Michael A. den Bakker

SDHB immunohistochemistry: a useful tool in the diagnosis of Carney–Stratakis and Carney triad gastrointestinal stromal tumors

Mutations in the tumor suppressor genes SDHB, SDHC, and SDHD (or collectively SDHx) cause the inherited paraganglioma syndromes, characterized by pheochromocytomas and paragangliomas. However, other tumors have been associated with SDHx mutations, such as gastrointestinal stromal tumors (GISTs) specifically in the context of Carney–Stratakis syndrome. Previously, we have shown that SDHB immunohistochemistry is a reliable technique for the identification of pheochromocytomas and paragangliomas caused by SDHx mutations. We hypothesized that GISTs in patients with SDHx mutations would be negative immunohistochemically for SDHB as well. Four GISTs from patients with Carney–Stratakis syndrome and six from patients with Carney triad were investigated by SDHB immunohistochemistry. Five GISTs with KIT or PDGFRA gene mutations were used as controls. In addition, SDHB immunohistochemistry was performed on 42 apparently sporadic GISTs. In cases in which the SDHB immunohistochemistry was negative, mutational analysis of SDHB, SDHC, and SDHD was performed. All GISTs from patients with Carney–Stratakis syndrome and Carney triad were negative for SDHB immunohistochemically. In one patient with Carney–Stratakis syndrome, a germline SDHB mutation was found (p.Ser92Thr). The five GISTs with a KIT or PDGFRA gene mutation were all immunohistochemically positive for SDHB. Of the 42 sporadic tumors, one GIST was SDHB-negative. Mutational analysis of this tumor did not reveal an SDHx mutation. All SDHB-negative GISTs were located in the stomach, had an epithelioid morphology, and had no KIT or PDGFRA mutations. We show that Carney–Stratakis syndrome- and Carney-triad-associated GISTs are negative by immunohistochemistry for SDHB in contrast to KIT- or PDGFRA-mutated GISTs and a majority of sporadic GISTs. We suggest that GISTs of epithelioid cell morphology are tested for SDHB immunohistochemically. In case of negative SDHB staining in GISTs, Carney–Stratakis syndrome or Carney triad should be considered and appropriate clinical surveillance should be instituted.

Brachyury expression in extra-axial skeletal and soft tissue chordomas: A marker that distinguishes chordoma from mixed tumor/myoepithelioma/parachordoma in soft tissue

Axial chordoma represents approximately 1% of malignant bone tumors. This tumor expresses cytokeratins, specifically cytokeratin 19, and commonly S100. More recently brachyury, a transcription factor important in mesodermal differentiation, including notochord development, has been detected by immunohistochemistry in axial chordomas and hemangioblastomas but not chondrosarcomas or other neoplasms. In this report, we describe 10 cases (6 men, 4 women: age 18 to 68 y; mean 44.6) of extra-axial tumors, 8 in bone and 2 in soft tissue, with morphologic and immunohistochemical features identical to those of axial chordoma. Imaging excluded metastases from axial chordoma. Three tumors occurred in the tibia, the others in the rib, metatarsal, ulna, femur, pubis: 2 intracortical, 6 intramedullary. Both soft tissue brachyury-positive tumors, one involving the thumb the other the wrist, were sited in the juxta-articular region. Seven of the tumors were widely excised and these patients are disease-free but of the 3 tumors that recurred, 1 was curetted, 1 was marginally excised, and 1 had a pathologic fracture on presentation. Metastases have not occurred in any of the patients. We also confirm the expression of brachyury in hemangioblastomas, and for the first time demonstrates its expression in spermatogonia and testicular germ cell tumors by immunohistochemistry. Brachyury was not detected in a wide range of tumors including carcinomas, lymphomas, and sarcomas. In conclusion, we describe the first series of extra-axial skeletal chordomas bringing the total number of such cases reported in the literature to 11, and present the first report of 2 soft tissue chordomas as defined by brachyury expression.

Seasonal and pandemic human influenza viruses attach better to human upper respiratory tract epithelium than avian influenza viruses.

Influenza viruses vary markedly in their efficiency of human-to-human transmission. This variation has been speculated to be determined in part by the tropism of influenza virus for the human upper respiratory tract. To study this tropism, we determined the pattern of virus attachment by virus histochemistry of three human and three avian influenza viruses in human nasal septum, conchae, nasopharynx, paranasal sinuses, and larynx. We found that the human influenza viruses-two seasonal influenza viruses and pandemic H1N1 virus-attached abundantly to ciliated epithelial cells and goblet cells throughout the upper respiratory tract. In contrast, the avian influenza viruses, including the highly pathogenic H5N1 virus, attached only rarely to epithelial cells or goblet cells. Both human and avian viruses attached occasionally to cells of the submucosal glands. The pattern of virus attachment was similar among the different sites of the human upper respiratory tract for each virus tested. We conclude that influenza viruses that are transmitted efficiently among humans attach abundantly to human upper respiratory tract, whereas inefficiently transmitted influenza viruses attach rarely. These results suggest that the ability of an influenza virus to attach to human upper respiratory tract is a critical factor for efficient transmission in the human population.

Identification of a putative protein-profile associating with tamoxifen therapy-resistance in breast cancer

Tamoxifen resistance is a major cause of death in patients with recurrent breast cancer. Current clinical factors can correctly predict therapy response in only half of the treated patients. Identification of proteins that are associated with tamoxifen resistance is a first step toward better response prediction and tailored treatment of patients. In the present study we intended to identify putative protein biomarkers indicative of tamoxifen therapy resistance in breast cancer using nano-LC coupled with FTICR MS. Comparative proteome analysis was performed on ∼5,500 pooled tumor cells (corresponding to ∼550 ng of protein lysate/analysis) obtained through laser capture microdissection (LCM) from two independently processed data sets (n = 24 and n = 27) containing both tamoxifen therapy-sensitive and therapy-resistant tumors. Peptides and proteins were identified by matching mass and elution time of newly acquired LC-MS features to information in previously generated accurate mass and time tag reference databases. A total of 17,263 unique peptides were identified that corresponded to 2,556 non-redundant proteins identified with ≥2 peptides. 1,713 overlapping proteins between the two data sets were used for further analysis. Comparative proteome analysis revealed 100 putatively differentially abundant proteins between tamoxifen-sensitive and tamoxifen-resistant tumors. The presence and relative abundance for 47 differentially abundant proteins were verified by targeted nano-LC-MS/MS in a selection of unpooled, non-microdissected discovery set tumor tissue extracts. ENPP1, EIF3E, and GNB4 were significantly associated with progression-free survival upon tamoxifen treatment for recurrent disease. Differential abundance of our top discriminating protein, extracellular matrix metalloproteinase inducer, was validated by tissue microarray in an independent patient cohort (n = 156). Extracellular matrix metalloproteinase inducer levels were higher in therapy-resistant tumors and significantly associated with an earlier tumor progression following first line tamoxifen treatment (hazard ratio, 1.87; 95% confidence interval, 1.25–2.80; p = 0.002). In summary, comparative proteomics performed on laser capture microdissection-derived breast tumor cells using nano-LC-FTICR MS technology revealed a set of putative biomarkers associated with tamoxifen therapy resistance in recurrent breast cancer.

Long-Term Results of Tumor Necrosis Factor α– and Melphalan-Based Isolated Limb Perfusion in Locally Advanced Extremity Soft Tissue Sarcomas

PURPOSE Because there is no survival benefit of amputation for extremity soft tissue sarcomas (STSs), limb-sparing surgery has become the gold standard. Tumor size reduction by induction therapy to render nonresectable tumors resectable or facilitate function-preserving surgery can be achieved by tumor necrosis factor α (TNF) -based and melphalan-based isolated limb perfusion (TM-ILP). This study reports the long-term results of 231 TM-ILPs for locally advanced extremity STS. PATIENTS AND METHODS We analyzed 231 TM-ILPs in 208 consecutive patients (1991 to 2005), who were all candidates for functional or anatomic amputation for locally advanced extremity STS. All patients had a potential follow-up of up to 5 years. TM-ILP was performed under mild hyperthermic conditions with 1 to 4 mg of TNF and 10 to 13 mg/L of limb-volume melphalan. Almost all patients (85%) had intermediate- or high-grade tumors. RESULTS The overall response rate (ORR) was 71% (complete response, 18%; partial response, 53%). Multifocal sarcomas had a significantly better ORR of 83% (P = .008). The local recurrence rate was 30% (n = 70); local recurrence rates were highest for multifocal tumors (54%; P = .001) and after previous radiotherapy (54%; P < .001). Five-year overall survival rate was 42%. Survival was poorest in patients with large tumors (P = .01) and with leiomyosarcomas (P < .001). Limb salvage rate was 81%. CONCLUSION We demonstrated that TM-ILP results in a limb salvage rate of 81% in patients with locally advanced extremity STS who would otherwise have undergone amputation. Whenever an amputation is deemed necessary to obtain local control of an extremity STS, TM-ILP should be considered.

SDHA mutations in adult and pediatric wild-type gastrointestinal stromal tumors

Most gastrointestinal stromal tumors (GISTs) harbor oncogenic mutations in KIT or platelet-derived growth factor receptor-α. However, a small subset of GISTs lacks such mutations and is termed ‘wild-type GISTs’. Germline mutation in any of the subunits of succinate dehydrogenase (SDH) predisposes individuals to hereditary paragangliomas and pheochromocytomas. However, germline mutations of the genes encoding SDH subunits A, B, C or D (SDHA, SDHB, SDHC or SDHD; collectively SDHx) are also identified in GISTs. SDHA and SDHB immunohistochemistry are reliable techniques to identify pheochromocytomas and paragangliomas with mutations in SDHA, SDHB, SDHC and SDHD. In this study, we investigated if SDHA immunohistochemistry could also identify SDHA-mutated GISTs. Twenty-four adult wild-type GISTs and nine pediatric/adolescent wild-type GISTs were analyzed with SDHB, and where this was negative, then with SDHA immunohistochemistry. If SDHA immunohistochemistry was negative, sequencing analysis of the entire SDHA coding sequence was performed. All nine pediatric/adolescent GISTs and seven adult wild-type GISTs were negative for SDHB immunohistochemistry. One pediatric GIST and three SDHB-immunonegative adult wild-type GISTs were negative for SDHA immunohistochemistry. In all four SDHA-negative GISTs, a germline SDHA c.91C>T transition was found leading to a nonsense p.Arg31X mutation. Our results demonstrate that SDHA immunohistochemistry on GISTs can identify the presence of an SDHA germline mutation. Identifying GISTs with deficient SDH activity warrants additional genetic testing, evaluation and follow-up for inherited disorders and paragangliomas.

Cells meeting our immunophenotypic criteria of endothelial cells are large platelets

Circulating endothelial cells (CEC) are shed from damaged vasculature, making them a rational choice to serve as surrogate marker for vascular damage. Currently, various techniques and CEC definitions are in use, and their standardization and validation is needed. A flow cytometric single platform assay defining CEC as forward light scatter (FSC)low‐to‐intermedate, sideward light scatter (SSC)low, CD45−, CD31++ and CD146+ is a promising approach to enumerate CEC because of its simplicity (Mancuso et al., Blood 2001;97:3658–3661). Here, we set out to confirm the endothelial nature of these cells.

Activating mutations in c-KIT and PDGFRα are exclusively found in gastrointestinal stromal tumors and not in other tumors overexpressing these imatinib mesylate target genes

Previous studies have shown that Imatinib mesylate (Gleevec), a selective tyrosine kinase inhibitor of c-KIT and platelet-derived growth factor receptors (PDGFR), is highly effective in c-KIT/CD117-positive gastrointestinal stromal tumors (GIST), especially in those having activating mutations in c-kit exon 11. In addition, gain-of-function mutations in the juxtamembrane domain (exon 12) and the kinase activation loop (exon 18) of PDGFRα were found in GISTs. Importantly, the presence and type of these mutually exclusive c-KIT or PDGFRα mutations were found to be associated with the response to imatinib. Here, we examined the prevalence of c-kit exon 11 and PDGFRα exons 12 and 18 mutations in other tumor types known to express these tyrosine kinase receptors in order to explore which other cancer types may potentially benefit from imatinib treatment. We determined the mutational status of these commonly mutated exons by direct sequencing in 11 different tumor types (in total: 215 unrelated cases), including GIST, chordoma, and various distinct tumors of lung, brain and its coverings, and skin cancer. Of the 579 exons examined (211 c-kit exon 11, 192 PDGFRα exon 12, 142 PDGFRα exon18, 17 PDGFRα exon 12 and 17 PDGFRβ exon 18), only 12 (all GIST) harbored mutations (10 c-kit exon 11 and 2 PDGFRα exon18). From these data we conclude that activating c-KIT and PDGFR mutations are sporadic in human cancers known to overexpress these tyrosine kinase receptor genes and suggest that, except in GIST, this overexpression is not correlated with activating mutations. The latter may imply that these wild-type c-KIT and PDGFR tumor types will probably not benefit from imatinib treatment.

NUT Midline Carcinoma of the Parotid Gland With Mesenchymal Differentiation

Nuclear protein in testis midline carcinomas (NMC) are highly aggressive carcinomas typically arising in midline structures in young individuals. These carcinomas are characterized by the presence of a chromosomal rearrangement of nuclear protein in testis the (NUT) gene on chromosome 15 (15q14), resulting from a chromosomal translocation most commonly involving the BRD4 gene on chromosome 19p13. Rarely, in about 1/3 of cases, other translocation partners are involved (termed NUT-variants). Most cases have involved midline structures and with few exceptions were located in the upper aerodigestive tract and the mediastinum. Except for a single case, all reported NMC have been fatal, proving resistant to multimodality treatment. We report an exceptional case of a NMC presenting outside of midline structures in the parotid gland and showing mesenchymal chondroid differentiation in a 15-year-old male. The presence of the t(15;19) chromosomal translocation in the chondroid component was confirmed by fluorescence in situ hybridization analysis and immunohistochemical staining, indicating mesenchymal transdifferentation of the tumor. The findings demonstrate the first case of NMC arising within salivary gland, and the first example of mesenchymal differentiation in this group of tumors.

Protease-Activated Receptor-2 Induces Myofibroblast Differentiation and Tissue Factor Up-Regulation during Bleomycin-Induced Lung Injury Potential Role in Pulmonary Fibrosis

Idiopathic pulmonary fibrosis constitutes the most devastating form of fibrotic lung disorders and remains refractory to current therapies. The coagulation cascade is frequently activated during pulmonary fibrosis, but this observation has so far resisted a mechanistic explanation. Recent data suggest that protease-activated receptor (PAR)-2, a receptor activated by (among others) coagulation factor (F)Xa, plays a key role in fibrotic disease; consequently, we assessed the role of PAR-2 in the development of pulmonary fibrosis in this study. We show that PAR-2 is up-regulated in the lungs of patients with idiopathic pulmonary fibrosis and that bronchoalveolar lavage fluid from these patients displays increased procoagulant activity that triggers fibroblast survival. Using a bleomycin model of pulmonary fibrosis, we show that bleomycin induces PAR-2 expression, as well as both myofibroblast differentiation and collagen synthesis. In PAR-2-/- mice, both the extent and severity of fibrotic lesions are reduced, whereas myofibroblast differentiation is diminished and collagen expression is decreased. Moreover, fibrin deposition in the lungs of fibrotic PAR-2-/- mice is reduced compared with wild-type mice due to differential tissue factor expression in response to bleomycin. Taken together, these results suggest an important role for PAR-2 in the development of pulmonary fibrosis, and the inhibition of the PAR-2-coagulation axis may provide a novel therapeutic approach to treat this devastating disease.