Page 167 - 2020_08-Haematologica-web

NPM-ALK quantification by RQ-PCR and dPCR in ALCL
Statistical analysis
Event-free survival and overall survival were analyzed using the Kaplan-Meier method with differences compared by the log-rank test. Cumulative incidence functions for relapse were constructed using the method of Kalbfleisch and Prentice. Functions were compared with the Gray test. Quantification by RQ-PCR and dPCR was compared using Spearman correlation. All analyses were performed using SAS (SAS-PC, version 9.4, SAS Institute Inc., Cary, NC, USA).
Results
Quantification of NPM-ALK fusion gene transcripts by quantitative real-time polymerase chain reaction (validation cohort)
Patients’ characteristics
MDD was quantified by RQ-PCR in initial BM samples from 91 NPM-ALK-positive ALCL patients. Parallel blood samples for quantification were available from 70 of those patients. The clinical and biological characteristics of the 91 patients are shown in Table 1. Twenty-six of the 91 patients relapsed, one patient died from initial tumor com- plications. The cumulative incidence of relapse at 3 years of the 91 patients was 29±5%, the event-free survival at 3 years was 70±5% and the overall survival 92±3%. More than 10 NCN NPM-ALK were measured in the BM of 18 patients and ≤10 NCN NPM-ALK were detected in the remaining 73 patients.
The detection of >10 NCN NPM-ALK in BM correlated with stage III/IV disease, mediastinal and visceral organ involvement, as well as low anti-ALK antibody titers (Table 1). No association of NPM-ALK copy numbers above 10 NCN and histological subtype was observed (Table 1).
Prognostic impact of quantitative minimal disseminated disease in bone marrow
The cumulative incidence of relapse of 18 patients with more than 10 NCN NPM-ALK in BM was 61±12% com- pared to 21±5% for the remaining 73 patients (P=0.0002), The event-free survival rates at 3 years were 33±11% and 79±5%, respectively (P<0.0001), the overall survival rates were 83±9% and 94±3%, respectively (P=0.099) (Online Supplementary Figure S1). Application of the cut-off of 10 NCN NPM-ALK allowed the separation of a group of patients with a very high risk of relapse in the validation cohort.
Prognostic impact of quantitative minimal disseminated disease in blood
In 70 of the 91 patients for whom MDD was measured in the BM, NPM-ALK transcripts could be measured in blood, as well. The results for blood and BM in the same patients correlated (r=0.74). Notably, more patients were MDD-positive and showed higher copy numbers in blood compared to BM (Figure 1).
At 3 years the cumulative incidence of relapse of the 70 patients for whom MDD measurements were available in both BM and blood was 26±5%, the event-free survival was 74±5% and the overall survival 94±3%. To analyze a possible influence of the biological medium used for the quantitative MDD measurement on the detection of very high-risk patients, outcome was compared according to quantitative MDD in blood and BM using the same cut-
off among these 70 patients. The cumulative incidence of relapse of the 17 patients with >10 NCN NPM-ALK meas- ured in blood was 59±13% compared to 15±5% in 53 patients with ≤10 NCN NPM-ALK (P=0.0004). In compar- ison, the cumulative incidence of relapse of the 13 patients with >10 NCN NPM-ALK measured in BM was 62±14% compared to 18±5% in the 57 patients with ≤10 NCN (P=0.0007) (Figure 2).
Establishment and validation of a digital droplet polymerase chain reaction assay
To overcome some limitations of RQ-PCR we tested an NPM-ALK-specific dPCR assay for fusion gene and refer- ence gene quantification.
A gradient PCR was performed to optimize the per- formance of the dPCR assays for NPM-ALK and ABL1.12 The amplification and elongation temperature was set to
Table 1. Association of the quantity of NPM-ALK transcripts in bone marrow with patients’ characteristics, clinical and biological risk fac- tors in the validation cohort.
All patients
≤10 NCN MDD >10 NCN P
Gender, n (%) Male
Female
Stage*, n (%) I
II III IV n.a.
Age, n (%)
Age <10 years Age ≥10 years
CNS, n (%) Negative Positive
Bone marrow, n (%) Negative
Positive
Bone, n (%) No
Yes
Skin, n (%) No
Yes
Mediastinum, n (%) No
Yes
Viceral organs**, n (%) No
Yes
Histology Non-common Common
Anti-ALK titer ≤750
>750
91
59
32
5 20 56 6 4
24
67
83 -
86
5
77 14
73
18
52 39
68
23
35 52
24
44
NPM-ALK
73
47 (64%)
26 (36%)
5 (7%) 20 (27%) 42 (58%) 2 (3%) 4 (5%)
19 (26%)
54 (74%)
67 (100%) -
72 (99%)
1 (1%)
64 (88%) 9 (12%)
58(80%)
15 (20%)
48 (66%) 25 (34%)
59 (81%)
14 (19%)
27 (39%) 43 (61%)
15 (28%)
38 (72%)
NPM-ALK
18
12 (67%) 1.0
6 (33%)
0 (0%)
0 (0%) 14 (78%) 4 (22%)
5 (28%)
13 (72%)
16 (100%) -
14 (78%)
4 (22%)
13 (72%) 5 (28%)
15 (83%)
3 (17%)
4 (22%) 14 (78%)
9 (50%)
9 (50%)
8 (47%) 9 (53%)
9 (60%)
8 (40%)
0.002
1.0
0.004
0.14
1.0
0.001
0.01
0.59
0.03
MDD: minimal disseminated disease; NCN: normalized copy number; n.a.: not avail- able; CNS: central nervous system. * St. Jude staging system; ** liver, spleen, lung.
haematologica | 2020; 105(8)
2143