Fluorescent in situ hybridization (FISH) in bone marrow and peripheral blood of leukemia patients: implications for occupational surveillance.

Although there has been a rapid rise in the application of fluorescent in situ hybridization (FISH) analysis of bone marrow tissue for the staging and prognosis determination of hematopoietic malignacies such as the chronic and acute leukemias, it's application as a surveillance tool for leukemogen exposed high risk occupational cohorts is understandably limited by the invasiveness of sample collection. While some small occupational studies have been performed using FISH in peripheral blood with promising results, some of the basic assumptions made in utilizing the FISH technique have not been fully explored. These include selection of the correct hematopoietic cell to assay (myeloid or lymphoid); selection of appropriate chromosomal markers and the sensitivity of peripheral blood FISH in detecting unbalanced genomic abnormalities. In this study, we performed a pilot 'validation' exercise utilizing the FISH technique and standard metaphase cytogenetics, comparing results in tandem pairs of peripheral blood with bone marrow cells, where clonal abnormalities arise. Samples were taken from patients with known chromosomal lesions associated with active leukemia. We carefully chose markers most frequently associated with leukemogen-inducing DNA damage and probes that have been utilized successfully in clinical practice. Ten de novo or therapy-related acute myeloid leukemia (t-AML) patients underwent bone marrow cell karyotyping and fluorescent in situ hybridization (FISH) analysis. Parallel peripheral blood samples were concommitently drawn and evaluated with FISH using the same probes. In six of eight paired samples treated with a 3-day phytohemagglutinin (PHA) stimulation, typically used to assay lymphocytes and their progenitors, we detected abnormal clones. In one of the two remaining cases, we identified an abnormal clone in both bone marrow and PHA-stimulated peripheral blood, although at a level in the peripheral blood sample that would typically be reported as "non-diagnostic" for clinical purposes. These results suggest that use of FISH in PHA stimulated peripheral blood samples with probes commonly employed in t-AML evaluations (chromosomes 5q, 7q, 8, 11q) to detect cytogenetic abnormalities in peripheral blood represents a potentially promising though as yet, under-utilized approach for the occupational surveillance of workers exposed to leukemogens, especially if it could be linked to automated high-throughput assays for increased sensitivity.

Distinct functions of two isoforms of a homeobox gene, BP1 and DLX7, in the regulation of the beta-globin gene.

Homeotic proteins are transcription factors that regulate the expression of multiple genes involved in development and differentiation. We previously isolated a cDNA encoding such a protein from the human leukemia cell line K562, termed Beta Protein 1 (BP1), which is involved in negative regulation of the human beta-globin gene. Sequence comparison revealed that BP1 is a member of the distal-less (DLX) family of homeobox genes and that it shares its homeodomain and 3' sequences with another DLX cDNA, DLX7. BP1 and DLX7 exhibit unique 5' regions, diverging at nucleotide 565 of BP1. We mapped this new distal-less family member BP1 to chromosome 17q21-22 by FISH and PCR, which is the same locus to which DLX7 has been mapped. These results strongly suggest that BP1 and DLX7 are isoforms (derived from the same gene). Since our previous data demonstrated that BP1 and DLX7 are frequently co-expressed, we determined whether DLX7 is also involved in the negative regulation of the beta-globin gene. Mobility shift assays demonstrated that both BP1 and DLX7 proteins, synthesized in vitro, bind to the same BP1 binding site. However, using transient assays, we showed that although BP1 represses activity of a reporter gene through either of two silencer DNA sequences upstream of the beta-globin gene, DLX7 did not show repressor activity against the beta-globin promoter. Further characterization of these apparent isoforms is of significance since they are jointly expressed in acute myeloid leukemia and in many leukemia cell lines.

Detection of diagnostically critical, often hidden, anomalies in complex karyotypes of haematological disorders using multicolour fluorescence in situ hybridization.

Multicolour fluorescence in situ hybridization (M-FISH) simultaneously detects all 24 human chromosomes in unique fluorescent colours. The identification of diagnostically critical gene rearrangement(s) in complex karyotypes of haematological disorders continues to be a challenge. We present five cases in which t(9;11), complex t(8;22), t(12;21) and t(11;14) were detected primarily using M-FISH and were confirmed using locus-specific probes. We conclude that M-FISH can be effective in complete characterization of critical gene rearrangements in haematological disorders.

Third Prader-Willi syndrome phenotype due to maternal uniparental disomy 15 with mosaic trisomy 15.

We report on a boy with mosaicism for trisomy 15 and Prader-Willi syndrome (PWS) due to maternal isodisomy for chromosome 15. His phenotype is consistent with PWS and trisomy 15 mosaicism. Although our patient is unusual in having maternal isodisomy rather than the more common maternal heterodisomy, we think that his more severe PWS phenotype is due to his trisomy 15 mosaicism rather than to homozygosity for deleterious chromosome 15 genes. We propose that individuals with PWS have one of three similar but distinctive phenotypes depending on the cause of their condition. Patients with paternal deletions have the typical PWS phenotype, patients with maternal UPD have a slightly milder phenotype with better cognitive function, and those with maternal UPD and mosaic trisomy 15 have the most severe phenotype with a high incidence of congenital heart disease. These phenotype-genotype differences are useful to guide the work-up of patients with suspected PWS and to provide prognostic counseling for families.

Interphase FISH for rapid identification of a down syndrome animal model.

Mouse models of Down syndrome (trisomy 21, DS) have been developed to study the consequences of triplication of regions syntenic to distal human chromosome 21. We report a technique for the identification of segmental trisomy 16 (Ts65Dn) and diploid progeny at any age using interphase fluorescence in situ hybridization (FISH) of uncultured tail fibroblasts. Our technique is faster and less expensive than blood karyotyping.

Patients with t(8;21)(q22;q22) and acute myeloid leukemia have superior failure-free and overall survival when repetitive cycles of high-dose cytarabine are administered.

PURPOSE: To examine the effect of single compared with repetitive (at least three) cycles of high-dose cytarabine after induction therapy for patients with acute myeloid leukemia (AML) who have the t(8;21)(q22;q22) karyotype. PATIENTS AND METHODS: Patients entered onto the study had AML and t(8;21) and attained a complete remission on four successive Cancer and Leukemia Group B studies. In these studies, either > or = three cycles of high-dose cytarabine or one cycle of high-dose cytarabine was administered, followed by sequential cyclophosphamide/etoposide and mitoxantrone/diaziquone with or without filgrastim support. Outcomes of these two groups of t(8;21) patients were compared. RESULTS: A total of 50 patients with centrally reviewed AML and t(8;21) were assigned to receive one (n = 29) or > or = three cycles (n = 21) of high-dose cytarabine as postinduction therapy. The clinical features of these two groups of patients were similar. Initial remission duration for t(8;21) patients assigned to one cycle of high-dose cytarabine was significantly inferior (P =.03), with 62% of patients experiencing relapse with a median failure-free survival of 10.5 months, compared with the group of patients who received > or = three cycles, in which only 19% experienced relapse and failure-free survival is estimated to be greater than 35 months. Furthermore, overall survival was also significantly compromised (P =.04) in patients assigned to one cycle of high-dose cytarabine, with 59% having died as a consequence of AML, compared with 24% of those who received > or = three cycles of high-dose cytarabine. CONCLUSION: These data demonstrate that failure-free survival and overall survival of patients with t(8;21)(q22;q22) may be compromised by treatment approaches that do not include sequential high-dose cytarabine therapy.

Quantitative measurement of telomerase activity in lymphadenopathy: correlation with histologic features and human immunodeficiency virus-1 infection.

Telomerase is a ribonucleoprotein that uses its internal RNA component as a template for synthesis of telomeric DNA on the ends of chromosomes after each round of cell division. It is expressed in approximately 90% of all human cancers tested to date, as well as in most immortal cell lines. Recently, telomerase activity was detected in normal proliferating lymphoid tissue and in non-Hodgkin's lymphomas (NHLs) by use of the telomeric repeat amplification protocol assay, a qualitative measure of telomerase activity. In this study, we modified the assay to measure quantitatively the telomerase activity in lymph node biopsy specimens obtained from patients with lymphadenopathy. The lymph nodes either contained benign reactive changes, were involved by NHL of B-cell lineage, or were involved by Hodgkin's disease. Telomerase activity was detected in all of our samples, benign as well as malignant. The levels of activity were unaffected by the patient's human immunodeficiency virus-1 status. Although the specimens involved by NHLs showed a range in telomerase activity from low to high, the levels did not correlate strictly with the histologic grade according to the Working Formulation. All of the cases of Hodgkin's disease also expressed telomerase activity, and the levels were similar regardless of histologic subtype. Our results showed that telomerase activity was expressed in both benign and malignant lymphoproliferative processes.

Patients with isolated trisomy 8 in acute myeloid leukemia are not cured with cytarabine-based chemotherapy: results from Cancer and Leukemia Group B 8461.

To date, neither the clinical significance of isolated trisomy 8, the most frequent trisomy in acute myeloid leukemia (AML), nor the effect of age within a single cytogenetic group has been examined. We report a large cohort of adult trisomy 8 patients and examine whether increasing age within a homogeneous cytogenetic group alters clinical outcome. Characteristics and outcome of patients with isolated trisomy 8 enrolled in the prospective Cancer and Leukemia Group B (CALGB) cytogenetic study CALGB 8461 are described. Isolated trisomy 8 was identified in 42 (3.03%) of 1387 patients enrolled in five CALGB treatment protocols. These patients had a median age of 64 (range, 16-79) years, 50% female proportion, and a low frequency of hepatomegaly (10%) or splenomegaly (10%). Laboratory features included a median white blood count of 7.3 x 10(9)/L, nonspecific French-American-British distribution, with 36% of patients having Auer rods. Treatment outcome was unsatisfactory with a complete remission (CR) rate of 59%, median CR duration of 13.6 months, and median survival of 13.1 months. Older age adversely affected outcome; trisomy 8 patients > or =60 years had both an inferior CR rate (40% versus 88%; P = 0.004) and overall survival (median, 4.8 versus 17.5 months; P = 0.01), as compared with those <60 years of age. Of the patients <60 years of age, only four remain alive, and all received noncytarabine-based intensive chemotherapy, followed in three cases by autologous (n = 2) or allogeneic (n = 1) stem cell transplant in CR1. Adults with AML and isolated trisomy 8 have a poor outcome that is accentuated by increasing age and is rarely cured with cytarabine-based therapy. Alternative investigational treatments should be considered for individuals with this AML subset.

Clinical characteristics of patients with de novo acute myeloid leukaemia and isolated trisomy 11: a Cancer and Leukemia Group B study.

Isolated trisomy 11 is the third most common sole trisomy in de novo acute myeloid leukaemia (AML). However, only 49 cases have been published, and for only a fraction of these cases has full description of clinical and haematological features been provided. As a result, little is known about the clinical characteristics of de novo AML patients with solitary trisomy 11. We have identified 13 patients (0.9%) with isolated trisomy 11 among a total of 1496 consecutive adult patients successfully karyotyped as part of a prospective Cancer and Leukemia Group B (CALGB) cytogenetic study (CALGB 8461). Nine patients (69%) were over the age of 60 (range 29-73 years). Eight patients (62%) were diagnosed with AML of FAB M2 subtype, three patients (23%) had FAB M1 AML and one patient each had AML of FAB M0 and M7, respectively. Seven patients (54%) had high, >100 x 10(9)/l, platelet counts (median 102 x 10(9)/l; range 17-207 x 10(9)/l). All patients received CALGB induction therapy with standard doses of cytarabine and daunorubicin. Six patients (46%) achieved a complete remission (CR). The median CR duration was 17.5 months (range 8.7-49.8). Only one patient, who underwent bone marrow transplantation in first CR, continues in initial CR. The median survival was 14.3 months (range 0.5-50.7); only one patient survives. We conclude that de novo AML with isolated trisomy 11 is predominantly associated with older age, M2 and M1 FAB subtypes, high platelet count and few long-term disease-free survivals, although it is currently unknown whether isolated trisomy 11 constitutes an independent prognostic factor.

Isochromosome 7q: the primary cytogenetic abnormality in hepatosplenic gammadelta T cell lymphoma.

Malignant lymphomas often have complex, nonrandom chromosomal abnormalities. Hepatosplenic gammadelta T cell lymphoma (gammadelta TCL) is an unusual post-thymic T cell lymphoma that primarily involves liver and spleen, often in young adult males. Few cases have had cytogenetic analysis. We report a consistent isochromosome 7q [i(7q)] abnormality in three cases of hepatosplenic gammadelta TCL, one with i(7q) as the sole abnormality at presentation. Three patients, 15-, 37- and 65-year-old males, presented with hepatosplenomegaly and fevers. Histopathologic, immunophenotypic, and molecular genetic studies supported the diagnosis. Spleen, liver, and bone marrow contained sinusoidal infiltrates of atypical lymphoid cells of T cell immunophenotype. PCR performed on two cases demonstrated clonal T cell receptor gamma gene rearrangements. Cytogenetic analysis of bone marrow showed i(7q) as the sole abnormality at presentation in one case. The second case showed i(7q) in addition to two normal chromosomes 7, and other structural and numerical abnormalities. The third case showed i(7q) and a deletion in the long arm of chromosome 11. These findings support the proposal that i(7q) represents the primary nonrandom cytogenetic abnormality in hepatosplenic gammadelta TCL, and plays a role in its pathogenesis.

Segregation of a familial balanced (12;10) insertion resulting in Dup(10)(q21.2q22.1) and Del(10)(q21.2q22.1) in first cousins.

An interchromosomal insertion in 3 generations of a family was ascertained through two developmentally delayed first cousins. Cytogenetic analysis using G-banding and chromosome painting showed an apparently balanced direct insertion of chromosome 10 material into chromosome 12, ins(12;10)(q15;q21.2q22.1), in the mothers and grandfather of these children. The proposita inherited only the derivative 10 chromosome, resulting in deletion of 10q21.2 --> 22.1 while her cousin inherited only the derivative 12, resulting in duplication of 10q21.2 --> 22.1. A comparison of the proposita with published deletion cases suggests a pattern of anomalies attributable to deletion of the 10q21 --> q22 region: developmental delay, hypotonia, a heart murmur, telecanthus, broad nasal root and ear abnormalities. This is the first report of a nontandem duplication of the 10q21 --> q22 region. The phenotype of the cousin with the duplication does not overlap greatly with published tandem 10q duplications. Finally, this report reaffirms the importance of obtaining family studies of patients with interstitial chromosomal abnormalities.

The NAD(P)H:quinone oxidoreductase locus in human colon carcinoma HCT 116 cells resistant to mitomycin C.

Previously, we reported an association of mitomycin C resistance and a deficiency of NAD(P)H:quinone oxidoreductase (NQO1) in HCT 116-R30A cells, a subline derived from mitomycin C-sensitive HCT 116 cells. In HCT 116 cells, we found two mRNAs coding full-length cDNAs of NQO1 differing at codon 139, one with arginine (wild type), and one with tryptophan. Only the tryptophan 139 form of mRNA was detected in HCT 116-R30A cells. In addition, an exon 4 deleted mRNA of NQO1, a product of alternative splicing, was detected in both cell lines. Analysis by semiquantitative reverse transcription-PCR showed that NQO1 mRNA coding full-length cDNAs in HCT 116-R30A cells was 15% of that present in HCT 116 cells. A Mr 26,000 protein, representing the exon 4 deleted mRNA, was not detected by polyclonal anti-NQO1 in HCT 116 sublines. Recombinant plasmids of exon 4 deleted cDNA generated a Mr 26,000 protein without enzymatic activity in Escherichia coli but not in Cos7 cells. The function of exon 4 deleted mRNA is yet unknown. The rates of decay of all NQO1 mRNAs in HCT 116 and HCT 116-R30A cells were similar. DNA sequences of the promoter regions of the NQO1 gene (-837 bp) from both cell lines did not differ from each other or from the same region of the human liver NQO1 gene. Sequences of cis elements in the 837-bp region and mRNA stability could not account for the low expression of full-length mRNA in HCT 116-R30A cells. Southern blot analysis showed the size and the intensity of the NQO1 gene in the two cell lines to be similar. This result was confirmed by semiquantitative PCR analysis of a 450-bp fragment in the NQO1 gene containing codon 139 and the exon 4 region. Digestion of this PCR-amplified fragment by restriction enzyme MspI revealed that HCT 116 cells have two heterozygous NQO1 alleles, a wild-type and a tryptophan 139 form. The functional wild-type NQO1 allele was not detected in HCT 116-R30A cells. Sensitive and resistant cell lines each contained one normal and one abnormal chromosome 16. Loss of the wild-type NQO1 allele in HCT 116-R30A cells did not result from a loss of chromosome 16 or copies of the NQO1 gene. Alteration of factor(s) such as trans-acting factors and DNA methylation may be involved in the down-regulation of NQO1 in the mitomycin C-resistant HCT 116-R30A cells.

Cytogenetic analysis of head and neck carcinomas.

Ten primary squamous cell carcinomas (SCC) of the head and neck were evaluated cytogenetically after 10-14 days of in vitro culture. Addition of 3% L-glutamine was essential for consistent epithelial growth of these carcinomas. Outgrowth of cells from tissue explants contained a mixture of chromosomally normal and abnormal cells; the abnormal cells had extensive changes including translocations, marker chromosomes, inversions, deletions, and duplications. In addition, all carcinomas contained cells with pulverization and double minute chromosomes (dmin). Chromosomes 11, 13, and 14 had "hotspots" of rearrangements.

Chromosomal analysis of recurrent laryngeal papillomas.

Recurrent laryngeal papillomas are tumors believed to be induced by human papillomaviruses. Severity of this disease varies due to the unpredictability of clinical remissions and recurrences. However, the severity of the disease does not affect the classification of these tumors as benign, and the rate of spontaneous conversion of recurrent laryngeal papillomas to carcinomas is very low. Laryngeal papillomas from six patients were evaluated cytogenetically after short-term culture. All six specimens were chromosomally normal, consistent with their classification as benign tumors with a low rate of malignant conversion. The presence of human papillomaviruses has no detectable effect on the chromosomes of these tumors.

Simultaneous study of karyotype and cell morphology in childhood erythroleukemia.

We followed a 2-year-old girl with erythroleukemia (EL) for 7 months, from the time of her initial diagnosis until her death. Immunophenotyping of bone marrow was negative for markers of myeloid and lymphoid lineages. Chromosome study of marrow at diagnosis revealed abnormalities in all mitotic cells, with a clonal karyotype of 48,XX,t(2;12)(p11.2; p13),+6,+21. Subsequent studies showed that the clone rapidly evolved and accumulated additional structural and numerical abnormalities, in spite of intensive chemotherapy during the final months. Simultaneous study of the karyotype and cell morphology of dividing bone marrow cells after 24 hours of in vitro culture, using a technique that preserves cell structure, showed that cells with the chromosome abnormalities were PAS-negative. The combined immunological, cytogenetic, and morphological data indicate that the original malignant cell type was neither myeloblast nor erythroblast but an early progenitor type that rapidly proliferated.

Duplication 17q mosaicism: an infant with features of Ellis-van Creveld syndrome.

We describe an infant with multiple dysmorphic features who is mosaic for duplication 17q21.1----qter, owing to a direct tandem duplication. He is the first case with mosaicism for a 17q duplication to be reported. His features are strikingly suggestive of Ellis-van Creveld syndrome.

Persistent chromosome damage induced by localized radiotherapy for lymphoma.

A fibroblast culture was established from a lymph node biopsy of a patient with non-Hodgkin lymphoma, 9 months after chemotherapy and intensive therapeutic x-irradiation of the area. In contrast with blood and bone marrow, which were chromosomally normal, all cells of the lymph node were chromosomally abnormal, with numerous clones having multiple structural abnormalities. Numerical abnormalities (trisomies and monosomies) were not found. Structural abnormalities included translocations, terminal deletions, and pericentric inversions, with an excess of centromeric breakpoints being the only apparent deviation from a random distribution of breakpoints. None of the rearrangements associated with malignant lymphoma were seen, indicating that the chromosome abnormalities in the lymph stroma were radiation-associated, not disease-associated. These acquired changes may be a cause of additional malignant transformation.

Chromosome abnormalities in erythroleukemia.

Erythroleukemia (EL) is a heterogeneous disease in terms of cell type affected, chromosome abnormalities found in the malignant clone, and clinical course. In this article, cases of erythroid EL from the recent medical literature are reviewed using cytogenetic criteria to distinguish such cases from those of myeloid EL. Although most patients with erythroid EL were elderly men, 20% of the cases occurred in the under-3 age group, where boys and girls were equally affected. Chromosomes 5 and 7 were found to be lost or partially deleted in two thirds of the adult patients only, but not in the pediatric patients; this suggests that EL is associated with cumulative mutagen exposure in adult patients only. It is proposed that cytogenetic criteria may be of use prospectively in distinguishing patients with erythroid EL from those with myeloid EL.

Mitotic recombination can explain the apparent polyclonal origin of some tumors.

Although most human tumors appear to be monoclonal in origin, a few express two G6PD types; on this basis they have been thought to be polyclonal in origin and exceptions to the general rule of monoclonality. In light of the recent discovery that mitotic recombination can cause a shift from genetic heterozygosity to homozygosity in tumor cells, we suggest that such a mechanism can also explain the occasional occurrence of two G6PD types in a monoclonal tumor.