Systematic review of the registered clinical trials for oncological hyperthermia treatment.

BACKGROUND: The use of heat to treat various diseases is called hyperthermia treatment (HT). Since the 1970s, the anti-cancer effects of HT have been investigated. Different HT techniques can be categorized as local, regional and whole-body hyperthermia treatment (WBHT). We aim to provide a summary of recent research done on HT to treat cancer. METHODS: In July 2020 ClinicalTrials.gov were systematically searched for all trials including hyperthermia and cancer registered between 2000 and 2020. Studies were excluded when they did not concern hyperthermal treatment, when they were not oncological studies, when they were observational or other non-interventional studies. RESULTS: Of 1654 identified trials, 235 were included. Of these 235 studies, 123 described the use of HIPEC (52.3%), 44 other types of regional HT (18.7%), 45 local HT (19.1%) and 15 WBHT (6.4%). A steady increase (720%) in research to hyperthermic intraperitoneal chemotherapy (HIPEC) can be observed in the last decade. Although HIPEC is the most researched HT modality, an evolution in other HT technologies could be observed during the past decade. CONCLUSIONS: Research to HT to treat cancer has expanded fast. Some techniques, for example HIPEC start to be used outside of research context, but overall, more research is needed to establish a clear effect of these HT techniques.

Tolerability of long-term temperature controlled whole-body thermal treatment in advanced cancer-bearing dogs.

Aim: In oncology, thermal therapy is the application of external heat to fight cancer cells. The goal of whole-body thermal treatment (WBTT) is to raise the patient's core temperature to 39-42 °C, and represents the only thermal treatment modality that can act on both the primary tumor and distant metastases. However, WBTT carries potential risks for toxicity when applied without accurate thermometry and monitoring.Methods: ElmediX has developed a medical device, HyperTherm, to deliver long-term controlled and accurate WBTT (41.5 °C, up to 8 h). The safety of the device and thermal treatment protocol was initially evaluated in minipigs, and we present the confirmation of tolerability of WBTT in dogs with advanced cancer, in combination with a reduced dose of radiotherapy or chemotherapy.Results: Thermometry in liver, rectum, and tumor confirmed a homogeneous heating of these body parts. Monitoring of clinical parameters showed acceptable and reversible changes in liver, cardiac, muscle and coagulation parameters, as was expected. Combination of WBTT with both radiotherapy and chemotherapy only caused some low-grade adverse events.Conclusion: We conclude that our findings support the safe use of HyperTherm-mediated WBTT for canine patients with advanced malignancies. They also tend to support a genuine therapeutic potential for long-term WBTT which needs to be confirmed on a larger dog patient population. Combined with previously reported safety results in minipigs, these contribute to support the ongoing clinical evaluation of WBTT in advanced human cancer patients.

Patterns of recurrence following definitive chemoradiation for patients with proximal esophageal cancer.

INTRODUCTION: The aim of this retrospective study was to determine the patterns of recurrence and overall survival (OS) in patients achieving clinical complete response after treatment with definitive chemoradiation (CRT) for proximal esophageal cancer. MATERIALS AND METHODS: Patients with proximal esophageal cancer treated with CRT between 2004 and 2014 in 11 centers in the Netherlands were included. OS and progression-free survival (PFS) were calculated using the Kaplan-Meier method. Cumulative incidence of first recurrence (locoregional or distant) and locoregional recurrence (LRR) were assessed using competing risk analyses. RESULTS: In 197 of the 200 identified patients, response was evaluated, 133 (68%) showed a complete response. In complete responders, median OS, three-year OS, and PFS were 45.0 months (95% CI 34.8-61.5 months), 58% (95% CI 48-66), and 49% (95% CI 40-57), respectively. Three- and five-year risk of recurrence were respectively 40% (95% CI 31-48), and 45% (95% CI 36-54). Three- and five-year risk of LRR were 26% (95% CI 19-33), and 30% (95% CI 22-38). Eight of 32 patients with an isolated LRR underwent salvage surgery, with a median OS of 32.0 months (95% CI 6.8-not reached). CONCLUSION: In patients with a complete response after definitive CRT for proximal esophageal cancer, most recurrences were locoregional and developed within the first three years after CRT. These findings suggest to shorten locoregional follow-up from five to three years.

A national study to assess outcomes of definitive chemoradiation regimens in proximal esophageal cancer.

Background: Proximal esophageal cancer (EC) is commonly treated with definitive chemoradiation (CRT). The radiation dose and type of chemotherapy backbone are still under debate. The objective of this study was to compare the treatment outcomes of contemporary CRT regimens.Material and Methods: In this retrospective observational cohort study, we included patients with locally advanced squamous cell cancer of the proximal esophagus, from 11 centers in the Netherlands, treated with definitive CRT between 2004 and 2014. Each center had a preferential CRT regimen, based on cisplatin (Cis) or carboplatin-paclitaxel (CP) combined with low (≤50.4 Gy) or high (>50.4 Gy) dose radiotherapy (RT). Differences in overall survival (OS) between CRT regimens were assessed using a fully adjusted Cox proportional hazards and propensity score (PS) weighted model. Safety profiles were compared using a multilevel logistic regression model.Results: Two hundred patients were included. Fifty-four, 39, 95, and 12 patients were treated with Cis-low-dose RT, Cis-high-dose RT, CP-low-dose RT, and CP-high-dose RT, respectively. Median follow-up was 62.6 months (95% CI: 47.9-77.2 months). Median OS (21.9 months; 95% CI: 16.9-27.0 months) was comparable between treatment groups (logrank p = .88), confirmed in the fully adjusted and PS weighted model (p > .05). Grades 3-5 acute adverse events were less frequent in patients treated with CP-low-dose RT versus Cis-high-dose RT (OR 3.78; 95% CI: 1.31-10.87; p = .01). The occurrence of grades 3-5 late toxicities was not different between treatment groups.Conclusion: Our study was unable to demonstrate a difference in OS between the CRT regimens, probably related to the relatively small sample size. Based on the superior safety profile, carboplatin and paclitaxel-based CRT regimens are preferred in patients with locally advanced proximal EC.

Loop Electrosurgical Excision Procedure Treats Cervical Intraepithelial Neoplasia 2/3 Among HIV- and HIV+ Women in Kenya.

OBJECTIVE: The aim of the study was to compare loop electrosurgical excision procedure (LEEP) as treatment for cervical intraepithelial neoplasia (CIN) 2/3 in HIV- versus HIV+ women. MATERIALS AND METHODS: Seventy-five HIV- and 75 HIV+ women at 6 months or more after LEEP for CIN 2/3 were enrolled between September 2013 and November 2014 in this prospective cohort study at the cervical cancer screening clinic in Eldoret, Kenya. Visual inspection with acetic acid (VIA), followed by cervical cytology with conventional cytology, was performed on all women. Women with positive VIA or abnormal cervical cytology underwent colposcopy/biopsy. Lesion progression, persistence, and regression were assessed to quantify the efficacy of LEEP. RESULTS: Post-loop electrosurgical excision procedure screening test showed both a negative VIA and normal cervical cytology in 64 (85%) of HIV- and 57 (77%) HIV+ women (risk difference = 8.3%, CI = -4.2% to 21%, p = .20). Eleven (15%) HIV- and 17 (23%) HIV+ (p = .20) women had positive VIA, abnormal cervical cytology, or both and were referred for colposcopy/biopsy. Twenty-one (8 HIV-, 13 HIV+) women were biopsied. Of the 8 HIV- women, 4 (50%) had CIN lesions that regressed, 3 (38.0%) persisted, and 1 (12%) progressed to invasive cancer after LEEP. Of the 13 HIV+ women, 6 (46%) had CIN lesions that regressed, 7 (54%) had CIN lesions that persisted, and no HIV+ women had CIN lesions that progressed after LEEP. There was no difference in estimated efficacies of LEEP for HIV- and HIV+ women (92.7% versus 89.4%, risk difference = 3.3%, CI = -4.8% to 15.3%, p = .85). CONCLUSIONS: Loop electrosurgical excision procedure for CIN 2/3 is effective treatment for HIV- and HIV+ women in low-resource settings. Future efforts should improve follow-up after treatment.

Using the VALGENT-3 framework to assess the clinical and analytical performance of the RIATOL qPCR HPV genotyping assay.

BACKGROUND AND OBJECTIVE: The VALGENT framework is developed to assess the clinical performance of HPV tests that offer genotyping capability. Samples from the VALGENT-3 panel are used to identify an optimal viral concentration threshold for the RIATOL qPCR HPV genotyping assay (RIATOL qPCR) to assure non-inferior accuracy to detect high-grade cervical intraepithelial neoplasia (CIN), compared to Qiagen Hybrid Capture 2 (HC2), a standard comparator test validated for cervical cancer screening. STUDY DESIGN: The VALGENT-3 panel comprised 1300 samples from women participating in the Slovenian cervical cancer screening programme, enriched with 300 samples from women with abnormal cytology. In follow- up, 126 women were diagnosed with CIN2+ (defined as diseased) and 1167 women had two consecutive negative Pap smears (defined as non-diseased). All 1600 samples were analyzed with the RIATOL qPCR. Viral concentration was expressed as viral log10 of the number of copies/ml. A zone of viral concentration cut-offs was defined by relative ROC analysis where the sensitivity and specificity were not inferior to HC2. RESULTS: The RIATOL qPCR had a sensitivity and specificity for CIN2+ of 97.6% (CI: 93.2-99.5%) and 85.1% (CI: 82.9-87.1%), respectively, when the analytical cut off was used. At a cut off of 6.5, RIATOL qPCR had a sensitivity of 96.0% (CI: 91.0-98.7%) and a specificity of 89.5% (87.6-91.2%). At optimized cut off, accuracy of the qPCR was non-inferior to the HC2 with a relative sensitivity of 1.00 [CI: 0.95-1.05 (p = 0.006)] and relative specificity of 1.00 [CI: 0.98-1.01 (p = 0.0069)]. CONCLUSIONS: The RIATOL qPCR has a high sensitivity and specificity for the detection of CIN2 + . By using a fixed cut-off based on viral concentration, the test is non-inferior to HC2. HPV tests that provide viral concentration measurements or other quantifiable signals allow flexibility to optimize accuracy required for cervical cancer screening.

VALHUDES: A protocol for validation of human papillomavirus assays and collection devices for HPV testing on self-samples and urine samples.

BACK GROUND: Systematic reviews have concluded that hrHPV DNA testing using target-amplification tests is as accurate on vaginal self-samples as on clinician-taken specimens for the detection of cervical precancer. However, insufficient evidence is available for specific HPV assay/self-sample device combinations. OBJECTIVES: The VALHUDES protocol is designed as a diagnostic test accuracy study that aims to compare the clinical sensitivity and specificity of particular hrHPV assay(s) on vaginal self-samples and first-void-urine, collected in agreement with standardized protocols, with hrHPV testing on matched clinician-taken samples. STUDY DESIGN: Five hundred enrolled women referred to a colposcopy clinic are invited to collect a first-void urine sample and one or more vaginal self-samples with particular devices before collection of a cervical sample by a clinician. Sample sets are subsequently analysed in a laboratory accredited for HPV testing. Disease verification for all enrolled patients is provided by colposcopy combined with histological assessment of biopsies. RESULTS: A first VALHUDES study has started in Belgium in December 2017 with enrolment from four colposcopy centres. The following assays are foreseen to be evaluated: RealTime High Risk HPV assay (Abbott), cobas-4800 and -6800 (Roche), Onclarity (BD), Xpert HPV (Cepheid) and Anyplex II HPV HR (Seegene). CONCLUSION: Given empirical evidence that the relative accuracy of HPV-testing on self- vs clinician-samples is robust across clinical settings, the VALHUDES protocol offers a framework for validation of HPV assay/self-sample device combinations that can be translated to a primary screening setting.

Transvaginal laparoscopy: A minimally invasive approach to obtain brush cytology of the Fallopian tube.

OBJECTIVE: To evaluate in a prospective pilot study the feasibility of cytobrushing of the fimbrial end using a transvaginal endoscopic access. STUDY DESIGN: Prospective feasibility study. The procedure was performed in a consecutive series of 15 infertile women referred for a transvaginal laparoscopy as part of their fertility investigation. Tubal cells were collected using a 5Fr cytobrush. Cytology and immunocytochemistry was done. RESULTS: In all patients enough cell material was obtained for analysis, without traumatizing the fimbrial end. Specimens showed the presence of a sufficient amount of cells enabling standard cytologic examinations and immunocytochemistry (Ki 67, p53). CONCLUSION: Fimbrial cytobrushing using the transvaginal approach is an easy and minimally invasive procedure. The easy accessibility of the fimbrial end and the distal ampullary part at TVL allows an accurate collection of tubal epithelial cells. In view of the recent data reporting the Fallopian tube and more specifically the fimbrial end as a possible origin of ovarian carcinoma, further research is needed to evaluate the potential of this technique as a possible screening method for patients at risk for ovarian cancer.

Optimization of HPV DNA detection in urine by improving collection, storage, and extraction.

The benefits of using urine for the detection of human papillomavirus (HPV) DNA have been evaluated in disease surveillance, epidemiological studies, and screening for cervical cancers in specific subgroups. HPV DNA testing in urine is being considered for important purposes, notably the monitoring of HPV vaccination in adolescent girls and young women who do not wish to have a vaginal examination. The need to optimize and standardize sampling, storage, and processing has been reported.In this paper, we examined the impact of a DNA-conservation buffer, the extraction method, and urine sampling on the detection of HPV DNA and human DNA in urine provided by 44 women with a cytologically normal but HPV DNA-positive cervical sample. Ten women provided first-void and midstream urine samples. DNA analysis was performed using real-time PCR to allow quantification of HPV and human DNA.The results showed that an optimized method for HPV DNA detection in urine should (a) prevent DNA degradation during extraction and storage, (b) recover cell-free HPV DNA in addition to cell-associated DNA, (c) process a sufficient volume of urine, and (d) use a first-void sample.In addition, we found that detectable human DNA in urine may not be a good internal control for sample validity. HPV prevalence data that are based on urine samples collected, stored, and/or processed under suboptimal conditions may underestimate infection rates.

Real-world echocardiography in patients referred for mitral valve surgery: the gap between guidelines and clinical practice.

BACKGROUND AND AIM OF THE STUDY: Mitral regurgitation (MR) is a common disorder for which mitral valve surgery is an established therapy. Although surgical indications are clearly defined for the management of valvular heart disease, a gap exists between current guidelines and their effective application. The study aim was to provide an insight into the diagnostic information provided for cardiac surgeons before performing mitral valve surgery. METHODS: The source documents and echocardiographic studies of 100 patients, referred by nine hospitals, were screened for arguments for MR severity justifying referral for surgery. Details of the documented MR mechanism, mitral annulus (MA) size, tricuspid regurgitation (TR) severity and annulus size were also noted. RESULTS: According to the referring physician, MR was severe in 83% and moderate-to-severe in 17%. In the great majority of patients (98%) the MR mechanism was mentioned, although specific information on the prolapsing scallops was available in only 17% of cases. The recommended primary determinants of MR severity, vena contracta and proximal isovelocity surface area (PISA) were measured in only 22% and 31% of patients, respectively. In 94% of patients with available PISA information this was described only qualitatively. Correct image expansion using the zoom mode was performed in only 25% of these patients, and a correct adaptation of the Nyquist limit in only 6%. Tricuspid annulus measurements guiding the need for concomitant tricuspid valvuloplasty in patients with less than severe TR were reported in only 6% of patients. CONCLUSION: These data demonstrate a clear and important gap between current guidelines and real-world practice with regards to the echocardiographic diagnostic information provided to the surgeon before performing mitral valve surgery.

Comparison of SPF10 real-time PCR and conventional PCR in combination with the INNO-LiPA HPV Genotyping Extra assay for the detection and typing of human papillomavirus in cervical samples.

The novel SPF10 real-time PCR assay allows the simultaneous amplification and detection of the HPV target. That way, LiPA analysis of the HPV-negative samples can be avoided, reducing workload and cost. This study aims to evaluate the performance of the SPF10 real-time PCR in combination with the LiPA assay for HPV detection and typing in cervical samples. Thirty-nine cervical samples were subjected to the SPF10 conventional PCR in combination with the LiPA assay. Subsequently, the SPF10 real-time PCR was performed to enable the comparison between the SPF10 conventional and the real-time PCR results. In case of discrepancy, the samples were subjected to the CLART HPV2 assay. As a result, 27 out of 39 samples were identified as HPV-positive by the SPF10 real-time PCR and were genotyped further by the LiPA assay. Twenty samples (74.1%) showed an absolute agreement between the conventional and real-time SPF10 PCR (concordant), three (11.1%) displayed additional or fewer types (compatible), two (7.4%) did not show any similarity between both assays (discordant) and the remaining two (7.4%) were LiPA-negative. The two assays showed an excellent strength of agreement for individual (κ=0.932) and multiple genotype detection (κ=0.834). In conclusion, the two SPF10 PCR methods are comparable. Therefore, the SPF10 real-time PCR with subsequent LiPA could be used for the detection and genotyping of HPV in cervical samples.

A real-time PCR approach based on SPF10 primers and the INNO-LiPA HPV Genotyping Extra assay for the detection and typing of human papillomavirus.

The SPF10 PCR targets a conserved 65bp region of the HPV L1 gene for broad-spectrum amplification. The LiPA assay allows subsequent genotyping of the HPV amplicons. This study aims to develop a SPF10 real-time PCR to achieve simultaneous amplification and detection of the HPV target. That way, LiPA analysis of the HPV-negative samples can be avoided, reducing workload and cost. The real-time PCR shows an analytical sensitivity of 29.7 copies for HPV 6, 16, 18 and 31 and an HPV-specific melting peak. Thirty-one HPV DNA plasmids were genotyped correctly using the SPF10 real-time PCR in combination with the LiPA. Here, the LiPA assay was performed at an increased hybridisation temperature (49.5°C) in combination with a reduced amplicon volume (1µl) to avoid cross-reactivity. In conclusion, the SPF10 real-time PCR proves to be very sensitive and generates amplicons, which are compatible with the LiPA.

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.

Human papillomavirus, lichen sclerosus and penile cancer: a study in Belgium.

PURPOSE: The prevalence of penile cancer varies between 1.5 (industrialized countries) and 4.5 per 100,000 men (non-industrialized countries). Predominant histological subtype is squamous cell carcinoma (SCC). Human papillomavirus (HPV) is found in 40-46% of cases: penile cancer is considered to behave as vulvar cancer. Non HPV related risk factors are lack of circumcision, phimosis, chronic inflammation, and smoking. The role of lichen sclerosus (LS) is unclear. Clinical diagnosis is difficult and treatment often mutilating. Preventive measures can be taken since the risk factors are known: the use of the prophylactic HPV vaccines may contribute. We measured the prevalence of HPV and LS in penile cancer in Belgium. MATERIALS AND METHODS: We found 76 samples of penile lesions in the archives of the departments of Histology of four university hospitals in Belgium. Real-time PCR of type-specific HPV DNA was performed targeting 18 HPV types. PRINCIPAL RESULTS: Patients with penile intraepithelial neoplasia (PeIN) were 56.1 years of age: patients with invasive penile cancer (IPC) 68.5 (p=0.009). Fifty-five samples (55/76) were adequate for HPV targeting. Overall HPV DNA was 70.9%: 89.5% in samples of PeIN (n=19) and 61.1% in samples of IPC (n=36). Invasive penile cancer samples were less likely to be HPV infected (p=0.028). HPV 16 was most prevalent: 48.3%: 20% PeIN, and 28.3% IPC. HPV DNA of the types, included in the prophylactic vaccines, was found in 33% of PeIN and 31.7% of IPC samples. Thrice, low risk HPV (lrHPV) types 6 (1 IPC) and 11 (1 PeIN, 1 IPC) were solely present. There was no difference in the presence of LS between HPV positive and HPV negative samples (p=0.944). CONCLUSIONS: Prevalence of HPV DNA in penile lesions in Belgium is high. However, the prophylactic vaccines may contribute to primary prevention of only a subset of cases. The role of LS remains unclear.

WITHDRAWN: A real-time PCR approach based on SPF10 primers and the INNO-LiPA HPV Genotyping Extra assay for the detection and typing of human papillomavirus.

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Detection of human papillomavirus DNA in urine. A review of the literature.

The detection of human papillomavirus (HPV) DNA in urine, a specimen easily obtained by a non-invasive self-sampling method, has been the subject of a considerable number of studies. This review provides an overview of 41 published studies; assesses how different methods and settings may contribute to the sometimes contradictory outcomes; and discusses the potential relevance of using urine samples in vaccine trials, disease surveillance, epidemiological studies, and specific settings of cervical cancer screening. Urine sampling, storage conditions, sample preparation, DNA extraction, and DNA amplification may all have an important impact on HPV DNA detection and the form of viral DNA that is detected. Possible trends in HPV DNA prevalence in urine could be inferred from the presence of risk factors or the diagnosis of cervical lesions. HPV DNA detection in urine is feasible and may become a useful tool but necessitates further improvement and standardization.

Small, smaller, smallest. Steps towards bloodless neonatal and infant cardiopulmonary bypass.

In open heart surgery in neonates and small children, the cardiopulmonary bypass circuit surface and the priming volume are relatively large in relation to patient size and blood volume. Therefore, the use of allogeneic blood is inevitable to maintain the optimal hematocrit level during bypass. To avoid the deleterious effects of blood transfusion, as well as to reduce the contact surface of blood with artificial materials, we stepwise reduced the bypass circuit size. Use of the commercially available minimized elements and an adjusted set-up of the system allowed us to reduce usage of allogeneic blood in the prime and during the bypass. However, other supplemental measures are needed to obtain asanguineous cardiopulmonary bypass for neonatal and infant patients.

Belgian consensus guidelines for follow-up of women with cervical cytological abnormalities.

In medical care cervical cancer screening is important because it enables the detection of precancer and cancer at an early stage. By adequate treatment after a screening-detected lesion it helps to reduce the mortality related to cervical cancer. Worldwide, many millions of women have smears taken at a more or less regular base and of these, approximately 7% are abnormal, and follow-up is thus required.As this represents an important cost in medical health care and has serious consequences for the affected women, it is important to have uniform and clear guidelines to allow an optimal follow-up and clinical management. A system for the uniform reporting of cervical cytology has been designed by the National Cancer Institute (U.S.A.) and resulted in the Bethesda System 1991. The present paper and the terminology used are based on the Bethesda System revised in 2001. It explains the guidelines, based on the 2001 Bethesda System and the 2004 consensus guidelines for the management of women with cervical cytological abnormalities, as developed by the members of the Board of the Belgian Society of Clinical Cytology, and adapted to the Belgian situation.

Cervical cytology biobanking: quality of DNA from archival cervical Pap-stained smears.

AIMS: Cervical cytology biobanking is a feasible concept in cervical pathology and could be an indispensable tool for fundamental and applied molecular biological research. PCR is a powerful molecular technique that can be performed on a variety of cervical sample types including Pap-stained cervical smears. However, since the quality of DNA from such specimens is inferior to that from fresh tissue, the correct processing methods are required. This study evaluates three commercial isolation methods and one digestion procedure for their ability to obtain DNA suitable for PCR from fixed and stained Pap smears. METHODS: The High Pure PCR Template Preparation kit, the NucliSENS easyMAG system, the QIAamp DNA Mini Kit and crude proteinase K digestion were used to obtain DNA for subsequent PCR applications. Amplification of beta-globin was performed to verify the presence and integrity of target DNA. The influence of PCR inhibitors and extent of DNA fragmentation were analysed. RESULTS: All commercial DNA isolation techniques provided DNA suitable for PCR amplification, and DNA isolated from 10-year-old archival smears yielded amplicons up to 400 base pairs. Conversely, crude proteinase K digestion limited the amplicon size to 300 bp and did not consistently yield amplifiable digests, as these were contaminated with PCR-inhibiting factors and debris. CONCLUSION: The study indicates that commercial DNA isolation techniques are suitable for PCR amplification of DNA isolated from archival smears, yielding amplicons up to 400 base pairs. Proteinase K digestion is not suitable to obtain amplifiable DNA from fixed and stained Pap-stained smears.

Effects of fixation on RNA integrity in a liquid-based cervical cytology setting.

AIMS: Despite many improvements, cervical cancer screening is still subject to shortcomings. Diagnostic accuracy may improve by using molecular biological techniques, requiring RNA of superior quality. This study determined the effect of SurePath fixation on RNA integrity to assess the suitability of clinical samples collected in this medium for RNA-based molecular assays. METHODS: RNA isolation was performed on fresh and fixed HeLa cells and exfoliated cervical cells fixed in SurePath. The RNA integrity was evaluated by analysis of ribosomal RNA as an indicator of quality. The effect of SurePath preservation on PCR amplification was evaluated by real-time reverse transcriptase (RT)-PCR. RESULTS: In contrast to unfixed cells, SurePath-fixed cells yielded less and severely degraded RNA, as shown by the absence of ribosomal RNA bands. RNA derived from SurePath-fixed cells showed poor real-time RT-PCR amplification characteristics, as evidenced by the absent correlation between threshold values and log cDNA concentration. CONCLUSIONS: Implementation of molecular biology in a clinical context is on the rise and may alleviate shortcomings in current screening and diagnostics. This study shows that SurePath fixation gives rise to highly fragmented RNA with insufficient quality for further reliable analysis by standard real-time RT-PCR applications. The increasing prominence of molecular screening stresses the importance of this finding, which must be considered in relation to choice of an appropriate liquid-based cytology system.