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