Novel detection methods for rare malignant cells in histologically benign surgical margins and lymph nodes

P80

Otolaryngology Head and Neck Surgery August 1997

ResearchForum-- Monday

patients with positive nodal status (n = 34). Strong immunoreactivity was observed in 20 patients with negative lymph node status (n = 36). Reduced expression of nm23 gene product was observed in patients with positive lymph node metastasis (p = 0.0006). Furthermore, patient survival was decreased (mortality rate, 50%) in the group with weak staining for nm23 gene product. The group with the strongest immunoreactivity for nm23 gene product had the lowest mortality rate of 19%. The nm23 gene exerts antimetastatic products in human squamous cell carcinoma of the head and neck region and perhaps contributes to the overall patient survival rate. Further investigations may identify the use of the gene in staging the aggressiveness of the tumor, as well as its use in follow-up surveillance for tumor recurrence. 9:10 AM

gical margins and lymph nodes are now prospectively collected for similar DNA analysis. The previously described techniques allow rapid and exact detection of p53 gene mutations in histologically "benign" surgical specimens. The mutations were identified by direct solid-phase sequencing of the PCR products. Conclusions: Molecular analysis of surgical margins and lymph nodes has identified patients with head and neck cancer who have a significantly greater local recurrence rate of their aerodigestive carcinoma (N Engl J Med 1995;332:42935). We now describe a novel assay that is automated, uses no radioactive labels, and can be completed within several hours. The new PCR-based procedure is thus superior to the previously described oligomer-specific hybridization technique and may eventually replace the intraoperative frozensection analysis of surgical margins and lymph nodes.

Discussion 10:40 AM 9:15 t o 10:00 AM

Posters (Session C) See pages P83 to P104 10:00 t o 10:30 AM

Award Ceremony 10:30 AM

Novel Detection Methods for Rare Malignant Cells in Histologically Benign Surgical Margins and Lymph Nodes NARENDRA P. SINGH, PhD, JOSEPH A. BRENNAN, MD (presenter), DEBRA M. NtEMEYER,PhD, KURTJ. SHULER,MD, GREGORY H, VELTRI,J. CRAIG EGAN, MD, and VITO G. DELVECCHIO, PhD, Travis Air Force Base, Calif.

Objective: Two of the most important prognostic factors for patients with head and neck squamous carcinoma (HNSC) are the complete removal of the neoplasm and the presence of metastatic disease to cervical lymph nodes. Molecular assays are currently able to detect one cancer cell among 10,000 normal cells in surgical margins and lymph nodes from patients with HNSC (N Engl J Med 1995;332:429-35). We now describe two novel techniques for detecting occult cancer cells, which are automated and require significantly less time than previously described oligomer-specific hybridization techniques. Methods: We have adapted the nonisotopic ribonuclease cleavase assay and the cleavase fragment length polymorphism for rapid detection of cells harboring p53 gene mutations in histologically benign surgical margins and lymph nodes. These new techniques are based on the polymerase chain reaction (PCR) and are exceedingly more sensitive than the standard technique of light microscopy. Results: The DNA from paraffin-embedded tissue has been isolated from approximately 100 surgical margins and lymph nodes removed from patients with HNSC. Fresh-frozen sur-

Expression of VIA (~I) Integrins in o Recurrent Pleomorphic Adenoma Cell Line MELISSA G. STEINER,PhD (presenter), SAMIEH S. RIZK,MD, WILLIAM I, KUHEL, MD, and W. SHAIN SCHLEY, MD, New York, N.Y.

lntegrins are heterodimers, comprised of tx and ~ subunits, which function in cell-cell and cell-extracellutar matrix interactions. Previous work in our laboratory has demonstrated varied expression of integrin receptor subunits in cell lines derived from pleomorphic adenoma (PA) of the parotid gland. The current study investigates the expression pattern of these same integrin subunits in a cell line derived from a recurrent pleomorphic adenoma (rPA). The rPA cell line was established from an enzymatically dissociated section of tissue obtained during surgical excision. The monolayer culture was generated and propagated in serum-free media. At confluence, the cell line was harvested by trypsinization (0.05% trypsin-EDTA); viability and cell counts were assessed, and the ceils were fixed in 100% methanol at -20 ~ C for at least 30 minutes. Immunofluorescence staining of the fixed cells was performed by utilizing antibodies to the ct2, ct3, ~5, c~V, and 91 integrin subunits and an FITC-conjugated secondary antibody; the expression of the integrin subunits was assessed by flow cytometric analysis, Mean fluorescence intensity values were normalized to an arbitrary channel of 10 for the isotype control; this assay was performed in triplicate. Comparisons among the percent of integrin§ cells or the expression levels of the integrin subunits were evaluated by using ANOVA (twoway; et = 0.05) and subsequent least-significant differences (LSD) tests to assess any specific differences between the subunits. All integrin subunits investigated were expressed in the rPA cell line to some extent. The mean percent of integrin§ cells ranged from approximately 6% to 92%, with the number of ~3 § cells being significantly greater than all other ct subunits (p < 0.0005); no other significant differences were