Oxygenation Gain Factor: A Novel Parameter Characterizing the Association between Hemoglobin Level and the Oxygenation Status of Breast Cancers

The introduction of the computerized pO₂-histography³ system (Eppendorf, Hamburg, Germany) for reliable measurements of tissue oxygen (O₂) tensions (pO₂) in the clinical setting allowed for the first time the systematic study of the oxygenation status in accessible, locally advanced tumors (1, 2). Over the last decade, assessment of tumor oxygen profiles determined with this O₂ microsensor technique repeatedly demonstrated that the presence of tissue hypoxia (i.e., tissue areas with critically reduced oxygen levels) or even anoxia (no measurable O₂ levels) are characteristic pathophysiological properties of solid tumors (3, 4, 5). In addition, hypoxia proved to be an independent, strong prognostic parameter in these malignancies (3, 4, 5, 6). Irrespective of the mode of treatment, patients with hypoxic tumors had a significantly poorer outcome than those with better oxygenated lesions of the same clinical size and stage. More important than its potential impact on the present treatment options are new insights into the fundamental role of tumor hypoxia in tumor propagation, malignant progression, acquired treatment resistance, and poor long-term prognosis (3, 4, 7).

Hypoxia is predominantly caused by structural and functional abnormalities of the newly formed tumor microvessels arising from neovascularization, by a compromised microcirculation, by enlarged diffusion distances, and by tumor-associated and/or therapy-induced anemia (3). The role of anemia in the development of tumor hypoxia as a consequence of a reduced O₂-carrying capacity of the blood has been demonstrated in an experimental tumor model (8, 9). These experimental data have provided strong evidence of a relationship between decreased Hb levels and a poor oxygenation status in solid tumors.

Using data from an ongoing prospective study examining the oxygenation status in breast cancers, this investigation is intended to evaluate a possible relationship between Hb levels and pretreatment tumor oxygenation in conscious patients. This is to our knowledge the first study relating blood Hb levels (anemia of grades I and II included, National Cancer Institute system) to the oxygenation status of breast cancers. Results obtained may have important implications for studies identifying inter alia the power of prognostic factors, the requirement of RBC transfusions, and/or the benefit of erythropoietin treatment of anemic patients.

The evaluation of intratumoral pO₂ distributions in locally advanced breast cancers was conducted at the Department of Obstetrics and Gynecology, University of Leipzig from January 1999 through December 2002. The study design was approved by the Local Human Ethics Committee, and all of the enrolled patients gave informed written consent. Pretreatment pO₂ measurements were performed on 37 conscious patients with breast cancer using the pO₂ histography system after a standard procedure, which has been described in detail previously (1, 7, 10). All of the pO₂ measurements were performed under ultrasound guidance of the O₂ sensor. For the description of the oxygenation status of the breast cancers, the median pO₂ and the fraction of pO₂ readings ≤2.5 mm Hg and ≤5 mm Hg were stated. In all of the patients, cHb was determined by standard procedures in venous blood samples before pO₂ measurements.

Results are expressed as means ± SE. Differences between groups were assessed by the Wilcoxon test. The significance level was set at α = 5% for all of the comparisons.

The age range of the enrolled patients was 42–90 years (median, 65 years). Twelve of the women were premenopausal, and 25 were postmenopausal. Largest tumor diameter ranged from 1.0 to 5.2 cm (median, 2.5 cm). All of the tumors were invasive and were classified according to the Tumor-Node-Metastasis staging system (Unio Internationale Contra Cancrum, 1997). pT-stages were pT1c (n = 10), pT2 (n = 19), pT3 (n = 5), and pT4b (n = 3). According to histopathologic classification, 28 cancers were ductal, 7 lobular, 1 was intracystic, and 1 apocrine. Histological grades were G1 (n = 5), G2 (n = 20), and G3 (n = 12). Nodal status was N0 (n = 10), N1 (n = 23), N2 (n = 1), and NX (n = 3). M stage was M0 (n = 8), M1 (n = 2), and MX (n = 27).

Approximately 63% of the breast cancers investigated exhibited hypoxic tissue areas (pO₂ ≤ 2.5 mm Hg), which were heterogeneously distributed within the tumor masses. The median pO₂ was 6 mm Hg. The fraction of pO₂ values ≤2.5 mm Hg was 25%, and 58.5% of the pO₂ values were ≤5 mm Hg. When tumors of different sizes were compared, there was no evidence of a correlation between the median pO₂ and the maximum tumor diameter. This implies that the oxygenation in breast cancers and the occurrence of hypoxia and/or anoxia do not correlate with the clinical and pathological stages. In addition, the oxygenation pattern does not correlate with tumor location within the breast, N stage, histological type, and grade, nor with a series of other clinically relevant parameters (e.g., hormone receptor status, parity, and menopausal status).

Pretreatment median cHb measured in our patient cohort was 13.0 g/dl (range, 8.0–15.3 g/dl). Larger tumors or higher stages tended to correlate with lower cHb values (12.95 g/dl in patients with tumors <2.5 cm diameter versus 12.05 g/dl in patients with cancers ≥2.5 cm). At presentation, 27% of our breast cancer patients were anemic. This value is in line with data published earlier (e.g., 28.7%; Ref. 11).

In patients with normal Hb levels (cHb ≥12 g/dl) the median pO₂ was significantly higher than in anemic patients (P = 0.015). Additionally, analysis of the oxygenation status as a function of baseline cHb was performed by dividing the patients into five groups based on “high” (above median cHb; median cHb of age-matched healthy women = 14.0 g/dl; Ref. 12), “intermediate” cHb values (13.0 g/dl < cHb ≤14.0 g/dl), “lower-normal” cHb values (12.0 g/dl ≤ cHb ≤13.0 g/dl), mild anemia (grade I, 10.0 g/dl < cHb <12.0 g/dl), and moderate anemia (grade II, 8.0 g/dl < cHb ≤10.0 g/dl).

On the basis of this separation, a correlation between Hb levels and median pO₂ values as shown in Fig. 1 was obtained. In moderately anemic patients all of the median pO₂ values were <6 mm Hg. At a mean Hb level of 8.5 ± 0.2 g/dl, the median pO₂ was 3 mm Hg. With increasing cHb values the median pO₂ exponentially increased (log pO₂ = 0.114 cHb − 0.472; r² = 0.985) reaching a maximum pO₂ of 15 mm Hg in the cHb range above the median (mean cHb = 14.7 ± 0.2 g/dl). The difference in the oxygenation status of breast cancer in moderately anemic patients and women with cHb values above the median (“high” cHb group) was statistically significant (P = 0.005; see Fig. 1). Accordingly, the hypoxic fraction of pO₂ values ≤5 mm Hg increased significantly from 53% ± 6% to 77% ± 7% (P = 0.020) when breast cancers of nonanemic patients (cHb = 13.6 ± 0.2 g/dl) were compared with anemic patients (cHb = 9.4 ± 0.5 g/dl).

pO₂ measurements performed in normal breast tissue, in the fatty tissue of the subcutis⁵ and in skeletal muscle (sternocleidomastoid muscle; Ref. 13) provided substantially different data (see Fig. 1). In normal breast tissue, the median pO₂ was 52 mm Hg over the cHb range from 8.4 to 14.8 g/dl with no cHb-related differences in pO₂ being evident. In the subcutis, the median pO₂ was 51 mm Hg in the cHb range from 10 to 16 g/dl with no differences in pO₂ being seen over this range. Only cHb values <10 g/dl (grade II anemia) were associated with decreased median tissue pO₂ values (P < 0.001). The relative decrease in the median pO₂ was only 23% compared with 80% in breast cancers. At a cHb of 9.5 g/dl, the median pO₂ was ∼40 mm Hg, which can still be considered as a “physiological” O₂ level and would not be expected to compromise the cellular energy status in any way (3). In skeletal muscle, the median pO₂ was 37 mm Hg in the cHb range from 10 to 17 g/dl. Here again, no pO₂ differences were observed between nonanemic and mildly anemic (grade I anemia) patients. As was the case with the subcutis, only cHb values <10 g/dl (moderate anemia, grade II anemia) coincided with a slightly lower muscle oxygenation status reaching a median pO₂ of 34 mm Hg at a mean cHb of 9.4 g/dl corresponding to a relative decrease in the median pO₂ of 9%. This latter O₂ status must also still be considered as being within the “physiological range.”

To characterize the association between cHb levels and the oxygenation status of primary breast cancers, we suggest a novel parameter, the tumor OGF, which is defined as follows:

The δs represent differences used to express a differential quotient and, thus, OGF is the approximation of the derivation of pO₂ by cHb. Hence, the calculation of OGF is based on a smooth nonlinear correlation function derived from individual values.

In breast cancer of anemic patients, OGF equals 1.5 mm Hg·dl/g. This indicates that the median pO₂ in anemic breast cancer patients should rise by ∼1.5 mm Hg for every 1 g/dl increment in cHb (see Table 1). Surprisingly, an even greater gain in tumor oxygenation (∼3 mm Hg per 1 g/dl rise in cHb) was seen over the normal cHb range (cHb ≥12 g/dl). A similar dependency was observed when improvement in overall QoL was assessed as a function of Hb levels. In 7724 patients QoL improved slightly in the anemic range, whereas at Hb levels >12 g/dl there was a more pronounced improvement in QoL (14, 15, 16).

In contrast, in normal tissues OGF is zero at cHb values ≥10 g/dl, i.e., in mildly anemic patients (10 g/dl ≤ cHb ≤12 g/dl) the reduced O₂ carrying capacity of the blood is fully compensated by a rise in local blood flow (and a possible increase in the O₂ extraction from the blood). In locally advanced solid tumors these two adaptive (compensatory) mechanisms may not work because flow regulation is limited, if not impossible, and O₂ extraction ratios are already high at physiological Hb levels (17).

In conclusion, this study clearly shows that the oxygenation status of primary breast cancers critically depends on the whole blood Hb level (cHb), with median cHb (over the range 8.5–14.7 g/dl) correlating with exponentially greater median pO₂ values (3–15 mm Hg) yielding an average OGF of 2 mm Hg·dl/g. In contrast, in normal tissues (subcutis and skeletal muscle) the median pO₂ values are substantially higher (51 mm Hg and 37 mm Hg, respectively) and are relatively constant over hemoglobin levels from 10 to 16 g/dl (grade I anemia and nonanemic patients). Only in moderate anemia (grade II anemia, 8 g/dl < cHb ≤10 g/dl) were relatively small decreases in the median pO₂ values observed (−9% in skeletal muscle and −23% in the subcutis), which is most probably without any biological relevance.

We thank Dr. Debra K. Kelleher for valuable editorial help during manuscript preparation.