Beata Bode

γ-Radiation Promotes Immunological Recognition of Cancer Cells through Increased Expression of Cancer-Testis Antigens In Vitro and In Vivo

Background γ-radiation is an effective treatment for cancer. There is evidence that radiotherapy supports tumor-specific immunity. It was described that irradiation induces de novo protein synthesis and enhances antigen presentation, we therefore investigated whether γ-radiation results in increased expression of cancer-testis (CT) antigens and MHC-I, thus allowing efficient immunological control. This is relevant because the expression of CT-antigens and MHC-I on tumor cells is often heterogeneous. We found that the changes induced by γ-radiation promote the immunological recognition of the tumor, which is illustrated by the increased infiltration by lymphocytes after radiotherapy. Methods/Findings We compared the expression of CT-antigens and MHC-I in various cancer cell lines and fresh biopsies before and after in vitro irradiation (20 Gy). Furthermore, we compared paired biopsies that were taken before and after radiotherapy from sarcoma patients. To investigate whether the changed expression of CT-antigens and MHC-I is specific for γ-radiation or is part of a generalized stress response, we analyzed the effect of hypoxia, hyperthermia and genotoxic stress on the expression of CT-antigens and MHC-I. In vitro irradiation of cancer cell lines and of fresh tumor biopsies induced a higher or de novo expression of different CT-antigens and a higher expression of MHC-I in a time- and dose-dependent fashion. Importantly, we show that irradiation of cancer cells enhances their recognition by tumor-specific CD8+ T cells. The analysis of paired biopsies taken from a cohort of sarcoma patients before and after radiotherapy confirmed our findings and, in addition showed that irradiation resulted in higher infiltration by lymphocytes. Other forms of stress did not have an impact on the expression of CT-antigens or MHC-I. Conclusions Our findings suggest that γ-radiation promotes the immunological recognition of the tumor. We therefore propose that combining radiotherapy with treatments that support tumor specific immunity may result in increased therapeutic efficacy.

Fluorescence In Situ Hybridization in the Definitive Diagnosis of Malignant Mesothelioma in Effusion Cytology

BACKGROUND Distinction of malignant mesothelioma (MM) from reactive mesothelial cells (RM) in effusions is notoriously difficult. The aim of our study was to test chromosomal aberrations detected by fluorescence in situ hybridization (FISH) in the diagnosis of MM in effusion cytology and to explore the potential role of p16, p14, and p15 gene methylation as an alternative mechanism of tumor suppressor gene inactivation. METHODS Fifty-two effusions of biopsy-proven MM and 28 benign effusions were retrospectively analyzed by multitarget FISH assay for aberrations of chromosomes 3, 7, 17, and 9p21. In case of a negative result, the corresponding MM biopsy specimen was analyzed. Methylation-specific polymerase chain reaction (MSP) for p16, p14, and p15 was performed on FISH-negative MM biopsy specimens. RESULTS Seventy-nine percent of effusions with biopsy-proven MM had chromosomal aberrations, with loss of 9p21 as the most common finding. All benign effusions were FISH negative. Sensitivity, specificity, and positive and negative predictive values for detection of MM by FISH were 79%, 100%, 100%, and 72%, respectively. Six of nine FISH-negative effusions with biopsy-proven MM were also FISH negative in the MM biopsy specimens. Four of five FISH-negative biopsy specimens showed promoter methylation in p16 and p14 as compared with one of 12 benign controls. CONCLUSIONS FISH is a sensitive and highly specific method for the definitive diagnosis of MM in effusion cytology. In the subset of FISH-negative MM, tumor suppressor genes on the chromosomal region 9p21 are often inactivated by promoter methylation.

Detection of ALK-Positive Non-Small-Cell Lung Cancers on Cytological Specimens High Accuracy of Immunocytochemistry with the 5A4 Clone

Introduction: Lung cancer is often diagnosed by cytology, necessitating predictive molecular marker analyses on cytological specimens. The gold standard for detection of predictive anaplastic lymphoma kinase (ALK)-rearrangements is fluorescence in situ hybridization (FISH), but FISH is both expensive and often challenging to interpret. The aim of our study was to investigate the accuracy of ALK immunocytochemistry (ICC) on cytological specimens of non–small-cell lung cancers (NSCLCs). Methods: Forty-one cytological specimens with available ALK FISH results were retrospectively analyzed with the 5A4 monoclonal antibody (Novocastra; Leica Biosystems) on a fully automated slide stainer. The specimens were enriched for ALK FISH-positive NSCLCs (14 of 41; 34.1%). Evaluation of the ICC staining was performed blinded to the FISH results. The staining intensity and the percentage of stained cancer cells were recorded. Any ICC staining was regarded as a positive result. The ALK ICC results were compared with the FISH results. In case of a discrepancy the ICC-stained slide and the FISH signals were reviewed. Results: ICC was evaluable on 40 of 41 specimens. Fifteen of 40 NSCLCs (37.5%) were ALK ICC-positive, with staining of the majority of cancer cells (median 100%; mean 82.3%). Twelve of the ICC-positive NSCLCs (80.0%) showed an intense staining (3+). Compared with the ALK FISH results, only one NSCLC was false-negative, and one false-positive by ICC, respectively. The sensitivity, specificity, and positive and negative predictive values for ALK ICC compared with ALK FISH were 93.3%, 96.0%, 93.3%, and 96%, respectively. Conclusion: ALK ICC is highly accurate for detecting ALK-rearranged NSCLCs.

The additional value of CT images interpretation in the differential diagnosis of benign vs. malignant primary bone lesions with 18F-FDG-PET/CT

ObjectiveTo evaluate the value of a dedicated interpretation of the CT images in the differential diagnosis of benign vs. malignant primary bone lesions with 18fluorodeoxyglucose-positron emission tomography/computed tomography (18F-FDG-PET/CT).Materials and methodsIn 50 consecutive patients (21 women, 29 men, mean age 36.9, age range 11–72) with suspected primary bone neoplasm conventional radiographs and 18F-FDG-PET/CT were performed. Differentiation of benign and malignant lesions was separately performed on conventional radiographs, PET alone (PET), and PET/CT with specific evaluation of the CT part. Histology served as the standard of reference in 46 cases, clinical, and imaging follow-up in four cases.ResultsAccording to the standard of reference, conventional 17 lesions were benign and 33 malignant. Sensitivity, specificity, and accuracy in assessment of malignancy was 85%, 65% and 78% for conventional radiographs, 85%, 35% and 68% for PET alone and 91%, 77% and 86% for combined PET/CT. Median SUVmax was 3.5 for benign lesions (range 1.6–8.0) and 5.7 (range 0.8–41.7) for malignant lesions.In eight patients with bone lesions with high FDG-uptake (SUVmax ≥ 2.5) dedicated CT interpretation led to the correct diagnosis of a benign lesion (three fibrous dysplasias, two osteomyelitis, one aneurysmatic bone cyst, one fibrous cortical defect, 1 phosphaturic mesenchymal tumor). In four patients with lesions with low FDG-uptake (SUVmax < 2.5) dedicated CT interpretation led to the correct diagnosis of a malignant lesion (three chondrosarcomas and one leiomyosarcoma). Combined PET/CT was significantly more accurate in the differentiation of benign and malignant lesions than PET alone (p = .039). There was no significant difference between PET/CT and conventional radiographs (p = .625).ConclusionDedicated interpretation of the CT part significantly improved the performance of FDG-PET/CT in differentiation of benign and malignant primary bone lesions compared to PET alone. PET/CT more commonly differentiated benign from malignant primary bone lesions compared with conventional radiographs, but this difference was not significant.

Cyr61 Expression in Osteosarcoma Indicates Poor Prognosis and Promotes Intratibial Growth and Lung Metastasis in Mice

Osteosarcoma is the most frequent primary malignant bone tumor in children and adolescents with a high propensity for lung metastasis, the major cause of disease‐related death. Reliable outcome‐predictive markers and targets for osteosarcoma metastasis‐suppressing drugs are urgently needed for more effective treatment of metastasizing osteosarcoma, which has a current mean 5‐year survival rate of approximately 20%. This study investigated the prognostic value and the biological relevance of the extracellular matrix‐associated growth factor Cyr61 of the CCN family of secreted proteins in osteosarcoma and metastasis. The prognostic value of Cyr61 was assessed with Kaplan‐Meier analyses based on Cyr61 immunostaining of a tissue microarray of osteosarcoma biopsies collected from 60 patients with local or metastatic disease. Effects of Cyr61 overexpression on intratibial tumor growth and lung metastasis of the low metastatic human SaOS‐2 osteosarcoma cell line were examined in severe combined immunodeficiency (SCID) mice. Cyr61‐provoked signaling was studied in vitro in nonmanipulated SaOS‐2 cells. Cyr61 immunostaining of osteosarcoma tissue cores correlated significantly (p = 0.02) with poor patient survival. Mice intratibially injected with Cyr61‐overexpressing SaOS‐2 cells showed faster tumor growth and an increase in number and outgrowth of lung metastases and consequently significantly (p = 0.0018) shorter survival than mice injected with control SaOS‐2 cells. Cyr61‐evoked PI‐3K/Akt/GSK3β signaling in SaOS‐2 cells resulted in a subcellular redistribution of the cell cycle inhibitor p21Cip1/WAF1. Cyr61 has considerable potential as a novel marker for poor prognosis in osteosarcoma and is an attractive target for primary tumor‐ and metastases‐suppressing drugs.

Solitary fibrous tumor of the orbit—two cases and a review of the literature

Solitary fibrous tumors of the orbit (SFT) are mesenchymal lesions that can develop either as malignant or benign neoplasias. We describe the histological features leading to the diagnosis in two females and review the current literature. Diagnosis of SFT only can be performed by histological examination, since clinical signs and radiological features are not specific enough. Even a malignant or benign course cannot be predicted, since clinical and radiological features do not correlate with histological signs of malignancy and vice versa. Radical resection is the treatment of choice, since no other treatment option has been proven to be efficient.

Fibrosis and Adventitious Bursae in Plantar Fat Pad of Forefoot: MR Imaging Findings in Asymptomatic Volunteers and MR Imaging–Histologic Comparison

PURPOSE To retrospectively evaluate plantar fat pad (PFP) signal intensity alterations in magnetic resonance (MR) imaging studies of asymptomatic volunteers and to compare PFP alterations with histopathologic findings in cadavers and patients. MATERIALS AND METHODS After appropriate institutional review board approval and any required informed consent were obtained, MR imaging studies of 70 asymptomatic volunteers (35 women, 35 men; mean age, 45 years; range, 21-69 years) obtained for another investigation were retrospectively analyzed by two musculoskeletal radiologists in consensus. The location, signal intensity, margin, extent, and size of PFP alterations were determined. MR imaging-histopathologic comparison was performed in six cadaveric feet and six feet of symptomatic patients (one woman, five men; mean age, 43 years; range, 31-60 years). For volunteers, the relationship between PFP alterations and Morton neuroma, age, and sex was analyzed by using the Fisher exact test, Spearman rank correlation, and the Wilcoxon rank sum test, respectively. Bonferroni correction was applied, and P < .01 was considered to indicate a significant difference. RESULTS Fifty-nine (84%) volunteers had PFP signal intensity alterations. Forty-nine (70%), six (9%), one (1%), four (6%), and 43 (61%) volunteers had alterations beneath the first, second, third, fourth, and fifth metatarsal heads, respectively. Ninety-four (91%) of 103 signal intensity alterations were in the form of hypointensity on T2-weighted images. Blurred margins were present in 90 (87%) alterations. Ninety percent of all PFP alterations in asymptomatic volunteers were 14 mm or smaller. The relationship between PFP alterations and Morton neuroma, age, and sex was not statistically significant. In cadaveric forefeet, PFP alterations corresponded histopathologically to a variable amount of fibrosis. In nine PFP alterations, development of fluid-containing spaces resembling bursae was present. Among the six patients with PFP alterations, histopathologic examination revealed fibrosis and adventitious bursae in two, fibrosis with inflammation in three, and a soft-tissue chondroma in one. CONCLUSION PFP signal intensity alterations are commonly seen in asymptomatic volunteers under the first and fifth metatarsal heads. At histologic examination, PFP signal intensity alterations correspond most commonly to fibrosis and adventitious bursae.

Mutations in the tyrosine kinase domain of the EGFR gene are rare in synovial sarcoma.

The prognosis of patients with synovial sarcomas is poor. New therapeutic strategies, such as target inhibition of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR) with erlotinib and gefinitib, could be effective, because most synovial sarcomas overexpress this protein. In lung cancer, the responsiveness to gefinitib is strongly related to the presence of mutations in the tyrosine kinase domain of the EGFR gene, while erlotinib sensitivity seems to be partly linked to chromosome 7 polysomy or gene amplification. To clarify the role of EGFR in synovial sarcoma and to explore the potential for a targeted therapy approach, we have examined 13 of these soft tissue tumors. We have analyzed the EGFR expression by immunohistochemistry, searched for polysomy and gene amplification with fluorescence in situ hybridization (FISH) and screened for EGFR mutations in exons 18–21 using PCR and direct sequencing. All 13 tumors showed strong diffuse or focal EGFR expression. No amplifications of the EGFR gene were found. In contrast, several point mutations were identified in exons 18–21 of two synovial sarcomas. Whereas one of these tumors carried only a synonymous mutation, two missense mutations in exons 19 and 21 of the EGFR gene (P733S and A840 T, respectively) could be demonstrated in the second sample. In conclusion, strong EGFR expression in synovial sarcomas is not related to gene amplification. The existence of mutations in the tyrosine kinase domain of the EGFR gene in a small subset of synovial sarcomas suggests that only few patients may profit from the tyrosine kinase inhibitor therapy.

Magic angle effect in MR imaging of ankle tendons: influence of foot positioning on prevalence and site in asymptomatic subjects and cadaveric tendons

The influence of foot positioning on prevalence of the magic angle effect (MAE) in ankle tendons was investigated. In 30 asymptomatic volunteers and five cadaveric feet, MR imaging of the ankle was performed in the supine (neutral position of the foot) and prone (plantar-flexed foot) position. MAE was considered if increased T1-weighted signal at a certain site was seen in one position only. Histological correlation was obtained at 25 sites of the cadaveric posterior tibialis tendons (PTT). MAE occurred in 6/30 vs 1/30 (supine vs prone) anterior tibialis tendons (ATT), 30/30 vs 0/30 extensor hallucis longus and 27/30 vs 0/30 extensor digitorum longus tendons, 29/30 vs 0/30 PTTs, 30/30 vs 0/30 flexor digitorum and flexor hallucis longus tendons, 30/30 vs 1/30 peroneus brevis and 23/30 vs 1/30 peroneus longus tendons. At 12/25 cadaveric PTT sites where MAE was exclusively responsible for the increased signal, histology revealed normal tissue (11/12) or minimal degeneration (1/12). In conclusion, the supine body position with neutral position of the foot, a high prevalence (77–100%) of MAE in ankle tendons except for the ATT (20%) is seen. MAE is almost absent in the prone body position with plantar flexion of the foot.

Is Dynamic Gadolinium Enhancement Needed in MR Imaging for the Preoperative Assessment of Scaphoidal Viability in Patients with Scaphoid Nonunion

PURPOSE To compare the accuracy of dynamic gadolinium-enhanced magnetic resonance (MR) imaging with that of standard MR imaging for assessing the viability of the proximal pole of the scaphoid in patients with nonunion. MATERIALS AND METHODS The study protocol was submitted to the institutional review board, and the need to obtain additional approval was waived for this retrospective study. Twenty-eight patients (mean age ± standard deviation, 24.3 years ± 6.4) with nonunion of a scaphoid fracture underwent dynamic gadolinium-enhanced MR imaging of the wrist 28 days ± 19 before surgery. Dynamic gadolinium-enhanced MR imaging consisted of acquisition of 40 consecutive coronal T1-weighted images over 1 minute. Two readers retrospectively evaluated MR images obtained with a standard protocol and rated the viability of the proximal scaphoid pole. The steepest upslope of gadolinium uptake was calculated in a region of interest placed in the proximal scaphoid pole by a third reader. Receiver operating characteristic curves were calculated, and the areas under the receiver operating characteristic curve (A(z) values) were compared. Diagnostic performance in determining scaphoid viability was calculated for readers 1 and 2. Histologic findings in 11 patients and surgical findings in all patients served as the standard of reference. RESULTS The sensitivity, specificity, and accuracy of standard MR imaging in the detection of scaphoid necrosis were 54%, 93%, and 75%, respectively, for reader 1 and 62%, 93%, and 78% for reader 2. Interreader reliability was excellent (κ = 0.92). The A(z) was 0.82 for reader 1 and 0.87 for reader 2. The diagnostic performance of dynamic gadolinium-enhanced MR imaging, determined with the steepest upslope value, was inferior to that of standard MR imaging, with an A(z) of 0.57. Findings at histologic examination (viable bone, necrotic bone, callus formation) did not correlate with those at dynamic gadolinium-enhanced MR imaging. CONCLUSION Because the diagnostic performance of dynamic gadolinium-enhanced MR imaging in the evaluation of scaphoid viability was inferior to that of a standard MR imaging protocol, dynamic acquisition may not be needed in patients with nonunion of scaphoid fractures.