Rhona Schreck

Transposition and amplification of oncogene-related sequences in human neuroblastomas

We have cloned a 2.0-kb EcoRI fragment of human genomic DNA (NB-19-21) which has homology to the v-myc oncogene but is distinct from the classical c-myc gene. This sequence is amplified from 25- to 700-fold in eight of nine tested human neuroblastoma cell lines which contain either homogeneously staining regions or double minutes (HSRs or DMs), the caryological manifestations of amplified genes. In the remaining line, the c-myc proto-oncogene is amplified approximately 30-fold. NB-19-21 hybridizes to a 3.2-kb cytoplasmic, poly(A)+ RNA species that is abundant only in lines in which the sequence is amplified. We propose that the gene encoding the NB-19-21-related RNA species may represent a new oncogene, which we call N-myc. NB-19-21 derives from chromosome 2; but in the five HSR-containing lines that have amplified this sequence, none has HSRs on chromosome 2. NB-19-21 is associated with DMs in a DM-containing line. A second, randomly cloned, amplified DNA segment from the HSR of one of the neuroblastoma lines is amplified in a subset of the lines in which NB-19-21 is amplified. In addition, this probe identifies a novel joint in the amplification unit of one line relative to that of the others. We suggest that, in the eight lines which have amplified NB-19-21, the amplification units are overlapping, but not identical, and that transposition of the common sequences may occur prior to amplification.

Sister chromatid exchanges.

Sister chromatid exchanges (SCEs) represent the interchange of DNA replication products at apparently homologous loci. These exchanges presumably involve DNA breakage and reunion, although little is known about the molecular basis of sister chromatid exchange formation, and information about the biological significance of exchanges is largely circumstantial. In spite of these uncertainties, analysis of sister chromatid exchange formation in cytological systems has already provided information about chromosome structure and has been used to detect the effects of clastogens and to differentiate between chromosome fragility diseases.

Mosaic trisomy 16 ascertained through amniocentesis: Evaluation of 11 new cases

Trisomy 16, once thought to result uniformly in early pregnancy loss, has been detected in chorionic villus samples (CVS) from on-going pregnancies and was initially ascribed to a second, nonviable pregnancy. Prenatally detected trisomy 16 in CVS and its resolution to disomy has led to the reexamination of the viability of trisomy 16. This study evaluates 11 cases of mosaic trisomy 16 detected through second trimester amniocentesis. In 9 of the 11 cases, amniocenteses were performed in women under the age of 35 because of abnormal levels of maternal serum alpha-fetoprotein (MSAFP) or maternal serum human chorionic gonadotropin (MShCG). The other two amniocenteses were performed for advanced maternal age. Five of the 11 pregnancies resulted in liveborn infants, and six pregnancies were electively terminated. The liveborn infants all had some combination of intrauterine growth retardation (IUGR), congenital heart defects (CHD), or minor anomalies. Two of them died neonatally because of complications of severe congenital heart defects. The three surviving children have variable growth retardation, developmental delay, congenital anomalies, and/or minor anomalies. In the terminated pregnancies, the four fetuses evaluated by ultrasound or autopsy demonstrated various congenital anomalies and/or IUGR. Cytogenetic and fluorescent in situ hybridization studies identified true mosaicism in 5 of 10 cases examined, although the abnormal cell line was never seen in more than 1% of cultured lymphocytes. Placental mosaicism was seen in all placentas examined and was associated with IUGR in four of seven cases. Maternal uniparental disomy was identified in three cases. Mosaic trisomy 16 detected through amniocentesis is not a benign finding but associated with a high risk of abnormal outcome, most commonly IUGR, CHD, developmental delay, and minor anomalies. The various outcomes may reflect the diversity of mechanisms involved in the resolution of this abnormality. As 80% of these patients were ascertained because of the presence of abnormal levels of MSAFP or MShCG, the increased use of maternal serum screening should bring more such cases to clinical attention.

Use of conditioned media in cell culture can mask cytogenetic abnormalities in acute leukemia

Conditioned media (CM) from a human lung adenocarcinoma cell line expressing interleukins 1 and 6 (IL-1, IL-6), granulocyte (G), macrophage (M), and GM colony-stimulating factors (G, M, GM-CSF) and transforming growth factor beta (TGF beta) were used to stimulate growth of bone marrow (BM) cells from 18 persons with leukemia, myelodysplastic syndrome, or lymphoma. The objective was to increase numbers of analyzable metaphases and to enhance the likelihood of detecting cytogenetic abnormalities. Although more mitotic cells were observed with CM, the detection rate of cytogenetic abnormalities decreased in 12 of 18 cases. These data indicate that use of CM for cytogenetic analyses may favor growth of normal versus leukemia cells and mask cytogenetic abnormalities.

Molecular and cytologic analysis of DNA amplification in retinoblastoma

Amplification of two distinct genomic DNA segments is observed in homogeneously staining regions in two sets of retinoblastoma cell lines derived from two different patients. One DNA segment was known to have sequence homology to the c-myc oncogene, and both DNA segments had previously been shown to be amplified in neuroblastoma cells. The absolute degree of amplification differed in all cytogenetically distinct retinoblastoma cell lines tested. Also, the relative amplification of these two DNA segments was unequal within a given cell line. Minimal amplification of both DNA segments was also detected in DNA directly isolated from one primary retinoblastoma. Based on these and previous results, it is concluded that assembly of amplifiable, relocatable units in many human retinoblastoma and neuroblastoma cells may involve a complex process of differential recruitment of separate DNA segments that are located on human chromosome #2.

Chronic phase of ETV6-ABL1 positive CML responds to imatinib

Imatinib acts as an effective agent for inhibiting the neoplastic clone in patients with BCR-ABL1positive CML by blocking the constitutively activated BCR-ABL1 kinase (Druker, 2003). Rare patients (<5%)withmorphological CMLdo not demonstrate this gene rearrangement (Onida et al., 2002; Druker, 2003). The presence of a novel gene fusion between ETV6 and ABL1 has been reported in patients with BCR-ABL1-negative CML (Andreasson et al., 1997). We report a patient who was diagnosed with BCR-ABL1-negative, ETV6-ABL1-positive chronic phase CML. Treatment with imatinib has produced an excellent hematological response. A 24-years-old female was referred in September, 2007 because of a 3 month history of leukocytosis. At consultation, her peripheral while blood cell (WBC) counts were 98,800/ll (blasts 2%, promeylocytes 6%, myelocyte 17%, metamyelocytes 1%, bands 15%, mature granulocytes 44%, lymphocytes 7%, monocytes 3%, eosinophils 4%, basophils 1%), Hb 11.3 g/ dl, and platelets 261,000/ll. Mild hepato-splenomegaly was detected. Her bone marrow was hyperplastic with normal differentiation. Chromosomal analysis revealed a normal karyotype (data not shown). Neither metaphase fluorescence in situ hybridization (FISH) analysis nor reverse transcription-polymerase chain reaction (RT-PCR) detected evidence of a BCR-ABL1 fusion (Fig. 1A and data not shown). However, the ASS gene, which is centromeric to ABL1, was missing from one copy of chromosome 9 (Fig. 1A). This deletion can be seen in association with the t(9;22) translocation (Sinclair et al., 2000). To explore the genetic abnormalities of this case, we performed single-nucleotide polymorphism genomic microarray (SNP-chip) analysis as previously described (Kawamata et al., 2008) and found duplication of the portion of 12p13 involving the 50 half of ETV6 gene and a deletion of 9q34 involving exon 1 of ABL1 (Fig. 1B). These findings were highly suggestive of a fusion between exons 1-5 of ETV6 and exons 2-11 of ABL1. Metaphase FISH analysis using ETV6, RUNX1, BCR, and ABL1 probes demonstrated a fusion of ETV6 and ABL1 (Fig. 1C) on chromosome 9, suggesting that the duplicated portion of 12q13 was inserted at 9q34.1 (Fig. 1D). RT-PCR analysis of the transcripts using primers specific to the ETV6 and ABL1 genes showed two sizes of PCR products (Fig. 2 A). Nucleotide sequencing of these PCR products demonstrated a fusion between exon 5 of ETV6 and exon 2 of ABL1, as well as a fusion of exon 4 of ETV6 and exon 2 of ABL1 (Fig. 2A). These two fusion products were probably generated by alternative splicing of exon 5 of ETV6. Imatinib treatment (400 mg/day) was started and the patient’s WBC decreased to normal range (7,800/ll) within 5 days (Fig. 2B). Treatment is continuing and the WBC has remained in the normal range for over 7 months (Fig. 2B). At three months after the treatment started, the abnormal cells were not detected by FISH in the peripheral blood (Fig. 2B and data not shown). Previously, a patient with myeloid blastic crisis of ETV6-ABL1-positive CML, who was treated with imatinib, but only responded for only 10 days has been described (O’Brien et al., 2002). Another report described an ETV6-ABL1-positive CML patient with blastic crisis who was placed in a second chronic phase using chemotherapy followed by maintenance treatment with imatinib (Barbouti et al., 2003). The patient remained in second chronic phase for 125 days, when second blastic crisis occurred (Barbouti et al., 2003). Our patient differed from these two cases by having imatinib treatment initiated during chronic phase CML with ETV6-ABL1 and receiving no chemotherapy prior to imatinib treatment, with an excellent response for over 7 months. This is the first report that ETV6-ABL1-positive chronic phase CML can be successfully treated with imatinib.

Blaschkolinear skin pigmentary variation due to trisomy 7 mosaicism.

Mosaic trisomy 7 is a rare condition that can be seen in individuals with Blaschkolinear skin pigmentary variation, somatic asymmetry, and variable other clinical anomalies. In any patient presenting with Blaschkolinear skin pigmentary variation, varying degrees of asymmetrical growth disturbance, developmental delay, and a normal lymphocytic karyotype, chromosomal mosaicism may be present. To rule out tissue-specific or occult chromosomal mosaicism, it is recommended to culture and karyotype skin fibroblasts, since lymphocyte cell lines may not demonstrate the abnormal cell line. Early diagnosis is of paramount importance, since early physical, occupational, and speech/language therapy can greatly improve the developmental outcome of these patients. We report on a fourth patient with trisomy 7 mosaicism in whom early diagnosis and developmental therapy contributed to an improved developmental outcome when compared with patients in previous reports. Early intervention can greatly benefit patients with this diagnosis, especially in minimizing the aggressive behavior associated with communication difficulties. Our patient has milder manifestations than the previously reported patients with no seizure activity or asymmetry and fewer cells with trisomy 7.

Single nucleotide polymorphism genomic arrays analysis of t(8;21) acute myeloid leukemia cells

Translocation of chromosomes 8 and 21, t(8;21), resulting in the AML1-ETO fusion gene, is associated with acute myeloid leukemia (AML). The findings of this study indicate that genomic alterations and KIT-D816 mutation confer a poor prognosis in t(8;21) AML patients. Translocation of chromosomes 8 and 21, t(8;21), resulting in the AML1-ETO fusion gene, is associated with acute myeloid leukemia. We searched for additional genomic abnormalities in this acute myeloid leukemia subtype by performing single nucleotide polymorphism genomic arrays (SNP-chip) analysis on 48 newly diagnosed cases. Thirty-two patients (67%) had a normal genome by SNP-chip analysis (Group A), and 16 patients (33%) had one or more genomic abnormalities including copy number changes or copy number neutral loss of heterozygosity (Group B). Two samples had copy number neutral loss of heterozygosity on chromosome 6p including the PIM1 gene; and one of these cases had E135K mutation of Pim1. Interestingly, 38% of Group B and only 13% of Group A samples had a KIT-D816 mutation, suggesting that genomic alterations are often associated with a KIT-D816 mutation. Importantly, prognostic analysis revealed that overall survival and event-free survival of individuals in Group B were significantly worse than those in Group A.

Using fluorescence in situ hybridization (FISH) in genome mapping

Fluorescence in situ hybridization (FISH) provides one of the most effective and rapid approaches for assigning and ordering DNA fragments within single eukaryotic chromosome bands. These techniques have wide applications not only for the mapping of the human genome and the genomes of other organisms, but also in clinical cytogenetics, somatic cell genetics, cancer diagnosis and gene expression studies.

Chromosome Banding Techniques

Chromosome banding techniques produce a series of consistent landmarks along the length of metaphase chromosomes that allow for both recognition of individual chromosomes within a genome and identification of specific segments of individual chromosomes. These landmarks facilitate assessment of chromosome normalcy, identification of sites of chromosome breaks and alterations, and location of specific genes. This unit covers these basic banding techniques (Q-banding, G-banding, and R-banding), which produce virtually identical patterns of bands along the length of human chromosomes, although the bands and polymorphic regions highlighted may differ with each technique. These techniques highlight reproducible landmarks along the length of the chromosome and specialized staining techniques can be used to highlight particular regions of chromosomes, such as heterochromatic and repeated-sequence segments. These specialized techniques, nucleolar organizer region (NOR) staining, centromeric heterochromatin staining (C-banding), methylated satellite DNA staining (distamycin-DAPI banding), and replication banding are also presented in this unit.