Gene length corrected trimmed mean of M-values (GeTMM) processing of RNA-seq data performs similarly in intersample analyses while improving intrasample comparisons.

BACKGROUND: Current normalization methods for RNA-sequencing data allow either for intersample comparison to identify differentially expressed (DE) genes or for intrasample comparison for the discovery and validation of gene signatures. Most studies on optimization of normalization methods typically use simulated data to validate methodologies. We describe a new method, GeTMM, which allows for both inter- and intrasample analyses with the same normalized data set. We used actual (i.e. not simulated) RNA-seq data from 263 colon cancers (no biological replicates) and used the same read count data to compare GeTMM with the most commonly used normalization methods (i.e. TMM (used by edgeR), RLE (used by DESeq2) and TPM) with respect to distributions, effect of RNA quality, subtype-classification, recurrence score, recall of DE genes and correlation to RT-qPCR data. RESULTS: We observed a clear benefit for GeTMM and TPM with regard to intrasample comparison while GeTMM performed similar to TMM and RLE normalized data in intersample comparisons. Regarding DE genes, recall was found comparable among the normalization methods, while GeTMM showed the lowest number of false-positive DE genes. Remarkably, we observed limited detrimental effects in samples with low RNA quality. CONCLUSIONS: We show that GeTMM outperforms established methods with regard to intrasample comparison while performing equivalent with regard to intersample normalization using the same normalized data. These combined properties enhance the general usefulness of RNA-seq but also the comparability to the many array-based gene expression data in the public domain.

Intestinal Bacteremia After Liver Transplantation Is a Risk Factor for Recurrence of Primary Sclerosing Cholangitis.

BACKGROUND: Primary sclerosing cholangitis (PSC) is a chronic progressive pathological process, related to inflammatory bowel disease and subsequent bacterial translocation. Liver transplantation (LT) is the only curative therapy, but outcomes are compromised by recurrence of PSC (rPSC). The aim of the study was to investigate a potential link between intestinal bacteremia, fucosyltransferase-2 (FUT2), and rPSC after LT. METHODS: LT recipients with PSC (n = 81) or without PSC (n = 271) were analyzed for clinical outcomes and positive bacterial blood cultures. A link between bacteremia and the genetic variant of the FUT2 gene was investigated. RESULTS: The incidence of inflammatory bowel disease was significantly higher in PSC recipients but not associated with rPSC. Bacteremia occurred in 31% of PSC recipients. The incidence of rPSC was 37% and was significantly more common in patients with intestinal bacteremia versus no bacteremia (82% versus 30%; P = 0.003). The nonsecretor polymorphism of the FUT2 gene was identified as a genetic risk factor for both intestinal bacteremia and rPSC. Combined FUT2 genotype and intestinal bacteremia in recipients resulted in the highest risk for rPSC (hazard ratio, 15.3; P < 0.001). CONCLUSIONS: Thus, in this article, we showed that bacterial translocation is associated with rPSC after LT and related to the FUT2 nonsecretor status.

Cut-off values for low skeletal muscle mass at the level of the third cervical vertebra (C3) in patients with head and neck cancer.

Background: Low skeletal muscle mass is associated with adverse outcomes in patients with cancer. For patients with head and neck cancer (HNC), skeletal muscle mass is often assessed at the third cervical vertebra on head and neck imaging. Due to the unavailability of standardized cut-off values for low skeletal muscle mass in patients with head and cancer, there is heterogeneousness of cut-off values for low skeletal muscle mass described in literature. Therefore, we aim to provide standardized cut-off values for low skeletal muscle mass in HNC patients. Methods: A retrospective cohort study was performed. Between 2008 and 2018, HNC patients with head and neck imaging were included. Skeletal muscle area (SMA) was manually delineated at the level of the third cervical vertebra and corrected for patients squared height to obtain the cervical skeletal muscle mass index. Gender and body-mass index specific cut-off values for low skeletal muscle mass were calculated based on mean cervical skeletal muscle mass index minus 2 standard deviations as suggested in literature. Results: Of the 1,415 included patients, the majority was male (69.8%) and had a body mass index below 25 kg/m2 (59.2%). A primary tumor localization in the oropharynx (35.3%) and a tumor, node, metastasis stage IV tumor (60.5%) were most frequently observed. Cervical skeletal muscle mass index was significantly correlated with gender (r2=0.4, P<0.01) and body mass index (r2=0.4, P<0.01). For male patients with a body mass index <25 and ≥25 kg/m2, a cervical skeletal muscle mass index of respectively ≤6.8 and ≤8.5 cm2/m2 was defined for low skeletal muscle mass. For female patients with a body mass index <25 and ≥25 kg/m2, a cervical skeletal muscle mass index of respectively ≤5.3 and ≤6.4 cm2/m2 was defined for low skeletal muscle mass. Conclusions: This study is the first to provide standardized cut-off values for low skeletal muscle mass at the level of the third cervical vertebra in patients with HNC. This information may aid in the uniformity of low skeletal muscle mass definition in research.

The association of pretreatment low skeletal muscle mass with chemotherapy dose-limiting toxicity in patients with head and neck cancer undergoing primary chemoradiotherapy with high-dose cisplatin.

BACKGROUND: Low skeletal muscle mass (SMM) is an adverse prognostic factor for chemotherapy dose-limiting toxicity (CDLT). In patients with locally advanced head and neck squamous cell carcinoma (HNSCC) undergoing chemoradiotherapy (CRT), low SMM is a predictor for CDLT. We aimed to validate these findings. METHODS: Consecutive LA-HNSCC patients treated with primary CRT with high-dose cisplatin were retrospectively included. SMM was measured on pre-treatment CT-imaging. A cumulative cisplatin dose below 200 mg/m2 was defined as CDLT. RESULTS: One hundred and fifty three patients were included; 37 (24.2%) experienced CDLT, and 84 had low SMM (54.9%). Patients with low SMM experienced more CDLT than patients with normal SMM (35.7% vs. 10.1%, p < 0.01). Low SMM (OR 3.99 [95% CI 1.56-10.23], p = 0.01) and an eGFR of 60-70 ml/min (OR 5.40 [95% CI 1.57-18.65], p < 0.01) were predictors for CDLT. CONCLUSION: Pre-treatment low SMM is associated with CDLT in LA-HNSCC patients treated with primary CRT. Routine SMM assessment may allow for CDLT risk assessment and treatment optimization.

Arterial calcification on preoperative computed tomography imaging as a risk factor for pharyngocutaneous fistula formation after total laryngectomy.

BACKGROUND: Research in esophageal surgery showed that computed tomography (CT) assessed arterial calcification (AC) is associated with postoperative complications. We investigated the association between AC and pharyngocutaneous fistula (PCF) formation after laryngectomy. METHODS: This was a retrospective cohort study of patients undergoing laryngectomy. AC was scored at 10 different anatomical locations on CT imaging, blinded for PCF occurrence. Association with PCF was investigated using logistic regression. RESULTS: The 224 patients were included; 62 (27.7%) developed a PCF. Moderate to severe AC was widespread in patients undergoing TL; 7.1% of patients had at most mild AC, of whom 1 experienced a PCF (p = 0.05). A higher cumulative calcification score was associated with PCF in univariable (OR 1.11, p = 0.04) and multivariable analysis (OR 1.14, p = 0.05). CONCLUSION: AC is widespread in patients undergoing laryngectomy and its burden is associated with PCF. Extensive AC on preoperative imaging may be considered a risk factor for PCF.

Preoperative low skeletal muscle mass as a risk factor for pharyngocutaneous fistula and decreased overall survival in patients undergoing total laryngectomy.

BACKGROUND: Low skeletal muscle mass (SMM) is associated with postoperative complications, prolonged hospital stay, and short overall survival (OS) in surgical oncology. We aimed to investigate this association in patients undergoing total laryngectomy (TL). METHODS: A retrospective study was performed of patients undergoing TL. SMM was measured using CT or MRI scans at the level of the third cervical vertebra (C3). RESULTS: In all, 235 patients were included. Low SMM was observed in 109 patients (46.4%). Patients with low SMM had more pharyngocutaneous fistulas (PCFs) than patients with normal SMM (34.9% vs 20.6%; P = .02) and prolonged hospital stay (median, 17 vs 14 days; P < .001). In multivariate analysis, low SMM (hazards ratio, 1.849; 95% confidence interval, 1.202-2.843) and high N stage were significant prognosticators of decreased OS. CONCLUSION: Low SMM is associated with PCF and prolonged hospital stay in patients undergoing TL. Low SMM is an independent prognostic factor for shorter OS.

Sarcopenia, a strong determinant for prolonged feeding tube dependency after chemoradiotherapy for head and neck cancer.

BACKGROUND: Sarcopenia might be a relevant lead for optimization of the condition of patients with head and neck cancer (HNC) before chemoradiotherapy (CRT) to prevent long-term functional swallowing impairment, such as feeding tube dependency. METHODS: Regression analyses were performed to assess the association between skeletal muscle mass index (SMI), as a measure of sarcopenia, and prolonged (>90 days) feeding tube dependency in 128 patients with HNC treated with primary CRT. RESULTS: Sixty-one patients (48%) became prolonged feeding tube-dependent. Lower SMI increased the risk of prolonged feeding tube dependency in multivariable analysis (risk ratio 1.08; 95% confidence interval 1.02-1.14, P = .01) adjusted for body mass index, abnormal diet, and socioeconomic status. CONCLUSIONS: Sarcopenia contributes to the risk of prolonged feeding tube dependency of patients with HNC treated with primary CRT. As sarcopenia might be a modifiable factor prior to treatment, it should be explored as a target for pretreatment patients' condition.

Dilation after laryngectomy: Incidence, risk factors and complications.

BACKGROUND: Neopharyngeal stenosis is a recognized sequela of total laryngectomy (TL). We aim to investigate the incidence of stenosis requiring dilation, risk factors for stenosis and complications of dilation. METHODS: Retrospective cohort study of patients undergoing TL in two dedicated head and neck centers in the Netherlands. RESULTS: A total of 477 patients, (81% men, median age of 64 at TL) were included. Indication for TL was previously untreated primary tumor in 41%, salvage following (chemo)radiotherapy (CRT) in 44%, dysfunctional larynx in 9% and a second primary tumor in 6%. The cumulative incidence of dilatation at 5 years was 22.8%, and in total 968 dilatations were performed. Median number of dilations per patient was 3 (range 1-113). Female gender, a hypopharynx tumor, and (C)RT before or after the TL were significantly associated with stenosis requiring dilation. We observed 8 major complications (0.8%) predominantly during the first dilation procedures. Use of general anesthesia is a risk factor for complications. The most frequent major complication was severe esophageal perforation (n = 6 in 5 patients). CONCLUSION: The cumulative incidence of pharyngeal stenosis needing dilation was 22.8% at 5 years. Roughly half of these patients could be treated with a limited number of dilations, the rest however needed ongoing dilations. Major complications are rare (0.8%) but can be life threatening. General anesthetics is a risk factor for complications, and complications occurred predominantly during the first few dilations procedures. This should alert the physician to be extra careful in new patients.

Confirmation of a metastasis-specific microRNA signature in primary colon cancer.

The identification of patients with high-risk stage II colon cancer who may benefit from adjuvant therapy may allow the clinical approach to be tailored for these patients based on an understanding of tumour biology. MicroRNAs have been proposed as markers of the prognosis or treatment response in colorectal cancer. Recently, a 2-microRNA signature (let-7i and miR-10b) was proposed to identify colorectal cancer patients at risk of developing distant metastasis. We assessed the prognostic value of this signature and additional candidate microRNAs in an independent, clinically well-defined, prospectively collected cohort of primary colon cancer patients including stage I-II colon cancer without and stage III colon cancer with adjuvant treatment. The 2-microRNA signature specifically predicted hepatic recurrence in the stage I-II group, but not the overall ability to develop distant metastasis. The addition of miR-30b to the 2-microRNA signature allowed the prediction of both distant metastasis and hepatic recurrence in patients with stage I-II colon cancer who did not receive adjuvant chemotherapy. Available gene expression data allowed us to associate miR-30b expression with axon guidance and let-7i expression with cell adhesion, migration, and motility.

Low skeletal muscle mass is a predictive factor for chemotherapy dose-limiting toxicity in patients with locally advanced head and neck cancer.

OBJECTIVES: Low skeletal muscle mass (SMM) or sarcopenia is emerging as an adverse prognostic factor for chemotherapy dose-limiting toxicity (CLDT) and survival in cancer patients. Our aim was to determine the impact of low SMM on CDLT in patients with locally advanced head and neck squamous cell carcinoma (LA-HNSCC) treated with primary radiochemotherapy (RCT). PATIENTS AND METHODS: Consecutive patients diagnosed with LA-HNSCC and treated with primary RCT between 2007 and 2011 in our center were included. Clinical variables were retrospectively retrieved and SMM was measured at the level of the third cervical vertebra using pre-treatment head and neck CT-scans. After determining a cut-off value for low SMM, multivariate analysis was performed to identify prognostic factors for CDLT. RESULTS: Of 112 patients included, 30.4% experienced CDLT. The optimal cut-off value for low SMM as a predictor of CDLT was ≤43.2cm2/m2. Using this cut-off, 54.5% patients had low SMM. Patients with low SMM experienced CDLT more frequently than patients with normal SMM (44.3% vs. 13.7%, p<0.001) and received a higher dose of chemotherapy/kg lean body mass (estimated from SMM, p=0.044). At multivariate analysis, low SMM was independently inversely associated with CDLT (OR 0.93, 95%CI: 0.88-0.98). Patients experiencing CDLT had a lower overall survival than patients who did not (mean 36.6vs. 54.2months, p=0.038). CONCLUSION: Low SMM is an independent risk factor for CDLT in LA-HNSCC patients treated with primary RCT. Pre-therapeutic estimation of SMM using routine CT-scans of the head and neck region may identify patients at risk of CDLT.

High mRNA expression of splice variant SYK short correlates with hepatic disease progression in chemonaive lymph node negative colon cancer patients.

OBJECTIVE: Overall and splice specific expression of Spleen Tyrosine Kinase (SYK) has been posed as a marker predicting both poor and favorable outcome in various epithelial malignancies. However, its role in colorectal cancer is largely unknown. The aim of this study was to explore the prognostic role of SYK in three cohorts of colon cancer patients. METHODS: Total messenger RNA (mRNA) expression of SYK, SYK(T), and mRNA expression of its two splice variants SYK short (S) and SYK long (L) were measured using quantitative reverse transcriptase (RT-qPCR) in 240 primary colon cancer patients (n = 160 patients with chemonaive lymph node negative [LNN] and n = 80 patients with adjuvant treated lymph node positive [LNP] colon cancer) and related to microsatellite instability (MSI), known colorectal cancer mutations, and disease-free (DFS), hepatic metastasis-free (HFS) and overall survival (OS). Two independent cohorts of patients with respectively 48 and 118 chemonaive LNN colon cancer were used for validation. RESULTS: Expression of SYK and its splice variants was significantly lower in tumors with MSI, and in KRAS wild type, BRAF mutant and PTEN mutant tumors. In a multivariate Cox regression analysis, as a continuous variable, increasing SYK(S) mRNA expression was associated with worse HFS (Hazard Ratio[HR] = 1.83; 95% Confidence Interval[CI] = 1.08-3.12; p = 0.026) in the LNN group, indicating a prognostic role for SYK(S) mRNA in patients with chemonaive LNN colon cancer. However, only a non-significant trend between SYK(S) and HFS in one of the two validation cohorts was observed (HR = 4.68; 95%CI = 0.75-29.15; p = 0.098). CONCLUSION: In our cohort, we discovered SYK(S) as a significant prognostic marker for HFS for patients with untreated LNN colon cancer. This association could however not be confirmed in two independent smaller cohorts, suggesting that further extensive validation is needed to confirm the prognostic value of SYK(S) expression in chemonaive LNN colon cancer.