Giorgio Cortesina

Somatic mutations of the MET oncogene are selected during metastatic spread of human HNSC carcinomas

A metastatic cancer develops by accumulation of mutations in genes that control growth, survival and spreading. The latter genes have not yet been identified. In lymph node metastases of head and neck squamous cell carcinomas (HNSCC), we found mutations in the MET oncogene, which encodes the tyrosine kinase receptor for Scatter Factor, a cytokine that stimulates epithelial cell motility and invasiveness during embryogenesis and tissue remodeling. We identified two somatic mutations: the Y1230C, known as a MET germline mutation which predisposes to hereditary renal cell carcinoma, and the Y1235D that is novel and changes a critical tyrosine, known to regulate MET kinase activity. The mutated MET receptors are constitutively active and confer an invasive phenotype to transfected cells. Interestingly, cells carrying the MET mutations are selected during metastatic spread: transcripts of the mutant alleles are highly represented in metastases, but barely detectable in primary tumors. These data indicate that cells expressing mutant MET undergo clonal expansion during HNSCC progression and suggest that MET might be one of the long sought oncogenes controlling progression of primary cancers to metastasis.

Treatment of recurrent squamous cell carcinoma of the head and neck with low doses of lnterleukin‐2 injected periiymphatically

Ten patients with recurrent squamous cell carcinoma of the head and neck received daily injections of interleukin‐2 (IL‐2) from the Jurkat T‐cell line purified by high pressure liquid chromatography for 10 days. Two hundred units of IL‐2 in 0.5 ml were injected 1.5 cm from the insertion of the sternocleidomastoid muscle on the mastoid. When possible, courses were repeated at 45‐day intervals. IL‐2 was ineffective in two patients who had already undergone functional or radical neck dissection. By contrast, in six patients with contralateral or bilateral cervical lymph nodes, complete or partial disappearance of the tumor was observed. The injections were occasionally followed by moderate local swelling and lymph node pain, but no systemic disturbances.

Pharyngocutaneous Fistula as a Complication of Total Laryngectomy: Review of the Literature and Analysis of Case Records:

Pharyngocutaneous fistula is the most common complication of total laryngectomy. The management of this problem increases hospitalization time and delays initiation of postoperative radiotherapy, where indicated. To identify factors predisposing to the development of pharyngocutaneous fistula, we reviewed the postoperative courses of 293 patients who underwent total laryngectomy at our clinic. General factors taken into account were concurrent diseases such as diabetes, liver diseases, or chronic anemia; local factors included radiotherapy before and after surgery, preoperative tracheostomy, type of cervical lymph node removal, and method of pharyngeal closure. We then compared our data with those reported in the literature by other authors. Last, we applied the Fisher exact test to a correlation we found between the higher incidence of fistula in patients with diabetes, liver diseases, or anemia. The local factor that turned out to be statistically most significant for the development of fistula was preoperative radiotherapy.

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.

Morphological Study of the Laryngeal Muscles in Man: Insertions and Courses of the Muscle Fibres, Motor End-Plates and Proprioceptors

Koelle and Friedenwalds test for acetylcholinesterase has been used in a study on the following laryngeal muscles in man: vocalis, cricothyroid, lateral crico-arytenoid and posterior crico-arytenoid. Serial sections were made with the freezing microtome and a number of muscles were submitted to microdissection. “Multi-motor end-plate” fibres were found in all four muscles studied (70 to 80 per cent in the vocalis muscle, about 50 per cent in the cricothyroid and lateral crico-arytenoid muscles and about 5 per cent in the posterior crico-arytenoid muscle). None of the fibres of the vocalis muscle was found to be inserted into the vocal ligament. Only in the vocalis muscle was it possible to demonstrate typical muscle spindles, whereas in all four muscles studied “spiral nerve endings” were observed.

Prognostic impact of HER-2/neu expression on squamous head and neck carcinomas.

HER‐2/neu gene amplification and protein overexpression have been identified in various solid tumors, but its prognostic relevance in head and neck squamous cell carcinoma (HNSCC) is still controversial.

Association Between Hypermethylated Tumor and Paired Surgical Margins in Head and Neck Squamous Cell Carcinomas

Purpose: Surgical margin status is reported to be a relevant prognostic factor in head and neck squamous cell carcinoma (HNSCC), associated with a high risk of local recurrence. This study examines whether gene-promoter hypermethylation could be detected in HNSCC surgical margins with no histologic evidence of malignancy, and if so, whether it reflects epigenetic events of primary tumors. Experimental Design: Promoter methylation status of MGMT, p16, and DAP-K genes was evaluated by methylation-specific PCR in 20 primary HNSCC tumors. Histopathologically negative surgical margins of hypermethylated tumors were collected, and their methylation status compared with the primary tumor status. Results: Promoter hypermethylation in at least one of the three tested genes was detected in 65% (13 of 20) of tumors. MGMT was hypermethylated in 50% (10 of 20), DAP-K in 45% (9 of 20), and p16 in 20% (4 of 20) of tumors. Methylation status was analyzed in 35 margins from 11 of 13 patients showing promoter hypermethylation in the tumor tissue. Identical methylation events were seen for at least one gene in primary tumor and surgical margins in 9 of 11 cases (82%). Association was found for gene-specific hypermethylation status in tumors and paired surgical margins, and gene-specific concordance was 63% for MGMT (κ = 0.24), 90% for DAP-K (κ = 0.74), and 90% for p16 (κ = 0.79). Conclusions: Our results support the hypothesis that detection of gene promoter hypermethylation in HNSCC tumor cells–free surgical margins may be a helpful biomarker to identify molecularly altered fields in areas adjacent to the tumor.

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.

Study of Ethmoidal Mucosa in a Population at Occupational High Risk of Sinonasal Adenocarcinoma

The occurrence of nasal adenocarcinomas was first described in 1965. Since 1988 these tumors have been accepted as an occupational disease for woodworkers in Italy. There are several underlying reasons why there is interest in studying the ways in which sinonasal adenocarcinomas occur. Often diagnosed at advanced stages of development because their symptoms are non-specific, these tumors are associated with a high mortality rate. A multidisciplinary study protocol was developed in this investigation. The aim was to identify the factors and conditions that promote sinonasal tumor growth in a population at risk due to occupational exposure to wood dust. Sixty-eight carpenters with a minimum of 10 years exposure to wood dust were studied. The control group comprised 81 volunteers. The patients underwent the following protocol: completion of a case report form, physical examination, evaluation of nasal cavity patency, clinical laboratory tests and histological study of the nasal mucosa. Our study provides significant evidence of the elevated incidence of pavimentous metaplasia in workers occupationally exposed to wood dust. In addition, it underscores a significant deficit of immunoglobulin A in such workers compared to the controls. However, we did not find, as reported elsewhere in the literature, a statistically significant difference between cases and controls as regards nasal symptoms and hyperemia of the nasal mucosa. Our study showed that, even in the absence of evident sinonasal lesions, it is still possible to determine an increased incidence of morphofunctional changes in subjects occupationally exposed to wood dust. Our findings may lead to the identification of occupational groups prone to elevated risk of the disease.