Two Ways to Complex Karyotype in MDS - the Role of Del(5q) and TP53
Authors: Haferlach Claudia , Huber Sandra , Hutter Stephan , Baer Constance Regina , Meggendorfer Manja , Hoermann Gregor , Kern Wolfgang , Haferlach Torsten 
Published: December 2024

Background:
Deletions (del) 5q in MDS typically occur either in MDS with low blasts and del(5q) (MDS-5q according to WHO) or in MDS with complex karyotype (CK). While MDS-5q are generally associated with a favorable prognosis and harbor TP53 alterations (alt) in 10-20% of these cases (most commonly single-hit), MDS with CK are associated with an unfavorable outcome, harbor additional cytogenetic abnormalities such as monosomy 7 or del(7q) and del(17p) and frequently show TP53 alt which are multi-hit in 80-90% of cases.
Aims:
To clarify based on hierarchy analysis whether MDS with CK arises from MDS-5q cases with del(5q) as primary abnormality.
Methods:
729 MDS patients harboring a del(5q) (median age: 76 years [32-98]; female: 58%) were collected between 2005 and 2023 and included in this study. All samples were analyzed by cytomorphology, cytogenetics, FISH and a targeted NGS panel. Diagnoses were established following WHO classification valid at time of sampling. We analyzed the clonal composition at initial diagnoses. Follow-up samples were available in 150/729 patients. To compare the variant allele frequencies (VAF) of mutations (mut) to those of cytogenetic abnormalities, the number of cells with aberrant nuclei were divided by two (CA-VAF). VAF estimates were used to evaluate clonal hierarchy of TP53 and del(5q) using a VAF difference cut-off of ≥5% in samples that harbored no del(17p) by FISH and had TP53 VAF &lt;55%, i.e. presumably unaffected by copy-neutral loss of heterozygosity (CN-LOH). TP53 multi-hit was defined as either ≥2 TP53 mut, one TP53 mut with VAF ≥55% or one TP53 mut accompanied by del(17p). For progression analysis only patients with a minimum follow-up time of 90 days were considered.
Results:
Of all del(5q) patients, 42% (308/729) had MDS-5q (median age: 77 years; female: 72%), 50% (365/729) had CK (median age: 74 years; female: 46%) and 8% (56/729) neither fulfilled criteria for MDS-5q or CK. Both age (p=0.001) and sex ratio (p&lt;0.0001) differed significantly between MDS-5q and CK.
The most frequently co-occurring somatic mut in cases with del(5q) was TP53, detected in 54% (397/729) of cases, out of which TP53 was the sole mutated gene in 63% (252/397). Notably, TP53 mut were more frequent in MDS with CK than in MDS-5q (87% vs 20%; p&lt;0.0001) and were also more commonly multi-hit in MDS with CK compared to MDS-5q (79% vs. 27% of TP53 alt cases; p&lt;0.0001).
Clonal hierarchy analysis of TP53 mut cases that neither harbored del(17p) nor CN-LOH showed that for MDS-5q (n=23) the median CA-VAF of del(5q) was higher than the VAF of TP53 mut (25% vs. 20%, p=0.48), while in CK cases (n=90) this was reversed (19% vs. 29%, p&lt;0.0001). Congruently, in MDS-5q the clear primary event (determined by CA-VAF del(5q) to VAF TP53 mut comparison in each case) was del(5q) in 43% (10/23) of cases compared to TP53 mut in 26% (6/23) of cases. For MDS with CK del(5q) was the clear primary event in 7% (6/90) and TP53 mut in 50% (45/90) of cases. In CK cases with del(17p) (n=104), del(5q) preferably occurred before del(17p) (27% vs. 13%; 60% hierarchy not determinable).
For a subset of patients data on karyotype and TP53 status was available from different time points (median follow-up time: 2.8 years). Out of 84 patients initially diagnosed with MDS-5q 4% (3/84) presented with normal karyotype following transplantation or therapy with lenalidomide, 4% (3/84) showed clonal evolution (acquisition of one additional cytogenetic alt), 24% (20/84) developed CK (acquisition of two or more additional cytogenetic alt) and 69% (58/84) showed a stable karyotype.
Out of the 20 cases acquiring CK during progression, 13 harbored TP53 alt (nine multi-hit, four single-hit), of these only six patients showed TP53 alt already in the MDS-5q phase (two multi-hit, four single-hit). TP53 alt were significantly more common at final follow-up in patients progressing to CK (13/20; 65%) than in those retaining a stable karyotype (22/58; 38%), as were multi-hit TP53 alt which were found in 45% (9/20) of CK acquiring patients and only in 19% (11/58) of stable MDS-5q patients (for both p=0.04).
Conclusion:
Clonal hierarchy analysis and serial genetic assessment suggest two separate routes to complex karyotypes in MDS: 1) deriving from MDS-5q which mainly harbor del(5q) within the ancestral clone and acquire TP53 alterations during the course of the disease, 2) more commonly arise from clones with a TP53 mutation as the primary abnormality acquiring del(5q) later.