorphism (SNP) rs2981582 (FGFR2) exhibited the biggest difference based on ER status (per-allele hazard ratio (HR) for ER-positive = 1.35, 95% CI: 1.17 to 1.56 vs HR = 0.91, 95% CI: 0.85 to 0.98 for ER-negative, P-heterogeneity = 6.5 &#215; 10-6). In contrast, SNP rs2046210 at 6q25.1 near ESR1 was primarily associated with ER-negative breast cancer risk for both BRCA1 and BRCA2 carriers. In BRCA2 carriers, SNPs in FGFR2, TOX3, LSP1, SLC4A7/NEK10, 5p12, 2q35, and 1p11.2 were significantly associated with ER-positive but not ER-negative disease. Similar results were observed when differentiating breast cancer cases by PR status. Conclusions The associations of the 12 SNPs with risk for BRCA1 and BRCA2 carriers differ by ER-positive or ER-negative breast cancer status. The apparent differences in SNP associations between BRCA1 and BRCA2 carriers, and non-carriers, may be explicable by differences in the prevalence of tumour subtypes. As more risk modifying variants are identified, incorporating these associations into breast cancer subtype-specific risk models may improve clinical management for mutation carriers

Comparison of quantification algorithms for circulating cell-free DNA methylation biomarkers in blood plasma from cancer patients

Abstract Background SHOX2 and SEPT9 methylation in circulating cell-free DNA (ccfDNA) in blood are established powerful and clinically valuable biomarkers for diagnosis, staging, prognosis, and monitoring of cancer patients. The aim of the present study was to evaluate different quantification algorithms (relative quantification, absolute quantification, quasi-digital PCR) with regard to their clinical performance. Methods Methylation analyses were performed in a training cohort (141 patients with head and neck squamous cell carcinoma [HNSCC], 170 control cases) and a testing cohort (137 HNSCC cases, 102 controls). DNA was extracted from plasma samples, bisulfite-converted, and analyzed via quantitative real-time PCR. SHOX2 and SEPT9 methylations were assessed separately and as panel [mean SEPT9/SHOX2 ] using the ΔCT method for absolute quantification and the ΔΔCT-method for relative quantification. Quasi-digital PCR was defined as the number of amplification-positive PCR replicates. The diagnostic (sensitivity, specificity, area under the curve (AUC) of the receiver operating characteristic (ROC)) and prognostic accuracy (hazard ratio (HR) from Cox regression) were evaluated. Results Sporadic methylation in control samples necessitated the introduction of cutoffs resulting in 61–63% sensitivity/90–92% specificity (SEPT9/training), 53–57% sensitivity/87–90% specificity (SHOX2/training), and 64–65% sensitivity/90–91% specificity (mean SEPT9/SHOX2 /training). Results were confirmed in a testing cohort with 54–56% sensitivity/88–90% specificity (SEPT9/testing), 43–48% sensitivity/93–95% specificity (SHOX2/testing), and 49–58% sensitivity/88–94% specificity (mean SEPT9/SHOX2 /testing). All algorithms showed comparable cutoff-independent diagnostic accuracy with largely overlapping 95% confidence intervals (SEPT9: AUCtraining = 0.79–0.80; AUCtesting = 0.74–0.75; SHOX2: AUCtraining = 0.78–0.81, AUCtesting = 0.77–0.79; mean SEPT9/SHOX2 : AUCtraining = 0.81–0.84, AUCtesting = 0.80). The accurate prediction of overall survival was possible with all three algorithms (training cohort: HR SEPT9  = 1.23-1.90, HR SHOX2  = 1.14-1.85, HRmeanSEPT9/SHOX2  =1.19-1.89 ; testing cohort: HR SEPT9  =1.22-1.67, HR SHOX2  = 1.15-1.71, HRmeanSEPT9/SHOX2  = 1.12-1.77). Conclusion The concordant clinical performance based on different quantification algorithms allows for the application of various diagnostic platforms for the analysis of ccfDNA methylation biomarkers

PD-1 (PDCD1) Promoter Methylation Is a Prognostic Factor in Patients With Diffuse Lower-Grade Gliomas Harboring Isocitrate Dehydrogenase (IDH) Mutations

Immune checkpoints are important targets for immunotherapies. However, knowledge on the epigenetic modification of immune checkpoint genes is sparse. In the present study, we investigated promoter methylation of CTLA4, PD-L1, PD-L2, and PD-1 in diffuse lower-grade gliomas (LGG) harboring isocitrate dehydrogenase (IDH) mutations with regard to mRNA expression levels, clinicopathological parameters, previously established methylation subtypes, immune cell infiltrates, and survival in a cohort of 419 patients with IDH-mutated LGG provided by The Cancer Genome Atlas. PD-L1, PD-L2, and CTLA-4 mRNA expression levels showed a significant inverse correlation with promoter methylation (PD-L1: p=0.005; PD-L2: p < 0.001; CTLA-4: p b 0.001). Furthermore, immune checkpoint methylation was significantly associated with age (PD-L2: p = 0.003; PD-1: p = 0.015), molecular alterations, i.e. MGMT methylation (PD-L1: p < 0.001; PD-L2: p < 0.001), ATRX mutations (PD-L2: p < 0.001, PD-1: p = 0.001), and TERT mutations (PD-L1: p= 0.035, PD-L2: p < 0.001, PD-1: p b 0.001, CTLA4: p < 0.001) as well a smethylation subgroups and immune cell infiltrates. In multivariate Cox proportional hazard analysis, PD-1 methylation qualified as strong prognostic factor (HR = 0.51 [ 0.34-0.76], p=0.001). Our findings suggest an epigenetic regulation of immune checkpoint genes via DNA methylation in LGG. PD-1 methylation may assist the identification of patients that might benefit from an alternative treatment, particularly in the context of emerging immunotherapies. (c) 2018 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY licens

Clinical performance validation of PITX2 DNA methylation as prognostic biomarker in patients with head and neck squamous cell carcinoma.

Despite advances in combined modality therapy, outcomes in head and neck squamous cell cancer (HNSCC) remain dismal with five-year overall survival rates of less than 50%. Prognostic biomarkers are urgently needed to identify patients with a high risk of death after initial curative treatment. Methylation status of the paired-like homeodomain transcription factor 2 (PITX2) has recently emerged as a powerful prognostic biomarker in various cancers. In the present study, the clinical performance of PITX2 methylation was validated in a HNSCC cohort by means of an independent analytical platform (Infinium HumanMethylation450 BeadChip, Illumina, Inc.).A total of 528 HNSCC patients from The Cancer Genome Atlas (TCGA) were included in the study. Death was defined as primary endpoint. PITX2 methylation was correlated with overall survival and clinicopathological parameters.PITX2 methylation was significantly associated with sex, tumor site, p16 status, and grade. In univariate Cox proportional hazards analysis, PITX2 hypermethylation analyzed as continuous and dichotomized variable was significantly associated with prolonged overall survival of HNSCC patients (continuous: hazard ratio (HR) = 0.19 [95%CI: 0.04-0.88], p = 0.034; dichotomized: HR = 0.52 [95%CI: 0.33-0.84], p = 0.007). In multivariate Cox analysis including established clinicopathological parameters, PITX2 promoter methylation was confirmed as prognostic factor (HR = 0.28 [95%CI: 0.09-0.84], p = 0.023).Using an independent analytical platform, PITX2 methylation was validated as a prognostic biomarker in HNSCC patients, identifying patients that potentially benefit from intensified surveillance and/or administration of adjuvant/neodjuvant treatment, i.e. immunotherapy

Loss of the LIM-only protein Fhl2 impairs inflammatory reaction and scar formation after cardiac ischemia leading to better hemodynamic performance

Aims: The pathogenesis of myocardial ischemia-reperfusion injury (MI/R) involves an inflammatory response. Since the four-and-a-half LIM domain-containing protein 2 (Fhl2) has been observed to modulate immune cell migration, we aimed to study the consequences of Fhl2(-/-) under MI/R with respect to immune reaction, scar formation, and hemodynamic performance. Material and methods: In a closed chest model of 1 h MI/R, immune cell invasion of phagocytic monocytes was characterized by flow cytometry and immunohistochemistry. In addition, infarct size was assessed by triphenyltetrazolium chloride/Masson trichrome staining 24 h/21 days after reperfusion and a set of hemodynamic parameters was recorded by catheterisation in Fhl2(-/-) mice and controls. Key findings: While flow cytometry did not reveal differences in myocardial CD45(high) immune cell infiltrate, histological analysis showed that infiltrating immune cells in Fhl2(-/-) animals were preferentially located in the perivascular area, whereas in wild type, immune cells were well dispersed within the area at risk. After 24 h and 21 days of reperfusion, infarct size was significantly reduced in Fhl2(-/-) compared to WT animals. In addition, hemodynamic performance was better in Fhl2(-/-) mice, compared to WT mice up to day 21 of reperfusion. The loss of Fhl2 leads to an altered immune response to myocardial ischemia, which results in smaller infarcts and better hemodynamic performance up to 21 days after myocardial ischemia reperfusion. Significance: Immune cell invasion plays a pivotal role in the context of MI/R. Fhl2 significantly influences immune cell function and immune cell interaction with injured cardiac tissue leading to altered scar composition. (C) 2016 Elsevier Inc. All rights reserved

Additional file 2: Table S2. of Diagnostic and prognostic value of SHOX2 and SEPT9 DNA methylation and cytology in benign, paramalignant, and malignant ascites

Specification of 25 cancer patients suffering from more than one primary tumor. Other existing primary tumors are listed for patients suffering from more than one primary tumor. (DOC 76 kb