J. D. Maass

p53 serum antibodies as prognostic indicator in head and neck cancer

Abstract p53 antibodies are a new serological parameter of unknown potential in patients with malignancies. Their occurrence has been described in various types of cancer patients. The mechanism underlying the immunization process is still unclear. We investigated the incidence of p53 serum antibodies in 143 head and neck cancer patients with an enzyme-linked immunosorbent assay. The post-therapy course of two matched study groups (n = 38 each), one p53-antibody-seropositive and one p53-antibody-seronegative, was followed up for 24 months. Thirty-nine head and neck cancer patients (27.3%) were seropositive for p53 antibodies. During the follow-up, the p53-antibody-seropositive patients accounted for more local tumor recurrences (n = 12 versus n = 8) and more tumor-related deaths (n = 11 versus n = 5) than did seronegative patients, and second primary tumors (n = 9 versus n = 0) occurred exclusively in seropositive patients. In total, therapy failures (recurrences, tumor-related deaths, second primaries) were observed in 17/38 cases (44.7%) in the p53-antibody-seropositive group and in 8/38 cases (21.1%) in the p53-antibody-seronegative group. These results, after a follow-up of 2 years, seem to indicate a prognostic value of p53 serum antibodies for therapy failure in patients with head and neck cancer.

Serum p53 Autoantibodies in the Follow-Up of Head and Neck Cancer Patients

p53 autoantibodies (AAB) are a fairly new serological parameter in patients with malignancies. Although the actual mechanism of how they develop is still unclear, it seems that these AAB could be of prognostic relevance. Very few studies demonstrated the usefulness of p53 AAB in the follow-up of cancer patients. In this study, 109 patients with head and neck cancer were investigated using an ELISA for the presence of p53 AAB in their serum and were followed-up for at least 36 months. In 21 of the cancer patients, p53 serum AAB were detected. In 5/21 p53-seropositive AAB patients, a correlation with the clinical course was observed. Sixteen of the p53-positive patients did not show any significant AAB titer changes during the follow-up, and no significant correlation with the clinical course was seen. According to these results, the clinical value of p53 AAB in the follow-up of patients with head and neck cancer seems to be limited.

Transcriptional repression of the human galactocerebrosidase gene in squamous cell carcinomas of the larynx

Alterations of gene expression in squamous cell carcinoma (SCC) cell lines derived from the larynx and keratinocytes derived from adjacent normal mucosa of the larynx have been studied using the mRNA differential display technique. Lane‐to‐lane comparison of reverse transcribed mRNA showed a strong repression of a 148 bp fragment in SCC cells. The fragment was reamplified and cloned. Sequencing revealed a 99.3% homology with a region in exon 17 of the human galactocerebrosidase (GALC) gene. Northern blot analysis confirmed the differential expression of this gene in both carcinoma cell lines and laryngeal SCC biopsies in contrast with corresponding normal mucosa. To provide further evidence for the differential expression rate, both types of cells were transiently transfected with a 152 bp (−176 to −24) high regulatory promoter element of the 5′ flanking region of the GALC gene. Results of 3 independent transfection experiments indicated a 16‐fold repression of the GALC gene expression in SCC cells compared with benign keratinocytes. However, neither mutation nor other alterations of the promoter sequence were detected. Expression of the GALC gene is thus greatly affected in SCCs of the larynx. Int. J. Cancer 83:750–754, 1999.

Antibodies to p53 may predict a high risk of treatment failure in head and neck cancer.

Overexpression of p53 as well as the occurrence of serum p53 antibodieshave been correlated to shorter survival and relapse-free intervals in head and neck squamous cell carcinoma (HNSCC). The aim of this study was to reconsider the clinical implications of serum p53 antibodies in HNSCC. Two matched study groups (n=78 patients with HNSCC each), one group positive and one negative for serum p53 antibodies, were followed-up (median of 3 years) regarding the occurrence of relapses, second primaries and tumor related deaths. Tumor relapses (n= 27 vs. 12), second primaries (n= 76 vs. 4) and tumor related deaths (n=21 vs. 9) occurred more frequently in the seropositive group. The results of this study indicate that the presence of serum p53 antibodies in patients with HNSCC points out a group of patients, who have a high risk of treatment failure.

Detection of human papillomavirus DNA sequences in squamous cell carcinomas of the head and neck

Soft tissue sarcomas of the extremities contribute to only 0.9% of all neoplasms. Even in experienced hands radical surgical resection is followed by a recurrency rate of 7 to 35%. The reasons for this high percentage of recurrent disease after radical surgery are not yet clear. Since angiogenesis is a key factor in tumor growth we have investigated concentrations of the angiogenic peptide bFGF in systemic blood and soft tissue sarcomas venous blood of 33 consecutive patients undergoing radical tumor resection: liposarcomas (6), malignant fibrous histiozytoma (6), fibrosarcomas (6), clear cell sarcomas (2), leiomyosarcomas (2), others (6). Systemic venous blood was obtained preoperatively, during surgery, lhour, I day and 1 week after operation. 10 cc of venous blood was collected from tumor veins during surgery. Samples wzre centifuged and stored at minus 20*Celsius. bFGF levels were measured with a monoclonal sandwich EL1SA (R&D Systems, USA). All samples were analyzed in duplicates on a Dynatech | ELISA plate reader. Preoperative bFGF values (mean• in serum were slightly but non-significantly elevated compared to normal subjects (6.4• pg/mll). Serum concentrations declined during surgery (4.6• pg/ml), and one hour (3.6-~.47 pg/ml) and one day post surgery (3.4• pg/ml, p=.046 compared to preoperative serum levels). An increase of bFGF in serum was found again after one week (5.1• pg/ml). Concentrations of bFGF determined in tumor blood were significantly higher than serum levels (190• pg/ml) but significantly lower (p=.003) than controls obtained from large soft tissue wounds (e.g. musculocutaneous flap donor sites; 586+227 pg/ml). Although no pathological bFGF serum levels could be detected in serum of soft tissue sarcoma patients, high bFGF concentrations were found in tumor blood. The fact that non-tumor soft tissue wounds expressed even higher bFGF levels suggests that a marked angiogenic potential is located within the wound bed which might promote tumor growth and development of recurrencies. The data support the importance of radical local tumor resection and anti-angiogenic treatment modalities.

More accurate P53 detection possible with advanced kind of tissue homogenisation

N~lans exposed to ethylene oxide vary widely in their susceptibility for ONA damage in white blood cells. A polynorphism in glutathi0ne transferase T1 was discussed to be responsible for the interiedividual differences. In tlle present study both, isolated white blood cells suspended in PM end totul venous blood from 33 donors were incubated with ethylens oxide in vitro. Both incubation procedures resulted in a statistically significant increase in DNA single strand breaks (ssb) in ~hite blood cells detected by alkaline filter elation. Interindividual differences were relatively small for incubation of white blood cells (coefficient of variation: 25 %1 and relatively large if venous blood was incubated (coefficient of variation: 53 t). These results suggest that the observed interiedividusl differences in htmans ere predominantly due to protective factors in the erythrocytes or the serum surrounding the white blood cells rather than in the white blood cells themselves. To differentiate between protective effects of erythrocytes and serum, white blood cells of I0 donors were isolated, suspended in the erythrocyteor senm-fractiun of respective donors or in PBS and ssb were detected after incubation with 0.4 ~ ethylene oxide for i h. An increase in the mean elutiom rates (which linearly correlate with the number of seb)of 0.16 _+ 0.024 (standard error) was induced by imenbatlon in PBS, 0.094 _+ 0.018 in erythroeytes, 0.063 • 0.018 in sertm and 0.066 .+ 0.013 if total venous blood was incubated. These results show that in both, sen= and erytltrocytes, protective factors against ethylens oxide =ere present. Those extraeelluler serum factors were at least as effective in protecting DNA against ethylene oxide induced damage as imtrasellular factors, e.g. glutathione transferese Tl in erythrecytes. Additionally we have genctyped 45 individuals for glutsthione transferase TI (GSTTI). Our results showed that the pulymorphism in GSTTI cannot satisfactorily explain the interindividuul differences in DNA damage. A 2 7

P53 gene polymorphisms occur in squamous cell carcinomas of the upper aerodigestive tract

In recent years the incident of aggressive nonmelanoma skin cancer is increasing and epidemiological data indicate exposure of UV-irradiati0n as a major risk factor caused by more extensive outdoor activities and/or stratospheric ozone depletion. Molecular evidence comes from sequence data of the p53 tumor suppressor gene. Approximately 80% of the nonmelanoma skin cancers contain mutations (C to T transitions in CC sites) indicative for UV-irradiation. In addition to a small fraction of skin carcinomas carrying the ras-oncogene, also chromosome #9 is postulated to be involved. From studies with our experimental in vitro skin carcinogenesis model HaCaT we postulate that in combination with mutational inactivation of the p53 gene functional loss of gene(s) on chromosome 3p are an early marker for skin cancer. As a late event in tumor progression since associated with malignancy we postulate loss of material from chromosome 15. For both alterations comparative genomic hybridization and fluorescence in situ hybridization studies provided evidence that they are also frequent in human skin cancer lines. Furthermore, functional studies by reintroducing a copy of the underrepresented chromosome suggest a distinct role in skin cancer in man and now allows to study mechanisms involved in tumor suppression in general. With a similar approach we also investigate at present genetic aberrations in UV-associated Merkel cell tumors which so far are genetically even less well understood as skin carcinomas.