Liquid biopsy: An emerging prognostic and predictive tool in Head and Neck Squamous Cell Carcinoma (HNSCC). Focus on Circulating Tumor Cells (CTCs).

Molecular diversity and continuing evolution of metastatic tumors are not easily captured by tissue biopsies. Development of non-invasive diagnostic tools, such asanalysis of circulating tumor DNA (ctDNA), Circulating Tumor Cells (CTCs) and exosomes provides the opportunity to assess a blood sample in order to monitor tumor change and extract molecular information from cancers at a given time. "Liquid biopsy", which refers to molecular analysis of tumor's genetic features based on circulating genetic material in the peripheral blood, is already used to monitor disease response and track mechanisms of drug resistance in solid tumors. Head and Neck Squamous Cell Carcinoma (HNSCC) is a malignancy associated with advanced disease at presentation and dismal outcomes; furthermore, there is lack of biomarkers to monitor disease burden. Incorporation of liquid biopsy in the management of HNSCC might help identify patients with occult metastatic disease earlier and in a non-invasive manner. Herein, we aim to review current knowledge regarding CTCs and ctDNA in HNSCC and address open questions in this fast-evolving field of research.

Development and validation of a multiplex methylation specific PCR-coupled liquid bead array for liquid biopsy analysis.

BACKGROUND: Liquid biopsy is based on minimally invasive blood tests and has the potential to characterize the evolution of a solid tumor in real time, by extracting molecular information from circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA). Epigenetic silencing of tumor and metastasis suppressor genes plays a key role in survival and metastatic potential of cancer cells. Our group was the first to show the presence of epigenetic alterations in CTCs. METHODS: We present the development and analytical validation of a highly specific and sensitive Multiplex Methylation Specific PCR-coupled liquid bead array (MMSPA) for the simultaneous detection of the methylation status of three tumor and metastasis suppressor genes (CST6, SOX17 and BRMS1) in liquid biopsy material (CTCs, corresponding ctDNA) and paired primary breast tumors. RESULTS: In the EpCAM-positive CTCs fraction we observed methylation of: a) CST6, in 11/30(37%) and 11/30(37%), b) BRMS1 in 8/30(27%) and 11/30(37%) c) SOX17 in 8/30(27%) and 13/30(43%) early breast cancer patients and patients with verified metastasis respectively. In ctDNA we observed methylation of: a) CST6, in 5/30(17%) and 10/31(32%), b) BRMS1 in 8/30 (27%) and 8/31 (26%) c) SOX17 in 5/30(17%) and 13/31(42%) early breast cancer patients and patients with verified metastasis respectively. CONCLUSIONS: Our results indicate a high cancerous load at the epigenetic level in EpCAM-positive CTCs fractions and corresponding ctDNA in breast cancer. The main principle of the developed methodology has the potential to be extended in a large number of gene-targets and be applied in many types of cancer.

Prognostic role of APC and RASSF1A promoter methylation status in cell free circulating DNA of operable gastric cancer patients.

Gastric carcinogenesis is a multistep process including not only genetic mutations but also epigenetic alterations. The best known and more frequent epigenetic alteration is DNA methylation affecting tumor suppressor genes that may be involved in various carcinogenetic pathways. The aim of the present study was to investigate the methylation status of APC promoter 1A and RASSF1A promoter in cell free DNA of operable gastric cancer patients. Using methylation specific PCR, we examined the methylation status of APC promoter 1A and RASSF1A promoter in 73 blood samples obtained from patients with gastric cancer. APC and RASSF1A promoters were found to be methylated in 61 (83.6%) and 50 (68.5%) of the 73 gastric cancer samples examined, but in none of the healthy control samples (p < 0.001). A significant association between methylated RASSF1A promoter status and lymph node positivity was observed (p = 0.005). Additionally, a significant correlation between a methylated APC promoter and elevated CEA (p = 0.033) as well as CA-19.9 (p = 0.032) levels, was noticed. The Kaplan-Meier estimates of survival, significantly favored patients with a non-methylated APC promoter status (p = 0.008). No other significant correlations between APC and RASSF1A methylation status and different tumor variables examined was observed. Serum RASSF1A and APC promoter hypermethylation is a frequent epigenetic event in patients with early operable gastric cancer. The observed correlations between APC promoter methylation status and survival as well as between a hypermethylated RASSF1A promoter and nodal positivity may be indicative of a prognostic role for those genes in early operable gastric cancer. Additional studies, in a larger cohort of patients are required to further explore whether these findings could serve as potential molecular biomarkers of survival and/or response to specific treatments.

Breast cancer metastasis suppressor-1 promoter methylation in cell-free DNA provides prognostic information in non-small cell lung cancer.

BACKGROUND: Breast-cancer metastasis suppressor 1 (BRMS1) gene encodes for a predominantly nuclear protein that differentially regulates the expression of multiple genes, leading to suppression of metastasis without blocking orthotropic tumour growth. The aim of the present study was to evaluate for the first time the prognostic significance of BRMS1 promoter methylation in cell-free DNA (cfDNA) circulating in plasma of non-small cell lung cancer (NSCLC) patients. Towards this goal, we examined the methylation status of BRMS1 promoter in NSCLC tissues, matched adjacent non-cancerous tissues and corresponding cfDNA as well as in an independent cohort of patients with advanced NSCLC and healthy individuals. METHODS: Methylation of BRMS1 promoter was examined in 57 NSCLC tumours and adjacent non-cancerous tissues, in cfDNA isolated from 48 corresponding plasma samples, in cfDNA isolated from plasma of 74 patients with advanced NSCLC and 24 healthy individuals. RESULTS: The BRMS1 promoter was highly methylated both in operable NSCLC primary tissues (59.6%) and in corresponding cfDNA (47.9%) but not in cfDNA from healthy individuals (0%), while it was also highly methylated in cfDNA from advanced NSCLC patients (63.5%). In operable NSCLC, Kaplan-Meier estimates were significantly different in favour of patients with non-methylated BRMS1 promoter in cfDNA, concerning both disease-free interval (DFI) (P=0.048) and overall survival (OS) (P=0.007). In advanced NSCLC, OS was significantly different in favour of patients with non-methylated BRMS1 promoter in their cfDNA (P=0.003). Multivariate analysis confirmed that BRMS1 promoter methylation has a statistical significant influence both on operable NSCLC patients' DFI time and OS and on advanced NSCLC patients' PFS and OS. CONCLUSIONS: Methylation of BRMS1 promoter in cfDNA isolated from plasma of NSCLC patients provides important prognostic information and merits to be further evaluated as a circulating tumour biomarker.

Clinical challenges in the molecular characterization of circulating tumour cells in breast cancer.

Blood testing for circulating tumour cells (CTC) has emerged as one of the hottest fields in cancer research. CTC detection and enumeration can serve as a 'liquid biopsy' and an early marker of response to systemic therapy, whereas their molecular characterisation has a strong potential to be translated to individualised targeted treatments and spare breast cancer (BC) patients unnecessary and ineffective therapies. Different analytical systems for CTC detection and isolation have been developed and new areas of research are directed towards developing novel assays for CTC molecular characterisation. Molecular characterisation of single CTC holds considerable promise for predictive biomarker assessment and to explore CTC heterogeneity. The application of extremely powerful next-generation sequencing technologies in the area of CTC molecular characterisation in combination with reliable single CTC isolation opens new frontiers for the management of patients in the near future. This review is mainly focused on the clinical potential of the molecular characterisation of CTC in BC.

Development of a multiplexed PCR-coupled liquid bead array assay for vascular endothelial growth factor (VEGF) splice variants.

OBJECTIVES: To develop a multiplex PCR-coupled liquid bead array assay for the expression of VEGF splice variants. DESIGN AND METHODS: The assay was based on the combination of multiplex PCR with liquid bead array technology, and optimized and evaluated in terms of analytical sensitivity, specificity, and reproducibility using the MCF-7 cell line. Clinical performance was evaluated in 16 pairs of fresh frozen cancerous and corresponding noncancerous adjacent tissues from NSCLC patients. RESULTS: The assay is highly sensitive, reproducible and can detect specifically VEGF splice variants in clinical samples. When applied in 32 clinical samples it gave comparable results to RT-qPCR (concordance of 81%, 75%, 88% and 81% for PBGD, VEGF(121), VEGF(165), and VEGF(189) respectively). CONCLUSIONS: The developed assay can simultaneously detect three VEGF splice variants with high specificity and sensitivity and can be further used to evaluate the role of each individual VEGF splice variant in molecular therapies targeted against VEGF.

Kallikrein 10 (KLK10) methylation as a novel prognostic biomarker in early breast cancer.

BACKGROUND: We evaluated the prognostic significance of KLK10 exon 3 methylation in patients with early-stage breast cancer since it has been shown to have a significant impact on biological characteristics of breast tumors. MATERIALS AND METHODS: Using methylation-specific PCR, we evaluated the specificity of KLK10 methylation in 10 breast tumors and matching normal tissues, 10 breast fibroadenomas, 11 normal breast tissues and in a testing group of 35 patients. The prognostic significance of KLK10 methylation was validated in an independent cohort of 93 patients. RESULTS: KLK10 was not methylated in normal breast tissues and fibroadenomas while it was in 5 of 10 breast tumors and in 1 of 10 matching normal tissues. In the testing group of 35 patients, KLK10 methylation was detected in 70.0% of patients who relapsed (P = 0.001) and in 77.8% of patients who died (P = 0.025). In the independent cohort, 53 of 93 (57.0%) patients were found positive for KLK10 methylation. During the follow-up period, 24 of 93 (25.8%) patients relapsed and 19 of 93 (20.4%) died. Disease-free interval (DFI) and overall survival (OS) were significantly associated with KLK10 methylation (P = 0.0025 and P = 0.003). Multivariate analysis revealed that KLK10 methylation was an independent prognostic factor for DFI and OS. CONCLUSION: KLK10 exon 3 methylation provides important prognostic information in early breast cancer patients.

Development of a quantitative luminometric hybridization assay for the determination of telomerase activity.

OBJECTIVES: To develop a quantitative luminometric hybridization assay for the determination of telomerase activity in tissue and cell extracts. DESIGN AND METHODS: Quantification is based on the coamplification of telomeric repeats synthesized by telomerase along with a specifically designed recombinant DNA-internal standard (DNA-IS). The DNA-IS has a similar size and the same primer recognition sites as the telomerase DNA products and differs from them only in a central 18 bp sequence. PCR products are captured on microtiter wells via the biotin-streptavidin system and hybridized with two distinct digoxigenin-labeled oligonucleotide probes that are designed to recognize specifically telomerase products and DNA-IS. The hybrids are quantified by a luminometric reaction using an antidigoxigenin antibody conjugated to alkaline phosphatase. The hybridization assay was validated with the MCF-7 breast carcinoma and leukemia K-562 cell lines and a synthetic telomerase product (R(8)). RESULTS: Luminescence ratios for telomerase products and DNA-IS were linearly related to the concentration of the pre-PCR product synthesized by telomerase (R(8)), in the range of 0.0005 to 10 pM. The overall reproducibility of the assay (between-run) varied between 11.3 and 15%. Application of the method in eleven breast tumors showed a great variation in the levels of telomerase enzymatic activity. CONCLUSIONS: The proposed luminometric hybridization assay for the quantitative determination of telomerase enzymatic activity is highly sensitive and can be used for a large-scale prospective evaluation of clinical samples.

BRCA1 mutation analysis in breast/ovarian cancer families from Greece.

Germline mutations in BRCA1 gene account for varying proportions of breast/ovarian cancer families, and demonstrate considerable variation in mutational spectra coincident with ethnic and geographical diversity. We have screened for mutations the entire coding sequence of BRCA1 in 30 breast/ovarian cancer women with family history of two or more cases of breast cancer under age 50 and/or ovarian cancer at any age. Genomic DNA from patient was initially analyzed for truncating mutations in exon 11 with PTT followed by DNA sequencing. In the cases where no frameshift mutation was observed in exon 11, all other exons were screened with direct sequencing. Two novel (3099delT, 3277insG) and three already described (3741insA, 1623del5-TTAAA, 5382insC-twice) truncating mutations were identified. In addition, 6 point mutations (L771L, P871L, E1038G, K1183R, S1436S, S1613G) which are already classified as polymorphisms were identified. Three unclassified intronic variants (IVS16-68 G>A, IVS16-92 G>A, IVS18+65G>A) were also detected. These results show that BRCA1 deleterious mutations are present in a fraction (20%) of Greek breast/ovarian cancer families similar to other European countries. Mutations were detected in high- (>/=3 members) as well as in moderate-risk (2 members) families. This is the first report of BRCA1 mutation analysis in Greece.

Determination of tumor necrosis factor-alpha (TNF-alpha) in serum by a highly sensitive enzyme amplified lanthanide luminescence immunoassay.

OBJECTIVES: To develop a highly sensitive enzyme amplified lanthanide luminescence (EALL) immunoassay for tumor necrosis factor-alpha (TNF-alpha). METHODS: The method is based on the use of two monoclonal antibodies against TNF-alpha, one "capture" antibody and one labeled with biotin, in a "sandwich type" assay format. Alkaline phosphatase (ALP) conjugated to an antibiotin-polyclonal antibody is used as the enzyme label. ALP cleaves phosphate from diflunisal phosphate (DIFP) to produce diflunisal (DIF). The detection system is based on the combination of enzymatic amplification introduced by ALP and the formation of a highly fluorescent terbium complex that can be monitored by time resolved or conventional fluorimetry. RESULTS: By using 50 microL of sample, the dynamic range of the assay extends up to 2000 ng/L of TNF-alpha, with a detection limit of 1 ng/L, within-run CVs ranging from 3 to 15% and recoveries of 97 +/- 2%. By using 100 microL of sample the dynamic range of the assay extends up to 1000 ng/L of TNF-alpha with a detection limit of 0.2 ng/L, recoveries of 94 +/- 13%, within-run CVs ranging from 2 to 6.5% and between-run CVs ranging from 5 to 15%, in a total incubation time of 3h. No interference by the presence of other cytokines (IL-1beta IL-2, IL-4, IL-6, IFN-gamma) or by rheumatoid factors has been observed. The results obtained by the proposed method and by a commercially available kit (Medgenix TNF-alpha EASIA) correlated well (n = 26, r = 0.934). CONCLUSION: The proposed method is highly sensitive, simple and rapid and can reliably measure TNF-alpha in the ng range in biological specimens.

Immunofluorometric assay of p53 protein versus sequencing of p53 exons 5 to 9 for the detection of p53 abnormalities in ovarian carcinoma.

p53 alteration, detected as mutation of the p53 gene or as accumulation of mutant p53 protein, is a common feature of most malignancies, including ovarian carcinoma, and may identify patients with unfavorable prognosis and resistance to chemotherapy. Tumor tissues from 55 patients with well or poorly differentiated (grades 1 or 3) primary epithelial ovarian carcinoma were assessed both for p53 protein overexpression by a sensitive time-resolved immunofluorometric assay employing DO-1 and CM-1 antibodies, and for genetic p53 abnormalities by direct sequencing of PCR-amplified exons 5 to 9. Sixteen p53 mutations (29%), including 3 deletions causing frameshifts as well as one nonsense and 12 missense point mutations were found in all exons except exon 9. Overexpression of p53 protein, defined as a concentration exceeding the 75th percentile, was found in 15 cases (27%), 10 of which had missense mutations (P < 0.01). Tumors with nonsense and frameshift mutations were p53-negative by immunoassay. Both p53 mutation (P = 0.04) and p53 protein accumulation (P < 0.01) were associated with stage III-IV disease, while p53 mutation was more closely related to grade 3 lesions (P = 0.04) and serous histotype (P = 0.01). These results indicate that p53 protein accumulation correlates well with missense point mutation in carcinoma of the ovary and, together with other evidence that p53 abnormality may be prognostic of outcome in this disease, suggest that the immunoassay of p53 protein may have clinical value.

Application of terbium sensitized fluorescence for the determination of fluoroquinolone antibiotics pefloxacin, ciprofloxacin and norfloxacin in serum.

A simple, rapid and sensitive spectrofluorimetric method for the determination of fluoroquinolone antibiotics, norfloxacin (NOR), ciprofloxacin (CIP) and pefloxacin (PEF) is described. The method is based on the radiative energy transfer from fluoroquinolones to terbium ions (Tb3+) in the presence of tri-n-octylphosphine oxide (TOPO) in weakly acidic (pH 5.5) micellar solution of cetylpyridinium chloride (CPCI). Optimum conditions for the formation of the fluoroquinolone-Tb(3+)-TOPO ternary complexes have been investigated. Under optimized conditions the detection limits are 1.7, 1.2 and 4.4 nM for NOR, CIP and PEF, respectively, while the range of application for all three drugs is 0.05-10 microM. The method has been successfully applied to the determination of NOR, CIP and PEF in serum samples after deproteinization with acetonitrile (serum-acetonitrile; 1:2, v/v). The mean recoveries from serum samples spiked with NOR, CIP and PEF (5.0-50.0 microM) were (90.3 +/- 4.9), (105.0 +/- 3.6), and (95.3 +/- 1.5)% respectively. Within-run and day-to-day s, values for 5.0, 25.0 and 50.0 microM of each fluoroquinolone varied from 1.7 to 5.4% and from 3.3 to 12.8%, respectively. The influence of several usually coadministered drugs on the determination of fluoroquinolones in serum has been investigated.

BRCA1 tumor suppressor gene product shares immunoreactive epitopes with a protein present in seminal plasma.

OBJECTIVES: To develop immunofluorometric procedures for measuring BRCA1 protein in various biological fluids and tissue extracts. DESIGN AND METHODS: Five commercially available monoclonal and polyclonal antibodies against BRCA1 were evaluated for developing competitive and non-competitive immunofluorometric procedures for BRCA1. Biotinylated and nonbiotinylated peptides were used to assess the specificity of the antibodies for blocking experiments and for the competitive immunoassay. Extensive studies to exclude cross-reactivity and non-specific effects in the non-competitive immunoassay were undertaken. Seminal plasmas as well as breast tumor extracts, amniotic fluids and cerebrospinal fluids were analyzed. RESULTS: We designed novel methods for measuring BRCA1 immunoreactivity. One configuration based on the "sandwich-type" immunoassay principle was used for further studies. We discovered that seminal plasma contains an immunoreactive protein which appears to possess the D-20 (aminoterminal) and C-20 (carboxyterminal) epitopes of BRCA1. Molecular weight identification using gel filtration chromatography has shown that the immunoreactive species has a molecular weight between 660 and 160 KDa. CONCLUSIONS: We identified for the first time a protein in seminal plasma that shares immunoreactive epitopes with the BRCA1 tumor suppressor protein. We are currently purifying this protein in order to examine if it is homologous or identical to BRCA1.

Simple spectrofluorometric determination of p-aminobenzoic and p-aminosalicylic acids in biological fluids by use of terbium-sensitized luminescence.

A novel, sensitive, and selective method has been developed for determination of p-aminobenzoic (PABA) and p-aminosalicylic (PAS) acids in the N-benzoyl-L-tyrosyl-PABA/ PAS test. PAS is measured as a ternary complex with terbium and EDTA (lambda(ex) = 324 nm, lambda(em) = 546 nm) in alkaline aqueous solution (pH approximately 12.6), whereas both compounds (PABA and PAS) are measured as ternary complexes with terbium and tri-n-octylphosphine oxide (lambda(ex) = 292 nm, lambda(em) = 546 nm) in weakly acidic aqueous solution (pH approximately 5.5). We inve stigated and implemented optimum conditions for formation of these complexes, yielding respective detection limits for PABA and PAS of 0.07 and 0.02 micromol/L and ranges of application of 0-10 and 0-40 micromol/L (final concentration). The method has been successfully applied to determinations of PABA and PAS in urine and, after alkaline hydrolysis, to determinations of PABA in serum that has been deproteinized with acetonitrile. Within-run imprecision of the PABA determination ranges from 0.8% to 4.2 % for urine samples and from 3.9% to 8.2% for serum samples; day-to-day imprecision varies from 3.2% to 10% for serum samples.

Assay of creatine kinase isoenzyme MB in serum with time-resolved immunofluorometry.

We describe the first time-resolved immunofluorometric assay for creatine kinase (EC 2.7.3.2) isoenzyme MB (CK-MB) in serum. The assay is based on the formation of the complex: solid-phase anti-CK-MB-CK-MB-biotinylated anti-CK-BB-streptavidin-BCPDA-Eu3+, where anti-CK-MB and anti-CK-BB are monoclonal antibodies against the CK isoenzymes MB and BB, respectively, and BCPDA is the europium chelator 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid. The solid-phase complex is fluorescent and is measured on the dry solid-phase (microtiter well) in a specially designed time-resolved fluorometer that uses laser excitation. The assay requires 25 microL of serum and is not affected by the presence of either CK-MM (up to 5000 micrograms/L) or CK-BB (up to 1000 micrograms/L) in the sample. Precision and accuracy indices for the assay were satisfactory.

Ultrasensitive time-resolved fluorescence method for alpha-fetoprotein.

We have examined the maximum sensitivity of a newly developed and optimized time-resolved fluorescence immunoassay system. The system, originally described elsewhere (Clin Biochem 1988:21:139-50), has undergone significant improvements in sensitivity through improvements of the labeled reagent used. We have chosen an alpha-fetoprotein (AFP) assay as a model and used monoclonal "capture" antibodies and monoclonal or polyclonal biotinylated antibodies in "sandwich-type" assay configurations. Streptavidin labeled with the europium chelator 4.7-bis(chlorosulfophenyl)-1,10-phenanthroline-2.9-dicarboxylic acid was used for detection. We can measure as few as 3 x 10(5) molecules of AFP with the optimized system. We have applied this assay to measure AFP in the serum of normal individuals after a 10-fold sample dilution. We conclude that this system is extremely sensitive and can be used in immunoassay or other applications where biotinylated reagents can be applied.

Determination of ursodeoxycholic acid in serum by a new fluorometric enzymatic method using 7 beta-hydroxysteroid dehydrogenase from Clostridium absonum.

A fluorometric enzymatic method for the determination of ursodeoxycholic acid (UDCA) and its glycine and taurine conjugates in human serum has been developed. A simple and fast purification and preconcentration procedure using Sep Pak C18 cartridges was employed for the UDCA extraction from human serum. UDCA and its conjugates were determined in the extracted sample by an equilibrium method based on the enzymatic conversion of the 7 alpha-hydroxy group into 7-oxo group by beta-nicotinamide adenine dinucleotide phosphate in the presence of 7 beta-hydroxysteroid dehydrogenase (7 beta-HSD) and the produced NADPH was monitored fluorometrically. The 7 beta-HSD, which is not yet commercially available, was isolated from Clostridium absonum cultures (ATCC No. 27555) and purified by affinity chromatography. The method has a limit of detection of 0.8 microM in serum and the precision varied from 6.1 to 2.0% for low and high concentrations, respectively. The recovery of UDCA from serum samples was about 99% (range 85-105%). The method was successfully applied to UDCA determination in serum samples from patients treated with UDCA for primary biliary cirrhosis.