Immunohistochemical detection of HIF-1alpha and CAIX in advanced head-and-neck cancer. Prognostic role and correlation with tumor markers and tumor oxygenation parameters.

BACKGROUND: Tumor hypoxia has an impact on the outcome of cancer patients treated with radiotherapy. The validity of endogenous markers such as hypoxia-inducible factor-1alpha (HIF-1alpha) and carbonic anhydrase isozyme IX (CAIX) to detect therapeutically relevant Levels of hypoxia within tumors is controversially discussed. Furthermore, the association of these hypoxia markers with tumor markers or tumor oxygenation parameters is of importance for understanding the relationship between the different factors. PATIENTS AND METHODS: Tumortissue sections of 34 patients with advanced head-and-neck cancertreated with radio(chemo)therapy were assessed by immunohistochemistry for the expression of HIF-1alpha and CAIX. The relationships of both markers with tumor oxygenation parameters, molecular factors like P53, OPN, VEGF, VHL, survivin, and Ki67 levels, and clinical parameters were studied. RESULTS: Bivariate analysis showed a significant correlation of HIF-1alpha expression with high P53 and high OPN expression, high serum VEGF Levels, and low VHL and low Ki67 expression. The CAIX expression was inversely correlated with pH value and directly correlated with T-stage. However, no correlation was found between HIF-1alpha and CAIX expression. Neither in a univariate Cox proportional hazard regression nor in a Kaplan-Meier analysis did expression of HIF-1alpha or CAIX have a significant impact on clinical outcome. However, in a Kaplan-Meier analysis, the combination of both factors showed that patients with intratumoral overexpression of either HIF-1alpha or CAIX or both markers died on average 2 years earlier than patients whose tumors had low expression of both factors (p < 0.05). CONCLUSION: Expression of HIF-1alpha and CAIX was correlated with different tumor parameters. Only combined HIF-1alpha and CAIX expression was significantly predictive of patients' overall survival.

Mitochondrial OXPHOS functions in R1H rhabdomyosarcoma and skeletal muscles of the rat.

The aim of the study was to determinate mitochondrial oxidative phosphorylation (OXPHOS) functions in rat rhabdomyosarcoma R1H (R1H) and rat skeletal muscles. For that purpose skinned fiber technique and multiple substrate inhibitor titration were adapted to tumor samples. In our animal tumor model (R1H) functional abnormalities of OXPHOS were found compared to skeletal muscles. In R1H the state 3 respiration of pyruvate + malate was decreased: 0.56 +/- 0.28 nmol O(2)/mg/min versus 2.32 +/- 1.19 nmol O(2)/mg/min, P < 0.001, whereas the state 3 respiration of succinate + rotenone was increased: 36 +/- 14% versus 19 +/- 11%, P < 0.001. In R1H the rotenone-insensitive respiration reached higher levels than the antimycin A-insensitive respiration, whereas in normal muscles the converse was observed. Additionally, the obvious difference between the CAT- and the antimycin A-independent respiration indicates an increased part of leak respiration in R1H. By now, the high feasibility of these techniques is appreciated for the investigation of muscles and prospectively for tumors, too.

Survivin protein expression and hypoxia in advanced cervical carcinoma of patients treated by radiotherapy.

BACKGROUND: Survivin is strongly overexpressed in the vast majority of cancers. Initial investigations suggest a role for Survivin in radiation resistance. In this study, we investigate the effect of Survivin expression on clinical outcome and its relationship to tumor oxygenation parameters, expression of Hif-1alpha and anemia in patients with advanced cervical cancers treated with radiotherapy. MATERIAL AND METHODS: Biopsies of 44 patients with cervical cancers (Stage IIB: n=9; Stage IIIB: n=31; Stage IVA: n=4) treated with radiotherapy were assessed by immunochemistry for expression of Survivin. Relation of Survivin to pretreatment tumor oxygenation parameters (HF5, pO(2)), hemoglobin (hb) level, Hif-1alpha expression and clinical parameters were investigated. RESULTS: Survivin expression was detected in all tumors of the 44 patients. Seven showed a strong expression and 37 have moderate Survivin expression. Patients whose tumors showed moderate Survivin expression had a 5-year overall survival of 66%. However, only one of the seven patients with strong Survivin expression was alive 45 months after treatment. In a Cox regression analysis, Survivin expression was correlated to poor overall survival (p=0.02, RR=3.3). There was no relationship between Survivin expression and pO(2) or HF5, but rather an inverse correlation with hemoglobin level (p=0.04). Furthermore, for six of the seven tumors with a high Survivin expression, Hif-1alpha was detected. CONCLUSION: Survivin protein expression is linked with anemia and prognosis in advanced cervical carcinoma of patients treated by radiotherapy.

Immunohistochemical detection of osteopontin in advanced head-and-neck cancer: prognostic role and correlation with oxygen electrode measurements, hypoxia-inducible-factor-1alpha-related markers, and hemoglobin levels.

PURPOSE: The tumor-associated glycoprotein osteopontin (OPN) is discussed as a plasma marker of tumor hypoxia. However, the association of immunohistochemical OPN expression in tumor sections with tumor oxygenation parameters (HF5, median pO(2)), the hypoxia-related markers hypoxia-inducible factor-1alpha (HIF-1alpha) and carbonic anhydrase IX (CAIX), or hemoglobin and systemic vascular endothelial growth factor (VEGF) levels has not been investigated. METHODS AND MATERIALS: Tumor tissue sections of 34 patients with advanced head-and-neck cancer treated with radiotherapy were assessed by immunochemistry for the expression of OPN, HIF-1alpha, and CA IX. Relationship of OPN expression with tumor oxygenation parameters (HF5, median pO(2)), HIF-1alpha and CA IX expression, hemoglobin and serum VEGF level, and clinical parameters was studied. RESULTS: Bivariate analysis showed a significant correlation of positive OPN staining with low hemoglobin level (p = 0.02), high HIF-1alpha expression (p = 0.02), and high serum vascular endothelial growth factor level (p = 0.02) for advanced head-and-neck cancer. Furthermore, considering the 31 Stage IV patients, the median pO(2) correlated significantly with the OPN expression (p = 0.02). OPN expression alone had only a small impact on prognosis. However, in a univariate Cox proportional hazard regression model, the expression of either OPN or HIF-1alpha or CA IX was associated with a 4.1-fold increased risk of death (p = 0.02) compared with negativity of all three markers. CONCLUSION: Osteopontin expression detected immunohistochemically is associated with oxygenation parameters in advanced head-and-neck cancer. When the results of OPN, HIF-1alpha, and CA IX immunohistochemistry are combined into a hypoxic profile, a strong and statistically significant impact on overall survival is found.

Plasma osteopontin levels in patients with head and neck cancer and cervix cancer are critically dependent on the choice of ELISA system.

BACKGROUND: The tumor-associated glycoprotein osteopontin (OPN) is discussed as a plasma surrogate marker of tumor hypoxia and as an indicator of the presence of pleural mesothelioma in asbestos-exposed individuals. The clinical introduction of plasma OPN measurements requires the availability of a reliable enzyme-linked immunosorbence assay (ELISA). METHODS: We compared previously described and currently available ELISA systems on 88 archival plasma samples obtained from patients with head and neck or cervix cancer between 20 days before and 171 after the start of radiotherapy. RESULTS: Median (range) plasma OPN levels were 667 (148.8-2095) ng/ml and 9.8 (3.5-189.5) ng/ml for a previously described and a newly marketed assay, respectively. Although results for different assays were significantly correlated (r = 0.38, p < 0.05, Spearman rank test), between-assay factors ranged from 2.0 to 217.9 (median 74.6) in individual patients. OPN levels in cervix cancer patients were comparable to those of head and neck cancer patients. CONCLUSION: Commercially available OPN ELISA systems produce different absolute plasma OPN levels, compromising a comparison of individual patient data with published results. However, different assays appear to have a similar capacity to rank patients according to plasma OPN level. A review of literature data suggests that plasma OPN levels measured even with identical ELISA systems can only be compared with caution.

[Presentation of a 3D conformal radiotherapy technique for head-and-neck tumors resulting in substantial protection of the parotid glands]. / Etablierung einer 3D-Konformationstechnik zur Radio therapie von Kopf-Hals-Tumoren unter Berücksichtigung der Parotisschonung.

PURPOSE: The aim of this study was to improve the irradiation technique for the treatment of head-and-neck tumors and, in particular, to make use of the advantages found in modern 3D planning to protect the parotid glands. PATIENTS AND METHODS: For this investigation the 3D dataset of a standard patient with oropharyngeal carcinoma of UICC stage IVA was used. In the CT scans (slice thickness 5 mm) the planning target volume (PTV), the boost volume and both parotids were delineated. Three different techniques were calculated for two different dose levels (50 Gy for PTV and 64 Gy for boost volume, using single doses of 2 Gy). For technique 1 (T1) a parallel opposed field photon/electron irradiation was designed, for technique 2 (T2) an opposed/arc field irradiation was employed, and for technique 3 (T3) a combination of a static coplanar and arc field irradiation was designed. The sum doses D(min), D(max) and D(mean) for PTV, boost volume, and ipsilateral and contralateral parotid gland were evaluated, and the time needed for calculation of the plans was also determined. RESULTS: For all techniques used, the calculated doses in the PTV (D(min) 5.6 +/- 0.1 Gy, D(max) 73.7 +/- 0.1 Gy, and D(mean) 57.9 +/- 0.5 Gy) and in the boost volume (D(min) 46.9 +/- 1.5 Gy, D(max) 73.8 +/- 0.12 Gy, and D(mean) 65.8 +/- 0.9 Gy) were equal. Significant differences were found regarding the three different techniques, e.g., for the ipsilateral parotid gland D(min) (T1 = 47.4, T2 = 50.6, and T3 = 38.4 Gy) as well as for the contralateral parotid gland D(min) (T1 = 42.1, T2 = 44.2, and T3 = 17.8 Gy) and D(mean) (T1 = 51.3, T2 = 52.8, and T3 = 32.6 Gy). Regarding the three different techniques, significant differences were found in favor of T3. The determined planning times were as follows: T1 = 90, T2 = 60, and T3 = 90 min. CONCLUSION: The combination of static coplanar and arc field technique (T3) resulted in a substantially better protection as compared to both other techniques. This was especially the case with regard to the contralateral parotid gland, when the dose distributions were calculated equally for PTV and boost volume. In this study, the D(mean) dose of the contralateral parotid gland was lower than the TD(50) of 37 Gy (95% confidence interval 32-43 Gy) previously assumed by the authors. Therefore, it can be concluded that in the present study a more intensive protection of this gland and a reduction in xerostomia were possibly obtained.

[Prognostic factors after surgery and adjuvant radiotherapy in cervical cancers. A retrospective analysis of 298 patients after surgery and adjuvant radiotherapy]. / Prognosefaktoren beim operierten und adjuvant bestrahlten Zervixkarzinom. Eine retrospektive Analyse von 298 operierten und nachbestrahlten Patientinnen.

PURPOSE: To determine prognostic factors in patients with cervical cancer treated with surgery followed by radiotherapy. PATIENTS AND METHODS: In a retrospective analysis, patients treated at the Department of Radiotherapy, University of Halle-Wittenberg, Germany, from 1980 through 1993 were evaluated for local control, survival and treatment sequelae with special emphasis on prognostic factors. 298 patients (age 23-81 years, median 46 years) with cervical cancers were treated with surgery followed by radiotherapy to a minimum of 50 Gy. 250 patients received radiotherapy after radical hysterectomy with pelvic lymphadenectomy and 48 patients after hysterectomy. Most of the cancers were squamous cell carcinomas (81%) with few adenocarcinomas (12.4%). Grading was G1/G2 in 28.9% of the patients and G3/G4 in 39.6%. RESULTS: The 5-year overall survival was 68.8% for the whole group, 70.5% for squamous cell carcinomas (n = 241), 64.4% for adenocarcinomas (n = 37; not significant), 80% for G1/G2 tumors (n = 86), and 69% for G3/G4 carcinomas (n = 118; p = 0.074). Survival dependent on tumor size and positive nodes was 76.2% (n = 94) for pT1N0, 65.8% for pT1N+ (n = 31), 68.2% for pT2N0 (n = 62), and 41.4% for pT2N+ (n = 35). An impact of grading on 5-year survival was only found in patients with positive nodes: G1/G2N0 84% (n = 62) versus G1/G2N+ 64.8% (n = 23); G3/G4N0 79.6% (n = 71) versus G3/G4N+ 39.9% (n = 31). The number of positive nodes was the strongest prognostic factor. Multivariate analysis (Cox regression) established a risk factor of 3.06 (p < 0.01) for positive nodes, of 1.7 for grading G1/G2 versus G3/G4 (p = 0.087), and of 1.3 for tumor size (pT1 vs. pT2; p = 0.079). There were no differences in the clinical outcome between patients in stage pT1 with or without lymphadenectomy (75% [n = 130] vs. 79% [n = 39]). CONCLUSION: In patients with cervical cancers treated with surgery and radiotherapy, positive nodes were an independent prognostic factor for local control and survival and should be an indication for additional chemotherapy. A value of lymphadenectomy for survival in this group of irradiated patients could not be established.

Aggressive simultaneous radiochemotherapy with cisplatin and paclitaxel in combination with accelerated hyperfractionated radiotherapy in locally advanced head and neck tumors. Results of a phase I-II trial.

BACKGROUND: Simultaneous radiochemotherapy (sRCT) is the treatment of first choice in locally advanced head and neck cancers. We have tested a very aggressive combination protocol with cisplatin and escalated paclitaxel in combination with accelerated hyperfractionated radiotherapy to assess the maximum tolerated dose (MTD), dose-limiting toxicity (DLT), overall toxicity, and response rate. PATIENTS AND METHODS: The trial recruited 24 patients (21 males, three females, mean age 57 years) treated at our department from 1998 through 2001. Irradiation was administered in daily doses of 2 Gy up to 30 Gy followed by 1.4 Gy twice daily up to 70.6 Gy to the primary tumor and involved nodes and 51 Gy to the clinically negative regional nodes. The chemotherapy schedule included cisplatin in a fixed dose of 20 mg/m(2) on days 1-5 and 29-33 and paclitaxel at increasing dose levels of 20, 25, 30 mg/m(2) twice weekly over the whole treatment time. Patients were recruited in cohorts of three to six, and the MTD was reached if two out of six patients in one cohort developed DLT. DLT was defined as any grade 4 toxicity or any grade 3 toxicity requiring treatment interruption or unplanned hospitalization or any grade 3 neurotoxicity. We recruited mainly patients with large tumors for this protocol; all patients were stage IV, and the mean tumor volume (primary + metastases) amounted to 72 +/- 61 cm(3). The mean follow-up was 30 months (range 4-39 months). RESULTS: One early death (peritonitis and sepsis at day 10) occurred, and 23 patients were evaluable for acute toxicity and response. The MTD of paclitaxel was reached at the third dose level (30 mg/m(2) paclitaxel twice weekly). The DLT was severe mucositis grade 3 (n = 1) and skin erythema grade 4 (n = 2). After determining the MTD, another 14 patients were treated at the recommended dose level of paclitaxel with 25 mg/m(2) twice weekly. In summary, 13/23 patients (57%) developed grade 3 and 10/23 (43%) grade 2 mucositis. Two patients (9%) had grade 4, five (22%) grade 3, and 16 (69%) grade 2 dermatitis. One patient died at day 30 of neutropenic infection. In one patient, a grade 2 nephrotoxicity appeared requiring cessation of cisplatin chemotherapy. 18/23 patients (78%) required blood transfusion (1-3 units) and 16/23 (70%) i.v. antibiotics. 14 patients (61%) achieved a complete and nine (39%) a partial remission, yielding an overall response rate of 100%. In summary, six patients died of local tumor progression (n = 2), distant metastases (n = 2), or therapy-related complications (n = 2) during follow-up. The 3-year overall survival was 71%. Tumor volume was not a risk factor for failure in this protocol (mean tumor volume in relapse-free vs. progressive patients 71 +/- 65 cm(3) vs. 64 +/- 38 cm(3)). All patients have, so far, developed only slight late effects (fibrosis, lymphedema) with no grade 3-4 late sequelae. CONCLUSIONS: This very aggressive sRCT protocol yielded excellent response and survival figures but was associated with a very high rate of acute toxicity (8% therapy-related deaths). A maximal supportive treatment is therefore required.

Tumor volume and tumor hypoxia in head and neck cancers. The amount of the hypoxic volume is important.

BACKGROUND: The prognostic impact of tumor volume and hypoxia is well established. We have investigated a possible prognostic impact of the hypoxic tumor volume which can be calculated as the product of tumor volume and hypoxia. PATIENTS AND METHODS: 125 patients with squamous cell cancer of the head and neck were investigated. All had locoregionally confined disease. The total tumor volume was calculated from pretreatment CT scans as the sum of all visible macroscopic tumor lesions (e.g., primary tumor plus neck nodes), and all patients underwent measurement of tumor oxygenation by pO2 histography. The hypoxic tumor volume was calculated as the product of the total tumor volume and the relative frequency of pO2 readings < 5 mmHg. The nonhypoxic volume was the difference between total tumor volume hypoxic volume. RESULTS: The total tumor volume ranged from 2 to 283 cm3 (mean 47 +/- 53 cm3), the hypoxic volume from 0 to 199 cm3 (mean 18 +/- 30 cm3), and the nonhypoxic volume from 1 to 237 cm3 (mean 29 +/- 34 cm3), and there was a strong correlation between the three parameters. 84 patients died and 41 survived in the observation period with a median survival of 12.5 months. Tumor volume and tumor oxygenation had a significant impact on survival. The tumor volume was significantly different in patients who had died as compared to surviving patients (mean 54 vs. 34 cm3; p = 0.017). The hypoxic volume was also different (11 vs. 22 cm3; p = 0.009), whereas the nonhypoxic volume was not significantly different (24 vs. 32 cm3; p = 0.2). If the impact of large versus small tumor volumes (total volume, hypoxic volume, and nonhypoxic volume, subdivision according to each median) on survival was analyzed, a significant impact of total tumor volume (median survival 298 vs. 485 days; p = 0.03) and a marginal impact of the hypoxic volume (342 vs. 404 days; p = 0.08), but no impact of the nonhypoxic volume were found (383 vs. 374 days; p = 0.6). In a multivariate Cox regression model, the hypoxic tumor volume was a strong and independent prognostic factor for survival (p = 0.001) and more important than the total tumor volume (p = 0.02) whereas the nonhypoxic volume had no impact on prognosis (p = 0.33). CONCLUSIONS: The total tumor volume is a major prognostic factor, but its impact mainly results from the hypoxic volume and can be explained by the strong correlation between total tumor volume and hypoxic volume. The nonhypoxic volume had no impact on survival. As a consequence, methods to measure and localize the hypoxic volume should be further developed.

[Adjuvant simultaneous radiochemotherapy after operated uterine cervix carcinoma in high risk situation. Results of a pilot study]. / Adjuvante simultane Radiochemotherapie nach operiertem Uteruszervixkarzinom in der High-Risk-Situation. Ergebnisse einer Pilotuntersuchung.

BACKGROUND: The most important factors for prognosis of cervical cancers are age and histological criteria such as the tumor size, the involvement of lymph nodes, lympho-vascular space involvement as well as microvessel involvement and poor tumor differentiation (grading 3). Here we present the results of concomitant chemo-radiation at high-risk situation of patients with cervical cancer after surgery. PATIENTS AND METHODS: The study comprised 34 patients with median age of 40 years (26-63 years) after Wertheim surgical technique for cervical cancer at the FIGO Stages IB (n = 19) and IIB (n = 15). All patients were treated between November 1995 and June 1999 by a schedule of concomitant chemoradiation. The indication for this treatment was given by the positive histological proof of lymph node metastasis, microvessel or lympho-vascular space involvement as well as a G3 grading. The chemo-therapy was given in week 1 and 5 (day 1-5 and day 29-33). The dosage of cisplatin was 20 mg/m2/d on every day and 5-FU was given as a 120-h infusion with 600 mg/m2/d. The external beam radiotherapy was applied to the pelvis with 1.8 Gy per fraction up to 50.4-54 Gy. In two patients the paraaortal region was irradiated too because of the involvement of these lymph nodes. RESULTS: The median observation time was 48 months (3-68 months). 30 patients are alive (88%) in complete response. Four patients died. The mean survival was 61 +/- 3 months. We have seen only slight acute toxicities of grade 1 and 2. Three patients suffered from a grade 3 diarrhea and three patients developed a grade 3 leukopenia. In seven patients we found a secondary lymphedema as a late toxicity. CONCLUSION: The concomitant chemoradiation containing cisplatin in high-risk situation for cervical cancer after surgery improves the outcome and survival in these patients.