High Tumor Mutational Burden Research Articles

The genomic, transcriptomic, and immunologic landscape of TEM8 (ANTXR1) in small-cell lung cancer (SCLC).

3128 Background: The TEM8 receptor (coded by ANTXR1) plays several roles in oncogenesis. Novel oncolytic therapies, such as the SVV-01 virus, uniquely bind this protein in tumor histologies such as small-cell lung cancer (SCLC). Emerging pre-clinical data suggest that TEM8-targeting therapies may convert immunologically “cold” tumor microenvironments (TME) into “hot” milieu with greater responses to immune checkpoint inhibitors (ICIs). Methods: NextGen sequencing of DNA (592 genes or whole exome)/RNA (whole transcriptome) was performed on SCLC ( N=1404) submitted to Caris Life Sciences (Phoenix, AZ). Mutations were defined as pathogenic SNVs/indels. ANTXR1H and ANTXR1L samples included those with >75th and <25th percentile expression, respectively. PD-L1 expression (22c3; Positive (+):TPS ³1%) was assessed by IHC. High tumor mutational burden (TMB-H) was defined as ≥10 mutations per MB. Cell infiltration in the TME was estimated by QuantiSEQ. Gene expression profiles were analyzed for transcriptional signatures predictive of response to immunotherapy (T cell-inflamed) and MAPK pathway activation score (MPAS). Real-world median overall survival (mOS) was assessed from insurance claims data, and Kaplan-Meier estimates were calculated for molecularly defined subpopulations of patients. Mann-Whitney U and X2/Fisher-Exact tests were applied where appropriate, with P-values adjusted for multiple comparisons ( p<0.05). Results: No pathogenic mutations were significantly associated with ANTXR1H vs L tumors ( p>0.05 for all), along with no differences in the prevalence of TMB-H (28.6% vs 36.3%, p=1) or PDL1+ (45.0%: vs 35.0%, p=1). However, a greater proportion of B cells (6.53 % vs 5.12%, p<0.001), M1 (1.63% vs 0.37%, p<0.001) and M2 macrophages (3.40% vs 1.50%, p<0.001) were observed in ANTXR1H TME, which were more frequently classified as T cell-inflamed compared to ANTXR1L TME (23% vs 5%, p<0.001). ANTXR1H tumors also had higher MPAS compared to ANTXR1L (1.36 vs -1.86 arbitrary units, p<0.001). There was no significant difference in mOS between ANTXR1H vs L tumors (12.2 vs 10.5 months, HR: 0.95, p=0.678). Conclusions: Increased B cell, M1 and M2 macrophage infiltrates, and increased prevalence of T-cell inflamed tumors in ANTXR1H TME suggest that these patients with SCLC may respond preferentially to ICI. A Phase 1 trial incorporating SVV-01 along with ICI is underway. Prospective investigation of molecular associations and clinical outcomes related to ANTXR1 expression in SCLC is warranted.

Ulcer-related cutaneous squamous cell carcinoma: New kid on the block.

e21559 Background: Cutaneous squamous cell carcinoma (cSCC) represents a favorable tumor microenvironment with high tumor mutational burden, and infiltration of immune cells that are highly responsive to immunotherapies. Yet, we encounter tumors arising on non-exposed skin, such as those developing from chronic ulcers in which the response to anti-PD1 is unknown. Methods: The authors reviewed the medical records of consecutive cSCC patients receiving Cemiplimab or Pembrolizumab at a single tertiary center. Results: Of the 84 eligible patients, 9 (11%) had ulcer-related cSCC. six out of the nine (67%) were located on the lower extremities. Only two out of these nine patients (22%) reached partial response (PR) and none reached complete response (CR). In contrast, of the 75 patients with non-ulcer-related cSCC, 40 had PR (53%), and 20 had CR (26%). The difference between the two subtypes was significant (P < 0.001). Interestingly, two of the seven non-responders in the ulcer-related subtype received second-line chemo-immunotherapy and experienced a partial response that continued for 21 and 8 months. Patients with ulcer-related SCC had a significantly shorter median progression-free survival (PFS) (2.1 months vs. non-reached, p < 0.001). Overall survival (OS) was also shorter, but didn't reach significance (17 vs. 30 months, p = 0.02). Multivariate analysis confirmed ulcer-related SCC as an independent risk factor for shorter PFS (hazard ratio [HR], 7.56; 95% confidence interval [CI], 1.26-40.74, p = 0.027). Conclusions: This study suggests immunotherapy is less beneficial in ulcer-related SCC. More work is needed to verify our findings and explore other treatment options including chemoimmunotherapy.

Evaluation of the genomic and immune profiles of patients with lung adenosquamous carcinoma (LUAS) and association of response to treatment.

8567 Background: LUAS is a rare subset of non-small cell lung cancer (NSCLC) comprising up to 4% of cases, characterized by at least 10% of both adenocarcinoma and squamous components. Limited studies exist on LUAS regarding the genomic/immune landscape and response to immunotherapy (IO). Methods: NSCLC samples (n = 35404) underwent NextGen Sequencing of DNA (592 genes or whole exome)/RNA (whole transcriptome) at Caris Life Sciences. PD-L1 expression was assessed using IHC (22c3; positive: TPS >1%). High tumor mutational burden (TMB-H) was defined as ≥10 mut/MB. Cell infiltration in the tumor microenvironment was estimated by quanTIseq. Gene expression profiles were analyzed for transcriptomic signatures (IFN-γ) predictive of IO response. Real-world overall survival (rwOS) was obtained from insurance claims data, calculated from time of biopsy to last contact. Time on treatment (TOT) was calculated from the first pembrolizumab (pembro) treatment to the last. Mann-Whitney U and X2/Fisher-Exact tests were applied where appropriate, with p-values adjusted ( p < .05). Results: Among NSCLC samples, 1.0% (n=374) were LUAS, 26.5% (n = 9365) were squamous (SCC) and 72.5% (n = 25665) were adenocarcinoma (AC). LUAS exhibited actionable alterations in 29% (n = 110/374) of cases, with MET exon 14 skipping alteration ( METex14) significantly higher in LUAS compared to SCC or AC (9.02% vs 1.21% vs 2.61%, p < 0.01). BRAF V600E (0.54% vs 0.13%, p = 0.041) and ALK-Fusion (1.13% vs 0.12%, p = 0.0145) were higher in LUAS compared to SCC, while PIK3CA mutation was higher in LUAS compared to AC (10.8% vs 4.1%, p <0.01). Notably, LUAS exhibited higher prevalence of KRAS, targetable EGFRand STK11 mutations compared to SCC but lower compared to AC ( KRAS G12C: 8.63% vs 1.37% vs 13.24%, p <0.01; EGFR: 13.1% vs 1.12% vs 17.53%, p<0.01; STK11: 6.97% vs 1.83% vs 17.22%, p<0.01). LUAS exhibited significantly higher PD-L1+ (65.5% vs 59.8% vs 53.7%, p <0.01), along with elevated expression of immune checkpoint genes and the IFN-γ signature compared to AC and SCC. Moreover, LUAS exhibited significantly higher neutrophils, CD8 T cells, M2 macrophages and Tregs compared to SCC. LUAS showed improved rwOS compared to SCC (HR 0.792, p<0.01), with no difference noted in the rwOS between LUAS and AC (HR = 0.98, p = 0.81). While PD-L1+ was not associated with TOT on pembro in LUAS, high TMB showed improved TOT on pembro in LUAS. Conclusions: This study provides a comprehensive genomic landscape of LUAS, highlighting actionable alterations in ~ 29% of cases. LUAS exhibits distinct molecular and clinical features compared to AC and SCC, with enrichment in actionable METex14, emphasizing the need for NGS testing. Although PD-L1 status may not significantly impact TOT in LUAS, it remains relevant in other NSCLC subtypes on IO and high TMB emerges as a potential biomarker for favorable TOT on pembro in LUAS, warranting further investigation.

Tumor mutation burden and FAT3 mutation influence long-term survival in surgically resected small cell lung cancer.

Small cell lung cancer (SCLC) is highly malignant and has a higher risk of recurrence even in patients who undergo early surgery. However, a subgroup of patients survived for many years. So far, the factors that determine the long-term survivorship remain largely unknown. To determine the genetic characteristics of long-term survival (LTS) after surgery in SCLC, we performed comprehensive comparative genomic profiling and tumor mutation burden (TMB) analysis of resected tumor tissues from patients with LTS and short-term survival (STS) after surgery. The present study screened 11 patients from 52 patients with SCLC who underwent surgery at Zhejiang Cancer Hospital from April 2008 to December 2017. A total of six LTS patients (≥4 years) with stage IIB or IIIA SCLC and five STS patients (<2 years) with stage IA or IB SCLC were included in the study. The STS patients were used as a control. All the patients underwent resection without neoadjuvant therapy. We assessed the genomic profiles of the resected tumor tissues and calculated the TMB using next-generation sequencing. We then analyzed and compared the molecular characteristics between the LTS and STS groups. Our data indicated that tumor tissues from patients with LTS harbor a high TMB. The median TMB for LTS patients was high (approximately 16.4 mutations/Mb), while that for STS patients was low (approximately 8.5 mutations/Mb). The median TMB of patients with LTS and STS showed a trend of significant difference (P=0.08). Gene alterations characterized the survival differences between the two groups. The FAT3 mutation was only found in the LTS group, and the P value determined by Fisher's exact test was 0.06. A high non-synonymous TMB and the FAT3 mutation could potentially influence LTS after SCLC resection. This study provides valuable information about the molecular differences between LTS and STS patients. Studies with larger sample sizes need to be conducted to confirm our findings in the future.

Comprehensive genomic profiling in solid tumors: Enhancing treatment selection and prognosis.

e13631 Background: The emergence of novel therapeutic targets, targeted drugs, and immune checkpoint inhibitors has catalyzed a paradigm shift from single-target testing to comprehensive genomic profiling in the treatment of solid tumors. However, the tangible benefits of this shift in terms of treatment optimization and patient prognosis improvement have yet to be fully substantiated. Methods: In this retrospective study, we analyzed genomic data from 1032 patients with solid tumors to identify actionable genetic alterations, calculate tumor mutation burden (TMB), measure PD-L1 expression via immunohistochemistry, and assess microsatellite instability (MSI) status. These assessments were utilized to evaluate potential therapeutic targets and immunotherapy-related markers for each patient. Results: Between January 2018 and July 2020, 1032 patients underwent genomic profiling, received treatment, and were followed up. Of 1156 treatment episodes, 423 involved targeted monotherapy or combination therapy, 114 involved immune checkpoint inhibitor therapy, and 619 involved chemotherapy or anti-angiogenic therapy. Patients receiving matched therapy (n=491) demonstrated significantly higher partial response rates (31.7% vs 10.4%, P&lt;0.001) and longer median progression-free survival (PFS) (159 days vs 96 days, P&lt;0.001) compared to those receiving unmatched therapy (n=665). Based on the highest level of mutation carried, there were 475, 8, 153, 405, and 115 patients with grade I, II, III, IV, and V alterations, respectively. Among patients with grade I-II alterations, 65.63% received targeted therapy, resulting in significantly longer PFS compared to those undergoing immunotherapy or chemotherapy (200 days vs 109 days vs 94 days). Conversely, only 16.8% of patients with grade III-IV alterations received targeted treatment, with marginal PFS improvement (112 days vs 114 days vs 92 days). Notably, an increase in the number of actionable variants was associated with a significant decrease in PFS among patients receiving targeted therapy. However, the number of actionable variants did not significantly affect PFS in patients receiving chemotherapy or immunotherapy. Within our cohort, 44 patients with cancer of unknown primary (CUP) underwent molecular diagnostic evaluation, leading to the reclassification of 32 patients based on their molecular profiles. Notably, 24 patients harbored actionable genetic variants, 9 patients had a high TMB (≥10 Muts/Mb), and 3 patients exhibited high PD-L1 expression (TPS ≥50%). Conclusions: Comprehensive genomic profiling demonstrates clinical utility in guiding treatment decisions and improving prognosis for patients with advanced solid tumors. These findings advocate for the integration of next-generation sequencing into the therapeutic regimens for these malignancies.

A blind retrospective analysis of a novel predictive marker to ICB response in NSCLC, calculated directly from histopathological slides.

2665 Background: Immune checkpoint blockers (ICB), and primarily PD-1/PD-L1 inhibitors, are in the forefront of contemporary clinical oncology and have become an integral part of treatment of many malignancies, including non-small cell lung cancer (NSCLC). Nevertheless, tumor response to ICB varies widely. Predictive markers commonly used to distinguish patients likely to respond to ICB, such as PD-L1 expression and tumor mutational burden (TMB) have limited predictive value, which calls for the development of practical and more accurate tests. We present results of a blind retrospective analysis of a novel predictive marker of ICB response in NSCLC, relying solely on histopathological slides. Methods: We obtained high resolution Hematoxylin and Eosin (H&amp;E) slides from tumor-tissue samples of 50 cases of metastatic NSCLC patients treated with first-line PD-1 inhibitors. We retrospectively applied our ENLIGHT-DeepPT (ENLIGHT-DP for short) pipeline to generate, in a blinded manner, an individual response score to PD-1 inhibition for each slide. ENLIGHT-DP is composed of two main steps: (i) predict mRNA expression directly from an H&amp;E slide using DeepPT, our digital-pathology based algorithm; and (ii) use these values as input to ENLIGHT, our transcriptome-based precision oncology platform, which generates a score that predicts response to targeted therapies and ICB (based on a 10-gene signature in this case). We then unblinded the clinical outcome (RECIST1.1), and evaluated ENLIGHT-DP’s performance vs. standard markers. Results: ENLIGHT-DP’s score is predictive of response in this cohort, which had an overall response rate of 68% (34 of 50), with ROC AUC = 0.69 (p = 0.01, one-sided permutation test). Using a predefined threshold for binary classification of response derived from independent data, all 15 patients that were predicted to respond by ENLIGHT-DP indeed responded (100% PPV, 44% sensitivity). In comparison, predicting response according to PD-L1 &gt; 1% achieves 68% PPV and 62% sensitivity, while PD-L1 &gt; 50% achieves 65% PPV and 38% sensitivity, i.e, both thresholds exhibit no predictive power (PPV &lt;= baseline response rate). Patients with high TMB (&gt;10) had 82% PPV and 26% sensitivity, showing lower predictive benefit than ENLIGHT-DP. ENLIGHT-DP was particularly good at stratifying patients with PD-L1 &lt; 1% (18 patients, ROC AUC = 0.8, p = 0.03). Conclusions: ENLIGHT-DP demonstrates high predictive power for response to ICB in NSCLC relying solely on accessible H&amp;E slides, outperforming the commonly used PD-L1 and TMB markers. ENLIGHT-DP is also able to identify responders within patients with PD-L1 &lt; 1%, for whom ICB is usually considered ineffective. Importantly, our approach does not require training on prior treatment outcomes, and can therefore be generalized to drugs for which such data is unavailable or scarce.

Efficacy of immunotherapy in first-line treatment for non-small cell lung cancer with HER2 mutation: Results from LC-SCRUM-Asia.

8629 Background: HER2 mutations occur in 2–4% of non-small cell lung cancer (NSCLC) patients. Previous studies with small cohorts have showed poor clinical outcomes in HER2-mutant NSCLC patients treated with immune checkpoint inhibitors (ICIs). However, in these previous studies, ICIs had not been used as the first line treatment in advanced NSCLC. Thus, the clinical outcomes in HER2-mutant NSCLC patients treated with ICIs in the first line treatment are still unclear. Methods: Using a large-scale clinical-genomic database of LC-SCRUM-Asia, the clinical-genomic characteristics and therapeutic outcomes of NSCLC patients harboring HER2 mutations were investigated. Tumor mutation burden (TMB) in tumors with HER2 mutations was compared to tumors with EGFR mutations or ALK fusions. Results: From March 2015 to June 2023, 15,251 NSCLC patients enrolled in LC-SCRUM-Asia. HER2 mutations were detected in 402 patients (2.6%). The most common subtype of HER2 mutations was exon 20 in-frame insertions (Ex20ins) (79%), followed by tyrosine kinase domain other than Ex20ins (10%), extracellular domain (7%) and transmembrane domain (3%). The most common Ex20ins was Y772_A775dupYVMA (66%), followed by G776delinsVC (14%), G778_P780dupGSP (7%). A total of 268 patients had received platinum-based chemotherapy with ICI (Chemo-ICI, n = 95) or without ICI (Chemo-alone, n = 173) until October 2022. The median follow-up periods were 18.5 and 19.9 months in the patients received Chemo-ICI and Chemo-alone, respectively. The patients received Chemo-ICI showed a significantly longer progression-free survival (PFS) compared to those received Chemo-alone (median 8.5 vs 6.3 months, HR [95%CI]: 0.66 [0.50–0.88], p &lt; 0.005). In multivariate analysis, the addition of ICI to platinum-based chemotherapy was an independent better prognostic factor for PFS. There was no significant difference in the overall survival between the patients received Chemo-ICI and Chemo-alone (median 31.1 vs 23.3 months, HR [95%CI]: 0.80 [0.57 – 1.12]). The overall response rates were 34.7% and 35.3% in the patients received Chemo-ICI and Chemo-alone, respectively. Tumors with HER2 mutations showed a higher TMB (median 4.22 mut/Mb) and a higher percentage of TMB≥10 mut/Mb (12.3%) compared to tumors with EGFR mutations (median 2.54 mut/Mb and 1.0%, respectively) or ALK fusions (median 2.52 mut/Mb and 0.8%, respectively). The subtype of HER2 mutations showed no significant difference in the clinical outcomes of Chemo-ICI and Chemo-alone. Conclusions: Our large cohort demonstrated that the addition of ICI to platinum-based chemotherapy as first line treatment may improve PFS in NSCLC patients with HER2 mutations. Relatively high TMB may contribute to prolongation of PFS with ICI.

Comprehensive whole genome and transcriptome analysis of patients with advanced solid tumor treated with immune checkpoint inhibitor therapy in the pan-cancer cohorts from the Marathon of Hope Cancer Centres Network Study (MOHCCN).

2645 Background: Immune checkpoint inhibitors (ICI) improve survival in multiple advanced solid tumors but many patients do not benefit. We conducted a comprehensive whole genome transcriptome sequencing (WGTS) analysis to identify predictors of immune sensitivity. Methods: Clinical and molecular data from archival or pre-treatment FFPE tumor tissue were available for analysis from The Marathon of Hope Cancer Centres Network Study (MOHCCN). It includes patients (Pts) with advanced solid tumors and ECOG PS 0 or 1 treated with ICIs targeting PD-1, PD-L1, or CTLA-4 in the INSPIRE (NCT02644369) and OCTANE (NCT02906943) cohorts. Response (R) to ICI was defined as radiological and clinical response without PFS event at 6 months; versus non-response (NR) as radiological or clinical progression within 6 months. Responders without evidence of progression for &gt;12 months were categorized as durable responders (DR). Nucleic acids were sequenced using Illumina NovaSeq 6000 system targeting minimum coverage of 80X and 30X for tumour and normal WGS, respectively and 80M reads for tumour RNA-seq. WGTS data were integrated with clinical data to identify associations with response to ICIs. Also, an analysis of immune cell types was conducted using multiplexed IHC and RNA-Seq deconvolution (CIBERSORT). Results: 59 Pts were included in this analysis: 28 (R) and 31 (NR). The most common tumor types were head and neck (n = 19) and melanoma (n = 7). Median age was 62 years (range 24-81); male 58% and median follow-up was 58 months (range 11-280). The most frequent ICI was pembrolizumab 76%, with 90% of all pts receiving ICI monotherapy and 10% combination. 75% of pts received chemotherapy prior to ICI. Higher tumor mutation burden (TMB) was observed in R vs NR (median 13 vs 5 coding mut/Mb, p=0.001), with the highest TMB in patients with DR (median 14 mut/Mb ). Differential RNA gene expression analysis showed NR had increased expression of several oncogenic pathway signatures, including MYC targets, G2M checkpoint, and E2F targets. Differential abundance analysis of immune cell types did not yield any differences of immune cell populations amongst R versus NR after correction for multiple comparisons. Responders had significant enrichment in mutations in WNT/β-catenin pathway genes in both coding (APC, AMER1, LZTR1, TCF7L2) and promoter regions (CTNNB1). Notably, the 6 Pts with CTNNB1 promoter mutations had significantly increased CTNNB1 gene expression (p = 0.005). Conclusions: ICI responders showed enrichment in coding mutations of several negative regulators of the Wnt/β-catenin pathway and non-coding promoter mutations in CTNNB1 compared with non-responders. Further investigation is ongoing to biologically validate how these mutations in the Wnt/β-catenin pathway may lead to improved response to ICI.

Axitinib plus avelumab for recurrent/metastatic adenoid cystic carcinoma (R/M ACC): Biomarker analysis of the phase II trial.

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.

Characterization of T-cell receptor repertoire and correlation with tumor mutational landscape in lung cancer.

2577 Background: T-cell receptor (TCR) repertoire represents an overall immune condition which is closely related to pathogens- or tumor-associated T cell responses. Increasing evidence suggests that TCR repertoire is expected to undergo cancer-specific changes during tumorigenesis, supporting that TCR characteristics can serve as novel markers to indicate early cancer progression. Here, we performed a systematic T cell repertoire analysis in lung cancer. We identified cancer-enriched TCR signatures and constructed a comprehensive lung cancer database integrating TCR diversity, tumor mutational profiles, immune markers expression and HLA genotypes. Methods: A total of 988 tissue samples and 3,360 blood samples were obtained from patients with lung cancer, in addition to 2,699 blood samples from healthy individuals. Multiplex-PCR-based sequencing of the CDR3 regions of TCR-β chains was applied to the tissue and blood samples. TCR repertoires were analyzed using MiXCR and VDJtools. Whole exome sequencing was performed on partial tumor tissues to profile the mutational landscape and HLA genotypes. An in-house pipeline, considering the frequency and distribution of CDR3, was utilized to identify cancer-enriched CDR3 sequences. The PD-L1 expression was evaluated and quantified using CPS. Furthermore, an enrich score was developed to measure the content of cancer-related TCRs by aligning against the built cancer-enriched CDR3 dataset. Spearman's rank correlation was used to assess relationships between variables. Results: Lung cancer exhibited significantly lower Shannon index, Simpson index and evenness index in TCR repertoire (p&lt;0.001), both in tissue and blood samples, when compared with healthy blood samples. Approximate 3% T-cell clonotypes within lung tissues were detected in blood TCR repertoires. A correlation was observed between the decreased TCR diversity and a higher tumor mutational burden (TMB)(r = -0.15, p &lt; 0.01), as well as a higher variant allele frequency (VAF) (r = -0.19, p &lt; 0.01). In this study, 3,652 and 3,840 CDR3 sequences were identified to be enriched in lung cancer tissue and blood respectively. A significant difference in cancer-enriched score was observed between cancer and healthy TCR repertoires (p &lt; 0.001). The cancer-enriched score showed a significant positive correlation with TMB, particularly in TP53- mutant tumors (r = 0.27, p &lt; 0.001). A higher CPS (PD-L1) was associated with a higher cancer-enriched score (r = 0.18 ,p &lt; 0.01). Conclusions: Cancer-related T-cell clonotypes in the tumor microenvironment and peripheral blood informs the changes in anti-tumor responses, supporting the potential application of TCR signatures in early cancer detection. This study identified the lung cancer-related TCR signatures and provided a comprehensive lung cancer TCR database, revealing the relationship between TCR characteristics and mutational profiles. Clinical trial information: ChiCTR2200055761.

A pan-cancer analysis of SMARCA4 alterations and the unique clinicogenomic characteristics associated with SMARCA4 mutation types.

3073 Background: SWI/SNF complexes are regulators of cell lineage and alterations in these proteins occur in &gt;20% of solid tumors with SMARCA4being the most common. SMARCA4 mutations are associated with worse clinical outcomes in various cancer types but are also vulnerable to novel SMARCA2 inhibitors currently being evaluated in clinical trials. We examined SMARCA4 alterations across cancer types to understand their interplay with other genomic abnormalities and clinical consequences. Methods: We analyzed the Memorial Sloan Kettering (MSK) institutional cohort profiled by MSK-IMPACT tumor-normal sequencing. SMARCA4 mutations were annotated based on OncoKB and literature. FACETS resolved zygosity and genomic metrics of complexity including tumor mutational burden (TMB) and fraction genome altered (FGA). The pan-cancer landscape of somatic SMARCA4 mutations was mapped by both category (Class I vs II) and zygosity (mono- vs biallelic). DISCOVER was employed to evaluate mutational signatures and frequency-adjusted co-occurring and mutually exclusive mutations. We then extracted progression-free (PFS) and overall survival (OS) to systemic therapy using natural language processing. Results: We identified 3,385 tumors from 3,049 patients across cancer types with a total 3,965 oncogenic somatic SMARCA4 alterations. Copy number estimation was successful for 2,343 (69%). Non-small cell lung cancer (NSCLC) was the most abundant SMARCA4-altered histology ( n=493, 7.1% of all NSCLC), and the majority (87%) were biallelic. Other common histologies were colorectal ( n=163, 2.9%), bladder ( n=122, 5.5%), uterine ( n=88, 4.3%), pancreatic ( n=81, 3.2%) and esophagogastric ( n=92, 4%). SMARCA4biallelic mutated tumors had higher chromosomal instability compared to WT (FGA = 0.53 vs 0.47; p&lt;0.001), while monoallelic had lower (FGA = 0.3; p&lt;0.001). Monoallelic SMARCA4 altered tumors had a higher TMB (10.5) than both WT (4.4) and biallelic cancers (6.1; p&lt;0.001). Monoallelic and biallelic SMARCA4 cancers exhibited distinct mutational signatures in most tumor types. Patients with biallelic SMARCA4 mutations had worse outcomes with both immune checkpoint blockade (ICB) and platinum chemotherapy across in certain tumor types, whereas monoallelic uterine cancers were associated with improved outcomes with ICB. Conclusions: This pan-cancer clinicogenomic analysis demonstrates that monoallelic and biallelic SMARCA4-mutated tumors have distinguishable genomic features. In addition, biallelic SMARCA4 alterations correlated with poor outcomes across different cancer-types and classes of therapy, while improved outcomes to ICB in monoallelic SMARCA4alterations were potentially due to higher representation of MSI-H tumors in this subset. Our findings suggest SMARCA4 zygosity may be important biomarkers for ongoing clinical trials in SMARCA4-deficient tumors.

Use of immunotherapy with check point inhibitors in soft tissue sarcoma: Analysis from single institution.

e23566 Background: Traditional treatments for soft tissue sarcomas include surgery, chemotherapy, and radiation. Effectiveness of Check point inhibitors for sarcoma has been unsatisfactory thus far. Major clinical trials PEMBROSARC and SARC028 revealed median PFS of 2 and 4.5 months, respectively. The FDA has approved three immunotherapy (IO) therapies for sarcoma. Atezolizumab was approved for alveolar soft part sarcoma. Dostarlimab is approved for sarcoma with DNA mismatch repair deficiency (dMMR), Sarcoma patients with high microsatellite instability (MSI-H), dMMR, or tumor mutational burden can receive pembrolizumab. A single-institution analysis of soft tissue sarcoma patients with IO is presented in this paper. Methods: All soft tissue sarcoma patients who have received IO from January 2021 to now have been included. At our institute, a total of seven patients received IO for soft tissue sarcoma. Observations were made regarding their fundamental characteristics and the progression free survival (PFS). The data was not statistically examined due to the small sample size and the heterogeneity of the data. Results: 2 of 7 trial patients were lost to follow-up. Recurrent autoimmune hepatitis terminated IO for another patient. Most of the patients are elderly with the median age of 78. Given their comorbidities and age, IO was chosen as the first line for 5 patients. Median PFS was 5.8 months. High PDL1 ( &gt; / = 5%) patients had a median PFS of 5.6 months, while low PDL1 patients had 4.5 months. However, one patient with low PDL1 had PFS of 7 months. 5/7 patients didn’t have IO related side effects. Two patients developed Autoimmune (AI) hepatitis. Conclusions: The limited sample size prevented definitive conclusions from being drawn. Despite the unsatisfactory outcomes, it is worth mentioning that IO provided a certain degree of PFS in older patients with comorbidities who are not suitable candidates for chemotherapy. Based on limited data, patients with high PDL1 status appear to benefit more. IO alone has not shown promising results. In clinical trials, IO is being tested with chemotherapy and Tyrosine kinase inhibitors. The findings from these trials will provide valuable insights into the potential benefits of combination therapy. [Table: see text]

Contrasting comprehensive genomic profiles of adenocarcinomas of the appendix in younger versus older patients.

3626 Background: Adenocarcinoma of the appendix (AAC) is a rare form of intestinal malignancy that can pursue an aggressive clinical course. Little is known about the clinical and biologic features of this disease when it presents in young patients under 40. Methods: 1,840 cases of clinically advanced AAC were selected and analyzed by hybrid capture based comprehensive genomic profiles (CGP) to evaluate all classes of genomic alterations (GA). MSI high status and tumor mutation burden (TMB) were determined from sequencing results and PD-L1 was measured by immunohistochemistry (Dako 22C3). Results: 135 (7.3%) of the AAC were identified in patients of 39 years of age or younger (“younger”). The mean age of the younger patients was 32.9 years and for the 40 and over (“older”) patients it was 60.4 years. The slight female gender preponderance was higher in the older patients than the younger patients (Table). The GA per sample were similar. The younger patients had a significantly higher frequency of KRAS GA (71.1% vs 60.4%; p=.017) and TP53 GA (56.3% vs 41.3%; p=.0008). The KRAS G12C mutation frequency was similarly low in both cohorts. Targetable GA in BRAF and ERBB2 were infrequent in both groups as were GA in PIK3CA, PTEN and FBXW7. GA in the APC gene was significantly more frequent in the older patients (10.8% vs 3.7%; p=.007). Biomarkers of immune-oncology (IO) drug efficacy were infrequently identified in both groups including MSI-High status, TMB levels and PD-L1 expression. Conclusions: At a 7.3% incidence, AAC is a relatively infrequent form of gastrointestinal malignancy in young patients. The data analysis shows significant differences in the genomics found in younger and older patients suffering from this disease. These results can have a crucial impact on current and future clinical trials used to design novel treatments. Further exploration into this subject can change the overall approach and even treatment guidelines for this type of cancer. [Table: see text]

Genomics to select treatment for patients (pts) with metastatic cancer: Final analysis of Molecular Tumor Board (MTB) evaluations in the ROME trial.

3157 Background: The ROME trial (NCT04591431) investigates the efficacy of a tailored treatment (TT), driven by extensive genomic tests and Molecular Tumor Board (MTB) evaluation, compared to standard treatment (SoC) in patients (pts) with refractory metastatic cancer. Here we report the prevalence of actionable variants identified and the personalized treatment received by the patients enrolled. Methods: A centralized extensive NGS test (FoundationOne CDx and Liquid CDx) was performed on both tissue and blood samples. Pts with at least one potentially targetable alteration matched to the available drugs were discussed at the MTB. MTB evaluation criteria were: genomic variant type, tumor mutational burden (TMB), VAF, availability of preclinical or clinical data of efficacy and patients’ clinical characteristics. COSMIC, ClinVar, OncoKB and VarSome datasets were used to evaluate the clinical actionability of genomic alterations. MTB indications were: randomization to TT vs SoC, screening failure (SF), or indication to other trial/access to the drug. Results: From November 2020 to August 2023, 1794 pts were screened. A total of 127 weekly MTB meetings were conducted and 899 cases were discussed (7 cases/meeting on average). After MTB discussion, a targetable genomic alteration was identified in 425 pts, who received an indication to be randomized. MTB assigned ICIs to 190 pts (155 to ipilimumab plus nivolumab; 8 to nivolumab) due to high (≥10 mut/mb) TMB. 234 pts had indications to target therapy: 86 pts (37%) to PI3K/AKT/mTOR inhibitors(i), 59 (25%) anti-HER2 agents, 34 (15%) FGFRi, 31 (13%) BRAF/MEKi, 7 (3%) PARPi, 5 (2%) CDK4/6i, 4 (2%) ALKi, 2 (&gt;1%) NTRKi, 2 (&gt;1%) RETi, 2 (&gt;1%) Hedgehog-i, 1 (&gt;1%) JAKi, 1 (&gt;1%) MEKi. 27 pts had indication to ICIs plus target therapy combinations. Additionally, 150 pts with potentially hereditary variants were addressed to genetic counselling and germline testing. Finally, according to the genomic evaluation, 63 pts were referred by MTB to other clinical trial even if considered SF for the ROME trial, 34 patients received from the MTB a modification of the initially proposed standard therapy and 4 had both suggestions. Overall, 652 pts (36%) received an indication following NGS test and MTB evaluation. Conclusions: Our study provides evidence that MTB discussion of comprehensive genomic profiling data identifies a personalized treatment in a large (36%) fraction of patients, thus outperforming the traditional histological approach. Clinical trial information: NCT04591431 .

Therapeutic implications of acquired high tumor mutational burden (TMB-H) after targeted therapy (TT) in metastatic colorectal cancer (mCRC).

3542 Background: mCRC treated w/ TT can acquire a high number of mutations at progression. TMB is used as a proxy of neoantigen load for predicting benefit from immune checkpoint blockade (ICB). In TMB-H CRC, benefit to ICB is restricted to microsatellite unstable (MSI-H) hypermutated or POL-D/E ultramutated tumors. We asked whether TT can sensitize microsatellite stable (MSS) CRCs to ICB by inducing an acquired TMB-H state. Methods: We screened the MSK-IMPACT dataset for mCRC patients (pts) who received TT and underwent pre-treatment (pre-TT) and post-progression (post-TT) tissue or liquid biopsies (LBx). We included pts who received TT against EGFR, BRAF V600E, KRAS G12C, or HER2. DNA sequencing of tissue and LBx was performed w/ MSK-IMPACT and Guardant 360. MSI status was assessed with algorithms measuring somatic changes in length of repetitive sequences (e.g., MSIsensor). Due to known discrepancies between tissue TMB (tTMB) and blood TMB (bTMB), we defined acquired TMB-H as: 1) tTMB &lt;10 mut/Mb or bTMB &lt;20 mut/Mb pre-TT; 2) ≥5-fold increase in TMB post-TT; 3) tTMB ≥10 mut/Mb or bTMB ≥40 mut/Mb post-TT. Results: We identified 26 pts w/ paired samples that had been sequenced w/ determination of TMB. 4/26 (15%) met criteria for acquired TMB-H. None had detectable pathogenic mismatch repair or DNA damage repair alterations pre-TT. All 4 post-TT samples were LBx only. One pt’s tumor acquired a somatic MLH1 mutation and converted from MSS to MSI-H. In all cases, the majority of acquired mutations were subclonal w/ many at variant allele frequencies (VAF) of ≤1% (Table). After detection of acquired TMB-H, 2/4 pts received ICB. Pt 1 had RAS wild type mCRC w/ pre-TT tTMB 6.9. She received irinotecan + panitumumab for 25.8 months. Post-TT bTMB was 40.19 and met criteria for MSI-H. She received pembrolizumab and experienced clinical + radiographic progression of disease (POD) within 49 days. Pt 2 had KRASG12C-mutant mCRC w/ pre-TT bTMB 13.4 and tTMB 4.9. She received sotorasib + panitumumab for 3.2 months w/ post-TT bTMB 145.34. She received pembrolizumab + regorafenib and experienced clinical + radiographic POD within 37 days. Conclusions: In CRC, acquisition of TMB-H at progression w/ predominantly low VAF mutations suggests activation of enhanced mutagenesis due to therapeutic pressure from exposure to TT. In this context, TMB may not be an accurate reflection of tumor immunogenicity as it does not distinguish between clonal and subclonal alterations. Our data suggest that pts w/ acquired TMB-H following TT are unlikely to benefit from ICB. [Table: see text]

Genomic profile of Japanese patients with rare ductal adenocarcinoma of the prostate has high grade and poor prognostic aspects.

e17046 Background: Ductal adenocarcinoma (DAC) is a rare subtype of prostate cancer with unknown etiology and poor prognosis. Genomic profile analysis using next-generation sequencing has the potential to elucidate the pathogenesis of rare cancers. To date, some studies have investigated the genetic profile of the DAC genotype; however, no definitive findings have been obtained due to the small number of patients and limited geographic coverage. The discovery of novel prognostic biomarkers and identification of new pharmacotherapeutic targets for DAC are currently of research interest. The aim of this study is to analyze the genomic profile of DAC in a Japanese population and to summarize its features. Methods: Clinical and histopathological records from our three hospitals were searched for radical prostatectomy, transurethral resection of the prostate, and prostate needle biopsy specimens containing a DAC component in the past 10 years. Twenty-eight patients diagnosed with DAC of the prostate were identified, of whom 15 were eligible for genomic profile analysis and 10 patients (66.7%) had the pure type. Genomic evaluation was performed using either the FoundationOne CDx or PleSSision testing platform. Results: At least one genomic alteration occurred in 14 patients (93.3%), and the most frequently mutated gene was tumor suppressor protein p53 ( TP53), which was found in seven cases (46.7%). Additionally, five cases (33.3%) had mutations in retinoblastoma transcriptional corepressor 1 ( RB1), and all these patients had the pure type. Four cases had both TP53 and RB1 alterations, and most of these possessed the pure type and had PSA levels of less than 4.0. Compared to previous study results, the frequency of genetic alterations in the phosphoinositide 3-kinase (PI3K) pathway (n = 3; 20.0%) and Wnt-β/catenin pathway (n = 6; 40.0%) was comparable, but alterations in the DNA damage repair (DDR) pathway were few (n=1; 6.7%). None of the patients presented high tumor mutation burden or microsatellite instability. Conclusions: We found that the Japanese cohort with DAC has unique features such as high frequency of alterations in tumor suppressor gene such as TP53 and RB1, and few DDR pathway alterations, suggesting the existence of regional and racial differences in genomic profiles. Genomic analysis using next-generation sequencing is expected to be useful in elucidating the pathogenesis of DAC, and further accumulation of cases is considered important.

Real-world outcomes and molecular correlates of incorporating immunotherapy (IO) and bevacizumab (Bev) in treating patients with peritoneal mesothelioma (PeM): A comparative analysis with pleural mesothelioma (PM).

e23300 Background: Nivolumab/Ipilimumab (IO) and Bev/Chemo are now considered standards of care for patients (pts) with advanced PM, based on randomized (CheckMate743 and MAPS) trials. By extrapolation, they are also endorsed by guidelines and used clinically in pts with PeM. Due to lack of any prospective data, we evaluated real-world effectiveness of these agents in PeM (vs PM) and investigated molecular correlates underlying their therapeutic impact. Methods: The study cohort was composed of mesothelioma pts (N = 723) who underwent DNA (592-gene panel or whole exome)/RNA (whole transcriptome) [PeM (221/224) and PM (493/499)] sequencing at Caris Life Sciences (Phoenix, AZ). Treatment and real-world survival (OS) were obtained from insurance claims and calculated from time of tissue collection to last contact. Hazard ratio (HR) was calculated using Cox proportional hazards model. Tumor microenvironment (TME) estimates were calculated from bulk RNA sequencing using QuanTIseq method. Significance was determined using Chi-square, Mann Whitney U and log-rank tests and corrected for multiple comparisons (q-value &lt; 0.05) when applicable. Results: PeM (vs PM) had a higher proportion of females (46% vs 29%) and younger age (62 vs 73 years; both p &lt; 0.001). 20% and 22% of PeM were treated with IO or Bev/Chemo, with similar utilization rates in PM of 22% and 24%, respectively. OS estimates and comparisons are shown in Table. Prevalence of mutations in key mesothelioma associated genes ( BAP1, NF2, TP53 and SETD2) was not significantly different between PeM and PM. NF2 (10.1 vs 51.3 m for wild type; p &lt; 0.001) and TP53 (10.4 vs 46.6 m for wild type; p &lt; 0.006) mutations were associated with worse OS in PeM but not in PM. Prevalence of high tumor mutation burden (≥10 mut/Mb: 1.0% vs 3.4%; q = 0.37) and PD-L1+ (18.7% vs 28.8%; q = 0.07) tumors was slightly lower in PeM than PM. Macrophage fractions, M2 were higher (1.5-fold) and M1 were lower (0.8-fold) in PeM (both q &lt; 0.05) compared to PM. Conclusions: In this large real-world analysis, use of IO appears to be associated with improved outcomes in PeM, despite a potentially immunosuppressive TME. Notably, addition of Bev did not appear to improve outcomes in PeM, in contrast to PM. These results highlight variances in clinical utility/value of these therapies. Molecular mechanisms underlying these outcome differences warrant further investigation. [Table: see text]

Predictive Impact of Tumor Mutational Burden on Real-World Outcomes of First-Line Immune Checkpoint Inhibition in Metastatic Melanoma.

The choice of threshold and reliability of high tumor mutational burden (TMB) to predict outcomes and guide treatment choice for patients with metastatic melanoma receiving first-line immune checkpoint inhibitor (ICI) therapy in the real world is not well known. Using a deidentified nationwide (US-based) melanoma clinicogenomic database, we identified a real-world cohort of patients with metastatic melanoma (N = 497) who received first-line monotherapy anti-PD-1 (n = 240) or dual anti-PD-1 and anti-CTLA-4 ICI (n = 257) and had a tissue-based comprehensive genomic profiling test TMB score. TMB-high (TMB-H; ≥10 mutations per megabase [muts/Mb], n = 352, 71%) was independently predictive of superior real-world progression-free survival and overall survival versus TMB-low (<10 mut/Mb, n = 145, 29%) in both mono ICI (hazard ratio [HR], 0.45 [95% CI, 0.32 to 0.63]; P < .001; HR, 0.61 [95% CI, 0.41 to 0.90]; P = .01, respectively) and dual ICI (HR, 0.67 [95% CI, 0.49 to 0.90]; P = .009; HR, 0.61 [95% CI, 0.42 to 0.88]; P = .007, respectively) patients. Dual ICI offered no significant advantage in BRAFwt patients and unexpectedly demonstrated greatest benefit in the TMB 10-19 mut/Mb group, identifying a TMB-very high (≥20 mut/Mb, n = 247, 50%) BRAFmut patient subgroup for whom mono ICI may be preferable. TMB-H predicts superior outcomes on ICI while coassessment of BRAF status and TMB may inform first-line regimen choice.

Whole Exome Sequencing Reveals Gene Mutation Characteristics of Primary Central Nervous System Lymphoma

To investigate gene mutation characteristics of primary central nervous system lymphoma (PCNSL) through whole exome sequencing (WES) to 18 patients with PCNSL. Tumor tissues from 18 patients with diffuse large B-cell lymphoma who were diagnosed with PCNSL in Department of Hematology, Lanzhou University Second Hospital from September 2018 to December 2020 and had normal immune function, no history of HIV or immunosuppressant therapy were collected. High-throughput-based WES was performed on the tumor tissues, with an average sequencing depth of >100×. After data processing and bioinformatics analysis of sequencing results, the mutation maps and mutation characteristics of 18 PCNSL patients were obtained. Obvious somatic mutations were detected in all 18 patients. The median number of somatic mutations was 321. Missense mutations were most prominent (accounting for about 90%), and the mutation type was dominated by C>T (50.2%), reflecting the age-related mutation pattern. Among the top 15 frequently mutated genes, PSD3, DUSP5, MAGEB16, TELO2, FMO2, TRMT13, AOC1, PIGZ, SVEP1, IP6K3, and TIAM1 were the driver genes. The enrichment results of driver gene pathways showed that RTK-RAS, Wnt, NOTCH, Hippo and Cell-Cycle pathways were significantly enriched. The tumor mutation burden was between 3.558 48/Mb and 8.780 89/Mb, and the average was 4.953 32/Mb, which was significantly higher than other cancer research cohorts in the TCGA database. PCNSL occurs somatic missense mutations frequently, mainly point mutations, and the mutation type is mainly C>T. The driver genes are mainly involved in RTK-RAS, Wnt, NOTCH and Hippo pathways, indicating that the above pathways may be related to the pathogenesis of PCNSL. PCNSL has a significantly high tumor mutation burden, which may explain the efficacy of PD-1 inhibitors in PCNSL.

Tumor purity as confounder for the determination of tumor mutational burden (TMB).

e14656 Background: TMB determination is a prerequisite to stratify advanced cancer patients for pembrolizumab based on the histology-agnostic FDA approval. The accuracy of TMB measurement is challenged by low tumor purity resulting in a lower number of detected somatic variants for a fixed threshold of variant allele frequency (VAF) and a lower measured TMB level as a consequence. Methods: We derived a mathematical formula describing the influence of tumor purity on the VAF of somatic variants, in which the DNA copy number of the corresponding region of the tumor genome is a parameter. We simulated the effect of tumor purity in a cohort of 4,900 tumors from the TCGA project with tumor purity of at least 80%. TMB was calculated as the number of missense mutations with VAF greater or equal than 5%. Tumors were classified as TMB-high when harboring at least 199 missense mutations. This cutpoint corresponds to 10 mut/Mb as derived in the TMB bridging study. We compared tumors with a simulated tumor purity of 70%, 60%, ..., and 10% with tumors with a tumor purity of 80%, which served as reference. Results: The median of TMB in the pan-cancer cohort was reduced by 2%, 10%, and 32% for tumor purity of 60%, 40%, and 20% compared to the reference of 80%. For the 10% tumors with the worst estimation, TMB was reduced even by 12%, 35%, and 77% or more, respectively. In the pan-cancer cohort, the sensitivity to detect high TMB was 98%, 90%, and 62% for tumor purity of 60%, 40%, and 20%, respectively. For head and neck cancer, lung adenocarcinoma, lung squamous cell carcinoma, and cutaneous melanoma, the sensitivity to detect high TMB was 80%, 89%, 92%, and 91%, respectively, for tumors of tumor purity 40%. Conclusions: High tumor purity is critical for accurate determination of the TMB and to reach a high sensitivity concerning the selection of patients for immunotherapy. As shown by the simulations, the sensitivity to detect high TMB is close to 100% for tumor purity of at least 60%, while a substantial proportion of patients that could receive immunotherapy is missed for tumors with purity of 40% or less. Potential ways out of the TMB underestimation include increasing the sequencing coverage and lowering the VAF threshold for mutation calling or using clonal TMB with an adapted cutpoint instead of total TMB as biomarker for immunotherapy guidance.