Franz X. Bosch

Involvement of intact HPV16 E6/E7 gene expression in head and neck cancers with unaltered p53 status and perturbed pRb cell cycle control.

We have identified parameters which define a causal role of HPV16 in head and neck cancer. Twenty-eight tumours which were typed positive for HPV16 DNA, were comprehensively analysed for expression of the viral oncogenes E6 and E7, the status of the p53 gene, and the protein status of pRb and p16INK4a. In a subset of cases, we have searched for integrated viral DNA, and have determined the genomic status of the E6 gene. Expression of E6/E7 was found in 12 tumours most of which were derived from the oropharynx, whereas p53 mutations were present in 13 tumours from various sites. The tumours either carried p53 mutations but did not express E6/E7, or they did express E6/E7 but were p53-wild-type. Coexistence of E6/E7 expression with a mutated p53 was found in only one case. Strikingly, in most p53-mutated tumours without E6/E7 expression, we found the E6 gene to be disrupted. E6/E7 expression was associated with reduced pRb and overexpressed p16INK4a. Viral-cellular fusion transcripts were found in two cases. Our data demonstrate that HPV16 DNA-positivity in head and neck cancers is not indicative of a causal role. A causal role of HPV16 in head and neck cancer is defined by: E6/E7 expression, viral integration with an intact E6 gene, and perturbation of pRb cell cycle control. Mostly, the p53 gene is wild-type.

TP53 DNA contact mutations are selectively associated with allelic loss and have a strong clinical impact in head and neck cancer

Recent studies have suggested that different mutation types within the core domain of the tumour suppressor protein p53, i.e. DNA contact mutations and structural mutations, confer different biological properties. We have analysed in 86 head and neck squamous cell carcinomas (HNSCC), whether these p53 mutation types have a differential clinical impact. Thirty-seven missense mutations were identified. Thirteen of these (36%) were DNA contact mutations, occurring in the L3 loop, in the H2 loop sheet helix motif, in the S10 β strand and in Zinc binding residues. Microsatellite marker analysis revealed a selective association between these mutations and the loss of wild-type alleles (100% LOH vs 50% LOH in tumours with structural mutations; P=0.0034, Fishers exact, 2-tailed). In comparison to structural mutations or to the absence of mutations in the core domain, DNA contact mutations were associated with higher tumour stages (84.6% vs 62%), a higher incidence of lymph node metastasis (91.7% vs 56%; P=0.014, Fishers exact, 2-tailed), a shortened recurrence-free survival (8.1 months vs 23.7 months, P=0.047, log rank test) and overall survival (11 months vs 29.2 months; P=0.003, log rank test). The latter was also the case when only stage IV tumours were analysed (P=0.0055, log rank test). These data indicate that in HNSCC, TP53 DNA contact mutations confer a strong selection pressure to eliminate wild-type alleles, and that they result in an accelerated tumour progression and reduced therapeutic responsiveness.

Two genetically defined trans-acting loci coordinately regulate overlapping sets of liver-specific genes

Mice homozygous for deletions around the albino locus fail to activate expression of a set of neonatal liver functions and die shortly after birth. This phenotype is thought to result from the loss of a positive transacting factor, denoted alf, in deletion homozygotes. Using differential cDNA screening, we isolated and characterized genes whose cell type-specific transcription is affected by alf and found as a common feature that expression of these genes is induced by glucocorticoids and cAMP. Surprisingly, a subset of these alf-responsive genes is negatively controlled by the tissue-specific extinguisher locus Tse-1. Administration of glucocorticoids and cAMP leads to reversal of Tse-1-mediated extinction of these genes. These results show that two trans-acting factors coordinately regulate expression of overlapping sets of liver-specific genes. We suggest that both the lethal phenotype and the extinguished state result from interference with hormone signal transduction.

Viral RNA patterns and high viral load reliably define oropharynx carcinomas with active HPV16 involvement.

Oropharyngeal squamous cell carcinomas (OPSCC) that are associated with human papilloma virus (HPV) infection carry a more favorable prognosis than those that are HPV-negative. However, it remains unclear which biomarker(s) can reliably determine which OPSCC specimens are truly driven by HPV infection. In this study, we analyzed 199 fresh-frozen OPSCC specimens for HPV DNA, viral load, RNA expression patterns typical for cervical carcinomas (CxCaRNA(+)), and the HPV-targeted tumor suppressor protein p16(INK4a) as markers for HPV infection. In this set of specimens, there was a 49% prevalence of DNA for the cancer-associated HPV type 16 (HPV(+)). However, there was only a 16% prevalence of high viral load and only a 20% prevalence of CxCaRNA(+), a marker of HPV16 carcinogenic activity. Among the CxCaRNA(+) tumors, 78% of the specimens exhibited overexpression of p16(INK4a), which also occurred in 14% of the HPV-negative tumors. Using a multivariate survival analysis with HPV negativity as the reference group, CxCaRNA(+) as a single marker conferred the lowest risk of death [HR = 0.28, 95% confidence interval (CI), 0.13-0.61] from oropharyngeal cancer, closely followed by high viral load (HR = 0.32, 95% CI, 0.14-0.73). In contrast, a weaker inverse association was found for OPSCC that were HPV(+) and p16(INK4a) high (HR = 0.55, 95% CI, 0.29-1.08). In summary, our findings argued that viral load or RNA pattern analysis is better suited than p16(INK4a) expression to identify HPV16-driven tumors in OPSCC patient populations.

Aberrant p21CIP1/WAF1 protein accumulation in head‐and‐neck cancer

p21CIP1/WAF1 is an inhibitor of cyclin‐dependent kinases and, in normal tissues including squamous epithelia, has been associated with cell‐cycle exit and differentiation. As shown in this pilot study, however, the majority of head‐and‐neck squamous‐cell carcinomas (HNSCC) display aberrant p21CIP1/WAF1 expression: of 42 tumors analyzed by immunohistochemical staining, 28 (67%) over‐expressed the p21CIP1/WAF1 protein. Accumulation of p21CIP1/WAF1 was independent of the histological grade of the tumors as well as the genetic status of the p53 gene. In many cases, most notably in poorly differ‐ entiated or undifferentiated HNSCC, p21CIP1/WAF1‐positive cells were actively proliferating tumor cells, since they also expressed proliferating‐cell nuclear antigen (PCNA) and Ki‐67. Accumulation of p21CIP1/WAF1 occurred through a post‐transcriptional mechanism since, in contrast to immunohistochemical analysis of the p21CIP1/WAF1 protein, in situ hybrid‐ ization showed no increase of mRNA levels as compared with cells in normal mucosa (n = 25). Clinically, among the patients with p21CIP1/WAF1‐over‐expressing tumors, there was increased recurring disease (p = 0.03; χ2‐test), shortened disease‐free survival (p = 0.0019; log‐rank test) and shortened overall survival (p = 0.0071; log‐rank test). These in vivo data indicate that in many HNSCC, accumulated p21CIP1/WAF1 is compatible with increased tumor‐cell proliferation, and they provide preliminary evidence that p21CIP1/WAF1 may be of prognostic and predictive significance. Int. J. Cancer 74:383–389, 1997.

Proteomic analysis reveals successive aberrations in protein expression from healthy mucosa to invasive head and neck cancer

Development of head and neck squamous cell carcinoma (HNSCC) is a multistep process and in many cases involves a phenomenon coined ‘field cancerization’. In order to identify changes in protein expression occurring at different stages of tumorigenesis and field cancerization, we analysed 113 HNSCCs and 73 healthy, 99 tumor-distant and 18 tumor-adjacent squamous mucosae by SELDI-TOF-MS on IMAC30 ProteinChip Arrays. Forty-eight protein peaks were differentially expressed between healthy mucosa and HNSCC. Calgizarrin (S100A11), the Cystein proteinase inhibitor Cystatin A, Acyl-CoA-binding protein, Stratifin (14-3-3 sigma), Histone H4, α- and β-Hemoglobin, a C-terminal fragment of β-hemoglobin and the α-defensins 1–3 were identified by mass spectrometry. The α-defensins showed various alterations in expression as validated by immunohistochemistry (IHC). Supervised prediction analysis revealed excellent classification of healthy mucosa (94.5% correctly classified) and tumor samples (92.9% correctly classified). Application of this classifier to the tumor-adjacent and tumor-distant mucosa samples disclosed dramatic changes: only 59.6% of the tumor-distant biopsies were classified as normal, 27.3% were predicted as aberrant or HNSCC. Strikingly, 72% of the tumor-adjacent mucosae were predicted as aberrant. These data provide evidence for the existence of genetically altered fields with inconspicuous histology. Comparison of the protein profiles in the tumor-distant-samples with clinical outcome of 32 patients revealed a significant association between aberrant profiles with tumor relapse events (P=0.018; Fishers exact test, two-tailed). We conclude that proteomic profiling in conjunction with protein identification greatly outperforms histopathological diagnosis and may have significant predictive power for clinical outcome and personalized risk assessment.

Extensive changes in cytokeratin expression patterns in pathologically affected human gingiva

SummaryThe stratified squamous epithelium of the oral gingiva and the hard palate is characterized by a tissue architecture and a cytoskeletal composition similar to, although not identical with, that of the epidermis and fundamentally different from that of the adjacent non-masticatory oral mucosa. Using immunocytochemistry with antibodies specific for individual cytokeratins, in situ hybridization and Northern blots of RNA with riboprobes specific for individual cytokeratin mRNAs, and gel electrophoresis of cytoskeletal proteins of microdissected biopsy tissue samples, we show changes in the pattern of expression of cytokeratins and their corresponding mRNAs in pathologically altered oral gingiva. Besides a frequently, although not consistently, observed increase in the number of cells producing cytokeratins 4 and 13 (which are normally found as abundant components in the sulcular epithelium and the alveolar mucosa but not in the oral gingiva) and a reduction in the number of cells producing cytokeratins 1, 10 and 11, the most extensive change was noted for cytokeratin 19, a frequent cytokeratin in diverse one-layered and complex epithelia. While in normal oral gingiva cytokeratin 19 is restricted to certain, sparsely scattered cells of- or near — the basal cell layer, probably neuroendocrine (Merkel) cells, in altered tissue of inflamed samples it can appear in larger regions of the basal cell layer(s) and, in apparently more advanced stages, also in a variable number of suprabasal cells. Specifically, our in situ hybridization experiments show that this altered suprabasal cytokeratin 19 expression is more extended at the mRNA than at the protein level, indicating that cytokeratin 19 mRNA synthesis may be a relatively early event during the alteration. These changes in cytokeratin expression under an external pathological influence are discussed in relation to other factors known to contribute to the expression of certain cytokeratins and with respect to changes occurring during dysplasia and malignant transformation of oral epithelia.

Head and neck tumor sites differ in prevalence and spectrum of p53 alterations but these have limited prognostic value.

The tumor site is a strong clinical factor in head and neck squamous cell carcinoma (HNSCC). To clarify the biologic and clinical role of p53 alterations in HNSCC, we have examined the prevalence and the nature of p53 alterations in a large cohort of tumors from the different sites. For immunohistochemical analysis of p53 protein expression, we introduced tyramide signal amplification immunohistochemistry (TSA‐IHC) on a tissue microarray. This allowed the discrimination between normal low‐level expression and reduced or lost expression. Two hundred fifty‐three tumors were subjected to mutational analysis by genomic DNA sequencing, employing also the p53 GeneChip from Affymetrix. The prevalence of all p53 alterations, i.e., mutations, overexpression and loss of expression, was significantly higher in hypopharyngeal tumors than in the other sites (p = 0.001). Laryngeal tumors showed the lowest rate of p53 alterations, but revealed a distinct mutation spectrum: most mutations affected exon 5 (p = 0.013) and the S2′ domain (p = 0.002), and most hot‐spot 248 mutations occurred in the larynx (p < 0.001). Sequencing by p53GeneChip technology was shown to be only insignificantly more sensitive than dideoxy sequencing. In agreement with p53 mutations occurring prior to invasiveness, their prevalence did not increase with tumor stage, and all mutation classes lacked prognostic significance. The large patient cohort of this study showed that p53 is differentially affected in the different tumor sites of the head and neck, but its mode of inactivation does not play a major role in tumor progression.

Biological evidence for a causal role of HPV16 in a small fraction of laryngeal squamous cell carcinoma

Background:Human papillomavirus (HPV) is a causal factor in virtually all cervical and a subset of oropharyngeal squamous cell carcinoma (OP-SCC), whereas its role in laryngeal squamous cell carcinoma (L-SCC) is unclear.Methods:Formalin-fixed paraffin-embedded (N=154) and deep-frozen tissues (N=55) of 102 L-SCC patients were analysed for the presence of 51 mucosal HPV types. HPV DNA-positive (HPV DNA+) cases were analysed for E6*I mRNA transcripts of all high risk (HR)/probably/possibly (p)HR-HPV identified, and for HPV type 16 (HPV16) viral load. Expression of p16INK4a, pRb, cyclin D1 and p53 was analysed by immunohistochemistry.Results:Ninety-two patients were valid in DNA analysis, of which 32 (35%) had at least one HPV DNA+ sample. Among the 29 single infections, 22 (76%) were HPV16, 2 (7%) HPV56 and 1 each (4%) HPV45, HPV53, HPV70, HPV11 and HPV42. Three cases harboured HPV16 with HPV33 (twice) or HPV45. Only 32% of HPV DNA+ findings were reproducible. Among HPV16 DNA+ L-SCC, 2 out of 23 (9%) had high viral loads, 5 out of 25 (21%) expressed E6*I mRNA and 3 out of 21 (14%) showed high p16INK4a and low pRb expression (all three HPV16 RNA-positive), immunohistochemical marker combination not identified in any other HPV DNA+ or HPV DNA-negative (HPV DNA−) L-SCC, respectively.Conclusion:HPV type 16 has a causative role in a small subgroup of L-SCC (<5% in this German hospital series).

Evidence that wild-type p53 in neuroblastoma cells is in a conformation refractory to integration into the transcriptional complex

Neuroblastoma (NB) cells reportedly accumulate wild-type p53 exclusively in the cytoplasm. However, immunofluorescence assays with five different antibodies showed that p53 accumulates in the nucleus of up to 10% of NB cells. PAb1801 detected cytoplasmic ‘punctate structures’ which were also found in p53-null cells, rendering this antibody unsuitable for p53 detection. A comparison of DO-1 and PAb1801 staining in NB tissue sections confirmed the results obtained with NB cells. Nuclear accumulation of p53 was induced in NB cells using substances which disturb p53s tertiary structure at its zinc finger motif, or by treatment with mitomycin C. Constitutive nuclear accumulation was observed in an SK-N-SH variant, AW-1, which has a point mutation in p53 at Cys176>Ser, disturbing the same motif. Even though p53 showed DNA-binding capability after mitomycin C treatment of NB cells, the target gene products MDM2 and p21WAF1,CIP1,SDI1 were not synthesized and no p53 transactivating activity measured in a reporter gene assay. Therefore we suggest that p53 in NB cells might be predominantly in a conformation refractory to integration into the transcriptional complex, resulting in at least partial transcriptional inactivity, hyperactive nuclear export and resistance to degradation by exogenously expressed MDM2.