A patient tumor-derived orthotopic xenograft mouse model replicating the group 3 supratentorial primitive neuroectodermal tumor in children

Background

Supratentorial primitive neuroectodermal tumor (sPNET) is a malignant brain tumor with poor prognosis. New model systems that replicate sPNET''s molecular subtype(s) and maintain cancer stem cell (CSC) pool are needed.

Methods

A fresh surgical specimen of a pediatric sPNET was directly injected into the right cerebrum of Rag2/SCID mice. The xenograft tumors were serially sub-transplanted in mouse brains, characterized histopathologically, and subclassified into molecular subtype through qRT-PCR and immunohistochemical analysis. CSCs were identified through flow cytometric profiling of putative CSC markers (CD133, CD15, CD24, CD44, and CD117), functional examination of neurosphere forming efficiency in vitro, and tumor formation capacity in vivo. To establish a neurosphere line, neurospheres were propagated in serum-free medium.

Results

Formation of intracerebral xenograft tumors was confirmed in 4 of the 5 mice injected with the patient tumor. These xenograft tumors were sub-transplanted in vivo 5 times. They replicated the histopathological features of the original patient tumor and expressed the molecular markers (TWIST1 and FOXJ1) of group 3 sPNET. CD133⁺ and CD15⁺ cells were found to have strong neurosphere-forming efficiency in vitro and potent tumor-forming capacity (with as few as 100 cells) in vivo. A neurosphere line BXD-2664PNET-NS was established that preserved stem cell features and expressed group 3 markers.

Conclusion

We have established a group 3 sPNET xenograft mouse model (IC-2664PNET) with matching neurosphere line (BXD-2664PNET-NS) and identified CD133⁺ and CD15⁺ cells as the major CSC subpopulations. This novel model system should facilitate biological studies and preclinical drug screenings for childhood sPNET.     

Establishment and characterization of clinically relevant models of ependymoma: a true challenge for targeted therapy

The development of new therapies for ependymoma is dramatically limited by the absence of optimal in vivo and in vitro models. Successful ependymoma treatment requires a profound understanding of the disease's biological characteristics. This study focuses on the establishment and characterization of in vivo and in vitro models of ependymoma to study the molecular pathways necessary for growth and progression in ependymoma. In addition, this study also emphasizes the use of these models for therapeutic intervention of ependymomas. We established optimal conditions for the long-term growth of 2 tumor xenografts and cultures of 2 human ependymoma cell lines. This study also describes the establishment of in vivo models. Histopathologic features of tumors from both intracranial and subcutaneous sites in mice revealed perivascular pseudorosettes and ependymal rosettes, which are typical morphologic features of ependymoma similar to those observed in human specimens. The in vitro models revealed glial fibrillary acidic protein and vimentin expression, and ultrastructural studies demonstrated numerous microvilli, caveolae, and microfilaments commonly seen in human ependymoma. To study signaling pathway alterations in ependymoma, we profiled established ependymoma models with Western blot analysis that demonstrated aberrant activation mainly of the phosphoinositide 3-kinase and epidermal growth factor receptor signaling pathways. Targeting phosphoinositide 3-kinase and epidermal growth factor receptor signaling pathways with small molecule inhibitors showed growth inhibitory effects. These models can also be used to study the standard therapies used for ependymomas, as shown by some of the drugs used in this study. Therefore, the models developed will assist in the biological studies and preclinical drug screening for ependymomas.     

Light microscopic demonstration of the microlumen of ependymoma: A study of the usefulness of antigen retrieval for epithelial membrane antigen (EMA) immunostaining

To determine the origin of dotlike epithelial membrane antigen (EMA) immunoreactivity of ependymoma, which is consistent with the eosinophilic globular body in hematoxylin and eosin (H&E) stain, an immuno-electron microscopic study was undertaken. The usefulness of antigen retrieval pretreatment in detecting the dotlike EMA immunoreactivity in ependymomas was also studied. The materials were 29 ependymomas, 7 autopsy brains as a normal control, and 50 brain tumors of various types. The study confirmed that most of the brown dots in EMA immunostain in ependymoma represented microlumina of tumor cells. In ependymomas, plain EMA immunostaining showed dotlike positivity in only six cases (21%), and antigen retrieval pretreatment increased the number of positives up to 26 cases (90%). Antigen retrieved CD99 detected 23 positive cases (80%) in ependymomas. On the basis of the results, although some false positive findings were raised by antigen retrieval pretreatment, the authors positively recommend adoption of the technique, especially when ependymoma remains as one of the differential diagnoses of the tumor.     

人肺癌组织免疫缺陷鼠移植瘤模型的建立

背景与目的：目前提倡对肿瘤实行靶向药物个性化治疗,建立不同通路异常肿瘤的动物模型成为必需。本研究探讨人非小细胞肺癌手术标本免疫缺陷鼠皮下移植瘤模型的建立及建模的适宜条件和方法。方法：选取BALB／c裸小鼠、SCID小鼠及NOD／scid小鼠各16只,采用套管针四点左边两块、右边一块式接种法,16例患者中每例患者的肿瘤组织标本分别接种于每个鼠种的一只。观察不同种免疫缺陷鼠移植瘤成瘤率、成瘤潜伏时间、肿瘤体积倍增时间、自然病亡率及左右两边接种点的成瘤率和头尾部成瘤点处死时大于1cm的比率,并鉴别移植瘤形态结构与来源组织的一致性及鉴定移植瘤是否为上皮来源。结果：总成瘤率为75％,其中4个病例只有SCID小鼠成瘤．2个病例只有BALB／c裸小鼠成瘤,NOD／scid小鼠无一例单独成瘤。不同种免疫缺陷鼠移植瘤成瘤率、成瘤潜伏时间和肿瘤体积倍增时间及左右两边接种点的成瘤率差异无统计学意义。SCID小鼠成瘤率最高,为56．25％;NOD／scid小鼠自然病亡率为25％,BALB／c裸小鼠、SCID小鼠在观察期内无一例病亡;处死时头部成瘤点大于1cm的比率（56．52％）与尾部成瘤点大于1cm的比率（25％）相比差异有统计学意义（P=0．037）;所有移植瘤苏木精．伊红染色显示其形态结构均与来源组织一致,所有移植瘤角蛋白免疫组化均为阳性,显示移植瘤均为上皮来源。结论：四点两块套管针接种BALB／c裸小鼠和SCID小鼠各一只的方法能很好地建立非小细胞肺癌手术标本免疫缺陷鼠皮下移植瘤模型,头部移植瘤的生长速度明显快于尾部。     

Radial glia cells are candidate stem cells of ependymoma

Tumors of the same histologic type often comprise clinically and molecularly distinct subgroups; however, the etiology of these subgroups is unknown. Here, we report that histologically identical, but genetically distinct, ependymomas exhibit patterns of gene expression that recapitulate those of radial glia cells in the corresponding region of the central nervous system. Cancer stem cells isolated from ependymomas displayed a radial glia phenotype and formed tumors when orthotopically transplanted in mice. These findings identify restricted populations of radial glia cells as candidate stem cells of the different subgroups of ependymoma, and they support a general hypothesis that subgroups of the same histologic tumor type are generated by different populations of progenitor cells in the tissues of origin.     

Eradication of tumors from a human colon cancer cell line and from ovarian cancer metastases in immunodeficient mice by a single-chain Ep-CAM-/CD3-bispecific antibody construct

Bispecific T-cell engager (BiTE) are a class of bispecific single-chain antibodies that can very effectively redirect cytotoxic T cells for killing of tumor target cells. Here, we have assessed the in vivo efficacy of one representative, called bscEp-CAMxCD3, with specificity for tumors overexpressing epithelial cell adhesion molecule (Ep-CAM) in human xenograft models. Cells of the human colon carcinoma line SW480 were mixed at a 1:1 ratio with unstimulated human peripheral mononuclear cells, s.c. injected in nonobese diabetes/severe combined immunodeficiency (NOD/SCID) mice, and animals were treated with bscEp-CAMxCD3. Five daily i.v. injections of as little as 100 ng per mouse of bscEp-CAMxCD3 completely prevented tumor outgrowth when treatment was started at the day of tumor cell inoculation. BscEp-CAMxCD3 was also efficacious when administered up to 8 days after xenograft injection. Established tumors could be eradicated in all animals by five 10 microg doses given between days 8 and 12 after tumor cell inoculation. To test the efficacy of bscEp-CAMxCD3 in a more physiologic model, pieces of primary metastatic tumor tissue from ovarian cancer patients were implanted in NOD/SCID mice. Partial tumor engraftment and growth was observed with four of six patient samples. Treatment of established tumors with daily 5 microg doses led to a significant reduction and, in some cases, eradication of human tumor tissue. These effects obviously relied on the tumor-resident T cells reactivated by bscEp-CAMxCD3. Our data show that the class of single-chain bispecific antibodies has very high antitumor efficacy in vivo and can use previously unstimulated T cells at low effector-to-target ratios.     

Establishment of human colon cancer cell lines from fresh tumors versus xenografts: comparison of success rate and cell line features

Obtaining representative human colon cancer cell lines from fresh tumors is technically difficult. Using 32 tumor fragments from patients with colon cancer, the present study shows that prior xenograft leads to more efficient cell line establishment compared with direct establishment from fresh tumors (P < 0.05). From 26 tumor specimens, we successfully established 20 tumor xenografts in nude mice (77%); among 19 of these xenografts, 9 (47%) led to cell lines, including four from liver metastases. Only 3 of 31 tumor specimens (9.7%) grew immediately in vitro, and all were derived from primary tumors. To compare major phenotypic and genotypic characteristics of human colon cancer cell lines derived from the same tumor fragment using two protocols, the two pairs of cell lines obtained from 2 of 32 tumor fragments were extensively studied. They displayed similar morphology and were able to form compact spheroids. Chemosensitivity to 5-fluorouracil, CPT11, and L-OHP differed between cell lines obtained from patient tumors and those derived from xenografts. Matched cell lines shared a common core of karyotype alterations and distinctive additional chromosomal aberrations. Expression levels of genes selected for their role in oncogenesis evaluated by real-time quantitative PCR were found to be statistically correlated whatever the in vitro culture model used. In conclusion, xenotransplantation in mice of tumor fragments before establishment of cell lines enables generation of more novel human cancer cell lines for investigation of colon cancer cell biology, opening up the opportunity of reproducing the diversity of this disease.     

Growth of Human Lymphoma Cells in SCID Mice

Two EBV-negative human lymphoma cell lines raised in this laboratory and peripheral blood cells of a patient with large cell lymphoma in leukemic phase were injected intravenously or intraperitoneally into C.B.17 SCID mice. One line (OCI-LY18) was derived from the pleural fluid of a patient with a large cell, immunoblastic malignant lymphoma. Cells of this line are of B cell origin and characterized by multiple rearrangements of the JH locus. The second line (OCI-LY17) was grown from the peripheral blood of a patient with a large cell lymphoma of T-cell phenotype which has characteristic rearrangement of the T-cell receptor beta chain. The cells directly obtained from a patient with large cell non-cleaved malignant lymphoma were of B-cell origin.

Animals carrying OCI-LY18 developed large tumor masses within 6-8 weeks of inoculation. The tumors were detected in the intestine, mesentery, retroperitoneum, lymph nodes, spleen, lung and kidney. The masses resembled the primary tumor with respect to histological appearance and immunological phenotype. It was possible to generate secondary cell lines from the tumors found in the inoculated SCID mice. Injection of one of these secondary cell lines into SCID mice resulted in the rapid development of lymphoma and as few as 10 cells were sufficient to establish the disease in the inoculated animals. In contrast cells of OCI-LY17 produced small tumor aggregates that did not appear to progress over time and did not cause death of the animals. The tumors were identified by the same phenotypic profile as that seen in the primary cell line and sections of the patient derived lymph node biopsy.

The sample of peripheral blood cells directly derived from the patient gave rise to tumor nodules in multiple locations. Their morphology and immunological phenotype was consistent with the original disease seen in the patient.     

Tumors arising in SCID mice share enhanced radiation sensitivity of SCID normal tissues.

We addressed the question of whether cancers arising in an abnormally radiation sensitive normal tissue are also abnormally sensitive to ionizing irradiation. Germ line mutation-carrying mice with an enhanced radiation sensitivity of the normal tissue, the severe combined immunodeficient (SCID), and normally radiation sensitive mice (C3H) were used to study the sensitivity of normal and tumor tissues in vivo and in vitro. The lethal dose for 50% of the irradiated animals after single dose whole body irradiation was 2.6-fold higher in C3H compared to SCID mice. The dose for an isoeffective acute skin reaction after single dose irradiation was end point dependent 1.7 to 3.7 times higher in C3H than in SCID mice. Embryonic fibroblast and methylcholanthrene induced soft tissue sarcomas derived from C3H and SCID mice were established in vitro and colony-forming assays after single dose irradiation were carried out. Choosing mean inactivation dose as the end point, SCID fibroblast lines were 3.0-fold and SCID tumor cell lines 2.7-fold more radiation sensitive than C3H fibroblast lines and C3H tumor cell lines. Tumor control and growth delay assays for 110-mm3 tumors were used to compare the radiation sensitivity of SCID and C3H tumors in vivo. The doses for 50% local tumor control and a growth delay of 40 days were 2.6 times higher in C3H tumors compared to SCID tumors. Tumors arising in an abnormally radiation sensitive normal tissue are also sensitive to irradiation. The difference in radiation sensitivity of normal tissues predicted the difference in tumor tissues in these two murine systems.     

EGFR tyrosine kinase inhibition radiosensitizes and induces apoptosis in malignant glioma and childhood ependymoma xenografts

Malignant gliomas and childhood ependymomas have a high rate of treatment failure. Epidermal growth factor receptor (EGFR) activation has been implicated in the tumorigenesis and radioresistance of many cancers, including brain tumors. Therefore, combining EGFR targeting with irradiation is a potentially attractive therapeutic option. We evaluated the tyrosine kinase inhibitor gefitinib for its antitumor activity and potential to radio-sensitize in vivo in two xenograft models: an EGFR amplified glioma and an EGFR expressing ependymoma, both derived from primary tumors. When administered at 100 mg/kg for 5 consecutive days, gefitinib-induced partial tumor regression in all treated EGFR amplified IGRG88 glioma xenografts. The addition of 1 Gy of irradiation prior to gefitinib administration resulted in 5 complete and 4 partial regressions for the 9 treated tumors as well as a significant tumor growth delay of 33 days for the combined treatment compared to 19 days for each therapy alone, suggesting additive antitumor activity. Tumor regression was associated with inhibition of AKT and MAPK pathways by gefitinib. In contrast, the ependymoma IGREP83 was sensitive to irradiation, but remained resistant to gefitinib. Combined treatment was associated with inhibition of radiation-induced MAPK phosphorylation and significant induction of apoptotic cell death though radiation-induced AKT phosphorylation was maintained. Depending on the scheduling of both therapies, a trend towards superior antitumor activity was observed with combined treatment. Thus, EGFR targeting through tyrosine kinase inhibition appears to be a promising new approach in the treatment of EGFR-driven glioma, particularly in combination with radiation therapy.     

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 a novel human anaplastic large cell lymphoma cell line tumorigenic in SCID mice

L82, a novel anaplastic large cell lymphoma (ALCL) cell line was established from the pleural effusion of a 24-year-old patient with recurrent ALCL. L82 cells showed the typical morphologic features of ALCL cells with irregular, often indented, nuclear profiles, prominent nucleoli, and abundant cytoplasm. The immunoprofile of L82 corresponds to that seen typically in primary ALCL cells, with positivity for CD30, EMA, CD3, CD4, CD25, CD71, TIA1, and granzyme B; the cells were negative for EBV-related antigens. Cytogenetic analysis showed a complex, near triploid karyotype with 72-77 chromosomes, including the ALCL specific translocation t(2;5)(p23;q35). Chromosomal analysis revealed a number of secondary structural alterations including amplification of 7q21-31, 1q, and 6p, and gain of chromosomal material in 8q (affecting the c-myc gene). The rearrangement of the T-cell receptor-gamma locus shows that L82 is clonally derived from T-lineage lymphoid cells. mRNAs for interleukin 7 (IL-7), IL-8, IL-9, IL-10, TNF-beta, and for the IL-7 and IL-9 receptor were found. These data show that the T-helper cell (Th)1/Th2 balance was polarized to Th2. L82 were inoculated intraperitoneally into 4 week-old SCID mice and produced a disseminated tumor within 4-6 weeks. Morphological, immunohistochemical, and molecular genetic investigation confirmed that the xenograft and the original ALCL tumor were identical. SCID mice xenografted with the human ALCL cell line, L82, provide a useful model system for the investigation of the biology of ALCL and of new therapeutic approaches, such as specific immunotherapy.     

A case of cervicomedullary junction tanycytic ependymoma associated with marked cyst formation

Tanycytic ependymomas are a subtype of ependymomas that were formally recognized as a new pathological entity in the latest World Health Organization (WHO) classification of 2000. They occur mostly in the spinal cord. Only a few reports have analyzed the proliferative potentials of these tumors; however, it has been reported that the MIB-1 labeling index of tanycytic ependymoma is lower than that of other subtypes of WHO grade II ependymomas. We report a rare case of cervicomedullary junction tanycytic ependymoma associated with marked cyst formation. A 62-year-old man had a history of progressive gait disturbance, diplopia, and swallowing disturbance over a one-month period prior to admission. Magnetic resonance imaging (MRI) showed a cystic mass with a mural nodule at the cervicomedullary junction with Gd-DTPA enhancement. Cyst-subarachnoid shunt was performed using a far lateral approach. After 6 years, however, the man was readmitted to the hospital because of reaccumulation of the cyst. Partial removal of a mural nodule and a cyst-subarachnoid shunt were performed simultaneously by a midline suboccipital approach. The pathological diagnosis was tanycytic ependymoma. Postoperatively, the patient recovered well and was discharged from the hospital without further treatment. Most of the tumor cells had small, round nuclei; pleomorphism was minimal. The cytoplasm was dilated. The tumor cells were positive for EMA and s-100, and negative for CD-34. GFAP was not determined due to difficulty caused by background glial processes. The MIB-1 labeling index was less than 1%. Ultrastructurally, the tumor cells had ependymal cell features, such as desmosomes and microvilli. Based on these findings, the pathological diagnosis was tanycytic ependymoma.     

Growth of human lymphoma cells in SCID mice.

Two EBV-negative human lymphoma cell lines raised in this laboratory and peripheral blood cells of a patient with large cell lymphoma in leukemic phase were injected intravenously or intraperitoneally into C.B.17 SCID mice. One line (OCI-LY18) was derived from the pleural fluid of a patient with a large cell, immunoblastic malignant lymphoma. Cells of this line are of B cell origin and characterized by multiple rearrangements of the JH locus. The second line (OCI-LY17) was grown from the peripheral blood of a patient with a large cell lymphoma of T-cell phenotype which has characteristic rearrangement of the T-cell receptor beta chain. The cells directly obtained from a patient with large cell non-cleaved malignant lymphoma were of B-cell origin. Animals carrying OCI-LY18 developed large tumor masses within 6-8 weeks of inoculation. The tumors were detected in the intestine, mesentery, retroperitoneum, lymph nodes, spleen, lung and kidney. The masses resembled the primary tumor with respect to histological appearance and immunological phenotype. It was possible to generate secondary cell lines from the tumors found in the inoculated SCID mice. Injection of one of these secondary cell lines into SCID mice resulted in the rapid development of lymphoma and as few as 10 cells were sufficient to establish the disease in the inoculated animals. In contrast cells of OCI-LY17 produced small tumor aggregates that did not appear to progress over time and did not cause death of the animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histological spectrum of ependymomas and correlation of p53 and Ki-67 expression with ependymoma grade and subtype

BACKGROUND: Clinical and histological criteria for ependymoma prognosis are well recognized. Recently few studies have been done based on Immunohistochemistry for prognostication of these tumours. In this study we have correlated the histological spectrum with immmunoexpression of p53 and Ki67 in these tumors. AIMS: To know the incidence of ependymomas; study their morphological spectrum and to evaluate expression of P53 and Ki 67 in different morphological subtypes. MATERIAL AND METHOD: A retrospective study was preformed on 70 ependymomas received in a period between 1994 and 2001. Entire tissue received was processed for routine paraffin embedded H&E stained sections. Immunocytochemistry was performed using antibodies to GFAP, EMA, Pancytokeratin and synaptophysin, to differentiate papillary ependymoma from choroid plexus papilloma; clear cell ependymoma from oligodendroglioma and central neurocytoma; ependymoblastoma from other embryonal tumours. p53 and Ki-67 immunohistochemistry was performed to correlate their expression with various tumour grades and subtypes. RESULTS: There were 3 cases (4.2%) of Grade I ependymoma (2 cases of myxopapillary ependymoma and 1 case of subependymoma); 57 cases (81.5%) of ependymoma grade II (43 of these were of classical variety, 11 of clear cell ependymoma, 2 of papillary and 1 case of cellular ependymoma). There were 9 cases (12.8%) of anaplastic ependymoma (one of these was a clear cell ependymoma and 1 case (1.5%) of ependymoblastoma CONCLUSION: p53 and Ki67 indices can be used in routine diagnostic laboratories to supplement the tumor grade on histology and more studies with follow up should be performed to analyse the prognosis of different subtypes. The expression of Ki 67 and p53 was significantly higher in anaplastic ependymomas. 4 out of 11 cases of clear cell ependymomas showed higher Ki 67 indices as compared to classical grade II ependymomas, thus further highlighting the importance of differentiating the various subtypes.     

Bcl-2 antisense oligonucleotides are effective against systemic but not central nervous system disease in severe combined immunodeficient mice bearing human t(14;18) follicular lymphoma.

The t(14;18) is present in 85-90% of follicular lymphomas. It results in overexpression of the Bcl-2 protein, which inhibits apoptosis and plays a role in lymphomagenesis. Bcl-2 antisense oligonucleotides (ODNs) down-regulate Bcl-2 expression and inhibit growth of the follicular lymphoma cell line WSU-FSCCL. In this study, we have established a human lymphoma xenograft model in severe combined immunodeficient (SCID) mice using the WSU-FSCCL cell line. s.c., i.v., or i.p. injection of WSU-FSCCL cells into SCID mice results in the development of disseminated tumors, with the liver, spleen, bone marrow, and lymph nodes as major sites of disease. Tumors were fatal in 7-14 weeks, depending on cell inoculum and route of administration. Immunohistochemistry, flow cytometry, and cytogenetic analysis confirmed the human B-cell origin of tumor cells in the xenograft. Phosphorothioate ODNs against the translation initiation site of bcl-2 mRNA in the antisense and mismatched antisense sequences were administered i.v. or i.p. to the xenograft models three times a week for 2 weeks, starting on day 7 after tumor injections. Antisense-treated animals had significantly longer survival (mean, 11.6 weeks) compared with 7.6 weeks for the control group and 7.5 weeks for the mismatched antisense-treated animals (P = 0.002 and 0.004, respectively). More significantly, a pathological examination showed no tumor in the liver, spleen, or bone marrow of the antisense group. However, subsequent experiments showed that the central nervous system was involved, causing mice to die although other sites were disease free. We conclude that bcl-2 antisense ODN therapy is effective against systemic FSCCL disease in SCID mice xenografts; however, it does not prevent disease dissemination into the central nervous system causing animal death.     

Specific chromosomal imbalances as detected by array CGH in ependymomas in association with tumor location,histological subtype and grade

Ependymomas are glial neoplasms originating from the wall of the ventricles or from the spinal canal. The significance of histopathological features in accurately predicting biological behavior is still debated. Moreover, key molecular events in the pathogenesis of ependymoma are yet to be defined. The main objective of the present study was to identify specific patterns of chromosomal aberrations that correlate with tumor location, histological subtype and grade. Forty-five ependymoma samples were analyzed by 1-megabase resolution array comparative genomic hybridization (CGH). Association between clinical or histopathological parameters and the genomic alterations identified was evaluated. The most frequently detected chromosome (chr) abnormalities were gain of chr 7, 9, 12 and 15 and loss of chr 22. Co-occurrence of those five alterations characterized spinal ependymomas (P = 0.01). Myxopapillary ependymomas displayed a specific genomic profile defined by concurrent gain of chr 5, 7, 9, 16 and 18 (P = 0.0007). Overall, the number of chromosomal abnormalities detected was inversely correlated with the malignancy grade. Gain of chr 1q correlated with intracranial high-grade tumors (P = 0.002). Loss of chr 6q was mainly observed in infratentorial (P = 0.02) and World Health Organization (WHO) grade III (P = 0.04) lesions. Chr 10q loss was associated with high-grade ependymomas (P = 0.01). The +7/+9/+12/+15/−22 genomic profile is significantly associated with WHO grade II spinal ependymomas, whereas the +5/+7/+9/+16/+18 genomic pattern is specific of myxopapillary ependymomas. Identification of specific genomic imbalances at a given tumor location suggests that ependymomas from different central nervous system (CNS) regions represent genetically distinct diseases. Detecting genomic alterations associated with aggressive biological behavior may help stratify patients in high- and low-risk disease.     

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.     

Laminin adhesion-selected primary human colon-cancer cells are more tumorigenic than the parenteral and nonadherent cells

Laminin has been shown to promote the malignant phenotype and the level of the 32/67 Kd laminin receptor has been found to correlate with Dukes' staging of colon cancer. A biopsy of a Dukes' stage B2 human colon carcinoma formed a tumor in a nude mouse after coinjection with Matrigel. The parental tumor and the murine tumor appeared identical at the histological level. A cell line LCC-C1 was established from the murine tumor. The cell line appeared moderately differentiated although it did not produce mucin in vitro; however, the xenograft in vivo did produce low levels of mucin. Laminin adherent and non-adherent cell lines were selected. The parental and the laminin-selected cell subclones adhered equally well to plastic and to fibronectin and showed similar growth rates on plastic. When injected subcutaneously into nude mice, the laminin-adherent cells formed relatively undifferentiated tumors that were twice as large as the parental cell tumors whereas the laminin non adherent cells formed very small, but highly differentiated tumors. These data demonstrate that subpopulations of tumor cells which differ in their tumorigenic properties can be selected based on their adhesion to laminin and thus provide models for studying the mechanisms of tumor growth.     

Characterization of a New Primary Human Pancreatic Tumor Line

A primary human pancreatic tumor line (BxPC-3) has been established from a biopsy specimen of a histologically confirmed adenocarcinoma of the body of the pancreas. Tumorigenicity was proven by xenograft in athymic nude mice. Upon re-establishment of tumor xenografts in tissue culture, the epithelial tumor cells retained their original morphology. Histopathologically, the tumors grown in nude mice exhibited the original characteristics of the primary adenocarcinoma in the patient, producing traceable mucin and displaying moderately well to poorly differentiated adenocarcinomas with occasional lymphocytic infiltrations at the tumor peripheries. Furthermore, the tumor xenografts differentially expressed carcinoembryonic antigen, human pancreas cancer-associated antigen, and human pancreas-specific antigen. Karyotyping and glucose-6-phosphate dehydrogenase isoenzyme characterization revealed that this tumor line was of human origin and devoid of HeLa cell contamination. The BxPC-3 tumor line has been maintained for more than four years in our laboratory and represents a valuable model for primary human pancreatic cancer.