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FISH Scoring in CLL

FISH Scoring in CLL. Stephanie Fink September 21, 2006. PAX5. CEN. CEN. CEN. CCND1. MDM2. c-MYB. c-MYC. ATM. D13S319. P53. MALT1. CEN. IgH. LAMP1. FISH for CLL. del(6)(q23). del(17)(p13). +12. idic 17. 6q-. +12. del(13)(q14)x1. del(11)(q23). 13q-. 11q-. Centromere 11.

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FISH Scoring in CLL

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  1. FISH Scoring in CLL Stephanie Fink September 21, 2006

  2. PAX5 CEN CEN CEN CCND1 MDM2 c-MYB c-MYC ATM D13S319 P53 MALT1 CEN IgH LAMP1

  3. FISH for CLL del(6)(q23) del(17)(p13) +12 idic 17 6q- +12 del(13)(q14)x1 del(11)(q23) 13q- 11q-

  4. Centromere 11 2R2G ATM Chromosome 11 Probe strategy to detect aneuploidy and deletions Interphase pattern

  5. Monosomy Upper limit of normal range Trisomy Monosomy 4.5% Trisomy 1.5% Deletion Deletions 5.0% Probe Strategies con’t

  6. False (-LSI) overlap overlap -LSI -LSI -LSI False (-LSI, -CEP) overlap +CEP -LSI overlap overlap -LSI -CEN +CEP -LSI False (-LSI) FALSE IMPRESSIONS +CEP

  7. Establish ND-FISH Scoring Criteria for c-MYB, ATM, Cen 12, D13S319 and p53 Control probe Green (G), Target probe Red (R) Normal 2R2G Abnormal 1R2G 6q-, 11q-, 13q-x1 or 17p- Abnormal 0R2G 13q-x2 Abnormal 3R3G +6, +11, +12, +17 Do not score overlapping cells Only score cells with at least 1R and 1G signal except for the D13S319 probe set. There it is ok to score 2G signals. Do not score cells with too many control signals

  8. Probes (chromosome locus) Upper limit of normal Chromosome anomaly SpectrumOrange SpectrumGreen deletion trisomy monosomy translocation 6q- c-MYB (6q23) D6Z1 (6 cen) 6.5% 1.5% 4.0% NA 11q- ATM (11q22.1) D11Z1 (11 cen) 5.0% 1.5% 4.5% NA +12 D12Z3 (12 cen) MDM2 (12q15) 7.5% 1.5% 5.0% NA 13q-x1 D13S319 (13q14) 13q34 7.0% 1.5% 6.5% NA 13q-x2 D13S319 (13q14) 13q34 1.5% 1.5% 6.5% NA 17p- p53 (17p13.1) D17Z1 (17 cen) 8.5% 1.5% 6.5% NA t(11;14) CCND1 (11q13) IgH (14q32) NA 7.6%* NA 2.5% Based on analysis of blood from 20 normal individuals and a one-sided 95% confidence interval for observing the maximum number of nuclei for each false-positive signal pattern seen in 200 scoreable nuclei using the binomial distribution. Cut-offs for DNA probes used in FISH studies

  9. Anomalies Pts % 1 97 57% 2 51 30% 3 17 10% 4 2 1% 5 1 1% 6 1 1% Total 169 100% 169/203 (83%) pts abnormal FISH resultsDistribution of 260 anomalies detected by FISH

  10. CLL status Pts Norm Abn % Stable 56 19 37 66 Progressive 57 7 50 88 Total 113 26 87 77 FISH for stable vs progressive CLL • Number of FISH anomalies is higher in progressive than stable • Mean % abnormal nuclei is higher in progressive (73+26) than stable (55+24)

  11. Blood versus Marrow in 25 patients with stable disease % abn nuclei is similar (p<0.0001)

  12. Figure 1 Pt. 4 Pt. 2 Pt. 11 Pt. 3 Pt. 1 Pt. 8 Pt. 9 Pt. 12 Pt. 6

  13. Normal Cell 1 Cell 2 Cell 3 Cell 4 Figure 2

  14. p53 13 13 13 12 12 12 11.2 11.2 11.2 11.1 11.1 11.1 11.1 11.1 11.1 Cen 11.2 11.2 11.2 12 12 12 21.1 21.1 21.1 21.2 21.2 21.2 21.3 21.3 21.3 22 22 22 23 23 23 24 24 24 25 25 25 SMS RARA Cen HNPP Sequential Probe Strategies to Identify Breakpoint Region of Chromosome 17 Anomalies Step 1 Step 2 Step 3 Localizes breakpoint to 17p arm Localizes breakpoint to 17cen-17p11.2 Localizes breakpoint to 17p11.2-17p12

  15. Pt 5 p53 SMS D17S122 Normal Pt 8 Localizes breakpoint to 17cen-17p11.2 Localizes breakpoint to 17p11.2-17p12 Pt 11

  16. IgH Translocations in 76 (7%) of 1032 Patients Referred for CLL FISH Testing 1/1/03 - 6/15/04 34 35 All specimens were negative for BCL6, BCL10, MALT1, PAX5, c-MAF, FGFR3 30 25 18 20 Number of patients 13 15 10 6 2 5 1 1 1 0 CCND1 BCL2 BCL3 MYC BCL11A CCND1 & BCL3 & Unknown BCL2 BCL11A IgH translocation partner locus

  17. Reflex To IgH/BCL2 and IgH/BCL3 IgH/BCL2 fus IgH/BCL2 IgH/BCL3 IgH/BCL3 fus Abnormal Normal IgH/CCND1 fus IGH x 3 IgH/CCND1 Normal cutoff >7.6% F Abnormal Normal

  18. IGHv Gene Expressed Enhancer Constant Region Joining Region 4-30.4 Diversity Region 3-74 7-4-1 3-72 1-69 4-59 4-39 3-21 3-48 4-34 5-51 3-11 3-15 3-30 3-53 3-13 3-23 3-33 3-7 1-2 4-4 2-5 1-8 3-9 6-1 D M 14qter Cen 14 FISH IGHv+ 1 1 1 1 4 1 2 1 1 1 1 1 4 1 1 3 1 1 4 1 2 FISH IGHv- 1 1 6 1 2 IGHv-

  19. Chromosome evolution as an indicator of disease progression in stable B-CLL • Cytogenetic and FISH studies suggest that patients with CLL acquire chromosome abnormalities during the course of their disease • There is minimal prospective data on the clinical utility of FISH to study clonal evolution • Between 1994 and 2002, we enrolled and collected baseline blood specimens from 160 patients with previously untreated CLL (83% Rai 0/1) on a prospective trial evaluating chromosome evolution by FISH

  20. Study Design/Results • Patients provided follow-up specimens for FISH testing up to 12 years post baseline specimen • Median time from diagnosis to study enrollment was 1.8 months • 18 patients experienced chromosome evolution during follow-up

  21. Time to chromosome evolution among 173 patients with B-CLL Number of patients Years from diagnosis

  22. c-MYB/CEN 6

  23. ATM/CEN 11

  24. CEN 12/MDM2

  25. D13S319/13q34

  26. p53/CEN 17

  27. IGH/CCND1

  28. Thanks! • Kim Stockero • Sarah Paternoster • Stephanie Smoley • Dr. Dewald

  29. CLL Unknown 2 Atypical CLL C-MYC 2 MCL BCL11A 1 FL Other LPD BCL-3 2 6 3 3 BCL-2 16 Cyclin D1 0 5 10 15 20 IgH Translocations in 33 (6%) of 542 Patients Referred for CLL FISH Testing • FISH studies using an IgH probe • improves the diagnosis of CLL and related disorders • provides prognostic information

  30. Table 1: Cytogenetic and FISH studies for 12 patients with 13q- and B-CLL Metaphase FISH (D13S319)1 Interphase FISH (200 nuclei)2 Pt Karyotype Cells 13q-x1 13q-x2 13q- Other 1 46,XY[17] 91 2 [2%] 0 13q-x1[82%] None 2 46,XY[20] 29 4 [14%] 0 13q-x1[66%]/13q-x2[12%] 11q-[19%] 3 46,XX[20] 86 2 [2%] 0 13q-x1[45%] 17p-[60%] 4 46,XY[20] 93 2 [2%] 0 13q-x1[37%] 11q-[44%] 5 46,XX[11] 11 0 0 13q-x1[17%]/13q-x2[66%] None 6 46,XY[11] 40 0 5 [13%] 13q-x2[90%] None 7 46,XX[13] 47 0 0 13q-x1[93%] +12[90%] 8 46,XX[7] 32 2 [6%] 0 13q-x1[72%] None 9 46,XX[30] 108 0 2 [2%] 13q-x2[13%] 17p-[11%] 10 46,XX[20] 181 0 0 13q-x2[25%] 11q-[30%] 11 46,XX[20] 61 2 [3%] 0 13q-x1[22%] None 12 46,XY[20] 21 0 2 [10%] 13q-x1[30%]/13q-x2[53%] +IGH[74%] Rai Stage IV III I NA 0 II 0 0 III 0 0 0 Abbreviations: 13q-, indicates an interstitial deletion involving 13q14; 13q-x1 indicates deletion of D13S319 in one chromosome 13; 13q-x2 indicates deletion of D13S319 in both chromosomes 13. 1Metaphase FISH studies were done only with D13S319 and 13q34 to detect 13q-. This precludes identification of other chromosome anomalies in the same metaphase. 2Panel FISH studies for anomalies of chromosomes 6, 11, 12, 13, 14 and 17.

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