IMP3, CDK4, MDM2 and ß-catenin expression in Enchondroma and Central Chondrosarcoma: Diagnostic and prognostic utility.

INTRODUCTION: The role of IMP3, CDK4, MDM2 and ß-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 ß-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 ß-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.

Right atrial cardiac myxoma with malignant transformation to undifferentiated sarcoma: A case report.

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.

DDIT3-amplified or low-polysomic pleomorphic sarcomas without MDM2 amplification: Clinicopathological review and immunohistochemical profile of nine cases.

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.

Atypical lipomatous tumor/well differentiated liposarcoma and related mimics with updates. When is molecular testing most cost-effective, necessary, and indicated?

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.

[Clinical value of fluorescence in situ hybridization with MDM2 and DDIT3 probe in diagnosis of liposarcoma].

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.

Liposarcoma of the pleural cavity.

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.

Comprehensive genomic profiling of a unique liposarcoma arising in a patient with Li-Fraumeni syndrome and the novel detection of c-myc amplification: a case report.

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.

Clinical biomarkers in soft tissue sarcoma A comprehensive review of current soft tissue sarcoma biomarkers.

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.

Akt Phosphorylation Orchestrates T11TS Mediated Cell Cycle Arrest in Glioma Cells.

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.

MDM2 amplification in malignant Brenner tumors may play a role in progression to malignancy and aid in separation from urothelial and other ovarian carcinomas.

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.

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.

Synchronous Renal Dedifferentiated Liposarcoma and Retroperitoneal Well-Differentiated Liposarcoma: A Case Report With Literature Review.

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.

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

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

The Utility of MDM2 and CDK4 Immunohistochemistry and MDM2 FISH in Craniofacial Osteosarcoma.

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.

Combined Effect of HPV and Several Gene SNPs in Laryngeal Cancer.

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. Conclusions: This finding suggests patients survival prognosis and tumor behavior are different according HPV status, SNP variants, and clinical characteristics of the LSCC.

Dual-channel surface plasmon resonance monitoring of intracellular levels of the p53-MDM2 complex and caspase-3 induced by MDM2 antagonist Nutlin-3.

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.

"Fat-Rich" (Spindle Cell-Poor) Variant of Atypical Spindle Cell/Pleomorphic Lipomatous Tumor: Striking Mimic of "Classical" Atypical Lipomatous Tumor/Well-Differentiated Liposarcoma.

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.

Lipomas of the Oral Cavity: Utility of MDM2 and CDK4 in Avoiding Overdiagnosis as Atypical Lipomatous Tumor.

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.