Comparative cytogenetic studies of normal and leukemic lymphoblastoid cell lines during the course of their establishment.

A comparative chromosomal analysis was made of 10 human lymphoblastoid cell lines, four of which originated from normal donor lymphocytes and six of which were from leukemic peripheral blood. For comparison of lymphoblastoid cells with respect to their normal or leukemic origin, cytogenetic studies have been carried out regularly since the beginning of the culture during more than 3 years. Samples were drawn during the three phases previously described for the establishment of these lines. The chromosome distribution remained diploid for at least 2 years in normal cell lines, and the cells were euploid. In contrast, an important variability of the chromosome set was demonstrated during the same period in leukemic cell lines. Moreover, in these lines, it was always possible to observe a nonsystemic pseudodiploidy. After 2 years, a clonal evolution was described in both types of cell lines that carried at least one marker. With a controlled-heating denaturation technique, it was possible to identify the markers as specific to each cell line. The cells with marker chromosomes appeared to have a selective advantage of growth.

Phase II trial of fludarabine monophosphate in patients with mantle-cell lymphomas.

PURPOSE: The aim of this phase II trial was to assess the efficacy of fludarabine monophosphate in untreated and pretreated mantle-cell lymphomas (MCL). PATIENTS AND METHODS: Fifteen patients with MCL were included in the study. In two cases, fludarabine was the first-line therapy, the second in four cases, the third in five cases, and the fourth in four cases. The diagnosis of MCL was based on the criteria of the European Lymphoma Task Force (ELTF), with morphologic, immunologic, and cytogenetic data. Patients were treated with intravenous fludarabine 25 mg/m2/d for 5 days every 4 weeks. RESULTS: Toxicity of fludarabine was mild: World Health Organization (WHO) grade 3 and 4 granulocytopenia occurred in 15 of 56 assessable cycles (cy) (27%), there was no grade 3 or 4 thrombocytopenia, one grade 3 bacterial lung infection, and no treatment-related death. There were five partial responses (33%) but no complete response. The duration of these responses was short and ranged from 4 to 8 months. CONCLUSION: These results suggest that fludarabine can be moderately effective in the treatment of MCL. Fludarabine appears to be far less effective than in chronic lymphocytic leukemia (CLL) and follicular non-Hodgkin's lymphoma (NHL). Therefore, fludarabine should be evaluated in association with other chemotherapeutic agents in MCL.

Activity of fotemustine in medulloblastoma and malignant glioma xenografts in relation to O6-alkylguanine-DNA alkyltransferase and alkylpurine-DNA N-glycosylase activity.

Fotemustine is a chloroethylnitrosourea with antitumor activity in disseminated melanoma and adult primary brain tumors. Because new drugs are required for the treatment of medulloblastoma in children, we evaluated the preclinical antitumor activity of fotemustine in four s.c. medulloblastoma xenografts, in comparison with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Both drugs were administered as a single i.p. injection to nude mice bearing advanced-stage tumor. Fotemustine displayed significant antitumor activity in three of four medulloblastoma xenografts; two, IGRM34 and IGRM57, were highly sensitive, with 37 and 100% tumor-free survivors, respectively, more than 120 days after treatment at the highest nontoxic dose (50 mg/kg). Fotemustine was also highly active in a malignant glioma xenograft (IGRG88; five of six tumor-free survivors on day 177). Fotemustine proved to be significantly more active than BCNU in IGRM34 and the glioma xenograft IGRG88. The DNA repair protein O6-alkylguanine-DNA alkyltransferase (ATase) was detected in all tumor xenografts, ranging in activity from 6 to 892 fmol/mg protein. The high in vivo sensitivity to fotemustine and BCNU observed in three xenografts was clearly associated with a low ATase activity (> 20 fmol/mg), whereas the two poorly sensitive or refractory medulloblastoma xenografts showed high ATase activity (> 500 fmol/mg). Alkylpurine-DNA N-glycosylase activity was detected in all tumor xenografts but at levels ranging only from 513 to 1105 fmol/mg/h; no consistent relationship was found between alkylpurine-DNA N-glycosylase activity and the in vivo sensitivity to the two chloroethylnitrosoureas. The improved activity and tolerance of fotemustine in comparison with BCNU in pediatric medulloblastoma xenografts strongly support the clinical development of this agent in children with brain tumors, in which ATase should be examined as a potential prognostic indicator.

Integration and loss of a single v-Ki-ras gene affects tumorigenic potential of human osteosarcoma cells.

The human osteosarcoma cell line Te85 clone F-5 is not tumorigenic in vivo. Its transformation with Kirsten murine sarcoma virus (KiMSV) (KHOS) confers full malignant properties and stable non-tumorigenic revertants of this KHOS cell line have been obtained. Here we show that integration and expression of a single copy of the KiMSV proviral DNA, which is totally lost in the HOS 240S revertant, is responsible for the acquisition of tumorigenicity. Cytogenetic analysis and the absence of a residual LTR copy in the revertant cellular genome suggest that the loss of KiMSV provirus is caused either by chromosomal segregation or by recombination not involving the LTR. In addition analysis of the expression of ras proteins revealed no changes in the pattern of c-ras products and the expression of v-ras only in the KHOS cells. All these data suggest that Te85 and HOS 240S cell lines could represent a human alternative recipient system to rodent cells in studies with oncogenes.

DNA-topoisomerase I, a new target for the treatment of neuroblastoma.

DNA-topoisomerase I is the nuclear target of new anticancer drugs, namely camptothecin and its derivatives. In order to establish the rational basis for their clinical development in paediatric oncology, the antitumour activity of irinotecan (CPT-11) and topotecan, two camptothecin water-soluble derivatives, was studied in nude mice bearing neuroblastoma xenografts. The panel was composed of 4 previously established subcutaneous xenograft lines (IGR-N835, IGR-N91, IGR-NB3, IGR-NB8) that exhibited the common biological markers of poor prognosis in children (MYCN amplification, 1p deletion, paradiploidy and/or MDR1 overexpression). Irinotecan and topotecan were administered i.v. or i.p. over 5 consecutive days in animals bearing tumours. Irinotecan (40 mg/kg/day) induced 20-100% complete regressions with tumour growth delays ranging from 20 to 46 days. Two out of 10 IGR-N91 bearing animals were tumour free more than 120 days after treatment with the top dose (50 mg/kg/day). Topotecan (2.7 mg/kg/day) induced 0-67% complete regressions with tumour growth delays ranging from 23 to 50 days. One out of 8 IGR-NB3 bearing mice was tumour free at the end of the experiment. The antitumour activity of both drugs was clearly sustained at a lower dose level. Topoisomerase I activity was assayed in 15 neuroblastomas, 3 ganglioneuroblastomas and 2 normal adrenal glands, using a DNA relaxation assay. Topoisomerase I activity ranged from 69 to 1304 arbitrary units/mg of protein, and was significantly higher in immature neuroblastomas than in ganglioneuroblastomas and adrenal glands. In conclusion, irinotecan and topotecan are active against neuroblastoma xenografts. Their target is expressed in patients' tumour samples. Clinical development of topoisomerase I inhibitors in children with neuroblastoma is warranted.

Genistein resistance in human leukaemic CCRF-CEM cells: selection of a diploid cell line with reduced DNA topoisomerase II beta isoform.

Genistein, an isoflavonoid derivative initially described as an in vitro protein tyrosine kinase inhibitor, also inhibits mammalian DNA topoisomerase II both in vitro and in vivo. From a human leukaemic T cell line (CCRF-CEM), two genistein-resistant cell lines, which grow in the presence of 50 and 150 microM genistein, respectively, were selected and designated CEM/GN50 and CEM/GN150. Flow cytometry and karyotype analyses revealed that more than 95% of the parental cells were tetraploid whereas both resistant sublines were essentially diploid and were likely derived from the diploid fraction in the initial population. The CEM/GN cells were 3- to 4-fold resistant to genistein, and highly cross-resistant to certain metabolic inhibitors such as cytosine-arabinoside (50-fold) and 5-fluoro-2'-deoxyuridine (5000-fold). This resistance was associated with a markedly decreased uptake of thymidine and a 10-fold reduction in thymidine kinase activity. The CEM/GM cells were also 15- to 30-fold cross-resistant to topoisomerase inhibitors (etoposide, m-AMSA, 2-Me-9-OH-ellipticinium). Comparison of topoisomerase II activities in the sensitive and resistant cells showed: (i) an approximately 2-fold reduced decatenation activity in nuclear extracts from the resistant cells; (ii) an approximate 30% reduction in DNA-protein cross-links in etoposide-treated resistant cells; and (iii) a markedly reduced expression of the topoisomerase II beta isoform. These data, consistent with our previous results, indicate that the cytotoxicity of genistein is at least in part related to its capacity to inhibit DNA topoisomerase II.

Cytogenetic abnormalities in a human null cell leukemia line (REH).

The chromosomes of a null cell line (REH6) derived from peripheral leukocytes of a patient with acute lymphoid leukemia (ALL) was examined with conventional and R-banding techniques on fresh and established cells bearing ALL-associated antigen(s). Five marker chromosomes were found in both fresh and established cells. The constitution of these abnormalities is related to and explained by breaks and translocations involving five chromosomes. Furthermore, one X chromosome is completely missing. The modal chromosome number in vitro was 45 up to 10 months, and 46 from 10 to 43 months after establishment of the cell line.

A translocation (7;10)(q35;q21) in a differentiated papillary carcinoma of the thyroid.

A case of differentiated thyroid carcinoma is reported. Cell culture techniques and cytogenetic investigations are described. A t(7;10)(q35;q21) appears to be the sole chromosome abnormality.

High catabolism of BrdU may explain unusual sister chromatid differentiation and replication banding patterns in cancer cells.

Two T-cell acute lymphoblastic leukemia (ALL) cell lines, PEER and CCRF-CEM, were studied by various chromosome banding techniques, including 5-bromodeoxyuridine (BrdU) incorporation methods. Although of very similar origin, these two cell lines behave quite differently. In particular, CEM cell line exhibited an abnormal replication banding pattern (RBP) and poor sister chromatid differentiation (SCD). Study of their thymidylate synthase and thymidine kinase activities indicated that CEM had a more active salvage pathway for thymidylate synthesis than did PEER cell line, which may suggest an efficient BrdU incorporation and its fast decrease in culture medium, resulting in the observed peculiarities. However, this was contradictory to the fact that CEM need a higher dose of BrdU than do PEER cells to induce SCD and RBP. Finally, the radioactivity from 3H-thymidine decreased in the culture medium much faster for PEER cell line than for CEM cell line, and about 50% of the remaining radioactivity was due to 3H-thymidine for CEM cell line. Thus, the abnormal SCD and RBP are explained by an active catabolism of thymidine and BrdU in CEM cell line.

Cytogenetic study of a case of synchronous bilateral seminoma.

Cytogenetic analysis of a case of synchronous bilateral seminoma revealed, for each tumor, a chromosomal hypotetraploid mode with gain and loss of chromosomes and only one structural rearrangement, del(1p) and i(12p), which were secondary chromosomal events for the left and right tumors, respectively. This study allowed us to discriminate between a sole or independent origin for these two tumors.

Cytogenetic characterization of a new human papillary thyroid carcinoma permanent cell line (GLAG-66).

A new permanent cell line (GLAG-66) has been established from the metastases of a papillary thyroid carcinoma in a male patient. Herein are reported the cytogenetic characteristics of this new cell line, which is tumorigenic in athymic mice. An aneuploid chromosomal pattern was observed (48 chromosomes) with various chromosomal abnormalities. The karyotype was: 48,XY,der(1)t(9;1;9), +der(8)t(1;8),der(9)t(1;9),der(9)t(1;9), +14. This cell line should prove to be of great value in the study of the biology of human papillary thyroid carcinomas.

Cytogenetic abnormalities in thyroid adenomas.

Cytogenetic investigations using short-term cultures are reported in six thyroid adenomas. Clonal and nonclonal numerical chromosomal changes were present in two tumors. In two of the four remaining, nonclonal numerical and structural abnormalities were observed, whereas only normal karyotypes were found in the other two. Chromosome 10 and 17 were involved in several cases, although no common clonal changes could be detected. The present studies show that thyroid adenomas, benign tumors of an endocrine gland, may have chromosomal abnormalities, as described for other benign tumors.

Phenotypic and genotypic diversity of human neuroblastoma studied in three IGR cell line models derived from bone marrow metastases.

Metastatic stage IV neuroblastoma tumors, as well as cell lines derived from them, are highly malignant and rapidly fatal. To determine whether malignant potential of these cells might be influenced by stromal tissue at sites frequently involved in metastasis, we initiated primary cultures from bone marrow of three patients (331, 337, and 91) with stage IV neuroblastoma. All three explants contained two distinct cell populations, malignant neuroblasts (Nb-type) and substrate adherent stromal-like (Str-type) cells. The cell types were separated at the first passage and studied by cytogenetic, molecular, and immunocytochemical methods. Karyotypic analyses after 3-6 passages in vitro revealed the presence of unique chromosomal abnormalities in Nb-type cells of all three lines: (1) der(1)t(1;7) (p32;q11) and der(5)t(5;17)(q35;q21) in pseudodiploid IGR-N-331 neuroblasts; (2) der(1)t(1;17)(p35;q21-22) x 2 and der(7)t(7;7)(p21;q21) in IGR-N-337 hyperdiploid neuroblasts; and (3) more than six rearranged chromosomes in two related subpopulations of hypodiploid IGR-N-91 neuroblasts. Neuroblastic cells from all three tumors amplified MYCN 25- to 50-fold (with amplified genes visible as dmin or, in one IGR-N-91 subline, as an hsr(14)[q32]) and expressed N-CAM. Str-type cells from tumors 331 and 337 had a normal diploid karyotype, did not express either N-CAM or S-100, and are probably normal bone marrow fibroblasts. By contrast, S-100 negative Str-type IGR-N-91 cells were hypodiploid and shared at least two unbalanced translocations, der(4)t(1;4)(q12;p15) and der(2)t(2;10;17)(p14;q11;q22), with neuroblastic counterparts, indicating that "stromal" cells and malignant neuroblasts had a common tumor cell origin. Thus, the Str-type cells of IGR-N-91 are examples of S-type phenotypic variants frequently described for long-term human neuroblastoma cells lines in vitro, but not previously observed in vivo.

Translocation t(4;11)(q21;q23) and MLL gene rearrangement in acute lymphoblastic leukemia secondary to anti topoisomerase II anticancer agents.

Secondary therapy-related, acute lymphoblastic leukemia (S-ALL) is less common than its myeloblastic counterpart. S-ALL with MLL gene rearrangements have only been reported on six previous occasions. Only three of these had t(4;11)(q21;23) S-ALL with MLL-AF4 fusion transcript has only been reported in one earlier case. In this report a rare case of S-ALL with MLL-AF4 transcript is described in a 36 year old woman treated for breast carcinoma with chemotherapy which included the topoisomerase II inhibitor, VP-16. The precise incidence of MLL gene rearrangement in S-ALL still remains to be clarified.

Mantle cell lymphomas: characteristics, natural history and prognostic factors of 45 cases.

We reviewed 77 cases considered as lymphocytic lymphomas of intermediate differentiation or diffuse centrocytic lymphomas. Forty-five cases were diagnosed as mantle cell lymphoma (MCL). The architectural pattern was diffuse in 95%, 8 cases presented large blastoid cells and CD5 positivity was observed in 28/34 cases. Of 20 cases studied, 8 presented a t(11;14)(q13;q32). Patient characteristics were: median age 59 years, B symptoms in 38%, 87% stages III-IV, bone marrow involvement in 67% with peripheral leukemic cells in 24%. Forty-four patients were treated with chemotherapy and 7 received radiotherapy. The complete response (CR) rate was 58%. Of the 26 CR, 19 relapsed at a median of 15 months. Disease-free survival was 42% and overall survival was 73% at 3 years. In a univariate analysis, overall survival was related to liver and bone marrow involvement, the presence of peripheral lymphomatous cells and achieving a complete response.

Revision of the chromosome anomalies of the T-cell malignant cell line peer.

A high resolution chromosome banding method was applied to define the karyotype of the PEER cell line. It was found significantly different from that previously described, and can be characterized as follows: 46,XX,-4, del(5) (q21q23), del(6)(q14q22), del(9)(p12p21), i(9p), +der(4) rea(4) involving a large duplication of 4q. The cell cycle duration varies in relation to the time after splitting, slow from 0 to 48 h and faster from 48 to 96 h. The average time found was 25 h with durations of 6 and 15 h for G2 and S-phases, respectively. This variable cell cycle led us to change the conditions of BrdU incorporation to obtain a convenient R-banding. According to our own experience, this can be transposed to many other malignant cells to obtain a high resolution chromosome banding.

[Does blastic crisis as revealed in chronic myeloid leukemia simulate either acute primary leukemia or acute primary leukemia with Philadelphia chromosome?]. / Crises blastiques révélatrices de leucémies myéloïdes chroniques et simulant des leucémies aiguës primaires, ou leucémies aiguës primaires à chromosome Philadelphie?

In ten cases of apparently primary acute leukaemia, the discovery of a Philadelphia chromosome at routine examination of the caryotype led a diagnosis of blastic crisis of chronic myeloid leukemia. The clinical, cytological and cytogenetic pictures varied and only routine caryotypic examination may be used in reaching the diagnosis. The prognosis appear to be less bad than in blastic crises occurring after a long course of chronic myeloid leukaemia and closer to that of primary acute myeloid leukaemia.