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Clinical Cancer Research 14, 3798, June 15, 2008. doi: 10.1158/1078-0432.CCR-07-5103
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Imaging, Diagnosis, Prognosis

RHAMM, p21 Combined Phenotype Identifies Microsatellite Instability-High Colorectal Cancers with a Highly Adverse Prognosis

Inti Zlobec1, Kristi Baker2, Luigi M. Terracciano1 and Alessandro Lugli1

Authors' Affiliations: 1 Institute of Pathology, University Hospital of Basel, Basel, Switzerland; and 2 Department of Pathology, McGill University, Montreal, Quebec, Canada

Requests for reprints: Inti Zlobec, Institute of Pathology, University Hospital of Basel, Schönbeinstrasse 40, CH-4031 Basel, Switzerland. Phone: 41-61-26-52895; Fax: 41-61-26-52966; E-mail: izlobec{at}uhbs.ch.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Purpose: The aim of this study was to identify prognostic subgroups of microsatellite instability-high (MSI-H) colorectal cancers by combined analysis of 10 well-established immunohistochemical tumor markers and 7 clinicopathologic features.

Experimental Design: Using a tissue microarray, immunohistochemistry was done on 223 cases of MSI-H cancers for the following protein markers: raf-1 kinase inhibitor protein, receptor for hyaluronic acid–mediated motility, apoptosis protease activating factor-1, mammalian sterile20-like kinase 1, p21, p27, p53, ephrin B2 receptor, Ki-67, and epidermal growth factor receptor. Seven clinicopathologic features and all tumor markers were evaluated in univariate and multivariable analyses.

Results: RHAMM overexpression [P < 0.001; hazard ratio [HR; 95% confidence interval (95% CI)], 3.86 (2.19-6.81)], loss of p21 [P = 0.002; 0.33 (0.16-0.67)], and higher N stage [P < 0.001; 3.31 (1.9-5.8)] were independent adverse prognostic factors. RHAMM/p21 combinations were evaluated by N stage. Significant differences in survival were observed with various RHAMM/p21 combinations (P < 0.001). Both node-negative and node-positive patients with RHAMM– tumors survived more than 120 months. Node-positive RHAMM+ patients had a strikingly worse prognosis [16.0 (10.0-63.0) months] and could further be divided into p21– patients [14.0 (9.0-27.0) months] and p21+ patients surviving 47.0 months. RHAMM+/p21– node-negative patients had a significantly shorter survival time than RHAMM+/p21+ tumors (P = 0.021).

Conclusion: These results suggest that the combined phenotype of RHAMM and p21 expression is an invaluable independent prognostic immunohistochemical profile in MSI-H colorectal cancer. Based on the prognostic subgroups identified in our cohort, node-negative patients overexpressing RHAMM but with loss of p21 may derive a potential benefit from postoperative treatment, whereas adjuvant chemotherapy should be reconsidered for MSI-H node-positive RHAMM– tumors.

Colorectal cancer is a heterogeneous disease that can develop through different genetic pathways. The most common is termed the chromosomal instability pathway and accounts for 70% to 85% of colorectal cancers (1). These tumors are characterized by mutations in APC, p53, and KRAS and by frequent allelic loss at 18q (1, 2). Aneuploidy, amplifications, and translocations are also common in these tumors. Familial adenomatous polyposis is the hereditary syndrome associated with these changes (3).

The microsatellite instability (MSI) pathway, comprising the remaining 15% of colorectal cancers, is characterized by loss of proficiency of the DNA mismatch repair (MMR) system and MSI (4). Microsatellites are repeated DNA sequences, usually 1 to 10 nucleotides long, present throughout the genome. Instability is mostly characterized by single base-pair insertions or deletions in these repeat loci, causing widespread genomic instability due to the failure of MMR. Sporadic high-level MSI (MSI-H) cases have been shown to arise through promoter hypermethylation and silencing of the hMLH1 gene (5). Nearly all cases of hereditary nonpolyposis colorectal cancer have MSI-H due to germ-line mutations in one of the DNA MMR genes (6). MSI-H tumors are often right-sided and poorly differentiated and of mucinous or medullary histologic type (4). A predilection for females has been shown as well as frequent mutation of TGFβRII and BRAF genes (4, 7, 8). Patients with MSI-H show a lower rate of both lymph node and distant metastases compared with patients with microsatellite-stable (MSS) tumors (9).

A survival advantage for MSI-H tumors has frequently been reported (10–12). A recent review by Popat et al. including over 7,000 cases, mostly from retrospective studies, reported a significant difference in overall survival between MSI-H and MSS tumors with a 35% relative improvement noted for MSI-H colorectal cancer (13). This improved clinical behavior has been attributed to infrequent mutation of p53, DCC, and KRAS genes and a lower pathologic stage at the time of diagnosis (14, 15). A considerable immune response, as evidenced by the significant presence of peritumoral and tumor-infiltrating lymphocytes (TIL) in MSI-H tumors, have also been underlined as possible mechanisms leading to improved outcome (16, 17).

In contrast, the largest prospective study to date evaluating survival differences in carriers and noncarriers of mutations in DNA MMR repair genes from 870 participants failed to confirm a stage-independent survival benefit in patients with hereditary nonpolyposis colorectal cancer tumors (18). The 5-year survival rate among patients with localized disease was 95% for carriers and 87% for noncarriers, whereas, in metastatic disease, a 5-year survival rate of 42% was noted for both carriers and noncarriers. A recent study by Malesci et al. evaluating survival in 893 patients reported significant findings between MSS and MSI-H tumors in univariate analysis, which were not maintained after adjusting for tumor stage (9).

Data from in vitro studies suggest a strong association between MSI-H and resistance to chemotherapeutic agents such as 5-fluorouracil (19, 20). Clinical studies investigating the effect of 5-fluorouracil and survival in MMR-deficient colorectal cancer have mainly been retrospective and derived from unselected nonrandomized groups and therefore potentially biased (21). However, Ribic et al. who analyzed 570 tissue specimens of stage II and III colorectal cancer from pooled randomized controlled trials described no advantage to 5-fluorouracil treatment in patients with MSI-H colorectal cancers (22). The updated guidelines from the European Group on Tumor Markers do not currently recommend MSI testing for determining prognosis or response to therapy in colorectal cancer but may be used as a prescreen for patients suspected of hereditary nonpolyposis colorectal cancer (23).

Although the prognostic and predictive value of MSI itself in colorectal cancer is under intense investigation, an evaluation of prognostic markers within the MSI-H subgroup has not yet been done. The aim of this study was to determine the independent prognostic power of 7 clinicopathologic features and 10 immunohistochemical tumor markers on a large cohort of 223 MSI-H colorectal cancers to identify subgroups of MMR-deficient tumors having adverse outcome and thus most likely to benefit from adjuvant therapy. The protein markers included in this study were selected for their involvement in cellular mechanisms or signaling pathways known to play a role in tumor progression and metastasis in colorectal cancer. These included mediators of cell cycle arrest (p21, p27, and p53) and metastasis [raf-1 kinase inhibitor protein (RKIP) and ephrin B2 receptor (EphB2)], members of mitogen-activated protein kinase signaling [epidermal growth factor receptor (EGFR) and receptor for hyaluronic acid–mediated motility (RHAMM)], apoptosis-related proteins [apoptosis protease activating factor-1 (APAF-1), mammalian sterile20-like kinase 1 (MST1)], and Ki-67, a marker of proliferation.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Patients. A previously described tissue microarray of 1,420 preoperatively untreated primary colorectal tumors was evaluated (24). Briefly, formalin-fixed, paraffin-embedded tissue blocks of colorectal cancer were retrieved. One tissue cylinder with a diameter of 0.6 mm was punched from morphologically representative tissue areas of each donor block and brought into one recipient paraffin block using a homemade tissue arrayer. Three tissue microarray blocks were generated and 5 µm sections of the tissue microarray blocks were transferred to an adhesive-coated slide (Instrumedics).

Cases (n = 1,197) were determined to be MMR proficient and displayed positive immunohistochemical staining of MHL1, MSH2, and MSH6. The remaining 223 tumors were defined as MSI-H and showed absence of expression of either nuclear MLH1 or MSH2 or MSH6. These MSI-H cases included both sporadic MLH1-negative tumors (n = 141) and 82 presumed hereditary nonpolyposis colorectal cancer cases with loss of MSH2 and/or MSH6 at any age or loss of MLH1 at <55 years (25). The clinicopathologic data included sex, age at diagnosis, tumor location, pT and pN stage, tumor grade, presence of vascular invasion, presence of peritumoral and intraepithelial lymphocytes, and complete follow-up. The median follow-up time was 60 months. For 101 patients, information on postoperative treatment was available. Thirteen (12.9%) patients received adjuvant therapy, including 12 treated only by chemotherapy and 1 receiving both adjuvant radiotherapy and chemotherapy. The remaining 88 (87.1%) patients underwent surgery alone and received subsequent treatment.

Immunohistochemistry. Immunohistochemistry was done for the following protein markers: RKIP, RHAMM, APAF-1, MST1, p21, p27, p53, EphB2, Ki-67, and EGFR according to protocols described elsewhere (16, 26–31). An overview of antibodies used for immunohistochemistry and their corresponding dilutions are summarized in Table 1 . Positive controls consisted of tissues known to express each protein. Negative controls were treated identically with primary antibody omitted.


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  		Table 1. Characteristics of protein marker used in immunohistochemical analysis

 
Evaluation of immunohistochemistry. The evaluation of immunoreactivity was conducted using a semiquantitative scoring method by determining the proportion of positive tumor cells over total number of tumor cells, ranging from 0% to 100%. Scores were reported in 5% increments. The interobserver agreement of this scoring method has been shown previously in tissue microarray punches (29, 30). Staining intensity was not assessed.

Statistical analysis. Immunohistochemical cutoff scores for tumor marker positivity were determined by receiver operating characteristic curve analysis (32, 33). Briefly, the sensitivity and (1 - specificity) for discriminating death or alive/censored was determined at each percentage score and plotted, thus generating a receiver operating characteristic curve. The cutoff score was determined to be the point on the receiver operating characteristic curve where (sensitivity + specificity) was maximized. In addition, 100-bootstrapped replications were done to resample the data and determine the reliability of the cutoff scores for each protein. Tumors with protein expression scores above the receiver operating characteristic curve-derived values were considered "positive" for the protein. Univariate survival analysis was carried out by the Kaplan-Meier method and log-rank test as well as by Cox proportional hazards regression for both univariate and multivariable analyses. Hazard ratios (HR) and 95% confidence intervals (95% CI) were used to determine the prognostic effect of each clinicopathologic feature and protein markers on survival time. A Bonferroni correction for multiple comparisons was done. To maintain an overall significance level of {alpha} = 0.05, only P values ≤ 0.003 after adjustment were considered significant. After obtaining the number of independent variables, a retrospective sample size calculation was done to determine whether the cohort of patients was adequately powered to provide sufficient evidence of a prognostic effect. In a first step, power calculations were done in a univariate setting using the HRs from proportional hazards analysis. In multivariable analysis, adequate sample size was established using the general rule-of-thumb for such analyses, that is, at least 10 outcomes must be observed per independent variable (34). Analyses were carried out with SAS (V9.1, The SAS Institute).


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
Clinicopathologic features. Compared with patients with MSS colorectal cancer, those in the MSI-H group were predominantly female (P < 0.001) and had significantly more right-sided tumors (P < 0.001). Furthermore, these tumors were more often of the mucinous subtype (P < 0.001) and more poorly differentiated (P < 0.001). The majority of MSI-H patients had pT3 (71.6%) or pT4 tumors (21.2%; P < 0.001). CD8+ TILs were found in significantly greater abundance in the MSI-H group (P < 0.001) and the presence of peritumoral lymphocytic infiltration was also considerably more frequent (P = 0.09). Median survival time in MSI-H patients exceeded 10 years and was significantly longer than for patients with MSS tumors (P < 0.001; Table 2 ).


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  		Table 2. Characteristics of patients with MSS and MSI-H colorectal cancer

 
Univariate survival analysis in MSI-H tumors. Seven clinicopathologic features were evaluated for their association with survival time in MSI-H patients, including N stage (node negative, N0, or any number of positive nodes, >pN0), tumor grade, tumor location, vascular invasion, age at diagnosis, and presence of peritumoral lymphocytes and TILs. Of these, higher N stage [P < 0.001; HR (95% CI), 3.69 (2.24-6.07)] and presence of vascular invasion [P < 0.001; HR (95% CI), 2.75 (1.68-4.53)] were significantly associated with poorer survival (Table 3 ). Adjuvant treatment was not linked with survival time in these patients [P = 0.786; HR (95% CI), 1.19 (0.35-4.08)].


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  		Table 3. Association of clinicopathologic features with survival time in patients with MSI-H colorectal cancer

 
Of the 10 immunohistochemical markers analyzed, positive expression of RHAMM [P < 0.001; HR (95% CI), 3.0 (1.81-4.96)] and loss of p21 [P = 0.003; HR (95% CI), 0.38 (0.2-0.71)] were the only proteins significantly associated with adverse survival time after adjustment for multiple comparisons (Table 4 ). Representative immunostains for RHAMM and p21 are illustrated in Fig. 1 .


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  		Table 4. Association of protein markers with survival time in patients with MSI-H colorectal cancer

 

Figure 1
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  		Fig. 1. Differential p21 and RHAMM expression in MSI-H colorectal cancer: nuclear p21 positivity (A) and negativity (B). Diffuse (C) and focally weak (D) cytoplasmic RHAMM expression.

 
Multivariable analysis. N stage, vascular invasion, and RHAMM and p21 expression were entered into multivariable analysis. N stage [P < 0.001; HR (95% CI), 3.31 (1.9-5.78)], positive RHAMM expression [P < 0.001; HR (95% CI), 3.86 (2.19-6.81)], and negative p21 expression [P = 0.002; HR (95% CI), 0.33 (0.16-0.67)] showed independent prognostic value in MSI-H tumors. Kaplan-Meier curves and corresponding P values from the log-rank test in Fig. 2 show the effect of each of these three features on survival time. In addition, the prognostic effect of each of these variables is highlighted in Table 5 , which describes the interquartile range of survival time for N stage, RHAMM and p21 expression, and incremental value of the addition of each of these features to poor outcome.


Figure 2
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  		Fig. 2. Kaplan-Meier survival plots illustrating survival time differences by lymph node status (N0, >N0; A) with the addition of RHAMM expression to lymph node status (B) and the addition of RHAMM and p21 in N0 (C) and >N0 (D) patients.

 

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  		Table 5. Interquartile ranges for survival time depicting the incremental value of the addition of each feature to poor outcome

 
Survival times were strikingly different with various multifeature combinations (P < 0.001). Three of the four node-negative tumor groups showed a median survival time exceeding 120 months. These included RHAMM– tumors as well as those with RHAMM+/p21+. In contrast, node-negative patients with simultaneous RHAMM+/p21– had a significantly worsened median survival time of 84 months.

Node-positive patients clustered into two groups based on their survival times, that is, those with RHAMM– tumors displaying a survival time exceeding 120 months and the second with RHAMM+ tumors with a median survival time of only 16.0 (10.0-63.0) months. p21 expression further subdivided node-positive RHAMM+ patients, with p21– tumors resulting in decreased survival time to 14.0 (9.0-27.0) months and p21+ expression considerably improving prognosis to 47.0 months (P = 0.021).

Survival times for the multifeature combinations are summarized in Fig. 3 , which highlights the significant adverse prognostic effect of RHAMM positivity in both node-negative and node-positive patients and the additional value of p21 to further categorize RHAMM+ tumors.


Figure 3
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  		Fig. 3. Multifeature combinations of lymph node status (N0, >N0), RHAMM (negative, positive) and p21 (negative, positive) expression, and median survival times in microsatellite-instable colorectal cancers. Top parentheses, number of deaths, number of survivors; bottom parentheses, percentage of deaths, percentage of survivors.

 
Sample size. To determine whether the cohort of MSI was adequately powered, a retrospective analysis was done based on the sample size, number of outcomes, and HRs for the independent variables, that is, N stage and RHAMM and p21 expression. In a univariate setting, the power was 99% for N stage, 98% for RHAMM, and 83% for p21. For multivariable analysis, at least 30 deaths from colorectal cancer were needed for the sample size to be judged as adequate. In this study, 61 outcomes were observed (Fig. 3), therefore indicating an appropriate size of the study cohort.

RHAMM and p21 in MSS colorectal cancers. Adjusting for the effects of RHAMM, p21 expression was not significantly associated with prognosis in MSS colorectal cancers [P = 0.099; HR (95% CI), 0.84 (0.68-1.03)]. In contrast to MSI-H cases, p21 was not found to have prognostic value independently of pT, pN, tumor grade, vascular invasion, and tumor border configuration [P = 0.365; HR, 0.91 (0.73-1.12)]. RHAMM expression maintained its significant effect on prognosis in multivariable analysis with these well-established prognostic features [P < 0.001; HR, 1.9 (1.6-2.3)].


   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
MSI-H colorectal cancers represent a small but important subgroup of colorectal tumors characterized by a generally favorable prognosis (4). Although many studies have compared survival between cancers with MSI and those with chromosomal instability, few studies have focused specifically on prognostic indicators within the MSI-H subset. The findings of this study on a large cohort of MSI-H colorectal cancers have resulted in an easily detectable immunohistochemical phenotype predictive of a decreased survival time in patients with these tumors. Specifically, patients expressing simultaneous positivity for RHAMM and negativity for p21 have a considerably shortened survival time.

Patients with node-positive colorectal tumors are typical candidates for adjuvant chemotherapy (35). Although several studies suggest that a subset of patients with node-negative tumors derive a positive effect from adjuvant treatment, chemotherapy is not yet recommended in the clinical management of stage II disease (36). A growing body of evidence suggests that MSI-H tumors are more chemoresistant than their MSS counterparts despite their comparatively prolonged survival (10, 22, 37–39). Hence, there is a need to identify patients with MSI-H colorectal cancers who not only may benefit significantly from adjuvant chemotherapy but also could be spared treatment.

Our findings using a multimarker combination of RHAMM and p21 have underlined the striking heterogeneity in outcome in patients with MSI-H tumors. Node-negative MSI-H patients with RHAMM+/p21– expression have a significantly more adverse prognosis than their node-negative RHAMM– or RHAMM+/p21+ counterparts. In contrast, patients with node-positive disease but RHAMM– tumors have a survival time exceeding 10 years and are therefore comparable with the majority of node-negative tumors. This result may have significant clinical implications for the treatment of MSI-H patients, that is, that node-positive patients with RHAMM negativity could be spared adjuvant treatment, whereas node-negative RHAMM+ patients who would otherwise not be considered for adjuvant treatment may in fact derive a survival benefit from postoperative therapy.

Our findings also outline the importance of evaluating multimarker phenotype combinations of immunohistochemical protein markers (40). According to our results, patients with highly adverse node-positive disease can be identified by immunostaining for RHAMM only, with RHAMM+ patients surviving little more than 1 year and RHAMM– patients surviving over 10 years. On the other hand, depending on their simultaneous p21 expression, node-negative RHAMM+ patients differ significantly in terms of survival time, with p21– tumors exhibiting a considerably shortened survival time of 83.0 months compared with p21+ patients.

RHAMM is a multifunctional glycoprotein that is often up-regulated in advanced malignancies (41). RHAMM exists in several variant forms and can be expressed both intracellularly and at the cell surface, where it may exert distinct functions (41, 42). RHAMM is a receptor for the extracellular matrix protein hyaluronan and both have been implicated in enhanced tumor cell motility (41–43). Additionally, RHAMM modulates the activity of several receptor tyrosine kinases and has been shown to interact directly with members of the mitogen-activated protein kinase pathway, leading to constitutive Erk activation (41, 44–46).

Overexpression of RHAMM has been identified as an adverse prognostic factor in studies of both breast and colorectal cancers (24, 46–48). Our findings are thus consistent with previous reports and suggest that the influence of RHAMM extends to MSI-H colorectal cancers despite the relatively high mutation rate of RHAMM reported in this subset of neoplasms (49). In most cases, the pathogenicity of RHAMM has been attributed to its ability to promote tumor cell migration. Both RHAMM and hyaluronan localize to the advancing lamellae of migrating cells and have been shown to be necessary players in transforming growth factor-β-induced cell migration (42, 50). This explanation likely extends to the results of the present study, particularly our findings with respect to node-positive cancers, where RHAMM expression was most discriminatory. The median survival time of all patients with RHAMM– tumors, regardless of whether they were positive or negative for lymph node spread, was greater than 120 months. In contrast, patients with RHAMM+ tumors fared much worse, particularly if they were also positive for lymph node spread. These results suggest that although it is not strictly necessary for baseline tumor cell motility, RHAMM is an important contributor to tumor cell migration and dispersal when it is expressed. Node-positive cancers with decreased RHAMM expression may be lacking an essential promoter of baseline tumor motility without which they are unable to seed distant metastases. Such an autocrine hyaluronan-mediated and RHAMM-dependent mechanism for basal tumor cell motility has been shown recently in invasive breast cancers (41).

Expression of p21 appears to act as a modifying factor on the survival time in RHAMM+ tumors. p21, also known as Waf1 and Cip1, is a potent inhibitor of cyclin-dependent kinases and acts as a checkpoint control at the G1-S phase of the cell cycle (51, 52). Both p53-dependent and p53-independent mechanisms are known to modulate the expression of p21. Among its other tumor-suppressing functions, p21 is known to inhibit cellular proliferation and to protect cells from apoptosis induction. In RHAMM+ MSI-H colorectal cancer, negativity for p21 has a particularly devastating effect on survival time. In node-positive, RHAMM+ cancers, p21 positivity increases median survival time from 14 to 47 months. It is likely that the absence of p21 is indicative of a tumor possessing a higher proliferative capacity and which has lost its G1 checkpoint control. Loss of p21 in cancers that also have a highly motile phenotype, such as those expressing high levels of RHAMM, would produce a tumor capable not only of easily spreading into the bloodstream but also of proliferating in new locations to permit the establishment of metastases. Further research will be needed to confirm the validity of this hypothesis in MSI-H cancers.

MSI testing by molecular analysis was not done in this study for several reasons. First, the clinicopathologic features described in our study are highly consistent with the well-established characteristics of MSI-H tumors. According to Jenkins et al., predictive features of MSI-H include tumors more frequently located on the right-side, of poor differentiation, and more often of mucinous histologic subtype. MSI-H tumors also tend to show a greater frequency of Crohn's-like reaction or inconspicuous peritumoral lymphocytic infiltration and an increased number of TILs compared with MSS tumors (53, 54). Preponderance for females is also recognized as an additional characteristic of MSI-H (55). Second, high sensitivity and specificity of immunohistochemical analysis for MSI has been reported by several studies (56–60). Lindor et al. evaluated more than 700 colorectal cancers and showed 92.3% sensitivity for detecting MSI via MLH1 and MSH2 immunostaining and a predictive value of 100% for MSI-H with absence of MLH1 or MSH2 staining (56). Third, MSI-low tumors display the same staining pattern as MSS tumors, that is, positivity for the MMR proteins (56). As defined in our study, a diagnosis of MSI-H can therefore be considered conclusive on discovery of immunohistochemical loss of MMR proteins. Finally, MSI testing is recognized as infeasible in routine pathology laboratories and evidence supporting its integration into routine practice is currently lacking (61).

The immunohistochemical analysis of RHAMM and p21 protein expression appears to provide invaluable prognostic information in patients with MSI-H colorectal cancers. Based on the prognostic subgroups identified in our cohort, node-negative patients overexpressing RHAMM but with loss of p21 may derive a potential benefit from postoperative treatment, whereas adjuvant chemotherapy should be reconsidered for node-positive MSI-H RHAMM– tumors.


   Disclosure of Potential Conflicts of Interest
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No potential conflicts of interest were disclosed.


   Footnotes
 
Grant support: Novartis Foundation (formerly Ciba-Geigy-Jubilee Foundation; I. Zlobec) and Canadian Institute of Health Research (K. Baker).

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.

Received 12/ 7/07; revised 2/29/08; accepted 3/12/08.


   References
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 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

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