Combined Analysis of Concordance between Liquid and ......2021/02/17 · Lilly, Merck Serono, Ni...

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Article type: Research article 1

Combined Analysis of Concordance between Liquid and Tumor Tissue Biopsies for RAS Mutations 2

in Colorectal Cancer with a Single Metastasis Site: The METABEAM Study 3

Yoshinori Kagawa1, Elena Elez Fernandez2, Jesus Garcia-Foncillas3, Hideaki Bando4, Hiroya Taniguchi5, 4

Ana Vivancos6, Kiwamu Akagi7, Ariadna Garcia2, Tadamichi Denda8, Javier Ros2, Tomohiro Nishina9, 5

Iosune Baraibar2, Yoshito Komatsu10, Davide Ciardiello2,11, Eiji Oki12, Toshihiro Kudo13, Takeshi Kato14, 6

Takeharu Yamanaka15, Josep Tabernero2, Takayuki Yoshino5 7

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1Department of Surgery, Kansai Rosai Hospital, Amagasaki, Hyogo, Japan 9

2Department of Medical Oncology, Vall d’Hebron University Hospital (HUVH), Vall d’Hebron Institute 10

of Oncology (VHIO); IOB-Quiron, UVic-UCC, Barcelona, Spain 11

3Oncology Department, Fundacion Jimenez Diaz University Hospital, Autonomous University; Madrid, 12

Spain 13

4Department of Clinical Oncology, Aichi Cancer Center Hospital, Nagoya, Japan 14

5Department of Gastrointestinal Oncology, National Cancer Center Hospital East, Kashiwa, Chiba, 15

Japan 16

6Cancer Genomics Group, Vall d’Hebron Institute of Oncology, Barcelona, Barcelona, Spain 17

7Division of Molecular Diagnosis and Cancer Prevention, Saitama Cancer Center, Ina, Saitama, Japan 18

8Division of Gastroenterology, Chiba Cancer Center, Chiba, Chiba, Japan 19

9Department of Gastrointestinal Medical Oncology, National Hospital Organization Shikoku Cancer 20

Center, Matsuyama, Ehime, Japan 21

10Department of Cancer Chemotherapy, Hokkaido University Hospital Cancer Center, Sapporo, 22

Hokkaido, Japan 23

11Department of Precision Medicine, University of Campania Luigi Vanvitelli, Naples, Italy 24

12Department of Surgery and Science, Graduate School of Medical Science, Kyushu University, 25

Fukuoka, Fukuoka, Japan 26

13Department of Frontier Science for Cancer and Chemotherapy, Graduate School of Medicine, 27

Osaka University, Suita, Osaka, Japan 28

14Department of Surgery, National Hospital Organization Osaka National Hospital, Osaka, Osaka, 29

Japan. 30

15Department of Biostatistics, Yokohama City University School of Medicine, Yokohama, Kanagawa, 31

Japan 32

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Running title: RAS Mutations in CRC Tested by Liquid and Tumor Biopsies 34

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Keywords 36

Research. on August 16, 2021. © 2021 American Association for Cancerclincancerres.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 February 18, 2021; DOI: 10.1158/1078-0432.CCR-20-3677

http://clincancerres.aacrjournals.org/

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Colorectal cancer, circulating tumor DNA, liquid biopsy, organ specificity, RAS mutations 37

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Funding 39

This work was funded and sponsored by Sysmex Corporation. 40

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Correspondence to: Dr. Takayuki Yoshino 42

Department of Gastrointestinal Oncology, National Cancer Center Hospital East 43

6-5-1 Kashiwanoha, Kashiwa-shi, Chiba 277-8577, Japan 44

Tel: +81-4-7133-1111 45

Fax: +81-4-7131-9960 46

E-mail: [email protected] 47

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Disclosure of Conflict of Interest 49

Y Kagawa reports personal fees received from Bayer, Chugai, Eli Lilly, Merck, Ono, and Taiho, grants 50 and personal fees from Takeda, and personal fees from Yakult Honsha outside the submitted work. E 51 Elez reports personal financial interests in the form of a scientific consultancy role for AbbVie, 52 Amgen, Array BioPharma, Boehringer Ingelheim, Bristol-Myers Squibb, GSK, Roche, MedImmune, 53 Merck Serono, MSD, Novartis, Pierre-Fabre, Sanofi-Aventis, and Servier. J Garcia-Foncillas reports 54 consultancies/advisories undertaken for Abbott, Amgen, Astellas, AstraZeneca, Bayer, Biocartis, 55 Boehringer Ingelheim, Bristol-Myers Squibb, Celgene, Eisai, Foundation Medicine, GSK, Hospira, 56 Janssen, Eli Lilly, Merck Serono, MSD, Novartis, Pfizer, Pharmamar, Roche, Sanofi, Servier, Sysmex, 57 and Tesaro. H Bando reports honoraria received from Chugai, Eli Lilly, Merck Serono, Taiho, Takeda, 58 Sanofi, and Yakult Honsha. H Taniguchi reports honoraria received from Bayer, Bristol-Myers Squibb, 59 Chugai, Daiichi Sankyo, Eli Lilly, MBL, Merck Serono, Mitsubishi Tanabe Pharma, MSD, Nippon 60 Kayaku, Novartis, Sanofi, Taiho, Takeda, and Yakult Honsha; research funding received from Array 61 BioPharma, Daiichi Sankyo, Dainippon Sumitomo Pharma, MSD, Novartis, Ono, Sysmex, and Takeda. 62 K Akagi reports honoraria received from MSD and Taiho. T Denda reports lecture fees received from 63 Sysmex; honoraria received from Sawai; research funding received from MSD and Ono. T Nishina 64 reports honoraria received from Bristol-Myers Squibb, Chugai, Eli Lilly, Merck Serono, Nippon Kayaku, 65 Taiho, and Takeda; research funding received from Chugai, Daiichi Sankyo, Dainippon Sumitomo 66 Pharma, Eisai, Eli Lilly, Merck Serono, MSD, Ono, and Taiho. Y Komatsu reports research funding 67 received from Astellas, A2 Healthcare, Bayer, Daiichi Sankyo, Dainippon Sumitomo Pharma, Eisai, Eli 68 Lilly, EP-CRSU, EPS, Incyte, IQVIA, Kyowa Kirin, Linical, Mediscience Planning, MSD, NanoCarrier, Ono, 69 Parexel, QuintilesIMS, Sanofi-Aventis, Syneos Health, Sysmex, Taiho, and Yakult Honsha; lecture fees 70 from Asahi Kasei Pharma, Bayer, Bristol-Myers Squibb, Chugai, CROee, Daiichi Sankyo, EA Pharma, Eli 71 Lilly, Kyowa Kirin, Mitsubishi Tanabe Pharma, Moroo, Nippon Kayaku, Nipro, Ono, Otsuka, Pfizer, 72 Sanofi-Aventis, Sanofi, Taiho, Takeda, and Yakult Honsha; honoraria received from Asahi Kasei, Bayer, 73 Chugai, Daiichi Sankyo, Eli Lilly, Kyowa Kirin, Medical Review, Merck, MSD, Nippon Kayaku, Nipro, 74 Novartis, Ono, Pfizer, Sanofi-Aventis, Sanofi, Sawai, Shiseido, Taiho, Takeda, and Yakult Honsha. E 75 Oki reports lecture fees received from Bayer, Chugai, Eli Lilly, Merck, Ono, Taiho, Takeda, and Yakult 76 Honsha. T Kato reports honoraria received from Bayer, Boehringer Ingelheim, Chugai, Ono, Taiho, 77 and Takeda; research funding from Chugai and Takeda. J Tabernero reports personal financial 78 interest in the form of scientific consultancy role for Array Biopharma, AstraZeneca, Bayer, BeiGene, 79




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Biocartis, Boehringer Ingelheim, Chugai, Foundation Medicine, Roche, Genentech, Genmab, HalioDX 80 SAS, Halozyme, Imugene, Inflection Biosciences, Ipsen, Kura, Eli Lilly, Menarini, Merck Serono, 81 Merrimack, Merus, Molecular Partners, MSD, Novartis, Peptomyc, Pfizer, Pharmacyclics, 82 ProteoDesign SL, Rafael Pharmaceuticals, Roche Diagnostics, Sanofi, SeaGen, Seattle Genetics, 83 Servier, Symphogen, Taiho, and VCN Biosciences. T Yoshino reports research funding received from 84 Chugai, Daiichi Sankyo, Dainippon Sumitomo Pharma, GSK, MSD, Novartis, Ono, Parexel, and Sanofi. 85 All remaining authors have declared no conflicts of interest. 86 87

Word count (main text): 2747 88

Number of Figures: 4 89

Number of Tables: 1 90




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Translational Relevance 91

The BEAMing technology with the OncoBEAMTM RAS CRC Kit can detect the plasma RAS status in 92

patients with metastatic colorectal cancer (mCRC). However, the concordance rate in circulating 93

tumor DNA (ctDNA) and tumor tissue RAS status varies by metastatic site. We conducted the first 94

international collaboration study on the concordance rate of liquid and tumor biopsy RAS status by 95

metastatic site. The utility of OncoBEAMTM may depend on tumor burden and/or the metastatic site. 96

There is no need to consider the cut-off for patients with only liver metastases. The baseline longest 97

diameter and number of lesions are associated with the concordance rate. Peritoneal metastases 98

alone with a lesion diameter <20 mm, lung metastases alone with a lesion diameter <20 mm, and 99

<10 lesions are discordant for ctDNA testing. Careful consideration should be given to patients with 100

mCRC with lung or peritoneal metastases only when using liquid biopsy. 101




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Abstract 102

Background: OncoBEAMTM is a circulating tumor DNA (ctDNA) test that uses the BEAMing digital 103

polymerase chain reaction technology. We clarified the association between the baseline tumor 104

burden and discordance in the RAS status by metastatic sites in patients with a single metastatic site. 105

Patients and methods: Data from previous Spanish and Japanese studies investigating the 106

concordance of the RAS status between OncoBEAMTM and tissue biopsy in 221 patients with 107

metastatic colorectal cancer (mCRC) were used. We collected data from patients with liver, 108

peritoneal, or lung metastases and evaluated the concordance rates according to the metastatic site 109

and the association between the concordance rate and tumor burden. 110

Results: Patients had metastases in the liver (n=151), peritoneum (n=25), or lung (n=45) with 111

concordance rates of 91% (95% confidence interval, 85–95%), 88% (68–97%), and 64% (49–78%), 112

respectively. Factors associated with concordance included the baseline longest diameter and lesion 113

number (P=0.004), and sample collection interval (P=0.036). Concordance rates ≥90% were observed 114

in the following groups: liver metastases alone, regardless of the baseline longest diameter and 115

lesion number; peritoneal metastases alone in patients with a baseline longest diameter ≥20 mm; 116

and lung metastases alone in patients with a baseline longest diameter ≥20 mm and/or number of 117

lesions ≥10. 118

Conclusion: Plasma ctDNA-based liquid biopsy in patients with mCRC may be useful depending on 119

the metastatic site. The maximum diameter and lesion number should be carefully considered when 120

determining patients' RAS status with only peritoneal or lung metastases. 121




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Introduction 122

Colorectal cancer (CRC) is the third most common cause of cancer death, and the fourth most 123

diagnosed cancer worldwide (1). Substantial progress has been made in treating metastatic CRC 124

(mCRC) through the development of anti-vascular endothelial growth factor and anti-epidermal 125

growth factor receptor (anti-EGFR) monoclonal antibodies (mAbs) (2, 3). However, several studies 126

have reported that patients with mCRC with tumors harboring RAS mutations are unlikely to benefit 127

from anti-EGFR antibodies (2, 3); therefore, such therapies are not recommended (4). 128

Almost all patients with mCRC have low but detectable levels of cell-free circulating tumor DNA 129

(ctDNA), in which tumor mutations can be characterized (5). Liquid biopsy is highly sensitive for 130

detecting mutations present at low frequencies. 131

The OncoBEAMTM RAS CRC Kit, which uses highly sensitive BEAMing digital polymerase chain 132

reaction (PCR) technology (6–8), is an in vitro diagnostic tool that has been CE-marked (conformity 133

with health, safety, and environmental protection standards for products sold within the European 134

Economic Area) and approved by the Pharmaceuticals and Medical Devices Agency in Japan for 135

detecting plasma RAS mutations in patients with mCRC. This kit has been widely used for patients 136

whose tissues are difficult to collect and who are planned to be treated with anti-EGFR antibody re-137

challenge. A comparison between the use of BEAMing technology to determine the RAS mutational 138

status in plasma ctDNA and the reference method of tumor tissue DNA in previous clinical trials 139

revealed concordance rates between 86% and 93% in all patients with mCRC regardless of the 140

metastatic sites (9–13). 141

Previous studies of small numbers of patients suggested that the concordance rates in ctDNA and 142

tissue RAS mutational status varied by metastatic site (9, 10). An adequate concordance between 143

the plasma and tissue RAS mutational status was observed in patients with mCRC with liver 144

metastases alone (9, 10). Although they were thought to be associated with a significant discordance 145

in patients with only lung metastases, the baseline longest diameter and number of lesions in the 146

lungs were significantly associated with concordance (9). Moreover, a range of concordance rates 147

depending on the metastatic site was suggested in previous studies (9, 10). 148

We integrated individual patient data from cases evaluated in previous studies (9–11) wherein the 149

RAS status in ctDNA using the OncoBEAMTM RAS CRC Kit was examined. Through a combined analysis, 150

we investigated the clinical factors associated with concordance between the plasma and tissue RAS 151

mutational status in patients with mCRC with liver, peritoneal, or lung metastases only to identify 152

individuals eligible for liquid biopsy analysis. 153

Materials and Methods 154

Study design and patients 155

Individual patient data from one Japanese (9) and two Spanish studies (10, 11) (n=659), which 156

examined the concordance rates between plasma and tissue RAS mutational status using the 157

OncoBEAMTM RAS CRC Kit and tissue reference method, respectively, were integrated. Patients who 158

met the following criteria were selected (n=261, CONSORT diagram, Supplementary Fig. S1): (1) 159

patients with pathologically confirmed metastatic colorectal adenocarcinoma; (2) patients who were 160

chemo-naïve or confirmed to have a progressive disease without having initiated subsequent 161

treatment; (3) patients with no prior treatment with anti-EGFR mAb or regorafenib; (4) patients with 162

only liver metastases, peritoneal metastases, or lung metastases; and (5) patients with no tumor 163




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removal before blood sample collection. The interval between plasma collection and CT was ≤ 3 164

months. 165

166

Tumor burden data for post-hoc analysis was obtained by investigating the baseline longest 167

diameter of the largest lesion and number of tumor lesions on computerized tomography (CT) within 168

three months before or after ctDNA sample collection. Scatterplots of concordant and discordant 169

cases based on the tumor burden data were used to confirm the significance of the cut-offs. This 170

combined study was approved by the ethics & review committees at each institution, and all 171

patients provided written informed consent. All procedures related to the study were performed in 172

accordance with the Helsinki Declaration and the Ethical Guidelines for Medical Health Research 173

Involving Human Subjects in Japan and Spain. 174

Study outcomes 175

This study's primary outcome was to investigate the clinical factors associated with concordance 176

between the plasma and tissue RAS mutational status. The secondary outcome was to determine 177

the optimal cut-offs for clinical factors to maintain a sufficient concordance greater than 90% at each 178

metastatic site. 179

Statistical analysis 180

Continuous variables were described using medians and ranges, whereas categorical variables were 181

presented as percentages. The concordance between the plasma and tissue RAS mutational status in 182

the overall cohort, RAS mutant concordance based on tissue RAS mutant cohort, and RAS wild 183

concordance based on tissue RAS wild cohort were calculated with 95% confidence intervals (CIs) 184

and tested using Fisher's exact test. 185

Factors associated with concordance were calculated using univariate and multivariate logistic 186

regression models. Multivariate analysis was performed for factors with P<0.1 based on univariate 187

analysis. Variables were entered directly if P<0.1 in the univariate analysis. Statistical analyses were 188

performed using R version 3.6.1. 189

Results 190

Of the 261 patients with mCRC who were initially enrolled (CONSORT diagram, Supplementary Fig. 191

S1), 40 were excluded because of missing measurements for the baseline longest diameter or 192

number of lesions (n=15) or due to an interval of more than three months between plasma 193

collection and CT (n=25). The full analysis set comprised 221 patients who had liver metastases 194

alone (n=151), peritoneal metastases alone (n=25), and lung metastases alone (n=45). Patient 195

characteristics categorized by metastatic sites are shown in Supplementary Table S1. The cut-offs for 196

the baseline longest diameter and number of lesions were provisionally set to 20 mm and 10 lesions, 197

respectively, based on a previous report (9). 198

The overall concordance rate in liver metastases alone was 91% (95% Cl 85–95), whereas those of 199

peritoneal and lung metastases alone were 88% (95% CI 68–97) and 64% (95% CI 49–78), 200

respectively. Similar trends were observed in the European and Asian subsets (Fig. 1). The 201

concordance rate of lung metastases alone was relatively low compared with that of liver or 202

peritoneal metastases alone. The mean of mutation allele fraction (MAF) of liver, peritoneum, and 203

lung was 6.8%, 7.2%, and 2.6%, respectively. The MAF is relatively low in lung metastases alone, as 204

shown in Supplementary Fig. S2. 205




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RAS mutations were detected in 39% and 47% of plasma and tissue samples, respectively. The 206

overall concordance between plasma- and tissue-based analyses was 85% (188/221; 95% CI 80–89), 207

with 76% (79/104; 95% CI 66–84) RAS mutant concordance based on tissue RAS mutant cohort and 208

93% (109/117; 95% CI 87–97) RAS wild concordance based on tissue RAS wild cohort. The RAS 209

mutant concordance was significantly lower than the RAS wild concordance(p<0.001). Almost no 210

difference in the concordance rate for each codon of RAS was observed (data not shown). 211

Univariate/multivariate analysis of the clinical findings between concordant and discordant cases 212

was conducted. The analysis for identifying factors associated with discordance is described in 213

Supplementary Table S2. On multivariate analysis, the most significant factor associated with 214

concordance was the baseline longest diameter of the lesion and the number of lesions (P=0.004) 215

(Table 1). Both factors are related to tumor burden and are evaluable factors in clinical practice; thus, 216

these values were adopted as cut-offs to select concordant cases in this study. 217

Furthermore, univariate and multivariate analyses of the clinical findings by tissue RAS mutational 218

status between concordant and discordant cases were conducted (Supplementary Tables S3 and S4). 219

In the tissue RAS mutant cohort, the analysis for identifying factors associated with discordance is 220

described in Supplementary Table S3. In multivariate analysis of the tissue RAS mutant cohort, the 221

significant factors associated with the RAS mutant concordance were collection interval (P=0.004), 222

baseline longest diameter, and number of lesions (P=0.005) as it is with the overall cohort. On the 223

other hand, there was no significant factor in univariate analysis of the tissue RAS wild cohort 224

(Supplementary Table S4). 225

The concordance rate in the liver metastases alone was over 90%, with any cut-off value used (Fig. 2). 226

Using the baseline longest diameter of lesion ≥20 mm as a cut-off, the concordance rate of 227

peritoneal metastases alone was 94%, providing a sufficient concordance and indication for 228

OncoBEAMTM testing, with a coverage of 68%, that is, this cut-off covered 68% of patients with liver 229

metastases. The reliability of using the cut-off of a baseline longest diameter ≥20 mm for patients 230

with mCRC with peritoneal metastases alone was reflected by a high RAS mutant concordance of 86% 231

(95% CI 42–99), along with 100% (95% CI 66–100) RAS wild concordance. Using 20 mm as the cut-off 232

value for the baseline longest diameter in peritoneal metastases alone, 67% (2/3) of discordant 233

cases were below the cut-off value, and 73% (16/22) of concordant cases were above the cut-off 234

(Supplementary Fig. S3). 235

The concordance rate for lung metastases alone using the cut-off of the baseline longest diameter 236

≥20 mm alone was 90%, but the coverage was poor (22%) (Fig. 2). Similarly, when the number of 237

lesions ≥10 alone was used as a cut-off, the concordance rate for lung metastases alone was 89%, 238

with poor coverage of 20% (Supplementary Fig. S4). However, the concordance rate of lung 239

metastases alone increased to 94%, with improved coverage of 38% when using the cut-off of a 240

baseline longest diameter ≥20 mm and ≥10 lesions (Fig. 3) with 88% RAS mutant concordance. Most 241

discordant cases (94%, 15/16) occurred in the baseline longest diameter of lung lesions<20 mm and 242

<10 lesions (Fig. 4, as shown in the black square). Concordance within and without the cut-offs were 243

46% (95% CI 28–66) and 94% (95% CI 69–100), respectively, showing a significant difference 244

(P<0.001). 245

Discussion 246

This is the first combined analysis of individual patient data from Europe and Asia on the 247

concordance of RAS mutational status by metastatic site between plasma ctDNA and tumor tissue 248

DNA testing (14). There were no differences between the European and Asian data. We found that 249




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the concordance rates differed by the metastatic site. Although there is currently no clear 250

explanation for this result, the effects may have been organ-specific. Therefore, it is necessary to 251

investigate the clinical factors associated with high concordance in each metastatic site so that 252

patients with these clinical factors can be selected for OncoBEAMTM-based detection of their RAS 253

mutational status. 254

In our multivariate statistical model, the most significant concordance factors were the baseline 255

longest diameter and the number of lesions (P=0.004), followed by the sample collection interval 256

(P=0.036). Tumor burden (that is, baseline longest diameter and number of lesions) impacts the 257

amount of ctDNA released into plasma (15). Thus, an increasing amount of ctDNA results in 258

increased concordance rates. The baseline longest diameter and number of lesions can be easily 259

measured and implemented in clinical practice. Additionally, the sample collection interval has been 260

associated with discordance, in which longer intervals between plasma and tissue collections may 261

contribute to clonal evolution (9, 16). 262

For patients with liver metastases alone, the concordance rate was high in Europe and Asia. As such, 263

the lesion diameter and number are not necessary to select patients for testing using the 264

OncoBEAMTM RAS CRC Kit. The median mutant allele fraction (MAF) was higher in liver metastases 265

than in peritoneal and lung metastases (9, 10, 12, 17). Bachet et al. reported that primary tumor 266

resection and the presence of liver metastases were significantly associated with the presence of 267

ctDNA, although liver metastases appeared to be the key factor (18). Elez et al. observed a high MAF 268

burden in liver metastases, which was not related to the volume and number of lesions in liver 269

metastases (17). ctDNA is thought to be easily released into the circulation regardless of the tumor 270

size or lesion number in the liver. 271

In previous studies, patients with peritoneal metastases alone showed a low MAF burden (12, 17). 272

MAF from patients with only peritoneal metastases was lower than in patients with peritoneal 273

metastases and at least one other metastatic site (12). The discordance was associated with the 274

presence of peritoneal metastases (18). However, our combined study revealed considerable high 275

concordance (88%) without setting a cut-off value. This may be because we enrolled cases in which 276

lesions could be detected by CT; a total of 68% (17/25) peritoneal metastases with the longest lesion 277

diameter ≥20 mm were included. Additionally, sufficient concordance and good coverage were 278

observed when we selected cases with the RAS wild concordance longest diameter ≥20 mm, with 279

high RAS mutant concordance and RAS wild concordance. In peritoneal metastases alone, the 280

maximum size may be related to the amount of ctDNA release. 281

In contrast, the concordance in patients with lung metastasis alone was extremely low in Europe and 282

Asia (9, 10), likely because of the low MAF (9, 10, 12, 17). Like previous studies, this study's results 283

also showed that the MAF with lung metastases tended to be lower than for other metastatic sites 284

(Supplementary Fig. S2). The release of ctDNA from lung metastases is thought to be extremely low; 285

thus, tumor burden plays a more important role in lung metastases. As such, the lesion size and 286

number must be determined to select appropriate patients with lung metastases alone for 287

OncoBEAMTM testing; that is, a closer evaluation of the tumor burden is required. These two factors 288

were also selected as cut-offs for patients with lung metastases alone in the Japanese study (9). 289

Under the cut-off value, a liquid biopsy may not detect RAS mutations in lung metastases. In such 290

cases, the tissue-based analysis would be the preferred choice. 291

In a post-hoc analysis, we reviewed the size and number of metastatic tumors of baseline CT in each 292

case. This is the first international collaborative study to evaluate the relationship between the 293

tumor burden and concordance by metastatic site between plasma- and tissue-based RAS 294




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mutational analysis. This study reveals the impact of the tumor burden and organ specificity on 295

concordance rates. 296

In our study, the presence of previous chemotherapy was significantly associated with a higher 297

discordance than its absence for the overall cohort (P=0.045) (Supplementary Table S2). In univariate 298

analysis of clinical findings between concordant and discordant in overall and tissue RAS mutant 299

cohorts, previous chemotherapy was indicated to be one of the significant factors, as described in 300

Table1 and Supplementary Table S3 (P=0.032 and P=0.019, respectively); however, it was not a 301

significant factor in multivariate analysis for overall and RAS mutant cohorts (P=0.500 and P=0.214, 302

respectively). This study included 50 patients who received chemotherapy between tissue and 303

plasma collection. The mutational state was changed in 12 cases, including 10 cases (1 case in the 304

liver, 3 in the peritoneum, and 6 in the lung), which changed from mutant to wild type, suggesting 305

below limit-of-detection level (called NeoRAS) (19). All of the 10 cases had tumors with sizes <20 mm, 306

and each case had <10 lesions, indicating a low tumor burden. The collection interval between tissue 307

and plasma sampling is also a significant factor in the multivariate analysis for the overall and tissue 308

RAS mutant cohorts shown in Table1 and the Supplementary Table S3 (p=0.036 and p=0.004, 309

respectively), indicating that the tumor volume reduction as a result of chemotherapy may affect the 310

discordance of the tissue RAS mutant. Only 2 cases (1 case each in the lung and the liver) changed 311

from wild type to mutant during chemotherapy. These might be due to clonal evolution or tumor 312

heterogeneity, even in cases that did not receive targeting agents such as an anti-EGFR antibody 313

and/or a regorafenib. 314

In conclusion, the OncoBEAMTM RAS CRC Kit's utility in patients with mCRC may depend on the 315

metastatic site. Although no further selection is required for patients with liver metastases alone, 316

careful attention should be given to patients with peritoneal metastases alone with lesion size <20 317

mm, as well as those with lung metastases alone with lesion size <20 mm and <10 metastatic lesions 318

when determining the RAS mutational status using the ctDNA-based assay. 319

Acknowledgments 320

We would like to express our special gratitude to all participating patients, their families, and all 321

participating investigators. The authors thank P4 Statistics Co. Ltd., Tokyo, for providing support 322

during the statistical analyses, and Celestina Chin and Yen May Ong of MIMS Pte Ltd., Singapore, for 323

providing medical writing support/editorial support. We also thank Editage (www.editage.com) for 324

English language editing, which was funded by Sysmex Corporation, Japan, in accordance with Good 325

Publication Practice (GPP3) guidelines (http://www.ismpp.org/gpp3). 326




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References 327

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RAS reversion from RAS mutated to RAS wild type. J Clin Oncol 2020;38(4_suppl):180. 377




13

Table 378

Table 1. Univariate and multivariate analyses of clinical findings between concordant and discordant 379

cases in the overall cohort. 380

Categorical variablesa

Univariate analysis Multivariate analysis

Odds ratio [95% CI] P value

b

Odds ratio [95% CI] P value

b

Age

1.0 [0.6, 1.7]

0.869 NA NA

Sex Male/female 1.4 [0.6, 3.3]

0.383 NA NA

Previous chemotherapy Yes/no 2.4 [1.1, 5.2]

0.032 0.7 [0.2, 2.0]

0.500

Location of primary tumorc Left/right 2.9

[1.0, 8.6] 0.059

2.0 [0.6, 6.8]

0.261

Metastatic site

Liver/other 0.3 [0.1, 0.6]

<0.001 0.9 [0.2, 3.9]

0.897

Peritoneum/other 0.8 [0.2, 2.7]

0.663 NA NA

Lung/other 5.2 [2.3, 11.4]

<0.001 1.8 [0.4, 8.1]

0.443

Collection interval (tissue vs. plasma)

2.2 [1.5, 3.3]

<0.001 1.9 [1.0, 3.3]

0.036

Source of tissue samples Metastasis/primary 1.9 [0.7, 5.7]

0.237 NA NA

Baseline longest diameter and number of lesions

<20 mm and <10, or ≥20 mm and ≥10

5.0 [2.3, 10.9]

<0.001 3.8 [1.5, 9.5]

0.004

Resection of primary tumor

Yes/no 3.8 [1.1, 13.1]

0.032 1.4 [0.3, 5.6]

0.633

CEA <5 ng/mL or ≥5 ng/mL 1.6 [0.7, 3.6]

0.296 NA NA

aThe second category for each variable was used as reference in the model to calculate the odds ratio.

381

bP values were derived using Fisher's exact test for categorical variables and the Kruskal–Wallis test 382

for continuous variables. 383 cLeft includes splenic flexure to rectum; right includes caecum to transverse colon. 384 CEA, carcinoembryonic antigen: NA, not available. 385 386




14

Figure Legends 387

388

Fig. 1. Combined analysis of concordance in the RAS mutational status between plasma- and tumor 389

tissue-based analyses for individual metastatic sites: (A) single-organ limited; (B) liver metastases 390

alone, (C) peritoneal metastases alone, and (D) lung metastases alone. CI, confidence interval. 391

392

Fig. 2. Concordance and coverage with an optimal cut-off of the baseline longest diameter of lesions 393

alone. Coverage refers to the percentage of patients having the specific metastatic lesions 394

(liver/peritoneal/lung) covered or included in the categories specified by lesion size, as defined by 395

computerized tomography. 396

397

Fig. 3. Concordance and coverage with optimal cut-offs of the baseline longest diameter of lesions 398

and number of lesions in lung metastases alone. The concordance rate of lung metastases increased 399

to 94%, with an improved coverage rate of 38%, when using the cut-off of baseline longest diameter 400

of ≥20 mm or ≥10 lesions. Coverage refers to the percentage of patients having the specific 401

metastatic lesions (liver/peritoneal/lung) covered or included in the categories specified by lesion 402

size and numbers, as defined by computerized tomography. 403

404

Fig. 4. Scatterplot of concordant and discordant cases based on baseline longest diameter and 405

number of lesions to confirm the optimal cut-offs in lung metastases alone. 406




Published OnlineFirst February 18, 2021.Clin Cancer Res Yoshinori Kagawa, Elena Elez Fernandez, Jesús García-Foncillas, et al. Cancer with a Single Metastasis Site: The METABEAM Study

Mutations in ColorectalRASTumor Tissue Biopsies for Combined Analysis of Concordance between Liquid and

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