As pointed out by Glynne Jones et al., prognostic factors have to be intended as specific measurable characteristics that can be easily obtained and quantified during observation within a certain population to be potentially correlated to measures of clinical outcomes [
11]. In anal cancer, prognostic factors have been described within retrospective series or randomized prospective phase III trials [
9,
11,
12]. Some depend on tumor characteristics such as primary tumor dimension and nodal involvement, while others are intrinsic to the patient as for example gender. Hemoglobin level is a patient-related clinical factor that has not been extensively explored in anal cancer patients [
15]. The correlation between Hb concentration and tumor oxygenation is well-established in several tumor types, with head and neck cancer being a paradigmatic example. [
21,
22]. Anemia may enhance tumor hypoxia, increasing tumor cell radio-resistance and leading to a potentially more aggressive tumor phenotype [
11]. This is well-known, for example, in cervical cancer, but evidence is also present for anal malignancies [
23,
24]. Interestingly, in the RTOG 98–11 phase III trial, which explored the role of cisplatin concurrent to radiation and that of maintenance therapy, patients having levels of Hb below 10 g/dl before randomization were excluded upfront from the study, because of the potential dismal prognosis [
7]. In this sense, international clinical guidelines (such as those of the National Comprehensive Cancer Network) suggest the use of blood transfusion in symptomatic patients with Hb levels below 10 g/dl, in order also to potentially enhance tumor re-oxygenation [
25]. Our data seems to confirm these findings. On univariate analysis, baseline Hb levels, considered as a continuous variable, had a significant correlation to both PFS (HR:0.57;95%CI:0.39–0.85;
p = 0.049) and OS (HR:0.53;95% CI:0.29–0.96;
p = 0.047). Conversely, on multivariate analysis, baseline Hb was significantly correlated only to OS (HR:0.50;95% CI:0.31–0.83;
p = 0.0051), but not to PFS (HR:0.80;95% CI:0.48–1.34;
p = 0.40). The discrepancy observed for the correlation between pre-treatment Hb and OS vs PFS prompts to interrogate whether baseline Hb is a real independent prognostic factor or if it is just a surrogate parameter for patient’s comorbid conditions or worse prognostic outcome. Nevertheless, it should be noted that the results seems to suggest the presence of a correlation trend between Hb and PFS and hence, our hypothesis is that this observation can be due to the slenderness of the sample size in our study. We also cannot rule out an effect related to competing causes of death, as low Hb levels could be related to a ‘frail’ patient phenotype or worse clinical conditions predisposing to a higher likelihood of death from any cause, not strictly related to cancer. We also have to notice that we could detect a significant correlation between nodal involvement and baseline Hb levels. Again, we cannot fully rule out the possibility that this correlation could explain, at least partially, the poor PFS and OS related to Hb levels. However, both nodal statuts and Hb levels were comprised within the multivariable model, showing independent statistical significance. Moreover, the VIF for both PFS and OS showed very low values (< 1.1), strongly suggesting a limited influence of this correlation on the final outcome results. In our analysis, we employed a cut-off point at Hb =12 g/dl, which was able to allocate patients to different prognostic classes for both 5-year PFS (82.2% for Hb ≥ 12 g/dl vs 29.3% for Hb < 12 g/dl) and OS (82.2% for Hb ≥ 12 g/dl vs 32.8.% for Hb < 12 g/dl). Our data are in line with those of Roldan et al. who found a significant correlation between pre-treatment Hb levels and PFS and OS on univariate analysis, in a series of 72 anal cancer patients treated with concurrent CT-RT [
15]. On multivariate analysis, lowest quartile pre-treatment Hb was a predictor for PFS, while pre-treatment Hb level was a significant predictor for OS. Interestingly, response to treatment at 3 months (together with nodal involvement and performance status) was confirmed as a significant predictor for both PFS and OS. Using baseline Hb values below 12 g/dl as a cut- off point (only 10% of patients were below the cut-off), Hb levels remained significantly associated to OS (log-rank
p = 0.003) and PFS (log-rank
p < 0.0001). In general, patients with pre-treatment Hb values in the lowest quartile had significantly worse PFS and OS than those whose values were in the 3 higher quartiles. A similar threshold was also found by Kapacee et al., in their series of 148 anal cancer patients treated CT-RT within the ACT-II trial (50.4 Gy/28 fractions delivered over 38 days concomitant to 5-FU and either MMC or DDP), where a pre-treatment Hb level < 13 g/dl was found to predict for lower distant metastasis and cancer-specific survival (
p < 0.05) [
26]. Given the impact of both CR and baseline Hb level on treatment outcomes, we tried to quantify the relationship between these 2 clinical variables. After clustering patients in different groups based on baseline Hb levels, we analyzed the slope of the linear regression curve and the intercept. The lowest Hb level category has a 55% chance to achieve a CR after CT-RT. Interestingly, this likelihood increases by 5.6% for every single-unit (g/dl) increase in Hb level. In our series response to treatment was found to be an independent predictor of PFS and OS, while baseline Hb level was found to independently predict OS but not PFS. Nevertheless, clinical response and baseline Hb seem to have a synergistic effect in determining survival, with higher pre-treatment Hb increasing the chance to achieve a CR and thus potentially affecting survival. This is in line with data coming from the ACT-I randomized phase III trial, which compared exclusive radiation to 5-FU/MMC-based concurrent CT-RT. In the analysis of prognostic factors performed within the study, baseline Hb level was shown as an independent prognostic factor for anal cancer-related death [
11]. After adjusting for sex and lymphnode status, Glynne-Jones et al. demonstrated that, on average, a single-unit (g/dl) increase in Hb was associated to a 19% reduction in the risk of anal cancer death [
11].