Changes in type-specific human papillomavirus load predict progression to cervical cancer.

Persistent high-risk human papillomavirus (HPV) infection is strongly associated with the development of high-grade cervical intraepithelial neoplasia or cancer (CIN3+). However, HPV infection is common and usually transient. Viral load measured at a single time-point is a poor predictor of the natural history of HPV infection. The profile of viral load evolution over time could distinguish HPV infections with carcinogenic potential from infections that regress. A case-cohort natural history study was set-up using a Belgian laboratory database processing more than 100,000 liquid cytology specimens annually. All cytology leftovers were submitted to real-time PCR testing identifying E6/E7 genes of 17 HPV types, with viral load expressed as HPV copies/cell. Samples from untreated women who developed CIN3+ (n = 138) and women with transient HPV infection (n = 601) who contributed at least three viral load measurements were studied. Only single-type HPV infections were selected. The changes in viral load over time were assessed by the linear regression slope for the productive and/or clearing phase of infection in women developing CIN3+ and women with transient infection respectively. Transient HPV infections generated similar increasing (0.21 copies/cell/day) and decreasing (-0.28 copies/cell/day) viral load slopes. In HPV infections leading to CIN3+, the viral load increased almost linearly with a slope of 0.0028 copies/cell/day. Difference in slopes between transient infections and infections leading to CIN3+ was highly significant (P < .0001). Serial type-specific viral load measurements predict the natural history of HPV infections and could be used to triage women in HPV-based cervical cancer screening.

Prior knowledge of HPV status improves detection of CIN2+ by cytology screening.

OBJECTIVE: The objective of the study was to investigate whether knowledge of human papillomavirus (HPV) deoxyribonucleic acid test results increases sensitivity of guided cytology screening for the detection of cervical intraepithelial neoplasia (CIN)-2 or higher-grade cervical lesions. STUDY DESIGN: This was a prospective colposcopy-controlled study of 2905 BD SurePath samples to identify cases with CIN2+ within a 24 month follow-up period. Sensitivity and specificity to detect CIN2+ was evaluated, comparing guided cytology screening with and without prior knowledge of HPV status. RESULTS: Prior knowledge of HPV status resulted in significantly higher detection rate of CIN2+ compared with screening blinded to HPV status (P = .005) with limited loss of specificity (P = .026). Gain in sensitivity is higher in older women (43.8%, P = .008) vs in younger women (10.2%, P = .317), whereas loss of specificity is more pronounced in younger women (P < .001) vs older women (P = .729). CONCLUSION: Guided cytological screening performed with prior knowledge of HPV status results in an improved detection of CIN2 or higher-grade lesions.

BD-ProExC as adjunct molecular marker for improved detection of CIN2+ after HPV primary screening.

BACKGROUND AND METHODS: We investigated the efficacy of 8 cervical cancer screening strategies relative to cytology with emphasis on immunocytochemical detection of high-risk human papillomavirus (hrHPV)-induced cell transformation (BD-ProExC) as a tool of triage following primary cytology or hrHPV testing. 3,126 women were tested with BD-SurePath liquid-based cytology, hrHPV PCR genotyping and BD-ProExC immunostaining, and colposcopy verification to calculate sensitivity and positive predictive value (PPV) in detecting cervical intraepithelial neoplasia (CIN2(+)). RESULTS: Compared to cytology screening, double testing with cytology and hrHPV resulted in the same sensitivity with a significant increase in the PPV (relative PPV: 1.83). However, twice as many tests were needed. Cytology with atypical squamous cells of undetermined significance (ASC-US) triage and hrHPV testing showed comparative results to double testing requiring only a small increase in number of tests. Screening for hrHPV subtypes 16/18, and ASC-US triage with hrHPV16/18 resulted in significant reductions in sensitivity (ratio: 0.74 and 0.96, respectively). Primary hrHPV/BD-ProExC screening was significantly more sensitive (ratio: 1.63/1.33), but had a significantly lower PPV (ratio: 0.64/0.88). ASC-US triage by BD-ProExC increased the PPV (ratio: 1.90) but decreased the sensitivity (ratio: 0.96). Primary hrHPV screening followed by BD-ProExC triage, led to significant increases in sensitivity (ratio: 1.30) and PPV (ratio: 2.89), and resulted in 55% fewer referrals for colposcopy. CONCLUSIONS: From the investigated screening strategies, primary hrHPV DNA-based screening followed by BD-ProExC triage was determined to be the best screening strategy. IMPACT: Immunocytological triage could be used to perfect hrHPV primary screening.

High-throughput detection, genotyping and quantification of the human papillomavirus using real-time PCR.

BACKGROUND: The establishment of the causal relationship between high-risk human papillomavirus (HR-HPV) infection and cervical cancer and its precursors has resulted in the development of HPV DNA detection systems. Currently, real-time PCR assays for the detection of HPV, such as the RealTime High Risk (HR) HPV assay (Abbott) and the cobas® 4800 HPV Test (Roche Molecular Diagnostics) are commercially available. However, none of them enables the detection and typing of all HR-HPV types in a clinical high-throughput setting. This paper describes the laboratory workflow and the validation of a type-specific real-time quantitative PCR (qPCR) assay for high-throughput HPV detection, genotyping and quantification. This assay is routinely applied in a liquid-based cytology screening setting (700 samples in 24 h) and was used in many epidemiological and clinical studies. METHODS: The TaqMan-based qPCR assay enables the detection of 17 HPV genotypes and ß-globin in seven multiplex reactions. These HPV types include all 12 high-risk types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59), three probably high-risk types (HPV53, 66 and 68), one low-risk type (HPV6) and one undetermined risk type (HPV67). RESULTS: An analytical sensitivity of ≤100 copies was obtained for all the HPV types. The analytical specificity of each primer pair was 100% and an intra- and inter-run variability of <6.4% was observed. CONCLUSIONS: The type-specific real-time PCR approach enables detection of 17 HPV types, identification of the HPV type and determination of the viral load in a single sensitive assay suitable for high-throughput screening.

Type-specific HPV geno-typing improves detection of recurrent high-grade cervical neoplasia after conisation.

The aim of this case-control study was to examine if type-specific human papillomavirus (HPV) DNA geno-typing before and after treatment of high-grade cervical intra-epithelial neoplasia (CIN) improves prediction of recurring or persisting CIN 2 or 3 compared with follow-up cytology or high-risk (hr)HPV testing. Women with biopsy-proven recurrence of CIN 2 or 3 (cases) in a follow-up period of at least 24 months after treatment of high-grade CIN were compared with women without recurrence (controls). These cohorts were identified by a database search of the Riatol Laboratoria (Antwerp, Belgium). In a cohort of 823 women treated with conisation for high-grade CIN between January 2001 and December 2007, 21 patients with a histologically proven recurrence of CIN2+ were identified. A group of women (n=42) from the same cohort without recurrence was randomly chosen. We found that hrHPV testing at 6 months post-treatment is significantly more sensitive compared with follow-up cytology (ratio: 1.31, 95% confidence interval (CI): 1.10-1.54), but less specific (ratio: 0.85, 95% CI: 0.81-0.90) to predict failure of treatment. When compared with hrHPV testing, HPV geno-typing is more efficient (equal sensitivity, but higher specificity, ratio: 1.43, 95% CI: 1.280-1.62). When compared with follow-up cytology, HPV geno-typing is more sensitive (ratio: 1.31, 95% CI: 1.10-1.54) and more specific (ratio: 1.22, 95% CI: 1.14-1.36). All women who developed a recurrence tested positive for hrHPV. The negative predictive value in the absence of hrHPV DNA was 100%. Six months after treatment HPV geno-typing is the most sensitive and specific method to predict recurrent or persistent CIN 2-3 in the next 24 months.

Nucleic acid sequence-based amplification assay for human papillomavirus mRNA detection and typing: evidence for DNA amplification.

Human papillomavirus (HPV) E6/E7 mRNA has been proposed as a more specific marker for cervical dysplasia and cancer than HPV DNA. This study evaluated the RNA specificity of nucleic acid sequence-based amplification (NASBA)-based HPV detection using HPV DNA plasmids (HPV type 16 [HPV16], HPV18, HPV31, HPV33, and HPV45) and nucleic acid extracts of several cell lines, which were systematically subjected to enzymatic treatments with DNase and RNase. HPV plasmid dilutions (10(6) to 10(0) copies/microl) and nucleic acid extracts (total DNA, RNA-free DNA, total RNA, and DNA-free RNA) of unfixed and fixed (PreServCyt and SurePath) HaCaT, HeLa, and CaSki cells were tested with the NucliSENS EasyQ HPV test. The RNA-free DNA extracts of HeLa and CaSki cells could be amplified by HPV18 and -16 NASBA, respectively. Fixation of the cells did not influence NASBA. All HPV plasmids could be detected with NASBA. Based on the plasmid dilution series, a lower detection limit of 5 x 10(3) HPV DNA copies could be determined. Our study identified viral double-stranded DNA as a possible target for NASBA-based HPV detection. The differences in diagnostic accuracy between the NASBA-based tests and conventional HPV DNA detection assays seem to be attributable not to the more specific amplification of viral mRNA but to the limited type range and the lower analytical sensitivity for HPV DNA.

Human papillomavirus 16 load and E2/E6 ratio in HPV16-positive women: biomarkers for cervical intraepithelial neoplasia >or=2 in a liquid-based cytology setting?

This retrospective case-control study assessed human papillomavirus 16 (HPV16) viral load and E2/E6 ratio as risk markers for cervical intraepithelial neoplasia (CIN) >or=2 lesions in HPV16-positive women in a routine liquid-based cytology setting. Triplex quantitative PCR for HPV16 E6, E2, and beta-globin was done to determine the HPV16 load and the E2/E6 ratio, as a surrogate marker for integration, for women with a negative histologic endpoint (200 controls: 83 normal histology and 117 CIN1) and women with a >or=CIN2 endpoint (180 cases: 41 CIN2, 122 CIN3, and 17 invasive carcinoma). Our analysis showed a significantly higher HPV16 load in the case group, which was completely attributable to the high viral load of samples with invasive carcinoma as histologic endpoint. There was no significant difference in viral load between the other histologic groups. The E2/E6 ratio proved to be lower for the cases. However, the E2/E6 ratio indicated the presence of HPV integration in a considerable amount of control samples (44.3%), which suggests that HPV integration occurs early in the development of cancer and undermines the clinical value of viral integration. Overall, the intrinsic heterogeneous nature of the cervical cytology samples caused a substantial overlap of the HPV16 load and the E2/E6 ratio between controls and cases, which precludes the determination of cutoff values for risk prediction and hampers the clinical applicability in a cervical screening setting.

Quality control for normal liquid-based cytology: rescreening, high-risk HPV targeted reviewing and/or high-risk HPV detection?

The objective of this prospective study was to compare the number of CIN2+cases detected in negative cytology by different quality control (QC) methods. Full rescreening, high-risk (HR) human papillomavirus (HPV)-targeted reviewing and HR HPV detection were compared. Randomly selected negative cytology detected by BD FocalPoint (NFR), by guided screening of the prescreened which needed further review (GS) and by manual screening (MS) was used. A 3-year follow-up period was available. Full rescreening of cytology only detected 23.5% of CIN2+ cases, whereas the cytological rescreening of oncogenic positive slides (high-risk HPV-targeted reviewing) detected 7 of 17 CIN2+ cases (41.2%). Quantitative real-time PCR for 15 oncogenic HPV types detected all CIN2+ cases. Relative sensitivity to detect histological CIN2+ was 0.24 for full rescreening, 0.41 for HR-targeted reviewing and 1.00 for HR HPV detection. In more than half of the reviewed negative cytological preparations associated with histological CIN2+cases no morphologically abnormal cells were detected despite a positive HPV test. The visual cut-off for the detection of abnormal cytology was established at 6.5 HR HPV copies/cell. High-risk HPV detection has a higher yield for detection of CIN2+ cases as compared to manual screening followed by 5% full review, or compared to targeted reviewing of smears positive for oncogenic HPV types, and show diagnostic properties that support its use as a QC procedure in cytologic laboratories.

Detection of circulating tumour cells in blood by quantitative real-time RT-PCR: effect of pre-analytical time.

BACKGROUND: We and others have recently explored the use of quantitative real-time RT-PCR analysis for the detection of circulating tumour cells in blood of patients with breast cancer (BC). One major problem in these experiments is the in vitro instability of the cellular RNA. The copy number of mRNA can change during storage and transport at room temperature and this may hamper accurate quantitative measurements of specific transcripts, especially when working with small numbers of target mRNAs. METHODS: Peripheral blood samples were obtained from two healthy volunteers and 13 patients with BC. Blood was stored at room temperature for 0, 1, 2, 4, 6, 24, 48 and 72 h. The potential alteration of gene expression for six target genes was investigated by quantitative real-time RT-PCR. RESULTS: For beta-actin, GAPDH, cytokeratin-19 (CK-19) and HER2, a significant decrease in expression level occurs after 4 h (CK-19 and HER2), 6 h (beta-actin) or 24 h (GAPDH). Mammaglobin expression was only measurable in two samples and seems to be stable for at least 6 h. For vascular endothelial growth factor (VEGF), a statistically significant increase in expression level is observed in samples processed 24 h after collection. CONCLUSIONS: Most transcripts were reduced in samples that were stored overnight at room temperature compared with fresh samples, but upregulation of transcripts, probably as an active response to cellular stress, also occurs when blood is removed from its in vivo environment and stored ex vivo. Optimally, blood samples and RNA should be processed or stabilised within 3 h after collection to avoid interference of the in vivo gene expression signature by ex vivo stress responses.

Prognostic significance of disseminated tumor cells as detected by quantitative real-time reverse-transcriptase polymerase chain reaction in patients with breast cancer.

BACKGROUND: In this study we have validated the feasibility of detecting disseminated tumor cells (DTC) by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) analysis. Bone marrow samples from a large cohort of patients with breast cancer were analyzed for the presence of DTC by immunocytochemistry (ICC) or a molecular-based method. PATIENTS AND METHODS: Bone marrow samples were collected from 170 patients with breast cancer with stage I-IV disease before the initiation of any local or systemic treatment. Staining for cytokeratin (CK)-positive cells was performed with the Epimet kit. Disseminated tumor cells were also quantified by measuring relative gene expression for CK19 and mammaglobin (MAM) using a quantitative RT-PCR detection method. The mean follow-up time was 30 months. Kaplan-Meier analysis was used for predicting overall survival. RESULTS: Despite an excellent quantitative correlation and qualitative concordance between ICC and RT-PCR, survival analysis suggested an improved prognostic significance of DTC as detected by quantitative RT-PCR. Univariate survival analysis computed a relative risk of death of 2.87 for women with ICC-positive cells in the bone marrow, as compared with those without positive cells. The relative risk for women with RT-PCR-positive bone marrow was even higher: 3.5 (CK19) and 3.39 (MAM). In multivariate analysis, bone marrow CK19 was a stronger prognostic factor than bone marrow ICC. CONCLUSION: Reverse-transcriptase polymerase chain reaction-detected DTC is shown to be prognostically significant in untreated patients with breast cancer. Furthermore, it seems to be a more sensitive method for detecting DTC in bone marrow samples when compared with ICC.

Relative microvessel area of the primary tumour, and not lymph node status, predicts the presence of bone marrow micrometastases detected by reverse transcriptase polymerase chain reaction in patients with clinically non-metastatic breast cancer.

About 50% of patients with breast cancer have no involvement of axillary lymph nodes at diagnosis and can be considered cured after primary locoregional treatment. However, about 20-30% will experience distant relapse. The group of patients at risk is not well characterised: recurrence is probably due to the establishment of micrometastases before treatment. Given the early steps of metastasis in which tumour cells interact with endothelial cells of blood vessels, and, given the independent prognostic value in breast cancer of both the quantification of tumour vascularisation and the detection of micrometastases in the bone marrow, the aim of this study was to determine the relationship between vascularisation, measured by Chalkley morphometry, and the bone marrow content of cytokeratin-19 (CK-19) mRNA, quantified by real-time reverse transcriptase polymerase chain reaction, in a series of 68 patients with localised untreated breast cancer. The blood concentration of factors involved in angiogenesis (interleukin-6 and vascular endothelial growth factor) and of factors involved in coagulation (D-dimer, fibrinogen, platelets) was also measured. When bone marrow CK-19 relative gene expression (RGE) was categorised according to the cut-off value of 0.77 (95th centile of control patients), 53% of the patients had an elevated CK-19 RGE. Patients with bone marrow micrometastases, on the basis of an elevated CK-19 RGE, had a mean Chalkley count of 7.5 +/- 1.7 (median 7, standard error [SE] 0.30) compared with a mean Chalkley count of 6.5 +/- 1.7 in other patients (median 6, SE 0.3) (Mann-Whitney U-test; P = 0.04). Multiple regression analysis revealed that Chalkley count, not lymph node status, independently predicted CK-19 RGE status (P = 0.04; odds ratio 1.38; 95% confidence interval 1.009-1.882). Blood parameters reflecting angiogenesis and coagulation were positively correlated with Chalkley count and/or CK-19 RGE. Our data are in support of an association between elevated relative microvessel area of the primary tumour and the presence of bone marrow micrometastases in breast cancer patients with operable disease, and corroborate the paracrine and endocrine role of interleukin-6 and the involvement of coagulation in breast cancer growth and metastasis.

Increased serum interleukin-8 in patients with early and metastatic breast cancer correlates with early dissemination and survival.

PURPOSE: The prognostic significance of serum interleukin (IL)-8 was evaluated in patients with metastatic breast cancer. The predictive value of serum IL-8 for the presence of occult metastatic tumor cells in bone marrow aspirates was evaluated in patients with operable and metastatic breast cancer. EXPERIMENTAL DESIGN: Serum IL-8 was measured in healthy controls, patients with operable breast cancer, and patients with untreated, progressive metastatic breast cancer. In 69 patients with either operable or advanced breast cancer, occult cytokeratin-positive cells were counted in bone marrow aspirates. RESULTS: Serum IL-8 levels are increased in 67% (52 of 77) of patients with advanced breast cancer. Overall, these levels are significantly higher in patients with breast cancer compared with healthy volunteers (P < 0.001). The IL-8 levels increase significantly in patients with more advanced disease. An elevated serum IL-8 is related to an accelerated clinical course, a higher tumor load, and the presence of liver or lymph node involvement. A multivariate analysis indicates that serum IL-8 is an independent significant factor for postrelapse survival. There was a significant difference between serum IL-8 levels in patients with or without occult cytokeratin-positive bone marrow cells (P < 0.04). Serum IL-8 levels also showed an association with the number of these cells (P < 0.01). CONCLUSIONS: Serum IL-8 is increased in patients with breast cancer and has an independent prognostic significance for postrelapse survival. The observations on the relationship between occult cytokeratin-positive bone marrow cells corroborate the concept of IL-8 acting as a contributor to the process of tumor cell dissemination. Similarly, the relationship between serum IL-8 and nodal stage at presentation deserves further study. These results further expand the concept that inflammation and inflammatory cytokines are critical components of tumor progression.