Diagnosis and management of tropomyosin receptor kinase (TRK) fusion sarcomas: expert recommendations from the World Sarcoma Network.

Sarcomas are a heterogeneous group of malignancies with mesenchymal lineage differentiation. The discovery of neurotrophic tyrosine receptor kinase (NTRK) gene fusions as tissue-agnostic oncogenic drivers has led to new personalized therapies for a subset of patients with sarcoma in the form of tropomyosin receptor kinase (TRK) inhibitors. NTRK gene rearrangements and fusion transcripts can be detected with different molecular pathology techniques, while TRK protein expression can be demonstrated with immunohistochemistry. The rarity and diagnostic complexity of NTRK gene fusions raise a number of questions and challenges for clinicians. To address these challenges, the World Sarcoma Network convened two meetings of expert adult oncologists and pathologists and subsequently developed this article to provide practical guidance on the management of patients with sarcoma harboring NTRK gene fusions. We propose a diagnostic strategy that considers disease stage and histologic and molecular subtypes to facilitate routine testing for TRK expression and subsequent testing for NTRK gene fusions.

Effect of process variables on the sulfate reduction process in bioreactors treating metal-containing wastewaters: factorial design and response surface analyses.

The individual and combined effect of the pH, chemical oxygen demand (COD) and SO4 (2-) concentration, metal to sulfide (M/S(2-)) ratio and hydraulic retention time (HRT) on the biological sulfate reduction (SR) process was evaluated in an inverse fluidized bed reactor by factorial design analysis (FDA) and response surface analysis (RSA). The regression-based model of the FDA described the experimental results well and revealed that the most significant variable affecting the process was the pH. The combined effect of the pH and HRT was barely observable, while the pH and COD concentration positive effect (up to 7 and 3 gCOD/L, respectively) enhanced the SR process. Contrary, the individual COD concentration effect only enhanced the COD removal efficiency, suggesting changes in the microbial pathway. The RSA showed that the M/S(2-) ratio determined whether the inhibition mechanism to the SR process was due to the presence of free metals or precipitated metal sulfides.

Morphology, mineralogy, and solid-liquid phase separation characteristics of Cu and Zn precipitates produced with biogenic sulfide.

The morphology, mineralogy, and solid-liquid phase separation of the Cu and Zn precipitates formed with sulfide produced in a sulfate-reducing bioreactor were studied at pH 3, 5, and 7. The precipitates formed at pH 7 display faster settling rates, better dewaterability, and higher concentrations of settleable solids as compared to the precipitates formed at pH 3 and 5. These differences were linked to the agglomeration of the sulfidic precipitates and coprecipitation of the phosphate added to the bioreactor influent. The Cu and Zn quenched the intensity of the dissolved organic matter peaks identified by fluorescence-excitation emission matrix spectroscopy, suggesting a binding mechanism that decreases supersaturation, especially at pH 5. X-ray absorption fine structure spectroscopy analyses confirmed the precipitation of Zn-S as sphalerite and Cu-S as covellite in all samples, but also revealed the presence of Zn sorbed on hydroxyapatite. These analyses further showed that CuS structures remained amorphous regardless of the pH, whereas the ZnS structure was more organized at pH 5 as compared to the ZnS formed at pH 3 and 7, in agreement with the cubic sphalerite-type structures observed through scanning electron microscopy at pH 5.

A proinflammatory cytokine inhibits p53 tumor suppressor activity.

p53 has a key role in the negative regulation of cell proliferation, in the maintenance of genomic stability, and in the suppression of transformation and tumorigenesis. To identify novel regulators of p53, we undertook two functional screens to isolate genes which bypassed either p53-mediated growth arrest or apoptosis. In both screens, we isolated cDNAs encoding macrophage migration inhibitory factor (MIF), a cytokine that was shown previously to exert both local and systemic proinflammatory activities. Treatment with MIF overcame p53 activity in three different biological assays, and suppressed its activity as a transcriptional activator. The observation that a proinflammatory cytokine, MIF, is capable of functionally inactivating a tumor suppressor, p53, may provide a link between inflammation and tumorigenesis.

Inhibition of medulloblastoma cell invasion by Slit.

Invasion of brain tumor cells has made primary malignant brain neoplasms among the most recalcitrant to therapeutic strategies. We tested whether the secreted protein Slit2, which guides the projection of axons and developing neurons, could modulate brain tumor cell invasion. Slit2 inhibited the invasion of medulloblastoma cells in a variety of in vitro models. The effect of Slit2 was inhibited by the Robo ectodomain. Time-lapse videomicroscopy indicated that Slit2 reduced medulloblastoma invasion rate without affecting cell direction or proliferation. Both medulloblastoma and glioma tumors express Robo1 and Slit2, but only medulloblastoma invasion is inhibited by recombinant Slit2 protein. Downregulation of activated Cdc42 may contribute to this differential response. Our findings reinforce the concept that neurodevelopmental cues such as Slit2 may provide insights into brain tumor invasion.

Twist is substrate for caspase cleavage and proteasome-mediated degradation.

Twist is a member of the basic helix-loop-helix family of transcription factors. An aberrant Twist expression has been found in diverse types of cancer, including sarcomas, carcinomas and lymphomas, supporting a role for Twist in tumor progression. Twist is known to be essential for mesodermal development. However, since a prolonged Twist expression results in a block of muscle, cartilage and bone differentiation, Twist has to be excluded from somites during late embryogenesis for terminal differentiation to occur. This implies that Twist expression must be target of a tight control. Here we provide evidence that Twist undergoes post-transcriptional regulation. Twist is substrate for cleavage by caspases during apoptosis and its cleavage results in ubiquitin-mediated proteasome degradation. Our findings suggest that Twist post-transcriptional regulation may play an important role in tissue determination and raise the possibility that alterations in the protein turnover may account for Twist overexpression observed in tumors.

Canadian recommendations for the treatment of glioblastoma multiforme.

RECOMMENDATION 1: Management of patients with glioblastoma multiforme (GBM) should be highly individualized and should take a multidisciplinary approach involving neuro-oncology, neurosurgery, radiation oncology, and pathology, to optimize treatment outcomes. Patients and caregivers should be kept informed of the progress of treatment at every stage. RECOMMENDATION 2: Sufficient tissue should be obtained during surgery for cytogenetic analysis and, whenever feasible, for tumour banking. RECOMMENDATION 3: Surgery is an integral part of the treatment plan, to establish a histopathologic diagnosis and to achieve safe, maximal, and feasible tumour resection, which may improve clinical signs and symptoms. RECOMMENDATION 4: The preoperative imaging modality of choice is magnetic resonance imaging (MRI) with gadolinium as the contrast agent. Other imaging modalities, such as positron emission tomography with [(18)F]-fluoro-deoxy-d-glucose, may also be considered in selected cases. Postoperative imaging (mri or computed tomography) is recommended within 72 hours of surgery to evaluate the extent of resection. RECOMMENDATION 5: Postoperative external-beam radiotherapy is recommended as standard therapy for patients with gbm. The recommended dose is 60 Gy in 2-Gy fractions. The recommended clinical target volume should be identified with gadolinium-enhanced T1-weighted mri, with a margin in the order of 2-3 cm. Target volumes should be determined based on a postsurgical planning MRI. A shorter course of radiation may be considered for older patients with poor performance status. RECOMMENDATION 6: During RT, temozolomide 75 mg/m(2) should be administered concurrently for the full duration of radio-therapy, typically 42 days. Temozolomide should be given approximately 1 hour before radiation therapy, and at the same time on the days that no radiotherapy is scheduled. RECOMMENDATION 7: Adjuvant temozolomide 150 mg/m(2), in a 5/28-day schedule, is recommended for cycle 1, followed by 5 cycles if well tolerated. Additional cycles may be considered in partial responders. The dose should be increased to 200 mg/m(2) at cycle 2 if well tolerated. Weekly monitoring of blood count is advised during chemoradiation therapy in patients with a low white blood cell count. Pneumocystis carinii pneumonia has been reported, and prophylaxis should be considered. RECOMMENDATION 8: For patients with stable clinical symptoms during combined radiotherapy and temozolomide, completion of 3 cycles of adjuvant therapy is generally advised before a decision is made about whether to continue treatment, because pseudo-progression is a common phenomenon during this time. The recommended duration of therapy is 6 months. A longer duration may be considered in patients who show continuous improvement on therapy. RECOMMENDATION 9: Selected patients with recurrent gbm may be candidates for repeat resection when the situation appears favourable based on an assessment of individual patient factors such as medical history, functional status, and location of the tumour. Entry into a clinical trial is recommended for patients with recurrent disease. RECOMMENDATION 10: The optimal chemotherapeutic strategy for patients who progress following concurrent chemoradiation has not been determined. Therapeutic and clinical-molecular studies with quality of life outcomes are needed.

Caspase activation and apoptosis in response to proteasome inhibitors.

Several studies have indicated that proteasome inhibitors (PIs) are promising anticancer agents. We have discovered that PIs have the unique ability to activate effector caspases through a mitochondrial Bcl-2 inhibitable but caspase-9 independent pathway. Stabilization of released Smac induced by blockade of the proteasome could explain the apoptosome-independent cell death induced by PIs. In fact, Smac/DIABLO critically supports this PIs-dependent caspase activation. By using a new assay, we confirm that at a single cell level both Smac and PIs can activate caspases in the absence of the apoptosome. Moreover, we have observed two PIs-induced kinetics of caspase activation, with caspase-9 being still required for the rapid caspase activation in response to mitochondrial depolarization, but dispensable for the slow DEVDase activation. In summary, our data indicate that PIs can activate downstream caspases at least in part through Smac/DIABLO stabilization.

Three discrete regions of deletion at 3p in head and neck cancers.

Alteration of the short arm of chromosome 3 is one of the most consistent cytogenetic abnormalities found in human head and neck cancers. These alterations, composed of translocations and deletions, have been associated with the presence of a tumor suppressor gene(s), but no clear evidence of the location of this presumptive gene(s) was available. We performed a molecular analysis of the 3p region using a polymerase chain reaction-based approach. Twenty-eight of the 38 cases analyzed (74%) showed the presence of single or multiple areas of allelic loss. Three commonly deleted regions, tentatively mapped to 3p24-ter, 3p21.3, and 3p14--cen, were identified. Our results suggest that at least three oncosuppressor genes mapping on 3p may be involved in head and neck cancer development and support a common oncogenic pathway with squamous cell lung cancer, for which a similar pattern of 3p deletion has been described recently.

In vivo growth of C6 glioma cells transfected with connexin43 cDNA.

In order to examine the possible role of intercellular communication via gap junctions in the control of tumor growth, we have transfected C6 glioma cells with connexin43 cDNA. We obtained several clones with variable expression of connexin43. The growth rate of these clones in culture was inversely related to the degree of expression of the transfected cDNA. To examine the growth of these transfected cells in vivo, cells were grown in spinner culture flasks to form spheroids 250-300 microns in diameter. Spheroids of nontransfected C6 cells produced large gliomas. Immunohistochemical and in situ hybridization analyses revealed relatively high levels of connexin43 protein and mRNA in the host tissue, while little of this protein was detected in the glioma. In contrast, spheroids of connexin43-transfected cells grew more slowly and exhibited elevated levels of connexin43 protein and mRNA. These findings suggest that the expression of connexin43 may be associated with the control of brain tumor growth in vivo.

Overexpression of CDC25A and CDC25B in head and neck cancers.

The deregulation of several cell cycle-related genes participates in neoplastic transformation. Cell cycle progression is driven by cyclin-dependent kinases, which are positively regulated by association with cyclins and negatively regulated by binding to inhibitory subunits. The activity of cyclin-dependent kinases is also regulated by the phosphorylation status, which is controlled by the antagonistic action of wee1 kinase and CDC25 phosphatases. Three CDC25 genes are present in human cells: CDC25A, CDC25B, and CDC25C. These three genes function at different phases of the cell cycle. Whereas CDC25A and CDC25B are expressed throughout the cell cycle, with peak expression in G1 for CDC25A and in both G1-S-phase and G2 for CDC25B, CDC25C is predominantly expressed in G2. Several lines of evidence suggest a role for CDC25s as oncogenes. CDC25A and CDC25B cooperate with Ha-ras or loss of Rb1 in the oncogenic transformation of rodent fibroblasts. Moreover, they are transcriptional targets of c-myc, and CDC25A in particular plays an important role as a mediator of myc functions. On the basis of the evidence that CDC25 phosphatases can act as oncogenes, we analyzed the expression of CDC25A, CDC25B, and CDC25C genes in 20 squamous cell carcinomas of the head and neck by quantitative reverse transcription-PCR. Our results show that whereas CDC25C is expressed at a low level with no relevant differences between neoplastic tissue and normal mucosa, CDC25A and CDC25B are overexpressed in a large fraction of tumors.

Chromosome 13q deletion mapping in head and neck squamous cell carcinomas: identification of two distinct regions of preferential loss.

Heal and neck squamous cell carcinomas show frequent cytogenetic alterations involving the long arm of chromosome 13. To define the extent of 13q deletions and to identify the minimal areas of chromosome loss, 48 primary squamous cell carcinomas of the head and neck were analyzed for loss of heterozygosity using 11 different polymorphic loci. About 67% of the tumors displayed loss of genetic material at 13q. Most of the cases showed loss of the entire long arm of the chromosome. However, the presence of partial deletions in 10 cases provided evidence of the existence of two preferential sites of chromosome loss at 13q32-ter and 13q14.2-q14.3. The colocalization of the 13q14 minimal region of deletion with the retinoblastoma (RB) gene, which has been proposed as an oncosuppressor in diverse tumor types, prompted us to verify the involvement of this gene in the development of head and neck cancer. No significant variation in RB protein or RB mRNA expression was detected, thus excluding a role for such a gene in the genesis of this type of tumor. Taken together, our data suggest the existence of two new tumor suppressor genes (one close to and one distal to RB), which play a role in the development and/or progression of head and neck squamous cell carcinomas.

N-myc activation by proviral insertion in MCF 247-induced murine T-cell lymphomas.

N-myc proto-oncogene rearrangement was found in three out of six AKR murine T-cell lymphomas induced by the highly oncogenic MCF 247 MuLV. Molecular analyses showed that structural modification of the proto-oncogene in all three lymphomas was in the consequence of MCF 247 proviral integration within the gene III exon. All integrated proviruses have the same transcriptional orientation as the N-myc gene. As a consequence of proviral insertion, the N-myc gene becomes transcriptionally active, producing an abnormal mRNA. These findings suggest a possible causative role of such an integrative event in murine T-cell lymphomagenesis.

High frequency of p53 gene alterations associated with protein overexpression in human squamous cell carcinoma of the larynx.

A series of 58 primary human squamous cell carcinomas of the larynx (LSCCs) was examined for the expression of the p53 tumor-suppressor gene by a combined immunohistochemical and molecular approach. About 60% of the cases displayed nuclear p53 overexpression as revealed by immunostaining with PAb1801, PAb122 and PAb240 monoclonal antibodies. This phenomenon was associated with the presence of structural and/or transcriptional alterations of the p53 gene. Our results provide evidence that p53 abnormalities constitute the most frequent genetic alteration identified so far in LSCC and indicate that the abnormal accumulation of the protein correlates with the presence of p53-mutated versions. These findings, taken together with the peculiar biochemical properties of p53, support the hypothesis of a possible pathogenetic relationship between smoke carcinogen exposure and p53 inactivation in the development of this tumor type.

Adhesion molecule expression does not influence the leukemic behavior of murine T-cell lymphomas.

Recirculation and homing properties of normal lymphocytes are controlled by interactions with high endothelial venules (HEVs), specialized vessels which mediate the extravasation into lymphoid tissues. The present study was aimed at elucidating whether lymphoma-derived leukemic cell spreading and peripheral lymph node invasion ability are mediated by the recognition mechanisms which physiologically regulate normal lymphocyte trafficking. For this purpose, we tested the HEV-binding ability and the expression of the lymphocyte homing receptor (LHR) for peripheral lymph nodes as well as Pgp-1/CD44, LFA-1 and ICAM-1 adhesion molecules by the highly leukemic cell line NQ22 in comparison with a series of non-leukemic murine T-lymphoma cell lines. Our results indicate that the hematogenous spreading as well as peripheral node invasion of lymphoma-derived leukemic cells may occur independently of LHR expression. In addition, our findings seem to rule out that gross quantitative modifications in LFA-1 or ICAM-1 antigen expression are associated with differential dissemination abilities of transformed lymphoid cells.

A quantitative assessment of microvessel ultrastructure in C6 astrocytoma spheroids transplanted to brain and to muscle.

Normal blood vessels invading a growing neoplasm undergo dramatic changes in morphology. Whether vessel characteristics are dictated entirely by the tumor, or from developmental restrictions in normal vessels from which tumor vessels originate is not known. To address this question we challenged two morphologically different types of capillaries (brain and muscle) with the same tumor environment (C6 astrocytoma), and quantified the invading vessel morphology. A vascular spheroids of C6 astrocytoma cells were implanted singly into rat cerebral cortex or iliacus muscle. Microvessels from the tumor, peritumoral tissue and control tissue were examined ultrastructurally and quantified. Tumor vessels differed significantly from host vessels but not from each other, regardless of implantation site. Neoplastic vessels were thick-walled relative to normal host vessels, had low densities of mitochondria and vesicular structures, and had both fenestrations and enlarged junctional clefts characteristic of highly permeable vessels. Control brain vessels were typically thin-walled, had a high density of mitochondria, a low density of endothelial vesicles and continuous tight junctions. Control muscle vessels were thin-walled with a low density of mitochondria, high density of vesicles and junctional zones with occasional enlarged clefts. Peritumoral vessel morphology was intermediate between that of tumor and the corresponding control tissue. We propose that C6 astrocytoma cells influence invading endothelial cells to develop a permeable phenotype radically different from host tissue endothelium, and host vessel phenotype does not influence tumor vessel morphology.

Expression of matrix metalloproteinases and their inhibitors in human brain tumors.

Sixty human brain tumors, classified according to the New World Health Organization (WHO) classification including, grade I schwannomas, meningiomas and pilocytic astrocytomas, grade II astrocytomas, grade III anaplastic astrocytomas, grade IV glioblastomas, grade III anaplastic oligodendrogliomas and grade IV glioblastomas and lung and melanoma metastases were analyzed for the expression of three matrix metalloproteinases (MMPs), two tissue inhibitors of MMPs (TIMPs) and for MMP activity. Some correlation was found between MMP expression and the degree of malignancy. Western blotting analysis revealed a more uniform pattern of distribution of MMP-2 (gelatinase A) than of MMP-9 (gelatinase B) and MMP-12 (metalloelastase) among tumors. MMP-9 levels were found to be significantly higher in grade III anaplastic astrocytomas and anaplastic oligodendrogliomas than those in grade I schwannomas and meningiomas. Anaplastic astrocytomas and Grade IV glioblastomas expressed significantly higher levels MMP-12 than grade I meningiomas. All sixty tumors showed a similar pattern of activity in zymography, proMMP-9 being the major species detected. Interestingly, TIMP-1 and TIMP-2 expression levels were especially low in tumors of grade II and grade III but significantly higher in tumors of grade I, particularly in schwannomas. Taken together, these data suggest that: 1) a balance between MMPs and TIMPs has an important role to play in human brain tumors; 2) TIMP expression may be valuable markers for tumor malignancy.

Subcellular localization of superoxide dismutases, glutathione peroxidase and catalase in developing rat cerebral cortex.

The levels of copper- and zinc-containing superoxide dismutase (CuZnSOD), manganese-containing superoxide dismutase (MnSOD), glutathione peroxidase (GSH-Px) and catalase (CAT) activity have been assessed in a nuclear fraction (NF), a mitochondrial fraction (MF) and a non-mitochondrial, non-nuclear fraction (NMNNF) isolated from developing rat cerebral cortex. The NF showed increasing CuZn and MnSOD activities and static, low activities of GSH-Px and CAT during development. The MF had increased levels of MnSOD and GSH-Px activities and a rapid decrease in CAT activity associated with development. Histochemical methods have localized greater CAT activity in mitochondria isolated from 2-day-old rat brain when compared to 77-day-old animals. Development was associated with increasing CuZnSOD activity, a decrease in CAT activity and, after an initial fall at 19 days, increasing GSH-Px activity in the NMNNF. Measurable activity of MnSOD were found in the NMNNF and appeared to be static during the time period assessed. A distinct ontogenetic pattern of oxidative enzyme activities and subcellular locations is associated with development in rat cerebral cortex.

Distribution of superoxide dismutase, glutathione peroxidase and catalase in developing rat brain.

This study was carried out to assess the developmental pattern of copper- and zinc-containing superoxide dismutase (CuZnSOD), manganese-containing superoxide dismutase (MnSOD), glutathione peroxidase (GSH-Px) and catalase (CAT) activity in rat brain. The enzymes studied were assayed in different brain regions (cerebral cortex, striatum, cerebellum and brainstem) and enzyme values were corrected for erythrocyte contamination. The cerebral ontogenetic pattern of these enzymes is characterized by increasing CuZnSOD activity, a progressive decrease in CAT activity and, after an initial 10-day fall, increasing GSH-Px activity. The activity of MnSOD appeared to be quite stable up to 40 weeks of age. Similar and comparable changes were seen in all brain regions studied.