Case Report and Review of Literature of Atypical Spindle Cell/Pleomorphic Lipomatous Tumor in the Paratesticular Region

Atypical spindle cell/pleomorphic lipomatous tumour (ASPLT) is an infrequently appreciated benign lipomatous neoplasm newly accepted into the most recent WHO classification of soft tissue tumours. Our cytopathology files were searched for examples of ASPLT and spindle cell/pleomorphic lipoma (SCPL) having histopathological verification. Conventional fine needle aspiration (FNA) biopsy smears were performed using standard techniques. Eleven patients including three cases of ASPLT and eight of SCPL (M:F=4.5:1; age range: 39-97 years, mean age=60 years) met the inclusion criteria. FNA biopsy sites included extremity (5, 45%), trunk (3, 27%), and head/neck (3, 27%). All aspirates were from primary neoplasms. FNA diagnoses of ASPLT cases were spindle cell lipomatous neoplasm, fibrotic low-grade SC neoplasm, and myxoid lipomatous neoplasm. Eight SCPL cases were diagnosed as spindle cell neoplasm (3), spindle cell lipoma (SCL) (1), pleomorphic lipoma (1), suspicious for SCL (1), benign adipose tissue (1), and benign spindle cells and connective tissue (1). Ancillary testing in two ASPLT cases showed positive CD34 and negative MDM2 immunostain in one, and negative FISH results for MDM2 and DDIT3 in another. ASPLT is a novel lipomatous neoplasm simulating primarily SCPL and atypical lipoma/well-differentiated liposarcoma. Diligent cytomorphological observation, clinical information, and ancillary testing may allow for its specific recognition using FNA biopsy.

DNA strand breaks are a prerequisite for many cancer-associated genomic alterations, including amplifications, deletions, inversions, and structural rearrangements. High-throughput technologies that measure genome-wide DNA quantities, such as DNA sequencing and single nucleotide polymorphism or comparative genomic hybridization arrays, enable researchers to accurately detect such lesions.1 Recently we reported a computational analysis of intragenic DNA breakpoints in glioblastoma (GBM), the most common adult tumor in the central nervous system.2 Using a cohort of more than 500 samples, we identified an expected increase in DNA strand breaks near genes commonly targeted by copy number gain and losses in GBM, such as EGFR on 7p, CDKN2A/B on 9q, and PTEN on 10q. Less anticipated was the enrichment of DNA breakpoints on an 18 megabase region encompassing cytoband 12q14–15. The 12q region, which we referred to as 12q14–15 breakpoint enriched region or BER, contains the well-known oncogenes murine double minute 2 (MDM2) and cyclin-dependent kinase 4 (CDK4), which are frequently amplified in GBM (7.6% and 14%, respectively).3 Detected in 24 of 493 (4.9%) GBMs, all BER samples showed a dense pattern of interspersed local copy number gains and losses. Among these small “copy number islands”, CDK4 and MDM2 were almost always amplified simultaneously (87.5% of BERs). Interestingly, each DNA gain appeared at similar dosage levels, suggesting that they were conjunctly amplified. Using matched RNA sequencing (RNAseq) data we observed direct gene fusions among the BER-related amplicons in 7 of 9 RNAseq available BER cases. This finding raises the question whether the frequent breaks in this relatively small region represent a mechanism aimed at simultaneous amplification of the CDK4 and MDM2 oncogenes. In our data set, the 21 of 33 (64%) of GBM with dual amplification of CDK4 and MDM2 also displayed a BER pattern, and this percentage may represent a lower boundary due to a stringent statistical threshold. Based on the pattern of revolving copy number gains and corroborating transcript fusions, we propose a hypothetical model that involves double minutes (Fig. 1), an extrachromosomal circular structure formed by DNA end-to-end joining.4 Using whole-genome sequencing data, more than 20 12q14–15 segments in a BER were shown to be connected and suggestive of a double minute-like structure.3 Double minutes have been reported as a means to amplify the epithelial growth factor receptor gene (EGFR) in gliomas.5 We speculate that glioma cells can similarly leverage this structure to jointly amplify CDK4 and MDM2. An unresolved issue is the mechanism by which this short DNA region is fragmented prior to the segments being joined. Figure 1. A double minute model to explain the 12q14–15 breakpoint enriched region (BER). Our survey of intragenic breakpoints identified a second unanticipated outlier of breakpoint enrichment in chromosome 1p. In this instance, the increased breakage frequency was related to the gene FAS1-associated factor 1 (FAF1). Intragenic breakpoints in FAF1, which we found to lead to mRNA depletion, is an epiphenomenon of the focal deletion of the 5′ adjacent tumor suppressor gene CDKN2C. However, transfection experiments demonstrated a proapoptotic role for FAF1 in cell culture and tumor-sphere formation. Our results suggest that converging breakpoints in one gene may provide increased cellular fitness, and thereby lead to proliferative advantages. In summary, our analysis revealed DNA breakpoints as a fingerprint of localized genome instability. In particular, we found a focal shattering pattern on 12q that was associated with adverse outcomes in a subset of glioblastoma patients.2 Related to the pattern, we reported orchestrated amplification of CDK4 and MDM2. Further studies are required to determine that the BER pattern relates to a synergistic interaction between the 2 oncogenes.

Atypical spindle cell/pleomorphic lipomatous tumour (AS/PLT) is a novel entity described under the benign lipomatous neoplasm recognised by the recent WHO classification of soft tissue tumours (fifth edition). In this report, we present a case of a 44-year-old male who with a swelling on dorsum of the hand measuring 8 × 8 cm. A Fine needle aspiration cytology (FNA) was performed which revealed adipocytic fragments with individual cells exhibiting mild to moderate nuclear atypia with floret like cells, multinucleated giant cells along with few pseudo-lipoblast-like cells. Based on cytomorphological findings a possibility of an Atypical spindle cell/Pleomorphic lipomatous tumour [AS/PLT] was given. An excision was performed which revealed features confirmed the diagnosis of an AS/PLT. MDM-2 Immunohistochemistry was performed and was negative ruling out a well differentiated Liposarcoma. Hence, the diagnosis was confirmed on histopathological examination. AS/PLT is a unique novel benign neoplasm with a slightly higher recurrence rate than a Pleomorphic lipoma. Here, we discussed the characteristic cyto-morphological features of an AS/PLT that help in the diagnosis and guide further surgical management.

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

Can MDM2 analytical tests performed on core needle biopsy be relied upon to diagnose well-differentiated liposarcoma?

Adipocytic tumors exist either as a benign or malignant form. The benign variant, lipoma, is composed of normal fat tissue. Lipomas typically develop from superficial fat cells beneath the skin or mucous membranes. Liposarcoma, the malignant counterpart, often develops in deeper tissues and is the most commonly diagnosed Soft Tissue Sarcoma (STS), comprising at least 20% of adult STS. However, malignant tumors of fatty origin exist as a spectrum of diagnoses, each carrying a unique risk of recurrence, metastasis, and longterm survival. The World Health Organization classifies liposarcomas into five categories: i) Atypical Lipomatous Tumors/Well Differentiated (ALT/WD); Ii) Dedifferentiated (ALT/DD); Iii) Myxoid; Iv) Round cell; and v) Pleomorphic. Lipomatous tumors often exhibit different immunohistochemical patterns. Benign lipomas are distinguished by the absence of Murine Double-Minute 2 (MDM2) amplification. Similarly, ALT/WD, classically defined as a low-grade and locally aggressive tumor, demonstrates consistent patterns of MDM2 amplification. Some studies suggest 10% of ALT/WD progress to the highgrade DD form, with others report a dedifferentiation rate of as high as 20% for primary ALT/WD based on location. The ALT/DD subtype is aggressive and has a high capacity to metastasize. While the mechanism of pathogenesis of ALT/DD metastasis is unknown, previous studies suggest that increased MDM2 amplification may play a role. This study sought to evaluate a single institutional experience treating the entire spectrum of lipomatous tumors and describe utilization patterns of MDM2 testing. The group hypothesized: i) Atypical Lipomatous Tumors (ALT), which include ALT/DD and ALT/WD, would exhibit a higher rate of local recurrence than lipomas with no significantly increased incidence of metastases; and ii) at least 50% of our MDM2 testing of ALT would prove positive for the MDM2 overamplification. This study retrospectively reviewed 105 cases (66 lipomas, 27 ALTs, 12 liposarcomas) of patients who underwent lipomatous tumor excision at our institution from 2013 to 2017. Twenty-five tumors (6 lipomas, 18 ALT, 1 liposarcoma) were tested for MDM2 amplification. Three of the tested tumors recurred (2 ALT, 1 liposarcoma), and each exhibited MDM2 overamplification. Five tumors (5 liposarcoma) developed late metastases. These data suggest that although ALT is associated with a higher rate of local recurrence, metastases are quite rare. Additionally, the data demonstrate a high rate of positive MDM2 testing (76%) based on clinical and imaging characteristics of the tumors.

Aggressive angiomyxoma with multinucleated giant cells: a lesion mimicking liposarcoma.

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.

Comments on the pathogenesis and medical treatment of central giant cell granulomas

ObjectiveLiposarcomas are rare tumors, and it is difficult to collect cases in less densely populated areas. Therefore, we aimed to document more cases over a relatively long period to provide more data about the characteristics of liposarcomas. In this study, the clinicopathological features of liposarcomas were investigated in 27 patients.Material and MethodsAll cases were confirmed by diagnosis through hematoxylin and eosin staining, immunohistochemistry (IHC), and fluorescence in situ hybridization (FISH). Combined IHC analysis was performed for murine double minute 2 (MDM2), cyclin-dependent kinase 4 (CDK4), multiple tumor suppressor 1 (P16), and Cyclin D1. FISH was performed to detect MDM2 amplification in atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDLPS) and dedifferentiated liposarcoma (DDLPS), and DNA damage inducible transcript 3 ( DDIT3) rearrangements in myxoid liposarcoma (MLPS).ResultsSeven cases of liposarcoma were located in the paratesticular region (25.9%, 7/27), 12 in the retroperitoneum (44.4%, 12/27), and eight in the limbs (29.6%, 8/27). Histological analysis showed that there were 13 cases of ALT/WDLPS (48.1%, 13/27), nine cases of DDLPS (33.3%, 9/27), three cases of MLPS (11.1%, 3/27), and two cases of pleomorphic liposarcoma (7.4%, 2/27). IHC analysis revealed that 26 cases were MDM2-positive (96.3%, 26/27), 22 were CDK4-positive (81.5%, 22/27), 26 were P16-positive (96.3%, 26/27), and 27 were cyclin D1-positive (100%, 27/27). FISH analysis revealed 20 cases of MDM2 positivity (90.9%, 20/22) and one case of DDIT3 positivity (50%, 1/2). The clinical outcomes were available for 21 patients. Four patients died (4/21, 19.0%), five experienced recurrence (5/21, 23.8%), and 12 (12/21, 57.1%) survived with no other disease.ConclusionA combined IHC examination of the four indicators may be used to diagnose ALT/WDLPS and DDLPS, and FISH is recommended as an important supporting method.

Atypical spindle cell/pleomorphic lipomatous tumor (ASPLT) is a new entity of benign adipocytic tumor that spans a wide spectrum of histology from adipocytic to spindle cell/pleomorphic tumors. The latter non-adipocytic component rarely shows sarcomatous features although ASPLTs are not thought to dedifferentiate. A 78-year-old woman with ASPLT in the left thigh had a sarcomatous component with high mitotic activity and Ki-67 labeling index (LI) mimicking dedifferentiated liposarcoma. The adipocytic component consisted of various-sized adipocytic cells with few lipoblasts. The sarcomatous component consisted of a fascicular proliferation of atypical spindle cells with scattered large bizarre and multinucleated giant cells. Mitotic figures including atypical mitoses were frequently observed. Immunohistochemically, the tumor cells were positive for cluster of differentiation 34 but not mouse double minute 2 homolog (MDM2), cyclin-dependent kinase 4 (CDK4), or retinoblastoma (Rb) protein. Ki-67 LI in the sarcomatous component reached 40%. MDM2 and CDK4 genes were not amplified and 13q14 including the RB1 locus was deleted according to fluorescence in situ hybridization. The patient is alive with no evidence of local recurrence or distant metastasis 3.5years after surgery. As ASPLT may exhibit morphological variation, it is important to rule out dedifferentiated liposarcoma with careful pathological examination.

A subset of fat-predominant angiomyolipomas label for MDM2: a potential diagnostic pitfall

Atypical lipomatous tumors (ALTs) are locally aggressive adipocytic malignancies that frequently occur in middle-aged adults. We report the rare case of an ALT of the thigh that occurred in a 4-year-old girl. Since the tumor was initially diagnosed as a lipoblastoma by incisional biopsy, marginal resection was performed. Histopathological findings of the surgical specimen revealed the proliferation of mature and variously sized adipocytes, as well as ectopic ossification; these features differ from the typical findings of lipoblastoma. Immunohistochemical findings showed nuclear positivity for a murine double minute 2 (MDM2) and cyclin-dependent kinase 4 (CDK4) and negativity for pleomorphic adenoma gene 1 (PLAG1). Fluorescence in situ hybridization showed abnormal amplification of the MDM2 gene. The patient was thus finally diagnosed as having an ALT. No signs of local recurrence or metastasis were noted 1 year postoperatively. This case is instructive in the differential diagnosis of primary adipocytic tumors. Lipoblastomas are the most common adipocytic tumors in children, but if a tumor is located in the deep tissue or imaging findings are not typical, the possibility of ALT should be considered and immunohistochemistry for MDM2 and CDK4 should be added.

Overexpression of p21 WAF1/C1P1 and MDM2 characterizes serous borderline ovarian tumors

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