Dynamic molecular analysis and clinical correlates of tumor evolution within a phase II trial of panitumumab-based therapy in metastatic colorectal cancer.

Background: Mutations in rat sarcoma (RAS) genes may be a mechanism of secondary resistance in epidermal growth factor receptor inhibitor-treated patients. Tumor-tissue biopsy testing has been the standard for evaluating mutational status; however, plasma testing of cell-free DNA has been shown to be a more sensitive method for detecting clonal evolution. Materials and methods: Archival pre- and post-treatment tumor biopsy samples from a phase II study of panitumumab in combination with irinotecan in patients with metastatic colorectal cancer (mCRC) that also collected plasma samples before, during, and after treatment were analyzed for emergence of mutations during/post-treatment by next-generation sequencing and BEAMing. Results: The rate of emergence of tumor tissue RAS mutations was 9.5% by next-generation sequencing (n = 21) and 6.3% by BEAMing (n = 16). Plasma testing of cell-free DNA by BEAMing revealed a mutant RAS emergence rate of 36.7% (n = 39). Exploratory outcomes analysis of plasma samples indicated that patients who had emergent RAS mutations at progression had similar median progression-free survival to those patients who remained wild-type at progression. Serial analysis of plasma samples showed that the first detected emergence of RAS mutations preceded progression by a median of 3.6 months (range, -0.3 to 7.5 months) and that there did not appear to be a mutant RAS allele frequency threshold that could predict near-term outcomes. Conclusions: This first prospective analysis in mCRC showed that serial plasma biopsies are more inclusive than tissue biopsies for evaluating global tumor heterogeneity; however, the clinical utility of plasma testing in mCRC remains to be further explored. ClinicalTrials.gov Identifier: NCT00891930.

Chondro/osteoblastic and cardiovascular gene modulation in human artery smooth muscle cells that calcify in the presence of phosphate and calcitriol or paricalcitol.

Vitamin D sterol administration, a traditional treatment for secondary hyperparathyroidism, may increase serum calcium and phosphorus, and has been associated with increased vascular calcification (VC). In vitro studies suggest that in the presence of uremic concentrations of phosphorus, vitamin D sterols regulate gene expression associated with trans-differentiation of smooth muscle cells (SMCs) to a chondro/osteoblastic cell type. This study examined effects of vitamin D sterols on gene expression profiles associated with phosphate-enhanced human coronary artery SMC (CASMC) calcification. Cultured CASMCs were exposed to phosphate-containing differentiation medium (DM) with and without calcitriol, paricalcitol, or the calcimimetic R-568 (10(-11)-10(-7) M) for 7 days. Calcification of CASMCs, determined using colorimetry following acid extraction, was dose dependently increased (1.6- to 1.9-fold) by vitamin D sterols + DM. In contrast, R-568 did not increase calcification. Microarray analysis demonstrated that, compared with DM, calcitriol (10(-8) M) + DM or paricalcitol (10(-8) M) + DM similarly and significantly (P < 0.05) regulated genes of various pathways including: metabolism, CYP24A1; mineralization, ENPP1; apoptosis, GIP3; osteo/chondrogenesis, OPG, TGFB2, Dkk1, BMP4, BMP6; cardiovascular, HGF, DSP1, TNC; cell cycle, MAPK13; and ion channels, SLC22A3 KCNK3. R-568 had no effect on CASMC gene expression. Thus, SMC calcification observed in response to vitamin D sterol + DM may be partially mediated through targeting mineralization, apoptotic, osteo/chondrocytic, and cardiovascular pathway genes, although some gene changes may protect against calcification. Further studies to determine precise roles of these genes in development of, or protection against VC and cardiovascular disease are required.

Exchangeable sodium and renin in hypertensive diabetic patients with and without nephropathy.

The objective of this study was to examine total exchangeable sodium, plasma-blood volume, and the status of the renin-angiotensin system in hypertensive diabetic patients with established nephropathy. We also evaluated hypertensive patients with diabetes who were free of clinically apparent nephropathy or other diabetic complications. Total exchangeable sodium (by 24Na dilution) was expressed as percentage predicted. Subjects were studied as inpatients receiving unrestricted sodium intake and in stable metabolic control. Total exchangeable sodium was 100 +/- 2% in controls (n = 42), higher (p less than 0.01) at 108 +/- 2% in normotensive patients with diabetes (n = 30), and higher still (p less than 0.005) in hypertensive patients with diabetic nephropathy (n = 16) 118 +/- 4% (p less than 0.05 vs normotensive diabetics). The value correlated with blood pressure only in diabetics with nephropathy (r = 0.61, p less than 0.01). Plasma renin activity, and blood and plasma volumes were similar in nephropathic diabetics and controls. Hypertensive patients with maturity-onset (type II) diabetes free of nephropathy (n = 18) were compared with nondiabetic controls (n = 16) and normotensive patients with type II diabetes (n = 18) of similar age. Total exchangeable sodium in the controls was 100 +/- 3%, higher (p less than 0.01) in normotensive diabetics at 109 +/- 2%, but not significantly elevated in hypertensive diabetics at 106 +/- 2%. Again, blood and plasma volumes did not differ among the groups. Plasma renin activity was suppressed (p less than 0.01) to a comparable degree in both normotensive and hypertensive patients with type II diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of inhibition of both diacylglycerol metabolism and phospholipase A2 activity on superoxide generation by human neutrophils.

A 'cocktail' consisting of an inhibitor of diacylglycerol kinase (R59022, 10 microM), an inhibitor of diacylglycerol lipase (RHC80267, 10 microM), and an inhibitor of phospholipase A2 (either 100 microM indomethacin, or 100 microM sodium meclofenamate) markedly enhanced superoxide production by human neutrophils stimulated with post-receptor stimuli, fluoride and gamma-hexachlorocyclohexane. On the other hand, the response to the C3b/Fc receptor stimulus, opsonized zymosan, was marginally decreased whilst that to the Fc receptor stimulus, aggregated IgG, was virtually unaffected. Since the inhibitors used are deemed to inhibit the main routes of arachidonate production, these results call into question the role of arachidonate in the transduction of O2- generation by post-receptor stimuli, but support a role for arachidonate in receptor-mediated transduction.

A novel conformationally restricted protein kinase C inhibitor, Ro 31-8425, inhibits human neutrophil superoxide generation by soluble, particulate and post-receptor stimuli.

A novel, bis-indolylmaleimide, Ro 31-8425, bearing a conformationally restricted side chain, inhibits protein kinase C isolated from rat brain and human neutrophils with a high degree of selectivity over cAMP-dependent kinase and Ca2+/calmodulin-dependent kinase. It also inhibits phorbol ester-induced intracellular events known to be mediated by protein kinase C (p47 phosphorylation in intact platelets, CD3 and CD4 down-regulation in T-cells). Ro 31-8425 inhibited superoxide generation in human neutrophils activated by both receptor stimuli (formyl-methionyl-leucylphenylalanine, opsonized zymosan, IgG and heat aggregated IgG) and post-receptor stimuli (1,2-dioctanoylglycerol and fluoride). The compound also blocked antigen driven, but not IL-2 induced, T-cell proliferation. These results support a central role for protein kinase C in the activation of the respiratory burst and antigen-driven T-cell proliferation.

The effect of putative protein kinase C inhibitors, K252a and staurosporine, on the human neutrophil respiratory burst activated by both receptor stimulation and post-receptor mechanisms.

1. Two compounds, reported to be potent inhibitors of protein kinase C (PKC), K252a and staurosporine, have been examined in order to gain further information as to the possible role played by PKC in the signal transduction sequence of the neutrophil respiratory burst as determined by superoxide (O2-) production. 2. A number of stimuli were used in the study, some acting at receptors i.e. fMet-Leu-Phe (fMLP), opsonized zymosan and heat-aggregated IgG (HAGG), one acting on a G-protein, fluoride, and two direct PKC activators, dioctanoylglycerol (diC8) and phorbol myristate acetate (PMA). 3. K252a and staurosporine inhibited the respiratory burst with all the stimuli but the order of agonist sensitivity was very different with the two inhibitors. 4. For K252a-induced inhibition of O2- release, the order of potency was fluoride greater than fMLP, HAGG greater than opsonized zymosan greater than PMA, DiC8. For staurosporine-induced inhibition of O2- release, the order of potency changed to fluoride greater than DiC8, PMA greater than HAGG, fMLP greater than opsonized zymosan. The significance of this unexpected difference in relative rank order of potency is discussed with reference to the reported mechanism of action of the two inhibitors and the events involved in the oxidative burst. 5. Staurosporine at low concentrations increased the fMLP-stimulated O2- response by 100%, the maximum effect occurring at 35 nM. 6. To the extent that the compounds used are specific inhibitors of PKC, these findings support a role for the enzyme PKC in stimulus-activation coupling in O2- generation with all the stimuli used in this study.

Cyclooxygenase-independent effects of non-steroidal anti-inflammatory drugs on the neutrophil respiratory burst.

A range of 12 non-steroidal anti-inflammatory drugs (NSAIDs), including members from each of the main chemical groups, were examined for their effects on the oxidative burst induced by the receptor stimulus, platelet-activating factor, and the two post-receptor stimuli, fluoride and dioctanoylglycerol. It was found that the NSAIDs fell into three categories: (1) those that increased the stimulated superoxide (O2-) response, (2) those that had no effect and (3) those that decreased O2- production. All the drugs were without effect in unstimulated cells. The mode of action of those drugs that caused enhancement of the O2- response is unlikely to be due to an inhibition of the cyclooxygenase pathway of arachidonate metabolism as not all NSAIDs caused the enhancement. This data could have clinical implications for the therapy of inflammatory disorders such as rheumatoid arthritis, in that those NSAIDs which cause an increased O2- response, while providing temporary relief of symptoms, could be exacerbating the underlying inflammatory condition and associated tissue damage.

The protein kinase C inhibitor, K252a, inhibits superoxide production in human neutrophils activated by both PIP2-dependent and -independent mechanisms.

We report that the putative protein kinase C inhibitor, K252a, at concentrations of 0.2 and 1 microM, inhibited the respiratory burst induced by fluoride and formyl-methionyl-leucyl-phenyl-alanine respectively, both in human neutrophils primed with a subthreshold dose of phorbol myristate acetate and in non-primed neutrophils. In addition, K252a effectively inhibited ConA-zymosan-mediated superoxide generation in Ca2(+)-depleted neutrophils, with virtually maximal inhibition seen at 1 microM. These results suggest that protein kinase C is involved in the putative phosphatidylinositol bisphosphate-independent signal transduction mechanism of the respiratory burst as well as the pathway dependent on phosphatidylinositol bisphosphate hydrolysis.

Transcription of the genes encoding the small heat shock protein ubiquitin is unchanged in patients with systemic lupus erythematosus.

The heat shock proteins hsp90 and hsp70 have been shown to be over-expressed in peripheral blood mononuclear cells from SLE patients. We show, however, that transcription of the three genes encoding the small hsp ubiquitin is not enhanced in these patients. The over expression of hsp90 and hsp70 in SLE is likely therefore to reflect cellular events resulting in the specific induction of these hsps rather than a generalized induction of all hsp synthesis due to the stress of the disease or the presence of fever.

Enhanced transcription of the gene encoding the SmN autoantigen in patients with systemic lupus erythematosus does not result in enhanced levels of the SmN protein.

The SmN, protein is closely related to the constitutively expressed SmB and SmB' autoantigens and can also act as a target for human autoimmune sera. In contrast to the single gene encoding SmB and SmB' which is expressed in all tissues, the distinct gene encoding SmN is expressed at high levels only in brain and heart tissue. We show that the SmN gene is transcribed at significantly elevated levels in peripheral blood mononuclear cells (PBMCs) from SLE patients compared to normal controls. In contrast no significant elevation in transcription of the genes encoding SmB/B' or the U1-associated 70kD RNP autoantigen is observed in these patients. The elevation in SmN gene transcription in patient PBMCs does not result however, in enhanced levels of the SmN protein in the PBMCs of these patients. The significance of transcriptional and post-transcriptional processes in regulating the expression in SLE patients of SmN and other autoantigens is discussed.

Calcification inhibitors and Wnt signaling proteins are implicated in bovine artery smooth muscle cell calcification in the presence of phosphate and vitamin D sterols.

Administration of active vitamin D sterols to treat secondary hyperparathyroidism in patients with chronic kidney disease receiving dialysis has been associated with elevated serum calcium and phosphorus levels, which may lead to increased risk of vascular calcification. However, calcimimetics, by binding to the parathyroid gland calcium-sensing receptors, reduce serum parathyroid hormone, calcium, phosphorus, and the calcium-phosphorus product. Using cultured bovine aorta vascular smooth muscle cells (BASMCs), an in vitro model of vascular calcification, we compared calcification levels and gene expression profiles after exposure to the phosphate source ss-glycerolphosphate (BGP), the active vitamin D sterols calcitriol and paricalcitol, the calcimimetic R-568, or BGP with the active vitamin D sterols or R-568. Cells exposed to BGP (10 mM) alone or with calcitriol or paricalcitol showed dose-dependent BASMC calcification. No change in calcification was observed in cultures exposed to BGP with R-568, consistent with the observed lack of calcium-sensing receptor expression. Microarray analysis using total cellular RNA from cultures exposed to vehicle or BGP in the absence and presence of 10(-8) M calcitriol or paricalcitol for 7 days showed that cells exposed to BGP with calcitriol or BGP with paricalcitol had virtually identical gene expression profiles, which differed from those of cells treated with BGP or vehicle alone. Several osteoblast- and chondrocyte-associated genes were modulated by BGP and vitamin D exposure. In this study, exposure of BASMCs to phosphate and active vitamin D sterols induced calcification and changes in expression of genes associated with mineralized tissue.

Ureteric stone displacement using a new technique.

Transcutaneous displacement of ureteric stones has not been widely employed. The technique described here is one in which four ureteric stones were actively displaced into the bladder using a balloon catheter. Major ureteric surgery was thus avoided. This new technique may be useful as an alternative to Dormia basket extraction of ureteric calculi.

Computed tomography in the diagnosis of extramedullary plasmacytoma.

Extramedullary plasmacytomas involving the retroperitoneum are rare. Review of the literature reveals only one case in which computed tomography (CT) findings were illustrated, and this case involved the pancreas. Described here is unusual case in which pancreatic and renal plasmacytomas occurred in a patient who 18 months previously had presented with a solitary plasmacytoma of bone. The clinically unsuspected renal masses was diagnosed by CT, allowing easy biopsy and radiotherapy planning.

The fatty meal and acalculous gall-bladder disease.

The practice of routinely obtaining after fatty meal films during oral cholecystography has been questioned. A retrospective analysis of 45 cases of adenomyomatosis or polyps revealed that, in 28% of patients with adenomyomatosis, the gall-bladder appeared entirely normal before fat. It is concluded that, for the detection of acalculous gall-bladder disease, the fatty meal should be a routine part of oral cholecystography.

Extrarenal arterial connections of the normal renal artery.

An angiographic study in cadavers has demonstrated connections between the renal artery and remote vessels including the intercostal, testicular, and inferior mesenteric arteries in subjects with no known history of renal disease. Such communications are a potential route by which embolic material introduced into the renal artery may inadvertently reach other organs.

Skin necrosis: a complication of alcohol infarction of a hypernephroma.

A case of skin infarction after transcatheter ablation of a hypernephroma using alcohol is described. Possible avenues for migration are discussed.

CT findings in obstructed upper moieties of duplex kidneys.

Obstructed upper moiety of a duplex kidney is an uncommon diagnostic problem in adults. This is a report on two cases examined by computed tomography (CT) for suspected abdominal and suprarenal masses, respectively. Although CT is not the primary investigative tool for this condition, the unusual presentations allowed us the opportunity of studying the CT appearance.

The small bowel follow-through: time to sit up.

It is common practice for patients to lie on their right side during small bowel barium follow-through examinations. The rationale is that this speeds up transit time and improves diagnostic usefulness. The results of a randomized, prospective study of 105 patients are presented. There was no significant difference in either transit time or diagnostic usefulness when the examination was carried out with the patient erect (sitting or ambulant) or when adopting the decubitus position. Abandoning the use of the right side down decubitus position allows a longstanding fiction to be discarded. Furthermore, lack of reclining space has been one factor which may limit the number of patients booked for this examination during a single session. Space for couches and trolleys need no longer be a barrier to the efficient organization of a small bowel screening session.