Murine Double Minute-2 Gene Amplification Research Articles

Abstract P6-10-10: Genetic alterations in breast cancer associated with MDM2 dependency and sensitivity to the MDM2 inhibitor milademetan

Abstract Background: Murine double minute 2 (MDM2), a potent negative regulator of p53, promotes tumorigenesis if dysregulated. MDM2 dysregulation occurs via different mechanisms, including MDM2 gene amplification, MDM2 overexpression, and loss of cyclin-dependent kinase inhibitor 2A (CDKN2A), which encodes the MDM2 regulator p14ARF. Combined inactivation of MDM2 and GATA3 in hormone receptor-positive (HR+) breast cancer is lethal to the cell. Pharmacologic inhibition of MDM2 is a rational therapeutic strategy for MDM2-dependent, TP53 wildtype (WT) tumors, including tumors with MDM2 amplification or CDKN2A loss, and GATA3-mutant HR+ breast cancers. We determined the frequency and associated characteristics of genetic alterations of MDM2-dependent breast cancers, and evaluated sensitivity of these tumors to the small-molecule MDM2 inhibitor milademetan (RAIN-32). Methods: Genomic data were obtained from three datasets: METABRIC; TCGA PanCancer Atlas, GDC v23.0 (April 2020); AACR Genie, v11. Among TP53 WT breast cancer samples from each dataset, the frequency of GATA3 frameshift mutations, MDM2 amplification (copy number [CN] ≥12), and CDKN2A homozygous loss was determined individually and as co-alterations. The antitumoral activity of milademetan was evaluated in a GATA3-mutant, TP53 WT HR+ breast cancer cell line (MCF7 GATA3 G336fs*17), a breast xenograft model (MCF7 GATA3 G335fs), and ex vivo in MDM2-amplified patient-derived breast cancer organoids (CTG-2810, ER+/PR+/HER2–, MDM2 CN 8). Results: Genetic alteration frequencies in TP53 WT breast cancers by dataset are shown in the Table. GATA3 frameshift mutations (7.3–11.7%), MDM2 amplification (0.3–1.1%), and CDKN2A loss (0.2–1.2%) occurred across breast tumors, but were found with highest frequencies in HR+ tumors. Co-alteration frequencies in TP53 WT breast cancers across the aforementioned datasets were < 1%: GATA3 mutations/MDM2 CN ≥12 (0.2–0.3%); GATA3 mutations/CDKN2A loss (0.1–0.2%); MDM2 CN ≥12/CDKN2A loss (0%). Mean MDM2 expression (log2 (TPM+1)) in HR+ breast cancers (TCGA) were: GATA3 mutations, 5.12; CDKN2A loss, 5.88; MDM2 CN ≥12, 8.13, TP53 WT without these alterations, 4.78; mutant TP53, 4.35. A GATA3-mutant ER+ breast cancer cell line was sensitive to milademetan in vitro (IC50 126 nM). Milademetan 100 mg/kg displayed antitumor activity in GATA3-mutant HR+ breast xenograft and PDX models (p< 0.05 vs. vehicle). Milademetan also displayed activity in MDM2-amplified HR+ breast cancer organoids (IC50 0.2 μM). In a phase I study (NCT01877382), a patient with heavily pretreated MDM2-amplified breast cancer (MDM2 CN 16.8) had tumor shrinkage (18.2%) and PFS of 7.3 months with milademetan (orally 260 mg 3/14 days). Conclusions: The frequency of genetic alterations potentially targetable by MDM2 inhibition among TP53 WT breast cancers (i.e., GATA3 mutations, MDM2 amplification, and CDKN2A loss) is greatest in the HR+ subset, and these genetic biomarkers are associated with higher MDM2 expression. Preclinical data show that the MDM2 inhibitor milademetan has antitumor activity in GATA3-mutant and MDM2-amplified HR+ breast cancers, and support the clinical evaluation of milademetan in these tumors. Two clinical trials of milademetan – MANTRA-2 (Phase 2 basket study in solid tumors with TP53 WT and MDM2 CN ≥12; NCT05012397) and MANTRA-4 (Phase 1 study of milademetan + atezolizumab in solid tumors with CDKN2A loss) – are enrolling patients or in planning. Table. Citation Format: Francois-Clement Bidard, Diana Bello Roufai, Arielle J. Medford, Vijaya Tirunagaru, Robert C. Doebele, Aditya Bardia. Genetic alterations in breast cancer associated with MDM2 dependency and sensitivity to the MDM2 inhibitor milademetan [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-10.

MDM2 gene a mplification in s kin c ancer p atients

Skin cancer: Skin cancer is the uncontrolled proliferation of abnormal skin cells; skin cancer is classified into non-melanoma (squamous cell carcinoma and basal cell carcinoma) and melanoma. Approximately, 90% of skin cancers are caused by ultraviolet radiation, because of ultraviolet radiation emits to damage of skin cells DNA resulting in genetic mutations and formation of skin cancer.
 Materials and methods: This study evaluated paraffin blocks from 17 specimen randomly selected from patients with skin cancer, and 6 specimens from patients non cancer patients as control group for comparison .This study was carried out in Laboratories of College of Science, Wasit University and AL Zahraa and ALKarama Teaching Hospitals, from October 2014 to April 2015. In this study MDM2( murine double minute 2) gene amplification was assessed by chromogenic in situ hybridization (CISH) technique.
 Results: This study showed that there was no significant difference between skin cancer patients and control group in relation to MDM2 amplification (p>0.05), but presence of highly significant difference between patients of skin cancer and control group in relation to amplification intensity of MDM2 (p<0.05). Moreover, and according to histological type, our finding found significant difference between the types of skin cancer in relation to MDM2 amplification (p<0.05). Additionally, there were significant differences between patients of basal cell carcinoma and squamous cell carcinoma and control group in relation to MDM2 amplification(p<0.05).
 Conclusion: We conclude that MDM2 amplification was high in types of skin cancer (BCC and SCC) because of MDM2 amplification in keratocytes of the epidermis may be associated with entry of these cells into the differentiation program.

Detection of MDM2 gene amplification on tissue microarray-based Fluorescence In-Situ Hybridization (FISH) in well-differentiated and dedifferentiated liposarcomas, displaying a wide morphological spectrum: A validation study at a tertiary cancer referral centre.

Liposarcomas including atypical lipomatous tumors (ALT)/well-differentiated liposarcomas (WDLPS) and dedifferentiated liposarcomas (DDLPSs) display a histomorphological spectrum with their several diagnostic mimics. Murine double minute 2(MDM2) gene amplification characterizes ALT/WDLPS and DDLPS. Presently, there is no documented study from our subcontinent on the validation of MDM2 gene testing in these tumors. Twenty-eight cases, diagnosed as ALT/WDLPS (n = 5) and DDLPSs (n = 23), along with 10 other tumors were tested for MDM2 gene amplification, using fluorescence in situ hybridization (FISH) on tissue microarrays (TMAs). Fourteen cases, diagnosed as ALT/WDLPS and DDLPS, along with 49 other tumors were tested for MDM2 immunostaining. Twenty tumors were tested for p16INK4a immunostaining. FISH was interpretable in 25 (89.2%) cases. Among the 20 cases diagnosed as DDLPSs, 19 displayed MDM2 gene amplification. Among the 5 cases diagnosed as ALT/WDLPS, four showed MDM2 gene amplification. Finally, 19 cases were confirmed as DDLPS and 4 as ALT/WDLPS. Furthermore, 7/19 cases confirmed as DDLPS and all 4 cases as ALT/WDLPS tested for MDM2 immunostaining, displayed its diffuse immunoexpression, while a single case of DDLPS showed its focal immunostaining. None of the 49 control cases displayed diffuse MDM2 immunoexpression. ALL 16 DDLPSs and 4 cases of ALT/WDLPS displayed p16INK4a immunostaining. The sensitivity for diffuse MDM2 immunostaining was 87.5% in cases of DDLPS, 100% in ALT/WDLPS, and specificity was 100%. The sensitivity for MDM2 gene amplification was 94.7% in cases of DDLPS and 100% in cases of ALT/WDLPS. The sensitivity for p16INK4a was 100%. This constitutes the first sizable study on MDM2 testing in ALT/WDLPS and DDLPS from our subcontinent using TMAs. MDM2 gene amplification testing continues as the diagnostic gold standard for ALTs/WDLPSs and DDLPSs and is useful in cases of diagnostic dilemmas. Diffuse MDM2 (IF2 clone) and p16INK4a immunostaining, together seem useful for triaging cases for FISH.

Pathological prognostic factors of retroperitoneal liposarcoma: comprehensive clinicopathological analysis of 124 cases.

Retroperitoneal liposarcoma (RLS) is a type of rare malignant tumor. The aim of the present study was to study the correlations between the clinicopathological characteristics and the prognostic factors of RLSso as to delineate independent pathological indicators affecting prognosis. The RLS pathological specimens were collected at Beijing Shijitan Hospital Affiliated to Capital Medical University from 2008 to 2018, and were used to study the histopathological features, including tumor size, histological types, lymphatic metastasis, tumor emboli in the blood and lymph vessels, and nerve invasion; immunohistochemical features, including Ki-67 and P53 proteins; and molecular pathological features, such as MDM2 (murine double minute2) gene amplification. Clinical treatment and follow-up data were collected, and the survival rate was calculated by Kaplan-Meier method. Univariate and multivariate survival analyses were performed to study the independent pathological factors. Of the 124 RLS cases, 63 were males (50.8%) and 61 were females (49.2%), with a median age of 52 years (range, 22-80 years). There were 51 cases (41.1%) of well-differentiated liposarcoma, 73 cases (57.3%) of dedifferentiated liposarcoma (DDL); 3 cases (2.4%) with lymph node metastases; 4 cases (3.2%) with tumor emboli in the vessels; 21 cases (16.9%) with Ki-67 label index <20% and 103 cases (83.1%) with Ki-67 label index ≥20%; and 81 cases (65.3%) with P53 <60% and 43 cases (24.7%) with P53 ≥60%. Fluorescence in situ hybridization studies revealed MDM2 gene amplification. Univariate analysis found the following pathological factors were correlated with prognosis: histopathological classification, recurrence, mitosis, tumor necrosis, Ki-67 label index, and P53. Multivariate analysis by Cox proportional hazard model found the following three independent pathological prognostic factors: age [hazard ratio (HR): 1.798, P=0.018], tumor site (HR: 1.263, P=0.002), tumor necrosis (HR: 1.984, P=0.024), and recurrence (HR: 2.161, P=0.049). Age, tumor necrosis, tumor site, and recurrence could be independent pathological prognostic factors for RLS.

MDM2 gene amplification as selection tool for innovative targeted approaches in PD-L1 positive or negative muscle-invasive urothelial bladder carcinoma

AimsAccording to The Cancer Genome Atlas (TCGA), around 9% of bladder carcinomas usually show abnormalities of the murine double minute 2 (MDM2) gene, but a few studies have been investigated...

Nuclear expression of MDM2 in hibernoma: a potential diagnostic pitfall.

Hibernoma is a rare benign adipocytic tumor composed of a proliferation of brown and white fat cells varying in their proportions. The tumor may also contain fat cells resembling lipoblasts, which makes it difficult to distinguish it from atypical lipomatous tumor/well differentiated liposarcoma (ALT/WDLS). Although nuclear expressions of murine double minute 2 (MDM2) and cyclin-dependent kinase 4 (CDK4) are widely used as immunohistochemical surrogate markers for ALT/WDLS, the utility of these proteins in distinguishing between hibernoma and ALT/WDLS still remains to be elucidated. We evaluated immunohistochemical expressions of MDM2 and CDK4 in 10 hibernomas expressing uncoupling protein-1 (UCP-1), a mitochondrial protein transporter consistently expressed in brown fat cells, and lacking MDM2 gene amplification, which was analyzed by fluorescence in situ hybridization (FISH). In contrast to the data previously obtained, nuclear expression of MDM2 was observed in 100% (10/10 cases) of the hibernomas irrespective of the proportion of brown fat cells, whereas no cases were positive for CDK4. The tumors also showed almost concurrent expression of p53 (in 9/10 cases) and ubiquitin-specific-processing protease 7 (USP7) (in 10/10 cases), which deubiquitinates and stabilizes MDM2, potentially resulting in its nuclear expression without MDM2 gene amplification. MDM2 expression may thus be a diagnostic pitfall for hibernoma particularly in differentiating it from ALT/WDLS.

Cardiac Intimal Sarcoma with Complete Response to Thoracic Radiotherapy- A Case Report

Objectives: Sarcomas are the most common primary, cardiac malignant tumours but are, nevertheless, rare (0.001-0.03% of all tumours [1-4]. Intimal sarcomas characterised by Murine Double Minute 2 (MDM2) gene amplification originate from large vessels, but from the heart only very rarely. The prognosis of primary cardiac sarcomas is very poor; no evidence-based management guidelines are available. Surgery is the first-line treatment but is not possible in over 50% of cases. Radiotherapy ensures efficient local control and is indicated as neoadjuvant, adjuvant, and palliative treatment. Materials and Methods: We report the case of a 59-year-old female with a metastatic, cardiac intimal sarcoma identified after symptomatic metastasis to the lower limbs. Imaging revealed a hypermetabolic cardiac mass emerging from the left atrium. Adductor muscle biopsy identified an intimal sarcoma metastasis exhibiting MDM2 gene amplification; the primary site was cardiac. We first prescribed anthracycline chemotherapy and irradiation of the cardiac mass. She received 50 Gy into the planned target volume including the atrial mass (2 Gy per day 5 days per week) delivered via three-dimensional external-beam radiotherapy. Results: The irradiated cardiac mass did not grow during follow-up, rather, stabilising and then gradually decreasing in size, associated with reduced hypermetabolism. Seven months after radiotherapy ceased, she achieved a complete cardiac response, but the metastatic lesions progressed. Her general condition improved. The heart failure signs disappeared and her symptoms became less serious. Conclusion: Radiotherapy should be favoured for patients with cardiac or intimal sarcomas featuring MDM2 amplification. The tumour seems to be radiosensitive, although anthracycline may also have been helpful. Radiotherapy is a valuable tool and could reduce mortality caused by local heart failure.

1732TiP - A phase II biomarker-driven study evaluating the clinical efficacy of an MDM2 inhibitor, milademetan, in patients with intimal sarcoma, a disease with a high unmet need

BackgroundIntimal sarcoma is an extremely rare, high-grade malignant neoplasm arising in the tunica intima of large blood vessels, most frequently the pulmonary arteries and aorta. In human tumors that retain wild-type p53 protein, p53 activity is often inhibited by interaction with murine double minute 2 (MDM2). As MDM2 amplification is found in over 70% of intimal sarcomas, inhibition of MDM2 could provide clinical benefit in this patient population. Milademetan is a novel specific small-molecule inhibitor of MDM2 that disrupts the MDM2-p53 interaction in tumor cells. A cell-free study using recombinant MDM2 and p53 proteins demonstrated inhibition of MDM2-p53 binding by milademetan (IC50: 5.57nM). Treatment with milademetan resulted in a concentration-dependent increase of p21 and PUMA mRNA levels in an osteosarcoma cell line harboring MDM2 gene-amplification. Milademetan also inhibited cell growth in different cell lines with wild-type p53 (GI50s: 0.043 - 0.276μM), but not p53 mutant or null cell lines (GI50s: >10μM). We focus on targeting MDM2-p53 axis with milademetan in patients with intimal sarcoma. Trial designThis is a phase II, open-label, 2-part study at a single site. Eligible patients will be at least 18 years old with intimal sarcoma who also have MDM2 amplification (≧4-fold). Part 1 (safety lead-in cohort) will enroll 3 subjects to evaluate the safety, tolerability, and pharmacokinetics (PK) of milademetan (260mg qdx3 every 14 days twice in a 28-days cycle). Part 2 of the study will enroll an open-ended number of subjects. Overall, enrollment of≥5 subjects with advanced intimal sarcoma is planned in the next 3 years. The primary endpoint of the study is objective response rate which will be assessed in all subjects enrolled in Parts 1 and 2 who have received milademetan. Due to the absence of any effective treatment for intimal sarcoma, at least 1 patient with objective response will be regarded as clinically meaningful. Secondary endpoints include safety, pharmacokinetics, disease control rate, progression-free survival, and overall survival. Enrollment began in Dec 2018. As of 1 Apr 2019, 3 subjects have been enrolled into Part 1. Clinical trial identificationJMA-IIA00402. Legal entity responsible for the studyNational Cancer Center. FundingJapan Agency for Medical Research and Development. DisclosureK. Yonemori: Honoraria (self): Taiho; Honoraria (self): Novartis; Honoraria (self): Eisai; Honoraria (self): Pfizer; Honoraria (self): ONO. T. Shimizu: Advisory / Consultancy: Takeda Oncology; Honoraria (self): Ono Pharmaceutical; Honoraria (institution): Ono Pharma Taiwan CO., LTD.; Honoraria (self): Boehringer Ingelheim; Honoraria (self): Taiho Pharmaceutical ; Honoraria (self): Chugai Pharmaceutical; Research grant / Funding (self): Takeda Oncology; Research grant / Funding (self): PharmaMar; Research grant / Funding (self): Bristol-Myers Squibb Japan; Research grant / Funding (self): Daiichi Sankyo,; Shareholder / Stockholder / Stock options: SymBio Pharmaceuticals; Research grant / Funding (self): Five Prime Therapeutics; Research grant / Funding (self): 3D Medicine; Research grant / Funding (self): Chordia Therapeutics; Research grant / Funding (self): AbbVie; Research grant / Funding (self): AstraZeneca; Research grant / Funding (institution): Eli Lilly; Research grant / Funding (self): Novartis. A. Hirakawa: Advisory / Consultancy: Ono Pharmaceuticals Co., Ltd.; Advisory / Consultancy: Kissei Co., Ltd.; Advisory / Consultancy: AbbVie GK; Advisory / Consultancy: Nippon Boehringer Ingelheim Co., Ltd.; Advisory / Consultancy: Astellas Pharm Inc.; Advisory / Consultancy: Nippon ShinyakuCo., Ltd.; Advisory / Consultancy: Sumitomo Dainippon Pharma Co., Ltd.; Advisory / Consultancy: Torii Pharmaceutical Co. Ltd. S. Fujitani: Full / Part-time employment: DaiichiSankyo. N. Yamamoto: Research grant / Funding (self): Astellas; Speaker Bureau / Expert testimony, Research grant / Funding (self): Chugai; Advisory / Consultancy, Research grant / Funding (self): Eisai; Research grant / Funding (self): Taiho; Speaker Bureau / Expert testimony, Research grant / Funding (institution): BMS; Speaker Bureau / Expert testimony, Research grant / Funding (self): Pfizer; Research grant / Funding (self): Novartis; Speaker Bureau / Expert testimony, Research grant / Funding (self): Eli Lilly; Research grant / Funding (self): AbbVie; Honoraria (self): Daiichi-Sankyo; Research grant / Funding (self): Bayer; Advisory / Consultancy, Research grant / Funding (self): Boehringer Ingelheim; Research grant / Funding (self): Kyowa-Hakko Kirin; Advisory / Consultancy, Research grant / Funding (self): Takeda; Speaker Bureau / Expert testimony, Research grant / Funding (self): ONO; Research grant / Funding (self): Janssen Pharma; Research grant / Funding (self): MSD; Research grant / Funding (self): Merck; Advisory / Consultancy: Otsuka; Advisory / Consultancy: Cimic. Y. Fujiwara: Honoraria (self): Astellas; Honoraria (self): Daiichi-Sankyo; Honoraria (self): BMS; Honoraria (self): SRL. All other authors have declared no conflicts of interest.

Duplication of chromosome segment 12q13-15 in a lipomatous tumor with minimal nuclear atypia: A case report.

Ordinary lipoma is cytogenetically characterized by structural rearrangements, particularly translocations, of 12q13-15. By contrast, atypical lipomatous tumors exhibit supernumerary ring or giant marker chromosomes that are composed mainly of amplified material from the 12q13-15 chromosome segment. The present study describes the cytogenetic and molecular cytogenetic findings from a lipomatous tumor with minimal nuclear atypia that was identified in a 49-year-old female patient. Magnetic resonance imaging of the right shoulder revealed a 13-cm fatty mass in the subcutaneous layer that possessed only pencil-line septa. Contrast-enhanced fat-suppressed T1-weighted images demonstrated faint enhancement. A marginal excision was performed. Histologically, the tumor was composed of lobules that consisted of mature adipocytes separated by thin fibrous septa. There was minimal nuclear atypia in certain cells, and a small number of binucleated cells were also observed within the tumor. Immunohistochemically, the tumor cells did not reveal the expression of murine double-minute 2 (MDM2). Cytogenetic analysis revealed a complex karyotype with several numerical and structural alterations, including 12q rearrangements. Spectral karyotyping demonstrated a duplication of chromosome segment 12q13-15. Interphase fluorescence in situ hybridization analysis revealed no MDM2 gene amplification. The present case indicates that duplication of 12q may be associated with minimal nuclear atypia in a subset of lipomatous tumors.

MDM2 gene amplification in esophageal carcinoma.

Esophageal cancer (EC) is one of the most common malignancies diagnosed in the Western world with an increasing incidence noted for esophageal adenocarcinoma (EAC). Despite improvements in staging, surgical procedures and postoperative treatments, the overall survival of patients with EC remains low. Murine double minute‑2 (MDM2) acts as an oncogene by inducing the degradation of the tumor‑suppressor protein TP53. In order to evaluate the MDM2 gene amplification status in EAC and squamous cell carcinoma (SCC), we established a quantitative PCR (qPCR) assay, screening a total of 127 esophageal carcinoma cases for MDM2 amplification. Esophageal carcinoma cases with enhanced MDM2 gene copy numbers were further analyzed by fluorescence in situ hybridisation (FISH) and MDM2 immunostaining. Among a total of 23 specimens (18%), identified by qPCR to possess elevated MDM2 gene copy numbers, one third (6.3%) showed a cluster‑like fluorescence pattern by FISH analyses and marked MDM2 protein immunostaining. MDM2 gene amplifications did not correlate with the occurrence of TP53 mutations. Due to the high therapeutic relevance of MDM2 overexpression, but the high cost of FISH, we suggest a primary screening of MDM2 copy number variations by qPCR, followed by detailed FISH analysis of the identified ECs.

Prognostic relevance of Fédération Nationale des Centres de Lutte Contre le Cancer grade and MDM2 amplification levels in dedifferentiated liposarcoma: a study of 50 cases

Dedifferentiated liposarcoma represents a form of liposarcoma composed of a non-lipogenic sarcoma associated with well-differentiated liposarcoma. The prognostic significance of histological grading of the dedifferentiated component remains to be elucidated due to vague grading criteria employed in previous studies. Molecular markers of tumor behavior, including amplification levels of murine double minute-2 (MDM2) and cyclin-dependent kinase-4 (CDK4) genes, have been explored in a limited number of cases. Here we investigate whether ‘Fédération Nationale des Centres de Lutte Contre le Cancer' (FNCLCC) grade and MDM2 gene amplification levels have prognostic value in dedifferentiated liposarcoma in terms of local recurrence and disease-specific survival. Fifty cases were retrieved, reviewed and FNCLCC grade was scored for the dedifferentiated component. Testing for MDM2 gene amplification was performed by fluorescence in situ hybridization. Amplification was categorized as high level (≥20 copies) and as low level (<20 copies). Follow-up data was obtained through chart review. Log-rank test and Cox proportional hazard models were used to determine the effect of grade and level of MDM2 amplification on outcomes. Our series includes 50 patients (male n=28, female n=22) with an average age of 63 years (range, 28–88) and a median follow-up of 28 months (range, 2–120). Tumors were graded as grade 1 (6%), grade 2 (58%), and grade 3 (36%). When adjusted for age, sex, site, tumor size, and margin status, grade 3 patients had a higher recurrence rate than grades 1 and 2 (HR=2.07, 95% CI: 1.24, 7.62; P=0.015). Patients with high-level MDM2 amplification had higher recurrence rate on univariate analysis (P=0.028), but not on multivariate analysis (HR=1.69, 95% CI: 0.73, 3.94; P=0.221). FNCLCC grade 3 dedifferentiation confers a worse prognosis in dedifferentiated liposarcoma in terms of local recurrence. MDM2 amplification level remains a useful diagnostic tool in dedifferentiated liposarcoma, but has no prognostic value in terms of local recurrence.

Correlation of TP53 and MDM2 genotypes with response to therapy in sarcoma

Relatively few sarcomas harbor TP53 (tumor protein p53) mutations, but in many cases, amplification of MDM2 (murine double minute 2) effectively inactivate p53. The p53 pathway activity can also be affected by normal genetic variation. The mutation status of TP53 and expression of MDM2, TP53, and their genetic variants SNP309 and R72P (Arg72Pro) were investigated in 125 sarcoma patient samples and 18 sarcoma cell lines. Association of the different genotypes and gene aberrations with chemotherapy response and survival, as well as response to MDM2 antagonists in vitro was evaluated. Twenty-two percent of the tumors had mutant TP53 and 20% MDM2 gene amplification. Patients with wild-type TP53 (TP53(Wt) ) tumors had improved survival (P < .001) and TP53(Wt) was an independent prognostic factor (hazard ratio = 0.41; 95% confidence interval = 0.23-0.74; P = .03). Interestingly, there was a trend toward longer time to progression after chemotherapy for tumors with the apoptosis-prone p53 variant R72 (P = .07), which was strongest with doxorubicin/ifosfamide-based regimens (P = .01). Liposarcomas had low R72 frequency (33% versus 56%), but increased levels of MDM2 and MDM4 (51% and 11%, P < .001). MDM2 overexpression on a TP53(Wt) background predicted better response to MDM2 antagonist Nutlin-3a, irrespective of R72P or SNP309 status. Improved survival after chemotherapy was found in patients with TP53(Wt) tumors harboring the R72 variant. MDM2 overexpression in TP53(Wt) tumors predicted good response to MDM2 antagonists, irrespective of R72P or SNP309 status. Thus, detailed TP53 and MDM2 genotype analyses prior to systemic therapy are recommended.

Evaluation of immunohistochemical expression of MDM2 protein in comparison with MDM2 gene amplification in diagnosing lipomatous tumors

ObjectiveOur goal was to compare the immunohistochemical detection of murine double minute 2 (MDM2) protein and MDM2 gene amplification in diagnosing adipose tissue tumors.Materials and methodsMDM2 protein expression was examined by immunohistochemistry in formalin-fixed paraffin-embedded tissue sam

MDM2 gene amplification and protein expressions in colon carcinoma: is targeting MDM2 a new therapeutic option?

The aim of this study was to investigate murine double minute-2 (MDM2) gene copy number changes in colon carcinoma and to correlate these findings with an immunohistochemical analysis of MDM2 protein expression and histopathologic prognostic indicators of the tumors. The study included 80 cases of sporadic colon carcinomas. MDM2 protein expression was assessed by immunohistochemistry, and MDM2 gene status by fluorescence in situ hybridization. MDM2 gene amplification was detected in 18% of the 80 cases examined. A strong correlation was found between MDM2 gene amplification and the presence, intensity, and staining proportion of cytoplasmic MDM2 protein expression (p = 0.01). No correlation was found between MDM2 gene amplification and the well-established histopathologic prognostic factors. Given the correlation with gene amplification, we clearly demonstrated that cytoplasmic expression of MDM2 protein is true and relevant and that this finding has to be taken into account when immunohistochemistry would be used as a screening for MDM2 gene amplification in the near future. Targeting MDM2 could be a new approach in colon cancer therapy. The amplification status could be a predictive factor of the response to MDM2-targeted therapy.

MDM2 gene amplification and transcript levels in human sarcomas: relationship to TP53 gene status.

Alterations of the TP53 tumor suppressor gene appear to be implicated in the tumorigenesis and progression of several types of human cancer, including different histologic subtypes of sarcomas. The MDM2 (murine double minute-2) gene encodes a nuclear phosphoprotein that may interact with both mutant and wild-type p53 proteins, thereby inhibiting p53-mediated transactivation in a dose-dependent manner. Recently it has been suggested that mdm2 and p53 proteins are components of an autoregulatory loop in which the MDM2 gene is transactivated by p53. Our purpose was to examine the frequency of MDM2 amplifications in larger panels of sarcomas, determine if the mRNA level could be elevated in tumors without concomitant gene amplification, and relate MDM2 findings to the TP53 status of the tumors. Sarcoma tissue of different histologic subtypes was obtained from 68 patients at the time of surgery and from 26 human xenografts in nude mice. In addition, two human sarcoma cell lines (OSA and U2OS) were studied. Genomic DNA from tumor tissue, in vitro cell lines, and peripheral blood cells were isolated by Southern-blot analysis methods to determine MDM2 gene amplification. Tumor DNA was analyzed for possible TP53 gene mutations in exons 5, 7, and 8 by constant denaturing gel electrophoresis. To determine the MDM2 and TP53 mRNA levels, Northern-blot analysis was performed. Amplification of the MDM2 gene was detected in 10 tumors (10.3%). Whereas MDM2 amplification and/or over-expression were found only in two (U2OS and OSA cell lines) of 18 osteosarcomas, one of 20 malignant fibrous histiocytomas (MFHs), and in none of 14 leiomyosarcomas, such alterations were observed in two of two fibrosarcomas, three of six malignant schwannomas, three of 19 liposarcomas, and in the one hemangiopericytoma examined. MDM2 overexpression was found in all nine examined cases with and in three tumors without amplification. TP53 mutations were detected in 12 cases (five osteosarcomas, four MFHs, and three leiomyosarcomas), of which none showed amplification, but one had increased levels of MDM2 mRNA. None of the fibrosarcomas, malignant schwannomas, and liposarcomas examined had mutated TP53. The six sarcomas that showed high TP53 mRNA expression in the absence of gene mutation also had elevated levels of MDM2 mRNA. The present data provide further indications that increased MDM2 expression level, caused by gene amplification or altered regulation of transcription, is involved in tumor progression of some, but not all, sarcoma subtypes.

Oncogene Amplification in Urothelial Cancers With p53 Gene Mutation or MDM2 Amplification

Previously, p53 (also known as TP53) gene mutations have been shown to be frequently detected in highly malignant urothelial cancers. Evidence has been accumulating that the disruption of the normal function of p53 may lead to genomic instability, including predisposition to gene amplification. Furthermore, the normal function of p53 may be abrogated by MDM2 (murine double minute-2) gene amplification in some human tumors. Our purpose was to investigate the relationship between protooncogene amplification and p53 alteration in urothelial cancers by examining the existence of amplification of MDM2 and 14 other protooncogenes in 50 urothelial tumors in which p53 gene status was known. We analyzed gene amplification by Southern-blot analysis in 50 urothelial cancer specimens. These tumors were previously examined for p53 mutations by polymerase chain reaction-single-strand conformation analysis, and 17 tumors contained p53 mutations. Two high-grade advanced tumors (4%) without p53 mutation harbored MDM2 amplification with concurrent int-2 gene amplification. As for other genes, amplification was detected for int-2 (also known as WNT2) (seven [14%] of 50), erbB-2 (also known as ERBB2) (three [6%] of 50), N-ras (also known as NRAS) (one [2%] of 50), L-myc (also known as MYCL1) (one [2%] of 50), and raf-1 (also known as RAF1) (one [2%] of 50). The amplification of at least one gene examined was observed in 11 (22%) of 50 tumors. The presence of p53 mutations was not significantly associated with the occurrence of gene amplification, since the amplification was detected in six (35%) of 17 tumors with p53 mutations and in five (15%) of 33 tumors without p53 mutations. However, eight (73%) of 11 tumors with proto-oncogene amplification harbored p53 mutations or MDM2 amplification. A subset of advanced urothelial cancers without p53 mutations may harbor MDM2 amplification. This finding should be taken into account when adopting p53 alteration as a marker of aggressiveness in urothelial cancers. Although the abrogation of normal p53 function may be one of the key steps to protooncogene amplification, the data further indicate that the predisposition to gene amplification in urothelial cancers was not determined by the presence of p53 alteration alone.