N-cadherin Research Articles

Silencing immune-infiltrating biomarker CCDC80 inhibits malignant characterization and tumor formation in gastric cancer

ObjectiveTumor immune infiltration leads to poor prognosis of gastric cancer patients and seriously affects the life quality of gastric cancer patients. This study was based on bioinformatics to screen prognostic biomarkers in patients with high degree of immune invasion of gastric cancer. Meanwhile, the action of biomarker CCDC80 was explored in gastric cancer by cell and tumorigenesis experiments, to provide reference for the cure of gastric cancer patients.MethodsData sets and clinical massage on gastric cancer were collected from TCGA database and GEO database. ConsensusClusterPlus was used to cluster gastric cancer patients based on the 28 immune cells infiltration in ssGSEA. R “Limma” package was applied to analyze differential mRNAs between Cluster 1 and Cluster 2. Differential expression genes were screened by single factor analysis. Stemness markers (SERPINF1, DCN, CCDC80, FBLN5, SPARCL1, CCL14, DPYSL3) were identified for differential expression genes. Prognostic value of CCDC80 was evaluated in gastric cancer. Differences in genomic mutation and tumor microenvironment immune infiltration were assessed between high or low CCDC80. Finally, gastric cancer cells (HGC-27 and MKN-45) were selected to evaluate the action of silencing CCDC80 on malignant characterization, macrophage polarization, and tumor formation.ResultsBioinformatics analysis showed that CCDC80, as a stemness marker, was significantly overexpressed in gastric cancer. CCDC80 was also related to the degree of gastric cancer immune invasion. CCDC80 was up-expressed in cells of gastric cancer. Silencing CCDC80 inhibited malignant characterization and subcutaneous tumor formation of gastric cancer cells. High expression of CCDC80 was positive correspondence with immune invasion. Silencing CCDC80 inhibited M2 polarization and promoted M1 polarization in tumor tissues. In addition, gastric cancer patients were likely to have mutations in CDH1, ACTRT1, GANAB, and CDH10 genes in the High-CCDC80 group.ConclusionSilencing CCDC80, a prognostic biomarker in patients with immune invasion of gastric cancer, could effectively inhibit the malignant characterization, M2 polarization, and tumor formation of gastric cancer.

Associations between CDH1 gene polymorphisms and the risk of gastric cancer: A meta-analysis based on 44 studies.

Numerous studies have investigated the association between CDH1 polymorphisms and gastric cancer (GC) risk. However, the results have been inconsistent and controversial. To further determine whether CDH1 polymorphisms increase the risk of GC, we conducted a meta-analysis by pooling the data. Relevant case-control studies were collected from PubMed, Embase, Web of Science and Cochrane databases up to January 7, 2024. Subsequently, odds ratios (ORs) with 95% confidence intervals (CIs) were used to evaluate the strength of correlations. A sensitivity analysis was performed to evaluate the robustness and reliability of these included studies. A total of 25 articles including 44 studies, were included in this meta-analysis, including 26 studies on rs16260, 6 studies on rs3743674, 7 studies on rs5030625, and 5 studies on rs1801552. The pooled results showed that rs16260 was remarkably associated with an increased GC risk of GC among Caucasians. Moreover, the rs5030625 variation dramatically enhanced GC predisposition in the Asian population. However, no evident correlations between CDH1 rs3743674 and rs1801552 polymorphisms and GC risk were observed. Our findings suggested that CDH1 gene polymorphisms were significantly correlated with GC risk, especially in rs16260 and rs5030625 polymorphisms.

Impact of pathogenic germline variants in HRR genes on gastroesophageal cancer in a large real-world Chinese population: A comprehensive study.

10588 Background: Genetic testing in gastroesophageal cancer has been limited to those with hereditary cancer syndromes, restricting understanding of the impact of germline variants in sporadic cancers. Our study expands this by exploring germline and somatic characteristics in a broad Chinese cohort, enhancing risk assessment and guiding treatment strategies in a wider population. Methods: From October 2017 to April 2023, we retrospectively analyzed 2596 Chinese gastroesophageal cancer (GEC) patients, including 2003 with gastric cancer (GC), 196 with gastroesophageal junction cancer (GEJC), and 397 with esophageal cancer (EC). A 520-gene panel including 62 genes associated with cancer predisposition, was employed for genomic profiling of paired tumor and white blood cell samples. The pathogenicity of germline variants was determined according to ACMG guidelines. Results: In this retrospective cohort, the proportion of patients carrying pathogenic/likely pathogenic (P/LP) germline variants was 5.3% in GC, 5.6% in GEJC, and 5.0% in EC, respectively. Notably, genes associated with HRR exhibited the highest rates of P/LP germline mutations in GEC (94, 3.7%), with the majority occurring in non-BRCA HRR genes (77, 3.0%). The top five genes with the highest mutation rates were ATM, PALB2, FANCA, CHEK2, and RAD54L. Compared to the East Asian population without cancer in the genomAD database, a higher proportion of P/LP germline mutations were found in the CDH1(Inf [6.46-Inf], p < 0.001), PALB2 (5.60 [1.61-13.09], p = 0.002), SDHA (8.99 [1.29-99.42], p = 0.012), ATM (2.15 [1.08-4.09], p = 0.02), and CHEK2 (3.83 [1.06-13.32], p = 0.02) genes in GC. Similarly, significant increases in PALB2 (10.36 [2.32-37.28], p = 0.002) and ATM (4.42 [0.86-14.33], p = 0.036) were also observed in EC and GEJC. The HRR P/LP variant group exhibited a higher incidence of biallelic inactivation (loss of wildtype allele) compared to the B/LB variant group (24.7% vs 6.6%, p<0.001). Moreover, the similar trend was observed for the moderately/uncertainly penetrant gene ATM and FANCA (38.5% vs 0.6%, p < 0.001; 42.9% vs 9.8%, p = 0.025), which suggests that ATM and FANCA may be associated with a high risk. The HRR genes ATM(56 vs 61, p = 0.052), CDH1(33 vs 61, p = 0.003), and FANCI(48 vs 61, p = 0.041) with P/LP variants were associated with a younger age of onset. Carriers of HRR P/LP variants had less prevalent somatic TP53 variants in GC and GEC and higher HRD score compared to non-carriers (p=0.005). Conclusions: Our research delineates the germline P/LP variant landscape in Chinese GEC, revealing the significant role of HRR P/LP variants. The presence of P/LP variants in HRR genes in GEC is linked to distinct disease characteristics and progression, underscoring their importance in guiding personalized treatment approaches and risk evaluation.

Abstract PO5-24-11: An integrated approach for comprehensive molecular and tumor microenvironment characterization of invasive lobular carcinoma

Abstract Introduction: Although recent studies have shown that invasive lobular carcinoma (ILC) is associated with worse long-term outcomes compared to invasive breast cancer of no special type (NST), they are treated in a similar manner. Therefore, classification approaches to help understand the molecular characteristics associated with the different BC subtypes are needed to personalize therapy. Here, we describe an integrated analytical approach to comprehensively characterize ILC samples. Methods: We collected genomic and transcriptomic data for 16,087 ILC and NST samples, with clinical and pathological annotation, from 59 datasets and used an internally developed algorithm based on clustering performed after gene expression analysis to classify them into 5 intrinsic subtypes: Basal, Luminal A (LumA), Luminal B (LumB), HER2 high, and HER2 low. We performed gene set enrichment analyses and utilized the DESeq2 algorithm to perform differential expression analysis of RNA-seq data. We performed molecular grade subtyping using methods described by Antysheva et al. (Cancer Res., 2022). Samples that were listed as ILC or had an inactivated CDH1 gene by mutation, deletion or low expression (< −2.5 MAD after median scaling) were defined as having an ILC histomolecular subtype. Using methods described by Bagaev et. al. (Cancer Cell, 2021), 29 functional gene expression signatures were selected, and unsupervised dense Louvain clustering was performed to identify TME subtypes. The Mann-Whitney U test was used for statistical analysis. The tertiary lymphoid structures (TLS) gene signature was evaluated using methods described by Sautès-Fridman et al. (Nat. Rev. Cancer, 2019). Results: In the ILC samples (n=1,887), we identified LumA (46%), LumB (40%), Basal (8%,), HER2 high (3%), and HER2 low (3%) subtypes. In contrast, the NST samples were classified as LumB (41%), LumA (25%), Basal (19%), HER2 high (9%), and HER2 low (5%). Molecular grade subtyping revealed that 67% of ILC samples were low grade and 33% were high grade, compared to NST where 55% were high grade and 45% were low grade. Notably, among high-grade ILC, LumB (64%) and Basal (23%) subtypes were predominant. Key differentially expressed genes included downregulated CDH1 and upregulated MMP23B, IGF1, and TFAP2B across LumA and LumB ILC samples. Further analysis revealed downregulation of TACSTD2 and CDH3 in the Basal subtype for ILC samples compared to NST samples. Transcriptomic-based TME subtyping revealed distinct TME distributions across the BC subtypes and molecular grades for ILC samples (Table 1). In high-grade ILC LumB samples, 38% had a Desert (D; predictive of poor immunotherapy response) and 39% had an Immune-Enriched (IE; predictive of favorable immunotherapy response) TME. In low-grade ILC LumB samples we found that while 51% had a D TME, only 14% had an IE TME. We found an overrepresentation of the TLS signature [Solid TLS] in ILC compared to NST samples, and higher matrix remodeling and CAF signatures in LumA compared to the LumB subtype among low-grade ILC samples. Samples with high CD274 expression levels had higher percentages of T cells and M1 macrophages, and LumA samples had significantly higher (p < 0.001) CD274 expression compared to the other BC subtypes. Conclusion: These results provide in-depth molecular and tumor microenvironment characterization of ILC. Further optimization of this analytical approach could lead to the development of more effective therapeutic strategies. Table 1. ILC subtypes classification Citation Format: Jason Mouabbi, Konstantin Chernyshov, Oleg Baranov, Vladimir Kushnarev, Polina Turova, Anna Butusova, Sofia Menshikova, Jessica Brown, Nikita Kotlov, Patrick Clayton, Krystle Nomie, Nathan Fowler, Debu Tripathy. An integrated approach for comprehensive molecular and tumor microenvironment characterization of invasive lobular carcinoma [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-24-11.

Abstract GS03-04: Novel Mechanisms of CDH1 Inactivation in Breast Invasive Lobular Carcinoma Unveiled by the Integration of Artificial Intelligence and Genomics

Abstract Background: Invasive lobular carcinoma (ILC) of the breast is the second most common histologic subtype of breast cancer (BC), following invasive ductal carcinoma of no special type (IDC-NST). The hallmark histologic feature of ILC is cellular discohesiveness, the result of bi-allelic inactivation of CDH1, and represents an important genotypic-phenotypic correlation in BC. Although most ILCs harbor CDH1 loss-of function mutations associated to loss-of-heterozygosity (LOH) of the wild type-allele, a subset of ILCs lack these alterations despite displaying a typical lobular phenotype. Here, we sought to identify alternative molecular mechanisms converging on CDH1 inactivation by employing an integrative artificial intelligence (AI) and genomics approach. Materials and Methods: A genomics-driven AI-based algorithm using hematoxylin and eosin (H&E) whole slide images (WSIs) as input, previously developed to detect bi-allelic CDH1 mutations (inactivating mutation associated to LOH) in BC was employed. WSIs of 1,057 BCs including ILCs (n=187) and non-lobular BCs (n=870) previously subjected to FDA-cleared tumor/normal targeted sequencing were subjected to analysis with the AI-based algorithm. Cases predicted to harbor CDH1 bi-allelic mutations by the AI-model but lacking CDH1 bi-allelic mutations by targeted sequencing were assessed through targeted sequencing data re-analysis, CDH1 gene promoter methylation evaluation and/or whole genome sequencing analysis. Results: AI-based analysis WSIs corresponding to 1,057 BCs resulted in the identification of 34 cases found to lack CDH1 bi-allelic mutations by targeted sequencing but predicted to harbor these genetic alterations by the AI-based model. CDH1 gene promoter methylation assessment revealed CDH1 promoter methylation in 18 cases. Targeted sequencing data reanalysis revealed other genetic mechanisms of CDH1 inactivation including CDH1 homozygous deletions (n=3), intragenic deletion with LOH (n=1), and likely pathogenic non-coding CDH1 alterations associated with LOH (n=2). WGS analysis of an ILC revealed a novel deleterious CDH1 fusion stemming from translocation t(13;16), resulting in loss of the 5’UTR, transcription start site and exons 1 and 2 of CDH1, associated with complete loss of E-cadherin protein expression. Taken together, we identified alternative/novel mechanisms of bi-allelic CDH1inactivation in 74% (25/34) cases analyzed. Conclusions: By applying an AI-based algorithm trained to detect a genetic alteration (i.e., CDH1 bi-allelic mutations), we were able to identify alternative epigenetic and genetic molecular mechanisms of CDH1 inactivation in ILCs, including novel non-coding CDH1 genetic alterations and a new inactivating CDH1 fusion gene. These findings indicate that molecular mechanisms affecting a single gene or process converging on the same phenotype can be unveiled by the integration of AI and genomics, highlighting the robustness of this approach for the discovery of novel biology. Citation Format: Fresia Pareja, Higinio Dopeso, Yikan Wang, Andrea Gazzo, David Brown, Pier Selenica, Jan Bernhard, Fatemeh Derakhshan, Edaise M. da Silva, Lorraine Colon-Cartagena, Thais Basili, Antonio Marra, Jillian Sue, Qiqi Ye, Arnaud Da Cruz Paula, Selma Yeni, Xin Pei, Hunter Green, Kaitlyn Gill, Yingjie Zhu, Matthew Lee, Ran Godrich, Adam Casson, Britta Weigelt, Nadeem Riaz, Hanna Y Wen, Edi Brogi, Matthew Hanna, Diana Mandelker, Jeremy Kunz, Brandon Rothrock, Sarat Chandarlapaty, Christopher Kanan, Gerard Oakley III, David Klimstra, Thomas Fuchs, Jorge Reis-Filho. Novel Mechanisms of CDH1 Inactivation in Breast Invasive Lobular Carcinoma Unveiled by the Integration of Artificial Intelligence and Genomics [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr GS03-04.

Exercise may be a Novel Strategy to Reduce Vascular Toxicity of Immune Checkpoint Inhibitors in a Mouse Melanoma Model

Background: Immune checkpoint inhibitors (ICIs) are a cornerstone in advanced melanoma treatment. Unfortunately, ICI-associated cardiovascular toxicity, particularly atherosclerotic events, poses a significant challenge. Identifying interventions to mitigate the cardiovascular toxicity of ICI is critical for enhancing the quality and duration of life in cancer survivors. Given the established benefits of exercise in atherosclerosis prevention, this study explores its potential to reduce ICI-induced vascular toxicity in the context of melanoma. We hypothesized that aerobic exercise would decrease ICI's vascular toxicity in a mouse melanoma model. The primary experimental outcome was endothelial mesenchymal transition (EndMT), an early atherosclerosis indicator. Methods: C57BL/6 mice were subcutaneously injected with 0.8 x 106 BP melanoma cells ( BrafV600E/WT and Pten−/−). Upon tumor palpability, mice were assigned into four groups: IgG, Exercise, anti-PD-1, and anti-PD-1+Exercise. Mice were treated with bi-weekly administration of anti-PD-1 or IgG antibody (150 μg) for three weeks, combined with or without treadmill exercise (12 meters/minute for 45 min per day, five days/week). Body weight was monitored weekly. Following the final treatment, aortic tissues were collected and en face immunofluorescence staining for endothelial marker VE-cadherin (VE-Cad) and vimentin was performed to assess EndMT, then analyzed via confocal microscopy. Results: No significant weight differences were observed between groups. Notably, the EndMT marker vimentin expression was significantly higher in anti-PD-1 treated tumor-bearing mice compared to IgG controls (3.8% vs 0.6%, p=.03) in the atheroprotective laminar flow area. This was reduced by exercise; the anti-PD-1+Exercise group showed significantly reduced endothelial vimentin levels relative to the anti-PD-1 alone (0.8% vs 3.8%, p=.04). Disturbed flow areas did not exhibit notable differences between any group. Conclusions: Aerobic exercise may be protective against vascular toxicity in mice treated with ICIs by promoting atheroprotective flow and potentially inhibiting EndMT. Although these initial results are promising, comprehensive studies are required to ascertain the impact of exercise on preventing ICI-induced EndMT and subsequent atherosclerosis in this mouse melanoma model. Future research will aim to more deeply characterize the process of EndMT following ICI treatment and evaluate the preventive capacity of exercise against atherosclerosis in this context. MD Anderson Cancer Center Melanoma SPORE grant (NCI P50CA221703-05). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Screening and construction of nanobodies against human CD93 using phage libraries and study of their antiangiogenic effects.

Cluster of Differentiation 93 (CD93) plays an important role in angiogenesis and is considered an important target for inhibiting tumor angiogenesis, but there are currently no therapeutic antibodies against CD93 in the clinic. Thus, we describe the screening of novel nanobodies (Nbs) targeting human CD93 from a phage library of shark-derived Nbs. Screening and enrichment of phage libraries by enzyme-linked immunosorbent assay (ELISA). Anti-CD93 Nbs were purified by expression in E. coli. The binding affinity of anti-CD93 Nbs NC81/NC89 for CD93 was examined by flow cytometry (FC) and ELISA. The thermal stability of NC81/NC89 was examined by ELISA and CD spectroscopy. Afterward, the anti-angiogenic ability of NC81/NC89 was examined by MTT, wound healing assay, and tube formation assay. The expression level of VE-cadherin (VE-Ca) and CD93 was detected by Western Blot (WB). The binding sites and binding forms of NC81/NC89 to CD93 were analyzed by molecular docking. The anti-CD93 Nbs were screened in a phage library, expressed in E. coli, and purified to >95% purity. The results of FC and ELISA showed that NC81/NC89 have binding ability to human umbilical vein endothelial cells (HUVECs). The results of ELISA and CD spectroscopy showed that NC81/NC89 retained the ability to bind CD93 at 80°C and that the secondary structure remained stable. In vitro, the results showed that NC81 and NC89 significantly inhibited the proliferation and migration of human umbilical vein endothelial cells (HUVECs) as well as tube formation on Matrigel. Western Blot showed that NC81 and NC89 also inhibited the expression of VE-Ca thereby increasing vascular permeability. It was found during molecular docking that the CDR regions of NC81 and NC89 could be attached to CD93 by strong hydrogen bonds and salt bridges, and the binding sites were different. We have successfully isolated NC81 and NC89, which bind CD93, and both Nbs significantly inhibit angiogenesis and increase vascular permeability. These results suggest that NC81 and NC89 have potential clinical applications in angiogenesis-related therapies.

TGFβ1 Regulates Cellular Composition of In Vitro Cardiac Perivascular Niche Based on Cardiospheres.

The cardiac perivascular niche is a cellular microenvironment of a blood vessel. The principles of niche regulation are still poorly understood. We studied the effect of TGFβ1 on cells forming the cardiac perivascular niche using 3D cell culture (cardiospheres). Cardiospheres contained progenitor (c-Kit), endothelial (CD31), and mural (αSMA) cells, basement membrane proteins (laminin) and extracellular matrix proteins (collagen I, fibronectin). TGFβ1 treatment decreased the length of CD31+ microvasculature, VE cadherin protein level, and proportion of NG2+ cells, and increased proportion of αSMA+ cells and transgelin/SM22α protein level. We supposed that this effect is related to the stabilizing function of TGFβ1 on vascular cells: decreased endothelial cell proliferation, as shown for HUVEC, and activation of mural cell differentiation.

Analysis of CDO1, PITX2, and CDH13 Gene Methylation in Early Endometrial Cancer for Prediction of Medical Treatment Outcomes.

To determine CDO1, PITX2, and CDH13 gene methylation levels in early endometrial cancer and atypical hyperplasia specimens obtained before organ-preserving treatment in the patients with adequate response and with insufficient response to hormonal treatment. A total of 41 endometrial specimens obtained during diagnostic uterine curettage in women with EC (n = 28) and AEH (n = 13), willing to preserve reproductive function, were studied; 18 specimens of uterine cancer IA stage G1 from peri- and early postmenopausal women (comparison group) were included in the study. The control group included 18 endometrial specimens from healthy women obtained by diagnostic curettage for missed abortion and/or intrauterine adhesions. Methylation levels were analyzed using the modified MS-HRM method. All 13 women with AEH had a complete response (CR) to medical treatment. In the group undergoing organ-preserving treatment for uterine cancer IA stage G1 (n = 28), 14 patients had a complete response (EC CR group) and 14 did not (EC non-CR group). It was found that all groups had statistically significant differences in CDO1 gene methylation levels compared to the control group (p < 0.001) except for the EC CR group (p = 0.21). The p-value for the difference between EC CR and EC non-CR groups was <0.001. The differences in PITX2 gene methylation levels between the control and study groups were also significantly different (p < 0.001), except for the AEH group (p = 0.21). For the difference between EC CR and EC non-CR groups, the p-value was 0.43. For CDH13 gene methylation levels, statistically significant differences were found between the control and EC non-CR groups (p < 0.001), and the control and EC comparison groups (p = 0.005). When comparing the EC CR group with EC non-CR group, the p-value for this gene was <0.001. The simultaneous assessment of CDO1 and CDH13 genes methylation allowed for an accurate distinction between EC CR and EC non-CR groups (AUC = 0.96). The assessment of CDO1 and CDH13 gene methylation in endometrial specimens from patients with endometrial cancer (IA stage G1), scheduled for medical treatment, can predict the treatment outcome.

Longitudinal Comparative Analysis of Circulating Tumor DNA and Matched Tumor Tissue DNA in Patients with Metastatic Colorectal Cancer Receiving Palliative First-Line Systemic Anti-Cancer Therapy.

This study aimed to compare tumor tissue DNA (ttDNA) and circulating tumor DNA (ctDNA) to explore the clinical applicability of ctDNA and to better understand clonal evolution in patients with metastatic colorectal cancer undergoing palliative first-line systemic therapy. We performed targeted sequencing analysis of 88 cancer-associated genes using germline DNA, ctDNA at baseline (baseline-ctDNA), and ctDNA at progressive disease (PD-ctDNA). The results were compared with ttDNA data. Among 208 consecutively enrolled patients, we selected 84 (41 males; median age, 59 years; range, 35 to 90 years) with all four sample types available. A total of 202 driver mutations were found in 34 genes. ttDNA exhibited the highest mutation frequency (n=232), followed by baseline-ctDNA (n=155) and PD-ctDNA (n=117). Sequencing ctDNA alongside ttDNA revealed additional mutations in 40 patients (47.6%). PD-ctDNA detected 13 novel mutations in 10 patients (11.9%) compared to ttDNA and baseline-ctDNA. Notably, seven mutations in five patients (6.0%) were missense or nonsense mutations in APC, TP53, SMAD4, and CDH1 genes. In baseline-ctDNA, higher maximal variant allele frequency (VAF) values (p=0.010) and higher VAF values of APC (p=0.012), TP53 (p=0.012), and KRAS (p=0.005) mutations were significantly associated with worse overall survival. While ttDNA remains more sensitive than ctDNA, our ctDNA platform demonstrated validity and potential value when ttDNA was unavailable. Post-treatment analysis of PD-ctDNA unveiled new pathogenic mutations, signifying cancer's clonal evolution. Additionally, baseline-ctDNA's VAF values were prognostic after treatment.

CDH1 Genotype Exploration in Women With Hereditary Lobular Breast Cancer Phenotype

Pathogenic or likely pathogenic (P/LP) germline CDH1 variants are associated with risk for diffuse gastric cancer and lobular breast cancer (LBC) in the so-called hereditary diffuse gastric cancer (HDGC) syndrome. However, in some circumstances, LBC can be the first manifestation of this syndrome in the absence of diffuse gastric cancer manifestation. To evaluate the frequency of germline CDH1 variants in women with the hereditary LBC (HLBC) phenotype, somatic CDH1 gene inactivation in germline CDH1 variant carriers' tumor samples, and the association of genetic profiles with clinical-pathological data and survival. This single-center, longitudinal, prospective cohort study was conducted from January 1, 1997, to December 31, 2021, with follow-up until January 31, 2023. Women with LBC seen at the European Institute of Oncology were included. Testing for germline CDH1, BRCA1, and BRCA2 genes was performed. Somatic profiling was assessed for germline CDH1 carriers. Accurate estimates of prevalence of germline CDH1 variants among patients with HLBC and the association of somatic sequence alteration with HLBC syndrome. The Kaplan-Meier method and a multivariable Cox proportional hazards regression model were applied for overall and disease-free survival analysis. Of 5429 cases of primary LBC, familial LBC phenotype accounted for 1867 (34.4%). A total of 394 women with LBC were tested, among whom 15 germline CDH1 variants in 15 unrelated families were identified. Among these variants, 6 (40.0%) were P/LP, with an overall frequency of 1.5% (6 of 394). Of the 6 probands with P/LP CDH1 LBC, 5 (83.3%) had a positive family history of BC and only 1 (16.7%) had sporadic juvenile early-onset LBC. No germline BRCA1 and BRCA2 variants were identified in CDH1 carriers. An inactivating CDH1 mechanism (second hit) was identified in 4 of 6 explored matched tumor samples (66.7%) in P/LP germline carriers. The P/LP CDH1 LBC variant carriers had a significantly lower age at diagnosis compared with the group carrying CDH1 variants of unknown significance or likely benign (42.5 [IQR, 38.3-43.0] vs 51.0 [IQR, 45.0-53.0] years; P = .03). In this cohort study, P/LP germline CDH1 variants were identified in individuals not fulfilling the classic clinical criteria for HDGC screening, suggesting that identification of these variants may provide a novel method to test women with LBC with early age at diagnosis and/or positive family history of BC.

Effects of Red Sorghum-Derived Deoxyanthocyanidins and Their O-β-D-Glucosides on E-Cadherin Promoter Activity in PC-3 Prostate Cancer Cells.

Although much less common than anthocyanins, 3-Deoxyanthocyanidins (3-DAs) and their glucosides can be found in cereals such as red sorghum. It is speculated that their bioavailability is higher than that of anthocyanins. Thus far, little is known regarding the therapeutic effects of 3-DAs and their O-β-D-glucosides on cancer, including prostate cancer. Thus, we evaluated their potential to decrease cell viability, to modulate the activity of transcription factors such as NFκB, CREB, and SOX, and to regulate the expression of the gene CDH1, encoding E-Cadherin. We found that 4',7-dihydroxyflavylium chloride (P7) and the natural apigeninidin can reduce cell viability, whereas 4',7-dihydroxyflavylium chloride (P7) and 4'-hydroxy-7-O-β-D-glucopyranosyloxyflavylium chloride (P3) increase the activities of NFkB, CREB, and SOX transcription factors, leading to the upregulation of CDH1 promoter activity in PC-3 prostate cancer cells. Thus, these compounds may contribute to the inhibition of the epithelial-to-mesenchymal transition in cancer cells and prevent the metastatic activity of more aggressive forms of androgen-resistant prostate cancer.

Integration of Pathological Criteria and Immunohistochemical Evaluation for Invasive Lobular Carcinoma Diagnosis: Recommendations From the European Lobular Breast Cancer Consortium

Invasive lobular carcinoma (ILC) is the second most frequent type of breast cancer (BC) and its peculiar morphology is mainly driven by inactivation of CDH1, the gene coding for E-cadherin cell adhesion protein. ILC-specific therapeutic and disease-monitoring approaches are gaining momentum in the clinic, increasing the importance of accurate ILC diagnosis. Several essential and desirable morphologic diagnostic criteria are currently defined by the World Health Organization, the routine use of immunohistochemistry (IHC) for E-cadherin is not recommended. Disagreement in the diagnosis of ILC has been repeatedly reported, but interpathologist agreement increases with the use of E-cadherin IHC. In this study, we aimed to harmonize the pathological diagnosis of ILC by comparing 5 commonly used E-cadherin antibody clones (NCH-38, EP700Y, Clone 36, NCL-L-E-cad [Clone 36B5], and ECH-6). We determined their biochemical specificity for the E-cadherin protein and IHC staining performance according to type and location of mutation on the CDH1 gene. Western blot analysis on mouse cell lines with conditional E-cadherin expression revealed a reduced specificity of EP700Y and NCL-L-E-cad for E-cadherin, with cross-reactivity of Clone 36 to P-cadherin. The use of IHC improved interpathologist agreement for ILC, lobular carcinoma in situ, and atypical lobular hyperplasia. The E-cadherin IHC staining pattern was associated with variant allele frequency and likelihood of nonsense-mediated RNA decay but not with the type or position of CDH1 mutations. Based on these results, we recommend the indication for E-cadherin staining, choice of antibodies, and their interpretation to standardize ILC diagnosis in current pathology practice.

RAC1-mediated integrin alpha-6 expression in E-cadherin-deficient gastric cancer cells promotes interactions with the stroma and peritoneal dissemination

Diffuse-type gastric cancer (DGC) is a subtype of gastric cancer that is prone to peritoneal dissemination, with poor patient prognosis. Although intercellular adhesion loss between cancer cells is a major characteristic of DGCs, the mechanism underlying the alteration in cell-to-extracellular matrix (ECM) adhesion is unclear. We investigated how DGCs progress and cause peritoneal dissemination through interactions between DGC cells and the tumour microenvironment (TME). P53 knockout and KRASG12V-expressing (GAN-KP) cells and Cdh1-deleted GAN-KP (GAN-KPC) cells were orthotopically transplanted into the gastric wall to mimic peritoneal dissemination. The GAN-KPC tumour morphology was similar to that of human DGCs containing abundant stroma. RNA sequencing revealed that pathways related to Rho GTPases and integrin-ECM interactions were specifically increased in GAN-KPC cells compared with GAN-KP cells. Notably, we found that Rac Family Small GTPase 1 (RAC1) induces Integrin Subunit Alpha 6 (ITGA6) trafficking, leading to its enrichment on the GC cell membrane. Fibroblasts activate the FAK/AKT pathway in GC cells by mediating extracellular matrix (ECM)-Itga6 interactions, exacerbating the malignant phenotype. In turn, GC cells induce abnormal expression of fibroblast collagen and its transformation into cancer-associated fibroblasts (CAFs), resulting in DGC-like subtypes. These findings indicate that Cdh1 gene loss leads to abnormal expression and changes in the subcellular localization of ITGA6 through RAC1 signalling. The latter, through interactions with CAFs, allows for peritoneal dissemination.

Mitochondrial calcium uniporter as biomarker and therapeutic target for breast cancer: Prognostication, immune microenvironment, epigenetic regulation and precision medicine

IntroductionMitochondrial calcium uniporter (MCU) is a central subunit of MCU complex that regulate the levels of calcium ions within mitochondria. A comprehensive understanding the implications of MCU in clinical prognostication, biological understandings and therapeutic opportunity of breast cancer (BC) is yet to be determined. ObjectivesThis study aims to investigate the role of MCU in predictive performance, tumor progression, epigenetic regulation, shaping of tumor immune microenvironment, and pharmacogenetics and the development of anti-tumor therapy for BC. MethodsThe downloaded TCGA datasets were used to identify predictive ability of MCU expressions via supervised learning principle. Functional enrichment, mutation landscape, immunological profile, drug sensitivity were examined using bioinformatics analysis and confirmed by experiments exploiting human specimens, in vitro and in vivo models. ResultsMCU copy numbers increase with MCU gene expression. MCU expression, but not MCU genetic alterations, had a positive correlation with known BC prognostic markers. Higher MCU levels in BC showed modest efficacy in predicting overall survival. In addition, high MCU expression was associated with known BC prognostic markers and with malignancy. In BC tumor and sgRNA-treated cell lines, enrichment pathways identified the involvement of cell cycle and immunity. miR-29a was recognized as a negative epigenetic regulator of MCU. High MCU levels were associated with increased mutation levels in oncogene TP53 and tumor suppression gene CDH1, as well as with an immunosuppressive microenvironment. Sigle-cell sequencing indicated that MCU mostly mapped on to tumor cell and CD8 T-cells. Inter-databases verification further confirmed the aforementioned observation. miR-29a-mediated knockdown of MCU resulted in tumor suppression and mitochondrial dysfunction, as well as diminished metastasis. Furthermore, MCU present pharmacogenetic significance in cellular docetaxel sensitivity and in prediction of patients’ response to chemotherapeutic regimen. ConclusionMCU shows significant implication in prognosis, outcome prediction, microenvironmental shaping and precision medicine for BC. miR-29a-mediated MCU inhibition exerts therapeutic effect in tumor growth and metastasis.

A novel imprinted locus on bovine chromosome 18 homologous with human chromosome 16q24.1.

Genomic imprinting is an epigenetic regulation mechanism in mammals resulting in the parentally dependent monoallelic expression of genes. Imprinting disorders in humans are associated with several congenital syndromes and cancers and remain the focus of many medical studies. Cattle is a better model organism for investigating human embryo development than mice. Imprinted genes usually cluster on chromosomes and are regulated by different methylation regions (DMRs) located in imprinting control regions that control gene expression in cis.There is an imprinted locus on human chromosome 16q24.1 associated with congenital lethal developmental lung disease in newborns. However, genomic imprinting on bovine chromosome 18, which is homologous with human chromosome 16 has not been systematically studied. The aim of this study was to analyze the allelic expressions of eight genes (CDH13, ATP2C2, TLDC1, COTL1, CRISPLD2, ZDHHC7, KIAA0513, and GSE1) on bovine chromosome 18 and to search the DMRs associated gene allelic expression. Three transcript variants of the ZDHHC7 gene (X1, X2, and X5) showed maternal imprinting in bovine placentas. In addition, the monoallelic expression of X2 and X5 was tissue-specific. Five transcripts of the KIAA0513 gene showed tissue- and isoform-specific monoallelic expression. The CDH13, ATP2C2, and TLDC1 genes exhibited tissue-specific imprinting, however, COTL1, CRISLPLD2, and GSE1 escaped imprinting. Four DMRs, established after fertilization, were found in this region. Two DMRs were located between the ZDHHC7 and KIAA0513 genes, and two were in exon 1 of the CDH13 and ATP2C2 genes, respectively. The results from this study support future studies on the molecular mechanism to regulate the imprinting of candidate genes on bovine chromosome 18.

The analysis of boric acid effect on epithelial-mesenchymal transition of CD133 + CD117 + lung cancer stem cells.

Targeting lung cancer stem cells (LC-SCs) for metastasis may be an effective strategy against lung cancer. This study is the first on epithelial-mesenchymal transition (EMT) properties of boric acid (BA) in LC-SCs. LC-SCs were isolated using the magnetic cell sorting (MACS) method. Tumor-sphere formation and flow cytometry confirmed CSC phenotype. The cytotoxic effect of BA was measured by MTT analysis, and the effect of BA on EMT was examined by migration analysis. The expression levels of ZEB1, SNAIL1, ITGA5, CDH1, ITGB1, VIM, COL1A1, and LAMA5 genes were analyzed by RT-qPCR. E-cadherin, Collagen-1, MMP-3, and Vimentin expressions were analyzed immunohistochemically. Boric acid slightly reduced the migration of cancer cells. Increased expression of transcription factor SNAIL (p < 0.001), but not ZEB1, was observed in LC-SCs. mRNA expression levels of ITGB1 (p < 0.01), ITGA5 (p < 0.001), COL1A1 (p < 0.001), and LAMA5 (p < 0.001) increased; CDH1 and VIM decreased in LC-SCs. Moreover, while E-cadherin (p < 0.001) and Collagen-1 (p < 0.01) immunoreactivities significantly increased, MMP-3 (p < 0.001) and Vimentin (p < 0.01) immunoreactivities decreased in BA-treated LC-SCs. To conclude, the current study provided insights into the efficacy and effects of BA against LC-SCs regarding proliferation, EMT, and cell death for future studies.

Abstract 966: Longitudinal comparative analysis of circulating tumor DNA and matched tumor tissue DNA in patients with metastatic colorectal cancer receiving palliative first-line systemic anti-cancer therapy

Abstract Background: Plasma circulating tumor DNA (ctDNA) analysis has become an attractive method for precision oncology. This study compared mutational landscapes of ctDNA with matched tumor tissue DNA (ttDNA) to explore clinical applicability and to better understand clonal evolution in patients with metastatic colorectal cancer receiving palliative first-line systemic anti-cancer therapy (SACT). Methods: We performed unique molecular identifier-based targeted sequencing of 88 cancer-associated genes using the following samples: ttDNA, germline DNA, ctDNA at baseline (baseline-ctDNA), and ctDNA at progressive disease (PD-ctDNA). Results: Of the 208 consecutively enrolled patients, 84 were selected (41 males; median age 59, ranging from 35 to 90) with access to all four samples. Excluding duplicated mutations, a total of 202 driver mutations were discovered in 34 genes. ttDNA exhibited the highest occurrence of mutations (N=232), followed by baseline-ctDNA (N=155) and PD-ctDNA (N=117). Genotype concordance for the ten most mutated genes was highest between baseline-ctDNA and PD-ctDNA, at 94.3%, while the concordance rates for ttDNA were 85.5% with baseline-ctDNA and 83.6% with PD-ctDNA. Overall, 40 of the 84 patients (47.6%) benefited from sequencing ctDNA in addition to ttDNA as it identified additional mutations. Compared to ttDNA and baseline-ctDNA, PD-ctDNA identified 13 novel mutations in ten patients (11.9%). Notably, 7 mutations in five patients (6.0%) were missense or nonsense mutations in APC, TP53, SMAD4, and CDH1 genes. In baseline-ctDNA, patients with maximal variant allele frequency (VAF) values of &amp;gt;0.059 showed significantly worse progression-free survival (median 10.5 vs. 17.1 months, P=0.036) and overall survival (median 18.1 vs. 40.3 months, P=0.010). Moreover, overall survival was significantly worse in those with higher VAF values of APC (median 18.1 vs. 36.8 months, P=0.012), TP53 (median 24.7 vs. 40.3 months, P=0.012), and KRAS (median 13.7 vs. 27.6 months, P=0.005) mutations. Conclusions: While ttDNA remains more sensitive than ctDNA, our unique molecular identifier-based ctDNA platform demonstrated the validity and potential value when ttDNA was unavailable. Furthermore, post-SACT analysis of PD-ctDNA revealed new pathogenic mutations, indicating the clonal evolution of cancer. In addition, the VAF values of baseline-ctDNA predicted prognosis after SACT. Citation Format: Ji-Won Kim, Seung-been Lee, Hong-Geun Kim, Sung-Hyun Hwang, Kui-Jin Kim, Ju Hyun Lee, Jeongmin Seo, Minsu Kang, Eun Hee Jung, Koung Jin Suh, Se Hyun Kim, Jin Won Kim, Yu Jung Kim, Jee Hyun Kim, Nak-Jung Kwon, Keun-Wook Lee. Longitudinal comparative analysis of circulating tumor DNA and matched tumor tissue DNA in patients with metastatic colorectal cancer receiving palliative first-line systemic anti-cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 966.

Abstract 2425: Pairwise analysis of plasma cell-free DNA before and after paclitaxel plus ramucirumab treatment in patients with metastatic gastric cancer

Abstract Background: Plasma cell-free DNA (cfDNA) analysis has emerged as an appealing alternative to detect somatic mutations. This study compared cfDNA at baseline (baseline-cfDNA) and progressive disease (PD-cfDNA) and tumor tissue DNA (ttDNA) in patients with metastatic gastric cancer who underwent palliative second-line paclitaxel+ramucirumab treatment. Methods: We conducted targeted sequencing of 106 cancer-related genes using germline DNA, baseline-cfDNA, and PD-cfDNA samples. The results were then compared with conventional cancer panel results using ttDNA. Results: Of 76 consecutively enrolled patients who received paclitaxel+ramucirumab treatment, 46 patients (27 males; median age 57.5 [range, 32-73] years) with access to all three samples were analyzed along with their ttDNA data. A total of 138, 145, and 80 mutations were detected in baseline-cfDNA, PD-cfDNA, and ttDNA respectively. Combined analysis of baseline-cfDNA and ttDNA revealed that TP53 (56.5%) was the most frequently mutated gene, followed by CDH1 (26.1%), KRAS (21.7%), and APC (13.5%). For the top four genes, the sensitivity and positive predictive value of baseline-cfDNA compared with ttDNA were 71.8% and 51.9%, respectively. Compared with ttDNA alone, 32 patients (70.0%) benefited from baseline-cfDNA in detecting novel mutations. When combined with baseline-cfDNA and PD-cfDNA, novel mutations were discovered in 34 patients (73.9%). Of note, PD-cfDNA analysis detected 9 novel pathogenic mutations in TP53, APC, PIK3CA, CDH1, RNF43, CTNNB1, and BRCA2 genes in 8 patients (17.4%) after treatment. In baseline-cfDNA, patients with a circulating ttDNA fraction concentration at 110-160 bp of &amp;gt;4.3777 ng/µL had significantly shorter progression-free survival (PFS) (median 3.5 vs. 5.3 months, P=0.016). In addition, maximal variant allele frequency (VAF) values of &amp;gt;0.1045 (median 3.4 vs. 5.2 months, P=0.022) and the sum of VAF values of &amp;gt;0.2071 (median 3.4 vs. 5.2 months, P=0.028) were significantly associated with shorter PFS. In patients with TP53 mutations, those with TP53 VAF values of &amp;gt;0.1014 significantly had worse PFS (median 4.6 vs. 6.4 months, P=0.022). Conclusions: Although cfDNA could not entirely take over the role of ttDNA, cfDNA analysis identified additional somatic mutations that were otherwise missed by ttDNA alone. Moreover, PD-cfDNA analysis detected novel pathogenic mutations that developed during treatment, implicating the clonal evolution of cancer. In addition, baseline cfDNA predicted PFS of patients receiving paclitaxel+ramucirumab therapy. Citation Format: Ji-Won Kim, Dong Soo Kyung, Won Yeong Ko, Hwang-Phill Kim, Sung-Hyun Hwang, Kui-Jin Kim, Ju Hyun Lee, Jeongmin Seo, Minsu Kang, Eun Hee Jung, Koung Jin Suh, Se Hyun Kim, Jin Won Kim, Yu Jung Kim, Jee Hyun Kim, Keun-Wook Lee. Pairwise analysis of plasma cell-free DNA before and after paclitaxel plus ramucirumab treatment in patients with metastatic gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2425.

Abstract 6156: Deciphering the genomic landscape of Chilean cancer: Unveiling driver pathway divergence, and novel germline and actionable somatic variants

Abstract Advances in understanding the genetic landscape of cancer have paved the way for more precise molecular profiling and the development of more efficacious therapeutic options. However, underrepresentation in clinical research endeavors has left the Latin American population uncharacterized, which may prevent them from reaping the benefits of these advancements. This study sought to identify the genetic drivers and actionable mutations in solid tumors from Chilean patients, enhancing our understanding of the mutational landscape in a population with a genetically diverse background shaped by admixture.Methods: 468 biopsy tissues from gastric (171), gallbladder (91), breast (72), Ovarian (69), and colorectal (65) Chilean cancer patients were collected in several pathology services and biobank at Universidad de Chile. Target-sequencing was performed using Oncomine Comprehensive Assay v.1 and v.3; and TumorSec cancer panel. Data from TCGA, MSK-IMPACT, and ACCR GENIE cohorts was accessed through cBioportal and used for comparison with non-Hispanic populations. Variants were annotated with CGI, CancerVar, OncoKB, and ClinVar tools.Results: Overall, tumors have mutations mainly in TP53 (42.4%), PIK3CA (12.5%), KRAS (9.6%), PTEN (4.7%), BRCA1/2 (9%). Driver mutations were present in TP53 (42%), PIK3CA (16%), KRAS (10%), ERBB2 (7%), PTEN (7%), BRCA1/2 (8%), ATM (7%), among others. Accionable mutations were found primarily in PIK3CA (17%), KRAS (10%), ERBB2 (9%), and NTRK1/2/3 (4.8%). Besides already-known mutations, many “novel” mutations were also found in all tumor types. About 5-15% of novel variants were predicted drivers, including variants in CDH1, NOTCH1, BRCA1/2, MTOR, TSC2, and MSH2 genes. Significantly, key driver mutations identified in non-Hispanic patients from international cohorts are also prevalent in admixed Chilean patients. However, variations at the driver pathway level were also observed. Notably, the PI3K and MTOR pathways were more frequently affected in Chilean CRC and GBC, respectively. Furthermore, mutations in specific genes, such as TP53 in GBC, were associated with Amerindian genetic ancestry.The observed differences could be attributed to the abundance of newly identified driver-predicted variants, though the possibility of the admixed genetic background influencing the oncogenic process in various tissue types cannot be excluded. Citation Format: Evelin González, Jessica Toro, Alejandro Blanco, Camilo Tapia, Solange Rivas, José Erices, Felipe Osorio, Gonzalo Sepúlveda-Hermosilla, Matias Freire, Valentina Gárate, Javier Cáceres-Molina, Iván Gallegos, Olga Barajas, Mónica Ahumada, Eva Bustamante, Jaime González, Carolina Ibañez, Alejandro Corvalan, Gareth Owen, Marcelo Garrido, Ricardo Armisen, Katherine Marcelain. Deciphering the genomic landscape of Chilean cancer: Unveiling driver pathway divergence, and novel germline and actionable somatic variants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6156.