Prognostic role and clinicopathological features of SMAD4 gene mutation in colorectal cancer: a systematic review and meta-analysis

DOI: https://doi.org/10.21203/rs.3.rs-379114/v1

Abstract

Background

Approximately 5.0%-24.2% of colorectal cancers (CRCs) have activating mutations in SMAD4, making it one of the frequently mutated genes in CRC. We thus carried out a comprehensive system review and meta-analysis investigating the prognostic significance and clinicopathological features of mutation of SMAD4 gene in CRC.

Methods

A detailed literature search was conducted in PubMed, Web of Science and Embase databases to study the relationship between mutations of SMAD4 gene and the demographic and clinicopathological characteristics in patients with CRC. The hazard ratios with 95% confidence intervals were used to evaluate the effect of SMAD4 mutations on overall survival (OS) and progression-free survival (PFS)/recurrence-free survival (RFS).

Results

Ten studies enrolling 4394 patients were eligible for inclusion. Data on OS were available from five studies. Comparing SMAD4-mutated CRC patients with SMAD4 wild-type CRC patients, the summary HR for OS was 1.46 (95% confidence interval [95% CI] 1.28–1.67, P=0.001), the summary HR for PFS/RFS was 1.59 (95% CI=1.14–2.22, P=0.006). In terms of clinicopathology parameters, SMAD4 mutations were associated with tumor location (odds ratio [OR]= 1.15, colon/rectum, 95% CI=1.01-1.31, P=0.042), TNM stage (OR=0.78, stage Ⅰ-Ⅲ/Ⅳ, [95% CI] 0.63-0.97, P=0.025), lymph node metastases (OR=1.42, N+/N0, 95% CI=1.20-1.67, P<0.001), mucinous differentiation (OR=2.23, 95% CI=1.85-2.70, P<0.001) and rat sarcoma viral oncogene homolog (RAS) status (OR=0.47, RAS wild-type/RAS mutation, 95% CI=0.30-0.73, P=0.001). No connection was found with age, gender, tumor grade, microsatellite instability (MSI) status and b-viral oncogene homolog B1(BRAF) status. Besides, publication bias was not observed in all studies.

Conclusions

This meta-analysis suggests that SMAD4 mutation was associated with OS, PFS/RFS, and clinicopathological parameters, including tumor site, disease stage, RAS status, lymph node metastases and tumor mucinous differentiation. It was indicated that SMAD4 mutations could predict the poor prognosis and aggressive clinicopathological characteristics of CRC. More large-sample cohort studies were needed to further confirm this conclusion. As SMAD4 mutation was found to be closely associated with RAS mutations, their relationship was worth further investigating. 

Background

Colorectal cancer (CRC) is the third most common cancer and the second most common cause of cancer-related death over the world[1]. Despite advances in early diagnosis and treatment, lymphatic metastasis and distant metastasis are still the main causes of death in newly diagnosed CRC patients, and the overall survival (OS) rate of advanced CRC is still unsatisfactory.

The Cancer Genome Atlas database revealed that the mutation frequency of SMAD4 is 10%, which is one of the most common mutated genes in CRC[2]. SMAD4 is the established tumor suppressor gene locating on chromosome 18q21, and one of the most commonly destroyed gene in cancer among SMAD family genes[3]. This gene encodes a member of the Smad family of signal transduction proteins, that is phosphorylated and activated by transmembrane serine-threonine receptor kinases in response to transforming growth factor beta (TGF-β) signal transduction. The product of this gene forms homomeric complexes and heteromeric complexes with other activated Smad proteins in the context of activating by TGF-β receptors, then accumulate in the nucleus and regulate the transcription of target genes[4]. Mutations or deletions in the SMAD4 gene have been shown to result in pancreatic cancer[5], juvenile polyposis syndrome[6], and hereditary hemorrhagic telangiectasia [7]. In the past two decades, many studies had shown that SMAD4 mutation can not cause tumorigenesis by itself, but promote tumor progression caused by other genes[8]. The role of SMAD4 in colorectal cancer is similar to that in pancreatic cancer. The prevalence of SMAD4 mutations have recently been reported in 5.0–24.2% of several retrospective studies of sporadic CRC from 1999 to 2020[914]. However, whether SMAD4 mutation  reduces the OS in all patients with CRC remains unclear. Therefore, we conducted a meta-analysis to assess the correlation between SMAD4 mutations and overall survival and clinicopathological characteristics of early and advanced CRC.

Methods

Search strategy

We conducted this study based on the preferred reporting items for Systematic Reviews and Meta-Analyses 2009 guidelines. Systematic review of several databases was conducted in December 2020 with no lower limit set for date of publication. Search for related articles published in English and Chinese in the following electronic databases: PubMed, Web of Science, and Embase. The keywords “SMAD4” or “DPC4” and “colorectal cancer” or “colon cancer” or “rectum cancer” were used for relative articles searching.

Study selection and inclusion criteria

All articles are limited to human studies published in English and Chinese that based on the following selection criteria: 1) Researches involved the prognostic of SMAD4 mutations in CRC patients, and provided sufficient information to obtain the Hazard ratios (HRs) and 95% confidence interval (CI) of OS directly or indirectly from the Kaplan-Meier curve. 2) Studies using surgical resection specimen of tumor in CRC to detect SMAD4 mutation. 3) The OR associated with clinicopathologic features directly gives or provides computable data. 4)The study does not include patients with CRC patients who received preoperative chemotherapy or radiotherapy. 4) Duplicate report results are unified by the latest or largest version.

Data extraction and quality assessment

The two authors (F.T. and L.T) extracted all data sets from the selected studies independently, if there are any objections, we resolved through consensus or consultation with the corresponding author. The following information was collected from each study: first author, year of publication, country, time of diagnosis, sample size, colorectal cancer cases with SMAD4 gene mutations, sequencing methods of SMAD4 gene, mean follow-up periods and participants’ characteristics, including median age, gender, lymph node metastasis status, rat sarcoma viral oncogene homolog (RAS), b-viral oncogene homolog B1 (BRAF), microsatellite instability (MSI) status and mucinous differentiation as well as tumor stage. HRs and 95%CI of OS and progression-free survival (PFS)/recurrence-free survival (RFS). we extracted from papers directly, if not, we choose to extract from Kaplan-Meier Curve via Engauge Digitizer Version 4.1(http://markummitchell.github. io/engauge-digitizer/). We used the Newcastle-Ottawa Scale to assess the methods and report quality of the included studies, and ranked them by score (8-9 points for high quality; 5-7 points for medium quality; less than 5 points for low quality)[15].

Statistical analyses

HRs and odds ratios (ORs) with their 95% confidence intervals were calculated. P value less than 0.05 was considered statistically significant. The Q statistic and I2 tests was used to estimate Heterogeneity among studies. The I2 statistic was ranged from 0 to 1. A random effect model was used for I2 >50%, which represented strong heterogeneity. Otherwise, fixed‐effect model would be applied. The analysis was performed to evaluate the impact of SMAD4 gene mutation on the prognosis of CRC. In addition, we evaluated the SMAD4 mutation in different tumor grades, tumor differentiation, and the correlation between SMAD4 status and lymph node metastasis status, as well as MSI/BRAF/RAS status. Sensitivity analysis was used to check data stability. The Egger’s test and Begg's test were used for detection of publication bias, and P < 0.05 indicated significant bias. All analysis was performed with STATA 16.0 (Stata Corporation, College Station, TX, USA).

Results

Selection of studies

The flowchart of the study selection is shown in Fig. 1. There were 465 articles identified from PubMed, 686 articles from Web of Science, 895 articles from Embase database. A total of 2045 articles were initially identified by the search strategy, and 657 full-text articles were retrieved after screening. Each selected article is tracked forward and backward, in case they contain another research of interest that has not yet been identified. Second, 1280 unrelated titles and abstracts were excluded from the study, and 108 full-text articles were evaluated for applicability, 7 articles were found to have no available outcome indicators or clinicopathologic features, 63 articles relate to SMAD4 protein expression and survival data, 12 articles were non-human trials and 15 articles were review, letter and case port. A total of 4394 patients were included in the final ten studies[9-12, 16-21]. The detailed features of these articles are listed in Table 1 and Table 2. The quality evaluation table of all articles is attached in Table 3.

Relationship between SMAD4 mutations and CRC prognosis

A total of 5 articles provided OS related data. Due to the moderate heterogeneity (I2=41.6%, P heterogeneity = 0.144), We use the fixed-effect model to pool HR. Comparing SMAD4 mutant CRC patients with SMAD4 wild-type CRC patients, the summary HR for OS was 1.46 (95% CI=1.28–1.67, P=0.001). (Fig 2.)

A total of 2 articles provided PFS/RFS related data. We Comparing SMAD4 mutant CRC patients with SMAD4 wild-type CRC patients, the summary HR for PFS/RFS was 1.59 (95% CI=1.14–2.22, P=0.006). (Fig 3.) and there was moderate heterogeneity between the studies (I2 = 48.2%, P heterogeneity = 0.145). So that we use the fixed-effect model to pool HR.

Relationship between SMAD4 mutations and clinicopathologic features of CRC

A total of 9 studies have data that can be extracted from clinicopathologic results, the specific characteristics of which are detailed in Table 2. The clinicopathologic OR values of the final merger are presented in Table 4. In terms of clinicopathology parameters, SMAD4 gene mutations were associated with tumor location (OR= 1.15, for colon versus rectum, [95% CI] 1.01-1.31, P=0.042), TNM stage (OR=0.78, for stage Ⅰ-Ⅲ vs Ⅳ, [95% CI] 0.63-0.97, P=0.025), lymph node metastases (OR=1.42, for N0 vs N+, [95% CI] 1.20-1.67, P<0.001), mucinous differentiation (OR=2.23, [95% CI]1.85-2.70, P<0.001) and RAS status (OR=0.47, for RAS wild-type vs mutant RAS, [95% CI] 0.30-0.73, P=0.001). And SMAD4 gene mutations has no connection with other clinicopathology parameters, including age, gender, tumor grade, MSI status and BRAF status.

Sensitivity Analysis and publication bias

Our analysis of publication bias using correlation test revealed that there is no obvious publication bias for OS (P=0.277 for Begg's test and 0.221 for Egger's test) (Fig.4) and PFS/RFS (P =0.235 for Begg's test and 1.000 for Egger's test) (Fig.5). In addition, the Sensitivity analysis confirmed that the results were reliable for OS (Fig.6) and PFS/RFS (Fig.7).

Discussion

Several studies have investigated the role of SMAD4 mutation of the prognosis and clinicopathological parameters in CRC, but the results are not consistent. Moreover, there is no meta-analysis to evaluate the impact of SMAD4 gene on the prognosis of CRC. Therefore, we conducted a meta-analysis and suggest that SMAD4 mutation is associated with poor prognosis in CRC. Compared with the SMAD4 wild-type controls, SMAD4 mutation is associated with worse OS (pooled HR = 1.46, 95% CI = 1.28–1.67, P < 0.001) and worse PFS/RFS (HR = 1.59, 95% CI = 1.14–2.22, P = 0.006). In order to further investigate the role of SMAD4 gene in colorectal cancer, we also analyzed the relationship between SMAD4 status with clinical pathological parameters of colorectal cancer, the results show that patients with SMAD4 mutation in colorectal cancer have higher TNM stages (I- III/IV; pooled OR = 0.78; 95%CI = 0.63–0.97; p = 0.025), that is, patients with SMAD4 mutation are more likely to occur distant metastasis. And SMAD4 mutant patients were more likely to feature mucinous differentiation (pooled OR = 2.23; 95%CI = 1.85–2.70, P = 0.000), tumors are more likely to occur in the colon (pooled OR = 1.15; 95%CI = 1.01–1.31; P = 0.042), more prone to lymph node metastasis (N0/N+; pooled OR = 1.42; 95%CI = 1.20–1.67; p = 0.000), and to harbor concurrent RAS mutations (pooled OR = 0.47; 95%CI = 0.30–0.73; P = 0.001). The important thing is all of these parameters often indicated a poor prognosis. Combined OR suggested that SMAD4 gene mutation has nothing to do with MSI and BRAF status. The role of SMAD4 gene affecting MSI or BRAF status remains to be elucidated. There is a meta-analysis showed that SMAD4 mutation (combined OR 2.04, 95% CI 1.41–2.95) were at a higher risk of distant metastasis[14], which is consistent with our results.

Over the past two decades, many studies have shown that SMAD4 mutation does not cause tumorigenesis by itself, but promote tumor progression caused by other genes[8].Ohtaki et al. reported that the frequency of SMAD4 mutations was significantly higher in tumors with liver metastasis than in those without such metastasis[22]. Inamoto et al. reported that SMAD4-deficient colorectal tumor cells secreted more CCL9 and CCL15, two chemokines that recruit CCR1 + myeloid cells through CCL9-CCR1 and CCL15-CCR1 axis, resulting in metastasis[23]. Vauthey et al. concluded that patients with SMAD4 mutations are less likely to undergo repeated hepatectomy for recurrent disease following initial tumor resection[24]. Alhopuro et al. showed that SMAD4 is a predictive biomarker for 5-fluorouracil (5-Fu) based chemotherapy in CRC patients[25]. Zhang et al. discovered a novel mechanism mediated by SMAD4 to trigger 5-Fu chemosensitivity through cell cycle arrest by inhibiting the PI3K/Akt/CDC2/survivin cascade[26]. Mei et al. suggested that SMAD4 mutations could be potential biomarkers for poor prognosis of cetuximab-based therapy [27], which needs to be further validated in a larger patient cohort. Lin et al. found that silencing SMAD4 reduces the sensitivity of colorectal cancer cells to cetuximab by promoting epithelial-mesenchymal transition(EMT), while the high expression of Smad4 may be clinically beneficial to cetuximab-based therapy[28].Mizuno et al. found that SMAD4 gene mutations were significantly associated with worse OS following hepatic resection, which was independent of RAS mutation status[11]. These findings indicate that SMAD4 genetic alteration has a key role in tumor progression and efficacy of target therapy for CRC patients.

In the current analysis, we found that SMAD4 gene alteration was significantly associated with loss of SMAD4 expression in CRC, and loss of SMAD4 disrupts canonical TGF-β signaling[29], because it is a transcription factor for signaling. In addition, it is reported that the loss of SMAD4 function is independently associated with the reduction of RFS and OS in CRC patients, especially patients with advanced disease[30]. In contrast, the median overall survival in CRC patients with high Smad4 expression is much longer than that with low Smad4 expression[31]. Germline mutations in genes in the TGF-β family signaling pathway strongly increase the risk of colonic neoplasia[32]. The canonical TGF-β/Smad4 signaling pathway acts as a tumor suppressor in the early stages, which is characterized by its anti-proliferative activity, ability to induce apoptosis and promote genome stability, while in the late stage of tumor, TGF-β acts as a tumor and metastasis promoter to stimulate tumor development[33].

Studies have suggested that EMT is a is a key step in tumor progression and metastasis, and theTGF-β1 signaling plays a key role in EMT[34]. EMT is a well-coordinated process in which epithelial cells lose cell connectivity and polarity and transform into mesenchymal cells with migration and invasion capabilities. Functional study results indicate that TGF-β-induced Smad4-dependent EMT followed by apoptosis in colorectal cancer cells[35, 36]. Siraj et al. identified TGF-β-induced EMT insufficient to obtain invasive potential, the activated Ras TGF-β will change the reaction, imparting tumorigenic and invasive potential[37]. Therefore, the synergistic effect between Ras-Raf-MAPK and TGF-β/Smad cascades are needed to obtain the aggressive phenotype in cancer.

At present, RAS has been recognized as tumor driver genes, predictive biomarkers and therapeutic targets in CRC. The expression of RAS up-regulates the expression of phosphotyrosine kinase receptors ERBB1 (EGFR) and ERBB2 (HER2) and induces an aggressive phenotype. Smad4-dependent signal transduction negatively regulates the expression of these receptors and inhibits the up-regulation of EGFR and ERBB2 induced by RAS, thereby play an anti-proliferative effect. The loss of oncogenic RAS and SMAD4 signals synergistically up-regulate the abnormal expression of EGFR and ERBB2, leading to the development of cancer and the metastasis and spread of the primary tumor[38, 39]. TGF-β can quickly activated RAS and ERK pathway[40], In contrast, the ERK pathway inhibits the TGF-β/Smad4 pathway by phosphorylating Smad2 and Smad3 at serine or threonine residues in the linker region, so epithelial cells with oncogenic RAS mutations usually exhibit loss of TGF-β antiproliferative response[8].Patients with RAS wild-type tumors and retained SMAD4 wild type had an overall survival longer than cases with mutations in both genes[41]. However, regardless of the RAS mutation status or other clinicopathological factors, SMAD4 mutations are significantly associated with poor OS. The precise cooperative mechanisms of SMAD4 with other genes of influence also requires further examination.

Given the relative frequency of SMAD4 mutations in CRC patients, routine SMAD4 testing may be appropriate. For individualized treatment of CRC, further research should be done for guiding clinical decision-making, which SMAD4 is a driver mutation and will be a novel target for precision medical treatment of CRC.

No heterogeneity or publication bias was found in this meta-analysis, and sensitivity analysis shows that our results are reliable. However, this analysis has several limitations. First, our meta-analysis included studies of qualified articles published in English and Chinese, did not include some relevant articles written in other languages or unpublished papers, which is likely to result in selection bias. Second, the use of specific therapies and tumor stage differed among the included articles. Third, the HR calculated from the data or extracted from the survival curve may not be as reliable as the HR calculated directly using the analysis of variance. Therefore, the results should be carefully interpreted. However, as far as we know, this is the first meta-analysis to demonstrate SMAD4 mutation by evaluating the pathological features and prognostication in CRC.

Conclusion

In conclusion, we found that SMAD4 mutation was associated with poor prognosis in CRC, but has nothing to do with MSI status, BRAF status and tumor grade. Further studies are needed to evaluate these findings and the clinical significance of SMAD4 status in CRC.

Abbreviations

CRC: Colorectal cancer

OS: overall survival

TGF-β: transforming growth factor beta

HRs: Hazard ratios

CI: confidence interval

RAS: rat sarcoma viral oncogene homolog

BRAF: b-viral oncogene homolog B1

MSI: microsatellite instability

PFS: progression-free survival

RFS: recurrence-free survival

ORs: odds ratios

5-Fu:5-fluorouracil

EMT: epithelial-mesenchymal transition

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analyzed during this study are included in this article.

Competing interests

The authors declare that they have no competing interests.

Funding

The authors declare that they did not receive funding for this research from any source.

Authors' contributions

All authors provided intellectual input into the study design and methodology. Tian Fang, Tingting Liang, and Yizhuo Wang screened texts, performed data extraction and risks of bias assessment. Tian Fang and Ting-Ting Liang drafted the manuscript. All authors provided comments and edited the manuscript to become the final version for submission. All authors approved the final version of the manuscript.

Acknowledgements

Not applicable.

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Tables

Table 1. Main characteristics of studies included.

Author

Year

Country

MA(Year)

TNM stage

time of diagnosis

MF (months)

Sample size

Sequencing methods

MutWT

Survival

endpoints

HRe

NOS

Sarshekeh

2017

UNITED STATES

52

II-Ⅳ

2000-2014

50 

734

HiSeq sequencing system

90:644

OS

PFS

HR

9

Mizuno

2018

USA

56 

2005-2015

22 

237

NGS

31:206

OS

RFS

HR

Curve

9

Oyanagi

2019

Japan

NR

I–IV

2009-2015

NR

201

NGS

56:145

OS

Curve

7

Liao 

2019

China

NR

I-Ⅳ

2013-2017

NR

84

NGS

12

NR

NR

8

Fleming

2013

Australia.

69

I-Ⅳ

NR

NR

744

Applied Biosystems

64:680

NR

NR

6

Stahler 

2020

Germany

NR

2007-2012 

40·3 

373

NGS

NR

OS

PFS

HR

8

Jia

2017

USA

NR

NR

NR

NR

53

NR

4:49

NR

NR

6

Ando

2005

Japan

66

A-D*

NR

NR

30

NR 

1:29

NR

NR

7

Miyaki

1999

Japan

NR

NR

NR

NR

61

NR

9:52

NR

NR

6

Khan

2018

US

NR

NR

2012-1016

51.6

1877

NGS

226:1599

OS

HR

7

Abbreviations: NR=not report; MA=mean age; MF=median follow-up; e=estimate. *Dukes’stage
 
 

Table 2. Data extracted from studies included.

study

SMAD4

status

Ageyears

Gender

Location

  Stage

Tumor grade

MSI status

RAS status

BRAF status

LN

Mucinous

65

≥65

Female

Male

Colon

Rectum

I-III

WMD

PD

Stable

Unstable

WT

Mut

WT

Mut

Yes

No

Yes

No

Sarshekeh

2017

Mut

WT

75

15

52

38

75

15

6

74

50

13

NR

NR

NR

NR

NR

561

83

286

358

410

234

85

526

357

114

Mizuno

2018

Mut

WT

NR

17

20

28

9

NR

34

3

NR

10

27

34

3

31

6

NR

110

131

218

60

214

27

135

106

238

3

148

93

Oyanagi

2019

Mut

32

24

19

37

NR

NR

18

38

NR

NR

NR

52

4

NR

17

11

WT

68

77

65

80

72

73

136

9

9

48

Liao

2019

Mut

NR

11

17

15

8

16

6

NR

20

2

6

22

26

2

16

6

NR

WT

30

26

34

23

53

3

40

14

27

29

45

11

26

30

Fleming

2013

Mut

NR

34

30

53

11

52

12

48

13

58

6

NR

NR 

NR

24

38

WT

296

384

501

178

593

87

498

158

588

92

133

540

Jia

2017

Mut

NR

NR

NR

3

1

NR

NR

NR

NR

NR

NR

WT

41

8

Ando

2005

Mut

0

1

0

1

1

0

1

0

1

0

NR 

NR

NR

NR

NR

WT

12

17

9

20

15

14

22

7

29

0

Miyaki

1999

Mut

NR

NR

NR

3

6

NR

NR

NR

NR

NR

NR

WT

41

11

Khan

2018

Mut

NR

NR

NR

NR

NR

NR

NR

NR

NR

65

161

WT

212

1387

Stahler

2020

Mut

NR

NR

NR

NR

NR

NR

NR

NR

NR

NR

WT
























Abbreviations: Mut=mutated; WT=wild type; WMD=Well to moderately differentiated; PD= Poorly differentiated; LN= Lymph node metastases; NR=not reported.
 

Table 3. Quality assessment according to the Newcastle–Ottawa scale of the included studies.

Author

Selection

Comparability

Exposure

Total score

Sarshekeh 2017

4

2

3

9

Mizuno 2018

4

2

3

9

Oyanagi 2019

3

2

2

7

Liao 2019

4

2

2

8

Fleming 2013

3

1

2

6

Jia 2017

3

2

                3       

8

Ando 2005

3

1

2

6

Miyaki 1999

3

2

2

7

Khan 2018

3

1

2

6

Stahler 2020

3

2

3

7

 

 

Table 4. Relationship of SMAD4 gene and Clinicopathologic characteristics of colorectal cancer.

Features

OR (95% CI)

Value

Age (65/≥65)

1.01 (0.91 ,1.12)

0.854

Gender (female/male)

1.09 (0.95,1.24)

0.212

Tumor site (colon/rectum)

1.15 (1.01,1.31)

0.042

TNM stage (Ⅰ-Ⅲ/Ⅳ)

0.78 (0.63,0.97)

0.025

Tumor grade (WMD/PD)

1.04 (0.96,1.12)

0.318

MSI (Stable/Unstable)

1.10 (0.95,1.28)

0.191

RAS (WT/Mut)

0.47 (0.30,0.73)

0.001

BRAF (WT/Mut)

1.00 (0.91,1.11)

0.976

Lymph node metastases (Yes/No)

1.42 (1.20,1.67)

0.000

Mucinous differentiation (Yes/No)

2.23 (1.85,2.70)

0.000

Abbreviations: WMD=Well to moderately differentiated; PD= Poorly differentiated.