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Keywords:

  • SMAD4;
  • colon cancer;
  • MSI;
  • CIMP;
  • high expression

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References

Colorectal cancer (CRC) is one of the most common causes of cancer-related deaths in western countries. CRC are commonly divided in cancers showing microsatellite stability (MSS) or microsatellite instability (MSI). A more novel classification is dependent on promoter hypermethylation of CpG islands (the CpG island methylator phenotype, CIMP), where cancers show high, low or negative methylation status. SMAD4, located on chromosome 18q, has been thoroughly investigated during the last years. Loss of SMAD4 expression has been reported to correlate with poor CRC patient prognosis. In this study, we analyze the impact of SMAD4 expression on prognosis in relation to MSI screening status and CIMP status. Four hundred and seventy-nine paraffin-embedded specimens of CRC were examined for nuclear SMAD4 expression using immunohistochemistry. The tumors were scored loss (−), moderate (+) and high (++) expressing tumors. Loss of SMAD4 correlated significantly with decreased survival in all colon cancer patients. High SMAD4 expression, however, was significantly associated with increased survival, especially in colon cancer patients, which has undergone potential curative surgery. In addition, in MSI tumors and CIMP-high tumors, high SMAD4 expression was significantly related to increase in survival, while loss of SMAD4 resulted in a significantly poorer prognosis. SMAD4 expression was not correlated to prognosis in rectal cancer cases. We conclude, loss of SMAD4 indicates a poor prognosis in colon cancer patients. The novel findings that high SMAD4 expression predicts a better prognosis suggests that SMAD4 immunohistochemistry could constitute a prognostic marker in combination with CIMP and MSI screening status.

Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths in the western world. Today, early detection is crucial for good prognosis, but roughly 40% of all patients with CRC will die from their disease within 5 years from diagnosis, despite having received the most recent treatment protocols. CRC is considered a highly heterogeneous group of cancers and deeper knowledge of the mechanisms behind colorectal tumorigenesis and progression towards advanced disease is needed.

CRC is commonly divided into specific groups according to genetic or epigenetic profile of the tumor, including characteristics such as hypermethylation and microsatellite stability, respectively. Whereas the topic of microsatellite stability is well established in research and clinical practice, hypermethylation of CpG-rich promoter regions and consequent gene silencing, as well as CpG hypermethylation throughout the genome of CRC cells, are of more recent interest. We and others have shown that patients with a tumor harboring the CpG-island methylator phenotype (CIMP) will have a worse prognosis, compared to those with a CIMP-negative tumor.1–3 Microsatellite instability (MSI) is characterized by widespread mutations in the genome.4 Mutations are enriched due to defects in mismatch repair genes (MMR), and might affect and inactivate tumor suppressor genes, thus driving malignant transformation. MSI has been reported to occur at a rate of 15% in sporadic CRC, but is also seen in hereditary cancers.4 It has often been associated with better prognosis than microsatellite stability (MSS) tumors.4

Many tumor suppressor genes are located on chromosome 18q, where loss of heterozygosity (LOH) frequently occurs late in CRC carcinogenesis.5 18q LOH has been shown to be positively correlated with CIMP-negative MSS cancers,6, 7 and generally results in a poorer patient prognosis.7 One gene that can be silenced by 18q LOH or mutations is SMAD4.8, 9 SMAD4 is an intracellular mediator of TGF-β superfamily signals, where TGF-β, activin and BMP are the activating ligands, regulating cell growth. SMAD4 forms a complex with receptor activated (R)-SMADs and translocates into the nucleus, where it activates transcription of responsive genes.10, 11 Down-regulation or loss of SMAD4 has been suggested to cause resistance to TGF-β superfamily growth inhibition and promote malignant progression in pancreatic tumors.12 Loss of SMAD4 has been reported to occur in CRC in frequencies ranging from 9 to 67%,13–16 and is associated with advanced disease, metastases in lymph node and liver and poor prognosis.9, 15, 17–19 In this investigation of a large set of patients, we analyzed the expression of SMAD4 in relation to MSI screening status and CIMP status in tumor tissue, and related the results to clinical outcome.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References

Patients

Archival paraffin-embedded CRC tissue samples from 493 patients from the CRC in Umeå Study (CRUMS) were included. All samples were collected during primary tumor surgery, over the period 1995–2003, at Umeå University Hospital, Sweden. All routinely stained sections were reviewed by one pathologist (RP), who performed all histopathological classifications including stage, grade and tumor type (mucinous or nonmucinous). Clinical and survival data were obtained by one surgeon (ÅÖ), by reviewing patient records. The survival data were collected at spring 2005. The handling of tissue samples and patient data in this study has been approved by the local ethics committee of Umeå University, Umeå, Sweden.

Immunohistochemistry–SMAD4

Specimens were fixed in 4% formaldehyde and embedded in paraffin, according to routine procedures at the Department of Clinical Pathology, Umeå University Hospital, Sweden. Sections of 4-μm were subjected to heat-mediated antigen retrieval using Diva solution (Biocare Medical) in a Decloaker™ pressure cooker. Anti-SMAD4 monoclonal antibody (B8, Santa Cruz) was used at a concentration of 1:100 in an intelliPATH FLX staining machine (Biocare Medical), where the slides were counterstained with hematoxylin. The specificity of this antibody has previously been confirmed in formalin fixed, paraffin embedded samples.14, 20

The prepared slides were evaluated twice, on separate occasions by one observer (MI-M), and the reviewer was blinded to the identity of the specimens between the first and second evaluation. The slides with differing SMAD4 grading between evaluations were interpreted once again followed by a conclusive judgment. The intra-observer variation between the two scoring occasions was low (<10%). To investigate the interobserver agreement we randomly selected 50 cases to be assessed and scored independently by a gastrointestinal pathologist (RP). The interobserver agreement was good with a kappa statistics of 0.86. To reduce risk of false-negative grading, staining in adjacent stromal lymphocytes, myocytes and fibroblasts were used as positive internal controls. Only areas where tumor cells were surrounded by positive stromal cells were evaluated. A subset of tumors, n = 68 (14%), showed heterogenic expression of SMAD4. In these cases, the parts of the tumor showing the lowest expression of SMAD4 were considered. Fourteen cases (3%) were excluded from further analysis due to poor staining, that is, no staining in internal control cells as described above, leaving 479 cases for further analysis.

Nuclear staining was evaluated according to a scale used in previous investigations of SMAD4 expression:14 −(0–5% cells showing positive staining), + (5–10% positivity), ++(10–33%) and +++(>33%). The + and ++ groups were combined to compare tumors with low, intermediate and high expression of SMAD4, hereafter referred to the novel categories: SMAD4loss (0–5% positivity), SMAD4moderate (5–33%) and SMAD4high (>33%).

MSI screening status

Microsatellite screening status was performed as previously described.1, 21 Briefly, immunohistochemical analyses were performed using standard procedures. Antigen retrieval was performed in EDTA (pH 8.0), followed by antibody treatment in a staining machine (Ventana ES; Ventana). Primary monoclonal antibodies directed towards MLH1 (clone G168-15, Pharmingen, 1:50), MSH2 (clone Ab2, Oncogene Research Products, 1:50), MSH6 (clone 44, BD Biosciences, 1:50) and PMS2 (clone A16-4, Pharmingen, 1:25) were used. For each antibody, samples were evaluated for nuclear staining. Cases without internal positive control staining, such as lymphocytes, were considered uninformative. Tissue samples with tumor cells lacking nuclear staining for MLH1, MSH2, MSH6 or PMS2 were considered to have a positive MSI screening status, hereafter referred to as MSI. Negative MSI screening status based on immunohistochemical staining is hereafter referred to as MSS. MSI screening status examined with immunohistochemistry for MMR protein expression reflects MSI-high status very well, with a sensitivity rate of ∼92% and specificity of ∼100%,22–24 but does not specify for MSI-low.25

CIMP status

To determine CpG island methylator phenotype (CIMP) status we used the MethyLight method (quantitative real-time PCR), as previously described.1 The methods and primer and probe sequences used have been described in detail elsewhere.1, 26, 27 Briefly, DNA was extracted and purified from formalin-fixed, paraffin-embedded CRC tissue. After bisulfate treatment, DNA was used in MethyLight reactions. The samples were run for each of the eight genes included in the CIMP panel (CDKN2A, MLH1, CACNA1G, NEUROG1, RUNX3, SOCS1, IGF2 and CRABP1). Samples were considered positive for methylation when an exponential amplification curve was present and generated a percent of methylated reference (PMR) > 10.26 Tumors with promoter hypermethylation in zero genes were classified as CIMP-negative, 1–5 genes as CIMP-low and 6–8 genes as CIMP-high.

Statistics

Clinicopathological characteristics in subgroups were compared using Kruskal-Wallis tests for continuous variables, and chi-square tests, or Fischer's exact test when expected or observed frequencies were less than five, for categorical variables. For cancer-specific survival analyses, Kaplan-Meier plots were used, and differences between groups were tested by log rank tests. Patients for whom complete follow up data were lacking or, who died with postoperative complications within 1 month after surgery (n = 37), were excluded from the survival analyses, leaving 442 patients for further analysis. In multivariate Cox proportional hazard models, 12 cases with missing values for the MSI screening status variable were treated as separate category. Other cases with missing values in any variable, very few in total were excluded from multivariate analyses. All statistical tests were conducted using the software PASW Statistics 18.0 (SPSS). Findings were considered statistically significant if p < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References

SMAD4 in relation to clinico-pathological variables

A total of 479 cases were evaluated for SMAD4 status with regard to nuclear staining. Clinicopathological characteristics of tumors with SMAD4loss, SMAD4moderate or SMAD4high expression are presented in Table 1. Of all cases, 139 (29%) were SMAD4loss and 247 (51.6%) showed moderate SMAD4 staining. Only 93 (19.4%) of the patients showed SMAD4high expression. When analyzing tumor site, we found that rectal tumors rarely showed high SMAD4 expression.

Table 1. SMAD4 expression in colorectal cancers in relation to clinical pathological variables
inline image

CIMP status and MSI screening status

Table 2 shows SMAD4 expression in relation to MSI screening status and CIMP status. Loss of SMAD4 was related to MSS, whereas SMAD4high was associated with positive MSI screening status. SMAD4high was also common among CIMP-high tumors. When combining MSI screening status with CIMP status, SMAD4high was associated with MSI/CIMP-high and MSI/CIMP-low CRC's.

Table 2. SMAD4 expression in colorectal cancers in relation to MSI screening status and CIMP
inline image

Survival analysis

Survival data were available in 442 cases. Cancer-specific survival was in general shortest for CRC cases with SMAD4loss and highest for SMAD4high (Fig. 1a). In colon cancer, a particularly poor prognosis was found for SMAD4loss compared to SMAD4moderate and SMAD4high (Fig. 1b). In patients who had undergone potentially curative surgery, SMAD4high was associated with longer survival for both colorectal (Fig. 1d) and colon (Fig. 1e) cancer, with a stronger result for colon cancer. SMAD4 expression was not correlated to prognosis in patients with rectal cancer (Figs. 1c and 1f).

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Figure 1. Cancer-specific survival in colorectal cancer. (a) all cases, (b) colon cancers, (c) rectum cancers, (d) all cases of patients undergone potentially curative surgery, (e) colon cancer patients undergone potentially curative surgery and (f) rectum cancers undergone potentially curative surgery. * = <0.05; ** = <0.01; *** = <0.001; ns = not statistically significant.

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Survival analysis and stage

Cancer-specific survival analyses in relation to tumor stage, suggested a longer survival for stage I–III patients with SMAD4high tumors, both for all CRC cases and specifically for colon cancer (Fig. 2). However, the finding was only statistically significant for stage II, in which SMAD4loss was associated with the shortest survival.

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Figure 2. Cancer-specific survival in colorectal cancer in relation to stage. (a) stage I, all cases, (b) stage II, all cases, (c) stage III all cases, (d) stage IV, all cases, (e) stage I, colon cancer, (f) stage II, colon cancer, (g) stage III, colon cancer and (h) stage IV, colon cancer. * = <0.05; ** = <0.01; *** = <0.001; ns = not statistically significant.

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Survival analysis in relation to MSI screening status and CIMP

MSI screening status alone is often found to predict better survival.4 In patients with MSI tumors, SMAD4loss was associated with the shortest, and SMAD4high with the longest, survival (Fig. 3b). In MSS tumors, SMAD4high did not indicate a better prognosis, but SMAD4loss was associated with a significantly shorter survival than SMAD4moderate (Fig. 3a).

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Figure 3. Cancer-specific survival in all cases of colorectal cancer in relation to microsatellite-instability screening status and CpG Island Methylator Phenotype (CIMP). (a) Microsatellite (MSS) stable tumors, (b) Microsatellite-instable (MSI) tumors, (c) CIMP negative tumors, (d) CIMP low tumors and (e) CIMP high tumors. * = <0.05; ** = <0.01; *** = <0.001; ns = not statistically significant.

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SMAD4loss indicate a shorter survival in both CIMP-low (Fig. 3d) and CIMP-high (Fig. 3e) tumors. In CIMP-low tumors, there were no difference in survival for SMAD4moderate, or SMAD4high patients (Fig. 3d), but SMAD4high patients had an increased cancer-specific survival in CIMP-high tumors (Fig. 3e). SMAD4 expression did not have any statistically significant effect on the survival of CIMP-negative patients (Fig. 3c).

Multivariate survival analyses

Based on our previous study on SMAD4 expression and prognosis,13 we performed a multivariate Cox proportional hazard model analysis including sex, age at diagnosis, site, tumor stage and SMAD4 (Table 3). Here, SMAD4loss was significantly associated with a poor prognosis in CRC (HR, 1.81; 95% CI, 1.09–3.00) compared with SMAD4high (referent category). The SMAD4moderate group also had an increased risk of cancer-specific deaths compared with SMAD4high (referent), although not statistically significant (HR, 1.46, 95% CI, 0.90–2.38). When stratifying the analyses by site, SMAD4loss was significantly associated with poorer survival in colon cancer (HR, 1.97; 95% CI, 1.13–3.42), but not in rectal cancer cases (HR, 1.24; 95% CI, 0.39–3.92).

Table 3. Results of a Cox proportional hazard model investigating survival in colorectal cancer patients
inline image

Further adjustment for MSI screening status in the multivariate model for CRC attenuated, and rendered nonsignificant, the HR for SMAD4loss (HR, 1.53; 95% CI, 0.93–2.51). For colon cancer in specific, however, the HRs remained unaltered when the model was further adjusted for MSI screening status (HR, 1.99; 95% CI, 1.13–3.51 for SMAD4loss and HR, 1.40; 95% CI, 0.80–2.44 for SMAD4moderate). Adding CIMP-status to this multivariate model had only minor effects on the risk estimates (data not shown).

Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References

In this study of CRC tissue samples in a large patient cohort, we analyzed the prognostic role of SMAD4 expression in relation to MSI screening status and CIMP status. The main findings were that loss of SMAD4 results in a shorter cancer-specific survival, and high SMAD4 expression tended to be related to an increase in patient survival. The results were largely consistent in both univariate and multivariate analyses. The prognostic impact of SMAD4 expression was most pronounced in patients with colon cancer, and in the patient groups with positive MSI screening status, and among CIMP-positive cases (both CIMP-high and CIMP-low). We did not observe any significant association to prognosis in the large group of MSS and CIMP-negative cases, respectively.

The relevance of TGFβ superfamily-signaling, which is impaired by deletions or mutations in the SMAD genes in CRC, has been thoroughly investigated in recent years. Loss or reduced expression of SMAD4 has been associated with poorer outcome in CRC patients.13–15, 28 It has also been associated with advanced stage, and lymph node- and liver metastases.9, 15, 17–19 In this study, cancer-specific survival analyses revealed a statistically significant reduction in long-time survival in SMAD4loss cases. SMAD4loss occurred more often in MSS CRCs, compared to MSI CRCs, where only 14.1% of MSI positive CRCs showed complete loss of SMAD4, compared to 31.5% of MSS CRCs. The cause of different distribution of SMAD4 expression in MSS and MSI CRCs cannot be fully explained here, but similar results have been reported previously by others.14, 29 Interestingly, the adverse prognosis for SMAD4 negative cases was found to be accentuated in the MSI subgroup in this study, while there was only a small statistically significant difference in survival in the MSS tumors. This is in line with recently reported findings showing that LOH in 18q has no impact on prognosis in MSS CRCs.6 These are interesting results, since SMAD4 is located on 18q, and a correlation between 18q LOH and loss of SMAD4 immunostaining has been reported.8, 9

In previously published articles, we and others have compared SMAD4loss with tumors expressing SMAD4.13, 17 In this study, we further explored the prognostic importance of the specific group of high SMAD4 expression, and found that these tumors might be prognostically distinguished from moderately expressing SMAD4 tumors. SMAD4high tumors show a significantly longer cancer-specific survival compared to SMAD4moderate and SMAD4loss, especially seen in colon cancer patients that have undergone potentially curative surgery. This is a novel evaluation form of SMAD4 expression, and clearly shows the clinical importance to analyze the different expression levels in the search for prognostic markers.

We further analyzed SMAD4 expression in relation to patient CIMP status. We used a well-established and thoroughly evaluated eight-gene panel to identify CIMP-negative, CIMP-low and CIMP-high (see Material and Methods). We chose this panel, since it has been shown that the three CIMP-groups have different characteristics,30–32 suggesting that they should be treated as separate patient groups. When analyzing SMAD4 expression in the different CIMP subclasses of CRC, we found that SMAD4high correlate with CIMP-high tumors. Cancer-specific survival analysis of the three CIMP-groups indicates that SMAD4high is a sign for a better prognosis only in the CIMP-high patient group. SMAD4loss, in contrast, signals bad prognosis in CIMP-low and CIMP-high patients. Together with the findings that SMAD4high tumors largely also are MSI, while SMAD4loss and SMAD4moderate are MSS, these results suggest that the group of SMAD4high tumors is genetically different from the other SMAD4 expression groups.

Furthermore, we found that SMAD4high correlates with increased survival in the group of stage II colon cancer patients. This patient group is not routinely treated with adjuvant chemotherapy, and the need for a good prognostic marker indicating risk of cancer-related death in this group is high. Our results suggest that differential expression of SMAD4 protein, detected by immunohistochemistry, could constitute a useful prognostic tool in stage II colon cancer patients.

The underlying reason for the different results received in colon and rectal cancer patients is somewhat unclear. Certainly, the frequencies of MSI- and CIMP-positive cases differ largely with respect to bowel site, but do not explain why the risk differences only within the group of colon cancers. A substantial part (47%) of our rectal cancer patients received preoperative radiotherapy, and since we used surgical specimens for our SMAD4 expression analyses, this might have influenced not only the expression scores but also the survival analyses. However, the SMAD4 expression scores were not at all correlated to whether preoperative radiotherapy were given (Table 1), which in turn do not support that radiotherapy was the underlying reason for survival differences. Due to low power, our patient material is not optimal for analyzing this prognostic issue further.

Expression of SMAD4 in MSI CRC tumors is interesting since the genes of its corresponding receptors [TGFβ receptor II (TGFβIIR), BMP receptor 2 (BMPR2) and Activin type 2 receptor (ACVR2)] contain microsatellite sequences and will be proned to replication errors in many of the MSI tumors.29, 33–36 Many studies have shown that TGFβ, BMP and activin serves as tumor suppressors,37–40 and mutations in the different molecular parts of their signaling pathways can give the cancer a chance to escape this suppression.9, 17 During the late stages of colorectal carcinogenesis, TGFβ has a different role, and will instead act as a tumor promoter and induce migration, invasion and metastasis.41, 42 It might even be so that it is the loss of SMAD4 that underlies this functional shift of TGFβ from tumor suppressor to tumor promoter.37 The exact mechanism of this is still unknown. With this in mind, and knowing that SMAD4 is translocated into the nucleus as a response to TGFβ superfamily signaling, one would expect that most of the MSI patients would lack SMAD4 in the cancer cells nuclei since so many should, statistically, have mutated receptors. However, in our patient cohort, 85.9% of MSI patients show SMAD4 expression in the nuclei, suggesting a functional signaling pathway. Of these 52.1% are SMAD4high, making us speculate that the higher survival rate in this group is, at least in part, due to growth suppressive signals, even though we have not analyzed TGFβIIR, BMPR2 or ACVR2 mutation or copy-number status. Further analysis to confirm this is needed.

In this study, we do not explore the mechanism behind the change in SMAD4 expression. Even though we cannot exclude the possibility that the effects we see from decreased SMAD4 levels are dependent on deletions of other oncogenes on chromosome 18q, we propose, with support from Miyaki et al.,9 that regulation of SMAD4 is a key event in CRC tumorigenesis.

To summarize, we found that the grading in three SMAD4 expression levels could give guidance for prognostic evaluation of CRC tumors, especially colon cancer. Loss of SMAD4 indicates a poor prognosis. The novel finding that high SMAD4 predicts a better prognosis suggests that SMAD4 immunohistochemistry could constitute a prognostic marker in combination with CIMP and MSI screening status.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References
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