Christiane Motsch

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.

PEG-Anlage bei Patienten mit HNO-Tumoren

ZusammenfassungDie Hälfte aller Tumorpatienten ist bereits zum Diagnosezeitpunkt mangelernährt. Als wesentliche Ursache der Malnutrition ist beim HNO-Karzinompatienten die verminderte bzw. unzureichende Energie- und Nährstoffaufnahme, bedingt durch Schmerzen beim Schlucken und durch Stenosierung der oberen Schluckstraße, anzusehen. Die über eine perkutan endoskopisch kontrollierte Gastrostomie (PEG) applizierte Ernährungssonde stellt zunehmend die Alternative zur nasogastralen Sonde dar, um die enterale Ernährung bei Stenosierung der oberen Schluckstraße aufrechtzuerhalten. Im Gegensatz zur in Deutschland am häufigsten ausgeführten Fadendurchzugsmethode haben wir uns an der Universitäts-HNO-Klinik Magdeburg zugunsten der PEG im Direktpunktionsverfahren entschieden. Zur Anwendung kam das Memosond-Direktpunktionsset (Fa. Pfrimmer Nutricia). Die Nährsonde wird dabei durch eine thermisch aktivierte Memory-Rückhaltespirale in situ gehalten. Von 1991–1996 wurden 415 Patienten mit einem fortgeschrittenen Karzinom der Mundhöhle, des Oro-/Hypopharynx, der Supraglottis und des Ösophagus mit einer PEG versorgt. Eine Dislokation der Sonde in die freie Bauchhöhle – eine schwere Komplikation, die im Schrifttum insbesondere dem PEG-Direktpunktionsverfahren angelastet wird – konnte nicht beobachtet werden. Ein methodenbedingter letaler Ausgang war nicht zu verzeichnen. Wegen Peritonitis in der Initialphase wurden 3 Patienten laparotomiert. Bei 160 der PEG-versorgten Patienten wurden Parameter zum Ernährungs-, Protein- und Immunstatus sowie die Compliance der Patienten zur PEG und Ernährungstherapie ermittelt. Ziel dieser Analyse war es, die Notwendigkeit einer konsequenten Ernährungstherapie im Gesamtbehandlungskonzept von HNO-Karzinompatienten zu belegen. Die PEG im Direktpunktionsverfahren erscheint bei fortgeschrittenen Karzinomen der oberen Schluckstraße sinnvoller, da mechanische Alterationen entlang der tumorösen Veränderungen bei nur einmaliger Gastroskopie wesentlich geringer sind, eine bakterielle Kontamination aus der Tumorregion ins Abdomen sowie eine iatrogene Verschleppung von Tumorzellen entlang des Fadens und der inneren Halteplatte mit Implantation in die Magenwand und dem damit verbundenen Risiko einer Metastasierung in den Magen oder in die Bauchwand vermieden wird.SummaryOral food intake in patients with obstructing pharyngeal and esophageal carcinomas is commonly insufficient because of tumor-induced dysphagia which gives rise to cachexia unless treated. While entailing an unfavorable prognosis, malnutrition is often a therapy-limiting factor. Tube feeding with liquid formula diets currently offers the most efficient and least-risky approach to long-term use and is best adopted even at a pre-treatment stage irrespective of the tumor therapy intended. A feeding tube placed by a percutaneous endoscopically controlled gastrostomy (PEG) increasingly offers an alternative to a nasogastric tube. After using diaphanoscopy, the stomach is punctured from outside under local anesthesia and a feeding tube inserted by means of a retrograde thread or a direct puncture method. A modification of the direct puncture method has been preferred at the Magdeburg University E.N.T. Department. The tube is held in place by thermally activated helical winding of a gastic tube end (using a memory-retaining helix). During the 1991–1996 period 415 patients with obstructing carcinomas of the upper digestive tract were treated with a feeding tube. No fatal complications were observed. Severe complications (peritonitis) occurred in three patients. In 160 patients with PEG the following parameters were recorded: weight-to-size index, body mass index, degree of dysphagia, nutrition status, lymphocyte count, total serum protein and patients’ compliance to PEG. The enteral nutrition therapy used was indicated in all of the patients treated with advanced carcinomas of the head and neck. In 81% of the patients the compliance to PEG was positive. Findings demonstrated that long-term intestinal nutrition via PEG was a safe and effective form of treatment. Inserting the tube by the direct puncture method was advantageous for patients with carcinomas in the upper digestive tract as only few mechanical alterations take place along tumorous tissues following PEG while contamination with bacteria and neoplastic cells from the tumor region into the abdomen are precluded.

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.