CYTOGENETICS OF HEMATOLOGIC DISORDERS

Abbreviation Definition
add -additional material of unknown origin cen -centromere del -deletion (loss of part of chromosome) dic -dicentric der -derivative chromosome dup -duplication fra -fragile site i (iso)- isochromosome idic -isodicentric chromosome Ins- insertion inv -inversion

mar marker chromosome; a structurally abnormal chromosome that cannot be identified with standard cytogenetics mos Mosaic; two or more cell lines present in one individual (two or more cell types are present, which differ in number of chromosomes or their structure) p short arm of chromosome ph Philadelphia chromosome

q -long arm of chromosome r -ring chromosome rcp -reciprocal Rea- rearrangement Rec- recombinant chromosome t -translocation Tel- telomere (end of chromosome arm) Ter- terminal end of chromosome

+ plus sign in front of chromosome number; gain of chromosome minus sign in front of chromosome number; loss of chromosome [ ] square brackets; number of cells in each clone

INTRODUCTION

Chromosome abnormalities in neoplasia are acquired during the process of tumorigenesis The abnormalities found are restricted to tumor tissue The distribution of abnormalities is nonrandom and clonal Both benign and malignant tumors have exhibited chromosome abnormalities.

INTRODUCTION CONT

Chromosome abnormalities in neoplasia result in:

Gain of whole chromosomes or chromosome segments. Loss of whole chromosomes or segments. Relocation of chromosomal segments within a chromosome or between chromosomes leading to recombination of genes located at the breaks.

INTRODUCTION CONT

Chromosome abnormalities can be classified into two groups

Primary abnormalities Secondary abnormalities

INTRODUCTION CONT

Primary abnormalities

Strongly correlates with a specific tumor type (nonrandom). May be the only cytogenetic abnormality. May play an important role in the earliest stages of tumor initiation.

INTRODUCTION CONT

Secondary abnormalities

Nonrandom Less disease specific Postulated to be later events contributing to the process of tumor progression

INTRODUCTION CONT

Leukemias are often separated into two major categories.

Acute a short term disease that is rapidly fatal with undifferentiated, or poorly differentiated, blast cells in the acute form. Chronic a long term, more slowly progressive illness with well-differentiated cells in the chronic form.

INTRODUCTION CONT

Subdivisions within each category, include myeloid and lymphoid, which allow for morphologic subtyping, indicating the predominant cell type (and presumably the cell of origin). Cytogenetic analysis is valuable in the diagnosis, prognosis and management of hematologic malignancies.

CHRONIC MYELOPROLIFERATIVE DISORDERS (CMD OR MPD)

Hallmark is the excessive, neoplastic proliferation of one or more lineages of the myeloid series. Maturation is less affected, with no maturation arrest or block

CHRONIC MYELOPROLIFERATIVE DISORDERS (CMD OR MPD)

Disorders include

Polycythemia vera (PV or PCV) Essential thrombocythemia (ET) Idiopathic myelofibrosis (IMF) Chronic myeloid leukemia (CML)

CHRONIC MYELOPROLIFERATIVE DISORDERS (CMD OR MPD)

Polycythemia vera (PV or PCV)

Overproduction of erythrocytes Relative rare disease (1 per 100,000) Median age of onset 60 years Chromosome abnormalities observed

del(20q) +8 +9 1q gain del(13q)

25% 15 20% 15 20% 10% 10%

CHRONIC MYELOPROLIFERATIVE DISORDERS (CMD OR MPD)

Essential thrombocythemia (ET)

Overproduction of platelets. Rarest of the subgroups. Chromosome abnormalities observed:

t(9;22)(q34;q11.2) +9

34% 10%

CHRONIC MYELOPROLIFERATIVE DISORDERS (CMD OR MPD)

Idiopathic myelofibrosis (IMF)

Myeloid metaplasia and reactive fibrosis 10 15% risk of progression to AML Monosomy 7 syndrome is classified as IMF

CHRONIC MYELOID LEUKEMIA (CML)

Traditionally recognized as a separate entity. Overproduction of granulocytes 15 20% of all leukemia cases. Most common in 40 to 50 year age group.

CHRONIC MYELOID LEUKEMIA (CML)

First neoplastic disease to be associated with a specific recurrent chromosome abnormality the t(9;22).

Discovered by Nowell and Hungerford in 1960 in Philadelphia. The der(22) chromosome has been described as the Philadelphia (Ph) chromosome.

CHRONIC MYELOID LEUKEMIA (CML)

Chromosome abnormalities observed:

Primary

t(9;22)(q34.1;q11.2)

Secondary

+Ph +8 i(17)(q10) Combination of these changes with +19 -7 Rearrangements of 3

46,XY,t(9;22)(q34;q11.2)

48,XY,+8,t(9;22)(q34;q11.2),+der(2 2)t(9;22)

CHRONIC MYELOID LEUKEMIA (CML)

More than 90% of patients exhibit a t(9;22) Variant translocation exist in 5 10% of CML cases

Simple Variant: Segment from 22 is translocated to a chromosome other than 9. Complex Variant: The translocation involves one or more chromosome in addition to the 9 and 22. All chromosome except Y have been involved in variant translocations. Variant Ph+ CML do not differ in hematology or prognosis.

CHRONIC MYELOID LEUKEMIA (CML)

The molecular result of the t(9;22) rearrangement is the translocation and subsequent fusion of the ABL oncogene on 9 to the breakpoint cluster region (BCR) gene on the 22.

BCR-ABL rearrangement

CHRONIC MYELOID LEUKEMIA (CML)

5 10% of CML cases will have no observable Ph chromosome.

FISH will show submicroscopic t(9;22) rearrangement. Differ from Ph+ patients

Older Male Lower platelet counts Lower WBC counts

ACUTE MYELOID LEUKEMIA (AML)

Excessive accumulation of immature nonlymphatic bone marrow precursor cells due to a block in cell maturation. Incidence is 2.5 cases/100,000 per year. Male predominance. Incidence rises sharply after 55 60 years of age.

ACUTE MYELOID LEUKEMIA (AML)

FAB Incidence(%) M0 <5% M1 20%

Description Acute myeloblastic leukemia without maturation Acute myeloblastic leukemia with minimal differentiation Acute myeloblastic leukemia with maturation Acute promyelocytic leukemia(hypergranular); (hypogranular) Acute myelomonocytic leukemia; M4 with eosinophilia Acute monoblastic leukemia; acute promonocytic monocytic leukemia Acute erythroblastic leukemia Acute megakaryoblastic leukemia

M2 30% M3; M3 variant 10%

M4;M4eo 25% M5a;M5b 10% M6 <5% M7 <5%

ACUTE MYELOID LEUKEMIA (AML)

Cytogenetic abnormalities are associated with hematologic subgroups M0 complex karyotypes, -5/5q-,-7/7q-, rearrangements of 5, 7, or 11, +8,+13,t(9;22), near tetraploidy, normal K-types in 25% M1 inv(3), t(3;3) M2 t(8;21) M3 t(15;17) M4(eo) inv(16), del(16q), t(16;16) M5 t(9;11) M6 complex karyotypes with multiple structural abnormalities, 5 and 7 most often affected. M7 Adults: complex aberrations, -5/5q-,-7/7q-,3q21 or q26 aberrations, t(9;22),+19,+21 Children: t(1;22),+21,+19,+8

ACUTE MYELOID LEUKEMIA (AML)

Secondary or therapy related AML (tAML)

-5/del(5q) and -7/del(7q)

Balanced rearrangements involving 11q23 and 21q22

Caused by medical exposure to alkylating agents Preceded by a myelodysplastic syndrome (MDS) phase. Poor response to chemotherapy.

Develops as a late effect after DNA-topoisomerase II inhibitors combined with alkylating agents and radiation. Begins earlier. Occurs without initial MDS More favorable response to chemotherapy

45,XY,del(5q),-7

AML M2

AML M2 FISH ETO-AML 1

AML M3

AML M3

AML M4 inv(16)

AML M4 CBFB(16q22) Break apart Probe

AML M5

AML M5 MLL (11q23)

MLL CEP 8 FISH

MYELODYSPLASTIC SYNDROMES (MDS)

Often termed preleukemias. Characterized by bone marrow. dysfunction involving all lineages. Incidence of 1/100,000 persons in the US. Mostly affects the elderly. 20 40% progress to AML.

MYELODYSPLASTIC SYNDROMES (MDS)

Most common chromosome abnormalities observed.

-5/del(5q) -7/del(7q) +8 del(20q) -Y 17p abnormalities

30% 25 35% 20% 5 10% 5 10% (LOH-p53)

46,XX,del(5)(q15q34)

EGR-1 = 5q31

45,XY,del(5q),-7

47,XX,r(7),+21c

46,XY,del(20q)

ACUTE LYMPHOCYTIC LEUKEMIA (ALL)

Characterized by the accumulation of immature lymphoid cells in the bone marrow and peripheral blood. More common in children than adults. Median occurrence at 3 5 years of age in children and under 30 years of age in adults. Incidence in childhood is 3/100,000. Male predominance. Clonal chromosome abnormalities are present in most ALL patients (>85% adults, >90% children). Chromosome ploidy in childhood ALL is predictive of clinical outcome and informative as to risk-specific therapy.

ACUTE LYMPHOCYTIC LEUKEMIA (ALL)

Five subtypes of ALL based on modal number of chromosomes.

Hyperdiploidy (47 50 chromosomes) Massive Hyperdiploidy (>50 chromosomes) Diploid with structural rearrangements Normal diploid Hypodiploidy (<46 chromosomes)

ACUTE LYMPHOCYTIC LEUKEMIA (ALL)

Hyperdiploidy

47 50 chromosomes Found in 25 30% of childhood ALL. Found in 7% of adult ALL Gains of 4*, 10, 21 *UNMC experience

ACUTE LYMPHOCYTIC LEUKEMIA (ALL)

Massive hyperdiploidy

> 50 chromosomes Good response to treatment Common gains of X, 4, 6, 10, 14, 17, 18, 20, and 21 Duplication of 1q i(17)(q10) Gain of 6 and the combination of +4, +10, and +17 have been associated with good outcome Presence of structural abnormality, especially i(17)(q10) in massively hyperdiploid cases has shown unfavorable prognosis.

Hyperdiploid ALL

TEL/AML DNA PROBE

ACUTE LYMPHOCYTIC LEUKEMIA (ALL)

Diploid chromosome number with structural rearrangements

Largest group in childhood ALL 40% of childhood ALL cases

ACUTE LYMPHOCYTIC LEUKEMIA (ALL)

Normal diploid chromosome complement.

Poor chromosome morphology hiding abnormality? Mitotically inactive clones? Submicroscopic genetic changes?

ACUTE LYMPHOCYTIC LEUKEMIA (ALL)

Hypodiploidy

<46 chromosomes 7 8% of childhood ALL 80% have modal number of 45 2% of adult ALL have 30 39 chromosomes <1% are near haploid

Contain at least one copy of each chromosome 90% have two sex chromosomes and two 21s Two copies of 10, 14, and 18 have been observed Associated with poor prognosis

ACUTE LYMPHOCYTIC LEUKEMIA (ALL)

Common ALL translocations

Rearrangements of the MYC protooncogene t(1;19)(q23;p13) t(9;22)(q34.1;q11.2) Translocations involving 11q23 t(12;21)(p13;q22) (cryptic)

ACUTE LYMPHOCYTIC LEUKEMIA (ALL)

Rearrangements of the MYC protooncogene

8q24 t(8;14)(q24;q32) t(2;8)(p12;q24) t(8;22)(q24;q11)

ACUTE LYMPHOCYTIC LEUKEMIA (ALL)

t(1;19)(q23;p13) and der(19)t(1;19)

Most common chromosomal translocation in childhood ALL Favorable prognosis if unbalanced [der(19)t(1;19)].

ACUTE LYMPHOCYTIC LEUKEMIA (ALL)

t(9;22)(q34.1;q11.2)

Most common of all ALL-associated chromosome abnormalities (15 20% of all cases). Rare in childhood ALL (2 5%) Poor prognosis in all age groups Often found with -7

ACUTE LYMPHOCYTIC LEUKEMIA (ALL)

Translocations involving 11q23

Most often exchanged with 4, 1, and 10. Poor prognosis

ACUTE LYMPHOCYTIC LEUKEMIA (ALL)

t(12;21)(p13;q22)

Undetectable cytogenetically FISH shows fusion of the TEL-AML 1 genes Common in pediatric ALL (22%) Generally good prognosis

ACUTE LYMPHOCYTIC LEUKEMIA (ALL)

Secondary changes

Numeric

Structural
del(22q) (9%) i(7q) (6%) dup(1q) (6%) del(9p) (5%)

+8 (10%) +21 (9%) -7 (9%) +X (7%) +4 (5%)

CHRONIC LYMPOPROLIFERATIVE DISORDERS (CLDs)

Characterized by clonal proliferation and accumulation in the bone marrow and peripheral blood of relatively mature cell of Band T-cell lineage. Most cases are B-cell (90%). Spontaneous mitotic activity of these cells is low. Mitogenic stimulation has improved cytogenetics analysis.

DSP/IL2

CHRONIC LYMPOPROLIFERATIVE DISORDERS (CLDs)

CLDs include:

Chronic lymphoproliferative leukemia (CLL) Hairy cell leukemia (HCL) Waldestrom macroglobulinemia (WM) Plasma cell leukemia (PCL) Multiple Myeloma (MM)

CHRONIC LYMPOPROLIFERATIVE DISORDERS (CLDs)

Chronic lymphoproliferative leukemia (CLL)

Most common leukemia in the US and Europe (30%) Extremely rare in the Orient Middle-aged to elderly patients Rarely seen in children Majority are B-cell origin

CHRONIC LYMPOPROLIFERATIVE DISORDERS (CLDs)

Common chromosome abnormalities in CLL

+12 (33%) 14q32 rearrangements

add(14)(q32) (20%) t(11;14)(q13;q32) t(2;14)(p13;q32) t(14;19)(q32;q13)

t/del(13q) del(6q)

47,XX,+12

CHRONIC LYMPOPROLIFERATIVE DISORDERS (CLDs)

Hairy cell leukemia (HCL)

Finger-like cytoplasmic projections on the cell surface give the cells their hairy appearance. Middle aged men (80%) Primarily B-cell Chromosome Abnormalities

14q+ 6q- (probably secondary abnormality) del(14q)

CHRONIC LYMPOPROLIFERATIVE DISORDERS (CLDs)

Waldenstrom macroglobulinemia (WM)

Characterized by the excessive proliferation of an IgM-producing clone of malignant plasmacytoid cells. 60-70 years of age. Indolent disease with long survival Chromosome changes

Numerical changes Unrelated clones.

CHRONIC LYMPOPROLIFERATIVE DISORDERS (CLDs)

Plasma cell leukemia (PCL)

Characterized by the infiltration of the bone marrow by plasma cells with an unusually high proliferative activity, leading to rapidly fatal outcome. Rare Chromosome abnormalities

Hypo- or hyperdiploid clones. 14q+ t(11;14)(q13;q32) Structural rearrangements of chromosome 1 (probably secondary).

CHRONIC LYMPOPROLIFERATIVE DISORDERS (CLDs)

Multiple Myeloma (MM)

Characterized by the proliferation of the plasma cell. Chromosome abnormalities

del(13)(q14)

Submicroscopic, seen utilizing FISH for a deletion of the Rb 1 gene. t(11;14)(q13;q32)

14q+

Structural abnormalities of chromosome 1 (secondary) 17p+ 6qNumerical abnormalities involving 3, 5, 7, 9, 11, 13, and 15.

Multiple Myeloma

Multiple Myeloma

Multiple Myeloma

CULTURING OF HEMATOLOGIC SPECIMENS

Cytogenetics of malignant disease must be based on the study of the tumor cells themselves.

CULTURING OF HEMATOLOGIC SPECIMENS

In leukemia the specimen of choice is usually a bone marrow aspirate.

If bone marrow cannot be obtained:

Bone marrow core biopsy Peripheral blood

Myeloproliferative disorder (CML) with difficult or dry tap. Blast crisis leukemia, (if WBC count is > 10,000 and 10% are immature myeloid or lymphoid cells. Collaborate bone marrow findings. Distinguish between constitutional or acquired abnormalities.

CULTURING OF HEMATOLOGIC SPECIMENS

Concentrate analysis on the spontaneously dividing malignant cells. Mitogens which stimulate normal lymphoid cells are normally not used. Mitogens may be used with certain disease indications however.

CLL/HCL,

B-cell mitogens may be used on 3 to 7 day cultures. DSP/IL2

CULTURING OF HEMATOLOGIC SPECIMENS

Specimen collection and transport.

Transport ASAP. Transport at room temperature. 1.5 cc of bone marrow.

5.0 cc of blood.

First aspirate is required for cytogenetics Aspirated into a syringe coated with preservative-free sodium heparin. Transport in the syringe if a short transport time or transfer into a sodium heparin vacutainer or a 15ml centrifuge tube containing culture media. Sodium heparin vacutainer >10% blasts CLL

CULTURING OF HEMATOLOGIC SPECIMENS

Clinical information

Complete clinical information allows for the selection of the appropriate processing or culture methods and alerts the laboratorian to the potential presence of specific abnormalities Important clinical information includes:

Patients name, age and sex Source of the specimen (BM or Blood) Referring diagnosis Clinical status (diagnosis, residual disease, remission, relapse, treated, transplanted, etc). Pertinent laboratory results (CBC, %circulation immature cells, bone marrow cellularity, % blasts).

CULTURING OF HEMATOLOGIC SPECIMENS

Bone Marrow Cultures same day samples.

Bone Marrow Cultures next day samples.

Direct culture 24-hr BMC 48-hr BMC

CLL

Direct Overnight (DON) 24-hr BMC 48-hr BMC

Always do 48-hr DSP/IL2

CULTURING OF HEMATOLOGIC SPECIMENS

Peripheral Blood Same day and next day samples.

DON 48 BC CLL always do a 48 DSP/IL2

CULTURING OF HEMATOLOGIC SPECIMENS

FISH Cultures

Identify small populations of cells.

Donor/Recipient Abnormal clone BCR/ABL [t(9;22)] Rb1 (13q14) TEL/AML 1 [t(12;21)] Trisomy 12 (CLL)

Identify submicroscopic abnormalities

Identify difficult to express abnormalities

CULTURING OF HEMATOLOGIC SPECIMENS

Harvesting

THC = Trypsin, Hypotonic (0.4% KCl), Colcemid 3/1 Methanol/Acetic acid Cold/Wet slides Hot/Dry environment

Slide dropping

CULTURING OF HEMATOLOGIC SPECIMENS

Analysis

Abnormal cells may represent only a small proportion of the total cell population. Adequate number of cells must be analyzed. All morphology of cells must be analyzed. Cells from different preparations (direct versus cultured) must be analyzed.