Targeted DNA Sequencing in Diagnosis of Malignant Phyllodes Tumors With Emphasis on Tumors With Keratin and p63 Expression.

The differential diagnosis of malignant spindle cell neoplasms in the breast most frequently rests between malignant phyllodes tumor (MPT) and metaplastic carcinoma (MBC). Diagnosis of MPT can be challenging due to diffuse stromal overgrowth, keratin (CK) and/or p63 immunopositivity, and absent CD34 expression, which can mimic MBC, especially in core biopsies. Distinction of MPT from MBC has clinical implications, with differences in surgical approach, chemotherapy, and radiation. In this study, we evaluated MPTs (78 tumors, 64 patients) for stromal CK, p63, and CD34 expression and profiled a subset (n = 31) by targeted next-generation DNA sequencing, with comparison to MBC (n = 44). Most MPTs (71%) were CK+ and/or p63+, including 32% CK+ (25/77 focal) and 65% p63+ (32/66 focal, 10/66 patchy, and 1/66 diffuse). Thirty percent of MPTs expressed both CK and p63 (20/66), compared with 95% of MBCs (40/42, P < .001). CK and/or p63 were positive in CD34+ and CD34- MPTs. Recurrent genetic aberrations in MPTs involved TERT, TP53, MED12, CDKN2A, chromatin modifiers, growth factor receptors/ligands, and phosphoinositide-3 kinase (PI-3K) and MAPK pathway genes. Only MED12 (39%, 12/31) and SETD2 (13%, 4/31) were exclusively mutated in MPTs and not MBCs (P < .001 and P = .044, respectively), whereas PIK3R1 mutations were only found in MBCs (37%, 13/35, P < .001). Comparative literature review additionally identified ARID1B, EGFR, FLNA, NRAS, PDGFRB, RAD50, and RARA alterations enriched or exclusively in MPTs vs MBCs. MED12 was mutated in MPTs with diffuse stromal overgrowth (53%, 9/17), CD34- MPTs (41%, 7/17), and CK+ and/or p63+ MPTs (39%, 9/23), including 36% of CD34- MPTs with CK and/or p63 expression. Overall, MED12 mutation and/or CD34 expression were observed in 68% (21/31) MPTs, including 61% (14/23) of CK+ and/or p63+ tumors. Our results emphasize the prevalence of CK and p63 expression in MPTs and demonstrate the diagnostic utility of next-generation DNA sequencing, especially in MPTs with confounding factors that can mimic MBC.

Diagnostic utility of FOXO1 immunohistochemistry for rhabdomyosarcoma classification.

AIMS: Rhabdomyosarcomas currently are classified into one of four subtypes (alveolar, embryonal, spindle cell/sclerosing, or pleomorphic) according to their morphological, immunohistochemical, and molecular genetic features. The alveolar subtype is characterised by a recurrent translocation involving PAX3 or PAX7 and FOXO1; identification of this translocation is important for appropriate classification and prognostication. In this study, we aimed to explore the diagnostic utility of FOXO1 immunohistochemistry for rhabdomyosarcoma classification. METHODS/RESULTS: A monoclonal antibody targeting a FOXO1 epitope retained in the fusion oncoprotein was used to study 105 rhabdomyosarcomas. FOXO1 was positive for expression by immunohistochemistry in all 25 alveolar rhabdomyosarcomas, with 84% showing diffuse expression in greater than 90% of neoplastic cells; the remainder of alveolar rhabdomyosarcomas displayed at least moderate staining in a minimum of 60% of lesional cells. Apart from three spindle cell rhabdomyosarcomas showing heterogeneous nuclear immunoreactivity in 40-80% of tumour cells, the 80 cases of embryonal, pleomorphic, and spindle cell/sclerosing rhabdomyosarcoma were negative for FOXO1 expression (96.3% specific) when using a threshold of nuclear staining in 20% of neoplastic cells to determine positivity. Variable cytoplasmic staining was present in a fraction of all rhabdomyosarcoma subtypes. Nonneoplastic lymphocytes, endothelial cells, and Schwann cells also showed variably intense nuclear anti-FOXO1 immunoreactivity. CONCLUSION: Taken together, our findings suggest that FOXO1 immunohistochemistry is a highly sensitive and relatively specific surrogate marker of the PAX3/7::FOXO1 fusion oncoprotein in rhabdomyosarcoma. Cytoplasmic immunoreactivity, expression in nonneoplastic tissues, and limited nuclear staining of nonalveolar rhabdomyosarcomas represent potential pitfalls in interpretation.

Contemporary diagnostic approach to atypical vascular lesion and angiosarcoma.

Vascular neoplasms account for a substantial fraction of cutaneous mesenchymal tumors, spanning from clinically indolent benign lesions to highly aggressive malignancies. These neoplasms present a distinctive challenge in terms of their diagnostic histopathology, both because of the breadth of their morphological manifestations and because of the significant histological overlap between different entities, even benign and malignant ones. The post-radiotherapy setting is particularly problematic diagnostically, insofar as radiation exposure predisposes not only to secondary angiosarcoma, but also to atypical vascular lesion, a largely benign proliferation of cutaneous blood vessels typically affecting the breast. To address these challenges, we explore the clinical, histological, and molecular features of malignant vascular neoplasia, including primary and secondary subtypes, through the comparative lens of atypical vascular lesion. In addition to highlighting the key morphological indicators of malignancy in superficial vasoformative tumors, we offer an approach that integrates clinical characteristics and molecular genetic profiling to facilitate accurate classification. With this current knowledge as our foundation, we also look ahead in an effort to frame some of the key unanswered questions regarding superficial vascular malignancies and their natural history, clinical management, and molecular underpinnings.

A Distal Loop Controls Product Release and Chemo- and Regioselectivity in Cytochrome P450 Decarboxylases.

Cytochrome P450 OleT utilizes hydrogen peroxide (H2O2) to catalyze the decarboxylation or hydroxylation of fatty acid (FA) substrates. Both reactions are initiated through the abstraction of a substrate hydrogen atom by the high-valent iron-oxo intermediate known as Compound I. Here, we specifically probe the influence of substrate coordination on OleT reaction partitioning through the combined use of fluorescent and electron paramagnetic resonance (EPR)-active FA probes and mutagenesis of a structurally disordered F-G loop that is distal from the heme-iron active site. Both probes are efficiently metabolized by OleT and efficiently trigger the formation of Compound I. Transient fluorescence and EPR reveal a slow product release step, mediated by the F-G loop, that limits OleT turnover. A single-amino acid change or excision of the loop reveals that this region establishes critical interactions to anchor FA substrates in place. The stabilization afforded by the F-G loop is essential for regulating regiospecific C-H abstraction and allowing for efficient decarboxylation to occur. These results highlight a regulatory strategy whereby the fate of activated oxygen species can be controlled at distances far removed from the site of chemistry.

The Enigmatic P450 Decarboxylase OleT Is Capable of, but Evolved To Frustrate, Oxygen Rebound Chemistry.

OleT is a cytochrome P450 enzyme that catalyzes the removal of carbon dioxide from variable chain length fatty acids to form 1-alkenes. In this work, we examine the binding and metabolic profile of OleT with shorter chain length (n ≤ 12) fatty acids that can form liquid transportation fuels. Transient kinetics and product analyses confirm that OleT capably activates hydrogen peroxide with shorter substrates to form the high-valent intermediate Compound I and largely performs C-C bond scission. However, the enzyme also produces fatty alcohol side products using the high-valent iron oxo chemistry commonly associated with insertion of oxygen into hydrocarbons. When presented with a short chain fatty acid that can initiate the formation of Compound I, OleT oxidizes the diagnostic probe molecules norcarane and methylcyclopropane in a manner that is reminiscent of reactions of many CYP hydroxylases with radical clock substrates. These data are consistent with a decarboxylation mechanism in which Compound I abstracts a substrate hydrogen atom in the initial step. Positioning of the incipient substrate radical is a crucial element in controlling the efficiency of activated OH rebound.

Acellular Dermal Matrix: Imaging Features With Histopathology Correlation.

Acellular dermal matrix (ADM) is an immunologically inert graft, typically from cadaveric skin, often used in postmastectomy breast reconstruction. Created from decellularized dermal tissues that have been treated to remove DNA and antigenic donor cells (leaving extracellular matrix), ADM is often used as a structural scaffold or sling to reinforce and support the structure and position of a breast implant during postoperative integration in implant-based breast reconstruction; ADM can also be used to fill cosmetic defects. Advantages of ADM use include improved cosmesis and reduced capsular contracture rates. On US, ADM can be seen as a subtle band with variable echogenicity adjacent to the implant. When folded on itself or redundant, ADM may present as a palpable oval mass with indistinct or circumscribed margins and variable echogenicity. On mammography, ADM can be seen as a circumscribed oval equal density mass when redundant and folded on itself; a layered appearance may be evident on tomosynthesis. On MRI, presence and absence of enhancement have been documented. Imaging findings likely vary depending on the degree of host tissue remodeling and incorporation, and when biopsied, histopathologically, ADM may be difficult to distinguish from scarring. Successful imaging diagnosis of ADM is aided by clinical knowledge of the intraoperative use and configuration of ADM, which may help differentiate ADM from new or recurrent malignancy and avoid unnecessary biopsy.

Enhancing ferryl accumulation in H2O2-dependent cytochrome P450s.

A facile strategy is presented to enhance the accumulation of ferryl (iron(IV)-oxo) species in H2O2 dependent cytochrome P450s (CYPs) of the CYP152 family. We report the characterization of a highly chemoselective CYP decarboxylase from Staphylococcus aureus (OleTSA) that is soluble at high concentrations. Examination of OleTSA Compound I (CpdI) accumulation with a variety of fatty acid substrates reveals a dependence on resting spin-state equilibrium. Alteration of this equilibrium through targeted mutagenesis of the proximal pocket favors the high-spin form, and as a result, enhances Cpd-I accumulation to nearly stoichiometric yields.

Mixed regiospecificity compromises alkene synthesis by a cytochrome P450 peroxygenase from Methylobacterium populi.

Intensive interest has focused on enzymes that are capable of synthesizing hydrocarbons, alkenes and alkanes, for sustainable fuel production. A recently described cytochrome P450 (OleTJE) from the CYP152 family catalyzes an unusual carbon-carbon scission reaction, transforming Cn fatty acids to Cn-1 1-alkenes. Here, we show that a second CYP152, CYP-MP from Methylobacterium populi ATCC BAA 705, also catalyzes oxidative substrate decarboxylation. Alkene production is accompanied with the production of fatty alcohol products, underscoring the mechanistic similarity of the decarboxylation reaction with canonical P450 monooxygenation chemistry. The branchpoint of these two chemistries, and regiospecificity of oxidation products, is strongly chain length dependent, suggesting an importance of substrate coordination for regulating alkene production.