A special fluorescent in situ hybridization technique to study peripheral blood and assess the effectiveness of interferon therapy in chronic myeloid leukemia

Using a highly sensitive fluorescence in situ hybridization method with probes for BCR and ABL1 (D-FISH), we studied 37 paired sets of bone marrow and blood specimens, collected within 24 to 96 hours of each other, from 10 patients before and during treatment for chronic myeloid leukemia (CML). The normal range for 500 interphase nuclei was 相似文献   

Genetic heterogeneity of primary and metastatic breast carcinoma defined by fluorescence in situ hybridization

Breast carcinoma is frequently associated with nonrandom chromosomal aberrations, but their identification by standard cytogenetics (SC) is often limited by technical difficulties. Fluorescence in situ hybridization (FISH) studies of interphase nuclei can circumvent some of these difficulties and has the potential to identify nonrandom molecular cytogenetic events occurring in breast cancer. FISH was performed on tumor nuclei isolated from 15 formalin-fixed, paraffin-embedded archival breast carcinomas using a panel of chromosome-specific alpha-satellite probes for enumerating chromosomes in interphase nuclei. Freshly isolated cells from these same cases had previously been studied by standard cytogenetics and FISH. In addition to archival primary carcinoma, archival metastases and normal tissue were also studied by FISH. Genetic numerical alterations were identified by standard cytogenetics or FISH in 14 of 15 carcinomas. Numeric alterations initially identified by standard cytogenetics were confirmed by FISH in 9 of 10 cases. Results of FISH performed on nuclei isolated from paraffin-embedded material were in agreement with FISH performed on freshly isolated cells. Clonal numeric alterations were observed in the archival primary tumor as well as in metastases. Archival normal tissue was consistently disomic.     

Inadequate measurement of urinary chloride

Fifteen years ago Talpaz and colleagues were the first to determine that natural interferon alpha (INF-alpha) induces hematologic remission in chronic phase patients with chronic myeloid leukemia (CML). Further research revealed that this agent, contrarily to conventional chemotherapy with busulfan or hydroxyurea, eliminates leukemic hematopoietic cells having Philadelphia chromosome (Ph1-positive) in about 20% of patients, leading to the phase of cytogenetic remission. Comparison of the efficiency of IFN-alpha with conventional chemotherapy was carried out in several randomized clinical trials. It was found, that IFN-alpha delays the occurrence of blastic phase of CML and prolongs patients life span. It does not create, however, a likely chance of full recovery. The results of the randomized trails, carried out in France, showed that the combination of IFN-alpha and cytarabine as compared with INF-alpha alone, increases the rate of major cytogenetic response and prolongs survival in the chronic phase of CML. IFN-alpha efficiency in acceleration and blastic transformation phases of CML has not been proven so far, although this drug may be of certain value in combination with hydroxyurea or other cytostatic agents. At present, it is more often considered that IFN-alpha should be a first line therapy in newly diagnosed CML in its chronic phase, if due to absence of appropriate donors or advanced age, allogenic bone marrow transplantation cannot be performed.     

Altered intracellular processing and enhanced secretion of procathepsin D in a highly deviated rat hepatoma

Using immunomagnetic cell separation and fluorescent in situ hybridization (FISH), we studied nine patients who had chronic granulocytic leukemia (CGL) for the proportion of interphase nuclei with Mbcr/abl fusion in a direct preparation of the bone marrow and also in the mononuclear cell (MNC), neutrophil, and B- and T-cell fractions of the peripheral blood. In five untreated patients, conventional cytogenetics revealed 97% to 100% Philadelphia chromosome (Ph)+ metaphases. In three of these five patients, FISH studies on bone marrow direct preparations and peripheral blood MNCs indicated that an Mbcr/abl fusion occurred in 62% to 69% of the cells. We observed 69% to 88% of nuclei with Mbcr/abl fusion within the neutrophil fractions. In contrast, the values were 12% to 39% within the T-cell fractions in the four patients we studied. B-cell fractions were studied in three patients, and only one had an abnormal value (58%). In the four patients receiving alpha-interferon therapy, the degree of conventional cytogenetic remission correlated best with the degree of FISH remission observed in the peripheral blood neutrophil fraction. Our results are in agreement with earlier studies in that both B and T lymphocytes may be involved with the clonal process in CGL. The FISH-based detection of Mbcr/abl fusion in the peripheral blood neutrophil compartment provided the best estimate for the proportion of Ph metaphases determined by conventional cytogenetics.     

Conventional cytogenetics and FISH evaluation of chimerism after sex-mismatched bone marrow transplantation (BMT) and donor leukocyte infusion (DLI)

BACKGROUND AND OBJECTIVES: Sensitive and quantitative cytogenetic methods to better assess the biological significance of post-BMT chimerism have been recently developed. In this study, we compared the results of chimerism analysis and evolution employing conventional cytogenetics and fluorescence in situ hybridization (FISH) in 16 patients after sex-mismatched BMT, and in 5 patients after donor lymphocyte infusion (DLI) to treat post-BMT relapse. DESIGN AND METHODS: FISH studies were performed using separate digoxigenin labeled centromeric DNA probes for the X (pDMX1) and Y (DYZ1/DYZ3) chromosomes. To this purpose, different types of samples were used: bone marrow (BM) and peripheral blood (PB) slides processed for conventional cytogenetics, and routine BM and PB smears. RESULTS: Results of chimerism studies performed on different types of samples showed no significant differences. No significant differences in the ability to identify the sex of each cell with both pDMX1 and DYZ1/DYZ3 probes were found and the results obtained from independent experiments showed a high linear correlation. Chimerism analysis by FISH showed initial mixed chimerism after BMT in 10 patients. Seven of these patients were also studied by conventional cytogenetics and 2 of these showed mixed chimerism. Seven of the former 10 patients evolved to complete donor chimera. 6 patients showed cytogenetic or hematologic bone marrow relapse, 3 of which were preceded by mixed chimaerism as revealed by FISH studies. FISH studies permitted an easy and accurate monitorization of the response to DLI in 5 relapsed patients, showing an increase in the proportion of donor cells in 4 patients as they reached a new complete remission. INTERPRETATION AND CONCLUSIONS: Both FISH and conventional cytogenetics are quantitative methods to assess chimerism. However, FISH is more sensitive, accurate and can even be applied on routine BM and PB smears. Furthermore, its combination with immunophenotyping approaches to quantify chimerism on cell subpopulations, will help to clarify post-BMT chimerism significance.     

Hyponatremia of cirrhosis: role of vasopressin and decreased ''effective'' plasma volume

Interphase FISH analysis, utilizing dual color XY probes, was performed on 27 patients following allogeneic sex-mismatched bone marrow transplantation and on 31 controls. Of the 123 167 examined interphase nuclei, 63 318 were from 19 of the 21 patients (54 specimens) who engrafted, 31 827 from five of the six patients (29 specimens) who relapsed (four) or failed to engraft (one) and 24 703 from the 31 control specimens. In patients who engrafted, the mean percentage of host cells was 0.26% between day 29 and 5 years following BMT. Microchimerism of 0.7% or less than 1-5 years following BMT was not predictive of relapse. Interphase FISH analysis predicted relapse or failure of engraftment in five of the six evaluable patients. In three of five patients both conventional cytogenetics and interphase FISH of bone marrow cells provided important information regarding engraftment status and degree of chimerism.     

Philadelphia chromosome-positive myeloid cells in the peripheral blood of chronic myelogenous leukemia patients: comparison with the frequency detected in cycling cells of the bone marrow

Monitoring the frequency of the Philadelphia (Ph) chromosome in chronic myelogenous leukemia (CML) is important in determining the effectiveness of treatment for patients during therapy. This can be done with high resolution by subjecting short-term bone marrow cultures (48 h) to 24 h of mitotic arrest before harvest and detecting Ph-positive (Ph+) metaphases by fluorescence in situ hybridization (FISH) in a procedure termed hypermetaphase FISH or HMF. Here, we compared procedures for detecting Ph+ interphase cells (interphase FISH or I-FISH) in peripheral blood polymorphonucleocytes (PMNs) with HMF results on the bone marrow of the same 26 CML patients in different stages of remission. The probes for I-FISH in these experiments were relatively large (200-300 kb) and sufficiently resolved in PMNs so that 97.5% of the cells were scorable. The correlation between the frequencies of Ph+ cells from the two different cell sources was excellent (r = 0.983, P < 0.0001); however, there was a consistently higher level of Ph+ cells observed in the cycling marrow cells than in the peripheral blood PMNs. This was discussed in terms of current theories of apoptosis in CML cells. The large number of PMNs analyzable by I-FISH (approximately 500/patient in this study) provided sufficiently narrow 99% confidence intervals to suggest the procedure as an effective and efficient method for monitoring the frequency of Ph+ cells in CML patients undergoing therapy. However, for detection and quantification of minimal residual disease, HMF is preferable to I-FISH because of the much lower frequency of false-positive readings with the former procedure.     

Phase II trial of recombinant interferon-alpha-2a and eflornithine in patients with recurrent glioma

The aim of this study was to investigate the influence of interferon-alpha (IFN-alpha) on bone marrow fibrosis associated with chronic myelogenous leukemia (CML). Eighty-two bone marrow biopsies from 41 patients in chronic phase CML were stained with Snook's reticulin stain for argyrophilic fibers. Grading of reticulin fibrosis (scale of 1 to 4) was according to the quantity and pattern of distribution of reticulin. The interval between biopsies was a median of 25 months (range 12 to 40 months). Patients had been treated with IFN-alpha for at least 12 months, were still on IFN-alpha therapy during the study, and had achieved at least a complete hematological response. Before the start of IFN-alpha therapy, reticulin fibrosis was grade 1-2 in 23 patients (56%) and grade 3-4 in 18 (44%). During IFN-alpha therapy, reticulin fibrosis in bone marrow did not worsen or was reduced in 33 patients (80%) and increased by one grade in eight patients (20%). Only five patients (12%) with limited fibrosis (grade 1-2) before start of IFN-alpha therapy showed an increase towards significant fibrosis (grade 3-4), while eight of the 18 patients (44%) with grade 3-4 fibrosis achieved a decrease of fiber content in bone marrow. In summary, IFN-alpha was not associated with an enhancing effect on myelofibrosis in patients with CML and may have prevented increasing fibrosis in patients who respond to therapy.     

A comparison of the antiproliferative properties of recombinant human IFN-alpha 2 and IFN-omega in human bone marrow culture

We compared the antiproliferative effects in vitro of recombinant preparations of interferon-alpha 2c (IFN-alpha 2) and IFN-omega on the formation of colonies from bone marrow progenitor cells. Both IFNs led to statistically indistinguishable dose-dependent inhibitory effects when tested on bone marrow cells derived from 8 normal donors or from 7 patients with chronic myelogenous leukemia (CML). With both IFNs, the cells from CML patients appeared slightly but not significantly more sensitive to inhibition than the cells from normal donors. These results suggest that under some circumstances, IFN-omega may prove an effective treatment for CML, for example, in those becoming resistant to IFN-alpha 2 because of the formation of neutralizing antibodies.     

Detection of monosomy 7 and trisomy 8 in myeloid neoplasia: a comparison of banding and fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) is a powerful tool for detection of numerical and structural chromosomal aberrations. We have compared conventional banding techniques and FISH for the detection of monosomy 7 (-7) and trisomy 8 (+8) in 89 patients with myeloid malignancies. Of these patients, 21 had -7, 30 had +8, four had both, and 34 had no aberrations or aberrations other than -7 or +8 as assessed by banding techniques. Sequential samples were available in 23 patients. Alphoid DNA probes specific for chromosomes no. 7 and 8 were used for FISH. As controls, 10 normal bone marrow (BM) samples were hybridized with the chromosomes no. 7 and 8 probes, and in addition all tumor samples were hybridized with a chromosome no. 1 specific probe. The cut-off value for -7 was 18% one-spot cells, and for +8 was 3% three-spot cells. FISH analysis of 44 samples with -7 or +8, and at least 10 metaphases evaluated, showed that the proportions of aberrant metaphase cells mirrored the interphase clone sizes. Most samples with nonclonal metaphase aberrations, including those with only a few metaphases, had increased numbers of aberrant interphase cells: 20% to 80% for -7, and 3% to 43% for +8. Interphase cytogenetics of the 34 samples without -7 or +8 did not show significant cell populations with -7 or +8. In four patients, -7 or +8 could not be confirmed by FISH due to additional structural aberrations, marker chromosomes, or wrongly interpreted banding results. As FISH will be used more and more in cytogenetic diagnosis, clinical follow-up, and therapy monitoring, it will be necessary to standardize FISH procedures and supplement the Standing Committee on Human Cytogenetic Nomenclature (ISCN) definitions of a clone with criteria specifically for in situ hybridization.     

Involvement of Fas-mediated apoptosis in the inhibitory effects of interferon-alpha in chronic myelogenous leukemia

Interferon-alpha (IFN-alpha) is an established treatment for chronic myelogenous leukemia (CML) in chronic phase, but the mechanism of its antileukemic activity is not clear. One possible mechanism of action might include the induction of apoptosis, and especially Fas-mediated cell killing may play an important role in the elimination of malignant cells. We investigated Fas receptor (Fas-R) expression and the consequences of Fas-R triggering in CML patients. Using two-color flow cytometry, we found a significantly higher number of Fas-R-expressing CD34+ cells in the bone marrow (BM) of CML patients compared with normal subjects. We have previously shown that IFN-gamma induces Fas-R expression on CD34+ cells; in this study, we investigated whether IFN-alpha induces Fas-R expression on CML progenitor cells. Dose-dependent induction of Fas-R expression was observed after IFN-alpha stimulation of CD34+ cells from CML BM. In methylcellulose culture, IFN-alpha alone at a therapeutic concentration showed only marginal antiproliferative effects on both normal and CML BM progenitors. In contrast, a Fas-R agonist, the anti-CD95 monoclonal antibody CH11, inhibited colony formation from normal progenitors, and the inhibition was even stronger on CML progenitors. When CML BM cells were cultured in the presence of IFN-alpha, Fas-R-mediated inhibition of colony growth was potentiated in a dose-dependent fashion, consistent with IFN-alpha induction of Fas-R expression. This functional effect did not require the presence of accessory cells, since similar results were obtained with purified CD34+ cells. In suspension cultures, we demonstrated that suppression of CML hematopoiesis by IFN-alpha and Fas-R agonist was exerted through Fas-R-mediated induction of apoptosis. Our findings suggest that the Fas-R/Fas-ligand system might be involved in the immunologic regulation of CML progenitor growth and that its effect can be amplified by IFN-alpha.     

Efficacy of fluorescence in situ hybridization for detecting PML/RARA gene fusion in treated and untreated acute promyelocytic leukemia

OBJECTIVE: To test the efficacy of fluorescence in situ hybridization (FISH) for detection of fusion of the promyelocytic leukemia (PML) and retinoic acid receptor alpha (RARA) genes in patients with treated or untreated acute promyelocytic leukemia (APL). DESIGN: We conducted a retrospective blind study on a series of stored bone marrow specimens from normal subjects and patients with APL. MATERIAL AND METHODS: Conventional cytogenetic and FISH analyses were done on interphase and metaphase cells in specimens from 31 normal subjects and 19 patients with untreated or treated APL. RESULTS: From 25 of the normal specimens, we calculated a normal cutoff of 10% for interphase cells and 0% for metaphase cells. With use of these criteria, the other six specimens from normal subjects showed normal findings, and each of the seven specimens from patients with untreated APL was abnormal by FISH analysis. The specimens from four patients in clinical relapse or with residual APL were abnormal. Of the eight specimens from patients in clinical remission, three were abnormal; two of these patients had a relapse within 8 months, and the other patient had received 1 month of chemotherapy and was entering remission. Of the other five patients in remission, four had normal FISH results and have now been in remission for 2.5 to 10 years. The other patient in remission with normal FISH results had a relapse within 6 months. PML/RARA fusion was detectable in three patients with hypogranular APL and in three with a cytogenetic variant of the t(15;17). CONCLUSION: The results of this study suggest that FISH with PML and RARA probes can be used to diagnose APL and may be useful for monitoring treated patients.     

Evidence from molecular genetic and cytogenetic analyses that bone marrow histopathology is reliable in the diagnosis of chronic myeloproliferative disorders

The reliability of histopathological diagnosis in bone marrow specimens from patients with chronic myeloproliferative disorders (CMPD) was evaluated by correlating the histological findings with molecular genetic and cytogenetic analyses of the Ph1-translocation. A rearrangement of m-bcr was detected only in patients (28/30) diagnosed histologically as chronic myeloid leukemia (CML). This finding was supported by the presence of a Ph1-chromosome in 24/26 patients with CML examined. All the patients with other types of CMPD, including polycythemia vera (PV), primary thrombocythemia (PTH) and chronic megakaryocytic-granulocytic myelosis (CMGM), as well as those with unclassifiable CMPD (CMPD.UC) were Ph1-negative (n = 38). The histopathological discrimination of CML from Ph1-negative varieties of CMPD was also reliable for patients with myelofibrosis complicating CML, CMGM and CMPD.UC. The results demonstrate that bone marrow histopathology allows a reliable diagnosis of CML. This is in contrast with hematological data such as high platelet counts which show considerable overlapping in the various forms of CMPD.     

Validation of primed in situ labeling (PRINS) for interphase analysis: comparative studies with conventional fluorescence in situ hybridization and chromosome analyses

Primed in situ labeling (PRINS) is a rapidly developing new technology with wide ranging clinical applications. To assess the sensitivity, specificity, and accuracy of PRINS, we carried out a retrospective study on cultured bone marrow cells to detect aneuploidy for chromosomes 7, 8, and 12. The results were then compared to the results of previous fluorescence in situ hybridization (FISH) and chromosome analyses (CA). In patients who showed aneuploidy with CA, both FISH and PRINS confirmed the aneuploidy in interphase cells. FISH and PRINS also showed excellent correlation with conventional cytogenetic analysis for the detection of mosaic aneuploidies. However, both FISH and PRINS showed significantly higher sensitivity in the detection of abnormal clones compared to CA. In 9 of the 17 cases, there were no significant differences in the detection rates between the two methods. Based on our studies, we conclude that PRINS is as sensitive as FISH in most cases for aneuploidy detection; and that PRINS, like FISH, is more sensitive than conventional CA for aneuploidy detection.     

Suppression of cytogenetic clonal evolution with interferon alfa therapy in patients with Philadelphia chromosome-positive chronic myelogenous leukemia

PURPOSE: To investigate whether cytogenetic clonal evolution can be suppressed with interferon alfa (IFN-alpha) therapy in patients with chronic myelogenous leukemia (CML). PATIENTS AND METHODS: Ninety patients with CML and cytogenetic clonal evolution who received IFN-alpha-based regimens were analyzed. Clonal evolution was defined as the presence of karyotypic abnormalities in addition to the Philadelphia (Ph) chromosome. Patients were evaluated for the suppression of cytogenetic clonal evolution after therapy, the cytogenetic response, and survival. RESULTS: The median age of the population was 39 years (range, 15 to 70 years), median time from diagnosis to clonal evolution 14 months (range, 0 to 145 months), and median percentage of abnormal metaphases 18% (range, 4% to 100%). Fifty six patients (62%) achieved some suppression of cytogenetic clonal evolution; in 41 patients (46%), the suppression was complete. The overall median survival was 51 months, with 43% alive at 5 years. Patients who achieved a complete suppression of cytogenetic clonal evolution had a median survival of 66 months, with 51% alive at 5 years. Characteristics associated with a better response include a lower percentage of abnormal metaphases, time to cytogenetic clonal evolution of 24 months or less, and absence of other features of accelerated disease. A prognostic classification for cytogenetic clonal evolution defined three groups with complete response (CR) rates of 85%, 34%, and 0% (P < .0001) and median survival times of 58, 31, and 30 months, respectively (P=.02). CONCLUSION: Patients with cytogenetic clonal evolution can respond to IFN-alpha therapy, and this response is associated with longer survival. A previously described prognostic model separates patients into subsets with different probabilities of response to IFN-alpha and survival.     

Tilt table testing in the evaluation and management of athletes with recurrent exercise-induced syncope

The development and application of a highly sensitive double-target fluorescence in situ hybridization (FISH) method in combination with immunohistochemistry for detection of chromosome 1 abnormalities in interphase nuclei of neuroblastoma samples is reported. An alpha-satellite probe specific for chromosome 1 and a VNTR probe that hybridizes to chromosome band 1p36.3 were hybridized to GD2 prestained neuroblastoma cells in double-target FISH experiments. The ratio of intact to deleted chromosome 1 homologs in the neuroblastoma cells was assessed. To demonstrate the reliability of the method described, four selected samples derived from different neuroblastoma stages are presented. FISH results correlated well with data obtained by conventional cytogenetic procedures. The technique described allows sensitive detection of chromosome 1 abnormalities in interphase nuclei and enables partial cytogenetic analysis of nondividing cells with a defined immunological phenotype.     

The hypereosinophilic variant of Ph''-positive chronic myeloleukemia

AIM: To confirm clonal nature of idiopathic hypereosinophilic syndrome (IHES), its relevance to Ph'-positive chronic myeloid leukemia. MATERIALS AND METHODS: 3 cases of idiopathic hypereosinophilic syndrome are reported with morphologic analysis of bone marrow cells and cytogenetic examinations. In one patient the presence of Ph'-chromosome was confirmed at fluorescent in situ hybridization (FISH) and molecular-genetic analysis (bcr/abl). Samples of bone marrow, spleen and liver were examined pathohistologically. RESULTS: The presence of chromosome anomaly t(9;22), i.e. Ph'-chromosome, associated with chronic myeloid leukemia (CML) was identified in all the 3 cases. There was also myeloid hyperplasia in the bone marrow (with primarily mature, eosinophilic granulocytes), spleen and liver, depression of megakaryocyto- and erythropoiesis. 2 patients had similar clinical symptoms which was not typical for CML in chronic phase: fever, elevated ESR, clear-cut anemia and thrombocytopenia. In the absence of hyperleukocytosis, blood and bone marrow eosinophils remained high (42.5, 21.5, 42.5% and 21.4, 7.1, 6.5%, respectively) due to "mature" forms. The number of blasts in the bone marrow was maximum 2.4%. CONCLUSION: The literature and the obtained data suggest closeness of idiopathic hypereosinophilic syndrome and Ph'-positive CML within myeloproliferative diseases.