Neoadjuvant radiotherapy (RT) with or without chemotherapy (chemoradiotherapy [CRT]) has been the gold standard treatment for locally advanced rectal adenocarcinoma for approximately 30 years. Ever since the publication of the results of the Swedish Rectal Cancer trial [1], short-course RT or long-course CRT has been utilized in the neoadjuvant setting with the goal of enhancing patients' prognosis and facilitating optimal surgical procedures. During the past 5 years, total neoadjuvant therapy—which incorporates a full preoperative chemotherapy course that is followed or preceded by RT or CRT—has emerged as the new standard treatment approach [2]. More recently, anti-PD1 immunotherapy is considered a first-line therapy for the treatment of mismatch-repair deficient locally advanced rectal adenocarcinoma [3], suggesting an important role of immunity in the control of this malignancy. Nevertheless, even in mismatch-repair proficient tumors preliminary results from clinical studies provide encouraging enhancement of pathological complete responses after combination of RT with immunotherapy [4].

Since a robust immune response is crucial for the outcome of CRT or immuno-CRT, immunocompromisation of any reason could potentially counteract treatment efficacy [5]. Strong experimental and clinical data support the concept of radio-vaccination, thus, the stimulation of the immune system through several pathways, including the type-I interferon response and HLA-class-I molecules up-regulation [6-8]. However, numerous studies reveal an additional ominous effect of RT, as irradiated patients experience profound lymphopenia depending upon the extent of field, fractionation, and dose levels [9-11]. Several clinical studies have documented the prognostic relevance of pre-treatment and treatment-related lymphopenia in solid tumors [12]. Regarding rectal cancer, Liu et al. reported that lower lymphocyte counts post-CRT define poor disease-free survival (DFS) [13]. Although a study by Lutsyk et al. [14] found no effect of lymphopenia on DFS, a significant association with poorer response to therapy was noted.

This retrospective study aimed to assess the effect of preoperative CRT on the peripheral blood lymphocytes of rectal adenocarcinoma patients and evaluate the impact of lymphopenia on treatment efficacy and patient prognosis.

Thirty-three rectal adenocarcinoma patients who were treated with preoperative CRT between 2014 and 2022 at the Radiation Oncology Unit of the Aretaieion University Hospital were included in this retrospective study. Table 1 summarizes patient demographics and imaging and histopathological disease characteristics. Patients who did not complete the treatment schedule or interrupted therapy for any reason for more than 1 week were excluded from the analysis.

Preoperative CRT is the standard treatment approach for locoregionally advanced rectal adenocarcinoma, while immunotherapy with the anti-PD1 monoclonal antibody dostarlimab has recently emerged as a potential active agent to further enhance prognosis for a subgroup of patients with mismatch repair deficiency [21]. RT has also been shown to activate in situ immune responses that may stimulate abscopal and in-field anti-tumor immunity which can eventually contribute to tumor eradication [22]. We recently summarized the effects of CRT on immune-checkpoint molecules up- or down-regulation on the cancer cell surface of rectal carcinomas, as well as its impact on the quantity and quality of tumor-infiltrating lymphocytes [23]. However, it is essential to underline that an intact systemic immune response is required in order to facilitate any kind of intratumoral immune response [24].

In this context, RT or CRT-related lymphopenia rises as a significant barrier to a potentially effective immune-mediated local or even distant—through the abscopal effects—disease eradication [25]. Multiple studies have reported on the severe lymphopenia documented post-treatment with RT or CRT for various tumor sites [26,27]. In fact, this adverse event becomes more prominent shortly after treatment completion [27], with a gradual restoration of LC being noted in the following months. Grossman et al. [26] further highlighted that the median LC count of patients 1 year after CRT was below 1,000/μL. As far as rectal adenocarcinoma is concerned, Campian et al. [28] demonstrated that severe lymphopenia (grade 3 or 4) was evident in 35% of 57 rectal adenocarcinoma patients 2 months after treatment with RT of 45 Gy and concurrent capecitabine. A more recent study by Lutsyk et al. [14] also confirmed significant CRT-induced lymphopenia in 198 patients with locally advanced rectal adenocarcinoma. In addition, a profound decrease in LC has been displayed in patients with rectal adenocarcinoma post-adjuvant CRT [29].

In the current study, we observed a statistically significant drop in the LC of 33 rectal adenocarcinoma patients who received 50.4 Gy of IMRT in combination with capecitabine. The median LC before CRT and at treatment completion were 2,184/μL and 517/μL, respectively, suggesting an incidence of grade 3 lymphopenia in 38% of patients, similar to the study by Campian et al. [28]. In the study by Li et al. [29], grade 3 lymphopenia was noted in 58% of patients, while Lutsyk et al. [14] reported lymphopenia—defined as LC less than 1,000/μL—in 75% of patients. The high intrinsic radiosensitivity of lymphocytic populations, the large radiation portals applied to cover pelvic lymph nodes, and the relatively high dose delivered to the bone marrow of the pelvic bones are the main contributing factors to RT-induced lymphopenia [25]. Fractionation of RT also appears to impact the severity of lymphopenia. Hypofractionated RT has been associated with less significant lymphopenia in two pancreatic and lung cancer studies, potentially due to the lower normalized total dose delivered to the lymphocytes and the shorter overall treatment time [30,31]. To our knowledge, there are no relevant data in rectal cancer patients comparing the long courses of CRT with short-course hypofractionated regimens. In the era of immune-RT, the potential incorporation of monoclonal antibodies in the preoperative setting brings forward the eventual value of reduced-length RT-fields to treat involved-only lymph nodes, while hypofractionation would further contribute to the protection of LCs and enhancement of immunotherapy’s efficacy [32].

In the next step, we assessed whether the graveness of treatment-related lymphopenia could affect response to CRT. Several studies have showcased a significant correlation between high LC after preoperative CRT for rectal adenocarcinoma patients and achieving a pathological CR [13,14,33,34]. Kitayama et al. [33] examined the lymphocyte ratio in the total white cell counts and found that this was increased in complete responders. In addition, Heo et al. [34] reported that patients who sustained LC above 35% of the pre-RT values experienced higher pathological CR rates, while Liu et al. [13] observed better tumor responses in patients with higher LC during CRT. On the other hand, Wu et al. [35] suggested that lymphopenia after neoadjuvant treatment could be linked with tumor regression, citing deprivation of regulatory T-cells as a potential explanation.

Although our study found no association between post-CRT LC and ypT stage or ypN status, a significant correlation was revealed between higher blood LC and better TRG. A larger cohort of patients could potentially allow the extraction of safer conclusions regarding the effect of CRT-related lymphopenia on treatment response. Moreover, the complexity of T-downstaging further hinders drawing out clear correlations; for example, downstaging of a cT3 stage to ypT2 and ypT1 stages are two different scenarios, possibly concealing a different effect of patients’ systemic immunity status and microenvironmental conditions that define the access of tumor-infiltrating lymphocytes. Hypoxia and low pH may suppress cytotoxic T-cell survival and anti-tumor activity in the tumor stroma, and this can counteract even strong systemic anti-tumor responses [36,37]. Poor lymphocytic presence within rectal carcinomas strongly affects the response to CRT and the outcome of therapy [23].

Complete response to CRT is associated with better prognosis for patients with rectal adenocarcinoma, as suggested by the majority of published data. While older data of a randomized phase III trial comparing preoperative CRT with adjuvant CRT for rectal adenocarcinoma support no correlation between response and survival [38], more recent retrospective studies and a meta-analysis suggest improved OS and DFS of patients with complete tumor regression [39-41]. Specifically, Ulusoy et al. reported that patients with better TRG(0,1) had significantly better OS [40]. In addition, a National Cancer Database analysis of 56,812 rectal adenocarcinoma patients who received neoadjuvant CRT demonstrated that patients with pathologic complete responses survived longer when compared to non-complete responders [42].

In our study, a higher ypT stage was significantly linked with shorter DMFS. This was also the case for patients with TRG2,3, although the statistical association was of marginal significance. VI and ypN+ status were also shown to define poor DMFS. Due to the relatively low number of patients and short-follow up in the current study, no significant correlations were found between some prognostic factors such as ypT stage, ypN status, VI, and TRG and survival outcomes including LPFS/OS. These findings are in accordance with previously published studies. Tang et al. [43] recently reported a significant association of ypN stage with distant metastasis in a series of 146 patients with rectal cancer. Similarly, VI has been suggested to adversely affect progression-free and overall survival [44,45].

Aside from tumor regression-related features, multiple studies have showcased the negative effect of RT- or CRT-induced lymphopenia on the prognosis of patients with carcinomas of various origins [11,46-48]. In a report by Liu et al. [13], it was demonstrated that lower LC post-CRT were associated with worse OS and DFS of rectal adenocarcinoma patients. A more recent study, however, found no effect of lymphopenia on DFS [14]. In the current retrospective analysis, a statistically significant impact of LC on DMFS was evident, since patients with more pronounced lymphopenia (LC ≤518/μL) developed distant metastases more frequently (44.5% vs. 0% of patients with LC >518/μL). Of note, Liu et al. [13] used a cutoff point of 440/μL for LC, as defined by ROC curve analysis, similar to the one proposed in our study. In contrast, a cutoff point of 1,000/μL was applied in the analysis by Lutsyk et al. [14].

Furthermore, Sung et al. [49] showed a significant association between an elevated NLR after preoperative CRT and worse prognosis of patients with locally advanced rectal adenocarcinoma. Ιn our study, this was not confirmed. It should be taken into account that the NLR incorporates neutrophil counts that are primarily affected by chemotherapy. Moreover, many neutropenic patients during CRT also receive granulocyte-colony stimulating factors; thus, the full blood count at treatment completion does not necessarily represent the actual effect of CRT. Thus, post-treatment NLR is suggested to harbor substantial bias related to chemotherapy.

Certain limitations of the study have already been addressed. The retrospective nature of our investigation, combined with the limited number of cases, does not allow the extraction of safe conclusions. Longer follow-up could also reveal associations of the examined parameters with OS. In addition, a comparative analysis of the current data with patients treated with hypofractionated short-course RT could further shed light on the impact of different fractionation on lymphopenia. However, we believe that our patient group represents a unique set with detailed pre- and post-CRT data. Moreover, central review of histopathology significantly contributes to the quality of the study.

Overall, lymphopenia has emerged as a significant adverse event that rectal adenocarcinoma patients face during treatment with neoadjuvant CRT, with subsequent implications on tumor response and prognosis. As suggested in the current study, LC reduction to levels in the range of profound grade 2 and grade 3 lymphopenia defines poorer pathological tumor responses and higher incidence of distant metastases. Further investigation on the effects of different RT schedules on lymphopenia (normal fractionation vs. hypofractionation or even ultra-hypofractionation), as well as the reduction of the RT fields that would spare functional lymph nodes in combination with targeted immunotherapy is required to understand better and overcome this prognosis-affecting toxicity.