Somatic MED12 mutations in uterine leiomyosarcoma and colorectal cancer.

BACKGROUND: Mediator complex participates in transcriptional regulation by connecting regulatory DNA sequences to the RNA polymerase II initiation complex. Recently, we discovered through exome sequencing that as many as 70% of uterine leiomyomas harbour specific mutations in exon 2 of mediator complex subunit 12 (MED12). In this work, we examined the role of MED12 exon 2 mutations in other tumour types. METHODS: The frequency of MED12 exon 2 mutations was analysed in altogether 1158 tumours by direct sequencing. The tumour spectrum included mesenchymal tumours (extrauterine leiomyomas, endometrial polyps, lipomas, uterine leiomyosarcomas, other sarcomas, gastro-intestinal stromal tumours), hormone-dependent tumours (breast and ovarian cancers), haematological malignancies (acute myeloid leukaemias, acute lymphoid leukaemias, myeloproliferative neoplasms), and tumours associated with abnormal Wnt-signalling (colorectal cancers (CRC)). RESULTS: Five somatic alterations were observed: three in uterine leiomyosarcomas (3/41, 7%; Gly44Ser, Ala38_Leu39ins7, Glu35_Leu36delinsVal), and two in CRC (2/392, 0.5%; Gly44Cys, Ala67Val). CONCLUSION: Somatic MED12 exon 2 mutations were observed in uterine leiomyosarcomas, suggesting that a subgroup of these malignant tumours may develop from a leiomyoma precursor. Mutations in CRC samples indicate that MED12 may, albeit rarely, contribute to CRC tumorigenesis.

Identification and cloning of a prethymic precursor T lymphocyte from a population of common acute lymphoblastic leukemia antigen (CALLA)-positive fetal bone marrow cells.

We have cloned common acute lymphoblastic leukemia (CALLA)-positive cells from human fetal bone marrow containing less than 1 in 10,000 E-RFC in round-bottomed microtiter wells (one cell per well) using the autocloning unit of an EPICS-V cell sorter. Expansion of such cells (with IL-2 and heavily irradiated autologous thymocytes as feeder cells) resulted in growth in 6-14% of the wells (mean, 11%) with cells with mature T lymphocyte phenotype. Two-color fluorescence analysis of outgrowing cultures furthermore ascertained that these cells had differentiated through a phase of simultaneous expression of T4 and T8 antigens and at the same time expression of the thymocyte-associated T6 antigens. Thus, given the fact that 10-20% of T cell acute lymphoblastic leukemia (T-ALLs) are CALLA+, we have been able to identify a human prethymic T lymphocyte population that might be the normal counterpart of precursor cell to the CALLA+ T-ALL cell.

Purification and characterization of fetal hematopoietic cells that express the common acute lymphoblastic leukemia antigen (CALLA).

Fetal hematopoietic cells that express the common acute lymphoblastic leukemia antigen (CALLA) were purified from both fetal liver and fetal bone marrow by immune rosetting with sheep erythrocytes coated with rabbit anti-mouse immunoglobulin and by fluorescence-activated cell sorting. Dual fluorescence techniques disclosed that these cells were heterogenous with respect to the expression of a series of differentiation and activation antigens defined by monoclonal antibodies. Thus, whereas all CALLA+ cells were Ia+ and expressed two activation antigens, J2 and T10, only 30-50% expressed B1 antigen. Furthermore, using methanol-fixed cells, it could be shown that approximately 20% contained intracytoplasmic mu chains (cyto-mu) and that approximately 15% were positive for the terminal transferase enzyme (TdT) marker. The CALLA+ fetal cells thus closely resemble the childhood acute lymphoblastic leukemia cell with respect to surface marker phenotype. A population of CALLA- cells devoid of mature erythroid and myeloid surface markers was found to contain higher numbers of TdT+ cells but lower numbers of cyto-mu, B1, and Ia+ cells than the CALLA+ subset. In vitro analysis of normal, purified CALLA+ cells demonstrated that incubation at 37 degrees C with J5 monoclonal antibody specific for CALLA resulted in the specific modulation of surface antigen. Similar results have previously been obtained with CALLA+ tumor cells. Although phenotypic analysis of CALLA+ cells suggests that these cells are relatively immature lymphoid cells, CALLA+ cells do not appear to contain either myeloid precursor cells (CFU-G/M) or the earliest lymphoid stem cells.

Fate of tumor cells injected into left ventricle of heart in BALB/c mice: role of natural killer cells.

The arrest, retention, and elimination (i.e., clearance) of radiolabeled YAC-1 lymphoma cells injected either iv or into the left ventricle (LV) of the heart were studied in male BALB/c mice, with special emphasis on the role of natural killer (NK) cells. After iv injection YAC-1 cells were arrested and, to a large extent, destroyed in the lungs, which contain the first capillary bed that iv injected tumor cells meet. After LV injection the initial distribution of the tumor cells, which depends on the distribution of cardiac output at the time of injection, was estimated by use of radiolabeled microspheres. Using this technique, we have shown that LV-injected tumor cells, in contrast to iv injected tumor cells, were not arrested in the first capillary bed that they encountered but passed viably through the microvasculature of the brain, heart, kidneys, intestinal tract, and to some extent, the bone, skin, and muscle. The only organs that could arrest the LV-injected tumor cells were the lungs and the liver. In the lungs clearance of YAC-1 cells began immediately after the cells were arrested. However, the rate of clearance could be almost abrogated by pretreatment of the recipients with anti-asialo GM1 antiserum, which destroys most of the NK cells in vivo and strongly depresses the in vitro NK cell activity. In contrast, YAC-1 cells arrested in the liver were not cleared from this organ during the first 1-2 hours after arrest. After this delay clearance of the cells commenced. Pretreatment of the recipients with anti-asialo GM1 also strongly depressed the clearance of tumor cells from the liver. Although pretreatment with polyinosinic-polycytidylic acid enhanced in vitro NK cell activity, it could augment only slightly the clearance of YAC-1 cells from the lungs and the liver. Thus these results strongly support the hypothesis that the rapid clearance of tumor cells from both the lungs and the liver depends, at least partially, on the NK cell activity within these organs.

Extensive intra- and interindividual heterogeneity of p15INK4B methylation in acute myeloid leukemia.

Silencing of the cyclin-dependent kinase inhibitor gene p15INK4B by cytosine methylation of the promoter region has been associated with some types of hematological malignancy. To study in detail the patterns of p15INK4B methylation in patients with acute myeloid leukemia, we adopted a novel approach based on PCR amplification of bisulfite-treated DNA followed by resolution of differentially methylated sequences by denaturing gradient gel electrophoresis. This method visually displays the degree and heterogeneity of DNA methylation and enables the isolation and characterization of distinct clonotypic epigenotypes. A surprisingly high degree of intra- and interindividual heterogeneity of p15INK4B methylation was observed in the 65 acute myeloid leukemia patients examined. Methylation was detected in 46 (71%) of the patients and was observed more frequently in the French-American-British subtypes M1/M2 than in M4/M5 (P < 0.025). Examination of the same panel of samples using a highly sensitive methylation-specific PCR method showed methylated p15INK4B alleles in 61 (94%) of the samples. We present evidence that the higher frequency of p15INK4B methylation determined by methylation-specific PCR may, at least in part, be due to the presence of a small fraction of p15INK4B-methylated lymphocytes in normal blood.

Immunoelectron microscopy studies on the modulation of common acute lymphoblastic leukemia antigen (CALLA) on NALM-6 cells: delineation of intracellular transport.

Even though much is known about the presence of the common acute lymphoblastic leukemia antigen (CALLA) with respect to its distribution in hematopoietic and non-hematopoietic tissues, its functional role in lymphoid cells is as yet unknown. Given the fact that CALLA is completely modulated on the surface of lymphoid cells, we have employed pre-embedding immunogold techniques at electron-microscopical level and demonstrated that J5 monoclonal antibody (MoAb)-mediated modulation of CALLA expression on the lymphoblastic cell line NALM-6 is a specific, rapid process, closely resembling receptor-mediated endocytosis. Furthermore, it was found that CALLA was internalized through plasmalemmal pits and cytoplasmic vesicles and processed intracellularly in multivesicular bodies and secondary lysosomes. In contrast, HLA-DR antigen remained at the cell surface upon contact with specific MoAb. These data suggest that CALLA might be a receptor for a hitherto unknown signal molecule.

A novel MLL-AF10 fusion mRNA variant in a patient with acute myeloid leukemia detected by a new asymmetric reverse transcription PCR method.

A novel variant of the chimerical MLL-AF10 mRNA transcript was detected in a pediatric patient with acute myeloid leukemia (AML) by a new asymmetric reverse-transcription polymerase chain reaction (ART-PCR) method. Sequence analysis of the fusion region on the amplified cDNA fragment showed an in-frame joining of exon e5 of the MLL gene and position 1931 of the cDNA sequence of the AF10 gene, giving rise to a new MLL-AF10 transcript. The presence of the new chimerical mRNA product in a sample from the patient was confirmed by classical RT-PCR.

Major proteins in normal human lymphocyte subpopulations separated by fluorescence-activated cell sorting and analyzed by two-dimensional gel electrophoresis.

We have compared the overall patterns of protein synthesis of normal human lymphocyte subpopulations taken from five volunteers using high resolution two-dimensional gel electrophoresis. The lymphocytes were isolated using density gradient centrifugation, labeled with subtype-specific MoAbs, and separated to a high degree of homogeneity by FACS into CD4+ helper T cells, CD8+ suppressor T cells, CD20+ B cells, and N901 (NHK-1)+ NK cells. The four lymphocyte subpopulations were labeled with [35S]methionine for 14 hr, solubilized in lysis buffer, and analyzed by two-dimensional gel electrophoresis (IEF). Of about 1000 proteins resolved in each case, most were found to be common to all subpopulations. However, eight putative markers for B1+ (proteins 5525, Mr = 63,700; 5621, Mr = 63,700; 8311, Mr = 36,900; 2202, Mr = 36,300; 6121, Mr = 30,300; 106, Mr = 29,300; 5009, Mr = 23,000; 8012, Mr = 11,600) and one for N901+ (protein 8129, Mr = 30,400) were identified. In contrast, no major protein markers were found that could differentiate T4+ and T8+ cells from each other or from B cells and NK cells. With the exception of two B1+ markers (proteins 5525 and 5621), lower but variable levels of the other markers were observed in all cell types. All the putative protein markers have been identified in the protein database of human peripheral blood mononuclear cells (PBMCs) (see accompanying article by Celis et al.). Comparison of the overall patterns of protein synthesis of the unsorted PBMCs with those of the four subpopulations showed that the synthesis of some major PBMC proteins decreased substantially in the sorted subsets. These proteins are most likely not of monocyte origin, as these cells constituted only about 15% of the total PBMCs. Also, the inhibition does not seem to be due to the addition of the single MoAbs or to cell cycle differences. Taken together, the data provide a background for further studies of protein profiles in normal (resting or activated) and malignant hematopoietic cells.

Evaluation of candidate control genes for diagnosis and residual disease detection in leukemic patients using 'real-time' quantitative reverse-transcriptase polymerase chain reaction (RQ-PCR) - a Europe against cancer program.

Real-time quantitative RT-PCR (RQ-PCR) is a sensitive tool to monitor minimal residual disease (MRD) in leukemic patients through the amplification of a fusion gene (FG) transcript. In order to correct variations in RNA quality and quantity and to calculate the sensitivity of each measurement, a control gene (CG) transcript should be amplified in parallel to the FG transcript. To identify suitable CGs, a study group within the Europe Against Cancer (EAC) program initially focused on 14 potential CGs using a standardized RQ-PCR protocol. Based on the absence of pseudogenes and the level and stability of the CG expression, three genes were finally selected: Abelson (ABL), beta-2-microglobulin (B2M), and beta-glucuronidase (GUS). A multicenter prospective study on normal (n=126) and diagnostic leukemic (n=184) samples processed the same day has established reference values for the CG expression. A multicenter retrospective study on over 250 acute and chronic leukemia samples obtained at diagnosis and with an identified FG transcript confirmed that the three CGs had a stable expression in the different types of samples. However, only ABL gene transcript expression did not differ significantly between normal and leukemic samples at diagnosis. We therefore propose to use the ABL gene as CG for RQ-PCR-based diagnosis and MRD detection in leukemic patients. Overall, these data are not only eligible for quantification of fusion gene transcripts, but also for the quantification of aberrantly expressed genes.

Integration of molecular methods for detection of balanced translocations in the diagnosis and follow-up of patients with leukemia.

Molecular methods are emerging as important tools for the diagnosis and stratification of patients with leukemia. Together with rearrangements in immunoglobulin and T-cell receptor genes, balanced translocations are the most important genetic lesions amenable to molecular diagnosis. Moreover, many publications have identified significant differences in the prognosis of acute leukemia patients with such balanced translocations. Because not all balanced translocations can be diagnosed by cytogenetic techniques, reverse-transcriptase polymerase chain reaction (RT-PCR)-based methods are increasingly employed. This method has the added advantage that it can also be used to monitor for minimal residual disease (MRD). A disadvantage is that the multitude of balanced translocations in leukemia would make efforts to detect all lesions at diagnosis by such standard techniques extremely labor-intensive. Furthermore, difficulties in optimizing semiquantitative PCR assays have limited the utility of these methods for MRD. Recent advances in the design of multiplex PCR, which detects a number of genetic aberrations simultaneously, may improve the diagnostic process. Accurate quantitation of the fusion transcript for balanced translocations has become possible by use of fluorogenic probes and real-time PCR. Together, such methodologies may constitute a novel platform for the integration of molecular methods in clinical decision-making.

Analysis of the lymphocyte distribution during Isopaque-Ficoll isolation of mononuclear cells from human peripheral blood.

Using chromium-labelled mononuclear cells the movements of lymphocytes in Isopaque-Ficoll (I-F) gradients were analysed. Even small percentages of ox or autologous human erythrocytes mixed with the lymphocytes caused a significant loss of lymphoid cells and this loss was augmented when higher numbers of red cells were added. Such losses could be prevented using I-F gradients of higher densities, but then neutrophils and erythrocytes were found to contaminate the mononuclear suspensions recovered. Purified B and T lymphocytes were found to move identically on the gradients, but when human erythrocytes were added to the lymphocyte suspensions to be separated, a small but significant preferential loss of T cells was observed.

An improved technique for obtaining E rosettes with human lymphocytes and its use for B cell purification.

The standard E rosette method and two previously described methods claimed to give improved E rosetting for enumeration of human T lymphocytes have been compared with respect to the speed of rosette formation, and the mechanical stability of the rosettes formed. Following rosette formation with the three methods, the lymphocyte-erythrocyte suspensions were sedimented on Ficoll-Isopaque to deplete them of rosette-forming cells and red cells. The purified lymphocyte preparations were tested for B and T cell markers to determine the degree of contamination with T cells. One of the improved rosetting methods was clearly better than the others, and led to the recovery of B lymphocytes with a contamination of only 2.0+/-1.9 per cent of T lymphocytes.

Natural killer function following allogeneic bone marrow transplantation. Very early reemergence but strong dependence of cytomegalovirus infection.

Natural killer (NK) cell function was followed sequentially after allogeneic bone marrow transplantation (BMT) using three approaches: (1) chromium-release assay with purified mononuclear effector cells, (2) chromium-release assay with whole blood effectors, and 3) enumeration of lymphocytes bearing the NK-associated antigen NKH-1 (N901). The two latter methods enabled us to demonstrate a very early reappearance (at day 4 posttransplant) of pre-NK cells, which after interferon-alpha enhancement effectively lysed K562 cells and carried the NKH-1 antigen. During the first month NK function steadily increased, and at day 28 activated NK cells, which lysed the otherwise resistant P815 cell line, could be demonstrated concomittant with a substantial over-shoot in the proportion of NKH-1+ cells. Furthermore, the increase in NK lysis was more pronounced in patients with cytomegalovirus (CMV) infections (primary or reactivated). In contrast, the presence of graft-versus-host (GVH) disease did not associate with consistent changes in the NK parameters measured here. After the first month of increase, NK declined reaching levels near those observed in their respective bone marrow donors at day 90. These data demonstrate a surprisingly early recovery after allogeneic BMT, which can largely be related to external factors among which CMV seems to be a prime candidate.

Immunomodulation of NK and ADCC by Corynebacterium parvum in acute myeloid leukaemia patients.

Natural killer (NK)- and antibody dependent cellular cytotoxicity assays were performed using cryopreserved effector cells in AML patients receiving i.c. and s.c. injections of Corynebacterium parvum. A dose related increase in NK could be demonstrated with peaks in NK at day 1 with full Cp dosage and at day 14 with 50% doses. This increase was attributable to the Cp vaccine since normal donors receiving tetanus toxoid or pneumococcal polysaccharide and AML patients randomized not to receive Cp did not show similar NK boosting. The increase was probably due to interferon induction in vivo and could be demonstrated with purified T- and non-T-lymphocyte subsets. However, longitudinal measurements showed that the ability of Cp to boost NK was gradually lost over 4-6 months. ADCC studies showed that while lymphocyte-ADCC was not consistently affected by Cp, monocyte-ADCC was enhanced with maximal cytotoxicity at day 14.

Monoclonal antibodies in myeloid diseases: prognostic use in acute myeloid leukaemia.

Bone marrow cells from 109 patients (median age 60) with newly diagnosed acute myeloid leukaemia (AML) were prospectively immunophenotyped (IP) and the prognostic value of monoclonal antibody (MAB) reactivities was analysed to detect differences in complete remission rates and survival, not only between groups of MAB + and - bone marrow cells, but also in cases with or without prominent MAB reactivity as compared to normal BM reactivity of the respective MABs. This approach was based on the assumption that the qualitative expression of antigens is not an all or none phenomenon, but that different degrees of expression of antigens exist. Patients with significantly elevated CD13 (MY7+) cells in bone marrows (CD13 greater than reference value + one standard deviation) (S.D.) showed decreased probability of entering CR (p less than 0.05) and a significantly shorter survival (p less than 0.05). Superior CR rates (p less than 0.05) without difference in long-term survival were seen in patients with low CD33 (MY9) or low HLA-DR expression, while high CD14 (MY4) expression showed a trend towards an adverse factor (p = 0.12). No other antibody reactivities showed differences in CR rates (CD3, CD20, CDw65 (VIM-2) and NAT-9). The more prominent bone marrow expression of CD33 antigen than CD13 (CD33/CD13 greater than 1) correlated to a better chance of entering CR (p = 0.01) and to improved survival (p = 0.002), while the expression of high numbers of VIM-2+ cells was a favourable prognostic factor regarding length of survival (p = 0.002). The importance of a high CD33/CD13 ratio as a positive prognostic factor was evaluated using stratified analysis according to age or leucocyte counts at presentation. In both cases, CD33/CD13 was associated with longer survival (age: p = 0.05, leucocyte counts: p = 0.03). A Cox multiparameter analysis revealed that the CD33/CD13 ratio was a favourable prognostic factor (p = 0.03) together with age (p = 0.001) and leucocyte counts in peripheral blood (p less than 0.01). We conclude that establishing the immunologic phenotype can be of prognostic value in cases of AML, especially with regard to the relationship between the CD33 and CD13 antigens.

Surface expression of the alpha 2-macroglobulin receptor on human malignant blood cells.

The surface expression of the alpha 2-macroblobulin receptor (alpha 2MR), detected by a monoclonal antibody, A2MR alpha-2, was determined on mononuclear blood cells from 90 cases of malignant blood disease. Flow cytometric analyses combined with immunoblotting and ligand binding experiments revealed that alpha 2MR was expressed on malignant cells from patients with acute and chronic myelomonocytic leukemias, while no significant expression was found on malignant cells from acute and chronic lymphatic leukemia, lymphomas, plasma cell leukemias or hairy cell leukemia. In acute myeloid leukemia, alpha 2MR was expressed in 50% of the M4-M5 cases, but only in three of thirty of the morphologically undifferentiated or non-monocytic cases (M1-M3 and M6). In chronic myelomonocytic leukemia five of seven cases were alpha 2MR-positive, while only one of seven cases of chronic myeloid leukemia was positive. The monocytic nature of the hematopoietic cells reacting with A2MR alpha 2 was further confirmed by a close correlation with CD14 surface expression.

A quantitative evaluation of erythropoiesis in myelodysplastic syndromes using multiparameter flow cytometry.

By staining human bone marrow cells with a monoclonal antibody reacting with erythroid precursor cells (AS-E1) and propidium iodide, we have evaluated the proliferative capacity of erythropoiesis in patients with myelodysplastic syndromes (MDS) using flow cytometry. Comparing 36 patients (13 RA/RAS, 13 RAEB, 10 RAEB-t) with 7 normal controls, significant differences in both the percentage of AS-E1+ cells and the fraction of AS-E1+ cells in the S or S-G2M-phase between the four groups were found. Since neither the percentage of AS-E1+ cells nor their fraction in S or S-G2M alone was found to characterize their proliferative activity, we introduced the proliferative fractions of the erythroid cell, i.e. the number of the AS-E1+ cells in S or S-G2M related to all bone marrow cells in S or S-G2M. Applying these parameters, we found significantly increased proliferative AS-E1 fractions in the RA/RAS group compared to the normal controls (p = 0.03 and 0.002) respectively, as well as a highly significant decrease with disease progression.

Natural killer (NK)-cell activity and antibody-dependent cellular cytotoxicity (ADCC) in primary preleukemic syndrome.

In 13 patients with a multi-parameter based diagnosis of primary acquired preleukemic syndrome (PPS), natural killer (NK) cell activity and antibody-dependent cellular cytotoxicity (ADCC) were investigated on peripheral blood mononuclear cell (MNC) fractions. In all but two patients a defective NK activity was found. Lymphocyte-monocyte mixture experiments demonstrated that this was not due to suppressor monocytes. Furthermore, NK activity was defective when both myeloid and non-myeloid target cell lines were used. Addition of human leukocyte interferon to the NK cultures augmented the cytotoxicity, which exhibited the same kinetics as that of normal controls, but NK activity levels remained subnormal. These data strongly indicate that the decreased NK activity seen in the patients is due to a decreased number of circulating NK cells. In contrast ADCC was within the normal range both when MNC suspensions as well as when purified peripheral blood lymphocytes were used as effector cells thus ruling out subnormal lymphocyte ADCC masked by the presence of monocyte ADCC. These results demonstrate that PPS patients have a selective NK defect with an intact lymphocyte ADCC function. Whether this defect will prove to be valuable in the assessment of a malignant transformation in a given patient will await further longitudinal NK studies and clinical follow-up of the patients.

13-cis retinoic acid treatment of myelodysplastic syndromes.

Of eight patients with primary myelodysplastic syndrome (MDS) treated with Roacutane (13-cis retinoic acid, Roche, Basel, (13-RA)) 20 mg/m2 for 6 weeks and an additional 100 mg/m2 for 4 weeks (3 patients), 4 responded either with a slight increase in peripheral blood neutrophil count or a decrease in myeloperoxidase deficient neutrophils. In agar cultures 2 patients showed a concurrent increase in growth of day 11 colonies and clusters. In 2 of the patients a decrease in the number of immature bone marrow cells positive for the myeloid antibody anti-My7 was observed. Only minor alterations were seen in natural killer cell activity. In 4 patients showing clonal chromosomal abnormalities before treatment a disappearance of minor clonal abnormalities during treatment was observed, and in 3 chromosomal abnormalities reappeared after cessation of therapy. Even though the overall impact of 13-RA on the hematopoietic system was minor, the increase in myeloperoxidase normal granulocytes in the blood and the decrease in My7 positive cells and in clonal chromosomal abnormalities warrants further interest in this agent as a treatment modality in MDS. The side effects, especially experienced by the patients receiving 100 mg/m2, were, in spite of symptomatic treatment, of such a degree that only low dose treatment (10-20 mg/m2) administered for prolonged periods of time (3-6 months) would seem recommendable.

Dynamic cell cycle kinetics in vitro and in vivo in myelodysplastic syndromes with special reference to the influence of hematopoietic growth factors.

We have investigated the effect of HGF in vivo and in vitro in MDS using a recently developed FCM assay involving the simultaneous measurement of cell surface antigens, DNA content, and BrdUrd or IodUrd incorporation. This allows for the determination of the dynamic cell kinetic parameters: LI, T(s), and T(pot) and we observed that in vitro HGF stimulation resulted in a significant decrease in mean T(pot) values from 6.6 to 3.5 days. Importantly, we demonstrated that in vivo GM-CSF administration to patients with RAEB resulted in a shortening of T(pot) within the 2 first weeks of GM-CSF treatment.