Severe phenotype in a girl with partial tetrasomy 7, karyotype 46,XX,trp(7)(q35q36).

On prenatal ultrasonography, polyhydramnion, internal hydrocephalus, hypoplasia of the corpus callosum, and dysmorphic features were detected in a fetus of a 22-year-old mother. Subsequent karyotyping of amniocytes revealed supernumerary material in distal 7q. The baby was delivered after 38+4 weeks of gestation, and postnatal array CGH analysis showed a triplication of 7q35-->q36, resulting in partial tetrasomy. The triplication was not distinguishable from a duplication by conventional and molecular cytogenetic methods, but was clearly identified by array CGH analysis. The phenotype was rather severe with limited cardiac contractility and subsequent respiratory problems, as well as progressive neurologic deterioration and several dysmorphic features. Triplications in general are rare, and this case is the first report of a microscopically visible triplication in 7q. Duplication patients of the same chromosomal segment also showed a severe phenotype, however, in our opinion there are no common features suggesting a clinically recognizable distal 7q duplication/triplication syndrome.

Trisomy 21 is a recurrent secondary aberration in childhood acute lymphoblastic leukemia with TEL/AML1 gene fusion.

TEL/AML1 gene fusion is the most frequent genetic lesion in pediatric acute lymphoblastic leukemia (ALL). It occurs as a consequence of the cryptic chromosomal translocation t(12;21)(p13;q22). In a cohort of 50 RT-PCR-positive TEL/AML1 patients, karyotype examination by GTG banding and fluorescence in situ hybridization (FISH) allowed us to identify chromosome anomalies in addition to the already existing t(12;21). Secondary aberrations were found in 29 out of 41 patients (71%) at initial diagnosis and in all 9 patients with relapse. Structural rearrangements affected chromosome arms 2p, 2q, 5q, 9p, 12p (n = 2), 6q, 11p (n = 3), and 21q (n = 4). An extra chromosome 21 was found to be the most frequent anomaly. It was detected in 6 out of 41 patients at initial diagnosis (15%) and in 7 out of the 9 patients at relapse. No karyotype with trisomy 21 exceeded 47 chromosomes. Gain of chromosome 21 was the sole anomaly in GTG-banding analysis in 2/41 patients at initial diagnosis and in 4/9 at relapse. Notably, chromosome painting analysis performed in 11 out of the 13 patients with an extra chromosome 21 revealed duplication of the normal chromosome 21 in 8, and duplication of der(21)t(12;21) in 3 patients. Furthermore, gain of der(21)t(12;21) chromosome was confined exclusively to the relapse patients.

RAS mutations and clonality analysis in children with juvenile myelomonocytic leukemia (JMML).

Juvenile myelomonocytic leukemia (JMML) is a malignant hematopoietic disorder of early childhood with excessive proliferation of the myeloid and monocytic lineage. Deregulation of the RAS signal transduction pathway is thought to play a key role in its pathogenesis. We examined peripheral blood or bone marrow cells of 36 children with JMML for activating point mutations in codons 12, 13 and 61 of the NRAS and KRAS proto-oncogenes by allele-specific restriction assay, single-strand conformation polymorphism and/or direct sequencing. Codons 12, 13 and 61 of HRAS were examined in 26 of these patients. We detected RAS mutations in six cases (17%) located at N12 (n = 2), N13 (n = 3) and K13 (n = 1). In addition, we performed clonality studies on different cell lineages in four of these patients applying the RAS mutation, the karyotype and X-chromosome inactivation patterns as clonal markers. Erythroid cells carried mutant RAS, indicating clonal origin. In EBV B cell lines, one of three patients studied harbored a RAS mutation, while the other two patients had polyclonal B cells with wild-type RAS. T lymphocytes were examined in one patient; they were polyclonal and had wild-type RAS. It is likely that JMML is a heterogeneous disease with respect to clonal involvement of different lineages.

Detection of hyperdiploid karyotypes (>50 chromosomes) in childhood acute lymphoblastic leukemia (ALL) using fluorescence in situ hybridization (FISH).

ALL patients with a hyperdiploid karyotype of more than 50 chromosomes (high hyperdiploidy) carry a better prognosis in contrast to patients presenting with other cytogenetic features, and an appropriate less intensive therapy protocol should be developed for these patients. For this reason it is desirable to have a quick screening method identifying those with this type of hyperdiploidy. We therefore studied the bone marrow and/or blood cells of 278 children with ALL using double target fluorescence in situ hybridization (FISH) on interphase. A combination of DNA probes (repetitive, centromere specific) was applied detecting chromosomes which are most frequently overrepresented in patients with hyperdiploidy (>50), at chromosomes 6, 10, 17 and 18. All patients showing hybridization signals differing from the normal signal distribution of two spots for each tested chromosome were analyzed cytogenetically as well. 102 children (102/278; 36.7%) were found to have a clone with aberrant FISH results. In 80 patients (80/278, 28.8%) the cytogenetic analysis detected a hyperdiploid karyotype >50 chromosomes, whereas the remaining patients (n=12) could be related to other ploidy subgroups, ie hyperdiploidy with 47-50 chromosomes, haploidy, triploidy/tetraploidy. Comparison of the FISH results with the measurements of the DNA content showed good agreement for 88.8% (208/234) of the investigated patients. The detected rate of 28.8% patients with a high hyperdiploid karyotype in our investigated cohort is comparable to the frequency of other studies. Only one patient was not identified as having a hyperdiploid karyotype with our combination of DNA probes. Our results indicate that FISH is a feasible and quick screening method for the detection of hyperdiploid karyotypes (>50 chromosomes) and other ploidy subgroups.

Incidence and clinical outcome of children with BCR/ABL-positive acute lymphoblastic leukemia (ALL). A prospective RT-PCR study based on 673 patients enrolled in the German pediatric multicenter therapy trials ALL-BFM-90 and CoALL-05-92.

A variety of oncogenes are activated by specific chromosomal translocations, which are associated with distinct subtypes of leukemia. The identification of these rearrangements provides critical diagnostic and prognostic information, which may contribute to the selection of specific anti-leukemic therapy. The translocation t(9;22), the equivalent of the BCR/ABL rearrangement, is associated with a poor prognosis. We therefore used RT-PCR to detect this molecular event in a prospective study including 890 children. 673 of them suffered from acute lymphoblastic leukemia (ALL) at primary diagnosis and a transcription of the chimeric gene was detected in 21 of 648 with a successful analysis (3.2%). All children were treated by one of the two German multicenter childhood ALL therapy studies ALL-BFM-90 or COALL-05-92, respectively. Comparison of clinical features between BCR/ABL-positive and -negative children showed no significant differences regarding WBC, percentage of blasts, splenomegaly, hepatomegaly and age. Immunophenotypic studies at diagnosis in 21 BCR/ABL-positive children identified common ALL in 16 patients (76.2%), pre-B-ALL in four (19.0%), and an early T-lineage ALL in one (4.8%). Coexpression of myeloid antigens (CD13 and/or CD33) was observed in six of 16 common ALL patients as well as in the one child with early T-lineage ALL phenotype. The type of breakpoint (m-BCR/ABL: n = 14; M-BCR/ABL: n = 7) showed no correlation with clinical parameters. A comparison of cytogenetic and molecular data was performed in 16 positive patients and was concordant in all of them. We analyzed the response to the prednisone pretreatment and found a higher incidence of poor responders among the BCR/ABL-positive children. Regarding the event-free survival (EFS) of BCR/ABL-positive (0.53) and -negative patients (0.79) after a follow-up of 2 years, significant differences (P < 0.05) between both groups could be demonstrated.

12q13, a new recurrent breakpoint in acute non-lymphoblastic leukemia.

The karyotypes of 312 successfully analyzed samples of children with acute non-lymphoblastic leukemia (ANLL), which were sent to us by 72 German hospitals, were examined in order to find new recurrent chromosome abnormalities of possible clinical relevance. Whereas most of the patients had one of the specific aberrations of ANLL or a normal karyotype, random numerical or structural changes were found in 61 children (20%). Four of them showed an abnormality involving band 12q13: t(12;17)(q13;q21), t(12;21)(q13;q21), t(2;12)(p13;q13), and t(5;12)(p11;q13). Despite the fact that FAB subtypes were different (M0, M1 M6, AHL), the blasts of all patients were characterized by immaturity and were difficult to classify. The breakpoint 12q13 might be of clinical importance in ANLL, because the four patients in our study, as well as the 21 patients with this aberration found in the literature, had a very poor prognosis.

Characterization of chromosome 8 abnormalities by fluorescence in situ hybridization in childhood B-acute lymphoblastic leukemia/non-Hodgkin lymphoma.

Using fluorescence in situ hybridization (FISH), we studied chromosome 8 abnormalities in 30 children with mature B-cell acute lymphoblastic leukemia (B-ALL) or B-cell non-Hodgkin lymphoma (B-NHL). FISH was performed on metaphase spreads and interphase nuclei with a whole chromosome 8 painting probe. Fifteen patients were studied retrospectively after metaphases from the malignant cell specimen had already been G-banded. When interphase nuclei were examined, FISH was able to confirm t(8;14)(q24;q32) in all nine patients positive by previous G-banding. FISH, however, was positive in metaphase spreads from only seven patients. Another 15 patients were included in a prospective study. In six of them (40%), a translocation involving chromosome 8 was shown by a split small segment (8q24-8qter) on interphase nuclei. Analysis of metaphase spreads showed only three positive cases each by FISH or G-banding, respectively, with corresponding results in two patients. By interphase FISH, trisomy of chromosome 8 also was detectable. In three patients shown by G-banding to have trisomy, interphase FISH study showed high scores of three chromosome 8 signal positive cells. There was no cross-hybridization to other chromosomes interfering with FISH analysis. FISH analysis on interphase nuclei using a whole chromosome 8 hybridization probe will supplement and can be more sensitive than metaphase cytogenetic techniques for detection of chromosome 8 rearrangements in B-ALL/NHL.

Spontaneous hematological remission in a boy with myelodysplastic syndrome and monosomy 7.

We report the case of a 14-month-old boy with myelodysplastic syndrome (refractory anemia with blast excess) and bone marrow monosomy 7. Within 2 years after presentation hematological remission gradually occurred without any chemotherapy. After the patient had received three transfusions within the first 4 months, red cell production normalized. However it took 18 more months for neutropenia to resolve. The patient is well 34 months after the first presentation. Molecular analysis of the bone marrow blasts showed no mutations of the ras genes or of the FLR-exon of the NF-1 gene. To our knowledge this is the first report of monosomy 7 syndrome with spontaneous hematological remission.

Double target in situ hybridization applied to the study of numerical aberrations in childhood acute lymphoblastic leukemia.

To test the feasibility of using fluorescent in situ hybridization (FISH) on interphase and metaphase cells to detect numerical aberrations in childhood acute lymphoblastic leukemia (ALL), we analyzed bone marrow of 15 patients with cytogenetically documented hyperdiploidy with more than 50 chromosomes at diagnosis. Patients were selected on the basis of being trisomic or tetrasomic for chromosomes 17 and/or 18 as determined by G-banded chromosome analysis. We performed a double target FISH using DNA probes specific for the centromeric region of chromosomes 17 and 18, respectively. The numerical changes regarding chromosome 17 and/or 18 identified by FISH on metaphases were found in all cases analyzed by FISH on interphase nuclei. In 8 of 15 patients, FISH on interphase nuclei demonstrated the presence of one or more groups of cells with different combinations of trisomy and tetrasomy of the two chromosomes investigated, beside the ones detected on metaphases. Overall our findings indicate that interphase FISH analysis could be a useful method to detect the presence of numerical aberrations of two chromosomes simultaneously in bone marrow and peripheral blood specimens of ALL as an adjunct to conventional cytogenetic investigation or metaphase FISH.

Blastic phase chronic myeloid leukemia with a four-break rearrangement: t(11;9)(9;22)(q23;p22q34;q11).

Chromosome analysis of bone marrow (BM) aspirate from a 36-year-old man with chronic myeloid leukemia (CML) in blastic phase (BP) showed a four-break rearrangement t(11;9)(9;22)(q23; p22q34;q11), which can be considered a t(9;22)(q34;q11) and a secondary t(9;11)(p22;q23). It is not surprising that additional chromosome abnormalities occur in patients with Ph-positive CML in BP, but it is of interest that t(9;11)(p22;q23), characteristic of acute myeloid leukemia French-American-British (FAB) type M5 (ANLL-M5) was observed. The possible meaning of this additional change in BP of CML is discussed.

Cytogenetic findings in acute leukaemias of infants.

Of 706 children, 528 with acute lymphoblastic leukaemia (ALL) and 178 with acute myelocytic leukaemia (AML), whose leukaemia karyotypes could be successfully analysed, 48 were infants less than 1 year of age, 28 with ALL (5% of ALL patients) and 20 with AML (11% of AML patients). In contrast to older children. ALL-leukaemocytogenetics in infants was characterised by lack of hyperdiploidy with over 50 chromosomes and higher incidence of pseudodiploidy. Thirteen (= 46%) infants had an 11q23 aberration, and 11 of them had t(4;11). In AML, nine (= 45%) infants also had an 11q23 abnormality, e.g. t(9;11). Thus, the 11q23 aberration was present in almost 50% of all leukaemia karyotypes of infants. In ALL of infants, the CALLA negative, pre-pre-B immunophenotype prevailed. In AML of infants, the monocytic subtype dominated. A biphenotypic morphology (lymphoid-monocytic) with the expression of lymphoid and myeloid antigens was seen in several ALL and AML cases. In conclusion, leukaemogenesis in infants is a rare event, arising in stem cells of very early hematopoietic differentiation (probably due to gene rearrangement errors, most frequently at FRA11B), and differs from leukaemogenesis in older age groups by unique clinical and cellular features.

The translocation t(1;22)(p13;q13) is a nonrandom marker specifically associated with acute megakaryocytic leukemia in young children.

We present the nonrandom occurrence, frequency, and degree of immunophenotype association of the t(1;22)(p13;q13) in children with acute nonlymphocytic leukemia (ANLL). This karyotype anomaly occurred in leukemia cells from five of 445 (1.1%) children with newly diagnosed ANLL who were successfully studied by cytogenetic analysis at four European centers between January 1987 and January 1992. The occurrence of the t(1;22) was restricted to the French-American-British classification (FAB) subtype M7. The overall incidence in children with acute megakaryocytic leukemia (AMKL) was 27.8% (5/18 cases); in infants with AMKL, the frequency of the t(1;22) was 66.7% (4/6 cases). Three of the patients carrying this anomaly had a diploid karyotype, whereas in two cases a hyperdiploid karyotype was found. However, in all five patients, the t(1;22) was the only translocation event present at diagnosis. All patients received aggressive chemotherapy for acute myelogenous leukemia (AML). Two patients died within 15 months of diagnosis without entering remission. One of three patients who entered remission died 7 months after diagnosis, most likely from intramedullar hemorrhage. Only two of the five children with the t(1;22) who received autologous bone marrow transplantation (BMT) are alive and in complete remission (CR) 23 and 40 months after diagnosis, respectively. At the time of diagnosis, the age of the oldest child carrying the t(1;22) was 18 months. The cases with this chromosome anomaly were compared with an age-matched group of five children with AMKL lacking this translocation. The patients with the t(1;22) had a lower median value of the peripheral white blood cell (WBC) count and a higher median hemoglobin level than the patients from the matched group. In the latter cases, normocellular or hypercellular bone marrow (BM) was detected at diagnosis. In contrast, all children with the t(1;22) in our series had a hypocellular BM. Histological BM analyses were available in three of these patients and showed marked fibrosis. Other clinical and laboratory parameters showed no obvious differences between the matched groups. Despite intensive chemotherapy, AMKL in children appears to be associated with a poor prognosis. The clinical courses of the children with AMKL and the t(1;22) presented may be indicative of a beneficial effect of autologous BMT in this subset of patients.

Frequency and DNA sequence of tal-1 rearrangement in children with T-cell acute lymphoblastic leukemia.

Using nested polymerase chain reaction (PCR) a gene rearrangement named tal-1 deletion was found in five of 56 leukemic bone marrow samples from children with T-cell acute lymphoblastic leukemia (ALL). The DNA sequences of the PCR fragments consisted of the known conserved germline sequences in addition to short DNA insertions at the breakpoint region, which were different in each patient. Moreover, one patient was examined at diagnosis and at relapse 11 months later, revealing identical DNA sequences at the rearrangement site. The recombination site of the tal rearrangement therefore may be used as a genetic marker for detecting minimal residual disease in about 10% of T-cell ALL in childhood.

[Chromosome aberrations in acute leukemia in childhood: analysis of 1009 patients]. / Chromosomenaberrationen bei akuten Leukämien im Kindesalter: Analyse von 1009 Patienten.

Leukemia karyotypes were analyzed in 792 children with acute lymphoblastic leukemia (ALL) and 217 patients with acute myelocytic leukemia (AML). These patients were registered and uniformly treated in German multicentre trials from 1984-01-01 to 1989-12-31. In distinct leukemia subgroups specific chromosome abnormalities were found: Numerical aberrations such as hyperdiploidy over 50 chromosomes in c-ALL or structural aberrations (translocations) such as t(8;14) in B-ALL, t(11;14) in T-ALL, t(4;11) in ppB-ALL, t(1;19) in pB-ALL, t(15;17) in AML-M3, t(8;21) in AML-M2. Prognostic significance of the leukemia karyotype probably can be changed by intensive cytotoxic chemotherapy. Unfavorable prognosis, however, still persists in t(9;22) and t(4;11); "favorable" prognosis can be seen in t(8;21) and t(15;17). Inherited or induced chromosome instability is discussed as a possible predisposing factor for the origin of chromosome aberrations.

Chromosome translocation (2;13)(q37;q14) in a disseminated alveolar rhabdomyosarcoma.

Chromosome analysis of tumour cells in the bone marrow of a 13.5-year-old girl (without a primary tumour) revealed a pseudo-diploid or pseudo-tetraploid karyotype with a translocation involving the long arms of chromosomes 2 and 13: t(2;13)(q37;q14). This finding enabled the diagnosis of a disseminated alveolar rhabdomyosarcoma (RMS) to be established. The patient was treated by cytotoxic chemotherapy, went into complete remission, but died of relapse 14 months after diagnosis. As several cases with this translocation have been described recently, this additional report confirms that t(2;13) is specific for the alveolar subtype of RMS.