Cytogenetics- Flow cytometry Correlation

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Cytogenetics- Flow cytometry Correlation

• May 19, 2004
• Dr. Alina Dulau
• Montefiore Medical Center

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CASE 1 88 year old female, presented with anemia
and leukopenia (11/ 2002)

• CBC Hgb 8.4, Hcr 28.4,WBC 3000/mmc Plt
  300.000/mmc
• Flow cytometry 7-10 CD11b, CD34
  hematopoietic precursors
• Bone marrow biopsy hypocellular (0-30), absent
  stainable iron

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Differential diagnosis of hypocellular BM

• Hypoplastic MDS
• - Cytogenetic abnormalities del(7q)
• - Blasts CD34, dim CD45 expression
• Aplastic anemia
• - Mecs immune destruction of CD34
• - HLA-DR, CD8, CD56

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FLOW CYTOMETRY

• Flow cytometry 3/2004

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Flow cytometry interpretation

• Blast population (58 of total cells) with
  intermediate intensity CD45 expression and low to
  intermediate side scatter
• CD7(partial), CD11b(partial), CD13,
  CD14(partial), CD33(partial), CD34,
  CD64(partial), CD117, HLA-DR-
• Dx Immature myelomonocytic phenotype c/w Acute
  myelomonocytic leukemia

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Chromosomal Analysis Unstimulated Peripheral
Blood

• Staining with GTG Band
  Resolution 450
• No. of cells analysed 20 No of cells
  karyotyped 2
• CYTOGENETIC DIAGNOSIS
• 46,XX, inv(3) (q21q26) 2 / 45, idem, -7 15 /
  46, idem, del (7) (p11.2p15) 3

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46,XX,inv(3)(q21q26),del(7)(p11.2p15)
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46,XX, inv(3)(q21q26),-7
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CASE 2 71 year old female with history of
circulating blasts (3) x 3 years

• CBC Hgb 11, Hcr 37.2, WBC 18,600
• Plt 61,000.
• Peripheral smear 7 blasts

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Flow Cytometry (Jan 2004)-Bone marrowNumber of
blasts, Immunophenotype
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• Flow cytometry interpretation
• 5 CD33, CD117 myeloblasts without
  expression of CD34
• Reduced side scatter of mature myeloid cells

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Chromosomal Analysis Unstimulated Peripheral
Blood

• Staining with GTG Band
  Resolution 450
• No. of cells analysed 20 No of cells
  karyotyped 2
• CYTOGENETIC DIAGNOSIS
• 46,XX, dup (1) (q12 q32), 8 20

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46,XX, dup(1)(q12q23),8
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MYELODYSPLASTIC SYNDROMES

• Clonal stem cell disorders
• Single or multilineage dysplasia
• Ineffective hematopoiesis
• - Impaired cellular differentiation
• - Increased apoptosis gtprogressive
  pancytopenia
• Accumulation of genetic abnormalities
• 30-75 evolution to acute leukemia

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Morphologic features

• Dyserythropoiesis
• º Peripheral blood nucleated RBC
• º BM erythroid hyperplasia, megaloblastic
  changes, nuclear budding, karyorrhexis,
  multinucleation, intense basophilia of the
  cytoplasm, vacuolization, poor hemoglobinization,
  PAS cytopl. granules

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BM Iron increased, coarse sideroblastic granules

• Dysgranulopoiesis hypogranularity (decreased
  reaction with MPX), abnormal nuclear segmentation
  and lobulation,
• ALIP (abnormal localization of immature
  precursors in the center vs paratrabecular or
  perivascular), immature granulocytes, monocytes,
  blasts in circulation

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Bone marrow
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Myloperoxidase in Bone marrow

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Clonality of hematopoietic elements involved

• Combined immunophenotyping and FISH (chromosome
  abnormalities)
• Neoplastic transformation of myeloid
  progenitors at the stem cell level
• Involvement of the B lymphoid lineage
• T lymphocytes and NK cells nonclonal

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Pathophysiology of MDS

• Stepwise accumulation of genetic lesion
• Unbalanced cytogenetic abnormalities
• ? unmasking of oncogenes
• ? inactivation or deletion of tumor
  suppressor genes
• Acquired defective DNA repair
• Genomic instability (telomere shortening)

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Evolution in MDS

• Early phase Apoptosis is up-regulated
  overweighing proliferation ? peripheral
  cytopenias (apoptosis in BM)
• Leukemic phase abrogated apoptosis (mecs loss
  of function of DAP-kinase due to
  hypermethylation)
• with proliferation of dysplastic clone

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Prognosis in MDS

• Bone marrow blasts, karyotype, cytopenias
• Good prognosis normal karyotype, Y- only,
• 5q-, 20q-
• Poor prognosis gt/3 karyotypic abnormalities,
  7q-, del(7q)
• Intermediate prognosis all other cytogenetic
  abnormalities

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Common cytogenetic abnormalities in MDS

• ABNORMALITY
  INCIDENCE
• Loss of all or part of chr 5
  13
• Loss of all or part of chr 7
  5
• Trisomy 8
  5
• del 17p
  lt1
• del 20q
  2
• Loss of X or Y
  2

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Other cytogenetic changes

• 1 secondary change in disease progression
• 13, 17, 19p, -13q
• Idic (X) (q13)
• Rings and dicentrics in more advanced MDS

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5q- syndrome

• MDS with del(5q) between bands q31-33
• Refractory anemia
• Middle age to old women
• Macrocytic anemia
• Elevated Platelet count (increased megakaryocytes
  in BM, hypolobated)
• Blasts lt 5

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Monosomy 7

• Most frequent cytogenetic abnormality of
  childhood MDS
• 20 of children with MDS have other chromosomal
  abnormalities
• Most commonly associated with Downs syndrome and
  Pearson syndrome
• Leukemic evolution similar to adult MDS

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AML with Myelodysplasia

• gt/ 2 cell lines with dysplastic features
• Myelodysplastic features in gt 10 of the bone
  marrow cells

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Refractory Anemia with Excess Blasts (RAEB)

• 40 of MDS
• RAEB 1 5-9 blasts in the BM (CD13,
  CD33,CD117)
• lt 5 blasts in the blood
• RAEB 2 10-19 blasts in the BM
• 5-19 blasts in the blood
• Presence of Auer rods
• Genetics8, -5, del(5q),-7, del(7q),del(20q)

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Molecular abnormalities

• N-ras oncogene
• RAS, FMS mutations aggressive course
• Increased WT1 gene expression evolution AML
• P53
• IRF-1 tumor-suppressor gene
• p15INK4b methylation is associated with del(7q)
• EVI 1, MLL transcription factor genes

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RAS Gene in MDS

• RAS gene family encodes signal-transducing
  proteins with role in cell growth and
  differentiation
• Normal bone marrow low, uniform levels of p21RAS
• In MDS, p21RAS is overexpressed
• Leukemogenesis in MDS

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Point mutations of AML1/RUN X1 gene

• Acute Myeloid Leukemia
• Myelodysplastic Syndrome
• AML/ MDS-t (post treatment with alkylating agents)

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Dysplastic abnormalities in the bone marrow

• Deficiencies of vitamin B12, folate
• Viral infections (HIV)
• Exposure to antibiotics
• Chemotherapeutic agents
• Ethanol
• Benzene
• Lead

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• Thank you.