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Association of CDX2 Expression With Survival in Early Colorectal Cancer: A Systematic Review and Meta-analysis
Accepted Manuscript Association of CDX2 expression with survival in early colorectal cancer: A Systematic Review and Meta-Analysis Gianluca Tomasello, Sandro Barni, Luca Turati, Michele Ghidini, Ezio Pezzica, Rodolfo Passalacqua, Fausto Petrelli PII:
S1533-0028(17)30507-8
DOI:
10.1016/j.clcc.2018.02.001
Reference:
CLCC 434
To appear in:
Clinical Colorectal Cancer
Received Date: 8 December 2017 Revised Date:
29 January 2018
Accepted Date: 8 February 2018
Please cite this article as: Tomasello G, Barni S, Turati L, Ghidini M, Pezzica E, Passalacqua R, Petrelli F, Association of CDX2 expression with survival in early colorectal cancer: A Systematic Review and Meta-Analysis, Clinical Colorectal Cancer (2018), doi: 10.1016/j.clcc.2018.02.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Association of CDX2 expression with survival in early colorectal cancer:
A Systematic Review and Meta-Analysis
Pezzica4, Rodolfo Passalacqua1 and Fausto Petrelli2 Running head:
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CDX2 in early colorectal cancer
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Gianluca Tomasello1, Sandro Barni2, Luca Turati3, Michele Ghidini1, Ezio
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Affiliations: 1
Oncology Unit, Oncology Department, ASST Ospedale di Cremona, Viale
Concordia 1, 26100, Cremona, Italy 2
Oncology Unit, Oncology Department, ASST Bergamo Ovest, Piazzale
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Ospedale 1, 24047, Treviglio (BG), Italy
(BG), Italy 4
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Surgery Unit, ASST Bergamo Ovest, Piazzale Ospedale 1, 24047, Treviglio
Pathology Unit, ASST Bergamo Ovest, Piazzale Ospedale 1, 24047, Treviglio
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(BG), Italy
Corresponding author: Fausto Petrelli, MD; Oncology Unit, Oncology Department, ASST Bergamo Ovest, Piazzale Ospedale 1, 24047, Treviglio (BG) Italy; tel. +390363424420, fax +390363424380, [email protected]
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Abstract Introduction: CDX2 is a homeobox gene encoding transcriptional factors for intestinal organogenesis and represents a specific marker of colorectal
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adenocarcinoma (CRC) differentiation. We have evaluated if CDX2 expression is associated with better overall- and disease-free survival (OS and DFS) in CRC patients.
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Material and methods: Pubmed, SCOPUS, EMBASE, The Cochrane Library and Web of Science (from inception to July 2017) were systematically reviewed
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for relevant studies on adult patients with CRC where OS and DFS were calculated according to CDX2 expression in uni- or multivariate analysis were included. Hazard ratio (HR) for mortality and/or disease progression was calculated.
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Results: The search produced 16 studies suitable for inclusion (6,291 individual patients). The meta-analysis showed a reduced risk of death for patients with CDX2 positive CRC in 14 studies (HR=0.5 [95%CI 0.38-0.66]; P<0.001
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according to random effect model). In 6 studies where only DFS data was
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available, CDX2 expression led to a 52% lower risk of relapse or death (HR=0.48 [95%CI 0.39-0.59]; P<0.001 according to random effect model). The results did not change as a function of ethnicity, type of study, CDX2 detection modality or stage. Interestingly, in stages II-III CDX2 expression was associated with a 70% lower risk of death (HR=0.3, 95%CI 0.12-0.77; P=0.01). CONCLUSIONS: CDX2 expression confirms to be a strong prognostic factor in stage II and III CRC. In this setting, along with other clinical and pathological
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ACCEPTED MANUSCRIPT factors, the lack of expression of CDX2 may be considered an important variable when deciding for adjuvant chemotherapy.
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Keywords: CDX2; colorectal cancer; prognosis; meta-analysis.
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Introduction Resected stage II and III colorectal cancers (CRCs) have 5-year disease-free survival (DFS) rates ranging from about 50 to more than 80%1. Survival is
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generally improved by adjuvant chemotherapy with most of the benefit being confined to stage III node positive disease2,3. In this specific setting, the addition of oxaliplatin to 5-Fluorouracil (5-FU)-based chemotherapy was shown to significantly reduce the risk of death at 10 years by about 10% as compared
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with bolus/infusional 5-FU plus leucovorin alone4.Among patients with resected
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node-negative (stage II) disease, the benefits of chemotherapy are much less evident. Likewise, the relative benefit of a doublet oxaliplatin-containing chemotherapy regimen has not been clearly demonstrated. Postoperative treatment decisions are commonly individualized. Major scientific societies
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guidelines recommend to take into consideration the presence of specific factors associated with a high-risk of recurrence including bowel obstruction, perforation, inadequate lymph node sampling, poorly-differentiated histology,
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lymphovascular or perineural invasion, mismatch repair (MMR) status or T4 disease2. No other clinical or histopathologic variables with adequate power to
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allow further risk stratification in resected stage II and III CRC are currently available.
Caudal-related homeobox transcription factor 2 (CDX2) is encoded by the CDX2 gene, a member of the caudal-related homeobox gene family5. In adults, it is specifically expressed in the nuclei of intestinal epithelial cells and is essential for intestinal development and differentiation6. CDX2 also regulates
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ACCEPTED MANUSCRIPT the expression of ubiquitous genes important in controlling specific cell functions, including adhesion, proliferation, cell cycle regulation, and apoptosis7. CDX2 is commonly used in diagnostic pathology as a tissue biomarker for
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gastrointestinal differentiation, especially for colorectal origin8. CDX2 expression is still present in most (more than 90%) adenocarcinomas of the large intestine. Currently, CDX2 is used as an accurate immune-histochemical marker of normal and neoplastic intestinal epithelium, necessary for the differential
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diagnosis of primary versus metastatic adenocarcinomas.
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By subgroup analysis of independent retrospective patient cohorts, Dalerba and colleagues recently evaluated the association of CDX2 with 5-year DFS and the benefit from adjuvant chemotherapy among patients with stage II and III colon cancer9. In the discovery data set, which included 466 patients, authors found
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that 5-year DFS was 6.9% compared to 93.1% in CDX2-negative and positive CRCs, respectively (hazard ratio [HR] for disease recurrence, 3.44; 95% confidence interval [CI], 1.60 to 7.38; P=0.002). A statistically significant
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difference in 5-year DFS was subsequently confirmed in the validation data set (314 patients). Interestingly, in a pooled database of all patient cohorts, 5-year
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DFS rate was 91% in stage II CRC patients who received adjuvant chemotherapy as compared with 56% in patients who did not (P=0.006).,. Therefore, according to PRISMA guidelines, we performed a systematic review of the literature and a meta-analysis with the aim to evaluate CDX2 expression as a potential prognostic factor in CRC (particularly in stage II and III) useful for risk stratification and selection for adjuvant chemotherapy.. Methods 5
ACCEPTED MANUSCRIPT Search strategy Electronic databases including PubMed, EMBASE, SCOPUS, The Cochrane Library and Web of Science were searched from inception to July 2017 for
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identification of abstracts and articles meeting selection criteria. The terms used were 'colorectal cancer' AND ('transcription factor cdx2' OR 'cdx2 transcription factor' OR 'cdx 2 protein' OR 'cdx2 homeobox transcription factor' OR 'caudal related homeobox protein 2' OR 'caudal related homeobox transcription factor 2'
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OR 'caudal related homeodomain protein 2' OR 'caudal type homeobox protein
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2' OR 'caudal type homeobox transcription factor 2' OR 'caudal type homeodomain protein 2' OR 'caudal type transcription factor 2' OR 'homeobox protein cdx2' OR 'homeodomain protein cdx2' OR 'protein cdx 2' OR 'protein cdx2' OR 'transcription factor cdx 2' OR 'transcription factor cdx2'). All original articles, previous reviews, and systematic reviews were evaluated for relevant
Inclusion criteria
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references. Only papers in English language were selected.
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All prospective and historical cohort studies or clinical trials measuring the
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outcome of patients with resected CRC (DFS and/or OS) were considered for inclusion. Selection criteria were: adult patients with a previous diagnosis of CRC, outcome data available according to immune-histochemistry CDX2 expression and studies with the hazard ratio (HR) and its 95% confidence interval (CI) according to multivariate analysis (or Kaplan Meier curve to calculate these data). If univariate analysis only was available, those HRs were considered. Case reports or letters, overlapping series or studies including less than 50 patients were excluded. Selection of studies was independently
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ACCEPTED MANUSCRIPT performed by two reviewers (FP and GT), and the final decision was reached after consultation with a third senior referee (SB) and team consensus. Data extraction and endpoints
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The following data was extracted: name of first author; publication year; patients’ number; country; ethnicity; median follow-up time; method of assessment and antibody source; rate of adjuvant chemotherapy; HR with
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95%CI for DFS and/or OS. The two reviewers checked the data again and discussed the data if the results differed to reach a consensus. A third author
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(SB) was invited to the discussion when the two primary authors could not reach an agreement. Study quality was evaluated independently by reviewers according to the Newcastle-Ottawa (NOS) quality assessment scale. Dagra software was used to ascertain survival data by digitizing figures if the
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information was not provided directly.
The primary outcome was OS defined as the time elapsed from diagnosis to death due to any cause. Secondary endpoint was DFS defined as survival from
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diagnosis of CRC to relapse or death whichever came first. If results of a study
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were reported in multiple publications, the most recent publication that included the relevant information was included. Statistical analysis
The impact of CDX2 expression on OS and DFS was estimated by pooling HRs and 95% CIs. In each analysis, heterogeneity was carried out with P<0.1 as level of significance, and I2 was also calculated with ≥50% standing for substantial heterogeneity. When P<0.10 or I2>50%, a random effects model
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ACCEPTED MANUSCRIPT was used to pool effect sizes of each study for heterogeneity. Otherwise, a fixed effects model was selected. The potential risk of publication bias was evaluated by Begg’s and Egger’s test,
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in which the P value <0.05 showed the significance. We also estimated the effect of individual studies on the summary HR by re-estimating and plotting the summary HR in the absence of each study (leave-one-out-procedure). Subgroup analyses were defined a priori and tested effect modification on OS
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according to stage (stage II-III vs all stages) and multivariate vs univariate (yes
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vs no). Sensitivity analysis was performed to explore the variation between studies by including or excluding studies based on study methodological quality (high/low quality). We also explored heterogeneity by CDX2 detection (IHC vs other
methods),
country
(Eastern
vs
Western),
chemotherapy
(rate),
microsatellite instability (MSI) status (i.e., deficient mismatch repair [dMMR] vs
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proficient [pMMR]), and type of study (prospective vs retrospective). Metaregression was performed to adjust for continuos variable when applicable.
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Meta-analysis of HRs and 95% CIs was generated by reference manager
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software (RevMan, version 5.3). Results
The search strategy originally retrieved 3,240 relevant clinical studies in English. Of these, 3,227 were eliminated because of various reasons. The remaining 23 articles were evaluated by a full-text review. Based on the inclusion and exclusion criteria, 16 studies were finally selected for this metaanalysis9–24. The detailed search and study selection process is shown in Figure 1. 8
ACCEPTED MANUSCRIPT Characteristics of included studies The 16 studies published between 2009 until 2017, included 6,291 patients at baseline, with a maximum sample size of 1,897 and a minimum sample size of
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82 participants. All studies except 1 were retrospective case series. Twelve included patients from Western countries, 4 from Asian countries. Nine studies included CRC patients with all stages of disease (stage I-IV), 4 only stages II-III,
patients had stages II or III CRC (mean 85%).
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1 stages III-IV, 1 stages I-III and 1 only metastatic patients. Overall, most
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CDX2 expression was evaluated in 14 studies with standard IHC technique. In 2 studies, mRNA expression from tissue microarray was applicated. CDX2 expression ranged from 57 to 94% of cases analysed. Information on adjuvant chemotherapy was available in 11 papers and ranged from 0 to 100% (mean
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50%). Data was obtained from multivariate or univariate analysis in n=9 and n=7 studies, respectively. The characteristics of the included studies are shown in Table 1.
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Overall survival and DFS
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Overall, 14 studies were available for OS analysis. Expression of CDX2 was associated with a 50% lower risk of death compared to poor or no protein expression (HR=0.5 [95%CI 0.38-0.66]; P<0.001 according to random effects model) (Fig.2). In 6 studies where DFS data was available, CDX2 expression was associated with a 52% lower risk of progression/relapse or death (HR=0.48 [95%CI 0.39-0.59]; P<0.001 according to random effects model and no heterogeneity was found [I2=0%, P=0.65]) (Figure 3). In primary analysis, heterogeneity was high: I2=71%, P<0.001. 9
ACCEPTED MANUSCRIPT Subgroup analysis for primary endpoint In studies were HR was calculated from univariate analysis, results were comparable to those coming from studies where the prognostic role of CDX2
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was achieved with multivariate analysis (HR=0.45 [95%CI 0.25-0.81]; P<0.001 vs HR=0.51 [95%CI 0.37-0.7]; P<0.001). In studies where CDX2 was evaluated through standard IHC analysis (n=9 studies), OS was significantly better when CDX2 was expressed (HR=0.5, 95%CI 0.35-0.7). In 4 studies where only stage
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II-III patients were included, CDX2 expression was strongly associated with a
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better OS (HR=0.3, 95%CI 0.12-0.77; P=0.01). In studies where patients with all CRC stages were included, HR for OS was 0.53 (95%CI 0.41-0.69; P<0.001). Administration of adjuvant chemotherapy had impact on the final results in meta-regression analysis (P=0.001). In studies including both Asian and Caucasian subjects, CDX2 expression maintained its prognostic role (HR=0.37
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[95%CI 0.19-0.72] and 0.54 [95%CI 0.39-0.73], respectively; P for difference 0.33). All studies except one had a retrospective design and HR for OS was
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comparable to that observed in the global population (HR=0.49, 95%CI 0.360.66; P<0.001). In both IHC and tissue microarray studies results remained
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significant for the primary endpoint analysis. Among types of IHC antibodies, only studies using Biogenex and Ventana kits showed a significant association in OS analysis. Finally, a further analysis adjusted for dMMR patients (n=7 studies with known data) through metaregression was performed. No significant association with OS was found, so that the prognostic role of CDX2 seems to be independent of MSI status. Additionally, in n=3 out of 9 studies with multivariate analysis available, a better survival associated with CDX2 expression was again independent of MSI status. 10
ACCEPTED MANUSCRIPT Publication bias We performed a sensitivity analysis in which one study was excluded at a time to determine the stability of our results. We found that the corresponding HRs
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were changed by Baba et al. 200910 and Zhang et al. 201724 (from 0.46 to 0.54), which indicated that these two studies contributed mainly to publication bias in the results. In this meta-analysis, publication bias was evaluated by Begg’s and Egger’s test. Primary analysis demonstrated a significant probability
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of publication bias with Begg’s Test (P=0.01) and Egger’s test (P=0.00028).
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Discussion
When diagnosed at early stages, colorectal cancer is often a curable disease. Adjuvant chemotherapy can further reduce the risk of relapse or death but most of the survival benefit is limited to stage III tumors2,3. Thus, scientific community
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efforts are constantly aimed at identifying new prognostic and predictive biomarkers to stratify patients into high and low risk groups and maximize the
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benefit from adjuvant chemotherapy.
Homeobox genes are often deregulated in cancer and are associated with both
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oncogenic and tumor-suppressing functions. CDX2 protein expression is seldom lost in colorectal cancer and the same gene is rarely mutated25. Low CDX2 protein expression is frequently accompanied by low CDX2 mRNA expression, which could reflect down regulation at the transcriptional level22. One of the mechanisms proposed for CDX2 silencing could rely on CDX2 promoter hyper-methylation, which has been described in up to 30% of cases with low CDX2 expression26. It has been hypothesized that CDX2 may act as a tumor suppressor gene whose reduced expression and/or function contributes 11
ACCEPTED MANUSCRIPT to initiation and progression of sporadic adenocarcinomas of the colon and rectum27. However, its precise role and clinical applicability as a prognostic biomarker must be still elucidated.
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Results of our study show that CDX2 may represent a reliable biomarker for risk stratification in early stages CRC. In this meta-analysis which included 6,291 subjects, CDX2 expression was associated with a statistically significant 50% lower risk of death compared to poor or no protein expression. By restricting the
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analysis to stages II and III (4 studies) the survival benefit was even more
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evident reaching an impressive 70% risk reduction. As for DFS, CDX2 positivity was also shown to be associated with a significant 52% lower risk of progression, relapse or death.
Our findings are consistent with previous reports demonstrating a clear
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prognostic role of CDX2 expression. Specifically, a recent meta-analysis of eight published studies containing 2,547 patients revealed that the low expression of CDX2 was associated with poorer outcomes for both OS
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(HR=1.99, 95% CI: 1.31-3.02; P=0.001) and DFS (HR=1.81, 95% CI: 1.08-3.05; P=0.025)28. Moreover, subgroup analysis showed that decreased CDX2 was a
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negative prognostic factor in Asian, but not in Caucasian patients. In our metaanalysis, however, 3000 more Caucasian patients were included and this likely increased our statistical power. By applying an original bioinformatics approach to the discovery of prognostic cancer biomarkers, Dalerba and colleagues published in early 2016 the results of a pivotal trial testing the relationship between CDX2 and survival of CRC patients9. Undoubtedly, investigators had the merit to establish the negative
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ACCEPTED MANUSCRIPT prognostic effect associated with the absence of CDX2 expression and to simultaneously identify stage II CRC cases who might significantly benefit from adjuvant chemotherapy. The study was also object of criticism especially due to retrospective nature of the analysis, the small number of patients with stage II
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colon cancer and CDX2-negative tumor, and the type of immunohistochemical analysis (performed on tissue microarrays) that may have underestimated the intra-tumoral
heterogeneity
of
CDX2
expression29.
Although
definitive
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conclusions cannot still be drawn, such trial has paved the way to a new wave of biomarker research on predictors of efficacy from adjuvant therapy in early
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stage CRC.
Among patients with stage II disease, 5-year survival rates can widely differ depending on depth of invasion through the intestinal wall (as low as 60 % for T4 tumors)30.To date, owing to the minimal advantage provided, the choice of
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recommending adjuvant treatment in stage II tumors relies on different clinicopathologic variables including stage, tumor grade and microsatellite
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instability. Alongside these classical parameters, novel prognostic tools mainly represented by gene expression assays (e.g., Coloprint, Oncotype-DX, etc.)
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have emerged in the last years and are currently available for clinical use in the United Stated. All these tests have been shown to perform well in independent patient series, though the design and sample numbers of the validation analyses varied considerably31. For this reason, translating such molecular signatures into regular clinical use has not yet been realized. Finally, the high costs of these procedures further limit their widespread application. CDX2 expression testing, as evaluated in all the trials included in our analysis (i.e., IHC), is generally not expensive and easy to perform. Also in light of these 13
ACCEPTED MANUSCRIPT aspects, we believe that findings of this meta-analysis are noteworthy. Importantly, as indicated by subgroup analysis, CDX2 positivity was shown to be a strong prognostic factor independent of other variables including ethnicity,
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type of study, MSI status, CDX2 detection modality or stage. Accumulating evidence suggests that CDX2 downregulation is associated with several adverse prognostic factors such as MMR deficiency (in advanced stages), BRAF mutations, CIMP positive status, right-sided tumors, and poor
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differentiation at both the mRNA and protein level22. If confirmed, CDX2 may
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therefore function as a single prognostic biomarker indicator of multiple adverse features. Of course, owing to the retrospective design of most studies analyzed, these findings need to be prospectively confirmed and validated in the context of appropriate randomized clinical trials projected to answer relevant translational research questions. To further investigate the sources of
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heterogeneity, we performed meta-regression analysis. We did not find any significant relationship between prognosis of CDX2+ (vs CDX2-) CRC and other
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confounding factors, except in patients that received chemotherapy. It is likely that the more disease is aggressive (high risk stage II or III) or advanced (stage
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IV), the higher is the survival difference between CDX2+ and CDX- CRC. Some limitations of this study must also be remarked. The heterogeneity among included studies cannot be ignored. Stages, receipt of adjuvant chemotherapy, ethnicity and retrospective nature of published series are the main reasons. Besides, these studies have used different anti-CDX2 antibodies and a variety of different scoring criteria to define positive CDX2 cases. Publication bias is the second possible reason. In fact, studies showing that CDX2 was not a prognostic factor could have been less likely published as compared with 14
ACCEPTED MANUSCRIPT studies showing positive results. Third, included studies were only Englishwritten publications. Fourth, individual patient’s data were not available, therefore adjustment for other prognostic covariates was not possible. Finally, studies with larger sample sizes, such as randomized multicenter trials
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evaluating adjuvant chemotherapy would be needed for more definitive conclusions.
To the best of our knowledge, this is the largest meta-analysis ever conducted
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to investigate the association between CDX2 expression and outcome in early
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stages CRC. CDX2 positivity confirms to be a strong prognostic determinant associated with an excellent survival in stage II and III CRC. In this specific stages, along with other clinical and pathological factors, lack of expression of CDX2 should be formally regarded as an important indicator for adjuvant
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Dawson H, Galván JA, Helbling M, et al. Possible role of Cdx2 in the serrated pathway of colorectal cancer characterized by BRAF mutation,
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high-level CpG Island methylator phenotype and mismatch repairdeficiency. Int J Cancer. 2014;134(10):2342-2351. doi:10.1002/ijc.28564. 27.
Abate-Shen C. Deregulated homeobox gene expression in cancer: cause
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or consequence? Nat Rev Cancer. 2002;2(10):777-785. doi:10.1038/nrc907.
Yu H, Zhang H, Cao Q, Zhu W. The prognostic value of CDX2 in
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28.
colorectal cancer: a meta-analysis. Int J Clin Exp Med. 2016;9(8):1595515960. www.ijcem.com. Accessed September 6, 2017. 29.
Boland CR, Goel A. Prognostic Subgroups among Patients with Stage II
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Colon Cancer. N Engl J Med. 2016;374(3):277-278. doi:10.1056/NEJMe1514353.
Meyerhardt JA, Mayer RJ. Systemic Therapy for Colorectal Cancer. N
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30.
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Engl J Med. 2005;352(5):476-487. doi:10.1056/NEJMra040958. 31.
Sveen A, Nesbakken A, Ågesen TH, et al. Anticipating the clinical use of prognostic gene expression-based tests for colon cancer stage II and III:
is Godot finally arriving? Clin Cancer Res. 2013;19(24):6669-6677. doi:10.1158/1078-0432.CCR-13-1769.
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Country
N° pts
Stage %
CT (%)
Baba 2009 Bae 2015 Bauer 2012
Prospective Retrospective Retrospective
USA Korea France
621 713 197
I-IV (II-III 59) I-IV (II-III 96) I-III (II-III 82)
0 65 -
Dalerba 2016
Retrospective
USA
1897
II-III (100)
74
Dawson 2013 Droy-Dupre 2015 Hong 2013 Karamitopolou 2010 Kim 2013 Knösel 2012 Lundberg 2016 Matsuda 2010 Olsen 2016 Pilati 2017
Retrospective Retrospective Retrospective Retrospective Retrospective Retrospective Retrospective Retrospective Retrospective Retrospective
Switzerland France Korea Greece/Switzerland Korea Germany Sweden Japan Denmark France
201 116° 207 82 109 402 445 97* 191 469
I-IV (100) I-IV (II-III 69) I-IV (II-III 76.9) III-IV (III 83.8) I-IV (II-III 89) II-III (100) I-IV (II-III 57.7) II-III (100) I-IV (85) II-III (100)
31.5 31 73.4 85.4 61.4 43
Zhang 2017 Nolte/2017
Retrospective Retrospective
USA Germany
145 612
IV (100) I-IV (II-III 61)
CDX2+ %/cutoff 70.5/weak to strong vs loss 94.1/retained vs loss 84/high vs low (15.7 and 10.5%)** 92.8/widespread vs loss/faint nuclear expression 57/diffuse vs loss 86.8/ >50% vs <50% 94.7/+ & strong+ vs weak + 61,3^/86.2/>5% vs <5% 58/high vs low 82.9/>5% vs <5% 78.7/ + vs 92/high/normal vs low/absent 84.4/> vs <6.5 log2 of normalized expression values 94.4/present vs absent -
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Type of study
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Author/year
100 39
CDX2 detection IHC/clone CDX2-88, Biogenex IHC/clone CDX2-88, Biogenex TMA (mRNA)/-
NOS score 9 8 7
IHC/clone CDX2-88, Biogenex
8
IHC/Novocastra IHC/clone CDX2-88, Biogenex IHC/Novocastra IHC TMA/ clone AMT28, AbCam IHC/Ventana Medical Systems IHC/MU 392A-UC BioGenex IHC/ clone CDX2-88,Biogenex IHC/BioGenex IHC/EPR2764Y, CellMarque TMA (mRNA)/-
7 6 7 6 7 7 6 7 7 7
IHC/IHC/MU392A-UC, Biogenex
6 7
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Tab. 1 characteristics of included trials
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*, only stage II-III patients included in survival analysis; °, with survival data available; ^, media n value; -, not reported; **, right and left colorectal cancer cutoffs; IHC: immunohistochemistry; pts: patients; CT: chemotherapy; NOS: Nottingham Ottawa Scale
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Records identified through Pubmed searching (n = 206 )
Additional records identified through other sources (n = 3034 )
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Records screened (n = 3240 )
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Full-text articles assessed for eligibility (n = 937)
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Included
Eligibility
Screening
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Records after duplicates removed (n = 3240 )
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Identification
Fig.1 Literature search flow diagram
Studies included in qualitative synthesis (n = 25 )
Records excluded because were duplicated (n = 2303) Full-text articles excluded, because they were review/letters/commentarie, included other cancer patients or did not analyse prognostic role of CDX2 (n = 912 ) Full-text articles excluded, because they did not reported hazard ratios or other survival data (n = 9)
Studies included in quantitative synthesis (meta-analysis) (n = 16 )
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Fig.3 association of CDX2 expression with disease-free survival
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Fig.2 association of CDX2 expression with overall survival
S1533-0028(17)30507-8
DOI:
10.1016/j.clcc.2018.02.001
Reference:
CLCC 434
To appear in:
Clinical Colorectal Cancer
Received Date: 8 December 2017 Revised Date:
29 January 2018
Accepted Date: 8 February 2018
Please cite this article as: Tomasello G, Barni S, Turati L, Ghidini M, Pezzica E, Passalacqua R, Petrelli F, Association of CDX2 expression with survival in early colorectal cancer: A Systematic Review and Meta-Analysis, Clinical Colorectal Cancer (2018), doi: 10.1016/j.clcc.2018.02.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Association of CDX2 expression with survival in early colorectal cancer:
A Systematic Review and Meta-Analysis
Pezzica4, Rodolfo Passalacqua1 and Fausto Petrelli2 Running head:
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CDX2 in early colorectal cancer
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Gianluca Tomasello1, Sandro Barni2, Luca Turati3, Michele Ghidini1, Ezio
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Affiliations: 1
Oncology Unit, Oncology Department, ASST Ospedale di Cremona, Viale
Concordia 1, 26100, Cremona, Italy 2
Oncology Unit, Oncology Department, ASST Bergamo Ovest, Piazzale
3
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Ospedale 1, 24047, Treviglio (BG), Italy
(BG), Italy 4
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Surgery Unit, ASST Bergamo Ovest, Piazzale Ospedale 1, 24047, Treviglio
Pathology Unit, ASST Bergamo Ovest, Piazzale Ospedale 1, 24047, Treviglio
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(BG), Italy
Corresponding author: Fausto Petrelli, MD; Oncology Unit, Oncology Department, ASST Bergamo Ovest, Piazzale Ospedale 1, 24047, Treviglio (BG) Italy; tel. +390363424420, fax +390363424380, [email protected]
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Abstract Introduction: CDX2 is a homeobox gene encoding transcriptional factors for intestinal organogenesis and represents a specific marker of colorectal
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adenocarcinoma (CRC) differentiation. We have evaluated if CDX2 expression is associated with better overall- and disease-free survival (OS and DFS) in CRC patients.
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Material and methods: Pubmed, SCOPUS, EMBASE, The Cochrane Library and Web of Science (from inception to July 2017) were systematically reviewed
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for relevant studies on adult patients with CRC where OS and DFS were calculated according to CDX2 expression in uni- or multivariate analysis were included. Hazard ratio (HR) for mortality and/or disease progression was calculated.
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Results: The search produced 16 studies suitable for inclusion (6,291 individual patients). The meta-analysis showed a reduced risk of death for patients with CDX2 positive CRC in 14 studies (HR=0.5 [95%CI 0.38-0.66]; P<0.001
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according to random effect model). In 6 studies where only DFS data was
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available, CDX2 expression led to a 52% lower risk of relapse or death (HR=0.48 [95%CI 0.39-0.59]; P<0.001 according to random effect model). The results did not change as a function of ethnicity, type of study, CDX2 detection modality or stage. Interestingly, in stages II-III CDX2 expression was associated with a 70% lower risk of death (HR=0.3, 95%CI 0.12-0.77; P=0.01). CONCLUSIONS: CDX2 expression confirms to be a strong prognostic factor in stage II and III CRC. In this setting, along with other clinical and pathological
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ACCEPTED MANUSCRIPT factors, the lack of expression of CDX2 may be considered an important variable when deciding for adjuvant chemotherapy.
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Keywords: CDX2; colorectal cancer; prognosis; meta-analysis.
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Introduction Resected stage II and III colorectal cancers (CRCs) have 5-year disease-free survival (DFS) rates ranging from about 50 to more than 80%1. Survival is
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generally improved by adjuvant chemotherapy with most of the benefit being confined to stage III node positive disease2,3. In this specific setting, the addition of oxaliplatin to 5-Fluorouracil (5-FU)-based chemotherapy was shown to significantly reduce the risk of death at 10 years by about 10% as compared
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with bolus/infusional 5-FU plus leucovorin alone4.Among patients with resected
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node-negative (stage II) disease, the benefits of chemotherapy are much less evident. Likewise, the relative benefit of a doublet oxaliplatin-containing chemotherapy regimen has not been clearly demonstrated. Postoperative treatment decisions are commonly individualized. Major scientific societies
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guidelines recommend to take into consideration the presence of specific factors associated with a high-risk of recurrence including bowel obstruction, perforation, inadequate lymph node sampling, poorly-differentiated histology,
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lymphovascular or perineural invasion, mismatch repair (MMR) status or T4 disease2. No other clinical or histopathologic variables with adequate power to
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allow further risk stratification in resected stage II and III CRC are currently available.
Caudal-related homeobox transcription factor 2 (CDX2) is encoded by the CDX2 gene, a member of the caudal-related homeobox gene family5. In adults, it is specifically expressed in the nuclei of intestinal epithelial cells and is essential for intestinal development and differentiation6. CDX2 also regulates
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ACCEPTED MANUSCRIPT the expression of ubiquitous genes important in controlling specific cell functions, including adhesion, proliferation, cell cycle regulation, and apoptosis7. CDX2 is commonly used in diagnostic pathology as a tissue biomarker for
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gastrointestinal differentiation, especially for colorectal origin8. CDX2 expression is still present in most (more than 90%) adenocarcinomas of the large intestine. Currently, CDX2 is used as an accurate immune-histochemical marker of normal and neoplastic intestinal epithelium, necessary for the differential
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diagnosis of primary versus metastatic adenocarcinomas.
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By subgroup analysis of independent retrospective patient cohorts, Dalerba and colleagues recently evaluated the association of CDX2 with 5-year DFS and the benefit from adjuvant chemotherapy among patients with stage II and III colon cancer9. In the discovery data set, which included 466 patients, authors found
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that 5-year DFS was 6.9% compared to 93.1% in CDX2-negative and positive CRCs, respectively (hazard ratio [HR] for disease recurrence, 3.44; 95% confidence interval [CI], 1.60 to 7.38; P=0.002). A statistically significant
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difference in 5-year DFS was subsequently confirmed in the validation data set (314 patients). Interestingly, in a pooled database of all patient cohorts, 5-year
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DFS rate was 91% in stage II CRC patients who received adjuvant chemotherapy as compared with 56% in patients who did not (P=0.006).,. Therefore, according to PRISMA guidelines, we performed a systematic review of the literature and a meta-analysis with the aim to evaluate CDX2 expression as a potential prognostic factor in CRC (particularly in stage II and III) useful for risk stratification and selection for adjuvant chemotherapy.. Methods 5
ACCEPTED MANUSCRIPT Search strategy Electronic databases including PubMed, EMBASE, SCOPUS, The Cochrane Library and Web of Science were searched from inception to July 2017 for
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identification of abstracts and articles meeting selection criteria. The terms used were 'colorectal cancer' AND ('transcription factor cdx2' OR 'cdx2 transcription factor' OR 'cdx 2 protein' OR 'cdx2 homeobox transcription factor' OR 'caudal related homeobox protein 2' OR 'caudal related homeobox transcription factor 2'
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OR 'caudal related homeodomain protein 2' OR 'caudal type homeobox protein
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2' OR 'caudal type homeobox transcription factor 2' OR 'caudal type homeodomain protein 2' OR 'caudal type transcription factor 2' OR 'homeobox protein cdx2' OR 'homeodomain protein cdx2' OR 'protein cdx 2' OR 'protein cdx2' OR 'transcription factor cdx 2' OR 'transcription factor cdx2'). All original articles, previous reviews, and systematic reviews were evaluated for relevant
Inclusion criteria
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references. Only papers in English language were selected.
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All prospective and historical cohort studies or clinical trials measuring the
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outcome of patients with resected CRC (DFS and/or OS) were considered for inclusion. Selection criteria were: adult patients with a previous diagnosis of CRC, outcome data available according to immune-histochemistry CDX2 expression and studies with the hazard ratio (HR) and its 95% confidence interval (CI) according to multivariate analysis (or Kaplan Meier curve to calculate these data). If univariate analysis only was available, those HRs were considered. Case reports or letters, overlapping series or studies including less than 50 patients were excluded. Selection of studies was independently
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ACCEPTED MANUSCRIPT performed by two reviewers (FP and GT), and the final decision was reached after consultation with a third senior referee (SB) and team consensus. Data extraction and endpoints
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The following data was extracted: name of first author; publication year; patients’ number; country; ethnicity; median follow-up time; method of assessment and antibody source; rate of adjuvant chemotherapy; HR with
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95%CI for DFS and/or OS. The two reviewers checked the data again and discussed the data if the results differed to reach a consensus. A third author
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(SB) was invited to the discussion when the two primary authors could not reach an agreement. Study quality was evaluated independently by reviewers according to the Newcastle-Ottawa (NOS) quality assessment scale. Dagra software was used to ascertain survival data by digitizing figures if the
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information was not provided directly.
The primary outcome was OS defined as the time elapsed from diagnosis to death due to any cause. Secondary endpoint was DFS defined as survival from
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diagnosis of CRC to relapse or death whichever came first. If results of a study
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were reported in multiple publications, the most recent publication that included the relevant information was included. Statistical analysis
The impact of CDX2 expression on OS and DFS was estimated by pooling HRs and 95% CIs. In each analysis, heterogeneity was carried out with P<0.1 as level of significance, and I2 was also calculated with ≥50% standing for substantial heterogeneity. When P<0.10 or I2>50%, a random effects model
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ACCEPTED MANUSCRIPT was used to pool effect sizes of each study for heterogeneity. Otherwise, a fixed effects model was selected. The potential risk of publication bias was evaluated by Begg’s and Egger’s test,
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in which the P value <0.05 showed the significance. We also estimated the effect of individual studies on the summary HR by re-estimating and plotting the summary HR in the absence of each study (leave-one-out-procedure). Subgroup analyses were defined a priori and tested effect modification on OS
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according to stage (stage II-III vs all stages) and multivariate vs univariate (yes
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vs no). Sensitivity analysis was performed to explore the variation between studies by including or excluding studies based on study methodological quality (high/low quality). We also explored heterogeneity by CDX2 detection (IHC vs other
methods),
country
(Eastern
vs
Western),
chemotherapy
(rate),
microsatellite instability (MSI) status (i.e., deficient mismatch repair [dMMR] vs
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proficient [pMMR]), and type of study (prospective vs retrospective). Metaregression was performed to adjust for continuos variable when applicable.
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Meta-analysis of HRs and 95% CIs was generated by reference manager
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software (RevMan, version 5.3). Results
The search strategy originally retrieved 3,240 relevant clinical studies in English. Of these, 3,227 were eliminated because of various reasons. The remaining 23 articles were evaluated by a full-text review. Based on the inclusion and exclusion criteria, 16 studies were finally selected for this metaanalysis9–24. The detailed search and study selection process is shown in Figure 1. 8
ACCEPTED MANUSCRIPT Characteristics of included studies The 16 studies published between 2009 until 2017, included 6,291 patients at baseline, with a maximum sample size of 1,897 and a minimum sample size of
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82 participants. All studies except 1 were retrospective case series. Twelve included patients from Western countries, 4 from Asian countries. Nine studies included CRC patients with all stages of disease (stage I-IV), 4 only stages II-III,
patients had stages II or III CRC (mean 85%).
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1 stages III-IV, 1 stages I-III and 1 only metastatic patients. Overall, most
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CDX2 expression was evaluated in 14 studies with standard IHC technique. In 2 studies, mRNA expression from tissue microarray was applicated. CDX2 expression ranged from 57 to 94% of cases analysed. Information on adjuvant chemotherapy was available in 11 papers and ranged from 0 to 100% (mean
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50%). Data was obtained from multivariate or univariate analysis in n=9 and n=7 studies, respectively. The characteristics of the included studies are shown in Table 1.
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Overall survival and DFS
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Overall, 14 studies were available for OS analysis. Expression of CDX2 was associated with a 50% lower risk of death compared to poor or no protein expression (HR=0.5 [95%CI 0.38-0.66]; P<0.001 according to random effects model) (Fig.2). In 6 studies where DFS data was available, CDX2 expression was associated with a 52% lower risk of progression/relapse or death (HR=0.48 [95%CI 0.39-0.59]; P<0.001 according to random effects model and no heterogeneity was found [I2=0%, P=0.65]) (Figure 3). In primary analysis, heterogeneity was high: I2=71%, P<0.001. 9
ACCEPTED MANUSCRIPT Subgroup analysis for primary endpoint In studies were HR was calculated from univariate analysis, results were comparable to those coming from studies where the prognostic role of CDX2
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was achieved with multivariate analysis (HR=0.45 [95%CI 0.25-0.81]; P<0.001 vs HR=0.51 [95%CI 0.37-0.7]; P<0.001). In studies where CDX2 was evaluated through standard IHC analysis (n=9 studies), OS was significantly better when CDX2 was expressed (HR=0.5, 95%CI 0.35-0.7). In 4 studies where only stage
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II-III patients were included, CDX2 expression was strongly associated with a
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better OS (HR=0.3, 95%CI 0.12-0.77; P=0.01). In studies where patients with all CRC stages were included, HR for OS was 0.53 (95%CI 0.41-0.69; P<0.001). Administration of adjuvant chemotherapy had impact on the final results in meta-regression analysis (P=0.001). In studies including both Asian and Caucasian subjects, CDX2 expression maintained its prognostic role (HR=0.37
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[95%CI 0.19-0.72] and 0.54 [95%CI 0.39-0.73], respectively; P for difference 0.33). All studies except one had a retrospective design and HR for OS was
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comparable to that observed in the global population (HR=0.49, 95%CI 0.360.66; P<0.001). In both IHC and tissue microarray studies results remained
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significant for the primary endpoint analysis. Among types of IHC antibodies, only studies using Biogenex and Ventana kits showed a significant association in OS analysis. Finally, a further analysis adjusted for dMMR patients (n=7 studies with known data) through metaregression was performed. No significant association with OS was found, so that the prognostic role of CDX2 seems to be independent of MSI status. Additionally, in n=3 out of 9 studies with multivariate analysis available, a better survival associated with CDX2 expression was again independent of MSI status. 10
ACCEPTED MANUSCRIPT Publication bias We performed a sensitivity analysis in which one study was excluded at a time to determine the stability of our results. We found that the corresponding HRs
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were changed by Baba et al. 200910 and Zhang et al. 201724 (from 0.46 to 0.54), which indicated that these two studies contributed mainly to publication bias in the results. In this meta-analysis, publication bias was evaluated by Begg’s and Egger’s test. Primary analysis demonstrated a significant probability
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of publication bias with Begg’s Test (P=0.01) and Egger’s test (P=0.00028).
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Discussion
When diagnosed at early stages, colorectal cancer is often a curable disease. Adjuvant chemotherapy can further reduce the risk of relapse or death but most of the survival benefit is limited to stage III tumors2,3. Thus, scientific community
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efforts are constantly aimed at identifying new prognostic and predictive biomarkers to stratify patients into high and low risk groups and maximize the
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benefit from adjuvant chemotherapy.
Homeobox genes are often deregulated in cancer and are associated with both
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oncogenic and tumor-suppressing functions. CDX2 protein expression is seldom lost in colorectal cancer and the same gene is rarely mutated25. Low CDX2 protein expression is frequently accompanied by low CDX2 mRNA expression, which could reflect down regulation at the transcriptional level22. One of the mechanisms proposed for CDX2 silencing could rely on CDX2 promoter hyper-methylation, which has been described in up to 30% of cases with low CDX2 expression26. It has been hypothesized that CDX2 may act as a tumor suppressor gene whose reduced expression and/or function contributes 11
ACCEPTED MANUSCRIPT to initiation and progression of sporadic adenocarcinomas of the colon and rectum27. However, its precise role and clinical applicability as a prognostic biomarker must be still elucidated.
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Results of our study show that CDX2 may represent a reliable biomarker for risk stratification in early stages CRC. In this meta-analysis which included 6,291 subjects, CDX2 expression was associated with a statistically significant 50% lower risk of death compared to poor or no protein expression. By restricting the
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analysis to stages II and III (4 studies) the survival benefit was even more
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evident reaching an impressive 70% risk reduction. As for DFS, CDX2 positivity was also shown to be associated with a significant 52% lower risk of progression, relapse or death.
Our findings are consistent with previous reports demonstrating a clear
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prognostic role of CDX2 expression. Specifically, a recent meta-analysis of eight published studies containing 2,547 patients revealed that the low expression of CDX2 was associated with poorer outcomes for both OS
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(HR=1.99, 95% CI: 1.31-3.02; P=0.001) and DFS (HR=1.81, 95% CI: 1.08-3.05; P=0.025)28. Moreover, subgroup analysis showed that decreased CDX2 was a
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negative prognostic factor in Asian, but not in Caucasian patients. In our metaanalysis, however, 3000 more Caucasian patients were included and this likely increased our statistical power. By applying an original bioinformatics approach to the discovery of prognostic cancer biomarkers, Dalerba and colleagues published in early 2016 the results of a pivotal trial testing the relationship between CDX2 and survival of CRC patients9. Undoubtedly, investigators had the merit to establish the negative
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ACCEPTED MANUSCRIPT prognostic effect associated with the absence of CDX2 expression and to simultaneously identify stage II CRC cases who might significantly benefit from adjuvant chemotherapy. The study was also object of criticism especially due to retrospective nature of the analysis, the small number of patients with stage II
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colon cancer and CDX2-negative tumor, and the type of immunohistochemical analysis (performed on tissue microarrays) that may have underestimated the intra-tumoral
heterogeneity
of
CDX2
expression29.
Although
definitive
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conclusions cannot still be drawn, such trial has paved the way to a new wave of biomarker research on predictors of efficacy from adjuvant therapy in early
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stage CRC.
Among patients with stage II disease, 5-year survival rates can widely differ depending on depth of invasion through the intestinal wall (as low as 60 % for T4 tumors)30.To date, owing to the minimal advantage provided, the choice of
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recommending adjuvant treatment in stage II tumors relies on different clinicopathologic variables including stage, tumor grade and microsatellite
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instability. Alongside these classical parameters, novel prognostic tools mainly represented by gene expression assays (e.g., Coloprint, Oncotype-DX, etc.)
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have emerged in the last years and are currently available for clinical use in the United Stated. All these tests have been shown to perform well in independent patient series, though the design and sample numbers of the validation analyses varied considerably31. For this reason, translating such molecular signatures into regular clinical use has not yet been realized. Finally, the high costs of these procedures further limit their widespread application. CDX2 expression testing, as evaluated in all the trials included in our analysis (i.e., IHC), is generally not expensive and easy to perform. Also in light of these 13
ACCEPTED MANUSCRIPT aspects, we believe that findings of this meta-analysis are noteworthy. Importantly, as indicated by subgroup analysis, CDX2 positivity was shown to be a strong prognostic factor independent of other variables including ethnicity,
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type of study, MSI status, CDX2 detection modality or stage. Accumulating evidence suggests that CDX2 downregulation is associated with several adverse prognostic factors such as MMR deficiency (in advanced stages), BRAF mutations, CIMP positive status, right-sided tumors, and poor
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differentiation at both the mRNA and protein level22. If confirmed, CDX2 may
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therefore function as a single prognostic biomarker indicator of multiple adverse features. Of course, owing to the retrospective design of most studies analyzed, these findings need to be prospectively confirmed and validated in the context of appropriate randomized clinical trials projected to answer relevant translational research questions. To further investigate the sources of
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heterogeneity, we performed meta-regression analysis. We did not find any significant relationship between prognosis of CDX2+ (vs CDX2-) CRC and other
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confounding factors, except in patients that received chemotherapy. It is likely that the more disease is aggressive (high risk stage II or III) or advanced (stage
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IV), the higher is the survival difference between CDX2+ and CDX- CRC. Some limitations of this study must also be remarked. The heterogeneity among included studies cannot be ignored. Stages, receipt of adjuvant chemotherapy, ethnicity and retrospective nature of published series are the main reasons. Besides, these studies have used different anti-CDX2 antibodies and a variety of different scoring criteria to define positive CDX2 cases. Publication bias is the second possible reason. In fact, studies showing that CDX2 was not a prognostic factor could have been less likely published as compared with 14
ACCEPTED MANUSCRIPT studies showing positive results. Third, included studies were only Englishwritten publications. Fourth, individual patient’s data were not available, therefore adjustment for other prognostic covariates was not possible. Finally, studies with larger sample sizes, such as randomized multicenter trials
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evaluating adjuvant chemotherapy would be needed for more definitive conclusions.
To the best of our knowledge, this is the largest meta-analysis ever conducted
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to investigate the association between CDX2 expression and outcome in early
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stages CRC. CDX2 positivity confirms to be a strong prognostic determinant associated with an excellent survival in stage II and III CRC. In this specific stages, along with other clinical and pathological factors, lack of expression of CDX2 should be formally regarded as an important indicator for adjuvant
1.
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Country
N° pts
Stage %
CT (%)
Baba 2009 Bae 2015 Bauer 2012
Prospective Retrospective Retrospective
USA Korea France
621 713 197
I-IV (II-III 59) I-IV (II-III 96) I-III (II-III 82)
0 65 -
Dalerba 2016
Retrospective
USA
1897
II-III (100)
74
Dawson 2013 Droy-Dupre 2015 Hong 2013 Karamitopolou 2010 Kim 2013 Knösel 2012 Lundberg 2016 Matsuda 2010 Olsen 2016 Pilati 2017
Retrospective Retrospective Retrospective Retrospective Retrospective Retrospective Retrospective Retrospective Retrospective Retrospective
Switzerland France Korea Greece/Switzerland Korea Germany Sweden Japan Denmark France
201 116° 207 82 109 402 445 97* 191 469
I-IV (100) I-IV (II-III 69) I-IV (II-III 76.9) III-IV (III 83.8) I-IV (II-III 89) II-III (100) I-IV (II-III 57.7) II-III (100) I-IV (85) II-III (100)
31.5 31 73.4 85.4 61.4 43
Zhang 2017 Nolte/2017
Retrospective Retrospective
USA Germany
145 612
IV (100) I-IV (II-III 61)
CDX2+ %/cutoff 70.5/weak to strong vs loss 94.1/retained vs loss 84/high vs low (15.7 and 10.5%)** 92.8/widespread vs loss/faint nuclear expression 57/diffuse vs loss 86.8/ >50% vs <50% 94.7/+ & strong+ vs weak + 61,3^/86.2/>5% vs <5% 58/high vs low 82.9/>5% vs <5% 78.7/ + vs 92/high/normal vs low/absent 84.4/> vs <6.5 log2 of normalized expression values 94.4/present vs absent -
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Type of study
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Author/year
100 39
CDX2 detection IHC/clone CDX2-88, Biogenex IHC/clone CDX2-88, Biogenex TMA (mRNA)/-
NOS score 9 8 7
IHC/clone CDX2-88, Biogenex
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IHC/Novocastra IHC/clone CDX2-88, Biogenex IHC/Novocastra IHC TMA/ clone AMT28, AbCam IHC/Ventana Medical Systems IHC/MU 392A-UC BioGenex IHC/ clone CDX2-88,Biogenex IHC/BioGenex IHC/EPR2764Y, CellMarque TMA (mRNA)/-
7 6 7 6 7 7 6 7 7 7
IHC/IHC/MU392A-UC, Biogenex
6 7
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Tab. 1 characteristics of included trials
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*, only stage II-III patients included in survival analysis; °, with survival data available; ^, media n value; -, not reported; **, right and left colorectal cancer cutoffs; IHC: immunohistochemistry; pts: patients; CT: chemotherapy; NOS: Nottingham Ottawa Scale
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Records identified through Pubmed searching (n = 206 )
Additional records identified through other sources (n = 3034 )
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Records screened (n = 3240 )
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Full-text articles assessed for eligibility (n = 937)
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Included
Eligibility
Screening
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Records after duplicates removed (n = 3240 )
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Identification
Fig.1 Literature search flow diagram
Studies included in qualitative synthesis (n = 25 )
Records excluded because were duplicated (n = 2303) Full-text articles excluded, because they were review/letters/commentarie, included other cancer patients or did not analyse prognostic role of CDX2 (n = 912 ) Full-text articles excluded, because they did not reported hazard ratios or other survival data (n = 9)
Studies included in quantitative synthesis (meta-analysis) (n = 16 )
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Fig.3 association of CDX2 expression with disease-free survival
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Fig.2 association of CDX2 expression with overall survival