RBM17 expression is associated with the efficacy of immune checkpoint inhibitor monotherapy in non-small cell lung cancer with low PD-L1 expression

doi:10.21203/rs.3.rs-2603216/v1

Immune checkpoint inhibitors (ICIs) are currently a standard treatment tool for non-small cell lung cancer (NSCLC). RNA-binding motif protein 17 (RBM17), a splicing factor,is frequently overexpressed in NSCLC, but little is known about the role of RBM17 in the efficacy of ICIs for NSCLC. Thus, we investigated the correlations between RBM17 expression and ICI efficacy in NSCLC. We collected biopsy or surgical specimens from patients with advanced or recurrent NSCLC who received ICI monotherapy or chemo-immunotherapy in a first-line setting. RBM17 expression was examined by immunohistochemistry. We evaluated the correlation between the efficacy of ICI monotherapy or chemo-immunotherapy and RBM17 expression. Among the 218 cases, 115 (52.8%) cases were positive for RBM17 expression. RBM17 expression was not associated with the objective response rate (ORR) or progression-free survival (PFS) in either of the ICI monotherapy or chemo-immunotherapy groups. However, among those with a low PD-L1 expression level (PD-L1 < 50%; n = 86), RBM17 expression was significantly associated with a better ORR (p = 0.045) and a better PFS (p < 0.001) in the ICI monotherapy group, and was significantly associated with a poor ORR in the chemo-immunotherapy group (p = 0.041). In conclusion, RBM17 might be a useful predictive marker for a higher efficacy of ICI monotherapy in NSCLC patients with a low PD-L1 expression level.

Biological sciences/Cancer

Health sciences/Oncology

Splicing factor

RBM17/SPF45

non-small cell lung cancer

immune checkpoint inhibitor

chemotherapy

predictive marker

chemosensitivity

Lung cancer is the leading cause of cancer death ¹. Currently, immune checkpoint inhibitors (ICIs) are a standard treatment for advanced or recurrent non-small cell lung cancer (NSCLC) ². Recently, the expression of programmed death-ligand 1 (PD-L1) has been used as a predictor of the efficacy of ICIs in NSCLC ^3–8. NSCLC patients with a high expression level of PD-L1 have shown a survival benefit from ICI treatment. However, some NSCLC patients with a low PD-L1 expression level have also shown a survival benefit from ICI treatment. Therefore, a new predictor of ICI efficacy other than PD-L1 expression is needed to identify NSCLC patients with a low PD-L1 expression level who would benefit from ICI treatment. Although ICI monotherapy and chemotherapy with ICI (chemo-immunotherapy) are currently standard treatments for NSCLC, there is still a lack of predictive markers that can guide whether ICI monotherapy or chemo-immunotherapy is indicated.

RNA-binding motif protein 17 (RBM17)/splicing factor 45 (SPF45) is a spliceosome-associated protein ⁹. RBM17 is not well expressed in normal tissues, but is frequently overexpressed in various cancers, including lung cancer ¹⁰. Although it has been reported that increased RBM17 expression is associated with resistance to cytotoxic chemotherapy ¹¹, it remains unclear whether RBM17 expression is related to the efficacy of immunotherapy in NSCLC.

Vascular endothelial growth factor receptor 2 (VEGFR2) expression in cancer cells regulates tumor angiogenesis and the tumor microenvironment through the VEGF/VEGFR2 axis, and it has been reported to be associated with the efficacy of immunotherapy ^12,13,14,15. RBM17 is a splicing factor that is thought to be associated with the expression of various factors that may be related to chemotherapy resistance and the immune environment in cancer; however, it remains unclear whether RBM17 expression is associated with VEGFR2 expression.

In this study, we evaluated the significance of RBM17 expression in the efficacy of ICI monotherapy or chemo-immunotherapy in NSCLC patients, particularly in NSCLC patients with a low PD-L1 expression level. In addition, we evaluated the association between RBM17 and VEGFR2 expression.

Patients and data collection

We conducted a retrospective, multicenter study of patients with advanced or recurrent NSCLC treated with ICI monotherapy or chemo-immunotherapy in a first-line setting at Osaka Metropolitan University Hospital, Ishikiriseiki Hospital, and Bell Land General Hospital between December 2015 and July 2020. Patients with insufficient residual tissue for creating tissue microarrays or insufficient tumor cell numbers for evaluating immunostaining results were excluded. We reviewed the subjects’ medical records, including age, sex, smoking status, Eastern Cooperative Oncology Group Performance Status (ECOG PS), histological type, tumor node metastasis stage, presence of metastasis (brain, liver, bone, and/or malignant pleural effusion), type of chemotherapy (immunotherapy or chemo-immunotherapy), tumor proportion score (TPS) of PD-L1 expression, response to immunotherapy or chemo-immunotherapy, and progression-free survival (PFS). Tumor responses were evaluated using the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 ¹⁶. PFS was calculated from the date of the first chemotherapy until disease progression or death from any cause. The cut-off date was May 31, 2022. The TPS of PD-L1 expression, which was measured in formalin-fixed tumor samples using the commercially available PD-L1 immunohistochemistry 22C3 pharmDx assay (Dako, Santa Clala, Ca, USA) at each institution, was assessed from clinical pathology reports. The TPS for PD-L1 expression was considered to be high when more than 50% of the cells expressed PD-L1.

Immunohistochemical staining

Microarrays were made using the surgical or biopsy specimens, and immunostained for RBM17 and VEGFR2. For RBM17 staining, the slides were deparaffinized with xylene, and rehydrated in gradually decreasing concentrations of ethyl alcohol. The slides were then reacted with methanol containing 3% hydrogen peroxide at room temperature for 15 min to remove endogenous peroxidase activity. Next, the slides were heated in an autoclave at 105°C for 10 min in Target Retrieval Solution (Dako, Santa Clara, CA, USA). Then, the slides were blocked with 10% normal rabbit serum for 10 min at room temperature, and incubated with anti-RBM17 antibody (monoclonal, sc-515587, 1:250; Santa Cruz, Dallas, TX, USA) overnight at 4°C. Subsequently, the samples were incubated with biotinylated secondary antibody for 10 min at room temperature, followed by treatment with streptavidin-peroxidase reagent for 5 min at room temperature, incubation in diaminobenzidine for 5 min at room temperature, and counterstaining with 100% Mayer hematoxylin for 20 s at room temperature. For VEGFR2 staining, the slides were deparaffinized and rehydrated, then the sections were incubated with citric acid (pH 6.0) for 20 min at 100°C. Next, the sections were stained with anti-VEGFR2 antibody (polyclonal, ab2349, 1:200; Abcam, Cambridge, UK) overnight at 4°C. Following incubation with the secondary antibody for 30 min at room temperature, the specimens were stained using the VECTASTAIN Elite ABC Universal PLUS Kit (Vector Laboratories, Inc., Newark, CA, USA), followed by counterstaining with 100% Mayer hematoxylin for 30 s at room temperature.

Immunohistochemical determination

The immunoreactivity of RBM17 staining was evaluated according to the intensity of the staining in the deepest level of the tumor. RBM17 was mainly expressed in the cytosol of cancer cells. The intensity of RBM17 expression was evaluated on a scale of 0 to 3 (0, negative; 1+, weak in the cytosol; 2+, moderate in the cytosol; 3+, strong in the cytosol; Fig. 1). The proportion of stained cells was evaluated on a scale of 0 to 3 (0, 0% staining; 1+, 1–30% staining; 2+, 31–60% staining; and 3+, 61–100% staining). The final score for RBM17 expression was calculated by multiplying each value. A final score of 2 or more was considered to be a positive result. VEGFR2 expression was considered to be positive when more than 5% of the tumor cells were stained.

Statistical analysis

We analyzed the associations of RBM17 positivity with the objective response rate (ORR), PFS in all patients, and expression level of PD-L1 in high or low PD-L1 expression groups. In addition, we analyzed the association between RBM17 and VEGFR2 expression. The significance of the differences in the patient characteristics and the response rate between the RBM17-positive and RBM17-negative groups was evaluated using the χ² test. Survival curves were created with Kaplan-Meier survival analysis using the log-rank test to compare the cumulative survival durations in the patient groups. In addition, the Cox proportional hazards model was used to compute the univariate and multivariate hazards ratios for the study parameters. Analyses were performed using EZR on R Commander version 1.54 (Saitama Medical Center, Jichi Medical University, Saitama, Japan). In all tests, p < 0.05 was considered to indicate statistical significance.

Ethical considerations

The study protocol conformed to the ethical guidelines of the Declaration of Helsinki. This study was approved by the institutional review boards and ethics committees of all participating institutions (approval numbers: Ethical Committee of Osaka City University Graduate School of Medicine, 2020–177; Ethical Committee of Ishikiriseiki Hospital, 20–26; and Certified Clinical Research Review Board of Bell Land General Hospital, 2020–020). The patient’s informed consent was waived for the retrospective nature of study, and we used an opt-out method so that patients and families could refuse to participate in the study.

Patient characteristics

In total, 218 patients with NSCLC were enrolled in this study. Table 1 shows the correlations between the patient characteristics and RBM17 expression. RBM17 expression was positive in 115 (52.8%) of the 218 patients. The median age was 73.0 (range, 33 to 90) years, and 172 (78.9%) patients were male. One hundred ninety (87.2%) patients had a smoking history, and 184 (84.4%) patients had an ECOG PS of 0 or 1. Forty-nine (22.5%) of the 218 patients had post-operative (45 patients) or post-radiotherapy (4 patients) recurrence. There were significantly more patients with recurrence in the RBM17-positive group than in the RBM17-negative group (p < 0.001). There were 142 (65.1%) patients with non-squamous cell carcinoma, and 76 (34.9%) patients with squamous cell carcinoma. PD-L1 was highly expressed in 126 (57.8%) patients, lowly expressed in 86 (39.4%) patients, and the PD-L1 expression status was unknown in 6 patients. One hundred eighteen (54.1%) patients received ICI monotherapy, and 100 (45.9%) patients received chemo-immunotherapy in a first-line setting. PD-L1 was more highly expressed in the RBM17-negative group than in the RBM17-positive group (50.4% vs. 66.0%, p = 0.035), and ICI monotherapy was administered significantly more frequently in the RBM17-negative group than in the RBM17-positive group (p < 0.001). Forty-two (19.3%) patients had brain metastasis, 24 (11.0%) patients had liver metastasis, 59 (27.1%) patients had bone metastasis, and 49 (22.5%) patients had malignant pleural effusion.

Table 1

Patient characteristics.
	No. of all patients	RBM17 positive	RBM17 negative	p-value
	n = 218	n = 115 (52.8%)	n = 103 (47.2%)
PD-L1 TPS (22C3)
Low expression (< 50%)	86 (39.4%)	53 (46.1%)	33 (32.0%)	0.035
High expression (≥ 50%)	126 (57.8%)	58 (50.4%)	68 (66.0%)
Chemotherapy
ICI monotherapy	118 (54.1%)	49 (42.6%)	69 (67.0%)	< 0.001
Chemo-immunotherapy	100 (45.9%)	66 (57.4%)	34 (33.0%)
Recurrence or Advanced
Recurrence	49 (22.5%)	39 (33.9%)	10 (9.7%)	< 0.001
Advanced	169 (77.5%)	76 (66.1%)	93 (90.3%)
Histology
Non Sq	142 (65.1%)	71 (61.7%)	71 (68.9%)	0.319
Sq	76 (34.9%)	44 (38.3%)	32 (31.1%)
Age
< 65	41 (18.8%)	24 (20.9%)	17 (16.5%)	0.488
≥ 65	177 (81.2%)	91 (79.1%)	86 (83.5%)
Sex
Male	172 (78.9%)	92 (80.0%)	80 (77.7%)	0.740
Female	46 (21.1%)	23 (20.0%)	23 (22.3%)
Smoking history
Former/Current	190 (87.2%)	100 (87.0%)	90 (87.4%)	0.837
Never	26 (11.9%)	13 (11.3%)	13 (12.6%)
ECOG PS
0–1	184 (84.4%)	102 (88.7%)	82 (79.6%)	0.092
2–4	34 (15.6%)	13 (11.3%)	21 (20.4%)
Brain metastasis
Negative	176 (80.7%)	94 (81.7%)	82 (79.6%)	0.733
Positive	42 (19.3%)	21 (18.3%)	21 (20.4%)
Liver metastasis
Negative	194 (89.0%)	101 (87.8%)	93 (90.3%)	0.666
Positive	24 (11.0%)	14 (12.2%)	10 (9.7%)
Bone metastasis
Negative	159 (72.9%)	84 (73.0%)	75 (72.8%)	1.000
Positive	59 (27.1%)	31 (27.0%)	28 (27.2%)
Malignant pleural effusion
Negative	169 (77.5%)	89 (77.4%)	80 (77.7%)	1.000
Positive	49 (22.5%)	26 (22.6%)	23 (22.3%)
RBM17, RNA binding motif protein 17; PD-L1, programmed death-ligand 1; TPS, Tumor Proportion Score; ICI, immune checkpoint inhibitor; Sq, squamous; ECOG PS, Eastern Cooperative Oncology Group Performance Status.
*Of the 142 non-squamous cell carcinoma, 119 patients had adenocarcinoma, 8 had large cell carcinoma 3 had pleomorphic carcinoma, 1 had clear cell carcinoma, 1 had spindle cell carcinoma, 1 had sarcomatoid carcinoma and 9 had not otherwise specified (NOS).

Relationship between RBM17 expression and the objective response rate

Table 2 shows the ORRs. Among the total patients, no significant difference in the ORR was observed between the RBM17-positive and RBM17-negative groups (p = 0.685). Similarly, no significant difference in the ORR was observed between the RBM17-positive and RBM17-negative groups among the patients who received ICI monotherapy (p = 0.188) or those who received chemo-immunotherapy (p = 0.142). In contrast, among patients with a low expression level of PD-L1, those who received ICI monotherapy in the RBM17-positive group had a significantly better ORR (p = 0.045), whereas those who received chemo-immunotherapy in the RBM17-positive group had a significantly poorer ORR (p = 0.041).

Table 2

Objective response rate
	RBM17 positive	RBM17 negative	p-value
	ORR % (n/N)	ORR % (n/N)	p-value
All patients (n = 218)	48.7 (56/115)	45.6 (47/103)	0.685
PD-L1 high (n = 126)	58.6 (34/58)	44.1 (30/68)	0.112
PD-L1 low (n = 86)	39.6 (21/53)	48.5 (16/33)	0.503
ICI monotherapy (n = 118)	49.0 (24/49)	36.2 (25/69)	0.188
PD-L1 high (n = 95)	52.8 (19/36)	40.7 (24/59)	0.292
PD-L1 low (n = 21)	41.7 (5/12)	0 (0/9)	0.045
Chemo-immunotherapy (n = 100)	48.5 (32/66)	64.7 (22/34)	0.142
PD-L1 high (n = 31)	68.2 (15/22)	66.7 (6/9)	1.000
PD-L1 low (n = 65)	39.0 (16/41)	66.7 (16/24)	0.041
ORR, Objective response rate; RBM17, RNA binding motif protein 17; ICI, immune checkpoint inhibitor; PD-L1, programmed death-ligand 1.

Relationship between RBM17 expression and progression-free survival

Figure 2 shows Kaplan-Meier curves of PFS. Among all patients, no significant difference in the PFS was observed between the RBM17-positive and RBM17-negative groups (p = 0.407; Fig. 2a). There was also no difference in PFS between the RBM17-positive and RBM17-negative groups among the patients who received ICI monotherapy (p = 0.178; Fig. 2b) or chemo-immunotherapy (p = 0.592; Fig. 2c). In contrast, when the high and low PD-L1 expression groups were evaluated separately, RBM17 positivity was associated with a better PFS (p < 0.001) in those who received ICI monotherapy in the low PD-L1 expression group (Fig. 2b). In the univariate analysis of PFS of the patients treated with ICI monotherapy, ECOG PS ≥ 2, presence of brain metastasis, presence of liver metastasis, presence of bone metastasis, and presence of malignant pleural effusion were significantly associated with a worse PFS (p < 0.001, p = 0.013, p = 0.003, p = 0.014, and p < 0.001, respectively), and in the multivariate analysis of PFS in the patients treated with ICI monotherapy, ECOG PS ≥ 2, presence of brain metastasis, and presence of malignant pleural effusion were significantly associated with a worse PFS (p = 0.001, p < 0.001, and p < 0.001, respectively). In the univariate analysis of PFS in the patients treated with ICI monotherapy in the low PD-L1 expression group, RBM17 positivity, ECOG PS < 2, and the absence of brain metastasis were significantly associated with a better PFS (p = 0.002, p = 0.041, and p = 0.004, respectively), and in the multivariate analysis of PFS in the patients treated with ICI monotherapy in the low PD-L1 expression group, no significant difference was seen for any of the factors (Table 3). In the univariate and multivariate analyses of PFS in the patients treated with chemo-immunotherapy, age ≥ 65 years (p = 0.040 and p = 0.017, respectively) and ECOG PS ≥ 2 (p = 0.002 and p < 0.001, respectively) were significantly associated with a worse PFS. In the univariate analysis of PFS in the patients treated with chemo-immunotherapy in the low PD-L1 expression group, ECOG PS ≥ 2, presence of liver metastasis, and presence of bone metastasis were significantly associated with a worse PFS (p = 0.033, p = 0.029, and p = 0.034, respectively), and in the multivariate analysis of PFS in the patients treated with chemo-immunotherapy in the low PD-L1 expression group, no factor was significantly associated with PFS (Table 4).

Table 3

Univariate and multivariate analysis of progression-free survival for ICI monotherapy.
Factor		PFS of all patients (n = 118)
		Univariate analysis			Multivariate analysis
		HR	95%CI	p-value	HR	95%CI	p-value
RBM17	Negative vs Positive	0.73	0.47–1.15	0.181
PD-L1 TPS (22C3)	High vs Low	1.62	0.95–2.77	0.075
Age	< 65 vs ≥ 65	1.12	0.56–2.24	0.753
Sex	Female vs Male	0.97	0.57–1.65	0.900
Smoking history	Ever vs Never	1.65	0.87–3.13	0.124
ECOG PS	0–1 vs 2–4	4.03	2.46–6.62	< 0.001	2.63	1.48–4.68	0.001
Recurrence or Advanced	Recurrence vs Advanced	1.04	0.63–1.73	0.883
Histology	Non Sq vs Sq	1.21	0.78–1.88	0.394
Brain metastasis	Negative vs Positive	1.98	1.15–3.39	0.013	2.77	1.57–4.86	< 0.001
Liver metastasis	Negative vs Positive	2.96	1.44–6.09	0.003	1.79	0.81–3.94	0.150
Bone metastasis	Negative vs Positive	1.82	1.13–2.94	0.014	1.57	0.94–2.63	0.085
Malignant pleural effusion	Negative vs Positive	3.04	1.83–5.06	< 0.001	2.88	1.63–5.07	< 0.001
Factor		PFS of PD-L1 low expression (n = 21)
		Univariate analysis			Multivariate analysis
		HR	95%CI	p-value	HR	95%CI	p-value
RBM17	Negative vs Positive	0.11	0.03–0.46	0.002	0.21	0.04–1.01	0.052
Age	< 65 vs ≥ 65	0.51	0.06–4.07	0.523
Sex	Female vs Male	0.91	0.32–2.63	0.864
Smoking history	Ever vs Never	2.02	0.64–6.33	0.228
ECOG PS	0–1 vs 2–4	3.15	1.05–9.47	0.041	2.66	0.78–9.04	0.118
Recurrence or Advanced	Recurrence vs Advanced	1.93	0.74–5.07	0.179
Histology	Non Sq vs Sq	1.46	0.50–4.33	0.491
Brain metastasis	Negative vs Positive	7.95	1.90-33.24	0.004	3.67	0.71–18.82	0.120
Liver metastasis	Negative vs Positive	3.22	0.86–12.1	0.082
Bone metastasis	Negative vs Positive	1.01	0.23–4.48	0.993
Malignant pleural effusion	Negative vs Positive	3.13	0.93–10.53	0.065
PFS, progression-free survival; ICI, immune checkpoint inhibitor; HR, hazard ratio; CI, confidence interval; RBM17, RNA binding motif protein 17; PD-L1, programmed death-ligand 1; TPS, Tumor Proportion Score; ECOG PS, Eastern Cooperative Oncology Group Performance Status; Sq, Squamous.

Table 4

Univariate and multivariate analysis of progression-free survival for chemo-immunotherapy
Factor		PFS of all patients (n = 100)
		Univariate analysis			Multivariate analysis
		HR	95%CI	p-value	HR	95%CI	p-value
RBM17	Negative vs Positive	1.15	0.69–1.90	0.593
PD-L1 TPS (22C3)	High vs Low	0.78	0.48–1.28	0.329
Age	< 65 vs ≥ 65	1.83	1.03–3.27	0.040	2.06	1.14–3.72	0.017
Sex	Female vs Male	1.11	0.61–2.04	0.732
Smoking history	Ever vs Never	1.15	0.58–2.25	0.692
ECOG PS	0–1 vs 2–4	5.40	1.90-15.32	0.002	7.36	2.47–21.87	< 0.001
Recurrence or Advanced	Recurrence vs Advanced	0.74	0.43–1.28	0.285
Histology	Non Sq vs Sq	0.96	0.56–1.65	0.895
Brain metastasis	Negative vs Positive	1.24	0.70–2.20	0.461
Liver metastasis	Negative vs Positive	1.88	0.98–3.59	0.057
Bone metastasis	Negative vs Positive	1.32	0.80–2.20	0.279
Malignant pleural effusion	Negative vs Positive	1.30	0.76–2.24	0.337
Factor		PFS of PD-L1 low expression (n = 65)
		Univariate analysis			Multivariate analysis
		HR	95%CI	p-value	HR	95%CI	p-value
RBM17	Negative vs Positive	1.34	0.74–2.43	0.341
Age	< 65 vs ≥ 65	1.34	0.68–2.64	0.400
Sex	Female vs Male	0.59	0.30–1.18	0.135
Smoking history	Ever vs Never	1.64	0.76–3.56	0.207
ECOG PS	0–1 vs 2–4	10.42	1.21–90.17	0.033	8.13	0.89–73.36	0.064
Recurrence or Advanced	Recurrence vs Advanced	0.76	0.41–1.41	0.382
Histology	Non Sq vs Sq	0.95	0.50–1.81	0.887
Brain metastasis	Negative vs Positive	1.09	0.49–2.44	0.836
Liver metastasis	Negative vs Positive	2.25	1.09–4.68	0.029	1.83	0.82–4.10	0.140
Bone metastasis	Negative vs Positive	1.91	1.05–3.48	0.034	1.53	0.78–2.99	0.215
Malignant pleural effusion	Negative vs Positive	0.81	0.42–1.57	0.539
PFS, progression-free survival; ICI, immune checkpoint inhibitor; HR, hazard ratio; CI, confidence interval; RBM17, RNA binding motif protein 17; PD-L1, programmed death-ligand 1; TPS, Tumor Proportion Score; ECOG PS, Eastern Cooperative Oncology Group Performance Status; Sq, Squamous.

Relationship between RBM17 and VEGFR2 expression

Since VEGFR2 expression could not be evaluated in 24 of the 218 patients, we evaluated it in 194 patients. VEGFR2 expression was positive in 90 (46.4%) patients. The VEGFR2 expression rate was significantly lower in the RBM17-positive group than in the RBM17-negative group (p = 0.021; Table 5).

Table 5

Relationship between RBM17 and VEGFR2 expression
		No. of patients n = 194	RBM17
			Positive	Negative	p-value
			n = 108	n = 86
VEGFR2	Positive	90 (46.4%)	42 (38.9%)	48 (55.8%)	0.021
VEGFR2	Negative	104 (53.6%)	66 (61.1%)	38 (44.2%)
RBM17, RNA binding motif protein 17; VEGFR2, vascular endothelial growth factor receptor 2.

In this study, RBM17 expression was associated with a better ORR and PFS in the ICI monotherapy group among the NSCLC patients with a low PD-L1 expression level. Although ICI had a limited effect in NSCLC patients with a low PD-L1 expression level, it showed beneficial effects in RBM17-positive NSCLC patients with a low PD-L1 expression level. In contrast, RBM17 expression was associated with a poorer ORR to chemo-immunotherapy in patients with a low PD-L1 expression level. In RBM17-positive NSCLC patients with a low PD-L1 expression level, the ORR and PFS were better in the ICI monotherapy group than in the chemo-immunotherapy group (41.7% vs. 39.0%; median PFS, 9.2 months vs. 6.5 months, respectively), indicating that ICI monotherapy may be sufficient for these patients. These findings suggest that RBM17 expression might be a useful predictive marker for a higher efficacy of ICI monotherapy in NSCLC cases with a low PD-L1 expression level. This is the first study to evaluate the relationship between RBM17 expression and the clinical efficacy of immunotherapy and chemotherapy in NSCLC.

RBM17 expression is related to VEGFR2 expression, which has recently been reported to be negatively correlated with the efficacy of ICIs in lung cancer ¹⁷. VEGFR2 expression has been reported to be associated with a poor response rate to ICI monotherapy in NSCLC patients with a PD-L1 expression level > 1% ¹⁷. Our data showed that RBM17 expression was associated with a low expression level of VEGFR2. In NSCLC, VEGFR2 expression stimulates VEGF production in tumor cells ¹⁸. VEGF not only promotes tumor angiogenesis ¹⁹, but also directly and indirectly affects the tumor immune environment ^20–24, which has been implicated in ICI resistance ²⁵. These findings might explain the better efficacy of ICI monotherapy in RBM17-positive patients as a result of the low expression level of VEGFR2.

RBM17 expression was associated with a poor response to chemo-immunotherapy in patients with a low PD-L1 expression level, even though ICI monotherapy was associated with a good response in RBM17-positive patients. RBM17 expression has been reported to be associated with a poor response to cytotoxic chemotherapy ^10,11. Recently, RBM17 was found to be a splicing factor that regulates the expression of some cancer-related genes ^26,27, including MYC, which has been reported to be associated with resistance to cytotoxic chemotherapy ^28,29,30,31. These findings suggest that RBM17 positivity is associated with a poor response to cytotoxic chemotherapy in NSCLC, which may be responsible for the poor efficacy of chemo-immunotherapy.

One of the limitations of our study was the lack of cases that received only chemotherapy, which prevented us from accurately assessing the relationship between the effect of cytotoxic chemotherapy and RBM17 expression. Another limitation was that our study was a retrospective study. A prospective study might be necessary in the future.

In conclusion, RBM17 expression is associated with a higher efficacy of ICI monotherapy and a poor efficacy of chemo-immunotherapy in NSCLC patients with a low PD-L1 expression level. RBM17 expression may thus be a novel indicator for guiding the therapeutic strategy in NSCLC patients with a low PD-L1 expression level.

Acknowledgment

We thank Akiko Tsuda (Molecular Oncology and Therapeutics, Osaka Metropolitan University Graduate School of Medicine) for technical support.

Author contributions

H.N. and M.Y. designed and performed the experiments, co-wrote the manuscript, and contributed equally; N.Y., M.I., A.S., K.N., K.O., Y.T., T.W., and K.A. collected clinical data; Y.M. and S.M. produced microarrays from biopsy and surgical specimens, and performed immunostaining; K.S. and H.K. analyzed the data; K.Y., K.F., A.M., and T.K. provided suggestions, and co-designed the study. All authors read and approved the final manuscript.

Data availability

The datasets generated and/or analyzed during the current study are not publicly available due to the inclusion of personal data, but they are available from the corresponding author upon reasonable request.

Competing interests

The authors declare no competing interests.

Ethics declarations

The study protocol conformed to the ethical guidelines of the Declaration of Helsinki. This study was approved by the institutional review boards and ethics committees of all participating institutions (approval numbers: Ethical Committee of Osaka City University Graduate School of Medicine, 2020–177; Ethical Committee of Ishikiriseiki Hospital, 20–26; and Certified Clinical Research Review Board of Bell Land General Hospital, 2020–020).

Informed consent: The patient’s informed consent was waived for the retrospective nature of study, and we used an opt-out method so that patients and families could refuse to participate in the study (Ethical Committee of Osaka City University Graduate School of Medicine).

Funding sources

This study was partially funded by KAKENHI (Grant-in-Aid for Scientific Research) 21H03008.

Sung, H. et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin 71, 209-249 https://doi.org:10.3322/caac.21660 (2021).
Miller, M. & Hanna, N. Advances in systemic therapy for non-small cell lung cancer. Bmj 375, n2363 https://doi.org:10.1136/bmj.n2363 (2021).
Borghaei, H. et al. Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer. N Engl J Med 373, 1627-1639 https://doi.org:10.1056/NEJMoa1507643 (2015).
Garon, E. B. et al. Pembrolizumab for the treatment of non-small-cell lung cancer. N Engl J Med 372, 2018-2028 https://doi.org:10.1056/NEJMoa1501824 (2015).
Garon, E. B. et al. Five-Year Overall Survival for Patients With Advanced Non‒Small-Cell Lung Cancer Treated With Pembrolizumab: Results From the Phase I KEYNOTE-001 Study. J Clin Oncol 37, 2518-2527 https://doi.org:10.1200/jco.19.00934 (2019).
Herbst, R. S. et al. Pembrolizumab versus docetaxel for previously treated, PD-L1-positive, advanced non-small-cell lung cancer (KEYNOTE-010): a randomised controlled trial. Lancet 387, 1540-1550 https://doi.org:10.1016/s0140-6736(15)01281-7 (2016).
Reck, M. et al. Pembrolizumab versus Chemotherapy for PD-L1-Positive Non-Small-Cell Lung Cancer. N Engl J Med 375, 1823-1833 https://doi.org:10.1056/NEJMoa1606774 (2016).
Mok, T. S. K. et al. Pembrolizumab versus chemotherapy for previously untreated, PD-L1-expressing, locally advanced or metastatic non-small-cell lung cancer (KEYNOTE-042): a randomised, open-label, controlled, phase 3 trial. Lancet 393, 1819-1830 https://doi.org:10.1016/s0140-6736(18)32409-7 (2019).
Neubauer, G. et al. Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex. Nat Genet 20, 46-50 https://doi.org:10.1038/1700 (1998).
Sampath, J. et al. Human SPF45, a splicing factor, has limited expression in normal tissues, is overexpressed in many tumors, and can confer a multidrug-resistant phenotype to cells. Am J Pathol 163, 1781-1790 https://doi.org:10.1016/s0002-9440(10)63538-9 (2003).
Perry, W. L., 3rd et al. Human splicing factor SPF45 (RBM17) confers broad multidrug resistance to anticancer drugs when overexpressed--a phenotype partially reversed by selective estrogen receptor modulators. Cancer Res 65, 6593-6600 https://doi.org:10.1158/0008-5472.Can-03-3675 (2005).
Hato, T., Zhu, A. X. & Duda, D. G. Rationally combining anti-VEGF therapy with checkpoint inhibitors in hepatocellular carcinoma. Immunotherapy 8, 299-313 https://doi.org:10.2217/imt.15.126 (2016).
Zhu, P., Hu, C., Hui, K. & Jiang, X. The role and significance of VEGFR2(+) regulatory T cells in tumor immunity. Onco Targets Ther 10, 4315-4319 https://doi.org:10.2147/ott.S142085 (2017).
Refae, S. et al. Hyperprogression under Immune Checkpoint Inhibitor: a potential role for germinal immunogenetics. Sci Rep 10, 3565 https://doi.org:10.1038/s41598-020-60437-0 (2020).
Cho, R. et al. Silencing of VEGFR2 by RGD-Modified Lipid Nanoparticles Enhanced the Efficacy of Anti-PD-1 Antibody by Accelerating Vascular Normalization and Infiltration of T Cells in Tumors. Cancers (Basel) 12 https://doi.org:10.3390/cancers12123630 (2020).
Eisenhauer, E. A. et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer 45, 228-247 https://doi.org:10.1016/j.ejca.2008.10.026 (2009).
Nakahama, K. et al. Vascular endothelial growth factor receptor 2 expression and immunotherapy efficacy in non-small cell lung cancer. Cancer Sci 113, 3148-3160 https://doi.org:10.1111/cas.15464 (2022).
Chatterjee, S. et al. Tumor VEGF:VEGFR2 autocrine feed-forward loop triggers angiogenesis in lung cancer. J Clin Invest 123, 1732-1740 https://doi.org:10.1172/jci65385 (2013).
Carmeliet, P. VEGF as a key mediator of angiogenesis in cancer. Oncology 69 Suppl 3, 4-10 https://doi.org:10.1159/000088478 (2005).
Voron, T. et al. VEGF-A modulates expression of inhibitory checkpoints on CD8+ T cells in tumors. J Exp Med 212, 139-148 https://doi.org:10.1084/jem.20140559 (2015).
Zhao, Y. et al. VEGF/VEGFR-Targeted Therapy and Immunotherapy in Non-small Cell Lung Cancer: Targeting the Tumor Microenvironment. Int J Biol Sci 18, 3845-3858 https://doi.org:10.7150/ijbs.70958 (2022).
Liu, X. et al. Endostatin reverses immunosuppression of the tumor microenvironment in lung carcinoma. Oncol Lett 15, 1874-1880 https://doi.org:10.3892/ol.2017.7455 (2018).
Bourhis, M., Palle, J., Galy-Fauroux, I. & Terme, M. Direct and Indirect Modulation of T Cells by VEGF-A Counteracted by Anti-Angiogenic Treatment. Front Immunol 12, 616837 https://doi.org:10.3389/fimmu.2021.616837 (2021).
Sun, L. et al. Clinicopathologic and prognostic significance of regulatory T cells in patients with hepatocellular carcinoma: a meta-analysis. Oncotarget 8, 39658-39672 https://doi.org:10.18632/oncotarget.17340 (2017).
Liu, Y. et al. Combined application of bevacizumab and PD-1 blockade displays durable treatment effects by increasing the infiltration and cytotoxic function of CD8(+) T cells in lung cancer. Immunotherapy 14, 695-708 https://doi.org:10.2217/imt-2021-0196 (2022).
Fukumura, K., Venables, J. P. & Mayeda, A. SPF45/RBM17-dependent splicing and multidrug resistance to cancer chemotherapy. Mol Cell Oncol 8, 1996318 https://doi.org:10.1080/23723556.2021.1996318 (2021).
Fukumura, K. et al. SPF45/RBM17-dependent, but not U2AF-dependent, splicing in a distinct subset of human short introns. Nat Commun 12, 4910 https://doi.org:10.1038/s41467-021-24879-y (2021).
Mei, Y., Liu, Y. B., Hu, D. L. & Zhou, H. H. Effect of RIF1 on response of non-small-cell lung cancer patients to platinum-based chemotherapy by regulating MYC signaling pathway. Int J Biol Sci 14, 1859-1872 https://doi.org:10.7150/ijbs.27710 (2018).
Tao, L. et al. MiR-451a attenuates doxorubicin resistance in lung cancer via suppressing epithelialmesenchymal transition (EMT) through targeting c-Myc. Biomed Pharmacother 125, 109962 https://doi.org:10.1016/j.biopha.2020.109962 (2020).
Wang, H. et al. Exosomes Derived From Macrophages Enhance Aerobic Glycolysis and Chemoresistance in Lung Cancer by Stabilizing c-Myc via the Inhibition of NEDD4L. Front Cell Dev Biol 8, 620603 https://doi.org:10.3389/fcell.2020.620603 (2020).
Di, Y. et al. The c-MYC-WDR43 signalling axis promotes chemoresistance and tumour growth in colorectal cancer by inhibiting p53 activity. Drug Resist Updat 66, 100909 https://doi.org:10.1016/j.drup.2022.100909 (2023).

No competing interests reported.

RBM17 expression is associated with the efficacy of immune checkpoint inhibitor monotherapy in non-small cell lung cancer with low PD-L1 expression

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Patients and data collection

Immunohistochemical staining

Immunohistochemical determination

Statistical analysis

Ethical considerations

Results

Patient characteristics

Relationship between RBM17 expression and the objective response rate

Relationship between RBM17 expression and progression-free survival

Relationship between RBM17 and VEGFR2 expression

Discussion

Declarations

Refferences

Additional Declarations

Status:

Version 1