A novel missense variant in the SDR domain of the WWOX gene leads to complete loss of WWOX protein with early-onset epileptic encephalopathy and severe developmental delay.

The human WWOX (WW domain-containing oxidoreductase) gene, originally known as a tumor suppressor gene, has been shown to be important for brain function and development. In recent years, mutations in WWOX have been associated with a wide phenotypic spectrum of autosomal recessively inherited neurodevelopmental disorders. Whole exome sequencing was completed followed by Sanger sequencing to verify segregation of the identified variants. Functional WWOX analysis was performed in fibroblasts of one patient. Transcription and translation were assessed by quantitative real-time PCR and Western blotting. We report two related patients who presented with early epilepsy refractory to treatment, progressive microcephaly, profound developmental delay, and brain MRI abnormalities. Additionally, one of the patients showed bilateral optic atrophy. Whole exome sequencing revealed homozygosity for a novel missense variant affecting the evolutionary conserved amino acid Gln230 in the catalytic short-chain dehydrogenase/reductase (SDR) domain of WWOX in both girls. Functional studies showed normal levels of WWOX transcripts but absence of WWOX protein. To our knowledge, our patients are the first individuals presenting the more severe end of the phenotypic spectrum of WWOX deficiency, although they were only affected by a single missense variant of WWOX. This could be explained by the functional data indicating an impaired translation or premature degradation of the WWOX protein.

Cell-Free Plasma DNA for Disease Stratification and Prognosis in Head and Neck Cancer.

BACKGROUND: Clinicians face many challenges in disease stratification and outcome prediction in head and neck squamous cancer cell (HNSCC) patients. Given the limitations of currently used clinical scoring, repetitive biopsies, and imaging techniques, liquid biopsy approaches may provide valuable additional diagnostic and prognostic information. METHODS: A noninterventional, single-center observational study was performed with clinical data and plasma samples from HNSCC patients. Cell-free tumor DNA-derived copy number aberrations (CNAs) were determined in 116 patients by low-coverage next-generation sequencing (NGS). Significant CNAs were combined in a genome-wide copy number instability score (CNI), which was evaluated with respect to conventional clinical staging and patient outcome. RESULTS: Receiver-operating characteristic (ROC) curve analysis comparing the presurgery CNI in patients (n = 103) with that in tumor-free controls (n = 142) yielded an area under the ROC curve of 87.2% (95% CI, 79.4%-93.3%). At a specificity of 95%, the sensitivity to detect tumors varied between 46% (pT1) and 94% (pT4). A CNI above the median (i.e., >72) had a positive predictive value of 90% (95% CI, 79%-96%) for lymph node involvement (LNI), while the negative predictive value was 57% (95% CI, 43%-70%). For a CNI >72, overall survival (OS) was worse (hazard ratio, 4.89; 95% CI, 1.39-17.17; P = 0.01) with 62% and 90% survivors 3 years after surgery for a CNI >72 and ≤72, respectively. In multivariable models, the CNI was a superior predictor of OS compared to established disease features, including LNI. CONCLUSIONS: The CNI may assist in predicting LNI and prognosis in HNSCC with direct therapeutic implications concerning the need for neck dissection or more aggressive treatment.

Reduction of nevirapine-driven HIV mutations by carbamazepine is modulated by CYP3A activity.

OBJECTIVES: The reduction in mother-to-child transmission of HIV-1 by single-dose nevirapine given at birth onset is achieved at the expense of de novo HIV-1 resistance mutations. In the VITA1 study, single-dose carbamazepine accelerated nevirapine elimination, but the accompanying trend towards fewer de novo HIV-1 mutations was statistically non-significant. METHODS: We investigated if the effect of carbamazepine was confounded by the individual variability in nevirapine metabolism and transport. RESULTS: Nine of 34 (26%) single-dose nevirapine-treated women had one or more nevirapine-associated resistance mutations, compared with 3 of 34 (9%) in the single-dose nevirapine/carbamazepine arm. The genetic polymorphisms in CYP2B6 and MRP7 affected neither nevirapine kinetics nor the development of HIV-1 resistance. In contrast, the reduction in HIV-1 mutations by single-dose carbamazepine reached statistical significance at P = 0.04 with an OR of 0.1 (95% CI 0.01-0.90) upon consideration of CYP3A activity, defined as the ratio of 4ß-hydroxycholesterol to cholesterol, and it was more likely in women with higher CYP3A activity. These findings were in agreement with CYP3A induction in carbamazepine-treated patients. Likewise, carbamazepine induced CYP3A4, but not CYP2B6, in vitro when combined with nevirapine. CONCLUSIONS: The induction of nevirapine elimination reduces HIV-1 resistance mutations, but this effect is modulated by individual CYP3A activity. The study suggests that CYP3A4 activity could be monitored using an endogenous marker and, if needed, boosted to improve clinical endpoints.

Evaluation of Prognosticators and Treatment-Related Side Effects in Patients Irradiated Postoperatively for Endometrial Cancer.

Numerous clinical trials sought to improve outcomes in endometrial cancer patients with multimodal treatment strategies. We tested the hypothesis that specific histopathological and clinical parameters are prognosticators for outcomes at our Gynecological Cancer Center. A total of 203 patients (median age, 69.5 years) was included. They were irradiated postoperatively (n = 184: Brachytherapy, n = 19: Teletherapy) between 05/2007 and 03/2020. The median follow-up was 37.2 months. As statistical methods, we used the univariable Cox proportional hazards regression, and log-rank statistics. First, we found a significant influence of grading and nodal stage on outcomes. These findings underline the recommendations of more intense treatment in these patient groups, as already reflected in current guidelines. Secondly, we found that patient age had a significant influence on survival be it due to comorbidities and/or due to too hesitant treatment regimen in the elderly. Thus, it should be aimed at particular strategies in treatment of these patients. Lastly, we found very low rates of treatment-related side effects in patients treated with brachytherapy and moderate rates of side effects in patients treated with teletherapy. Overall, our study serves as basis for further improvement of treatment strategies and for conceptualization of clinical trials.

The impact of rectal/bladder filling and applicator positioning on in vivo rectal dosimetry in vaginal cuff brachytherapy using an enhanced therapy setting.

PURPOSE: The impact of rectal filling and bladder volume on in vivo rectal dosimetry (IVD) in vaginal cuff brachytherapy (VCBT) is unknown. The purpose of this study was to compare rectal doses from IVD with those calculated from treatment planning and to identify influencing factors. MATERIALS AND METHODS: We collected data of 80 VCBT sessions, four for each of 20 patients. Each was retrospectively compared with doses determined by the treatment planning system. Factors potentially predicting the IVD rectum dose were analyzed. RESULTS: For a series of 80 brachytherapy applications, the calculated mean dose to the rectum was 2.52 Gy. The mean difference between all calculated and measured doses for the 80 applications with five probe positions each was 0.09 Gy (p = 0.952) proving high overall accordance between IVD and calculated doses at the rectum. The mean volume of the rectum was 119 ± 57 cm³. The rectal volume was not statistically significantly associated with the IVD or the calculated rectum doses. At the third and fourth rectal probe position in craniocaudal ordering, increased filling of the urinary bladder resulted in decreased measured and calculated doses (p < 0.05 for both). A rectum pointing position of the applicator significantly increased the maximum rectum dose compared with a bladder-oriented position (p < 0.05). CONCLUSIONS: IVD provided valuable data for rectal exposure in VCBT. Increased bladder filling and vaginal applicator positioning off the rectum elicited related with less rectal radiation exposure, whereas rectal filling did not. Further confirmation including assessment of IVD in bladder is pending to define optimal dosimetric conditions in VCBT.

KRAS mutation status concordance between the primary tumor and the corresponding metastasis in patients with rectal cancer.

INTRODUCTION: Oncogenic mutation within the KRAS gene represents a negative predictor for treatment response to anti-epidermal growth factor receptor (EGFR) in patients with colorectal cancer. Recently, we have shown no relevant heterogeneity for KRAS mutation status within and between pre- and posttherapeutic samples from the primary tumor in patients with locally advanced rectal cancer. The aim of this study was to evaluate the intertumoral heterogeneity of KRAS mutation status between the primary tumor and the corresponding metastasis or local recurrence in the similar cohort and to evaluate the ideal representative tissue for KRAS mutation testing. MATERIALS AND METHODS: KRAS mutation status was analyzed from 47 patients with locally advanced rectal cancer, which were enrolled in the CAO/ARO/AIO-94 or CAO/ARO/AIO-04 trial. Mutations in KRAS codons 12, 13, and 61 were analyzed by using the KRAS RGQ PCR Kit (therascreen® KRAS test). Six patients needed to be excluded due to incomplete follow up data. 11 patients showed a relapse of the disease during the follow up presented by distant metastases or local recurrence. DNA from representative areas of metastatic tissue was obtained from formalin-fixed paraffin-embedded specimens. RESULTS: The mean patient age was 64.13 ± 10.64 years. In total, 19 patients showed a KRAS mutation (46.34%) in the primary tumor. Of the eleven patients with a metastatic disease or local recurrence, five patients showed a KRAS mutation whereas six patients had a KRAS wildtype status. Metastatic localizations included the liver (n = 2), lung (n = 4), local recurrence (n = 1), liver + lung (n = 3), lung + local recurrence (n = 1). For these eleven patients with paired data available for the primary tumor and metastatic tissue, a significant KRAS mutation status concordance was detected in 81.18% (9/11) of the patients (p = 0.03271). Only two patients showed intertumoral heterogeneity, which harbored in one patient a KRAS G12C mutation status in the primary tumor, but a G12V KRAS mutation status in the corresponding lung lesion, and in the other patient a G12A mutation in the primary lesion and a WT in the lung metastasis. CONCLUSIONS: We show a significant concordance of the KRAS mutation status between tumor samples obtained from the primary tumor and the corresponding metastasis and/ or local recurrence in patients with rectal cancer indicating no relevant intertumoral heterogeneity. Our data suggest that sampling either the primary (pre- or posttherapeutical tumor tissue) or metastatic lesion may be valid for the initial evaluation of KRAS mutation status predicting the response to anti-EGFR treatment and guiding clinical decisions.

High Expression of EpCAM and Sox2 is a Positive Prognosticator of Clinical Outcome for Head and Neck Carcinoma.

Locally advanced head and neck squamous cell carcinomas (HNSCC) have limited prognosis due to frequent treatment failure. Currently, TNM-classification and human papillomavirus (HPV) infection are the sole clinical prognosticators of outcome. Tumor heterogeneity and stemness based on epithelial-mesenchymal-transition reportedly associate with therapy resistance. The capacity of epithelial marker EpCAM (EpEX), stemness regulator Sox2 and mesenchymal marker vimentin to predict clinical outcome of HSNCC patients was assessed upon immunohistochemistry staining in two cohorts of HNSCC patients treated with surgery and adjuvant radio (chemo) therapy (n = 94) and primary radio (chemo) therapy (n = 94), respectively. Prognostic values with respect to overall, disease-free and disease-specific survival were assessed in uni- and multivariate cox proportional hazard models to generate integrated risk scores. EpEX, Sox2 and vimentin displayed substantial inter- and intratumoral heterogeneity. EpEXhigh and Sox2high predicted improved clinical outcome in the discovery cohort and in the HPV-negative sub-cohort. EpEXhigh and Sox2high were confirmed as prognosticators of clinical outcome in the validation cohort treated with definitive radio(chemo)therapy. Importantly, EpEXhigh identified patients with improved survival within the HPV-negative subgroup of the validation cohort. Hence, Sox2high and particularly EpEXhigh have potential as tools to predict clinical performance of HNSCC patients, foremost HPV-negative cases, in the frame of molecular-guided treatment decision-making.

T cell abundance in blood predicts acute organ toxicity in chemoradiotherapy for head and neck cancer.

Treatment of head and neck squamous cell carcinoma (HNSCC) by chemoradiotherapy (CRT) often results in high-grade acute organ toxicity (HGAOT). As these adverse effects impair the patients' quality of life and the feasibility of the planned therapy, we sought to analyze immunological parameters in tumor material and blood samples obtained from 48 HNSCC patients in order to assess the potential to predict the individual acute organ toxicity. T cells in the tumor stroma were enriched in patients developing HGAOT whereas levels of soluble factors in the plasma and gene expression in whole blood did not coincide with the occurrence of acute organ toxicity. In contrast, the frequency and absolute numbers of selected leukocyte subpopulations measured in samples of peripheral blood mononuclear cells (PBMCs) directly before the beginning of CRT were significantly different in patients with HGAOT as compared to those without. When we validated several potential markers including the abundance of T cells in a small prospective study with 16 HNSCC patients, we were able to correctly predict acute organ toxicity in up to 81% of the patients. We conclude that analysis of PBMCs by fluorescence-activated cell sorting (FACS) might be a convenient strategy to identify patients at risk of developing HGAOT caused by CRT, which might allow to adapt the treatment regimen and possibly improve disease outcome.

Relevance of Sp Binding Site Polymorphism in WWOX for Treatment Outcome in Pancreatic Cancer.

BACKGROUND: A genome-wide association study (GWAS) suggested inherited genetic single-nucleotide polymorphisms (SNPs) affecting overall survival (OS) in advanced pancreatic cancer. To identify robust clinical biomarkers, we tested the strongest reported candidate loci in an independent patient cohort, assessed cellular drug sensitivity, and evaluated molecular effects. METHODS: This study comprised 381 patients with histologically verified pancreatic ductal adenocarcinoma treated with gemcitabine-based chemotherapy. The primary outcome was the relationship between germline polymorphisms and OS. Functional assays addressed pharmacological dose-response effects in lymphoblastoid cell lines (LCLs) and pancreatic cancer cell lines (including upon RNAi), gene expression analyses, and allele-specific transcription factor binding. All statistical tests were two-sided. RESULTS: The A allele (26% in Caucasians) at SNP rs11644322 in the putative tumor suppressor gene WWOX conferred worse prognosis. Median OS was 14 months (95% confidence interval [CI] = 12 to 15 months), 13 months (95% CI = 11 to 15 months), and nine months (95% CI = 7 to 12 months) for the GG, GA, and AA genotypes, respectively (P trend < .001 for trend in univariate log-rank assuming a codominant mode of inheritance; advanced disease subgroup P trend < .001). Mean OS was 25 months (95% CI = 21 to 29 months), 19 months (95% CI = 15 to 22 months), and 13 months (95% CI = 10 to 16 months), respectively. This effect held true after adjustment for age, performance status according to Eastern Cooperative Oncology Group classification, TNM, grading, and resection status and was comparable with the strongest established prognostic factors in multivariable analysis. Consistently, reduced responsiveness to gemcitabine, but not 5-fluorouracil, along with lower WWOX expression was demonstrated in LCLs harboring the AA genotype. Likewise, RNAi-mediated WWOX knockdown in pancreatic cancer cells confirmed differential cytostatic drug sensitivity. In electrophoretic mobility shift assays, the A allele exhibited weaker binding of Sp family members Sp1/Sp3. CONCLUSIONS: WWOX rs11644322 represents a major predictive factor in gemcitabine-treated pancreatic cancer. Decreased WWOX expression may interfere with gemcitabine sensitivity, and allele-specific binding at rs11644332 might be a causative molecular mechanism behind the observed clinical associations.

A putatively functional haplotype in the gene encoding transforming growth factor beta-1 as a potential biomarker for radiosensitivity.

PURPOSE: To determine whether genetic variability in TGFB1 is related to circulating transforming growth factor-ß1 (TGF-ß1) plasma concentrations after radiotherapy and to radiosensitivity of lymphoid cells. PATIENTS AND METHODS: Transforming growth factor-ß1 plasma concentrations (n=79) were measured in patients 1 year after radiotherapy and chromosomal aberrations (n=71) ex vivo before therapy start. Furthermore, TGF-ß1 secretion and apoptosis were measured in isolated peripheral blood mononuclear cells of 55 healthy volunteers. These phenotypes were analyzed in relation to five germline polymorphisms in the 5' region of the TGFB1 gene. Because of high linkage disequilibrium, these five polymorphisms reflect frequent genetic variation in this region. A presumed impact of TGF-ß1 on DNA damage or repair was measured as micronucleus formation in 30 lymphoblastoid cell lines. RESULTS: We identified a hypofunctional genetic haplotype termed H3 tagging the 5' region of the TGFB1 gene encoding TGF-ß1. H3 was associated with lower TGF-ß1 plasma concentrations in patients (p=0.01) and reduced TGF-ß1 secretion in irradiated peripheral blood mononuclear cells (p=0.003). Furthermore, cells with H3 were less prone to induction of chromosomal aberrations (p=0.001) and apoptosis (p=0.003) by irradiation. The hypothesis that high TGF-ß1 could sensitize cells to DNA damage was further supported by increased micronuclei formation in 30 lymphoblastoid cell lines when preincubated with TGF-ß1 before irradiation (p=0.04). CONCLUSIONS: On the basis of TGF-ß1 plasma levels and radiation sensitivity of lymphoid cells, this study revealed a putatively hypofunctional TGFB1 haplotype. The significance of this haplotype and the suggested link between TGF-ß1 function and DNA integrity should be further explored in other cell types, as well as other experimental and clinical conditions.

A functional polymorphism in the NAD(P)H oxidase subunit CYBA is related to gene expression, enzyme activity, and outcome in non-Hodgkin lymphoma.

NAD(P)H oxidase is a major endogenous source of reactive oxygen species (ROS). ROS may not only be involved in carcinogenesis but also in efficacy of chemotherapeutic agents like doxorubicin. By a comprehensive genotyping approach covering 48 genetic polymorphisms (single-nucleotide polymorphisms) in five subunits of phagocytic NAD(P)H oxidase, we asked whether they affect gene expression, enzymatic activity, and outcome of CHO(E)P chemotherapy. A highly consistent effect was observed for the CYBA 640A>G variant. In peripheral blood granulocytes of 125 healthy volunteers, the G allele of 640A>G was associated with lower NAD(P)H oxidase activity (P = 0.006). Moreover, the G allele was associated with lower mRNA and protein expression (both P = 0.02). Of clinical importance, the outcome of patients suffering from non-Hodgkin lymphoma and treated with CHO(E)P regimen was dependent on the CYBA 640A>G polymorphism. In an exploratory study (n = 401), carriers of 640GG had an event-free survival (EFS) risk ratio of 1.95 [95% confidence interval (95% CI), 1.31-2.90; P = 0.001] compared with 640AA. In a confirmatory set (n = 477), the risk ratios were 1.53 (1.04-2.25, P = 0.03). The complete set of 878 patients showed a relative risk of 1.72 (1.30-2.26) and 1.59 (1.14-2.21) for EFS and overall survival, respectively. Further molecular-biological experiments showed lower expression and reduced stability of transcripts with the G allele in lymphoblastoid cell lines. Transfection of allele-specific plasmids into HEK293 cells elicited lower activity for the G allele in a luciferase reporter gene construct. Thus, CYBA 640A>G was shown to be a functional polymorphism with possible consequences for patients receiving CHO(E)P chemotherapy and might have further implications for other ROS-mediated modalities.

Bioinformatic and functional analysis of TGFBR1 polymorphisms.

OBJECTIVES: The transforming growth factor-beta (TGFB) pathway has substantial impact on cellular functions, cell proliferation, and apoptosis. We used bioinformatics, gene expression, and cell biological assays to evaluate the functionality of frequent inherited germline polymorphisms in the TGFB receptor 1 (TGFBR1). METHODS: In an exploratory (n=55) and confirmatory (n=106) study, we analyzed the TGFB1 pathway after incubation with TGFbeta1 ligand and after exposure to X-rays in peripheral blood human mononuclear cells. Expression of TGFB pathway genes was assessed by real-time PCR, and cellular viability was analyzed by flow cytometry. A total of six polymorphisms including the deletion variant (*6A) were identified to tag currently known common genetic variations in TGFBR1 and were analyzed in relation to the phenotypes. RESULTS: In accordance with a negative feedback mechanism, incubations with the ligand TGFbeta1 was followed by up-regulation of the intracellular SMAD7 and down-regulation of the SMAD3 mRNA molecules. The TGFBR1*6A deletion variant attenuated the suppression of SMAD3 in response to TGFbeta1 (P=0.02, in both studies). Moreover, cells harboring *6A were more sensitive toward cytotoxic effects of irradiation (P=0.001 after adjustment for age and sex). Cells were particularly prone toward radiation toxicity when carrying, in addition to *6A, the variant allele of rs11568785, which exhibits a strong genetic selection signature. CONCLUSION: The *6A deletion and the linked rs11568785 polymorphisms seem to attenuate TGFB signaling. This should be considered not only for clinical-epidemiological studies on cancer susceptibility but may also be relevant for side effects from drugs or radiotherapy.