Radioligand therapy of metastatic prostate cancer using 177 Lu-PSMA-617 after radiation exposure to 223 Ra-dichloride

Radioligand therapy with 177Lu-PSMA-617 is an innovative and effective therapy for castrate-resistant metastatic prostate cancer patients. For patients with symptomatic bone metastases without visceral metastases, the guidelines recommend radionuclide therapy with 223Ra-dichloride as a single therapeutic agent or in combination with hormone therapy. The aim of this study was to evaluate the safety of repeated cycles of 177Lu-PSMA-617 after exposure to more cycles of 223Ra. Forty-nine patients were treated with three cycles of Lu-PSMA-617 divided into two groups subjected to a history of therapy with 223Ra. Group 1 included 20 patients, who had received therapy with 223Ra prior to Lu-PSMA-617 therapy. Group 2, which was the control group regarding hematotoxicity, comprised 29 patients without any history of a bone-targeted radionuclide therapy. No CTC 4° hematotoxicity was observed in the entire study population. There was no CTC 3° or CTC 4° leucopenia in either group. One and three patients from group 1 and 2, respectively, showed CTC 3° anemia. In group 1 there was significantly more CTC 2° anemia (50% vs. 6.9%) (p=0.008). One patient from group 1 (5%) showed a CTC 3° thrombocytopenia without any concurrent anemia, and two patients from group 2 (7%) showed a CTC 3° thrombocytopenia, one with CTC 3° anemia and one without any anemia. There were no significant differences between the two groups regarding leucopenia and thrombocytopenia. These results confirmed that performing repeated cycles of Lu-PSMA-617 after 223Ra seems to be safe with a very small probability of hematotoxicity.


INTRODUCTION
Prostate-specific membrane antigen (PSMA) is an attractive target for the diagnosis and therapy of metastasized prostate cancer (mPC) [1][2][3][4]. So far, all of the published papers about radioligand therapy (RLT) with 177 Lu-PSMA-617 (Lu-PSMA-617) have demonstrated that this therapy is safe and has a low toxicity profile [5][6][7][8]. For patients with symptomatic bone metastases without visceral metastases the international guidelines recommend radionuclide therapy with 223 Radichloride ( 223 Ra, Xofigo ® ) as a single therapeutic agent or in combination with hormone therapy [9]. Although, according to our recently published results, performing Lu-PSMA-617 therapy after therapy with 223 Ra seems safe with a low hematotoxicity profile [5,7,10,11], because of the limited number of patients in these studies, as well as the low number of performed therapy cycles (max. 2

Clinical Research Paper
Oncotarget 55568 www.impactjournals.com/oncotarget cycles), there was a need to evaluate hematotoxicity in patients after exposure to 223 Ra who received 3 cycles of RLT compared to patients without a history of therapy with 223 Ra.

RESULTS
Altogether 147 cycles of RLT were performed. The mean follow-up time after the third cycle was 3.4 months (2-10 months). Patients in groups 1 and 2 were treated with a mean cumulative activity of 18 GBq and 17.8 GBq, respectively (p = 0.55) ( Table 1).
Although there were no significant differences regarding the number of blood cell counts between the two groups prior to the first cycle of RLT, there was more CTC 1° and 2° thrombocytopenia in patients in group 1 (p = 0.04) with a history of therapy with 223 Ra (Table 2).

Hematotoxicity in all patients
The blood parameters according to the common toxicity criteria prior to the first cycle of therapy are listed in Table 3. Relevant anemia, thrombocytopenia and leucopenia (CTC 3°) occurred during the observation period after the third cycle in 4 (8.2%), 3 (6.1%) and 0 patients, respectively. No CTC 4° hematotoxicity was observed in the entire study population. More than 60% of patients did not show any hematotoxicity. Two patients with CTC 3° thrombocytopenia showed no anemia and one had a concurrent CTC 3° anemia. Three patients with CTC 3° anemia had no thrombocytopenia (CTC 0°). Table 4 shows the hematotoxicity in each group according to the CTC criteria. There was no CTC 3° or CTC 4° leucopenia in either group. One and three patients from group 1 and 2, respectively, showed CTC 3° anemia. In group 1 there was significantly more CTC 2° anemia (50% vs. 6.9%) (p = 0.008). One patient from group 1 (5%) showed a CTC 3° thrombocytopenia without any concurrent anemia (CTC 0°), and two patients from group 2 (7%) showed a CTC 3° thrombocytopenia, one with CTC 3° anemia and one without any anemia (CTC 0°). There were no significant differences between the two groups regarding leucopenia and thrombocytopenia. There was no significant correlation between the number of performed 223Ra therapies and higher hematotoxicity in group 1. There was also no significant increase in hematotoxicity rate in patients with a history of external radiotherapy compared to patients without this therapy in either group (groups 1&2).

Hematotoxicity in patients who underwent both chemotherapy and radionuclide therapy with 223 Ra
Eleven patients from group 1 (55%) had received chemotherapy prior to or after 223 Ra. The median time interval between the last cycle of chemotherapy and the first cycle of the PSMA therapy was 11 months (2 -38 months). There was no significant increase in hematotoxicity rate in these patients compared to patients in group 1 who had not received any chemotherapy ( Table  5). Table 6 shows the grade of toxicity according to the extent of bone involvement in each group and in all patients together. There was a significant correlation between the extent of bone involvement and thrombocytopenia 1°-3° in all patients (p = 0.03); however, there was just a trend toward more thrombocytopenia 1°-3° in patients in group 2 with SuperScan (p = 0.08). Otherwise, there was no significant increase in hematotoxicity according to the extent of bone involvement in this patient cohort.
A combination of the mentioned therapies, especially chemotherapeutic agents and radionuclides, may be hematotoxic, i.e. inducing a bone marrow failure (BMF) . BMF may be the result of anemia, thrombocytopenia, Oncotarget 55569 www.impactjournals.com/oncotarget leucopenia, or a combination of 2 or more of these factors. However, depending on the extent of bone and bone marrow metastases, bone marrow function might become compromised, resulting in anemia and thrombocytopenia [25], independently of therapeutic regimen.
Apart from this, there are some other factors that may influence the bone marrow function in patients with PC, such as castration and androgen blockage, which have been shown to cause anemia and could be corrected with recombinant erythropoietin [26,27]. Nieder et al. showed that about half of PC patients experience low hemoglobin ( < 10 g/dL) unrelated to adverse effects of the previously mentioned therapeutic agents, and this in turn leads to a short survival time [28]. In the case of repeated radionuclide therapies with one or more radiopharmaceuticals the probability of a BMF increases, and in patients with a high tumor involvement of bone marrow, this toxicity may occur earlier with more serious consequences [29][30][31][32].
In this study no patient showed a CTC 3° leucopenia and only in 3 patients (6.1%) was a CTC 3° thrombocytopenia detected, two of them (4.1%) without a concurrent anemia (CTC 0°), which was most likely induced by RLT, and one patient with concurrent CTC 3° anemia, which was possibly induced by RLT (Table 3). In the subgroup analysis, despite there being more patients with CTC 1°/2° thrombocytopenia in group 1 prior to the first cycle of RLT, which was most likely because of the therapies performed with 223 Ra, only one patient from group 1 showed a CTC 3° thrombocytopenia without a concurrent anemia (CTC 0°), which was probably induced by RLT, and two patients in group 2 showed a CTC 3° thrombocytopenia, one with concurrent CTC 3° anemia and one without anemia. There was no significant difference between these two groups regarding radiationinduced hematotoxicity (p = 0.88; Table 4). These results reinforce the fact that RLT using Lu-PSMA can be performed after 223 Ra safely.
All of these three patients with CTC 3° thrombocytopenia had a diffuse bone and bone marrow involvement (SuperScan), which shows the higher probability of hematotoxicity in this group of patients (Table 6); however, five and four other patients with a SuperScan showed CTC 0° and CTC 1°/2° thrombocytopenia, respectively, which indicates the safety of RLT in patients in such an advanced stage of the disease. Three patients (6.1%) with CTC 3° anemia had no thrombocytopenia (CTC 0°), which was most likely due to disease progression in these patients.
According to the results of ALSYMPCA trial [33] in patients not treated with docetaxel, the hematoxicity of Ra-223 was same as that in Placebo arm showing clearly     that 223 Ra alone is not bone marrow toxic. In the current study, we did not detect any higher hematotoxicity in patients with a history of 223 Ra and chemotherapy prior to Lu-PSMA therapy. In the group 1, eight patients received Docetaxel and three patients Docetaxel as well as cabazitaxel as the second line chemotherapy. In the group 2, eleven patients received docetaxel and 6 patients docetaxel as well as cabazitaxel as the second line chemotherapy. There was no significant increase in hematotoxicity rate because of prior chemotherapy in either group.

.0) in both groups prior to RLT Group 1 (Hx of Ra-223) Group 2 CTC 0° (%) CTC 1°-2° (%) CTC 3° (%) CTC 0° (%) CTC 1°-2° (%) CTC 3° (%) p-value
In our recently published paper we showed that a prior therapy with 223 Ra had no negative impact on therapy response of a RLT [34]. In the current study a significant response after 3 cycles of RLT, defined as a PSA decline > 50%, was detected in 45% and 58.6% of patients in group 1 and 2, respectively. It seems that more patients in group 2 showed a significant response to the therapy, however this difference was not significant.
Although this study is a retrospective study and should be confirmed by prospective studies with more patients, these results encourage us to perform RLT in patients with a history of 223 Ra therapy. Performing repeated cycles of Lu-PSMA-617 after 223 Ra seems to be safe, with a very small probability of hematotoxicity and at the same time a significant therapeutic response. In conclusion RLT with Lu-PSMA-617 after radiation exposure to 223 Ra is a safe with no increased probability of radiation-induced hematotoxicity same as patients without a history of treatment with 223 Ra.

Patients
Forty-nine hormone refractory mPC patients with distant metastases and progressive disease according to the PSA level were treated with three cycles of Lu-PSMA-617 with 8-week intervals between the cycles. All of these patients had at least 2 months follow-up after the third cycle. The patients were divided into two groups. Group 1 included 20 patients (40.8%), who had received between 1 and 6 cycles of therapy with 223 Ra (median: 6 cycles; mean: 4.9) 30-365 days (mean: 106 days; median: 75 days) prior to Lu-PSMA-617 therapy. Group 2, which was the control group regarding hematotoxicity, comprised 29 patients (59.2%) without any history of a bone-targeted radionuclide therapy. There were no significant differences between these two groups of patients regarding age, Gleason score, complete blood counts, the number of red blood cell transfusions prior to the first cycle of RLT, previous therapies, the extent of bone and other metastases and the ECOG status, as well as the amount of administered activity in each cycle and cumulatively ( Table 1). The local ethics committee approved this retrospective study, and all subjects signed a written informed consent.

Laboratory tests
One day prior to each cycle, the hematological status was evaluated in all patients. The baseline blood values of each group according to the common toxicity criteria are shown in Table 2. Laboratory examinations for at least up to 8 weeks after the third cycle were obtained in all patients.
The treatment solution was administered by slow intravenous injection over 1 minute followed by 1000 ml of NaCl or Ringer's solution. All patients were discharged 48 hours after therapy in accordance with the rules of the Federal Office for Radiation Protection in Germany (BfS).

Toxicity
Toxicity was recorded using the Common Terminology Criteria for Adverse Events (CTCAE), version 4.0, and was analyzed according to the recommendation of the NCI guidelines for investigators [35]. Etchebehere et al. [32] used the following classification for classifying the reasons for bone marrow failure (BMF), which is adopted in the current study according to CTC criteria: 1) BMF is most likely due to disease progression in patients who developed CTC 3°/4° anemia with only CTC 1°/2° thrombocytopenia; 2) it is most likely induced by RLT in patients who had CTC 3°/4° thrombocytopenia with only CTC 1°/2° anemia; and 3) it is possibly induced by RLT in patients who developed CTC 3°/4° anemia associated with CTC 3°/4° thrombocytopenia.

Tumor response evaluation
The tumor marker PSA was used as the main marker for the response evaluation. We classified the changes in PSA level as a decrease of more than 50% and any decline. Any increase in PSA was considered disease progression.

Statistical analysis
Variables of interest were calculated using descriptive statistics. The chi-square test (χ2) was used to compare different variables in both groups. For the comparison of variables with a normal distribution, a t-test was used. Statistical analysis was performed using a commercially available software package (SPSS 22, IMB, Armonk, NY, USA). Values of P < 0.05 were considered significant.