Centrosomal Protein 55 Regulates Chromosomal Instability in Cancer Cells by Controlling Microtubule Dynamics.

Centrosomal Protein 55 (CEP55) exhibits various oncogenic activities; it regulates the PI3K-Akt-pathway, midbody abscission, and chromosomal instability (CIN) in cancer cells. Here, we analyzed the mechanism of how CEP55 controls CIN in ovarian and breast cancer (OvCa) cells. Down-regulation of CEP55 reduced CIN in all cell lines analyzed, and CEP55 depletion decreased spindle microtubule (MT)-stability in OvCa cells. Moreover, recombinant CEP55 accelerated MT-polymerization and attenuated cold-induced MT-depolymerization. To analyze a potential relationship between CEP55-controlled CIN and its impact on MT-stability, we identified the CEP55 MT-binding peptides inside the CEP55 protein. Thereafter, a mutant with deficient MT-binding activity was re-expressed in CEP55-depleted OvCa cells and we could show that this mutant did not restore reduced CIN in CEP55-depleted cells. This finding strongly indicates that CEP55 regulates CIN by controlling MT dynamics.

Applications of Nanopore sequencing in precision cancer medicine.

Oxford Nanopore Technologies sequencing, also referred to as Nanopore sequencing, stands at the forefront of a revolution in clinical genetics, offering the potential for rapid, long read, and real-time DNA and RNA sequencing. This technology is currently making sequencing more accessible and affordable. In this comprehensive review, we explore its potential regarding precision cancer diagnostics and treatment. We encompass a critical analysis of clinical cases where Nanopore sequencing was successfully applied to identify point mutations, splice variants, gene fusions, epigenetic modifications, non-coding RNAs, and other pivotal biomarkers that defined subsequent treatment strategies. Additionally, we address the challenges of clinical applications of Nanopore sequencing and discuss the current efforts to overcome them.

Thymic Atrophy and Immune Dysregulation in Infants with Complex Congenital Heart Disease.

Congenital heart disease (CHD) is the most common birth defect, and up to 50% of infants with CHD require cardiovascular surgery early in life. Current clinical practice often involves thymus resection during cardiac surgery, detrimentally affecting T-cell immunity. However, epidemiological data indicate that CHD patients face an elevated risk for infections and immune-mediated diseases, independent of thymectomy. Hence, we examined whether the cardiac defect impacts thymus function in individuals with CHD. We investigated thymocyte development in 58 infants categorized by CHD complexity. To assess the relationship between CHD complexity and thymic function, we analyzed T-cell development, thymic output, and biomarkers linked to cardiac defects, stress, or inflammation. Patients with highly complex CHD exhibit thymic atrophy, resulting in low frequencies of recent thymic emigrants in peripheral blood, even prior to thymectomy. Elevated plasma cortisol levels were detected in all CHD patients, while high NT-proBNP and IL-6 levels were associated with thymic atrophy. Our findings reveal an association between complex CHD and thymic atrophy, resulting in reduced thymic output. Consequently, thymus preservation during cardiovascular surgery could significantly enhance immune function and the long-term health of CHD patients.

Liquid Biopsy in Diagnosis and Prognosis of Non-Metastatic Prostate Cancer.

Currently, sensitive and specific methods for the detection and prognosis of early stage PCa are lacking. To establish the diagnosis and further identify an appropriate treatment strategy, prostate specific antigen (PSA) blood test followed by tissue biopsy have to be performed. The combination of tests is justified by the lack of a highly sensitive, specific, and safe single test. Tissue biopsy is specific but invasive and may have severe side effects, and therefore is inappropriate for screening of the disease. At the same time, the PSA blood test, which is conventionally used for PCa screening, has low specificity and may be elevated in the case of noncancerous prostate tumors and inflammatory conditions, including benign prostatic hyperplasia and prostatitis. Thus, diverse techniques of liquid biopsy have been investigated to supplement or replace the existing tests of prostate cancer early diagnosis and prognostics. Here, we provide a review on the advances in diagnosis and prognostics of non-metastatic prostate cancer by means of various biomarkers extracted via liquid biopsy, including circulating tumor cells, exosomal miRNAs, and circulating DNAs.

Circulating DNA and Liquid Biopsies in the Management of Patients with Cancer.

Forty-five years ago, Cancer Research published the study by Leon and colleagues in which the authors described the observation of increased levels of cell-free DNA (cfDNA) in the serum of patients with cancer as compared with healthy individuals, with the highest serum levels in patients with metastatic cancer. Most interestingly was the correlation between serum DNA concentrations and therapy outcome: Increased serum DNA levels were associated with poor response to treatment, whereas decreases in DNA levels during treatment appeared to be a sign of better prognosis. Since the discovery of the prognostic value of blood DNA, much research has been focused on the characterization of cfDNA to understand its origins and increase the sensitivity and specificity of using cfDNA as a prognostic and predictive marker in the battle against cancer. Tumor-specific cfDNA markers that were discovered include genetic alterations, chromosomal aberrations, epigenetic modifications, and DNA fragmentation size. In recent years, due to the development of highly sensitive molecular technologies, cfDNA-based assays are now being introduced into the clinic as the so called "liquid biopsy." The advantages of a liquid biopsy over traditional biopsy and imaging have led to the implementation in the clinic for early cancer detection, improved cancer staging, early detection of relapse, real-time monitoring of therapeutic efficacy, and detection of therapeutic targets and resistance mechanisms. Despite Leon and colleagues' initial skepticism about the potential diagnostic value of serum DNA, cfDNA-based liquid biopsy has become one of the most important tools for personalized cancer treatment. See related article by Leon and colleagues, Cancer Res 1977;37:646-50.

Detection and Characterization of Estrogen Receptor α Expression of Circulating Tumor Cells as a Prognostic Marker.

CTCs have increasingly been used as a liquid biopsy analyte to obtain real-time information on the tumor through minimally invasive blood analyses. CTCs allow for the identification of proteins relevant for targeted therapies. Here, we evaluated the expression of estrogen receptors (ER) in CTCs of patients with metastatic breast cancer. From sixty metastatic breast cancer patients who had ER-positive primary tumors (range of 1−70% immunostaining) at initial cancer diagnosis, 109 longitudinal blood samples were prospectively collected and analyzed using the CellSearch System in combination with the ERα monoclonal murine ER-119.3 antibody. Prolonged cell permeabilization was found to be required for proper staining of nuclear ER in vitro. Thirty-one cases were found to be CTC-positive; an increased number of CTCs during endocrine and chemotherapy was correlated with disease progression, whereas a decrease or stable amount of CTC number (<5) during treatment was correlated with a better clinical outcome. Survival analyses further indicate a positive association of CTC-status with progression-free survival (HR, 66.17; 95%CI, 3.66−195.96; p = 0.0045) and overall survival (HR, 6.21; 95%CI, 2.66−14.47; p < 0.0001). Only one-third of CTC-positive breast cancer patients, who were initially diagnosed with ER-positive primary tumors, harbored ER-positive CTCs at the time of metastasis, and even in those patients, both ER-positive and ER-negative CTCs were found. CTC-positivity was correlated with a shorter relapse-free survival. Remarkably, ER-negative CTCs were frequent despite initial ER-positive status of the primary tumor, suggesting a switch of ER phenotype or selection of minor ER-negative clones as a potential mechanism of escape from ER-targeting therapy.

Circulating Cellular Communication Network Factor 1 Protein as a Sensitive Liquid Biopsy Marker for Early Detection of Breast Cancer.

BACKGROUND: Despite recent progress in liquid biopsy technologies, early blood-based detection of breast cancer is still a challenge. METHODS: We analyzed secretion of the protein cellular communication network factor 1 (CCN1, formerly cysteine-rich angiogenic inducer 61) in breast cancer cell lines by an enzyme-linked immunosorbent assay (ELISA). Soluble CCN1 in the plasma (2.5 µL) of 544 patients with breast cancer and 427 healthy controls was analyzed by ELISA. The breast cancer samples were acquired at the time of primary diagnosis prior to neoadjuvant therapy or surgery. A classifier was established on a training cohort of patients with breast cancer and age-adapted healthy controls and further validated on an independent cohort comprising breast cancer patients and healthy controls. Samples from patients with benign breast diseases were investigated as additional controls. Samples from patients with acute heart diseases (n = 127) were investigated as noncancer controls. The diagnostic accuracy was determined by receiver operating characteristic using the parameters area under the curve, sensitivity, and specificity. RESULTS: CCN1 was frequently secreted by breast cancer cell lines into the extracellular space. Subsequent analysis of clinical blood samples from patients with breast cancer and age-adjusted healthy controls revealed an overall specificity of 99.0% and sensitivity of 80.0% for cancer detection. Remarkably, 81.5% of small T1 cancers were already CCN1-positive, while CCN1 concentrations in patients with benign breast lesions were below the threshold for breast cancer detection. CONCLUSIONS: Circulating CCN1 is a potentially novel blood biomarker for the detection of breast cancer at the earliest invasive stage.

Transcriptome Analysis in Vulvar Squamous Cell Cancer.

To date, therapeutic strategies in vulvar squamous cell carcinoma (VSCC) are lacking molecular pathological information and targeted therapy hasn't been approved in the treatment of VSCC, yet. Two etiological pathways are widely accepted: HPV induced vs. HPV independent, associated with chronic skin disease, often harboring TP53 mutations (mut). The aim of this analysis was to analyze the RNA expression patterns for subtype stratification on VSCC samples that can be integrated into the previously performed whole exome sequencing data for the detection of prognostic markers and potential therapeutic targets. We performed multiplex gene expression analysis (NanoString) with 770 genes in 24 prior next generation sequenced samples. An integrative data analysis was performed. Here, 98 genes were differentially expressed in TP53mut vs. HPV+ VSCC, in the TP53mut cohort, where 56 genes were upregulated and 42 were downregulated in comparison to the HPV+ tumors. Aberrant expression was primarily observed in cell cycle regulation, especially in HPV+ disease. Within the TP53mut group, a distinct cluster was identified that was correlated to a significantly worse overall survival (p = 0.017). The RNA expression profiles showed distinct patterns with regard to the known VSCC subtypes and could potentially enable further subclassification in the TP53mut groups.

BRCA1 promoter hypermethylation on circulating tumor DNA correlates with improved survival of patients with ovarian cancer.

Methylation of the BRCA1 promoter is an epigenetic gene expression regulator and is frequently observed in ovarian cancer; however, conversion of methylation status is thought to drive disease recurrence. Therefore, longitudinal monitoring of methylation status by liquid biopsy in cell-free DNA may be a predictive marker. In total, 135 plasma samples were collected from 69 ovarian cancer patients before and during systemic treatment. Our liquid biopsy assay could detect down to a single molecule of methylated DNA in a high background of normal DNA (0.03%) with perfect specificity in control samples. We found that 60% of the cancer patients exhibited BRCA1 promoter hypermethylation at one point, although 24% lost hypermethylation during treatment. Multivariate survival analyses indicate that relapses are independent events and that hypermethylation and methylation conversion are independently correlated to longer relapse-free survival. We present a highly sensitive and specific methylation-specific quantitative PCR-based liquid biopsy assay. BRCA1 promoter hypermethylation is frequently found in ovarian cancer and is often reversed upon recurrence, indicating the selection of therapy-resistant clones and unfavorable clinical outcome.

Emerging Insights into Keratin 16 Expression during Metastatic Progression of Breast Cancer.

Keratins are the main identification markers of circulating tumor cells (CTCs); however, whether their deregulation is associated with the metastatic process is largely unknown. Previously we have shown by in silico analysis that keratin 16 (KRT16) mRNA upregulation might be associated with more aggressive cancer. Therefore, in this study, we investigated the biological role and the clinical relevance of K16 in metastatic breast cancer. By performing RT-qPCR, western blot, and immunocytochemistry, we investigated the expression patterns of K16 in metastatic breast cancer cell lines and evaluated the clinical relevance of K16 expression in CTCs of 20 metastatic breast cancer patients. High K16 protein expression was associated with an intermediate mesenchymal phenotype. Functional studies showed that K16 has a regulatory effect on EMT and overexpression of K16 significantly enhanced cell motility (p < 0.001). In metastatic breast cancer patients, 64.7% of the detected CTCs expressed K16, which was associated with shorter relapse-free survival (p = 0.0042). Our findings imply that K16 is a metastasis-associated protein that promotes EMT and acts as a positive regulator of cellular motility. Furthermore, determining K16 status in CTCs provides prognostic information that helps to identify patients whose tumors are more prone to metastasize.

A Comprehensive Molecular Analysis of in Vivo Isolated EpCAM-Positive Circulating Tumor Cells in Breast Cancer.

BACKGROUND: Circulating tumor cell (CTC) analysis is highly promising for liquid biopsy-based molecular diagnostics. We undertook a comprehensive molecular analysis of in vivo isolated CTCs in breast cancer (BrCa). METHODS: In vivo isolated CTCs from 42 patients with early and 23 patients with metastatic breast cancer (MBC) were prospectively collected and analyzed for gene expression, DNA mutations, and DNA methylation before and after treatment. 19 healthy donor (HD) samples were analyzed as a control group. In identical blood draws, CTCs were enumerated using CellSearch® and characterized by direct IF staining. RESULTS: All 19 HD samples were negative for CK8, CK18, CK19, ERBB2, TWIST1, VEGF, ESR1, PR, and EGFR expression, while CD44, CD24, ALDH1, VIM, and CDH2 expression was normalized to B2M (reference gene). At least one gene was expressed in 23/42 (54.8%) and 8/13 (61.5%) CTCs in early BrCa before and after therapy, and in 20/23 (87.0%) and 5/7 (71.4%) MBC before and after the first cycle of therapy. PIK3CA mutations were detected in 11/42 (26.2%) and 3/13 (23.1%) in vivo isolated CTCs in early BrCa before and after therapy, and in 11/23 (47.8%) and 2/7 (28.6%) MBC, respectively. ESR1 methylation was detected in 5/32 (15.7%) and 1/10 (10.0%) CTCs in early BrCa before and after therapy, and in 3/15(20.0%) MBC before the first line of therapy. The comprehensive molecular analysis of CTC revealed a higher sensitivity in relation to CellSearch or IF staining when based on creatine kinase selection. CONCLUSIONS: In vivo-CTC isolation in combination with a comprehensive molecular analysis at the gene expression, DNA mutation, and DNA methylation level comprises a highly powerful approach for molecular diagnostic applications using CTCs.

CD74 and CD44 Expression on CTCs in Cancer Patients with Brain Metastasis.

Up to 40% of advance lung, melanoma and breast cancer patients suffer from brain metastases (BM) with increasing incidence. Here, we assessed whether circulating tumor cells (CTCs) in peripheral blood can serve as a disease surrogate, focusing on CD44 and CD74 expression as prognostic markers for BM. We show that a size-based microfluidic approach in combination with a semi-automated cell recognition system are well suited for CTC detection in BM patients and allow further characterization of tumor cells potentially derived from BM. CTCs were found in 50% (7/14) of breast cancer, 50% (9/18) of non-small cell lung cancer (NSCLC) and 36% (4/11) of melanoma patients. The next-generation sequencing (NGS) analysis of nine single CTCs from one breast cancer patient revealed three different CNV profile groups as well as a resistance causing ERS1 mutation. CD44 and CD74 were expressed on most CTCs and their expression was strongly correlated, whereas matched breast cancer BM tissues were much less frequently expressing CD44 and CD74 (negative in 46% and 54%, respectively). Thus, plasticity of CD44 and CD74 expression during trafficking of CTCs in the circulation might be the result of adaptation strategies.

Genome-wide methylation profiling of glioblastoma cell-derived extracellular vesicle DNA allows tumor classification.

BACKGROUND: Genome-wide DNA methylation profiling has recently been developed into a tool that allows tumor classification in central nervous system tumors. Extracellular vesicles (EVs) are released by tumor cells and contain high molecular weight DNA, rendering EVs a potential biomarker source to identify tumor subgroups, stratify patients and monitor therapy by liquid biopsy. We investigated whether the DNA in glioblastoma cell-derived EVs reflects genome-wide tumor methylation and mutational profiles and allows noninvasive tumor subtype classification. METHODS: DNA was isolated from EVs secreted by glioblastoma cells as well as from matching cultured cells and tumors. EV-DNA was localized and quantified by direct stochastic optical reconstruction microscopy. Methylation and copy number profiling was performed using 850k arrays. Mutations were identified by targeted gene panel sequencing. Proteins were differentially quantified by mass spectrometric proteomics. RESULTS: Genome-wide methylation profiling of glioblastoma-derived EVs correctly identified the methylation class of the parental cells and original tumors, including the MGMT promoter methylation status. Tumor-specific mutations and copy number variations (CNV) were detected in EV-DNA with high accuracy. Different EV isolation techniques did not affect the methylation profiling and CNV results. DNA was present inside EVs and on the EV surface. Proteome analysis did not allow specific tumor identification or classification but identified tumor-associated proteins that could potentially be useful for enriching tumor-derived circulating EVs from biofluids. CONCLUSIONS: This study provides proof of principle that EV-DNA reflects the genome-wide methylation, CNV, and mutational status of glioblastoma cells and enables their molecular classification.

Sensitive Blood-Based Detection of Asbestos-Associated Diseases Using Cysteine-Rich Angiogenic Inducer 61 as Circulating Protein Biomarker.

BACKGROUND: Detection of asbestos-associated diseases like asbestosis or mesothelioma is still challenging. We sought to improve the diagnosis of benign asbestos-associated disease (BAAD) by detection of the protein cysteine-rich angiogenic inducer 61 (Cyr61) in human plasma. METHODS: Plasma Cyr61 was quantified using an enzyme-linked immunosorbent assay. Plasma samples from males diagnosed with BAAD, but without a malignant disease (n = 101), and malignant mesothelioma (n = 21; 15 males, 6 females), as well as nonasbestos-exposed healthy control participants (n = 150; 58 males, 92 females) were analyzed. Clinical sensitivity and specificity of Cyr61 were determined by receiver operating characteristic analysis. RESULTS: The median plasma Cyr61 concentration for healthy control participants was 0.27 ng/mL. Cytoplasmic Cyr61 in peripheral blood mononuclear cells from healthy control participants was evenly distributed, as detected by immunofluorescent staining. The increase in plasma Cyr61 concentrations in the BAAD study group was statistically significant compared to the healthy control participants (P < 0.0001). For the detection of BAAD vs male healthy control participants, clinical sensitivity was 88% and clinical specificity 95% with an area under the curve of 0.924 at maximal Youden Index. For a predefined clinical specificity of 100%, the clinical sensitivity was 76%. For male mesothelioma patients vs male healthy control participants, the clinical sensitivity at maximal Youden Index was 95% with a clinical specificity of 100% (area under the curve, 0.997) and for a predefined clinical specificity of 100%, the clinical sensitivity was 93%. CONCLUSIONS: In our study, plasma Cyr61 protein concentrations showed to be a new biomarker for asbestos-associated diseases like BAAD and mesothelioma in men, which deserves further investigation in large-scale cohort studies.

Cell-Free HPV-DNA as a Biomarker for Oropharyngeal Squamous Cell Carcinoma-A Step Towards Personalized Medicine?

Global incidences of oropharyngeal squamous cell carcinoma (OPSCC) are rising due to an association with high-risk human papillomavirus (HPV). Although there is an improved overall survival of HPV-related OPSCC; up to 25% of the patients develop recurrent or distant metastatic disease with a fatal outcomes. Biomarkers to monitor this disease are not established. This meta-analysis reviews the role of cell-free HPV DNA in liquid biopsy (LB) as a biomarker for HPV-related OPSCC. Pubmed, Livivo, and Cochrane Library databases were searched from inception to August, 2020. All studies were analyzed by Meta-DiSc 1.4 and Stata 16.0 statistical software. In total, 16 studies were considered for systematic review, whereas 11 studies met inclusion criteria for meta-analysis, respectively. Pooled sensitivity of cfHPV-DNA at first diagnosis and during follow-up was 0.81 (95% CI; 0.78-0.84) and 0.73 (95% CI; 0.57-0.86), while pooled specificity was 0.98 (95% CI; 0.96-0.99) and 1 (95% CI; 0.99-1). The diagnostic odds ratio (DOR) at first diagnosis was 200.60 (95% CI; 93.31-431.22) and 300.31 (95% CI; 60.94-1479.88) during follow-up. The area under the curve (AUC) of summary receiver operating characteristic (SROC) was 0.99 at first diagnosis and 1.00 during follow-up, respectively. In conclusion, cfHPV-DNA presents a potential biomarker with high specificity in patients with HPV-related OPSCC.

Characterization of circulating breast cancer cells with tumorigenic and metastatic capacity.

Functional studies giving insight into the biology of circulating tumor cells (CTCs) remain scarce due to the low frequency of CTCs and lack of appropriate models. Here, we describe the characterization of a novel CTC-derived breast cancer cell line, designated CTC-ITB-01, established from a patient with metastatic estrogen receptor-positive (ER+ ) breast cancer, resistant to endocrine therapy. CTC-ITB-01 remained ER+ in culture, and copy number alteration (CNA) profiling showed high concordance between CTC-ITB-01 and CTCs originally present in the patient with cancer at the time point of blood draw. RNA-sequencing data indicate that CTC-ITB-01 has a predominantly epithelial expression signature. Primary tumor and metastasis formation in an intraductal PDX mouse model mirrored the clinical progression of ER+ breast cancer. Downstream ER signaling was constitutively active in CTC-ITB-01 independent of ligand availability, and the CDK4/6 inhibitor Palbociclib strongly inhibited CTC-ITB-01 growth. Thus, we established a functional model that opens a new avenue to study CTC biology.

Genomic characterization of vulvar squamous cell carcinoma.

BACKGROUND: Despite increasing incidence, vulvar squamous cell carcinoma (VSCC) is still a rare disease. Until now, two etiological pathways have been described: a high-risk human papillomavirus (HPV)-dependent route and an HPV-independent pathway often associated with lichen sclerosus. To date, therapeutic strategies in VSCC are not influenced by molecular pathological information and therapeutic options for advanced or recurrent disease are limited. METHODS: Whole exome sequencing of DNA, isolated from 34 VSCC samples and matched normal tissue for each individual was performed on an Illumina HiSeq4000. Short variant discovery was carried out using BWA mem and FreeBayes. Variants were annotated using ANNOVAR. RESULTS: FIGO stages were: IB (n = 7), II (n = 11), III (n = 8), and IVA (n = 3), (n = 5 unknown). TP53 missense mutations were most commonly detected with 56% (19/34). 12/34 (35.3%) samples were HPV positive (all HPV16), HPV positivity and TP53 mutations were mutually exclusive (p < .0001). Additionally, we observed mutations in known cancer relevant genes, like NBPF1 (n = 7), MACF1 (n = 5), SYNE2 (n = 5), DOCK2 (n = 4), KMT2D (n = 4), MAP2 (n = 4), NACA (n = 4), PIK3CA (n = 4), SYNE1 (n = 4), FBWX7 (n = 3), MSH6 (n = 3), NSD1 (n = 3), POLE (n = 3), TSC2, (n = 3) and CDKN2A (n = 2), but at considerably lower frequencies. For the total cohort 1848 cancer related mutations were detected (median of 54.4 per sample). CONCLUSIONS: The key mutation in HPV negative vulvar carcinoma affects TP53. While a multitude of cancer related mutations was detected in various samples, only few mutations recur and/or affect concurrent signaling pathways.

BRCA1 Promoter Methylation and Clinical Outcomes in Ovarian Cancer: An Individual Patient Data Meta-Analysis.

BACKGROUND: BRCA1 methylation has been associated with homologous recombination deficiency, a biomarker of platinum sensitivity. Studies evaluating BRCA1-methylated tubal and ovarian cancer (OC) do not consistently support improved survival following platinum chemotherapy. We examine the characteristics of BRCA1-methylated OC in a meta-analysis of individual participant data. METHODS: Data of 2636 participants across 15 studies were analyzed. BRCA1-methylated tumors were defined according to their original study. Associations between BRCA1 methylation and clinicopathological characteristics were evaluated. The effects of methylation on overall survival (OS) and progression-free survival (PFS) were examined using mixed-effects models. All statistical tests were 2-sided. RESULTS: 430 (16.3%) tumors were BRCA1-methylated. BRCA1 methylation was associated with younger age and advanced-stage, high-grade serous OC. There were no survival differences between BRCA1-methylated and non-BRCA1-methylated OC (median PFS = 20.0 vs 18.5 months, hazard ratio [HR] = 1.01, 95% CI = 0.87 to 1.16; P = .98; median OS = 46.6 vs 48.0 months, HR = 1.02, 95% CI = 0.87 to 1.18; P = .96). Where BRCA1/2 mutations were evaluated (n = 1248), BRCA1 methylation displayed no survival advantage over BRCA1/2-intact (BRCA1/2 wild-type non-BRCA1-methylated) OC. Studies used different methods to define BRCA1 methylation. Where BRCA1 methylation was determined using methylation-specific polymerase chain reaction and gel electrophoresis (n = 834), it was associated with improved survival (PFS: HR = 0.80, 95% CI = 0.66 to 0.97; P = .02; OS: HR = 0.80, 95% CI = 0.63 to 1.00; P = .05) on mixed-effects modeling. CONCLUSION: BRCA1-methylated OC displays similar clinicopathological features to BRCA1-mutated OC but is not associated with survival. Heterogeneity within BRCA1 methylation assays influences associations. Refining these assays may better identify cases with silenced BRCA1 function and improved patient outcomes.

Evaluation of Microfluidic Ceiling Designs for the Capture of Circulating Tumor Cells on a Microarray Platform.

The capture of circulating tumor cells (CTCs) is still a challenging application for microfluidic chips, as these cells are rare and hidden in a huge background of blood cells. Here, different microfluidic ceiling designs in regard to their capture efficiency for CTCs in model experiments and more realistic conditions of blood samples spiked with a clinically relevant amount of tumor cells are evaluated. An optimized design for the capture platform that allows highly efficient recovery of CTCs from size-based pre-enriched samples under realistic conditions is obtained. Furthermore, the viability of captured tumor cells as well as single cell recovery for downstream genomic analysis is demonstrated. Additionally, the authors' findings underline the importance of evaluating rational design rules for microfluidic devices based on theoretical models by application-specific experiments.