J. Donald Chapman

Hypoxia: importance in tumor biology, noninvasive measurement by imaging, and value of its measurement in the management of cancer therapy.

PURPOSE The Cancer Imaging Program of the National Cancer Institute convened a workshop to assess the current status of hypoxia imaging, to assess what is known about the biology of hypoxia as it relates to cancer and cancer therapy, and to define clinical scenarios in which in vivo hypoxia imaging could prove valuable. RESULTS Hypoxia, or low oxygenation, has emerged as an important factor in tumor biology and response to cancer treatment. It has been correlated with angiogenesis, tumor aggressiveness, local recurrence, and metastasis, and it appears to be a prognostic factor for several cancers, including those of the cervix, head and neck, prostate, pancreas, and brain. The relationship between tumor oxygenation and response to radiation therapy has been well established, but hypoxia also affects and is affected by some chemotherapeutic agents. Although hypoxia is an important aspect of tumor physiology and response to treatment, the lack of simple and efficient methods to measure and image oxygenation hampers further understanding and limits their prognostic usefulness. There is no gold standard for measuring hypoxia; Eppendorf measurement of pO(2) has been used, but this method is invasive. Recent studies have focused on molecular markers of hypoxia, such as hypoxia inducible factor 1 (HIF-1) and carbonic anhydrase isozyme IX (CA-IX), and on developing noninvasive imaging techniques. CONCLUSIONS This workshop yielded recommendations on using hypoxia measurement to identify patients who would respond best to radiation therapy, which would improve treatment planning. This represents a narrow focus, as hypoxia measurement might also prove useful in drug development and in increasing our understanding of tumor biology.

Incorporating clinical measurements of hypoxia into tumor local control modeling of prostate cancer: Implications for the α/β ratio

Abstract Background and purpose The recently obtained low value of ∼1.5 for the α/β of prostate cancer has led us to reexamine the optimal prostate tumor biology parameters, while taking into account everything known about the radiation response of prostate clonogens for use in a predictive dose–response model. Methods and materials Averages of the literature values of the α- and β-inactivation coefficients for human prostate cancer cell lines were calculated. A robust tumor local control probability (TLCP) model was used that required average α and β, as well as σ α , for the interpatient variation in single-hit killing (α). Median P o 2 values ≤1 mm Hg in the prostates of Fox Chase Cancer Center brachytherapy patients had been found in 21% of 115 cases. The oxygen enhancement ratios of 1.75 and 3.25 for α- and β-inactivation, respectively, measured for tumor cells in vitro , were incorporated into the TLCP model, together with a clonogen density of ∼10 5 cells/cm 3 . Severe hypoxia and radioresistance were estimated for a proportion of tumors that was increased with PSA level. Results For asynchronous human prostate cell lines irradiated in air, α mean was 0.26 ± 0.07 (standard error) Gy −1 , σ α = 0.06 Gy −1 , and β mean was 0.0312 Gy −2 ± 0.0064 (standard error) Gy −2 . The TLCP data indicated that most tumors that contained aerobic cells would be cured, whereas most tumors that contained hypoxic cells would not be cured by total doses of 76 to 80 Gy. Clinical response data from the literature for external beam dose escalation, stratified by PSA value, and for low-dose-rate brachytherapy, were well predicted by the model, where the α/β ratio was 8.5 and 15.5 for well-oxygenated and hypoxic clonogens, respectively. Conclusions Neither α/β ratio nor clonogen number need be extremely low to explain the response of prostate cancer to brachytherapy and external beam therapy, contradicting other recent analyses. It is strongly suggested that severe hypoxia in the prostates of certain patients limits the overall cancer cure rate by conventional radiation therapy.

Hypoxic prostate/muscle Po2 ratio predicts for biochemical failure in patients with prostate cancer: Preliminary findings

OBJECTIVES To investigate whether low partial pressure of oxygen (PO2) in prostate cancer (CaP) predicts for biochemical outcome after radiotherapy. We previously reported that hypoxic regions exist in human CaP. METHODS Custom-made Eppendorf PO2 microelectrodes were used to obtain approximately 100 PO2 readings from both pathologically involved regions of the prostate (as determined by sextant biopsies) and normal muscle (as an internal control). Fifty-seven patients with localized disease were prospectively studied; all received brachytherapy implants (48 low dose rate and 9 high dose rate) under spinal anesthesia. Nine patients had received prior hormonal therapy. Biochemical failure was defined as two consecutive rises in prostate-specific antigen level, without a return to baseline. Cox proportional hazards regression analysis was used to evaluate the influence of hypoxia on biochemical control, while adjusting for prostate-specific antigen, Gleason score, stage, implant type (low dose rate versus high dose rate), perineural invasion, hemoglobin level, use of hormonal therapy, average (mean) of the median prostate PO2, average median muscle PO2, and prostate/muscle PO2 (P/M) ratio. RESULTS With a median follow-up of 19 months (range 4 to 31), 9 patients developed biochemical failure. A threshold analysis of the P/M ratio demonstrated that biochemical control at 2 years differed significantly at a ratio of less than 0.05 versus 0.05 or greater (31% versus 92%, P <0.0001). However, the classic prognosticators were similar in these two groups. On multivariate analysis, the P/M ratio was the only predictor of biochemical control (P = 0.0002). CONCLUSIONS To our knowledge, this is the first study to correlate the degree of hypoxia in CaP with treatment outcome after radiotherapy. The P/M PO2 ratio was the strongest predictor for biochemical control on stepwise multivariate analysis. Longer follow up with more patients is planned to confirm this result.

Measuring hypoxia and predicting tumor radioresistance with nuclear medicine assays

Tumor cells at low oxygen tension are relatively radioresistant. The hypoxic fraction of individual tumors before, during and after radiotherapy is likely to have prognostic value but its diagnosis still awaits an accurate and acceptable assay. The recent indications that hypoxia can also induce the expression of specific genes and promote a more aggressive tumor phenotype makes its diagnosis even more important. Over 15 years ago, misonidazole, an azomycin-based hypoxic cell radiosensitizer, was found to link covalently to cellular molecules at rates inversely proportional to intracellular oxygen concentration. The use of bioreducible markers to positively label zones of viable hypoxic cells within solid tumors and to predict for tumor radioresistance was proposed. Several hypoxic markers have now been identified and their selective binding within tumors has been measured by both invasive and non-invasive assays. Research from our laboratory has emphasized both mechanistic and preclinical studies associated with nuclear medicine procedures for measuring tumor hypoxia and predicting tumor radioresistance. This report updates radiation oncologists about the status of nuclear medicine hypoxic marker research and development as of mid-1997. While several potential imaging agents have been identified, their testing and validation in appropriate human tumors will require focused research efforts by individual academic departments and, possibly, by clinical trials performed through cooperative groups. Since the prediction of hypoxia in individual tumors could strongly impact radiotherapy treatment planning, the radiation oncology research community is best positioned to execute the validation studies associated with these markers.

Hypoxic regions exist in human prostate carcinoma

OBJECTIVES The purpose of this study was to characterize, by use of the Eppendorf microelectrode, the extent of hypoxia (range/heterogeneity) in human prostate carcinomas. METHODS Custom-made Eppendorf pO2 microelectrodes were used to obtain PO2 measurements from the pathologically involved side of the prostate, as well as from a region of normal muscle for comparison. Each set of measurements comprised approximately 100 separate readings of pO2, for a total of 2145 individual measurements. Twelve patients were studied, 7 of whom underwent brachytherapy, 3 a radical prostatectomy, and 2 a cystoprostatectomy. The pO2 measurements were obtained in the operating room, using sterile technique, under spinal anesthesia for the brachytherapy group patients and under general anesthesia for the surgery group patients. The Eppendorf histograms were recorded and described by the median pO2, mean pO2, and percentage of measurements less than 5 mm Hg and less than 10 mm Hg. RESULTS Because of differences in patient characteristics and the anesthesia employed, control measurements were obtained from nearby normal muscle as an internal control in all but 2 patients. This internal comparison showed that the oxygen measurements from the pathologically involved portion of the prostate were significantly lower than those from normal muscle. Similarly, higher pO2 readings were obtained from the pathologically normal prostates (in the patients with bladder cancer) than from the prostates of patients with prostate carcinoma. Increasing levels of hypoxia were observed with increasing clinical stage. Significant predictors of oxygenation include the type of tissue (pathologically involved prostate versus normal muscle or normal prostate), clinical stage, and type of anesthesia. CONCLUSIONS This report, to our knowledge, represents the first study to obtain in vivo electrode measurements of oxygen levels in patients with prostate cancer and suggests that hypoxic regions exist in human prostate carcinoma. More patients will be accrued to this prospective study to correlate the oxygenation status of prostate carcinoma with known prognostic factors and treatment outcome.

Hypoxia in human prostate carcinoma: an Eppendorf PO2 study.

The purpose of this study was to characterize the extent of hypoxia in human prostate carcinoma using the Eppendorf Po2 microelectrode. Custom-made Eppendorf Po2 microelectrodes were used to obtain Po2 measurements from the pathologically involved region of the prostate (as determined by the pretreatment sextant biopsies), as well as from a region of normal muscle for comparison. Fifty-nine patients with localized prostate cancer were studied, all of whom received brachytherapy implants under spinal anesthesia. A multivariate mixed effects analysis for prediction of tumor oxygenation was performed including the following covariates: type of tissue (prostate versus muscle), prostatic-specific antigen, disease stage, patient age and race, tumor grade, volume, perineural invasion, and hormonal therapy. Because of differences in patient characteristics, control measurements were obtained from normal muscle in all patients. This internal comparison showed that the oxygen measurements from the pathologically involved portion of the prostate were significantly lower (average median Po2 = 2.4 mm Hg) compared with the measurements from normal muscle (average median Po2 = 30.0 mm Hg), p < 0.0001. A multivariate, linear, mixed analysis demonstrated that the only significant predictor of oxygenation was the type of tissue (prostate versus muscle). This study, using in vivo electrode oxygen measurements, suggests that hypoxia exists in human prostate carcinoma. More patients will be accrued to this study to ultimately correlate the oxygenation status in prostate carcinoma tumors with treatment outcome.

Increased hypoxia correlates with increased expression of the angiogenesis marker vascular endothelial growth factor in human prostate cancer.

OBJECTIVES To test the hypothesis that increasing levels of hypoxia are associated with increased expression of vascular endothelial growth factor (VEGF) in prostate cancer by correlating the level of median tissue oxygenation in human prostate tumors with the immunohistochemically determined level of VEGF expression. METHODS Custom-made Eppendorf oxygen microelectrodes were used to quantitate the pO(2) levels in prostate tumors of 13 men undergoing radical prostatectomy. All pO(2) measurements were performed under fluorine-based general anesthesia. Paraffin-embedded tumor tissue from these men was analyzed to measure the level of VEGF expression by immunohistochemical staining. The significance of the associations between the pO(2) levels and VEGF staining were determined by the Pearson correlations. RESULTS The range of the median pO(2) levels (based on between 97 and 129 individual measurements) among 13 prostate tumors was 0.5 to 44.9 mm Hg. The blinded comparison of pO(2) levels and VEGF staining intensity demonstrated a significant correlation between increasing hypoxia and the percentage of cells staining positive for VEGF (r = -0.721, P = 0.005). This correlation was also significant when pO(2) levels were compared with the overall immunoreactive score, which takes into account staining intensity (r = -0.642, P = 0.018). CONCLUSIONS To our knowledge, this is the first study demonstrating a significant association between increasing levels of hypoxia and increased expression of the angiogenesis marker VEGF in human prostate carcinoma. The results of our study further support the exploration of antiangiogenesis strategies for the treatment of human prostate cancer.

Polarographic needle electrode measurements of oxygen in rat prostate carcinomas : accuracy and reproducibility

PURPOSE The oxygenation status of tumors may be important for predicting tumor response to therapy. Previous studies with the anaplastic (R3327-AT) and well-differentiated (R3327-H) Dunning rat prostate tumors using indirect assays of tumor oxygenation indicated the relative hypoxic and radioresistant nature of the anaplastic tumor. We now report direct measurements of oxygen in these tumors made with the pO2 histograph to determine: (a) whether a significant difference in oxygenation status could be detected between them: (b) whether sequential measurements on the same tumor gave similar values; and (c) whether tumor oxygenation correlated with tumor volume. METHODS AND MATERIALS R3327-AT and R3327-H tumors were grown in Fischer X Copenhagen rat to volumes of 1.0-7.0 cm3. Electrode measurements (100-200) were made in tumors in anesthetized animals along two parallel tracks. Repeat measurements were made at 1-5 days along different parallel tracks. Oxygen partial pressures of muscle tissue were measured and served as a normal tissue control. Statistical analyses were applied to determine whether tumor oxygen levels were different between the two tumor histologies, whether sequential measurements in the same tumor were reproducible, and whether tumor oxygenation correlated with tumor volume. RESULTS The average median pO2 of the well-differentiated (n = 15) and the anaplastic (n = 15) tumors was 6.0 mmHg (SE +/- 1.3) and 2.2 mmHg (SE +/- 0.3), respectively. The average median pO2 of normal rat muscle (n = 15) was 23.6 mmHg (SE +/- 2.0). These values represent highly significant differences in oxygen concentration between the two tumors and rat muscle. The differences in average mean pO2 values were also highly significant. Repeat measurements in the same tumors on different days gave average median values of 4.7 and 2.2 mmHg in the R3327-H (n = 15) and R3327-AT (n = 15) tumors, respectively. For these repeat measurements, median pO2 values decreased in 15 and increased in 15 tumors, and were not significantly different from the first measurements. The average differences observed in median pO2 were 37% (SE +/- 7) and 58% (SE +/- 10) for the R3327-H and R3327-AT tumors, respectively. No significant correlation was observed between pO2 levels and the tumor volumes investigated in this study. CONCLUSIONS The median pO2 values of the anaplastic Dunning tumors were significantly lower than those of the well-differentiated tumors (p < 0.001). Oxygen levels in both tumors were significantly lower than those measured in normal rat muscle (p < 0.00005). Repeat measurements of median pO2 in the same tumors were not significantly different for either tumor model (p > 0.5). The changes observed in pO2 distributions within individual tumors from day to day may indicate true dynamics of its oxygenation status and/or the limits of electrode measurements, by sampling along only two insertion sites. The electrode measurements of pO2 in these tumor models are reproducible and confirm previously detected oxygenation differences between the anaplastic and well-differentiated tumors.

Proceedings of the Oxygen Homeostasis/Hypoxia Meeting

The first Oxygen Homeostasis/Hypoxia Meeting was held on February 12, 2003, at the Sheraton National Hotel, Washington, D.C. The meeting was hosted by Drs. S. Percy Ivy and Giovanni Melillo of the National Cancer Institute, NIH. The purpose of the meeting was to stimulate collaborations among the participants who are engaged in different areas of hypoxia research and application, including basic research on hypoxia, and its induction and consequences; the development of drugs targeting hypoxia and factors involved in pathways leading to (or controlled by) hypoxia; and the development and application of hypoxia imaging techniques and reagents.

The power of radiation biophysics-let's use it.

Two recent editorials in this Journal about the linear quadratic (LQ) model (1, 2) have debated its use on essentially empirical grounds. Missing are its roots in biophysics and what they state about how it can (and cannot) be used. In the 1970s, 2 roads led to analysis of in vitro cell killing by ionizing radiation in terms of the underlying mechanisms. Kellerer and Rossi (3) explored the statistics of energy deposition in small volumesdthe microdosimetric approach. Chadwick and Leenhouts (4) related cell killing to lesions in 2-stranded DNAdthe biophysical approach. Both approaches postulate that radiation can kill cells by 2 distinct processes, a 1-hit mechanism and another that requires 2 independent events. We began to use the LQ equation in 1973 for the pragmatic reason that it fits the survival data better than the other models we tested (5). It soon emerged that, if key factors were controlled, this equation offers insights into the mechanisms involved in cell death and survival during and after irradiation. Here, we reviewed 4 forms of this equation that describe cell survival in specific circumstances and how they might help those working to improve radiotherapeutic protocols. The basic LQ equation states that the cell surviving fraction (S) is the product of 2 Poisson escape probabilities. The mean numbers of events for the underlying mechanisms are proportional to the first and second powers of dose, D, respectively.