Minimal residual disease (MRD) in remission t(8;21) AML and in vivo differentiation detected by FISH and CD34+ cell sorting.

Many patients with t(8;21) AML have residual positive cells during remission. We previously developed D-FISH probes that detect both derivative chromosomes and the normal alleles. In negative controls, only 2/44,000 (0.0045%) positive signals were observed. To investigate MRD, we examined specimens from 29 patients who had initially obtained CR. In remission patients, 61% had 1-4/2000 positive cells (0.05-0.19%). Higher frequencies were found in two patients in early relapse and in one patient in early remission. However, a negative test did not exclude relapse. Since false positives were negligible and because most t(8;21) AMLs express CD34, we asked whether cell sorting combined with FISH would increase the sensitivity. In one patient, we observed that 80% of CD34+ cells were t(8;21)+ at 2 months from initial clinical and cytogenetic remission. However, by 5 months the pre- and post-sorted populations contained 0.15% and 0.06% t(8;21) cells, respectively. Whereas essentially all t(8;21) cells in the initial specimen expressed CD34, only 0.6% were subsequently CD34+. These results are consistent with in vitro assays showing that residual t(8;21) cells undergo differentiation. Thus, FISH can identify MRD in a majority of t(8;21) patients and, combined with CD34+ selection, may provide an indirect assessment of the differentiation state of residual t(8;21) cells.

Fusion AML1 transcript in a radiation-associated leukemia results in a truncated inhibitory AML1 protein.

AML1 is a transcription factor that is essential for normal hematopoietic development. It is the most frequent target for translocations in acute leukemia. Recently, fluorescence in situ hybridization was used to identify a novel syndrome of radiation-associated secondary acute myelogenous leukemia that had AML1 translocations. Using polymerase chain reaction, the AML1 fusion transcript was isolated from the patient who had a t(19;21) radiation-associated leukemia. The AML1 gene is fused out of frame to chromosome 19 sequences, resulting in a truncated AML protein bearing the DNA binding domain but not the transcriptional activation domain. This fusion AML1 protein functions as an inhibitor of the normal AML1 protein. (Blood. 2001;97:2168-2170)

Identification of two new translocations that disrupt the AML1 gene.

The AML1 gene, located at chromosome 21q22, encodes a component (CBFalpha2) of a heterodimeric transcription factor complex termed core binding factor (CBF), which binds to DNA and activates gene expression. Chromosomal rearrangements may lead to disruption of this gene and development of acute leukemia. Twelve AML1 translocations have been identified to date, and include sites on chromosomes 1, 2, 3, 5, 8, 12, 14, 15, 16, 17, 18, and 19. Here we report two new translocations involving AML1 in acute myeloid leukemia, in which the disruption of the AML1 gene was documented by GTG banding cytogenetic studies and metaphase and interphase FISH analysis. These chromosomal breakpoints identified as harboring new fusion partners for AML1 are at 2p11.2 and 20q13.1. The two patients in who these translocation were identified were elderly males with newly diagnosed AML. These patients shared the same poor outcomes reported for other rare AML1 translocations.

A novel syndrome of radiation-associated acute myeloid leukemia involving AML1 gene translocations.

AML1 is a transcriptional activator that is essential for normal hematopoietic development. It is the most frequent target for translocations in acute leukemia. We recently identified 3 patients in whom pancytopenia developed almost 50 years after high-level radiation exposure from nuclear explosions during or after World War II. In all 3 patients, acute myeloid leukemia (AML) eventually developed that had similar characteristics and clinical courses. Cytogenetics from the 3 patients revealed a t(1;21)(p36;q22), a t(18;21)(q21;q22), and a t(19;21)(q13.4;q22). By fluorescent in situ hybridization (FISH), all 3 translocations disrupted the AML1 gene. Two of these AML1 translocations, the t(18;21) and the t(19;21), have not been reported previously. It is possible that the AML1 gene is a target for radiation-induced AML. (Blood. 2000;95:4011-4013)

A group of previously not recognized cytogenetic abnormalities in myeloid hematological malignancies.

We have identified a group of previously not reported chromosome abnormalities related to myeloid hematological malignancies. Cases 1 and 2 were observed to have an additional i(4)(p10) as the sole anomaly with similar clinical features of myeloid disorders; that is, acute nonlymphocytic leukemia (ANLL-M2) and myelodysplastic syndrome (MDS)-refractory anemia with an excess of blasts in transformation, respectively. Fluorescence in situ hybridization studies with the use of a 4p-specific microdissection probe further confirmed the presence of an i(4)(p10) in these patients. Case 3 was diagnosed with ANLL-M1 and had an additional i(8)(p10) as the only change, also confirmed by a whole-chromosome painting procedure. In cases 4-6, deletions of 18q at breakpoints q12, q23, and q21 were identified as the sole anomaly in a myeloproliferative disorder (MPD), MPD, and MDS, respectively. X-autosome translocations other than t(X;10)(p11;p11) and t(X;11)(q13;q23) have not been reported as recurrent or primary changes in hematological disorders. In the present study, a t(X;9)(q26;q22) and t(X;5)(q13;q33) as the sole anomaly were found in cases 7 and 8, respectively. Both cases had the same diagnosis of MDS. Considering that trisomies 4 (+4) and 8 (+8) are common anomalies in MDS and ANLL, our findings strongly indicate that amplification of genes on 4p and 8p, but not on 4q and 8q, may play a crucial role in the pathogenesis of MDS and ANLL. In addition, genes on 18q12-23 and on Xq13-26 may be involved in the pathogenesis of myeloid disorders.

PHA/IL2: an efficient mitogen cocktail for cytogenetic studies of non-Hodgkin lymphoma and chronic lymphocytic leukemia.

This study documents the utility of a mitogen/cytokine cocktail composed of phytohemagglutinin and Interleukin 2 (PHA/IL2) used to stimulate cultures from patients with chronic lymphoproliferative disorders. We report the results of a selected series of 57 patients with non-Hodgkin lymphoma (NHL) or chronic lymphocytic leukemia (CLL), in which only the culture stimulated with PHA/IL2 demonstrated the presence of an abnormal clone. On average, cells in the abnormal clone comprised 40% of the mitotic cells in this culture. The most common abnormalities observed in these patients were trisomy 12, present in 39% of the cases, and t(14;18), seen in 14% of cases.

Gain of 9p in the pathogenesis of polycythemia vera.

Polycythemia vera (PV) is a clonal stem cell disorder characterized by excessive erythrocyte production, resulting in absolute erythrocytosis. No specific structural chromosomal abnormalities have been reported in PV to date. We have observed two cases of PV with an extra i(9)(p10) as the sole anomaly, and FISH analysis using a 9p-specific chromosome microdissection probe showed that two other PV patients previously identified as having an add(18p) and an add(1p) as the primary changes actually carried a der(18)t(9;18)(p12;p11.2) and a der(1)t(1;9)(p12;p12), respectively. The same FISH assay was employed to evaluate domain signals on interphase cells of 15 more cases of PV with normal karyotypes and five normal controls. Two patients were observed with a significant increase in the percentage of cells with three domain signals. Our results strongly indicate that an additional i(9)(p10) is a new and recurrent primary chromosome anomaly in PV, and, in consideration of trisomy 9 being one of the most common anomalies in PV, amplification of a gene or genes on 9p, but not on 9q, may play a crucial role in the pathogenesis of PV.

Identical cytogenetic clones and clonal evolution in pediatric monozygotic twins with acute myeloid leukemia: presymptomatic disease detection by interphase fluorescence in situ hybridization and review of the literature.

PURPOSE: Observation of identical acquired genetic changes in infant monozygotic (MZG) twins with acute leukemia has provided strong evidence for in utero twin-twin transfusion as the cause of concordance. Documentation of similar phenomenon in older MZG twins offers insight into the latency period for leukemia and may provide the opportunity for presymptomatic disease detection in one twin. DESIGN: The literature describing leukemia in MZG twins is reviewed and the results of classical and molecular cytogenetic studies of one pair of MZG twins at 3 and 4 years with acute nonlymphocytic leukemia-FAB type M1 are reported. RESULTS: The twins studied had cytogenetically identical neoplastic clones with identical clonal evolution. Retrospective fluorescence in situ hybridization studies demonstrated the presence of the abnormal clone in the asymptomatic twin at the time of bone marrow transplant of the first twin. CONCLUSIONS: These observations support in utero twin-twin transfer as the origin of leukemic clones in pediatric and infant leukemia, demonstrate that clonal evolution of a leukemic clone may occur years before onset of overt disease, and indicate that knowledge of acquired genetic change(s) in one twin may provide markers to assess disease in the asymptomatic twin.

Pre-clinical evaluation of probes to detect t(8;21) AML minimal residual disease by fluorescence in situ hybridization.

The 8;21 translocation in acute myeloid leukemia (AML) results in a consistent fusion transcript, AML1/ETO. Long-term clinical remission occurs in some patients despite incomplete eradication of AML1/ETO as demonstrated by RT-PCR, thus limiting the usefulness of this assay. An important future goal will be to determine if there is a level of minimal residual disease (MRD) in patients below which relapse is unlikely. For the detection of MRD, we have developed reagents for fluorescence in situ hybridization (FISH) that identify both derivative 8 and 21 chromosomes with a high analytical sensitivity. In t(8;21) AML cells, two fused signals were detected in addition to the normal 8 and 21 alleles. The sensitivity and specificity of this probe mixture were analyzed in cell lines and patient bone marrows. One and two randomly juxtaposed signals were observed in 2.4 and 0.04% of normal cells, respectively. However, these were easily differentiated from t(8;21) cells by the absence of signals from the normal alleles. Using as criteria the presence of two fused signals plus the normal alleles, we observed no false positives among 5,000 normal cells. The probe correctly identified 20/20 patients with t(8;21) AML and 10/10 non-t(8;21) patients. In cell dilution experiments, the analytical sensitivity of this reagent was equal to that of the X chromosome and Y chromosome alpha-satellite probes. These optimized probes should facilitate the quantitative assessment and study of MRD in t(8;21) AML.

t(4;19)(q35;q13.1): a recurrent change in primitive mesenchymal tumors?

We report an apparently balanced t(4;19)(q35;q13.1) as the sole cytogenetic change in a highly malignant extraskeletal sarcoma in a 12-year-old-boy. Tumor cells were negative for all immunocytochemical markers except vimentin and neuron-specific enolase. Electron microscopy indicated chondroblastic differentiation. The tumor was categorized as a malignant sarcoma with differentiation toward extraskeletal mesenchymal chondrosarcoma. Reports of a similar translocation in an embryonal rhabdomyosarcoma (RMS) and in a dedifferentiated sarcoma with both rhabdomyosarcomatous and osteosarcomatous elements suggest that this translocation can arise in a primitive mesenchymal stem cell that can differentiate along at least these three pathways.

Incidence and significance of chromosome mosaicism involving an autosomal structural abnormality diagnosed prenatally through amniocentesis: a collaborative study.

Among 179,663 prenatal diagnosis cases collected from ten institutions and two publications, 555 (0.3 per cent) were diagnosed as having chromosome mosaicism. Of these, 57 (10.3 per cent) were mosaic for an autosomal structural abnormality, 28 (5 per cent) for a sex chromosome structural abnormality, and 85 (15.3 per cent) were mosaic for a marker chromosome. Ninety-five cases of prenatally diagnosed mosaicism with a structural abnormality in an autosome and a normal cell line, and with a known phenotypic outcome, were collected for karyotype-phenotype correlations through our collaboration (40 cases), a prior survey (26 cases), and published reports (29 cases). They included 13 balanced reciprocal translocations, one unbalanced reciprocal translocation, four balanced Robertsonian translocations, four unbalanced Robertsonian translocations, four inversions, 17 deletions, three ring chromosomes, 19 i(20q), seven +i(12p), six other isochromosomes, and 17 partial trisomies resulting from a duplication or other rearrangement. All cases mosaic for a balanced structural rearrangement resulted in a normal phenotype. All cases of 46/46,i(20q) resulted in normal liveborns. Five of seven cases with 46/47,+i(12p) had an abnormal phenotype compatible with Killian-Pallister syndrome. The overall risk for an abnormal outcome for a mosaic case with an unbalanced structural abnormality, excluding 46/46,i(20q) and 46/47,+i(12p), is 40.4 per cent. In the same category, the study also suggested a correlation between the percentage of abnormal cells and an abnormal phenotype. For mosaicism involving a terminal deletion, the possibility of a familial fragile site should be considered.

Cardiac myxoma characterized by clonal telomeric association.

We report on the cytogenetic analysis of a case of cardiac myxoma of the "syndrome myxoma" type, in which clonal telomeric associations between chromosomes 13 and 15, as well as nonclonal telomeric associations between chromosomes 12 and 17 and between chromosome 2 and an unidentified chromosome were observed. Nonclonal structural abnormalities were also present. This is the second reported case of cytogenetic abnormalities of syndrome cardiac myxoma, and the fourth case in which telomeric associations have been described.

The use of giant cell tumor conditioned media in cytogenetic studies of hematologic malignancies.

The use of conditioned media produced from solid tumor cell lines has been beneficial in the study of hematologic malignancies. Conditioned media from giant cell tumors (GCT), human lung adenocarcinoma, and human bladder carcinoma express growth factors that have been used to stimulate growth of bone marrow cells and improve the quality of the preparations. It has been reported that addition of Lu-CSF1-conditioned media from a lung adenocarcinoma cell line masks abnormalities in cases of acute leukemia [1.] Because we routinely use GCT-CM in bone marrow and leukemic blood cultures for chromosome analysis in our lab, we investigated this potential effect on our case analysis. We have performed a serial study of a 100 cases of hematologic malignancies received for analysis in our lab to determine the effect of the addition of GCT-CM to our culture media with respect to 1) mitotic index, 2) quality of preparation, and 3) differential selection of either chromosomally normal or abnormal cell lines. Our results indicate that the mitotic index and quality of metaphases is enhanced with the addition of GCT media and that there is no difference in the rate of abnormality detection with or without the addition of GCT media.

Prenatal diagnosis and outcomes of five cases of mosaicism for an isochromosome of 20q.

Five cases of mosaicism for an isochromosome of 20q have been detected from a total of 50,000 cases analysed for prenatal diagnosis by amniocentesis. Karyotypes were designated mos 46,X-/46,X-,i(20q). In all cases, the abnormal cell line was detected in more than one primary culture, thus fulfilling the criterion for true (level III) mosaicism. Indications for prenatal diagnosis were parental anxiety (two cases), low maternal serum alpha-fetoprotein (AFP) (two cases), and high maternal serum AFP (one case). Level II ultrasounds on all five fetuses were normal, and the abnormal cell line was never detected in fetal blood and/or cord blood. All five pregnancies were continued and had normal outcomes, with birth weights ranging from 2.4 to 3.8 kg. The development of all five children has been normal, with the oldest child in the study now 4 years of age. We suggest that the abnormal cell line in each case was of extrafetal origin, and that this may be one of the more common examples of this phenomenon, occurring in approximately 1/10,000 prenatal diagnoses. Mosaicism i(20q) may have been missed in the past because of the higher resolution necessary to detect this subtle change.

Sensitivity of chromosomal mosaicism detection by different tissue culture methods.

In the field of prenatal cytogenetic diagnosis, two tissue culture methodologies are currently in use: the flask method, which examines mixed populations of cells, and the in situ method, which examines distinct colonies of cells. These two methods provide inherently different levels of sensitivity which can be made comparable by adjusting the number of cells examined depending on the methodology used and the number of colonies formed per ml of specimen. Assuming that there are 2 colonies per ml of amniotic fluid in a 20 ml specimen, in order to detect 10, 20, and 30 per cent mosaicism with 95 per cent confidence, 29, 14, and 9 colonies should be examined respectively by the in situ method. Similarly, 50, 17, and 10 cells must be analysed by the flask method.

Translocation 1;7 in four cases of myeloid disorders.

A der(1)t(1;7)(p11;p11) was observed in bone marrow chromosome analyses of four patients with myeloproliferative disorders. Three patients had developed secondary leukemias or preleukemias following chemotherapeutic exposure, and one patient was diagnosed with M4 de novo.