UTILIZATION OF A SNP MICROARRAY FOR CHRONIC LYMPHOCYTIC LEUKEMIA: EFFICACY, INFORMATIVE FINDINGS AND PROGNOSTIC CAPABILITIES S Schwartz, Z Hosseini, S Schepis, P Papenhausen Laboratory Corporation of America Holdings
OBJECTIVES Effectiveness of array analysis if initial CLL FISH was normal Effectiveness if initial CLL FISH was abnormal Does the array add information Delineating the usefulness as a diagnostic and prognostic tool Is the array as sensitive as FISH Determining best way to approach CLL Benefits and limitations to array and FISH
CLL CURRENT ANALYSIS In many laboratories both cytogenetic analysis and FISH may be used to study patients with Chronic Lymphocytic Leukemia Cytogenetic studies are often of limited value FISH will target 13q (mir 15/16), 11q (ATM), 17p (TP53) and the centromere of chromosome 12 FISH will detect aberrations in ~80% of patients FISH with a CCND1/IGH fusion probes may also be used to rule/out Mantle Cell Lymphoma 3
SNP Microarray Genome-wide Affymetrix CytoScan HD Array More than 2.69 million markers Across the entire genome 743,304 SNPs (single nucleotide polymorphisms) Polymorphic probes Assess copy number changes Assess genotypes (e.g., UPD and identity by descent) 1,953,246 structural probes (copy number probes) Non-polymorphic Assess copy number change (e.g., deletions and duplications) Affymetrix and CytoScan are Registered Trademarks of Affymetrix, Inc.
CLL NORMAL FISH RESULTS Array can be used to study CLL patients that have normal FISH results 775 patients studied by microarray with documented CLL Of the 21.2% of patients with normal FISH results However, approximately ~55% had a detectable abnormality by the array Overall with array over 90% of the patients have a detectable abnormality
ARRAY NORMAL FISH CLL patient - no abnormality detected from routine FISH analysis Array duplication of 2p Marker of progression and indicative of poor prognosis Copy-neutral loss of heterozygosity (CN- LOH) 17 Confirmed homozygous p53 mutation Both findings suggestive of poor prognosis Data on File (March, 2017)
FISH VERSUS ARRAY FISH, as is currently utilized will detect deletions in 11q, 13q and 17p and gain of 12 Poor prognosis 11q, 17p deletions Intermediate prognosis trisomy 12 Better prognosis 13q Array can detect other abnormalities Recent literature suggests that complexity (3 or more abnormalities) associated with poorer prognosis Some suggested 13q with Rb1 deletion poorer prognosis 7
ADDITIONAL ABNORMALITIES DETECTED BY ARRAY (COMPLEXITY) In numerous patients with CLL, abnormalities might be detected by FISH but the array may provide additional (prognostic) information In CLL, FISH detected complexity (> 3 abnormalities) in ~9% of patients with an abnormality detected by FISH Complexity detected by the array in ~46% of patients; poorer prognosis Complexity in 38% of the 13q deletion patients Data on File (March, 2017)
ADDITIONAL ABNORMALITIES DETECTED BY THE ARRAY FISH RESULTS SINGLE ABNORMALITY ADDITIONAL ABNORMALITIES % OVERALL WITH COMPLEXITY 13q Deletion (with RB1) 13q Deletion (no RB1) 36.1% 63.9% 51.9% 54.1% 45.9% 28.9% 11q Deletion 8.6% 91.4% 72.0% Trisomy 12 40.0% 60.0% 29.8% 17p Deletion 0% 100% 100%
CLL PATIENT GENOME COMPLEXITY 87.5 year old male FISH 13q deletion (87%) Flow: 91% of leukocytes with a B-cell chronic lymphocytic leukemia/small lymphocytic leukemia (CLL/SLL) phenotype Array 13q deletion 8q duplication (MYC) Xq duplication CN-LOH 11q
CLL PATIENT WITH CHROMOSOME COMPLEXITY ADDED INFORMATION - PROGNOSTIC
11Q COPY-NEUTRAL LOSS OF HETEROZYGOSITY ATM INVOLVEMENT?
CHROMOTHRIPSIS Phenomena involving multiple gains/losses in a chromosome/specific region With arrays suggested to involve 10 or more gains and/or losses Seen in all different types of neoplasia Associated with a poorer prognosis Seen in ~1.6% of patients with CLL 13
CLL PATIENT GENOME COMPLEXITY CHROMOTHRIPSIS 89 year old male FISH No 17p deletion Flow: 90% of leukocytes with a B-cell chronic lymphocytic leukemia/small lymphocytic leukemia (CLL/SLL) phenotype ZAP70 Positive, CD38 Negative Array Numerous abnormalities Poor prognosis
CHROMOSOME 13 GENOME INSTABILITY NUMEROUS DELETIONS - CHROMOTHRIPSIS
COPY-NEUTRAL LOSS OF HETEROZYGOSITY Finding with no gain or loss of chromosome Telomeric homozygosity (rescue event) Associated with doubling of a mutation of deletion in affected chromosome Will often be associated with a poor outcome Seen in ~15.3% of patients with CLL Several chromosome involved Mostly 1p, 11q, 13q, 17p and 20q 16
HOMOZYGOUS 13Q DELETION AND CN-LOH ALLELE DIFFERENCE SMOOTH SIGNAL LOG2 RATIO
17P COPY-NEUTRAL LOSS OF HETEROZYGOSITY p ALLELE DIFFERENCE TP53 MUTATION SMOOTH SIGNAL = 2.0 LOG2 RATIO
DELINIATION OF A DIFFERENT DISORDER Laboratory testing may be ordered prior to result from either flow cytometry or morphology studies. If only CLL FISH has been utilized, it may be non-informative, especially regarding information for other disorders. Utilization of the array has allowed detection of a disorder, other than CLL, in 2.5% of patients referred for CLL 19
FUTURE DIRECTIONS - CLL FISH for 11q-, +12, 13q-, 17p-, t(11;14) are currently initial studies Should consider array as 1 st tier test Have compared ~ 192 patients [FISH and Array] Overall efficacy of array compared with FISH ~611 hybridizations studied Overall concordance of FISH and array results ~99.2% at 10% or greater mosaicism FISH is done for t(11;14) FISH is done for low level mosaicism (residual disease) [INTERNAL DATA MARCH, 2017]
CONCLUSIONS The array is clearly more effective in detecting abnormalities than FISH alone When FISH is normal When FISH was abnormal Detection of abnormalities not available by FISH and not seen in chromosome analysis The array may be slightly less sensitive than FISH but will detect abnormalities present at least 10% of the time 21
CONCLUSIONS The array detected genome complexity 90% of the time that FISH did not Allow delineation of clonal information Some studies have suggested an association with poorer outcomes The array was effective in subdividing 13q deletions into two different groups One with a more likely chance of being an aggressive disease 22
CONCLUSIONS The array was effective in detecting chromothripsis The array could also detect of copy-neutral loss of heterozygosity CN-LOH 17p Ability to provide better prognostic information The array was effective in detecting a second disorder, such as myelodysplasia, not initially suggested by the referring physician 23
ACKNOWLEDGEMENTS LabCorp Directors/Counselors Peter Papenhausen Jim Tepperberg Inder Gadi Rachel Burnside Karen Phillips Hiba Risheg Katie Rudd Rao Potluri Justin Schleede Romela Pasion Huong Cabral Jennifer Shafer Laura Kline Margriet Johansen Sharon Molinari Michelle Pierce LabCorp Array Lab Brian Williford Carolyn Bullen Jessica Whaley-Davis Savanna Schepis Jesse Soileau Brent Gessner Jeremy Pierce Andrew Endicott Caitlin Jordan Audra Baker Paul Colacicco Laura Dewitt Jarrett Glass Keli Rodriguez Nicolette Rossmell Danielle Wright LabCorp FISH Lab Holly Goode Amanda Felder Shorne Cox Renee Royster Treasure White Tracy Hummel