High- vs regular-dose recombinant human thrombopoietin plus cyclosporine A in patients with newly diagnosed non-severe aplastic anemia: a retrospective cohort study

ABSTRACT

Background:

Cyclosporine A (CsA) and regular doses of recombinant human thrombopoietin (rhTPO) can accelerate platelet recovery in patients with non-severe aplastic anemia (NSAA). However, it is unclear whether CsA plus rhTPO at a higher dose can further increase the efficacy.

Methods:

Data from patients with newly diagnosed NSAA, who had been treated with CsA in combination with different doses of rhTPO between February 2021 and August 2021 at Peking Union Medical College Hospital, were reviewed. All the enrolled patients had been treated with CsA at 3–5 mg/(kg/d), and patients were further classified into high-dose (with rhTPO 30000U qd × 14 days for 2 months) group or regular-dose (with rhTPO 15000U qd × 7days for 3 months) group. The treatment response and therapy-related adverse events were compared.

Results:

36 patients including 16 (44.4%) in the high-dose and 20 (55.6%) in the regular-dose group were enrolled. The baseline characteristics were compatible between the two groups. The platelet counts were significantly higher at 1/3/6 months in the high-dose group (p = 0.028, 0.0063 and p = 0.040, respectively). The high-dose group had a significantly shorter time to platelet transfusion independence ([1 (0.5–6) months vs 2.5 (1–12) months, p = 0.040]). There was no significant difference in overall response and complete response rate between the two groups at 1/3/6/12 months (p > 0.05). Treatment-related morbidities were similar between the two groups (p > 0.05).

Conclusions:

Adding a higher dose of rhTPO can further accelerate platelet recovery and platelet transfusion independence in patients with newly diagnosed NSAA.

KEYWORDS:

• Non-severe aplastic anemia
• recombinant human thrombopoietin
• cyclosporine A
• response
• platelet recovery

Introduction

Aplastic anemia (AA), a bone marrow disease, is characterized by pancytopenia and bone marrow hypocellularity [1], which can be classified into severe aplastic anemia (SAA) and non-severe aplastic anemia (NSAA) according to the severity of cytopenia [1,2]. Replacement of failed bone marrow is curative of the AA [1]. In the absence of leukocyte antigen (HLA)-matched related sibling donors, immunosuppressive therapy, in combination with or without eltrombopag, is a useful first-line therapeutic option for AA patients [1].

By binding to the TPO receptor, c-MPL, thrombopoietin (TPO) is an essential regulator of megakaryocyte development and platelet formation [3]. Consequently, thrombopoietin receptor agonists (TPO-RAs), including eltrombopag, romiplostimm avatrombopag, etc. have been developed and approved for the treatment of chronic immune thrombocytopenia (ITP), and/or AA [4–6]. Peffault de Latour. et al. [7] found that eltrombopag plus immunosuppressive therapy, compared with immunosuppressive therapy alone, was beneficial in untreated patients with severe aplastic anemia, without additional toxic effects. Furthermore, eltrombopag is effective in up to 40% of patients who fail or relapse after standard immunosuppression and shows promising efficacy as a frontline combination [7,8].

In nearly half of patients with lower-risk myelodysplastic syndrome (LR-MDS), the platelet counts increased subsequent therapy with thrombopoietin agonists such as romiplostim or eltrombopag [9,10]. Moreover, after receiving eltrombopag or romiplostim, patients with chronic immunological (idiopathic) thrombocytopenic purpura experienced a rise in mean and peak platelet counts in a dose-dependent manner [11,12]. These results demonstrated that platelet counts can be recovered in patients with bone marrow diseases by using thrombopoietin agonists. Nonetheless, approximately 10% of patients with LR-MDS or thrombocytopenia experienced transient increases in the number of circulating blasts [13]. Furthermore, TPO-RAs must be taken for an extended period to sustain the therapeutic effects, which adds to the financial burden and increases the danger of leukemia transformation and bone marrow fibrosis [14].

Recombinant human thrombopoietin (rhTPO) is a glycosylated full-length peptide TPO produced by 3SBIO (Shenyang, China), which has been demonstrated as an effective treatment for patients with corticosteroid-resistant or relapsed immune thrombocytopenia (ITP) [15–18]. During rhTPO therapy, patients developed temporary anti-TPO antibodies, yet these antibodies could neutralize endogenous TPO [19]. Additionally, recent data [18–20] also have demonstrated that rhTPO with rituximab works well to treat ITP by reducing the response time and improving the complete response (CR) rate. Furthermore, our recent study [21] also demonstrated that adding short-term rhTPO in patients with LR-MDS can reduce platelet transfusion and speed up the early platelet response without apparent adverse effects.

rhTPO is superior to eltrombopag at 25 mg/day for quickly raising platelet counts in patients with ITP [22]. Furthermore, we previously discovered that CsA combined with regular-dosage rhTPO can accelerate the recovery of the platelet level with tolerable side effects in NSAA patients [23].

Taken together, these results show that rhTPO at regular dosages is beneficial in treating bone marrow disorders such as ITP, LR-MDS and NSAA. Nevertheless, it is unclear if rhTPO exhibited dose-dependent effects similar to those observed in TPO-RAs. Here we conducted a retrospective analysis of the data from NSAA patients receiving either high- or regular doses of rhTPO treatment.

Methods

Patient selection

Patients with newly diagnosed NSAA at Peking Union Medical College Hospital between February 2021 and August 2021, who had received treatment with cyclosporin A (CsA) in combination with rhTPO at different dose, were retrospectively analysed in this study. The eligible patients were assigned to the high-dose or regular-dose group based on their economic situation or their willingness. The diagnosis of NSAA was performed according to the previously described criteria [24]. Those who met the following criteria was included: (1) 18 years or older; (2) with confirmed diagnoses of NSAA; (3) had been treated with CsA for at least 6 months, meanwhile, had been treated with rhTPO at the dose of 30000U qd × 14 days for 2 months or at 15000U qd × 7days for 3 months; (4) with no history of hematopoietic stem cell transplantation (HSCT); (5) had not been treated with antithymocyte globulin (ATG); (6) patients had been followed-up for at least 6 months; (7) with complete clinicopathological data and were unwilling to provide their medical history; and (8) without COVID-19 infection.

Treatment regimen

All patients had been treated with CsA at the initial dose of 3–5 mg/(kg/d) in combination with different dosages of rhTPO and the trough plasma blood concentration was maintained at 100–200 ng/ml. In the high-dose group, patients had been treated in combination with 30,000 U rhTPO qd subcutaneously for 14 days per month ×2 months if not responded. In the regular-dose group, patients were treated in combination with 15,000 U rhTPO qd for 7 days per month ×3 months if not responded. The supportive therapy included transfusion if hemoglobin was <60 g/L, platelet < 20 × 10⁹/L or G-CSF (5ug/kg/d) if neutrophil was less than 0.5 × 10⁹/L. Clinical data, including gender, age, symptoms, signs, complete blood cell count, serum biochemistry, such as liver and kidney functions and ferritin level, bone marrow smear and biopsy, chromosomal analysis, and myeloid malignancy gene mutation, paroxysmal nocturnal hemoglobinuria (PNH) clone, laboratory inspection results and final outcome, were collected before and after therapy. All side effects related to the treatment and the state of the disease were found in the medical records, or very occasionally, from the telephone interview with the patients and their relatives.

Response criteria

Treatment response was defined as follows [1,25]: complete response (CR) was defined as absolute neutrophil counts (ANC) > 1.5 × 10⁹/L, hemoglobin >110 g/L and platelet counts > 100 × 10⁹/L for 2 months. Partial response (PR): patients who met the response criteria for one or more lineages at 12 weeks but did not meet the criteria of CR. Clonal evolution was defined as a new clonal cytogenetic abnormality or characteristic changes in bone marrow consistent with the myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Relapse was defined as declining blood counts that warranted the reintroduction of full-dose cyclosporine.

Safety was evaluated by analyzing the incidence and severity of adverse events, which was classified according to the National Cancer Institute Common Toxicity Criteria for Adverse Events version 5.0 [26].

Statistics analysis

All statistical analyses were performed with SPSS (version 21.0). Data for patient demographics and laboratory measurements were presented as the median plus range or percentage. Comparison between two groups was tested by the Fisher Exact test or the Chi-Square test or the student t-test. Covariate effects on the response rate were evaluated using the univariable logistic regression with statistical inference presented using the corresponding standard errors. A p-value < 0.05 was considered statistically significant.

Results

Basic patient characteristics

Based on the inclusion criteria, a total of 36 patients were included in our final analysis. For the high-dose group (n = 16), the median age (range) was 59 (19–73) years and 7 patients (43.8%) were males. For the regular-dose group (n = 20), the median age was 56 (36–91) years and 11 (55%) were males. The median baseline hemoglobin, neutrophil and platelet count were 92 (46–99) g /L, 1.19 (0.63–9.52) and 16 (3–30) × 10⁹/L for the high-dosage group and 90.5 (72–100) g/L, 2.34 (0.52–6.63) × 10⁹/L and 17 (6–29) × 10⁹/L for the regular-dose group, respectively. There was no difference in concomitant treatment during the study period. The median follow-up time was 12 (9–18) months in the high-dose group and 12 (6–18) months in the regular-dose group, respectively (p = 0.876). The baseline values were compatible between the two groups and are outlined in Table 1.

Table 1. Baseline clinical characteristics of patients at the time of diagnosis.

	All patients (n = 36)	High-dose group (n = 16)	Regular-dose group (n = 20)	P value
Age, median (range)	57.5 (19–91)	59 (19–73)	56 (36–91)	0.775
Male, n (%)	18 (50.0%)	7 (43.8%)	11 (55%)	0.724
Ret (×10^9/L)	50.5 (10.8–98.7)	51.1 (20.4–98.7)	47.5 (10.8–87.10)	0.418
Hemoglobin, g/L	91.5 (46–100)	92 (46–99)	90.5 (72–100)	0.129
NEUT# (×10^9/L)	1.94 (0.52–9.52)	1.91 (0.63–9.52)	2.34 (0.52–6.63)	0.283
PLT (×10^9/L)	17 (3–30)	16 (3–30)	17 (6–29)	0.727
Cr, μmol/L	73 (39–91)	71.5 (39–87)	74.5 (50–91)	0.887
LDH (U/L)	226 (97–301)	227 (97–301)	211.5 (101–291)	0.607
Ferroprotein, ng/ml	355.5 (174–2192)	344 (176–2035)	367 (174–2192)	0.063
Plt TD (n, %)	11 (30.6%)	5 (31.2%)	6 (30.0%)	0.978
EPO (n, %)	5 (13.9%)	2 (12.5%)	3 (15.0%)	0.847
G-CSF (n, %)	7 (19.4%)	4 (25.0%)	3 (15.0%)	0.694
Follow-up (months)	12 (6–18)	12 (9–18)	12 (6–18)	0.876

Notes: Ret: reticulocyte; NEUT: neutrophil; Cr: creatinine; LDH: lactate dehydrogenase; Plt: platelet; TD: transfusion dependence; EPO: erythropoietin; G-CSF: granulocyte-colony-stimulating factor.

Response

In the high-dose group, 6 patients discontinued rhTPO at one month because platelet counts were higher than 50 × 10⁹/L. The rest had been treated with rhTPO for 2 months. In the regular-dose group, 4 patients discontinued rhTPO treatment when platelet counts were higher than 50 × 10⁹/L at one month. 4 patients stopped rhTPO when platelet counts were higher than 50 × 10⁹/L at 2 month, and the rest had been treated with rhTPO for 3 months.

The response rate at 1/3/6/12 months is summarized in Table 2. The overall response rate (ORR) at 1/3/6/12 months was 18.8%, 37.5%, 56.3% and 68.8% for the high-dose group and 15.0%, 30.0%, 50.0%, 65.0% for the regular-dose group, respectively (p > 0.05). Meanwhile, the complete response rate (CRR) at 1/3/6/12 months was 0%, 6.3%, 6.3% and 12.6% for the high-dose group and 0%, 5.0%, 5.0% and 10.0% for the regular-dose group, respectively (p > 0.05). Furthermore, the platelet count levels at 1/3/6 months’ post-treatment were all significantly higher in the high-dose group as compared with the regular-dose group (p = 0.028, 0.0063 and 0.040, respectively). Furthermore, the time of platelet transfusion independence was significantly shorter in the high-dose group compared with the regular-dose group [1 (0.5–6) months vs 2.5 (1–12) months, p = 0.040].

Table 2. Summary the response rate of groups.

Month	CR		P- value	ORR		P-value
	High-dose group	Regular-dose group		High-dose group	Regular-dose group
1	0%	0%	-	3 (18.8%)	3 (15.0%)	0.987
3	1(6.3%)	1(5%)	0.850	6(37.5%)	6 (30.0%)	0.867
6	1(6.3%)	1 (5%)	0.850	9(56.3%)	10(50.0%)	0.940
12	2(12.6%)	2 (10%)	0.863	11(68.8%)	13(65.0%)	0.984

Notes: CRR: complete response rate; ORR: overall response rate.

We also checked the megakaryocyte count in the high-dose (n = 9) and the regular-dose group (n = 13) before and 6 months after treatments, respectively. With the compatible baseline value, megakaryocyte count was 13 (8–18) and 11 (2–16) at 6 months for patients in the high-dose and regular-dose group, respectively (p = 0.105). Meanwhile, there was also no increase in reticulin or fibrosis in any examined bone marrow after 6 and 12 months of treatment in either group.

Safety and relapse

No severe bleeding events were observed in either group for the first three months after treatment. However, in the high-dose group, one patient developed obvious subhyaloid hemorrhage at 5 months, which disappeared after platelet transfusion; in the regular-dose group, one patient who did not respond had intracranial hemorrhage (ICH) at 6 months after rhTPO discontinuation.

No other serious adverse events occurred in the two groups (Table 3). Most of the adverse events were grade one or two. Fatigue (6.3%), infection (6.3%), drug-related renal injury (18.6%), drug-related liver injury (12.5%) and gastrointestinal disorders (12.5%) were the most common adverse events in the high-dose group, whereas drug-related renal injury (10.0%), drug-related liver injury (5.0%), infection (10.0%) and gastrointestinal disorders (15.0%) were the main adverse events in the regular-dose group. The incidence of drug-related adverse events was similar between the two groups (p > 0.05).

Table 3. Adverse events.

Adverse events (n, %)	High-dose group (n = 16)	Regular-dose group (n = 20)	P value
Infection rate	1 (6.3%)	2 (10.0%)	0.980
Drug-related liver injury	2 (12.5%)	1 (5.0%)	0.991
Drug-related renal injury	3 (18.6%)	2 (10.0%)	0.979
Fatigue	1 (6.3%)	1 (5.0%)	0.809
Gastrointestinal disorders	2 (12.5%)	3 (15.0%)	0.907
Intracranial hemorrhage	1 (6.3%)	1 (5.0%)	0.999

The median follow-up time was 12 (9–18) months in the high-dose group and 12 (6–18) months in the regular-dose group (p = 0.876). In the high-dose group, two patients who responded declined in platelet count and became transfusion-dependent again at 6 months, making the relapse rate 18.2% (2/11), in the regular-dose group, two patients (15.4%, 2/13) experienced relapse at 7 months (p = 0.906).

Clone evolution and final outcomes

During the follow-up period as mentioned above, for high-dose group, one patient who had no response to the full-dose CsA therapy transformed to SAA and was switched to ATG therapy at 9 months. No progression to paroxysmal nocturnal hemoglobinuria (PNH), myelodysplastic syndromes (MDS), acute myeloid leukemia (AML) or deaths were documented.

For the regular-dose group, one patient presented with a PNH clone at 6 months. No other clone evolution was noticed. Only one patient who had no response died from intracranial hemorrhage at 6 months.

Discussion

We previously discovered that CsA plus rhTPO is better than CsA alone with regard to the recovery speed of platelet in newly diagnosed NSAA [23]. However, in that study, patients were treated with rhTPO at 15000U qd × 7 days only for 3 months. Based on the experience [8,27] in newly diagnosed AA patients, TPO-RAs can provide a better response if the increase dose is according to the platelet level, it is possible that increasing the dose of rhTPO may also improve the reaction rate. In the present study, we discovered that high-dose rhTPO is more effective than regular-dose rhTPO in terms of speeding up platelet recovery and promoting platelet transfusion independence, which was in accordance with our hypothesis.

Regarding the recovery of the platelet count, we observed that the platelet count level at 1/3/6-month was significantly higher in the high-dose group, but there was no significant difference in the platelet counts between the two groups at 12 months. Since the majority of the bleeding occurred during the early stages, when CsA was still ineffective, the high-dose rhTPO may, therefore, be meaningful. At the time of 12 months, rhTPO for either group had been discontinued for quite a long time and the effects of CsA may be dominant.

In terms of therapy response, similar to our previous report [23] that adding rhTPO at 15000U qd × 7 for 3 months to CsA did not improve the ORR or CRR compared with CsA alone, there was no significant difference in ORR and CRR between the higher-dose and the regular-dose group, either. This result may be also verified by our previous finding [21] in LR-MDS that adding rhTPO to stanozolol did not influence the reaction rate at 6 months although it accelerated the early recovery of platelet. Unlike TPO-RA, which may stimulate hematopoiesis at an earlier stage, rhTPO may work on the late stage of megakaryopoiesis, which may show quicker early effects of platelet improvement, as found in ITP [22,27]. Unfortunately, the maintenance of rhTPO effects is illusive, in part, due to its subcutaneous administration, which makes it challenging to employ over a long period. Nevertheless, our study suggested that CsA plus rhTPO can increase the early platelet reaction in a dose-dependent manner.

Adverse events in this study were mild and reversible with or without medications, as has been shown in earlier research on rhTPO [18,19]. One patient in either group developed severe bleedings, one recovered and another died. Thromboembolic risk is another important aspect which needs special attention with rhTPO treatment. We did not notice any such events at either dose group so far. However, the risk of thrombosis should be closely monitored, particularly in rapid responders, elderly patients or those with thromboembolic risks.

For TPO-RAs, one concern was the possibility of clone evolution [9,13]. In our study, we did not observe significant clues of clone evolution in either group compared with the previous reports in the literature [18,21] although our follow-up time was relatively short. Nevertheless, further observation and a longer period of follow-up were required. Furthermore, we did not find bone marrow fibrosis, which was previously reported in earlier rhTPO-related investigations.

There were some limitations in our study. First, the dosage and frequent of rhTPO usage are different between the two groups, which may cause some potential bias for our data interpretation. Second, this study only enrolled 36 patients, some of the findings can be underestimated due to the small sample size. Third, this is a retrospective cohort study, some patients lacked some clinical data and missed some regular follow-ups. Finally, given the potential impact of COVID-19 infection on patients such as the thrombocytopenia [28–30] post-COVID-19 infection or COVID-19 mRNA vaccination, the follow-up time was relatively short for assessing long-term effectiveness. Even with those limitations, our results indicated that adding of higher dose of rhTPO to CsA may further accelerate the platelet recovery and platelet transfusion independence in patients with newly diagnosed NSAA.

Author contributions

Yuan Yang and Bing Han: study design and study concept. Yuan Yang, Qinglin Hu: data collection. Yuan Yang and Qinglin Hu: statistical analysis, data visualization. Yuan Yang: manuscript drafting. Yuan Yang, Chen Yang, Miao Chen, and Bing Han: data interpretation.

Consent for publication

The publication of this manuscript has been approved by all authors.

Acknowledgements

We would like to thank all the patients who participated in this study and their supportive families, as well as the investigators and clinical research staff from our centers.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

All data are available in the main text. All detailed metadata are available upon reasonable request to the corresponding author.

Additional information

Funding

This retrospective study was supported by fundings from CAMS Innovation Fund for Medical Sciences (CIFMS 2021-I2M-1-003), National High Level Hospital Clinical Research Funding (2022-PUMCH-C-026, 2022-PUMCH-D-002, 2022-PUMCH-B-046). The funders played no roles in the study design, the collection, analysis, and interpretation of data, the writing of the report, and the decision to submit the article for publication. All authors have no financial or non-financial conflicts to disclose in this study.