Value of patient-reported symptoms in the follow up of patients potentially cured of laryngeal carcinoma.

OBJECTIVE: To evaluate the significance of patients' ability to recognise symptoms that signify recurrence. METHODS: A retrospective analysis was conducted in Norway of demographic, clinical and follow-up data for patients with laryngeal carcinoma considered free of disease following treatment. The study included clinical data from 732 patients with glottic tumours and 249 patients with supraglottic tumours who were considered cured of disease. Data on the site, time and type of recurrence (symptomatic or asymptomatic) were retrieved. RESULTS: Recurrence was observed in 127 patients with glottic tumours and 71 with supraglottic tumours. A total of 103 glottic recurrences and 53 supraglottic recurrences were symptomatic. For patients with glottic carcinoma, recurrence detection through symptoms was associated with a favourable post-salvage survival rate compared with asymptomatic recurrences (p = 0.003). CONCLUSION: A patient's ability to self-detect 'red flag' symptoms and self-initiate visits represents a previously ignored prognostic factor, and may rationalise follow up and improve survival.

Magnetic resonance imaging of the wrist and finger joints in patients with inflammatory joint diseases.

OBJECTIVE: To study magnetic resonance imaging (MRI) features in the wrist and metacarpophalangeal (MCP), proximal interphalangeal (PIP), and distal interphalangeal (DIP) joints in 4 patient groups: early rheumatoid arthritis (RA) (< 3 yrs); established RA (> 3 yrs); other arthritis; arthralgia. METHODS: MRI was obtained before and after contrast (gadodiamide) injection of the wrist and finger joints in 103 patients and 7 controls. The study included: (1) 28 patients with disease duration < 3 yrs who fulfilled the American College of Rheumatology (ACR) criteria for RA; (2) 25 patients with RA disease duration > 3 yrs who fulfilled the ACR criteria. (3) 25 patients with reactive arthritis, psoriatic arthritis, or mixed connective tissue disease; and (4) 25 patients with arthralgia. The following MRI variables were assessed: number of joints with enhancement after contrast injection, number of joints with joint fluid, and number of bones with edema in the wrist and fingers. The volume of the enhancing synovial membrane after contrast injection in the MCP, PIP, and DIP joints was manually outlined. MR images were scored independently under blinded conditions. RESULTS: Bone marrow edema was found in 68% of the patients with established RA, and the number of bones with edema was significantly higher in patients with established RA compared to patients with early RA, other arthritis, and arthralgia (Mann-Whitney p < 0.04). Bone edema was not found in patients with arthralgia. There was marked overlap within and between the patient groups. No differences in MRI features were found between patients with early RA and patients with other arthritis. The volumes of the synovial membrane in the MCP, PIP, and DIP joints were significantly higher in patients with arthritis compared to patients with arthralgia. CONCLUSION: Although there was marked overlap between the arthritis patient groups, MRI determined bone marrow edema and synovial membrane volumes provided additional information about disease activity and may be used as a marker of it. Bone marrow edema appeared with the highest percentage in patients with long duration of RA (> 3 yrs) and is probably secondary to changes in inflammatory activity.

MRI of the arthritic small joints: comparison of extremity MRI (0.2 T) vs high-field MRI (1.5 T).

The aim of this study was to compare the diagnostic capabilities of extremity MRI (E-MRI) with high-field MRI in arthritic small joints, and to evaluate the patients' acceptance and perceptions of the two MR systems. One hundred three patients (group 1 = 28 patients with RA < 3 years, group 2 = 25 patients with reactive and psoriatic arthritis and mixed connective tissue disease, group 3 = 25 patients with rheumatoid arthritis (RA) more than 3 years and group 4 = 25 patients with arthralgia) underwent dedicated E-MRI and high-field MRI of the wrist and finger joints. Coronal short tau inversion recovery and transversal 3D T1-weighted images before and after gadodiamide (Gd) were performed in both cases to outline the volume of the synovial membrane (Vsm) and to evaluate joints with enhancement, effusion, bone edema, and erosions. Investigators blinded to the clinical findings evaluated the images. Patients' compliance and acceptance of E-MRI and high-field MRI were evaluated. The median Vsm obtained on E-MRI did not differ significantly from that obtained on high-field MRI. Vsm = 1 ml (E-MRI) and 1.1 ml (high-field MRI) before Gd and Vsm = 0.1 ml (E-MRI) and 0 ml (high-field MRI) after Gd (Wilcoxon test, p > 0.05). The difference in agreement was 8% for joint enhancement, 2% for joint effusion, 3% for bone edema, and 4% for bone erosions. Of the patients, 64% preferred E-MRI due to more comfortable positioning and less claustrophobia and noise. Extremity MRI of the small arthritic joints is comparable to high-field MRI and more readily accepted than high-field MRI by this patient group.

MRI of the plantar structures of the foot after falanga torture.

Falanga is an ancient form of punishment or torture but is still commonly reported by our refugees. The late result of caning the heel and ball of the foot is a chronic painful condition with few clinical signs. The aim of the present study was to assess, by MRI, possible morphologic characteristics of the heel and ball of the foot, related to falanga and pain in correlation to clinical findings. Magnetic resonance imaging of the foot was obtained in 12 victims exposed to falanga torture and 9 healthy volunteers. Sagittal T1-weighted spin-echo images (TR 616-840 ms, TE 20 ms), T2-weighted spin-echo images (TR 1900 ms, TE 90 ms), and short tau inversion recovery (STIR) images (TR 1200 ms, TE 15 ms, TI 100 ms) were performed. The central portion of the plantar aponeurosis was generally significantly thicker in victims exposed to falanga torture as compared with that of controls (P < 0.05). In all except one of the victims, MRI demonstrated two layers of the thickened plantar aponeurosis: a deeper portion with normal homogeneous low signal intensity (SI) appearance, and a superficial layer with characteristic areas of mixed SI on both T1- and T2-weighted images. There were no signs of chronic muscular compartment syndromes, and the thickness of the plantar pad did not differ between the two groups. Magnetic resonance imaging may demonstrate morphologic characteristics of the plantar aponeurosis which may confirm falanga torture. Further imaging with more specific sequences is warranted to demonstrate the supposed injuries in the compartmental fat tissue chambers and the vascularity of the ball pad of the foot.

Long-term genetic modification of rhesus monkey hematopoietic cells following transplantation of adenoassociated virus vector-transduced CD34+ cells.

We have explored the potential of recombinant adenoassociated virus (AAV) vectors for gene transfer of the human beta-globin gene and the genetic modification of primate pluripotent hematopoietic stem cells (P-PHSCs). Transduction of P-PHSCs was tested in a preclinical bone marrow transplantation model in rhesus monkeys. CD34+ cells were transduced ex vivo and autologously transplanted without prior selection into irradiated rhesus monkeys. Vector-transduced peripheral blood mononuclear cells and granulocytes were present in the circulation for more than 15 months after transplantation. Approximately 1 in 10(5) cells in the circulation was vector modified. The vector was detected in the bone marrow, in granulocytes, and in purified populations of B and T cells, thus demonstrating multilineage repopulation by vector-transduced stem cells. Comparison of transduction protocols suggested that short-term culture of P-PHSCs enhances transduction and subsequent repopulation by rAAV-transduced cells. These results demonstrate that rAAV vectors can be used for the permanent genetic modification of a rhesus monkey hematopoietic system in the absence of selective pressure.

Determinants of musculoskeletal flexibility: viscoelastic properties, cross-sectional area, EMG and stretch tolerance.

Cross-sectional area, stiffness, viscoelastic stress relaxation, stretch tolerance and EMG activity of the human hamstring muscle group were examined in endurance-trained athletes with varying flexibility. Subjects were defined as tight (n = 10) or normal (n = 8) based on a clinical toe-touch test. Cross-sectional area was computed from magnetic resonance imagining (MRI) images. Torque (Nm) offered by the hamstring muscle group, electromyographic (EMG) activity, knee joint angle and velocity were continuously monitored during two standardized stretch protocols. Protocol 1 consisted of a slow stretch at 0.087 rad/s (dynamic phase) to a pre-determined final angle followed by a 90-s static phase. In the dynamic phase final angle and stiffness was lower in tight (28.0+/-2.9 Nm/rad) than normal subjects (54.9+/-6.5 Nm/rad), P<0.01. In the static phase tight subjects had lower peak (15.4+/-1.8 Nm) and final torque (10.8+/-1.6 Nm) than normal subjects (31.6+/-4.1 Nm, 24.1+/-3.7 Nm, respectively)(P<0.01), but torque decline was similar. Protocol 2 consisted of a slow stretch to the point of pain and here tight subjects reached a lower maximal angle, torque, stiffness and energy than normal subjects (P<0.01). On the other hand, stiffness was greater in tight subjects in the common range (P<0.01). Cross-sectional area of the hamstring muscles and EMG activity during the stretch did not differ between the groups. However, lateral hamstring cross-sectional area was positively related to mid-range stiffness (P<0.05), but inversely related to final stiffness, peak torque and the toe-touch test (P<0.01). Final angle and peak torque in protocol 1 combined to improve the predictability of the toe-touch test (R2=0.77, P<0.001). These data show that the toe-touch test is largely a measure of hamstring flexibility. Further, subjects with a restricted joint range of movement on a clinical toe-touch test have stiffer hamstring muscles and a lower stretch tolerance.

Radiologic features compared to clinical findings in a prospective study of 153 patients with metastatic spinal cord compression treated by radiotherapy.

Plain radiography, myelography and post-myelographic CT-scan are described and related to clinical findings in a prospective study of 153 consecutive patients with myelographic signs of spinal cord compression. The majority of the metastatic tumours arise in the vertebral body or the pedicles. In 80% of the patients with total blockage to the contrast medium on myelography the post-myelographic-CT showed passage of the contrast medium. Ambulatory function at time of diagnosis was correlated to the degree and the localization of the epidural block. In 64 patients who underwent a second myelography, the post-treatment findings of sensory function were correlated to radiological regression.

Isolation, purification and biological activity of major docetaxel metabolites from human feces.

We have developed a procedure suited for the isolation of metabolites of docetaxel (Taxotere) from human feces. The compounds were extracted from the feces with diethyl ether and further purified by (semipreparative) HPLC. Four metabolic products were obtained in submilligram quantities. Analytical HPLC with photodiode array detection showed that the purity of each compound was higher than 98%. There structures have been characterized by UV absorption and FAB/MS. All four compounds were oxidation products of the tert-butyl group attached to the C13-side chain, and corresponded to structures identified previously. The purified products were used for evaluating their cytotoxic activities against a human ovarian cancer (A2780) and a rat colon cancer (CC531) cell line, and their myelosuppressive effects in a hematopoietic progenitor toxicity assay. Although distinctions in biological activities between the compounds were evident, all metabolites showed a marked reduction in both cytotoxic and myelotoxic properties.

Frequency analysis of multidrug resistance-1 gene transfer into human primitive hematopoietic progenitor cells using the cobblestone area-forming cell assay and detection of vector-mediated P-glycoprotein expression by rhodamine-123.

Transfer of the multidrug resistance-1 (MDR1) gene into hematopoietic progenitor cells may reduce myelotoxicity of MDR1-related cytotoxic agents and therefore allow dose intensification. Mobilized peripheral blood progenitor cells (PBPC) can be obtained in ample quantity and are a suitable target cell population. CD34-selected PBPC samples (n = 6) were transduced with cell-free supernatant (SNT) of a cell line producing recombinant retrovirus containing the human MDR1 gene. Limiting-dilution long-term cultures were employed that allow continuous monitoring of stroma-adherent cobblestone areas (CA) and comparison of their frequency in a 5-log range over time. MDR1 provirus integration in CA-containing wells followed single-hit kinetics. According to Poisson statistics, proviral DNA was contained in 22% of unselected cobblestone area-forming cells (CAFC) at week 6, which represent primitive hematopoietic precursors. In comparison, 1.0 +/- 0.44% (mean +/- SEM) of week-6 CAFC were expressing P-glycoprotein at sufficient levels to convey vincristine resistance, suggesting low expression of the retroviral vector or splicing of the vector-drived mRNA in hematopoietic progenitor cells. Next we analyzed lineage-committed progenitors. The proviral DNA was detectable in 20-66% of colony-forming units granulocyte-macrophage (CFU-GM) while corresponding percentages (25-52%) of CD34+ PBPC were in the S/G2M phase of the cell cycle at the end of the transduction period. The proportion of vincristine-resistant CFU-GM was similar to the CAFC data and no significant differences were found between various MDR1-SNT transduction schedules whereas MDR1 co-cultivation, which served as a positive control, yielded significantly higher proportions of resistant colonies (5.3 +/- 1.4%, IL-3, 96 hr, p < or = 0.05). Assessment of rhodamine-123 (Rh-123) efflux in the myelo-monocytic progeny of MDR1-transduced cells mirrored the colony assay results in the SNT and co-cultivation groups. Less culture effort was required in the Rh-123 assay and functional characterization of the transferred P-glycoprotein was possible using cyclosporin A. Further development toward an effective MDR1 gene therapy should be facilitated by the CAFC assay, which allows estimation of the retroviral gene transfer frequency into primitive hematopoietic cells, and by the Rh-123 assay, which permits tractable side-by-side assessments of numerous MDR1 transduction protocols or different MDR1-SNT lots.

Retroviral transfer of the multidrug resistance-1 gene into lineage-committed and primitive hemopoietic cells.

Transfer of the multidrug resistance-1 (MDR1) gene to hemopoietic cells for myeloprotection against cytostatic agents is a new and rapidly developing field in "cancer gene therapy." Before clinical application, safety and efficacy criteria need to be met. The retroviral producer cell lines and the retroviral supernatant need to be tested for replication-competent retrovirus and contamination with adventitious agents. The cell source needs to contain sufficient hemopoietic cells with repopulating ability. We used CD34(+)-selected mobilized peripheral blood progenitor cells (PBPC) for MDR1 transductions in order to obtain a favorable vector to target cell ratio. An analysis of 249 patients who had undergone PBPC harvesting revealed that primarily solid tumor and non-Hodgkin's lymphoma patients are eligible for CD34+ selection. They can be expected to retain sufficient CD34+ cells for rapid and sustained engraftment after myeloablative therapy if the CD34+ cell loss (approximately 50%) during the procedure is taken into account. Clinical MDR1 gene therapy protocols focus on these two patient groups. Next we characterized MDR1 gene transfer into lineage-committed and primitive hemopoietic cells. Provirus-specific polymerase chain reactions showed a high efficiency gene transfer into colony-forming-units granulocyte-macrophage and long-term culture cells. The level of the conferred P-glycoprotein expression was estimated by fluorescence-activated cell sorting analysis to be up to 3 log above mock-transduced controls. The cobblestone area forming cell assay, which is a stroma-dependent long-term culture assay measuring frequencies of stem cell subsets in a limiting-dilution set-up, allowed demonstration of sustained expression of the MDR1 gene in the progeny of primitive hemopoietic cells. This is a favorable basis for a clinical MDR1 gene therapy trial.

Isolation, purification, and biological activity of mono- and dihydroxylated paclitaxel metabolites from human feces.

Three metabolites of the cytotoxic drug paclitaxel (Taxol) were isolated and purified from the feces of cancer patients receiving the agent as an intravenous infusion. The procedures involved sample homogenization in water followed by liquid-liquid extraction with diethyl ether and high-performance liquid chromatography (HPLC). Approximately 1-3.5 mg of each metabolite was obtained from 100 g of feces. As judged from the chromatographic traces of analytical HPLC with ultraviolet (UV) detection at 227 nm, the purity of each compound was > 97%. On-line photodiode-array detection demonstrated that the UV spectrum of the isolated compounds closely resembles that of the parent drug. Mass spectrometry provided evidence that these metabolites are mono- and dihydroxy-substituted derivatives, namely, 6 alpha-hydroxypaclitaxel, 3'-p-hydroxypaclitaxel, and 6 alpha, 3'-p-dihydroxypaclitaxel. The two 6 alpha-hydroxy-substituted metabolites were shown to have lost their cytotoxicity in in vitro clonogenic assays using the A2780 human ovarian carcinoma and the CC531 rat colon-carcinoma tumor cell lines. In addition, the metabolites showed reduced myelotoxic effects as compared with paclitaxel in an in vitro hemopoietic progenitor toxicity assay. Our procedure for the isolation and purification of paclitaxel metabolites in milligram quantities should be useful for testing the biological activities of these compounds and for the preparation of calibration standards essential for pharmacokinetics studies.

[Magnetic resonance imaging compared to myelography and CT. Diagnostic value in patients with suspected medullary spinal compression syndrome]. / Magnetisk resonans-billeddannelse sammenlignet med myelografi og CT. Diagnostisk vaerdi hos patienter mistoenkt for medulloert tvoersnitssyndrom.

Comparison between myelography (MY) and magnetic resonance imaging (MRI) was carried out in 36 patients with clinical suspicion of spinal cord or root compression due to metastatic disease in the spinal canal. In three patients metastatic lesions were visualized on MY but not on MRI, while there were no cases with a negative MY and a positive MRI. In 44% of the cases MY alone or combined with postmyelographic CT (pm-CT) showed a larger tumour extension than did MRI, while the opposite occurred in 25%. As for detection of bony metastases and tumour masses localized outside the spine there was no difference between MRI and MY + pm-CT. The results indicate that the choice between MRI and MY + pm-CT still can be based on the availability and quality of the procedure at a given institution.

How variable is a spontaneous mutation rate in cultured mammalian cells?

The Luria-Delbrück fluctuation analysis provides a method to estimate mutation rates and is commonly applied in somatic cell genetics and in cancer biology. We developed an assay for a Luria-Delbrück fluctuation analysis using the mouse lymphoma cell line, GRSL13. As these cells grow in suspension, one can handle hundreds of parallel cultures using multiwell dishes and dispensers. This assay thereby allows not only an accurate determination of the mutation rate per cell generation but also makes it possible to determine at which time after seeding mutations take place. Using approx. 8000 parallel cultures it has been possible to test whether the mutation rate is constant during the assay. It has been found that the spontaneous mutation rate of GRSL13 cells decreases in the course of a fluctuation test from 2 x 10(-6) to about 2 x 10(-7)/cell/generation. It was shown that this increased replication fidelity may partly be caused by cell density: maintenance of cells at high cell density resulted in a spontaneous mutation rate of 0.7 +/- 4.0 x 10(-7) compared to 4.0 +/- 3.1 x 10(-7) for the standard protocol. In contrast, growing the cells at extremely low cell density resulted in an enhanced mutation rate of 7.7 +/- 1.3 x 10(-7). Thus altogether the mutation rate can vary from 2 x 10(-6) to 0.7 x 10(-7) (approx. 30-fold). These results show that the spontaneous mutation rate is not constant, but highly dependent on experimental conditions. As incomplete expression and metabolic cooperation cannot explain the findings, the data suggest that the fidelity of DNA replication is not fixed but open to variation. Hence, determination of replication infidelity in cultured cells needs rigorous standardization or/and application of controlled variation in culture conditions.

Circumvention of chemotherapy-induced myelosuppression by transfer of the mdr1 gene.

Drug-induced myelosuppression is a frequent reason for curtailing chemotherapy in cancer patients. 'Rescue' of myelosuppressed patients with autologous marrow transplants is reasonably advanced and permits an increase in the dose of anticancer drugs. Despite this improvement, patients often relapse with drug resistance disease. The human multidrug resistance (mdr1) gene might make it possible to render hemopoietic stem cells resistant to anticancer drugs after transfer of this gene. By introducing resistant stem cells into patients it might be possible to treat these patients repeatedly with otherwise ablative therapy. This review explores the feasibility of mdr1 gene therapy.

A possible factor in genetic instability of cancer cells: stress-induced secreted proteins lead to decrease in replication fidelity.

Ultraviolet irradiation triggers a response in mammalian cells known as the UV response. Part of the UV response forms the enhanced synthesis of various extracellular proteins able to transmit the response to non-irradiated cells. Because several cancer-prone syndromes with enhanced genetic instability also have an enhanced synthesis of the same set of proteins without prior stimulation it is possible that induction of these stress responsive proteins may be involved in the process of carcinogenesis and genetic instability. To test this hypothesis mouse T lymphoma cells, GRSL13, were treated with the conditioned medium of UV-induced cells under various experimental conditions. Overall the mutation rate is enhanced 1.8-fold (P < 0.01). However, the degree of enhancement is strongly influenced by culture conditions. UV-induced factors only lead to an enhanced mutation rate when cells, both for the production and response to those factors, originate from a similar cell density. In addition, it was found that fresh medium interferes with this response. To eliminate the hindrance of these factors on the effect of the conditioned medium on the mutation rate, serum-starved cells at high density were treated with serum-free medium derived from high-density UV-irradiated cultures. Using these conditions a 2.8-fold (P < 0.002) enhancement of the mutation rate was found. Fluctuation analysis indicated that the enhancement is 10-fold during the first five generations after treatment. UV-induced factors have also been found to induce cell growth, and the degree of induction was linearly correlated with the enhancement in mutation rate. These experiments are in agreement with the hypothesis that induction of stress responses leads to genetic instability.

Stress response induced by DNA damage leads to specific, delayed and untargeted mutations.

Cells of the mouse T-lymphoma line GRSL13 were treated with 8-methoxy-psoralen plus longwave ultraviolet light (PUVA) under conditions where the biological effects are mainly due to non-persistent DNA cross-links (PUVA-CL treatment). Fluctuation analysis showed that PUVA-CL treatment resulted in an enhancement of the mutation rate in the progeny of treated cells, which persisted until the eleventh generation after treatment. Since only 5 cross-links are available to account for 52 mutational events observed in the coding region, about 90% of the induced mutational events must have been untargeted. This was confirmed by molecular analysis of these mutations, which showed that 53% of the point mutations arose at sites which are not a target for psoralens. This supports the hypothesis that stress responses may give rise to untargeted mutagenesis. Further support for this hypothesis is provided by the observation that 8-methoxy-psoralen (8-MOP) or UVA alone (both of which are known to induce many pleiotropic effects) each acted as indirect mutagen by enhancing the mutation rate 2-4 fold in the progeny of treated cells.

Comparison of myelography combined with postmyelographic spinal CT and MRI in suspected metastatic disease of the spinal canal.

Comparison between myelography (MY) and magnetic resonance imaging (MRI) was carried out in 36 patients with clinical suspicion of spinal cord or root compression due to metastatic disease in the spinal canal. In 3 patients metastatic lesions were visualized on MY but not on MRI, while there were no cases with a negative MY and a positive MRI. In 44% of the cases MY alone or combined with postmyelographic CT (pm-CT) showed a larger tumor extension than did MRI, while the opposite occurred in 25%. As for detection of bony metastases and tumor masses localized outside the spine there was no difference between MRI and MY + pm-CT. The results indicate that the choice between MRI and MY + pm-CT still can be based on the availability and quality of the procedure at a given institution.

Concomitant induction of signal transduction pathways and genetic instability by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate.

The hypothesis that activation of the signal transduction pathways by environmental stress may lead to genetic instability was tested. Mouse T-lymphoma cells, GRSL13, were treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). The induction of transcription of c-fos, fosB, c-jun, junB and collagenase was studied as well as the mutation rate in the progeny of treated cells. It was found that mRNA levels of fosB, junB and collagenase, all known to be involved in the growth factor signal transduction pathway, were enhanced. No transcription of c-fos and c-jun was observed in control and TPA-treated cells. These results suggest that transcription of c-fos is not a prerequisite for the induction of transcription of collagenase. The degree of induction of the signal transduction pathway was dependent on culture conditions of the treated cells, growing cells having less response than stationary cells. The mutation rate was significantly enhanced in the progeny of TPA-treated cells from 4.2 X 10(-7) to 9.8 X 10(-7)/cell/generation. Fluctuation analysis showed that TPA leads to a temporary enhancement of the mutation rate up to the eighth generation after treatment. The enhancement of the mutation rate is less apparent in growing cells than in stationary cells (1.8- and 2.9-fold respectively) which, because the signal transduction pathways are less induced in growing cells than in stationary cells, is in agreement with the hypothesis that induction of the signal transduction pathway leads to genetic instability.