Evolution of MET and NRAS gene amplification as acquired resistance mechanisms in EGFR mutant NSCLC.

EGFR mutant non-small cell lung cancer patients' disease demonstrates remarkable responses to EGFR-targeted therapy, but inevitably they succumb to acquired resistance, which can be complex and difficult to treat. Analyzing acquired resistance through broad molecular testing is crucial to understanding the resistance mechanisms and developing new treatment options. We performed diverse clinical testing on a patient with successive stages of acquired resistance, first to an EGFR inhibitor with MET gene amplification and then subsequently to a combination EGFR and MET targeted therapies. A patient-derived cell line obtained at the time of disease progression was used to identify NRAS gene amplification as an additional driver of drug resistance to combination EGFR/MET therapies. Analysis of downstream signaling revealed extracellular signal-related kinase activation that could only be eliminated by trametinib treatment, while Akt activation could be modulated by various combinations of MET, EGFR, and PI3K inhibitors. The combination of an EGFR inhibitor with a MEK inhibitor was identified as a possible treatment option to overcome drug resistance related to NRAS gene amplification.

Short communication: Circulating serotonin (5-HT) concentrations on day 1 of lactation as a potential predictor of transition-related disorders.

The monoamine serotonin (5-hydroxytryptamine; 5-HT) has been described as a homeostatic regulator of lactation. Recently, our laboratory determined that 5-HT is involved in the regulation of calcium and glucose homeostasis during the transition period in rodents. More specifically, we demonstrate that 5-HT is responsible for calcium mobilization from bone and upregulation of hepatic gluconeogenic enzymes and mammary gland glucose transporters. Our objective was to investigate the correlation between circulating 5-HT concentrations and circulating ionized calcium, parathyroid hormone-related protein (PTHrP), and glucose concentrations on d 1 postpartum. We also investigated the correlation between circulating 5-HT and milk fever and ketosis incidence and severity in multiparous Holstein cows at the onset of lactation. Blood samples were collected from 42 multiparous cows on d 1 of lactation and analyzed for 5-HT, calcium, glucose, and PTHrP. Milk fever (determined subjectively for each cow on d 1 postpartum) and ketosis incidence and severity (scale 1 to 4, determined objectively for each cow during the first 10 d postpartum) were recorded for all animals. Serum 5-HT was positively correlated with serum calcium and with plasma PTHrP (r>0.37). Serum 5-HT was negatively correlated with milk fever incidence and with ketosis severity (most severe ketosis incidence recorded during the first 10 d postpartum; r<-0.33). Serum calcium and plasma glucose concentrations were negatively correlated with milk fever and ketosis severity, respectively (r<-0.39). These data indicate that 5-HT potentially plays a role in the regulation of calcium and glucose homeostasis during the transition period in cattle, which we previously demonstrated in rodents. Increased circulating concentrations of 5-HT might decrease milk fever at the onset of lactation and ketosis severity during the first 10 d postpartum in dairy cows. Understanding this physiological axis could help describe the underlying mechanisms associated with these periparturient metabolic disorders in dairy cows.

Feeding 5-hydroxy-l-tryptophan during the transition from pregnancy to lactation increases calcium mobilization from bone in rats.

An increasing demand for calcium during pregnancy and lactation can result in both clinical and subclinical hypocalcemia during the early lactation period in several mammalian species, in particular the dairy cow. Serotonin (5-HT) was recently identified as a regulator of lactation and bone turnover. The purpose of this study was to determine whether supplementation of the maternal diet with a 5-HT precursor would increase maternal bone turnover and calcium mobilization to maintain appropriate circulating maternal concentrations of ionized calcium during lactation. Female Sprague-Dawley rats (n = 30) were fed either a control diet (n = 15) or a diet supplemented with the 5-HT precursor 5-hydroxytryptophan (5-HTP, 0.2%; n = 15) from day 13 of pregnancy through day 9 of lactation. Maternal serum and plasma (day 1 and day 9 of lactation), milk and pup weight (daily), mammary gland and bone tissue (day 9 of lactation) were collected for analysis. The 5-HTP diet elevated circulating maternal concentrations of 5-HT on day 1 and day 9 of lactation and parathyroid hormone related-protein (PTHrP) on day 9 of lactation (P < 0.033). In addition, 5-HTP supplementation increased total serum calcium concentrations on day 1 of lactation and total milk calcium concentration on day 9 of lactation (P < 0.032). Supplemental 5-HTP did not alter milk yield, maternal body weight, mammary gland structure, or pup litter weights (P > 0.05). Supplemental 5-HTP also resulted in increased concentrations of mammary 5-HT and PTHrP, as well as increased mRNA expression of rate-limiting enzyme in 5-HT synthesis, tryptophan hydroxylase 1, and Pthrp mRNA on day 9 of lactation (P < 0.028). In addition, supplementation of 5-HTP resulted in increased mRNA expression of maternal mammary calcium transporters and resorption of bone in the femur, indicated by increase osteoclast number and diameter as well as mRNA expression of classical markers of bone resorption on day 9 of lactation (P < 0.048). These results show that increasing 5-HT biosynthesis during the transition from pregnancy to lactation could be a potential therapeutic target to explore for prevention of subclinical and clinical hypocalcemia.

Inhibition of articular cartilage degradation by glucosamine-HCl and chondroitin sulphate.

Glucosamine and chondroitin sulphate in many animal and human trials has improved joint health. In vitro studies are beginning to clarify their mode of action. The objective of this research was to: 1) determine at what concentrations glucosamine-HCl (GLN) and/or chondroitin sulphate (CS) would inhibit the cytokine-induced catabolic response in equine articular cartilage explants and 2) to determine if a combination of the 2 was more effective at inhibiting the catabolic response than the individual compounds. Articular cartilage was obtained from carpal joints of horses (age 1-4 years). Cartilage discs (3.5 mm) were biopsied and cultured. Explants were incubated with lipopolysaccharide (LPS) in the presence of varying concentrations of GLN, CS, or both. Control treatments included explants with no LPS and LPS without GLN or CS. Media were analysed for nitric oxide (NO), prostaglandin E2 (PGE2) and keratan sulphate. Cartilage was extracted for analysis of metalloproteinases (MMP). Four experiments were conducted. In all experiments, GLN at concentrations as low as 1 mg/ml decreased NO production relative to LPS stimulated cartilage without GLN over the 4 day period. In general, CS at either 0.25 or 0.5 mg/ml did not inhibit NO production. The addition of CS to GLN containing media did not further inhibit NO production. GLN at concentrations as low as 0.5 mg/ml decreased PGE2 production, whereas CS did not effect on PGE2. The combination of GLN/CS decreased MMP-9 gelatinolytic activity but had no effect on MMP-2 activity. The combination in 2 experiments tended to decrease MMP-13 protein concentrations and decreased keratan sulphate levels in media. Overall, the combination of GLN (1 mg/ml) and CS (0.25 mg/ml) inhibited the synthesis of several mediators of cartilage degradation. These results further support the effort to understand the role of GLN and CS in preserving articular cartilage in athletic horses.

Effect of antibiotics on in vitro and in vivo avian cartilage degradation.

Antibiotics are used in the livestock industry not only to treat disease but also to promote growth and increase feed efficiency in less than ideal sanitary conditions. However, certain antibiotic families utilized in the poultry industry have recently been found to adversely affect bone formation and cartilage metabolism in dogs, rats, and humans. Therefore, the first objective of this study was to determine if certain antibiotics used in the poultry industry would inhibit in vitro cartilage degradation. The second objective was to determine if the antibiotics found to inhibit in vitro cartilage degradation also induced tibial dyschondroplasia in growing broilers. Ten antibiotics were studied by an avian explant culture system that is designed to completely degrade tibiae over 16 days. Lincomycin, tylosin tartrate, gentamicin, erythromycin, and neomycin sulfate did not inhibit degradation at any concentration tested. Doxycycline (200 microg/ml), oxytetracycline (200 microg/ml), enrofloxacin (200 and 400 microg/ml), ceftiofur (400 microg/ml), and salinomycin (10 microg/ml) prevented complete cartilage degradation for up to 30 days in culture. Thus, some of the antibiotics did inhibit cartilage degradation in developing bone. Day-old chicks were then administered the five antibiotics at 25%, 100%, or 400% above their recommended dose levels and raised until 21 days of age. Thiram, a fungicide known to induce experimental tibial dyschondroplasia (TD), was given at 20 ppm. Birds were then killed by cervical dislocation, and each proximal tibiotarsus was visually examined for TD lesions. The results showed that none of these antibiotics significantly induced TD in growing boilers at any concentration tested, whereas birds given 20 ppm thiram had a 92% incidence rate.

Recombinant equine interleukin-1beta induces putative mediators of articular cartilage degradation in equine chondrocytes.

Interleukin-1 is considered a central mediator of cartilage loss in osteoarthritis in several species, however an equine recombinant form of this cytokine is not readily available for in vitro use in equine osteoarthritis research. Equine recombinant interleukin-1beta was cloned and expressed and its effects on the expression and activity of selected chondrocytic proteins implicated in cartilage matrix degradation were characterized. Reverse transcriptase polymerase chain reaction methods were used to amplify the entire coding region of the equine IL-1beta mRNA, which was cloned into an expression vector, expressed in E. coli, and purified using a Ni2+ chromatographic method. The effects of the recombinant peptide on chondrocyte gene expression were determined by Northern blotting using RNA from equine chondrocyte cultures hybridized to probes for matrix metalloproteinases (MMP 1, MMP 3, MMP 13), tissue inhibitor of matrix metalloproteinases 1 (TIMP 1) and cyclooxygenase 2 (COX 2). Effects on selected mediators of cartilage degradation (nitrite concentrations and MMP activity) were determined using conditioned medium from reIL-1beta-treated equine cartilage explant cultures. A recombinant peptide of approximately 21 kd was obtained. Northern blotting analyses revealed a marked up-regulation of expression of all MMPs, TIMP 1, and COX 2 in mRNA from treated chondrocytes. Furthermore, cartilage explants exposed to reIL-1beta had augmented collagenase/gelatinase and stromelysin activities as well as increased concentration of nitrite in conditioned media. The development of a biologically active, species-specific IL-1beta provides a valuable tool in the study of osteoarthritis pathophysiology and its treatment in horses.

Should ultrasound criteria of the prostate be redefined to better evaluate when and where to biopsy.

During the past 10 years, it has been suggested, and accepted by some, that transrectal ultrasound (TRUS) of the prostate should be used to identify a hypoechoic lesion or, if needed, guide biopsy into nonspecific areas. Retrospectively, the authors attempted to evaluate the need to identify areas that were on pathologic analysis, prostate cancer, but were not hypoechoic, but would require random/systematic biopsy to exclude prostate cancer. Six-hundred fifteen consecutive men were referred to the authors because of a concern found on digital rectal examination or because of increase in prostate-specific antigen. All patients underwent TRUS-guided biopsy of the prostate using either the four-quadrant or sextant biopsy technique. Each area undergoing biopsy was characterized as: 1) normal-appearing; 2) hypoechoic; 3) mixed echogenic (containing both hypoechoic and hyperechoic elements); 4) subtly hyperechoic (containing no calculi); or 5) isoechoic (lesion was seen because of distortion of the normal architecture). A diagnosis of carcinoma was made in 197 patients (32%). Of these, 99 (50.2%) patients had a hypoechoic lesion as the primary site, corresponding to their highest Gleason grade. Twenty-five (12.7%) had mixed echogenicity, nine (4.6%) had hyperechoic foci, and 23 (11.7%) had isoechoic biopsy-proven foci of prostate cancer. Forty-one (20.8%) patients with adenocarcinoma had normal ultrasound findings. The median Gleason grade for cancer in visible mixed echogenic and hyperechoic areas were generally higher than that for cancer in hypoechoic sites. Hypoechoic cancer sites had a Gleason grade range of 2 to 10 (median 5); mixed echogenic foci had a Gleason range of 2 to 10 (median 6); hyperechogenic cancers had a Gleason range of 2 to 8 (median 6); isoechoic cancers had a Gleason range of 2 to 7 (median 5); normal foci had a Gleason range of 2 to 8 (median 5). Results of this study suggest that 50% of clinically significant prostate cancers are not purely hypoechoic, and 37% of all diagnosed cancers contain no hypoechoic elements.

Visual inspection of the prostate biopsy specimen: aid to diagnosis and management.

The purpose of this article is to assess if the visual inspection of the prostate biopsy specimen can be used as a guide when deciding whether to attempt to sample another core of tissue from the same area if a less than adequate specimen was obtained during the first attempt. Five hundred thirty-seven specimens from 84 patients, referred because of an increased prostate-specific antigen (PSA) level and/or a suspicious result on digital rectal examination (DRE), were sampled and prospectively graded based on the lack of formation and amount of liquid in the specimen (grade I) compared to a highly rigid, solid core (grade V). Specimens were then fixed in formalin and retrospectively compared, and the pathologic diagnosis was compared with the subjective visual grade assigned to the specimen. Receiver-operator curve techniques were used to quantify the results and to test for statistical significance. Rigid biopsy specimens were cancer, and liquid, formless specimens were benign. Most biopsy specimens were solid, with moderate consistency, and could not be diagnosed accurately by visual inspection. Diagnosis of prostate cancer, despite the use of PSA, DRE, or diagnostic endorectal ultrasound, requires biopsy for definitive confirmation. Although the use of spring-loaded biopsy needles routinely yields good-quality cores of tissue for pathologic analysis, there are many occasions when a less than optimal specimen is obtained. The question of whether a repeat biopsy in that region is indicated always arises. These data suggest that if the initial specimen is grade I or II, repeat biopsy is probably not indicated. If the initial specimen is grade IV or V, repeat biopsy is recommended.

The effects of glucosamine derivatives on equine articular cartilage degradation in explant culture.

OBJECTIVE: To determine whether glucosamine-3-sulfate, glucose-3-sulfate (control) and N-acetyl glucosamine inhibit experimentally induced degradation of equine articular cartilage explants similar to glucosamine HCl. DESIGN: Articular cartilage was obtained from the antebrachio-carpal and middle joints of horses (2-8 years old) killed for reasons unrelated to lameness. Cartilage discs were harvested from the weight-bearing region of the articular surface and cultured. Media were exchanged daily and the recovered media stored at 4 degrees C. On days 1 and 2 lipopolysaccharide (LPS, 10 microg/ml) was added to induce cartilage degradation. To evaluate the effects of different sources of glucosamine (on an equal molar basis), varying concentrations of glucosamine HCl (0.25, 2.5, or 25 mg/ml), glucosamine-3-sulfate (0.304, 3.04, or 30.4 mg/ml), or N-acetyl-glucosamine (0.256, 2.56, or 25.6 mg/ml) were added to the cultures. The glucose-3-sulfate control was added at 0.3075, 3.075 or 30.75 mg/ml. Nitric oxide and proteoglycan released into conditioned media and tissue proteoglycan synthesis and total tissue PG content were measured as indicators of cartilage metabolism. RESULTS: Glucosamine-3-sulfate consistently inhibited cartilage degradation in a manner similar to glucosamine HCl, while the effects of N-acetyl-glucosamine were highly variable and did not inhibit cartilage degradation. Glucose-3-sulfate did not inhibit cartilage degradation. CONCLUSION: Our results indicate that glucosamine sulfate also has the potential to prevent or reduce articular cartilage degradation similar to glucosamine HCl in vitro. The amine group at the carbon-2 position appears important for the effectiveness of the glucosamine derivative. The therapeutic value of N-acetyl-glucosamine remains questionable.

Cartilage turnover in embryonic chick tibial explant cultures.

Growth plate cartilage regulates the rate of growth and ultimate length of several bones in the skeleton. Chondrocytes within the growth plate proliferate, differentiate, enlarge, and die. The extracellular matrix undergoes synthesis, reorganization, and eventually degradation. The majority of research in growth plate physiology has focused on the proliferation and differentiation of chondrocytes as well as proteins they produce for the extracellular matrix. However, little is known about the transition from hypertrophic to apoptotic chondrocytes or the regulation of terminal degradation of cartilage prior to bone formation. An explant culture has been developed to study cartilage differentiation using 12-d-old embryonic chick tibiae. We have modified the explant culture and are using it to further elucidate mechanisms involved in the regulation of growth plate cartilage turnover. In our cultures, chondrocytes mature and then die, completely degrading the cartilage in approximately 16 d. The matrix undergoes a predictable pattern of degradation in which proteoglycans followed by collagen are removed. Increases in matrix metalloproteinase activity and nitric oxide production are detected in cartilage concurrently with release of proteoglycans into media. Inhibitors of nitric oxide inhibit nitric oxide production and proteoglycan degradation, suggesting that nitric oxide, at least in part, regulates growth plate cartilage turnover in the explant culture. Information gained from using this explant culture will aid in understanding the regulation of growth plate cartilage turnover in vivo and potentially help determine the cause of bone growth diseases such as tibial dyschondroplasia.

Glucosamine HCl reduces equine articular cartilage degradation in explant culture.

Objective To determine whether glucosamine inhibits experimentally induced degradation of equine articular cartilage explants. Methods Articular cartilage was obtained from the antebrachio-carpal and middle joints of horses (2-8 years old) killed for reasons unrelated to lameness. Cartilage discs were harvested from the weight-bearing region of the articular surface and cultured. Media were exchanged daily and the recovered media stored at 4 degrees C. Explants were maintained in basal media 2 days prior to the start of four treatment days. On days 1-4 lipopolysaccharide (LPS, 10 microg/ml) or recombinant human interleukin-1 (rhIL-1, 50 ng/ml) were added to induce cartilage degradation. To test the potential protective effects of glucosamine, the compound was added in three concentrations (0.25, 2.5, or 25 mg/ml) and treatments were performed in triplicate. Controls included wells without LPS, rhIL-1beta, or glucosamine. Nitric oxide, proteoglycan and matrix metalloproteinases (MMP) released into conditioned media and tissue proteoglycan synthesis were measured as indicators of cartilage metabolism. Results Maximal nitric oxide production, proteoglycan release, and MMP activity were detected 1 day after the addition of LPS or rhIL-1beta to the media. The addition of 25 mg/ml of glucosamine prevented the increase in nitric oxide production, proteoglycan release and MMP activity induced by LPS or rhIL-1. Conclusions These data indicate that glucosamine can prevent experimentally induced cartilage degradation in vitro.

Effect of injection rate of contrast medium on pancreatic and hepatic helical CT.

PURPOSE: To assess the effect of the injection rate of contrast medium on pancreatic and hepatic enhancement at abdominal helical computed tomography (CT). MATERIALS AND METHODS: Sixty-four contrast medium-enhanced abdominal helical CT scans (64 adult patients) were obtained with 150 mL of contrast medium. The injection rate was 2.5 mL/sec for the first 32 scans and 5.0 mL/sec for the remaining 32. Scans were obtained at 5-sec intervals, with an intermediate 8-sec breathing interval. Hepatic and pancreatic enhancement levels were measured and averaged, and time-attenuation curves were plotted for both groups. Differences in weight, age, time to peak pancreatic and hepatic enhancement, and peak enhancement were assessed with the Student t test. RESULTS: Both peak enhancement and time to peak enhancement were significantly different between the two injection rates (P < or = .002), with faster, more intense hepatic and pancreatic enhancement at the higher rate. At 2.5 mL/sec, the pancreas reached a peak attenuation level of 65 HU at 69 sec, and the liver reached a peak of 58 HU at 87 sec. At 5.0 mL/sec, the pancreas reached a peak attenuation of 84 HU at 43 sec, and the liver reached a peak of 75 HU at 63 sec.

Diagnostic test comparisons in patients with deep venous thrombosis.

UNLABELLED: New diagnostic modalities are often judged relative to accepted standard procedures. These comparisons are influenced by the accuracy of the standard test and the prevalence of disease in the study population. We evaluated the importance of these factors in the assessment of antifibrin scintigraphy when used to detect deep venous thrombosis. METHODS: Scintigraphy is compared to contrast venography in two populations of patients with different disease prevalence. We calculate the sensitivity and specificity by limb site (calf, knee, thigh) and the overall diagnosis for each modality. The sensitivity and specificity results obtained using venography as a gold standard are compared to those obtained using a maximum likelihood statistical procedure that does not require comparison to a standard test. RESULTS: A significant variation in the apparent sensitivity, specificity and accuracy is found for antifibrin scintigraphy as related to limb site, disease prevalence and use of a gold standard. The value of antifibrin scintigraphy sensitivity (84.7%) and specificity (75.8%) predicted by the maximum likelihood analysis are substantially higher than those obtained from the estimates based on the use of venography as a gold standard for both high and low disease prevalence populations. The sensitivities and specificities of antifibrin scintigraphy (84.7% and 75.8%, respectively) and venography (71.7% and 80.7%, respectively) are comparable for the combined study group of 268 patients. CONCLUSION: To obtain unbiased evaluations of a new diagnostic modality, it is essential to take into account the errors of the standard reference test and disease prevalence in the study population. The results of our analysis suggest that it may not be appropriate to use contrast venography as a gold standard in the assessment of new diagnostic imaging procedures for DVT.

Value of selective second-look sonography by radiologists.

PURPOSE: To assess the usefulness of sonography performed by radiologists after a review of the sonographer's findings. MATERIALS AND METHODS: A total of 398 sonograms were obtained in 392 patients. Sonographers presented preliminary images and impressions to radiologists, who performed additional imaging and recorded their conclusions. Radiologists also attempted to predict in which cases their scan was likely to show new findings or to refute the sonographer's findings. Follow-up data were obtained whenever the sonographer's and the radiologist's findings disagreed. RESULTS: In 28 cases, the radiologist made important new findings. Positive initial findings were refuted in 24 cases. Discrepant findings were seen in 22% of cases in which additional scanning was predicted to be beneficial, compared with only 6% of cases in which second-look sonography was predicted not to be of value. This difference was statistically significant (P<.0001). CONCLUSION: Second-look sonography by radiologists provides a valuable check of the sonographer's findings.

Fat-suppressed spoiled GRASS imaging of knee hyaline cartilage: technique optimization and comparison with conventional MR imaging.

OBJECTIVE: We studied healthy volunteers with fat-suppressed three-dimensional (3D) spoiled gradient-recalled acquisition in the steady state (SPGR) to determine parameters that maximize positive contrast between knee articular cartilage and fluid, marrow fat, and muscle; and we compared the technique with conventional MR imaging sequences. The purpose was to determine if fat-suppressed 3D SPGR imaging is useful for detecting abnormalities of the articular cartilages. SUBJECTS AND METHODS: The knees of 10 healthy volunteers were imaged in the axial plane. Fat-suppressed 3D SPGR imaging was performed with a TR of 60 msec, a TE ranging from 5 to 15 msec, and a flip angle ranging from 20 degrees to 80 degrees. This was followed by a similar set of fat-suppressed two-dimensional (2D) SPGR images, and conventional T1- and T2-weighted spin-echo and multiplanar gradient studies. Contrast-to-noise (C/N) ratios were determined for cartilage versus a saline fluid phantom, marrow fat, and muscle. Optimal parameters were determined both quantitatively and by a blinded subjective analysis. RESULTS: A TE of 5 msec and a flip angle of 40 degrees demonstrated the greatest C/N ratio between the signals for cartilage and for fluid, marrow, and muscle. C/N ratios in the 3D sequences were higher than in the 2D, spin-echo, and gradient series, although the absolute C/N ratio values for the T2-weighted spin-echo sequence were higher than those for the 3D fat-suppressed SPGR sequence. Subjective analysis showed articular cartilage to have a consistent trilaminar appearance, and independent interpreters favored a 3D fat-suppressed SPGR sequence with a TE of 5 msec and a flip angle of 40 degrees. Three subjects with incidental joint fluid had C/N ratios within a 95% confidence range for cartilage versus the fluid phantom. CONCLUSION: When a fat-suppressed 3D SPGR sequence of 60/5/40 degrees (TR/TE/flip angle) is used, MR images can show high positive contrast between articular hyaline cartilage and adjacent structures. This convenient technique is different from standard MR imaging sequences because it demonstrates greater signal intensity in cartilage than in fluid, marrow fat, and muscle, and because it consistently shows an organized internal architecture of hyaline cartilage. Fat-suppressed 3D SPGR imaging therefore has promise for detecting abnormalities of the articular cartilage.