Variation in RNA expression and genomic DNA content acquired during cell culture.

Specific chromosomal abnormalities are increasingly recognised to be associated with particular tumour subtypes. These cytogenetic abnormalities define the sites of specific genes, the alteration of which is implicated in the neoplastic process. We used comparative genomic hybridisation (CGH) to examine DNA from different breast and ovarian cancer cell lines for variations in DNA sequence copy number compared with the same normal control. We also compared different sources of the MCF7 breast line by both CGH and cDNA expression arrays. Some of the differences between the subcultures were extensive and involved large regions of the chromosome. Differences between the four subcultures were observed for gains of 2q, 5p, 5q, 6q, 7p, 7q, 9q, 10p, 11q, 13q, 14q, 16q, 18p and 20p, and losses of 4q, 5p, 5q, 6q, 7q, 8p, 11p, 11q, 12q, 13q, 15q, 19p, 19q, 20p, 21q, 22q and Xp. However, few variations were found between two subcultures examined, 5 months apart, from the same initial source. The RNA arrays also demonstrated considerable variation between the three different subcultures, with only 43% of genes expressed at the same levels in all three. Moreover, the patterns of the expressed genes did not always reflect our observed CGH aberrations. These results demonstrate extensive genomic instability and variation in RNA expression during subculture and provide supportive data for evidence that cell lines do evolve in culture, thereby weakening the direct relevance of such cultures as models of human cancer. This work also reinforces the concern that comparisons of published analyses of cultures of the same name may be dangerous.

Genomic imbalances associated with acquired resistance to platinum analogues.

During the past several years, a panel of human tumor cell lines (predominantly ovarian) with acquired resistance to cisplatin, the orally bioavailable analogue JM216, and the structurally hindered analogue AMD473, has been established and characterized for underlying mechanisms of resistance. We have examined these resistant cell lines for gains and losses of DNA associated with the acquisition of resistance using the molecular cytogenetic technique of comparative genomic hybridization. Our comparison of three analogues has shown the most frequently observed changes to include amplification of 4q (5/7) and 6q (5/7), followed by amplification of 5q (3/7). We have defined four minimal common overrepresented regions, two each on 4q and 6q, which are potential loci of genes associated with platinum analogue resistance. Additional consistent abnormalities appear to be associated with cell lines sharing specific resistance mechanisms. For example, amplification of 12q was observed in the CH1 lines made respectively resistant to JM216 and AMD473 in which increased DNA repair appears to be a major mechanism of resistance for both agents. Hence, these comparative genomic hybridization studies have identified distinct chromosomal aberrations which may correlate with defined mechanisms of resistance and contain hitherto unrecognized genes that may provide targets for future therapeutic intervention.

A molecular cytogenetic approach to studying platinum resistance.

The technique of comparative genomic hybridisation (CGH) has until recently been used to screen for common genomic abnormalities in fresh tumour material; it has identified previously unrecognised regions of amplification associated with poor prognosis subtypes of breast cancer and lymphoma. Our group has applied this technique to resistant cell lines and their sensitive counterparts in order to define chromosomal abnormalities associated with acquired drug resistance. We have demonstrated the applicability of this technique to the study of drug resistance using cell lines with known mechanisms of resistance. The ability to detect novel genomic alterations in cell lines with novel mechanisms of resistance was also demonstrated. We subsequently examined the CGH profiles of seven different cell lines made resistant to three platinum analogues and showed the most consistent abnormalities to involve over-representation of regions 4q and 6q. More recently, we have applied the CGH technique to a series of testicular germ cell tumours (TGCTs) collected as formalin-fixed paraffin-embedded biopsy specimens from patients, both pre- and post-therapy using a platinum-based regimen (POMB/ACE). Previous reports have shown over-representation of X, 7q, 8q and 12p and loss of 13q to occur in 25% of primary TGCTs. Over-representation of 12p was confirmed in the majority of these biopsy samples; deletion of 13q was noted in the initial biopsies of several patients. We also demonstrated alterations of 4p, 4q, 5q and 6q in this series of patients. Newly acquired deletions of 2q and 18q and amplifications of 8q were frequently observed in post-chemotherapy samples from resistant tumours. The CGH studies on these patients with TGCT will not only enable us to correlate our observations on clinical material with those from long-term cell lines, but should also identify sites of key genes involved in clinical platinum resistance.

Abnormalities of chromosomes 8, 11, 14, and X in T-prolymphocytic leukemia studied by fluorescence in situ hybridization.

Twenty-one patients with T-prolymphocytic leukemia (T-PLL) were studied by FISH to characterize abnormalities of chromosomes 8, 11, 14, and X. A higher percentage of abnormalities of these chromosomes was detected by FISH than by cytogenetics. Seventy-one percent had inv(14) (q11q32)/t(14;14)(q11;q32). Four patients had abnormalities involving Xq28 (MTCP-1 locus) resulting from t(X;14)(q28;q11) or t(X;7)(q28;q35). These abnormalities have also been described in persistent expanding pre-malignant T-cell clones in patients with ataxia telangiectasia (AT). We have previously reported that in T-PLL and AT developing T-cell leukemia, the above abnormalities occur with additional abnormalities, mainly trisomy for 8q resulting predominantly from an i(8)(q10) and an increased expression of MYC. In this series, 81% of cases had chromosome 8 abnormalities including i(8)(q10)[43%]/t(8;8)(p12;q11)[14%], + 8[14%], and 8p + [14%]. The use of probes for MYC (8q24) and chromosome 8 centromere on metaphase chromosomes revealed that cases with i(8)(q10) were dicentric and t(8;8) monocentric. These abnormalities are not only associated with increase in dosage of 8q and the MYC gene, but also involved 8p. 8p is known to have several suppressor genes associated with solid tumors. Our findings suggest that the possible loss of a tumor suppressor gene plus the increased dosage of the q arm and/or the high expression of TCL-1/MTCP-1, which results from inv(14)/t(14;14), allows the malignant phenotype to emerge.

Bilineal acute leukemia of B and T lineage with a novel translocation t(9;17)(p11;q11).

We describe a case of bilineal leukemia in a 5-year old boy with a rare immunophenotype and the novel translocation t(9;17)(p11;q11) as the sole chromosomal abnormality. Two immunologically distinct blast cell subsets expressed T-markers (CD2, CD5, CD7) and common ALL markers (TdT, CD19, CD22, CD10), respectively. Both cell populations were CD34 negative. The patient, who presented with CNS leukemia, responded promptly to standard chemotherapy for lymphoblastic leukemia and remains in complete remission 20 months from diagnosis. Other translocations between chromosomes 9 and 17 have been infrequently reported in a variety of leukemias but as yet their biologic significance is unknown. The clinical course of this case suggests that t(9;17)(p11;q11) may not have an adverse influence on the disease outcome. However, the role of t(9;17) in the pathogenesis of this unusual lymphoid phenotype remains unresolved.

Additional chromosome abnormalities confer worse prognosis in acute promyelocytic leukaemia.

Acute promyelocytic leukaemia (APL) has been associated with a favourable prognosis in many studies of acute myeloid leukaemia. A series of 54 patients treated at the Royal Marsden Hospital between 1979 and 1996, with APL and the t(15;17) chromosome translocation at presentation, was examined for the effect of additional chromosome abnormalities in their presentation karyotype on survival. The patients were aged between 2 and 62 years with a median age of 31 years. There were approximately equal numbers of males and females. Presentation white cell count ranged from O.7 to 156 x 10(9)/l with a median of 1.0 x 10(9)/l. 39% of patients (21/54) had additional chromosome abnormalities at presentation. Statistical analyses were performed for factors thought to influence survival such as age, sex, white cell count, and number of courses of chemotherapy required to enter remission. These showed that the presence of additional chromosome abnormalities has an adverse effect on prognosis, independent of other prognostic indicators, reducing it to the level of patients with AML from less-favourable cytogenetic subgroups. These data indicate that additional therapeutic strategies may be required in patients with APL who demonstrate cytogenetic aberrations over and above the t(15;17) at presentation. The biological basis for the more aggressive nature of these cases remains to be determined.

Heterogeneity of breakpoints at the transcriptional co-activator gene, BOB-1, in lymphoproliferative disease.

Chromosome 11q23 is frequently a site of chromosomal translocation in both acute leukemias and chronic lymphoproliferative disorders. In the former, an 8 kb region within the MLL gene is consistently involved, whereas in the latter breakpoints appear to be heterogeneous. In a B cell acute leukemia cell line with t(14;18)(q32.3;q21.3) we have previously demonstrated a reciprocal translocation between the LAZ3/BCL6 gene at 3q27 and the B cell specific transcriptional coactivator gene BOB-1 at 11q23.1, implicating BOB-1 as a potential proto-oncogene. To confirm the chromosomal localization of BOB-1 we have mapped it by FISH to 11q23.1. It lay immediately telomeric of the ATM gene. We have also investigated the frequency of BOB-1 rearrangements in a panel of 32 cell lines and 71 patient samples. In one case of T cell prolymphocytic leukemia-a disease where 11q23 abnormalities are observed-a chromosomal rearrangement was identified 3.3-0.9 kb centromeric of the 3' end of the gene. Thus, there is a heterogeneity of breakpoints associated with BOB-1 while the frequency of the gene's involvement in lymphoproliferative diseases is low.

Interleukin-4 enhances the survival of severe combined immunodeficient mice engrafted with human B-cell precursor leukemia.

Human interleukin-4 (huIL-4) has been shown to inhibit the growth in vitro of cells from patients with acute lymphoblastic leukemia (ALL). With the aim of determining whether this cytokine might be useful in the treatment of patients with ALL, the effects of huIL-4 on human B-cell precursor ALL engrafted in severe combined immunodeficient (SCID) mice were examined. The inhibition of [3H] thymidine uptake of primary ALL cells by huIL-4 was maintained following engraftment and passage of leukemia in SCID mice. Five of seven xenograft leukemias showed significant inhibition in vitro by huIL-4 at concentrations as low as 0.5 ng/mL; furthermore, huIL-4 counteracted the proliferative effects of IL-7. When used to treat two human leukemias engrafted in SCID mice, huIL-4 200 microgram/kg/d, as a continuous 14-day subcutaneous infusion, suppressed the appearance of circulating lymphoblasts and extended survival of mice by 39% and 108%, respectively, the first demonstration of IL-4 activity against human leukemia in vivo. The mean steady-state huIL-4 level in mouse plasma during the infusion was 1.46 ng/mL (SEM +/- 0.14 ng/mL), which was similar to concentrations found to be effective in vitro. ALL cells obtained from mice relapsing after huIL-4 treatment continued to show inhibition by the cytokine in vitro. These data suggest that IL-4 may be useful in the treatment of patients with ALL.

Expression of c-myc oncoprotein in chronic T cell leukemias.

T cell clones in patients with ataxia telangiectasia (AT) and T cell prolymphocytic leukemia (T-PLL) have identical chromosome abnormalities, namely inv(14)(q11q32), t(14;14)(q11;q32) and t(X;14)(q27;q11). In T-PLL and AT developing T cell leukemia, the above abnormalities occur frequently together with trisomy for 8q. We postulated that the additional abnormalities of chromosome 8, where the c-myc oncogene is mapped to 8q24, may play a role in the development of overt leukemia. DNA analysis using the CD1A c-myc probe did not reveal rearrangements of the c-myc gene by Southern blotting. We have used a monoclonal antibody for the c-myc protein to investigate the level of expression in 11 patients with T-PLL and two with Sezary cell leukemia and compared it with levels seen in normal lymphocytes. Significantly higher levels were observed in patients compared with controls (P < 0.0001). The highest levels of c-myc were seen in eight cases with trisomy for 8q resulting from an i(8q). One patient was investigated before and after treatment. In the active state, c-myc showed a level of 64.36 units (range 20-200). After treatment a residual population of malignant cells showed a c-myc level of 155 (range 90-280). This study suggests that the increased expression of c-myc as a result of trisomy for 8q may have a role in the pathogenesis of de novo T-PLL and T cell leukemia supervening AT and that there may be a correlation between c-myc levels and resistance to therapy.

An eight-way variant t(15;17) in acute promyelocytic leukemia elucidated using fluorescence in situ hybridization.

A complex eight-way translocation was identified, with the aid of fluorescence in situ hybridization (FISH), in a patient diagnosed as having acute promyelocytic leukemia (APL). The balanced translocation was defined as 46,XY,t(1;6;7;6;17;15;12;3) (p22;q27;p15;q13;q21;q22;q13;p13), which includes a der(15) chromosome consistent with the der(15) chromosome of the t(15;17)(q22;q21) typically found in APL. The patient was treated with all-trans retinoic acid (ATRA) and had a clinical course typical of the disease, which is currently in remission after an autologous bone marrow transplant. The other structural rearrangements appeared to have little effect on the biology of the neoplasia.

Interstitial insertion of retinoic acid receptor-alpha gene in acute promyelocytic leukemia with normal chromosomes 15 and 17.

The translocation t(15;17)(q22;q21) is seen exclusively in patients with acute promyelocytic leukemia (APL) and in the promyelocytic blast crisis of chronic myeloid leukemia (CML). This translocation juxta-poses the promyelocytic leukemia (PML) gene on chromosome 15 and the retinoic acid receptor-alpha (RARA) gene on chromosome 17, resulting in the formation of a chimeric mRNA transcript. We describe a patient with the microgranular variant form of APL, with no detectable cytogenetic abnormality of either chromosomes 15 or 17, who nevertheless had juxtaposition of PML and RARA genes and expressed a chimeric transcript. Conventional cytogenetics showed the karyotype 46,XY,d-er(3)t(3;8)(p25;q12). Fluorescent in situ hybridization (FISH) with paints for chromosomes 8, 15, and 17 confirmed the presence of structurally intact chromosomes 15 and 17 and trisomy for chromosome 8q. Nevertheless, FISH using cosmid probes for PML and RARA showed their juxtaposition on one chromosome 15 homolog. Both genes were also present on their normal homologs; in addition, part of the RARA gene was still present on the remaining chromosome 17. DNA analysis by Southern blotting, performed with a variety of probes including PML, RARA and retinoic acid receptor-beta (RARB), showed a rearrangement in PML. Reverse transcriptase polymerase chain reaction (RT-PCR) confirmed the existence of hybrid transcripts of 276, 455 bp and 623 bp, from PML-RARA on the der(15) chromosome, consistent with alternate exon splicing of the long form of the transcript occurring in 50% to 60% of patients with APL. Our results show that APL patients with cytogenetically normal chromosomes 15 and 17 may, nevertheless, have involvement of both PML and RARA genes defining a subgroup of APL, t(15;17)-negative/PML-RARA-positive which is analogous to Philadelphia chromosome-negative/BCR-ABL-positive CML. In this case, the presence of chimeric transcripts suggests that treatment with all-trans RA may be warranted in APL, even in the absence of detectable cytogenetic change, showing the usefulness of RT-PCR or FISH to aid diagnosis.

Peripheral blood involvement in non-Hodgkin's lymphoma detected by clonal gene rearrangement as a biological prognostic marker.

Peripheral blood from 67 patients with non-Hodgkin's lymphoma was examined at initial diagnosis for the presence of circulating lymphoma cells by DNA hybridisation using immunoglobulin and T-cell receptor gene probes. Clonal gene rearrangement was found in 31% (21/67) of patients and correlated with clinical stage, histological grade and bone marrow involvement. Clinical stage and the presence of lymphoma cells in peripheral blood were prognostic factors for progression-free survival in all patients on univariate analysis, but the detection of lymphoma cells was not independent of stage. It was also not a significant predictor for survival. In patients with intermediate- and high-grade lymphoma, the detection of lymphoma cells in peripheral blood was a significant prognostic factor for progression-free survival (PFS) and survival only on univariate analysis. The 3-year PFS was 17% in patients with circulating lymphoma cells compared with 75% if these were absent (P < 0.05). The presence of lymphoma cells in peripheral blood is associated with extensive disease and may be a biological marker of poor disease control. Sensitive techniques of detection should form part of large prospective studies in non-Hodgkin's lymphoma.

Ki-ras oncogene mutations in non-HPV-associated anal carcinoma.

Human papillomavirus 16 (HPV 16) DNA is found in a high proportion of anal squamous cell carcinomas in whose genesis it is thought to play an important role. In addition, it can be shown to cooperate in vitro with activated ras oncogenes in cellular transformation. We have therefore screened a series of such tumours for activating mutations of the ras oncogene family using DNA amplified in vitro by the polymerase chain reaction (PCR) and a series of synthetic oligonucleotide probes. Mutations were seen in only two cases (both Ki-ras codon 12), neither of which was HPV-associated. Our results suggest that ras activation is not a common event in the genesis of these tumours and, when it does occur, it does not appear to cooperate with HPV.

Co-incident N and K ras gene mutations in a case of AML, restricted to differing cell lineages.

Peripheral blood from a patient with acute myeloid leukaemia (AML) of M5 FAB classification, was shown to have mutations to both the N and K ras genes. Leucophoresed blood was separated on a discontinuous Percoll density gradient to provide fractions enriched for different cell lineages. DNA extracted from these fractions was amplified using the polymerase chain reaction (PCR) technique, and hybridized with oligonucleotide probes specific for the single base mutations previously demonstrated. The N-ras mutation was shown to be restricted to the blast and monocytic cell fractions, concordant with the FAB subtype of M5. The K-ras mutation, however, was present in all fractions, suggesting it had occurred in a multi-potential stem cell representing an earlier stage in the generation of the leukaemia, or possibly an incidental background phenomenon.

Chromosomal assignments of the genes coding for human types II, III, and IV collagen: a dispersed gene family.

The human type II collagen gene, COL2A1, has been assigned to chromosome 12, the type III gene, COL3A1, to chromosome 2, and one of the type IV genes, COL4A1, to chromosome 13. These assignments were made by using cloned genes as probes on Southern blots of DNA from a panel of mouse/human somatic cell hybrids. The two genes of type I collagen, COL1A1 and COL2A1, have been mapped previously to chromosomes 17 and 7, respectively. This family of conserved genes seems therefore to be dispersed throughout the genome.

Nucleotide sequence of cDNA encoding human alpha 2-macroglobulin and assignment of the chromosomal locus.

Six alpha 2-macroglobulin (alpha 2M) cDNA clones were isolated from a human liver cDNA library by using synthetic oligonucleotides as hybridization probes. One of these, p alpha 2M1, carries a 4.6 kilobase-pair insert, which was sequenced. The insert contains the coding sequences for the mature alpha 2M polypeptide (1451 amino acids) and for a 23-amino acid signal peptide at the NH2 terminus of the precursor pro-alpha 2M. At the 3' end of the insert a poly(A) addition signal A-A-T-A-A-A and part of the poly(A) tail of the messenger RNA were found. The protein sequence deduced from the nucleotide sequence agrees with the published alpha 2M amino acid sequence for all except three residues. The alpha 2M locus was assigned to human chromosome 12 by Southern blot analysis with DNA from a panel of mouse/human somatic cell hybrids, using alpha 2M cDNA as a hybridization probe.

Genetic analysis of the 15;17 chromosome translocation associated with acute promyelocytic leukemia.

Somatic cell hybrids have been constructed between a thymidine kinase-deficient mouse cell line and blood leukocytes from a patient with acute promyelocytic leukemia showing the 15q+;17q- chromosome translocation frequently associated with this disease. One hybrid contains the 15q+ translocation chromosome and very little other human material. We have shown that the c-fes oncogene, which has been mapped to chromosome 15, is not present in this hybrid and, therefore, probably is translocated to the 17q- chromosome. Analysis of the genetic markers present in this hybrid has enabled a more precise localization of the translocation breakpoints on chromosomes 15 and 17. Our experiments also have enabled an ordering and more precise mapping of several genetic markers on chromosomes 15 and 17.