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Extraskeletal myxoid chondrosarcoma (EMC) is a rare malignant soft tissue tumor of unpredictable clinical behavior. The morphological spectrum of EMC based on histology alone can be difficult. There is no precise immunohistochemical (IHC) profile that together with the clinical parameters is able to predict the clinical outcome. We studied 31 cases confirmed as EMC. Clinical and follow-up data were recorded. Histopathological, molecular, and IHC studies were performed. Association among histopathological parameters was assessed using a chi-square test to determine homogeneity or linear trend for ordinal variables. The Kaplan-Meier proportional risk test (log rank) was used to study the impact of the histological, IHC, and molecular factors on progression-free survival (PFS) and disease-specific survival (DSS). Most EMCs showed a typical architectural pattern. Only a few cases presented an atypical histology (higher cellularity and solid pattern). IHC positivity (focal or diffuse) was present for CDK4 (100%), STAT-6 (90%), CD117 (84%), HNK-1 (81%), SATB2 (68%), and S-100 (58%). Synaptophysin and INSM1 were expressed in 22.6% and 38.7% of cases respectively. The EWSR1::NR4A3 rearrangement was found in 19 cases and 7 tumors presented the TAF15::NR4A3 fusion. Positive surgical margins together with atypical histology and expression of p53 and Ki67 correlated with worse clinical prognosis. EMCs express several IHC markers which are also seen in other soft tissue sarcomas. The molecular detection of NR4A3 rearrangement supports the differential diagnosis. Positive surgical margins together with atypical histology and positive expression of p53 and Ki-67 seem to predict a poor clinical outcome with worse prognosis, increased rate of recurrence, metastasis, and poor overall survival.

Drug resistance is one of the major factors associated with poor outcome of cancer patients. Treatment of Ewing sarcoma (EwS), an aggressive neoplasm mainly affecting children, adolescents and young adults, is associated with therapy failure and tumor relapse in 30-80% of the cases. Thus, it supports the need to explore the mechanisms modulating drug activity. Here, we describe a novel mechanism of drug sensitivity based on the role of EWS::FLI1 in R-loop metabolism. Our results demonstrate that EWS::FLI1 promotes R-loop formation favoring the interaction between DHX9 and elongating RNA polymerase II. In addition, we discovered that EWS::FLI1 kidnaps DHX9 preventing the resolution of TOP1 poisoning-associated R-loops. Our findings indicate that R-loops accumulation promotes replicative stress, genome instability and cell sensitivity to SN-38. Collectively, these results uncover a novel mechanism behind EwS sensitivity to genotoxic agents, with relevant implications for EwS treatment.

Intimal sarcomas (IS) are rare malignant mesenchymal tumors arising in large blood vessels of the systemic and pulmonary circulation and also in the heart. They are morphologically similar to other spindle cell, poorly differentiated sarcomas. The prognosis is poor and depends mainly on surgical options. Three cases of IS were collected from two institutions. Clinical data were retrieved and histological study was performed. A wide immunohistochemical panel was analyzed. FISH of MDM2 gene was performed, and a molecular study with NGS was implemented in all cases. The mean age of our cases was 54 years. Histologically, the tumors presented a diffuse growth pattern with heterogeneous atypical epithelioid or spindle cells and extensive thrombosed areas. All cases presented intense immunoexpression for MDM2, CDK4, CD117, c-myc, PDGFRA, and p16. PDGFRA, HTERT, and pan-TRK gained expression, while p16 lost intensity, being weaker in both the local recurrences and xenografts. The three cases showed amplification of MDM2 by FISH. NGS analysis revealed amplifications in the CDK4, PDGFRA, and KIT genes, together with BRAF mutation and KRAS amplification. P16 was expressed in all cases, losing intensity in local recurrence and xenografts. Two new alterations, a BRAF mutation and a KRAS amplification, were detected by NGS in different tumors, opening up new therapeutic options for these patients.

Supplementary Figures S1-S5. MDM4 modulation by HEY1 occurs downstream of TP53 (S1); Modulation of SIRT1 and TP53 induction upon ectopic expression of Flag-tagged HEY1 in the B-cell malignancy cell lines "697" and NALM6, and in primary keratinocytes (S2); The degree of SIRT1 modulation depends on HEY1 levels (S3); Flow-diagram of samples analysed on tissue micro arrays (TMA1, TMA2, TMA3), and FFPE sections used in this study (S4); Kaplan-Meier survival estimates for patients analysed for SIRT1 expression on TMA3 (S5).

Supplementary Tables S1-S4. Identification of HEY1 regulated genes downstream of EWS-FLI1 silencing in Ewing sarcoma (S1); Combined immunohistochemical staining results for samples from patients with metastases at diagnosis on TMA1 and 2 according to site of metastasis (S2); Clinical characteristics and SIRT1 staining results of primary tumors from 43 ES patients treated at St. Jude Children´s Hospital between 1979 and 1987 using vincristine, doxorubicin, and cyclophosphamide-based regimens, with subsequent addition of ifosfamide and etoposide (S3); Materials used in the study (S4).

Supplementary Figure Legends. Legend for Supplementary Figures S1-S5.

ObjectivesWe explored features of the angiosarcoma (AS) tumor microenvironment to discover subtypes that may respond to immunotherapy.MethodsThirty-two ASs were included. Tumors were studied by histology, immunohistochemistry (IHC), and gene expression profile using the HTG EdgeSeq Precision Immuno-Oncology Assay.ResultsComparing cutaneous and noncutaneous ASs, the second group showed 155 deregulated genes, and unsupervised hierarchical clustering (UHC) delineated two groups: the first mostly cutaneous AS and the second mainly noncutaneous AS. Cutaneous ASs showed a significantly higher proportion of T cells, natural killer cells, and naive B cells. ASs without MYC amplification revealed a higher immunoscore in comparison with ASs with MYC amplification. PD-L1 was significantly overexpressed in ASs without MYC amplification. UHC showed 135 deregulated genes differentially expressed when comparing ASs from the non–head and neck area with patients who had AS in the head and neck area. ASs from the head and neck area showed high immunoscore. PD1/PD-L1 content was significantly more highly expressed in ASs from the head and neck area. IHC and HTG gene expression profiling revealed a significant correlation between PD1, CD8, and CD20 protein expression but not PD-L1.ConclusionsOur HTG analyses confirmed a high degree of tumor and microenvironment heterogeneity. Cutaneous ASs, ASs without MYC amplification, and ASs located in the head and neck area seem to be the most immunogenic subtypes in our series.

Ewing sarcoma (ES) is an aggressive neoplasm with variable morphology. It has no specific immunoprofile or molecular signature. Neither CD99, NKX2.2 nor PAX7 immunoreactivity alone is completely specific, although diagnostic specificity improves when combined. The purpose of the present study was to investigate the immunohistochemical (IHC) expression of PAX7 in a large series of genetically confirmed ES. Existing results for CD99 and NKX2.2 immunoexpression, morphological findings and molecular studies (fusion gene subtypes) were retrieved from a previous study. Survival analyses were performed in cases with available clinical follow-up. PAX7 was positive in 95.5% of ES with diffuse staining ( 50%) in all positive cases and moderate or strong intensity for most cases. Nineteen ES displayed both PAX7 and CD99 immunoreactivity but lacked NKX2.2 immunoexpression. No relationships could be found between PAX7 expression and the histological types or ES gene fusion subtypes. Univariant/multivariate analysis showed that lack of PAX7 and/or NKX2.2 immunoexpression constitute independent poor prognostic factors for progression free survival (PFS) and overall survival (OS). In conclusion, IHC for CD99, NKX2.2, and PAX7 may be useful in daily practice for ES diagnosis, particularly in hospitals lacking facilities for molecular studies. In addition, the combination of strong CD99 membranous positivity and nuclear PAX7 and NKX2.2 immunoreactivity seems to be very reliable for ES diagnosis when supported by a corroborating histomorphologic and clinical picture. Although PAX7 is not entirely specific for ES, it seems to have a more extensive and strong nuclear immunoreactivity than NKX2.2 expression, even in tumors with decalcification artifact. Considering the prognostically significant data herein reported, we strongly recommend validation in prospective ES series that include localized and disseminated tumors.

We report the histopathological, immunohistochemical (IHC), and molecular findings in 3 patients with adult pancreatoblastoma, including 2 with autopsy features. The tumors were located in the tail and body of the pancreas, and the 2 autopsy examinations revealed liver and lung metastases. Histopathologically the neoplasms were composed of solid epithelial elements with nested or trabecular growth patterns, fibrous stroma, and squamoid clusters. Keratin 19 was positive mainly in squamoid corpuscles, and trypsin or chymotrypsin was positive in the acinar component. Neuroendocrine differentiation was observed in all tumors, and nuclear β-catenin expression in 2 tumors. Despite nuclear β-catenin expression, CTNNB1 mutation was found only in tumor 2. APC mutation was detected in tumor 1, and SMAD4 as well as MEN1 mutations in tumor 3. This last tumor also revealed chromosomal instability with many chromosomal losses and gains. The follow-up showed regional or distant metastases in all patients. Two patients died of disease after 3 and 26 months of follow-up and 1 patient is alive with no evidence of disease 6 years and 2 months after surgery. Adult pancreatoblastoma can display genetic heterogeneity, diverse histological appearance, and overlapping IHC findings. As a result, the differential diagnosis with other adult pancreatic tumors, such as acinar cell carcinoma, neuroendocrine neoplasm, solid pseudopapillary neoplasm, and mixed tumors may be challenging, especially when dealing with limited tumor tissue. The identification of squamoid corpuscles is essential for diagnosis. Although molecular findings might provide useful information, the integration of clinical, radiological, and histopathological findings is essential in pancreatoblastoma diagnosis.

The clinical evolution of solitary fibrous tumors (SFTs) is often uncertain and several risk stratification systems (RSS) have been proposed. The Demicco et al. RSS is the most frequently implemented. In this study we aim to validate two alternative RSS (Sugita et al. and G-Score) using results for the Demicco RSS from a previous study of 97 SFTs. In addition, we aim to determine whether reclassified cases had any distinctive molecular features. As the Sugita et al. system substitutes mitotic count with Ki-67 index we also investigated whether Ki-67 results for tissue microarrays are comparable to those obtained using whole tissue sections. In the present study we detected that many cases classified by Demicco RSS as low-risk were reclassified as intermediate risk using the new system (G-score RSS). Kaplan-Meier survival plots for G-Score RSS showed that the low-risk and intermediate-risk SFTs had a similar evolution that contrasted with the more aggressive high-risk group. Moreover, the similar evolution in both low and intermediate-risk groups occurred despite the G-score system being stricter in classifying low-risk tumors. We observed that Sugita RSS does not provide any better risk stratification in comparison with the Demicco RSS, and testing both RSS in our series produced similar Kaplan-Meier survival data. We found some discordant results when comparing whole sections and the corresponding tissue microarrays samples, finding the hotspot areas easier to locate in whole sections. Forty-one SFTs with initial low-risk assigned by the Demicco RSS were reclassified as intermediate-risk by G-score finding both TP53 and HTER mutations in four cases, only HTER mutation in 11 cases, and only TP53 mutation in 2 cases. All six cases of SFT classified as high-risk by both the Demicco and G-score RSS suffered recurrence/metastasis, and half showed both TP53 and HTER mutations. Five SFTs were categorized as low-risk by both Demicco and G-score, of which 4 cases revealed HTER mutation. Regarding the outcome of these 5 patients, two were lost to follow-up, and one of the remaining three patients suffered recurrence. We believe that although the presence of both TP53 and HTER mutations may confer or be related to poor evolution, the isolated presence of HTER mutation alone would not necessarily be related to poor outcome. The G-score RSS more accurately identified low-risk patients than the other two risk models evaluated in the present series. Late recurrence/metastasis may occasionally be observed even in low-risk SFTs categorized by stricter classification systems such as the G-score RSS. These findings support the possibility that additional, as yet unknown factors may influence the clinical evolution of SFTs. In conclusion, given the possibility of late recurrence, long-term follow-up is recommended for all SFT patients, even in cases classified as low risk by the stricter G-score system. An integration of clinical, radiological, pathological, and molecular findings may improve SFT risk stratification and better predict patient outcome.

Resumen La histiocitosis de celulas de Langerhans (HCL) es una enfermedad heterogenea caracterizada por una proliferacion de celulas de Langerhans y la presencia de la mutacion BRAF en aproximadamente la mitad de los casos. La afectacion osea es comun pero la presencia de grandes masas en partes blandas no es habitual. Reportamos un caso de un paciente pediatrico que comenzo con una gran masa tumoral en hueso iliaco izquierdo con extension a partes blandas adyacentes. La tomografia axial computarizada mostraba una lesion osteolitica con gran extension a partes blandas. Se realizo una biopsia quirurgica por curetaje y el diagnostico histopatologico fue de HCL con positividad intensa para CD1a en las celulas neoplasicas. El estudio molecular detecto la presencia de mutacion BRAF V600E. Se discute el diagnostico diferencial histopatologico e inmunohistoquimico con otras histiocitosis.

The clinical evolution of solitary fibrous tumor (SFT) remains unclear. Although various clinical, morphological and molecular criteria may indicate increased risk of malignancy, some SFT can still progress despite having a clearly benign appearance. Various risk stratification systems have been proposed, but unfortunately they are not sufficient to precisely determine the malignant potential. In this review, we discuss current knowledge on SFT, focusing on the following controversial issues: (i) the diverse morphologic spectrum: 'the great simulator;' (ii) malignant transformation or dedifferentiation; (iii) current risk stratification systems; and (iv) molecular factors associated with clinical evolution. The morphological spectrum of SFT and the list of differential diagnoses continue to expand. Both have increased considerably since the first descriptions of specific molecular alterations. A classification of malignant SFT should not be based on histology alone. The correlation of all pathological and molecular factors is recommended; its inclusion in risk stratification systems may help to improve diagnosis and prognosis.

Solitary fibrous tumors (SFTs) are a rare type of mesenchymal lesion in which specific clinicopathologic factors have been related to patient outcome. We collected clinical, pathological, and molecular data of 28 patients with histologically confirmed SFT having at least one pathological factor associated with aggressive behavior. Molecular analysis to detect NAB2/STAT6 gene fusion, TP53, and/or TERT promoter mutation was performed. We analyzed the pathological factors predictive of recurrence/metastasis and compared with clinical outcome. The risk of metastasis was calculated using four previously described scoring systems. Histopathologically, all tumors revealed hypercellularity, 11 had ≥ 4 mitoses/10 HPF, and 12 showed necrosis. Dedifferentiation was observed in three tumors. STAT6 was positive in all cases. Desmin, p16, INSM1, and HTER immunoexpressions were detected in 14, 18, 21, and 46% of the SFT, respectively. The NAB2/STAT6 gene fusion was detected in 16 tumors. After a median follow-up of 34 months, 32.0% recurred, 32.1% metastasized, and 35.7% died of disease. TERT mutations were detected in almost half the tumors. Tumors with TP53 mutations or with TP53 and TERT promoter mutations were almost always classified as high risk, and the patients developed metastases and/or died of disease. Tumors with intermediate-risk and TERT mutation had a worse evolution. SFTs with adverse pathological parameters were not always related with a poor outcome, thus confirming the unpredictable clinical behavior of SFT. The inclusion of molecular factors (TP53 and TERT promoter status) may provide new prognostic indicators for future risk stratification systems, especially in the intermediate-risk group.

Background Osteosarcoma is the most common primary malignant tumour of bone occurring in children and young adolescents and is characterised by complex genetic and epigenetic changes. The miRNA miR-486-5p has been shown to be downregulated in osteosarcoma and in cancer in general. Results To investigate if the mir-486 locus is epigenetically regulated, we integrated DNA methylation and miR-486-5p expression data using cohorts of osteosarcoma cell lines and patient samples. A CpG island in the promoter of the ANK1 host gene of mir-486 was shown to be highly methylated in osteosarcoma cell lines as determined by methylation-specific PCR and direct bisulfite sequencing. High methylation levels were seen for osteosarcoma patient samples, xenografts and cell lines based on quantitative methylation-specific PCR. 5-Aza-2′-deoxycytidine treatment of osteosarcoma cell lines caused induction of miR-486-5p and ANK1, indicating common epigenetic regulation in osteosarcoma cell lines. When overexpressed, miR-486-5p affected cell morphology. Conclusions miR-486-5p represents a highly cancer relevant, epigenetically regulated miRNA in osteosarcoma, and this knowledge contributes to the understanding of osteosarcoma biology.

Ewing sarcoma (EWS) is an aggressive bone and soft tissue tumor of children and young adults in which the principal driver is a fusion gene, EWSR1-FLI1. Although the essential role of EWSR1-FLI1 protein in the regulation of oncogenesis, survival, and tumor progression processes has been described in-depth, little is known about the regulation of chimeric fusion-gene expression. Here, we demonstrate that the active nuclear HDAC6 in EWS modulates the acetylation status of specificity protein 1 (SP1), consequently regulating the SP1/P300 activator complex binding to EWSR1 and EWSR1-FLI1 promoters. Selective inhibition of HDAC6 impairs binding of the activator complex SP1/P300, thereby inducing EWSR1-FLI1 downregulation and significantly reducing its oncogenic functions. In addition, sensitivity of EWS cell lines to HDAC6 inhibition is higher than other tumor or non-tumor cell lines. High expression of HDAC6 in primary EWS tumor samples from patients correlates with a poor prognosis in two independent series accounting 279 patients. Notably, a combination treatment of a selective HDAC6 and doxorubicin (a DNA damage agent used as a standard therapy of EWS patients) dramatically inhibits tumor growth in two EWS murine xenograft models. These results could lead to suitable and promising therapeutic alternatives for patients with EWS., Research in the E.D.A. lab is supported by Asociación Española Contra el Cáncer (AECC), the Ministry of Science of Spain-FEDER (CIBERONC, PI1700464, PI2000003, RD06/0020/0059)S. D.G.D. and L.H.P. are supported by CIBERONC (CB16/12/00361). D.G.D., M.J.R. and L.H.P. are PhD researchers funded by the Consejería de Salud, Junta de Andalucía (PI-0197-2016, ECAI F2-0012-2018 and PI-0013-2018, respectively).

Based on the French Federation Nationale des Centers de Lutte Contre le Cancer (FNCLCC) grading system, this study assesses the accuracy of conventional and modified core biopsy (CB) systems in predicting the final grade (low vs high) assigned to the resected specimen. Substituting Ki-67 immunoexpression for mitotic count, and radiological for histological assessment of necrosis, we used two modified FNCLCC CB grading systems: (1) Ki-67 immunoexpression alone, and (2) Ki-67 plus radiological assessment of necrosis. We graded 199 soft tissue sarcomas (STS) from nine centers, and compared the results for the conventional (obtained from local histopathology reports) and modified CB systems with the final FNCLCC grading of the corresponding resected specimens. Due to insufficient sample quality or lack of available radiologic data, five cases were not evaluated for Ki67 or radiological assessment of necrosis. The conventional FNCLCC CB grading system accurately identified 109 of the 130 high-grade cases (83.8%). The CB grading matched the final FNCLCC grading (low vs high) in 175 (87.9%) of the 199 resected tumors; overestimating the final grade in three cases and underestimating in 21 cases. Modified system 1 (Ki-67) accurately identified 117 of the 130 high-grade cases (90.0%). The CB grading matched the final FNCLCC grading (low vs high) in 175 (89.7%) of the 195 evaluated cases; overestimating seven and underestimating 13 cases. Modified system 2 (Ki-67 plus radiological necrosis) accurately identified 120 of the 130 high-grade cases (92.3%). This last matched the final FNCLCC grading (low vs high) in 177 (91.2%) of the 194 evaluated cases; overestimating seven and underestimating 10 cases. Modified system 2 obtained highest area under ROC curves, although not statistically significant. Underestimated CB grades did not correlate with histological subtypes, although many of the discrepant cases were myxoid tumors (myxofibrosarcomas or myxoid liposarcomas), leiomyosarcomas or undifferentiated pleomorphic/spindle cell sarcomas. Using modified FNCLCC CB grading systems to replace conventional mitotic count and histologic assessment of necrosis may improve the distinction between low and high-grade STS on CB. Our study confirms that classifying grade 1 as low grade and grades 2 and 3 as high grade improves correlation between CB and final grade by up to 21%, irrespective of CB system used. A higher than expected Ki-67 score in a low-grade sarcoma diagnosed on CB should raise concern that a higher-grade component may not have been sampled. Furthermore, correlation of all clinicopathological and radiological findings at multidisciplinary meetings is essential to assess the histological grade on CB as accurately as possible.

The diagnosis of Ewing sarcoma requires the integration of the information generated from numerous techniques, some of them being very sophisticated. However, the first steps of the diagnostic process are crucial to achieve the maximum possible diagnostic performance. In this chapter we will review how to handle the diagnostic specimen from its collection, how to prepare it for diagnosis, how to make a complete pathology report, and provide guidance for the reasonable use of immunohistochemical techniques in this malignancy.

The diagnosis of Ewing sarcoma requires the integration of the information generated from numerous techniques, some of them being very sophisticated. However, the first steps of the diagnostic process are crucial to achieve the maximum possible diagnostic performance. In this chapter we will review how to handle the diagnostic specimen from its collection, how to prepare it for diagnosis, how to make a complete pathology report, and provide guidance for the reasonable use of immunohistochemical techniques in this malignancy.

Osteosarcoma is the most common primary malignant tumour of bone occurring in children and young adolescents, and is characterised by complex genetic and epigenetic changes. The miRNA miR-486-5p has been shown to be downregulated in osteosarcoma and in cancer in general. To investigate if the miR-486 locus is epigenetically regulated, we integrated DNA methylation and miR-486-5p expression data using cohorts of osteosarcoma cell lines and patient samples. An upstream CpG island of mir-486 was shown to be highly methylated in osteosarcoma cell lines as determined by methylation-specific PCR and direct bisulfite sequencing. High methylation levels were seen for osteosarcoma patient samples, cell xenografts and cell lines based on quantitative methylation-specific PCR. 5-Aza-2’-deoxycytidine treatment of osteosarcoma cell lines caused induction of miR-486-5p in osteosarcoma cell lines. When overexpressed, miR-486-5p affected cell morphology. miR-486-5p represents a highly cancer relevant, epigenetically regulated miRNA in osteosarcoma.

Angiosarcomas (AS) represent a heterogenous group of tumors with variable clinical presentation. AS share an important morphologic and immunohistochemical overlap with other sarcomas, hence the differential diagnosis is challenging, especially in poorly-differentiated tumors. Although molecular studies provide significant clues, especially in the differential diagnosis with other vascular neoplasms, a thorough hematoxylin and eosin analysis remains an essential tool in AS diagnosis. In this review, we discuss pathological and molecular insights with emphasis on implications for differential diagnosis in cutaneous, breast, soft tissue and visceral AS.

Our main goal was to investigate the potential utility of MDM2/CDK4 immunohistochemistry to act as surrogate for FISH to identify a subset of lipoma-like ALT/WDL that might, otherwise, be underdiagnosed on initial screening. Lack of cytologic atypia in lipomatous tumors with negative expression for MDM2/CDK4 IHC does not fully exclude the possibility of underlying MDM2/CDK4 amplification. Present study identified that isolated CDK4/p16 positive expression, in the absence of MDM2 expression, may have potential utility during the initial screening of these tumors but the proportion of conventional lipomas, which may also exhibit low levels of CDK4/p16 expression remains uncertain.

Background Despite extensive immunohistochemical (IHC) and molecular studies combined with morphologic findings, a group of round/ovoid cell tumors histologically similar to Ewing sarcomas (ES) but lacking EWSR1-rearrangements may remain unclassifiable. Design We retrospectively analyzed 41 Ewing-like tumors (formalin-fixed, paraffin-embedded) previously determined as negative or non-informative for EWSR1-rearrangements by FISH and/or RT-PCR. A new histopathology revision and additional IHC and molecular analyses were carried out in order to investigate whether additional IHC and/or molecular testing in combination with the morphological findings may help in reaching a definitive diagnosis. Results Almost all the tumors (n = 40) involved soft tissue and/or bone and half the patients died of disease. In the archival cases all diagnoses were Ewing sarcoma (ES), Ewing-like sarcoma (ELS), myoepithelial tumor and undifferentiated sarcoma (US). In the new review all the tumors were re-classified as, ES (n = 16), Ewing-like tumor with EWSR1 rearrangement and amplification and possible EWSR1-NFATC2 gene fusion (n = 1), CIC-rearranged sarcomas or undifferentiated sarcoma, most consistent with CIC-rearranged sarcoma (n = 7), sarcoma with BCOR-alteration or undifferentiated sarcoma, consistent with BCOR-associated sarcoma (n = 3), neuroblastoma (n = 2), unclassifiable neoplasm with neuroblastic differentiation (n = 1), malignant rhabdoid tumor (n = 2), lymphoblastic lymphoma (n = 1), clear cell sarcoma of the gastrointestinal tract (n = 1), small cell carcinoma (n = 1), sclerosing rhabdomyosarcoma (n = 1), desmoplastic small round cell tumor (n = 1), malignant peripheral sheath nerve tumor (n = 1), poorly-differentiated synovial sarcoma (n = 1), Possible gastrointestinal stromal tumor/GIST with predominant round cells (n = 1) and possible SMARCA4-deficient-sarcoma (n = 1). NKX2.2, ETV4 and BCOR immunoreactivity was observed in all ES, CIC-rearranged sarcomas and sarcomas with BCOR alteration, respectively. CIC-rearrangement by FISH was observed in many of the CIC-rearranged sarcomas. Conclusion Our analysis of 41 Ewing-like tumors confirms that there may be a significant pathological and IHC overlap among Ewing-like tumors, with prognostic and therapeutic impacts. Additional IHC (NKX2.2, ETV4 and BCOR) and molecular studies including FUS, CIC or BCOR analysis may support the final diagnosis when FISH or RT-PCR fail to detect EWSR1-rearrangements. Any molecular findings should always be interpreted in relation to the specific clinical and pathological context.

Ewing's sarcoma family of tumors (ESFT) are aggressive neoplasms with scant tumor-infiltrating lymphocytes. We analyzed the immunohistochemical (IHC) expression of PD-L1 and PD-1 and their prognostic significance in clinically localized neoplasms in a cohort of 370 ESFT. Slides prepared from tissue microarrays were stained for PD-L1, PD-1, and CD8. Membranous/cytoplasmic staining over 5% of tumor cells was regarded as positive for PD-L1 and PD-1. Prognostic analysis was done considering only clinically localized tumors (n = 217). PD-L1 expression was present in 19% of ESFT, while PD-1 was expressed in 26%. Forty-eight percent of tumors were negative and 12% were positive for both PD-L1 and PD-1. Metastatic tumors displayed higher expression of PD-L1 (p

Ewing sarcoma is an aggressive neoplasm of pediatric and adolescent patients. Immunohistochemistry (IHC) can be used to support the morphologic diagnosis of Ewing sarcoma family of tumors (ESFT) in a convincing clinical/radiological context. Although neither NKX2.2 nor CD99 alone are entirely specific, when combined, the diagnostic specificity is high. The aim of the present study was to investigate the IHC expression of NKX2.2, ETV4 and BCOR in a large series of genetically confirmed ESFT. The results for CD99 and CAV-1 immunoreactivity, and the histological and fusion gene subtypes were retrieved from our previous study. NKX2.2 demonstrated moderate or strong nuclear positivity in 91.2% of the tumors. The staining intensity was heterogeneous. Many of the ESFT with negative NKX2.2 immunoreactivity were in bone. Strong/moderate ETV4 nuclear expression was detected in two small round cell tumors, both were negative for NKX2.2. No relationships could be found between expression of NKX2.2 and the histological subgroups or ESFT gene fusion subtypes. BCOR was negative in all ESFT. In conclusion, NKX2.2, ETV4 and BCOR IHC may be helpful in daily practice for distinguishing ESFT from CIC or BCOR-associated sarcomas, especially in hospitals without access to molecular assays. In addition, the combination of strong CD99 membranous positivity and nuclear NKX2.2 positivity seems to be very reliable for ESFT diagnosis in an appropriate clinicoradiological setting. So far no antibody is entirely specific for ESFT diagnosis, and the IHC or molecular results in round cell tumors of bone may be strongly influenced by decalcification processes.

Desmoplastic melanoma (DM) and cutaneous malignant peripheral nerve sheath tumors (MPNST) reveal histological and immunohistochemical similarities, including S100 positivity and negative staining for conventional melanocytic markers. We present 3 cases of cutaneous S100-positive spindle cell tumors in elderly patients, in which first findings led to initial misdiagnoses as cutaneous MPNST and benign peripheral sheath nerve tumor (neurofibroma). The identification of adjacent atypical melanocytic hyperplasia in the overlying skin along with tumor cell proliferation, also in the superficial dermis, the neurotropic component and the absence of any relationship between the tumor and a major nerve, pre-existing neural benign tumor or the existence of stigmata suggestive of neurofibromatosis raised consideration of a DM. Careful attention should be paid to the presence of a firm dermal nodule and atypical scar lesions especially in sun-exposed areas (mainly head and neck region) in elderly patients associated with S100-positive spindle cell proliferation, solar elastosis and adjacent atypical melanocytic proliferation. In such cases, the possibility of a DM should be excluded with caution, especially if the tumor reveals a paucicellular morphology resembling various non-melanocytic neoplasms including malignant or benign peripheral sheath nerve tumors.

Given the potential for neuroendocrine differentiation in Ewing's sarcoma family of tumors (ESFT), we aimed to determine neuroendocrine expression in a large series of genetically-confirmed ESFT and its prognostic significance in clinically-localised neoplasms (n = 176). Slides prepared from tissue microarrays were stained for Insulinoma-associated protein 1 (INSM1), CD56, chromogranin-A and synaptophysin. INSM1 expression was present in 59% of ESFT, while synaptophysin, chromogranin-A and CD56 were expressed in only 13%, 8% and 5% of ESFT, respectively. Histological subtypes were only significantly correlated with INSM1 (p = 0.032) or CD56 (p = 0.016) immunoexpression. Regarding prognosis, no significant association was found between INSM1, synaptophysin or chromogranin-A immunoexpression and progression-free survival (PFS) or overall survival (OS). Despite the low proportion of tumors with CD56 immunoreactivity, CD56 expression was shown to correlate with both poor PFS (p < 0.001) and poor OS (p < 0.001) in the present series. In conclusion, neuroendocrine differentiation is often present in ESFT, and in the present study INSM1 expression in particular was found to be higher than previously described in Ewing's tumors. Nevertheless, this finding does not distinguish these tumors from other round cell tumors that may show focal or diffuse neuroendocrine differentiation. CD56 expression could be used as a prognostic factor in ESFT, although given the results herein obtained, we recommend a prospective validation in independent series including localized and disseminated tumors in ESFT.

SATB2 is commonly expressed in osteosarcomas. Although apparently being a valuable diagnostic marker for differentiating between small cell osteosarcoma (SCO) and other small round cell tumors of bone, for instance Ewing sarcoma family of tumors (ESFT), it has not been tested in a large series of ESFT and chondrosarcomas so far. We studied the immunohistochemical expression of SATB2 in 42 osteosarcomas, 31 chondrosarcomas, and 371 genetically confirmed ESFT. SATB2 positivity was detected in 90.4% of osteosarcomas, 87.5% of SCO, 91.3% of osteoblastic osteosarcomas, and in all chondroblastic and parosteal osteosarcomas. The osteoblastic and SCO subtypes expressed SATB2 more intensely than other histological types. SATB2 was expressed in 46.6% of chondrosarcomas, and in 1.3% of ESFT. Sensitivity and specificity of SATB2 immunoexpression were 90.4% and 95.3%, respectively. The positive and negative predictive values in osteosarcoma diagnosis were 66.6% and 98.9%, respectively. In chondrosarcoma, SATB2 immunoexpression was more frequent and intense in high-grade chondrosarcoma (Grade III) and uncommon in chondrosarcoma grade I. SATB2 positivity was detected in 55.6% of chondrosarcomas grade II. SATB2 apparently cannot distinguish between chondroblastic osteosarcoma and high-grade chondrosarcoma. Nevertheless, SATB2 is frequently expressed in osteogenic tumors, but is rarely positive in ESFT, and with the support of CD99 expression and specific molecular studies, it is very useful for distinguishing between these two lesions. Although SATB2 immunoexpression helps to distinguish osteosarcoma from their mimickers, the identification of malignant osteoid matrix formation and the integration of clinical and radiological data remain the corner stone of osteosarcoma diagnosis and as yet no antibody has equalled the diagnostic value of this important morphologic hallmark.

Hyaline globules and papillary fragments in cytologic samples from two intra-abdominal tumors in young females are presented including the cytological features and the correlation with the histopathologic and immunohistochemical findings. In the first case a cytologic study from an ovarian mass showed papillary structures and isolated tumor cells with epithelioid morphology, irregular reniform-like nuclear contour, pale or vacuolated cytoplasm, abundant hyaline globules and occasional glomeruloid structures resembling Schiller-Duval bodies. Yolk sac tumor (YST) was the diagnosis on the histological slides. Tumor cells showed positivity for cytokeratin (AE1/AE3), epithelial membrane antigen (EMA), alpha-fetoprotein (AFP) and Sal-like protein 4 (SALL4). In case number two the cytologic study from a liver metastasis displayed papillary and rosette-like clusters composed of uniform and bland cells showing occasional long cytoplasmic tails, hyaline globules and nuclear grooves. A diagnosis of hepatic metastasis from solid pseudopapillary neoplasm of the pancreas (SPNP) was rendered from the histology. Tumor cells revealed immunoreactivity for cytokeratin (AE1/AE3), Vimentin, Galectin-1 (GAL-1), Neuron specific-enolase, CD10, progesterone and β-catenin (nuclear stain). Regarding differential diagnosis, in the patient with the ovarian mass an ovarian clear cell carcinoma was considered, as well as other germ cell tumors or metastatic carcinoma, while in the patient with a liver metastasis a neuroendocrine carcinoma was taken into account. YST and SPNP share some cytological findings, including hyaline globules, papillary structures, clear cells and intercellular eosinophilic basement membrane deposits. Thus, a detailed study and careful interpretation of the cytological, histological and immunohistochemical findings may be worthwhile to avoid a potential misdiagnosis, particularly in the cytologic specimens of the ovarian and/or intra-abdominal mass, when involving young females. Diagn. Cytopathol. 2016;44:935-943. © 2016 Wiley Periodicals, Inc.

Background Differentiation of Ewing sarcoma family of tumors (ESFT) and Ewing-like tumors remains problematic. Certain ESFT with morphological and immunohistochemical (IHC) profiles lack the EWSR1-ETS transcript. To improve diagnostic accuracy we investigated the presence of several specific transcripts in 200 small round cell tumors (SRCT) displaying ESFT morphology and immunophenotype in which EWSR1 FISH analysis was non-informative or negative. Design 200 tumors (formalin-fixed, paraffin-embedded) were analyzed by RT-PCR. All tumors were tested for EWSR1-ETS , EWSR1 / WT1 , PAX3 / 7-FOX01 or SYT / SSX transcripts, and the negative tumors were subsequently analyzed for CIC / DUX4 , BCOR / CCNB3 and CIC / FOX04 transcripts. Results 133 (66.5%) ESFT displayed one of the above EWSR1-ETS translocations. Three cases (1.5%) revealed the SYT-SSX transcript for Synovial sarcoma, and one (0.5%) a EWSR1-WT1 transcript for Desmoplastic Small Round Cell tumor. The CIC-DUX4 translocation was found in six Ewing-like tumors (3%) with CD99 positivity. The BCOR-CCNB3 gene fusion was observed in 5 tumors (2.5%) displaying round or spindle cells with strong CCNB3 IHC expression in 3 tumors. Moreover, RT-PCR failed to detect any gene fusion transcripts in 19 tumors (9.5%) and were considered "undifferentiated small round cell sarcoma" (SRCS). Molecular biology results were non-informative in 33 SRCTs (16.5%) due to RNA degradation through inadequate fixation and/or decalcification. Conclusion Our analysis of 200 SRCTs confirms the molecular heterogeneity of neoplasms with ESFT morphology and highlight that molecular studies with RT-PCR including new emerging gene fusion transcripts are mandatory for the diagnosis when EWSR1 FISH is negative or non-informative. The incidence of CIC-DUX4 , BCOR-CCNB3 and CIC-FOX04 transcripts was relatively low. A small group of Ewing-like sarcomas or undifferentiated SRCS remains unclassified. Adopting appropriate tissue fixation and processing protocols is important to avoid degradation of fixed/embedded tissue when no frozen tumor is available.

Background Ewing sarcoma (EWS) is the second most common bone cancer in pediatric patients. Angiogenesis is a major factor for tumor growth and metastasis. Our aim was to carry out a histological, immunohistochemical, and molecular characterization of the neovascularization established between xenotransplanted tumors and the host during the initial phases of growth in nude mice in three angiogenesis experiments (ES2, ES3, and ES4). Methods The original human EWS were implanted subcutaneously on the backs of three nude mice. Tumor pieces 3 mm–4 mm in size from early passages of Nu432, Nu495, and Nu471 were also implanted subcutaneously on the backs of three sets (ES2, ES3, and ES4) of athymic Balb-c nude mice (n = 14 each). The animals were sacrificed at 24, 48, and 96 hours and at 7, 14, 21, and 28 days after implantation to perform histological, immunohistochemical, and molecular studies (neovascularization experiments). Results We observed histological, ultrastructural, and immunohistochemical changes in the xenografted tumor at different times after implantation. Chemokine ligand expression peaked twice, once during the first 48 hours and again in the second week. We observed that tumor cells in contact with murine peritumoral stroma presented higher expression of chemokine ligands as well as more tumor cells around the capillary vessels. Mouse serum vascular endothelial growth factor levels peaked twice, once in the first hours and then in the second week after tumor implantation. Conclusion Chemokines and other angiogenic factors may be relevant in the angiogenic mechanism during tumor growth. This model provides information on the early stages of the angiogenic process and could be a useful tool in researching anti-angiogenic drugs for new therapeutic strategies in EWS.

Langerhans cell histiocytosis (LCH) is a heterogeneous disease characterized by proliferation of Langerhans cells and BRAF mutation in almost half of the cases. Bone involvement is common but large soft tissue disease is uncommon. We report a pediatric patient with a large tumor mass involving the left iliac bone and the adjacent soft tissue. The computed tomography scan showed an osteolytic lesion with soft tissue extension. Surgical curettage of the lesion was performed and the final histopathologic diagnosis was LCH with CD1a immunoreactivity in tumor cells. The molecular analysis revealed a BRAF V600E mutation. We discuss the histopathological and immunohistochemical differential diagnosis with histiocytosis other than LCH.

Neuroendocrine differentiation or aberrant expression of neuroendocrine markers is very uncommon in angiosarcomas (AS) and creates a challenging differential diagnosis with other superficial or soft tissue tumors. Herein, we report a new case of superficial AS presenting as a tumor lesion on the little finger of the right hand of a 52-year-old man. The tumor displayed CD56, chromogranin-A, and synaptophysin immunoreactivity. Tumor cells were positive for vascular markers (CD31, FLI1, ERG, D2-40, VE-cadherin, VEGR1,2, and 3), CD99, and EMA, but were negative for S100, CK (AE1/AE3), CK20, polyomavirus, and myogenic (desmin and myogenin) and melanocyte markers (melan-A and HMB45). Ki67 immunostains indicated high proliferative activity (>50%). The whole-body computed tomography did not reveal distant disease. The initial assessment considered several tumor subtypes as possible histological diagnoses, including Ewing sarcoma, Ewing-like sarcoma, Merkel cell carcinoma, and undifferentiated "small round cell sarcoma". Fluorescence in situ hybridization analysis was negative for EWSR1 translocation and molecular analysis failed to detect any EWSR1, CIC, SYT or BCOR rearrangement. As a follow-up investigation, we tested 17 cutaneous/superficial AS for neuroendocrine markers; however, only one of these showed focal CD56 and synaptophysin expression. In conclusion, the present findings indicate that neuroendocrine differentiation is a very infrequent feature in AS. We report an AS of the finger with an uncommon histological appearance and immunohistochemical profile: predominant round cell tumor proliferation and neuroendocrine differentiation. Pathologists should be aware of these potential histological and immunohistochemical pitfalls in AS.

Epithelioid hemangioma (EH) is a rare benign vascular lesion, characterized by endothelial cells with epithelioid/histiocytoid appearance. Heart involvement is extremely rare. We present an unusual case of cardiac EH in a young woman with supraventricular arrhythmia, nephrotic syndrome (membranous glomerulopathy), and peripheral eosinophilia after a pregnancy and normal partum resembling Kimura disease. Echocardiogram showed a large tumor mass in the right cardiac ventricle. The cardiac tumor was removed and the histopathology revealed an endothelial proliferation associated with abundant eosinophils. The neoplastic endothelial cells were eosinophilic and polygonal with epithelioid/histiocytoid morphology. Lymphoid nodules were occasionally seen. The neoplastic cells were positive for CD34, CD31, ERG, and factor VIII and negative for CK. A diagnosis of EH was rendered. The patient was alive and well after surgical resection. EH and Kimura disease represent separate entities, but clinical and/or histological overlapping can be observed. Epithelioid/histiocytoid endothelial cells constitute the hallmark feature that favors a final diagnosis of EH.

Resumen Los angiosarcomas son neoplasias malignas agresivas con diferenciacion de celulas endoteliales. La hemorragia espontanea debida a una coagulacion intravascular diseminada con coagulopatia de consumo es una forma poco comun de presentacion de estos tumores denominada sindrome de Kasabach-Merritt. En el presente estudio describimos 2 casos con estudio necropsico de angiosarcomas complicado con sindrome de Kasabach-Merritt. El primer caso con diagnostico de angiosarcoma mediastinico asociado a un tumor de celulas germinales y el segundo caso con angiosarcoma primario hepatico y multiples metastasis.

A new case of soft tissue myoepithelial carcinoma (MEC) with rhabdoid-like differentiation is presented including cytologic, histopathologic, immunohistochemical, and molecular biologic features. A 45-year-old woman was admitted to the Hospital with nodular mass involving the lower part of the abdominal wall. Fine-needle aspiration cytology showed a round cell tumor with abundant cytoplasm in the myxoid background. The nuclei were uniform, round to ovoid, with finely distributed chromatin, nucleoli, and pale, vacuolated, or eosinophilic cytoplasm with rhabdoid-like appearance resembling a soft tissue malignant rhabdoid tumor. The surgically removed tumor was poorly demarcated, yellow, soft, and myxoid. The histopathology revealed sheets of poorly differentiated round malignant cells with focal myxoid stroma and rhabdoid-like morphology. Immunohistochemistry showed positivity for CK (AE1/AE3), EMA, S100, vimentin, CD99, and SMA; however desmin, CD34, and gliofibrilar acid protein (GFAP) were negative. Tumor cells revealed loss of INI1 expression. The EWSR1 gene rearrangement was detected by fluorescence in situ hybridization (FISH), but molecular biology failed to detect EWSR1/ETS, EWSR1/NR4A3, EWSR1/DDIT3, EWSR1/ATF1, EWSR1-POU5F1, EWSR1/ZNF444, EWSR1-PBX1 gene fusions. The final diagnosis was soft tissue malignant myoepithelioma with rhabdoid changes and EWSR1 gene rearrangement. The differential diagnosis included soft tissue malignant rhabdoid tumor, cellular extraskeletal myxoid chondrosarcoma, proximal epithelioid sarcoma, and other soft tissue tumor with EWSR1 rearrangement. To our knowledge, this is the first case of MEC with rhabdoid features and description of fine-needle aspiration cytology.

Sinonasal tract (SNT) alveolar rhabdomyosarcoma (ARMS) are frequently misdiagnosed, especially in adults. Fifty-two adult (≥18 years) patients with SNT ARMS were reviewed and characterized by immunohistochemistry and molecular studies. Twenty-six females and 26 males (18–72 years; mean 43.2 years), presented after a short duration (mean 2.6 months) with a large (mean 5.5 cm) destructive nasal cavity mass, involving multiple contiguous paranasal sites (n = 46) and with cervical adenopathy (n = 41). The tumors showed an alveolar, nested to solid growth pattern below an intact, but often involved (n = 9) epithelium with frequent necrosis (n = 37), destructive bone invasion (n = 30), and lymphovascular invasion (n = 25). The neoplastic cells were dyshesive and dilapidated, with crush artifacts. Rhabdoid features (n = 36) and tumor cell multinucleation (n = 28) were common. Mitotic counts were high (mean 17/10 HPFs). The neoplastic cells showed the following immunohistochemical positive findings: desmin (100%), myogenin (100%), MYOD1 (100%), MSA (96%), SMA (52%), CAM5.2 (50%), AE1/AE3 (36%); other positive markers included S100 protein (27%), CD56 (100%), synaptophysin (35%), and chromogranin (13%). Overall, 54% show epithelial marker reactivity. Molecular studies showed FOXO1 translocations (81%) with PCR demonstrating PAX3 in 72.7% tested. Patients presented with high stage (IV 24; III 26) and metastatic disease (lymph nodes n = 41; distant metastases n = 25) (IRSG grouping). Surgery (n = 16), radiation (n = 41) and chemotherapy (n = 45) yielded an overall survival of 36.1 months (mean; range 2.4–286); 18 alive without disease (mean 69.6 months); 7 alive with disease (mean 11.0 months); 1 dead without disease (63.7 months); and 26 dead with disease (mean 18.5 months). SNT ARMS frequently present in adults as a large, destructive midline mass of short symptom duration, with high stage disease. The alveolar to solid pattern of growth of cells with rhabdoid-plasmacytoid features suggests the diagnosis, but epithelial immunohistochemistry markers are present in 54% of cases, leading to misdiagnosis as carcinomas if muscle markers are not also performed. Overall survival of 36.1 months is achieved with multimodality therapy, but 64% have incurable disease (16.9 months). Mixed anatomic site (p = 0.02) was a significant adverse prognostic indicator, while stage (0.06) and tumor size >5 cm (0.06) approached marginal significance.