Supplemental figure demonstrating relationship of mean fluorescence intensity for normal tissues compared to tumor.

Supplemental figure correlating biological variance ratio (BVR) to contrast-to-variance ratio (CVR).

Supplemental figure correlating biological variance ratio (BVR) to tumor to background ratio (TBR).

Low-grade fibromyxoid sarcoma (LGFMS) arising at acral sites is exceedingly rare, and its clinicopathologic features and clinical outcomes remain poorly characterized. Herein, we report a cohort of ten cases of acral LGFMS. The cohort included six males and four females, with a mean age at diagnosis of 23 years (median, 14 years; range, 2–67 years). Tumors arose in the foot (n = 7) or hand (n = 3). All cases were superficially located and exhibited classic morphologic features of LGFMS. Diffuse MUC4 expression was observed in all nine cases tested. Fluorescence in situ hybridization (FISH) and next-generation sequencing (NGS) demonstrated FUS gene rearrangements in all five cases analyzed. Clinical follow-up was available for six patients, with a mean duration of 37 months (range, 6–91 months). One patient developed a local recurrence and subsequently underwent digital amputation. No patients have developed metastases to date. Recognition of this rare, deceptively bland neoplasm in acral locations is critical, given its potential for local recurrence and metastatic spread.

Ossifying fibromyxoid tumor (OFMT) is a rare mesenchymal neoplasm first described in 1989. It typically arises in the superficial soft tissues of the extremities as a slow-growing, painless mass. Histologically, it is commonly characterized by a multilobular architecture composed of uniform epithelioid cells embedded in a fibromyxoid matrix, often surrounded by a rim of metaplastic bone. While classic cases are readily identifiable, the tumor's histopathological heterogeneity can mimic a range of benign and malignant neoplasms, posing significant diagnostic challenges. Molecularly, most OFMTs harbor PHF1 rearrangements, commonly involving fusion partners such as EP400, MEAF6, or TFE3. This review underscores the importance of an integrated diagnostic approach- incorporating histopathological, immunohistochemical, and molecular data- to accurately classify OFMT and distinguish it from its mimics. Expanding awareness of its morphologic and molecular spectrum is essential for precise diagnosis, optimal patient management, and a deeper understanding of this enigmatic neoplasm.

Supplemental methods and materials for testing the binding of ABY-029 to EGFR in A431 cells.

Since the original description of CRTC1::TRIM11 fusion cutaneous tumors (CTCT) in 2018, an increasing number of cases with metastatic behavior have been reported, yet there are limited studies analyzing prognostic parameters. This expanding data set presents an opportunity for reappraisal of clinical behavior. We present a series of 21 cases with detailed morphologic, molecular, clinical outcomes, and therapeutic response data. We utilize this data and a meta-analysis of all the cases in the literature to assess potential prognostic parameters to assist in clinical management. Primary tumors resulting in metastasis were larger ( P =0.038), had higher mitotic counts ( P <0.001), were more frequently ulcerated ( P =0.001), and exclusively harbored TERT promoter mutations ( P =0.005). A functional analysis of mixed data demonstrated a clear clustering pattern in which metastatic cases had larger diameters and were more mitotically active compared with nonmetastatic cases. Inclusion of TERT promoter mutational status further improved the model. There were no metastatic events among cases meeting all the following criteria: size <1.2 cm, <7 mitoses/mm 2 , and absent TERT promoter mutation. Metastatic events frequently involved regional lymph nodes, and all patients with distant metastasis had pulmonary involvement. Considering metastatic events in 23% of cases, sentinel lymph node biopsy and imaging studies may be considered in some cases, while, in cases with smaller size, low mitotic counts, and no evidence of a TERT promoter mutation, excision alone may be adequate. Given poor therapeutic responses in reported metastatic cases, more therapeutic options should be explored.

Dermatofibrosarcoma protuberans (DFSP) is a locally aggressive fibroblastic neoplasm characterized by recurrent COL1A1::PDGFB fusions that drive autocrine PDGFR-β activation. Recent sequencing studies have revealed rare DFSPs with alternative noncanonical PDGFB or PDGFD rearrangements, underscoring the genetic diversity of PDGF-ligand activation in this tumor. Here, we describe two conventional DFSPs harboring previously unreported fusions: FGL2::PDGFD and TGFBI::PDGFB. In the first case, FGL2(ex1)::PDGFD(ex6) replaces the PDGFD N-terminal CUB domain with the FGL2 signal peptide, a configuration predicted to generate a constitutively active, secreted PDGFD ligand. In the second case, TGFBI(ex14)::PDGFB(ex3) juxtaposes PDGFB to the highly expressed extracellular matrix gene TGFBI, providing an alternative promoter context for PDGFB overexpression. Both cases showed classic DFSP morphology and diffuse CD34 expression. These findings expand the molecular landscape of DFSP and illustrate convergent mechanisms of PDGFR-β activation achieved through diverse yet functionally equivalent genomic rearrangements.

PURPOSE Irinotecan and temozolomide (iT) have antitumor activity in relapsed Ewing sarcoma (ES). The purpose of this phase II trial was to evaluate whether iT added to an alkylator-intensified five-drug regimen (Ewing Family of Tumors [EFT]) improved the 4-year event free survival (EFS) for patients with newly diagnosed localized ES. METHODS Patients with localized ES received six cycles of iT consolidation following completion of 21 weeks of EFT chemotherapy; patients with metastatic disease received 10 intercalated cycles of iT during EFT. The primary end point was 4-year EFS for patients with localized disease, with 85% and 70% of patients without events deemed promising and not promising, respectively. Secondary end points included EFS for metastatic patients, overall survival (OS), toxicity, and exploratory circulating tumor DNA analyses. RESULTS Eighty-three patients were enrolled (46 localized, 37 metastatic; median age 16 years). The 4-year EFS for patients with localized disease was 76% (95% CI, 64% to 90%), which did not exceed the predefined threshold for unpromising efficacy. Among patients with metastatic disease, 4-year EFS was 56% (95% CI, 37% to 84%) for those with lung-only metastases and 43% (95% CI, 25% to 74%) for those with extrapulmonary metastases. Febrile neutropenia occurred in 96% of patients, most frequently following alkylator-containing cycles. Secondary malignancies developed in four patients. No unexpected toxicities were observed. CONCLUSION The addition of iT to high-dose, alkylator-based up-front chemotherapy did not exceed the historical EFS benchmark in patients with radiographically localized ES.

Supplemental tables: Table S1, S2, and S3.

This article highlights selected cutaneous mesenchymal tumors, emphasizing histopathology, immunophenotype, and molecular alterations. It outlines diagnostic pitfalls, distinguishing features, and clinical behavior of indolent and malignant lesions. Awareness ensures accurate and appropriate treatment, and prevents overtreating benign mimics.

PURPOSE Desmoplastic small round cell tumor (DSRCT) is a rare sarcoma which remains almost universally fatal despite multimodality therapy. Human epidermal growth factor receptor 2 (HER2) has been identified as a potential therapeutic target prompting HER2-specific investigations and subsequent off-label treatment with fam-trastuzumab deruxtecan (T-DXd) in our DSRCT clinical cohort. METHODS Fifty-two and 61 DSRCT patient samples were analyzed by immunohistochemical (IHC) assays and RNAseq, respectively. In addition, 19 patients at a single institution with DSRCT in need of therapy underwent IHC testing and RNAseq when feasible and received off-label T-DXd (monotherapy [n = 14], alternating with another agent [n = 2], concomitantly in combination [n = 2]; measurable disease [n = 17], adjuvant setting [n = 2]). RESULTS HER2 expression levels by RNAseq were analyzed in the context of 346 patients with solid tumor and 23 histologies, with DSRCT having the third highest HER2 expression level overall and a median transcripts per million (TPM) expression level of 41.8 (range, 6.3-116.3). While TMA IHC analyses noted minimal HER2 IHC positivity using 4B5, IHC with clones 29D8 and CB11 uncovered 8.7- to 12.7-fold more HER2 reactivity, respectively. All 17 patients with measurable disease experienced clinical benefit (stable disease or partial response [PR]) with minimal toxicity. In addition, 9 of 17 patients achieved a RECIST PR (53% overall response rate). The response rate did not correlate with available IHC or RNAseq data, and some patients with no membranous IHC expression achieved PR. CONCLUSION These data show that T-DXd is active in DSRCT and support the planned biomarker-agnostic formal clinical trial with the Children's Oncology Group.

Understanding the molecular underpinnings of oncogenesis has led to an expansion of the spectrum of superficial mesenchymal neoplasms. The present review aims to provide an overview of the clinicopathologic and molecular characteristics of emerging entities with disease-defining molecular alterations. These include benign and indolent tumors such as hemangioma with epithelioid features harboring TPM3/4::ALK fusions, OGT-rearranged mesenchymal neoplasm, acral fibrochondromyxoid tumor, superficial FET::ETS neurocristic tumor, as well as hybrid peripheral nerve sheath tumors (hPNSTs), a subset of which harbor recurrent VGLL3 rearrangements or alternative rare and unique fusions. A concise review of the main characteristics of recently described biologically aggressive to overtly malignant tumors is also presented, including nodular necrotizing fibroblastic tumor, superficial GLI1-altered mesenchymal tumor, CRTC1::TRIM11 cutaneous tumor, and TFCP2-rearranged rhabdomyosarcoma. The review concludes with an update on dermatofibrosarcoma protuberans (DFSP). A subset of DFSPs harbor PDGFD rearrangements, and approximately 5% of both PDGFB- and PDGFD-rearranged DFSPs show S100 expression, which may pose a diagnostic challenge for practicing pathologists. These findings have further elucidated the molecular background and associated morphologic characteristics of superficial mesenchymal tumors, leading to the recognition of novel entities and further subclassification of existing ones, with important implications for diagnosis and patient management.

With the first series of PRRX1-rearranged tumors published in 2019, the spectrum of these so-called fibroblastic tumors has been expanded. Since then, several smaller case series have been published; however, our understanding of them continues to be quite limited given their rarity. We herein studied 18 additional cases, the largest series to date. Eighteen tumors present in 9 male, 8 female, and 1 nonbinary patient with a median age of 35 years (range: 11 to 70 y) and involved the neck (5), the chest region (4), thigh (3), back (1), shoulder (1), forehead (1), lower leg (1), axilla (1), and the parapharyngeal region (1). Clinical follow-up (9/18 tumors; 50%; median: 10 mo; range: 4 to 40 mo) showed consistent indolent behavior without local recurrences or distant metastases. On morphology, these tumors were characterized by well-circumscription and distinctive peripheral crescent-shaped vessels. They were composed of uniform spindle and round cells growing in short fascicles within often densely hyalinized collagen lacking significant mitotic activity, necrosis, or cytologic atypia. Immunohistochemically, about half of the tested tumors expressed focal to rarely diffuse S100 with occasional co-expression of SOX10. Interestingly, almost half of the tested cases also showed complete loss of RB expression. All but 1 tumor harbored a PRRX1::NCOA1 fusion, while 1 case harbored a novel PRRX1::EP300 fusion. We herein provide additional data on these exceptionally uncommon tumors, expand their molecular spectrum, and compare them to their close morphologic mimics to aid in accurate diagnosis and avoid confusion with potentially more aggressive neoplasms.

Cribriform tumor of the skin (formerly primary cutaneous cribriform carcinoma/primary cutaneous cribriform apocrine carcinoma) is a rare adnexal neoplasm of uncertain malignant potential. Recent studies have identified recurrent co-deletion of the long arm of chromosomes 6 and 9 and CD38 overexpression as potential diagnostic features, but their sensitivity and specificity remain incompletely defined. We identified 11 cribriform tumors with classic morphologic features, including several previously reported molecular characterized cases and additional unpublished cases, from multiple institutions. CD38 immunohistochemistry was performed on all tumors and compared with a panel of morphologic mimics, including adenoid cystic carcinoma (n = 8), digital papillary adenocarcinoma (n = 8), eccrine/apocrine adenomas (n = 7), hidradenoma (n = 2), and endocrine mucin-producing sweat gland carcinoma (n = 3). SNP array analysis was available in nine cases. Patients had a median age of 47 years with a slight female predominance (64

Hybrid peripheral nerve sheath tumors (PNSTs) are rare mesenchymal neoplasms with dual differentiation, most often combining two of the three main PNST types: schwannoma, neurofibroma, and perineurioma. Recognized by the WHO since 2013, these tumors can also exhibit fewer common combinations, such as hybrid granular cell tumor/perineurioma. We herein report two rare cases of hybrid granular cell tumor perineurioma with molecular analysis. Both tumors presented as dermal to subcutaneous, well-circumscribed lesions composed of a combination of spindled and granular cell components. By immunohistochemistry, the granular cells were positive for S100, SOX10, and CD68, while the perineurial cells were highlighted by EMA and GLUT1 stains. Subsequent molecular testing revealed pathogenic mutations involving PIK3CA in both cases. Our study expands on the clinical and pathologic spectrum and provides the first molecular data on these unusual neoplasms. Further, we provide a comprehensive review of the literature of all previously reported cases and briefly discuss relevant differential diagnoses and their molecular features.

Background: The 23-gene expression signature (GES) assay (myPath Melanoma) is a well-established molecular test for analyzing challenging melanocytic lesions, alongside fluorescence in situ hybridization (FISH) and single nucleotide polymorphism (SNP) array. However, routine use of these tests is often limited by high costs, long turnaround times, significant tissue requirements, and limited accessibility. This study aimed to evaluate the diagnostic concordance of PRAME immunohistochemistry (IHC) and the GES assay in difficult melanocytic lesions to determine whether PRAME IHC, widely available in pathology laboratories, could serve as a surrogate for GES. In addition, we correlated these methods with SNP array and FISH analyses, where available, to assess their diagnostic value in challenging melanocytic lesions. Methods: We conducted a single-institution retrospective analysis of 56 diagnostically ambiguous melanocytic lesions that underwent ancillary GES testing. All 56 cases were evaluated for PRAME IHC, while 35 had FISH results, 16 had SNP array results, and 3 had both FISH and SNP results. Two board-certified dermatopathologists independently reviewed hematoxylin and eosin (H&E)-stained slides, PRAME IHC slides, and other available immunostains, along with GES results, FISH, and/or SNP array results, classifying each lesion as benign or malignant. Results: Fifty-six cutaneous melanocytic lesions with challenging histopathologic features were evaluated. Diagnostically ambiguous categories included dysplastic nevi versus melanoma (29 cases, 51.8%), spitzoid lesions (19 cases, 33.9%), nevoid lesions (7 cases, 12.5%), and blue nevus-like lesions (1 case, 1.8%). Of these, 38 cases (67.9%) were ultimately classified as benign, while 18 cases (32.1%) were classified as malignant. PRAME IHC showed a significant association with malignancy, with an 83.9% concordance rate (χ 2 = 21.37, P = 0.0001), accurately classifying 47 out of 56 cases. GES demonstrated an 82.1% concordance rate (χ 2 = 18.68, P = 0.0001), accurately classifying 46 out of 56 cases. FISH showed a 77.1% agreement with the final diagnosis (χ 2 = 7.63, P = 0.005), correctly categorizing 27 out of 35 cases. Although the number of cases were relatively small, SNP array analysis correctly identified all 16 cases (χ 2 = 16, P = 0.0001). Conclusions: This study supports PRAME IHC as a useful surrogate for the GES assay in the evaluation of challenging melanocytic lesions. PRAME IHC offers a cost-efficient, accessible, and practical ancillary tool that can be rapidly integrated into routine clinical practice for diagnostically ambiguous melanocytic lesions.