Cytogenetics

CYTOGENETICS OF SOFT TISSUE TUMORS:
PRINCIPLES AND DIAGNOSTIC CONSIDERATIONS
WITH SPECIAL REFERENCE TO PEDIATRIC NEOPLASIA

John Hicks
Texas Children’s Hospital and
Baylor College of Medicine, Houston, Tx

INDEX:
PRINCIPLES OF CYTOGENETICS
TERMINOLOGY OF NEOPLASIA
IMPRINTED GENE ALTERATIONS IN TUMORS
CHIMERIC TRANSCRIPTIONAL CONTROL GENES IN CHILDHOOD SOLID TUMORS
TUMOR SUPPRESSOR GENES IN NEOPLASMS
RECURRENT ONCOGENES AMPLIFIED IN TUMORS
ONCONGENES: TYPES AND NEOPLASIA
RECURRING 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 and 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 Kaposi’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