Herein we report the clinicopathologic and molecular features of a cohort of uterine tumours harbouring the KAT6B/A::KANSL1 gene fusion. The major novel finding of our study is that KAT6B/A::KANSL1 uterine tumours are defined by a specific DNA methylation signature that is distinct from other uterine mesenchymal neoplasms, indicating they constitute a distinct type of uterine mesenchymal tumour. These findings are in line with a recent study indicating that KAT6B::KANSL1 uterine tumours are characterized by a distinct expression profile. In that study, Trecourt et al. demonstrated that, based on unsupervised clustering of RNA expression profiles, KAT6B::KANSL1 uterine tumours grouped homogeneously and were distinct from ESN/LGESS, HGESS, and uterine smooth muscle tumours [6]. In addition, KAT6B/A::KANSL1 uterine tumours lack evidence of Wnt/β-catenin pathway activation that is typically seen in LGESS [15]. It is worth noting that while a significant subset of KAT6B/A::KANSL1 uterine tumours exhibits focal sex cord differentiation, they displayed a methylation profile that was also distinct from UTROSCTs. Given the distinct global DNA methylation and gene expression profile, we advocate for KAT6B/A::KANSL1 uterine tumours to be recognized as a distinct type of uterine sarcoma—KAT6B/A::KANSL1 uterine sarcomas.

Perhaps surprisingly, in our study, KAT6B/A::KANSL1 uterine sarcomas with high-grade histological features exhibited a variable DNA methylation profile distinct from the core KAT6B/A::KANSL1 cluster tumours. These outliers clustered near other known genotypes and phenotypes of uterine mesenchymal tumours. Nevertheless, a review of the immunohistochemical profiles of the 5 outliers showed that they did not align with the specific tumor groups which they clustered near. The reason for this divergence between DNA methylation profiles and bona fide tumor differentiation remains unclear. Typically, the DNA methylation profiles of cancer cells reflect the characteristics of their cell of origin. While this could indicate that morphologically high-grade KAT6B/A::KANSL1 uterine sarcomas originate from different progenitor cells in the uterus, in the light of the divergent immunohistochemical profiles, it is perhaps more likely that high-grade histologic transformation in KAT6B/A::KANSL1 uterine sarcomas represents a shift in cellular context to either a more primitive cellular state or a transdifferentiated cellular state. This shift may coincide with an altered DNA methylation profile, the presence of a greater degree of CNVs, and genomic instability. Moreover, while uterine sarcomas with recurrent and "simple" genetic alterations, such as gene fusions, are typically considered genomically stable tumours, recent studies of various high-grade, fusion-driven uterine tumours have shown that co-occurring and likely secondary CNVs may occur in a significant number of cases. For example, in YWHAE::NUTM2A/B HGESS and inflammatory myofibroblastic tumour, CDKN2A deletions are associated with aggressive behaviour [16, 17]. In the current study, KAT6B/A::KANSL1 uterine sarcomas falling into the core DNA methylation cluster were consistently histologically bland and showed no significant CNVs. In contrast, three outlier cases showed various CNVs, including deletions at the CDKN2A/B and/or NF1 loci. This raises the question of whether secondary genomic alterations could play a role in high-grade transformation of KAT6B/A::KANSL1 uterine sarcomas, potentially correlating with an aggressive clinical course. To address these questions, it is essential to conduct studies on larger cohorts KAT6B/A::KANSL1 uterine sarcomas with low-grade and high-grade histologic features, particularly cases that contained both components synchronous or metachronously.

To gain a better understanding of the clinical behaviour of KAT6B/A::KANSL1 uterine sarcoma, we performed a meta-analysis of the cases reported herein, as well as previously published cases with available follow-up. The results highlight that approximately one-third of tumors harboring the KAT6B/A::KANSL1 gene fusion recur, and some patients ultimately die of disease. It is, however, possible that, as with other rare tumour types, initial series reported may be skewed by tertiary referral centre bias and that the actual clinical behaviour of KAT6B/A::KANSL1 uterine sarcomas may be less aggressive. Future studies looking at population-based series are needed to provide further insights.

Though preliminary, our findings suggest that histological grading of KAT6B/A::KANSL1 uterine sarcomas may result in a more meaningful diagnostic classification of these tumours. For instance, tumours showing ESN/LGESS and/or leiomyoma-like features with bland nuclei and low mitotic activity could be classified as low-grade KAT6B/A::KANSL1 uterine sarcomas, while tumours showing concurrent monomorphic nuclear atypia (large, sometimes epithelioid, nuclei) and elevated mitotic activity (readily identified mitotic figures) could be classified as high-grade KAT6B/A::KANSL1 uterine sarcomas. Future studies examining an expanded series of clinically annotated KAT6B/A::KANSL1 uterine sarcomas showing low-grade and high-grade histologic features are needed to evaluate the clinical need for histologic grading. Moreover, the value of molecular studies described herein—including DNA methylation profiling, analysis of the degree of genomic instability, and evaluation of specific secondary genomic alterations, such as CDKN2A/B deletions—in classifying KAT6B/A::KANSL1 uterine sarcoma into clinically relevant groups needs to be studied in larger cohorts.

Given the incomplete current understanding of these neoplasms, we suggest that all uterine mesenchymal neoplasms with overlapping morphology and immunophenotype between endometrial stromal and smooth muscle neoplasms undergo molecular testing to identify a KAT6B/A::KANSL1 fusion. More specifically, we recommend testing for tumors exhibiting the following histologic features, particularly in the context of a “myomectomy-type’ resection: 1) Tumours displaying histologic and immunophenotypic features intermediate between an ESN with classic histology and cellular or highly cellular leiomyoma. This includes tumors resembling ESN but containing scattered thick-walled vessels with perivascular hyalinization throughout, as well as those suspected to be a highly cellular leiomyoma primarily due of the presence of scattered thick-walled vessels. 2) Tumours showing whorled spindle cell proliferations with fibrous to fibromyxoid stroma (fibroblastic/fibromyxoid LGESS-like) and a prominent hemangiopericytomatous vascular network with scattered thick-walled vessels and perivascular hyalinization throughout. 3) Tumours with the aforementioned features that also contain a high-grade round cell component or are suspected to have recurred as a high-grade round cell malignancy. In these scenarios, molecular testing should ideally cover not only the KAT6B/A::KANSL1 fusion but also other relevant fusions associated with endometrial stromal tumors.

In terms of clinical management, despite their typically bland cytologic features, KAT6B/A::KANSL1 uterine sarcomas ideally require at least total hysterectomy and ideally bilateral salpingo-oophorectomy also in ER-positive cases for definitive surgical management. “Myomectomy-type” resection does not appear to be sufficient as the tumour can recur locally [5, 6]. When exhibiting malignant cytologic features either in the primary or recurrent tumours, KAT6B/A::KANSL1 uterine sarcomas can pursue a rapidly progressive clinical course, and there is currently no known effective systemic therapy (chemotherapy or targeted therapy) for this tumour type.

In conclusion, KAT6B/A::KANSL1 uterine sarcoma is a molecularly unique tumour that should be recognized as a distinct entity. While most tumours display low-grade histologic features, a subset have high-grade histologic features that is accompanied by a divergent methylation profile and a higher number of CNVs, which appears to correlate with a more aggressive clinical trajectory.