Radiotherapy and Oncology

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1 Radiotherapy and Oncology 98 (2011) Contents lists available at ScienceDirect Radiotherapy and Oncology journal homepage: Phase III randomised trial The importance of haemoglobin level and effect of transfusion in HNSCC patients treated with radiotherapy Results from the randomized DAHANCA 5 study Camilla Molich Hoff a,, Hanne Sand Hansen b, Marie Overgaard c, Cai Grau c, Jørgen Johansen d, Jens Bentzen e, Jens Overgaard a a Department of Experimental Clinical Oncology, Aarhus University Hospital, Denmark; b Department of Oncology, The Finsen Centre, Rigshospitalet, Copenhagen, Denmark; c Department of Oncology, Aarhus University Hospital, Denmark; d Department of Oncology, Odense University Hospital, Denmark; e Department of Oncology, Herlev Hospital, Denmark article info abstract Article history: Received 15 July 2010 Received in revised form 23 September 2010 Accepted 24 September 2010 Available online 20 October 2010 Keywords: Transfusion Radiotherapy Randomized trial Head and neck squamous cell cancer Haemoglobin Hypoxia Background and purpose: Patients with head and neck squamous cell carcinoma (HNSCC) and a low level of haemoglobin (Hb) often have a poor response to radiation which may be related to hypoxia induced radioresistance. The aim of the study was to evaluate the prognostic significance of low Hb level and its modification by transfusion in HNSCC patients treated with radiotherapy. The study was performed as a subrandomization in the DAHANCA 5 trial. Material and methods: Patients were randomized to treatment with the hypoxic radiosensitizer nimorazole or placebo, and in addition, patients with low pre-irradiation Hb values (females < 13 g/dl; males < 14.5 g/dl) were subrandomized to plus or minus transfusion. Transfusion was given with packed red blood cells with the aim to achieve a Hb level in the high value range. Results: A total of 414 patients were included, 243 patients had high Hb levels and 171 patients had low Hb levels. Of the low Hb patients, 82 were randomized to receive transfusion and 89 not to receive transfusion. The treatment arms were well balanced. In the majority of patients, transfusion resulted in increased Hb levels although this tended to decline throughout treatment. Patients with high Hb levels had a significantly better probability of locoregional control, disease-specific survival and overall survival compared to low Hb no transfusion patients. In the low Hb group, transfusion did not improve the outcome in locoregional control, disease-specific survival or overall survival. In multivariate analyses, T and N classifications were significant for all outcome measures, whereas there was no significant influence of transfusion or Hb level on endpoints. Conclusion: The univariate prognostic significance of high Hb level was demonstrated in patients with HNSCC treated with radiotherapy; however, transfusion prior to and during treatment did not improve the outcome in patients with low Hb values. Ó 2010 Elsevier Ireland Ltd. All rights reserved. Radiotherapy and Oncology 98 (2011) Patients with squamous cell carcinomas of the head and neck (HNSCC) and a low level of haemoglobin (Hb) often have a poor response to radiotherapy (RT) [1 4]. Tumours are heterogenic and hypoxic radioresistant cells are often present [5,6]. A correlation between hypoxia and haemoglobin level has been shown, but the precise relationship remains uncertain [4,7,8]. The recent use of erythropoietin stimulating agents (ESA) and the uncertainty in safety when using these agents [9 13] have again opened the discussion on how to raise haemoglobin levels before and during treatment. Corresponding author. Address: Department of Experimental Clinical Oncology, Aarhus University Hospital, Noerrebrogade 44, bld 5 2nd floor, DK-8000 Århus C, Denmark. address: camilla@oncology.dk (C.M. Hof. Several studies, both experimental and clinical, have shown that low haemoglobin levels and hypoxia are related [14,15]. Transfusions to tumour bearing anaemic mice have suggested that tumours, otherwise radioresistant, could regain radiosensitivity [16 19]. Correction of anaemia in clinical studies has resulted in improvement in tumour oxygenation and a subsequent increase in the therapeutic efficacy of irradiation [2,20 28]. As part of the randomized trial DAHANCA 5, evaluating the role of nimorazole as a hypoxic radiosensitizer, the role of transfusion to low haemoglobin patients was evaluated [29,30]. The hypothesis being that in HNSCC patients with low haemoglobin levels, an increase in haemoglobin level by transfusion may improve the effect of radiotherapy irrespective of hypoxic modification [29] /$ - see front matter Ó 2010 Elsevier Ireland Ltd. All rights reserved. doi: /j.radonc

2 C.M. Hoff et al. / Radiotherapy and Oncology 98 (2011) Methods Patients and treatment The Danish Head and Neck Cancer Study, DAHANCA protocol 5 85 was activated in January 1986 as a multicenter randomized and balanced double-blind trial with the purpose of assessing (1) the efficacy of nimorazole in conjunction with radiotherapy, (2) the tolerance and toxicity of nimorazole and (3) the influence of haemoglobin concentration on tumour response to irradiation. Detailed study design has been published previously [29 31]. In brief, patients were prior to randomization stratified according to gender, institution, tumour site, tumour stage and haemoglobin concentration. In the main study the patients were randomized to radiotherapy with nimorazole or placebo. In addition to the randomization to nimorazole, patients with low pre-irradiation haemoglobin (females < 13.0 g/dl; males < 14.5 g/ dl) were offered subrandomization to receive or not to receive transfusion prior to and during radiotherapy. Transfusions were given with packed red blood cells (one unit approximately equivalent to 500 ml full blood) to achieve a haemoglobin concentration in the high value range. Transfusion was given before radiotherapy and if during radiation the haemoglobin level fell below the values indicated above, the transfusion was repeated. The haemoglobin level was measured at least every 2 weeks [29]. Radiotherapy was given according to DAHANCA guidelines ( Primary target was given a total of Gy, 2 Gy/Fx, 5 Fx/week over a period of 6.5 weeks, including a boost to the primary tumour, maximum 18 Gy while elective regions received Gy. Nimorazole was given according to protocol guidelines. No elective neck dissection was performed [29]. The study was done in accordance with the Helsinki declaration and approved by relevant ethical committees. All patients gave written informed consent [29]. Follow up Patients were followed at the oncological centres for at least 5 years or until death. They were evaluated with clinical examination weekly during treatment, 2 months after treatment, 3 month intervals for the first year, 4 month intervals for the second year, and then twice annually for up to 5 years after randomization [29]. Endpoints The primary endpoint was locoregional control after radiotherapy. The definition of this endpoint was complete and persistent disappearance of the disease in the primary tumour (T-site) and regional lymph nodes (N-site) after radiotherapy. Failure was recorded in case of recurrent tumour, or if the primary tumour never completely disappeared. In the latter situation, the tumour was assumed to have failed at the time of randomization. The primary endpoint did not include the effect of successful procedure with salvage surgery. Secondary endpoints include disease-specific survival and overall survival. The endpoint used for diseasespecific survival was death from or with the actual cancer. The endpoint for overall survival was any death, irrespective of cause. All time estimates were done using the date of randomization as the initial value. The treatment effect was evaluated using the intention to treat principle and patients were included in their randomization group irrespective of whether or not they had completed the planned treatment. Time for evaluation of locoregional control, diseasespecific survival and overall survival was 5 years after randomization since patients were followed regularly only for that period. Statistical analyses First patients with high haemoglobin and patients with low haemoglobin without transfusion (high, low t) were compared to determine the effect of haemoglobin level. The effect of transfusion was evaluated by comparing the two low groups, with and without transfusion (low+t, low t), respectively. Statistical analyses were done using the STATA 10 software package. Patient characteristics were compared with chi 2 -test and significance level was chosen to be 5%. The actuarial values of endpoints were evaluated by the Kaplan Meier plots and compared with log-rank test for equality of survivor functions. The p-values estimated are those for a two tailed test and the significance level was chosen to be 5%. A multivariate Cox proportional hazards analysis was used to evaluate prognostic factors and treatment with respect to the risk of locoregional failure, disease-specific survival and overall survival. Parameters were included when statistically significant in univariate analysis. Data are presented as 5-year actuarial hazard ratios (HR) with 95% confidence intervals, unless otherwise mentioned. The study was not dimensioned with regard to the present analysis of transfusion effect, but with approximately 50 events in each of the low groups and an expected locoregional control rate of 50%, a difference of approximately 27% with alpha 0.05 and a power of 0.8 can be detected. Results Study flow chart and patient characteristics are shown in Fig. 1 and Table 1. The analysis included 414 eligible and evaluable patients, 243/414 (59%) in the high haemoglobin and 171/414 (41%) in the low haemoglobin group. No patients were excluded from the analysis. The high haemoglobin group contained relatively more male patients and more supraglottic cancers, beside from this, the patient and tumour characteristics in three groups were in general similar. There were no differences between the transfused and the non-transfused low Hb groups (Table 1). Pre-treatment measurements showed that 171 patients had low haemoglobin levels, 25/110 (23%) of females and 146/304 (48%) of males. These patients were subsequently randomized to transfusion 82/171 (48%) or no transfusion 89/171 (52%). The group given transfusion consisted of 11/25 (44%) females and 71/ 146 (49%) males. There was no difference in the distribution of haemoglobin levels in the transfused and non-transfused group before treatment. Compliance to radiotherapy was good and overall 97% completed the planned radiotherapy treatment, irrespective of treatment group. Transfusion In the majority of the cases, transfusion was able to raise haemoglobin values. The haemoglobin level during radiotherapy decreased throughout the course of radiotherapy, despite an obvious effect of transfusion at the beginning of treatment (Fig. 2, Appendices 1 and 2). The number of packed red blood cells received per patient ranged between 0 6 units (median 2). A total of 55/82 (67%) patients received transfusion at one occasion, 20/82 (24%) received more than one transfusion and 7/82 (9%) never received any transfusions despite their randomization group. Most patients received their first transfusion immediately prior to start of radiotherapy (median 1 day, range: 13 to 43 days).

3 30 DAHANCA 5 - Effect of transfusion in HNSCC Fig. 1. Study flow chart. Table 1 Patient and tumour characteristics. Parameter Low Hb transf Low Hb + transf High Hb All patients p-value * p-value # Age <60 years 42 47% 32 39% % >60 years 47 53% 50 61% % 213 Gender Female 14 16% 11 13% 85 35% Male 75 84% 71 87% % 304 Site Supraglottis 20 22% 21 26% 84 35% Pharynx 69 78% 61 74% % 289 T-classification T1 + T % 35 43% % T3 + T % 47 57% % 217 N-classification N % 28 34% % N % 54 66% % 227 Stage I + II 15 17% 17 21% 59 24% III + IV 74 83% 65 79% % 323 Treatment Nimorazole 44 49% 48 59% % Placebo 45 51% 34 41% % 195 Total * Comparing difference between the two low Hb groups (low t vs low+t). # Comparing difference between the two non-transfused Hb groups (low t vs high). Treatment outcome At the time of clinical assessment, 225/414 (54%) patients had experienced locoregional failure. A total of 204/414 (49%) had died of disease and 272/414 (66%) had died overall (Fig. 1). The frequency of patients with distant metastases in the three groups low t, low+t and high were 11% (10/89), 15% (12/82) and 9% (22/243), respectively. Only 9 patients experienced an isolated distant recurrence, distributed in the groups as one, three and five patients, respectively. Univariate analysis showed statistical significance of age at inclusion, gender, site, T- and N-classifications, stage, nimorazole treatment and haemoglobin level at inclusion (Table 2). The high haemoglobin group had a better outcome regarding locoregional control probability compared to low non-transfused patients (HR 0.71; CI ; p = 0.03). However, no difference between transfusion and no transfusion in the low haemoglobin group was observed (HR 0.99; CI ; p = 0.9) (Fig. 3a and Table 2). Similar results were found using the endpoint of dis-

4 C.M. Hoff et al. / Radiotherapy and Oncology 98 (2011) Fig. 2. Haemoglobin level during RT treatment as a function of gender. ease-specific survival probability between high and low t (HR 0.66; CI ; p = 0.01), and a lack of effect of transfusion (HR 1.07; CI ; p = 0.8) (Fig. 3b and Table 2). Finally the same pattern was found for overall survival probability in high haemoglobin and low non-transfused patients (HR 0.69; CI ; p = 0.01), and again no benefit from transfusion (HR 1.10; CI ; p = 0.6) (Fig. 3c and Table 2). The importance of haemoglobin level and lack of effect of transfusion were found irrespective of whether the patients had received nimorazole treatment or not (Appendix 3). Multivariate analysis confirmed the importance of T- and N- classifications and gender for all endpoints whereas neither haemoglobin value nor transfusion could be identified as independent factors of outcome. Age at inclusion was significant for overall and disease-specific survival and nimorazole treatment for locoregional control (Table 2). Discussion The study confirmed the prognostic importance of haemoglobin level in HNSCC patients receiving radiotherapy. In patients with a low haemoglobin level transfusion was able to raise the haemoglobin level during radiotherapy, however, this increase in haemoglobin level did not improve the effect of radiotherapy. The haemoglobin level was found to be a significant prognostic factor in univariate analysis for all outcome measurements, this significance however did not appear in the multivariate analysis. This final conclusion is therefore in accordance with the initial haemoglobin results of the DAHANCA 5 study, which also demonstrated univariate prognostic importance of haemoglobin level and no benefit of transfusion [29]. The use of transfusion to improve hypoxic radioresistance has only been investigated in one small randomized trial in patients with cancer of the uterine cervix. Bush et al. concluded that patients with a haemoglobin level during treatment below 12 g/dl had a significantly higher recurrence rate and lower cure rate and that a correction of anaemia by transfusion could decrease the recurrence and increase the cure rate, this being consistent with tumour hypoxia being greater in anaemic patients [26]. However, a reanalysis of this trial by Fyles et al. using the intention-totreat principle could not confirm the convincing evidence of the original report [27]. Other non-randomized uterine cervix studies showing an importance of haemoglobin level and effect of transfusion have been published [12,28,32,33], but cancers of the uterine cervix differ from head and neck cancers since they often suffer from bleeding from the tumour and bleeding could also be a sign of more advanced disease, poorer performance status, etc. There are several potential reasons to explain the lack of effect of transfusion, but they are not examined by the present analysis and still subject to discussion. Hirst et al. has in several preclinical studies investigated the importance of anaemia in radiation resistance [16,34]. Using rodent tumours they not only described a clear benefit of blood transfusion to improve outcome in radiotherapy [19], but also that the timing between transfusion and irradiation was of outmost importance, since increased haemoglobin concentration may induce tumour cell proliferation. Although head and neck tumour volume doubling time is relatively slow [35], tumour cells around a small blood vessel may show a very fast cellular proliferation [36]. We cannot exclude that some patients in our study may have had a too long interval between transfusion and irradiation, in such a way that the tumours may have adapted to the increased blood supply by growth [37,38]. It can also be argued that the aim for haemoglobin level was set too high since it has been suggested that the optimal range in haemoglobin levels for achieving maximum oxygenation status in solid tumours are g/dl for women and g/dl for men [15]. We attempted to keep females above 13.0 g/dl and males Table 2 Univariate and multivariate analyses. Locoregional control Disease-specific survival Overall survival HR CI p-value HR CI p-value HR CI p-value Univariate parameter Age >60 vs < ( ) ( ) ( ) Gender Female vs male 0.67 ( ) ( ) ( ) Site Pharynx vs supragl 1.24 ( ) ( ) ( ) T-classification T1 + T2 vs T3 + T ( ) ( ) ( ) N-classification N0 vs N ( ) < ( ) < ( ) <0.001 Stage I + II vs III + IV 0.58 ( ) ( ) ( ) 0.03 Treatment Nimo vs placebo 0.70 ( ) ( ) ( ) 0.7 Differentiation Moderate + well vs UK + poor 1.19 ( ) ( ) ( ) 0.5 Haemoglobin level Low + t vs low t 0.99 ( ) ( ) ( ) 0.6 High vs low t 0.71 ( ) ( ) ( ) 0.01 Multivariate analysis Age >60 vs < ( ) ( ) ( ) Gender Female vs male 0.73 ( ) ( ) ( ) Site Pharynx vs supragl 1.04 ( ) ( ) ( ) 0.1 T-classification T1 + T2 vs T3 + T ( ) ( ) ( ) 0.01 N-classification N0 vs N ( ) < ( ) < ( ) Treatment Nimo vs placebo 0.72 ( ) ( ) ( ) 0.9 Haemoglobin level Low + t vs low t 0.93 ( ) ( ) ( ) 0.9 High vs low t 0.81 ( ) ( ) ( ) 0.2

5 32 DAHANCA 5 - Effect of transfusion in HNSCC haemoglobin level and the oxygen carrying capacity of haemoglobin. Unfortunately, such data are not available in the present study, but a prospective recording has subsequently been initiated. The influence of other tobacco related health factors may indirectly be seen when looking at the survival curves in the present study. The largest influence of haemoglobin concentration is found in overall survival. This might be an indication of other factors (e.g. tobacco related diseases, secondary cancers) than the cancer in question may influence the outcome. The study confirmed that LRC was significantly improved by nimorazole irrespective of haemoglobin concentration. However, in some cases these differences did not reach statistical significance, probably due to a small number of patients in each group, and the haemoglobin level was therefore important despite hypoxic modification. The present study has been performed as a subrandomization in the DAHANCA 5 trial, with a risk that the study does not have the necessary power to detect clinical relevant differences. The subrandomization was therefore continued in the DAHANCA 7 protocol and will be reported later. This will also allow a more detailed analysis of the haemoglobin levels during treatment. Despite efforts to maintain high haemoglobin level following transfusion there was a general trend of reduction of the peak level. This may account for the lack of response in the transfused patients, since a continuous increased gradient in the haemoglobin concentration is a part of the theoretical mechanism behind improved tumour oxygenation. Such decrease may be avoided by using treatment with erythropoietin stimulating agents, and several clinical trials (including DAHANCA 10) have been initiated to investigate this hypothesis, without showing any significant benefit [9 13]. Conclusion The study confirmed the univariate prognostic importance of haemoglobin level in HNSCC patients receiving radiotherapy. Transfusion was able to raise the haemoglobin level during radiotherapy, however, this increase in haemoglobin level by transfusion was not maintained during the entire treatment period, and did not result in improved effect of radiotherapy. The study was however too small to ultimately determine the role of transfusion, and the randomization has consequently been continued in the DAHANCA 7 protocol. Conflicts of interest statement No conflicts of interest declared by the authors. Fig. 3. Locoregional control (a), disease-specific (b) and overall survival (c) probability curves (Kaplan Meier method) according to haemoglobin group. above 14.5 g/dl based on previous observations of the relationship between haemoglobin level and locoregional control [1,39,40]. Only few patients reached such high haemoglobin levels, and the present study did not have enough patients to make a proper subanalysis of different haemoglobin levels. The benefit of high haemoglobin concentration relates to the assumption that the oxygen carrying capacity of the blood is associated with tumour cell oxygenation. Disturbing this oxygen carrying capacity by binding carbon monoxide to the haemoglobin may strongly alter this mechanism as it may happen in smokers [41,42]. Animal studies have shown that the oxygen availability is decreased to 30 40% of the normal air-breathing controls [43]. Therefore, smoking status should also be known when considering the Acknowledgements Supported by CIRRO The Lundbeck Foundation Center for Interventional Research in Radiation Oncology and the Danish Council for Strategic Research. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi: /j.radonc References [1] Overgaard J, Hansen HS, Jorgensen K, Hjelm HM. Primary radiotherapy of larynx and pharynx carcinoma an analysis of some factors influencing local control and survival. Int J Radiat Oncol Biol Phys 1986;12: [2] Overgaard J. Sensitization of hypoxic tumour cells clinical experience. Int J Radiat Biol 1989;56:

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