Rise of oxygenation in cervical lymph node metastasis during the initial course of radiochemotherapy

Rise of oxygenation in cervical lymph node metastasis during the initial course of radiochemotherapy ANDREAS DIETZ, MD, VOLKER RUDAT, MD, BERNHARD VANSELOW, MD, PETRA WOLLENSACK, CandMed, CLAUDIA BETTSCHEIDER, MD, CHRISTIAN CONRADT, PhD, and MICHAEL J. EBLE, MD, Heidelberg, Germany

It has been hypothesized that during radiation treatment a reoxygenation of hypoxic tumor tissue takes place. To test this hypothesis, we have investigated whether reoxygenation in lymph node metastases could be determined by invasive PO2 measurements. Through a hypodermic needle inserted transcutaneously into tumor-positive lymph nodes, polarographic oxygen determinations were made in 18 patients with advanced squamous cell carcinomas of the oropharynx and hypopharynx. These measurements were performed before therapy and a week after the onset of radiotherapy or radiochemotherapy, respectively. Low PO2 values before treatment (mean value of the patient’s median was 12.6 mm Hg PO2) and a mean hypoxic fraction (PO2 < 5 mm Hg) of 39.6% indicated manifest tumor hypoxia. After 1 week of treatment, a significant increase in the median PO2 (mean value of shift: 7.3 mm Hg) and a reduction in the hypoxic fraction (mean value of shift: 13.4% PO2 < 5 mm Hg, P < 0.03) were observed after both radiotherapy and radiochemotherapy. Thus invasive PO2 histography fulfills the requirements for a method to confirm tumor hypoxia in head and neck tumors. The results obtained indicate that reoxygenation occurs during the initial phases of radiotherapy and radiochemotherapy, and they will form the basis for future comparative investigations on the possible influence of hypoxic parameters on tumor responsiveness toward radiation and radiochemotherapy. (Otolaryngol Head Neck Surg 1999;121: 789-96.)

From the Departments of Otolaryngology–Head and Neck Surgery (Drs Dietz, Vanselow, and Wollensack) and Radiooncology (Drs Rudat, Bettscheider, and Eble) and the Institute of Medical Biometry (Dr Conradt), University of Heidelberg. Reprint requests: Andreas Dietz, MD, Department of Otolaryngology–Head and Neck Surgery, University of Heidelberg, Universitäts-Hals-Nasen-Ohrenklinik, Im Neuenheimer Feld 400, 69120 Heidelberg, Germany. Copyright © 1999 by the American Academy of Otolaryngology– Head and Neck Surgery Foundation, Inc. 0194-5998/99/$8.00 + 0 23/77/91545

T

issue oxygenation is known to be one of the key factors in the microenvironment of solid tumors influencing tumor biology (ie, tumor growth and response to therapy). Factors that modulate tumor tissue oxygenation are vascularization and perfusion. Impaired tumor perfusion caused by altered vessel morphology results in impaired oxygenation and metabolism of tumor tissue.1 The p53 tumor-suppressor gene plays a central role in the regulation of hypoxia-dependent apoptosis.2,3 Experimental investigations have shown that under conditions of hypoxia, cellular cytotoxicity is suppressed.4 It could be shown that hypoxia causes changes in gene expression including the induction of “hypoxic stress proteins” (oxygen-related protein) or of transcription factors including proto-oncogenes.2 Hypoxia also influences regulation of angiogenesis.5 Concerning radiotherapy, oxygenation is an important determinant of therapeutic success. Studies using animal models have shown that the radiosensitivity of tumor tissue in hypoxic conditions is reduced.6 After experimental reduction of PO2 to 3 to 4 mm Hg, radiosensitivity was reduced to 50% compared with normal tissue oxygenation (>30 mm Hg PO2).7 Because of the exponential character of this relation, a dramatic decrease of radiosensitivity in tissue below 3 mm Hg PO2 has to be expected. Several authors have described a relationship between low pretherapeutic tumor oxygenation and the high rate of recurrent disease or poor response to therapy in different tumor sites.8-11 Hypoxia also plays a role in the response to antineoplastic chemotherapy. Interestingly, hypoxia is able to augment the effect of alkylating substances such as mitomycin D but reduces the response to bleomycin and actinomycin D.12 It has been suggested that hypoxic tumor cells with retained clonogenicity are responsible for recurrent disease after radiotherapy.1,13 Such tumor cells with increased resistance to apoptosis might have been selected for under hypoxic conditions. Several authors have shown that squamous cell carcinomas of the head and neck have a low oxygenation status.10,14-16 Investigations in murine models have shown that radiation causes reoxygenation of hypoxic tumor tissue.17-19 In vivo measurements in head and neck malignancies with regard to oxygenation would be very helpful in answering questions as to the role of the 789

790

Otolaryngology– Head and Neck Surgery December 1999

DIETZ et al

Table 1. Protocol of accelerated hyperfractionated concomitant boost radiochemotherapy Week of treatment 1 2 (hospitalized) (ambulant)

Treatment

Carboplatin (infusion 15 min IV 70 mg/m2 BS/day) 5-Fluorouracil (infusion 24 hr IV 600 mg/m2 BS/day) Radiation (total = 69.9 Gy) 18 Gy/day Boost (1.5 Gy/day)*

3 4 5 6 (ambulant) (hospitalized) (hospitalized) (hospitalized)

Days 1-5 —

— —

— —

— —

Days 1-5 —

— —

Days 1-5 No

Days 1-5 No

Days 1-5 No

Days 1-5 Yes

Days 1-5 Yes

Days 1-3 Yes

In case of radiotherapy only, the same protocol without application of chemotherapy was conducted. This multicenter trial was designed as a 2-arm study: first-line radiotherapy versus radiochemotherapy (principal investigator Prof. Dr. H. P. Müller, Dr. S. Staar, Department of Radiooncology of the University of Cologne). IV, Intravenous; BS, body surface. *Boost: Concomitant boost of 1.5 Gy 6 hours after first radiation dose of 1.8 Gy was applied daily in weeks 4, 5, and 6.

Table 2. Invasive PO2 histography in cervical lymphatic node metastases of 18 patients with stage IV carcinomas Patient Age no. Sex (yr)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

M M M F M M F M M M M M M M F M M M

41 55 58 51 56 68 56 48 59 63 65 53 45 45 74 63 54 40

Tumor site

Oropharynx Oropharynx Oropharynx Oro/hypopharynx Oropharynx Hypopharynx Oropharynx Oro/hypopharynx Hypopharynx Hypopharynx Oropharynx Hypopharynx Oropharynx Oropharynx Oropharynx Oro/hypopharynx Oropharynx Oro/hypopharynx

Staging* Grading Therapy

T4N2bM0 T4N2cM0 T4N2bM0 T4N2bMo T4N2bM0 T4N2bM0 T4N2bM0 T2N2bM0 T4N2bM0 T4N2bM0 T4N2bM0 T4N2cM0 T4N2cM0 T4N2cM0 T3N2cM0 T4N2cM0 T4N2cM0 T4N2cM0

1 2 2 2 2 4 2 2 4 3 3 3 2 2 2 3 3 3

RC RC R R R RC RC R R R R RC RC R RC R RC RC

R, Radiotherapy only, RC, radiochemotherapy. *TNM classification according to UICC 4th edition (1987).

oxygenation status in the response to therapy. Only a few publications are available at this time.10,20-23 Different methods to measure tumor oxygenation in vivo are recommended: positron emission tomography, P31-MR-spectroscopy, cryophotometry, C14-MISOscan, PQM, FRIM,24 radiobiologic methods, and PO2 histography.25-29 PO2 histography with the Kimoc 6650, Sigma PO2 histograph (Eppendorf, Hamburg, Germany) is an invasive method to measure tissue oxygenation with a polarographic needle electrode inserted in tumor tissue. The major advance of this method is that oxygenation in up to 200 different locations within a single

lesion can be determined. Former techniques used glass electrodes and lacked computer-assisted manipulation.16,20 It has been shown that the Kimoc is well tolerated and valid for measuring tumor oxygenation.9,10,22,26,27,29-31 Because of its superficial location, cervical lymph node metastases of head and neck tumors are particularly suitable for this method. In this article we present results of PO2 histography of patients with advanced squamous cell carcinoma of head and neck in the initial round of Methods radiotherapy or radiochemotherapy. METHODS AND PATIENTS The Kimoc 6650, Sigma PO2 histograph was used for the polarographic PO2 measurements. The measurements took place at the top of a steel-covered needle with a 0.3 mm outside diameter, transcutaneously introduced into tumor tissue. Movement and insertion of the needle into tumor tissue was performed with a computer-assisted micromanipulator. Under ultrasound guidance, the needle was inserted into the periphery of a lymphatic node metastasis of the neck. By computerassisted micromanipulation, up to 200 measurements were performed in a stepwise manner. Importantly, before each measurement the needle was drawn back 0.3 mm to avoid compression defects of the tissue. At this point, the PO2 was measured at the tip of the needle and displayed on the histograph (reaction time < 0.5 seconds). The actual polarographic measurement used a galvanic element recording the flow of electrodes from an Ag/AgCl skin anode toward a 12-µm gold cathode. The flow of electrodes at the tip of the needle is directly proportional to the concentration of molecular oxygen. In this way, up to 200 different measurements per metastasis could be evaluated in about 10 minutes. The placement and movement of the needle were monitored by ultrasound. Before each measurement, the needle was calibrated with air and pure nitrogen saturated in physiologic sodium solution, thus resulting a PO2 of 0 mm Hg. Analysis of the data includ-

Otolaryngology– Head and Neck Surgery Volume 121 Number 6

DIETZ et al

791

Table 3. Interval between first and second measurements of tumor oxygenation and volume of the cervical lymph node metastasis pretherapeutically Patient no.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Total Mean Median SD

Therapy

Days of therapy*

Radiation dose at 2nd measurement (Gy)

Volume of nodes†

No. of 1st single measurements

No. of 2nd single measurements

RC RC R R R RC RC R R R R RC RC R RC R RC RC — — — —

5 5 4 3 6 5 5 5 5 6 5 6 6 5 6 5 6 5 — — — —

9.0 9.0 7.2 5.4 10.8 9.0 9.0 9.0 9.0 10.8 9.0 12.0 10.8 9.0 10.8 9.0 10.8 9.0 — 9.4 9.0 1.5

10.8 4.8 0.8‡ 0.3‡ 19.0 2.7 8.5 3.3 37.1 5.0 2.0 1.6‡ 4.6 1.8‡ 13.4 3.0 21.7 4.9 — 8.1 4.7 9.5

172 149 167 40 189 163 200 200 200 200 200 200 200 200 200 200 200 200 3280 — — —

200 200 159 132 189 200 200 200 200 200 200 200 200 200 200 200 190 200 3470 — — —

*Interval

between first and second measurements. of volume with rotations ellipsoid: V = D1 × D2 × D3 × π/2 (pretherapeutic). ‡Nodes smaller than 2.0 mL showed pathologic solar Doppler findings. There was no cytologic confirmation of malignancy in these nodes. †Estimation

ed calculation of mean and median values and determination of the hypoxic fraction. All PO2 values lower than 10, 5, and 2.5 mm Hg were documented separately as hypoxic fractions. Eighteen patients with cervical lymphatic node metastases of advanced head and neck squamous cell carcinomas (oropharyngeal and hypopharyngeal carcinomas exclusively) were investigated. Nine patients were given combined radiochemotherapy with fluorouracil and carboplatin, and 9 patients were given radiotherapy without chemotherapy according to a multicenter trial protocol. The schedule followed is shown in Table 1. All patients were treated in the Departments of Otolaryngology–Head and Neck Surgery and Radiooncology, University of Heidelberg. The median age of patients was 55 years (range 42 to 68 years). Recruiting of patients was done in accordance with the exclusion and inclusion criteria of the trial. All patients had carcinomas of UICC stage IV. Table 2 shows the distribution of age, tumor site, staging, grading, and therapy (radiotherapy or radiochemotherapy). All measurements were conducted at superficially located cervical lymph node metastases with a diameter larger than 1.5 cm. The first series of measurements was performed before the start of therapy and the second series after the first

week of therapy (after application of a radiation dose of about 9 Gy). The location and volume of the lymph node metastases were estimated by ultrasound with the calculations of a rotation ellipsoid. The patients were placed in a supine position with the upper body elevated 30 degrees. The skin electrode was attached at the upper thorax. The polarographic needle was inserted under aseptic conditions with a 14-G vein catheter under ultrasound control. Forty to 200 measurements per node were collected. In addition, respiratory and circulatory parameters of the patients were monitored. All participants of the trial were informed by the investigator about possible complications (infection, bleeding, pain) and signed the acceptance form. The ethics committee of the University of Heidelberg gave approval to this protocol. For statistical calculations, the SAS package (SAS Institute Inc., Cary, NC) was used. The data from each series of measurements were evaluated and recorded as mean and median values with the standard deviation and as quantiles. Differences between values of the first and second series were examined with the Mann-Whitney-Wilcoxon test. No adjustment for multiple testing was done because of the basic character of this trial.

792

Otolaryngology– Head and Neck Surgery December 1999

DIETZ et al

Fig 1. Distribution (frequency histogram) of oxygenation in all 18 patients before start of therapy (n = 3280 single measurements) and after 1 week of therapy (n = 3470).

Table 4. Values of PO2 measurements at beginning of therapy and distribution of hypoxic fraction PO2 measurements

Distribution of hypoxic fraction

Patient no.

Mean (mm Hg)

Median (mm Hg)

<10 mm Hg (%)

<5 mm Hg (%)

<2.5 mm Hg (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Mean Median SD

22.1 19.8 21.2 17.1 11.2 20.6 32.0 18.0 21.3 10.0 35.7 24.3 8.2 14.9 11.0 19.7 13.0 14.5 18.6 18.9 7.3

20.6 8.2 16.7 16.6 7.8 21.0 27.0 7.9 13.0 3.9 36.3 15.0 0.7 6.6 2.9 14.7 2.5 5.2 12.6 10.6 9.4

26.2 52.9 41.9 32.5 53.4 32.5 37.0 53.5 48.5 58.5 13.0 47.5 74.0 57.0 63.0 39.0 73.5 61.0 48.1 50.7 16.1

17.4 45.3 38.3 32.5 41.8 22.1 32.0 46.5 41.0 52.5 12.0 39.5 66.5 32.5 53.5 25.0 67.5 47.5 39.6 40.3 15.2

0.0 19.8 34.1 30.0 32.3 14.7 30.0 40.0 3.0 43.5 10.5 33.5 59.0 0.0 47.5 5.5 50.0 19.0 26.2 30.0 18.1

RESULTS

Measurements have been well tolerated by all patients. A few patients reported some pain and a feeling of pressure caused by insertion of the needle into the cervical node. No local or systemic complications such as infections, bleeding, or severe pain were noticed in any of the patients after a follow-up of 6 months. In

total, 3280 measurements were performed in the first and 3470 in the second series (Table 3). Figure 1 shows the respective histograms of PO2 values (pretherapeutic and after 1 week of therapy). The pretherapeutic histogram shows that an increased concentration of values in hypoxic areas with PO2 values lower than 10 mm Hg were found. In contrast, the second histogram after 1

Otolaryngology– Head and Neck Surgery Volume 121 Number 6

DIETZ et al

793

Fig 2. Oxygenation (median value) of first (white bars) and second (black bars) measurement in all 18 patients.

Table 5. PO2 measurements after first week of therapy and distribution of hypoxic fraction PO2 measurements

Distribution of hypoxic fraction

Patient no.

Mean (mm Hg)

Median (mm Hg)

<10 mm Hg (%)

<5 mm Hg (%)

<2.5 mm Hg (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Mean Median SD

14.6 23.1 35.8 36.4 29.1 31.8 29.2 26.2 16.6 30.5 31.5 40.2 24.7 24.4 14.1 4.9 12.1 9.1 24.1 25.5 10.1

7.0 11.5 39.8 36.0 32.6 31.2 28.0 22.8 4.6 29.1 28.4 36.7 19.7 20.9 3.5 0.5 2.6 2.7 19.9 21.9 13.7

65.5 48.5 13.8 0.0 17.5 14.0 26.0 31.5 59.0 33.5 32.5 0.0 2.0 24.0 77.0 87.5 72.1 78.5 37.9 32.0 29.0

16.5 12.5 8.2 0.0 11.1 9.5 18.5 19.0 52.0 29.0 4.5 0.0 0.0 4.0 68.5 87.0 64.7 66.5 26.2 14.5 28.2

0.0 0.5 3.8 0.0 7.9 8.0 13.5 10.0 25.0 24.5 0.5 0.0 0.0 0.0 29.5 85.0 48.9 45.0 16.8 8.0 23.1

week of radiation shows a decrease of hypoxic values less than 10 mm Hg and increase of PO2 values around 30 to 40 mm Hg. For statistical evaluation, all measurements per patient were summarized as 1 value (median). Figure 2 shows the comparison of median values of PO2 measurements between the first and second series of mea-

surements. The mean value of (median) PO2 before the beginning of therapy was 12.6 ± 9.4 mm Hg (Table 4). In 50.7% of all measurements in the first series (before therapy, median value), a PO2 of less than 10 mm Hg was registered (Table 4). That means there is a high concentration of values in the hypoxic fraction lower 10 mm Hg PO2. After 1 week of therapy the medi-

794

Otolaryngology– Head and Neck Surgery December 1999

DIETZ et al

Table 6. Reoxygenation in initial phase of radiochemotherapy: Distribution of changes of PO2 from all 18 patients

Shift† Shift of hypoxic fraction (%) <10 mm Hg <5 mm Hg <2.5 mm Hg

Mean ± SD (mm Hg)

25% Q

50% Q (median)

75% Q

P value*

7.3 ± 13.5

–2.5

6.7

19.4

0.03

–10.1 ± 31.0 –13.4 ± 28.5 –9.5 ± 30.0

–32.5 –30.7 –30.0

–14.8 –18.5 –17.5

14.0 –0.9 0

0.18 0.03 0.08

Q, Quantile. *P-value calculated with Wilcoxon test for linked samples. †Shift: difference between median value of first and second (after 1 week) measurement.

an value of the hypoxic fraction lower than 10 mm Hg decreased to 32% (Table 5). In 2 patients, the second measurement did not show any values under 10 mm Hg. On average, the second measurement showed increased PO2 values. In contrast to the first measurement (12.6 ± 9.4) , the mean value of the medians of PO2 increased to 19.9 ± 13.7 mm Hg (Tables 4 and 5). In 13 patients there was an increase of median PO2 during the initial phase of therapy (Fig 2). In 3 cases values of the second trial were found to be twice as high as in the first trial. In 5 patients a decrease of oxygenation under therapy was noticed (Fig 2). The mean difference between the median PO2 values of first and second measurements in all 18 patients (Table 6) was 7.3 ± 13.5 mm Hg (P = 0.03). The median shift of the hypoxic fraction under 10 mm Hg in the initial phase of therapy decreased by 14.8%, but this difference was not significant (Table 6). The comparison between the median hypoxic fractions of less than 5 mm Hg PO2 showed significant results (reduction to 13.5% in the second trial, P = 0.03; Table 6). The difference between the hypoxic fractions of less than 2.5 mm Hg showed no significant difference (Table 6). In this study, we have not attempted to analyze the relationship between oxygenation and therapeutic response. DISCUSSION

In our trial the distribution of the oxygenation values showed a wide range of variation, both in individual patients and between patients (Tables 4 and 5). Possibly this variation was caused by an uneven distribution of hypoxic and nonhypoxic areas in tumor tissues. A similar variation has also been found in other studies using the Kimoc histograph. In a study of 31 patients with head and neck tumors, Nordsmark et al32 found that variation in oxygenation between different tumors was significantly greater than within the same tumor. Also, the variation in oxygenation of subcutaneous tissue was significantly greater between different patients than

within 1 patient. These results and the results presented in our study indicate that the PO2 histography with Kimoc is a valid method to determine the level of oxygenation in cervical lymph node metastases of head and neck tumors. It is clear, however, that for the individual patient the data should be interpreted very cautiously. The lymph node metastases examined in our study showed an extended hypoxia before therapy began. In 50.7% of all measurements in the first series, a PO2 of less than 10 mm Hg was registered (Table 5). Vaupel et al1 reported that the lowest physiologic median PO2 of 24 mm Hg occurs in brain tissue, as compared with spleen (66 mm Hg), skin (50 mm Hg), stomach mucosa (47 mm Hg), cervix uteri, skeletal muscle (28 mm Hg), heart muscle (25 mm Hg), and liver (24 mm Hg). Sutherland et al5 have stated that the ratio of mean oxygenation between normal tissue and tumor tissue in the case of squamous cell carcinoma ranges from 5.1 to 1.8. The median PO2 in squamous cell carcinoma has been reported to be in the range of 15 mm Hg.1 These data suggest that in squamous cell carcinomas huge areas of hypoxia exist. This highly reduced mean oxygenation status is one of the main obstacles for successful radiotherapy. Oxygenation of tumor tissue directly correlates with response to radiotherapy.7 In experimental tumor models it was shown that after reduction of PO2 to about 3 to 4 mm Hg, the relative cellular radiosensitivity was only half that of normal tissue oxygenation.7 It has been suggested that radiotherapy by itself can improve oxygenation of tumor tissue. Recent investigations of Zywietz et al19 in transplanted rat rhabdomyosarcoma showed reoxygenation in the early phase of irradiation. The total dose in this study was 60 Gy, given in 20 fractions during a 4-week period. PO2 was measured at weekly intervals with the Kimoc PO2 histograph. Doses lower than 45 Gy resulted in a decrease of the hypoxic fraction (<5 mm Hg). The investigations of Zywietz et al indicated that improved oxygenation in the initial phase of radiation occurred. Radiation doses above 45 Gy led

Otolaryngology– Head and Neck Surgery Volume 121 Number 6

to a considerable decrease in tumor oxygenation during the later phase of irradiation. Polarographic measurements in head and neck cancer during radiotherapy by Badip and Webster20 revealed a time-related increase of PO2 under therapy. Because there was only 1 measurement per tumor unit, results of this investigations have to be discussed critically. Another investigation by Füller et al21 on the oxygenation of cervical lymph node metastases under radiotherapy showed in 2 cases an increase and in 1 case a decrease of mean PO2. The problem with this study is that measurements were done at different doses (20 to 50 Gy). In our investigation, the increase of PO2 in the early phase of therapy was statistically significant and can be interpreted as reoxygenation. This improvement of oxygenation in the microenvironment of these cells appears to depend directly on radiotherapy and radiochemotherapy. We propose that better blood circulation in the first days of radiotherapy may be the mechanism underlying this phenomenon, which is in line with findings by Reinhold.33 Radiotherapy may cause reduction of pathologically increased interstitial pressure because of reopening of compressed tumor vessels.14 Radiotherapy-dependent reoxygenation in vivo was investigated in several tumors, mainly in mammary and cervix carcinomas.34-36 All authors remark that there are interindividual differences and a high variation in the levels of reoxygenation at different tumor sites. This could be confirmed in our investigations. In 6 patients, there was decreased oxygenation under therapy, in contrast to the main trend (Fig 2). Nordsmark et al10 stated in a study of 34 head and neck cancer patients that pretreatment tumor oxygenation status is predictive of radiation response when the fraction of PO2 values less than 2.5 mm Hg is used as an end point. This statement was based on a hypoxic fraction hypothetically defined as clinically relevant. On the basis of the literature concerning the role of oxygenation of head and neck tumors, it is at present not possible to define the clinical characteristics of PO2 histography. The wide range of distribution of PO2 values in the individual histogram of 1 lymphatic node suggests that an estimation of predictive character for the single patient based on PO2 histography is problematic. Because of the small number of patients (9 patients treated with radiation only and 9 patients treated with combined radiotherapy and chemotherapy), no conclusion can be drawn as to a possible differential influence of these treatments on the level of reoxygenation. Therefore investigations with a large number of patients are clearly required to work out the relevance of oxygenation and hypoxic fraction in head and neck tumors

DIETZ et al

795

in relation to radiotherapy and radiochemotherapy. Further follow-up investigations concerning the relationship between response and survival and tumor oxygenation must be done. Furthermore, it seems to be of high practical consequence for radiation protocols to find out how long it takes after radiation for maximal reoxygenation and supposedly highest radiosensitivity to be reached. CONCLUSION

Invasive PO2 histography with the Kimoc PO2 histograph is suitable to examine the role of tumor hypoxia, as shown here for metastatic carcinoma of the head and neck. The results obtained in this study indicate that reoxygenation occurs even during the initial phases of radiotherapy and radiochemotherapy, and they will form the basis for future comparative investigations of the possible influence of hypoxic parameters on tumor responsiveness toward radiation and radiochemotherapy. We thank Franz Xaver Bosch, PhD, molecular biology laboratory, Department of Otorhinolaryngology–Head and Neck Surgery, University of Heidelberg, Germany, for his assistance in interpretation of the data and helpful discussions. REFERENCES 1. Vaupel P, Kallinovski F, Okunieff P. Blood flow, oxygen and nutrient supply and metabolic microenvironment of human tumors: a review. Cancer Res 1989;49:6449-65. 2. Giaccia AJ. Hypoxia stress proteins: survival of the fittest. Semin Radiat Oncol 1996;6:46-58. 3. Graeber TG, Osmanian C, Jacks T, et al. Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumors. Nature 1996;379:88-91. 4. Loeffler DA, Juneau PL, Masserant S. Influence of tumour physico-chemical conditions on interleukin-2–stimulated lymphocyte proliferation. Br J Cancer 1992;66:619-22. 5. Sutherland RM, Ausserer WA, Murphy BJ, et al. Tumor hypoxia and heterogeneity: challenges and opportunities for the future. Semin Radiat Oncol 1996;6:59-70. 6. Hall J. The oxygen effect and radiation. In: Hall J, editor. Radiobiology for the radiologist. 4th ed. Philadelphia (PA): JB Lippincott; 1994. p. 133-50. 7. Alper T, Howard-Flanders P. Role of oxygen in modifying the radiosensitivity of E. coli. Nature 1956;178:978-9. 8. Gatenby RA, Kessler HB, Rosenblum JS, et al. Oxygen distribution in squamous cell carcinoma metastases and its relationship to outcome of radiation therapy. Int J Radiat Oncol Biol Phys 1988;14:831-8. 9. Höckel M, Knoop C, Schlenger K, et al. Intratumoral PO2 predicts survival in advanced cancer of the uterine cervix. Radiother Oncol 1993;26:45-50. 10. Nordsmark N, Overgaard M, Overgaard J. Pretreatment oxygenation predicts radiation treatment response in advanced squamous cell carcinoma of the head and neck. Radiother Oncol 1996;41:31-9. 11. Okunieff P, Höckel M, Dunphy EP, et al. Oxygen tension distributions are sufficient to explain the local response of human breast tumors treated with radiation alone. Int J Radiat Oncol Biol Phys 1993;26:631-6. 12. Teicher BA, Lazo JS, Sartorelli AC. Classification of antineoplastic agents by their selective toxicities towards oxygenated and hypoxic tumor cells. Cancer Res 1981;41:73-81.

796

Otolaryngology– Head and Neck Surgery December 1999

DIETZ et al

13. Powers WE, Tolmach LJ. A multicomponent x-ray survival curve for mouse lymphosarcoma cells irradiated in vivo. Nature 1963;197:710-1. 14. Cater DB, Silver IA. Quantitative measurements of oxygen tensions in normal tissues and in tumors of patients before and after radiotherapy. Acta Radiol 1960;53:233-56. 15. Lartigau E, Le Ridant AM, Lambin P et al. Oxygenation of head and neck tumors. Cancer 1993;71:2319-25. 16. Gatenby RA, Cioa LR, Richter MP, et al. Oxygen tension in human tumors: in vivo mapping using CT-guided probes. Radiology 1985;165:211-4. 17. Kallmann RF, Jardine LJ, Johnson CW. Effects of different schedules of dose fractionation on the oxygen status of a transplantable mouse sarcoma. J Natl Cancer Inst 1970;44:369-77. 18. Van Putten LM, Kallmann RF. Oxygenation status of a transplantable tumor during fractionated radiation therapy. J Natl Cancer Inst 1968;40:441-51. 19. Zywietz F, Reeker W, Kochs E. Tumor oxygenation in a transplanted rat rhabdomyosarcoma during fractionated irradiation. Int J Radiat Oncol Biol Phys 1995;32:1391-400. 20. Badip AO, Webster JH. Changes in tumor oxygen tension during radiation therapy. Acta Radiol Ther Phys Biol 1969;8:247-57. 21. Füller J, Feldmann HJ, Molls M, et al. Untersuchungen zum Sauerstoffpartialdruck im Tumorgewebe unter Radio- und Thermoradiotherapie. Strahlenther Onkol 1994;170:453-60. 22. Terris DJ, Dunphy EP. Oxygen tension measurements of head and neck cancers. Arch Otolaryngol Head Neck Surg 1994;120:283-7. 23. Guichard M. Oxygenation of head and neck tumors. Cancer 1993;71:2319-25. 24. Helmlinger G, Yuan F, Dellian M, et al. Interstitial pH and PO2gradients in solid tumors in vivo: high-resolution measurements reveal a lack of correlation. Nature Med 1997;3:177-82. 25. Chapman JD. The detection and measurement of hypoxic cells in solid tumors. Cancer 1984;54:2441-9.

26. Höckel M, Schlenger K, Knoop C, et al. Oxygenation of carcinomas of the uterine cervix: evaluation by computerized O2-tension measurements. Cancer Res 1991;51:6098-102. 27. Kallinowski F, Zander R, Höckel M, et al. Tumor tissue oxygenation as evaluated by computerized-PO2-histographie. Int J Radiat Oncol Biol Phys 1990;19:953-61. 28. Khalil AA, Horsman MR, Overgaard J. The importance of determining necrotic fraction when studying the effect of tumour volume on tissue oxygenation. Acta Oncol 1995;34:297-300. 29. Vaupel P, Schlenger K, Knoop C, et al. Oxygenation of human tumors: evaluation of tissue oxygen distribution in breast cancers by computerized O2-tension measurements. Cancer Res 1991;51: 3316-22. 30. Eble MJ, Lohr F, Wenz F, et al. Tissue oxygen tension distribution in two sublines of the Dunning prostate tumor R3327. In: Vaupel P, Kelleher DK, Günderoth M, editors. Tumoroxygenation, Reihe Funktionsanalyse Biologischer Systeme Nr. 42. Stuttgart: Gustav-Fischer Verlag; 1995. p. 95-105. 31. Lartigau E, Lespinasse F, Vitu L, et al. Does the direct measurement of oxygen tension in tumors have any adverse effects? Int J Radiat Oncol Biol Phys 1992;22:949-51. 32. Nordsmark M, Bentzen SM, Overgaard J. Measurement of human tumour oxygenation status by a polarographic needle electrode. Acta Oncol 1994;33:383-9. 33. Reinhold HS. Improved microcirculation in irradiated tumors. Eur J Cancer 1971;7:273-80. 34. Du Sault LA. Reoxygenation of tumors during radiotherapy. Radiology 1969;92:626-8. 35. Goda F, O’Hara J, Rhodes ES, et al. Changes of oxygen tension in experimental tumors after a single dose of x-ray radiation. Cancer Res 1995;55:2249-52. 36. Pappova N, Siracka E, Vacek A, et al. Oxygen tension and prediction of the radiation response. Polarographic study in human breast cancer. Neoplasma 1982;29:669-74.

AVAILABILITY OF JOURNAL BACK ISSUES As a service to our subscribers, copies of back issues of Otolaryngology–Head and Neck Surgery for the preceding 5 years are maintained and are available for purchase from Mosby at a cost of $16.00 per issue until inventory is depleted. The following quantity discounts are available: 25% off on quantities of 12 to 23, and one third off on quantities of 24 or more. Please write to Mosby, Inc., Subscription Services, 11830 Westline Industrial Dr, St. Louis, MO 63146-3318, or call 800-453-4351 or 314-453-4351 for information on availability of particular issues. If unavailable from the publisher, photocopies of complete issues may be purchased from Bell & Howell Information and Learning, 300 N Zeeb Rd, Ann Arbor, MI 481061346; phone, 734-761-4700 or 800-521-0600.