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

During cancer development, tumor cells may elicit cytotoxic T-lymphocyte (CTL)-mediated immune responses–partly a consequence of accumulated gene mutations that are translated into altered peptides [1]. Tumor cell expression of HLA class I-antigen complexes is essential for CTL recognition of aberrant peptides and subsequent activation [2]. Consequently, alteration of HLA class I cell surface expression provides an effective mechanism by which tumors can escape immune detection [3, 4]. Multiple mechanisms have been shown to underlie defects in HLA class I expression by tumor cells. They include mutations in the individual HLA class I genes HLA-A, -B and -C, located on chromosome 6p21.3) [5]; mutations in β2-microglobulin (β2m) [6‐9], molecule required for cell surface expression of HLA class I antigens; and defects in components of the HLA class I-associated antigen-processing machinery (APM) [9‐11]. The APM consists of proteasome components delta, MB1 and Z; the immunoproteasome components LMP2, LMP7 and LMP10; peptide transporters TAP1 and TAP2; and chaperones Calnexin, Calreticulin, ERp57, and Tapasin. The immunoproteasome generates peptides mostly, although not exclusively from endogenous proteins, TAP1 and TAP2 facilitate peptide translocation from the cytosol into the lumen of the endoplasmic reticulum, where the peptides are loaded onto the HLA class I molecules with the aid from the several chaperones [12].

Chromosomal instability (CIN) and microsatellite instability (MIN) are the two major forms of genetic instability in colorectal cancer. Combined with distinct somatic mutation patterns and epigenetic modifications, CIN and MIN lead to the development of sporadic colorectal cancer [13]. MIN sporadic tumors, which constitute approximately 15% of all colorectal cancer cases and up to 40% of the tumors localized on the right side (preceding the splenic flexure) of the colon [14], have a phenotype resulting from the epigenetic inactivation of the mismatch repair gene hMLH1. Its inactivation destroys a cell's ability to repair base-base mismatches and small insertions or deletions in repetitive stretches, leading to an accumulation of frameshift mutations that get translated into abnormal peptide sequences. When these mutations are accumulated to large extent in the cell genome the tumors are said to possess high-microsatellite instability (MSI-H) [15]. Hence, it is expected that genes containing microsatellite sequences within their coding regions are more susceptible to somatic mutations, as seen in the TGFβ-RII gene. TGFβ-RII's third exon contains a microsatellite repeat of 10 adenines that is frequently targeted by frameshift mutations in MSI-H tumors [16]. MSI-H is also the hallmark of hereditary non-polyposis colorectal cancer (HNPCC), in which germline mutations of hMLH1, hMSH2, hMSH6 and PMS2 can be found. HNPCC constitutes approximately 2–4% of all CRC cases [17]. Tumors with MSI-H are thought to be more able to stimulate a CTL-mediated immune response due to their frequent generation of the aberrant frameshift peptides [18]. Therefore, these tumors are subjected to a greater selective pressure which favors the outgrowth of tumor cells with the ability to escape from recognition and destruction by host immune system.

Various studies have identified HLA alterations in colorectal cancer [19‐21], including the prevalence of HLA class I alterations in MSI-H tumors [8, 22]. However, the latter studies 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. It was suggested that MSI-H sporadic and hereditary tumors follow parallel evolutionary pathways during tumorigenesis in terms of both genotype and phenotype [23]. As far as HLA class I defects are concerned it was never investigated whether these different tumors present distinct escape mechanisms from the immune system. In the present study, we compared the frequency of defects in HLA class I expression in right-sided sporadic (MSI-H and microsatellite-stable (MSS) sub-groups) colon tumors and in HNPCC tumors and studied the mechanisms underlying any abnormalities in these subgroups.

Abnormalities in HLA class I cell surface expression are commonly observed in tumors and are interpreted as a mechanism by which tumor cells evade the host immune system [1]. In colorectal cancer, especially in MSI-H tumors, the high degree of lymphocytic infiltrate in some cases may suggest an active immune response during tumor development [41, 42]. Moreover, MSI-H tumors might cause increased immune reactivity as a consequence of the high amounts of aberrant frameshift peptides they generate [8, 18]. A selective pressure by CTLs upon these tumors would favor the outgrowth of tumor cells that lost HLA class I expression at the cell surface allowing them to surpass the action of the immune system.

Applying immunohistochemistry on tissue arrays, we compared HLA class I expression in both sporadic RST (MSI-H and MSS sub-groups) and HNPCC tumors. RST were chosen because of the high percentage of MSI-H cases in this specific tumor type [43]. Indeed, immunohistochemical staining with mAb showed that HLA class I loss was frequent in the MSI-H cases analyzed when compared to their MSS counterpart. This finding supports the hypothesis that MSI-H tumors face greater selective pressure to lose HLA class I expression, as described by Kloor et al[8]. However, we have shown for the first time that distinct molecular mechanisms underlie HLA class I loss in sporadic MSI-H and HNPCC colon cancers. In the latter, HLA class I loss was preferentially associated with that of β2m, while in the former HLA class I loss was associated with that of one or more APM components (p < 0.0001).

We investigated the genetic abnormalities underlying the HLA class I loss of expression. They included LOH on chromosome region 6p21.3 (encompassing HLA class I and TAP genes), mutations in APM components and mutations in β2m.

Loss of heterozygosity at 6p21.3 was most prevalent in MSS tumors. This is consistent with the observation that these tumors frequently possess gross chromosomal aberrations and are often aneuploid [13]. Moreover, since LOH events in MSS tumors normally comprise large areas of a chromosome, LOH on 6p21 might not be a direct consequence of selective pressure directed to the loss of HLA expression but instead to other genes within the same chromosomal region. The general absence of LOH in MSI-H tumors suggests that this is not the major mechanism by which the cells abrogate HLA class I expression.

The genome's coding regions contain multiple microsatellite repeats, which are considered hotspots for mutations in mismatch repair-deficient tumors [43]. Such repeats are also present within the exons of the APM components, β2m, HLA-A and HLA-B genes. In about half of the MSI-H cases, loss of expression of HLA class I was concordant with the detection of one or more mutations in these genes. We have discovered novel mutations in the antigen presenting machinery genes; Tapasin, Erp57, Calreticulin and Calnexin in colorectal cancer. Previous reports associated the loss of HLA class I expression in MSI-H tumors with defects on β2m molecule [7, 9]. However, the authors did not distinguish the sporadic/hereditary nature of the tumors that were studied. We cannot exclude that the MSI-H cases included in these studies were mainly HNPCC tumors.

The reason sporadic MSI-H tumors would target APM members for inactivation and HNPCC would target the β2m chaperon is unclear. One possibility worth further exploration is that the various mutations suggest different immune-escape mechanisms for thwarting distinct anti-tumor responses. HNPCC tumors can have an age of onset before the 5^th decade of life while sporadic MSI-H tumors appear generally around the 7^th decade of life [43]; one would therefore predict that the alertness and robustness of the immune system would be higher in HNPCC patients leading to a stronger, or at least different selective pressure on the latter. Furthermore it has been recently suggested that the JC polyoma virus plays a role in the oncogenicity of colon tumors with an identical phenotype to sporadic MSI-H tumors [44]. Although speculative, the presence of the JC virus might be implicated in a different immune response between sporadic MSI-H and HNPCC tumors.

The advantages of different escape mechanisms (loss of APM members vs. abrogation of β2m) are not understood. The only known function of APM members is facilitating the expression of HLA classical molecules in complex with endogenous peptides. Thus, one would expect that only these HLA molecules would be affected by failure of the antigen processing machinery. On the other hand, it is accepted that cell surface expression of non-classical HLA molecules (e.g. HLA -G, -E) also depends on β2m, so the function of these highly specialized molecules would be compromised if β2m were mutated or lost. These molecules might play an important role in regulation of immune cell activity by inhibiting or activating its function. Therefore, MSI-H sporadic tumors that have lost expression of both HLA and an APM component and HNPCC tumors with lost β2m expression might behave differently or present a different kind of interaction with cells from the immune system. For instance, Yamamoto et al. have described a correlation between β2m mutations and unfavorable prognosis in colorectal cancer [45].

We separately analyzed the presence of the characteristic BRAF V600E somatic mutations in the RST cohort (data not shown). Forty-percent of MSI-H sporadic tumors presented with this mutation which was absent in the MSS tumors. It was previously described that this mutation is also absent in HNPCC tumors [46]. V600E was distributed equally between tumors that lost vs. retained expression of HLA class I in the sporadic MSI-H cases.

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.