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Introduction: Prostate cancer hypoxia is a negative prognostic biomarker. A promising MRI-based tool to assess hypoxia is the 'Consumption and Supply based Hypoxia' (CSH) model based on diffusion-weighted imaging (DWI). The aim of the study was to validate the association between the CSH hypoxia fraction (HFDWI) with pathological Grade Group (pGG) and pathological T-staging (pTstage) in an external prostatectomy cohort. Methods: Apparent diffusion coefficient (ADC) and fractional blood volume (fBV) maps were assessed from DWI data from 291 prostatectomies and combined by the CSH model. HFDWI was calculated for each lesion after median scaling of ADC and fBV to address differences in acquisition and analysis between centers. The absolute HFDWI values and the associations of HFDWI between pGG < 3 versus ≥ 3, and pTstage = 2 versus = 3 in the Netherlands Cancer Institute (NKI) cohort were compared to the obtained by original cohort (Oslo cohort). Statistical T- and Mann-Whitney tests (p<0.05) were performed. Pearson correlation was determined between HFDWI and individual pGG groups. Results: The HFDWI showed comparable absolute values and similar metric performance as in the original published cohort. Higher HFDWI values were observed for higher pGG (Oslo: 0.27; NKI: 0.24) compared to lower pGG (Oslo: 0.11; NKI: 0.17). Similar results were obtained for pTstage. Furthermore, HFDWI demonstrated a significant positive correlation with pGG groups 1-5 (ρ = 0.41, p<0.001). Conclusion: The CSH model exhibited sufficient robustness in the external cohort, suggesting a plausible reflection of true hypoxia and enabling the use of the HFDWI metric for further research into prostate cancer and hypoxia.

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BACKGROUND: The hypoxia marker pimonidazole is a candidate biomarker of cancer aggressiveness. We investigated the transcriptional programme associated with pimonidazole staining in prostate cancer. METHODS: Index tumour biopsies were taken by image guidance from an investigation cohort of 52 patients, where 43 patients received pimonidazole before prostatectomy. Biopsy location within the index tumour was verified for 46 (88%) patients, who were included for gene expression profiling and immunohistochemistry. Two independent cohorts of 59 and 281 patients were used for validation. RESULTS: Expression of genes in proliferation, DNA repair and hypoxia response was a major part of the transcriptional programme associated with pimonidazole staining. A signature of 32 essential genes was constructed and showed positive correlation to Ki67 staining, confirming the increased proliferation in hypoxic tumours as suggested from the gene data. Positive correlations were also found to tumour stage and lymph node status, but not to blood prostate-specific antigen level, consistent with the findings for pimonidazole staining. The association with aggressiveness was confirmed in validation cohorts, where the signature correlated with Gleason score and had independent prognostic impact, respectively. CONCLUSIONS: Pimonidazole staining reflects an aggressive hypoxic phenotype of prostate cancer characterised by upregulation of proliferation, DNA repair and hypoxia response genes.

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Increased knowledge of the physiological basis behind the signal enhancement in tumors during dynamic contrast-enhanced magnetic resonance (MR) imaging may be useful in development of predictive assays based on this technique. In the present work, the relative signal intensity (RSI) increase in gadopentetate dimeglumine (Gd-DTPA)-enhanced MR images of patients with cervical carcinoma was related to tumor perfusion, vascular density, cell density, and oxygen tension (pO(2)). The patients were subjected to MR imaging before the start of treatment (N = 12) and after two weeks of radiotherapy (N = 8). Perfusion was determined from the kinetics of contrast agent in tumors and arteries, vascular density and cell density were determined from tumor biopsies, and pO(2) was determined by polarographic needle electrodes. The maximal RSI was correlated to perfusion (P = 0.002) and cell density (P = 0.004), but was not related to vascular density. There was also a correlation between pO(2) and perfusion (P < 0.001). Moreover, pO(2) tended to be correlated to cell density (P = 0.1), but was not related to vascular density. There was a significant correlation between RSI and pO(2), regardless of whether the median pO(2) (P < 0.001) or the fraction of pO(2) readings below 2.5 mmHg (P < 0.001), 5 mmHg (P < 0.0001), or 10 mmHg (P < 0.001) was considered. Our results suggest that the Gd-DTPA-induced signal enhancement in MR images of cervical tumors is influenced by both perfusion and cell density. These parameters are also of major importance for tumor oxygenation, leading to a correlation between signal enhancement and oxygenation. Dynamic contrast-enhanced MR imaging may therefore possibly be useful in prediction of treatment outcome.

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Knowledge of the intratumor heterogeneity in blood perfusion may lead to increased understanding of tumor response to treatment. In the present work, absolute perfusion values, in units of ml/g.min, were determined in 20 tumor subregions of patients with cervical cancer before treatment (n = 12) and after 2 weeks of radiotherapy (n = 8), by using a method based on contrast-enhanced magnetic resonance imaging. The aims were to evaluate the intratumor heterogeneity in perfusion in relation to the intertumor heterogeneity and to search for changes in the heterogeneities during the early phase of therapy. The intra- and intertumor heterogeneity in perfusion were estimated from components of one-way analyses of variance. The mean perfusion differed significantly among the patients before treatment, ranging from 0.044 to 0.12 ml/g x min. Large differences in perfusion were also observed within individual tumors. The heterogeneity was largest in the best perfused tumors, perfusion values ranging, e.g., from 0.055 to 0.29 ml/g x min were observed. The intratumor heterogeneity was similar to the intertumor heterogeneity. The mean perfusion generally increased or tended to increase during radiotherapy, ranging from 0.064 ml/g x min to 0.13 ml/g x min after 2 weeks of treatment. There was a tendency of increased intratumor heterogeneity in perfusion after therapy, consistent with the higher mean value; a difference in perfusion of more than a factor of 10 was seen within some tumors. These results suggest that cervix tumors contain a significant amount of poorly perfused subregions with high treatment resistance. Moreover, the perfusion and perfusion heterogeneity may increase during the early phase of radiotherapy and influence tumor response.

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The perfusion in tumors shows substantial spatial heterogeneity compared to that in normal tissues. The aim of the present study was to evaluate the intratumor heterogeneity in perfusion in tumors of two amelanotic human melanoma xenograft lines, A-07 and R-18, grown intradermally in Balb/c nu/nu mice. A non-invasive contrast-enhanced magnetic resonance imaging method yielding results in absolute values was applied. The perfusion was determined in manually defined regions of interest, corresponding to a whole tumor or to subregions of a tumor. The mean perfusion and the intertumor heterogeneity in perfusion were similar for the two tumor lines. For whole A-07 tumors, the perfusion ranged from 0.089 mL/(g . min) to 0.20 mL/(g . min) [mean: 0.15 mL/(g . min)], and for whole R-18 tumors, from 0.030 mL/(g . min) to 0.17 mL/(g . min) [mean: 0.13 mL/(g . min)]. The intratumor heterogeneity, on the other hand, was estimated to be 6.4 times larger in A-07 tumors than in R-18 tumors. The highest perfusion values, up to 0.69 mL/(g . min), were found in subregions of A-07 tumors. The intratumor heterogeneity was substantially larger than the intertumor heterogeneity in A-07 tumors, whereas in R-18 tumors, the intratumor heterogeneity was similar to the intertumor heterogeneity. These observations imply that measurements of mean tumor perfusion may have limited value as a predictive assay for outcome of treatment.

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Poor outcome of treatment in advanced cervix carcinoma has been shown to be associated with poor oxygenation of the primary tumour. Hypoxia may cause radiation resistance and promote lymph-node metastasis. The purpose of the study reported here was to investigate whether hypoxia-induced treatment failure in advanced cervix carcinoma is primarily a result of hypoxia-induced radiation resistance or the presence of hypoxia-induced lymph-node metastases at the start of treatment. Thirty-two patients with squamous cell carcinoma of the uterine cervix were included in the study. Radiation therapy was given with curative intent as combined external irradiation and endocavitary brachytherapy. The oxygenation status of the primary tumour was measured prior to treatment using the Eppendorf PO2 Histograph. Pelvic and para-aortal lymph-node metastases were detected by magnetic resonance imaging at the time of initial diagnosis. The primary tumours of the patients with metastases (n = 18) were significantly more poorly oxygenated than those of the patients without metastases (n = 14). Multivariate Cox regression analyses involving biological and clinical parameters identified the tumour subvolume having PO2 values below 5 mmHg (HSV (pO2 < 5 mmHg) as the only significant, independent prognostic factor for locoregional control, disease-free survival and overall survival. The probabilities of locoregional control, disease-free survival and overall survival were significantly lower for the patients with HSV (PO2 < 5 mmHg) above the median value than for those with HSV (PO2 < 5 mmHg) below the median value. On the other hand, the outcome of treatment was not significantly different for the patients with metastases and the patients without metastases at the start of treatment, irrespective of clinical end-point. Consequently, treatment failure was primarily a result of hypoxia-induced radiation resistance rather than hypoxia-induced lymph-node metastasis, suggesting that novel treatment strategies aiming at improving tumour oxygenation or enhancing the radiation sensitivity of hypoxic tumour cells may prove beneficial in attempts to improve the radiation therapy of advanced cervix carcinoma.

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The aim of this study was to investigate whether apparent diffusion coefficients (ADCs) could be used as measures of cell density and necrotic fraction of tumors. Tumors of four human melanoma xenograft lines were subjected to diffusion-weighted magnetic resonance imaging (DWI). ADCs were calculated from the images and related to cell density and necrotic fraction, as determined from histological sections. A significant correlation was found between the ADC of the viable tissue and cell density, regardless of whether tumors of different lines or different regions within individual tumors were considered. Necrosis was found in two of the lines. A single region of massive necrosis that could be differentiated from the viable tissue in ADC maps was found in one line, whereas a number of smaller necrotic regions that could not be identified in ADC maps were found in the other line. Tumor ADC was significantly correlated with the necrotic fraction of the former, but not of the latter line. Our results suggest that ADCs can be used as measures of cell density and necrotic fraction of some but not of all tumors, depending on whether the individual necrotic regions are large enough to be differentiated from the viable tissue with the obtained spatial resolution of the DW images. Magn Reson Med 43:828-836, 2000.

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Identification of biological parameters of major importance for the control of malignant diseases can be useful for the design of optimal treatment regimes for individual patients. Tumor oxygen tension (pO2), vascular density, cell density, and frequency of mitosis and apoptosis were measured before treatment (40 patients) and after 2 weeks of radiotherapy (22 patients) in patients with uterine cervical cancer. The aim was to investigate whether one of the parameters was more important for disease control than the others. Three sets of data were considered; the pretreatment parameters, the parameters measured after 2 weeks of radiation, and the changes in the parameters during this time. The pO2 was measured polarographically; the other parameters were determined by histological analyses of tumor biopsies. Hypoxic subvolume (HSV5), ie., the fraction of pO2 readings <5 mm Hg multiplied with tumor volume, showed the strongest correlation to control. Patients with a small HSVs before treatment had a higher control probability after a median follow-up time of 50 months than patients with a large HSV5 (P < 0.001). All other parameters or changes in parameters showed impaired correlation to control compared with pretreatment HSV5. The present results suggest that pretreatment oxygenation is more important for disease control of cervical cancer than the oxygenation after 2 weeks of radiotherapy or the changes in oxygenation during this time. Moreover, vascular density, cell density, and frequency of mitosis and apoptosis before treatment or after 2 weeks of therapy are probably not as important as pretreatment oxygenation as well. Although significant correlations between disease control and some of the parameters other than pretreatment oxygenation can occur in studies based on a large number of patients, the specificity of these parameters in the prediction of control is probably not as high as for oxygenation.

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Poor outcome of treatment was found to be associated with low oxygen tension in the primary tumor and not with high intratumor microvessel density in 40 patients with advanced squamous cell carcinoma of the uterine cervix. Multivariate Cox regression analysis identified the total volume of the hypoxic tumor regions, i. e. the tumor subvolume having pO(2) values below 5 mmHg, as a significant prognostic factor for locoregional control, disease-free survival, and overall survival. Other important prognostic factors, identified by univariate Cox regression analysis, were tumor volume, the fraction of pO(2) readings giving pO(2) values below 5 mmHg, and tumor stage.

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PURPOSE: Changes in oxygen tension (pO(2)) during the early phase of fractionated radiotherapy were studied in 22 patients with uterine cervical cancer. The aims were to investigate (a) whether possible changes in pO(2) differed among and within tumors and (b) whether the changes could be attributed to changes in vascular density, cell density, and frequency of mitosis and apoptosis. METHODS AND MATERIALS: The pO(2) was measured polarographically in four regions of the tumors before treatment and after 2 weeks of radiotherapy. The vascular density, cell density, and frequency of mitosis and apoptosis were determined from biopsies taken from the tumor regions after each pO(2) measurement. RESULTS: The changes in pO(2) during therapy differed among the tumors and were correlated to pO(2) before treatment (p < 0.001). The direction of the changes was consistent throughout the tumors; all regions in tumors with increased oxygenation had increased or no change in pO(2) and vice versa. The tumors with increased pO(2) (n = 10) had a large decrease in cell density and a significant increase in apoptotic frequency. In contrast, the tumors with decreased pO(2) (n = 10) had a smaller decrease in cell density (p = 0.014) and no significant increase in apoptotic frequency. Vascular density and mitotic frequency showed no change during therapy; however, vascular damage other than decreased vascular density was observed. CONCLUSION: These results indicate that the oxygenation of cervix tumors generally changes during the early phase of radiotherapy. The change depends on the balance between the factor leading to an increase and that leading to a decrease in oxygenation; i.e., decreased cell density and vascular damage, respectively. Increased apoptotic frequency may contribute to a large decrease in cell density and hence increased oxygenation during therapy.

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Tumor proliferation may be predictive for malignant progression and response to fractionated therapy of cancer. The purpose of the present work was to investigate whether the proliferation activity of solid tumors can be assessed in vivo from the proton relaxation times, T1 and T2. Tumors of four amelanotic human melanoma xenograft lines were studied. Three parameters were used to represent tumor proliferation activity; the volume doubling time, Tvol, the potential doubling time, Tpot, and the fraction of cells in S-phase. Tvol was determined from volumetric growth data. Tpot and S-phase fraction were determined by flow cytometric analysis of tumor cells after bromodeoxyuridine (BrdU) incorporation in vivo. T1 and T2 were measured by 1H-MRI in vivo, using spin-echo pulse sequences. The proliferation parameters and relaxation times differed considerably among the tumor lines. Significant correlations were found between the proliferation parameters and the relaxation times, regardless of whether Tvol, Tpot, or S-phase fraction was considered. Tumors with short Tvol and Tpot and high S-phase fraction had long T1 and T2 compared to tumors with long Tvol and Tpot and low S-phase fraction. The elongated T1 and T2 of fast growing tumors were probably due to increased interstitial and/or intravascular water content. The present results suggest that in vivo spin-echo 1H-MRI can be used to discriminate between tumors of high and low proliferation activity.

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Increased knowledge about changes that occur in tumour oxygenation during radiotherapy and the biological factors causing these changes can be useful in the development of optimal radiation treatments. The aims of this study were a) to study changes in the oxygen tension (pO2) of human head and neck tumours during radiotherapy in relationship to changes in cell density and vascular density, and b) to investigate whether the pO2, measured before or during therapy, can be used to predict the therapeutic outcome. Preliminary data from the first 11 patients included in the study are reported. The pO2 was measured before treatment (11 patients) and once a week during therapy (8 patients), using polarographic needle electrodes. Cell density and vascular density were determined from biopsies taken after each pO2 measurement in 5 patients. Significant fluctuations in pO2 occurred during therapy. Changes in hypoxic fraction; i.e., fraction of pO2 readings below 2.5 mm Hg, 5 mm Hg or 10 mm Hg, coincided with changes in cell density, but not with changes in vascular density, which suggests that the changes in hypoxic fraction were caused by changes in oxygen consumption rather than supply. Response evaluation after a median follow-up time of 19 months showed that progressive disease occurred among the patients with highly hypoxic tumour, regardless of whether hypoxic fraction before treatment or after two weeks of radiotherapy was considered.

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Some clinical studies involving several histological types of cancer have suggested that high vascular density in the primary tumour promotes metastasis. Other studies have suggested that a high incidence of metastases is associated with low oxygen tension in the primary tumour. The purpose of the study reported here was to search for correlations between incidence of metastases and oxygen tension or vascular density in the same population of patients. Thirty-eight consecutive patients with squamous cell carcinoma of the uterine cervix were included in a prospective study. Pelvic, iliac and retroperitoneal lymph node metastases were detected by magnetic resonance imaging at the time of initial diagnosis. Oxygen tension was measured polarographically using the Eppendorf pO2 Histograph 6650. Vascular density was determined by histological examination of tumour biopsies. The primary tumours of the patients with metastases (n = 19) were more poorly oxygenated than those of the patients without metastases (n = 19). Thus, the fractions of the pO2 readings resulting in values below 5 mmHg and 10 mmHg were significantly higher for the former group of patients than for the latter (P = 0.03 and 0.02 respectively). In contrast, the vascular density of the primary tumour was not significantly different for the two groups of patients. The present study suggests that a high incidence of metastases in squamous cell carcinoma of the uterine cervix is associated with poor oxygenation of the primary tumour and not with a high vascular density.

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Reliable methods based on MRI for measurement of the perfusion rate in human tumors are highly warranted. Tumors of two amelanotic human melanoma xenograft lines were subjected to dynamic 1H MRI after i.v. administration of gadopentetate dimeglumine (Gd-DTPA). The aim was to investigate to what extent different perfusion parameters determined from the Gd-DTPA kinetics, i.e., the initial uptake rate, the maximal uptake, the decay rate, and the perfusion rate calculated by use of the Kety equation, can be used as a reliable estimate of tumor perfusion rate. Each parameter was calculated in dual; one calculation was based on relative signal intensity increase (RSII) in T1-weighted MR images and the other on Gd-DTPA concentration determined from the images. The perfusion parameters were compared with the perfusion rates determined from measurement of tumor uptake of 86Rb or [14C]iodoantipyrine. The results showed that reliable estimates of tumor perfusion rate can be achieved from analysis of Gd-DTPA kinetics by use of the Kety equation. Gd-DTPA kinetics based on concentration might be used to achieve reliable estimates of absolute tumor perfusion rate, whereas reliable estimates of the relative perfusion rate might also be achieved from Gd-DTPA kinetics based on RSII. The initial uptake rate, the maximal uptake, and the decay rate of Gd-DTPA, however, are not reliable estimates of tumor perfusion rate, mainly because these parameters are highly influenced by the tumor extracellular volume fraction in addition to the perfusion rate.

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Studies of interrelationships between physiological parameters of tumours are sparse. The possibility that vascular density might be related to the rate of angiogenesis, the rate of perfusion and/or the development of necrosis was examined in the work reported here. Xenografted tumours of four human melanoma cell lines (A-07, D-12, R-18 and U-25) grown orthotopically in BALB/c nu/nu mice were included in the study. Vascular density and fraction of necrotic tissue were determined by stereological analysis of histological sections. The rate of angiogenesis was measured by using the intradermal angiogenesis assay. The rate of perfusion was studied by using the 86Rb uptake method. A-07 showed a higher vascular density, a higher rate of angiogenesis and a higher rate of perfusion than the other lines. D-12, R-18 and U-25, which differed significantly in the rate of angiogenesis, showed similar vascular densities and similar perfusion rates. Consequently, vascular density is not a sensitive measure of the rate of angiogenesis in the melanoma lines studied here, but might adequately reflect the perfusion rate. Significant necrotic regions developed in D-12 and U-25, but not in A-07 and R-18, presumably because large-diameter vessels, possibly arteriovenous shunts, occurred more frequently in D-12 and U-25 than in A-07 and R-18.

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The prognosis of patients with carcinoma of the uterine cervix has been shown to depend on the oxygenation and vascularization status of the tumors. The purpose of the study reported here was to search for possible differences in oxygen tension and vascular density between adenocarcinomas and squamous cell carcinomas. Ten patients with adenocarcinoma and forty patients with squamous cell carcinoma were included in the study. Oxygen tension was measured polarographically using the Eppendorf pO2 Histograph 6650. Vascular density was determined by histological examination of tumor biopsies. The adenocarcinomas were significantly better oxygenated than the squamous cell carcinomas. The squamous cell carcinomas and the adenocarcinomas did not differ significantly in vascular density. The difference in prognosis between patients with adenocarcinoma and patients with squamous cell carcinoma is probably not attributable to differences in tumor oxygenation or vascularization.

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BACKGROUND AND PURPOSE: The use of polarographic needle electrodes for measurement of oxygen tension (pO2) in tumours requires documentation of the validity of the method. In the present work the pO2 values measured polarographically with the Eppendorf pO2 histograph in human tumours were compared with the histological appearance of the tumour tissue, i.e. vascular density, fraction of necrosis and fraction of hypoxic tissue, to investigate whether the measurements reflected the expected pO2. MATERIALS AND METHODS: The pO2 was measured in cervix tumours in patients and in human melanoma xenografted tumours in athymic mice. Vascular density was determined in the cervix tumours by histological analysis of biopsies from the pO2 measurement tracks. Fraction of necrosis and fraction of hypoxic tissue, i.e. tissue binding the hypoxia marker pimonidazole, were determined in the melanomas by analysis of histological sections from the tumour planes in which the pO2 measurements were performed. RESULTS: The pO2 distributions showed large intratumour heterogeneity. In cervix tumours, tumour regions with vascular density (vascular length per unit tissue volume) in the range of 47-77 mm/mm3 showed higher pO2 than tumour regions with vascular density in the range of 20-47 mm/mm3, which in turn showed higher pO2 than tumour regions with vascular density in the range of 0-20 mm/mm3. In melanomas, tumour regions in which necrosis and hypoxia constituted more than 50% of the tissue showed lower pO2 than other tumour regions. CONCLUSIONS: The pO2 measured in the tumours was consistent with the histological appearance of the tissue in which the measurements were performed, suggesting that reliable pO2 distributions of tumours can be obtained with polarographic needle electrodes.

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The outcome of radiation therapy of cervix carcinoma might depend on the oxygenation of the tumor tissue. An adequate method for measurement of tumor oxygen tension (pO2) is therefore needed. The purpose of the work reported here was dual: (1) to investigate whether polarographic pO2 measurements with the Eppendorf pO2 Histograph 6650 are sufficiently sensitive to detect differences in tumor pO2 before and after blood transfusion of anemic patients and between poorly and well-vascularized tumor tissue, and (b) to investigate whether accurate tumor, pO2 measurements require extensive mapping of tumor temperature and the avoidance of anesthesia. Nineteen patients with squamous cell carcinoma of the uterine cervix FIGO stages Ib to IVb were included in the study. Vascular density was determined by histological examination of tumor biopsies. Propofol was used as a single anesthetic agent. Tumor pO2 distributions recorded before and after the administration of propofol were not different (P > 0.05). The temperatures measured in the tumor periphery and center did not differ from the rectal temperature (P > 0.05), suggesting that tumor pO2 measurements can be based on the rectal temperature. Increased hemoglobin concentrations after blood transfusion resulted in increased tumor oxygenation in 50% of the patients (P < 0.001). The pO2 frequency distributions of the susceptible tumors showed increased 50th percentiles but unchanged 10th percentiles, suggesting that transfusion cannot reduce the fraction of radiation-resistant hypoxic tumor cells extensively. Tumor tissue with high vascular density showed higher pO2 values than tumor tissue with low vascular density (P < 0.001). In conclusion, polarographic measurement of tumor pO2 with the Eppendorf pO2 Histograph 6650 is a sensitive method for assessment of the oxygenation of cervix carcinoma. Reliable tumor pO2 measurements can be performed in patients given propofol anaesthesia and without extensive mapping of tumor temperature.

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