FISH IN MYELOMA

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  • 1. BTG 2013 FISH in Myeloma Dr Edmond S K Ma Department of Pathology Hong Kong Sanatorium & Hospital
  • 2. BTG 2013 Agenda • Review HKS&H experience • CG abnormalities: East meets West • Diagnostic applications
  • 3. BTG 2013 CG abnormalities in myeloma • Conventional cytogenetics 30% • Interphase FISH ~90% – Drach J et al Cancer Res 1995; 55: 3854 – 9 • Reasons: – Low plasma cell % in BM – Low proliferative index of plasma cells – Translocations may be morphologically cryptic e.g. t(4;14)(p16;q32)
  • 4. BTG 2013 Prognostic significance
  • 5. Prognostic significance Black: No 13q-, t(4;14) and 17p- and low B2M Blue: Positive for 13q-, No t(4;14) and 17p- and low B2M Green: No 13q-, t(4;14) and 17p- and high B2M Red: Positive for 13q-, No t(4;14) and 17p- and high B2M Gray: Either t(4;14) of 17p-, and low B2M Pink: Either t(4;14) of 17p-, and high B2M Avet-Loiseau et al, Blood 2007; 109: 3489 - 95
  • 6. BTG 2013 International Myeloma Workshop Consensus • Risk stratification (panel 2) Blood 2011; 117: 4696 – 4700 – Cytogenetically detected chromosomal 13 or 13q deletion, t(4;14) and del(17p), and detection by FISH of t(4;14), t(14;16) and del(17p) – Repeat at relapse • Standard investigative workup (panel 3) Blood 2011; 117: 4701 – 5 – Standard metaphase cytogenetics and FISH for t(4;14), t(14;16) and del(17p)
  • 7. BTG 2013 Agenda • Review HKS&H experience • CG abnormalities: East meets West • Diagnostic applications
  • 8. BTG 2013 Myeloma FISH • Aim at same day processing • Examination of bone marrow smears • Reject if < 5% plasma cells • Cell sorting by CD138 immunomagnetic beads (Miltenyi Biotec) – For samples with < 50% plasma cells – Aim at > 90% purity • Pre-analytical variables: sample adequacy, freshness, plasma cell %
  • 9. BTG 2013 CD138 immunomagnetic bead sorting Before After
  • 10. BTG 2013 CD138 immunomagnetic bead sorting Before After
  • 11. BTG 2013 FISH probes • 13q, t(4;14), 17p, CEP 5/9/15 • t(4;14), t(14;16), 17p, CEP 5/9/15 • Current panel – t(4;14)(p16;q32) (IGH/FGFR3 DF probe) – t(14;16)(q32;q23) (IGH/MAF DF probe) – TP53/CEP17 – CKS1B/CDKN2C(p18) amplification/deletion
  • 12. BTG 2013 Gain of 1q: new high-risk CG marker in myeloma From Cytocell website Red (1q) = CKS1B Green (1p) = CDKN2C (p18) Avet-Loiseau H et al, J Clin Oncol 30: 1949 – 52, 2012
  • 13. BTG 2013 FISH analysis • Analyze at least 100 cells • Two observers • Laboratory threshold – Mean + 3SD of normal BM controls • Positive cutoff levels Ross FM et al 2012; Avet-Loiseau H et al 2012 – 10% for fusion or break-apart probes – 20% for numerical abnormalities – 30% for chromosome 1q gain – 40% for del(17p)/p53
  • 14. BTG 2013 Summary of HKS&H data • n = 105 cases – From April 2011 – Dec 2012 • Male: female = 73 : 32 • Median age = 62 years (range: 42 – 88) • Median % plasma cells = 53% – Range 5 – 95% • Cell sorting performed in 44/105 (42%) – i.e. < 50% plasma cells
  • 15. Summary of HKS&H data CG abnormality Frequency (%) Number Range of +ve cells 13q- 24% 5/21 60 – 90% t(4;14) 16% 17/105 10 – 90% t(14;16) 3% 2/80 50 – 80% 17p- 6% 6/105 20 – 80% 1q gain 42% 5/12 40 – 90% Polysomy 5/9/15 36% 31/87 20 – 95%
  • 16. BTG 2013 Summary of HKS&H data • Secondary FISH abnormalities – 6/17 t(4;14)+ve showed atypical (1Y1O1G) signal pattern – Polysomy / amplification 4, 14 or 16 • Common – Monosomy / deletion 17, 4, 14 or 16 • Occasional
  • 17. BTG 2013 Agenda • Review HKS&H experience • CG abnormalities: East meets West • Diagnostic applications
  • 18. Frequency of FISH abnormalities across studies Reference t(4;14) t(14;16 ) t(11;14 ) del(17p) del(13q) Hyper- diploidy MYC 1q gain Fonseca 2003 (ECOG) 12.7% 4.6% 15.8% 10.7% 54.2% NA NA NA Avet-Loiseau 2007 (IFM) 14% NA 21% 11% 48% 39% 13% NA Huang S-Y 2005 (Taiwan) NA NA NA 6.8% 34.1% ~20% NA NA Tan 2010 (Singapore) 14.9% 5.4% 18.9% 20.3% 29.7% NA NA NA Lai Y-Y 2012 (China) IgH translocation 57.6% 34.7% 60.4% NA NA 49% Yu H 2011 (China) NA NA NA 22.2% ~50% NA NA 45.8% HKS&H experience 16% 3% NA 6% 24% 36% NA 42%
  • 19. Frequency of FISH abnormalities across studies Reference t(4;14) t(14;16 ) t(11;14 ) del(17p) del(13q) Hyper- diploidy MYC 1q gain Fonseca 2003 (ECOG) 12.7% 4.6% 15.8% 10.7% 54.2% NA NA NA Avet-Loiseau 2007 (IFM) 14% NA 21% 11% 48% 39% 13% NA Huang S-Y 2005 (Taiwan) NA NA NA 6.8% 34.1% ~20% NA NA Tan 2010 (Singapore) 14.9% 5.4% 18.9% 20.3% 29.7% NA NA NA Lai Y-Y 2012 (China) IgH translocation 57.6% 34.7% 60.4% NA NA 49% Yu H 2011 (China) NA NA NA 22.2% ~50% NA NA 45.8% HKS&H experience 16% 3% NA 6% 24% 36% NA 42%
  • 20. Frequency of FISH abnormalities across studies Reference t(4;14) t(14;16 ) t(11;14 ) del(17p) del(13q) Hyper- diploidy MYC 1q gain Fonseca 2003 (ECOG) 12.7% 4.6% 15.8% 10.7% 54.2% NA NA NA Avet-Loiseau 2007 (IFM) 14% NA 21% 11% 48% 39% 13% NA Huang S-Y 2005 (Taiwan) NA NA NA 6.8% 34.1% ~20% NA NA Tan 2010 (Singapore) 14.9% 5.4% 18.9% 20.3% 29.7% NA NA NA Lai Y-Y 2012 (China) IgH translocation 57.6% 34.7% 60.4% NA NA 49% Yu H 2011 (China) NA NA NA 22.2% ~50% NA NA 45.8% HKS&H experience 16% 3% NA 6% 24% 36% NA 42%
  • 21. BTG 2013 Agenda • Review HKS&H experience • CG abnormalities: East meets West • Diagnostic applications
  • 22. IgM paraprotein: WM or myeloma? • F/57 Caucasian • Bone lesion • Hb 11.3 g/dL • WBC 6.7 x 109 /L • Platelets 285 x 109 /L • RFT normal • Paraprotein – IgM kappa at 10.4 g/L
  • 23. BTG 2013 IgM paraprotein: WM or myeloma? CD20 CD138 Kappa PAX-5
  • 24. BTG 2013 IgM paraprotein: WM or myeloma? • t(11;14) positive • MYD88 L256P negative • Conclusion CD20+ IgM myeloma Cyclin D1
  • 25. BTG 2013 IgG paraprotein: myeloma or WM? • F/68 Chinese • Nephrotic syndrome, renal impairment • IgG kappa paraprotein – increasing trend • First BM in 2010: not myeloma Peripheral blood
  • 26. BTG 2013 Plasma cell myeloma with t(11;14)(q13;q32) • Around 20% of myeloma • Cyclin D1 over-expression • Lymphoplasmacytic morphology (40 – 50%) • CD138+ 19+ 20+ PAX5+ cyclin D1+ • Mostly IgG, IgA and light chain myeloma • 80% IgM-myeloma associated with t(11;14) – but CD20-ve in contrast to t(11;14) non-IgM PCM
  • 27. BTG 2013 IgG paraprotein: myeloma or WM? BM BM Trephine x 100 Trephine x 600
  • 28. BTG 2013 IgG paraprotein: myeloma or WM? CD20 CD138 Kappa ISH Lambda ISH
  • 29. BTG 2013 IgG paraprotein: myeloma or WM? • t(11;14) -ve • MYD88 L256P +ve • Conclusion: IgG WM T → C
  • 30. BTG 2013 Disease progression • M/72 Chinese • Light chain myeloma • Achieved CR • Sudden relapse as plasma cell leukaemia 10 months later • Acquired del(17p) that was negative at diagnosis Diagnosis Relapse
  • 31. BTG 2013 Acknowledgements • Prof. C S Chim • Mr. Anthony Wong • Ms. Candy Wang • Referring clinicians