Approach to early-onset colorectal cancer: Clinicopathological, familial, molecular and immunohistochemical characteristics

Table 1 Antibodies, suppliers and clones used for immunohistochemical analysis

Marker	Supplier	Clone
Mismatch repair system proteins
Mlh1	BD PharMingen	G168-15
Msh2	Oncogene	FE11
Msh6	BD Transduction Lab.	44
Apoptosis
Bcl-2	DAKO	124
Cell adhesion
β-catenin	BD Transduction Lab.	14
E-cadherin	Zymed	4A2C7
Cell cycle markers
Chk2	Novocastra	DCS 270.1
Cyclin A	Novocastra	6E6
Cyclin D1	NeoMarkers	SP4-rabbit
Cyclin D3	Novocastra	DCS-22
Cyclin E	Novocastra	13A3
p16	Santa Cruz	F-12
p21 (WAF1)	Oncogene	EA10
p27	BD Transduction Lab.	57
RB-P	Santa Cruz	Poli-rabbit
Skp2	Zymed	1G12E9
Cdk2	NeoMarkers	8D4
Proliferation
p53	Novocastra	DO-7
Ki-67	DAKO	MIB-1
Others
CK20	DAKO	Ks20.8
RAD50	Abcam	2C6
SMAD4	Santa Cruz	B8

Table 2 Clinical, pathological, and familial characteristics of the global group and the microsatellite stable and microsatellite instability groups n (%)

	Global	MSS	MSI	P (χ2)
Patients	45 (100)	29 (69)1	13 (31)1
Median age of onset (range, yr)	39 (25-45)	41 (25-45)	37 (32-42)	0.032
Sex
Male	23 (51)	13 (45)	7 (54)
Female	22 (49)	16 (55)	6 (46)	NS
Location
Right colon	20 (45)	11 (38)	9 (69)
Left colon	15 (33)	11 (38)	2 (16)
Rectum	10 (22)	7 (24)	2 (15)	NS
Tumor differentiation
Well	2 (4)	2 (7)	0 (0)
Moderate	36 (80)	23 (79)	10 (77)
Poor	7 (16)	4 (14)	3 (23)	NS
Mucin production	15 (33)	5 (17)	5 (39)	NS
“Signet ring” cells	4 (9)	0/29 (0)	4/13 (30)	0.006
Modified Astler Coller stage
A	3 (7)	2 (7)	0 (0)
B	17 (38)	12 (41)	5 (39)
C	15 (33)	7 (24)	6 (46)
D	10 (22)	8 (28)	2 (15)	NS
Associated polyps during follow-up	15 (33)	10 (35)	4 (31)	NS
Synchronous or metachronous CRCs	3 (7)	1 (3)	1 (8)	NS
Familial history of cancer
Lynch-related neoplasms in family	18 (40)	5 (17)	11 (85)	< 0.01
Lynch unrelated neoplasms	12 (27)	8 (28)	7 (54)	0.02
Sporadic cases	27 (60)	24 (83)	2 (15)	< 0.001

¹Proportions were calculated for 42 tumors;

²Statistical comparison between microsatellite stable (MSS) and microsatellite instability (MSI) groups was performed using Student´s t test. CRC: Colorectal cancer; NS: Not significant.

Table 3 Immunohistochemical study of the global group¹n (%)

	Normal expression	Lack of expression
Mismatch repair system proteins
Mlh1	24 (86)	4 (14)
Msh2	24 (86)	4 (14)
Msh6	23 (88)	3 (12)
Apoptosis
Bcl-2	7 (25)	21 (75)
Cell adhesion
β-catenin
Membrane	24 (86)	4 (14)
Nucleus	10 (36)	18 (64)
E-cadherin	19 (73)	7 (27)
Cell cycle markers
Chk2	13 (54)	11 (46)
Cyclin A	13 (46)	15 (54)
Cyclin D1	12 (43)	16 (57)
Cyclin D3	12 (43)	16 (57)
Cyclin E	8 (30)	19 (70)
p16	13 (48)	14 (52)
p21	14 (56)	11 (44)
p27	12 (52)	11 (48)
RB-P	13 (48)	14 (52)
Skp2	12 (46)	14 (54)
Cdk2	13 (48)	14 (52)
Proliferation
p53	15 (54)	13 (46)
Ki-67	17 (63)	6 (37)
Others
CK20	10 (59)	7 (41)
RAD50	10 (63)	6 (37)
SMAD4	13 (54)	11 (46)

¹Not all tumors could be studied by immunohistochemical.

Table 4 Comparison of the immunohistochemical analyses of microsatellite stable and microsatellite instability groups n (%)

	IHC expression				P
	MSS		MSI
	Normal	Lack	Normal	Lack
MMR system proteins
Mlh1	20 (100)	0 (0)	4 (50)	4 (50)	0.010
Msh2	21 (100)	0 (0)	3 (43)	4 (57)	0.002
Ms6	21 (100)	0 (0)	2 (40)	3 (60)	0.004
Apoptosis
Bcl-2	5 (24)	16 (76)	2 (29)	5 (71)	NS
Cell adhesion
β-catenin
Membrane	18 (86)	3 (14)	6 (86)	1 (14)	NS
Nucleus	8 (38)	13 (62)	2 (29)	5 (71)	NS
E-cadherin	15 (75)	5 (25)	4 (60)	2 (40)	NS
Cell cycle markers
Chk2	10 (53)	9 (47)	4 (60)	2 (40)	NS
Cyclin A	8 (38)	13 (62)	5 (71)	2 (29)	NS
Cyclin D1	7 (33)	14 (67)	5 (71)	2 (29)	NS
Cyclin D3	8 (38)	13 (62)	4 (57)	3 (43)	NS
Cyclin E	3 (14)	18 (86)	5 (83)	1 (17)	0.004
p16	9 (43)	12 (57)	4 (67)	2 (33)	NS
p21	10 (53)	9 (47)	4 (67)	2 (33)	NS
p27	8 (44)	10 (56)	4 (80)	1 (20)	NS
RB-P	11 (55)	9 (45)	2 (29)	5 (71)	NS
Skp2	9 (43)	12 (57)	3 (60)	2 (40)	NS
Cdk2	9 (43)	12 (57)	4 (67)	2 (33)	NS
Proliferation
p53	11 (52)	10 (48)	5 (57)	3 (43)	NS
Ki-67	14 (67)	7 (33)	3 (50)	3 (50)	NS
Others
CK20	9 (75)	3 (25)	1 (20)	4 (80)	NS
RAD50	9 (75)	3 (25)	1 (25)	3 (75)	NS
SMAD4	11 (55)	9 (45)	2 (50)	2 (50)	NS

IHC: Immunohistochemical; MSS: Microsatellite stable; MSI: Microsatellite instability; MMR: Mismatch repair; NS: Not significant.