AGK-BRAF is associated with distant metastasis and younger age in pediatric papillary thyroid carcinoma.

BACKGROUND: The incidence of thyroid carcinoma has increased in most populations, including pediatric patients. The increase is almost exclusively due to an increase in the incidence of papillary thyroid carcinoma (PTC). Genetic alterations leading to mitogen-activated protein kinase (MAPK) pathway activation are highly prevalent in PTC, with BRAF V600E mutation being the most common event in adult PTC. Although a lower prevalence of BRAF V600E had been reported among pediatric patients, a higher prevalence of BRAF fusion has been identified in both radiation-exposed and sporadic pediatric PTC. However, little is known about the prognostic implications of BRAF fusions in pediatric PTC. PROCEDURE: In this study, we investigated the prevalence of BRAF alterations (AGK-BRAF fusion and BRAF V600E mutation) in a large set of predominantly sporadic pediatric PTC cases and correlate with clinicopathological features. Somatic AGK-BRAF fusion was investigated by RT-PCR and confirmed by FISH break-apart. The BRAF V600E mutation was screened using Sanger sequencing. RESULTS: AGK-BRAF fusion, found in 19% of pediatric PTC patients, was associated with distant metastasis and younger age. Conversely, the BRAF V600E, found in 15% of pediatric PTC patients, was correlated with older age and larger tumor size. CONCLUSION: Collectively, our results advance knowledge concerning genetic bases of pediatric thyroid carcinoma, with potential implications for diagnosis, prognosis, and therapeutic approaches.

Targeting Histone Deacetylase Activity to Arrest Cell Growth and Promote Neural Differentiation in Ewing Sarcoma.

There is an urgent need for advances in the treatment of Ewing sarcoma (EWS), an aggressive childhood tumor with possible neuroectodermal origin. Inhibition of histone deacetylases (HDAC) can revert aberrant epigenetic states and reduce growth in different experimental cancer types. Here, we investigated whether the potent HDAC inhibitor, sodium butyrate (NaB), has the ability to reprogram EWS cells towards a more differentiated state and affect their growth and survival. Exposure of two EWS cell lines to NaB resulted in rapid and potent inhibition of HDAC activity (1 h, IC50 1.5 mM) and a significant arrest of cell cycle progression (72 h, IC50 0.68-0.76 mM), marked by G0/G1 accumulation. Delayed cell proliferation and reduced colony formation ability were observed in EWS cells after long-term culture. NaB-induced effects included suppression of cell proliferation accompanied by reduced transcriptional expression of the EWS-FLI1 fusion oncogene, decreased expression of key survival and pluripotency-associated genes, and re-expression of the differentiation neuronal marker ßIII-tubulin. Finally, NaB reduced c-MYC levels and impaired survival in putative EWS cancer stem cells. Our findings support the use of HDAC inhibition as a strategy to impair cell growth and survival and to reprogram EWS tumors towards differentiation. These results are consistent with our previous studies indicating that HDis can inhibit the growth and modulate differentiation of cells from other types of childhood pediatric tumors possibly originating from neural stem cells.

Molecular Characterization of Pediatric Acute Myeloid Leukemia: Results of a Multicentric Study in Brazil.

BACKGROUND AND AIMS: The biological characterization of childhood acute myeloid leukemia (c-AML) is an important outcome predictor. In Brazil, very little is known about the frequency of AML subgroups, although c-AML accounts for about 18% of leukemias. We carried out this study to investigate the contribution of type I and II gene mutations in the probability of overall survival (pOS) of c-AML in Brazil. METHODS: Seven hundred and three de novo pediatric AML cases (2000-2015) were assessed throughout a multicentric network study. Mutations in hotspot regions of FLT3, NRAS, KRAS, PTPN11, and c-KIT genes were analyzed as well as fusion genes (RUNX1-RUNX1T1, MLL/KMT2A-r, CBFß-MYH11, and PML-RARα) associated with AML. Patients were treated out of the clinical trial although following the BFM-AML2004 protocol. Acute promyelocytic leukemia (APL) was treated differently. AML with Down syndrome was excluded. RESULTS: There were significant differences in gene mutations among age ranges (≤2 years-old; >2-10 years old and ≥11 years old) and the nonrandom association between type I/II mutations. Lower white blood cell count (≤50 × 109/L) was associated with RUNX1-RUNX1T1, whereas higher WBC with CBFß-MYH11 (p <0.05). Cumulative pOS in 5 years was 37.7 ± 2.8% for total AMLs and 59.8 ± 6.2% for APL (p = 0.03). pOS differences were observed between Brazilian regions. The South-Southeast regions had a better 5-year pOS, whereas the Midwest region presented the poorest pOS (23.7 ± 4.9%). PTPN11 mutations conferred an adverse prognosis as an independent prognostic factor. CONCLUSIONS: Identification of genetic subgroups contributes to the molecular epidemiology and biology of AML worldwide, reflecting the profile of pediatric AML cases in Brazil.

A pediatric case of mammary analogue secretory carcinoma within the parotid.

Mammary analogue secretory carcinoma (MASC) is a recently described entity in the differential diagnosis of salivary gland tumors. It is notable for a characteristic t(12;15)(p13;q25) translocation that results in a unique fusion protein, ETV6-NTRK3. While several studies have retrospectively identified this translocation in cases previously diagnosed as a different salivary malignancy, there have been relatively few cases where this translocation was identified on initial pathology results, and fewer still in a pediatric population. We present a case of a 15 year old female with a slowly enlarging, painless, left facial mass. MRI demonstrated a cystic mass extending into the deep lobe of the parotid, and she underwent parotidectomy. The tumor cells stained positive for S100 and CK19. ETV6 translocation was present, confirming the diagnosis. Mammary analogue secretory carcinoma is a recently described tumor of the salivary glands, which often masquerades as more common primary salivary gland tumors and cysts. More research is needed to characterize the typical behavior of this neoplasm and the optimal treatment regimen. With identification of its characteristic translocation, mammary analogue secretory carcinoma can be easily differentiated from its more prevalent counterparts, and should therefore remain within the differential of the pathologist and head and neck surgeon.

Identification of ANLN as ETV6 partner gene in recurrent t(7;12)(p15;p13): a possible role of deregulated ANLN expression in leukemogenesis.

The ETV6 gene encodes an ETS family transcription factor that is involved in a myriad of chromosomal rearrangements found in hematological malignancies and other neoplasms. A recurrent ETV6 translocation, previously described in patients with acute myeloid leukemia (AML) (Genes Chromosomes Cancer 51:328-337,2012, Leuk Res 35:e212-214, 2011), whose partner has not been identified is t(7;12)(p15;p13). We herein report that the t(7;12)(p15;p13) fuses ETV6 to ANLN, a gene not previously implicated in the pathogenesis of hematological malignancies, and we demonstrate that this translocation leads to high expression of the fusion transcript in the myeloid and lymphoid lineages.

[Molecular biology and childhood leukemia: E2A-PBX1 and central nervous system relapse]. / Biología molecular y leucemia: E2A-PBX1 y recaída al sistema nervioso central.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children. The inclusion of molecular biology techniques in the diagnosis and prognostic stratification of these patients has allowed major treatment achievements in developed countries. One of the best studied gene rearrangements is E2A-PBX1, which predicts isolated central nervous system relapse in patients with ALL. However, further research on the search for new molecular markers related to prognosis of patients with childhood leukemia is required. Such studies need the integration of different disciplines, including epidemiology. Epidemiological studies are needed not only to accelerate the discovery of new molecular markers and new biological signals as to the etiology and pathophysiology of cancer, but also to evaluate the clinical impact of these findings in well-defined populations.

La leucemia linfoblástica aguda (LLA) es el cáncer más frecuente en niños. La inclusión de técnicas de biología molecular en el diagnóstico y la estratificación pronóstica de estos pacientes ha permitido que se logren avances importantes del tratamiento en países desarrollados. Uno de los rearreglos génicos más estudiados es el E2A-PBX1, el cual predice la recaída aislada al sistema nervioso central (SNC) en pacientes con LLA. Es necesaria una mayor investigación acerca de la búsqueda de nuevos marcadores moleculares relacionados con el pronóstico de los pacientes con leucemia infantil. Este tipo de estudios requieren de la integración de diferentes disciplinas del campo de la investigación, entre ellas la epidemiología. Los estudios epidemiológicos son necesarios no solo para acelerar los descubrimientos de nuevos marcadores moleculares y nuevas señales biológicas en cuanto a la etiología y la fisiopatología del cáncer, sino también para para evaluar el impacto clínico de esos descubrimientos en poblaciones bien definidas.

Expression differences in TEL-AML1 fusion gene in leukemia glucocorticoid-sensitive and -resistant cell lines.

We investigated the expression differences of the TEL-AML1 fusion gene in a leukemia glucocorticoid (GC)-sensitive cell line (CEM) and a GC-resistant cell line (Jurkat). Changes in TEL-AML1 expression before and after GC exposure were analyzed. Expression of GC-sensitive and GC-resistant leukemia cells following initial diagnosis and during treatment was simulated. Leukemia cells were divided into a GC-unexposed or a GC-exposed group. A methyl thiazolyl tetrazolium assay was used to detect cell proliferation inhibition, flow cytometry was used to observe cell apoptosis, reverse transcription-polymerase chain reaction was used to detect the mRNA expression of TEL-AML1 before and after exposure, and western blotting was used to analyze protein levels of TEL-AML1 before and after exposure. Inhibitory concentrations of 50% of cells in the Jurkat and CEM cells at 24 h were 382 and 9 mM, respectively, and at 48 h they were 216 and 2 mM. The proliferation inhibition effect of dexamethasone sodium phosphate on Jurkat cells was much lower than that on CEM cells. Jurkat cells showed obvious apoptosis after exposure to 100 mM dexamethasone sodium phosphate for 48 h. In the exposed group, Jurkat cells showed higher TEL-AML1 expression than did CEM cells (P < 0.05). In the unexposed group, TEL-AML1 gene expression in Jurkat cells was not affected by GC exposure (P > 0.05), while the CEM cells presented significant differences before and after exposure (P < 0.05). Sustained high expression of TEL-AML1 participated in and maintained the occurrence of GC resistance. Inhibition of TEL-AML1 may provide a new therapeutic approach to reverse GC resistance.

The CATS (FAM64A) protein is a substrate of the Kinase Interacting Stathmin (KIS).

The CATS protein (also known as FAM64A and RCS1) was first identified as a novel CALM (PICALM) interactor that influences the subcellular localization of the leukemogenic fusion protein CALM/AF10. CATS is highly expressed in cancer cell lines in a cell cycle dependent manner and is induced by mitogens. CATS is considered a marker for proliferation, known to control the metaphase-to-anaphase transition during the cell division. Using CATS as a bait in a yeast two-hybrid screen we identified the Kinase Interacting Stathmin (KIS or UHMK1) protein as a CATS interacting partner. The interaction between CATS and KIS was confirmed by GST pull-down, co-immunoprecipitation and co-localization experiments. Using kinase assay we showed that CATS is a substrate of KIS and mapped the phosphorylation site to CATS serine 131 (S131). Protein expression analysis revealed that KIS levels changed in a cell cycle-dependent manner and in the opposite direction to CATS levels. In a reporter gene assay KIS was able to enhance the transcriptional repressor activity of CATS, independent of CATS phophorylation at S131. Moreover, we showed that CATS and KIS antagonize the transactivation capacity of CALM/AF10.In summary, our results show that CATS interacts with and is a substrate for KIS, suggesting that KIS regulates CATS function.

Functional analysis of the two reciprocal fusion genes MLL-NEBL and NEBL-MLL reveal their oncogenic potential.

MLL gene aberrations are frequently diagnosed in infant acute myeloid leukemia (AML). We previously described the MLL-NEBL and NEBL-MLL genomic fusions in an infant AML patient with a chromosomal translocation t(10;11)(p12;q23). NEBL was the second Nebulin family member (LASP1, NEBL) which was found to be involved in MLL rearrangements. Here, we report on our attempts to unravel the oncogenic properties of both fusion genes. First, RT-PCR analyses revealed the presence of the MLL-NEBL and NEBL-MLL mRNAs in the diagnostic sample of the patient. Next, expression cassettes for MLL-NEBL and NEBL-MLL were cloned into a sleeping beauty vector backbone. After stable transfection, the biological effects of MLL-NEBL, NEBL-MLL or the combination of both fusion proteins were investigated in a conditional cell culture model. NEBL-MLL but also co-transfected cells displayed significantly higher growth rates according to the data obtained by cell proliferation assay. The focus formation experiments revealed differences in the shape and number of colonies when comparing MLL-NEBL, NEBL-MLL- and co-transfected cells. The results obtained in this study suggest that the reciprocal fusion genes of the Nebulin gene family might be of biological importance.

(+)α-Tocopheryl succinate inhibits the mitochondrial respiratory chain complex I and is as effective as arsenic trioxide or ATRA against acute promyelocytic leukemia in vivo.

The vitamin E derivative (+)α-tocopheryl succinate (α-TOS) exerts pro-apoptotic effects in a wide range of tumors and is well tolerated by normal tissues. Previous studies point to a mitochondrial involvement in the action mechanism; however, the early steps have not been fully elucidated. In a model of acute promyelocytic leukemia (APL) derived from hCG-PML-RARα transgenic mice, we demonstrated that α-TOS is as effective as arsenic trioxide or all-trans retinoic acid, the current gold standards of therapy. We also demonstrated that α-TOS induces an early dissipation of the mitochondrial membrane potential in APL cells and studies with isolated mitochondria revealed that this action may result from the inhibition of mitochondrial respiratory chain complex I. Moreover, α-TOS promoted accumulation of reactive oxygen species hours before mitochondrial cytochrome c release and caspases activation. Therefore, an in vivo antileukemic action and a novel mitochondrial target were revealed for α-TOS, as well as mitochondrial respiratory complex I was highlighted as potential target for anticancer therapy.

Synovial sarcoma of the tongue confirmed by molecular detection of the SYT-SSX2 fusion gene transcript.

Involvement of the tongue by a synovial sarcoma (SS) is an extremely rare event; there have only been 13 cases previously reported. The authors present herein a case of monophasic SS arising in the tongue in a 32-year-old woman. The neoplasm expressed cytokeratins AE1-3, OSCAR, and EMA as well as Bcl-2 and TLE1. Molecular analysis indicated that the patient tested positive for the SYT/SS2 fusion transcript.

Halofuginone has anti-proliferative effects in acute promyelocytic leukemia by modulating the transforming growth factor beta signaling pathway.

Promyelocytic leukemia-retinoic acid receptor alpha (PML-RARα) expression in acute promyelocytic leukemia (APL) impairs transforming growth factor beta (TGFß) signaling, leading to cell growth advantage. Halofuginone (HF), a low-molecular-weight alkaloid that modulates TGFß signaling, was used to treat APL cell lines and non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice subjected to transplantation with leukemic cells from human chorionic gonadotrophin-PML-RARα transgenic mice (TG). Cell cycle analysis using incorporated bromodeoxyuridine and 7-amino-actinomycin D showed that, in NB4 and NB4-R2 APL cell lines, HF inhibited cellular proliferation (P<0.001) and induced apoptosis (P = 0.002) after a 24-hour incubation. Addition of TGFß revealed that NB4 cells were resistant to its growth-suppressive effects and that HF induced these effects in the presence or absence of the cytokine. Cell growth inhibition was associated with up-regulation of TGFß target genes involved in cell cycle regulation (TGFB, TGFBRI, SMAD3, p15, and p21) and down-regulation of MYC. Additionally, TGFß protein levels were decreased in leukemic TG animals and HF in vivo could restore TGFß values to normal. To test the in vivo anti-leukemic activity of HF, we transplanted NOD/SCID mice with TG leukemic cells and treated them with HF for 21 days. HF induced partial hematological remission in the peripheral blood, bone marrow, and spleen. Together, these results suggest that HF has anti-proliferative and anti-leukemic effects by reversing the TGFß blockade in APL. Since loss of the TGFß response in leukemic cells may be an important second oncogenic hit, modulation of TGFß signaling may be of therapeutic interest.

Pax7-FKHR transcriptional activity is enhanced by transcriptionally repressed MyoD.

Alveolar rhabdomyosarcoma (ARMS) are characterized by the expression of chimeric transcription factors Pax3-FKHR and Pax7-FKHR, due to chromosomal translocations fusing PAX3 or PAX7 with the FKHR gene. Although ARMS exhibits a muscle lineage phenotype, the cells evade terminal differentiation despite expressing the potent myogenic transcriptional regulator MyoD. Here we show that while Pax7-FKHR inhibits MyoD-dependent transcription, MyoD enhances Pax7-FKHR activity in myogenic cell cultures. Importantly, this effect is not recapitulated by close related transcription factor myogenin and involves specific MyoD functional domains, distinct from those required for Pax7 to regulate MyoD during muscle formation. Together, these results suggest that although repressed as a myogenic regulatory factor, MyoD can play an active role in ARMS by augmenting Pax7-FKHR function.

BCL6, MUM1, and CD10 expression in mantle cell lymphoma.

Mantle cell lymphoma (MCL) characteristically express CD20, CD5, and cyclin-D1, carries the translocation t(11;14) (q13;q32) and typically has no expression of germinal center cell markers. So-called aberrant phenotypes such as CD5 negative and cyclin-D1-negative MCL have been described. Also few cases with CD10 and/or BCL-6 protein expression have been reported. We analyzed 127 MCL looking for the frequency of aberrant immunophenotype, CD10, BCL-6, and MUM1 expression. All cases were CD20 and cyclin-D1 positive, 96% expressed CD5, and 98% showed the t(11;14). BCL-6 expression was observed in 12% of the cases and MUM1 in 35%. No one case showed CD10 positivity in 30% or more neoplastic cells. Only 3 cases showed 10% to 20% of tumoral cells positive for CD10. MUM1 expression was observed in 67% of the BCL-6 positive cases. Thirty-two percent of the cases showed a MUM1+/BCL-6-/CD10- phenotype and 56% had a triple-negative-pattern. Aberrant phenotype is infrequent but not rare, and does not rule out a diagnosis of MCL in an otherwise typical case.

Detection of SS18-SSX fusion transcripts in formalin-fixed paraffin-embedded neoplasms: analysis of conventional RT-PCR, qRT-PCR and dual color FISH as diagnostic tools for synovial sarcoma.

Synovial Sarcoma consistently harbors t(X;18) resulting in SS18-SSX1, SS18-SSX2 and rarely SS18-SSX4 fusion transcripts. Of 328 cases included in our study, synovial sarcoma was either the primary diagnosis or was very high in the differential diagnosis in 134 cases: of these, amplifiable cDNA was obtained from 131. SS18-SSX fusion products were found in 126 (96%) cases (74 SS18-SSX1, 52 SS18-SSX2), using quantitative and 120 by conventional reverse transcriptase-polymerase chain reaction (RT-PCR). One hundred and one cases in a tissue microarray, analyzed by fluorescence in situ hybridization (FISH), revealed that 87 (86%) showed SS18 rearrangement: four RT-PCR positive cases, reported as negative for FISH, showed loss of one spectrum green signal, and 15 cases had multiple copies of the SS18 gene: both findings are potentially problematic when interpreting results. One of three cases, not analyzed by RT-PCR reaction owing to poor quality RNA, was positive by FISH. SS18-SSX1 was present in 56 monophasic and 18 biphasic synovial sarcoma: SS18-SSX2 was detected in 41 monophasic and 11 biphasic synovial sarcoma. Poorly differentiated areas were identified in 44 cases (31%). There was no statistically significant association between biphasic, monophasic and fusion type. Five cases were negative for SS18 rearrangement by all methods, three of which were pleural-sited neoplasms. Following clinical input, a diagnosis of mesothelioma was favored in one case, a sarcoma, not otherwise specified in another and a solitary fibrous tumor in the third case. The possibility of a malignant peripheral nerve sheath tumor could not be excluded in the other two cases. We concluded that the employment of a combination of molecular approaches is a powerful aid to diagnosing synovial sarcoma giving at least 96% sensitivity and 100% specificity but results must be interpreted in the light of other modalities such as clinical findings and immunohistochemical data.

Detection of TLS/FUS-CHOP fusion transcripts in a case of oral liposarcoma.

OBJECTIVE: The aim of this study was to detect the chromosomal translocation t(12;16)(q13;p11) that leads to a gene fusion encoding a FUS-CHOP chimeric protein and has been shown to be highly characteristic of myxoid and round cell subtypes of liposarcoma, in a case of oral myxoid liposarcoma. METHOD AND MATERIALS: Nested reverse transcriptase-polymerase chain reaction to detect the TLS/FUS-CHOP fusion gene transcript was performed. A case of inflammatory fibrous hyperplasia and a case of oral lipoma were included as negative controls. RESULTS: Only the myxoid oral liposarcoma showed a 103-base pair product, specific of TLS/FUS-CHOP fusion type II transcript. CONCLUSION: The identification of FUS-CHOP transcript is potentially useful in the diagnosis and research of oral liposarcomas.