Halo 2

The ACD RNAscope 2.5 HD Assay Kit 322360 was used. Five μm section slides were deparaffinized by xylene followed by 100% ethanol, then incubated with hydrogen peroxide for 10 minutes at room temperature and washed with distilled water. Antigen retrieval was performed by boiling slides in 1× Target Retrieval solution (ACD 322000) for 10 minutes, washing with distilled water, and then dehydrating with ethanol and air-drying. Protease Plus was added to each section, incubated at 40°C for 30 minutes, and washed with distilled water. The slides were incubated with a custom-made hFMR1-Codon-C1RNAscope probe in a HybEZ oven for 2 hours at 40°C and washed with 1× wash buffer, followed by incubating with AMP 1–6 for 30 or 15 minutes and the use of the RNAscope 2.5 HD Detection Kit protocol. The G-quartet motif in the RGG domain of the endogenous FMR1 sequence was not codon-optimized to ensure retention of FMRP binding to FMR1 mRNA. The slides were then incubated with kit-provided RED solution for 10 minutes, counterstained with Mayer’s hematoxylin, and imaged with an Aperio ImageScope; histology images were analyzed using custom analysis settings in the HALO image analysis platform (HALO2.2, Indica Labs).

Hematoxylin and eosin staining and immunohistochemistry (IHC) were performed on 4 μm FFPE pre and post-treatment tissue samples. IHC analysis on subsequent sections was performed using antibodies against CD3 (Agilent Dako, cat#A0452, 1:100), CD20 (Agilent Dako, cat#M075501-2, 1:1400), CD8 (Thermo Scientific, cat# MS-457-S, 1:25), Granzyme B (Leica Microsystems, cat# PA0291, RTU) and PD-1 (Abcam, cat# ab137132, 1:250). Sections were processed with peroxidase-conjugated avidin/biotin and 3’-3-diaminobenzidine substrates (Leica Biosystems, cat# DS9800), and the IHC slides were scanned and digitalized using the Scanscope XT system (Aperio/Leica Technologies). Single stain IHC quantification analysis was performed by the pathologist using the HALO 2.3.2089.70 software (Indica Labs). The number of marker positive cells for each analysis area were calculated and expressed as density (number of positive cells/mm²) and densities were plotted using Prism V8.4.3 (GraphPad). TLS quantification was done as previously described and the total number of TLS per mm² of tumor area was plotted^{18 (link)}. Statistical analysis was done using a two-tailed, unpaired Mann–Whitney test, and p values <0.05 were considered statistically significant. P values were adjusted for multiple comparisons using the Benjamini–Hochberg procedure.

Hematoxylin and eosin (H&E) staining, and IHC were performed on 4 μm formalin-fixed paraffin-embedded (FFPE) pre- and post-treatment tissue samples. Stromal TILs were quantified manually by H&E-stained FFPE sections and scored as a percentage of stromal area according to the International Immuno-Oncology Working Group method for assessing TILs [23 (link)]. IHC staining on subsequent sections was performed using anti-CD4 (Abcam, cat#ab133616, 1:250), anti-Gr-B (11F1) (Leica Microsystems, cat#PA0291, ready-to-use), and anti-CD57 (BD Biosciences, cat# 347390, 1:40) antibodies. Sections were stained using the BondRX instrument and the Bond Polymer Refine Detection kit (Cat. #DS9800), and the IHC slides were scanned and digitized using the ScanscopeXT system (Aperio/Leica Technologies). Single stain IHC quantification analysis was performed by the pathologist using the HALO 2.3.2089.70 software (Indica Labs). The number of marker positive cells for each analysis area were calculated and expressed as density (number of positive cells/mm2) and densities were plotted using Prism V8.4.3 (GraphPad). Statistical analysis was done using a two-tailed, paired t-test, and P < 0.05 was considered statistically significant.