Patient characteristics
From January 2017 to January 2019, there were altogether 156 NSAA patients treated with FK. 55 patients were excluded either due to lost follow-up (32 patients), withdrawal of FK (12 patients) within 6 months after FK, follow-up less than 12 months (10 patients) or denying the consent form (3patients). There were 101 patients enrolled for the final analysis, including 49 males (48.5%) and 52 females (51.5%), with a median age of 46 (range 14~83) years. Among them, 58 (57.4%) patients were under the age of 50-year-old. PUMCH is known for difficult and complicated cases so most of the refractory NSAA are referred to our clinic which make our cohort highly selective---about 20% of younger patients discontinue CsA due to renal disfunction after repeating use of CsA in long period of time. Before FK treatment, patients’ hematological and biochemistry parameters were as shown in Table 1. As for the chromosome abnormalities, +8 was found in 5 patients, -Y in 3 patients, 20q- in 2 patient and monosomy 7 in 1 patient. 7 patients had notable PNH clone(percentage of flaer negative neutrophil from 3.5%~18.3%). 50 (48.1%) patients were red blood cell transfusion dependent and the median transfusion requirements were 6 (range 4~10) units/eight weeks and 26 (25.0%) patients were platelet transfusion dependent and the median transfusion requirements were 4 (range 1~5) units/eight weeks before FK treatment (Table 1) .
Table 1 Patients’ baseline demographic and clinical characters
Baseline characters
|
n=101
|
Age (year) median (min,max)
|
46(14, 83)
|
Sex
|
|
Male n (%)
|
49 (48.5)
|
Female n (%)
|
52 (51.5)
|
Hemoglobin (g/L) mean (SD)
|
61.88(28.13)
|
Reticulocyte (1012/L) mean (SD)
|
6.13 (10.01)
|
White cell count (109/L) mean (SD)
|
3.08 (1.03)
|
Neutrophil count (109/L) mean (SD)
|
1.32 (0.80)
|
Platelet (109/L) mean (SD)
|
38.26 (62.50)
|
SGPT (U/L) mean (SD)
|
26.88 (24.48)
|
Scr (μmol/L) mean (SD)
|
98.56 (58.66)
|
SF (μg/L) mean (SD)
|
1761.23 (1937.18)
|
Glu (mmol/L) mean (SD)
|
6.51 (3.97)
|
Notable PNH clone n (%)
|
7 (6.9)
|
RBC transfusion dependent n (%)
|
50 (49.5)
|
Platelet transfusion dependent n (%)
|
26 (25.7)
|
Chromosome abnormalities
|
|
+8 n (%)
|
5 (5.0)
|
-Y n (%)
|
3 (3.0)
|
20q- n (%)
|
2 (2.0)
|
Monosomy 7 n (%)
|
1 (1.0)
|
Patients had been treated with CsA alone before FK treatment. Among them, 45 patients (refractory patients) had no response and the median time for CsA treatment was 8 (6~10) months, 36 patients (relapsed patients) relapsed when CsA was tapered or stopped but did not response when CsA were added again or dosage increased and the median time for CsA treatment was 18(12~60)months. 20 patients (intolerant patients) could not tolerate CsA due to severe side effects like gingival hyperplasia, muscle tremor, kidney impairment, gastrointestinal disturbance, etc. The median time of CsA exposure for those patients was 4 (3~7) months. None of them had reached PR before FK treatment. Of all patients, 50(53.2%) patients had FK concentration of 4~12 ng/mL within three months of tacrolimus treatment, while others did not reach the aim concentration due to the kidney limitation.
Efficacy
The median follow-up time was 19 (14~36) months since FK started. At the end of follow-up, the median time on FK treatment was 14 ( 6~30) months. Of all the 101 patients, the median time to response (at least partial response, PR) was 4 (1~6) months, the medium time to optimal response was 6 (3~10) months. There was 10 (9.9%) CR, 29 (28.7%) PR, 62 (61.4%) NR, with OR (CR+PR) rate of 38.6%.
Of the 39 patients who achieved OR, 9(23.1%)were solely erythroid response ( 6 of them became transfusion independent), 11(28.2%)were solely platelet response , 3(7.7%)with neutrophil response, 9 (23.1%) patients had bilineage responses and 7(17.9%) patients had trilineage response.
Moreover, 14 patients with liver or kidney impairment improved their liver and kidney function after FK treatment, among them 10 patients with elevated creatinine level and the other 4 patients with abnormal bilirubin level prior FK treatment returned to normal after 6 months of medication. 10 patients’ gingival hyperplasia and gastrointestinal symptoms were significantly improved.
Subgroup analysis showed that the OR rate (ORR) for patients younger than 50 years old was 50.0%, significantly higher than that of patients over 50 years old (23.3%, P=0.004). Patients with FK concentration 4.0~12.0 ng/ml had higher ORR (49.2%) compared with those with concentration<4.0 ng/ml (22.7%, P= 0005). Females had higher ORR than males (P= 0.0442). There was no significant difference in ORR between patients with refractory/relapsed and intolerant to CsA (35.8% vs 50.0%, P=0.2429, Table 2). However, multiple logistic regression result indicated that age (P=0.0005),FK concentration (4.0~12ng/ml) (P=0.0005) and intolerance to CsA(P=0.0142) were the independent risk factors for ORR (Table 3).
Table 2 Patients’ response in different groups
|
|
Number
|
CR(N/%)
|
P* value
|
PR(N/%)
|
P& value
|
ORR(N/%)
|
P# value
|
Gender
|
Male
|
49
|
2(4.1)
|
0.09338
|
12(24.5)
|
0.3625
|
14(28.6)
|
0.0442
|
|
Female
|
52
|
8(15.4)
|
|
17(32.7)
|
|
25(48.1)
|
|
Age (range)years
|
≤50
|
58
|
6(10.3)
|
1
|
23(39.7)
|
0.0033
|
29(50.0)
|
0.004
|
|
>50
|
43
|
4(9.3)
|
|
6(14.0)
|
|
10(23.3)
|
|
CsA
|
refractory/relapsed
|
81
|
7 (8.6)
|
0.4103
|
22(27.2)
|
0.4877
|
29(35.8)
|
0.2429
|
|
intolerant
|
20
|
3 (15.0)
|
|
7(35.0)
|
|
10 (50.0)
|
|
FK concentration
|
<4.0ng/ml
|
44
|
2(4.5)
|
0.0977
|
8(18.2)
|
0.0126
|
10(22.7)
|
0.0005
|
|
4.0~12ng/ml
|
50
|
8(13.6)
|
|
21(35.6)
|
|
29(49.2)
|
|
*P value indicate the comparison of CR among different groups, &P value indicate the comparison of PR among different groups, #P value indicate the comparison of ORR among different groups
Table 3 Multivariate analysis for the factors associated with ORR
|
|
Coefficient
|
Standard error
|
OR
|
lower bound 95% CI
|
upper bound 95% CI
|
P
|
Male(ref=Female)
|
-0.7727
|
0.5187
|
0.462
|
0.167
|
1.276
|
0.1363
|
Age (year)
|
-0.0660
|
0.0191
|
0.936
|
0.902
|
0.972
|
0.0005
|
FK (4~12 ng/ml)
|
1.9729
|
0.5636
|
7.192
|
2.383
|
21.706
|
0.0005
|
refractory/relapse (ref=intolerant)
|
-1.7391
|
0.7090
|
0.176
|
0.044
|
0.705
|
0.0142
|
Safety
In a total of 101 patients, there were 32 (31.7%) cases of elevated creatinine level (9 of them had increased Scr level before FK treatment), 8 cases (7.9%) with elevation of AST/ALT, 6 (5.9%)cases of hypertension, 5 (5.0%) cases of elevated bilirubin, and 2 (2.0%) cases of drug allergy, most of them were grade I-II in CTCAE criteria 4.0 which recovered after symptomatic treatment. Severe adverse events resulting in drug withdraw or hospitalization included the followings: 4 cases of grade III creatinine increase, 4 episodes of fever with grade III~IV neutropenia, one of which linked to culture-confirmed infection, occurred in one patient who did not have a response.
Relapse, survival and clonal evolution
For the 39 patients who had reached CR or PR, 25.6% (10/39) patients relapsed at the median of 12 (10~16) months before FK tapering. Clonal evolution to acute myeloid leukemia (AML) was observed in one patient with monosomy 7 who did not response to FK. Two patients who did not response evolved into myelodysplastic syndrome (MDS) one year after treatment and lived with supportive care. No increase of PNH clone, nor clonal evolution to MDS or AML developed in other patients in our cohort. Four young patients who did not response underwent HSCT and achieved CR.
There were two deaths during the follow-up period in the no response cohort, one was due to severe infections as mentioned above, and the other was the patient transforming to AML who died of infections 2 months after chemotherapy. Other patients with no response lived with supportive care. No other deaths were observed in the cohort.
Regulatory T cells in peripheral blood before and after FK
Thirty-one patients had tested Treg cells before and after FK, and they all achieved at least PR and the blood was taken after six months of treatment, meanwhile the level of Treg cells from eight age and sex-matched normal volunteers was taken as controls. Level of Treg cells pre FK was much lower compared with that of healthy controls (Treg/CD4+T: 3.7±0.6% vs 6.8±0.7%, P=0.0004). As expected, the level of Treg cells increased significantly after FK treatment (Treg/CD4+T, before: 3.7±0.6% vs. after: 7.1±0.8%,P=0.0039, Fig 1).