PVR and TIGIT expression relative to clinical factors
We investigated 125 bone marrow biopsy samples for expression of PVR in plasma cells and clinical parameters, with PVR expression examined via double-staining IHC. Figure 1 shows the representative images based on the H-score, and Figure 2a shows the distribution of PVR expression in patients with MM. We then examined the 14 available bone marrow samples to assess TIGIT expression in CD8+ T and NK cells using flow cytometric analysis (Fig. 2b), with the original flow cytometric data shown in Table S1.
We then compared PVR and TIGIT expression between categorical clinical parameters, including age, Eastern Cooperative Oncology Group (ECOG) performance status (< 2 vs. ≥ 2), serum free-light chain ratio [high (≤ 0.01 or ≥ 100) vs. low], cytogenetic abnormalities (high risk vs. others), Revised-International Staging System (R-ISS) stage (I vs. II vs. III), osteolytic bone lesion (presence vs. absence), and extramedullary plasmacytoma (EMP) (presence vs. absence). High-risk cytogenetics were defined as t(4;14), t(14;16), del(17/17p), TP53 deletion, or chromosome 1 abnormalities, including gain(1q) and del(1p). Patients with R-ISS stage III showed significantly higher PVR-expression levels than those with stage I (P = 0.004) and II (P = 0.001) (Fig. 2c). Additionally, patients with bone lesions (P = 0.034) (Fig. 2d) and EMP (P = 0.002) (Fig. 2e) showed significantly higher PVR expression than those without bone lesions and EMP. Analysis of continuous clinical parameters based on PVR- and TIGIT-expression status included age, serum M-protein levels, percentage of bone marrow plasma cells, β2-microglobulin levels, and lactate dehydrogenase (LDH) levels. Patients with positive PVR expression (H-score > 0) showed significantly higher bone marrow plasma cell (mean, 29.10% vs. 46.15%; P < 0.001) (Fig. 2f) and β2-microglobulin (mean, 5.064 mg/L vs. 7.642 mg/L; P = 0.005) (Fig. 2g) levels than patients with negative PVR expression (H-score = 0). Moreover, patients with high TIGIT expression in CD8+ T cells (cut-off point, median) showed a significantly higher percentage of bone marrow plasma cells (P = 0.037) (Fig. 2h) than patients with low TIGIT expression. No significant differences were found among other factors.
Analysis of PVR expression using different detection methods
For validation of the results of PVR-expression analysis by the H-score, PVR protein levels were analyzed using ELISA and PVR mRNA levels using qPCR in the available 22 bone marrow-aspiration specimens (Table 1). The PVR protein levels measured via ELISA showed a significant correlation with the H-score from IHC analysis (r = 0.513, P = 0.015) (Fig. 3a). Moreover, the PVR mRNA levels evaluated using qPCR showed a significant correlation with PVR protein levels determined by the H-score from IHC analysis (r = 0.458, P = 0.032) (Fig. 3b); however, we observed no significant correlations between PVR mRNA levels and the protein levels determined by ELISA (r = 0.293, P = 0.307) (Fig. 3c).
Table 1. Expression of PVR using different detection methods
Patient
|
IHC
(H-score)
|
ELISA
(ng/mL)
|
qPCR
(fold, PVR/GAPDH)
|
#1
#2
#3
#4
#5
#6
#7
#8
#9
#10
#11
#12
#13
#14
#15
#16
#17
#18
#19
#20
#21
#22
|
2
0
4
3
0
0
2
2
2
6
6
4
2
0
2
2
2
0
0
0
2
5
|
0.251
0.078*
0.311
0.181
0.173
0.078*
0.243
0.250
0.276
0.333
0.166
0.246
0.167
0.078*
0.218
0.188
0.199
0.182
0.237
0.180
0.166
0.242
|
0.520
0.348
2.815
1.643
2.362
5.528
0.143
0.751
1.170
6.619
1.248
0.443
0.475
0.227
0.109
0.373
0.729
0.223
0.199
0.285
0.061
6.176
|
* Values were below the detection limit range and replaced with half of the limit of detection values for statistical analysis.
ELISA, enzyme-linked immunosorbent assay; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IHC, immunohistochemistry; PVR, poliovirus receptor; qPCR, quantitative polymerase chain reaction.
Analysis of soluble PVR and TIGIT levels
Because PVR and TIGIT proteins can be found in soluble forms, we quantified soluble PVR protein levels in bone marrow plasma from patients with MM, MGUS, and patients without cancer. Although we found soluble PVR in both MGUS and patients with MM, we detected no significant differences in levels between the two patients subsets, although soluble PVR levels were significantly higher in patients with MM relative to those in patients without cancer (P = 0.042) (Fig. 4a). We identified soluble TIGIT in patients with MM but no significant correlations between soluble TIGIT and PVR protein levels (Fig. 4b).
We then examined the relationship between soluble PVR levels and surface PVR expression using various detection methods. Soluble PVR levels were significantly correlated with the surface PVR expression data obtained using ELISA (r = 0.525, P = 0.012) (Fig. 4c), IHC (r = 0.425, P = 0.049) (Fig. 4d) as well as mRNA levels obtained with qPCR (r = 0.565, P = 0.006) (Fig. 4e). We then examined the relationship between soluble TIGIT level and surface TIGIT-expression levels. Soluble TIGIT levels also positively correlated with surface TIGIT expression in CD8+ T cells (r = 0.757, P = 0.002) (Fig. 4f) and NK cells (r = 0.887, P < 0.001) (Fig. 4g). ELISA data for soluble PVR and TIGIT protein levels are shown in Table S2.
Patient characteristics according to PVR-expression status
We determined the optimal cut-point value for high or low PVR expression based on the H-score of 3. Based on an H-score ≥ 3, we classified 27 patients as having high PVR expression and 98 as having low PVR expression. Table 2 summarizes patient characteristics according to PVR-expression status. We found no significant difference in median age between the high PVR expression [66.0; interquartile range (IQR): 58.0–72.0 years] and low PVR expression (66.0; IQR, 58.0–72.3 years) groups (P = 0.865). In total, 16 patients (59.3%) with high PVR expression and 37 (37.8%) with low PVR expression showed high LDH levels (greater than the upper limit of normal [ULN]; P = 0.045). Thirteen (48.1%) and 28 (28.6%) patients in the high and low PVR groups, respectively, had high-risk cytogenetic abnormalities (P = 0.055), and patients in the high PVR group showed a higher percentage of bone marrow plasma cells (46.5; IQR, 25.9–77.0) as compared with those in the low PVR group (30.4; IQR, 5.39–10.5) (P = 0.018). Additionally, patients in high PVR group had high β2-microglobulin levels (6.63; IQR, 25.9–77.0 mg/L) as compared with patients in low PVR group (4.44; IQR, 2.66–7.87 mg/L) (P = 0.004), and hemoglobin levels were lower in the high PVR group (8.30; IQR, 7.40–9.30 g/dL) than those observed in the low PVR group (9.65; IQR, 8.70–11.4 g/dL) (P = 0.002). Furthermore, most patients in the high PVR group were in stage III according to the R-ISS classification (n = 15; 55.6 %), whereas most patients in the low PVR group were in stage II (n = 66; 67.3 %) (P < 0.001).
Table 2. Comparison of clinical factors between patients with low and high PVR expression
|
Total
(n = 125)
|
Low
(n = 98)
|
High
(n = 27)
|
P
|
Age, y
Sex, female
ECOG PS, ≥ 2
BM plasma cells, %
Serum M-protein, g/dL
Albumin, g/dL
< 3.5 g/dL
β2-microglobulin, mg/L
≥ 5.5 mg/L
LDH, IU/L
> ULN
Calcium, mg/dL
> 11 mg/dL
Creatinine, mg/dL
> 2 mg/dL
Hb, g/dL
< 10 g/dL
Cytogenetic abnormalities
High risk*
ISS
Stage I
Stage II
Stage III
R-ISS
Stage I
Stage II
Stage III
Treatment regimen
VTD
TD or RD
VMP
Others
Supportive only
Transplantation
Auto-SCT
Allo-SCT
None
|
66.0 (58.0–72.0)
56 (44.8)
8 (6.4)
32.6 (15.6–61.3)
2.20 (0.45–4.75)
3.30 (2.75–3.90)
71 (56.8)
4.87 (3.07–8.35)
53 (42.4)
390 (303–480)
53 (42.4)
9.10 (8.35–9.75)
12 (9.6)
1.07 (0.81–1.84)
24 (19.2)
9.50 (8.30–11.3)
75 (60.0)
41 (32.8)
26 (20.8)
46 (36.8)
53 (42.4)
14 (11.2)
78 (62.4)
33 (26.4)
26 (20.8)
24 (19.2)
57 (45.6)
7 (5.6)
11 (8.8)
30 (24.0)
0
95 (76.0)
|
66.0 (58.0–72.3)
46 (46.9)
6 (6.1)
30.4 (12.8–51.9)
2.12 (0.39–4.63)
3.40 (2.80–3.90)
54 (55.1)
4.44 (2.66–7.87)
35 (35.7)
363 (297–463)
37 (37.8)
9.20 (8.48–9.70)
4 (6.7)
1.03 (0.80–1.53)
10 (16.7)
9.65 (8.70–11.4)
28 (46.7)
28 (28.6)
25 (25.5)
38 (38.8)
35 (35.7)
14 (14.3)
66 (67.3)
18 (18.4)
22 (22.4)
17 (17.3)
45 (45.9)
4 (4.1)
10 (10.2)
22 (22.4)
0
76 (77.6)
|
66.0 (58.0–72.0)
10 (37.0)
2 (7.4)
46.5 (25.9–77.0)
3.30 (0.50–5.00)
2.90 (2.50–3.70)
17 (63.0)
6.63 (5.39–10.5)
18 (66.7)
434 (391–570)
16 (59.3)
8.50 (7.80–10.5)
8 (12.3)
1.20 (1.01–2.21)
14 (21.5)
8.30 (7.40–9.30)
47 (72.3)
13 (48.1)
1 (3.7)
8 (29.6)
18 (66.7)
0 (0.0)
12 (44.4)
15 (55.6)
4 (14.8)
7 (25.9)
12 (44.4)
3 (11.1)
1 (3.7)
8 (29.6)
0
19 (70.4)
|
0.865
0.360
0.682
0.018
0.350
0.088
0.465
0.004
0.004
0.070
0.045
0.886
0.285
0.123
0.490
0.002
0.003
0.055
0.006
<0.001
0.381
0.439
|
Data are shown as number (percentage) or median (interquartile range).
* High-risk cytogenetics were defined as t(4;14), t(14;16), del(17/17p), TP53 deletion, or chromosome 1 abnormalities, including gain(1q) and del(1p).
BM, bone marrow; ECOG, Eastern Cooperative Oncology Group; ISS, International Staging System; Hb, hemoglobin; LDH, lactate dehydrogenase; PS, performance status; PVR, poliovirus receptor; RD, lenalidomide/dexamethasone; R-ISS, revised international staging system; SCT, stem cell transplantation; TD, thalidomide/dexamethasone; VMP, bortezomib/melphalan/prednisone; VTD, bortezomib/thalidomide/ prednisone; ULN, upper limit of normal.
Survival analysis
For investigating whether PVR expression is associated with the survival of patients with MM, we analyzed the OS and PFS of patients in relation to PVR-expression status. For PFS analysis, patients receiving only supportive treatment (n = 11) were excluded in accordance with the definition. Among all patients, those with high expression of PVR showed significantly inferior OS compared to those with low expression (median, 24 vs. 68 months; P = 0.02) (Fig. 5a), and those with high PVR expression also had significant inferior PFS compared to patients with low PVR expression (median, 15 vs. 29 months; P = 0.04) (Fig. 5b). In the subgroup of patients who received immunomodulatory drug (IMiD)-based therapy, we observed no significant differences in OS (Fig. 5c) or PFS (Fig. 5d) between the two groups. Analysis of patients who received non-IMiD therapy revealed that those with high PVR expression had significantly shorter OS (median, 24 vs. 54 months; P = 0.02) (Fig. 5e) and PFS (median, 8 vs. 24 months; P = 0.03) (Fig. 5f) relative to those with low PVR expression. We then assessed whether TIGIT expression was associated with survival outcomes of patients with MM; results showed that the median OS of the high TIGIT expression group (cut-off, median) was 14 months, whereas low expression group did not reach the median level (P = 0.06) (Fig. 5g). Moreover, patients with high TIGIT expression showed a shorter PFS than those with low PVR expression (P = 0.03) (Fig. 5h).
Prognostic implication of PVR expression
For evaluating the prognostic effects of PVR expression, we used the Cox proportional hazard model (Table 3). In univariate analysis, inferior prognosis was significantly correlated to an ECOG performance status ≥ 2, LDH levels > ULN, high-risk cytogenetics, and high PVR expression. Additionally, poor prognosis was associated with older age with borderline significance (P = 0.052). Multivariate analysis, using the backward stepwise elimination method that included all of the variables used in univariate analysis, showed that poor OS was independently predicted by high PVR expression [hazard ratio (HR), 2.029; 95% confidence interval (CI): 1.003–4.103; P = 0.048], ECOG performance status ≥ 2 (HR, 3.768; 95% CI: 1.185–11.983; P = 0.025), LDH levels > ULN (HR, 2.069; 95% CI: 1.040–4.113; P = 0.038), and high-risk cytogenetics (HR, 2.373; 95% CI: 1.165–4.843; P = 0.017). Collectively, these findings suggested PVR expression as a possible independent poor prognostic factor for survival in patients with MM.
Table 3
Univariate and multivariate analyses for OS
Prognostic factors | Univariate | | Multivariate |
HR | 95% CI | P | HR | 95% CI | P |
Age, y ECOG performance status, ≥ 2 BM plasma cells, % Serum M-protein, mg/dL Albumin, g/dL β2-microglobulin, mg/L LDH, > ULN Cytogenetics, high-risk* PVR expression, high | 1.032 3.142 1.004 0.950 0.900 1.041 2.912 2.072 2.127 | 1.000, 1.065 1.097, 8.994 0.992, 1.016 0.835, 1.080 0.595, 1.359 0.987, 1.099 1.545, 5.488 1.086, 3.956 1.114, 4.065 | 0.052 0.033 0.525 0.434 0.615 0.139 0.001 0.027 0.022 | 1.032 3.768 0.887 2.069 2.373 2.029 | 0.997, 1.068 1.185, 11.983 0.765, 1.028 1.040, 4.113 1.165, 4.834 1.003, 4.103 | 0.075 0.025 0.111 0.038 0.017 0.048 |
* High-risk cytogenetics were defined as t(4;14), t(14;16), del(17/17p), TP53 deletion, or chromosome 1 abnormalities, including gain(1q) and del(1p). |
BM, bone marrow; CI, confidence interval; ECOG, Eastern Cooperative Oncology Group; HR, hazard ratio; LDH, lactate dehydrogenase; OS, overall survival; PVR, poliovirus receptor; ULN, upper limit of normal. |