Primary Tumor Tissue Research Articles

Abstract 267: Xenograft models of non-metastatic non-small cell lung cancer reveals micrometastasis-associated single cell composition

Abstract Background: Circulating tumor cells (CTCs) represent micrometastatic disease and may offer unique insights into future recurrences in lethal malignancies, including non-small cell lung cancer (NSCLC). Due to CTC rarity and limited stability, no CTC-derived xenograft (CDX) models have ever been generated from non-metastatic NSCLC patients directly. Alternative strategies are needed to molecularly characterize CTCs in this potentially curable patient group. Methods: Surgically resected NSCLC primary tumor tissues were implanted in immunodeficient mice to establish ten patient-derived xenografts (PDXs). CTCs from 2/10 PDX models led to generation of two stable metastatic models that were studied by single cell sequencing. Results: Single cell analysis revealed an additional alveolar epithelial type II (AT2) population in metastatic tumors, besides a common AT2 cluster in PDX/metastatic tumors. This was consistent with an external validation set analysis in primary and metastatic NSCLC patient tumors. Further, AT2 clusters of metastatic tumors expressed higher cancer stemness genes versus primary PDX tumor that was recapitulated in patients primary and metastatic tumors. Conclusions: Stable metastatic models from early stage NSCLC patients can be generated with CTCs from PDX models. The distinct AT2 population identified in CDX tumors with cancer stemness features might be critical mediator of metastasis that deserves further study to discover personalized strategies against NSCLC micrometastases. Citation Format: Kanve Nagaraj Suvilesh, Yulia I. Nussbaum, Vijay Radhakrishnan, Yariswamy Manjunath, Diego M. Avella, Kevin F. Staveley-O’Carroll, Eric T. Kimchi, Aadel A. Chaudhuri, Chi-Ren Shyu, Guangfu Li, Klaus Pantel, Wesley C. Warren, Jonathan B. Mitchem, Jussuf T. Kaifi. Xenograft models of non-metastatic non-small cell lung cancer reveals micrometastasis-associated single cell composition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 267.

Programmed Death-Ligand 1 Expression in Lymphovascular Tumor Emboli in Lung Cancer

IntroductionProgrammed death-ligand 1 (PD-L1) expression determined by immunohistochemistry is the most widely used biomarker for predicting response to immune checkpoint inhibitors. The characteristics of PD-L1 expression in tumor cells inside lymphovascular spaces are largely unknown. Although PD-L1 expression in circulating tumor cells within vascular spaces had been studied, results were conflicting due to lack of standardized PD-L1 expression assessment. MethodsWe investigated PD-L1 expression in lung cancer primary tumor tissue, lymphovascular tumor emboli, and lymph node metastasis using the standard PD-L1 immunohistochemistry 22C3 pharmDx assay. PD-L1 expression was scored in the primary tumor, lymphovascular emboli, and lymph node metastasis by a pathologist using the tumor proportion score (TPS). ResultsWe collected and analyzed surgical specimens from 36 patients with lung cancer with lymph node metastasis. In the primary tumor, 64% of cases were PD-L1 negative (TPS < 1%), 25% were PD-L1 low (TPS 1%–49%), and 11% were PD-L1 high (TPS ≥ 50%). In contrast, in lymphovascular tumor emboli, 89% of cases were PD-L1 negative, 11% were PD-L1 low, and none were PD-L1 high. In lymph node metastases, 72% of cases were PD-L1 negative, 17% were PD-L1 low, and 11% were PD-L1 high. ConclusionsWe observed a significant decrease of PD-L1 expression in lymphovascular tumor emboli compared with that in primary tumors (p = 0.002). Whether such differences are related to intrinsic tumor cell heterogeneity or extrinsic factors such as the microenvironment warrants further investigation.

Circulating Tumor DNA and Late Recurrence in High-Risk Hormone Receptor-Positive, Human Epidermal Growth Factor Receptor 2-Negative Breast Cancer.

To examine the prevalence and dynamics of circulating tumor DNA (ctDNA) and its association with metastatic recurrence in patients with high-risk early-stage hormone receptor-positive breast cancer (HR+ BC) more than 5 years from diagnosis. We enrolled 103 patients with high-risk stage II-III HR+ BC diagnosed more than 5 years prior without clinical evidence of recurrence. We performed whole-exome sequencing (WES) on primary tumor tissue to identify somatic mutations tracked via a personalized, tumor-informed ctDNA test to detect minimal residual disease (MRD). We collected plasma at the time of consent and at routine visits every 6-12 months. Patients were followed for clinical recurrence. In total, 85 of 103 patients had sufficient tumor tissue; of them, 83 of 85 (97.6%) patients had successful whole-exome sequencing. Personalized ctDNA assays were designed targeting a median of 36 variants to test 219 plasma samples. The median time from diagnosis to first sample was 8.4 years. The median follow-up was 10.4 years from diagnosis and 2.0 years from first sample. The median number of plasma samples per patient was two. Eight patients (10%) had positive MRD testing at any time point. Six patients (7.2%) developed distant metastatic recurrence, all of whom were MRD-positive before overt clinical recurrence, with median ctDNA lead time of 12.4 months. MRD was not identified in one patient (1.2%) with local recurrence. Two of eight MRD-positive patients had not had clinical recurrence at last follow-up. In this prospective study, in patients with high-risk HR+ BC in the late adjuvant setting, ctDNA was identified a median of 1 year before all cases of distant metastasis. Future studies will determine if ctDNA-guided intervention in patients with HR+ BC can alter clinical outcomes.

MODL-10. Tumor-brain-organoids as a model for pediatric brain tumors research

Abstract BACKGROUND: Embryonal brain neoplasms like atypical teratoid rhabdoid tumor (ATRT) or embryonal tumor with multilayered rosettes (ETMR) still have a very poor outcome despite intensive treatment including chemotherapy, irradiation and surgery. To date, precision oncology has identified clinically relevant innovative therapeutic targets only for a minor subpopulation of pediatric brain tumor patients, which may be due to current in vitro screens not recapitulating the cellular heterogeneity and cellular interactions in vivo. As cellular heterogeneity and cellular interactions majorly influence the response of tumor cells to treatment, we established an innovative 3D screening platform that combines human neural tissue surrounding primary tumor tissue. METHODS: We established a model of tumor-brain-organoids (TBO) by incorporating embryonal tumor cells (ATRT and ETMR tumor cells) into hiPSC-derived forebrain organoids. Using whole mount immunostaining (WMI), we evaluated cancer-phenotype, the neuronal and progenitor cell distribution in brain organoids, and we performed drug screening analysis. Furthermore, we used single-cell RNA-sequencing to characterize the cellular heterogeneity and the effect of tumor-organoid cell-cell communication on transcriptional programs. RESULTS: ATRT as well as ETMR tumor cells incorporated extensively into the organoid tissue. We observed remarkable differences in the invasiveness of ATRT-MYC cells into TBO in comparison with ATRT-SHH and ETMR cells via high content imaging. Moreover, tumor cells affected the gene expression of different cell types of the organoid by upregulating genes of important signaling/growth related pathways (e. g. MAP2K2, IGFBP2) and epigenetic regulators (like BRD7). Screening through a 300 compound FDA-approved drug library in these TBO, we identified potential innovative therapeutic approaches against these embryonal tumors. CONCLUSION: Tumor-brain-organoids can be used as a platform to study tumor biology, tumor interactions with its neural tissue microenvironment, as well as for high-throughput drug and toxicity screening in pediatric brain tumor precision oncology.

Establishment and characterization of NCC-GCTB5-C1: a novel cell line of giant cell tumor of bone

Giant cell tumor of bone (GCTB), is a rare intermediate malignant bone tumor with high local infiltrative ability, and is genetically characterized by mutation in the H3-3A gene. Standard treatment is curative surgical tumor resection. GCTB demonstrates both local recurrence and pulmonary metastasis after surgical treatment, and effective systematic chemotherapy is yet to be established. Therefore, development of novel chemotherapies for GCTB is necessary. Although patient-derived tumor cell lines are potent tools for preclinical research, 15 GCTB cell lines have been reported to date, and only four are publicly available. Thus, this study aimed to establish and characterize a novel GCTB cell line for preclinical studies on GCTB. Herein, we described the establishment of a cell line, NCC-GCTB5-C1, from the primary tumor tissue of a patient with GCTB. NCC-GCTB5-C1 was shown to harbor a mutation in the H3-3A gene, which is typical of GCTB; thus, it has useful properties for in vitro studies. We conducted the largest integrated screening analysis of 214 antitumor agents using NCC-GCTB5-C1 along with four GCTB cell lines. Romidepsin (a histone deacetylase inhibitor), camptothecin, and actinomycin D (topoisomerase inhibitors) demonstrated remarkable antitumor effects, suggesting that these antitumor agents are potential therapeutic candidates for GCTB treatment. Therefore, the NCC-GCTB5-C1 cell line could potentially contribute to the elucidation of GCTB pathogenesis and the development of novel GCTB treatments.

DNA Damage Repair Proteins, HSP27, and Phosphorylated-HSP90α as Predictive/Prognostic Biomarkers of Platinum-based Cancer Chemotherapy: An Exploratory Study.

Platinum analogs are commonly used for cancer treatment. There is increasing interest in finding biomarkers which could predict and overcome resistance, because to date there is no reliable predictive/prognostic marker for these compounds. Here we studied the immunohistochemical expression of proteins involved in DNA damage response and repair (γH2AX, 53BP1, ERCC1, MLH1, and MSH2) in primary tumor tissues from patients treated with platinum-based chemotherapy. Levels and localization of Heat Shock Protein (HSP)27 and phospho-(Thr5/7)-HSP90α (p-HSP90α) were also determined. The implications in clinical response, disease-free survival and overall survival were analyzed. High γH2AX and 53BP1 expressions were associated with poor clinical response. Nuclear p-HSP90α, as well as nuclear absence and low cytoplasmic expression of HSP27 correlated with good response. Patients with high γH2AX and high cytoplasmic HSP27 expressions had shorter overall survival and disease-free survival. MLH1, MSH2, or ERCC1 were not associated with clinical response or survival. We report the potential utility of p-HSP90α, HSP27, γH2AX, and 53BP1 as predictive/prognostic markers for platinum-based chemotherapy. We present the first study that evaluates the predictive and prognostic value of p-HSP90α in primary tumors. Our research opens new possibilities for clinical oncology and shows the usefulness of immunohistochemistry for predicting chemotherapy response and prognosis in cancer.

BASECAMP-1: Leveraging human leukocyte antigen (HLA) loss of heterozygosity (LOH) in solid tumors by next-generation sequencing (NGS) to identify patients with relapsed solid tumor for future logic-gated Tmod CAR T-cell therapy.

TPS2676 Background: Solid tumors comprise &gt; 90% of cancers. Metastatic colorectal cancer, non-small cell lung cancer, and pancreatic cancer are among the leading causes of cancer-related mortality (5-year overall survival: 14%, 6%, and 3%, respectively) (ACS. 2021). Chimeric antigen receptor (CAR) T-cell therapy has demonstrated clinical efficacy in hematologic malignancies (Neelapu S. et al. N Engl J Med. 2017). Translating engineered T-cell therapies to solid tumors has proven to be challenging due to a lack of tumor-specific targets that can discriminate cancer cells from normal cells. Previous studies using carcinoembryonic antigen (CEA) T-cell receptors and mesothelin (MSLN) CARs resulted in dose-limiting on-target, off-tumor toxicities (Parkhurst M, et al. Mol Ther. 2011; Tanyi J. Cellicon Valley '21). To create a therapeutic safety window, Tmod CAR T-cell therapy utilizes dual-signaling receptors to create a robust NOT logic gate capable of killing tumor cells, while leaving healthy cells intact (Hamburger A, et al. Mol Immunol. 2020). The 2 receptors in Tmod CAR T-cell therapy comprise an activator that recognizes an antigen on the surface of tumor cells that may also be present on normal cells, such as CEA and MSLN, and a blocker that recognizes a second surface antigen from an allele lost only in tumor cells. The frequency of HLA LOH among advanced GI solid tumor cancers in the Tempus real-world dataset is 16.3% with a range of 15.6%-20.8% between colorectal, pancreatic, and gastroesophageal tumors (Hecht R. et al. ASCO-GI 2022. Abstract #190). As such, HLA LOH offers a definitive tumor versus normal discriminator target for CAR T-cell therapy. Different activator/blocker combinations can be engineered with the Tmod platform technology and may be applied to T cells and natural killer cells in autologous and allogeneic settings. BASECAMP-1 is a currently enrolling observational study with key objectives of 1) To identify patients with somatic HLA LOH eligible for Tmod CAR T-cell therapy, and 2) To obtain leukapheresis and feasibility for the future EVEREST Tmod CAR T-cell trial. Methods: BASECAMP-1 (NCT04981119) patient eligibility has 2 parts: 1) Patients will be initially screened to identify germline HLA-A*02 heterozygosity by central NGS. If HLA-A*02 heterozygosity is confirmed, primary archival tumor tissue will be analyzed for somatic mutations by xT-Onco NGS testing. 2) If the tumor demonstrates HLA-A*02 LOH and the patient is eligible after screening, the patient will undergo leukapheresis. Banked T cells will be available for the autologous EVEREST Tmod CAR T-cell therapy interventional study to reduce waiting time at relapse. Clinical trial information: NCT04981119.

Patient-derived organoids to predict the drug response in locally advanced or metastatic lung cancer: A real-world study.

9136 Background: Lung cancer organoids (LCOs) were expected to be the potential precision medicine approach for clinical response prediction. However, the clinical applications of both tissue and malignant serous effusions (MSE) derived LCOs were rarely reported. Our previous work demonstrated that MSE-derived LCOs maintain the genomic signature of the original tumor. In this study, we aimed to create LCOs using tissue or MSE, then validate the reliability of the model by comparing LCOs and their origin from the pathological and molecular levels. Furthermore, drug sensitivity tests of LCOs were also performed to evaluate the feasibility of LCO drug test as an approach for personalized medicine. Methods: Primary or metastatic tumor tissues were obtained from advanced lung cancer patients through core biopsy or surgically resected biopsy at the Guangdong Provincial People’s Hospital. MSEs were also collected. LCOs were generated from the obtained tissue and MSE, and the pathological features and genomic profiles were verified by analyzing the consistency with their origin. Then, the drug sensitivity scheme was formulated to follow the principles of clinical medication. In addition, proteomics analysis by 4D LC-MS/MS was also performed to analysis the molecular details of combinational therapy. Results: In our study, we generated 213 LCOs from 106 patients, mainly from MSE. The success rate to generate LCOs derived from MSE was 81.4% (131/161). The concordance rate of pathological phenotypes of LCOs samples verified by immunohistochemistry with clinical samples was 75% (63/84). In our cohort, LCO based drug sensitivity tests (LCO-DST) of targeted therapies were performed to predict the tumor response, and the AUC value of ROC analysis of osimertinib in EGFR-mutant adenocarcinoma reached 0.94(LCOs samples = 15, p= 0.0047). There were 2 patients with advanced lung adenocarcinoma, one with de novo EGFR mutation /MET amplification and the other with EGFR mutation combined with acquired RET fusion. The results of LCO based drug tests of 2 patients showed that combined targeted therapy (osimertinib plus savolitinib/cabozantinib) showed high tumor inhibition rate validated in clinical treatment and made differences. Then, 4D label-free high through-put proteomic analysis was performed in the patient with EGFR mutation and acquired RET fusion, demonstrating caspase 3 increased dramatically in combination of osimertinib and BLU-667 and the downstream proteins of EGFR and RET were down-regulated. Conclusions: LCOs derived from MSE faithfully reflected the pathological and genomic features of their original patients. The LCOs based drug test results are remarkably consistent with the tumor response. These results suggested the important prospects of LCO as an in vitro model for lung cancer precision medicine.

Prognostic values of immune landscape in Ras-mutant colorectal cancer peritoneal metastases.

e15583 Background: The peritoneal metastasis (PM) in patients with colorectal cancer (CRC) is associated with poor survival, especially in those with Ras mutations. However, knowledge on the mechanism of molecular biology in CRC peritoneal metastasis (CRCPM) is limited, and the impact of tumor immune microenvironment (TME) on PM pathogenesis and the prognosis of CRCPM remain unclear. Therefore, we characterized the TME of CRCPM and evaluated its potential diagnostic and prognostic values. Methods: This study involved 21 patients with metastatic CRC (mCRC), of whom 11 with CRCPM were classified into the experimental group and 10 with mCRC without PM (noCRCPM) were classified into the control group. Formalin-fixed and paraffin-embedded tissue of primary tumors from all patients was examined using the NanoString RNA sequencing system. The clinicopathological variables and TME biomarkers were compared using the chi-square test and Cox regression analysis. According to the results of multivariate analysis, a prognostic nomogram was generated, and its prediction ability was measured using the concordance index (C-index). Survival curves were generated using the Kaplan–Meier method, and survival comparison between groups was conducted using the log-rank test. Results: The TME in the CRCPM and noCRCPM groups was different, especially the expression of NOS2, TNSF9, KIR3DL2, MAGEA12, MRC1, KIR3DL1, and CD244 and the content of DC cells, macrophages, exhausted CD8+T cells, NK CD56dim cells, and M2 macrophages. Univariate analysis revealed that the expression of PTPN11, TIE1, MAGEA4, PDGFB, and PMS2 was significantly correlated with progression-free survival (PFS) time. These genes were subjected to the least absolute shrinkage and selection operator regression analysis and established a risk score model. Multiple Cox regression analysis showed that the risk score was a significant independent prognostic factor. However, considering the particularity of serum carcinoembryonic antigen (CEA), we built a combined model that included the CEA level and risk score. It could perform well in predicting the PFS time in patients with CRCPM (C-index 0.77). Conclusions: The TME of the primary lesion was significantly different between the CRCPM and noCRCPM groups. The model that combined the risk score and CEA level was a better outcome predictor among patients with CRCPM.

P-175 Local inflammatory biomarkers and their association with systemic inflammation as well as overall survival in primary metastatic gastroesophageal cancer patients

Immunotherapy has recently led to a major breakthrough in the treatment of metastatic gastroesophageal cancer patients. Yet, further analyses of large trials investigating checkpoint inhibitor monotherapy or combination with standard chemotherapy suggest, that only a subgroup of patients might benefit from addition of immunotherapy to chemotherapy. Despite these advances, prognosis remains poor, especially in patients with Caucasian ethnicity. Thus, novel prognostic as well as predictive biomarkers are desperately needed to improve patient management. Further knowledge on the complex local inflammatory mechanisms might help to improve our understanding on the efficacy of immunotherapy. Thus, the aim of this study was to further characterize local inflammatory processes in patients with advanced gastroesophageal cancer and investigate their association with systemic inflammation as well as the overall survival (OS). We analyzed local inflammatory biomarkers for T-cells (CD3, CD8), macrophages (CD68) and immune checkpoint inhibition (Lymphocyte-activation gene 3 (LAG3)) in previously untreated, primary tumor tissue samples of advanced gastroesophageal cancer patients treated at the Medical University of Vienna between 2003 and 2016. FFPE tissue was stained using an automated immunohistochemistry slide staining system (Roche Ventana Medical Systems Inc., Tucson, AZ, USA). Analyses of the immunohistochemical staining was performed by Definiens Tissue Studio 4.0 software. Systemic serum inflammatory parameters including leucocyte levels (WBC), C-reactive protein levels (CRP) and albumin (Alb) as well as the OS was evaluated retrospectively by hospital chart review. Local inflammatory parameters were then associated with systemic parameters and the OS using log-rank test and Kruskal-Wallis-test. Primary tumor tissue samples (localization: 29% esopheageal, 25% gastroesophageal junction, 46% stomach; histological subtype: 77% adenocarcinoma, 23% squamous cell carcinoma) of 48 patients (65% male) were analyzed. CD3 was positive in 5.6%, CD8 in 1.8%, CD68 in 2.4% and LAG3 in 0.24% of analyzed cells. Slides were then divided by the median expression into samples with more positive and less positive cells. The median OS of the cohort was 7.6 months (95%CI 5.3-9.9). Local inflammatory biomarker could not be statistically significantly associated with the OS (CD3: p=0.317; CD8: p=0.905, CD68: p=0.369; LAG3: p=0.428). In addition, no clinically significant association with systemic inflammatory parameters could be identified (CD3: CRP p=0.168, WBC p=0.706, Alb p=0.849; CD8: CRP p=0.214, WBC p=0.275, Alb p=0.340; CD68: CRP p=0.499, WBC p=0.992, Alb p=0.181; LAG3: CRP p=0.766, WBC p=0.384), only Alb was associated with LAG3 expression (p=0.048). Local inflammation might play an important role as a prognostic and predictive tool. However, the understanding of local inflammation in gastroesophageal cancer is still scarce and, thus, further research is warranted to identify promising novel biomarkers. Additional staining for further local inflammatory markers as well as expansion of sample size are underway to further characterize the local inflammatory microenvironment in advanced gastroesophageal cancer patients.

Integrated analysis of transcriptome profiles in lung adenocarcinoma with spread through air spaces.

e21103 Background: Spread through air spaces (STAS) is a recently described invasive pattern of lung cancer with a relatively poor prognosis. The current study aims to evaluate and compare the transcriptome patterns and tumor microenvironment (TME) of STAS positive and negative lung adenocarcinoma (LUAD) cases. Methods: Nineteen LUAD patients who underwent radical surgery were enrolled in the current study, of which 11 were STAS-positive and 8 were STAS-negative. We performed RNA sequencing and data analyzes of the patient’s resected primary tumor tissue samples in a College of American Pathologists accredited laboratory (Genecast Biotechnology Co., Ltd, Wuxi). Results: A total of 841 differently expressed genes (DEGs) were identified between STAS positive and negative groups, including 442 up-regulated and 399 down-regulated DEGs in STAS positive tumors. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses indicated that the 841 DEGs were significantly enriched in humoral immune response, cell adhesion molecules, and IL-17 signaling pathways, and so on. Moreover, hypoxia VHL targets up was exceedingly up-regulated in STAS positive tumors through gene set enrichment analysis (GSEA). In addition, the abundance of regulatory T cells (Tregs) showed a borderline significant trend to be higher in STAS positive cases. Conclusions: Our study is the first to report the transcriptome profiles and TME of LUAD patients with STAS. These novel findings may add to further understanding underlying biological mechanisms of STAS occurrence and help developing rationale treatments to deliver more survival benefits for STAS positive LUAD patients.

Peripheral blood mononuclear cells (PBMCs), an ideal liquid biopsy approach to evaluate systematic immunity and predict response of neoadjuvant chemo-immunotherapy in resectable NSCLC.

e20618 Background: Immune checkpoint inhibitors (ICIs) dramatically improved clinical outcomes of patients with resectable non-small cell lung cancer (NSCLC). However, ICIs are not the scenario of “One-size fits to all”. It is of great importance to explore biomarkers, especially via liquid biopsy-based approach, to predict or monitor treatment response. Besides comprehensive genomic profiling of primary tumor tissue, this study focused on gene expression profile of PBMCs at baseline as well as one week post 1st immunotherapy administration, and its association with major pathological response (MPR) of neoadjuvant chem-immunotherapy in resectable NSCLC patients. Methods: Pre-treatment tumor biopsies and PBMC at baseline (D0) and 7 days after neoadjuvant chem-immunotherapy (D7) were collected from 27 patients with stage IB-IIIB NSCLC from two prospective clinical trials (NCT04422392 and NCT04762030) between Aug 2019 and July 2021. Tumor biopsies were subjected for comprehensive genomic profiling by an NGS panel covering 571 cancer related genes, and RNA from PBMC was subjected to an RNA sequencing panel covering transcripts of 2660 genes. (Amoy Diagnostics, Xiamen, China). Single sample gene set enrichment analysis (ssGSEA) was used to assess enrichment of inflammatory-related gene sets. CIBERSORT and in-housed established DAISM-DNN algorithm were applied to estimate immune cell populations from PBMC-derived gene expression profile. Results: Patients received chemo-immunotherapy achieved 59.3% of MPR. Stronger signatures including effector T cell and IFN-γ/Effector T-cell were significantly enriched in baseline PBMC samples in MPR patients (p = 0.028, p = 0.042 respectively). In parallel, significant increasing Naïve CD8 positive T cells was observed in PBMC at D7 in MPR patients by both CIBERSORT (p = 0.047) and DAISM-DNN algorithm (p = 0.018). Interestingly, at D7, signature of M0 macrophage was enriched in PBMC from patients with non-MPR. In addition, comprehensive genomic profiling indicated a numerically higher level of tumor mutation burden (TMB) in pretreatment tumor samples in MPR patients (Median TMB, MPR 11.51 mut/Mb vs Non-MPR 7.19 mut/Mb, p = 0.074). Conclusions: Expression profile of baseline PBMC as well as dynamics of PBMC profile in a short period (7 days) may predict the clinical efficacy of neoadjuvant chemo-immunotherapy. More patients are being enrolled into this study. Moreover, to validate these findings, large-scale and prospective investigations are warranted. Clinical trial information: NCT04422392 and NCT04762030.

EA8185: Phase 2 study of bladder-sparing chemoradiation (chemoRT) with durvalumab in clinical stage III, node-positive urothelial carcinoma (INSPIRE), an ECOG-ACRIN/NRG collaboration.

TPS4617 Background: Patients [pts] withlymph node positive (LN+), non-metastatic bladder cancer (BC) have a better prognosis than those with metastatic (M1) disease. However, this population is under-represented in advanced bladder trials and ineligible for bladder-sparing trials. Therefore, there have been no larger prospective trials establishing the standard of care in LN+ BC. Given the promise of immunotherapy in advanced BC and potential synergy between immunotherapy and radiation, INSPIRE was designed to determine the role of concurrent and adjuvant durvalumab (durva) in this patient population when treated with induction chemotherapy (IC) followed by concurrent chemoRT. Methods: This is a randomized phase II study that is enrolling BC pts with stage III [N1-3 M0], pure or mixed urothelial cancer. Pts must have received ≥3 cycles of IC [either before or after registration, prior to randomization] without progression. LN+ is defined as radiologically LN ≥1.0 cm in short axis, with or without biopsy prior to IC. As long as pts do not progress on induction chemotherapy, they will be randomized to chemoRT+/- durva using 5 stratification factors (Simon Pocock minimization method) a) IC prior vs. post registration b) cisplatin vs non-cisplatin regimen during RT c) LN size d) response to IC e) extent of TURBT. Pts on the chemoRT+durva arm will get chemotherapy per physician choice + IMRT + 3 x doses of Q3wk durva for 6.5-8 wks, whereas those on the control arm will get chemoRT alone. The primary end point is clinical complete response [CR], defined as no radiologically measurable disease in the LNs and negative cystoscopy and bladder biopsy 8-10 weeks post-chemoRT +/- durva. Pts on the chemoRT + durva arm who have a CR or clinical benefit ( &gt; T0 and ≤T2 in bladder per cystoscopy, biopsy + CR/PR/SD in LN by imaging) will get adjuvant Q4wk durva for 9 doses, while those on the chemoRT arm will undergo observation. Secondary end points include OS, PFS, bladder-intact event-free survival, rate of toxicity and salvage cystectomy. This study is designed to detect an improvement of 25% in clinical CR between both arms (37.5% to 62.5%). A total accrual of 114 pts (in order to enroll 92 evaluable pts) will provide 81% power to detect this difference using a Fisher’s exact test (assuming 10% drop out + anticipating that 20% chemotherapy-naïve pts will progress post IC). We are banking blood and primary tumor tissue pre- and post-chemoRT in both groups. The study was activated in August 2020 and accrual is ongoing. We expanded eligibility to include N3 in 9/2021. INSPIRE is the first prospective study designed for only LN+ BC and will define both short-term and long-term outcomes for bladder sparing in this patient population and has the potential to define a new treatment strategy for stage III BC. Clinical trial information: NCT04216290.

Circulating tumor DNA (ctDNA) and late recurrence in high-risk, hormone receptor–positive, HER2-negative breast cancer (CHiRP).

103 Background: Hormone receptor–positive breast cancer (HR+ BC) is the most common cause of BC-related death. Over half of metastatic recurrences occur ≥ 5 years (y) from diagnosis. Detection of minimal residual disease (MRD) via circulating tumor DNA (ctDNA) can identify cancer recurrence months to years in advance and may be an important tool to guide therapy. Little is known about ctDNA in the late adjuvant setting. We investigated ctDNA dynamics and clinical outcomes in pts ≥ 5 y from diagnosis of high-risk early-stage HR+ BC. Methods: Patients with high-risk HR+ BC (T3-4 or N2-3 or T1 with 3+ lymph nodes, or T2N1 [and Oncotype RS ≥ 26; grade 3; or Ki-67 ≥ 20%]) with no evidence of recurrence 5 y after diagnosis were prospectively identified and consented. Plasma samples were collected at time of consent and at routine visits every 6-12 mos. Whole-exome sequencing (WES) was performed on primary tumor tissue to identify somatic mutations and design for each patient a RaDaR assay, a tumor-informed liquid biopsy test to detect plasma ctDNA. Per current practice standards, pts did not undergo regular surveillance imaging. All pts were followed for development of local and/or distant metastatic recurrence, as determined by their clinical provider. Results: Of 103 pts enrolled, 85 had sufficient tumor tissue, and 83 pts had successful WES. Personalized RaDaR assays were designed targeting 12-51 variants (median, 36), and used to test 219 plasma samples from 83 pts. The number of plasma samples per patient ranged from 1-7 (median, 2). 57 pts (68.7%) had stage 3 disease, and most (75, 90.4%) received curative-intent chemotherapy. All pts received endocrine therapy (ET). 39 (47%) remained on adjuvant ET at time of last follow up. Of 44 pts who completed adjuvant ET, 41 (93.2%) received &gt; 5 y of treatment. Time from diagnosis to first sample ranged from 4.9-20 y (median, 8.4 y). Median (range) follow up was 10.2 (6.7-22.3) y from diagnosis and 1.8 (0-3.6) y from first sample. 5 pts (6%) developed distant metastatic recurrence and 2 pts (2.4%) had locoregional recurrence. 4/83 (5%) pts were MRD+ at study entry and 8/83 (10%) pts were MRD+ at any time point. 5/5 (100%) pts with metastatic recurrence were MRD+, with ctDNA lead times up to 37.6 mos. ctDNA was detected at tumor fractions of 0.0027-26.84% (median, 0.396%). 2/8 (25%) MRD+ pts had not had clinical recurrence at latest follow up, one with no follow up since detection and one 15.4 mos from ctDNA detection (with ctDNA levels 0.045% and 26.84%). Conclusions: Here we report—to our knowledge—the first data on ctDNA detection in late adjuvant HR+ BC. 10% of pts had MRD at ≥ 5 y from diagnosis. ctDNA analysis identified MRD in all cases of distant recurrence. ctDNA was detected in 2 pts who have not yet experienced recurrence. Longer follow up is necessary for these pts at high risk. Additional studies will determine if ctDNA-guided intervention can alter clinical outcomes.

Clinical and prognostic features of BRAF V600E-mutant resectable colorectal cancer: A single-center retrospective analysis of Chinese patients.

e15505 Background: Molecular subtyping provides a fundamental basis for the selection of strategies for colorectal cancer treatment. Unresectable metastatic colorectal cancer (CRC) with BRAF V600E mutation have received great attentions due to the poor prognosis. Nevertheless, clinical characteristics and prognosis of Chinese patients with BRAF V600E-mutant (BM) resectable CRC remain poorly reported. Methods: Patients with BM resectable CRC were included according to the following criteria: 1) Diagnosed with colorectal adenocarcinoma at Zhongshan Hospital Fudan University from June 2015 to December 2018. 2) Underwent surgery for the primary tumor of CRC without any anti-tumor treatment before surgery. 3) With BRAF V600E mutation confirmed by the genetic test of the primary tumor tissue. 4) No other primary malignant tumors or inherited tumor history. 5) Written informed consent was obtained. Clinical information was collected by consulting electronic medical records with the approval of the Ethics Committee of Zhongshan Hospital Fudan University (Approval code: B2021-739). KRAS exon2 mutant (KM) and KRAS/BRAF wildtype (WT) patients were selected in a 1:1 ratio by matching gender, age, location and the year of surgery with the BM group. Overall survival was defined as the time from surgery to death for any reason or last follow-up (September, 2021). Categorical data were assessed by chi-square test. Survival analysis was performed using Kaplan-Meier survival curve with log-rank test, univariate and multivariate Cox regression analyses. Results: A total of 214 patients with BM resectable CRC met the inclusion criteria. Among them, 111 (51.9%) patients were females and 103 (48.1%) were males, 114 (53.3%) patients were younger than age 65 years while 100 (46.7%) were &gt; = 65 years old, 108 (50.5%) patients’ primary lesions sited on the left side and 106 (49.5%) on the right side. Numbers of patients with resectable metastasis and with mismatch-repair- deficient (dMMR) were both 48 (22.4%). 171 KM patients and 165 WT patients were matched with BM patients and the three groups were balanced in gender, age, and location by matching. The BM group had the worst overall survival ( P &lt; 0.001) with significant higher proportions of stage IIĨIV ( P = 0.002) and dMMR patients ( P &lt; 0.001). Survival analysis in subgroups suggested that the inferior prognosis of the BM group was more pronounced in patients with pMMR or Stage IV CRC. Conclusions: BRAF V600E-mutant resectable CRC presented a higher incidence of dMMR but a worse prognosis than KRAS exon2 mutant or KRAS/BRAF wildtype patients.

When “No-Smoking” is not enough: Hypoxia and nicotine acetylcholine receptor signaling may drive lung adenocarcinoma progression in never-smokers

The question of how lung cancer progresses in never-smokers remains largely unanswered. In our analysis of data from 1727 lung cancer patients, we observed a difference of only 47 days in the overall survival between lung adenocarcinoma patients who were smokers vis-a-vis never-smokers - the disease has a poor prognosis irrespective of the smoking status, or gender. We have investigated the possible collaboration between the nAChR and hypoxia signaling pathway to explicate a mechanism of disease progression in never-smokers using patient-derived tumor cells. We found a previously unidentified increase in both acetylcholine and nAChR-α7 levels in non-small cell lung cancer cells in hypoxia. A similar increase in ubiquitously expressed nAChR-α7 transcripts was also observed in other cancer lines and primary tumor tissues. A direct binding of HIF-1α with the hypoxia-response element (HRE) present at −48 position preceding the transcriptional start site in nAChR-α7 promoter region was established. Crucially, the increased acetylcholine levels in hypoxia drove a feedback loop via modulation of PI3K/AKT pathway to stabilize HIF-1α in hypoxia. Further, hypoxia-mediated metastasis and induction of HIF-1α in these cells was significantly reversed by bungarotoxin, an antagonist of nAChR-α7. The nAChR-AKT-HIF network needs to be further investigated to conclusively prove its mechanism and to explore its therapeutic potential. Our study gives a plausible explanation for the equally worse prognosis of lung adenocarcinoma in never-smokers wherein the nAChR signaling is enhanced in hypoxia by acetylcholine in the absence of nicotine.

Tumor-derived IL-8 facilitates lymph node metastasis of gastric cancer via PD-1 up-regulation in CD8+ T cells

BackgroundThe pretherapeutic serum interleukin-8 (sIL-8) levels have been revealed to be increased in about half of patients with locally advanced gastric cancer. However, the roles of IL-8 in lymph node metastasis (LNM) and the underlying mechanisms remain unclear.Methods146 patients with primary gastric carcinoma were enrolled in this study. ELISA was used to measure IL-8 levels. The CD4/CD8 ratio and programmed cell death-1 (PD-1) expression of T cells in primary tumor tissues, tumor-draining lymph nodes (TDLNs) and non-draining lymph nodes (NDLNs) were assayed with flow cytometry. Protein expression of the molecules was determined with immunohistochemistry, western blotting or immunoprecipitation. The gastric cancer mouse tumor model with LNM was utilized to determine the role of IL-8 in regulation of tumor metastasis and progression.ResultsThe elevated sIL-8 levels were associated with LNM and poor prognosis in gastric cancer. Furthermore, sIL-8 was identified to be prominently produced by gastric cancer-associated fibroblasts (CAFs). Elevated IL-8 can up-regulate PD-1 expression in CD8+ T cells, resulting in immunosuppression in primary tumors and TDLNs, which enhances LNM of gastric cancer. Molecularly, IL-8 increases PD-1 expression through JAK2/STAT3 signaling activation, and inhibits its ubiquitination via Fbxo38 down-regulation. In addition, the in vivo studies in mouse gastric cancer model demonstrated that IL-8 promotes LNM via PD-1 up-regulation in CD8+ T cells.ConclusionThe present study elucidates the pro-metastatic role of elevated IL-8 in gastric cancer, and provides novel insights to enhance immune checkpoint blockade therapy for anti-PD-1 in gastric cancer.

Knockdown of the stem cell marker Musashi-1 inhibits endometrial cancer growth and sensitizes cells to radiation

BackgroundEndometrial carcinoma is the most common gynecological cancer in Europe. Musashi-1 is known to be a key regulator of endometrial cancer stem cells and a negative prognostic marker. In the present study, we aimed to understand growth and gene expression patterns in endometrial carcinoma after Musashi-1 knockdown in vitro and in vivo. Changes in therapeutic resistance were also assessed.MethodsFirst, we performed analyses to understand Musashi-1 expression patterns using The Cancer Genome Atlas database. We then proceeded to assess effects of small interfering RNA-based Musashi-1 targeting in two endometrial carcinoma cell lines, Ishikawa and KLE. After quantifying baseline changes in cell metabolism, we used MTT tests to assess chemotherapy effects and colony formation assays to understand changes in radioresistance. For mechanistic study, we used quantitative polymerase chain reaction (qPCR) and western blotting of key Musashi-1 target genes and compared results to primary tissue database studies. Finally, xenograft experiments in a mouse model helped understand in vivo effects of Musashi-1 knockdown.ResultsMusashi-1 is aberrantly expressed in primary tumor tissues. In vitro, silencing of Musashi-1 resulted in a strong decline in cell proliferation and radioresistance, while chemoresistance remained unchanged. Loss of Musashi-1 led to downregulation of telomerase, DNA-dependent protein kinase, the Notch pathway and overexpression of cyclin-dependent kinase inhibitor p21, the latter of which we identified as a key mediator of Msi-1 knockdown-related anti-proliferative signaling. In vivo, the anti-proliferative effect was confirmed, with Msi-1 knockdown tumors being about 40% reduced in size.ConclusionsMusashi-1 knockdown resulted in a strong decrease in endometrial cancer proliferation and a loss of radioresistance, suggesting therapeutic potential.

Prognostic Value of Stromal Type IV Collagen Expression in Small Invasive Breast Cancers.

Breast cancer is the most common cause of cancer death among women worldwide. Localized breast cancer can be cured by surgery and adjuvant therapy, but mortality remains high for tumors that metastasize early. Type IV collagen is a basement membrane protein, and breach of this extracellular matrix structure is the first step of cancer invasion. Type IV collagen is found in the stroma of many cancers, but its role in tumor biology is unclear. Here, expression of type IV collagen in the stroma of small breast cancers was analyzed, correlated to clinically used prognostic biomarkers and patient survival. The findings were further validated in an independent gene expression data cohort. Tissue samples from 1,379 women with in situ and small invasive breast cancers (≤15 mm) diagnosed in 1986-2004 were included. Primary tumor tissue was collected into tissue microarrays. Type IV collagen expression in tissues was visualized using immunohistochemistry. Gene expression data was extracted from the Cancer Genome Atlas database. Out of 1,379 women, 856 had an invasive breast cancer and type IV collagen staining was available for 714 patients. In Kaplan-Meier analysis high type IV collagen expression was significantly associated (p = 0.026) with poorer breast cancer specific survival. There was no correlation of type IV collagen expression to clinically used prognostic biomarkers. High type IV collagen expression was clearly associated to distant metastasis (p = 0.002). In an external validation cohort (n = 1,104), high type IV collagen mRNA expression was significantly (p = 0.041) associated with poorer overall survival, with overexpression of type IV collagen mRNA in metastatic tissue. Stromal type IV collagen expression in the primary tumor correlates to poor breast cancer specific survival most likely due to a higher risk of developing distant metastasis. This ECM protein may function as biomarker to predict the risk of future metastatic disease in patients with breast cancers.

Double-Matched Matrix Decomposition for Multi-View Data

We consider the problem of extracting joint and individual signals from multi-view data, that is, data collected from different sources on matched samples. While existing methods for multi-view data decomposition explore single matching of data by samples, we focus on double-matched multi-view data (matched by both samples and source features). Our motivating example is the miRNA data collected from both primary tumor and normal tissues of the same subjects; the measurements from two tissues are thus matched both by subjects and by miRNAs. Our proposed double-matched matrix decomposition allows us to simultaneously extract joint and individual signals across subjects, as well as joint and individual signals across miRNAs. Our estimation approach takes advantage of double-matching by formulating a new type of optimization problem with explicit row space and column space constraints, for which we develop an efficient iterative algorithm. Numerical studies indicate that taking advantage of double-matching leads to superior signal estimation performance compared to existing multi-view data decomposition based on single-matching. We apply our method to miRNA data as well as data from the English Premier League soccer matches and find joint and individual multi-view signals that align with domain-specific knowledge. Supplementary materials for this article are available online.