FISH and Karytotype

We have come to rely on FISH to tell us about the chromosomes in CLL, but it is not a very satisfactory test. It will tell us about the abnormalities that we know exist in CLL - del 13q, del 11q, trisomy 12 and del 17p - but not about ones that we don't look for. In particular we know that isolated del 13q usually has a good prognosis, but unless we look at other chromosomes how do we know it is isolated.

The standard way of looking at chromosomes is to make cells divide, but in CLL this is very difficult. You need a mitogen and the standard mitogen is phorbol ester. Unfortunately although our lab gets over 80% of CLL cells to divide, nobocy else in the world can match our results. This has led other labs to experiment with cytokines to stimulate the CLL cells to divide.

In this month's Leukemia Research, a paper from Poland describes the use of a new mitogen, DSP30. DSP30 is a CpG-oligonucleotide (CpG-ODN). CpG-ODNs act as danger signals and stimulate the immune system. It induces proliferation
of both normal and leukemic B lymphocytes, stimulates production of cytokines, including IL-2 and TNF- , and regulates expression of surface antigens CD25 and CD86 in B lymphocytes. It also stimulates some other immune system cells, such as T lymphocytes, NK cells, dendritic cells and monocytes, to proliferate, secrete cytokines and/or differentiate.

Using DSP30, a sufficient number of metaphases suitable for analysis was obtained in 56/62 (90%) patients, and clonal chromosomal aberrations were detected in 42/56 (75%) patients. Normal karyotype was established in 14/56 (25%). In 9/14 (64%) cases with normal karyotype, aberrations were detected by FISH which revealed anomalies in 52/62 (84%) patients. Using DSP30 and/or FISH, aberrations were detected in 56/62
(90%) patients. DSP30 disclosed del 13q in only 10/56 (18%) cases, while FISH showed this anomaly in 41/62 (66%) subjects. Del 13q was cryptic and not detected by DSP30 in 28 patients. In those cases the presence of submicroscopic del 13q was confirmed by FISH on metaphases. In 4/41 (9.7%) patients del 13q were biallelic or concomitant monoallelic and biallelic. In 26/41 (63%) patients del 13q detected by FISH was a sole anomaly. Del 11q was detected by both FISH and DSP30 in 14 patients, whereas in two patients it was detected only by FISH due to an unsuccessful karyotyping. DSP30 enabled the detection of additional aberrations, especially translocations, in 7/14 (50%) patients with del 11q observed in FISH. DSP30 revealed cytogenetic anomalies which were not searched by FISH in 33/56 (59%) patients. The most frequently observed numerical aberrations were: monosomy 9 (n = 2), monosomy 10 (n = 2), monosomy 13 (n = 2), monosomy 14 (n = 2), monosomy 18 (n = 2) and loss of chromosome Y (n = 2).

It seems to me that complete karyotyping of CLL cells is now possible for all cytogenetics labs, and that it should be added to FISH for patients undergoing clinical trials.