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Appropriate Use of Genetic Testing in the Diagnosis of Myeloid Neoplasms

DR ROBERT HASSERJIAN

TUESDAY HEMATOPATHOLOGY

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Appropriate Use of Genetic Testing in the Diagnosis of Myeloid Neoplasms

Robert P Hasserjian MDMassachusetts General Hospital

Harvard Medical School

Ineffective hematopoiesisIntact maturation

Effective hematopoiesisIntact maturation

MDS/MPNMDS MPN

Arrested maturation

AML

Mastocytosis

BPDCN

MLN‐Eo

MLN‐Eo

MLN‐Eo

Genetic aberrations queried in evaluating myeloid neoplasms

• Conventional karyotype (global)–Chromosomal rearrangements and segmental or whole chromosome gains and losses

• FISH/CSH/SNP arrays (targeted)–Chromosomal rearrangements

– Smaller copy number alterations, e.g. amplifications or deletions

• PCR and next‐generation sequencing (NGS)(targeted)– Sequence alterations, including single nucleotide variants (SNVs) and small insertions/deletions

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How does genetic testing help us in diagnosing myeloid neoplasms?

• Disease‐defining alteration: a “slam dunk” classifier

– BCR‐ABL1 in a patient with neutrophilic leukocytosis

– PML‐RARA in an acute leukemia

• Disease‐supporting alteration: strongly associated with a particular disease pattern, but not entirely specific; requires appropriate morphologic findings

– JAK2 supporting a diagnosis of a myeloproliferative neoplasm in a patient with thrombocytosis

– Del(5q) on karyotype supporting a diagnosis of MDS with isolated del(5q)

• Prognostic or predictive marker: does not affect the disease category, but associated with patient outcome and may influence choice of therapy

– IDH2 or FLT3‐ITDmutation in AML

– TP53mutation in MDS or AML

• Proving a clonal myeloid process

– Beware ‘incidental’ clonal hematopoiesis, germline mutation mimickers, and mutations in non‐myeloid cells in the marrow

Genetic tests performed on myeloid disorders at MGH (blood or bone marrow)

•Conventional karyotype (+/‐ FISH as indicated)•NGS and PCR‐based tests

• Global myeloid gene panel (Heme SnapShot NGS): 103 genes• Abbreviated panels

• JAK2, CALR, MPL panel

• NPM1 and FLT3‐ITD panel (PCR‐based; rapid)

• IDH1/IDH2 panel (PCR‐based; rapid)• RNA‐based NGS Heme Fusion Panel• RNA‐based RT‐PCR for single gene fusions

• BCR‐ABL1, PML‐RARA, KMT2A (MLL)

MGH Snapshot Heme NGS panel: 103 genes

ABL1 (4-10)ALK (22-25)ANKRD26 (1)ASXL1 (1-12)ATM (1-63)ATRX (8-11, 17-32)BCOR (2-15)BCORL1(1-12)BCR (1-5)BIRC3 (2-9)BRAF (3, 10-15)BTK (15)CALR (1-9)CARD11 (5-9)CBL (2-5,7-9,16)CBLB (3,9,10)CBLC (9,10)CD79A (4, 5)CD79B (5, 6)CDKN2A (1-3)CEBPA (1)CREBBP (1-31)CSF3R (10, 14-18)CUX1(1-24)CXCR4 (1,2)DCK(2,3)

DDX41 (1-17)DNM2 (17,19)DNMT3A (1-23)EP300 (1-31)ETV6 (1-8)EZH2 (2-20)FBXW7(1-11)FLT3 (8-17, 19-21)GATA1 (2)GATA2 (2-6)GNAS (8-11)HRAS (2-4)IDH1 (3,4)IDH2 (4, 6)IKZF1 (2-6, del 1-3)IKZF3 (5,8)JAK1 (14-16)JAK2 (12-16, 19-25)JAK3 (3,11,13,15,18,19)KDM5A(8,11,13,14,18,21,23,25) KDM6A (1-29)KIT (1,2, 5, 8-15, 17, 18)KLF2 (1-3)KMT2A (1-36)KMT2C (14,25,27,36,38,43,44,55) KMT2D (8,11,15,31,34,39,44,53)

KMT2E (14,15,21)KRAS (2-4)LUC7L2 (1-10)MAP2K1 (1-11)MEF2B (1, 2)MPL (10, 12)MYC (1-3)MYD88 (3-5)NF1 (1-57)NFKBIE (1)NOTCH1 (UTR,26-28,34)NOTCH2 (34)NPM1 (11)NRAS (2- 5)NT5C2 (9,11,13,15,17-19)PDGFRA (12,14,15,18)PHF6 (2-10)PLCG2 (19,24)PML (1-9)PPM1D (6)PRPF40B (1-26)PTEN (1-9)PTPN11 (3,4,7,8,11-13)RAD21 (2-14)RARA (5-7,9)RB1 (1-27)

RBBP6 (16)RHOA (2)RPS15 (4)RUNX1 (2-9)SETBP1 (4)SETD2 (1-21)SF3B1 (13-21)SH2B3 (2-8)SLC29A1 (4,13)SMC1A (1-25)SMC3 (10,13,19,23,25,28) SRC (10) SRSF2 (1,2)STAG2 (2-33)STAT3 (2-24)STAT5B (15-17)TET2 (3-11)TNFAIP3 (1-9)TNFRSF14 (1-6)TP53 (2-11)U2AF1 (2,6,7)U2AF2 (1-12)WT1 (1-9)XPO1 (15,16,18)ZRSR2 (1-11).

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Integration of genetic testing into bone marrow diagnosis process at MGH

Biopsy, aspirate smear, peripheral blood smear morphology

Flow cytometry from aspirate smear

Cytogenetics on aspirate

NGS on aspirate

2‐3 days: Issue pathology report

Stored fresh marrow aspirate (4o)

2 weeks: Genetic reports

3 weeks: addendum with final classification

Hematopathology fellows and faculty, molecular pathologist, clinicians

Myeloproliferative neoplasms (WHO 2016)

• Chronic myeloid leukemia, Ph+

• Polycythemia vera

• Essential thrombocythemia

• Primary myelofibrosis

• Rarer entities• Chronic neutrophilic leukemia• Chronic eosinophilic leukemia/hypereosinophilic syndrome• Myeloproliferative neoplasm, unclassifiable

Genetically defined eosinophilic neoplasms

BCR‐ABL

CSF3R

JAK2MPLCALR

PDGFRA

PDGFRBFGFR1

PCM1‐JAK2

“Ph‐ MPNs”

What genetic studies should be done to diagnose and classify bone marrow with probable MPN?

• Essential in all cases–Conventional karyotype– FISH and/or RT‐PCR for BCR‐ABL1– JAK2 V617F

• Case‐by‐case basis– If negative for JAK2 V617F: Reflex to NGS for JAK2, CALR, MPL– If eosinophilia: FISH for FIP1L1‐PDGFRA, consider FISH/NGS‐based fusion tests for PDGFRB, FGFR1, JAK2 & broad NGS mutation panel

– If leukocytosis and BCR‐ABL1 negative (suspected CNL): CSF3R–Consider broad NGS panel for prognosis

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Genetic testing in suspected MPN

Leukocytosis

t(9;22) on karyotypeBCR‐ABL1 on FISH or RT‐PCR

CML, BCR‐ABL1+ Broad NGS Panel• CSF3R: CNL• JAK2/MPL/CALR: Ph‐ MPN• Other: Possible MDS/MPN or reactive

Yes No

Polycythemia

Broad NGS Panel• JAK2 exon12: PV• Negative: Possible reactive

No

PVYes

JAK2 V617F

Ph‐ MPN or MDS/MPN‐RS‐TYes

Thrombocytosis

JAK2CALRMPL

Broad NGS Panel: ‘Triple negative’ MPN or other

No

The results of these tests must always be interpreted in the context of the bone marrow morphology!

Swerdlow SH et al. ed. Revised 4th edition WHO Classification, 2017

New genetic criteria defining AP in CML

CML with chronic‐phase morphology. . . but accelerated phase genetics

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• Adverse mutations: ASXL1, SRSF2, DNMT3A, EZH2, IDH1/2

Rumi et al, Blood 2014, Klampfl T et al. NEJM 2013;369:2379

JAK‐STAT pathway mutations in Ph‐ MPN: relevant both to diagnosis and prognosis

52 year‐old man diagnosed with PV 2 years ago, on ruxolitinibwith progressive splenomegaly and new circulating blasts

• NGS 1 year ago

– JAK2 V617F (43%)

– JAK2 C618R (43%)

– IDH2 R140Q (51%)

– SRSF2 R94dup (38%)

BMA: 39% blasts

WBC 26.4K with 9% blasts

Genetic testing in eosinophilias

Eosinophilia

CytogeneticsBCR‐ABL1 by FISH and/or RT‐PCRFIP1L1‐PDGFRA FISH

CML, BCR‐ABL1+

MLN with PDGFRA rearrangementMLN with PDGFRB rearrangementMLN with FGFR1 rearrangementMLN with PCM1‐JAK2

Most are cytogenetically cryptic!

CEL (other clonal genetic abnormality)

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2016 WHO Criteria for CEL, NOS

Wang SA, et al. Mod Pathol. 2016:854‐64.

Most commonly ASXL1, EZH2, TET2, DNMT3A, NOTCH1, SETBP1, CBL

Genetic testing in eosinophilia

Eosinophilia

CytogeneticsBCR‐ABL1 by FISH and/or RT‐PCRFIP1L1‐PDGFRA FISH

CML, BCR‐ABL1+

MLN with PDGFRA rearrangementMLN with PDGFRB rearrangementMLN with FGFR1 rearrangementMLN with PCM1‐JAK2

CEL (other clonal genetic abnormality)

All negative

Broad NGS PanelHeme Fusion assay

Negative

Idiopathic hypereosinophilic syndromeReactive eosinophilia

Consider mastocytosisif KIT mutation

MGH Heme Fusion Assay (Archer)

•Targeted RNA next generation sequencing (NGS) using Anchored Multiplex PCR

•Validated/reportable genes• ABL1, CBFB, JAK2, KMT2A, PAX5, RARA, RUNX1

• Other genes in panel not yet validated: PDGFRA, PDGFRB, FGFR1, others

• Can pick up rare partners or variant rearrangements missed by conventional FISH probes

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What genetic studies should be done to diagnose and classify MDS?

• Essential in all cases–Conventional karyotype

• Evaluate for isolated del(5q) and MDS‐defining cytogenetic aberrations

• ”MDS FISH panel” not indicated if 20 metaphases are obtained

• Critical for prognosis

• Recommended for WHO classification– SF3B1 mutation: can diagnose MDS‐RS in cases with 5‐15% ring sideroblasts

– TP53mutation testing in MDS with isolated del(5q)

• Broad NGS panel also helpful for diagnosis and prognosis

Cytogenetic findings that can define MDS in a cytopenic patient even in the absence of morphologic dysplasia

MDS‐U

What about finding mutations on NGS in a patient with unexplained cytopenia and no or borderline dysplasia?

• Revisit morphology and discuss with clinician and molecular pathologist

– How compelling are the cytopenia, and mutation results? Flow cytometry evidence of dysmaturation or abnormal blasts?

• Make sure that a germline polymorphism has been excluded

• Isolated DNMT3A, TET2, or ASLX1 mutations may be incidental “clonal hematopoiesis” mutations unrelated to the cytopenia

– Is the bone marrow sample really adequate to evaluate dysplasia?

• Consider repeat biopsy (and genetic testing) at a later date if cytopenias persist and remain unexplained

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Powerful influence of karyotype on prognosis in MDS

Risk group Cytogenetic abnormality

Very good Single del(11q) or ‐Y

Good Normaldel(5q)(single or with 1 other)Single del(12p) or del(20q)

Intermediate +8, i(17q), +19, single del(7q)Any other single or double

Poor ‐7, inv(3), t(3q), del(3q)del(7q) with 1 other3 separate abnormalities

Very poor 4 or more separate abnormalities (complex)

Schanz et al, J Clin Oncol, 2012 30:820‐9

• Recommend testing for TP53mutation or p53 IHC

• Identify patients with poor response to lenalidomide

TP53 mutation influence on MDS with isolated del(5q)

Mallo M Leukemia 2011;25:110, Jadersten M JCO 2011;29:1971, Germing U Leukemia 2012;26:1286

What genetic studies should be done to diagnose and classify MDS/MPN? • Essential in all cases

– Conventional karyotype

– BCR‐ABL1 by FISH and/or qualitative RT‐PCR to exclude CML

Boiocchi L et al. Mod Pathol 2013;26:204

• Consider a broad NGS panel– JAK2, MPL and CALRmay suggest progression from a prior MPN

– JAK2 + SF3B1 common pattern in MDS/MPN‐RS‐T

– Mutation pattern can suggest CMML in cases borderline with MDS

– Mutation pattern can help separate CNL (CSF3R) from atypical CML (SETBP1 or ETNK1)

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Molecular basis of MDS/MPN‐RS‐T

Cazzola et al. Hematology Am Soc Hematol Educ Program. 2011;2011:264‐72

Normal hematopoietic cell

Ring sideroblasts and ineffective erythropoiesis

(myelodysplastic features of RARS)

Ring sideroblasts and thrombocytosis

(myelodysplastic & myeloproliferative

features of RARS-T)

Somatic mutation of SF3B1determining mitochondrial iron

overload, ineffective erythropoiesis and anemia

Somatic mutation of JAK2or MPL or CALR

determining gain of signaling and

thrombocytosis

Use of extended molecular testing in MDS and MDS/MPN

• Mutation profile adds prognostic power to existing (IPSS, IPSS‐R) MDS risk‐stratification schemes

– Poor prognosis mutations: SF3B1, ASXL1, RUNX1, EZH2, TP53

– Number of mutations also prognostic

– Unclear how to incorporate mutation results into clinical management

• Beware of using single mutations to establish ‘clonality’

– Overlap with CHIP/CCUS (especially DNMT3A, TET2, ASXL1)

– Less common variants may be uncatalogued germline polymorphisms

– Multiple mutations and “high‐risk” mutation combinations may be more reliable at predicting MDS or MDS/MPN

How do I use mutations in day‐to‐day diagnosis of MDS?

• 64 year‐old man presented with anemia and fatigue

• WBC 3.8 x 109/L

–66% polys (ANC 2.5 x 109/L, 23% lymphs, 3% monos, 7% eos, 1% immature granulocytes, 1 nRBC/100 WBC

• HGB 7.3 g/dL (MCV 76.9 fL)

• PLT 162 x 109/L

Illustrative Case

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Peripheral smear

Bone marrow aspirate

Bone marrow biopsy

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Balancing differential diagnosis

MDS Reactiveerythroid

hyperplasia

• Normal iron studies

• HGB electrophoresis 6/26/13

– HGB A: 92.1% [95.8‐98.0%]

– HGB A2: 5.5% [2.0‐3.3%]

– HGB F: 2.4% [0‐0.9%]


– HGB A 91.9% [95.8‐98.0%]

– HGB A2: 5.4% [2.0‐3.3%]

– HGB F: 2.7% [0‐0.9%]

• Consistent with beta thalassemia minor

Thank you for doing the bone marrow biopsy, this is a great teaching case of beta‐thalassemia mimicking MDS!

Additional results

Hematologist: ”Sorry I did this bone marrow, I now realize that this patient probably just has thalassemia”



hyperplasia

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Additional results



– HGB A: 92.1% [95.8‐98.0%]

– HGB A2: 5.5% [2.0‐3.3%]

– HGB F: 2.4% [0‐0.9%]


– HGB A 91.9% [95.8‐98.0%]

– HGB A2: 5.4% [2.0‐3.3%]

– HGB F: 2.7% [0‐0.9%]


• Resident: “I found rare ring sideroblasts on the iron stain”

• Iron‐stained aspirate smear was very hemodilute

We asked the lab to stain another aspirate smear for iron

Iron stain on bone marrow aspirate (repeated)



hyperplasia

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Additional results



– HGB A: 92.1% [95.8‐98.0%]

– HGB A2: 5.5% [2.0‐3.3%]

– HGB F: 2.4% [0‐0.9%]


– HGB A 91.9% [95.8‐98.0%]

– HGB A2: 5.4% [2.0‐3.3%]

– HGB F: 2.7% [0‐0.9%]


• Iron stain: 15% ring sideroblasts

However, ring sideroblasts can be found in patients with thalassemia!Pediatr Blood Cancer. 2017 May;64(5).



hyperplasia

Additional results



– HGB A: 92.1% [95.8‐98.0%]

– HGB A2: 5.5% [2.0‐3.3%]

– HGB F: 2.4% [0‐0.9%]


– HGB A 91.9% [95.8‐98.0%]

– HGB A2: 5.4% [2.0‐3.3%]

– HGB F: 2.7% [0‐0.9%]


• Iron stain: 15% ring sideroblasts

• Cytogenetics

– 46,XY,del(20)(q11.2q13.3)[18]/46,XY[2]

• NGS Heme SnapShot Panel

– SF3B1 p.Lys700Glu c.2098A>G

– VAF 8.2%

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Cardiac surgery

MCV

Hemoglobin



hyperplasia

Final diagnosis

1. MDS with ring sideroblasts and single lineage dysplasia (MDS‐RS‐SLD)

2. Beta‐thalassemia minor

In this case, the pathogenic SF3B1 mutation helped establish that the ring sideroblasts truly reflected a myeloid neoplasm superimposed on the patient’s lifelong thalassemia

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2016 WHO AML Classification

AML

“De novo”

AML with recurrent genetic abnormalities

AML, not otherwise specified

“Secondary”

Therapy‐related AML

AML with myelodysplasia‐related changes

Myeloid neoplasms with germline predisposition

Tend to be less genetically complexInclude entities with more favorable prognosis

– APML with PML‐RARA– CBF AML: inv(16)/t(16;16), t(8;21)– AML with mutated NPM1– AML with double‐mutated CEBPA– Others with less favorable prognosis

Tend to be more genetically complexInclude entities with poorer prognosis

AML Classification Toolbox

• Clinical history

• Morphology evaluation (bone marrow biopsy and aspirate)

• Flow Cytometry

• Cytogenetics by conventional karyotyping on bone marrow

• Mutation testing

– NPM1, CEBPA, RUNX1 Required for WHO Classification

– FLT3 and others recommended for further risk stratification

Use of genetic tests to classify AML

Clinically de novo AML

AML with recurrent genetic

abnormality

Recurrent cytogenetic abnormality?

MDS‐associated cytogenetic abnormality

NPM1, RUNX1 or double CEBPAmutation?

AML with myelodysplasia‐related changes

AML with mutated NPM1AML with biallelic CEBPA mutationAML with mutated RUNX1

No

Yes

No

t(8;21);RUNX1‐RUNX1T1inv(16);CBFB‐MYH11PML‐RARAt(9;11);KMT2A‐MLLT3t(6;9);DEK‐NUP214inv(3);GATA2, MECOMt(1;22);RBM15‐MKL1BCR‐ABL1

Complex, ‐7/del(7q) del(5q)/t(5q) i(17q)/t(17p) ‐13/del(13q), del(11q) del(12p)/t(12p), others

Yes Yes

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PML‐RARAinv(16) or t(16;16)

CBFB‐MYH11t(8;21)

RUNX1‐RUNX1T1

Cytogenetic abnormalities that define AML even if blasts are < 20%

t(15;17)

These abnormalities also have up‐front treatment implications; perform rapid FISH or 24‐hour karyotype if suspected on morphology

Gemtuzumab ozagomycin(anti‐CD33) in induction

Gemtuzumab ozagomycin(anti‐CD33) in induction ATRA prior to induction

Cytogenetic abnormalities that define AML‐MRC

–Can be used to diagnose AML‐MRC even in patients with no history of MDS or MDS/MPN

– Supercede NPM1 or double‐CEPBAmutation status

–Confer adverse risk

– Important treatment implications as of 2018: patients are eligible for CPX‐351 (Vyxeos)

Arber DA et al. Blood 2016;127:2391

Mutations refine cytogenetic risk in AML

Cytogenetics alone is good at risk‐stratifying AML. . .

Patel JP et al. NEJM 2012;366:1079

but gets better when we add NGS. . .

NPM1FLT3‐ITDCEBPAIDH1IDH2ASXL1MLL‐PTDTET2DNMT3APHF6

Byrd JC et al. Blood 2002;100:4325

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ASH/CAP guidelines for genetic testing in AML

•WHO classify all cases• Conventional karyotype• NPM1, CEBPA, RUNX1 testing for cases without defining cytogenetics and lacking features of secondary AML

• FLT3‐ITD testing for all cases (up front treatment implications: midostaurin [FLT3 inhibitor] during induction)

•KIT testing for AML with t(8;21) and inv(16)/t(16;16)

•Consider testing for IDH1, IDH2, TET2, WT1, DNMT3A, TP53

Arber DA et al. Arch Pathol Lab Med 2017

Rapid tests for AML‐associated genetic lesions• FLT3‐ITD: Multiplex PCR sizing assay•NPM1 exon 12: Multiplex PCR sizing assay• IDH1: Quantitative allele‐specific PCR

• p.R132C, p.R132G, p.R132H, p.R132S• IDH2: Quantitative allele‐specific PCR

• p.R140Q, p.R172K•Recently instituted policy to rush all new AML karyotypes (24‐48 hours TAT)• RT‐PCR and/or FISH testing for the critical AML‐associated rearrangements also provide rapid results

Likely more therapeutic targets to come in AML

• Challenges: how fast can we do the testing?

– Should induction be delayed pending molecular results?

• If multiple targetable mutations are present, which treatment(s) should be used?

– E.g. FLT‐ITD and IDH1/2mutations

Dohner et al Blood 2017;129:424

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Another challenge: germline mutations that predispose to myeloid neoplasms

Czuchlewski DR et al. Surg Pathol Clin 2016:9:165, West AH et al. Ann NY Acad Sci 2014;1310:111, Wlodarski MW et al. Blood 2016;127:1387, Lewinsohn M et al. Blood 2016:127:1017.

– Congenital syndromes Down, Noonan, Emberger, dyskeratosis congenita, Fanconi anemia, “mono‐mac” syndrome (GATA2)

– Platelet disordersGermline RUNX1, ANKRD26, ETV6 mutations

–Without pre‐existing abnormalitiesDDX41, CEBPA, TP53

–Diagnosis requires careful family and personal history and use of genetic counselors

– Important implications on disease management and prognosis

Summary and take‐home messages

• The recent decade has seen a barrage of data on the significance of mutations in myeloid neoplasms

• NGS‐based genetic testing is now widespread; panels that simultaneously test multiple relevant genes are routinely employed

• Even in this current ‘NGS era’, conventional karyotype should always be performed on the bone marrow of any possible new myeloid neoplasm

– Karyotype still forms the major basis of disease classification and risk stratification

• The results of cytogenetics and molecular genetic testing must always be interpreted in the context of the clinical features and morphology and ideally should be incorporated into the final diagnosis

Ineffective hematopoiesisIntact maturation

Effective hematopoiesisIntact maturation

MDS/MPNMDS MPN

Arrested maturation

AML

Mastocytosis

BPDCN

MLN‐Eo

MLN‐Eo

MLN‐Eo BCR‐ABL1

JAK2MPLCALR

CSF3R

KIT

RAS pathwayTET2/ASLX1/SRSF2

SETBP1JAK2+SF3B1

SF3B1

Isolated del(5q)

NPM1Double CEBPARUNX1Cytogenetically‐defined

PDGFRA‐vPDGFRB‐vFGFR1‐vPCM1‐JAK2

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WHO and Beyond: Practical Strategies for Myeloid Neoplasms

DR KATHYRN FOUCAR


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WHO and Beyond: Practical Strategies for Myeloid

Neoplasms

Kathryn Foucar, [email protected]

2019 Hawaii HemepathConference

Objectives:• Define the Pathologists’ expanding

role in disease classification• Discuss blood and bone marrow

features that are clues to subtypes ofmyeloid neoplasms

• Discuss new WHO 2016classification criteria

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Myeloid Neoplasms- WHO 2016

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AML: 25 subtypes; 3 new genetic entities(numerous prognostic “types”)(new criteria for blast enumeration)(new familial/germline predisposition category)

MDS: 7 subtypes(all new names; some integration of molecular)

MDS/MPN: 5 subtypes; 1 new entity(new molecular genetic criteria)

MPN: 8 subtypes(new molecular genetic criteria)

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Complexity of Classification• WHO 2016: Many exclusionary criteria within

some entities • Elaborate morphologic criteria for MDS despite

evidence of limited reproducibility (Major issue when genetic studies normal)

• 25 subtypes of AML with many additionalgenetic features contributing prognostic information

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Diagnostic Approach • Morphology and clinicopathologic

correlation are still step 1• CBC and blood smear review • Count blasts, assess dysplasia • Determine lineage of blasts by flow

cytometry, especially when increased• Integrate unique morphologic features

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Blast Enumeration • Morphology is gold standard for

blast enumeration • Cytochemical stains uniquely

helpful in some circumstances• Flow blast percent does not replace

morphologic blast present 6

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Blast Lineage Determination • Flow cytometry required for all

acute leukemias to confirm lineage

• IHC can be used for blast lineage determination in selected circumstances

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Systematic Approach• Recognize blasts and blast equivalents • Promonocytes always included in blast percentage• Promyelocytes only included in blast percentage in

APL• Blast percentage based on total BM cells for all

AML subtypes (revised erythroleukemia criteria)• Blast enumeration based on morphologic

differential cell count (not flow cytometry percents)8

Myeloid Blasts

9Morphologic Assessment/Enumeration

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Blast Lineage?

10Acute megakaryoblastic leukemia

Clumps of Abnormal Cells

11Acute megakaryoblastic leukemia

Systematic Approach: Dysplasia• What morphologic features constitute dysplasia?• Dysplastic cells must exceed 10% in a lineage in MDS;

≥50% in AML-MRC• Dysplasia assessment very challenging

– lack of consensus at 10% threshold– better consensus at 40% threshold, especially for

megakaryocytes • Dysplasia assessment based on blood and BM aspirate

smears for erythroid and granulocytic lineages• Megakaryocyte dysplasia based on evaluation of at

least 30 megakaryocytes on core biopsy sections 12

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Dysplasia in Each Lineage

Percent Dysplastic cells critical

Megakaryocyte Assessment for Dysplasia

14Increased, hypolobated megakaryocytes

Dysplasia Caveats• Many benign causes of RBC

pathology in blood and bone marrow

• Excellent stain quality essential to assess neutrophils, identify blasts

• Adequacy of BMA and Bx key• Know key MDS mimics 15

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Routine Assessment for Myeloid Neoplasms Blood: CBC

Morphology (dysplasia assessment)Blast percent

BMA: Morphology (dysplasia assessment)Blast percentPossible MPO, NSEIron stain essential

BM bx, clot section:

CellularityBone Confirm BMA findingsAssess megakaryocytes morphology and distribution

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CBC findings at presentationAML Hematopoietic failure (markedly reduced RBC,

absolute neutrophil and platelet counts);usually no maturation

Variable % blasts; highly variable WBC

MDS Cytopenia(s) requiredDysplasia (≥ 10% required)Virtually never have leukocytosis at presentation Variable blast % (<20%)

MDS/MPN Hybrid blood pictureAt least one elevated and one reduced HP lineage Variable blast % (<20%)

MPN At least one elevated lineage (cytosis) No cytopenias in stable phaseLow blast % in stable phase

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MDS MPN AML

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Comparison of Blood Features

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MDS CML AML

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Comparison of Bone Marrow Features

Atypical CML

2073y/o F: WBC 160, Hgb 8, PLT 112

Integration of CBC, Blast %, and Dysplasia Assessment

• Reasonable prediction of correct WHO category (exceptions)

• Allows for upfront determination of appropriate specialized testing

• Allows pathologist to alert clinician regarding potential medical emergencies (e.g. APL)

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Reporting Requirements • WHO Subtype of Myeloid Neoplasm• All standard items (Bld, CBC,

morphology, special stains, IP and cytochemical stains)

• Percent blasts in blood and BM for all myeloid neoplasms

• Dysplasia percent (each lineage): MDS, MDS/MPN, AML

• Flow cytometry findings 22

Specialized Testing Goals• Lineage of blasts and potential MRD

monitoring by FCI• Confirmation of specific myeloid

neoplasm subtypes by genetic testing, also used in MRD monitoring• Prognosis assessment by genetic testing;

clonal evolution assessment23

Final Integrated Report• Once all specialized (often

referral) testing completed

• ASH-CAP CPG requirement for all acute leukemias

• Most feasible a referral centers24

8/8/19

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In the world of Myeloid Neoplasms, CML has always led

the way25

Battle between Bennett

and Virchow

Leukemia First Described in 1845

26

Clinico-Pathologic Correlation

Blood:Buffy Coat CML:WBC > 900,000WBC’s

27

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Unstained

130 years ago150 years ago 28

1960Nowell and Hungerford

291st Neoplasm linked to cytogenetic abnormality

Philadelphia Chromosome

Courtesy J. Anastasi

1973

30

t(9;22) (q34.1;q11.2)

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31Courtesy J. AnastasiDavid Baltimore, 1980’s

1980’s; different groups

31

t(9;22) (q34.1;q11.2)

Ph1: reciprocal translocationBCR-ABL1 fusion gene

1982-1985

Translocation results in constitutive tyrosine kinase activity à CML 32

CML

•1st genetically defined leukemia

•Must document BCR-ABL1fusion gene for diagnosis

33

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Source: Kalidas, et al. NEJM 2001; 286:895-898

Leukemogenic Effects

of Constitutive

Non-Receptor Tyrosine

Kinase Activation

34

Source: Kalidas, et al. NEJM 2001;286:895-898

Therapy to Block Tyrosine Kinase Activity (1987-1998 )

35

Blast-Phase in CML: 1983-present

Source: Hehlmann, R. How I treat CML blast crisis. Blood 2012;120:737.

36

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CML•First genetically defined

distinct clinicopathologicentity

•Diagnosis cannot be made without genetic confirmation of BCR-ABL1.

37

Clincopathology Entity Model Applied to all WHO Neoplasms

• Entities based on clinical features, morphology, IP, cytogenetics or molecular (2001-Present)

• Entire new group of germline predisposition neoplasms added (family history and molecular genetic confirmation)

• Entitles based on Clinical Advisory Committee discussions

38

Acute Myeloid Leukemia• Blast enumeration, dysplasia assessment and

lineage confirmation by flow cytometry still essential

• Progressively greater role of molecular genetic testing in defining entities, refining prognosis assessment, minimal residual disease monitoring, and identifying patients for possible targeted therapies.

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AML: Many Diseases• 198 recurrent mutations (molecular)• 819 recurrent structural chromosomal

abnormalities (CC)•Ongoing recognition of additional

mutations or relevant combinations of mutations

40Watt, Knowles Neoplastic Hematopathology, 3rd edition, 2014

AML – Types of Mutations

41Source: CDWatt,et al. Knowles Neoplastic Hematopathology. 2014.

Class I: Non-specificClass II: AML-definingClass III: Epigenetic

Source: NEJM 366(12):1079-89, 2012

Molecular Fine Tuning of Prognostic Group

42Integration of molecular with karyotype

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Additional Reporting Requirements

•Integrate cytogenetics, FISH and molecular results in terms of diagnosis, prognosis and targeted therapy

43

Myelodysplasia and MDS/MPN

44

• Key CBC parameters, morphologic features and blast percentage

• Lesser role of genetics in defining entitles (some exceptions)

• Major role of genetics in risk stratification, possible targeted therapy

CMML

45WBC 24.3, Hgb 8.1, PLT 349

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CMML-BX

46Hypercellular; Gran.pred, abnormal megas

Other Myeloid Neoplasms

47

• Similar clinicopathologic approach to define specific entities in neoplasms with <20% blasts

• MPN: Key CBC parameters, BM morphology, evidence of splenomegaly and additional genetic testing beyond BCR-ABL1 are key (e.g. JAK2, CALR, MPL, and CSF3R)

48

•59a36

BM: hyperlobulated megas; Bld: ↑ ↑ plts

Essential Thrombocythemia1.7 million plts

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Role of the Pathologist

49

Primary care setting1. Recognition of myeloid neoplasm and general

neoplasm category2. Exclusion of myeloid neoplasm lookalikes3. Rapid diagnosis or recognition of possible

APL4. Oversee acquisition of specimens for all

necessary specialized testing (unless patient transferred)

Role of the Pathologist

50

Tertiary care setting

1. Integrate routine and esoteric testing

2. Provide comprehensive risk stratification information

3. Monitor treatment response and minimal residual disease testing

Acute Lymphoblastic Leukemia

DR ROBERT MCKENNA


1

Notice of Faculty Disclosure

In accordance with ACCME guidelines, any individual in a position to influence and/or control the content of this CME activity has disclosed all relevant financial relationships within the past 12 months with commercial interests that provide products and/or services related to the content of this CME activity.

The individual below has responded that they have no relevant financial relationship with commercial interest to disclose:

Robert W. McKenna, MD

Lymphoblastic Neoplasms

• Acute Lymphoblastic Leukemia (>90%)– Children

• 85% B lymphoblastic

• 15% T lymphoblastic

– Adults• 75% B lymphoblastic


• Lymphoblastic Lymphoma (<10%)– 70% Children

• 10% B lymphoblastic


• 6,000 new cases in USA annually• 75% occur in kids < 6‐years‐old• 80% of all leukemia in kids

B Lymphoblastic Leukemia in Bone Marrow

1

2

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T lymphoblastic Lymphoma in Mediastinum

WHO Classification of Lymphoblastic Leukemia/Lymphoma‐‐2017

• B lymphoblastic leukemia/lymphoma, NOS

• B lymphoblastic leukemia/lymphoma with recurrent genetic abnormalities (9 categories)

• T lymphoblastic leukemia/lymphoma (1 subset)

• Natural killer cell leukemia/lymphoma (provisional)

B Lymphoblastic Leukemia/Lymphoma (B LL/L) With Recurrent Genetic Abnormalities

• B LL/L with t(9;22)(q34.1;q11.2); BCR‐ABL1

• B LL/L with t(v;11q23.3); KMT2A/MLL rearranged

• B LL/L with t(12;21)(p13.2;q22.1); ETV6‐RUNX1/TEL‐AML1

• B LL/L with t(5;14)(q31.1;q32.1); IGH/IL3

• B LL/L with (1;19)(q23;p13.3); TCF3‐PBX1/E2A‐PBX1

• B LL/L with hyperdiploidy

• BLL/L with hypodiploidy*

• Near haploid

• Low hypodiploid

• B LL/L BCR‐ABL1‐like (provisional)*

• B LL/L with iAMP21 (provisional)*

4

5

6

3

T Lymphoblastic Leukemia/Lymphoma (T LL/L)

• T lymphoblastic leukemia/lymphoma (T LL/L)

– Early T‐cell precursor LL/L*

• Natural killer cell leukemia/lymphoma (provisional)*

Diagnosis and Risk‐Stratification

• Clinical presentation

• Blood counts‐‐Blood smear

• Bone marrow examination (morphology)

• Immunophenotyping

– Flow cytometry

– Immunohistochemistry

• Genetic studies

– Cytogenetics/FISH

– Molecular genetics

• Post‐induction MRD studies

Typical Lymphoblasts in 2 Patients with B-ALL

7

8

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Lymphoblasts in B-ALL

Down Syndrome-B ALLBCR/ABL1 (+) B ALL

Mature Appearing Lymphoblasts in 2 Patients with B-ALL

Cytoplasmic Vacuoles in Lymphoblasts in B-ALL

10

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5

Differential Diagnosis of ALL

• Neoplastic

– Acute myeloid leukemia

– Mature lymphoid neoplasms

– Metastatic small blue cell tumors

• Non‐neoplastic

– Hematogones

– Aplastic anemia

– Reactive lymphocyte proliferations

B-ALL AML with t(8;21)(q22;q22.1)

13

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6

Morphologically Indeterminate

Acute Megakaryoblastic Leukemia

Hematogones

• Bone marrow B‐cell precursors

• Size and morphology bridge mature lymphocytes and neoplastic lymphoblasts

• Large percentages seen in healthy infants and young children

• Increased in • Regenerating marrows• Autoimmune or congenital

cytopenias• Lymphoma, neuroblastoma• AIDS

Hematogones

• Bone marrow B‐cell precursors

• Size and morphology bridge mature lymphocytes and neoplastic lymphoblasts

• Large percentages seen in healthy infants and young children

• Increased in • Regenerating marrows• Autoimmune or congenital

cytopenias• Lymphoma, neuroblastoma• AIDS

16

17

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7

Aplastic Anemia with Increased Hematogones

Immunophenotyping

• Distinguishes ALL from AML and B from T ALL and other lymphoid neoplasms

• Identifies subsets of both B and T ALL

• Distinguishes neoplastic lymphoblasts from hematogones

• Immunophenotypic prognostic groups of ALL

• MRD detection

B ALL

T ALL

Genetics of ALL

• Defines prognostic and treatment groups

• One of the most important factors in risk‐stratification treatment

• Defines categories of B lymphoblastic leukemia in WHO classification

Pui C et al. JCO 2011;29:551-565

Frequency of Specific Genotypes in Childhood ALL

19

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8

Genetic Risk Groups of B ALL

• Low Risk

Hyperdiploid > 50

t(12;21)(p13;q22), ETV6/RUNX1

• Intermediate Risk

Hyperdiploid 47‐50

Normal (diploid)

del(6q)

• High Risk

Near haploid; Low hypodiploid

t(9;22)(q34l1;q11.2), BCR/ABL1

11q23, KMT2A, rearangements

t(5;14)(q31;q32), IL3/IgH

t(17;19)(q21‐22;p13), E2A/HLF

9p abnormalities

del(17p), (TP53 both alleles

iAMP21

Alterations of IKZF1 and CRLF2

BCR/ABL1‐like

COG Risk Classification of B ALL

• First assigned to “standard” or “high risk”– Patient age

– White blood cell count

• Cytogenetic abnormalities and MRD then refine risk classification

– Low• High hyperdiploidy, t(12;21)

– Standard/intermediate

– High• Age, WBC

– Very high• Hypodiploidy, BCR/ABL1+ ALL, BCR/ABL1‐like ALL, iAMP21, t(17;19)

• High level MRD after induction and persistent MRD at later time points

ETV6/RUNX1 ( )

B-ALL with t(12;21)(p13;q22)/ETV6/RUNX1

22

23

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9

B Lymphoblastic Leukemia with t(9;22)(q34;q11.2), BCR‐ABL1

High‐resolution genome‐wide analysis

–Has provided new insights into pathobiology of ALL

– Identifies novel subtypes of leukemia, especially markers of high‐risk disease

–Potential targets for molecular based therapy

B Lymphoblastic Leukemia/Lymphoma (B LL/L) With Recurrent Genetic Abnormalities

• B LL/L with t(9;22)(q34.1;q11.2); BCR‐ABL1

• B LL/L with t(v;11q23.3); KMT2A/MLL rearranged

• B LL/L with t(12;21)(p13.2;q22.1); ETV6‐RUNX1/TEL‐AML1

• B LL/L with t(5;14)(q31.1;q32.1); IGH/IL3

• B LL/L with (1;19)(q23;p13.3); TCF3‐PBX1/E2A‐PBX1

• B LL/L with hyperdiploidy

• BLL/L with hypodiploidy*

• Near haploid

• Low hypodiploid

• B LL/L BCR‐ABL1‐like (provisional)*

• B LL/L with iAMP21 (provisional)*

25

26

27

10

B‐ALL with Hypodiploidy <40 Chromosomes

• Near haploid (23‐29 chromosomes)

– 0.5% of ALL in kids (med. age‐5yrs.)

– Never seen in adults

– Somatic mutations targeting tyrosine kinase and RAS signaling

• Low hypodyploid ALL (33‐39 chroms.)

– 0.5% of ALL in kids (med. age‐11.5)

– 3‐4% of ALL in adults

– >90% TP53 mutations (~50% germline in kids); IKZF2; RB1

– Low hypodiploid ALL in kids is associated with Li‐Fraumeni synd.

Blood 2010;115:5312-21

N Engl J Med 2009; 360:470-80

A subtype of childhood acute lymphoblastic leukaemia with poor treatment outcome: a genome-wide classification study Monique L. Den Boer, PhD1,#, Marjon van Slegtenhorst, PhD1,#, Renée X. De Menezes, PhD1,2, Meyling H. Cheok, PhD3, Jessica G.C.A.M. Buijs-Gladdines1, Susan T.C.J.M. Peters1, Laura J.C.M. Van Zutven, PhD4, H. Berna Beverloo, PhD4, Peter J. Van der Spek, PhD5,$, Gaby Escherich, MD6, Martin A. Horstmann, PhD6,$, Gritta E. Janka-Schaub, PhD6, $, Willem A. Kamps, PhD7,8,$, William E. Evans, PhD3,$, and Rob Pieters, PhD1,8,$

Lancet Oncol. 2009 February ; 10(2): 125–134. doi:10.1016/S1470-2045(08)70339-5.

B Lymphoblastic Leukemia/Lymphoma, “Ph+‐like ALL” or “BCR‐ABL1‐like”

• Lack the BCR‐ABL1 translocation but gene expression profile is very similar to ALL with BCR‐ABL1

• 7‐25% of B ALL

–Frequency lowest in children with standard risk ALL (7‐10%)

–Higher in those with high risk ALL, adolescents, and adults (20‐25%)

–Higher in children with Downs, Hispanics and native Americans

28

29

30

11

Genetics of B ALL “BCR‐ABL1‐like”

• Different types of chromosomal rearrangements– Many different genes and partners

• CRLF2 rearrangements account for about half – [t(14;X or Y) or interstitial deletions]

– Half of these have mutations of JAK2 or JAK1

• Tyrosine kinase‐type translocations, ABL1 or others– Over 30 different partner genes

• Many show deletions or mutations in genes important to leukogenesis– IKZF1 and CDKN2A/B

Treatment and Prognosis of B Lymphoblastic Leukemia/lymphoma, “BCR‐ABL1‐like”

• Overall poor prognosis

– Other high risk features• Older age; High WBC; MRD +

• High risk of relapse

• Most have targetable lesions involving ABL or JAK‐STAT signaling pathways

– ABL, PDGFRB, etc‐‐‐‐Dasatinib

– CRLF2, JAK2, etc‐‐‐‐‐‐Ruxolitinib

B ALL with iAMP21

• 2% of B ALL; more common in older children; low WBCs

• Detected with FISH probe to RUNX1

• 5 or more copies of RUNX1 or 3 or more on a single abnormal chromosome

• Many secondary genetic abns.

• Gains of X, 10, 14, ‐7

• High number of somatic mutations

• Frequent mutations of RAS pathway gene

31

32

33

12

B ALL with iAMP21

• Pts. with constitutional Robertsonian translocation, rob(15;21)q10;q10)c, have 2700 fold increase in this leukemia

• High relapse rate and poor EFS and OS with standard risk therapy

• Requires intensive treatment

• Potential for targeted therapies

T lymphoblastic Leukemia

T Lymphoblastic leukemia/Lymphoma

• Improved prognosis with intensive chemotherapy regimens

• 65% to 75% overall survival in children

• Conventional cytogenetics studies are less contributory in identifying risk groups

• Immunophenotype and gene expression profile identify a sub‐set of TLL/L: Early T‐Cell Precursor Leukemia (ETP‐ALL)

34

35

36

13

Lancet Oncol. 2009; 10: 147-156

Early T‐Cell Precursor (ETP) Leukemia

• 10% to 15% of T ALL

• Derived from a subset of thymocytes that retain stem cell‐like features

• Distinctive phenotype

– CD1a‐, CD8‐,CD5weak with stem cell/myeloid agns.

• ETP related gene expression signature

Early T‐Cell Precursor LeukemiaCoustan‐Smith MS, etal. Lancet Oncol. 2009, 10:147‐56

37

38

39

14

Prognosis of Early T cell Precursor ALL (ETP‐ALL)

• Initial descriptions reported a very poor outcome compared with other T ALL

• More recent larger studies with more effective therapy show little or no effect on outcome for ETP‐ALL

• This despite higher rates of MRD following induction therapy

Five‐year survival rates for children less than 15 years old with ALL: 1960‐2004. SEER Cancer Statistics Review.

Prognosis for Children with ALL‐‐2019

• 10 to 20% of children with B ALL and 25 to 35% with T ALL are not cured

• Relapsed ALL remains the 4th commonest childhood malignancy

• Most common cause of cancer deaths in kids

• Further improvement by dose‐escalation will be limited by toxicities

40

41

42

15

New Therapies for ALL

• To improve cure rates further less toxic targeted approaches are necessary

– Tyrosine kinase inhibitors, JAK inhibitors, etc.

– T cell engaging immunotherapies

• Chimeric antigen receptor T‐cells (CAR T‐cells) – Anti‐CD19 and anti‐CD22 engineered T‐cells

– BCR‐ABL specific cytotoxic T‐cell therapy

– Anti‐TSLPR/CRLF2 antibodies (preclinical testing)

– Other

43

Flow Cytometry of Lymphoblastic & Acute Leukemia of Ambiguous Lineage

DR HORATIU OLTEANU

MONDAY SURGICAL PATHOLOGY

9/16/2019

1

©2019 MFMER | slide-1

Flow Cytometric of Lymphoblastic Leukemias and Acute Leukemias of Ambiguous Lineage

Horatiu Olteanu, MD, PhDProfessor and Medical Director of Flow CytometryMayo Clinic, Rochester, MN


Flow Cytometry in Acute Leukemias

• Roles:• Differentiates ALL from AML• Distinguishes B-ALL from T-ALL• Identifies subtypes of AML: megakaryocytic,

monocytic, etc.• Treatment and prognostic groups determined partly

by immunophenotype• Fingerprint for MRD assessment


ALL: Immunophenotypic Features

• 80-85% B-ALL• B-cell antigens such as CD19, CD20, CD22• CD10 in 90%• Markers of immaturity (CD34 and/or TdT) in most cases

• 15-20% T-ALL• T-cell antigens such as CD2, CD3, CD5, CD7, CD4, and CD8• Markers of immaturity such as CD1a, CD34, and TdT

• Aberrant expression of myeloid antigens seen in many cases

1

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B LymphoblasticLeukemia/Lymphoma


Hematogone Maturation


Kroft SH AJCP 2004; 122: S19-S32

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Typical B-ALL Immunophenotype


Typical B-ALL Immunophenotype


Case #1

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66-year-old F with h/o B-ALL, 3 months s/p alloSCT; pancytopenia


Other results

• CBC: WBC=1,700/uL, Hb=8.9 g/dL, Plt=168,000/uL• Differential count: 5% segs, 52% lymphocytes, 32%

monocytes, 9% eosinophils; 2% basophils

• Morphology:• BM: 11% blasts; 11% lymphs (including

hematogones)

• Cytogenetics:• 47,XX,+8,t(9;22)(q34;q11.2)[1]/46,XX[19]• t(9;22) translocation in 1% of 200 cells analyzed


H&E

TdT

W-G

CD34

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Diagnosis:

Recurrent B Lymphoblastic Leukemia/Lymphoma (B-ALL) with

Hematogone Hyperplasia


B-Lymphoblasts and Hematogones

• Hematogones show a reproducible maturation pattern• B-lymphoblasts essentially always demonstrate

immunophenotype aberrancies• Beware of hematogone hyperplasia in the setting of B-

ALL

• Flow cytometry and morphology usually provide concordant blast percentages

• Hemodilution may underestimate blast counts by flow cytometry


Case #2

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55-year-old M with leukoctyosis

• Presented to PCP with 2-week history of myalgias, night sweats, and 10-15 lbs weight loss

• CBC: Leukocytosis (14,100/uL) and 16% blasts• Hgb 15 g/dL, plt 76,000/uL, LDH >2,500 U/L

• PB flow cytometry performed prior to BM biopsy

• BM biopsy: MPO and NSE cytochemical stains (-)



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• Red (78% blasts): Small cells, CD1a (-), CD2 (-), surface CD3 (-), cytoplasmic CD3 (-), CD4 (-), CD5 (-), CD7 (-), CD8 (-), CD10 (+), CD11b (-), CD13 (-), CD14 (-), CD15 (-), CD16 (-), CD19 (+), CD20 minor subset (+), CD22 dim (+), CD33 (-), CD34 (-), CD36 (-), CD38 (+), CD45 moderate to slightly bright (+), CD45RO (-), CD56 (-), CD64 (-), CD79a dim (+), CD117 (-), HLA-DR (+), MPO (-), TdT (+), and surface immunoglobulin (-).


Cytogenetics

• 47,XY,+i(1)(q10),t(8;14)(q24;q32)[20]

• All 20 metaphase cells analyzed had an extra copy of an abnormal chromosome 1 composed of two copies of 1q resulting in tetrasomy 1q, and what appears to be a balanced translocation between the long arms of chromosomes 8 and 14 (MYC-IGH). No normal cells were observed. FISH performed on the same specimen also revealed an IGH rearrangement in 61.5% of 200 interphase cells analyzed.


Diagnosis:

B-LymphoblasticLeukemia/Lymphoma with t(8;14)

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B-ALL with t(8;14)

• Very rare variant; raises differential diagnosis with Burkitt lymphoma

• Morphology: blasts (immature cells) rather than typical Burkitt morphology

• Immunophenotype:• Blasts are positive for Tdt and negative for sIg

• Genetics:• May be associated with t(9;22), +21, or complex

karyotype


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


B-Lymphoblastic Leukemia/Lymphoma, BCR-ABL1-like

• 2016 WHO new provisional entity

• B-ALL with translocations involving TKs or cytokine receptors (e.g. CRLF2 and JAK mutations)

• Gene expression profiles similar to cases of B-ALL with BCR-ABL1

• Associated with adverse prognosis• May respond to TKI therapy in some cases

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MRD Analysis in B-ALL

• MRD-based risk stratification has become standard of care for B-ALL in several ongoing clinical trials

• Negative MRD status (<0.01%) at the end of induction therapy has be proven to be the most reliable indicator of favorable outcome

• Conversely, a high level of MRD early after induction chemotherapy is a poor prognostic factor

• The absence of MRD is being considered as a surrogate therapeutic endpoint for drug approval in clinical trials


MRD Analysis in B-ALL

• COG antibody panel is standardized in North America• Tube 1: CD20FITC/CD10PE/CD38PerCPCy5.5/CD19PC7/

CD58APC/CD45APCH7• Tube 2: CD9FITC/CD13 + 33PE/CD34PerCPCy5.5/CD19PC7/

CD10APC/CD45APCH7• Tube 3: Syto16*/CD3PerCPCy5.5/CD19PC7/CD45APCH7

• May be performed on PB (day 8) and BM (day 9) post-induction chemotherapy, respectively

• There is broad variation in number/type of antibody combinations and analysis software in different laboratories

• Examples of single-tube panels:• CD66c/CD9/CD34/CD19/CD10/CD20/CD38/CD45• CD10/CD19/CD20/CD22/CD24/CD34/CD38/CD45/CD58/CD66c


EFS of all patients enrolled on 9900 series therapeutic studies with satisfactory end-induction MRD.

Michael J. Borowitz et al. Blood 2008;111:5477-5485©2008 by American Society of Hematology

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B-ALL MRD (+) (0.24%)


B-ALL MRD (+) (0.02%)


B-ALL MRD (+) (0.03%), s/p CAR-T cells

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B-ALL MRD (+) (0.03%), s/p CAR-T cells


T LymphoblasticLeukemia/Lymphoma


Typical T-ALL Immunophenotype

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Typical T-ALL Immunophenotype


Case #3


72-year-old M with pancytopenia

• Presented to ED with 3-week history of fevers, fatigue, night sweats, and 15-20 lbs weight loss

• CBC: Leukopenia (640/uL) and 25% blasts• Hgb 9.1 g/dL, plt 123,000/uL, LDH >2,500 U/L

• PB flow cytometry performed prior to BM biopsy


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Isotype Control

MPO TdT and Cytoplasmic CD3

Isotype Control

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Diagnosis:

Early T-Cell PrecursorLymphoblastic Leukemia


2016 WHO: T-ALL Categories

• Early T-precursor (ETP) ALL

• Has unique IP and genetic profile• Blasts express CD7, but lack CD1a and CD8• Are positive for myeloid/stem cell antigens: CD34,

CD117, HLA-DR, CD13, CD33, CD11b, CD65• Typically express CD2, cytoplasmic CD3, and/or CD4

(not required for definition)• Frequent FLT3, NRAS/KRAS, DNMT3A, IDH1, and

IDH2 mutations


ETP-ALL

Must show definite evidence of T-cell differentiation

CD7 positive CD3 positive(can be either surface or

cytoplasmic)

PLUS

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ETP-ALL

Must not show evidence of myeloid lineage differentiation

MPO negative No evidence of mono-cytic differentiation

PLUS


CD8 negative(less than 5% of total

population can be positive)

ETP-ALL

Must show features of early thymocyte precursors

CD1a negative(less than 5% of total

population can be positive)

CD5 dim/ (-)(expressed by <75% blasts)

orMFI is at least 1 log dimmer than normal T lymphocytes)

Note: CD4 is often negative


ETP-ALL

Must express at least 1 stem cell or myeloid-associated antigen (positive in >10% of blasts)

CD34 (+) > 10%CD117 (+) > 10%

HLA-DR (+) > 10% CD13 (+) > 10% CD33 (+) < 10%

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Identifying ETP-ALL

• As with any hematolymphoid malignancies, identification of ETP-ALL and typical T-ALL requires knowledge of the immunophenotypic features of the normal cell counterpart, i.enormal thymocytes.


Normal Thymocyte Maturation

10 10 1 0 10 100 1 2 3 4

CD7 FITC10 10 1 0 10 100 1 2 3 4

CD45 PerCP

10 10 1 0 10 100 1 2 3 4

Surface CD3 PerCP

10 10 1 0 10 100 1 2 3 4

CD5 PE10 10 1 0 10 100 1 2 3 4

CD2 FITC

0 256 512 768 1024

Forward Scatter

10 10 1 0 10 100 1 2 3 4

CD1a PE

10 10 1 0 10 100 1 2 3 4

CD8 FITC10 10 1 0 10 100 1 2 3 4

TdT FITC

10 10 1 0 10 100 1 2 3 4

TdT FITC10 10 1 0 10 100 1 2 3 4

CD10 FITC


Nearly 100% of neoplastic immature T cells can be detected by examining

patterns of expression for CD1a vs. sCD3 and

CD4 vs. CD8

Identifying T-LL and ETP-LL

Thymocytes

T-ALL

ETP-ALL

The remaining IP features can then be used to

differentiate typical T-ALL from ETP-LL

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Identifying T-ALL and ETP-ALL

Thymocytes

T-ALL

ETP-ALL


Thymocytes

T-ALL

ETP-ALL

Identifying T-ALL and ETP-ALL


Case #4

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26-year-old M with diffuse LAD

• Presented with nausea, vomiting, night sweats• Diffuse lymphadenopathy and mediastinal mass

on CT

• CBC: Mild leukocytosis (12,200/uL) and 25% blasts

• PB flow cytometry performed in 02/2016



• Red (25% blasts): Medium to large sized cells with increased SSC(consistent with cytoplasmic vacuoles) that are CD1a (-), CD2 (+), surface CD3 partial dim (+), cytoplasmic CD3 bright (+), CD4 minor subset (+), CD5 minor subset (+), CD7 (+), CD8 (-), CD10 (-), CD11b partial (+), CD13 (-), CD14 (-), CD15 (-), CD16 (-), CD19 (-), CD20 (-), CD22 (-), CD33 variably (+), CD34 (+), CD36 (-), CD38 (+), CD45 moderately (+), CD45RO (-), CD56 (-), CD64 minor subset (+), CD79a (-), CD117 (-), MPO (-), TdT (-), and surface immunoglobulin (-)

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Cytogenetics - Diagnosis

• 58-59,XY,+1,+4,+6,+8,del(9)(p21),+10,+18,+19,+19,+20,+20,+22, +22[cp5]/46,XY[26]

• Twenty-six cells were normal, while five cells had a hyperdiploid complement of 58-59 chromosomes in a pattern often observed in hyperdiploid ALL. This pattern included trisomy 4 and trisomy 10 as well as trisomies for 1, 6, 8, 18, tetrasomies for 19, 20 and 22, supported by FISH. In addition deletion 9p (also supported by FISH) was observed; deletion 9p in ALL may be an unfavorable prognostic finding.


Diagnosis:

T-LymphoblasticLeukemia/Lymphoma


Interval history

• Induction chemotherapy CALGB 10403 protocol• BM biopsy from 03/2016: MRD• BM biopsy from 05/2016: CR

• Consolidation and maintenance chemotherapy

• Recurrent disease: 05/2017• Pancytopenia and 90% blasts• PB flow cytometry

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• Red (91% blasts; relapse): Medium to large sized cells CD1a (-), CD2 variably (+), surface CD3 (-), cytoplasmic CD3 (-), CD4 variably (+), CD5 (-), CD7 (-), CD8 (-), CD10 (-), CD11b (-), CD13 (-), CD14 (-), CD16 (-), CD19 (-), CD20 (-), CD22 (-), CD33 variably (+), CD34 variably (+), CD36 (+), CD38 variably dim (+), CD41 (-), CD45 moderately (+), CD45RO (-), CD56 partial (+), CD61 (-), CD64 (-), CD71 (+), CD79a (-), CD117 (-), HLA-DR partial dim (+), glycophorin A (+), MPO (-), TdT (-), and surface immunoglobulin (-).

vs.

• Red (25% blasts; diagnosis): Medium to large sized cells with increased side scatter (consistent with cytoplasmic vacuoles) that are CD1a (-), CD2 (+), surface CD3 partial dim (+), cytoplasmic CD3 bright (+), CD4 minor subset (+), CD5 minor subset (+), CD7 (+), CD8 (-), CD10 (-), CD11b partial (+), CD13 (-), CD14 (-), CD15 (-), CD16 (-), CD19 (-), CD20 (-), CD22 (-), CD33 variably (+), CD34 (+), CD36 (-), CD38 (+), CD45 moderately (+), CD45RO (-), CD56 (-), CD64 minor subset (+), CD79a (-), CD117 (-), HLA-DR (-), MPO (-), TdT (-), and surface immunoglobulin (-).

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What is your diagnosis?

• A. T-lymphoblastic leukemia/lymphoma

• B. Acute myeloid leukemia

• C. Acute undifferentiated leukemia

• D. Acute erythroid leukemia


Cytogenetics - Relapse

• 52-55,XY,+X,-2,+6,+8,add(11)(p15),-12,add(13)(p11.2),-13,-17, add(17)(p11.2),+18,+add(19)(p13.3),+20,add(21)(p11.2),+22,+22, +3-6mar [cp10]

• All ten cells had a hyperdiploid complement of 52-55 chromosomes in a pattern often observed in hyperdiploid ALL. This pattern included monosomy 2 and trisomies or tetrasomies for 6, 8, 18, 19, 20 and 22. This appears to be a considerably evolved version of a clone that was observed on 02/2016; fewer chromosomes are present (52-55 instead of 58-59), and many more structural abnormalities are present, affecting chromosomes 11, 13, 17, 18, 19, 21.


Diagnosis:

Recurrent T-LymphoblasticLeukemia/Lymphoma with

Evidence of Clonal Evolution

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Acute Leukemias of Ambiguous Lineage


Acute Leukemias of Ambiguous Lineage: WHO 2016

• Acute undifferentiated leukemia

• Mixed phenotype acute leukemia with

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

• Mixed phenotype acute leukemia with t(v;11q23);

KMT2A rearranged

• Mixed phenotype acute leukemia, B/myeloid, NOS• Mixed phenotype acute leukemia, T/myeloid, NOS• Mixed phenotype acute leukemia, NOS, rare types• Acute leukemias of ambiguous lineage, NOS


Case #5

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42-year-old M with syncopal episode

• Presented to ED after fall at work• Small scalp hematoma on back of head

• CBC: Leukocytosis (50,800/uL) and 90% blasts

• PB flow cytometry and BM biopsy was performed




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• Red (91% blasts): Small to medium sized cells, CD1a (-), CD2 (-), surface CD3 (-), cytoplasmic CD3 (-), CD4 (-), CD5 (-), CD7 partial dim (+), CD8 (-), CD10 (-), CD11b (-), CD13 (-), CD14 (-), CD15 (-), CD16 (-), CD19 (-), CD20 (-), CD22 (-), CD33 (-), CD34 (+), CD36 (-), CD38 variably dim (+), CD45 (-) to dim (+), CD45RO (-), CD56 (-), CD64 (-), CD79a (-), CD117 (+), HLA-DR (+), MPO (-), TdT (-), and surface immunoglobulin (-).


Cytogenetics

• 45,XY, t(5;12)(q13;q24.1),-7, del(12)(p11.2), del(21)(q11.2)[19]/ 46,XY[1]

• 19 cells each have multiple structural and numerical abnormalities including what appears to be a balanced translocation between the long arms of chromosomes 5 and 12; monosomy 7; and terminal deletions of 12p and 21q. FISH performed on the same specimen also revealed deletion 12p, deletion 21q, and monosomy 7 in 61.5-72.5% of 200 interphase cells analyzed, which is consistent with classical cytogenetic findings.


Diagnosis:

Acute Undifferentiated Leukemia

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Acute Leukemias of Ambiguous Lineage

• Contentious topic• Somewhat arbitrary requirements for assigning

more than one lineage to a single blast population:• Myeloid: MPO• T cell: Cytoplasmic CD3 (bright, i.e. same

intensity as normal T cells)

• Acute undifferentiated leukemia shows no lineage-specific markers

• Blasts are often positive for HLA-DR, CD34, CD38• May be positive for Tdt


• Myeloid lineage• MPO (FC, IHC or cytochemistry) or• Monocytic differentiation (at least 2 of the following: NSE,

CD11c,CD14, CD64, lysozyme)

• T lineage• Strong cytoplasmic CD3 (with antibodies to CD3 epsilon chain)

or Surface CD3

• B lineage• Strong CD19 with at least one of the following strongly

expressed: CD79a, cytoplasmic CD22 or CD10 or • Weak CD19 with at least two of the following strongly

expressed: CD79a, cytoplasmic CD22 or CD10

Criteria for Lineage Assignment for Mixed Phenotype Acute Leukemia (MPAL)


MPAL - New Emphases in 2016 WHO

• For MPAL cases, if there are two distinct blast populations, and each individual population meets a definition for either a B, T or myeloid leukemia, it is not necessary that the specific markers be present.

• If ALL or AML is NOT MPAL, it is not necessary to meet the more strict MPAL criteria in order to assign lineage.

• Some typical B-ALL cases with homogeneous expression of lymphoid markers on a single blast population may express low-level MPO using IP methods without other evidence of myeloid differentiation. Because the clinical significance of this finding has not yet been established, it is recommended that care be taken before making a diagnosis of B/Myeloid MPAL when low intensity MPO is the only myeloid-associated feature.

• Multi-parameter FC is the preferred method for recognizing MPAL.

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


Cross-Lineage Antigen Expression in AML

Partial CD7 and CD56 expression


Cross-Lineage Antigen Expression in AML

Partial CD2 and CD7 expression

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MPAL with Two Distinct Blast Populations


Summary

• Routine flow cytometry in ALL requires in-depth knowledge of maturation patterns of normal immature cells (hematogones and T cells)

• Flow cytometry has applications in the differential diagnosis, prognosis, and treatment of ALL

• MRD analysis by flow cytometry is a powerful predictor of outcome in B-ALL, in the clinical trial setting


Thank you for participating!

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Acute Myeloid Leukemia

DR KATHYRN FOUCAR

MONDAY SURGICAL PATHOLOGY

8/8/19

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Acute MyeloidLeukemia

Kathryn [email protected]

2019 Hawaii Hemepath Conference

Objectives• Review diagnostic process for AML• Apply new WHO criteria and ASH-

CAP guidelines for acute leukemia• Highlight key problem areas• Integrate molecular genetic features

in diagnosis of AML and riskstratification 2

3

Outline• Definition, epidemiology• Key steps in AML dx (ASH-CAP CPG’s)• Hematologic parameters• Identification of blasts• Genetic/biologic features/Prognostic factors• Classification (WHO 2016 revised criteria)• Distinctive types

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AML: Definition

• Clonal HP disorder• Autonomous proliferation• Single/multilineage involvement• Minimal (if any) maturation

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5

AML: Epidemiology• All ages, predominates in adults• Increased incidence in elderly• Linked to genetic/acquired BM

disorders• Linked to environmental, toxic,

therapeutic exposures

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Key Steps in AML Dx (ASH/CAP CPG)• Recognize hematologic profile of CBC• Blast enumeration (≥ 20% threshold)• Determine lineage of blasts (flow)

(cytochem)

• Assess all lineages for dysplasia• Integrate with clinical, genetic/ biologic

parameters (cytogenetics, molecular)• Risk stratification (integrated report)

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Early Steps in Diagnosis • Clinicopathologic correlation/CBC review (Prior hematologic disorder, prior CBC data, antecedent therapy)• Blood: CBC and Morphologic Review

– Blast % in blood (differential count)– Blast % in bone marrow (differential count)– Any unique features of blasts– MPO/NSE stains– Dysplasia Assessment– Lineage confirmation by FCI 7

Diagnostic Traps• Failure to rapidly recognize APL• Exceptions to ≥ 20% threshold

APL, t(8;21), inv(16)• Mis-identification of promonocytes

(blast equivalents)• Classification of erythroid predominant

neoplasms• Identification of pronormoblasts and

megakaryoblasts 8

9

Hematologic Parameters; MorphologyBlood:

– Pancytopenia (RBC’s, platelets, neutrophils)– Highly variable WBC– % blasts/blast equivalents– Variable single/multilineage dysplasia

Bone Marrow:– Hypercellular; rarely hypocellular– Variable proportion blasts (³ 20%)– Limited maturation (usually)– Variable single/multilineage dysplasia

Tip: HP Failure; Percent Blasts

Tip: Percent Blasts, Dysplasia

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10

Criteria for BlastMorphology: nuclear featuresIP: CD34, CD117, w CD45, TdTOther Blastic Neoplasms:

– Childhood tumors – neuroblastoma, retinoblastoma

– Blastic variants of mantle cell lymphoma, HCL, myeloma

Normal Immature Cells:– Hematogones

11AML, Auer rod

76b13

Blasts and Blast Equivalents †

• Myeloblasts• Promyelocytes (only in APL)• Monoblasts• Promonocytes• Erythroblasts (only in PEL)• Megakaryoblasts

†Blast % derived from morphologic cell count, not flow cytometry12

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Myeloid Blasts

13Morphologic Assessment/Enumeration

MPO

Acute myeloid leukemia 14

MPO

Acute promyelocytic leukemia 15

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Acute monoblastic leukemia16

NSE

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MPO

Acute megakaryoblastic leukemia

neut

Blast Lineage Determination •Flow cytometry required for all acute leukemias for dx and eventual MRD assessment‡

•IHC can be used for blast lineage determination

‡ ASH-CAP CPG 18

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MPO

Side

Sca

tter

CD45

Side

Sca

tter

Forward Scatter CD33

CD34

AML

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Morphologic, Cytochemical and Immunophenotypic Features of AML

Blasts/ Immature Cells‡

CytochemicalProperties‡

Immunophenotype‡

Myeloblasts MPO+ CD34, HLA-DR, CD33, CD13, anti-MPO, weak CD45

Promyelocytes MPO+ CD33, CD13, anti-MPOMonoblasts/ Promonocytes

NSE+ HLA-DR, CD33, CD13, CD36, CD64

Erythroblasts PAS+ eCadherin, CD71, Glycophorin A, Hgb A

Megakaryoblasts CD61, CD41, CD42b, CD3120‡ ASH-CAP-CPG

Systematic Approach: Dysplasia• Dysplastic cells must exceed 10% in a lineage in

MDS; 50% in AML-MRC• Dysplasia assessment challenging; use strict

criteria• Dysplasia assessment based on blood and BM

aspirate smears for erythroid and granulocytic lineages

• Megakaryocyte dysplasia based on evaluation of at least 30 megakaryocytes on core biopsysections 21

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Assess Each Lineage

22AML with t(3;3)

Abnormal Megakaryocytes

23AML with t(3;3)

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AML Classification: Biologic Groups2008 2016/2017

AML with recurrent genetic abnormalities

9 typest(1;22), NPM1, CEBPA,

inv(3), t(6;9)

11 TypesBCR-ABL1+/RUNX1+

Biallelic CEBPA

AML with MDS-related changes

AML after MDS, MDS/MPN AML with

multi. dysplasia AML with MDS karyotypes

Revised criteria for multilineage dysplasia

cases

Myeloid Neoplasm (t-MN)

T-AML, MDS, MPN T-AML(no major changes)

Familial AML/MDSNew

Germline predisposition disorders

(many subtypes)Blast enumerationNew Criteria

Based on total cells for allAML subtypes

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New Acute Myeloid Leukemia 2016/2017• AML with RUNX1 mutation (provisional)• AML with BCR-ABL1 mutation (provisional)• AML with biallelic CEBPA mutations• Familial AML/MDS (Germline predisposition)• New blast percent definition for erythroid

predominant neoplasms • Revised AML-MRC criteria

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Ref: Arber DA, et al Blood 2016; 127:2391/Arber DA 2017 WHO revised classification

WHO 2001:AML--Overall Survival by Karyotype*

Years from Start of Therapy

*Source: J Clin Oncol 21:256, 2003

P < 0.0001

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t(8;21), inv(16), t(15;17)

Source: NEJM 366(12):1079-89, 2012

Molecular Fine Tuning of Prognostic Group

27Integration of molecular karyotype

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AML Genomic Classification and Prognosis

28Ref: Papaemmanuil E et al. N Engl J Med 2016;374:2209-2221.

AML Risk StratificationFavorable: Core binding factor, (APL)

NPM1m, FLT3wt , FLT3low

Biallelic CEBPAm

Intermediate: CC not favorable or adverseNPM1m, FLT3high

NPM1wt, FLT3wt , FLT3low

t(9;11), MLLT3-KMT2A

Unfavorable: Complex, monosomal-5, del5q, -7, -17, -17/abnl(17p)t(6;9), inv(3), t(9;22), t(v;11q23.3)NPM1wt and FLT3high

RUNX1m or ASXL1m or TP53m

29Sources: Dohner 2017, NCCN 2019

Required Testing for all AML Cases*

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• Morphologic blast enumeration/dysplasia assessment

• Flow cytometry for blast lineage confirmation and MRD monitoring

• Conventional karyotype (targeted FISH for rapid dx of APL, etc)

• Molecular assessment for FLT3, NPM1, CEBPA, RUNX1, KIT, TP53 (myeloid gene panel)

* ASH/CAP-2016 (Arch Pathol Lab Med)

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CBF-AML:t(8;21)(q22;q22.1)(RUNX1-RUNX1T1)

Morphol: Usually AML with maturation; blasts may be <20%

Clinical: Young adults, occasional childrenMyeloid sarcoma

IP: Coexpression of CD19, CD56, PAX5Molecular: RUNX1/RUNX1T1 , KIT (impacts prognosis)Outcome: High CR rate

Favorable outcome (KIT adverse)Other genetic abnls influence survivalMastocytosis in 10% (KITm)• concurrent SM-may be masked by AML at dx.

31CBF = Core Binding Factor

AML with t(8;21) 32

AML with t(8;21) 33

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AML: t(8;21)(q22;q22.1)

Immunophenotypic clues

Morphologic clues

Bone marrow

SSC

CD19

CD33

Courtesy K. ReichardCD45 CD10 CD71

25-year-old femaleEcchymoses

Subcutanous nodulesCC: InadequateFISH: + t(8;21)

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Low Blast Count AML• Cases of AML with t(8;21) or inv(16) may

present with < 20% blasts ‡

• Clinical course is that of overt AML and AML therapy warranted

• Assess for morphologic “clues” for t(8;21) and inv(16)

• Clue: Auer rods in cases with <20% blasts (May be MDS but must be sure)

‡ Note APL often <20% blasts

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Acute Promyelocytic Leukemia t(15;17)(q24.1;q21.2)

Clinical: Constant rate over lifetime Profound thrombocytopenia; coagulopathyMedical emergency!

Morphology: Major: hypergranular promyelocytes (low WBC) Microgranular: 1) folded nuclei (high WBC) 2) inconspicuous granulesLittle, if any, maturation beyond promyelocyte

Cytogenetics/ Molecular: t(15;17) PML/RARA fusion gene

IP: My antigen + , HLA/DR - , CD34 - ,MPO+ , High side scatter! CD11a, b- , CD18-

Outcome: Begin ATRA; Manage coagulopathy; favorable risk

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APL• Cure rates exceed 80% (90%)‡

• New therapy: ATRA plus arsenic trioxide

(no chemotherapy!)

37Source: Lo-Coco, et al. NEJM 369:111, 2013.‡ Blood 2016

APL with t(15;17) 38

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Acute Promyelocytic Leukemia“Morphogenotype”

Classic APL: morphology highly correlated with PML/RARA fusion gene detection (95% sensitivity; 92% specificity)*

* Bennett, Leukemia 14: 1197-1200, 2000.

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40Microgranular APL AMoL

Classic APL: rare promyelocyte in thick area of smear

High Index of Suspicion!

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61a01

MPO

APLt(15;17)

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APL Diagnostic Emergency: Alert Clinician!!• Early deaths from coagulopathy not prevented by ATRA • Morphology High index of suspicion

– If WBC low: Search for hypergranular promyelocytes(feather edge, thick areas)

– If WBC high: Consider microgranular APL every time there are folded nuclei, monocyticappearanceSearch for hypergranular formsSearch for stacks of Auer rods

• Rapid MPO Intense, uniformly positive (cytochem)• Rapid DIC screen Coagulopathy supports APL

• Rapid FISH Rare cases false negative

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MDS AML with MRC

WHO 2016: (≥ 20% blasts) (any 1 of 3 criteria)1. Multilineage dysplasia (> 50% in at least 2 lineages)

(NPM1, CEBPA, exceptions)2. Hx of MDS or MDS/MPN3. MDS-related chromosomal abnormalities: -5/del(5q),

-7/del(7q), many others (specific WHO list)Usual features:• Advanced age, environmental exposures• Overlap with alkylating agent therapy-induced t-MN;

(Excludes AML-MRC)

biologic continuum

45AML with MDS-related changes

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63a21 with 63a16 inset

AML: Multilineage dysplasia 46

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AML: Key Tips/Comfort Zone• Low blast count AML• Rapid diagnosis of APL• Morphologic vs IP blasts:

All CD34+ cells are blasts but NOT all blasts are CD34+

• Morphology + IP + genotype

WHO Revisions• Two new genetic subtypes;

RUNX1, BCR-ABL1 (provisional)• Biallelic CEBPA• Blast enumeration changes eliminating

AEL myeloid/erythroid designation• Revised criteria for AML-MRC• Germline predisposition (familial)

neoplasms 48

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AML: Key tips/New Challenges• Application of WHO criteria requires CC and

molecular integration in diagnosis• Emphasis on prognostication, MRD, and

identification of potential targeted therapy options

• Apply ASH-CAP CPG• Identify cases of AML with “wrinkles”:

<20% blasts• Negative PML-RARA FISH 49

Challenging Hematopathology Cases

DR ROBERT MCKENNA

MONDAY HEMATOPATHOLOGY

9/17/2019

1

Challenging Cases in Hematopathology

Rob McKenna

09/24/2019

Notice of Faculty DisclosureIn accordance with ACCME guidelines, any individual in a position to influence and/or control the content of this CME activity has disclosed all relevant financial relationships within the past 12 months with commercial interests that provide products and/or services related to the content of this CME activity.

The individual below have responded that they have no relevant financial relationship with commercial interest to disclose:

Robert W. McKenna, MD

Case 1‐‐Clinical History

• 52 yr. old man

• Parasthesias in feet and lower legs

• Back pain

• 15 lb weight loss

• Increasing symptomatology for 3 months

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Physical Examination

• Areas of hyperpigmentation of skin

• Moderate gynecomastia

• ? Mild hepatomegaly

• Decreased sensation over feet and lower extremity, ? weakness

Relevant Laboratory Findings

• Blood counts:• Hemoglobin ‐ 14.7 gm/dl

• Leukocyte count ‐ 6.8 X 109/L

• Platelet count 640 X 109/L

• Serum testosterone ‐ 120 ng/dl (normal 300‐1200 ng/dl)

• Serum protein electrophoresis was performed

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Lambda

What is the Diagnosis?

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Questions?

Comments!


• 12 Year old boy with no significant PMH

• URI symptoms for 2‐3 weeks

• Emesis, diarrhea, leg pain

• Acute chest pain and SOB

• Rash on hands

• Hepatosplenomegaly

• Tachycardia, gallop rhythm

Radiographic Studies

• Chest

– No infiltrates

• Mild cardiomegaly

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Cardiac Studies

• Troponin I ‐ 14.0 ng/ml, CK‐MB 7 U/ml, LDH 1562 IU/L

• EKG ‐ left axis deviation, ST‐T wave changes

• Echocardiogram– Tricuspid and mitral regurgitation

– Thrombus on MV with decreased mobility of MV

– Thickened RV wall with septal dyskinesis

Blood Counts

• Leukocyte count‐‐142 X109/L

– Eosinophils‐‐84%

– Neutrophils—5%

– Monocytes—1%

– Lymphocytes‐‐5%

– Others—5%

• Hemoglobin‐‐12.3 gm/dl

• Platelet count‐‐57 X109/L

Preliminary Diagnosis is:

Marked eosinophilia resulting in systemic manifestations of

hypereosinophilia

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Causes of Eosinophilia

• Parasitic and other infections

• Drug reactions

• Allergic/immunologic disorders

• Neoplastic diseases

• Miscellaneous syndromes

Complications of Hypereosinophilia

• Myocardial infarctions

• Congestive heart failure

• Mural thrombus/arterial thrombus

• Pulmonary infiltrates

• Skin rash

• Thrombophlebitis

• Death due to cardiac failure 30%

Primary/Clonal Neoplastic Causes of Eosinophilia

• Myeloid and lymphoid neoplasms with abns. of PDGFRA (4q12), PDGFRB (5q31)

or FGFR1 (8p11)

• AML with inv(16)(p13.1;q22)

• Other

• Leukemia originates in a multipotential stem cell

• Leads to a clonal neoplasm of more than one lineage including eosinophils

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Secondary/Reactive Neoplastic Causes of Eosinophilia

• Lymphomas, eg., Hodgkin lymphoma, T cell, etc

• B Lymphoblastic leukemia with t(5;14)(q31;q32)

• Non‐hematopoietic neoplasms

• T cells activated by tumor associated antigens elaborate IL‐3, IL‐5 or GM‐CSF causing eosinophilia

• Neoplastic cells elaborate growth factors causing secondary eosinophilia

Bone Marrow Findings

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10901


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Questions?

Comments!


• 1 day old, 32 week gestation female neonate with:

• Microcephaly• Respiratory distress• Hepatosplenomegaly• Acites• Enlarged kidneys

• Leukocyte count ‐ 329,000/L

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CD61

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Cytogenetics

Clinical Course

• Double volume exchange transfusion• Leukocyte count to 38,000/L

• Liver failure at day 14• Bilirubin 18.7 mg/dl• Worsening acites

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Clinical Course

• Lung abscess, respiratory distress, sepsis, coagulopathy at day 20

• Leukocyte count 60,000/L with more maturation

• Died on day 24 – Autopsy performed

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CD34 CD61

Reticulin stain Collagen Stain

What is the diagnosis?

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Questions?

Comments!


• 81 year old man with compression fractures of the thoracic spine

• Renal dysfunction

• Pancytopenia

• Hypogammaglobulinemia with decreased IgG, IgA and IgM

• Elevated serum kappa free light chains (8580 mg/L) and Bence‐Jones proteinuria

• No lymphadenopathy organomegaly

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Flow Cytometry Results

• CD20 (+)

• CD23 (partial +)

• CD117 (+)

• CD19 (‐)

• CD5 (‐)

• CD10 (‐)

• (s) kappa (‐)

• (s) lambda (‐)

• CD56 (‐)

CD138

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KappaKappa Lambda

CD20

Cyclin D1

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Immunophenotype by Immunohistochemistry

• CD138 (+)

• (c)kappa (+)

• CD20 (+)

• Cyclin D1 (+)

• MUM‐1 (+)

• CD56 (‐)

• CD5 (‐)

• CD10 (‐)

• (c)lambda (‐)

• IgM (‐), IgD (‐)

• IgG (‐), IgA (‐)

Cytogenetics: 46,XY,t(11;14)(q13;q32),del(13)(q14q22)

Differential Diagnosis

• Plasma cell myeloma

– CD20+, cyclin D1+ with small mature PCs

– Light chain only myeloma

– IgD or IgE myeloma

• B cell lymphoma with extreme plasma cell differentiation

– Lymphoplasmacytic lymphoma

– Marginal zone lymphoma

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Questions?

Comments!

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