F.I.S.H. in PATHOLOGY;F.I.S.H. in PATHOLOGY;Applications,Answers..and Applications,Answers..and

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Allan KennedyAllan KennedyLead Biomedical Scientist (Molecular Lead Biomedical Scientist (Molecular

Pathology)Pathology)Glasgow Royal InfirmaryGlasgow Royal Infirmary

SamplesSamples

Tissue ImprintsTissue Imprints Blood Blood Formalin-fixed, paraffin embedded Formalin-fixed, paraffin embedded

sections-usually 1sections-usually 1µm thick!µm thick!

TECHNIQUETECHNIQUE

ACCESS THE TARGETACCESS THE TARGET DENATUREDENATURE HYBRIDISEHYBRIDISE COUNTERSTAINCOUNTERSTAIN VIEWVIEW

F.I.S.H. WORK STATIONF.I.S.H. WORK STATION

Probe useProbe use

Enumeration; Loss or AmplificationEnumeration; Loss or Amplification Breakapart; specific gene targetedBreakapart; specific gene targeted Dual Fusion; specific translocations Dual Fusion; specific translocations

detecteddetected

ENUMERATION ENUMERATION PROBESPROBES

e.g. Her2, CLLe.g. Her2, CLL

Ideogram

                                                                                                                                                                      

Her2 probe set

Normal Her2

AMPLIFIED HER2

Her2 ; Polysomy

Monosomy 17 with Her2 Amplification

Chronic Lymphocytic Chronic Lymphocytic LeukemiaLeukemia(C.L.L.)(C.L.L.)

CLL PROBE SET

CLLP53 (17p13.1) Red ATM (11q22.3) Green;

CLLP53 (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 Oligodendroglial TumoursTumours

Loss of Heterozygosity 1p/19q ?

1p36 Red1q25 GreenLoss of 1p36

19p13 green19q13 redLoss of 19q

DUAL COLOURDUAL COLOURDUALDUAL

FUSION FUSION PROBESPROBES

e.g.(t11:14, t14:18)e.g.(t11:14, t14:18)

Molecular Pathogenetic Mechanisms in Molecular Pathogenetic Mechanisms in Rhabdomyosarcoma and Ewing’s SarcomaRhabdomyosarcoma and Ewing’s Sarcoma

Normal Cells

Ch 14Ch 11

Ch 14Ch 11

Lymphoma Cells

t14:18 Negative

Chr14 Green (IgH)Chr18 Red (bcl2)CEP18 Aqua

T14:18 Positive

Chr14 Green (IgH)Chr18 Red (bcl2)CEP18 AquaTranslocations Yellow

BREAKAPART BREAKAPART PROBESPROBES

e.g. cMyce.g. cMyc

BreakapartBreakapart probeprobe

b/apcmyc

Ch 8

Lymphoma Cells

cMyc Negative

C-Myc (8q24) Positive

ANSWERSANSWERS

? Burkitt’s Lymphoma? Burkitt’s Lymphoma?DLBCL?DLBCL

LCA,CD10,CD20,bcl6LCA,CD10,CD20,bcl6 ;Positive ;Positive

TdT,CD5,MUM-1;TdT,CD5,MUM-1; Negative Negative FISH REQUEST FOR FISH REQUEST FOR

c-Mycc-Myc

t8:14t8:14

t14:18t14:18

C-Myc (8q24) Positive

t8:14 Positive

t14:18 Negative - Burkitt’s Lymphoma

? Burkitt? Burkitt?Follicular Lymphoma?Follicular Lymphoma

CD20,CD10,CD20,CD10,; Positive; Positive CD5,Cyclin D1,CD23,bcl2CD5,Cyclin D1,CD23,bcl2; Negative; Negative

Myc negative

t14:18 (IgH / bcl 2) positive

Bcl6 (3q27) translocation negative; Follicular Lymphoma

SarcomasSarcomas

Molecular Diagnosis of SarcomasMolecular Diagnosis of Sarcomas

TumorTumor TranslocationTranslocation Fusion GeneFusion Gene

Ewing/PNETEwing/PNET t(11;22)(q24;q12)t(11;22)(q24;q12) EWS/FLI1EWS/FLI1

t(21;22)(q22;q12)t(21;22)(q22;q12) EWS/ERGEWS/ERG

Alveolar Alveolar RhabdomyosarcomaRhabdomyosarcoma

t(2;13)(q35;q14)t(2;13)(q35;q14) PAX3/FKHRPAX3/FKHR

t(1;13)(p36;q14)t(1;13)(p36;q14) PAX7/FKHRPAX7/FKHR

Desmoplastic small round Desmoplastic small round cell tumorcell tumor

t(11;22)(p13;q12)t(11;22)(p13;q12) EWS/WT1EWS/WT1

Synovial SarcomaSynovial Sarcoma t(X;18)t(X;18)(p11.2;q11.2)(p11.2;q11.2)

SYT/SSX1+2SYT/SSX1+2

Congenital FibrosarcomaCongenital Fibrosarcoma t(12;15)(p13;q25)t(12;15)(p13;q25) ETV6/NTRK3ETV6/NTRK3

Clear Cell SarcomaClear Cell Sarcoma t(12;22)(q13;q12)t(12;22)(q13;q12) EWS/ATF1EWS/ATF1

SarcomaSarcoma

? Ewings Sarcoma? Ewings Sarcoma ? Synovial sarcoma? Synovial sarcoma

Syt-Negative

EWS Positive-Ewings Sarcoma

? ALVEOLAR ? ALVEOLAR RHABDOMYOSARCOMARHABDOMYOSARCOMA

TEST FOR;TEST FOR;

t1:13t1:13

t2:13t2:13

FKHR breakapartFKHR breakapart

t1:13 (Pax7 green/FKHR red ) Negative

t2:13 (Pax 3 green /FKHR red) Positive

FKHR (13q14) Positive

ANSWERS…and MOREANSWERS…and MORE

CLLP53 (17p13.1) Red ATM (11q22.3) Green;

t11:14IgH 14q32 GreenBcl2 18q21 RedCEP11 aqua

Amplified Her2Ratio 2.3

Amplified Her2Ratio 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 Need to record all observations-whether expected or not!expected or not!

Need to collaborate across centresNeed to collaborate across centres Need close co-operation with PathologistsNeed close co-operation with Pathologists Boundaries between Pathology, Genetics Boundaries between Pathology, Genetics

and Haematology rapidly blurringand Haematology rapidly blurring

Thank You