Emanuela Galeazzi

Staging of head and neck squamous cell carcinoma using the MET oncogene product as marker of tumor cells in lymph node metastases.

In head and neck squamous cell carcinomas (HNSCC), metastasis to cervical lymph nodes is a major determinant of patient outcome. To detect metastases, we used the MET oncogene as marker, which encodes the receptor for hepatocyte growth factor/scatter factor, mediating epithelial cell motility and invasiveness. The MET gene is expressed in epithelia and over‐expressed in carcinomas of specific histotypes, but not in lymphatic tissue. A total of 151 lymph nodes from 20 squamous cell carcinomas were studied with both in‐depth histology and end‐point and real‐time quantitative RT‐PCR. MET‐encoded sequences were found in 61 of 151 nodes (40%), of which 24 (16%) were found metastatic by in‐depth histopathology. Parallel routine histopathologic analysis of 654 lymph nodes from the same cases identified 36 metastases (5%). Real‐time quantitative RT‐PCR was used to measure MET gene‐specific mRNA in normal tissues, primary tumors and lymphatic metastases and showed a 2–8‐fold increased expression in tumor cells which metastasize. RT‐PCR for 3 cytokeratins expressed in HNSCC (K4, K10 and K13) proved to be less sensitive in detecting occult lymphatic metastases. Western blot analysis demonstrated the presence of the full‐size MET receptor in primary tumors and lymph node metastases; immunohistochemistry showed receptor localization in tumor cells. Altogether, these data demonstrate that the MET gene product is a valuable marker with which to detect occult tumor cells in lymph nodes, thanks to its high expression in metastatic cells. After RT‐PCR analysis we were able to attribute a more advanced stage to 10 out of 20 HNSCC cases, including 5 cases classified as tumor‐free after routine histopathology. Int. J. Cancer 89:286–292, 2000.

Temporary regression of recurrent squamous cell carcinoma of the head and neck is achieved with a low but not with a high dose of recombinant interleukin 2 injected perilymphatically

The efficacy of ten daily injections of 500 or 500,000 U of recombinant interleukin 2 (IL-2) day-1 given 1.5 cm from the insertion of the sternocleidomastoid muscle on the mastoid was evaluated in 31 patients with recurrent head and neck squamous cell carcinoma. No toxic effects were noted. One complete response (CR) and three partial responses (PRs) were observed in the 16 patients who received 500 U of IL-2, whereas the higher dose was not effective. The CR was recorded in one of the seven patients with a oropharyngeal recurrence. Partial responses were obtained in 1/5 patients with hypopharyngeal recurrences, in 1/5 patients with oral cavity recurrences and 1/7 patients with laryngeal recurrences. The duration of the responses was 3-5 months and additional courses of ten injections of IL-2 had no further effect.

Detection of MET oncogene/hepatocyte growth factor receptor in lymph node metastases from head and neck squamous cell carcinomas.

The c-MET oncogene encodes the receptor for hepatocyte growth factor/scatter factor (HGF/SF), which is known to stimulate the invasive growth of epithelial cells cultured in vitro. TheMet/HGF receptor is a heterodimeric transmembrane tyrosine kinase, which is a prototype for a new family of growth factor receptors. The c-MET oncogene is expressed in several types of epithelial tissue including keratinocytes and is over-expressed in a number of human carcinomas. Studies on various carcinoma cell lines have shown that over-expression and structural alteration of the receptor result in its activation and confer tumorigenesis. We have studiedMet/HGF receptor expression in tissue specimens from 34 patients with head and neck squamous cell carcinomas (HNSCC) and in 17 regional lymph node metastases. Western blot analysis was employed, using monoclonal antibodies directed against either the intracellular or extracellular domain of the receptor. Each sample was compared to its normal counterpart. The receptor did not show any major structural alterations in HNSCC tissues, but its expression was increased from 2- to 50-fold in about 70% of tumors. Immunohistochemistry then showed that the same antibodies stained only a few cells in the basal layer of normal squamous epithelium but intensely marked tumor cells. In the lymph node metastases of Met-positive tumors, receptor expression was maintained and sometimes increased with respect to primary tumors. Immunohistochemical analysis of the metastatic lymph nodes showed that cells were negative in the normal lymphatic tissue and strongly stained in tumor cells. Over-expression of theMet/HGF receptor was found at all tumor stages but was more significant in those associated with enlarged or multiple (N2–N3) lymph node metastases. These data show that expression of theMet/HGF receptor may be involved in the progression of HNSCC towards a metastatic phenotype and may be a useful marker of head and neck tumor cell spread to regional lymph nodes.

The Effect of Preoperative Local Interleukin-2 (IL-2) Injections in Patients with Head and Neck Squamous Cell Carcinoma: An Immunological Study

Clinical, immunological, immunophenotypical, pathological and molecular biological studies were performed on tumor infiltrating lymphocytes (TIL) and lymph node lymphocytes (LN-ly) of 8 patients with squamous cell carcinoma of the oral cavity and oropharynx treated with 10 daily locoregional injections of low doses of IL-2 before surgery. No complications were seen during or after surgery. In 3 cases the LN-ly showed a moderate LAK activity, higher in the LN-ly omolateral to the tumor and near the IL-2 injection site; in 2 of these 3 patients a good LAK activity was induced after 6-day culture with IL-2. The LN-ly derived from nodes next to the tumor showed a decreased NK activity and proliferative ability both in basal conditions and after in vitro lymphokine challenging. LN-ly of 2 IL-2 treated patients showed high levels of mRNA encoding for IL-2-R, while it was absent in 2 untreated cases. Immunophenotypical studies on TIL showed statistically improved levels of CD25+ and LAK1+ cells in treated cases. Clusters of CD11c+ (macrophages) cells were seen close to the neoplastic sheets.

Interleukin 2 Receptors on Squamous Cell Carcinomas of the Head and Neck: Characterization and Functional Role

IL2Rs, in the presence of IL2, mediate activation and proliferation of human normal lymphocytes and modulate functional changes of some human leukocytic malignancies. IL2Rs have been demonstrated also on human neoplastic and fetal non-hematologic cells in vitro, although their functional role has not been described. We describe the presence of high affinity IL2Rs on squamous cell carcinoma of the head and neck (SCCHN) lines in vitro. Expression of 200 binding sites for IL2 with an affinity of 17 x 10(-12) was demonstrated by radiobinding experiments. When cytocentrifuged SCCHN cells were studied by immunoperoxidase staining, strong positive staining was repeatedly obtained using a monoclonal antibody to the p70 subunit of the IL2Rs. Experiments in vitro and in vivo, in a nude mouse model, showed a functional role for these receptors. In fact, low doses (8-500 U/ml) of IL2 were able to inhibit growth of 11 of the 16 SCCHN lines tested. Our observations may have broad implications for the immunotherapy of cancer in general, showing the complementary immunomodulatory and direct effects of IL2.

Immunological Investigations on Tonsillar and Peripheral Blood Lymphocytes after Adeno-tonsillectomy: Possible Suggestions for Phenotypical and Functional Differences

The phenotypical and functional aspects of tonsillar (TMNC) and peripheral blood mononuclear cells (PBMNC) obtained from 40 patients who had undergone adeno-tonsillectomy for different tonsillar diseases were investigated. TMNC and PBMC were separately tested at the time of surgery; then a further investigation on the peripheral blood lymphocytes was performed three months after surgery. The most interesting findings are presented and discussed with particular attention to their clinical implications.