Arjen Brink

Molecular characterization of p16‐immunopositive but HPV DNA‐negative oropharyngeal carcinomas

Recent studies have reported that p16 protein overexpression qualifies as a surrogate marker identifying an oncogenic human papillomavirus (HPV) infection in oropharyngeal squamous cell carcinoma (OPSCC). However, there is still a percentage of OPSCCs that are positive for p16 immunohistochemistry (p16 IHC) but lack HPV DNA. The objective of this study was to characterize this group at the molecular level by performing sensitive HPV DNA‐ and RNA‐based PCR methods and genetic profiling. All patients diagnosed with an OPSCC in the period 2000–2006 in two Dutch university medical centers were included (n = 841). The presence of HPV in a tumor sample was tested by p16 IHC followed by an HPV DNA GP5+/6+ PCR. p16 IHC scored positive in 195 samples, of which 161 were HPV DNA‐positive and 34 (17%) HPV DNA‐negative. In the latter group, a SPF10‐LiPA25 assay, an HPV16 type‐specific E7 PCR and an E6 mRNA RT‐PCR were performed. Next, ten of these cases were further analyzed for loss of heterozygosity (LOH) of 15 microsatellite markers at chromosome arms 3p, 9p and 17p. Of the 34 p16‐positive but PCR‐negative OPSCCs, two samples tested positive by SPF10 assay, HPV16 E7 PCR and HPV16 E6 mRNA RT‐PCR. Three samples tested positive by SPF10 assay but negative by the HPV16‐specific assays. Nine of ten cases that were tested for LOH showed a genetic pattern comparable to that of HPV‐negative tumors. This study categorizes p16‐positive but HPV DNA‐negative OPSCCs as HPV‐negative tumors based on genetic profiling. This study highlights the importance of performing HPV testing in addition to p16 IHC for proper identification of HPV‐associated OPSCCs.

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.

Screening for Oral Precancer with Noninvasive Genetic Cytology

Oral squamous cell carcinomas develop in precancerous fields consisting of genetically altered mucosal epithelial cells. These precancerous fields may appear as clinically visible lesions, in particular, oral leukoplakia, but the large majority remains clinically undetectable. The aim of this study was to assess the potential value of a noninvasive screening approach to detect precancerous fields. As a first step, we developed a suitable assay and investigated 25 leukoplakia patients and 20 noncancer control subjects. Exfoliated cells were removed by a brush from multiple small areas of the oral mucosa, including the leukoplakia. Brushed samples were investigated for allelic imbalance (AI) at chromosomes 3p, 9p, 11q, and 17p using microsatellite markers known to show frequent alterations in oral precancer. AI was absent in all (137) of the samples of the 20 control subjects, yielding a specificity of 100%. AI was detected in exfoliated cell samples of 40% (10 of 25) of the leukoplakia lesions studied. Genetic changes were also found outside the leukoplakia lesions. Most frequent was AI at 9p (9 of 10). The noninvasive assay was validated against the biopsy results of the leukoplakia lesions yielding an estimate of sensitivity of 78% (7 of 9) and a positive predictive value of 100% (7 of 7). Altogether, these results show the feasibility of a noninvasive genetic screening approach for the detection and monitoring of oral precancer. This assay could therefore contribute to the secondary prevention of oral squamous cell carcinoma. The assay also shows promise for the detection of precancerous changes that are not macroscopically visible.

Cancer stem cell enrichment marker CD98: A prognostic factor for survival in patients with human papillomavirus-positive oropharyngeal cancer

PURPOSE Several hypotheses have been proposed to explain the relatively good prognosis of patients with a human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) and one of these is a higher sensitivity to (chemo)radiation. Previous studies have suggested that treatment failure in OPSCC patients is caused by resistance of cancer stem cells (CSCs). The purpose of this study was to evaluate the association between the number of CSCs and prognosis in HPV-positive OPSCC patients. EXPERIMENTAL DESIGN All OPSCC patients (n=711) treated between 2000 and 2006 in two Dutch university hospitals were included. Presence of HPV in a tumour tissue specimen was tested by p16-immunostaining followed by HPV DNA GP5+/6+polymerase chain reaction (PCR). The presence and intensity of tumour CSC markers CD44 and CD98 were determined by immunohistochemistry and semiquantitative scoring was performed. Overall survival (OS) and progression-free survival (PFS) rates were compared between patients with low and high CD44/CD98 expression in relation to HPV status. RESULTS HPV-positive tumours showed a lower percentage of cells with CD44 and CD98 expression than HPV-negative tumours (p<0.001, χ(2)-test). Within the group of patients with HPV-positive OPSCC, a high percentage of CD98-positive tumour cells was associated with a significantly worse 5-year OS and PFS (OS: 36.4% and PFS: 27.3%) compared to patients with a low percentage of CD98-positive cells (OS: 71.9% and PFS: 70.5%, respectively) (p<0.001). CONCLUSIONS HPV-positive OPSCCs harbour fewer cells expressing the CSC enrichment markers CD44 and CD98. Furthermore, OS and PFS were significantly worse for patients with HPV-positive OPSCC with a high percentage of CD98-positive cells.

Comparative evaluation of genetic assays to identify oral pre-cancerous fields

BACKGROUND Oral squamous cell carcinomas often develop in a pre-cancerous field, defined as mucosal epithelium with cancer-related genetic alterations, and which may appear as a clinically visible lesion. The test characteristics of three genetic assays that were developed to detect pre-cancerous fields were investigated and compared to histology. METHODS In total, 10 pre-cancerous fields that were not visible at clinical inspection and gave rise to malignant transformation based on an identical TP53 mutation in tumor and mucosal epithelium in the surgical margin, as well as 10 normal oral mucosa specimens were analyzed for numerical chromosomal changes with multiplex ligation-dependent probe amplification (MLPA), for loss of heterozygosity (LOH), with microsatellite PCR and for DNA index alterations with DNA image analysis. RESULTS No alterations were detected in normal tissue by either of the assays. Both MLPA and LOH assays detected all pre-cancerous fields. DNA cytometry identified aneuploidy in four of 10 pre-cancerous fields, while the corresponding tumors that developed in these fields were shown to be aneuploid. CONCLUSIONS Both the MLPA and LOH assay seem suitable for screening pre-cancerous fields in subjects at high risk for oral cancer even in the absence of clinically abnormal appearing oral mucosa. Measurements of DNA index might be valuable to determine the time to progression.

Molecular screening of oral precancer

OBJECTIVES Early detection and treatment of high risk premalignant mucosal changes of the oral cavity, will expectedly improve survival and reduce treatment-related morbidity. Aims of this study were to evaluate a non-invasive screening approach and to assess the value of molecular markers to identify patients at risk for oral cancer. MATERIALS AND METHODS Exfoliated cells and biopsies were obtained from oral leukoplakia lesions of 43 patients, of whom six developed oral cancer. All samples were investigated for loss of heterozygosity (LOH) at chromosomes 3p, 9p, 11q and 17p using microsatellite markers. On the biopsy specimen additional immunohistochemical staining for p53, TP53 mutation analysis and histopathological grading were performed. RESULTS The analytical sensitivity of the non-invasive assay using exfoliated cells to detect genetic changes present in the lesions was 45% (9 of 20), the specificity was 100% (19 of 19), and the positive predictive value was also 100% (9 of 9). LOH was present in 20 of 39 (51%) of the biopsies with uniformly LOH at 9p. Mutated TP53 and LOH at 9p in the biopsy, as single markers and in combination, were significant risk factors for malignant progression of leukoplakia to oral cancer (Kaplan-Meier analysis, p<0.05). CONCLUSION A non-invasive genetic screening approach using LOH in exfoliated cells has limited value for monitoring patients with leukoplakia. However, LOH at 9p, but also mutated TP53 in biopsies of oral leukoplakia have a significant association with malignant transformation and are promising candidate biomarkers to predict the risk for malignant progression.

DNA-Bound Platinum Is the Major Determinant of Cisplatin Sensitivity in Head and Neck Squamous Carcinoma Cells

Purpose The combination of systemic cisplatin with local and regional radiotherapy as primary treatment of head and neck squamous cell carcinoma (HNSCC) leads to cure in approximately half of the patients. The addition of cisplatin has significant effects on outcome, but despite extensive research the mechanism underlying cisplatin response is still not well understood. Methods We examined 19 HNSCC cell lines with variable cisplatin sensitivity. We determined the TP53 mutational status of each cell line and investigated the expression levels of 11 potentially relevant genes by quantitative real-time PCR. In addition, we measured cisplatin accumulation and retention, as well as the level of platinum-DNA adducts. Results We found that the IC50 value was significantly correlated with the platinum-DNA adduct levels that accumulated during four hours of cisplatin incubation (p = 0.002). We could not find a significant correlation between cisplatin sensitivity and any of the other parameters tested, including the expression levels of established cisplatin influx and efflux transporters. Furthermore, adduct accumulation did not correlate with mRNA expression of the investigated influx pumps (CTR1 and OCT3) nor with that of the examined DNA repair genes (ATR, ATM, BRCA1, BRCA2 and ERCC1). Conclusion Our findings suggest that the cisplatin-DNA adduct level is the most important determinant of cisplatin sensitivity in HNSCC cells. Imaging with radio-labeled cisplatin might have major associations with outcome.

CD98 marks a subpopulation of head and neck squamous cell carcinoma cells with stem cell properties

Patients with advanced head and neck squamous cell carcinomas (HNSCCs) are often treated with concomitant chemotherapy and radiotherapy, but only 50% is cured. A possible explanation for treatment failure is therapy resistance of the cancer stem cells (CSCs). The application of compounds specifically targeting these CSCs, in addition to routinely used therapeutics, would likely improve clinical outcome. We demonstrate that the previously described monoclonal antibody K984 recognizes the CD98 cell surface protein, which is specifically expressed by cells forming the squamous basal cell layer, the region where the squamous stem cells reside. Moreover, CD98 is highly resistant to the proteolytic enzymes required for CSC enrichment procedures. We show that CD98(high) cells, in contrast to CD98(low) cells, are able to generate tumors in immunodeficient mice, indicating that CD98(high) cells have stem cell characteristics. Furthermore, the CD98(high) subpopulation expresses high levels of cell cycle control and DNA repair genes, while the CD98(low) fraction shows expression patterns that represent the more differentiated cells forming the bulk of the tumor. CD98 is a promising CSC enrichment marker in HNSCC. Our data support the CSC concept in head and neck cancer and the potential relevance of these cells for treatment outcome.

Defects in the Fanconi Anemia Pathway and Chromatid Cohesion in Head and Neck Cancer

Failure to repair DNA damage or defective sister chromatid cohesion, a process essential for correct chromosome segregation, can be causative of chromosomal instability (CIN), which is a hallmark of many types of cancers. We investigated how frequent this occurs in head and neck squamous cell carcinoma (HNSCC) and whether specific mechanisms or genes could be linked to these phenotypes. The genomic instability syndrome Fanconi anemia is caused by mutations in any of at least 16 genes regulating DNA interstrand crosslink (ICL) repair. Since patients with Fanconi anemia have a high risk to develop HNSCC, we investigated whether and to which extent Fanconi anemia pathway inactivation underlies CIN in HNSCC of non-Fanconi anemia individuals. We observed ICL-induced chromosomal breakage in 9 of 17 (53%) HNSCC cell lines derived from patients without Fanconi anemia. In addition, defective sister chromatid cohesion was observed in five HNSCC cell lines. Inactivation of FANCM was responsible for chromosomal breakage in one cell line, whereas in two other cell lines, somatic mutations in PDS5A or STAG2 resulted in inadequate sister chromatid cohesion. In addition, FANCF methylation was found in one cell line by screening an additional panel of 39 HNSCC cell lines. Our data demonstrate that CIN in terms of ICL-induced chromosomal breakage and defective chromatid cohesion is frequently observed in HNSCC. Inactivation of known Fanconi anemia and chromatid cohesion genes does explain CIN in the minority of cases. These findings point to phenotypes that may be highly relevant in treatment response of HNSCC.

Comparison of oncolytic adenoviruses for selective eradication of oral cancer and pre-cancerous lesions

Oncolytic adenoviruses are being investigated as potential anti-cancer agents. Selective lytic replication in cancer cells is essential for an effective and safe treatment. In this study, we compared 11 oncolytic adenoviruses in relevant cell cultures to assess their use for treating oral cancer and pre-cancerous lesions. We determined the cytotoxicity of oncolytic adenovirus infection and calculated selectivity indices for cytotoxicity to cancer cells compared with normal oral keratinocytes and fibroblasts. Keratinocytes were very sensitive to wild-type adenovirus serotype 5 (Ad5); 1- to 3-log more than head and neck squamous cell carcinoma (HNSCC) cells. The potencies of oncolytic adenoviruses to kill HNSCC cells within 7 days after infection ranged from approximately 10 times less potent to approximately 10 times more potent than Ad5. The selectivity indices determined on fibroblasts and keratinocytes differed markedly. Two oncolytic adenoviruses were more selective than Ad5 for HNSCC cells compared with fibroblasts; and five viruses showed selective replication on HNSCC cells compared with keratinocytes. Overall, CRAd-S.RGD with E1A driven by the survivin promoter and an infectivity-enhancing capsid modification showed the most favourable cytotoxicity pattern; being very potent in killing HNSCC cells, only slightly less effective than Ad5 in killing pre-neoplastic keratinocytes and the least toxic to normal keratinocytes.