Single-tube multiplex PCR using type-specific E6/E7 primers and capillary electrophoresis genotypes 21 human papillomaviruses in neoplasia

<p>Abstract</p> <p>Background</p> <p>Human papillomavirus (HPV) <it>E6/E7 </it>type-specific oncogenes are required for cervical carcinogenesis. Current PCR protocols for genotyping high-risk HPV in cervical screening are not standardized and usually use consensus primers targeting HPV capsid genes, which are often deleted in neoplasia. PCR fragments are detected using specialized equipment and extra steps, including probe hybridization or primer extension. In published papers, analytical sensitivity is typically compared with a different protocol on the same sample set.</p> <p>A single-tube multiplex PCR containing type-specific primers was developed to target the <it>E6/E7 </it>genes of two low-risk and 19 high-risk genotypes (HPV6, 11 and 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73 and 82) and the resulting short fragments were directly genotyped by high-resolution fluorescence capillary electrophoresis.</p> <p>Results</p> <p>The method was validated using long oligonucleotide templates, plasmid clones and 207 clinical samples of DNA from liquid-based cytology, fresh and formalin-fixed specimens and FTA Microcards^®imprinted with cut tumor surfaces, swabbed cervical cancers or ejected aspirates from nodal metastases of head and neck carcinomas. Between one and five long oligonucleotide targets per sample were detected without false calls. Each of the 21 genotypes was detected in the clinical sample set with up to five types simultaneously detected in individual specimens. All 101 significant cervical neoplasias (CIN 2 and above), except one adenocarcinoma, contained <it>E6/E7 </it>genes. The resulting genotype distribution accorded with the national pattern with HPV16 and 18 accounting for 69% of tumors. Rare HPV types 70 and 73 were present as the sole genotype in one carcinoma each. One cervical SCC contained DNA from HPV6 and 11 only. Six of twelve oropharyngeal cancer metastases and three neck metastases of unknown origin bore <it>E6/E7 </it>DNA; all but one were HPV16. One neck aspirate contained atypical squames with HPV26.</p> <p>Analytical sensitivity in dilute plasmid mixes was variable.</p> <p>Conclusions</p> <p>A primer-rich PCR readily detects the <it>E6/E7 </it>oncogenes of 21 HPV types in cellular and fixed tissue specimens. The method is straightforward, robust and reproducible and avoids post-PCR enzymatic and hybridization steps while detecting HPV with high clinical sensitivity in significant HPV-related neoplasia regardless of specimen type.</p

Ascitic complement system in ovarian cancer

Ovarian cancer spreads intraperitoneally and forms fluid, whereby the diagnosis and therapy often become delayed. As the complement (C) system may provide a cytotoxic effector arm for both immunological surveillance and mAb-therapy, we have characterised the C system in the intraperitoneal ascitic fluid (AF) from ovarian cancer patients. Most of the AF samples showed alternative and classical pathway haemolytic activity. The levels of C3 and C4 were similar to or in the lower normal range when compared to values in normal sera, respectively. However, elevated levels of C3a and soluble C5b-9 suggested C activation in vivo. Malignant cells isolated from the AF samples had surface deposits of C1q and C3 activation products, but not of C5b-9 (the membrane attack complex; MAC). Activation could have become initiated by anti-tumour cell antibodies that were detected in the AFs and/or by changes on tumour cell surfaces. The lack of MAC was probably due to the expression of C membrane regulators CD46, CD55 and CD59 on the tumour cells. Soluble forms of C1 inhibitor, CD59 and CD46, and the alternative pathway inhibitors factor H and FHL-1 were present in the AF at concentrations higher than in serum samples. Despite the presence of soluble C inhibitors it was possible to use AF as a C source in antibody-initiated killing of ovarian carcinoma cells. These results demonstrate that although the ovarian ascitic C system fails as an effective immunological surveillance mechanism, it could be utilised as an effector mechanism in therapy with intraperitoneally administrated mAbs, especially if the intrinsic C regulators are neutralised

OT1-02-12: Early Detection of Cardiotoxicity by Advanced Cardiac Imaging and a Novel Biomarker in Breast Cancer Patients Undergoing Chemotherapy.

Abstract Background The survival rate of breast cancer patients has increased due to improvements in cancer treatment. However, many survivors develop irreversible or reversible cardiotoxicity associated with anthracycline or trastuzumab therapy, respectively. To detect cardiac damage, the currently accepted method is to measure left ventricular ejection fraction (LVEF) by echocardiography, which lacks the sensitivity to predict early cardiac dysfunction. Early identification of cardiotoxicity is essential to cancer survivors, as development of cardiomyopathy carries a worse outcome independent of cancer prognosis. Currently, there are no accepted guidelines for the early detection of myocardial injury. The use of cardiac biomarkers and more sensitive echocardiographic techniques have expanded options for monitoring, but have yet to reach a consensus. Hence, our study will evaluate the potential predictive value of novel cardiac biomarkers and advanced echocardiographic and cardiac magnetic resonance imaging (CMR) techniques to detect subclinical myocardial damage. Our findings may be applicable for monitoring new antineoplastic agents during food and drug administration (FDA) clinical trials. Trial Design Prospective cohort study with internal control of 20 patients newly diagnosed with breast cancer. The trial will assess endpoints at baseline, 2 weeks after initiation of therapy, and 2 weeks and 6 months after chemotherapy completion. 1. Primary Endpoint a. Decline in left ventricular ejection fraction assessed by CMR and 3D-echo not detected by conventional methods b. Presence of either myocardial fibrosis or edema detected by CMR c. Changes in myocardial deformation detected by echo or CMR strain d. Increase in cardiac biomarkers (Serum caspase-3 p17 peptide, Troponin I, B-type natriuretic peptide) and possible correlation with imaging parameters 2. Secondary Endpoint a. Development New York Heart Association class 1 to 4 symptoms b. Decrease in LVEF of ≥5% to ≤50% with or without symptoms Eligibility Criteria Inclusion Criteria: 1. Newly diagnosed stage I, II, or III breast cancer 2. Age between 18 and 75 years old. 3. Treatment with trastuzumab or anthracycline-based chemotherapy Exclusion Criteria: 1. History of cardiovascular disease 2. Pacemaker 3. History of mediastinal radiotherapy 4. Creatinine clearance &amp;lt;30 ml/min 5. Serum bilirubin &amp;gt;2.0 mg/dl, ALT and AST &amp;gt; 100 U/1) 6. Hypertension, uncontrolled &amp;gt;140/90 7. LVEF &amp;lt;55% per 2-D echocardiogram 8. Claustrophobia Specific Aims 1. Detect early myocardial injury. 2. Evaluate early predictors of left ventricular dysfunction. 3. Evaluate timing of monitoring during or post treatment Statistical Method This is a pilot study and 20 patients are required to reach statistical significance with 85% power. All values will be analyzed as mean±SD or n (%). Categorical indicators will be analyzed using nonparametric statistics such as Cochran's Q. Changes in imaging and biomarker parameters will be assessed using analysis of variance, while correlation between the two will be assessed using mixed models appropriate for binary outcome. Significance will be accepted at p ≤0.05 for all tests. Present accrual and target accrual Nine subjects are enrolled with a goal of 20. Contact for people interested in trial: 1. Dr. Erick Avelar, [email protected] 2. Dr. Susan Tannenbaum, [email protected] Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr OT1-02-12.</jats:p