Fifty patients with histological confirmation of metastatic NETs (WHO II) were enrolled in the study. At the time of treatment all patients showed PD confirmed by CT examination, somatostatin receptor scintigraphy (SRS) and/or increasing blood concentration of chromogranin A (CgA).

Patients were divided into two groups. Group A consisting of 25 patients (11 men and 14 women) with a mean age (± SD) of 57.3 ± 10.6 years was treated with ⁹⁰Y-DOTATATE. Group B consisting of 25 patients (9 men and 16 women) with a mean age (± SD) of 56 ± 11.6 years was treated with a combination treatment including ⁹⁰Y and ¹⁷⁷Lu in one i.v. application.

The major criterion for inclusion into this study was the benefit from PRRT therapy. Because of the fact that it was the first PRRT study in Poland, patients had extended disease. The Karnofsky index was used for estimating the patients’ general condition.

The following inclusion criteria for therapy were used:

• SRS-positive disease with uptake in the tumour and metastases higher that the liver, evaluated within 3 months before inclusion (qualitative analysis)
• Histological confirmation of NET; inoperable or metastatic disease
• Haemoglobin level (Hb) ≥ 10 g/dl; leucocytes (WBC) ≥ 2×10⁹/l; thrombocytes (PLT) ≥ 90×10⁹/l
• Calculated creatinine clearance (CrCl) > 40 ml/min
• Karnofsky performance status ≥ 60
• Life expectancy > 3 months
• No pregnancy or lactation

All patients underwent blood tests (full blood count, kidney, liver functions tests and CgA) and staging prior to therapy with the use of CT and SRS using ^99mTc-HYNIC-TATE (^99mTc-HYNIC-Tyr³-octreotate) [¹⁴].

For patients receiving cold long-acting somatostatin analogues, the radionuclide therapy was performed 5 weeks after the completion of octreotide (Sandostatin LAR, Novartis) and 3 weeks after lanreotide (Somatuline, Ipsen). Patients who received chemotherapy were not treated with PRRT until after a period of 3 months.

Patients were given a follow-up examination in the clinic post PRRT at 3, 6, 12, 24 and 36 months using blood tests, CT and ^99mTc-HYNIC-TATE following the same protocols as the ones used prior to therapy.

^99mTc-HYNIC-TATE: the detailed method of kit preparation and labelling with ^99mTc was presented before [¹⁴, ¹⁵]. Briefly, the peptide conjugate HYNIC-Tyr³-octreotate (piChem, Graz, Austria) was prepared in the dried kit form under aseptic conditions (Institute of Atomic Energy POLATOM, Poland) after adding commercially available stannous chloride, mannitol, tricine, and N,N′-ethylenediaminediacetic acid. Quality control was performed by instant thin-layer chromatography silica gel (ITLC-SG) strips (Pall) developed in 0.9% saline for the determination of non-peptide-bound radioactivity.

The labelling yield efficiency exceeded 90% in all cases. Tests for sterility and bacterial endotoxins were routinely performed.

⁹⁰Y-DOTATATE and ⁹⁰Y/¹⁷⁷Lu-DOTATATE dried kits containing 100 μg (DOTA-Tyr³-octreotate) (piChem, Graz, Austria) and 50.0 mg of commercially available ascorbic acid were prepared under aseptic conditions. For labelling the content of each kit vial was dissolved in not more than 0.5 ml of ⁹⁰Y no-carrier-added chloride of desired radioactivity (max. 6.5 GBq per kit) or ¹⁷⁷Lu carrier-added (specific activity of around 555 GBq/mg Lu) chloride solution, both isotopes provided by the Institute of Atomic Energy POLATOM, Poland. In case of low volume 0.9% NaCl was used to fill up to 0.5 ml. Incubation was carried out at 95°C for 25 min. After cooling down to room temperature, the volume was increased up to 1.5–2.0 ml with 0.9% NaCl. The preparation was sterilized by filtration with 0.22-μm filters (Millipore) to sterile glass vials followed by filter wash with 50 mg/ml ascorbic acid to the final radioactive concentration of 740 MBq/ml. The radiochemical purity (RCP) was assessed by HPLC (column: Synergy 4 Fusion RP 80A 150 × 4.6 mm, flow rate 0.6 ml/min, UV detection at 220 nm and radiometric detection, solvent A: 0.1% trifluoroacetic acid in water, solvent B: acetonitrile, gradient: 0 min 18% B; 9 min 60% B; 12 min 60% B; 15 min 18% B; 21 min 18% B) and Sep-Pak C18 (Waters) mini-column separation according to the supplier’s instructions. The limit for RCP was over 99.0% for each ⁹⁰Y- and ¹⁷⁷Lu-labelled DOTATATE. The specific activities obtained were on average 74.7 GBq/μmol DOTATATE (in the range from 55.86 to 98.89 GBq/μmol) and 38.20 GBq/μmol (in the range from 23.55 to 44.83 GBq/μmol) for ⁹⁰Y- and ¹⁷⁷Lu-labelled DOTATATE, respectively. ⁹⁰Y-DOTATATE was administered as such while the mixed doses of ⁹⁰Y/¹⁷⁷Lu-DOTATATE were prepared from each ⁹⁰Y- and ¹⁷⁷Lu-labelled DOTATATE solution with equal radioactivity (1 GBq ¹⁷⁷Lu/1 GBq ⁹⁰Y).

Therapy was performed on an outpatients basis. Mixed amino acid (1,000 ml Vamin 18, Fresenius Kabi) and Ringer’s solutions (500 ml) were infused over 8 h for kidney protection, with infusion of 200 ml prior to administration of the treatment [¹⁶–¹⁹]. Before administration of the radiopharmaceutical, ondansetron (8 mg, Zofran, Glaxo Wellcome, Atossa, Anpharm SA) was injected intravenously to prevent nausea and vomiting.

Treatment sessions were repeated, up to a total calculated dose of 7.4 GBq/m². The injected activity per one course was 2.2–3.7 GBq. With respect to ⁹⁰Y/¹⁷⁷Lu-DOTATATE treatment, 50% activity of ⁹⁰Y-DOTATATE and 50% activity of ¹⁷⁷Lu-DOTATATE were injected. The median period between the treatment courses was 40 and 49 days, respectively.

A whole-body scan (256 × 256 matrix, 8-min acquisition per cm) and single photon emission computed tomography (SPECT) acquisition of the abdomen (128 × 128 matrix, 6º × 60 s) were performed using the dual-head Varicam camera (GE) with parallel-hole, high-energy, general purpose collimators [energy window centred on ¹⁷⁷Lu photopeak (216 keV) and a ±10% window width].

Bremsstrahlung imaging whole-body scans in 256 × 256 matrix, 8 cm/min was performed in ten patients 24 h post ⁹⁰Y-DOTATATE therapy again using the dual-head Varicam camera (GE) with a parallel-hole, low-energy, high-resolution collimator (energy window centred on 100 keV with a window width of ±35%) [²⁰].

The staging of disease and treatment response were evaluated at 3, 6, 12, 24 and 36 months follow-up. Blood tests were repeated every 7 days, others between 14 and 21 days after each cycle of therapy, and finally 3, 6, 12, 24 and 36 months after the completion of the therapy. Response to treatment on CT was defined according to Response Evaluation Criteria in Solid Tumors (RECIST). Side effects were scored according to the WHO criteria.

Event-free survival (EFS) was defined as the time from PRRT to the first evidence of progression or relapse, or to death. OS was defined as the time from PRRT to death from any cause.

The following progression criteria were defined:

• In patients with clinical symptoms of carcinoid syndrome, aggravation of flushing, diarrhoea or lacrimation occurrence
• Increased level of CgA of more than 50% above a previously measured value on follow-up (3, 6 and 12 months following therapy)
• The increase of two-dimensional tumour diameters of more than 30% on enhanced three-phase CT
• The detection of new tumour foci on SRS with ^99mTc-HYNIC-TATE on planar whole-body and SPECT imaging of the abdomen (if needed, SPECT of the chest)

Means and standard deviations, medians and quartiles or frequencies depending on the parameters’ distribution were used to summarize patient characteristics. The difference between comparable parameters was checked on the basis of Mann-Whitney and Wilcoxon tests.

OS, EFS and probability of 24-month survival were calculated on Kaplan-Meier estimator and compared via the log-rank test [²¹]. The multiple proportional hazards regression model (Cox analysis) was used for the analysis of OS and EFS. The association of chosen clinical predictors with OS and EFS was verified using the multiple proportional hazards regression model. No model reduction was performed to allow the adjustment of the effect of quality measurements for the effect of possible clinical predictors.

Model assumptions were tested on the basis of Schoenfeld residuals and in cases of violation the stratified model was fitted [²²].

Calculations were done in Stata v.10.1 software (Stata Statistical Software: Release 10,Stata Corporation LP, College Station, TX, USA).

In the group treated with ⁹⁰Y-DOTATATE alone, 12 patients were diagnosed with foregut NETs (9 pancreas NET: 3 gastrinoma, 1 insulinoma, 5 non-functioning tumours; 3 bronchial NET), 11 patients with midgut NETs (8 carcinoid, 3 small intestinal), 1 patient with hindgut NET (rectal NET) and 1 patient with unknown primary tumours.

In the group treated with a combination of ⁹⁰Y/¹⁷⁷Lu-DOTATATE, 13 patients were diagnosed with foregut NETs (9 pancreas NET: 1 glucagonoma, 4 gastrinoma, 4 non-functioning tumours; 3 bronchial NET, 1 epiglottal NET), 8 patients with midgut NETs (7 carcinoids, 1 small intestinal), 1 patient with hindgut NET (rectal NET), 2 patients with other tumours [1 patient with multiple endocrine neoplasia (MEN 1), 1 patient with von Hippel-Lindau syndrome] and 1 patient with unknown primary tumours.

There was no statistical difference in comparable parameters (age, sex, origin of tumours, size of metastases on CT, CgA, number and localization of metastases and treatment before PRRT). A Karnofsky performance status ≥ 80 was seen in the majority of patients; only two patients in both groups had a status ≥ 60.

Detailed patient characteristics are listed in Table 1.

Post-therapeutic scans were performed to evaluate the biodistribution of tracer. In all cases increased uptake in the tumours and metastases was observed, compared to the liver background on qualitative analysis. The results of post-therapeutic images in all cases were comparable with the distribution of disease on ^99mTc-HYNIC-TATE imaging (Figs. 1 and 2). Following post-therapeutic scans uptake in the majority of patients was unchanged. Only in two patients (one in group A and one in group B) were decreasing uptake and decrease in size of the liver metastatic lesions observed.

The median follow-up observation period in group A was 37.7 months and the interquartile range (IQR) for the observation period was 20.5–46.7 months. In group B the median follow-up observation period was 34.6 months and the IQR was 25.2–39.5 months.

In the ⁹⁰Y-DOTATATE group of patients OS was 26.2 months. In patients treated with ⁹⁰Y/¹⁷⁷Lu-DOTATATE, median survival was not reached, because less than 50% patients died. The survival plot depicting OS and the log-rank test (p < 0.027) demonstrate that the OS in the group treated with ⁹⁰Y/¹⁷⁷Lu-DOTATATE was statistically significantly longer (p < 0.027) (Fig. 3). The calculated probability of 24-month survival was 62% in the group treated with ⁹⁰Y-DOTATATE and 89% in those treated with ⁹⁰Y/¹⁷⁷Lu-DOTATATE.

Median EFS in the ⁹⁰Y-DOTATATE and ⁹⁰Y/¹⁷⁷Lu-DOTATATE groups was 21.4 and 29.4 months, respectively; the difference in EFS was not statistically significant (Fig. 4). The probability of 24-month PFS was 44 and 57%, respectively.

At the 12-month follow-up in the ⁹⁰Y-DOTATATE group vs the ⁹⁰Y/¹⁷⁷Lu-DOTATATE group, the following was observed: SD in 13 vs 16 patients, disease regression (RD) in 5 vs 3 patients, and PD in 3 vs 4 patients; 4 and 2 patients died, respectively (Fig. 5a).

At the 24-month follow-up: SD was observed in nine vs ten patients, RD in patients in the group treated with ⁹⁰Y/¹⁷⁷Lu-DOTATATE and progression in four patients in both groups; five and four patients died, respectively (Fig. 5b).

At the 36-month follow-up, SD was noted in six vs eight patients and PD in one patients in both groups; four and four patients died, respectively (Fig. 5c).

CT showed regression in size of the biggest lesions in the group treated with ⁹⁰Y-DOTATATE from median 50 to 44 mm and in the group treated with ⁹⁰Y/¹⁷⁷Lu-DOTATATE from 47 to 33 mm (Fig. 6). The changes in both groups are not statistically significant. Correlation between tumour size before and after therapy is shown in Fig. 7.

The patients in our study were not randomized to either group A or B but were treated first with ⁹⁰Y-DOTATATE (group A) and patients who presented later were treated with ⁹⁰Y/¹⁷⁷Lu-DOTATATE. For this reason we used the Cox analysis to evaluate the effects of the relevant prognostic factors on the survival times in each group of patients and the independence of these variables.

In the proportional hazards regression model for OS the radionuclides used, age of patients, sex, CgA level, number of lesions and size of maximal lesion were included. The model for OS was stratified by number of lesions due to a violated proportional hazards assumption.

Treatment is statistically significantly associated with OS, but not with EFS (Tables 2 and 3). Statistical analysis led to lower probability of death in the group treated with tandem ⁹⁰Y/¹⁷⁷Lu-DOTATATE than with ⁹⁰Y-DOTATATE alone [hazard ratio (HR) = 5.74, 95% confidence interval (CI) 1.63–20.2, p = 0.006] (Table 2). Effects of treatment on SRS are shown in Fig. 8.

The treatment was well tolerated. No severe adverse events occurred. Nausea and vomiting during administration of treatment and amino acids were observed in 20 of 50 patients (40%) with the same frequency in both groups. All cases of nausea and vomiting were successfully treated with ondansetron. Mild pain (no treatment required) at the site of the tumour was observed in 4 of 25 patients (16%) treated with ⁹⁰Y-DOTATATE within the first 48 h post-treatment and in 3 of 25 patients (12%) treated with ⁹⁰Y/¹⁷⁷Lu-DOTATATE within the first 72 h after treatment. In 2 of 50 patients (4%) chest pain immediately after administration was observed (1 patient treated with ⁹⁰Y-DOTATATE and 1 patient treated with ⁹⁰Y/¹⁷⁷Lu-DOTATATE). Liver tests were stable. According to WHO haematological toxicity criteria, haematological toxicity grade 3 was seen only in one patient treated with ⁹⁰Y-DOTATATE (patient received chemotherapy before PRRT). Toxicity grades 1 and 2 were seen with nearly the same frequency in both groups (grade 1 in six patients in the group treated with ⁹⁰Y-DOTATATE vs seven patients in the group treated with ⁹⁰Y/¹⁷⁷Lu-DOTATATE and grade 2 in three patients vs four patients, respectively) and without clinical symptoms. The decrease of WBC, RBC and PLT after therapy was similar in both groups. During treatment CrCl was stable in 15 patients treated with ⁹⁰Y-DOTATATE and in 13 patients treated with ⁹⁰Y/¹⁷⁷Lu-DOTATATE. Deterioration in kidney function was observed in three patients in each group, measured by creatinine level and calculated CrCl (the maximum observed changes in CrCl in the group treated with ⁹⁰Y-DOTATATE was from 111 ml/min prior to therapy which declined to 54 ml/min at the 36-month follow-up and in the group treated with ⁹⁰Y/¹⁷⁷Lu-DOTATATE from 106 ml/min prior to therapy to 67 ml/min at the 36-month follow-up). These patients received chemotherapy (etoposide+cisplatin) prior to PRRT. In two patients treated with ⁹⁰Y/¹⁷⁷Lu-DOTATATE transient deterioration in kidney function was observed.

Totally, for all patients at the 36-month follow-up the mean creatinine level increase was 0.07 mg/dl per year and CrCl decrease 5.1 ml/min per year.

The results indicate that therapy with tandem radioisotopes (⁹⁰Y/¹⁷⁷Lu-DOTATATE) provides longer OS than with a single radioisotope (⁹⁰Y-DOTATATE) and the safety of both methods is comparable. It seems that OS time and EFS time rather than WHO criteria for determining tumour burden should be used to assess the response to therapy.

However, more extensive studies with a larger number of patients are required both to establish the proportion of each isotope to be used in this dual therapy and to evaluate EFS over a longer period of follow-up.