HNPCC versus sporadic microsatellite-unstable colon cancers follow different routes toward loss of HLA class I expression

Abstract

Background: Abnormalities in Human Leukocyte Antigen (HLA) class I expression are common in colorectal cancer. Since HLA expression is required to activate tumor antigen-specific cytotoxic T-lymphocytes (CTL), HLA class I abnormalities represent a mechanism by which tumors circumvent immune surveillance. Tumors with high microsatellite instability (MSI-H) are believed to face strong selective pressure to evade CTL activity since they produce large amounts of immunogenic peptides. Previous studies identified the prevalence of HLA class I alterations in MSI-H tumors. However, those reports did not compare the frequency of alterations between hereditary and sporadic MSI-H tumors neither the mechanisms that led to HLA class I alterations in each subgroup.

Methods: To characterize the HLA class I expression among sporadic MSI-H and microsatellite-stable (MSS) tumors, and HNPCC tumors we compared immunohistochemically the expression of HLA class I, beta2-microglobulin (beta2m), and Antigen Processing Machinery (APM) components in 81 right-sided sporadic and 75 HNPCC tumors. Moreover, we investigated the genetic basis for these changes.

Results: HLA class I loss was seen more frequently in MSI-H tumors than in MSS tumors (p < 0.0001). Distinct mechanisms were responsible for HLA class I loss in HNPCC and sporadic MSI-H tumors. Loss of HLA class I expression was associated with beta2m loss in HNPCC tumors, but was correlated with APM component defects in sporadic MSI-H tumors (p < 0.0001). In about half of the cases, loss of expression of HLA class I was concordant with the detection of one or more mutations in the beta2m and APM components genes.

Conclusion: HLA class I aberrations are found at varying frequencies in different colorectal tumor types and are caused by distinct genetic mechanisms. Chiefly, sporadic and hereditary MSI-H tumors follow different routes toward HLA class I loss of expression supporting the idea that these tumors follow different evolutionary pathways in tumorigenesis. The resulting variation in immune escape mechanisms may have repercussions in tumor progression and behavior.

Figure 1

Example of immunohistochemical analysis performed on the RST and HNPCC tumors (Amplification 10×). A, Positive expression of HLA class I antigens detected with the HCA2 antibody. The epithelial (large arrow) membranous expression of HLA class I antigens is identical to the lymphocytic infiltrate (small arrow). B, Loss of expression of HLA class I identified with the HCA 2 antibody. The lymphocytic infiltrate (small arrow) was used as a positive control to determine the loss of expression on the epithelial cells. C, Loss of expression of β2m in a HNPCC case. D, Loss of expression of one of the APM members (Tapasin) in a RST case.

Figure 2

Loss of expression of β2m and different APM members was detected by immunohistochemistry in the RST that presented with HLA loss. The shadowing (in black) is indicative for loss of expression of the respective molecules. (tpsn – Tapasin, calnx – Calnexin, crtcln – Calreticulin)

Figure 3

LOH and frameshift analysis was performed on sporadic RST that lost HLA class I expression. Only the tumors that presented with loss of one of the APM molecules or β2m were subjected to fragment analysis in their respective genes. The following repeats were analyzed for frameshift mutations: HLA A: 1 – 4^th exon 7(C), 2 – 5^th exon 3 (GGA); HLA – B: 3(GA) & 3(CA);β2m: 1 – 1^st exon 4(CT), 2 – 2^nd exon 4(GA) & 5(A), 3 – 2^nd exon 5(A); TAP1: 1 – 1^st exon 5(C), 2 – 3^rd exon 5(T), 3 – 8^th exon 5(G), 4 – 10^th exon 5(G), 5 – 11^th exon 6(G) & 5(A); TAP2: 1 – 2^nd exon 6(C), 2 – 9^th exon 5(G); Tapasin: 1 – 2^nd exon 5(G), 2 – 3^rd exon 5(C), 3 – 4^th exon 6(C), 4 – 5^th exon 5(G); Calnexin: 1 – 7^th exon 5(A), 2 – 8^th exon 5(A), 3 – 11^th exon 8(T); Calreticulin: 1 – 3^rd exon 5(G), 6^th exon 5(C); ERp57: 1 – 5^th exon 6(T), 2 – 6^th exon 6(A), 3 – 13^th exon 6(C); LMP2: 1 – 2^nd exon 5(G), 2 – 6^th exon 5(G); LMP7: 1^st exon 6(C) (key: ins – insertion; del – deletion; 0 – no mutation). LOH analysis of the 6p chromosome was also performed with the following markers: 1 – MOGc, 2 – D6S510, 3 – C125, 4 – C141, 5 – D6S2444, 6 – TAP1, 7 – M2426 (Key: Black – Loss of heterozygosity; Striped – non informative marker; White – Retention of heterozygosity).

Figure 4

LOH and frameshift analysis was performed on HNPCC tumors that simultaneously lost HLA class I and β2m expression. LOH markers: (see legend from Figure 3 for key). Frameshift markers:HLA A: 1 – 4^th exon 7(C) 2 – 5^th exon 3 (GGA); HLA – B: 3(GA) & 3(CA); β2m: 1 – 1^st exon 4(CT), 2 – 2^nd exon 4(GA). & 5(A), 3 – 2^nd exon 5(A) (key: ins – insertion; del – deletion; 0 – no mutation; IHC -immunohistochemistry)

Figure 5

Genetic analysis performed on tumors that have lost HLA class I expression. The different peaks correspond to different sizes from the PCR-amplified products. Peaks corresponding to the normal samples are represented in green whereas tumor samples are represented in blue. A, LOH analysis performed on the RST 41 sample with the polymorphic marker C141. The total loss of a normal allele (on top) illustrates the technical advantage of using flow cytometric sorting to identify LOH events. B, Frameshift mutations identified in different members of the APM machinery. On top a homozygous deletion in the sample RST 65 on the 4^th exon of the Tapasin gene is shown. On the bottom, a heterozygous deletion in the sample RST 18 on the second exon of the TAP2 gene is shown. C, Frameshift mutation identified in one HNPCC case (h4) in the 2^nd exon of the β2m gene. Because flow sorting was not performed in the HNPCC cases, we cannot determine whether the frameshifts are homo- or heterozygous due to contamination with normal DNA.