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Background: The identification of oncogenic mutations in diffuse large B-cell lymphoma (DLBCL) has led to the development of drugs that target essential survival pathways, but whether targeting multiple survival pathways may be curative in DLBCL is unknown., Methods: We performed a single-center, phase 1b-2 study of a regimen of venetoclax, ibrutinib, prednisone, obinutuzumab, and lenalidomide (ViPOR) in relapsed or refractory DLBCL. In phase 1b, which included patients with DLBCL and indolent lymphomas, four dose levels of venetoclax were evaluated to identify the recommended phase 2 dose, with fixed doses of the other four drugs. A phase 2 expansion in patients with germinal-center B-cell (GCB) and non-GCB DLBCL was performed. ViPOR was administered every 21 days for six cycles., Results: In phase 1b of the study, involving 20 patients (10 with DLBCL), a single dose-limiting toxic effect of grade 3 intracranial hemorrhage occurred, a result that established venetoclax at a dose of 800 mg as the recommended phase 2 dose. Phase 2 included 40 patients with DLBCL. Toxic effects that were observed among all the patients included grade 3 or 4 neutropenia (in 24% of the cycles), thrombocytopenia (in 23%), anemia (in 7%), and febrile neutropenia (in 1%). Objective responses occurred in 54% of 48 evaluable patients with DLBCL, and complete responses occurred in 38%; complete responses were exclusively in patients with non-GCB DLBCL and high-grade B-cell lymphoma with rearrangements of MYC and BCL2 or BCL6 (or both). Circulating tumor DNA was undetectable in 33% of the patients at the end of ViPOR therapy. With a median follow-up of 40 months, 2-year progression-free survival and overall survival were 34% (95% confidence interval [CI], 21 to 47) and 36% (95% CI, 23 to 49), respectively., Conclusions: Treatment with ViPOR was associated with durable remissions in patients with specific molecular DLBCL subtypes and was associated with mainly reversible adverse events. (Funded by the Intramural Research Program of the National Cancer Institute and the National Center for Advancing Translational Sciences of the National Institutes of Health and others; ClinicalTrials.gov number, NCT03223610.)., (Copyright © 2024 Massachusetts Medical Society.)

Peripheral T-cell lymphomas (PTCL) are morphologically and biologically heterogeneous and a subset expresses CD30, including anaplastic large cell lymphomas (ALCL) and a minority of PTCL, not otherwise specified (PTCL, NOS). ALCL with ALK translocations (ALCL, ALK+) are readily identified by routine diagnostic methods, but differentiating ALCL without ALK translocation (ALCL, ALK-) and PTCL, NOS expressing CD30 (PTCL CD30+) can be challenging. Furthermore, rare PTCL co-express CD30 and CD15 (PTCL CD30+CD15+); some resemble ALCL, ALK- while others resemble classic Hodgkin lymphoma. To explore the relationship between PTCL CD30+CD15+ and ALCL, ALK-, we analysed 19 cases of PTCL with CD30 expression, previously diagnosed as ALCL, ALK- (nine cases) and PTCL CD30+CD15+ (10 cases) for DUSP22/IRF4 rearrangements, coding RNA expression and selected transcriptome analysis using the NanoString nCounter gene expression analysis platform. Unsupervised clustering showed no clear segregation between ALCL, ALK- and PTCL CD30+CD15+. Three cases previously classified as PTCL CD30+CD15+ showed DUSP22/IRF4 rearrangements, favouring a diagnosis of ALCL, ALK-. Our results suggest that cases previously designated PTCL CD30+CD15+, likely fall within the spectrum of ALCL, ALK-; additionally, a subset of ALCL, ALK- with DUSP22/IRF4 rearrangement expresses CD15, consistent with previous reports and expands the immunophenotypic spectrum of this lymphoma subgroup., (© 2024 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Primary cutaneous follicle center lymphoma has been distinguished from nodal follicular lymphoma (FL) based on genomic and clinical features. The nature of other extranodal FLs is not well defined. We report 15 cases of follicle center lymphoma involving the lower female genital tract. Cases were evaluated using an immunohistochemical panel for B-cell lymphoma, B-cell clonality, fluorescence in situ hybridization for BCL2 gene rearrangement, and next-generation sequencing. All patients had localized disease with no evidence of bone marrow involvement. Most cases (12/15, 80%) had a follicular pattern, at least focally. Large centrocytes were a prominent feature leading to concern for diffuse large B-cell lymphoma by referring pathologists. Neoplastic cells were positive for CD20 and BCL-6, while BCL-2 was positive in 2/15 (13%) cases. Fluorescence in situ hybridization for BCL2 gene rearrangement was negative in 10/11 (91%) cases. Next-generation sequencing performed in 10 cases revealed TNFRSF14 as the most frequently mutated gene in 6/10 (60%) cases. No case had CREBBP or KMT2D mutations as seen in nodal FL. None of the patients had progressive disease with durable complete remission achieved in 10/12 (83%) cases. The median follow-up period was 7.8 years (range: 0.2 to 20.5 y) with a 5-year overall survival of 100%. We conclude that follicle center lymphoma of the lower female genital tract is a novel variant of primary cutaneous follicle center lymphoma. Despite a frequent component of large cells, it is characterized by localized disease and low risk for dissemination. Awareness and recognition are important to distinguish these lesions from aggressive B-cell lymphomas., Competing Interests: Conflicts of Interest and Source of Funding: Supported by the Intramural Research Program of the Center for Cancer Research, National Cancer Institute. The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Epstein-Barr virus (EBV)-positive plasmacytoma is a rare plasma cell neoplasm. It remains unclear whether EBV-positive plasmacytoma represents a distinct entity or a variant of plasmacytoma. It shares morphologic features with plasmablastic lymphoma (PBL) and may cause diagnostic uncertainty. To better understand EBV-positive plasmacytoma and explore diagnostic criteria, this study describes 19 cases of EBV-positive plasmacytoma, compared with 27 cases of EBV-negative plasmacytoma and 48 cases of EBV-positive PBL. We reviewed the clinicopathologic findings and performed immunohistochemistry, in situ hybridization for EBV, fluorescence in situ hybridization for MYC , and next-generation sequencing. We found that 63.2% of patients with EBV-positive plasmacytoma were immunocompromised. Anaplastic features were observed in 7/19 cases. MYC rearrangement was found in 25.0% of them, and extra copies of MYC in 81.3%. EBV-positive and EBV-negative plasmacytomas possessed similar clinicopathologic features, except more frequent cytologic atypia, bone involvement and MYC aberrations in the former group. The survival rate of patients with EBV-positive plasmacytoma was comparable to that of patients with EBV-negative plasmacytoma. In comparison to PBL, EBV-positive plasmacytoma is less commonly associated with a "starry-sky" appearance, necrosis, absence of light chain expression, and a high Ki67 index (>75%). The most recurrently mutated genes/signaling pathways in EBV-positive plasmacytoma are epigenetic regulators, MAPK pathway, and DNA damage response, while the most frequently reported mutations in PBL are not observed. Collectively, EBV-positive plasmacytoma should be regarded as a biological variant of plasmacytoma. Thorough morphologic examination remains the cornerstone for distinguishing EBV-positive plasmacytoma and PBL, and molecular studies can be a valuable complementary tool., Competing Interests: Conflicts of Interest and Source of Funding: This work was supported by funding from the Intramural Research Program of the Center for Cancer Research, National Cancer Institute, NIH. The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Spindle cell rhabdomyosarcoma (RMS) is a rare variant of RMS accounting for up to 10% of cases in infants. In older children and adults, spindle cell RMS is associated with MYOD1 mutations and a poor prognosis. In infants, it is associated with recurring fusions involving NCOA2 and VGLL2. Reports in the literature suggest a favorable prognosis for this subset, however, little is known about treatment and outcome data of infants with spindle cell RMS., Methods: Characteristics, treatment, and outcome of an international cohort of 40 patients aged ≤ 12 months with spindle cell RMS treated from 1997 to 2018 were evaluated., Results: Localized disease (LD) was diagnosed in 39 patients. The median age at diagnosis was 2.5 months (range 0-12 months). Expert pathologic review confirmed the diagnosis of spindle cell RMS in all patients. Among 26 tumors that had molecular evaluation, 13 had rearrangements of NCOA and/or VGLL. Multimodal treatment of infants with LD included conventional (age adjusted) chemotherapy (n = 37), resection (n = 31) and radiotherapy (RT) (n = 5, brachytherapy in 3). Complete remission was achieved in 37/39 patients. Progressive disease occurred in two infants, relapsed disease in three. Microscopically complete surgical resection was associated with five-year event-free survival (EFS) and overall survival (OS) of 100%. Two patients with tumors ≤ 5 cm were treated with microscopically complete resection only and were alive 1 and 4.2 years after diagnosis. The 5-year EFS and OS for infants with LD were 86% (±11; CI 95%) and 91% (±9; CI 95%), respectively. One patient had metastatic disease (NCOA fusion positive) with primary tumor in head and neck and brain metastases. This patient died despite chemotherapy and delayed resection of the primary tumor due to respiratory failure secondary to cytomegalovirus infection 1.2 years after diagnosis., Conclusion: Infants with spindle cell RMS have an excellent prognosis. Multimodal treatment including microscopically complete resection of the tumor is strongly recommended., Competing Interests: Conflict of interest statement The authors declare no conflict of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Adverse prognosis in Ewing sarcoma (ES) is associated with the presence of metastases, particularly in bone, tumor hypoxia and chromosomal instability (CIN). Yet, a mechanistic link between these factors remains unknown. We demonstrate that in ES, tumor hypoxia selectively exacerbates bone metastasis. This process is triggered by hypoxia-induced stimulation of the neuropeptide Y (NPY)/Y5 receptor (Y5R) pathway, which leads to RhoA over-activation and cytokinesis failure. These mitotic defects result in the formation of polyploid ES cells, the progeny of which exhibit high CIN, an ability to invade and colonize bone, and a resistance to chemotherapy. Blocking Y5R in hypoxic ES tumors prevents polyploidization and bone metastasis. Our findings provide evidence for the role of the hypoxia-inducible NPY/Y5R/RhoA axis in promoting genomic changes and subsequent osseous dissemination in ES, and suggest that targeting this pathway may prevent CIN and disease progression in ES and other cancers rich in NPY and Y5R., (© 2022. The Author(s).)

Background: Pheochromocytoma and paraganglioma (PPGL) are neuroendocrine tumors with frequent mutations in genes linked to the tricarboxylic acid cycle. However, no pathogenic variant has been found to date in succinyl-CoA ligase (SUCL), an enzyme that provides substrate for succinate dehydrogenase (SDH; mitochondrial complex II [CII]), a known tumor suppressor in PPGL., Methods: A cohort of 352 patients with apparently sporadic PPGL underwent genetic testing using a panel of 54 genes developed at the National Institutes of Health, including the SUCLG2 subunit of SUCL. Gene deletion, succinate levels, and protein levels were assessed in tumors where possible. To confirm the possible mechanism, we used a progenitor cell line, hPheo1, derived from a human pheochromocytoma, and ablated and re-expressed SUCLG2., Results: We describe 8 germline variants in the guanosine triphosphate-binding domain of SUCLG2 in 15 patients (15 of 352, 4.3%) with apparently sporadic PPGL. Analysis of SUCLG2-mutated tumors and SUCLG2-deficient hPheo1 cells revealed absence of SUCLG2 protein, decrease in the level of the SDHB subunit of SDH, and faulty assembly of the complex II, resulting in aberrant respiration and elevated succinate accumulation., Conclusions: Our study suggests SUCLG2 as a novel candidate gene in the genetic landscape of PPGL. Large-scale sequencing may uncover additional cases harboring SUCLG2 variants and provide more detailed information about their prevalence and penetrance., (Published by Oxford University Press 2021. This work is written by a US Government employee and is in the public domain in the US.)

Background: Despite recent advances, there is an urgent need for agents targeting HER2-expressing cancers other than breast cancer. We report a phase I study (NCT01730118) of a dendritic cell (DC) vaccine targeting HER2 in patients with metastatic cancer or bladder cancer at high risk of relapse., Patients and Methods: Part 1 of the study enrolled patients with HER2-expressing metastatic cancer that had progressed after at least standard treatment and patients who underwent definitive treatment for invasive bladder cancer with no evidence of disease at the time of enrollment. Part 2 enrolled patients with HER2-expressing metastatic cancer who had progressed after anti-HER2 therapy. The DC vaccines were prepared from autologous monocytes and transduced with an adenoviral vector expressing the extracellular and transmembrane domains of HER2 (AdHER2). A total of five doses were planned, and adverse events were recorded in patients who received at least one dose. Objective response was evaluated by unidimensional immune-related response criteria every 8 weeks in patients who received at least two doses. Humoral and cellular immunogenicity were assessed in patients who received more than three doses., Results: A total of 33 patients were enrolled at four dose levels (5 × 10 6 , 10 × 10 6 , 20 × 10 6 , and 40 × 10 6 DCs). Median follow-up duration was 36 weeks (4-124); 10 patients completed five doses. The main reason for going off-study was disease progression. The main adverse events attributable to the vaccine were injection-site reactions. No cardiac toxicity was noted. Seven of 21 evaluable patients (33.3%) demonstrated clinical benefit (1 complete response, 1 partial response, and 5 stable disease). After ≥3 doses, an antibody response was detected in 3 of 13 patients (23.1%), including patients with complete and partial responses. Lymphocytes from 10 of 11 patients (90.9%) showed induction of anti-HER2 responses measured by the production of at least one of interferon-gamma, granzyme B, or tumor necrosis factor-alpha, and there were multifunctional responses in 8 of 11 patients (72.7%)., Conclusions: The AdHER2 DC vaccine showed evidence of immunogenicity and preliminary clinical benefit in patients with HER2-expressing cancers, along with an excellent safety profile. It shows promise for further clinical applications, especially in combination regimens., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Maeng, Moore, Bagheri, Steinberg, Inglefield, Dunham, Wei, Morris, Terabe, England, Roberson, Rosing, Sachdev, Pack, Miettinen, Barr, Weiner, Panch, Stroncek, Wood and Berzofsky.)

We report a novel group of clinically aggressive spinal cord ependymomas characterized by Grade III histology, MYCN amplification, an absence of NF2 alterations or other recurrent pathogenic mutations, and a unique methylation classifier profile. Seven cases were found to have MYCN amplification in the course of routine mutational profiling of 552 patients with central nervous system tumors between December 2016 and July of 2019 and an eighth patient was identified from an unrelated set of cases. Methylation array analysis revealed that none of the 8 cases clustered with any of the nine previously described ependymoma methylation subgroups, and 7 of 8 formed their own tight unique cluster. Histologically all cases showed grade III features, and all demonstrated aggressive clinical behavior. These findings are presented in the context of data from three other studies describing similar cases. Therefore, a combined total of 27 MYCN amplified spinal cord ependymoma cases have now been reported in the literature, warranting their consideration as a distinctive subtype of spinal cord ependymoma (SP-EPN-MYCN) with their unique molecular characteristics and aggressive clinical behavior.

Metastasis is responsible for 90% of human cancer mortality, yet it remains a challenge to model human cancer metastasis in vivo. Here we describe mouse models of high-grade serous ovarian cancer, also known as high-grade serous carcinoma (HGSC), the most common and deadliest human ovarian cancer type. Mice genetically engineered to harbor Dicer1 and Pten inactivation and mutant p53 robustly replicate the peritoneal metastases of human HGSC with complete penetrance. Arising from the fallopian tube, tumors spread to the ovary and metastasize throughout the pelvic and peritoneal cavities, invariably inducing hemorrhagic ascites. Widespread and abundant peritoneal metastases ultimately cause mouse deaths (100%). Besides the phenotypic and histopathological similarities, mouse HGSCs also display marked chromosomal instability, impaired DNA repair, and chemosensitivity. Faithfully recapitulating the clinical metastases as well as molecular and genomic features of human HGSC, this murine model will be valuable for elucidating the mechanisms underlying the development and progression of metastatic ovarian cancer and also for evaluating potential therapies., Competing Interests: The authors have declared that no competing interests exist.

The presence of multiple BAP1-negative melanocytic neoplasms is a hallmark of familial cancer susceptibility syndrome caused by germline mutations in BAP1. Melanocytic tumors lacking BAP1 expression may also present as sporadic lesions in patients lacking a germline BAP1 mutation. Here, we report histomorphologic and clinical characteristics of cutaneous melanomas with loss of BAP1 expression in 4 patients with no known history of BAP1-associated cancer susceptibility syndrome. The lesions were nodular melanomas composed predominantly of intradermal large epithelioid (Spitzoid) melanocytes with nuclear pseudoinclusions as well as scattered multinucleated cells, arising in association with a typical intradermal nevus. Of the 4 patients, only 1 had recurrence. This patient had multiple recurrences with in-transit and regional lymph node metastases. To the best of our knowledge, this is the first reported series of cutaneous melanomas with loss of BAP1 expression arising in patients without a family history of cancer.

Background: The role of the hypoxia signaling pathway in the pathogenesis of pheochromocytoma/paraganglioma (PPGL)-polycythemia syndrome has been elucidated. Novel somatic mutations in hypoxia-inducible factor type 2A (HIF2A) and germline mutations in prolyl hydroxylase type 1 and type 2 (PHD1 and PHD2) have been identified to cause upregulation of the hypoxia signaling pathway and its target genes including erythropoietin (EPO) and its receptor (EPOR). However, in a minority of patients presenting with this syndrome, the genetics and molecular pathogenesis remain unexplained. The aim of the present study was to uncover novel genetic causes of PPGL-polycythemia syndrome., Case Presentation: A female presented with a history of JAK2 V617F positive PV, diagnosed in 2007, and right adrenal pheochromocytoma diagnosed and resected in 2011. Her polycythemia symptoms and hematocrit levels continued to worsen from 2007 to 2011, with an increased frequency of phlebotomies. Postoperatively, until early 2013, her hematocrit levels remained normalized. Following this, the hematocrit levels ranged between 46.4 and 48.9% [35-45%]. Tumor tissue from the patient was further tested for mutations in genes related to upregulation of the hypoxia signaling pathway including iron regulatory protein 1 (IRP1), which is a known regulator of HIF-2α mRNA translation. Functional studies were performed to investigate the consequences of these mutations, especially their effect on the HIF signaling pathway and EPO. Indel mutations (c.267-1_267delGGinsTA) were discovered at the exon 3 splicing site of IRP1. Minigene construct and splicing site analysis showed that the mutation led to a new splicing site and a frameshift mutation of IRP1, which caused a truncated protein. Fluorescence in situ hybridization analysis demonstrated heterozygous IRP1 deletions in tumor cells. Immunohistochemistry results confirmed the truncated IRP1 and overexpressed HIF-2α, EPO and EPOR in tumor cells., Conclusions: This is the first report which provides direct molecular genetic evidence of association between a somatic IRP1 loss-of-function mutation and PHEO and secondary polycythemia. In patients diagnosed with PHEO/PGL and polycythemia with negative genetic testing for mutations in HIF2A, PHD1/2, and VHL, IRP1 should be considered as a candidate gene.

GATA2 deficiency is a recently described genetic disorder affecting hematopoietic stem cells and is associated with immunodeficiency, hematologic malignancy, and various cutaneous pathologies including cutaneous tumors. To explore the incidence and clinical course of melanoma in patients with germline GATA2 deficiencies, we conducted a retrospective chart review of 71 such patients and identified two with invasive melanoma. One melanoma was diagnosed early because it was associated with pruritus due to a graft-versus-tumor effect following bone marrow transplantation. The other one, a lentigo maligna melanoma, was locally excised but progressed to widespread metastasis and death several years later. Our observations and published studies of melanoma biology suggest an association between decreased GATA2 expression and melanoma progression. These findings suggest that GATA2 deficient patients may have an increased risk of melanoma and should be observed closely for new or changing skin lesions., (Published 2017. This article is a U.S. Government work and is in the public domain in the USA.)

Background: Recent discovery of gene rearrangements have brought a new look to the molecular pathogenesis of cancer. Gene fusions occur in nearly 60% of prostate adenocarcinoma, being the TMPRSS2-ERG one of the most common. Evidence supports the role of ERG fusion in tumorigenesis, progression and invasion via effecting pathways such as WNT , MYC , uPA , PI3K/AKT/PTEN , RAS/RAF/MAPF , NKX3.1 , GST-pi and androgen receptor (AR) mediated signaling. Most of the ERG fusions involve 5'-partners androgen responsive. Therefore, we aimed to evaluate AR and ERG fusion protein expression on prostate tissue to find clinicopathological applications and possible role in therapy. Methods: One hundred three samples, including prostate core biopsies and radical prostatectomy specimens, were evaluated for ERG and AR expression by immunohistochemistry (IHC). ERG rearrangement was done by fluorescence in situ hybridization (FISH) on 11 randomly selected cases and correlated with IHC results. Results: From the total of 103 samples, eight (8/103) were benign, fourteen (14/103) had atypical glands, two (2/103) had prostatic intraepithelial neoplasia (PIN), and seventy nine (79/103) showed prostate adenocarcinoma. Forty four (44/79) tumor cases were Gleason score (GS) 6-7 (lower GS), and thirty five (35/79) were GS of 8-10 (higher GS). ERG immunoreaction was observed in 27.8% (22/79) of the tumor cases, showing higher expression in those with lower GS (68.2%, 15/22) compared to higher GS (31.8%, 7/22). Neither benign glands nor PIN stained with ERG . AR expression was observed in 75% of benign samples, 78.5% of atypical glands, 100% of PIN, and in 87.3% of tumor cases with no significant difference based on GS. Co-expression of ERG and AR was evaluated on all the tumor samples. ERG +/AR+ was seen in 77.3% (17/22) of the ERG + tumor cases, with higher frequency in lower GS (64.7%, 11/17) compared to those with higher GS (35.3%, 6/17). All but five corresponding ERG + tumor samples were negative for AR. Only 5 samples were ERG -/AR- corresponding to adenocarcinoma GS of 6. Presence or absence of ERG rearrangement was confirmed by FISH and correlated with IHC results. Conclusions: Characterization of ERG status by IHC in prostate tissue has an excellent correlation with FISH. It may also assist in diagnosis since none of the benign glands stained with ERG . Co-expression of ERG+ /AR+ in prostate tumor by IHC may suggest gene fusion between ERG and a 5'-partner driven by androgen signaling such as TMPRSS2 , which it could represent an important ancillary test for clinical management and development of new therapeutic targets., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Purpose: Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma and includes a PAX3- or PAX7-FOXO1 fusion-positive subtype. Amplification of chromosomal region 12q13-q14, which contains the CDK4 proto-oncogene, was identified in an aggressive subset of fusion-positive RMS. CDK4/6 inhibitors have antiproliferative activity in CDK4-amplified liposarcoma and neuroblastoma, suggesting CDK4/6 inhibition as a potential therapeutic strategy in fusion-positive RMS., Experimental Design: We examined the biologic consequences of CDK4 knockdown, CDK4 overexpression, and pharmacologic CDK4/6 inhibition by LEE011 in fusion-positive RMS cell lines and xenografts., Results: Knockdown of CDK4 abrogated proliferation and transformation of 12q13-14-amplified and nonamplified fusion-positive RMS cells via G1-phase cell-cycle arrest. This arrest was mediated by reduced RB phosphorylation and E2F-responsive gene expression. Significant differences in E2F target expression, cell-cycle distribution, proliferation, or transformation were not observed in RMS cells overexpressing CDK4. Treatment with LEE011 phenocopied CDK4 knockdown, decreasing viability, RB phosphorylation, and E2F-responsive gene expression and inducing G1-phase cell-cycle arrest. Although all fusion-positive cell lines showed sensitivity to CDK4/6 inhibition, there was diminished sensitivity associated with CDK4 amplification and overexpression. This variable responsiveness to LEE011 was recapitulated in xenograft models of CDK4-amplified and nonamplified fusion-positive RMS., Conclusions: Our data demonstrate that CDK4 is necessary but overexpression is not sufficient for RB-E2F-mediated G1-phase cell-cycle progression, proliferation, and transformation in fusion-positive RMS. Our studies indicate that LEE011 is active in the setting of fusion-positive RMS and suggest that low CDK4-expressing fusion-positive tumors may be particularly susceptible to CDK4/6 inhibition., (©2015 American Association for Cancer Research.)

Dysembryoplastic neuroepithelial tumours (DNET) are a common cause of tumour-associated epilepsy, and are usually located in the temporal lobes. We present a case of multifocal DNETs in both infra- and supra-tentorial locations, in a 23-year-old man with a coincident Type I Chiari malformation, presenting with medically refractory focal seizures. The extensive anatomical distribution of the lesions suggests a genetic component in their tumourigenesis.

Context: Carcinoids have rarely been described in von Hippel-Lindau (VHL) disease., Objective: We describe the first reported case of a patient with VHL who developed a pulmonary carcinoid that subsequently metastasized to a pre-existent cranial hemangioblastoma., Results: Histological and immunohistochemical features of the metastatic lesion were similar to the primary carcinoid. Both lesions demonstrated heterozygous VHL gene deletions with fluorescence in situ hybridization analysis., Conclusions: This case provides direct molecular genetic evidence of an association between VHL and carcinoids.

Gene rearrangements involving the Ewing sarcoma breakpoint region 1 (EWSR1) gene are seen in a broad range of sarcomas and some nonmesenchymal neoplasms. Ewing sarcoma is molecularly defined by a fusion of the EWSR1 gene (or rarely the related FUS gene) to a member of the E26 transformation-specific (ETS) family of transcription factors, frequently the EWSR1-FLI1 fusion. More recently, EWSR1 gene fusion to non-ETS family members, including the nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 2 (NFATC2) gene, has been reported in a histological variant of Ewing sarcoma. Here, we report a malignant round cell tumor of bone with an EWSR1-NFATC2 fusion gene. This report builds upon the unusual morphological and clinical presentation of bone neoplasms containing an EWSR1-NFATC2 fusion gene.

Ewing sarcoma is described classically as a small, round cell tumor of bone and soft tissue in children and young adults. Ewing sarcoma most often is characterized by a fusion of the Ewing sarcoma breakpoint region 1 (EWSR1) and the Friend leukemia virus integration 1 (FLI1) genes, forming an EWSR1-FLI1 fusion transcript. We report an exceptional case of primary subcutaneous Ewing sarcoma in a 16-year-old female composed entirely of spindle cells with focal fascicular growth and exhibiting strong, diffuse immunohistochemical reactivity for S100, unlike classic Ewing sarcoma. However, reverse transcription-polymerase chain reaction (RT-PCR) analysis confirmed the presence of a rare variant of the EWSR1-FLI1 fusion transcript, featuring fusion of EWSR1 exon 10 to FLI1 exon 6. To our knowledge, the combined histologic, molecular, and clinical features have not been reported previously in Ewing sarcoma, and raise a broad differential diagnosis emphasizing the importance of molecular techniques in the diagnosis of this tumor.

Introduction: There remains a significant therapeutic need for small-cell lung cancer (SCLC). We and others have reported high frequency of copy number gains in cytogenetic bands encoding fibroblast growth factor receptor 1 (FGFR1) in SCLC tumors and cell lines., Methods: Thirteen SCLC cell lines and 68 SCLC patient tumor samples were studied for FGFR1 amplification. Growth inhibition assays were performed using PD173074, a pan-FGFR inhibitor to determine the correlation between FGFR1 expression and drug sensitivity., Results: We did not detect FGFR1 mutations in SCLC cell lines. Focal amplification of FGFR1 gene was found in five tumor samples (7%), with high-level focal amplification in only one tumor sample (1%). Amplification owing to polysomy of chromosome 8, where FGFR1 locates, was observed in 22 tumor samples (32%). There was no correlation between FGFR1 gene copy number and messenger RNA expression or protein expression in SCLC cells. FGFR inhibitor sensitivity correlated with FGFR1 copy number determined by real-time polymerase chain reaction assay (r= -0.79; p = 0.01)., Conclusion: FGFR1 gene mutations and focal amplification are rare in SCLC, but polysomy of chromosome 8 is relatively common. FGFR1 copy number gain predicts sensitivity to FGFR inhibition, and FGFR expression correlates inversely with chemosensitivity.

von Hippel-Lindau disease (VHL) patients develop highly vascular tumors, including central nervous system hemangioblastomas. It has been hypothesized that the vascular nature of these tumors is the product of reactive angiogenesis. However, recent data indicate that VHL-associated hemangioblastoma neoplastic cells originate from embryologically-arrested hemangioblasts capable of blood and endothelial cell differentiation. To determine the origin of tumor vasculature in VHL-associated hemangioblastomas, we analyzed the vascular elements in tumors from VHL patients. We demonstrate that isolated vascular structures and blood vessels within VHL-associated hemangioblastomas are a result of tumor-derived vasculogenesis. Further, similar to hemangioblastomas, we demonstrate that other VHL-associated lesions possess vascular tissue of tumor origin and that tumor-derived endothelial cells emerge within implanted VHL deficient UMRC6 RCC murine xenografts. These findings further establish the embryologic, developmentally arrested, hemangioblast as the tumor cell of origin for VHL-associated hemangioblastomas and indicate that it is also the progenitor cell for other VHL-associated tumors.

ERG transcription factor is constitutively expressed in endothelial cells. Because benign and malignant vascular endothelia retain the ERG expression, ERG is considered a useful marker for angiosarcomas and related tumors. ERG is also expressed in a subset of prostate carcinomas and Ewing sarcomas due to ERG-involved translocations; therefore, this marker is also of high interest in the study of these malignancies. In this study, we evaluated 109 epithelioid sarcomas for ERG expression, on the basis of an initial observation of an ERG-positive case. We also studied expression of other endothelial antigens in epithelioid sarcoma. ERG was expressed in 38% of epithelioid sarcomas (41/109), usually with a uniform nuclear staining, similar to that seen in angiosarcomas. However, all epithelioid sarcomas were negative for ERG gene rearrangement indicating that ERG expression is not likely related to ERG-involving translocations in epithelioid sarcoma. Other endothelial markers, CD31, claudin 5, and Prox1, were absent in epithelioid sarcomas. The only exception was a pulmonary metastasis of epithelioid sarcoma showing focal CD31 expression, which probably resulted from antigen adsorption onto tumor cell surfaces. However, podoplanin was commonly (7/9) expressed in epithelioid sarcoma; therefore, this marker is not useful in distinguishing epithelioid sarcoma from angiosarcoma. INI1/SMARCB1 gene product was absent in all epithelioid sarcomas (considered here a definitional feature) but was absent from only 1 epithelioid angiosarcoma, indicating its relative specificity for epithelioid sarcoma in this differential diagnostic setting. ERG expression is fairly common in epithelioid sarcoma and should be recognized as a diagnostic pitfall in the differential diagnosis of epithelioid sarcoma and epithelioid angiosarcoma. General lack of endothelial cell-specific markers in epithelioid sarcoma helps in this distinction.

Follicular lymphoma in situ (FLIS) was first described nearly a decade ago, but its clinical significance remains uncertain. We reevaluated our original series and more recently diagnosed cases to develop criteria for the distinction of FLIS from partial involvement by follicular lymphoma (PFL). A total of 34 cases of FLIS were identified, most often as an incidental finding in a reactive lymph node. Six of 34 patients had prior or concurrent FL, and 5 of 34 had FLIS composite with another lymphoma. Of patients with negative staging at diagnosis and available follow-up (21 patients), only one (5%) developed FL (follow-up: median, 41 months; range, 10-118 months). Follow-up was not available in 2 cases. Fluorescence in situ hybridization for BCL2 gene rearrangement was positive in all 17 cases tested. PFL patients were more likely to develop FL, diagnosed in 9 of 17 (53%) who were untreated. Six patients with PFL were treated with local radiation therapy (4) or rituximab (2) and remained with no evidence of disease. FLIS can be reliably distinguished from PFL and has a very low rate of progression to clinically significant FL. FLIS may represent the tissue counterpart of circulating t(14;18)-positive B cells.

Background: BORIS/CTCFL is a paralogue of CTCF, the major epigenetic regulator of vertebrate genomes. BORIS is normally expressed only in germ cells but is aberrantly activated in numerous cancers. While recent studies demonstrated that BORIS is a transcriptional activator of testis-specific genes, little is generally known about its biological and molecular functions., Methodology/principal Findings: Here we show that BORIS is expressed as 23 isoforms in germline and cancer cells. The isoforms are comprised of alternative N- and C-termini combined with varying numbers of zinc fingers (ZF) in the DNA binding domain. The patterns of BORIS isoform expression are distinct in germ and cancer cells. Isoform expression is activated by downregulation of CTCF, upregulated by reduction in CpG methylation caused by inactivation of DNMT1 or DNMT3b, and repressed by activation of p53. Studies of ectopically expressed isoforms showed that all are translated and localized to the nucleus. Using the testis-specific cerebroside sulfotransferase (CST) promoter and the IGF2/H19 imprinting control region (ICR), it was shown that binding of BORIS isoforms to DNA targets in vitro is methylation-sensitive and depends on the number and specific composition of ZF. The ability to bind target DNA and the presence of a specific long amino terminus (N258) in different isoforms are necessary and sufficient to activate CST transcription. Comparative sequence analyses revealed an evolutionary burst in mammals with strong conservation of BORIS isoproteins among primates., Conclusions: The extensive repertoire of spliced BORIS variants in humans that confer distinct DNA binding and transcriptional activation properties, and their differential patterns of expression among germ cells and neoplastic cells suggest that the gene is involved in a range of functionally important aspects of both normal gametogenesis and cancer development. In addition, a burst in isoform diversification may be evolutionarily tied to unique aspects of primate speciation.

The role of repetitive DNA sequences in pericentromeric regions with respect to kinetochore/heterochromatin structure and function is poorly understood. Here, we use a mouse erythroleukemia cell (MEL) system for studying how repetitive DNA assumes or is assembled into different chromatin structures. We show that human gamma-satellite DNA arrays allow a transcriptionally permissive chromatin conformation in an adjacent transgene and efficiently protect it from epigenetic silencing. These arrays contain CTCF and Ikaros binding sites. In MEL cells, this gamma-satellite DNA activity depends on binding of Ikaros proteins involved in differentiation along the hematopoietic pathway. Given our discovery of gamma-satellite DNA in pericentromeric regions of most human chromosomes and a dynamic chromatin state of gamma-satellite arrays in their natural location, we suggest that gamma-satellite DNA represents a unique region of the functional centromere with a possible role in preventing heterochromatin spreading beyond the pericentromeric region.

Animal models of human diseases are widely used to address questions of tumor development. Selection of a particular animal model depends upon a variety of factors, among them: animal cost, species lifespan and hardiness; availability of biomolecular and genetic tools for that species; and evolutionary distance from humans. In spite of the growth in genomic data in the past several years, many animal models cannot yet be studied extensively due to gaps in genetic mapping, sequencing and functional analyses. Thus, alternative molecular genetic approaches are needed. We have designed an interspecies comparative genomic hybridization approach to analyze genetic changes in radiation-induced brain tumors in the non-human primate, Macaca mulatta. Using homologies between the primate and human genomes, we adapted widely-available CGH techniques to generate cytogenetic profiles of malignant gliomas in four monkey tumors. Losses and gains were projected onto the corresponding homologous chromosomal regions in the human genome, thus directly translating the status of the monkey gliomas into human gene content. This represents a novel method of comparative interspecies cytogenetic mapping that permits simultaneous analysis of genomic imbalances of unknown sequences in disparate species and correlation with potential or known human disease-related genes.

Brother of the regulator of imprinted sites (BORIS) was previously described as a transcription factor for epigenetic reprogramming the expression of which is strictly confined to germ cells of adult testes but is aberrantly activated in the vast majority of neoplastic cells. Considering the critical role of BORIS in cancerogenesis and the fact that its expression pattern may preclude thymic tolerance, we generated DNA- and protein-based mouse BORIS antitumor vaccines using a non-DNA-binding version of the BORIS molecule. Clinical use of BORIS as a vaccine Ag would require that certain safety concerns be met. Specifically, administration of the functional BORIS protein would hypothetically pose a risk of BORIS accelerating the progression of cancer. To alleviate such safety concerns, we have developed vaccines based on the BORIS molecule lacking the DNA-binding zinc fingers domain. To enhance anti-BORIS cellular immune responses, we used a standard molecular adjuvant approach. It consisted of plasmids encoding murine IL-12 and IL-18 for a DNA-based vaccine and conventional Th1 type adjuvant, Quil A, for a protein-based vaccine. Both DNA- and protein-based vaccines induced Ag-specific CD4(+) T cell proliferation with Th1 and Th2 cytokine profiles, respectively. Protein-based, but not DNA-based, BORIS vaccine induced a significant level of Ab production in immunized animals. Importantly, potent anticancer CD8(+)-cytotoxic lymphocytes were generated after immunization with the DNA-based, but not protein-based, BORIS vaccine. These cytolytic responses were observed across a wide range of different mouse cancers including mammary adenocarcinoma, glioma, leukemia, and mastocytoma.

Neurons, once committed, exit the cell cycle and undergo maturation that promote specialized activity and are believed to operate upon a stable genome. We used fluorescence in situ hybridization, selective cell microdissection, and loss of heterozygosity analysis to assess degree of aneuploidy in patients with a neurodegenerative disease and in normal controls. We found that aneuploidy occurs in approximately 40% of mature, adult human neurons in health or disease and may be a physiological mechanism that maintains neuronal fate and function; it does not appear to be an unstable state. The fact that neuronal stem cells can be identified in adult humans and that somatic mosaicism may be found in neuronal precursor cells deserves further investigation before using adult neural stem cells to treat human disease.

Regulatory sequences recognized by the unique pair of paralogous factors, CTCF and BORIS, have been implicated in epigenetic regulation of imprinting and X chromosome inactivation. Lung cancers exhibit genome-wide demethylation associated with derepression of a specific class of genes encoding cancer-testis (CT) antigens such as NY-ESO-1. CT genes are normally expressed in BORIS-positive male germ cells deficient in CTCF and meCpG contents, but are strictly silenced in somatic cells. The present study was undertaken to ascertain if aberrant activation of BORIS contributes to derepression of NY-ESO-1 during pulmonary carcinogenesis. Preliminary experiments indicated that NY-ESO-1 expression coincided with derepression of BORIS in cultured lung cancer cells. Quantitative reverse transcription-PCR analysis revealed robust, coincident induction of BORIS and NY-ESO-1 expression in lung cancer cells, but not normal human bronchial epithelial cells following 5-aza-2'-deoxycytidine (5-azadC), Depsipeptide FK228 (DP), or sequential 5-azadC/DP exposure under clinically relevant conditions. Bisulfite sequencing, methylation-specific PCR, and chromatin immunoprecipitation (ChIP) experiments showed that induction of BORIS coincided with direct modulation of chromatin structure within a CpG island in the 5'-flanking noncoding region of this gene. Cotransfection experiments using promoter-reporter constructs confirmed that BORIS modulates NY-ESO-1 expression in lung cancer cells. Gel shift and ChIP experiments revealed a novel CTCF/BORIS-binding site in the NY-ESO-1 promoter, which unlike such sites in the H19-imprinting control region and X chromosome, is insensitive to CpG methylation in vitro. In vivo occupancy of this site by CTCF was associated with silencing of the NY-ESO-1 promoter, whereas switching from CTCF to BORIS occupancy coincided with derepression of NY-ESO-1. Collectively, these data indicate that reciprocal binding of CTCF and BORIS to the NY-ESO-1 promoter mediates epigenetic regulation of this CT gene in lung cancer cells, and suggest that induction of BORIS may be a novel strategy to augment immunogenicity of pulmonary carcinomas.

Brother of the Regulator of Imprinted Sites (BORIS) is a mammalian CTCF paralog with the same central 11Zn fingers (11ZF) that mediate specific interactions with varying approximately 50-bp target sites. Regulated in vivo occupancy of such sites may yield structurally and functionally distinct CTCF/DNA complexes involved in various aspects of gene regulation, including epigenetic control of gene imprinting and X chromosome inactivation. The latter functions are mediated by meCpG-sensitive 11ZF binding. Because CTCF is normally present in all somatic cells, whereas BORIS is active only in CTCF- and 5-methylcytosine-deficient adult male germ cells, switching DNA occupancy from CTCF to BORIS was suggested to regulate site specificity and timing of epigenetic reprogramming. In addition to 11ZF-binding paternal imprinting control regions, cancer-testis gene promoters also undergo remethylation during CTCF/BORIS switching in germ cells. Only promoters of cancer testis genes are normally silenced in all somatic cells but activated during spermatogenesis when demethylated in BORIS-positive germ cells and are found aberrantly derepressed in various tumors. We show here that BORIS is also expressed in multiple cancers and is thus itself a cancer-testis gene and that conditional expression of BORIS in normal fibroblasts activates cancer-testis genes selectively. We tested if replacement of CTCF by BORIS on regulatory DNA occurs in vivo on activation of a prototype cancer-testis gene, MAGE-A1. Transition from a hypermethylated/silenced to a hypomethylated/activated status induced in normal cells by 5-aza-2'-deoxycytidine (5-azadC) was mimicked by conditional input of BORIS and is associated with complete switching from CTCF to BORIS occupancy at a single 11ZF target. This site manifested a novel type of CTCF/BORIS 11ZF binding insensitive to CpG methylation. Whereas 5-azadC induction of BORIS takes only few hours, derepression of MAGE-A1 occurred 1 to 2 days later, suggesting that BORIS mediates cancer-testis gene activation by 5-azadC. Indeed, infection of normal fibroblasts with anti-BORIS short hairpin RNA retroviruses before treatment with 5-azadC blocked reactivation of MAGE-A1. We suggest that BORIS is likely tethering epigenetic machinery to a novel class of CTCF/BORIS 11ZF target sequences that mediate induction of cancer-testis genes.

Here we describe and characterize a small serine/threonine kinase (SSTK) which consists solely of the N- and C-lobes of a protein kinase catalytic domain. SSTK protein is highly conserved among mammals, and no close homologues were found in the genomes of nonmammalian organisms. SSTK specifically interacts with HSP90-1beta, HSC70, and HSP70 proteins, and this association appears to be required for SSTK kinase activity. The SSTK transcript was most abundant in human and mouse testes but was also detected in all human tissues tested. In the mouse testis, SSTK protein was localized to the heads of elongating spermatids. Targeted deletion of the SSTK gene in mice resulted in male sterility due to profound impairment in motility and morphology of spermatozoa. A defect in DNA condensation in SSTK null mutants occurred in elongating spermatids at a step in spermiogenesis coincident with chromatin displacement of histones by transition proteins. SSTK phosphorylated histones H1, H2A, H2AX, and H3 but not H2B or H4 or transition protein 1 in vitro. These results demonstrate that SSTK is required for proper postmeiotic chromatin remodeling and male fertility. Abnormal sperm chromatin condensation is common in sterile men, and our results may provide insight into the molecular mechanisms underlying certain human infertility disorders.

Twenty-four pituitary adenomas, both the sporadic type (n = 18) and the type arising in association with either multiple endocrine neoplasia, type 1 (MEN1; n = 2), or Carney complex (CNC, n = 4) were analyzed by comparative genomic hybridization. Twenty-one (88%) tumors displayed chromosomal alterations. The number of chromosomal aberrations in each tumor varied from 2 to greater than 10. Several recurrent chromosomal abnormalities were identified in this study. The most frequently detected losses of chromosomal material involved 1p (14 of 24, 58%), 11p (11 of 24, 46%), 17 (10 of 24, 42%), 16p (9 of 24, 38%), 4 (8 of 24, 33%), 10p (6 of 24, 25%), 12 (6 of 24, 25%), 20 (6 of 24, 25%), 22q (6 of 24, 25%), 13q (5 of 24, 21%), and 9p (4 of 24, 17%). Copy number increases were detected on 4q (7 of 24, 29%), 17 (8 of 24, 33%), 19 (7 of 24, 29%), 1p (6 of 24, 25%), 5 (6 of 24, 25%), 20 (6 of 24, 25%), 6q (5 of 24, 21%), 13q21-qter (5 of 24, 21%), and 16p (5 of 24, 21%). Chromosome 11 loss, which involved 11p in all cases, was the most significant finding and was common to tumors arising sporadically and in association with MEN1 and CNC. In addition, the majority of the tumors (18 of 24, 75% overall and 86% of all tumors with chromosomal abnormalities) showed involvement of chromosome 1. Tumors had either loss (14 of 24, 58%) or gain (6 of 24, 25%) in the 1p32-1pter region. Finally, changes on chromosome 17, either loss or gain, occurred in 71% (17) of all 24 patients. In summary, all the tumors with chromosomal rearrangements (21 of 24, 88%), whether sporadic pituitary adenomas or those associated with MEN1 or CNC, had alteration(s) of 1p32, 11p, or 17., (Copyright 2005 Wiley-Liss, Inc.)

Tal1/SCL is a basic helix-loop-helix transcription factor critical for normal hematopoiesis. To understand the mechanisms underlying transcriptional regulation by Tal1/SCL, we combined an in vitro DNA binding strategy and an in vivo chromatin immunoprecipitation analysis to search for Tal1/SCL target regions in K562 erythroleukemia cells. A 0.4-kb genomic DNA clone containing two Tal1/SCL binding E-boxes and GATA- and SATB1-binding motifs (EEGS) was identified that localized to the pericentromeric region with high homology to satellite 2 DNA. Pericentric DNA is related to heterochromatin and gene inactivation. We found that Tal1/SCL could complex with the histone H3 lysine 9 (H3K9)-specific methyltransferase Suv39H1. Binding of Tal1/SCL to EEGS chromatin correlated with hypermethylation of H3K9 and the association of heterochromatin protein HP1 to this region. In Rep4 reporter gene assays, EEGS affected repression in a manner dependent on the expression level of Tal1/SCL that was accompanied by increased H3K9 methylation in chromatin associated with EEGS and a linked promoter. A specific histone deacetylase inhibitor, trichostatin A, relieved Tal1/SCL-mediated repression by EEGS. In addition, SATB1 bound EEGS chromatin and promoted Tal1/SCL EEGS-dependent repression. We expand the list of potential interacting partners for Tal1/SCL by demonstrating direct associations of Tal1/SCL with SATB1 and with Suv39H1. These results reveal a novel mechanism of action for Tal1/SCL and implicate heterochromatin-like silencing via a cis-acting binding motif for transcriptional repression.

Coexpression of epidermal growth factor receptor (EGFR) and c-erbB-2 in 47-68% of ovarian cancer cells indicate their strong association with tumor formation. We examined the effects of simultaneous antisense- or immunosuppression of EGFR and c-erbB-2 expression on the invasive phenotype, aneuploidy, and genotype of cultured human ovarian carcinoma cells (NIH:OVCAR-8). We report here that suppression of both EGFR and c-erbB-2 results in regression of aneuploidy and genomic imbalances in NIH:OVCAR-8 cells, restores a more normal phenotype, and results in a more normal gene expression profile. Combined with cytogenetic analysis, our data demonstrate that the regression of aneuploidy is due to the selective apoptosis of double antisense transfected cells with highly abnormal karyotype.

Generally, von Hippel-Lindau (VHL) disease is caused by a germline mutation of the VHL gene (chromosome 3p), and tumorigenesis is initiated from a "second-hit" deletion. A subset of VHL patients have a germline deletion of the VHL gene, and the molecular events leading to tumorigenesis are not fully understood. To determine the molecular pathogenesis of tumor formation in this setting, we analyzed five central nervous system hemangioblastomas from three patients of a single VHL germline deletion kindred, all displaying mild clinical phenotype. Rather than loss of heterozygosity (the "second hit" in VHL germline mutation patients), all tumors from this kindred showed "second-hit" point mutations on the wild-type allele. Moreover, in two patients who each had two hemangioblastomas resected each tumor contained a unique mutation. The specific germline deletion and the overall genetic makeup of the patient did not predict these random "second-hit" point mutations. These results suggest that in patients with germline deletion of a tumor suppressor gene there is a unique genetic mechanism underlying tumorigenesis. This unique genetic mechanism correlates with and may help to understand the mild clinical phenotype seen in these patients.

Inflammatory myofibroblastic tumor (IMT) is a rare childhood neoplasm. The natural history of this disease is poorly understood. Recently chromosomal rearrangements involving the anaplastic lymphoma kinase (ALK) gene have been implicated in this tumor. We have studied a case of ALK-positive soft tissue IMT showing clinical and morphologic features of malignancy. Interphase fluorescence in situ hybridization demonstrated ALK rearrangements in both primary and metastatic lesions. Rapid amplification of cDNA ends (5'RACE) identified cysteinyl-tRNA synthetase (CARS) gene fused to ALK, which predicts an in-frame chimeric protein with the preserved functional catalytic domain of ALK at the C terminus. Amplification and sequencing of tumor DNA confirmed the breakpoint at the genomic level. Restriction analysis of DNA from primary soft tissue and recurrent lung tumors showed identical patterns, indicating the same clonal origin of both lesions. Western blot analysis with C-terminus ALK antibody showed expression of an aberrantly sized chimeric protein of approximately 130 kd in tumor tissue. This is the second case of IMT demonstrating CARS as the ALK fusion partner, which confirms the recurring involvement of ALK in IMT by a common genetic mechanism. Moreover, identical clonality of separate lesions involving different sites supports metastasis in IMT.

We have previously identified two second hit mechanisms involved in the development of multiple endocrine neoplasia type 2 (MEN 2)-associated tumors: trisomy 10 with duplication of the mutant RET allele and loss of the wild-type RET allele. However, some of the MEN 2-associated tumors investigated did not demonstrate either mechanism. Here, we studied the TT cell line derived from MEN 2-associated medullary thyroid carcinoma with a RET germline mutation in codon 634, for alternative mechanisms of tumorigenesis. Although we observed a 2:1 ratio between mutant and wild-type RET at the genomic DNA level in this cell line, fluorescence in situ hybridization analysis revealed neither trisomy 10 nor loss of the normal chromosome 10. Instead, a tandem duplication event was responsible for amplification of mutant RET. In further studies we could for the first time demonstrate that the genomic chromosome 10 abnormalities in this cell line cause an increased production of mutant RET mRNA. These findings provide evidence for a third second hit mechanism resulting in overrepresentation and overexpression of mutant RET in MEN 2-associated tumors.

Object: To determine the acute and long-term effects of a therapeutic dose of brain radiation in a primate model, the authors studied the clinical, laboratory, neuroimaging, molecular, and histological outcomes in rhesus monkeys that had received fractionated whole-brain radiation therapy (WBRT)., Methods: Twelve 3-year-old male primates (Macaca mulatta) underwent fractionated WBRT (350 cGy for 5 days/week for 2 weeks, total dose 3500 cGy). Animals were followed clinically and with laboratory studies and serial magnetic resonance (MR) imaging. They were killed when they developed medical problems or neurological symptoms, lesions appeared on MR imaging, or at study completion. Gross, histological, and molecular analyses were then performed. Nine (82%) of 11 animals that underwent long-term follow up (> 2.5 years) developed neurological symptoms and/or enhancing lesions on MR imaging, which were defined as glioblastoma multiforme (GBM), 2.9 to 8.3 years after radiation therapy. The GBMs were categorized as either unifocal (three) or multifocal (six), and were located in the supratentorial (six), infratentorial (two), or both (one) cranial regions. Histological examination revealed distant, noncontiguous tumor invasion within the white matter of all nine animals harboring GBMs. Novel interspecies comparative genomic hybridization (three animals) uniformly showed deletions in the GBMs that corresponded to chromosome 9 in humans., Conclusions: The high rate of GBM formation (82%) following a therapeutic dose of WBRT in nonhuman primates indicates that radioinduction of these neoplasms as a late complication of this therapy may occur more frequently than is currently recognized in human patients. The development of these tumors while monitoring the monkeys' conditions with clinical and serial MR imaging studies, and access to the tumor and the entire brain for histological and molecular analyses offers an opportunity to gather unique insights into the nature and development of GBMs.