Interphase FISH and Comparative Genomic Hybridization Performed in Addition to Chromosome Banding Analysis in AML with Normal Karyotype Detect Prognostically Relevant Chromosome Abnormalities.
Authors: Schoch Claudia , Klaus Mirjam , Schnittger Susanne , Hiddemann Wolfgang , Kern Wolfgang , Haferlach Torsten 
Published: October 2019

Abstract
In AML karyotype abnormalities are not detected in 40 to 45% of cases using classical chromosome banding analysis. For several reasons false negative results might occur in chromosome banding analysis: 1. no proliferation of the aberrant clone in vitro, 2. low resolution due to technical problems or limitations of the method itself, 3. real cryptic rearrangements. In order to determine the proportion of “false negative” karyotypes by chromosome banding analysis we conducted a study using interphase-FISH and comparative genomic hybridization in addition to chromosome banding analysis. In total, chromosome banding analysis have been performed in 3849 AML at diagnosis. Of these 1748 showed a normal karyotype (45.4%). Out of these in 3 cases cytomorphology revealed an APL and in 2 cases an AML M4eo. Using interphase FISH with a PML-RARA or CBFB probe we detected cryptic PML-RARA or CBFB-rearrangements, respectively, in all 5 cases, which were cytogenetically invisible due to submicroscopic insertions. 480 cases of AML with normal karyotype were analyzed for MLL gene rearrangements using FISH with an MLL-probe. 11 cases with a cryptic MLL-rearrangement were detected (FAB-subtypes: M5a: 7, M2: 2, M0: 2). In 273 patients interphase-FISH screening with probes for ETO, ABL, ETV6, RB, P53, AML1 and BCR was performed. In 6 out of 273 (2.2%) pts an abnormality was detectable. In two cases the aberrant clone did not proliferate in vitro: 1 case each with monosomy and trisomy 13. Due to limitations of resolution in chromosome banding analysis translocations or deletions of very small chromosome fragments were only detected with FISH in n=4 cases (ETV6 rearrangements: t(11;12)(q24;p13), t(12;22)(p13;q12), ETV6 deletions: del(12)(p13), n=2). Like interphase-FISH comparative genomic hybridization (CGH) does not rely on proliferating tumor cells but in contrast to interphase-FISH allows the detection of all genomic imbalances and not only of selected genomic regions. Therefore, we selected 48 cases with normal karyotype and low in vitro proliferation (less than 15 analyzable metaphases in chromosome banding analysis). In 8 of 48 cases (16.7%) an aberrant CGH-pattern was identified which was verified using interphase-FISH with suitable probes. In 3 cases a typical pattern of chromosomal gains and losses observed in complex aberrant karyotypes was detected. In one case each a trisomy 4 and 13 was observed, respectively. In one case trisomy 13 was accompanied by gain of material of the long arm of chromosome 11 (11q11 to 11q23). One case each showed loss of chromosome 19 and gain of the long arm of chromosome 10, respectively. In conclusion, CGH in combination with interphase-FISH using probes for the detection of balanced rearrangements is a powerful technique for identifying prognostically relevant karyotype abnormalities in AML assigned to normal karyotype by chromosome banding analysis. Especially this is true in cases with a low yield of metaphases and in AML with a high probability of carrying a specific, cytogenetically cryptic fusion-gene. Thus, in these cases interphase-FISH and CGH should be performed in a diagnostic setting to classify and stratify patients best.