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Generally, sarcomas arising from benign soft tissue are rare. Cardiac myxoma (CM) is a benign tumor, and few reports have described its malignant transformation. Herein, we documented a case of an 89-year-old man with prostate cancer and a 5-year history of a right atrium tumor without Carney complex. The tumor was resected surgically and had a myxomatous or gelatinous appearance. Microscopically, the tumor had two components: a sarcomatous area and myxomatous area. In the myxomatous area, typical myxoma cells were demonstrable and were strongly immunoreactive for immunohistochemistry (IHC) of calretinin. In the sarcomatous area, the epithelioid- to spindle-shaped cells with prominent atypia proliferated densely. The IHC profile of cells in the sarcomatous area was different from that of cells in the myxomatous area; MDM2-positive cells were found only in the sarcomatous area. Especially, the Ki-67 index and number of p53-positive cells in the sarcomatous area were higher than those in the myxomatous area. The transition of the two components was seamless. Thus, we made a diagnosis of CM with malignant transformation corresponding to undifferentiated pleomorphic sarcomas. This case suggests that CM may transform into sarcoma, albeit rarely., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Introduction: The role of IMP3, CDK4, MDM2 and β-catenin proteins in Enchondroma and Central Chondrosarcoma is not totally understood. The aim of this study is to evaluate the immunoexpression of these proteins, associating histological grade, clinical data and prognosis to these tumors., Methods: This is a retrospective-analytical study of 32 Enchondroma and 70 Central Chondrosarcoma., Results: IMP3, CDK4, MDM2 and β-catenin expression was observed in 22.82 %, 13.82 %, 17.17 % and in 8.8 % of cases, respectively. All Enchondromas positive for these immunomarkers were located in short tubular bones. The positivity for these antibodies is directly proportional to Chondrosarcoma's histological grade increase. No difference was found between Enchondroma and Chondrosarcoma, Grade 1 for IMP3, CDK4 and ß-catenin positivity. Significant metastasis outcome was observed for IMP3, CDK4, MDM2 and death for MDM2 expression., Conclusion: IMP3, CDK4, MDM2 and β-catenin expression in Enchondromas of short bones phenotypically characterizes these tumors. Their expression has not proven to be useful either as diagnostic markers of these neoplasms or in distinguishing between Enchondroma and Chondrosarcoma, Grade 1. The significant immunoexpression of IMP3, CDK4 and MDM2 in metastatic Chondrosarcoma and the lower survival in those with positivity for MDM2 suggest a possible association of these proteins with tumor aggressiveness., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 HCFMUSP. Published by Elsevier España, S.L.U. All rights reserved.)

The classification and work-up of adipocytic neoplasms remains challenging and sometimes controversial. Since its initial description by Dr. Enterline, the variety of subtypes and morphological appearances considered to represent the spectrum of atypical lipomatous tumor/well differentiated liposarcoma (ALT/WDL) has expanded, resulting in significant morphologic overlap with other entities, including the recently described atypical spindle cell/pleomorphic lipomatous tumor (ASPLT), conventional spindle cell/pleomorphic lipoma (SPL), and so-called "low-grade" forms of dedifferentiated liposarcoma (DL). Nevertheless, the distinction of most examples of ALT/WDL from lipomas/lipoma-like lesions is easily performed on routine histologic examination but can be problematic if the characteristic atypical cells are poorly represented, particularly in small biopsy specimens, obscured by other cellular elements (inflammation), or simply not recognized. The discovery that lipomatous tumors harbor specific and unique karyotypes and molecular events has resulted in ancillary tests that can help provide more accurate diagnoses, especially in less-than-optimal scenarios. Confirmation of MDM2 immunohistochemical over-expression and detection of the MDM2 gene rearrangement via fluorescent in situ hybridization (FISH) have proven particularly reliable and useful. While FISH analysis for MDM2 gene amplification may be helpful for confirming (or excluding) ALT/WDL, it also can lead to overutilization and overdependence. Furthermore, a small subset of otherwise typical ALT/WDL lack MDM2 gene amplification, employing alternative molecular pathways. The recent recognition of ASPLT has introduced a tumor easily mistaken morphologically for ALT/WDL, often exhibiting bizarre and pleomorphic lipoblasts, but lacking the underlying molecular abnormalities and subsequent risk of dedifferentiation. ASPLT also have overlapping features with the better-established SPL but with a greater tendency to locally recur and more frequent involvement of the distal extremities. The precise criteria separating cellular forms of ALT from what some consider "low grade" forms of DL remains controversial and inconsistently applied, even among individual pathologists within institutions. Given their underlying shared cytogenetic abnormality, molecular testing has no utility in this distinction. Herein is a comprehensive historical overview of ALT/WDL, with updates on its distinction from other similar lipomatous tumors and DL, including practical evidence-based criteria for the appropriate cost-effective use of MDM2 testing., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Several high-grade pleomorphic sarcoma cases that cannot be classified into any existing established categories have been reported. These cases were provisionally classified into undifferentiated pleomorphic sarcoma (UPS). Some dedifferentiated liposarcoma (DDLS) cases may also have been classified into the UPS category due to the absence of MDM2 amplification or an atypical lipomatous tumor/well-differentiated liposarcoma component. We retrieved and reviewed 77 high-grade pleomorphic sarcoma cases, initially diagnosed as UPS in 66 cases and DDLS in 11 cases. Fluorescence in situ hybridization (FISH) analyses of DDIT3 and MDM2 were performed for available cases. Of the cases successfully subjected to DDIT3 FISH (n = 56), nine (7 UPS and 2 DDLS) showed DDIT3 amplification but no MDM2 amplification. Two UPS cases showed both telomeric (5') and centromeric (3') amplification of DDIT3 or low polysomy of chromosome 12, whereas 5 UPS and 2 DDLS cases showed 5'-predominant DDIT3 amplification. Histopathologically, all cases showed UPS-like proliferation of atypical pleomorphic tumor cells. Immunohistochemically, only one case showed focal nuclear positivity for DDIT3, supporting the previous finding that DDIT3 expression was not correlated with DDIT3 amplification. All three cases with focal MDM2 expression involved 5'-predominant amplification, two of which showed DDLS-like histological features. The majority of cases (7/9) showed decreased expression in p53 staining, suggesting that DDIT3 amplification regulates the expression of TP53 like MDM2. From a clinicopathological perspective, we hypothesize that DDIT3-amplified sarcoma, especially with 5'-predominant amplification, can be reclassified out of the UPS category., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to disclose., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Liposarcoma rarely occurs in the pleura or thoracic cavity, and few reports appear in the literature. We hypothesized that combining clinicopathologic, immunohistochemical, and fluorescence in situ hybridization methods would allow definite diagnoses. Using formalin-fixed, paraffin-embedded blocks, we examined 6 atypical lipomatous tumor/well-differentiated liposarcomas (ALT/WDLPS), 5 dedifferentiated liposarcomas (DDLPSs), 2 pleomorphic liposarcomas, and 1 myxoid liposarcoma (MLPS). We used the Kaplan-Meier method and the Wilcoxon test for survival analysis for prognostic factor evaluation. Histologically, ALT/WDLPS was composed of a relatively mature adipocytic proliferation, accompanied by some lipoblasts. DDLPS exhibited round-to-oval tumor cells with a high nucleus-to-cytoplasm ratio that had proliferated in nests, accompanied in case 10 by some giant cells but no fatty cells. The pleomorphic type contained a varying proportion of pleomorphic lipoblasts. MLPS displayed uniform round- to oval-shaped cells and small signet-ring lipoblasts in a myxoid stroma. Immunohistochemically, 11 (79%), 11 (79%), and 10 (71%) of 14 cases were positive for S-100, p16, and CDK4, respectively. Six of the 14 cases (43%) were positive for MDM2 and adipophilin. One case of ALT/WDLPS and 3 cases of DDLPS exhibited MDM2 amplification by fluorescence in situ hybridization (Vysis LSI MDM2 SpectrumGreen Probe plus Vysis CEP 12 SpectrumOrange probe). ALT/WDLPS was the most favorable type for survival, while adipophilin tended to be a negative prognostic factor for pleural liposarcoma. For a firm diagnosis of liposarcoma in the pleura, immunohistochemistry for CDK4, MDM2, and adipophilin together with MDM2 gene amplification by fluorescence in situ hybridization may be an important diagnostic tool., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Objective: To investigate the value of using MDM2 amplification probe and DDIT3 dual-color, break-apart rearrangement probe fluorescence in situ hybridization (FISH) technique in the diagnosis of liposarcoma., Methods: In the study, 62 cases of liposarcoma diagnosed in Peking University First Hospital from January 2015 to December 2019 were analysed for clinicopathological information. Of these 62 cases of liposarcoma, all were analysed for MDM2 amplification and 48 cases were analysed for DDIT3 rearrangement using a FISH technique. Our study aimed to evaluate the status of MDM2 and DDIT3 by FISH in liposarcoma and correlate it with diagnosis of different subtypes of liposarcoma. The subtypes of liposarcoma were classified according to the FISH results, combined with the relevant clinicopathological features., Results: The patients aged 31-89 years (mean: 59 years) with a 1.75:1 male to female ratio. Histologically, there were 20 cases of atypical lipomatous tumour/well-differentiated liposarcoma (ALT/WDLPS), 26 cases of dedifferentiated liposarcoma (DDLPS), 13 myxoid liposarcoma (MLPS) and 3 pleomorphic liposarcoma (PLPS). Tumors with DDLPS (23/26) and WDLPS (8/20) were localized retroperitoneally, while both tumours of MLPS and PLPS were localized extra-retroperitoneally, and the difference of sites among the four subtypes of liposarcoma was statistically significant ( P < 0.05). Histologically, varied mucoid matrix could be observed in the four subtypes of liposarcoma, and the difference was statistically significant ( P < 0.05). MDM2 gene amplification was demonstrated in all cases of ALT/WDLPS and DDLPS (100%, 20/20 and 26/26 respectively); DDIT3 gene rearrangement was noted only in MLPS (100%, 13/13); most cases of DDLPS (96.2%, 25/26) and ALT/WDLPS (83.3%, 5/6, 6 cases selected for detection) demonstrated the picture of amplification of the DDIT3 telomeric tag. According to the instructions of DDIT3 break-apart rearrangement probe, the 5' telomere probe and 3' centromere probe spanned but did not cover the DDIT3 gene itself, on the contrary, the 5' telomere probe covered the CDK4 gene, while the DDIT3 and CDK4 gene were located adjacent to each other on chromosome, therefore, when the amplification signal appeared on the telomeric tag of the DDIT3 rearrangement probe, it indeed indicated the CDK4 gene amplification rather than the DDIT3 gene rearrangement. Then the 10 cases with DDIT3 telomeric tag amplification were selected for CDK4 and DDIT3 gene amplification probe FISH tests, and all the cases showed CDK4 gene amplification (100%, 10/10) and two of the 10 cases demonstrated co-amplification of CDK4 and DDIT3 (20%, 2/10); DDIT3 polysomy detected by DDIT3 gene rearrangement probe was found in 1 case of DDLPS and 2 cases of PLPS (66.7%, 2/3) with morphology of high-grade malignant tumour and poor prognosis., Conclusion: Our results indicate that a diagnosis of different subtype liposarcoma could be confirmed based on the application of MDM2 and DDIT3 FISH, combined with clinicopathological findings. It is also noteworthy that atypical signals should be correctly interpreted to guide correct treatment of liposarcomas.

Background: Germline TP53 mutations have been frequently reported in patients with Li-Fraumeni syndrome (LFS), resulting in a predisposition to various malignancies. Mutations other than germline TP53 mutations can also cause LFS-associated malignancies, but their details remain unclear. We describe a novel c-myc amplification in a unique liposarcoma in a patient with LFS., Case Presentation: A female patient with LFS developed breast cancer twice at the age of thirty; both were invasive ductal carcinomas harboring HER2 amplifications. Computed tomography revealed an anterior mediastinal mass, which was surgically resected. Histological analysis revealed three different lesions corresponding to myxoid liposarcoma-, pleomorphic liposarcoma-, and well-differentiated liposarcoma-like lesions. Fluorescence in-situ hybridization (FISH) analysis did not detect MDM2 amplification, Rb1 deletion, break apart signals of EWS, FUS, DDIT3, or c-myc, or c-myc-IGH fusion signals, but it did detect more c-myc signals. Further FISH analysis and comprehensive genomic profiling revealed c-myc amplification. We considered two differential diagnoses, dedifferentiated liposarcoma lacking MDM2 amplification and myxoid pleomorphic liposarcoma (MPLPS), and determined that this case is most likely MPLPS. However, definite diagnosis could not be made because a clear-cut differentiation of the case from liposarcomas was not possible., Conclusions: A previous study demonstrated that c-myc amplification could not be detected in various liposarcomas, but the present unique liposarcoma showed c-myc amplification, so the c-myc amplification may indicate that the present liposarcoma is an LFS-related tumor. The present case further clarifies the pathological features of MPLPS and LFS-related liposarcomas by broadening their histopathological and genetic diversities., (© 2022. The Author(s).)

Soft tissue sarcomas (STS) are a heterogeneous group of tumors that arise from mesenchymal tissue. Investigation at the molecular level has been challenging due to the rarity of STS and the number of histologic subtypes. However, recent research has provided new insight into potential genomic, proteomic, and immunological biomarkers of STS. The identification of biomarkers can improve diagnosis, prognosis, and prediction of recurrence and treatment response. This review provides an understanding of biomarkers, discussing the current status of biomarker research in STS., (© 2021 Wiley Periodicals LLC.)

Malignant Brenner tumor (MBT) is diagnosed in the setting of invasive high-grade carcinoma with urothelial-like morphology and the presence of an adjacent benign Brenner tumor (BBT) or borderline Brenner tumor (BLBT). MDM2 amplification was recently detected by next-generation sequencing on a small number of MBTs, potentially significant for future targeted therapy. Experience is limited, however, and evaluation of widely available MDM2 immunohistochemistry (IHC) has not been performed to determine clinical utility. After confirming all diagnoses morphologically and immunohistochemically, we performed MDM2 IHC on 4 MBTs, 3 BLBTs, 26 BBTs, 142 high-grade serous carcinomas (HGSC), 6 ovarian endometrioid carcinomas (OEC) with urothelial-like morphology, and 49 high-grade urothelial carcinomas (HGUC). MDM2 IHC was considered positive with diffuse (>25%) nuclear reactivity; in cases of patchy staining (10-25% nuclear reactivity), MDM2 was considered equivocal. Positive staining in <10% of cells was considered negative. In cases with positive or equivocal staining, MDM2 amplification was evaluated by fluorescence in-situ hybridization (FISH). Three MBTs (75%) showed diffuse nuclear reactivity for MDM2 by IHC, a finding corroborated by amplification of MDM2 in all three cases. One MBT and 2 BLBTs showed equivocal MDM2 IHC, but all three were negative for MDM2 amplification. The final BLBT, as well as all BBTs, HGSC, OEC, and HGUC, were negative for MDM2. In conclusion, our limited cohort confirms MDM2 amplification in MBT and suggests that MDM2 IHC may have an influence in rare diagnostically challenging cases., (Copyright © 2021. Published by Elsevier Inc.)

The novel anti-neoplastic glycopeptide T11TS retards glioma both in in-vitro clinical samples and in-vivo models. This study investigates the correlation between altering the glioma microenvironment with glioma arrest and death. Flow cytometry, immunoblotting, ELISA, and co-immunoprecipitation were employed to investigate glioma cell arrest and death. Results include a decline in phosphorylation of Akt and attenuation of p21 phosphorylation (Thr145,Ser146) and disassociation of p-Akt-Mdm2 and p-Akt-BAD facilitating death by Akt>BAD. T11TS influence phosphorylation patterns in two focal axes Akt>p21 and Akt>Mdm2>p53. The current article provides crucial insight in deciphering the mechanism of T11TS induced glioma cell arrest and death.

Liposarcoma is the most common soft tissue malignancy and usually occurs in the retroperitoneum or the extremities but rarely in the kidney. In this article, we report a case of a 71-year-old female patient who presented with abdominal lump and left flank pain for 1 month. An abdominal contrast-enhanced computed tomography scan demonstrated a 12 cm × 7 cm solid mass arising from the upper pole of left kidney and another 8 cm × 6 cm low-density retroperitoneal mass with fat density. Radical nephrectomy of the left kidney and resection of the retroperitoneal mass were performed. Surprisingly, pathological examination revealed a high-grade sarcoma (with minor lipomatous component) in the left kidney and a retroperitoneal well-differentiated liposarcoma. MDM2 gene amplification was identified by fluorescence in situ hybridization in both tumors, supporting final diagnosis of dedifferentiated liposarcoma of the kidney and well-differentiated liposarcoma of the retroperitoneum.

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.

Craniofacial osteosarcoma is rare (2-10% of all osteosarcomas). Most low grade fibroblastic osteosarcomas of the long bones are characterized by amplification of chromosome12q including MDM2 and CDK4 genes. This study aims to investigate the utility of MDM2 and CDK4 immunostains as well as MDM2 FISH in craniofacial osteosarcomas as a means of distinguishing them from benign fibro-osseous lesions. Cases of primary osteosarcoma and benign fibro-osseous lesions of the craniofacial bones were identified in the diagnostic pathology archives. MDM2 (SMP14 and/or IF2) and CDK4 (D9G3E and/or DCS-31) immunostains were performed on a representative block from each osteosarcoma and benign case. Fluorescence in situ hybridization (FISH) for MDM2 was performed on non-decalcified osteosarcomas. In osteosarcomas, the rate of expression of either MDM2 IF2, MDM2 SMP14, CDK4 DCS-31, or CDK4 D9G3E was 72.7% (8/11 cases), usually focal and weak. Using the MDM2 IF2 clone and the CDK4 DCS-31 clone, MDM2 and CDK4 were negative in lesional cells in all 14 benign fibro-osseous lesions. Using the IF2 and SMP14 clones, MDM2 nuclear expression was present in associated osteoclast-like giant cells in both benign and malignant cases. Of 4 successful cases, 1 high grade osteosarcoma was positive for MDM2 amplification. MDM2 or CDK4 expression or MDM2 amplification may aid in a diagnosis of head and neck osteosarcoma. However, when absent, sarcoma is not excluded. Due to focal weak expression of MDM2 in tumor cells in conjunction with nuclear expression in associated giant cells, caution should be exercised when interpreting positive stains.

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 highlight the potential utility and pitfalls of using MDM2 RNA-ISH in the distinction of atypical lipomatous tumor and related liposarcomas from dysplastic lipoma., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background and objectives: Laryngeal squamous cell carcinoma (LSCC) is one of the most common head and neck tumors. The molecular mechanism of LSCC remains unclear. The aim of this study was to evaluate the prevalence of Human papillomavirus (HPV) and single nucleotide polymorphisms (SNPs) of TP53, MDM2, MDM4, MTHFR, CASP8, and CCR5 genes in LSCC, and to assess their correlations with patient survival. Materials and Methods: 49 LSCC patients were enrolled in this study. PCR and qRT-PCR were used to detect, identify, and quantify HPV. SNPs were genotyped using PCR and PCR-RFLP. Results: By analyzing the interactions of the SNPs of the genes with clinical parameters, the majority of patients with lymph node status (N1,2) were identified as carriers of MDM2 T/G , CASP8 ins/del , CCR5 wt/wt SNP. Cluster analysis showed that patients with MDM2 T/T SNP survive longer than patients identified as CASP8 ins/ins , MTHFR C/C , and MDM4 A/A variant carriers; meanwhile, LSCC patients with MDM2 T/T polymorphic variant had the best survival. Multivariate analysis showed that HPV-positive patients without metastasis in regional lymph nodes (N0) and harboring CASP8 ins/del variant had the best survival. Meanwhile, HPV-negative patients with identified metastasis in lymph nodes (N1 and N2) and CASP8 ins/del variant had poor survival. C onclusions: This finding suggests patients survival prognosis and tumor behavior are different according HPV status, SNP variants, and clinical characteristics of the LSCC., Competing Interests: Ethics approval and consent to participate: All patients included in this study have signed an Informed patient consent approved by the Vilnius Regional Committee of Biomedical Research (Lithuania, 2013-06-11 permission No. 158200-13-638-204). Consent for publication: Not Applicable. Availability of data and materials: All data generated or analyzed during this study are included in this published article and its supplementary information files. Competing interests: The authors declare that they have no competing interests.

We report the case of a "fat-rich" (spindle cell-poor) variant of an atypical spindle cell/pleomorphic lipomatous tumor in a 63-year-old female patient presenting with a firm, painless soft tissue mass on the right hip. Atypical spindle cell/pleomorphic lipomatous tumor is a very recently described low-grade adipocytic neoplasm, which occurs predominantly in adults with a predilection for the limbs and limb girdles. In the present case, the diagnosis of an atypical spindle cell/pleomorphic lipomatous tumor was challenging because the tumor was almost exclusively composed of an atypical adipocytic component (resembling "classical" atypical lipomatous tumor/well-differentiated tumor) with only very focal presence of the diagnostic atypical morphologic features (atypical spindle-shaped cells, floret-like multinucleated cells, and "bizarre" pleomorphic [multinucleated] cells). The possibility of a "classical" atypical lipomatous tumor/well-differentiated liposarcoma was ruled out by immunohistochemistry (lack of MDM2 expression and loss of Rb expression) and molecular genetic testing (no amplification of MDM2 and presence of monoallelic deletion of RB1 ). Another interesting morphologic observation in this case was the striking perivascular location of the atypical spindle/pleomorphic cells in some areas (so-called "pericytic mimicry"). To our knowledge, pericytic mimicry has not been reported in the setting of an atypical spindle cell/pleomorphic lipomatous tumor.

MDM2 can mediate the degradation of tumor suppressor p53 through an autoregulatory feedback loop, in which MDM2 abolishes wild-type p53 function and accelerates malignant transformation. However, the incorporation of MDM2 antagonist Nutlin-3 could reactivate the transcriptional activity of p53, up-regulate caspase-3, and induce apoptosis. In this work, the simultaneous and label-free monitoring of p53-MDM2 complex and caspase-3 levels in cancer cells before and after Nutlin-3 treatment was conducted using dual-channel surface plasmon resonance (SPR). The p53-MDM2 complex was captured in one fluidic channel covered with consensus double-stranded (ds)-DNA, while the other channel was pre-immobilized with caspase-3-specific biotinylated DEVD-containing peptides. To amplify the SPR signals, the attachment of streptavidin (SA)-conjugated anti-MDM2 antibody in both channels was achieved. The signal diversity before and after Nutlin-3 treatment is indicative of the difference in the levels of the intracellular p53-MDM2 complex and caspase-3. The limit of detection for p53-MDM2 and caspase-3 down to 4.54 pM and 0.03 ng mL -1 , respectively, was attained. Upon treatment with Nutlin-3, MCF-7 cancer cells with wild-type p53 showed decreased expression of the p53-MDM2 complex and an increased caspase-3 level, while MDA-MB-231 cancer cells with mutant p53 exhibited an elevated caspase-3 level and unchanged p53-MDM2 complex expression. The apoptosis of MCF-7 and MDA-MB-231 cancer cells upon Nutlin-3 treatment follows a p53-dependent and a p53-independent pathway, respectively. The proposed method is sensitive, selective and label-free, holding great promise for assaying intracellular p53-MDM2 complex and caspase-3 levels and differentiating Nutlin-3-mediated p53-dependent or p53-independent apoptotic pathways.

Traumatized lipomas with degenerative change may demonstrate histopathologic features that mimic atypical lipomatous tumor (ALT). Previously reported series of ALT involving the oral cavity preceded routine use of MDM2 and CDK4 immunohistochemistry. Our aim is to evaluate MDM2 and CDK4 immunohistochemical expression in adipocytic tumors arising in this site, in conjunction with the histiocytic marker PU.1, to determine whether MDM2 and CDK4 impacts classification. 17 cases originally diagnosed as ALT were retrieved and immunohistochemical studies for MDM2, CDK4 and PU.1 were performed. FISH analysis for MDM2 amplification was performed in select cases. For this study group, the male:female ratio was 9:8 and the median age was 62 (range 41-88). All 17 cases presented as well- or predominantly well-circumscribed proliferations of variably sized, mature adipocytes exhibiting uni- or multi-vacuolation with occasional scalloped nuclei and mild nuclear atypia. Variable amounts of fibrous stroma with focal myxoid change and bland spindle cells were identified in 14/17 cases. Lipoblasts or atypical hyperchromatic stromal cells were not identified in any cases. 14 of 17 cases were negative for MDM2 and CDK4 in tumor cells and 11 of these 14 showed weak nuclear positivity for MDM2 in histiocytes. 3 of 17 cases showed weak, multifocal immunohistochemical expression of MDM2 and CDK4. PU.1 highlighted histiocytes in all 17 cases. FISH analysis for MDM2 amplification was negative in all 3 cases with weak MDM2/CDK4 expression. All cases were reclassified as lipoma with degenerative changes. ALT, in all likelihood, is less common than previously thought in this anatomic location and best diagnosed with ancillary studies. MDM2 expression in histiocytes is best interpreted in conjunction with CDK4 immunohistochemistry and confirmatory FISH for MDM2 amplification.

The distinction of atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDL) from its benign counterpart, lipoma, may represent a challenge. MDM2 DNA amplification is used as the gold standard as MDM2 immunohistochemistry lacks specificity and sensitivity. Herein, we investigate the diagnostic utility of MDM2 RNA in situ hybridization (RNA-ISH) and compare the test with MDM2 immunohistochemistry and MDM2 DNA fluorescence in situ hybridization (FISH) in benign and malignant lipomatous neoplasms. We evaluated 109 neoplasms including 27 lipomas, 25 spindle cell lipomas, 32 ALTs/WDLs, and 25 dedifferentiated liposarcomas (DDL). The validation cohort included 14 lipoma-like neoplasms that lacked unequivocal features of ALT/WDL and in which MDM2 immunohistochemistry was either equivocal, negative or falsely positive. Immunohistochemistry, automated RNA-ISH and DNA-FISH for MDM2 were performed. Tumors with diffuse nuclear staining or >50 dots per cell on RNA-ISH were considered positive. All lipomas and lipoma variants were negative for RNA-ISH while all ALTs/WDLs and DDLs were positive. Eighty percent (24/30) and 92% (22/24) of ALTs/WDLs and DDLs were positive for MDM2 immunohistochemistry. Lipomas and its variants were negative for MDM2 amplification; 92% and 100% of ALTs/WDLs and DDLs showed MDM2 DNA amplification. The mean percentage of ALT/WDL tumor cells showing MDM2 RNA-ISH positivity was 73% compared with 24% on MDM2 immunohistochemistry. RNA-ISH correctly classified all 10 ALTs/WDLs and all 4 lipomas in the validation cohort. The performance of MDM2 RNA-ISH and MDM2 DNA-FISH are equivalent. MDM2 RNA-ISH can be of diagnostic value in histologically challenging lipomatous neoplasms. The automated MDM2 RNA-ISH assay should allow for more widespread use of MDM2 testing and for a more sensitive and specific diagnosis of ALT/WDL.

Background: Pulmonary arterial hypertension (PAH) is a complex pulmonary vasculature disease characterized by remodeling of the pulmonary vessels and a persistent increase in the pulmonary vascular resistance (PVR) with a poor prognosis. Serotonin increases the expression of S100A4/Mts1, which in turn stimulates the proliferation and migration of human pulmonary artery smooth muscle cells through the interaction with RAGE (receptor for advanced glycation end products) and thus S100A4/Mts1 has been implicated in the development of PAH in vitro. Fluoxetine, a selective serotonin re-uptake inhibitor has been shown to protect against PAH. The current study was designed to test whether S100A4 and its associated proteins connected in the development of PAH in vivo as well as to investigate the involvement of those proteins in the protective effect of fluoxetine against PAH., Methods: MCT-induced PAH models were established in Wistar rats by a single intraperitoneal injection of MCT (60 mg/kg). Fluoxetine (2 and 10 mg/kg/day) was intragastrically administered once a day for 3 weeks along with controls. The detection methods followed include Hematoxylin and Eosin (H&E) staining, immunohistochemistry, western blotting and real-time reverse transcription-polymerase chain reaction (RT-PCR)., Results: MCT induced pulmonary hypertension, pulmonary vascular remodeling, and right ventricular hypertrophy significantly increased the expressions of S100A4 and RAGE in the pulmonary arteries, lungs and right ventricle (RV). Fluoxetine dose-dependently inhibited MCT-induced pulmonary arterial hypertension, pulmonary vascular remodeling, and right ventricular hypertrophy and reduced the S100A4 and RAGE. Further analysis revealed that fluoxetine alleviated both the increase of p53, MMP13, MMP2 and MMP9 and the decrease of pp53Ser15 and MDM2 in lungs and RV tissues of MCT-induced PAH rats., Conclusion: From the present investigation it could be concluded that S100A4/Mts1 and its associated proteins are involved in the evolution of MCT-induced PAH in rats and fluoxetine inhibits MCT-induced PAH in rats mainly through S100A4/RAGE signaling axis and involved factors., (Copyright © 2018. Published by Elsevier Taiwan LLC.)

Venetoclax (ABT-199) and idasanutlin (RG7388) are efficient anticancer drugs targeting two essential apoptosis markers, Bcl-2 and MDM2, respectively. Recent studies have shown that the combination of these two drugs leads to remarkable enhancement of anticancer efficacy, both in vitro and in vivo. In an attempt to disclose the relationships of their protein targets, competitive affinity-based proteome profiling coupled with bioimaging was employed to characterize their protein targets in the same cancer cell line and tumor tissue. A series of protein hits, including ITPR1, GSR, RER1, PDIA3, Apoa1, and Tnfrsf17 were simultaneously identified by pull-down/LC-MS/MS with the two sets of affinity-based probes. Dual imaging was successfully carried out, with the simultaneous detection of Bcl-2 and MDM2 expression in various cancer cells. This could facilitate the novel diagnostic and therapeutic strategies of dual targeting of Bcl-2/MDM2., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Hibernoma is a benign adipocytic tumor with predilection for subcutaneous tissue of the thigh, upper trunk, and neck of middle-aged adults. 11q13 rearrangement resulting in MEN1/AIP codeletion is characteristic. Hibernomas are composed, in varying proportions, of brown fat cells, mature adipocytes, and microvacuolated lipoblast-like cells. Examples containing predominantly multivacuolated lipoblast-like cells are uncommon and distinction from atypical lipomatous tumor (ALT) is important for clinical management. We herein present the clinicopathologic features of 64 hibernomas histologically mimicking ALT. MDM2 and CDK4 immunohistochemistry as well as MDM2 fluorescence in situ hybridization were performed in a subset of cases. Clinical and follow-up information were obtained from referring pathologists. Thirty-four patients were male and 30 female, with a median age of 43 years (range, 24 to 78 y). The tumors were well circumscribed and mostly deeply located (53/64 cases, 83%) with a median tumor size of 12.9 cm (range, 3.5 to 23 cm) and predilection for the thigh (42/64 cases, 66%). Histologically, large cells with prominent lipoblast-like cytoplasmic fatty vacuoles and small central nuclei were present to a prominent degree in all cases, along with mature univacuolated adipocytes and smaller numbers of large, finely vacuolated cells with eosinophilic granular cytoplasm. Nuclear atypia and mitoses were absent. None of the 39 cases tested showed CDK4 and MDM2 overexpression or MDM2 amplification. Follow-up, available for 16/64 cases (median, 47 mo; range, 1 to 165 mo), revealed no recurrences or metastases. Hibernoma mimicking ALT shows predilection for deep soft tissue, especially in the thigh. These tumors behave in a benign manner and MDM2/CDK4 negativity may be useful in excluding ALT.

MDM2 is a key negative regulator of the p53 tumor suppressor. Direct binding of MDM2 to p53 represses the protein's transcriptional activity and induces its polyubiquitination, targeting it for degradation by the proteasome. Consequently, small molecule inhibitors that antagonize MDM2-p53 binding, such as RG7388, have progressed into clinical development aiming to reactivate p53 function in TP53 wild-type tumors. Here, we describe the design, synthesis, and biological evaluation of a trans-cyclooctene tagged derivative of RG7388, RG7388-TCO, which showed high cellular potency and specificity for MDM2. The in-cell reaction of RG7388-TCO with a tetrazine-tagged BODIPY dye enabled fluorescence imaging of endogenous MDM2 in SJSA-1 and T778 tumor cells. RG7388-TCO was also used to pull down MDM2 by reaction with tetrazine-tagged agarose beads in SJSA-1 lysates. The data presented show that RG733-TCO enables precise imaging of MDM2 in cells and can permit a relative assessment of target engagement and MDM2-p53 antagonism in vitro.

Malignant peripheral nerve sheath tumor (MPNST) and dedifferentiated liposarcoma (DDLPS) are 2 major types of pleomorphic spindle cell sarcoma. The differentiation of MPNST and DDLPS by histomorphology alone can be problematic. Although MDM2 amplification and PRC2 alteration leading to H3K27me3 deficiency are genetic hallmarks of DDLPS and MPNST, respectively, a small number of MDM2-amplified MPNSTs and H3K27me3-deficient DDLPSs have been reported in the literature. We systematically compared MDM2 and H3K27me3 status in 68 MPNSTs and 47 DDLPSs. Of the 62 MPNSTs, 22 were immunopositive for MDM2, mostly in a weak and/or focal manner. Of the 21 MDM2-positive MPNSTs successfully tested by fluorescence in situ hybridization, high-level MDM2 amplification was observed in 1 case. In contrast, MDM2 staining and high-level MDM2 amplification were positive in all the DDLPS tested (28/28 and 20/20). Of the 68 MPNSTs, 42 cases (62%) exhibited complete loss of H3K27me3. All the 13 MPNSTs that showed heterologous differentiation were deficient in H3K27me3. Of the 47 DDLPSs, 3 cases (6%) had complete loss of H3K27me3, all of which exhibited heterologous differentiation. One case of H3K27me3-deficient DDLPS exhibited homozygous loss of EED according to targeted next-generation sequencing, whereas there were no alterations in NF1 and CDKN2A. In conclusion, high-level MDM2 amplification strongly suggests DDLPS over MPNST. Although a good marker for MPNST, H3K27me3 deficiency also uncommonly occurs in DDLPS in association with PRC2 mutational inactivation. Because both markers are imperfectly specific, rare sarcomas with dual features could be encountered, and their classification should integrate other parameters.

Angiomyolipoma (AML) arises primarily from the kidney but may grow into the retroperitoneal space mimicking a primary retroperitoneal tumor. Fine needle aspiration (FNA) and core needle biopsy of AML, particularly the fat-predominant variant, may be difficult to distinguish from retroperitoneal well-differentiated liposarcoma (WDLS) or lipoma. Commonly used immunomarkers, MDM2 and p16, have proven useful in diagnosing WDLS and dedifferentiated liposarcoma (DDLS), while HMB45 and Melan-A are melanocyte-related markers characteristically expressed in AML. In this study, we investigated the utility of MDM2 and p16 along with HMB45 and Melan-A immunohistochemical analysis in distinguishing AML from WDL/DDLS or lipoma. Immunohistochemically, AMLs demonstrated focal MDM2 expression (40% of cases) and focal/diffuse expression of p16 (60%). AMLs marked focally or diffusely with HMB45 (76% of cases) and Melan-A (96%). These latter two immunomarkers were not expressed in any of the WDLS/DDLSs or lipomas tested. WDLS/DDLSs showed focal/diffuse expression of MDM2 (91% of cases) and p16 (97%). While focal expression of MDM2 and p16 was observed in 14% and 67% of lipomas, respectively, no lipoma exhibited diffuse MDM2 positivity. In our hands, MDM2 expression by itself cannot exclude the diagnosis of AML or lipoma, and p16 alone is not helpful in separating AML and conventional lipoma from WDLS/DDLS. However, along with morphology, an immunohistochemical battery including HMB45, Melan-A, MDM2 and p16 are useful in distinguishing AML from WDLS/DDLS or lipoma. For equivocal cases, fluorescence in situ hybridization for MDM2 should be performed., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Murine double minute 2 (MDM2) is an oncoprotein mediating the degradation of the tumor suppressor p53 protein. The physiological levels of MDM2 protein are closely related to malignant transformation and tumor growth. In this work, the simultaneous and label-free determination of free and p53-bound MDM2 proteins from sarcoma tissue extracts was conducted using a dual-channel surface plasmon resonance (SPR) instrument. Free MDM2 protein was measured in one fluidic channel covered with the consensus double-stranded (ds)-DNA/p53 conjugate, while MDM2 bound to p53 was captured by the consensus ds-DNA immobilized onto the other channel. To achieve higher sensitivity and to confirm specificity, an MDM2-specific monoclonal antibody (2A10) was used to recognize both the free and p53-bound MDM2 proteins. The resultant method afforded a detection limit of 0.55 pM of MDM2. The amenability of the method to the analysis of free and p53-bound MDM2 proteins was demonstrated for normal and sarcoma tissue extracts from three patients. Our data reveal that both free and total MDM2 (free and bound forms combined) proteins from sarcoma tissue extracts are of much higher concentrations than those from normal tissue extracts and the p53-bound MDM2 protein only constitutes a small fraction of the total MDM2 concentration. In comparison with enzyme-linked immunosorbent assay (ELISA), the proposed method possesses higher sensitivity, is more cost-effective, and is capable of determining free and p53-bound MDM2 proteins in clinical samples.

Context: Urothelial carcinoma (UC) is one of the most frequent epithelial tumors worldwide., Aims: We aimed to investigate the protein expressions of caspase-8, p53, murine double minute 2 (mdm2), and p14 ARF in nonmuscle UCs and to correlate the findings with clinicopathological characteristics., Settings and Design:: A total of 50 patients who had pTa and pT1 tumors were analyzed., Subjects and Methods: The protein expressions of caspase-8, p53, mdm2, and p14 ARF were analyzed by immunohistochemistry., Statistical Analysis Used: Chi-square test was done using SPSS version 16.0 (SPSS, Inc., Chicago, IL, USA)., Results: 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 p14 ARF was not also related with pathological stage or grade (P > 0.05 for all). The p14 ARF 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)., Conclusions: 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., Competing Interests: There are no conflicts of interest

Nuclear magnetic resonance spectroscopy (NMR) is a powerful technique for characterizing the structural and dynamic properties of intrinsically disordered proteins and protein regions (IDPs & IDRs). However, the application of NMR to IDPs has been limited by poor chemical shift dispersion in two-dimensional (2D) 1 H- 15 N heteronuclear correlation spectra. Among the various detection schemes available for heteronuclear correlation spectroscopy, 13 C direct-detection has become a mainstay for investigations of IDPs owing to the favorable chemical shift dispersion in 2D 13 C'- 15 N correlation spectra. Recent advances in cryoprobe technology have enhanced the sensitivity for direct detection of both 13 C and 15 N resonances at high magnetic field strengths, thus prompting the development of 15 N direct-detect experiments to complement established 13 C-detection experiments. However, the application of 15 N-detection has not been widely explored for IDPs. Here we compare 1 H, 13 C, and 15 N detection schemes for a variety of 2D heteronuclear correlation spectra and evaluate their performance on the basis of resolution, chemical shift dispersion, and sensitivity. We performed experiments with a variety of disordered systems ranging in size and complexity; from a small IDR (99 amino acids), to a large low complexity IDR (185 amino acids), and finally a ∼73 kDa folded homopentameric protein that also contains disordered regions (133 amino acids/monomer). We conclude that, while requiring high sample concentration and long acquisition times, 15 N-detection often offers enhanced resolution over other detection schemes in studies of disordered protein regions with low complexity sequences., (Copyright © 2018 Elsevier Inc. All rights reserved.)

We present a rare case of retroperitoneal dedifferentiated liposarcoma (DDLPS) masquerading as rhabdomyosarcoma. The patient was a 74-year-old man, complaining a loss of appetite. Abdominal computed tomography revealed a retroperitoneal mass, 10 cm in diameter, between the liver and the right adrenal gland. The tumor was resected and histologically diagnosed as conventional DDLPS, in which dedifferentiated component was highly cellular and composed of pleomorphic anaplastic cells. After 3 years, the tumor recurred in the right retroperitoneal space. The recurrent tumor consisted of 2 components: lipogenic and nonlipogenic. The latter differ from the dedifferentiated component of the primary tumor. The tumor cells were small, round to ovoid cells with monomorphous, round, hyperchromatic nuclei, and scant cytoplasm. Interestingly, they were diffusely positive for myogenin and desmin. To rule out the possibility of the second primary, we performed fluorescence in situ hybridization to detect FOXO1 rearrangement. We failed to demonstrate splits of the probes. In contrast, high-level amplification of MDM2 was detected by dual-color in situ hybridization. Given the morphologic and molecular findings, the neoplasm was identified as a peculiar DDLPS mimicking rhabdomyosarcoma. Retroperitoneal rhabdomyosarcoma-like tumors of adults, therefore, should be distinguished carefully from DDLPS. It could be challenging when lipogenic component was absent, but in situ molecular analyses can be helpful.

Diffuse-type tenosynovial giant cell tumor (D-T TSGCT) is regarded as a benign but locally aggressive neoplasm with significant recurrent potential. We report a case of malignant D-T TSGCT with pleural metastases arising in the left knee in a 57-year-old man. The tumor demonstrated atypical features, including a solid infiltrative pattern with spindling of the tumor cells, nuclear pleomorphism with prominent nucleoli, and markedly increased mitotic activity (>20 mitoses/10 high-power fields). The immunoprofile demonstrated clusterin+, D2-40+, CD68+, p63+, MDM2+, and p16+ tumor. The next-generation sequencing-based assay demonstrated loss of the CDKN2A/B gene. Pleural metastases with identical histologic and immunohistochemical features were identified 2 years later after primary tumor resection. To the best of our knowledge, this is the first reported case of D-T TSGCT with CDKN2A/B genomic alteration, MDM2 expression, and p16 loss. Clinicians and pathologists should be aware of the morphologic variability and the metastatic propensity of this entity., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Extraskeletal osteosarcoma (ESOS) is an uncommon malignant neoplasm. Most ESOSs are high grade, although some low-grade cases have been reported. A few cases of ESOS with MDM2 amplification have also been reported, suggesting some similarity to skeletal low-grade osteosarcoma such as parosteal osteosarcoma, where MDM2 is often amplified. However, the frequency of low-grade cases and cases with MDM2 amplification among ESOSs remains unknown, and their relationship is unclear. To clarify this, we examined 18 primary ESOS cases clinically, pathologically, and genetically, focusing on their MDM2 amplification status. Our cases comprised 10 men and 8 women whose mean age was 58.6 years; the most common site of the lesion was the thigh and buttock. There were one histologically low-grade case evaluated by biopsy specimen with an aggressive course and 2 relatively low-grade cases whose lesions were of low grade for the most part. MDM2 amplification status was revealed by fluorescence in situ hybridization in all 18 cases; 2 patients-histologically intermediate- and high-grade cases-were found to have MDM2 amplification. In conclusion, this study indicates that histologically low-grade and relatively low-grade cases of ESOS are not always associated with MDM2 amplification. The ESOS case with MDM2 amplification could be high grade, although MDM2-amplified dedifferentiated liposarcoma with osteogenic differentiation should be ruled out in making the diagnosis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: The development of thoracic aortic dissection (TAD) is attributed to a broad range of degenerative, genetic, structural, oxidative, apoptotic, and acquired disease states. In this study, we examined the role of the disturbed p53-MDM2 (murine double minute 2) feedback loop in the formation of TAD, and one of a potential feedback loop regulator, TRIM25 (tripartite motif protein-25)., Methods: Surgical specimens of the aorta from TAD patients (n = 10) and controls (n = 10) were tested for α-smooth muscle actin (α-SMA), p53, MDM2, and TRIM25 by western blot, immunohistochemical staining, and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), respectively., Results: When compared with controls, western blot shows that the protein levels of p53, MDM2, and TRIM25 were increased significantly in the aortic media of TAD patients. qRT-PCR further verified that the mRNA expression of MDM2 and TRIM25 was also increased 6- and 4-folds, respectively, in the TAD media of the aortic wall. Immunohistochemistry results showed significantly decreased staining of α-SMA, smooth muscle cells, and more collagen deposition in the media of the aortic wall from patients with TAD., Conclusion: This study provided a new insight into the disturbed p53-MDM2 feedback loop in the pathogenesis of TAD, and this may be because of the TRIM25 overexpression., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Stress-induced activation of p53 is an essential cellular response to prevent aberrant cell proliferation and cancer development. The ubiquitin ligase MDM2 promotes p53 degradation and limits the duration of p53 activation. It remains unclear, however, how p53 persistently escapes MDM2-mediated negative control for making appropriate cell fate decisions. Here we report that TBP-like protein (TLP), a member of the TBP family, is a new regulatory factor for the p53-MDM2 interplay and thus for p53 activation. We found that TLP acts to stabilize p53 protein to ensure long-lasting p53 activation, leading to potentiation of p53-induced apoptosis and senescence after genotoxic stress. Mechanistically, TLP interferes with MDM2 binding and ubiquitination of p53. Moreover, single cell imaging analysis shows that TLP depletion accelerates MDM2-mediated nuclear export of p53. We further show that a cervical cancer-derived TLP mutant has less p53 binding ability and lacks a proliferation-repressive function. Our findings uncover a role of TLP as a competitive MDM2 blocker, proposing a novel mechanism by which p53 escapes the p53-MDM2 negative feedback loop to modulate cell fate decisions., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: In prior retrospective studies, we assessed a number of prostate tumor tissue biomarkers that were associated independently with the clinical outcome of men treated with radiotherapy (RT) ± androgen deprivation therapy (ADT). In this report, the associations of selected biomarkers with biochemical or clinical disease failure (BCDF) were prospectively evaluated in men with T1-T3 prostate cancer on a randomized hypofractionation trial., Methods: Biomarkers were analyzed in 263 of 303 men randomly assigned to standard vs moderate hypofractionation. Median follow-up was 65.9 months. Archival tissue was analyzed for Ki-67 (n = 231), MDM2 (n = 209), p16 (n = 195), Cox-2 (n = 126), p53 (n = 206), bcl2 (n = 223), bax (n = 210), and PKA (n = 160). The base model for multivariable Fine-Gray regression analysis included treatment assignment and risk groups. All statistical tests were two-sided., Results: Each biomarker was tested one at a time relative to the base model and selected for inclusion in multivariable analysis. Ki-67 (hazard ratio [HR] = 2.31, 95% confidence interval [CI] = 1.19 to 4.48, P = .01) and bcl2&bax (HR = 2.19, 95% CI = 1.08 to 4.46, P = .03) were statistically significantly related to higher BCDF and were independently statistically significant when considered jointly (Ki-67: HR = 2.26, 95% CI = 1.12 to 4.58, P = .02; bcl2&bax: HR = 2.14, 95% CI = 1.03 to 4.41, P = .04). At 2.5 years postradiotherapy, the C-index of Ki-67 was 73.2%, while for the base model was only 46.2%; Ki-67 was the most statistically significant when tested without bcl2&bax., Conclusions: In this prospective multiple biomarker analysis in men with prostate cancer treated with RT±ADT, both Ki-67 and bcl2&bax were independently related to early BCDF; however, Ki-67 alone is indicated to be the most clinically meaningful by C-index analysis and is universally available., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com)

The effect of smokeless tobacco (gutkha) was investigated by treating male and female Swiss Albino mice with an aqueous extract of smokeless tobacco (AEST). AEST was administered at a dose of 25 mg kg-1 body weight per day for different time periods (6, 12, 16, and 24 weeks), and control animals were provided only drinking water without AEST for the same period. Control and AEST-treated mice were observed for different oxidative stress parameters, nitric oxide (NO) release, and myeloperoxidase (MPO) release, and they were evaluated for alterations in tumor suppressor and DNA repair responses in the liver and spleen. Both male and female mice treated with AEST showed significant increase in lipid peroxidation, protein carbonylation, and NO and MPO release in the liver and spleen compared to age- and gender-matched controls. The significant decline in tumor suppressor p53 protein levels, likely mediated by concomitantly upregulated levels of Mdm2, was observed. We also observed a significant decline in the levels of DNA repair proteins Brca2 and Ape-1 compared to the respective controls. Thus, AEST induces oxidative stress, inflammation, and significantly lowers tumor suppressor and DNA repair responses. These factors may work in conjunction to increase the risk for certain diseases, including cancer.

The differential diagnosis between atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDLPS) and dedifferentiated liposarcoma (DDLPS) from their morphologic counterparts is challenging. Currently, the diagnosis is guided by MDM2 and CDK4 immunohistochemistry (IHC) and is confirmed by the amplification of the corresponding genes. Recently, p16 IHC has been proposed as a useful diagnostic biomarker. The objective was to assess the utility of p16 IHC in the differential diagnosis of ALT/WDLPS and DDLPS. Our series included 101 tumors that were previously analyzed using fluorescence in situ hybridization for MDM2 and CDK4 amplification. We compared sensitivity and specificity of p16 IHC to MDM2 and CDK4 IHC in the differential diagnosis of ALT-WDLPS (n=19) versus benign adipocytic tumors (n=44) and DDLPS (n=18) versus mimicking sarcomas (n=20). In the differential diagnosis of ALT-WDLPS, p16 had a sensitivity of 89.5% but a specificity of 68.2%, which was impaired by false-positive lipomas with secondary changes, especially in biopsies. Likewise, in the differential diagnosis of DDLPS, p16 had a sensitivity of 94.4% and a specificity of 70%, which hampered its use as a single marker. However, adding p16 to MDM2 and/or CDK4 increased diagnostic specificity. Indeed, MDM2+/p16+ tumors were all ALT-WDLPS, and MDM2-/p16- tumors were all benign adipocytic tumors. Moreover, all MDM2+/CDK4+/p16+ tumors were DDLPS, and the MDM2-/CDK4-/p16- tumor was an undifferentiated sarcoma. Although the use of p16 as a single immunohistochemical marker is limited by its specificity, its combination with MDM2 and CDK4 IHC may help discriminate ALT-WDLPS/DDLPS., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Angiomyolipomas (AMLs) are typically benign mesenchymal tumors with variable histologic composition. Fat-predominant AMLs can mimic well-differentiated liposarcomas (WDLSs) both radiographically and histologically because of the abundance of fat with admixed atypical cells resembling lipoblasts. However, the treatment and prognosis of AMLs and WDLSs are vastly different. Immunohistochemistry for murine double minute 2 (MDM2) has been used to support a diagnosis of WDLS; however, MDM2 labeling has not been specifically evaluated in fat-predominant AMLs. Here, we evaluated MDM2 immunohistochemistry in 36 AMLs (including 14 conventional AMLs, 13 fat-predominant AMLs, 6 fat-rich AMLs, 3 epithelioid AMLs) and 10 WDLSs. In addition, we labeled cases for HMB45, calponin, or actin, which are immunostains traditionally used to label AML. We performed fluorescence in situ hybridization (FISH) for MDM2 amplification on selected cases. By immunohistochemistry, 14% (5/36) of AMLs were MDM2+, including 23% (3/13) of fat-predominant AMLs. All MDM2+ AMLs evaluated by FISH (n=4) were negative for MDM2 amplification. By immunohistochemistry, 90% of WDLSs were MDM2+, and both MDM2+ WDLSs evaluated by FISH (n=2) were MDM2 amplified. All 36 AMLs labeled with HMB45 and calponin or actin. No WDLS labeled with HMB45; however, 80% of WDLSs labeled with calponin or actin. Although uncommon, MDM2 labeling is seen in a subset of fat-predominant AMLs and is a potential diagnostic pitfall in the evaluation of fatty tumors of the retroperitoneum. HMB45 is more sensitive and specific for AML than calponin or actin, and an immunopanel containing both HMB45 and MDM2 may be warranted to distinguish between fat-predominant AML and WDLS in histologically ambiguous cases., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Purpose: p53 tumor suppressor protein (17p13.1) regulates critically the cell cycle and thus it is involved in cancer initiation and prevention. The gene is frequently mutated in breast cancer patients and the mutations have been associated with poor prognosis and response rates to chemotherapy. The purpose of this study was to correlate p53 expression with MDM2, a proto-oncogene (12q14.3), which acts as a major negative regulator in p53-MDM2 auto-regulatory pathway., Methods: Seventy breast adenocarcinoma cases were included in the study. Sixty tumors were pathologically categorized as invasive ductal adenocarcinomas, whereas the rest of them were diagnosed as pure in situ carcinomas. Immunohistochemistry (IHC) was applied using anti-p53 and anti-MDM2 antibodies in the corresponding tissue sections., Results: Overexpression of p53 protein was observed in 39/60 (65%) invasive cases, while 40/60 (66.7%) expressed MDM2 protein. Interestingly, in 26/60 (43%) cases a combined p53/ MDM2 co-expression was detected, whereas in 7/60 (11%) a combined loss of expression was identified (overall co-expression: p=0.999). Concerning in situ carcinomas, co-expression of p53/MDM2 was observed in 7/10 (70%) cases., Conclusions: MDM2 oncogene overexpression - predominantly due to gene amplification - is a frequent and critical genetic event in both in situ and invasive breast adenocarcinomas. Accumulation of p53 protein in the nucleus of tumor cells harboring mutant p53 - as the result of its overexpression - does not mean necessarily decreased expression of MDM2. MDM2 directly binds to p53 and represses its transcriptional activity promoting p53 degradation. So targeting the molecule, p53's crucial tumor suppressor function is normally regulated.

Pericytes are modified smooth muscle cells that closely enwrap small blood vessels, regulating and supporting the microvasculature through direct endothelial contact. Pericytes demonstrate a distinct immunohistochemical profile, including expression of smooth muscle actin, CD146, platelet-derived growth factor receptor β, and regulator of G-protein signaling 5. Previously, pericyte-related antigens have been observed to be present among a group of soft tissue tumors with a perivascular growth pattern, including glomus tumor, myopericytoma, and angioleiomyoma. Similarly, malignant tumor cells have been shown to have a pericyte-like immunoprofile when present in a perivascular location, seen in malignant melanoma, glioblastoma, and adenocarcinoma. Here, we examine well-differentiated liposarcoma specimens, which showed some element of perivascular areas with the appearance of smooth muscle (n = 7 tumors). Immunohistochemical staining was performed for pericyte antigens, including smooth muscle actin, CD146, platelet-derived growth factor receptor β, and regulator of G-protein signaling 5. Results showed consistent pericytic marker expression among liposarcoma tumor cells within a perivascular distribution. MDM2 immunohistochemistry and fluorescence in situ hybridization for MDM2 revealed that these perivascular cells were of tumor origin (7/7 tumors), whereas double immunohistochemical detection for CD31/CD146 ruled out an endothelial cell contribution. These findings further support the concept of pericytic mimicry, already established in diverse malignancies, and its presence in well-differentiated liposarcoma. The extent to which pericytic mimicry has prognostic significance in liposarcoma is as yet unknown., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In contrast to long bone osteosarcoma, mandibular osteosarcoma is highly heterogeneous and morphologically overlaps with benign tumors, obscuring diagnosis and treatment selection. Molecular characterization is difficult due to the paucity of available specimens of this rare disease. We aimed to characterize the spectrum of mandibular osteosarcoma using immunohistochemistry and molecular techniques (quantitative polymerase chain reaction and sequencing) and compare them with benign fibro-osseous lesions. Forty-nine paraffin-embedded mandible osteosarcoma tissue samples were collected retrospectively and compared with 10 fibrous dysplasia and 15 ossifying fibroma cases. These were analyzed for molecular markers thought to differ between the different diseases and subtypes: MDM2 (murine double-minute type 2) overexpression, GNAS (guanine nucleotide-binding protein/α subunit) mutations, and amplification of MDM2 and/or RASAL1 (RAS protein activator like 1). Five fibroblastic high-grade osteosarcoma subtypes showed MDM2 amplification, including 2 with a microscopic appearance of high-grade osteosarcoma with part low-grade osteosarcoma (differentiated/dedifferentiated osteosarcoma) and MDM2 overexpression. The other 3 contained a coamplification of MDM2 and RASAL1, a signature also described for juvenile ossifying fibroma, with no overexpression of MDM2. These were of the giant cell-rich high-grade osteosarcoma, with areas mimicking juvenile ossifying fibroma (ossifying fibroma-like osteosarcoma). Our results show that some diagnosed high-grade osteosarcomas are differentiated/dedifferentiated osteosarcomas and harbor an overexpression and amplification of MDM2. In addition, juvenile ossifying fibromas can potentially evolve into giant cell-rich high-grade osteosarcomas and are characterized by a RASAL1 amplification (osteosarcoma with juvenile ossifying fibroma-like genotype). Thus, the presence of a RASAL1 amplification in ossifying fibroma may indicate a requirement for closer follow-up and more aggressive management., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Purpose: To evaluate the rate of overexpression of matrix metalloproteinase-2 (MMP2), mouse double minute 2 homolog (MDM2) and epidermal growth factor receptor (EGFR) in patients with non-small cell lung cancer (NSCLC), and evaluate their correlation with clinicopathological parameters and prognosis., Methods: This was a prospective cohort study conducted from 2003 to 2008 among 184 NSCLC patients who underwent tumor resection. Each patient's clinical history and tumor characteristics were obtained from histopathology reports and medical records. EGFR, MDM2 and MMP2 expression were assessed by immunohistochemical (IHC) staining of the tissue specimens., Results: MDM2 overexpression was observed in 70 (38%) of the patients studied, and was significantly higher in younger patients (p=0.01). Only 46 (25%) of patients had overexpression of MMP2. EGFR positive staining occurred in 105 (57%percnt;) of the evaluated tumor specimens and was more frequent in specimens with squamous cell carcinoma (p<0.001), the elderly (p<0.001), and in smokers (p<0.001). Independent risk factors for mortality were older age (adjusted odds ratio/aOR 1.3=), being a smoker (aOR 10), having stage II disease (aOR 10.8) or stage III/IV disease (aOR 28.3), expression of EGFR (aOR 5.9) and MMP2 (aOR 4.1). However, the expression of MDM2 independently predicted a reduced risk of death (aOR 0.3)., Conclusion: Overexpression of MMP2 and EGFR were independent risk factors for mortality in NSCLC patients, while overexpression of MDM2 independently predicted a reduced risk of death.

The GLI1 and MDM2 genes are amplified or exhibit copy number gains in rhabdomyosarcoma (RMS). Here, we used immunohistochemistry to determine the relationships between GLI1 and MDM2 protein expression and several clinicopathological variables of RMS. GLI1 and MDM2-positivity rates were 61.36% and 13.64%, respectively. GLI1 expression correlated with presence of the PAX3-FOXO1 fusion gene (P=0.040) and lymph node metastasis (P=0.034), and a significant association was found between GLI1 expression and overall survival (OS) (P=0.008). However, there was no association between MDM2 expression and any of the clinicopathological parameters or OS. Thus, GLI1 may be a biomarker of poor prognosis in RMS patients, and could itself be a therapeutic target. This contrasts with the apparent lack of clinical importance of MDM2 in RMS pathology, at least in the cohorts we examined.