Clinical Cytogenetics Flashcards
Chromosome and FISH studies are important for investigating the causes of (2)
Leukemia and Lymphoma
Chromosome microarray is standard of complimentary to chromosome and FISH studies for who?
Individuals with developmental delays (including multiple congenital anomalies, autism, intellectual disabilities)
Specimens for Cytogenetic Studies (4)
Bone Marrow
Blood
Tissue
Fluids
What tissues are used in cytogenetic studies for cancer diagnostics (5)
Bone marrow Unstimulated Blood Lymph node tissue Solid tumor tissue CNS fluid
When we are looking and leukemia and lymphoma, we usually see block in 3 points of differentiation in hematopoietic lineages, what are they?
L blast –> Lymphocyte
Pro-myelocyte –> myelocyte (neutrophil lineage)
Megakaryocyte –> platelet
What are two common cytogenetic findings in childhood B-cell leukemia?
Low hypodiploidy
High hyperdiploidy
Low hypodiploidy or near triploidy in Childhood B-cell acute lymphocytic leukemia is associated with what prognosis
Poor Prognosis
Poor prognosis in Childhood B-Cell ALL is associated with what cytogenic finding?
Low hypodiploidy or near triploidy
In cases of pediatric B-cell acute lymphocytic leukemia (ALL) what is associated with a good prognosis?
High hyperdiploidy (e.g. >50) - specifically 4,10,17
*but not that near triploidy is associated with poor
What childhood disease has cytogenetic studies helped to improve dramatically (i.e. 95% remission)
Childhood ALL
So in addition to diagnosis, cytogenetics is important in prognosis and treatment protocol
One example being Childhood ALL
If Hypodiploid ?
If Hyperdiploid?
Hypo - poor prog –> high dose chem
Hyper - good prog –> low dose chem
When we do cytogenetic studies, in addition to finding cells with abnormal chromosomal constitutions we also find cells with normal 46XXorY constitutions, what is this called?
Somatic mosaicism
Suppose a 3 year old presented with leg and arm pain / fever / abdomen distension / and 48% BLASTS? What would this be indicative of?
ALL
Blasts should be no more than 1%
FISH =
Florescence in situ hybridization
What does FISH do?
Specific, cloned DNA sequences can enumerate number of a specific chromosome OR identify translocation!
Types of FISH probes (5)
Centromere Locus specific Dual fusion/fusion Break apart Whole chromosome paint
Chromosome analysis of ______________ is performed to detect common anomalies associated with leukemia and lymphoma
Bone marrow
What studies can reveal the high hyperdiploidy in Childhood B-Cell ALL?
chromosome and FISH analysis
What are two of the most common leukemia translocations?
t(9;22) is diagnostic for chronic myelogenous leukemia (CML) - which can be treated with tryrosine kinase inhibitors
t(15;17) is diagnostic for a specific acute promyeloid leukemia (APML), which can be treated with retinoic acid
Centromere FISH probes are used for___________
example?
Enumeration
All panel
Locus specific FISH probes are used for?
Deletion / duplication detection
e.g. p53
Dual fusion / fusion (DF, F) FISH probes are used for?
example (2)
Translocation detection
BCR;ABL
PML;RAR
Break apart FISH probes are used for?
Translocation rearragement
MLL
Whole chormosome paint FISH probes are used for?
Identifying markers / translocations
WCP 1-22,X,Y (all studies)
Two significant effects of translocations
Altered gene expression (e.g. translocated next to a strong enhancer or promoter)
Creation of novel fusion product
What kind of probe would we use for BCR;ABL FISH?
Dual fusion
What does it mean for a translocation to be cryptic and what do we need to detect?
Cryptic translocations result in normal looking karyotypes because translocated regions were equal in size and I would assume had similar banding
Thus, we need FISH to detect these
e.g. t(12;21)(p13;q22) in ALL
Acute Myelogenous Leukemia
Do we see this in adults or kids?
More frequently adults but also in children/young adults
What are the two chromosomal aberrations that we focused on?
t(15;17)(q22;q21) PML-RARalpha (4.1%)
t(9;22)(q34;q11) ABL1;BCR (0.7%)
What are auer rods?
They show up on path slides, they are suggestive of myeloid disorder - acute promyelocytic leukemia (APML)
What is the 9;22 tranlocation called?
Philidelphia chromosome
What is the t(9;22) consistent with?
Diagnosis of chronic myelogenous leukemia
What chromosomal aberration is characteristic of APML (Acute promyelocytic Leukemia)?
PML/RAR rearrangement
t(15;17)
What is the consequence of PML/RARa rearrangement?
Myelodi Hematopoietic precursor differentiation blockage
The PML-RARa fusion protein seems to interrupt the differentiation of myeloid hematopoietic precursors past the promyelocytic stage
How do we treat patients with PML/RARa
Treatment of APML patients with retinoic acid apparently overcomes the inhibition of the fusion protein and allows differentiation of myeloid precursors
What is a common secondary cytogenetic abnormality in cases with t(15;17)
Trisomy 8
What common secondary cytogenetic abnormalities do we see in CML
Trisomy 8
Why does BCR;ABL yield cancer?
The activity of tyrosine kinases is typically controlled by other molecules, but the mutant tyrosine kinase of the BCR-Abl transcript codes for a protein that is “always on” or continuously activated, which results in unregulated cell division (i.e. cancer).
Imantinib (Gleevac)
Treats CML
Binds ATP binding site in abl tyrosine kinase and bcr/abl tyrosine kinase
Inhibits cell proliferation
DS infants and children are at elevated risk for which cancers?
20-100x for ALL or AML
500x for AMKL
Note: FISH studies only demonstrate gain of 21 sequences - so maybe something else going on?
CMA - what is on slide?
Target DNA - single stranded oligomers
CMA - what does it detect?
Gains and Losses (ONLY!)
Can CMA detect re-arrangements?
NO
Can CMA detect mosaicism
Limited (10-15%)
Compare and Contrast
Cytogenetics and CMA
What are we screening?
Both screen the genome
Compare and Contrast
Cytogenetics and CMA
What phase are we screening?
Cytogenetics - mitotic
CMA - interphase
Compare and Contrast
Cytogenetics and CMA
What cells do we screen?
Cyto - selected cells
CMA - all cells
Compare and Contrast
Cytogenetics and CMA
What are we looking for?
Cyto - Gain/Loss and Balanced Rearrangement
CMA - Gain/Loss only
Compare and Contrast
Cytogenetics and CMA
Accuracy
Cyto - Technologist expertise
CMA - technology
What are runs of homozygosity?
Runs of homozygosity (ROH) are long stretches of consecutive homozygous genotypes probably reflecting segments shared identically by descent as the result of processes such as consanguinity, population size reduction, and natural selection.
CMA can detect what?
Runs of homozygosity because they contain SNPs
What is the advantage of chromosomal microarrays?
Detects chromosomal gains and losses, some of which my be “Submicroscopic” -
One array = 850,000 FISH studies
Intensity and allelic imbalance
Chromosomal microarrays detects abnormalities in known hot spots - what are known hot spots?
Areas of known genetic disease
e.g. 22q11.2 deletion
Chromosomal microarrays can be used to characterize chromosome abnormalities detected by karyotyping - what does this mean?
Can detect specific size of imbalance and genes involved
Current CMAs use ________ based platform to interrogate over 850,000 regions of the genome
SNP
CMA methods
What is our sample, usually?
Peripheral blood
CMA methods - what are we comparing
Spots/Color intensity of sample DNA to 50 normal individuals
CMA methods - the SNP platform provides information on intensity and on runs of homozygosity, which might possibly reveal?
Autosomal recessive conditions
CMA frequently reveals ________-and _____________ of genetic material that cannot be seen by standard cytogenetics and light microscopy
deletions and duplications
Copy number variants
duplications or deletions within the DNA for which there can be associated pathology or no consequence - depending on location and size
Can you detect copy number variants at the chromosomal level?
NO
CNV are there regions of known clinical significance?
Yes
There are regions known to be del/dup associated with Mendelian disorders
Also Tumor susceptible loci
CMA Reports
Determine what?
Size and location of deletion/duplication (most >200Kbp) –> leads to further investigation of data base
CMA reports - look for genomic content of region
Copy number variants?
Gene/functions
Known phenotypes
CMA reports and heritability
Is de novo or inherited?
Chromosomal microarray - drop in florescent signal at given chromosomal region indicates?
Dropout - deletion
Loss of segment of chromosome DNA
If possible, what do we always follow up CMA test with?
FISH Confirmation
So if a child comes in with developmental delay, autism, and dysmorphic features, we first do A and then do B lastly C
A. CMA
B. Metaphase FISH confirmation
C. Look at database of genomic variants for relevant deletions / diseases
CMA reports determine _ and _ and are supported by multiple levels of __
size and location of deletions and duplication and are supported by multiple levels of SNP
CMA lab reports thresholds
Deletion >200kb
Duplications>400 kb
smaller findings in clinically significant regions
CMA lab reports ROH
Evaluated at >5Mb with reporting threshold 10Mb
Total % ROH reported if >5%
CMA cannote detect (3)
low level mosaicism
heterodisomy
balanced chromosome rearrangements
CMA reports
Genomic content of regions (2)
Copy number varians
Genes/functions
CMA reports
Heredity?
De novo or inherited
Can we see point mutations with fish?
No, probes are 200kb so would still hyrbidize? strange since it is SNP platform
