Differential responses of cellular immunity in patients undergoing neoadjuvant therapy followed by surgery for carcinoma of the oesophagus

Major surgery is known to be associated with immune suppression. Several studies, investigating alterations in immune defence after oesophageal resection, have shown that oesophagectomy depresses host immune response [41, 42]. However, little is known about the immunological effects of preoperative neoadjuvant treatment in these patients [40].

The present study describes a broad spectrum of both numerical and functional immunological parameters in a series of surgical patients, some of whom received preoperative treatment, while others were directly operated on. The study population was uniform in that all patients had potentially curable disease based on preoperative investigations, and clinical parameters were comparable in both groups.

In brief, patients who received neoadjuvant thermochemoradiation therapy demonstrated a significant decrease in the number of granulocytes and lymphocyte subsets. T cell cytokine release was decreased after 2 weeks of neoadjuvant therapy, while production per T helper cell remained the same or showed a modest increase. CD8+ (cytotoxic) T cells but not CD4+ (helper) T cells recovered in the period between neoadjuvant therapy and operation. CD4+ T (helper) cell cytokine production levels were almost recovered, preoperatively.

All lymphocyte subsets that had not recovered after neoadjuvant therapy (i.e., CD4+ T cells, NK and B cells but not CD8+ T cells) had persistent lower counts as compared to control patients (operated without neoadjuvant therapy) at 1 week postoperatively.

Hyperthermia causes a transient stimulation of the cell-mediated immune response [38]. Recent research has shown that, when cancer cells are heated to approximately 41.6°C, they form highly specific protein structures on their surface known as heat-shock proteins [27]. These proteins activate the patient’s own NK cells to attack the cancer cells and stimulate the immune system [27]. Studies, evaluating the immune response after chemotherapy and/or radiation therapy, showed profound negative effects on the immune system [18, 22, 29, 36, 43]. In the present study, the neo patients underwent a combination of chemotherapy, radiotherapy and hyperthermia. The cumulative effects of this combination therapy led to a reduction of the white blood cell count which resulted in an overall reduced capacity to produce cytokines after 2 weeks of therapy. Apparently, the immunosuppressive impact of chemoradiation surpasses the immunostimulatory effect of hyperthermia.

In patients with sepsis, the loss of lymphocytes, contributing to immunosuppression by decreasing the number of available immune cells, is a well-known phenomenon. However, relatively recent work implicates that, not only a decrease in the number of effector cells, but also lymphocyte apoptosis plays a potential factor in the immunosuppression in sepsis [16]. The apoptotic cells appear to have important adverse effects on immune function by actively suppressing the inflammatory response [15]. It is unknown at present whether lymphocyte apoptosis plays a role in thermochemoradiation-induced immunosuppression, and may be an important subject for further study.

The differential recovery of CD8+ cells in contrast to CD4+ cells leading to a prolonged T cell imbalance after thermochemoradiation therapy has been described by others after intensive chemotherapy and after peripheral blood stem cell transplantation [19, 31]. As most peripheral CD4+ en CD8+ T cells share a common primary developmental pathway until relatively late in their development, this significant difference in the rate of recovery is surprising. In short, T cell precursors move from the bone marrow to the thymus, and develop to T cells. In the thymus various T cell precursors can be distinguished by the expression of specific cell surface markers: first TCR (T cell receptor), second CD3 (which serves as the signal transduction component of TCR), and finally CD4/CD8. It could be hypothesised that the regeneration of both CD4+ and CD8+ T cells occurs exclusively via thymic dependent pathways [19]. However, there is no existing evidence to suggest that a suboptimal thymic regenerative capacity would preferentially affect CD4+ populations, which could explain the differential recovery rate [3, 32, 37]. Alternatively, extrathymic generation of CD8+ cells from haematopoietic precursors could be involved and/or peripheral expansion of mature CD8+ T cells, which remain in the secondary lymphoid tissues after completion of chemotherapy. Mackall et al. [19] suggested that extrathymic lymphopoiesis is involved, as in their study the predominance of CD8+ cells lacked CD28 expression. This lack of CD28 expression has been associated with impaired thymic function in HIV infection [4], post-bone marrow transplantation [2, 24], and aged hosts [34]. Expansions of CD8+CD57+ cells have also been described in combination with restricted T cell repertoire diversity [11]. Thus, the relatively rapid regeneration of CD8+ cells compared to the slow CD4+ regeneration might be due to thymic independent pathways that efficiently and selectively induce regeneration of distinct CD8+ subsets.

Furthermore, differences in sensitivity of the factors that regulate homoeostatic proliferation of memory CD8+ T cells as compared to memory CD4+ T cells may explain the slower recovery of the CD4+ T cell-population from thermochemoradiation. Homoeostatic proliferation of CD8+ T cells seems to depend on IL-15 and IL-7, whereas these cytokines are probably not required for homoeostatic proliferation of CD4+ T cells [39].

Further work is necessary to be able to specify the origin of regenerated CD8+ cells and either reject or sustain the explanation of biological distinctions between CD8+ and CD4+ T cell regenerative pathways. It may be of clinical importance to determine if a predominance of CD8+ cells exists of CD8+CD28^- and CD8+CD57+ subsets, since a limited effectiveness of these populations has been described in vivo [30]. A significant depression in cell-mediated immunity in conjunction with possible alterations of CD8+ T cell subsets in neo patients as compared to control patients may have implications for the host defence against foreign pathogens as well as in the immunological control of disseminated tumour cells.

Although the role of the immune response in controlling tumour growth and cancer recurrence is controversial, there is evidence in oesophageal cancer suggesting that T cell infiltrates have a beneficial prognostic impact [14]. It was shown that CD8+ T cell infiltrations within the tumour specimen have a favourable outcome [35]. And a recent research in patients with colorectal cancers indicated that type, density and location of immune cells within tumour tissues were a better predictor of patient survival than the current methods to stage colorectal cancers [10, 28]. Interestingly, colorectal tumours from patients without recurrence had higher immune cell densities of CD3+ cells within the tumour and at the infiltrating tumour margin. Among CD4+ T lymphocytes, Th1 and Th2 phenotypes can be distinguished [25]. Activation of these two functionally distinct subsets of mature T helper cells is of great importance for an effective immune response against infection. Th1 cells are mainly characterised by secretion of IL-2 and IFN-γ and induce cell-mediated immune responses. Th2 cells preferentially secrete the cytokines IL-4 and IL-13 which favour humoural immune responses [7, 33]. Previously published results from our group showed that oesophagectomy severely depressed host immune responses. In the present study, neoadjuvant therapy caused both reduced T cell counts and decreased absolute levels of both Th1 and Th2 derived cytokines. This is in line with previous investigations, studying alterations in immune defence after neoadjuvant treatment [12]. However, in the present study it was shown that after neoadjuvant therapy production of the Th1 and Th2 cytokines per T helper cell was not decreased and for IL-13 even moderately increased, indicating that reduced cell counts rather than functional deficits contributed to the post-therapy reduction of T cell cytokine production.

At present, novel therapeutic strategies in oesophageal cancer mainly focus on applying new chemotherapeutic drugs, varying drug dosages, optimising the dose of radiotherapy or on adding hyperthermia to optimise long-term survival. However, the role of the host immunocompetence in the inhibition of disease progression and the way it is altered by the various new therapeutic strategies have only been partially investigated. The present study shows significant disturbances of host cellular immunity induced by preoperative thermochemoradiation compared to surgery alone in oesophageal cancer patients. This neoadjuvant therapy caused reduced T, NK and B cell counts, reduced overall Th1 and Th2 cytokine production, and differential recovery of T cytotoxic and T helper cells, leading to prolonged T cell imbalance that extends beyond the time of surgery. However, the study group was to small to conclude on the basis of the immune response whether to stop or continue neoadjuvant therapy, although it indicates that for individual patients it is worthwhile monitoring their immune status, having a vigilant attitude toward emerging infections. The oncological consequences of these immunological changes need further investigation, as recovery of T helper cytokine production towards surgery was less impaired than T helper cell counts.