CDX2 Loss Research Articles

Association of CDX2 expression with clinical, pathologic, and molecular features in MSI-H colorectal cancer.

e15583 Background: Microsatellite unstable (MSI) colorectal cancers (CRCs) are due to DNA mismatch repair (MMR) deficiency and are seen in 15% of early stage and 5% of metastatic disease. CRC with the MMR-D/MSI-H phenotype carries unique characteristics that are associated with increase mutational burden. Specifically, loss of CDX2 expression is associated with aggressive carcinomas and correlates with worse disease-free survival in the general population. However, the impact of loss of CDX2 expression in MMR-D/MSI-H phenotype is not well known. In this study, we investigated the association of loss of CDX2 expression with clinical and pathological outcomes. Methods: We retrospectively reviewed 543 patients who were diagnosed with colorectal cancer with pathological findings for the presence of MMR-D/MSI-H phenotype, and who had been seen at UPMC Hillman Cancer Center from 2013-2020. CDX2 expression was evaluated across all patients for the association with various molecular, pathological features and survival. In our investigations of association with loss of CDX2, coded as a binary variable in an H score, we used Mann-Whitney or two-sample (Welch’s) t-test for continuous variables, and Chi-square or Fisher’s exact tests for categorical variables. For RFS and OS, we compared H score groups with log-rank tests at two-sided alpha = 0.05 and plotted respective Kaplan-Meier curves. Results: A total of 99 patients were identified with adequate tissue sample to assess for CDX2 expression and with consistent clinical follow-up from date of diagnosis to present. When examining loss of CDX2 expression and association with pathological factors, we observed a significant association with a higher T4 stage (43% vs 14%; p = 0.008), and high grade tumor (86% vs 22%; p = 1.086 x 10-8 ) as well as presence of PNI (peri-neural invasion) (36% vs 17%; p = 0.030). There was no association observed between CDX2 expression and N stage or location of tumor. When examining loss of CDX2 expression and association with molecular factors, we observed that patients with Lynch syndrome were more likely to preserve CDX2 expression (0% vs 27%; p = 0.001) while patients with BRAF mutation were more likely to present with loss of CDX2 expression. Finally, patients with loss of CDX2 expression had numerically lower median survival (8.2 years vs. 9.3 years) though this did not reach statistical significance (p = 0.120). Conclusions: Overall, loss of CDX2 expression correlates with worse pathological features as it was significantly seen in patients with a diagnosis of CRC at a higher T stage, grade, presence of PNI and presence of BRAF mutation. Although no significant association was observed in terms of survival outcomes due to likely insufficient power, a notable separation of survival curves was noted, which indicates overall lower survival.

Concomitant expression patterns of CDX2 and SATB2 as prognostic factors in stage III colorectal cancers

CDX2 and SATB2 are often used as biomarkers for identification of colorectal origin in primary or metastatic adenocarcinomas. Loss of CDX2 or SATB2 expression has been associated with poor prognosis in patients with colorectal cancer (CRC). However, little is known regarding clinicopathological features, including prognosis, of CRCs with concomitant loss of CDX2 and SATB2.A total of 431 stage III CRCs were analyzed for their expression status in CDX2 and SATB2 using tissue microarray-based immunohistochemistry and expression status was correlated with clinicopathological variables, molecular alterations, and survival.CDX2-negative (CDX2–) CRCs and SATB2-negative (SATB2–) CRCs were found in 8.1 % and 17.2 % of CRCs, respectively, whereas both CDX2-negative and SATB2-negative (CDX2–/SATB2–) CRCs comprised 3.2 % of the CRCs. On survival analysis, neither CDX2–/SATB2+ nor CDX2+/SABT2– CRCs but CDX2–/SATB2– CRCs were associated with poor prognosis. CDX2–/SATB2– CRCs showed significant associations with tumor subsite of right colon, poor differentiation, decreased expression of CK20, aberrant expression of CK7, CIMP-high, MSI-high, and BRAF mutation.In summary, our results suggest that concomitant loss of CDX2 and SATB2 is a prognostic biomarker but isolated loss of CDX2 or SATB2 is not a prognostic biomarker for stage III CRCs.

CDX2 dose-dependently influences the gene regulatory network underlying human extraembryonic mesoderm development.

Loss of Cdx2 in vivo leads to stunted development of the allantois, an extraembryonic mesoderm-derived structure critical for nutrient delivery and waste removal in the early embryo. Here, we investigate how CDX2 dose-dependently influences the gene regulatory network underlying extraembryonic mesoderm development. By engineering human induced pluripotent stem cells (hiPSCs) consisting of wild-type (WT), heterozygous (CDX2-Het), and homozygous null CDX2 (CDX2-KO) genotypes, differentiating these cells in a 2D gastruloid model, and subjecting these cells to single-nucleus RNA and ATAC sequencing, we identify several pathways that are dose-dependently regulated by CDX2 including VEGF and non-canonical WNT. snATAC-seq reveals that CDX2-Het cells retain a WT-like chromatin accessibility profile, suggesting accessibility alone is not sufficient to drive this variability in gene expression. Because the loss of CDX2 or TBXT phenocopy one another in vivo, we compared differentially expressed genes in our CDX2-KO to those from TBXT-KO hiPSCs differentiated in an analogous experiment. This comparison identifies several communally misregulated genes that are critical for cytoskeletal integrity and tissue permeability. Together, these results clarify how CDX2 dose-dependently regulates gene expression in the extraembryonic mesoderm and reveal pathways that may underlie the defects in vascular development and allantoic elongation seen in vivo.

Tissue-location-specific transcription programs drive tumor dependencies in colon cancer

Cancers of the same tissue-type but in anatomically distinct locations exhibit different molecular dependencies for tumorigenesis. Proximal and distal colon cancers exemplify such characteristics, with BRAFV600E predominantly occurring in proximal colon cancers along with increased DNA methylation phenotype. Using mouse colon organoids, here we show that proximal and distal colon stem cells have distinct transcriptional programs that regulate stemness and differentiation. We identify that the homeobox transcription factor, CDX2, which is silenced by DNA methylation in proximal colon cancers, is a key mediator of the differential transcriptional programs. Cdx2-mediated proximal colon-specific transcriptional program concurrently is tumor suppressive, and Cdx2 loss sufficiently creates permissive state for BRAFV600E-driven transformation. Human proximal colon cancers with CDX2 downregulation showed similar transcriptional program as in mouse proximal organoids with Cdx2 loss. Developmental transcription factors, such as CDX2, are thus critical in maintaining tissue-location specific transcriptional programs that create tissue-type origin specific dependencies for tumor development.

Primary Papillary Colonic Adenocarcinoma With PDGFRA Mutation. A New Morphological Subtype? A Case Report and Review of Literature.

Classic colon carcinomas are defined as adenocarcinomas, characterized by groups of medium/large cells with basophilic and polymorphous nuclei and an eosinophilic elongated cytoplasm, that rearrange on glandular structures. Signs of poor prognosis include high tumor budding, lymphovascular and perineural invasion, poor differentiation, positive margins, and CDX2 loss. Less frequent colon carcinoma subtypes are: mucinous, medullary, signet-ring cell, squamous cell, small cell and undifferentiated carcinoma, among others. In the following case report, we present a 65-year-old woman with a T2N0Mx colon carcinoma with a remarkable papillary and follicular histological appearance. The immunohistochemical stains confirmed an intestinal origin (CDX2+) and excluded a thyroid, gynecological, and urological metastasis, with tumor cells negative for GATA3, PAX8, TTF-1, and thyroglobulin. There was no loss of mismatch repair proteins and p53 showed a wild-type staining. next generation sequencing showed a platelet-derivated growth factor receptor alpha (PDGFRA) mutation. To the best of our knowledge, there have been only two examples of primary papillary colon carcinoma reported in the literature, and neither of them with a PDGFRA mutation. We describe one tumor and discuss its pathological features.

Genetic and immune landscape evolution in MMR-deficient colorectal cancer.

Mismatch repair-deficient (MMRd) colorectal cancers (CRCs) have high mutation burdens, which make these tumours immunogenic and many respond to immune checkpoint inhibitors. The MMRd hypermutator phenotype may also promote intratumour heterogeneity (ITH) and cancer evolution. We applied multiregion sequencing and CD8 and programmed death ligand 1 (PD-L1) immunostaining to systematically investigate ITH and how genetic and immune landscapes coevolve. All cases had high truncal mutation burdens. Despite pervasive ITH, driver aberrations showed a clear hierarchy. Those in WNT/β-catenin, mitogen-activated protein kinase, and TGF-β receptor family genes were almost always truncal. Immune evasion (IE) drivers, such as inactivation of genes involved in antigen presentation or IFN-γ signalling, were predominantly subclonal and showed parallel evolution. These IE drivers have been implicated in immune checkpoint inhibitor resistance or sensitivity. Clonality assessments are therefore important for the development of predictive immunotherapy biomarkers in MMRd CRCs. Phylogenetic analysis identified three distinct patterns of IE driver evolution: pan-tumour evolution, subclonal evolution, and evolutionary stasis. These, but neither mutation burdens nor heterogeneity metrics, significantly correlated with T-cell densities, which were used as a surrogate marker of tumour immunogenicity. Furthermore, this revealed that genetic and T-cell infiltrates coevolve in MMRd CRCs. Low T-cell densities in the subgroup without any known IE drivers may indicate an, as yet unknown, IE mechanism. PD-L1 was expressed in the tumour microenvironment in most samples and correlated with T-cell densities. However, PD-L1 expression in cancer cells was independent of T-cell densities but strongly associated with loss of the intestinal homeobox transcription factor CDX2. This explains infrequent PD-L1 expression by cancer cells and may contribute to a higher recurrence risk of MMRd CRCs with impaired CDX2 expression. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The Prognostic Role of Mismatch Repair Status and CDX-2 Expression with Inflammatory Markers and Pathological Risk Factors in Stage II and III Colon Cancer: Multicenter Real-Life Data.

Colorectal cancer is common worldwide, and adjuvant treatment's benefit is still controversial. We designed this study to determine the role of MSI and CDX-2 status determined by immunohistochemistry (IHC) combined with the inflammatory markers and pathological parameters in predicting disease recurrence in stage II and III colon cancer. A total of 226 stage II/III colon cancer patients with a median age of 59years who underwent initial surgery were included in this retrospective study. The pathologic assessment of MSI and CDX-2 was performed twice by immunohistochemistry (IHC) and two different pathologists. No staining/weak staining below 10% of the tumor was accepted as CDX-2 negative, and any MSI clones with weak staining below 10% were accepted as MSI-H. The laboratory parameters were noted at the initial diagnosis. One hundred twenty-one and 105 patients were diagnosed with stage III and II colon cancer. 58.0% of patients were male, 46 (20.4%) of tumor tissue were MSS, and 17 (7.5%) were CDX-2 negative. One hundred twenty-nine tumors were localized in the right colon. Disease recurrence was significantly correlated with tumor localization, CDX-2 status, stage at diagnosis, and preoperatively median CRP and CEA levels. DFS rates for MSS patients with CDX-2 negative and positive were 36.7% and 98.1%, respectively [p < 0.001]. There was no significant correlation between MSI status and CDX-2 status. MSI status, the presence of adjuvant treatment, and systemic inflammatory markers were not significant prognostic factors for DFS. CDX-2 status [HR:0.08, CI 95% 0.03-0.17, p < 0.001 HR: 1.7, CI 95% 1.1-3.0, p = 0.03], disease stage [HR:2.6, CI 95% 1.43-4.74], and preoperatively CEA levels [HR:4.1 CI 95% 2.18-785, p < 0.001 were independent significant prognostic factors for DFS. CDX-2 loss was an independent prognostic factor for DFS and disease recurrence in early-stage colon cancer. MSS patients with CDX-2 loss had significantly worse survival outcomes, and this might be the reason for deciding on adjuvant chemotherapy.

Mucin-Phenotype and Expression of the Protein V-Set and Immunoglobulin Domain Containing 1 (VSIG1): New Insights into Gastric Carcinogenesis.

In gastric cancer (GC), intestinal metaplasia (IM) is a common precursor lesion, but its relationship to the MUC2/MUC5AC/CDX2 axis is not completely understood. Although V-set and immunoglobulin domain containing 1 (VSIG1) is supposed to be a specific marker for gastric mucosa and GC, respectively, no data about its relationship with IM or mucin phenotype have been published. The aim of our study was to explore the possible linkage between IM and these four molecules. The clinicopathological features of 60 randomly selected GCs were examined in association with VSIG1, MUC2, MUC5AC and CDX2. Two online database platforms were also used to establish the transcription factors (TFs) network involved in MUC2/MUC5AC/CDX2 cascade. IM was more frequently encountered in females (11/16 cases) and in patients below 60 years old (10/16 cases). Poorly differentiated (G3) carcinomas tended to show a loss of CDX2 (27/33 cases) but not of MUC2 and MUC5AC. MUC5AC and CDX2 were lost in parallel with the depth of invasion of the pT4 stage (28/35 and 29/35 cases), while an advanced Dukes-MAC-like stage was only correlated with CDX2 and VSIG1 loss (20/37 and 30/37 cases). VSIG1 was directly correlated with MUC5AC (p = 0.04) as an indicator of gastric phenotype. MUC2-negative cases showed a propensity towards lymphatic invasion (37/40 cases) and distant metastases, while CDX2-negative cases tended to associate with hematogenous dissemination (30/40 cases). Regarding the molecular network, only 3 of the 19 TFs involved in this carcinogenic cascade (SP1, RELA, NFKB1) interacted with all targeted genes. In GC, VSIG1 can be considered an indicator of gastric phenotype carcinomas, where carcinogenesis is mainly driven by MUC5AC. Although infrequently encountered in GC, CDX2 positivity might indicate a locally advanced stage and risk for vascular invasion, especially in tumors developed against the background of IM. The loss of VSIG1 indicates a risk for lymph node metastases.

Abstract 314: Intratumor genetic heterogeneity dictates metastatic subclones in stage II colon cancer

Abstract The high incidence of colon cancer (CC) worldwide is a major health concern. Although surgery and adjuvant chemotherapy are effective to a high extent in lymph-node negative colon cancers, there are still some 10-15% of patients that show disease relapse within the next 5-year period after intended curative surgery. We hypothesize that genomic instability and intratumor heterogeneity fuel the formation of subclonal populations in the primary tumor that might promote metastasis. In the current study, we assessed the presence of intratumor heterogeneity in CC using multiplex interphase FISH (miFISH) and whole-exome sequencing (WES) to evaluate samples obtained from primary stage II colon tumors and their patient-matched associated liver metastases using multi-region sampling (N=9). In combination with genome-wide copy-number changes obtained from WES analysis, miFISH allowed the simultaneous quantification of copy-numbers for nine CC relevant genes and a centromeric ploidy control probe in intact tumor nuclei derived from archival patient material. Our preliminary data on the four cases analyzed so far, revealed high similarities regarding copy-number changes and ploidy between primary tumor and metastatic samples for three of the cases. While two of these cases showed either triploid or highly aneuploid genomes with different degrees of genomic instability and subclonality among them, the third case displayed a diploid content for both matched lesions. Intriguingly, as seen by miFISH and WES, loss of CDX2 in the primary tumor might have originated the main clonal population giving rise to metastasis in this latter case. Conversely, the fourth case analyzed exhibited a triploid baseline for the primary tumor while the metastasis revealed a diploid population. Interestingly, despite higher absolute copy numbers in the primary tumor, there was an overlap of five of the gains and losses observed for the major clone populations of the tumor and metastasis by miFISH. However, two of the highly clonal gains observed in the tumor, namely the EGFR and MYC gain, were not present in the metastasis. Altogether, in addition to the ploidy change, results indicated major discrete copy-number changes from primary tumor to metastasis in this patient. In summary, we show here that the combined analysis of WES and miFISH is able to describe the subclonal composition of copy-number alterations in primary tumors and their patientmatched liver metastases. Future perspectives also include the assessment of subclonality affecting single-nucleotide variants to complete the genomic landscape of tumor evolution and clonal development in this patient cohort with the goal to better understand the nature of these early metastases. Citation Format: Sanam Yaghoubi, Kerstin Heselmeyer-Haddad, Ivan Archilla, Carolina Parra, Darawalee Wangsa, Giancarlo Castellano, Sara Lahoz, Veronica Pablo-Fontecha, Daniela Hirsch, Wei-Dong Chen, Thomas Ried, Miriam Cuatrecasas, Paul S. Meltzer, Jordi Camps. Intratumor genetic heterogeneity dictates metastatic subclones in stage II colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 314.

Abstract 1233: Depletion of Apc gene in Cdx2 gastro-intestinal cells facilitates metastasis of colorectal adenocarcinoma Lgr5 cells to distant organs

Abstract Background: CDX2 is a transcription factor expressed in the gastro-intestinal (GI) epithelial (IEC) and stromal cells. CDX2 along with APC can regulate Lgr5 intestinal stem cell (ISC) differentiation to control GI development or neoplasia. CDX2 or APC loss function is associated with CRC advance. However, whether APC directly regulates CRC metastasis is not clear. We aim to determine the role of inactive Apc gene in Cdx2 GI cells on Lgr5 ISC metastasis. Methods: Tissue sections were collected from CRC or CRC hepatic-metastatic patients, CDX2, CTNNB1, α-SMA, APC and LGR5 expressions were examined. Apc flox mice were crossed with Lgr5CreER and/or Cdx2CreER mice to inactivate Apc gene in Lgr5 ISCs, Cdx2 IECs or stromal cells. Cdx2CreER;Apc mice were then crossed with tgfr2 flox mice to deplete tgfr2 in the Apc-deficient Cdx2 cells. GI tract and liver histology was evaluated and the expression of Lgr5, APC, α-SMA, β-catenin, and CDX2 were examined. The colorectal polyps induced by Apc inactivation were dissected, total RNA was extracted to perform RNA-Seq and quantitative PCR analyses. The colorectal organoids were differentiated and transplanted into abdominal cavity of recipient mice. Meanwhile, HT-29 cells were transfected with APC and/or CDX2 gRNAs. Results: CTNNB1 and LGR5 expression were increased in human CRC and hepatic-metastatic CRC while interstitial CDX2 and SMA colocalization were robustly pronounced compared to normal colorectum. TCGA analysis showed the positive correlation of APC lower and CDX2 higher with metastatic rectal cancer. Apc depletion in Lgr5 cells in 6-week-old mice led to Lgr5+ rectal adenocarcinoma and Lgr5 crypts in liver while depletion of Apc in Lgr5 cells in 3-month-old mice only resulted in Lgr5 intestinal adenoma. Apc depletion in Cdx2 cells led to rectal fibroma and intestinal adenoma as well as undifferentiated metastatic Lgr5 cells in liver. Notably, Apc depletion decreased crypt CDX2 while increased colocalization of CDX2 and α-SMA in the stromal cells of adenocarcinoma, and increased expression of TGFBβr2, β-catenin, and epithelial-mesenchymal transition (EMT) markers (Fibronectin and Vimentin). Finally, tgfr2 depletion in Apc-inactive Cdx2 cells impaired GI cancer cell metastasis to liver. Colorectal orgnoids exhibited cancer phenotypes upon Apc depletion in CDX2 or Lgr5 cells. Intra-abdominal xenotransplantation exhibited that organoids with Apc deficiency in Lgr5 cells appeared no colonization on or invasion of liver tissues whereas the organoids with Apc deficiency in Cdx2 cells invaded liver. Conclusion: Reduced Apc in Cdx2 cells are required for Lgr5 cell transformation to Lgr5- cancer stem cells. Inactive intestinal Apc increases Cdx2-α-SMA stromal niche cells and facilitates Lgr5 ISC metastasis to liver partially through TGF-β and/or wnt-dependent EMT mechanisms. Citation Format: Ahmed Bakheet, Wen Gao, Dan Cai, Nathan Berger, William Tse, Xiaonan Han. Depletion of Apc gene in Cdx2 gastro-intestinal cells facilitates metastasis of colorectal adenocarcinoma Lgr5 cells to distant organs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1233.

353P Loss of CDX2 and high COX2 (PTGS2) expression in stage IV colorectal cancer

Lack of expression of the tumor suppressor CDX2 is associated with stage II/III colorectal cancer (CRC) poor outcomes. Its potential prognostic and predictive values in metastatic CRC (mCRC), and correlation with other potential biomarkers are of great interest. Overexpression of the PTGS2 gene encoding cyclooxygenase 2 (COX2) has shown a significantly direct association with advanced CRC stages. Reduced CDX2 in CRC has been suggested to enhance NF-κB-mediated inflammatory response, upregulating COX2 expression.

360P Epigenetic engineering on CDX2 allows to determine the interplaying consequences with other genetic and transcriptomic events in colorectal cancer

The loss of the caudal homeobox 2 protein (Cdx2) is associated with poor prognosis in colorectal cancer (CRC) patients. However, the mechanisms that generate the loss of expression and the interplay with other genetic and transcriptomic aberrations remain unknown. Understanding the regulatory mechanism and interaction processes of CDX2, through a multi-omic approach, would help to change the genomic/transcriptomic tumor pattern, favoring sensitivity to standard treatment. CDX2 promoter methylation and mutations were studied by Miseq in both 7 CRC cell lines and 50 CRC patients. Azaytidine was used as demethylation treatment. A parallel study using CRISPR-dCAS9 fused with the catalytic TET and DNMT domains was required for the expression reversal studies. Determination of CDX2 status has been performed using a new approach developed in-house. A custom gene panel was used to study other genomic aberrations. Transcriptomic analysis by RNAseq was assessed through differential expression by DESeq2. Among the analyzed CG positions, a promoter island with an increased methylation rate tumor/normal and inverse correlation with expression was identified. Azacytidine treatment showed a dose-dependent correlation between methylation and expression (p<0.001). We also demostrated a specific reversion of CDX2 expression using CRISPR-dCAS9, subsequently changes in FOLFOX sensitivity was observed (p<0.05). Patients with CDX2- had higher levels of methylation in the studied CpG island than those with CDX2+. NGS showed no functional mutations or copy-number changes on CDX2-. Upstream mutations displayed an interaction between CDX2- and mutations in FLNA. Transcriptomic analysis revealed changes in INFa response pathway and MAPK proliferation genes. CDX2 promoter hypermethylation is the main regulatory mechanism of CDX2 expression. Loss of CDX2 interacts with immune and proliferation pathways at genomic and transcriptomic levels, conferring an agresive phenotype. Epigenetic engineering modifications of CDX2 could favor increase the sensitivity to standar therapy in this setting.

Single-cell chromatin profiling of the primitive gut tube reveals regulatory dynamics underlying lineage fate decisions

Development of the gastrointestinal system occurs after gut tube closure, guided by spatial and temporal control of gene expression. However, it remains unclear what forces regulate these spatiotemporal gene expression patterns. Here we perform single-cell chromatin profiling of the primitive gut tube to reveal organ-specific chromatin patterns that reflect the anatomical patterns of distinct organs. We generate a comprehensive map of epigenomic changes throughout gut development, demonstrating that dynamic chromatin accessibility patterns associate with lineage-specific transcription factor binding events to regulate organ-specific gene expression. Additionally, we show that loss of Sox2 and Cdx2, foregut and hindgut lineage-specific transcription factors, respectively, leads to fate shifts in epigenomic patterns, linking transcription factor binding, chromatin accessibility, and lineage fate decisions in gut development. Notably, abnormal expression of Sox2 in the pancreas and intestine impairs lineage fate decisions in both development and adult homeostasis. Together, our findings define the chromatin and transcriptional mechanisms of organ identity and lineage plasticity in development and adult homeostasis.

Transcriptome of sessile serrated adenoma/polyps is associated with MSI-high colorectal cancer and decreased expression of CDX2.

The objective of this study was to elucidate the molecular background of sessile serrated adenoma/polyp (SSA/P) endoscopically resected with comprehensive gene expression analysis. Gene expression profiling was performed for 10 tumor-normal pairs of SSA/P. Cluster analysis, gene set enrichment analysis (GSEA), and consensus molecular subtype (CMS) classification of colorectal cancer (CRC) were applied to our transcriptome analysis. Unsupervised cluster analysis showed that the gene expression profile of SSA/Ps is different from that of adjacent normal epithelial cells, even in the very early stage of tumorigenesis. According to the CMS classification, our microarray data indicated that SSA/Ps were classified as CMS1. GSEA demonstrated a strong association between SSA/P and microsatellite instability-high (MSI-H) CRC (p < 10-5 ). Transcriptome analysis of five MSI-related genes (MSH2, MSH6, MLH1, PMS1, and PMS2) and five CRC-related genes (BRAF, KRAS, APC, TP53, and CDX2) showed that CDX2 expression was most severely decreased in SSA/P. Immunohistochemical staining confirmed that CDX2 protein was reduced compared with the surrounding mucosa. Direct sequencing of the BRAF gene showed that the BRAF V600E mutation was detected in only nine of 36 cases. In a mouse model, BRAF, APC, or CDX2 deficiency indicated that the gene expression pattern with loss of CDX2 is more similar to our SSA/Ps compared with those induced by BRAF or APC mutation. Transcriptome analysis of SSA/Ps showed characteristic gene expression with a strong resemblance to MSI-H CRC. Downregulation of CDX2 expression is an essential molecular mechanism involved in the initial stage of SSA/P tumorigenesis. (UMIN000027365).

Dedifferentiation-like tubular and solid carcinoma of the stomach shows phenotypic divergence and association with deficient SWI/SNF complex.

Gastric carcinoma showing an abrupt transition from a tubular to solid pattern is an unusual phenomenon reminiscent of dedifferentiation. The phenotypic and molecular characteristics of this transition are still unclear. We retrospectively collected 41 gastric carcinomas exhibiting dedifferentiation-like tubular to solid transition and applied an array of immunohistochemical stains, including neuroendocrine and hepatocytic markers, to delineate their lineage. The status of Epstein-Barr virus (EBV) infections, mismatch repair proteins, SWI/SNF complex proteins and p53 expression levels were examined. The clinicopathologic differences were assessed by statistical analysis. Except for 10 cases with neuroendocrine differentiation and 2 EBV-associated carcinomas, we identified 8 hepatoid carcinomas and 21 solid adenocarcinomas with loss of CDX2 and/or hep-par1 expression in solid part (12/29). A subset of solid adenocarcinoma was associated with MSI (8) and mutant p53 expression was frequent in non-MSI cases (10/13). We found hepatoid carcinomas usually harbored SMARCA2 loss (5/8), MSI-associated cases commonly had ARID1A loss (6/8), and non-MSI solid adenocarcinomas frequently showed SMARCA2/A4 loss (7/13) with a high rate of concurrent ARID1A loss (4/7). Spatial correlation between solid transition and loss of SWI/SNF complex subunits were seen in 63% of tumors (12/19). Dedifferentiation-like tubular and solid carcinoma was associated with a propensity to inferior survival outcomes (p = 0.034), especially hepatoid carcinoma and in the non-MSI/EBV intestinal subgroup. In conclusion, gastric cancer exhibiting dedifferentiation-like tubular to solid transition is a phenotypically divergent group that shares common alterations in the SWI/SNF complex.

Loss of SATB2 Occurs More Frequently Than CDX2 Loss in Colorectal Carcinoma and Identifies Particularly Aggressive Cancers in High-Risk Subgroups

Simple SummaryThe immunohistochemical analysis of Special AT-rich sequence-binding protein 2 (SATB2) is increasingly being used to detect colorectal differentiation. Our study aimed to investigate SATB2 expression levels and the prognostic relevance of SATB2 loss in colorectal carcinoma (CRC), especially in comparison with CDX2, the standard marker of colorectal differentiation. We tested SATB2 expression in 1039 CRCs and identified SATB2 as a strong prognosticator in the overall cohort as well as in specific subcohorts, including high-risk subgroups. Compared to CDX2, SATB2 showed a higher prognostic power but was lost at a much higher frequency, generally rendering SATB2 as the less sensitive marker for colorectal differentiation compared to CDX2.Background: Special AT-rich sequence-binding protein 2 (SATB2) has emerged as an alternative immunohistochemical marker to CDX2 for colorectal differentiation. However, the distribution and prognostic relevance of SATB2 expression in colorectal carcinoma (CRC) have to be further elucidated. Methods: SATB2 expression was analysed in 1039 CRCs and correlated with clinicopathological and morphological factors, CDX2 expression as well as survival parameters within the overall cohort and in clinicopathological subgroups. Results: SATB2 loss was a strong prognosticator in univariate analyses of the overall cohort (p < 0.001 for all survival comparisons) and in numerous subcohorts including high-risk scenarios (UICC stage III/high tumour budding). SATB2 retained its prognostic relevance in multivariate analyses of these high-risk scenarios (e.g., UICC stage III: DSS: p = 0.007, HR: 1.95), but not in the overall cohort (DSS: p = 0.1, HR: 1.25). SATB2 loss was more frequent than CDX2 loss (22.2% vs. 10.2%, p < 0.001) and of higher prognostic relevance with only moderate overlap between SATB2/CDX2 expression groups. Conclusions: SATB2 loss is able to identify especially aggressive CRCs in high-risk subgroups. While SATB2 is the prognostically superior immunohistochemical parameter compared to CDX2 in univariate analyses, it appears to be the less sensitive marker for colorectal differentiation as it is lost more frequently.

Prognostic role of tumour-infiltrating lymphocytes and macrophages in relation to MSI, CDX2 and BRAF status: a population-based study of metastatic colorectal cancer patients.

Tumour-infiltrating CD3, CD8 lymphocytes and CD68 macrophages are associated with favourable prognosis in localised colorectal cancer, but the effect in metastatic colorectal cancer (mCRC) is not established. A Scandinavian population-based cohort of non-resectable mCRC patients was studied. Tissue microarrays (n = 460) were stained with CD3, CD8 and CD68 using fluorescence-based multiplex immunohistochemistry. Associations with clinicopathological variables, overall survival (OS) and progression-free survival were estimated. Two-thirds of microsatellite instable (MSI) and one-fourth of microsatellite stable (MSS) tumours displayed the highest quartile density of CD8. For CD3 high vs low cases, median OS was 20 vs 16 months (HR: 0.76, 95% CI: 0.59, 0.76, p = 0.025) with 3-year OS of 27 vs 13%. For CD68 high vs low cases, median OS was 23 vs 15 months (HR: 0.69, 95% CI: 0.54, 0.88, p = 0.003) with 3-year OS of 28 vs 12%. MSI, BRAF mutation and CDX2 loss were negative prognostic markers independent of tumour immune infiltration. In mCRC, high lymphocyte infiltration was found in proportions of MSI and MSS tumours-potential subgroups of immunotherapy response. Tumour-infiltrating CD3 lymphocytes and CD68 macrophages were associated with median and long-term survival. MSI was a significant negative prognostic marker despite high immunogenicity.

Loss of CDX2 in colorectal cancer is associated with histopathologic subtypes and microsatellite instability but is prognostically inferior to hematoxylin\u2013eosin-based morphologic parameters from the WHO classification

BackgroundImmunohistochemical loss of CDX2 has been proposed as a biomarker of dismal survival in colorectal carcinoma (CRC), especially in UICC Stage II/III. However, it remains unclear, how CDX2 expression is related to central hematoxylin–eosin (HE)-based morphologic parameters defined by 2019 WHO classification and how its prognostic relevance is compared to these parameters.MethodsWe evaluated CDX2 expression in 1003 CRCs and explored its prognostic relevance compared to CRC subtypes, tumour budding and WHO grade in the overall cohort and in specific subgroups.ResultsCDX2-low/absent CRCs were enriched in specific morphologic subtypes, right-sided and microsatellite-instable (MSI-H) CRCs (P < 0.001) and showed worse survival characteristics in the overall cohort/UICC Stage II/III (e.g. DFS: P = 0.005) and in microsatellite stable and left-sided CRCs, but not in MSI-H or right-sided CRCs. Compared with CDX2, all HE-based markers showed a significantly better prognostic discrimination in all scenarios. In multivariate analyses including all morphologic parameters, CDX2 was not an independent prognostic factor.ConclusionCDX2 loss has some prognostic impact in univariate analyses, but its prognostic relevance is considerably lower compared to central HE-based morphologic parameters defined by the WHO classification and vanishes in multivariate analyses incorporating these factors.

Cdx2 regulates immune cell infiltration in the intestine

The intestinal epithelium is a unique tissue, serving both as a barrier against pathogens and to conduct the end digestion and adsorption of nutrients. As regards the former, the intestinal epithelium contains a diverse repertoire of immune cells, including a variety of resident lymphocytes, macrophages and dendritic cells. These cells serve a number of roles including mitigation of infection and to stimulate regeneration in response to damage. The transcription factor Cdx2, and to a lesser extent Cdx1, plays essential roles in intestinal homeostasis, and acts as a context-dependent tumour suppressor in colorectal cancer. Deletion of Cdx2 from the murine intestinal epithelium leads to macrophage infiltration resulting in a chronic inflammatory response. However the mechanisms by which Cdx2 loss evokes this response are poorly understood. To better understand this relationship, we used a conditional mouse model lacking all intestinal Cdx function to identify potential target genes which may contribute to this inflammatory phenotype. One such candidate encodes the histocompatability complex protein H2-T3, which functions to regulate intestinal iCD8α lymphocyte activity. We found that Cdx2 occupies the H3-T3 promoter in vivo and directly regulates its expression via a Cdx response element. Loss of Cdx function leads to a rapid and pronounced attenuation of H2-T3, followed by a decrease in iCD8α cell number, an increase in macrophage infiltration and activation of pro-inflammatory cascades. These findings suggest a previously unrecognized role for Cdx in intestinal homeostasis through H2-T3-dependent regulation of iCD8α cells.

MRNA profiling of a well-differentiated G1 pancreatic NET correlates with immunohistochemistry profile: a case report

BackgroundNeuroendocrine neoplasms (NENs) are a complex group of tumours that occur in many organs. Routinely used IHC markers for NEN diagnosis include CgA, synaptophysin, Ki67 and CD56. These have limitations including lack of correlation to clinical outcomes and their presence in non-tumour tissue. Identification of additional markers and more quantitative analyses of tumour tissue has the potential to contribute to improved clinical outcomes. We used qRT-PCR to profile the expression levels of a panel of markers in tumour and matched non-tumour tissue from a patient with a G1 pancreatic neuroendocrine tumour. Differences in mRNA levels between tumour and non-tumour tissue were compared with IHC analyses of the same sample.Case presentationAn elderly man presented with lower abdominal pain for 6 months. Histological analysis identified a low grade, well differentiated pancreatic endocrine neoplasm. Twenty-seven tumour markers for neuroendocrine status, proliferation, stem cell phenotype, angiogenesis, epithelial to mesenchymal transition, cell adhesion, differentiation and tumour suppression were selected from previous studies and mRNA levels of these markers were measured in tumour and adjacent non-tumour tissue sample using qRT-PCR. IHC was carried out on the same tissue to detect the corresponding marker proteins. Of the markers analysed, seven showed higher mRNA levels in tumour relative to non-tumour tissue while thirteen had lower expression in tumour relative to non-tumour tissue. Substantial differences in mRNA levels were a gain of CgA, CD56, β-catenin, CK20, PDX1 and p53 and loss of Ki67, PCAD, CK7, CD31, MENA, ECAD, EPCAM, CDX2 and CK6. Comparison of qRT-PCR data with IHC showed correlation between fifteen markers.ConclusionOur study is unique as it included matched controls that provided a comparative assessment for tumour tissue analysis, whereas many previous studies report tumour data only. Additionally, we utilised qRT-PCR, a relatively quantitative diagnostic tool for differential marker profiling, having the advantage of being reproducible, fast, cheap and accurate. qRT-PCR has the potential to improve the defining of tumour phenotypes and, in combination with IHC may have clinical utility towards improving tumour stratification or distinguishing tumour grades. The results need to be validated with different grades of NENs and related to clinical outcomes.