Detection of minimal residual cells carrying the t(14;18) by DNA sequence amplification.

By means of the polymerase chain reaction (PCR) technique, DNA sequences were amplified that flank the crossover sites of a characteristic chromosomal translocation for follicular lymphomas, t(14;18)(q32;q21). This technique permitted the detection of cells carrying the t(14;18) hybrid DNA sequences at a dilution of 1:100,000. The remission marrow and blood samples of a patient with follicular lymphoma and the t(14;18) failed to show any abnormality by morphological examination and conventional Southern blot analysis. However, the t(14;18) hybrid DNA sequences were detected by the PCR technique. Thus, this technique is a highly sensitive tool to detect minimal residual cells carrying the t(14;18) and has the potential to identify a subpopulation of patients with subclinical disease.

Identification of putative oncogenes in lung adenocarcinoma by a comprehensive functional genomic approach.

Amplification and overexpression of putative oncogenes confer growth advantages for tumor development. We used a functional genomic approach that integrated simultaneous genomic and transcript microarray, proteomics, and tissue microarray analyses to directly identify putative oncogenes in lung adenocarcinoma. We first identified 183 genes with increases in both genomic copy number and transcript in six lung adenocarcinoma cell lines. Next, we used two-dimensional polyacrylamide gel electrophoresis and mass spectrometry to identify 42 proteins that were overexpressed in the cancer cells relative to normal cells. Comparing the 183 genes with the 42 proteins, we identified four genes - PRDX1, EEF1A2, CALR, and KCIP-1 - in which elevated protein expression correlated with both increased DNA copy number and increased transcript levels (all r > 0.84, two-sided P < 0.05). These findings were validated by Southern, Northern, and Western blotting. Specific inhibition of EEF1A2 and KCIP-1 expression with siRNA in the four cell lines tested suppressed proliferation and induced apoptosis. Parallel fluorescence in situ hybridization and immunohistochemical analyses of EEF1A2 and KCIP-1 in tissue microarrays from patients with lung adenocarcinoma showed that gene amplification was associated with high protein expression for both genes and that protein overexpression was related to tumor grade, disease stage, Ki-67 expression, and a shorter survival of patients. The amplification of EEF1A2 and KCIP-1 and the presence of overexpressed protein in tumor samples strongly suggest that these genes could be oncogenes and hence potential targets for diagnosis and therapy in lung adenocarcinoma.

Characterization of a fusion cDNA (RARA/myl) transcribed from the t(15;17) translocation breakpoint in acute promyelocytic leukemia.

A nonrandom chromosomal translocation breakpoint, t(15;17)(q22;q21), is found in almost all patients with acute promyelocytic leukemia (APL). Most of these breakpoints occur within the second intron of the retinoic acid receptor-alpha (RARA) gene. We screened a cDNA library of APL and have identified and sequenced a cDNA transcribed from the t(15;17) translocation breakpoint. The 5' end of cDNA p1715 consists of 503 bp of the RARA exon II sequence. A 1.76-kb cDNA without homology to any known gene available in GenBank was found truncated downstream. This cDNA sequence was assigned to chromosome 15 by dot blot hybridization of the flow cytometry-sorted chromosomes. We designate this fusion cDNA RARA/myl, which is different from myl/RARA reported by de The et al. (H. de The, C. Chomienne, M. Lanotte, L. Degos, and A. Dejean, Nature (London) 347:558-561, 1990). This result demonstrates that the two different types of hybrid mRNA can be transcribed from this breakpoint. We screened a non-APL cDNA library and identified a 2.8-kb myl cDNA. This cDNA is able to encode a polypeptide with a molecular weight of 78,450. Alternative splicing of the myl gene which resulted in myl proteins with different C terminals was found. Southern blot analysis of the genomic DNA isolated from 17 APL patients by using the myl DNA probe demonstrated that the myl gene in 12 samples was rearranged. Northern (RNA) blot analysis of RARA gene expression in two APL RNA samples showed abnormal mRNA species of 4.2 and 3.2 kb in one patient and of 4.8 and 3.8 kb in another patient; these were in addition to the normal mRNA species of 3.7 and 2.7-kb. The myl DNA probe detected a 2.6-kb abnormal mRNA in addition to the normal mRNA species of 3.2, 4.2, and 5.5 kb. Using the polymerase chain reaction, we demonstrated that both RARA/myl and myl/RARA were coexpressed in samples from three different APL patients. From this study, we conclude that the t(15;17) translocation breakpoint results in the transcription of two different fusion transcripts which are expected to be translated into fusion proteins.

Branched co-polymers of histidine and lysine are efficient carriers of plasmids.

We previously determined that a linear co-polymer of histidine and lysine (HK) in combination with liposomes enhanced the transfection efficiency of cationic liposomes. In the current study, we designed a series of HK polymers with increased branching and/or histidine/lysine ratio to determine if either variable affects transfection efficiency. In the presence of liposomes, the branched polymer with the highest number of histidines, HHK4b, was the most effective at enhancing gene expression. Furthermore, when serum was added to the medium during transfection, the combination of HHK4b and liposomes as a gene-delivery vehicle increased luciferase expression 400-fold compared to liposomes alone. In contrast to linear HK polymers, the higher branched HHK polymers were effective carriers of plasmids in the absence of liposomes. Without liposomes, the HHK4b carrier enhanced luciferase expression 15-fold in comparison with the lesser branched HHK2b carrier and increased expression by 5-logs in comparison with the HHK or HK carrier. The interplay of several parameters including increased condensation of DNA, buffering of acidic endosomes and differential binding affinities of polymer with DNA have a role in the enhancement of transfection by the HK polymers. In addition to suggesting that branched HK polymers are promising gene-delivery vehicles, this study provides a framework for the development of more efficient peptide-bond-based polymers of histidine and lysine.

Uropod-bearing lymphocytes (hand mirror cells) in a virus-induced murine lymphoma.

A Moloney murine leukemia virus-induced T-cell preleukemic thymic lymphoma tissue culture from an inbred C3H/HeJ mouse contained numerous hand mirror cells (HMC). The cells were studied by light and phase-contrast microscopy, special stains, indirect immunofluorescence for terminal deoxynucleotidyl transferase, and scanning and transmission electron microscopy. The uropods of the mouse and human HMC were similar. In contrast, viruses were noted on the tip of the mouse HMC uropod by transmission electron microscopy. These observations, reported for the first time in an animal model, will enable investigators to study the HMC under controlled conditions.

Liposomes complexed to plasmids encoding angiostatin and endostatin inhibit breast cancer in nude mice.

Gene therapy transfer of angiostatin and endostatin represents an alternative method of delivering angiogenic polypeptide inhibitors. We examined whether liposomes complexed to plasmids encoding angiostatin or endostatin inhibited angiogenesis and the growth of MDA-MB-435 tumors implanted in the mammary fat pads of nude mice. We determined that plasmids expressing angiostatin (PCI-Angio) or endostatin (PCI-Endo) effectively reduced angiogenesis using an in vivo Matrigel assay. We then investigated the efficacy of these plasmids in reducing the size of tumors implanted in the mammary fat pad of nude mice. Both PCI-Angio and PCI-Endo significantly reduced tumor size when injected intratumorally (P < 0.05). Compared to the untreated control group, the mice treated with PCI-Angio and PCI-Endo exhibited a reduction in tumor size of 36% and 49%, respectively. In addition, we found that i.v. injections of liposomes complexed to PCI-Endo reduced tumor growth in the nude mice by nearly 40% when compared to either empty vector (PCI) or untreated controls (P < 0.05). These findings provide a basis for the further development of nonviral delivery of antiangiogenic genes.

Detection of the AML1/ETO fusion transcript in the t(8;21) masked translocation in acute myelogenous leukemia.

The fusion transcript AML1/ETO was detected in the bone marrow of two t(8;21)-negative acute myelogenous leukemia (AML) patients by means of reverse transcription-polymerase chain reaction. This fusion transcript is identical to the one transcribed from the t(8;21) translocation base, as deduced from (a) the size and restriction pattern of the amplified DNA fragment and (b) the DNA sequence analysis of the fusion junction. We also showed that the ETO gene is highly expressed in these patients, much as it is in the t(8;21)-positive AML. Southern blot analysis showed rearrangement of the AML1 gene in one of the patients. Together, our results demonstrate that there is a masked t(8;21) translocation in AML that is not detectable by cytogenetic analysis but is able to transcribe an AML1/ETO fusion transcript similar to that transcribed in t(8;21)-positive AML-M2 patients.

Expression of AML1-ETO fusion transcripts and detection of minimal residual disease in t(8;21)-positive acute myeloid leukemia.

The t(8;21) translocation breakpoint, which is observed in acute myeloid leukemia (AML), has recently been cloned and a fusion transcript identified. We have now designed primer sets capable of amplifying the breakpoint junction of the fusion transcript by the reverse transcription-polymerase chain reaction (RT-PCR). Primer set 821U/821D1 amplified a 200-bp DNA fragment, and primer set 821U/821D2 amplified a 1.2-kb DNA fragment in all t(8;21)-positive AML tested. Sequence analysis of the amplified DNA fragments demonstrated that all fusion transcripts were fused at exactly the same site, indicating that this translocation breakpoint occurs within a single intron of the AML1 and ETO genes. Forty-five cycles of RT-PCR were used to detect residual t(8;21)-positive leukemia cells in three patients who had been in complete remission for 1, 3 and 5 years. Minimal residual disease was found in all three samples. Northern blot analysis demonstrated that two fusion transcripts of 7 and 10 kb were expressed in the t(8;21)-positive AML and that the ETO gene is not normally expressed in the hematopoietic system. Expression of a normal 5.5-kb ETO mRNA was found in the lung. From these results we concluded that expression of the ETO gene in t(8;21)-positive AML was activated as a result of the translocation.

Change in karyotype between diagnosis and first relapse in acute myelogenous leukemia.

We compared karyotype at first relapse with presenting karyotype in 212 patients with AML seen at MD Anderson Cancer Center (Houston, TX, USA) between 1975 and 1994. In 38% the karyotypes at diagnosis and relapse were identical. A stable karyotype was most frequent (70%) among patients who presented without cytogenetic abnormalities, suggesting that the finding of a normal karyotype is usually not due to sampling error. In contrast, a finding of insufficient metaphases at diagnosis was repeated at relapse in only 6% of cases. A change in karyotype occurred in 67% of 101 patients who presented with an abnormal karyotype and had sufficient metaphases for evaluation at relapse. The great majority of changes involved clonal evolution, clonal devolution (regression), or both; a purely normal karyotype and unrelated clones were seen in 11 and three of the 101, respectively. Change in karyotype between diagnosis and relapse and type of change were unrelated to remission duration. The only group in which karyotype at relapse vs that at diagnosis had a possible bearing on achievement of second CR were patients who presented with abnormalities other than inv(16), t(8;21) or t(15;17) and who at relapse had only normal metaphases; such patients had higher CR rates than comparable patients who retained their presenting abnormalities.

Cytogenetic evolution following the transformation of myelodysplastic syndrome to acute myelogenous leukemia: implications on the overlap between the two diseases.

Cytogenetic evolution in the myelodysplastic syndrome (MDS) has been associated with an abrupt shift to acute myelogenous leukemia (AML). To investigate the 'evolution' of MDS to AML we compared the karyotypes of MDS patients at presentation and at development of AML. Of 170 patients with MDS who developed AML, 63 had banded karyotypes done at both time points. Fifteen patients had refractory anemia (RA) or RA with ringed sideroblasts (RARS), 27 had RA with excess blasts (RAEB), and 21 had RAEB in transformation (RAEBT). Patients had MDS for at least 12 weeks prior to developing AML. Thirty-one patients received cytotoxic therapy for MDS. Seventeen of 63 patients (27%) acquired a cytogenetic change when they developed AML. This percentage was significantly higher in RA/RARS patients compared to RAEB/RAEBT (53.3 vs. 18.8%, p < 0.01). Age, prior malignancy, cytotoxic therapy for MDS, and time to development of AML did not influence the probability of acquiring a cytogenetic change. The small number of patients did not allow testing for the effect of specific karyotypes on the incidence of cytogenetic change or the effect of this change on AML prognosis. The only recurring cytogenetic change was addition of chromosome 13 which occurred in four of 17 patients who changed. These data suggest that a cytogenetic change may be partly responsible for the transformation of RA/RARS, but not RAEB or RAEBT, to AML. This supports the concept that RAEB, RAEBT, and AML are different manifestations of the same disease, whereas RA/RARS are conditions that predispose to acute leukemia.

Localization of an abundant myeloid mRNA to individual leukocytes in mixed cell populations.

Differential screening of cDNA libraries with radiolabeled RNAs isolated from various sources provides a convenient way to identify cDNA clones representing RNAs that are more (or less) abundant in selected tissues. This strategy was previously used to isolate cDNA clones representing poly(A+)RNAs (mRNA) that are abundant in leukocytes from chronic myelogenous leukemia (CML) patients. One limitation with the initial experiments was that the RNAs were isolated from heterogenous cell populations and it was impossible to distinguish whether all of the cells were producing the abundant mRNAs or if a subset of the cell population was responsible for the majority of the RNA species. To resolve this important issue, we have directly hybridized radiolabeled cDNAs to the cellular RNAs of intact, morphologically distinguishable, primary hematopoietic cells. In the present study clone pC-A3, which represents an mRNA species that is abundant in the chronic phase of CML, was used to examine three samples from normal bone marrow, one sample from normal peripheral blood, and four samples from peripheral blood of leukemic patients (one Ph1 + AL, two CML in compensated phase, and one CML in accelerated phase). The results show that while C-A3 gene expression is detectable in cells from the granulocytic, monocytic, and lymphoid lineage, its relative abundance peaks at the level of the neutrophilic myelocytes and promyelocytes. Earlier myeloid precursors like myeloblasts or mature neutrophilic granulocytes show less labeling. Further, all maturation stages of eosinophils (Eos) and basophil (Baso) are highly labeled. This finding reinforces recent evidence that Eos and Baso share a common progenitor and suggests that these two cell types may have a stronger role than previously noted in the prominent myeloproliferative response that is characteristic of CML.

Developmental and differential regulation of human MPO gene in leukemic cells.

Northern blot analysis of RNA isolated from HL-60 cells before and after differentiation induction by TPA and DMSO showed that four MPO mRNA species (3.3, 3.1, 2.7, and 2.5 kb, respectively designated alpha 1, beta 1, alpha 2, and beta 2) are expressed in HL-60 cells. However, alpha 2 and beta 2 lack part of the 3' end sequence due to different polyadenylation sites. The steady state levels of alpha 2 and beta 2 MPO mRNA increase significantly after 1 hr of induction, while all four MPO mRNA species decrease dramatically after 10 hr of induction. Our results demonstrate that MPO gene expression is developmentally and differentially regulated. Northern blot analysis of RNA isolated from blast samples of acute myelogenous leukemia (M0-M5) and chronic lymphocytic leukemia (CLL) patients indicate that four MPO mRNA species are expressed in M1-M4 but are undetectable in M5 and CLL. Primer extension and S1 nuclease protection analysis of the MPO mRNA revealed a single transcription initiation site for the MPO gene.

Determination of clonality in acute nonlymphocytic leukemia by restriction fragment length polymorphism and methylation analysis.

Determination of cellular clonality in hematological malignancies provides fundamental information that is important in understanding the pathogenesis of these disorders. We present here an extension of one approach to accomplish this that is based on the interpretation of different methylation patterns on active and inactive X chromosomes within the region of the hypoxanthine-guanine phosphoribosyltransferase gene spanned by a restriction fragment length polymorphism. The successful application of the method to determine clonality is described for three female patients with acute nonlymphocytic leukemia.

Cytogenetics for detection of minimal residual disease in acute myeloblastic leukemia.

Bone marrow samples collected from acute myeloblastic leukemia (AML) patients in complete clinical and hematological remission were studied for the persistence of cytogenetic abnormalities. AML patients from the three favorable cytogenetic categories [inv 16, t(8;21) and t(15;17)] and patients from the unfavorable cytogenetic categories (+8, -5, -7 and Philadelphia-positive) were studied. Seventy-one patients had evaluable metaphase spreads in remission marrows and 20 (28%) had one or more abnormal metaphases identical to that present in the pretreatment marrow. All 20 of these patients relapsed within 78 weeks, thus there were no false positive studies. Fifty-one patients had only diploid metaphases in their complete remission marrow, 25 relapsed, and 21 remained in continuous complete remission. Thus there was a 49% false negative rate of this study. These data indicate that the failure to detect residual chromosomally abnormal cells in the bone marrow does not guarantee continuous complete remission. Cytogenetic study was most useful in the favorable cytogenetic groups and least useful in the unfavorable groups. The persistence of normal metaphases in pretreatment marrows did not affect outcome or risk of recurrence. Twenty-five of 34 evaluable patients who relapsed after remission had either the identical cytogenetic abnormality present in the pretreatment marrow or showed the identical abnormality with additional chromosomal changes. Thus study indicates that cytogenetic examinations of complete remission bone marrow samples in patients with AML provides an objective method for detecting residual leukemia, and identifies patients with a potential for prolonged disease-free survival.

Childhood monosomy 7 syndrome: clinical and in vitro studies.

The clinical and cell growth characteristics of 11 children with monosomy 7 presenting as preleukemia (eight cases) or acute nonlymphoblastic leukemia (three cases) were studied. Anemia was common to all patients, with nine showing leukocytosis, seven thrombocytopenia, and one thrombocytosis. There was a striking predominance of males (M/F ratio, 10:1) and a young median age (3 years). Preleukemia evolved to acute nonlymphoblastic leukemia in five patients and to myelofibrosis in one. In vitro studies of bone marrow progenitor cells cultured in leukocyte feeder-stimulated agar revealed abnormal cell proliferative patterns, most often an increased number of small clusters, for all 11 subjects. The cells of some preleukemic patients showed increased growth even in the absence of an exogenous source of colony-stimulating factor, suggesting autonomous growth or possibly autocrine stimulation. Combination chemotherapy or bone marrow transplantation failed to induce complete remission in the seven patients who were treated. Our findings in these 11 cases confirm the poor prognosis of monosomy 7 presenting as preleukemia in children. The in vitro studies suggest an association between altered cell growth in vitro and clinical evolution to frank leukemia.

Rearrangement of the retinoic acid receptor gene in acute promyelocytic leukemia.

The retinoic acid receptor-alpha (RAR-alpha) gene was previously localized to chromosome 17q21, a region close to the t(15;17) (q22;q21) abnormality in acute promyelocytic leukemia (APL). We used the RAR-alpha gene as a probe and found that eight of nine APL patient samples with t(15;17) (q22;q21) showed rearranged bands. A tenth APL patient was diploid and demonstrated no rearrangement. One patient who had rearrangement as an acute leukemia did not have rearrangement in remission. The results obtained from intron/exon mapping of the RAR-alpha gene demonstrated that breakpoints of seven of the eight patients occurred within intron 1. Northern blot analysis of leukemic samples indicated the expression of two RAR-alpha mRNA of 2.7 and 3.7 kb. However, two additional mRNA of 4.1 and 3.2 kb were found in an APL patient. We conclude that the RAR-alpha gene is directly involved in the t(15;17) translocation in APL and may transcribe aberrant messages.

Down regulation of myeloperoxidase gene associated with specific nuclease hypersensitive sites during TPA induced differentiation of HL-60.

The level of myeloperoxidase (MPO) mRNA is reduced significantly after HL-60 induced differentiation with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). We examined the chromatin structural changes of the MPO gene during TPA induction. Before TPA induction about nine DNase I hypersensitive sites (HS) were found on the 5' upstream and at various intron regions of the MPO gene. A new HS was found on intron 8 within 4 h of induction; its appearance preceded down regulation of the MPO gene. At the same time DNase I HS found in 0.3 and 1-1.5 kb upstream of the MPO CAP site, were significantly reduced or disappeared after TPA induction. These chromatin structural changes could be closely linked to the mechanism which regulates the MPO gene expression.

Abnormalities in the long arm of chromosome 11 (11q) in patients with de novo and secondary acute myelogenous leukemias and myelodysplastic syndromes.

Leukemias with abnormalities in chromosome 11q23 occur frequently after exposure to topoisomerase II-reactive drugs. We investigated the characteristics and outcome of patients with de novo or secondary acute myelogenous leukemia (AML) or myelodysplastic syndrome (MDS) with abnormalities in chromosome 11q. Sixty-one patients had 11q abnormalities. Alterations involved 11q23 in 38 patients and other 11q abnormalities in 23. Sixteen patients had secondary disease, 12 involving 11q23, and four with other 11q abnormalities; 26 patients with de novo disease had 11q23 abnormalities and 19 other 11q abnormalities. The most common 11q23 abnormality was t(9;11), significantly more common in secondary (9/12) than in de novo (6/26) leukemias (p = 0.003). There were no significant differences in clinical characteristics between de novo and secondary groups involving 11q23. Five of 12 patients (42%) with secondary and 20/26 (77%) with de novo disease achieved complete remission (p = 0.05). Median survival was 6 weeks in the secondary group and 71 weeks in the de novo group (p = 0.001). There were no long-term survivors in either group. Results are similar when other 11q abnormalities are included. Adults with AML or MDS with 11q abnormalities secondary to prior chemotherapy have a worse prognosis than patients presenting de novo. However, 11q abnormalities define a population with a poor prognosis even when presenting de novo.

Detection of AML1/ETO fusion transcript as a tool for diagnosing t(8;21) positive acute myelogenous leukemia.

The nonrandom chromosomal translocation t(8;21)(q22;q22) can be found frequently in acute myelogenous leukemia with maturation (AML-M2). The breakpoint of this translocation has been cloned and characterized, and fusion transcript AML1/ETO has been identified. Reverse transcription polymerase chain reaction (RT-PCR) can be used to amplify the breakpoint site of AML1/ETO in t(8;21)-positive AML-M2 patients. The chimeric transcript can be detected in all 16 (100%) t(8;21)-positive AML-M2 patients. In all samples, the size of the amplified DNA fragments and pattern of restriction digest were identical, indicating that the t(8;21) translocation breakpoint occurs within a single intron of the AML1 and ETO genes. Interestingly, this fusion transcript was also detected in one of 13 AML-M2 patients without the t(8;21) translocation, indicating that a masked translocation involving chromosomes 8 and 21, exists in AML. Minimal residual disease was detected by semi-nested RT-PCR in all four patients tested, who had been in complete remission for 12, 15, 34, and 52 months, respectively. These results indicate that RT-PCR amplification of the AML1/ETO fusion transcript is a powerful tool for diagnosing and monitoring minimal residual disease in AML-M2 patients.

Myeloid surface antigen-positive acute lymphoblastic leukemia (My+ ALL): immunophenotypic, ultrastructural, cytogenetic, and molecular characteristics.

Leukemic blasts from 40 consecutively admitted adults with untreated acute lymphoblastic leukemia (ALL) were examined for myeloid surface antigen expression. Of these, 14 (35%) were reactive with one or more myeloid monoclonal antibodies. Each example of myeloid surface antigen-positive (My+ ALL) met the standard morphologic and cytochemical criteria for ALL. In addition, none of the 13 samples studied for ultrastructural evidence of myeloperoxidase met the criteria for acute myelocytic leukemia (AML). All patient samples reacted with lymphoid monoclonal antibodies: CD10+ (8 patients), CD19+ CD10- (2 patients), T cell+ (2 patients), and T cell+ CD10+ (2 patients). Coexpression of myeloid and lymphoid determinants was established by two-color immunofluorescence studies using flow cytometry in five of five samples analyzed. Cytogenetic abnormalities that have been associated with myeloid and mixed leukemias were common, including t(9;22), 7q-, abnormalities of 11q with or without a translocation, 20q-, and -5. Blasts from seven patients were studied at the molecular level. Immunoglobulin heavy chain gene rearrangements were detected in five of five samples with B cell+ T cell- phenotypes. One sample that was T cell+ CD10+ was germline for the immunoglobulin heavy chain and the T cell receptor gamma- and beta-chain genes. The other patient with T cell+ CD10+ blasts relapsed with AML following allogeneic bone marrow transplantation. The leukemia cells at the time of diagnosis and the cells at relapse demonstrated similar cytogenetics and the same immunoglobulin gene rearrangement, suggesting a clonal relationship. As a group, the My+ ALL patients had a significantly decreased complete remission rate when compared to My- ALL patients. Further studies at the molecular level will be required to determine the significance of karyotype abnormalities in My+ ALL.