Myeloid Leukemia

112 Campbell
4. Notes
1. The diagnostic marrow or blood sample for all new CML cases should have two
cultures established for conventional cytogenetic analysis; at the Victorian Cancer
Cytogenetics Service (VCCS), two synchronized cultures are set up, one inoculated
with less sample than the other. A marrow or blood sample from a patient
with untreated CML may grow so aggressively in culture that as little as one drop
of marrow added to a 10-mL culture is sufficient to produce several milliliters of
suspension. CML cells are relatively robust, and a successful cytogenetic result
may be obtained from a specimen taken 2 to 3 d before cultures are initiated.
2. The quantities of probe and hybridization buffer described are recommended for
Vysis ® probes. When using probes from other suppliers, check the product insert
for exact quantities.
3. There are a number of probes available that produce signals to identify the presence of
both the BCR/ABL fusion on der(22) and the ABL/BCR fusion on der(9). The use of a
dual fusion probe is preferred, because it will identify the cases with loss of only chromosome
9 or chromosome 22 sequences on the der(9). Most cases show loss of both 9
and 22 sequences, indicating that an extensive deletion has occurred (see Fig. 1).
4. Ideally, metaphase spreads should be identified and the location of the fusion
signals confirmed on the der(22) and der(9). In patients with a deletion of the
der(9), only the der(22) BCR/ABL fusion signal will be observed, together with
signals identifying the normal copies of ABL and BCR on chromosomes 9 and 22,
respectively. However, if metaphases cannot be located, the pattern of two fusion
signals/one red (ABL) signal/one green (BCR) signal indicates the presence of the
t(9;22) without a deletion of the der(9); the pattern of one fusion signal/one red
(ABL) signal/one green (BCR) signal indicates the presence of the t(9;22) with a
deletion of the der(9). The latter pattern of signals in interphase may also reflect
a false-positive cell with incidental co-localization of one red and one green signal
to simulate a fusion signal. Thus, this pattern is useful only in confirming the
presence of the t(9;22) when it is seen in 10% or more of interphase cells (2).
5. The standard karyotype for an individual with chronic myeloid leukemia as
assessed by conventional cytogenetics is 46,XX or XY,t(9;22)(q34;q11·2). When
metaphase FISH with a dual fusion probe is added, the karyotype becomes
46,XX,t(9;22)(q34;q11·2).ish t(9;22)(ABL+,BCR+;BCR+,ABL+), indicating that
there are positive fluorescent signals for both ABL and BCR on the der(9) and the
der(22). When a deletion has been identified, the karyotype becomes
46,XX,t(9;22)(q34;q11).ish t(9;22)(ABL–,BCR–;BCR+,ABL+), showing that the
expected ABL and BCR signals on the der(9) are missing. There is considerable
controversy surrounding the interpretation of the FISH nomenclature guidelines
(13). However, any changes must await the next edition of the International
System for Human Cytogenetic Nomenclature (ISCN).
6. If a deletion has been identified, studies performed after therapy to assess
response to therapy cannot be reliably interpreted using any of the dual-color
dual-fusion or extra signal probes, unless an additional probe is added to the
probe mixture (see Fig. 2).