RESUMEN
The NUP98 gene is fused with 19 different partner genes in various human hematopoietic malignancies. In order to gain additional clinico-hematological data and to identify new partners of NUP98, the Groupe Francophone de Cytogénétique Hématologique (GFCH) collected cases of hematological malignancies where a 11p15 rearrangement was detected. Fluorescence in situ hybridization (FISH) analysis showed that 35% of these patients (23/66) carried a rearrangement of the NUP98 locus. Genes of the HOXA cluster and the nuclear-receptor set domain (NSD) genes were frequently fused to NUP98, mainly in de novo myeloid malignancies whereas the DDX10 and TOP1 genes were equally rearranged in de novo and in therapy-related myeloid proliferations. Involvement of ADD3 and C6ORF80 genes were detected, respectively, in myeloid disorders and in T-cell acute lymphoblastic leukemia (T-ALL), whereas the RAP1GDS1 gene was fused to NUP98 in T-ALL. Three new chromosomal breakpoints: 3q22.1, 7p15 (in a localization distinct from the HOXA locus) and Xq28 were detected in rearrangements with the NUP98 gene locus. The present study as well as a review of the 73 cases previously reported in the literature allowed us to delineate some chromosomal, clinical and molecular features of patients carrying a NUP98 gene rearrangements.
Asunto(s)
Neoplasias Hematológicas/genética , Proteínas de Complejo Poro Nuclear/genética , Translocación Genética/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Análisis Citogenético , Femenino , Francia , Proteínas de Homeodominio/genética , Humanos , Hibridación Fluorescente in Situ , Lactante , Masculino , Persona de Mediana Edad , Receptores Citoplasmáticos y Nucleares/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Sociedades MédicasRESUMEN
Pediatric acute myeloid leukemia (AML) is a rare disease whose prognosis is highly variable according to factors such as chromosomal abnormalities. Recurrent genomic rearrangements are detected in half of pediatric AML by karyotype. NUcleoPorin 98 (NUP98) gene is rearranged with 31 different fusion partner genes. These rearrangements are frequently undetected by conventional cytogenetics, as the NUP98 gene is located at the end of the chromosome 11 short arm (11p15). By screening a series of 574 pediatric AML, we detected a NUP98 rearrangement in 22 cases (3.8%), a frequency similar to CBFB-MYH11 fusion gene (4.0%). The most frequent NUP98 fusion gene partner is NSD1. These cases are homogeneous regarding their biological and clinical characteristics, and associated with bad prognosis only improved by bone marrow transplantation. We detailed the biological characteristics of these AML by exome sequencing which demonstrated few recurrent mutations (FLT3 ITD, WT1, CEBPA, NBPF14, BCR and ODF1). The analysis of the clonal structure in these cases suggests that the mutation order in the NUP98-rearranged pediatric AML begins with the NUP98 rearrangement leading to epigenetic dysregulations then followed by mutations of critical hematopoietic transcription factors and finally, activation of the FLT3 signaling pathway.
Asunto(s)
Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Proteínas de Complejo Poro Nuclear/genética , Translocación Genética , Alelos , Biomarcadores de Tumor , Proteínas Potenciadoras de Unión a CCAAT/genética , Niño , Preescolar , Epigénesis Genética , Exoma , Femenino , Regulación Leucémica de la Expresión Génica , Frecuencia de los Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Hibridación Fluorescente in Situ , Lactante , Recién Nacido , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidad , Masculino , Mutación , Proteínas de Fusión Oncogénica/genética , Pronóstico , Transducción de Señal , Proteínas WT1/genética , Tirosina Quinasa 3 Similar a fms/metabolismoRESUMEN
The MTCP1 gene is involved in the t(X;14)(q28;q11) translocation associated with T-cell prolymphocytic leukemia and related conditions. This gene is unusual in that it codes for two distinct proteins: a small mitochondrial protein, p8MTCP1, and a putative oncogenic protein, p13MTCP1. Scarcity of material from t(X;14)-associated proliferations and very low levels of mRNA expression have so far prevented a thorough description of p13MTCP1-encoding transcripts. Here, we characterize two additional t(X;14) bearing leukemias allowing this analysis. In one case, with a breakpoint located 5' to the MTCP1 gene, the level of transcription of previously described p13MTCP1-encoding transcripts is enhanced. In the second case, with a breakpoint within the MTCP1 intron I, an alternative transcription initiation site is demonstrated in the tumor cells at 229 bp upstream to exon II. The identification of this internal promoter, together with the similarity between TCL1 and MTCP1 genomic structures, allow us to propose a model in which the duplication of an ancestral gene was followed by the insertion of one copy within the intron of a p8-encoding gene, accounting for the unusual feature of the MTCP1 gene.
Asunto(s)
Leucemia de Células T/genética , Transcripción Genética , Translocación Genética , Anciano , Secuencia de Bases , División Celular/genética , Femenino , Humanos , Leucemia Prolinfocítica/genética , Persona de Mediana Edad , Datos de Secuencia Molecular , Proteínas Proto-Oncogénicas/genética , Linfocitos T/fisiologíaRESUMEN
Nodal mantle cell lymphoma (MCL) is a well-defined entity, but non-nodal leukemic cyclin D1 positive lymphoproliferative disorders have been reported and their relationship with MCL remains controversial and their prognosis heterogeneous. We prospectively studied the expression of cyclin D1 in CD5 positive leukemic B lymphoproliferative disorders at diagnosis and identified 65 cases overexpressing cyclin D1. We did not distinguish any clinical or biological criteria allowing one to identify a non-MCL group. Multivariate analysis identified age, anemia and p27kip1 expression as independent prognostic factors of survival. By univariate analysis, p27kip1 high expression proved to be the strongest predictor of prolonged survival. The median survival of p27 low expressors was 30 months, while it was not reached for p27 high expressors. A high level of p27 expression was often found associated with the absence of nodal involvement and the presence of somatic mutations, but neither of them was restricted to the p27 high expression group. In conclusion, we hypothesize that MCL and these cyclin D1 positive leukemic lymphoproliferative disorders represent a continuous spectrum of diseases. Determination of p27 expression level appears as a routine applicable test allowing identification of a subset of patients who could be considered for different therapeutic approaches.
Asunto(s)
Proteínas de Ciclo Celular/análisis , Ciclina D1/análisis , Trastornos Linfoproliferativos/metabolismo , Proteínas Supresoras de Tumor/análisis , Adulto , Anciano , Aberraciones Cromosómicas , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Femenino , Genes de Inmunoglobulinas , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Inmunofenotipificación , Trastornos Linfoproliferativos/genética , Trastornos Linfoproliferativos/inmunología , Masculino , Persona de Mediana Edad , PronósticoRESUMEN
Abnormal CCND1 expression is found in the majority of mantle cell lymphomas (MCL) and in a minority of other mature B cell malignancies. Its evaluation can therefore aid diagnostic classification, in conjunction with clinical, morphological, immunophenotypic and cytogenetic analysis. We describe a rapid slot-blot hybridization technique allowing quantitative assessment of CCND1 expression relative to beta-actin, with a sensitivity cut-off of approximately 10%. This allowed clear separation (P < 0.01) of CCND1 MCL (0.89 +/- 0.4; range 0.23-1.81; n = 25) from control samples (0.02 +/- 0.04; range 0-0.09; n = 22) on limited quantities of RNA (1-3.5 microg). Of nine samples in which a potential diagnosis of MCL lymphoma was based on morphological analysis of paraffin-embedded material, without adequate immunophenotype analysis, all were CCND1 negative and subsequent immunophenotype demonstrated features compatible with chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) (CD5+, CD23+, FMC7-) in all cases tested. This study demonstrates the feasibility of slot-blot CCND1 quantification and the importance of the availability of cryopreserved material.
Asunto(s)
Ciclina D1/biosíntesis , Linfoma no Hodgkin/patología , Adulto , Anciano , Anciano de 80 o más Años , Autorradiografía , Northern Blotting/métodos , Médula Ósea/patología , Criopreservación , Ciclina D1/análisis , Diagnóstico Diferencial , Femenino , Humanos , Inmunofenotipificación , Ganglios Linfáticos/patología , Linfocitos/inmunología , Linfocitos/patología , Linfoma no Hodgkin/sangre , Linfoma no Hodgkin/inmunología , Masculino , Persona de Mediana Edad , Derrame Pleural Maligno/inmunología , Derrame Pleural Maligno/patología , Sensibilidad y EspecificidadRESUMEN
The translocation (8;21)(q22;q22) is associated with acute myeloblastic leukemia (AML M2). The accurate detection of this chromosomal rearrangement is vital due to its association with a favorable prognosis. Variant translocations exist; these may be hidden within an unusual or complex karyotype. In such cases, it is often difficult to confirm the presence of t(8;21)(q22;q22) by conventional cytogenetic analysis alone. The molecular detection of the AML1/ETO fusion gene is possible by reverse transcriptase polymerase chain reaction (RT-PCR) or dual-color fluorescence in situ hybridization (FISH) using probes specific for AML1 and ETO. Four cases of AML M2, with unusual or complex structural chromosomal abnormalities, without cytogenetic evidence of the classical t(8;21)(q22;q22), were studied by FISH. Two were AML1/ETO positive by RT-PCR, one showed a rearrangement by AML1 by Southern analysis, and the fourth had morphological features characteristic of t(8;21). The FISH results showed a co-localization of one AML1 and one ETO signal in interphase and metaphase nuclei in all four cases, demonstrating the presence of variant t(8;21)(q22;q22) rearrangements. Therefore, FISH analysis with the AML1 and ETO probes is extremely valuable, in cases of AML M2, because of its ability to reveal masked t(8;21)(q22;q22) translocations and thus quickly confirm the diagnosis, allowing patients to be assigned to the correct risk group in terms of treatment.
Asunto(s)
Cromosomas Humanos Par 21 , Cromosomas Humanos Par 8 , Leucemia Mieloide Aguda/genética , Adulto , Niño , Femenino , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , Masculino , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Banding karyotype is a routine technique, which allows the identification of numerous aneusomy and/or aneuploïdy in congenital diseases and cancers. However, this analysis fails to detect small or complex chromosome rearrangements. Molecular cytogenetic techniques like fluorescence in situ hybridization (FISH) analysis can overlap these limitations. Particularly, multicolor karyotyping by spectral karyotyping (SKY) may rectify or precise the conventional karyotype results. With two examples, we present here, the principle, the indications and the limits of this technique for constitutional and cancer chromosomal abnormalities characterization. Moreover, we present an easy way to build efficient sky probes with a best sensitivity than the probes classically used.
Asunto(s)
Aberraciones Cromosómicas , Marcadores Genéticos , Discapacidad Intelectual/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Cariotipificación Espectral , Adulto , Factores de Edad , Secuencia de Bases , Niño , Cromosomas Humanos/genética , Cromosomas Humanos Par 11/genética , ADN/genética , Humanos , Inmunofenotipificación , Hibridación Fluorescente in Situ , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Investigación , Sensibilidad y Especificidad , TrisomíaRESUMEN
Several groups have published flow cytometry scores useful for the diagnosis or prognosis of myelodysplastic syndromes (MDS), mainly based on the detection of immunophenotypic abnormalities in the maturation of granulocytic/monocytic and lymphoid lineages. As anemia is the most frequent symptom of early MDS, the aim of this study was to identify markers of dyserythropoiesis relevant for the diagnosis of MDS analyzed by selecting erythroblasts in a whole no-lysis bone marrow strategy by using a nuclear dye. This prospective study included 163 patients, including 126 with cytopenias leading to MDS suspicion and 46 controls without MDS. In a learning cohort of 53 unequivocal MDS with specific markers, there was a significant difference between the coefficients of variation of mean fluorescence intensities of CD71 and CD36 in MDS patients compared with controls. These two parameters and the hemoglobin level were used to build a RED-score strongly suggestive of MDS if ≥ 3. Using the RED-score in the whole cohort, 80% of MDS or non-MDS patients were correctly classified. When combined with the flow score described by Ogata et al., this strategy allowed to reach a very high sensitivity of 88% of patients correctly classified.
Asunto(s)
Antígenos CD/metabolismo , Eritroblastos/patología , Citometría de Flujo/métodos , Síndromes Mielodisplásicos/diagnóstico , Receptores de Transferrina/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/metabolismo , Pronóstico , Estudios Prospectivos , Curva ROC , Adulto JovenRESUMEN
Familial haemophagocytic lymphohistiocytosis (FHLH) is a genetic disorder caused by defective lymphocyte cytotoxicity, resulting in impaired lymphocyte homeostasis and macrophage infiltration of solid tissues and bone marrow, with extensive haemophagocytosis. It is invariably fatal unless treated by allogeneic haematopoietic stem cell transplantation (HSCT). In a retrospective analysis of 11 cases of FHLH, transplanted in one centre between January 1999 and December 2003, it was found that host T cell expansion occurred early after HSCT in a setting of a viral infection (cytomegalovirus and Epstein-Barr virus respectively) in two cases who received T cell-depleted HSCT. Transient recurrence of clinical and biological manifestations of FHLH was observed, despite evidence for donor cell engraftment. Secondary development of donor T cells led to stable mixed chimaerism and sustained remission of FHLH. Detection of host-derived T cells soon after HSCT in a patient with FHLH should thus not mistakenly be taken as a manifestation of graft rejection.
Asunto(s)
Antígenos CD34 , Trasplante de Células Madre Hematopoyéticas , Histiocitosis de Células no Langerhans/inmunología , Proliferación Celular , Humanos , Lactante , Masculino , Periodo Posoperatorio , Recurrencia , Estudios Retrospectivos , Linfocitos T/patología , Quimera por Trasplante , Trasplante HomólogoAsunto(s)
Cromosomas Humanos Par 11/genética , Cromosomas Humanos Par 14/genética , Cadenas Pesadas de Inmunoglobulina/genética , MicroARNs/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Translocación Genética/genética , Adulto , Secuencia de Bases , Femenino , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Homología de Secuencia de Ácido NucleicoRESUMEN
Nijmegen breakage syndrome (NBS) is characterized by growth retardation, microcephaly, mental retardation, immunodeficiency, and predisposition to malignancies, especially B-cell lymphomas. In contrast, leukemia is rare. A 23-year-old NBS patient presented with anemia, thrombocytopenia, and hyperlymphocytosis. The diagnosis of T-cell prolymphocytic leukemia (T-PLL) was confirmed by cytological and immunological assays (TdT(-), CD2(+), CD5(+), CD3m, and CD7(+)). Biological assays also showed a hemolytic anemia and a clotting factor V decrease. The patient was first treated by methylprednisone for 3 weeks. During this period the lymphocyte count decreased. The simultaneous normalization of the hemolysis and of factor V suggested that both could be related to T-PLL. Since T-PLL is refractory to conventional therapies with a poor prognosis, an intensive chemotherapy such as 2'-deoxycoformycin with anti-CDw52 monoclonal antibodies is usually favored. In the present case, however, because of the specific context (i.e., NBS-induced immunodepression, severe hemolytic anemia, and acquired factor V deficiency), he received pentostatin weekly during 1 month and in maintenance during 6 months. At last follow-up (7 months) he showed a persistent control of the lymphocytosis with no side effect.
Asunto(s)
Anemia Hemolítica Autoinmune/etiología , Trastornos del Crecimiento/complicaciones , Síndromes de Inmunodeficiencia/complicaciones , Discapacidad Intelectual/complicaciones , Leucemia Prolinfocítica/etiología , Leucemia de Células T/etiología , Microcefalia/complicaciones , Adolescente , Anemia Hemolítica Autoinmune/tratamiento farmacológico , Antibióticos Antineoplásicos/uso terapéutico , Análisis Citogenético , Deficiencia del Factor V/etiología , Genes Recesivos , Glucocorticoides/uso terapéutico , Trastornos del Crecimiento/genética , Humanos , Síndromes de Inmunodeficiencia/genética , Recién Nacido , Discapacidad Intelectual/genética , Leucemia Prolinfocítica/tratamiento farmacológico , Leucemia de Células T/tratamiento farmacológico , Masculino , Metilprednisolona/uso terapéutico , Microcefalia/genética , Pentostatina/uso terapéutico , SíndromeRESUMEN
We report a case of spontaneous remission of lymphoid blast crisis in chronic myelogenous leukaemia (CML) which returned to chronic phase, without the use of cytostatic chemotherapy, following an episode of viral infection and blood transfusion. Although complete remissions of acute leukaemia have been described, this evolution is extremely rare and has never been reported in CML blast crisis. The role of hypothetical factors leading to such a rare event are briefly discussed.
Asunto(s)
Crisis Blástica/terapia , Transfusión Sanguínea , Leucemia Mielógena Crónica BCR-ABL Positiva/terapia , Virosis/complicaciones , Anciano , Femenino , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/complicaciones , Regresión Neoplásica EspontáneaRESUMEN
Danazol has been used with success in some hematological diseases, but there is no report of this treatment in acute leukemia. We report here a case of remission of myelodysplastic syndrome with myelofibrosis in transformation after danazol therapy in a 72-yr-old man. The role of danazol in remission induction is briefly discussed.
Asunto(s)
Danazol/uso terapéutico , Antagonistas de Estrógenos/uso terapéutico , Síndromes Mielodisplásicos/tratamiento farmacológico , Síndromes Mielodisplásicos/patología , Mielofibrosis Primaria/tratamiento farmacológico , Anciano , Humanos , Masculino , Inducción de RemisiónAsunto(s)
Cromosomas Humanos Par 8 , Trastornos Mieloproliferativos/genética , Proteínas Proto-Oncogénicas/genética , Proteínas de Unión al ARN/genética , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN , Humanos , Cariotipificación , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Expression of NG2 has been reported in the majority of paediatric acute leukaemia (AL) cases with MLL rearrangement. We demonstrated 7. 1 positivity in 2/3 paediatric and 4/11 adult MLL rearranged acute myeloid leukaemia (AML) but in 0/28 adult AML without MLL rearrangement, thus extending the 100% specificity to adult cases. Positivity correlated with stage of maturation arrest since it was found in 0/6 immature AML but in 6/8 monoblastic cases. These data demonstrate that, if NG2 expression in AL is the (in)direct result of MLL rearrangement, such activation is restricted to a monoblastic population in AML. They also have practical implications for NG2 diagnostic screening strategies.
Asunto(s)
Antígenos/genética , Proteínas de Unión al ADN/genética , Leucemia Mieloide/genética , Proteoglicanos/genética , Proto-Oncogenes , Factores de Transcripción , Enfermedad Aguda , Adolescente , Adulto , Antígenos/metabolismo , Preescolar , Reordenamiento Génico , N-Metiltransferasa de Histona-Lisina , Humanos , Inmunofenotipificación , Leucemia Mieloide/inmunología , Persona de Mediana Edad , Proteína de la Leucemia Mieloide-Linfoide , Proteoglicanos/metabolismo , Células Tumorales CultivadasRESUMEN
The rapid detection of chromosome band 8q24 rearrangements, including classical translocations involving MYC and variant 3' translocations, is important for the accurate diagnosis and appropriate treatment of lymphoid malignancies. We have identified and characterized a CEPH YAC, 934e1, which extends from at least 190 kbp upstream to over 280 kbp downstream to MYC, allowing detection of classical t(8; 14)(q24;q32) and variant t(8;22)(q24;q11) and t(8;14)(q24;q11), extending distal to PVT1 and therefore, by extrapolation, to BVR1. This YAC also allowed clarification of complex chromosome 8 abnormalities and the identification of translocations in interphase nuclei. A second CEPH YAC, 904c3, previously shown to contain the PVT1 locus but not MYC, allowed distinction between translocations occurring centromeric and telomeric to MYC. Use of the 934e1 YAC will aid classification of a variety of lymphoid proliferations and further characterization of rearranged cases with the 904c3 YAC will simplify mapping of their diverse breakpoints.
Asunto(s)
Genes de Inmunoglobulinas/genética , Genes myc/genética , Regiones Constantes de Inmunoglobulina/genética , Hibridación Fluorescente in Situ/métodos , Linfoma/genética , Translocación Genética/genética , Cromosomas Artificiales de Levadura , Cromosomas Humanos Par 14/genética , Cromosomas Humanos Par 22/genética , Cromosomas Humanos Par 8/genética , Humanos , Cariotipificación , Proto-Oncogenes/genética , Mapeo Restrictivo , Células Tumorales CultivadasRESUMEN
We studied a stratified cohort of 51 childhood B-lineage acute lymphoblastic leukemias (B-ALLs) to evaluate the efficiency of spectral karyotyping (SKY) in the detection of chromosome aberrations previously diagnosed using chromosome banding and/or reverse transcriptase polymerase chain reaction. Despite the small number of cases analyzed, several important features emerge from the study: (a) The result of banding analysis was revised in two-thirds of the cases. Eighty-three chromosome anomalies previously undetected or not characterized using chromosome banding were identified by spectral karyotyping, even in patients with apparently normal karyotypes. (b) All hyperdiploidy cases showed one or more extra copies of chromosomes X, 14, and 21. (c) Two hidden rearrangements, a t(7;12)(?p12;p13), and a new translocation, a t(9;12)(q31;p13), both involving the TEL gene, were characterized. (d) Some cryptic rearrangements, such as the der(21) t(12;21) translocation, remained undetected. (e) No new recurrent chromosome anomalies were discovered with this technique. In conclusion, the present study confirms the efficiency of the SKY technique in resolving and characterizing many complex chromosome anomalies seen in childhood B-ALLs, but it raises questions about the ability of this technique to detect cryptic rearrangements, such as the t(12;21) translocation.
Asunto(s)
Linfoma de Burkitt/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Linfoma de Burkitt/patología , Niño , Aberraciones Cromosómicas/genética , Bandeo Cromosómico , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador , Hibridación Fluorescente in Situ , Cariotipificación , Masculino , Microscopía Fluorescente , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Translocación Genética/genéticaRESUMEN
The rate of detection of chromosome abnormalities in T-cell proliferations is lower than that observed in B-cell malignancies. The former frequently involve the TCR alpha/delta locus at chromosome band 14q11. We have identified a YAC encompassing 70% of the TCR alpha/delta locus, which has been used as a fluorescence in situ hybridization probe to detect chromosome rearrangements involving 14q11, both at metaphase and within interphase nuclei, in patients with a variety of T-lymphoproliferative disorders. Its use allowed detection of previously unsuspected TCR alpha/delta rearrangements in 4/13 (30%) immature T-lineage acute leukemias, including two t(10;14) and 2 minor inversion 14s. It also clarified interpretation of complex chromosome 14 abnormalities in mature T-cell proliferations (T-prolymphocytic leukemia and ataxia telangiectasia). Use of this probe will aid the detection and characterization of abnormalities involving the TCR alpha/delta locus, particularly in cases with normal or complex karyotypes and in those proliferations for which mitoses are difficult to obtain.