A. J. C. van den Brule

The BRAF V600E mutation is an independent prognostic factor for survival in stage II and stage III colon cancer patients

BACKGROUND Molecular markers in colon cancer are needed for a more accurate classification and personalized treatment. We determined the effects on clinical outcome of the BRAF mutation, microsatellite instability (MSI) and KRAS mutations in stage II and stage III colon carcinoma. PATIENTS AND METHODS Stage II colon carcinoma patients (n = 106) treated with surgery only and 258 stage III patients all adjuvantly treated with 5-fluorouracil chemotherapy were included. KRAS mutations in codons 12 and 13, V600E BRAF mutation and MSI status were determined. RESULTS Older patients (P < 0.001), right-sided (P = 0.018), better differentiated (P = 0.003) and MSI tumors (P < 0.001) were significantly more frequent in stage II than stage III. In both groups, there was a positive association between mutated BRAF and MSI (P = 0.001) and BRAF mutation and right-sided tumors (P = 0.001). Mutations in BRAF and KRAS were mutually exclusive. In a multivariate survival analysis with pooled stage II and stage III data, BRAF mutation was an independent prognostic factor for overall survival (OS) and cancer-specific survival [hazards ratio (HR) = 0.45, 95% confidence interval (CI) 0.25-0.8 for OS and HR = 0.47, 95% CI 0.22-0.99]. KRAS mutation conferred a poorer disease-free survival (HR = 0.6, 95% CI 0.38-0.97). CONCLUSIONS The V600E BRAF mutation confers a worse prognosis to stage II and stage III colon cancer patients independently of disease stage and therapy.

Local recurrence in rectal cancer can be predicted by histopathological factors

AIM The main cause of local recurrence (LR) in rectal cancer is involvement of the circumferential resection margin (CRM). However, patients with a negative CRM can also develop LR, suggesting that additional factors are important for LR. The aim of this study was to identify histopathological factors predictive for the development of LR after primary rectal cancer treatment. METHODS T x N x M0 patients treated for locally recurrent rectal cancer at the Catharina hospital from 1994 to 2006 (n=92) were matched with a control group of patients who did not develop LR after primary rectal cancer treatment for at least 2 years (n=185) based on the type of neoadjuvant treatment in a 1:2 ratio. The pathology of all primary rectal cancers was reviewed. Patient, treatment and histopathological characteristics were studied in relation to the development of LR with logistic regression. RESULTS Logistic regression indicated the presence of lymphovascular invasion (LVI, OR 4.66, P<0.001), extramural venous invasion (EMVI, OR 4.54, P<0.001), positive CRM (OR 2.56, P=0.032), serosal involvement (OR 6.74, P=0.035) and poor differentiation (OR 2.59, P=0.012) as factors with an increased risk to develop LR. Older age was a protective factor (OR 0.95, CI 0.93-0.98, P=0.001). CONCLUSION Apart from a positive CRM and serosal involvement, LVI, EMVI and poor differentiation are important independent predictive factors for the development of LR. Adjuvant therapy may be considered in the presence of these features in order to decrease the risk of a local recurrence.

Comparative Study Using Phenotypic, Genotypic, and Proteomics Methods for Identification of Coagulase-Negative Staphylococci

ABSTRACT Five methods were compared to determine the most accurate method for identification of coagulase-negative staphylococci (CoNS) (n = 142 strains). Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) showed the best results for rapid and accurate CoNS differentiation (99.3% of strains correctly identified). An alternative to this approach could be Vitek2 combined with partial tuf gene sequencing (100% of strains correctly identified when both methods are performed simultaneously).

PIK3CA kinase domain mutation identifies a subgroup of stage III colon cancer patients with poor prognosis

BackgroundPIK3CA mutations in the helical domain (in exon 9) and in the kinase domain (exon 20) cause tumor formation by different means. We aimed to determine the effects of each of these mutations on survival of colon carcinoma patients.MethodsA large cohort of 685 colon carcinoma patients was tested for PIK3CA mutations in exons 9 and 20 by single nucleotide primer extension (N = 428) or by real time PCR (N = 257).ResultsPIK3CA mutation rate was 13%. 66 of 83 (79.5%) were in exon 9 and 17 of 83 (20.5%) in exon 20. In survival analysis, PIK3CA mutations in exon 9 and 20 had different effects on patient outcome. The PIK3CA exon 20 mutation conferred a poorer disease free survival compared to patients with wild type alleles and exon 9 mutations (Log rank p = 0.04 and p = 0.03 respectively) and cancer specific survival (Log rank p = 0.03 and p = 0.056 respectively) in stage III patients. In stage I and II this negative effect on outcome was not seen.ConclusionsPIK3CA mutation in exon 20 is a negative prognostic factor in stage III colon cancer patients. Moreover, this negative effect is not present in stage I and II patients.

Developments for improved diagnosis of bacterial bloodstream infections

Bloodstream infections (BSIs) are associated with high mortality and increased healthcare costs. Optimal management of BSI depends on several factors including recognition of the disease, laboratory tests and treatment. Rapid and accurate identification of the etiologic agent is crucial to be able to initiate pathogen specific antibiotic therapy and decrease mortality rates. Furthermore, appropriate treatment might slow down the emergence of antibiotic resistant strains. Culture-based methods are still considered to be the “gold standard” for the detection and identification of pathogens causing BSI. Positive blood cultures are used for Gram-staining. Subsequently, positive blood culture material is subcultured on solid media, and (semi-automated) biochemical testing is performed for species identification. Finally, a complete antibiotic susceptibility profile can be provided based on cultured colonies, which allows the start of pathogen-tailored antibiotic therapy. This conventional workflow is extremely time-consuming and can take up to several days. Furthermore, fastidious and slow-growing microorganisms, as well as antibiotic pre-treated samples can lead to false-negative results. The main aim of this review is to present different strategies to improve the conventional laboratory diagnostic steps for BSI. These approaches include protein-based (MALDI-TOF mass spectrometry) and nucleic acid-based (polymerase chain reaction [PCR]) identification from subculture, blood cultures, and whole blood to decrease time to results. Pathogen enrichment and DNA isolation methods, to enable optimal pathogen DNA recovery from whole blood, are described. In addition, the use of biomarkers as patient pre-selection tools for molecular assays are discussed.

Cyclooxygenase 2 Expression in Rectal Cancer Is of Prognostic Significance in Patients Receiving Preoperative Radiotherapy

Purpose: To determine the effect of cyclooxygenase (COX)-2 expression on clinical behavior in irradiated and nonirradiated rectal carcinomas. Experimental Design: Tumor samples were collected from 1,231 patients of the Dutch TME trial, in which rectal cancer patients were treated with standardized surgery and randomized for preoperative short-term (5 × 5 Gy) radiotherapy or no preoperative radiotherapy. Tissue microarrays were constructed from primary tumor material, and COX-2 expression was assessed by immunohistochemistry. Tumor cell apoptosis was determined by M30 immunostaining. Results: A high level of COX-2 expression after radiotherapy was associated with low levels of tumor cell apoptosis (P = 0.001). COX-2 expression had no significant effect on patient survival or tumor recurrence in nonirradiated tumors. However, in patients receiving preoperative radiotherapy, high level of COX-2 expression was associated with higher incidence of distant recurrences [P = 0.003; hazard ratio (HR), 1.7; 95% confidence interval (95% CI), 1.2-2.5] and shorter disease-free survival (P = 0.002; HR, 1.8; 95% CI, 1.2-2.5) and overall survival (P = 0.009; HR, 1.5; 95% CI, 1.1-2.0), independent of patient age, tumor stage, tumor location, or the presence of tumor cells in the circumferential resection margin. Conclusions: A high level of COX-2 expression after preoperative radiotherapy in resection specimens is associated with apoptosis resistance, high distant recurrence rates, and a poor prognosis in rectal cancer.

Preoperative radiochemotherapy is successful also in patients with locally advanced rectal cancer who have intrinsically high apoptotic tumours

BACKGROUND Not all patients with locally advanced rectal cancer (LARC) respond equally to neo-adjuvant radiochemotherapy (RCT). Patients with highly apoptotic less advanced rectal cancers do not benefit from short-term radiotherapy. This study investigates whether this is also the case in the setting of RCT for LARC. PATIENTS AND METHODS Tissue microarrays were constructed of biopsy and resection specimens of 201 LARC patients. Apoptosis (M30) and several apoptosis-regulating proteins [p53, Bcl-2, Bax, cyclooxygenase-2 (Cox-2) and mamma serine protease inhibitor (maspin)] were studied with immunohistochemistry. Subsequently, predictive values for local recurrence (LR), overall survival (OS) and histological tumour regression were analysed. RESULTS Apoptotic levels, quantified as the number of apoptotic cells/mm(2) tumour epithelium, were higher in posttherapy tissues compared with biopsies (P < 0.001). Biopsies from clinical T4 stage tumours demonstrated significantly higher levels of apoptosis than clinical T3 stage tumours (P = 0.020). Therapy-induced apoptosis was higher when the interval between the last day of irradiation and surgery increased (P < 0.001, correlation coefficient = 0.355). Pre- and posttherapy apoptosis, p53, Bcl-2, Bax and Cox-2 were not associated with LR, OS or tumour regression. Intense pretherapy cytoplasmatic staining of maspin indicated a higher risk on LR (P = 0.009) only. CONCLUSION Combined RCT is also successful in highly apoptotic tumours and is therefore independent of intrinsic apoptosis.

High-risk human papillomavirus detection in self-sampling compared to physician-taken smear in a responder population of the Dutch cervical screening: Results of the VERA study

In 2017 the cervical cancer screening program in The Netherlands will be revised. Cervical smears will primarily be tested for the presence of high-risk human papillomavirus (hrHPV) instead of cytology, and vaginal self-sampling will be offered to non-responders. This includes a potential risk that part of the women who would otherwise opt for a cervical smear will wait for self-sampling. However, self-sampling for hrHPV in a responder population has never been studied yet. The aim of this study was to investigate the applicability and accuracy of self-sampling in detecting hrHPV in a screening responder population. A total of 2049 women, aged 30-60years, participating in the screening program in The Netherlands were included from April 2013 to May 2015. After they had their cervical smear taken, women self-collected a cervicovaginal sample with a brush-based device, the Evalyn Brush. Both the cervical smear and self-sample specimen were tested with the COBAS 4800 HPV platform. The hrHPV prevalence was 8.0% (95% CI 6.9-9.2) among the physician-taken samples, and 10.0% (95% CI 8.7-11.3) among the self-samples. There was 96.8% (95% CI 96.0-97.5) concordance of hrHPV prevalence between self-samples and physician-taken samples. Women in our study evaluated self-sampling as convenient (97.1%), user-friendly (98.5%), and 62.8% preferred self-sampling over a physician-taken sampling for the next screening round. In conclusion, self-sampling showed high concordance with physician-taken sampling for hrHPV detection in a responder screening population and highly acceptable to women. Implementation of HPV-self-sampling for the responder population as a primary screening tool may be considered.

Multiple-type human papillomavirus infection in younger uncircumcised men.

A cohort of 388 young men enrolled for military service in the Danish army was established and the participants underwent a clinical examination with human papillomavirus (HPV) testing. In addition, a questionnaire containing questions regarding sociodemographic variables, sexual habits and lifestyle factors was completed. The prevalence of HPV was 33.4% in this cohort of uncircumcised men aged 18–29 years. Multiple HPV types were prevalent with one-third of the HPV-positive men being positive for more than one HPV type. Number of recent sexual partners and infrequent condom use were strong risk factors, particularly in men having multiple HPV types. Our findings re-emphasize the importance of sexual transmission and also point to a role of factors that may be related to individual susceptibility as genital warts, alcohol intake and, to a lesser extent, smoking were strongly associated with having multiple HPV types.

Correlation of haloperidol concentration in blood and cerebrospinal fluid: a pharmacokinetic study.

To the Editors: Haloperidol is often prescribed to older patients for the treatment of acute or chronic psychotic symptoms, such as delirium or behavioral symptoms in dementia. Its use is associated with adverse effects such as antipsychotic-induced parkinsonism (AIP) and tardive dyskinesia. Antipsychotic-induced parkinsonism is characterized by the presence of tremor, rigidity, and bradykinesia and is associated with impaired quality of life. In a study with 150 elderly patients, 46% of the patients treated with haloperidol in a dose varying from 0.3 to 5.0 mg/d developed AIP. In this group, a significant (but moderate) relationship was found between dose and serum concentration. Neither the dose nor the serum concentrations of haloperidol however were associated with the occurrence of AIP. It is unclear why some older patients develop AIP at a given dosage of haloperidol and others do not. There are different hypotheses that could explain the differences in sensitivity to antipsychotics. One possible explanation could be that interindividual variation in transport crosses the blood-brain barrier (BBB). The relationship between blood and cerebrospinal fluid (CSF) concentration of haloperidol has not been described earlier in an older population. The aim of this crosssectional study, therefore, was to determine this relationship in older patients. The study was conducted in a population of patients visiting the preoperative screening and delirium prevention outpatient clinic from the Department of Geriatric Medicine of the Jeroen Bosch Hospital, a large teaching hospital in ’s-Hertogenbosch, the Netherlands, between January 2012 and January 2013. In case of an increased risk of delirium, 1 mg/d of haloperidol for 5 days preoperative was prescribed by the geriatrician, according to hospital protocol. Inclusion criteria were age older than 64 years, elective surgery under spinal anesthesia, adequately started with 1 mg/d of haloperidol according to hospital protocol, mentally competent, and written informed consent. Approval was obtained from the regional Medical Research Ethics Committee. Informed consent was asked by 1 of the researchers. Elective surgery under spinal anesthesia made it possible to obtain 2 mL of CSF for research goals without patient burden such as a lumbar puncture. A blood sample (2 mL) was drawn by the anesthesiologist in addition. A published liquid chromatography-mass spectometrie method was adapted to perform the analysis of the samples. The quantification limit of haloperidol levels was 0.02 ng/mL. Only 1 patient had a haloperidol CSF concentration below this quantification limit. We changed the CSF concentration for this patient in 0.01 ng/mL. Using the Statistical Package for the Social Sciences (IBM, SPSS 20), frequencies and distributions were extracted. The correlation coefficient (R) was calculated by linear regression. R was used to express the percentage of explained variance between CSF and serum. We corrected in multiple linear regression analysis for age and sex. Twenty patients were included with an average age of 78.9 years (range, 68Y91 y). Four patients were women. Eighteen patients used a dosage of 1 mg/d of haloperidol for a 5-day period before CSF and serum concentrations were measured. Two patients deviated from this scheme; 1 patient used 2 mg of haloperidol instead of 1 mg on day 5. One patient used double dosage of 2 mg/d of haloperidol for a 5-day period. Haloperidol is metabolized by the cytochrome P450 enzyme 2D6 (CYP2D6). No patients used strong CYP2D6 inhibitors or strong CYP2D6 inducers. Patients 2 and 15 used darifenacin, patient 6 used mirtazepine, patient 11 used venlafaxine, and patient 13 used citalopram, all weak CYP2D6 inhibitors. Serum concentrations of haloperidol were averaged 0.52 Kg/L (range, 0.17Y0.99 Kg/L) and CSF concentrations were averaged 0.04 Kg/L (range, G0.017Y0.09 Kg/L; ratio average, 11.45%). The serum concentration at the same dose of haloperidol has a large, factor 6, variation. A strong and significant correlation between CSF and serum concentrationswas found (r = 0.85, r = 0.73, P G 0.01; Fig. 1); the CSF concentrations of haloperidol were 73% explained by the serum haloperidol concentration. The explained variance increased with only 4% when age was added to the modelVr 2 = 0.77, P G 0.01. Sex did not explain differences in CSF concentration nor did comedication explain differences in serum concentrations of haloperidol. DISCUSSION This study shows a strong and significant correlation between the serum concentration and CSF concentration of haloperidol in older patients. Sex or age hardly influenced this relation nor did comedication explain the serum concentration. As far as we know, only 1 study was published studying haloperidol concentrations in CSF and in serum, however, not in older patients, but in younger schizophrenic patients. Rimón et al analyzed 12 chronic neuroleptic nonresponsive schizophrenic patients (mean age, 39 y) after 1 month on 60 mg of haloperidol daily. Cerebrospinal fluid concentrations of haloperidol were significantly correlated (r = 0.55 and P G 0.01) and averaged 4.3% of the serum concentrations. In our study, CSF concentrations of haloperidol were stronger correlated to the plasma concentration (r = 0.85); however, twice as high relative concentrations were measured (11.5% of serum concentration). One of the hypotheses of variability of response to haloperidol, variability in transport over the BBB, seems to be not an explanatory variable given these results. A possible explanation for a higher ratio between CSF and serum concentrations of haloperidol in our study (11.5% compared with 4.3%) could be age. It might be possible that older patients have a more permeable BBB than younger people; although no correlation of age (in our study aged 68Y91 y) and CSF concentrations (P = 0.09) was found and adding age to the model did hardly increase correlation, we could not underline that hypothesis. It should be taken into account that the range in age in this study population was quite small to do a proper analysis on age, so this could still be a possible hypothesis. The higher ratio could also be related to the much lower doses used, 1 versus 60 mg/d of haloperidol. This seems less likely to be the explanation because haloperidol is not a substrate of P-glycoprotein. P-glycoprotein is an efflux pump expressed at the BBB and limits drug access into the central nervous system. A previous study found that AIP could not be explained at all by dose or blood concentration of haloperidol. Taken together, although, by age, still, some variation in CSF concentration might be explained by future research, the main explanation seems to be plasma concentration of haloperidol, which is in line with a previous study. So, the interindividual variability of CSF LETTERS TO THE EDITORS