FINALS LEC: MOLECULAR DETECTION: ONCOLOGY Flashcards by Chin Lys

Study of tumors/neoplasm  growth of tissue that exceeds and is not coordinated with normal tissue

ONCOLOGY

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not recurrent

benign

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invasive & tending to recur at multiple sites (cancer)

malignant

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movement of tumor cells from the original (primary) site of the tumor to other locations

matastasis

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suffix, to the tissue of origin

-Oma

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study of cancer at the molecular level

Molecular oncology

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An abnormal mass of tissue that usually does not contain cysts/liquid areas

SOLID TUMORS

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tumor of epithelial origin

Carcinoma

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tumor of bone, cartilage, muscle, blood vessels, or fat

sarcoma

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multiple cell types

teratocarcinoma

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Abbormal cells in the blood grow out of control

HEMATOLOGICAL MALIGNANCIES

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large nos. of WBCs populate the bone marrow & peripheral blood

Leukemia

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neoplasm of lymphocytes that form discrete tissue masses

Lymphoma

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Plasma cell neoplasms: abnormal plasma cells/cells form tumors in the bones/soft tissues of the body

HEMATOLOGICAL MALIGNANCIES

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is caused by nonlethal mutations in DNA affecting 2 types of genes that control the cell division cycle and cell survival

cancer

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2 types of genes that control the cell division cycle and cell survival:

Oncogenes
Tumor-supressor genes

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● Promote cell division ● Include cell membrane receptors, that are bound by growth factors, hormones, and other extracellular signals
● Support cell survival by inhibiting apoptosis
● >100 in the human genome

Oncogenes

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● Factors that control transcription/translation of genes required for cell division
● Participate in repairing DNA damage & in promoting apoptosis
● Slow down/stop cell division by counteracting the movement of the cell from G1-to S (G1 checkpoint) or G2 to M phase (G2 checkpoint)
● >30 in the human genome

Tumor-supressor genes

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IN CANCER CELLS

Gain-of-function mutations resulting from:
 Amplification/translocation of DNA regions containing the genes
 Activating mutations that cause aberrant activity of the proteins

Oncogenes

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IN CANCER CELLS

Loss-of-function genes mutations  Inactivation of the gene products (deletion, translocation, mutation of the genes)

Tumor-supressor genes

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Molecular diagnosis and treatment of cancer cells:

Detection of abnormalities in specific tumor suppressors or oncogenes

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ANALYTICAL TARGETS OF MOLECULAR TESTING:

Tissue specific targets
Tumor-specific targets

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● Molecular characteristics of the tissue from which a tumor arose
● Detection and monitoring the presence of tumor  abnormal amounts or locations of DNA, RNA, proteins or other molecules from these targets
● Examples: DNA/RNA from
1. Cytokeratin genes in gastric cancer 2. Carcinoembryonic antigen in breast cancer

Tissue specific targets

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● Genetic structures resulting from mutations in oncogenes & tumorsuppressor genes that are associated with tumor development
● Solid tumors, leukemias, lymphomas ● Mutated cell free nucleic acid/circulating tumor cells can be detected in blood & other body fluids
● Examples: surface receptors, mutated proteins, tumor-associated antigen, metabolic markers

tumor-specific targets

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 Tyrosine-kinase activity  Normal activity: cell growth and division  Overexpressed in 25%-30% of human breast cancers

Human epidermal growth factor receptor 2, HER/neu/erb-b2 1 (17q21.1)

Detection methods of Human epidermal growth factor receptor 2, HER/neu/erb-b2 1 (17q21.1)

IHC SOUTHERN, NORTHERN, WESTERN BLOT FISH Chromogenic in situ hybridization (CISH) Silver-enhanced in situ hybridization (SISH)

uses antibodies (fresh/frozen tissues)

IHC

gene amplification  increased RNA & proteins

Southern, northern, western blots

direct detection of increased copy nos. of the gene in DNA

FISH

standard bright-field microscope, genome & chromosome mutation detection

Chromogenic in situ hybridization (CISH)

 Normal activity: cell signaling pathways that control cell division and survival  Overexpressed in solid tumors

Epidermal growth factor receptor, EFGR (7p12)

Detection methods of Epidermal growth factor receptor, EFGR (7p12)

IHC qPCR SSP-PCR, SSCP, direct sequencing, NGS

to assess gene copy no. through increased gene expression

qPCR

mutations

SSP-PCR, SSCP, direct sequencing, NGS

 Small GTP-binding proteins that receive signals from the cell surface proteins & activate the initial steps of the mitogen-activated protein kinase (MAPK) pathway cascade  Gain-of-function mutations in ras protooncogenes  cancers  Mutations in these genes are the most common oncogene mutations in human cancers (codons 12, 13, 22, & 61 in exons 2 & 3 of the KRAS gene)  Detection methods of mutation: direct sequencing, pyrosequencing

Kirsten rat sarcoma viral oncogene homolog, K-ras (12p12); neuroblastoma ras, Nras (1p13); & Harvey rat sarcoma viral oncogene homolog, H-ras (11p15)

 Ewing sarcoma: group of tumors arising from primitive neuroectodermal tissue (PNET)  Diagnosis and prognosis: a. cytogenetic methods b. molecular methods: RT-PCR, with amplification control (GAPDH or 18S RNA) on tissue/liquid biopsies

Ewing sarcoma, EWS (22q12)

group of tumors arising from primitive neuroectodermal tissue (PNET)

Ewing sarcoma

 Detection methods: a. FISH b. RT-PCR c. Semi-nested PCR d. Agarose gel electrophoresis & EtBr staining (PCR product detection)

Synovial sarcoma translocation, chromosome 18 – Synovial sarcoma breakpoint 1 & 2, SYT-SSX1, SYT-SSX2 t(X;18) (p11.2;q11.2)

rare type of cancer of the muscle, fat, fibrous tissue, blood vessels, or other supporting tissue of the body

Synovial sarcoma

 Detection methods: FISH, RT-PCR, qPCR, RNA sequencing

Paired box-fokhead in Rhabdomyosarcoma, PAX3-FKHR, PAX7-FKHR, t(1;13), t(2;13)

most common soft tissue sarcoma of childhood (10% of all solid tumors in children)  alveolar RMS, embryonal (RMS-E), & primitive (RMS-P)

Rhabdomyosarcoma (RMS)

 Mutations in this gene is found in all types of cancers and also aids in the diagnosis of LiFraumeni syndrome  Detection methods: IHC, microarrays/panel sequencing, SSCP, direct sequencing

Tumor protein 53, TP53 (17p13)

gene product of TP53, DNA-binding protein that controls expression of other genes and participates also in the arrest of cell division in the event of DNA damage

p53

 Carriers (autosomal recessive mutations)  develops leukemia (B-cell lymphocytic leukemia, T-cell prolymphocytic leukemia), lymphoma (mantle cell lymphoma), or other types of cancers  Detection methods: direct DNA sequencing, SSCP, functional test for the repair of dsbreaks induced by irradiation, test for other family members

Ataxia telangiectasia mutated gene, ATM (11q22)

DNA repair and/or control of the cell cycle

ATM protein

neurological disorder that affects the part of the brain that controls motor movement & speech; predisposition to cancer

Ataxia telangiectasia (AT)

 Gene products are involved in DNA dsbreak repair by homologous recombination  Screening tests for mutation: SSCP, protein truncation testes, chromosome breakage tests, etc.  Clinical application method: direct sequencing  Other detection methods: SSP-PCR, allelespecific oligomer hybridization

Breast cancer 1 gene, BRCA1 (17q21), & breast cancer 2 gene, BRCA2 (13q12)

mutation in BRCA1 (60%-80% lifetime risk of breast/ovarian cancer)

Women

mutation in BRCA2 (increased risk of breast/colon/prostate cancer)

Men

Functions as a tumor-suppressor gene, promoting cell differentiation

von Hippel-Lindau gene, VHL (3p26)

 genetic condition involving abnormal growth of blood vessels in organs (eyes, kidneys, adrenal glands, etc.)  predisposition for renal cell carcinoma & other cancers  deletions, point mutations, splice-site mutations  Somatic mutations: renal cell carcinoma & tumors of the adrenal glands  Detection method: direct sequencing

Von Hippel-Lindau syndrome

 Myc family proteins: increase the expression of several genes a. c-myc oncogene (8q24.21): amplified in breast & ovarian cancer, lymphomas, & leukemias b. l-myc (1p34.2): amplified in oral cancer c. n-myc (2p24): amplified in neuroblastoma & retinoblastoma  Detection methods: IHC, FISH, sequencing, array analysis, transcription of n-myc through qPCR

V-myc myelocytomatosis viral-related oncogene, neuroblastoma-derived, MYCN or n -myc (2p24)

coding for a membrane receptor tyrosine kinase  rearranged in a variety of human cancers (1%-3% lung adenocarcinomas)

ROS1 oncogene

its gene product participates in sending signals to the nucleus

RET proto-oncogene

 ALK: receptor tyrosine kinase  Chromosomal rearrangements (most common), mutations, or amplifications tumors (lymphomas, neuroblastoma, & nonsmall cell lung cancer)  Detection methods: FISH & sequencing

Anaplastic lymphoma receptor tyrosine kinase (ALK) proto-oncogene, 2p23.1

receptor tyrosine kinase

ALK

 Kit protein: transmembrane receptor with tyrosine kinase activity  Gene mutation: gastrointestinal stromal tumors (GISTs), mast cell disease, & AML  Detection methods: a. IHC: increased KIT protein b. Sequencing: missense mutations

V-Kit Hardy-Zuckerman 4 feline sarcoma oncogene homolog, KIT, c-KIT (4q12)

transmembrane receptor with tyrosine kinase activity

Kit protein

gastrointestinal stromal tumors (GISTs), mast cell disease, & AML

Gene mutation

 Contraction & expansion of nucleotide repeat sequences in DNA MICROSATELLITE INSTABILITY (MSI)  Cause: dysfunction of 1 or more components of mismatch repair (MMR) systems  Lynch syndrome or hereditary nonpolyposis colorectal cancer (HNPCC)  Inherited cancer predisposition syndrome, 3%-5% of colon cancers & 3% endometrial cancers  Increased risk of developing other cancers  MMR mutations in MSH2 & MLH1 genes  Detection methods: direct sequencing, IHC, MSI analysis, PCR, gel/capillary gel electrophoresis (more bands/peaks in the tumor tissue than the normal)  National Cancer Institute: recommended the screening of BAT25 & BAT26; & D5S346, D2S123, & D17S250 for MSI determination

MICROSATELLITE INSTABILITY (MSI)

 Inherited cancer predisposition syndrome, 3%-5% of colon cancers & 3% endometrial cancers  Increased risk of developing other cancers  MMR mutations in MSH2 & MLH1 genes

Lynch syndrome or hereditary nonpolyposis colorectal cancer (HNPCC)

 Deletion/inactivation of a functional allele, leaving a mutated allele  Detection methods: PCR & capillary electrophoresis (amplification of heterozygous STR/VNTR loci closely linked to the disease gene)

LOSS OF HETEROZYGOSITY (LOH)

A laboratory test done on a sample of blood (plasma/serum), urine, or other body fluid to look for cells & nucleic acids released by the tumors into a person's body fluids

LIQUID BIOPSY

LIQUID BIOPSY 2 approaches:

1. Cell-free or circulating free nucleic acids 2. Circulating tumor cells (CTC)

1. Cell-free or circulating free nucleic acids Targeted:

directed at specific mutations (sequencing/PCR methods)

1. Cell-free or circulating free nucleic acids Untargeted:

whole exomes or gene panels (sequencing & CGH)

most frequent source material

Plasma DNA

Mechanism of tumor metastases from 1 tissue site to another (2 to >50 cells)

Circulating tumor cells (CTC)

2. Circulating tumor cells (CTC) Isolation methods:

antibody capture, negative depletion of leukocytes

2. Circulating tumor cells (CTC) DNA:

allele-specific PCR or other sensitive methods

Series of intrachromosomal recombination events mediated by recombinase enzymes that recognize specific sequences flanking the gene segments

GENE REARRANGEMENTS

Normal intrachromosomal breaking & specific sequences flanking joining of DNA in the genes coding for immunoglobulins and T-cell receptors

V(D)J RECOMBINATION

 Gene encoding the immunoglobulin heavy chain is located on chromosome 14  As B lymphocytes mature, selected gene segments are joined together so that the rearranged gene contains only 1 of each V (variable), D (diversity) & J (joining) gene segments

Immunoglobulin heavy-chain gene rearrangement in B cells