Trisomy 8 and monosomy 7 detected in bone marrow using primed in situ labeling, fluorescence in situ hybridization, and conventional cytogenetic analyses. A study of 54 cases with hematological disorders

Trisomy 8 and monosomy 7 are the two most frequent aneuploidies found in hematological disorders such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In this study, primed in situ labeling (PRINS), fluorescence in situ hybridization (FISH) and conventional cytogenetic approaches were used to investigate 54 cases of hematopoietic disorders. Of these cases, there were 22 cases of trisomy 8, 2 cases of tetrasomy 8, 14 cases of monosomy 7, and 16 cases with two copies of both chromosomes 7 and 8. PRINS was carried out in interphase nuclei of bone marrow samples using primers that can specifically detect alpha-satellite DNA sequences of chromosomes 7 and 8. In parallel, using the alpha-satellite probes for chromosomes 7 and 8, FISH was performed for all the cases. PRINS and FISH techniques showed similar specificity and sensitivity. In comparison to FISH, PRINS is more advantageous since it is a faster, easier, and more cost-effective technique. Additionally, for most of the cases, a higher proportion of aneuploidy was detected in metaphases using conventional cytogenetics, as compared to the one found in interphase nuclei identified with PRINS and FISH techniques. In other words, for these cases, the cells with trisomy 8 or monosomy 7, had a distinct proliferative advantage compared to the disomic cell population. Therefore, to better quantify the proportion of aneuploid cells in bone marrow, we recommend to investigate the frequency of aneuploidy in nuclei using PRINS, rather than studying only the dividing cells as detected by conventional cytogenetic techniques.     

Fanconi anemia: myelodysplasia as a predictor of outcome

The adverse potential of the development of myelodysplastic syndrome (MDS) in Fanconi anemia (FA) was examined in a retrospective study of 41 FA patients who had bone marrow morphology and chromosomes reviewed by a single group. Thirty-three patients had adequate cytogenetic studies, and 16 (48%) had one or more abnormal studies: nine initially, and seven more on follow-up. Cytogenetic clonal variation was frequent, including disappearance of clones, clonal evolution, and appearance of new clones. The estimated five-year survival with a cytogenetic clone is 0.40, compared to 0.94 without a clone. Morphologic myelodysplasia (MDS), independent of a cytogenetic clone, was found in 13/41 patients (32%). The estimated five-year survival with MDS is 0.09, versus 0.92 without MDS. Leukemia developed in three patients whose initial cytogenetic clones prior to leukemia were t(1;18), t(5;22) and monosomy 7; the one with t(1;18) also had MDS. Our results focus on marrow morphology, and suggest that morphologic MDS may be more important than classical cytogenetics in prediction of an adverse outcome.     

Monosomy 21 in hematologic diseases

Monosomy 21 mosaicism as a sole cytogenetic abnormality is very uncommon, with 47 cases described in the literature. We identified five cases of low-level monosomy 21 mosaicism since 1998, none of which were confirmed by fluorescence in situ hybridization (FISH) analysis or follow-up cytogenetic studies. These five cases, and many of the previously reported cases, probably represent the random appearance of several monosomy 21 cells as artifacts of cell culture or microscope slide preparation. The most convincing reported cases of monosomy 21 mosaicism suggest a rare association of monosomy 21 with acute myelocytic leukemia and chronic lymphocytic leukemia. Future cases suggestive of monosomy 21 mosaicism should be confirmed by analysis of additional metaphase cells and by FISH analysis of interphase cells.     

Clinical implications of monosomy 7 in acute nonlymphocytic leukemia

A group of 18 patients with acute nonlymphocytic leukemia and the chromosomal aberration monosomy 7 in their bone marrow cells was compared to a group of control patients with the same disease but normal bone marrow chromosomes. The monosomy 7 group of patients had a higher incidence of fever and infections, and a higher white blood cell and granulocyte count compared to the control group at the time of diagnosis. The clinical difference between the groups continued over the first month of hospitalization. Complete remission was obtained in 12% of the monosomy 7 group and in 59% of the control group. Survival was clearly longer in the control group of patients. Monosomy 7 of the bone marrow in acute nonlymphocytic leukemia is therefore to be considered a bad prognostic sign.     

Myelodysplastic syndrome associated with monosomy 7 in childhood: a retrospective study

Myelodysplastic syndrome (MDS) is a clonal hematopoietic stem cell disorder characterized by ineffective hematopoiesis, peripheral cytopenia, and dysplastic changes in the bone marrow. Monosomy 7 or partial loss of 7q is a common cytogenetic abnormality in MDS patients and is associated with poor prognosis. This study examined eight patients with monosomy 7 and MDS. Five MDS patients with monosomy 7 progressed to acute leukemia: three cases transformed into acute myelogenous leukemia (AML) in a mean time of only 4.6 months and two cases into acute lymphoblastic leukemia (ALL) in a mean time of 9 months. To our knowledge, this is the first report showing progression of monosomy 7 associated with MDS to ALL in the childhood period.     

Spurious monosomy 7 in leukemia due to centromeric heteromorphism

We report heteromorphism of the centrometric region of human chromosome 7, which was observed in the cytogenetic assessment of a complete remission of a pre-B acute lymphoblastic leukemia in the bone marrow cells of a 25-year-old woman. Classical cytogenetic study was performed, as well as metaphase and interphase fluorescence in situ hybridization (FISH) carried out with an alphoid DNA probe specific for the chromosome 7 centromere for detection of leukemic clones with monosomy 7 found at the initial diagnosis. We show an important centromeric heteromorphism of this chromosome detected by FISH and clearly visible on all metaphases and nuclei analyzed. This heteromorphism is observable as a fluorescent signal five- or sixfold larger than that on the homologue. To our knowledge, this heteromorphism of chromosome 7 has not been reported in the literature. However, with the use of FISH analysis, it could be easily mistaken for a mosaicism of monosomy 7, which can be misleading in the interpretation of the results.     

Conventional and molecular cytogenetic findings of myelodysplastic syndrome patients

Abstract Myelodysplastic syndrome (MDS) involves myeloid cells of the bone marrow, which is important in progressive bone marrow insufficiency. Of all MDS patients, 40%–50% have at least one chromosomal rearrangement. Loss of specific chromosomal regions like 5q– and 7q– are usually the secondary cytogenetic abnormalities associated with MDS. In order to detect chromosome abnormalities associated with MDS, bone marrow samples from 26 patients diagnosed as MDS were obtained prior to chemotherapy. Both conventional cytogenetic analyses and fluorescence in situ hybridisation (FISH) methods were performed and locus–specific probes for 5q and 7q were used. Results obtained were compared. Twenty–one patients had normal karyotypes and four patients had abnormal karyotypes, while in one patient we could not obtain metaphases from cultures. Three patients with normal karyotypes revealed del (5q), two patients had del (7q) and one patient had monosomy (7). A total of 10 of 26 patients had chromosome changes visualised by either conventional or molecular cytogenetics (~38.5%). Our results show that both methods are important in diagnosis and follow up of MDS patients. When used together, conventional cytogenetics and FISH detect clinically significant chromosome abnormalities in MDS patients.     

Necessity of bilateral bone marrow biopsies for ancillary cytogenetic studies in the pediatric population

The need for bilateral pediatric bone marrow biopsies for cytogenetic and fluorescent in situ hybridization (FISH) studies has not been clearly delineated. We retrospectively identified 166 pediatric bilateral bone marrow biopsy specimens obtained from patients with a variety of clinical diagnoses, including solid tumors, lymphoma, leukemia, and other hematologic conditions. The cases included all pediatric bilateral bone marrow biopsies performed at our hospital spanning the years of 1992 to 2008. Agreement of FISH and classical cytogenetic results between the 2 sides was assessed. Of a total of 166 bilateral cases, 2 cases showed disagreement (1.2%), both from patients with solid tumors. One case was a rhabdomyosarcoma, in which FISH only was performed; the second was a neuroblastoma in which FISH and cytogenetics were performed (both FISH and classical cytogenetic results disagreed). The remainder of the cases showed complete agreement between the 2 sides (total 98.8%). We conclude that it is usually not necessary to perform bilateral bone marrow biopsies for FISH and cytogenetics in the pediatric population outside of the setting of solid tumor staging.     

Is monosomy 5 an uncommon aberration? Fluorescence in situ hybridization reveals translocations and deletions in myelodysplastic syndromes or acute myelocytic leukemia

Acquired loss of material from chromosome 5 in bone marrow cells is common in myelodysplastic syndromes (MDS) and acute myelocytic leukemia (AML). In this study, we have applied fluorescence in situ hybridization (FISH) analyses with probes for the three regions 5p15.2, 5q31, 5q33-q34, and whole chromosome 5 painting probes (WCP 5) to investigate what further information could be gained regarding the cytogenetic abnormalities of chromosome 5 in 35 patients with MDS or AML. With FISH, a del(5q) was found in all patients except for two. Translocations of material from chromosome 5 were found in 10 patients. Among 16 patients with clones of monosomy 5 seen by cytogenetics, 14 had deletions or translocations. Different breakpoints on chromosome 5 were observed. In conclusion, the extended FISH analyses yielded additional information about chromosome 5 abnormalities in 60% of the patients. Of interest is the finding of a high proportion of translocations and that monosomy 5 occurs less often than is generally believed.     

BCR-ABL探针在慢性粒细胞白血病中的应用

目的用常规细胞遗传学（conventional cytogenetics,CC）和荧光原位杂交（fluorescence chromosomal in situ hybridization,FISH）技术检测Ph染色体。方法常规细胞遗传学分析（CC）,荧光原位杂交（FISH）技术。结果7例患者4例Ph染色体阴性,其中2例分别伴有t（18;22）和t（17;22）异常,其余2例为异基因造血干细胞移植后正常核型。一例培养后无中期分裂相。2例Ph染色体阳性,FISH结果bcr/abl（＋）细胞检出率分别为63.87%,84.51%,7.56%,4.0%,74.45%,67%,47%。结论常规细胞遗传学与荧光原位杂交技术相结合对CML患者诊断治疗过程中肿瘤负荷动态检测有显著意义。     

Aplastic anemia and monosomy 7-associated dysmegakaryocytopoiesis

Aplastic anemia (AA) is marrow failure due to an inadequate number of hematopoietic cells in the marrow. Prior reports have described a more aggressive clinical course in aplastic anemia with monosomy 7. We report 3 pediatric cases of AA with normal cytogenetics followed by acquisition of monosomy 7. Bone marrow biopsies were initially diagnostic of AA but later showed monosomy 7 and an increased number of megakaryocytes with small hypolobated nuclei. Immunohistochemical stains for CD61 highlighted the marked dysmegakaryocytopoiesis. The marrow blast percentage was increased in only 1 patient with 4.6% blasts. The 3 patients underwent bone marrow transplantation, and each has remained disease free for 7 to 18 months after transplantation. Pediatric patients with AA and normal cytogenetics may develop monosomy 7 with a myelodysplastic syndrome, unclassified. Patients with AA and monosomy 7 should be evaluated for dysmegakaryocytopoiesis.     

Interphase fluorescence in situ hybridization overcomes pitfalls of G-banding analysis with special reference to underestimation of chromosomal aberration rates

Fluorescence in situ hybridization (FISH) is suitable for detecting different types of chromosome aberrations on interphase nuclei even in specimens with no or few chromosome metaphases. However, it is not known why FISH is superior to conventional G-banding analysis. The sensitivity of interphase FISH was compared to that of G-banding analysis in 288 leukemia/lymphoma patients for 10 different types of chromosome aberrations: t(9;22) (M- and m-BCR), t(8;21), 11q23 abnormalities, t(15;17), del(5)/-5, del(13)/-13, +8, -7, and +12. The results revealed that t(15;17) positive cells could not proliferate well in culture, leading to underestimation of abnormality by G-banding. Monosomy 7 in acute myelocytic leukemia (AML) and myelodysplastic syndrome (MDS) as well as trisomy 12 and deletion chromosome 13 in chronic lymphocytic leukemias (CLL) were also severely underestimated by G-banding. On the other hand, no discrepancies were observed in t(8;21), t(9;22), translations involving 11q23, or in trisomy 8. These findings indicate the superiority of interphase FISH over conventional cytogenetics for detecting chromosome abnormalities in small clones, especially for monosomy 7 or (15;17) translocations.     

Detection of monosomy 7 by fluorescenceIn situ hybridization in acute nonlymphocytic leukemia and myelodysplastic syndrome

Summary Fluorescencein situ hybridization (FISH) with a chromosome 7 specific alpha satellite DNA probe was used to detect monosomy 7 in interphase and metaphase cells obtained from patients with myelodysplastic syndrome (MDS) and acute nonlymphocytic leukemia (ANLL). Chromosome analysis revealed monosomy 7, either alone or as part of a complex chromosome abnormality, in all cell samples. FISH analyses of 12 marrow samples and a blood sample using a chromosome 7 specific alpha satellite DNA probe revealed a single fluorescence spot in 80.5–97.5% of interphase cells indicating monosomy 7. In contrast, 83.5–92.0% of the same cells had two copies of chromosome 17 as two fluorescent spots were detected using a chromosome 17 specific alpha satellite DNA probe used as a positive control. The proportion of interphase cells with monosomy 7 did not correlated with the percentage of metaphase cells with monosomy 7 detected by conventional karyotyping or with the percentage of blast cells in the bone marrow.     

RAS, FLT3, and TP53 mutations in therapy-related myeloid malignancies with abnormalities of chromosomes 5 and 7

Oncogenic mutations in the KRAS2, NRAS, or FLT3 gene are detected in more than 50% of patients with de novo acute myeloid leukemia (AML). RAS mutations are also prevalent in de novo myelodysplastic syndrome (MDS), especially chronic myelomonocytic leukemia and juvenile myelomonocytic leukemia. However, few studies have examined these genetic lesions in therapy-related myeloid malignancies. Monosomy 7/del(7q) and monosomy 5/del(5q) represent the most common cytogenetic abnormalities in therapy-related MDS and AML (t-MDS/t-AML) and are strongly associated with prior exposure to alkylating agents. Mutational analysis of bone marrow specimens from a well-characterized cohort of 26 t-MDS/t-AML patients with abnormalities of chromosomes 5 and/or 7 revealed 3 with RAS mutations. Further analyses of 23 of these cases uncovered one FLT3 internal tandem duplication and five TP53 mutations. The four patients with RAS or FLT3 mutations had monosomy 7, including one with abnormalities of chromosomes 5 and 7. One specimen demonstrated mutations in both KRAS2 and TP53. RAS and FLT3 mutations, which are thought to stimulate the proliferation of leukemia cells, appear to be less common in t-MDS/t-AML than in de novo AML, whereas TP53 mutations are more frequent.     

Numerical chromosomal changes and risk of development of myelodysplastic syndrome--acute myeloid leukemia in patients with Fanconi anemia

Fanconi Anemia (FA) is an inherited bone marrow failure syndrome characterized by congenital abnormalities, progressive marrow failure and predisposition to myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), and solid tumors. The most common acquired chromosomal aberrations in FA patients are trisomy of 1q and monosomy of chromosome 7; the latter is known to be associated with poor prognosis. A few reports also suggest that gains of 3q are associated with progression to MDS–AML and overall poor prognosis. It is not uncommon for patients with Fanconi anemia to have easily detectable (oligoclonal) chromosomal alterations in their still normal (nonmalignant) marrow, which makes it even more challenging to determine the import of such alterations. We conducted a retrospective longitudinal analysis of fluorescent in situ hybridization (FISH) analysis for gains in 1q and 3q and for monosomy 7 and 7q deletions on 212 bone marrow samples from 77 children with FA treated at our institution between 1987 and 2007. Given the baseline increased chromosomal instability and defective DNA repair in patients with FA, which leads to unbalanced chromosomal aberrations such as deletions, insertions, and translocations, for the purpose of this analysis an abnormal clone was defined as ≥10% abnormal cells. Chromosome 3 and 7 aberrations were associated with increased risk of developing MDS–AML (P = 0.019 and P < 0.001 respectively), although the significance of chromosome 3 aberrations disappeared when different observation times were accounted for. Gain of 1q alone did not predict development of MDS–AML. In conclusion, children with FA should be followed closely with FISH analyses, because some of the clonal chromosomal abnormalities may be early indicators of progression toward MDS–AML and thus also of the need for hematopoietic stem cell transplantation.     

Use of fluorescence in situ hybridization for retrospective detection of aneuploidy in multiple myeloma

In malignancies with a low mitotic index such as multiple myeloma (MM), conventional cytogenetic studies may not be informative. This study's purpose was to assess specific numerical chromosomal aberrations in non-dividing MM cells by fluorescence in situ hybridization (FISH) of DNA chromosome probes on bone marrow smears. Old air-dried bone marrow smears from 18 MM patients were probed with alpha satellite DNA sequences for chromosomes 7, X, and Y, and a whole painting probe for chromosome 11. Plasma cells were identified by their morphologic characteristics so that counts of fluorescent signals in the nuclei of MM cells could be differentiated from those of normal marrow cells. Numerical chromosome aberrations were found in 66.7% of the cases (12 of 18), including 5 cases of trisomy 7, 2 cases of tetraploidy, 2 cases of monosomy X in females, 2 cases of disomy X in males, and 1 case of nullisomy Y. In addition, 2 of the 7 cases probed with chromosome 11 paint demonstrated 3 signals in about 15% of the cells. This study illustrates the advantages of FISH for interphase analysis of chromosome aberrations in slowly dividing cells, as well as the ability to use old slides for retrospective studies. © 1993 Wiley-Liss, Inc.     

Significance of cytogenetic and fluorescence in situ hybridization analysis in evaluating antichronic myeloid leukemia efficacy of different immune effector cells

We report on the antileukemia effect of interleukin 2 (IL2) on different immune cells from 22 patients with chronic myeloid leukemia (CML). Bone marrow cells from these patients were first cultured in modified long-term bone marrow culture medium for several days, then separately cultured with lymphokine activated killer cells (LAK), cytokine-induced killer cells (CIK), and dendritic cell cocultured CIK (DC-CIK) for another 1-2 days. They were then detected for presence of the Philadelphia chromosome (Ph) by cytogenetic analysis and fluorescence in situ hybridization (FISH). The percentage of Ph-chromosome-positive cells in the bone marrow mononuclear cells after culturing with CIK and DC-CIK was significantly lower than that after culturing with IL2 or LAK. Our results demonstrate that cytogenetics and FISH are useful techniques for the evaluation of the anti-CML effect of immune cells and that CIK or DC-CIK can be appropriate candidates for adoptive immune cell therapy in vivo or for leukemia cell purging ex vivo.     

Secondary acute non-lymphocytic leukemia with monosomy 7 arising 9 years after acute lymphoblastic leukemia in childhood

We report a case of pediatric acute non-lymphocytic leukemia (ANLL) with monosomy 7 occurring in a child successfully treated for acute lymphoblastic leukemia (ALL) nine years earlier. Acquired monosomy 7 is currently recognized as a distinct therapy-related cytogenetic abnormality which nonrandomly occurs as a late complication of cytotoxic therapy used in the treatment of both malignant and nonmalignant disease. Most commonly, this occurs as a disorder of bone marrow morphology and function characterized as a myelodysplastic syndrome (MDS) or ANLL. This case report emphasizes the need for continued evaluation of long-term survivors of childhood cancer to identify and minimize therapy-related side effects without compromising successful management.     

High frequency of tissue-specific mosaicism in Turner syndrome patients

Interphase fluorescent studies of X chromosome aneuploidy in cultured and uncultured blood lymphocytes and oral mucosa epithelial cells using X centromere-specific DNA probe in addition to standard karyotype analysis were performed in 50 females with a clinical suspicion of Turner syndrome. All the patients were previously screened for the presence of 'hidden' Y chromosome mosaicism, using the primers DYZ3 and DYZ. The use of fluorescence in situ hybridization (FISH) analysis of interphase nuclei of tissues from different germ layers (lymphocytes from mesoderm and buccal epithelial cells from ectoderm) improves the accuracy of detection of low-level mosaicism. FISH studies on interphase nuclei revealed that 29% of patients with a pure form of monosomy X detected by metaphase analysis are, in fact, mosaics. The level of cells with the normal chromosomal constitution in lymphocytes of these cases as a rule was low, ranging from 3 to 18%, with an average of 7%. Two false-positive cases and one false-negative case of X monosomy mosaicism determined by standard cytogenetic approach were detected using FISH analysis. The majority of patients (92%) with mosaic form of Turner syndrome have considerable tissue-specific differences in levels of X aneuploidy. Our data indicate that in cases when mosaic aneuploidy with low-level frequency is questionable (approximately 10% and lower), the results of standard metaphase analysis should be supplemented with additional FISH studies of interphase nuclei. Tissue-specific differences in contents of different cell lines in the same patients point to the necessity of studying more than one tissue from each patient.     

Fluorescence in situ hybridization for the study of cell lineage involvement in myelodysplastic syndromes with chromosome 5 anomalies

Fluorescence in situ hybridization (FISH) with a locus-specific dual DNA probe (LSI EGR-1SO/D5S23SG) for chromosome 5 was used in combination with morphology to study bone marrow cell lineage involvement of the abnormal chromosomal clone in 13 patients with deletion 5q [del(5q)], either as a sole aberration or as part of a complex karyotype, and in six cases with monosomy 5 by metaphase cytogenetics, all with complex karyotypes including 2-6 marker chromosomes. In the monosomy 5 group, only one case displayed the expected one orange and one green (1O + 1G) FISH pattern in a majority of the cells. The other five patients instead showed 1O + 2G FISH signals in 17-86% of the bone marrow cells, which is the typical pattern for del(5q). In the del(5q) group, 26-98% of the bone marrow cells exhibited 1O + 2G FISH signals. All patients showed clonal involvement of the myeloid cell lineages, including the megakaryocytes in a few cases, whereas lymphoid cells generally exhibited the normal 2O + 2G FISH pattern. No difference was seen between patients with 5q- syndrome, those with del(5q) and a complex karyotype, and the monosomy 5 group. We were thus unable to confirm the recent suggestion that B-cells are a part of the abnormal clone in MDS with del(5q). Furthermore, true monosomy 5 seems to be rare in MDS.