Cytogenetics of Hematologic Disorders

CYTOGENETICS OF HEMATOLOGIC DISORDERS

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- insertioninv -inversion

Abbreviation Definition

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 CON’T 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 CON’T

Chromosome abnormalities can be classified into two groups Primary abnormalities Secondary abnormalities

INTRODUCTION CON’T

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 CON’T

Secondary abnormalities Nonrandom Less disease specific Postulated to be later events

contributing to the process of tumor progression

INTRODUCTION CON’T

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 CON’T

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) 25% +8 15 – 20% +9 15 – 20% 1q gain 10% del(13q) 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) 34% +9 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(22)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(%) Description M0 <5% Acute myeloblastic leukemia without

maturation M1 20% Acute myeloblastic leukemia with

minimal differentiation M2 30% Acute myeloblastic leukemia with

maturation M3; M3 variant 10% Acute promyelocytic

leukemia(hypergranular); (hypogranular) M4;M4eo 25% Acute myelomonocytic leukemia; M4

with eosinophilia M5a;M5b 10% Acute monoblastic leukemia; acute

promonocytic monocytic leukemia

M6 <5% Acute erythroblastic leukemia M7 <5% Acute megakaryoblastic leukemia

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) Caused by medical exposure to alkylating agents Preceded by a myelodysplastic syndrome (MDS)

phase. Poor response to chemotherapy.

Balanced rearrangements involving 11q23 and 21q22

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 M5MLL (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) 30% -7/del(7q) 25 – 35% +8 20% del(20q) 5 – 10% -Y 5 – 10% 17p abnormalities (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 21’s 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 proto-

oncogene 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 proto-oncogene 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

+8 (10%) del(22q) (9%) +21 (9%) i(7q) (6%) -7 (9%) dup(1q) (6%) +X (7%) del(9p) (5%) +4 (5%)

CHRONIC LYMPOPROLIFERATIVE DISORDERS (CLDs) Characterized by clonal proliferation

and accumulation in the bone marrow and peripheral blood of relatively mature cell of B- and 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. 14q+

t(11;14)(q13;q32) Structural abnormalities of chromosome 1 (secondary) 17p+ 6q- Numerical 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.

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.

5.0 cc of blood. 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: Patient’s 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. Direct culture 24-hr BMC 48-hr BMC

Bone Marrow Cultures – next day samples. Direct Overnight (DON) 24-hr BMC 48-hr BMC

CLL 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

Identify submicroscopic abnormalities BCR/ABL [t(9;22)] Rb1 (13q14) TEL/AML 1 [t(12;21)]

Identify difficult to express abnormalities Trisomy 12 (CLL)

CULTURING OF HEMATOLOGIC SPECIMENS

Harvesting THC = Trypsin, Hypotonic (0.4% KCl),

Colcemid 3/1 Methanol/Acetic acid

Slide dropping Cold/Wet slides Hot/Dry environment

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.