Anne Hagemeijer

Diagnosis of acute promyelocytic leukaemia by RT-PCR: detection of PML-RARA and RARA-PML fusion transcripts

Summary. Acute promyelocytic leukaemia (APL; AML M3) is identified by a unique t(15;17) translocation which fuses the PML gene to the retinoic acid receptor alpha gene (RARA). Reverse transcription coupled with the polymerase chain reaction (RT‐PCR) has been used to develop a diagnostic test for APL based on the PML‐RARA fusion message. Separate PCR assays were designed to amplify either PML‐RARA (15q+ derived) or RARA‐PML (17q‐ derived) chimaeric transcripts. PML‐RARA transcripts were detected in every case from a series of 18 APL patients with cytogenetically confirmed t(15;17) translocations, whereas RARA‐PML messages were detected in only 67% (12/18) of these patients. This suggests that it is the 15q + derivative which mediates leukaemogenesis. Furthermore the PCR approach (or Southern analysis) may be used to identify in which of the alternative PML introns the breakpoint occurs; 52% of cases (15/29 patients) utilize a 5′ PML intron and 48% the 3′ intron (14/29 cases). Neither the choice of PML intron nor the expression of the 17q‐derivative could be correlated with the microgranular variant of APL (M3V), overall survival rate, age, sex or presence of coagulopathy. Finally, the fusion message is undetectable in five remission samples. This indicates a possible use for RT‐PCR in monitoring remission patients for evidence of relapse.

Cytogenetic follow-up of patients with nonlymphocytic leukemia. II. Acute nonlymphocytic leukemia

Bone marrow (BM) karyotypes of 86 patients with acute nonlymphocytic leukemia (ANLL) were studied at the time of diagnosis: 39 of them (45%) were normally diploid and 47 (55%) showed acquired abnormalities. The median survival was no longer in the diploid group than in the aneuploid one. Nonrandom aberrations were often found: trisomy 8 (15 times), monosomy 7 (7 times), and t(8;21) (7 times). Two patients with acute promyelocytic leukemia presented with the t(15;17) in BM cells. Serial cytogenetic studies performed in 17 cases showed that karyotypic evolution closely followed the clinical evolution. Complete remission, obtained in 10 cases, was characterized by BM metaphases with a normal karyotype. Relapse after a period of complete remission was documented four times; the BM metaphases then showed the original abnormal karyotype with additional changes that, in three cases, were limited to a new structural aberration.

Cytogenetic, Molecular Genetic and Pathological Analyses in 126 Meningiomas

Abstract. In a series of 126 meningiomas, tumor and patient charactersitics were investigated and statistically analyzed. A combined cytogenetic and molecular genetic approach was used to study chromosomal abnormalities and loss of markers on chromosome 22q. This apprach was successfully applied to 93 meningiomas. In 66 cases, complete or partial loss of chromosome 22 was observed and in at least 12 of them this chromosome was involved in structural aberrations. In addition to chromosome 22 changes, chromosomes 1, 6, 11, 13, 14, 18, 19, X, and Y were also frequently involved in structural and numerical aberrations. Statistical analysis revealed a significant association between the number of choromosomal abnormalities and tumor grade. Complex karyotypes predominated in the group of grade II/III meningiomas. Furthermore, other variables showed statistically (or marginally statistically) significant differences. Meningiomas from the convexity were more ofthen grade II/III, displayed predominantly (partial) loss of chromosome 22 and had complex karyotypes more often. These fetures were frequently found in meningiomas from males. Base meningiomas, on the other hand, occurred more often in females; they were usually grade I, showed loss of (parts of) chromosome 22 less often and displayed fewer additional chromosomal abnormalities.

Establishment and characterization of primary and metastatic uveal melanoma cell lines

We report on the establishment and characterization of 2 primary (EOM‐3, EOM‐29) and 3 metastatic uveal melanoma cell lines (OMM‐1, OMM‐2, OMM‐3), and further cytogenetic characterization of a previously described primary uveal melanoma cell line (OCM‐1). Only a few long‐term growing primary uveal melanoma cell lines have as yet been established, while of metastatic uveal melanoma cell lines we have found no descriptions. The morphology of the in vitro cultured cells varied from spindle to epithelioid. The cell lines were characterized by immunocytochemistry, electron microscopy and cytogenetical analysis. The relative growth rate was determined by bromode‐oxyuridine (BUdR) incorporation. The melanocytic origin of the cell lines was determined by positive staining with antibodies identifying melanoma‐associated antigens. Melanosomes and pre‐melanosomes were indeed observed by electron microscopy in all cell lines. The stem‐cell karyotype was found to be normal in 3 cell lines (EOM‐29, OMM‐2, OMM‐3) and abnormal in 3 others (EOM‐3, OCM‐1, OMM‐1) showing a net loss of chromosome 6. The OCM‐1 and the OMM‐1 cell lines even demonstrated a large amount of structural chromosomal aberrations, the former being near‐tetraploid and the latter triploid. The EOM‐29 cell line, cultured from an ciliary body melanoma, did not show the previously described chromosome 3 and 8 abnormalities.

The 5q‐ chromosome abnormality in haematological disorders:|a collaborative study of 34 cases from the Netheralands

Summary. Clinical, haematological and cytogenetic data of all patients with an acquired 5q ‐ abnormality, diagnosed in four cytogenetic laboratories, were studied. The 5q‐ arose de novo in 24 patients, while 10 patients exhibited it after radiation and/or chemotherapy. Sixteen cases with a dysmyelopoietic syndrome (DMPS) and one case with acute nonlymphocytic leukaemia (ANLL) had no chromosome abnormalities other than 5q‐ . All nine patients with refractory anaemia (RA) belonging to the de novo group had a normal cell line. So far none of them has transformed to acute leukaemia, while three patients with refractory anaemia with excess of blasts (RAEB), pure red cell aplasia (PRCA) and acquired idiopathic sideroblastic anaemia (AISA), having the 5q‐ in 100% of the cells showed such a transformation. The second group consisted of nine patients with a 5q‐ and additional chromosome abnormalities: three of them died of progression of the disease, while four died of unrelated causes. Eight out of nine patients with a 5q‐ and acute leukaemia, either de novo (five) or secondary (four) to radiation and/or chemotherapy, had additional chromosome abnormalities. Among the changes (partial) monosomy of chromosome 7 was most frequently found.

PHA-induced colony formation in acute non-lymphocytic and chronic myeloid leukemia

Abstract A method which has previously been introduced for growing colonies in vitro from T-lymphocytic cells in normal subjects, has been applied to studying the growth of colonies from the bone marrow and blood of patients with acute non-lymphocytic leukemia and chronic myeloid leukemia. The technique consists of an underlayer of agar containing irradiated leukocytes and a liquid overlayer containing PHA. In leukemic patients, the assay permitted the formation of T-lymphocytic as well as leukemic colonies. Removal of the E-rosette forming cells before culture resulted in the appearance of mainly leukemic colonies. Cytogenetic analysis demonstrated the presence of the acquired karyotypic changes characteristic of the leukemic cells of these patients in the colony cells. Study of eight cases of acute non-lymphocytic leukemia, four cases of blast crisis of CML, and seven cases of CML chronic phase revealed the assay to be efficient in growing large numbers of leukemic blast colonies, as compared to the Robinson culture assay where large colony formation is only found in chronic leukemia [10]. Removal of the progenitors of lymphocytic colonies was sometimes incomplete in acute leukemia but not in CML.

Inhibition of the growth of cultured human meningioma cells by recombinant interferon-α

In this paper the results of investigations on the effect of interferon-alpha (IFN-alpha) on the growth of meningioma cells in culture is reported. A consecutive series of six meningiomas and one meningioma/neurofibroma derived from a patient with neurofibromatosis type 2 was investigated and it was found that the growth of all seven tumours in response to mitotic stimuli (fetal bovine serum or epidermal growth factor) is strongly inhibited by IFN-alpha. Maximal response varied between 100% and 70% inhibition of the incorporation of tritiated thymidine. In some cases an inhibitory response was obtained already at very low doses (less than or equal to 10 U of IFN-alpha per ml). These results indicate that further clinical investigation of the application of IFN-alpha to the treatment of meningioma is warranted.

Bone Marrow Karyotypes of Children with Nonlymphocytic Leukemia

Summary: Bone marrow (BM) karyotypes from 16 consecutive children presenting with nonlymphocytic leukemia were established with the use of banding techniques, before therapy. The two patients with chronic myeloid leukemia (CML) showed the Philadelphia (PhI) translocation (9q+;22q−). Five of the 14 patients with an acute nonlymphocytic leukemia (ANLL) presented no acquired cytogenetic abnormalities, but one of these five showed a high level of hypodiploidy. One patient with AML evidenced a variant of the Ph1 chromosome originated as a translocation (12p+;22q−). Nonrandom abnormalities (−7; 7q−; +8; t(8;21); −21) were found in six patients, isolated or in association with other aberrations. Among the random abnormalities, apparently balanced translocations and chromosomal deletions were observed.In ANLL, no correlation could be found between morphologic diagnosis and cytogenetic findings. On the other hand, the presence of BM cells with a normal karyotype at diagnosis was associated with an improved remission rate and survival time. Followup studies were performed in four ANLL patients with an abnormal cell clone at diagnosis. Three of them achieved hema-tologic remission; their BM karyotype was found to be normal at that stage. In the 4th patient, generalization of the abnormal karyotype in BM cells was seen in the terminal phase of the disease.Speculation: Chromosome identification techniques have greatly improved our knowledge of acquired BM aneuploidy found in leukemia. Nonrandom patterns of abnormalities have been found. Analyses of larger series of patients using advanced banding techniques and systematic followup studies will ascertain the significance of these cytogenetic abnormalities for diagnosis, prognosis, and neoplastic transformation.

CDKN2 deletions have no prognostic value in childhood precursor-B acute lymphoblastic leukaemia

CDKN2 deletions have no prognostic value in childhood precursor-B acute lymphoblastic leukaemia

Cytogenetic follow-up of patients with nonlymphocytic leukemia I. Philadelphia chromosome-positive chronic myeloid leukemia☆

Abstract Chromosomes of blood and bone marrow cells were studied in 53 patients with Philadelphia chromosome (Ph 1 )-positive chronic myeloid leukemia (CML). Fifty patients presented with t(9;22) and three with variant translocations: t(17p+;22q−); t(17q+;22q−), and t(1;9;22). Serial studies were carried out in 27 patients during both the chronic and the blastic phase of the disease. Five patients in the chronic phase developed cytogenetic changes in addition to the Ph 1 . In two of these cases the changes preceded a transformation into acute leukemia, but in three cases no acceleration of the disease has occurred 1 to 4 years after the emergence of the subclones. Blastic transformation occurred in 15 instances; 11 of these patients acquired additional nonrandom chromosomal changes: trisomy 8 (9 times), i(17q) (4 times), trisomy 17 (2 times), trisomy 19 (7 times), and duplication of the 22q− (6 times). Clonal evolution was found in eight cases, either at the onset of blastic transformation or later in follow-up cultures. The differences between karyotypic evolution during the chronic and blastic phases of CML are discussed, together with their prognostic significance.