Influence of BRAF and PIK3CA mutations on the efficacy of FOLFIRI plus bevacizumab or cetuximab as first-line therapy in patients with RAS wild-type metastatic colorectal carcinoma and <3 baseline circulating tumour cells: the randomised phase II VISNÚ-2 study.

BACKGROUND: We explored the influence of BRAF and PIK3CA mutational status on the efficacy of bevacizumab or cetuximab plus 5-fluorouracil/leucovorin and irinotecan (FOLFIRI) as first-line therapy in patients with RAS wild-type metastatic colorectal cancer (mCRC). PATIENTS AND METHODS: VISNÚ-2 was a multicentre, randomised, phase II study. Patients with RAS wild-type mCRC and <3 circulating tumour cells/7.5 ml blood were stratified by BRAF/PIK3CA status (wild-type versus mutated) and number of affected organs (1 versus >1), and allocated to bevacizumab (5 mg/kg every 2 weeks) or cetuximab (400 mg/m2 then 250 mg/m2 weekly) plus FOLFIRI [irinotecan 180 mg/m2, leucovorin 400 mg/m2, 5-fluorouracil 400 mg/m2 (bolus) then 2400 mg/m2 (46-h continuous infusion) every 2 weeks]. The primary endpoint was progression-free survival (PFS). All analyses were exploratory. RESULTS: Two hundred and forty patients with BRAF/PIK3CA wild-type (n = 196) or BRAF- and/or PIK3CA-mutated tumours (n = 44) were enrolled. Median PFS was 12.7 and 8.8 months in patients with BRAF/PIK3CA wild-type and BRAF/PIK3CA-mutated tumours, respectively [hazard ratio (HR) = 1.22; 95% confidence interval (CI) 0.80-1.85; P = 0.3602]. In the BRAF- and/or PIK3CA-mutated cohort, median PFS was 2.8, 8.8 and 15.0 months in patients with BRAF/PI3KCA-mutated (n = 8), BRAF-mutated/PI3KCA wild-type (n = 16) and BRAF wild-type/PI3KCA-mutated (n = 20) tumours, respectively (P = 0.0002). PFS was similar with bevacizumab plus FOLFIRI versus cetuximab plus FOLFIRI in BRAF/PIK3CA wild-type (HR = 0.99; 95% CI 0.67-1.45; P = 0.9486) and BRAF/PIK3CA-mutated tumours (HR = 1.11; 95% CI 0.53-2.35; P = 0.7820). The most common grade 3/4 treatment-related adverse events were neutropenia, diarrhoea and asthenia in both treatment groups. CONCLUSIONS: BRAF/PIK3CA status influences outcomes in patients with RAS wild-type mCRC but does not appear to assist with the selection of first-line targeted therapy.

Ultra-selection of metastatic colorectal cancer patients using next-generation sequencing to improve clinical efficacy of anti-EGFR therapy.

BACKGROUND: Extended RAS analysis is mandatory in metastatic colorectal cancer (mCRC) patients. The optimal threshold of RAS mutated subclones to identify patients most likely to benefit from antiepidermal growth factor receptor (EGFR) therapy is controversial. Our aim was to assess the clinical impact of detecting mutations in RAS, BRAF, PIK3CA and EGFRS492R in basal tissue tumour samples by using a highly sensitive next-generation sequencing (NGS) technology in mCRC patients treated with chemotherapy plus anti-EGFR or anti-vascular endothelial growth factor. PATIENTS AND METHODS: Five hundred and eighty-one tumour samples from untreated mCRC patients from 7 clinical studies were collected. Mutational analysis was carried out by standard-of-care (therascreen pyro) with a sensitivity detection of 5% mutant allele fraction (MAF), and compared with NGS technology using 454GS Junior platform (Roche Applied Science, Germany) with a sensitivity of 1%. Molecular results were correlated with clinical outcomes. RESULTS: After quality assessment, 380 samples were evaluable for molecular analysis. Standard-of-care mutational analysis detected RAS, BRAFV600E or PIK3CA mutations in 56.05% of samples compared with 69.21% by NGS (P = 0.00018). NGS identified coexistence of multiple low-frequency mutant alleles in 96 of the 263 mutated cases (36.5%; range 2-7). Response rate (RR), progression-free survival (PFS) and overall survival (OS) were increasingly improved in patients with RAS wild-type, RAS/BRAF wild-type or quadruple (KRAS/NRAS/BRAF/PIK3CA) wild-type tumours treated with anti-EGFR, assessed by standard-of-care. No additional benefit in RR, PFS or OS was observed by increasing the detection threshold to 1% by NGS. An inverse correlation between the MAF of the most prevalent mutation detected by NGS and anti-EGFR response was observed (P = 0.039). EGFRS492Rmutation was not detected in untreated samples. CONCLUSIONS: No improvement in the selection of patients for anti-EGFR therapy was obtained by adjusting the mutation detection threshold in tissue samples from 5% to 1% MAF. Response to anti-EGFR was significantly better in patients with quadruple wild-type tumours.

SEOM Clinical Guideline of localized rectal cancer (2016).

Localized rectal adenocarcinoma is a heterogeneous disease and current treatment recommendations are based on a preoperative multidisciplinary evaluation. High-resolution magnetic resonance imaging and endoscopic ultrasound are complementary to do a locoregional accurate staging. Surgery remains the mainstay of treatment and preoperative therapies with chemoradiation (CRT) or short-course radiation (SCRT) must be considered in more locally advanced cases. Novel strategies with induction chemotherapy alone or preceding or after CRT (SCRT) and surgery are in development.

Inhibitory action of dopamine involves a subthreshold Cs(+)-sensitive conductance in neostriatal neurons.

Intracellular recordings in in vitro slice preparations of rat brain were used to compare the actions of dopamine and dopamine receptor agonists on the subthreshold membrane properties of neostriatal neurons. A reproducible response for dopaminergic agonists was evoked after firing produced by current ramp injections that induced a subthreshold voltage displacement. Dopamine (10-100 microM) decreased both firing rate and membrane slope input resistance in virtually all cells tested. Input resistance change appeared as an increase in inward rectification. Approximate reversal potential was around -87 mV. The D1 receptor agonists SKF 38393 and Cl-APB (1-10 microM) mimicked both dopamine effects with a reversal potential around -89 mV. The effects were blocked by the presence of 5-10 mM caesium (Cs+) but not by 1 microM tetrodotoxin, suggesting that main D1 effects on input resistance are due to subthreshold Cs(+)-sensitive conductances. cAMP analogues mimicked the actions of D1 receptor agonists. The D2 agonist, quinpirole (1-10 microM), did not produce any input resistance change, nonetheless, it still produced a decrease in firing rate. This suggests that the main D2 effect on firing is due to actions on suprathreshold ion conductances. All effects were blocked by D1 and D2 antagonists, respectively. D1 or D2 effects were found in the majority of cells tested.

Relationship between P-glycoprotein expression and cyclosporin A in kidney. An immunohistological and cell culture study.

P-glycoprotein (P-gp), encoded in humans by the mdr-1 gene, acts physiologically as an efflux pump to expel hydrophobic substances from cells. This glycoprotein is closely related to multidrug resistance in tumor cells and can be modulated by cyclosporin A (CsA). We investigated the relationship between CsA and P-gp in 52 renal allograft biopsies and in cultures of Madin-Darby canine kidney (MDCK) renal tubule cells to determine whether the intrarenal accumulation of CsA or chronic stimulation with the drug modified the expression of P-gp. Expression of P-gp and CsA was analyzed by immunohistochemistry. Immunostaining was evaluated semiquantitatively. Modulation of P-gp in MDCK cells after chronic stimulation with CsA for 7, 30, and 60 days was analyzed by flow cytometry. P-gp and CsA immunostaining in renal post-transplant biopsies showed considerable overlap in all cases (Spearman's test, r = 0.577, P < 0.001). After 7 days in vitro, the number of cells expressing P-gp increased progressively; a further increase in mean fluorescence was found after 60 days (P < 0.001, Student's t-test). Our findings suggest that in non-neoplastic cells, CsA may stimulate P-gp as a mechanism of detoxification. Individual differences in the adaptive responses to glycoprotein may be responsible for the appearance of nephrotoxicity or a CsA-resistant rejection reaction in cases of overexpression on lymphocytes and macrophages.

Influence of intrarenal deposits of ciclosporin A on acute renal transplant rejection.

Immunohistochemical techniques were used to study the presence of ciclosporin A (CsA) and leukocyte subsets in 36 posttransplant renal biopsy specimens histologically diagnosed as acute graft rejection. Glomeruli from patients with CsA deposits contained more leukocytes (p < 0.05) than glomeruli from tissues without deposits. In contrast, the interstitium from patients without deposits contained significantly more B lymphocytes than interstitia from kidneys with CsA deposits. In both glomeruli and interstitia, the CD4/CD8 ratios were higher in tissues without deposits, although the difference was not significant. The plasma levels of creatinine increased with the intensity of renal CsA deposits, and significantly more patients on hemodialysis had deposits as compared with patients not on hemodialysis. Our findings suggest two types of acute nonvascular rejection: (1) predominantly interstitial, with a good prognosis, characterized by low numbers of intrarenal CsA deposits and a predominance of B lymphocytes and (2) predominantly glomerular, with a poor prognosis, characterized by high levels of intrarenal CsA and a predominance of CD8-positive cells and macrophages.

Immunohistochemical study of 30 cases of cyclosporin A-induced gingival overgrowth.

Immunohistochemical techniques were used to study the presence of cyclosporin A (CsA) and leukocyte subsets in 30 gingival biopsies of renal transplant subjects with gingival overgrowth (GO). Statistical analysis revealed significant differences in the total number of inflammatory cells determined by monoclonal antibody CD45, the monocyte/macrophage (CD68) subset, the plasmatic cells (EMA), and the total of T-lymphocytes (CD3) (P < 0.001, Student t test) between the treated subjects and the healthy control group. Differences were found in the helper/inducer T lymphocytes CD4 (P < 0.001 Student t test) and cytotoxic/suppressor T lymphocyte (CD8) (P < 0.01, Student t test) subsets between both groups. The CD4/CD8 ratio was greater in the transplant subjects than in the control group (1.82 +/- 0.16 versus 1.35 +/- 0.05 respectively) (P < 0.05 Student t test). There was no significant difference in the populations CD16+, CD57+, and CD20+. The CD45+ CD4+, and CD68+ cells increased in number along with the degree of GO. The number of epithelial cells/mm2 which displayed a deposit of CsA increased in accordance with the degree of GO (P < 0.05, Kruskal-Wallis's test). Likewise, the intraepithelial deposit of CsA in the GO region was found to be related to the inflammatory infiltrate CD4+, CD8+, and CD68+ (r = 0.7432; r = 0.7346; r = 0.77005, respectively). Our findings suggest that the intraepithelial deposit of CsA and the inflammatory infiltrate play a predominantly pathogenic role and are both related to the degree of GO.

Subthreshold rectification in neostriatal spiny projection neurons.

Intracellular recordings from slice preparations were used to assess the subthreshold electrophysiological behavior of rat neostriatal projection neurons. Both current steps and ramp currents were used to estimate the current-voltage relationship (I-V plot). Inward rectification in the subthreshold range was a characteristic of most neurons. The amount of rectification varied greatly, and it was complex: membrane voltage trajectories in response to ramps were made up by almost piece-wise changes in the rate of voltage rise, suggesting that multiple conductances contribute to the subthreshold range. Inward current blockers such as tetrodotoxin (TTX) or Cd2+ decreased inward rectification, whereas outward current blockers such as tetraethylammonium (TEA) or 4-aminopyridine (4-AP) increased inward rectification. However, most inward rectification was due to TEA- and Cs(+)-sensitive conductances and not to TTX- or Cd(2+)-sensitive conductances. Cs(+)-sensitive conductances predominated at more negative membrane potentials, whereas 4-AP-sensitive conductances predominated at just +/- 10 mV below the firing threshold. In spite of a very slow activation, there was evidence for transient outward currents modulating the response, i.e., 4-AP-sensitivity, and voltage-sensitivity for firing frequency and threshold. TEA-sensitive conductances also contributed toward fixing the firing threshold. These results imply the contribution of various ion conductances on the shaping of the characteristic physiological firing recorded in vivo. Modulation of these responses by transmitters or peptides may help to understand neural processing in the neostriatum.

Reduction of motor impairment by adrenal medulla transplants in aged rats.

Recently we have reported that drugs that enhance dopaminergic transmission, such as L-dopa and D-amphetamine, substantially improve the age-related deterioration of extrapyramidal motor functions, as assessed by the narrow uphill beam test. Here we report the effect of fetal adrenal medullary transplants upon the motor performance of aged rats in such a test. Rats were grafted with 300,000 cultured adrenal medullary cells, placed into the head of either the left caudate nucleus, or into both caudates. A third group was grafted with one freshly dissected adrenal medulla placed as a block of tissue into the lateral ventricle, whereas the control group sustained sham grafting. Evaluation of the motor performance of cultured and sham-grafted rats showed no improvement along the testing phase. Only adrenal block-grafted rats exhibited a significant recovery of motor coordination. Histologically, cultured cell grafts had a deteriorated appearance with poor survival rates, while block grafts exhibited chromaffin cells with round and neuron-like shapes. The results suggest that cultured adrenal grafts may not induce motor improvement due to their extremely low survival and poor integration, at least in the aged host brain, while fresh adrenal transplants may improve motor coordination for as long as 84 days.

[Suprarenal carcinoma of an unusual size]. / Carcinoma suprarrenal de tamaño inusual.

Suprarenal carcinoma is an unusual entity, rarely surpassing 4,000 gr. In general, tumors of such big size are not functional. We present a case of a 31-years-old woman with a suprarenal carcinoma weighting 5,200 gr, in which hormonal production was clinically and biochemically demonstrated.

[Lymphomatoid papulosis evolving into malignant lymphoma. Clinico-pathological and immunohistochemical study of 3 cases]. / Papulosis linfomatoide con evolución a linfoma maligno. Estudio clinicopatológico e inmunohistoquímico de 3 casos.

Lymphomatoid papulosis is a disorder characterized by recurrent skin lesions with histological features suggestive of malignant lymphoma. In most cases the cutaneous lesions heal spontaneously but the course of the disease is long-lasting and an evolution into a Hodgkin's disease and non-Hodgkin lymphomas may be seen. We report herein the clinical, pathological and immunohistochemical study of three patients having a long-standing lymphomatoid papulosis, which turned into Hodgkin's disease, mycosis fungoides and nodular paragranuloma, respectively. Immunohistochemical analysis showed that the immunophenotype of atypical cells in lymphomatoid papulosis was similar to that observed in Reed-Sternberg cells in Hodgkin's disease and the neoplastic cells of mycosis fungoides. However, the immunohistochemical profile of cells in lymphomatoid papulosis differed from those observed in cells of nodular paragranuloma, developed by one of the three patients. The relationship between lymphomatoid papulosis and malignant lymphomas associated to lymphomatoid papulosis is discussed. The results show that no definitive criteria can be infered from an immunohistochemical study in lymphomatoid papulosis, in predicting the clinical evolution of the disease.

[Gastric emptying of solids in chronic alcoholic hepatopathy: role of autonomic insufficiency]. / Vaciamiento gástrico de sólidos en la hepatopatía etílica crónica: responsabilidad de la insuficiencia autonómica.

Thirty one subjects are studied, 15 healthy (controls) and 16 presenting chronic alcoholic hepatopathy, in whom the existence of an autonomic insufficiency syndrome (AIS) was evaluated measuring the variations in heart rate while breathing (R index) and after standing up [O(B) index]. Vagal functioning was also studied when evaluating gastric emptying of digestible solids (VGS) and non digestible (VGSND). VGS after 45, 75 and 105 minutes in alcoholic patients was slower than in healthy controls (p less than 0.05) and in 50% of them it was slow after 105 minutes. VGSND was similar in both groups. R and O(B) indexes in alcoholic patients was smaller than in the control group (p less than 0.001), presenting 94% of them some index altered. An elevated percentage of patients suffering chronic alcoholic hepatopathy present a AIS which involves several organs, being R and O(B) indexes a better diagnostic method than VGS study.

Dibutyryl cAMP stimulates analgesia in rats bearing a ventricular adrenal medulla transplant.

In the present study, a significant increase in pain threshold (current to elicit vocalization to tail shock) was found 15 and 60 min after injection of dibutyryl cyclic AMP (db cAMP) (30 micrograms) into the lateral ventricle in rats bearing a transplant of fetal adrenal medulla (AM). By contrast, no effect on pain threshold was observed in rats bearing an AM transplant but receiving no db cAMP, or in rats receiving db cAMP but not bearing an AM transplant. In primary cultures of rat fetal chromaffin cells, db cAMP increased the number of neuron-like cells that showed both vasoactive intestinal polypeptide (VIP)- and tyrosine hydroxylase (TH)-like immunoreactivity. These findings indicate that db cAMP exerts a pharmacological modulation of the functional activity (i.e. elevation in pain thresholds) of fetal adrenal AM transplants, and induces phenotypic changes in cultured chromaffin cells with expression of a peptide that elevates pain threshold.

Vasoactive intestinal polypeptide induces REM recovery in insomniac forebrain lesioned cats.

Basal forebrain (BF) lesions in cats produces insomnia by reducing both slow wave sleep (SWS) and rapid-eye-movement (REM) sleep time. Recently it has been shown that vasoactive intestinal polypeptide (VIP) may be a specific REM inductor in the parachlorophenylalanine (PCPA) insomniac model. The purpose of this study was to test the hypnogenic properties of VIP in a nonpharmacological model of insomnia. Cats were rendered insomniac by delivering a DC current through stainless steel tripolar electrodes implanted in the basal forebrain area (BFA). Sleep-waking cycle recordings were done prior to lesions and on days 7, 9, 10, 11, 14, and 21 days after BF lesion. On day 10 after the lesion, 200 ng of VIP was injected into the 4th ventricle. Results showed that on postlesion days 7 and 9, SWS and REM sleep total times decreased, while waking time increased significantly. VIP restored REM sleep total time and frequency for almost 48 h, and SWS sleep total time for 24 h. On days 14 and 21 postlesion, insomnia was reestablished. Results are discussed in terms of the possible anatomical and neurochemical substrates whereby VIP can induce the recovery of sleep-waking control values.

In vitro effects of androgens upon the spontaneous rat uterine contractility.

The effectiveness of ring A reduced (5 alpha and 5 beta) testosterone (T) derivatives upon the rat uterus spontaneous contractility was tested in vitro. Compounds with the 3 alpha-hydroxy-5 alpha reduced configuration, such as androsterone and androstanediol, and one 5 beta reduced (5 beta-dihydrotestosterone) elicited a remarkable inhibitory effect upon the myometrial activity. Although steroids with 5 beta reduction were less potent than 3 alpha-hydroxy-5 alpha reduced compounds for depressing the myometrial activity, they were somewhat more potent than T, DHEA, androstenedione and the remaining 5 alpha reduced compounds tested. Therefore, T could act as a "prehormone", accounting for the maintenance of the physiological myometrial activity through its 5-reduced derivatives.