Introduction

As a key screening test for the NCI myeloMATCH clinical trial, karyotyping detects aberrations at single-cell resolution and uniquely delineates clonal evolution in hematologic malignancies. Its success starts with robust cell culturing methodologies that reveal chromosomal aberrations in dividing cells. While planning for 72-hour turn-around time (TAT) without compromising quality, we evaluated culturing conditions for 139 diagnostic acute myeloid leukemia (AML) and high-grade myelodysplasia (MDS) patient samples from Fred Hutchinson Cancer Center (McClung et al, 2023 JAM). We concluded that 48-hour culture with MarrowMax (48MM) was better than 24-hour cultures, especially for peripheral blood (PB). A similar trend was observed for bone marrow (BM) samples although 48MM appeared less critical. This study aims to evaluate prospectively collected AML/MDS patient samples undergoing karyotype analysis for myeloMATCH as the evidence base to confirm the best culturing condition allowing for rapid TAT.

Methods We investigated 249 myeloMATCH specimens from 215 patients [median age 65.6 (Range 18.4 to 95.8); 45% female]. These were sent for diagnostic screening (191 BM and 24 PB) and reassessment (30 BM and 1 PB), together with 3 samples that canceled testing. Culturing conditions tested included media type [RPMI (n=244 ) vs MarrowMax (n=85)] and duration [24-hour only (n=64) vs 24- and 48-hour (n=180)].

To compare the effectiveness of various culturing conditions, we investigated the number of metaphase images captured per slide, the cytogenetic findings from 24- vs 48-hour cultures, and the clinical significance of the additional results contributed by 48MM.

Results

  • The 48MM culture improved mitotic index of the sample. The number of metaphase images captured per slide was measured as an indication for the mitotic index. The number of metaphase cells per slide from the 24-hour RPMI culture was set as the reference, where the performance of other cultures was presented as the log 2 value of the ratio of the metaphase cells per slide divided by that of the reference. The 48MM showed improved mitotic index, consistently observed from BM (n=160) with a median log2 ratio of 2.10 (95% CI 1.73-2.33) and PB (n=25, median 2.31, 95% CI 1.28-3.47). Further delineation showed consistent observation from different flask sizes (2.5, 5, 7.5, and 10 F). In 24-hour cultures, MarrowMax is not significantly better than the RPMI for BM (n=222, median=0.95, 95% CI 0.86-1.22) or PB (n=25, median=1.37, 95% CI 0.80-2.32).

  • The 48MM improved success of the study. Karyotype results were compared for samples where analysis for both 24- and 48-hour cultures were attempted (n=184). One sample failed the the 24h cultures but yielded a full study with 20 cells from 48MM. A high number of samples had less than 20 analyzable cells (n=81; 33%) from 24-hour cultures and showed a normal result, including two samples with less than 5 cells for analysis, six with 6-10 cells, 72 with 11-15 cells, and one with 17 cells. Per SWOG cytogenetics guideline, when less than 20 cells are analyzed and the result is normal, the study is considered failure or limited. The 48MM culture yielded more metaphases to analyze and made 79 of the 81 (98%) workups into a full 20-cell study and the remaining two completed with 10 and 17 cells. Furthermore, qualitative assessment indicates 48MM cultures tend to have better chromosomal morphology compared to that of the 24-hour cultures.

  • The 48MM revealed more abnormalities that improved diagnostic yield and produced prognostically significant findings and more thoroughly identified markers for disease follow-up. Two samples changed from normal results at 24h to abnormal from 48MM, representing 0.8% of the specimens. One of these is a reassessment sample that showed inv(3) and -7, key markers of the patient's disease, in one cell from 48MM. Fourteen samples showed additional abnormalities in 48MM compared with the 24-hour cultures, including 12 samples with clonal evolution and two with new independent clones in 48MM.

Conclusions Our systematic assessment using the 249 NCI myeloMatch samples demonstrated that the inclusion of 48MM culture produces better quality karyotyping studies for AML/MDS than 24-hour cultures only. We therefore strategize that the 48MM to be incorporated routinely, especially when the 24-hour cultures yield a normal result or an abnormal result with intermediate risk.

This content is only available as a PDF.
2025
Sign in via your Institution