Rapid, Reliable, and Comprehensive Identification of MDS-Defining Cytogenetic Changes By a FISH-Panel Containing Six Probes in a Real-World Population of Patients with Suspected AML
Authors: Shirneshan Katayoon , Treiber Hannes , Rittscher Katharina , Germing Ulrich , Hildebrandt Barbara , Strupp Corinna , Schanz Julie , Windolph Ute , Ganster Christina , Haase Detlef 
Published: December 2024

Background: Genetically distinct hematologic entities such as Ph+ ALL or APL with PML::RARA-fusion require promptly initiated targeted therapy. In this context, rapid FISH-analysis has become the gold standard for fast diagnosis. CPX351 (Vyxeos®) is approved as a tailored therapy in pts. with t-AML/AML-MRC. A relevant portion of these cases is identified by MDS-defining cytogenetic changes (MDCC) according to WHO 2022 criteria. Referring to a large published AML cohort, we designed a panel of 10 FISH probes that would have detected 99.6% of all MDCC. In a retrospective pilot study of 121 pts. from our center, all 10 FISH probes were evaluable in 86% of pts., with no cases being completely uninformative and 96% of all probes could be analyzed. Encouraged by these results, we initiated a dicentric (Universities of Düsseldorf and Göttingen) prospective diagnostic study. Further optimization of the panel revealed that only 6 probes were necessary to identify the vast majority (97%) of MDCC in AML.
Aim: This study aims to prove that a rapid identification of AML-MRC cases with MDCC is practicable and reliable using a streamlined 6-probes FISH panel (5p15/EGR1, CEN7/7q31, KMT2A, ETV6/RUNX1, 13q14/13q34, TP53/NF1). We assess practicability, success rate, turnaround times (TAT), correlation with centralized morphology, correlation of FISH and chromosomal banding analysis (CBA), and the reliability of analysis of peripheral blood (pb) vs. bone marrow (bm).
Methods: After obtaining informed consent and approval from the ethics committees, 96 pts. with suspected AML were screened between 11/22 and 07/24 (median age 63 years, range 20-89, m:f ratio 1:0.8). FISH-panel analyses were performed in 91 pts. Corresponding CBA results were available for 71 and NGS panel analyses (49 to 51 genes) for 50 pts. TAT between arrival of the sample in the lab and delivery of the FISH report were documented for all patients.
Results: The morphology and histology results were as follows: ALL (n=5, excluded), AML de novo (35), AML-MRC (29), AML n.o.s. (14), MDS-IB2 (11), MDS-LB (1), Myelosarcoma (1). Among the 91 FISH analyses performed, none failed completely. The entire panel of 6 probes was evaluable in 88/89 pts, with 576/579 probes successfully analyzed. Bm only was examined in 33 pts, pb only in 18 and pb + bm in 39 pts. The results were well comparable between bm and pb. No abnormal bm clone was missed in pb, although clone size in pb (median 39%, 5.8%-98%) was non-significantly (p=0.21) smaller than in bm (60%, 5.8%-99%). Clonal FISH anomalies were uncovered in 40/91 pts. (44.4%), clonal CBA changes were found in 35/71 pts. (49.3%). FISH analyses detected the following anomalies: -5/5q- in 40%, -7/7q- in 45%, 11q23-anomalies in 43%, ETV6/12p-anomalies in 35%, 13q14/13q34-anomalies in 43%, 17p13/TP53-deletions in 30% and RUNX1/21q22-anomalies in 30%. Complex changes (&gt;3 anomalies) were seen in 47.5%, and very complex (&gt;4) in 37.5% of abnormal cases. TAT for FISH analysis ranged from 4.6 hours (h) to 27.2 h, with a median of 7.9 h. Comparing FISH with CBA (n=69 pts.), identical results were found in 35 cases (50.7%). Additional abnormalities were detected by FISH in 6 and by CBA in 21 pts. In 8 cases, FISH but not CBA was possible due to lack of dividing cells. Complex anomalies were missed by FISH in 6 cases and by CBA in 4. MDCC were identified in 22 cases by FISH and CBA, in 4 pts. by FISH only, and in 3 cases by CBA only, resulting in a sensitivity of 86% for detecting MDCC by CBA and 90% by FISH. Mutations were detected in 49/50 pts, with a median number of 3 mutations per patient (range: 0-7). The most frequently mutated genes were DNMT3A (16 pts.), FLT3-(ITD, 14 pts.), and NPM1 (12 pts). We observed a rapid initiation of CPX351 based therapy in our study cohort compared to standard of care diagnostic workup. The median time from the arrival of samples at the laboratory to the commencement of treatment was 5.7 days vs. 12.9 days (p=0.0017).
Conclusions: Our proposed FISH panel demonstrated high reliability, detecting clonal changes in 44.4% of pts. It was well applicable in pb, allowed a rapid TAT (median of 7.9 h), and was not inferior to CBA in detecting MDCC and complex changes. Thus, this panel has the potential to improve and expedite the diagnosis of MDCC in pts. with AML.