Whole-Genome SNP Array Analysis Genomic Complexity and Clinical Relevance in Prenatal, Postnatal, and Oncology Testing

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1 The Evolution of Cytogenetics Testing 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. 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 Karyotyping has been the Gold Standard A subjective whole-genome test of limited resolution (5-10 Mb) 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 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 (Molecular) Karyotype The Virtual Karyotype is Produced by Single Nucleotide Polymorphism (SNP) Array Analysis Ch. 2q deletion (12.3 Mb) Ch.5p duplication (41.8 Mb) Ch. 6q deletion (48.5 Mb) Ch. 9p (10.4 Mb) and 9q (25.5Mb) with LOH Ch. 11q deletions (1.9 and 5.5Mb) Genotyping 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 bp 9.2 million SNPs have been reported 1

2 SNP Arrays Detect Copy Number Neutral Loss of Heterozygosity/Acquired Uniparental Disomy A B A A SNP C T C C Mitotic Recombination Error Resulting in Segmental UPD Evolution of Cytogenetics Into Cytogenomics Advantages of DNA Microarray Analysis 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 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, , Genet Med 15(2), 2013 Genet Med 13(7), 2011 Genet Med advance online publication 25 April

3 Case #1 Case #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 11p13-11p14.2 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 Case #1 Why is this SNP Array Diagnosis Important in this Child? PAX 6 gene WT1 gene It provides a unifying genetic diagnosis WAGR Syndrome It provides an etiology for the child s present phenotype (aniridia and cryptorchidism) It provides important guidelines for medical management including: - Consultation with a pediatric oncologist for Wilms tumor surveillance by abdominal/renal sonography until the 8 th 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 Case #2 Case #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 of the SCN1A gene on chromosome 2q24.3 Deletion of exons

4 Case #2 Case #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 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. Case #3 Case #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 kb chromosome 2p24.3 duplication involving four genes including MYCN. The child s mother demonstrated the same 560kb duplication of chromosome 2p 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. Prenatal Diagnostics Using CMA Recommendations are Forthcoming for the Use of CMA in Prenatal Diagnostics 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 % of pregnancies with omphalocele - >50% with nasal bone absence - 40% of pregnancies with nuchal fold (2 nd 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? N ENGL J MED 2012; 367;23 Prenatal Diagnosis 2012, 32 N ENGL J MED 2012; 367;23 4

5 Prenatal Diagnosis Using CMA Prenatal Diagnostics Using CMA LabCorp (n=>1600) Signature Genomics (n=3,876) U/S Finding Positive Findings 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 Considerations in Prenatal CMA Amniocentesis for ventriculomegaly and agenesis of the corpus callosum 46,XX 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 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? Applications of SNP Array Testing in Oncology Chronic lymphocytic leukemia Myelodysplastic syndrome/myeloproliferative neoplasm Plasma cell myeloma Acute lymphoblastic leukemia Acute myeloid leukemia Renal cell carcinoma Meduloblastoma Glial tumors Neuroblastoma Malignant melanoma SNP Array Analysis in CLL CLL is a highly variable disease with life expectancies from a few months to many decades CLL FISH panel detects abnormalities that may not be found by karyotype CLL SNP array useful as the disease is characterized by genomic gain or loss CLL SNP array has high concordance with other studies; however, SNP array can identify 30-76% more abnormalities 5

6 SNP Array Identifies Large Genomic Aberrations in CLL With Normal FISH and Predicts Clinical Outcome CLL Case -FISH Positive for Trisomy 12 Time to First Treatment Overall Survival Ch. 11 ATM gene deletion not identified by FISH Ch. 18p deletion Ch. 19p duplication Ch. 19q 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 6

7 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. 6q deletion (48.5 Mb) Ch. 9p (10.4 Mb) and 9q (25.5Mb) with LOH Ch. 11q deletions (1.9 and 5.5Mb) Chromosome 1q deletions (15.9 Mb) Ch.1q deletion (15.9kb) with LOH Ch. 17q duplication (36.8Mb) Low-level mosaic trisomy 8p Complex ch. 13 rearrangements with loss and gain Chromosome 1 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 MYB gene Case Report: B-Cell Non-Hodgkin Lymphoma SNP Array Analysis in Renal Cell Carcinoma UPD (CNNLOH) 4 or more copies Clear cell RCC Papillary RCC Normal disomy Deletion * RB1 gene LAMP1 gene * FISH misidentified this as monosomy 13. In fact, it s far more complicated! Mucinous, tubular, and spindle cell carcinoma Chromophobe RCC Oncocytoma 7

8 SNP Molecular Karyotyping in Malignant Melanoma 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 Conclusions 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, 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 (Tier 1) Interrogation of genomic regions for copy number and single nucleotide polymorphism (SNP) variants for chromosomal abnormalities CPT (Tier 2) Molecular pathology procedure, level 7 (eg. analysis of exons by DNA sequence analysis, mutation scanning, or duplication/deletion variants of exons, cytogenomic array analysis for neoplasia) Thank You 8