Total circulating cell-free DNA as a prognostic biomarker in metastatic colorectal cancer before first-line oxaliplatin-based chemotherapy.

BACKGROUND: Metastatic colorectal cancer (mCRC) is a heterogeneous disease where prognosis is dependent both on tumor biology and host factors. Total circulating cell-free DNA (cfDNA) has shown to harbor prognostic information in mCRC, although less is known about the biological correlates of cfDNA levels in this patient group. The primary objective was to evaluate the prognostic value of pretreatment cfDNA in patients receiving the first-line oxaliplatin-based chemotherapy for mCRC, by using a predefined upper limit of normal (ULN) from a cohort of presumed healthy individuals. The secondary objective was to model cfDNA levels as a function of predefined tumor and host factors. PATIENTS AND METHODS: This was a retrospective post hoc study based on a prospective multicenter phase III trial, the NORDIC-VII study. DNA was purified from 547 plasma samples and cfDNA quantified by a droplet digital PCR assay (B2M, PPIA) with controls for lymphocyte contamination. Main clinical end point was overall survival (OS). RESULTS: cfDNA was quantified in 493 patients, 54 were excluded mainly due to lymphocyte contamination. Median cfDNA level was 7673 alleles/ml (1050-1 645 000) for B2M and 5959 alleles/ml (555-854 167) for PPIA. High cfDNA levels were associated with impaired outcome; median OS of 16.6 months for levels above ULN and 25.9 months for levels below ULN (hazard ratio = 1.83, 95% confidence interval 1.51-2.21, P < 0.001). The result was confirmed in multivariate OS analysis adjusting for established clinicopathological characteristics. A linear regression model predicted cfDNA levels from sum of longest tumor diameters by RECIST, the presence of liver metastases and systemic inflammatory response as measured by interleukin 6 (F(6, 357) = 62.7, P < 0.001). CONCLUSION: cfDNA holds promise as a minimally invasive and clinically relevant prognostic biomarker in mCRC before initiating first-line oxaliplatin-based chemotherapy and may be a complex entity associated with tumor burden, liver metastases and systemic inflammatory response. TRIAL REGISTRATION: ClinicalTrials.gov, NCT00145314.

Results of a phase 1 trial combining ridaforolimus and MK-0752 in patients with advanced solid tumours.

BACKGROUND: The phosphatidylinositol 3-kinase/protein kinase-B/mammalian target of rapamycin (PI3K-AKT-mTOR) signalling pathway is aberrantly activated in several cancers. Notch signalling maintains cell proliferation, growth and metabolism in part by driving the PI3K pathway. Combining the mTOR inhibitor ridaforolimus with the Notch inhibitor MK-0752 may increase blockade of the PI3K pathway. METHODS: This phase I dose-escalation study (NCT01295632) aimed to define the dose-limiting toxicities (DLTs) and maximum tolerated dose (MTD) of combination oral ridaforolimus (rising doses starting at 20 mg, 5 days/week) and oral MK-0752 (1800 mg once weekly) in patients with solid tumours. No intrapatient dose escalation was permitted. RESULTS: Twenty eight patients were treated on study. Ridaforolimus doses were escalated from 20 to 30 mg/day. Among 14 evaluable patients receiving ridaforolimus 20 mg, one DLT (grade 2 stomatitis, second episode) was reported. Among eight evaluable patients receiving ridaforolimus 30 mg, three DLTs were reported (one each grade 3 stomatitis, grade 3 diarrhoea, and grade 3 asthenia). The MTD was 20 mg daily ridaforolimus 5 days/week+1800 mg weekly MK-0752. The most common drug-related adverse events included stomatitis, diarrhoea, decreased appetite, hyperglycaemia, thrombocytopenia, asthenia and rash. Two of 15 (13%) patients with head and neck squamous cell carcinoma (HNSCC) had responses: one with complete response and one with partial response. In addition, one patient experienced stable disease ⩾6 months. CONCLUSIONS: Combined ridaforolimus and MK-0752 showed activity in HNSCC. However, a high number of adverse events were reported at the MTD, which would require careful management during future clinical development.

Role of diacylglycerol (DAG) in hormonal induction of S phase in hepatocytes: the DAG-dependent protein kinase C pathway is not activated by epidermal growth factor (EGF), but is involved in mediating the enhancement of responsiveness to EGF by vasopressin, angiotensin II, and norepinephrine.

The role of diacylglycerol (DAG) in hormonal induction of S phase was investigated in primary cultures of rat hepatocytes. In this model, several agonists that bind to G protein-coupled receptors act as comitogens when added to the cells soon after plating (i.e., in Go/early Gl phase), while the cells are most responsive to the mitogenic effect of epidermal growth factor (EGF) at 24-48 h of culturing (i.e., mid/late Gl). It was found that the cellular concentration of DAG rose markedly and progressively during the first 24 h of culturing. Exposure of the hepatocytes at 3 h to alpha1-adrenergic stimulation (norepinephrine with timolol), vasopressin, or angiotensin II further increased this rise, producing a sustained increase in the DAG level. Norepinephrine, which was the most efficient comitogen, produced the most prolonged DAG elevation. In contrast, no significant increase of DAG was found in response to EGF, neither at 3 nor at 24 h, using concentrations that markedly stimulated the ERK subgroup of the mitogen-activated protein kinases (MAPK) and DNA synthesis. Addition of Bacillus cereus phosphatidylcholine-specific phospholipase C (PC-PLC) strongly elevated DAG, while Streptomyces phospholipase D (PLD) increased phosphatidic acid (PA) but not DAG. B. cereus PC-PLC and the protein kinase C (PKC) activator tetradecanoyl phorbol-acetate (TPA), like norepinephrine, vasopressin, and angiotensin II, stimulated MAPK and enhanced the stimulatory effect of EGF on DNA synthesis. The PKC inhibitor GF109203X did not diminish the effect of EGF on MAPK or DNA synthesis, but strongly inhibited the effects of norepinephrine, vasopressin, angiotensin II, TPA and B. cereus PC-PLC on MAPK and almost abolished the enhancement by these agents of EGF-stimulated DNA synthesis. These results suggest that although generation of DAG is not a direct downstream response mediating the effects of the EGF receptor in hepatocytes, a sustained elevation of DAG with activation of PKC markedly increases the responsiveness to EGF. Mechanisms involving DAG and PKC seem to play a role in the comitogenic effects of various agents that bind to G protein-coupled receptors and activate the cells early in Gl, such as norepinephrine, angiotensin II, and vasopressin.

Growth-promoting effects of Ca(2+)-mobilizing agents in hepatocytes: lack of correlation between the acute activation of phosphoinositide-specific phospholipase C and the stimulation of DNA synthesis by angiotensin II, vasopressin, norepinephrine, and prostaglandin F2 alpha.

Although several hormones that promote hepatocyte proliferation also activate phosphoinositide-specific phospholipase C (PI-PLC) and mobilize Ca2+, the role of PI-PLC in the growth-stimulating effect of these agents is not clear. We have investigated this issue further, by exposing freshly isolated adult rat hepatocytes to vasopressin, angiotensin II, norepinephrine (in the presence of the beta-adrenoceptor blocker timolol) or PGF2 alpha, and examined both acute responses and the subsequent DNA synthesis when the cells were grown in monolayer culture. All the agonists elevated the level of inositol 1,4,5-trisphosphate (InsP3) and enhanced the DNA synthesis, amplifying the response to epidermal growth factor (EGF), and this comitogenic effect could be exerted by a single exposure of the cells 24 h prior to the addition of EGF. The acute activation of PI-PLC, measured as the early rise (peak 15-60 s) in InsP3, was 8-10-fold with vasopressin or angiotensin II, 3-4-fold with norepinephrine, and approximately 2-fold with PGF2 alpha. For all the agonists, a rise in cytosolic free Ca2+ in 100% of the cells and a maximal increase in glycogen phosphorylase activity were evoked at concentrations that approximately doubled the level of InsP3. However, the growth-stimulatory effects of these agonists showed a different order of efficacy as compared to the activation of PI-PLC; in terms of the maximal stimulation of DNA synthesis, the effects were: norepinephrine approximately PGF2 alpha > angiotensin II > vasopressin. Also, norepinephrine, PGF2 alpha, and angiotensin II, but not vasopressin, further enhanced the DNA synthesis when their concentrations were increased above those yielding maximal elevation of InsP3. In experiments where vasopressin and angiotensin II were combined, their effects on the DNA synthesis were additive while the InsP3 responses were not. The results show that the extent of the initial activation of PI-PLC is not the determinant for the magnitude of the growth effects of Ca(2+)-mobilizing hormones in hepatocytes. This suggests either (a) that the proliferative response to these agents is determined by the activity of PI-PLC at a later time, or its integral over an extended part of the prereplicative period, rather than by the acute activation, or (b) that additional, PI-PLC-independent, mechanisms are required.

On the mechanisms of the growth-promoting effect of prostaglandins in hepatocytes: the relationship between stimulation of DNA synthesis and signaling mediated by adenylyl cyclase and phosphoinositide-specific phospholipase C.

While many observations indicate that prostaglandins may act as positive regulators of hepatocyte proliferation, the underlying mechanisms are not known. We have examined some of the signal pathways in the growth response induced by prostaglandins in hepatocytes, with particular focus on adenylyl cyclase and phosphoinositide-specific phospholipase C. Adult rat hepatocytes were cultured as primary monolayers in serum-free medium in the presence of EGF and insulin. PGE2 or PGF2 alpha (added 0-3 h after plating) enhanced the incorporation of [3H]-thymidine into DNA (measured at 50 h); at 100 microM the stimulation was about threefold PGI2 and PGD2 also showed significant but smaller stimulatory effects. No significant increase in the level of cyclic AMP (cAMP) was detected in response to any of the prostaglandins. Low concentrations of glucagon (0.1-10 nM), a potent activator of hepatic adenylyl cyclase, or 8-bromo-cAMP (0.1-10 microM) enhanced the DNA synthesis. When 8-bromo-cAMP was used in maximally effective concentrations, no further stimulation was obtained by combining it with glucagon, whereas the effects of PGE2 and 8-bromo-cAMP were completely additive. All the prostaglandins also showed additivity with the effect of glucagon on the DNA synthesis. PGE2, PGF2 alpha, PGI2, and PGD2 increased intracellular inositol-1,4,5-trisphosphate (InsP3), with a relative order of efficacy roughly corresponding to their activity as stimulators of DNA synthesis. Increases in cytosolic free Ca2+, as measured in single cells, were elicited in a majority of the hepatocytes by all these prostaglandins at 1 microM. Supramaximal concentrations of vasopressin, a strong activator of phospholipase C in hepatocytes, acted additively with PGE2 on the DNA synthesis. Pretreatment of the hepatocytes with a concentration of pertussis toxin that prevented the inhibitory effect of PGE2 on glucagon-induced cAMP accumulation did not abolish the ability of PGE2 to stimulate the DNA synthesis. The results do not support a role for adenylyl cyclase activation in the stimulatory effect of prostaglandins on hepatocyte growth. While the data are compatible with an involvement of phosphoinositide-specific phospholipase C in the growth-promoting effect of prostaglandins in cultured rat hepatocytes, they suggest this may not be the sole mechanism.

Elevated glucose concentrations inhibit DNA synthesis and expression of c-myc in cultured hepatocytes.

Glucose depressed DNA synthesis in rat hepatocytes in primary culture. As compared to controls cultured with 5.6 mM glucose, maximal (> or = 75%) inhibition was obtained at 20-30 mM, and half-maximal effect at 10-15 mM. Comparison of D- and L-glucose showed that the effect was specific for the D-form. Maximal inhibition required the presence of glucose during the first 24 hours of culture. The expression of the c-myc gene was reduced when the hepatocytes were cultured in the presence of elevated glucose. The responses to epidermal growth factor, insulin and vasopressin, in terms of percentual stimulation of DNA synthesis, were qualitatively similar at 5.6 and 16.8 mM glucose, while glucagon stimulated more strongly when the glucose concentration was increased; glucagon at concentrations > or = 1 nM reversed the inhibition by glucose. 8-Br-cAMP mimicked the effect of glucagon. These results suggest that an increase in the level of glucose depresses hepatocyte DNA synthesis. The effect is associated with lowered expression of the c-myc gene and is counteracted by cAMP.

Stimulatory and inhibitory effects of catecholamines on DNA synthesis in primary rat hepatocyte cultures: role of alpha 1- and beta-adrenergic mechanisms.

Previous studies suggest that catecholamines may be involved in the regulation of liver growth. Considerable evidence implicates alpha 1-adrenergic mechanisms in the initiation of hepatocyte proliferation, while the role of beta-adrenoceptors is less clear. We have examined further the adrenergic regulation of hepatocyte DNA synthesis, using primary monolayer cultures. In hepatocytes that were also treated with epidermal growth factor and insulin, epinephrine or norepinephrine added early after the seeding strongly accelerated the rate of S phase entry. The beta-adrenergic agonist isoproterenol and the alpha-adrenergic agonist phenylephrine also stimulated the DNA synthesis, but were less efficient than epinephrine and norepinephrine. Experiments with the alpha 1-receptor blocker prazosine and the beta-receptor blocker timolol showed that the stimulatory effect of norepinephrine consisted of both an alpha 1- and a beta-adrenergic component. The alpha 1-component was most prominent in terms of maximal response at high concentrations of the agonist, but the beta-component contributed significantly and predominated at low concentrations (less than 0.1 microM) of norepinephrine. At later stages (about 40 h) of culturing norepinephrine strongly but reversibly inhibited the cells, acting at a point late in the G1 phase. This inhibition was mimicked by isoproterenol and abolished by timolol but was unaffected by prazosine, suggesting a beta-adrenoceptor-mediated effect. The results confirm the alpha 1-adrenoceptor-mediated stimulatory effect, but also show that beta-adrenoceptors may contribute to the growth stimulation by catecholamines. Furthermore, catecholamines, via beta-adrenoceptors and cyclic AMP, inhibit the G1-S transition, and may thus play a role in the termination of hepatic proliferation.

Dual effects of glucagon and cyclic AMP on DNA synthesis in cultured rat hepatocytes: stimulatory regulation in early G1 and inhibition shortly before the S phase entry.

Although several lines of evidence implicate cyclic AMP in the humoral control of liver growth, its precise role is still not clear. To explore further the role of cyclic AMP in hepatocyte proliferation, we have examined the effects of glucagon and other cyclic AMP-elevating agents on the DNA synthesis in primary cultures of adult rat hepatocytes, with particular focus on the temporal aspects. The cells were cultured in a serum-free, defined medium and treated with epidermal growth factor (EGF), insulin, and dexamethasone. Exposure of the hepatocytes to low concentrations (10 pM-1 nM) of glucagon in the early stages of culturing (usually within 6 h from plating) enhanced the initial rate of S phase entry without affecting the lag time from the plating to the onset of DNA synthesis, whereas higher concentrations inhibited it. In contrast, glucagon addition at later stages (24-45 h after plating) produced only the inhibition. Thus, if glucagon was added at a time when there was a continuous EGF/insulin-induced recruitment of cells to S phase, the rate of G1-S transition was markedly decreased within 1-3 h. This inhibitory effect occurred at low glucagon concentrations (ID50 less than 1 nM) and was mimicked by cholera toxin, forskolin, isobutyl methylxanthine, and 8-bromo cyclic AMP. The results indicate that cyclic AMP has dual effects on hepatocyte proliferation with a stimulatory modulation early in the prereplicative period (G0 or early G1), and a marked inhibition exerted immediately before the transition from G1 to S phase.

Injury to the femoral vessels--the Lebanese War experience.

Over a 10-year-period extending from January 1976 to September 1986, 800 peripheral vascular injuries were surgically treated at the American University of Beirut Medical Centre. Of these, 150 were operated on because of injury to the femoral vessels. Bullets were the commonest wounding agents (60% of cases). Thirty-seven patients had a femoral artery injury, 27 a femoral vein injury and 86 combined femoral artery and vein damage. Twenty-three per cent of the patients had an associated fracture of the femur. Patients with fractures had an 11% amputation rate compared with 1% for those without fractures. Our data reveals the importance of venous reconstruction in the presence of an arterial injury instead of its ligation (P less than 0.05), particularly when there is an associated fracture of the femur. Thirty-seven per cent of patients were in shock on admission (systolic BP 90 mmHg). The amputation rate in these patients was 6.8% in contrast to 1% in the haemodynamically stable group (P less than 0.05). The overall amputation rate was 3.3%. Complications occurred in 36.6% of patients and the mortality was 4.0%.

Acquired hypoganglionosis after soave endorectal pull-through procedure--a case report.

A case of acquired neurogenic megacolon in a child is reported. The condition resulted after a Soave endorectal pull-through procedure for Hirschsprung's disease and was secondary to hypoganglionosis which developed in the pulled-through segment. The clinical, radiological, and pathologic features as well as the successful surgical management of this case are described.