Fluorescence In Situ Hybridization (FISH) Reveals Unexpected Cryptic Chromosome 11 Defects in MDS/AML Patients.
Authors: Bernasconi Paolo , Dambruoso Irene , Boni Marina , Cavigliano Paola Maria , Giardini Ilaria , Zappatore Rita , Calatroni Silvia , Rocca Barbara , Caresana Marilena , Lazzarino Mario 
Published: April 2020

Conventional cytogenetics (CC) discovers clonal chromosomal defects in about 40–80% of de novo MDS/AML. However, due to the poor quality of metaphases, CC is often unable to reveal cryptic defects, precisely define chromosomal breakpoints and establish the nature of marker chromosomes. All these drawbacks may be overcome by FISH, a powerful technique with high sensitivity and specificity. Abnormalities of chromosome 11 long arm and of band 11p15 are seen in 5–7% and in 0.5% of de novo MDS/AML. Herein we report two patients diagnosed as AML evolved from MDS. On CC the karyotype of the first patient was 47,XX,+3,t(15;20)(q15;p11),add(11)(p15) [20] [case 1] and that of the second patient was 45,XX,t(1;?)(q12;?),−5,−7,+8,add(11)(q23),add(12)(p13),add(17)(p13),add(21)(q22) [7]/46,XX,t(1;?)(q12;?),add(5)(q11),−7,+8,add(17)(p13),add(21)(q22) [8] [case 2]. In both the patients FISH was performed with commercial probes (LSI and TCP from Vysis and QBiogene, applied following manufacturer’s guidelines) and Bacterial Artificial Chromosome (BAC) probes (kindly provided by the Wellcome Trust Sanger Institute, Cambridge UK) in order to better define chromosome 11 rearrangements. The BAC probes were cultured in LB broth for a night and the following day the DNA was extracted after lysis. Labelling with biotin and digoxigenin was carried out by nick translation reaction. Signal detection was obtained by fluorescein avidin and anti-digoxigenin.

Case one was at first investigated with a BAC probe specific for the NUP98 gene, mapped at 11p15. One signal was expected on the normal chromosome 11 and a split signal on add(11)(p15). Instead, two spots on the short arm and the other on the long arm of a marker chromosome were seen. The investigation with the Cyclin D1/CEP11 probe, mapped at 11q13.3, allowed us to establish that this marker was a number 11 and, at the same time to identify the other chromosome 11, which did not show any signal corresponding to NUP98. This same chromosome showed two signals corresponding to MLL and two other signals corresponding to ATM when these probes were applied. A TCP 11 probe did not identify any other chromosome 11 fragment. Therefore, we realized that chromosomes 11 were rearranged with each others. Additional BAC probes lead us to fix the possible breakpoint area on the chromosome 11 rearranged in the long arm in between bands q21 and q13 and on the other chromosome 11 under the NUP98 gene.

Case two was at first investigated with TCP 1 and 11 probes that showed chromosome 11 material on the rearranged chromosome 1 and a fragment of chromosome 1 inserted in the middle of chromosome 11 long arm, which was not totally painted by the two probes. This datum lead us to hypothesize the involvement of a third chromosome in the rearrangement. A TCP 5 probe demonstrated that the unpainted material under der(11) belong to chromosome 5. When MLL and ATM probes were applied the former was on der(1) and the second was on der(11), under the inserted chromosome 1 fragment, but over chromosome 5 material. Therefore, the breakpoint region between der(11) and chromosome 5 material lies in between band 11q22 and 11q23.

In conclusion, FISH:

reveals unexpected rearrangements absolutely cryptic on CC, is a very effective tool to map chromosome breakpoints and identifies new genes involved in leukemogenesis.