Myelodysplastic Syndromes (MDS) and the Acute

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					           Myelodysplastic Syndromes (MDS) and the Acute
           Leukemias: Pathophysiology, Classification and
                         Practical Diagnosis
                                    Pathology 7140
                                     February 2012
                                  Ronald L Weiss, M.D.
                                 Department of Pathology


Objectives
   1.      Provide an overview of the pathophysiology of MDS and the acute leukemias.
   2.      Provide an overview of the current classification of these disorders.
   3.      Provide an understanding of the practical laboratory diagnosis of these
           disorders and the importance of certain modalities in precisely classifying and
           stratifying patients for treatment.

                       Myelodysplastics Syndromes (MDS)

Introduction

       MDS occurs at all ages with a non-age adjusted annual incidence of 3-5/100,000,
        but is most common in patients >60 years of age with the highest incidence in
        patients >70 years of age (20/100,000) where it is the most common malignant
        hematologic disorder
       MDS is either de novo (most common form), i.e. without known etiology, or is
        secondary to known environmental, occupational or iatrogenic exposures (e.g.,
        ionizing radiation, chemicals like benzenes, and certain chemotherapeutic agents
        used for other cancers)
       Symptoms and signs are generally non-specific, including gradual onset of
        fatigue, dyspnea on exertion, recurrent infections and bleeding, reflective of one
        or more cytopenias (anemia, leukopenia and thrombocytopenia)
       These are disorders are caused by ineffective hematopoiesis characterized by
        peripheral blood cytopenias and paradoxical hypercellularity of the bone marrow
       “De-regulation of apoptosis” (programmed cell death) is the leading explanation
        for MDS; resulting in accelerated cell death, in diminished cell death, or a
        combination of both
       Genetic alterations are commonly found in many cases and likely accumulate in
        number (“multi-hit”) as the disease progresses, including transformation into
        acute leukemia; affecting oncogenes, tumor suppressor genes and genes affected
        by ineffective DNA repair mechanisms




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      Differential diagnosis:
           o Nutritional deficiencies (especially folate and/or vitamin B12), certain
              autoimmune diseases, heavy metal intoxication (arsenic), copper
              deficiency, congenital dyserythropoietic anemia, certain infectious
              diseases (e.g., HIV), and drugs that impair normal hematopoiesis should
              all be considered in the initial evaluation of suspected cases of MDS

Classification

      The first formal classification scheme for MDS was proposed by the French-
       American-British (FAB) working group in 1982, and was based on the
       morphologic assessment of the number of blasts in blood and bone marrow (30%)
      In 2001, the World Health Organization (WHO) published a revised classification
       scheme for all hematolymphoid neoplasms, including MDS where changes in the
       absolute blast percentage criteria was changed to 20% to reflect the observations
       that cases with 20-30% blasts clinically behaved like acute myeloid leukemias
       (AML), and a cytogenetically define entity—5q- Syndrome—was added
      In 2008, a revision of the WHO classification was introduced; these disorders are
       subdivided based upon peripheral blood findings, the percentage of blasts in blood
       and bone marrow, the presence of Auer rods and the presence of lineage-specific
       dysplasia (in 10% or more of the cells of that lineage):
           o Refractory cytopenia with unilineage dysplasia
                    Refractory anemia (RA)
                    Refractory neutropenia (RN)
                    Refractory thrombocytopenia (RT)
           o Refractory anemia with ring sideroblasts (RARS)
           o Refractory cytopenia with multilineage dysplasia (RCMD)
           o Refractory anemia with excess blasts-1 (RAEB-1)
           o Refractory anemia with excess blasts-2 (RAEB-2)
           o MDS-unclassified (MDS-U)
           o MDS associated with isolated del(5q)
           o Childhood myelodysplastic syndrome
                    Very uncommon, unless it is present in the background of patients
                       with congenital bone marrow failure syndromes (e.g., Fanconi’s
                       anemia), or following exposure to know cytotoxins
                    20-25% of childhood MDS occurs in patients with Down
                       syndrome, considered a unique category of diseases
                           Transient abnormal myelopoiesis
                           AML associated with DS

      Bone marrow morphology is the most important tool for classifying most cases of
       MDS. However, concordance between observers may vary making MDS of the
       most difficult categories of myeloid disorders to diagnose and classify.




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Secondary MDS—Therapy-Related Myelodysplasia (t-MDS)

     MDS and AML can be late complications of the use of cytotoxic therapy for a
      number of malignancies and following myeloablation treatment prior to bone
      marrow transplantation
         o The most common agents are the alkylators (e.g., melphalan,
             cyclophosphamide, chlorambucil, busulfan, etc.) and the topoisomerase II
             inhibitors (e.g., etoposide, teniposide, doxorubicin, actinomycin, etc.)
                  Latency is usually 2-11 years post-alkylator treatment and <1-3
                     years for the topoisomerase inhibitors
     Multiply genetic “hits” are theorized as the explanation for t-MDS
         o >90% of patients have one or more cytogenetic abnormalities, usually
             involving chromosomes 5 and/or 7 in alkylator-associated MDS and
             11q23 abnormality in topoisomerase-associated MDS
     Characterized by being refractory to treatment and having particularly poor
      prognoses, with median survival of ~8 months and <10% 5 year survival

Laboratory Diagnosis—General Findings in MDS

     MDS is characterized by one or more peripheral blood cytopenias: anemia,
      leukopenia, and thrombocytopenia
     Blasts may be present in the peripheral blood, but should be <5% of leukocytes
     Morphologic evidence of disorderly maturation (dyspoiesis or dysplasia) is found
      in one or more cell lines: erythroid, granulocytic and megakaryocytic
          o Dyserythropoiesis: anisocytosis and poikilocytosis in RBCs;
              megaloblastic changes and nuclear abnormalities (e.g., nuclear budding,
              nuclear blebbing, and multinucleation) in nucleated RBCs (NRBC);
              presence of abnormal iron stores in NRBCs (ringed sideroblasts)
          o Dysgranulopoiesis (of neutrophils, eosinophils and/or monocytes):
              cytoplasmic hypogranulation, nuclear segmentation abnormalities
              (hyposegmentation and hypersegmentation); loss of cytoplasmic
              myeloperoxidase activity
          o Dysmegakaryocytopoiesis: abnormal appearing platelets in peripheral
              blood (hypogranular, hypergranular); circulating megakaryocyte
              fragments or micromegakaryocytes; megakaryocyte abnormalities
              (hypolobated forms, hyperlobated forms)

Laboratory Diagnosis—Practical diagnostic procedures for MDS

     Perform a complete blood cell count (CBC) with differential and peripheral blood
      smear review in any patient suspected of having MDS
     Perform a bone marrow aspiration and biopsy procedure to determine blast
      percentage and to assess for evidence of ineffective and dyspoietic hematopoiesis
          o Usually hypercellular for age and in stark contrast to the degree of
             peripheral cytopenias



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      Submit a bone marrow aspirate sample for cytogenetic analysis
           o Cytogenetics of MDS;
                    Clonal cytogenetic abnormalities are found in at least 50% of de
                      novo cases MDS and nearly all secondary cases
                    The most common abnormalities are: del (5q), del (7q), -7, +8 and
                      -Y
                    A variety of other translocations and deletions have been described
                    In general, the more clonal abnormalities present, the worse the
                      prognosis
                    In those patients with refractory cytopenia(s) but without
                      diagnostic morphology, specific clonal cytogenetic abnormalities
                      are presumptive evidence of MDS.
      Flow cytometric immunophenotyping of blood and/or bone marrow has limited
       value in the workup of suspected cases of MDS
           o Useful in enumerating the number of blasts (CD34-positive myeloblasts,
               for example), and/or identifying immunophenotypic evidence of
               disordered granulocyte maturation (e.g., loss of normal antigens,
               expression of aberrant antigens such as lymphoid antigens) which can be
               followed over time and with treatment
      Rule out other causes of dysplasia, including nutritional deficiencies, with
       appropriate laboratory tests

Prognosis

      International Prognostic Scoring System (IPSS) stratifies MDS patients into 4 risk
       categories, based on the summation of scores for: % of bone marrow blasts,
       cytogenetic abnormalities and the number of peripheral blood cytopenias
           o IPSS predicts duration of survival plus the propensity to progress to acute
               leukemia

Score = 0              =0.5                =1               =1.5               =2
Blasts <5%            5-10%                                11-19%            20-30%
Good karyotype     Intermediate           Poor
0-1 cytopenias          2-3

          o Low grade (score=0): median survival of 5.7 yr [RA, RARS, 5q-]
          o Intermediate-1 grade (score=0.5-1.0): median survival of 3.5 yr [RCMD]
          o Intermediate-2 grade (score=1.5-2.0): median survival of 1.2 yr [RAEB-
            1]
          o High grade (score=2.5 or greater): median survival of 0.4 yr [RAEB-2]

Treatment of MDS

      Most patients have a protracted course of progressive cytopenias that require
       supportive transfusions but who eventually become refractory to these



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      Death is usually due to the results of severe cytopenia (hemorrhage, infection,
       exacerbation of underlying chronic diseases) or transformation into AML
      The goal of therapy varies by IPSS category:
           o Low/Intermediate-1 grades: improve hematopoiesis with growth factors
               (EPO, GM-CFS, Thrombopoietin)
           o Intermediate-2/High grades: improve survival and delaying AML
      For selected patients bone marrow transplantations may be attempted for long-
       term remissions
      Lenalinomide (and other new agents, including 5-azacytidine, decitabine, and
       deferasirox) may improve cytopenias and reduce transfusion requirements in low
       grade MDS cases


               Acute Leukemias of Myeloid and Lymphoid
                                Origin
Introduction

      Acute myelogenous leukemia (AML) is a clonal disorder derived from either a
       bone marrow hematopoietic stem cell (HSC) or a myeloid lineage-specific
       progenitor cell; acute lymphoblastic leukemia (ALL) is a clonal disorder derived
       from B-cell or T-cell precursor cells
      The incidence of AML increases with age from 2-4/100,000 per year in childhood
       and early adulthood to representing 70-80% of acute leukemias in older adults;
       ALL is the predominate acute leukemia in childhood with an incidence of
       3/100,000 per year and 75% of the acute leukemias in children <15 years of age
      AML is divided into de novo and secondary types, with de novo cases usually
       seen in younger patients; as with MDS, secondary cases occur subsequent to
       known environmental, occupational or iatrogenic exposures; there are no known
       specific etiologic factors in ALL
      Both AML and ALL usually present clinically with non-specific signs and
       symptoms, including fatigue, fever, weight loss, bleeding, infection and
       exacerbation of underlying chronic diseases, due to the presence of peripheral
       blood cytopenias; both AML and ALL can present with soft tissue, organ and
       lymph node enlargement due to infiltration of leukemic cells

Classification

      The first classification scheme for AML and ALL was proposed by the FAB
       Working Group in 1976 based upon morphology and the few laboratory-based
       methods then available; with the publication of the WHO 2001 and WHO 2008
       schemes, classification has progressed to the point where multiple
       clinicopathologically defined forms of acute leukemia are now well characterized
       morphologically, immunophenotypically and genotypically:



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o AML
      AML with recurrent genetic abnormalities
              AML with t(8;21) RUNX1-RUNX1T1
              AML with inv(16) or t(16;16) CBFA-MYH11
              Acute promyelocytic leukemia with t(15;17) PML-RARA
              AML with t(9;11) MLLT3-MLL
              AML with t(6;9) DEK-NUP214
              AML with inv(3) or t(3;3) PRN1-EVl1
              Megakaryoblastic AML with t(1;22) RBM15-MKL1
              AML with mutated NPM1
              AML with mutated CEBPA
      AML with myelodysplasia-related changes
      Therapy related MDS/AML
      Acute myeloid leukemia, NOS
              AML with minimal differentiation
              AML without maturation
              AML with maturation
              Acute myelomonocytic leukemia
              Acute monoblastic and monocytic leukemia
              Acute erythroid leukemia
              Acute megakaryoblastic leukemia
              Acute basophilic leukemia
              Acute panmyelosis with myelofibrosis
      Myeloid sarcoma
              An extra-medullary proliferation of myeloid blasts
              Diagnostic of AML regardless of blood or bone marrow
                    o Uncertain overall prognostic significance
              Skin is the most common site, mucosal membranes, orbit,
                potentially any other site
      Myeloid proliferations related to Down syndrome
              Transient abnormal myelopoiesis
              Myeloid leukemia associated with Down syndrome
o Precursor lymphoid neoplasms (ALL)
      B lymphoblastic leukemia/lymphoma
              B lymphoblastic leukemia/lymphoma/NOS
              B lymphoblastic leukemia/lymphoma with recurrent
                genetic abnormalities
                    o with t(9;22) BCR-ABL1
                    o with t(v;11q23) MLL rearranged
                    o with t(12;21) TEL-AML1
                    o with hyperdiploidy
                    o with hypodiploidy
                    o with t(5;14) IL3-IGH
                    o with t(1;19) E2A-PBX1
      T lymphoblastic leukemia/lymphoma


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         o Acute leukemias of ambiguous lineage
               Acute undifferentiated leukemia
               Mixed phenotype acute leukemia with t(9;22) BCR-ABL1
               Mixed phenotype acute leukemia with t(v;11q23); MLL rearranged
               Mixed phenotype acute leukemia, B/myeloid, NOS
               Mixed phenotype acute leukemia, T/myeloid, NOS
               Natural killer (NK) lymphoblastic leukemia/lymphoma

Laboratory Diagnosis and Prognosis—AML

     Perform a CBC with differential and peripheral blood smear review; identify the
      percentage, if any, of blasts and assess the granulocyte and monocyte populations
      for any evidence of dyspoiesis/dyplasia
     Perform a bone marrow aspirate and biopsy to determine the percentage of blasts
      and the presence of any differentiation and/or dyspoiesis of myeloid, monocytic,
      erythroid and megakaryocytic cell lines
          o AML is defined by finding >20% blasts in bone marrow and/or peripheral
              blood
     Submit a bone marrow aspirate sample for karyotyping and/or molecular genetic
      analysis
          o Clonal cytogenetic abnormalities (numeric and structural) are used to
              stratify patients into risk groups
          o Approximately 40-50% of patients <60 years old have normal karyotypes
              by conventional cytogenetics
     Submit a bone marrow aspirate sample for flow cytometric immunophenotyping
      to establish cell lineage, to assess the percentage of blasts and to determine the
      presence of any aberrant antigen expression; this aids not only in diagnostic
      classification, but also in identifying markers to subsequently follow through the
      course of treatment to assess effectiveness and identify minimal-residual disease
      and/or relapse
     Myeloblasts can have characteristic morphologic features, including fine nuclear
      chromatic with one or more large nucleoli, moderately abundant cytoplasm and
      the presence of Auer rods (abnormal condensation of primary granules) in most
      cases; in addition, the finding of dysmyelopoiesis in surrounding myeloid
      precursors (if present) provides confidence in the diagnosis of AML rather than
      ALL; some cases, however, can be morphologically indistinguishable from ALL
      lymphoblasts
     Certain simple cytochemical stains can be performed to help in establishing
      lineage specificity:
          o Myeloperoxidase (MPO) is specific for myeloid differentiation and will
              variably stain myeloblasts
          o Sudan Black B (SBB) is similar in specificity to MPO
          o Non-specific Esterase (NSE) is generally specific for monocytes and their
              precursors



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     Genomic abnormalities are critical for the precise classification of AML; such
      abnormalities not only define specific subtypes of AML, but are also important in
      predicting biologic behavior, appropriate treatment and prognosis
         o For example, AML with t(15;17) is a distinct entity that requires different
              treatment (initial induction with all-trans-retinoic acid, ATRA) and has a
              more favorable prognosis than other forms of AML (80-90% CR, 70-80%
              cure rates)
         o Certain molecular genetic abnormalities are also important markers of
              disease behavior, including NPM1 mutations which are present in 50-60%
              of those cases with normal karyotypes (NK-AML) by cytogenetics and are
              associated with better complete response rates, CEBPA mutations which
              are present in up to 20% of NK-AML and are associated with better
              outcomes, and FLT3 tyrosine kinase mutations found in 30% of NK-
              AML, including FLT3-Internal Tandem Deletions (ITD) which are
              associated with poor outcome even in the presence of NPM1 mutations


Laboratory Diagnosis and Prognosis—ALL

     ALL is a spectrum of disease due to the expansion of lymphoblasts in blood, bone
      marrow and/or other tissues; it includes, as part of this spectrum, cases that
      predominately involve lymph nodes—lymphoblastic lymphoma (LBL)
          o When >25% of the bone marrow is involved, by convention this is
              classified as ALL regardless of the coincidental involvement of lymph
              nodes
     Perform CBC with differential, peripheral smear examination and bone marrow
      aspirate and biopsy examination
          o Determine the presence of blasts with morphologic features consistent
              with lymphoblasts
                   Blast with fine nuclear chromatin with or without nuclear
                      convolutions, variably prominent nucleoli and variably abundant
                      cytoplasm with or without vacuolization
          o There should be no evidence of myelodysplasia
          o When cytochemical stains are performed, lymphoblasts are negative for
              MPO, SBB and NSE, but are usually positive for the presence of Terminal
              Deoxytidyltransferase (TDT), a nuclear enzyme found in precursor cells
     Submit a bone marrow aspirate for flow cytometric immunophenotyping to
      establish lineage specific markers of either precursor B-cells or precursor T-cells,
      and to determine antigen expression patterns that can be used to monitor disease
      treatment
     Submit a bone marrow aspirate for cytogenetic karyotyping and/or molecular
      genetic analysis for specific structural abnormalities of diagnostic and prognostic
      importance
     Prognosis is stratified by lineage, age, total peripheral blood leukocyte count and
      genetic abnormalities:
          o Precursor T-cell ALL is a high risk disease


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         o Precursor B-cell ALL is generally a good prognostic disease
               >95% complete remission and high cure rates
         o Age 1-10 years is associated with better prognosis
         o Leukocytes count >50,000 is a poor sign
         o Genetics
               Favorable genetic abnormalities
                       B-ALL with t(12;21)
                       Hyperdiploidy
               Unfavorable genetic abnormalities
                       B-ALL with t(9;22)
                       B-ALL with t(v;11q23)
                       Hypodiploidy

Summary and Key Concepts

     MDS and AML are related clonal disorders of bone marrow stems cells
     In some patients MDS and AML represent a longitudinal spectrum of disease
      evolution
     The etiology of MDS and AML involve cytogenetic abnormalities and/or
      exposures to environmental, occupational and iatrogenic toxins
     MDS, AML and ALL are classified morphologically, immunophenotypically and
      genetically
     It is clinically important to identify specific subtypes of MDS, AML and ALL

References
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9. Harrison CJ. The detection and significance of chromosomal abnormalities in
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