Patients’ characteristics and responses.
For this study, fifty-seven melanoma patients were analyzed. Considering the iBOR, 28 patients responded positively to CPI therapy with an iCR (n = 11) or an iPR (n = 17), 16 patients had no objective response (iSD), and 13 patients developed an iPD. The median follow-up time for PFS was 12.2 months (mean 17.1 month, ranging from 1.1 to 61.7 months). At 1 year, PFS was 57.7%, with a median PFS of 33.4 months for iCR, 20.7 for iPR, 9.7 for iSD, and 2.8 for iPD patients. There were no patients lost to follow-up. Fifteen patients developed leukoderma at a mean time of 6.8 months after CPI therapy starting (Table 1).
Table 1
Characteristics and treatment outcomes of enrolled patients.
Patient
|
Stagea
|
Checkpoint inhibitora
|
iBORb
|
PFSc
|
Progressionc
|
Leukodermad
|
1
|
IIIc
|
nivo
|
iCR
|
10.8
|
no
|
8.7
|
2
|
M1a
|
nivo
|
iCR
|
40.7
|
no
|
2.1
|
3
|
M1a
|
nivo
|
iCR
|
13.5
|
no
|
-
|
4
|
M1a
|
nivo
|
iCR
|
12.2
|
no
|
-
|
5
|
M1b
|
pembro
|
iCR
|
21.9
|
no
|
7.6
|
6
|
M1c
|
nivo
|
iCR
|
33.4
|
no
|
4.7
|
7
|
M1c
|
pembro
|
iCR
|
37.3
|
no
|
11.1
|
8
|
M1c
|
pembro
|
iCR
|
37.3
|
no
|
9.7
|
9
|
M1c
|
pembro
|
iCR
|
48.1
|
no
|
-
|
10
|
M1c
|
nivo
|
iCR
|
13.3
|
no
|
-
|
11
|
M1d
|
pembro
|
iCR
|
62.6
|
no
|
-
|
12
|
M1a
|
nivo
|
iPR
|
40.5
|
no
|
13.1
|
13
|
M1a
|
pembro
|
iPR
|
26.2
|
no
|
-
|
14
|
M1a
|
pembro
|
iPR
|
20.7
|
no
|
4.3
|
15
|
M1a
|
pembro
|
iPR
|
19.1
|
no
|
-
|
16
|
M1a
|
pembro
|
iPR
|
7.1
|
yes
|
-
|
17
|
M1a
|
nivo
|
iPR
|
11.5
|
no
|
-
|
18
|
M1a
|
nivo
|
iPR
|
10.8
|
no
|
-
|
19
|
M1b
|
pembro
|
iPR
|
33.6
|
no
|
2.1
|
20
|
M1b
|
pembro
|
iPR
|
9.2
|
no
|
6.7
|
21
|
M1c
|
nivo
|
iPR
|
8.9
|
yes
|
-
|
22
|
M1c
|
nivo
|
iPR
|
42.3
|
no
|
-
|
23
|
M1c
|
nivo
|
iPR
|
36.8
|
no
|
9.7
|
24
|
M1c
|
pembro
|
iPR
|
39.1
|
no
|
-
|
25
|
M1c
|
pembro
|
iPR
|
38.0
|
no
|
7.7
|
26
|
M1c
|
pembro
|
iPR
|
36.8
|
no
|
6.9
|
27
|
M1c
|
nivo
|
iPR
|
13.9
|
yes
|
-
|
28
|
M1c
|
nivo
|
iPR
|
12.9
|
no
|
-
|
29
|
M1a
|
pembro
|
iSD
|
7.7
|
yes
|
-
|
30
|
M1a
|
nivo
|
iSD
|
12.9
|
no
|
-
|
31
|
M1b
|
nivo
|
iSD
|
18.2
|
yes
|
-
|
32
|
M1b
|
nivo
|
iSD
|
11.7
|
yes
|
-
|
33
|
M1c
|
nivo
|
iSD
|
5.9
|
yes
|
-
|
34
|
M1c
|
nivo
|
iSD
|
5.3
|
yes
|
-
|
35
|
M1c
|
nivo
|
iSD
|
36.4
|
no
|
4.3
|
36
|
M1c
|
nivo
|
iSD
|
6.8
|
yes
|
-
|
37
|
M1c
|
pembro
|
iSD
|
5.2
|
yes
|
-
|
38
|
M1c
|
pembro
|
iSD
|
4.1
|
yes
|
-
|
39
|
M1c
|
pembro
|
iSD
|
32.5
|
yes
|
-
|
40
|
M1c
|
pembro
|
iSD
|
6.3
|
yes
|
-
|
41
|
M1c
|
pembro
|
iSD
|
26.4
|
yes
|
-
|
42
|
M1c
|
pembro
|
iSD
|
15.8
|
no
|
-
|
43
|
M1d
|
pembro
|
iSD
|
4.8
|
no
|
2.8
|
44
|
M1d
|
pembro
|
iSD
|
17.3
|
no
|
-
|
45
|
IIIc
|
pembro
|
iPD
|
2.8
|
yes
|
-
|
46
|
M1a
|
pembro
|
iPD
|
2.7
|
yes
|
-
|
47
|
M1a
|
pembro
|
iPD
|
2.7
|
yes
|
-
|
48
|
M1c
|
nivo
|
iPD
|
3.6
|
yes
|
-
|
49
|
M1c
|
nivo
|
iPD
|
3.7
|
yes
|
-
|
50
|
M1c
|
nivo
|
iPD
|
4.1
|
yes
|
-
|
51
|
M1c
|
nivo
|
iPD
|
1.8
|
yes
|
-
|
52
|
M1c
|
pembro
|
iPD
|
2.0
|
yes
|
-
|
53
|
M1c
|
pembro
|
iPD
|
1.1
|
yes
|
-
|
54
|
M1c
|
pembro
|
iPD
|
4.0
|
yes
|
-
|
55
|
M1c
|
pembro
|
iPD
|
3.1
|
yes
|
-
|
56
|
M1c
|
pembro
|
iPD
|
1.4
|
yes
|
-
|
57
|
M1c
|
nivo
|
iPD
|
3.9
|
yes
|
-
|
aStaging before starting therapy: nivo, nivolumab; pembro, pembrolizumab. |
biBOR, best overall response according to the iRECIST criteria: iCR, complete response; iPR, partial response; iSD, stable disease; iPD, progressive disease. |
cData cut off on 11th November 2020. PFS, progression-free survival (months). |
dPatients who had developed (months) or not (-) leukoderma at the cut off data. |
We analyzed patient characteristics and tumor-associated factors at the beginning of the treatment with anti-PD-1 antibodies. As shown in Table 2, 37 patients were males (64.9%) and 20 females (35.1%). The mean age at treatment start was 67.3 years. Levels of circulating LDH were found high in 13 patients (26.5%) and were not assessed for 8 patients. The M staging of the extent of metastatization was stage IIIc-M1b for 21 patients (36.8%), and M1c-M1d for 36 patients (63.2%). In particular, stage IIIC for 2 patients, stage M1a for 12 patients (M1a(0) for 8, M1a(1) for 3, LDH not assessed for 1), stage M1b for 7 patients (M1b(0) for 5, M1b(1) for 2), stage M1c for 33 patients (M1c(0) for 20, M1c(1) for 8, LDH not assessed for 5), stage M1d for 3 patients (M1d(0) for 2, LDH not assessed for 1). BRAF gene mutation was detected in 15 patients (26.8%). Thirty-one patients (54.4%) were treated with pembrolizumab and 26 patients (45.6%) with nivolumab. First-line treatment was performed by 32 patients (56.1%), and second-to-fourth line by 25 patients (43.9%). Leukoderma was developed by 15 patients (26.3%). Of those, n = 6 iCR patients (40.0%), n = 7 iPR patients (46.7%), n = 2 iSD patients (13.3%), none iPD patient. Clinical characteristics of the response groups were similar for sex, age, metastasis stage, serum LDH amount, BRAF mutation, drug administrated, and line of treatment. Patients who developed leukoderma were associated with a good response to CPI therapy (p = 0.003). Out of 15 patients with leukoderma, 12 (80%) developed it during a first-line treatment.
Table 2
Patient characteristics, treatments, and leukoderma development.
Characteristics
|
All
(n = 57)
|
|
iPD
(n = 13)
|
|
iSD
(n = 16)
|
|
iPR
(n = 17)
|
|
iCR
(n = 11)
|
|
p valuea
|
Sex
|
|
|
|
|
|
|
|
|
|
|
|
Male
|
37 (64.9)
|
|
9 (69.2)
|
|
9 (56.3)
|
|
12 (70.6)
|
|
7 (63.6)
|
|
|
Female
|
20 (35.1)
|
|
4 (30.8)
|
|
7 (43.7)
|
|
5 (29.4)
|
|
4 (36.4)
|
|
0.866
|
Age, years
|
|
|
|
|
|
|
|
|
|
|
|
mean (SD)
|
67.3 (12.8)
|
|
62.0 (12.0)
|
|
67.1 (13.5)
|
|
73.2 (12.2)
|
|
64.7 (11.3)
|
|
|
median (IQR)
|
67 (59–78)
|
|
65 (54–68)
|
|
67 (60–79)
|
|
76 (67–81)
|
|
65 (56–72)
|
|
0.084b
|
≤ 65
|
22 (38.6)
|
|
6 (46.1)
|
|
7 (43.7)
|
|
4 (23.5)
|
|
5 (45.4)
|
|
|
> 65
|
35 (61.4)
|
|
7 (53.9)
|
|
9 (56.3)
|
|
13 (76.5)
|
|
6 (54.6)
|
|
0.509
|
Serum LDH
|
|
|
|
|
|
|
|
|
|
|
|
Normal
|
36 (73.5)
|
|
8 (80.0)
|
|
10 (71.4)
|
|
11 (68.8)
|
|
7 (77.8)
|
|
|
Elevated
|
13 (26.5)
|
|
2 (20.0)
|
|
4 (28.6)
|
|
5 (31.2)
|
|
2 (22.2)
|
|
0.970
|
Stage
|
|
|
|
|
|
|
|
|
|
|
|
IIIc-M1b
|
21 (36.8)
|
|
3 (23.1)
|
|
4 (25.0)
|
|
9 (52.9)
|
|
5 (45.4)
|
|
|
M1c-M1d
|
36 (63.2)
|
|
10 (76.9)
|
|
12 (75.0)
|
|
8 (47.1)
|
|
6 (54.6)
|
|
0.263
|
BRAF status
|
|
|
|
|
|
|
|
|
|
|
|
Mutation
|
15 (26.8)
|
|
5 (38.5)
|
|
4 (25.0)
|
|
3 (18.7)
|
|
3 (27.3)
|
|
|
No mutation
|
41 (73.2)
|
|
8 (61.5)
|
|
12 (75.0)
|
|
13 (81.3)
|
|
8 (72.7)
|
|
0.702
|
Drug
|
|
|
|
|
|
|
|
|
|
|
|
Pembrolizumab
|
31 (54.4)
|
|
5 (38.5)
|
|
7 (43.7)
|
|
8 (47.1)
|
|
6 (54.6)
|
|
|
Nivolumab
|
26 (45.6)
|
|
8 (61.5)
|
|
9 (56.3)
|
|
9 (52.9)
|
|
5 (45.4)
|
|
0.907
|
Line of treatment
|
|
|
|
|
|
|
|
|
|
|
|
1
|
32 (56.1)
|
|
5 (38.5)
|
|
7 (43.7)
|
|
11 (64.7)
|
|
9 (81.8)
|
|
|
2–4
|
25 (43.9)
|
|
8 (61.5)
|
|
9 (56.3)
|
|
6 (35.3)
|
|
2 (18.2)
|
|
0.117
|
Leukoderma
|
|
|
|
|
|
|
|
|
|
|
|
No
|
42 (73.7)
|
|
13 (100.0)
|
|
14 (87.5)
|
|
10 (58.8)
|
|
5 (45.4)
|
|
|
Yes
|
15 (26.3)
|
|
-
|
|
2 (12.5)
|
|
7 (41.2)
|
|
6 (54.6)
|
|
0.003
|
Totals may vary because missing values. Data are n (%) unless otherwise stated. |
Abbreviation: iPD, progressive disease; iSD, stable disease; iPR, partial response; iCR, complete response. SD, Standard Deviation; IQR, Interquartile Range; LDH, lactate dehydrogenase. |
p value was calculated using the Fisher's exact test (a) or Kruskal-Wallis test (b). |
In a sub-group of 44 patients for whom blood analysis data were available (patients no. 1–5, 7, 10, 14–16, 18–28, 30–39, 42–44, 46, 47, 49–55, 57 of Table 1), we observed higher neutrophil count at the beginning of the therapeutic plan in iPD patients in respect with iCR patients (p = 0.043; Table 3). Moreover, our data indicated higher baseline values of NLR and derived NLR (dNLR) in iPD patients rather than in iCR patients, suggesting that these higher basal levels could associate with a tendency towards progression disease after treatment with CPIs (Table 3).
Table 3. Analysis of the relationship between neutrophils and lymphocytes before the start of immunotherapy with CPIs.
Neutrophil count (µL); NLR, neutrophil-to-lymphocyte ratio (neutrophils / lymphocytes); dNLR, derived neutrophil-to-lymphocyte ratio [neutrophils / (leukocytes-neutrophils)]. Best overall response according to iRECIST criteria: iPD, progressive disease (n. 10 patients); iSD, stable disease (n. 13 patients); iPR, partial response (n. 14 patients); complete response, iCR (n. 7 patients). Data are expressed as mean value ± standard error of the mean (SEM). (*) p = 0.043 iCR versus iPD, assessed by Mann-Whitney U test.
CD25 and CXCL9 as potential early biomarkers of response to CPI therapy.
To search for potential biomarkers that could predict patient clinical response or that could represent early indicators of response to CPIs, selected biomarkers of the vitiligo active phase were analyzed in serum samples from patients before (T0) and after 1 month (T1) and 3 months (T2) of treatment. We analyzed circulating levels of CD25, CD27, CXCL9, CXCL10, CXCL11, S100B, and IL-17A. For the patients examined, the circulating amount of either S100B or IL-17A was below the ELISA detection level (data not shown).
Regarding CD25, iSD and iPR patients had significantly higher levels of CD25 at baseline than iPD patients (Fig. 1a, b). A tendency to have higher serum amounts of CD25 was observed also for iCR patients, but it did not reach statistical significance (Fig. 1a, b). Basal CD25 levels (T0) significantly increased at T1 and T2 for iPD patients, but values remained lower than the other patient groups at the same time point.
As far as CD27, iCR patients tended to have less circulating protein amount than other groups at all time-point analyzed. Statistical significance was observed only at T1 between iCR and iPD patients (Fig. 1c, d).
CXCL9 was undetectable at T0 in the sera of iPD compared to other patients who had baseline higher chemokine circulating levels (significance was achieved at T0 for iSD and iCR versus iPD patients; Fig. 2a, b). Moreover, the trend of lower circulating amount of CXCL9 in iPD patients compared to the other groups was maintained after 1 and 3 months of therapy (significance was achieved at T1 and T2 for iPR versus iPD patients). All three groups of patients showed CXCL9 serum basal levels (T0) that significantly increased after 1 month of therapy (T1). Interestingly, while for iPR and iCR patients CXCL9 levels continued to significantly growth at T2, for iPD and iSD patients the levels of CXCL9 decreased at T2.
CXCL10 amounts significantly increased at T1 compared to T0 for iSD, iPR and iCR patients, and a significative increment was also present for iCR patients at T2 (Fig. 2c, d). No significant differences in serum concentration among the three response groups of patients was observed. Similarly, CXCL11 concentrations among the patient groups was significantly different only at T1 for iCR versus iPD patients (Fig. 2e, f). CXCL11 levels increased between T0 and T1 for iPD and iPR patients, and between T0 and T2 for iSD patients (Fig. 2e, f). In iPD patients, the increment observed at T1 was followed by a reduction in CXCL11 levels at T2 (Fig. 2e, f).
Since both CD25 and CXCL9 levels could represent early biomarkers of response to CPI therapy, we also observed that no correlation was found between the two proteins levels (Spearman’s ρ = 0.03, p = 0.821).
miR-19b, miR-25 and miR-16 as promising indicators of response to CPIs therapy.
For a subset of 29 patients, for whom plasma samples were available, circulating miRNAs, previously identified as biomarkers of vitiligo, were analyzed. Patients no. 1, 3–5, 9–11, 13–18, 20, 27–30, 32, 40–47, 56, 57 of Table 1 were examined (iPD, 5 patients; iSD, 8 patients; and iPR/iCR, 16 patients; iPR and iCR were assembled due to the small number of patients analyzed). As shown in Fig. 3 and in Additional Table 1, although statistical significance was not achieved in the comparisons between patient groups or times of drugs administration, some trends towards significance were observed. Higher levels of miR-19b and miR-25 were present at all time points of treatment in the plasma of iSD and iPR/iCR patients in respect to iPD individuals. For miR-16, a higher amount was present in iSD patients at T1 ant T2 and in iPR/iCR patients at T2 compared to iPD patients at the corresponding time points. Furthermore, miR-19b, miR-25, and miR-16 showed an increment at T1 versus T0 in iSD and iPR/iCR patients, but not in iPD patients, whose values remained low. No major differences in the amount of miR-574 among the three patient groups and at any time of treatment were observed. Interestingly, regarding miR-19b, miR-25 and miR-16, most of the iPR/iCR outliers that fall outside the distribution of miRNA levels, were the same patients who developed leukoderma during CPI therapy (stars in Fig. 3).