Research Article

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Concordance of acquired mutations between
metastatic lesions and liquid biopsy inmetastatic colorectal cancer
Fumitaka Taniguchi1 , Akihiro Nyuya2 , Toshiaki Toshima1 , Kazuya Yasui1 , Yoshiko Mori3 ,Makoto Okawaki2 , Hiroyuki Kishimoto1 , Yuzo Umeda1 , Toshiyoshi Fujiwara1 , Hiroaki
Tanioka2 , Yoshiyuki Yamaguchi2 , Ajay Goel4& Takeshi Nagasaka*,1,2

1Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry & Pharmaceutical
Sciences, Okayama, 700-8558, Japan2
Department of Clinical Oncology, Kawasaki Medical School, Kurashiki, 701-0192, Japan3
Department of Clinical Genetics & Digestive Tract & General Surgery, Saitama Medical Center, Saitama Medical University,
Kawagoe, Saitama, 350-8550, Japan
4
Department of Molecular Diagnostics & Experimental Therapeutics, Beckman Research Institute of City of Hope Comprehensive
Cancer Center, CA 91016, USA*Author for correspondence: Tel.: +81 86 462 1111; takeshin@med.kawasaki-m.ac.jp

Aim: To evaluate whether PCR-reverse sequence-specific oligonucleotide can examine the concordance
between liquid biopsy and metastatic lesions with acquired resistance. Materials & methods: We examinedacquired mutations in chemoresistant lesions and blood obtained from four patients with RAS wildtype metastatic colorectal cancer who underwent treatment with anti-epidermal growth factor receptor
antibodies. Results: In one patient, metastatic lesions harbored diverse acquired mutations in KRAS in
all seven metastases; the two acquired mutations were detectable in blood collected after the patientacquired resistance. None of the other patients exhibited liquid biopsy mutations, except one, with a BRAF
mutation confirmed in primary tumor and peritoneal dissemination. Conclusion: Liquid biopsy based on
PCR-reverse sequence-specific oligonucleotide is a successful procedure for capturing acquired mutations
with precise information on the RAS mutational spectrum.
First draft submitted: 4 May 2021; Accepted for publication: 7 October 2021; Published online:
8 November 2021
Keywords: Acquired mutations • BRAF • colorectal cancer • liquid biopsy • PCR-rSSO • RAS

Acquired resistance and primary resistance play major roles in anticancer treatment [1–3]. The epidermal growth
factor receptor (EGFR)-targeted antibodies cetuximab and panitumumab can treat metastatic colorectal cancer(mCRC) negative for mutations in KRAS and NRAS exons 2–4 [4–6]. Although patients with metastatic CRC
without activated RAS mutations show a clinical response to anti-EGFR antibodies, acquired resistance may
develop. Indeed, several studies have identified acquired genetic alterations, including KRAS, HER2 or MET
amplification, and KRAS, NRAS, BRAF or EGFR mutations [1,3,7–12].
Recent studies have suggested characterizing genomic alterations in solid tumors by analyzing circulating tumor
DNA (ctDNA) released from cancer cells into the plasma [13]. Currently, OncoBEAM-based liquid biopsy is astandard procedure for detecting RAS mutations in plasma [14,15]. Indeed, a previous study using OncoBEAM
technology demonstrated that the mutant allele frequency (MAF) is as low as 0.1% of the acquired RAS mutant
alleles [1,16]. However, approximately 10–18% of patients harbored RAS mutations in tissue that could not be
detected in plasma [14,15]. The reason for this discordance may be attributed to tumor heterogeneity, lower
circulating tumor DNA shedding, or lower tumor burden. Clinically, patients with mCRC at advanced stages
possess multiple metastatic lesions in multiple organs. Therefore, in such cases, the heterogeneity of RAS mutations
must be considered. However, a limited number of studies have evaluated the concordance of RAS mutational
status in all metastatic lesions located in multiple organs and ctDNAs.

C 2021 The Authors
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Taniguchi, Nyuya, Toshima et al.

More importantly, although the OncoBEAM-based liquid biopsy has higher sensitivity for RAS mutant alleles
and can demonstrate which exon is mutated, it cannot provide a precise RAS mutational spectrum, for example,
G12C or G12A. KRAS mutations are often associated with resistance to targeted therapies and poor outcomesin patients with cancer; however, no selective KRAS inhibitor has yet been approved despite more than three
decades of scientific effort [17–23]. Recent advances have led to the development of a small molecule, sotorasib,
which specifically and irreversibly inhibits KRAS G12C through a unique interaction with a pocket of the switch
II region. Sotorasib has demonstrated encouraging anticancer activity in patients with heavily pretreated advanced
solid tumors harboring KRAS G12C mutations [24–27].
Thus, in the clinical setting, the RAS mutational spectrum identification will continue to improve as itsrole in multiple cancers is further recognized. Besides OncoBEAM technology, a PCR-reverse sequence-specificoligonucleotide (PCR-rSSO) method for detecting RAS mutations is now used in the clinical setting to detect RAS
mutations in tumor tissues. The PCR-rSSO approach has a lower sensitivity for minor mutant alleles (the PCRrSSO can detect >1% MAF) than OncoBEAM technology, but it can simultaneously identify all RAS mutational
spectra. In this study, the PCR-rSSO method was examined for its concordance for identifying RAS mutations
present in multiple metastatic lesions in multiple organs and ctDNAs in plasma throughout anti-EGFR therapy inpatients with mCRC [15].
Patients & methods
Tumor samples

Four patients with mCRC who had primary tumors without activated RAS mutations were analyzed in this study.
The patients were treated between 2011 and 2017 at the Okayama University Hospital, Japan. Each patient
enrolled as a research subject in clinical trials (University Hospital Medical Information Network Center; IDs:
8377, 9698 and 11954).
Patient 1’s primary tumors were obtained through biopsy before initiating any treatment (Tb) and by surgicalresection before acquiring resistance during anti-EGFR treatment (Ts). Metastatic lesions were excised at morbid
autopsy and included those of the liver (middle segment [MS], S2 and S3), hepatic lymph node (HN), lung(Lu) and kidney (Kd) (Figure 1A).
The primary tumors from patient 2 (Figure 2A) and patient 3 (Figure 2D) were obtained by surgical resection
before chemotherapy. A metastatic liver lesion from patient 2 was obtained after subsequent liver resection, andone from patient 3 was obtained at the initial liver resection.
The primary tumor of patient 4 was obtained by biopsy before initiating any treatment. A metastatic lesion of
peritoneal dissemination was obtained by surgical resection after anti-EGFR treatment (Figure 2G).
Tissues obtained from autopsy or surgical resection were immediately stored at -80◦ C.
Blood samples

Blood samples from patient 1 were collected before the development of progressive disease (PD) during first-line
chemotherapy (time point 1) and after the patient acquired resistance to second-and third-line chemotherapies(time points 2 and 3, Figure 1A). The blood sample at time point 1 was collected from patient 2 after surgicalresection of liver metastases at the right lobe. Blood samples from time points 2 and 3 were collected after confirmed
recurrence in the liver and after a second resection of liver metastasis (Figure 2A). The time point 1 blood sample
was collected from patient 3 before initiating treatment. The time point 2 blood sample was collected after PD
development during bevacizumab (an antivascular endothelial growth factor antibody)-based chemotherapy. The
time point 3 blood sample was collected after cetuximab treatment with tumor shrinkage (Figure 2D). In patient 4,
the time point 1 blood sample was collected before PD development, the time point 2 blood sample was collected
after surgical resection, and the time point 3 blood sample was collected after the patient acquired resistance tocetuximab treatment (Figure 2G). Plasma was separated immediately and stored at -80◦ C.
Extraction of genomic DNA

Genomic DNA was extracted from fresh-frozen samples using the QIAamp DNA Mini Kit (Qiagen NV, Hilden,
The Netherlands). Tumor DNA of several metastatic lesions was microscopically extracted from formalin-fixedparaffin-embedded (FFPE) specimens, which included metastases located in the liver (RL) and HN. DNA derived
from FFPE specimens was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen NV). Circulating cell-free

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Concordance of acquired mutations between metastatic lesions & liquid biopsy in mCRC

Liver
metastases
S3
MS

Lung
metastasis
Lu

S2

MS

HN

16,000S3

S2

RL

Kidneymetastasis

N

14,000

Kd

Tb

Ts

RL

MS

S2

S3

HN

Lu

Kd

Lu

RL

HN
Lymph
node
metastasis

12,000Kd
Regorafenib

CEA (ng/ml)

10,000Frozen sample
FFPE sample

Panitumumab (PANI)

PANI

Bevacizumab (BEV)PANI

FOLFOXsLV5FU2

CapelRIsLV5FU2

Passedaway

10,000

TAS102

Index

Tb
Ts
Primary lesion

1000
Autopsy
Surgery

RL
MS
S2
S3

100
Ts

BiopsyTb

10

Point 1

HN
Lu
Kd

6000

4000

Point 3

Point 2

8000

1
0

20

40

60

80

100

120

2000

140

Weeks after initiation of therapy
0 Kd
KRAS
Exon 212

13

59

Exon 4

60

61

117

Exon 2
146

12

Exon 3
13

59

60

Exon 15

Exon 4
61

117

146

599

600

601

N

G12S
G12C
G12R
G12D
G12V
G12A
G13S
G13C
G13R
G13D
G13V
G13A
A59T
A59G
Q61K
Q61E
Q61L
Q61P
Q61R
Q61Ht
Q61Hc
K117Nc
K117Nt
A146T
A146P
A146V
G12S
G12C
G12R
G12D
G12V
G12A
G13S
G13C
G13R
G13D
G13V
G13A
A59T
A59G
Q61K
Q61E
Q61L
Q61P
Q61R
Q61Ht
Q61Hc
K117Nc
K117Nt
A146T
A146P
A146V

11

Exon 3

Lu
HN
S3
S2
MS
RL
Ts
Tb

BRAF

NRAS

N

KRAS

NRAS

Tb

RL
G12V

3000

MS

Point 1

2500

Point 2

Point 3

Q61Hc

2000

Index

S2

G12R

1500
1000

S3

500

G13D

0Point 3

HN

Pointt 2

A11Ag G12C

KRAS
KRAS

Q61Hc
K117Nc
K117Nt
A146T
A146P
A146V

Lu

G12S
G12C
G12R
G12D
G12V
G12A
G13S
G13C
G13R
G13D
G13V
G13A
A59T
A59G
Q61K
Q61E
Q61L
Q61P
Q61R
Q61Ht
Q61Hc
K117Nc
K117Nt
A146T
A146P
A146V
G12S
G12C
G12R
G12D
G12V
G12A
G13S
G13C
G13R
G13D
G13V
G13A
A59T
A59G
Q61K
Q61E
Q61L
Q61P
Q61R
Q61Ht

oint 1
Point

Q61Hc

NRAS
NRAS

Kd

Figure 1. Summary of a patient with rectosigmoid cancer (patient 1) with unresectable multiple liver metastases. (A) Timeline of thetreatment course of patient 1. The line graph abscissa indicates time, and the ordinate indicates CEA levels, scaled logarithmically. The
computed tomography scan of the liver metastases (above) and chemotherapy regimen (below) are described for each time point. Blood
was collected before the administration of chemotherapy at time points 1, 2 and 3 after the initiation of first-line chemotherapy. (B)Status of RAS and BRAF mutational metastases by Sanger sequencing. Mutational status of KRAS, NRAS, and BRAF. Sanger sequencing
results are described for all samples, including primary lesions with distant focal metastasis. Numbers in red and gray boxes indicate thecodon numbers, codons in red boxes indicate the hot spots and red arrows indicate the observed missense mutations. (C) Status of RAS
mutational metastases by PCR-rSSO. Panels show index values of 12 types of RAS exon 2 (G12S, G12C, G12R, G12D, G12V, G12A, G13S,
G13C, G13R, G13D, G13V and G13A), eight types of RAS exon 3 (A59T, A59G, Q61K, Q61E, Q61L, Q61P, Q61R and Q61H), and four types of
RAS exon 4 (K117N, A146T, A146P and A146V) mutations by PCR-rSSO indices of metastatic specimens in the patient. (D) Detection of
circulating RAS mutant DNA. The detection of circulating RAS mutant alleles in plasma by PCR-rSSO. The KRAS G12R mutant allele index
value increased to 2600 in plasma obtained at time point 3. The index values of the KRAS G13D and Q61Hc mutant alleles also increased
to 159 and 471, respectively, in plasma obtained at time point 3.
CEA: Carcinoembryonic antigen; PCR-rSSO: Polymerase chain reaction-reverse sequence-specific oligonucleotide.

DNA was extracted from 200 μl of plasma using the QIAamp Blood Kit (Qiagen) per to the manufacturer’s
instructions.
Conventional sequencing

Sanger sequencing was performed to confirm mutations in KRAS, NRAS exons 2–4, and BRAF exon 15 (includingcodon 600) in all samples. The primer sequences for KRAS, NRAS exons 2–4, and BRAF exon 15, and the PCR
conditions are described in Supplementary Table 1. The PCR products were purified using the QIAquick PCR
R
3100-Avant and SeqStudio Genetic
Purification Kit (Qiagen) and were directly sequenced using the ABI PRISM
Analyzer (Thermo Fisher Scientific, OH, USA).
PCR-rSSO

Extensive RAS mutations (both KRAS and NRAS mutations) of DNA purified from FFPE, fresh-frozen tissues, andR
technology
plasma were evaluated using the MEBGEN™ RASKET or RASKET-B KIT based on the Luminex(MBL, Nagoya, Japan). The assay was performed according to the manufacturer’s protocol. The MEBGEN RAS-

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Patient 2

Patient 3
Livermetastasis

10Surgery

Point 1

0.1
0Point 2

Point 3

Primary tumor

Liver metastases

Surgery

10

Peritoneal
dissemination

Primary tumor

1
Point 1

0.1
0

50

400

600
400

G12S
G12C
G12R
G12D
G12V
G12A
G13S
G13C
G13R
G13D
G13V
G13A
A59T
A59G
Q61K
Q61E
Q61L
Q61P
Q61R
Q61Ht
Q61Hc
K117Nc
K117Nt
A146T
A146P
A146V
G12S
G12C
G12R
G12D
G12V
G12A
G13S
G13C
G13R
G13D
G13V
G13A
A59T
A59G
Q61K
Q61E
Q61L
Q61P
Q61R
Q61Ht
Q61Hc
K117Nc
K117Nt
A146T
A146P
A146V
V600E

BRAF

Point 2

1200

Point 3

lndex

lndex

400

600
400

400

0
G12S
G12C
G12R
G12D
G12V
G12A
G13S
G13C
G13R
G13D
G13V
G13A
A59T
A59G
Q61K
Q61E
Q61L
Q61P
Q61R
Q61Ht
Q61Hc
K117Nc
K117Nt
A146T
A146P
A146V
G12S
G12C
G12R
G12D
G12V
G12A
G13S
G13C
G13R
G13D
G13V
G13A
A59T
A59G
Q61K
Q61E
Q61L
Q61P
Q61R
Q61Ht
Q61Hc
K117Nc
K117Nt
A146T
A146P
A146V
V600E

0
G12S
G12C
G12R
G12D
G12V
G12A
G13S
G13C
G13R
G13D
G13V
G13A
A59T
A59G
Q61K
Q61E
Q61L
Q61P
Q61R
Q61Ht
Q61Hc
K117Nc
K117Nt
A146T
A146P
A146V
G12S
G12C
G12R
G12D
G12V
G12A
G13S
G13C
G13R
G13D
G13V
G13A
A59T
A59G
Q61K
Q61E
Q61L
Q61P
Q61R
Q61Ht
Q61Hc
K117Nc
K117Nt
A146T
A146P
A146V
V600E

200

0

KRAS
BRAF

Point 3

600

200

NRAS

Point 2

800

200

KRAS

Point 1

1000

800

800

Patient 4

1400

Patient 3
Point 1

NRAS

KRAS

BRAF

1000

Peritoneal dissemination

G12S
G12C
G12R
G12D
G12V
G12A
G13S
G13C
G13R
G13D
G13V
G13A
A59T
A59G
Q61K
Q61E
Q61L
Q61P
Q61R
Q61Ht
Q61Hc
K117Nc
K117Nt
A146T
A146P
A146V
G12S
G12C
G12R
G12D
G12V
G12A
G13S
G13C
G13R
G13D
G13V
G13A
A59T
A59G
Q61K
Q61E
Q61L
Q61P
Q61R
Q61Ht
Q61Hc
K117Nc
K117Nt
A146T
A146P
A146V
V600E

G12S
G12C
G12R
G12D
G12V
G12A
G13S
G13C
G13R
G13D
G13V
G13A
A59T
A59G
Q61K
Q61E
Q61L
Q61P
Q61R
Q61Ht
Q61Hc
K117Nc
K117Nt
A146T
A146P
A146V
G12S
G12C
G12R
G12D
G12V
G12A
G13S
G13C
G13R
G13D
G13V
G13A
A59T
A59G
Q61K
Q61E
Q61L
Q61P
Q61R
Q61Ht
Q61Hc
K117Nc
K117Nt
A146T
A146P
A146V
V600E

NRAS

BRAF

600

Primary tumor

1000
0

Patient 2

Point 3100 120 140 160 180 200

1500
500

Point 3

80

2000

0

Point 2

Point 2
60

Patient 4
2500

0Point 1

40

3000

200

1000

20

Days after initiation of therapy

Liver metastasis

KRAS

BiopsyPrimary tumor

10
Point 1

200

NRAS

100

0

lndex

lndex

400

Nivolumab

Primary tumor/peritoneal dissemination

1

800

600

CETU
FOLFlRl
Surgery

1000

450

Patient 2
Primary tumor

CETU
FOLFlRl

10,000

Point 3

Point 2100 150 200 250 300 350
Days after initiation of therapy

1000

Liver metastasis

KRAS

lndex

Primary tumor

Surgery

Patient 2
Primary tumor

CETU
FOLFlRl

100

100 200 300 400 500 600 700 800 900
Days after initiation of therapy

1000

lndex

Liver metastasis

Liver metastases

1

Primary tumor

800

Surgery

Surgery

Bevacizumab (BEV)
FOLFOX

1000

Recurrence100

CEA (ng/ml)

CEA (ng/ml)

CETU
FOLFOX

NRAS

G12S
G12C
G12R
G12D
G12V
G12A
G13S
G13C
G13R
G13D
G13V
G13A
A59T
A59G
Q61K
Q61E
Q61L
Q61P
Q61R
Q61Ht
Q61Hc
K117Nc
K117Nt
A146T
A146P
A146V
G12S
G12C
G12R
G12D
G12V
G12A
G13S
G13C
G13R
G13D
G13V
G13A
A59T
A59G
Q61K
Q61E
Q61L
Q61P
Q61R
Q61Ht
Q61Hc
K117Nc
K117Nt
A146T
A146P
A146V
V600E

Cetuximab (CETU)
FOLFOX

1000

CEA (ng/ml)

Livermetastasis

Primary tumor

Patient 4

KRAS
BRAF

NRAS
BRAF

Figure 2. Summary of the three metastatic colorectal cancer patients. (A–C) Timeline of the treatment course of patients 2, 3 and 4. Theline graph abscissa indicates time, and the ordinate indicates CEA levels, scaled logarithmically. Magnetic resonance imaging (patient 2,A), computed tomography (patient 3, B), and positron emission tomography (patient 4, C) scan of the liver metastases or peritoneal
dissemination are described for each time point. Blood was collected at time points 1, 2 and 3. (D–F) RAS and BRAF mutational status inprimary tumor and metastases by PCR-rSSO. Panels show the index values of 12 types of RAS exon 2 (G12S, G12C, G12R, G12D, G12V,
G12A, G13S, G13C, G13R, G13D, G13V and G13A), eight types of RAS exon 3 (A59T, A59G, Q61K, Q61E, Q61L, Q61P, Q61R and Q61H), andfour types of RAS exon 4 (K117N, A146T, A146P and A146V) mutations by PCR-rSSO indices of the primary tumor and metastatic
specimens in the patients. (G–I) Detection of Circulating RAS and BRAF mutant DNA. The detection of circulating RAS and BRAF mutant
alleles in plasma by PCR-rSSO. Patients 2 (G) and 3 (H) showed no mutation in liquid biopsy. In patient 4 (I), the BRAF V600E mutant allele
index value increased to 1242 in plasma obtained at time point 3.
CEA: Carcinoembryonic antigen; PCR-rSSO: Polymerase chain reaction-reverse sequence-specific oligonucleotide.

KET KIT can simultaneously examine 12 types of RAS exon 2 (G12S, G12C, G12R, G12D, G12V, G12A, G13S,
G13C, G13R, G13D, G13V and G13A), eight types of RAS exon 3 (A59T, A59G, Q61K, Q61E, Q61L, Q61P,
Q61R and Q61H), and four types of RAS exon 4 (K117N, A146T, A146P and A146V) mutations [16]. The
MEBGEN RASKET-B KIT was used to examine 12 types of RAS exon 2, eight types of RAS exon 3, four types of
RAS exon 4 and the BRAF V600E mutation.
Results
Identification of acquired RAS mutations by conventional Sanger sequencing
In patient 1, there was no activating RAS mutation in the primary tumor biopsy specimen obtained before treatment,in the primary tumor tissue obtained after 1 year of panitumumab administration, or in a metastatic tumor in theright liver lobe (RL) that continuously responded to panitumumab with first-line systemic chemotherapy. The tumor
shrank in response to a third-line panitumumab rechallenge (Figure 1A). KRAS sequences in metastatic specimens
obtained during autopsy revealed diverse acquired mutations at different metastatic sites, indicating resistance tosystemic chemotherapy, including anti-EGFR antibody treatment. Sanger sequencing in liver segments II (S2), III(S3), and MS detected acquired activating KRAS mutations resulting in G61Hc, G12R and G12V, respectively.
The KRAS G12C, G13D and G61Hc mutations were also detected in metastases in the left kidney (Kd), a HN,
and the left lung (Lu), respectively. No samples from primary and metastatic lesions harbored mutations in NRAS
or BRAF (Figure 1B).
Identification of acquired RAS mutations by a PCR-rSSO method

Sanger sequencing detected acquired mutations in six (87%) of the seven metastatic lesions in patient 1. Given thelower sensitivity for detecting mutant alleles by Sanger sequencing (∼20%), we could not exclude the possibilityof associated metastatic lesions caused by low numbers of resistant cells already existing in the primary lesion.

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Concordance of acquired mutations between metastatic lesions & liquid biopsy in mCRC

We further analyzed all samples using the PCR-rSSO method, Rasket, which has high sensitivity in detecting
extended RAS mutant alleles at lower frequencies (1–5%). A sample was mutation-positive when the index was
estimated over the cut-off value and the sensitivity for each mutant allele was 1–5% [16].
There were no KRAS or NRAS mutations with indices higher than the cut-off values in DNA purified frompretreated primary tumor cells (Figure 1C & Supplementary Table 2). Additionally, PCR-rSSO analysis revealed
the same mutation spectrum as Sanger sequencing in the patient except for the Kd and RL metastatic lesion. The
index of the KRAS G12C allele captured from the Kd by PCR-rSSO analysis was 114, in other words, lower thanthe conventional cut-off value (index value: 300). This low index may have been influenced by the immediatesynonymous mutation of A11Ag demonstrated by Sanger sequencing, as previously reported (Figure 1A) [16].
Interestingly, the metastatic tumor in the RL, which consistently responded to panitumumab and showed no
mutation by Sanger sequencing, revealed multiple KRAS mutations, Q61Ht, G12A and G12R, with indices higher
than the cut-off values. Although these mutant alleles were not sufficiently frequent to be detected by Sanger
sequencing, heterogeneity in RAS mutant cancer cells may exist in the RL tumor mass.
In summary, by Sanger sequencing and PCR-rSSO, the seven metastatic lesions were found to harbor diverse
acquired mutations in the KRAS gene: Q61Ht, G12A and G12R in RL; G12V in MS; Q61Hc in S2; G12R in
S3; G12C in Kd; G13D in HN; and Q61Hc in Lu.
Detection of acquired mutations in circulating cell-free DNA

We investigated whether acquired RAS mutations could be detected in the plasma samples using PCR-rSSO(Figure 1D). None of the seven diverse acquired mutations found in the resistant tumors were detectable before orafter PD during the initial course of panitumumab treatment (time points 1 and 2 in Figure 1A & Supplementary
Table 3), but two of the seven diverse acquired mutations were detectable at a significant level following PD
confirmation during third-line panitumumab rechallenge (time point 3). The KRAS G12R and Q61Hc frequency
confirmed in the resistant tumor in S3 and S2, respectively, were strikingly elevated in circulating cell-free DNA
in a plasma sample collected at time point 3. None of the acquired mutations in the time point 3 plasma sample
were detectable in the plasma of time points 1 and 2.
Detection of acquired mutations in circulating cell-free DNA in patients 2, 3 & 4

We next evaluated the three patients with mCRC patients without activating RAS mutations. Patient 2 was initiallyresected for the primary tumor and then treated with FOLFOX plus cetuximab. After 1.5 years of chemotherapy
administration, metastatic tumors in the RL that had continuously responded were resected (Figure 2A). Following
liver resection, patient 2 was carefully followed without chemotherapy. However, the patient experienced a recurrenceof single metastasis at the MS of the liver. FOLFOX plus cetuximab was re-introduced, which led to effective
shrinkage of the metastatic lesion. Thus, second-time liver resection was performed. Neither the primary tumors
obtained before treatment initiation nor the metastatic liver lesion after the second-time liver resection showed
activated RAS mutations by the PCR-rSSO method (Figure 2B). Importantly, as patient 2 never experienced
acquired resistance during the treatment course, the liquid biopsies among the three-time points showed no
activating RAS mutations (Figure 2C).
Patient 3 was initially resected for the primary tumor and then treated with FOLFOX plus bevacizumab. Tenmonths after treatment, this first-line systemic chemotherapy failed, and FOLFIRI plus cetuximab was selected as
the second-line treatment. The metastatic tumors in the RL responded rapidly to cetuximab, and liver resection
was performed. Neither the primary tumor obtained before the initiation of treatment nor a metastatic liver lesion
obtained after response to cetuximab showed activating RAS mutations by the PCR-rSSO method (Figure 2E).
Similar to patient 2, patient 3 never experienced acquired resistance to cetuximab; therefore, it is reasonable thatliquid biopsies among the three-time points showed no activating RAS mutations (Figure 2F).
Patient 4 was initially diagnosed with a primary tumor that had the BRAF V600E mutation and underwent
treatment with FOLFIRI plus cetuximab as the first-line chemotherapy (Figure 2G & H). The primary tumor andperitoneal dissemination responded rapidly to cetuximab. After tumor shrinkage, the primary tumor and peritoneal
dissemination were resected. After surgery, FOLFIRI plus cetuximab was continuously administered. Approximately
3 months after surgery, residual metastatic lesions grew, indicating resistance to systemic chemotherapy. Liquidbiopsies at time points 1 and 2 were collected when the tumors responded rapidly to cetuximab. However, theliquid biopsy at time point 3 was collected after confirmation of acquired resistance to cetuximab. No BRAF V600E
mutation nor RAS mutations were confirmed in plasma DNA (time points 1 and 2) while the tumors responded tofuture science group10.2144/fsoa-2021-0059

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Taniguchi, Nyuya, Toshima et al.

cetuximab. In contrast, the BRAF V600E mutation was detectable in the plasma DNA collected after the patient
became refractory to cetuximab (time point 3, Figure 2I).

Discussion
Sanger sequencing is less sensitive for detecting minor cancer cell populations. In the current study, we aimed todetermine the efficacy of a PCR-rSSO liquid biopsy method. Initially, we examined whether acquired mutations
were detectable in metastatic sites collected after an autopsy by Sanger sequencing, which was confirmed by our
results. Therefore, this study showed, for the first time, that acquired mutations can be detected in liquid biopsy
by PCR-rSSO.
PD is generally determined by radiological evaluation. Liquid biopsy could be useful for early identification ofindividuals at risk of developing drug resistance before radiographic documentation of PD, as well as individuals
who already have certain mutations in tumor burden [1,13–15]. Here, PCR-rSSO analysis was conducted on serial
plasma samples and all metastatic lesions in multiple organs from patients who were treated with chemotherapies,
including panitumumab and cetuximab.
In patient 1, none of the seven acquired mutations found in the resistant tumors were detectable before orafter PD during the initial panitumumab treatment course (time points 1 and 2). However, three (G12R, G13D,
and Q61Hc) of the seven acquired mutations (G12C, G12R, G12V, G12A, G13D, Q61Ht and Q61Hc) weredetectable after PD was confirmed during third-line panitumumab rechallenge (time point 3). Indeed, two of thethree acquired RAS mutations (G12R and Q61Hc) confirmed in the PCR-rSSO were beyond the cut-off values
estimated by tumor tissue analysis. Of note, although the metastatic lesion at the RL was confirmed to possess
three acquired mutations (G12R, G12A and Q61Ht) at autopsy, this lesion demonstrated an effective response to
chemotherapies, including anti-EGFR antibody rechallenge.
In support of the results obtained from patient 1, among the liquid biopsies obtained from patient 4, only
the time point 3 blood sample, collected after confirmation of radiological PD, demonstrated the BRAF V600E
mutation that the primary tumor and peritoneal dissemination possessed. In contrast, as all blood specimens ofpatients 2 and 3 were collected when chemotherapies effectively caused tumor regression, these specimens did notshow any activated mutations causing acquired resistance.
A previous study detected acquired mutation in plasma as early as 10 months before radiological PD [1]. Therefore,
we also attempted to confirm whether acquired RAS variants were detectable in plasma before radiological PD.
Unfortunately, given the conventional cut-off value of the PCR-rSSO, we could not detect acquired mutations
before radiological PD. Our inability to detect ctDNA in the plasma samples might be partially explained by thedifferences in analytical technologies. The PCR-rSSO method used in this study can detect MAFs >1%, whereas
the previous study with OncoBEAMing technology considered 0.1% MAFs mutation-positive [1,16]. The optimal
cut-off value of OncoBEAMing is still debatable. Moreover, the PERSEIDA study reported an association between
cut-off values of MAF of RAS mutant alleles in patients treated with panitumumab and their overall response
rate [28]. If the cut-off value increases the sensitivity for lower MAF of RAS mutant alleles, the overall response ratein patients diagnosed as RAS wild-type was increased. However, similarly, the number of patients diagnosed withRAS mutant also showed an increased overall response rate to panitumumab treatment. Thus, patients who respondto anti-EGFR antibody treatment may be excluded to increase the sensitivity for lower MAF of RAS mutant alleles.
Indeed, the OncoBEAM-based liquid biopsy has a high sensitivity for RAS mutant alleles and can identify
which exon is mutated. Still, it cannot provide a precise RAS mutational spectrum, for example, G12C or G12A.
In contrast, although the PCR-rSSO strategy had less sensitivity for MAF, and even though the index values aresemi-quantitative, the advantage of the PCR-rSSO is that it confirms all RAS mutations simultaneously. Thus, thePCR-rSSO may represent a useful and complementary tool for liquid biopsy to detect RAS mutations.
Even if we use the highest sensitivity method to detect mutant alleles of resistance in liquid biopsy, we missed themutant alleles from the blood in 10–18% of patients with RAS mutations in tumor tissue [14,15]. This is likely due
to tumor heterogeneity, lower ctDNA shedding, or lower tumor burden. In particular, the location of metastatic
tumors may be important for detecting mutant alleles. Discrepancies of mutations between tumor tissue and ctDNAin patients with lung-only metastases occurred, but a higher agreement in liver metastases was found [15,29,30]. In linewith this, our results from patient 1 also showed that the degree of RAS mutational concordance varied according to
the metastatic site, e.g., KRAS G12R detected in ctDNA was confirmed in resistant tumors in S3, which appeared
to be more aggressively progressed by radiographic findings.

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Concordance of acquired mutations between metastatic lesions & liquid biopsy in mCRC

At least two explanations could account for the development of acquired mutations [3,9]. First, resistant cells
harboring these acquired mutations may be present in low numbers upon treatment initiation. Second, cells may
have acquired a de novo activating mutation in response to the continued molecularly-targeted therapy. In the firstmodel, the metastatic CRC response to EGFR-targeted therapies accompanies a selection of pre-existing resistantclones metastasized to the initial metastatic lesion. Therefore, if the acquired RAS mutations in this study were
present at treatment initiation but at a low enough frequency to be undetectable by the two different procedures, atleast some resistant metastatic lesions that grew after treatment should harbor multiple RAS mutations. However,almost all resistant metastatic lesions (except for the RL in patient 1) harbored one acquired RAS mutation, as
demonstrated by conventional Sanger sequencing and the sensitive PCR-SSO procedure. In the RL in patient 1,
multiple KRAS mutants were displayed by PCR-rSSO. However, the proportion of mutants was low and was notdetected by Sanger sequencing, suggesting the existence of many types of cancer cells. As visualized by computed
tomography, the lower proportion of multiple KRAS mutants could explain why the RL continued to shrink withcalcification during sequential chemotherapy treatment.
As only a small part of the tumor lesions could be examined, we cannot exclude sampling bias regarding theexistence of mutant alleles precluding the assessment of genetic heterogeneity within or among lesions. However, in
CRC, RAS mutations are believed to occur in the early phase of tumorigenesis, such as developing a small adenoma
into a larger adenoma [31]. Thus, an RAS mutation spreads homogeneously within the tumor mass, resulting in
more than 95% concordance in RAS mutation status across different sites of a tumor mass [32]. Therefore, RAS
mutation-based drug resistance may be attributable to new mutations arising rather than the selection and clonal
amplification of an exceedingly small number of pre-existing RAS mutant cells. This scenario was also supported
by previously reported in vitro studies [1,33]. Indeed, Shaffer et al. presented that in a BRAF-mutant melanoma
cell line, the population of resistant cells may arise upon selecting multiple clones that were already present before
BRAF inhibitor treatment [34]. Of note, these resistant cells arise from profound transcriptional variability at thesingle-cell level, which involves infrequent, semi-coordinated transcription of several resistance markers at highlevels in a small percentage of cells. The addition of the drug then induces epigenetic reprogramming in these cells,
converting the transient transcriptional state to a stably resistant state with acquired mutations.
In this study, although we only analyzed four cases and were unable to identify acquired mutations via liquid biopsy
before radiographic documentation of PD, we could detect acquired drug resistance-inducing mutations in liquidbiopsy collected after radiographical PD by the PCR-rSSO method. Interestingly, patients 1 and 3 experienced
radiographic PD during chemotherapy with bevacizumab. Moreover, the blood obtained after confirmation of
bevacizumab resistance showed no RAS acquired mutations. Moreover, even if acquired mutations occur, they do
not always indicate that the drug is ineffective, as demonstrated by the metastatic lesion of the RL in patient 1.
Our results strongly emphasize the clinical utility of liquid biopsy in the detection of RAS mutations for decisions
regarding anti-EGFR antibody administration to individuals at risk of drug resistance.

Conclusion
In this study, we intended to establish whether PCR-rSSO can be used to detect acquired mutations in liquidbiopsy. Although the sample size is too small to reach significance, our results demonstrated that acquired mutations
observed in metastatic lesions with acquired resistance were detected in plasma by the PCR-rSSO method.

Executive summary• We evaluated whether a PCR-reverse sequence-specific oligonucleotide (PCR-rSSO) method can examine theconcordance between liquid biopsy and metastatic lesions with acquired resistance.
• Liquid biopsy based on the PCR-rSSO is a successful procedure for capturing acquired mutations with precise
information of mutational spectrum that may lend us to reach selective target agents for RAS mutations.
• Specimen and liquid biopsy analyses revealed that the patient acquired multiple secondary activating KRAS
mutations that differed among the metastatic sites following treatment with panitumumab, suggesting acontinued process of mutagenesis and the need for alternative treatment strategies in cases treated with
anti-EGFR antibodies.

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Taniguchi, Nyuya, Toshima et al.

Supplementary data
To view the supplementary data that accompany this paper please visit the journal website at: www.future-science.com/doi/suppl/10.2144/fsoa-2021-0059
Author contributions
F Taniguchi performed genetic analyses and drafted the manuscript. A Nyuya, T Toshima, K Yasui and M Okawaki extracted DNA
and analyzed the genetic mutations. Y Mori, H Kishimoto, Y Umeda, T Fujiwara and H Tanioka treated patients, provided thepatient samples, and summarized all clinicopathological data. Y Yamaguchi and A Goel assisted with the interpretation of all data
and drafted the manuscript. T Nagasaka designed the project, performed analyses, assisted with the interpretation of all data,
secured the funding and drafted the manuscript. All authors read and approved the final manuscript.
Acknowledgments
The authors would like to thank F Uno, A Watanabe, T Nakai, T Yamanishi and K Tokuda for their technical assistance.
Financial & competing interests disclosureThis work was supported by KAKENHI (20590572, 25860409, 26462016 and 15H03034) and in part by a Research Project Grant(R02B046) from Kawasaki Medical School. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript
apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
Ethical conduct of researchThis study was approved by the ethics committee of Okayama University (Institutional Review Board [IRB] no.: ge270, 1591, ge271,
and 80) and Kawasaki Medical School (IRB no.: 3193, 3194, and 3196-1). The patients provided written informed consent and all
studies were conducted in accordance with the Declaration of Helsinki.
Data sharing statement
The datasets used during the current study are available from the corresponding author on reasonable request. All data generated
by the PCR-rSSO during this study are included in this published article and its supplementary information files.
Open accessThis work is licensed under the Creative Commons Attribution 4.0 License. To view a copy of this license, visit http://creativecomm
ons.org/licenses/by/4.0/

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