Proteomic Characterization of Undifferentiated Small Round Cell Sarcomas With EWSR1 and CIC::DUX4 Translocations Reveals Diverging Tumor Biology and Distinct Diagnostic Markers.

Undifferentiated small round cell sarcomas (USRS) of bone and soft tissue are a group of tumors with heterogenic genomic alterations sharing similar morphology. In the present study, we performed a comparative large-scale proteomic analysis of USRS (n = 42) with diverse genomic translocations including classic Ewing sarcomas with EWSR1::FLI1 fusions (n = 24) or EWSR1::ERG fusions (n = 4), sarcomas with an EWSR1 rearrangement (n = 2), CIC::DUX4 fusion (n = 8), as well as tumors classified as USRS with no genetic data available (n = 4). Proteins extracted from formalin-fixed, paraffin-embedded pretherapeutic biopsies were analyzed qualitatively and quantitatively using shotgun mass spectrometry (MS). More than 8000 protein groups could be quantified using data-independent acquisition. Unsupervised hierarchical cluster analysis based on proteomic data allowed stratification of the 42 cases into distinct groups reflecting the different molecular genotypes. Protein signatures that significantly correlated with the respective genomic translocations were identified and used to generate a heatmap of all 42 sarcomas with assignment of cases with unknown molecular genetic data to either the EWSR1- or CIC-rearranged groups. MS-based prediction of sarcoma subtypes was molecularly confirmed in 2 cases where next-generation sequencing was technically feasible. MS also detected proteins routinely used in the immunohistochemical approach for the differential diagnosis of USRS. BCL11B highly expressed in Ewing sarcomas, and BACH2 as well as ETS-1 highly expressed in CIC::DUX4-associated sarcomas, were among proteins identified by the present proteomic study, and were chosen for immunohistochemical confirmation of MS data in our study cohort. Differential expressions of these 3 markers in the 2 genetic groups were further validated in an independent cohort of n = 34 USRS. Finally, our proteomic results point toward diverging signaling pathways in the different USRS subgroups.

Diagnosis of joint invasion in patients with malignant bone tumors: value and reproducibility of direct and indirect signs on MR imaging.

OBJECTIVES: To evaluate the performance and reproducibility of MR imaging features in the diagnosis of joint invasion (JI) by malignant bone tumors. METHODS: MR images of patients with and without JI (n = 24 each), who underwent surgical resection at our institution, were read by three radiologists. Direct (intrasynovial tumor tissue (ITT), intraarticular destruction of cartilage/bone, invasion of capsular/ligamentous insertions) and indirect (tumor size, signal alterations of epiphyseal/transarticular bone (bone marrow replacement/edema-like), synovial contrast enhancement, joint effusion) signs of JI were assessed. Odds ratios, sensitivity, specificity, PPV, NPV, and reproducibilities (Cohen's and Fleiss' κ) were calculated for each feature. Moreover, the diagnostic performance of combinations of direct features was assessed. RESULTS: Forty-eight patients (28.7 ± 21.4 years, 26 men) were evaluated. All readers reliably assessed the presence of JI (sensitivity = 92-100 %; specificity = 88-100%, respectively). Best predictors for JI were direct visualization of ITT (OR = 186-229, p < 0.001) and destruction of intraarticular bone (69-324, p < 0.001). Direct visualization of ITT was also highly reliable in assessing JI (sensitivity, specificity, PPV, NPV = 92-100 %), with excellent reproducibility (κ = 0.83). Epiphyseal bone marrow replacement and synovial contrast enhancement were the most sensitive indirect signs, but lacked specificity (29-54%). By combining direct signs with high specificity, sensitivity was increased (96 %) and specificity (100 %) was maintained. CONCLUSION: JI by malignant bone tumors can reliably be assessed on preoperative MR images with high sensitivity, specificity, and reproducibility. Particularly direct visualization of ITT, destruction of intraarticular bone, and a combination of highly specific direct signs were valuable, while indirect signs were less predictive and specific. KEY POINTS: • Direct visualization of intrasynovial tumor was the single most sensitive and specific (92-100%) MR imaging sign of joint invasion. • Indirect signs of joint invasion, such as joint effusion or synovial enhancement, were less sensitive and specific compared to direct signs. • A combination of the most specific direct signs of joint invasion showed best results with perfect specificity and PPV (both 100%) and excellent sensitivity and NPV (both 96 %).

Soft tissue masses: distribution of entities and rate of malignancy in small lesions.

BACKGROUND: Small soft tissue masses are often falsely assumed to be benign and resected with failure to achieve tumor-free margins. Therefore, this study retrospectively investigated the distribution of histopathologic diagnosis to be encountered in small soft tissue tumors (≤ 5 cm) in a large series of a tertiary referral center. METHODS: Patients with a soft tissue mass (STM) with a maximum diameter of 5 cm presenting at our institution over a period of 10 years, who had undergone preoperative Magnetic resonance imaging and consequent biopsy or/and surgical resection, were included in this study. A final histopathological diagnosis was available in all cases. The maximum tumor diameter was determined on MR images by one radiologist. Moreover, tumor localization (head/neck, trunk, upper extremity, lower extremity, hand, foot) and depth (superficial / deep to fascia) were assessed. RESULTS: In total, histopathologic results and MR images of 1753 patients were reviewed. Eight hundred seventy patients (49.63%) showed a STM ≤ 5 cm and were therefore included in this study (46.79 +/- 18.08 years, 464 women). Mean maximum diameter of the assessed STMs was 2.88 cm. Of 870 analyzed lesions ≤ 5 cm, 170 (19.54%) were classified as superficial and 700 (80.46%) as deep. The malignancy rate of all lesions ≤ 5 cm was at 22.41% (superficial: 23.53% / deep: 22.14%). The malignancy rate dropped to 16.49% (20.79% / 15.32%) when assessing lesions ≤ 3 cm (p = 0.007) and to 15.0% (18.18% / 13.79%) when assessing lesions ≤ 2 cm (p = 0.006). Overall, lipoma was the most common benign lesion of superficial STMs (29.41%) and tenosynovial giant cell tumor was the most common benign lesion of deep STMs (23.29%). Undifferentiated pleomorphic sarcoma was the most common malignant diagnosis among both, superficial (5.29%) and deep (3.57%) STMs. CONCLUSIONS: The rate of malignancy decreased significantly with tumor size in both, superficial and deep STMs. The distribution of entities was different between superficial and deep STMs, yet there was no significant difference found in the malignancy rate.

[Parosteal low-grade osteosarcoma with focal differentiation]. / Parossales Low-grade-Osteosarkom mit fokaler Dedifferenzierung.

This case study deals with the case of a 16-year-old male patient with a low-grade parosteal osteosarcoma of the distal femur with focal differentiation. Case history, disease course, and surgery as well as the pathological workup with final diagnosis are presented. Relevant radiologic and pathologic differential diagnoses und diagnostic pitfalls are explained in detail and discussed. Additionally, postoperative treatment options are illustrated.

Circulating cKIT and PDGFRA DNA indicates disease activity in Gastrointestinal Stromal Tumor (GIST).

This prospective trial aimed to investigate whether tumor-specific cKIT and PDGFRA mutations can be detected and quantified in circulating tumor (ct)DNA in patients with active GIST, and whether detection indicates disease activity. We included 25 patients with active disease and cKIT or PDGFRA mutations detected in tissue. Mutant ctDNA was detected in the peripheral blood plasma using allele-specific ligation (L-)PCR and droplet digital (d)PCR. CtDNA harboring tumor-specific cKIT or PDGFRA mutations was detected at least once in 16 out of 25 patients using L-PCR (64%) and in 20 out of 25 patients with dPCR (80%). Using dPCR, the absolute numbers of ctDNA fragments (DNA copies/ml) and the mutant allele frequency (MAF; in percent of wild-type control) strongly correlated with tumor size expressed as RECIST1.1 sum of diameter (SOD) in mm (ρ = 0.3719 and 0.408, respectively, p < 0.0001) and response status (ρ = 0.3939 and 0.392, respectively, p < 0.0001 and p < 0.001). Specificity of dPCR for detection of progression was 79.2% with a sensitivity of 55.2% and dPCR discriminated CR from active disease with a specificity of 96% and s sensitivity of 44.7%. With L-PCR, correlations of MAF with tumor size and response status were less prominent. Serial ctDNA measurement reflected individual disease courses over time. Targeted panel sequencing of four patients detected additional driver mutations in all cases and secondary resistance mutations in two cases. Thus, ctDNA indicates disease activity in patients with GIST and should be incorporated as companion biomarker in future prospective trials.

Differentiating atypical lipomatous tumors from lipomas with magnetic resonance imaging: a comparison with MDM2 gene amplification status.

BACKGROUND: To evaluate the diagnostic value of MR imaging for the differentiation of lipomas and atypical lipomatous tumors (ALT) in comparison with histology and MDM2 amplification status. METHODS: Patients with well-differentiated lipomatous tumors (n = 113), of which 66 were diagnosed as lipoma (mean age 53 years (range, 13-82); 47% women) and 47 as atypical lipomatous tumor (ALT; mean age 60 years (range, 28-88); 64% women), were included into this study using histology and MDM2 amplification status by fluorescence in situ hybridization (FISH) as standard of reference. Preoperative MR images were retrospectively assessed by two radiologists for the following imaging features: maximum tumor diameter (mm) as well as the affected compartment (intramuscular, intermuscular or subcutaneous), septa (absent, thin (< 2 mm) or thick septa (> 2 mm) with nodular components); contrast enhancing areas within the lipomatous tumor (< 1/3 of the tumor volume, > 1/3 of the tumor volume); RESULTS: Of the 47 patients with ALT, 40 (85.1%) presented thick septa (> 2 mm) and this finding significantly increased the likelihood of ALT (OR 6.24, 95% CI 3.36-11.59; P < 0.001). The likelihood of ALT was increased if the tumor exceeded a maximum diameter of 130.0 mm (OR 2.74, 95% CI 1.82-4.11, P < 0.001). The presence of contrast enhancement in lipomatous tumors significantly increased the likelihood of ALT (Odds ratio (OR) 2.95, 95% confidence interval (CI) 2.01-4.31; P < 0.001). Of the lipomas, 21.1% were located subcutaneously, 63.6% intramuscularly and 15.2% intermuscularly. On the other hand, none of the ALTs were located subcutaneously, the majority was located intermuscularly (87.3%) and a small number of ALTs was located intramuscularly (12.7%). CONCLUSIONS: Our results suggest that using specific morphological MR imaging characteristics (maximum tumor diameter, thick septa and contrast enhancement) and the information on the localization of the lipomatous tumor, a high sensitivity and substantial specificity can be achieved for the diagnosis of lipomas and ALTs.

Milk cloud appearance-a characteristic sign of fibrous dysplasia on contrast-enhanced MR imaging.

OBJECTIVE: To evaluate contrast-enhanced T1-weighted magnetic resonance (MR) images in histologically proven fibrous dysplasia (FD) for the prevalence of "milk cloud appearance" and its association with ground-glass appearance (GGA) on radiography or computed tomography (CT). METHODS: For this retrospective cohort study, 37 patients with histologically proven FD imaged preoperatively with contrast-enhanced MR imaging and radiography or CT were identified at our institution. Three radiologists independently evaluated MR images for the presence of milk cloud appearance on T1-weighted contrast-enhanced images, sites of skeletal involvement, type of bone involved, uni- vs. multifocality, mono- vs. polyostotic disease, maximum diameter, proportion of bone involved, expansile remodeling, and T2 homogeneity. The presence or absence of GGA on radiography or CT was determined in consensus. Inter-reader agreement was evaluated for milk cloud appearance using Cohen's kappa, and associations between milk cloud appearance and other imaging parameters were tested using Spearman's rho. RESULTS: Among the 37 histologically proven FD lesions, GGA was identified in 70% of the lesions, while milk cloud appearance was found in 82% of the lesions. Inter-reader agreement for milk cloud appearance on MR imaging was good to excellent (κ 0.65, 0.82, and 0.8). A significant correlation was found between milk cloud appearance and GGA (ρ = 0.31, p < 0.001). CONCLUSION: Milk cloud appearance is a characteristic sign of FD on contrast-enhanced T1-weighted MR images. Recognition of this feature may be helpful when radiographs are equivocal or unremarkable or when MR imaging is performed as the primary imaging modality in cases of FD. KEY POINTS: • Fibrous dysplasia displays a characteristic feature on contrast-enhanced T1-weighted MR imaging: milk cloud appearance. • Milk cloud appearance correlates well with the radiographic or CT finding of ground-glass appearance. • Recognition of milk cloud appearance on contrast-enhanced MR imaging may be helpful when radiographs are equivocal or unremarkable or when MR imaging is performed as the primary imaging modality in cases of fibrous dysplasia.

Liver transplantation followed by autologous stem cell transplantation for acute liver failure caused by AL amyloidosis. Case report and review of the literature.

 Hepatic involvement in AL amyloidosis may present as acute liver failure. Historically, liver transplantation in these cases has achieved poor outcomes due to progress of amyloidosis and non-hepatic organ damage. In the era of bortezomib treatment, the prognosis of AL amyloidosis has been markedly improved and may also result in better post-transplant outcomes. We present a case of isolated acute liver failure caused by AL amyloidosis, bridged to transplantation with bortezomib and treated with sequential orthotopic liver transplantation (OLT) and autologous stem cell transplantation. The patient is in stable remission 3 years after OLT.

Erdheim-Chester disease with bilateral Achilles tendon involvement.

Erdheim-Chester disease (ECD) is a rare non-Langerhans cell histiocytosis with a broad spectrum of organ manifestations, presenting with characteristic radiological and histological findings. Soft tissue manifestations (xanthogranulomas) have been reported to be most commonly found in the region of the orbits. We report bilateral Achilles tendon xanthogramlomas in a 36-year-old male with biopsy-proven and B-RAF V600E-positive ECD. Although rare, ECD should be considered in the differential diagnosis of intratendinous masses.

TFAP2E-DKK4 and chemoresistance in colorectal cancer.

BACKGROUND: Chemotherapy for advanced colorectal cancer leads to improved survival; however, predictors of response to systemic treatment are not available. Genomic and epigenetic alterations of the gene encoding transcription factor AP-2 epsilon (TFAP2E) are common in human cancers. The gene encoding dickkopf homolog 4 protein (DKK4) is a potential downstream target of TFAP2E and has been implicated in chemotherapy resistance. We aimed to further evaluate the role of TFAP2E and DKK4 as predictors of the response of colorectal cancer to chemotherapy. METHODS: We analyzed the expression, methylation, and function of TFAP2E in colorectal-cancer cell lines in vitro and in patients with colorectal cancer. We examined an initial cohort of 74 patients, followed by four cohorts of patients (total, 220) undergoing chemotherapy or chemoradiation. RESULTS: TFAP2E was hypermethylated in 38 of 74 patients (51%) in the initial cohort. Hypermethylation was associated with decreased expression of TFAP2E in primary and metastatic colorectal-cancer specimens and cell lines. Colorectal-cancer cell lines overexpressing DKK4 showed increased chemoresistance to fluorouracil but not irinotecan or oxaliplatin. In the four other patient cohorts, TFAP2E hypermethylation was significantly associated with nonresponse to chemotherapy (P<0.001). Conversely, the probability of response among patients with hypomethylation was approximately six times that in the entire population (overall estimated risk ratio, 5.74; 95% confidence interval, 3.36 to 9.79). Epigenetic alterations of TFAP2E were independent of mutations in key regulatory cancer genes, microsatellite instability, and other genes that affect fluorouracil metabolism. CONCLUSIONS: TFAP2E hypermethylation is associated with clinical nonresponsiveness to chemotherapy in colorectal cancer. Functional assays confirm that TFAP2E-dependent resistance is mediated through DKK4. In patients who have colorectal cancer with TFAP2E hypermethylation, targeting of DKK4 may be an option to overcome TFAP2E-mediated drug resistance. (Funded by Deutsche Forschungsgemeinschaft and others.).

Local Staging of Soft-Tissue Sarcoma: Emphasis on Assessment of Neurovascular Encasement-Value of MR Imaging in 174 Confirmed Cases.

PURPOSE: To evaluate utility of magnetic resonance (MR) imaging in local staging of soft-tissue sarcoma, with an emphasis on assessment of neurovascular encasement. MATERIALS AND METHODS: Institutional review board approval was obtained; informed consent requirement was waived. Preoperative MR images in 174 patients with soft-tissue sarcoma were analyzed by two readers. Tumor staging according to the American Joint Committee on Cancer/Union International Contre le Cancer and Enneking staging systems and analysis of osseous and articular invasion were performed. To assess neurovascular encasement, contact between tumor and arteries, between tumor and veins, and between tumor and nerves was classified (no contact, contact ≤90°, 91°-180°, 181°-270°, >271°). Interobserver agreement was determined; imaging findings were correlated with intraoperative findings and/or histopathologic findings (Pearson correlation coefficient [r] and Cohen κ coefficient). RESULTS: Intraoperative evaluation and/or histopathologic evaluation confirmed osseous, articular, and neurovascular invasion in 8.6%, 2.9%, and 25.3% of patients. Interobserver agreement was excellent for tumor staging (American Joint Committee on Cancer/Union International Contre le Cancer staging, κ = 0.811; Enneking staging, κ = 0.943) and osseous invasion (κ = 1.000). It was substantial for articular invasion (κ = 0.794). Sensitivity and specificity for osseous invasion were 100% and 98.7%, respectively (both readers). For articular invasion, sensitivity was 80% (both readers); specificities were 100% and 98.8% for readers 1 and 2, respectively. Interobserver agreement in quantifying contact between tumor and vessels and between tumor and nerves was excellent for arteries, veins, and nerves (κ = 0.845, 0.892, 0.893, respectively). Receiver operating characteristic analysis revealed optimal threshold of greater than 180° for prediction of arterial and venous encasement (both readers). For neural encasement, optimal threshold was greater than 180° (reader 1) and greater than 270° (reader 2). Sensitivities in diagnosing encasement for arteries, veins, and nerves were 84.6%, 84.6%, and 77.8% (reader 1) and 84.6%, 84.6%, and 72.2% (reader 2). Specificities for encasement of arteries, veins, and nerves, respectively, were 97.5%, 97.5%, and 93.2% (reader 1) and 93.8%, 94.7%, 97.3% (reader 2). CONCLUSION: MR imaging allows reliable and accurate local staging of soft-tissue sarcoma. Encasement of arteries, veins, and nerves should be diagnosed, if the contact between tumor and vascular or neural circumference exceeds 180°.

Study of Preoperative Radiotherapy for Sarcomas of the Extremities with Intensity-Modulation, Image-Guidance and Small Safety-margins (PREMISS).

BACKGROUND: The aim of the trial is to demonstrate that with the use of modern IMRT/IGRT and reduction of safety margins postoperative wound complications can be reduced. METHODS/ DESIGN: The trial is designed as a prospective, monocentric clinical phase II trial. The treatment is performed with helical IMRT on the Tomotherapy HiArt System© or with RapidArc© IMRT as available. All treatments are performed with 6 MV photons and daily online CT-based IGRT. A dose of 50 Gy in 2 Gy single fractions (5 fractions per week) is prescribed. Restaging including MRI of the primary tumor site as well as CT of the thorax/abdomen is planned 4 weeks after RT. PET-examinations or any other imaging can be performed as required clinically. In cases of R1 resection, brachytherapy is anticipated in the 2nd postoperative week. Brachytherapy catheters are implanted into the tumor bed depending on the size and location of the lesion. Surgery is planned 5-6 weeks after completion of neoadjuvant RT. All patients are seen for a first follow-up visit 2 weeks after wound healing is completed, thereafter every 3 months during the first 2 years. The endpoints of the study are evaluated in detail during the first (2 weeks) and second (3 months) follow-up. Functional outcome and QOL are documented prior to treatment and at year 1 and 2. Treatment response and efficacy will be scored according to the RECIST 1.1 criteria. A total patient number of 50 with an expected 20% rate of wound complications were calculated for the study, which translates into a 95% confidence interval of 10.0-33.7% for wound complication rate in a binomial distribution. DISCUSSION: The present study protocol prospectively evaluates the use of IMRT/IGRT for neoadjuvant RT in patients with soft tissue sarcomas of the extremity with the primary endpoint wound complications, which is the major concern with this treatment sequence. Besides complications rates, local control rates and survival rates, as well as QOL, functional outcome and treatment response parameters (imaging and pathology) are part of the protocol. The data of the present PREMISS study will enhance the current literature and support the hypothesis that neoadjuvant RT with IMRT/IGRT offers an excellent risk-benefit ratio in this patient population. TRIAL REGISTRATION: NCT01552239.

Distinct transcriptional signature and immunoprofile of CIC-DUX4 fusion-positive round cell tumors compared to EWSR1-rearranged Ewing sarcomas: further evidence toward distinct pathologic entities.

Round cell sarcomas harboring CIC-DUX4 fusions have recently been described as highly aggressive soft tissue tumors of children and young adults. Due to partial morphologic and immunohistochemical overlap with Ewing sarcoma (ES), CIC-DUX4-positive tumors have generally been classified as ES-like and managed similarly; however, a systematic comparison at the molecular and immunohistochemical levels between these two groups has not yet been conducted. Based on an initial observation that CIC-DUX4-positive tumors show nuclear immunoreactivity for WT1 and ETS transcription factors, FLI1 and ERG, we performed a detailed immunohistochemical and molecular analysis including these markers, to further investigate the relationship between CIC-DUX4 tumors and ES. The study group included 21 CIC-DUX4-positive sarcomas and 20 EWSR1-rearranged ES. Immunohistochemically, CIC-DUX4 sarcomas showed membranous CD99 positivity in 18 (86%) cases, but only 5 (24%) with a diffuse pattern, while WT1 and FLI1 were strongly positive in all cases. ERG was positive in 18% of cases. All ES expressed CD99 and FLI1, while ERG positivity was only seen in EWSR1-ERG fusion positive ES. WT1 was negative in all ES. Expression profiling validated by q-PCR revealed a distinct gene signature associated with CIC-DUX4 fusion, with upregulation of ETS transcription factors (ETV4, ETV1, and ETV5) and WT1, among top overexpressed genes compared to ES, other sarcomas and normal tissue. In conclusion, the distinct gene signature and immunoprofile of CIC-DUX4 sarcomas suggest a distinct pathogenesis from ES. The consistent WT1 expression may provide a useful clue in the diagnosis in the context of round cell sarcomas negative for EWSR1 rearrangement. © 2014 Wiley Periodicals, Inc.

Intraductal papillary neoplasms of the bile duct: stepwise progression to carcinoma involves common molecular pathways.

Intraductal papillary neoplasms of the bile duct are still poorly characterized regarding (1) their molecular alterations during the development to invasive carcinomas, (2) their subtype stratification and (3) their biological behavior. We performed a multicenter study that analyzed these issues in a large European cohort. Intraductal papillary neoplasms of the bile duct from 45 patients were graded and subtyped using mucin markers and CDX2. In addition, tumors were analyzed for common oncogenic pathways, and the findings were correlated with subtype and grade. Data were compared with those from 22 extra- and intrahepatic cholangiocarcinomas. Intraductal papillary neoplasms showed a development from preinvasive low- to high-grade intraepithelial neoplasia to invasive carcinoma. Molecular and immunohistochemical analysis revealed mutated KRAS, overexpression of TP53 and loss of p16 in low-grade intraepithelial neoplasia, whereas loss of SMAD4 was found in late phases of tumor development. Alterations of HER2, EGFR, ß-catenin and GNAS were rare events. Among the subtypes, pancreato-biliary (36%) and intestinal (29%) were the most common, followed by gastric (18%) and oncocytic (13%) subtypes. Patients with intraductal papillary neoplasm of the bile duct showed a slightly better overall survival than patients with cholangiocarcinoma (hazard ratio (cholangiocarcinoma versus intraductal papillary neoplasm of the bile duct): 1.40; 95% confidence interval: 0.46-4.30; P=0.552). The development of biliary intraductal papillary neoplasms of the bile duct follows an adenoma-carcinoma sequence that correlates with the stepwise activation of common oncogenic pathways. Further large trials are needed to investigate and verify the finding of a better prognosis of intraductal papillary neoplasms compared with conventional cholangiocarcinoma.

Detection of truncated HER2 forms in formalin-fixed, paraffin-embedded breast cancer tissue captures heterogeneity and is not affected by HER2-targeted therapy.

Truncated forms of HER2, previously identified in subsets of HER2-positive breast cancer, originate from proteolytic extracellular domain (ECD) cleavage or alternative translation initiation. They lack ECD but may retain intracellular domain functionality, potentially associated with unfavorable prognosis, metastasis, and decreased sensitivity to antibody-based HER2-targeted therapy. To study the distribution of truncated HER2 in breast cancer, we detected loss of membrane-bound ECD independently of its molecular origin in paraffin sections, combining multispectral unmixing of chromogenic duplex IHC for HER2 ECD and intracellular domain with advanced image analysis. HER2 C-terminal fragment 611-transfected MCF7 and 4-aminophenylmercuric acetate-treated SKBR3 cell lines were used as controls. Applying a prototype work flow to whole sections, paired surgical resection/core needle biopsy samples, and paired samples from 69 patients of a phase 2 neoadjuvant clinical trial, we observed unexpected heterogeneity of ECD loss at the single-cell level, and in different areas of individual tumors, indicating that extent and localization of HER2 ECD loss add relevant information to averaging truncated HER2 across whole sections. We show acceptable run-to-run variation (coefficient of variation, <0.15), image analysis results in moderate agreement with conventional slide assessment (Cohen's κ = 0.59), and no obvious interference with previous HER2-ECD-targeted therapy. We conclude that duplex IHC and digital image processing extend current approaches of truncated HER2 detection.

Bone marrow oedema on MR imaging indicates ARCO stage 3 disease in patients with AVN of the femoral head.

OBJECTIVES: To test the hypothesis that bone marrow oedema (BME) observed on MRI in patients with avascular necrosis (AVN) of the femoral head represents an indicator of subchondral fracture. METHODS: Thirty-seven symptomatic hips of 27 consecutive patients (53% women, mean age 49.2) with AVN of the femoral head and associated BME on magnetic resonance (MR) imaging were included. MR findings were correlated with computed tomography (CT) of the hip and confirmed by histopathological examination of the resected femoral head. Imaging studies were analysed by two radiologists with use of the ARCO classification. RESULTS: On MR imaging a fracture line could be identified in 19/37 (51%) cases, which were classified as ARCO stage 3 (n = 15) and stage 4 (n = 4). The remaining 18/37 (49%) cases were classified as ARCO stage 2. However, in all 37/37 (100%) cases a subchondral fracture was identified on CT, indicating ARCO stage 3/4 disease. The extent of subchondral fractures and the femoral head collapse was graded higher on CT as compared to MRI (P < 0.05). Histopathological analysis confirmed bone necrosis and subchondral fractures. CONCLUSIONS: In patients with AVN, BME of the femoral head represents a secondary sign of subchondral fracture and thus indicates ARCO stage 3 disease. KEY POINTS: BME on MRI in AVN of femoral head indicates a subchondral fracture. BME in AVN of the femoral head represents ARCO stage 3/4 disease. CT identifies subchondral fractures and femoral head collapse better than MR imaging. This knowledge helps to avoid understaging and to trigger adequate treatment.

Hepatic angiosarcoma: cross-sectional imaging findings in seven patients with emphasis on dynamic contrast-enhanced and diffusion-weighted MRI.

OBJECTIVE: Primary hepatic angiosarcoma is a very rare and aggressive malignancy of vascular origin. We describe cross-sectional imaging findings of this entity with emphasis on dynamic contrast-enhanced (DCE) and diffusion-weighted (DWI) MR imaging. METHODS: Seven cases of pathologically confirmed hepatic angiosarcoma were retrospectively reviewed (CT and MRI examinations were available in seven and six patients, respectively). Two radiologists evaluated lesion growth patterns, attenuation, signal intensity characteristics, contrast enhancement patterns, and apparent diffusion coefficients (ADCs). RESULTS: Multifocal hepatic disease was present in six patients by means of a mixed pattern of large dominant masses and multiple small nodules; one patient had a solitary large mass. Unenhanced images depicted hemorrhagic areas and a markedly heterogeneous internal architecture within large tumors. Contrast-enhanced early phase images showed variable patterns including patchy peripheral or bizarre shaped intralesional foci of enhancement, peripheral rim enhancement, and small lesions without enhancement. On DCE images, the majority of lesions presented with varying degrees of progressive enhancement. Small nodules frequently displayed homogeneous enhancement on delayed phase images due to complete fill-in. DWI revealed a high interlesional variability of ADC values (range 0.57-2.41 × 10(-3 )mm(2)/s, mean 1.37 × 10(-3 )mm(2)/s). CONCLUSION: Cross-sectional imaging findings of hepatic angiosarcoma reflect the varied histopathological composition of the tumors. Multifocal disease, hemorrhage within large lesions, as well as progressive enhancement on DCE images are typical features of hepatic angiosarcoma. The mean ADC of lesions was found to be slightly elevated in comparison with other hepatic malignancies.

Effects of cold ischemia and inflammatory tumor microenvironment on detection of PI3K/AKT and MAPK pathway activation patterns in clinical cancer samples.

The accuracy of common markers for PI3K/AKT and MAPK pathway activation in preclinical and clinical cancer biomarker studies depends on phosphoepitope stability and changes of phosphorylation under ischemia. Herein, we define conditions under which phosphoepitope-specific duplex immunohistochemistry (IHC) on formalin-fixed, paraffin-embedded tumor tissues reflects pathway activation in situ as accurately as possible, and identify activation patterns linked to mutational status, pathway dependency and tumor microenvironment in clinical tumor samples, cell culture and xenograft tissues. Systematically assessing robustness of pAKT, pERK1/2, pMEK1/2 and pmTOR detection and related markers in xenograft tissues exposed to ischemia, we show that control of preprocessing and ischemia times allows accurate interpretation of staining results. Phosphorylation patterns were then analyzed in 33 xenograft models and in 58 cases with breast cancer, including 21 paired samples of core-needle biopsies with corresponding mastectomy specimens, and 37 mastectomy samples obtained under rigorously controlled conditions minimizing ischemia time. Patterns of pAKT and pERK1/2 staining (predominant PI3K/AKT, predominant MAPK and concomitant activation) were associated with sensitivity to pathway inhibition and partially with the mutational status in cell lines and corresponding xenograft tumors. In contrast, no clear correlation between mutational status and staining patterns was observed in clinical breast cancer samples, suggesting that interaction with the human tumor microenvironment may interfere with the use of phosphoepitope-specific IHC as potential markers for pathway dependency. In contrast to core needle biopsies, surgically resected breast cancer samples showed evidence of severe signal changes comparable to those effects observed in xenograft tumors exposed to controlled ischemia.

Histopathologic features of ovarian borderline tumors are not predictive of clinical outcome.

OBJECTIVES: Ovarian borderline tumors (BOTs) generally have an excellent prognosis, although recurrences and malignant transformation can occur. Our aim was to compare clinicopathologic features of BOT with clinical outcome. METHODS: In seventy consecutive BOTs clinicopathologic parameters, tumor cell proliferation (Ki67) and in selected cases KRAS, BRAF and p53 mutational status were analyzed with recurrence-free and overall survival as the endpoints. RESULTS: Sixty-one (87%) patients presented with FIGO stage I, 3 stage II, and 6 stage III. Thirty-four patients had serous and 36 mucinous BOT (30 intestinal and 6 endocervical subtypes). Non-invasive peritoneal implants occurred in 9 patients, and no invasive implants were observed. Recurrence-free and overall survival rates were 91% and 99%, respectively, at a mean follow-up of 63 months. Disease recurrence occurred in 6 cases (all FIGO stage I) including 3 serous, 1 mucinous-intestinal, and 2 mucinous-endocervical subtypes. Mean time to recurrence was 27 months (range 8-68). The recurrence rate following fertility-conserving surgery was 31% (5/16) compared to 2% (1/54) after bilateral salpingo-oophorectomy. Neither peritoneal implants (9/70), micropapillary pattern (2/34), microinvasion (4/70), nor increased tumor cell proliferation was associated with a higher recurrence rate. The frequency of KRAS or BRAF mutations was 50% (3/6 recurrences and 3/6 controls; 4 KRAS, 2 BRAF mutations). No p53 mutations (0/12) were detected in primary or recurrent BOTs. CONCLUSIONS: Histopathologic parameters were not predictive of BOT recurrence including previously suggested risk factors such as micropapillary pattern and microinvasion. However, fertility-conserving surgery and incomplete surgical staging were associated with a higher risk for recurrence.