20150926 PNWSP Talk BMBx MDS MPN Printout.ppt · 2015-09-26 2 4 How to Perform a BMBx •Always have concurrent blood specimen, CBC printout, bone marrow aspirate and Bx • Site:

2015-09-26

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Bone Marrow Biopsy in Myelodysplastic Syndromes & Myeloproliferative Neoplasms

A Review for Anatomic Pathologists

Bakul I. Dalal MD FRCPC DABP FACP FASCP

Clinical Professor, Department of Pathology, UBC

Hematopathologist, Vancouver General Hospital

[email protected]

(email me for PDF of this presentation. Also available online)

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Objectives• This session focuses on BMBx histopathology… H&E, IHC and special

stains.

– The blood and bone marrow aspirate findings will not be covered.

At the end of this session…

• You will know the general issues related to BMBx: indications, procedure, processing, examination, writing interpretive reports.

• You will have basic concept of most recent WHO classification of MPN, MDS and other chronic myeloid neoplasms.

• The learners will know the common and uncommon morphologic features of MPN and MDS in BMBx.

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Indications of BMBx in Patient Suspected of Having MPN/MDS

• Persistent cytopenia with dysplastic features

• Blastemia

• Persistent increase in counts

• Leukocytosis with left shift with occasional blast, eosinophilia, basophilia, myelocyte bulge

• Thrombocytosis (>450x109/L), with megathrombocytes

• Persistent erythrocytosis, with features of iron deficiency

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How to Perform a BMBx• Always have concurrent blood specimen, CBC printout, bone marrow

aspirate and Bx

• Site: Posterior Iliac spine

• Occasionally anterior iliac spine, tibia etc

• Video of the procedure available…

• https://www.youtube.com/watch?v=NkdsLHBCreI (NEJM)

• https://www.youtube.com/watch?v=qfOeGWRmyl0 (UofT)

• Keep the BM procedure control with you to maintain the quality

Lee et al, IJLH 2008; www.cap.org/apps/docs/reference/bone-marrow-comments.pdf

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Processing of Bone Marrow Specimens• Aspirate (Romanowski stains):

• Push film (spread). Make 6 films. At bedside. Un‐anticoagulated. Stain 3 films. 100 cell differential in each slide. Fix rest.

• Squash. Stain 1‐2

• Touch imprints of biopsy. Stain 2

• Buffy coat preparations. As necessary.

• Unstained: Archive for FISH studies, extracting DNA etc.

• Bx/clot etc (H&E):

• BMBx: Cut three levels. One of them 1u thick. Paraffin or plastic (methacrylate). Fixative: Formalin better. B5 denatures nucleic acids. FISH does not work.

• Clot (filter) preparations: Better for IHC

Lee et al, IJLH 2008 (ICSH); www.cap.org/apps/docs/reference/bone-marrow-comments.pdf

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How to Examine a BMBx• Always review CBC, blood film and asp concurrently

• Not possible in some centers. Only biopsy available!

• Headings:

• Quality

• Cellularity

• ME ratio

• Erythropoiesis (incl iron stain)

• Granulopoiesis (incl blasts)

• Megakaryocytes

• Lymphoplasmacytic complement

• Stromal elements

• Other infiltrates

• Cortical and trabecular bone

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Thiele et al, Hematologica 2005 7

Quality• Length: 1.5 or 2 cms.

• Fragmentation, crushing, hemorrhage

• Number of hematopoietic spaces

• On a scale of 0‐4

• 0: no inter‐trabecular spaces

• 1: inter‐trabecular spaces, but very little hematopoietic tissue

• 2: <10 inter‐trabecular spaces/w hem tissue

• 3: Adquate. >10 spaces/w hem. tissue. 1‐2 cm long.

• 4: Excellent. As above, no fragmentation, no hemorrhage, longer than minimum req.

Thiele et al, Hematologica 2005; Friebert et al, J Ped Hem Onc 1998 8

Cellularity• Hypo‐, normo‐ or hyper‐ for the age

• Ignore subcortical empty spaces (upto 5) and crushed areas

• Calculate hematopoietic vs all other space (fat, fibrosis, vascularity etc)

Age Upper limit

• <2 yrs 80%

• 2‐4 yrs 70%

• 5‐30 yrs 60%‐70%

• 40‐60 yrs 40%‐50%

• ≥70 30%‐40%

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Stromal Complement• Reticulin and collagen network: Grade away from edges, blood

vessels and trabeculae

• Grading: 0‐3, WHO 2008

• MF‐0: Normal. Scattered linear reticulin with no intersections (cross‐overs)

• MF‐1: Loose network of reticulin with intersections

• MF‐2: Diffuse and dense increase in reticulin with extensive intersections;

• collagen and/or osteosclerosis absent or focal

• MF‐3: Coarse bundles of collagen, often with significant osteosclerosis

• Vascularity: CD34 IHC

Thiele et al, Haematologica 2005

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Cortical and Trabecular Bone• Osteopenia and osteosclerosis

• Osteoclastic and osteoblastic activity

• Bone remodelling and post traumatic repair

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Reporting a BMBx• BMBx and aspirate are the same tissue, so issue ONE report covering

both.

• Several guidelines available

• Vancouver General Hospital format:

• Specimens / tests requested

• Clinical summary / diagnosis / indication for BMBx

• Lab findings, including CBC

• Gross description of aspirate and bx (length, fragmentation, color)

• Nucleated differential count: No diff / 300 / 500, …

• Microscopy: Quality, cellularity, M:E ratio, E/G/M/LP, iron stores, stroma/microenvironment, infiltrates, trabecular bone, special tests (flow, IHC, silver etc)

11Lee et al, IJLH 2008 (ICSH); www.cap.org/apps/docs/reference/bone-marrow-comments.pdf

WHO Classification of MPNs/MDS etc..• MPN or MPD?

• Detailed dx criteria available

12Vardiman et al, Blood 2009

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WHO Classification of MPNs/MDS etc.. 2

• STI responsive



• Bridge and provisional entities




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Usefulness of BMBx in MPN / MDS• Precise quantitation of cellularity

• Precise quantitation of blasts (no hemodilution, clotting etc)

• Blast % by BMAsp vs flow vs BMBx‐CD34 does not correlate!

• MDS patients

• Asp vs BMBx: discrepancy 65/243 cases (27%)

• Asp over‐estimating 22%, under‐estimating 5%

• BMBx vs flow: discrepancy 29/89 cases (33%)

• Flow over‐estimating 10%, under‐estimating 23%

• Asp vs flow: discrepancy 39%

• Prognostic value – flow best prognostic factor for non‐RAEB

• Morphologic findings in BMBx in MPN and MDS

Levi et al, ASH 2004

BMBx in CML• Dx: Ph1 pos , BCR/ABL pos (can be done on blood before the BM

procedure is done)

• BMBx:

– Hypercellular

– M: E ratio markedly increased

– Granulocyte hyperplasia, left shift, myelocyte bulge, eosinophilia, basophilia

– Megakaryocytes: increased, smaller (dwarf)

– Paratrabecular granulocyte cuff >4 cells thick

– Fibrosis 1‐2/3

• AP: Blasts increased (CD34), increasing fibrosis, increasing and crowding of abnormal megakaryocytes.

• BP: Blasts markedly increased, fibrosis disappears.17

PV


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BMBx in PV• Pre‐polycythemic and polycythemic (Cellular phase):

– Hypercellular marrow, panmyelosis, reticulin normal, megakaryocytes increased, pleomorphic (normal + ET like)

• Post‐polycythemic (spent) phase:

– Looks like fibrotic phase of PMF or ET.

– Hypocellular marrow, grade 3/3 fibrosis (rarely osteosclerosis), clusters of hyperchromatic megakaryocytes, intra‐sinusoidal hematopoiesis.

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ET

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BMBx in ET• BMBx one of 4 major criteria.

– Normo or hypercellular

– Megakaryocyte hyperplasia

– Enlarged hyperlobulated megakaryocytes, staghorn, bunch of grapes

– Granulopoiesis, erythropoiesis not hyperplastic

– Fibrosis

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PMF

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BMBx in PMF• BMBx morphology one of three major criteria.

• Cellular/prefibrotic phase:

– Hypercellular marrow

– Megakaryocytes markedly increased and abnormal, pleomorphic large and small, prominent crowding, endosteal / perisinusoidal; bulbous or cloud‐like (plump lobulation) nuclei.

– Fibrosis 1‐2/3.

• Fibrotic phase:

– Hypocellular marrow

– Fibrosis 3/3, osteosclerosis

– Very abnormal megakaryocytes similar to cellular phase.

23Thiele, Sem Hematol 2005; Vardiman, Blood 2009

Megakaryocyte Morphology in MPNsCML PMF ET PV

Number N > ↑↑↑↑ ↑↑↑↑ ↑↑↑↑ N > ↑

Localization Scatteredor clustered

clustered, paratrabecular, perisinusoidal

Scatteredor occ

clustered

Scattered

Size N > ↓ (dwarf) N > ↑ ↑↑↑ N

Nucleus Hypolobated bizzare, hyperchromatic,

cloud-like, baloon shaped,

bare nuclei

hyperlobulated, stag-horn, bunch of grapes

N

Cytoplasm Scant/moderate

moderate abundant moderate

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Gianelli et al, Leuk Res 2015

BMBx Morphology in MPNs Does Not Always Correlate with Clinical/Lab findings

• 29 patients with MPN and thrombosis

• Detailed BMBx morphology

– WHO classification: N=29; ET 6, PV 11, PMF 11, NOS 1

• Correlation with clinical / lab criteria: ET 2/6, PV 3/11, PMF 11/11

• Remainder called NOS

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BMBx Morph in JAK2-mut vs JAK2-wt MPNs (ET & PV)

Campbell et al, Lancet 2005

Tefferi et al, Leuk 2014

BMBx IHC for Molecular Abnormalities: CALR

• JAK2 and MPL donot work by IHC.

• Calreticulin is endoplasmic reticulum based Ca binding protein.

• CALR gene is mutated in 50‐80% of patients with JAK2 neg, MPL neg ET or PMF patients.

• Expected to be included in the WHO 2015/6.

• Together, JAK2, MPL and CALRmolecular markers account for 80‐90% of ET/PMF group.

• 10‐20% are “triple negative”.

• Antibodies to mutated CALR are commercially available.

• Mark megakaryocytes.

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BMBx in Mastocytosis• BMAsp: Abnormal shaped mast cells

• BMBx:

– Pseudogranulomatous lesions

– IHC

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Myelodysplastic Syndromes• Diagnosis and classification of MDS is based mainly on blood and

bone marrow aspirate findings:

– % of blasts

– Dysplastic morphology

– Extent of dysplasia: uni‐ or multi‐lineage

– Other findings

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BMBx in MDS• BMBx has a limited role:

– ALIP is powerful prognostic marker

– Classification

• Staging: Precise blast quantitation

– tissue distribution of blasts

• Hypocellular MDS from SAA

• 5q‐: Megakaryocyte morphology

• RARS / RARS‐T: Iron stores, ringed sideroblasts

• MDS‐F: MDS/w fibrosis

– FISH possible (In C/O dry tap), except in B5 fixed tissue.

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ALIP• Abnormal localization of immature precursors

• GCSF secreted by stromal cells in para‐trabecular location

• Three aggregates in each level of >5 immature cells, >100u from the bony trabecula

• Can be seen in regenerative marrow.

• Should differentiate from aggregates of megaloblastic erythroid precursors, and from monocyte aggregates.

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Association of ALIP with OS

32Verburgh et al, JCO 2003

Iron – Perl’s reaction

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CD34 IHC in MDS• CD34 pos in 80‐90% of blasts in MDS.

– 1/3 the expression is partial, so quantitation inaccurate

– CD117 second choice; however, also positive in promyelocytes

• Precise staging RA vs RAEB1 vs RAEB2

– Discrepancy.

– BMAsp diff still rules

– Blast aggregates indicate aggressive course

• Hypocellular MDS vs Aplastic Anemia

– H&E morph helps in 10‐20% of cases. Cytogenetics helps in 25%.

– CD34pos cells (IHC) in 10 HP fields 10 in hypocellular MDS (0‐45) vs 3 in SAA (0‐8).

– p53pos cells 3 (0‐36) vs 0 (0‐7).

34Orazi et al, AJCP 1997; Cha et al, AnnLabMed 2014

CD34 IHC in MDS … 2• CD34+ megakaryocytes, >20%. Seen in 29/202 (15%). More blasts,

cytopenia, bad cytogenetics, short survival

35Tang et al, Leuk Res 2011

MDS-F• N=79 (13%)

• Shorter LFS, OS

• 25% were JAK2mutated.

• If developed during the course of disease, indicative of leukemic transformation

36Fu et al, Mod Pathol 2013

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Approach to a BMBx in MDS• H&E

• CD34, CD117, p53

• Reticulin, trichrome

• Perl’s reaction for iron stores

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Summary:• BMBx morphology is an integral part of diagnostic algorithm of MPN

and MDS.

• BMBx should be performed on every case of suspected MPN or MDS.

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20150926 PNWSP Talk BMBx MDS MPN Printout.ppt · 2015-09-26 2 4 How to Perform a BMBx •Always have concurrent blood specimen, CBC printout, bone marrow aspirate and Bx • Site:

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