Bernd Freigang

Retention of the Arginine Allele in Codon 72 of the p53 Gene Correlates with Poor Apoptosis in Head and Neck Cancer

The allele constitution at codon 72 of the p53 gene (CGC-arginine or CCC-proline) plays a major role in inducing apoptosis in p53 mutant cells. To verify this, we determined GC-status, p53-mutations, and p53-loss of heterozygosity (LOH) in a group of 54 squamous cell carcinomas of the head and neck (SCCHN). A novel approach, using a one-step real-time PCR analysis with fluorescent hybridization probes, was applied to detect the GC status in tumors and corresponding blood samples. p53 mutations in exons 4 to 8 were detected by PCR-SSCP-sequencing analysis. Apoptosis was determined immunohistochemically using antibodies against Fas, FasL, p53, Bcl2, and terminal deoxy-transferase-mediated dUTP nick end labeling (TUNEL) staining. The overall frequency of p53-LOH in SCCHN was 45.2%. In cases of LOH, there was a preferential loss of the proline allele, which was associated with an up-regulation of Bcl2 and lack of co-expression of Fas/FasL and, thus, impaired apoptosis (P < 0.001). Apoptosis was not observed in tumors carrying the arginine allele. p53 mutations were detected in 29.6% of SCCHN and preferentially occurred at the arginine allele (P = 0.01). p53 alterations were more frequently observed in tumors of the oral cavity, oropharynx and hypopharynx, whereas they were rare in larynx carcinomas (P = 0.07). The p53-LOH status was not found to be significantly correlated with sex, age, TNM-status, or tumor grading. We conclude that apoptosis is correlated with the allelic status of codon 72 in SCCHN. Homozygous proline 72 appears to be an important regulator of apoptosis via the Fas/FasL pathway in SCCHN.

Prognostic value of MMP-2, -9 and TIMP-1,-2 immunoreactive protein at the invasive front in advanced head and neck squamous cell carcinomas.

In head and neck cancer as well as in other carcinomas, tumor expansion and spread to distant sites require the secretion of destructive enzymes that degrade the extracellular matrix. A variety of proteases contribute to matrix destruction. Characteristics of the invasive tumor front may reflect tumor prognosis better than do other parts of the tumor. Therefore, it was the aim of the present study to (i) compare central and peripheral tumor zones for differences in the expression of matrix-metalloproteinases (MMP) -2 and -9 and their naturally occurring inhibitors (tissue inhibitor of matrix-metalloproteinases (TIMP) -1 and -2), (ii) examine the morphological potential of malignancy, and (iii) correlate these findings with clinicopathological parameters. The study population consisted of 106 surgical specimens of advanced head and neck squamous cell carcinomas. The invasive front was graded for malignancy, and immunohistochemical staining with MMP-2, MMP-9, TIMP-1 and TIMP-2 antibodies was performed. Both MMP-2 and MMP-9 were found to be significantly overexpressed at the tumor front. The MMP-2-positive invasive front exhibited diminished overall survival times. In multivariate analysis, MMP-2 expression retained its correlation with overall survival in addition to nodal status and total malignancy score. Expression of TIMP-2 correlated with local tumor invasion. We conclude that the expression of MMP-2 at the invasive front is a marker of poor survival and appears to be associated with early recurrence in initially lymph node-negative patients.

Hereditary p16-Leiden Mutation in a Patient with Multiple Head and Neck Tumors

To the Editor: Recent studies have shown that most Dutch families with atypical multiple-mole melanoma (FAMMM) have a 19-bp germline deletion (p16-Leiden) in exon 2 of the p16 gene (p16 [MIM 600160]) (Gruis et al. 1995; van der Velden et al. 1999, 2001). Incomplete penetrance and variable clinical expression in p16-Leiden carriers point to the fact that other genetic mechanisms can compensate for the p16 loss of function (Gruis et al. 1995). Indeed, van der Velden et al. (2001) reported in the Journal that variants in the melanocortin-1 receptor modify the risk of melanoma in p16-Leiden carriers. It is interesting that, apart from reports on simultaneous pancreatic tumors, other cancer types have never been found in such families with p16-Leiden (Gruis et al. 1995). Recently, we found a hereditary heterozygous p16-Leiden mutation in a man who neither smoked more than five cigarettes daily nor abused alcohol, initially diagnosed at age 54 years, who simultaneously developed three carcinomas of the pharynx and oral cavity. The patient showed a familial accumulation of tumor diseases. Both of his parents and his only sister died of cancer very early (the mother of gynecologic cancer, the father of liver carcinoma, and the sister of leukemia). Dutch relatives are not known. DNA from tumors and blood was extracted according to a standard protocol (Sambrook et al. 1989). Mutation analysis of exon 1 and exon 2 of the p16 gene was done by PCR-SSCP sequencing as described by Schneider-Stock et al. (1998). The p16-Leiden mutation was found in the heterozygous constitution in all three tumors and in the blood of the patient. We investigated all DNA samples for p16 promotor methylation to check the status of the retained wild-type allele and, thus, to assess the real functional significance of this finding. The methylation status of the p16 promotor was determined by methylation-specific PCR (Herman et al. 1996). The primer sequences for the unmethylated reactions were (sense) 5′-TTA TTA GAG GGT GGG GTG GAT TGT-3′ and (antisense) 5′-CAA CCC CAA ACC ACA ACC ATA A-3′, which amplify a 151-bp product. The primer sequences for the methylated reaction were (sense) 5′-TTA TTA GAG GGT GCG GAT CGC-3′ and (antisense) 5′-GAC CCC GAA CCG CGA CCG TAA-3′, which amplify a 150-bp product. The two forward primers were labeled with FAM dye. PCR was done using 1/10 of bisulfite reaction and a MasterAmp Optimization Kit (Biozym) in an automated thermocycler (PTC 200) according to the manufacturers instructions. PCR products were analyzed using an ABI310 sequencer (injection time between 20–30 s, Pop 4, dRhodamine Matrix standard ABI Prism [Perkin Elmer]). The p16 promotor was methylated in all three tumor samples but not in the blood of the patient (fig. 1). Figure 1 Analysis of p16INK4 promotor methylation on ABI310 Prism. Methylated (left arrow) and unmethylated (right arrow) signals can be detected between the 150-bp and the 160-bp ROX matrix standards. There is a near-equal amplification of unmethylated and methylated ... This result led us to suggest a loss of p16 protein expression and, thus, a complete loss of p16 tumor suppressor function. For p16 immunohistochemistry, a monoclonal mouse antibody to p16 (1:100 dilution, Quartett) was used according to the manufacturers instructions. Indeed, all tumor sections were immunohistochemically p16-negative. The third tumor showed an additional gross rearrangement in the p53 gene (75-bp insertion in exon 4). This is the first report on p16-Leiden mutation in orolaryngeal carcinomas, although p16 alterations are very common in this tumor type (50% p16 promotor methylation [Bittencourt Rosas et al. 2001], 30% p16 mutations [Esteller et al. 2001a; Poi et al. 2001]). Because of the heterozygous p16-Leiden constitution, our proband was a suitable model for studying the type of p16 inactivation in the three metachronous carcinomas. To date, the methylation status of p16-Leiden carriers has never been checked. To the best of our knowledge, this is one of the only a few clear examples that aberrant promotor methylation might function as the “second genetic hit” in a familial cancer syndrome. Whereas no methylation could be recognized in the blood of the patients DNA, all three tumors showed inactivation of the retained wild-type allele, with the somatic event being aberrant promotor methylation. There are only a few reports demonstrating the role of promotor methylation for biallelic gene inactivation (Grady et al. 2000; Esteller et al. 2001b). Furthermore, it has to be mentioned that the p16-Leiden mutation should also affect the p14ARF transcript, because alternative splicing of the first exon and common downstream exons permit this locus to encode two different products that regulate the cell cycle via two distinct pathways. It is noteworthy that contact with the patient began five months before the actual date of examination when the patient attended a talk. His presence was not because of symptoms of a tumor but because of genetic findings. This early meeting led to discovery of the tumor of the tongue while the tumor was still in a less-advanced stage. Therefore, the tumor could be easily resected, leaving the tongue almost unaffected. Consequently, the patient is now in good physical condition (Karnofsky 70%–80%). The patient was informed about the hereditary mutation and was included in the risk program for upper-abdominal screening because p16-Leiden mutation has been reported to be a risk factor for developing pancreatic carcinoma (Lal et al. 2000; Vasen et al. 2000; Lynch et al. 2002). We think that regarding head and neck cancer, our data is novel and may have consequences for further studies of families with FAMMM.

Fasciitis ossificans of the paranasal sinus.

We report a case of fasciitis ossificans of the nasal cavity and paranasal sinus in an infant who was surgically treated in our department. Fasciitis ossificans is a rare benign reactive lesion and a variant of the more commonly known entity nodular fasciitis. We present the radiographic appearance and discuss the surgical resection, which was performed by a paranasal section, as well as the histologic and immunohistochemical results. To the best of our knowledge, no other case of fasciitis ossificans of the paranasal sinus has yet been reported. It is important to publish cases such as this, because their recognition as benign entities can prevent aggressive surgical procedures.

Rolle des Operationszeitpunktes bei rhinobasaler Fraktur und Komplikationsraten

In einer retrospektiven Studie wurden 148 Patienten, die von 1993 bis 1996 wegen einer rhinobasalen Fraktur Uber einen subkranialen Zugang operiert wurden, hinsichtlich der Beziehung von entzUndlich bedingten endokraniellen Komplikationen zum Operationszeitpunkt untersucht. Bei 44% der Verletzten war die Rhinobasisfraktur mit einer Mittelgesichtsfraktur kombiniert. Intraoperativ Hess sich in drei Viertel der FAlle eine DuralAsion nachweisen. Die operative Versorgung erfolgte bei 71 Patienten am Unfalltag, bei 24 Patienten am 1. Tag nach dem Trauma, bei 28 Patienten am 2.–5. Tag nach dem Trauma, bei 20 Patienten zwischen dem 5. Tag und der 3. Woche nach dem Trauma, bei 5 Patienten ab 3. Monat nach dem Trauma. Schwerste perforierende Gesichtsweichteilverletzungen (n = 18) sowie massive Blutungen aus Nase und Nasenrachen (n = 6) bei bestehender kombinierter Mittelgesichts-Rhinobasis-Fraktur zwangen in 16% der FAlle zum sofortigen operativen Eingreifen. Eine HAufung von FrUhkomplikationen nach rhinobasaler Fraktur war in der Patientengruppe zu verzeichnen, die zwischen dem 5. Tag und der 3. Woche nach dem Trauma definitiv versorgt wurde (FrUhmeningitis n = 4, beginnende Sepsis n = 3). Bei 5 Patienten, bei denen das Zeitintervall zwischen Unfallereignis und Operation 3 Monate bis 22 Jahre betrug, handelte es sich ausschliesslich um schwere endokranielle Komplikationen (SpAtmeningitis, Subduralabszess, Frontalhirnabszess, schUttende Rhinoliquorrhoe, Sinus-cavernosus-Fistel zur Arteria carotis interna). Zusammenfassend kann man sagen, dass die One-stage-Versorgung bei kombinierten Mittelgesichts-Rhinobasis-Frakturen Uber einen subkranialen Zugang innerhalb der ersten 48 h die besten Asthetischen und funtionellen Resultate bietet. Das Risiko einer ent-zUndlichen endokraniellen Komplikation ist bei FrUhversorgung ebenfalls als sehr gering einzuschAtzen.