Characterization of the cryoablation-induced immune response in kidney cancer patients

Figures & data

Table 1. Clinical information of 22 kidney cancer patients performed cryoablation

Case	Age at cryoablation (yr)	Gender	Affected side	Tumor size (mm)	Stage	Histology	pre-eGFR (mL/min/1.73m^2)	post-eGFR (mL/min/1.73m^2)	Recurrence	Status	Follow-up (mo)
CK1	80	M	Lt	17	pT1a	Clear Cell RCC	71.4	66.5	—	Alive	26.7
CK3	69	M	Lt	22	pT1a	Clear Cell RCC	38.3	35.4	—	Alive	24
CK4	80	M	Lt	20	pT1a	Clear Cell RCC	98.2	86.6	—	Alive	24
CK5	75	F	Lt	17	pT1a	Clear Cell RCC	67.7	63.1	—	Alive	21.5
CK6	81	M	Rt	28	pT1a	Clear Cell RCC	63.2	45	—	Alive	19.6
CK7	76	M	Lt	25	pT1a	Clear Cell RCC	44.2	30.7	—	Alive	15.7
CK8	78	M	Lt	21	pT1a	Clear Cell RCC	30.7	20.8	—	Alive	18.2
CK9	74	M	Rt	22	pT1a	Clear Cell RCC	46.2	40.6	—	Alive	13.8
CK11	60	M	Autologous	22	pT1a	Clear Cell RCC	50.9	39.6	—	Alive	18.4
CK12	67	M	Lt	30	pT1a	Clear Cell RCC	34.9	27.6	—	Alive	15.3
CK13	77	M	Lt	38	pT1a	Clear Cell RCC	49.8	44.9	—	Alive	12.5
CK14	71	M	Rt	29	pT1a	Clear Cell RCC	61.8	64.1	—	Alive	6.2
CK15	77	M	Lt	32	pT1a	Clear Cell RCC	73.2	63.9	—	Alive	15.5
CK17	73	F	Lt	14	pT1a	Chromophobe RCC	83.8	78.9	—	Alive	3.8
CK19	87	M	Rt	32	pT1a	Clear Cell RCC	65.2	44.8	—	Alive	12.4
CK22	82	F	Rt	35	pT1a	Clear Cell RCC	68.0	66.9	—	Alive	12.6
CK23	68	M	Lt	22	pT1a	Clear Cell RCC	46.1	48.7	—	Alive	9.4
CK28	66	M	Lt	20	pT1a	Clear Cell RCC	52.8	53.4	—	Alive	3.8
CK29	88	M	Rt	26	pT1a	Clear Cell RCC	28.7	28.5	—	Alive	3.7
CK30	62	F	Rt	23	pT1a	Clear Cell RCC	51.7	50.2	—	Alive	3.9
CK31	80	M	Lt	38	pT1a	Clear Cell RCC	31.5	24.5	—	Alive	8.6
CK32	52	M	Lt	20	pT1a	Clear Cell RCC	65.7	62.8	—	Alive	4.0

M: male, F: female, Lt: left kidney, Rt: right kidney, Autologous: A kidney after autologous transplantation, pT: pathologic primary tumor stage, RCC: renal cell carcinoma, eGFR: estimated glomerular filtration rate.

Figure 1. Clonal T cell expansion in post-cryoablation tissues. (A) The distribution of the abundant TCRB CDR3 clonotypes with the read frequency of 1% or higher is presented in pie charts (green color: pre-cryoablation; blue color: post- cryoablation). Gray color indicates portion of CDR3 clonotypes below the read frequency of 1%. (B) The total proportion of the abundant TCRB CDR3 clonotypes with the frequency of 1% or higher was significantly increased in post-cryoablation tissues (p = 0.024). (C) After cryoablation, tissue samples revealed significantly lower TCRB diversity index compared with that in pre-cryoablation (p = 0.021). The Mann—Whitney U test was performed for the comparison of total proportion of the most abundant CDR3 clonotypes (frequency of ≥ 1%) and the diversity index of TCRB between pre- and post-cryoablation.

Figure 2. Cryoablation stimulated expansion of unique T cell repertoires in tumor tissues. (A) The 20 most abundant TCRB CDR3 clonotypes newly appeared or drastically increased in the tissues after cryoablation (green bars: clonotypes that were rarely present in pre- cryoablation tissues; red bars: clonotypes that were completely absent in pre-cryoablation tissues; blue bars: 20 most abundant clonotypes observed in post-cryoablation tissues). Figures are from 15 representative cases that revealed a drastic increase of oligoclonal CDR3 sequences in post-cryoablation tissues in Fig 1A. (B) One TCRB CDR3 clonotype was shared in two patients with HLA-A24:02 genotype. Yellow-color bars indicate the identical CDR3 clonotype.

Figure 3. A set of T cell clones identified in both tumor tissues and systemic circulation. Heatmaps showed the distribution of TCRB CDR3 clonotypes in PBMC samples (the top 20 CDR3 clonotypes of post-cryoablation tissue were selected) of five patients who had dominant T cell clones (frequency of ≥ 10%) in post-cryoablation tissues. The asterisks indicate CDR3 clonotypes that were statistically significantly increased at 3 mo post-cryoablation compared with pre-cryoablation samples of PBMC. +, ++ and +++ indicate CDR3 clonotypes that had frequency of 10—20%, 20—30% and more than 30%, respectively.

Figure 4. Expression of immune-related genes in tumor tissues. (A) Expression level of each gene was calculated relative to that of GAPDH. The x-axis indicates the ratio of post-cryoablation /pre-cryoablation in the log₂ transformed values. *p < 0.01, **p < 0.05 indicates the significant difference by the Mann—Whitney U test. (B) and (C) Correlation analysis between HLA-A and CD8 expression or HLA-DRB1 and CD4 expression in pre-cryoablation tumor tissues. HLA-A and HLA-DRB1 represent one of the major HLA class I and class II molecule, respectively. (D) Correlation analysis between HLA-A expression in pre-cryoablation tumor tissues and the post/pre-cryoablation ratio of the sum of the abundant TCRB CDR3 clonotypes (frequency of ≥ 1%). (E) Comparison of basal HLA-A expression levels in the pre-cryoablation tumor tissues where total proportion of the abundant TCRB CDR3 clonotypes (frequency of ≥ 1%) were decreased (left) or increased (right) by cryoablation.

Figure 5. Drastic increase of CD8⁺ T cells and CD11c⁺ cells in post-cryoablation tissues. Representative immunohistochemistry results for CD8 (A) and CD11c (B) staining in tumor tissues of case CK3 before and after cryoablation. Magnification: 200 × for upper row, 400 × for lower row. Scale bars = 200 μm.