CDX2 Expression Research Articles

Extensive CXCR4-MAPK Signaling Activation in the Novel B Cell Acute Lymphoblastic Subtype of CDX2/Ubtf

Background B-cell acute lymphoblastic leukemia (B-ALL) is a genetically heterogeneous group of acute leukemia with novel subtypes emerging. Since we first reported a novel subgroup B-ALL with half cases possessing CXCR4 truncated mutations and abnormal expression of CDX2 in all cases, the genomic features were further elucidated by different research groups that concurrent focal deletions lead to both upstream enhancer hijacking by CDX2 and UBTF::ATXN7L3 fusion transcript. But the molecular characterization and underlying mechanism beyond the novel subtype B-ALL remain ambiguous. Methods In order to comprehensively elucidate the molecular features, we adopt whole genome sequencing to reveal the genomic abnormalities, including snv, indel, copy number variant, and structural variation. Then, the signaling transduction and functional effect of differentially expressed genes were conducted by transcriptome analysis. Furthermore, we constructed a weighted gene co-expression network (WGCNA) and transcriptional regulons network to unravel the hub genes or transcriptional factors in the subtype. Results We retrospectively identify 2.4% (n=12) cases belonging to a novel subtype among 503 B-ALL cases with available transcriptome data by unsupervised clustering, and this subtype is characterized by CXCR4 truncated mutations or high expression of FLNA which could enhance CXCR4 signaling. Among these patients, only one male was affected showing a strong sex tendency towards females. Uniformly, all cases harbor both focal deletions encompassing between PAN3 and FLT3 located upstream of CDX2, and between UBTF and ATXN7L3, unraveled by whole genome sequencing (n=10). The focal deletions lead to overexpression of CDX2 by enhancer hijacking of upstream PAN3 and fusion transcript UBTF::ATXN7L3, respectively. In addition, 80% (n=8) cases also occurred with 1q+ which is regarded as a factor contributing to poor prognosis in leukemia and lymphoma. Transcriptomic analysis of the differentially expressed genes (DEGs) for this subtype confirmed the critical role of oncogenic MAPK signaling. Enhanced MAPK signaling was further supported by the function of the hub genes including KIAA1549 and KIAA1549L, which belong to oncogenic MAPK signaling, derived from WGCNA analysis. According to the functional enrichment analysis of DEGs, axon guidance was the only significant enriched KEGG pathway. The hub genes NTN3 from WGCNA analysis and regulon CDX2 both confirmed the functional involvement of axon guidance in this subtype. Furthermore, we prospectively identify 8 more cases by RT-PCR screening of the UBTF::ATXN7L3 transcript. Totally, twenty patients have been found in our cohort, and interestingly, there exist two cases among this subtype accumulated with multiple subclones characterized by different CXCR4 mutations, which strengthen the genotoxic stress over CXCR4 signaling. Discussion Although genomic characterization of this novel subtype has been revealed by our and others' research. The functional effect and underlying mechanism have never been elucidated. Here, we emphasize that enhanced CXCR4 signaling in this subtype may act as an upstream driver event, and it could be supplied as a therapeutic target.

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.

SMAD1 Is Dispensable for CDX2 Induction but Required for the Repression of Ectopic Small-Intestinal Gene Expression in Human-Pluripotent-Stem-Cell-Derived Colonic Organoids

The generation of gastrointestinal tissues from human pluripotent stem cells has provided unprecedented insight into the molecular mechanisms that drive the patterning of the primitive gut tube. Previous work has identified bone-morphogenetic-protein (BMP) signaling as an important mediator of mid/hindgut versus foregut and hindgut versus midgut cell fate choice. Inhibition of BMP signaling during gut tube morphogenesis inhibits the expression of the pan-intestinal transcription factor CDX2. Treatment of CDX2+ mid/hindgut cultures with BMP patterns them into hindgut, which gives rise to colonic organoids (HCOs). While the role for BMP signaling is clear, the molecular mechanisms through which BMP signaling patterns the mid/hindgut and colon remain unclear. BMPs bind to BMP receptors, activating a signaling cascade that results in the activation of SMADs, which function as transcription factors. We hypothesized that one of these factors, SMAD1, would be necessary for establishing the CDX2 domain and the colon domain. Unexpectedly, endoderm derived from SMAD1-deficient induced pluripotent stem cells was capable of inducing CDX2 in response to WNT and FGF signaling. In addition, CDX2+ gut tube cultures could activate posterior HOX genes in response to BMP. However, examination of HCOs following cytodifferentiation revealed that SMAD1-deficient HCOs ectopically expressed small-intestinal markers despite expressing posterior HOX genes. These results indicate that there is redundancy of SMADs during early hindgut patterning but that SMAD1 is required for the inhibition of small-intestinal gene expression in HCOs.

A Case of Medullary Carcinoma of the Jejunum Combined with the Intestinal Lymphangiectasia Accompanied by the Malabsorption Syndrome

Aim: to present a clinical and morphological observation of an extremely rare combination of medullary carcinoma of the jejunum and intestinal lymphangiectasia in a 33-year-old patient with clinical features of malabsorption syndrome over the 10 years.Key points. An autopsy revealed a tumor formation spreading from the wall of the jejunum to the mesentery, with metastases to the mesenteric lymph nodes. The medullary carcinoma with positive expression of СD117, DOG1, EMA, PanCK, PDL-1, vimentin, mosaic non-intense expression of CA19-9, calretinin, CD10, CDX2, CEA, MUC-5AC, SATB2, and negative reaction to ALK, CD3, CD8, CD20, CD30, CD31, CD34, CD45, CD56, chromogranin, CK7, CK20, desmin. The proliferative index was high: Ki-67 > 80 %. Moreover, during the histological examination of the intestinal wall, intestinal lymphangiectasia complicated by the malabsorption syndrome was revealed.Conclusion. The uniqueness of this clinical and morphological case is in the combination of medullary carcinoma of the jejunum metastasized to the mesenteric lymph nodes with the underlying intestinal lymphangiectasia accompanied by the development of malabsorption syndrome.

Dendrobium officinale Polysaccharides Inhibit CDCA-Induced Gastric Intestinal Metaplasia through Activating NRF2/HO-1 and Modulating HNF4α/CDX2 Signaling Pathway

Bile reflux (BR) was considered to be an independent risk factor for the development of precancerous gastric lesions and GC. Dendrobium officinale polysaccharides (DOP) show a novel potential in preventing the progress of gastric cancer. However, the specific mechanism of DOP that causes such activities remains a mystery. This study aimed to investigate the effects of DOP on chenodeoxycholic acid (CDCA)-induced gastric intestinal metaplasia and explore the underlying mechanisms. Different concentrations of DOP had no significant damage to normal GSE-1 cells and gastric intestinal metaplasia model cells by CCK-8 assay. After DOP treatment, the mRNA and protein expression of CDX2 ( p < 0.01) and HNF4α ( p < 0.01) were decreased, and HO-1 ( p < 0.05) and TFF2 ( p < 0.01) were increased. The NRF2 protein expression was slightly upregulated ( p < 0.05), and H-DOP further promoted NRF2 protein expression in the nucleus ( p < 0.05). Hence, our findings reveal that DOP could be used as a potential anti-inflammation supplement by activating NRF2/HO-1 and modulating the HNF4α/CDX2 signaling pathway to inhibit the progress of CDCA-induced gastric intestinal metaplasia.

Maternal exposure to hyperbaric oxygen at the preimplantation stages increases apoptosis and ectopic Cdx2 expression and decreases Oct4 expression in mouse blastocysts via Nrf2-Notch1 upregulation and Nf2 downregulation.

The environmental oxygen tension has been reported to impact the blastocyst quality and cell numbers in the inner cell mass (ICM) during human and murine embryogenesis. While the molecular mechanisms leading to increased ICM cell numbers and pluripotency gene expression under hypoxia have been deciphered, it remains unknown which regulatory pathways caused the underweight fetal body and overweight placenta after maternal exposure to hyperbaric oxygen (HBO). The blastocysts from the HBO-exposed pregnant mice revealed significantly increased signals of reactive oxygen species (ROS) and nuclear Nrf2 staining, decreased Nf2 and Oct4 expression, increased nuclear Tp53bp1 and active caspase-3 staining, and ectopic nuclear signals of Cdx2, Yap, and the Notch1 intracellular domain (N1ICD) in the ICM. In the ICM of the HBO-exposed blastocysts, both Nf2 cDNA microinjection and Nrf2 shRNA microinjection significantly decreased the ectopic nuclear expression of Cdx2, Tp53bp1, and Yap whereas increased Oct4 expression, while Nrf2 shRNA microinjection also significantly decreased Notch1 mRNA levels and nuclear expression of N1ICD and active caspase-3. We show for the first time that maternal exposure to HBO at the preimplantation stage induces apoptosis and impairs ICM cell specification via upregulating Nrf2-Notch1-Cdx2 expression and downregulating Nf2-Oct4 expression.

N4-acetylcytidine of Nop2 mRNA is required for the transition of morula-to-blastocyst.

N-acetyltransferase 10 (NAT10)-mediated N4-acetylcytidine (ac4C) modification is crucial for mRNA stability and translation efficiency, yet the underlying function in mammalian preimplantation embryos remains unclear. Here, we characterized the ac4C modification landscape in mouse early embryos and found that the majority of embryos deficient in ac4C writer-NAT10 failed to develop into normal blastocysts. Through single-cell sequencing, RNA-seq, acetylated RNA immunoprecipitation combined with PCR (acRIP-PCR), and embryonic phenotype monitoring, Nop2 was screened as a target gene of Nat10. Mechanistically, Nat10 knockdown decreases the ac4C modification on Nop2 mRNA and reduces RNA and protein abundance by affecting the mRNA stability of Nop2. Then, depletion of NOP2 may inhibit the translation of transcription factor TEAD4, resulting in defective expression of the downstream lineage-specific gene Cdx2, and ultimately preventing blastomeres from undergoing the trophectoderm (TE) fate. However, exogenous Nop2 mRNA partially reverses this abnormal development. In conclusion, our findings demonstrate that defective ac4C modification of Nop2 mRNA hinders the morula-to-blastocyst transition by influencing the first cell fate decision in mice.

Immunohistochemical Expression of CDX2 as Early Biomarkers for Gastric Intestinal Metaplasia among Sudanese Patients with Chronic Gastritis

Background: Chronic gastric infection with Helicobacter pylori can cause intestinal metaplasia, a pathologic change that is frequently a precursor of gastric cancer; CDX2 has an essential role in the development and maintenance of intestinal differentiation in the gut and ectopic sites such as intestinal metaplasia of the stomach. Objective: This study aimed to investigate caudal-type home box (CDX2) Immuno expression as an early indicator for gastric intestinal metaplasia among Sudanese patients with chronic gastritis. Materials and methods: This is a descriptive retrospective cross-sectional study conducted at Atbara Medical Complex and El-MakNimr University Hospital at River Nile State in the period from 2019 to 2022. 140 archival paraffin tissue blocks were collected from patients diagnosed with chronic gastritis, immunohistochemistry was used to detect (CDX2 expression as an early indicator for gastric intestinal metaplasia. Results: From 140 samples, 80 were females (57.1 %) and 60 were males (42.9%), the patient's ages ranged from (12 to 97) with a mean age of (53.27) years, and age was categorized into three groups, less than 40 (27) (19.3%), 40- 60 (64) (45.7%), more than 60 (49) (35.0%). The prevalence of gastric intestinal metaplasia was (17.9%, while no statistically significant correlation of gastric intestinal metaplasia with gender and age as the P.value was (0.308 and 0.256) respectively. Conclusion: The development of gastric intestinal metaplasia as a premalignant condition of gastric carcinoma is important to detect this change at an early point to prevent and control gastric cancer.

Kat8-mediated H4K16ac is essential in maintaining trophoblast stemness by regulating CDX2 expression

Kat8-mediated H4K16ac is essential in maintaining trophoblast stemness by regulating CDX2 expression

Association of CDX2 and mucin expression with chemotherapeutic benefits in patients with stage II/III gastric cancer.

Better predictors of patients with stage II/III gastric cancer (GC) most likely to benefit from adjuvant chemotherapy are urgently needed. This study aimed to assess the ability of CDX2 and mucin markers to predict prognosis and fluorouracil-based adjuvant chemotherapy benefits. CDX2 and mucin protein expressions were examined by immunohistochemistry and compared with survival and adjuvant chemotherapy benefits in a prospective evaluation cohort of 782 stage II/III GC patients. Then, the main findings were validated in an independent validation cohort (n = 386) and an external mRNA sequencing dataset (ACRG cohort, n = 193). In the evaluation cohort, CDX2, CD10, MUC2, MUC5AC, and MUC6 expressions were observed in 59.7%, 26.7%, 27.6%, 55.1%, and 57.7% of patients, respectively. However, only the expression of CDX2 was found to be associated with adjuvant chemotherapy benefits. Most importantly, CDX2-negative patients had a poorer prognosis when treated with surgery only, while the prognosis of CDX2-negative and CDX2-positive patients was similar when receiving postoperative adjuvant chemotherapy. Further analysis revealed that patients with CDX2 negative tumors benefited from chemotherapy (5-year overall survival rates: 60.0% with chemotherapy vs. 23.2% with surgery-only, p < 0.001), whereas patients with CDX2 positive tumors did not (pinteraction = 0.004). Consistent results were obtained in the validation and ACRG cohorts. Negative expression of CDX2 is an independent risk factor for survival in stage II/III GC, but subsequent adjuvant chemotherapy is able to compensate for this unfavorable effect. Therefore, active chemotherapy is more urgent for patients with negative CDX2 expression than for patients with positive CDX2 expression.

Loss of SATB2 and CDX2 expression is associated with DNA mismatch repair protein deficiency and BRAF mutation in colorectal cancer.

The relationship between the expression of the SATB2 and CDX2 proteins and common molecular changes and clinical prognosis in colorectal cancer (CRC) still needs further clarification. We collected 1180 cases of CRC and explored the association between the expression of SATB2 and CDX2 and clinicopathological characteristics, molecular alterations, and overall survival of CRC using whole-slide immunohistochemistry. Our results showed that negative expression of SATB2 and CDX2 was more common in MMR-protein-deficient CRC than in MMR-protein-proficient CRC (15.8% vs. 6.0%, P = 0.001; 14.5% vs. 4.0%, P = 0.000, respectively). Negative expression of SATB2 and CDX2 was more common in BRAF-mutant CRC than in BRAF wild-type CRC (17.2% vs. 6.1%, P = 0.003; 13.8% vs. 4. 2%; P = 0.004, respectively). There was no relationship between SATB2 and/or CDX2 negative expression and KRAS, NRAS, and PIK3CA mutations. The lack of expression of SATB2 and CDX2 was associated with poor histopathological features of CRC. In multivariate analysis, negative expression of SATB2 (P = 0.030), negative expression of CDX2 (P = 0.043) and late clinical stage (P = 0.000) were associated with decreased overall survival of CRC. In conclusion, the lack of SATB2 and CDX2 expression in CRC was associated with MMR protein deficiency and BRAF mutation, but not with KRAS, NRAS and PIK3CA mutation. SATB2 and CDX2 are prognostic biomarkers in patients with CRC.

Wei-fu-chun tablet halted gastric intestinal metaplasia and dysplasia associated with inflammation by regulating the NF-κB pathway

Ethnopharmacological relevanceChi006Eese herbal medicine Weifuchun Tablets (WFC) approved by the State Food and Drug Administration in 1982 has been widely used in treating a variety of chronic stomach disorders including Chronic atrophic gastritis (CAG) and Gastric precancerous lesions in China clinically. This study aimed to investigate the efficacy and potential mechanism of WFC in treating Gastric intestinal metaplasia (GIM) and Gastric dysplasia (GDys). Materials and methodsRat GIM and GDys established by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) combined with hot paste, ethanol injury, and intermittent fasting were intervened by WFC. Body weight, histopathology, pH of gastric acid, pepsin activity, intestinal metaplasia index and inflammation were detected. Rat bone marrow derived macrophages (BMDMs) pretreated with WFC were stimulated by LPS. Inflammatory factors and the nuclear factor-kappa B (NF-κB) pathway were assessed. GES-1 cells pretreated by WFC were stimulated by MNNG and TNF-α, intestinal metaplasia index, the NF-κB pathway and interaction between P65 and CDX2 were detected. ResultsWFC improved rat body weight, histopathology, pH value of gastric acid, activity of gastric pepsin, intestinal metaplasia (CDX2), inflammation (IL-1β, IL-6 and TNF-α), macrophage aggregation (CD68) in gastric mucosa in rat GIM and GDys. WFC inhibited inflammation (IL-1β and TNF-α) by inactivating the NF-κB pathway. WFC reduced the expression of CDX2 by inhibiting the binding of CDX2 promoter TSS upstream region with p65. ConclusionWFC blocked GIM and GDys associated with inflammation by regulating the NF-κB pathway.

CDX2 Is a Prognostic Biomarker for Unresectable Metastatic Colorectal Cancer.

Colorectal cancer (CRC) with reduced expression of the homeobox transcription factor CDX2, a master gene essential for the development and maintenance of the intestinal tract, is known as a poor prognosis subtype of CRC. The recurrence rate is high in patients with CDX2low CRC. However, the prognostic significance of CDX2 in advanced CRC is unclear. This study aimed to elucidate the prognostic significance of CDX2 in unresectable metastatic CRC (mCRC). Twenty-nine patients with unresectable mCRC who underwent primary site resection at the Kobe University Hospital during a 6-year period from January 2008 to January 2015 were included. The tissues from those patients were immunohistochemically stained with anti-CDX2 antibody (clone: CDX2-88). The patients were divided into CDX2high CRC group and CDX2low CRC group and their prognoses were analyzed. There were no clear differences in background between the two groups. A low CDX2 expression was associated with reduced overall survival (37.67 months vs. 25.32 months, p=0.03) and tended to associate with reduced progression-free survival (17.4 months vs. 12.9 months, p=0.37). Two patients received chemotherapy after resection of the primary lesion and obtained pathological complete response. CDX2 expression might be a possible prognostic biomarker for unresectable mCRC.

Clinical and Pathological Characteristics and Prognosis of Lung Adenocarcinoma With High-Grade Fetal Features: A Retrospective Analysis.

Fetal adenocarcinoma of the lung is a rare tumor. The clinical and pathological characteristics, treatment, and prognosis of patients with lung adenocarcinoma with fetal lung-like morphology were retrospectively investigated. The tumors of 9 patients with lung adenocarcinoma contained fetal lung-like morphology. One patient had pure-type high-grade fetal adenocarcinoma. Two patients had more than 50% high-grade fetal adenocarcinoma. Six specimens accounted for < 50% of the high-grade fetal features. It occurred in 7 men and 2 women. The median age at diagnosis was 62.0 years. Thyroid transcription factor-1 was frequently expressed in 8 specimens. All 9 specimens showed high rates of immunopositivity for β-catenin and E-cadherin. Three specimens showed nuclear β-catenin staining. Some patients showed immune expression of CDX2, α-fetoprotein (AFP), SALL4, and Glypican-3. Three of these specimens were diffusely strongly positive for p53, including 1 mixed-type high-grade fetal adenocarcinoma and 2 lung adenocarcinomas with high-grade fetal features. However, the other 6 patients had wild-type p53, including 1 pure-type high-grade fetal adenocarcinoma. PD-L1 was not expressed in all patients. Epidermal growth factor receptor mutations were detected in 1 patient. All patients were diagnosed using surgical samples. During the follow-up period of 36 months (range: 1-92 months), 3 patients received chemotherapy. One patient underwent radiotherapy. Two patients experienced recurrences. No patient died. PD-L1 expression status suggests a poor response to immune checkpoint therapy. The prognosis of the patient was relatively good.

Hungatella hathewayi impairs the sensitivity of colorectal cancer cells to 5-FU through decreasing CDX2 expression.

Hungatella hathewayi (H. hathewayi), also known as Clostridium hathewayi, has been reported to be accumulated in the colorectal cancer (CRC) samples. In addition, evidence has demonstrated that inoculation with H. hathewayi promotes the proliferation of colonic epithelial cells in mice. Herein, we explored H. hathewayi role in regulating the 5-fluorouracil (5-FU) resistance in CRC cells, and investigated the underlying mechanisms. H. hathewayi abundance in CRC tissues and the corresponding adjacent normal tissues was tested using qRT-PCR. Both parental and 5-FU resistance CRC cell lines were used to assess H. hathewayi role in regulating the 5-FU resistance of CRC cells using CCK-8, flow cytometry and animal experiments. H. hathewayi abundance was significantly increased in CRC tissues, and the high level of H. hathewayi was linked to lower overall survival rate. H. hathewayi treatment significantly weakened 5-FU effects on inhibiting cell growth and inducing cell apoptosis in CRC HCT116 and HT29 cells. In addition, H. hathewayi enhanced the 5-FU resistance of HCT116/5-FU and HT29/5-FU cells (the 5-FU resistance cell lines). In mechanism, H. hathewayi decreased the expression of CDX2, and increased the expression of nuclear accumulation of β-catenin. Overexpression of CDX2 abolished H. hathewayi-mediated enhancement in cell growth and inhibition in cell apoptosis in HCT116/5-FU and HT29/5-FU cells, as well as inhibited the expression and nuclear accumulation of β-catenin. In conclusion, H. hathewayi abundance was increased in CRC tissues, and the high level of H. hathewayi was linked to lower overall survival rate. In mechanisam, H. hathewayi treatment enhanced the 5-FU resistance of CRC cells through modulating CDX2/β-catenin signaling.

High-fat diet plus HNF1A variant promotes polyps by activating β-catenin in early-onset colorectal cancer.

The incidence of early-onset colorectal cancer (EO-CRC) is rising and is poorly understood. Lifestyle factors and altered genetic background possibly contribute. Here, we performed targeted exon sequencing of archived leukocyte DNA from 158 EO-CRC participants, which identified a missense mutation at p.A98V within the proximal DNA binding domain of Hepatic Nuclear Factor 1 α (HNF1AA98V, rs1800574). The HNF1AA98V exhibited reduced DNA binding. To test function, the HNF1A variant was introduced into the mouse genome by CRISPR/Cas9, and the mice were placed on either a high-fat diet (HFD) or high-sugar diet (HSD). Only 1% of the HNF1A mutant mice developed polyps on normal chow; however, 19% and 3% developed polyps on the HFD and HSD, respectively. RNA-Seq revealed an increase in metabolic, immune, lipid biogenesis genes, and Wnt/β-catenin signaling components in the HNF1A mutant relative to the WT mice. Mouse polyps and colon cancers from participants carrying the HNF1AA98V variant exhibited reduced CDX2 and elevated β-catenin proteins. We further demonstrated decreased occupancy of HNF1AA98V at the Cdx2 locus and reduced Cdx2 promoter activity compared with WT HNF1A. Collectively, our study shows that the HNF1AA98V variant plus a HFD promotes the formation of colonic polyps by activating β-catenin via decreasing Cdx2 expression.

Immunohistochemical phenotype of colorectal carcinoma in patients with KRAS mutation and mismatch repair status.

Aberrant expression of CK7/CK20/CDX2 is reported in percentage of colorectal carcinomas (CRC).

Циторедуктивне хірургічне лікування гігантського метастазу колоректального раку в яєчник. Клінічний випадок

Purpose - to present and analyze the case of surgical treatment of a patient with secondary giant adenocarcinoma of the right ovary. It is presented a clinical case of a giant metastasis of colorectal cancer to the ovary in a 48-year-old woman, measuring 30×20×15 cm, weighing about 9 kg, which occupied almost the entire volume of the abdominal cavity, pushing the stomach and intestines under the diaphragm with compression of the intestines, right ureter and the presence of ascites with a volume of 1.5 liters. A right-sided hemicolectomy with lymphodissection was performed, and an end-to-end ileotransversoanastmosis was applied. A right adnexectomy, extirpation of the uterus, omentectomy were performed. During the immunohistochemical study, membrane-cytoplasmic expression of cytokeratin (CK) 20 and nuclear expression of CDX2 were revealed. CDX2 is a defining marker for the diagnosis of adenocarcinoma of the gastrointestinal tract, as well as for the differentiation of primary and metastatic ovarian carcinomas. The patient has a lack of expression of receptors for estrogen and CK7 and positive nuclear expression of CDX2, which indicates metastasis of colorectal adenocarcinoma to the ovary. According to the results of the histological examination, which were of decisive importance, the origin of the tumor was determined and, accordingly, the patient’s diagnosis - the tumor of the cecum is G2 adenocarcinoma with germination into the serous membrane, metastases to the right ovary and regional lymphatic nodes 10 out of 10, and areas of tumor growth in the mesentery. Conclusions. In the case of detection of an ovarian neoplasm, the differential diagnosis of primary and secondary tumors is extremely important. Reproductive age at the time of presentation should not interfere with the diagnosis of primary or secondary ovarian malignancy. Metastases of primary colorectal cancer in the ovaries can manifest as pelvic tumors, so preoperative examination of the gastrointestinal tract and removal of the ovarian tumor with immunohistochemical examination is the leading option for choosing an appropriate treatment strategy. Immunohistochemical study of cytokeratin expression markers CK7, CK20 are appropriate for the differential diagnosis of synchronous cancer of the cecum and ovary, and CDX2 protein is a defining marker for the diagnosis of adenocarcinoma of the gastrointestinal tract and for distinguishing between primary and metastatic ovarian carcinomas. Cytoreductive surgery appears to be an important treatment option for metastatic ovarian neoplasia to improve prognosis. A multidisciplinary approach remains key in further examination, determination of the main diagnosis and development of an optimal treatment strategy. The research was carried out in accordance with the principles of the Helsinki Declaration. The study protocol was approved by the Local Ethics Committee of all participating institutions. The informed consent of the patient was obtained for conducting the studies. No conflict of interests was declared by the authors.

P-540 Improper placentation due to skewed Oct4-Cdx2 expression may be a leading factor in idiopathic recurrent pregnancy loss (RPL) cases from Assam, India.

Abstract Study question Does the differential expression of key embryonic stem cell (ESC) markers due to upstream signaling cascade(s) deregulation influence RPL pathogenesis in the studied population? Summary answer Imbalanced Oct4-Cdx2 expression linked differential ESCs polarization due to β-catenin signaling deregulation during the peri-implantation period impacts RPL pathogenesis in the studied population. What is known already RPL affects approximately 2-5% of women globally and around 7% Indian women in reproductive age group; with 50% of case etiology being unclear. Proper placentation is indispensable for establishment, continuation, and success of a pregnancy; and it depends largely on a healthy balance between apoptosis and stem cell differentiation that aids proper trophoblast invasion. These differentiation events are tightly controlled by the interplay of oxygen tension, hormones, growth factors, other signaling molecules and most importantly by transcription factors including those deciding the stem cell lineage like Oct4, NANOG, and Cdx2, wherein, β-catenin is known to enhance Oct-4 activity in ESCs. Study design, size, duration The sample size was predicted using Raosoft software utilizing the prevalence rate of RPL. Overall 87 women with 2 or more pregnancy losses before completion of 20 weeks of gestation were enrolled as RPL cases with informed consent, whereas, for the control group, 107 women volunteers undergoing medical termination of pregnancy (MTP) were enrolled as gestation-week-matched comparative study group for experimental data analysis. Participants/materials, setting, methods Products of conception (POC) was collected from the MTP and RPL cases. POCs were collected in 2vials for every subject: (i) 4% buffered formalin for fixation and preparation of microscopic slides for immunofluorescence (IF) based protein expression of Oct4 and Cdx2; (ii) RNA later solution for site specific transcriptional analysis of Oct4, NANOG, Cdx2, and β-catenin utilizing Realtime PCR. The difference in expression was analyzed using SPSS statistical software. Main results and the role of chance IF based site specific protein expression data confirmed higher expression of OCT4 in RPL cases compared to MTP controls, whereas CDX2 expression was found to be low in RPL compared to MTP subjects. The Realtime PCR amplification of cDNA using β-actin as internal normalization control for Oct4 (25.76 ± 10.17 folds) and NANOG (18.32 ± 6.67folds) showed increased expression whereas the Cdx2 expression was downregulated (0.487 ± 0.186 folds) in RPL cases compared to MTP controls, indicating a shift towards pluripotency which is a non-favorable environment for the ongoing pregnancy since cell polarization for initiation of differentiation to different cell lineages is required at that time of gestation. The cdx2 and oct4 expression correlated inversely and significantly {Pearson’s correlation= −0.838, p = 0.009; Spearman’s rho= −0.786; p = 0.016}. The realtime PCR amplification of β-catenin, the key molecule involved in direct enhancing of Oct4 expression during gestation was found to be increased in RPL cases compared to MTP controls. Statistically, the β-catenin-Oct4 expression correlated positively significantly. Limitations, reasons for caution Being a patient based study, the present study is limited to being an associative study only; and therefore secondary validation of the data for causal association using cell lines or suitable model system futuristically may add valuable scientific inputs and aid clinical intervention modalities for controlling RPL. Wider implications of the findings As the majority of RPL pathogenesis etiology is undefined limiting the possibilities of clinical interventions therefore we presume the present data can layout possible therapeutic interventions by regulating key regulators influencing skewed ESC profile aiding proper placentation leading to successful pregnancy in a subpopulation of cases with RPL history. Trial registration number Not applicable

P-802 TFAP2C is required for proper segregation of developmental markers regulating trophectoderm commitment during mouse preimplantation development

Abstract Study question What is the effect of CRISPR/Cas9-mediated knock-out of trophectoderm markers TFAP2A/C on polarization and lineage commitment in mouse? Summary answer CRISPR/Cas9-mediated KO revealed discrepancies in TFAP2A/C function, wherein TFAP2C is dispensable for blastocyst formation but controls the segregation of transcription factors in the mouse embryo. What is known already TFAP2C is an important transcription factor that is expressed solely in trophectoderm (TE) in mouse, and both TE and epiblast in human. Recently, genetic ablation of both TFAP2C and TEAD4 (player of the HIPPO-pathway) completely abolished polarization in mouse, which indicates that TFAP2C might already act before the first lineage segregation takes place. In addition, TFAP2C RNA depletion experiments in mouse embryos revealed a compensatory upregulation of TFAP2A, which indicates functional redundancy. Nonetheless, the question remains how exactly TFAP2C regulates polarization on a molecular level in mouse and whether there is also a functional implication of its isoform TFAP2A. Study design, size, duration Guide RNAs were designed targeting exon 5 of Tfap2c and exon 2 of the Tfap2a gene. CRISPR/Cas9 ribonucleoprotein complexes were delivered into mouse zygotes via electroporation. Additionally, appropriate non-targeted and scramble (inactive crRNA) control groups were included. Morphological analysis, immunofluorescence and next-generation sequencing (NGS) were applied to check for gene editing efficiency and the impact of KO on embryonic development and lineage segregation. Participants/materials, setting, methods Targeted mouse embryos and controls were cultured for a maximum of 4.5 days in vitro. They were stained for different developmental markers, including CDX2 (TE), SOX2 (early ICM), NANOG (epibast, EPI) and SOX17 (hypoblast, PrE) at different stages of development, such as polarization (E2.5), first (E3.5) and second (E4.5) lineage segregation. Immunostaining was used to determine cell number, TE/ICM fraction, marker localization and fluorescence intensity. Embryos were subjected to genetic analysis to determine on-target efficiency. Main results and the role of chance CRISPR/Cas9-mediated electroporation of mouse zygotes generated efficiently complete knock-out embryos. Of the 39 mouse zygotes targeted for TFAP2C, 38 (97%) of them were edited. From the 38 embryos edited for TFAP2C, 24 (62%) embryos displayed 100% frameshift mutations, and were considered KO. TFAP2C KO mouse embryos cultured until E4.5 were still able to form blastocysts (13/15, 87%), but were associated with inferior blastocyst quality compared to the control groups. Furthermore, TFAP2c-null blastocysts could hatch but herniated at multiple places, in contrast to wild-type blastocysts, which typically herniate at one place. In full frameshift TFAP2C KO mouse embryos, we observed delayed CDX2 expression at E2.5 (n = 7). At E3.5 (n = 5), however, CDX2 expression could still be pertained in KO embryos, whereas nuclear localization of SOX2 was disturbed. At E4.5 (n = 6), some primitive endoderm cells were observed in the presumed ICM (SOX17-positive), whereas no NANOG-positive (EPI) cells could be detected. However, some cells of presumed TE of E4.5 TFAP2C-KO embryos displayed erroneous SOX17 or NANOG expression, indicating that they adopted another cell fate irrespective of cell localization. Secondly, we generated TFAP2A KO mouse embryos (3/5, 60%) which exhibited complete morula arrest (100%; n = 3) at E4.5. Limitations, reasons for caution CRISPR/Cas9 is limited by the occurrence of mosaicism (more than one genotype present in an embryo) and potential off-target editing, which we will assess at in silico predicted off-target sites via NGS in mouse embryonic stem cells. The observations of the study will be consolidated by increasing the sample size. Wider implications of the findings Gene editing studies enable us to unravel the molecular interactions that are required for human preimplantation development. Obtaining novel insights into the molecular networks of the TFAP2-transcription factor family could improve our fundamental understanding on the spatial segregation of key transcription factors, which is crucial for successful implantation. Trial registration number Not applicable