Prognostic Factors in Epithelioid Hemangioendothelioma: Analysis of a Nationwide Molecularly/Immunohistochemically Confirmed Cohort of 57 Cases.

Epithelioid hemangioendothelioma (EHE) is an extremely rare vascular sarcoma with variable aggressive clinical behavior. In this retrospective study, we aimed to investigate prognostic factors based on clinicopathologic findings in a molecularly/immunohistochemically confirmed nationwide multicenter cohort of 57 EHE cases. Patients had unifocal disease (n = 29), multifocal disease (n = 5), lymph node metastasis (n = 8) and/or distant metastasis (n = 15) at the time of diagnosis. The overall survival rate was 71.4% at 1 year and 50.7% at 5 years. Survival did not correlate with sex, age or histopathological parameters. No survival differences were observed between multifocal and metastatic disease, suggesting that multifocality represents early metastases and treatment options are limited in comparison to unifocal disease. In unifocal tumors, survival could be predicted using the risk stratification model of Shibayama et al., dividing the cases into low- (n = 4), intermediate- (n = 15) and high- (n = 3) risk groups. No clinical or histopathological parameters were associated with progressive unifocal disease course. Lymph node metastases at the time of diagnosis occurred in 14.0% of the cases and were mainly associated with tumor localization in the head and neck area, proposing lymph node dissection. In conclusion, our results demonstrate the aggressive behavior of EHE, emphasize the prognostic value of a previously described risk stratification model and may provide new insights regarding tumor focality, therapeutic strategies and prognosis.

EWSR1-The Most Common Rearranged Gene in Soft Tissue Lesions, Which Also Occurs in Different Bone Lesions: An Updated Review.

EWSR1 belongs to the FET family of RNA-binding proteins including also Fused in Sarcoma (FUS), and TATA-box binding protein Associated Factor 15 (TAF15). As consequence of the multifunctional role of EWSR1 leading to a high frequency of transcription of the chromosomal region where the gene is located, EWSR1 is exposed to aberrations such as rearrangements. Consecutive binding to other genes leads to chimeric proteins inducing oncogenesis. The other TET family members are homologous. With the advent of widely used modern molecular techniques during the last decades, it has become obvious that EWSR1 is involved in the development of diverse benign and malignant tumors with mesenchymal, neuroectodermal, and epithelial/myoepithelial features. As oncogenic transformation mediated by EWSR1-fusion proteins leads to such diverse tumor types, there must be a selection on the multipotent stem cell level. In this review, we will focus on the wide variety of soft tissue and bone entities, including benign and malignant lesions, harboring EWSR1 rearrangement. Fusion gene analysis is the diagnostic gold standard in most of these tumors. We present clinicopathologic, immunohistochemical, and molecular features and discuss differential diagnoses.

Myxoid pleomorphic liposarcoma-a clinicopathologic, immunohistochemical, molecular genetic and epigenetic study of 12 cases, suggesting a possible relationship with conventional pleomorphic liposarcoma.

Myxoid pleomorphic liposarcoma is a recently defined subtype of liposarcoma, which preferentially involves the mediastinum of young patients and shows mixed histological features of conventional myxoid liposarcoma and pleomorphic liposarcoma. While myxoid pleomorphic liposarcoma is known to lack the EWSR1/FUS-DDIT3 fusions characteristic of the former, additional genetic data are limited. To further understand this tumor type, we extensively examined a series of myxoid pleomorphic liposarcomas by fluorescence in situ hybridization (FISH), shallow whole genome sequencing (sWGS) and genome-wide DNA methylation profiling. The 12 tumors occurred in 6 females and 6 males, ranging from 17 to 58 years of age (mean 33 years, median 35 years), and were located in the mediastinum (n = 5), back, neck, cheek and leg, including thigh. Histologically, all cases consisted of relatively, bland, abundantly myxoid areas with a prominent capillary vasculature, admixed with much more cellular and less myxoid foci containing markedly pleomorphic spindled cells, numerous pleomorphic lipoblasts and elevated mitotic activity. Using sWGS, myxoid pleomorphic liposarcomas were found to have complex chromosomal alterations, including recurrent large chromosomal gains involving chromosomes 1, 6-8, 18-21 and losses involving chromosomes 13, 16 and 17. Losses in chromosome 13, in particular loss in 13q14 (including RB1, RCTB2, DLEU1, and ITM2B genes), were observed in 4 out of 8 cases analyzed. Additional FISH analyses confirmed the presence of a monoallelic RB1 deletion in 8/12 cases. Moreover, nuclear Rb expression was deficient in all studied cases. None showed DDIT3 gene rearrangement or MDM2 gene amplification. Using genome-wide DNA methylation profiling, myxoid pleomorphic liposarcomas and conventional pleomorphic liposarcomas formed a common methylation cluster, which segregated from conventional myxoid liposarcomas. While the morphologic, genetic and epigenetic characteristics of myxoid pleomorphic liposarcoma suggest a link with conventional pleomorphic liposarcoma, its distinctive clinical features support continued separate classification for the time being.

Loss of H3K27me3 occurs in a large subset of embryonal rhabdomyosarcomas: Immunohistochemical and molecular analysis of 25 cases.

Loss of histone 3 lysine 27 trimethylation (H3K27me3) has been described as a diagnostic marker for malignant peripheral nerve sheath tumor (MPNST), also discriminating MPNST with rhabdomyoblastic differentiation (malignant Triton tumor) from rhabdomyosarcoma (RMS). We studied the immunohistochemical expression of H3K27me3 in embryonal RMSs (ERMSs), performed methylation profiling in order to support the diagnosis and RNA-sequencing for comparison of the transcriptome of H3K27me3-positive and -negative cases. Of the 25 ERMS patients, 17 were males and 8 were females with an age range from 1 to 67 years (median, 6 years). None were known with neurofibromatosis type 1. One patient had Li-Fraumeni syndrome. Tumor localization included paratesticular (n = 9), genitourinary (n = 6), head/neck (n = 5), retroperitoneal (n = 4) and lower arm (n = 1). Five MPNSTs served as reference group. All ERMS had classical features including a variable spindle cell component. Immunohistochemical loss (partial or complete) of H3K27me3 was detected in 18/25 cases (72%). Based on methylation profiling, 22/22 cases were classified as ERMS. Using RNA sequencing, the ERMS group (n = 14) had a distinct gene expression profile in contrast to MPNSTs, confirming that the H3K27me3 negative ERMS cases do not represent malignant Triton tumors. When comparing H3K27me3-negative and -positive ERMSs, gene set enrichment analysis revealed differential expression of genes related to histone acetylation and normal muscle function with H3K27me3 negative ERMSs being associated with acetylation. Conclusion: Loss of H3K27me3 frequently occurs in ERMSs and correlates with H3K27 acetylation. H3K27me3 is not a suitable marker to differentiate ERMS (with spindle cell features) from malignant Triton tumor.

USP6-Associated Neoplasms: A Rapidly Expanding Family of Lesions.

Nearly 20 years ago, the first description of a translocation involving chromosome 17 on which USP6 resides was described. Since then, not only the culprit gene but also many fusion partners, leading to transcriptional activation of USP6, have been detected. The first neoplasm known to harbor USP6 rearrangements was aneurysmal bone cyst. Since then, other entities like nodular fasciitis, myositis ossificans, fibro-osseous pseudotumor of digits, and a subgroup of fibromas of tendon sheath, probably representing tenosynovial nodular fasciitis, have been added to the list of USP6-rearranged lesions. Remarkably, all of them share clinical as well as morphological characteristics, and authors have suggested that these entities actually belong to the same spectrum. This review summarizes the current knowledge regarding USP6-rearranged lesions and further elaborates on how these neoplasms relate to one another. We propose to call these lesions UAN (Usp6-associated neoplasm).

Kaposiform hemangioendothelioma and tufted angioma - (epi)genetic analysis including genome-wide methylation profiling.

Kaposiform hemangioendothelioma (KHE) is a locally aggressive vascular condition of childhood and is clinicopathologically related to tufted angioma (TA), a benign skin lesion. Due to their rarity molecular data are scarce. We investigated 7 KHE and 3 TA by comprehensive mutational analysis and genome-wide methylation profiling and compared the clustering, also with vascular malformations. Lesions were from 7 females and 3 males. The age range was 2 months to 9 years with a median of 10 months. KHEs arose in the soft tissue of the thigh (n = 2), retroperitoneum (n = 1), thoracal/abdominal (n = 1), supraclavicular (n = 1) and neck (n = 1). One patient presented with multiple lesions without further information. Two patients developed a Kasabach-Merritt phenomenon. TAs originated in the skin of the shoulder (n = 2) and nose/forehead (n = 1). Of the 5 KHEs and 2 TAs investigated by DNA sequencing, one TA showed a hot spot mutation in NRAS, and one KHE a mutation in RAD50. Unsupervised hierarchical clustering analysis indicated a common methylation pattern of KHEs and TAs, which separated from the homogeneous methylation pattern of vascular malformations. In conclusion, methylation profiling provides further evidence for KHEs and TAs potentially forming a spectrum of one entity. Using next generation sequencing, heterogeneous mutations were found in a subset of cases (2/7) without the presence of GNA14 mutations, previously reported in KHE and TA.

Calcifying fibrous tumor and inflammatory myofibroblastic tumor are epigenetically related: A comparative genome-wide methylation study.

Based on histological findings, calcifying fibrous tumor (CFT) may be a late (burned out) stage of inflammatory myofibroblastic tumor (IMT). This concept, however, has not been proven by molecular means. Five CFTs were analyzed for IMT-related rearrangements in ALK, ROS1 and RET using fluorescence in situ hybridization (FISH). Additionally, genome-wide methylation patterns were investigated and compared with IMT (n = 7), leiomyoma (n = 7), angioleiomyoma (n = 9), myopericytoma (n = 7) and reactive soft tissue lesions (n = 10) using unsupervised hierarchical cluster analysis and t distributed stochastic neighbor embedding. CFT patients, 4 females and 1 male, had a median age of 20 years ranging from 7 to 43 years. Two patients were younger than 18 years old. The tumors originated in the abdomen (n = 4) and axilla (n = 1). Histologically, all lesions were (multi) nodular and hypocellular consisting of bland looking (myo)fibroblasts embedded in a collagenous matrix with calcifications. FISH analysis brought up negative results for ALK, RET and ROS1 rearrangements. However, genome-wide methylation analysis revealed overlapping methylation patterns of CFT and IMT forming a distinct homogeneous methylation cluster with exception of one case clustering with myopericytoma/angioleiomyoma. In conclusion, DNA methylation profiling supports the concept that CFT and IMT represent both ends of a spectrum of one entity with CFT being the burn out stage of IMT.

Surgical Treatment of Chronic Parotitis.

Introduction chronic parotitis (CP) is a hindering, recurring inflammatory ailment that eventually leads to the destruction of the parotid gland. When conservative measures and sialendoscopy fail, parotidectomy can be indicated. Objective to evaluate the efficacy and safety of parotidectomy as a treatment for CP unresponsive to conservative therapy, and to compare superficial and near-total parotidectomy (SP and NTP). Methods retrospective consecutive case series of patients who underwent parotidectomy for CP between January 1999 and May 2012. The primary outcome variables were recurrence, patient contentment, transient and permanent facial nerve palsy and Frey syndrome. The categorical variables were analyzed using the two-sided Fisher exact test. Alongside, an elaborate review of the current literature was conducted. Results a total of 46 parotidectomies were performed on 37 patients with CP. Near-total parotidectomy was performed in 41 and SP in 5 cases. Eighty-four percent of patients was available for the telephone questionnaire (31 patients, 40 parotidectomies) with a mean follow-up period of 6,2 years. Treatment was successful in 40/46 parotidectomies (87%) and 95% of the patients were content with the result. The incidence of permanent and transient facial nerve palsy was 0 (0%) and 12 (26.1%), respectively. Frey syndrome manifested in 20 (43.5%) patients. Neither this study nor careful review of the current literature resulted in evident difference between SP and NTP regarding the primary outcome variables. Conclusion parotidectomy is a safe and effective treatment for CP in case conservative therapy fails. There is no evidence of a distinct difference between SP and NTP regarding efficiency, facial nerve palsy or Frey syndrome.

Fibro-osseous pseudotumor of digits - Expanding the spectrum of clonal transient neoplasms harboring USP6 rearrangement.

Fibro-osseous pseudotumors of the digits (FOPD) is a rare self-limiting lesion composed of bland looking hypercellular fibrous tissue and bone. USP6 rearrangement is a consistent genetic finding in aneurysmal bone cyst, nodular fasciitis, myositis ossificans and giant cell lesions of small bones. We report herein the occurrence of USP6 rearrangement in fibro-osseous pseudotumors of the digits using fluorescence in situ hybridization analysis (FISH). Of the five patients included, three were female and two were male. The age ranged from 33 to 72 years (mean 48 years). Lesions arose in the palm (n = 2), thenar (n = 1), middle finger (n = 1) and great toe (n = 1). All patients underwent resection. Four cases (80%) harbored USP6 rearrangements showing that fibro-osseous pseudotumors of digits belongs to the spectrum of clonal transient neoplasms including aneurysmal bone cyst, nodular fasciitis, myositis ossificans and giant cell lesion of small bones.

Conventional Pathology Versus Gene Signatures for Assessing Luminal A and B Type Breast Cancers: Results of a Prospective Cohort Study.

In this study, in estrogen receptor positive (ER+) early stage breast cancer patients who were considered candidates for 70-gene signature (70-GS, "MammaPrint") use, we compared molecular subtyping (MS) based on the previously validated 80-gene signature (80-GS, "BluePrint") versus surrogate pathological subtyping (PS). Between 1 January 2013 and 31 December 2015, 595 clinical intermediate risk ER+ early stage breast cancer patients were enrolled. Hormone receptor (HR) and HER2 receptor status were determined by conventional pathology using immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH). Ki67 was assessed in a subset of patients. The overall concordance between PS and MS for luminal type cancers (A and B together) was 98%. The concordance between PS and MS for luminal A and luminal B type cancers based on the Bloom Richardson histological grade (BR) (n = 586) or Ki67 (n = 185) was low: 64% (Kappa 0.20 [95% CI 0.11⁻0.28]) and 65% (Kappa 0.22 [95% CI 0.062⁻0.37]), respectively. In this prospective study (NCT02209857) of a selection of ER+ and predominantly HER2- early-stage breast cancer patients, the additional ability of the 80-GS to distinguish between luminal, HER2-type and basal-like cancers was inherently very limited. The distinction of luminal-type tumors into A and B according to Ki67 status or BR grade versus the 70-GS revealed poor concordance.

Myositis ossificans - Another condition with USP6 rearrangement, providing evidence of a relationship with nodular fasciitis and aneurysmal bone cyst.

Myositis ossificans is defined as a self-limiting pseudotumor composed of reactive hypercellular fibrous tissue and bone. USP6 rearrangements have been identified as a consistent genetic driving event in aneurysmal bone cyst and nodular fasciitis. It is therefore an integral part of the diagnostic workup when dealing with (myo)fibroblastic lesions of soft tissue and bone. Two cases of myositis ossificans with USP6 rearrangement were published so far. We determine herein the incidence of USP6 rearrangement in myositis ossificans using USP6 fluorescence in situ hybridization analysis (FISH). Of the 11 cases included, seven patients were female and four were male. Age ranged from 6 to 56 years (mean 27 years). Lesions were located in the thigh (n = 5), knee (n = 1), lower leg (n = 1), lower arm (n = 1), perineum (n = 1), gluteal (n = 1) and thoracic wall (n = 1). All assessable cases except one (8/9) showed rearrangement of USP6 providing evidence that myositis ossificans is genetically related to nodular fasciitis and aneurysmal bone cyst.

Multifocal occurrence of extra-abdominal desmoid type fibromatosis - A rare manifestation. A clinicopathological study of 6 sporadic cases and 1 hereditary case.

Desmoid-type fibromatosis, also called desmoid tumor, is a locally aggressive myofibroblastic neoplasm that usually arises in deep soft tissue with significant potential for local recurrence. It displays an unpredictable clinical course. ß-Catenin, the genetic key player of desmoid tumors shows nuclear accumulation due to mutations that prevent its degradation leading to activation of Wnt signaling and myofibroblastic cell proliferation. The corresponding hot spot mutations are located in exon 3 of the CTNNB1 gene or alternatively, in the APC tumor suppressor gene, most often as a germline mutation. Multifocal desmoid tumors are very rare and clinical characteristics are poorly understood. Here we present six sporadic and one familial case of multifocal desmoid tumors. Four female and three male patients, aged between 7 and 30 years (mean 18.4 years) were identified in a cohort of 1392 cases. Tumors were located in (distal) extremities, thorax, breast, abdominal wall, shoulder, and neck. Four cases showed a CTNNB1 mutation and one an APC germline mutation. In two sporadic cases no CTNNB1 mutation was identified. Four patients showed (multiple) recurrences and one patient was lost to follow-up. In conclusion, multifocal desmoid tumors are a very rare disease and may occur in sporadic cases that are characterized by recurrent CTNNB1 mutations. However, the underlying pathogenesis of multifocal desmoid tumors remains poorly understood with often aggressive clinical behavior and challenging therapeutical management.

Mutational analysis using Sanger and next generation sequencing in sporadic spindle cell hemangiomas: A study of 19 cases.

Spindle cell hemangioma (SCH) is a distinct vascular soft-tissue lesion characterized by cavernous blood vessels and a spindle cell component mainly occurring in the distal extremities of young adults. The majority of cases harbor heterozygous mutations in IDH1/2 sporadically or rarely in association with Maffucci syndrome. However, based on mosaicism and accordingly a low percentage of lesional cells harboring a mutant allele, detection can be challenging. We tested 19 sporadic SCHs by Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), conventional next generation sequencing (NGS), and NGS using a single molecule molecular inversion probes (smMIP)-based library preparation to compare their diagnostic value. Out of 10 cases tested by Sanger sequencing and 2 analyzed using MLPA, 4 and 1, respectively, revealed a mutation in IDH1 (p.R132C). The 7 remaining negative cases and additional 6 cases were investigated using smMIP/NGS, showing hot spot mutations in IDH1 (p.R132C) (8 cases) and IDH2 (3 cases; twice p.R172S and once p.R172G, respectively). One case was negative. Owing to insufficient DNA quality and insufficient coverage, 2 cases were excluded. In total, in 16 out of 17 cases successfully tested, an IDH1/2 mutation was found. Given that IDH1/2 mutations were absent in 161 other vascular lesions tested by smMIP/NGS, the mutation can be considered as highly specific for SCH.

Soft tissue angiofibroma: Clinicopathologic, immunohistochemical and molecular analysis of 14 cases.

Soft tissue angiofibroma is rare and has characteristic histomorphological and genetic features. For diagnostic purposes, there are no specific antibodies available. Fourteen lesions (6 females, 8 males; age range 7-67 years) of the lower extremities (12) and trunk (2) were investigated by immunohistochemistry, including for the first time NCOA2. NCOA2 was also tested in a control group of other spindle cell lesions. The known fusion-genes (AHRR-NCOA2 and GTF2I-NCOA2) were examined using RT-PCR in order to evaluate their diagnostic value. Cases in which no fusion gene was detected were additionally analysed by RNA sequencing. All cases tested showed nuclear expression of NCOA2. However, this was not specific since other spindle cell neoplasms also expressed this marker in a high percentage of cases. Other variably positive markers were EMA, SMA, desmin and CD34. STAT6 was negative in the cases tested. By RT-PCR for the most frequently observed fusions, an AHRR-NCOA2 fusion transcript was found in 9/14 cases. GTF2I-NCOA2 was not detected in the remaining cases (n = 3). RNA sequencing revealed three additional positive cases; two harbored a AHRR-NCOA2 fusion and one case a novel GAB1-ABL1 fusion. Two cases failed molecular analysis due to poor RNA quality. In conclusion, the AHRR-NCOA2 fusion is a frequent finding in soft tissue angiofibroma, while GTF2I-NCOA2 seems to be a rare genetic event. For the first time, we report a GAB1-ABL1 fusion in a soft tissue angiofibroma of a child. Nuclear expression of NCOA2 is not discriminating when compared with other spindle cell neoplasms.

Myxoid liposarcoma of the foot: a study of 8 cases.

INTRODUCTION: Myxoid liposarcoma is the only translocation-associated liposarcoma subtype. It classically originates in the deep soft tissues of the thigh. At distal sites of the extremities, this tumor is exceedingly rare. We present a series of 8 cases occurring in the foot/ankle. RESULTS: Two female and 6 male patients, aged between 32 and 77 years (mean, 54.3 years), were identified. Tumor size ranged from 1.1 to 10 cm (mean, 6.8 cm). Two lesions eroded bone. All tumors were treated by excision and 7 by (neo)adjuvant radiotherapy. R0 status was reached in 2 cases with 1 case followed by metastasis in the groin. All other cases were documented with R1 (n=2) or R2 (n=4) resection status. In 1 patient, the follow-up status was unknown. All other patients were alive 15-135 (mean, 55.8) months after initial diagnosis. We conclude that myxoid liposarcoma at acral sites are exceedingly rare, and in this series, prognosis was good irrespective of resection status. Clinicians and pathologists have to be aware because this sarcoma type shows a peculiar clinical behavior with high radio- and chemosensitivity and metastatic spread to extrapulmonary sites.

First clinical experience with a dedicated MRI-guided high-intensity focused ultrasound system for breast cancer ablation.

OBJECTIVES: To assess the safety and feasibility of MRI-guided high-intensity focused ultrasound (MR-HIFU) ablation in breast cancer patients using a dedicated breast platform. METHODS: Patients with early-stage invasive breast cancer underwent partial tumour ablation prior to surgical resection. MR-HIFU ablation was performed using proton resonance frequency shift MR thermometry and an MR-HIFU system specifically designed for breast tumour ablation. The presence and extent of tumour necrosis was assessed by histopathological analysis of the surgical specimen. Pearson correlation coefficients were calculated to assess the relationship between sonication parameters, temperature increase and size of tumour necrosis at histopathology. RESULTS: Ten female patients underwent MR-HIFU treatment. No skin redness or burns were observed in any of the patients. No correlation was found between the applied energy and the temperature increase. In six patients, tumour necrosis was observed with a maximum diameter of 3-11 mm. In these patients, the number of targeted locations was equal to the number of areas with tumour necrosis. A good correlation was found between the applied energy and the size of tumour necrosis at histopathology (Pearson = 0.76, p = 0.002). CONCLUSIONS: Our results show that MR-HIFU ablation with the dedicated breast system is safe and results in histopathologically proven tumour necrosis. KEY POINTS: • MR-HIFU ablation with the dedicated breast system is safe and feasible • In none of the patients was skin redness or burns observed • No correlation was found between the applied energy and the temperature increase • The correlation between applied energy and size of tumour necrosis was good.

Prognostic value of CTNNB1 gene mutation in primary sporadic aggressive fibromatosis.

BACKGROUND: Aggressive fibromatosis (AF) comprises tumors with a varying biological behavior. Genetic tumor characteristics may be predictive of recurrence; hence, the prognostic value of three specific mutations on the CTNNB1 gene was evaluated in relation to known clinicopathologic risk factors in patients with primary, sporadic AF. METHODS: In a multi-institutional retrospective cohort study of patients with primary extra-abdominal and abdominal wall AF who underwent surgical treatment, the original pathology specimens were reviewed for the presence of a T41A, S45F, and 45P mutations on the CTNNB1 gene. For these mutations, the risk of recurrence was analyzed by the Kaplan-Meier method with log-rank test. Univariable and multivariable Cox regression was performed to calculate hazard ratios. RESULTS: A total of 101 patients were analyzed. During a median follow-up of 41 months, 17 recurrences were detected; the cumulative 5-year recurrence rate was 22.8 %. A specific CTNNB1 mutation was found in 76 patients, with the majority of patients having a T41A mutation (n = 49). CTNNB1 mutations were associated with the risk of recurrence: the presence of a S45F mutation was associated with a 5-year cumulative risk of recurrence of 63.8 % (P < 0.001). Multivariable analysis showed that young age and S45F mutation were independent risk factors (P = 0.011 and P < 0.001). CONCLUSIONS: The presence of specific CTNNB1 mutations was predictive for recurrence in patients after surgical treatment for primary, sporadic extra-abdominal and abdominal AF. A S45F mutation increased the risk of recurrence significantly.

Epithelioid Hemangioendothelioma: clinicopathologic, immunhistochemical, and molecular genetic analysis of 39 cases.

BACKGROUND: Epithelioid hemangioendothelioma is a malignant, often indolent vascular tumor which occurs at various anatomic sites. Based on a reciprocal translocation t (1;3)(p36;q25), a consistent WWTR1-CAMTA1 fusion gene has been found. An alternate YAP1-TFE3 fusion has been detected in a small and distinct subset of cases. METHODS: Thirty-nine tumors, from 24 females and 15 males with an age range 9-85 years, were located in soft tissue (head and neck [8], trunk [5], upper extremities [3], lower extremities [2], mediastinal [1], and paratesticular [1]), lymph node (1), breast (1), skin (2), bone (6), lung (7), and liver (2). The cases were investigated using a panel of immunohistochemical markers. The aforementioned fusion-genes were examined using RT-PCR and/or FISH in order to validate their diagnostic value. RESULTS: Follow-up available for 17 patients ranged from 3 months to 7 years (median interval 1.5 years). Eleven patients were alive without disease, 2 patients were alive with disease after 1.5 and 2 years, respectively. Four patients died of disease after 4 months (n = 1), 5 months (n = 2), and 1.5 years (n = 1).The size, known for 30 lesions, was >3 cm in 9 of them. Histologically, all lesions had classical features, at least focally. Four tumors counted >3 mitoses/50 HPF. Immunohistochemically, all cases tested stained positive for ERG (21), FLI1 (5) and CD31 (39). CD34 and D2-40 positivity was seen in 81% and 71% of the examined cases, respectively. 11/35 cases expressed pan-keratin and 6/20 cases CK8.18. TFE3 showed a nuclear reaction in 21/24 cases, irrespective of TFE3 rearrangement.Molecular genetically, 35/35 cases revealed one of the fusion genes by FISH and/or RT-PCR with WWTR1-CAMTA1 in 33 cases and YAP1-TFE3 in 2 cases. CONCLUSIONS: These results demonstrate the high diagnostic value of FISH and RT-PCR in detecting the fusion genes of EHE. The immunohistochemical utility of TFE3 appears questionable in this study. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4010279141259481.

HER2 gene amplification in patients with breast cancer with equivocal IHC results.

AIMS: Equivocal human epidermal growth factor receptor 2 protein (HER2) (2+) immunohistochemistry (IHC) is subject to significant interobserver variation and poses a challenge in obtaining a definitive positive or negative test result. This equivocal test result group accounts for approximately 15% of all tumours, and for optimal guidance of HER2 targeted therapy, a further analysis of quantification of gene copy number and amplification status is needed for patients with early or metastatic breast cancer. METHODS: 553 breast-cancer specimens with equivocal HER2 IHC(2+) test results were collected and subsequently centrally retested by chromogenic in situ hybridisation (CISH), and HER2 gene copy numbers per tumour cell nucleus were determined. RESULTS: Using CISH, 77 of 553 equivocal HER2 IHC(2+) test result cases (13.9% of total) showed high levels of HER2 gene amplification (≥10.0 gene copies per nucleus), and 41 of 553 (7.4% of total) showed low-level HER2 gene amplification (6.0-9.9 gene copies per nucleus). In 73.6% of cases, no amplification of the HER2 gene was shown, and in only 4.9% of cases was an equivocal test result by CISH observed (4.0-5.9 gene copies per nucleus). CONCLUSIONS: Testing by CISH of all equivocal HER2 IHC(2+) test result provides a definitive guidance in HER2 targeted therapy in 95.1% of cases. A significant proportion (21.3%) of patients with equivocal IHC(2+) test results show amplification of the HER2 gene.