High β-1,4-Galactosyltransferase-I expression in peripheral T-lymphocytes is associated with a low risk of relapse in germ-cell cancer patients receiving high-dose chemotherapy with autologous stem cell reinfusion

Figures & data

Figure 1. Gating strategy applied for cell sorting with further RT-PCR analysis of B4GALT1 and IL10 expression levels in compartments of MNC from leukapheresis products. (A) Forward scatter versus side scatter gating for exclusion of debris. (B) CD14 versus vital stain gating for the exclusion of dead cells (grey gate). CD14 versus CD3 plotting in order to identify CD3-positive T-cells (upper left black gate) and CD14-positive monocytes (lower right black gate). (C) B4GALT1 levels in peripheral blood T-lymphocytes are significantly higher in patients with no relapse compared to those patients with relapse (Mann-Whitney-test, p = 0.008). Neither B4GALT1 levels in (D) monocytes (p = 0.251), and (E) unsorted MNC samples (p = 0.650), nor (F) IL10 expression levels in T-cells (p = 0.178) are significantly different upon Mann-Whitney-testing. Plots show mean and standard deviation, respectively. FSC: forward scatter; MNC: mononuclear cells; ns: not significant; SSC: side scatter.

Table 1. Difference in relapse-free survival was calculated with the Cox regression model.

	Univariate Cox-Models		Multivariate Cox-Model for B4GALT1 T-cells		Multivariate Cox-Model for IL10 T-cells
Covariate	HR (95% CI)	p-value	HR (95% CI)	p-value	HR (95% CI)	p-value
B4GALT1 T-cells	0.62 (0.42,0.91)	0.02	0.66 (0.45,0.97)	0.03
log IL10 T-cells	0.62 (0.39,1)	0.05			0.3 (0.14,0.65)	0.002
NoTx				0.21		0.008
1			reference		reference
2			3.02 (0.3,30.87)		0.22 (0.02,2.35)
3			0.51 (0.17,1.47)		0.04 (0.01,0.3)
IPFSG score class				0.53		0.24
good			reference		reference
intermediate			2.54 (0.26,24.74)		5.9 (0.42,83.77)
poor			3.25 (0.4,26.15)		7.43 (0.71,77.44)
Further PCR measurements
log BaGALT1 Mono	1 (0.99,1.01)	0.77
log B4GALT1 MNC	1.02 (0.98,1.05)	0.37
log IL10 Mono	0.93 (0.76,1.16)	0.54
Cytometric measurements
log CD31 T-cells	1.27 (0.84,1.93)	0.26
log CD49 d T-cells	1 (0.99,1.01)	0.93
log CD184 T-cells	1^1^,^1	0.43
log Cd31 Mono	0.45 (0.25,0.79)	0.01
log Cd49 d Mono	0.97 (0.58,1.63)	0.92
log CD184 Mono	0.6 (0.23,1.61)	0.31

In the multivariate analysis B4GALT1 mRNA expression levels in T-lymphocytes had a significant influence on the RFS (p = 0.03). The IPFSG score and the number of transplantations did not influence survival in our patient cohort. For IL10 mRNA expression levels in T-lymphocytes the multivariate analysis also showed a significant impact on the RFS (p = 0.002).

CI: confidence interval of the hazard ratio; HR: hazard ratio; IPFSG: International Prognostic Factor Study Group; Tx: transplantation.

Table 2. Patients' characteristics.

		Relapse
	[ALL] N = 46	No N = 27	Yes N = 19	N
Patients
Follow-up time months (median) [min; max]	29 [1; 168]	90 [15; 168]	6 [1; 22]	44
IPFSG Score Class:				40
Good (0)	5 (12.5%)	4 (10.0%)	1 (2.5%)
Intermediate^1	10 (25.0%)	6 (15.0%)	4 (10.0%)
Poor^2-3 and 2nd relapse	25 (62.5%)	13 (32.5%)	12 (30.0%)
No. of HDCT/SCT				46
1	17 (37.0%)	7 (25.9%)	10 (52.6%)
2	2 (4.3%)	1 (3.7%)	1 (5.3%)
3	27 (58.7%)	19 (70.4%)	8 (42.1%)
Gene expression levels
B4GALT1_T-cells	2.57 ± 1.98	3.40 ± 2.22	1.60 ± 1.08	37
log.IL10_T-cells	0.58 ± 1.40	1.08 ± 1.36	0.04 ± 1.30	25
log.B4GALT1_Mono	0.61 ± 0.99	0.42 ± 0.95	0.86 ± 1.01	44
log.IL10_Mono	3.02 ± 1.41	3.02 ± 1.40	3.02 ± 1.47	38
log.B4GALT1_MNC	0.64 ± 0.72	0.68 ± 0.75	0.58 ± 0.70	32
Protein expression levels
log.CD31_T-cells	4.36 ± 0.89	4.33 ± 0.59	4.39 ± 1.21	46
log.CD49 d_T-cells	3.65 ± 0.50	3.68 ± 0.50	3.61 ± 0.51	46
log.CD184_T-cells	3.91 ± 1.02	3.81 ± 1.24	4.05 ± 0.58	46
log.CD31_Mono	6.39 ± 0.58	6.33 ± 0.44	6.46 ± 0.73	46
log.CD49 d_Mono	4.26 ± 0.43	4.20 ± 0.46	4.33 ± 0.38	46
log.CD184_Mono	5.68 ± 1.06	5.54 ± 1.33	5.89 ± 0.44	46

Number of samples analyzed, median follow-up period, IPFSG scores, number of HDCT cycles, gene and protein expression levels are depicted. The patients were divided into groups with and without relapse. Score calculation was not possible for 6/46 patients: For 4/46 patients no follow-up data were available, 2/46 patients underwent primary HDCT.

HDCT: high-dose chemotherapy; IPFSG: International Prognostic Factor Study Group; SCT: stem cell transplantation.

Figure 2. Relapse-free survival rates in 46 relapsed germ cell cancer patients with respect to the number of HDCT and ASCR, the international prognostic factor score, B4GALT1 and IL10 expression levels. (A) RFS is not significantly different in patients with one (black line), two (grey line) or three (dotted line) SCT cycles (log-rank test, p = 0.055). (B) Analysis of patients’ RFS according to the IPFSG categories. Patients with a good prognosis score (i.e. 0; black line) were compared to those with an intermediate (i.e. 1; grey line) and poor prognosis score (i.e. 2–3 and 2^nd relapse; dotted line). RFS is not significantly different between these groups (log-rank test, p = 0.420). (C) RFS is significantly higher in patients with high T-cell B4GALT1 mRNA expression levels (grey line) compared to those with low levels (black line) (log-rank test, p = 0.044). High and low refer to values above and below the median value, respectively. (D) RFS is significantly higher in patients with high IL10 mRNA expression levels (grey line) compared to those with low levels (black line) (log-rank test, p = 0.015). IPFSG: International Prognostic Factor Study Group; RFS: relapse-free survival; SCT: stem cell transplantation.

Lorch A, Neubauer A, Hackenthal M, Dieing A, Hartmann JT, Rick O, Bokemeyer C, Beyer J. High-dose chemotherapy (HDCT) as second-salvage treatment in patients with multiple relapsed or refractory germ-cell tumors. Ann Oncol. 2010;21(4):820–5. doi:10.1093/annonc/mdp366.

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