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Personalized Medicine : Genetic

Testing and the Implications forfuture therapiesDr. Mohammad Omar Hussaini, MD

Assistant Member, Moffitt Cancer Center

Assistant Professor, University of South Florida

Hematopathology and Molecular Pathology

The body is made of many different types of

cells.

n itrogenous b ases:

- adenine

c:r: thym ine

...guanine

cytosinek,,....._

sugar

phosphate

b ackbone

(a)

base

pair

major

groove

m inor

groove

3' 5 '

5 ' 3 '

(b) (c)

The DNA code consists of 4 letters

• A= adenine

• T= thymine

• C = cytosine

• G= guanine

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Basics

• DNA contains 3 Gb (1 bp= 1 byte)

• 1% represents coding regions

• 25,000-30,000 genes

• Contain introns and exons

• Why sequence DNA?

• Central dogma of biology

Koboldt. Cell. 2013 Sep 26;155(1):27-38.

• DNA is the way that the cell stores information

• Provides the blueprint to make amino acids

• Amino acids come together to make proteins

• Proteins are important for the structure of cells, function of cells, and

regulation of cells

• If everything is working fine, the DNA contains all the information for

the cell to work normally

• However, if the something goes wrong in the DNA, then somethingcan go wrong in the cells and this results in disease

• Have to keep in mind that the whole story does not lie in the genome

only.

• Changes can be genetic (in genes) or

• Epigenetic

• Leave the gene sequence untouched

• Alters the gene activity (for example by methylation, acetylation, phosphorylation, ubiquitylation, chromatin modification and sumolyation)

• The changes can be inherited by daughter cells

• Can be influence by environment to turn on and off genes

Weinhold. Environ Health Perspect. 2006 Mar; 114(3):A160–A167.

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• Not every change in DNA is bad

• Gene mutations can either:

• Germline

• Acquired (somatic)

• As we age we accumulate mutations (or changes in the DNA code)

• Some of these can be deleterious (pathogenic) and some not (benign)

• By sequencing DNA we can find pathogenic mutations in BMF

syndromes.

Cell. 2012 Jul 20; 150(2): 264–278

T

sp

•

he wrong mutation in the wrong cell c

ell trouble.

an

Cell. 2012 Jul 20; 150(2): 264–278

Bone marrow failure syndromes

• BMF are thought to be due to DNA mutation in stem cells

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Domen et al. Bone Marrow (Hematopoietic) Stem Cells. stemcells.nih.gov/

The idea is that if we had some way to look at the DNA, we could get very useful information about

disease.

http://www.fidelitysystems.com/Unlinked_DNA.html

What type of information could we get from

DNA?

• Diagnostic (BRAF V600E mutation in hairy cell leukemia)

• Prognostic (TP53 mutations in MDS)

• Therapeutics

• Mutations that can be targeted (FLT3 ITD and midastaurin)

• Pathways that can be targeted

• Mutations that can make one resistant to certain therapies (ABL1 kinase domain mutation in imatinib)

• Markers that we can follow for minimal residual disease

Yang and Press. The Hematologist. May-June 2016, Volume 13,Issue 3

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A Modee of Clonal Ex1>1tnsion and Clonal Evoeution from N ounal Hematopoeis is toMyelod'ys,plasia a n d Myelo id Leuke'm ia

MDS

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The diagnostic piece is particularly relevant in

BMF syndromesAcronym Condition Description/Definition

ARCH Aging related clonal hematopoiesis Describes the presence of detectable, benign clonal

hematopoiesis (defined by the presence of somatic mutations

in the blood or bone marrow) whose incidence increases with

age. No formal definition involving clonal abundance or types

of mutations. No clinical significance is implied.

CHIP Clonal hematopoiesis of indeterminate potential Defined by somatic mutations of myeloid malignancy-

associated genes in the blood or bone marrow present

at2% variant allele frequency in individuals without a

diagnosed hematologic disorder.

CHOP Clonal hematopoiesis of oncogenic potential Describes clonal hematopoiesis in a clinical context where it is

associated with a significant likelihood of progressing to a

frank malignancy.

IDUS Idiopathic dysplasia of undetermined significance Individuals with unexplained morphologic dysplasia of blood

cells who are not cytopenic. Can occur with or without clonal

hematopoiesis.

ICUS Idiopathic cytopenia of undetermined significance Patients with one or more unexplained cytopenias who do not

meet diagnostic criteria for myelodysplastic syndrome or

another hematologic disorder. Can occur with or without

clonal hematopoiesis although often used to refer to

cytopenias without evidence of clonal hematopoiesis.

CCUS Clonal cytopenia of undetermined significance Patients with one or more unexplained cytopenias who do not

meet diagnostic criteria for myelodysplastic syndrome or

another hematologic disorder, but who have somatic

mutations of myeloid malignancy-associated genes in the

blood or bone marrow present at2% variant allele frequency.

Can be considered as the intersection between CHIP and ICUS.

• So, if we are able to detect a mutation that is present in a group of

cells we now have evidence of clonal hematopoeisis.

• Cancer is a clonal disease- a cell of origin undergoes unrestrictedgrowth and replication.

• We now know that all clones are not cancer

• For example:

• All dogs are four legged animals but that does not mean that all four leggedanimals are dogs

What is in a name- implications beyond

diagnosis

Bejar. Leukemia (2017) 31, 1869–1871

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Bejar. Haematologica. 2014 Jun; 99(6): 956–964.Haematologica. 2014 Jun; 99(6): 956–964.

Because of the prognostic implications, genemutations are integrated into professionalguidelines

There are several different type of genetics

tests

• Cytogenetics testing- looking at whole chromosome

• Biochemical testing- looking at protein (quantity or quality)

• Even if there is a change at DNA level we want to make sure that it is making a difference at protein level

• Molecular genetic testing

• Can look at single gene

• Panel testing

• Whole exome

• Whole genome

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Cytogenetics

• 1956- Tjio and Levan demonstrated we have 46 chromosomes • 1969- chromosome banding was discovered

• These involved staining protocols that were able to producereproducible patterns of dark and light bands along the length of a chromosome

Nature Reviews Genetics 3, 769-778 (October 2002)

• Several days of cell culture

• Chromosomes are fixed

• Chromosomes are spread on microscope slides

• Chromosomes are stained.

• Distinct bands of each chromosome revealed by staining allow foranalysis of chromosomal structure.

GeneticAlliance; The New York-Mid-Atlantic Consortium

for Genetic and Newborn Screening Services.

Washington (DC): GeneticAlliance; 2009 Jul 8. https://ghr.nlm.nih.gov/chromosome

Allowed scientists to1. identify chromosomes2. Detect subtle deletions3. Inversions4. Insertions5. Translocations6. Fragile sites7. More complex rearrangements

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FISH

O'Connor, C. (2008) Fluorescence in situ hybridization(FISH). Nature Education 1(1):171

Rafael Bejar

Haematologica June 2014 99: 956-964

Advantages

Provides global information in a single assay

Includes primary and secondary anomalies

Knowledge of anticipated anomaly or histological diagnosis not necessary

Variants undetectable by interphase FISH or RT-PCR may be uncovered

Diagnostically useful

Sensitive and specific

Can be performed on fine-needle aspirates

Provides direction for molecular studies of pathogenetically important genes

Limitations

Requires fresh tissue

Although direct preparations can be performed, cell culture is typically required (1–10 days)

May encounter complex karyotypes with suboptimal morphology

Submicroscopic or cryptic rearrangements may result in a false-negative result

Normal karyotypes may be observed following therapy-induced tumor necrosis or overgrowth of normal

supporting stromal cells

Difficulties encountered with bone tumors include low cell density and the release of cells from bone matrix

Advantages and limitations of conventional cytogenetic analysis

FISH, fluorescence in situ hybridization; RT-PCR, reverse transcription-polymerase chain reaction

Bridges.J Orthop Sci. 2008 May; 13(3): 273–282.

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Biochemical Testing

• There can be a lot of mutations in a particular gene

• May not be cost effective, feasible, or reliable to detect all mutations

that may damage the protein

• We can measure the amount of protein or inability of protein tofunction

• Infer that there must be a mutation underlying that aberration

• Protein is derived from various tissue sample types

• For example:• Blood• Urine• Amniotic fluid

• Cerebrospinal fluid.

• Various methods can then be employed to analyze the protein function or amount:• high performance liquid chromatography (HPLC)• gas chromatography/mass spectrometry (GC/MS)• tandem mass spectrometry (MS/MS)

• Bioassays are functional and can employ flourometric, radioisotopic, or thin-layer chromatography methodologies.

GeneticAlliance; The New York-Mid-Atlantic Consortium

for Genetic and Newborn Screening Services.

Washington (DC): GeneticAlliance; 2009 Jul 8.

Genetic Molecular Testing

• Direct analysis of DNA

• Can be performed on almost any tissue

• Require very low amount of input DNA

• Actually can read DNA sequence

Polymerase chain reaction-based assays (PCR)

• Denaturation

• Annealing

• Elongation

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Sanger sequencing

• Developed in 1977

• Heat to denature and form ssDNA

• Primer anneals to template strand

• DNA polymerase, template, dNTP, and ddNTP are added

• ddNTP are irreversible chain terminators

• Different size fragments can be separate by PAG eletrophoresis or CE

Comparative genomic hybridization (CGH) DNA microarray analysis

Wiestner, et al. Blood 2003 101:4944-4951

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What is Next Generation Sequencing (NGS)?

• Post-Sanger sequencing technologies are referred to as NGS

• They share the following characteristics

• High throughput

• Massively parallel sequencing

• Lower Cost

Stronger

ER Mardis. Nature 470, 198-203 (2011) doi:10.1038/nature09796

Cheaper

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Welch. Genomics of AML: Clinical Applications of Next-Generation

Sequencing. ASH. 2011.

--

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Many Faces of NGS

Direct-read Sequencing by TEM

Semiconductor-based pH

sequencing

Sequencing-by-hybridization

Technology Amplification Read length Throughput Sequence by synthesis

Currently available

Roche/GS-FLX Titanium Emulsion PCR 400–600 bp 500 Mbp/run Pyrosequencing

Illumina/HiSeq 2000, HiScan Bridge PCR (Cluster PCR) 2 × 100 bp 200 Gbp/run Reversible terminators

ABI/SOLiD 5500xl Emulsion PCR 50–100 bp >100 Gbp/run Sequencing-by-ligation (octamers)

Polonator/G.007 Emulsion PCR 26 bp 8–10 Gbp/run Sequencing-by-ligation (monomers)

Helicos/Heliscope No 35 (25–55) bp 21–37 Gbp/run True single-molecule sequencing

(tSMS)

In development

Pacific BioSciences/RS No 1000 bp N/A Single-molecule real time (SMRT)

Visigen Biotechnologies No >100 Kbp N/A Base-specific FRET

U.S. Genomics No N/A N/A Single-molecule mapping

Genovoxx No N/A N/A Single-molecule sequencing by

synthesis

Oxford Nanopore Technologies No 35 bp N/A Nanopores/exonuclease-coupled

NABsys No N/A N/A Nanopores

Electronic BioSciences No N/A N/A Nanopores

BioNanomatrix/nanoAnalyzer No 400 Kbp N/A Nanochannel arrays

GE Global Research No N/A N/A Closed Complex/nanoparticle

IBM No N/A N/A Nanopores

LingVitae No N/A N/A Nanopores

Complete Genomics No 70 bp N/A DNA nanoball arrays

base4innovation No N/A N/A Nanostructure arrays

CrackerBio No N/A N/A Nanowells

Reveo No N/A N/A Nano-knife edge

Intelligent BioSystems No N/A N/A Electronics

LightSpeed Genomics No N/A N/A Direct-read Sequencing by EM

Halcyon Molecular No N/A N/A Direct-read Sequencing by EM

ZS Genetics No N/A N/A

Ion Torrent/PostLight No N/A N/A

Genizon BioSciences/CGA No N/A N/A

How NGS works:

• Library Preparation

• Massively Parallel Sequencing

• Sequence

• Image

• Alignment

• Variant Calling

• Annotation

Jason M. Rizzo, and Michael J. Buck Cancer Prev Res 2012;5:887-900

©2012 by American Association for Cancer Research

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Spencer, et al. The Journal of Molecular Diagnostics, Vol. 15, No. 1, January 2013

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Read alignments are the raw data ofNGS

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©2012 by American Association for Cancer Research

Jason M. Rizzo, and Michael J. Buck Cancer Prev Res2012;5:887-900

METHODOLOGY

Experimental Methodology: This test uses targeted next-generation sequencing to analyze coding regions of the most inclusive annotated RefSeq transcript for

each of the targeted genes. TruSeq Custom Amplicon assay was conducted for resequencing of enriched targeted regions. Sequenc ing of enriched libraries was

performed in multiplex on the Illumina MiSeq with paired-end, 150 base-pair configuration.

Informatics Methodology: There are four informatics tools used and relevant parameters used for each tool are detailed as follows:

1. Novoalign

Version 3.02.00

Parameters: --r none --softclip 9999 -l 30 -e 100 -i 230 140 -t 254 --amplicons <amplicon file> -H

2. samtools

Version 0.1.19

Parameters: -B -d 1000000

3. Varscan

Version 2.3.6

Parameters: --min-freq 0.01 -strand-filter 0 --min-avg-qual 0

4. Freebayes

Version 0.9.7

Parameters: --use-duplicate-reads --min-alternate-count 10 --min-alternate-fraction 0.03 --min-coverage 10 --min-base-quality 20 --min-mapping-quality 30 --min-

supporting-quality 30,20 --min-alternate-qsum 40

Novoalign is an alignment tool. Samtools provides an input for Varscan. Varscan is a variant caller used to identify SNVs. Freebayes is a variant caller used to

identify insertions and deletions.

For single base-pair substitutions, an evaluation of this gene panel found a sensitivity of 99.32% for variant allele frequencies of 10-20% and a sensitivity of

100% for variant allele frequencies >20%. Specificity and positive predictive value were found to be 100% for substitutions with a variant allele frequency >10%.

Cutoff criteria were set such that a minimum variant allele frequency of 10% and a depth of 1000x were required to call single nucleotide variants.

For insertions and deletions, an evaluation of this gene panel identified 24 of 24 insertions and deletions with a variant allele frequency > 10%

Cutoff criteria were set such that a minimum variant allele frequency of 10% and a depth of 1000x were required to call insertions and deletions.

Note that it is possible that pathogenic variants may not be reported by one or more of the tools because of the parameters used. However, tool parameters were

optimized to maximize specificity and sensitivity.

Now that we know how to look; what do we

look for?Germline mutation testing

Dohner et al. Blood 2016 :blood-2016-08-733196

WHO classification

Classification*

Myeloid neoplasms with germ line predisposition without a preexisting disorder or organ dysfunction

AML with germ line CEBPA mutation

Myeloid neoplasms with germ line DDX41 mutation†

Myeloid neoplasms with germ line predisposition and preexisting platelet disorders

Myeloid neoplasms with germ line RUNX1 mutation†

Myeloid neoplasms with germ line ANKRD26 mutation†

Myeloid neoplasms with germ line ETV6 mutation†

Myeloid neoplasms with germ line predisposition and other organ dysfunction

Myeloid neoplasms with germ line GATA2 mutation

Myeloid neoplasms associated with bone marrow failure syndromes

Juvenile myelomonocytic leukemia associated with neurofibromatosis, Noonan syndrome, or Noonan syndrome-like disorders

Myeloid neoplasms associated with Noonan syndrome

Myeloid neoplasms associated with Down syndrome†

Guide for molecular genetic diagnostics‡

Myelodysplastic predisposition/acute leukemia predisposition syndromes

CEBPA, DDX41, RUNX1, ANKRD26, ETV6, GATA2, SRP72, 14q32.2 genomic duplication (ATG2B/GSKIP)

Cancer predisposition syndromes§

Li Fraumeni syndrome (TP53)

Germ line BRCA1/BRCA2 mutations

Bone marrow failure syndromes

Dyskeratosis congenita (TERC, TERT)

Fanconi anemia

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Testing in AML 2017 ELN risk stratification by genetics

Risk category* Genetic abnormality

Favorable t(8;21)(q22;q22.1); RUNX1-RUNX1T1

inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB-

MYH11

Mutated NPM1 without FLT3-ITD or with FLT3-ITDlow†

Biallelic mutated CEBPA

Intermediate Mutated NPM1 and FLT3-ITDhigh†

Wild-type NPM1 without FLT3-ITD or with FLT3-

ITDlow† (without adverse-risk genetic lesions)

t(9;11)(p21.3;q23.3); MLLT3-KMT2A‡

Cytogenetic abnormalities not classified as favorable

or adverse

Adverse t(6;9)(p23;q34.1); DEK-NUP214

t(v;11q23.3); KMT2A rearranged

t(9;22)(q34.1;q11.2); BCR-ABL1

inv(3)(q21.3q26.2) or

t(3;3)(q21.3;q26.2); GATA2,MECOM(EVI1)

−5 or del(5q); −7; −17/abn(17p)

Complex karyotype,§ monosomal karyotype||

Wild-type NPM1 and FLT3-ITDhigh†

Mutated RUNX1¶

MDS

• 2017 NCCN guidelinesSpl ic ing factors

DNA methylat ion

H istone modif ication

Cohesin components

SF3B 1,SRSF2, U2AF1, ZRSR2

100

Transcription factors

Signal transduction

p53 pathway

TET2, DNMT3A, IDH1/2

ASXL 1,EZH2, BCOR, EP300

STAG2, RAD21, SMC1A, SMC3

RUNX1, ETV6, CUX1, GATA2

CBL, JAK2, NRAS, KRAS, MPL, NF1, PTPN11, KIT, FLT3

TP53, PPM1D

0 20 40 60

Mutation Frequency (%)

80

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I

I

I

I

I

I

I

I

I

II

- Ua>)Cytopenias + + +

caCJ . .

·c: .... Dysplasia + +·- cQa)

( . )

LL BM b lasts < 5% < 5o/o up to 20% > 20%

Summary on Impact on prognosis

• As number of driver mutation increases, the prognosis gets worse

• Somatic mutations can predict patient outcomes

• Many studies showTP53, EZH2, ETV6, RUNX1, ASXL1, and SRSF2 mutations predict poor overall survival

• SF3B1 mutations are associated with better outcomes

• In univariate analysis

• Can predict OS independent of clinical prognostic scoring systems

• In multivariate analysis, contribution is small (due to close link

between mutations and clinical factors)

Kennedy and Ebert. Clinical Implications of Genetic

Mutations in Myelodysplastic Syndrome. JCO. 2017

Therapeutic Implications in MDS

• del(5q) MDS

• Treatment with lenolidamide cytogentic CR and lower transfusion reqs

• HMAs

• 50% show hematologic response

• TET2 mutated consistently show association with better response

• TP53, RUNX1, and ASXL1 mutated patients do worse with transplant so may need to look at other options

• DNMT3A inhibitors, Spliceosome inhibitor, IDH inhibitors etc. arepresent and identification of mutation can assist triage to clinical trial

Kennedy and Ebert. Clinical Implications of Genetic

Mutations in Myelodysplastic Syndrome. JCO. 2017

AA

• Can be acquired or somatic

• Acquired BMF syndromes:

• Fanconi anemia

• Shwachman-Diamond syndrome

• Dyskeratosis congenita

• Diamond-Blackfan anemia

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Dokal. Haematologica. 2010Aug; 95(8): 1236–1240.Dokal. Haematologica. 2010 Aug; 95(8): 1236–1240

N Engl J Med 2015; 373:35-47N Engl J Med 2015; 373:35-47

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PNH

National Center for Biotechnology Information (US).

Bethesda (MD): National Center for Biotechnology

Information (US); 1998-.

•Nature Reviews Disease Primers 3,

Article number: 17028 (2017)

•Nature Reviews Disease Primers 3, Article number: 17028

(2017)

Medical Genetics versus Direct to Consumer

Genetics

• Quality of the data

• Does it occur in a CAP/CLIA accredited environment

• Is it FDA approved

• What are the implications of the data?

• Interpretation

• Correlation is not causation

• Other factors (diet, environment, ethnicity, etc.) may influence expression

• Emotional toll- what if your adopted? What if you have only small risk for

cancer but think that you have it?

• May be hard to get some types of insurance

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What does medical genetic testing involve?

• Informed Consent

• Discussing the patient's role in the decision-making process

• Describing the clinical issue and suggested treatment;

• Discussing alternatives to the suggested treatment (including the option of no

treatment)

• Discussing risks and benefits of the suggested treatment (and comparing them to the risks and benefits of alternatives)

• Discussing related uncertainties

• Assessing the patient's understanding of the information provided

• Eliciting the patient's preference

Evidence Reports/Technology Assessments, No. 211.

Rockville (MD): Agency for Healthcare Research and

Quality (US); 2013 Mar.

Genetic Counseling

• Aimed to asssist in

• Assessing inherited cancer risk (family history)

• Seeing what testing may be needed

• Look for opportunities for risk reduction, early detection and/or targeted

treatment

• Insurance coverage

• Discussing ethical or potential emotional impact of results

Important because

• Genetics are not destiny

• No way to predict whether any given individual will develop cancer or not

• What to do with variants of unknown significance

• Our ability to detect aberration has outstripped our ability to determine what they mean

• Genetic testing is not cheap (1000-7000 USD)

• Help make a good decision about

• Whether to get tested or nor

• What test to get

• Determine what the test means

How common are hereditary disposition to

leukemia?• 11% of MDS/AML patients have two or more first-degree relatives

with acute leukemia, myelodysplastic syndrome, or aplastic anemia

• Approximately 8% of patients with myeloproliferative neoplasms havetwo or more affected family members

10/31/2017

21

Bannon et al. Hereditary predispositions to myelodysplastic syndrome. IJMS 2016.

Table 1. Familial myelodysplastic syndromes (MDS)/acute leukemia (AL) predisposition syndromes.

Syndrome Gene Inheritance Heme Malignancy Other Associated

Abnormalities

Reference

Familial platelet disorder

with propensity to myeloid

malignancies

RUNX1 AD MDS/AML/T-cell ALL Thrombocytopenia, bleeding

propensity, aspirin-like

platelet dysfunction

[3]

Thrombocytopenia 2 ANKRD26 AD MDS/AML Thrombocytopenia, bleeding

propensity

[4]

Familial AML with mutated

DDX41

DDX41 AD MDS/AML, CMML None [5]

Thrombocytopenia 5 ETV6 AD MDS/AML, CMML, B-cell ALL,

multiple myeloma

Aplastic anemia [6]

Familial MDS/AML with

mutated GATA2

GATA2 AD MDS/AML/CMML Neutropenia,

monocytopenia, MonoMAC

syndrome, Emberger

syndrome

[7]

Familial aplastic anemia with

SRP72 mutation

SRP72 AD MDS Aplastic anemia [8]

Familial AML with mutated

CEBPA

CEBPA AD AML None [9]

Fanconi anemia Complementation Groups AR, X-linked MDS, AML Pancytopenia, macrocytic

anemia, congenital

malformations

[10]

Telomeropathies

(dyskeratosis congenita)

TERC, TERT, others AD, AR MDS/AML Macrocytosis, aplastic

anemia, oral leukoplakia,

dysplastic nails, lacy skin rash

[11]

When would one start thinking that their

cancer is hereditary?

• Two or more first degree relatives with blood cancer

• Acute leukemia aggregating in close relatives

• Family history of SCC of H&N or anogenital

• If someone in family has multiple cancer or develops cancer at young

age (e.g., Li-Fraumeni)

• Have signs a susceptibility syndrome (abnormal nails, liver/lung

fibrosis, birth defects, etc.)

What to do if there one has a susceptibility

syndrome?

• Monitor CBC every 6 months

• May have to get skin biopsy, if find certain mutations in PBL to

determine if somatic or germline

• Baseline BMBx and then monitor esp. if new or worsening cytopenia

• If get dysplasia, can think about BMT

• Keep in mind that early detection does not always mean that there is

something to do if detect early

• Also if one does have a mutation, not much one can do to reduce risk other than observe closely.

Churpek JE et al, Leuk & Lymph 2012

What on the horizon?

• We do NGS on every new MDS and AML and sequential testing

• Cases with cytopenia of uncertain etiology will get NGS

• Cases with potential germline variant will get referred to genetic

counseling (referral guidelines are in place)

• Have developed various germline panels to assess for BMFsyndromes, familial MDS, familial AML