The Role of KMT2A-PTDs in Myeloid Malignancies

KMT2A (also known as MLL) rearrangements are among the most significant genomic alterations in myeloid malignancies, particularly acute myeloid leukemia (AML). These alterations include both KMT2A gene fusions and KMT2A partial tandem duplications (PTDs), which have distinct biological implications. Historically, detection of KMT2A-PTDs has relied on RT-qPCR assays, which are limited in multiplexing capacity and breadth of variant detection.¹

Recent advances in next-generation sequencing (NGS) have enabled simultaneous detection of multiple key biomarkers within a single assay. A study presented at the American Association of Cancer Research (AACR) Annual Meeting reviews data generated using the Oncomine Myeloid Assay GX v2, demonstrating robust detection of KMT2A-PTDs in both myeloid malignancy samples and healthy donors, while highlighting meaningful differences in PTD read counts between these populations.

Figure 1: Estimated new cases (%) of Leukemia, Lymphoma and Myeloma in USA, 2021

The importance of KMT2A alterations

KMT2A rearrangements play a central role in leukemogenesis through dysregulation of HOX gene expression and epigenetic control. KMT2A fusions are well-established oncogenic drivers, while KMT2A-PTDs represent intragenic duplications involving specific exons of the KMT2A gene.²

KMT2A-PTDs occur in approximately 5–10% of AML cases and prior studies have shown that low-level KMT2A-PTDs may also be detectable in healthy individuals, raising important questions about their biological relevance.^3,4

Study overview: Detection of KMT2A-PTDs in healthy donor and myeloid malignant samples using next-generation sequencing

This multi-site study analyzed a total of 8,503 samples using targeted RNA sequencing:

8,483 myeloid malignancy samples processed at Sonora Quest Laboratories and Thermo Fisher Scientific
20 healthy donor whole blood samples, representing 127 technical replicates, sequenced across three Thermo Fisher Scientific sites

All samples were processed on the Ion Torrent Genexus System and analyzed using Oncomine Myeloid Assay GX v2, which targets:

6 known KMT2A-PTD variants
199 KMT2A fusion isoforms
A broad panel of DNA mutations and RNA fusions relevant to myeloid malignancies

Figure 2: Oncomine Myeloid assay enables rapid and efficient multi-biomarker testing

Assay design and technical performance

The Oncomine Myeloid Assay GX v2 is designed to deliver a comprehensive molecular profile within approximately two days, consolidating multiple traditionally independent tests into a single workflow. Sequencing metrics in this study demonstrated:

Mean read length of 90–120 bp
20,000–30,000 mapped fusion reads per sample
Reliable detection of both low-level and high-abundance fusion transcripts

Variant	Length (bp)
KMT2A::KMT2A.nt51.K11K2	178
KMT2A::KMT2A.K9K2	172
KMT2A::KMT2A.K7K2	169
KMT2A::KMT2A.K11K2	116
KMT2A::KMT2A.K8K2	105
KMT2A::KMT2A.K10K2	105

Table 1: Oncomine Myeloid assay is designed to detect six KMT2A-PTD variants

Prevalence of KMT2A fusions and PTDs

Across the full cohort:

~1.9% of samples contained at least one KMT2A-PTD
~1.2% of samples harbored at least one KMT2A fusion
~3.1% of all samples contained one or more KMT2A fusion or PTD

Among fusion-positive cases, the most prevalent fusion partners were KMT2A-MLLT1 and KMT2A-MLLT3, consistent with known epidemiology in myeloid malignancies.

Category	Gene Pair	Found in # of unique samples	Found in % of all samples (N=8503)
KMT2A fusions	KMT2A::AFF4	2	0.02%
	KMT2A::CASC5	2	0.02%
	KMT2A::CBL	4	0.05%
	KMT2A::ELL	11	0.13%
	KMT2A::EPS15	4	0.05%
	KMT2A::MLLT1	30	0.35%
	KMT2A::MLLT10	9	0.11%
	KMT2A::MLLT3	29	0.34%
	KMT2A::MLLT4	14	0.16%
	Total	105	1.23%
KMT2A-PTDs	KMT2A::KMT2A	162	1.91%
KMT2A-PTDs	Total	162	1.91%

Samples w/ ≥ 1 KMT2A fusion or PTD

Total

265

3.12%

Table 2: KMT2A fusions & PTDs existed in ~3% samples

Differentiating myeloid malignancy from healthy donors

A key objective of this study was to determine whether quantitative differences in PTD read counts could distinguish malignant from non-malignant samples.

The results showed a clear separation:

Healthy donor PTD read counts were consistently below 2,000 reads
Mean PTD read counts in healthy donors were approximately one-third of those observed in myeloid malignancies
~33% of myeloid samples exhibited PTD read counts higher than any healthy donor replicate

These findings suggest that read count magnitude, rather than simple presence or absence, may be critical for interpreting KMT2A-PTD results in a research setting.

Figure 3: Read count differentiates myeloid cancer PTDs

Orthogonal verification and structural confirmation

To confirm the biological validity of detected PTDs, representative samples were evaluated using:

Integrative Genomics Viewer (IGV) alignment
BLAT (BLAST-like Alignment Tool) analysis

Both approaches demonstrated clean exon-to-exon junctions consistent with a true KMT2A-PTD in both healthy donor and myeloid samples. An 18-bp insertion at the duplication breakpoint was traced to KMT2A intron 1, further supporting accurate structural resolution by the assay.

Figure 4: IGV view of KMT2A-KMT2A.K9K2 in healthy donor samples vs myeloid samples

Figure 5: BLAT view of KMT2A-KMT2A.K9K2 in healthy donor samples vs myeloid samples

On IGV view (Figure 4 above), the

alignment of KMT2A-KMT2A.K9K2 looks clean with some insertions and mismatches in both healthy donor and myeloid malignant samples.

This is confirmed by BLAT (Figure 5b left). One insertion of 18 bps is observed at the break point of KMT2A-KMT2A.K9K2. BLAT shows that it is from intron 1 (Figure 5c left).

Conclusion

This study describes the detection of KMT2A fusions in myeloid malignancy samples by RNA sequencing using the Oncomine Myeloid Assay GX v2. The study data shows the presence of KMT2A-PTDs in both healthy donor and malignant myeloid samples, with the malignant cases showing significantly higher PTD read counts. Additional prospective and retrospective studies are required to understand the significance of this finding and determine whether PTD levels can be used as a future early indicator for myeloid malignancy development.

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