Cell kinetics analysis in patients affected by squamous cell carcinoma of the head and neck treated with primary surgery and adjuvant radiotherapy.

BACKGROUND: The increasing complexity of management strategies for patients with head and neck squamous cell carcinoma (HN-SCC) calls for the investigation of new objective prognostic parameters to subdivide patients according to the tumor's biological aggressiveness. METHODS: We evaluated in 35 HN-SCC patients the pretreatment cell kinetics parameters and DNA ploidy after in vivo infusion of bromodeoxyuridine and flow cytometric analysis. Patients were treated with radical surgery followed by conventional radiation therapy. Locoregional control data are available for follow-up times above five years. RESULTS: We found that the likelihood of locoregional control for patients with rapidly proliferating HN-SCC characterized by a short potential doubling time (Tpot <5 days) was significantly smaller than for HN-SCC patients with slow tumor proliferation (Tpot >5 days). Moreover, when patients were stratified according to DNA ploidy and Tpot value, we found that the locoregional failure rate for rapidly proliferating tumors was significantly higher for diploid HN-SCCs than for aneuploid HN-SCCs. CONCLUSION: The present data suggest that patients with resectable HN-SCC characterized by fast growth might have a worse prognosis after surgery and adjuvant conventional radiotherapy and might benefit from more aggressive radiotherapeutic modalities.

Evidence of cell kinetics as predictive factor of response to radiotherapy alone or chemoradiotherapy in patients with advanced head and neck cancer.

PURPOSE: The aim of this study was to investigate the potential clinical relevance of cell kinetics parameters to the locoregional control (LRC) and overall survival of patients affected by head and neck squamous cell carcinoma (HN-SCC) treated by conventional radiotherapy, partly accelerated radiotherapy, or alternating chemoradiotherapy. METHODS AND MATERIALS: Between January 1993 and June 1996,115 patients with HN-SCC at Stage III and IV entered the study. Multiple primary tumor biopsies were obtained 6 h after in vivo infusion of bromodeoxyuridine (BrdUrd), an analogue of thymidine that is incorporated in DNA-synthesizing cells. In vivo S-phase fraction labeling index (LI), duration of S-phase (Ts), and potential doubling time (Tpot) were obtained by analysis of the flow cytometric content of BrdUrd and DNA. Eighty-two patients were randomly assigned to receive either alternating chemoradiotherapy or partly accelerated radiotherapy, whereas 33 other matching patients received conventional radiotherapy. RESULTS: Univariate LRC analysis showed that LI value was a prognostically significant factor, independent of type of therapy. Multivariate analysis failed to show cell kinetics parameters as statistically significant factors affecting LRC probability and overall survival. However, subgroup analysis showed that LRC probability at 4 years for fast proliferating tumors characterized by a LI >/= 8% was significantly better for patients treated either with alternating chemoradiotherapy or partly accelerated radiotherapy than it was for those treated with conventional radiotherapy. Conversely, LRC probability for slow proliferating tumors (LI < 8%) treated with the three treatment modalities was similar. CONCLUSIONS: These results showed that, independent of type of treatment, pretreatment cell kinetics provided only a weak prognostic role of outcome in HN-SCC. However, this report raises the hypothesis that fast growing HN-SCC may be more likely to benefit from intensified therapy, as given in this series. Cell kinetics parameters studied by the in vivo BrdUrd/flow cytometry method might be considered predictive factors of response, providing information on which type of treatment may be selected according to tumor proliferation rate.

Ki-ras activation in vitro affects G1 and G2M cell-cycle transit times and apoptosis.

Mutant ras genes occur frequently in human neoplasia and, in particular, in pancreatic, colorectal, and lung adenocarcinomas. Recent evidence suggests that G-->T and G-->C transversions of the Ki-ras gene in codon 12 may lead to biological effects in vitro and in vivo that may be associated with an abnormal cell cycle and increased tumour aggressiveness. The role of Ki-ras activation (a G-->C transversion in codon 12, arginine for glycine) in the cell cycle and apoptosis was investigated using control and permanently transfected NIH3T3 mouse fibroblasts. Flow cytometry was used to evaluate the G1-, S- and G2M-phase transit times, the potential doubling time, the growth fraction, and the cell loss factor during asynchronous exponential growth. Apoptosis was induced in both cell lines by absence of growth factors for an extended period of time (72 h) and quantitatively evaluated using the TUNEL method coupled with flow cytometry. It was found that codon 12 G-->C Ki-ras transfected cells compared with controls, had a significant prolongation of G1 by about 50%, a reduction of the G2M transit time by 30%, and a decrease of the cell loss factor by about 90%. Apoptotic cells were about 10% in control and less than 0.5% in Ki-ras transfected cells after 72 h starvation-confluency. These data suggest that codon 12 G-->C Ki-ras activation in mouse NIH3T3 fibroblasts is associated with deregulation of checkpoint controls in the G1 and G2M phases of the cell cycle and inhibition of apoptosis. It appears plausible that these cell mechanisms are related to a proliferative advantage and that they may also be important in the progression of human tumours characterized by specific Ki-ras mutations.

Analysis of erythrocyte glycophorin-A variants by flow cytometry in lung disease patients detects the effect of tobacco smoke.

The glycophoryn A (GPA) assay evaluates somatic in vivo mutations. It is considered a cumulative biodosimeter for genotoxic exposures and is under evaluation in cancer risk assessment. GPA, a polymorphic membrane protein of the erythrocytes, determines the MN blood groups. The NO and NN variant frequencies (VF) may be detected in MN subjects (about 50% of the population) by flow cytometry using two differently labelled antibodies. We explored if GPA NO and NN VF might be relevant to the assessment of individual lung cancer risk and susceptibility, in a small population with a high prevalence of heavy tobacco smokers: 8 lung cancer patients and 16 subjects with non-malignant lung diseases associated with increased risk of lung cancer. There was a wide interindividual variability and complete overlap between non-neoplastic and neoplastic patients. A significant positive correlation was seen with smoking duration in NO VF (p = 0.04, age-adjusted). Current smokers (n = 12) displayed higher NO values than never (n = 1) or ex-smokers (n = 11), 36.3 +/- 18.2 and 21.0 +/- 13.2, respectively (p < 0.01). No association was shown with occupational exposure. The present exploratory study suggests that assessment of individual lung cancer risk and susceptibility by the GPA assay does not seem to be feasible. The assay appears to provide a biomarker of longterm exposure to tobacco smoke.

Intratumor heterogeneity of k-ras and p53 mutations among human colorectal adenomas containing early cancer.

The molecular pathways and the timing of genetic events during human colorectal carcinogenesis are still not fully understood. We have addressed the intratumor heterogeneity of the mutational status of the k-ras oncogene and of the p53 oncosuppressor gene during the adenoma-carcinoma sequence by investigating 26 human colorectal adenomas containing early cancer. An intratumor comparative analysis was obtained among the adenomatous and carcinomatous component pairs. Additionally, we have analyzed 17 adenomas having cancer in the near vicinity. The adenomatous components of the adenomas containing early cancer and the adenomas having cancer in the near vicinity had comparable frequencies for k-ras mutations (28 and 47%) but different for p53 mutations (52 and 7%, p-value = 0.01). Interestingly, the adenomatous and carcinomatous components of the adenomas containing early cancer were rarely heterogeneous for the k-ras mutational status (only in 13% of the cases) but were characterized by heterogeneity of the p53 status in 59% of the cases (p-value < 0.01). In addition, the mutations of p53 for the adenomatous components of the adenomas containing early cancer were statistically significantly associated with severe dysplasia (p-value = 0.01). Intratumor homogeneity of k-ras status during the human colorectal adenoma-carcinoma sequence suggests that the role of k-ras is more related to tumor initiation than to tumor progression. On the contrary, intratumor heterogeneity of p53 mutations indicates that the type of the p53 mutations may also be relevant for selection and expansion of new subclones leading to tumor progression.

Cell cycle variations in azoxymethane-induced rat colorectal carcinogenesis studied by flow cytometry.

Cell cycle variations and DNA aneuploidy, were investigated in different phases of azoxymethane (AOM)-induced colon carcinogenesis in rats by flow cytometry. K-ras gene mutations (transitions Gright curved arrow A) were frequently detected in aberrant crypt foci (ACF) initial pre-neoplastic lesions. The fraction of cells in the G2M-phase of the cell cycle was higher in ACF compared to the normal mucosa of control rats. A similar modification of the cell cycle was found in adenomas and adenocarcinomas but, unexpectedly, also in morphologically normal mucosa from AOM-treated animals indicating that AOM treatment permanently modifies cell cycle control in rat colon mucosa. These alterations, however, were not associated with DNA aneuploidy as reported in human sporadic colorectal cancer, suggesting that tumour development in AOM-treated rats is less dependent on aneuploidy.

Intratumor heterogeneity of chromosome 1, 7, 17, and 18 aneusomies obtained by FISH and association with flow cytometric DNA index in human colorectal adenocarcinomas.

BACKGROUND: The origin and evolution of somatic chromosome aberrations in colorectal cancer is still poorly understood. The data in the literature suggest that some specific chromosome aberrations are more common. It is not known, however, if there is a correlation of these with near-diploid and high aneuploidy previously proposed to be a characteristic of the adenoma-carcinoma sequence. METHODS: Chromosome 1, 7, 17 and 18 numerical aberrations and 1p deletions were evaluated by fluorescence in situ hybridization analysis for 20 human sporadic colorectal adenocarcinomas in 70 distinct tumor sectors and correlated with flow cytometric DNA index (DI) values. RESULTS: Aneusomy for at least one of the investigated chromosomes was observed in 60 of 70 tumor sectors corresponding to 19 of 20 adenocarcinomas (95%). Deletions at 1p, observed in 8 of 18 adenocarcinomas (44%), were intratumor homogeneous in 7 of 8 tumors. In contrast, the other aberrations were intratumor heterogeneous. Aneusomies of chromosomes 1, 7, and 17 were strongly associated with DNA high aneuploidy (DI > or = 1.4), whereas aneusomy of chromosome 18 and 1p deletions were equally common among DNA diploid and near-diploid tumors (DI < 1.4 and DI not equal to 1). CONCLUSIONS: Overall, these data suggest the existence of different aneuploidization routes correlated with specific chromosome aberrations. In addition, intratumor homogeneity of 1p deletions appears to be an indication of early occurrence or strong selection. We also suggest that tumors with monosomies and in particular monosomies-trisomies for the same chromosomes support a model of aneuploidization and chromosome instability during the colorectal tumor progression based on loss of symmetry during chromosome segregation (Giaretti: Lab Invest 71:904-910, 1994).

K-ras2 activation and genome instability increase proliferation and size of FAP adenomas.

The possible role of K-ras2 mutations and aneuploidy toward increase of proliferation and adenoma size in Familial Adenomatous Polyposis (FAP) adenomas is not known. The present study addresses these issues by investigating 147 colorectal adenomas obtained from four FAP patients. The majority of adenomas had size lower than or equal to 10 mm (86%), low grade dysplasia (63%), and were preferentially located in the right colon (60%). Normal mucosa samples were obtained from 19 healthy donors. Three synchronous adenocarcinomas were also investigated. K-ras2 mutation spectrum was analysed by PCR and Sequence Specific Oligonucleotide (SSO) hybridization, while flow cytometry (FCM) was used for evaluating degree of DNA ploidy and S-phase fraction. Overall, incidences of K-ras2 mutations, DNA aneuploidy and high S-phase values (>7.2%) were 6.6%, 5.4% and 10.5%, respectively. In particular, among the adenomas with size lower than 5 mm, K-ras2 mutation and DNA aneuploidy frequencies were only slightly above 1%. Statistically significant correlations were found between K-ras2 and size, DNA ploidy and size and K-ras2 and S-phase (p < 0.001). In particular, among the wild type K-ras2 adenomas, high S-phase values were detected in 8% of the cases versus 57% among the K-ras2 mutated adenomas (p = 0.0005). The present series of FAP adenomas indicates that K-ras2 activation and gross genomic changes play a role toward a proliferative gain and tumour growth in size.

p53 mutations and DNA ploidy in colorectal adenocarcinomas.

The p53 tumour suppressor gene has an important role in the the maintenance of genome stability and its mutational inactivation may be at the origin of aneuploidy in cancer cells. The aim of this study was to determine whether p53 mutations were associated to DNA aneuploidy, as assessed by flow cytometry, in colorectal adenocarcinomas. Analysis of p53 mutations spectrum of the sorted nuclei was done by Denaturing Gradient Gel Electrophoresis (DGGE) and DNA sequencing. Overall, we studied 20 adenocarcinomas, the corresponding control mucosa, and 7 lymph node metastases. Five tumours (25%) were DNA diploid, while 15 tumours (75%) were composed of DNA aneuploid and diploid subpopulations. DNA diploid control mucosa and adenocarcinomas showed no p53 mutations, while 60% of the tumours with DNA aneuploidy had p53 mutations. Therefore, p53 mutations occurred significantly more often in DNA aneuploid than in DNA diploid tumours (p < 0.04, Fisher's exact test). Incidences of DNA aneuploidy and p53 mutations in lymph node metastases were 60 and 86%, respectively. In all tumours showing a p53 mutation, the wild-type allele was not or only bearly visible in DNA aneuploid cells suggesting that, in such cells, aneuploidy is accompanied by complete p53 functional inactivation. The present observations suggest that p53 mutations may have a role in the origin of aneuploidy at late stages of colorectal carcinogenesis.

Specific K-ras2 mutations in human sporadic colorectal adenomas are associated with DNA near-diploid aneuploidy and inhibition of proliferation.

Recent studies indicate that p21ras proteins mediate their multiple cell functions through interactions with multiple effectors and that the number of new effectors is growing. We recently reported that K-ras2 mutations in human colorectal adenomas were associated with chromosome instability and proliferation changes. In the present study, we extend these previous observations. Hereditary and multiple (n > or = 5) adenomas and adenomas with early cancer were excluded. Dysplasia was moderate in 91 cases and high in 25, and the median adenoma size was 1.5 cm. K-ras2 spectrum analysis was done by sequence-specific oligonucleotide hybridization using nuclear suspensions provided by analysis and sorting of multiparameter flow cytometry. In particular, tissue inflammatory cells were separated for DNA diploid tumors, whereas DNA aneuploid epithelial subclones were analyzed separately. K-ras2 mutations and DNA aneuploidy were both detected in 29 of 116 (25%) cases. DNA aneuploid index was in the near-diploid region in the majority of cases. DNA aneuploidy was strongly associated with G-->C/T transversions. An association was also found between low S-phase values and G-->A transitions. These findings were confirmed using multivariate logistic regression analysis to account for the effects of size, dysplasia, site, type, age, and sex. These data suggest that specific K-ras2 mutations in a subgroup of human sporadic colorectal adenomas play a role in chromosome instability and, contrary to expectations, are associated with inhibition of proliferation.

Correlation between 1p deletions and aneusomy in human colorectal adenomas.

Human sporadic colorectal adenomas are characterized by a relatively high occurrence of aneuploidy. Similarly, 1p deletions have been reported to be an early event in colorectal tumorigenesis, while chromosome 7, 17 and 18 gain/losses were also found. The present study investigated 1p deletions, the numerical aberrations of chromosomes 1, 7, 17 and 18, and the nuclear DNA content as obtained by flow cytometry in a series of 34 human sporadic colorectal adenomas. From these adenomas, 51 intra-adenoma regions were microdissected according to 2 degrees of dysplasia and presence of foci of early cancer. Isolated epithelial nuclei were analyzed by fluorescence in situ hybridization interphase cytogenetics using centromeric probes for chromosomes 7, 17 and 18 and, in a double-target analysis, a centromeric probe for chromosome 1 simultaneously with a telomeric probe mapping to the 1p36 band. Aneuploidy incidence due to presence of numerical aberrations for at least one among the investigated chromosomes and/or abnormal flow-cytometric DNA content was 35%, while 1p deletion incidence was 38%. The correlation of 1p deletions with aneuploidy was statistically highly significant (p = 0.003), suggesting that loss of genes in this region may be implicated in chromosome instability.

Preliminary characterization of a monoclonal antibody (AS-2) against cell cycle related proteins.

A monoclonal antibody (AS-2) raised by using isolated nuclei from a human erythroleukemia cell line as immunogen is described. AS-2 was of IgM type and recognized proteins present in both isolated cytoplasms and nuclei. The molecular weight of the AS-2 recognized proteins in the cytoplasm was 200 kDa and 70 and 60 kDa in the nucleus. The relative amount of these proteins were measured simultaneously with DNA content by flow cytometry. We found the highest protein content (or stainability) for both cells and nuclei in late-G1, S and G2, at approximately the same level, and the lowest content in M and early-G1. Sorting based on DNA content and AS-2 associated fluorescence helped identifying the staining pattern of cells and nuclei. Interphase isolated nuclei and cell cytoplasms were characterized by interdispersed staining over the entire surfaces while mitoses showed two dots only. The present preliminary data indicate that the proteins recognized by the AS-2 monoclonal are cell cycle related and suggest that in mitoses they are associated with the centrosomes.

Evidence of apoptosis in neuroblastoma at onset and relapse. An analysis of a large series of tumors.

Neuroblastoma (NB) is a tumor of pediatric age that is associated with high mortality in metastatic stages, although stage IVS patients undergo frequent spontaneous regression. Since apoptosis has been proposed as a possible cause of remission among cancer patients, we tested this hypothesis among both localized and metastatic NB and, in particular, NB metastatic stage IVS. We have assayed 36 localized and 117 metastatic neuroblastomas for evidence of internucleosomal DNA degradation and confirmed DNA fragmentation by the flow cytometric Terminal deoxynucleotidyl Transferase method, which also allowed us to measure DNA content and cell cycle phases. These techniques provided evidence of apoptosis in 18 out of 153 samples (11.8%), that were equally distributed among all stages except IVS, i.e. 11.1% in stage I (2/18), 11.1% in stage II (2/18), 13.2% in stage III (5/38), 13.4% in stage IV (9/67), and 0% in stage IVS (0/12). Tumor tissue samples collected at onset and also at relapse for the same patients showed that apoptosis may occur at relapse. In addition, cells appear to undergo apoptosis independently from N-myc amplification, cell cycle phase and DNA ploidy. In conclusion, apoptosis seems to take place with about an equal frequency for both favourable and unfavourable stages with an exception for IVS. Since DNA fragmentation remained undetected in stage IVS, we suggest that apoptosis is not a mechanism of spontaneous regression for these patients. A better basic understanding of the complex molecular mechanisms and biochemical pathways that control apoptosis in neuroblastoma appears to be necessary.

Chemoradiotherapy as an alternative to radiotherapy alone in fast proliferating head and neck squamous cell carcinomas.

The aim of this pilot study was to explore the prognostic relevance of cell kinetics parameters on the local control of patients affected by head and neck squamous cell carcinoma (HN-SCC), randomly assigned to receive either alternating chemoradiotherapy or partly accelerated radiotherapy. Between 1992 and 1995, 40 patients with HN-SCC at stages III and IV entered the study. Multiple primary tumor biopsies were obtained 6 h after in vivo infusion of bromodeoxyuridine, an analogue of thymidine that is incorporated in DNA-synthesizing cells. In vivo S-phase fraction labeling index (LI), duration of S-phase (TS), and potential doubling time (Tpot) were obtained by analysis of the flow cytometric content of bromodeoxyuridine and DNA. Twenty patients were treated by alternating chemotherapy and conventional radiotherapy (arm A), whereas 20 other matching patients received partly accelerated radiotherapy alone (arm B). Univariate local control analysis showed that LI, TS, and Tpot were not prognostically significant in either arm. However, local control probability at 2 years for fast growing tumors, characterized by a LI of 9%, was higher for patients treated with alternating chemoradiotherapy than it was for those treated with partly accelerated radiotherapy alone (68 versus 39%). Conversely, local control probabilities for slow proliferating tumors (LI, <9%) treated in the two arms were similar. These results suggest a potential role for alternating chemotherapy and radiotherapy in HN-SCC patients with fast growing tumors.

Cell kinetics and tumor regression during radiotherapy in head and neck squamous-cell carcinomas.

Head and neck squamous-cell carcinoma (HN-SCC) patient management is mainly based on TNM classification and needs be improved by considering other potentially useful prognostic factors. We examined the pre-radiotherapy tumor potential doubling time (Tpot) evaluated after in vivo infusion of bromodeoxyuridine and flow-cytometric analysis and the early clinical tumor regression after 40 Gy (40 Gy-TR). Tpot values and clinical 40 Gy-TR classes (minor and major) were available for 82 HN-SCC patients. Radiation therapy completion was done either with 1 dose per day (conventional regimen) or 2 doses per day (accelerated regimen). Local control was also available for follow-up times above 4 years. We found that major 40 Gy-TR was strongly correlated with fast tumor growth, characterized by Tpot values below 5 days, and that patients with major 40 Gy-TR showed better local control than those with minor 40 Gy-TR, independently from the radiotherapy regimen type. We also found that treatment completion with accelerated radiotherapy gave better local control for patients with major 40 Gy-TR and fast tumor growth than conventional radiotherapy. Multivariate analysis, performed on all patients, assigned an independent prognostic value to Tpot, tumor classification and 40 Gy-TR.

Transfection of human mutated K-ras in mouse NIH-3T3 cells is associated with increased cloning efficiency and DNA aneuploidization.

The aim of the study was to test the hypothesis that a human mutated K-ras protein induces abnormalities in mitosis and development of sub-clones characterized by changes in DNA ploidy and proliferation. For this purpose, we used control and NIH-3T3 mouse cells transfected with the human codon 12 G-C-mutated K-ras oncogene. We found that abnormal mitoses, mainly characterized by lagging chromosomes in prometaphase or anaphase, had a significantly higher frequency in transfected cells than in control cells. The generation of sub-clones was screened by limiting-dilution experiments followed by cell expansion. Cloning efficiency was much higher for the K-ras transfected cells with 858/2112 (41%) successful sub-clones than for control, which provided 564/2592 (22%) sub-clones. DNA flow cytometry of 4.6-diamidino-2-phenilindole-2-hydrochloride-stained nuclei from randomly selected sub-clones was performed in order to evaluate DNA index and S-phase fraction values. We found 9 out of 100 DNA aneuploid sub-clones generated by the K-ras-transfected cells vs. 1 out of 100 for the controls. Overall, our data indicate that high expression of the mutationally activated human K-ras product in NIH-3T3 cells was associated with abnormal mitoses, increase of cloning efficiency and DNA aneuploidization.

Intratumor heterogeneity of K-ras2 mutations in colorectal adenocarcinomas: association with degree of DNA aneuploidy.

Detailed information about intratumor K-ras2 mutations in colorectal adenocarcinomas and a possible association with DNA content heterogeneity is still lacking. DNA diploid and aneuploid subclones, detected among multiple histologically selected primary sectors (57 superficial and 40 deep) and 9 lymph node metastases, were flow cytometrically sorted and separately submitted to codons 12-13 K-ras2 mutation spectrum analysis. DNA aneuploidy was absent among 20 near and 20 distant mucosa sites and present in 7/9 lymph node metastases and in 17/19 primary tumors (90%). Primary intratumor DNA multiclonality was approximately 50%. Degree of DNA aneuploidy (DNA Index) distribution was nonrandom and showed peaks at approximate mean DNA Index values 1.2, 1.5, and 1.8. K-ras2 mutations were detected in 0/20 mucosa cases, in 2/9 lymph node metastases, and in 9/19 adenocarcinomas (47%). No more than one mutation type per tumor was detected. Intratumor distribution of K-ras2 mutations was homogeneous in 6 and heterogeneous in 3 cases. Homogeneous distribution was associated with DNA near-diploid aneuploidy. K-ras2 mutations were strongly associated with DNA Index in the near-diploid region (83%) and almost absent (5%) among DNA near-triploid subclones (P = 0.0001). K-ras2 mutation intratumor heterogeneity indicates that sampling of the tumor may be a critical step and suggests that K-ras2 activation may be a late event in a subgroup of tumors. Our data also suggest the existence of an early process of the colorectal carcinogenesis that favors both K-ras2 mutations and DNA near-diploid aneuploidy. Onset of DNA near-triploid subclones appears, instead, to be independent from K-ras2 activation.

In vivo cell kinetics in head and neck squamous cell carcinomas predicts local control and helps guide radiotherapy regimen.

PURPOSE: To determine whether pretherapy cell kinetics can predict local control for patients affected by head and neck squamous cell carcinomas (HN-SCCs) to be treated by primary radiotherapy and, moreover, guide to a choice between conventional and accelerated radiotherapy. PATIENTS AND METHODS: Between 1989 and 1993, 83 patients with stage II to IV HN-SCC entered the study. Multiple primary tumor biopsies were obtained 6 hours after in vivo infusion of bromodeoxyuridine (BrdUrd). In vivo S-phase fraction labeling index (LI), duration of S phase (Ts), and potential doubling time (Tpot) were obtained by analysis of multivariate flow-cytometric data. Between April 1989 and January 1991, 49 patients were treated by conventional radiotherapy (70 Gy in 35 fractions over 7 weeks), whereas, afterwards, 34 patients entered an accelerated radiotherapy regimen with the concomitant boost technique (75 Gy in 40 fractions over 6 weeks). RESULTS: Univariate analysis showed that, among patients treated by conventional radiotherapy, local control probability was affected by tumor stage (P = .02), Tpot (P < .001), and LI (P = .04). Similarly, among patients treated with accelerated radiotherapy, we found that local control probability was related to tumor stage (P = .03) and primary tumor site (P = .05). For the subgroup of patients with tumors characterized by fast growth (Tpot < or = 5 days), accelerated radiotherapy gave a better local control rate than conventional radiotherapy (P = .02). Cox multivariate analysis of the total number of patients showed that the only significant independent prognostic factors related to local control were tumor stage (P = .002) and Tpot (P = .004). Moreover, when the Cox analysis was restricted to the subgroup of patients treated with conventional radiotherapy, Tpot was the most significant factor to predict local outcome (P < .01). CONCLUSION: Pretreatment tumor Tpot appears to be an important independent prognostic factor for local control of HN-SCC treated by primary radiotherapy.