Diagnosis Greg Wolgamot, MD PhD Workup of leukemia: 1.Blood - - PowerPoint PPT Presentation

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Diagnosis Greg Wolgamot, MD PhD Workup of leukemia: 1.Blood - - PowerPoint PPT Presentation

Oct 20, 2023 209 likes •790 views

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

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Diagnosis

Greg Wolgamot, MD PhD

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Workup of leukemia: 1.Blood 2.Lymph node 3.Marrow 4.Prognostic indicators

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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

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

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The Power of Flow Cytometry

  • Single cell analysis
  • Multiparametric
  • Rapid
  • Quantitative
  • Flexible
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Flourochrome-tagged antibodies

flourochrome

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Flow Cytometry

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Flow Cell

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Flow Cytometry

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Flow Cytometry

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CD45/SS

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

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Normal Granulocytic Maturation

Wood and Borowitz (2006) Henry’s Laboratory Methods

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Normal Granulocytic Maturation

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

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Normal B cell Maturation

Wood and Borowitz (2006) Henry’s Laboratory Medicine

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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

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Case 1: Reactive

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Case 3: CLL

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WOLGAMOT

Lymphoma WOLGAMOT

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

Further Workup

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Core biopsy of cervical lymph node

Case 3

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Case 3: CLL

Mutated CLL = good prognosis Unmutated CLL = poor prognosis

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

Case 3: CLL

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Case 2: Mantle cell lymphoma

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Bone marrow aspirate

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Case 3: CLL

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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

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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

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Cytogenetics & Molecular Studies

  • 1. Cytogenetics
  • 2. FISH (flourescence in situ hybridization)
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Case 1: Would be normal

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Case 2: Mantle cell lymphoma

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Case 3: CLL

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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

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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:

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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)

. . . . . . . .

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ISH Detection Methods:

Fluorochrome-labeled (FISH) Chromogenic (CISH)

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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

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Case 2: Mantle cell lymphoma

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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

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Case 2: Mantle cell lymphoma

Fusion probes

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Case 3: CLL

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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

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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.

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WOLGAMOT

Lymphoma WOLGAMOT

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WOLGAMOT

Lymphoma WOLGAMOT

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Ryan Fortna , MD PhD NW Pathology Brent Wood, MD PhD U of Washington Cancer Program at SJH

Special thanks to: