Soni, Sneha

198 Background: The Hedgehog (Hh) signaling pathway regulates tissue patterning, growth and differentiation, including within gastrointestinal tract. Mammalian homologs include Sonic (SHH), Indian (IHH) and Desert (DHH) Hedgehog, which signal through PTCH and SMO and the GLI transcription factors. In the digestive tract, both SHH and IHH are expressed, but the functional role of Hh signaling in mCRC remains incompletely defined. Methods: We analyzed 433 mCRC patients treated with either bevacizumab (bev = 226) or cetuximab (cet = 207) plus first line chemotherapy in CALGB/SWOG 80405 (NCT00265850). RNA expression (Illumina HiSeq 2500) of 9 Hh pathway genes (SHH, IHH, DHH, PTCH1, PTCH2, SMO, GLI1, GLI2, GLI3) was evaluated. Median overall survival (mOS) and progression free survival (mPFS) in months (mo) were compared across expression tertiles (low [L], medium [M], high [H]). Likelihood ratio tests, hazard ratios and 95% confidence intervals were computed from multivariable Cox proportional hazards models, adjusting for age, sex, ethnicity, ECOG PS, tumor location, number of metastatic sites, KRAS , microsatellites instability status and treatment. Prognostic effects were modeled as marginal terms; predictive effects were modeled as multiplicative interactions. Results: Overall, IHH and GLI2 demonstrated prognostic value for OS after adjusting for multiple testing (Benjamini-Hochberg False Discovery Rate (FDR) < 0.05). High IHH expression (H group, n = 143) correlated with improved mOS (H: 36.0 mo vs M: 32.8 mo vs L: 23.5 mo; p= 0.031), whereas low GLI2 expression (L group, n = 145) was associated with longer mOS (H: 25.6 mo vs M: 33.9 mo vs L: 33.7 mo; p= 0.0016). Moreover, IHH showed predictive value for cet benefit in both OS (mOS H: 38.1 mo vs M: 34.0 mo vs L: 18.4 mo; p= 0.0032) and PFS (mPFS H: 12.6 mo vs M: 12.9 mo vs L: 8.8 mo; p= 0.0025) with significant treatment interaction for OS and PFS (p= 0.0046 and p= 0.0049, respectively; FDR< 0.05). No significant associations were found in bev treated pts. No other Hh genes showed significant prognostic or predictive effects after FDR correction. Conclusions: These results highlight a dual role of Hh pathway in mCRC. Consistent with prior preclinical evidence, reduced IHH expression appears to mark poor prognosis, likely reflecting the loss of IHH-mediated restraint on Wnt signaling, which drives proliferation and dedifferentiation. Conversely, low GLI2, a downstream transcription factor, was linked with favorable outcomes, underscoring context-specific effects of Hh signaling. The strong predictive association of IHH with cet efficacy supports its potential as a biomarker in RAS wild type mCRC. These results suggest that Hh pathway modulation, particularly IHH signaling, may influence disease biology and treatment response. Validation in independent cohorts and mechanistic studies is warranted to clarify implications.

179 Background: Neuroligins are a family of postsynaptic cell adhesion molecules that play a crucial role in synapse formation and function. Recent studies suggest Neuroligin family genes (NLGN1,2,3,4X) may promote cancer cell growth and metastasis in various cancers, including colorectal cancer. Here, we investigated the association between neuroligin family gene expression and first-line treatment outcomes in mCRC. Methods: We evaluated the expression of 4 genes in the neuroligin family (NLGN1, NLGN2, NLGN3 and NLGN4X) on treatment outcomes of 433 pts with mCRC treated with either bevacizumab (Bev, n = 226) or cetuximab (Cet, n = 207) in combination with first-line chemotherapy within the CALGB/SWOG 80405 trial. mRNA was isolated from FFPE tumor samples and sequenced on the HiSeq 2500 (Illumina). Overall survival (OS) and progression-free survival (PFS) were compared between groups of pts categorized by low expression (Q1) (≤25 percentile), medium expression (Q2-3) (25th-75th percentile) and high expression (Q4)(>75th percentile) of NLGN family genes. The associations between gene expression and clinical outcomes were assessed via the multivariable Cox model and plotted with Kaplan-Meier curve. Treatment-gene interactions were evaluated using the likelihood ratio test. False discovery rate (FDR) was controlled through the Benjamini-Hochberg procedure. Results: High NLGN2 expression was associated with shorter PFS (median 10.0 mos, 95% CI (8.3, 11.3)) and OS (median 25.2mos, 95%CI (20.9, 30.0)) compared to low NLGN2 expression (PFS: median 11.2mos, 95% CI (9.8,14.3))(HR 1.41, 95% CI (1.06, 1.89)), P = 0.029). (OS: (median 31.8mos, 95%CI (26.8, 37.5)( HR 1.49, 95% CI (1.10, 2.01) P =0.0095) in multivariate analysis. Patients treated in the cetuximab arm(n=207), high HLGN2 expression significantly associated with shorter PFS (median 8.0 vs 13.4, HR 2.02, 95% CI (1.35, 3.03), P = 0.0021) and OS (median19.6 vs 36.3, HR 2.30, 95% CI (1.50, 3.53), P =8.3e-05). High NLGN3 expression was associated with shorter OS (23.9 vs 36.0 mos, P =0.0083) in cetuximab treatment arms, no significance found in PFS. We found significant treatment interaction for OS with NLGN2 and NLGN3 favoring cetuximab. In pts treated with cetuximab, High NLGN2 expression was significantly associated with shorter OS (median 29.1vs 36.3) (p=0.024) compared to pts treated with bevacizumab. For NLGN3, pts treated with cetuximab, high NLGN3 expression was significantly associated with shorter OS (median 25.0 vs 36.0) (p=0.018) compared to pts treated with bevacizumab. No significant association was found in NLGN1 and NLGN4X. Conclusions: Our results suggest NLGN2/3 gene expression may predict mCRC treatment outcomes. Potential therapeutic drug target NLGN in combination with anti-EGFR treatment in mCRC is warranted.