MPNs

Question 1

CML Chronic Phase

Answer 1

<10% blasts (usually less than 2%)

Often anemia and thrombocytosis

Paratrabecular cuff immature grans 5-10

Megas tend to cluster, unlike AML t(3;3), inv(3)

40% have mildly increased reticulin fibrosis

Question 2

CML Accelerated Phase

Answer 2

One or more of the following:

Persistent or increasing WBC (>10 x 10⁹/L) or splenomegaly

Persistant thrombocytosis (>1000 x 10⁹/L)

Persistant thrombocytopenia (<100 x 10⁹/L)

Cytogenetic evidence of clonal evolution

PB basophils ≥ 20%

10-19% blasts in PB or BM

Question 3

CML Blast Phase

Answer 3

One or more of the following:

PB or BM blasts ≥ 20% (70% AML, 25% B-ALL, rare T-ALL)

Extramedullary blast proliferation (i.e. myeloid sarcoma)
Large foci or clusters of blasts in the BM bx (entire intertrabecular region)

*Still continue to treat with TKIs, not chemo*

Question 4

CML vs Leukemoid Rxn

Answer 4

CML:
decreased alkaline phosphatase (decreased LAP score)

myelocyte bulge

Leukemoid Rxn:

increased alkaline phosphatase (increased LAP score)

no myelocyte bulge

Question 5

BCR-ABL1

Answer 5

• 90-95% have the Ph (del22) chromosome
• Most of the remaining cases have variant genetic abnormalities that resutl in the BCR-ABL1 fusion gene but involve other chromosomes in addition to 9 and 22
• Very few have cryptic BCR-ABL1 translocations that cannot be identified by routine karyotyping and requite molecular or FISH
• Remember BCR is on chr.22 and ABL1 is on chr.9

Question 6

Sensitivities of Techniques for BCR-ABL1

Answer 6

karyotype= 90-95%

RT-PCR= 99%

FISH= >99%

Question 7

p210

Answer 7

BCR exons 12-16

Transcripts:

b2a2

b3a2 (most common translocation)

Question 8

p230

Answer 8

BCR exons 17-20

Transcript: e19a2

Marked thrombocytosis or neutrophilia (resembling CNL)

Question 9

p190

Answer 9

BCR exons 1-2

Transcript: e1a2 (less commonly e1a3)

B-ALL
CML with increased monocytes (resembling CMML)

***A small amount of the p190 transcript can be detectedin >90% of pts with p210 CML due to alternative splicing of the BCR gene***

Question 10

Cytogenetic changes seen in transformation

Answer 10

extra Ph

+8

+19

i(17q)

Question 11

Which pts should have kinase domain mutation testing?

Answer 11

1) all high risk pts
2) standard risk pts who fail to achieve complete cytogenetic response by 6mos
3) pts showing loss of response to imatinib, relapse to Ph+, or increased BCR-ABL1 transcript by ≥1 log
4) at time of progression to accelerated or blast phase

Question 12

Complete Hematologic Response

Answer 12

PB counts completely return to normal, including plt count

No blasts or immature cells circulating

No signs/sx of disease including no enlarged spleen

Question 13

Complete Cytogenetic Response

Answer 13

No Ph chromosome detected with BM cytogenetics

Question 14

Partial Cytogenetic Response

Answer 14

1-35% of cells have the Ph chromosome on BM cytogenetics

Question 15

Major Cytogenetic Response

Answer 15

0-15% of cells have the Ph chromosome on BM cytogenetics

(complete + partial response)

Question 16

Complete Molecular Response

Answer 16

No BCR-ABL1 copies detectable by QPCR using the IS

Question 17

Major Molecular Response

Answer 17

≥3 log reduction in BCR-ABL1 levels

OR

BCR-ABL1 0.1% by QPCR using IS

Question 18

Relapse

Answer 18

• Any sign of loss of response: defined as hematologic or cytogenetic relapse
• A 1-log increase in BCR-ABL1 levels with loss of major molecular response should prompt BM evaluation (but is not alone defined as relapse)

Question 19

Most important prognostic indicator

Answer 19

Response to TKI at the hematologic, cytogenetic, and molecular level

Question 20

Minor Cytogenetic Response

Answer 20

>35% of cells have the Ph chromosome on BM cytogenetics

Question 21

Cytogenetic Response Rate to Imatinib

Answer 21

70-90%, with 5 year progression free survival/overall survival 80-95%

Question 22

BCR gene

Answer 22

25 exons, including two putative alternative first (e1’) and second (e2’) exons

Question 23

ABL1 breakpoints

Answer 23

Almost invariably occur:

1) upstream of exon Ib
2) b/w exon Ib and Ia
3) b/w Ia and a2

Question 24

Mechanisms of Drug Resistance

Answer 24

• kinase domain mutations
• BCR-ABL1 gene amplification or protein overexpression
• alterations in drug efflux kinetics
• upregulation of other kinase pathways
• rare BCR-ABL1 mutations outside of the kinase domain

Question 25

Drugs for pts with Imatinib resistance

Answer 25

dasatinib nilotinib bosutinib

Question 26

Relapsed Ph+ ALL

Answer 26

80-90% will have a BCR-ABL kinase domain mutation

Question 27

Kinase Domain Mutation Testing

Answer 27

- Direct sequencing of BCR-ABL1 gene by Sanger method (because detection of low level mutant clones may not be clinically significant)- detects a mutation in 1 in 5 BCR/ABL1 transcripts - low level imatinib resistance in M351T, with probable response to dose escalation - high level resistance T315I, Y253H, E255K, with need for change in therapy - T315I mutation is relatively common and the worst (resistant to almost all TKIs)

Question 28

Diagnostic Criteria for CNL

Answer 28

- WBC ≥25 x 10⁹/L (\>80% PMNs & bands, \<10% pros/myelos/metas, \<1% blasts) - hypercellular BM - hepatosplenomegaly (most have splenomegaly) - no BCR-ABL1 fusion gene - no rearrangement of PDGFRA, PDGFRB, FGFR1 - no evidence of PV, ET, or PMF - no evidence of MDS or MDS/MPN (no dysplasia, monos\<1 x 10⁹/L) - no physiologic cause for neutrophilia or if so, demonstration of myeloid clonality

Question 29

Morphology of CNL

Answer 29

- Marked increase in PMNs and bands, almost never myeloblasts - PMNs appear toxic - Up to 20% of cases are associated with another neoplasm, usually myeloma (where CNL is thought to be 2º to abnormal cytokine release from neoplastic plasma cells) - If plasma cell dyscrasia is present, clonality of PMN lineage shoudl be proven by cytogenetics or molecular studies before diagnosing CNL

Question 30

Mutation Associated with CNL

Answer 30

- many cases show a mutation of the CSF3R gene - cytogenetics normal in 90% cases - JAK2 mutations have also been described - ?SETBP1?

Question 31

3 Phases of Polycythemia Vera

Answer 31

1) _Prodromal (prepolycythemic) phase_: symptoms suggestive of PV, borderline-mild erythrocytosis 2) _Overt polycythemic phase_: significant ↑ RBC mass 3) _"Spent"/post-polycythemic phase_: cytopenias (including anemia), PB leukoerythroblastosis, BM fibrosis, splenomegaly, and extramedullary hematopoiesis

Question 32

Signs and Symptoms of PV

Answer 32

HTN vascular abnormalities (venous/arterial thrombosis, MI/stroke, Budd-Chiari) plethora/pruritis HA/dizziness visual changes gout (hyperuricemia from high cell turnover) hepatosplenomegaly (usually mild)

Question 33

PV Diagnostic Criteria

Answer 33

Requires both major + 1 minor OR first major + 2 minor _Major_: 1. Hgb \>18.5 g/dL in men, \>16.5 g/dL in women (or other evidence of ↑ red cell volume) 2. JAK2 V617F or JAK2 exon 12 mutation _Minor:_ 1. Hypercellular marrow showing panmyelosis 2. Low serum EPO 3. Endogenous erythroid colony formation *in vivo*

Question 34

Pre-polycythemic and Polycythemic Stages

Answer 34

PB shows increase in all 3 lineages (thrombocytosis in about 50%) BM shows: - left shifted granulocytes - pleomorphic megas (less than PMF, more than ET) dispersed or loosely clustered - enlarged erythroid islands that tend to form sheets - absent stainable iron\*\*\*

Question 35

"Spent"/Post-polycythemic Phase

Answer 35

PB shows leukoerythroblastosis, poikilocytosis with frequent dacrocytes BM shows reticulin & collagen fibrosis ↑ splenomegaly 2º/2 EMH \*\*\*Lymphoid aggregates are seen in 20%\*\*\*

Question 36

JAK2

Answer 36

- cytoplasmic tyrosine kinase acts through JAK-STAT family of nuclear receptors - V617F is most common mutation, due to G→T point mutation in exon 12 (valine to phenylalanine) - mutation happens in pseudokinase domain (usually turns of TK activity), leads to constitutive activation of JAK2 - mutually exclusive with CALR mutations

Question 37

Cytogenetic Abnormalities PV

Answer 37

10-20% of patients +8 +9 del(20q) del(13p) del(1p)

Question 38

PV Treatment & Prognosis

Answer 38

Phlebotomy Pegylated interferon effective in reducing risk of thrombosis and progression to fibrosis Most pts die from thrombosis/hemorrhage (median survival 10yrs) Up to 20% pts develop MDS or AML

Question 39

Primary Myelofibrosis Clinical

Answer 39

90% of patients have splenomegaly

Question 40

BCR/ABL1 Drug Resistance Mutations- General

Answer 40

- among pts with chronic phase CML, who develop secondary resistance to imatinib, 30-50% will have one or more BCR/ABL1 kinase domain mutations detectable by DNA sequencing - mutation frequency higher in those with accelerated/blast phase (especially lymphoid blast phase) - 80-90% pts w/relapsed Ph+ ALL will have BCR/ABL1 KD mutation - absence of mutation does not exclude resistance by other mechanisms

Question 41

BCR/ABL1 Drug Resistance Mutations- Indications for Testing

Answer 41

- chronic phase for those with inadequate initial response to TKIs or those with evidece of loss of response (10-fold or greater increase in transcript levels) - at time of progression to accelerated or blast phase