CYTOGENETICS OF SOFT TISSUE TUMORS :

Principles and Diagnostic Considerations

with Special Reference to Pediatric Neoplasia

 

INDEX:

    Principles of Cytogenetics
    Terminology of Neoplasia
    Imprinted Gene Alterations in Tumors
    Chimeric Transcriptional Control Genes in Childhood Solid Tunors
    Tumor Suppressor Genes in Neoplasia
    Recurrent Oncogenes Amplified in Tumors
    Oncogenes: Types and Neoplasia
    Recurrent Karyotypic Abnormalities in Solid Tumors (Malignant)
    Recurring Karyotypic Abnormalities in Solid Tumors (Benign)
    Altered Transcriptional Control Genes in Leukemia and Lymphoma


PRINCIPLES OF CYTOGENETICS

  • Normal Expression of Growth Regulating Genes
  • Gain of Function Mutation (activating gene mutation)
  • Loss of Function Mutation (predisposing gene mutation)
  • Dominant Negative Mutation
  • Imprinting
  • Tumor Suppressor Mutation

TERMINOLOGY OF NEOPLASIA

  • Proto-Oncogene
    - Normal positive regulation of cell growth/proliferation, survival and cell death
  • Oncogene
    - Inappropriate activation of normal growth promoting genes or cellular proto-oncogenes
    - Retroviral insertion
    - Insertion, deletion, or substitution of nucleotides
    - Chromosomal translocation(s)
  • Tumor Suppressor Gene
    -Normal negative regulation of cell growth/proliferation, survival and cell death
  • Mutated Tumor Suppressor Gene
    - Malignant transformation requires disruption of both gene copies
    - Deletion or inactivation of gene(s)
    - One functional gene leads to normal phenotype
    - Both genes mutated leads to unregulated growth, and then to malignant transformation
    - Knudson "two-hit" model of carcinogenesis (retinoblastoma):
    a) Development of tumor at young age
    b) Familial predisposition
    c) Amplification and penetrance increased
  • Novel Fusion Protein
    - Chromosomal translocation
    - Involve activation of transcription factors leads to:
    a) Disrupted control of transcription
    b) Activation of inappropriate gene
  • Genes Controlling DNA Replication and Repair
    - Mismatch DNA repair system gene (hMSH2, hMLH1, hPMS1, hPMS2):
    a) Scan newly formed DNA
    b) Remove erroneously incorporated base pairs
    c) Reconstruct corrected DNA molecule
    - Inactivation of both genes leads to tumorigenesis:
    a) Hereditary nonpolyposis colon cancer
  • Gene Amplification
    - Increased copies of a gene (normal 2 copies per cell)
    - Detected by Southern blot, quantitative DNA-PCR, FISH, RT-PCR,
    a) Comparative genomic hybridization, Spectral karyotyping (SKY)
  • Structural or Numerical Chromosomal Abnormalities
    - Chromosome number
    - Abnormalities of individual chromosomes (monosomy, trisomy)
    - Deletions of portions of chromosome or entire chromosome
    - Translocations of portions of one chromosome to another chromosome
    - Double minutes: small extrachromosomal segments of DNA existing in pairs in metaphase spread
    - Homogeneous staining regions: tandem repeats of DNA segments incorporated into chromosome, lacks banding pattern on staining
  • Loss of Heterozygosity
    - Detected only when two copies of particular allele or genetic sequence can be distinguished with respect to parental origin (heterozygosity)
  • Imprinting
    - Normal event by an epigenetic modification (imprint) placed on DNA of each allele on a specific gene during gametogenesis
    - Only one parental gene activated, while other gene inactivated, i.e. X-linked inactivation in female
    - In a particular cell type expression of paternal copy of particular gene, maternal gene transcriptionally silent
    - Many imprinted genes: growth factors or growth control factors
  • Loss of Imprinting
    - Expression of parental allele that is normally transcriptionally silent
    - Re-expression of normally silent maternal IGF-2 gene with altered pattern in methylation of IGF-2 (embryonal rhabdomyosarcoma and sporadic Wilms tumor)

 

 

 

 IMPRINTED GENE ALTERATIONS IN TUMORS

Tumor Chromosome  Alteration  Allele
Wilms tumor
Rhabdomyosarcoma
Osteosarcoma
Acute Myeloid Leukemia
Neuroblastoma
Neuroblastoma
11
11
13
7
1
2
LOH
LOH
LOH
LOH
LOH
Amplified
maternal
maternal
maternal
paternal
maternal
paternal

 11p15 Gene (imprinting abnormality) and Tumor Formation
 IGF2 Wilms Tumor, Rhabdomyosarcoma, Hepatoblastoma, Bladder Cancer, Cervical Cancer, Prostate Cancer, Testicular Cancer, Breast Cancer, Choriocarcinoma, Ovarian Cancer

 

CHIMERIC TRANSCRIPTIONAL CONTROL GENES IN CHILDHOOD SOLID TUMORS

Rhabdomyosarcoma (Alveolar)

t(2;13)(q35:q14)
t(1;13)(p36;q14 )

PAX3/FKHR
PAX7/FKHR

Ewings Sarcoma Family
of Tumors

t(11;22)(q24;q12)
t(21;22)(q22;q12)
t(7;22)(p22;q12)
 
t(17;22)(q21;q22)
t(2;22)(q33;q12)

EWS/FLI
EWS/ERG
EWS/EVT1
EWS/E1A-F
EWS/FEV

Desmoplastic Round Cell Tumor

t(11;22)(p13;q12)

EWS/WT1 

Melanoma of Soft Parts

t(12;22)(q13;q22) 

EWS/ATF1 

Congenital Fibrosarcoma

t(12;15)(p13;q25)

ETV6/NTRK3

Congenital Mesoblastic Nephroma

t(12;15)(p13;q25)

ETV6/NTRK3

Liposarcoma (Myxoid and Round Cell)

t(12;16)(q13;p11)
t(12;22)(q13;q12)
 

FUS/CHOP
EWS/CHOP

Myxoid Chondrosarcoma, Extraskeletal

t(9;22)(q22;q31) 

EWS/CHN 

Synovial Sarcoma 

t(X;18)(p11.2;q11.2)
t(X;18)(p11;q11)
t(5;18)(q11;q11)

SYT/SSX1,3
SYT/SSX2
SYT/?

Hemangiopericytoma

t(12;19)(q13;13) 

  ?

Adenoid Cystic Carcinoma

t(6;9)(q21-23;p13-22) 

  ?

Mucoepidermoid Carcinoma

t(11;19)(q14-21;p12-13)

  ?


 

TUMOR SUPPRESSOR GENES IN NEOPLASMS

APC

5q21

Colorectal Cancer, Leukemia, Lymphoma, FAP

NF1

17q11

Schwannoma, PNET, Glioma, Melanoma, Neuroblastoma, Pheochromocytoma, Juvenile CML, Sarcomas, NF-type 1

NF2

22q12

Schwannoma, Meningioma, Melanoma, Astrocytoma, NF-type 2

p16ink41

9p21

Astrocytoma, Glioma, Pheochromocytoma, Osteosarcoma, Leukemia, Familial Melanoma, Dysplastic Nevus, Pancreatic Cancer

Rb-1

13q14

Retinoblastoma, Sarcomas  

p53

17p13

Glioma, Sarcoma, CML, Burkitts Lymphoma,
Leukemia, Anaplastic Wilms Tumor, Li-Fraumeni Syndrome
 

VHL

3p25.5

Hemangiopericytoma, Pheochromocytoma, VHL

WT1

11p13 

Wilms Tumor, WAGR Syndrome, Deny-Drash Syndrome

WT2

11p15

Wilms Tumor, Hepatoblastoma

 ??

11p15

Embryonal Rhabdomyosarcoma

CDC2L1

1p36

Neuroblastoma 

p16arf

9p21

Familial Melanoma/Dysplastic Nevus

PITSLRE

1p36

Familial Melanoma/Dysplastic Nevus

CDK4

12q14

Familial Melanoma/Dysplastic Nevus

PTEN

 

Cowden Syndrome, Breast Carcinoma, Prostate
Carcinoma, Follicular Thyroid Carcinoma, Glioma

PTC

9q22

Gorlin Syndrome, Basal Cell Carcinoma, Medulloblastoma

BRAC-1

17q21

Familial Breast and Ovarian Cancer; Colon, Endometrial and Gastric Carcinomas

RET

10q11

MEN type 2, Thyroid and parathyroid carcinomas, pheochromocytoma

ATM

11q22-23

Lymphoma, Lymphoid Leukemia, Breast Cancer

BLM

16q26.1

Leukemia, Lymphoma, Gastric & Colon Cancer, Bloom Syndrome

MSH2

2p21-22

Colorectal, Endometrial, Ovarian, and Gastric Carcinomas, HNPCC Syndrome 

MLH1

3p21

Colorectal, Endometrial, Ovarian, and Gastric Carcinomas, Glioblastoma, HNPCC Syndrome

PMS1

2q31-33

Colorectal, Endometrial, Ovarian, and Gastric carcinomas, HNPCC Syndrome

PMS2

7p22

Colorectal, Endometrial, Ovarian, and Gastric Carcinomas, Glioblastoma, HNPCC Syndrome

TSC1
TSC2

9q34.3
16p13.3

Angiomyolipomas, Cardiac Rhabdomyoma, Subependymal Astrocytoma, Cortical Tubers, Retinal Hamartoma, Tuberous Facial Angiofibroma, Sclerosis 

PPP2R1B

11q22-24

Colon and Lung Carcinoma

 

RECURRENT ONCOGENES AMPLIFIED IN TUMORS

    Oncogene

    Locus

Tumor

 Frequency

MYCN

2p24

Neuroblastoma
Rhabdomyosarcoma, Alveolar

 20-25%

MYC

8q22

Small Cell Cancer, Lung
Esophageal Cancer
Cervical Cancer
Ovarian Cancer
Head & Neck Cancer

 15-20%
38%
25-50%
20-30%
7-10%

MYB

6q23

Colorectal Cancer

 15-20%

EGFR (erb-b1)

7p12

Glioblastoma  
Head and Neck Cancer

 33-50%
10%

EGFR2

17q11

Breast Cancer 
Ovarian Cancer

20%
15-30%

FGFR1

8p12

Breast Cancer

12%

FGFR2

10q25

Breast Cancer

12%

Cyclin D1

11q13

Breast Cancer
Esophageal Cancer
Hepatocellular Cancer
Head and Neck Cancer

15-20%
25%
13%
50%

Cyclin E

19q12

Gastric Cancer

15%

kRAS

12q13

Colorectal Cancer
Gastric Cancer

22%
10%

hRAS

 11p15

Colorectal Cancer

29%

AKT2

 19q13

Ovarian Cancer

12%

MDM2

12q14

Sarcoma

10-30%

cdk4

12q14

Sarcoma

11%


 

ONCONGENES: TYPES AND NEOPLASIA

ONCOGENE
TUMOR(S) PROTO-ONCOGENE

Growth Factors

    v-sis
    int 2
    KS3
    HST
    int I

    Glioma/Fibrosarcoma
    Breast Cancer
    Kaposií»s Sarcoma
    Gastric Cancer
    Breast Cancer

    PDGF
    FGF
    FGF
    FGF
    ? growth factor

Non-Protein Kinase Activity Receptor

mas
Breast Cancer  Angiotensin receptor 

Tyrosine Kinase (integral membrane proteins, growth factor receptors)

    EGFR
    v-fms
    v-kit
    v-ros
    MET
    TRK

    NEU

    RET

    Squamous Carcinoma
    Sarcoma
    Sarcoma
    Sarcoma
    Osteosarcoma
    Colon cancer
    Neuroblastoma
    Breast Cancer
    Neuroblastoma
    MEN 2A, 2B, Thyroid Cancer

    protein kinase-EGFR
    protein kinase-CSF1R
    protein kinase-stem cell
    protein kinase
    protein kinase-HGF/SFR
    protein kinase - NGFR

    protein kinase

    protein kinase-GDNFR

Tyrosine Kinases (non-receptor)

    SRC
    v-yes
    v-fgr
    v-fps
    v-fes
    BCR/ABL

    Colon Cancer
    Sarcoma
    Sarcoma
    Sarcoma
    Sarcoma
    CML (leukemia)

    protein kinase
    protein kinase
    protein kinase
    protein kinase
    protein kinase
    protein kinase

Membrane Associate G-Proteins

    H-Ras
    K-Ras
    N-Ras
    gsp
    gip

    Colon, Lung, Pancreas Cancer
    AML, Thyroid Cancer, Melanoma
    Carcinoma, Melanoma
    Thyroid Carcinoma
    Ovary, Adrenal Carcinoma

    GTPase
    GTPase
    GTPase
    GTPase
    GTPase
    GTPase

GEF Family of Proteins

    Dbl
    Ost
    Tiam-1
    Vav
    Lbc

    Diffuse B-Cell Lymphoma
    Osteosarcoma
    T-Cell Lymphomas Hematopoietic Neoplasia
    Myeloid Leukemias

    GEF- Rho, CDC42H
    GEF- RhoA, CDC42H
    GEF- Rac, CDC42H
    GEF- Ras
    GEF- Rho

Serine/Threonine Kinases (cytoplasmic)

    v-mos
    v-raf
    pim-1

    Sarcoma
    Sarcoma
    T-Cell Lymphoma

    protein kinase
    protein kinase
    protein kinase

Cytoplasmic Regulators

    v-crk

     

    SH2/SH3 adaptor 

Nuclear Protein Family

    v-myc
    MYCN
    L-MYC
    v-myb
    v-fos
    v-jun
    v-ski
    v-rel
    v-ets
    v-erbA

    Myeloid neoplasia
    Neuroblastoma, Lung Cancer
    Lung Cancer
    Myeloblastosis
    Osteosarcoma
    Sarcoma
    Carcinoma
    Lymphoid Leukemia
    Myeloblastosis
    Erythroblastosis

    transcription factor
    transcription factor
    transcription factor
    transcription factor
    API transcription factor
    API transcription factor
    transcription factor
    Mutant NFKB
    transcription factor
    Mutant Thioredoxine Reductase



RECURRING KARYOTYPIC ABNORMALITIES IN SOLID TUMORS

Malignant Tumors
Karyotypic Abnormality
Bladder Cancer
Rhabdoid, brain
Breast, adenocarcinoma
Colon Cancer
Fibrosarcoma
Giant Cell Tumor
Gliomas
Renal Cell Carcinoma
Papillary Renal Carcinoma
Larynx (squamous carcinoma)
Liposarcoma
Liposarcoma, myxoid
Liposarcoma, well differentiated
Lung (squamous carcinoma)
Fibrous Histiocytoma
Melanoma, uveal
Meningioma
Nasopharynx (squamous carcinoma)
Neuroblastoma
Ovarian cancer
Prostate cancer
Retinoblastoma
Adenocarcinoma, Salivary Gland
Squamous Carcinoma, Oral
Testicular (seminoma, teratoma)
Thyroid Adenocarcinoma
Adenocarcinoma, uterus
Wilms Tumor
Mucoepidermoid Carcinoma

+7, del(10)(q22-24), del(21)(q22)
-22
-17, I(1q), der(16)t(1;16)(q10;p10)
+7, +20
-Y
+8
+7, -10; -22, -X, +X, -Y
del(3)(p14-21), del(3)(p11-14)
+7, +17, t(X;1)(p11;q21)
-Y
t(12q13-14)
t(12;16)(q13;p11)
+r(12)
del(3)(p14p23), +7
-Y
-3, partial deletion 3, +8; I(8q)
-22, +22, -Y, del(22)(q11-q13)
-Y
del(1)(p32-36)
+12, +7, +8, -X
del(10)(q24), +7, -Y
I(6p); del(13)(q14.1q14.1)
del(6)(q22-25); +8; -Y
+7
I(12p), +12p
I(10)(q11q21)
+10
del(11)(p13p13); del(11)(p15p15)
del(6q)



RECURRING KARYOTYPIC ABNORMALITIES IN SOLID TUMORS

Benign Tumors
Karyotypic Abnormality
Kidney Adenoma
Thyroid Adenoma
Pleomorphic Adenoma, Salivary
Ovarian Thecoma/Adenoma/Fibroma
Lipoma

Atypical Lipoma
Lipoblastoma
Spindle Cell Lipoma
Leiomyoma



Myofibroma (myofibroblastic tumors)
Giant Cell Tumor
Barrett Esophagus
Neurinoma

+7, +17, -Y
+5, +7, +12, Structural Changes in 19q
8q12, 12q15, Structural changes 3p21
+12
t(12q13-15), t(6p),
Structural Changes 13q
+r(12)
der(8)(q11-13)
-16q13->qter
t(12q13-15)
Rearrangement of 6p
del(7)(q21q31)
+12
+8
+8
-Y
Structural Changes 22; -Y



ALTERED TRANSCRIPTIONAL CONTROL GENES IN LEUKEMIA AND LYMPHOMA

LYMPHOID LEUKEMIA

 

B-Cell ALL/Burkitts

t(8;14)(q24;q32)
t(2;8)(p12;q24)
t(8;22)(q24;q11)

MYC
MYC
MYC

 

Pre-B Cell ALL

t(17;19)(q22;p13)
t(1;19)(q23;p13)
t(4:11)(q21;q23)
t(9;22)(q34;q11)
t(12;21)(p13;q22)

E2A-HLF
E2A-PBX1
MLL-AF4
BCR-ABL
TEL-AML1

 

T-Cell ALL

t(8;14)(q24;q11)
t(7;19)(q35;p13)
t(1;14)(p32;q11)
t(7;9)(q35;q34)
t(11;14)(p15;q11)
t(11;14)(p13;q11)
t(7;11)(q35;p13)
t(10;14)(q24;q11)
t(7:10)(q35;q24)
t(9;22)(q34;q11)

MYC
LYL1
TAL1 (SCL)
TAL2
LMO1 (RBTN1)
LMO2 (RBTN2)
LMO2 (RBTN2)
HOX11
HOX11
BCR-ABL

DIFFUSE B-CELL LYMPHOMA

 

(Large Cell)

t(3;14)(q27;q32)
t(3;4)(q27;p11)

BCL6
BCL6
 

Anaplastic

t(2;5)(p23;q35)

NPM-ALK

 

t(10;14)(q24;q32)

LYT10

B-CLL

t(14;19)(p32;p13)

BCL3

B-CELL LYMPHOMA

t(10;14)(q24;q32)

LYT10

LYMPHOPLASMACYTOID B-CELL LYMPHOMA

t(9;14)(p13;q32)

PAX5

MYELOID LEUKEMIA

AML, granulocytic

t(8:21)(q22;q22)
t(9;22)(q34;q11)

AML1-ETO
BCR-ABL

Myelodysplasia

t(3;21)(q26;q22)

AML-EAP

CML, Blast Crisis

t(3;21)(q26;q22)
t(9;22)(q34;q11)
 

AML-EV11
BCR-ABL

AML, undifferentiated

t(3;v)(q26;v)
t(9;22)(q34;q11)

EV11
BCR-ABL

AML, myelomonocytic

inv(16)(p13;q22) 

CBFbeta-MYH11

AML, monocytic

t(9;11)(p21;q23)

MLL-AF9

AML, promyelocytic

t(15;17)(q21;q21)
t(11;17)(q23;q21)
t(5;17)(q32;q12)

PML-RARalpha
PLZF-RARalpha
NPM-RARalpha

AML, undifferentiated

t(16;21)(p11:q22)
t(12;22)(p12;q11)

FUS-ERG
TEL-MN1

MIXED LINEAGE LEUKEMIAS

Pro-B Cell ALL

t(4;11)(q21;q23)

MLL-AF4

AML (monocytic)

t(9;11)(p21;q23)

MLL-AF9

ALL/AML

t(11;19)(q23;p13.3)

MLL-ENL

AML

t(11;19)(q23;p13.1)
t(1;11)(q21;q23)
t(1;11)(p32;q23)
t(6;11)(q27;q23)
t(10;11)(p12;q23)
t(11;17)(q23;q21)
t(X;11)(q13;q23)

MLL-ELL
MLL-AF1Q
MLL-AFIP
MLL-AF6
MLL-AF10
MLL-AF17
MLL-ARX1