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Biomedical subjects

Ruud H Brakenhoff

Publications and source records attributed to Ruud H Brakenhoff.

At least 19 recordsLinked to original sources
PubMed2006

Human lymphoblastoid proteome analysis reveals a role for the inhibitor of acetyltransferases complex in DNA double-strand break response.

A DNA double-strand break (DSB) is highly cytotoxic; it emerges as the type of DNA damage that most severely affects the genomic integrity of the cell. It is essential that DNA DSBs are recognized and repaired efficiently, in particular, prior to mitosis, to prevent genomic instability and eventually, the development of cancer. To assess the pathways that are induced on DNA DSBs, 14 human lymphoblastoid cell lines were challenged with bleomycin for 30 and 240 minutes to establish the fast and more prolonged response, respectively. The proteomes of 14 lymphoblastoid cell lines were investigated to account for the variation among individuals. The primary DNA DSB response was expected to occur within the nucleus; therefore, the nuclear extracts were considered. Differential analysis was done using two-dimensional difference in gel electrophoresis; paired ANOVA statistics were used to recognize significant changes in time. Many proteins whose nuclear levels changed statistically significantly showed a fast response, i.e., within 30 minutes after bleomycin challenge. A significant number of these proteins could be assigned to known DNA DSB response processes, such as sensing DSBs (Ku70), DNA repair through effectors (high-mobility group protein 1), or cell cycle arrest at the G(2)-M phase checkpoint (14-3-3 zeta). Interestingly, the nuclear levels of all three proteins in the INHAT complex were reduced after 30 minutes of bleomycin challenge, suggesting that this complex may have a role in changing the chromatin structure, allowing the DNA repair enzymes to gain access to the DNA lesions.

Amino Acid Sequence↗
PubMed2006

BAC to the future! or oligonucleotides: a perspective for micro array comparative genomic hybridization (array CGH).

The array CGH technique (Array Comparative Genome Hybridization) has been developed to detect chromosomal copy number changes on a genome-wide and/or high-resolution scale. It is used in human genetics and oncology, with great promise for clinical application. Until recently primarily PCR amplified bacterial artificial chromosomes (BACs) or cDNAs have been spotted as elements on the array. The large-scale DNA isolations or PCR amplifications of the large-insert clones necessary for manufacturing the arrays are elaborate and time-consuming. Lack of a high-resolution highly sensitive (commercial) alternative has undoubtedly hindered the implementation of array CGH in research and diagnostics. Recently, synthetic oligonucleotides as arrayed elements have been introduced as an alternative substrate for array CGH, both by academic institutions as well as by commercial providers. Oligonucleotide libraries or ready-made arrays can be bought off-the-shelf saving considerable time and efforts. For RNA expression profiling, we have seen a gradual transition from in-house printed cDNA-based expression arrays to oligonucleotide arrays and we expect a similar transition for array CGH. This review compares the different platforms and will attempt to shine a light on the 'BAC to the future' of the array CGH technique.

Chromosome Aberrations↗
PubMed2006

Microarray analysis of bleomycin-exposed lymphoblastoid cells for identifying cancer susceptibility genes.

The uncovering of genes involved in susceptibility to the sporadic cancer types is a great challenge. It is well established that the way in which an individual deals with DNA damage is related to the chance to develop cancer. Mutagen sensitivity is a phenotype that reflects an individual's susceptibility to the major sporadic cancer types, including colon, lung, and head and neck cancer. A standard test for mutagen sensitivity is measuring the number of chromatid breaks in lymphocytes after exposure to bleomycin. The aim of the present study was to search for the pathways involved in mutagen sensitivity. Lymphoblastoid cell lines of seven individuals with low mutagen sensitivity were compared with seven individuals with a high score. RNA was isolated from cells exposed to bleomycin (4 hours) and from unexposed cells. Microarray analysis (19K) was used to compare gene expression of insensitive and sensitive cells. The profile of most altered genes after bleomycin exposure, analyzed in all 14 cell lines, included relatively many genes involved in biological processes, such as cell growth and/or maintenance, proliferation, and regulation of cell cycle, as well as some genes involved in DNA repair. When comparing the insensitive and sensitive individuals, other differentially expressed genes were found that are involved in signal transduction and cell growth and/or maintenance (e.g., BUB1 and DUSP4). This difference in expression profiles between mutagen-sensitive and mutagen-insensitive individuals justifies further studies aimed at elucidating the genes responsible for the development of sporadic cancers.

Adolescent↗
PubMed2005

Genetic reprogramming of tumor cells by zinc finger transcription factors.

Cancer arises by the accumulation of genetic alterations in DNA leading to aberrant gene transcription. Expression-profiling studies have correlated genomewide expression signatures with malignancy. However, functional analysis elucidating the contribution and synergy of genes in specific cancer cell phenotypes remains a formidable obstacle. Herein, we describe an alternative genetic approach for identification of genes involved in tumor progression by using a library of zinc finger artificial transcription factors (ATFs) and functional screening of tumor cells as a source of genetic plasticity and clonal selection. We isolated a six-zinc finger transcriptional activator (TF 20-VP, TF 20 containing the VP64 activator domain) that acts to reprogram a drug-sensitive, poorly invasive, and nonmetastatic cell line into a cell line with a drug-resistant, highly invasive, and metastatic phenotype. Differential expression profiles of cells expressing TF 20-VP followed by functional studies, both in vitro and in animal models, revealed that invasion and metastasis requires co-regulation of multiple target genes. Significantly, the E48 antigen, associated with poor metastasis-free survival in head and neck cancer, was identified as one specific target of TF 20-VP. We have shown phenotypic modulation of tumor cell behavior by E48 expression, including enhanced cell migration in vitro and tumor cell dissemination in vivo. This study demonstrates the use of ATFs to identify the group of genes that cooperate during tumor progression. By co-regulating multiple targets, ATFs can be used as master genetic switches to reprogram and modulate complex neoplastic phenotypes.

Animals↗
PubMed2005

Generation and molecular characterization of head and neck squamous cell lines of fanconi anemia patients.

Patients with Fanconi anemia (FA) are prone to develop malignancies at an early age. Besides hematologic malignancies, squamous cell carcinomas in the anogenital region and head and neck are also frequently found in these patients. The aim of this study was to generate a panel of head and neck squamous cell carcinoma (HNSCC) cell lines and xenografts of FA HNSCC, and to characterize these cell lines in comparison with a panel of seven cell lines from patients with sporadic HNSCC. Analyses have been done on sensitivity to DNA cross-linking agents, loss of heterozygosity profile, TP53 mutations, TP53 polymorphisms and the presence of human papillomavirus. Four FA HNSCC cell lines were established. Sensitivity to DNA cross-linking agents (cisplatin) in the FA HNSCC cell lines was on average 10 times higher as compared with the sporadic HNSCC cell lines. Human papillomavirus was not detected in any of the FA or sporadic cell lines. No differences were found in loss of heterozygosity pattern, TP53 mutation frequency and TP53 polymorphism between FA and sporadic HNSCC cell lines. This is the first report on the generation of squamous cell lines of FA patients. The FA HNSCC cell lines we have generated may be utilized for future studies and might aid in the development of new preventive therapies for FA patients. The genetic characteristics of these cell lines suggest that FA HNSCC are not very different from sporadic HNSCC, except for the sensitivity to cisplatin which is consistent with the known cellular FA phenotype.

Adult↗
PubMed2005

Histopathologic validation of the sentinel node concept in oral and oropharyngeal squamous cell carcinoma.

BACKGROUND: In patients with head and neck squamous cell carcinoma (HNSCC), the presence of lymph node metastases is the most important prognosticator. Sentinel node (SN) biopsy has been shown to be an accurate staging technique for patients with breast cancer and melanoma and might also be suited for patients with HNSCC. This study was undertaken to determine whether the SN concept holds true for HNSCC and could be exploited for SN biopsy. METHODS: In 22 patients with T2 to T4 N0 oral or oropharyngeal squamous cell carcinoma (SCC) who were scheduled to undergo combined primary tumor excision and elective unilateral (n = 17) or bilateral (n = 5) neck dissection, SN identification was performed the day before surgery by use of lymphoscintigraphy after peritumoral injections of 99mTc-labeled colloidal albumin. After the neck dissection specimens were removed, all SNs, all other radioactive lymph nodes, and all nonradioactive lymph nodes were retrieved for histopathologic analysis, including serial sectioning at 250-microm intervals and immunohistochemical analysis (IHC). RESULTS: Overall, in 21 (78%) of 27 neck sides, an SN was identified by scintigraphy. Of the six neck sides in which SNs were not identified by scintigraphy, four were from three patients who underwent bilateral neck dissection. In another patient treated by bilateral neck dissection, the SN identified by scintigraphy could not be found in the specimen. In the remaining 20 neck dissection specimens, 23 SNs and 30 additional radioactive lymph nodes could be found. At histologic examination of the 20 neck specimens in which the SN was found, at least one SN was tumor positive in eight cases. In one neck specimen, a metastasis was detected in a nonradioactive lymph node, whereas the SN was tumor free, also at serial sectioning and IHC. In the remaining 11 neck sides in which the SN was tumor negative, none of the other radioactive (n = 13) and none of the nonradioactive (n = 279) lymph nodes contained tumor at histopathologic analysis, including serial sectioning and IHC. The sensitivity of the SN procedure for predicting lymph node metastases, therefore, was 89% (eight of nine neck specimens) when an SN was identified by scintigraphy and found in the specimen. The overall accuracy of the SN procedure for predicting the presence or absence of lymph node metastases in the neck was 95% (19 of 20 neck specimens). CONCLUSIONS: Our study seems to validate the SN hypothesis for oral and oropharyngeal cancer. The role of SN biopsy in the management of the N0 neck in such patients has yet to be established through prospective trials. SN identification (and thus biopsy) does not seem to be reliable in patients with tumors located in or close to the midline.

Adult↗
PubMed2005

Expanding fields of genetically altered cells in head and neck squamous carcinogenesis.

Recent molecular pathology studies in head and neck cancer support a carcinogenesis model in which the development of a field with genetically altered cells plays a central role. This preneoplastic field is of monoclonal origin and expands non-invasively superseding normal epithelium. Clonal divergence and selection within the field ultimately leads to the development of cancer. These fields can be large (over 7 cm diameter) and are often not visible for the surgeon explaining that they may remain after resection of the primary tumor. When not removed, a field is an important risk factor for secondary cancer.

Carcinoma, Squamous Cell↗
PubMed2005

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.

Carcinoma, Squamous Cell↗
PubMed2005

Second field tumors: a new opportunity for cancer prevention?

Recent molecular genetic studies provide evidence that the majority of, if not all, head and neck squamous cell carcinomas (HNSCCs) develop within a contiguous field of preneoplastic cells. Cells of a field show genetic alterations associated with the process of carcinogenesis. A subclone in a field gives rise to an invasive carcinoma. An important implication of this knowledge is that, after surgery of the initial carcinoma, part of the field may remain in the patient. A field with preneoplastic cells that share genetic alterations with cells of the excised tumor has been detected in the resection margins of at least 25% of patients, indicating that this frequently occurs. Fields can be much larger than the actual carcinoma, sometimes having a diameter >7 cm. When a field remains after resection of the tumor, the risk for another carcinoma, designated as a second field tumor (SFT), is considerably greater. It is important to realize that an SFT develops from preneoplastic cells clonally related to the initial tumor. In this respect, it should be discriminated from a recurrent carcinoma that has developed from minimal residual cancer that was left behind and from a second primary tumor that independently develops from the initial carcinoma. Patients at risk for SFTs belong to a unique patient group for whom intense surveillance is indicated and chemoprevention is an attractive option. The priorities are to identify the patients in whom a remaining field will progress to cancer and to find the genes involved. With this knowledge, highly efficient clinical prevention trials, including those using the local application of therapeutic agents, can be designed. It is important to note that SFTs also may occur after treatment of various other cancers, including those of the bladder, skin, esophagus, lung, cervix, breast, and colon.

Carcinoma, Squamous Cell↗
PubMed2004

Clinical significance of micrometastatic cells detected by E48 (Ly-6D) reverse transcription-polymerase chain reaction in bone marrow of head and neck cancer patients.

PURPOSE: Despite improvements in locoregional treatment of head and neck squamous cell carcinoma (HNSCC), local and distant failure rates remain high. The strongest prognostic indicator of HNSCC is the presence of lymph node metastases in the neck, but the value of this indicator has limitations when using for the individual patient. The presence of micrometastatic cells in bone marrow has been shown to be a putative prognostic indicator in HNSCC and other epithelial malignancies, which might allow more accurate staging and selection of patients for whom adjuvant or experimental therapy is recommended. The gene encoding the E48 antigen is selectively expressed by HNSCC, and the detection of E48 transcripts in bone marrow by reverse transcription-polymerase chain reaction (RT-PCR) presumably represents the presence of micrometastatic cells. The purpose of this study was to determine the association between the presence of micrometastatic cells in bone marrow of HNSCC patients and clinical outcome. EXPERIMENTAL DESIGN: A total of 162 patients treated surgically for primary HNSCC underwent a single bone marrow aspiration from the upper iliac crest for detection of micrometastatic cells using E48 RT-PCR. In total, 139 patients were evaluable. The primary statistical endpoints were disease-free survival and distant metastasis-free survival. In addition, bone marrow samples of 30 noncancer controls were evaluated. RESULTS: E48 RT-PCR indicated the presence of micrometastatic cells in the bone marrow in 56 of 139 (40%) of the HNSCC patients and 0 of 30 of the noncancer controls (P < 0.0001). The presence of micrometastatic cells had no significant influence on disease-free survival or distant metastasis-free survival for the whole group of HNSCC patients (P = 0.1460 and P = 0.2912, respectively). For patients with >or=2 lymph node metastases, however, the presence of micrometastatic cells was associated with a poor distant metastasis-free survival (P = 0.0210). CONCLUSIONS: The presence of micrometastatic cells in bone marrow of HNSCC patients with >or=2 lymph node metastases is correlated with a poor distant metastasis-free survival. In this subgroup of HNSCC patients, E48 RT-PCR seems to be a valuable tool to identify patients who are at increased risk for development of distant metastases and therefore might benefit from experimental adjuvant systemic therapy.

Antigens, Ly↗
PubMed2004

Genetic patterns in head and neck cancers that contain or lack transcriptionally active human papillomavirus.

BACKGROUND: Transcriptionally active high-risk human papilloma viruses (HPVs), particularly HPV type 16 (HPV16), are found in a subset of head and neck squamous-cell carcinomas (HNSCCs). HPV16-associated carcinogenesis is mediated by expression of the viral E6 and E7 oncoproteins, which cause deregulation of the cell cycle by inactivating p53 and pRb, respectively. We tested the hypothesis that HPV-associated HNSCCs display a pattern of genetic alterations different from those of HNSCCs without HPV DNA. METHODS: Polymerase chain reaction-based assays were used to examine 143 consecutive HNSCCs (106 of the oral cavity and 37 of the oropharynx) for the presence of HPV DNA and for viral E6 and/or E7 messenger RNA (mRNA) expression. The HPV DNA-and E6 and E7 mRNA-positive HNSCCs and an equal number of HPV DNA-negative HNSCCs were further analyzed for mutations in TP53, the gene encoding p53, and for allelic loss of 28 microsatellite markers at chromosome arms 3p, 6q, 8p, 9p, 13q, 17p, and 18q, including markers located in regions of chromosome arms 9p and 17p that harbor genes involved the p53 and pRb pathways. All statistical tests were two-sided. RESULTS: Twenty-four (16.7%) of the 143 HNSCCs were positive for HPV16 DNA, and 12 of these HNSCCs (8.4% of total number) expressed E6 and E7 mRNAs. None of the HPV DNA-and E6/E7 mRNA-positive tumors had TP53 gene mutations, whereas nine (75%) of the 12 HPV DNA-negative tumors had such mutations (P<.001). Compared with the HPV DNA-negative HNSCCs, the E6/E7 mRNA-positive HNSCCs had statistically significantly lower levels of allelic loss for 13 of the 15 markers on 3p, 9p, and 17p. CONCLUSIONS: HNSCCs with transcriptionally active HPV16 DNA are characterized by occasional chromosomal loss, whereas HNSCCs lacking HPV DNA are characterized by gross deletions that involve whole or large parts of chromosomal arms and that already occur early in HNSCC development. These distinct patterns of genetic alterations suggest that HPV16 infection is an early event in HNSCC development.

Adult↗
PubMed2004

Molecular diagnosis of surgical margins and local recurrence in head and neck cancer patients: a prospective study.

PURPOSE: Approximately 10-30% of surgically treated head and neck cancer patients develop local recurrences while the resection margins are histologically tumor free. These recurrences may arise from cancer cells left behind but not detected by the pathologist, or they may develop from precursor lesions adjacent to the tumor that were not completely resected. We have investigated whether TP53-mutated DNA in the surgical margins is suitable to identify patients with head and neck squamous cell carcinoma at risk for local and locoregional recurrence. EXPERIMENTAL DESIGN: In a prospective cohort study of 76 patients with histologically tumor-free margins, the presence of TP53-mutated DNA was determined in the surgical margins using the phage plaque assay and correlated to clinical outcome. Immunostaining of the molecular-positive margins for mutated p53 protein was used to identify whether unresected precursor lesions or residual tumor cells were left behind. RESULTS: The absence of TP53-mutated DNA in surgical margins was significantly associated with remaining free of local and locoregional recurrence (P = 0.027 and P = 0.028, respectively). Moreover, the presence of TP53-mutated DNA in the surgical margins was an independent prognosticator for locoregional recurrence (relative risk = 7.1; P = 0.021; 95% confidence interval, 0.9-56). In 20% of the cases, the presence of TP53-mutated DNA in the surgical margins was found to be related to the presence of tumor-related precursor lesions. CONCLUSIONS: This study shows the value of TP53-mutated DNA as a molecular marker to predict locally recurrent head and neck squamous cell carcinoma. The observation that all patients who were negative for TP53-mutated DNA in the surgical margins remained free of local recurrence raises hope that molecular analysis of histologically tumor-free surgical margins can be exploited to decide on postoperative radiotherapy. Furthermore, our data provide evidence that local recurrences originate mainly from tumor cells left behind but also originate, in part, from unresected precursor lesions.

Adult↗
PubMed2004

Genetically altered fields as origin of locally recurrent head and neck cancer: a retrospective study.

PURPOSE: Surgeons treating patients with head and neck squamous cell carcinoma (HNSCC) rely heavily on histology to decide whether the resection margins are tumor free and subsequent adjuvant treatments can be omitted. However, despite the presence of tumor-free margins, 10-30% of HNSCC patients still develop a locally recurrent tumor. Evidence is available that recurrent cancer develops from either (a). outgrowth of a relatively small number of tumor cells that have not been detected by the pathologist or (b). a precursor lesion in which additional genetic alterations have led again to invasive cancer. EXPERIMENTAL DESIGN: In a retrospective study on 13 HNSCC cases, we analyzed the primary tumor, its surrounding histologically tumor-free resection margins, and local recurrences for loss of heterozygosity (22 microsatellite markers on 6 chromosomes) and TP53 mutations to determine the origin of the recurrent cancer. RESULTS: A precursor lesion was absent in 5 of 13 (39%) cases, and the genetic similarity of the primary and recurrent cancer was high, providing evidence that residual cancer cells were the origin of recurrence. For the remaining eight cases (61%) a genetically related precursor lesion (field) was detected, and for five of these cases, evidence was found that both the primary and recurrent carcinoma originated from this field. The remaining three cases were less conclusive. CONCLUSIONS: This study explains the pathobiology of locally recurrent HNSCC in patients with histologically tumor-free resection margins and indicates that the development of novel therapies to decrease the local recurrence rates in HNSCC should not only be focused on eradicating residual cancer cells but also on the precursor lesions that are left behind.

Adult↗
PubMed2004

Using tissue adjacent to carcinoma as a normal control: an obvious but questionable practice.

When carcinoma tissue is investigated using biochemical, immunohistochemical, and genetic techniques, adjacent tissue that is macroscopically normal is frequently used as a control, since cancer-related pheno- and geno-typic alterations are assumed to be absent. However, a field that contains genetically abnormal cells surrounds a significant proportion of carcinomas (for example, over 30% of head and neck cancers). These fields can be large (>7 cm in diameter) and consist of cells that are clonally related to the carcinoma. This indicates that adjacent epithelium must be checked for genetic abnormalities before it is considered normal and used as a control for comparison with carcinoma.

Biomarkers, Tumor↗
PubMed2004

A genetic progression model of oral cancer: current evidence and clinical implications.

Based on recent genetic studies, we propose a progression model for the development of oral squamous cell carcinoma. In the initial phase, a stem cell acquires a genetic alteration; subsequently a patch is formed, a clonal unit consisting of the stem cell with its daughter cells that all share the DNA alteration. The next critical step is the conversion of a patch into an expanding field as a result of additional genetic alterations. This mucosal field replaces the normal epithelium and in the oral cavity such fields have been detected with dimensions of over 7 cm in diameter. Sometimes these fields are visible as leukoplakia. Ultimately, clonal selection leads to the development of carcinoma within this contiguous field of pre-neoplastic cells. An important clinical implication of this model is that fields often remain after surgery of the primary tumor and may lead to new cancers, presently designated by clinicians as second primary tumors or local recurrences.

Carcinoma, Squamous Cell↗