Impairment of lymphocyte function following yttrium-90 DOTATOC therapy

Abstract

The radiolabeled somatostatin analogue, yttrium-90 DOTA-d-Phe(1)-Tyr(3)-octreotide (DOTATOC), is currently applied to treat advanced somatostatin receptor-positive tumors, e.g., neuroendocrine tumors of the pancreas, lung or gut. However, effects of this treatment on antimicrobial immune responses are not yet defined. In 20 patients treated with DOTATOC, cellular in vitro immune function was determined. Their antimicrobial lymphocyte responses were assessed by lymphocyte transformation test and enzyme-linked immunospot—measuring lymphocyte proliferation and on a single cell level production of pro- and anti-inflammatory cytokines (interferon-γ and interleukin-10)—prior to therapy, at day 1, day 7 and day 90 post-therapy. Proliferative lymphocyte responses and interferon-γ production after in vitro stimulation with microbial antigens were non-significantly suppressed at day 1 and significantly (p < 0.05) at day 7 versus pre-therapy. In vitro immune responses did not fully recover until day 90. In contrast, at day 1 interleukin-10 production was significantly (p < 0.05) increased. Taken together, we observed a decrease in pro-inflammatory immune responses after DOTATOC therapy. Patients with versus without bone metastases displayed significantly (p < 0.05) lower cellular immune responses toward several microbial antigens. Progressive disease and higher tumor burden could also be defined as factors associated with impaired immune function. Spearman correlation analysis indicated that cellular in vitro immunity was positively correlated with kidney function; better kidney function led to stronger immune responses. In conclusion, DOTATOC therapy caused a decrease in in vitro immune responses against microorganisms. The clinical impact needs to be evaluated in further studies.

Introduction

The number of indications for the application of radionuclides in tumor therapy is constantly growing. In particular, peptide receptor radionuclide therapy is increasingly used as a treatment modality for certain neoplasias such as neuroendocrine tumors. These tumors often exhibit an overexpression of somatostatin receptors compared to normal tissues. Those receptors can be targeted with different synthetic somatostatin analogues. Many of these peptidic analogues can be conjugated with chelators and labeled with radiometals. The basis of this treatment is then the binding of the radiolabeled compound to the receptor, the subsequent internalization of the receptor–ligand complex and the intracellular emission of ionizing radiation.

A somatostatin analogue that is frequently used in peptide receptor radionuclide therapy of neuroendocrine tumors is yttrium-90 DOTA-d-Phe(1)-Tyr(3)-octreotide (DOTATOC). It contains the chelating agent 1,4,7,10-tetraazacyclododecane-N,N′, N″,N‴-tetraacetic acid (DOTA) to label Tyr(3)-octreotide (TOC) with yttrium-90 (⁹⁰Y) [1]. ⁹⁰Y is an energetic beta particle emitter that requires enhanced stability of the radionuclide complex to minimize in vivo release of high levels of free ⁹⁰Y and, in turn, bone marrow toxicity [2, 3]. Conjugates of ⁹⁰Y and octreotide can be stabilized, e.g., by DOTA [4, 5]. Octreotide is an octapeptide that mimics natural somatostatin pharmacologically, though it is a more potent inhibitor of growth hormone, glucagon and insulin than the natural hormone. ⁹⁰Y DOTATOC is considered a safe tool for receptor-mediated radiopeptide therapy of somatostatin receptor-overexpressing tumors [1]. In these tumors, intravenous administration of ⁹⁰Y DOTATOC is a highly targeted and effective therapy where minimal side effects are described [1, 6].

Recently, it could be demonstrated that radioiodine therapy in patients with thyroid carcinoma led to a short-term increase in pro-inflammatory immune responses [7]. However, radioiodine-induced changes were moderate and only observed at day 1 post-therapy. We now addressed the question whether treatment with DOTATOC alters antimicrobial immune responses in a similar way.

We are not aware of any study investigating the effect of DOTATOC on immune defense in a clinical setting. But it has been known for many years that the immune system is highly sensitive to gamma rays [8–12] which cause, e.g., chromosomal aberrations [10], reduced reagibility of lymphocytes [11] or reduced levels of all lymphocyte subsets, with B lymphocytes and naïve (unprimed) T lymphocytes being most sensitive [12]. To assess the effect of irradiation, the kind of radiation, its specific activity and the activity distribution have to be considered [7, 12].

In the present study, DOTATOC induces continuous but inhomogeneous irradiation that acts not only locally but to some extent also on the whole body. The target tissue expressing the somatostatin receptor is exposed to a high dose of DOTATOC, whereas the exposure of the remaining tissue is low. Particularly, patient-specific bone marrow dosimetry is challenging [13]. Current literature suggests that DOTATOC therapy as applied in our study (4.0–5.9 GBq) leads to anemia, leukopenia and thrombocytopenia in approximately 60, 30 and 30 % of patients, respectively [14]. The ⁹⁰Y dose considered the generally accepted maximum bone marrow dose of 2 Gy [15]. With a dose of 2 Gy, the probability for developing leukemia is approximately 2 % [16].

To measure immunoreactivity after intense gamma irradiation, the highly sensitive lymphocyte transformation test (LTT) has been used as a gold standard [7, 10, 11]. This test measures cell proliferation and determines the uptake of ³H thymidine by peripheral blood mononuclear cells (PBMCs) after in vitro stimulation. Various groups of stimuli can be used, e.g., mitogens which can stimulate naïve as well as memory T lymphocytes or microbial antigens (recall antigens) which can only stimulate memory T lymphocytes. Response to the LTT mitogen can determine the overall reactivity of lymphocytes (almost all T/B lymphocytes should proliferate if stimulated by mitogens), and the LTT antigen can determine whether microorganisms are recognized adequately by T lymphocytes. A proper in vitro reaction toward antigens indicates that in vivo infections should also be controlled effectively by proliferating T lymphocytes.

Recently, it was shown that the enzyme-linked immunospot (ELISpot), a sensitive method which determines cytokine production on a single cell level, is an excellent tool to quantify effects of irradiation on the immune system [7]. The pattern of cytokines produced allows suggesting whether the immune system is more in an inflammatory or an immunosuppressed condition. An inflammatory state is characterized by the production of, e.g., interferon (IFN)-γ, an immunosuppressed by the production of, e.g., interleukin (IL)-10. IFN-γ production is a prerequisite for the control of microorganisms, whereas IL-10 production facilitates the persistence of infections.

It was the aim of the present study to define for the first time how DOTATOC therapy affects lymphocyte function in patients with advanced somatostatin receptor-positive tumors in the short- and long-term (day 1–7 and day 90 post-treatment, respectively). Further, we considered potential influencing factors of cellular in vitro immunity.

Methods and materials

Patients

Twenty patients receiving DOTATOC therapy because of advanced somatostatin receptor-positive malignant tumors were consecutively included into this study from January to August 2010. Patient characteristics are detailed in Table 1. For treatment planning with ⁹⁰Y DOTATOC, hybrid imaging of positron emission tomography together with computed tomography (PET/CT) was performed prior to therapy with gallium-68 (⁶⁸Ga) DOTATOC (Fig. 1). Because of the disseminated nature of the disease, the tumor burden was estimated visually on an ordinal scale. The glomerular filtration rate was determined with technetium-99m diethylene-triamine-pentaacetate (DTPA) using the two-sample, two-compartment technique described by Russel [17]. The applied activity of ⁹⁰Y DOTATOC was calculated according to the national guidelines (in press) and was influenced by neither tumor burden nor kidney function. Assays to detect cellular immunity were performed immediately prior to therapy (day 0) and at day 1, 7 and 90 post-therapy.

Table 1.

Characteristics of 20 patients with advanced somatostatin receptor-positive tumors receiving DOTATOC therapy

Female/male	8/12
Median age (range), years	66 (23–74)
Median activity of current therapy cycle (range), GBq	4.8 (4.0–5.9)
Therapy cycle, no.
1st	13
2nd	2
3rd	5
Activity of all therapy cycles, no.
>4 and ≤5 GBq	11
>5 and ≤10 GBq	3
>10 and ≤16 GBq	6
Primary tumor
Pancreas	5
Lung	4
Gut	5
Paraganglioma	2
Thyroid	2
Other	2
With/without bone metastases	13/7
Glomerular filtration rate prior to therapya
Within/below age-dependent norm	17/3
Median (range), (ml/min/1.73 m2)	95 (55–112)
Response to therapy
Stable disease	10
Progression	10

^aThe glomerular filtration rate was determined by radioisotope nephrography

Fig. 1.

⁶⁸Ga DOTATOC positron emission tomography (PET) in 20 patients prior to ⁹⁰Y DOTATOC therapy. The images were arranged according to the tumor burden

Lymphocyte transformation test

Cellular in vitro responses toward four mitogens [phytohemagglutinin, concanavalin A, pokeweed mitogen and anti-CD3 monoclonal antibody (OKT3)] and twelve microbial antigens (tuberculin, tetanus toxoid, diphtheria toxoid, Candida albicans, cytomegalovirus, Herpes simplex virus type 1, Varicella zoster virus, rubella virus, mumps virus, measles virus, influenza A and B virus) were measured using standardized assay formats as described previously in greater detail [7, 18]. In brief, PBMCs were collected after density gradient centrifugation, and 50,000 cells per 200 μL were incubated with mitogens for 3 days and antigens for 5 days, respectively. For the last 16 h, the cultures were labeled with 37 kBq ³H thymidine per culture. Cells were then harvested onto filter pads, and the incorporated radioactivity was quantified by liquid scintillation counting.

ELISpot assay

The secretion of IFN-γ and IL-10 was detected by ELISpot as also detailed previously [7]; 200,000 PBMCs per 200 μL culture were stimulated by four mitogens (same as for LTT) and 400,000 PBMCs per 200 μL culture by three microbial antigens [tuberculin, tetanus toxoid and Candida albicans]. After 2 days of cell culture, spot formation was detected by adding avidin–biotin peroxidase complex and the substrate 3-amino-9-ethyl-carbazole. Numbers of spots were analyzed by an ELISpot plate reader.

Statistical analysis

LTT data were presented as counts per minute (cpm), representing the uptake of ³H thymidine, and ELISpot data as spot numbers per cell culture. Cpm increment or spots increment was generated as mitogen stimulated or antigen stimulated minus autologous (unstimulated) reaction. In addition, the cumulative antigen score was generated by an algorithm described previously [18]; it sums up reactions toward twelve recall antigens. Reactions pre- and post-DOTATOC therapy were compared by paired t test or Wilcoxon matched pairs test as appropriate. Correlation of numeric clinical parameters and immune function was analyzed using Spearman correlation analysis. The influence of categorical parameters was tested by Mann–Whitney U test. All these data were analyzed by GraphPad Prism software (version 3.02).

Results

Immune response after DOTATOC therapy

In 20 patients receiving DOTATOC therapy, inflammatory T lymphocyte responses toward mitogens and microbial antigens continuously decreased until day 7 post-therapy (Fig. 2a, b). The data indicate a DOTATOC-induced impairment of antimicrobial immune responses. In detail, at day 1 after therapy lymphocyte responses were moderately suppressed. At day 7, lymphocyte proliferation (LTT) after stimulation with all four mitogens tested and with the microbial antigens Herpes simplex virus type 1, influenza A and influenza B virus was significantly lower than at day 0 (p < 0.05 each). Thereafter, at day 90, responses still tended to be lower than pre-therapy. This means that therapy induced a short-term decrease in specific lymphocyte proliferation.

Fig. 2.

Cellular in vitro reaction toward mitogens and recall antigens in patients with somatostatin receptor-positive tumors receiving ⁹⁰Y DOTATOC therapy. Data represent mean and standard error of the mean (SEM), either pre-therapy (day 0, white bars), at day 1 (black bars), at day 7 (hatched bars) or at day 90 (striped bars) post-therapy. LTT mitogen responses (a, n = 18–20) and LTT antigen responses (b, n = 18–20) are given as counts per minute of ³H thymidine uptake (cpm), IFN-γ and IL-10 secretion to the ELISpot (c–f, n = 15–20) as spots per culture. The Wilcoxon matched pairs test was used for comparisons; the level of significance is indicated in comparison with day 0 (*p < 0.05, **p < 0.01). LTT lymphocyte transformation test, Aut. autologous, PHA phythohemagglutinin, ConA concanavalin A, PWM pokeweed mitogen, OKT3 anti-CD3 monoclonal antibody, PPD purified protein derivate (tuberculin), TET tetanus toxoid, CAN Candida albicans, HSV-1 Herpes simplex virus type 1, VZV Varicella zoster virus, INV-A influenza virus A, INV-B influenza virus B, IFN interferon, IL interleukin

IFN-γ secretion as determined by ELISpot was also significantly (p < 0.05 each) decreased at day 7 for the mitogens concanavalin A and pokeweed mitogen and the antigen Candida albicans (Fig. 2c, d). Furthermore, at day 1 IL-10 secretion to the ELISpot was increased (p < 0.05 each for phytohemagglutinin, pokeweed mitogen and tuberculin) (Fig. 2e, f).

Influence of DOTATOC therapy on white cell counts

DOTATOC therapy led to a significant (p < 0.05) decrease in PBMC numbers on day 1 and day 7 post-therapy [day 0: 9.7 ± 1.0, day 1: 8.2 ± 0.9, day 7: 7.1 ± 0.8 million PBMC per 10 mL peripheral blood, data represent the mean and standard error of the mean (SEM), n = 19–20]. On day 90, cell numbers were comparable to those on day 1 (8.2 ± 0.9 million PBMC per 10 mL blood, n = 18). White blood counts at day 7 versus day 0 indicated that lymphocytes were significantly reduced (0.7 ± 0.1 vs. 1.2 ± 0.2/nL, n = 15, p = 0.0002), whereas leukocyte and granulocyte numbers did not change significantly (5.8 ± 0.3 vs. 6.1 ± 0.3/nL and 4.3 ± 0.3 vs. 4.2 ± 0.3/nL). However, leukocyte numbers tended to decrease and the percentage of granulocytes tended to increase (74 vs. 69 %).

Correlation analysis of clinical parameters and cellular in vitro immune responses

To assess the dependency of immune function on clinical parameters, all patient characteristics as given in Table 1 were included into the analysis. Significant results are displayed in Fig. 3 and Table 2.

Fig. 3.

Immune responses toward microbial antigens are dependent on the presence of bone metastases in patients with ⁹⁰Y DOTATOC therapy. In vitro immune responses toward tetanus toxoid and Candida albicans were determined immediately prior to therapy (day 0), at day 1, day 7 and day 90 post-therapy. Patients with bone metastases (n = 13, black bars) displayed significantly lower immune responses as compared to patients without bone metastases (n = 7, white bars). Immunity was determined by lymphocyte transformation test as proliferation (LTT, a, b) and by ELISpot as interferon (IFN)-γ production per cell (c, d). The Mann–Whitney test was used for comparisons between patient groups (*p < 0.05, **p < 0.005). Not tested at day 7: n = 1 and at day 90: n = 2. Cpm: counts per minute; increment: The background reaction (autologous value) was subtracted from the specific reaction toward tetanus toxoid or Candida albicans

Table 2.

Spearman correlation analysis of kidney function^a and cellular in vitro immunity in 20 patients receiving DOTATOC therapy

Parameter of immune function	Time point (day)	r	p
ConA LTT	1	0.47	0.04
	90	0.59	0.009
PWM LTT	1	0.45	<0.05
	90	0.61	0.008
OKT3 LTT	90	0.70	0.001
PPD LTT	1	0.61	0.004
	7	0.69	0.001
	90	0.63	0.005
CAN LTT	1	0.49	0.03
	7	0.61	0.005
	90	0.48	<0.05
INV-A LTT	1	0.45	<0.05
	7	0.54	0.02
INV-B LTT	1	0.51	0.02
	7	0.55	0.02
	90	0.61	0.008
Cum. antigen score	1	0.50	0.03
	7	0.65	0.003
PWM IFN-γ ELISpot	90	0.60	0.009
PPD IFN-γ ELISpot	0	0.54	0.02
	1	0.77	<0.0001
	7	0.60	0.007
	90	0.61	0.007
CAN IFN-γ ELISpot	7	0.59	0.008
	90	0.51	0.03
ConA IL-10 ELISpot	0	0.50	0.02
	1	0.51	0.03

ConA concanavalin A, LTT lymphocyte transformation test, PWM pokeweed mitogen, OKT3 anti-CD3 monoclonal antibody, PPD purified protein derivate (tuberculin), CAN Candida albicans, INV-A influenza virus A, INV-B influenza virus B, Cum. antigen score cumulative antigen score (sum of reactions toward antigens) [18], IFN interferon, IL interleukin

^aKidney function was determined by radioisotope nephrography and was measured as glomerular filtration rate

Patients with bone metastases had significantly (p < 0.05) lower cellular immune reactivity toward several microbial antigens (tetanus toxoid, Candida albicans, Herpes simplex virus type 1, Varicella zoster virus and influenza A virus) and toward a set of twelve recall antigens (cumulative antigen score). Figure 3 depicts reactions toward two of these recall antigens, tetanus toxoid and Candida albicans. Differences were observed at all time points; i.e., patients with bone metastases had lower immune function pre- and post-therapy.

A similar result was observed for patients with progressive versus stable disease; reduced T cell responses were detectable in patients with progressive disease pre- and post-therapy. These patients had lower T lymphocyte proliferation after stimulation with microbial antigens. However, differences only reached statistical significance (p < 0.05) for Herpes simplex virus type 1.

Patients with higher versus lower tumor burden had impaired immune function. This was also observed pre- and post-therapy. Summing up reactions toward twelve recall antigens (cumulative antigen score), significant differences between patients with high and low tumor burden could be observed (p < 0.05). Differences for single antigens were all non-significant.

Interestingly, we also observed a significant correlation (p < 0.05) between activity of the current therapy cycle and immune function. T cell proliferation after stimulation with various antigens (tetanus toxoid, Candida albicans, Varicella zoster virus, influenza A and B virus), IFN-γ and IL-10 production were positively (r values between 0.45 and 0.66) correlated with the current activity (Fig. 4).

Fig. 4.

Immune responses toward microbial antigens are dependent on the activity of the current therapy cycle in patients with ⁹⁰Y DOTATOC therapy. T cell proliferation as determined by lymphocyte transformation test (LTT) at day 7 post-therapy was significantly correlated with the activity of the current therapy cycle. Spearman correlation analysis is shown for immune responses toward tetanus toxoid (a) and Candida albicans (b) (n = 19 each). Cpm: counts per minute; increment: The background reaction (autologous value) was subtracted from the specific reaction toward tetanus toxoid or Candida albicans

As expected, Spearman correlation analysis indicated that cellular in vitro immunity was positively correlated with kidney function; i.e., better kidney function led to better immune function (Table 2). Kidney function was determined by radioisotope nephrography and was measured as glomerular filtration rate.

Finally, gender, age, therapy cycle number, activity of all therapy cycles and primary tumor had no significant influence on immune function.

Summarizing, factors suppressing immune function comprised the presence of bone metastases, progressive disease, higher tumor burden and impaired kidney function.

Discussion

DOTATOC therapy led to a decrease in antimicrobial immune responses in vitro. We could observe a moderate immunosuppression at day 1 and a more pronounced suppression at day 7 after treatment. In contrast, a previous study on radioiodine therapy [7] showed that immune function was increased at day 1 after treatment and returned to baseline at day 7. Notably, we used exactly the same assay conditions for the radioiodine and DOTATOC study, and age and sex distribution were similar in both cohorts. Thus, the underlying disease and treatment appeared as the major differences. A direct comparison between the immunosuppressive effects of iodine-131 in thyroid cancer patients described in an earlier paper [7] and the current results for peptide receptor radionuclide therapy with ⁹⁰Y DOTATOC is problematic because the biodistribution and kinetics of these radiopharmaceuticals differ substantially. Although ⁹⁰Y DOTATOC has a substantially faster blood clearance than iodine-131 [19–21], only 60 % of the injected activity is excreted after 24 h and little more after 48 h [21], whereas the whole-body retention of iodine-131 is only 14 % after 48 h [22]. ⁹⁰Y DOTATOC is retained with half-life >2 days in well-perfused organs such as liver, spleen and kidneys and irradiates circulating cells [20, 21]. Furthermore, the contribution to the dose of circulating immune cells is related to fractional cardiac output, mean transit time and activity concentration, whereas the dose to sessile cells is dominated by the binding and kinetics in the host organ and the circulating blood. The kinetics of the exchange between the pools of sessile and circulating cells is unknown. The biological radiation effects may be further confounded by changes in the biological effectiveness due to the described time-varying dose rates from different sources [23]. The development and validation of a rigorous dosimetric model of these effects are beyond the scope of this study.

At day 90 after treatment, lymphocyte responses were still slightly lower than pre-therapy. This finding is line with the long life span of CD4- and CD8-positive T lymphocytes, i.e., helper T and cytotoxic T lymphocytes. Using mathematical modeling, the median half-life time for naïve CD4- and CD8-positive T lymphocytes (stimulable with mitogens) was estimated as 4.2 and 6.5 years, and the median half-life time for CD4- and CD8-positive memory T lymphocytes (stimulable with mitogens and recall antigens) as 155 and 244 days, respectively [24]. Moreover, following total body irradiation and hematopoietic stem cell transplantation, it takes approximately 2.5 years until T lymphocyte responses recover [25]. Thus, it appears as likely that lymphocytes have not been completely replaced at day 90 after DOTATOC therapy.

Currently, there are no data on the effect of ⁹⁰Y DOTATOC treatment on immune function. However, a previous study [26] analyzed the short- and long-term effects of another tumor therapy containing ⁹⁰Y. It was shown that intra-arterial application of ⁹⁰Y microspheres in patients with hepatocellular carcinoma was associated with profound and prolonged lymphopenia. Lymphocytes (and not granulocytes, platelets or NK cells) were sensitive to treatment with ⁹⁰Y microspheres. Using ⁹⁰Y microspheres, predominantly circulating cells are irradiated from ⁹⁰Y in the liver as opposed to ⁹⁰Y DOTATOC where the bone marrow is also exposed. Despite the different distribution of the radiopharmaceuticals, similar results on leukocyte subpopulations were observed after ⁹⁰Y microsphere and ⁹⁰Y DOTATOC therapy. Our study also showed that the percentage of lymphocytes was reduced after therapy, whereas the percentage of granulocytes tended to increase. Also in line with our data, no fungal or bacterial infections were noted after therapy with ⁹⁰Y microspheres. Unfortunately, the authors of the ⁹⁰Y microsphere study [26] did not present data on viral infections. In our study, an increase in viral infection/reactivation was not observed. Whereas fungal and bacterial infections are mainly controlled by granulocytes, viral infection/reactivation is especially controlled by lymphocytes. Presumably, the number of patients in our study was too small to elucidate whether viral control was impaired after ⁹⁰Y DOTATOC treatment.

In addition to this clinical study, early mouse experiments [27] indicated that intravenous treatment with a mixture of strontium-90 (⁹⁰Sr) and ⁹⁰Y [2.5 − 10 µCi (=92.5 − 370 kBq)/mouse] either depressed or increased the response of splenocytes to mitogens within the first week after irradiation. The long-term irradiation led to higher splenocyte reactivity. However, it is hard to compare this study with ours because of different biodistributions. Presumably, the effect in our patients was similar to that of the high-dose irradiations in mice; within the first week, we also observed a decrease in lymphocyte reactivity. But long-term effects post-treatment (32 days in mice and 90 days in man) differed. We could not observe enhanced lymphocyte reactivity. Further, a previous study on hemodialysis patients [28] indicated that extracorporeal irradiation of the blood using a portable ⁹⁰Sr–⁹⁰Y source (beta-ray emitter) resulted in selective lymphocytopenia, but not in a definite effect on lymphocyte response, as measured in vitro with lymphocyte transformation tests (n = 6). In this study, low transit doses (100–600 rad = 1–6 Gy) were applied over a long period. Data on lymphocyte function can hardly be compared with that previous study due to the field of irradiation (in vivo vs. extracorporal) and duration and dose of irradiation.

To answer the question whether there was impairment not only in lymphocyte numbers but also in function per cell, the percentage of reduction in number and in function at day 7 versus day 0 was compared. For example, considering lymphocyte proliferation after stimulation with twelve microbial antigens—which is most likely the best test to mimic in vivo immune responses—the reduction in numbers was 27 % and in function 46 % (mean value for all antigens). According to these data, number and function per cell were both reduced. Of note, this is only a rough estimate as PBMCs were used for the functional assays—and not pure lymphocyte preparations. For example, PBMCs contain approximately 14 % CD14+ cells [29] that “dilute” the lymphocytes, indicating that the effect on lymphocyte function may be even stronger than calculated.

The presence of bone metastases could be identified as an important influencing factor of baseline immune function and of radiation effects. Apparently, patients with bone metastases are in a worse clinical condition. This also applies for patients with progressive disease, higher tumor burden and impaired kidney function. Previously, it was described that adverse clinical condition and disease progress were accompanied by decreased immune function [30]. Moreover, the radiopharmaceutical ⁹⁰Y DOTATOC accumulates in bone metastases which consequently results in higher bone marrow doses. Thus, it is expectable that the lymphocyte function was more severely impaired in patients with bone metastases.

In conclusion, our findings demonstrate for the first time that ⁹⁰Y DOTATOC therapy in patients with advanced somatostatin receptor-positive tumors led to a significant reduction in cellular antimicrobial T cell responses in vitro. We could observe immunosuppression at days 1 and 7 after therapy; but it was nearly undetectable at day 90. The clinical relevance of that finding and the duration of immunosuppression, however, have to be further defined.

Abbreviations

Cpm

Counts per minute

DOTATOC

DOTA-d-Phe(1)-Tyr(3)-octreotide

DTPA

Diethylene-triamine-pentaacetate

ELISpot

Enzyme-linked immunospot

⁶⁸Ga

Gallium-68

IFN

Interferon

IL

Interleukin

LTT

Lymphocyte transformation test

OKT3

Anti-CD3 monoclonal antibody

PBMCs

Peripheral blood mononuclear cells

PET/CT

Positron emission tomography together with computed tomography

⁹⁰Sr

Strontium-90

⁹⁰Y

Yttrium-90