Background: Transcriptomic subtyping holds promise for personalized therapy in SCLC, but intratumoral transcriptomic heterogeneity and its clinical significance remain poorly defined. In this study, we aimed to assess transcription factor defined subtypes within multiple regions of intact tissues and identify gen sets associated with long-term chemo-immunotherapy benefit. Methods: We assessed baseline FFPE tumors from 32 ES-SCLC patients enrolled in the IMfirst clinical trial (EudraCT: 2019-002784-10). We used GeoMx DSP to perform transcriptomic analysis from multiple intratumoral regions of interest (ROIs). We used an assay with +1800 genes (GeoMx CTA) plus custom-designed mRNA probes targeting subtype-defining transcription factors not included in the CTA assay (POU2F3, NEUROD1, and YAP1). Custom probes were quantitatively validated using FFPE cell lines. Results: We profiled 175 ROIs from 32 tumors. Cluster analysis based on the expression of ASCL1, NEUROD1, POU2F3, and YAP1 showed that all samples clustered within 4 groups: SCLC-A (76 ROIs [43%]), SCLC-N (31 ROIs [18%]), SCLC-P (18 ROIs [10%]), and SCLC-Y (50 ROIs [29%]). ASCL1 was the most abundantly expressed transcription factor, prevailed in the SCLC-A subtype, and showed inverse correlation with POU2F3 (r=-0.55, p<0.0001) and YAP1(r=-0.70, p<0.0001). YAP1 was expressed at low levels and was more broadly distributed across all 4 subtypes. Differential expression and gene set enrichment analysis (GSEA) using linear mixed models revealed that SCLC-A subtype was enriched in cell cycle and DNA damage repair genes, and showed repression of multiple gene sets associated with anti-tumor immune response. In contrast, SCLC-Y subtype showed the opposite pattern. The SCLC-P subtype was also enriched in genes related to cancer antigens and T-cell checkpoints. Most patients (n=18, 56%) had tumors with coexistence of more than one subtype, not evident through morphological inspection. Combined SCLC-A and SCLC-Y subtypes was the most common mixed phenotype (n=8, 25%). Four patients (13%) had tumors with co-existence of three subtypes. Transcriptional subtypes, subtype-defining transcription factors as single genes, or the presence of subtype heterogeneity, were not associated with outcomes. Gene sets involved in mitochondrial metabolism and MHC class I antigen presentation were the highest enriched pathways in tumors from patients with sustained benefit (time to progression ³ 12 months). Conclusions: This study reveals substantial intratumoral transcriptomic subtype heterogeneity in human SCLC. In this limited sample set, we did not observe outcome association for transcriptional subtypes. Our findings related to long-term chemo-immunotherapy benefit require validation in independent cohorts.