The Evolution of Cytogenetics Testing
• Karyotyping has been the “Gold Standard”
• A subjective whole-genome test of limited resolution (5-10 Mb)
Whole-Genome SNP Array Analysis –
Genomic Complexity and Clinical Relevance
in Prenatal, Postnatal, and Oncology Testing
Mark Micale, Ph.D., F.A.C.M.G.
• Identifies larger chromosome abnormalities; low yield for detecting
small rearrangements associated with intellectual disability,
multiple congenital anomalies, and autism/ASD
• Identifies balanced abnormalities associated with many malignancies
• FISH is more sensitive than karyotyping, but only interrogates
a specific genomic region of interest
• Useful for suspected microdeletion/microduplications, prenatal
aneuploidy detection, detection of common
rearrangements in neoplasia, and FISH panels for
hematolymphoid disorders
Medical Director, Clinical Cytogenomics Laboratory
Department of Pathology and Laboratory Medicine
Beaumont Health System
Associate Professor of Pathology and Laboratory Medicine
Oakland University William Beaumont School of Medicine
• Chromosome microarray analysis (CMA)
with oligonucleotides or SNPs is a
whole-genome test with much
greater sensitivity than karyotyping
• Higher diagnostic yield in many patient
groups
• Does not detect balanced rearrangements
The Virtual Karyotype is Produced by Single
Nucleotide Polymorphism (SNP) Array Analysis
The Virtual (Molecular) Karyotype
Ch. 2q deletion (12.3 Mb)
Ch.5p duplication (41.8 Mb)
Genotyping
Ch. 9p (10.4 Mb) and 9q
(25.5Mb) with LOH
Ch. 6q deletion (48.5 Mb)
Ch. 11q deletions
(1.9 and 5.5Mb)
Ch.1q deletion (15.9kb) with LOH
Ch. 17q duplication
(36.8Mb)
Low-level mosaic
trisomy 8p
Copy Number
Analysis
Complex ch. 13 rearrangements with loss
and gain
B-cell non-Hodgkin lymphoma
with a normal karyotype
SNP Array Workflow
Genotyping Analysis Utilizing SNPs
• Estimated one SNP
every 100- 300 bp
• 9.2 million SNPs have
been reported
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SNP Arrays Detect Copy Number Neutral Loss
of Heterozygosity/Acquired Uniparental Disomy
Evolution of Cytogenetics Into Cytogenomics
Advantages of DNA Microarray Analysis
SNP
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B
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• Virtual karyotype for the entire genome at a very high resolution
• Does not rely on cell culture so no clonal selection; Can utilize fresh or archived tissue
• Interpretation of raw data using objective biostatistical algorithms
Mitotic
Recombination
Error Resulting
in Segmental
UPD
• Significantly higher sensitivity for detecting genomic imbalance (~25% yield at Beaumont)
• With SNP arrays, can detect constitutional or acquired uniparental disomy (cnLOH)
• A ready interface of the digital data with genome browsers and Web-based genome-annotated
databases to determine genomic content involved in imbalance permitting genotype:
phenotype correlations
Limitations of DNA Microarray Analysis
• Copy number variants of uncertain significance (an important problem for prenatal diagnostics)
• Inability to detect molecularly-balanced chromosome rearrangements (more of a problem with neoplasia)
• Inability to detect tumor-specific changes with a low ratio of tumor cells to normal cells (MRD detection)
• Inability to determine the chromosomal mechanisms of genetic imbalance (may require karyotype or
FISH studies)
• Inability to characterize clonal and subclonal populations
• With SNP arrays, can identify consanguinity
Determination of CNV Pathogenicity
SNP Array Significantly Increases the Diagnostic Yield
of Constitutional Chromosome Abnormalities
Copy Number Variant (CNV)
Benign or Pathogenic?
Recommendations Have Been Established for
the Use of CMA in Routine Clinical Testing
Most Clinically Significant “Balanced”
Chromosome Rearrangements Are Not Balanced
Genet Med 12(11), 2010
Am J Hum Genet 86, 749-764, 2010.
Genet Med 15(2), 2013
Genet Med 13(7), 2011
Genet Med advance online publication 25 April 2013
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Pediatric Genetic Evaluation Using SNP ArrayCase #1
Pediatric Genetic Evaluation Using SNP ArrayCase #1
• 20 day old male with multiple congenital anomalies including aniridia,
arthrogryposis, and cryptorchidism
• No unifying clinical genetic diagnosis
• SNP array identified an 8.6 Mb deletion involving chromosome segment 11p1311p14.2 that contains 51 genes including WT1 and PAX6.
• The deleted segment lies within the Wilms tumor-aniridia-genitourinary
anomalies-retardation (WAGR) syndrome critical region and also
overlaps the proximal portion of the 11p15-p14 deletion syndrome critical
region.
Pediatric Genetic Evaluation Using SNP Array –
Case #1
Pediatric Genetic Evaluation Using SNP ArrayCase #1
Why is this SNP Array Diagnosis Important in this Child?
• It provides a unifying genetic diagnosis – WAGR Syndrome
PAX 6 gene
• It provides an etiology for the child’s present phenotype (aniridia and
cryptorchidism)
• It provides important guidelines for medical management including:
WT1 gene
- Consultation with a pediatric oncologist for Wilms tumor surveillance by
abdominal/renal sonography until the 8th birthday
- Recognition of potential developmental issues with appropriate early
intervention
- Parental evaluation as a means for providing precise recurrence risk genetic
counseling
• It makes this child eligible for services provided by the Michigan Department of
Community Health
Pediatric Genetic Evaluation Using SNP ArrayCase #2
Pediatric Genetic Evaluation Using SNP ArrayCase #2
• 4 year-old male referred to pediatric neurology for neurological examination and
evaluation of seizures. The seizures began at 6 months of age (febrile, tonicclonic). Seizures now occur once a week and are precipitated by sickness and
lack of sleep.
• He was been seen by neurology and clinical genetics in two local institutions with no
specific diagnosis rendered.
• The child also has profound sensorineural hearing loss and presents with moderate
cognitive impairment and speech delay.
• SNP array demonstrated a 10kb deletion resulting in removal of exons 15-18 of the
SCN1A gene on chromosome 2q24.3
Deletion of
exons 15-18
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Pediatric Genetic Evaluation Using SNP ArrayCase #2
Pediatric Genetic Evaluation Using SNP ArrayCase #3
Why is this SNP Array Diagnosis Important in this Child?
• The SCN1A gene, which codes for the alpha-1 subunit of neuronal voltage-gated
sodium channels, is one of the most clinically relevant of all known epilepsy
genes. Mutations in this gene are associated with a variety of seizure disorders.
• The diagnosis of an SCN1A mutation aids in the selection of antiepileptic drugs,
as some drugs such as sodium-channel blocking agents may exacerbate
symptoms – Great example of Personalized Medicine.
• Approximately 90% of cases are de novo, while in the other 10%, the mutation is
inherited from a parent who may manifest a much milder phenotype or be a
gonadal mosaic for the mutation.
• It makes this child eligible for services provided by the Michigan Department
of Community Health
Pediatric Genetic Evaluation Using SNP ArrayCase #3
• A three year old male with congenital
hydrocephalus, partial agenesis
of the corpus callosum, and a
small ventricular septal defect.
• At the age of four months, bilateral
renal masses were identified
and thought to represent
Wilms tumor. He underwent
right radical nephrectomy
which revealed triphasic
Wilms’ tumor with favorable
histology. A needle core
biopsy on one of two lesions
on the left kidney revealed
Wilms’ tumor.
Pediatric Genetic Evaluation Using SNP ArrayCase #3
• Tumor karyotype: 46,XY,t(7;8) (q36;p11)
Constitutional karyotype: 46,XY
suggests that the t(7;8)(q36;p11) is associated with the malignancy.
• A partial left nephrectomy was subsequently performed. Margins were positive,
necessitating left flank radiotherapy.
• Following completion of treatment, MRI and CT scans revealed no evidence of
residual disease or metastatic disease. Imaging studies performed at 22 and 25
months of age revealed no evidenceof disease recurrence. He continues to be
disease free to date.
• Clinical genetic evaluation was performed at eleven months of age. The exam
revealed only borderline macrocephaly. The phenotype was not consistent
with most Wilms tumor-associated genetic syndromes.
• SNP array revealed a 560.49 kb chromosome 2p24.3 duplication involving four
genes including MYCN.
• The child’s mother demonstrated the same 560kb duplication of chromosome 2p24.3.
Prenatal Diagnostics Using CMA

Conventional karyotyping detects chromosome abnormalities in:
- 35% of pregnancies with fetal ultrasound abnormalities (All)
- 2-3% of pregnancies presumed to be “at-risk”
- 40% of prenatally ascertained heart defects (22q11.2 FISH)
- 50% of pregnancies with cystic hygroma
- 4.5-35% of pregnancies with omphalocele
- >50% with nasal bone absence
- 40% of pregnancies with nuchal fold (2nd trimester)

The clinical sensitivity of prenatal interphase FISH (for rapid aneuploidy detection) in
the AMA population approaches 80%, but falls to 65-70% for all prenatal
patients

Classical cytogenetics (karyotyping) is not good at detecting small (<5Mb) deletions
or duplications that may be clinically significant. FISH is great at detecting
such imbalance, but only if there are “clinical cues” to guide appropriate choice
of DNA probe used

But what about a fetus with U/S anomalies and a normal karyotype?
560.49 kb chromosome
2p24.3 duplication
• The child’s health insurer initially denied authorization for CMA on the basis that
“there is insufficient evidence in the peer-reviewed literature to demonstrate the
clinical/therapeutic utility” of CMA, and it was more than one year before such
authorization was granted. That initial decision to deny coverage could have had
untoward health implications for this child, as the identification of constitutional
MYCN duplication necessitated surveillance for neuroblastoma.
Recommendations are Forthcoming for
the Use of CMA in Prenatal Diagnostics
N ENGL J MED 2012; 367;23
N ENGL J MED 2012; 367;23
Prenatal Diagnosis 2012, 32
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Prenatal Diagnosis Using CMA
Prenatal Diagnostics Using CMA
LabCorp (n=>1600)
U/S Finding
Positive Findings
Signature Genomics (n=3,876)
U/S Finding
Positive Findings
Single abn.
5.6%
Single abn.
5.6%
Multiple abns.
15.3%
Multiple abns.
9.8%
Brain
9.6%
Abn. Of growth
2.6%
Kidney
10.9%
> One soft sign
2.6%
Cardiac
6.8%
Holoprosencephaly
11%
Posterior fossa defect 14.6%
Skeletal anomalies
10.8%
VSD
11.4%
Hypoplastic left heart
16.2%
Based on these studies, ACOG will likely recommend that array should be
used as a first-tier test in certain prenatal clinical situations
Case Report: Fetus with Ventriculomegaly
and Agenesis of the Corpus Callosum
Amniocentesis for ventriculomegaly
and agenesis of the corpus callosum
CMA done on 9 week old child for
multiple congenital anomalies, ACC, and
lack of normal development
2.88Mb chromosome 1q44
deletion which includes the 1qter
critical region for corpus callosum
agenesis/hypogenesis
Considerations in Prenatal CMA

The parental anxiety created by identifying CNVs of unknown significance

The need to do parental CMA studies to further characterize uncertain results

Familial variants are not always benign due to reduced penetrance and
variable expressivity

The need for pretest and posttest genetic counseling

The identification of a genomic imbalance that is not related to the reason for
referral nor to the observed phenotype, but rather to late-onset
disorders, infertility, neurological, or cancer predisposition loci

Should CMA replace standard karyotyping and FISH, and when?
46,XX
Applications of SNP Array Testing in Oncology
• Chronic lymphocytic leukemia
SNP Array Analysis in CLL
• CLL is a highly variable disease with life expectancies from a few
months to many decades
• Myelodysplastic syndrome/Myeloproliferative neoplasm
• CLL FISH panel detects abnormalities that may not be found by
karyotype
• Plasma cell myeloma
• CLL SNP array useful as the disease is characterized by genomic gain or
loss
• Acute lymphoblastic leukemia
• Acute myeloid leukemia
• CLL SNP array has high concordance with other studies; however, SNP
array can identify 30-76% more abnormalities
• Renal cell carcinoma
• Meduloblastoma
• Glial tumors
• Neuroblastoma
• Malignant melanoma
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SNP Array Identifies Large Genomic Aberrations in
CLL With Normal FISH and Predicts Clinical Outcome
Time to First Treatment
CLL Case -FISH Positive for Trisomy 12
Overall Survival
Ch. 11 – ATM gene deletion
not identified by FISH
Ch. 18p deletion
Ch. 19q duplication
Ch. 19p duplication
Metaphase Cytogenetics and SNP Array Provides the
Greatest Diagnostic Yield in Myeloid Malignancies
Case Report: Myeloproliferative Neoplasm
With Acquired UPD (cnLOH)
• Patient with hx of MPN positive for JAK 2 mutation, negative for BCR/ABL
• Normal karyotype
cnLOH includes the JAK2 gene
resulting in homozygous JAK2
mutation
Multiple
clones with
different
sized
segments of
9p cnLOH
The presence and number of SNP array-detected chromosome abnormalities
are independent predictors of overall and event free survival in MDS and can
define a group of patients with much higher risk of transformation to AML
JAK2 gene
The Significance of Acquired UPD as
Detected by SNP Array Analysis
Case Report: B-Cell Non-Hodgkin Lymphoma
• Copy neutral LOH is known to constitute between 50-80% of LOH in all human
cancers
• aUPDs are found in:
- 20% of AML
- 30% of MDS
- 80% of lymphomas
O’Shea et al (2009):
Regions of aUPD at
diagnosis of follicular
lymphoma are associated
with both overall survival
and risk of transformation.
Blood 113(10).
• UPDs are associated with homozygous mutations of a number of tumor suppressor
genes including TET2, CDKN2A/B, TP53, NF1, Rb, CEBPa, and RUNX1
• UPDs are also associated with gain-of-function alleles of oncogenes including
JAK2, NRAS, c-CBL, and FLT3
• Normal karyotype
• Positive for IGH/CCND1
rearrangement and for
monosomy 13 by FISH
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Case Report: B-Cell Non Hodgkin Lymphoma
CaseReport: B-Cell Non Hodgkin Lymphoma
Ch. 2q deletion (12.3 Mb)
Ch.5p duplication (41.8 Mb)
Ch. 9p (10.4 Mb) and 9q
(25.5Mb) with LOH
Ch. 6q deletion (48.5 Mb)
Ch. 11q deletions
(1.9 and 5.5Mb)
Ch.1q deletion (15.9kb) with LOH
Ch. 17q duplication
(36.8Mb)
Chromosome 1q deletions (15.9 Mb)
Low-level mosaic
trisomy 8p
Chromosome 1
Complex ch. 13 rearrangements with loss
and gain
CaseReport: B-Cell Non Hodgkin Lymphoma
CaseReport: B-Cell Non Hodgkin Lymphoma
Chromosome 5p duplication (41.8Mb)
Chromosome 6q (48.5Mb)
Chromosome 6q deletion (48.5 Mb)
Chromosome 5p
Chromosome 6q
Case Report: B-Cell Non-Hodgkin Lymphoma
UPD (CNNLOH)
MYB gene
SNP Array Analysis in Renal Cell Carcinoma
4 or more copies
Clear cell RCC
Papillary RCC
Mucinous, tubular,
and spindle cell
carcinoma
Normal disomy
Deletion
* RB1 gene
Chromophobe RCC
LAMP1 gene *
FISH misidentified this as monosomy 13. In fact,
it’s far more complicated!
Oncocytoma
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SNP Molecular Karyotyping in
Malignant Melanoma
Conclusions
• SNP array analysis provides a genetic approach to predicting outcome in
malignant melanoma.
• Good prognosis was defined as survival throughout the observation interval
(median 14.8 years) without local relapse or metastasis
• Chromosome microarray analysis provides a highly-sensitive whole-genome analysis
of the human genome that detects, at present, only genomic imbalance.
• SNP array karyotyping is the most sensitive of all CMA technologies, as it detects
very small but clinically significant abnormalities as well as uniparental
disomy.
• In the pediatric population, CMA significantly increases the diagnostic yield of
constitutional chromosome abnormalities resulting in better patient (and
family) management. Recommendations have been established for
the use of CMA in routine clinical testing
• Recommendations are forthcoming for the use of CMA in prenatal diagnostics. It is
now accepted that CMA should be offered in any pregnancy with ultrasound
anomalies and a normal karyotype.
Hirsch et al (2013): Cancer Res
73, 1454-1460
Melanomas with greater genomic instability
as assessed by SNP array are associated with
a poorer prognosis
Conclusions
• It is now well documented that CMA, especially SNP array analysis, can
identify clinically significant genomic abnormalities in a variety of
hematolymphoid disorders and solid tumors. The use of SNP array in the
characterization of malignancies is in its infancy and will see
significant growth over the next several years.
• Microarrays that can detect both balanced and unbalanced genomic alterations
will likely replace conventional karyotyping in many instances;
however, FISH will continue to play an important role in the
Cytogenomics Laboratory.
• Scare Me!! - What will CMA reimbursement utilizing the new Molecular
Pathology codes be:
CPT 81229 (Tier 1) – Interrogation of genomic regions for copy number
and single nucleotide polymorphism (SNP) variants for chromosomal
abnormalities
CPT 81406 (Tier 2) – Molecular pathology procedure, level 7
(eg. analysis of 11-25 exons by DNA sequence analysis, mutation
scanning, or duplication/deletion variants of 26-50 exons,
cytogenomic array analysis for neoplasia)
Thank You
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