Repeated mutKRAS ctDNA measurements represent a novel and promising tool for early response prediction and therapy monitoring in advanced pancreatic cancer.

Background: The presence of mutated KRAS (mutKRAS ctDNA) in plasma samples has been consistently shown to be a negative prognostic indicator in pancreatic cancer (PC). Only small pilot studies have evaluated the value of serial mutKRAS ctDNA-measurements in PC. Patients and methods: The aim of the present study was to explore the potential of repeated mutKRAS ctDNA measurements for response prediction and therapy monitoring in advanced PC patients. We used the BEAMing technology to determine levels of mutKRAS ctDNA, CA 19-9, CEA and CYFRA 21-1 in 284 plasma samples of 54 patients with advanced PC receiving gemcitabine-based chemotherapy. Absolute levels and kinetics of mutKRAS ctDNA, CA 19-9, CEA and CYFRA 21-1 were correlated to radiological response, progression-free and overall survival. Results: mutKRAS ctDNA was present in a majority of advanced PC patients (n = 36/54, 67%) and indicated tissue KRAS mutation status with a high sensitivity (75%) and specificity (100%). The presence of mutKRAS ctDNA, as well as higher levels of CA 19-9, CEA and CYFRA 21-1 before initiation of the first-line chemotherapy, was significantly correlated to an adverse overall survival. During therapy, changes in mutKRAS ctDNA levels were more rapid and pronounced than changes in protein-based tumor markers. A decrease in mutKRAS ctDNA levels during therapy was an early indicator of response to therapy, while there was no significant correlation between kinetics of CA 19-9, CEA or CYFRA 21-1 and response to chemotherapy during the first four weeks of treatment. Repeated mutKRAS ctDNA measurements during follow-up appeared to be superior to protein-based tumor markers in detecting progressive disease (sensitivity: 83%, specificity: 100%). Conclusion: mutKRAS ctDNA kinetics appear to be a powerful and highly specific tool in early response prediction and therapy monitoring of advanced PC patients receiving chemotherapy.

Characterizing the patterns of clonal selection in circulating tumor DNA from patients with colorectal cancer refractory to anti-EGFR treatment.

INTRODUCTION: KRAS and EGFR ectodomain-acquired mutations in patients with metastatic colorectal cancer (mCRC) have been correlated with acquired resistance to anti-EGFR monoclonal antibodies (mAbs). We investigated the frequency, co-occurrence, and distribution of acquired KRAS and EGFR mutations in patients with mCRC refractory to anti-EGFR mAbs using circulating tumor DNA (ctDNA). PATIENTS AND METHODS: Sixty-two post-treatment plasma and 20 matching pretreatment archival tissue samples from KRAS (wt) mCRC patients refractory to anti-EGFR mAbs were evaluated by high-sensitivity emulsion polymerase chain reaction for KRAS codon 12, 13, 61, and 146 and EGFR 492 mutations. RESULTS: Plasma analyses showed newly detectable EGFR and KRAS mutations in 5/62 [8%; 95% confidence interval (CI) 0.02-0.18] and 27/62 (44%; 95% CI 0.3-0.56) samples, respectively. KRAS codon 61 and 146 mutations were predominant (33% and 11%, respectively), and multiple EGFR and/or KRAS mutations were detected in 11/27 (41%) cases. The percentage of mutant allele reads was inversely correlated with time since last treatment with EGFR mAbs (P = 0.038). In the matching archival tissue, these mutations were detectable as low-allele-frequency clones in 35% of patients with plasma mutations after treatment with anti-EGFR mAbs and correlated with shorter progression-free survival (PFS) compared with the cases with no new mutations (3.0 versus 8.0 months, P = 0.0004). CONCLUSION: Newly detected KRAS and/or EGFR mutations in plasma ctDNA from patients refractory to anti-EGFR treatment appear to derive from rare, pre-existing clones in the primary tumors. These rare clones were associated with shorter PFS in patients receiving anti-EGFR treatment. Multiple simultaneous mutations in KRAS and EGFR in the ctDNA and the decline in allele frequency after discontinuation of anti-EGFR therapy in a subset of patients suggest that several resistance mechanisms can co-exist and that relative clonal burdens may change over time. Monitoring treatment-induced genetic alterations by sequencing ctDNA could identify biomarkers for treatment screening in anti-EGFR-refractory patients.

Manufacturing DNA microarrays of high spot homogeneity and reduced background signal.

Analyses on DNA microarrays depend considerably on spot quality and a low background signal of the glass support. By using betaine as an additive to a spotting solution made of saline sodium citrate, both the binding efficiency of spotted PCR products and the homogeneity of the DNA spots is improved significantly on aminated surfaces such as glass slides coated with the widely used poly-L-lysine or aminosilane. In addition, non-specific background signal is markedly diminished. Concomitantly, during the arraying procedure, the betaine reduces evaporation from the microtitre dish wells, which hold the PCR products. Subsequent blocking of the chip surface with succinic anhydride was improved considerably in the presence of the non-polar, non-aqueous solvent 1,2-dichloroethane and the acylating catalyst N:-methylimidazole. This procedure prevents the overall background signal that occurs with the frequently applied aqueous solvent 1-methyl-2-pyrrolidone in borate buffer because of DNA that re-dissolves from spots during the blocking process, only to bind again across the entire glass surface.

Comparison of two triphasic oral contraceptives containing either gestodene or norethindrone: a randomized, controlled trial.

Two triphasic oral contraceptives containing either gestodene or norethindrone as the progestogenic compound combined with ethinyl estradiol were compared in a randomized clinical trial to assess their contraceptive reliability, clinical tolerance and cycle control. Both preparations were effective in preventing pregnancy. The gestodene preparation proved significantly superior regarding cycle control and general tolerance.

PIP: In Germany, researchers randomly placed 126 women into a group taking a triphasic oral contraceptive (OC) with gestodene and 128 women into another group taking triphasic norethindrone to do a 1-year controlled, multicenter open trial examining contraceptive efficacy, cycle control, and general tolerance. No one became pregnant during the study, despite mistakes in pill intake in 32 (2.5%) and 44 (3.7%) pill cycles of the gestodene group and norethindrone group, respectively. Women in the gestodene group experienced spotting at a higher rate than those in the norethindrone group (27% vs. 15.8%; p .05). The difference remained significant even when the researchers examined only 1st-time OC users (10.2% vs. 5%; p .05). On the other hand, breakthrough bleeding occurred less often in women in the gestodene group (19.3% vs. 29.6%). Over time, women in the norethindrone group were more likely to gain more than 2 kg than those in the gestodene group. Neither group experienced changes in blood pressure. Neither OC caused any serious adverse events, e.g., thromboembolism. The most common subjective adverse events were painful menstrual periods, breast tension, and headache. The incidence of acne increased in the norethindrone group. Adverse events were responsible for 3.5% of women in the gestodene group and 11.3% in the norethindrone group dropping out, sometimes citing more than 1 adverse event. Women in the norethindrone group were more likely to drop out because of adverse events than were those in the gestodene group (50% of all dropouts vs. 25%; p .05). Triphasic gestodene had better cycle control and was better tolerated than triphasic norethindrone. Weight gain and acne were less common in the gestodene group than the norethindrone group. In conclusion, triphasic gestodene is superior to that of triphasic norethindrone.

Triphasic combination of ethinyl estradiol and gestodene. Long-term clinical trial.

A triphasic oral contraceptive containing ethinyl estradiol and gestodene was used by 1933 women for up to 36 cycles in order to evaluate contraceptive efficacy, general tolerance and cycle control. Altogether, 30,763 treatment cycles were recorded. Two pregnancies were seen, both of them classified as user failure. The rates of spotting and breakthrough bleeding fell below the rates of the pre-treatment cycle after the first three months of treatment. Adverse events were mostly minor with breast tension and headaches the most common complaints. After the first three cycles, the incidence of most complaints was the same as or even lower than in the pre-treatment cycle. Blood pressure and body weight remained practically unchanged. The trial preparation provided reliable contraception with good general tolerance and cycle control when taken for up to three years.

The effect on blood pressure of a monophasic oral contraceptive containing ethinylestradiol and gestodene.

To obtain an overview of the effect of monophasic gestodene on blood pressure and to determine the frequency of "OC elevated BP/hypertension," the results of blood pressure monitoring from four clinical studies of contraceptive efficacy and safety have been retrospectively analyzed. A total of 1930 women took part in the studies, which recorded BP for up to 24 cycles. Analysis of results revealed that 97 women (5.0%) showed an increase in blood pressure from previously normal to elevated values while taking monophasic gestodene, with only 26 (1.35%) fulfilling the criteria of "OC elevated BP/hypertension." Only four women dropped out of the trials due to hypertensive blood pressure values, while 67 women (3.5%) experienced a normalization of previously elevated blood pressure measurements. In conclusion, this analysis has confirmed that gestodene has a negligible effect on blood pressure, with increased BP a relatively rare event.

PIP: Schering AG (manufacturer of a monophasic oral contraceptive [OC] containing 75 mcg gestodene plus 30 mcg ethinyl estradiol) in Berlin, Germany, conducted a retrospective analysis of blood pressure measurements from 4 clinical trials of the contraceptive efficacy and safety of monophasic gestodene to examine gestodene's effect on blood pressure and the incidence of OC-related blood pressure/ hypertension. (OC-related blood pressure/hypertension is defined as: women with neither history of hypertension nor elevated blood pressure before OC use develop increased blood pressure or hypertension that is reversible once OC use ceases.) The clinical trials recorded the blood pressure of 1930 women for up to 24 cycles. Most women (89.9%) experienced no change in their blood pressure during OC use. 97 women (5%) experienced an increase in blood pressure. 26 women (1.35%) had OC-elevated blood pressure/hypertension. Four women left the trials due to hypertension. 67 women (3.5%) who had elevated blood pressure before OC use attained normalization of blood pressure during OC use. These results show that the gestodene-containing OC had an insignificant effect on blood pressure and that elevated blood pressure rarely occurred.

Anchoring cells (Desmocytes) in the hydrozoan polyp Cordylophora.

Desmocytes or anchoring cells are present on the upright stolons of the athecate hydroid Cordylophora caspia and function to support the soft coenosarc within the rigid tube of perisarc by linking the perisarc with the mesoglea. These cells are characterized by accumulations of 70 A filaments which aggregate into dense rods at the apical end and contact the perisarc. At the base of the desmocytes the filaments are distributed within large cytoplasmic processes which interdigitate with an extension of the mesoglea. Desmocytes in Cordylophora are temporally and spatially formed in sequence as the upright elongates. Depending on their location and structure they can be categorized as forming, functional, or remnant desmocytes. The youngest, forming desmocytes are found in the distal end of the stolon 0.5-1.0 mm from the base of the hydranth. In this region coenosarc is just beginning to separate from the perisarc. Functional desmocytes are scattered 1-3 mm from the base of the hydranth and are associated with perpendicular extensions of the mesoglea. Remnants have lost their mesogleal connection and are located in more proximal, older regions of upright stolon. Support provided by the desmocytes to the upright stolon is limited by three factors that characterize the athecate hydroid: distribution of perisarc, pattern of growth, and extent of movement. The distal location of forming desmocytes is coincident with the hardening of new perisarc. The temporary nature of attachment sites is directly related to upright elongation. It is probable that the orientation of filaments within the cell and the mesogleal extension provide an addition feature of flexibility necessary to permit feeding, growth, and rhythmic pulsation movements characteristic of these hydroids.

Multiple and antagonistic effects of water on intrinsic physical properties of model Fischer-Tropsch cobalt catalysts evidenced by in situ X-ray diffraction.

Direct evidence for cobalt oxidation under water treatment at 230 °C was revealed using in situ XRD, even in the presence of small amounts of H2. Oxidation kinetics was proved to be particle size dependent. The resulting Co3O4 showed a firmly smaller coherent domain size than the initial calcined catalyst.

Opposite action of hippocampal CB1 receptors in memory reconsolidation and extinction.

Retrieval of a consolidated memory triggers a number of processes which depend, among other factors, on the duration of the reactivation session: reconsolidation requires a brief reactivation session, and extinction, a prolonged one. The scope of this study is to explore the potential role of the hippocampal endocannabinoid system on reconsolidation and extinction processes. Bilateral infusion of the CB1 cannabinoid receptor antagonist, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) into the CA1 region of the dorsal hippocampus of Wistar rats after memory reactivation facilitated the reconsolidation of the contextual fear conditioning memory. The inhibition of protein synthesis with DRB in the same brain region blocked memory reconsolidation. Both effects were persistent, lasting up to 7 days after the first retrieval experience. In contrast, the local infusion of anandamide blocked memory reconsolidation, an effect that was antagonized by the combined administration of anandamide with a subthreshold dose of a CB1 antagonist, supporting a CB1-mediated role of the hippocampal endocannabinoid system in the modulation of the memory reconsolidation. Local infusion of AM251 into CA1 blocked memory extinction whereas the administration of anandamide facilitated it; however, when combined with a subthreshold concentration of the CB1 antagonist, anandamide did not affect the extinction process. The clear-cut, opposite effects observed in each situation suggest a possible role of the hippocampal endocannabinoid system as a switching mechanism deciding which processes will take place, either maintaining the original memory (reconsolidation) or promoting a new learning (extinction).

Identification of the binding site for a novel class of CCR2b chemokine receptor antagonists: binding to a common chemokine receptor motif within the helical bundle.

Monocyte chemoattracant-1 (MCP-1) stimulates leukocyte chemotaxis to inflammatory sites, such as rheumatoid arthritis, atherosclerosis, and asthma, by use of the MCP-1 receptor, CCR2, a member of the G-protein-coupled seven-transmembrane receptor superfamily. These studies identified a family of antagonists, spiropiperidines. One of the more potent compounds blocks MCP-1 binding to CCR2 with a K(d) of 60 nm, but it is unable to block binding to CXCR1, CCR1, or CCR3. These compounds were effective inhibitors of chemotaxis toward MCP-1 but were very poor inhibitors of CCR1-mediated chemotaxis. The compounds are effective blockers of MCP-1-driven inhibition of adenylate cyclase and MCP-1- and MCP-3-driven cytosolic calcium influx; the compounds are not agonists for these pathways. We showed that glutamate 291 (Glu(291)) of CCR2 is a critical residue for high affinity binding and that this residue contributes little to MCP-1 binding to CCR2. The basic nitrogen present in the spiropiperidine compounds may be the interaction partner for Glu(291), because the basicity of this nitrogen was essential for affinity; furthermore, a different class of antagonists, a class that does not have a basic nitrogen (2-carboxypyrroles), were not affected by mutations of Glu(291). In addition to the CCR2 receptor, spiropiperidine compounds have affinity for several biogenic amine receptors. Receptor models indicate that the acidic residue, Glu(291), from transmembrane-7 of CCR2 is in a position similar to the acidic residue contributed from transmembrane-3 of biogenic amine receptors, which may account for the shared affinity of spiropiperidines for these two receptor classes. The models suggest that the acid-base pair, Glu(291) to piperidine nitrogen, anchors the spiropiperidine compound within the transmembrane ovoid bundle. This binding site may overlap with the space required by MCP-1 during binding and signaling; thus the small molecule ligands act as antagonists. An acidic residue in transmembrane region 7 is found in most chemokine receptors and is rare in other serpentine receptors. The model of the binding site may suggest ways to make new small molecule chemokine receptor antagonists, and it may rationalize the design of more potent and selective antagonists.

Identification of surface residues of the monocyte chemotactic protein 1 that affect signaling through the receptor CCR2.

The CC chemokine, monocyte chemotactic protein, 1 (MCP-1) functions as a major chemoattractant for T-cells and monocytes by interacting with the seven-transmembrane G protein-coupled receptor CCR2. To identify which residues of MCP-1 contribute to signaling though CCR2, we mutated all the surface-exposed residues to alanine and other amino acids and made some selective large changes at the amino terminus. We then characterized the impact of these mutations on three postreceptor pathways involving inhibition of cAMP synthesis, stimulation of cytosolic calcium influx, and chemotaxis. The results highlight several important features of the signaling process and the correlation between binding and signaling: The amino terminus of MCP-1 is essential as truncation of residues 2-8 ([1+9-76]hMCP-1) results in a protein that cannot stimulate chemotaxis. However, the exact peptide sequence may be unimportant as individual alanine mutations or simultaneous replacement of residues 3-6 with alanine had little effect. Y13 is also important and must be a large nonpolar residue for chemotaxis to occur. Interestingly, both Y13 and [1+9-76]hMCP-1 are high-affinity binders and thus affinity of these mutants is not correlated with ability to promote chemotaxis. For the other surface residues there is a strong correlation between binding affinity and agonist potency in all three signaling pathways. Perhaps the most interesting observation is that although Y13A and [1+9-76]hMCP are antagonists of chemotaxis, they are agonists of pathways involving inhibition of cAMP synthesis and, in the case of Y13A, calcium influx. These results demonstrate that these two well-known signaling events are not sufficient to drive chemotaxis. Furthermore, it suggests that specific molecular features of MCP-1 induce different conformations in CCR2 that are coupled to separate postreceptor pathways. Therefore, by judicious design of antagonists, it should be possible to trap CCR2 in conformational states that are unable to stimulate all of the pathways required for chemotaxis.