Pathologic Characteristics of Spitz Melanoma With MAP3K8 Fusion or Truncation in a Pediatric Cohort.

Spitz melanoma is a rare variant of melanoma defined by distinct clinical, histologic, and genetic features and affecting patients of all ages. Half of these tumors are driven by fusion of kinase genes including ALK, NTRK1/3, ROS1, RET, MET, or BRAF. We recently reported recurrent fusion or truncation of the potentially targetable serine-threonine kinase gene MAP3K8 in 33% of Spitz melanomas. Here we describe the histologic features of these MAP3K8-rearranged tumors (16 pediatric Spitz melanomas; 1 atypical Spitz tumor), using hematoxylin-eosin slides, p16 immunohistochemistry, and CDKN2A fluorescence in situ hybridization. The lesions consisted of a compound melanocytic proliferation, ranging in thickness from 1.5 to 13.4 mm (median, 3.1 mm), with 8 having a predominant dermal and 3 having a predominant junctional component. The predominant cell type was epithelioid (94%). The epithelioid melanocytes were generally monomorphic and amelanotic, arranged in expansile epithelial aggregates, confluent hypercellular nests, or enlarged syncytial nodules in the dermis. Ulceration was present in 9 of 17 tumors (53%) and deep mitotic figures were seen in 15 of 17 tumors (88%). Complete loss of p16 expression and homozygous CDKN2A deletion were observed in 82% and 70% of tumors, respectively. Recognition of MAP3K8-altered Spitz melanoma may thus be facilitated by these morphologic features, most notably presence of cohesive cellular nodules in the dermis and an epithelioid-cell phenotype.

BRAF VE1 immunoreactivity patterns in epithelioid glioblastomas positive for BRAF V600E mutation.

Epithelioid glioblastomas (E-GBMs) manifest BRAF V600E mutation in up to 50% of cases, compared with a small percentage of ordinary GBMs, suggesting that they are best considered variants rather than a different pattern of GBM. Availability of a targeted therapy, vemurafenib, may make testing BRAF status important for treatment. It is unclear whether BRAF VE1 immunohistochemistry (IHC) can substitute for Sanger sequencing in these tumors. BRAF VE1 IHC was correlated with Sanger sequencing results on our original cohort of E-GBMs, and then new E-GBM cases were tested with both techniques (n=20). Results were compared with those in similarly assessed giant cell GBMs, anaplastic pleomorphic xanthoastrocytomas. All tumors tested showed 1:1 correlation between BRAF V600E mutational results and IHC. However, heavy background immunostaining in some negatively mutated cases resulted in equivocal results that required repeat IHC testing and additional mutation testing using a different methodology to confirm lack of detectable BRAF mutation. Mutated/BRAF VE1 IHC E-GBMs and anaplastic pleomorphic xanthoastrocytomas tended to manifest strong, diffuse cytoplasmic immunoreactivity, compared with previously studied gangliogliomas, which demonstrate more intense immunoreactivity in the ganglion than in the glial tumor component. One of our E-GBM patients with initial gross total resection quickly recurred within 4 months, required a second resection, and then was placed on vemurafenib; she remains tumor free 21 months after second resection without neuroimaging evidence of residual disease, adding to the growing number of reports of successful treatment of BRAF-mutated glial tumors with drug. E-GBMs show good correlation between mutational status and IHC, albeit with limitations to IHC. E-GBMs can respond to targeted therapy.

Epithelioid inflammatory myofibroblastic sarcoma treated with ALK inhibitor: a case report and review of literature.

Epithelioid inflammatory myofibroblastic sarcoma is extremely rare and belongs to a variant of inflammatory myofibrobalstic tumor with aggressive clinical course. We describe a case of a 22 years old man presented with an abdominal huge tumor. Microscopically, the neoplasm cells were rounded and epithelioid in shape. Abundant interstitial edema and less myxoid stroma were also present together with an inflammatory infiltrate. Fluorescence in situ hybridization revealed that ALK gene presented mutation. After surgery the patient received chemotherapy with an anaplastic lymphoma kinase (ALK) inhibitor, crizotinib. The patient continues to be alive with disease for 16 months and has no recurrence. Although EIMS has a poor prognosis, this is the few successful case with sustained response of targeted therapy.

Carboxypeptidase-M is regulated by lipids and CSFs in macrophages and dendritic cells and expressed selectively in tissue granulomas and foam cells.

Granulomatous inflammations, characterized by the presence of activated macrophages (MAs) forming epithelioid cell (EPC) clusters, are usually easy to recognize. However, in ambiguous cases the use of a MA marker that expresses selectively in EPCs may be needed. Here, we report that carboxypeptidase-M (CPM), a MA-differentiation marker, is preferentially induced in EPCs of all granuloma types studied, but not in resting MAs. As CPM is not expressed constitutively in MAs, this allows utilization of CPM-immunohistochemistry in diagnostics of minute granuloma detection when dense non-granulomatous MAs are also present. Despite this rule, hardly any detectable CPM was found in advanced/active tubercle caseous disease, albeit in early tuberculosis granuloma, MAs still expressed CPM. Indeed, in vitro both the CPM-protein and -mRNA became downregulated when MAs were infected with live mycobacteria. In vitro, MA-CPM transcript is neither induced remarkably by interferon-γ, known to cause classical MA activation, nor by IL-4, an alternative MA activator. Instead, CPM is selectively expressed in lipid-laden MAs, including the foam cells of atherosclerotic plaques, xanthomatous lesions and lipid pneumonias. By using serum, rich in lipids, and low-density lipoprotein (LDL) or VLDL, CPM upregulation could be reproduced in vitro in monocyte-derived MAs both at transcriptional and protein levels, and the increase is repressed under lipid-depleted conditions. The microarray analyses support the notion that CPM induction correlates with a robust progressive increase in CPM gene expression during monocyte to MA maturation and dendritic cell (DC) differentiation mediated by granulocyte-MA-colony-stimulating factor+IL-4. M-CSF alone also induced CPM. These results collectively indicate that CPM upregulation in MAs is preferentially associated with increased lipid uptake, and exposure to CSF, features of EPCs, also. Therefore, CPM-immunohistochemistry is useful for granuloma and foam MA detections in tissue sections. Furthermore, the present data offer CPM for the first time to be a novel marker and cellular player in lipid uptake and/or metabolism of MAs by promoting foam cell formation.

TGF-ß-mediated sustained ERK1/2 activity promotes the inhibition of intracellular growth of Mycobacterium avium in epithelioid cells surrogates.

Transforming growth factor beta (TGF-ß) has been implicated in the pathogenesis of several diseases including infection with intracellular pathogens such as the Mycobacterium avium complex. Infection of macrophages with M. avium induces TGF-ß production and neutralization of this cytokine has been associated with decreased intracellular bacterial growth. We have previously demonstrated that epithelioid cell surrogates (ECs) derived from primary murine peritoneal macrophages through a process of differentiation induced by IL-4 overlap several features of epithelioid cells found in granulomas. In contrast to undifferentiated macrophages, ECs produce larger amounts of TGF-ß and inhibit the intracellular growth of M. avium. Here we asked whether the levels of TGF-ß produced by ECs are sufficient to induce a self-sustaining autocrine TGF-ß signaling controlling mycobacterial replication in infected-cells. We showed that while exogenous addition of increased concentration of TGF-ß to infected-macrophages counteracted M. avium replication, pharmacological blockage of TGF-ß receptor kinase activity with SB-431542 augmented bacterial load in infected-ECs. Moreover, the levels of TGF-ß produced by ECs correlated with high and sustained levels of ERK1/2 activity. Inhibition of ERK1/2 activity with U0126 increased M. avium replication in infected-cells, suggesting that modulation of intracellular bacterial growth is dependent on the activation of ERK1/2. Interestingly, blockage of TGF-ß receptor kinase activity with SB-431542 in infected-ECs inhibited ERK1/2 activity, enhanced intracellular M. avium burden and these effects were followed by a severe decrease in TGF-ß production. In summary, our findings indicate that the amplitude of TGF-ß signaling coordinates the strength and duration of ERK1/2 activity that is determinant for the control of intracellular mycobacterial growth.

Epithelioid inflammatory myofibroblastic sarcoma: An aggressive intra-abdominal variant of inflammatory myofibroblastic tumor with nuclear membrane or perinuclear ALK.

Inflammatory myofibroblastic tumor (IMT) is a mesenchymal neoplasm of intermediate biological potential, which may recur and rarely metastasize. Pathologic features do not correlate well with behavior. Approximately 50% of conventional IMTs harbor ALK gene rearrangement and overexpress ALK, most showing diffuse cytoplasmic staining. Rare IMTs with a distinct nuclear membrane or perinuclear pattern of ALK staining and epithelioid or round cell morphology have been reported. These cases pursued an aggressive clinical course, suggesting that such patterns may predict malignant behavior. We describe 11 cases of IMT with epithelioid morphology and a nuclear membrane or perinuclear pattern of immunostaining for ALK. Ten patients were male and 1 was female, ranging from 7 months to 63 years in age (median, 39 y). All tumors were intra-abdominal; most arose in the mesentery or omentum, measuring 8 to 26 cm (median, 15 cm). Six tumors were multifocal at presentation. The tumors were composed predominantly of sheets of round-to-epithelioid cells with vesicular nuclei, large nucleoli, and amphophilic-to-eosinophilic cytoplasm. In all cases, a minor spindle cell component was present. Nine tumors had abundant myxoid stroma. In 7 cases neutrophils were prominent and in 3 cases lymphocytes were prominent. Plasma cells were often absent. Median mitotic rate was 4/10 HPF; 6 tumors had necrosis. By immunohistochemistry, all tumors were positive for ALK, 9 tumors showing a nuclear membrane staining pattern and 2 tumors showing a cytoplasmic pattern with perinuclear accentuation. Other positive markers were desmin (10 of 11), focal smooth muscle actin (4 of 8), and CD30 (8 of 8). All tumors were negative for MYF4, caldesmon, keratins, EMA, and S-100. Fluorescence in situ hybridization was positive for ALK gene rearrangement in 9 cases, and in 3 cases tested, a RANBP2-ALK fusion was detected by reverse transcription polymerase chain reaction. Ten patients underwent surgical resection; 1 patient was inoperable. Follow-up was available for 8 patients and ranged from 3 to 40 months (median, 13 mo). All patients experienced rapid local recurrences; 4 patients had multiple recurrences. Eight patients were treated with postoperative chemotherapy; 2 patients received additional radiotherapy. Two patients also developed metastases (both patients developed metastases to the liver; 1 patient developed metastases to the lung and lymph nodes as well). Thus far, 5 patients died of disease, 2 patients are alive with disease, and 1 patient, treated with an experimental ALK inhibitor, has no evidence of disease. In summary, the epithelioid variant of IMT with nuclear membrane or perinuclear ALK is a distinctive intra-abdominal sarcoma with a predilection for male patients. Unlike conventional IMT, abundant myxoid stroma and prominent neutrophils are common. These tumors pursue an aggressive course with rapid local recurrences and are frequently fatal. We propose the designation "epithelioid inflammatory myofibroblastic sarcoma" to convey both the malignant behavior of these tumors and their close relationship with IMT.

Inhibition of cell survival, invasion, tumor growth and histone deacetylase activity by the dietary flavonoid luteolin in human epithelioid cancer cells.

Phytochemical compounds and histone deacetylase (HDAC) inhibitors are emerging as a new generation of anticancer agents with limited toxicity in cancer patients. We investigated the impact of luteolin, a dietary flavonoid, on survival, migration, invasion of cancer cells in vitro, and tumor growth in vivo. Luteolin (25-200µM) decreased the viability of human cancer cell lines originating from the lung (LNM35), colon (HT29), liver (HepG2) and breast (MCF7/6 and MDA-MB231-1833). Luteolin effectively increased the sub-G1 (apoptotic) fraction of cells through caspase-3 and -7 dependent pathways. We provide evidence that luteolin at sub-lethal/non-toxic concentrations inhibited the invasive potential of LNM35, MCF-7/6 and MDA-MB231-1833 cancer cells using Matrigel as well as the chick heart and Oris invasion assays. Moreover, we demonstrate for the first time that luteolin is a potent HDAC inhibitor that potentiates the cytotoxicity of cisplatin in LNM35 cells and decreases the growth of LNM35 tumor xenografts in athymic mice after intraperitoneal injection (20mg/kg/day for 18days) Thus, luteolin, in combination with standard anticancer drugs such as cisplatin, may be a promising HDAC inhibitor for the treatment of lung cancer.

Quantification of alternative mRNA species and identification of thioredoxin reductase 1 isoforms in human tumor cells.

The human selenoenzyme thioredoxin reductase 1 (TrxR1) is a very important enzyme for cell growth, differentiation, and the defense against oxidative stress. Several studies have shown that TrxR1 is up-regulated in tumor cells. The regulation of TrxR1 is very complex and involves the expression of different transcript forms of mRNA. We have, by quantitative polymerase chain reaction, investigated the total expression of TrxR1 mRNA and quantified the expression of alternative mRNA forms (alpha1/2, alpha6, alpha7/8, alpha10/11, alpha13, gamma2-4, and beta1) in six different human malignant mesothelioma cell lines of epithelioid, sarcomatoid, or mixed phenotype. The most abundant alpha-form was surprisingly alpha1/2 and not the expected alpha7/8. Selenium treatment resulted in increased expression of all alpha-variants, except the alpha10/11, where the levels were unaffected. The expression of protein isoforms was studied and the less abundant forms TrxR1v.2, TrxR1v.3, and TrxR1v.5 were detected in cell lysates and in human tumor tissue, using specific peptide antibodies. Furthermore, TrxR1v.3 and TrxR1v.5, previously not identified in human cells, were detected by mass spectrometry. Our data show differential expression of TrxR1 mRNA forms in malignant mesothelioma of different phenotype, and investigation of alternative transcript variants of TrxR1 could be a valuable tool in the diagnostics and characterization of tumors.

Cathepsin K--a marker of macrophage differentiation?

Cathepsin K is a cysteine protease with high matrix-degrading activity. Initially, cathepsin K was described as being expressed exclusively by osteoclasts. It was suggested that cathepsin K expression is a specific feature of cells involved in bone remodelling. The aim of this study was to investigate the hypothesis that cathepsin K is expressed not only in bone-resorbing macrophages, but also more generally in specifically differentiated macrophages, such as epithelioid cells and multinucleated giant cells in soft tissues. Specimens obtained from different organs and anatomical locations of patients suffering from sarcoidosis, tuberculosis, granulomas caused by foreign materials, and sarcoid-like lesions were investigated for the expression of cathepsins B, K, and L. Immunohistochemistry and in situ hybridization showed cathepsin K in epithelioid cells and multinucleated giant cells irrespective of the pathological condition and anatomical location, but not in normal resident macrophages. By immunoelectron microscopy, cathepsin K was discovered in cytoplasmic granules of multinucleated giant cells. In contrast, cathepsin B and cathepsin L were expressed ubiquitously in CD68-positive tissue macrophages, epithelioid cells, and multinucleated giant cells. The results demonstrate that cathepsin K, but not cathepsin B or cathepsin L, differentiates specific phenotypes of macrophages independently of the anatomical site. Its enzymatic characteristics, particularly its high matrix-degrading activity, suggest that cathepsin K-positive epithelioid cells and multinucleated giant cells are characterized by an enhanced specific proteolytic capability.

Cavitary tuberculosis produced in rabbits by aerosolized virulent tubercle bacilli.

Liquefaction of solid caseous tuberculous lesions and the subsequent cavity formation are probably the most dangerous processes in the pathogenesis of human pulmonary tuberculosis. In liquefied caseum, the tubercle bacilli grow extracellularly for the first time since the onset of the disease and can reach such large numbers that mutants with antimicrobial resistance may develop. From a cavity, the bacilli enter the bronchial tree and spread to other parts of the lung and also to other people. Of the commonly used laboratory animals, the rabbit is the only one in which cavitary tuberculosis can be readily produced. This report is the first to describe and analyze the complete course of cavitary tuberculosis, produced by aerosolized virulent bovine-type tubercle bacilli in commercially available New Zealand white rabbits. After the inhalation of 220 to 880 bacillary units, all of the rabbits were overtly well until they were sacrificed at 33 weeks. After the inhalation of 3,900 to 5,800 bacillary units, half of the rabbits died of progressive tuberculosis between 5 and 9 weeks and the other half lived until they were sacrificed at 18 weeks. Pulmonary cavities developed in both low- and high-dose groups, some beginning as early as 6 weeks. Bacilli from primary cavities sometimes caused nearby secondary cavities, but more frequently, they ascended the bronchial escalator, were swallowed, and caused secondary tubercles in the lymphoid tissue of the appendix and ileocecal junction. Histologically, and by culture, the number of bacilli found in the liquefied caseum varied from many to comparatively few. Strong tuberculin reactions at 4 weeks after infection were associated with fewer primary lesions, while strong tuberculin reactions at 33 weeks were associated with more cavitary lesions. In the tuberculous granulation tissue surrounding caseous and liquefied pulmonary foci and cavities, we found many mature epithelioid macrophages that contained high levels of the proteinase cathepsin D. Therefore, cathepsin D probably plays a major role in the liquefaction of solid caseous material and in the subsequent cavity formation.

Epithelioid cell histiocytoma: a report of two cases.

Epithelioid cell histiocytoma is a rarely reported tumor derived from factor-XIIIa-positive dermal dendrocytes. Two additional cases are presented including their clinical, histologic and immunohistochemical features.

[Induction of atypical interferon after heat shock]. / Induction d'un interféron atypique après choc thermique.

Hyperthermia (45 degrees C) induced the release of heat-shock induced factor(s) (HSIF) in the culture medium of Madin-Darby bovine kidney cells (MDBK) during their recovery period at 37 degrees C. HSIF is capable of inducing an increase in the 2'5' oligoadenylate synthetase activity in fresh MDBK cells without any detectable antiviral activity (Chousterman et al. J. Biol. Chem. 1987, 262, 4806-4811). We demonstrated here that even though HSIF did not crossreact antigenically with alpha, beta or gamma bovine interferons, it was still also able to induce an antiviral activity which copurified with its capacity of inducing 2'5' oligoadenylate synthetase activity. Therefore we concluded that HSIF is an atypical bovine interferon induced in response to heat shock.