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PDF file - 285K, Identification of TP53-G245C mutation in the germline, primary ACC and xenograft tumor

Purpose: Pediatric adrenocortical carcinoma (ACC) is a rare and highly aggressive malignancy. Conventional chemotherapeutic agents have shown limited utility and are largely ineffective in treating children with advanced ACC. The lack of cell lines and animal models of pediatric ACC has hampered the development of new therapies. Here we report the establishment of the first pediatric ACC xenograft model and the characterization of its sensitivity to selected chemotherapeutic agents.Experimental Design: A tumor from an 11-year-old boy with previously untreated ACC was established as a subcutaneous xenograft in immunocompromised CB17 scid−/− mice. The patient harbored a germline TP53 G245C mutation, and the primary tumor showed loss of heterozygosity with retention of the mutated TP53 allele. Histopathology, DNA fingerprinting, gene expression profiling, and biochemical analyses of the xenograft were conducted and compared with the primary tumor and normal adrenal cortex. The second endpoint was to assess the preliminary antitumor activity of selected chemotherapeutic agents.Results: The xenograft maintained the histopathologic and molecular features of the primary tumor. Screening the xenograft for drug responsiveness showed that cisplatin had a potent antitumor effect. However, etoposide, doxorubicin, and a panel of other common cancer drugs had little or no antitumor activity, with the exception of topotecan, which was found to significantly inhibit tumor growth. Consistent with these preclinical findings, topotecan as a single agent in a child with relapsed ACC resulted in disease stabilization.Conclusion: Our study established a novel TP53-associated pediatric ACC xenograft and identified topotecan as a potentially effective agent for treating children with this disease. Clin Cancer Res; 19(7); 1740–7. ©2013 AACR.

PDF file - 73K, Expression of p53 and IGF2 in normal adrenal cortex and xenograft tumor

Signaling through the type 1 insulin-like growth factor receptor (IGF-1R) occurs in many human cancers, including childhood sarcomas. As a consequence, targeting the IGF-1R has become a focus for cancer drug development. We examined the antitumor activity of CP-751,871, a human antibody that blocks IGF-1R ligand binding, alone and in combination with rapamycin against sarcoma cell lines in vitro and xenograft models in vivo. In Ewing sarcoma (EWS) cell lines, CP751,871 inhibited growth poorly (In vivo CP-751,871, rapamycin, or the combination were evaluated against EWS, osteosarcoma, and rhabdomyosarcoma xenografts. CP751871 induced significant growth inhibition [EFS(T/C) >2] in four models. Rapamycin induced significant growth inhibition [EFS(T/C) >2] in nine models. Although neither agent given alone caused tumor regressions, in combination, these agents had greater than additive activity against 5 of 13 xenografts and induced complete remissions in one model each of rhabdomyosarcoma and EWS, and in three of four osteosarcoma models. CP751,871 caused complete IGF-1R down-regulation, suppression of AKT phosphorylation, and dramatically suppressed tumor-derived vascular endothelial growth factor (VEGF) in some sarcoma xenografts. Rapamycin treatment did not markedly suppress VEGF in tumors and synergized only in tumor lines where VEGF was dramatically inhibited by CP751,871. These data suggest a model in which blockade of IGF-1R suppresses tumor-derived VEGF to a level where rapamycin can effectively suppress the response in vascular endothelial cells. [Cancer Res 2009;69(19):7662–71]

Supplementary Materials and Methods, Tables 1-3, Figures 1-2 from The Insulin-like Growth Factor-1 Receptor–Targeting Antibody, CP-751,871, Suppresses Tumor-Derived VEGF and Synergizes with Rapamycin in Models of Childhood Sarcoma

Pulmonary involvement in Crohn's disease (CD) may precede the development of intestinal inflammation, but in most cases occurs during the course of treatment, either as an extra-intestinal manifestation, because of secondary infections, or as a side effect of the therapy itself. This case highlights the differential diagnosis and work up for multiple pulmonary nodules that developed in a patient with CD who had been in remission on infliximab therapy. Even though infectious causes, such as Mycobacteria and Fungi, account for majority of these cases, the possibility of non-infectious conditions such as autoimmune disorders should also be considered.

Purpose: Pediatric adrenocortical carcinoma (ACC) is a rare and highly aggressive malignancy. Conventional chemotherapeutic agents have shown limited utility and are largely ineffective in treating children with advanced ACC. The lack of cell lines and animal models of pediatric ACC has hampered the development of new therapies. Here we report the establishment of the first pediatric ACC xenograft model and the characterization of its sensitivity to selected chemotherapeutic agents. Experimental Design: A tumor from an 11-year-old boy with previously untreated ACC was established as a subcutaneous xenograft in immunocompromised CB17 scid−/− mice. The patient harbored a germline TP53 G245C mutation, and the primary tumor showed loss of heterozygosity with retention of the mutated TP53 allele. Histopathology, DNA fingerprinting, gene expression profiling, and biochemical analyses of the xenograft were conducted and compared with the primary tumor and normal adrenal cortex. The second endpoint was to assess the preliminary antitumor activity of selected chemotherapeutic agents. Results: The xenograft maintained the histopathologic and molecular features of the primary tumor. Screening the xenograft for drug responsiveness showed that cisplatin had a potent antitumor effect. However, etoposide, doxorubicin, and a panel of other common cancer drugs had little or no antitumor activity, with the exception of topotecan, which was found to significantly inhibit tumor growth. Consistent with these preclinical findings, topotecan as a single agent in a child with relapsed ACC resulted in disease stabilization. Conclusion: Our study established a novel TP53-associated pediatric ACC xenograft and identified topotecan as a potentially effective agent for treating children with this disease. Clin Cancer Res; 19(7); 1740–7. ©2013 AACR.

Juvenile xanthogranuloma is a rare histiocytic disorder of childhood mainly affecting skin and rarely deep soft tissues and viscera. We report a 2-month-old infant who presented with respiratory distress secondary to a large pericardial effusion associated with an epicardial mass. Excisional biopsy was performed and the mass was diagnosed as juvenile xanthogranuloma. The child is well without evidence of disease 8 months following the excision. The corresponding literature on juvenile xanthogranuloma with cardiac manifestations is reviewed.

Background There are limited data regarding the differences in clinical presentation and outcome of liposarcomas between adult and pediatric patients. The role of adjuvant radiotherapy in the treatment of childhood liposarcoma is unclear.

Renal cell carcinoma represents a model for contemporary classification of solid tumors; however, unusual and unclassifiable cases exist and are not rare in children and young adults. The anaplastic lymphoma kinase (ALK) gene has recently been implicated in subsets of pulmonary, esophageal, breast, and colon cancers. These findings strengthen the importance of molecular classification of carcinomas across different organ sites, especially considering the evolving targeted anticancer therapies with ALK inhibitors. In the current study of six pediatric renal cell carcinomas, two cases exhibited structural karyotypic abnormalities involving the ALK locus on chromosomal band 2p23. Fluorescence in situ hybridization (FISH) studies were positive for an ALK rearrangement in one case, and subsequent 5' rapid amplification of cDNA ends analysis of this tumor revealed that the 3' portion of the ALK transcript encoding for the kinase domain was fused in frame to the 5' portion of vinculin (VCL, NM_003373). The new fusion gene is predicted to have an open reading frame of 4122 bp encoding for a 1374-aa oncoprotein; its expression was shown by immunoblotting with anti-VCL and anti-ALK antibodies in tumor tissue lysates. Immunohistochemistry with the same antibodies demonstrated cytoplasmic and subplasmalemmal localization of the oncoprotein determined by its N-terminal VCL portion. FISH with a custom-designed VCL-ALK dual-fusion probe set confirmed the presence of the fusion in neoplastic cells and demonstrated the potential clinical utility of this approach for detecting VCL-ALK in routinely processed tissue. The five remaining pediatric renal cell carcinomas did not show ALK rearrangement by FISH or ALK expression by immunohistochemistry. The data identify the kidney as a new organ site for ALK-associated carcinomas and VCL as a novel ALK fusion partner. The results should prompt further studies to advance the molecular classification of renal cell carcinoma and help to select patients who would benefit from appropriate targeted therapies.

The spectrum of lipomatous tumors differs in the adult and pediatric populations, with liposarcoma being rare in children. Nearly 10% of individuals with Li-Fraumeni syndrome develop sarcomas in the first 2 decades of life; however, the frequency of sarcoma types and subtypes in this syndrome is unknown. Two atypical lipomatous tumors/well-differentiated liposarcomas (ALT/WDLS) were identified in the pathology files of our institution in young children from “classical” Li-Fraumeni and Li-Fraumeni variant kindreds with a known germline TP53 mutation (Y220C) in one of the families. The patients were 5 and 6 years of age and the ALT/WDLSs were the first expression of the syndrome. The tumors had a high degree of cellular atypia and differed from sporadic ALT/WDLS by strong nuclear immunoreactivity for p53 and absent mdm2 expression. This is the first report of 2 ALT/WDLSs presenting in children before 10 years of age, both in association with Li-Fraumeni syndrome/variant. ALT/WDLS in a young child should raise the possibility of a cancer predisposition syndrome and, in this setting, the p53+/mdm2− immunophenotype might be characteristic. Recognition of this lesion and its association is important for early diagnosis and subsequent tumor surveillance in the proband and affected family members.

Signaling through the type 1 insulin-like growth factor receptor (IGF-1R) occurs in many human cancers, including childhood sarcomas. As a consequence, targeting the IGF-1R has become a focus for cancer drug development. We examined the antitumor activity of CP-751,871, a human antibody that blocks IGF-1R ligand binding, alone and in combination with rapamycin against sarcoma cell lines in vitro and xenograft models in vivo. In Ewing sarcoma (EWS) cell lines, CP751,871 inhibited growth poorly (2] in four models. Rapamycin induced significant growth inhibition [EFS(T/C) >2] in nine models. Although neither agent given alone caused tumor regressions, in combination, these agents had greater than additive activity against 5 of 13 xenografts and induced complete remissions in one model each of rhabdomyosarcoma and EWS, and in three of four osteosarcoma models. CP751,871 caused complete IGF-1R down-regulation, suppression of AKT phosphorylation, and dramatically suppressed tumor-derived vascular endothelial growth factor (VEGF) in some sarcoma xenografts. Rapamycin treatment did not markedly suppress VEGF in tumors and synergized only in tumor lines where VEGF was dramatically inhibited by CP751,871. These data suggest a model in which blockade of IGF-1R suppresses tumor-derived VEGF to a level where rapamycin can effectively suppress the response in vascular endothelial cells. [Cancer Res 2009;69(19):7662–71]

Giant cell-rich lesions of bone, including giant cell tumor of bone, giant cell reparative granuloma (GCRG), and aneurysmal bone cyst (ABC), may have overlapping clinical, radiologic, and histopathologic features. In fact, GCRG and solid ABC are currently differentiated solely based on skeletal location. Prior cytogenetic studies have reported that telomeric associations are present in the majority of giant cell tumors of bone, whereas translocations involving 16q22 and/or 17p13 are characteristic of ABCs. There is only one previously published karyotype of a GCRG, which revealed a reciprocal translocation, t(X;4)(q22;q31.3). We report 3 cases of giant cell-rich bone lesions with novel karyotypes: one lesion located in the first metacarpal, a typical location for GCRG, was histologically consistent with a giant cell tumor and showed the following karyotype [46,XX,inv(2)(p13q21),t(inv2;11)(q21;q13)]; the second lesion, also a giant cell tumor of bone, in the sacrum showed the following karyotype [46,XX,r(9)(p24q34)[cp7]/46,idem,?r(16)(p13.3q24)[cp10]/46,XX]. The third lesion, a hard palate mass, had the histopathologic features of a GCRG and a karyotype showing a reciprocal translocation, 46,XY,t(2;10)(q23;q24). These findings suggest that at least a subset of GCRGs may be neoplastic and that these lesions differ cytogenetically from classic giant cell tumors of bone or solid ABC, although the latter entity is otherwise indistinguishable from reparative granuloma. Further cytogenetic characterization of giant cell-rich bone lesions may improve the utility of karyotyping as a tool in their differential diagnosis and may shed light on the pathogenetic relationship between these lesions.

Kaposiform hemangioendothelioma is a distinctive vascular neoplasm affecting predominantly children and neonates. In neonates it needs to be differentiated from common infantile hemangioma and other vascular lesions of infancy. Kaposiform hemangioendothelioma immunoreacts with vascular endothelial growth factor receptor 3, and partial lymphothelial differentiation of this lesion has been suggested. D2-40 has been recently proposed as a selective marker of lymphatic endothelium. We performed immunohistochemical analysis with the D2-40 antibody on 24 kaposiform hemangioendotheliomas and 48 other pediatric vascular lesions including common infantile hemangioma (n=10), rapidly involuting congenital hemangioma (n=10), non-involuting congenital hemangioma (n=9), verrucous hemangioma (n=9), and pyogenic granuloma (n=10) to define whether this marker can be applied in the diagnosis of vascular lesions of infancy. In all, 23 of 24 (96%) kaposiform hemangioendotheliomas exhibited a distinct staining, while none of the other lesions immunoreacted with D2-40. D2-40 stained the neoplastic spindled cells and lymphatic channels adjacent to vascular lobules of kaposiform hemangioendothelioma. These findings support D2-40 as a new determinate marker for kaposiform hemangioendothelioma, useful in differentiating it from other vascular lesions of infancy and suggest lymphothelial differentiation of the neoplastic component of kaposiform hemangioendothelioma. Further studies are necessary to define the identity of the D2-40 antigen and to elucidate the biologic significance of its selective lymphothelial reactivity..

Reply to ‘Distinct ALK-rearranged and VCL-negative papillary renal cell carcinoma variants in two adults without sickle cell trait’

Anaplastic lymphoma kinase (ALK) break-apart fluorescent in situ hybridization (FISH) is currently used in diagnostics for the selection of non-small cell lung cancer (NSCLC) patients to receive crizotinib. We evaluated ALK status in NSCLC with a novel ALK mRNA test based on the break-apart FISH concept, which we called break-apart transcript (BAT) test. ALK5' and ALK3' transcript patterns were established with qPCR for ALK-expressing controls including fusion-negative neuroblastomas, as well as fusion-positive anaplastic large cell lymphomas and NSCLC. The BAT test was evaluated on 271 RNA samples from routinely processed paraffin NSCLC tissues. Test results were compared with ALK FISH (n=121), immunohistochemical (IHC) analysis (n=86), and automated quantitative analysis (AQUA, n=83). On the basis of the nonoverlapping ALK BAT patterns in ALK-expressing controls (P0.0001), 8/174 adenocarcinomas (4.6%) among 259 informative NSCLC were predicted as fusion positive. Overall concordance for paired method results was high (94.1% to 98.8%) but mainly concerned negative prediction because of the limited availability of positive-matched cases. Tumors with 100% cytoplasmic IHC staining of any intensity (n=3) were positive for AQUA, FISH, and BAT test; tumors with lower IHC positivity and different staining patterns were AQUA-negative. Upon multiple reevaluations, ALK gene status was considered as originally misinterpreted by FISH in 3/121 cases (2.5%). Tumors with4 ALK gene copies were associated with longer overall survival upon first-line chemotherapy. In conclusion, application of the ALK BAT test on routinely processed NSCLC tissues yields the same fusion partner independent information as ALK break-apart FISH but is more robust and cost-effective. The BAT concept may be considered for the development of further drug-predictive translocation tests.

Neuroendocrine tumors of the lung consist of a spectrum of neoplasms, including typical carcinoids, atypical carcinoids, large-cell neuroendocrine carcinomas (LCNEC), and small-cell lung carcinomas (SCLC). We previously reported frequent inactivation of the gene responsible for multiple endocrine neoplasia type 1 (MEN1) in both typical and atypical carcinoid tumors. In the present study, we extend the analysis of human NE lung tumors to include 9 primary SCLCs, 36 SCLC cell lines, and 13 primary LCNECs for MEN1 gene inactivation. In SCLC, loss of heterozygosity (LOH) at the MEN1 gene on chromosome band 11q13 was detected in one primary tumor and two cell lines. The coding sequence and splice junctions of the MEN1 gene were screened for mutations in all 44 tumors and cell lines, and no mutations were detected. Northern blot analysis of 13 SCLC cell lines showed the MEN1 transcript to be present and of normal size. In LCNECs, a somatic frameshift in the MEN1 gene (1226delC) was found in one of 13 tumors, representing the first mutation observed outside the spectrum of neoplasms associated with MEN1. Interestingly, neither a deletion nor a mutation was detected in the other allele, and wild-type mRNA sequence was expressed in the tumor, suggesting that the MEN1 gene was not inactivated by a conventional two-hit mechanism. The data support the hypothesis that SCLC and lung carcinoids develop via distinct molecular pathways; however, further investigation is necessary to determine the significance of the MEN1 gene mutation observed in a single case of LCNEC. Genes Chromosomes Cancer 28:58–65, 2000. Published 2000 Wiley-Liss, Inc.

Dideoxyfingerprinting was used to screen for germline and somatic MEN1 mutations. This method, applied to a panel of germline DNA from 15 probands with multiple endocrine neoplasia type 1 (MEN-1), allowed confident discovery of the MEN1 gene. Germline MEN1 mutation has been found in 47 out of 50 probands with familial MEN-1, in 7 out of 8 cases with sporadic MEN-1, and in 1 out of 3 cases with atypical sporadic MEN-1. Germline MEN1 mutation was not found in any of five probands with familial hyperparathyroidism. Somatic MEN1 mutations were found in 7 out of 33 parathyroid tumours not associated with MEN-1. Allowing for repeating mutations, a total of 47 different germline or somatic MEN1 mutations have been identified. Most predict inactivation of the encoded 'menin' protein. supporting expectations that MEN1 is a tumour suppressor gene. The 16 observed missense mutations were distributed across the gene, suggesting that many domains are important to its as yet unknown functions.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder that manifests as varying combinations of tumors of endocrine and other tissues (parathyroids, pancreatic islets, duodenal endocrine cells, the anterior pituitary and others). The MEN1 gene is on chromosome 11q13; it was recently identified by positional cloning. We previously reported 32 different germline mutations in 47 of the 50 familial MEN1 probands studied at the NIH. Eight different germline MEN1 mutations were encountered repeatedly in two or more apparently unrelated families. We analyzed the haplotypes of families with recurrent MEN1 mutations with seven polymorphic markers in the 11q13 region surrounding the MEN1 gene (from D11S1883 to D11S4908). Disease haplotypes were inferred from germline DNA and also from tumors with 11q13 loss of heterozygosity. Two different disease haplotype cores were shared by apparently unrelated families for two mutations in exon 2 (five families with 416delC and six families with 512delC). These two repeat mutations were associated with the two founder effects that we reported in a prior haplotype analysis. The disease haplotypes for each of the other six repeat mutations (seen twice each) were discordant, suggesting independent origins of these recurrent mutations. Most of the MEN1 germline mutations including all of those recurring independently occur in regions of CpG/CpNpG, short DNA repeats or single nucleotide repeat motifs. In conclusion, recurring germline mutations account for about half of the mutations in North American MEN1 families. They result from either founder effects or independent occurrence of one mutation more than one time. Hum Mutat 12:75–82, 1998. Published 1998 Wiley-Liss, Inc.1

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder in which affected individuals develop tumors primarily in the parathyroids, anterior pituitary, endocrine pancreas, and duodenum. The locus for MEN1 is tightly linked to the marker PYGM on chromosome 11q13, and linkage analysis has previously placed the MEN1 gene within a 2-Mb interval flanked by markers D11S1883 and D11S449. Loss of heterozygosity (LOH) studies in MEN1 and sporadic tumors have helped narrow the location of the gene to a 600-kb interval between PYGM and D11S449. Eighteen new polymerase chain reaction (PCR)-based polymorphic markers were generated for the MEN1 region, with ten mapping to the PYGM-D11S449 interval. These new markers, along with 14 previously known polymorphic markers, were precisely mapped on a 2.8-Mb (D11S480-D11S913) high-density clone contig-based, physical map generated for the MEN1 region.

Loss of heterozygosity (LOH) for polymorphic markers flanking the multiple endocrine neoplasia type 1 (MEN-1) gene in parathyroid and pancreatic islet tumors from subjects with familial MEN-1 (FMEN-1) has been well documented and has led to the hypothesis that the MEN-1 gene functions as a tumor suppressor. To assess the role of the MEN-1 gene in the pathogenesis of tumors less commonly associated with MEN-1, we employed a large number of highly informative polymorphic markers closely linked to the MEN-1 gene to study a series of 13 such tumors from subjects with FMEN-1 for LOH at 11q13. We were able to identify LOH for 1 or more 11q13 markers in 2 of 3 pituitary tumors, 3 lung carcinoids, and 1 of 2 lipomas. In every case studied, the allele lost represented the normal allele inherited from the unaffected parent. No LOH was detected in 3 skin angiofibromas, an esophageal leiomyoma, or a renal angiomyolipoma despite the presence of at least 2 informative markers for each tumor. Our results suggest that, like that for parathyroid and pancreatic islet tumors, the pathogenesis of pituitary tumors, lung carcinoids, and lipomas occurring in subjects with FMEN-1 probably involves loss of the normal tumor suppressor function of the MEN-1 gene. Our inability to detect 11q13 LOH in skin angiofibromas, leiomyoma, and angiomyolipoma from subjects with FMEN-1 is consistent with the possibility that these neoplasms arose independently by a mechanism unrelated to the MEN-1 gene, but a role for the MEN-1 gene in the pathogenesis of these tumors cannot be definitively excluded until the gene itself is identified and evaluated for small intragenic deletions or point mutations in such tumors.

We describe the case of a 9-year-old boy with encephalitis associated with histiocytic necrotizing lymphadenitis (HNL), also known as Kikuchi-Fujimoto disease. The child presented with unilateral cervical lymphadenopathy and fever that evolved to encephalitis in 3 weeks. Brain MRI showed bilateral temporal lobe hyperintense signal on T2 and FLAIR, hyperintense FLAIR signal in the periaqueductal gray matter, medial walls of the third ventricle, and mammillary bodies, multiple diffusion restriction foci in a central perivascular distribution and central perivascular enhancement. The perivascular distribution and nodularity of the diffusion restriction seen in this case has not been previously reported in HNL encephalitis.

Pediatric adrenocortical tumors (ACTs) are rare and are frequently associated with tumor predisposition syndromes. Somatic GNAS1 mutations are associated with adrenocortical hyperplasia, but have not typically been reported in ACTs.We report on genetic and histopathological findings in a 3-month-old infant presenting with a unilateral cortisol-producing ACT with malignant features.We performed a detailed clinical evaluation of the patient along with molecular genetic testing of genes associated with ACTs in both tumor tissue and peripheral lymphocytes. We also performed a histopathological analysis of the tumor tissue.The patient was found to have a p.R201C-activating mutation in exon 8 of the GNAS1 gene in adrenocortical tumor tissue but not peripheral lymphocytes. This mutation is the characteristic genetic change in McCune-Albright syndrome. In contrast to previously reported GNAS1-positive tumors characterized by bimodal diffuse and nodular adrenocortical hypertrophy, our patient had a single adrenocortical mass that showed features of malignancy, including areas of necrosis, microcystic degeneration, and venous and capsular microinvasion-changes that have been seen previously in Beckwith-Wiedemann syndrome. However, our patient did not have clinical features of Beckwith-Wiedemann syndrome. Further analysis revealed abnormal allele-specific hypomethylation of the KCNQ1OT1 gene in the tumor sample but not peripheral lymphocytes.This is a novel case of an activating GNAS1 mutation associated with an epigenetic alteration that may be related to adrenocortical tumorigenesis. Our findings may have implications in the molecular pathogenesis of pediatric ACTs.

Juvenile xanthogranuloma is a rare histiocytic disorder of childhood mainly affecting skin and rarely deep soft tissues and viscera. We report a 2-month-old infant who presented with respiratory distress secondary to a large pericardial effusion associated with an epicardial mass. Excisional biopsy was performed and the mass was diagnosed as juvenile xanthogranuloma. The child is well without evidence of disease 8 months following the excision. The corresponding literature on juvenile xanthogranuloma with cardiac manifestations is reviewed.

Cellular morphology of small cell osteosarcoma, an aggressive variant of osteosarcoma, is similar to Ewing sarcoma, but its molecular pathogenesis is largely unknown. We report the case of a 12-year-old girl with multifocal small cell osteosarcoma positive for the Ewing sarcoma breakpoint region 1 (EWSR1) gene rearrangement by interphase fluorescent in situ hybridization and negative for EWSR1-FLI1, EWSR1-ERG, and EWSR1-WT1 fusion transcripts by reverse transcriptase PCR. Rapid amplification of cDNA ends revealed exon 6 of the cAMP-responsive element binding protein 3-like 1 gene (CREB3L1, also known as “OASIS,” NM_52854.2) fused in-frame to the EWSR1 exon 11, consistent with the EWSR1-CREB3L1 fusion transcript expressed in tumor tissue. The corresponding chimeric gene was confirmed by amplification and subsequent sequencing of the genomic breakpoint between introns 11 and 5 of EWSR1 and CREB3L1, respectively. An ∼70 kDa product in the tumor tissue lysate reacted with the CREB3L1 carboxyterminal antibody, consistent with a 656-amino acid predicted chimeric protein. Immunohistochemistry with the same antibody showed signal translocation from the physiologic perinuclear compartment observed in glia and unrelated osteoblasts to nuclei of tumor cells, consistent with the likely function of EWSR1-CREB3L1 as a transcriptional regulator predicted by its structure. This is the first report of a fusion transcript in osteogenic sarcoma; it demonstrates a relation between molecular mechanisms of small cell osteogenic and Ewing sarcomas. The 3′-end partner and the inferred structure of EWSR1-CREB3L1, however, are different from those of Ewing sarcoma, suggesting different targets of the new oncogene. © 2011 Wiley Periodicals, Inc.

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.

Multiple endocrine neoplasia type 1 (MENI) is a promising model to understand endocrine and other tumors. Its most common endocrine expressions are tumors of parathyroids, entero-pancreatic neuro-endocrine tissue, and anterior pituitary. Recently, collagenomas and multiple angiofibromas of the dermis also have been recognized as very common. MEN1 can be characterized from different perspectives: (a) as a hormone (parathyroid hormone, gastrin, prolactin, etc.) excess syndrome with excellent therapeutic options; (b) as a syndrome with sometimes lethal outcomes from malignancy of entero-pancreatic neuro-endocrine or foregut carcinoid tissues; or (c) as a disorder than can give insight about cell regulation in the endocrine, the dermal, and perhaps other tissue systems. The MEN1 gene was identified recently by positional cloning, a comprehensive strategy of narrowing the candidate interval and evaluating all or most genes in that interval. This discovery has opened new approaches to basic and clinical issues. Germline MEN1 mutations have been identified in most MEN1 families. Germline MENI mutations were generally not found in families with isolated hyperparathyroidism or with isolated pituitary tumor. Thus, studies with the MENI gene helped establish that mutation of other gene(s) is likely causative of these two MEN1 phenocopies. MEN1 proved to be the gene most frequent L4 mutated in common-variety, nonhereditary parathyroid tumor, gastrinoma, insulinoma, or bronchial carcinoid. For example, in common-variety parathyroid tumors, mutation of several other genes (such as cyclin D1 and P53) has been found, but much less frequently than MEN1 mutation. The majority of germline and somatic MEN1 mutations predicted truncation of the encoded protein (menin). Such inactivating mutations strongly supported prior predictions that MEN1 is a tumor suppressor gene insofar as stepwise mutational inactivation of both copies can release a cell from normal growth suppression. Menin is principally a nuclear protein; menin interacts with junD. Future studies, such as discovery of menin's metabolic pathway, could lead to new opportunities in cell biology and in tumor therapy.

We analyzed constitutional and tumor DNA from 27 MEN1 kindreds not known to be related to each other. Disease allele haplotypes were constructed for each pedigree based on shared alleles from two or more affected members and from determination of allelic loss patterns in their tumors. Analysis of disease allele haplotypes showed unexpected linkage disequilibrium at marker PYGM. Further haplotype analysis indicated this could be explained by the presence of two founder chromosomes, one in four families, the other in three. A shared disease haplotype was not observed among two MEN1 kindreds with the prolactinoma phenotype of MEN1.

Lung carcinoids occur sporadically and rarely in association with multiple endocrine neoplasia type 1 (MEN1). There are no well defined genetic abnormalities known to occur in these tumors. We studied 11 sporadic lung carcinoids for loss of heterozygosity (LOH) at the locus of the MEN1 gene on chromosome 11q13, and for mutations of the MEN1 gene using dideoxy fingerprinting. Additionally, a lung carcinoid from a MEN1 patient was studied. In four of 11 (36%) sporadic tumors, both copies of the MEN1 gene were inactivated. All four tumors showed the presence of a MEN1 gene mutation and loss of the other allele. Observed mutations included a 1 bp insertion, a 1 bp deletion, a 13 bp deletion and a single nucleotide substitution affecting a donor splice site. Each mutation predicts truncation or potentially complete loss of menin. The remaining seven tumors showed neither the presence of a MEN1 gene mutation nor 11q13 LOH. The tumor from the MEN1 patient showed LOH at chromosome 11q13 and a complex germline MEN1 gene mutation. The data implicate the MEN1 gene in the pathogenesis of sporadic lung carcinoids, representing the first defined genetic alteration in these tumors.

BACKGROUND & AIMS: Both gastrin and genetic factors were suggested to underlie the pathogenesis of multiple gastric enterochromaffin-like (ECL) cell carcinoids. To assess the role of genetic alterations in carcinoid tumorigenesis, loss of heterozygosity (LOH) at the locus of the multiple endocrine neoplasia type 1 (MEN-1) gene was studied in gastric carcinoids of patients with MEN-1 and chronic atrophic type A gastritis (A-CAG), as well as in sporadically arising intestinal carcinoids. METHODS: DNA extracted from archival tissue sections of 35 carcinoid tumors was assessed for LOH with eight polymorphic markers on chromosome 11q13. A combined tumor and family study was performed in 1 patient with MEN-1-Zollinger-Ellison syndrome (ZES). RESULTS: LOH at 11q13 loci was detected in 15 of 20 (75%) MEN-1-ZES carcinoids, and each ECL-cell carcinoid with LOH showed deletion of the wild-type allele. Only 1 of 6 A-CAG carcinoids displayed LOH at the MEN-1 gene locus, and none of the 9 intestinal and rectal carcinoids showed 11q13 LOH. CONCLUSIONS: Gastric ECL-cell carcinoid is an independent tumor type of MEN-1 that shares a common developmental mechanism (via inactivation of the MEN-1 gene) with enteropancreatic and parathyroid MEN-1 tumors. Further analysis of sporadic and A-CAG carcinoids is needed to elucidate genetic factors involved in their tumorigenesis. (Gastroenterology 1997 Sep;113(3):773-81)

Primary hyperparathyroidism is a common disorder with an annual incidence of approximately 0.5 in 1,000 (ref. 1). In more than 95% of cases, the disease is caused by sporadic parathyroid adenoma or sporadic hyperplasia. Some cases are caused by inherited syndromes, such as multiple endocrine neoplasia type 1 (MEN1; ref. 2). In most cases, the molecular basis of parathyroid neoplasia is unknown. Parathyroid adenomas are usually monoclonal, suggesting that one important step in tumour development is a mutation in a progenitor cell. Approximately 30% of sporadic parathyroid tumours show loss of heterozygosity (LOH) for polymorphic markers on 11q13, the site of the MEN1 tumour suppressor gene. This raises the question of whether such sporadic parathyroid tumours are caused by sequential inactivation of both alleles of the MEN1 gene. We recently cloned the MEN1 gene and identified MEN1 germline mutations in fourteen of fifteen kindreds with familial MEN1 (ref. 10). We have studied parathyroid tumours not associated with MEN1 to determine whether somatic mutations in the MEN1 gene are present. Among 33 tumours we found somatic MEN1 gene mutation in 7, while the corresponding MEN1 germline sequence was normal in each patient. All tumours with MEN1 gene mutation showed LOH on 11q13, making the tumour cells hemi- or homozygous for the mutant allele. Thus, somatic MEN1 gene mutation for the mutant allele. Thus, somatic MEN1 gene mutation contributes to tumorigenesis in a substantial number of parathyroid tumours not associated with the MEN1 syndrome.

Small cell osteosarcoma (SCOS) is an aggressive variant of osteogenic sarcoma that has some morphologic similarities to Ewing sarcoma. Little is known about the molecular biology of SCOS and no specific molecular abnormality has been reported in this tumor subtype to date. We performed a molecular biology study of a SCOS that was positive for the Ewing sarcoma breakpoint region 1 (EWSR1) gene rearrangement by interphase fluorescent in situ hybridization and negative for EWSR1-FLI1, EWSR1-ERG and EWSR1-WT1 fusion transcripts by reverse transcriptase polymerase chain reaction. Rapid amplification of cDNA ends with the EWSR1 gene-specific forward primers revealed the exon 6 of the cAMP responsive element binding protein 3-like 1 gene (CREB3L1) fused in-frame to the EWSR1 exon 11, consistent with the EWSR1-CREB2L1 fusion transcript expressed in tumor tissue. The corresponding chimeric gene in the tumor DNA was confirmed by amplification and subsequent sequencing of the genetic breakpoint located in introns 11 and 5 of the EWSR1 and CREB3L1, respectively. No reciprocal fusion transcript or gene could be amplified in the tumor. An approximately 68 kDa product detected in tumor tissue lysate by immunoblotting with the CREB3L1 carboxyterminal antibody was consistent with a 656 aa predicted chimeric protein. This is the first report of a fusion transcript in osteosarcoma that demonstrates the relation of molecular mechanism of SCOS subtype to that of Ewing sarcoma. The 3'end fusion partner and the inferred structure of the new EWSR1-CREB3L1 chimera, however, are different from those underlying Ewing sarcoma, predicting different functions of the novel fusion product. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 253.

Desmoid tumor (musculoaponeurotic fibromatosis) is a rare locally aggressive soft tissue tumor. A subset of cases is associated with familial adenomatous polyposis (FAP), governed by the APC (adenomatous polyposis coli) gene. Typically, an intra-abdominal desmoid is a painless lump or mass with a slow progression. We report a case of 14 year old male with Asperger syndrome who presented to our hospital with acute onset of progressively worsening abdominal pain. On the physical examination a large abdominal mass was palpated in the left upper quadrant. An abdominal CT (computerized tomography) scan confirmed the mass. The tumor was resected with free tumor cell margin. The histological diagnosis was desmoid fibromatosis. The nuclear β-catenin immunohistochemistry was positive. The tumor had encroached the splenic vessels and infarcted spleen. This splenic infarction was most likely the cause of the sudden abdominal pain. The patient is alive with no postoperative symptoms or evidence of the disease 6 months after the surgery. Family history and genetic analysis of the APC gene were negative for FAP.