Genetic Testing and the Implications for Future Therapies  Genetic Testing for Inherited Marrow Failure

4/24/2017

Personalized Medicine: What This Talk Will Cover Genetic Testing and the Implications for Future Therapies  Genetic testing for inherited marrow failure . General background: What are gene mutations? . Risk and severity: Do all people with inherited mutations develop disease? National Patient and Living with . How does medical genetic testing differ from commercially available testing? Aplastic Anemia, Family Conference MDS or PNH Las Vegas, NV April 22, 2017  Cytogenetic testing in Bone Marrow Failure

 Genomic profiling of bone marrow cells in MDS and AA for acquired mutations Implications for diagnosis, prognosis and therapies Katherine R. Calvo, M.D., Ph.D. Department of Laboratory Medicine, Clinical Center National Institutes of Health  Benefits and Limitations of Genetic Testing and Genomic Profiling Bethesda, Maryland

Bone marrow failure syndromes: How can genetic testing help to distinguish and guide therapy?

AA/PNH Part One: PNH LGL Autoimmune SDS Disease: AA MS, IBD, uveitis, HYPOCELLULAR Genetic Testing for DM type 1, etc. TELOMERE MDS (DKC) GATA2 MDS AML Inherited Marrow Failure FA Acquired AA SDS

AA, aplastic anaemia; AID, autoimmune disease; AML, acute myelogenous leukemia; DKC, dyskeratosis congenita; IBD, inflammatory bowel disease; LGL, large granular lymphocyte leukemia; GATA2, Gata2 deficiency; FA, Fanconi anemia; SDS, Shwachman–Diamond syndrome; MDS, myelodysplastic syndrome; MS, multiple sclerosis; DM, diabetes mellitus; PNH, paroxysmal nocturnal hemoglobinuria Modified from Young NS, et al. Blood 2006;108:2509–19

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GENETIC TESTING – Background GENETIC TESTING – Background The Basics: GENES are made of DNA The Basics: DNA is made up of Nucleotides (building blocks)  Each person has a unique “Genome” made up of DNA is made up of four types of SNPs= Single Nucleotide Polymorphisms DNA that forms individual genes building blocks (called nucleotides)  Humans have around 30,000 genes  These spell out the genetic “code”  Genes encode the directions for making: used in our genes  Proteins and everything that is you  We all have normal “variations” in  Hair color/texture, eye color, skin color our genetic code that make each one  All the cells in the blood, bone marrow, and of us different or unique https://neuroendoimmune.wordpress.com/2014/03/27/dna-rna-snp-alphabet-soup-or-an- every organ of the body introduction-to-genetics/

http://prn.fm/wp-content/uploads/2015/05/DNA.jpg A=Adenosine C=Cytosine G=Guanine T=Thymidine

GENETIC TESTING – Background What is a mutation? WHAT IS A MUTATION?  A mutation is a change in the genetic code that can increase your risk of getting:

 Disease or Conserved region  A major change in whatever the gene is responsible for

Mutations usually occur in conserved regions of genome

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GENETIC TESTING – Background GENETIC TESTING – Background WHAT ARE CHROMOSOMES? WHAT ARE CHROMOSOMES? DNA CHROMOSOMES FROM ONE - We normally have 23 pairs of BONE MARROW CELL => NORMAL MALE KARYOTYPE  In the nucleus of cells DNA is chromosomes tightly wound and forms Chromosomes - Each chromosome contains thousands of genes

 TELOMERES = protective caps - Chromosomes are numbered at the ends of each chromosome according to size

Chromosome Telomeres - Sex Chromosomes:

- XX female https://ghr.nlm.nih.gov/primer/basics/howmanychromosomes - XY male http://www.yourgenome.org/facts/what-is-a-chromosome

GENETIC TESTING – Background GENETIC TESTING – Background WHAT ARE CHROMOSOMES and HOW ARE THEY INHERITED? HOW DO WE GET MUTATIONS? INHERITED (Germline) MUTATIONS - Most cells have two copies of each chromosome => Inherited mutations are present at birth and are in all of the cells of the body - One is inherited from your father and the other from Recessive mutations – Need two copies of the your mother mutation (one from mom and one from dad) - Each child has a unique for disease combination of parents’ DNA Dominant mutations – Only need one copy of the mutation (from mom or dad) for disease

https://scratchcradle.wordpress.com/2012/07/29/gms5-chicken-chromosomes/

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How does medical genetic testing ordered by your doctor for bone GENETIC TESTING marrow failure differ from commercially available DNA testing? Examines your DNA to look for mutations in the genetic code

“Next Generation Sequencing” Ted.com

 Panel of genes related to hereditary marrow failure

 Usually testing is only needed once

=> Samples sent for testing can be: Saliva, Buccal Swab, Blood, Bone Marrow, Skin biopsy, or Hair follicle

Ancestry DNA testing Commercially Available DNA testing – Genetic Traits Similar: 23andMe.com => DNA sequencing techniques Asparagus Odor Detection Hair Curliness Different: Back Hair (available for men only) Light or Dark Hair The genes tested Bald Spot (available for men only) Male Hair Loss (available for men only) Bitter Taste Perception Newborn Hair Amount Nuclear or mitochondrial DNA Cheek Dimples Photic Sneeze Reflex genes that vary based on: Cleft Chin Red Hair . patterns of human migration Earlobe Type Skin Pigmentation . ethnic populations Earwax Type Sweet Taste Preference Eye Color Toe Length Ratio Examples: Finger Length Ratio Unibrow National Geographic Genographic Project Freckles Widow's Peak Ancestry.com 23andMe https://www.geni.com/blog/dna-testing-for-genealogy-getting-started-part-two-376163.html

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Commercially available DNA testing: Health Genetic Mutations Causing Hereditary Bone Marrow Failure Disorders

23andMe - 35 Health related Genes currently • tested including: Limited amount of information Inheritance FANCC (3 variants related to Ashkenazi Jewish decent) related to small number of gene Gene Pattern Cystic Fibrosis mutations well studied by Fanconi anemia over 19 genes encoding FANC proteins AR, XLR Sickle Cell Anemia scientists Telomeropathies, Dyskeratosis cong. TERT, TERC, DKC and others AD, AR, XLR Alpha-1 antitrypsin deficiency (lung and liver disease) Diamond Blackfan anemia RPS19, RPS24, RPL11 AD Schwachman Diamond SBDS AR Late-onset Alzheimer’s disease • Nearly all of the genes related Celiac disease (a digestive disorder, can’t eat gluten) Familial MDS/AML GATA2, RUNX1, DDX41 AD to BMF and MDS are not Early-onset primary dystonia (a movement disorder) Familial AA SRP72 AD Factor XI deficiency (a blood-clotting disorder) included (with the exception of FANCC) Familial Thrombocytopenia/AA/MDS ANKRD26, ETV6 AD Gaucher disease type 1 (an organ and tissue disorder) Familial AML, ET, CMML, PMF ATG2B, SKIP duplications AD Glucose-6-phosphate dehydrogenase deficiency • Not sufficient for diagnosing Hereditary hemochromatosis (an iron overload disorder) hereditary marrow failure Hereditary thrombophilia (a blood clot disorder) disorders Parkinson’s disease

Bone marrow failure syndromes: How can genetic testing help to distinguish and guide therapy? Medical Genetic Testing for Bone Marrow Failure

AA/PNH PNH LGL Purpose:

Autoimmune SDS Disease: AA => To diagnose hereditary bone marrow failure MS, IBD, uveitis, HYPOCELLULAR DM type 1, etc. TELOMERE MDS disorders (DKC) GATA2 MDS AML FA Acquired AA SDS => Sequence genes known to cause BMF, looking for mutations

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GENETIC TESTING – Reasons Your Doctor May Order Genetic Testing Why is it important to identify an inherited BMF gene mutation? If other family members have: • Proper diagnosis Bone marrow failure MDS • Guides therapy Leukemia Physical malformations: small height, abnormal fingers or nail growth, etc. • Implications for family members Stiffening (fibrosis) of the lung and liver Early hair graying • Implications for future pregnancies

=> Evaluate for an inherited genetic mutation • Donor selection and screening for bone marrow transplantation Most common onset of disease is in children and young adults

Genetic Testing for Inherited Mutations Genetic Testing - Informed Consent Examples of Genes Commonly Tested in Sequencing Panels for Bone Marrow Failure RPL11 SBDS VPS45 BRCA2 FANCA G6PC3 RPL3A SBF2 WAS Patient or guardian must fully understand: CSFR3 FANCB GATA2 RPL5 SLX4 WRAP53 CTC1 FANCC* GFI1 RPS10 SRP72 DKC1 FANCD2 HAX1 - Testing procedure RPS19 TERC - Benefits and limitations of the test ELANE FANCE MPL FANCF NHP22 RPS24 TERT - Possible consequences of the test ERCC4 FANCG NOP10 RPS26 TINF2 - Implications for family members and future pregnancies FANCI PALB2 RPS7 USB1 - Voluntary agreement to have the test done FANCL RAD51C RTEL1 XRCC2 FANCM RBM8A RUNX1 * The only gene included in 23andMe testing

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Example of Genetic Testing Results What does “Variant of unknown significance” mean? Patient 1 Result Patient 1 Result, cont. DNA Protein

“Variant of Unknown Significance” VUS: Zygosity: Not sure if the DNA change is a normal Heterozygous - one copy of gene is mutated and the other copy is normal variation, or if it is a mutation. Homozygous – both copies of the gene are mutated

Inheritance (pattern for the specific gene): Recessive - need both copies of the gene mutated to have increased risk of disease Dominant – only need one copy of the gene mutated for increased risk of disease

Question: Do all family members with an inherited mutation develop disease? Do all family members with an inherited mutation develop disease? Answer: Not necessarily. It depends on the gene.

RISK: Proportion of people with a mutation that have signs of disease Not all gene mutations have the same risk of disease Some persons with mutations may be normal and not develop disease

SEVERITY: Variation in the degree of signs and symptoms of the disease - very mild vs. severe - symptoms differ among affected persons

Bottom Line: It’s complicated! Need genetic counselors and geneticists to help educate and advise

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What are acquired cytogenetic abnormalities in the bone marrow? Part Two

Cytogenetic Analysis

Cytogenetic Analysis Cytogenetic Analysis

Karyotype - During the process of cell Other cytogenetic abnormalities that can be detected in the bone marrow division in the bone marrow, problems can arise leading to loss or gain of chromosomes

Examples: - monosomy 7 - trisomy 8

20 bone marrow cells analyzed Abnormalities are only in BM cells Abnormal Chromosomes can Function like Mutations Not inherited or passed Increasing Risk of Disease Ordered many times to monitor ds 46, XY

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Cytogenetic Analysis – how can this help diagnose FISH (Fluorescence In Situ Hybridization) disease in bone marrow cells? also detects cytogenetic abnormalities

TRISOMY 8 In General

AA and PNH: Normal cytogenetics Bone marrow cell (blue)

MDS: Abnormal cytogenetics in 50% Each pink dot inside the cell is one copy of chromosome 8 (too many)

Cytogenetic Abnormalities in MDS and Prognosis What is “Clonal Evolution” and what does it mean for patients with BMF? Very Good del (11q), -Y

Good Normal, del (5q), del (12p), del(20q)

Intermediate del(7q), +8, i(17q), +19, other

Poor inv(3), t(3q), del(3q), -7, 3 abnormalities

Very poor greater than 3 abnormalities

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What is “Clonal Evolution” and CYTOGENETIC EVOLUTION IN TREATED APLASTIC ANEMIA what does it mean for patients with BMF? Incidence: 100 Clonal evolution => detection of new total prevalence 12% 10-20% of AA actuarial risk at 5 years 15% cytogenetic abnormality or mutation that 75 at 10 years 20% patients developed was not previously present 50

Cytogenetic 25 Monitored by cytogenetic analysis, FISH, or (%) evolution abnormalities genomic testing for acquired mutations 0 50 100 150 200 Time (months) at 10 yrs May be insignificant, or may indicate progression or transformation of disease

Weissman, I http://rstb.royalsocietypublishing.org/content/370/1680/20140364

CYTOGENETIC EVOLUTION IN TREATED APLASTIC ANEMIA All cytogenetic abnormalities are not created equal Part Three Prognosis: Trisomy 8 Genomic profiling of bone marrow cells Vs. in AA and MDS for acquired mutations Monosomy 7

0 20 40 60 80 100 120 Time (months)

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What are ACQUIRED (aka SOMATIC) MUTATIONS? What are acquired mutations and ACQUIRED MUTATIONS: how do we get them? Not present at birth Only present in certain cells, not in all cells Can not be passed on to offspring Can be related to toxic exposure (smoking, radiation, benzene, asbestos, etc.) Environment (e.g. UV radiation from sun) Random Normal in aging population PNH – PIG-A mutations May increase your risk for disease: MDS Neoplasia

GENOMIC PROFILING for Acquired Mutations Mutations and Cytogenetic Abnormalities are common in MDS Examines your DNA to look for mutations in the genetic code

=> Genomic profiling of cells in bone marrow to characterize disease

 Sequence panels of genes related myeloid disease (MDS, AML, CMML)

 Repeated over time as disease changes, monitor for disease progression

 Major Area of Research and recently available for clinical testing

 Prognostic implications for outcome & response to therapy in MDS and AA • Somatic point mutations in bone marrow cells are common in myelodysplastic syndromes (~90%) and are often associated with specific clinical features.

Bejar R et al. N Engl J Med 2011;364:2496-2506

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Distribution of recurrent mutations and karyotypic abnormalities in MDS. Clonal cells from ∼50% of MDS patients harbor a splicing factor (SF) mutation, and a similar fraction carry ≥1 mutated epigenetic regulator (ER). Specific Acquired Gene Mutations in BMF => Area of active investigation

=> Some doctors are using mutations profiles to help make decisions about treatment or determine eligibility on clinical trials

 Potentially useful in MDS for new inhibitors and clinical trials

=> Not yet used for diagnosis ------WHY?

 Not specific for MDS, can be seen in other diseases (MPN, AML, etc.) Rafael Bejar, and David P. Steensma Blood 2014;124:2793-2803 AND……..

Acquired Mutations in MDS are also found Effects of Time in Healthy Aging Population

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MDS-associated mutations are also in healthy people What about acquired mutations in AA or PNH?

• 10% of people >70 years carry at least one Genovese et al NEJM 2014; Jaiswal et al. NEJM 2014)

• Present in fewer % of blood cells than MDS (<10% vs. 30%)

• Increased risk of subsequent hematologic malignancy (factor of 11)

• Increased risk of higher all- cause mortality (cardiovascular disease, factor 2-2.6) Hallmark of PNH => acquired mutations in PIG-A in bone marrow cells

Jaiswal S et al. N Engl J Med 2014;371:2488-2498

Mutations in Aplastic Anemia A SUBSET OF MUTATIONS in AA CORRELATE % of Patients with Gene Type 12.0% 8.0% 4.0% 0.0% WITH SURVIVAL - FREE FROM CLONAL EVOLUTION

PIGA

DNMT3A

Splicing JAKs Good outcome TP53

SETBP1 Mutation Status in AA PIGA PRC2 (n=256) BCOR/BCORL1 LAMB4 5% negative 7% 1 mutation DNMT3A TERF1/TERT 2 mutations PHF6 3 mutations ASXL1 23% RIT1

IDH2 65% RBBP4

PRPF8

MPL One-third of AA have at least 1 mutation POT1 Yoshizato T et al. N Engl J Med 2015;373:35-47 multiple STAT3 missense nonsense DIS3 frameshift Yoshizato T et al. N Engl J Med 2015;373:35-47 splicing

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GENETIC TESTING and GENOMIC PROFILING SUMMARY Benefits: - Knowledge of underlying disease factors Part Four - Making informed decisions about health care - Inherited mutations: - Implications for family planning Benefits and Limitations of Genetic - Screening donor family members for patients undergoing BMT Testing and Genomic Profiling - Acquired mutations: - Prognostic implications for outcome and response to therapy (BMT, HSCT, HMAs, etc) - Monitoring for disease progression over time, clonal evolution - Availability of targeted therapy for specific mutations and clinical trials with new drugs

GENETIC TESTING SUMMARY What is on the Horizon? Drawbacks and Limitations: - Financial implications - More research needed to understand the significance of acquired - Emotional implications mutations and “VUS”s in MDS, AA and other myeloid diseases - Limitations of genetic testing: For diagnosis, treatment and prognosis - Inherited mutations: - Not everyone with a mutation may develop disease => value of discussing with genetic counselor - Discovery: Growing list of genes/mutations related to BMF - Acquired mutations: - Not necessarily specific for MDS/BMF - May be seen in other diseases - Development of new targeted therapies in the pipeline against - May be present in healthy individuals specific mutations in MDS and other myeloid diseases

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Specific Gene Mutations and Associations a few examples Thank you! based on research studies • SF3B1 and TET2 - each mutated in 20-25% of MDS cases, most common • TET2 and DNMT3A predicts higher likelihood of response to HMAs • SF3B1 – ring sideroblasts • RUNX1 – thrombocytopenia • TP53 – complex karyotype, therapy related disease, higher relapse after HSCT • SRSF2, CBL - MDS/MPN overlap • ASXL1 - CMML • SETBP1 – atypical CML • JAK2 and SF3B1 – RARS-T