This document discusses cytogenetic abnormalities in myeloma detected by fluorescence in situ hybridization (FISH). It summarizes the experience at Hong Kong Sanatorium & Hospital using FISH to detect abnormalities including t(4;14), t(14;16), del(17p), and 1q gain. Key findings from 105 cases are reported, such as frequencies of 16% for t(4;14), 3% for t(14;16), 6% for del(17p), and 42% for 1q gain. Comparative data from other studies on frequencies of FISH abnormalities are also presented. Examples are provided showing how FISH can help distinguish between myeloma and related disorders in difficult cases.

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

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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)

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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

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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

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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 %

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CD138 immunomagnetic bead sorting
Before After

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CD138 immunomagnetic bead sorting
Before After

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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

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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

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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

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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%

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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

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IgM paraprotein: WM or myeloma?
CD20 CD138
Kappa PAX-5

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IgM paraprotein: WM or myeloma?
• t(11;14) positive
• MYD88 L256P negative
• Conclusion
CD20+ IgM myeloma
Cyclin D1

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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

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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

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IgG paraprotein: myeloma or WM?
BM
BM
Trephine x
100
Trephine x
600

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IgG paraprotein: myeloma or WM?
CD20 CD138
Kappa
ISH
Lambda
ISH

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IgG paraprotein: myeloma or WM?
• t(11;14) -ve
• MYD88 L256P +ve
• Conclusion: IgG WM
T → C

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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

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Acknowledgements
• Prof. C S Chim
• Mr. Anthony Wong
• Ms. Candy Wang
• Referring clinicians