The potential of liquid biopsy in the management of cancer patients.

Over the last decade, liquid biopsy has gained much attention as a powerful tool in personalized medicine, since it enables monitoring cancer evolution and follow-up of cancer patients in real time. Through minimally invasive procedures, liquid biopsy provides important information through the analysis of Circulating Tumor Cells (CTCs), and circulating tumor-derived material like circulating tumor DNA (ctDNA), circulating miRNAs (cfmiRNAs) and extracellular vehicles (EVs). CTCs and ctDNA analysis has already an important impact on the prognosis, detection of minimal residual disease (MRD), treatment selection and monitoring of cancer patients, while recent data show also its potential for early cancer diagnosis (Figure 1). Numerous clinical trials include now a liquid biopsy arm, and functional studies mainly based on CTC derived cell-lines and CTC derived explants (CDx) provide important insight on the metastatic process. The recent findings in the field of liquid biopsy and the benefits and main clinical applications of CTC and ctDNA analysis in solid tumors are summarized in this review.

Prognostic significance of PD-L1 expression on circulating tumor cells in patients with head and neck squamous cell carcinoma.

BACKGROUND: Successful application of programmed death 1 (PD1) checkpoint inhibitors in the clinic may ultimately benefit from appropriate patient selection based upon predictive biomarkers. Molecular characterization of circulating tumor cells (CTC) is crucial for the investigation of molecular-targeted therapies while predictive biomarkers for response to PD1 checkpoint inhibitors are lacking. We sought to assess whether overexpression of PD-L1 in CTCs could be detected at baseline and at different timepoints during treatment in a prospective cohort of head and neck squamous cell carcinoma (HNSCC) patients and used to predict clinical outcome after treatment with curative intent. PATIENTS AND METHODS: We developed a highly sensitive, specific and robust RT-qPCR assay for PD-L1 mRNA expression in EpCAM(+) CTCs. In a prospective cohort of 113 locally advanced HNSCC patients treated with curative intent we evaluated PD-L1 expression in the EpCAM(+) CTC fraction at baseline, after 2 cycles of induction chemotherapy (week 6) and at the end of concurrent chemoradiotherapy (week 15). RESULTS: PD-L1 overexpression was found in 24/94 (25.5%) patients at baseline, 8/34 (23.5%) after induction chemotherapy and 12/54 (22.2%) patients at the end of treatment. Patients with CTCs overexpressing PD-L1 at end of treatment had shorter progression-free survival (P = 0.001) and overall survival (P < 0.001). Multivariate analysis revealed that PD-L1 overexpression at end of treatment was independent prognostic factor for progression-free survival and overall survival. The absence of PD-L1 overexpression at the end of treatment was strongly associated with complete response with an odds ratio = 16.00 (95% CI = 2.76-92.72, P = 0.002). CONCLUSIONS: We demonstrate that detection of CTCs overexpressing PD-L1 is feasible and may provide important prognostic information in HNSCC. Our results suggest that adjuvant PD1 inhibitors deserve evaluation in HNSCC patients in whom PD-L1(+) CTCs are detected at the end of curative treatment.

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.

Preoperative Mutational Analysis of Circulating Tumor Cells (CTCs) and Plasma-cfDNA Provides Complementary Information for Early Prediction of Relapse: A Pilot Study in Early-Stage Non-Small Cell Lung Cancer.

PURPOSE: We assessed whether preoperativemutational analyses of circulating tumor cells (CTCs) and plasma-cfDNA could be used as minimally invasive biomarkers and as complimentary tools for early prediction of relapse in early-stage non-small -cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: Using ddPCR assays, hotspot mutations of BRAF, KRAS, EGFR and PIK3CA were identified in plasma-cfDNA samples and size-based enriched CTCs isolated from the same blood samples of 49 early-stage NSCLC patients before surgery and in a control group of healthy blood donors (n= 22). Direct concordance of the mutational spectrum was further evaluated in 27 patient-matched plasma-cfDNA and CTC-derived DNA in comparison to tissue-derived DNA. RESULTS: The prevalence of detectable mutations of the four tested genes was higher in CTC-derived DNA than in the corresponding plasma-cfDNA (38.8% and 24.5%, respectively).The most commonly mutated gene was PIK3CA, in both CTCs and plasma-cfDNA at baseline and at the time of relapse. Direct comparison of the mutation status of selected drug-responsive genes in CTC-derived DNA, corresponding plasma-cfDNA and paired primary FFPE tissues clearly showed the impact of heterogeneity both within a sample type, as well as between different sample components. The incidence of relapse was higher when at least one mutation was detected in CTC-derived DNA or plasma-cfDNA compared with patients in whom no mutation was detected (p =0.023). Univariate analysis showed a significantly higher risk of progression (HR: 2.716; 95% CI, 1.030-7.165; p =0.043) in patients with detectable mutations in plasma-cfDNA compared with patients with undetectable mutations, whereas the hazard ratio was higher when at least one mutation was detected in CTC-derived DNA or plasma-cfDNA (HR: 3.375; 95% CI, 1.098-10.375; p =0.034). CONCLUSIONS: Simultaneous mutational analyses of plasma-cfDNA and CTC-derived DNA provided complementary molecular information from the same blood sample and greater diversity in genomic information for cancer treatment and prognosis. The detection of specific mutations in ctDNA and CTCs in patients with early-stage NSCLC before surgery was independently associated with disease recurrence, which represents an important stratification factor for future trials.

Metabolism-Related Gene Expression in Circulating Tumor Cells from Patients with Early Stage Non-Small Cell Lung Cancer.

PURPOSE: Metabolic reprogramming is now characterized as one of the core hallmarks of cancer, and it has already been shown that the altered genomic profile of metabolically rewired cancer cells can give valuable information. In this study, we quantified three Metabolism-Related Gene (MRG) transcripts in the circulating tumor cells (CTCs) of early stage NSCLC patients and evaluated their associations with epithelial and EMT markers. EXPERIMENTAL DESIGN: We first developed and analytically validated highly sensitive RT-qPCR assays for the quantification of HK2, MCT1 and PHGDH transcripts, and further studied the expression of MRGs in CTCs that were isolated using a size-dependent microfluidic device (Parsortix, Angle) from the peripheral blood of: (a) 46 NSCLC patients at baseline, (b) 39/46 of these patients one month after surgery, (c) 10/46 patients at relapse and (d) 10 pairs of cancerous and adjacent non-cancerous FFPE tissues from the same NSCLC patients. Epithelial and EMT markers were also evaluated. RESULTS: MCT1 and HK2 were differentially expressed between HD and NSCLC patients. An overexpression of MCT1 was detected in 15/46 (32.6%) and 3/10 (30%) patients at baseline and at progression disease (PD), respectively, whereas an overexpression of HK2 was detected in 30.4% and 0% of CTCs in the same group of samples. The expression levels of all tested MRGs decreased in CTCs one month after surgery, but a significant increase was noticed at the time of relapse for PHGDH and MCT1 only. The expression levels of HK2 and MCT1 were associated with the overexpression of mesenchymal markers (TWIST-1 and VIM). CONCLUSION: An overexpression of MRGs was observed at a high frequency in the CTCs isolated from early NSCLC patients, thereby supporting the role of MRGs in metastatic processes. The glycolytic and mesenchymal subpopulation of CTCs was significantly predominant compared to CTCs that were glycolytic but not mesenchymal-like. Our data indicate that MRGs merit further evaluation through large and well-defined cohort studies.

Evaluation of Monocarboxylate Transporter 4 (MCT4) Expression and Its Prognostic Significance in Circulating Tumor Cells From Patients With Early Stage Non-Small-Cell Lung Cancer.

Purpose: Monocarboxylate transporter 4 (MCT4) can influence the amount of lactate in the tumor microenvironment and further control cancer cell proliferation, migration, and angiogenesis. We investigated for the first time the expression of MCT4 in circulating tumor cells (CTCs) derived from early stage Non-Small Cell Lung Cancer patients (NSCLC) and whether this is associated with clinical outcome. Experimental Design: A highly sensitive RT-qPCR assay for quantification of MCT4 transcripts was developed and validated and applied to study MCT4 expression in CTC isolated through the Parsortix size-dependent microfluidic device from 53 and 9 peripheral blood (PB) samples of NSCLC patients at baseline (pre-surgery) and at relapse, respectively, as well as the "background noise" was evaluated using peripheral blood samples from 10 healthy donors (HD) in exactly the same way as patients. Results: MCT4 was differentially expressed between HD and NSCLC patients. Overexpression of MCT4 was detected in 14/53 (26.4%) and 3/9 (33.3%) patients at baseline and at progression disease (PD), respectively. The expression levels of MCT4 was found to increase in CTCs at the time of relapse. Kaplan-Meier analysis showed that the overexpression of MCT4 was significantly (P = 0.045) associated with progression-free survival (median: 12.5 months, range 5-31 months). Conclusion: MCT4 overexpression was observed at a high frequency in CTCs from early NSCLC patients supporting its role in metastatic process. MCT4 investigated as clinically relevant tumor biomarker characterizing tumor aggressiveness and its potential value as target for cancer therapy. We are totally convinced that MCT4 overexpression in CTCs merits further evaluation as a non-invasive circulating tumor biomarker in a large and well-defined cohort of patients with NSCLC.

What do we need to obtain high quality circulating tumor DNA (ctDNA) for routine diagnostic test in oncology? - Considerations on pre-analytical aspects by the IFCC workgroup cfDNA.

The analysis of circulating cell free DNA is an important tool for the analysis of tumor resistance, tumor heterogeneity, detection of minimal residual disease and detection of allograft rejection in kidney or heart transplant patients. The proper use of this technique is important, and starts with considering pre-analytic aspects. The current paper addresses some important technical considerations to ensure the proper and harmonized use of cfDNA techniques.

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.

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.

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.

Germ line BRCA1 & BRCA2 mutations in Greek breast/ovarian cancer families: 5382insC is the most frequent mutation observed.

BRCA1 and BRCA2 genes were screened for loss-of-function mutations in a series of 85 patients having at least one first- or second-degree relative affected by breast and/or ovarian cancer. All BRCA1 exons and BRCA2 exons 10 and 11 were screened with a combination of methods including SSCP, PTT and direct sequencing. We have found disease-associated mutations in 14 families (16.5%), eleven in BRCA1 and three in BRCA2. The known founder mutation 5382insC of BRCA1 was identified in seven unrelated families. The other mutations identified include the non-sense R1751X, the splice junction variant 5586G>A of BRCA1 and three frameshifts, 2024del5, 3034del4, and 6631del5, of BRCA2. Nine out of these 14 families had a family history of three or more breast/ovarian cancer cases. A large number of polymorphic or unclassified variants is also reported. Combined with our previously published data 5382insC was found in nine out of 20 families (45%), suggesting that this mutation may represent a common founder mutation in the Greek population.

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.

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 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.

Quantitative RT-PCR luminometric hybridization assay with an RNA internal standard for cytokeratin-19 mRNA in peripheral blood of patients with breast cancer.

OBJECTIVES: To develop a highly sensitive quantitative RT-PCR hybridization assay for the determination of CK-19 mRNA in peripheral blood of patients with breast cancer. PATIENTS AND METHODS: Quantification of CK-19 mRNA was based on the coamplification of CK-19 mRNA with a recombinant CK-19 RNA internal standard (CK-19 RNA-IS) through RT-PCR. The biotinylated amplification products were immobilized on steptavidin coated wells, hybridized with digoxigenin labeled probes and determined through an antidigoxigenin antibody conjugated to alkaline phosphatase by luminometric detection. The developed luminometric hybridization assay was validated with samples containing total RNA of known amounts from CK-19 expressing cells (MCF-7) in the presence of 1 microg total RNA isolated from peripheral blood mononuclear cells (PBMC) of healthy controls and a constant amount of CK-19 RNA-IS. The method was applied for the quantitative determination of CK-19 mRNA in the peripheral blood of 26 healthy volunteers, 14 patients with stage IV breast cancer and 37 patients with stage I/II breast cancer before chemotherapy. RESULTS: Luminescence ratios for CK-19 mRNA and CK-19 RNA-IS were linearly related to the number of MCF-7 cells within the range of 1 to 2000 cells. The overall reproducibility of the assay (between-run) varied between 8.9% and 13.4%. The method can clearly detect CK-19 mRNA from 1 MCF-7 cell in the presence of 10(6) normal PBMC and is highly specific as none of the 26 healthy controls tested had detectable CK-19 mRNA levels, while 10 out of 14 (71.4%) and 9 out of 37 (24.3%) patients with stage IV and stage I/II breast cancer, respectively, were tested positive. CONCLUSION: The developed quantitative RT-PCR hybridization assay for CK-19 is reproducible, highly sensitive and specific, and can be used for a large-scale prospective evaluation of clinical samples.

Molecular detection of cancer cells in the peripheral blood of patients with breast cancer: comparison of CK-19, CEA and maspin as detection markers.

PURPOSE: To investigate and compare the diagnostic value of the detection of cytokeratin 19 (CK-19), carcinoembryonic antigen (CEA) and maspin mRNA by nested RT-PCR in the peripheral blood of women with breast cancer. MATERIALS AND METHODS: The tumor cell lines MCF-7 and LOVO were used in an experimental tumor cell dilution model to determine the sensitivity of the nested RT-PCR for the 3 detection markers. RT-PCR analysis was performed in the peripheral blood of 54 healthy female blood donors, 28 patients with hematological malignancies, 31 with metastatic colorectal cancer, 75 with operable and 50 with metastatic breast cancer before receiving any cytotoxic chemotherapy, as well as in the bone marrow aspirates of 61 breast cancer patients. RESULTS: Nested RT-PCR for CK-19 mRNA presented the highest sensitivity by detecting 1 tumor cell amongst 10(6) PBMC in 4 out of 5 experiments. CK-19 mRNA was detected in the peripheral blood of 3.7% of female blood donors, 14.3% of hematological malignancies, 32% of operable and 42% of metastatic breast cancer patients. CEA mRNA was undetectable in the blood of female blood donors but was detected in blood samples of 3.5% of hematological malignancies, 19.3% of colorectal cancer and 10% of breast cancer patients. Maspin mRNA was undetectable in the blood of female blood donors, patients with hematological malignancies and colorectal cancer but was detected in 9.3% of operable and 14% of metastatic breast cancer patients. Maspin mRNA positivity correlated with tumor size in patients with early stage breast cancer (p = 0.057). The detection rates of CK-19 and maspin mRNA in bone marrow aspirates were 33% and 11% for operable and 62% and 9% for metastatic breast cancer, respectively. During follow-up, 27.4% of blood samples were positive for CK-19 mRNA versus 10.7% for maspin mRNA in patients with operable breast cancer with a concordance rate of only 12.7% for positives and 86% for negatives. CONCLUSION: RT-PCR positivity for CK-19 mRNA is the most sensitive detection marker for occult tumor cells in operable and metastatic breast cancer, although nested RT-PCR for maspin mRNA appears to be more specific.