New Approaches to

Lymphoid Disorders in

Blood and Bone

Marrow

Kathryn Foucar, MD

kfoucar@salud.unm.edu

July 2013

Colorado Society

for Clinical

Pathologists

New Problems Require New

Approaches

• Atypical lymphocytosis

• CLL vs. monoclonal B

lymphocytosis

• Prognostic markers in CLL

• “Wolf in Sheep’s Clothing”

(2013 version) 2

Atypical Lymphocytosis

• Not really new but comes in different ways:

Sometimes arises from med tech

referral criteria or sometimes from

“weird” flow results

• Be aware that sensitive flow (6 or 8 color

or more) finds lots of “abnormal”

populations 3

4

Atypical Lymphocytes

5

CD4’s predominate

Atypical Lymphocytes

Diagnosis?

1. Leukemia?

2.Lymphoma?

3. Benign? 6

What information will

help?

1. Age?

2. CBC data?

3. Clinical? 7

Yes

Yes

Yes

Data:

1. Child

2. Severe cough

3. High WBC, high platelet

count, normal H/H

8

Final Diagnosis?

9

Pertussis (Bordetella pertussis)

Why lymphocytosis in bacterial

infection?

Bacterial toxin inhibits normal

lymphocyte migration from blood

10 Non-Activated Lymphs: CD4’s predominate

Age, Clinical Features Key Child-Pertussis

Adult- Leuk/Lymphoma

Pertussis

• Child

• Severe cough

(whoop)

• Defect in

migration (lymphs

not reacting to

bacterial infection)

• Flow: CD4> CD8

Leuk/Lymphoma

• Usually adult

• May have cytopenias

• Variable symptoms

• Monotypic B or

Aberrant T by flow

• Various

molecular/genetic

features

11

New Approaches/New Issues

1. Much more “integrative”

2. “Too” much reliance on flow is

costly and generates potential

“false positives”

3. Morphol + clinical + other CBC

parameters + flow (sometimes) +

genetic (highly selected) 12

Monoclonal B Lymphocytosis (<3%)

13

Absolute monoclonal B cell = 300

Small Monoclonal B cell

population detected by flow

• Monoclonal B lymphocytosis vs. “early” CLL vs.

minimal blood involvement in patient with SLL

• Interpretation of flow cytometry in a vacuum is

problematic

• Integration with clinical, CBC data, and

morphology essential

• Recognize that FISH and molecular will not help

in distinguishing MBL from CLL and SLL 14

Monoclonal B Lymphocytosis

(>15% of Healthy Elderly)

• May be CLL type (CD5+ CD23+) or non-CLL type

(CD5 -, sometimes CD 10+)

• If CLL type can show same FISH and molecular

abnormalities as overt CLL

• Substantial evidence that MBL is precursor to CLL

• Distinction between MBL and overt CLL is arbitrary

set point of absolute monoclonal B cell count

≥ 5, 000

• Rate of progression to overt CLL is 1% per year 15

16

3 yr old—normal

Absolute Lymphocyte Count 9,000

65 yr old—CLL

Absolute Lymphocyte Count by Age*

Lymphocytosis Lymphopenia

Newborn > 10,000 < 2,500

< 1year > 9,000 < 4,000

Child > 7,000 < 2,800

Adult > 4,000 < 1,500

*Conventional units per µL

17

B-Chronic LPN

Definition:

Clonal proliferation of morphologically and immunophenotypically mature B-lymphocytes. These cells are generally characterized by a low proliferation rate and prolonged cell survival. 18

CLL:

Incidence

by Age

Source: Cheson (ed) Chronic Lymphoid Leukemias, 2001 19

Approach to CLPN • Clinical- Age, physical exam

• CBC- all lineages

• Morphology of lymphocytes

• IP profile, genotype (some cases)

• BM findings

• Prognostic factors (esp. CLL) 20

Elderly female;

mild

lymphadenopathy

Courtesy J. Saiki

21

Most CLL patients have mild adenopathy

CLL: Preserved Hematopoiesis 22

CLL, smudge cell 23

CLL: Diagnostic Immunophenotypic

Profile

Mature B Monoclonal SIg (weak)

B-cell

antigens

CD19, weak CD20, weak CD22

Coexpression CD5, CD23

Absent CD10, FMC7, CD11c (weak to

neg.), CD79b, CD103

Prognosis CD38, ZAP 70 24

CLL, CD38-

67 y.o. female

WBC 25.9 (22 L)

Hgb 9.6

Hct 29%

Plt 158K FSC

SS

C

Lam

bda

Kappa

CD5 CD23 CD20

CD

23

CD

38

CD

3

25

CLL, CD38+

55 yr-old male

with autoimmune

hemolytic anemia

Forward Scatter S

ide

Sca

tter

Lam

bda

Kappa

CD5

CD

23

CD5

CD

38

CD20 C

D3

26

CLL: Survival Data • Median survival times exceed 10-15 yrs

• 1/3 patients never require therapy

(median survival: 25 years)

• 1/3 patients have prolonged indolent

phase but eventually progress

• 1/3 have more aggressive disease

requiring upfront therapy (median

survival: 8 years) 27

Prognostic Factors in CLL • Rai, Binet stage; age; performance status

• CD38 expression

• ZAP 70 expression

• Genetic features; mutation status

• Pattern of BM infiltration

• Thymidine kinase, b2 microglobulin

• Transformation

• Many others 28

CLL: IGHV@ Mutations • IgH variable chain mutations

normally occur during germinal center stage of B-cell maturation ‡

• Normal process linked to diversity of antibody production vs. antigens

• Mutation status is significant prognostic factor

‡ May occur outside germinal center 29

Progression Risk in CLL

30

Source: Morabito, et al. Br J Haematol 2009; 146:44

Genetics and Prognosis in CLL

Favorable

del13q as sole

abnormality

Intermediate

Normal

Trisomy 12

Unfavorable

p53 deletion (17p)

ATM deletion

(11q) 31

del17p del11q

tri 12

del13q

normal

Dohner et al. Genomic Aberrations and Survival in Chronic

Lymphocytic Leukemia. NEJM. 2000;343:1910-16.

Courtesy K. Reichard

Flow (typical): CD5+, CD23+, weak CD20, weak SIg

FISH: del(17p13)

CLL with Atypical Morphology

32

CLL: How Many Prognostic

Factors are Enough? • Flow Cytometry (6 color) with CD38 and ZAP70

• CLL FISH panel

(tri 12, del 13q, del 11q, del 17p)

• Somatic mutation assessment

(mutated vs. unmutated IGHV@)

• BME required to assess pattern of infiltration

• Multiple lab parameters – e.g. β 2 microglobulin

33

Cadillac = all of the above

Yugo = none of the above (morphology)

34

CLL: Independent Prognostic

Factors

• Rai, Binet stage

• CD38

• ZAP 70 (Problematic)

• Mutation status

• Cytogenetics (FISH) 35

When to assess prognostic

factors?

• At initial diagnosis

• When patient requires therapy

• Discuss with clinician

36

Hairy Cell Leukemia

Clinical: Middle age to elderly,

splenomegaly

Hemogram: Pancytopenia, monocytopenia

Morphology: Oval to round nuclei with “spongy” chromatin; abundant cytoplasm with hairy projections

Cytochemistry: TRAP (strong)

Bone Marrow: Distinct pattern of infiltration

Molecular: BRAF mutations in all cases 37

HCL: Rare cells in blood

38

Hairy Cell Leukemia

Forward Scatter

Sid

e S

catt

er

CD

11c

CD22

CD

22

CD103 CD25

CD

22

39

HCL: Minute (0.3%) Population Detected

Sid

e S

catter

CD

11c A

PC

-A

CD

25 P

E-C

y7-A

S

SC

-A

Forward Scatter

CD103 FITC-A

CD22 PE-A

CD22 PE-A

All Events

CD22+

Primary

CD22+

CD22+

CD22+ 11c+ CD22+ 11c+ 25+ 103+

(x1

00

0)

40

HCL, diffuse, interstitial 41

HCL, diffuse, interstitial 42 Fried egg inset

HCL Immunohistochemistry

TRAP CD20

ALSO: Annexin, CD123, t-bet 43

HCL: IHC and Caveats

• Annexin 1—Myeloid cells+, T cells+

• CD123—Dendritic cells+

• DBA .44—Only subset+

• TRAP—Histiocytes TRAP+

• Cyclin D1-weak—Confuse with MCL

44

Wolf in Sheep’s

Clothing (WSC #1)

• Hypocellular

neoplastic BM

biopsy, especially

with accompanying

lousy aspirate (due

to reticulin fibrosis) 45

Hypocellular HCL; rare cell in blood 46

47 Hypocellular HCL; Rare cell in blood

retic

WSC #1: Hypocellular

Neoplasms

• HCL *

•T-LGL

•MDS/AML

48

WSC #1: Key Strategy

• Always do CD20, CD3, and

CD34 before making a

diagnosis of aplastic anemia

• Note HCL is highly responsive

to 2-CDA; very short course of

therapy

(cannot miss this diagnosis)

49

WSC #2: Case

54 year old female asymptomatic;

routine CBC (July 2010 WBC 6.5)

CBC: WBC 35.9 (ANC 4.8,

RBC 5.0 ALC 30.9)

H/H 14.9/47

MCV 93

Plt 180 50

Key Features

•Marked absolute

lymphocytosis

•Preserved hematopoiesis

• What is likely diagnosis?

51

CLL

54 year old female; ALC 30.9 52

54 year old female; ALC 30.9 53

WSC#2: Flow Cytometric IP: Not CLL

• Mature helper T-cell (96:1)

• Normal Pan T-cell antigen expression (85%T’s)

WSC#2: Call to Clinician:

Question Answer

Hepatosplenomegaly No

Lymphadenopathy No

Skin lesions No

Symptoms None

55

What Next?

• T- cell clonality (strong TCRα peak)

• Cytogenetics – must use T- cell

mitogen

56

WSC#2: Karyotype:

44,XX,add(5)(q34),-8,del(11)(q21),

-13,inv(14)(q11.2q32),add(19)(q13.3)

-22,+mar[5]/45,idem,del(9)(q22),add(18)

(p11.1),+mar[cp12]46,XX[4]

FISH for t(2;5), ALK rearrangement of

ALCL-negative

57

WSC#2: FISH/Cytogenetics

Must stimulate T-cells 58

Wolf in Sheep’s Clothing #2

• Should be CLL but is not

• Diagnosis?

59

- T-prolymphocytic leukemia

Summary

•Focus on more common

diagnostic problem areas

• Outlined key strategies to

keep the “wolves” at bay!

60