Dirk J. Kuik

Effect of tissue-harvesting site on yield of stem cells derived from adipose tissue: implications for cell-based therapies

The stromal vascular fraction (SVF) of adipose tissue contains an abundant population of multipotent adipose-tissue-derived stem cells (ASCs) that possess the capacity to differentiate into cells of the mesodermal lineage in vitro. For cell-based therapies, an advantageous approach would be to harvest these SVF cells and give them back to the patient within a single surgical procedure, thereby avoiding lengthy and costly in vitro culturing steps. However, this requires SVF-isolates to contain sufficient ASCs capable of differentiating into the desired cell lineage. We have investigated whether the yield and function of ASCs are affected by the anatomical sites most frequently used for harvesting adipose tissue: the abdomen and hip/thigh region. The frequency of ASCs in the SVF of adipose tissue from the abdomen and hip/thigh region was determined in limiting dilution and colony-forming unit (CFU) assays. The capacity of these ASCs to differentiate into the chondrogenic and osteogenic pathways was investigated by quantitative real-time polymerase chain reaction and (immuno)histochemistry. A significant difference (P = 0.0009) was seen in ASC frequency but not in the absolute number of nucleated cells between adipose tissue harvested from the abdomen (5.1 ± 1.1%, mean ± SEM) and hip/thigh region (1.2 ± 0.7%). However, within the CFUs derived from both tissues, the frequency of CFUs having osteogenic differentiation potential was the same. When cultured, homogeneous cell populations were obtained with similar growth kinetics and phenotype. No differences were detected in differentiation capacity between ASCs from both tissue-harvesting sites. We conclude that the yield of ASCs, but not the total amount of nucleated cells per volume or the ASC proliferation and differentiation capacities, are dependent on the tissue-harvesting site. The abdomen seems to be preferable to the hip/thigh region for harvesting adipose tissue, in particular when considering SVF cells for stem-cell-based therapies in one-step surgical procedures for skeletal tissue engineering.

Distress in Spouses and Patients After Treatment for Head and Neck Cancer

Background: The objective of this study is to obtain insight into distress in spouses and patients treated for head and neck cancer.

Hearing status, need for recovery after work, and psychosocial work characteristics: results from an internet-based national survey on hearing

The main aim of the study is to address the relationship between hearing status and need for recovery. In addition, the role of hearing status in the relationship between psychosocial work characteristics (i.e. job demands and job control) and need for recovery was assessed. The sample comprised 925 normally-hearing and hearing-impaired working adults (aged 18–65 years) participating in the National Longitudinal Study on Hearing. Hearing status was determined using the national hearing (speech-in-noise) test over the internet. Psychosocial work characteristics and need for recovery were assessed using the job content questionnaire and the Dutch questionnaire on the experience and assessment of work. Regression models revealed a significant association between hearing status and need for recovery after work, poorer hearing leading to an increasing need for recovery. Additionally, poorer hearing led to a higher odds for risky levels of need for recovery. Hearing status did not influence the significant relationship between psychosocial work characteristics (i.e. job demand and job control) and need for recovery after work. Implications for clinical practice, such as the necessity of having adequate enablement programs for this specific group of patients, are discussed.

Prospective effects of hearing status on loneliness and depression in older persons: Identification of subgroups

Abstract Objective: To determine the possible longitudinal relationships between hearing status and depression, and hearing status and loneliness in the older population. Design: Multiple linear regression analyses were used to assess the associations between baseline hearing and 4-year follow-up of depression, social loneliness, and emotional loneliness. Hearing was measured both by self-report and a speech-in-noise test. Each model was corrected for age, gender, hearing aid use, baseline wellbeing, and relevant confounders. Subgroup effects were tested using interaction terms. Study sample: We used data from two waves of the Longitudinal Aging Study Amsterdam (2001–02 and 2005–06, ages 63–93). Sample sizes were 996 (self-report (SR) analyses) and 830 (speech-in-noise test (SNT) analyses). Results: Both hearing measures showed significant adverse associations with both loneliness measures (p < 0.05). However, stratified analyses showed that these effects were restricted to specific subgroups. For instance, effects were significant only for non-hearing aid users (SR-social loneliness model) and men (SR and SNT-emotional loneliness model). No significant effects appeared for depression. Conclusions: We found significant adverse effects of poor hearing on emotional and social loneliness for specific subgroups of older persons. Future research should confirm the subgroup effects and may contribute to the development of tailored prevention and intervention programs. Sumario Objetivo: Determinar las posibles relaciones longitudinales entre la condición auditiva y la depresión, y la condici n auditiva y la soledad, en adultos mayores. Diseño: Se usaron múltiples análisis de regresión lineal para evaluar las asociaciones entre la audición basal y el seguimiento a 4 años con la depresiún, la soledad social y la soledad emocional. La audición se midió tanto por auto-reporte como por la prueba de audición en ruido. Cada modelo fue corregido por edad, g nero, uso del auxiliar auditivo, bienestar basal y elementos relevantes de confusión. El efecto de subgrupo fue evaluado usando términos de interacción. Muestra del Estudio: Usamos datos de dos etapas del Estudio Longitudinal de Envejecimiento e Amsterdam (2001-02 y 2005-06, edades 63-93). El tamaño de las muestras fue 996 (análisis de auto-reporte (SR) y 830 (análisis de la prueba de audición en ruido). Resultados: Ambas mediciones auditivas mostraron asociaciones adversas significativas con ambas medidas de soledad (p < 0.05). Sin embargo, los análisis estratificados mostraron que estos efectos eran restringidos a subgrupos específicos. Por ejemplo, los efectos fueron significativos solo para quienes no usaban auxiliar auditivo (modelo SR – soledad social) y para hombres (SR y SNT – modelo de soledad emocional). No hubo efecto significativo para la depresión. Conclusiones: Encontramos efectos adversos significativos de un audición pobre sobre la soledad emocional y social para subgrupos específicos de personas mayores. Investigaciones futuras deberán confirmar el efecto de subgrupo y podrán contribuir al desarrollo de programas de prevenci n e intervenci n a la medida.

Perioperative arginine-supplemented nutrition in malnourished patients with head and neck cancer improves long-term survival

BACKGROUND Plasma arginine concentrations are lower in patients with cancer, which indicates that arginine metabolism may be disturbed in these patients. Arginine supplementation has been associated with positive effects on antitumor mechanisms and has been shown to reduce tumor growth and to prolong survival. Furthermore, the prognosis of patients with head and neck cancer remains disappointing. Insufficient intake frequently leads to malnutrition, which contributes to high morbidity and mortality rates. OBJECTIVE The aim of this study was to assess the long-term effects of perioperative arginine supplementation in severely malnourished patients with head and neck cancer. DESIGN In this double-blind, randomized, controlled trial, we randomly assigned 32 severely malnourished patients with head and neck cancer to receive 1) standard perioperative enteral nutrition (n = 15) or 2) arginine-supplemented perioperative enteral nutrition (n = 17). The primary outcome was long-term (≥10 y) survival. Secondary outcomes included the long-term appearance of locoregional recurrence, distant metastases, and second primary tumors. RESULTS No significant differences in baseline characteristics were observed between groups. The group receiving arginine-enriched nutrition had a significantly better overall survival (P = 0.019) and better disease-specific survival (P = 0.022). Furthermore, the arginine-supplemented group had a significantly better locoregional recurrence-free survival (P = 0.027). No significant difference in the occurrence of distant metastases or occurrence of a second primary tumor was observed between the groups. CONCLUSION Perioperative arginine-enriched enteral nutrition significantly improved the long-term overall survival and long-term disease-specific survival in malnourished patients with head and neck cancer.

Prognostic Significance of Truncating TP53 Mutations in Head and Neck Squamous Cell Carcinoma

Purpose: TP53 is a key gene in cellular homeostasis and is frequently mutated in head and neck squamous cell carcinoma (HNSCC). There is a variety of TP53 mutations, each with its own biological and clinical implication. Aim of the study was to assess the prognostic significance of TP53 mutations in HNSCCs and to identify the most relevant mutation. Experimental Design:TP53 mutation status was investigated in 141 consecutive HNSCCs treated by surgery with radiotherapy when indicated and with a known human papilloma virus status. The type of mutation was correlated with overall and progression-free survival in a multivariate two-sided Cox regression analysis with wild type as reference. Results: A TP53 mutation was found in 88 (62.4%) of the carcinomas and was not significantly associated with overall survival (HR = 1.65, P = 0.11). Patients with a mutation resulting in a truncated protein (n = 36, 25.5%) had a significantly worse overall survival (HR = 2.54, P = 0.008) and progression-free survival (HR = 2.65, P = 0.002). Four of these mutations were at a splice site, 13 were nonsense mutations (produces stop codon), and 19 were insertions or deletions resulting in a frameshift. After multivariate analysis, a truncating mutation remained a significant prognosticator. A missense (i.e., nontruncating) mutation did not influence prognosis. Other ways of classification (disruptive vs. nondisruptive, hotspot vs. nonhotspot, and DNA binding vs. non–DNA binding) were less discriminative. Conclusion: In HNSCCs, a truncating TP53 mutation is associated with a poor prognosis. This patient group seems as a target population for adjuvant therapy with chemoradiation or viral vector–mediated TP53 gene transfer. Clin Cancer Res; 17(11); 3733–41. ©2011 AACR.

The transplanted liver graft is capable of clearing asymmetric dimethylarginine

Asymmetric dimethylarginine (ADMA) has been recognized as an endogenous inhibitor of the arginine–nitric oxide (NO) pathway. Its concentration is tightly regulated by urinary excretion and degradation by the enzyme dimethylarginine dimethylaminohydrolase (DDAH), which is highly expressed in the liver. Considering the liver as a crucial organ in the clearing of ADMA, we hypothesized increased ADMA levels during hepatic failure and, consequently, a decline of ADMA concentrations after successful liver transplantation. The aim of the present study was to investigate the role of the liver in the metabolism of ADMA in patients undergoing liver transplantation. In this prospective study, we investigated the course of ADMA concentrations in 42 patients undergoing liver transplantation and results showed that preoperative ADMA concentrations were higher in patients with acute (1.26 μmol/L, P < .001) and in patients with chronic (.69 μmol/L, P < .001) hepatic failure compared with healthy volunteers (.41 μmol/L). In addition, ADMA concentrations decreased from the preoperative day to the first postoperative day in both the acute (ΔADMA: −.63 μmol/L, P = .005) and the chronic hepatic failure group (ΔADMA: −0.15 μmol/L, P < .001). Furthermore, in patients who experienced acute rejection, ADMA concentrations were higher during the whole first postoperative month compared with nonrejectors (P = .012). Moreover, in 11 of 13 rejectors (85%) a clear increase in ADMA concentration preceded the onset of the first episode of rejection, which was confirmed by liver biopsy. In conclusion, our results indicate that the transplanted liver graft is quickly capable of clearing ADMA, suggesting preservation of DDAH. In addition, increased ADMA concentrations in the posttransplantation period reflect serious dysfunction of the liver graft during acute rejection. (Liver Transpl 2004;10:1524–1530.)

A noninvasive genetic screening test to detect oral preneoplastic lesions

Early diagnosis of oral squamous cell carcinoma (OSCC) may have a major impact on survival and quality of life. Recent studies have shown that the majority of OSCC is preceded by precursor lesions characterized by genetic alterations. The aim of this study was to develop and evaluate a noninvasive screening test for oral preneoplastic lesions, based on genetic alterations as marker. Various methods to obtain a high yield of cells by brushing a small area of the oral mucosa were compared. A novel genetic assay, multiplex ligation-dependent probe amplification (MLPA), was applied that enables the measurement of gains and losses at 40 different chromosomal locations in one PCR reaction using 150 ng DNA. MLPA was performed on DNA of normal and dysplastic oral mucosa as well as of OSCC with the intention to select a specific probe set for accurate detection of precursor lesions in the oral cavity. The assay was correlated to loss of heterozygosity analysis using microsatellite markers, and evaluated on noncancer subjects and patients with oral leukoplakia. A noninvasive sampling method was developed with DNA yields ranging from 150 to 600 ng. Using 120 probes, we could detect large differences with MLPA in the number of alterations between normal vs dysplastic and dysplastic vs tumor tissue with P-values <0.001. A significant correlation was found between the number of alterations as detected by MLPA and the analysis for allelic loss. The available data enabled the selection of a set of 42 MLPA probes, which had the power to optimally discriminate between normal and dysplastic tissue. Our data show that MLPA is a sensitive, reliable, high-throughput and easy-to-perform technique, enabling the detection of genetic alterations on small noninvasive samples and can be considered a promising method for population-based screening of preneoplastic lesions in the oral cavity.

Expression profiling and prediction of distant metastases in head and neck squamous cell carcinoma

Background: For breast and prostate cancer, a gene expression signature of the tumour is associated with the development of distant metastases. Regarding head and neck squamous cell carcinoma (HNSCC), the only known risk factor is the presence of ⩾3 tumour-positive lymph nodes. Aim: To evaluate whether a HNSCC gene expression signature can discriminate between the patients with and without distant metastases. Methods: Patients with HNSCC with and without distant metastases had >3 tumour-positive lymph nodes, and did not differ with respect to other risk factors. Statistical analysis was carried out using Student’s t test, as well as statistical analysis of microarrays (SAM), to assess the false discovery rate for each gene. These analyses were supplemented with a newly developed method that computed deviations from gaussian-order statistics (DEGOS). To validate the platform, normal mucosa of the head and neck was included as control. Results: 2963 genes were differently expressed between HNSCC and normal mucosa (t test; p<0.01). More rigorous statistical analysis with SAM confirmed the differential expression of most genes. The comparison of genes in HNSCC with and without metastases showed 150 differently expressed genes (t test; p<0.01), none of which, however, could be confirmed using SAM or DEGOS. Conclusions: No evidence for a metastasis signature is found, and gene expression profiling of HNSCC has seemingly no value in determining the risk of developing distant metastases. The absence of such a signature can be understood when it is realised that, for HNSCC in contrast with breast cancer, the lymph nodes are a necessary in-between station for haematogenous spread.

Loss of heterozygosity at 9p and p53 immunopositivity in surgical margins predict local relapse in head and neck squamous cell carcinoma.

A major problem in head and neck cancer surgery is the high rate of local relapse (LR). In at least 25% of the surgically treated head and neck squamous cell carcinoma (HNSCC) patients, a genetically defined preneoplastic lesion, also known as “field,” can be detected in the surgical margins. A remaining field may be an important cause for the development of LR. The aims of our study are (i) to investigate whether HNSCC patients with an unresected field are more likely to develop LR, and (ii) to identify molecular risk factors that predict malignant transformation of field. We retrospectively studied 35 HNSCC patients of whom 16 patients developed LR and 19 patients remained disease‐free for at least 4 years. Loss of heterozygosity (LOH) at chromosomes 3p, 9p and 17p, p53 immunostaining, Ki‐67 immunostaining and histopathological grading of all available paraffin‐embedded surgical margins was performed, and related to LR. Significant associations were determined by Kaplan‐Meier analysis and Cox‐proportional hazard models. We show that presence of field is significantly associated with LR and that LOH at 9p and p53 immunostaining have the most predictive potential (hazard ratios 3.17 and 3.46, and p values 0.027 and 0.017, respectively). The combination of LOH at 9p and/or a large p53 positive field is most predictive (hazard ratio 7.06 and p = 0.01). Presence and grade of dysplasia was not associated with LR. These data may have major impact for future diagnostic workup of surgically treated HNSCC patients.