Effect of preoperative chemotherapy on the histopathological classification of gastric cancer.

BACKGROUND: In the era of individualized gastric cancer (GC) treatment, accurate determination of histological subtype becomes increasingly relevant. As yet, it is unclear whether preoperative chemotherapy may affect the histological subtype. The aim of this study was to assess concordance in histological subtype between pretreatment biopsies and surgical resection specimens before and after the introduction of perioperative treatment. METHODS: Histological subtype was centrally determined in paired GC biopsies and surgical resection specimens of patients treated with either surgery alone (SA) in the Dutch D1/D2 study or with preoperative chemotherapy (CT) in the CRITICS trial. The histological subtype as determined in the resection specimen was considered the gold standard. Concordance rates and sensitivity and specificity of intestinal, diffuse, mixed, and "other" subtypes of GC were analyzed. RESULTS: In total, 105 and 515 pairs of GC biopsies and resection specimens of patients treated in the SA and CT cohorts, respectively, were included. Overall concordance in the histological subtype was 72% in the SA and 74% in the CT cohort and substantially higher in the diffuse subtype (83% and 86%) compared to the intestinal (70% and 74%), mixed (21% and 33%) and "other" subtypes (54% and 54%). In the SA cohort, sensitivities and specificities were 0.88 and 0.71 in the intestinal, 0.67 and 0.93 in the diffuse, 0.20 and 0.98 in the mixed, and 0.50 and 0.93 in the "other" subtypes, respectively. CONCLUSION: Our results suggest that accurate determination of histological subtype on gastric cancer biopsies is suboptimal but that the impact of preoperative chemotherapy on histological subtype is negligible.

Clinically decisive (dis)agreement in multidisciplinary team assessment of esophageal squamous cell carcinoma; a prospective, national, multicenter study.

BACKGROUND: Decisions regarding tumor staging, operability, resectability, and treatment strategy in patients with esophageal cancer are made at multidisciplinary team (MDT) conferences. We aimed to assess interobserver agreement from four national MDT conferences and whether this would have a clinical impact. METHODS: A total of 20 patients with esophageal cancer were included across all four upper gastrointestinal (GI) cancer centers. Fully anonymized patient data were distributed among the MDT conferences which decided on TNM category, resectability, operability, curability, and treatment strategy blinded to each other's decisions. The interobserver agreement was expressed as both the raw observer agreement and with Krippendorff's α values. Finally, a case-by-case evaluation was performed to determine if disagreement would have had a clinical impact. RESULTS: A total of 80 MDT evaluations were available for analysis. A moderate to near-perfect observer agreement of 79.2%, 55.8%, and 82.5% for TNM category was observed, respectively. Substantial agreement for resectability and moderate agreement for curability were found. However, an only fair agreement was observed for the operability category. The treatment strategies had a slight agreement which corresponded to disagreement having a clinical impact in 12 patients. CONCLUSIONS: Esophageal cancer MDT conferences had an acceptable interobserver agreement on resectability and TM categories; however, the operability assessment had a high level of disagreement. Consequently, the agreement on treatment strategy was reduced with a potential clinical impact. In future MDT conferences, emphasis should be on prioritizing the relevant information being readily available (operability, T & M categories) to minimize the risk of disagreement in the assessments and treatment strategies, and thus, delayed or suboptimal treatment.

Adjuvant chemotherapy is superior to chemoradiation after D2 surgery for gastric cancer in the per-protocol analysis of the randomized CRITICS trial.

BACKGROUND: The Intergroup 0116 and the MAGIC trials changed clinical practice for resectable gastric cancer in the Western world. In these trials, overall survival improved with post-operative chemoradiotherapy (CRT) and perioperative chemotherapy (CT). Intention-to-treat analysis in the CRITICS trial of post-operative CT or post-operative CRT did not show a survival difference. The current study reports on the per-protocol (PP) analysis of the CRITICS trial. PATIENTS AND METHODS: The CRITICS trial was a randomized, controlled trial in which 788 patients with stage Ib-Iva resectable gastric or esophagogastric adenocarcinoma were included. Before start of preoperative CT, patients from the Netherlands, Sweden and Denmark were randomly assigned to receive post-operative CT or CRT. For the current analysis, only patients who started their allocated post-operative treatment were included. Since it is uncertain that the two treatment arms are balanced in such PP analysis, adjusted proportional hazards regression analysis and inverse probability weighted analysis were used to minimize the risk of selection bias and to estimate and compare overall and event-free survival. RESULTS: Of the 788 patients, 478 started post-operative treatment according to protocol, 233 (59%) patients in the CT group and 245 (62%) patients in the CRT group. Patient and tumor characteristics between the groups before start of the post-operative treatment were not different. After a median follow-up of 6.7 years since the start of post-operative treatment, the 5-year overall survival was 57.9% (95% confidence interval: 51.4% to 64.3%) in the CT group versus 45.5% (95% confidence interval: 39.2% to 51.8%) in the CRT group (adjusted hazard ratio CRT versus CT: 1.62 (1.24-2.12), P = 0.0004). Inverse probability weighted analysis resulted in similar hazard ratios. CONCLUSION: After adjustment for all known confounding factors, the PP analysis of patients who started the allocated post-operative treatment in the CRITICS trial showed that the CT group had a significantly better 5-year overall survival than the CRT group (NCT00407186).

Esophageal stenting for benign and malignant disease: European Society of Gastrointestinal Endoscopy (ESGE) Clinical Guideline

This Guideline is an official statement of the European Society of Gastrointestinal Endoscopy (ESGE), endorsed by the European Society for Radiotherapy and Oncology (ESTRO), the European Society of Digestive Endoscopy (ESDO), and the European Society for Clinical Nutrition and Metabolism (ESPEN). The Grading of Recommendations Assessment, Development, and Evaluation (GRADE) system was adopted to define the strength of recommendations and the quality of evidence. Main recommendations for malignant disease 1 ESGE recommends placement of partially or fully covered self-expandable metal stents (SEMSs) for palliative treatment of malignant dysphagia over laser therapy, photodynamic therapy, and esophageal bypass (strong recommendation, high quality evidence). 2 For patients with longer life expectancy, ESGE recommends brachytherapy as a valid alternative or in addition to stenting in esophageal cancer patients with malignant dysphagia. Brachytherapy may provide a survival advantage and possibly a better quality of life compared to SEMS placement alone. (Strong recommendation, high quality evidence.) 3 ESGE recommends esophageal SEMS placement as the preferred treatment for sealing malignant tracheoesophageal or bronchoesophageal fistula (strong recommendation, low quality evidence). 4 ESGE does not recommend the use of concurrent external radiotherapy and esophageal stent treatment. SEMS placement is also not recommended as a bridge to surgery or prior to preoperative chemoradiotherapy. It is associated with a high incidence of adverse events and alternative satisfactory options such as placement of a feeding tube are available. (Strong recommendation, low quality evidence.) Main recommendations for benign disease 1 ESGE recommends against the use of self-expandable stents (SEMSs) as first-line therapy for the management of benign esophageal strictures because of the potential for adverse events, the availability of alternative therapies, and costs (strong recommendation, low quality evidence). 2 ESGE suggests consideration of temporary placement of SEMSs as therapy for refractory benign esophageal strictures (weak recommendation, moderate evidence). Stents should usually be removed at a maximum of 3 months (strong recommendation, weak quality evidence). 3 ESGE suggests that fully covered SEMSs be preferred over partially covered SEMSs for the treatment of refractory benign esophageal strictures, because of their lack of embedment and ease of removability (weak recommendation, low quality evidence). 4 For the removal of partially covered esophageal SEMSs that are embedded, ESGE recommends the stent-in-stent technique (strong recommendation, low quality evidence). 5 ESGE recommends that temporary stent placement can be considered for treating esophageal leaks, fistulas, and perforations. The optimal stenting duration remains unclear and should be individualized. (Strong recommendation, low quality evidence.) 6 ESGE recommends placement of a SEMS for the treatment of esophageal variceal bleeding refractory to medical, endoscopic, and/or radiological therapy, or as initial therapy for patients with massive esophageal variceal bleeding (strong recommendation, moderate quality evidence).

PET hypoxia imaging with FAZA: reproducibility at baseline and during fractionated radiotherapy in tumour-bearing mice.

PURPOSE: Tumour hypoxia is linked to treatment resistance. Positron emission tomography (PET) using hypoxia tracers such as fluoroazomycin arabinoside (FAZA) may allow identification of patients with hypoxic tumours and the monitoring of the efficacy of hypoxia-targeting treatment. Since hypoxia PET is characterized by poor image contrast, and tumour hypoxia undergoes spontaneous changes and is affected by therapy, it remains unclear to what extent PET scans are reproducible. Tumour-bearing mice are valuable in the validation of hypoxia PET, but identification of a reliable reference tissue value (blood sample or image-derived muscle value) for repeated scans may be difficult due to the small size of the animal or absence of anatomical information (pure PET). Here tumour hypoxia was monitored over time using repeated PET scans in individual tumour-bearing mice before and during fractionated radiotherapy. METHODS: Mice bearing human SiHa cervix tumour xenografts underwent a PET scan 3 h following injection of FAZA on two consecutive days before initiation of treatment (baseline) and again following irradiation with four and ten fractions of 2.5 Gy. On the last scan day, mice were given an intraperitoneal injection of pimonidazole (hypoxia marker), tumours were collected and the intratumoral distribution of FAZA (autoradiography) and hypoxia (pimonidazole immunohistology) were determined in cryosections. RESULTS: Tissue section analysis revealed that the intratumoral distribution of FAZA was strongly correlated with the regional density of hypoxic (pimonidazole-positive) cells, even when necrosis was present, suggesting that FAZA PET provides a reliable measure of tumour hypoxia at the time of the scan. PET-based quantification of tumour tracer uptake relative to injected dose showed excellent reproducibility at baseline, whereas normalization using an image-derived nonhypoxic reference tissue (muscle) proved highly unreliable since a valid and reliable reference value could not be determined. The intratumoral distribution of tracer was stable at baseline as shown by a voxel-by-voxel comparison of the two scans (R = 0.82, range 0.72-0.90). During treatment, overall tracer retention changed in individual mice, but there was no evidence of general reoxygenation. CONCLUSION: Hypoxia PET scans are quantitatively correct and highly reproducible in tumour-bearing mice. Preclinical hypoxia PET is therefore a valuable and reliable tool for the development of strategies that target or modify hypoxia.

Hypoxia in human soft tissue sarcomas: adverse impact on survival and no association with p53 mutations.

Clinical and experimental studies have suggested that tumour hypoxia is associated with poor treatment outcome and that loss of apoptotic potential may play a role in malignant progression of neoplastic cells. The tumour suppressor gene p53 induces apoptosis under certain conditions and microenvironmental tumour hypoxia may select for mutant tumour cells with diminished apoptotic potential due to lack of p53 function. The aim of this study was to evaluate the prognostic relevance of oxygenation status for treatment outcome and to compare pre-treatment tumour oxygenation measurements were done in 31 of those by PCR using DNA extracted from paraffin-embaedded sections (n = 2) or frozen biopsies (n = 29). The overall median of the tumour median pO(2)was 19 mmHg (range 1-58 mmHg). Only 6 tumours had functional p53 mutations and no association was found between mutant p53 and tumour hypoxia. Five out of 6 STS with lower histopathological grade were well-oxygenated whereas high-grade STS were both hypoxic and well-oxygenated. At a median follow-up of 74 months, 16 patients were still alive among 28 available for survival analysis. When stratifying into hypoxic and well-oxygenated tumours patients with the most hypoxic tumours has a statistically poorer disease-specific and overall survival at 5 years. In conclusion hypoxia was an indicator for both a poorer disease specific and overall survival in human STS but hypoxic tumours were not characterized by mutations in the p53 gene.

Invasive oxygen measurements and pimonidazole labeling in human cervix carcinoma.

PURPOSE: This study was designed to compare tumor hypoxia assessed by invasive O2 sensitive electrodes and pimonidazole labeling in primary human cervix carcinomas. METHODS AND MATERIALS: Twenty-eight patients with primary cervix carcinomas (FIGO Stage Ib-IVa) were investigated. Both invasive pO2 measurements and pimonidazole labeling were obtained in all patients. Before treatment, patients were given pimonidazole as a single injection (0.5 g/m2 i.v.). Ten to 24 h later, oxygenation measurements were done by Eppendorf histography, and after this procedure biopsies were taken for pimonidazole-binding analysis. Tumor oxygen partial pressure (pO2) was evaluated as the median tumor pO2 and the fraction of pO2 values < or = 10 mmHg (HF10). Biopsies were formalin fixed and paraffin embedded, and hypoxia was detected by immunohistochemistry using monoclonal antibodies directed against reductively activated pimonidazole. Pimonidazole binding was evaluated by a semiquantitative scoring system. RESULTS: Both Eppendorf measurements and pimonidazole binding showed large intra-and intertumor variability. A comparison between pimonidazole binding expressed as the fraction of fields at the highest score and HF10 showed a trend for the most well-oxygenated tumors having a low fraction of fields; however, the correlation did not reach statistical significance (p = 0.43, r = 0.165; Spearman's rank correlation test). CONCLUSION: Hypoxia measured in human uterine cervix carcinomas is heterogeneously expressed both within and between tumors when assessed by either invasive pO2 measurements or pimonidazole binding. Despite a trend that tumors with high pO2 values expressed less pimonidazole binding, no correlation was seen between the two assays in this preliminary report.

A confirmatory prognostic study on oxygenation status and loco-regional control in advanced head and neck squamous cell carcinoma treated by radiation therapy.

INTRODUCTION: In a previous report of 35 patients we suggested that pretreatment tumour pO(2) measured by Eppendorf electrodes was predictive of loco-regional control after primary radiotherapy in advanced head and neck squamous cell carcinomas. Radiother Oncol 41 (1996) 31. MATERIALS AND METHODS: The aim of the present study was to test this hypothesis. Therefore, pretreatment tumour pO(2) was measured using the same assay in a new cohort of 35 patients that received an identical treatment being primary radiation along with a total dose of 66-68 Gy as 2 Gy per fraction over 5.5 or 6.5 weeks. Treatment outcome was evaluated as loco-regional control probability at 2 years using the same oxygenation parameter and applying the cut-off value from the first study. RESULTS: In agreement with the hypothesis generating study we found that the loco-regional tumour control probability was significantly higher (90%) among well oxygenated tumours as compared with the hypoxic subgroup (45%) (P=0. 04). CONCLUSION: This study confirmed that pretreatment tumour oxygenation status was prognostic of loco-regional tumour control after primary radiation alone in advanced head and neck squamous cell carcinoma.

Techniques to assess the oxygenation of human tumors. State of the art.

There is now good evidence that the oxygenation status of certain types of human tumors plays an important role in determining the response of those tumors to therapy. More recent evidence suggests that oxygenation status can also influence local tumor aggressiveness and metastatic spread. As a consequence significant effort is being made to find relevant methods for assessing tumor oxygenation status and thereby allowing for the selection of patients that should be given additional/alternative therapies to improve response. In this paper we will review the importance of tumor oxygenation status and discuss the potential clinical methods for monitoring that are currently available.

The effect of hypoxia and hyperoxia on nucleoside triphosphate/inorganic phosphate, pO2 and radiation response in an experimental tumour model.

This study has evaluated the effect of breathing 100% oxygen, carbogen and carbon monoxide (at 660 p.p.m.) on the bioenergetic and oxygenation status and the radiation response of 200-mm3 C3H mammary carcinomas grown in the feet of CDF mice. Bioenergetic status was assessed by 31P magnetic resonance spectroscopy (MRS) using a 7-tesla spectrometer with both short (2 s) and long (6 s) pulse repetition times. Tumour partial pressure of oxygen (PO2) was measured with an Eppendorf polarographic electrode; the oxygenation parameters were the median pO2 and fraction of pO2 values < or = 2.5 mmHg. The radiation response was estimated using a tumour growth delay assay (time to grow three times treatment volume). Carbon monoxide breathing decreased tumour pO2 and compromised the radiation response, but the beta-nucleoside triphosphate (NTP)/Pi ratio was unchanged. Both carbogen and oxygen (100%) increased tumour pO2 and beta-NTP/Pi and enhanced the radiation response, the effects being similar under the two gassing conditions and dependent on the gas breathing time. Thus, in this tumour model, 31P-MRS can detect hyperoxic changes, but because cells can remain metabolically active even at low oxygen tensions the beta-NTP/Pi did not correlate with low tissue oxygenation. An analysis of variance showed that gas breathing time induced a significant systematic effect on beta-NTP/Pi, the MRS pulse repetition time had a significant effect on beta-NTP/Pi change under hypoxic but not under hyperoxic conditions and the type of gas that was inhaled had a significant effect on beta-NTP/Pi.

Tumour oxygenation assessed by polarographic needle electrodes and bioenergetic status measured by 31P magnetic resonance spectroscopy in human soft tissue tumours.

The purpose of this study was to evaluate the feasibility of polarographic oxygen electrode measurements and phosphorus magnetic resonance spectroscopy (31P-MRS) in extravisceral soft tissue tumours, designated to receive preoperative radiotherapy. Pretreatment tumour oxygenation was determined in 41 cases and 31P-MRS was amenable to lesions in 34 patients. Biopsies were characterized histopathologically as 25 primary soft tissue sarcomas (STS), 2 recurrent STS, 9 benign and 5 other malignancies. Evaluation of phosphorus (31P) spectra was possible in 11 cases. The oxygenation status of normal tissue was higher than that of tumours, whereas no difference was found between oxygenation status of benign lesions and that of STS. There was substantial variation between tumours in the median pO2 and the bioenergetic status (beta-NTP/Pi). No correlation was found between tumour pO2 and volume (n = 25). Moreover, there was no correlation between beta-NTP/Pi and the median tumour pO2, the fraction of pO2 values < or =2.5 mmHg or tumour volume (n = 10), respectively. In conclusion, oxygen electrode assessment was found to be a clinically applicable and feasible technique for measuring tumour oxygenation status, whereas the success of 31P-MRS in human neoplasms was limited by a very poor resolution in the phosphorus signal that allowed analysis of 31P spectra in 11 tumours out of 34 cases.

Pretreatment oxygenation predicts radiation response in advanced squamous cell carcinoma of the head and neck.

BACKGROUND AND PURPOSE: Hypoxic tumor cells are known to be relatively radioresistant. The aim of the study was to correlate oxygenation status and radiation response in advanced squamous cell carcinomas of head and neck. METHODS AND PATIENTS: Pretreatment oxygenation status was measured in 34 lymph nodes and one primary tumor neck using oxygen electrodes. The primary oxygenation endpoint was the fraction of pO2 values less than 2.5 mmHg. Patients received standardized, conventional, external radiotherapy 66-68 Gy in 33-34 fractions. RESULTS: Sixteen patients had loco-regional failure. Among these 16 patients the median of the fraction of pO2 values less than 2.5 mmHg was 22% (range 0-95%) as compared to 6% (range 0-51%) among patients with loco-regional tumor control. When separating all 35 patients by the median of the fraction of pO2 values less than 2.5 mmHg and comparing the 2 years actuarial tumor control probability using Kaplan-Meier estimates, the most hypoxic subgroup had significantly lower loco-regional tumor control (P = 0.013, Logrank test). By univariate regression analysis the fraction of pO2 values less than 2.5 mmHg was found to be significant as continuous variable (P = 0.010). Finally, by Cox multiple regression analysis the fraction of pO2 values less than 2.5 mmHg was found to be the strongest independent variable in predicting radiation response when using tumor control in the site of pO2 assessment as treatment endpoint (P = 0.018). CONCLUSION: These results suggest that pretreatment tumor oxygenation status is predictive of radiation response, when using the fraction of pO2 values less than 2.5 mmHg as endpoint.

Effect of hydralazine in spontaneous tumours assessed by oxygen electrodes and 31P-magnetic resonance spectroscopy.

Hydralazine can substantially decrease the blood flow, oxygen status and energy metabolism of transplanted tumours. In spontaneous tumours, two recent studies reported no effects of hydralazine on energy metabolism measured by 31P-magnetic resonance spectroscopy (31P-MRS), although another study saw changes in oxygen partial pressure (pO2) distributions measured with electrodes. We have now performed 31P-MRS and pO2 measurements in the same T138 spontaneous tumours, before and after intravenously (i.v.) injecting anaesthetised mice with 5 mg kg-1 hydralazine. Tumour pO2 was measured with an Eppendorf oxygen electrode and 31P-MRS spectra obtained with a 7-tesla SISCO magnet. Of 12 tumours all showed an increase in the percentage of pO2 values < or = 5 mmHg after hydralazine treatment and 10/12 showed a decrease in median pO2. The average (+/-1 s.e.) values for the percentage < or = 5 mmHg went from 45% (+/-9) to 79% (+/-5) and the median from 9 mmHg (+/-2) to 2 mmHg (+/-1). With the 31P-MRS 8/12 tumours showed an increase in the ratio of the peaks of inorganic phosphate to beta-nucleoside triphosphate with the average (+/-1 s.e.) values going from 1.1 (+/-0.2) to 2.4 (+/-0.9). Thus spontaneous tumours can respond to hydralazine and do so in a way consistent with that reported for transplanted tumours.

The relationship between tumor oxygenation and cell proliferation in human soft tissue sarcomas.

PURPOSE: In malignant tumors the oxygenation status and tumor cell proliferation are known to influence local tumor control after radiotherapy. However, the relationship between oxygenation status and tumor cell kinetics in human tumors has not yet been described. Newly developed clinically applicable techniques such as oxygen electrode measurements and assessment of tumor cell proliferation rates have been suggested as promising predictive assays. The purpose of the present study was to characterize tumor oxygenation status in soft tissue sarcomas and to compare this with tumor cell kinetics and clinical parameters. METHODS AND MATERIALS: Pretreatment tumor oxygenation status was measured by polarographic oxygen needle electrodes and evaluated as the median pO2 and the percentage of pO2 values < or = 5 mmHg and < or = 2.5 mmHg in 22 patients with primary soft tissue sarcomas. All tumors were characterized by histology, grade of malignancy, the level of microscopic necrosis, the level of effective hemoglobin, and magnetic resonance imaging estimation of tumor volume. The tumor cell potential doubling time and labeling index were measured by flow cytometric and immunohistochemical analysis of tumor biopsy specimens after in vivo incorporation of iododeoxyuridine. RESULTS: There was a significant correlation between the median pO2 and the tumor cell potential doubling time (p = 0.041), whereas no correlation was found between the level of hypoxia expressed by the percentage of pO2 values < or = 2.5 and < or = 5 mmHg, respectively, and tumor cell potential doubling time. Furthermore, no correlation was found between either of the three tumor oxygenation parameters and labeling index. The material represented large intertumor heterogeneity in oxygenation status, cell kinetics, and tumor volume, and no correlation was found between oxygenation status and either volume, histopathology, grade of malignancy, or effective hemoglobin. CONCLUSION: This report is the first to suggest a correlation between tumor oxygenation and tumor cell doubling time, as the fastest proliferating tumor cells were found in the poorest oxygenated soft tissue sarcomas. More data are needed to clarify if this relation is really a true biological phenomenon. Furthermore, tumor oxygenation status of soft tissue sarcomas was heterogeneous and independent of clinical and histopathological parameters.

Direct evidence that hydralazine can induce hypoxia in both transplanted and spontaneous murine tumours.

Hydralazine can substantially decrease blood flow and increase hypoxia in transplanted tumours. Previous indirect studies have suggested that hydralazine does not induce such effects in spontaneous tumours. We have now directly investigated the ability of hydralazine to increase hypoxia in both transplanted and spontaneous murine tumours by measuring tumour oxygen partial pressure (pO2) distributions using an Eppendorf oxygen electrode. Spontaneous tumours arose at different sites in CDF1 mice, while transplanted tumours were produced by implanting a C3H mouse mammary carcinoma on the backs of the same mouse strain. Measurements of pO2 were made in anaesthetised mice immediately before and 45 min after an intravenous injection of 5 mg kg-1 hydralazine. In the transplanted tumours hydralazine significantly decreased tumour oxygenation, such that the percentage of pO2 values < or = 5 mmHg increased from 45% to 87%, and median pO2 decreased from 5 to 3 mmHg. Similar significant changes were induced by hydralazine in the spontaneous tumours, the percentage of pO2 values < or = 5 mmHg increasing from 60% to 94% while the median pO2 values decreased from 8 to 2 mmHg. These results clearly show that there is no difference in the response of transplanted and spontaneous mouse tumours to hydralazine.

Relationship between tumour oxygenation, bioenergetic status and radiobiological hypoxia in an experimental model.

Tumour oxygenation and bioenergetic status were measured in the same tumour and these results related to radiobiological hypoxia. A C3H mouse mammary carcinoma grown in the feet of CDF1 mice was used. Bioenergetic status was assessed by 31P MRS using a SISCO 7 Tesla magnet, oxygen measurements were done by a polarographic electrode and the hypoxic fraction was determined from direct analysis of the radiation dose-response data. During all examinations restrained, non-anaesthetized mice were allowed to breathe either 100% oxygen, carbogen, normal air, carbon monoxide (CO) at 75, 220, or 660 ppm or had blood flow occluded by clamping. Results showed a significant correlation between the radiobiological hypoxic fraction and % pO2 < or = 5 mmHg under the different treatment conditions, whereas no correlation was found between beta nucleosidetriphosphate/inorganic phosphate (beta-NTP/Pi) ratio and either the hypoxic fraction or the % of pO2 values < or = 5 mmHg under the different treatment conditions. In conclusion, oxygen electrode measurements were sensitive to changes in tumour hypoxia whereas the bioenergetic status alone seemed to be a less precise measure of hypoxia in this tumour model. Furthermore, the present study demonstrated that tumour cells in vivo can actually maintain the bioenergetic status during a period of severe hypoxia.

Effect of carbon monoxide breathing on hypoxia and radiation response in the SCCVII tumor in vivo.

PURPOSE: To study the influence of a clinically relevant concentration of carbon monoxide (CO) on tumor oxygenation and response to irradiation. METHODS AND MATERIALS: The murine tumor model was the SCCVII squamous cell carcinoma transplanted to the feet of C3H/Km mice. RESULTS: Sixty minutes of breathing CO at 200 ppm resulted in a carboxyhemoglobin level of 15%. This resulted in a reduction in p50 (the oxygen partial pressure at which hemoglobin is 50% saturated) to 78% of the control value, and a decrease in tumor blood perfusion to 73% of the control value. The combined effect of a decrease in effective hemoglobin and blood perfusion resulted in a reduction in tumor oxygen supply to 62% of the control value. In agreement with this, intratumoral pO2 measurements showed a significant increase in tumor hypoxia, such that the percentage of measurements with low pO2 (< or = 5 mmHg) increased from 33% to 62%. The fraction of clonogenic hypoxic cells, measured radiobiologically by paired cell survival curves, similarly increased from 0.2% to 3.8%. Radiation sensitivity, evaluated from in vivo-in vitro excision assay, was significantly decreased by CO breathing with both single dose and fractionated irradiation. The observed enhancement ratios for radiation given in 1, 4, 8, and 12 fractions were 0.71, 0.77, 0.83, and 0.71, respectively. CONCLUSION: The present SCCVII tumor data confirm the general experimental observation that CO breathing significantly increases tumor hypoxia and reduces the effectiveness of ionizing irradiation.