The effects of telomere shortening on cancer cells: a network model of proteomic and microRNA analysis.

Previously, we have shown that shortening of telomeres by telomerase inhibition sensitized cancer cells to cisplatinum, slowed their migration, increased DNA damage and impaired DNA repair. The mechanism behind these effects is not fully characterized. Its clarification could facilitate novel therapeutics development and may obviate the time consuming process of telomere shortening achieved by telomerase inhibition. Here we aimed to decipher the microRNA and proteomic profiling of cancer cells with shortened telomeres and identify the key mediators in telomere shortening-induced damage to those cells. Of 870 identified proteins, 98 were differentially expressed in shortened-telomere cells. 47 microRNAs were differentially expressed in these cells; some are implicated in growth arrest or act as oncogene repressors. The obtained data was used for a network construction, which provided us with nodal candidates that may mediate the shortened-telomere dependent features. These proteins' expression was experimentally validated, supporting their potential central role in this system.

Androgen levels during adjuvant endocrine therapy in postmenopausal breast cancer patients.

OBJECTIVE: To investigate plasma steroid hormone levels in postmenopausal breast cancer patients with and without adjuvant endocrine therapy and in healthy postmenopausal women. METHODS: Steroid hormone levels in postmenopausal breast cancer patients treated with aromatase inhibitors (n = 32) were compared with breast cancer patients treated with tamoxifen (n = 34), breast cancer patients without adjuvant endocrine therapy (n = 15), and healthy postmenopausal women (n = 56). Pregnenolone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol, cortisol, cortisone, dehydroepiandrosterone (DHEA), androstenedione, total testosterone, dihydrotestosterone, estrone and estradiol were measured using liquid chromatography-tandem mass spectrometry. Sex hormone binding globulin was measured by solid-phase chemiluminescent immunometric assays, and the free androgen index was calculated. RESULTS: Aromatase inhibitor users did not differ in dihydrotestosterone, total testosterone, androstenedione, DHEA, or free androgen index levels from healthy controls or untreated breast cancer patients. The highest total testosterone levels were found in tamoxifen-treated women, who had significantly higher plasma concentrations than both women treated with aromatase inhibitors and breast cancer patients without adjuvant treatment. Concentrations of cortisol and cortisone were significantly greater in aromatase inhibitor users as well as tamoxifen users, in comparison with healthy controls and untreated breast cancer patients. Aromatase inhibitor users had lower estrone and estradiol plasma concentrations than all other groups. CONCLUSION: Adjuvant treatment with aromatase inhibitors or tamoxifen was associated with increased cortisol and cortisone plasma concentrations as well as decreased estradiol concentrations. Androgen levels were elevated in tamoxifen-treated women but not in aromatase inhibitor users.

Higher than expected estradiol levels in aromatase inhibitor-treated, postmenopausal breast cancer patients.

OBJECTIVE: Vaginal estradiol is considered contraindicated in aromatase inhibitor (AI)-treated patients because of the risk of elevated estrogen levels. This leaves limited treatment options for patients experiencing gynecological symptoms. However, in clinical practice, no precise estimation has been performed of circulating estrogens and aromatase index in postmenopausal breast cancer patients on long-lasting AI or tamoxifen treatment. METHODS: Steroid hormones were measured using liquid chromatography tandem mass spectrometry (LC-MS/MS) and extraction radioimmunoassay (RIA). Postmenopausal AI-treated patients (n =33) were compared with tamoxifen-treated patients (n =34) and controls without vaginal treatment (n =56), with vaginal estradiol (n =25), or with estriol (n =11) treatment. RESULTS: By use of LC-MS/MS, median (range) estradiol plasma concentrations were 16.7 (2.4-162.6), 31.0 (13.4-77.1), 27.2 (7.8-115.8) and 33.3 (20.3-340.1) pmol/l in AI-treated breast cancer patients, tamoxifen-treated breast cancer patients, postmenopausal controls and postmenopausal controls on vaginal estradiol, respectively. The AI-treated group and subgroups had significantly lower estradiol and estrone concentrations than all other groups (p <0.05). There was extensive interindividual variation in estradiol concentration within the AI-treated group, measured using both LC-MS/MS (2.3-182.0 pmol/l) and extraction RIA (2.4-162.6 pmol/l). The AI-treated group had lower aromatase index compared to all other groups (p <0.05-0.001). CONCLUSION: Circulating estrogen levels may have been underestimated in previous longitudinal studies of AI-treated breast cancer patients. Additional studies are required to further evaluate the role of circulating estrogens in breast cancer patients suffering from gynecological symptoms.

Clinical utility of comprehensive circulating tumor DNA genotyping compared with standard of care tissue testing in patients with newly diagnosed metastatic colorectal cancer.

BACKGROUND: Comprehensive biomarker testing is essential in selecting optimal treatment for patients with metastatic colorectal cancer (mCRC); however, incomplete genotyping is widespread, with most patients not receiving testing for all guideline-recommended biomarkers, in part due to reliance on burdensome sequential tissue-based single-biomarker tests with long waiting times or availability of only archival tissue samples. We aimed to demonstrate that liquid biopsy, associated with rapid turnaround time (TAT) and lower patient burden, effectively identifies guideline-recommended biomarkers in mCRC relative to standard of care (SOC) tissue testing. PATIENTS AND METHODS: Prospectively enrolled patients with previously untreated mCRC undergoing physician discretion SOC tissue genotyping submitted pretreatment blood samples for comprehensive circulating tumor DNA (ctDNA) analysis with Guardant360 and targeted RAS and BRAF analysis with OncoBEAM. RESULTS: Among 155 patients, physician discretion SOC tissue genotyping identified a guideline-recommended biomarker in 82 patients, versus 88 identified with comprehensive ctDNA (52.9% versus 56.8%, noninferiority demonstrated down to α = 0.005) and 69 identified with targeted PCR ctDNA analysis (52.9% versus 44.5%, noninferiority rejected at α = 0.05). Utilizing ctDNA in addition to tissue increased patient identification for a guideline-recommended biomarker by 19.5% by rescuing those without tissue results either due to tissue insufficiency, test failure, or false negatives. ctDNA median TAT was significantly faster than tissue testing when the complete process from sample acquisition to results was considered (median 10 versus 27 days, P < 0.0001), resulting in accelerated biomarker discovery, with 52.0% biomarker-positive patients identified by ctDNA versus 10.2% by SOC tissue 10 days after sample collection (P < 0.0001). CONCLUSIONS: Comprehensive ctDNA genotyping accurately identifies guideline-recommended biomarkers in patients with mCRC at a rate at least as high as SOC tissue genotyping, in a much shorter time. Based on these findings, the addition of ctDNA genotyping to clinical practice has significant potential to improve the care of patients with mCRC.

Evolving pattern of Guillain-Barre syndrome in a community hospital in Israel.

OBJECTIVES: To investigate the frequency of axonal Guillain-Barre syndrome (GBS) in our ward over 6 years (1999-2005). MATERIALS AND METHODS: Clinical and electrophysiological findings of 40 patients admitted to neurology with abnormalities compatible with acute motor axonal neuropathy (AMAN), acute motor sensory axonal neuropathy (AMSAN) and acute inflammatory demyelinating polyneuropathy (AIDP) were reviewed. RESULTS: Electrophysiological findings showed that 25 (63%) patients had AIDP, nine (22%) AMAN and six (15%) AMSAN. There were significant differences in disease severity. Most axonal patients (87%) were hospitalized with moderate or severe symptoms (3-4 Hughes grade score) and progressed to severe grade (4-6) in comparison with AIDP patients (64% admitted with mild forms) (1-2 Hughes grade score) and progressed to severe in 44% of cases. Cranial nerve involvement was more frequent in AIDP (56%) in comparison with the axonal type (13%). Raised cerebrospinal fluid protein at the time of hospitalization was observed in 76% of demyelinating and 33% of axonal patients. CONCLUSIONS: Axonal GBS occurred more frequently in Israel compared with other Western countries.

Diagnostic value of vestibular evoked myogenic potentials in cerebellar and lower-brainstem strokes.

BACKGROUND: Vestibular evoked myogenic potentials (VEMPs) provide assessment of vestibular function. They consist in picking up compound muscle action potentials in the sternocleidomastoid (SCM) muscles in response to auditory stimulation of the vestibulum. VEMP testing has found application mainly in peripheral vestibular disorders, whereas reports about VEMPs in central vestibular lesions are rather scarce. AIMS OF THE STUDY: Based on the physiological connections between the cerebellum and the vestibular nuclei, we investigated the influence on VEMPs of cerebellar and lower-brainstem strokes. We examined whether or not this method may be suitable as a clinical tool for the evaluation of the extent of cerebellar strokes. PATIENTS AND METHODS: Nineteen patients with cerebellar ischemic stroke and 15 patients with lower-brainstem ischemic stroke (11 in the pons, four in the medulla) were included. The latencies and amplitudes of P13 and N23 in both groups of patients were compared with those obtained in a control group of 53 normal individuals. RESULTS: VEMP responses were obtained in all patients and controls. At the group level, mean peak latencies and amplitudes, and the number of subjects with significantly deviant values did not differ between patients and controls. There were no latency or amplitude differences ipsilaterally or contralaterally to the lesion. At the individual level, there was no correlation between laterality of lesion and that of P13 or N23 abnormalities in patients with cerebellar strokes; however, there were two patients (one pontine, one medullar stroke) who presented P13 and N23 latency abnormalities ipsilaterally to the lesion. CONCLUSION: Cerebellar strokes do not influence VEMPs. Moreover, despite previous reports, we were unable to find at a group level any statistically significant VEMP changes in patients with lower-brainstem strokes as compared with controls. Therefore, VEMPs do not appear a suitable tool for assessment of brainstem integrity in patients with posterior fossa strokes. However, they could constitute a sensitive method for documentation of involvement of the central vestibular pathways in patients with brainstem stroke.

Effects of testosterone replacement in human immunodeficiency virus-infected women with weight loss.

The objective of this study was to determine whether physiological testosterone replacement increases fat-free mass (FFM) and muscle strength and contributes to weight maintenance in HIV-infected women with relative androgen deficiency and weight loss. Fifty-two HIV-infected, medically stable women, 18-50 yr of age, with more than 5% weight loss over 6 months and testosterone levels below 33 ng/dl were randomized into this double-blind, placebo-controlled trial of 24-wk duration. Subjects in the testosterone group applied testosterone patches twice weekly to achieve a nominal delivery of 300 mug testosterone over 24 h. Data were evaluable for 44 women. Serum average total and peak testosterone levels increased significantly in the testosterone group, but did not change in the placebo group. However, there were no significant changes in FFM (testosterone, 0.7 +/- 0.4 kg; placebo, 0.3 +/- 0.4 kg), fat mass (testosterone, 0.3 +/- 0.7 kg; placebo, 0.6 +/- 0.7 kg), or body weight (testosterone, 1.0 +/- 0.9 kg; placebo, 0.9 +/- 0.8 kg) between the two treatment groups. There were no significant changes in leg press strength, leg power, or muscle fatigability in either group. Changes in quality of life, sexual function, cognitive function, and Karnofsky performance scores did not differ significantly between the two groups. High-density lipoprotein cholesterol levels decreased significantly in the testosterone group. The patches were well tolerated. We conclude that physiological testosterone replacement was safe and effective in raising testosterone levels into the mid to high normal range, but did not significantly increase FFM, body weight, or muscle performance in HIV-infected women with low testosterone levels and mild weight loss. Additional studies are needed to fully explore the role of androgens in the regulation of body composition in women.

Verapamil inhibits B-cell proliferation and tumor necrosis factor release and induces a clinical response in B-cell chronic lymphocytic leukemia.

Four B-CLL patients, treated with verapamil for cardiac problems, showed substantial reduction of lymphadenopathy in one, a 3- and 5-year stabilization of B-CLL in two patients, and a dramatic decrease in lymphocyte count, lymphadenopathy and splenomegaly in one stage IV patient. We therefore studied the effects of verapamil on B-CLL cells in vitro. In 13 samples we observed that verapamil strongly inhibited in vitro proliferation of pokeweed mitogen (PWM) stimulated and unstimulated cells. Using a cytotoxic bioassay, we found that verapamil markedly inhibited the spontaneous and PMW-induced release of tumor necrosis factor (TNF) by B-CLL cells. These findings suggest that verapamil may block B-CLL cell proliferation through inhibition of TNF release and thereby may contribute to the management of B-CLL.

[Opsoclonus myoclonus with ataxia].

Opsoclonus myoclonus with ataxia (OMA) is a rare neurological disorder. The syndrome is characterized by involuntary, conjugate, multidirectional eye movements accompanied by involuntary movements of limb or face, and sometimes ataxia, dysarthria, irritability, dementia, altered level of consciousness and even death. OMA is associated with various etiologies including infectious, toxic, drug-related, vascular and paraneoplastic conditions. Paraneoplastic opsoclonus myoclonus with ataxia (POMA) is more common in patients over 40 years of age and is usually associated with lung (especially small cell), breast and ovarian cancer but has also been reported with many other cancers. The syndrome is thought to be mediated by autoantibodies directed against onconeural antigens that are expressed by the tumor as well as by neurons. Studies from several laboratories were able to demonstrate a role for the cellular response in the pathogenesis of POMA. The results for treatment of this syndrome have been disappointing, although aggressive multimodality immunosuppressive treatments have been used. This is a case study of a patient with POMA who clearly demonstrates the difficulties in the diagnosis and treatment of this syndrome.

Antineurofilament antibodies in postpolio syndrome.

We determined the levels of antineurofilament antibodies in 29 patients with postpolio syndrome (PPS), 26 stable postpolio (PP) patients, 22 patients with ALS, and 20 normal controls (NCs). Patients with PPS had higher antibody levels to cholinergic neurofilaments than did all other groups. PP patients and those with ALS had antibody levels similar to those of NCs. The antibody binding level showed no relation to the age of the patients, duration of disease, or motor score.

Pharmacological basis for functional selectivity of partial muscarinic receptor agonists.

Muscarinic receptor agonists activate phosphoinositide hydrolysis and adenylate cyclase in Chinese hamster ovary cells transfected with cDNAs encoding the human muscarinic ml and m3 receptors. Whereas carbachol activates similarly both receptor subtypes, 4-[3-chlorophenyl-carbamoyloxy]-2-butynyltrimethyl ammonium chloride (McN-A-343) preferentially activates the m1 subtype over m3, in regard to both phosphoinositide hydrolysis and adenylate cyclase activity. On the other hand, oxotremorine activates phosphoinositide hydrolysis to a similar extent in both cell lines, but it activates preferentially adenylate cyclase in m1 versus m3 receptor expressing cells. Relative to carbachol, both McN-A-343 and oxotremorine activate preferentially phosphoinositide hydrolysis over adenylate cyclase in both cell lines. Prolonged incubation of cells with either carbachol, McN-A-343, or oxotremorine down-regulated the m1 receptors. This was accompanied by a parallel decrease in adenylate cyclase activity, whereas phosphoinositide hydrolysis remained relatively high. Inactivation of the receptors by alkylation with acetylethylcholine mustard, or by blocking with atropine, reduced carbachol-stimulated adenylate cyclase activity more effectively than carbachol-induced phosphoinositide hydrolysis in both m1 and m3 receptor expressing cells. These findings imply that the receptor reserve in these cell lines is greater for phosphoinositide hydrolysis response than for adenylate cyclase response. Yet, the receptor reserve for each of these responses is similar in both m1 and m3 receptor expressing cells. Since the binding affinities of McN-A-343 and of oxotremorine to m1 and m3 receptors are very similar, and since both cell lines contain similar amounts of spare receptors, we propose that the preferential activation of muscarinic m1 over m3 receptor by partial agonists is related to differences in the abilities of the two receptor subtypes to undergo conformational changes following agonist binding. This hypothesis is supported by results showing that the muscarinic m1 but not m3 receptor exhibits two affinity states in a competition binding assay.

H reflex behavior in Parkinson's disease patients and patients with extrapyramidal and pyramidal signs combined.

The H reflex was elicited in 21 normal subjects, 48 patients with Parkinson's disease (PD) and 22 patients with pyramidal and extrapyramidal signs combined (PESC). In most normal subjects (90.5%), in 29.2% of PD patients and 54.5% of patient with PESC the threshold for sensory fibers was lower than for motor fibers, and the H reflex was obtained before the M response for all duration stimuli in both legs. In 9.5% of normal subjects, 39.6% of PD patients with mild and moderate rigidity (according to the motor part of UPDRS) and 31.8% of patients with PESC, the threshold for the H reflex and M response was the same or the M response threshold was lower in at least one of the legs for short stimulus duration (0.1-0.2 ms). In 31.2% of PD patients (most of them with severe rigidity) and 13.7% of patients with PESC, the threshold for M response was lower for all stimulus duration in at least one of the legs, and it was obtained before H reflex. These very significant differences in behavior of the H reflex in PD patients (Fisher exact test, p<0.0001) that almost disappear in patients with PESC, could be possibly explained by changes in agonist-antagonist inhibition.

Somatosensory and autonomic neuropathy as the only manifestation of long standing leprosy.

The clinical and neuropathological findings in a patient with "neuritic" leprosy are described. In this rare form of leprosy, skin changes are only minimal or absent and the diagnosis can be established only by nerve biopsy.

Schizophrenia and fatal self-induced water intoxication with appropriately-diluted urine.

A 31-year-old woman with untreated chronic schizophrenia developed extreme polydipsia which rapidly led to coma and death due to cerebral edema. Hyponatremia (120 mEq/liter) and serum hypo-osmolality (260 mOsm/kg) were associated with marked polyuria (up to 1850 ml/hour) and appropriately low urinary osmolality (90 mOsm/kg) which responded to treatment. This case and few qualifying previous reports which are reviewed support the possibility that pure self-induced water intoxication with no major contribution of inadequate release of antidiuretic hormone may occur, and that extreme polydipsia can sometimes overwhelm normal renal diluting capacity in psychotic patients.

Immediate dressing of the burn wound--will it change its natural history?

Thirty deep dermal burns were inflicted on six domestic pigs. On the treated animals, the epidermis was removed and immediately replaced with Omiderm, Xeroform or Mettalin dressings. Burns in which the blister was left intact or not dressed after epidermis removal, served as controls. Macroscopic and microscopic assessment of the healing process was then carried out. Surprisingly, the no-epidermis controls healed somewhat faster than did the untreated controls (those with blister intact). On day 7, 83 per cent of the treated lesions showed initiation of epidermal regeneration, compared with 58 per cent in the no-treatment and no-epidermis controls. All dressing materials were found equal in this model, and differences between control and treatment groups were not significant after 12 days.

Improving ion mass ratio performance at low concentrations in methamphetamine GC-MS assay through internal standard selection.

In federally regulated drug testing, laboratories must identify and quantitate drugs and their breakdown products by gas chromatography-mass spectrometry (GC-MS) to a concentration that is at least 60% below the cutoff concentration for reconfirmation purposes. Use of methamphetamine-d5 as an internal standard in routine testing with derivatization by HFBA was found to contribute m/z 91 and 118 ions to the same ions from the nondeuterated methamphetamine in the specimen. This resulted in poor chromatography and occasionally caused the 91/254 and 118/254 ion mass ratios to exceed the +20% acceptance limit established in the calibration process at low concentrations. The analogues methamphetamine-d8 and -d11 were evaluated for contributions to the nondeuterated methamphetamine ion fragments. Methamphetamine-d8 produced m/z 91 and 118 ions, but in less abundance than methamphetamine-d5. Methamphetamine-d11 was found to produce little or no detectable m/z 91 or 118. Replacing methamphetamine-d5 with methamphetamine-d11 eliminates this problem and allows the assay to consistently produce ion mass ratios and acceptable chromatography sufficient for identifying and quantitating methamphetamine at 60% below the 500-ng/mL cutoff concentration.

Analysis of gabapentin in serum and plasma by solid-phase extraction and gas chromatography-mass spectrometry for therapeutic drug monitoring.

A simple method for the determination of gabapentin (Neurontin) is described. The method uses solid-phase extraction by disk column and derivatization followed by gas chromatographic-mass spectrometric analysis. The single-step derivatization with MTBSTFA produces a t-BDMS derivative of both the carboxylic and amine moieties of the molecule. Each step of the procedure was optimized to assure reliable performance of the method. The assay limit of detection was 0.1 microg/mL with a linear range from 1.0 to 35 microg/mL. Within-run (n = 3) and between-run (n = 40) coefficients of variation were less than 8.2 and 15.9%, respectively. The method has proven reliable in routine production for more than a year, producing clean chromatography with unique ion fragments, consistent ion mass ratios, and no interferences. Statistical analysis of the gabapentin concentrations measured in 1020 random specimens over a 2-month period showed a mean concentration of 6.07 microg/mL with a standard deviation of 5.28.

Comparison of four derivatizing reagents for 6-acetylmorphine GC-MS analysis.

The propionyl, trimethylsilyl, trifluroacetyl, and heptafluoroacyl derivatives of 6-acetylmorphine (6-AM) were evaluated with respect to optimal method performance, derivative stability, and methods characterization for use in gas chromatographic-mass spectrometric (GC-MS) analysis with electron ionization mode and selected ion monitoring. The most common potential interferences and compatibility with other derivatives when used on the same GC-MS were determined for the derivatizing reagents. The propionyl, trimethylsilyl, and trifluroacetyl derivatives produced adequate stability, accuracy, and precision for the method. The 6-AM derivatization with commercially available propionic anhydride generated a relatively small amount of 6-AM-propionyl derivative from the free morphine present in a specimen. The trimethylsilyl derivative obtained by the reaction with MSTFA did not require incubation, was the easiest to prepare, and had the highest potential for use on an automated sample-preparation device. An important advantage of derivatization with MSTFA is elimination of the possibility of heroin decomposition to 6-AM that is due to incubation at elevated temperature.

A rapid gas chromatographic method quantitating clozapine in human plasma or serum for the purpose of therapeutic monitoring.

A gas chromatographic method using nitrogen-phosphorus detection was developed to quantitate clozapine in plasma or serum. Methyl clonazepam was used as an internal standard. Sample preparation included a single-step extraction with ethyl acetate, which was injected directly onto a wide-bore capillary column. Within-run and total precision, measured as percent coefficient of variation, were determined at low, therapeutic, and high clozapine plasma concentrations. The within-run precision for the low, therapeutic, and high clozapine plasma samples was 5.2, 2.7, and 2.4%, respectively. The total precision for the low, therapeutic, and high clozapine plasma samples was 10.0, 2.6, and 2.0%, respectively. Analytical accuracy was evaluated by comparing quantitative results with those obtained from a reference laboratory. Those samples containing therapeutic or high concentrations agreed within 3%; the sample containing a subtherapeutic concentration differed by 11.9%. The limit of quantitation was determined to be 35 ng/mL, and the upper limit of linearity was 3000 ng/mL. No significant interferences were detected after testing more than two dozen drugs and metabolites.

Nitrite adulteration of workplace urine drug-testing specimens. I. Sources and associated concentrations of nitrite in urine and distinction between natural sources and adulteration.

The active ingredient in the commercial workplace urine drug-testing adulterant, Klear, was previously determined to be nitrite ion. Nitrite adulteration compromises the confirmation of some drugs, notably the marijuana metabolite. A previously reported bisulfite step overcomes some nitrite adulteration, but it cannot do so in every case, which leaves the laboratory to report the specimen as not suitable for testing. Unlike many other adulterants, nitrite is found in normal urine at low concentrations. In order to defend a report of nitrite adulteration, it is necessary to provide evidence that the amount of nitrite in a workplace urine specimen could not arise by normal means. The objectives of this study were to identify all sources of nitrite in urine and the range of concentrations associated with these sources and to determine if nitrite adulteration can be supported based upon a quantitative result. The scientific literature was reviewed for internal and external sources of nitrite and their concentration ranges and are reported. The following specimens were obtained and nitrite concentrations measured by a spectrophotometric method: clinical specimens nitrite positive by test strip (< 15 micrograms/mL); specimens culture positive for nitrate-reducing microorganisms (< 36 micrograms/mL); specimens from patients on medications that may metabolize to nitrite (< 6 micrograms/mL); and drug-test specimens, both negative (< 130 micrograms/mL) and others that appeared to be adulterated with nitrite (range 1910-12,200 micrograms/mL, mean 5910). The literature and the nitrite measurements of this study indicate a substantial difference between concentrations from natural sources compared with adulteration. A quantitative measurement of nitrite by a well-structured assay can provide scientifically valid and forensically defensible proof of adulteration with a nitrite-containing substance.