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Aims: We investigated key signalling pathways' activity and mutational status of early-stage breast carcinomas with low and intermediate 21-gene recurrence score (RS) to identify molecular features that may predict recurrence., Methods: This is a retrospective case-control study of 18 patients with recurrent breast carcinoma with low and intermediate 21-gene RS (<25) and control group of 15 non-recurrent breast cancer patients. DNA and mRNA were extracted from tumour tissue. mRNA expression of genes involved in oestrogen receptor (ER), androgen receptor (AR), PI3K and MAPK signalling pathways was measured by real-time quantitative reverse transcription-qPCR (OncoSIGNal G4 test, InnoSIGN). Tumour mutational landscape was assessed by targeted DNA sequencing (Oncomine Precision Assay)., Results: There were no statistical differences between the groups' demographic and clinicopathological characteristics. PI3K pathway showed significantly higher activity in cases compared with controls (p=0.0014). Receiver operating characteristic curve analysis showed an area under the curve of 0.79 for PI3K pathway activity in the prediction of recurrent disease in low and intermediate 21-gene RS breast cancer. There was no difference in ER, AR and MAPK pathway activity. PIK3CA alterations were the most common driver mutations, but no difference was found between the groups (p=0.46) and no association with PI3K pathway activity (p=0.86). Higher Ki67 gene expression was associated with recurrences (p=0.042) CONCLUSION: Increased PI3K pathway activity, independent of PIK3CA mutations, may play a role in the recurrence of early-stage breast cancer with low and intermediate 21-gene RS. Pathway analysis can help to identify high-risk patients in this setting., Competing Interests: Competing interests: YW-R, DvS, SN and PAvdW are employees or contractors of InnoSIGN and own InnoSIGN shares and/or options. MS is scientific advisor and shareholder of C2i Genomics, Heidelberg Epignostix and Halo Dx, and a scientific advisor of Arima Genomics, and InnoSIGN, and received research funding from Lilly USA., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Androgen deprivation therapy (ADT) is first-line palliative treatment in androgen receptor-positive (AR+) salivary duct carcinoma (SDC), and response rates are 17.6-50.0%. We investigated potential primary ADT resistance mechanisms for their predictive value of clinical benefit from ADT in a cohort of recurrent/metastatic SDC patients receiving palliative ADT (n = 30). We examined mRNA expression of androgen receptor (AR), AR splice variant-7, intratumoral androgen synthesis enzyme-encoding genes AKR1C3, CYP17A1, SRD5A1 and SRD5A2, AR protein expression, ERBB2 (HER2) gene amplification and DNA mutations in driver genes. Furthermore, functional AR pathway activity was determined using a previously reported Bayesian model which infers pathway activity from AR target gene expression levels. SRD5A1 expression levels and AR pathway activity scores were significantly higher in patients with clinical benefit from ADT compared to those without benefit. Survival analysis showed a trend toward a longer median progression-free survival for patients with high SRD5A1 expression levels and high AR pathway activity scores. The AR pathway activity analysis, and not SRD5A1 expression, also showed a trend toward better disease-free survival in an independent cohort of locally advanced SDC patients receiving adjuvant ADT (n = 14) after surgical tumor resection, and in most cases a neck dissection (13/14 patients) and postoperative radiotherapy (13/14 patients). In conclusion, we are the first to describe that AR pathway activity may predict clinical benefit from ADT in SDC patients, but validation in a prospective study is needed., (© 2019 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Objectives: To investigate the added value of assessing transcripts for the long cAMP phosphodiesterase-4D (PDE4D) isoforms, PDE4D5 and PDE4D9, regarding the prognostic power of the 'CAPRA & PDE4D7' combination risk model to predict longitudinal postsurgical biological outcomes in prostate cancer., Patients and Methods: RNA was extracted from both biopsy punches of resected tumours (606 patients; RP cohort) and diagnostic needle biopsies (168 patients; DB cohort). RT-qPCR was performed in order to determine PDE4D5, PDE4D7, and PDE4D9 transcript scores in both study cohorts. By RNA sequencing, we determined the TMPRSS2-ERG fusion status of each tumour sample in the RP cohort. Kaplan-Meier survival analyses were then applied to correlate the PDE4D5, PDE4D7 and PDE4D9 scores with postsurgical patient outcomes. Logistic regression was then used to combine the clinical CAPRA score with PDE4D5, PDE4D7, and PDE4D9 scores in order to build a 'CAPRA & PDE4D5/7/9' regression model. ROC and decision curve analysis was used to estimate the net benefit of the 'CAPRA & PDE4D5/7/9' risk model., Results: Kaplan-Meier survival analysis, on the RP cohort, revealed a significant association of the PDE4D7 score with postsurgical biochemical recurrence (BCR) in the presence of the TMPRSS2-ERG gene rearrangement (logrank p<0.0001), compared to the absence of this gene fusion event (logrank p=0.08). In contrast, the PDE4D5 score was only significantly associated with BCR in TMPRSS2-ERG fusion negative tumours (logrank p<0.0001 vs. logrank p=0.4 for TMPRSS2-ERG+ tumours). This was similar for the PDE4D9 score although less pronounced compared to that of the PDE4D5 score (TMPRSS2ERG- logrank p<0.0001 vs. TMPRSS2ERG+ logrank p<0.005). In order to predict BCR after primary treatment, we undertook ROC analysis of the logistic regression combination model of the CAPRA score with the PDE4D5, PDE4D7, and PDE4D9 scores. For the DB cohort, this demonstrated significant differences in the AUC between the CAPRA and the PDE4D5/7/9 regression model vs. the CAPRA and PDE4D7 risk model (AUC 0.87 vs. 0.82; p=0.049) vs. the CAPRA score alone (AUC 0.87 vs. 0.77; p=0.005). The CAPRA and PDE4D5/7/9 risk model stratified 19.2% patients of the DB cohort to either 'no risk of biochemical relapse' (NPV 100%) or the 'start of any secondary treatment (NPV 100%)', over a follow-up period of up to 15 years. Decision curve analysis presented a clear, net benefit for the use of the novel CAPRA & PDE4D5/7/9 risk model compared to the clinical CAPRA score alone or the CAPRA and PDE4D7 model across all decision thresholds., Conclusion: Association of the long PDE4D5, PDE4D7, and PDE4D9 transcript scores to prostate cancer patient outcome, after primary intervention, varies in opposite directions depending on the TMPRSS2-ERG genomic fusion background of the tumour. Adding transcript scores for the long PDE4D isoforms, PDE4D5 and PDE4D9, to our previously presented combination risk model of the combined 'CAPRA & PDE4D7' score, in order to generate the CAPRA and PDE4D5/7/9 score, significantly improves the prognostic power of the model in predicting postsurgical biological outcomes in prostate cancer patients.

Purpose. To further validate the prognostic power of the biomarker PDE4D7, we investigated the correlation of PDE4D7 scores adjusted for presurgical clinical variables with longitudinal postsurgical biological outcomes. Methods. RNA was extracted from biopsy punches of resected tumors (550 patients; RP cohort) and diagnostic needle biopsies (168 patients; DB cohort). Cox regression and survival were applied to correlate PDE4D7 scores with patient outcomes. Logistic regression was used to combine the clinical CAPRA score with PDE4D7. Results. In univariate analysis, the PDE4D7 score was significantly associated with PSA recurrence after prostatectomy in both studied patient cohorts' analysis (HR 0.53; 95% CI 0.41-0.67; p<1.0E-04 and HR 0.47; 95% CI 0.33-0.65; p<1.0E-04, respectively). After adjustment for the presurgical clinical variables preoperative PSA, PSA density, biopsy Gleason, clinical stage, percentage tumor in the biopsy (data only available for RP cohort), and percentage of positive biopsies, the HR was 0.49 (95% CI 0.38-0.64; p<1.0E-04) and 0.43 (95% CI 0.29-0.63; p<1.0E-04), respectively. The addition of the PDE4D7 to the clinical CAPRA score increased the AUC by 5% over the CAPRA score alone (0.82 versus 0.77; p=0.004). This combination model stratified 14.6% patients of the DB cohort to no risk of biochemical relapse (NPV 100%) over a follow-up period of up to 15 years. Conclusions. The PDE4D7 score provides independent risk information for pretreatment risk stratification. Combining CAPRA with PDE4D7 scores significantly improved the clinical risk stratification before surgery.

Sequencing the genomes of individual cells enables the direct determination of genetic heterogeneity amongst cells within a population. We have developed an injection-moulded valveless microfluidic device in which single cells from colorectal cancer derived cell lines (LS174T, LS180 and RKO) and fresh colorectal tumors have been individually trapped, their genomes extracted and prepared for sequencing using multiple displacement amplification (MDA). Ninety nine percent of the DNA sequences obtained mapped to a reference human genome, indicating that there was effectively no contamination of these samples from non-human sources. In addition, most of the reads are correctly paired, with a low percentage of singletons (0.17 ± 0.06%) and we obtain genome coverages approaching 90%. To achieve this high quality, our device design and process shows that amplification can be conducted in microliter volumes as long as the lysis is in sub-nanoliter volumes. Our data thus demonstrates that high quality whole genome sequencing of single cells can be achieved using a relatively simple, inexpensive and scalable device. Detection of genetic heterogeneity at the single cell level, as we have demonstrated for freshly obtained single cancer cells, could soon become available as a clinical tool to precisely match treatment with the properties of a patient's own tumor.

Background: The clinical metrics used to date to assess the progression risk of newly diagnosed prostate cancer patients only partly represent the true biological aggressiveness of the underlying disease., Objective: Validation of the prognostic biomarker phosphodiesterase-4D7 (PDE4D7) in predicting longitudinal biological outcomes in a historical surgery cohort to improve postsurgical risk stratification., Design, Patients, and Methods: RNA was extracted from biopsy punches of resected tumors from 550 patients. PDE4D7 was quantified using one-step quantitative reverse transcription-polymerase chain reaction. PDE4D7 scores were calculated by normalization of PDE4D7 to reference genes. Multivariate analyses were adjusted for clinical prognostic variables. Outcomes tested were: prostate-specific antigen relapse, start of salvage treatment, progression to metastases, overall mortality, and prostate cancer-specific mortality. The PDE4D7 score was combined with the clinical risk model Cancer of the Prostate Risk Assessment Postsurgical Score (CAPRA-S) using multivariate regression modeling; the combined score was tested in post-treatment progression free survival prediction., Outcome Measurements and Statistical Analysis: Correlations with outcomes were analyzed using multivariate Cox regression and logistic regression statistics., Results and Limitations: The PDE4D7 score was significantly associated with time-to-prostate specific antigen failure after prostatectomy (hazard ratio [HR]: 0.53, 95% confidence interval [CI]: 0.41-0.67 for each unit increase, p<0.0001). After adjustment for postsurgical prognostic variables the HR was 0.56 (95% CI: 0.43-0.73, p<0.0001). The PDE4D7 score remained significant after adjusting the multi-variate analysis for the CAPRA-S model categories (HR=0.54, 95% CI=0.42-0.69, p<0.0001). Combination of the PDE4D7 score with the CAPRA-S demonstrated a significant incremental value of 4-6% in 2-yr (p=0.004) or 5-yr (p=0.003) prediction of progression free survival after surgery. The combined model of PDE4D7 and CAPRA-S improves patient selection with very high risk of fast disease relapse after primary intervention., Conclusions: The PDE4D7 score has the potential to provide independent risk information and to restratify patients with clinical intermediate- to high-risk characteristics to a very low-risk profile., Patient Summary: In this report, we studied the potential of a novel biomarker to predict outcomes of a cohort of prostate cancer patients who underwent surgery more than 10 yr ago. We found that a gene called phosphodiesterase-4D7 added extra information to the available clinical data. We conclude that the measurement of this gene in tumor tissue may contribute to more effective treatment decisions., (Copyright © 2017 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

Large variation exists in mitochondrial DNA (mtDNA) not only between but also within individuals. Also in human cancer, tumor-specific mtDNA variation exists. In this work, we describe the comparison of four methods to extract mtDNA as pure as possible from frozen tumor tissue. Also, three state-of-the-art methods for sensitive detection of mtDNA variants were evaluated. The main aim was to develop a procedure to detect low-frequent single-nucleotide mtDNA-specific variants in frozen tumor tissue. We show that of the methods evaluated, DNA extracted from cytosol fractions following exonuclease treatment results in highest mtDNA yield and purity from frozen tumor tissue (270-fold mtDNA enrichment). Next, we demonstrate the sensitivity of detection of low-frequent single-nucleotide mtDNA variants (≤1% allele frequency) in breast cancer cell lines MDA-MB-231 and MCF-7 by single-molecule real-time (SMRT) sequencing, UltraSEEK chemistry based mass spectrometry, and digital PCR. We also show de novo detection and allelic phasing of variants by SMRT sequencing. We conclude that our sensitive procedure to detect low-frequent single-nucleotide mtDNA variants from frozen tumor tissue is based on extraction of DNA from cytosol fractions followed by exonuclease treatment to obtain high mtDNA purity, and subsequent SMRT sequencing for (de novo) detection and allelic phasing of variants.

Objective: The endothelium has a crucial role in wound healing, acting as a barrier to control transit of leukocytes. Endothelial barrier function is impaired in atherosclerosis preceding myocardial infarction (MI). Besides lowering lipids, statins modulate endothelial function. Here, we noninvasively tested whether statins affect permeability at the inflammatory (day 3) and the reparative (day 7) phase of infarct healing post-MI using contrast-enhanced cardiac magnetic resonance imaging (MRI)., Approach and Results: Noninvasive permeability mapping by MRI after MI in C57BL/6, atherosclerotic ApoE -/- , and statin-treated ApoE -/- mice was correlated to subsequent left ventricular outcome by structural and functional cardiac MRI. Ex vivo histology, flow cytometry, and quantitative polymerase chain reaction were performed on infarct regions. Increased vascular permeability at ApoE -/- infarcts was observed compared with C57BL/6 infarcts, predicting enhanced left ventricular dilation at day 21 post-MI by MRI volumetry. Statin treatment improved vascular barrier function at ApoE -/- infarcts, indicated by reduced permeability. The infarcted tissue of ApoE -/- mice 3 days post-MI displayed an unbalanced Vegfa (vascular endothelial growth factor A)/ Angpt1 (angiopoetin-1) expression ratio (explaining leakage-prone vessels), associated with higher amounts of CD45 + leukocytes and inflammatory LY6C hi monocytes. Statins reversed the unbalanced Vegfa/Angpt1 expression, normalizing endothelial barrier function at the infarct and blocking the augmented recruitment of inflammatory leukocytes in statin-treated ApoE -/- mice., Conclusions: Statins lowered permeability and reduced the transit of unfavorable inflammatory leukocytes into the infarcted tissue, consequently improving left ventricular outcome., (© 2017 American Heart Association, Inc.)

We demonstrate on-chip, differential DNA and RNA extraction from a single cell using a microfluidic chip and a two-stage lysis protocol. This method enables direct use of the whole extract, without additional washing steps, reducing sample loss. Using this method, the tumor driving pathway in individual cells from a colorectal cancer cell line was determined by applying a Bayesian computational pathway model to sequences obtained from the RNA fraction of a single cell and, the mutations driving the pathway were determined by analyzing sequences obtained from the DNA fraction of the same single cell. This combined functional and mutational pathway assessment of a single cell could be of significant value for dissecting cellular heterogeneity in tumors and analyzing single circulating tumor cells.

Phosphodiesterase 4D7 was recently shown to be specifically over-expressed in localized prostate cancer, raising the question as to which regulatory mechanisms are involved and whether other isoforms of this gene family (PDE4D) are affected under the same conditions.We investigated PDE4D isoform composition in prostatic tissues using a total of seven independent expression datasets and also included data on DNA methylation, copy number and AR and ERG binding in PDE4D promoters to gain insight into their effect on PDE4D transcription.We show that expression of PDE4D isoforms is consistently altered in primary human prostate cancer compared to benign tissue, with PDE4D7 being up-regulated while PDE4D5 and PDE4D9 are down-regulated. Disease progression is marked by an overall down-regulation of long PDE4D isoforms, while short isoforms (PDE4D1/2) appear to be relatively unaffected. While these alterations seem to be independent of copy number alterations in the PDE4D locus and driven by AR and ERG binding, we also observed increased DNA methylation in the promoter region of PDE4D5, indicating a long lasting alteration of the isoform composition in prostate cancer tissues.We propose two independent metrics that may serve as diagnostic and prognostic markers for prostate disease: (PDE4D7 - PDE4D5) provides an effective means for distinguishing PCa from normal adjacent prostate, whereas PDE4D1/2 - (PDE4D5 + PDE4D7 + PDE4D9) offers strong prognostic potential to detect aggressive forms of PCa and is associated with metastasis free survival. Overall, our findings highlight the relevance of PDE4D as prostate cancer biomarker and potential drug target.

Background: There is an acute need to uncover biomarkers that reflect the molecular pathologies, underpinning prostate cancer progression and poor patient outcome. We have previously demonstrated that in prostate cancer cell lines PDE4D7 is downregulated in advanced cases of the disease. To investigate further the prognostic power of PDE4D7 expression during prostate cancer progression and assess how downregulation of this PDE isoform may affect disease outcome, we have examined PDE4D7 expression in physiologically relevant primary human samples., Methods: About 1405 patient samples across 8 publically available qPCR, Affymetrix Exon 1.0 ST arrays and RNA sequencing data sets were screened for PDE4D7 expression. The TMPRSS2-ERG gene rearrangement status of patient samples was determined by transformation of the exon array and RNA seq expression data to robust z-scores followed by the application of a threshold>3 to define a positive TMPRSS2-ERG gene fusion event in a tumour sample., Results: We demonstrate that PDE4D7 expression positively correlates with primary tumour development. We also show a positive association with the highly prostate cancer-specific gene rearrangement between TMPRSS2 and the ETS transcription factor family member ERG. In addition, we find that in primary TMPRSS2-ERG-positive tumours PDE4D7 expression is significantly positively correlated with low-grade disease and a reduced likelihood of progression after primary treatment. Conversely, PDE4D7 transcript levels become significantly decreased in castration resistant prostate cancer (CRPC)., Conclusions: We further characterise and add physiological relevance to PDE4D7 as a novel marker that is associated with the development and progression of prostate tumours. We propose that the assessment of PDE4D7 levels may provide a novel, independent predictor of post-surgical disease progression.

A new mechanism is described for DNA amplification using nucleic acid sequence-based amplification (NASBA) including a restriction enzyme digestion and P1 primer binding directly upstream of the digestion. For hepatitis B virus (HBV), herpes simplex virus (HSV) and methicillin resistant Staphylococcus aureus (MRSA) DNA, which all show very poor amplification with normal NASBA, assay sensitivity was improved by a factor 100-1000 when restriction enzyme digestion was performed prior to amplification. For the quantitative HBV assay, in combination with the NucliSENS Extractor (bioMérieux, Boxtel, The Netherlands), a 95% target detection rate of 242 WHO IU/ml and 50% detection rate of 35 WHO IU/ml was achieved. The lowest detectable HBV concentration was 10 WHO IU/ml. HBV DNA could be quantified with an algorithm comparable to that used for RNA quantitation and by using a two step approach a dynamic range of 10(2)-10(9)WHO IU/ml (>6 log) was shown to be quantifiable. For the qualitative HSV assay, in combination with the NucliSENS miniMAG (bioMérieux, Boxtel, The Netherlands), the 95% detection rate was determined to be 84 and 138 copies/isolation for HSV 1 and HSV 2, respectively, which corresponds to approximately 10 copies per amplification for both targets. For MRSA, the limit of detection was <10 equivalent CFU per amplification.

We have developed a hepatitis B virus (HBV) DNA detection and quantification system based on amplification with nucleic acid sequence-based amplification (NASBA) technology and real-time detection with molecular beacon technology. NASBA is normally applied to amplify single-stranded target RNA, producing RNA amplicons. In this work we show that with modifications like primer design, sample extraction method, and template denaturation, the NASBA technique can be made suitable for DNA target amplification resulting in RNA amplicons. A major advantage of our assay is the one-tube, isothermal nature of the method, which allows high-throughput applications for nucleic acid detection. The homogeneous real-time detection allows a closed-tube format of the assay, avoiding any postamplification handling of amplified material and therefore minimizing the risk of contamination of subsequent reactions. The assay has a detection range of 10(3) to 10(9) HBV DNA copies/ml of plasma or serum (6 logs), with good reproducibility and precision. Compared with other HBV DNA assays, our assay provides good sensitivity, a wide dynamic range, and high-throughput applicability, making it a viable alternative to those based on other amplification or detection methods.

A new method to quantify two individual mRNAs in a single NASBA reaction is described. In this study, tissue factor and CD14 mRNAs were used as a model system. RNA ratios of -4 to +4 log units were determined with good precision (within 0.3 log) and accuracy (within 0.2 log). By measuring both mRNAs in human monocytes that were stimulated with LPS, the multiplex Q-NASBA proved to be a successful tool to monitor the expression levels of two individual mRNAs in a single-tube amplification system. The method has potential in all fields in which quantitative information is needed on two individual RNAs.

APC resistance is a common and strong hereditary risk factor for venous thrombosis. This plasma abnormality appears to be almost always caused by the same defect in the coagulation factor V gene (a G --> A transition at nucleotide 1691 leading to replacement of 506 Arg by Gln; factor V Leiden). Therefore, it is possible to consider a simple and specific genetic test as an alternative to a plasma APC resistance test that is compromised by treatment and other factors. We have investigated whether a new amplification procedure, NASBA, together with the detection procedure ELGA would provide a simple protocol for the nucleotide specific detection of the factor V mutation.

Serum HIV-1 RNA and p24 antigen levels were examined in 28 seropositive asymptomatic individuals participating in a trial on the efficacy of zidovudine. Sixteen individuals remained asymptomatic until 4 years after the onset of the trial, whereas 12 individuals were diagnosed with an AIDS-defining event. The serum HIV-1 RNA load and p24 antigen levels were determined before the onset of therapy and during the first 8 weeks of therapy to establish whether the patterns of change were predictive of clinical outcome. Among the 28 participants 43% had measurable pretreatment concentrations of p24 antigen. Initiation of zidovudine therapy was followed by a similar decline of p24 antigen levels in nonprogressors as well as progressors and, therefore, these groups could not be distinguished on the basis of this parameter. HIV-1 RNA was detected in the pretreatment samples of 82% of the individuals and could be detected in p24 antigen-positive as well as p24 antigen-negative individuals. Similar changes in HIV-1 RNA load during zidovudine therapy were observed in p24 antigen-positive and -negative individuals. Analysis of the HIV-1 RNA response according to clinical outcome demonstrated that HIV-1 RNA copy numbers had declined significantly after 4 weeks of therapy in both nonprogressors and progressors, but the decline in RNA load was much stronger in the nonprogressors. Our data show that the HIV-1 RNA load in serum can be used to monitor the response to antiviral therapy in p24 antigen-positive as well as -negative individuals. Posttreatment changes in p24 antigen levels are not indicative for clinical outcome, whereas RNA copy numbers are.(ABSTRACT TRUNCATED AT 250 WORDS)

Virus load and virus phenotype have both been indicated as major determinants of disease progression in HIV-1 infection. In this study HIV-1 RNA copy numbers in serum, virus phenotype, and CD4+ cell counts were analyzed longitudinally in a group of 20 seroconverters progressing to AIDS within 5.5 years. In this group 12 individuals developed AIDS without syncytium-inducing (SI) viruses ever being isolated, while 8 individuals showed a non-SI (NSI) to SI phenotypic switch prior to AIDS development. HIV-1 RNA copy numbers in sera of all progressors were stable and high from seroconversion until development of AIDS. Twenty-one seroconverters remaining asymptomatic for more than 5.5 years were selected as nonprogressing controls, and both progressors and nonprogressors were evaluated at seroconversion and early in infection (3 years post seroconversion). Comparative analysis revealed that at the point of seroconversion HIV-1 RNA copy numbers in sera from NSI progressors, SI progressors, and nonprogressors were not significantly different, nor were their CD4+ cell counts. At seroconversion all individuals harbored viruses with an NSI phenotype. In contrast to the progressors, HIV-1 RNA copy numbers in sera of nonprogressors had declined significantly during the early period of infection. At the second time point RNA copy numbers in the sera of NSI progressors and nonprogressors differed significantly (P = 0.0005), while RNA copy numbers in the sera of SI progressors and nonprogressors did not. However, at this time point the CD4+ cell counts of SI progressors were significantly lower than those from nonprogressors (P = 0.002), while the CD4+ cell counts of NSI progressors and nonprogressors did not differ significantly. These results show that early in HIV-1 infection progressors and nonprogressors are distinguishable. NSI progressors can be distinguished from nonprogressors on the basis of serum HIV-1 RNA load and S1 progressors on the basis of CD4+ cell decline. In addition, a significant decrease in the number of HIV-1 RNA copies in the early phase of infection seems to postpone the development of AIDS.

Quantification of HIV-1 viral RNA based on co-amplification of an internal standard Q-RNA dilution series requires a number of NASBA nucleic acid amplification reactions. For each internal standard Q-RNA concentration a separate NASBA amplification has to be performed. The development of an electrochemiluminescent (ECL) detection system with a dynamic signal detection range over 5 orders of magnitude enabled simplification of the Q-NASBA protocol. Three distinguishable Q-RNAs (QA, QB and QC) are mixed with the wild-type sample at different amounts (i.e. 10(4) QA, 10(3) QB and 10(2) QC molecules) and co-amplified with the wild-type RNA in one tube. Using ECL-labelled oligonucleotides the wild-type, QA, QB and QC amplificates are separately detected with a semi-automated ECL detection instrument and the ratio of the signals determined. The amount of initial wild-type RNA can be calculated from the ratio of wild-type signal to QA, QB and QC signals. This one-tube Q-NASBA protocol was compared to the earlier described protocol with six amplifications per quantification using model systems and HIV-1 RNA isolated from plasma of HIV-1-infected individuals. In all cases the quantification results of HIV-1 RNA were comparable between the two methods tested. Due to the use of only one amplification per quantification in the one-tube Q-NASBA protocol the QA, QB and QC internal standard RNA molecules can be added to the sample before nucleic acid isolation. The ratio of QA:QB:QC:WT RNAs, from which the initial input of WT-RNA is calculated, will remain constant independent of any loss that might occur during the nucleic acid isolation.

Quantification of HIV-1 viral RNA in plasma was achieved by competitive co-amplification of a dilution series of in vitro generated RNA using the nucleic acid sequence based amplification (NASBA) technology. This 1.5 kilobase in vitro RNA, comprising the gag and part of the pol region, differs only by sequence-randomization of a 20 nt fragment from the wild-type RNA, ensuring equal efficiency of amplification. In model systems the accuracy of this method is within one log. Application of the Q-NASBA to plasma samples of a patient with a primary HIV-1 infection shows good concordance of the HIV-1 RNA profile with the p24 antigen profile. However, the HIV-1 RNA determination is more sensitive than the p24 antigen determination. Peak values of HIV-1 RNA are around 10(8) RNA molecules per ml plasma at the moment of seroconversion. Quantitative nucleic acid detection methods, like Q-NASBA, allow the monitoring of HIV-1 RNA during the course of infection which might have predictive value for disease development.

A new amplification procedure, NASBA (nucleic acid sequence-based amplification), was used together with the polymerase chain reaction (PCR) to detect HIV-1 sequences in different blood fractions of both HIV-infected and uninfected samples. We tested whole blood, plasma, peripheral blood mononuclear cells (PBMCs), and platelets. No HIV-1 sequences were found in blood fractions of 37 uninfected persons either by PCR, reverse transcriptase-PCR (RT-PCR), or NASBA. We found that none of the infected plasma samples contained HIV-1 DNA sequences. However, a high percentage of these plasma samples was positive for HIV-1 RNA: 86% by NASBA and 80% by RT-PCR. The concordance on a sample-to-sample basis of NASBA and RT-PCR was 91%. Only 33% of the plasma samples was HIV-1 p24-antigen positive, demonstrating the superior sensitivity of amplification procedures. We found that almost all PBMC fractions were positive for HIV-1 (pro)viral sequences (99% HIV-1 DNA positive, 91% HIV-1 RNA positive). A large proportion of the platelet fractions contained HIV-1 RNA, as demonstrated by positive RT-PCR and NASBA results. We found an inverse relation between CD4+ T cell count and T cell reactivity on the one hand and detection of HIV-1 sequences by PCR, RT-PCR, and NASBA on the other hand in all blood fractions. Quantification of the HIV-1 PCR signal in PBMCs revealed an inverse relation of proviral titers with CD4+ levels. This finding supports earlier observations that clinical disease and low CD4+ cell counts are related to an increased viral burden.

Isothermal nucleic acid amplification of target RNA or DNA sequences is accomplished by the simultaneous enzymatic activity of AMV reverse transcriptase, T7 RNA polymerase and RNase H. Amplification factors of the nucleic acid sequence based amplification (NASBA) method range from 2 x 10(6) to 5 x 10(7) after 2.5 h incubation at 41 degrees C. During NASBA there is a major accumulation of specific single stranded RNA. RNA:DNA hybrid and double stranded DNA are also synthesized, although to a minor extent. The system is optimized for the detection of HIV-1 sequences in in vitro infected cells, blood and plasma. Detection levels are 10 molecules of HIV-1 in a model system with in vitro generated HIV-1 RNA as input and 5 infected cells on a background of 5 x 10(4) non-infected cells. Blood and plasma can also be used as the source of nucleic acid for detection of HIV-1 sequences using a specifically developed sample preparation method. Using NASBA it is possible to amplify specifically RNA or DNA from a pool of total nucleic acid, which permits the investigation of the expression of specific genes involved in pathogenesis of infectious agents. The combination of NASBA with a rapid and user-friendly nucleic acid extraction method makes the whole procedure suitable for large scale diagnosis of infectious agents (e.g. HIV-1).