- Molecular Cytogenetics in Hematologic Malignancies -

 


I. INTRODUCTION

  • Components of the Hematopoietic System
  • Hematologic malignancies
  • Leukemia - bone marrow derived neoplasm with peripheral blood involvement
    • Acute -- fulminant clinical onset with "blast" cells; may be lymphocytic or myeloid derived
    • Chronic -- more indolent clinical presentation; malignant cells have more mature morphology; also may be lymphocytic or myeloid
  • Lymphoma - malignant tumor mass usually arising in lymph nodes but sometimes in extranodal sites
    • nonHodgkin's lymphoma - malignancy of B and T lymphocytes
    • Hodgkin's disease - malignant cell is called the Reed-Sternberg cell, which is believed to arise from a mutated B lymphocyte

II. TECHNIQUES FOR STUDYING GENETIC ALTERATIONS

  • Cytogenetics -- A clinical laboratory test in which cellular samples (amniotic fluid, bone marrow, peripheral blood, etc.) are cultured and stimulated to undergo mitosis. While in metaphase, the mitosis is chemically stopped and a picture of the chromosomes (karyotype) is made and examined for abnormalities (translocations, deletions, etc.). This tecnique is adequate for detecting "gross" and numerical chromosomal abnormalities.
  • Molecular Genetics -- This includes a wide variety of techniques (PCR, reverse PCR, Southern Blotting, etc.) in which genetic material is examined at the gene level. Among other things, these techniques can detect point mutations and abnormal fusion of normally unrelated genes (i.e., from translocation).

III. MOLECULAR PATHOLOGY OF HEMATOLOGIC MALIGNANCIES

Many genetic changes occur in these neoplasms. For many, the most important changes are chromosomal translocations. A number of acute and chronic leukemias and nonHodgkin's lymphomas subtypes are associated with specific translocations. The genes located at breakpoints are often involved in lymphocytic or myeloid cell proliferation and/or differentiation. When these genes are translocated into a new genetic "environment", they are upregulated and overexpressed or mutated. These changes result in loss of growth control and malignancy. Some specific examples follow.

  • Leukemia
    • Chronic Myelogenous Leukemia (CML)
      • Clinical presentation -- middle age, indolent presentation, splenomegaly
      • Laboratory findings -- elevated peripheral blood leukocyte count due to proliferation of neutrophils and precursors
      • Cytogenetics -- t(9;22) Philadelphia chromosome
        abnormal chromosome 22 resulting from a translocation with chromosome 9; acquired mutation occurring in a hematopoietic stem cell
      • Molecular genetics
        • chromosome 9 breakpoint: abl (abelson) oncogene ; codes for tyrosine kinase with low levels of expression in hematopoietic cells
        • chromosome 22 breakpoint: breakpoint cluster region (bcr) gene; function unknown
        • new fusion gene on chr 22 made up partly by abl now fused to a portion of bcr. The resultant fusion protein, abnormal in structure compared to the normal abl protein, has markedly increased tyrosine kinase activity. this is believed to interact with signal transduction pathways and turn on proliferation signals to the nucleus
    • Acute Myelogenous Leukemia (AML)
      • Clinical presentation -- wide age range, but incidence increases with advancing age; often fulminant presentation: fatigue, fever, malaise
      • Laboratory features
        • in peripheral blood, anemia, thrombocytopenia; variable WBC count with circulating blasts
        • in bone marrow, hypercellularity with arrest of hematopoietic development at the blast stage
      • Cytogenetics -- t(8;21) 5 to 12% of all AML
      • Molecular genetics
        • chromosome 21 breakpoint: AML1 gene; protein product is the alpha subunit of core binding factor (CBFa), a critical transcription factor controlling differentiation in hematopoietic cells
        • chromosome 8 breakpoint: ETO gene; function largely unknown
        • translocation occurs in an early myeloid precursor cell. The t(8,21) fusion gene product AML/ETO is unable to normally interact with the ususal AML1 partner, core binding factor beta; and, therefore, normal transcription of genes driving differentiation in hematopoietic precursor cells is disrupted; diffierentiation is halted and maturation is arrested, producing the clinical syndorme and morphological features of AML
    • Other molecular cytogenetic changes in leukemia
      • acute promyelocytic leukemia
        t(15;17)      15: PML oncogene, 17: retinoic acid receptor
      • acute myelomonocytic leukemia
        inv (16)       core binding factor beta
      • therapy related AML
        11q23         MLL gene
  • NonHodgkin's Lymphoma (NHL)
    • Follicular lymphoma (22% of all NHL)
      • Clinical presentation -- typically 50' to 60's, lymphadenopathy, often multiple lymph node groups with mild symptomatology
      • Morphology -- usual lymph node architecture replaced by homogenous nodules of malignant lymphocytes
      • Cytogenetics -- (t(14;18) seen in 80 to 90% of these lymphomas
      • Molecular genetics
        • chromosome 14: immunoglobin heavy chain (IgH) gene
        • chromosome 18: bcl-2 oncogene, prevents cellular apoptosis
        • translocation in which the bcl-2 oncogene is moved and fused to the heavy chain gene. This event occurs in a B lymphocyte, and since the function of these cells is to synthesize antibody, the IgH gene is normally heavily transcribed. In its new transcriptional environment, the bcl-2 oncogene is now under the reulatory control of the IgH gene promoter; and, therefore, it becomes overexpressed, inducing cell immortality by preventing apoptosis.
    • Other molecular cytogenetic changes in NHL
      • Burkitt's lymphoma        t(8;14)      8: c-myc oncogene, 14: IgH gene
      • Mantle cell lymphoma    t(11;14)   11: bcl-1 (cell cycle regulator), 14: IgH gene

IV. CLINICAL SIGNIFICANCE OF GENETIC ABNORMALITIES

  • Prognosis -- Not only do the cytogenetic and molecular genetic abnormalities provide insight onto the pathogenesis of hematologic neoplasia, it has emerged that, especially in acute leukemia, they are the most important factor in predicting clinical outcome for these diseases.
    • Good prognostic group:
      AML:                       t(15,17), inv 16, t(8;21)
      ALL (childhood):      hypodiploidy, t(1;19)
    • Adverse prognostic group:
      AML:                      11q abnormalities, -5, -7
      ALL (childhood):      hypodiploidy, t(1:19)
  • Targeted therapies -- genetic alterations produce abnormal quantities of normal gene products or abnormal protein gene products. These may serve as therapeutic targets.
    • CML    bcr/abl fusion protein, drug: STI571/Gleevec
    • Follicular lymphoma   bcl-2 oncoprotein, drug: bcl-2 antisense

V. SUMMARY

  • Hematologic malignancies are all the result of genetic mutations.
  • Specific mutations are consistently and reporducibly found in particular diseases and in fact are often diagnostic of them. These mutations can be studied clinically by cytogenetic and molecular genetic techniques.
  • Many of these mutations are translocations that involve genes which
    • are oncogenic and promote uncontrolled growth or diminished apoptosis
      OR
    • control hematologic differentialtion.
  • Genetic changes convey important prognostic information, especially in acute leukemia.
  • Currently, and increasingly so in the future, some of the genetic alterations and their gene products will be the point of attack of specific, targeted therapies.