Hematology Exam III

M stage

Mitosis (prophase, metaphase, anaphase, telophase)

G1 stage

Growth and synthesis of components for replication

S stage

DNA synthesis and repair of any damage to DNA

G2 stage

Rapid cell growth and protein synthesis

How cyclin-dependent kinases (CdKs) regulate the transition between the stages of the cell cycle

Act to phosphorylate target molecules important in cellular control.

CdK must bind with regulatory subunits (cyclins) in order to be active

Cyclin / CdK associated with the M stage

Cyclin B / CdK 1

Cyclin / CdK associated with the G1 stage

Cyclin D1, D2, D3, E / CdK 4,6

Cyclin / CdK associated with the S stage

Cyclin A / CdK 2

Cyclin / CdK associated with the G2 stage

No cyclin / CdK 1

Goals the cell must achieve to effectively reproduce itself

Faithfully replicate DNA once during S phase to produce identical copies.

Distribute identical chromosomal copies equally to daughter cells in M phase.

Embark on mitosis only after DNA duplication is complete.

Never separate sister chromatids until all pairs are aligned on mitosis spindle at metaphase.

Never reduplicate chromosomes before sister chromatids have been have been separated at the previous mitosis.

G1 checkpoint

Checks for DNA damage and will prevent progression into S phase if integrity of the genome is compromised

G2-M checkpoint

Checks for damaged or unduplicated DNA and can block mitosis if any is found

M checkpoint

Ensures that all chromosomes are properly aligned on the spindle apparatus prior to initiating chromosome separation and segregration

Caspases

Act in a cascade that tells the cell to die

p53

Acts in the G1 checkpoint. Tumor suppressor gene (can induce apoptosis if DNA is damaged)

pRB

Retinoblastoma tumor suppressor protein: prevents excess cell growth by inhibiting cell cycle progression until a cell is ready to divide

Apoptosis

Orderly, regulated process of cell death under direction of caspases.

Bax-Bax signals apoptosis, Bax-Bcl-2 prevents apoptosis.

Default cellular status for hematopoietic stem cells and progenitor cells.

Involved in T and B cell recognition repertories and cytotoxic T cells

Neoplasm

Abnormal formation of new tissue (such as a tumor) that serves no useful purpose. May be benign or malignant

Benign neoplasm

Neoplasm formed from highly organized, differentiated cells and do not spread or invade surrounding tissue. May or may not progress to a malignant form

Malignant neoplasm

Clone of identical, anaplastic, proliferating cells which can metastasize and are harmful or deadly

Dysplasia

Common term to describe malignant neoplasm of epithelial tissue but is commonly used to refer to all malignant neoplasms

Myeloproliferative disorder (MPD)

Group of disorders characterized by autonomous hypercellular proliferation of one or more hematopoietic elements in the BM, often accompanied by enlarged spleen or liver

Myelodysplastic syndrome (MDS)

Group of primary hematologic disorders associated with abnormal division, maturation, and production of RBCs, monocytes, and PLTs. Usually progresses to severe BM failure with infections and bleeding. Acute leukemia forms in 30-40% of cases. Blast crisis is mostly pure myeloid or myelomonocytoid phenotypes

Leukemia

Progressive, malignant disease of the hematopoietic system characterized by unregulated, clonal proliferation of the HSC

Lymphoma

Malignant proliferation of lymphs, usually arising in LNs, but can begin at many extranodal sites

Proto-oncogene

Normal, unaltered counterpart of oncogene

Oncogene

Genes within normal cells that have potential to cause cancer if they become altered or activated

General means of activation of oncogenes

Gene mutation, gene rearrangement, gene amplification, viral infection, genetic susceptibility, immunologic dysfunction, MPD/MDS

Information learned by: differential

Cell morphology

Information learned by: flow cytometry

Immunophenotyping, retic counting, DNA analysis

Information learned by: cell staining

Cell differentiation based on chemical composition

Information learned by: cytogenetics

Study of variants in chromosome number and morphology as it relates to a normal or abnormal state

Information learned by: molecular diagnostics

DNA and RNA analysis to detect and identify normal and abnormal genes

Stains that myeloblasts are positive for

MPO, SBB, chloroacetate esterase, PAS (variable), TdT (10-20%)

Stains that lymphoblasts are positive for

PAS (variable), TdT (>90%)

Interpret location on a chromosome: 7q32

7: chromosome number

q: arm

3: region

2: band

Advantages of molecular diagnostics for the study of hem neoplasms

Don't require viable cells capable of mitotic activity.

Can be used on current and archival specimens

Disadvantages of molecular diagnostics for the study of hem neoplasms

Give information only about a single gene mutation as opposed to other chromosome aberrations that may be present.

May not be able to detect all mutations with current technology because multiple mutations can occur as disease progresses

Host risk factors for leukemia

Hereditary, congenital chromosomal abnormality, immunocompromised status, chronic marrow dysfunction, secondary leukemia

Environmental risk factors for leukemia

Ionizing radiation, chemicals and drugs, viruses

Acute leukemia

Children and young adults.

Rapid onset.

<6 months untreated.

Treated with chemo and BM transplant.

Cause of death is infection or hemorrhage.

Diagnosis based on PB, BM, stains, flow cytometry, cytogenetics, electron microscopy.

Immature cells / blasts.

Mild to severe anemia.

Low to very low PLT count.

Variable or increased WBC count.

Mild organomegaly.

>20-95% blasts in BM.

>5-95% blasts in PB.

Chronic leukemia

Mid-age and elderly.

Insidious onset.

2-6 years untreated.

Treated with chemo and BM transplant.

Cause of death is infection or hemorrhage.

Diagnosis based on PB, BM, and cytogenetics (Philadelphia chromosome)

Mature cells.

Mild anemia.

N/increased PLTs early, decreased later on.

Increased WBC count.

Prominent organomegaly.

<10% blasts in BM/PB

AML

Adults (around 65), rare in children.

Anemia, neutropenia, thrombocytopenia, myeloblasts (20-95%).

M:E increased.

Medium to large blasts, Auer rods, fine chromatin, distinct nucleoli.

MPO, SBB, specific and nonspecific esterase positive.

Can have extramedullary disease in spleen, liver, LNs, CNS

ALL

Children (1-5 years).

Anemia, neutropenia, thrombocytopenia, lymphoblasts.

M:E increased.

Small to medium blasts, scant cytoplasm, no granules, fine chromatin, indistinct nucleoli.

TdT positive.

Can have extramedullary disease in spleen, liver, LNs, CNS, gonads

Auer rod

Reddish blue staining needle-like inclusions within the cytoplasm of leukemic myeloblasts that occur because of abnormal cytoplasmic granule formation.

Presence excludes ALL

Phi body

A smaller version of an Auer rod

SLUR cell

A cell in which there is a large collection of Auer rods and/or phi bodies

MPO

Stains peroxidase in primary granules.

Promyelocytes and stages after are positive.

Sources of error: needs fresh specimen (peroxidase is labile)

SBB

Most sensitive for granulocyte precursors and is useful with specimens that aren't fresh.

Sources of error: rare cases of ALL can be positive

Specific esterase / Naphthol AS-D Chloroacetate

Can be used on paraffin-embedded tissues

Nonspecific esterase / Alpha-Naphthyl Acetate or Butyrate

Positive for monocytic cells, negative for granulocytic cells

PAS

Stains glycogen, polysaccharides, and glycoproteins.

Is helpful in supporting erythroleukemia diagnoses.

Not helpful in differentiating acute leukemias

TdT

Stains DNA polymerase present in stem cells and precursor B and T lymphoid cells

Myeloid antigens

CD13, 33, 117, MPO

B lymphoid antigens

CD10, 19, 22, 79a

T lymphoid antigens

CD2, 3, 4, 5, 7, 8

Hairy Cell Leukemia antigens

CD11c, 19, 22, 25, 103

CLL antigens

CD5, 19, 20, 21, 23

PLL antigens

CD19, 22

M1: Myeloblastic without maturation

>30% blasts are peroxidase pos. +/- Auer rods

M2: Myeloblastic with maturation

>30% of cells are myeloblasts and promyelocytes. Maturation beyond promyelocytes present

M3: Hypergranular promyelocytic

>30% of the cells are abnormal promyelocytes. Auer rods are common

M4: Myelomonocytic

>20% are monocytic. >30% myeloblasts and promyelocytes

M5: Monocytic

>30% of cells are blasts. >80% are promonocytes or monocytes. With/without differentiation

M6: Erythroleukemia

>50% of cells are abnormal erythroid

M7: Megakaryocytic

Abnormal mononuclear (<30%) megakaryoblasts predominate. BM is very fibrotic

Acute Promyelocytic Leukemia (APL)

t(15:17)(q22:q12). Chimera formed from PML and RARa (retinoic acid receptor alpha gene).

Therapy is a form of vitamin A called all-trans retinoic acid (ATRA), which binds and offsets effect of the chimeric PML/RARa.

Resistance to therapy can be caused by other mutations

Erythroleukemia (Di Guglielmo's syndrome)

Indistinguishable from AML.

Low M:E.

PAS helpful in diagnosis.

Megaloblastic erythropoiesis, giant bizarre-shaped multi-nucleated erythroid precursors, ringed sideroblasts, nuclear budding and fragmentation, cytoplasmic vacuoles, HJ bodies

Prognosis of ALL

Poor if <1 year old, >13 years old, WBC > 20 X 10^9 /L, or structural chromosomal abnormalities

L1 ALL

Small uniform lymphoblasts with homogenous, lacy chromatin. Nuclear membrane is regular in shape. Rare nucleoli. Cytoplasm is scanty in quantity and moderately basophilic

L2 ALL

Large lymphoblasts with variable chromatin pattern, irregularly shaped nuclear membrane. Nucleoli are present. Cytoplasm size is moderate with variable cytoplasmic basophilia

L3 ALL / Burkitt's

Lymphoblast is large and homogenous, with open, delicate chromatin. Nuclear membrane is round to oval in shape. 1-3 nucleoli are usually seen. Cytoplasm is moderate in size and shows vacuoles and intense basophilia

Precursor B cell ALL

Most frequently encountered form of lymphoblastic leukemia.

Occurs in children 3-5 years old.

L2 morphology more common in adults, L1 more common in children

B cell ALL (Burkitt's) % frequency

2-5%

T cell ALL % frequency

15-20%

Examples of MPD

CML, PV, ET, Primary Myelofibrosis

Chronic stage of CML

Lasts 2-5 years.

<10% blasts in BM, <2% blasts in PB.

Generally responds well to treatment with Imatinib/Gleevec

Blast stage of CML

Lasts 3-4 months.

>20% blasts in BM/PB.

Unresponsive to treatment with Imatinib/Gleevec

Diagnostic criteria for differential identification of CML

Leukocytosis with absolute neutrophilia and L shift maturation.

Absolute basophilia and eosinophilia.

Thrombocytosis or thrombocytopenia.

BM hypercellularity with granulocytic proliferation (M:E is 10:1)

LAP activity decreased

Leukemoid reaction

Transient, increased WBCs, L shift, increased LAP

Philadelphia chromosome

Translocation between long arms of chromosomes 22 and 9.

t(9;22)(q34;q11).

BCR and ABL gene sites are translocated.

Results in formation of transgene p210.

Present in 90-95% CML, 33% ALL, <1% AML, and in very low levels in healthy children/adults

Imatinib / Gleevec

First-line therapy for CML. Targets the enzymatically active portion of the transfusion protein p210.

Competes with ATP for the site and thus prevents activation of the molecule and prevents the development of abnormal cells

Mechanisms of resistance to Imatinib (BCR/ABL1 dependent)

Tumor cells express more protein, mutation in drug-binding site, other mutations in p210 or other proteins

Diagnostic criteria for Polycythemia Vera (PV)

Middle age population, higher frequency in males.

Hypercellular BM across all 3 lineages, normal morphology, decreased or absent iron stores. Normal M:E. Megakaryocytes variably sized, can be in loose clusters.

Increased RBC, HH, WBC, PLT (with abnormal function), histamine and uric acid.

EPO increased or normal.

B12 binding capacity decreased.

LAP score increased.

Oval macrocytes, large HJ bodies, hyper-segmented neutrophils, large agranular PLTs.

Arterial O2 saturation >92%

PV associated with renal disease

Patient has tumor that concentrates EPO, leading to production of increased RBC mass

PV in severely burned patients

Dehydration reduced amount of plasma, leading to relative increase in RBCs, iron deficiency, microcytic, hypochromic anemia

PV associated with emphysema

Hypoxia triggers increase in EPO which leads to increased production of RBCs. Arterial O2 saturation <92%

Essential Thrombasthenia (ET) / Idiopathic Thrombocythemia

Rare chronic MPD marked by increase in PLTs, associated with abnormal PLT function.

PLT >450 X 10^9 /L. Megakaryocyte hyperplasia in BM. Generally asymptomatic

Idiopathic myelofibrosis / Agnogenic Myeloid Metaplasia (AMM)

Doesn't fit WHO criteria for other myeloid neoplasms.

Fibrosis of BM.

Myeloid metaplasia of spleen and liver.

Giant and bizarre megakaryocytes, teardrops, anisocytosis

Chronic Neutrophilic Leukemia (CNL)

PB leukocytosis with L shift. Increase in % and number of neutrophilic granulocytes. Hypercellular BM with increase in myeloblasts. Decreased monocytes

Chronic Eosinophilic Leukemia (CEL)

Eosinophilia. Organ dysfunction from degranulation, can involve the heart. BM hypercellularity with eosinophil proliferation, can have Charcot-Leyden crystals

Myeloproliferative neoplasm, unclassifiable (MPN-U)

Does not meet specific criteria or overlaps with one or more MPD

Dyserythropoiesis of PB in MDS

Anemia, macrocytes, ovalocytes, basophilic stippling, nRBCs, vacuoles, HJ bodies, sideroblasts

Dyserythropoiesis of BM in MDS

Nuclear budding, internuclear bridging, karyorrhexis, multiple nuclei, megaloblastoid changes, ringed sideroblasts, vacuolization, diffuse or granular PAS+

Dysgranulopoiesis of PB in MDS

Neutropenia, hypogranulation, abnormal granulation, left shift, nuclear abnormalities, pseudo Pelger-Huet, ring nuclei, monocytosis

Dysgranulopoiesis of BM in MDS

Pseudo Pelger-Huet, hypersegmentation, pseudo Chediak-Higashi granules, small size

Megakaryocyte dysplasia of PB in MDS

Thrombocytopenia or thrombocytosis, giant platelets, hypogranulation, micromegakaryocytes, functional abnormalities

Megakaryocyte dysplasia of BM in MDS

Micromegakaryocytes, hypo- or non-lobate nuclei, multiple widely separated nuclei

Refractory

Not readily responding to treatment

FAB classification of MDS

Based on blast count and amount of dyspoiesis in PB and BM