“Genes, Chromosomes and “Genes, Chromosomes and

Cancer”

Carlos A. Tirado, Ph.D., FACMG

• 1890, David von Hansemann speculated that the abnormal mitotic figures in cancer biopsies were important to the origin and that the abnormal mitotic figures in cancer biopsies were important to the origin and development of maligancies.

• In 1914, Theodor Boveri published a systematic somatic mutation theory of cancer stating that chromosome abnormalities were responsible for cellular cancer stating that chromosome abnormalities were responsible for cellular changes that caused normal cells to become malignant.

Chronic Myeloid Leukemia

NOWELL 1960

Molecular modification

in Ph chromosomeJanet Rowley in 1974

22

9

Ph

9+

bcr

abl

bcr-

abl

ABL1

BCR ABL1/

BCR

• The catalytic domain of ABL1 is fused to a domain on BCR that mediates constitutive oligomerization of the fusion protein in the absence of physiological activating signals, thereby promoting aberrant tyrosine kinasethereby promoting aberrant tyrosine kinaseactivity.

JAK2JAK2BCRBCR ABL1ABL1

MYCMYC

BCL2BCL2

Where does it go wrong?

G1

TP53

M

GoG2

DNA

Repair Tumor

OncogenesOncogenesOncogenesOncogenes

GoTP53

Rb1GoGo

G1G1

Rb

S

Repair

genesTumor

Suppressor

GenesSynthesis

DNA replication

Rb

-P04Cyclin

B-P04

Cyclin D

Rb

+P04:

Cyclin

A

B

TP53

+P04:

Free E2

Cyclin E

CYCLIN D1: CCND1 (11q13)

• It shows amplification in Burkitt’s• It shows amplification in Burkitt’s

lymphoma, breast cancer.

• CCND1 would also cooperate to

activate RAS and MYC. activate RAS and MYC.

CYCLIN D1: CCND1 (11q13)

• t(11;14) (q13;q32) is a recurrent

abnormality detected in 50-70% mantle cell

lymphoma cases & 10-20% plasma cell

myeloma (PCM).

• According to the literature, PCM patients

with the t(11;14)(q13;q32) are characterized with the t(11;14)(q13;q32) are characterized

by cyclin D1 up-regulation and a

lymphoplasmacytic morphology.

Mantle cell lymphoma

• No fusion protein, but promoter exchange.• No fusion protein, but promoter exchange.

• The immunoglobulin gene enhancer

stimulates the expression of BCL1

(CCND1).

• Oncogenesis: Overexpression of BCL1 • Oncogenesis: Overexpression of BCL1

accelerates passage through the G1 phase.

Loss of tumor suppressor genes:

1963: Retinoblastoma1963: Retinoblastoma

Cavenee et alCavenee et al: Deletion of a : Deletion of a

DD--group chromosomegroup chromosome

Deletion 13q

• Plasma cell myeloma

• B Chronic lymphocytic leukemia

• Non Hodgkin’s lymphomas

• Acute Myeloid leukemia

• Myelodysplastic syndromes (Refractory

anemia)

45,X,-Y,add(3)(p21),r(7)?(p22q32),add(8)(q24)x2,

add(10)(q24),-13,+17,dic(1;21)(q11;q22)[8]/

46,XX[12]

t(4;14)(p16;q32)

Poor prognosis

t(14;16)(q32;q23)

Poor prognosis

Plasma Cell Myeloma

• 13 q deletions:

Adverse/intermediate Adverse/intermediate

prognosis

• t(11;14): Intermediate

prognosis

• t(4;14): Adverse prognosis

• t(14;16): Adverse prognosis

• +1q or amplification of • +1q or amplification of

CKS1B: High standard risk in

PCM

Chromothripsis

Chromosome catastrophes involve

replication mechanisms generating complex genomic rearrangements.

Poor outcome in PCM.

Cell 148, January 2012:29-32Cell 148, January 2012:29-32

CKS1B in Plasma cell myeloma (PCM)

• CKS1B amplification is seen in monoclonal

gammopahy of undetermined signficance and PCM.gammopahy of undetermined signficance and PCM.

• British Journal of Haematology (2006) Vol 134,

Issue 6: 613-615 .

• A specific role for increased expression of CKS1B,

as a consequence of 1q21 copy number gain has

been postulated as both pathogenic as well as a been postulated as both pathogenic as well as a

powerful clinical prognostic factor in PCM.

• Leukemia (2006) 20: 2034-2040.

Deletion 13q in CLL

• ~ly 50% of CLL cases.

nuc ish (D12Z3x2,D13S319x0,D13S1020x2)[200/200]

CLL• Trisomy 12 : Adverse/Intermediate prognosis. It

can be associated with poor survival.can be associated with poor survival.

• Del(11)(q22-q23): Disease progression and

decreased survival.

• Del(13)(q14): Favorable. The common deleted

region is around 1.6 cm telomeric to the Rbregion is around 1.6 cm telomeric to the Rb

gene.

• Del(17)(p13): Shorter survival & treatment

failure.

Deletion 13q in CLL

• CLL carrying 70% of 13q deleted nuclei with

deletions not comprising the RB1 locus are deletions not comprising the RB1 locus are

characterized by a particular long time to treatment.

• CLL with 13q deletions in < 70% of nuclei

involving the RB1 locus or carrying 13q deletions

(mono allelic or bi allelic) in > 70% with or without

the RB1 are associated with a shorter time to the RB1 are associated with a shorter time to

treatment (Intermediate to unfavorable prognosis).

• Genes, Chromosomes & Cancer 50: 633-643 (2011)

miRNAs on 13q14.3 and 11q22

• 13q14.3 & 11q22-23 are cancer associated • 13q14.3 & 11q22-23 are cancer associated

genomic regions (CAGR) frequently deleted in

CLL harbors the micro-RNAS clusters

miR-15a/miR-16-1 in 13q

miR34b/miR34c in 11q

miR-9 and miR-223 (potential importance as miR-9 and miR-223 (potential importance as

biomarkers in recurrent ovarian cancer)

miR-15a & miR-16-1

• Downregulate BCL2 in a normal conditions with • Downregulate BCL2 in a normal conditions with

repression of its expression & promote apoptosis.

• Deletions or a downregulation of these mi-RNAs

would allow an overexpression of

BCL-2 and loss of APOPTOSIS.

Therefore these mi-RNAs could be promissingTherefore these mi-RNAs could be promissing

antisense BCL-2 molecules able to suppress the

BCL-2 overexpression in CLL & B-cell neoplasias.

miR-9, a MYC/MYCN-activated microRNA,

regulates E-cadherin and cancer metastasis

• It is upregulated in breast cancer cells, directly • It is upregulated in breast cancer cells, directly

targets CDH1, the E-cadherin-encoding messenger

RNA, leading to increased tumor angiogenesis.

• Significantly, in human cancers, miR-9 levels

correlate with MYCN amplification, tumor grade

and metastatic status.and metastatic status.

• Inhibiting miR-9 in highly malignant cells

inhibits metastasis formation.

• Nature Cell Biology (21 February 2010)

SF3B1 and other Novel Cancer Genes

in CLL

• SF3B1, which functions at the catalytic core of the • SF3B1, which functions at the catalytic core of the

splicesome, was the second most frequently mutated

gene (15% cases).

• TP53 (15%), ATM (9%), MYD88 (3p22) [10%] &

NOTCH1 (9q34.3) [4%]. Others were ZMYM3,

MAPK1, FBXW7, and DDX3X. MAPK1, FBXW7, and DDX3X.

• The New England Journal of Medicine (2011) 365(26): 2497-2506

SF3B1 in CLL

• SFB31 (2q33.1) mutations in CLL occur

primarily with deletions 11q involving the primarily with deletions 11q involving the

ATM gene: Poor prognosis.

• Mutations in SF3B1 had alterations in pre-

messenger RNA (mRNA) splicing of a

narrow spectrum of transcripts derived from

genes involved in cancer related processes, genes involved in cancer related processes,

including cell cycle control, angiogenesis and

apoptosis. • The New England Journal of Medicine 2011

CLL

• Mutations in NOTCH1 and FBXW7 were

associated with trisomy 12.

• NOTCH1 mutations were consistently associated

with unmutated IGHV.

• MYD88 mutations were present in samples that

were heterozygous for del (13q) and mutated IGHV

status.

• Splicing function in CLL is altered as a result of

mutations in SF3B1 rather than deletion 11q.

• The New England Journal of Medicine (2011) 365(26): 2497-2506

“Multiple-hit hypothesis”

Normal tissue “Normal” tissue

(hemizygous BRCA1

deletion)

Hyperproliferative cells

(homozygous BRCA1

deletion)

Tumor suppressor

loss (ie. TP53)

Atypical

hyperplasiaInvasive cancer

loss (ie. TP53)

Oncogene activation

(ie. MYC amplification)

GENES INVOLVED IN

CANCER

• ONCOGENES:

MYC (8q24), ABL1 (9q34)

• TUMOR SUPPRESSOR GENES:

RB1(13q14), TP53(17p13),

BRCA1(17q21), BRCA2(13q12)

• DNA REPAIR GENES: MSH2(2p21-22), PMS2 (3p21).PMS2 (3p21).

• APOPTOSIS REGULATING GENES:BCL2 (18q21), TP53(17p13).

• CELL CYCLE REGULATORS: CDKN2 (9p21), MDM2(12q14)

CANCER CELLS SHOW :

A.- Loss of differentiation

B.- Increased invasiveness

C.- Decreased drug sensitivity

D.- Karyotypic alterations: Whole D.- Karyotypic alterations: Whole chromosome gains and loss, ploidy changes.

Fanconi anemia (FA)

• A chromosome instability syndrome with progressive bone marrow failure and an increased risk of cancers.risk of cancers.

• Cases of MDS and AML arising in patients with FA appear to be preceded by isolated focal gains or cryptic rearrangements of 3q26 that cause overexpression of the EVI1 (ecotropic viral integration site 1) gene.

• This early genetic event may have a role in the • This early genetic event may have a role in the development of cancers that result from a constitutional imbalance between genotoxic stress and DNA repair.

Fanconi anemia

• Spontaneous

chromatid/chromosome chromatid/chromosome

breaks, triradials,

quadriradials with

mitomycin C (MMC) &

diepoxybutane (DEB).

• Abnormalities found in

MDS or AML:MDS or AML:

• -5/del(5q), and -7/del(7q),

in 10 % of cases

.

Fanconi anemia (FA)

Cryptic inversion

• inv(2)(p22-p21p23) in 6.7% of Japanese

patients with non–small-cell lung patients with non–small-cell lung

cancer, will result in the formation of a

fusion gene comprising portions of

EML4 and the gene encoding the ALK

receptor tyrosine kinase.

Juvenile myelomonoytic

leukemia (JMML)

• Association of JMML and NF1 (200-500x).• Association of JMML and NF1 (200-500x).

• Occasionally infants with Noonan syndrome develop a JMML-like disorder.

• In 35% of Noonan patients, a germline mutation in the PTPN11 (12q24) : The gene encoding the protein tyrosine phosphatase (SPH2).protein tyrosine phosphatase (SPH2).

• NRAS, KRAS and NF1.

Nomenclature - some rules

• Use standard abbreviations (del, inv, dup, • Use standard abbreviations (del, inv, dup,

etc.)

• List chromosomes in order from X, Y,

1,2,…22

• If multiple abnormalities, keep in same • If multiple abnormalities, keep in same

order, but list numerical before structural

abnormalities (eg. 47,XY,+21,del(21)

ISCN (Nomenclature)

46, XY,t(9;22)(q34;q11)[12]/

48,idem,+8,+12[8]

46,XX,add(19)(p13)

46,XX,der(19)t(19;?)(p13;?) 46,XX,der(19)t(19;?)(p13;?)

46,XX,del(6)(q13q23)[8]/46,XX[12]

Types of probes:

Centromeric WCP probes

Locus

specific

Dual Color, Break Apart

Rearrangement Probes

• If normal:

46,XY[20].nuc ish (5’MLL,3’MLL)x2

(5’MLL con 3’MLLx2)[200]

• If Abnormal:

nuc ish (MLLx2)(5’MLL sep nuc ish (MLLx2)(5’MLL sep

3’MLLx1)[196/200]

TXL3 on 5q35

RGF

46,XY[20].ish (TXL3x2)(5’TLX3 sep

3’TLX3x1)[20]

In T-ALL with t(5;14)(q35;q32)

Dual Color, Dual Fusion

Translocation Probes

LSI BCR/ABL

t(9;22) in CML

RGFF pattern

RGFF

46,XY,t(9;22)(q34;q11.2)[20].nuc ish

(ABL1,BCR)x3,(ABL1 con

BCRx2)[179/200]

Myelodysplastic syndromes • Clinically heterogeneous disorders for which

treatments are tailored to the predicted prognosis for each patient.each patient.

• Current prognostic scoring systems consider karyotypic abnormalities and certain clinical features to stratify patients with MDS into risk groups.

• > ½ of patients with MDS: Normal karyotype.

• An understanding of the clinical effects of mutations • An understanding of the clinical effects of mutations in various genes could improve the prediction of prognosis for patients with MDS and inform the selection of specific therapies.

Recurring chromosomal abnormalites and

their frequency in MDS at diagnosis

Unbalanced

+8+8

-7/del7q 10% 50% therapy related

-5/del5q 10% 40% therapy related

del(20q) 5-8%

-Y 5%

i(17q) or (i17p) 3-5%

Del(11q) 3%

Del(12p) or t(12p) 3%

Del(9) 1-2%

Idic(X)(q13) 1-2%

Recurring chromosomal abnormalites and

their frequency in MDS at diagnosis

Balanced

t(11;16)(q23;p13.3) 3% (therapy related)t(11;16)(q23;p13.3) 3% (therapy related)

t(3;21)(q26.2;q22.1) 2% (therapy related)

t(1;3)(p36.3;q21.2) 1%

t(2;11)(p21;q23) 1%

inv(3)(q21q26.2) 1%

t(6;9)(p23;q34) 1%

Myeloid malignancies

recurrent abnormalities

• MDS, MPD, • MDS, MPD,

AML

• Prognostic value

• (5q-, -7, 7q-, +8,)

del(5q)

del(5)(q13q33)

del(7)(q22) or

monosomy 7

t(3;21)(q26;q22)

EVI1 (Ecotropic Viral Integration Site 1 (EVI1) on 3q26

inv(3)(q21q26.2)

t-MDS and t-AML

5q- Syndrome

• Severe macrocytic anemia

• Normal or elevated platelet counts, normal or • Normal or elevated platelet counts, normal or

reduced neutrophil counts.

• Defect in erythroid differentiation.

• Hypolobulated micromegakaryocytes.

• Tendency to AML but slower than other forms of

MDS.

• Response to Revlimid

• ~ 1.5 Mb : 40 genes

• EGR1 gene

• PDGFRB

• RPS14 encodes a protein that is a component

of the 40S subunit of the ribosome.of the 40S subunit of the ribosome.

• ? Function but its deficiency blocks

preribosomal RNA processing and the

polymer formation of the 40S

• A link between the 5q- syndrome (Deletion

of one allele of RPS14) & congenital bone

marrow failure syndromes (Diamond-Black

Anemia) caused by the heritable mutation of

other ribosome-associated proteins.

Deletion 11qnuc ish(MLLx1)[20/200]

• Deletion of 11q23 are observed in treatment • Deletion of 11q23 are observed in treatment

related MDS (RARS, RAEB-1)/MPN

(CMML, JMML), AML, ALL,

biphenotypic leukemias.

• Therapy with topoisomerase II inhibitors is

associated with balanced abnormalities:

MLL rearrangements.

Myelodysplastic syndromes

• Mutations of TET2, RUNX1, TP53, NRAS, JAK2 ,

ETV6, PTPN11, BRAF, PTEN & CDKN2A have

been reported to be associated with MDS.

• Mutations in TP53, EZH2, ETV6, RUNX1 and

ASXL1 are predictors of poor overall survival,

independently of established risk factors.

• The mutated genes most strongly associated with a

specific karyotype group were TET2 and TP53.

• N Eng J Med June 29, 2011

Schanz et al. Journal of Clinical

Oncology (2012)

The 17p- chromosome• Loss of TP53 gene.

• 10-15% of follicle centre cell lymphoma • 10-15% of follicle centre cell lymphoma

(FCCL) & mantle cell lymphoma (MCL).

• Minority of marginal zone B-cell

lymphomas may be associated with 17p

deletion deletion

• Plasma Cell Myeloma and CLL.

• Poor prognosis in low grade lymphomas.

i(17q) in myeloid malignancies

dic(17;20) in myeloid malignancies

Case A1

• 57 year old patient with a history of stage • 57 year old patient with a history of stage IV diffuse large B-cell lymphoma status post 6 cycles of combination chemotherapy six years ago.

• In August 2002 presented with enlarged lymph nodes, anemia and leukocytosis.lymph nodes, anemia and leukocytosis.

• Peripheral blood smear from patient showing • Peripheral blood smear from patient showing

anemia, leukocytosis with increased granulocytes

and precursors. No increase in circulating blasts,

basophils, or platelet abnormalities was observed.

• Patient bone marrow showing hypercellularity

with marked myeloid hyperplasia and mild with marked myeloid hyperplasia and mild

eosinophilia (13%), with no increase in blasts.

BCR/ABL probe

RRGGG pattern VARIANT signal pattern

46,XY,t(4;22)(q12;q11.2)

CASE A1: t(4;22)(q12;q11.2)

BCR with Platelet derived growth factor receptor alpha

GEF Ser-Thr

tyrosine

177 Grb-2

site

CDC BCR TK1 TK2Olig

BCR exons 1-17

(1005 AA)

GEF

dbl-l

Ser-Thr

kinase

CDC

24

BCR

PHTK1 TK2

PDGFRA exons 12-23

(520 AA)

Diagrammatic representation of BCR and PDGFRA functional

domains

of the predicted approximately 4.6 kb full-length mRNA fusion

transcript

which is expected to encode a 1525 aa 169 kDa protein

Myeloid & lymphoid neoplasms with

eosinophilia and abnormalities of PDGFRA

rearrangement (MPN) :Deletion at 4q12

FIP1L1/PDGFRA FUSION GEN

Deletion at 4q12

Dysregulates tyrosine kinase activity

& transforms hematopoietic

CellsCells

BUT it is inhibited

by GLEEVEC

• Presentation as CEL (chronic

FIP1L1 and PDGFRA fusion

• Presentation as CEL (chronic

eosinophilia leukemia) but can also

present as AML, precursor T-

lymphoblastic lymphoma (T-LBL) .

• Rare cases of fusion of BCR with • Rare cases of fusion of BCR with

PDGRA resulting from a

t(4;22)(q12;q11).

Myeloid neoplasms with PDGFRB

rearrangement :

5q33

Chronic myelomonocytic leukemia (CMML)

with eosinophilia

t(5;12)(q31-q33;q24) KIAA15009-PDGRB

t(5;17)(q33;p11.2) SPECC1-PDFGRB

Myeloid neoplasms with FGFR1Myeloid neoplasms with FGFR1

rearrangement :

8p11.2

Platelet derived growth factor receptor beta:

PDGFRB (5q32-q33)

• Rearrangements of

the PDGFRB gene

can result from a

gene fusion to one of

as many as 15

different known different known

partner genes.

Myeloproliferative Neoplasms:

Chronic myelogenous leukemia, BCR-

ABL1 positive

Distinct forms of BCR-ABL1 from

alternative chromosome 22

breakpoints

• Deletion of any part of the BCR or ABL1 gene on the derivative chromosome 9 is a poor prognostic marker that derivative chromosome 9 is a poor prognostic marker that indicates rapid disease progression in chronic myeloid leukemia [Cancer Genet Cytogenetics: 2006 Apr 1;166(1):65-73].

• All the patients with BCR deletion, except for one, had a concomitant ABL1 deletion, suggesting that BCR deletion occurs in conjunction with ABL1 deletion. occurs in conjunction with ABL1 deletion.

• Deletion of any of the BCR or ABL1 genes on derivative chromosome 9 was associated with poor prognosis in CML.

22

9

Ph

der 9

der 99

22Ph

Myeloproliferative Neoplasms

Chronic neutrophilic leukemia: +8, +9,+21, Chronic neutrophilic leukemia: +8, +9,+21, del(20q),del(11q), del(12p)

Polycythaemia vera: del(20q)

Primary myelofibrosis: del(20q), partial trisomy 1q, del(13q), der(1;6)(q21-q23;p21.3)

Essential thrombocythaemia: +8, 9q Essential thrombocythaemia: +8, 9q rearrangements, del(20q) and del(5q)

Myeloproliferative neoplasms:

del(20q)

JAK2 Mutations in

Myeloproliferative

Neoplasms:

A valine to phenylalanine A valine to phenylalanine

substitution at position 617

(V617F) in the pseudokinase

kinase-like domain of JAK2.

Deregulation of the kinase activity, and

thus to constitutive tyrosine

phosphorylation activity.phosphorylation activity.

The JAK2 allele burden decreases

with successful therapy, disappears

in some patients, and reappears

during relapse.

V617F mutation

PV (~ 95%), essential thrombocythemia & idiopathic myelofibrosis idiopathic myelofibrosis

~ 20% of Ph-negative (ATYPICAL CML)

Minority of hypereosinophilic syndrome

> than 10% of CMML

~ 15% of (Acute megakaryoblastic leukemia) ~ 15% of (Acute megakaryoblastic leukemia)

& 1/5 patients with JMML.

Activating mutations of MPL (1p34)

exon 10

• Idiopathic myelofibrosis (IMF) or essential

thrombocythemia (ET). thrombocythemia (ET).

• Mutations in the myeloproliferative leukemia gene (MPL)

exon 10:

W515L and W515K are associated with severe

anemia and have been detected in patients with ET

or MF but not in patients with PV. or MF but not in patients with PV.

• An inherited MPL mutation (S505N; exon 10) has also

been found in a Japanese pedigree with familial ET.

Exon 12 mutations

A2

• A 14-year-old Hispanic male presented with increasing • A 14-year-old Hispanic male presented with increasing

weakness and decreased appetite. Laboratory examination

revealed hyperleukocytosis (white blood cell count of

253,000×109/L), anemia (hemoglobin 3.3 g/L) and

thrombocytopenia (platelet count of 36,000×109/L).

BCR (green), ABL1 (red), WNL

46,XY,t(9;22)(p24;q13)[14]/46,XY[7]

9

22

der(22)

der(9)

DiGeorge

Probe

der9

der22

ARSA signal

der22

Normal 9der9

Normal 9der9

der 22

Normal 9

der 9

• Induce the dimerization or

Translocations of

JAK2

• Induce the dimerization or

oligomerization of JAK2 without ligand

binding, which also results in constitutive

activation of JAK2.

Primary structure of JAK1, JAK2, and JAK3 showing the

location of missense (▼) and insertion/deletion (▲) mutations

Mullighan C G et al. PNAS 2009;106:9414-9418

©2009 by National Academy of Sciences

R683S and R683G � JAK-STAT activation.

R867Q and D873N in JH1 also cause

JAK mutations in pediatric ALL patients

R867Q and D873N in JH1 also cause

enhanced kinase activity of JAK2� JAK-STAT

activation.

S798P in JAK3 and L624_R629>W, S646F

and V658F in JAK1.and V658F in JAK1.

PNAS (2009) Vol 106 no. 23 p.9414-18.

JAK2R683

JAK2 exon 16 mutations in 18% cases

of Down Syndrome with ALL

JAK2R683

Arginine 683 mainly point mutations,

Complex in-frame insertion and deletion

events proximal to R683

JAK2 constitutive activation and JAK2 constitutive activation and

cytokine-independent growth in vitro.

Br J Haematol. 2009 144(6): 930-932.

Deletion of IKZF1 (encoding the lymphoid

Pediatric BCR-ABL1-negative

ALL patients

Deletion of IKZF1 (encoding the lymphoid

transcription factor IKAROS) &

CDKN2A/CDKN2B.

A poor prognosis similar to BCR-ABL1-

positive ALL, raising the possibility of

activated tyrosine kinase signaling within this

leukemia subtype.

PNAS (2009) Vol 106 no. 23 p.9414-18.

Hyperdiploidy

>50 chromosomes

25.0%

Hypodiploidy

<45 chromosomes

1.0%Others

22.0%

BCR/ABL

t(9;22)

Common anomalies in ALL (pediatric)

Hyperdiploidy

(25%)

Hypodiploidy

(1%)

t(9;22)

(3%)

Others

(22%)

TEL/AML1

t(12;21)

22.0%

3.0%

MLL

rearrangements

8.0%HOX11L2

5q35

2.5%

LYL1

(25%)

t(12;21)

(22%)

(3%)

MLL

(8%)

MYC

t(8;14)

2.0%TCF3/PBX1

t(1;19)

5.0%

HOX11

10q24

0.7%

LYL1

19p13

1.5%

MLL-ENL

0.3%

TAL1

1p32

7.0%

(22%)

t(8;14)

(2%)MLL-ENL

(0.3%)

t(1;19)

(5%)

19p13 (1.5%)

1p32 (7%)

4 and 10

Hyperdiploidy: ALL

Hyperdiploidy in ALL

favorable prognosis

Bone MarrowBone Marrow

57 Chromosomes

ABNORMALITYABNORMALITY ADULTSADULTS CHILDRENCHILDREN

t(9;22)t(9;22) 3030--37%37% 22--6%6%

t(12;21)t(12;21) 00--3%3% 2020--25%25%

Abnormalities seen in >5% of adults and/or

children with ALL

t(12;21)t(12;21) 00--3%3% 2020--25%25%

AbnAbn(9p)(9p) 66--30%30% 77--11%11%

High High hyperdiploidyhyperdiploidy 22--11%11% 2323--26%26%

t(4;11)t(4;11) 33--7%7% 2%2%

Del(6q)Del(6q) 33--16%16% 66--9%9%Del(6q)Del(6q) 33--16%16% 66--9%9%

Del(7p/q)Del(7p/q) 66--11%11% 4%4%

+8+8 1010--12%12% 2%2%

Abn(12p)Abn(12p) 44--6%6% 77--9%9%

Low hyperdiploidyLow hyperdiploidy 1010--15%15% 1010--11%11%

ALL FISH requirements

B - lineage patients:B - lineage patients:

If very young: TEL/AML1

If older than 10 years:

BCR/ABL1 dual fusion and MLL

CEN 4 and 10 CEN 4 and 10

dup(21q) amplified (RUNX1)

• Acute lymphoblastic leukemia : B-lineage immunophenotype (mostly common or Pre-B), FAB L1/L2

Precursor B cell Acute Lymphoblastic

Leukemia

Unfavorable prognosis

– t(9;22)(q34;q11.2); (BCR/ABL) (e1a2)

– t(4;11)(q21;q23); (AF4/MLL)

– Hypodiploid

Intermediate prognosis

t(1;19)(q23;p13); (E2A/PBX1)t(1;19)(q23;p13); (E2A/PBX1)

Favorable prognosis

– t(12;21); (TEL-AML1)

– Hyperdiploid >50

AML FISH requirements

• If normal karyotype results:• If normal karyotype results:

CBFß inv(16)/t(16;16)

11q23: MLL

t(8;21): ETO/AML1t(8;21): ETO/AML1

5q-,7q-

AML with balanced rearrangements

AML with (8;21)(q22;q22);RUNX1-RUNXT1RUNXT1

AML with inv(16)(p13.1;q22);CBFB-MYH11

AML with (15;17)(q22;q12);PML-RARA

Variants

AML with (9;11)(p22;q23);MLLT3-MLLAML with (9;11)(p22;q23);MLLT3-MLL

Intermediate survival better than any other MLL rearrangement

AML with balanced rearrangements

AML with (6;9)(p23;q34); DEK-NUP214AML with (6;9)(p23;q34); DEK-NUP214

Anemia & thrombocytopenia. Poor prognosis

AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2);RPN1-EVI1 :

Poor prognosisPoor prognosis

AML with (1;22)(p13;q13);RBM15-MKL1

Case A4

• WBC 11.1X109/L with 69% blasts. • WBC 11.1X109/L with 69% blasts.

• The bone marrow aspirate : Increased marrow

cellularity (90%).

• Cytochemistry demonstrated that the blasts were

positive for myeloperoxidase and negative for

non-specific esterasenon-specific esterase

• Flow cytometry: Aberrant population of • Flow cytometry: Aberrant population of

myeloblasts expressing CD45, CD34,

CD117, HLA-DR, MPO, CD13, CD33,

CD15, CD19 and CD56.

ETO/AML1 t(8;21)(q22;q22)ETO/AML1 t(8;21)(q22;q22)

2121

ETO on chrom 1

21

88

8

t(8;21)

8

Chr 1

Chr 21

der 8

Chr 21

Chr 8

Chr 21

P58 probe (1p36)

Chromosome 21 Chromosome 21Chromosome 1

Chromosome 1

AML with t(8;21)

• RUNX1 and RUNX1/RUNXT1 recognize the • RUNX1 and RUNX1/RUNXT1 recognize the

same binding sequence in the DNA, but the

hybrid RUNX1/RUNXT1 recruits additional

cofactors, mostly repressors, working as a

transcriptional repressor for the RUNX1 WT

target genes & ARREST MYELOID target genes & ARREST MYELOID

DIFFERENTIATION.

RUNX1/RUNXT1 recruits the NCoR/HD

Co-Repressor Complex to CBF

promoters

RUNX1T1RUNX1T1

RUNX1RUNX1

NCoRNCoR

HDHD

NO NO

TRANSCRIPTIONTRANSCRIPTION

Case A5

• By In January 2009, a 30-year-old man was

taken for urgent surgery due to bruising and taken for urgent surgery due to bruising and

ecchymoses in his lower extremities. A chest

computed tomography revealed a right atrial

mass.

• By

• By

Case A5

• By

Case A5

• By

• He was then treated with ATRA/ATO and local radiation therapy to all sites of disease.

• In September 2009, he had an MS relapse in a • In September 2009, he had an MS relapse in a supraclavicular mass.

• PET scan again showed multiple sites of disease in soft tissue, but bone marrow was normal.

• He is now being treated with ATRA/GO, but • He is now being treated with ATRA/GO, but response is yet to be determined.

Met1/FISH1

Case 2009

• By

• PML gene

Chromosomal rearrangements that

entail transcriptional repression: APL

• PML gene

Encodes a protein involved in tumor suppression & apoptosis.

• RARα gene

Encodes a protein

PML RARαααα

t(15;17)(q22;q21)

Encodes a protein involved in myeloid differentiation

PML/RARα Fusion Gene

• Interferes with the normal RARα function• Interferes with the normal RARα function

– Blocking the differentiation of promyelocytes(Maturation block): PML/RARA is a transcriptional repressor.

Interferes with normal PML

– Deactivates the tumor-suppression and pro-apoptotic functionsfunctions

– Promoting growth and inhibiting apoptosis

• Dual action gives APL cells a survival advantage and leads to cell proliferation

Variant Translocations

NPM1 ZBTB1ZBTB1

66

NUMA

66

NPM1: Nuclear protein

nucleophosmin

NUMA:

Nuclear mitotic apparatus protein 1

ZBTB16:ZBTB16:

Zinc finger and BTB domain Zinc finger and BTB domain

containing 16containing 16

APL

• Three regions of the PML locus are involved in

the translocation breakpoint cluster region (bcr):

• Intron 6 (bcr1)

• Exon 6 (bcr2)

• Intron 3 (bcr3)

• Whereas RARA breakpoints always occur in

intron 2, which is 17 kb in length.intron 2, which is 17 kb in length.

Cancer Genetics and Cytogenetics 188(2009) 103 107.

Leukemia Research 26 (2002): 439-442

PML/RARA Isoforms

• Long or bcr1• Long or bcr1

• Variant (V or bcr2)

• Short (S or bcr3): POOR OUTCOME.

• MOST OF THESE TRANSCRIPTS

CAN BE DETECTED BY CAN BE DETECTED BY

COMMERCIALLY AVAILABLE RT-

PCR kits.

AML with normal karyotype

• NPM1 and no FLT3 mutations or with • NPM1 and no FLT3 mutations or with

CEBPA (19 p13.1) mutation :

Favorable prognosis

FLT3: 13q12.2

• FLT3 is a member of the class III receptor tyrosine kinase (RTK) family:tyrosine kinase (RTK) family:

24 exons and at least 96 kb.

Expressed in many cell types including hematological stem cells.

• FLT3 mutations ���� constitutive ligand • FLT3 mutations ���� constitutive ligand independent autophosphorylation of receptor � Inhibition of APOPTOSIS & proliferation of leukemic cells.

FLT3: 13q12.2• 20-25% are internal tandem duplication (FLT3/ITD) or FLT3-

LM (length mutation)

(Juxtamembrane domain ) (Juxtamembrane domain )

In 20-25% AML, 5-10 % MDS.

• 7-9% mutations in the second tyrosine kinase domain, mostly point mutations in codon 835 (D835) or deletions of codon 836

In 7-8% of all AML and 3-15% of MDS.In 7-8% of all AML and 3-15% of MDS.

• Inhibition of APOPTOSIS and proliferation of leukemic cells� Unfavorable prognosis, high relapse rate.

AML with MDS related changes

-7, Del (7q) Del(11q)

-5/del(5q) -13/Del(13q)

i(17q) or i(17p)

Del(12p)/t(12p)

Del(9q)

Idic(Xp)

AML with MDS related changes

t(11;16)(q23;p13.3) t(5;17)(q33;q21)t(11;16)(q23;p13.3)

t(3;21)(q26.2;q22.1)

t(1;3)(p36.3;q21.1)

t(5;17)(q33;q21)

t(5;10)(q33;q21)

t(3;5)(q25;q34)

t(5;12)(q33;p12)

Complex karyotypes are seen

in lymphomas

t(8;14)(q24;q32)

t(2;8)(p12;q24)

t(8;22)(q24;q11)

Aqua – centromere on chromosome 8

Red – MYC locus on chromosome 8

Green – IGH locus on chromosome 14

MYC/IGH

MYC/IGH

• B-cell malignant neoplasias. • B-cell malignant neoplasias.

• No hybrid gene but

• Constitutive expression of C-MYC induces

proliferation even in the absence of growth

factorsfactors

t(14;18)(q32;q21)

t(2;18)(p11;q21)

t(18;22)(q21;q11)

• Extra nodal marginal B-cell lymphoma:t(11;18)(q21;q21) API2 (BIRC3) with MALT1

(Fusion protein 5’API2 with 3’MALT1)

• Anaplastic Large B-cell lymphoma: t(2;5)(p23;q35) • Anaplastic Large B-cell lymphoma: t(2;5)(p23;q35) ALK/NPM1

• Follicular lymphoma: t(14;18)(q32;q21) IGH/BCL2Promoter exchange.

The IgH gene enhancer stimulates the expression of The IgH gene enhancer stimulates the expression of BCL2. Upregulates APOPTOSIS. As BCL2 is an apoptosis inhibitor, cell death is delayed.

Gene amplificationGene amplification

• Concurrent FISH analysis • Concurrent FISH analysis

confirmed amplification of

the MYCN gene.

Amplification of MYCN is

found in various tumors, in

particular neuroblastoma;

the level of amplification the level of amplification

increases with tumor

progression.

46,XX,add(1)(p22),add(9)(p22),add(13)(q34),add(14)(q22),a

dd(15)(q26.1),1-16dmin[cp20]

47~49,XY,+r,+1-4mar[cp9]. ish

r(12)(MDM2+),mar(12)(MDM2+)

Well-differentiated liposarcoma

nuc ish(EWSR1x2)( 5’EWSR1 sep

3’EWSR1x1)[290/300]

96.7%

(290/300)

Her-2/neu

• It plays a key role in the regulation of cell growth

• It has been shown to be amplified in human breast, • It has been shown to be amplified in human breast, ovarian & other cancers

• It is a prognostic factor in

STAGE II + breast cancer patients. It is further indicated as an aid to predict disease-free & overall survival in these patients (treated with overall survival in these patients (treated with cyclosphosphamide chemotherapy).

ERBB2 (HER2/neu) FISH: POSITIVE FOR HER-2/neu

AMPLIFICATION

Ratio R/G: 8.3

• A ratio of greater than or equal to 2.2 is • A ratio of greater than or equal to 2.2 is

considered amplified.

• A ratio of less than or equal to 1.8 is

considered not amplified.

• A ratio between 1.8 and 2.2 is considered

"equivocal" and the results must be "equivocal" and the results must be

interpreted with caution.

UROVYSION

• DETECTION of ANEUPLOIDY :

3, 7, 17, loss of p16 (9p21) in urine specimens 3, 7, 17, loss of p16 (9p21) in urine specimens from subjects with transitional cell carcinoma of the bladder.

• The results are intended for use as a non-invasive method for monitoring TUMOR RECURRENCE in conjunction with cytoscopyRECURRENCE in conjunction with cytoscopyin patients previously diagnosed with bladder cancer.

UROVYSION TEST

• Detects chromosomal abnormalities associated

with the development and progression of bladder with the development and progression of bladder

cancer.

• UroVysion in conjunction with cystoscopy

delivers the best balance of sensitivity (100%) and

specificity (94.5%).

• Detects bladder cancer recurrence up to 6 months • Detects bladder cancer recurrence up to 6 months

sooner than current diagnostic methods.

• Early detection of high grade disease is critical to

increased survival.

UroVysion

Bladder Cancer FISH Probe Panel

UroVysion Bladder Cancer FISH

(a) normal cell

(c) homozygous 9p21 loss(b) Trisomy 7

Patient D

• Patient born 2/17/1998• Patient born 2/17/1998

• Chromosomes: 46,XY[20]

• Medulloblastoma

(undifferentiated/classic (undifferentiated/classic

type)(WHO grade IV)

RARA 17q21.1

73%

P53

73%

Microarray analysis overview

• Allow us to identify microduplications,

microdeletions and unbalanced translocations.

Microarray analysis

microdeletions and unbalanced translocations.

• It is a great tool to detect the presence of new

and possibly significant chromosomal

abnormalities in cancer, some of which might

be cryptic at the chromosomal level. be cryptic at the chromosomal level.

We can determine in

a single experiment,

the expression levels

of hundreds or thousands

of genes within a cell

Microarray

analysis

of genes within a cell

by measuring the amount

of mRNA bound to each

site on the array.

With the aid of a computer,

the amount of mRNA the amount of mRNA

bound to the spots on the

microarray is precisely

measured, generating a

profile of gene expression in the cell.

SNP array

• Will allow the very small copy-number • Will allow the very small copy-number

aberrations (CNAs) at high accuracy.

• Detect small deletions (< 1 Mb).

• Detection of CNLOH or a UPD

• Reliable detection of low grade mosaicism.• Reliable detection of low grade mosaicism.

Cancer Genetics and Cytogenetics 2008 Vol 180,

Issue 1: 56-59

• A 62-year-old man with gait and speech disturbances. • A 62-year-old man with gait and speech disturbances.

• Computed tomography and magnetic resonance: MASS.

• Tumor was surgically removed .

• Histopathology: Transitional type of meningioma with partially necrotic foci.

• After ~ 1 year, the patient was readmitted due to seizure and left-side hemiparesis.

• Tumor recurrence was discovered and resected. As of • Tumor recurrence was discovered and resected. As of writing, he had remained disease-free for 7 months. The recurrent tumor was diagnosed as a transitional meningioma with necrosis.

del(1)(p36.33p32.1 dup(6)(p25.3p23)

del(12)(p13.33p11.21 del(6)(q16.2q27)

Monosomy 22

Monosomy 18

Monosomy 22

• Monosomy 22 or deletion 22q : The most common chromosomal abnormality in meningiomas.

alterations in addition to

chromosome 22 losses.

• In contrast, complex meningiomas.

• Deletion 1p : 2nd most frequently deleted chromosome arm after 22q in meningiomas.

• In contrast, complex

karyotypes are seen in

aggressive tumor

behavior.

• Benign meningiomas only rarely harbor structural chromosomal

• Malignant progression

of meningiomas: Losses

on 1p, 9p, 10q, and 14q.

Recommendations

• Appropriate specimen processing depends on the • Appropriate specimen processing depends on the

information that accompanies the sample. Critical

information includes patient demographics,

differential diagnosis, symptoms, and other

laboratory findings.

• For hematological tumors, 2-3 ml of bone marrow • For hematological tumors, 2-3 ml of bone marrow

aspirate collected in sodium heparin is the

specimen of choice.

Recommendations

• When a BM aspirate cannot be obtained

because of marrow fibrosis, a BM trephine because of marrow fibrosis, a BM trephine

biopsy or a peripheral blood (PB) specimen

might be successful.

• For PB specimen to yield information about

clonal chromosomal aberrations, the PB clonal chromosomal aberrations, the PB

white cell count must be abnormal.

• Although one of the oldest methods,

remains a powerful tool for genetic remains a powerful tool for genetic

diagnosis and classification of

hematological malignancies and solid

tumors.

• Molecular cytogenetics (FISH) analysis

targets breakpoints specific for a particular targets breakpoints specific for a particular

disease.

The Role of Cytogenetics

• Cytogenetics now standard of care for leukemia • Cytogenetics now standard of care for leukemia patients

• Implementation of cytogenetic subgroups in WHO classification of tumors of hematopoietic and lymphoid tissues in 2008

• Many of the fusion genes associated with identified translocationsidentified translocations

• Prognosis

• Targets for therapy