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[Cancer Research 65, 6418-6424, July 15, 2005]
© 2005 American Association for Cancer Research

Immunology

Immunoselective Pressure and Human Leukocyte Antigen Class I Antigen Machinery Defects in Microsatellite Unstable Colorectal Cancers

Matthias Kloor1, Christina Becker1, Axel Benner2, Stefan M. Woerner1, Johannes Gebert1, Soldano Ferrone3 and Magnus von Knebel Doeberitz1

1 Institute of Molecular Pathology, University of Heidelberg; 2 Central Unit Biostatistics, German Cancer Research Center, Heidelberg, Germany; and 3 Department of Immunology, Roswell Park Cancer Institute, Buffalo, New York

Requests for reprints: Magnus von Knebel Doeberitz, Institute of Molecular Pathology, University of Heidelberg, Im Neuenheimer Feld 220/221, 69120 Heidelberg, Germany. Phone: 49-6221-562876; Fax: 49-6221-565981; E-mail: knebel{at}med.uni-heidelberg.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In colorectal cancer, the immune response is particularly pronounced against tumors displaying the high microsatellite instability (MSI-H) phenotype. MSI-H tumors accumulate mutations affecting microsatellites located within protein encoding regions (coding microsatellites, cMS), which lead to translational shifts of the respective reading frames. Consequently, novel tumor-specific frameshift-derived neopeptides (FSP) are generated and presented by MSI-H tumor cells, thus eliciting effective cytotoxic immune responses. To analyze whether the immunoselective pressure was reflected by the phenotype of MSI-H colorectal cancer cells, we compared here the expression of antigen processing machinery (APM) components and human leukocyte antigen (HLA) class I antigen subunits in 20 MSI-H and 20 microsatellite-stable (MSS) colorectal cancer using a panel of newly developed APM component-specific monoclonal antibodies. In addition, we did a systematic analysis of mutations at cMS located within APM genes and ß2-microglobulin (ß2m). Total HLA class I antigen loss was observed in 12 (60.0%) of the 20 MSI-H lesions compared with only 6 (30.0%) of the 20 MSS colorectal cancer lesions. Moreover, total loss of membraneous HLA-A staining was significantly more frequent in MSI-H colorectal cancer (P = 0.0024). Mutations at cMS of ß2m and genes encoding APM components (TAP1 and TAP2) were detected in at least 7 (35.0%) of 20 MSI-H colorectal cancers but in none of the MSS colorectal cancers (P = 0.0002). These data show that defects of HLA class I antigen processing and presentation seem to be significantly more frequent in MSI-H than in MSS colorectal cancer, suggesting that in MSI-H colorectal cancer the immunoselective pressure leads to the outgrowth of cells with defects of antigen presentation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Antigen presentation of the human leukocyte antigen (HLA) class I pathway is mediated by a network of several proteins. Briefly, peptide fragments that are generated from cellular proteins by the proteasome complex are transported to the endoplasmatic reticulum (ER) by TAP1 and TAP2 proteins. There, peptides are loaded on ß2-microglobulin (ß2m)–associated HLA class I heavy chain molecules, a process that is supported by several chaperone molecules (for a detailed review, see ref. 1).

Malignant tumors may generate a variety of antigens that may be presented to and recognized by the host's immune system and may eventually lead to destruction of the respective tumor cells by CTLs (reviewed in refs. 2, 3). Abnormalities of antigen presentation have been found in various malignancies and may reflect the immune selective pressure imposed by the continuous antitumoral immune response of the host (reviewed in ref. 4). The immunoselection hypothesis has been supported by the increased frequency of HLA class I antigen presentation defects observed in tumors upon application of T cell–based immune therapies (5, 6). Different mechanisms may eventually lead to impairments of HLA class I antigen presentation (7). Inactivation of both alleles of the gene encoding ß2m, most frequently because of mutation in one allele in combination with loss of heterozygosity of the ß2m gene locus seems to be the most frequent mechanism leading to complete loss of HLA class I expression on the cell surface (8). Alterations of proteasome subunits (LMP proteins) and the transporter proteins TAP1 and TAP2 also play an important role, and have been observed in different tumor entities (9–11).

In colorectal cancer, there is growing evidence that the extent of the antitumoral immune response correlates with the type of genetic instability. Colorectal cancer may emerge through two principally different carcinogenic pathways. The majority of colorectal cancers are characterized by numerical and structural chromosomal alterations (chromosomal instability). In about 15% of colorectal cancers, defects of the DNA mismatch repair system result in rapid accumulation of deletions or insertions of single nucleotides particularly in repetitive DNA sequences (microsatellites), a process commonly referred to as high microsatellite instability (MSI-H). There are several characteristics of MSI-H colorectal cancer which indicate a particularly high immunogenicity of these tumors, for example, a dense lymphocytic infiltration (12, 13) and the comparably low frequency of distant metastases (14). Recent studies suggest that the high immunogenicity of MSI-H colorectal cancer is induced by the abundant expression of frame-shift neopeptides (FSP) that are generated as a consequence of insertion/deletion mutations at coding microsatellites (cMS; refs. 15, 16). The immunogenicity of several MSI-associated FSPs has been shown in vitro (15–18).

Taken together, there is strong evidence for a tight immunologic surveillance particularly of MSI-H colorectal cancer. MSI-H colorectal cancer cells with defects of the antigen processing machinery (APM) might therefore be insistently selected for during tumorigenesis. Indeed, ß2m mutations leading to total HLA class I loss have been found to be closely associated with the MSI-H phenotype in colorectal cancer (19, 20).

In this study, we analyzed in detail whether the different level of the immunoselective pressure on colorectal cancer of the MSI-H or the microsatellite-stable (MSS) phenotype was reflected by a differential pattern of defects in HLA class I antigen processing and presentation. Therefore, we systematically examined the APM profile of 20 MSI-H and 20 MSS colorectal cancers by immunohistochemistry using a panel of newly developed monoclonal antibodies (mAb). To obtain additional information, mutation analysis was done, focusing on cMS located in APM component-encoding genes. Our data suggest, that MSI-H colorectal cancers are significantly more often affected by alterations of HLA class I antigen processing and presentation. In particular, total HLA class I and selective HLA-A antigen loss on tumor cell surface was closely linked to the MSI-H phenotype. Furthermore, we found that transporter subunits TAP1 and TAP2 may be directly inactivated by MSI-related frame-shift mutations at cMS.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue samples and preparation. Forty formalin-fixed, paraffin-embedded colorectal cancer lesions (MSS, n = 20; MSI-H, n = 20) were randomly selected from tumors previously tested for their microsatellite status according to the recommendations of the National Cancer Institute/ICG-HNPCC (21). No patient included in this study had received neoadjuvant therapy. Blocks were cut into 2-µm sections for immunohistochemical staining with mAb and into 5-µm sections for the preparation of genomic DNA. Informed consent was obtained from all patients included in this study.

Antibodies. The mAb HC-10 which recognizes a determinant expressed on ß2m-free HLA-A10, HLA-A28, HLA-A29, HLA-A30, HLA-A31, HLA-A32, and HLA-A33 heavy chains and on all ß2m-free HLA-B and HLA-C heavy chains (22, 23); the mAb HC-A2 which recognizes a determinant expressed on ß2m-free HLA-A (excluding HLA-A24), HLA-B7301, and HLA-G heavy chains (22, 24); the anti-ß2m mAb L368 (25); the anti-calnexin mAb TO-5; the anti-ERp57 mAb TO-2; the anti-calreticulin mAb TO-11; and the anti-tapasin mAb TO-3 were developed and characterized as described (26). The anti-LMP2 mAb SY-1 and the anti-TAP1 mAb TO-1 were developed and characterized using the strategy described elsewhere (26). Briefly, the mAb-secreting hybridomas were derived from BALB/c mice immunized with synthetic peptides derived from the amino acid sequence of the native protein and with recombinant proteins. Antibodies of the desired specificity were identified by their specific binding to the immunizing peptides in ELISA. The specificity of the selected mAb was proven by their reactivity with the corresponding antigens when tested with lymphoid cell lysates with the appropriate phenotype in Western blotting. The specificity of anti-LMP2 mAb SY-1 and anti-TAP1 mAb TO-1 was corroborated further by their lack of reactivity with a lysate of the T2 cell line, which does not express these molecules (27). Second antibodies (biotinylated anti-mouse IgG) were purchased from Vector (Burlingame, CA).

Immunohistochemistry. Tissue sections were stained using the Vectastain elite avidin-biotin complex detection system (Vector) according to the manufacturer's instructions. Briefly, 2-µm sections were deparaffinized with xylene and passaged through decreasing concentrations of ethanol. Subsequently, antigens were retrieved by heating the slides in a microwave oven (thrice, 5 minutes, 560 W) at pH 6.0. Tissue sections were incubated with an optimal amount of first antibody at 4°C overnight (5 µg/mL of mAb HC-10, TO-1, TO-5, and SY-3; 6.7 µg/mL of mAb HC-A2, L368, SY-1, SY-2, SY-4, SJJ-3, TO-3, TO-11, and NB1). After washing and incubation with an optimal amount of biotinylated anti-mouse IgG antibodies as the secondary antibody (Vector) for 1 hour at 37°C, tissue sections were stained using 3-amino-9-ethylcarbazole (DakoCytomation, Glostrup, Denmark) or diaminobenzidine (DakoCytomation) as substrates.

Tissue sections were scored as positive, heterogeneous, and negative when the percentage of stained tumor cells was >75, 25 to 75, and <25, respectively. All slides were scored by two observers independently (C.B. and M.K.). Staining of normal cells in each tissue section was used as an internal positive control; slides that exhibited no positive staining of normal cells were not included in the evaluation.

Identification and mutation analysis of coding microsatellites. A systematic European Molecular Biology Laboratory (EMBL) database search for translated (coding) microsatellites in human was done (ref. 28; update based on EMBL Rel. 71, June 2002). Mononucleotide repeat tracts consisting of six or more nucleotides were considered for further analysis.

For mutation analysis, tumor and normal tissue DNA was isolated from microdissected tissue sections according to the manufacturer's instructions (DNeasy Tissue Kit, Qiagen, Hilden, Germany). Oligonucleotide primers used for APM gene fragment analysis are listed in Table 1. Fragment length analysis was done on an ABI 3100 genetic analyzer (Applied Biosystems, Darmstadt, Germany). Genescan Analysis Software (Applied Biosystems) was used for data evaluation. Exonwise sequencing of ß2m gene was carried out as described (29).


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  		Table 1. Oligonucleotide primers used for mutation analysis of cMS in APM component-encoding genes

 
Statistical analysis. For the comparison of immunohistochemical staining results and frequencies of mutational events at cMS, Fisher's exact test was applied. In addition, a logistic regression model (30) was applied for the comparison of MSI-H and MSS colorectal cancer.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Human leukocyte antigen class I heavy chains and ß2m expression in colorectal cancer lesions. MSI-H and MSS colorectal cancer lesions were examined for HLA class I subunit expression. The ß2m-specific mAb L368 and the mAb HC-10 and HC-A2 that recognize different epitopes expressed on HLA class I heavy chains were used as probes. Because HLA class I complexes are denatured during formalin fixation, total and membraneous localization of staining signals were documented separately to distinguish between free heavy chains or ß2m molecules and assembled HLA class I heavy chains/ß2m complexes transferred to the cell surface.

Staining results are summarized in Table 2. A loss of membraneous heavy chain signals was observed in all tumors exhibiting total loss of ß2m staining (except two cases with regionally retained membraneous HC-A2 signals; data not shown).


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  		Table 2. HLA class I antigen expression in MSI-H, MSS, and total colon carcinoma specimens

 
Loss of ß2m expression accompanied by total HLA class I antigen loss on the cell surface (Fig. 1A) was observed in 12 (60.0%) of the 20 MSI-H colorectal cancer lesions and in only 6 (30.0%) of the 20 MSS colorectal cancer lesions (Table 2). In addition, MSI-H colorectal cancers frequently displayed a specific loss of certain HLA class I heavy chains detected by the mAb HC-10 (9 of 20, 45.0%) and HC-A2 (8 of 20, 42.1%). In detail, the frequency of reduced staining with mAb HC-10 was significantly higher in MSI-H tumors than in MSS tumors (9 of 20, 45.0% versus 2 of 20, 10%; P = 0.05). Loss of membrane staining with mAb HC-A2 was significantly more often observed in MSI-H than in MSS colorectal cancer lesions (17 of 19, 89.5% versus 6 of 18, 33.3%; P = 0.0024).



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  		Figure 1. A, immunohistochemical stainings with mAbs L368 (ß2m, left), HC-A2 (middle), and HC-10 (right). Tumor U13 (MSI-H, top row): tubular tumor crypts on the right are ß2m positive and retain membrane staining with mAbs specific for HLA class I heavy chains. Carcinoma cells (left) present complete loss of ß2m expression. In the ß2m-negative region, reactivity with mAbs HC-10 (reduced in carcinoma compared with surrounding stroma cells) and HC-A2 is exclusively cytoplasmatic. Tumor U18 (MSI-H, bottom row): Complete loss of ß2m expression in tumor cells; note positive staining in tumor-infiltrating lymphocytes. Weak cytoplasmatic staining with mAb HC-10 indicates reduction of HLA class I heavy chain expression; HC-A2 reactivity is lost completely. Surrounding stromal cells serve as internal positive control. B, immunohistochemical staining of tumors that harbor mutations at coding microsatellites of APM genes. Tumor U5 (TAP1 mutation positive, top row) and U10 (TAP2 mutation positive, bottom row) with mAbs TO-1 and SY-2, respectively. Two regions of each tumor. Expression of TAP1 and TAP2 is regionally retained in both tumors (right) but lost or markedly reduced in regions near the infiltration front (left). Infiltrating lymphocytes serve as internal positive controls.

 
Mutations affecting the ß2m gene. Exonwise sequencing of the ß2m gene detected mutations in 5 (29.4%) of 17 MSI-H colorectal cancer samples (Table 3). In three cases, ß2m sequencing was not possible due to poor DNA quality of the paraffin-embedded lesions. All tumors that displayed ß2m mutations were not stained by ß2m-specific mAb L368 in immunohistochemistry (Fig. 1A). All identified mutations were localized at coding repeats of the ß2m gene, the (CT)4 repeat in exon 1 (three samples) and two A5 repeats in exon 2 (four samples; Table 3B). Two of the ß2m-negative tumors had two mutations, one in exon 1 and one in exon 2 (U5 and U17). We could not determine whether these mutations were localized on the same or on different alleles of the ß2m gene. No ß2m mutations were detected in the 20 MSS tumor samples.


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  		Table 3.

 
Expression and mutation analysis of antigen processing machinery components. To identify APM genes that harbor coding mononucleotide repeats and thus may be targeted by mutations during MSI-H tumorigenesis, a database search was done. One T8 mononucleotide repeat was found in the calnexin gene, nine additional repeats composed of six nucleotides were detected in the ER chaperone ERp57, tapasin, the proteasome subunit Delta, the immunoproteasome subunit LMP7, and the TAP subunits TAP1 and TAP2 (Table 1).

Fragment length analysis of these loci revealed frame-shift mutations in the transporter genes TAP1 and TAP2 in 2 of 18 (11.1%) and 1 of 17 (5.9%) of the lesions, respectively. Coding repeats of proteasome subunits Delta and LMP7 and of chaperone molecules calnexin, ERp57, and tapasin were not found to be mutated in the present collective (Table 3).

In addition, the expression of APM components was evaluated by immunohistochemistry. No significant differences were observed between MSI-H and MSS colorectal cancer (Table 4). Interestingly, the three carcinoma lesions that encompassed mutant TAP1 and TAP2 alleles all retained regional immunoreactivity with the respective antibodies and were scored heterogeneous (Fig. 1B).


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  		Table 4. Expression of APM components in MSI-H and MSS colorectal cancer lesions

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Human cancer cells accumulate various structural alterations that may give rise to translation of modified or novel peptide sequences that may be presented to the immune system. Immune cells may recognize these peptides as foreign and attack the respective tumor cells (4). Colorectal cancer cells with deficient DNA mismatch repair that display the MSI-H phenotype are characterized by a pronounced local infiltration with CTL, a phenomenon that has been proposed as a histopathologic criterion to identify MSI-H colorectal cancer (13). This suggested that MSI-H cancer cells may present particularly antigenic peptides. In line with this hypothesis, recent studies indeed revealed a novel class of antigenic peptides generated by frame-shift mutations of genes encompassing coding repetitive sequences. These peptides are apparently abundantly expressed and presented by MSI-H colorectal cancer cells (15–18). This might well explain the selective pressure favoring the outgrowth of ß2m-negative MSI-H tumor cells which are incapable of HLA class I antigen-mediated presentation.

In the present study, we therefore examined in detail the type and frequency of APM component alterations that may be found in MSI-H colorectal cancer cells as a consequence of the immunoselective pressure and compared them with MSS colorectal cancer. Previous studies that examined alterations of antigen presentation in colorectal cancer either did not distinguish between MSI-H and MSS samples (e.g., refs. 31, 32), or relied upon a collection of colorectal cancer specimens containing only a very small proportion of MSI-H tumors (e.g., refs. 19, 20). In this study, we examined for the first time a cohort of colorectal carcinomas that had been typed for their microsatellite status a priori. Thus, equal numbers of MSI-H and MSS samples could be included in the analysis.

Due to the denaturation of the HLA class I complex during the formalin fixation process, the immunohistochemical detection of functionally active HLA heavy chain/ß2m complexes was not feasible. Therefore, ß2m positivity and membraneous localization of anti-HLA heavy chains antibody stainings were used as the criterion indicative for the expression of intact HLA class I complexes on tumor cell surface. Using this approach, a complete loss of membraneous ß2m staining accompanied by loss of membraneous HLA class I heavy chain staining was observed in 60.0% of the MSI-H carcinoma lesions compared with only 30.0% of the MSS carcinomas. Mutations of the ß2m gene were detected in five of these ß2m-negative MSI-H colorectal cancers, in the remaining seven cases, no mutations were detected, suggesting, for example, large deletions which could not be detected by gene sequencing or loss of ß2m expression not related to mutational events. No ß2m mutations were detected in any of the MSI-H tumors displaying positive ß2m staining in immunohistochemistry or any of the MSS tumors. Thus, in a larger collection of MSI-H cases, our study confirms previous studies assuming a close correlation of ß2m mutations with the MSI-H phenotype in colorectal cancer (19, 20). The elevated mutation frequency of the ß2m gene most probably indicates that ß2m represents a relevant target gene during MSI carcinogenesis. In addition, we observed that selective HLA-A down-regulation was significantly more frequent in MSI-H colorectal cancers. Menon et al. (33) have previously reported an association of HLA-A negativity with a better prognosis in colorectal cancer patients; however, MSI typing had only been done in six HLA-negative tumors, with MSI present in three of six (50%) samples analyzed. The authors therefore speculated that there might be a relation of HLA-A negativity with MSI. Our data prove that down-regulation or loss of HLA-A heavy chains is closely associated with the MSI-H phenotype, thus validating the assumption of Menon et al. (33) in a collective that has been MSI-typed a priori.

Notably, significant differences between MSI-H and MSS colorectal cancer were restricted to membraneous HLA-A staining, whereas general HLA-A staining differences did not reach statistical significance. These data may point to the functional relevance of HLA-A loss in antigen presentation breakdown, suggesting that ß2m-positive tumors, potentially capable of HLA class I antigen presentation, are particularly affected by loss of HLA-A expression, thereby reflecting the particular immunoselective pressure exerted on these tumors. In contrast, HLA-B and HLA-C staining results showed significant differences only when regarding general but not membraneous staining. Hence, the significance of loss of HLA-B and HLA-C expression in MSI-H colorectal cancer awaits further clarification.

Previous studies suggested the involvement of proteasome subunits and transporters of antigen presentation in immune evasion of colorectal cancer cells. TAP1 has been reported to be lost in ≥14% of colorectal cancer (32, 34), whereas LMP7 and TAP2 down-regulation has been found to impair antigen presentation predominantly in MSS colorectal cancer (20). We were interested to define whether these APM components or ER chaperones might be targets of microsatellite instability and hit by mutations in cMS. Whereas no mutations were detected in proteasome subunits and ER chaperones, frame-shift mutations in transporter genes TAP1 and TAP2 were identified in 3 of 20 MSI-H carcinomas. These mutations were not detected in corresponding normal tissue samples, underlining that they were not polymorphisms but really somatic mutations occurring during MSI tumorigenesis. TAP1 mutations were detected in two cases; one case harbored a mutation at the C6 repeat of TAP2. Interestingly, none of the tumors with mutations at these loci presented a complete immunohistochemical loss of the corresponding protein. This observation may be explained either by retained protein translation from an intact second allele, or by a positive staining reaction due to the use of antibodies recognizing epitopes located upstream of the mutated repeat. In this case, immunohistochemical staining reactivity may be preserved, even if the corresponding proteins have lost their functional activity in the tumor cells.

Using immunohistochemistry, TAP1 and TAP2 were not detected in 41.0% and 15.0% of the lesions analyzed, respectively. Although not statistically significant, we observed a trend towards a higher frequency of TAP2 loss in MSS colorectal cancer. This observation is in agreement with the data of Cabrera et al. (20) who described dysregulation of TAP2 as one important factor contributing to APM impairment in MSS colorectal cancer.

In summary, our data show that the MSI-H phenotype in colorectal cancer is associated with a high frequency of defects in HLA class I antigen presentation. Most APM components did not reveal significant differences between MSI-H and MSS colorectal cancer, underlining that frame-shift mutations of the ß2m gene represent the predominant cause of antigen presentation breakdown in MSI-H colorectal cancer. In addition, a specific loss of HLA-A heavy chains is significantly associated with the MSI-H phenotype. Moreover, we provide evidence that genes coding for TAP subunits TAP1 and TAP2 may be immediate mutational targets of MSI carcinogenesis. Further studies examining the prognostic effect of APM alterations on the survival of MSI-H colorectal cancer patients are currently in progress.


   Acknowledgments
 
Grant support: Deutsche Krebshilfe and National Cancer Institute, Department of Health and Human Services/USPHS grant CA67108.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank I. Voehringer and B. Kuchenbuch for excellent technical assistance and Y. Schwitalle for critical reading of the article and for helpful discussion.


   Footnotes
 
Note: M. Kloor and C. Becker contributed equally to this article.

Received 1/ 6/05. Revised 4/ 6/05. Accepted 4/28/05.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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