Background While the administration of tyrosine-kinase inhibitors (TKIs) in ALK rearranged non-small cell lung cancer (NSCLC) has revolutionized precision medicine, detection of gene rearrangements from liquid biopsies remains challenging. RNA-based detection has demonstrated promising sensitivity for rearrangement detection and thus we hypothesize that a liquid biopsy assay analyzing circulating-tumor RNA (ctRNA) in addition to circulating-tumor DNA (ctDNA) will improve detection. Furthermore, we hypothesize that detection of gene fusions at baseline will correlate with clinical outcome. Methods We retrospectively analyzed 86 plasma samples from 33 patients enrolled in the BRIGHTSTAR clinical trial assessing local consolidative therapy (LCT) and brigatinib in patients with stage IV or recurrent NSCLC and confirmed ALK rearrangement (NCT03707938) using a targeted NGS assay that analyzes ctDNA to detect gene rearrangements and mutations in 80 genes and ctRNA to detect gene arrangements in 36 genes. Results ALK rearrangements were detected in 15/28 patients (54%) at baseline, of which eight were detected in both ctDNA and ctRNA. ALK rearrangements were detected in two patients pre-LCT, exclusively in ctRNA, but cleared completely post-LCT. The detection of ALK fusion at baseline was associated with significantly worse progression-free survival (p = 0.033). Plasma cell-free DNA (cfDNA) concentrations for patients with detectable ALK rearrangements at baseline were significantly higher than for those without detectable gene fusions (12.3ng/ml versus 20.2ng/ml; p = 0.0046). Conclusions The inclusion of ctRNA in liquid biopsies increased detection of ALK rearrangements and detection at baseline was associated with significantly worse progression-free survival highlighting the added benefit of ctRNA.

A hallmark of small cell lung cancer (SCLC) is its recalcitrance to therapy. While most SCLCs respond to frontline therapy, resistance inevitably develops. Identifying phenotypes potentiating chemoresistance and immune evasion is a crucial unmet need. Previous reports have linked upregulation of the DNA damage response (DDR) machinery to chemoresistance and immune evasion across cancers. However, it is unknown if SCLCs exhibit distinct DDR phenotypes. To study SCLC DDR phenotypes, we developed a new DDR gene analysis method and applied it to SCLC clinical samples, in vitro, and in vivo model systems. We then investigated how DDR regulation is associated with SCLC biology, chemotherapy response, and tumor evolution following therapy. Using multi-omic profiling, we demonstrate that SCLC tumors cluster into three DDR phenotypes with unique molecular features. Hallmarks of these DDR clusters include differential expression of DNA repair genes, increased replication stress, and heightened G2/M cell cycle arrest. SCLCs with elevated DDR phenotypes exhibit increased neuroendocrine features and decreased “inflamed” biomarkers, both within and across SCLC subtypes. Clinical analyses demonstrated treatment naive DDR status was associated with different responses to frontline chemotherapy. Using longitudinal liquid biopsies, we found that DDR Intermediate and High tumors exhibited subtype switching and coincident emergence of heterogenous phenotypes following frontline treatment. We establish that SCLC can be classified into one of three distinct, clinically relevant DDR clusters. Our data demonstrates that DDR status plays a key role in shaping SCLC phenotypes and may be associated with different chemotherapy responses and patterns of tumor evolution. Future work targeting DDR specific phenotypes will be instrumental in improving patient outcomes.

Current treatment guidelines refer to small cell lung cancer (SCLC), one of the deadliest human malignancies, as a homogeneous disease. Accordingly, SCLC therapy comprises chemoradiation with or without immunotherapy. Meanwhile, recent studies have made significant advances in subclassifying SCLC based on the elevated expression of the transcription factors ASCL1, NEUROD1, and POU2F3, as well as on certain inflammatory characteristics. The role of the transcription regulator YAP1 in defining a unique SCLC subset remains to be established. Although preclinical analyses have described numerous subtype-specific characteristics and vulnerabilities, the so far non-existing clinical subtype distinction may be a contributor to negative clinical trial outcomes. This comprehensive review aims to provide a framework for the development of novel personalized therapeutic approaches by compiling the most recent discoveries achieved by preclinical SCLC research. We highlight the challenges faced due to limited access to patient material as well as the advances accomplished by implementing state-of-the-art models and methodologies.

Abstract Small-cell lung cancer (SCLC) remains the deadliest histologic lung cancer subtype with a median survival of only 12.3 months among trial-eligible patients with extensive-stage (ES-SCLC) disease, as demonstrated in the IMPower133 trial. Heavy tobacco use, >40 pack-years, is strongly associated with developing SCLC, which near universally harbors RB1 and TP53 co-mutations. However, there is a paucity of information regarding SCLC tumors that occur in patients with minimal tobacco use. This represents an unmet need given an increasing appreciation for low pack-year and never-smoker SCLC with some studies, like the CAPSTONE-1 trial, demonstrating an incidence of never-smoking SCLC >20%. To address this, we clinically characterized the treatments and outcomes of a 164 SCLC patient cohort with ≤10 pack year smoking histories treated at the University of Texas MD Anderson Cancer Center; 22 of which underwent whole-exome sequencing (WES). The overall survival of limited-stage (LS-SCLC) and extensive-stage (ES-SCLC) disease in the low pack-year cohort were 33.6 and 16.6 months, respectively (P=0.003); superior to historic medians of roughly 17.0 and 12.3 months, respectively. Among those with ES-SCLC, those who reported 1-10 pack-years had significantly improved median overall survival as compared to never-smokers; 19.5 vs 15.8 months, respectively (P=0.02). This trend was observed in LS-SCLC as well with 1-10 pack-year and never-smokers demonstrating median overall survivals of 88.6 vs 26.2 months, respectively (P=0.2). Demographic differences including age were similar across these cohorts. WES of tumors from 22 patients demonstrated significantly fewer mutations in RB1 and TP53 than was observed in previously reported cohorts of SCLC from smokers. Among the low-pack year cohort, 7/22 (32% vs 98.2%:P<0.001) harbored an RB1 alteration, 7/22 (32% vs 90.9%: P<0.001) harbored a TP53 mutation, and 4/22 (18% vs 90.9%: P<0.001) had co-occurring RB1/TP53 mutations. No patients harbored mutations in EGFR, HER2, or RET. Mutations in potentially targetable pathways were observed throughout the cohort including the DNA-damage repair pathways, RTK/RAS, and PI3K. Taken together, these data demonstrate that patients with low pack-year SCLC have improved overall survival compared to historic averages for SCLC patients with a history of heavy tobacco use. Paradoxically, however, light-smokers with SCLC live longer than never-smokers, and these patients may harbor a higher frequency of targetable alterations than typical SCLC patients, highlighting the importance of molecular profiling in SCLC patients with minimal tobacco use. Citation Format: Kyle Concannon, Moushumi Sahu, Whitney Lewis, Hai Tran, Lixia Diao, Yuanxin Xi, Benjamin Morris, Simon Heeke, Ximing Tang, John Stewart, Ivan Valiev, Gabriela Raso, Jing Wang, Koji Sasaki, Carl Gay, John Heymach, Lauren Byers. Improved survival and unique mutational signatures in small cell lung cancer arising in patients with limited tobacco use [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5061.

8088 Background: SCLC is an aggressive cancer with a poor prognosis. B7-H3, a transmembrane protein showing low expression in normal tissues but overexpression in SCLC and other tumors, is a promising target for antibody–drug conjugates. We performed a multidimensional analysis of B7-H3/ CD276 expression in SCLC molecular subtypes (SCLC-A, -N, -P, and -I) and its relationship with expression of other immune-related genes. Methods: Clinical and molecular data for a cohort of patients (pts) with SCLC were derived from a real-world database (Caris Life Sciences, Irving, TX, USA). Tumor RNA expression was used to assign the SCLC molecular subtype derived from non-negative matrix factorization. Programmed death ligand-1 (PD-L1) protein expression was assessed by immunohistochemistry (IHC; antibody 22c3; positive cut-off: tumor proportion score >1%). Demographic, clinical, and molecular data were summarized by SCLC subtype or B7-H3 expression quartile (Q). Spearman’s r evaluated correlations between expression of B7-H3 and other immune-related genes/signatures. Results: The cohort included 1721 pts (52.5% female; 98.9% [434/439] smokers; median age, 67 years [range, 18–90+]). Across the subtypes, B7-H3 expression was high and remarkably consistent ( q>0.05; Table). Other biomarker genes, including DLL3 (q<0.05), had lower and more variable expression. Median (range) B7-H3 expression across all samples was 16.75 (0–68.2) transcripts per million (TPM). Across B7-H3 expression Qs, the proportion of PD-L1-positive pts by IHC was similar (Q1, 39.8%; Q2, 46.5%; Q3, 40.7%; Q4, 39.2%). Median (95% confidence interval) B7-H3 expression was similar between pts with prior immunotherapy ( n=24; 14.43 [11.64–21.17] TPM) and pts without ( n=208; 14.20 [13.00–15.91] TPM); treatment information was not available for the remaining samples in this cohort. B7-H3 expression was not strongly correlated with any other immune-related genes/signatures, although several had moderate correlation ( HAVCR2/ TIM3, r=0.59; PDCD1LG2/ PD-L2, r=0.56; M2 macrophages, r=0.56; CD86, r=0.56). Conclusions: In pts with SCLC, B7-H3 showed high and consistent expression across subgroups defined by molecular subtype or prior immunotherapy. The relative expression of B7-H3 was higher and less variable among molecular subtypes than for other key molecular targets in this tumor type, including DLL3. B7-H3 expression had limited association with PD-L1 expression, supporting a role as a distinct therapeutic target. [Table: see text]

Activated T cells undergo a metabolic shift to aerobic glycolysis to support the energetic demands of proliferation, differentiation, and cytolytic function. Transmembrane glucose flux is facilitated by glucose transporters (GLUT) that play a vital role in T cell metabolic reprogramming and anti-tumour function. GLUT isoforms are regulated at the level of expression and subcellular distribution. GLUTs also display preferential selectivity for carbohydrate macronutrients including glucose, galactose, and fructose. GLUT5, which selectively transports fructose over glucose, has never been explored as a genetic engineering strategy to enhance CAR-T cells in fructose-rich tumour environments. Fructose levels are significantly elevated in the bone marrow and the plasma of acute myeloid leukaemia (AML) patients. Here, we demonstrate that the expression of wild-type GLUT5 restores T cell metabolic fitness in glucose-free, high fructose conditions. We find that fructose supports maximal glycolytic capacity and ATP replenishment rates in GLUT5-expressing T cells. Using steady state tracer technology, we show that 13C6 fructose supports glycolytic reprogramming and TCA anaplerosis in CAR-T cells undergoing log phase expansion. In cytotoxicity assays, GLUT5 rescues T cell cytolytic function in glucose-free medium. The fructose/GLUT5 metabolic axis also supports maximal migratory velocity, which provides mechanistic insight into why GLUT5-expressing CAR-Ts have superior effector function as they undergo "hit-and-run" serial killing. These findings translate to superior anti-tumour function in a xenograft model of AML. In fact, we found that GLUT5 enhances CAR-T cell anti-tumour function in vivo without any need for fructose intervention. Accordingly, we hypothesize that GLUT5 is sufficient to enhance CAR-T resilience by increasing the cells' competitiveness for glucose at physiologic metabolite levels. Our findings have immediate translational relevance by providing the first evidence that GLUT5 confers a competitive edge in a fructose-enriched milieu, and is a novel approach to overcome glucose depletion in hostile tumour microenvironments (TMEs).

EGFR status assessment is mandatory for adjuvant decision-making of resected stage IB-IIIA non-squamous non-small cell lung cancer (NS-NSCLC). It is questionable whether single-gene RT-PCR versus next-generation sequencing (NGS) should be used for this evaluation. Moreover, co-occurring mutations have an impact on tumor behavior and may influence future therapeutic decision-making. We aimed to describe the clinico-pathological and molecular features, as well as the prognostic factors of resected EGFR-mutant NS-NSCLC evaluated with reflex NGS and RT-PCR, so as to compare the results of the two methods. We retrospectively included and collected data from patients with resected EGFR-mutant NS-NSCLC diagnosed in our institution between 2005 and 2024. Additional cases from another center were included. Tumors were analyzed using targeted NGS and RT-PCR. A total of 153 patients were selected. The median follow-up after surgery was 22 months. The positive percent agreement of RT-PCR compared to NGS for the detection of an EGFR mutation was 88

Navigating change in tumor naming. Balance organ-based and molecular classifications for optimal treatment.

ERBB2 (HER2) represents a newly recognized actionable oncogenic driver in non-small cell lung cancer (NSCLC), with approved targeted therapy available. Understanding the landscape of ERBB2 alterations and co-occurring mutations is essential for guiding treatment decisions. We conducted an analysis involving 3000 NSCLC patients with all types of ERBB2 alterations, drawn from two extensive retrospective cohorts: 1281 from Geneplus (Chinese) and 1719 from Guardant360 (the United States, US). The incidence of all types of ERBB2 alterations was found to be 5.6% in the Chinese group and 5.2% in the US group. In both cohorts, among oncogenic alterations of ERBB2, exon 20 insertion Y772_A775dupYVMA was the most frequent alteration (58% vs 41.6% in the Chinese vs the US), followed by G776delinsVC/LC/VV/IC (10.7% vs 9.7%), and S310X (10.5% vs 15.4%). EGFR ex20 insertions were identified in the A767-V774 region, whereas ERBB2 ex20 insertions were observed in the Y772-P780 region. Notably, EGFR ex20 insertions exhibited greater insertion diversity. Clinical characteristics of EGFR and ERBB2 ex20 NSCLC were similar, characterized by low tumor mutation burden (TMB), a predominant never-smoker population, and a majority of lung adenocarcinoma cases.

Introduction NRG1 gene fusions are clinically actionable alterations identified in NSCLC and other tumors. Previous studies have reported that NRG1 fusions signal through HER2 and HER3 but, thus far, strategies targeting HER3 specifically or HER2-HER3 signaling have exhibited modest activity in patients with NSCLC bearing NRG1 fusions. Although NRG1 fusion proteins can bind HER4 in addition to HER3, the contribution of HER4 and other HER family members in NRG1 fusion-positive cancers is not fully understood. Methods We investigated the role of HER4 and EGFR-HER3 signaling in NRG1 fusion-positive cancers using Ba/F3 models engineered to express various HER family members in combination with NRG1 fusions and in vitro and in vivo models of NRG1 fusion-positive cancer. Results We determined that NRG1 fusions can stimulate downstream signaling and tumor cell growth through HER4, independent of other HER family members. Moreover, EGFR-HER3 signaling is also activated in cells expressing NRG1 fusions, and inhibition of these receptors is also necessary to effectively inhibit tumor cell growth. We observed that cetuximab, an anti-EGFR antibody, in combination with anti-HER2 antibodies, trastuzumab and pertuzumab, yielded a synergistic effect. Furthermore, pan-HER tyrosine kinase inhibitors were more effective than tyrosine kinase inhibitors with greater specificity for EGFR, EGFR-HER2, or HER2-HER4, although the relative degree of dependence on EGFR or HER4 signaling varied between different NRG1 fusion-positive cancers. Conclusions Our findings indicate that pan-HER inhibition including HER4 and EGFR blockade is more effective than selectively targeting HER3 or HER2-HER3 in NRG1 fusion-positive cancers.

Mutations (single nucleotide variants), deletions, and insertions in the kinase domain of EGFR gene are oncogenic drivers in NSCLC. These alterations can co-occur with one another (compound mutations) or exist as single mutations. The compound vs single mutation frequencies for classical and EGFR P-loop and αC-helix compressing (PACC) mutations have not been characterized and the impact of compound mutations on treatment outcomes are not understood. Guardant Health liquid biopsy database was queried for NSCLC samples for their genetic profiling. Incidences EGFR mutations as single and compound mutations (excluding T790M and C797S) were analyzed and compared. A systematic literature search was performed and analyzed for esponse to different TKIs. Of the 104,393 lung cancer samples queried, 32,700 had an EGFR SNV/indel, of which, 17,488 (16.8%) had at least one SNV/indel within the kinase domain. The most frequent mutations were ex19del (6,670, 38.1%), L858R (4,700, 26.9%), G719 (712, 4.1%), S768 (335, 1.9%), and G709 (207, 1.2%). Classical EGFR mutations occur more frequently as a single mutation, each at 89.6% (5,970/6,665) and 76.7% (3,606/4,700) respectively. In comparison, PACC mutations occur more frequently as compound mutations (Table), G719 at 73.2% (521/712), S768 at 86.9% (291/335) and G709 at 97.1% (201/207). The most frequent compound mutation with G719 is G709 and S768, with S768 is V769, and with G709 is G719. A total of 852 cases with clinical response to EGFR TKIs from a clinical cohort were identified and analyzed by PACC mutation single (693) vs compound (159) (Table). As expected, PACC mutations responded better to 2nd-generation TKIs than 1st- or 3rd-generation TKIs. Compound mutations responded better than single mutations for each PACC mutation.Table: 24PPACC mutationsGuardant360Retrospective clinical response dataCase #PercentFirst-gen TKISecond-gen TKIThird-gen TKIORRNORRNORRNG719Single19126.8%39%31056%24833%36Compound52173.2%42%5277%7759%27S768Single4413.1%31%2948%4633%9Compound29186.9%54%1163%5259%17G709Single62.9%0%550%650%4Compound20197.1%43%762%1683%6 Open table in a new tab Compared to classical EGFR mutations, PACC mutations frequently occur as compound mutations, and tend to have improved response to EGFR TKIs than single PACC mutations.

While we recognize the prognostic importance of clinicopathological measures and circulating tumor DNA (ctDNA), the independent contribution of quantitative image markers to prognosis in non-small cell lung cancer (NSCLC) remains underexplored. In our multi-institutional study of 394 NSCLC patients, we utilize pre-treatment computed tomography (CT) and 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) to establish a habitat imaging framework for assessing regional heterogeneity within individual tumors. This framework identifies three PET/CT subtypes, which maintain prognostic value after adjusting for clinicopathologic risk factors including tumor volume. Additionally, these subtypes complement ctDNA in predicting disease recurrence. Radiogenomics analysis unveil the molecular underpinnings of these imaging subtypes, highlighting downregulation in interferon alpha and gamma pathways in the high-risk subtype. In summary, our study demonstrates that these habitat imaging subtypes effectively stratify NSCLC patients based on their risk levels for disease recurrence after initial curative surgery or radiotherapy, providing valuable insights for personalized treatment approaches. Predicting recurrence risk in non small cell lung cancer can help to guide treatment decisions. Here, the authors use CT and PET imaging to develop predictive imaging subtypes, which can be integrated with existing ctDNA methods to predict recurrence.

6104 Background: In the phase II axitinib (VEGFR inhibitor) plus avelumab (PD-L1 inhibitor) trial in R/M ACC, response rate was 18% with a median progression-free survival (PFS) of 7.3 months, with subsequent inclusion of the combination in NCCN guidelines as a possible treatment for ACC (category 2B). We sought to identify potential biomarkers predictive of axitinib plus avelumab benefit in R/M ACC. Methods: The phase II trial included 28 R/M ACC patients (pts). Pre-treatment tumors were analyzed by whole exome sequencing (n=24), using Twist Human Core Exome V2 kit, and transcriptome profiling (n=26), using HTG Transcriptome Panel (19,616 probes). For 17 pts, imaging mass cytometry (IMC) was performed with 35 metal-tagged markers and analyzed with Visiopharm software. Microbiome composition was assessed in tumor (n=20), oral rinse (n=20) and fecal (n=19) samples via 16s rRNA gene sequencing. All results were assessed for association with PFS on therapy with a Cox proportional hazards model, maintaining ACC subtype (ACC-I vs. ACC-II) as a covariate. Results: Only 1 (6%) of 17 assessed tumors were PD-L1 positive. By WES, tumor mutational burden (TMB) was overall low (median 1.4 mut/Mb, range 0.7 – 18.7), but higher TMB was associated with worse PFS. Four mutational signatures significantly correlated with worse PFS, including SBS86, associated with previous chemotherapy exposure. Immune deconvolution of RNA-seq data revealed that a higher neutrophil-to-lymphocyte ratio and more T follicular helper cells associated with worse PFS, while presence of cytotoxic cells, T effector memory cells, and mast cells associated with longer PFS. Notably, a 167-gene predictive signature was developed and validated in the phase II ipilimumab plus nivolumab salivary gland cancer trial. This 167-gene signature was not prognostic in ACC pts who did not receive immunotherapy. IMC single-cell immune mapping revealed that pts with longer PFS had increased presence of CD8 T cells, CD73+ macrophages in tumor, a higher stromal CD8 T cell:Regulatory T cell ratio, increased stromal SIGLEC15 positive cells, and a higher tumor-to-stroma ratio of fibroblasts. Conversely, Ki67-positive T cells, cancer stem cells, and M2 macrophages were associated with worse PFS. Through microbiome analysis, 13 bacterial genera in tumor samples, 25 in stool, and 8 in oral rinse were significantly associated with therapy outcomes. Of note, in stool microbiome, presence of Akkermansia spp. and Bifidobacterium spp. were significantly correlated with improved PFS, with the latter association also seen in oral rinse. Conclusions: Correlative analysis of the phase II axitinib plus avelumab trial revealed potential biomarkers predictive of combination clinical benefit in ACC, including a gene-expression signature. These findings may guide patient stratification for combinatorial therapy.

In preclinical studies, EGFR TKI resistant cells displayed vulnerabilities to aurora kinase inhibitors. LY3295668 is an oral, selective aurora kinase A inhibitor with monotherapy RP2D at 25mg twice-daily (BID). The most common adverse events (AEs) were mucositis, diarrhea and corneal deposition. This phase I/II trial (NCT05017025) was designed to evaluate the safety and establish osimertinib combination RP2D in EGFR-mutant NSCLC patients (pts); and preliminarily evaluate the clinical efficacy of the combination in pts whose tumors progress on osimertinib. Pts with NSCLC harboring EGFR mutations and progressed on EGFR TKIs were enrolled. Up to 3 prior lines of therapies were allowed. LY3295668 was administered at 25mg BID with osimertinib 80mg daily. The primary endpoint for safety run-in was DLT; co-primary endpoints for the efficacy cohort were progression-free survival (PFS) at 6 months and best objective response rate (BRR). 30 pts were enrolled (Mar 2022 - Feb 2023) and received at least one dose of treatment (intent-to-treat [ITT] cohort); 27 pts had at least one efficacy scan (efficacy cohort). Median age in ITT was 60, 77% female, 60% whites, 27% Asians. In the safety (n=10) and the entire cohort, there was no DLT. The RP2D was determined at LY3295668 25mg BID plus osimertinib 80mg daily. In the efficacy cohort (n=27), 6-month PFS rate was 37% (10/27). Three partial responses (PRs, 11%), 17 stable diseases (SD, 63%) and 7 progressive diseases (PD, 26%) were observed, with a disease control rate (DCR 74%). At the data cut-off on 12/12/2023, 5 pts were still on treatment. In the ITT cohort, 100% of pts had any AEs. Treatment-related AEs (TRAEs) were 80%, including 3% grade 4 (n=1, thrombocytopenia), 13% grade 3 (thrombocytopenia, lymphopenia, neutropenia, diarrhea, abdominal pain), and 63% grade 1-2. The most common TRAEs are diarrhea (73%), thrombocytopenia (60%), anemia (47%) and elevated transaminases (47%). The combination of LY3295668 25mg BID plus osimertinib 80mg daily was found to be safe in EGFR-mutant NSCLC pts. The combination yields a 6-month PFS rate of 37% with 11% PR and 63% SD, demonstrating moderate clinical efficacy.

Studying lung adenocarcinoma (LUAD) early carcinogenesis is challenging, primarily due to the lack of LUAD precursors specimens. We amassed multi-omics data from 213 LUAD and LUAD precursors to identify molecular features underlying LUAD precancer evolution. We observed progressively increasing mutations, chromosomal aberrations, whole genome doubling and genomic instability from precancer to invasive LUAD, indicating aggravating chromosomal instability (CIN). Telomere shortening, a crucial genomic alteration linked to CIN, emerged at precancer stage. Moreover, later-stage lesions demonstrated increasing cancer stemness and decreasing alveolar identity, suggesting epithelial de-differentiation during early LUAD carcinogenesis. The innate immune cells progressively diminished from precancer to invasive LUAD, concomitant with a gradual recruitment of adaptive immune cells (except CD8+ and gamma-delta T cells that decreased in later stages) and upregulation of numerous immune checkpoints, suggesting LUAD precancer evolution is associated with a shift from innate to adaptive immune response and immune evasion mediated by various mechanisms.

Background: Several studies have reported the predictive and prognostic value of novel transcriptional factor-based molecular subtypes in small-cell lung cancer (SCLC). We conducted an in-depth analysis using multi-omics data to elucidate the underlying characteristics that lead to differences in clinical outcomes between subtypes. Patients and Methods: Immunohistochemistry (IHC, n=252), target exome sequencing (n=422), and whole transcriptome sequencing (WTS, n=189) data generated from 427 patients with SCLC patients were comprehensively analyzed. The differences in the mutation profile, gene expression profile were analyzed according to the IHC-based molecular subtype. Clinical implication was evaluated based on treatment outcomes of individual patients. Results: IHC based molecular subtyping revealed a high incidence of ASCL1 subtype (SCLC-A, 56.3%) followed by ASCL1/NEUROD1 co-expressed (SCLC-trans, 17.9%), NEUROD1 (SCLC-N, 12.3%), POU2F3 (SCLC-P, 9.1%), triple-negative (SCLC-TN, 4.4%) subtypes showing high concordance with WTS-based subtyping. We delineated the SCLC-trans subtype resembling SCLC-A rather than SCLC-N in terms of both gene expression profiles and clinical outcomes. SCLC-TN type was defined as non-significant expression of A, N, P. Favorable overall survival (OS) was observed in SCLC-A compared to SCLC-N (adjusted HR 2.4, 95% CI 1.5-3.9, p < 0.001) and SCLC-P (adjusted HR 1.7, 95% CI 0.9-2.9, p = 0.087). SCLC-TN showed a similar OS with SCLC-A (adjusted HR 1.2, 95% CI 0.6 -2.7, p = 0598). The clinical outcome based on inflamed phenotype, clustered by effector cell gene expression profile which are only found in 5% of SCLC-N but 60% of SCLC-P, was more likely to benefit from first-line immunotherapy treatment than non-inflamed phenotype (p = 0.013). Inflammed phenotype demonstrated longer progression-free survival to the first line immunotherapy compared to the non-inflammed phenotype. (10.5 vs 4.3, p = 0.013) Conclusions: This study provides fundamental data, including the incidence and basic demographics of molecular subtypes of SCLC using both IHC and WTS from a comparably large cohort including potential differences in the distribution of subtypes based on ethnicity. Additionally, our results reveal differences in the underlying biological pathway activities and immunogenicity based on molecular subtype, possibly related to the difference in clinical outcomes, including immunotherapy response. Citation Format: Sehhoon Park, Tae Hee Hong, Soohyun Hwang, Hyun-Ae Jung, Jong-Mu Sun, Jin Seok Ahn, Myung-Ju Ahn, Jong Ho Cho, Yong Soo Choi, Jhingook Kim, Young Mog Shim, Hong Kwan Kim, Yoon-La Choi, Se-hoon Lee, Keunchil Park. Comprehensive analysis using transcriptional factor based molecular subtypes and correlation to clinical outcomes in small-cell lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4546.

The identification of ALK fusions in advanced non-small-cell lung carcinoma (aNSCLC) is mandatory for targeted therapy. The current diagnostic approach employs an algorithm using ALK immunohistochemistry (IHC) screening, followed by confirmation through ALK FISH and/or next-generation sequencing (NGS). Challenges arise due to the infrequency of ALK fusions (3–7% of aNSCLC), the suboptimal specificity of ALK IHC and ALK FISH, and the growing molecular demands placed on small tissue samples, leading to interpretative, tissue availability, and time-related issues. This study investigates the effectiveness of RNA NGS as a reflex test for identifying ALK fusions in NSCLC, with the goal of replacing ALK IHC in the systematic screening process. The evaluation included 1246 NSCLC cases using paired techniques: ALK IHC, ALK FISH, and ALK NGS. ALK IHC identified 51 positive cases (4%), while RNA NGS detected ALK alterations in 59 cases (4.8%). Of the 59 ALK-positive cases identified via NGS, 53 (89.8%) were confirmed to be positive. This included 51 cases detected via both FISH and IHC, and 2 cases detected only via FISH, as they were completely negative according to IHC. The combined reporting time for ALK IHC and ALK FISH averaged 13 days, whereas ALK IHC and RNA NGS reports were obtained in an average of 4 days. These results emphasize the advantage of replacing systematic ALK IHC screening with RNA NGS reflex testing for a more comprehensive and accurate assessment of ALK status.

Small cell lung cancer (SCLC) is an aggressive malignancy composed of distinct transcriptional subtypes, but implementing subtyping in the clinic has remained challenging, particularly due to limited tissue availability. Given the known epigenetic regulation of critical SCLC transcriptional programs, we hypothesized that subtype-specific patterns of DNA methylation could be detected in tumor or blood from SCLC patients. Using genomic-wide reduced-representation bisulfite sequencing (RRBS) in two cohorts totaling 179 SCLC patients and using machine learning approaches, we report a highly accurate DNA methylation-based classifier (SCLC-DMC) that can distinguish SCLC subtypes. We further adjust the classifier for circulating-free DNA (cfDNA) to subtype SCLC from plasma. Using the cfDNA classifier (cfDMC), we demonstrate that SCLC phenotypes can evolve during disease progression, highlighting the need for longitudinal tracking of SCLC during clinical treatment. These data establish that tumor and cfDNA methylation can be used to identify SCLC subtypes and might guide precision SCLC therapy.

Abstract Background: Small cell lung cancer (SCLC) is the most lethal form of lung cancer. A key driver of this near universal lethality is SCLC’s recalcitrance to therapy. The standard of care for SCLC is a chemotherapy doublet of etoposide and cis/carboplatin in combination with immunotherapy (EP+IO). While most tumors respond to these therapies, >80% of SCLCs progress within one year of treatment. Additionally, the aggressive clinical course of SCLC has historically precluded collection of patient-matched treatment naïve and recurrent tumor tissue traditionally required to study evolution. In lieu of tissue, several groups have used circulating tumor DNA (ctDNA) to study SCLC evolution. However, these studies used targeted sequencing panels that analyze <0.04% of the SCLC genome. Using new methods to comprehensively trace how SCLCs rapidly evolve to therapy resistance is a significant unmet need. Methods: Medical records were reviewed to identify treatment naïve SCLC patients treated at MD Anderson that had both pre- and post-EP+IO liquid biopsies collected under IRB approved protocols. ctDNA was isolated from plasma using a MiniMax High Efficiency Cell-Free DNA Isolation Kit (Apostle Bio). Germline DNA was extracted from buffy coat samples using a QIAamp DNA Blood Midi Kit (Qiagen). Deep whole genome sequencing (WGS) (>100X) of ctDNA and WGS of germline DNA (>60X) was performed using an Illumina NovaSeq X sequencer. Reads were aligned to the hg38 reference genome using BWA-mem. Somatic mutations were called using MuTect2, and clonal and subclonal copy number alterations were identified using Battenberg. DPClust was used to reconstruct cancer cell populations, leveraging mutation and copy number calls. Results: We identified several deleterious genomic alterations impacting TP53 and RB1 tumor suppressor genes, known genetic drivers of SCLC development. We also detected recurrent, whole chromosome arm loss of heterozygosity events impacting many chromosomes in treatment naïve samples, some of which are known to be present in >80% of SCLCs. Additionally, we found that both treatment naïve and recurrent SCLCs were composed of multiple subclones, each defined by dozens to thousands of unique somatic mutations. COSMIC signature analysis identified high activity of tobacco carcinogens, APOBEC, and replicative-based processes across ctDNA samples. We also identified emergence of novel subclones carrying unique copy number alterations following EP+IO recurrence. Importantly, the affected regions encode key effectors that regulate anti-tumor immune responses and cell survival following DNA damage, both of which are intimately related to the mechanism of action of EP+IO. Conclusions: Our data shows that deep WGS of ctDNA recapitulates known genetics of SCLC and allows for sensitive characterization of therapy resistant clones. Using this method, we have captured snapshots of both intrinsic and acquired resistance mechanisms to frontline EP+IO. Deep WGS of ctDNA is a promising approach to comprehensively dissect routes of SCLC evolution and therapy resistance. Citation Format: Benjamin B. Morris, Zhihui Zhang, Simon Heeke, Kyle Concannon, Bingan Zhang, Waree Rinsurongkawong, Vadeerat Rinsurongkawong, J. Jack Lee, Jianjun Zhang, Don L. Gibbons, Ara A. Vaporciyan, Carl M. Gay, Hai T. Tran, John V. Heymach, Lauren A. Byers, Peter Van Loo. Dissecting patterns of small cell lung cancer evolution using deep whole genome sequencing of circulating tumor DNA [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A022.