Association of Interphase Fluorescence in Situ Hybridization (IP-FISH) Response Criteria with Conventional Metaphase Cytogenetics in CML Patients On Therapy.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4707-4707
Author(s):  
Thomas Schenk ◽  
Martin C Müller ◽  
Alice Fabarius ◽  
Philipp Erben ◽  
Thomas Ernst ◽  
...  
Keyword(s):  
Bone Marrow ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Conflicts Of Interest ◽  
False Negative ◽  
Response Criteria ◽  
Interphase Cells ◽  
Definition Of ◽  
Fish Response

Abstract Abstract 4707 The Philadelphia (Ph) chromosome and its molecular equivalent, the BCR-ABL fusion gene, represent the pathogenetic cause and a useful marker for diagnosis and follow up monitoring of chronic myeloid leukemia (CML). Cytogenetic analysis of bone marrow metaphases (Cy) has been established as the standard method. In contrast, interphase fluorescence in situ hybridization (IP-FISH) has been increasingly applied in many studies due to recent optimization of the technique but is not represented in current treatment guidelines. We therefore sought to define IP-FISH response criteria which correspond best with complete (CCyR) and major cytogenetic responses (MCyR). In order to quantitatively compare results of both methods 1,749 consecutive non selected bone marrow samples from 748 CML patients at different stages of CML were analyzed in parallel with Cy and IP-FISH. 5 patients with Ph negative/BCR-ABL positive CML were excluded from the analysis. 643 patients in chronic phase (CP) were analyzed during imatinib based therapy, ten patients received interferon alpha. 74 patients at different stages of the disease received 2nd generation tyrosine kinase inhibitors: nilotinib, n=18 (CP, n=13; accelerated phase, AP, n=2; blast crisis, BC, n=3); dasatinib, n=56 (CP, n=41; AP, n=4; BC, n=11). 21 patients received no therapy or the therapy was not evaluable. The correlation between Ph positive metaphases and the proportion of FISH positive interphase cells was determined using the Spearman's rank correlation coefficient. The chi-square test was used to compare IP-FISH and Cy data. The optimally separating threshold value between Cy and IP-FISH was chosen as cut-off point. Cy and IP-FISH data correlated well (r=0.89; p<0.0001). The following cut-off values were defined: '30% IP-FISH positivity was found to correspond best with MCyR ('35% Ph+ metaphases); <6% IP-FISH positivity was concordant with CCyR (0% Ph+ metaphases). Of 1,163 samples of patients in CCyR, 99.1% showed a percentage of <6% IP-FISH positive cells. 82 of 1,163 samples (7.0%) with 0% Ph+ metaphases by Cy were IP-FISH positive (median 3%, range, 1-21% positive interphases). IP-FISH showed false negative results in 10 of 1,090 samples (0.9%) with a median of 8% Ph+ metaphases (range, 4-40%). Using these IP-FISH cut-off points, the diagnostic specificity for the definition of CCyR was 93.8% for all patients and 93.7% for CP pts only and for the definition of MCyR 89.4% for all patients and 88.4% for CP patients only, respectively. In conclusion, BCR-ABL FISH data derived from bone marrow interphase cells are comparable with metaphase cytogenetics but the cut-off points differ. IP-FISH might be used instead of Cy in order to assess the achievement of response milestones in CML patients during therapy. The prognostic value of IP-FISH data, however, should be analyzed in prospective controlled trials. Disclosures: No relevant conflicts of interest to declare.

Study On the Clonal Origin of Dysplastic Cells by Fluorescence in Situ Hybridization in Myelodysplastic Syndromes.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4858-4858
Author(s):  
Jun Zhang ◽  
Yongquan Xue ◽  
Jinlan Pan ◽  
Yafang Wu ◽  
Juan Shen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Myelodysplastic Syndromes ◽  
Chromosomal Abnormalities ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Banding Technique ◽  
Clonal Origin

Abstract Abstract 4858 Objective To determining the clonal origin of dysplatic cells in Myelodysplastic syndromes (MDS) . Methods Karyotypic analyses of bone marrow cells using R-banding technique were carried out to determine the chromosomal abnormalities. Interphase fluorescence in situ hybridization (FISH) and morphologic analysis of bone marrow aspirates were performed in the same cells to investigate the clonal origin of dysplatic cells in 8 MDS patients. Result All patients had clonal karyotypic abnormalities: simple abnormality in 1 patient, complex abnormalities in 6 patients, coexistent of two unrelated clones in 1 patient. Most of dysplastic cells in 7 of 8 MDS patients derived from neoplasia clone while 1 patient had a reverse result,no matter what cell lineage was involved. Some of non-dysplastic cells of all patients derived from malignant clone; in 7 patients, the proportion of dysplastic cells in malignant clone were significantly higher than that of non- malignant clone. Conclusion Most of dysplastic cells in MDS derived from malignant clones, while the minority of them derived from non-malignant clones. Thus, it is reasonable to expect that in most cases myelodysplasia is present in malignant clone and can be taken as an important diagnostic evidence for MDS. Disclosures No relevant conflicts of interest to declare.

Multiple Myeloma, Detection of Four Unfavorable Genetic Pronostic Factor by Interphase Two-Color Fluorescence in Situ Hybridization Analyses: A Study of 33 Patients At Diagnosis or Relapse

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5004-5004 ◽  
Author(s):  
Carlo Sergio Barragan ◽  
Silvia B. Hansson ◽  
Silvina A Cicchini ◽  
Federico J Hidalgo ◽  
Claudia E Ochoa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Poor Prognosis ◽  
Conflicts Of Interest ◽  
Autologous Bone Marrow Transplantation ◽  
Chromosome Band ◽  
Chemotherapy Drugs ◽  
Genomic Aberrations

Abstract Abstract 5004 Background: Interphase Fluorescence in situ hybridization (FISH) has improved the detection of early bad prognostic genomic aberrations in Myeloma (MM) is a fast and reliable method. We used this assay in myeloma patients at diagnosis, after relapse or treatment failure. Methods: Interphase Mononuclear cells from Bone Marrow of 36 Caucasian patients, 13 men and 23 women, age ranged from 40 to 84 years (median, 64) with MM. were analyzed by LEXEL Live FISH probes, for deletions in chromosome band 13q14. 3, 17p13. 1; IGH/CCND1 translocation dual fusion and cMYC breakapart 8 Chromosome. Results: Chromosomal aberrations were detected in 16 of 33 evaluable patient samples of a total 36 patients population. The most frequent aberrations were 7/16 in 13p14. 3 band deletion (43. 75%) 7/33 (21%); 5/16 (31, 25%)patients with deletion had 17p13. 15/33 (15, 15%); 3 cMYC breakapart 3/16 (18. 75%) 3/33 (9%); IGH/CCND1 translocation in 3 patients 3/16 (18. 75%) 3/33 (9%), two of them associated one with cMYC and the other with 13p14. 3. We found other association: A)13p14. 3; 17p13. 1 and cMYC in (1/33). B) 13p14. 3 and cMYC (1/33) C) 17p13. 1; cMYC (1/33). Two patients had 8 chromosome trisomy 2/33 (6%). We foun that 13p14. 3, 17p13. 1 and cMYC association was correlated with a worst and fast evolution. Who had 13p14. 3 band deletion had a worst evolution in our patients. cMyc was associated with a poor prognosis. Conclusions: Genomic aberrations are independent prognosis factors of disease progression, chemotherapy respond and survival in Myeloma. Novel DNA molecular target and bone marrow micro environment modification drugs such as bortezomib and thalidomide or lenalidomide must be use in first line associated perhaps with standar chemotherapy drugs for improving result and consolidate this patients with autologous bone marrow transplantation avoiding the dilate of this strategic in patient with poor prognosis. Future trials must be performed to include a more complete panel of probes in Myeloma. Disclosures: No relevant conflicts of interest to declare.

Combination Of Fluorescence In SITU Hybridization (FISH) and Cytogenetic Techniques Optimize The Diagnostic Process Of Patients With Myelodysplastic Syndrome (MDS)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5210-5210
Author(s):  
Drorit Merkel ◽  
Ilia Navikov ◽  
Abraham Avigdor ◽  
Ninette Amariglio ◽  
Arnon Nagler ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Conflicts Of Interest ◽  
False Negative ◽  
Diagnostic Process ◽  
Minimal Amount ◽  
Structural Abnormality ◽  
Minimal Size ◽  
Gold Standard Method

Abstract Background Cytogenetic analysis in myelodysplastic syndromes (MDS) is the “gold standard” method for identification of clonal chromosomal abnormalities, dependent on culturing the bone marrow cells and analyzing at least 20 metaphases. According to the International System for Human Cytogenetic Nomenclature (ISCN 2009) definition of a clone requires at least two metaphases with the same structural abnormality or the same extra chromosome, or alternatively, the presence of three or more metaphases missing the same chromosome. The minimum number of metaphases to detect a clone is calculated according to the formula presented in Fig.1. To avoid missing a clone present in 14% of cells, 20 metaphases must be analyzed, and to avoid missing a clone present in only 10% of cell 30 metaphases must be analyzed, with 95% confidence. Fluorescence in situ hybridization (FISH) on cultured cells is used as a complementary test, permitting analysis of large numbers of cells. The protocol in our lab includes analyzing, independently, both cytogenetic and FISH, on uncultured BM cells. Aims The aim of our study is to determine the minimal amount of mitoses that must be analyzed by cytogenetic combined with FISH to avoid false-negative results thus optimizing the diagnosis process of MDS patients. Methods Retrospective study was performed on cytogenetic and FISH results of a group of 325 suspected MDS patients analyzed at the Chaim Sheba Medical Center in the years 2009-2011. Results FISH was normal, for the most common MDS abnormalities, (-5,del(5q); -7,del(7q); +8; del(20q), as well as -Y for male patients), in 258 (79.4%) of patients 95.3% of them with normal cytogenetic. One or two abnormal clones, from the intermediate risk group in IPSS, were found in 10 patients (3.9%), at least in 30% of the mitoses. Complex karyotypes with 3 abnormalities, defined as poor risk, were found in two (0.8%) patients in 87.5% of the mitoses. Importantly, when FISH is normal, 9 metaphases are enough to detect minimal size of an abnormal clone with 95 % confidence and 14 metaphases with 99% confidence (Fig.1). Abnormal FISH was detected in 67 of patients. The same abnormalities were found by cytogenetic in 42 (62.6%), at least in 20%of the mitoses. In 25 patients, cytogenetic yielded additional chromosomal aberrations, not detected by FISH, at least in 20% of the mitoses, of them 20 patients (29.9%) with more than one aberration. Notably, in patients with abnormal FISH, analyses of 14 metaphases are enough to detect minimal size of CCA clone present in 20% of mitoses with 95 % confidence and 21 metaphases with 99% confidence (Fig.1). In addition cryptic deletions were detected by FISH, and not by cytogenetic, in 5 patients: del(7q) (n=3),del(5q) and del(20q), one each. Furthermore in 17 of 20 patients (85%) with complex karyotypes del(5q) was involved. Finally, in patients in whom -Y was detected by FISH, cytogenetic did not detect any other chromosomal abnormality implying to the benign nature of this lesion. Summary / Conclusion The combination of FISH, for the five most common abnormalities, and cytogenetic may improve the accuracy and speed of the diagnostic process, and may permit significant reduction of the number of mitoses to be analyzed in suspected MDS patients. The minimal size of mitoses to be analyzed to detect an abnormal clone has to be defined in each laboratory. We suggest that search for cryptic del(5q), del(7q) and del(20q) by FISH be added to the routine BM assessment in MDS patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Detection of trisomy 12 in chronic lymphocytic leukemia by fluorescence in situ hybridization to interphase cells: a simple and sensitive method

Blood ◽  
1992 ◽  
Vol 79 (7) ◽  
pp. 1796-1801 ◽  
Author(s):  
J Anastasi ◽  
MM Le Beau ◽  
JW Vardiman ◽  
AA Fernald ◽  
RA Larson ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Chronic Lymphocytic Leukemia ◽  
Fluorescence In Situ Hybridization ◽  
Cytogenetic Analysis ◽  
Normal Karyotype ◽  
Lymphocytic Leukemia ◽  
Chromosome 12 ◽  
Interphase Cells ◽  
Trisomy 12

Abstract Trisomy 12 is the most common cytogenetic abnormality in chronic lymphocytic leukemia (CLL), and a number of studies have suggested that it may be an adverse prognostic indicator. We have evaluated the usefulness of fluorescence in situ hybridization with a chromosome 12- specific probe as a simple means for detecting trisomy 12 in interphase cells. Forty cases of B-cell CLL previously studied with conventional cytogenetic techniques were analyzed with a biotinylated probe to the centromeric region of chromosome 12. Thirty of these retrospective cases could be reevaluated with in situ hybridization. Our analysis showed three hybridization signals (ie, trisomy 12) in interphase cells from seven of seven cases found previously to have trisomy 12. Trisomy 12 was also detected in five additional cases: in one case thought to have a normal karyotype, in two cases that had been inadequate for routine cytogenetic analysis, and in two cases that had been found to have an abnormal karyotype without trisomy 12. In a prospective series of 20 newly accrued CLL cases, all cases were analyzed successfully by in situ hybridization and six (30%) showed trisomy 12. We were able to perform the analysis on routinely prepared and previously Wright- stained peripheral blood smears. We conclude that fluorescence in situ hybridization is a simple means for the detection of trisomy 12 in CLL. The technique is more sensitive than conventional cytogenetic analysis and would be a useful tool in clinical studies.

Early Detection of Relapse by Hypermetaphase Fluorescence In Situ Hybridization After Allogeneic Bone Marrow Transplantation for Chronic Myeloid Leukemia

2000 ◽  
Vol 18 (9) ◽  
pp. 1831-1836 ◽  
Author(s):  
Chy-Myong Seong ◽  
Sergio Giralt ◽  
Hagop Kantarjian ◽  
Jingping Xu ◽  
Jolynn Swantkowski ◽  
...  
Keyword(s):  
Bone Marrow ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Low Frequency ◽  
Allogeneic Bone Marrow ◽  
Myelogenous Leukemia ◽  
Mixed Chimerism ◽  
Allogeneic Bone ◽  
Time Points

PURPOSE: Standard G-band cytogenetic analysis (CG) provides information on approximately 25 metaphases for monitoring the presence of Philadelphia chromosome positive (Ph+) cells in chronic myelogenous leukemia (CML) patients, making the detection of a low frequency of Ph+ cells problematic. The purpose of this study was to improve the detection of a low frequency of Ph+ cells. PATIENTS AND METHODS: We combined fluorescence in situ hybridization (FISH) with long-term colcemid exposure, capturing several hundred metaphases in bone marrow cultures (hypermetaphase FISH [HMF]). Using probes that identify Ph+ cells, HMF was compared with CG analysis in the follow-up evaluations of 51 patients with CML at various time points after allogeneic bone marrow transplant (BMT). RESULTS: Thirty-five patients never showed the presence of Ph+ cells by either method. In four patients, high frequencies of Ph+ cells were detected by both methods. In the remaining 12 patients, Ph+ cells were detected by HMF at time points after BMT when they were not detected by CG. In seven of the 12 patients, low but statistically significant frequencies of Ph+ cells (0.37% to 5.20%) were detected 3 months or later after BMT, and when no intervention was initiated, all seven patients later relapsed. Based on those data, an eighth patient with mixed chimerism and a similar HMF-detected Ph+ frequency (1.8% at 27 months after BMT) was reinfused with donor lymphocytes and achieved remission with 0% Ph+ cells studied by HMF (up to 50 months after BMT). Ph+ cells detected by HMF but not by CG less than 3 months after BMT disappeared on later examination in two of four patients. After detection of Ph+ cells by HMF only, the median time to cytogenetic progression (detection of Ph+ cells by CG) was 101 days. CONCLUSION: The results demonstrate the ability of HMF to detect low but clinically relevant levels of leukemic cells not detected by CG in transplant patients. The data indicate that HMF can detect low levels of Ph+ cells before standard cytogenetics at a time that may be useful in monitoring disease status and planning clinical interventions.

scholarly journals Detection of 11q13 rearrangements in hematologic neoplasias by double- color fluorescence in situ hybridization

Blood ◽  
1996 ◽  
Vol 87 (4) ◽  
pp. 1512-1519 ◽  
Author(s):  
LJ Coignet ◽  
E Schuuring ◽  
RE Kibbelaar ◽  
TK Raap ◽  
KK Kleiverda ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Hematologic Malignancies ◽  
Current Data ◽  
Lymphocytic Leukemia ◽  
Cell Nuclei ◽  
Interphase Cell ◽  
Interphase Cells ◽  
Chromosomal Breaks

Rearrangements within the chromosome 11q13 region are frequent in hematologic malignancies. 50% of 75% of mantle cell lymphomas (MCLs) carry a translocation t(11;14) (q13;q32). Using Southern blot analysis, a BCL1 breakpoint can be detected in approximately 50% of MCLs. It is not known whether other MCLs harbor also breakpoints at 11q13. Breakpoints in this region not involved in t(11;14), are detected in chronic lymphocytic leukemia and acute myeloid leukemia. To detect and localize breakpoints at 11q13 more accurately, we have developed fluorescence in situ hybridization using two probe sets of differently labeled cosmids, symmetrically localized at either side of the major translocation cluster of BCL1. These probes span a region of 450 to 750 kb. We applied this assay to a series of hematologic malignancies with 11q13 abnormalities identified by classical cytogenetics. All four samples with a t(11;14) (q13;q32) showed dissociation of the differently colored signals in metaphase and interphase cells, thereby indicating a chromosomal break in the region defined by the probe sets. The frequency of abnormal metaphase and interphase cells was comparable with that observed in any of the 13 malignancies with other chromosomal 11q13 abnormalities, indicating that these chromosomal breaks occurred outside the 450- to 750-kb region covered by the probes. One patient showed triplication and one patient showed monoallelic loss of this region. The current data show that double-color fluorescence in situ hybridization is a simple and reliable method for detection of the t(11;14)(q13;q32) in interphase cell nuclei and that is can be used to distinguish this translocation from other 11q13 rearrangements in hematologic malignancies.

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