Sentinel node in head and neck cancer: use of size criterion to upstage the no neck in head and neck squamous cell carcinoma.

BACKGROUND: Anatomical imaging tools demonstrate poor sensitivity in head and neck squamous cell carcinoma (HNSCC) patients with clinically node-negative necks (cN0). This study evaluates nodal size as a staging criterion for detection of cervical metastases, utilizing sentinel node biopsy (SNB) and additional pathology (step-serial sectioning, SSS; and immunohistochemistry, IHC). METHODS: Sixty-five patients with clinically N0 disease underwent SNB, with a mean of 2.4 nodes excised per patient. Nodes were fixed in formalin, bisected, and measured in 3 axes before hematoxylin-eosin staining. Negative nodes were subjected to SSS and IHC. SNB-positive patients underwent modified radical neck dissection. RESULTS: Maximum diameter was larger in levels II and III (13.1 and 13.2 mm) when compared with level I (10.5 mm; p = .004, p = .018), while minimum diameter was constant. Positive nodes were larger than negative nodes (p = .007), but nodes found positive by SSS/IHC were not significantly larger than negative nodes for either measurement (p = .433). Sensitivity and specificity were poor for all measurements. CONCLUSIONS: Nodal size is an inaccurate predictor of nodal metastases and should not be regarded as an accurate means of staging the clinically N0 neck.

Divergent routes to oral cancer.

Most head and neck squamous cell carcinoma (HNSCC) patients present with late-stage cancers, which are difficult to treat. Therefore, early diagnosis of high-risk premalignant lesions and incipient cancers is important. HNSCC is currently perceived as a single progression mechanism, resulting in immortal invasive cancers. However, we have found that approximately 40% of primary oral SCCs are mortal in culture, and these have a better prognosis. About 60% of oral premalignancies (dysplasias) are also mortal. The mortal and immortal tumors are generated in vivo as judged by p53 mutations and loss of p16(INK4A) expression being found only in the original tumors from which the immortal cultures were derived. To investigate the relationships of dysplasias to SCCs, we did microarray analysis of primary cultures of 4 normal oral mucosa biopsies, 19 dysplasias, and 16 SCCs. Spectral clustering using the singular value decomposition and other bioinformatic techniques showed that development of mortal and immortal SCCs involves distinct transcriptional changes. Both SCC classes share most of the transcriptional changes found in their respective dysplasias but have additional changes. Moreover, high-risk dysplasias that subsequently progress to SCCs more closely resemble SCCs than nonprogressing dysplasias. This indicates for the first time that there are divergent mortal and immortal pathways for oral SCC development via intermediate dysplasias. We believe that this new information may lead to new ways of classifying HNSCC in relation to prognosis.

High levels of telomere dysfunction bestow a selective disadvantage during the progression of human oral squamous cell carcinoma.

Human epithelial cells experience multiple barriers to cellular immortality in culture (mortality mechanisms 0, 1, and 2). Mortality mechanism 2 (M2) is termed crisis and involves telomere dysfunction due to lack of telomerase. However, proliferating normal keratinocytes in vivo can express telomerase, so it is unclear whether human squamous cell carcinomas (SCCs), which usually have high telomerase levels, develop from preexisting telomerase-positive precursors or by the activation of telomerase in telomerase-deficient somatic cells. We show that 6 of 29 oral SCCs show characteristics of M2 crisis in vivo, as indicated by a high anaphase bridge index (ABI), which is a good correlate of telomere dysfunction, and that 25 of 29 tumors possess some anaphase bridges. ABIs in excess of 0.2 in the primary tumor showed a decrease in the corresponding lymph node metastases. This suggests that high levels of telomere dysfunction (>0.2) and, by inference, M2 crisis bestow a selective disadvantage on SCCs during progression stages of the disease. Supporting this, SCCs with high levels of telomere dysfunction grow poorly in culture, and the ectopic expression of telomerase corrects this, together with other features of M2 crisis. Our data suggest that a substantial proportion of oral SCCs in vivo ultimately arise from telomerase-deficient keratinocytes rather than putative telomerase-proficient cells in the undifferentiated parts of the epithelium. Furthermore, the presence of significant levels of telomere dysfunction in a high proportion of SCCs at diagnosis but not in the normal epithelium implies that the therapeutic inhibition of telomerase should selectively compromise the growth of such tumors.

The use of sentinel node biopsy to upstage the clinically N0 neck in head and neck cancer.

OBJECTIVE: To investigate the possible role of sentinel node biopsy (SNB) alone to upstage the clinically N0 neck in patients with oral and oropharyngeal squamous cell carcinoma. DESIGN: Prospective clinical study. SETTING: Head and neck referral center. PATIENTS: Patients with primary untreated oral and/or oropharyngeal squamous cell carcinoma accessible to injection and with clinically N0 necks were enrolled in the study. INTERVENTION: An SNB was performed after radiocolloid and blue dye injection. Preoperative lymphoscintigraphy and the perioperative use of a gamma probe identified radioactive sentinel nodes and visualization of blue-stained lymphatics identified blue sentinel nodes. If the sentinel node was found negative, there was no further treatment to the neck. If the sentinel node tested positive, a therapeutic neck dissection was performed. All patients underwent regular follow-up at the outpatient clinic to identify possible recurrence. MAIN OUTCOME MEASURES: Upstaging of the clinically N0 neck by SNB and development of subsequent disease in SNB-negative necks. RESULTS: An SNB was performed on 57 clinically N0 necks in 48 patients. Sentinel nodes were harvested in 43 (90%) of 48 patients. Fifteen (35%) of 43 patients were upstaged by SNB and 28 (65%) of 43 were staged SNB negative. There was a mean follow-up of 18 months. One patient developed subsequent disease after having been staged negative with SNB. The overall sensitivity of the procedure using the full pathologic protocol was 94% (15/16). CONCLUSIONS: Sentinel node biopsy can be used to upstage the N0 neck in patients with early subclinical nodal disease. However, before it becomes the standard of care in head and neck squamous cell carcinoma, longer follow-up observational trials are needed.

Molecular changes associated with oral dysplasia progression and acquisition of immortality: potential for its reversal by 5-azacytidine.

This study has identified molecular changes characteristic of early oral cancer progression. We reported previously that acquisition of the immortal phenotype is an early event in oral cancer development (F. McGregor et al., Cancer Res., 57: 3886-3889, 1997); our current data indicate that about half of oral dysplasia cultures are immortal, and this is associated with loss of expression of retinoic acid receptor (RAR)-beta and the cell cycle inhibitor p16(ink4a) (p16), p53 mutations, and increased levels of telomerase/human telomerase reverse transcriptase mRNA. In contrast, increased expression of the epidermal growth factor receptor, known to be a characteristic of oral cancer, does not occur until after the dysplasia stage in squamous cell carcinomas. Acquisition of invasive properties as judged by an in vitro Matrigel invasion assay also does not occur until the carcinoma stage and is further increased in metastases. Interestingly, one atypical mortal dysplasia with a considerably extended life span has lost expression of RAR-beta and p16, but it still expresses only wild-type p53 (albeit at a higher level than normal) and has not activated telomerase. RAR-beta and/or p16 re-expression can be induced by treatment with 5-aza-2-deoxycytidine (Aza-C) in some immortal dysplasias, and this has been shown to be due to silencing of gene expression by promoter methylation. Aza-C treatment also down-regulated telomerase activity and human telomerase reverse transcriptase mRNA. Interestingly, with one dysplasia, Aza-C was able to reverse its immortal phenotype, as judged by morphological criteria and expression of the senescence-associated acid beta-galactosidase activity during terminal growth arrest; this immortal dysplasia was the only one in which Aza-C treatment not only down-regulated telomerase activity but also induced re-expression of both RAR-beta and p16. The possibility of reversing the immortal phenotype of some dysplasias by Aza-C may be of clinical usefulness.