Benefits of combined modality treatment of Merkel cell carcinoma of the head and neck: single institution experience.

BACKGROUND: There is ongoing debate surrounding the roles of surgery and adjuvant radiotherapy in the management of primary and recurrent Merkel cell carcinoma of the head and neck. This study assessed the influence of local excision, margin status, adjuvant radiotherapy and chemotherapy on locoregional recurrence and survival. METHOD: A retrospective review of 54 consecutive cases of head and neck Merkel cell carcinoma at a single institution. RESULTS: Median disease-specific survival time was 120 months. Forty-four per cent of patients developed locoregional recurrence. Combined treatment with surgery and locoregional radiotherapy improved diseasespecific survival. Radiotherapy was associated with longer time to recurrence and regional recurrence. Irradiation of the regional nodes improved regional control, irrespective of clinical status. Margin-negative excision was not associated with improved local control. Combined modality treatment of recurrent disease resulted in a four-fold improvement of local control, but small numbers prevented this trend from reaching statistical significance. CONCLUSION: Surgical excision of the primary disease and clinically involved regional nodes, plus adjuvant radiotherapy to the surgical bed and regional nodes are recommended for all patients with Merkel cell carcinoma of the head and neck, irrespective of clinical status. Recurrent disease should be aggressively treated with combined modality treatment.

Palliative surgery for neoplastic unilateral vocal cord paralysis.

BACKGROUND: Locally advanced, recurrent or metastatic neoplasms are the commonest causes of unilateral vocal cord paralysis (UVCP). The aim of the present study was to evaluate both survival and results of treatment of vocal cord medialization procedures in this group of patients. METHODS: Fifty-seven patients (36 male, 21 female) with UVCP considered to be due to advanced malignancy who underwent medialization (Teflon injection or type I thyroplasty) between January 1994 and July 2000 were retrospectively reviewed. RESULTS: The malignancy responsible for UVCP was non-small-cell lung carcinoma (NSCLC) in 43 patients, small-cell lung carcinoma (SCLC) in four patients, thyroid carcinoma in three patients and metastatic lower cervical lymph nodes in seven patients. All patients complained of dysphonia and 29 patients had symptoms of aspiration. Teflon injection was performed in 44 patients and thyroplasty in 13. Improvement in voice occurred in 51 patients (89%) and resolution of aspiration in 28 patients (97%) after 2 months. The median time from onset of symptoms of UVCP to death in NSCLC was 170 days; SCLC, 69 days; thyroid carcinoma, 783 days; and metastatic lower cervical lymph nodes, 304 days. CONCLUSION: Surgical treatment of neoplastic UVCP provides satisfactory palliation of symptoms, and management decisions should be based on patient survival expectations.

Laryngo-pharyngeal carcinoma in childhood.

Laryngo-pharyngeal carcinoma is rare in children. We present two cases of squamous cell carcinoma of the laryngopharynx in children less than 15 years of age. Both patients presented with a prolonged history of symptoms and extensive disease at diagnosis. Early visualisation the vocal cords with flexible larygnoscopy is important in children presenting with symptoms suggestive of laryngeal pathology. Long-term complications of definitive local therapy for laryngopharyngeal carcinoma are important in young children. Evidence from studies in adult patients suggests that adjuvant chemotherapy may play a role in laryngeal preservation in a select group of patients.

Heterozygous mice for the transforming growth factor-beta type II receptor gene have increased susceptibility to hepatocellular carcinogenesis.

The transforming growth factor-beta (TGF-beta) receptor complex and its downstream signaling intermediates constitute a tumor suppressor pathway. In many cancers, expression of TGF-beta type II receptor (TbetaR-II) is markedly decreased. In the present study, we show that the hepatocytes isolated from 15-day-old, but not 9-month-old, mice heterozygous for the deletion of the TbetaR-II gene are slightly less sensitive to the growth-inhibitory effect of TGF-beta when compared with wild-type littermates of same age. In addition, the proliferation index of hepatocytes as indicated by bromodeoxyuridine incorporation is mildly increased in the heterozygous mice. These subtle changes in cellular phenotype did not result in either gross or microscopic abnormality of the liver. The treatment of these mice with the chemical carcinogen, diethylnitrosamine, results in a significantly enhanced tumorigenesis in the liver when compared with the wild-type littermates. Our results demonstrate the gene-dosage effect of TbetaR-II and indicate that the reduced expression of TbetaR-II in mice increases susceptibility to tumorigenesis in the liver.

Epistaxis. Strategies for management.

BACKGROUND: Epistaxis is a problem frequently encountered in general practice and may present as an emergency, as a chronic problem of recurrent bleeds or may be a symptom of a generalised disorder. OBJECTIVE: To provide practical guidelines for the management of the patient with epistaxis, with emphasis on the important aspects of patient history, examination and whether investigations are necessary. DISCUSSION: Given adequate knowledge of anatomy and clinical technique, epistaxis is easily managed. A focus on important aspects of history and examination assists in determining the site of bleeding and whether further investigations are necessary. Management includes resuscitation, if necessary, followed by cautery and/or packing of the site.

Midfacial tumours: a review of 72 cases.

We review 72 midfacial tumours managed during the 10-year period between 1985 and 1995. We describe presenting features, sites of lesions and histology, treatment regimens and outcomes, as well as the various surgical approaches for the resection of midfacial tumours, and their indications and contraindications. The choice of approach should be based on type of tumour, its site, and extent.

Radiation with concurrent late chemotherapy intensification ('chemoboost') for locally advanced head and neck cancer.

The aim of this study was to review our experience with a treatment regimen that combined conventionally fractionated radiation therapy (70 Gy over 7 weeks) with chemotherapy (cisplatin and fluorouracil), given concurrently in the last 2 weeks of radiation therapy in patients with previously untreated advanced squamous cell cancer of the head and neck region.Twenty-eight patients, all but two having UICC stage IV disease, were treated at the Peter MacCallum Cancer Institute between November 1995 and April 1998. Planned chemotherapy consisted initially of continuous infusion at 10 mg/m(2) per day of cisplatin and 400 mg/m(2) per day of fluorouracil on days 1-5 of weeks 6 and 7 of a conventionally fractionated course of radiotherapy. After the first 14 patients, the dose of fluorouracil was reduced to 360 mg/m(2) per day because of acute toxicity.36.8 months), with an estimated 50% surviving at 2 years (CI, 29-71%). Sixteen patients (57%) developed confluent mucositis and 11 (39%) developed patchy mucositis. The median duration of mucositis for these 27 patients was 1.5 months. Seventeen patients (61%) required nutritional support for a median duration of 1.4 months. Fourteen patients (50%) had grade three skin reactions, and 12 (43%) had one or more other significant (Grade 3) toxicities, predominantly infective. Grade 3 late toxicity has been observed in three patients to date (three xerostomia, including one with severe depression), and one patient had chronic ulceration of the oral tongue (grade 4). This chemoradiation regimen achieved an excellent complete response rate and good locoregional control at 2 years in patients with a poor initial prognosis. Acute toxicity was significant but manageable. The regimen offers an alternative to surgery and postoperative radiation therapy in locally advanced head and neck cancer.

Smad7 differentially regulates transforming growth factor beta-mediated signaling pathways.

Smad7 has been identified as a negative regulator of transforming growth factor beta (TGF-beta) signaling by interfering with the phosphorylation of other Smad proteins by TGF-beta receptor type I (TbetaRI). We established a mink lung epithelial (Mv1Lu) cell line where ectopic expression of Smad7 is tightly controlled by doxycycline using an improved Tet-on system. Once induced by doxycycline, the recombinant Smad7 was localized predominantly in the perinuclear region and in the cytoplasm. However, the type of culture surface alters the subcellular localization of Smad7: on plastic or on fibronectin-coated glass, Smad7 was localized in the cytoplasm; but when the cells were cultured on glass, nuclear localization was observed. TGF-beta stimulation did not alter substantially the cellular distribution of Smad7. Importantly, the expression of recombinant Smad7 differentially inhibited TGF-beta signaling pathways. Consistent with previous studies, Smad7 inhibited TGF-beta-stimulated induction of type 1 plasminogen activator inhibitor as measured by p3TP-Lux reporter. However, expression of Smad7 had little effect on TGF-beta-induced growth inhibition.

Extracellular domain of the transforming growth factor-beta receptor negatively regulates ligand-independent receptor activation.

We have previously proposed that transforming growth factor (TGF)-beta receptor activation occurs via a relative rotation between the receptors. This model suggests that in the absence of the ligand the receptor extracellular domain negatively regulates the activation of the receptor complex. To investigate this proposition, four TGF-beta type I and II receptor extracellular/transmembrane-cytoplasmic and extracellular-transmembrane/cytoplasmic chimeras, TbetaRII-I-I and TbetaRI-II-II as well as TbetaRII-II-I and TbetaRI-I-II, and two extracellular domain truncated receptors TbetaRI-STC and TbetaRII-STC were generated. In either mutant mink lung R1B (lacking functional type I receptor) or DR26 (where the type II receptor is nonfunctional) cells, coexpression of two chimeric receptors, which are complementary in extracellular and cytoplasmic domains, transduced TGF-beta induced signaling, as measured by the transcriptional activation of a p3TP-Lux reporter gene. Coexpression of this type of chimeric receptor with a wild-type receptor containing the opposite cytoplasmic domain exhibited a varied level of constitutive activity depending on the particular combination of the extracellular domains. In general, the type I-type I extracellular domain combination gave higher constitutive activity than the type I-type II or type II-type II combinations. Furthermore, coexpression of the extracellular domain truncated receptor with any receptor containing the opposite cytoplasmic domain always resulted in ligand independent receptor signaling. Immunoprecipitation studies showed that the formation of the receptor complexes paralleled the ligand independent activation of p3TP-Lux. Our results support the conclusion that the TGF-beta receptor extracellular domain plays a negative regulatory role in receptor activation in the absence of ligand.

The use of titanium mandibular reconstruction plates in patients with oral cancer.

A series of 21 patients, who were selected for primary mandibular stabilisation with titanium plates following excision of advanced malignant tumours, is reported. Success was defined as a plate that did not have to be removed due to fracture, exposure or infection. The overall success rate was 71%, with follow-up ranging from 7-53 months. The majority of plate losses were experienced when either anterior or large lateral defects which included the condyle were bridged and when patients were subjected to either pre- or postoperative radiotherapy.

A pivotal role for the transmembrane domain in transforming growth factor-beta receptor activation.

Transforming growth factor-beta (TGF-beta) delivers diverse growth and differentiation signals by binding two distantly related transmembrane serine/threonine kinase receptors: the type I receptor (TbetaRI) and the type II receptor (TbetaRII). In an attempt to establish the role of the transmembrane domain in receptor signaling, two chimeric TGF-beta receptors, TbetaRI-II-I and TbetaRII-I-II, containing the opposite transmembrane domain were generated. When transfected into a mutant mink lung epithelial cell line R1B, which lacks functional TbetaRI, TbetaRI-II-I restored TGF-beta1-induced transcriptional activation of a TGF-beta reporter p3TP-Lux to approximately 25% of the levels restored by wild-type TbetaRI. In the mutant mink lung epithelial cell line DR26, which contains a truncated, nonfunctional TbetaRII, wild-type receptor TbetaRII restored the TGF-beta responsiveness, while the TbetaRII-I-II cDNA was inactive. When both TbetaRI and TbetaRII were transfected into R1B, DR26, or Mv1Lu cells, a low level of constitutive p3TP-Lux activity was observed. However, cotransfection of both transmembrane chimeric receptors, TbetaRI-II-I and TbetaRII-I-II, or the wild-type TbetaRI with the transmembrane chimeric TbetaRII-I-II resulted in high levels of ligand-independent receptor activation. These results suggest that the transmembrane domains of both TGF-beta receptors are essential and play a pivotal role in receptor activation. To investigate the role of the transmembrane domain further, four type II transmembrane mutants were generated: TbetaRIIDelta-1, TbetaRIIDelta-2, TbetaRIIDelta-3, and TbetaRIIDelta-4, which have one, two, three, or four amino acids deleted at the N terminus of the transmembrane domain, respectively. Interestingly, co-expression of TbetaRIIDelta-1 with the wild-type TbetaRI in DR26 cells resulted in high levels of constitutive activation, while only low levels of the activation were observed when TbetaRIIDelta-2, TbetaRIIDelta-3, or TbetaRIIDelta-4 were co-expressed with the wild-type TbetaRI. However, TbetaRIIDelta-1 restored very little the TGF-beta responsiveness in DR26cells. Expression of TbetaRIIDelta-2, TbetaRIIDelta-3, and TbetaRIIDelta-4 resulted in a progressive increase in TGF-beta responsiveness, with TbetaRIIDelta-4 reaching the level of activity of the wild-type TbetaRII. Furthermore, like TbetaRII-I-II, co-expression of TbetaRIIDelta-1 with TbetaRI-II-I also resulted in high levels of constitutive activation. These results are consistent with an important role for the transmembrane region of the receptors. We further propose a model of receptor activation in which receptor activation occurs via relative orientational rotation.

Restricted expression of type-II TGF beta receptor in murine embryonic development suggests a central role in tissue modeling and CNS patterning.

The type-II TGF beta receptor mediates many of the biological responses to TGF beta. An examination of the expression of the type-II TGF beta receptor during mouse embryogenesis therefore provides specific information about the role of TGF beta during embryogenesis than has been available to date. We have isolated the genomic murine homologue of the human type-II TGF beta receptor corresponding to exon 2. The murine and human sequences show a high degree of homology. Using the murine probe, we found that type-II TGF beta receptor expression is regulated in both a spatial and a temporal fashion by using in situ hybridization and ribonuclease protection assays. Type-II TGF beta receptor expression is localized to the mesenchyme during critical interactions with adjacent epithelium such as developing hair follicles, whisker follicles and tooth anlage. In the central nervous system, type-II TGF beta receptor expression is highly restricted to the floor plate. Strong expression is also detected in migrating neural crest cells, meninges, and choroid plexus. Specific mesenchymal localization of type-II TGF beta receptor is also observed in lung, kidney, intestine, stomach, and bladder. The restricted expression of type-II TGF beta receptor in mesenchymal cells at sites of epithelial-mesenchymal interactions suggests that type-II TGF beta receptor plays a major role in mediating the establishment of embryonic organ systems. The highly restricted expression of type-II TGF beta receptor in the developing CNS suggests an important role for a serine/threonine kinase in patterning of the nervous system.

Association of Sos Ras exchange protein with Grb2 is implicated in tyrosine kinase signal transduction and transformation.

The proteins Grb2-Sem-5, Shc and Sos have been implicated in the signalling pathway from tyrosine kinase receptors to Ras. Grb2-Sem-5 binds directly to murine Sos1, a Ras exchange factor, through two SH3 domains. Sos is also associated with ligand-activated tyrosine kinase receptors which bind Grb2-Sem-5, and with the Grb2-Sem-5 binding protein, Shc. Ectopic expression of Drosophila Sos stimulates morphological transformation of rodent fibroblasts. These data define a pathway by which tyrosine kinases act through Ras to control cell growth and differentiation.

Anti-sense transforming growth factor alpha oligonucleotides inhibit autocrine stimulated proliferation of a colon carcinoma cell line.

Many carcinoma cells secrete transforming growth factor alpha (TGF alpha). A 23 base anti-sense oligonucleotide that recognizes the TGF alpha mRNA inhibits both DNA synthesis and the proliferation of the colon carcinoma cell line LIM 1215. The effects of the anti-sense TGF alpha oligonucleotide are reversed by epidermal growth factor (EGF) at 20 ng/ml. When the LIM 1215 cells are grown under serum free conditions, the anti-sense TGF alpha oligonucleotides have their greatest effects at high cell density (2 x 10(5) cells/cm2), indicating that the secreted TGF alpha is acting as an exogenous growth stimulus. In addition, at higher cell densities, the kinase activity of the EGF receptor is activated and the receptor is down-modulated. The cell density dependent activation of the EGF receptor is inhibited by the application of the antisense TGF alpha oligonucleotides.

Asparagine 26, glutamic acid 31, valine 45, and tyrosine 64 of Ras proteins are required for their oncogenicity.

Ras and Rap1 proteins are related GTP-dependent signal transducers which require Gly-12, the effector domain (residues 32-40), and Ala-59 for stimulation of their GTPase activities by GAP1 and GAP3, respectively. The replacement of Gly-12 by Val or Ala-59 by Thr potentiates the Ras oncogenicity and Rap1A antioncogenicity. However, the mutations in the effector domain, in particular the replacement of Thr-35 by Ala, abolish both Ras oncogenicity and Rap1A antioncogenicity, indicating that the effector domain is involved in interactions of these signal transducers with their targets as well as the GAPs. In this paper, we demonstrate that (i) replacement of Tyr-64 of the Ha-Ras protein or Phe-64 of the Rap1A protein by Glu or other non-hydrophobic amino acids reduces their intrinsic GTPase activities and abolishes their stimulation by GAP1 or GAP3, respectively, (ii) replacement of Tyr-64 by Gly and other non-hydrophobic amino acids results in complete loss of the oncogenicity of the v-Ha-Ras protein, indicating that the hydrophobic residue 64, in addition to the known effector domain, is essential for the Ras protein to interact with its target as well as GAP1. In addition we have found that Asn-26, Glu-31, and Val-45 of the v-Ha-Ras protein are required for its oncogenicity. Replacement of the Ras residues at either positions 26, 31, or 45 by the corresponding Rap1A residues abolishes the Ras oncogenicity.

The proliferative and morphologic responses of a colon carcinoma cell line (LIM 1215) require the production of two autocrine factors.

The role of autocrine growth factors in tumor cell growth has been difficult to prove. Our results indicate that more than one autocrine factor is required for the autonomous growth of the LIM 1215 colonic carcinoma cell line. Furthermore, the morphologic changes induced by epidermal growth factor (EGF) are also density dependent and appear to require a synergistic autocrine factor. The serum-free proliferation of the colonic carcinoma cell line LIM 1215 depends on cell density and the presence of EGF (A. Sizeland, S. Bol, and A.W. Burgess, Growth Factors 4:129-143, 1991). At cell densities below 10(4)/cm2, conditioned medium (from cells at a density of 10(5)/cm2) was required for the cells to elicit a mitogenic response to exogenous EGF. At higher cell densities (10(5)/cm2), the cells were independent of both exogenous EGF and conditioned medium. In addition, the EGF receptor was found to be phosphorylated on tyrosine in LIM 1215 cells proliferating at high density, suggesting that the autocrine production of transforming growth factor alpha (TGF alpha) and subsequent ligation to the EGF receptor was occurring. The proliferation of cells at high density was partly inhibited by TGF alpha antibodies but was almost completely inhibited by an antisense oligonucleotide to TGF alpha. The antisense inhibition could be overcome by the addition of EGF, indicating that the effect of the antisense TGF alpha oligonucleotide was on the production of autocrine TGF alpha. LIM 1215 cells were also observed to undergo morphologic changes (spreading and actin cable organization) in response to EGF. These changes were density dependent, but they occurred with a cell density dependence different from that of the proliferative response. These results suggest two possibilities: that the morphologic changes and proliferative responses have different sensitivities to the autocrine factors or that the actions of the autocrine factors are mediated through different signal transduction pathways.

The residues of Ras and Rap proteins that determine their GAP specificities.

The oncogenic transformation of a normal fibroblast by mutated Ras genes can be reversed by overexpression of a Ras-related gene called Rap1A (or Krev1). Both Ras and Rap1A proteins are G proteins and appear to serve as signal transducers only in the GTP-bound form. Therefore, GAP1 and GAP3, which stimulate the intrinsic GTPase activities of normal Ras and Rap1A proteins, respectively, serve as attenuators of their signal transducing activities. In this paper, we describe the enzymatic properties of several mutated Rap1A and chimeric Ras/Rap1A (or -1B) proteins which lead to the following conclusions: (i) the GAP3-dependent activation of both Rap1A and -1B GTPases requires Gly12, but neither Thr61 nor Gln63; (ii) residues 64 to 70 of the Rap1 GTPases are sufficient to determine their specificities for GAP3; and (iii) residues 61 to 65 of the Ras GTPases are sufficient for determining their specificities for GAP1. Thus, the domains of the Ras or Rap1 proteins that determine whether their signals are attenuated by GAP1 or GAP3 are distinct from the N-terminal domain (residues 21 to 54) that determines whether their signals are oncogenic or antioncogenic. The Arg12 mutant of chimeric HaRas(1-54)/Rap1A(55-184) protein has been previously reported to be oncogenic (Zhang, K., Noda, M., Vass, W. C., Papageorge, A.G., and Lowy, D.R. (1990) Science 249, 162-165). In this paper, we show that the Val12 mutant of chimeric HaRas(1-54)/Rap1B(55-184) protein is also oncogenic, suggesting that the C-terminal geranylgeranylation of the Rap 1B protein can replace functionally the C-terminal farnesylation of the Ras protein to allow the G protein to be oncogenic.

The action of epidermal growth factor (EGF) is limited to specific phases of the cell cycle in an EGF dependent colonic cell line.

In the presence of epidermal growth factor (EGF) a human colon cell line, LIM 1215, proliferates in serum-free medium. Under these culture conditions the cells are dependent on the presence of EGF for both proliferation and survival. In order to study the action of growth factors at different stages of the LIM 1215 cell cycle, pure populations of G1, S and G2/M cells were obtained by cell sorting after supravital staining of the DNA with Hoechst 33342. Conditions were established for Hoechst 33342 staining which produced satisfactory DNA histograms and greater than 80% survival of cells. The kinetics of passage for sorted S or G2/M cells into G1 were not affected by EGF or fetal calf serum. After sorting there appeared to be a 4 h delay before the cells proceeded in the cell cycle. Sorted S cells entered G2 over an 8 h period and maintained this same transition period from G2 into G1. If EGF or serum was present, these cells then re-entered the cell cycle after a variable delay and in an asynchronous manner. EGF was applied to S phase and G2/M phase LIM 1215 cells for periods of 2-10 h at various times after replating in serum-free conditions. Cells in S phase only responded to EGF as they passed from G2/M into G1. Exposure to EGF in S phase resulted in little growth stimulus once the cells returned to G1. For cells in G2/M phase, EGF was required immediately to give the maximum stimulus for re-entering the cell cycle. If the EGF was delayed for more than 8 h, the cells did not re-enter the cycle within the following 20 h. Exposure to EGF for less than 2 h failed to stimulate proliferation. These results indicate that EGF must be present as cells enter G1 from mitosis. Once the cells have entered G1, EGF is required for a 10 h period for a large number of cells to re-enter the cycle from G1.