Anal squamous cell carcinoma (ASCC) arises from the anal canal and is a rather rare malignancy with an incidence of two to three in 100,000 per year in the general population (1). While organ-preserving definitive chemoradiotherapy (CRT) remains standard of care, locoregional persistence or relapse still occurs in up to 30% of all patients (2, 3). Large randomized trials have already established T-stage, N-stage, and gender as prognostic factors (4, 5). Nevertheless, due to the heterogeneity in treatment outcomes, there is still a need for reliable biomarkers to further stratify patients to treatment escalation or de-escalation.

The development of ASCC is associated with infection with high-risk strains of human papilloma virus (HPV) in 70–90% of cases (6, 7). HPV positivity, as measured by immunohistochemistry or polymerase chain reaction, is associated with better treatment outcome after (chemo)radiotherapy (7, 8). Furthermore, there seems to be a link between HPV and immune contexture, as HPV positivity is associated with a more favorable tumor microenvironment (TME) (9).

The role of the immune TME in mediating cancer development and progression gained attraction in recent years. The positive prognostic impact of tumor-infiltrating lymphocytes (TILs) has been reported in several malignancies (10), including ASCC (9, 11–13). CD8+ T-cells have cytotoxic capabilities due to granules secreting perforin and granzyme B, which are released upon detection of antigens via the major histocompatibility complex I and T-cell receptor (TCR) (14). In contrast, myeloid cells like neutrophils remain unexplored in ASCC. Neutrophils aid tumor progression and resistance to conventional anticancer treatments by mediating angiogenesis and suppression of CD8+ TIL-mediated antitumor immunity via secretion of inhibitory factors, e.g., inducible nitric oxide synthase (iNOS), arginase 1 (ARG1), and transforming-growth-factor beta (TGFβ) (15–17).

The negative impact of elevated pretreatment white blood cell (WBC) count in blood samples has already been reported in cervical and colorectal cancer (18–20). In ASCC, the same effect was described in recent retrospective studies (21–23). Leukocytosis was also correlated with adverse cancer-specific survival and overall survival (OS) in the ACT-I clinical trial (24). Leukocytosis in solid tumors can be caused directly by tumor cells via secretion of stimulatory factors such as granulocyte-colony-stimulating factor (G-CSF), granulocyte-macrophage-colony-stimulating factor, and upregulation of C-X-C chemokine receptor 2 (CXCR2) ligands (17). Nevertheless, no explanation for this observation has been provided so far.

Platelets play an important role in cancer progression. Clinical and preclinical evidence suggests that platelets can help tumor cells resist apoptosis, induce angiogenesis, facilitate chemoresistance, and help immune evasion during generation of metastasis (25, 26). Elevated pretreatment platelets are associated with worse outcome in colorectal cancer patients (27) and a higher propensity for metastasis (28).

In this study, we provide insight on the worse clinical outcome in patients with leukocytosis in correlation CD8+ TILs and tumor-associated neutrophils (TAN) in ASCC.

In the present study, we aimed to investigate the clinical impact of peripheral blood leukocytosis in the context of intratumoral immune profile. Patients with ASCC that have baseline leukocytosis presented a worse outcome after CRT in line to previous observation in ASCC (21, 23) and cervical cancer (18, 20). Our data confirm these results using the established cutoff point of 10/nl, but we found worse DFS and DMFS also in patients with moderately elevated WBC, based on median cutoff value.

In our work, patients with leukocytosis had a significantly lower intratumoral CD8+ TIL expression and vice versa. Leukocytes are important in acute inflammation to eliminate pathogens but, in a chronically activated state, they can promote tumor progression by secretion of TGFβ, epidermal-growth-factor, and proteolytic enzymes (33). Importantly, leukocytosis as part of chronic inflammation can lead to inhibition of CD8+ T-cells via upregulation of programmed death 1 on T-cells and myeloid cells (34) that is in line to our findings. To the best of our knowledge, this phenomenon has not been reported yet and it proposes an additional explanation for the adverse role of leukocytosis in malignancies.

The role of TAN in ASCC has not been previously investigated. Notably, we found an inverse correlation between peritumoral TAN and CD8+ TIL densities. Several mechanisms have been implicated in the immunosuppressive effect of TAN. First, secretion of ARG1 lowers the expression of the ζCD3 chain of the TCR leading to reduced activity (35, 36). Second, nitration of CC-chemokine ligand 2 by neutrophil-derived reactive nitrogen species reduces T-cell infiltration (37). Third, iNOS released by neutrophils inhibits proliferation of CD8 lymphocytes (16). Fourth, TGFβ1 signaling downregulates interferon gamma (IFNγ) in CD8+ T-cells (38). The positive prognostic impact of high CD8+ TIL density has already been demonstrated in ASCC (9, 11–13).

Previous studies have reported strong association of leukocytosis with peripheral blood neutrophilia in ASCC (23) and cervical cancer (18, 20, 39). The impact of leukocytosis on DFS and OS in our study was mainly due to distant metastases, and experimental models showed that neutrophils play a major role in early metastatic spread (40). Tumors promote neutrophil release via G-CSF and CXCR2 ligands (41, 42) that can lead to chemo- and radioresistance (39, 43). Of note, we failed to identify a significant correlation between total peripheral WBC and intratumoral neutrophils, which could be attributed to the fact that WBC count incorporates various peripheral blood cell populations.

The negative impact of elevated platelets on DMFS is in line with preclinical evidence that suggests an important role for platelets in metastasis (26). Similar data have been reported before in other tumor types, such as colorectal cancer (27, 28). Also, prophylactic aspirin that inhibits platelet activation has been shown to decrease metastatic incidence and cancer mortality (44).

We did not observe any significant prognostic influence of TAN on treatment outcome. Of note, a high variance has been reported regarding the prognostic value of neutrophils (45–50). One possible explanation for these discrepancies in literature could be the heterogeneous treatments and cohorts, and the use of different markers to evaluate TAN (CD11b, CD15, MPO). Another explanation could be different compositions of the TME between malignancies. In cervical cancer, a negative prognostic impact of TAN is reported (51), whereas in squamous cell carcinoma of the head and neck, studies have reported positive and negative prognostic value (52, 53). Several approaches for targeting TAN are under clinical evaluation, for example, by blocking CXCR2 using the inhibitor reparixin (54). Interestingly, preclinical evidence has also suggested a synergistic effect of combining CXCR2 blockade with immune checkpoint inhibition (55).

Our study has several limitations. First, differential baseline blood count values, including neutrophils and lymphocytes, were unavailable in a large proportion of patients. Second, the retrospective design could have resulted in selection bias. Third, although our median follow-up of 26 months was relatively long, randomized trials have been reported with longer follow-up time. Fourth, immunohistochemical staining scoring was performed using a non-automated system due to the lack of international consensus and pathology standardization that constitutes another limitation of our work. These results clearly need to be validated in other cohorts, preferably in a prospective manner. In that context, we and others have previously investigated additional potential biomarkers relevant to the immune microenvironment, such as EGFR, surviving, Plk3, FOXP3, and PD-1/PD-L1 (9, 56–60). These could be potentially exploited in the future to generate robust prognostic models with several biomarkers to guide treatment stratification for future trials.

In conclusion, we here demonstrate that peripheral leukocytosis was associated with adverse clinical outcome in patients with ASCC treated with definitive CRT. This is the first study to show an inverse correlation between peripheral blood leukocytosis and intratumoral CD8+ TIL as well as between peritumoral MPO+ TAN and stromal CD8+ TIL that provide important mechanistic insight on the adverse impact of peripheral leukocytosis and the interplay between different immune cell populations in ASCC. These findings add to the previous literature on the role of leukocytosis and immune contexture in mediating response to treatment in ASCC and should lead to further investigations into the immune microenvironment of this disease.

This study was carried out in accordance with the recommendations of the ethics committee of the University Hospital Frankfurt am Main and approval from the institutional review board. All patients gave written informed consent in accordance with the Declaration of Helsinki.

All authors designed and drafted the project. Clinical data collection and scoring of histochemical staining was done by DM, FR, PB, CR, and EF; RW performed pathological evaluation. All authors contributed to statistical analyses and DM, PB, and EF drafted the final manuscript with support from all co-authors. All authors performed final evaluation and agreed to be accountable for the content of the study.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.