Post on 13-Aug-2020
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Article type: Cancer Immunology Miniatures 1
Association of probiotic Clostridium butyricum therapy with 2
survival and response to immune checkpoint blockade 3
In patients with lung cancer 4
5
Running title: Probiotic therapy impacts cancer immunotherapy 6
Yusuke Tomita1*†, Tokunori Ikeda2†, Shinya Sakata1, Koichi Saruwatari1, Ryo Sato1, 7
Shinji Iyama1, Takayuki Jodai1, Kimitaka Akaike1, Shiho Ishizuka1, Sho Saeki1, and 8
Takuro Sakagami1 9
10
1 Department of Respiratory Medicine, Graduate School of Medical Sciences, 11
Kumamoto University, Honjo 1-1-1, Chuo-ku, Kumamoto-shi, Kumamoto, 860–12
8556, Japan 13
2 Department of Clinical Investigation, Kumamoto University Hospital, Honjo 1-1-1, 14
Chuo-ku, Kumamoto-shi, Kumamoto, 860–8556, Japan 15
†Yusuke Tomita and Tokunori Ikeda contributed equally to this work. 16
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*Address for correspondence: Yusuke Tomita M.D., Ph.D., Department of Respiratory 18
Medicine, Graduate School of Medical Sciences, Kumamoto University, Honjo 1-1-1, 19
Chuo-ku, Kumamoto-shi, Kumamoto, 860-8556 Japan. 20
(*Email: y-tomita@kumadai.jp) 21
Phone: +81-96-373-5012. Fax: +81-96-373-5328. 22
Total Number of Figures and Tables: Two figures, two tables, one supplementary 23
figure, and three supplementary tables. 24
Text Word Count: 2886 words 25
Keywords: Antibiotics, Immune checkpoint inhibitor, Probiotics, Clostridium butyricum, 26
Lung cancer 27
Disclosure of Potential Conflict of interest: The authors declare no potential conflicts 28
of interest. 29
Financial support: This work was supported by JSPS KAKENHI Grant Number 30
JP18K15928. 31
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Abbreviations: CI, confidence interval; HR, hazard ratio; ICB, Immune checkpoint 32
blockade; NSCLC, Non-small cell lung cancer; Probiotic CBT, Probiotic Clostridium 33
butyricum therapy 34
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Cancer Immunology Miniature 36
37
ABSTRACT 38
Gut dysbiosis caused by antibiotics impairs response to immune checkpoint blockade 39
(ICB). Gut microbiota is becoming an attractive therapeutic target for cancer. The 40
Clostridium butyricum MIYAIRI 588 strain is a probiotic therapy used to improve 41
symptoms related to antibiotic-induced dysbiosis in Japan. We hypothesized that 42
probiotic Clostridium butyricum therapy (CBT) may affect the therapeutic efficacy of 43
ICBs. We retrospectively evaluated 118 advanced non-small cell lung cancer patients 44
treated with ICBs at Kumamoto University Hospital. Survival analysis comparing 45
patients given CBT before and/or after ICB was conducted using univariate analyses 46
and Cox proportional hazards regression models using propensity score. Propensity 47
score analyses confirmed that probiotic CBT treatment significatnly prolonged PFS and 48
and OS. Probiotic CBT significantly associated with longer PFS and OS even in 49
patients who received antibiotic therapy. This study suggests that probiotic CBT may 50
have a positive impact on therapeutic efficacy of ICB in cancer patients. 51
52
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Introduction 54
Immune checkpoint blockade (ICB) has led to a paradigm shift in cancer therapy, 55
yet clinical benefit from ICB is restricted to only a proportion of patients (1-4). The gut 56
microbiota play a role in the response and resistance to immunotherapy (2,4-6). Gut 57
dysbiosis caused by antibiotics impairs response to ICB, suggesting that an intact gut 58
microbiota is essential to improve the efficacy of ICB and an attractive therapeutic target 59
for cancer treatment (1,3,4,7). Manipulating commensal microbiota enhances the 60
efficacy of ICB in murine models (2,4,5). However, the clinical value of modulating gut 61
microbiota by administration of specific bacterial species in cancer patients receiving 62
ICB remains largely unknown (2). 63
Clostridium butyricum is a spore-forming bacillus named for its capacity to 64
produce high amounts of butyric acid and is found in soil. Clostridium butyricum 65
MIYAIRI 588 strain (MIYA-BM®
) is widely used as probiotic therapy to improve 66
symptoms related to dysbiosis such as constipation, non-antimicrobial-diarrhea and 67
antimicrobial-associated diarrhea in Japan and China (8-12). Clostridium butyricum 68
increases beneficial bacteria, especially lactobacilli and bifidobacteria (9,13-15). 69
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Bifidobacterium promotes antitumor immunity and facilitates efficacy of ICB (5,6). 70
Thus, we hypothesized that probiotic Clostridium butyricum therapy (CBT) may 71
enhance the therapeutic efficacy of ICB through the modulation of gut microbiota. 72
Here, we found that probiotic CBT compared with no probiotic CBT was 73
significantly improved PFS and OS in non-small cell lung cancer (NSCLC) patients 74
treated with ICB. PFS and OS were significantly improved in patients treated with 75
probiotic CBT than those not treated with probiotic CBT even in patients who received 76
antibiotic therapy prior to ICB. These findings suggest that manipulating commensal 77
microbiota by probiotic CBT has the potential to enhance the efficacy of ICB and 78
probiotic CBT may improve the diminished efficacy of ICB during antibiotic treatment. 79
80
81
Materials and Methods 82
Patients 83
We retrospectively evaluated 118 advanced NSCLC patients consecutively treated 84
with ICB therapy in routine clinical practice at Kumamoto University Hospital between 85
January 1, 2016 and May 31, 2019. The medical records of patients who had received 86
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nivolumab (3 mg/kg every 2 weeks), pembrolizumab (200 mg every 3 weeks), or 87
atezolizumab (1200 mg every 3 weeks) were reviewed. Treatments were provided until 88
disease progression, unacceptable toxicity, or consent withdrawal. All patients enrolled 89
in this study were Japanese. 90
To investigate whether probiotic CBT (Miyarisan Pharmaceutical 91
Co., Ltd., Tokyo, Japan) before and/or during ICI therapy affected progression-free 92
survival (PFS), overall survival (OS), and response to ICI therapy in patients with 93
NSCLC treated with ICB, those receiving probiotic CBT within 6 months before 94
beginning ICB and/or concurrently with ICB until cessation were compared with those 95
who did not. The history of probiotic CBT was extracted by using prescription database 96
and also manually checked from medical records. Attending physician and/or 97
pharmacists have confirmed that all patients have taken Clostridium butyricum 98
MIYAIRI 588 strain (MIYA-BM®
) as prescribed. Patient characteristics are 99
summarized in Table 1. Tumor responses of 108 patients were objectively assessed by 100
pulmonary physicians according to Response Evaluation Criteria in Solid Tumors, 101
version 1.1. Diarrhea and immune-related enterocolitis were graded using the National 102
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Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE), Version 103
5.0. This study was conducted in accordance with the amended Declaration of Helsinki. 104
The present study was performed after approval by the Kumamoto University 105
Institutional Review Board (IRB number, 1825, Approval Date, 24 October 2019), which 106
also waived the need to obtain informed consent because the data were analyzed 107
retrospectively and anonymously. 108
109
Statistical analysis 110
We presented patient characteristics as medians as appropriate. Patient 111
characteristics were compared using Fisher’s exact test for categorical data and 112
Wilcoxon rank sum test for continuous data. The Kaplan–Meier method was used to 113
obtain estimates of PFS and OS. We compared the curves with a two-tailed log-rank test. 114
PFS was measured from the date ICB started to the date of documented progression or 115
death. Patients who were alive and not known to have progressed were censored. OS 116
was measured from the date ICB started to the date of death or last follow-up. The data 117
cutoff date was October 1, 2019. Survival analysis was conducted using univariate 118
analyses and Cox proportional hazards regression models using propensity score to 119
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correct for potential confounding factors that may affect the treatment assignment. For 120
multivariable modeling, we used propensity score adjustment for sex, age, body weight, 121
Eastern Cooperative Oncology Group (ECOG) performance status, histology, smoking 122
history, programmed cell death-ligand 1 (PD-L1) status, initial stage, ICI therapy line, 123
ICI monotherapy/combination therapies, antibiotic use within the 60 days before the start 124
of ICI therapy, and immune-related enterocolitis. Each factor was categorized as shown 125
in Table 1 and Supplementary Table S1. The method of propensity score adjustment 126
preserved statistical power by reducing covariates into a single variable. To evaluate the 127
adjusted effect of probiotic CBT, propensity score was estimated through a binary 128
logistic regression providing the predicted probability with making probiotic CBT have a 129
function above background factors. Next, we performed survival analyses using Cox 130
proportional hazard models with inverse probability of treatment weighting (IPTW) 131
using the propensity score that balances the relevant characteristics between probiotic 132
CBT group vs no probiotic CBT group. To confirm the statistical robustness, we 133
performed another method using the propensity score as covariate in Cox proportional 134
hazard models. Statistical analyses were performed with R version 3.5.3 (The R 135
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Foundation for Statistical Computing, Vienna, Austria). Statistical significance was 136
indicated by P < 0.05. 137
138
139
Results 140
Patient Characteristics 141
99 men and 19 women (median [range] age, 68 [37-83] years) with advanced 142
NSCLC were included in this study, most patients had a performance status of 0 to 1 143
(93 [79%]), and patients had received anti–programmed cell death 1 (PD-1)/ PD-L1 144
ICB as first or second-line therapy (80 [68%]). Thirty-nine of 118 patients (33%) 145
received probiotic CBT within 6 months before beginning ICI and/or concurrently with 146
ICI (Table 1). Among the 39 patients, 9 (23%) received probiotic CBT within 6 months 147
before beginning ICB, 12 (31%) received concurrently with ICB, and 18 (46%) 148
received before and during ICB therapy (Table 1). Probiotic CBT was administered to 149
improve symptoms of constipation, non-antimicrobial-diarrhea or 150
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antimicrobial-associated diarrhea. The indications and characteristics of probiotic CBT 151
are shown in Supplementary Table S2. 152
153
Probiotic CBT enhanced the efficacy of ICB in NSCLC patients. 154
Univariate survival analyses confirmed that probiotic CBT compared with no 155
probiotic CBT associated with longer PFS (median, 250 versus 101 days, P=0.009) and 156
OS (median, not reached (NR) versus 361 days, P=0.005) (Figure 1). We applied Cox 157
proportional hazard models with IPTW and propensity score was estimated by a logistic 158
regression model. Sex, age, ECOG performance status, histology, smoking history, 159
PD-L1 status, initial stage, ICB therapy line, ICB monotherapy/combination therapies, 160
and antibiotic use, and immune-related enterocolitis were used as the background 161
factors (Table 1 and Supplementary Table S1). The propensity score analysis 162
confirmed that probiotic CBT compared with no probiotic CBT prolonged PFS [median, 163
250 versus 111 days; hazard ratio (HR) 0.37, P=0.001, 95% confidence interval (CI) 164
0.21–0.65] and OS (median, not reached versus 361 days; HR 0.2, P<0.001, 95% CI 165
0.07–0.44). To confirm statistical robustness, we performed another method using the 166
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propensity score as covariate in Cox proportional hazards regression models, which 167
confirmed that probiotic CBT was independently associated with longer PFS (HR 0.41, 168
P=0.002, 95% CI 0.23–0.71) and OS (HR 0.27, P=0.004, 95% CI 0.11–0.66). 169
Among the 118 patients who received ICB therapy, five patients (4.2%) 170
developed endoscopically confirmed immune-related enterocolitis and 34 patients 171
(28.8%) developed diarrhea during ICB therapy (Supplementary Table S1). 172
Immune-related adverse events associate with efficacy of ICB in NSCLC (16). Both 173
immune-related enterocolitis and diarrhea during ICB therapy associate with improved 174
survival outcomes (17). In our cohort, twelve patients (31%) among 39 patients who 175
received probiotic CBT developed diarrhea during ICB therapy (Supplementary Table 176
S1). Probiotic CBT was started to improve diarrhea during ICI therapy for six patients 177
and diarrhea due to endoscopically confirmed immune-related enterocolitis for one 178
patient (Supplementary Table S2). Thus, we considered not only immune-related 179
enterocolitis but also diarrhea during ICB therapy as confounding factors. As the result 180
of survival analyses using Cox proportional hazards regression models using propensity 181
score, we confirmed that probiotic CBT was independently associated with improved 182
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survival outcomes (PFS, HR 0.37, P=0.001, 95% CI 0.21–0.68; OS, HR 0.20, P<0.001, 183
95% CI 0.08–0.50) (Supplementary Table S3). 184
185
Probiotic CBT improved ICB efficacy in antibiotic treated patients 186
Antibiotic therapy before the start of ICB therapy reduces response to ICB (1,3,4,7). 187
However, no treatment which can improve the patients’ survival deteriorated by 188
antibiotics are available. We hypothesized that probiotic CBT would improve the 189
response to ICB even in patients who received antibiotics prior to ICB. In this study, 46 190
of 118 patients (39%) received antibiotic therapy within 60 days before the start of ICI 191
therapy. Twenty two of 39 patients (56%) received antibiotic therapy in probiotic CBT 192
group. Twenty four of 79 patients (30%) received antibiotic therapy in no probiotic 193
CBT group. The indications and characteristics of antibiotic therapy within 60 days 194
before the start of ICI therapy are shown in Table 2. β-lactam and quinolone–based 195
antibiotic therapy were the most common antibiotics used for both groups. 196
First, we evaluated the association of antibiotic therapy within 60 days before the 197
start of ICB therapy with survival in patients with NSCLC. In contrast to the results 198
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previously reported, antibiotic therapy prior to initiation of ICB was not significantly 199
associated with worse clinical outcomes with ICB therapy in total 118 patients’ cohort 200
(Supplementary Figure S1). Next, we evaluated the impact of probiotic CBT on 201
survival in those who received or those who did not receive antibiotics within 60 days 202
of ICI therapy. In patients with no antibiotic therapy, probiotic CBT did not improve 203
PFS (P =0.18), but probiotic CBT was associated with improved OS (median OS, NR 204
versus NR, P =0.039) (Figure 2A). Patients who received antibiotic therapy had 205
improved PFS and OS when given probiotic CBT compared to those not given probiotic 206
CBT (median PFS, 216 days versus 71 days, P =0.003; median OS, NR versus 182 days, 207
P=0.02) (Figure 2B). 208
209
Discussion 210
This study reports the potential positive impact of probiotic CBT on ICB efficacy in 211
lung cancer patients. Probiotic CBT associated with favorable clinical outcomes in 212
NSCLC patients treated with ICB. In addition, probiotic CBT significantly associated 213
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with improved therapeutic outcomes in patients who received antibiotic therapy before 214
the start of ICB therapy. 215
Antibiotic use is negatively associated with PFS and OS in cancer patients treated 216
with ICI (7). In current study 46 of 118 patients (39%) received antibiotic therapy . We 217
considered that comorbidities which required the use of antibiotics could be one of the 218
confounding factors, and antibiotic use was used as one of the background factor for 219
propensity score analysis, which confirmed that probiotic CBT compared with no 220
probiotic CBT was independently associated with improved survival outcomes. 221
Clostridium butyricum increases Bifidobacterium and reduces intestinal epithelial 222
damage (8,13-15). Intriguingly, Sivan et al., report that commensal Bifidobacterium 223
promotes dendritic cell function and T-cell-directed antitumor immunity leading to 224
improved ICB efficacy in a tumor-bearing murine model (5). These data suggest that 225
Clostridium butyricum may improve the efficacy of ICB through increasing commensal 226
Bifidobacterium. 227
Gut microbiota modulate immune responses (4,5,8,18). Clostridium butyricum 228
reduce systemic T helper 17 (Th17) cells in a murine model of inflammatory disease (8). 229
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Th17 cells promote invasion and migration of cancer cells, and cancer stem cell-like 230
properties via signal transducer and activator of transcription 3 signaling in NSCLC, 231
suggesting Clostridium butyricum may inhibit the aggressiveness through the reduction 232
of Th17 cells in NSCLC treated with ICB (19). 233
In preclinical murine models of probiotic CBT, modulation of intestinal flora 234
balance and gut metabolic alterations are observed 14-21 days after probiotic CBT 235
administration (13,14). However, the duration of the effects of probiotic CBT on 236
systemic immunity have not been elucidated in cancer patients who receive ICB therapy. 237
Thus, we used 6 months prior to start of ICB as a cutoff of defining use of probiotic CBT 238
before ICB initiation to maximally detect the impact of probiotic CBT on clinical 239
outcomes of lung cancer patients treated with ICB. 240
In subgroup analysis of patients with or without antibiotic therapy prior to initiation 241
of ICB, the impact of probiotic CBT on patients’ survival were more significant in 242
cancer patients who receiving antibiotic therapy than those who did not receiving 243
antibiotic therapy , suggesting that a clinical setting of probiotic CBT under the condition 244
of antibiotic use may be necessary to maximize the benefit from probiotic CBT and 245
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detect a beneficial effect of probiotic CBT. Probiotic CBT improve dysbiosis under the 246
condition of antibiotic therapies (8-11,13-15). Bifidobacterium, which promotes 247
antitumor immunity and facilitates efficacy of ICB, is significantly increased in 248
combination with probiotic CBT and clindamycin when compared with only probiotic 249
CBT (13,15), suggesting that a combination therapy of probiotic CBT with antibiotics to 250
deplete microbial communities could potentially improve responses to ICB therapy (20). 251
We speculate that a beneficial impact on gut microbiome by probiotic CBT might be 252
enhanced under the condition of antibiotic use and have led to significant survival 253
benefit coupled with the reduced patients’ survival due to antibiotic-related gut 254
dysbiosis in group not treated with probiotic CBT. However, our results from subgroup 255
analysis should be interpreted with caution due to small sample sizes. Profiling of the gut 256
microbiome in cancer patients who receiving probiotic CBT with/without antibiotics 257
during ICB therapy is essential to elucidate the mechanism of impact of probiotic CBT on 258
clinical outcomes. 259
Human gut mucosal probiotic colonization is significantly enhanced by antibiotics 260
(21), suggesting that gut mucosal colonization of probiotic Clostridium butyricum might 261
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be enhanced by antibiotics in cancer patients. Post-antibiotic gut mucosal microbiome 262
reconstitution is impaired by probiotics (21). Dysbiosis of the gut microbiota is 263
implicated in carcinogenesis and impaired response to cancer therapies, indicating an 264
unfavorable microbiota already exists in advanced cancer patients (20,22,23). These 265
results suggest that probiotic CBT in combination with antibiotics may shift the 266
pre-existing gut dysbiosis in cancer patients to a favorable microbiota and delay an 267
unfavorable gut microbiome reconstitution. This hypothesis should be validated by 268
correlative analyses of the gut microbiota in prospective studies. 269
In contrast to the results previously reported (1,3,4,7), antibiotic therapy prior to 270
initiation of ICB was not significantly associated with worse clinical outcomes with ICB 271
therapy. We consider that this discrepancy in the results of impact of antibiotic therapy 272
on clinical outcomes between our study and previous studies might occur because 22 of 273
46 patients (47%) had received probiotic CBT in antibiotic therapy group. In thus study, 274
we found that probiotic CBT was significantly associated with improved survival 275
outcomes in the cohort of NSCLC patients who received antibiotic therapy. These 276
results support our hypothesis that probiotic CBT may improve the decreased efficacy 277
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of ICB in patients treated with antibiotics. 278
Due to the lack of objective criteria to evaluate whether probiotic CBT was 279
effective or not effective, we could not assess the effectiveness of probiotic CBT in this 280
retrospective study. The association of the efficacy of probiotic CBT for symptoms 281
related to dysbiosis with survival benefits in cancer patients treated with ICB need to be 282
assessed in prospective studies. 283
Characteristics such as diet, lifestyle, or genetics can affect the composition of the gut 284
microbiota. The ethnic origin of individuals are an important factor to consider in 285
microbiome research (24). In our study, only Japanese patients have been analyzed, 286
which is a limitation. 287
Our study has another limitation. We speculate that Clostridium butyricum may 288
modulate gut microbiota and shift an unfavorable antimicrobial-associated or 289
non-antimicrobial-associated dysbiosis to a favorable microbiota, leading to increase 290
ICI clinical activity. However, we did not characterize the mechanism by which 291
probiotic CBT exerts a positive effect on clinical outcomes. Profiling of the gut 292
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microbiome during ICI therapy with or without probiotic CBT is essential to elucidate 293
the mechanism of impacts of probiotic CBT on clinical outcomes. 294
In conclusion, our findings support the hypothesis that probiotic CBT may have a 295
positive impact on the therapeutic efficacy of ICB, providing rationale for combining 296
probiotic therapy with immunotherapies. Despite being limited by the small sample size, 297
retrospective study, heterogeneity of study cohort, and the lack of correlative analyses on 298
patients’ gut microbiota and impacts of probiotic CBT on systemic immunity, our 299
findings provide the first evidence that manipulating commensal microbiota by probiotic 300
CBT may enhance the efficacy of ICB and also suggest that probiotic CBT may 301
improve the efficacy of ICB deteriorated by antibiotics in patients with lung cancer. 302
303
Acknowledgments 304
We are very grateful to our patients for participation in this study. 305
306
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398
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Table 1. Baseline characteristics of 118 advanced NSCLC patients receiving 399
anti-PD-1/PD-L1 blockade 400
401
Probiotic CBT
N=39
No probiotic CBT
N=79 P value
Median age (IQR) 68.0 (62.0-71.0) 67.0 (60.0-72.0) 0.83
Sex, No. (%)
Male 33 (85%) 66 (84%) 1.00
Female 6 (15%) 13 (16%)
ECOG performance status, No. (%)
0 10 (26%) 23 (29%)
0.07
1 15 (38%) 45 (57%)
2 11 (28%) 11 (14%)
3 2 (5%) 0 (0%)
4 1 (3%) 0 (0%)
Body weight, median (IQR) 61.9 (55.6-68.7) 59.4 (51.4-68.5) 0.24
Smoking history, No. (%)
Current 5 (13%) 9 (11%)
0.77 Former 30 (77%) 57 (72%)
Never 4 (10%) 13 (17%)
Stage at initial diagnosis, No. (%)
I-III 16 (41%) 32 (41%) 1.00
IV 23 (59%) 47 (59%)
Histology, No. (%)
Adenocarcinoma 25 (64%) 56 (71%) 0.53
Squamous/NOS 14 (36%) 23 (29%)
EGFR mutation status, No. (%)
wild-type 25 (64%) 64 (81%)
0.10 mutant 3 (8%) 3 (4%)
Unknown 11 (28%) 12 (15%)
PD-L1 status, No. (%)
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TPS ≥50% 16 (41%) 24 (30%)
0.019 TPS 1-49% 5 (13%) 14 (18%)
TPS <1% 14 (36%) 15 (19%)
Unknown/Undeterminable 4 (10.5%) 26 (33%)
ICB therapy line, No. (%)
1st line 14 (36%) 23 (29%)
0.33 2nd line 16 (41%) 27 (34%)
≥3rd line 9 (23%) 29 (37%)
Immune checkpoint blockade, No. (%)
Nivolumab 12 (31%) 39 (49%)
0.038 Pembrolizumab 20 (51%) 36 (46%)
Atezolizumab 7 (18%) 4 (5%)
ICB monotherapy/
combination therapy, No. (%)
Monotherapy 33 (85%) 74 (94%) 0.18
Combination therapy 6 (15%) 5 (6%)
Antibiotic use within 60 days before
the start of ICB therapy, No. (%) 22 (56%) 24 (30%) 0.009
Time point of administration of
probiotic Clostridium butyricum
MIYAIRI 588 strain, No. (%)
Before ICI initiation 9 (23%) -
During ICI therapy 12 (31%) -
Before and during ICI therapy 18 (46%) -
Response to ICB, No. (%) N=37 N=69
CR 3 (8%) 1 (1%)
0.08
PR 15 (40%) 17 (25%)
SD 11 (30%) 31 (45%)
PD 8 (22%) 20 (29%)
ORR 49% 26%
DCR 78% 71%
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402
Pembrolizumab/pemetrexed/platinum (N=6), pembrolizumab/nab-paclitaxel/carboplatin 403
(N=4), and atezolizumab/bevacizumab/carboplatin/paclitaxel (N=1) were used as 404
combination therapies with ICB and chemotherapies. Tumor response to therapy was 405
objectively assessed by pulmonary physicians according to Response Evaluation 406
Criteria in Solid Tumors, version 1.1. Abbreviations: IQR, interquartile; CBT, 407
Clostridium butyricum therapy; CR, complete response; ECOG, Eastern Cooperative 408
Oncology Group; EGFR, Epidermal Growth Factor Receptor; DCR, disease control rate; 409
ICI, immune checkpoint inhibitor; No., number; ORR, objective response rate; PD, 410
progression disease; PD-L1, Programmed cell death ligand1; PR, partial response; SD, 411
stable disease; TPS, tumor proportion score. 412
413
414
415
416
417
418
419
420
421
422
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Table 2. Characteristics of antibiotic therapy within 60 days before the start of 423
ICB therapy in 118 advanced NSCLC patients receiving anti-PD-1/PD-L1 424
blockade 425
426
Antibiotic class Probiotic CBT
(N=39)
No probiotic CBT
N=79
Antibiotic
therapy
N=22 (56%)
No antibiotic
therapy
N=17 (44%)
Antibiotic
therapy
N=24 (30%)
No antibiotic
therapy
N=55 (70%)
-lactams ± -lactamase inhibitors 6 - 6 -
Carbapenems 1 - - -
Macrolides 1 - 2 -
Quinolones 9 - 14 -
Quinolones + -lactams ±
-lactamase inhibitors 3 - - -
Quinolones + Glycopeptide +
-lactams +-lactamase inhibitors 1 - - -
Quinolones + Sulfonamides - - 1 -
Tetracyclines - - 1 -
Sulfonamides 1 - - -
Indication for antibiotic therapy N=22 N=24
Respiratory tract infection 12 9
Gastrointestinal infection 2 0
Skin/Soft tissue infection 2 1
Unclear source 6 14
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28
FIGURE LEGENDS 427
Figure 1. Association between probiotic Clostridium butyricum therapy (CBT) 428
and survival in NSCLC patients treated with ICB. A, PFS in NSCLC patients 429
treated with ICB, stratified by administration of probiotic CBT is shown. B, OS in 430
NSCLC patients treated with ICB, stratified by administration of probiotic CBT is 431
shown. 432
433
Figure 2. Association between probiotic Clostridium butyricum therapy (CBT) 434
and survival in patients who received antibiotic therapy within 60 days before the 435
start of ICB therapy. A, PFS and OS in NSCLC patients who did not receive 436
antibiotic therapy within 60 days before the start of ICB therapy, stratified by 437
administration of probiotic CBT is shown. B, PFS and OS in NSCLC patients who 438
received antibiotic therapy within 60 days before the start of ICB therapy, stratified by 439
administration of probiotic CBT is shown. 440
441
442
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Figure 1
Tomita Y. et al.
Pro
gre
ssio
n-F
ree S
urv
ival (%
)
Time since first dose of ICI (days) Number at risk
Time since first dose of ICI (days)
Overa
ll S
urv
ival (%
)
100
80
60
40
20
0
Probiotic CBT
No probiotic CBT
Probiotic CBT
No probiotic CBT
100
80
60
40
20
0
0 50 100 150 200 250 300 350
0 50 100 150 200 250 300 350
39 35 28 22 19 14 11 10 Probiotic CBT
No probiotic CBT 79 58 41 33 28 19 16 10
Number at risk
39 37 35 30 27 21 20 19 Probiotic CBT
No probiotic CBT 79 65 59 54 45 38 33 27
Log-rank P=0.005
Log-rank P=0.009
A
B
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Time since first dose of ICI (days)Number at risk
0
0 50 100 150 200 250 300 350
Pro
gre
ssio
n-F
ree S
urv
ival (%
) 100
80
60
40
20
24 14 8 6 6 3 2 2No Probiotic CBT
Probiotic CBT 22 20 14 11 9 6 6 5
Figure 2
Tomita Y. et al.
No antibiotic therapy group
Antibiotic therapy group
No probiotic CBT
Probiotic CBT
Log-rank P=0.003
Overa
ll S
urv
ival (%
)
100
80
60
40
20
Time since first dose of ICI (days)Number at risk
0
0 50 100 150 200 250 300 350
24 18 15 13 12 11 8 5No Probiotic CBT
Probiotic CBT 22 21 20 15 13 10 10 10
No probiotic CBT
Probiotic CBT
Log-rank P=0.020
Overa
ll S
urv
ival (%
)
100
80
60
40
20
Time since first dose of ICI (days)Number at risk
0
0 50 100 150 200 250 300 350
No probiotic CBT
Probiotic CBT
Log-rank P=0.039
55 47 44 41 33 27 25 22No Probiotic CBT
Probiotic CBT 17 16 15 15 14 11 10 9
Pro
gre
ssio
n-F
ree S
urv
ival (%
) 100
80
60
40
20
Time since first dose of ICI (days)Number at risk
0
0 50 100 150 200 250 300 350
No probiotic CBT
Probiotic CBT
Log-rank P=0.18
55 44 33 27 22 16 14 8No Probiotic CBT
Probiotic CBT 17 15 14 11 10 8 5 5
A
B
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Published OnlineFirst July 14, 2020.Cancer Immunol Res Yusuke Tomita, Tokunori Ikeda, Shinya Sakata, et al. patients with lung cancersurvival and response to immune checkpoint blockade in Association of probiotic Clostridium butyricum therapy with
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