Genetic (in)stability in tomato

In the present study tomato lines carrying unstable alleles of the loci yv or sulfurea were characterized. In addition, we aimed at the isolation of an endogenous transposable element supposedly active in the unstable lines. Since the unstable loci were not cloned, we developed a transposon target system that enabled us to select for putative insertion mutants of the Adh-1 gene. While investigating the genetic characteristics of the Yv msand Sulfurea line it became apparent that the instability of their marker loci was not necessarily related to the activity of transposition elements. Also, the instability could not be related to the activity of tomato sequences homologous to transposable elements from related species. This invited speculation about the stability of DNA sequences in tomato. It is proposed that mechanisms which are known to underlie genetic instability in other organisms, have a role in the unstable expression of yv and sulfurea . These alternative mechanisms have been summarized in Chapter 1. Chapter 2 describes the isolation of an EMS induced Adh-1 null mutant and its use in crosses with the unstable tomato lines. In the resulting F 1 populations additional Adh-1 null mutants were selected through the resistance of seeds or pollen grains to allyl alcohol (Chapter 4). The cloning of the entire Adh-1 gene allowed the molecular analysis of the Adh-1 null mutants (Chapter 3). Chapter 5 describes the cytogenetic and genetic characterization of the tomato line Yvms. 'Me characteristics of a newly isolated unstable allele of the sulfurea locus are presented in chapter 6. Finally, chapter 7 describes the hybridization of tomato DNA to alien transposable element probes. These experiments emphasize the stability of transposable element-like sequences in the tomato genome

RAB25 expression is epigenetically downregulated in oral and oropharyngeal squamous cell carcinoma with lymph node metastasis

Oral and oropharyngeal squamous cell carcinoma (OOSCC) have a low survival rate, mainly due to metastasis to the regional lymph nodes. For optimal treatment of these metastases, a neck dissection is required; however, inaccurate detection methods results in under- and over-treatment. New DNA prognostic methylation biomarkers might improve lymph node metastases detection. To identify epigenetically regulated genes associated with lymph node metastases, genome-wide methylation analysis was performed on 6 OOSCC with (pN+) and 6 OOSCC without (pN0) lymph node metastases and combined with a gene expression signature predictive for pN+ status in OOSCC. Selected genes were validated using an independent OOSCC cohort by immunohistochemistry and pyrosequencing, and on data retrieved from The Cancer Genome Atlas. A two-step statistical selection of differentially methylated sequences revealed 14 genes with increased methylation status and mRNA downregulation in pN+ OOSCC. RAB25, a known tumor suppressor gene, was the highest-ranking gene in the discovery set. In the validation sets, both RAB25 mRNA (P = 0.015) and protein levels (P = 0.012) were lower in pN+ OOSCC. RAB25 mRNA levels were negatively correlated with RAB25 methylation levels (P < 0.001) but RAB25 protein expression was not. Our data revealed that promoter methylation is a mechanism resulting in downregulation of RAB25 expression in pN+ OOSCC and decreased expression is associated with lymph node metastasis. Detection of RAB25 methylation might contribute to lymph node metastasis diagnosis and serve as a potential new therapeutic target in OOSCC

Shared Neuroanatomical Substrates of Impaired Phonological Working Memory Across Reading Disability and Autism

Background Individuals with reading disability and individuals with autism spectrum disorder (ASD) are characterized, respectively, by their difficulties in reading and social communication, but both groups often have impaired phonological working memory (PWM). It is not known whether the impaired PWM reflects distinct or shared neuroanatomical abnormalities in these two diagnostic groups. Methods White-matter structural connectivity via diffusion weighted imaging was examined in 64 children, age 5 to 17 years, with reading disability, ASD, or typical development, who were matched on age, gender, intelligence, and diffusion data quality. Results Children with reading disability and children with ASD exhibited reduced PWM compared with children with typical development. The two diagnostic groups showed altered white matter microstructure in the temporoparietal portion of the left arcuate fasciculus and in the occipitotemporal portion of the right inferior longitudinal fasciculus (ILF), as indexed by reduced fractional anisotropy and increased radial diffusivity. Moreover, the structural integrity of the right ILF was positively correlated with PWM ability in the two diagnostic groups but not in the typically developing group. Conclusions These findings suggest that impaired PWM is transdiagnostically associated with shared neuroanatomical abnormalities in ASD and reading disability. Microstructural characteristics in left arcuate fasciculus and right ILF may play important roles in the development of PWM. The right ILF may support a compensatory mechanism for children with impaired PWM

Discovery of new methylation markers to improve screening for cervical intraepithelial neoplasia grade 2/3

Background: Assessment of DNA promoter methylation markers in cervical scrapings for the detection of cervical intraepithelial neoplasia (CIN) and cervical cancer is feasible, but finding methylation markers with both high sensitivity as well as high specificity remains a challenge. In this study, we aimed to identify new methylation markers for the detection of high-grade CIN (CIN2/3 or worse, CIN2+) by using innovative genome-wide methylation analysis (MethylCap-seq). We focused on diagnostic performance of methylation markers with high sensitivity and high specificity considering any methylation level as positive. Results: MethylCap-seq of normal cervices and CIN2/3 revealed 176 differentially methylated regions (DMRs) comprising 164 genes. After verification and validation of the 15 best discriminating genes with methylation-specific PCR (MSP), 9 genes showed significant differential methylation in an independent cohort of normal cervices versus CIN2/3 lesions (p < 0.05). For further diagnostic evaluation, these 9 markers were tested with quantitative MSP (QMSP) in cervical scrapings from 2 cohorts: (1) cervical carcinoma versus healthy controls and (2) patients referred from population-based screening with an abnormal Pap smear in whom also HPV status was determined. Methylation levels of 8/9 genes were significantly higher in carcinoma compared to normal scrapings. For all 8 genes, methylation levels increased with the severity of the underlying histological lesion in scrapings from patients referred with an abnormal Pap smear. In addition, the diagnostic performance was investigated, using these 8 new genes and 4 genes (previously identified by our group: C13ORF18, JAM3, EPB41L3, and TERT). In a triage setting (after a positive Pap smear), sensitivity for CIN2+ of the best combination of genes (C13ORF18/JAM3/ANKRD18CP) (74 %) was comparable to hrHPV testing (79 %), while specificity was significantly higher (76 % versus 42 %, p <= 0.05). In addition, in hrHPV-positive scrapings, sensitivity and specificity for CIN2+ of this best-performing combination was comparable to the population referred with abnormal Pap smear. Conclusions: We identified new CIN2/3-specific methylation markers using genome-wide DNA methylation analysis. The diagnostic performance of our new methylation panel shows higher specificity, which should result in prevention of unnecessary colposcopies for women referred with abnormal cytology. In addition, these newly found markers might be applied as a triage test in hrHPV-positive women from population-based screening. The next step before implementation in primary screening programs will be validation in population-based cohorts

Identification of a methylation panel as an alternative triage to detect CIN3+ in hrHPV-positive self-samples from the population-based cervical cancer screening programme

Background: The Dutch population-based cervical cancer screening programme (PBS) consists of primary high-risk human papilloma virus (hrHPV) testing with cytology as triage test. In addition to cervical scraping by a general practitioner (GP), women are offered self-sampling to increase participation. Because cytological examination on self-sampled material is not feasible, collection of cervical samples from hrHPV-positive women by a GP is required. This study aims to design a methylation marker panel to detect CIN3 or worse (CIN3+) in hrHPV-positive self-samples from the Dutch PBS as an alternative triage test for cytology.Methods: Fifteen individual host DNA methylation markers with high sensitivity and specificity for CIN3+ were selected from literature and analysed using quantitative methylation-specific PCR (QMSP) on DNA from hrHPV-positive self-samples from 208 women with CIN2 or less (&lt; CIN2) and 96 women with CIN3+. Diagnostic performance was determined by area under the curve (AUC) of receiver operating characteristic (ROC) analysis. Self-samples were divided into a train and test set. Hierarchical clustering analysis to identify input methylation markers, followed by model-based recursive partitioning and robustness analysis to construct a predictive model, was applied to design the best marker panel.Results: QMSP analysis of the 15 individual methylation markers showed discriminative DNA methylation levels between &lt; CIN2 and CIN3+ for all markers (p &lt; 0.05). The diagnostic performance analysis for CIN3+ showed an AUC of ≥ 0.7 (p &lt; 0.001) for nine markers. Hierarchical clustering analysis resulted in seven clusters with methylation markers with similar methylation patterns (Spearman correlation&gt; 0.5). Decision tree modeling revealed the best and most robust panel to contain ANKRD18CP, LHX8 and EPB41L3 with an AUC of 0.83 in the training set and 0.84 in the test set. Sensitivity to detect CIN3+ was 82% in the training set and 84% in the test set, with a specificity of 74% and 71%, respectively. Furthermore, all cancer cases (n = 5) were identified.Conclusion: The combination of ANKRD18CP, LHX8 and EPB41L3 revealed good diagnostic performance in real-life self-sampled material. This panel shows clinical applicability to replace cytology in women using self-sampling in the Dutch PBS programme and avoids the extra GP visit after a hrHPV-positive self-sampling test.</p

Telomere length in breast cancer patients before and after chemotherapy with or without stem cell transplantation

High-dose chemotherapy and peripheral blood stem cell transplantation (PBSCT) may accelerate telomere length loss in haematopoietic stem cells. As data including pre-and post-treatment samples are lacking, we studied leukocyte telomere length and telomerase activity before and after treatment in breast cancer patients randomized to receive 5 adjuvant courses FEC (5-FU, epirubicin and cyclophosphamide) (n= 17), or 4 × FEC followed by high-dose cyclophosphamide, thiotepa, carboplatin and autologous PBSCT (n= 16). Haemoglobin, MCV, leukocyte-and platelet numbers were assessed prior to (t0), 5 months after (t1) and 9 months after chemotherapy (t2); these parameters were decreased at t1 and t2 compared to t0(high-dose: all parameters; standard-dose: leukocytes and platelets), and all parameters were lower after high-dose than standard-dose treatment at t1. Paired individual leukocyte samples of t0 and t1 showed telomere length change (determined by telomere restricted fragment (TRF) assay) ranging from +0.8 to –2.2 kb, with a decreased TRF length in 9 patients of both groups. Telomerase activity (determined by TRAP assay) was below detection limit in leukocyte samples of t0 and t1. Thus, standard-and high-dose chemotherapy negatively affect haematological reconstitution in this setting. In individual patients, telomere length can be remarkably changed following haematological proliferative stress after treatment. © 2001 Cancer Research Campaign www.bjcancer.co

Abnormalities in reparative function of lung-derived mesenchymal stromal cells in emphysema

Mesenchymal stromal cells (MSCs) may provide crucial support in the regeneration of destructed alveolar tissue (emphysema) in COPD. We hypothesized that lung-derived MSCs (LMSCs) from emphysema patients are hampered in their repair capacity, either intrinsically or due to their interaction with the damaged micro-environment. LMSCs were isolated from lung tissue of controls and severe emphysema patients, and characterized at baseline. Additionally, LMSCs were seeded onto control and emphysematous decellularized lung tissue scaffolds and assessed for deposition of extracellular matrix (ECM). We observed no differences in surface markers, differentiation/proliferation potential and expression of ECM genes between control- and COPD-derived LMSCs. Notably, COPD-derived LMSCs displayed lower expression of FGF10 and HGF mRNA, and HGF and decorin protein. When seeded on control decellularized lung tissue scaffolds, control and COPD-derived LMSCs showed no differences in engraftment, proliferation or survival within 2 weeks, with similar ability to deposit new matrix on the scaffolds. Moreover, LMSC numbers and ability to deposit new matrix was not compromised on emphysematous scaffolds. Collectively, our data show that LMSCs from COPD patients compared to controls show less expression of FGF10 mRNA, HGF mRNA and protein and decorin protein, while other features including the mRNA expression of various ECM molecules are unaffected. Furthermore, COPD-derived LMSCs are capable of engraftment, proliferation and functioning on native lung tissue scaffolds. The damaged, emphysematous micro-environment as such does not hamper the potential of LMSCs. Thus, specific intrinsic deficiencies in growth factor production by diseased LMSCs may contribute to impaired alveolar repair in emphysema

Prognostic image-based quantification of CD8CD103 T cell subsets in high-grade serous ovarian cancer patients

CD103-positive tissue resident memory-like CD8+ T cells (CD8CD103 TRM) are associated with improved prognosis across malignancies, including high-grade serous ovarian cancer (HGSOC). However, whether quantification of CD8, CD103 or both is required to improve existing survival prediction and whether all HGSOC patients or only specific subgroups of patients benefit from infiltration, remains unclear. To address this question, we applied image-based quantification of CD8 and CD103 multiplex immunohistochemistry in the intratumoral and stromal compartments of 268 advanced-stage HGSOC patients from two independent clinical institutions. Infiltration of CD8CD103 immune cell subsets was independent of clinicopathological factors. Our results suggest CD8CD103 TRM quantification as a superior method for prognostication compared to single CD8 or CD103 quantification. A survival benefit of CD8CD103 TRM was observed only in patients treated with primary cytoreductive surgery. Moreover, survival benefit in this group was limited to patients with no macroscopic tumor lesions after surgery. This approach provides novel insights into prognostic stratification of HGSOC patients and may contribute to personalized treatment strategies in the future

The phosphatase and tensin homologue deleted on chromosome 10 mediates radiosensitivity in head and neck cancer

BACKGROUND: For locally advanced squamous cell carcinoma of the head and neck (HNSCC), the recurrence rate after surgery and postoperative radiotherapy is between 20 and 40%, and the 5- year overall survival rate is similar to 50%. Presently, no markers exist to accurately predict treatment outcome. Expression of proteins in the human epidermal growth factor receptor (EGFR) pathway has been reported as a prognostic marker in several types of cancer. METHODS: The aim of this study was to investigate the prognostic value of proteins in the EGFR pathway in HNSCC. For this purpose, we collected surgically resected tissue of 140 locally advanced head and neck cancer patients, all treated with surgery and postoperative radiotherapy. RESULTS: In a multivariate analysis, expression of the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was significantly related to worse locoregional control (LRC; HR: 2.2, 95% CI: 1.1-4.6; P = 0.03), independent of lymph node metastases (HR: 5.6, 95% CI: 1.2-27.4; P = 0.03) and extranodal spread (HR: 2.7; 95% CI: 1.2- 6.5; P = 0.02). In vitro clonogenic radiosensitivity assays confirmed that overexpression of PTEN resulted in increased radioresistance. CONCLUSION: Our study is the first report showing that expression of PTEN mediates radiosensitivity in vitro and that increased expression in advanced HNSCC predicts worse LRC. British Journal of Cancer (2010) 102, 1778-1785. doi: 10.1038/sj.bjc.6605707 www.bjcancer.com Published online 25 May 2010 (C) 2010 Cancer Research U