Classification of patients with metastatic colorectal cancer into consensus molecular subtypes into real-world: A pilot study

Table 1 Primers employed for reverse transcription-quantitative polymerase chain reaction experiments to determine the expression of β-catenin, c-Myc and TFG-β, and the genes used as reference genes

Name	Primer	Sequence	Product Lenght	Ref.
TGF-β	Forward	5’- TACCTGAACCCGTGTTGCTCTC-3’	122	[10]
	Reverse	5’- GTTGCTGAGGTATCGCCAGGA-3’
β-catenin	Forward	5’- CACAAGCAGAGTGCTGAAGGTG-3’	146	[11]
	Reverse	5’- GATTCCTGAGAGTCCAAAGACAG-3’
c-MYC	Forward	5’-GCCACGTCTCCACACATCAG-3’	132	[12]
	Reverse	5’-TGGTGCATTTTCGGTTGTTG-3’
B2M	Forward	5’-GTGCTCGCGCTACTCTCTC-3’	150	[13]
	Reverse	5’-GTCAACTTCAATGTCGGAT-3’
PPIA	Forward	5’-GCAAATGCTGGACCCAACACAAAT-3’	174	[14]
	Reverse	5’-AATGGTGATCTTCTTGCTGGTCTTG-3’
RPLP0	Forward	5’-GCAATGTTGCCAGTGTCTG-3’	142	[15]
	Reverse	5’-GCCTTGACCTTTTCAGCAA-3’

Table 2 Clinical characteristics, overall survival, and reverse transcription-quantitative polymerase chain reaction results of the n = 26 patients included in the final analysis

Patient number	Age	Gender	Site of primary cancer	Overall survival (mo)	Miss-match repair proteins expression	β-catenin expression (RT-qPCR) (relative expression with respect to reference gene average)	c-MYC expression (RT-qPCR) (relative expression with respect to reference gene average)	TGF-β expression (RT-qPCR) (relative expression with respect to reference gene average)	CMS
1	69	Male	Sigmoid	5	Proficient	0.185	2.864	0.201	CMS2
2	85	Female	Right colon	31	Proficient	0.100	0.352	0.169	CMS1
3	68	Female	Rectal and sigmoid	12	Proficient	0.042	0.384	0.076	CMS3
4	57	Male	Rectal and sigmoid	34	Proficient	2.684	18.817	9.778	CMS3
5	45	Female	Transverse	40	Proficient	1.812	19.445	5.231	CMS2
6	62	Male	Rectum	28	Proficient	0.010	4.401	0.973	CMS3
7	54	Male	Rectum	20	Proficient	0.301	3.234	1.433	CMS2
8	55	Male	Sigmoid	53	Proficient	0.080	1.870	11.718	CMS4
9	73	Male	Sigmoid	62	Proficient	0.038	0.645	0.461	CMS1
10	79	Male	Rectum	40	Proficient	0.121	2.080	3.513	CMS3
11	56	Female	Right colon	29	Proficient	0.235	3.799	14.700	CMS4
12	66	Female	Right colon	10	Proficient	0.351	6.004	76.116	CMS4
13	53	Male	Sigmoid	52	Proficient	0.233	3.863	2.688	CMS4
14	75	Male	Sigmoid	35	Proficient	0.089	0.760	0.205	CMS3
15	63	Male	Right colon	32	Proficient	0.089	0.760	0.466	CMS3
16	48	Female	Sigmoid	28	Proficient	0.038	1.110	0.498	CMS3
17	53	Female	Rectum	20	Proficient	0.084	1.124	0.801	Not classifiable
18	71	Female	Right colon	12	Proficient	0.083	1.540	0.897	CMS1
19	61	Female	Sigmoid	45	Proficient	0.106	9.208	6.820	CMS4
20	71	Male	Rectum	10	Proficient	0.013	0.855	0.065	CMS3
21	49	Female	Sigmoid	6	Deficient	0.063	2.968	1.871	CMS1
22	74	Male	Right colon	11	Deficient	0.047	0.552	0.249	CMS1
23	65	Female	Rectum	39	Proficient	0.059	0.828	0.084	Not classifiable
24	59	Female	Sigmoid	8	Proficient	0.045	1.324	0.152	CMS2
25	54	Male	Sigmoid	5	Deficient	0.036	0.543	0.127	CMS1
26	69	Male	Sigmoid	22	Proficient	0.192	5.025	2.654	CMS2

Each patient is individually identified in the first column on the left with a sequential number ranging from 1 to 26. Additionally, the consensus molecular subtype assigned based on the Tumour Board analysis is provided. RT-qPCR: reverse transcription-quantitative polymerase chain reaction; TGF-β: Transforming growth factor beta; CMS: consensus molecular subtype.

Table 3 Mutations identified in the n = 26 patients included in the final analysis through massive genomic sequencing using the TumorSec panel

Patient number	Mutation	Mutation variant classification	Affected protein	Variant type
1	TSC2	Missense	p.R1729C	SNV
	TP53	Missense	p.R175H	SNV
2	KRAS	Missense	p.G12C	SNV
3	KRAS	Missense	p.G12V	SNV
	TP53	Missense	p.R175H	SNV
4	KRAS	Missense	p.Q61H	SNV
	PIK3CA	Missense	p.E545G	SNV
5	TP53	Missense	p.P152L	SNV
6	KRAS	Missense	p.G12D	SNV
7	BRCA2	Missense	p.K584E	SNV
	ARID1A	Nonsense	p.Q1584	SNV
8	KRAS	Missense	p.N116H	SNV
	TP53	Missense	p.R175H	SNV
	PIK3CA	Missense	p.H1047R	SNV
	BRAF	Missense	p.N581Y	SNV
9	BRCA2	Frameshift (deletion)	p.N863Ifs11	SNV
	ARID1A	Frameshift (deletion)	p.P1326Rfs155	SNV
	PIK3CA	Missense	p.H1047R	SNV
10	PTEN	Nonsense	p.Y225	SNV
	KRAS	Missense	p.G12C	SNV
	TP53	Frameshift (insertion)	p.Q317Pfs20	SNV
11	KRAS	Missense	p.Q61H	SNV
12	KRAS	Missense	p.G12D	SNV
	TP53	Missense	p.R280K	SNV
13	TP53	Missense	p.R273H	SNV
14	KRAS	Missense	p.G12D	SNV
	TP53	Missense	p.P278L	SNV
15	KRAS	Missense	p.K117N	SNV
	TP53	Missense	p.R282W	SNV
16	KRAS	Missense	p.G12D	SNV
	TP53	Frameshift (deletion)	p.S260Qfs3	Deletion
17	KRAS	Missense	p.Q61L	SNV
	BRCA2	Missense	p.S3147Y	SNV
	TP53	Missense	p.R249G	SNV
18	KRAS	Missense	p.G12C	SNV
	ARID1A	Frameshift (deletion)	p.Q611Hfs7	Deletion
19	TP53	Missense	p.Y220C	SNV
20	KRAS	Missense	p.A59G	SNV
	KRAS	Missense	p.G12D	SNV
	TP53	Missense	p.H214R	SNV
21	NRAS	Missense	p.Q61R	SNV
	ARID1A	Frameshift (deletion)	p.K1072Nfs21	SNV
22	TP53	Missense	p.R273C	SNV
23	TP53	Nonsense	p.E51	SNV
	ARID1A	Frameshift (deletion)	p.Q372Sfs19	SNV
24	TP53	Missense	p.R248W	SNV
	PIK3CA	Missense	p.E545K	SNV
25	PTEN	Nonsense	p.Q149	SNV
	KRAS	Missense	p.G13D	SNV
	TSC2	Missense	p.R1713C	SNV
	TP53	Missense	p.R273C	SNV
	TP53	Missense	p.R158H	SNV
	ARID1A	Nonsense	p.R1335	SNV
26	BRCA2	Missense	p.E3002K	SNV
	TP53	Missense	p.C176Y	SNV

SNV: Single nucleotide variant.