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

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

Lymphoma WOLGAMOT WOLGAMOT

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

Case 3 Core biopsy of cervical lymph node

Case 3: CLL Mutated CLL = good prognosis Unmutated CLL = poor prognosis

Case 3: CLL FINAL DIAGNOSIS: Cervical lymph nodes, core biopsy: Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). Camilla T. Allen, MD Pathologist

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

Prognostic Indicators CLL Abnormality Prognosis Very High risk High risk low risk Very low risk same as 10 year survival ------> 29% 37% 57% 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 intermediate risk. Bulky nodes, faster growth, unmutated IgH, requires del 11q22.3 DCI FISH 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) ?

Cytogenetics & Molecular Studies 1. Cytogenetics 2. FISH (flourescence in situ hybridization)

Case 1: Would be normal

Case 2: Mantle cell lymphoma

Case 3: CLL

Prognostic Indicators CLL Abnormality Prognosis Very High risk High risk low risk Very low risk same as 10 year survival ------> 29% 37% 57% 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 intermediate risk. Bulky nodes, faster growth, unmutated IgH, requires del 11q22.3 DCI FISH 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) ?

In Situ Hybridization (ISH) Key features: 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) 3) Detection method Peroxidase or FITC phosphatase (radiolabel) (fluorochrome: FISH) (enzyme: CISH) S35 S35 S35 * * * . . . . . . . . 2) Probe (DNA) A A C C T A G T T C T G T G G C A G T A G T C A A T A G A A A G T A G T . . . . . . . . 1) Target (DNA or RNA)

ISH Detection Methods: Fluorochrome-labeled Chromogenic (FISH) (CISH)

Uses of In Situ Hybridization  Chromosome enumeration  Locus-specific copy number alterations  Translocation detection t(11;14) translocation by single-fusion FISH  Detection of specific transcripts (RNA) Trisomy 21 HER2 amplification CISH for kappa and by FISH by FISH lambda light chain mRNA  Detection of foreign DNA/RNA Detection of EBV RNA by CISH

Case 2: Mantle cell lymphoma

Fusion Probes vs. Break-Apart Probes for Translocation Detection  Target one locus  Target two loci  Useful when there are  Identifies both partners numerous potential involved in a translocation partner genes

Case 2: Mantle cell lymphoma Fusion probes

Case 3: CLL

Prognostic Indicators CLL Abnormality Prognosis Very High risk High risk low risk Very low risk same as 10 year survival ------> 29% 37% 57% 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 intermediate risk. Bulky nodes, faster growth, unmutated IgH, requires del 11q22.3 DCI FISH 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) ?

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.

Lymphoma WOLGAMOT WOLGAMOT

Lymphoma WOLGAMOT WOLGAMOT

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