Loss of heterozygosity on 6q, 16q, and 17p in human central nervous system primitive neuroectodermal tumors.

The loss of genetic material from specific chromosomal locations in a given tumor type has been taken for evidence of the importance of tumor suppressor genes at these loci in the genesis of the tumor. The primitive neuroectodermal tumor of the central nervous system has such a loss on 17p in one-third of tumors. In this report, a detailed analysis of 17p loss in 23 tumors has been performed using 10 probes mapping to this region. In addition, an analysis for allelic deletion on chromosomes 6q, 16q, and 22q has been performed. Six of the 23 tumors showed loss of markers on 17p, and the area of common loss spanned 17p11.2 to 17pter. Five of 23 tumors showed loss of markers on 6q, and 3 showed loss on 16q. No tumor lost markers on 22q. Only one tumor showed loss at more than one location. These data suggest that primitive neuroectodermal tumors either are a heterogeneous group of tumors with more than one mechanism leading to a tumor or that more than one recessive oncogene may play a role in the genesis of these tumors.

Reduction to homozygosity and gene amplification in central nervous system primitive neuroectodermal tumors of childhood.

The loss of genetic material from specific chromosomal locations has been identified for a number of pediatric tumors. This loss has been taken as evidence for the importance of tumor suppressor genes at these loci in the genesis of these tumors. One of these pediatric tumors, the primitive neuroectodermal tumor of the central nervous system, has not been well studied. In this report, an analysis of primitive neuroectodermal tumors for allelic deletions on chromosomes 1p, 7q, 10, 11p, 13q, and 17p has been performed. One of ten tumors was found to have increased copies of c-myc. Three different patients were found to reduce to homozygosity at one of three different locations. Significantly, however, three of nine informative patients showed a reduction to homozygosity on chromosome 17p. Thus, primitive neuroectodermal tumor is one of a growing number of tumor types in which deletions in the short arm of chromosome 17 might be important in oncogenesis.

Sporadic medulloblastomas contain oncogenic beta-catenin mutations.

The beta-catenin, glycogen synthase kinase 3beta (GSK-3beta), and adenomatous polyposis coli (APC) gene products interact to form a network that influences the rate of cell proliferation. Medulloblastoma occurs as part of Turcot's syndrome, and patients with Turcot's who develop medulloblastomas have been shown to harbor germ-line APC mutations. Although APC mutations have been investigated and not identified in sporadic medulloblastomas, the status of the beta-catenin and GSK-3beta genes has not been evaluated in this tumor. Here we show that 3 of 67 medulloblastomas harbor beta-catenin mutations, each of which converts a GSK-3beta phosphorylation site from serine to cysteine. The beta-catenin mutation seen in the tumors was not present in matched constitutional DNA in the two cases where matched DNA was available. A loss of heterozygosity analysis of 32 medulloblastomas with paired normal DNA samples was performed with four microsatellite markers flanking the GSK-3beta locus; loss of heterozygosity with at least one marker was identified in 7 tumors. Sequencing of the remaining GSK-3beta allele in these cases failed to identify any mutations. Taken together, these data suggest that activating mutations in the beta-catenin gene may be involved in the development of a subset of medulloblastomas. The GSK-3beta gene does not appear to be a target for inactivation in this tumor.

Sporadic medulloblastomas contain PTCH mutations.

Nevoid basal cell carcinoma syndrome (NBCCS), or Gorlin's syndrome, is an autosomal dominant disorder that predisposes to developmental defects and various forms of cancer. PTCH was recently proposed as a candidate gene for NBCCS due to its frequent mutation in basal cell carcinomas, the cancer most often associated with this syndrome. Another NBCCS-associated cancer is medulloblastoma, a common central nervous system tumor in children. Most medulloblastomas, however, occur without indication of an inherited predisposition. We have examined 24 sporadic medulloblastomas for loss of heterozygosity (LOH) at loci flanking as well as within PTCH. In cases with LOH, single-strand conformational polymorphism and sequencing analysis were performed to determine the status of the remaining PTCH allele. Microsatellite analysis indicated LOH of PTCH in 5 of 24 tumors, and in three of these cases a mutation of the remaining allele was identified. Two of the mutations were duplication insertions, and the third consisted of a single base deletion. It is interesting that all three mutations occur in exon 17 of the PTCH gene. These data suggest that inactivation of PTCH function is involved in the development of at least a subset of sporadic medulloblastomas.

Deletion within the D17S34 locus in a primitive neuroectodermal tumor.

Loss of heterozygosity on chromosome 17p13.3 is frequently observed in solid tumors, and the presence of a tumor suppressor gene has been predicted in this region of chromosome 17. We have analyzed a primitive neuroectodermal tumor sample exhibiting loss of heterozygosity at the D17S34 locus, a commonly used telomeric marker on the short arm of chromosome 17. The remaining allele showed a rearrangement. Cosmids spanning the D17S34 locus and probes from that region were used to demonstrate a 9-kb deletion within the D17S34 locus and were found to contain evolutionary, conserved sequences. Genetic alterations in this region may also affect expression of immediately adjacent genes, such as ABR, and could be a common mechanism in the causation of primitive neuroectodermal tumors.

Effective chemotherapy for advanced CNS embryonal tumors in adults.

PURPOSE: Embryonal tumors of the CNS include, among others, medulloblastoma, cerebral neuroblastoma, pineoblastoma, and primitive neuroectodermal tumors (PNETs). Almost all data on the treatment of embryonal CNS tumors are derived from the pediatric population, since these tumors are uncommon in adulthood. The purpose of this study was to examine the rate and duration of response to chemotherapy of advanced embryonal CNS tumors in adults. PATIENTS AND METHODS: We retrospectively studied all adult (> 18 years of age) patients with advanced embryonal tumors of the CNS who received chemotherapy at our institution between 1976 and 1994. Seventeen consecutive patients were treated with regimens that contained either nitrosourea or cisplatin or both sequentially, with no patients having received the combination of nitrosourea and cisplatin concurrently. RESULTS: In patients who received cisplatin-based chemotherapy, responses were observed in 84.5% (26% complete response [CR] rate), 10.5% remained stable, and 5% progressed. The median time to progression was 18 months for patients who had a CR, 6 months for those with partial response (PR), and 10 months for stable patients. Among patients who received nitrosourea-based chemotherapy, PR was observed in 27%, 36.5% remained stable, and 36.5% progress. The median time to progression was 6 months for patients who had a PR and 6.5 months for stable patients. CONCLUSION: In adults with advanced embryonal CNS tumors, conventional-dose intravenous cisplatin-based chemotherapy regimens are able to produce responses in the majority of the patients (84.5%), even as second- or third-line regimens. Nitrosourea-based regimens less frequently produce responses (27%).

Phase II trial of primary chemotherapy followed by reduced-dose radiation for CNS germ cell tumors.

PURPOSE: A prospective phase II study was initiated to assess the response rate, survival, and late effects of treatment in patients with newly diagnosed CNS germ cell tumors (GCT), using etoposide plus cisplatin followed by radiation therapy prescribed by extent of disease, histology, and response to chemotherapy. PATIENTS AND METHODS: Seventeen patients aged 8 to 24 years with histologically proven CNS GCT received etoposide (100 mg/m2/d) plus cisplatin (20 mg/m2/d) daily for 5 days every 3 weeks for four cycles, followed by radiation therapy. Nine patients had germinomas; eight had mixed GCT. Four patients (three with germinomas and one with mixed GCT) presented with leptomeningeal dissemination. RESULTS: Radiographically, 14 of 17 patients were assessable for response; 11 patients experienced complete regression, and three had major partial regression before radiation. Six of seven assessable patients with elevated CSF levels of alpha-fetoprotein or betahuman chorionic gonadotropin had normalization with chemotherapy alone; all normalized with combined chemotherapy and radiation therapy. All 17 patients are alive without evidence of disease (median follow-up, 51 months). One patient developed a relapse in the spinal leptomeninges and was rendered free of disease with spinal radiation more than 5 years ago. One patient developed carotid stenosis requiring surgery. Thus far, only minimal long-term deterioration in neurocognitive function has been detected as a consequence of protocol treatment. CONCLUSION: Conventional-dose intravenous chemotherapy with etoposide and cisplatin can effect tumor regression in a high proportion of patients with CNS GCT, including those with leptomeningeal metastases. Acute and long-term toxicities are acceptable. Progression-free survival and overall survival are excellent.

Mutations of the INI1 rhabdoid tumor suppressor gene in medulloblastomas and primitive neuroectodermal tumors of the central nervous system.

Germ-line and somatic mutations of the hSNF5/INI1 gene have been reported in atypical teratoid/rhabdoid tumors (AT/RTs) of the brain, consistent with its role as a tumor suppressor gene. In the present study, we determined the frequency of deletions and mutations of INI1 in 52 children whose original diagnosis was medulloblastoma (MB) or primitive neuroectodermal tumor (PNET) of the central nervous system. Mutations were detected in DNA isolated from four tumors, all from children less than 3 years of age at diagnosis. Two of the four were reviewed and reclassified as atypical teratoid tumor, whereas there was insufficient material to establish this diagnosis in the two remaining cases. The relatively low frequency of mutations, even in a large series of infants, suggests that loss of sequences from chromosome 22 and/or mutations of INI1 do not account for the poor prognosis of children with MB or PNET who are less than 3 years of age at diagnosis. Nevertheless, chromosome 22 deletion and INI1-mutation analysis of infants with MB/PNET should be considered for all children who are less than 1 year of age. Detection of these mutations suggests that the child has an AT/RT, rather than a MB/PNET, a finding with important prognostic value.

Prognostic significance of chromosome 17p deletions in childhood primitive neuroectodermal tumors (medulloblastomas) of the central nervous system.

Deletions in the short arm of chromosome 17 (17p) are the most common genetic abnormality in primitive neuroectodermal tumors of the posterior fossa/medulloblastoma (PNET/Mb). The biological consequences of these deletions are not known for children with PNET/Mb; however, the presence of a tumor suppressor gene located in 17p, distinct from p53, has been implicated in tumorigenesis. Two recent studies suggest that 17p deletions in PNET/Mb are associated with a poor prognosis. To address this question, we identified deletions of chromosome 17p by cytogenetic and/or molecular biology methods in tumor biopsy samples from 56 patients with PNET/Mb. Associations between clinical characteristics or survival outcomes and 17p status were examined by multivariate analysis. Forty-one percent of PNET/Mb cases had a deletion of 17p. No significant association was found between 17p deletion and shorter survival duration or higher metastatic stage. Multivariate analysis did not find independent prognostic significance for 17p deletions after accounting for the effects of significant clinical variables. A larger study of the prognostic value of 17p deletion should be considered; however, clinical use of this factor to distinguish high-risk from standard-risk PNET/Mb populations is not warranted at this time.

Analysis of oncogene and tumor suppressor gene alterations in pediatric malignant astrocytomas reveals reduced survival for patients with PTEN mutations.

Although common among adult intracranial neoplasms, pediatric malignant astrocytomas (PMAs) comprise a relatively small proportion of the brain tumors that occur in children. The scarcity of such cases generally requires that molecular analyses of PMAs are based on the utilization of paraffin-embedded material, and here we have used 39 such specimens to examine the incidence and prognostic significance of oncogene and tumor suppressor gene alterations (including amplifications of EGFR, CDK4, and MDM2 as well as inactivating mutations of CDKN2A, TP53, and PTEN) in these tumors. In general, the frequency of alteration for the genes we have studied fell within ranges that have been reported for adult astrocytomas. However, EGFR amplification, which is usually observed in approximately 40% and 15% of adult grade 4 and grade 3 astrocytomas, respectively, was not detected in any member of this series. With regard to prognosis, PTEN mutations were significantly associated with decreased survival among grade 3 and grade 4 PMA patients, a potentially important observation because neither patient age nor tumor malignancy grade was correlated with outcome for these individuals. In total, our data suggest at least one significant distinction between the genetic etiology of pediatric and adult astrocytomas and additionally reveal that analysis of PTEN mutations in PMA patients may be useful in the differential diagnosis of these tumors.

Symptomatic hydrocephalus: initial findings in brainstem gliomas not detected on computed tomographic scans.

In a retrospective review of 85 patients younger than 18 years of age with a diagnosis of brainstem glioma treated between 1974 and 1987, seven (8.3%) initially had hydrocephalus and no evidence of tumor on CT scans. Intrinsic brain tumors, six in the pons and one in the diencephalon, were discovered later, either on follow-up CT scans or on magnetic resonance images obtained despite persistently normal CT scan findings. The initial radiologic study of choice for children and adolescents with hydrocephalus should be magnetic resonance imaging, including axial and sagittal T1- and T2-weighted images. If a CT scan is obtained first and hydrocephalus but not tumor is found, magnetic resonance image should be obtained to rule out the possible presence of an intrinsic brainstem tumor.

The effect of bromodeoxyuridine and ultraviolet light on 9L rat brain tumor cells.

Incorporation of the thymidine analog bromodeoxyuridine (BrdUrd) into DNA increases the sensitivity of a cell to uv light. We have examined the effect of uv light on cell killing and alkaline elution profiles in 9L rat brain tumor cells pretreated with BrdUrd. Combination treatment with BrdUrd and uv irradiation produced a dose enhancement ratio of 3.8 at the 10% survival level compared with uv-radiated control cells; cell killing depended on both the time of treatment and the concentration of BrdUrd used for incubation. Sequential treatment caused single-strand breaks and DNA-protein crosslinks in the portion of DNA containing BrdUrd; uv irradiation alone caused very few strand breaks and no DNA-protein crosslinks. Because of the presence of both lesions in cells treated with BrdUrd and uv light, it was possible to calculate crosslinking factors without using a charging X-ray dose to induce strand breaks, the method commonly used with crosslinking drugs. Results of repair studies suggested that single-strand breaks are repaired more rapidly than are DNA-protein crosslinks.

Cytogenetic analysis of 39 pediatric central nervous system tumors.

Consistent cytogenetic abnormalities have been described in many pediatric solid tumors, including Ewing's sarcoma, Wilms' tumor, and neuroblastoma. Similar analysis of pediatric central nervous system (CNS) tumors has been hampered by technical problems. We report chromosome results from 39 pediatric CNS tumors. Abnormalities of chromosome 17 were noted in 3 of 11 primitive neuroectodermal tumors (including i(17q) in 2 tumors), confirming data observed by other investigators. Cells from 2 of 11 primitive neuroectodermal tumors (PNET) exhibited loss or structural abnormalities involving chromosome 11. Loss or distal deletion of chromosome 7q was noted in cells from two PNETs. Because other investigators have shown loss of heterozygosity on 17p in about one-third of PNET, we propose that chromosome regions 7q and 11 are areas worthy of further study in pediatric PNET. Numerical abnormalities were noted in 6 of 21 astrocytomas. Hyperdiploidy was demonstrated in 1 of 4 pilocytic astrocytomas and pseudopolyploidy was demonstrated in 4 of 13 anaplastic astrocytomas. Structural chromosome abnormalities (translocations, deletions) were noted in 4 of 13 anaplastic astrocytomas. Complex structural anomalies were observed in one craniopharyngioma. A rhabdoid tumor of the brain exhibited multiple complex structural rearrangements but did not exhibit the monosomy 22 observed in some rhabdoid tumors. Hypodiploidy and loss of chromosome 22 were noted in a clinically aggressive meningioma, corroborating observations by other investigators.

Assessment of functional MR imaging in neurosurgical planning.

BACKGROUND AND PURPOSE: Presurgical sensorimotor mapping with functional MR imaging is gaining acceptance in clinical practice; however, to our knowledge, its therapeutic efficacy has not been assessed in a sizable group of patients. Our goal was to identify how preoperative sensorimotor functional studies were used to guide the treatment of neuro-oncologic and epilepsy surgery patients. METHODS: We retrospectively reviewed the medical records of 46 patients who had undergone preoperative sensorimotor functional MR imaging to document how often and in what ways the imaging studies had influenced their management. Clinical management decisions were grouped into three categories: for assessing the feasibility of surgical resection, for surgical planning, and for selecting patients for invasive functional mapping procedures. RESULTS: Functional MR imaging studies successfully identified the functional central sulcus ipsilateral to the abnormality in 32 of the 46 patients, and these 32 patients are the focus of this report. In epilepsy surgery candidates, the functional MR imaging results were used to determine in part the feasibility of a proposed surgical resection in 70% of patients, to aid in surgical planning in 43%, and to select patients for invasive surgical functional mapping in 52%. In tumor patients, the functional MR imaging results were used to determine in part the feasibility of surgical resection in 55%, to aid in surgical planning in 22%, and to select patients for invasive surgical functional mapping in 78%. Overall, functional MR imaging studies were used in one or more of the three clinical decision-making categories in 89% of tumor patients and 91% of epilepsy surgery patients. CONCLUSION: Preoperative functional MR imaging is useful to clinicians at three key stages in the preoperative clinical management paradigm of a substantial percentage of patients who are being considered for resective tumor or epilepsy surgery.

To shunt or to fenestrate: which is the best surgical treatment for arachnoid cysts in pediatric patients?

The treatment options for intracranial arachnoid cysts are either craniotomy and fenestration of the cyst into the cerebrospinal fluid spaces or shunting of the cyst contents extracranially. Fenestration may eliminate the need to shunt, but it is a major operative procedure and is not always successful. To determine which treatment provides the greatest benefit with the fewest complications, the records of 31 patients with 34 arachnoid cysts treated at the Children's Hospital of Los Angeles between 1976 and 1986 were reviewed. The mean age of the patients was 4.4 years, with a range of 0 to 15.5 years. The most common location was the middle fossa (14 cases), followed by the posterior fossa (7 cases), the suprasellar region (5 cases), and hemispheric (5 cases) and other locations (3 cases). Signs and symptoms were related to abnormally rapid head growth in infants and to increased intracranial pressure and seizures in older children. The initial treatment of 29 cysts was fenestration. Twenty-two (76%) procedures were successful, with no additional treatment needed for the cyst. The other 7 cysts required the subsequent placement of a cystoperitoneal shunt. In 5 cases, the cysts were treated initially with cystoperitoneal shunts. Of the total 12 cystoperitoneal shunts, 5 have required revisions on one or more occasions. No significant difference in morbidity was noted between the two treatment options. Because we consider shunt independence to be a major goal of therapy, we suggest that patients with arachnoid cysts be divided into two categories, those presenting with associated hydrocephalus and those without hydrocephalus.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrylic cranioplasty with alginate molding: technical note.

OBJECTIVE: Acrylic cranioplasty for cranial defects is a common neurosurgical procedure that is performed when the original bone flap becomes infected or is unusable for other reasons. We developed a simple technique to produce a complete copy of the original bone flap from acrylic, using alginate impression material as a mold. METHODS: Alginate impression material was used to form a mold of the patient's original bone flap. Methylmethacrylate was then placed inside the mold to create an exact duplicate of the bone flap. The acrylic flap was sterilized with gamma irradiation and used for cranioplasty. Two patients who had cranial defects secondary to infections of their craniotomy bone flaps underwent cranioplasty with this technique. RESULTS AND CONCLUSION: A perfect copy of the patient's original bone flap was easily and quickly created with this technique, and excellent cosmetic results were obtained. Operative time was shortened because the prosthesis was preformed before the operation. This technique can also be used to mold large or complex cranial defects as long as the original bone flap is available and there is no major cranial remodeling around the defect.

The lack of a role for p53 in astrocytomas in pediatric patients.

Mutations in the p53 gene, which codes for a cell division regulatory protein, have been identified in approximately one-third of adult astrocytomas. We evaluated 35 astrocytic tumors (17 pilocytic, 4 diffuse low grade, 12 anaplastic, and 2 glioblastoma) in pediatric patients for p53 mutations, using polymerase chain reaction-single-stranded conformation polymorphism analysis as a screening technique. Additionally, those tumors identified with homozygosity in the area of the p53 gene on chromosome 17 by Southern blotting were sequenced to look for p53 mutations. No tumors were identified with polymerase chain reaction-single-stranded conformation polymorphism analysis shifts indicative of mutations in the p53 gene. Five of 21 tumors were homozygous in the region of the p53 gene on chromosome 17; no mutations in exons 5 to 8 were found in any of these tumors. The frequency of p53 mutation in pediatric astrocytomas is significantly less than the frequency for adult tumors, regardless of tumor grade. Furthermore, the frequency of p53 mutations in high-grade astrocytomas is significantly lower in pediatric tumors than in adult tumors. These results suggest that p53 is not important in the oncogenesis of pediatric astrocytomas. Oncogenesis in pediatric astrocytomas may occur by different mechanisms than those of similar tumors in adults.

Management of symptomatic chronic extra-axial fluid collections in pediatric patients.

The records of 103 pediatric patients having symptomatic chronic extra-axial fluid collections treated at Children's Hospital of Los Angeles from 1977 to 1988 were reviewed. Patients were treated with observation, serial percutaneous needle drainage, drainage through burr holes, drainage into a closed external drainage system, or subdural to peritoneal shunt. If the initial treatment was not effective, additional forms of treatment were instituted. Shunts, ultimately used in 73% of the patients, proved to be the most effective treatment. Of the group with shunts, the extra-axial fluid was unilateral in 20% and bilateral in 80%. In those patients with bilateral effusions, no difference in efficacy of shunts was seen in patients treated with bilateral versus unilateral shunts. Of the 75 patients with shunts, 12% required a shunt revision for progressive or recurrent symptoms. Shunt infections occurred in 3% of the patients, necessitating removal of the shunt and treatment with antibiotics. Eosinophilia in the subdural fluid was associated with shunt obstruction requiring revision. The shunt was never removed in 51% of patients with no untoward effects. This study demonstrates that the most efficacious treatment of symptomatic chronic extra-axial fluid collections in children is a unilateral subdural to peritoneal shunt. The shunt need not be removed after resolution of the fluid collections.

Benign cerebellar astrocytomas of childhood.

Benign cerebellar astrocytomas of childhood are potentially surgically curable lesions. Histologically, these neoplasms can be divided into pilocytic and diffuse astrocytomas. Whether there is a difference in the recurrence rate between these two tumor types after a surgical resection is not clear. In addition, the role of immediate postoperative imaging in predicting a recurrence has not been established. To answer these questions, we have reviewed the charts of 23 patients with benign cerebellar astrocytomas treated at Childrens Hospital of Los Angeles over a 10-year period (1977-1987). Of the 23 tumors, 15 were pilocytic and 8 were diffuse. All patients underwent an attempted gross total surgical removal of the tumor, and all patients had a postoperative computed tomographic (CT) scan with and without intravenously administered contrast material performed within 72 hours of the operation. Based on the postoperative CT scan, 12 patients had residual tumors. Seven of the subtotally resected tumors were pilocytic (7 of 15), and 5 were diffuse (5 of 8). Interestingly, the surgeon believed that a gross total resection had been obtained in 9 of these patients. There have been 4 recurrences in these 23 patients, with a mean follow-up of 4.9 years. All recurrences were in patients with subtotal resections. Of the 11 patients with a total resection of the tumor, 7 developed a small rim of enhancement on subsequent scans an average of 5 months after the operation.(ABSTRACT TRUNCATED AT 250 WORDS)

Two primitive neuroectodermal tumor cell lines require an activated insulin-like growth factor I receptor for growth in vitro.

OBJECTIVE: To determine the expression of the insulin-like growth factors (IGFs) and the IGF-I receptor in primitive neuroectodermal tumor cell lines and to assess the importance of these proteins in the growth of cell lines in vitro. METHODS: Ribonucleic acid blotting and reverse transcriptase-polymerase chain reaction were used for detection of IGF and IGF-I expression. Ribonucleic acid blotting was used for detection of up-regulation of c-fos in the presence of exogenous growth factor. Immunoprecipitation was used to demonstrate autophosphorylation of the receptor in the presence of exogenous growth factor. Ligand binding analysis was used to determine the binding affinity of the receptor and the number of receptors per cell. Growth of curves in the presence of monoclonal antibody that blocks binding of ligand to receptor was measured to determine the requirement for an activated receptor during growth. RESULTS: Expression of IGF-II was identified in one cell line. No expression of IGF-I was seen in any cell line. Expression of IGF-I receptor was detected in all three cell lines. Immunoprecipitation experiments demonstrated autophosphorylation of the receptor after addition of IGF-I to growing cells. Ligand binding analysis revealed 9.2 x 10(4) and 4 x 10(4) receptors per cell in the Daoy and PFSK cell lines, respectively. Addition of either IGF alone or in combination to serum-starved cells was not able to restore growth of the cell lines. A blocking monoclonal antireceptor antibody decreased growth of Daoy and PFSK cells in a dose-dependent fashion. Complete arrest of growth occurred at 1 microgram/ml antibody in both cell lines. CONCLUSION: The IGF-I receptor is expressed by primitive neuroectodermal tumor cell lines in vitro. An activated receptor is important for cell proliferation in vitro. Additional work will establish the importance of these findings for tumors in vivo.