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Publications and source records attributed to Ruud H Brakenhoff.
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The concept of "field cancerization" was first introduced by Slaughter et al. [D. P, Slaughter et al., Cancer (Phila.), 6: 963-968, 1953] in 1953 when studying the presence of histologically abnormal tissue surrounding oral squamous cell carcinoma. It was proposed to explain the development of multiple primary tumors and locally recurrent cancer. Organ systems in which field cancerization has been described since then are: head and neck (oral cavity, oropharynx, and larynx), lung, vulva, esophagus, cervix, breast, skin, colon, and bladder. Recent molecular findings support the carcinogenesis model in which the development of a field with genetically altered cells plays a central role. In the initial phase, a stem cell acquires genetic alterations and forms a "patch," a clonal unit of altered daughter cells. These patches can be recognized on the basis of mutations in TP53, and have been reported for head and neck, lung, skin, and breast cancer. The conversion of a patch into an expanding field is the next logical and critical step in epithelial carcinogenesis. Additional genetic alterations are required for this step, and by virtue of its growth advantage, a proliferating field gradually displaces the normal mucosa. In the mucosa of the head and neck, as well as the esophagus, such fields have been detected with dimensions of >7 cm in diameter, whereas they are usually not detected by routine diagnostic techniques. Ultimately, clonal divergence leads to the development of one or more tumors within a contiguous field of preneoplastic cells. An important clinical implication is that fields often remain after surgery of the primary tumor and may lead to new cancers, designated presently by clinicians as "a second primary tumor" or "local recurrence," depending on the exact site and time interval. In conclusion, the development of an expanding preneoplastic field appears to be a critical step in epithelial carcinogenesis with important clinical consequences. Diagnosis and treatment of epithelial cancers should not only be focused on the tumor but also on the field from which it developed.
The E48 antigen is a successfully explored molecular marker for the diagnosis and therapy of HNSCC. The applicability of E48 as an HNSCC-associated antigen, however, is restricted due to its heterogeneous expression in 30% of tumors; and identification of additional target antigens is therefore desired. E48 belongs to the Ly-6 antigen family, comprising a group of highly homologous, low m.w., GPI-anchored surface proteins, of which some show tissue-restricted expression patterns. To identify novel human HNSCC-associated Ly-6 members with squamous cell-associated expression patterns, we performed comprehensive gene-screening consisting of BLAST searches within GenBank databases, followed by expression analysis. Using this approach, the Ly-6K gene could be annotated as a novel member of the human Ly-6 family. Expression of the human Ly-6 genes E48, Ly-6K, PSCA, GML, RIG-E, G6C and Ly-6H was prescreened by qualitative RT-PCR and subsequently analyzed by quantitative RT-PCR in normal keratinocytes, HNSCC cell lines, normal mucosa, HNSCC tumors as well as normal peripheral blood and bone marrow cells. PSCA was highly expressed in normal mucosa, but 100-fold decreased expression was seen in HNSCC. For Ly-6H, GML and G6C, no or very low expression was observed in keratinocytes and HNSCC. Expression of RIG-E was high in normal and malignant squamous cells and in peripheral blood and bone marrow cells, thus limiting its applicability as an HNSCC-associated marker. In contrast, besides the E48 gene, the Ly-6K gene also appeared to be selectively expressed in HNSCC and normal squamous cells. Moreover, expression of Ly-6K was shown in HNSCC cell lines, in which no E48 expression could be detected. These data justify further evaluation of Ly-6K as potential target antigen for the diagnosis and therapy of HNSCC.
Histological grading of epithelial dysplasia in the oral cavity and oropharynx is used to predict the risk for cancer and to determine the treatment strategy. This grading, however, is subjective and not well reproducible. Recent publications have shown that molecular markers are promising in cancer risk assessment. The aim of the present study was to compare classical histological and molecular grading and to relate these to the proliferation rate by quantitative assessment of Ki-67 staining. Forty-three samples were analysed from the margins of patients who had undergone resection of their squamous cell carcinoma in the oral cavity/oropharynx. Three experienced pathologists performed the histological grading. With the consensus score, 12 samples were classified as normal and 31 as dysplastic (21 mild, six moderate, and four severe). Loss of heterozygosity (LOH) was assessed in the same samples with 15 microsatellite markers at chromosomes 3p, 9p, 17p, 8p, 13q, and 18q, and was present in 28 of the 43 samples. Twenty-four of the 28 cases (86%) with LOH were classified as dysplastic and four as normal. All ten samples with moderate and severe dysplasia and 14 of 21 samples with mild dysplasia contained LOH. In four of 12 biopsies classified as normal, LOH was found. A very striking and significant difference of the Ki-67 index was observed between LOH-positive and LOH-negative cases, 36.6 +/- 11.1% versus 19.4 +/- 2.8% positive cells, respectively. In mild dysplasia, 13 of 14 lesions containing LOH had a higher Ki-67 index than all seven lesions without LOH. Thus, in the oral cavity/oropharynx, LOH is more frequently found in the histologically higher-grade lesions (moderate dysplasia or worse) and in the lower grade lesions when a high proliferation rate is present. Assessment of proliferation with Ki-67 is a better surrogate for LOH than histological grading.
The presence of lymph node metastases is the major determinant for prognosis in head and neck squamous cell carcinoma (HNSCC). It is at present unknown whether the same holds true for the presence of histologically undetectable micrometastases. We analyzed 456 histologically tumor-negative lymph nodes of 23 HNSCC patients without (pN0) and 18 patients with one or two tumor-positive lymph nodes (pN+) in their neck dissection specimens at histopathologic examination. To detect the presence of disseminated tumor cells and micrometastases in these lymph nodes, we used real-time quantitative RT-PCR with E48 (Ly-6D) transcripts as a squamous cell-specific molecular marker. The results were compared with histopathologic examination and clinical outcome. E48 transcripts were detected in lymph nodes of 5 (22%) of 23 patients in the pN0 group, and in histologically negative lymph nodes of 10 (56%) of 18 patients in the pN+ group. In the pN0 group, the presence of E48-positive lymph nodes was significantly associated with a distinctly poor cause-specific survival as compared with those with E48-negative lymph nodes. Our results indicate that E48 real-time quantitative RT-PCR is a suitable method for the detection of micrometastases in lymph nodes of patients with HNSCC. Moreover, detection of micrometastases seems clinically relevant but should be confirmed in a large multicenter trial.
PURPOSE: Staging of the clinically N(0) neck in patients with head and neck squamous cell carcinoma (HNSCC) using ultrasound-guided, fine needle aspiration cytology (USgFNAC) has a false-negative rate of approximately 20% that might be caused by inaccurate cytology. Molecular analysis of aspirate residues might reduce the false-negative rate, and we therefore set up a quantitative reverse transcription-PCR (Q-RT-PCR) assay based on TaqMan technology using the squamous cell-specific antigen E48 (Ly-6D) as molecular marker. EXPERIMENTAL DESIGN: The detection limit of the assay was determined in reconstruction experiments. The sensitivity of the assay was tested on cytological tumor-positive aspirate residues and the specificity on lymph node aspirate residues of noncancer controls. Subsequently, 235 lymph node aspirate residues of 64 HNSCC patients staged with USgFNAC were examined for the presence of E48 mRNA. E48 Q-RT-PCR results of the aspirated lymph nodes were compared with cytology and clinical outcome. RESULTS: The detection limit of E48 Q-RT-PCR was a single tumor cell in a background of 10(6) peripheral blood mononuclear cells. From the 41 aspirates that were not evaluable at cytology, 24 (59%) could be diagnosed with E48 Q-RT-PCR. In the 191 aspirates that were tumor negative or not evaluable at cytology, 8 samples from 6 patients were E48 positive. These results were confirmed by histology or clinical outcome in 3 of 6 patients. E48 Q-RT-PCR showed an increase in sensitivity from 56 to 67% and an increase in frequency of reached diagnosis from 97 to 100% compared with cytology. The specificity decreased from 100 to 92%. CONCLUSIONS: Real-time E48 Q-RT-PCR is an accurate technique for squamous cell detection in lymph node aspirates of HNSCC patients. The assay shows an increase in sensitivity and frequency of reached diagnosis compared with cytology. The test could be implemented routinely in USgFNAC to diagnose cases for which cytological examination is not conclusive.
Ample molecular data are available on the progression from normal mucosa to invasive head and neck squamous cell carcinoma (HNSCC), but information on further genetic progression to metastatic disease is scarce. To obtain insight into the metastatic process, we compared 23 primary HNSCCs with 25 corresponding lymph node metastases (LNMs) and 10 corresponding distant metastases (DMs) with respect to TP53 mutations and patterns of loss of heterozygosity (LOH) based on 26 microsatellite markers on six chromosome arms (3p, 9p, 17p, 13q, 8p, and 18q). In 18 of the 23 patients, a TP53 mutation was detected in the primary tumor, and in all cases the same TP53 mutation was present in the corresponding LNM or DM. In nine of 20 patients with LNMs and three of seven patients with DMs, the LOH pattern of metastasis differed from that of the corresponding primary tumor by at least one marker. Microsatellite markers located on chromosome arms 13q, 8p, and 18q were most frequently discordant, providing evidence that alterations at these chromosomes occur late in HNSCC carcinogenesis. Moreover, evidence was found that DMs had developed directly from the primary tumor and not from LNMs. Remarkably, we observed that the mutational status of the TP53 gene is associated significantly with the degree of genetic differences between primary HNSCCs and corresponding metastases. All patients with TP53 wild-type primary tumors showed significantly more discordant LOH patterns in the corresponding LNMs and DMs than patients with TP53-mutated tumors. The percentages were 100% versus 27% (LNMs) and 100% versus 0% (DMs), respectively (P = 0.008 and P = 0.029; two-sided Fisher exact test). This finding suggests that TP53-mutated tumors need fewer additional genetic alterations to develop metastases compared with TP53 wild-type primary tumors.
BACKGROUND: Management of the N0 neck in patients with head and neck squamous cell carcinoma (SCC) remains controversial. We describe the outcome of patients who underwent transoral tumor excision and a wait-and-see policy for the neck staged N0 by ultrasonography-guided cytology (USgFNAC). Because selection of lymph nodes for USgFNAC is currently based on size criteria, we investigated the additional value of sentinel node (SN) identification. METHODS: The outcome of 161 patients with T1-T2 oral/oropharyngeal SCC was determined. In a subgroup of 39 patients the SN was identified and aspirated in addition. RESULTS: SN identification and aspiration was possible in 38 of 39 patients but without decreasing the false-negative rate of USgFNAC. During follow-up (12-99 months) 34 of 161 (21%) patients developed lymph node metastases. After therapeutic neck dissection and postoperative radiotherapy, 27 of 34 (79%) could be salvaged (88% regional control). CONCLUSIONS: Wait-and-see seems justified in case of negative USgFNAC. Strict follow-up with USgFNAC is required. SN identification and aspiration is feasible but did not improve lymph node selection.
Second primary tumors (SPTs) are a significant problem in treating oral and oropharyngeal squamous cell carcinoma and have a negative impact on survival. In most studies the definition of SPT is based on the criteria of Warren and Gates, published in 1932. These criteria, however, are ill-defined and lead to confusion. Recent molecular studies have shown that a tumor can be surrounded by a mucosal field consisting of genetically altered cells. Furthermore, evidence has been provided that SPTs (defined by classical criteria) can share some or even all genetic markers with the index tumor, indicating that both tumors have arisen from a common cell clone. We propose that these secondary neoplastic lesions should not be considered SPTs, implying that the present concept of SPT needs revision. This review describes a novel classification of the secondary tumors that develop after treatment of a carcinoma in the oral cavity or oropharynx. On the basis of the molecular analysis of the tumors and the genetically altered mucosal field in between, we propose definitions for a "true SPT," a local recurrence, a "SFT" (second field tumor derived from the same genetically altered mucosal field as the primary tumor), and a metastasis. Considering the etiologic differences of these lesions, we believe that an accurate molecular definition is essential to make headway with the clinical management of oral and oropharyngeal cancer.
In total, 10-30% of patients with head and neck squamous cell carcinoma (HNSCC) develop local recurrences despite seemingly adequate tumour resection. This may result from minimal residual cancer (MRC): small numbers of tumour cells left behind in the surgical margins, undetectable by routine histopathology. In recent studies, p53 mutations have been considered as selective and sensitive DNA markers of cancer cells. There are two potential problems in using mutated-p53 DNA as a marker. Firstly, p53 mutations occur early in progression and might therefore detect unresected precursor lesions besides tumour cells. Secondly, DNA is a very stable biomolecule that might lead to false-positive results. These two potential problems have been evaluated in this study. Fifty patients with a radical tumour resection were included, of whom 30 showed a p53 mutation in the primary tumour. Histopathologically tumour-free surgical margins were quantitatively analysed for mutated p53 by molecular diagnosis (plaque assay) and subsequent (immuno)histopathology. p53 mutated DNA was detected in the surgical margins of 19/30 patients. Immunohistochemistry confirmed the presence of small tumour foci in 2/19 mutated p53-positive cases. In 7/19 cases, the tumour-specific p53 mutation was found in unresected dysplastic mucosal precursor lesions. Moreover, in a number of cases small p53-immunostained patches were detected, but the mutations found were never tumour-related. By screening contralateral exfoliated cells and plaque assays on RNA it was shown that detection of mutated-p53 DNA is prone to false-positive results. In conclusion, using p53 mutations as a marker, both MRC and unresected mutated p53-positive mucosal precursor lesions are detected within surgical margins. Molecular assessment of surgical margins using p53 mutations enables the selection of HNSCC patients at high risk for tumour recurrence, but tumour RNA seems at present to be a more specific biomolecule for analysis than tumour DNA.
The development of second primary tumors has a negative impact on the prognosis of head and neck squamous cell carcinoma. Previously, we detected genetically altered and tumor-related mucosal lesions in the resection margins in 25% of unselected head and neck squamous cell carcinoma patients (Tabor MP, Brakenhoff RH, van Houten VMM, Kummer JA, Snel MHJ, Snijders PJF, Snow GB, Leemans CR, Braakhuis BJM: Persistence of genetically altered fields in head and neck cancer patients: biological and clinical implications. Clin Cancer Res 2001, 7: 1523-1532). The aim of this study was to determine whether first and second primary tumors are clonally related and originate from a single genetically altered field. From 10 patients we analyzed the first tumor of the oral cavity or oropharynx, the >3-cm remote second primary tumor, and the mucosa from the tumor-free margins from both resection specimens. We compared TP53 mutations and loss of heterozygosity profiles using 19 microsatellite markers at chromosomes 3p, 9p, 13q, and 17p. In all patients, genetically altered mucosal lesions were detected in at least one resection margin from both first and second primary tumor. Evidence for a common clonal origin of the first tumor, second primary tumor, and the intervening mucosa was found for at least 6 of 10 patients. Our results indicate that a proportion of multiple primary tumors have developed within a single preneoplastic field. Based on different etiology and clinical consequences, we propose that independent second primary tumors should be distinguished from second field tumors, that arise from the same genetically altered field the first tumor has developed from.
BACKGROUND AND OBJECTIVE: Retinoids show promise in the treatment of various (pre)malignancies, including head and neck squamous cell carcinoma (HNSCC). It has been shown that metabolic pathways of retinoids are important in their anticancer effect and that these pathways may change during HNSCC carcinogenesis. We have previously reported that HNSCC cells have a 17-fold greater turnover rate of retinoic acid (RA) than normal oral keratinocytes from noncancer controls, and that the formation of polar metabolites such as 4-oxo-RA and 4-hydroxy-RA is only seen in HNSCC cell lines. We aimed to establish whether this altered retinoid metabolism is an intrinsic characteristic of HNSCC patients. METHODS: The normal mucosa of cancer and noncancer patients was the source of keratinocyte cultures. The cells were exposed to RA for various time periods, and the levels of various retinoids were measured in the culture medium and cell pellets with reverse-phase liquid chromatography. RESULTS: Cells from cancer patients were morphologically normal and showed no genetic aberrations (i.e. loss of heterozygosity). The RA turnover rate in normal oral keratinocytes of cancer patients was 15 times higher (p = 0.003) than that in normal oral keratinocytes of noncancer controls, with average turnover rates of 218.6 and 14.8 pmol/mg protein/h, respectively. Specific profiles of RA metabolites were similar. CONCLUSION: The observed higher RA metabolism in noncancer cells of HNSCC patients suggests that individuals with a relatively high RA turnover have an increased risk of developing HNSCC.