Transcript Document

F.I.S.H. in PATHOLOGY;
Applications,Answers..and
More
Allan Kennedy
Lead Biomedical Scientist (Molecular
Pathology)
Glasgow Royal Infirmary
Samples
 Tissue
Imprints
 Blood
 Formalin-fixed,
paraffin embedded
sections-usually 1µm thick!
TECHNIQUE
 ACCESS
THE TARGET
 DENATURE
 HYBRIDISE
 COUNTERSTAIN
 VIEW
F.I.S.H. WORK STATION
Probe use
 Enumeration;
Loss or Amplification
 Breakapart; specific gene targeted
 Dual Fusion; specific translocations
detected
ENUMERATION
PROBES
e.g. Her2, CLL
Her2 probe set
Ideogram
Normal Her2
AMPLIFIED HER2
Her2 ; Polysomy
Monosomy 17 with Her2 Amplification
Chronic Lymphocytic
Leukemia
(C.L.L.)
CLL PROBE SET
CLL
P53 (17p13.1) Red
ATM (11q22.3) Green;
CLL
P53 (17p13.1) Red
ATM (11q22.3) Green;
Loss of ATM associated with progressive disease
CLL:Loss of p53 associated with progressive disease and Fludarabine
resistance
P53 (17p13.1)Red
ATM (11q22.3) Green
Oligodendroglial
Tumours
Loss of Heterozygosity 1p/19q ?
1p36 Red
1q25 Green
Loss of 1p36
19p13 green
19q13 red
Loss of 19q
DUAL COLOUR
DUAL
FUSION
PROBES
e.g.(t11:14, t14:18)
Molecular Pathogenetic Mechanisms in
Rhabdomyosarcoma and Ewing’s Sarcoma
Ch 11
Ch 14
Normal Cells
Ch 11
Ch 14
Lymphoma
Cells
t14:18 Negative
Chr14 Green (IgH)
Chr18 Red (bcl2)
CEP18 Aqua
T14:18 Positive
Chr14 Green (IgH)
Chr18 Red (bcl2)
CEP18 Aqua
Translocations Yellow
BREAKAPART
PROBES
e.g. cMyc
Breakapart probe
Ch 8
b/ap
cmyc
Lymphoma
Cells
cMyc
Negative
C-Myc (8q24) Positive
ANSWERS
? Burkitt’s Lymphoma
?DLBCL
LCA,CD10,CD20,bcl6 ;Positive
TdT,CD5,MUM-1;
Negative
 FISH REQUEST FOR
c-Myc
t8:14
t14:18
C-Myc (8q24) Positive
t8:14 Positive
t14:18 Negative - Burkitt’s Lymphoma
? Burkitt
?Follicular Lymphoma
 CD20,CD10,;
Positive
 CD5,Cyclin D1,CD23,bcl2; Negative
Myc negative
t14:18 (IgH / bcl 2) positive
Bcl6 (3q27) translocation negative;
Follicular Lymphoma
Sarcomas
Molecular Diagnosis of Sarcomas
Tumor
Translocation
Fusion Gene
t(11;22)(q24;q12)
EWS/FLI1
t(21;22)(q22;q12)
EWS/ERG
t(2;13)(q35;q14)
PAX3/FKHR
t(1;13)(p36;q14)
PAX7/FKHR
Desmoplastic small round cell
tumor
t(11;22)(p13;q12)
EWS/WT1
Synovial Sarcoma
t(X;18)(p11.2;q11.2)
SYT/SSX1+2
Congenital Fibrosarcoma
t(12;15)(p13;q25)
ETV6/NTRK3
Clear Cell Sarcoma
t(12;22)(q13;q12)
EWS/ATF1
Ewing/PNET
Alveolar Rhabdomyosarcoma
Sarcoma
?
Ewings Sarcoma
 ? Synovial sarcoma
Syt-Negative
EWS Positive-Ewings Sarcoma
? ALVEOLAR
RHABDOMYOSARCOMA
TEST FOR;
t1:13
t2:13
FKHR breakapart
t1:13 (Pax7 green/FKHR red ) Negative
t2:13 (Pax 3 green /FKHR red) Positive
FKHR (13q14) Positive
ANSWERS…and MORE
CLL
P53 (17p13.1) Red
ATM (11q22.3) Green;
t11:14
IgH 14q32 Green
Bcl2 18q21 Red
CEP11 aqua
Amplified Her2
Ratio 2.3
Amplified Her2
Ratio 10
Her2 ; Fusions?
Chromosome 17
Her2 gene
Her2 ; Triple Bandpass filter
Her2 gene
Chromosome 17
Her2 ;
Homogeneously Stained Regions (HSR’s)
Her2;Double Minutes
 Need
to record all observations-whether
expected or not!
 Need to collaborate across centres
 Need close co-operation with Pathologists
 Boundaries between Pathology, Genetics
and Haematology rapidly blurring
Thank You