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Impact of Personalized Medicine for the Practice of Hematopathology
Impact of Personalized Medicine for the Practice of Hematopathology
Adam Bagg MDProfessor
Director, HematologyUniversity of Pennsylvania
Kojo S.J. Elenitoba‐Johnson MDHenry C Bryant Endowed Professor Director of Molecular Diagnostics
University of Michigan
Megan S. Lim MD PhDProfessor
Director of HematopathologyUniversity of Michigan 2011 College of American Pathologists. Materials are used with the permission of the faculty.
1
DisclosuresDisclosures
None
2
Course ObjectivesCourse Objectives
• Be familiar with concepts of personalized medicine in the context of hematopathology
• Understand the appropriate diagnostic and prognostic value of specific molecular tests in the evaluation of hematopoietic neoplasms
• Appreciate how novel genetic alterations in myeloid and lymphoid neoplasms impact patient management.
3
AgendaTopic Speaker Time
I. Introduction Lim 5’
II. Principles of personalized medicine and evolution of molecular hematopathology-Overview of commonly used molecular technologies in hematopathology-Update in new technologies including array CGH, next generation sequencing
Elenitoba-Johnson
40’
III. Approach to myeloid neoplasms in the era ofpersonalized medicine-Chronic myeloid leukemia-Acute myeloid leukemia
Bagg 65’
IV. Approach to NHL in the era of personalized medicine-Aggressive B-cell lymphomas-Splenic Lymphoma-ALCL
Lim 60’
IV. Summary and Closing Lim 10’4
Impact of Personalized Medicine for the Practice of Hematopathology
Impact of Personalized Medicine for the Practice of Hematopathology
Kojo S.J. Elenitoba‐Johnson MDHenry C Bryant Endowed Professor
Director of Molecular Diagnostics LaboratoryUniversity of Michigan
2011 College of American Pathologists. Materials are used with the permission of the faculty.5
Personalized medicine and evolution of molecular hematopathology
1. Principles of personalized medicine
2. Evolution of molecular diagnostics
3. Update in new technologies including array CGH, next-generation sequencing
6
Concepts of Personalized Medicine
Personalized Medicine (2013) 10(1), 13-15
7
Personalized Medicine
DefinitionForm of medicine that uses information about a person’s genes, proteins, and environment to prevent diagnose and treat disease National Cancer Institute http://www.cancer.gov/
Not a new concept
“The new language of genomics, as applied to medicine, is less a revolution than an evolution“ Steele 2009 Personalized medicine: something old, something new. Pers Med 6:1‐5
8
Genetic testing for personalized medicine in hematopathology
9
Pre-symptomatic risk assessment
Diagnosis Prognosis Treatment
Pharmacogenomics
Classification Disease monitoring
Response to treatment
Prediction of response
1980 1985 19901988 1994 2001 2008 …
Morphologic evaluation
Cytogenetics
Introduction of immunopathology, flow cytometry immunohistochemistry
Rapid growth in molecular genetic application
Gleevecapproved by FDA
New therapies
•Combination of small molecules
Clinical sequencing for detection of structural alterations/mutations
2015
Evolution of Molecular Diagnostics in Hematopathology
PCR FFPE tissues REAL classification
WHO classification
10
MOLECULAR HEMATOPATHOLOGY
11
Genetic alterations in hematologic neoplasms
12
Gene rearrangements
Gene Mutations
Gene Mutations
AdditionsAdditions
LossesLosses
Different types of gene rearrangements
13
VDJ/TCR
Homogeneous Heterogeneous
Monoclonal Polyclonal
Physiologic
Diagnosis
Qualitative Quantitative
Chromosomaltranslocations
Yes or No How much
Pathologic
MinimalResidual disease
Diagnosis
t(11;14)t(14;18)t(2;5)
V D J C Germline(lane A)
18 kb
V DJ C DJ rearrangement(lane B)
12 kb
V DJ C VDJ rearrangement(lane C)
21 kb
A B C21 kb
18 kb12 kb
Southern blot
JB1B2 Probe
JB1B2 Probe
JB1B2 Probe
D
D
5´
5´
5´ 3´
3´
3´
B cells undergo immunoglobulin gene rearrangements
14
Detection of clonal rearrangements
15
Southern blot hybridization PCR/gel
electrophoresis PCR/capillarygel electrophoresis
Applications of Molecular Approaches to Clonality Testing
• Benign versus malignant
• Lymphoma versus carcinoma
• Minimal residual disease
• Lineage: B or T
• Subclassification
16
Common Recurrent Chromosomal Aberrations in Human Malignant Lymphoma
Cytogenetic Abnormality Genes Involved Disease Clinical Features Frequency
(%)t(14;18)(q32;q21) IGH@/BCL2 FL Indolent ~90
t(2;18)(p12;q21) IGK@/BCL2 FL Indolent <5
t(3;14)(q27;q32) IGH@/BCL6 FL Indolent ~10
t(11;14)(q13;q32) IGH@/CCND1 MCL Aggressive >90
Trisomy 3 Unknown MZBCL Indolent Variable
t(11;18)(p21;q21) BIRC3(API2)/MALT1 MZBCL Indolent 40
t(1;14)(p22;q32) IGH@/BCL10 MZBCL Indolent *
t(14;18)(q32;q21) IGH@/MALT1 MZBCL Indolent *
t(3;14)(p22;q32) IGH@/FOXP1 MZBCL Indolent *
t(8;14)(q24;q32) IGH@/MYC Burkitt lymphoma Highly Aggressive 75
t(2;8)(p12;q24) IGK@/MYC Burkitt lymphoma Highly Aggressive 15
t(8;22)(q24;q11) IGL@/MYC Burkitt lymphoma Highly Aggressive 10
t(3;14)(q27;q32) IGH@/BCL6 DLBCL Aggressive ~30
t(14;18)(q32;q21) IGH@/BCL2 DLBCL Aggressive 30
Amplification 2p REL PMLBCL Aggressive *
del 13q14 DLEU2/mir-15a/16-1 CLL Indolent 25-50
Trisomy 12 Unknown CLL Indolent 30
t(2;5)(p23;q35) NPM1/ALK ALCL Aggressive ~40
inv 14(q11;q32) TRA@/TCL1 T-PLL Aggressive 75
17
18
Fluorescence in situ hybridization detection of chromosomal translocations
TranslocationNormal
Gene 1probe
Gene 2probe
Gene 1Bprobe
Gene 1Aprobe
FusionAssay
SplittingAssay
Mantle Cell Lymphoma
BCL-1/IGH PCR
Cyclin D1
CD20+CD43+CD23-
19
FISH detecting t(11;14)
Molecular genetics of hematologic neoplasms
Technologies
• Clonal populations• Rearrangements• Chimeric fusions• Mutations• Splice variants
20
• PCR • Cytogenetics• FISH • CGH arrays• SNP arrays• NGS
Genetic events
21
Current role of molecular diagnostics in hematopathology
WHO 2008 Classification
Diagnosis Prognosis Therapy
Traditional “Genomic” Technology: Metaphase Cytogenetics
• Drawbacks– Requires actively dividing cells for analysis– Cannot be performed on archived tissue– Lower limit of resolution of approximately 2,500 kb for
abnormalities
• Benefits– Routinely performed in many centers– Excellent for detecting large numerical and structural
abnormalities– Can detect balanced abnormalities– Allows distinction between different clones
22
Impact of New Genomic Technologies on Hematopathology
High density aCGH/SNP arrays
Next generation sequencing
23
Array Comparative Genomic Hybridization
24
High Density SNP Array
25
aCGH and SNP Array• Benefits
– Allows resolution of ~10 ‐ 50kb, depending upon coverage– Far better than metaphase karyotyping to identify small copy
number changes– Can be performed on archival tissue, with some loss in quality– SNP arrays allow detection of copy‐neutral loss of
heterozygosity (acquired uniparental disomy)
• Drawbacks– Are unable to identify balanced translocations– Identify many benign CNVs—paired normal tissue helpful to
identify somatic changes in tumors
26
BM
CD3
SNP array detection of aquireduniparental disomy
27
PRIVATE SECTOR PUBLIC SECTOR
29
Next generation sequencing
Human Genome Project
Rapid development in cost and quality trends of DNA sequencing
30
Sanger sequencing
PCR followed by cycle sequencing with dNTPs/ddNTPS
Electrophoretic separation of chain termination products
31
Traditional DNA “Sequencing” MethodsMethod Basic Technique Sensitivity Advantages DisadvantagesSanger “chain‐terminationmethod”
Fluorescent dye–labeled bases; DNA fragments separated by capillary electrophoresis
High “Gold standard”; complete sequence
Time‐consuming; cannot detect deletions, translocations, copy number changes
Pyrosequencing“sequencing by synthesis method”
Chemiluminescentdetection; DNA polymerase synthesizes DNA complementary to a target template; pyrophosphate release detected at each base addition
Higher More sensitive than Sanger; provides percentage of mutated vs wild‐type DNA; works well with fragmented DNA from FFPE samples
Short length reads limit technique to hot spots; limited accuracy detecting changes in homopolymer runs; scalability limited com‐pared with other NGS methods
Mass spectroscopy–based mutation analysis (MALDI‐TOF)
Primer extension with chain termination using PCR amplicon templates identifies variant alleles
Higher High sensitivity; high resolution of DNA fragments; detects frame‐shift mutations; readily identifies germline SNPs
Mass spectrometry resolution window balanced with PCR amplicon design requirements combine to limit scalability
Allele‐specific RT‐PCR
Primers span DNA sites of interest; probes detect specific mutations
Higher Very high sensitivity; widely used for clinical testing for oncogene mutations in CRC, NSCLC
Scalability constraints limit application to hot spots
RT‐PCR melting curve analysis
Heterogeneous DNA PCR products melt at temperatures different from those for homogeneous DNA PCR products
High High sensitivity; provides percentage of mutated vs wild‐type DNA
Often difficult to resolve differences in melt curves; difficult to standardize; multiplex capability limited
32
Next‐generation sequencing
• Massively parallel sequencing
• Powerful approach to DNA sequencing
• Dramatic reduction of cost‐per‐base and time
• Disruptive technology resulting in dramatic change in rate of new discoveries
33
NEXT GENERATION SEQUENCING
DNA sequencingTranscriptomesequencing
Gene regulation and control analyses
Whole genome/Exomesequencing/resequencing
De novo sequencing/Targeted
resequencing
SNP, CNV, deletion, Translocations‐Chromosomal
rearrangement
Tag profiling
Small RNA discovery
mRNA seq
Mapping reads Non mapping reads
Gene expression
SNP discovery
Fusion transcripts
Alternate splicing
Viral genome
ChIP Seq
DNA protein interaction
Genome wide methylation
34
Comparison of Sanger and Next‐Generation Sequencing Methods
High‐throughput Sanger• Multiple copies of single
fragment produced• ~100 fragments sequenced
in parallel• Each fragment ~1kb• 0.1 Mb of sequence per
“run”
Next‐generation• Multiple copies of multiple
fragments produced• Millions of fragments
sequenced in parallel• Each fragment 50‐100bp• >100 Gb of sequence per
“run”
35N Engl J Med. 2010 May 27;362(21):2001-11
A number of technical platformsbut similar strategy
Miniaturization of individual sequencing chemical reactions to:
– overcome limited scalability of Sanger sequencing
– overcome bottlenecks of library preparation and template preparation
– allows millions of individual sequencing reactions to occur in parallel
– results are high volumes of short read sequence data in unprecedented detail and single‐nucleotide resolution
36
Next Generation Sequencingsequence DNA fragment library in situ
A T C G
Massively paralleled configuration of sequences
37
Read= A short length of sequence
Example: Illumina Hi‐ Seq
• Each read is 100 bp
• 160M of these reads!
• Need to connect sequences to each other
38
Disruptive Technology in Drivers of Medical Genomics
Time Period Genomes Turn‐around time FTEs Cost per genome
1990‐2003 1.NIH reference ~5 years ~2,000 ~$2‐3 billion2.Celerareference
2003‐2009 ~10 additional ~6 months Dozens $300,000→38,000
2010‐2014 103‐105 4 weeks 3‐4 $ 6,000 exome$ 9,500 genome
2015‐2020 Millions 6‐0.5 hr <<1 $1000‐250
39
Whole Genome Sequencing
Human genome = 3.1 billion base‐pairs
WGS – determining the sequence of an individuals genome
Includes sequence of the genes – exons & introns
Includes sequence of regions between genes
40
Adapter ligation
Flow cellcDNA Fragmented
cDNA
QC analysis
mRNA
Whole Transcriptome Sequencing
Paired end sequencing
41
RNAseq
Exome sequencing: sequencing the coding region of all genes
• Human Genome: 20K to 25K genes
• Genes composed of exons that code for AA of a protein
• Introns are spacer regions that are spliced out
• Can interpret a change in AA sequence such as an Arg to a stop codon
42
Whole Exome Sequencing
• Exons are shown as colored part of gene
• Capture array has complementary sequence of each exon bound to solid support
• Single strand DNA of exons hybridize • Selected DNA sequenced • 1% of the genome
43
WES vs WGS vs RNAseq
Whole exome Whole genome Whole transcriptome
Coverage of genome 1% Better coverage mRNA, tRNA,
Cost* $600‐800 $1000‐3000 $700‐800
Genome Only coding region
Coding and noncoding mRNA, tRNA, alternativetranscripts
Bioinformatics Better developed Challenging Better developed
Applications SNV, indels Rearrangements, SNV, indels
Rearrangements, inversions, SNV, indels,GEP, alternative spliced variants
44
Commercial Next‐Generation Sequencing Platforms
Technologic Basis Application Time RequiredInstrument Cost($, thousands)
Illumina Flow cell‐based, reversible dye termination, and4‐color optical imaging
Whole exomesequencing; whole genome sequencing; SNP detection
Intermediate, 4 d fragment; 9 dpaired end
500‐900
454 Pyrosequencing
Emulsion PCR with bead‐based pyrosequencing and CCD light imaging
Targeted exon sequencing; confirmatory sequencing; SNP detection
Fast, <1 d 500‐700
Helicos Oligo‐dT captured PolyA‐tailed DNA fragments; flow cell 4‐color dNTP optical imaging
Single molecule sequencing; whole genome sequencing
Slowest, 8 d 999
SOLiD Sequential dinucleotide ligation; flow cell‐based 4‐color optical imaging
Whole exome sequencing; whole genome sequencing; SNP detection
Slow, 7 d fragment run; 14 d paired end
600‐700
Ion Torrent Semiconductor‐based nonoptical detection; standard dNTP sequencing chemistry
Targeted sequencing projects not demanding deep sequencing
Fast, <1 d 50
45
Performance characteristics of NGS platforms
100 1000 10000 100000
1 Mb
100 Mb
1 Gb
10 Gb
100 Gb
Read Length bp
Total datayield
Sanger
Roche454 PacBioRS
Ion Torrent
IlluminaHiSeq2000
Ion Proton
IlluminaHiSeq2500
White = frozen, orange = FFPE
MR Schweiger et al. PLoS ONE 2009;4:e5548 M Kerick et al. BMC Medical Genomics 2011;4:68
Targeted high throughput sequencing of FFPE tissuesis feasible
47
Novel alterations in lymphoid neoplasia identified by NGS
• Chronic lymphocytic leukemia
• Splenic marginal zone lymphoma
• Hairy cell leukemia
• Follicular lymphoma
• Diffuse large B‐cell lymphoma
• Burkitt lymphoma
• Peripheral T cell lymphoma, NOS
• Angioimmunoblastic T cell lymphoma
• Cutaneous anaplastic large cell lymphoma
• Clonal evolution
• Tumor heterogeneity
48
2015
Bioinformatics Analysis of Genome Sequencing Data
The steps from sample processing to result interpretation
Sample
DNA
FASTQ
BAM
VCF
aVCF
Interpretation
ATGACGATGATAGGATGA
chr19: 17,949,097 ATGACGATATAAGATGA
chr19: 17,949,108 G A Ref: 35 Alt:39 GQ: 99
chr19: 17,949,108 G A JAK3 p.M511I MISSENSE NM_000215
Known pathogenic mutation in T-cell leukemia with targeted therapeutic
What are the barriers to automated genome sequencing interpretation?
Genotype-phenotype correlations are known for 500+ conditions
Increasingly, these data are available in public databases…
True Positives and
True Negatives
Diagnosis
Genome Sequencing DataThe challenges associated with automated interpretation
Negative Positive
False FN FP
True TN TP
False Negativespotentially missed diagnoses
True Negativeswhat can safely be ignored
False Positivestoo much to look at
True Positiveswhat needs to be acted on
Sensitivity how often positive when it should be?
Specificity how often negative when it should be?
Positive Predictive Value how often positive if test result is positive?
Negative Predictive Value how often negative if test result is negative?
Practical Aspects of NGS in the Clinical Laboratory
Rigorous Validation Required!
• Verify all secondary mutations identified during validation using known standards
• Run normals – Tissues and HAPMAP cell line
• Orthogonal and parallel platform validations
• Bioinformatics Validation
• Variant database construction
• External Proficiency and User Community Groups
Targeted Sequencing Assays
Recommendations for Development and Validation
Ion Torrent 50 gene panel – depth & uniformity
8 specimens on 316v2
8 specimens on 318v2
Ion Variant Caller Report
Mixed Mutation Control
Cell block mix of 4 engineered cell linesWe extract DNA from scrollsCost effectiveMutation levels determined/verified via digital droplet PCR
KRAS – G13D (16.5%) and G12D (5.5%)FFPE reference cell line mixture; mutations quantified via digital PCR8 specimens on Ion Torrent 316v2 chip
KRAS G12D
KRAS G13D
KRAS – G12D low‐level
KRAS G12D
FFPE tissue sequenced via Sanger at ~7% mutation8 specimens on Ion Torrent 318v2 chip
MPL W515L (low‐level)
MPL W515L
Peripheral blood tested positive for W515L via allele‐specific PCRTested negative via Sanger sequencing
DL‐12‐16233
Conclusions
• NGS is a disruptive technology
• NGS is transforming the routine practice in molecular pathology and personalized diagnostics
• Robust bioinformatics infrastructure is essential in NGS implementation
• Pathologists should embrace this platform for integration in all aspects of Diagnostic Pathology
QUESTIONS?
AgendaTopic Speaker Time
I. Introduction Lim 5’
II. Principles of personalized medicine and evolution of molecular hematopathology-Overview of commonly used molecular tests in hematopathology-Update in new technologies including array CGH, next generation sequencing
Elenitoba-Johnson
40’
III. Approach to myeloid neoplasms in the era ofpersonalized medicine-Chronic myeloid leukemia-Acute myeloid leukemia
Bagg 65’
IV. Approach to NHL in the era of personalized medicine-Aggressive B-cell lymphomas-Splenic Lymphoma-ALCL
Lim 60’
IV. Summary and Closing Lim 10’61
Impact of Personalized Medicine for the Practice of Hematopathology
62
Approach to myeloid neoplasms in the era of personalized medicine
Adam Bagg MDUniversity of Pennsylvania
Case #1
30-year-old previously healthy female patient
- nonspecific abdominal pain - noted to have a leukocytosis on a routine CBC- a bone marrow biopsy was performed
- digital whole slide images of PB and BM Bx
• What is the favored diagnosis based upon the clinical
presentation and peripheral blood smear morphology?
What is the role of performing a bone marrow biopsy in
this case?
• What additional studies would you perform to confirm
the diagnosis?
Case #1 – Questions
• The patient read on the internet that the presence of
ABL1 mutations might affect her response to therapy,
and wants to be tested for these; should she be
tested now? What are the indications for testing for thesemutations?
• How should the patient be monitored after initiation of
therapy?
Case #1 – Questions
CML - a peripheral blood diagnosis
Major flavors of myeloid neoplasms
AML
MPN
MDS
MPN/MDS
MALNWEAAOPPOF
Myeloproliferative -vs- Myelodysplastic
bone marrowhypercellularity
MPNhigh
peripheralcounts
MDSlow
peripheralcounts
quantitativelyincreased
hematopoiesis
effective
ineffective
neoplastichematopoietic
stem celldisorders
CEL Mastocytosis
UnclassifiableCNL
Flavors of myeloid neoplasms (non-AML, non-MDS)
CML
Ph-neg MPNs“Classical”
“Non-classical”
MPN
CMML
JMML
aCML
Unclassifiable
PDGFRA PDGFRB FGFR1
MPN/MDS
MALNWEAAOPPOF
PV
ET
PMF
Mast cell
SM
MALNWEAAOPPOF
CML
PV ET PMF
BCR-ABL1
JAK2JAK2
MPL
KIT
PDGFRAPDGFRBFGFR1
CEL
CNL
CMML JMML aCML
TET2ASXL1
SRSF2
PTPN11
RASNF1
CALR
CSF3R
SETBP1
The (other) Philadelphia story
• median age ~ 67 years
• non-specific symptoms- fatigue/weight loss/LUQ discomfort- routine CBC (~40%)
• splenomegaly (~90%)
• is a peripheral blood diagnosis [but defined by genetics]- marked leukocytosis (> 50 -100 x 109/l) - granulocytes in all stages of maturation (but < 10% blasts)- [need to rule out a “leukemoid reaction”]- basophils- platelets , N or - hemoglobin mildly
Chronic myelogenous leukemia
Blast phase
CML is (was) a triphasic disease
Accelerated phase
Chronic phase
Genetic testing in CML
Diagnosis
Monitoring
Resistance
Diagnosis
Monitoring
Resistance
? CML cytogenetics
CML ~2.5% -~2.5% + CML
Avoid term“Ph-neg” CML
(no such thing!)
? CMML? aCML
? BCR-ABL1
~95% +
CML
~5% -
moleculargenetics
? t(9;22)
CML diagnosis: t(9;22) and BCR-ABL1
? CML cytogenetics
CML ~2.5% +
? moleculargenetics
YES!molecular target for:
1] Rx2] MRD
? cytogenetics
YES!- clonal “evolution”
[Ph+; Ph- with imatinib]
? BCR-ABL1
~95% +
CML
~5% -
moleculargenetics
? t(9;22)
CML diagnosis: t(9;22) and BCR-ABL1
Woessner et aCancer J 201117; 477-486
ROS
Genomic Instability
Progression
TKDmutations
Monitoring
Molecular testing in CML
Diagnosis
Monitoring
ResistanceResistance
CML monitoring
Two major forms of therapy …
TKI
SCT
• Initial therapy of choice• Does not eradicate/cure CML (?)• ? Long-term outcome• Minimal toxicity1
1 cytopenias, GI/pancreas, fluid retention, myalgia
• No longer 1st line Rx2
• Only Rx that cures CML3
• Major toxicity and mortality4
• Not available to all5
BCR-ABL1reduction
BCR-ABL1negativity
Rx goal (?)
2 indicated when [i] very young, [ii] (selected)TKI failure, [iii] AP an3 10-yr survival ~65%4 10-20% mortality even when low risk 5 need to have a donor!
CML monitoring
sensitivities…modalities…
CBC ~10% [~10-1]
conventional cytogenetics
~5% [~10-2]
D-FISH ~0.5% [~10-3]
RT-PCR [quantitative] ~0.001% [~10-5]
RQ-PCR [qualitative] ~0.001%* [~10-5]* non-nested; can reach 10-8 with nested RT-PC
CML monitoring: definitions of response
complete hematologic
• platelet: < 450• WBC: < 10• diff: no immature granulocytes• basos: < 5%• clinical: non-palpable spleen
cytogenetic # Ph+
• none: >95% • minimal: 66-95%• minor: 36-65% • partial: 1-35% • complete: 0%
molecular
• next slide please …{major
diagnosis
complete hematologic remission
complete cytogenetic remission
major molecular response(MR3.0)
“complete molecular remission”undetectable transcript
(MR4.5)
1012
<1010
<109
<107-8
100
<0.1
<0.01
responses BCR-ABL1ratio
… yet overall survival ~85%
imatinibresponses
~98%
~85%
~75%
~10%
logreduction
<1011
>3
# cells
24% @ 6 mo39% @ 1 yr55% @ 2 yr86% @ 8 yr
? up to 40-50%800mg vs 400mg
but imatinib failure-free survival ~60% (SE, LOR)
The sooner the better …
• predicting adverse outcomes if don’t reach landmarks:- 18 months <3 log reduction (> 0.1%) = MMR- 12 months <2 log reduction (>1%)- 6 months <1 log reduction (>10%)
• likelihood of an adverse event, if achieve MMR by:- 12-18 months ~15%
- 6-12 months ~8%
- 6 months ~0%
• thus, the quicker the response, the better the outcome
- testing as early as 3 months (or even 1 month) may be predictive!level >10% at 3 months (ie, < 1 log reduction) particularly poor
RQ-PCR: Ongoing attempts at harmonization
international scale
conversionfactor
internationalstandard
• standardized baseline from original IRIS trial• pool of 30 CML cases
• sample exchange between your lab and IRIS lab• analogous to INR
• WHO initiative—freeze dried preparations • 4 serial dilutions of K562 into HL60:
10%, 1%, 0.1%, 0.01% • also insufficient amounts of this!• use restricted to:
reference laboratoriesmanufacturers of secondary reference materi
rapidly exhausted
Molecular testing in CML
Diagnosis
Monitoring
Resistance
Resistance to imatinib (and other TKIs …)
primary • failure to achieve an initial response
• uncommon--seen in ~5% of pts
• bioavailability; not due to mutations
secondary • commoner--usually due to mutations
• response loss of response (especially early on)- 1st 3 years on Rx: ~5%/year
- 2nd 3 years on Rx: ~1%/year
• hence 1st 3 years most important monitoring perio- most likely to encounter mutations
• incidence of mutations: depends on disease phas
Mechanisms of resistance to imatinib
BCR-ABL1independent
BCR-ABL1dependent
1. Kinase domain mutations:- most common cause of resistance [~60% overall]
- spans ~240 aa’s
2. BCR-ABL1 amplification:- genomic > transcriptional [~10%]
3. Clonal evolution:- other genetic/cellular pathways [LYN/JAK-
STAT/SRC/GAB2/PI3K]4. ↓Bioavailability:- absorption- metabolism [hepatic]- plasma binding [1acid glycoprotein sequestration]- ↓influx ↑efflux [MDR1, PGP, BCRP2/ABCG2, hOCT1,
MRP1]5. 7. BIM6. TKI BCL6
T315I
Kinase domain mutations
P = P loop- ATP-binding site- ? worst mutations
B = Binding domain- where imatinib binds
C = Catalytic doma
A = Activation loop- conformation altered- affects imatinib bindi- closed: inactive- open: active
2-10% of patients
>10% of patientsgreen
red
> 100 different mutations[ABL1 exons 4-10]
these 6 accountfor > ~65% ofall mutations
~15-20%
T315I
Kinase domain mutations
• MUST test for specific mutations before changing Rx to
determine appropriate action
1. do nothing … may be innocuous (SNPs)2. increase dose of imatinib3. switch to 2nd (or 3rd) generation kinases
inhibitor ** type of mutation will dictate which one to use
4. stem cell transplant5. alternative Rx/clinical trail
• If a mutation is found, options include:
• If a mutation is NOT found:1. consider compliance failure 2. if due to other biologic causes problematic
Indication for mutation testing
treatment failure/suboptimal response
loss of response
anyone in AP or BP
• no CHR by 3 months• no PaCR by 6 months• no CCR by 12 months• no MMR by 18 months
• hematologic relapse• cytogenetic relapse• ↑ing BCR-ABL1 levels [1-log]
No role at CP diagnosis* NEVER change Rx based upon a single result:(1) short-term lapse in compliance(2) assay variation
P h l i i ti ith l f
*
• BCR/ABL1 >10% at 3 month
Methods of mutation testing
Technology Sensitivity
Specificity Bias
Sanger sequencing 15-25 ++ no
Subcloning and sequencing 10 +++ noD-HPLC 0.1-10 ++ noPyrosequencing 5 ++ noDouble-gradient denat. electroph.
5 ++ no
Fluorescence PCR – PNA clamping
0.2 ++ yes
ASO-PCR 0.01 ++ yesSmall clones (<20%) may not be clinically signific
Sanger sequencing (current) method of chto detect, then pyrosequencing to quantify
Emerging data to support more sensitive detectio if have T315I @ 10-5 precludes MMR!
High resolution meltingNanofluidicsNext gen. sequencingMass Spec
Overcoming resistance
• Alternative kinase inhibitors:- 2nd generation TKIs: nilotinib- dual SRC-ABL inhibitors: dasatinib
bosutinib - multikinase (KIT/PDGFRA/FLT3/FGFR1) inhibs: ponatinib- aurora kinase inhibitors: XL-228
MK-0457
• Others:- protein synthesis inhibitors: omacetaxine- switch pocket-control inhibitors: rebastinib (DCC-2036)
• ImmunoRx:- vaccines against: BCR-ABL1, PR1, WT1, HSPs- 1o role in MRD Rx
• Targeting the stem cell:- not dependent on BCR-ABL1 WNT-CTN/HH/JAK2-PP2A/TGF-
**
* FDA approved
**
∞ active vs T315I
∞
∞
∞
∞
∞
Overcoming resistance
• Alternative kinase inhibitors:- 2nd generation TKIs: nilotinib- dual SRC-ABL inhibitors: dasatinib
bosutinib - multikinase (KIT/PDGFRA/FLT3/FGFR1) inhibs: ponatinib- aurora kinase inhibitors: XL-228
MK-0457
• Others:- protein synthesis inhibitors: omacetaxine- switch pocket-control inhibitors: rebastinib (DCC-2036)
• ImmunoRx:- vaccines against: BCR-ABL1, PR1, WT1, HSPs- 1o role in MRD Rx
• Targeting the stem cell:- not dependent on BCR-ABL1 WNT-CTN/HH/JAK2-PP2A/TGF-
**
* FDA approved
**
∞ active vs T3151
∞
∞
∞
∞
∞® ~20% vascular/
thrombotic problems®
80 -
100 -
60 -
20 -
40 -
0 -
CCR (1 yr) MMR (1 yr)
66%
28%
77%
46%
CCR (1 yr) MMR (1 yr)
65%
22%
80%
44%
2nd generation TKIs as 1st line therapy
imatinib 400mg
dasatinib 100mg
imatinib 400mg
nilotinib 300mg x 2
Genetic testing in CML: summary
Diagnosis
Monitoring
Resistance
CC BM PB FISH instead is not ideal
RT-PCR PB qualitative (not quantitative) with characterization
CC BM 3-6 monthly until CCR6-12 monthly thereafter
FISH PB ? before achieve CCR (not ideal)
RQ-PCR PB 3 monthly until MMR, then 6 monthly (ELN)
directsequencing
PB vBM
Rx failure, loss of response, accelerated & blast phase
Survival in newly-diagnosed CP CML by year of Rx
<19751975-1982
>2001
1991-2000
1983-1990
Kantarjian H et al. Blood 2012;119:1981-1987
• What is the favored diagnosis based upon the clinical
presentation and peripheral blood smear morphology?
What is the role of performing a bone marrow biopsy in
this case?
• What additional studies would you perform to confirm
Case #1 – Answers
-- chronic myelogenous leukemia, BCR-ABL1positive
-- best shot at getting metaphases
-- qualitative RT-PCR (FISH if negative or complex)
• The patient read on the internet that the presence of
ABL1 mutations might affect her response to therapy,
and wants to be tested for these; should she be
tested now?
• Would you be surprised if cytogenetics by metaphase
analysis showed a normal karyotype?-- a little (~ 2-3% missed)
-- probably not, but things may change
Case #1 – Answers
What are the indications for testing for thesemutations?
• How should the patient be monitored after initiation of
therapy?
Case #1 – Answers
-- failure to reach milestones-- loss of response-- accelerated phase or blast phase
-- CBC-- BM cytogenetics-- RQ-PCR frequency depends on attainment of
milestones
• How will the finding of a mutation affect subsequent
therapy?
Case #1 – Answers
-- do nothing … may be innocuous-- increase dose of imatinib-- switch to 2nd (or 3rd) generation kinases inhibitor
** type of mutation will dictate which one to use-- stem cell transplant-- alternative Rx/clinical trail
Case #2
57-year-old male patient--recent onset of bleeding gums
- CBC: HGB 10.2 g/dLWBC 7.4 x 109/L PLT 21 x 109/L
- abnormal cells (79 %) on peripheral blood sme- bone marrow aspirate/biopsy performed
- digital whole slide images of PB and BM Bx
• What is the favored diagnosis based upon the
clinical presentation and morphology?
• What standard additional studies would youperform to confirm and refine the
diagnosis, andwhich of these are likely to have the
greatestimpact on prognosis and therapy?
Case #2 – Questions
• Would you be surprised if cytogenetics bymetaphase analysis showed a normalkaryotype?
Case #2 – Questions
• How might the finding of a normal karyotype
affect what additional genetic tests you might
order?• In addition to the molecular studies ordered as
per questions 2 and 4 above, what other genetic
analyses are likely to be useful in terms ofprognosis and therapy?• What technology might be best to test for
allthese mutations?
Acute myeloid leukemia
morphology
cytochemistry
immunophenotype
the most important diagnostic test inacute leukemia
(cyto)genetics
Acute leukemia
Risk group Genetics Frequency CR 5yr Sur
t(8;21)Favorable t(15;17) ~25% ~85% ~60%
inv(16)
normalIntermediate +8, +21 ~50% ~80% ~40%
t(9;11)
-5, -7Unfavorable inv(3)
complex ~25% ~60% ~15%monosomal11q23t(6;9)
M2M3M4Eo
M5
dysmeg
basophilia
AML: Cytogenetic stratification
AML: Cytogenetic stratification
100
60
80
40
20
1 32 4 5 6 7 years
surv
ival
intermediate
unfavorable
favorable
AML: Cytogenetics
The big 4 sine qua non for WHO
• t(15;17) [M3] ~10%• t(8;21) [M2] ~10% GOOD• inv(16) [M4Eo] ~10%
• t(11q23)* [M4/M5] ~5 - 10% not so go
© 2001 2008 = big 7 [added t(1;22), t(6;9) and inv(3); t(9;11) specifically]
* promiscuous (>60 partners)
}}
AML: Predicting the genetics
Translocation Partner Frequency ATRA-responsive
t(15;17)(q24;q21) PML >99% +t(11;17)(q23;q21) ZBTB16 0.5% -t(11;17)(q13;q21) NUMA <0.5% ?+t(5;17)(q32;q21) NPM <0.5% ?+t(17;17)(q21;q21) STAT5B <0.5% ?
The various genetic flavors of APL
Non-t(15;17)-positive cases not “APL”
Another morphologic flavor of APL
AML with t(8;21)(q22;q22); RUNX1-RUNX1T1
AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB-MYH11
AML with t(6;9)(p23;q34); DEK-NUP214
AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2); RPN1-EVI1
AML: Cryptic abnormalities
t(8;21)RUNX1-RUNX1T1
inv(16)CBFB-MYH11
t(15;17)PML-RARA
• ~ 30% of positive cases lack M4Eo morphology• upto 30% with M4Eo morphology are:
- RT-PCR/FISH- cytogenetics
• some M4Eos do not have inv(16) at all
• upto 30% are:- RT-PCR/FISH- cytogenetics
• upto 15% are:- RT-PCR/FISH
t ti
+-
+-
+-
Mutations in AML
15 1614 20191817
ECD JM TK2 TM
*
400bp
300bp
350bp
FLT3ITD heterozygous FLT3ITD hemizygous
+ -FLT3WT
FLT3 : genotyping
FLT3 : multiplex PCR on CE
ITDe14/e15
e20/ECoRV D835
• One of the most common known molecular targets in
• Possibly most prognostically relevant molecular lesion- quantification is important (mutant:wt)
• A potentially useful MRD target- patient specific primers vs ITD- ITD may [rarely] not be stable between presentation and re
[+ -] [- +] [+A +B] [+ ++]
• A therapeutic target- a la imatinib mesylate in CML
FLT3
NPM1
• most commonly mutated gene in AML- ~60% of cytogenetically normal AML- ~30% of all AML- shuttles between nucleus and cytoplasm (lives in nucleus)- mutations (typically 4bp insertion):
* lose nucleolar localization signal/creates export signal
- good prognosis:* but only if FLT3 wild type (and perhaps if IDH2 mutant?)
without IHC IHC—wild type IHC—mutant
AML: Genetics – WHO 2008
Karyotype
Genes Morphology
Frequency
Prognosis
t(8;21) RUNX1/RUNX1T1
M2 ~5% good
t(15;17) PML/RARA M3 ~5-8% good
inv(16) CBFB/MYH11 M4Eo ~5-8% good
t(9;11) MLLT3/KMT2A M4/M5 ~2% intermediate
t(1;22) RBM15/MKL1 M7 <1% good
t(6;9) DEK/NUP214 Basophila ~1% poor
inv(3) RPN1/EVI1 MLD ~1% poorNPM1mutations*
M4/M5 ~30% good
CEBPAmutations*
M1/M2 ~10% good* AMLs with mutated NPM1 and CEBPA = provisional entitiesAMLs with FLT3 mutations not an “entity” but testing “strongly recommend
AML genetics – but wait, there’s more…
• explosion of discoveries (NGS, SNPa …)
• enriched in cytogenetically normal (CN) AML• unlike most “AML-defining” translocations:
- may be (variably) seen in other myeloid malignancies
(MDS, MPN, MDS/MPN, 20 AML)• relevant in prognosis may dictate Rx
AML mutations: the NKOTB
TET2
CBL
DNMT3A
IDH1/2
EZH2ASXL1
MLL
WT1
RUNX1SF3B1BCOR
PTPN11
JAK3
ND4
NPM1
CEBPA
FLT3
KIT
Meme K K
me K
hmeC
me
me
IDH1/2
IDH1/2
MLL
EZH2
DNMT3
TET2
gain-of-function loss-of-function
me
ASXL1KMT2A
AML
AML: Cooperative mutations
FLT3RASKIT
PTPN11JAK2
Class I mutations
Signal transducers:• [+] proliferation• [+] survival
Class II mutations
PML-RARARUNX1-RUNX1T1
CBFB-MYH11KMT2A fusions
?NPM1
Transcription factors:• [-] differentiation• [-] apoptosis
Class III mutations
Epigenetics
IDH1/2TET
DNMT3A
AML: But wait, there’s more … 99.5% of cases
Activated signalling
DNA methylation
NPM1 mutation
Chromatin modifiers
Transcription factor fusions
Transcription factor mutations
Tumor suppressors
Cohesin complex
Spliceosome
FLT3 RASs PTPs
IDH1/2 TET2 DNMT2A
KMT2A ASXL1 EZH2
PML-RARA CBFB-MYH11
RUNX1 CEBPA
TP53 WT1 PHF6
RAD21 SMC3 STAG2
SF3B1 SRSF2 U2AF1
~60%
~45%
~30%
~30%
~20%
~20%
~15%
~15%
~15%
AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB-MYH11
+8mKIT
FLT3-TKD
+22
mRAS
15%
35%
15%
20%
50%
}
AML with FLT3 or NPM1 or IDH1/2 mutation
AML: how to manage the nascent data
• has become extraordinarily complex
• reaching the point of TMI
• discovery outpacing clinical trials
• not all are mutually exclusive- some associations and interactions
• how to handle?- algorithms- multiplexing
AML: associations and interactions
Patel JP et al. Integrated genetic profiling in acute myeloid leukemia. N Engl J Med, 2012;366:1079 1089
AML: Cytogenetic stratification
Risk group Genetics Frequency CR 5yr Sur
t(8;21)Favorable t(15;17) ~25% ~85% ~60%
inv(16)
normalIntermediate +8, +21 ~50% ~80% ~40%
t(9;11)
-5, -7Unfavorable inv(3)
complex ~25% ~60% ~15%monosomal11q23t(6;9)
Normalcytogenetics
Cytogeneticallyintermediateprognostic
group
mutation
NPM1 But only if FLT3 WT(and ? IDH2 mutated)
CEBPA But only if biallelic
FLT3 But only if ITD and no WTAnd no effect on t(15;17) TET2
But only if CEBPA WTAnd only if R882But benefit from hi dose dauna
DNMT3A
PHF6
ASXL1
AML: Cytogenetic molecular stratification
• What is the favored diagnosis based upon the
clinical presentation and morphology?-- acute leukemia
• What standard additional studies would youperform to confirm and refine the
diagnosis, andwhich of these are likely to have the
greatestimpact on prognosis and therapy?
-- flow cytometry-- cytochemistry [?]
Case #2 – Answers
Case #2 – Answers
• Would you be surprised if cytogenetics bymetaphase analysis showed a normalkaryotype?
-- no, they are normal in ~45%
• How might the finding of a normal karyotype
affect what additional genetic tests you might
order?-- FISH or RT-PCR for recurrent,
disease
Case #2 – Answers
• In addition to the molecular studies ordered as per
questions 2 and 4 above, what other genetic analyses are likely to be useful in terms ofprognosis and therapy?
-- mutational analysis of ~10-30 other genes [IDH1, IDH2, TET2, ASXL1, DNMT3A, RUNX1,
KIT, TP53, NRAS]
• What technology might be best to test for all these
mutations?
QUESTIONS?
AgendaTopic Speaker Time
I. Introduction Lim 5’
II. Principles of personalized medicine and evolution of molecular hematopathology-Overview of commonly used molecular tests in hematopathology-Update in new technologies including array CGH, next generation sequencing
Elenitoba-Johnson
40’
III. Approach to myeloid neoplasms in the era ofpersonalized medicine-Chronic myeloid leukemia-Acute myeloid leukemia
Bagg 65’
IV. Approach to NHL in the era of personalized medicine-Aggressive B-cell lymphomas-Splenic Lymphoma-ALCL
Lim 60’
IV. Summary and Closing Lim 10’142
Impact of Personalized Medicine for the Practice of Hematopathology
Approach to non-Hodgkin lymphoma in the era of personalized medicine
Aggressive B-cell lymphomasSplenic lymphoma
Anaplastic large cell lymphoma
Megan S. Lim MD PhDUniversity of Michigan
143
Case 3
• A 40 year old woman presented with enlarging neck lesion. The biopsy of the right neck lymph node revealed a soft tan lymph node with dimensions of 3.2 x 1.6 x 0.6 cm.
• An initial immunohistochemical evaluation reveals a B cell lymphoma (CD20 and CD10 positive). A virtual slide of the sections of the lymph node is provided.
144
Questions • What are the differential diagnostic considerations?• What additional stains would you perform to refine the
diagnosis?• What additional molecular studies may help refine the
diagnosis?• What is the role of karyotypic analysis, FISH and gene
rearrangement in the workup of this class of lymphoma? • What other lymphoid neoplasms demonstrate MYC
aberrations? • What genetic abnormalities have been recently described
in the group of aggressive B‐cell lymphomas?
145
Case 3: H&E
146
BCL2 BCL6
CD3
CD10
CD20 Ki67
Diagnosis ?
Diffusely + Background T cells >95%
Faintly variably + Diffusely +Small subset +147
WHO classification 2008Aggressive B‐cell Lymphoma
• Diffuse large B‐cell lymphoma, not otherwise specified
• Diffuse large B‐cell lymphoma, subtypesT‐cell/histiocyte‐rich large B‐cell lymphoma• Primary mediastinal (thymic) large B‐cell lymphoma• Intravascular large B‐cell lymphoma• DLBCL associated with chronic inflammation (New entity)• ALK‐positive B‐cell lymphoma• Plasmablastic lymphoma• Large B‐cell lymphoma arising in HHV8‐associated with Castleman disease• Primary effusion lymphoma
• Burkitt lymphoma• B‐cell lymphoma, unclassifiable, with features between DLBCL and Burkitt lymphoma
148
Differential Diagnosis of Aggressive B cell Lymphomas
Histology Immunophenotype Proliferative Index
MolecularGenetics
DLBCL Large centroblasticcells
CD5+/-, CD10+/-CD20+, BCL2+, BCL6+, CCND1-
<90% t(14;18) ~20%, t MYC (10%) 3q27 (~20%)
Burkittlympoma
Intermediate monotonous cells multiple nucleoli
CD5-, CD10+, CD20+, BCL2-,
BCL6+, CCND1 -
>95% t MYC ~100%
Blastoid mantle cell lymphoma
Intermediate lymphoblast like cells
CD5+, CD10-, CD20+, BCL2+, BCL6-, CCND1+
<90% t(11;14)~100%
B-cell lymphoma, unclassifiable, with
features between DLBCL and Burkitt
lymphoma
Intermediate CD5-, CD10+, CD20+, BCL2-,
BCL6+, CCND1 -
>90-95 t(14;18) ~20%, t MYC (10%) 3q27 (~20%)
149
Classic Burkitt lymphoma
150
EndemicSporadicImmunodeficiency
associated
MYC rearrangements in BL
1518q24
8q24
t(8;14)/t(8;22)/t(2;8) low complexity genotype; no coexisting translocations involving bcl2 or bcl6 or bcl1
B‐cell lymphoma, unclassifiable, with features between DLBCL and Burkitt
lymphoma
• 4% of NHLs• Extranodal disease• High proliferative index• Low median survival• Ig/Myc rearrangements or variant myc partners • Includes “double‐hit” lymphomas
152
Double Hit Lymphomas
• Aggressive B‐cell lymphomas harboring MYC/8q24 gene rearrangement with another recurrent breakpoint
• BCL2/IGH + MYC/8q24 DHL 60%• BCL6/IGH + MYC/8q24 DHL 8%• CCND1/IGH + MYC/8q24 DHL 10%• BCL2/IGH + BCL6/IGH DHL Rare
153 Aukema SM et al., BLOOD 2010
DLBCL BL BLU
Age at presentation Usually older but can occur at any age
Children, young adults Older adults
PathogenesisMay be related to the germinal center (GCB), activated B cell or other pathway
GCB derived GCB derived
Growth rate RapidExtremely rapid, Ki67 approaching 100%
Extremely rapid but usually less than 100%
Stage Even distribution, 50% stage 1 or 2
Usually high stage Usually advanced III/IV
Bone marrow involvement
Uncommon, often terminal Common Common
CNS involvement UnusualLeptomeningeal disease common at presentation in children and adults60
Common
EBVUncommon in the absence of immunodeficiency or age‐related senescence
>90% in endemic BL 40% in sporadic and HIV‐related BL
Negative
MYC translocationUncommon, usually a secondary event associated with a complex karyotype
Almost always present as initiating event and single abnormality (MYC simple)
Often double hits with translocations involving MYC, plus BCL2 and/or sometimes BCL6
DLBCL, BL and B-cell lymphoma unclassifiable, with features intermediate between DLBCL and
BL (BLU)
Modern Pathology (2013) 26, S42–S56;154
Is it important to make the distinction between DLBCL, BL and DHL?
155
MYC positive diffuse large B‐cell lymphoma and poor survival
156
CD20
Savage et al. Blood 2009
Ki-67
“Double Hit” Lymphoma
Snuderel et al. AJSP 2010
BL
DLBCLDHL
BACK TO OUR CASE
158
Diagnostic Algorithm
Aggressive B‐cell lymphoma FISH panel• t(14;18) BCL2/IgH
• 3q27 rearrangement BCL6/IgH
• 8q24 rearrangement CMYC/IgH
• t(11;14) BCL1/IgH
159
DDX of BL or BCLU
HistologyImmunohistochemistry
Normal
2G2R
Follicular Lymphomat(14;18) IGH/BCL2
GRFF
BCL2IGH
IGH/BCL2 dual‐color dual‐fusion FISH
160
Abbott MYC Dual Color Break Apart Probe
161
Normal MYC rearrangement
Breakpoint Region
Abbott Molecular
Normal cell (FF) BCL6 translocation +VE (FGR)
telChr 3q27 cenBCL6 gene
TranslocationBreakpoints
Red Probe Green Probe
BCL6 Break-Apart FISH
Integrated interpretation
163
Diagnosis: B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and Burkitt lymphoma (Double-hit)
Aggressive B-cell lymphoma FISH panel• 8q24 rearrangement MYC/IgH +• 3q27 rearrangement BCL6/IgH +• t(14;18) BCL2/IgH -• t(11;14) BCL1/IgH -
Integrated interpretation
164
Diagnosis: B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and Burkitt lymphoma (Double-hit)
Aggressive B-cell lymphoma FISH panel• 8q24 rearrangement MYC/IgH +• 3q27 rearrangement BCL6/IgH -• t(14;18) BCL2/IgH +• t(11;14) BCL1/IgH -
Integrated interpretation
165
Diagnosis: Mantle cell lymphoma, t(11;14) positive with MYC rearrangement
Aggressive B-cell lymphoma FISH panel• 8q24 rearrangement MYC/IgH +• 3q27 rearrangement BCL6/IgH -• t(14;18) BCL2/IgH -• t(11;14) BCL1/IgH +
Integrated interpretation
166
Diagnosis: Burkitt lymphoma
Aggressive B-cell lymphoma FISH panel• 8q24 rearrangement MYC/IgH +• 3q27 rearrangement BCL6/IgH -• t(14;18) BCL2/IgH -• t(11;14) BCL1/IgH -
BCL6‐breakapart FISH
167
C‐MYC (8q24) breakapart probe
168
Integrated interpretation (Diagnosis)
• 8q24 rearrangement (MYC) –Positive
• 3q27 rearrangement (BCL6) – Positive
• B‐cell lymphoma, unclassifiable, with features intermediate between DLBCL and Burkitt lymphoma (Double‐hit)
• Predicted prognosis ‐ Poor
169
Conclusions• Aggressive B‐cell lymphomas are
clinically, histologically and genetically diverse neoplasia.
• Molecular genetics is necessary to convey important prognostic information relevant for therapy.
• Evaluate all DLBCL with greater than 95% PI and other aggressive B cell lymphomas with the panel of FISH probes.
Double HitLymphoma
DLBCL
BL
MYC BCL2
DHS = 1
DHS = 2Green TM et al., JCO 30:3460-3467: 2012171
MYC/BCL2 immunohistochemistry in DLBCL
MYC/BCL2 immunohistochemistry in DLBCL
• MYC/BCL2 coexpression was reasonably sensitive and specific for MYC/BCL2 double‐hit rearrangements: sensitivity 79%, specificity 81%
• Much better than Ki‐67 index (recent study used 75% as threshold, Maitong‐Kalaw et al., Histopathology 2012;61:1214‐8)
172Johnson NA et al., J Clin Oncol. 2012
Green TM et al., J Clin Oncol. 2012
• Coexpression of MYC and BCL2 identifies a subset of patients with DLBCL (18‐29%) with inferior OS and PFS: “biologic double‐hit”
• Independent of IPI and cell of origin (ABC and GCB)
• Co‐expression of MYC and BCL2 can identify the ABC‐type and IPI‐high DLBCL cases with worse outcomes
• May identify the majority of patients with refractory or resistant DLBCL
173
MYC/BCL2 immunohistochemistry in DLBCL
Impact of novel genetic alterations identified by next generation
sequencing
Aggressive B‐cell lymphomasin 2015
Mutations of ID3 and TCF3 cooperatewith IG‐MYC in BL
ID3 and TCF3mutations in 70% of sporadic BL6/47 (13%) other BCL with MYC‐IG translocation
Cyclin D3 in mutations in 38% sporadic BL .
175
Cooperation between MYC and ID3/TCF3/Cyclin D3 in BL
Schmitz R et al., Nature 2012176
Relative frequency of gene alterations in GCB and ABC DLBCL
Courtesy of Bailey N 2013177
178
Impact of genetic aberrations on pathophysiology of B‐cell NHL
TAKE HOME MESSAGES• True BL has low genetic complexity
• Identification of MYC positive mature lymphoma is important to guide chemo (CVAD vs. R‐CHOP)– mBL signature (t(8;14), low complexity,benefit from CVAD
– Double hit or MYC+ DLBCL (high complexity), less responsive to either
179
Take home messages• Aggressive B‐cell lymphomas are clinically, histologically and genetically diverse neoplasms
• Molecular genetics is necessary to convey important prognostic information relevant for therapy
• Algorithmic approach using immunohistochemistry and FISH analysis will allow subclassification for better risk stratification and treatment
• NGS identifies novel genetic events that may have a role in diagnosis and therapy
180
Answers for Case 3• What are the differential diagnostic considerations?
DLBCL, BL, Double hit lymphoma, MCL, BCL Unc
• What additional stains would you perform to refine the diagnosis?CD20, BCL2, BCL6, MIB1, MUM1
• What additional molecular studies may help refine the diagnosis?FISH for MYC, BCL2 and BCL6 rearrangements
• What is the role of karyotypic analysis, FISH and gene rearrangement in the workup of this class of lymphoma?
Gene rearrangement study would not be helpfulKaryotypic and FISH studies will aid in determining genetic complexity and presence of MYC, BCL2, BCL6 rearrangements
181
Answers for Case 3
What other lymphoid neoplasms demonstrate MYC aberrations?
ALCL, MCL, NK/T cell lymphomasWhat genetic abnormalities have been recently described in the group of aggressive B‐cell lymphomas?
ID3, TCF3, Cyclin D3 mutations in BLCARD11,CD79a,A20 mutations in DLBCL
182
Case 4
• A 53 year old man presented with abdominal discomfort. Physical examination and radiologic studies revealed an enlarged spleen which was removed. The spleen weighed 3.55 kg with dimensions of 25 x 19.5 x11.7 cm.
• A virtual slide of a section of the splenectomyspecimen is provided.
183
Case 4: H&E
184
Case 4• What are the differential diagnostic considerations?• What additional stains would you perform to refine the
diagnosis?• What additional molecular studies may help refine the
diagnosis?• What is the role of karyotypic analysis, FISH and gene
rearrangement in the workup of splenic lymphomas? • What genetic abnormalities have been recently described
in lymphomas that present primarily in the spleen?• How will genetic abnormalities contribute to patient
management such as prognosis, therapy and disease monitoring?
185
Case 4: Differential diagnosis
• Splenic marginal zone lymphoma• Splenic B‐cell lymphoma, unclassifiable• Follicular lymphoma• Mantle cell lymphoma• Chronic lymphocytic leukemia/SLL• Hairy cell leukemia
• T cell lymphoma
186
Case 4: Immunophenotype
CD5 negativeCD43 negativeCyclin D1 negativeCD10 negative
187
Splenic Marginal Zone Lyphoma
• SMZL uncommon indolent lymphoma involving splenic white pulp, blood, and bone marrow
• First-line therapies• splenectomy• anti-B-cell biologicals
• Median survival 10yr
188 Kiel MJ, .. Elenitoba-Johnson KSJ J Exp Med. 2012 Aug27;209(9):1553‐65.
PathoPic
EH&O
Molecular genetics of splenic lymphoma
189
• del7q, +3/+3q [+18, +12] • no recurrent translocation• no known genetic etiology
• del7q, +3/+3q [+18, +12] • no recurrent translocation• no known genetic etiology
Experiment Design: Whole Genome and Targeted Sequencing
190
del7q
del7q
-19
del13q
+8q
SMZL Genome Complexity
191
NOTCH2 Frameshift Mutation
192
Recurrent NOTCH2 Nonsense Mutations
193
Recurrent NOTCH2 Mutations in SMZL
Additional 93 SMZL specimens sequenced in validation cohort.22 additional cases with NOTCH2 mutations identified
Kiel MJ, .. Elenitoba-Johnson KSJ J Exp Med. 2012 Aug27;209(9):1553‐65. 194
Frequency of NOTCH2 Mutations in Various Lymphoma Subtypes and Reactive Lymph Nodes
Kiel MJ, .. Elenitoba-Johnson KSJ J Exp Med. 2012 Aug27;209(9):1553‐65. 195
Recurrently targeted pathways in SMZL
Rossi D et al. J Exp Med 2012;209:1537-1551 196
Decreased Relapse-free Survival in NOTCH2-mutated SMZL
Kiel MJ, .. Elenitoba-Johnson KSJ J Exp Med. 2012 Aug27;209(9):1553‐65. 197
SMZL
SMZL
delta jagged/serrate
γ-secretase
NICD
CSLCoR CSL
NICDCoA
Transcription of target genes:HES, HEY, NF-κB, PPAR
CoR
Notch heterodimer
Notch signaling can be inhibited by gamma secretase inhibitors
DAPT
Cell survival and differentiation
198
ConclusionsConclusions
• NOTCH2 is recurrently mutated in SMZL• Mutations cluster in C-terminus
causing gain-of-function of NOTCH2• NOTCH2 mutations are specific to MZL• NOTCH2 mutations confer an adverse
prognosis
• KLF2 is recurrently mutated in SMZL
199Piva R et al., Leukemia 2014 KLF2
Molecular testing in small B cell lymphomas of the spleen
200
• Splenic marginal zone lymphoma
• Splenic B‐cell lymphoma, unclassifiable
• Nonsplenic MALT• Follicular lymphoma• Mantle cell lymphoma• Chronic lymphocytic leukemia/SLL
• Hairy cell leukemia
Novel alterations in lymphoid neoplasia identified by NGS
201
Recurrently mutated genes in CLL
202
Gene Protein Mutation Mutated cases /
total
Overall frequency
(%)
Frequency in IGHV-
unmutated(%)
Frequency in IGHV-
mutated (%)
NOTCH1 Notch 1 P2515Rfs*4Q2503*F2482Ffs*2
29/2551/2551/255
12.2 20.4 7
MYD88 Myeloid differentiation primary response gene 88
L265P 9/310 2.9 0.8 5.6
XPO1 Exportin 1 E571KE571G
3/1651/165
2.4 4.6 0
KLHL6 Kelch-like 6 F49L/L65PL90FL58P/T64A/Q81P
3/160 1.8 0 4.5
Puente XS et al. Nature, 2011; 475: 101-05.
N‐Ras
B‐Raf
MEK1/2
ERK1/2
A MOLECULAR DIAGNOSTIC APPROACH
BRAF V600E is strongly associated with classic HCL
203
BRAF exon‐15 mutation is sensitive and specific for hairy cell leukemia
204
Cases (n=243)Tumor Entity
analyzed mutated % mutatedHairy Cell Leukemia 48 48 100Splenic Marginal Zone Lymphoma 22 0 0Splenic Lymphoma/Leukemia, Unclassifiable* 16 0 0Chronic Lymphocytic Leukemia 21 0 0Follicular Lymphoma 35 0 0Diffuse Large B-cell Lymphoma 71 0 0Mantle Cell Lymphoma 18 0 0Burkitt Lymphoma 12 0 0
Tiacci E et al. N Engl J Med 2011;364:2305-2315.
N‐Ras
B‐Raf
MEK1/2
ERK1/2
MAP2K1 (MEK) mutations are associated with HCL‐v
205
RAF kinase is a molecular target
206Vakiani and Solit J Pathol 2011: 223:219-229
Improved survival with vemerafanib in melanoma patients with BRAF
mutations
207Chapman PB et al., N Eng J Med 2011: 364:2507-2516
Immunohistochemistry for detection of B‐RAF V600E (clone VE1) mutant protein
208Andrulis M et al., Am J Surg Pathol 2012
B‐RAF V600E (clone VE1) mutant protein IHC for minimal residual disease
209Akarca AU et al., Br J Haematol Jul 2013
Discovery of novel genetic alterations in B‐NHL
210
Traditional NGSBL MYC rearrangements ID3, TCF3, Cyclin D3
DLBCL, GC MYC, BCL2, BCL6 rearrangementsSomatic hypermutations
Chromatin remodelling genes(MLL2, EP300, CREBBP)Epigenetic modifiers (EZH2)TBL1XR1/TP63
DLBCL, activated MYC, BCL2, BCL6 rearrangements CARD11, CD79B, A20,BLIMP1. MYD88Chromatin remodelling genes(MLL2, EP300, CREBBP)
Pediatric DLBCL MYC IRF4 translocations
Primary mediastinal B‐cell lymphoma JAK2 amplifications MHC) class II transactivator CIITA
Hodgkin lymphoma JAK MHC) class II transactivator CIITA
Splenic marginal zone lymphoma del7q, +3/+3q [+18, +12] NOTCH2, NFKB pathway genes, chromatin remodelling genes
Hairy cell leukemia IGH‐CyclinD1 B‐RAF/ MAP2K1
Follicular lymphoma IGH‐BCL2 EZH2, MLL, TBL1XR1/TP63
CLL TrisomyDel 17p, del11q, del13q
NOTCH1, MYD88, SF3B1
Waldenstrom macroglobulinemia MYD88
Answers for Case 4• What are the differential diagnostic considerations?
Splenic marginal zone lymphomaSplenic B‐cell lymphoma, unclassifiableNonsplenic MALTFollicular lymphomaMantle cell lymphomaChronic lymphocytic leukemia/SLLHairy cell leukemia
• What additional stains would you perform to refine the diagnosis?CD43, CD5, Cyclin D1, CD25
• What additional molecular studies may help refine the diagnosis?B‐RAF for HCL, MYD88 for WM
• What is the role of karyotypic analysis, FISH and gene rearrangement in the workup of splenic lymphomas?
FISH for CLL associated genetic alterationsFISH for t(11;14)
211
Answers for Case 4
• What genetic abnormalities have been recently described in lymphomas that present primarily in the spleen?
NOTCH2mutations in 25% KLF2mutations in 40%
• How will genetic abnormalities contribute to patient management such as prognosis, therapy and disease monitoring?
SMZL with NOTCH2mutations may have worse survivalNOTCH2 signaling may represent a therapeutic target
212
Take home messagesTake home messages
• Many subtypes of B‐cell lymphomas can present in the spleen
• SMZL is a B‐cell malignancy that has not been associated with a recurrent genetic abnormality
• NGS studies identified NOTCH2mutations in 25% of SMZL
• Gene mutations in NFKB pathway, chromatin remodeling are also present in SMZL
• Molecular studies may help in subclassification of other B‐cell lymphomas that present in the spleen
213
Case 5
• A 14 year old boy presented with a one year history of diffuse lymphadenopathy of cervical, axillary, abdominal regions. He complained of fevers, weight loss, night sweats.
• An excisional biopsy of the cervical lymph node was performed. A virtual slide of the lymph biopsy is provided.
214
Case 5: Questions• What are the differential diagnostic considerations?• What additional stains would you perform to refine the diagnosis?
• What additional molecular studies may help refine the diagnosis?
• What is the role of karyotypic analysis, FISH and gene rearrangement in the workup of T cell lymphomas?
• What genetic abnormalities have been recently described in T cell lymphomas?
• How will genetic abnormalities contribute to patient management such as prognosis, therapy and disease monitoring?
215
Case 5: H&E
216
Differential diagnostic considerations
Hematopoietic • Non‐Hodgkin lymphoma
‐diffuse large B‐cell lymphomas with anaplastic features, ‐anaplastic large cell lymphoma (ALK‐positive and ALK‐negative)Peripheral T cell lymphomas, NOS
• Extramedullary myeloid tumors, • Hodgkin lymphoma • Anaplastic myeloma
Non‐hematopoietic • Melanoma • Carcinoma (anaplastic
variants)
217
Immunophenotype
218
CD30
ALK1
Immunophenotype
Antibody ResultsCD45 Negative
CD20 Negative
CD3 Negative
CD2 Negative
CD4 Positive
CD7 Negative
CD8 Negative
CD30 Positive +++
Perforin Positive
ALK‐1 Positive +++ N/C
Anaplastic large cell lymphoma, ALK+
Diagnosis
219
ALKALK
ALKALK
Y Y YYJAK2/3 PI3K
AKTPDK
STAT3/5 PKCα
NPM
AUTOPHOSPHORYLATION
NPM
Apoptosis Proliferation ??
t(2;5)(p23;q35)
PLC
Signaling Cascades Induced byNPM-ALK
Activation
Localization
Expression
Aberrant
ALK
220
t(2;5)(p23;q35) ~75% N/C 80kd
t(1;2)(q25;p23 ~18% C 104kd
t(2;3)(p23;q21) ~1% C 97kd
t(2;3)(p23;q21) ~1% C
inv(2)(p23q35) ~2% C 96kd
t(2;17)(p23;q23) ~2% C 250ld
t(2;19)(p23;p13.1) - C
t(2;2)(p23;q11-13)?or inv (2)(p23p11-13)?
- C
ALK Kinase
ALK Kinase
ALK Kinase
ALK Kinase
ALK Kinase
ALK Kinase
ALK Kinase
ALK Kinase
NPM
TPM3
TFGL
TFGS
ATIC
CLTC
TPM4
RanBP2
117 680
221 784
193 756
138 701
229 792
1634 2197
221 784
867 1430
Variant ALK Translocation Partner Genes
CARS, Moesin, MYH9, SEC31A221
Diagnosis by FISH Cytogenetics
222
Intracellular localization of ALK expression is dependent on the partner gene
Nuclear and cytoplasmic Cytoplasmic
223
Other tumors that express ALK protein
• Lymphomas
• Soft tissue tumors
• Carcinomas
• Neuroblastomas
• Chromosomal translocations
• Gene amplifications• Kinase activating
mutations• Overexpression
MechanismsNeoplasms
224
ALK 75%
15%
ALCL2p235q35
NPM1
TMP3
TFG
ATIC
CLTCL
~10%
IMT
Diffuse large B-cell lymphoma
TPM4
ALK
ALK
ALK
ALK
ALK
MYH
MSN
ALO17
ALK
ALK
ALK
RanBP2 ALK
ATIC ALK
CLTCL ALK
TPM4 ALK
TMP3 ALK
CARS ALK
SEC31L1 ALK
EML4
KIF5B
ALK
ALK
CLTCL ALK
ALKNPM1
Non-small-cell lung cancer
ALK translocations in human cancer
SQSTM1 ALK
225
ALK is a therapeutic target
Phase 1/2 study of PF-2341066, oral small molecule inhibitor of ALK and C-MET in children with relapsed/refractory solid tumors and anaplastic large cell lymphoma
ADVL0912
Children’s Oncology Group
Butrynski JE et al: N Engl J Med 2010;363:1727-33Kwak EL, et al: N Engl J Med 2010;363:1693-703
Phase II study of crizotinib in children with newly diagnosed anaplastic large cell lymphoma
ANDHL12P1Children’s Oncology Group
226YP Mosse , MS Lim et al Lancet Oncology 2013 May;14(6):472‐80
227
580
4126
77 8 3 60
500
1000
1500
2000
2500
3000
3500
4000
4500
0 1 2 3 4
Copies NPM
‐ALK/10 00
0 AB
L
Months post treatment
Molecular monitoring of NPM-ALK transcript in bone marrowand peripheral blood samples of ALCL patients before and after Crizotinib
38
59
23
913 13
6 71 3 3 1
0
10
20
30
40
50
60
70
0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Copies NPM
‐ALK/10 00
0 AB
L
Months post treatment
YP Mosse , MS Lim et alLancet Oncology 2013 May;14(6):472-80
Update on genomic studies of mature T cell lymphoma/leukemia
228
• JAK-STAT mutations in 76% of T-PLL
• IL2RG implicated in human cancer for the first time
• Inhibition of JAK or pSTAT5 may represent a therapeutic strategy for T-PLL patients
229
Integrated genomic analysis of T-PLL identifies novel highly recurrent activating mutations
Kiel M et al., Blood. 2014 Aug 28;124(9):1460‐72
JAK1 JAK3JAK3
STAT5B
IL2
IL2R
IL2R
IL2R
Transcriptional activation
230BLOOD, 11 Dec 2014: 124: 3768
TYK2 gene fusions in cutaneous CD30+ LPDs
CD30
TYK2TYK2
NPM1‐TYK2
Recurrent structural abnormalities in T‐cell neoplasia
231
Disease entity Structural Abnormality Frequency
ALK+ ALCL NPM1‐ALK
Various‐ALK
84%
16%
T‐PLL TRA‐TCL1A
TRA‐MTCP1
70‐80%
10%
HSTL i(7q)(q10) >80%
EATL 9q gains
16q12.1 loss
~70%
~30%
ALK‐ ALCL, c‐ALCL IRF4/DUSP22 translocations
TYK2/ NPM1‐TYK2
25%
17%/4%
PTCL P53‐related genes 6%
F‐PTCL ITK‐SYK 18‐38%
AITL ITK‐SYK rare
Recurrent somatic gene mutations in T‐cell neoplasia
232
Leukemias
Lymphomas
Gene Disease entity FrequencyJAK1/JAK3 T‐PLL
ENKTCL
40%
20‐35%
STAT3 T‐LGLLGD HSTCLCLPD‐NK
27‐40%9%30%
STAT5B T‐PLLGD HSTCLT‐LGLL
36%36%rare
RHOA AITL; PTCL, NOS 67%; 18%
FYN AITL; PTCL, NOS 3% rare
TET2 AITL
F‐PTCL
33‐47%
58%
IDH2 AITL ~25%
DNMT3A AITL; PTCL, NOS 11% overall
Molecular studies will impact therapeutic decisionsin T‐cell neoplasia
Lymphoma Genetic features Therapeutic relevance
ALCL ALK ALK inhibitorcALCL TYK2 TYK2 inhibitorAITL ITK/SYK SYK inhibitorT‐PLL/ENKTCL JAK3 JAK3 inhibitorT‐PLL STAT5B STAT5 inhibitorT‐LGLL, NK‐LPD STAT3 STAT3 inhibitor
AITL TET2, IDH2, DNMT3A Epigenetic modulators
PTCL, NOS DNMT3A Epigenetic modulators
F‐PTCL TET2 Epigenetic modulators
233
Genomic Classification of Mature T‐cell Lymphoma/Leukemia
Cutaneous
Primary Cutaneous CD30+ T‐cell Disorders
Mycosis Fungoides
Extranodal
NK/TCL Nasal Type Adult T‐cell Leukemia/Lymphoma
T‐cell Large Granular
Lymphocytic Leukemia
Subcutaneous Panniculitis‐like TCL
Leukemic
Enteropathy‐associated TCL
Hepatosplenic TCL
Aggressive NK‐Cell Leukemia
T‐PLL
Mature T‐/NK‐cell
Primary Cutaneous Gamma/Delta TCL
Sézary Syndrome
PTCL,NOS
Nodal
AITLAnaplastic Large Cell Lymphoma (ALK +)
Anaplastic Large Cell Lymphoma (ALK‐)
STAT3RHOA/IDH2/TET2/DNMT3A
TYK2
?????
ALK
IRF4/DUSP
JAK3
JAK/STAT5B
STAT3
234
TP53/ARID1A/MLL
Case 5: Answers• What are the differential diagnostic considerations?
Other ALK+ neoplasms, ALCL ALK neg, • What additional stains would you perform to refine the diagnosis?
Pan T cell antigens • What additional molecular studies may help refine the diagnosis?
ALK FISH, TCR gene rearrangement• What is the role of karyotypic analysis, FISH and gene
rearrangement in the workup of T cell lymphomas?TCR gene rearrangement may be necessary for “null cell phenotype”, FISH may be necessary in cases where ALK staining is weak
235
Case 5: Answers
• What genetic abnormalities have been recently described in T cell lymphomas?
TYK2 fusions in cALCLJAK/STAT5B mutations in T‐PLLIDH1/2, TET2, RhoAmutations in AITL
• How will genetic abnormalities contribute to patient management such as prognosis, therapy and disease monitoring?
Molecular methods for disease monitoringDetection of ALK mutations for monitoring disease response to therapy
236
SUMMARY AND CONCLUSIONS
237
IMPLICATIONS OF MOLECULAR GENETICS FOR HEMATOPATHOLOGY?
238
Translating New Genetic Findings into Clinical Practice
• Genomic profiling technologies have identified an explosion of genes associated with various hematolymphoid malignancies
• More clinical studies are needed to sort out which genes are most important prognosticallyand therapeutically.
239
Molecular studies will impact therapeutic decisions in lymphoma
Lymphoma Genetic features Therapeutic relevanceBurkitt MYC/IGH CVAD vs RCHOPDouble Hit DLBCL MYC/IGH; BCL2/IGH Not responsive
BCL6/IGHGastric MALT API2/MALT1 MALT1 inhibitorsHairy cell leukemia B‐RAF mutation B‐RAF inhibitorsCLL/SLL IgH SHM No need for
aggressive TxCLL/SLL; SMZL NOTCH mutation Gamma secretase inhibitorFCL/DLBCL EZH2, MLL, p300 Demethylating agentsALCL ALK Tyrosine kinase inhibitorT cell lymphoma ITK/SYK SYK inhibitor
240
Target Year Target Discovered
Disease(s) and Proportions
Estimated Total # Pts Annually (US)
Drug(s) Clinical Outcomes
Outcomes from Conventional Chemotherapy
Year Mutation-Targeted Treatment Documented
BCR-ABL 1960 CML (100%) 5,000ImatinibDasatinibNilotinib
RR 90%5y PFS 80%5y OS 90%
RR 35%5y OS 70% 2001
EGFR 1978EGFR mutated NSCLC (10% of NSCLC)
17,000 ErlotinibGefitinib
RR 75%Median PFS 11 mosMedian OS 31 mos
RR 30%Median PFS 5 mosMedian OS 24 mos
2004
KIT 1998 GIST 6,000 Imatinib
RR 55%Median PFS 27 mosMedian OS 58 mos
RR 5%Median OS 20 mos 2002
BRAF 2002 50% of melanoma100% HCL 34,000 PLX4032
RR 77%Median PFS 7 mosOS not yet determined
RR 10-20%PFS 1.5 mosOS 8 mos.
2010
ALK 2007EML4-ALK NSCLC (5% of NSCLC)ALK+ ALCL
8,500 Crizotinib
RR 55%6 month PFS 70%OS not yet determined
RR 25%Median PFS 4-6 mosMedian OS 12 mos
2010
Changing Pace of Target Discovery to Therapy
Gerber DE and Minna JD Cancer Cell 2010241
242
Multiplexed targeted platforms
243
Impact on WHO Classification?
WHO 2008 Classification
Diagnosis Prognosis Therapy
CONCLUSIONSCONCLUSIONS• Molecular techniques are routinely being employed to
provide adjunctive results critical to patient management
• Molecular techniques also provide opportunities for improved diagnosis, early disease detection and prognosis of hematopoietic diseases
• Molecular techniques e.g. ABL1 kinase sequencing now offer opportunities for therapeutic refinement and adaptation to diseases specific to each patient (personalized medicine)
244
• New genomic tools have led to discovery of novel mutations in hematopoietic neoplasms.
• Certain genetic abnormalities previously underappreciated as important lesions in hematopoietic malignancies.
• As costs of sequencing continue to fall, additional novel mutations likely to be identified at a rapid rate.
245
CONCLUSIONSCONCLUSIONS
1980 1985 19901988 1994 2001 2008 …
Morphologic evaluation
Cytogenetics
Introduction of immunopathology, flow cytometry immunohistochemistry
Rapid growth in molecular genetic application
Gleevecapproved by FDA
New therapies
•Combination of small molecules
Multi-panel detection of structural alterations/mutations with prognostic and therapeutic implications
2015
Evolution of Molecular Diagnostics in Hematopathology
PCR FFPE tissues REAL classification
WHO classification
246
NEED FOR INTEGRATED HEMATOPATHOLOGY REPORTS
247
1) What disease does the patient have? (diagnosis)
1) How much of the disease is there? (residual disease)
2) What drug will the disease respond to? (therapy)
3) Who needs treatment? (prognosis)
4) What dose? (pharmacogenomics, dynamics)
Mass spectrometry
Integrated evaluation of molecular abnormalities in hematologic disease
New insights into pathogenesis
Altered gene expressionAltered gene expression
Gene rearrangementsGene rearrangements Gene mutationsGene mutationsChromosomal translocationsChromosomal translocations
Early Detection Diagnostics Prognostics New Therapeutics 248
Next Generation Pathologist
SNP arrayaCGH NGS
EpigenomeEpigenome
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249