Molecular Subtypes of Primary SCLC Tumors and Their Associations With Neuroendocrine and Therapeutic Markers

Abstract

Introduction: A new molecular subtype classification was recently proposed for SCLC. It is necessary to validate it in primary SCLC tumors by immunohistochemical (IHC) staining and define its clinical relevance.

Methods: We used IHC to assess four subtype markers (ASCL1, NEUROD1, POU2F3, and YAP1) in 194 cores from 146 primary SCLC tumors. The profiles of tumor-associated CD3⁺ and CD8⁺ T-cells, MYC paralogs, SLFN11, and SYP were compared among different subtypes. Validation was performed using publicly available RNA sequencing data of SCLC.

Results: ASCL1, NEUROD1, POU2F3, and YAP1 were the dominant molecular subtypes in 78.2%, 5.6%, 7%, and 2.8% of the tumors, respectively; 6.3% of the tumors were negative for all four subtype markers. Notably, three cases were uniquely positive for YAP1. Substantial intratumoral heterogeneity was observed, with 17.6% and 2.8% of the tumors being positive for two and three subtype markers, respectively. The non-ASCL1/NEUROD1 tumors had more CD8⁺ T-cells and manifested more frequently an "inflamed" immunophenotype. L-MYC and MYC were more often associated with ASCL1/NEUROD1 subtypes and non-ASCL1/NEUROD1 subtypes, respectively. SLFN11 expression was absent in 40% of the tumors, especially those negative for the four subtype markers. SYP was often expressed in the ASCL1 and NEUROD1 subtypes and was associated with less tumor-associated CD8⁺ T-cells and a "desert" immunophenotype.

Conclusions: We validated the new molecular subtype classification in primary SCLC tumors by IHC and identified several intriguing associations between subtypes and therapeutic markers. The new subtype classification may potentially assist treatment decisions in SCLC.

Keywords: CD8(+) T-cells; Intratumoral heterogeneity; L-MYC; MYC; SLFN11; Synaptophysin.

Figure 1.. Intertumoral and intratumoral heterogeneity of molecular subtype markers in primary SCLC tumors.

A) Representative images of IHC staining in primary SCLC tumors positive for one single molecular subtype marker. B) The pie chart showing the percentage of each dominant molecular subtype tumor (Total=142). Four tumors were excluded in this analysis: one was lost because of detachment during IHC staining; three had discrepancies in molecular subtype classification between different cores of the same tumors. C) The pie chart showing the percentages of the SCLC tumors (N=142) positive for none, single, or multiple molecular subtype marker(s). D) A case with spatially separated populations of tumor cells positive for either ASCL1 (dominant) or NEUROD1. The arrowheads mark an island of tumor cells that were negative for ASCL1 but positive for NEUROD1. E) A case with spatially separated populations of tumor cells positive for either POU2F3 (dominant) or YAP1. F) A case with spatially separated populations of tumor cells either dually positive for ASCL1 (dominant) and NEUROD1 (distal from the cleft) or singly positive for YAP1 (adjacent to the cleft). Scale bar: 40 μM.

Figure 2.. Profiles of tumor-associated CD8⁺ T-cells in different molecular subtypes of SCLC.

A) Comparison of CD8⁺ T-cell counts among the SCLC tumors grouped by dominant molecular subtypes. Red horizontal bars represent medians. B) Comparison of CD8⁺ T-cell counts among the tumors positive for single or multiple molecular subtype markers. ‘NEUROD1 + other(s)’ and ‘YAP1 + other(s)’ groups had one single case in each group and were not shown. The Mann-Whitney test was used to assess the statistical significance of two-group comparison in (A–B). C) Representative images of three different CD8⁺ T-cell immunophenotypes in primary SCLC tumors. Black dotted lines mark the boundary between tumors cells and their neighboring stromal cells, and orange dotted lines show the margins of the nearby groups of tumor cells. Scale bar: 40 μM. D) A breakdown of CD8⁺ T-cell immunophenotypes in all tumors (N=142). Four tumors were excluded in this analysis: one tumor was lost because of detachment during IHC staining; three tumors had a discrepancy in CD8⁺ T-cell immunophenotype classification between different cores of the same tumors. E) Comparison of dominant molecular subtype composition (ASCL1/NEUROD1 subtypes versus non-ASCL1/NEUROD1 subtypes) among three different CD8⁺ T-cell immunophenotypes. Statistical significance was calculated using Fisher exact test. Where indicated, ns, not significant; *, P<0.05; **, P<0.01.

Figure 3.. Expression patterns of MYC paralogs in various molecular subtypes of SCLCs.

A) Representative images of the tumors stained positive for MYC, L-MYC, and/or N-MYC. Scale bar: 40 μM. B) The piechart showing the percentages of the SCLC tumors predominantly positive for MYC, L-MYC, or N-MYC. Two tumors were excluded because of discrepancies in MYC paralogue classification between multiple cores of the same tumors. All-negative: negative for all three MYC paralogs. C) The piechart showing the percentages of the tumors positive for a single MYC paralogue or multiple MYC paralogs. D) The molecular subtype composition in the SCLC tumors that were predominantly positive for MYC. E) MYC positively correlated with POU2F3 in the 81 SCLC tumors from the George dataset. F) The molecular subtype composition of the SCLC tumors that were predominantly positive for L-MYC. G) MYCL positively correlated with ASCL1 in the SCLC tumors from the George dataset. H) The molecular subtype composition of the SCLC tumors that were negative for all MYC paralogs. Two tumors were excluded because of discrepancies in molecular subtype classification between multiple cores of the same tumors. I) A breakdown of MYC paralogue classification in the tumors of single or multiple molecular subtypes. Statistical significance was assessed using a chi-square test.

Figure 4.. Expression patterns of SLFN11 in various molecular subtypes of SCLCs.

A) Representative images of SLFN11 staining at different H-scores. Far-right panel: The black dotted line marks the boundary between tumors cells and their neighboring stromal cells. Scale bar: 40 μM. B) A histogram showing frequency distribution of the SCLC tumors across the whole spectrum of SLFN11 H-score in an interval (bin width) of 50. C) Comparison of SLFN11 H-scores among the SCLC tumors grouped by dominant molecular subtypes. Red horizontal bars represent medians. D) Comparison of SLFN11 H-score among the SCLC tumors positive for single or multiple molecular subtype markers. The Mann-Whitney test was used to calculate statistical significances in (C) and (D). Where indicated, *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. E) t-SNE feature plots with select tumor cell populations of MDA-SC4 CDX (treatment naïve) highlighted as specified. Left panel, SLFN11-positive fraction (red; SLFN11⁺) and SLFN11-negative fraction (blue; SLFN11⁻) were highlighted in the tumor cells expressing one of the four molecular subtype markers (NAPY⁺); right panel, SLFN11⁺ fraction (dark red) and SLFN11⁻ fraction (green) in the tumor cells lacking expression of the four subtype markers (NAPY⁻). F) Comparison of SLFN11 (top), INSM1 (second from the top), CKB (second from the bottom), and ASCL1 (bottom) expression in the NAPY⁺ and NAPY⁻ tumor cells of MDA-SC4 CDX (treatment-naïve). Right-side y-axis shows the scale of gene expression.

Figure 5.. Associations between NE marker synaptophysin and molecular subtype markers, MYC paralogs, or SLFN11.

A) SYP H-scores were compared among the tumors grouped by dominant molecular subtypes. B) Comparison of SYP H-scores among the tumors positive for single or multiple molecular subtype markers. C) The CD8⁺ T-cell counts (log10-transformed) were compared between the SCLC tumors positive and negative for SYP. D) Comparison of the CD8⁺ T-cell counts (log10-transformed) among the SCLC tumors negative for SYP, those with low SYP H-scores (1–150), and those with high SYP H-scores (151–300). E) The CD8⁺ T-cell immunophenotype composition was compared between the tumors positive and negative for SYP. F) Comparisons of CD8⁺ T-cell immunophenotype compositions in the tumors grouped by SYP H-score. G) Comparisons of MYC paralogue compositions in the tumors grouped by SYP H-score. H) Comparison of SLFN11 H-scores in the tumors grouped by SYP H-score. The statistical significances between two specified groups were calculated using the Mann-Whitney test in (A–B), Student t-test in (C–D, H), and chi-square test in (E–G). Where indicated, ns, not significant; *, P<0.05; ****, P<0.0001.