Amniocentesis was performed at the 19 week of gestation because of advanced maternal age and a presumed risk of 1/14 for Down syndrome as determined by the triple test. Chromosomal analysis of the fetus revealed a karyotype of 46,XX,inv(17)(p11.2;q25.1). This chromosomal breakpoint can be associated with a microdeletion on chromosome 17 and be related with Smith-Magenis syndrome and shown by cytogenetic observations. That is why, we have examined the fetus with the Smith-Magenis probe by the use of fluorescence in situ hybridization (FISH) technique to identify the microdeletion of band p11.2 of chromosome 17. After it is confirmed that the inverted chromosome is familial and no deletion has occurred, the pregnancy continued for a healthy carrier baby. These findings have implications for prenatal counseling of carriers of pericentric inversions, who typically are considered to bear minimal reproductive risk. This report shows that, FISH provides a reliable means for prenatal detection of the chromosome 17 rearrangements in this family.

Ileri anne yasi ve uclu testte 1/14 risk ile Down sendromu dusunulen gebelige 19. haftasinda amniyosentez uygulandi. Fetusun kromozom analizi sonucu karyotipi 46,XX,inv (17) (p11.2;q25.1) olarak belirlendi. Sitogenetik olarak tespit edilen bu kromozomal kirik noktasi mikrodelesyon sendromu olan Smith-Magenis sendromu ile iliskili olabilecegi icin, Smith Magenis probu kullanilarak FISH yontemi uygulandi. Delesyon olmadigi ve familyal inversiyon 17 oldugu tesbit edilerek gebelige devam edildi ve saglikli bir bebek dogdu. Bu bulgularin isigi altinda, perisentrik inversiyon tasiyicilarinin prenatal genetik danismasinin onemli olabilecegi dikkate alinmalidir. Bu makalede, FISH yonteminin prenatal olarak inversiyon sonucu kromozom 17'de olusabilecek mikrodelesyon ve yeniden duzenlenmelerin analizi acisindan guvenilir oldugu vurgulanmaktadir. Anahtar Kelimeler: Kromozom 17, Perisentrik inversiyon, Fluoresans in situ hibridizasyon

Chromosome aberrations are frequently observed in precursor-B-acute lymphoblastic leukemias (ALL) and T-cell acute lymphoblastic leukemias (T-ALL). These translocations can form leukemia-specific chimeric fusion proteins or they can deregulate expression of an (onco)gene, resulting in aberrant expression or overexpression. Detection of chromosome aberrations is an important tool for risk classification. We developed rapid and sensitive split-signal fluorescent in situ hybridization (FISH) assays for six of the most frequent chromosome aberrations in precursor-B-ALL and T-ALL. The split-signal FISH approach uses two differentially labeled probes, located in one gene at opposite sites of the breakpoint region. Probe sets were developed for the genes TCF3 (E2A) at 19p13, MLL at 11q23, ETV6 at 12p13, BCR at 22q11, SIL-TAL1 at 1q32 and TLX3 (HOX11L2) at 5q35. In normal karyotypes, two colocalized green/red signals are visible, but a translocation results in a split of one of the colocalized signals. Split-signal FISH has three main advantages over the classical fusion-signal FISH approach, which uses two labeled probes located in two genes. First, the detection of a chromosome aberration is independent of the involved partner gene. Second, split-signal FISH allows the identification of the partner gene or chromosome region if metaphase spreads are present, and finally it reduces false-positivity.

The proto-oncogene EVI1 encodes a DNA binding protein and is located on chromosome 3q26. The gene is aberrantly expressed in acute myeloid leukemia (AML) patients carrying 3q26 abnormalities. Two mRNAs are transcribed from this locus: EVI1 and a fusion of EVI1 with MDS1 (MDS1-EVI1), a gene located 5' of EVI1. The purpose of this study was to investigate which of the 2 gene products is involved in transformation in human AML. To discriminate between EVI1 and MDS1-EVI1 transcripts, distinct real-time quantitative polymerase chain reaction (PCR) assays were developed. Patients with 3q26 abnormalities often showed high EVI1 and MDS1-EVI1 expression. In a cohort of 319 AML patients, 4 subgroups could be distinguished: EVI1(+) and MDS1-EVI1(-) (6 patients; group I), EVI1(+) and MDS1-EVI1(+) (26 patients; group II), EVI1(-) and MDS1-EVI1(+) (12 patients; group III), and EVI1(-) and MDS1-EVI1(-) (275 patients; group IV). The only 4 patients with a 3q26 aberration belonged to groups I and II. Interestingly, high EVI1 and not MDS1-EVI1 expression was associated with unfavorable karyotypes (eg, -7/7q-) or complex karyotypes. Moreover, a significant correlation was observed between EVI1 expression and 11q23 aberrations (mixed lineage leukemia [MLL] gene involvement). Patients from groups I and II had significantly shorter overall and event-free survival than patients in groups III and IV. Our data demonstrate that high EVI1 expression is an independent poor prognostic marker within the intermediate- risk karyotypic group.

Expression of the multidrug resistance ( MDR 1) phenotype, encoded by the MDR 1 gene, is an adverse prognostic factor for CR and survival in acute myeloid leukemia (AML). Other prognostic factors, such as specific cytogenetic abnormalities, have been identified in AML. We have investigated the expression of the MDR 1 gene in untreated AML patients with monosomy 7 ( n = 12), and partial deletions ( n = 7) of the long arm of chromosome 7 (respectively −7/7q−), because of the extremely bad prognosis associated with these cytogenetic abnormalities and because of the fact that the MDR 1 gene is located on chromosome 7q21.1. The findings were compared with the level of MDR 1 expression in a group of 42 other AML patients, matched for age with favourable, neutral or complex cytogenetic abberations. MDR 1 mRNA expression, as measured by the RNase protection assay was significantly higher in the −7/7q− group vs other AML patients (median 1.3 vs 0.1 arbitrary units, P = 0.02). Protein expression of MDR 1 in the −7/7q− group, as determined with the monoclonal antibody MRK16, was found to be similar to the levels found in the control group. With a functional rhodamine retention assay using the modulator PSC833, increased MDR 1 activity was observed in the −7/7q− group as compared to the control group of patients ( P = 0.05). Considering the higher MDR 1 mRNA expression and equal or slightly elevated level of protein expression of MDR 1, we studied the presence of MDR 1 genes in this group of −7/7q− patients. Fluorescence in situ hybridization (FISH) studies, using a specific MDR 1 probe revealed no loss of an MDR 1 allele in any of the deleted q− arms of the seven patients with 7q−, whereas all monosomy 7 patients lacked one MDR 1 gene homologue. To determine whether there was selective loss of the MDR 1 gene in the −7/7q− patients, the genetic polymorphism of the MDR 1 gene was used. Both allelic variants (G and T) were represented in the −7/7q− and in the control group, showing a predominance for GT at position 2677 of the MDR 1 gene in the control group. In the 12 monosomy 7 patients loss of the MDR 1 allele was random. Methylation studies of the CpG island of the MDR 1 gene revealed no hypermethylation in any of the −7/7q− patients. We conclude that MDR 1 expression in −7/7q− AML patients is upregulated at transcriptional, but not at translational level, suggesting that mechanisms other than MDR 1 are responsible for the poor prognosis in these patients.

Our retrospective karyotype review revealed two rare recurrent translocations affecting ETV6 (TEL) : t(7;12)(q36;p13) and t(7;12)(q32;p13). Five patients with a t(7;12) were from a group of 125 successfully karyotyped pediatric patients enrolled in consecutive clinical AML trials of the Dutch Childhood Leukemia Study Group over a period of 7 years. During a search of available cytogenetic databases, we found 7q and 12p abnormalities in two additional Dutch patients and in three participants in Pediatric Oncology Group trials. A del(12p) had been initially identified in four of these patients and re-examination of the original karyograms revealed a t(7;12)(q36;p13) in two instances and a probable t(7;12) in the other two. FISH confirmed the presence of a t(7;12)(q36;p13) in the latter. Most ( n = 7) also had trisomy 19. The t(7;12)(q36;p13) ( n = 9) was more common than the t(7;12)(q32;p13) ( n = 1). These subtle translocations were found only in children 18 months of age or younger. A literature search revealed that the t(7;12) with breakpoints at 7q31-q36 and 12p12-p13 had been reported in six children with myeloid disorders and in two with acute lymphoblastic leukemia; all were 12 months of age or younger. Only two of the 17 for whom survival data were available, were alive after at least 22 months of continuous complete remission. Our findings suggest that ETV6 rearrangements due to a t(7;12) may play an adverse role in myeloid disorders in children 18 months of age or younger. Therefore, children in this age group with myeloid disorders should be screened for both MLL and ETV6 rearrangements.

The RAD18 gene of the yeast Saccharomyces cerevisiae encodes a protein with ssDNA binding activity that interacts with the ubiquitin-conjugating enzyme RAD6 and plays an important role in postreplication repair. We identified and characterized the putative mouse homolog of RAD18, designated mRAD18Sc. The mRAD18Sc open reading frame encodes a 509-amino-acid polypeptide that is strongly conserved in size and sequence between yeast and mammals, with specific conservation of the RING-zinc-finger and the classic zinc-finger domain. The degree of sequence conservation between mRAD18Sc, RAD18, and homologous sequences identified in other species (NuvA from Aspergillus nidulans and Uvs-2 from Neurospora crassa) is entirely consistent with the evolutionary relationship of these organisms, strongly arguing that these genes are one another's homologs. Consistent with the presence of a nuclear translocation signal in the amino acid sequence, we observed the nuclear localization of GFP-tagged mRAD18Sc after stable transfection to HeLa cells. mRNA expression of mRAD18Sc in the mouse was observed in thymus, spleen, brain, and ovary, but was most pronounced in testis, with the highest level of expression in pachytene-stage primary spermatocytes, suggesting that mRAD18Sc plays a role in meiosis of spermatogenesis. Finally, we mapped the mRAD18Sc gene on mouse chromosome 6F.

BACKGROUND:Chromosome banding techniques and in situ hybridization reveal the majority of chromosomal aberrations. However, difficulties remain in cases of highly contracted chromosomes, poor quality of the metaphases or the presence of markers with the involvement of several chromosomes. Here, it is demonstrated that reverse painting can be applied successfully starting with bone marrow cells from primary acute myelocytic leukemias (AML).METHODS:This was accomplished by culturing the leukemic cells with a cocktail of various growth factors, which yielded sufficient numbers of cells in cycle to harvest chromosomes for sorting. Aberrant chromosomes were flow-sorted and amplified by degenerate oligonucleotide-primed PCR. The resulting products were labeled by nick-translation and hybridized on normal metaphase spreads.RESULTS:Two patients with marker chromosomes in their leukemia cells were analyzed in detail. The hybridization pattern displayed the composition of the aberrant sorted chromosome. Results were compared with conventional cytogenetic analyses that were performed on material obtained from the same aspirate. The reverse-painting technique enabled identification of aberrations that were not detected by conventional cytogenetic analysis.CONCLUSIONS:Primary AML cells can be cultured in vitro, using optimal culture conditions, facilitating the production of high quality flow karyotypes, suitable for sorting of marker chromosomes to produce DOP-PCR derived chromosome painting probes for reverse painting. Valuable additional cytogenetic information can thus be obtained about complex chromosomal rearrangements or structural aberrations that could not be completely resolved by conventional cytogenetic analysis.

The combined use of retinoic acid and chemotherapy has led to an important improvement of cure rates in acute promyelocytic leukemia. Retinoic acid forces terminal maturation of the malignant cells and this application represents the first generally accepted differentiation-based therapy in leukemia. Unfortunately, similar approaches have failed in other types of hematological malignancies suggesting that the applicability is limited to this specific subgroup of patients. This has been endorsed by the notorious lack of response in acute promyelocytic leukemia bearing the variant t(11;17) translocation. Based on the reported synergistic effects of retinoic acid and the hematopoietic growth factor granulocyte colony-stimulating factor (G-CSF), we studied maturation of t(11;17) positive leukemia cells using several combinations of retinoic acid and growth factors. In cultures with retinoic acid or G-CSF the leukemic cells did not differentiate into mature granulocytes, but striking granulocytic differentiation occurred with the combination of both agents. At relapse, the patient was treated with retinoic acid and G-CSF before reinduction chemotherapy. With retinoic acid and G-CSF treatment alone, complete granulocytic maturation of the leukemic cells occurred in vivo, followed by a complete cytogenetical and hematological remission. Bone marrow and blood became negative in fluorescense in situ hybridization analysis and semi-quantitative polymerase chain reaction showed a profound reduction of promyelocytic leukemia zinc finger-retinoic acid receptor-alpha fusion transcripts. This shows that t(11;17) positive leukemia cells are not intrinsically resistant to retinoic acid, provided that the proper costimulus is administered. These observations may encourage the investigation of combinations of all-trans retinoic acid and hematopoietic growth factors in other types of leukemia.

The inv(16)(p13q22) and t(16;16)(p13;q22) in acute myeloid leukaemia are associated with a relatively good prognosis but are difficult to detect using classic cytogenetics. We have designed a two‐colour fluorescence in situ hybridization approach that uses two DNA probes that map close to and on either side of the inv(16) p‐arm breakpoint region. This new strategy clearly detected the inv(16)(p13q22)/t(16;16)(p13;q22) on both metaphase chromosomes and in interphase nuclei, even when they are of poor quality. This procedure also detected the inv(16) in cases with an additional deletion of sequences proximal to the 16p‐arm breakpoint which is present in 20% of all cases.

In order to analyze the efficiency of interphase FISH for the detection and monitoring of Ph + cells in chronic myelogenous leukemia (CML) under interferon (IFN) treatment, the following experiments were performed: (1) 98 specimens derived from 32 patients were analyzed in parallel by dual-color FISH and by conventional chromosome analysis (CCA). A 300/200 kb BCR/ABL probe was used in all tests and a smaller 35.5/39 kb probe was tested in parallel in 22 BM samples; (2) 30 BM samples were prepared by direct harvest and by 24-h culture and were analyzed in parallel; (3) PB and BM samples obtained simultaneously from 11 patients were analyzed. The cut-off point for the recognition of BCR/ABL fusion was set at 2.4%, calculated as the mean percent of false positivity in 11 controls plus 3 s.d. A very close correlation was observed ( r = 0.994, r 2 = 0.988, P < 0.0001) between the percentages of ph + cells as assessed by CCA and by interphase FISH in 98 samples (26 at diagnosis). There was a moderate overestimation of the frequency of Ph + cells by FISH with respect to CCA, that was more evident at low-to-medium values of Ph positivity. Seven specimens without Ph + metaphases (17–50 cells analyzed) were shown to carry 2.5–8% interphase cells with BCR/ABL fusion. Similar percentages of BCR/ABL + nuclei were recorded in 22 samples hybridized using the 300/200 kb and the 35.5/39 kb probe-sets (variation range: 0–5%, mean 2.3%). A very good correlation between the frequency of Ph + interphase cells was observed when analyzing in parallel BM preparations after direct harvest and after 24-h culture. Underestimation of the percentage of BCR/ABL + cells was noted to occur in 2/11 PB samples, compared to BM samples, the remaining nine cases showing superimposable results at either sites. We arrived at the following conclusions: (1) dual-color FISH enables an accurate detection and monitoring of the size of the Ph-positive clone in CML at diagnosis and after IFN-therapy; (2) FISH is more accurate than CCA, especially at low levels of Ph-positive cells; (3) testing of directly harvested BM samples is feasible and accurate, giving the opportunity to perform centralized FISH analysis in the context of multicentre trials; (4) the percentage of BCR/ABL + PB cells usually, though not invariably, reflects the frequency of mutated cells in the BM.

PURPOSE:To correlate abnormal magnetic resonance (MR) imaging signal patterns in cartilage with the effectiveness of radiation treatment. MATERIALS AND METHODS:Eighty previously untreated patients underwent MR imaging and radiation therapy with a curative intent. Cartilage was considered to have an abnormal signal pattern if it had intermediate signal intensity on T1-weighted spin-echo (SE) MR images and high signal intensity on T2-weighted SE MR images. The minimum follow-up was 2 years. RESULTS:Abnormal MR imaging signal patterns of the thyroid cartilage (P < .001; P < .04) were more ominous than those of other cartilage. Abnormal signal patterns in cartilage of patients with small tumors (< 5 cm3 and especially < 1 cm3) were less significant. Abnormal signal patterns in cartilage combined with a large tumor (> 5 cm3) worsened the prognosis significantly (P < .05). CONCLUSION:Abnormal MR imaging signal patterns in cartilage may not indicate a poor prognosis in every case. Abnormal signal intensity in the thyroid cartilage combined with a tumor volume of > 5 cm3, however, appears to indicate an adverse prognosis with regard to tumor recurrence.

The cell morphology and karyotype of bone marrow samples from 24 patients with myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) were studied simultaneously with a combined technique of May-Grünwald-Giemsa (MGG) staining and fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes. This enabled us to investigate cell lineage involvement in three malignant conditions: MDS (n = 12), leukaemia-transformed MDS (LT-MDS) (n = 5) and de novo AML (n = 7). In MDS we found blasts and often significant proportions of mature granulocytic and erythroid cells to be cytogenetically abnormal. Percentages of granulocytic and erythroid cells with cytogenetic aberrations were generally less than those of blasts. These data support the involvement of a transformed pluripotent stem cell that has retained maturation abilities. In two patients with chronic myelomonocytic leukaemia (CMMoL) the clonal involvement of monocytes was predominant. Results in the five patients with LT-MDS were similar to those in MDS. In the bone marrow of five of the seven de novo AML patients the cytogenetic abnormalities were restricted to the blasts and did not include the more mature granulocytic or erythroid populations. In the other two patients with AML, both with a t(8;21) and a loss of the Y chromosome, high percentages of mature neutrophils were cytogenetically abnormal. These patterns of clonal lineage involvement in MDS, LT-MDS, t(8;21) AML and AML appear typical and may be of clinical use, for example, for distinguishing LT-MDS from de novo AML in newly presenting patients.

Bone marrow and blood from three patients with myelodysplastic syndrome (MDS) and monosomy 7 were studied for cell lineage involvement of the chromosomal abnormality. Cytogenetic involvement of the myeloid and erythroid cell lineages in MDS with monosomy 7 has been shown before. Lymphoid subpopulations have also been investigated but generally with negative results. A combined technique of May-Grünwald-Giemsa (MGG) for cell cytology and interphase fluorescence in situ hybridization (FISH) using a chromosome 7 specific DNA probe was applied. Further, immunophenotype and genotype of the cells were simultaneously examined with alkaline phosphatase anti-alkaline phosphatase (APAAP) immunostaining and FISH. The monosomy 7 was found in the blasts and in all or in subpopulations of myeloid and erythroid cells. T cells (CD3+, CD5+) did not appear to be involved. B cells (CD19+, CD22+) showed a normal distribution of FISH spots in two patients. In one patient however the loss of a chromosome 7 was found in approximately 70% of the cells positive for B cell markers including CD79a. The results of this study show that in some cases MDS is a disease arising in a progenitor cell with repopulative abilities restricted to myelopoiesis and erythropoiesis. In other cases, the pluripotent progenitor cells in MDS may show the capacities to differentiate into B lineage lymphoid cells, as well as suggesting that in those instances MDS represents a condition of more primitive transformed hematopoietic ancestor cells.

Species: Mouse Locus name: Zinc finger protein 15 Locus symbol: Zfp15 Map position: Zfp15 is localized on mouse Chromosome (Chr) 4: centromere-D4Bir11-7.4 + 2.7-Zfp15-1.1 + 1.1-D4Mit4-17.0 + 3.9-D4Bir16-7.4 +_ 2.7-(D4Mit9, D4Bir20)--4.3 + 2.1-D4Bir2110.6 + 3.2-D4Mit11-5.4 + 2.4-D4Bir23-3.2 + 1.8-D4Bir24-8.5 + 2.9-(D4Mit13, Pnd, D4Bir25, D4Mit14)-l.1 + 1.1-D4Mit42-6.4 + 2.5-(Dvl, Rnr4)-telomere. Method of mapping: Zfp15 was localized by haplotype analysis of 94 progeny from an interspecific backcross, (C57BL/6J x M. spretus)F 1 x C57BL/6J [1]. Molecular reagents used for mapping: The Zfp15 probe was obtained by polymerase chain reaction amplification of a 552-bp fragment from mouse pituitary cDNA with primers designed on the basis of the rat Zfpl5 cDNA sequence [2]. The primers amplify 465 bp of coding sequence, outside of the conserved zinc finger regions, corresponding to the last 154 amino acids, and 87 bp of the 3' untranslated region. The RT-PCR product was cloned into the pGEM4Z vector and sequenced to confirm its correspondence with the rat Zfpl5 cDNA. Allele detection: A BglI polymorphism was detected in mouse genomic DNA with the Zfp15 probe, resulting in an M. spretusspecific restriction fragment of 15 kb and a common restriction fragment of 24 kb. The mapping was confirmed with a BamHI polymorphism, resulting in two M. spretus-specific restriction fragments of 8.9 and 6.6 kb and two common fragments of 18 and 5.5 kb.

PURPOSE: To determine the predictive value of several clinical and radiologic parameters for recurrence of laryngeal cancer. MATERIALS AND METHODS: Eighty previously untreated patients underwent magnetic resonance (MR) imaging before radiation therapy with curative intent. Tumor volume was calculated from T1-weighted MR images. Cartilage was considered invaded by pathologic tissue if it had intermediate signal intensity on T1-weighted spin-echo (SE) MR images and high signal intensity on T2-weighted SE MR images. The minimum follow-up was 2 years. RESULTS: Parameters such as age, sex, histopathologic findings, and invasion of the vocal muscle or pre-epiglottic space were not significantly correlated with tumor recurrence. Logistic regression analysis showed three relevant contributors: cord mobility, as judged clinically, and tumor volume and, more significantly, cartilage invasion, as seen at MR imaging. CONCLUSION: For untreated laryngeal cancer, MR imaging findings of tumor volume and cartilage invasion allow better patient selection for either radiation therapy or surgery. MR imaging is mandatory for T staging of laryngeal cancer.

Three recombinant phages containing hamster interferon-alpha-encoding genes (Ha Ifa) were isolated from a Ha genomic library, using a murine (Mu) Ifa probe. The phage inserts contained overlapping genomic fragments which span a total length of approx. 30 kb, on which four Ha Ifa genes are localized. The Ifa gene cluster could be assigned to hamster chromosome 2q. The nt sequences of the four Ifa genes were determined. Two of the genes are functional (Ha Ifa-1 and Ifa-3) and two are pseudogenes (Ifa-ps2 and Ifa-ps4). Ha Ifa-1 and -3 were transiently expressed in COS cells and they gave rise to protein products (A1 and A3, respectively) with antiviral properties on hamster CHO cells. In addition, Ha A1 revealed high antiviral activity on murine L929 cells.

The interferon-alpha (IFN-alpha) regulated mouse Ifi54/Ifi56 gene family, which is composed of at least four members (Ifi54, Ifi56, Ifi56-ps1, and Ifi56-ps2), was isolated and characterized. In addition, the chromosomal localization of the four genes was determined. The Ifi54 and Ifi56 genes show an identical organization. Both are composed of a very small first exon and a second exon, which contains the complete open reading frame, except for the ATG start codon and the first two nucleotides of the second codon. In both genes, the two exons are separated by a small intron (5 and 2.5 kb, respectively). Expression of both genes is rapidly induced by IFN-alpha (within 2 h). The Ifi54 promoter region contains two sequences, which are closely related to the interferon stimulated response element (ISRE) consensus sequence (ISRE 1, GGTTTCAATTTCT, and ISRE 2, AGTGTTACTTTCT). The two elements are located directly adjacent to each other. A similar organization was recently established for the hamster Ifi54 promoter (Bluyssen et al., 1994). However, the mouse promoter is 70% less active than the hamster promoter. It turned out that ISRE 2 is hardly active, due to the G at position 4, which is a T in the hamster Ifi54 ISRE 2 and in the ISRE consensus sequence. The Ifi56 promoter region contains at a similar position two functional ISREs of identical strength (ISRE 1, AGTTTCAGTTTCT, and ISRE2, AGTTTCACTTTCC). In the Ifi56 promoter, the two ISRE motifs are separated by 6 bp. In addition to the Ifi56 gene, parts of two closely related genes (Ifi56-ps1 and Ifi56-ps2) were isolated. Both fragments contain an Ifi56-related open reading frame. However, we were unable to isolate the presumed first exon of Ifi56-ps1 and Ifi56-ps2, nor could we show expression of the genes. The Ifi54, Ifi56, Ifi56-ps1, and Ifi56-ps2 genes could all be assigned to mouse chromosome 19D1, suggesting a tight clustering.