The Common Carotid Artery in the Ultrasound Evaluation of Giant Cell Arteritis.

OBJECTIVES: Vascular ultrasound is commonly used to diagnose giant cell arteritis (GCA). Most protocols include the temporal arteries and axillary arteries, but it is unclear which other arteries should be included. This study investigated whether inclusion of intima media thickness (IMT) of the common carotid artery (CCA) in the ultrasound evaluation of GCA improves the accuracy of the examination. METHODS: We formed a fast-track clinic to use ultrasound to rapidly evaluate patients with suspected GCA. In this cohort study, patients referred for new concern for GCA received a vascular ultrasound for GCA with the temporal arteries and branches, the axillary artery, and CCA. RESULTS: We compared 57 patients with GCA and 86 patients without GCA. Three patients with GCA had isolated positive CCA between 1 and 1.49 mm, and 21 patients without GCA had isolated positive CCA IMT. At the 1.5-mm CCA cutoff, 4 patients without GCA had positive isolated CCA, and 1 patient with GCA had a positive isolated CCA. The sensitivity of ultrasound when adding carotid arteries to temporal and axillary arteries was 84.21% and specificity 65.12% at an intima media thickness (IMT) cutoff of ≥1 mm and 80.70% and 87.21%, respectively, at a cutoff of ≥1.5 mm. CONCLUSION: Measurement of the CCA IMT rarely contributed to the diagnosis of GCA and increased the rate of false-positive results. Our data suggest that the CCA should be excluded in the initial vascular artery ultrasound protocol for diagnosing GCA. If included, an IMT cutoff of higher than 1.0 mm should be used.

The Usefullness of Subclavian Artery Ultrasound Assessment in Giant Cell Arteritis Evaluation.

OBJECTIVE: Vascular ultrasound has been increasingly used to diagnose giant cell arteritis (GCA). The temporal and axillary arteries are commonly evaluated. However, the usefulness of including the subclavian artery remains unclear. This study investigated whether inclusion of the subclavian artery in addition to the temporal and axillary arteries in the ultrasound evaluation of GCA improves the accuracy of the examination beyond ultrasonography of the temporal and axillary arteries alone. METHODS: We formed a fast-track clinic to use ultrasound to rapidly evaluate patients with suspected GCA. In this cohort study, patients referred for new concern for GCA received a vascular ultrasound for GCA. Subclavian intima-media thickness (IMT) cutoffs of 1.0 and 1.5 mm were retrospectively assessed. RESULTS: Two hundred thirty-seven patients were referred to the fast-track clinic from November 2017 to August 2021. One hundred sixty-eight patients received an ultrasound for concern for new GCA. With a subclavian IMT cutoff of 1.5 mm, inclusion of the subclavian artery did not identify any patients with GCA who were not otherwise found to have positive temporal and/or axillary artery examinations, and at this cutoff, there was 1 false-positive result. A subclavian IMT cutoff of 1.0 mm identified several subjects diagnosed with GCA who had otherwise negative ultrasounds, but most subjects with an isolated subclavian IMT greater than 1.0 mm had false-positive results, and the specificity of this cutoff was poor. CONCLUSION: Inclusion of the subclavian artery in the ultrasound assessment of GCA at 2 different cutoffs rarely contributed to the accurate diagnosis of GCA and increased the rate of false-positive results.

Ultrasound-guided adductor canal block: a cadaver study investigating the effect of a thigh tourniquet.

BACKGROUND: Placement of local anaesthetic within the adductor canal using ultrasonography is an alternative to femoral nerve blocks for postoperative pain relief after knee joint replacement surgery. However, the effect of an inflated thigh tourniquet on the distribution of local anaesthetic within the adductor canal is unknown. The aim of this cadaveric study was to compare the distribution of radio-opaque dye within the adductor canal in the presence or absence of an inflated thigh tourniquet. METHODS: Bilateral ultrasound-guided adductor canal blocks were performed on the thawed lower limbs of five fresh frozen human cadavers. The left and right lower cadaver limbs were randomised to receive or not receive a thigh tourniquet inflated to 300 mm Hg for 1 h. X-rays with iohexol radio-opaque dye were obtained in four views, and fiducial markers inserted as reference points. Virtual editing technology was used to recreate outlines representing the distribution of the radio-opaque dye and superimpose these on anatomical images. RESULTS: Radio-opaque dye was distributed on the medial aspect of the thighs with entire and well circumscribed margins. The majority of the radio-opaque dye was confined within the adductor canal. Superior-inferior dye distribution was 315 mm [95% confidence intervals (CI) 289-342] and 264 mm (95% CI 239-289) in the presence and absence of an inflated thigh tourniquet, respectively (diff 95% CI -80.46 to -22.22, P=0.0081). Image analysis using the recreated radio-opaque outlines suggested that the most proximal point of the radio-opaque dye was 100 mm (95% CI 82-117) or 117 mm (95% CI 62-171) below the inguinal ligament in the presence and absence of an inflated thigh tourniquet, respectively (diff 95% CI -38 to 72, P=0.456). CONCLUSIONS: Application and inflation of thigh tourniquets significantly increased the combined superior-inferior dye distribution within the adductor canal of cadaveric limbs. There was insufficient evidence to suggest significant proximal spread of 25 ml of local anaesthetic to involve the motor branches of the femoral nerve. In some patients, the local anaesthetic may reach the popliteal fossa in close approximation to the sciatic nerve.

Loss of Rearranged L-Myc Fusion (RLF) results in defects in heart development in the mouse.

Recently we reported that Rearranged L-Myc Fusion, RLF, acts as an epigenetic modifier maintaining low levels of DNA methylation at CpG island shores and enhancers across the genome. Here we focus on the phenotype of Rlf null mutant mice generated via an ENU mutagenesis screen, to identify genes required for epigenetic regulation. RLF is expressed in a range of fetal mouse tissues, including the fetal heart. Comprehensive timed-mating studies are consistent with our previously reported findings that Rlf homozygous mutant mice rarely survive to adulthood, with the majority dying shortly after birth. Histological analysis of two independent Rlf ENU mutant lines at E11.5-E14.5 showed heart defects resembling those present in humans with Left Ventricular Non-Compaction (LVNC). In situ hybridisation analysis localized expression of Rlf to the endocardium and epicardium of embryonic and postnatal hearts, and transiently to cardiomyocytes during heart looping and early chamber formation stages. RNA-seq analysis of Rlf mutant hearts highlighted defective NOTCH pathway signalling, recently describe as one cause of LVNC. This study provides the first evidence that RLF is required for normal heart development in the mouse. The heart morphological defects present at high penetrance in Rlf mutants are consistent with features of LVNC in humans, and molecular analysis identified attenuated JAGGED 1 expression and NOTCH signalling as likely contributors to these defects. Our study highlights the importance of RLF-dependent epigenetic modifications to DNA for maintaining correct gene regulatory network and intercellular signalling interactions during heart chamber and septal development. Further investigations are needed to define the biochemical role of RLF in the developing heart, and whether RLF mutations are a cause of heart defects in humans.

Markers of the p53 pathway further refine molecular profiling in high-risk endometrial cancer: A TransPORTEC initiative.

BACKGROUND: The morphological classification of high-risk endometrial cancer is of limited prognostic value. Recent attempts to stratify tumours according to molecular signatures have shown considerable promise. Here we attempted to further refine molecular classifications using markers of the p53 pathway. METHODS: We analysed the expression of p53 as well as three downstream markers of the p53 pathway, p21, mdm2 and phospho-p63 (pp63), by immunohistochemistry in a series of 114 endometrial cancers (86 endometrioid, 28 non-endometrioid subtype) with high-risk features (such as high tumour grade and deep myometrial invasion) and correlated results with clinical outcome. The Cancer Genome Atlas (TCGA) data were used to analyse TP63 mutations and copy-number alterations using cBioPortal. TP53 was silenced in two endometrial cancer cell lines to study its effect on p21 and p63. RESULTS: About half of the tumours showed a p53 mutant phenotype and there was a strong negative correlation with p21 expression. Being marker positive for pp63 or mdm2 was associated with a significantly increased likelihood of dying, [hazard ratios 5.93 (95% CI 2.37-7.27) and 7.48 (95% CI 3.04-9.39), respectively]. These findings were seen in both p53 wildtype and p53 mutant tumours. Only 11% of TCGA endometrial cancers had a functional TP63 alteration. Upon silencing of TP53, p21 expression was decreased in one cell line, but no effects on p63 were observed. CONCLUSION: Markers of the p53 pathway improve stratification of endometrial cancers and provide novel insights into the role of this pathway in the disease.

Treatment of septic arthritis of the coxofemoral joint in 12 foals.

OBJECTIVE: To describe the clinical signs, surgical treatment, and outcome of septic arthritis of the coxofemoral joint in foals. STUDY DESIGN: Retrospective clinical study. SAMPLE POPULATION: Foals (n = 12) with confirmed sepsis of the coxofemoral joint. METHODS: Lameness was localized to the coxofemoral joint based on physical examination. Sepsis was confirmed by cytological analysis of synovial fluid obtained under ultrasonographic guidance, during general anesthesia or standing sedation. Intra-articular analgesia was used as an adjunct diagnostic modality in 2 foals. Surgical lavage of the affected joint was performed via arthroscopy or needle lavage, with repeated lavage performed in 7 foals. RESULTS: Synovial fluid contained 4.4 to 173 × 109 /L white blood cells (WBCs), and 38-63 g/L total protein. Cultures were positive in 10/12 foals. Isolated organisms included Salmonella spp., Streptococcus spp., Rhodococcus spp., Enterococcus spp., Escherichia spp., Staphylococcus spp., Acinetobacter spp., Methicillin-resistant Staphylococcus aureus and Bacillus spp. Ten foals were discharged from hospital (83%). One of these was euthanized 15 days later due to chronic intestinal salmonellosis and renal failure, and 9 foals survived with no residual lameness detected 1 year after discharge from hospital. CONCLUSIONS: Sepsis of the coxofermoral joint can be effectively treated with a combination of arthroscopic lavage and the use of systemic and local antimicrobials.

Vascular Ultrasound for Giant Cell Arteritis: Establishing a Protocol Using Vascular Sonographers in a Fast-Track Clinic in the United States.

OBJECTIVE: We developed a fast-track clinic (FTC) to expedite the evaluation of patients suspected of having giant cell arteritis (GCA) using vascular ultrasound. Though FTCs have demonstrated efficacy in Europe, no protocolized clinic in the United States has been developed. This study introduces a new FTC model unique to the United States, using vascular sonographers, and describes the protocols used to develop reliable findings. We evaluate clinical outcomes using vascular ultrasound and temporal artery biopsy (TAB). METHODS: A retrospective review included all subjects referred to the University of Washington FTC aged 50 years old or older who received both ultrasound and TAB between November 2017 and November 2019. Ultrasound was performed by a vascular sonographer trained in GCA detection. Ultrasound results were read by a vascular surgeon and reviewed by four rheumatologists certified in musculoskeletal ultrasound who had completed a course in vascular ultrasound use in GCA and large-vessel vasculitis. RESULTS: A total of 43 subjects underwent both vascular ultrasound and TAB. Six subjects had both positive ultrasound and TAB results. There were also seven positive ultrasound results in patients with negative TAB results, most due to detection of large-vessel GCA (LV-GCA). All 29 subjects with negative ultrasound results had negative TAB results. CONCLUSION: This is the first study in the United States to demonstrate a reliable FTC protocol using vascular sonographers. This protocol demonstrated good agreement between ultrasound and TAB and allowed for the detection of additional cases of LV-GCA by vascular ultrasound. Vascular ultrasound improved the rate of GCA diagnosis primarily by detecting additional cases of LV-GCA.

Association of owner-reported noise with findings during dynamic respiratory endoscopy in Thoroughbred racehorses.

REASONS FOR PERFORMING STUDY: To determine the association between owner-reported noise and findings during dynamic respiratory endoscopy (DRE) in a large case series. HYPOTHESIS: The sensitivity of owner-reported noise for dynamic upper respiratory tract obstructions in horses is low, and the specificity is high. METHODS: One hundred horses underwent DRE for the investigation of abnormal respiratory noise and/or poor performance. The association of abnormal noise with findings during DRE was evaluated. RESULTS: Eighty-five horses underwent DRE for the investigation of abnormal respiratory noise. Of these, 82% were found to have one or more obstructive upper respiratory tract abnormalities during DRE. Forty-eight percent of horses reported to gurgle, rattle or make a rough noise were diagnosed with solitary palatal dysfunction. A further 24% with this history showed palatal dysfunction in combination with an additional abnormality. Twenty-seven percent of horses with a history of whistling or roaring showed some degree of recurrent laryngeal neuropathy. Seven percent of horses with a history of whistling or roaring had vocal cord collapse as a solitary condition, whereas 40% had vocal cord collapse and another abnormality. The sensitivity of abnormal respiratory noise for any obstruction of the upper portion of the respiratory tract was high (84%), while the specificity was low (25%). Characteristic owner reported noise patterns showed moderate to low sensitivity for specific conditions. Whistling and roaring showed the highest specificity (≥80%) for laryngeal dysfunction. CONCLUSION: Diagnosis of upper respiratory tract obstructions based solely on owner-reported noise and performance history may result in incomplete diagnoses. CLINICAL RELEVANCE: DRE should be performed in horses with abnormal respiratory noise to rule out complex conditions of the upper portion of the respiratory tract.

The U.S. Veterans Health Administration national giant cell arteritis (GCA) database cohort: incident ophthalmic complications in biopsy-negative GCA patients.

INTRODUCTION/OBJECTIVES: This study aimed to identify the incidence of ophthalmic complications of giant cell arteritis (GCA) among subjects with negative temporal artery biopsy (TAB) and to determine if duration of prednisone exposure relative to GCA diagnosis was associated with ophthalmic complications in TAB-negative subjects. METHOD: The U.S. Veterans Health Administration (VHA) national database was queried for subjects between 1999 and 2017 with ICD-9/-10 diagnosis code for GCA, procedure code for TAB, and ICD-9/-10 diagnosis code for blindness, anterior or posterior ischemic optic neuropathy, or branch or central retinal artery occlusion. Pharmacy data regarding prednisone dispensation were collected. A Cox proportional hazard model was performed using ophthalmic complication by 1 year as the outcome variable in TAB-negative subjects, adjusting for age, TAB length, TAB laterality, and prednisone dose relative to GCA diagnosis date. RESULTS: Incident ophthalmic complication occurred by 1 year in 9.6% with positive TAB and in 6.2% with negative TAB. The majority of complications occurred within the first month for both groups. Compared to a reference group of prednisone initiation 0-14 days prior to GCA diagnosis, ophthalmic complications in TAB-negative subjects were significantly higher when prednisone initiation was delayed 14-28 days after GCA diagnosis. CONCLUSIONS: A substantial number of TAB-negative subjects accrued an incident ICD-9/-10 code for ophthalmologic complication within a year after diagnosis, most occurring within the first month. Delaying prednisone initiation 14-28 days after GCA diagnosis in TAB-negative subjects led to a 3.5-fold higher rate of ophthalmic events occurring by 1 year. Key Points • This study provides an incidence rate of ophthalmic complication by one year in biopsy-negative subjects suspected of having GCA. • Delaying prednisone initiation 14-28 days after GCA diagnosis in TAB-negative GCA subjects led to a 3.5-fold higher rate of ophthalmic events occurring by 1 year.

Physical activity recommendations for cancer survivors living with bony metastases: views of oncologic healthcare providers.

PURPOSE: The potential of physical activity to improve function and quality of life of an individual with advanced cancer is now established. The purpose of this survey of oncologic healthcare providers (OHPs) is to understand their attitude towards physical activity for individuals living with bony metastases and to assess requirements to confidently provide physical activity recommendations. METHODS: A web-based survey administered through Qualtrics™ included questions on participant demographics and attitude questions ranked on a Likert scale. Eligibility was a physician or nurse practitioner currently providing care in the cancer care system of a public healthcare system in Canada. 338 participants were identified and invited to participate in this survey. RESULTS: The response rate was 34.6%. The majority of OHPs believed physical activity is important (89%) and safe (82%) in individuals living with bony metastases. OHPs agreed that these individuals looked to them for physical activity recommendations (74%) and that these recommendations would be followed (58%). Yet, 86% of OHPs felt they required more information before they could recommend physical activity to individuals living with bony metastases, and less than half (43%) of OHPs felt confident enough to prescribe physical activity. CONCLUSIONS: OHPs agree that physical activity for individuals living with bony metastatic cancer is beneficial and safe. However, OHPs are not confident in their ability to recommend physical activity to this population. IMPLICATIONS FOR CANCER SURVIVORS: There is a need to create physical activity guidelines for individuals living with bony metastases and improve access to rehabilitation services.

Dynamic respiratory endoscopy in 67 Thoroughbred racehorses training under normal ridden exercise conditions.

REASONS FOR PERFORMING STUDY: There are potential advantages to imaging the upper portion of the respiratory tract (URT) of horses during ridden exercise. With the advent of a wireless endoscope, this is now possible. However, there has been no detailed validation of the technique and findings have not been compared to ridden speed. OBJECTIVES: To assess the combined use of a Dynamic Respiratory Scope (DRS) and global positioning system (GPS) receiver for examining the URT of a group of Thoroughbred racehorses randomly selected from a single flat racehorse training establishment. METHODS: Horses were selected randomly from a population of Thoroughbred horses in training at the same training yard. Endoscopic images of the URT were recorded during a ridden exercise test on an 'all-weather-gallop' and were reviewed post testing. Speed was measured using a wrist mounted GPS receiver. RESULTS: A total of 67 (34%) of the 195 horses in training were examined. Endoscopic findings included: normal URT function (44 cases); dorsal displacement of the soft palate (DDSP) (13 cases); laryngeal asymmetry (4 cases); and axial deviation of the aryepiglottic folds (3 cases). Maximum speed obtained by individual horses ranged from 41.8-56.3 km/h. Ridden speed was variably affected by DDSP. CONCLUSIONS: The DRS provides a safe effective system for imaging the equine URT during ridden exercise at speed. The abnormalities of the URT identified were similar to those observed during treadmill endoscopy studies reported in the literature. The effect of URT abnormalities on ridden speed requires further investigation. POTENTIAL RELEVANCE: This technique can be used to diagnose common causes of URT associated with poor performance in horses during normal training. This has substantial implications for future clinical diagnosis and treatment of URT pathology.

Frequent activating FGFR2 mutations in endometrial carcinomas parallel germline mutations associated with craniosynostosis and skeletal dysplasia syndromes.

Endometrial carcinoma is the most common gynecological malignancy in the United States. Although most women present with early disease confined to the uterus, the majority of persistent or recurrent tumors are refractory to current chemotherapies. We have identified a total of 11 different FGFR2 mutations in 3/10 (30%) of endometrial cell lines and 19/187 (10%) of primary uterine tumors. Mutations were seen primarily in tumors of the endometrioid histologic subtype (18/115 cases investigated, 16%). The majority of the somatic mutations identified were identical to germline activating mutations in FGFR2 and FGFR3 that cause Apert Syndrome, Beare-Stevenson Syndrome, hypochondroplasia, achondroplasia and SADDAN syndrome. The two most common somatic mutations identified were S252W (in eight tumors) and N550K (in five samples). Four novel mutations were identified, three of which are also likely to result in receptor gain-of-function. Extensive functional analyses have already been performed on many of these mutations, demonstrating they result in receptor activation through a variety of mechanisms. The discovery of activating FGFR2 mutations in endometrial carcinoma raises the possibility of employing anti-FGFR molecularly targeted therapies in patients with advanced or recurrent endometrial carcinoma.

Fractures of the cervical vertebral odontoid in four horses and one pony.

Four horses and one pony, ranging in age from one to 11 years, were diagnosed with two different types of odontoid peg fractures. Their clinical signs included reluctance to move the neck and head, dullness, and abnormalities of gait. Radiography was essential for the diagnosis, and the method of treatment varied depending on the severity of the neurological signs, the intended use of the horse, and financial constraints. Optimal treatment requires a technique that allows decompression, anatomical alignment, and stabilisation of the odontoid fracture. If the clinical (neurological) signs are not too severe and the animal shows signs of feeling peripheral pain, conservative treatment can be applied, as is common practice in human surgery. All except the pony made a full recovery.

Reduced brain glucose with normal plasma glucose in salicylate poisoning.

After the intraperitoneal injection into young mice of 700-800 mg/kg of salicylate, brain glucose fell to one-third or less of control values despite normal plasma glucose levels; brain lactate was nearly doubled and there were small decreases in phosphocreatine (18%) and in glycogen (17%). ATP, pyruvate, alpha-ketoglutarate, and glutamate were unchanged. In liver, glycogen was reduced 79% and lactate was five times higher than in control animals; glucose, glucose-6-phosphate, and ATP were unchanged. Since salicylate uncouples oxidative phosphorylation, it is postulated that high energy phosphate in the brain is maintained near normal levels by a compensatory increase in cerebral glycolysis. Apparently the brain glucose level falls because the rate of utilization exceeds the rate at which glucose can be supplied from the blood. Concurrent administration of glucose with salicylate elevated brain glucose concentration and was associated with striking improvement in the condition and the increased survival of the animals. These findings stress the fact that in salicylate poisoning the supply of glucose to the brain may be inadequate even when blood glucose levels are normal.

Evidence for three tumor suppressor loci on chromosome 9p involved in melanoma development.

Cytogenetic and loss of heterozygosity (LOH) studies have long indicated the presence of a tumor suppressor gene (TSG) on 9p involved in the development of melanoma. Although LOH at 9p has been reported in approximately 60% of melanoma tumors, only 5-10% of these tumors have been shown to carry CDKN2A mutations, raising the possibility that another TSG involved in melanoma maps to chromosome 9p. To investigate this possibility, a panel of 37 melanomas derived from 35 individuals was analyzed for CDKN2A mutations by single-strand conformation polymorphism analysis and sequencing. The melanoma samples were then typed for 15 markers that map to 9p13-24 to investigate LOH trends in this region. In those tumors demonstrating retention of heterozygosity at markers flanking CDKN2A and LOH on one or both sides of the gene, multiplex microsatellite PCR was performed to rule out homozygous deletion of the region encompassing CDKN2A. CDKN2A mutations were found in tumors from 5 patients [5 (14%) of 35], 4 of which demonstrated LOH across the entire region examined. The remaining tumor with no observed LOH carried two point mutations, one on each allele. Although LOH was identified at one or more markers in 22 (59%) of 37 melanoma tumors corresponding to 20 (57%) of 35 individuals, only 11 tumors from 9 individuals [9 (26%) of 35] demonstrated LOH at D9S942 and D9S1748 the markers closest to CDKN2A. Of the remaining 11 tumors with LOH 9 demonstrated LOH at two or more contiguous markers either centromeric and/or telomeric to CDKN2A while retaining heterozygosity at several markers adjacent to CDKN2A. Multiplex PCR revealed one tumor carried a homozygous deletion extending from D9S1748 to the IFN-alpha locus. In the remaining eight tumors, multiplex PCR demonstrated that the observed heterozygosity was not attributable to homozygous deletion and stromal contamination at D9S1748, D9S942, or D9S974, as measured by comparative amplification strengths, which indicates that retention of heterozygosity with flanking LOH does not always indicate a homozygous deletion. This report supports the conclusions of previous studies that a least two TSGs involved in melanoma development in addition to CDKN2A may reside on chromosome 9p.

CDKN2A/p16 is inactivated in most melanoma cell lines.

The CDKN2A gene maps to chromosome 9p21-22 and is responsible for melanoma susceptibility in some families. Its product, p16, binds specifically to CDK4 and CDK6 in vitro and in vivo, inhibiting their kinase activity. CDKN2A is homozygously deleted or mutated in a large proportion of tumor cell lines and some primary tumors, including melanomas. The aim of this study was to investigate the involvement of CDKN2A and elucidate the mechanisms of p16 inactivation in a panel of 60 cell lines derived from sporadic melanomas. Twenty-six (43%) of the melanoma lines were homozygously deleted for CDKN2A, and an additional 15 (25%) lines carried missense, nonsense, or frameshift mutations. All but one of the latter group were shown by microsatellite analysis to be hemizygous for the region of 9p surrounding CDKN2A. p16 was detected by Western blotting in only five of the cell lines carrying mutations. Immunoprecipitation of p16 in these lines, followed by Western blotting to detect the coprecipitation of CDK4 and CDK6, revealed that p16 was functionally compromised in all cell lines but the one that carried a heterozygous CDKN2A mutation. In the remaining 19 lines that carried wild-type CDKN2A alleles, Western blot analysis and immunoprecipitation indicated that 11 cell lines expressed a wild-type protein. Northern blotting was performed on the remaining eight cell lines and revealed that one cell line carried an aberrantly sized RNA transcript, and two other cell lines failed to express RNA. The promoter was found to be methylated in five cell lines that expressed CDKN2A transcript but not p16. Presumably, the message seen by Northern blotting in these cell lines is the result of cross-hybridization of the total cDNA probe with the exon 1beta transcript. Microsatellite analysis revealed that the majority of these cell lines were hemi/homozygous for the region surrounding CDKN2A, indicating that the wild-type allele had been lost. In the 11 cell lines that expressed functional p16, microsatellite analysis revealed loss of heterozygosity at the markers immediately surrounding CDKN2A in five cases, and the previously characterized R24C mutation of CDK4 was identified in one of the remaining 6 lines. These data indicate that 55 of 60 (92%) melanoma cell lines demonstrated some aberration of CDKN2A or CDK4, thus suggesting that this pathway is a primary genetic target in melanoma development.

Tamoxifen in men: a review of adverse events.

Tamoxifen is an off-label option to treat men for breast cancer, infertility, and idiopathic gynecomastia. Lately, tamoxifen has been proposed as a treatment to prevent gynecomastia in prostate cancer patients receiving antiandrogen therapy. We reviewed the adverse events (AEs) reported in studies of men prescribed tamoxifen for these conditions to better understand its side-effect profile. We searched PubMed for randomized controlled trials (RCTs) that included safety data of tamoxifen treatment in men with prostate cancer, breast cancer, infertility, and idiopathic gynecomastia. Non-RCTs were also reviewed. The results demonstrate that the AE profile in tamoxifen-treated male populations varied. Excluding breast events, gastrointestinal, and cardiovascular problems were the most commonly reported AEs in prostate cancer patients, whereas more psychiatric disorders were reported in male breast cancer patients. Few AEs have been documented in men receiving tamoxifen for infertility and idiopathic gynecomastia. Less than 5% of men withdrew from tamoxifen therapy because of toxicity. This suggests that for most men, tamoxifen is well-tolerated. Of those who discontinued tamoxifen, the majority were male breast cancer patients, and cardiovascular events were the most common reason for stopping tamoxifen treatment. Unfortunately, in many cases, the reasons for withdrawing tamoxifen were unspecified. Based on the available evidence, tamoxifen's AE profile appears to vary depending upon which male population is treated. Also, the frequency at which AEs occur varies - less AEs in men with infertility and idiopathic gynecomastia compared to men with prostate cancer or breast cancer. Long-term studies that rigorously document the side-effect profile of tamoxifen in men are lacking.

Evidence for u.v. induction of CDKN2 mutations in melanoma cell lines.

The CDKN2 gene, encoding the cyclin dependent kinase inhibitor p16, is a tumour suppressor gene involved in melanoma and maps to chromosome band 9p22. Mutations or interstitial deletions of this gene have been found both in the germline of familial melanoma cases and somatically in melanoma cell lines. Previous mutation analyses of melanoma cell lines have indicated a high frequency of C:G to T:A transitions, with all of these mutations occurring at dipyrimidine sites. Including three melanoma cell lines carrying tandem CC to TT mutations, the spectrum of mutations so far reported indicates a possible role for u.v. radiation in the mutagenesis of this gene in some tumours. To further examine this hypothesis we have characterised mutations of the CDKN2 gene in 30 melanoma cell lines. Nineteen lines carried complete or partial homozygous deletions of the gene. Of the remaining cell lines, eight were shown by direct sequencing of PCR products from exon 1 and exon 2 to carry a total of nine different mutations of CDKN2. Two cell lines carried tandem CC to TT mutations and a high rate of C:G to T:A transitions was observed. This study provides further evidence for the role of u.v. light in the genesis of melanoma, with one target being the CDKN2 tumour suppressor gene.

Analysis of the CDKN2A, CDKN2B and CDK4 genes in 48 Australian melanoma kindreds.

Germline mutations within the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene and one of its targets, the cyclin dependent kinase 4 (CDK4) gene, have been identified in a proportion of melanoma kindreds. In the case of CDK4, only one specific mutation, resulting in the substitution of a cysteine for an arginine at codon 24 (R24C), has been found to be associated with melanoma. We have previously reported the identification of germline CDKN2A mutations in 7/18 Australian melanoma kindreds and the absence of the R24C CDK4 mutation in 21 families lacking evidence of a CDKN2A mutation. The current study represents an expansion of these efforts and includes a total of 48 melanoma families from Australia. All of these families have now been screened for mutations within CDKN2A and CDK4, as well as for mutations within the CDKN2A homolog and 9p21 neighbor, the CDKN2B gene, and the alternative exon 1 (E1beta) of CDKN2A. Families lacking CDKN2A mutations, but positive for a polymorphism(s) within this gene, were further evaluated to determine if their disease was associated with transcriptional silencing of one CDKN2A allele. Overall, CDKN2A mutations were detected in 3/30 (10%) of the new kindreds. Two of these mutations have been observed previously: a 24 bp duplication at the 5' end of the gene and a G to C transversion in exon 2 resulting in an M531 substitution. A novel G to A transition in exon 2, resulting in a D108N substitution was also detected. Combined with our previous findings, we have now detected germline CDKN2A mutations in 10/48 (21%) of our melanoma kindreds. In none of the 'CDKN2A-negative' families was melanoma found to segregate with either an untranscribed CDKN2A allele, an R24C CDK4 mutation, a CDKN2B mutation, or an E1beta mutation. The last three observations suggest that these other cell cycle control genes (or alternative gene products) are either not involved at all, or to any great extent, in melanoma predisposition.