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1.
Oncogene ; 35(14): 1797-810, 2016 Apr 07.
Article in English | MEDLINE | ID: mdl-26119939

ABSTRACT

Primary effusion lymphoma (PEL) is an aggressive type of non-Hodgkin lymphoma localized predominantly in body cavities. Kaposi's sarcoma-associated herpes virus (KSHV) is the causative agent of PEL. PEL is an incurable malignancy and has extremely poor prognosis when treated with conventional chemotherapy. Immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide are Food and Drug Administration-approved drugs for the treatment of various ailments. IMiDs display pronounced antiproliferative effect against majority of PEL cell lines within their clinically achievable concentrations, by arresting cells at G0/G1 phase of cell cycle and without any induction of KSHV lytic cycle reactivation. Although microarray examination of PEL cells treated with lenalidomide revealed activation of interferon (IFN) signaling, blocking the IFN pathway did not block the anti-PEL activity of IMiDs. The anti-PEL effects of IMiDs involved cereblon-dependent suppression of IRF4 and rapid degradation of IKZF1, but not IKZF3. Small hairpin RNA-mediated knockdown of MYC enhanced the cytotoxicity of IMiDs. Bromodomain (BRD) and extra-terminal domain (BET) proteins are epigenetic readers, which perform a vital role in chromatin remodeling and transcriptional regulation. BRD4, a widely expressed transcriptional coactivator, belongs to the BET family of proteins, which has been shown to co-occupy the super enhancers associated with MYC. Specific BRD4 inhibitors were developed, which suppress MYC transcriptionally. Lenalidomide displayed synergistic cytotoxicity with several structurally distinct BRD4 inhibitors (JQ-1, IBET151 and PFI-1). Furthermore, combined administration of lenalidomide and BRD4 inhibitor JQ-1 significantly increased the survival of PEL bearing NOD-SCID mice in an orthotopic xenograft model as compared with either agent alone. These results provide compelling evidence for clinical testing of IMiDs alone and in combination with BRD4 inhibitors for PEL.


Subject(s)
Ikaros Transcription Factor/genetics , Interferon Regulatory Factors/genetics , Lymphoma, Primary Effusion/drug therapy , Nuclear Proteins/genetics , Peptide Hydrolases/genetics , Proto-Oncogene Proteins c-myc/genetics , Transcription Factors/genetics , Adaptor Proteins, Signal Transducing , Animals , Apoptosis/drug effects , Azepines/administration & dosage , Cell Cycle Proteins , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Synergism , Gene Knockdown Techniques , Humans , Ikaros Transcription Factor/biosynthesis , Immunologic Factors/administration & dosage , Interferon Regulatory Factors/biosynthesis , Lenalidomide , Lymphoma, Primary Effusion/genetics , Lymphoma, Primary Effusion/pathology , Mice , Proto-Oncogene Proteins c-myc/antagonists & inhibitors , Thalidomide/administration & dosage , Thalidomide/analogs & derivatives , Triazoles/administration & dosage , Ubiquitin-Protein Ligases , Xenograft Model Antitumor Assays
2.
Prostate Cancer Prostatic Dis ; 17(1): 64-9, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24145624

ABSTRACT

BACKGROUND: Due to their varied outcomes, men with biochemical recurrence (BCR) following radical prostatectomy (RP) present a management dilemma. Here, we evaluate Decipher, a genomic classifier (GC), for its ability to predict metastasis following BCR. METHODS: The study population included 85 clinically high-risk patients who developed BCR after RP. Time-dependent receiver operating characteristic (ROC) curves, weighted Cox proportional hazard models and decision curves were used to compare GC scores to Gleason score (GS), PSA doubling time (PSAdT), time to BCR (ttBCR), the Stephenson nomogram and CAPRA-S for predicting metastatic disease progression. All tests were two-sided with a type I error probability of 5%. RESULTS: GC scores stratified men with BCR into those who would or would not develop metastasis (8% of patients with low versus 40% with high scores developed metastasis, P<0.001). The area under the curve for predicting metastasis after BCR was 0.82 (95% CI, 0.76-0.86) for GC, compared to GS 0.64 (0.58-0.70), PSAdT 0.69 (0.61-0.77) and ttBCR 0.52 (0.46-0.59). Decision curve analysis showed that GC scores had a higher overall net benefit compared to models based solely on clinicopathologic features. In multivariable modeling with clinicopathologic variables, GC score was the only significant predictor of metastasis (P=0.003). CONCLUSIONS: When compared to clinicopathologic variables, GC better predicted metastatic progression among this cohort of men with BCR following RP. While confirmatory studies are needed, these results suggest that use of GC may allow for better selection of men requiring earlier initiation of treatment at the time of BCR.


Subject(s)
Prostatic Neoplasms/diagnosis , Prostatic Neoplasms/genetics , Aged , Disease Progression , Genomics , Humans , Male , Middle Aged , Neoplasm Grading , Neoplasm Metastasis , Prognosis , Prostate-Specific Antigen/metabolism , Prostatectomy , Prostatic Neoplasms/surgery , ROC Curve , Recurrence , Reproducibility of Results
3.
Cell Death Differ ; 19(3): 501-10, 2012 Mar.
Article in English | MEDLINE | ID: mdl-21941369

ABSTRACT

Suppression of anoikis after detachment of cancer cells from the extracellular matrix is a key step during metastasis. Here we show that, after detachment, mouse embryonic fibroblasts (MEFs) transformed by K-Ras(V12) or ETV6-NTRK3 (EN) activate a transcriptional response overrepresented by genes related to bioenergetic stress and the AMP-activated protein kinase (AMPK) energy-sensing pathway. Accordingly, AMPK is activated in both transformed and non-transformed cells after detachment, and AMPK deficiency restores anoikis to transformed MEFs. However, AMPK activation represses the mTOR complex-1 (mTORC1) pathway only in transformed cells, suggesting a key role for AMPK-mediated mTORC1 inhibition in the suppression of anoikis. Consistent with this, AMPK-/- MEFs transformed by EN or K-Ras show sustained mTORC1 activation after detachment and fail to suppress anoikis. Transformed TSC1-/- MEFs, which are incapable of suppressing mTORC1, also undergo anoikis after detachment, which is reversed by mTORC1 inhibitors. Furthermore, transformed AMPK-/- and TSC1-/- MEFs both have higher total protein synthesis rates than wild-type controls, and translation inhibition using cycloheximide partially restores their anoikis resistance, indicating a mechanism whereby mTORC1 inhibition suppresses anoikis. Finally, breast carcinoma cell lines show similar detachment-induced AMPK/mTORC1 activation and restoration of anoikis by AMPK inhibition. Our data implicate AMPK-mediated mTORC1 inhibition and suppression of protein synthesis as a means for bioenergetic conservation during detachment, thus promoting anoikis resistance.


Subject(s)
AMP-Activated Protein Kinases/metabolism , Anoikis/physiology , Embryo, Mammalian/metabolism , Fibroblasts/metabolism , Protein Biosynthesis , TOR Serine-Threonine Kinases/metabolism , AMP-Activated Protein Kinases/genetics , Animals , Cell Line, Transformed , Cell Line, Tumor , Embryo, Mammalian/cytology , Fibroblasts/cytology , Mechanistic Target of Rapamycin Complex 1 , Mice , Mice, Knockout , Multiprotein Complexes , NIH 3T3 Cells , Proteins/genetics , Proteins/metabolism , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/genetics
4.
Oncogene ; 29(19): 2772-83, 2010 May 13.
Article in English | MEDLINE | ID: mdl-20190807

ABSTRACT

Although retinoic acid (RA) is a potent agent that coordinates inhibition of proliferation with differentiation of many cell types, RA-mediated signaling pathways in osteosarcoma cell differentiation are uncharacterized. In this study, we show that in human U2OS osteosarcoma cells, decreased phosphorylation of RA receptor alpha (RARalpha) by RA treatment or overexpressing a phosphorylation-defective mutant RARalphaS77A results in the inhibition of proliferation and induction of differentiation, and that U2OS cells transduced with RARalphaS77A suppresses tumor formation in nude mice. Moreover, using different human primary osteosarcoma cells and human mesenchymal stem cells for gene expression analysis, we found that either RA or RARalphaS77A induces many of the same differentiation response pathways and signaling molecules involved in U2OS cell differentiation. In addition, overexpression of the fibroblast growth factor 8f (FGF8f), one of the downstream targets induced by both RA and RARalphaS77A in U2OS cells, inhibits proliferation and induces expression of osteoblastic differentiation regulators. Hence, these data strongly suggest that RA-suppressed phosphorylation of RARalpha induces FGF8f expression to mediate differentiation response pathway in U2OS osteosarcoma cells.


Subject(s)
Cell Differentiation/drug effects , Osteosarcoma/pathology , Receptors, Retinoic Acid/metabolism , Signal Transduction/drug effects , Tretinoin/pharmacology , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin-Dependent Kinases/metabolism , Fibroblast Growth Factor 8/genetics , G1 Phase/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Humans , Mice , Mice, Inbred BALB C , Mutation , Osteoblasts/drug effects , Osteoblasts/pathology , Osteosarcoma/genetics , Osteosarcoma/metabolism , Phosphorylation/drug effects , Receptors, Retinoic Acid/genetics , Retinoic Acid Receptor alpha , Cyclin-Dependent Kinase-Activating Kinase
5.
Eur J Clin Invest ; 39(5): 384-94, 2009 May.
Article in English | MEDLINE | ID: mdl-19309323

ABSTRACT

BACKGROUND: Development of betulinic acid derivatives for clinical use has been hampered by adverse pharmacological and physico-chemical characteristics of this class of compounds. We here present a novel semi-synthetic betulinic acid-derived drug candidate well suited for further clinical development. MATERIALS AND METHODS: In vitro activity and mode of action of NVX-207 were determined using normal as well as cancer cell lines. Gene expression profiling was performed with Affymetrix U133 microarrays. NVX-207 binding partners were identified using a heterobifunctional chemical crosslinker system. Potential binding proteins were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis. Clinical studies were conducted in canine cancer patients suffering from spontaneously arising pre-treated tumours. RESULTS: NVX-207 showed anti-tumour activity (mean IC(50) = 3.5 microM) against various human and canine cell lines. NVX-207-induced apoptosis was associated with activation of the intrinsic apoptotic pathway via cleavage of caspases -9, -3, -7 and of poly (ADP-ribose) polymerase (PARP). Global gene expression profiling demonstrated regulation of genes associated with lipid metabolism, most notably an upregulation of genes coding for insulin-induced gene 1 (Insig-1), low-density lipoprotein receptor (LDL-R) and of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA). NVX-207 bound to apolipoprotein A-I, a major regulator of lipid metabolism and cholesterol transport. A phase I/II study in dogs suffering from naturally occurring cancer receiving local treatment of NVX-207 (10 mg mL(-1)) showed excellent clinical responses including a complete remission in so far 5/5 treated animals. CONCLUSIONS: NVX-207 is well tolerated and has significant anti-cancer activity in vitro and in vivo in dogs with treatment-resistant malignancies.


Subject(s)
Anticarcinogenic Agents/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Propanolamines/pharmacology , Triterpenes/pharmacology , Animals , Cell Line, Tumor/drug effects , Dogs , Female , Humans , Mice , Models, Animal , Pentacyclic Triterpenes , Betulinic Acid
6.
Oncogene ; 27(23): 3282-91, 2008 May 22.
Article in English | MEDLINE | ID: mdl-18084326

ABSTRACT

Ewing family tumors (EFT), classically Ewing's sarcoma and peripheral primitive neuroectodermal tumor, share a common class of tumor-specific fusion genes thought to be key mediators of tumor biology. Here we demonstrate that the most common Ewing's fusion, EWS/FLI1, produces transcriptional upregulation of GLI1 and its direct transcriptional target PATCHED1 in a model transformation system. This deregulation of GLI1 is common to other EWS/ets chimera and depends on the functional transcriptional regulatory domains. Inhibition of GLI1 via RNAi or via overexpression of endogenous inhibitors results in a reduction of EWS/FLI1 transformation activity. Activation of GLI1 appears to occur in a Hedgehog-independent fashion as blockade of Hedgehog signaling has only a modest effect on EFT cells. We present evidence that EWS/FLI1 upregulation of cMYC may play a role in the upregulation of GLI1 in EWS/FLI1-transformed NIH3T3 cells. Finally, we demonstrate that observations made in a model transformation system translate to an Ewing cellular background. EFT cell lines express GLI1 and PATCHED and this expression is EWS/FLI1 dependent. Inhibition of GLI1 expression via RNAi results in reduced anchorage-independent growth in an EFT cell line. GLI1 appears to be a transcriptionally deregulated target of EWS/FLI1 that mediates a portion of its tumorigenic phenotype.


Subject(s)
Kruppel-Like Transcription Factors/genetics , Oncogene Proteins, Fusion/physiology , Proto-Oncogene Protein c-fli-1/physiology , Animals , Bone Neoplasms/genetics , Cell Line, Tumor , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Gene Expression Regulation, Neoplastic , Genes, myc/physiology , Hedgehog Proteins/physiology , Kruppel-Like Transcription Factors/physiology , Mice , NIH 3T3 Cells , Phenotype , RNA-Binding Protein EWS , Sarcoma, Ewing/genetics , Transcription Factors/physiology , Transfection , Zinc Finger Protein GLI1
7.
Cancer Res ; 61(16): 6185-93, 2001 Aug 15.
Article in English | MEDLINE | ID: mdl-11507071

ABSTRACT

Neuroblastomas can acquire a sustained high-level drug resistance during chemotherapy and especially myeloablative chemoradiotherapy. p53 mutations are rare in primary neuroblastomas, but a loss of p53 function could play a role in multidrug resistance. We determined p53 function by measuring induction of p21 and/or MDM2 proteins in response to melphalan (L-PAM) in seven L-PAM-sensitive and 11 L-PAM-resistant neuroblastoma cell lines. p53 was functional in seven/seven drug-sensitive but in only 4/11 drug-resistant cell lines (P = 0.01). In four of the seven cell lines lacking p53 function, mutations of p53 were detected by the microarray GeneChip p53 Assay and automated sequencing, whereas six cell lines with functional p53 had no evidence of p53 mutations. All of the cell lines with wild-type (wt) p53 showed a strong transactivation of the p53-HBS/CAT reporter gene, whereas the four cell lines with mutant p53 failed to transactivate p53 HBS/CAT. Overexpression of MDM2 protein (relative to p53 functional lines) was seen in two p53-nonfunctional cell lines with wt p53; one showed genomic amplification of MDM2. Nonfunctional and mutated p53 was detected in a resistant cell line, whereas a sensitive cell line derived from the same patient before treatment had functional and wt p53. Loss of p53 function was selectively achieved by transduction of human papillomavirus 16 E6 (which degrades p53) into two drug-sensitive neuroblastoma cell lines with intact p53, causing high-level drug resistance to L-PAM, carboplatin, and etoposide. These data obtained with neuroblastoma cell lines suggest that the high-level drug resistance observed in some recurrent neuroblastomas is attributable to p53 mutations and/or a loss of p53 function acquired during chemotherapy. If confirmed in patient tumor samples, these data support development of p53-independent therapies for consolidation and/or salvage of recurrent neuroblastomas.


Subject(s)
Drug Resistance, Multiple/physiology , Neuroblastoma/drug therapy , Nuclear Proteins , Repressor Proteins , Tumor Suppressor Protein p53/physiology , Antineoplastic Agents/pharmacology , Carboplatin/pharmacology , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/biosynthesis , Cyclins/genetics , Etoposide/pharmacology , Gene Amplification , Humans , Melphalan/pharmacology , Mutation , Neuroblastoma/genetics , Neuroblastoma/metabolism , Oncogene Proteins, Viral/genetics , Proto-Oncogene Proteins/biosynthesis , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-mdm2 , Transcriptional Activation , Transduction, Genetic , Tumor Cells, Cultured , Tumor Suppressor Protein p53/biosynthesis , Tumor Suppressor Protein p53/genetics
9.
Cancer J ; 7(1): 2-15, 2001.
Article in English | MEDLINE | ID: mdl-11269644

ABSTRACT

Childhood cancer, like all cancer, is at heart a genetic disease. Consequently, fundamental understanding of the oncogenic process is likely to be beneficially addressed by genetic methodology. Current methods have largely focused on single-gene defects, like chimeric genes, which are present in many sarcomas and leukemias. Real understanding is more likely to derive from a genome-wide analysis of these malignancies. Recent technologic advances have made it possible to simultaneously assess the entire expressed gene profile, or transcriptome, of a given cancer. Foremost among these methods is gene expression profiling using DNA microarrays. Two basic approaches predominate: spotted arrays and photolithography arrays. Regardless of the method, the resulting information can be used to create disease profiles, but only if appropriate bioinformatic solutions are employed. Common analytic approaches include two-way expression comparisons, or scatter analyses; outlier gene analysis, to identify significantly dysregulated genes; dendrogram analyses, as pioneered by Eisen; cluster analyses to identify diagnostic or biologic groups; and various forms of functional analyses to identify relevant genes and biologic pathways. Studies of both adult and pediatric cancer have demonstrated the feasibility of such analyses to identify both diagnostic and prognostic groups of tumors. Acute childhood leukemias have been grouped into myelogenous and lymphoid, and even B- and T-cell subsets. Breast cancer prognostic groups have been identified on the basis of a small subset of expressed genes. In addition, preliminary data on childhood sarcomas appear to identify both diagnostic and prognostic subsets. Specifically, embryonal rhabdomyosarcoma could be distinguished from alveolar rhabdomyosarcoma, and even morphologically mixed embryonal and alveolar rhabdomyosarcoma showed similar gene expression profiles in both histologies. Further, collaborative studies using clustering analyses appear to identify prognostic groups of diverse sarcomas. Larger institutional and cooperative group studies are currently underway to validate these preliminary findings.


Subject(s)
Gene Expression Profiling/methods , Leukemia/diagnosis , Oligonucleotide Array Sequence Analysis/methods , Sarcoma/diagnosis , Animals , Bone Neoplasms/diagnosis , Bone Neoplasms/genetics , Child , Child, Preschool , Computational Biology , Humans , Leukemia/genetics , Osteosarcoma/diagnosis , Osteosarcoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/diagnosis , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Prognosis , Rhabdomyosarcoma, Alveolar/diagnosis , Rhabdomyosarcoma, Alveolar/genetics , Rhabdomyosarcoma, Embryonal/diagnosis , Rhabdomyosarcoma, Embryonal/genetics , Sarcoma/genetics , Statistics as Topic
10.
Mol Cell Biol ; 21(1): 260-70, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11113200

ABSTRACT

The cyclin-dependent kinase (CDK)-activating kinase (CAK) is involved in cell cycle control, transcription, and DNA repair (E. A. Nigg, Curr. Opin. Cell. Biol. 8:312-317, 1996). However, the mechanisms of how CAK is integrated into these signaling pathways remain unknown. We previously demonstrated that abrogation of MAT1 (ménage à trois 1), an assembly factor and targeting subunit of CAK, induces G(1) arrest (L. Wu, P. Chen, J. J. Hwang, L. W. Barsky, K. I. Weinberg, A. Jong, and V. A. Starnes, J. Biol. Chem. 274:5564-5572, 1999). This result led us to investigate how deregulation of CAK by MAT1 abrogation affects the cell cycle G(1) exit, a process that is regulated most closely by phosphorylation of retinoblastoma tumor suppressor protein (pRb). Using mammalian cellular models that undergo G(1) arrest evoked by antisense MAT1 abrogation, we found that deregulation of CAK inhibits pRb phosphorylation and cyclin E expression, CAK phosphorylation of pRb is MAT1 dose dependent but cyclin D1/CDK4 independent, and MAT1 interacts with pRb. These results suggest that CAK is involved in the regulation of cell cycle G(1) exit while MAT1-modulated CAK formation and CAK phosphorylation of pRb may determine the cell cycle specificity of CAK in G(1) progression.


Subject(s)
G1 Phase , Neoplasm Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Cell Division , Cyclin-Dependent Kinases/metabolism , Cyclins/metabolism , Gene Expression Regulation, Enzymologic , Humans , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , Phosphorylation , Protein Binding , Recombinant Fusion Proteins , Retinoblastoma Protein/metabolism , Substrate Specificity , Transduction, Genetic , Transfection , Tumor Cells, Cultured , Cyclin-Dependent Kinase-Activating Kinase
11.
Med Pediatr Oncol ; 35(6): 563-8, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11107118

ABSTRACT

BACKGROUND: Neuroblastomas often acquire sustained drug resistance during therapy. Sensitivities to carboplatin, etoposide, or melphalan were determined for 18 neuroblastoma cell lines; eight were sensitive and ten were resistant. As p53 mutations are rare in neuroblastomas studied at diagnosis, we determined if acquired p53 mutations and loss of function conferred multidrug resistance. RESULTS: Loss of p53 function (p53-LOF), defined as a failure to induce p21 and/or MDM2 in response to melphalan, was seen in 1/8 drug-sensitive and 6/10 drug-resistant cell lines. In four cell lines p53-LOF was associated with mutations in the DNA binding region of p53, while three cell lines with LOF and four cell lines with functional p53 had no evidence of p53 muta-tions. Nonfunctional and mutated p53 was detected in one resistant cell line, while a sensitive cell line derived from the same patient prior to treatment had functional and wild type (wt) p53. We transfected HPV 16 E6 (which mediates degradation of p53, causing LOF) into two drug-sensitive neuroblastoma cell lines with functional p53. LC(90) values of HPV 16 E6 transfected cell lines were 3-7-fold (melphalan), 8-109-fold (carboplatin), and 2-158-fold (etoposide) greater than that of LXSN-transfected controls. CONCLUSIONS: These data suggest that some neuroblastomas acquire p53 mutations during therapy, which is associated with a loss of p53 function, and can confer high-level multidrug resistance.


Subject(s)
Genes, p53/genetics , Neuroblastoma/drug therapy , Neuroblastoma/genetics , Nuclear Proteins , Chromosome Deletion , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/genetics , Drug Resistance, Neoplasm/genetics , Humans , Mutation , Neoplasm Proteins/genetics , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-mdm2 , Tumor Cells, Cultured
12.
J Pediatr Hematol Oncol ; 22(2): 106-11, 2000.
Article in English | MEDLINE | ID: mdl-10779022

ABSTRACT

The presence of well-differentiated rhabdomyoblasts at the end of therapy for rhabdomyosarcoma has been noted. This study was undertaken to investigate the therapeutic implications of the presence of well-differentiated rhabdomyoblasts at the end of therapy for pelvic rhabdomyosarcoma. Six patients with pelvic rhabdomyosarcoma (bladder-prostate, 4; vulvovaginal, 2) with disease diagnosed between the years 1974 and 1992 were sequentially investigated by cystoscopic or vaginoscopic examination and biopsy during and after completing therapy. All six patients received treatment according to prevailing therapeutic protocols. Biopsy material from all six patients at the end of therapy documented the presence of well-differentiated rhabdomyoblasts. Repeated biopsies demonstrated the presence of rhabdomyoblasts; however, they appeared to decrease in number with time. Mitotic activity was not observed in the biopsy materials obtained. All six patients are alive without evidence of disease from 37 to 233 months after therapy ended. The presence of well-differentiated rhabdomyoblasts at the end of therapy for pelvic rhabdomyosarcoma is a common finding. The biologic nature of these well-differentiated rhabdomyoblasts is not completely known, but they do not appear to connote the persistent presence of malignant disease and are not an indication for the continuation of therapy.


Subject(s)
Pelvic Neoplasms/pathology , Rhabdomyosarcoma/pathology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cell Differentiation , Child, Preschool , Cyclophosphamide/therapeutic use , Dactinomycin/therapeutic use , Female , Humans , Male , Outcome Assessment, Health Care , Pelvic Neoplasms/drug therapy , Pelvic Neoplasms/radiotherapy , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/pathology , Prostatic Neoplasms/radiotherapy , Radiography , Rhabdomyosarcoma/drug therapy , Rhabdomyosarcoma/radiotherapy , Urinary Bladder Neoplasms/drug therapy , Urinary Bladder Neoplasms/pathology , Urinary Bladder Neoplasms/radiotherapy , Vaginal Neoplasms/diagnostic imaging , Vaginal Neoplasms/drug therapy , Vaginal Neoplasms/pathology , Vincristine/therapeutic use , Vulvar Neoplasms/diagnostic imaging , Vulvar Neoplasms/drug therapy , Vulvar Neoplasms/pathology
13.
Genes Chromosomes Cancer ; 27(4): 337-44, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10719362

ABSTRACT

A comparative genomic hybridization (CGH) approach provides identification of genomic gains and losses in a tumor specimen in a single experiment. Only 11 embryonal rhabdomyosarcomas (E-RMS) have previously been subjected to CGH. The underlying genetic events in this histologic subtype are not well defined. In this investigation, 12 E-RMS specimens from 10 patients entered into Intergroup Rhabdomyosarcoma Study (IRS) I-IV and two local patients were analyzed by CGH and fluorescence in situ hybridization (FISH). Gains of chromosomes or chromosomal regions 2 (50%), 7 (42%), 8 (67%), 11 (42%), 12 (58%), 13q21 (33%), and 20 (33%) and losses of 1p35-36.3 (42%), 6 (33%), 9q22 (33%), 14q21-32 (25%), and 17 (25%) were most prominent. Chromosomal regions 1p35-36.3 and 9q22 represent novel regions of loss. Importantly, loss of 9q22 corresponds to the locus of a putative tumor suppressor gene (PTCH), which has been shown to play a role in rhabdomyosarcoma in a mouse model of Gorlin syndrome. Loss of 1p36 corresponds to the locus for PAX7, a paired box containing gene characteristically altered in alveolar rhabdomyosarcoma. Moreover, loss of 1p36 is prominent in another common pediatric soft tissue tumor, neuroblastoma. Gains of 2, 7, 8, 12, and 13 and loss of 14 were seen in the sole prior E-RMS CGH series; thus, these data provide important confirmatory results. In contrast to this previous study, however loss, not gain, of chromosome 17 was observed in the current study. Chromosome 17 loss correlates well with previous descriptions of frequent allelic loss of 17p (TP53) in E-RMS. In summary, CGH and FISH analyses of 12 E-RMS specimens revealed novel genomic imbalances that may be useful in directing further molecular studies for the determination of E-RMS critically involved genes.


Subject(s)
Chromosome Aberrations/genetics , Rhabdomyosarcoma, Embryonal/genetics , Adolescent , Child, Preschool , Female , Head and Neck Neoplasms/genetics , Humans , In Situ Hybridization, Fluorescence/methods , Infant , Male , Nucleic Acid Hybridization/methods , Prostatic Neoplasms/genetics , Urinary Bladder Neoplasms/genetics , Uterine Neoplasms/genetics
14.
Clin Cancer Res ; 6(2): 551-8, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10690538

ABSTRACT

Small round cell tumors of childhood can be histologically ambiguous, can require tumor markers for an accurate diagnosis, and include neuroblastoma, peripheral primitive neuroectodermal tumor (pPNET), Ewing's sarcoma (ES), lymphoma, and rhabdomyosarcoma. Because the cell type of origin for ES remains controversial, characterizing gene expression in ES can provide diagnostic markers and lead to better understanding of tumor biology. We studied RNA expression of the neuronal genes protein gene product 9.5 (PGP 9.5) and tyrosine hydroxylase (TH) by Northern analysis in cell lines and tissue from small round cell tumors. PGP 9.5 showed strong expression in 17 of 17 neuroblastoma cell lines, 9 of 9 pPNET cell lines, and 11 of 11 ES cell lines. PGP 9.5 was weakly expressed in 1 of 1 alveolar rhabdomyosarcoma cell lines but not in 1 of 1 embryonal rhabdomyosarcomas, and weak expression was seen in 1 of 7 leukemia cell lines. In tumor tissue, all 12 neuroblastomas expressed PGP 9.5, as did all 7 pPNET and all 7 ES. PGP 9.5 was very weakly expressed in 6 of 9 rhabdomyosarcomas and 1 of 9 lymphomas. TH was expressed only in neuroblastomas, and no TH expression was seen in cell lines or tissue from other tumors. As high expression of PGP 9.5 was only found in neural tumors; PGP 9.5 expression by ES provides further evidence for a neural origin of this tumor, whereas TH expression is highly specific for neuroblastomas. PGP 9.5 expression should allow sensitive detection of minimal residual disease for ES and pPNET using reverse transcription-PCR, and the variability in TH and PGP 9.5 expression levels in neuroblastomas indicates that expression of both genes should be used for monitoring minimal residual disease by reverse transcription-PCR.


Subject(s)
Lymphoma/pathology , Neuroblastoma/pathology , Neuroectodermal Tumors, Primitive/pathology , Rhabdomyosarcoma/pathology , Thiolester Hydrolases/genetics , Transcription, Genetic , Tyrosine 3-Monooxygenase/genetics , Adolescent , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Child , Child, Preschool , Humans , Infant , Lymphoma/genetics , Neoplasm Staging , Neuroblastoma/genetics , Neuroectodermal Tumors, Primitive/genetics , Rhabdomyosarcoma/genetics , Thiolester Hydrolases/analysis , Tumor Cells, Cultured , Tyrosine 3-Monooxygenase/analysis , Ubiquitin Thiolesterase
15.
Pediatr Res ; 45(4 Pt 1): 552-8, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10203148

ABSTRACT

Accumulated clinical evidence suggests that alveolar rhabdomyosarcoma (ARMS) is more aggressive than embryonal rhabdomyosarcoma (ERMS). Here, we study six childhood rhabdomyosarcoma cell lines, three ERMS and three ARMS. We have assayed the ability of the tumor cells to grow in culture and in nude mice as well as their propensity for pulmonary metastasis formation by tail vein injection. We also compared levels of c- and N-myc oncogene expression and DNA copy number. We find no correlation of histologic tumor type (i.e. ERMS versus ARMS) with growth rate in culture, but we do find suggestive correlations of histologic type with tumorigenicity (mean tumor diameter in millimeters at 6 wk: ARMS 30, ERMS 10; p1 = 0.1) and metastasis formation (ARMS 12, ERMS 0; p1 = 0.1). These properties also correlate with uniform greater overexpression of c-myc in ARMS (mean 39.3-fold, range 16-83) compared with ERMS (mean 5.3, range 4-8) (p1 = 0.05, control fibroblasts = 1). Although c-myc was often amplified in vitro (four of six lines), there was no correlation with histologic type (2/3 ARMS, 2/3 ERMS). These data on rhabdomyosarcoma cell lines derived from verified ERMS and ARMS tumors support the impression from previous clinicopathologic observations that ARMS is a more malignant form of rhabdomyosarcoma than ERMS.


Subject(s)
Gene Expression Regulation, Neoplastic , Genes, myc , Proto-Oncogene Proteins c-myc/genetics , Rhabdomyosarcoma, Alveolar/genetics , Rhabdomyosarcoma, Alveolar/pathology , Rhabdomyosarcoma, Embryonal/genetics , Rhabdomyosarcoma, Embryonal/pathology , Animals , Cell Division , Child , Child, Preschool , Female , Gene Amplification , Humans , Infant , Kinetics , Male , Mice , Mice, Nude , Neoplasm Metastasis , Transplantation, Heterologous , Tumor Cells, Cultured
16.
Am J Surg Pathol ; 23(3): 309-15, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10078922

ABSTRACT

Prompt and accurate diagnosis of small round cell tumors warrants ancillary studies. Recently, two-color fluorescence in-situ hybridization (FISH) using probes for specific gene rearrangements has gained wide acceptance. EWS gene rearrangements, present in essentially 100% of Ewing's Sarcoma/peripheral primitive neuroectodermal tumor, were evaluated by FISH on frozen sections (FS) of tumor biopsies from 10 patients, plus a negative control, and in seven other malignant neoplasms of childhood. 4mu FS were hybridized overnight, using a single EWS gene-specific probe spanning the EWS breakpoint. We identified EWS rearrangements in 8 of 10 cases (80%) of Ewing's Sarcoma/pPNET. There are no known false positives in diploid or near-diploid tumors, or in any of the non-EWS tumors tested; the uncommon false negative can be confirmed by RT-PCR. Hyperdiploid cases with multiple copies of chromosome 22 may be better evaluated by two-color FISH. This is the first use on FS biopsy material of a single probe for EWS, capable of detecting all known EWS rearrangements, in ES and other tumors. Utilization of this ancillary technique on FS for ES/pPNET and other tumors with distinctive chromosomal translocation is highly specific, reliable, expeditious (24-36 hours) and cost-effective.


Subject(s)
Neuroectodermal Tumors/genetics , Sarcoma, Ewing/genetics , Translocation, Genetic , Chromosomes, Human, Pair 11/genetics , Chromosomes, Human, Pair 22/genetics , Frozen Sections , Humans , In Situ Hybridization, Fluorescence , Reverse Transcriptase Polymerase Chain Reaction
17.
Lab Invest ; 79(12): 1535-43, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10616204

ABSTRACT

Ewing's sarcoma is the least differentiated member of the peripheral primitive neuroectodermal (pPNET) tumor family. Chromosomal translocations involving the EWS gene and five different Ets family transcription factor genes create fusion genes encoding aberrant transcription factors and are implicated in the vast majority of Ewing's sarcoma cases. Here, NIH 3T3 fibroblasts were infected with control (tk-neo or RAS) and two different EWS/ETS-expressing retroviruses. In vitro studies of established polyclonal lines expressing the two EWS/ETS genes, either EWS/FLI1 or EWS/ETV1, showed induction of cytokeratin 15 gene expression. Both fusion genes also caused characteristic gross morphologic, histologic, and ultrastructural changes in NIH 3T3 cells when transformed cell lines were injected into CB-17-scid mice. Native NIH 3T3 cells with a spindled cell morphology were converted to polygonal cells with high nucleo-cytoplasmic ratios that continued to express abundant cytokeratin. Extracellular collagen deposition was abolished, rough endoplasmic reticulum was markedly diminished, and rudimentary cell-cell attachments appeared. Most strikingly, neurosecretory-type dense core granules like those seen in pPNET were now evident. This murine model, created in mesenchyme-derived NIH 3T3 cells, demonstrated new characteristics of both neuroectodermal and epithelial differentiation and resembled small round cell tumors microscopically.


Subject(s)
Cell Differentiation , Oncogene Proteins, Fusion/genetics , Proto-Oncogene Proteins/genetics , Ribonucleoproteins/genetics , Transcription Factors/genetics , 3T3 Cells , Animals , Cell Line, Transformed , Ectoderm/cytology , Epithelial Cells/cytology , Heterogeneous-Nuclear Ribonucleoproteins , Immunohistochemistry , Mice , Mice, SCID , Neoplasm Invasiveness , Neuroectodermal Tumors, Primitive/genetics , Neuroectodermal Tumors, Primitive/pathology , Neuroectodermal Tumors, Primitive/ultrastructure , Proto-Oncogene Proteins c-ets , RNA-Binding Protein EWS , Sarcoma, Ewing/genetics , Sarcoma, Ewing/pathology , Sarcoma, Ewing/ultrastructure
18.
Pediatr Dev Pathol ; 1(6): 550-61, 1998.
Article in English | MEDLINE | ID: mdl-9724344

ABSTRACT

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood, and 75% of such cases in the United States are reviewed at the Pathology Center for the Intergroup Rhabdomyosarcoma Study Group (IRSG). The first four generations of IRSG therapeutic trials (IRS I-IV) and supportive pathologic studies have generated a new International Classification of Rhabdomyosarcoma (ICR) that offers new morphologic concepts to the practicing pathologist. The objective of this report is to clearly define emerging histopathologic categories of RMS as defined by the ICR, and to emphasize correlative immunohistochemical or molecular studies. Emerging ICR variants of RMS place the patient in widely divergent prognostic categories (superior, botryoid or spindle cell variants; poor, solid alveolar or diffusely anaplastic variants). The cardinal histopathologic features of the ICR combined with results of studies of fusion genes seen with t(1;13) and t(2;13) will help delineate therapeutic subgroups of RMS for the fifth generation (IRS V) of IRSG studies. Consequently, it is imperative for the practicing pathologist to be familiar with the practical workup and diagnosis of RMS in childhood.


Subject(s)
Rhabdomyosarcoma/pathology , Soft Tissue Neoplasms/pathology , Biomarkers, Tumor/analysis , Humans , Immunoenzyme Techniques , Neoplasm Staging/methods , Prognosis , Rhabdomyosarcoma/chemistry , Rhabdomyosarcoma/classification , Rhabdomyosarcoma/genetics , Soft Tissue Neoplasms/chemistry , Soft Tissue Neoplasms/classification , Soft Tissue Neoplasms/genetics
19.
Cancer ; 83(3): 575-81, 1998 Aug 01.
Article in English | MEDLINE | ID: mdl-9690552

ABSTRACT

BACKGROUND: The Quebec Neuroblastoma Screening (QNS) Project completed a 5-year program for measuring urinary vanillylmandelic acid (VMA)/homovanillic acid (HVA) levels at age 3 weeks and/or 6 months in 89% of 476,603 Quebec-born infants from 1989-1994; 45 screening positive preclinical cases (S-positive cases) and 20 congenital/neonatal (C/N) cases were identified. As of April 1997, an additional 59 cases in the same birth cohort were diagnosed clinically; these neuroblastomas developed after screening verified normal VMA/HVA levels (S-negative cases). METHODS: Pathology specimens from 45 of 59 S-negative cases were reviewed centrally and classified according to the Shimada system. Results were compared with clinical data and also with S-positive and C/N cases. RESULTS: Of 45 S-negative cases, 27 tumors had favorable histology (FH) and 18 had unfavorable histology (UH). Approximately 52% of FH tumors were diagnosed before age 1 year, whereas UH tumors were nearly exclusively (94%) diagnosed after age 1 year (P < 0.01). Approximately 89% of FH tumors were Stage I, II, or IV-S, whereas 72% UH tumors were Stage III or IV (P < 0.001). All children with FH tumors were alive at last follow-up (range of follow-up period: 9-79 months; median, 35 months), whereas 8 children with UH tumors died of disease even after limited follow-up (range of follow-up period: 0-60 months; median, 20 months). By contrast, S-positive and C/N cases were predominantly (97%) FH tumors, often (76%) Stage I, II, or IV-S, with excellent clinical outcome (survival rate of 98%). CONCLUSIONS: The majority of the UH neuroblastomas that developed in the birth cohort of the QNS Project were included in the group of S-negative cases and could not be detected by the screening at age 3 weeks and/or 6 months.


Subject(s)
Neuroblastoma/pathology , Child , Child, Preschool , Humans , Infant , Infant, Newborn , Mass Screening , Neuroblastoma/mortality
20.
Med Pediatr Oncol ; 29(3): 181-9, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9212842

ABSTRACT

UNLABELLED: The occurrence of rhabdomyosarcoma (RMS) primary in or metastatic to breast has been regarded as an uncommon event, associated with an unfavorable outcome. Records of 26 patients with diagnoses of breast RMS, either primary or secondary, entered in the Intergroup Rhabdomyosarcoma Study (IRS) (1972-1992) were reviewed and compared with data regarding 47 similar patients in published reports. Of the 26 IRS cases, the histologic subtype was alveolar in 24, embryonal in 1, and not determined in 1. All were female with ages ranging from 11.5 to 20.2 years (median, 15.2 years; mode, 14-16 years). This compact age distribution of both primary (n = 7) and metastatic (n = 19) breast RMS was seen in previously reported series. Among the 19 cases of RMS with initial dissemination to breast, primary tumor sites, were extremity (n = 8), nasopharynx/paranasal sinuses (n = 7), and trunk (n = 4). IRS treatment was risk-based according to site and extent of disease. Four of 7 patients with primary RMS remain disease free 2.9 to 7 years post diagnosis. Among 19 patients with RMS initially metastatic to breast, including 7 in IRS clinical group IV at original diagnosis, three are disease free at 7.6, 15.7 and 17.0 years. CONCLUSIONS: primary or metastatic RMS in breast is almost confined to adolescent females having tumors with alveolar histology. Approximately one-half of the patients with primary breast disease and 15% of those with metastatic breast disease as an initial recurrence are long-term survivors.


Subject(s)
Breast Neoplasms/pathology , Rhabdomyosarcoma/pathology , Adolescent , Adult , Age Factors , Breast Neoplasms/secondary , Child , Disease-Free Survival , Extremities/pathology , Female , Follow-Up Studies , Humans , Nasopharyngeal Neoplasms/pathology , Neoplasm Recurrence, Local/pathology , Paranasal Sinus Neoplasms/pathology , Retrospective Studies , Rhabdomyosarcoma/secondary , Rhabdomyosarcoma, Alveolar/pathology , Rhabdomyosarcoma, Alveolar/secondary , Rhabdomyosarcoma, Embryonal/pathology , Rhabdomyosarcoma, Embryonal/secondary , Survival Rate , Thoracic Neoplasms/pathology , Treatment Outcome
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