181P - Intratumoral heterogeneity of immune infiltrate in leiomyosarcomas

Background

Leiomyosarcoma (LMS) is a highly aggressive and rare malignancy, often presenting as large tumors with potentially heterogeneous tumor microenvironment (TME). This TME heterogeneity may contribute to poor responsiveness to immunotherapy and its overall unfavorable prognosis. We aimed to provide a detailed characterization of the intratumoral heterogeneity and possible mechanisms of non-responsiveness to immune checkpoint inhibitors in large LMS.

Methods

We investigated four large treatment-naïve LMS (ten samples/tumor). We analyzed four regions: vital and necrotic centers (VC, NC), organ-adjacent and free margins (O-AM, FM), where we evaluated T cells, macrophages, immune inhibitory molecules, and tertiary lymphoid structures (TLS) by IHC and flow cytometry. Moreover, we performed unsupervised clustering of flow cytometry data and analyzed cytokine secretion after anti-LAG-3 blockade in each tumor sample. Lastly, factorial analysis of mixed data (FAMD) analysis revealed the drivers of tumor sample variability.

Results

The expression of PD-1 and PD-L1 profoundly varied within the same tumor tissue. TLS were detected only at the tumor margins. Interestingly, TLS were present in one out of ten samples in one LMS tissue. Moreover, shared immune patterns were detected in specific tumor regions, such as higher levels of IDO, absence of specific macrophage subpopulation, and lower levels of PD-1 and LAG-3 expression in the O-AM. Following the findings about LAG-3 expression, we tested the cell's reactivity to anti-LAG-3 blockade. This blockade enhanced IL-8 in the NC and PD-L1 in the O-AM compared to other tumor regions. FAMD showed that samples can be separated by the tumor regions, where VC and NC displayed distinct patterns.

Conclusions

Our findings emphasize the importance of multiple biopsies to accurately assess the immune heterogeneity in large LMS. The intratumoral heterogeneity may explain varied responses to immunotherapy, even with low or absent expression of these molecules. A single biopsy may not capture the full spectrum of immune cell infiltration, potentially leading to incomplete evaluations. Advanced immunoprofiling offers deeper insights into the TME, improving diagnostic accuracy and treatment selection. AZV NU23J-08–00031, GA UK 94323.

Legal entity responsible for the study

University Hospital Motol, Prague, Czech Republic.

Funding

Czech Health Research Council NU23J-08-0031, Charles University in Prague - 94323.

Disclosure

J. Bartunkova: Financial Interests, Personal, Full or part-time Employment and a minority shareholder: Sotio Biotech, a.s. All other authors have declared no conflicts of interest.