association of probiotic clostridium butyricum therapy ... · 14/07/2020 · 39 gut dysbiosis...

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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

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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

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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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Research. on November 14, 2020. © 2020 American Association for Cancercancerimmunolres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 14, 2020; DOI: 10.1158/2326-6066.CIR-20-0051

http://cancerimmunolres.aacrjournals.org/

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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: [email protected]) 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



mailto:[email protected]


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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

35




4

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




5

53

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




6

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




7

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




8

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




9

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




10

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




11

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




12

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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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. (％)




25

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%




26

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




27

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




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




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




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


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


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


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




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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