Highly Sensitive Telomerase Assay Insusceptible to Telomerase and Polymerase Chain Reaction Inhibitors for Cervical Cancer Screening Using Scraped Cells

A sensitive telomerase assay based on asymmetric-polymerase chain reaction (A-PCR) on magnetic beads and subsequent application of cycling probe technology, STAMC, which is insusceptible to DNase and PCR inhibitors, was for the first time applied to clinical specimens in addition to a conventional telomeric repetitive amplification protocol (TRAP). The electrophoresis results showed that an increase in scraped cervical cancer cells not only reduced TRAP products but also increased smaller products, suggesting the unreliability of TRAP for clinical samples. To achieve the required sensitivity of STAMC for clinical application, the sequence and concentration conditions were explored for the forward and reverse primers for A-PCR, which resulted in a detection limit of only two HeLa cells with 1 μM TS primer (5′-AATCC­GTCGAGC­AGAGTT-3′) and 0.04 μM ACX primer (5′-GCGCGGC­TTACCCTTA­CCCTTACC­CTAACC-3′). Under the same primer conditions, the fluorescence signal of STAMC increased as scraped cervical cancer cells increased despite showing a negligible intensity for benign tumors. Furthermore, STAMC showed no signal for a cervical cancer patient treated with irradiation therapy. These results indicate that STAMC is useful for not only cervical cancer screening but also investigating the effect of cancer treatments such as radiation therapy and drug administration

Arterial Transit Time Mapping Obtained by Pulsed Continuous 3D ASL Imaging with Multiple Post-Label Delay Acquisitions: Comparative Study with PET-CBF in Patients with Chronic Occlusive Cerebrovascular Disease

<div><p>Arterial transit time (ATT) is most crucial for measuring absolute cerebral blood flow (CBF) by arterial spin labeling (ASL), a noninvasive magnetic resonance (MR) perfusion assessment technique, in patients with chronic occlusive cerebrovascular disease. We validated ASL-CBF and ASL-ATT maps calculated by pulsed continuous ASL (pCASL) with multiple post-label delay acquisitions in patients with occlusive cerebrovascular disease. Fifteen patients underwent MR scans, including pCASL, and positron emission tomography (PET) scans with ¹⁵O-water to obtain PET-CBF. MR acquisitions with different post-label delays (1.0, 1.5, 2.0, 2.5 and 3.0 sec) were also obtained for ATT correction. The theoretical framework of 2-compartmental model (2CM) was also used for the delay compensation. ASL-CBF and ASL-ATT were calculated based on the proposed 2CM, and the effect on the CBF values and the ATT correction characteristics were discussed. Linear regression analyses were performed both on pixel-by-pixel and region-of-interest bases in the middle cerebral artery (MCA) territory. There were significant correlations between ASL-CBF and PET-CBF both for voxel values (r = 0.74 ± 0.08, slope: 0.87 ± 0.22, intercept: 6.1 ± 4.9) and for the MCA territorial comparison in both affected (R² = 0.67, y = 0.83x + 6.3) and contralateral sides (R² = 0.66, y = 0.74x + 6.3). ASL-ATTs in the affected side were significantly longer than those in the contralateral side (1.51 ± 0.41 sec and 1.12 ± 0.30 sec, respectively, p <0.0005). CBF measurement using pCASL with delay compensation was feasible and fairly accurate even in altered hemodynamic states.</p></div

The ASL signal changes for model parameters.

<p><b>a)</b> ASL signal dependency to T_1e. The single-compartment model is constant along with T_1e value, since labeled spins are retained in the vascular space and correspond to the relaxation of T_1b, while the other model assumption with venous outflow has an increasing trend along with the increase of T_1e. <b>b)</b> ASL signal dependency to CBV. ASL signal has an increasing trend along with CBV increase. Changes are quite stable in the normal brain CBV range (2.0 to 5.0 ml/100 g) (see text). <b>c)</b> ASL signal dependency to PS. PS is also constant despite the wide range of 20 to 200 ml/min/g, which adequately covers the physiological cerebral blood flow range.</p

Independent component analysis (ICA) results.

<p>The average components for females and males are shown in the left and right columns, respectively. (A) Independent components of the anterior default mode network (DMN). (B) Independent components of posterior DMN. The maps depict the statistical threshold for both results set at <i>p</i> = 0.05 with whole-brain FWE correction. The coordinates for the panels above are (A) (4, -52, 32) and (B) (4, -58, 42). Color bars denote the t-statistic range.</p

Patient characteristics, clinical data, and MRI findings.

<p>Patient characteristics, clinical data, and MRI findings.</p

Comparison between MR and PET in a typical case of left ICA obstruction.

<p><b>a)</b> Comparison between PET-CBF and ASL-CBF. From top to bottom rows, PET-CBF, ASL-CBF, ASL-ATT (transit time map) are calculated from the multiple PLD data and ASL-CBF calculated from single PLD (1.5 s) data, based on the simple single-compartment model (see text). The decreased signal in the right frontal cortex corresponds to cystic change after infarction. <b>b)</b> MRA revealing the complete obstruction of left ICA suggests the left MCA territory is fed through collaterals of A-Com and/or left IC-PC. <b>c)</b> 2D-plots of PET and ASL-CBF on pixel-by-pixel basis. The plotted CBF images through ventricle body level correspond to the third column images from the right side in the 1^st and 2^nd rows. Abscissa and ordinate axes represent PET and ASL CBF, respectively. Scale bars and units as indicated.</p

Seed-based analysis functional connectivity results.

<p>(A) Comparison of aMPFC seed ROI functional connectivity between groups. Females showed stronger connectivity to the ANG, but males showed stronger connectivity to the SFG, TempP, MOG, and SOG. (B) Comparison of PCC seed ROI functional connectivity. Females showed stronger connectivity to the MOG, MPFC/OFC, MTG, and ANG, but males exhibited stronger connectivity in the SFG. The maps depict the statistical threshold at <i>p</i> < 0.001 uncorrected for height and a cluster at <i>p</i> < 0.05 uncorrected for extend. The bar graph indicates the mean and standard error of beta weights in each group. The red and blue bars indicate females and males, respectively. Color bars denote the t-statistic range. aMPFC, anterior prefrontal cortex; ANG, angular gyrus; MOG, middle occipital gyrus; MPFC, medial prefrontal cortex; MTG, middle temporal gyrus; OFC, orbital frontal cortex. PCC, posterior cingulate cortex; SFG, superior frontal gyrus; TempP, temporal pole.</p

Linear regression analysis of PET-CBF and ASL-CBF.

<p>The ROI values from affected and contralateral normal sides are plotted using a diamond shapes (◊) and crosses (x), respectively. <b>a)</b> 2D-plots of PET and ASL CBF using gray and white matter ROIs in both affected and contralateral cerebral hemispheres. The regression lines and the coefficient of determination (R²) are shown as insets on the graph. <b>b)</b> Bland-Altman plots of ROI-based CBF comparison between ASL-CBF and PET-CBF. Bias and mean ± 2 SD precision lines are drawn on the graph as thick lines and dashed lines, respectively. <b>c)</b> Extracted Bland-Altman plots of affected ROI of ASL-CBF data calculated from multi-PLD data. There is no significant regressed line. The regression lines and the coefficient of determination (R²) are shown as insets of the graph, but were not significant statistically.</p

Regional measure results.

<p>(A) fALFF Z maps for females and males are shown on the left and right, respectively. Females showed significantly stronger fALFF results in the PCC/PreC compared with males, but males fALFF results were stronger in the CERE and IFG. The maps depict the statistical threshold at <i>p</i> < 0.001 uncorrected for height and a cluster at <i>p</i> < 0.05 uncorrected for extend. Color bars denote the t-statistic range. CERE, cerebellum; IFG, inferior frontal gyrus; L, left; PCC, posterior cingulate cortex; PreC, precuneus; R, right. (B) ReHo fALFF Z maps for females and males are shown on the left and right, respectively. Males showed significantly stronger ReHo results in the CERE and IFG compared with females. The maps depict the statistical threshold at <i>p</i> < 0.001 uncorrected for height and a cluster at <i>p</i> < 0.05 uncorrected for extend. The bar graph indicates the mean and standard error of Z scores in each group. The red and blue bars indicate females and males, respectively. Colored bars denote the t-statistic range. CERE, cerebellum; fALFF, fractional amplitude of low-frequency fluctuation; IFG, inferior frontal gyrus; L, left; PCC, posterior cingulate cortex; R, right; ReHo, regional homogeneity.</p

Comparison between MR and PET in a typical case of left MCA severe stenosis.

<p><b>a)</b> Comparison between PET-CBF and ASL-CBF. From top to bottom rows, PET-CBF, ASL-CBF, ASL-ATT (transit time map) are calculated from the multiple PLD data and ASL-CBF calculated from single PLD (1.5s) data based on the simple single-compartment model (see text). <b>b)</b> MRA reveals that left MCA branches are less bright than those of the contralateral side. <b>c)</b> 2D-plots of PET and ASL-CBF on a pixel-by-pixel basis. The plotted CBF images through the ventricle body level correspond to third column images from the right side in 1st and 2nd rows. Abscissa and ordinate axes represent PET and ASL CBF, respectively.</p