Genomic and clinical characterization of a familial GIST kindred intolerant to imatinib.

Familial gastrointestinal stromal tumors (GIST) are rare. We present a kindred with multiple family members affected with multifocal GIST who underwent whole genome sequencing of the germline and tumor. Affected individuals with GIST harbored a germline variant found within exon 13 of the KIT gene (c.1965T>G; p.Asn655Lys, p.N655K) and a variant in the MSR1 gene (c.877 C > T; p.Arg293*, pR293X). Multifocal GISTs in the proband and her mother were treated with preoperative imatinib, which resulted in severe intolerance. The clinical features of multifocal GIST, cutaneous mastocytosis, allergies, and gut motility disorders seen in the affected individuals may represent manifestations of the multifunctional roles of KIT in interstitial cells of Cajal or mast cells and/or may be suggestive of additional molecular pathways which can contribute to tumorigenesis.

SATB2 enhances migration and invasion in osteosarcoma by regulating genes involved in cytoskeletal organization.

Osteosarcoma (OS) is the most common malignant bone tumor and the majority of recurrences are due to metastasis. However, the molecular mechanisms that regulate OS metastatic spread are largely unknown. In this study, we report that special AT-rich-binding protein 2 (SATB2) is highly expressed in OS cells and tumors. Short hairpin RNA-mediated knockdown of SATB2 (sh-SATB2) decreases migration and invasion of OS cells without affecting proliferation or viability. Microarray analysis identified genes that were differentially regulated by SATB2 including the actin-binding protein Epithelial Protein Lost In Neoplasm (EPLIN), which was upregulated in sh-SATB2 cells. Silencing EPLIN rescues the decreased invasion observed in sh-SATB2 cells. Pathway analyses of SATB2-regulated genes revealed enrichment of those involved in cytoskeleton dynamics, and increased stress fiber formation was detected in cells with SATB2 knockdown. Furthermore, sh-SATB2 cells exhibit increased RhoA, decreased Rac1 and increased phosphorylation of focal adhesion kinase (FAK) and paxillin. These findings identify SATB2 as a novel regulator of OS invasion, in part via effects on EPLIN and the cytoskeleton.

High frequency of de novo mutations in Li-Fraumeni syndrome.

BACKGROUND: Li-Fraumeni syndrome is an autosomal dominant cancer predisposition syndrome caused by germline mutations in the TP53 gene. The frequency of germline de novo TP53 mutations is largely unknown; few unequivocal de novo mutations have been reported. METHODS AND RESULTS: Of 341 patients with early onset cancer sent for clinical testing to a national reference laboratory, 75 patients had TP53 germline mutations. Five (7%) de novo mutations were identified, as well as an additional 10 TP53 germline mutations likely to be de novo by family history. The frequency of de novo TP53 mutations in this patient sample is at least 7% and may be as high as 20%. CONCLUSIONS: The possibility that de novo germline TP53 mutations are relatively common has implications for testing and the identification of potential Li-Fraumeni syndrome in patients with little or no family history of cancer.

Telomere maintenance and dysfunction predict recurrence in paediatric ependymoma.

We have recently described the enzymatic subunit of telomerase (hTERT) as an important prognostic marker for paediatric ependymoma. Because of the lack of good, representative pre-clinical models for ependymoma, we took advantage of our large cohort of ependymoma patients, some with multiple recurrences, to investigate telomere biology in these tumours. Our cohort consisted of 133 ependymomas from 83 paediatric patients and included 31 patients with recurrences. Clinical outcome was measured as overall survival, progression-free survival and response to therapy. In all 133 tumours, hTERT expression correlated with proliferative markers, including MIB-1 index (P<0.0001) and mitotic index (P=0.005), as well as overall tumour grade (P=0.001), but not with other markers of anaplasia. There was no correlation between telomere length and hTERT expression or survival. Surprisingly, prior radiation or chemotherapy neither induced sustained DNA damage nor affected telomere maintenance in recurrent tumours. There was an inverse correlation between hTERT expression and telomere dysfunction as measured by gamma H2AX expression (P=0.016). Combining gamma H2AX and hTERT expressions could segregate tumours into three different survival groups (log rank, P<0.0001) such that those patients whose tumours expressed hTERT and showed no evidence of DNA damage had the worst outcome. This study emphasises the importance of telomere biology as a prognostic tool and telomerase inhibition as a therapeutic target for paediatric ependymoma. Furthermore, we have demonstrated that analysing tumours as they progress in vivo is a viable approach to studying tumour biology in humans.

Fatal peri-operative acute tumour lysis syndrome precipitated by dexamethasone.

A 3-year-old patient presented for elective adenotonsillectomy to treat symptomatic obstructive sleep apnoea. The patient had not been assessed at a pre-operative anaesthesia clinic but had undergone uneventful general anaesthesia twice in the previous two years. An uneventful operative course was complicated by the development of clinical instability over the first 6 h postoperatively culminating in cardiorespiratory arrest. Subsequent investigation demonstrated the acute development of tumour lysis syndrome in the setting of a new onset, undiagnosed acute leukaemia. The patient died on the third postoperative day. The use of dexamethasone for prophylaxis against postoperative nausea and vomiting was the likely aetiology of the acute tumour lysis syndrome in this case. This is the first documented peri-operative death due to tumour lysis syndrome after administration of dexamethasone. We discuss the various problems encountered with this case and review the recent literature and case reports on tumour lysis syndrome in the operating theatre.

Cisplatin treatment increases survival and expansion of a highly tumorigenic side-population fraction by upregulating VEGF/Flt1 autocrine signaling.

The cellular and molecular mechanisms of tumor progression following chemotherapy are largely unknown. Here, we demonstrate that cisplatin (CDDP) treatment upregulates VEGF and Flt1 expression leading to the survival and expansion of a highly tumorigenic fraction of side-population (SP) cells in osteosarcoma (HOS), neuroblastoma (SK-N-BE2) and rhabdomyosarcoma (RH-4) cell lines. In all three lines, we show that CDDP treatment increases levels of VEGF and Flt1 expression, and induces enhanced clonogenic capacity and increased expression of the 'stemness'-associated genes Nanog, Bmi-1 and Oct-4 in the SP fraction. In HOS, these changes are associated with the transformation of a non-tumorigenic osteosarcoma SP fraction to a highly tumorigenic phenotype. Inhibition of Flt1 led to complete reduction of tumorigenicity in the HOS SP fraction, and reduction of clonogenic capacity and expression of stemness genes in the SK-N-BE(2) and RH-4 SP fractions. Treatment with U0126, a specific inhibitor of MAPK/ERK1,2 completely downregulates CDDP-induced VEGF and Flt1 expression and induction/expansion of SP fraction in all three cell lines, indicating that these effects are mediated through MAPK/ERK1,2 signaling. In conclusion, we report a novel mechanism of CDDP-induced tumor progression, whereby the activation of VEGF/Flt1 autocrine signaling leads to the survival and expansion of a highly tumorigenic SP fraction.

Adenovirus-mediated p53 gene therapy in osteosarcoma cell lines: sensitization to cisplatin and doxorubicin.

The poor prognosis for patients with metastatic osteosarcoma (OS) indicates that new therapeutic options should be explored. Studies with adenoviral-mediated p53 gene transfer have been conducted in many cancer types including cervical, ovarian, prostatic and head and neck tumors. However, limited work has been carried out with pediatric cancers, including OS. Using three viral constructs containing cDNA for wild-type p53, mutant p53 (Cys135Ser) and lacZ, we studied the effect of adenoviral-mediated gene therapy in four OS cell lines: Saos-2 (p53-/-), HOS (R156P), KHOS/NP (R156P) and MNNG (R156P, F270L). We demonstrated that the virus efficiently enters the cells using the beta-galactosidase assay. Using the MTT assay, we have shown a dose-dependent decrease in cell viability 72 h post-treatment that occurs with Ad-wtp53 but not with Ad-mutp53. We have also shown that treatment with Ad-wtp53 significantly increases sensitivity of the cell lines to cisplatin and doxorubicin, chemotherapeutic agents commonly used in the treatment of OS. Our results indicate that restoration of wt p53 function in OS cells provides a basis for novel approaches to treatment of this disease.

Mutant, wild type, or overall p53 expression: freedom from clinical progression in tumours of astrocytic lineage.

Abnormalities of the p53 tumor-suppressor gene are found in a significant proportion of astrocytic brain tumours. We studied tumour specimens from 74 patients evaluated over 20 years at the Massachusetts General Hospital, where clinical outcome could be determined and sufficient pathologic material was available for immunostaining. p53 expression studies employed an affinity-purified p53 monoclonal antibody, whose specificity was verified in absorption studies and, in a minority of cases, a second antibody recognising a different epitope of p53. Significant overexpression of p53 protein was found in 48% of the 74 tumours included in this series and high levels of expression were associated with higher mortality from astrocytic tumours (P<0.001, log rank). Multivariate analyses revealed that immunohistochemically detected p53 was an independent marker of shortened progression-free and overall actuarial survival in patients with astrocytic tumours, suggesting that increased expression of p53 plays an important role in the pathobiology of these tumours. In a subset of 36 cases, coding regions of the p53 gene were completely sequenced via SSCP and direct DNA sequencing, revealing that overexpression of p53 protein is not always associated with point mutations in conserved exons of the p53 gene. Finally, we confirmed p53 protein expression in early-passage human glioma cell lines of known p53 mutational status and immunostaining scores. Although grade continues to be the strongest prognostic variable, the use of p53 staining as a prognostic indicator, in contrast to mutational DNA analyses, may be a useful adjunct in identifying patients at higher risk of treatment failure.

Isochromosome (17)(q10) and translocation (4;12)(q12;p13) in a child with acute myeloid leukemia.

Isochromosome 17q is a commonly observed cytogenetic aberration in hematologic malignancies. Isolated isochromosome 17q usually presents as a marker of a chronic myeloid disorder, with a high propensity for transformation into acute nonlymphoblastic leukemia (ANLL). t(4;12)(q11-12;p13) is a recently described translocation, associated with ANLL, predominantly in adults. In this article, we present a case of acute myeloblastic leukemia (AML) in a 14-year-old female in which i(17q) and t(4;12)(q12;p13) were found in the leukemic clone at diagnosis. We briefly review the literature and hypothesize as to the significance of the coexistence of these cytogenetic changes.

Absence of germline and somatic p53 alterations in children with sporadic brain tumors.

Cancers of the central nervous system are the most common solid tumors of childhood. Although somatic alterations of the p53 tumor suppressor gene have been implicated in brain tumorigenesis, the role of germline p53 mutations in the development of childhood brain tumors has not been well defined. As a component of an ongoing extensive study of the epidemiology of childhood brain tumors, we prospectively examined the germline and tumor p53 gene status in 85 children without a family history of cancer who were diagnosed with a sporadic malignant central nervous system tumor. Using PCR/single-strand conformational polymorphism analysis and direct DNA sequencing, 85 children were screened for the presence of constitutional p53 sequence alterations in exons 2 and 4 through 11. No mutations were identified. Commonly reported sequence polymorphisms were observed at codon 72, as well as in 2 other previously described nucleotide residues. Forty-four brain tumor samples were available for analysis and of these 40 were paired with peripheral blood. Once again, no p53 mutations were found. Of the 5 germline samples with the 2 common polymorphisms, only one had a paired tumor sample for comparison and the tumor contained the same alteration as the germline. Of note, one tumor, a PNET of the cerebellum (medulloblastoma), showed loss of heterozygosity at codon 72. We can conclude that the frequency of germline and somatic p53 mutations in sporadic childhood brain tumors is very low, probably less than 1%, and there is no need to screen these patients routinely for their germline p53 status. However, the potential significance of LOH at codon 72 remains to be elucidated.

Tissue-specific expression of SV40 in tumors associated with the Li-Fraumeni syndrome.

Inactivation of wild-type p53 tumor suppressor function is the primary mechanism of tumor initiation in Li-Fraumeni syndrome (LFS) individuals with germline p53 mutations. Tumors derived from LFS patients frequently retain the normal p53 allele, suggesting that alternative mechanisms in addition to gene deletion must be involved in inactivating wild-type p53 protein. DNA tumor viruses, such as SV40, target p53 for inactivation through the action of viral oncoproteins. We studied the probands from two unrelated LFS families, each of whom presented with multiple malignant neoplasms. Patient 1 developed an embryonal rhabdomyosarcoma (RMS) and a choroid plexus carcinoma (CPC), while patient 2 developed a CPC and subsequently presented with both an osteosarcoma (OS) and renal cell carcinoma (RCC). We utilized DNA sequence analysis and immunohistochemistry to determine p53 gene status in the germline and tumors, as well as evidence for SV40 T-antigen oncoprotein expression. Each patient harbored a heterozygous germline p53 mutation at codons 175 and 273, respectively. In patient 1, the normal p53 gene was lost while the mutant p53 allele was reduced to homozygosity in the RMS. Both normal and mutant genes were maintained in the CPC. In patient 2, normal and mutant p53 alleles were retained in both the CPC and RCC. Both specific PCR and immunostaining detected SV40 T-antigen in both CPCs and the RCC. In addition to chromosomal alterations, epigenetic mechanisms may disrupt p53 function during tumorigenesis. In two LFS patients, we found SV40 DNA sequences and viral T-antigen expression that could account for inactivation of the normal p53 protein. Inactivation of p53 or other tumor suppressors by viral proteins may contribute to tumor formation in specific tissues of genetically susceptible individuals.

The role of p53 in human cancer.

In the two decades since its original discovery, p53 has found a singularly prominent place in our understanding of human cancer. Although the biochemistry of p53 has been worked out in some detail, our knowledge of the biologic consequences of p53 dysfunction is still quite rudimentary. Over the next several years, it will be important to determine how best to harness the complex properties of p53's ability to induce cellular growth arrest and cell death to generate novel, effective approaches to cancer therapy. Furthermore, a clearer appreciation of the direct interaction of epigenetic factors with p53 will lead to development of strategies to inhibit tumour initiation and progression. The next decade promises to offer exciting opportunities to apply our vast knowledge of this intriguing tumor suppressor to clinical advantage.

Germ-line p53 mutations predispose to a wide spectrum of early-onset cancers.

Germ-line p53 mutations are associated with dominantly inherited Li-Fraumeni syndrome (LFS), which features early-onset sarcomas of bone and soft tissues, carcinomas of the breast and adrenal cortex, brain tumors, and acute leukemias. However, carriers of germ-line p53 mutations may also be at increased risk of other cancers. To clarify the tumor spectrum associated with inherited p53 mutations, we examined cancer occurrences among our series of 45 families, plus 140 other affected cases and kindreds reported in the literature. The analyses included all cancers in patients with a germ-line p53 mutation and their first-degree relatives with nearly 50% likelihood of being a carrier. Data were abstracted on tumor types and ages at diagnosis in eligible family members, and duplicate reports were excluded. Among 738 evaluable cancers, 569 (77%) were the six tumor types (breast and adrenocortical carcinomas, sarcomas of the bone and soft tissues, brain tumors, and leukemias) associated with LFS. The remaining 169 (23%) cancers included diverse carcinomas of the lung and gastrointestinal tract, lymphomas, and other neoplasms that occurred at much earlier ages than expected in the general population. Unusually early ages at diagnosis are characteristic of hereditary cancers and suggest that carriers of germ-line p53 mutations are at increased risk of a wide range of neoplasms. Future studies addressing age-specific penetrance and site-specific cancer risks can increase the utility of LFS as a model for understanding the role of p53 alterations in carcinogenesis and for designing diagnostic and preventive interventions for the broad array of neoplasms in this syndrome.

Choroid plexus carcinomas and rhabdoid tumors: phenotypic and genotypic overlap.

Five of six poorly differentiated choroid plexus carcinomas identified at our institution contained cells displaying a rhabdoid phenotype. Immunoperoxidase stains showed focal positivity for cytokeratin, epithelial membrane antigen, glial fibrillary acidic protein, S100, and vimentin. The MIB-1 proliferative index ranged from 7.0% to 27.1%. All six tumors were p53 positive. Only the one child with Li-Fraumeni syndrome had a p53 germline mutation. Electron microscopy verified choroid plexus differentiation and the co-existence of rhabdoid cells. Of the five studied, four had deletions of chromosome 22 [three with monosomy 22 and one with del(22)(q12)]. Thus, there was a phenotypic and genotypic overlap between choroid plexus carcinomas and rhabdoid tumors.

Identification of a novel PTEN intronic deletion in Li-Fraumeni syndrome and its effect on RNA processing.

Germline mutations of the TP53 tumor suppressor gene account for the predisposition to cancer observed in many Li-Fraumeni syndrome (LFS) families. A causative genetic factor in the remaining families that harbor no TP53 mutations remains to be elucidated. The PTEN phosphatase tumor suppressor gene is mutated in human cancers observed in LFS. There also exists some phenotypic overlap in the occurrence of cancers in LFS and Cowden's disease (CD), for which germline PTEN mutations are believed to be responsible. We hypothesized that PTEN may be altered in some TP53-wild-type LFS families. We examined LFS primary patient lymphocytes for PTEN alterations using SSCP and sequence analysis. A novel intronic deletion was found in two unrelated individuals, adjacent to the splice acceptor site of PTEN exon 4. Based on an in vitro mRNA processing assay this alteration is predicted to be a polymorphism. The in vivo effects of this proximal splice site deletion are unknown and a genetic cause for the cancers in these families remains to be elucidated. Germline mutations of PTEN were not detected in other families, suggesting that alterations of this tumor suppressor gene do not account for the cancers observed in the subset of LFS individuals with wild-type germline TP53.

The effects of prebiopsy corticosteroid treatment on the diagnosis of mediastinal lymphoma.

BACKGROUND/PURPOSE: For children with probable mediastinal lymphoma and a high risk of cardiorespiratory morbidity, many centers recommend delaying the diagnostic biopsy for 24 to 48 hours while corticosteroids are administered to reduce tumor size and morbidity. This study was undertaken to determine the effect of preoperative steroid use on the accuracy of the pathological diagnosis and incidence of perioperative cardiorespiratory morbidity. METHODS: From 1988 to 1998, 86 children were treated for mediastinal lymphoma. Twenty-three received steroid before biopsy (study group) because of clinical evidence of respiratory compromise, and the remaining 63 served as controls. Clinical parameters, steroid use, and detailed pathological findings obtained at initial and subsequent biopsies were reviewed. Steroid treatment was considered to have had an adverse effect on the pathological diagnosis if (1) a definitive diagnosis was delayed more that 1 month, (2) a definitive diagnosis could not be made, or (3) the extent of disease could not be staged with certainty. RESULTS: Steroid treatment had an adverse effect on the pathological diagnosis in 5 of 23 (22%) children: 1 diagnostic delay, 3 failures of a definitive diagnosis, and 1 possible failure of staging. A definitive diagnosis was made in all control patients. Perioperative survival was 100% in both groups. At biopsy, only 3 children in the steroid treatment group and 2 children in the control group had moderate, nonfatal cardiorespiratory instability. Parameters of steroid use among children who had inaccurate pathological diagnoses or cardiorespiratory morbidity were not significantly different from those who did not. CONCLUSIONS: Steroid treatment before biopsy of mediastinal lymphoma may adversely affect the pathological accuracy or cause a delay in definitive diagnosis in a minority of cases. The dose and duration of steroid use was not related to outcome. Prebiopsy steroid appears to minimize the likelihood of cardiorespiratory morbidity in high-risk patients.

Assessment of systemic toxicity in children receiving chemotherapy with cyclosporine for sarcoma.

BACKGROUND: Overexpression of P-glycoprotein in malignant tumors has been associated with poor responses to chemotherapy. It appears biologically plausible that addition of the P-glycoprotein inhibitor cyclosporine (CsA) to standard chemotherapy may improve the outcome. The protective functions of P-glycoprotein in healthy tissues, however, have not been fully elucidated. Addition of CsA may lead to increased systemic chemotherapy toxicity, so we compared the rate and severity of chemotherapy-associated systemic toxicity in the presence and absence of CsA. PROCEDURE: Standard chemotherapy consisted of etoposide/ifosfamide (VP16/IFOS) cycles, alternating with vincristine/dactinomycin/cyclophosphamide (VAC) cycles. CsA was given at a median dose of 20 mg/kg with unaltered doses of the antineoplastic drugs. The analysis of toxicity was performed by comparing clinically significant toxicity events recorded during and after chemotherapy cycles with and without CsA. RESULTS: Toxicity-related hospital admissions occurred after 93% of VAC cycles with CsA compared to 40% of the cycles without CsA (P < 0. 0001); 29% of VP16/IFOS cycles with CsA led to admissions vs. 12% with non-CsA cycles (P = 0.04). Infections or fever and neutropenia were the main reasons for these admissions. Thirty-seven percent of the VAC cycles with CsA were complicated by culture-proved sepsis, which did not occur in cycles without CsA (P < 0.0001). Requirements for blood and platelet transfusions were greatly increased after VAC cycles with CsA compared to VAC cycles without CsA. CONCLUSIONS: The chemosensitizer CsA increases the systemic toxicity of VAC chemotherapy in patients with sarcomas. Future trials of chemotherapy with chemosensitizers will have to take into account a potential increase in systemic toxicity. Careful monitoring of chemotherapy-related toxicity becomes mandatory in such studies.

The role of p53 in megakaryocyte differentiation and the megakaryocytic leukemias of Down syndrome.

Sequence analysis of the p53 tumor suppressor gene was performed on diagnostic blood and bone marrow samples from nine children with Down syndrome and acute megakaryoblastic leukemia. p53 sequence alterations were not observed in any of seven cases of transient leukemia; however, samples from two of three patients with acute megakaryoblastic leukemia harbored sequence alterations. The acquisition of both mutations (Gly245Val and Arg72Pro) in the transformation from transient leukemia to overt acute megakaryoblastic leukemia suggests a functional role of mutant p53 in the evolution of this disease.