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Cytogenetics Analysis 3
1
Cytogenetics Analysis
Avery A. Sandberg and Zhong Chen
1. Introduction
1.1. Principles
The establishment of reliable and meaningful chromosomal (cytogenetic,
karyotypic) changes in hematological disorders, primarily the leukemias and
lymphomas, must be based on the examination of the involved cells or tissues.
Thus, in the case of the leukemias bone marrow (BM) aspirations yield optimal
results in the preponderant number of patients, whereas in the lymphomas
affected tissues, usually lymph nodes, are the best source of cells carrying
cytogenetic anomalies. Generally, BM is not a good source of cells for cytoge-
netic analysis in lymphoma. Not only is the marrow often not affected by the
lymphoma, but also when it is, the number of abnormal cells is relatively small
and/or the abnormal cells are not in division and, hence, do not yield a suffi-
cient number of metaphases for cytogenetic analysis. In some situations, blood
cells can be utilized as a source of metaphases affected by karyotypic changes,
e.g., in cases with about 10% immature cells in the peripheral blood (PB), in
chronic lymphocytic leukemia (CLL), in cases where the marrow is fibrotic or
extremely hypocellular, or in determining the presence of Ph+ cells in estab-
lished cases of chronic myelocytic leukemia (CML) (1,2).
Paramount for a successful cytogenetic study is the presence of metaphases
suitable for analysis. In the normal BM, a significant number of dividing cells,
and hence metaphases, are usually present in sufficient number for cytogenetic
analysis without having to resort to culture or lengthy incubation. However, in
some leukemias the number of dividing cells (especially the leukemic ones) is
very low and, hence, incubation of the marrow specimen for a number of days
(2–5 d) may be necessary to generate a significant number of metaphases for
From:Methods in Molecular Medicine, vol. 55: Hematologic Malignancies: Methods and Techniques
Edited by: G. B. Faguet © Humana Press Inc., Totowa, NJ
3
4 Sandberg and Chen
cytogenetic study. The statements just made apply in particular to acute
promyelocytic leukemia (APL) and following chemotherapy and/or radiation
therapy for the leukemia (1,2).
In cases where cytogenetic analysis reveals only abnormal metaphases,
especially those with a balanced translocation, it may be necessary to rule out
a constitutional chromosomal anomaly. This is best established through the
cytogenetic examination of phytohemagglutinin (PHA) stimulated lympho-
cytes of the PB.
A number of mitogenic agents capable of stimulating lymphoid or, less fre-
quently, myeloid cells have been introduced over the years. Outstanding among
these has been PHA capable of stimulating the growth and division of lympho-
cytes of T-cell origin. However, PHA is not routinely added to BM or PB cul-
tures in acute leukemias because PHA may interfere with the evaluation of
spontaneously dividing malignant cells.
The quality of chromosome preparations has been significantly improved
with some new techniques, such as the use of amethopterin for cell synchroni-
zation, the use of short exposures to mitosis-arresting agents, the use of DNA-
binding agents to elongate chromosome (3,4), improved staining procedures,
and the use of conditioned culture medium containing hematopoietic growth
factors [e.g., GCT (giant cell tumor)-conditioned medium primarily for
myeloid disorders (4,5) and PHA/IL-2 (interleukin-2) for both B- or T-cell
lymphoid diseases (6,7)].
The rate of successful cytogenetic analysis varies with the specific type of
disease, and is also related to the adjustment of variables in each laboratory,
such as serum concentration, medium pH, and cell concentration.
1.2. Clinical Applications
The common and recurrent chromosome changes seen in the leukemias and
lymphomas are shown in Tables 1–3 and Figs. 1 and 2.
CML is a pluripotent stem cell disorder characterized cytogenetically by the
Philadelphia chromosome (Ph), the first consistent abnormality observed in a
human cancer. The Ph arises from a reciprocal translocation, t(9;22)(q34;q11)
(8). It is characterized molecularly by the fusion of parts of the C-ABLgene (at
9q34) and the BCR gene (at 22q11), generating an abnormal BCR/ABL fusion
gene (9). Cytogenetically, more than 85% of patients with CML are found to
have the Ph in the CML cells, even during remission, unlike the Ph in acute
leukemia, which is not seen during complete remission. When CML progresses,
additional changes, such as +8, +Ph, i(17q), +19, and +21 are noted in 75–80%
of cases. These changes may precede hematologic progression by 2–6 mo or
occur at the blast phase; therefore, they are valuable prognostic indices. How-
ever, there is no evidence that these additional changes correlate with response
Cytogenetics Analysis 5
Table 1
Common Chromosome Changes in Acute Nonlymphocytic Leukemia
(ANLL)(1,12)
der(1;7)(q10;p10)a t(9;22)(q34;q11) M1(M2)
t(1;22)(p13;q13) M7 t(11;V)(q23;V)b M5(M4)
ins(3;3)(q26;q21q26)a,d M1(M7) del(11)(q23) M5(M4)
inv(3)(q21q26)a,d M1(M7) +11
t(3;3)(q21;q26)a,d M1(M7) del(12)(p11p13)a
t(3;21)(q26;q22)a +13a
+4 M2, M4 +14a
–5 or del(5)(q12–13 or q31–35)a M1–M4 t(15;17)(q22;q11–21) M3
+6 del(16)(q22)c M4EO
t(6;9)(p23;q34)a M2(M4) (basophilia) inv(16)(p13q22)c M4EO
–7 or del(7)(q22)a M1–M5 t(10;16)(p13;q22)c M4EO
+8a t(16;21)(p11;q22)
t(8;16)(p11;p13) M5b (erythrophagocytosis) i(17)(q10)a
t(8;21)(q22;q22) M2 (Auer rods+) +19
+9 del(20)(q11–13)a
del(9)(q22) +21
idic(X)(q13)a
aChange also seen in myelodysplastic syndromes.
bV = chromosomes 6, 9, 17, and 19.
cAssociated with marrow eosinophilia.
dAssociated with platelet and/or megakaryocytic anomalies.
Where appropriate, the type of ANLL or other information associated with a particular chro-
mosome change is also shown.
to presently used therapy during the acute phase of CML. Clinically, treatment
strategies for CML should include, in addition to the hematologic criteria, the cyto-
genetic findings and the molecular genetic criteria of the BCR/ABLfusion gene
obtained using Southern blotting or polymerase chain reaction (PCR) techniques.
Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal
hematopoietic stem cell disorders characterized by dysplastic and ineffective
hematopoiesis and a high risk of transformation to ANLL. Clonal chromo-
somal abnormalities can be detected in 40–70% of MDS patients at presenta-
tion (see Table 4). Additional aberrations may evolve during the course of
MDS and appear to portend its transformation to leukemia. To confirm the
diagnosis of MDS, morphologic examination of BM aspirate and cytogenetic
analysis should be performed. Moreover, the chromosomal findings have been
shown to be an independent prognostic indicator second only to the French-
American-British (FAB) classification subtype as a predictor of progression to
leukemia and survival (seeTable 5).
6 Sandberg and Chen
Table 2
Common Chromosome Changes in B-Lineage
Acute Lymphocytic Leukemia (ALL) (1,12)
t(1;9)(q23;p13) Pre-B-cell
t(2;8)(p12;q24) L3 (B-cell)
t(4;11)(q21;q23) Biphenotypic, early
B-precursor
t(5;14)(q31;q32)
del(6)(q13–14 or q21–27)
t(8;14)(q24;q32) L3 (B-cell)
t(8;22)(q24;q11) L3 (B-cell)
del(9)(p13–22)
t(9;22)(q34;q11) B-lineage
del(11)(q14–23)
t(11;19)(q23;p13) Mixed, biphenotypic
t(12;V)(p12;V)a B-lineage
t(14;19)(q32;q13)
t(14;22)(q32;q11)
aV = Chromosomes 7, 9, and 17.
Table 3
Common Chromosome Changes in B-Cell
Non-Hodgkin Lymphoma (NHL) (1,12)
Chromosome changes Histology
t(2;3)(p12;q27) Diffuse large cell
t(2;8)(p12;q24) (Burkitt) small noncleaved cell
t(3;14)(q27;q32) Diffuse large cell
t(3;22)(q27;q11) Diffuse large cell
+3 Follicular large cell, immunoblastic
del(6q) Follicular small cleaved cell
t(8;14)(q24;q32) (Burkitt) small noncleaved
t(8;22)(q24;q11) (Burkitt) small noncleaved
t(11;14)(q13;q32) Centrocytic (variable zone) with CD5+ cells
+12 Diffuse small cell
t(14;18)(q32;q21) Mixed, small cleaved, and large cell follicular
t(18;22)(q21;q11) Follicular
The acute leukemias, which are classified either as lymphoblastic (ALL) or
nonlymphocytic (ANLL), result from neoplastic transformation of uncommit-
ted or partially committed hematopoietic stem cells. About two thirds of ANLL
and ALL patients have recognizable clonal chromosomal anomalies. These
Cytogenetics Analysis 7
Fig. 1. G-banded karyotype of a marrow cell showing the Philadelphia (Ph) translo-
cation, t(9;22)(q34;q11) (arrows point to breakpoints). This was the only change
present in the affected cells of this case with CML.
Fig. 2. G-banded karyotype showing trisomy 12 (+12) as the only change in a case
of CLL. This change (+12) is seen in a significant number of CLL cases and is usually
associated with a poor prognosis.
8 Sandberg and Chen
Table 4
Frequency of Chromosomal Changes and Evolution to ANLL
in Myelodysplasia
Evolution to Chromosomal
FAB subtypes and distribution ANLL changes mo
Refractory anemia (30%) 11% 48% 37
Refractory anemia
With ring sideroblasts (18%) 15% 12% 49
With excess blasts (25%) 25% 57% 19
With excess blasts & transformation (12%) 50% 93% 16
Chronic myelomonocytic leukemia (15%) 15% 29% 22
Table 5
Prognosis in Myelodysplastic Syndromes According
to Cytogenetic Findings
Prognostic category Karyotype Median survival (mo)
Good Normal >24
Deletion 5q
Intermediate Trisomy 8 >18
Poor Monosomy 7
Deletion 7q
Isochromosome 17q <12
Deletion 20q
Complex changes
may fall into a specific category that characterizes unique clinical and cytoge-
netic entities. Survival, as a function of cytogenetic findings in ANLL and
ALL, is shown in Table 6. The determination of the chromosomal changes in
acute leukemia serves a number of practical purposes, for example, the estab-
lishment of the exact diagnosis, prediction of prognosis, and as a guide to the
treatment and monitoring phases of therapy or BM transplantation, as well as
some basic purposes, such as supplying the molecular biologist with possible
information on the location or nature of the genes affected by translocations, dele-
tions, and inversions. A case in point is the t(15;17)(q22;q21), seen in APL, which
has been shown to affect a gene related to the α-retinoic acid receptor. This has
led to the use of retinoic acid in the therapy of APL with remarkable results.
More than 90% of non-Hodgkin lymphomas (NHL) have clonal chromo-
somal changes; t(8;14)(q24;q32), t(8;22)(q24;q11) and t(2;8)(p12;q24) have
Cytogenetics Analysis 9
Table 6
Chromosomal Abnormalities and Survival in ANLL and ALL
Chromosomal abnormality Median survival (mo)
ANLL
Rearrangements of 16q22 18
Translocation (8;21)(q22;q22) (seeFig. 3) 14
Normal karyotype 10
Abnormal 11q 18
Abnormal 5 and/or 7 13
Translocation (15;17)(q22;q21)a (seeFig. 4) a
ALL
More than 50 chromosomes 58
Deletion 6 (q15q21) 30
Normal karyotype 29
Translocation (9;22)(q34;q11) 12
Less than 46 chromosomes 12
Rearrangements of 14q32 18
Translocation (4;11)(q21;q23) 17
Rearrangements 15
aSurvival for patients with a 15;17 translocation is markedly improved with aggressive treat-
ment and trans-alpha retinoic acid therapy compared with the median survival previously
reported (2 mo)
been found in 75–80%, 10–17%, and 5–8% of Burkitt lymphomas (BL) of
both African and non-African origin, respectively (10). Molecularly, fusion of
theMYCgene to immunoglobulin genes has been identified in all BL cases. In
non-Burkitt NHL, a 14q+ marker characterizes about 50% of the cases. Many
of the nonrandom anomalies correlate with histology and immunologic pheno-
type, such as t(14;18)(q32;q21) with follicular (nodular) B-cell lymphomas,
del(6q) with large-cell lymphomas, and t(8;14)(q24;q32) with either small,
noncleaved cell or diffuse large-cell lymphomas.
Approximately 50% of CLL patients have chromosomal abnormalities, the
most common of which are trisomy 12, 14q+, 13q, and 11q abnormalities. An
abnormal karyotype is a poor prognostic sign in CLL, and trisomy 12 and pos-
sibly 14q+ are the least favorable abnormalities. Three factors are of impor-
tance in CLL: lymphocyte doubling time, diffuse lymphocyte infiltration of
BM and lymph nodes, and the chromosomal pattern. Combining these
three factors with the current clinical staging of CLL may optimize therapeutic
decisions.
10 Sandberg and Chen
Fig. 3. G-banded karyotype with the translocation (8;21)(q22;q22) as the only
anomaly. This change is seen almost exclusively in M2 type of ANLL associated with
a relatively good prognosis, Auer bodies in the cells, and a high remission rate.
2. Materials
2.1. Specimens
1. BM aspirate or bone core biopsy: One to 3 mL of BM should be aseptically aspi-
rated into a sodium-heparinized syringe and transferred to a sterile sodium
vacutainer tube. The specimen can be transported with or without culture
medium (RPMI 1640 + 5–10% fetal calf serum [FCS] + 1% penicillin[pen]/
streptomycin[strep]). If the specimen cannot be delivered immediately, it may
be stored at room temperature or in a refrigerator overnight. Do not freeze the
specimen. Cell viability drops off sharply by 72 h after collection. If a marrow
aspirate cannot be achieved, a BM biopsy may be accepted for cytogenetic
analysis.
2. Peripheral blood: Five to 10 mL of PB should be aseptically collected and trans-
ferred, transported, and preserved in the same way as for a BM specimen.
3. Lymph node and spleen: Lymph node and spleen biopsies or samples should be
collected aseptically and transferred to a sterile sodium vacutainer tube contain-
ing culture medium (RPMI 1640 + 5–10% FCS + 1% penicillin/streptomycin).
The specimen should be transported and preserved in the same manner as for BM
specimen.
Cytogenetics Analysis 11
Fig. 4. R-banded karyotype of a cell from a patient with APL (M3) containing the
translocation (15;17)(q22;q11–21) as the only chromosome change. This transloca-
tion is characteristic of APL (M3).
2.2.Reagents and Instruments
1. RPMI 1640/MEM Alpha/FBS complete media: 100 mL RPMI 1640, 70 mL
MEM alpha, 30 mL fetal bovine serum (15%), 2 mL 3% L-glutamine (1%), 2 mL
penicillin/streptomycin (1%) (10,000 units pen/mL; 10,000 µg strep/mL).
2. Colcemid (Gibco Karyomax colcemid solution—10 µg/mL):
a. 1/20 Solution: Dilute 10 mL of the stock solution with 10 mL of sterile deion-
ized water to a final concentration of 5 µg/mL). Store at 4°C.
b. 1/200 Colcemid solution: Dilute 5 mL of 1/20 colcemid with 45 mL of sterile
deionized water to a final concentration of 0.5 µg/mL). Store at 4°C.
3. Potassium chloride—0.068 M:
a. Stock solution: 5.6 g KCl in 100 mL of deionized water.
b. Working solution: 10 mL KCl of stock solution in 100 mL of deionized water.
Prewarm to 37°C before use.
