Serial cytogenetic studies of nonendemic Burkitt's lymphoma cell lines.

Cytogenetic study of nonendemic Burkitt's lymphoma (BL) cell lines showed a translocation of chromosomes #8 and #14, t(8;14), present in 16 of the 18 lines examined. Serial cytogenetic studies of nine of these lines showed the t(8;14) to be stable and present in all cells examined. Although many other chromosome aberrations were present, they did not demonstrate the stability or the pervasiveness of the t(8;14). The significance of these results and previously reported cytogenetic studies on the etiology of BL was discussed.

t(11;22) and other chromosomal rearrangements in Ewing's sarcoma.

Abnormal karyotypes from 13 human cases of Ewing's sarcoma are reported in this paper. The t(11;22) was seen in 9 cases, with 2 additional cases containing only a del(22). Other abnormalities included trisomy of 1q, translocations to 19p13, deletions of 3p and 6q, and homogeneously staining regions.

Chromosome 1 abnormalities: a common feature of pediatric solid tumors.

Abnormalities of chromosome 1 were found in 32 of 46 pediatric solid tumors including Ewing's sarcoma, Wilms' tumor, rhabdomyosarcoma, primitive neuroectodermal tumor, and hepatoblastoma. Trisomy of 1q was the most common abnormality, and breakpoints were most frequent in the region 1cen to 1p22. Abnormalities of chromosome 1 are not specific to any type of tumor. However, their frequent occurrence indicates that they may endow a clonal advantage in the development of cancer.

Ability of circular extrachromosomal DNA molecules to carry amplified MYCN proto-oncogenes in human neuroblastomas in vivo.

Amplification of the proto-oncogene MYCN (also known as N-myc) in neuroblastomas has been shown to correlate with both disease stage and prognosis, yet little is known about the DNA structures that carry amplified MYCN genes in neuroblastomas in vivo. We have used DNA irradiation and pulsed-field gel electrophoresis to analyze MYCN amplification structures in eight neuroblastomas from separate patients (four primary tumors and four metastatic lesions exhibiting MYCN amplification). Six of the eight neuroblastomas (three primary tumors and three metastatic lesions) exhibited MYCN DNA irradiation profiles consistent with the presence of circular extrachromosomal DNA amplification structures. Five neuroblastomas possessed amplification structures within the size range of double minute chromosomes, and one contained smaller DNA circles. Two neuroblastomas exhibited MYCN DNA irradiation patterns consistent with larger (presumably chromosomal) amplification structures. Multiple sizes of DNA circles were observed in the neuroblastomas of four different patients, implying in vivo multimerization of amplification structures. The presence of circular MYCN amplification structures in six of eight neuroblastomas examined suggests that circular DNA molecules are important structures in in vivo gene amplification.

Characterization of lymphoma-derived cell lines: comparison of cell lines positive and negative for Epstein-Barr virus nuclear antigen. I. Physical, cytogenetic, and growth characteristics.

Sixteen lymphoid cell lines were derived from patients with undifferentiated lymphoma of Burkitt's or non-Burkitt's type. They were obtained directly from tumor biopsies, from serous effusions, or from bone marrow. In 10 of the cell lines, the Epstein-Barr virus (EBV) nuclear antigen (EBNA) was undetectable; the remaining 6 lines were EBNA-positive (EB-pos). Of the 16 lines, 15 were aneuploid, with detectable chromosome "14q+ markers (11 had +8;14 translocations). These 15 lines, which included the EBNA-negative (EB-neg) lines, were believed to be of tumor cell origin. The remaining line consisted predominantly of diploid cells derived from normal lymphocytes, but some cells of tumor origin were present. Four EB-pos cell lines derived from EB-neg tumors had an aneuploid karyotype consistent with an origin from tumor cells (including no.8;14 translocation in two), which suggested that either tumor cells were infected with EBV in vitro or a tiny fraction of EB-pos tumor cells (or potential tumor cells) present in vivo gave rise to the predominant cell of the line. EB-neg B-cell lines and EB-pos cell lines established from undifferentiated lymphomas differed greatly. EB-neg lines had consistently smaller electronic mean cell volumes and narrow-angle light scatter than did EB-pos lines. This finding correlated with a lower nuclear:cytoplasmic ratio in EB-pos lines. EB-neg lines also had higher saturation cell densities than did EB-pos lines under standard culture conditions. The data indicate either that EBV influences the morphologic and physiologic characteristics of lymphoid cell lines or that EB-neg B-cell lines and EB-pos cell lines are derived ultimately from different lymphocyte subpopulations or that both may apply.

Receptor characteristics of the rat mammary carcinoma cell line 64-24.

Saturable binding of androgens, glucocorticoids, and triiodothyronine was found in the 64-24 hormone-responsive rat mammary carcinoma cell line. Androgen receptors had a dissociation content (Kd) for methyltrienolone of 3.4 X 10(-10) M and a binding capacity of approximately 10,000 sites/cell in whole cells. 5 alpha-[3H]dihydrotestosterone (DHT) was specifically taken up into approximately 2,150 nuclear sites with an affinity of 8.3 X 10(-10) M when nuclei were isolated from whole cells incubated with [3H]DHT. Sucrose gradient centrifugation of cytosol prepared from these cells revealed a displaceable [3H]DHT-binding component which migrated at 8S. Sedimentation analysis with high salt gradients of nuclear extracts from cells incubated with [3H]DHT revealed a peak of radioactivity in the 4S region which was abolished by coincubation of the cells with excess nonradioactive methyltrienolone. Receptors for [3H]dexamethasone were more abundant (approximately 50,000 sites/cell) in whole cells and had a Kd of 7.5 X 10(-9) M, but the number of nuclear binding sites was similar to that for androgens. Specificity studies using unlabeled steroids showed that each of the two classes of steroid receptors had greater affinities for their appropriate hormones. High affinity receptors for estrogens and progestins were not detectable in these cells. Triiodothyronine receptors were demonstrable but at a very low binding capacity (1,100 sites/cell). The Kd of these receptors was 0.6 X 10(-10) M. Cytogenetic studies revealed 44 chromosomes/mitosis with several unique markers. These receptor and karyotypic features suggest that the 64-24 cells may be useful in studying androgen action on breast cancer independently of estrogen or progestin influence, as well as the effects of thyroid hormone and glucocorticoids on breast cancer cells.

In situ selection of a human rhabdomyosarcoma resistant to vincristine with altered beta-tubulins.

In order to simulate more closely conditions in which resistance to vincristine (VCR) is selected in human solid tumors, a human rhabdomyosarcoma grown as a xenograft in immune-deprived mice has been selected for resistance in situ. Karyotype analysis showed the resistant line, HxRh18/VCR-3, to have a diploid modal number, with no apparent translocations, whereas the predominant population in the parental, sensitive HxRh18 xenograft demonstrated a modal number near-tetraploid with many marker chromosomes. From the rapid rate at which resistance was selected and from karyotypic evidence, data strongly suggest that HxRh18/VCR-3 was a subpopulation within the parent tumor. When grown in the same host, HxRh18/VCR-3 tumors accumulated less drug, and the rate of [3H]VCR loss was 5-fold greater than in HxRh18 tumors. Thus, accumulation and retention of [3H]VCR in HxRh18/VCR-3 resistant tumors was identical to that of [3H]vinblastine (VLB) in HxRh18 xenografts. HxRh18 xenografts are intrinsically resistant to VLB. Analysis by high-performance liquid chromatography of [3H]VCR:protein complexes in HxRh18 cytosols indicated one binding species (Mr 95,000 to 116,000), probably the tubulin heterodimer. Of interest was the observation that beta-tubulin species, identified on Western blots by monoclonal antibody, differed in these tumors. In HxRh18/VCR-3, less acidic beta-tubulins of HxRh18 were decreased or absent, with three additional more acidic isoforms present in the resistant line. As vincristine may bind to the beta-subunit of tubulin, this may have importance to vincristine resistance in vivo.

Amplification of the gli gene in childhood sarcomas.

The gli gene, originally identified by its amplified copy number in cells from a human malignant glioma, has a predicted translation product that contains five tandem DNA-binding zinc finger motifs related to those of Krüppel, a developmentally important Drosophila segmentation gene. Because of the potential importance of overproduction of this protein in neoplastic development, we examined DNAs from 29 cases of childhood sarcoma for evidence of amplification of the gli gene. In one of the 13 rhabdomyosarcomas studied, genomic DNA restriction fragments containing the gli gene were amplified approximately 30-fold, and expression of the 4.0-kilobase gli mRNA transcript was identified. The tumor with gli gene amplification lacked the usual histological features of alveolar or embryonal rhabdomyosarcoma; however, ultrastructural analysis of tumor cells established in culture revealed attenuated sarcomeres, resembling those found in primitive rhabdomyoblasts. Cytogenetic analysis of this cell line disclosed double-minute chromatin bodies, with no apparent rearrangements in the region of the gli locus on the long arm of chromosome 12, bands q13 to q14.3. A 15-fold level of gli amplification and gli mRNA transcripts were also detected in an established cell line from a patient with a rare form of osteosarcoma characterized by multipotential histological features. A similar level of gli gene amplification was observed in cryopreserved primary tumor cells from this patient, confirming that gene amplification took place during tumor development and not during in vitro cell culture. Amplified gli sequences were cytogenetically localized by in situ hybridization to a homogeneously staining region contained on a derivative chromosome 7. Of eight osteosarcomas and seven Ewing's sarcomas with typical histopathological features, none had detectable rearrangements or amplification of gli sequences. Thus, gli gene amplification in childhood sarcomas appears restricted to tumors with primitive histopathological features, perhaps reflecting overproduction of a gene product able to influence gene expression during early mesenchymal cell development.

Development and characterization of human ependymoma xenograft HxBr5.

We describe the successful heterotransplantation of a human ependymoma in CBA/CaJ mice immune deprived by infant thymectomy and whole-body irradiation. The xenograft, HxBr5, was established from a fourth ventricular ependymoma, locally recurrent in an 11-yr-old girl who had been treated with radiation therapy to the posterior fossa. HxBr5 retains histological and ultrastructural fidelity to the tumor from which it was derived as does the DNA content, as confirmed by flow cytometric analysis. The karyotype of the xenograft, which is pseudodiploid and exhibits trisomy 1q and deletion of 1p, is the first human ependymoma banded karyotype to be reported. Growth rates of the xenograft tumors are similar to the primary tumor as clinically observed with a doubling time of approximately 42 days. Cell kinetic parameters indicate that this slow-growing tumor has a relatively high growth fraction of 70.8% with a high cell loss of approximately 91%. We anticipate that HxBr5 may be useful as one component of a more complex model for studying the biology and differentiation of human ependymoma.

Characterization of cell lines derived from xenografts of childhood rhabdomyosarcoma.

Three human rhabdomyosarcoma cell lines (Rh10, Rh18, and Rh28) have been established from three independently derived xenografts. These lines have been characterized as mesenchymal in origin (reactivity to desmin and vimentin antibodies) and as expressing a human fetal muscle surface antigen recognized by monoclonal antibody 5.1 H11. Measurable levels of creatine phosphokinase have been detected in the cell lines. Rh10 and Rh28 exhibit the same chromosomal translocation and express an atypical lactate dehydrogenase isoenzyme which may be homologous to those previously reported in other tumor types. The karyotype analysis has confirmed that each cell line was derived from its respective tumor and thus provides a unique model for future investigations.

Nonlinkage of 16q markers to familial predisposition to Wilms' tumor.

Wilms' tumor (WT), a childhood cancer of the kidney, occurs in both familial and sporadic forms. Chromosome 11 genes have been implicated in the etiology of WT, and mutations in a gene at chromosomal band 11p13, WT1, have been identified in a few WT cases. However, 11p13 has been excluded as the site of the predisposition mutation segregating in several large WT families, which implies the existence of a non-11p familial predisposition gene. Recently, loss of heterozygosity for 16q markers located between chromosomal bands 16q13 and 16q22 has been reported in approximately 20% of sporadic Wilms' tumors. To determine if this region of 16q harbors the non-11p familial WT gene, a genetic linkage study of five WT families was undertaken. Using multipoint analyses, we ruled out genetic linkage of familial WT predisposition to 16q.

Evidence for genetic heterogeneity in familial Wilms' tumor.

Wilms' tumor (WT), a childhood kidney cancer, occurs both sporadically and, less frequently, in a familial context. Genetic linkage studies of several large WT families have excluded the one cloned WT gene, WT1, as the locus responsible for familial predisposition. These data demonstrate the existence of a familial predisposition gene distinct from WT1 and, more broadly, imply that the genetic etiology of WT is heterogenous. However, it has been unknown whether the predisposition observed in large WT families is also heterogenous or perhaps is due to mutations at a single locus. Recently, examination of a large French-Canadian WT family has demonstrated genetic linkage to 17q12-q21. We report here the results from a genetic linkage study of six WT pedigrees. Analyses of genotype data from eight loci within the 17q12-q21 region in these families resulted in cumulative lod scores of <-4.0 through the region, thereby excluding linkage. The ability to rule out the 17q region as the site of a predisposition gene in several of these pedigrees individually demonstrates the existence of more than one gene that predisposes to WT in large pedigrees and again emphasizes that the etiology of WT is genetically heterogenous.

Linkage of familial Wilms' tumor predisposition to chromosome 19 and a two-locus model for the etiology of familial tumors.

Familial predisposition to Wilms' tumor (WT), a childhood kidney tumor, is inherited as an autosomal dominant trait. For most WT families studied, the 11p13 gene WT1 and genomic regions implicated in tumorigenesis in a subset of tumors can be ruled out as the site of the familial predisposition gene. Following a genome-wide genetic linkage scan, we have obtained strong evidence (log of the odds ratio = 4.0) in five families for an inherited WT predisposition gene (FWT2) at 19q13.3-q13.4. In addition, we observed loss of heterozygosity at 19q in tumors from individuals from two families in which 19q can be ruled out as the site of the inherited predisposing mutation. From these data, we hypothesize that alterations at two distinct loci are critical rate-limiting steps in the etiology of familial WTs.

Development and characterization of pediatric osteosarcoma xenografts.

Of 33 surgical specimens of osteosarcoma obtained from 24 patients, eight were established as transplantable tumor lines in immune-deprived CBA/CaJ inbred mice. Each line retained the histological characteristics of the corresponding primary tumor and produced human lactate dehydrogenase isozymes. Volume doubling times, which ranged from a mean of 12.3 +/- 5.6 to 39.3 +/- 9.8 days, were stable for individual lines over multiple passages. Flow cytometric analysis indicated similar cellular DNA content values in the primary human tumors and established xenograft lines; the presence of two separate stem lines, as in the original tumors, was observed in the laboratory models. Comparison of two methods of immune deprivation indicated that thymectomy, whole-body irradiation, and bone marrow reconstitution was associated with a higher rate of successful engraftment than was thymectomy, 1-beta-D-arabinofuranosylcytosine treatment, and whole-body irradiation. Bone marrow-reconstituted mice also showed less variability in tumor volume doubling time. We conclude that osteosarcoma can be heterotransplanted into bone marrow-reconstituted mice with a relatively high success rate and that the xenografts retain features characteristic of the tumors of origin. The availability of these models should prove useful in the development of new therapeutic regimens and in understanding the biology of osteosarcoma.

Oncogene amplification in pediatric brain tumors.

Despite a considerable amount of information concerning chromosomal and molecular abnormalities found in gliomas in adults, relatively little is known regarding these abnormalities in pediatric brain tumors. We have analyzed DNA from 37 primary brain tumors and 4 tumor-derived cell lines for oncogene amplification. Probes utilized represent 11 known oncogenes (erbB1, gli, neu, myc, L-myc, N-myc, H-ras, K-ras, N-ras, sis, and src). Of 20 primary medulloblastomas studied, only one tumor was found to have erbB1 amplification. In contrast, of the 4 medulloblastoma cell lines studied, 1 had c-myc amplification, 1 had erbB1 amplification, and 1 had amplification of N-myc. Twelve glial brain tumors were analyzed, and only 1 case with amplification of the erbB1 oncogene was found. Other tumors studied include 1 meningioma, 2 ependymomas, 1 anaplastic ependymoma, and 1 cerebral primitive neuroectodermal tumor, none of which had oncogene amplification. These results suggest that oncogene amplification is relatively uncommon in primary medulloblastomas, but the frequency and diversity of oncogene amplification is greater in tumors that can be established as cell lines. The lower frequency of erbB1 amplification in glial brain tumors in children compared to adults is consistent with the generally lower grade of glial tumor histology seen in pediatric patients. However, the case with amplification of the erbB1 oncogene represented 1 of 2 cases of glioblastoma multiforme we studied, which suggests that pediatric glioblastoma multiforme may have a similar frequency of erbB1 oncogene amplification to glioblastomas seen in adults. Our results suggest that oncogene amplification is a relatively uncommon mechanism of oncogene activation in pediatric brain tumors, and they provide molecular evidence for heterogeneity in tumors classified as medulloblastomas.

Unique expression of the human Evi-1 gene in an endometrial carcinoma cell line: sequence of cDNAs and structure of alternatively spliced transcripts.

Retroviral insertional activation of the expression of the Evi-1 is one of the most common events associated with transformation in murine myeloid leukemia. The murine Evi-1 gene encodes a 145 kDa nuclear, DNA binding protein that contains two domains containing seven and three sets of repeats of the zinc finger motif. During studies to determine the role of the Evi-1 gene in the transformation of human cells, we have found that the Evi-1 gene is uniquely expressed at low levels in HEC-1-A cells and at high levels in HEC-1-B cells, two related human endometrial carcinoma cell lines. cDNA clones were isolated and sequenced from the HEC-1-B cell line. The human gene is highly homologous to the murine gene and shows 91% and 94% homology in nucleotide or amino acid sequence respectively. In addition an alternatively spliced form of the gene was identified that encodes a protein with an internal deletion 315 amino acids including two of the zinc finger repeats. The possible basis for the unique expression of the Evi-1 gene by HEC-1 cells could not be determined by karyotype or Southern blot analysis.

Splice-site mutation of the p53 gene in a family with hereditary breast-ovarian cancer.

Germline mutations within evolutionary conserved exons of the p53 gene predispose to tumor development in several familial cancer syndromes. We now report identification of a novel p53 mutation affecting the splice acceptor site of exon 6 in the germline DNA of a family with hereditary breast-ovarian cancer. This splice-site mutation, which results in omission of exon 6 and creates a frame-shift and premature stop codon in transcripts from the mutant allele, was found in seven family members--four of whom have developed breast, ovarian or choroid plexus tumors before age 35. Our finding suggests the need to examine the entire p53 gene for splice-site, frame-shift, and nonsense (as well as missense) mutations in families with early-onset hereditary breast and breast-ovarian cancers not linked to the BRCA1 gene on chromosome 17q. We propose that the term 'p53 familial cancer syndrome' be applied to clusters of tumors in families with documented germline p53 mutations, regardless of the histopathologic findings or pattern of tumor development.

Cisplatin, vincristine, and fluorouracil therapy for hepatoblastoma: a Pediatric Oncology Group study.

PURPOSE: To estimate the disease-free survival rate in children with grossly resected hepatoblastoma treated with cisplatin, vincristine, and fluorouracil (CDDP/VCR/FU) and to assess the disease-response rate and disease-free survival (DFS) rate in children with unresectable or metastatic tumors treated with this combination. PATIENTS AND METHODS: Sixty assessable patients with hepatoblastoma received therapy with five (stage I and II) to seven (stage III and IV) courses of CDDP (90 mg/m2), day 1, and VCR (1.5 mg/m2), and FU (600 mg/m2), day 3. RESULTS: Nineteen of 21 patients with stage I or II disease survive free of disease (actuarial survival, 90% at 5 years). Twenty-four of 31 patients with stage III disease achieved a complete remission (CR) after chemotherapy and surgical excision; actuarial DFS at 4 years is 67%. Only one of eight patients with stage IV disease achieved a remission and survives. CONCLUSION: Relatively brief exposure to chemotherapy with CDDP/VCR/FU provided excellent disease control to patients with grossly resected tumors. In patients with initially unresectable disease, this therapy provides a response rate and DFS rate comparable to regimens that contain doxorubicin (DOX).

Significance of pulmonary computed tomography at diagnosis in Wilms' tumor.

Pulmonary lesions were found by computed tomography (CT) despite normal chest roentgenograms (CXR) at diagnosis in 11 of 124 patients with Wilms' tumor. All patients were entered on a treatment protocol at St Jude Children's Research Hospital from 1978 to 1986. The 11 patients all had favorable histology Wilms' tumor. Staging and therapy were based on interpretation of the CXR and abdominal findings. Excluding CT findings, one patient had stage I disease, two stage II, seven stage III, and one stage IV on the basis of multiple liver metastases. Four patients have relapsed: one with stage II and three with stage III. All relapses have been pulmonary. Overall, 4/11 (36%) relapsed. This relapse rate is considerably greater than the 20% overall relapse rate of patients treated according to this protocol though not statistically significant. These relapses suggest that such patients may be at increased risk for pulmonary recurrence. The results also indicate that small lesions initially noted only on CT scans of the chest in children with Wilms' tumor frequently represent metastatic tumor. Further studies of larger numbers of patients will be necessary to confirm these findings.

Relationship of tumor-cell ploidy to histologic subtype and treatment outcome in children and adolescents with unresectable rhabdomyosarcoma.

Clinical and histopathologic features are often inadequate for accurate prediction of relapse or survival of individual patients with rhabdomyosarcoma (RMS). We therefore studied the cellular DNA content (ploidy) of RMS cells in relation to histology and response to therapy in 37 patients with unresectable tumors. Using flow cytometric techniques, we found that about one third of patients had diploid tumor stem lines, regardless of the histologic subtype. In the group with abnormal ploidy, a hyperdiploid classification (1.10 to 1.80 times the DNA content of normal diploid cells) was exclusively associated with embryonal histology (P = .001). By contrast, near-tetraploidy (1.80 to 2.60 times the DNA content of normal cells) was strongly associated with alveolar histology (P = .001). Thus, in these histologic subtypes of RMS, abnormal ploidy appears to arise through different mechanisms. Tumor-cell ploidy had a significant impact on survival that was especially apparent in patients with unresectable, nonmetastatic (group III) tumors. In this subgroup, hyperdiploidy conferred the best prognosis and diploidy the worst (P less than .0001). None of the eight patients with diploid tumors survived for more than 18 months. Tumor-cell ploidy was the best predictor of treatment outcome for patients with either embryonal (P less than .001; relative risk, 25.5) or alveolar (P = .073; relative risk 7.1) RMS and contributed significantly after adjustment for disease stage and anatomic site. Patients with unresectable diploid RMS have an unacceptably high risk of treatment failure, justifying new therapeutic approaches for this distinct subgroup.