Diagnosis

Greg Wolgamot, MD PhD

Workup of leukemia:

1.Blood

2.Lymph node

3.Marrow

4.Prognostic indicators

Result Name Result Abnl Normal Range Units WBC 14.7 H 4.0-11.0 K/mm3 RBC 4.51 4.31-5.77 M/mm3 HGB 12.9 L 13.2-17.5 g/dL HCT 38.7 L 38.9-49.9 % MCV 85.8 80.0-100.0 fL MCH 28.6 27.8-33.8 pg MCHC 33.3 31.5-36.5 g/dL RDW 14.0 11.5-14.2 % PLATELET COUNT 126 L 150-400 K/mm3 NEUTRO% 23.6 % LYMPH.% 69.8 % MONO% 5.1 % EOS% 0.6 % BASO% 0.4 % IMM GRAN% 0.5 0-0.5 % Includes myelocytes, metamyelocytes and promyelocytes. NEUTRO# 3.5 1.5-8.0 K/mm3 LYMPH# 10.3 H 1.0-3.5 K/mm3 MONO# 0.7 0.2-1.0 K/mm3 EOS# 0.1 0-0.5 K/mm3 BASO# 0.1 0-0.2 K/mm3 Smudge cells present

PATHOLOGIST INTERPRETATION: Lymphocytosis suggestive of chronic lymphocytic leukemia Flow cytometry would be contributory for further workup John W. Hoyt, MD Pathologist

The Power of Flow Cytometry

• Single cell analysis

• Multiparametric

• Rapid

• Quantitative

• Flexible

Flourochrome-tagged antibodies

flourochrome

Flow Cytometry

Flow Cell

Flow Cytometry

Flow Cytometry

CD45/SS

Borowitz et al (1993) AJCP 100:534-40. Steltzer et al (1993) Ann NY Acad Sci 667:265-280

Normal Granulocytic Maturation

Wood and Borowitz (2006) Henry’s Laboratory Methods

Normal Granulocytic Maturation

Wood (2004) Methods Cell Biology 75:559-576

Normal B cell Maturation

Wood and Borowitz (2006) Henry’s Laboratory Medicine

Abnormal population identification

• Normal – Antigens expressed in consistent and

reproducible patterns with maturation

• Neoplastic – Increased or decreased normal antigens

– Asynchronous maturational expression

– Aberrant antigen expression

– Homogeneous expression

Case 1: Reactive

Case 3: CLL

WOLGAMOT Lymphoma

WOLGAMOT

1. Cervical lymph node 2. Bone marrow 3. Prognostic indicators

Further Workup

Core biopsy of cervical lymph node

Case 3

Case 3: CLL

Mutated CLL = good prognosis Unmutated CLL = poor prognosis

FINAL DIAGNOSIS: Cervical lymph nodes, core biopsy: Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). Camilla T. Allen, MD Pathologist

Case 3: CLL

Case 2: Mantle cell lymphoma

Bone marrow aspirate

Case 3: CLL

Result Name Result Abnl Normal Range Units WBC 14.7 H 4.0-11.0 K/mm3 RBC 4.51 4.31-5.77 M/mm3 HGB 12.9 L 13.2-17.5 g/dL HCT 38.7 L 38.9-49.9 % MCV 85.8 80.0-100.0 fL MCH 28.6 27.8-33.8 pg MCHC 33.3 31.5-36.5 g/dL RDW 14.0 11.5-14.2 % PLATELET COUNT 126 L 150-400 K/mm3 NEUTRO% 23.6 % LYMPH.% 69.8 % MONO% 5.1 % EOS% 0.6 % BASO% 0.4 % IMM GRAN% 0.5 0-0.5 % Includes myelocytes, metamyelocytes and promyelocytes. NEUTRO# 3.5 1.5-8.0 K/mm3 LYMPH# 10.3 H 1.0-3.5 K/mm3 MONO# 0.7 0.2-1.0 K/mm3 EOS# 0.1 0-0.5 K/mm3 BASO# 0.1 0-0.2 K/mm3 Smudge cells present

CLL Abnormality Prognosis Very High risk High risk low risk Very low risk

10 year survival ------> 29% 37% 57% same as controls

del 17p13.1 (p53) DCI FISH poor; chemo resistance; consider BMT X

p53 mutation poor; chemo resistance X

BIRC3 very poor; chemo resistance, mutually exclusive to p53 X

ZAP70 expression poor X

CD38 expression poor X

NOTCH associated with Richter's X

del 11q22.3 DCI FISH intermediate risk. Bulky nodes, faster growth, unmutated IgH, requires alkylating drugs (cytoxan, bendamustine) X

SF3B1 poor. Resistance to fludarabine X

trisomy 12 DCI FISH good; low risk. If no NOTCH mutation, low risk X, if NOTCH X, if no NOTCH

IgH mutation present good (unmutated is poor) X

normal cytogenetics good X

del 13q14.3/13q34 DCI FISH good, if isolated X

13p if isolated 13p, good; very low risk X, if isolated

t(14;19) ?

t(2;14) ?

Prognostic Indicators

Cytogenetics & Molecular Studies

1. Cytogenetics 2. FISH (flourescence in situ hybridization)

Case 1: Would be normal

Case 2: Mantle cell lymphoma

Case 3: CLL

CLL Abnormality Prognosis Very High risk High risk low risk Very low risk

10 year survival ------> 29% 37% 57% same as controls

del 17p13.1 (p53) DCI FISH poor; chemo resistance; consider BMT X

p53 mutation poor; chemo resistance X

BIRC3 very poor; chemo resistance, mutually exclusive to p53 X

ZAP70 expression poor X

CD38 expression poor X

NOTCH associated with Richter's X

del 11q22.3 DCI FISH intermediate risk. Bulky nodes, faster growth, unmutated IgH, requires alkylating drugs (cytoxan, bendamustine) X

SF3B1 poor. Resistance to fludarabine X

trisomy 12 DCI FISH good; low risk. If no NOTCH mutation, low risk X, if NOTCH X, if no NOTCH

IgH mutation present good (unmutated is poor) X

normal cytogenetics good X

del 13q14.3/13q34 DCI FISH good, if isolated X

13p if isolated 13p, good; very low risk X, if isolated

t(14;19) ?

t(2;14) ?

Prognostic Indicators

1) Probe a specific sequence of DNA or RNA

2) Visualize ‘in situ’ – within the context of tissue

3) Can be performed on interphase cells

4) Can be performed on non-living/fixed cells

In Situ Hybridization (ISH)

Key features:

In Situ Hybridization (ISH)

A C G T A A A G T A C G T A A A G T G G T A G T . . . . . . . .

A A G T A G T T C C C T

1) Target (DNA or RNA)

2) Probe (DNA)

3) Detection method S35 S35 S35 * * *

(radiolabel) (fluorochrome: FISH) FITC Peroxidase or phosphatase (enzyme: CISH)

. . . . . . . .

ISH Detection Methods: Fluorochrome-labeled

(FISH) Chromogenic

(CISH)

Uses of In Situ Hybridization

Chromosome enumeration

Locus-specific copy number alterations

Translocation detection

Detection of specific transcripts (RNA)

Detection of foreign DNA/RNA

Trisomy 21 by FISH HER2 amplification by FISH

CISH for kappa and lambda light chain mRNA

t(11;14) translocation by single-fusion FISH

Detection of EBV RNA by CISH

Case 2: Mantle cell lymphoma

Fusion Probes vs. Break-Apart Probes for Translocation Detection

Target two loci Identifies both partners

involved in a translocation

Target one locus Useful when there are

numerous potential partner genes

Case 2: Mantle cell lymphoma

Fusion probes

Case 3: CLL

CLL Abnormality Prognosis Very High risk High risk low risk Very low risk

10 year survival ------> 29% 37% 57% same as controls

del 17p13.1 (p53) DCI FISH poor; chemo resistance; consider BMT X

p53 mutation poor; chemo resistance X

BIRC3 very poor; chemo resistance, mutually exclusive to p53 X

ZAP70 expression poor X

CD38 expression poor X

NOTCH associated with Richter's X

del 11q22.3 DCI FISH intermediate risk. Bulky nodes, faster growth, unmutated IgH, requires alkylating drugs (cytoxan, bendamustine) X

SF3B1 poor. Resistance to fludarabine X

trisomy 12 DCI FISH good; low risk. If no NOTCH mutation, low risk X, if NOTCH X, if no NOTCH

IgH mutation present good (unmutated is poor) X

normal cytogenetics good X

del 13q14.3/13q34 DCI FISH good, if isolated X

13p if isolated 13p, good; very low risk X, if isolated

t(14;19) ?

t(2;14) ?

Prognostic Indicators

Question 2: FISH: 1. Include both osteichthyes and chondrichthyes. 2. Is a molecular technique that can detect

specific sequences of DNA.

3. Allows classification of leukemias based on the proteins the cells express.

WOLGAMOT Lymphoma

WOLGAMOT

WOLGAMOT Lymphoma

WOLGAMOT

Ryan Fortna , MD PhD NW Pathology Brent Wood, MD PhD U of Washington

Cancer Program at SJH

Special thanks to: