MDM2 Research Articles

Establishment and characterization of NCC-DDLPS1-C1: a novel patient-derived cell line of dedifferentiated liposarcoma.

Dedifferentiated liposarcoma (DDLPS) is one of the four subtypes of liposarcomas; it is characterized by the amplification of the 12q13-15 region, which includes MDM2 and CDK4 genes. DDLPS has an extremely high local recurrence rate and is refractory to chemotherapy and radiation, which leads to poor prognosis. Therefore, a novel therapeutic strategy should be urgently established for improving the prognosis of DDLPS. Although patient-derived cell lines are important tools for basic research, there are no DDLPS cell lines available from public cell banks. Here, we report the establishment of a novel DDLPS cell line. Using the surgically resected tumor tissue from a patient with DDLPS, we established a cell line and named it NCC-DDLPS1-C1. The NCC-DDLPS1-C1 cells contained 12q13-15, 1p32, and 1q23 amplicons and highly expressed MDM2 and CDK4 proteins. NCC-DDLPS-C1 cells exhibited constant growth, spheroid formation, aggressive invasion, and tumorigenesis in mice. By screening a drug library, we identified that the proteasome inhibitor, bortezomib, had inhibitory effects on the proliferation of NCC-DDLPS1-C1 cells. We concluded that the NCC-DDLPS1-C1 cell line may serve as a useful tool for basic and pre-clinical studies of DDLPS.

The MDM2 ligand Nutlin-3 differentially alters expression of the immune blockade receptors PD-L1 and CD276

BackgroundThe links between the p53/MDM2 pathway and the expression of pro-oncogenic immune inhibitory receptors in tumor cells are undefined. In this report, we evaluate whether there is p53 and/or MDM2 dependence in the expression of two key immune receptors, CD276 and PD-L1.MethodsProximity ligation assays were used to quantify protein-protein interactions in situ in response to Nutlin-3. A panel of p53-null melanoma cells was created using CRISPR-Cas9 guide RNA mediated genetic ablation. Flow cytometric analyses were used to assess the impact of TP53 or ATG5 gene ablation, as well as the effects of Nutlin-3 and an ATM inhibitor on cell surface PD-L1 and CD276. Targeted siRNA was used to deplete CD276 to assess changes in cell cycle parameters by flow cytometry. A T-cell proliferation assay was used to assess activity of CD4+ T-cells as a function of ATG5 genotype.ResultsCD276 forms protein-protein interactions with MDM2 in response to Nutlin-3, similar to the known MDM2 interactors p53 and HSP70. Isogenic HCT116 p53-wt/null cancer cells demonstrated that CD276 is induced on the cell surface by Nutlin-3 in a p53-dependent manner. PD-L1 was also unexpectedly induced by Nutlin-3, but PD-L1 does not bind MDM2. The ATM inhibitor KU55993 reduced the levels of PD-L1 under conditions where Nutlin-3 induces PD-L1, indicating that MDM2 and ATM have opposing effects on PD-L1 steady-state levels. PD-L1 is also up-regulated in response to genetic ablation of TP53 in A375 melanoma cell clones under conditions in which CD276 remains unaffected. A549 cells with a deletion in the ATG5 gene up-regulated only PD-L1, further indicating that PD-L1 and CD276 are under distinct genetic control.ConclusionGenetic inactivation of TP53, or the use of the MDM2 ligand Nutlin-3, alters the expression of the immune blockade receptors PD-L1 and CD276. The biological function of elevated CD276 is to promote altered cell cycle progression in response to Nutlin-3, whilst the major effect of elevated PD-L1 is T-cell suppression. These data indicate that TP53 gene status, ATM and MDM2 influence PD-L1 and CD276 paralogs on the cell surface. These data have implications for the use of drugs that target the p53 pathway as modifiers of immune checkpoint receptor expression.

BL1391: an established cell line from a human malignant peripheral nerve sheath tumor with unique genomic features.

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive tumors, accounting for around 5% of all soft tissue sarcomas. A better understanding of the pathogenesis of these tumors and the development of effective treatments are needed. In this context, established tumor cell lines can be very informative, as they may be used for in-depth molecular analyses and improvement of treatment strategies. Here, we present the genomic and transcriptomic profiling analysis of a MPNST cell line (BL1391) that was spontaneously established in our laboratory from a primary tumor that had not been exposed to genotoxic treatment. This cell line shows peculiar genetic features, such as a large marker chromosome composed of high-copy number amplifications of regions from chromosomes 1 and 11 with an embedded neocentromere. Moreover, the transcriptome profiling revealed the presence of several fusion transcripts involving the CACHD1, TNMA4, MDM4, and YAP1 genes, all of which map to the amplified regions of the marker. BL1391 could be a useful tool to study genomic amplifications and neocentromere seeding in MPNSTs and to develop new therapeutic strategies.

Relationship between Chromosomal Aberrations and Gene Expressions in the p53 Pathway in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is a neoplasm characterized by excessive accumulation of B lymphocytes in the peripheral blood, bone marrow and lymph nodes. We assessed the expressions of 22 genes in the p53 pathway in 30 CLL patients and 15 healthy subjects by a RT2 Profiler PCR (polymerase chain reaction) Array technique and their relation to cytogenetic aberrations detected by fluorescent in situ hybridization (FISH). Our Student’s t-test results indicated that ATM, ATR, BAX, CASP9, CDK4, CDKN2A, CHEK1, CHEK2, E2F3, MCL1, MDM2, MDM4, PCNA, RB1, P53 and BCL2 genes were statistically significant (p <0.001). For six genes (APAF1, CDKN1A, E2F1, GADD45A, PTEN and PTX3) were not statistically significant. The ATM, ATR, BAX, CASP9, CDK4, CDKN1A, CDKN2A, CHEK1, CHEK2, MDM2, MDM4, PCNA, RB1, P53, E2F1, GADD45A and BCL2 genes were found to be upregulated by the 2-ᐃᐃCt (relative fold change in gene expression) method. The highest up-regulation was detected in CDKN2A and BCL2 genes, 10.22- and 8.51-fold, respectively. On the other hand, the PTX3 gene with a fold regulation of 1.84 was found to the highest downregulation. Overall, the CDNK2A BCL2 and PTX3 genes are related to the mechanism of the disease in the p53 pathway and may be an important predictor of the prognosis of the disease. The BCL2 gene may be associated with increased risk of developing CLL. We suggest that the PTX3 gene may be considered as a marker associated with CLL disease. The CDKN2A gene expression seems to play a protective role in CLL.

Next-Generation Sequencing Analysis of ctDNA for the Detection of Glioma and Metastatic Brain Tumors in Adults.

Background and aims: The next-generation sequencing technologies and their related assessments of circulating tumor DNA in both glioma and metastatic brain tumors remain largely limited.Methods: Based largely on a protocol approved by the institutional review board at Peking University International Hospital, the current retrospective, single-center study was conducted. Genomic DNA was extracted from blood samples or tumor tissues. With the application of NextSeq 500 instrument (Illumina), Sequencing was performed with an average coverage of 550-fold. Paired-end sequencing was employed utilized with an attempt to achieve improved sensitivity of duplicate detection and therefore to increase the detection reliability of possible fusions.Results: A total of 28 patients (21 men and 7 women) with brain tumors in the present study were involved in the study. The patients enrolled were assigned into two groups, including glioma group (n = 21) and metastatic brain tumor group (n = 7). The mean age of metastatic brain tumor group (59.86 ± 8.85 y), (43.65 ± 13.05 y) reported significantly higher results in comparison to that of glioma group (45.3 ± 12.3 years) (P < 0.05). The mutant genes in metastatic brain tumor group included ALK, MDM2, ATM, BRCA1, FGFR1, MDM4 and KRAS; however, there were no glioma-related mutant genes including MGMT, IDH1, IDH2, 1p/19q, and BRAF et al. Interesteringly, only two patient (28.3%) was detected blood ctDNA in metastatic brain tumor group; In contrast, blood ctDNA was found in ten glioma patients (47.6%) including 1p/19q, MDM2, ERBB2, IDH1, CDKN2A, CDK4, PDGFRA, CCNE1, MET. The characterizations of IDH mutations in the glioma included IDH1 mutation (p.R132H) and IDH2 mutation (p.R172K). The mutation rate of IDH in tumor tissues was 37.06 ± 8.32%, which was significantly higher than blood samples (P < 0.05).Conclusion: The present study demonstrated that the mutant genes among glioma and metastatic brain tumors are shown to be different. Moreover, the ctDNAs in the metastatic brain tumors included ALK and MDM2, and glioma-related ctDNAs included 1p/19q and MDM2 followed by frequencies of ERBB2, IDH1, CDKN2A, CDK4, PDGFRA, CCNE1, MET. These ctDNAs might be biomarkers and therapeutic responders in brain tumor.

Abstract 6213: Extracellular vesicles MDM2-DNA derived from dedifferentiated liposarcoma induces MMP2 production from preadipocytes

Abstract Dedifferentiated liposarcoma (DDLPS) is molecularly characterized by wt p53 and MDM2 gene amplification causing MDM2 protein over-production, the key oncogenic process in DDLPS. Commonly located in fat-bearing retroperitoneal areas, almost 60% of DDLPS patients undergo multifocal recurrence, typically amenable to palliative treatment only, and occasionally develop distant metastasis. These factors lead to an abysmal 10% 10 year overall survival rate. Tumor cell-derived extracellular vesicles (EVs) can facilitate loco-regional malignancy dissemination by depositing molecular factors that participate in the development of pre-metastatic niches for tumor cell implantation and growth. High number of MDM2 DNA molecules was identified within EVs from DDLPS patient serum (ROC vs normal; 0.95) as well as from DDLPS cell lines. This MDM2 DNA could be transferred to preadipocytes (P-a), a major and ubiquitous cellular component of the DDLPS tumor microenvironment (TME), with subsequent P-a production of matrix metalloproteinase 2 (MMP2), a critical component in the metastatic cascade. Since multifocal loco-regional DDLPS spreading is the main cause of the remarkably high lethality of this disease, a better understanding of the underlying oncogenic processes and their regulatory mechanisms is essential to improve the outcome of this devastating disease. Citation Format: Lucia Casadei, Federica Calore, Ameya Deshmukh, Abeba Zewdu, Paolo Fadda, Danielle Braggio, Martin Wabitsch, Carlo M. Croce, Dina Lev, Raphael Pollock. Extracellular vesicles MDM2-DNA derived from dedifferentiated liposarcoma induces MMP2 production from preadipocytes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6213.

MDM2 Antagonists Induce a Paradoxical Activation of Erk1/2 through a P53-Dependent Mechanism in Dedifferentiated Liposarcomas: Implications for Combinatorial Strategies.

The MDM2 gene is amplified in dedifferentiated liposarcoma (DDLPS). Treatment with MDM2 antagonists is a promising strategy to treat DDLPS; however, drug resistance is a major limitation when these drugs are used as a single agent. This study examined the impact of MDM2 antagonists on the mitogen-activated protein kinase (MAPK) pathway in DDLPS and investigated the potential synergistic activity of a MAPK kinase (MEK) inhibitor in combination with MDM2 antagonists. We identified a synergistic effect and identified the mechanism behind it. Combination effects of MDM2 antagonists and a MEK inhibitor were analyzed in a patient-derived xenograft mouse model and in DDLPS and leiomyosarcoma cell lines using different cell proliferation assays and immunoblot analysis. MDM2 antagonist (RG7388)-resistant IB115 [P4] cells and p53-silenced DDLPS cells were also established to understand the importance of functional p53. We found that MDM2 antagonists induced an upregulation of phosphorylated extracellular signal-regulated kinase (p-ERK) in DDLPS cells. The upregulation of p-ERK occurred due to mitochondrial translocation of p53, which resulted in increased production of reactive oxygen species, causing the activation of receptor tyrosine kinases (RTKs). Activated RTKs led to the activation of the downstream MEK/ERK signaling pathway. Treatment with a MEK inhibitor resulted in decreased expression of p-ERK, causing significant anti-tumor synergy when combined with MDM2 antagonists. Our results provide a framework for designing clinical studies of combination therapies in DDLPS patients.

Association of Molecular Genetic Markers of TP53, MDM2, and CDKN1A Genes with Progression-Free Survival of Patients with Ovarian Cancer after Platinum-Based Chemotherapy.

We studied the association of polymorphic markers of cell cycle control genes (Arg72Pro of the TP53 gene, T(-410)G of the MDM2 gene, and Ser31Arg of the CDKN1A gene) in ovarian cancer and progression-free survival following platinum-based chemotherapy. Tumor tissue samples obtained from 49 patients who had undergone chemotherapy were examined. Patients received standard platinum-based chemotherapy and were observed until disease progression. Polymorphic markers of genes were evaluated by PCR-RFLP and real-time PCR. In patients carrying the G allele of the T(-410)G marker of the MDM2 gene, a decreasing trend was observed in median progression-free survival. An increase in the median progression-free survival was observed in carriers of the Pro allele of the TP53 gene (p=0.045). Furthermore, a stronger association was noted with carriers of the minor Pro/Pro homozygous genotype relative to the Arg/Arg genotype (p=0.007). In the subgroup of patients who underwent optimal or complete cytoreductive surgery, carriage of the minor Arg allele of the Ser31Arg marker (CDN1A gene) was associated with a decrease in the median progression-free survival time (p=0.004).

Correlation between P53 Arg72Pro and MDM4 gene rs4245739 polymorphisms in breast cancer

BackgroundMDM4 is a negative regulator of p53 tumor suppression pathway. Various studies reveal the fact that the rs4245739A>C polymorphism of MDM4 in 3′-untranslated region is targeted by miR-191 which leads to lower MDM4 expression. On the other hand, the importance of Arg72Pro polymorphism of p53 in breast cancer is proven. The aim of this study is to present the correlation between the aforementioned SNPs and the risk of breast cancer. The statistical society of the study includes 199 healthy women as the control group and 206 women suffering from breast cancer included from Turkish population of the East Azarbaijan. ResultsAlleles of both positions are detected through Tetra-ARMS PCR while SPSS and the SHEsis, online software was exerted for allele typing, genotyping, and haplotype analysis. Different alleles of both MDM4 rs4245739 and p53 Arg72Pro had no significant frequency in patients (P > 0.05). Furthermore, genotypes of neither MDM4 rs4245739 nor p53 Arg72Pro could increase or decrease the risk of breast cancer in patients compared to healthy women. Additionally, a significant factor affecting the risk of breast cancer is gene-gene interaction. The results of the ongoing study revealed that two genetic variants, MDM4 rs4245739 and p53 Arg72Pro polymorphisms, failed to be associated, individually and combined together, with the risk of breast cancer. However, additional well-designed studies on large populations are required to validate such an association. ConclusionVarious alleles of both SNPs had no significant frequency difference between patients and controls. Moreover, genotypes of neither MDM4 nor p53 were significantly distributed in women with breast cancer compared to the healthy controls. Meanwhile, haplotype analysis failed to be significant. Last but not least, all clinical manifestations, other than association between tumor size and MDM4 genotypes could not be correlated with either various alleles or genotypes.

Abstract B45: Therapeutic potential of splicing in RMS: SRSF2 binding modulation controls MDM2 alternative splicing

Abstract MDM2 undergoes complex alternative splicing and one of its spliced isoforms, MDM2-ALT1, comprised of coding exons 3 and 12, is highly expressed in pediatric rhabdomyosarcoma, and its expression correlates with a poor disease prognosis. MDM2-ALT1 expression is also upregulated under conditions of cellular genotoxic stress, and this alternative splicing is conserved in the mouse Mdm2 gene. Furthermore, previous research from our lab has reported that the splicing factors SRSF1, FUBP1, and PTBP1 are involved in this regulation; nevertheless, the way that MDM2 splicing is controlled is not entirely understood. In this work, we explore the role of the splicing factor SRSF2/SC35 in the alternative splicing of MDM2, hypothesizing that this factor acts as a positive regulator, in both human and mouse, facilitating the production of the full-length MDM2 mature transcript. This splicing factor has been reported to be mutated in several cancers, including myelodysplastic syndromes and rhabdomyosarcoma. Moreover, using a minigene system that mimics the endogenous Mdm2 splicing in response to UV and cisplatinum-induced DNA damage and the CRISPR-Cas9 technology, we show that mutations in SRSF2 predicted binding sites alter Mdm2 alternative splicing. The CRISPR-engineered mutant cells that express this splice isoform show increased proliferation in an Arf/p19 null context. On the other hand, we show that by using antisense oligonucleotides working as splice-switching molecules that target the SRSF2 binding sites in the MDM2 pre-mRNA, we are able to shift the expression of the MDM2 splice isoforms to drive p53 target gene expression in p53 wild-type cells. Furthermore, in an effort to understand better the effect of the MDM2-ALT1 isoform in carcinogenesis, we performed the CRISPR-Cas9 technique to generate a mouse model expressing the cancer-associated spliced isoform. We expect the mouse to be a cancer-sensitized model that will give rise to RMS tumors and provide a preclinical model for testing splice-switching oligonucleotide therapies. Citation Format: Matias I. Montes, Daniel F. Comiskey, Safiya Khurshid, Dawn S. Chandler. Therapeutic potential of splicing in RMS: SRSF2 binding modulation controls MDM2 alternative splicing [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B45.

Dynamics of p53 regulatory network in DNA damage response

The tumor suppressor p53 plays a central role in the DNA damage response by inducing cell cycle arrest or apoptosis and several recent experiments have shown interesting dynamics in p53 protein expression during the repair cycle. In this paper, based on the three-module model proposed by Chickermane et al., a general model of delayed p53 regulatory network in the DNA damage response is formulated by introducing ubiquitous transcription and translation delays in the p53-Mdm2 oscillator module proposed by Zhang et al.. The dynamics of p53 regulatory network in DNA damage response are investigated through analytic analysis and numerical methods. It turns out that the pulsatile behavior can be generated due to initial DNA damage signals and ATM shows a switch-like behavior and relays the DNA damage signal. Moreover, the delays required for transcription and translation in Mdm2 gene expression can drive p53 to be oscillatory and an optimal rate of model parameters is also essential to these oscillations. Furthermore, the length of these delays can determine the amplitude and period of the oscillations. These results may provide a perspective for the development of anti-cancer therapy.

Association of KLK3, VAMP8 and MDM4 Genetic Variants within microRNA Binding Sites with Prostate Cancer: Evidence from Serbian Population.

A growing number of studies have suggested that genetic variants affecting the micro-RNA- binding mechanisms (miRSNPs) constitute a promising novel class of biomarkers for prostate cancer (PCa) biology. Among the most extensively studied miRSNPs in the context of cancer is the variation rs4245739 in the MDM4 gene, while a recent large-scale analysis revealed significant differences in genotype distributions between aggressive and non-aggressive disease for rs1058205 in KLK3 and rs1010 in VAMP8. In this study, we examined a total of 1083 subjects for these three variants using Taqman® SNP Genotyping Assays. Three hundred and fifty-five samples of peripheral blood were obtained from patients with PCa and 358 samples from patients with benign prostatic hyperplasia (BPH). The control group consisted of 370 healthy volunteers. Comparisons of genotype distributions among PCa and BPH patients, as well as between PCa patients and healthy controls, yielded no evidence of association between the analyzed genetic variants and the risk of developing PCa. However, all three tested genetic variants have shown the association with the parameters of PCa progression. For KLK3 variant rs1058205, minor allele C was found to associate with the lower serum PSA score in PCa patients (PSA > 20ng/ml vs. PSA < 10ng/ml comparison, Prec = 0.038; ORrec = 0.20, 95%CI 0.04-1.05). The obtained results point out the potential relevance of the tested genetic variants for the disease aggressiveness assessment.

The genes CNV features in the tumor cells in patients with ovarian serous adenocarcinoma.

e13641 Background: Serous adenocarcinoma is the most common subtype of ovarian cancer. Early diagnosis of this disease can significantly improve the prognosis. Development of more effective methods for early diagnosis and identification of more reliable markers require understanding the molecular mechanisms underlying the malignant behavior of ovarian epithelial cells. The aim of the study was to analyze the copy number variation (CNV) of genes that regulate apoptosis, DNA repair, cell proliferation, metabolism and estrogen reception in tumor and normal ovarian cells. Methods: For the study, we used tissue sections from FFPE blocks of 65 patients with serous ovarian adenocarcinoma diagnosis. Tumor and normal ovarian epithelial cells were isolated using laser microdissection with non-contact capture (Palm MicroBeam, Carl Zeiss). DNA was extracted from cells using the phenol-chloroform method. CNV of 33 genes (BRAF, KRAS, EGFR, PIK3CA, PTEN, TP53, BRCA1, BRCA2, PRKCI, NOTCH1, AKT1, BAX, CASP7, CASP3, CASP8, CASP9, MDM2, BCL2, CYP1A1, CYP1A1, CYP1A2, CYP1A2, CYP1A2, CYP1A1, CYP1A2, CYP1, ESR2, GPER, STS, SULT1A, SULT1E1, OCT4, SOX2, C-MYC, SOX18, SCNN1A) were determined by Real-Time qPCR method (reference B2M, GAPDH). Statistical analysis was performed using the Mann-Whitney test. For cluster analysis (Hierarchical Clustering, Euclidean distance) scripts in R were used. Results: A statistically significant (p &lt; 0.005) increase in the CNV of PTEN, MDM2, SOX2, CYP1B1, ESR1, and SULT1E1 by 2.0, 2.0, 1.8, 2.5, 3.0 and 2.0 times, respectively, was found, as well as a 2.0-fold decrease in the CNV of CASP3 and CASP8 in tumor cells relatively to normal ones. Cluster analysis allowed us to distinguish 2 groups of serous adenocarcinoma samples that differed in gene CNV (p &lt; 0.005): in group 1 (n = 40), the copy number of the SOX2, MDM2, ESR1, CYP1B1 and SULT1E1 genes was increased and the copy number of the TP53 and BRCA2 genes was reduced, in group 2 (n = 25), copy number of PTEN, PIK3CA, BCL2 was increased and copy number of BAX, CASP3 and CASP8 was lower. Conclusions: CNV analysis revealed the most characteristic markers of ovarian serous adenocarcinoma cells. Based on the differential CNV, 2 molecular subtypes of serous adenocarcinoma were distinguished.

Study of the MDM2 -410T-G polymorphism (rs2279744) by pyrosequencing in mothers of Down Syndrome subjects.

Trisomy 21 or Down syndrome (DS) is the most frequent genetic etiology of intellectual disability in humans. MDM2 gene expression has a potential role as a risk factor for human aneuploidy. -410T-G (rs2279744) functional polymorphism in MDM2 gene impacts on the mechanisms of chromosomal non-disjunction. We analyzed, within a case-control study, such polymorphism in mothers of subjects with DS. Nucleotide polymorphism was detected by pyrosequencing technology. The distribution of MDM2-410T-G polymorphism showed no significant difference among mothers of subjects with DS and controls. Our results suggest that MDM2 -410T-G polymorphism is not a risk factor for DS in mothers.

Dysplastic lipoma: potential diagnostic pitfall of using MDM2 RNA in situ hybridization to distinguish between lipoma and atypical lipomatous tumor

The distinction between lipoma and atypical lipomatous tumor can be challenging in some cases. While detection of MDM2 gene amplification via fluorescence in situ hybridization (FISH) has been well established as a diagnostic tool to distinguish atypical lipomatous tumor and well-differentiated liposarcoma from benign mimics, MDM2 RNA in situ hybridization (RNA-ISH) has recently been proposed as an alternative diagnostic assay. During clinical workup for lipomatous tumors using MDM2 RNA-ISH, we noticed several dysplastic lipomas that were positive for MDM2 RNA-ISH but negative for MDM2 amplification by FISH. In this study, we examined a series of 11 dysplastic lipomas, all confirmed to be negative for MDM2 amplification by FISH. Positive MDM2 RNA-ISH was noted in 10 (91%) dysplastic lipomas. Single-nucleotide polymorphism array on one dysplastic lipoma identified the presence of homozygous deletion of 13q, including the RB1 gene locus with no evidence of MDM2 copy number gain. Our findings on the discordance between MDM2 FISH and MDM2 RNA-ISH highlightthe potential utility and pitfalls of using MDM2 RNA-ISH in the distinction of atypical lipomatous tumor and related liposarcomas from dysplastic lipoma.

Dedifferentiated Liposarcoma With Meningothelial-Like Whorls: Five Additional Cases and Review of the Literature.

Background. Diagnosis of dedifferentiated liposarcoma (DDL) can sometimes be challenging due to a wide variety of histological features. "Meningothelial-like" whorl is an uncommon histological feature of DDL, which is also observed in neural tumors and follicular dendritic cell sarcoma. This feature is frequently associated with metaplastic bone formation. We conducted this study to describe the clinicopathological features of DDL with meningothelial-like whorls that would aid in establishing accurate diagnosis. Material and Methods. Microscopic glass slides of 5 cases of DDL with meningothelial-like whorls, diagnosed between January 2010 and December 2019, were reviewed. Results. Paratesticular region was the most common site. Whorls occupied 10% to 75% of tumor area and ranged in size from <0.1 cm to >2 cm. In 1 case, these whorls coalesced to form large areas of dedifferentiation. The cells forming whorls were spindle to epithelioid shaped and lacked significant nuclear pleomorphism and increased mitoses. Metaplastic bone formation was observed in 4 cases and cartilage formation in 3 cases. p16 and α-smooth muscle actin (α-SMA) immunohistochemical stains were positive in 2 cases, when performed. MDM2 gene amplification was observed in all cases by fluorescence in situ hybridization technique. These tumors showed aggressive behavior, similar to that of DDL without meningothelial-like whorls. Two patients died, 1 developed recurrence, 1 presented as recurrent tumor, and 1 developed metastasis. Conclusion. Meningothelial-like whorls in DDL most likely represent an early stage of dedifferentiation. Presence of well-differentiated liposarcoma areas, metaplastic bone formation, positive expressions for p16 and α-SMA immunohistochemical stains, and MDM2 gene amplification are useful diagnostic clues. These tumors have the potential to behave aggressively.

Selected CDKN2A and MDM2 polymorphisms in oral cavity cancer.

The head and neck squamous cell carcinoma (HNSCC) is an aggressive human malignancy diagnosed in more than 800 000 new cases worldwide, and mostly arises in the oral cavity, oropharynx, larynx, hypopharynx, and nasopharynx. The study presented here aimed to determine a possible association of rs11515 and rs3088440 gene polymorphisms in the CDKN2A gene (cyclin dependent kinase inhibitor 2A), as well as rs769412 and rs937283 in the MDM2 gene (murine double minute 2), with oral cavity cancer in a sample of Polish population; CDKN2A is crucial in regulating the cell cycle while MDM2 is an oncogene involved in regulating cell proliferation and apoptosis. The study included 95 primary tumor samples following surgical resection from patients, and the control group consisted of 100 healthy individuals. DNA samples were genotyped by employing the 5' nuclease assay for allelic discrimination using TaqMan SNP Genotyping Assays (Applied Biosystems, USA). There was no significant association between any of the polymorphism (rs11515, rs3088440, rs769412 and rs937283) and the oral cavity cancer risk. We found that the AA homozygotes for rs3088440 were significantly more frequent in the control group (OR=0.046, p<0.0001). In addition, the GG genotype of rs769412 was not found in any group. We found no influence of the examined genotypes on clinicopathological parameters, such as T, N and grading values in patients with oral cavity cancer. The results of this study indicate that none of the investigated polymorphisms were associated with the risk of oral cavity cancer in the examined sample of the Polish population.

Gene-to-gene interactions and the association of TP53, XRCC1, TNFα, HMMR, MDM2 and PALB2 with breast cancer in Kyrgyz females.

At present, little is known about the genetic background of breast cancer (BC) in Kyrgyz. Therefore, the aim of this study was to assess gene-to-gene interactions and the contribution of p.Arg72Pro (TP53 gene), p.Gln399Arg (XRCC1 gene), p.Arg194Trp (XRCC1 gene), g.4682G > A (TNFα gene), p.Val353Ala (HMMR gene), c.14 + 309T > G (MDM2 gene) and g.38444T > G (PALB2 gene) polymorphic loci in breast cancer (BC) risk in females of Kyrgyz ethnicity. The case-control study comprised 103 females with histologically verified BC and 102 controls with no cancer. We used polymerase chain reaction-based restriction fragment length polymorphism to genotype polymorphic loci. Gln/Arg heterozygous variant of XRCC1 gene's p.Gln399Arg locus, as well as combined carriage of Arg/Gln//Arg/Pro of XRCC1/TP53; Arg/Gln//T/T of XRCC1/MDM2; Arg/Gln//G/G and Arg/Gln//G/A of XRCC1/TNFα, Arg/Gln//T/T of XRCC1/PALB2; Arg/Gln//Arg/Arg and Arg/Gln//Arg/Trp for p.Gln399Arg and p.Arg194Trp polymorphic loci of XRCC1 were associated with BC in Kyrgyz females. TP53, XRCC1, TNFα, HMMR, MDM2 and PALB2 genes' polymorphic site combinations appear to be candidate markers of genetic predisposition to BC in Kyrgyz population and prompt targeted personalized care.

Genetic alterations in epidermal growth factor receptor-tyrosine kinase inhibitor-naïve non-small cell lung carcinoma.

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) are an approved first-line therapy against unresectable or advanced non-small cell lung cancer (NSCLC) harboring EGFR gene activating mutations. However, the majority of tumors develop acquired resistance against EGFR-TKIs and some tumors exhibit natural resistance. A number of resistance mechanisms against the latest third-generation EGFR-TKIs have been reported, including tertiary EGFR C797S mutation and several gene alterations activating EGFR or other signaling pathways. The current study aimed to identify the frequency of natural EGFR-TKI resistance in pretreatment NSCLC and to predict the therapeutic effect of EGFR-TKIs. A total of 246 EGFR-TKI-naïve NSCLC patients harboring known EGFR gene mutations were identified. The presence of EGFR C797S and T790M mutations were determined using the peptide nucleic acid-locked nucleic acid PCR clamp method. ERBB2, MET, EGFR, ALK, BRAF, FGFR1, MYC, RET, CCND1, CCND2, CDK4, CDK6, MDM2 and MDM4 gene amplification, which can lead to resistance against any generation EGFR-TKIs, was determined using the multiplex ligation-dependent probe amplification assay. No concurrent C797S mutation with known EGFR mutations were identified. T790M mutation was identified in 12 patients (4.9%). ERBB2 or MET gene amplification was found in some patients (0.0-0.4%). MDM2 gene amplification was associated with tumor recurrence and shorter progression-free survival (PFS) for first- or second-generation EGFR-TKIs. De novo EGFR C797S mutation was not identified. Other resistance mechanisms against EGFR-TKIs were indicated in some patients with EGFR-TKI-naïve NSCLC. MDM2 gene amplification, which can lead to altered cell cycle, was associated with tumor recurrence and shorter PFS in EGFR-TKI therapy.

Manipulating Senescence of Cardiac Fibroblasts In Vivo and In Vitro to Investigate Arrhythmogenic Effect of Senescence

Cellular senescence is a stress response characterized by irreversible cell cycle arrest, secretion of inflammatory cytokines and growth factors termed the “senescence associated secretory phenotype” (SASP), and resistance to apoptosis. Senescence plays beneficial roles in tumor suppression, wound healing, and development in young animals, but is thought to underlie aging‐associated pathology in numerous tissues, mainly via the SASP. Our previous work in the aged rabbit myocardial infarction (MI) model demonstrated an increase and persistence of senescence, which we hypothesize to drive increased risk of arrhythmogenesis with age post‐MI. Here, we investigate the molecular mechanisms by which we believe senescence drives arrhythmogenesis. Specifically, we hypothesize that inflammatory cytokines in the SASP locally produced by senescent myofibroblasts acts on border zone myocytes to disrupt cellular‐level electrophysiology. Concordantly, we hypothesize that genetically or pharmacologically eliminating senescent cells post‐MI will mitigate arrhythmogenesis in vivo and in vitro, and that genetically increasing senescence post‐MI in a mouse Cre‐Lox model will increase arrhythmogenesis.Here, we show that commercially‐available aged human cardiac fibroblasts and primary adult rabbit cardiac fibroblasts can be induced into senescence via treatment with low doses of Etoposide in a time‐dependent manner. Additionally, via a senolytic drug screening, we found that high doses of Dasatinib, Quercetin, and Navitoclax effectively eliminate senescent cells but not non‐senescent cells. However, we found combinations of Dasatinib and Quercetin or Dasatinib and Venetoclax did not perform better than either drug in solo. Additionally, we verify that calcium linescan imaging as well as patch clamp measurements of the IKr current can be measured in primary 3‐week‐old rabbit cardiomyocytes.Using genetically altered mice to conditionally knock out the pro‐senescence p53 gene in myofibroblasts under control of the Periostin gene (p53 CKO mice), we find that p53 CKO mice have significantly less senescence post‐MI compared to littermate controls, and the effect is stronger in females compared to males. We also report that fibroblast‐specific knockout of the MDM2 gene in the same condition is embryonic lethal, as Periostin is expressed in the developing valve leaflets and endocardial cushion cells, where senescence plays a vital role in pruning transient fetal structures.In conclusion, we demonstrate methods to genetically and pharmacologically manipulate senescence in vivo and in vitro, which will allow us to investigate the molecular mechanisms through which senescence might play a role in promoting arrhythmogenesis with age post‐MI.Support or Funding InformationThe authors would like to acknowledge our supporting NIH funding sources, R21 grant 1R21AG049608‐01 and R01 grant R01HL139467