Clinical validation of a type-specific real-time quantitative human papillomavirus PCR against the performance of hybrid capture 2 for the purpose of cervical cancer screening.

To be acceptable for use in cervical cancer screening, a new assay that detects DNA of high-risk human papillomavirus (hrHPV) types must demonstrate high reproducibility and performance not inferior to that of a clinically validated HPV test. In the present study, a real-time quantitative PCR (qPCR) assay targeting the E6 and E7 genes of hrHPV was compared with Hybrid Capture 2 (hc2) in a Belgian cervical cancer screening setting. In women >30 years old, the sensitivity and specificity for intraepithelial neoplasias of grade 2 or worse (93 cases of cervical intraepithelial neoplasias of grade 2 or worse (CIN2+) and 1,207 cases of no CIN or CIN1) were 93.6% and 95.6%, respectively, and those of hc2 were 83.9% and 94.5%, respectively {relative sensitivity of qPCR/hc2 = 1.12 [95% confidence interval (CI), 1.01 to 1.23]; relative specificity = 1.01 [95% CI, 0.99 to 1.03]}. A score test showed that the sensitivity (P < 0.0001) and specificity (P < 0.0001) of the qPCR assay were not inferior to those of hc2 at the required thresholds of 90% and 98%, respectively. The overall agreement of hrHPV positivity between the two runs of the qPCR tests was 98.7% (95% CI, 97.5 to 99.4%), with a kappa value of 0.96 (95% CI, 0.83 to 1.00). The qPCR assay used in this study can be considered a reliable HPV assay that fulfills the clinical validation criteria defined for use in cervical cancer screening.

Circulating tumour cells in the central and the peripheral venous compartment in patients with metastatic breast cancer.

BACKGROUND: The enumeration of circulating tumour cells (CTC) has prognostic significance in patients with metastatic breast cancer (MBC) and monitoring of CTC levels over time has considerable potential to guide treatment decisions. However, little is known on CTC kinetics in the human bloodstream. METHODS: In this study, we compared the number of CTC in both 7.5 ml central venous blood (CVB) and 7.5 ml peripheral venous blood (PVB) from 30 patients with MBC starting with a new line of chemotherapy. RESULTS: The number of CTC was found to be significantly higher in CVB (median: 43.5; range: 0-4036) than in PVB (median: 33; range: 0-4013) (P=0.001). When analysing samples pairwise, CTC counts were found to be significantly higher in CVB than in PVB in 12 out of 26 patients with detectable CTC. In contrast, only 2 out of 26 patients had higher CTC counts in PVB as compared with CVB, whereas in 12 remaining patients no significant difference was seen. The pattern of CTC distribution was independent of the sites of metastatic involvement. CONCLUSION: A substantial difference in the number of CTC was observed between CVB and PVB of patients with MBC. Registration of the site of blood collection is warranted in studies evaluating the role of CTC assessment in these patients.

Circulating tumour cell detection: a direct comparison between the CellSearch System, the AdnaTest and CK-19/mammaglobin RT-PCR in patients with metastatic breast cancer.

BACKGROUND: The detection, enumeration and isolation of circulating tumour cells (CTCs) have considerable potential to influence the clinical management of patients with breast cancer. There is, however, substantial variability in the rates of positive samples using existing detection techniques. The lack of standardisation of technology hampers the implementation of CTC measurement in clinical routine practice. METHODS: This study was designed to directly compare three techniques for detecting CTCs in blood samples taken from 76 patients with metastatic breast cancer (MBC) and from 20 healthy controls: the CellSearch CTC System, the AdnaTest Breast Cancer Select/Detect and a previously developed real-time qRT-PCR assay for the detection of CK-19 and mammaglobin transcripts. RESULTS: As a result, 36% of patients with MBC were positive by the CellSearch System, 22% by the AdnaTest, 26% using RT-PCR for CK-19 and 54% using RT-PCR for mammaglobin. Samples were significantly more likely to be positive for at least one mRNA marker using RT-PCR than using the CellSearch System (P=0.001) or the AdnaTest (P<0.001). CONCLUSION: We observed a substantial variation in the detection rates of CTCs in blood from breast cancer patients using three different techniques. A higher rate of positive samples was observed using a combined qRT-PCR approach for CK-19 and mammaglobin, which suggests that this is currently the most sensitive technique for detecting CTCs.

Comparison of MY09/11 consensus PCR and type-specific PCRs in the detection of oncogenic HPV types.

The causal relationship between persistent infection with high-risk HPV and cervical cancer has resulted in the development of HPV DNA detection systems. The widely used MY09/11 consensus PCR targets a 450bp conserved sequence in the HPV L1 gene, and can therefore amplify a broad spectrum of HPV types. However, limitations of these consensus primers are evident, particularly in regard to the variability in detection sensitivity among different HPV types. This study compared MY09/11 PCR with type-specific PCRs in the detection of oncogenic HPV types. The study population comprised 15, 774 patients. Consensus PCR failed to detect 522 (10.9%) HPV infections indicated by type-specific PCRs. A significant correlation between failure of consensus PCR and HPV type was found. HPV types 51, 68 and 45 were missed most frequently. The clinical relevance of the HPV infections missed by MY09/11 PCR was reflected in the fraction of cases with cytological abnormalities and in follow-up, showing 104 (25.4%) CIN2+ cases. The MY09/11 false negativity could be the result of poor sensitivity, mismatch of MY09/11 primers or disruption of L1 target by HPV integration or DNA degradation. Furthermore, MY09/11 PCR lacked specificity for oncogenic HPVs. Diagnostic accuracy of the PCR systems, in terms of sensitivity (MY09/11 PCR: 87.9%; type-specific PCRs: 98.3%) and specificity (MY09/11 PCR: 38.7%; type-specific PCRs: 76.14%), and predictive values for histologically confirmed CIN2+, suggest that type-specific PCRs could be used in a clinical setting as a reliable screening tool.

Improved endocervical sampling and HPV viral load detection by Cervex-Brush Combi.

OBJECTIVE: Liquid-based cytology (LBC) for cervical screening is becoming increasingly used. Together with SurePath LBC, various collecting devices can be utilized, among which the Cervex-Brush is the most widely used. The new Rovers Cervex-Brush Combi combines the advantages of the Cervex-Brush with the EndoCervex-Brush increasing sampling of the endocervical canal. The objective of this study was to analyse and to compare the Cervex-Brush Combi with the Cervex-Brush for the collection of squamous and endocervical cells, human papillomavirus (HPV) typing/quantification and disease detection in SurePath LBC. METHODS: Using either the Cervex-Brush or the Cervex-Brush Combi 100 consecutive SurePath LBC samples were collected using each brush type. All 200 slides were read by the FocalPoint and screened by guided screening using slide wizards. The viral load of HPV type 16 E7, 18 E7, 31 E6, 33 L1, 33 E6, 35 E4, 39 E7, 45 E7, 51 E6, 52 L1, 52 E7, 53 E6, 56 E7, 58 L1, 58 E6, 59 E7, 66 E6 and 68 E7 was determined using a TaqMan-based real-time quantitative PCR analysis. RESULTS: The mean number of sampled squamous cells did not differ between the two brush types (54 963 versus 54 595 cells). The use of the Cervex-Brush Combi, however, resulted in a two- to threefold increase in the number of sampled endocervical cells (P < 0.00001). Using the Cervex-Brush Combi slightly more lesions were detected (three versus two low-grade squamous intraepithelial lesions), and resulted in the detection of more atypical squamous cells of undetermined significance (six versus three). In the Cervex-Brush group, 60% (3/5) of abnormal smears were positive for oncogenic HPV types, whereas 66.7% (6/9) of abnormal smears in the Cervex-Brush Combi group tested positive. The median HPV viral load for samples taken with the Cervex-Brush Combi was 0.1825 copies/cell and was significantly higher than in samples taken with the Cervex-Brush (0.0042 copies/cell) (P = 0.02). CONCLUSION: Sampling with the Cervex-Brush Combi resulted in the collection of the same amount of squamous cells, but in a two to threefold harvest of endocervical cells. This led to the detection of a higher viral load for oncogenic HPV and an increase in the number of detected abnormal smears.

Real-time RT-PCR detection of disseminated tumour cells in bone marrow has superior prognostic significance in comparison with circulating tumour cells in patients with breast cancer.

This study assessed the ability of real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis to detect disseminated epithelial cells (DEC) in peripheral blood (PB) and bone marrow (BM) of patients with breast cancer (BC). Detection of DEC in BM is an obvious choice in BC, but blood sampling is more convenient. The aim of this study was to evaluate whether the detection of DEC in either PB or BM predicts overall survival (OS). Peripheral blood and BM samples were collected from 148 patients with primary (stage M0, n=116/78%) and metastatic (stage M+, n=32/21%) BC before the initiation of any local or systemic treatment. Peripheral blood of healthy volunteers and BM of patients with a nonmalignant breast lesion or a haematological malignancy served as the control group. Disseminated epithelial cells was detected by measuring relative gene expression (RGE) for cytokeratin-19 (CK-19) and mammaglobin (MAM), using a quantitative RT-PCR detection method. The mean follow-up time was 786 days (+/- 487). Kaplan-Meier analysis was used for predicting OS. By taking the 95 percentile of the RGE of CK-19 (BM: 26.3 and PB: 58.7) of the control group as cutoff, elevated CK-19 expression was detected in 42 (28%) BM samples and in 22 (15%) PB samples. Mammaglobin expression was elevated in 20% (both PB and BM) of the patients with BC. There was a 68% (CK-19) and 75% (MAM) concordance between PB and BM samples when classifying the results as either positive or negative. Patients with an elevated CK-19 or MAM expression in the BM had a worse prognosis than patients without elevated expression levels (OS: log-rank test, P=0.0045 (CK-19) and P=0.025 (MAM)). For PB survival analysis, no statistical significant difference was observed between patients with or without elevated CK-19 or MAM expression (OS: log-rank test, P=0.551 (CK-19) and P=0.329 (MAM)). Separate analyses of the M0 and M+ patients revealed a marked difference in OS according to the BM CK-19 or MAM status in the M+ patient group, but in the M0 group, only MAM expression was a prognostic marker for OS. Disseminated epithelial cells, measured as elevated CK-19 or MAM mRNA expression, could be detected in both PB and BM of patients with BC. Only the presence of DEC in BM was highly predictive for OS. The occurrence of DEC in the BM is probably less time-dependent and may act as a filter for circulating BC cells. The use of either larger volumes of PB or performing an enrichment step for circulating tumour in blood cells might improve these results.

Real-time RT-PCR correlates with immunocytochemistry for the detection of disseminated epithelial cells in bone marrow aspirates of patients with breast cancer.

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) is a technique with the potential of improving the quantification of disseminated epithelial cells (DEC) in haematological tissues due to its exquisite sensitivity. This sensitivity may lead to false positivity. Immunocytochemistry (ICC) is regarded as the standard methodology to diagnose DEC. In this study, detection with ICC was compared with quantitative real-time RT-PCR for CK-19 and mammaglobin (hMAM) mRNA in bone marrow (BM) of patients with metastatic breast cancer (MBC). Bone marrow was aspirated from 14 control patients and from 29 patients with MBC. Mononuclear cells (MNC) were isolated. Immunostaining was carried out with the Epimet kit. Quantitative PCR was performed on the ABI Prism 7700. The CK-19 and hMAM mRNA quantities were normalised against beta-Actin and calculated relative to a calibrator sample (relative gene expression). All controls were negative by ICC and for hMAM expression measured by RT-PCR, whereas the median RGE value for CK-19 was 0.57. For the MBC patients, the median RGE for hMAM was 0 and 10 out of 25 (40%) tested positive. Median RGE for CK-19 was 2.9 and 20 out of 25 (80%) tested positive. With ICC, the median value was 1 stained cell per sample, and 15 out of 24 (62%) samples were positive. A correlation was observed between CK-19 and hMAM expression (r=0.7; P=0.0003), and between hMAM expression and ICC (r=0.6; P=0.003). CK-19 expression and ICC (r=0.9; P<0.0001) showed the strongest correlation. Reverse transcriptase-polymerase chain reaction for CK-19 resulted in a higher number of positive BM samples of patients with MBC than ICC. Since an excellent correlation is observed between ICC and RT-PCR, and RT-PCR is probably more sensitive with the advantage of being less observer dependent and thus also more easy to automate, we consider our quantitative real-time RT-PCR method as validated for the detection of DEC in the bone marrow of breast cancer patients.