MDM2 Status Research Articles

Therapy-relevant MDM2 amplification in cholangiocarcinomas in Caucasian patients

Background: Cholangiocarcinomas (CCA) are a group of aggressive malignancies with poor prognosis. The distinct subtypes are related to different etiologies and genetic aberrations that are subject to targeted therapies. Mouse double minute 2 homolog (MDM2) is a potent inhibitor of tumor suppressor p53 and is proven to be altered in certain carcinomas. Novel targeted drugs, such as the MDM2-p53 antagonist Brigimadlin, have shown promising results for therapeutic efficacy in patients with MDM2 amplification and wild-type TP53. Objectives: This study therefore aimed to characterize CCAs regarding their MDM2 status, compare the concordance between fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) methods, and elucidate the role of MDM2 amplification in prognosis and other clinicopathological characteristics. Design: Retrospective cohort study. Methods: All patients ( n = 52) were diagnosed with CCA and received surgical resection with curative intention at the University Hospital of Cologne. Samples were analyzed retrospectively for MDM2 amplification with FISH and IHC. We correlated results with pre-existing molecular as well as clinical data. Results: We included 52 patients with primary CCA, three of which showed positive MDM2 amplification (5.8%). MDM2 amplification was present only in the intrahepatic CCA type and all patients with positive MDM2 amplification exhibited normal p53 status. Among the large-duct subtypes of intrahepatic CCAs, patients with positive MDM2 amplification demonstrated better survival than patients with negative MDM2 amplification ( p = 0.041). Of the patients with MDM2 amplification, two underwent adjuvant therapy post-surgery (66.7%). There was a strong correlation between MDM2 amplification and positive protein expression in IHC. There were no identifiable molecular co-alterations of MDM2 with FGFR2 or SWI/SNF complex alterations. Conclusion: Real-world evidence in our Caucasian patient population confirmed that a significant number of intrahepatic CCAs showcase MDM2 amplification, qualifying for a personalized therapy option with Brigimadlin. MDM2 amplification must therefore be considered in the context of personalized molecular testing in CCA.

MDM2 Amplification Status in a Cohort of Well-Characterized Myxofibrosarcoma: A Clinicopathologic Analysis of 22 Tumors.

Myxofibrosarcomas (MFS) present as slowly enlarging superficial masses in elderly patients. Even though these tumors fail to exhibit a distinct immunophenotype, diagnosis is straightforward when they present in subcutaneous tissue. Intramuscular MFS, however, are more challenging to diagnose as the differential also includes dedifferentiated liposarcoma with myxoid features. The vast majority of dedifferentiated liposarcomas show MDM2 amplification, whereas limited data exists as to the MDM2 status of MFS. We sought to explore the rate of MDM2 amplification in cases of classic MFS. Our archives were searched for MFS; only subcutaneous well-sampled resections were included. FISH for MDM2 amplification was performed on each tumor. A cohort of myxoid dedifferentiated liposarcoma resections was studied for comparison. Twenty-two MFS arose in patients aged 44 to 85 years. All tumors contained an infiltrative population of atypical cells embedded in a myxoid stroma with curvilinear blood vessels. MDM2 amplification by FISH was identified in 3 (of 22; 14%) tumors. Available follow up on 17 patients (range 1-96 months; median 13 months) revealed 6 patients with local recurrence and 1 with distant metastasis. Of 3 patients with MDM2- amplified MFS, 1 experienced recurrence and died of unrelated causes, while the second was alive without disease 12 months after diagnosis. Even though the rate of MDM2 amplification by FISH in MFS appears to be low, a subset of cases may show this genetic alteration, which pathologists should be aware of to avoid misclassification as myxoid dedifferentiated liposarcomas. Further studies are necessary to determine if amplification status adds prognostic value.

HCINAP alleviates senescence by regulating MDM2 via p14ARF and the HDAC1/CoREST complex.

Cellular senescence is a major process affected by multiple signals and coordinated by a complex signal response network. Identification of novel regulators of cellular senescence and elucidation of their molecular mechanisms will aid the discovery of new treatment strategies for aging-related diseases. In the present study, we identified human coilin-interacting nuclear ATPase protein (hCINAP) as a negative regulator of aging. Depletion of cCINAP significantly shortened the lifespan of Caenorhabditis elegans and accelerated primary cell aging. Moreover, mCINAP deletion markedly promoted organismal aging and stimulated senescence-associated secretory phenotype in the skeletal muscle and liver from mouse models of radiation-induced senescence. Mechanistically, hCINAP functions through regulating MDM2 status by distinct mechanisms. On one hand, hCINAP decreases p53 stability by attenuating the interaction between p14ARF and MDM2; on the other hand, hCINAP promotes MDM2 transcription via inhibiting the deacetylation of H3K9ac in the MDM2 promoter by hindering HDAC1/CoREST complex integrity. Collectively, our data demonstrate that hCINAP is a negative regulator of aging and provide insight into the molecular mechanisms underlying the aging process.

The Efficacy of Molecular Analysis in the Diagnosis of Bone and Soft Tissue Sarcoma: A 15-Year Mono-Institutional Study

The histological diagnosis of sarcoma can be difficult as it sometimes requires the combination of morphological and immunophenotypic analyses with molecular tests. A total of 2705 tissue samples of sarcoma consecutively collected from 2006 until 2020 that had undergone molecular analysis were assessed to evaluate their diagnostic utility compared with histological assessments. A total of 3051 molecular analyses were performed, including 1484 gene fusions tested by c/qRT-PCR, 992 gene rearrangements analysed by FISH, 433 analyses of the gene status of MDM2, 126 mutational analyses and 16 NGS analysis. Of the samples analysed, 68% were from formalin-fixed, paraffin-embedded tissue and 32% were from frozen tissue. C/qRT-PCR and FISH analyses were conclusive on formalin-fixed, paraffin-embedded tissue in 74% and 76% of samples, respectively, but the combination of the two methods gave us conclusive results in 96% and 89% of frozen and formalin-fixed, paraffin-embedded tissues, respectively. We demonstrate the utility of c/qRT-PCR and FISH for sarcoma diagnosis and that each has advantages in specific contexts. We conclude that it is possible to accurately predict the sarcoma subtype using a panel of different subtype-specific FISH probes and c/qRT-PCR assays, thereby greatly facilitating the differential diagnosis of these tumours.

MDM2 analysis in the management of benign lipomas versus atypical lipomatous tumors/well-differentiated liposarcomas: A useful prognostication tool?

IntroductionWell-differentiated liposarcomas (WDLS) are low-grade lipomatous tumors with low malignant potential. Previous review identified controversy on whether upfront wide resection is necessary when they occur on the trunk or the extremities. MDM2 amplification is a genetic mutation typically present in WDLS and absent in benign lipomas (BL). We aimed to study the influence of MDM2 status on the management/recurrences of lipomatous tumors in the trunk or the extremities. MethodsAll patients with lipomatous tumors with MDM2 testing in the Province of Alberta between 2015 and 2020 were identified from the Cancer Cytogenetics Laboratory dataset. High grade sarcomas, retroperitoneal, head/neck, or groin tumors were excluded. Primary outcome measures including MDM2 status, surgical margin, local recurrence, reoperation rate, dedifferentiation, and metastasis were abstracted from chart review. Descriptive statistics were used to analyse treatment patterns and recurrence rates according to MDM2 status. ResultsTotal of 764 charts were retrieved, and 282 were included for analysis. 33 showed MDM2 amplification (11.7%), and 2 of them had local recurrence (6.1%). Two patients with recurrent tumors underwent limb-salvaging reoperation (6.1%), but no dedifferentiation or metastasis was seen. ConclusionFindings in this study confirmed the benign behaviour of truncal/extremities lipomas with no MDM2 amplification. Given we found a 6.1% recurrence rate in MDM2 amplified tumors, a prolong follow up of this subset of patients is warranted. Overall, regardless of the MDM2 status, we believe an initial marginal excision is a reasonable surgical approach as recurrences are rare, and they can be managed with re-excision when they occur.

The utility of fluorescence in situ hybridization (FISH) in determining DNA damage-inducible transcript 3 (DDIT3) amplification in dedifferentiated liposarcomas – an important diagnostic pitfall

Background and objectiveDedifferentiated liposarcoma (DDLPS) is characterized by non-lipogenic sarcoma fields coexisting with adipocyte-rich well-differentiated areas. Amplification of the 12q13–15 region includes the MDM2 and DDIT3 genes. MDM2 amplification is considered a genetic hallmark of DDLPS, while DDIT3 is typically rearranged in myxoid liposarcoma. Recent studies showed that DDIT3 amplification is associated with myxoid liposarcoma-like (LPS-like) morphology in DDLPS. Our study aimed to evaluate the status of MDM2 and DDIT3 by FISH in DDLPS and correlate it with MLPS-like features. Material and methodsSix patients with MLPS-like morphology DDLPS were investigated pathologically, immunohistochemically, and genetically. The control groups of patients with classical DDLPS morphology and well-differentiated liposarcoma (WDLPS) were established and molecularly assessed as well. Fluorescence in situ hybridization (FISH) used in routine diagnostics was performed to determine the status of MDM2 and DDIT3 genes. ResultsThe patient's mean age was 64 (range from 43 to 85 years) with a 5:4 male to female ratio. Tumors were localized retroperitoneally (15) and extra-retroperitoneally (3). All cases demonstrated amplification of the 12q15 region containing MDM2 gene and co-amplification of the 5′ DDIT3 FISH Probe representing DDIT3 telomeric tag. However, we did not find the relation of myxoid LPS-like morphology with DDIT3 amplification as previously reported. ConclusionsThe biopsy material from DDLPS with myxoid areas can be misclassified as myxoid liposarcoma. Indeed, according to the histological image, DDIT3 status may be evaluated first. In these cases, we show that the DDIT3 telomeric tag amplification assessed by FISH, is a common, nonspecific feature, which is also found in classical DDLPS and WDLPS. Therefore, we believe that co-amplification of DDIT3 and MDM2 may be considered a spectrum of the 12q13–15 region amplification due to the specification of FISH methodology.

Abstract 2163: Molecular characteristics of CDK4 and/or MDM2 amplification in Chinese soft tissue sarcoma (STS) patients

Abstract Background: Cyclin-dependent kinase 4 (CDK4) and mouse double minute 2 homolog (MDM2) amplification occur at high frequency in well-differentiated liposarcoma and dedifferentiated liposarcoma, which are generally considered to be diagnostic criteria for these tumors. Studies have also reported that CDK4 and/or MDM2 amplification can emerge in STS subtypes, such as Ewing's sarcoma (ES) and fibrosarcoma (FS). However, the clinical pathological and molecular characteristics of CDK4 and/or MDM2 amplification in STS are still unclear. We systematically depicted the clinical characteristics of MDM2 and CDK4 gene amplification in Chinese STS patients (pts), aiming to assist the precise diagnosis and treatment of STS. Methods: Totally 184 Chinese pts with different STS subtypes were included in this study. In terms of subtypes distribution, the cohort contained multiple STS subtypes including myofibroblastic sarcoma (MFS, n = 23), liposarcoma (LPS, n = 21), FS (n = 20), rhabdomyosarcoma (RMS, n = 19), liomyosarcoma (LMS, n = 12), undifferentiated pleomorphic sarcoma (n = 11), clear cell sarcoma (n = 10), synovial sarcoma (n = 8), ES (n = 7), hemangiosarcoma (n=7), other rare subtypes (n = 37) and unknown types (n = 9). The amplification status of CDK4 and MDM2 in the tumor tissues of these pts was reviewed by the DNA and RNA next generation sequencing method. Results: Totally 16.8% (31/184) of the Chinese STS pts harbored CDK4 and/or MDM2 amplification. Among the 31 pts, 17 were with co-amplification of CDK4 and MDM2, including 12 LPSs, two RMSs, one MFS, one LMS and one FS. Fourteen pts were with single amplification of MDM2 or CDK4, including LPS (n = 4), RMS (n = 3), MFS (n = 3), FS (n = 2), ES (n = 1), and malignant peripheral nerve sheath tumor (n = 1). In LPS, the ratios of co-amplification and single amplification of CDK4/MDM2 were 57.1% (12/21) and 19.0% (4/21) respectively. Other STS subtypes with high frequency of CDK4 and/or MDM2 amplification included RMS (26.3%, 5/19) and MFS (19.4%, 4/23). Meanwhile, our study revealed that co-amplification of CDK4 and MDM2 appeared to be a strong driver event in STS, which mutated exclusively with other driver genes, such as TP53, MYOD1, ATRX and PTEN. Pts with co-amplification of CDK4 and MDM2 were likely to benefit from the treatment of CDK4 and/or MDM2 inhibitors. However, more than half of the pts with single amplification of CDK4 or MDM2 were accompanied by mutations in other driven genes, such as FLI1-EWSR1 fusion, PAX3-FOXO1 fusion, MYOD1 p.L122R, TP53 p.R273S, and NRAS p.G13C. In these pts, single amplification of CDK4 or MDM2 may not be the main driver event, who may be less likely to benefit from CDK4 or MDM2 inhibitor monotherapy. Conclusion: Our study revealed the clinicopathologic and molecular features of CDK4 and/or MDM2 amplification in Chinese STS pts, which can guide the selection of personalized treatment strategies in STS pts. Citation Format: Gu Jin, Chunyang Wang, Xiaojuan Wang, Qifan He, Tonghui Ma. Molecular characteristics of CDK4 and/or MDM2 amplification in Chinese soft tissue sarcoma (STS) patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2163.

Molecular classification and diagnostics of upper urinary tract urothelial carcinoma.

Upper urinary tract urothelial carcinoma (UTUC) is one of the common urothelial cancers. Its molecular pathogenesis, however, is poorly understood, with no useful biomarkers available for accurate diagnosis and molecular classification. Through an integrated genetic study involving 199UTUC samples, we delineate the landscape of genetic alterations in UTUC enabling genetic/molecular classification. According to the mutational status of TP53, MDM2, RAS, and FGFR3, UTUC is classified into five subtypes having discrete profiles of gene expression, tumor location/histology, and clinical outcome, which is largely recapitulated in an independent UTUC cohort. Sequencing of urine sediment-derived DNA has a high diagnostic value for UTUC with 82.2% sensitivity and 100% specificity. These results provide a solid basis for better diagnosis and management of UTUC.

¿Lipoma gigante o liposarcoma retroperitoneal? Controversias en su diagnóstico y tratamiento

BackgroundRetroperitoneal lipomas are extremely rare tumors that must be differentiated from well-differentiated liposarcomas (WD-LPS). ObjectivesTo summarize the evidence about giant retroperitoneal lipomas or liposarcomas; and to elaborate recommendations for their management. Data sourcesA systematic literature search from January 1985 to December 2019 and a review of our own cases was performed. ResultsOur series comprises four patients, two females and two males. The diagnosis was incidental in two cases. The medium size was 26 cm, being two cases located exclusively in the retroperitoneum, one in the inguinal region and one in the buttock via pelvic space. All cases were surgically removed being confirmed the initial diagnosis of retroperitoneal lipomas in two cases, as the rest two cases were classified as WD_LPS after MDM2/CDK4 genetic analysis.The review of the available literature plus our own cases revealed 30 cases, of which 58% were woman. Only two cases were asymptomatic. The main symptom was abdominal mass (53%) followed by abdominal pain (40,6%). The median size of the lesions was 24,9 cm with a median weight of 4.576,3 g. All cases were surgically removed, being necessary to remove contiguous organs in only four cases (12,5%). ConclusionsRetroperitoneal lipoma is a rare tumor which must be differentiated from WD-LPS. This is a very difficult task, being necessary to determinate MDM2 status (by FISH or MLPA), present in liposarcoma but not in lipomas, for its correct diagnosis. The treatment must be based on a complete surgical resection with negative margins.

Abstract A49: Combined targeting of the p53 and pRb pathway in neuroblastoma

Abstract Neuroblastomas account for approximately 15% of all childhood cancer deaths. Clinical complete remission is achieved in many stage 4 neuroblastoma patients, but the high risk of relapse and the accompanying treatment-resistant nature of these tumors is still a challenge. We have previously identified higher frequencies of mutations that affect both the p53 and the pRb pathway, such as the homozygous loss of CDKN2A or co-amplification of MDM2 and CDK4. In addition, both cyclin D1 and MDM2 overexpression is a common characteristic of primary neuroblastoma. These genes act upstream of pRb and p53 and hence play a major role in cell cycle regulation. To study whether these G1 checkpoint aberrations render cells more sensitive to CDK4 or MDM2 inhibition, we exposed a series of 11 cell lines to the most promising CDK4 (ribociclib, palbociclib, and abemaciclib) and MDM2 (SAR405838, HDM-201, and idasanutlin) inhibitors. These cell lines have well-defined mutational properties: homozygous CDKN2A deletion, co-amplification of CDK4 and MDM2, TP53 mutation, and MYCN amplification. Whereas sensitivity for the MDM2 inhibitors inversely correlated with TP53 mutation status, it did not correlate to other G1 checkpoint aberrations. Similarly, there was no clear correlation seen for the CDK4 inhibitors. To further explore this, we generated cell lines with inducible overexpression of CDK4, MDM2, or both. The overexpression did indeed not increase sensitivity to CDK4 or MDM2 inhibitors. Next, we were interested whether combined treatment with the most potent CDK4 and MDM2 inhibitors, abemaciclib and idasanutlin respectively, would be of added value. Cells were exposed to 10 different concentrations of both compounds and Bliss Independence values were calculated as a measure of synergy. Interestingly, combined treatment resulted in slight antagonism in the range of clinically relevant doses in most cell lines. This adverse effect was supported by cell cycle analyses, which showed a lower apoptotic fraction, as well as PARP and caspase 3 cleavage, after combined treatment, as opposed to MDM2 inhibition alone. As was previously suggested in the first clinical trials with CDK4 inhibitors, neither CDK4 nor CDKN2A status is a clear biomarker for CDK4 inhibitor sensitivity. Our results suggest that MDM2 and CDKN2A status also fail as biomarkers for MDM2 inhibitor sensitivity. Further testing is necessary to identify (other) biomarkers for these inhibitors. Moreover, the logical rationale to combine CDK4 and MDM2 inhibitors in neuroblastoma patients with both pRb and p53 pathway disturbances should be taken with precaution, as first results do not show a beneficial response. In vivo experiments to confirm this finding are currently ongoing. Citation Format: Nil Schubert, Linda Schild, Stijn van Oirschot, Kaylee Keller, Lindy Alles, Lindy Vernooij, Marloes Nulle, Emmy Dolman, Marlinde van den Boogaard, Jan Molenaar. Combined targeting of the p53 and pRb pathway in neuroblastoma [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 A49.

MDM2 dual-color in situ hybridization (DISH) aids the diagnosis of intimal sarcomas.

Intimal sarcoma is a rare malignant mesenchymal tumor arising from the intima of the great vessels and the heart, and is associated with poor outcomes. As clinico-radiological findings and pathological features are often non-specific, the diagnosis of intimal sarcoma is challenging. Recently, MDM2 amplification was reported to be a characteristic genetic event in this tumor. In the present study, we examined MDM2 status by immunohistochemistry, and by fluorescence and dual-color in situ hybridization (FISH and DISH) using intimal sarcoma (10 tumors), angiosarcoma (5), pulmonary sarcomatoid carcinoma (p-SC) (14) and chronic pulmonary thrombosis (CPT) (3) to investigate MDM2 amplification for the diagnosis of intimal sarcoma. MDM2 and CDK4 were immunopositive in all 10 intimal sarcoma tumors, and high-level amplification of MDM2 was detected in eight tumors by both FISH and DISH. The other two tumors had polysomy of chromosome 12 and overexpression of p53 protein. Although MDM2 aberrations were observed in three p-SCs (two with amplification and one with polysomy), angiosarcomas and CPTs lacked MDM2 amplification. Furthermore, there was high concordance between FISH and DISH. In conclusion, we found that MDM2 amplification strongly supports the diagnosis of intimal sarcoma, and MDM2 DISH was a concordant method and an acceptable alternative to FISH. As MDM2 amplification and p53 overexpression were mutually exclusive, disruption of the MDM2-p53 pathway may be an essential genetic event for this malignant tumor.

Abstract 3084: BI-907828: A novel, potent MDM2 inhibitor, inhibits GBM brain tumor stem cells in vitro and prolonged survival in orthotopic xenograft mouse models

Abstract Glioblastoma (GBM) is characterized by an aggressive clinical course, therapeutic resistance and striking molecular heterogeneity. GBM remains incurable with a median survival of 12-15 months post-surgery even with standard of care chemotherapy and radiation. The tumor suppressor p53 (TP53) is frequently mutated in cancer and along with its downstream effectors is inactivated in more than half of all cancers. The remaining 50% of tumors have TP53 wild-type status. However, TP53 function is frequently attenuated in these cancers by other mechanisms including amplification and overexpression of its key negative regulator MDM2. Inhibition of the protein-protein interaction between p53 and MDM2 is still a novel concept for cancer therapy. MDM2 inhibitors are designed to restore and maintain p53 activity in p53 wild-type tumors. BI-907828, currently in Phase I clinical trials, is a novel and potent MDM2 inhibitor with good oral bioavailability and pharmacokinetic properties. We investigated the efficacy of BI-907828 in p53 wild-type GBM patient-derived brain tumor stem cell (BTSC) lines with different amplification status of MDM2. BI-907828 potently reduced proliferation and viability in BTSC lines with IC50 values in the picomolar range. All BTSC lines, including lines resistant to the standard of care for GBM, temozolomide (TMZ), were sensitive to the MDM2 inhibitor irrespective of the copy number (CN) status of the MDM2 gene. Consistently, induction of cell death by BI-907828 was demonstrated in a BTSC line with an MDM2 gene amplification, as well as in another line with wild-type MDM2 CN status. PK-PD studies in SCID mice, orthotopically xenografted with BTSC lines, demonstrated that a single dose of BI-907828 resulted in strong activation of target genes. Human CDKN1a and GDF15 mRNA levels were significantly increased in brain tumor tissue in both the MDM2-amplified and non-amplified orthotopic models, suggesting that BI-907828 is able to pass the blood-brain-barrier and demonstrates on-target MDM2 inhibition in vivo. Efficacy of BI-907828 was assessed in Kaplan Meier survival studies as monotherapy or in combination with TMZ in two orthotopic BTSC xenograft models with MDM2 amplified or wild-type CN status, respectively and both with p53 wild-type status and promoter methylation of the MGMT gene. Long-term treatment with BI-907828 as monotherapy or in combination with TMZ was well tolerated in both these orthotopic BTSC xenograft models for several months. BI-907828 treatment demonstrated striking improved efficacy over vehicle control animals in both models. There was also significant survival benefit for the combinatorial treatment with TMZ in the MDM2 wild-type CN model over monotherapies alone. BI-907828 thus provides an extremely promising new therapeutic option for patients with primary brain tumors. Citation Format: Hema A. Luchman, Danielle Bozek, Xiaoguang Hao, Dorothea Rudolph, Sophia Blake, Jörg Rinnenthal, Samuel Weiss. BI-907828: A novel, potent MDM2 inhibitor, inhibits GBM brain tumor stem cells in vitro and prolonged survival in orthotopic xenograft mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3084.

Exploring ceramide metabolism as a potential target in dedifferentiated liposarcoma.

e22558 Background: Dedifferentiated liposarcomas (DDLPS) are highly aggressive mesenchymal tumors characterized by MDM2 amplification. Elevated MDM2 amplification levels are associated with tumor growth, chemoresistance, and changes in cellular metabolism. Previous work from our laboratory has demonstrated that lipid metabolism, particularly increased glycosylated ceramides (Gly-Cer), is profoundly altered as a function of MDM2 amplification. Importantly, ceramides have been identified to play a key role in proliferation arrest and apoptosis or autophagy by stabilizing p53 through the binding and disrupting the MDM2-p53 interaction. Gly-Cer clears out ceramides, thereby preventing the ceramides from performing their anti-proliferative activity. We hypothesized that restoring Gly-Cer levels would induce anti-proliferative effects in DDLPS. Methods: 6 DDLPS cell lines (MDM2-high: Lipo224, Lipo141, Lipo224B, Lipo246; MDM2-low: Lipo815, Lipo863) were selected for in vitro assessment. MDM2 levels were ascertained via Western blot and RNA-sequencing. Metabolomic (Metabolon) and lipidomic (SCIEX 5600 TripleTOF-MS) profiling were conducted on all cell lines. Inhibitory concentration 50% (IC50) of the C6-ceramide (C6C) and nanoliposomal C6-Ceramides (C6CNL) were tested in vitro by XTT . Adjusted t-tests were used to determine metabolite relevance. Results: Metabolomic/lipidomic analyses identified a correlation between MDM2 amplification status and elevated levels of glycosylated ceramides (nom: Pval < 0.001), including GlcCer_NS(d18:1/24:1), GlcCer_NS(d19:1/24:1), GlcCer_NS(d18:1/25:0), glycosyl-N-palmitoyl-sphingosine, and glycosyl-N-stearoyl-sphingosine. Further, the ratio of glycosylated to un-glycosylated ceramides in DDLPS cell was also associated with MDM2 status for Cer(d19:1/24:1) (Pval < 0.01) and borderline associated for Cer(d18:1/16:0) and Cer(d18:1/25:0) (Pval < 0.2). In vitro treatment of DDLPS cell lines with C6C resulted in a reduction of cellular viability most prominent in MDM2-high cell lines (IC50: 17 vs. 55 µM). When treated with C6CNL, all DDLPS cell lines exhibited equal sensitivity (IC50: ~9 µM). Western blots demonstrate C6CNL treatment resulted in a reduction in MDM2 expression and restoration of p53 function. Conclusions: Exogenous ceramide treatment reduces MDM2 expression, restores TP53 status, and manifests anti-proliferative activity in DDLPS. C6CNL exhibited lower effective concentrations relative to free C6 ceramide. Prospective in vivo studies are underway to confirm these findings.

MDM2 copy number increase: a poor prognostic, molecular event in esophageal squamous cell carcinoma

The present study aimed to elucidate the clinicopathological significance of molecular alterations in MDM2 in esophageal squamous cell carcinoma (ESCC). A total of 399 resected cases of ESCC were examined by dual-color in situ hybridization for MDM2 and immunohistochemistry for p53 using tissue microarrays. Clinicopathological features were correlated with the MDM2 status. Among 362 cases with a successful dual-color in situ hybridization analysis, 19 (5%) and 13 (4%) had MDM2 amplification and chromosome 12 polysomy, respectively, and these were examined as an MDM2-positive group. A comparison between amplified and polysomic cases revealed that the latter were more strongly associated with preoperative chemotherapy than the former. Sixteen (50%) of 32 MDM2-positive cases had positive results in all tissue cores examined, indicating diffuse MDM2 alterations. Cases with the diffuse alteration of MDM2 were characterized by an advanced pT stage and extensive vascular infiltration. The relationship between MDM2 copy number increases and p53 mutations was weak, with the overexpression of p53 being similarly detected in MDM2-positive and MDM2-negative cases (59% versus 49%; P = .267). Overall survival was shorter in patients with MDM2-positive ESCC than in those without MDM2 alterations (P = .033). The poor prognostic value of MDM2 alterations became more obvious when only diffusely altered cases were counted (P = .005). In conclusion, the present study revealed that MDM2 copy number increases occurred in 9% of ESCC cases, and MDM2 alterations, particularly diffuse abnormalities, were associated with a poor prognosis. MDM2-altered ESCC may achieve beneficial effects from MDM2-targeted therapy.

Extraskeletal osteosarcoma: MDM2 and H3K27me3 analysis of 19 cases suggest disease heterogeneity.

Extraskeletal osteosarcoma (ESOS) is a sarcoma in the non-skeletal tissue that directly produces neoplastic osteoid or bone. De-differentiated liposarcoma (DDLPS) and malignant peripheral nerve sheath tumour (MPNST) are the two most common types of sarcoma that can harbour heterologous osteosarcomatous differentiation. We aimed to determine the potential relationship of ESOS to DDLPS and MPNST. We investigated MDM2 and H3K27me3 status in 19 cases of ESOS, two of which contained a low-grade component. The ESOS affected deep soft tissues (n = 10), superficial soft tissues (n = 3) and organs (n = 6). Among 10 deep soft-tissue ESOS, six showed MDM2 amplification, four of which also harboured CDK4 co-amplification. Both ESOS with a low-grade component showed co-amplification for MDM2 and CDK4. Among the six organ-based ESOS three giant cell-rich ESOS showed an H3K27me3 deficiency (one in primary and two in metastatic sites). Using targeted next generation sequencing, an H3K27me3-deficient ESOS showed EED homozygous deletion, while none of the three showed alterations in NF1, CDKN2A or SUZ12 genes. During median follow-up of 20 months, all six patients with MDM2-amplified ESOS lived for 3-103 months, while two of the three patients with H3K27me3-deficient ESOS died from this disease in 4 and 20 months, respectively. We demonstrate that ESOS may include at least two small subsets: an MDM2-amplified deep soft-tissue ESOS (which may be related to DDLPS) and an H3K27me3-deficient organ-based ESOS (which is probably unrelated to MPNST). Larger studies are required to validate the present observations and investigate the clinical implications of such subcategorisation.

The determination of stage in nonmuscle urothelial carcinoma: Staining pattern of caspase-8.

Urothelial carcinoma (UC) is one of the most frequent epithelial tumors worldwide. We aimed to investigate the protein expressions of caspase-8, p53, murine double minute 2 (mdm2), and p14ARF in nonmuscle UCs and to correlate the findings with clinicopathological characteristics. A total of 50 patients who had pTa and pT1 tumors were analyzed. The protein expressions of caspase-8, p53, mdm2, and p14ARF were analyzed by immunohistochemistry. Chi-square test was done using SPSS version 16.0 (SPSS, Inc., Chicago, IL, USA). Cytoplasmic caspase-8 expression was significantly higher in pT1 UCs while nuclear caspase-8 expression was significantly higher in pTa UCs (P = 0.005 and P = 0.011, respectively). Cytoplasmic caspase-8 expression was also higher in high-grade UCs (P = 0.035). The expression of p53, mdm2, and p14ARF was not also related with pathological stage or grade (P > 0.05 for all). The p14ARF expression was related with nuclear caspase-8 expression in most of the patients. Complete agreement among nonmuscle UCs for immunohistochemical expression of p14 and nuclear caspase-8 was seen in 41 cases, and the pairwise kappa agreement value was substantial (κ =0.614). The patients who had recurrence were positive for both p53 and mdm2 or either p53 or mdm2 (P = 0.025). These results suggested that the staining pattern of caspase-8 might be helpful for determining of the stages in nonmuscle UC. It was also showed that the expression status of p53 and mdm2 were related with the recurrence.

Abstract 3861: Development of testicular cancer patient derived xenograft models to test combination therapies targeting PI3K/Akt and MDM2

Abstract Metastatic testicular cancer (TC) is highly sensitive to cisplatin-based chemotherapy. However, patients with advanced disease in the poor prognosis group only have a 50% 5-year survival resulting from chemoresistance. Previous data showed that both the PI3K/Akt pathway and the MDM2/p53 axis are involved in cisplatin resistance of TC cells. In this study we aim to establish and characterize TC patient-derived xenografts (PDX) in order to investigate PI3K/Akt and/or MDM2 inhibition as possible treatment options for TC. Excised and minced (8mm3) primary TC tissue was implanted subcutaneously into male NSG mice to obtain a first generation PDX model (F1). Animals were sacrificed when tumor volume exceeded 1500 mm3. The tumor was harvested and minced. Small pieces (8mm3) were implanted in a second generation (F2), as well as stored in FCS-5% DMSO to establish a biobank. In a panel of TC cell lines, combination treatment of Akt inhibition with or without cisplatin or Nutlin-3a was performed. Cleaved caspase-3 staining was assessed to measure apoptosis, and western blot was used to determine the effect of different treatments on sub-cellular localization and phosphorylation status of MDM2. We have established 3 TC PDX models. Histology of the primary tumors included the following subtypes: mixed germ cell tumors with embryonal- and yolk sac carcinoma and teratoma components. Engraftment efficacy was 100% and tumor growth initiated within 4-5 weeks after implantation. Interestingly, we successfully implanted a biopsy taken from a metastatic lesion of a patient presenting with progressive refractory disease after receiving standard chemotherapy. Histology of the different PDX generations was comparable to the patient material, although a loss of the teratoma component was observed. In our cell line panel, Akt inhibition using MK2206 sensitized the acquired cisplatin resistant cell line TeraCP towards cisplatin treatment, whereas an additive effect was observed in other cell lines. Combination of Akt and MDM2 inhibition was highly synergistic in apoptosis induction in all cell lines. No relation was observed between the synergistic effect of Akt combined with MDM2 inhibition and sub-cellular localization or phosphorylation levels of MDM2. Taken together, we have successfully established 3 TC PDX models, including a chemo-resistant model. The biobank is currently being expanded. Combined Akt and MDM2 inhibition resulted in synergistic induction of apoptosis, and these combinations or other novel therapies will be tested in established TC PDX models. Supported by Dutch Cancer Society grant RUG 2014-6691 and CONACYT grant 381543 Citation Format: Gerda de Vries, Ximena Rosas-Plaza, Marcel A. van Vugt, Albert J.H. Suurmeijer, Jourik A. Gietema, Steven de Jong. Development of testicular cancer patient derived xenograft models to test combination therapies targeting PI3K/Akt and MDM2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3861. doi:10.1158/1538-7445.AM2017-3861

P53 expression and relationship with MDM2 amplification in breast carcinomas.

Carcinoma of the breast, like other malignancies, is a genetic disease with multiple genetic events leading to the malignant phenotype. p53 mutations are the most common genetic events in human cancer. Inactivation of p53 can be a result of mutation in gene sequence. One of the main structures that regulate p53 stabilization is MDM2. It suppresses p53 transcriptional activation by recognizing transactivation domain of p53. The loss of MDM2 function on p53 regulation results in deprivation of p53 tumor suppressor ability. Single nucleotide polymorphisms (SNP309 T->G exchange) or MDM2 amplification has been proposed to play a role in this issue. In the present study, our aim is to analyze p53 and MDM2 status and investigate their interactions in human sporadic breast carcinoma. The study groups were separated according to their molecular classifications. In each group, histologic type of the tumor, conventional prognostic parameters, p53, and MDM2 interactions were compared statistically. Tumors are divided into 4 subtypes due to estrogen and progesterone receptor status, HER-2, and Ki-67 proliferation index results. According to this classification, 23 cases are in the luminal A, 32 cases are in the luminal B, 15 cases are in the HER-2 positive, and 22 cases are in the triple-negative group, with a total of 92 cases. p53 expression is low in luminal breast carcinomas than HER-2 and triple-negative subtypes. MDM2 amplification frequency was found to be 5.4% in total. MDM2 gene amplification does not have a significant role in breast carcinogenesis, but other possible mechanisms may play a role in its inactivation.

P53 and MDM2 Over-expression and Five-year Survival of Kidney Cancer Patients Undergoing Radical Nephrectomy--Iranian Experience.

Relatively little is known with certainty about the status and role of p53 or MDM2 in predicting prognosis and survival of renal cell carcinoma. The present study aimed to determine the value of P53 and MDM2 over-expression, alone and simultaneously, to predict five-year survival of patients with kidney cancer in Iran. Patients with kidney cancer referred to Hasheminejad Kidney Center between 2007 and 2009, underwent radical nephrectomy and had pathology reports of clear cell, papillary or chromophobe renal cell carcinoma were included in our cohort study. Other histological types of renal cell carcinoma were not included. The patients with missed, incomplete or poor quality paraffin blocks were also excluded. Overall ninety one patients met the inclusion and exclusion criteria. To assess the histopathological features of the tumor, immunohistochemical (IHC) staining of formalin fixed, paraffin-embedded tumor samples were performed. The five-year survival was determined by the patients' medical files and telephone following-up. In total, 1.1% of all samples were revealed to be positive for P53. Also, 20.8% of all samples were revealed to be positive for MDM2.The patients were all followed for 5 years. In this regard, 5-year mortality was 30.5% and thus 5-year survival was 85.3%. According to the Cox proportional hazard analysis, positive P53 marker was only predictor for patients' 5-year survival that the presence of positive p53 increased the risk for long-term mortality up to 2.8 times (HR=2.798, 95%CI: 1.176-6.660, P=0.020). However, the presence of MDM2 could not predict long-term mortality. In this regard, analysis by the ROC curve showed a limited role for predicting long-term survival by confirming P53 positivity (AUC=0.610, 95%CI: 0.471-.750, P=0.106). The best cutoff point for P53 to predict mortality was 0.5 yielding a low sensitivity (32.0%) but a high specificity (97.9%). In similar analysis, measurement of MDM2 positivity could not predict mortality (AUC=0.449, 95%CI: 0.316-.583, P=0.455). The simultaneous presence of both P53 and MDM2 markers in our population is a rare phenomenon and the presence of these markers may not predict long-term survival in patients who undergoing radical nephrectomy.

Pre-clinical evaluation of the MDM2-p53 antagonist RG7388 alone and in combination with chemotherapy in neuroblastoma

Neuroblastoma is a predominantly p53 wild-type (wt) tumour and MDM2-p53 antagonists offer a novel therapeutic strategy for neuroblastoma patients. RG7388 (Roche) is currently undergoing early phase clinical evaluation in adults. This study assessed the efficacy of RG7388 as a single-agent and in combination with chemotherapies currently used to treat neuroblastoma in a panel of neuroblastoma cell lines. RG7388 GI50 concentrations were determined in 21 p53-wt and mutant neuroblastoma cell lines of varying MYCN, MDM2 and p14(ARF) status, together with MYCN-regulatable Tet21N cells. The primary determinant of response was the presence of wt p53, and overall there was a >200-fold difference in RG7388 GI50 concentrations for p53-wt versus mutant cell lines. Tet21N MYCN+ cells were significantly more sensitive to RG7388 compared with MYCN- cells. Using median-effect analysis in 5 p53-wt neuroblastoma cell lines, selected combinations of RG7388 with cisplatin, doxorubicin, topotecan, temozolomide and busulfan were synergistic. Furthermore, combination treatments led to increased apoptosis, as evident by higher caspase-3/7 activity compared to either agent alone. These data show that RG7388 is highly potent against p53-wt neuroblastoma cells, and strongly supports its further evaluation as a novel therapy for patients with high-risk neuroblastoma and wt p53 to potentially improve survival and/or reduce toxicity.