miRNA-214 modulates radiotherapy response of non-small cell lung cancer cells through regulation of p38MAPK, apoptosis and senescence.

BACKGROUND: Radio- and chemotherapy (RT/CT) resistance hampers success in combating small and non-small cell lung cancers (SCLC/NSCLC). The underlying molecular mechanisms of RT/CT resistance of LCs are multifactorial and have been understood in part hitherto. miRNAs, key regulators of mRNAs, are well-recognised oncomirs; however, their role in regulating RT response remains poorly understood. METHODS: Six human NSCLC and five SCLC cell lines with different SF2 values were investigated. Using microarray we examined whether expression of miRNAs is linked to the RT resistance of NSCLCs or SCLCs. Obtained data were validated by quantitative real-time PCR. Apoptosis and senescence were analysed using siRNA transfection, western blot and flow cytometry. RESULTS: miRNA-21, miRNA-1827, miRNA-214, miRNA-339-5p, miRNA-625, miRNA-768-3p, miRNA-523-3p, miRNA-1227, miRNA-324-5p, miRNA-423-3p, miRNA-1301 and miRNA-1249 are differentially expressed in LC cells. miRNA-214 is upregulated in RT-resistant NSCLC cells relative to radiosensitive counterparts. Considering miRNA-214 as a putative regulator of RT resistance, we demonstrate that knockdown of miRNA-214 in radioresistant NSCLCs sensitised them to RT by stimulation of senescence. Consistently, overexpression of miRNA-214 in radiosensitive NSCLCs protected against RT-induced apoptosis. Protection was mediated by p38MAPK, as downregulation of this kinase could reverse the miRNA-214 overexpression-induced resistance of NSCLC cells. CONCLUSION: miRNA profiling of LC revealed putative RT resistance signalling circuits, which might help in sensitisation of LC to RT.

Radioresistant cervical cancer shows upregulation of the NHEJ proteins DNA-PKcs, Ku70 and Ku86.

BACKGROUND: Radiotherapy is central in the treatment of cervical cancer. The formation of DNA double-strand breaks is considered to be critical for the radiotherapeutic effect. The non-homologous end joining (NHEJ) proteins DNA-PKcs, Ku70 and Ku86 have a major role in repairing DNA lesions. The objective of this study was to analyse if the expression of DNA-PKcs, Ku70 and Ku86 and their downstream signalling molecules p53, p21 and Mdm-2 are altered in residual cervical tumours after radiotherapy. METHODS: Retrospective analysis of 127 patients with cervical cancer stage IB-IIA treated with preoperative radiotherapy and radical surgery, revealed residual tumour in the cervical specimen in 30 patients. In 22 cases tumour material from residual and corresponding primary tumour were retrieved and the expression of DNA-PKcs, Ku86, Ku70, p53, p21 and Mdm-2 were assessed by immunohistochemistry. RESULTS: Residual tumours showed increased frequency of DNA-PKcs (P=0.037), Ku70 (P=0.018), Ku86 (P=0.008) positive cells. A correlation in DNA-PKcs expression between primary and residual tumours was found. The frequency of p21-positive cells was decreased (P=0.007) in residual tumours whereas no change in p53 or Mdm-2-positive cells were observed. CONCLUSION: Our results show that cervical carcinoma surviving radiotherapy have an increased DNA-PK expression. Studies on larger patient cohorts are needed to allow an interpretation that an upregulation of DNA-PK function may be part of a radioresistance mechanism within this tumour type.

Significantly higher levels of vascular endothelial growth factor (VEGF) and shorter survival times for patients with primary operable triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) lacking expression of steroid receptors and human epidermal growth factor receptor 2, having chemotherapy as the only therapeutic option, is characterised by early relapses and poor outcome. We investigated intratumoural (i.t.) levels of the pro-angiogenic cytokine vascular endothelial growth factor (VEGF) and survival in patients with TNBC compared with non-TNBC. PATIENTS AND METHODS: VEGF levels were determined by an enzyme immunosorbent assay in a retrospective series consisting of 679 consecutive primary breast cancer patients. RESULTS: Eighty-seven patients (13%) were classified as TNBC and had significantly higher VEGF levels; median value in TNBC was 8.2 pg/microg DNA compared with 2.7 pg/microg DNA in non-TNBC (P < 0.001). Patients with TNBC had statistically significant shorter recurrence-free survival [hazard ratio (HR) = 1.8; P = 0.0023], breast cancer-corrected survival (HR = 2.2; P = 0.004) and overall survival (HR = 1.8; P = 0.005) compared with non-TNBC. Patients with TNBC relapsed earlier than non-TNBC; mean time from diagnosis to first relapse was 18.8 and 30.7 months, respectively. The time between first relapse and death was also shorter in TNBC: 7.5 months versus 17.5 months in non-TNBC (P = 0.087). CONCLUSIONS: Our results show that TNBC have higher i.t. VEGF levels compared with non-TNBC. Ongoing clinical trials will answer if therapy directed towards angiogenesis may be an alternative way to improve outcome in this poor prognosis group.

Ionizing radiation activates IGF-1R triggering a cytoprotective signaling by interfering with Ku-DNA binding and by modulating Ku86 expression via a p38 kinase-dependent mechanism.

Ionizing radiation exposure results in the activation of several tyrosine kinase receptors that participate in radiation-induced DNA damage response and radioresistance. We previously showed that insulin-like growth factor 1 receptor (IGF-1R) inhibition enhanced radiosensitivity of non-small-cell lung cancer (NSCLC) cells. In this paper, we demonstrate that in U1810 NSCLC cells gamma-radiation activates IGF-1R within 10 min, with a maximal activation effect 2 h post-irradiation. Impairment of IGF-1R tyrosine kinase activity enhances human lung cancer cells radiosensitivity by a mechanism that involves phosphatidylinositol 3-kinase (PI3-K) and p38 kinase. In an active form, IGF-1R binds and activates p38 kinase, promoting receptor signaling. Conversely, inhibition of IGF-1R phosphorylation results in IGF-1R/p38 complex disruption and p38 kinase inactivation. We have also demonstrated that in insulin-like growth factor-1-stimulated cells, Ku-DNA-binding activation is induced by ionizing radiation within 4 h, reaches a maximum level at 12 h and remains active up to 72 h. Blockade of IGF-1R activity or its downstream signaling through p38 kinase induces a decrease in radiation-mediated Ku-DNA-binding activation and downregulates the level of Ku86, without affecting Ku70 expression in the nucleus of U1810 cells. The IGF-1R signaling via PI3-K does not interfere with the p38 signaling, the Ku-DNA-binding activity or the level of Ku86. Our present study demonstrates for the first time that ionizing radiation activates IGF-1R. Inhibition of IGF-1R signaling via p38 kinase induces radiosensitivity by a novel mechanism involving nuclear Ku86.

Current status of prognostic immunohistochemical markers for urothelial bladder cancer.

The management and prognostication of patients with urothelial carcinomas (UCs), the most common histological type of bladder cancer, is mainly based on clinicopathological parameters. Several markers have been proposed to monitor this disease, including individual cell cycle-related proteins such as p53, pRb, p16, p21 and p27. Other putative markers are the oncogene products of FGFR3 and the ErbB family, proliferation markers including Ki-67, Aurora-A and survivin and different components within the immune system. In this review, a total of 12 parameters were evaluated and their discriminatory power compared. It is concluded that, in single-marker analyses, the proliferation markers Ki-67, survivin and Aurora-A offer the best potential to predict disease progression since they were all able to demonstrate independent prognostic power in repeated studies. Markers related to the immune system (e.g. CD8+ cells, regulatory T cells and cyclooxygenase-2 expression) or oncogene products of the ErbB family and FGFR3 are less powerful predictors of outcome or have not been equally well studied. The cell cycle-related proteins p53, pRb, p16, p21 and p27 have been extensively studied, but their usefulness as single prognostic markers remains unclear. However, in multimarker analyses, these markers appear to add prognostic information, indicating that they may contribute to more accurate treatment of UC.

COORDINATION OF MANAGEMENT OF THE ACUTE RADIATION SYNDROME.

The acute radiation syndrome (ARS) constitutes the most challenging, immediate medical consequence of exposure to high doses of ionizing radiation in an emergency situation. This report highlights some of the currently available medical guidelines and recommendations on the clinical management of ARS, comments recent trends regarding the approval of targeted pharmaceuticals for ARS, and suggests further initiatives for international collaboration aiming at continuously updating the medical knowledge base of this syndrome.

Mitochondrial dysfunction is an essential step for killing of non-small cell lung carcinomas resistant to conventional treatment.

This corrects the article DOI: 10.1038/sj.onc.1205018.

Different activities of unscheduled DNA synthesis in human melanoma and bone marrow cells.

Unscheduled DNA synthesis (UDS) indicated by melphalan was studied in freshly collected tumor cells from human melanoma metastases. Comparative studies were done on human bone marrow blast cells. Significant levels of UDS comparable with those in myeloblasts were found in only two of eight melanoma cell populations. This difference between melanoma and blast cells was not related to different cellular uptake of melphalan. When UDS was induced by ultraviolet irradiation, significant levels of UDS were found in all melanoma and blast cell populations studied. Also, in a human melanoma cell line, high levels of UDS were found after exposure to ultraviolet irradiation, while treatment with melphalan did not result in detectable levels of UDS. Possible explanations for the divergent results of UDS in melphalan-exposed melanoma cells are discussed.

Formation and removal of DNA cross-links induced by melphalan and nitrogen mustard in relation to drug-induced cytotoxicity in human melanoma cells.

The formation and removal of nitrogen mustard (HN2)- and melphalan-induced DNA cross-links (DNA interstrand and DNA-protein cross-links) in a human melanoma cell line (RPMI 8322), as determined by alkaline elution of DNA, was compared and related to the cytotoxic effect of each drug. HN2 was considerably more cytotoxic than melphalan as determined by inhibition of colony formation. Immediately following exposure to HN2 maximum levels of DNA cross-links were found. Melphalan, in contrast, caused a protracted induction of DNA cross-links with maximum levels obtained 6-12 h following drug exposure. HN2 induced approximately 13 times higher peak levels of DNA cross-links compared to equal concentrations of melphalan. Removal of DNA cross-links following exposure to both drugs followed an exponential time course. The rate of removal of HN2-induced DNA cross-links was, however, 1.5-2.4 times more rapid than that of melphalan-induced cross-links. A strong correlation was obtained between the cytotoxicity of both drugs and the total area under the curve for DNA interstrand cross-links, indicating that both the initial induction of as well as the rate of removal of DNA interstrand cross-links are important for the cytotoxic effects of bifunctional alkylating agents.

Frequent amplification of the telomerase reverse transcriptase gene in human tumors.

Activation of telomerase is a crucial step during cellular immortalization and malignant transformation of human cells and requires the induction of the catalytic component, human telomerase reverse transcriptase (hTERT), encoded by the hTERT gene. It is poorly understood how the hTERT gene is activated in human cancer cells. In the present study, we examined the hTERT gene copy number in human cancer cell lines and in primary tumor tissues. Amplification of the hTERT gene was observed in 8 of 26 (31%) tumor cell lines and 17 of 58 (30%) primary tumors examined (8 of 21 lung tumors, 3 of 10 cervical tumors, 5 of 19 breast carcinomas, and 1 of 8 neuroblastomas). In addition, 13 of 26 (50%) cell lines and 13 of 58 (22%) primary tumors displayed gain of hTERT gene copies with 3-4 copies/cell. The present findings imply that the hTERT locus may be a frequent target for amplification during tumorigenesis and that this genetic event probably contributes to the dysregulation of telomerase activity occurring in human tumors.

Defective caspase-3 relocalization in non-small cell lung carcinoma.

Many anticancer drugs exert their cytotoxicity through DNA damage and induction of apoptosis. Small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) have different sensitivity to treatment with radiation and chemotherapeutic agents with SCLC being more sensitive than NSCLC both in vitro and in vivo. This difference might be related to the different susceptibility of small and non-small cell lung carcinoma to undergo apoptosis. The aim of this study was to investigate if deficiencies in the apoptotic pathways can explain the intrinsic resistance of NSCLC to anti-cancer treatment. Three different triggers were used to induce apoptosis. Etoposide and gamma-radiation, which are important parts of clinical lung cancer treatment, induce DNA-damage, whereas Fas ligation induces receptor-mediated apoptotic pathways. NSCLC cells were cross-resistant to all treatments, whereas SCLC cells, which do not express pro-caspase-8, were resistant to alphaFas-, but not to DNA-damage-induced apoptosis. Cytochrome c release, activation of caspase-9 and the executioner caspase-3 were observed in both types of lung cancer cells. However, cleavage of known nuclear substrates for caspase-3, such as PARP and DFF45/ICAD, was documented only in the sensitive SCLC cells but not in the resistant NSCLC cells. Moreover, relocalization of active caspase-3 from the cytosol into the nucleus upon treatment was observed only in the SCLC cell line. These results indicate that the inhibition of apoptosis in NSCLC occurs downstream of mitochondrial changes and caspase activation, and upstream of nuclear events.

Phase III trial of modulation of cisplatin/fluorouracil chemotherapy by interferon alfa-2b in patients with recurrent or metastatic head and neck cancer. Head and Neck Interferon Cooperative Study Group.

PURPOSE: In preclinical experiments, interferon alfa modulates the anticancer activity of fluorouracil (5-FU) and cisplatin (CDDP). To test this effect clinically in patients with recurrent or metastatic head and neck cancer (RMHNC), a multicenter randomized controlled trial with CDDP and 5-FU with or without interferon alfa-2b (IFNalpha) was performed. PATIENTS AND METHODS: Eligible patients had histologically confirmed RMHNC; a good performance status; measurable disease; adequate bone marrow, hepatic, and renal function; no prior chemotherapy for recurrent or metastatic disease; only one chemotherapy regimen administered with previous local therapy; and a treatment-free interval of at least 3 months following previous local therapy. Patients were randomized and stratified according to treatment center, and prior radiotherapy and chemotherapy. The treatment regimen consisted of CDDP 100 mg/m2 on day 1 and 5-FU 1,000 mg/m2/d by continuous infusion for 96 hours (days 1 to 4), without (arm A) or with (arm B) IFNg alpha 3 x 10(6) U/d subcutaneously on days 1 to 5. Cycles were repeated every 21 days. RESULTS: One hundred twenty-two patients were entered on each arm. The response rate (RR) was similar in both arms (arm A: complete response [CR] 10.7%, partial response [PR] 36.4%; arm B: CR 6.8%, PR 31.6%) (.70 < P < .50). There was no difference in median survival between the two arms (arm A 6.3 months v arm B 6.0 months; P = .49). Anorexia, fever, leukopenia, and thrombocytopenia grade III to IV were significantly more frequent in the IFNalpha arm. CONCLUSION: Modulation of CDDP and 5-FU with IFNalpha as used in this study does not improve the RR or the median survival in patients with RMHNC. Patients on both study arms had a poor prognosis, which indicates the need for novel therapies.

Decreased UV-induced DNA repair synthesis in peripheral leukocytes from patients with the nevoid basal cell carcinoma syndrome.

The UV-induced DNA repair synthesis in peripheral leukocytes from 7 patients with the nevoid basal cell carcinoma syndrome was compared to that in peripheral leukocytes from 5 patients with basal cell carcinomas and 39 healthy subjects. A dose response curve was established for each individual, and maximum DNA repair synthesis was used as a measure of the capacity for DNA repair. The patients with the nevoid basal cell carcinoma syndrome had about 25% lower level of maximum DNA repair synthesis as compared to the patients with basal cell carcinomas and control individuals. The possibility that DNA repair mechanisms may be involved in the etiology to the nevoid basal cell carcinoma syndrome is discussed.

Cell cycle-dependent regulation of the DNA-dependent protein kinase.

Human DNA-dependent protein kinase (DNA-PK) is a nuclear-localized serine/threonine protein kinase. The holoenzyme consists of a catalytic subunit with a molecular mass of 465 kDa and a DNA-binding heterodimer Ku86/70. The kinase has been implicated in a variety of nuclear processes including V(D)J recombination, double-strand break repair, and transcription. Cells with defective DNA-PK activity show increased radiosensitivity and lack of V(D)J recombination. To study DNA-PK activity during the cell cycle, HeLa cells were separated by elutriation centrifugation into different cell cycle compartments based on cellular size. DNA-PK activity was found to vary during the cell cycle. The kinase activity was lowest during G1 phase and increased dramatically as the cells entered S phase and remained high during the G2-phase. The subcellular distribution of DNA-PKcs is relocalized from the cytoplasm during M and G1 phases to the nucleus during G1-S phase transition and S phase. Expression of both the catalytic subunit and the Ku86/70 heterodimer was found to be constant throughout the cell cycle. This study demonstrates that DNA-PK activity as well as its subcellular localization fluctuates during the cell cycle. In addition, the distribution of DNA-PK during M phase corresponds with low DNA-PK activity.

DNA-dependent protein kinase content and activity in lung carcinoma cell lines: correlation with intrinsic radiosensitivity.

Intrinsic radiosensitivity and rejoining of radiation-induced DNA double-strand breaks (DNA-dsb) were analysed in five lung carcinoma cell lines: U-1285, U-1906, H-69, H-82 and U-1810. RS correlated with both the initial phase of DNA-dsb rejoining, at 15 min (r2 = 0.818) and the late phase, at 120 min postirradiation (r2 = 0.774), the most sensitive cell line (U-1285) showing least dsb rejoining and the most resistant (U-1810) showing most dsb rejoining of all five cell lines studied. As DNA-PK has been recognised as an important molecular component involved in DNA-dsb repair, we analysed content and activity of this kinase. We found that DNA-PK content and activity correlated with RS (r2 = 0.941 and r2 = 0.944, respectively). The lowest DNA-dependent content/activity was found in the most radiosensitive cells, U-1285 and H-69, whilst the highest content/activity was found in the most radioresistant cells U-1810. These results suggest a correlation between RS and DNA-PK content/activity in lung carcinoma cell lines.

Oral dosage of melphalan and response to treatment in multiple myeloma.

Forty-nine consecutive patients with multiple myeloma were analysed for treatment response in relation to dose of orally administered melphalan (induction therapy). All patients were given at least six courses of melphalan-prednisone. Treatment response, defined as a reduction of the myeloma protein of greater than 50%, was seen in 26 patients while 23 were non-responders. When treatment response was related to the dosage of melphalan given by mg/kg of body weight, the numbers of responding and non-responding patients were similar in the group of patients without dose reduction as well as in that with dose reduction. Drug-induced suppression of white blood cell and platelet count was similar in the responding as well as in the non-responding group indicating that the reason for non-response is not simply explained by deficient drug absorption. When the cumulative dose of melphalan given during the induction therapy was analysed, however, a positive correlation (r = 0.47, P less than 0.001) was seen between the cumulative dose and the degree of response. Thus, the cumulative dose of melphalan given during induction therapy seems to be of importance for the response, but other factors as intrinsic differences in cell sensitivity may also explain the individual responsiveness.

Response to radiotherapy of human uterine cervix carcinoma is not correlated with rearrangements of the Ha-ras-1 and/or c-myc genes.

An association between the presence of the activated form of Ha-ras-1 and c-myc genes and increased cellular radioresistance has been shown in several cell lines. The aim of this study was to determine whether such an association could be observed in clinical tumour biopsies. We examined 70 tumour specimens and 51 samples of peripheral blood obtained from untreated patients with carcinoma of the uterine cervix mainly stage II and III. In addition to initial clinical tumour response to radiotherapy, radiosensitivity was also analysed by the subrenal capsule assay (SRCA). Mutations in exons 1 and 2 of the Ha-ras-1 gene were examined using PCR single-strand conformation polymorphism (PCR-SSCP) and direct sequencing; and restriction fragment length polymorphism of the Ha-ras-1 gene was analysed using Southern hybridisation. Eight (11%) out of 70 tumours contained mutations in exons 1 and 2 of the Ha-ras-1 gene. Eleven (22%) out of the 51 tumours displayed rearrangements of the Ha-ras-1 gene (six tumours (12%) showed alterations of allele length, one amplification and four (8%) loss of one Ha-ras-1 allele). In addition, 12 (17%) out of 70 patients demonstrated the presence of rare alleles. Only one of the 70 tumours contained an amplified c-myc gene. There was no significant correlation between either rearrangements of the structure of the Ha-ras-1 and/or c-myc genes or presence of rare alleles in tumours and tumour response to radiotherapy.

Hypothyroidism after external radiotherapy for head and neck cancer.

PURPOSE: To study the development of thyroid hypofunction in patients with head and neck cancers admitted for external radiotherapy. METHODS AND MATERIALS: Between November 1990 and July 1996, thyroid function was measured in 264 consecutive patients, where the entire thyroid gland or part of it was included in the target volume. The time to development of hypothyroidism (HT) was calculated from the start of the radiotherapy. RESULTS: The median follow-up period was 19 months. Seventeen patients (6%) developed elevated serum thyroid-stimulating hormone levels with depressed (free) thyroxine levels (i.e., clinical HT). Elevated serum thyroid-stimulating hormone level with normal (free) thyroxine levels (i.e., chemical HT) developed in 57 (22%). The median time to clinical HT was 15 months (range: 7 to 32). The median time to chemical HT was also 15 months (range: 2 to 28). The actuarial risk of developing clinical or chemical HT 3 years after treatment was 15 and 40%, respectively. The incidence of chemical HT was significantly higher (p = 0.041) when the whole thyroid was included in the target volume compared to patients where only part of the thyroid was irradiated. The same trend was seen as regards clinical HT (p = 0.063). For those 20 patients who underwent laryngectomy, there was an increased risk of both chemical and clinical HT (p = 0.011 and 0.019, respectively). Increasing age was associated with an increased risk of chemical HT (p = 0.001), but not of clinical HT (p = 0.553). Sex, tumor site, radiation dose, and combination of radiotherapy and chemotherapy were not significant factors for thyroid hypofunction. CONCLUSION: Depressed thyroid function is common after external radiotherapy for cancers of the head and neck. Routine testing for possible thyroid hypofunction should be included in the follow-up procedures, even many years after end of radiotherapy.

DNA double-strand break repair, DNA-PK, and DNA ligases in two human squamous carcinoma cell lines with different radiosensitivity.

PURPOSE: Variation in sensitivity to radiotherapy among tumors has been related to the capacity of cells to repair radiation-induced DNA double-strand breaks (DSBs). DNA-dependent protein kinase (DNA-PK) and DNA ligases may affect DNA dsb rejoining. This study was performed to compare rate of rejoining of radiation-induced DSBs, DNA-PK, and DNA ligase activities in two human squamous carcinoma cell lines with different sensitivity to ionizing radiation. METHODS AND MATERIALS: Cell survival of two human squamous carcinoma cell lines, UM-SCC-1 and UM-SCC-14A, was determined by an in vitro clonogenic assay. DSB rejoining was studied using pulsed field gel electrophoresis (PFGE). DNA-PK activity was determined using BIOTRAK DNA-PK enzyme assay system (Amersham). DNA ligase activity in crude cell extracts was measured using [5'-33P] Poly (dA) x (oligo (dT) as a substrate. Proteolytic degradation of proteins was analyzed by means of Western blotting. RESULTS: Applying the commonly used linear-quadratic equation to describe cell survival, S = e-alphaD-betaD2, the two cell lines roughly have the same alpha value (approximately 0.40 Gy(-1)) whereas the beta value was considerably higher in UM-SCC-14A (0.067 Gy(-2)+/-0.007 Gy(-2) [SEM]) as compared to UM-SCC-1 (0.013 Gy(-2)+/-0.004 Gy(-2) [SEM]). Furthermore, UM-SCC-1 was more proficient in rejoining of X-ray-induced DSBs as compared to UM-SCC-14A as quantified by PFGE. The constitutive level of DNA-PK activity was 1.6 times higher in UM-SCC-1 as compared to UM-SCC-14A ( < 0.05). The constitutive level of DNA ligase activity was similar in the two cell lines. CONCLUSIONS: The results suggest that the proficiency in rejoining of DSBs is associated with DNA-PK activity but not with total DNA ligase activity.

Prognostic impact of complete remission after preoperative irradiation of tonsillar carcinoma: a retrospective analysis of the radiumhemmet data, 1980-1995.

PURPOSE: This retrospective study was done to determine the outcome of patients with tonsillar carcinoma treated at Radiumhemmet, Karolinska Hospital, between January 1980 and December 1995 with radiotherapy alone or in combination with surgery. In addition the importance of tumor remission for patient survival was analyzed. METHODS AND MATERIALS: The analysis is based on 167 previously untreated patients with biopsy-proven, invasive tonsillar squamous cell carcinoma of the tonsillar region. All patients were consecutively admitted to the Department of General Oncology, Radiumhemmet, and treated with curative intent. The median follow-up time was 20 months. The median target dose was 64 Gy, delivered in fractions of 2 Gy 5 times weekly. Twenty-eight percent of the patients underwent surgery of the primary site and/or neck dissection after radiotherapy (RT). RESULTS: The overall local control rate for the whole patient group after radiotherapy was 79%. Probability of survival after 5 years for patients responding with complete remission (CR) was 43% and for patients with incomplete response (non-CR) 9%, (p<0.0001). The survival in the non-CR group treated with combination therapy was 20 months longer than in patients receiving radiotherapy alone (p<0.0001). There was no statistically significant difference in prediction of long-term survival when the patient population was stratified according to tumor differentiation grade, age, sex, nodal status, or treatment time. CONCLUSION: The strongest clinical predictor of survival was the degree of tumor remission after RT. For the non-CR group receiving combination treatment including surgery there was a survival benefit as compared to patients treated with RT alone (p<0.0001) although there were few long-term survivors in this patient group.