Tumor Samples Research Articles

Utility of circulating tumor DNA (ctDNA) from cerebrospinal fluid (CSF) for prognosis of patients with recurrent high grade glioma.

2010 Background: High-Grade Gliomas (HGGs) are the most aggressive primary brain tumors in adults and molecular characterization is crucial for diagnosis and optimal disease treatment. Due to the eloquent location of many HGGs, upfront or repeat tumor sampling may be sub-optimal. Hence, there is an urgent need to develop alternative, minimally invasive means to obtain diagnostic information and determine the expected clinical behavior. Here we report that circulating tumor DNA (ctDNA) from cerebrospinal fluid (CSF) can be used to identify disease defining genomic alterations and to track clonal evolution. Additionally, we demonstrate that detection of CSF ctDNA is positively correlated with leptomeningeal disease and overall survival suggesting that CSF ctDNA should be integrated into clinical decision making in the clinic. Methods: Our study includes 313 samples from 253 patients with recurrent glioma treated at Memorial Sloan Kettering Cancer Center who underwent CSF collection for routine clinical care and were sequenced using the MSK-IMPACT targeted clinical sequencing assay (468 and 505 genes). Alterations were classified as oncogenic based on OncoKB. 17% of genomic alterations detected were identified using a secondary bioinformatics analysis, following an informed approach with less stringent mutation calling criteria and Gaussian Mixture Model to call copy number events. CSF ctDNA positivity was determined by the presence of at least one oncogenic disease defining alteration or any shared alteration with the tumor. Results: Within this cohort, we found 193 CSF ctDNA positive (62%) and 120 CSF negative (38%) samples. Of note, CSF ctDNA positivity was the highest amongst histone mutant tumors with 94% CSF ctDNA positivity compared to 56% CSF ctDNA positivity for IDH-WT tumors. We noticed 44% of alterations shared between the tumor and the CSF, additionally, we noted considerable tumor evolution particularly within the growth signaling pathways (e.g. PDGFRA). The majority of the shared alterations were clonal, and we noticed the emergence of new clonal events in the CSF. Conclusions: Our cohort demonstrated that patients with positive CSF ctDNA had a significantly shorter overall survival compared to those who were CSF ctDNA negative (4.83 months vs 11.83 months, HR 2.1, p < 0.001). In tandem with secondary clinical analysis, radiographic findings also correlated with CSF ctDNA positivity. Specifically, the presence of enhancing disease and contact of the tumor with the ventricular space were positively associated with detection of CSF ctDNA. Additionally, CSF ctDNA was associated with positive cytology in 21/21 cases (100%). With our improved bioinformatics pipeline, we hypothesize ctDNA from CSF may be used as a prognostic biomarker for survival, but confirmation requires further validation in a prospective study.

Clinical application of individualized tumor-informed circulating tumor DNA for therapeutic response and relapse prediction in patients with neuroblastoma.

10053 Background: Patients with high-risk neuroblastoma (NB) generally present with widely metastatic disease and often relapse despite intensive therapy. At present, circulating tumor DNA (ctDNA)-based minimal residual disease (MRD) has been studied in various tumors and proven to be valuable in guiding treatment and predicting recurrence, but its application in NB has rarely been reported. This study aimed to assess the effectiveness of tumor-informed ctDNA monitoring for evaluating the response to induction therapy in NB patients, as well as its role in predicting the risk of relapse. Methods: We conducted a prospective cohort study of NB patients. Primary tumor samples were collected at baseline and blood samples were collected sequentially from baseline until disease progression or the last follow-up. Tumor tissue samples were subjected to whole exome sequencing (WES), and clonal mutations were selected for the individualized MRD panel. Sequencing was conducted at OrigiMed, a College of American Pathologists accredited and Clinical Laboratory Improvement Amendments certified laboratory. Results: Our cohort included 43 individuals diagnosed with NB, with a median age of 3.5 years (0.42-40 years). WES revealed that 75% (2054/2753) of the mutated genes were unique to each patient, and 98.9% (704/712) of the selected tumor-informed single nucleotide variants were variants of unknown significance, suggesting that individualized tumor-informed MRD (n = 23) is superior to fixed tumor-agnostic panel (n = 20) MRD in NB. A successful individualized MRD panel was constructed for 53.5% (23/43) of patients, with a remarkable 92.3% success rate for those older than 5 years (p < 0.01). The initial positivity rate for ctDNA detection was 68%, with the initial positivity rate being associated with the stage (p < 0.01). During treatment and follow-up, 0/6 of the patients whose ctDNA levels remained negative experienced a relapse, whereas 2/5 patients who had a negative conversion or an increase in positivity experienced a relapse. In addition, ctDNA changes matched radiological evaluations in 94% of patients. Notably, ctDNA was detected prior to radiological relapse, with a lead time of 6 months in one patient. Patients can be divided into four groups according to the mutation landscape: ALK mutation, MYCN amplification, chromosome amplification, and other groups. Among them, the rate of ctDNA clearance was the highest in the chromosome amplification group, indicating that patients in this group were the most sensitive to chemotherapy. Conclusions: Individualized tumor-informed ctDNA monitoring shows promise for evaluating therapeutic response and forecasting relapse in NB patients. This approach proves more effective than fixed tumor-agnostic panel-based MRD due to high genetic mutation uniqueness. Clinical trial information: NCT05076071 .

YB-800, a monoclonal antibody targeting the human neuronal pentraxin receptor (NPTXR), as a potential candidate for the design of a first in class antibody drug conjugate (ADC) as novel therapeutic intervention in gastro-intestinal cancers.

e15022 Background: The human neuronal pentraxin receptor (NPTXR) gene encodes a type II transmembrane protein that functions as a trans-synaptic organizer and anchors neuronal pentraxin complexes to plasma membranes. Beside a subset of neuronal cells (Hippocampus and Cerebral Cortex), expression of NPTXR is not detected in all other tissues [ The Human Protein Atlas database]. In recent studies [Kanda et al. (2020) Mol. Cancer. doi: 10.1186/s12943-020-01251-0], transcriptome and bioinformatics analysis of gastric cancer (GC) tissues from patients with or without metastasis have revealed that NPTXR expression was associated with disease progression. NPTXR expression was also correlated negatively with survival in GC patients. It appears that NPTXR does not have a major physiological function; e.g., NPTXR−/− mice showed no abnormalities in reproduction, development, metabolism, or motor function. NPTXR is expressed in a number of GI cancers, i.e., gastric, esophageal, colorectal, pancreatic, bladder, liver, as well as breast and thyroid cancer. YB-800 is a fully humanized monoclonal antibody against NPTXR (identified by in silico analysis). Methods: The present studies were conducted to provide a rationale for the design of a YB-800-based antibody drug conjugate (ADC) for therapeutic intervention in oncology. Hence, YB-800 binding to and internalization in selected tumor cells, as well as immunohistochemistry (IHC) in tumor biopsies, were evaluated. Results: Taken together experimental data confirm that: (i) YB-800 binds to a new target (NPTXR) on selected tumor cells; confirmed by FACS analysis; (ii) IHC analysis in tumor samples demonstrates specific immunoreaction in bladder, gastric, prostate, hepatocellular carcinoma, colorectal cancer and cervical cancer, but not in adjacent healthy tissues; and (iii) once YB-800 binds to NPTXR, the conjugate is internalized in selected tumor cells in a time-dependent manner. Conclusions: In conclusion, YB-800 represents a first in class ADC candidate that could be used as novel treatment for gastro-intestinal cancers. Hence, as initial proof of concept, tesirine (DNA intercalating agent) and deruxtecan (topoisomerase I inhibitor) containing protease-sensitive linkers were coupled to YB-800 by stochastic maleimide conjugations to interchain cysteines. Initial results with YB-800-based ADCs will be discussed.

Her-2/neu gene amplification and its correlation with other prognostic factors in Iranian breast cancer patients.

Breast cancer is one of the most common diseases in females, arising from overexpression of a variety of oncogenes like HER2/neu. The amplification rate of this gene is variable in different breast cancer patients. In this study, the amplification of the HER2/neu oncogene was distinguished in breast cancer patients and its correlation with prognostic factors. Also, the simultaneous effect of prognostic factors on the occurrence of a specific prognostic factor was investigated. The multiplex PCR technique was used to assay the amplification of the HER2/neu oncogene in breast cancer patients. After extracting DNA from 100 tumor tissue and 8 normal breast tissue samples, the amplification of the HER2/neu gene was distinguished by the co-amplification of a single-copy reference gene, γ-IFN, and the target gene HER2/neu in the PCR reaction and using the Gel analyzer software. SPSS 23 and STATA 9.1 software were used for statistical analysis. The HER2/neu gene was amplification in 30% of the tumor samples. The statistical analysis showed a statistically significant relationship between HER2/neu gene amplification and progesterone receptors. Amplification of the HER2/neu gene significantly increases the chance of lymph node involvement. Also, the amplification of this gene in tumors with histological grade II tissue is more than grade I. The amplification of the HER2/neu gene can be used as an independent prognostic factor in predicting lymph node involvement and histological grade in breast cancer patients.

Tumor origin identification through machine learning and gene expression profiling.

e13597 Background: Accurate identification of tumor origin is crucial for effective diagnosis and treatment, particularly in cases of metastatic tumors. Interpretable machine learning models have displayed significant potential in addressing this problem when imaging and immunohistochemistry (IHC) examinations are ineffective. In this study, we developed a panel using gene expression profiles from various tumors and constructed a robust machine learning model for precise identification of tumor origin. Methods: RNA sequencing (RNA-seq) data of 9462 tumor samples originating from 21 different organs were collected from The Cancer Genome Atlas (TCGA). We conducted feature engineering through unsupervised clustering and differential gene expression analysis, selecting a refined panel of 164 genes from a pool of over 60,000 identifiers. Subsequently, a machine learning classifier grounded in Logistic regression (LR) was trained on 9462 samples with the constructed 164-gene panel. To enhance the adaptability of our model to this task, 10-fold cross-validation was employed in the multi-class mode. Two independent test sets, the Primary tumor set (PT, n=3420, including 19 tumor types) and the Metastatic tumor set (MTP, n=100, all originating from the prostate and spread to bone, liver, etc.), were established with samples from Gene Expression Omnibus (GEO) and other published studies. Notably, all samples underwent FPKM normalization and there was no overlap between training and testing samples. Model performance was assessed using accuracy, specificity, and sensitivity metrics. Results: The 164-gene expression panel achieved a cross-validation accuracy of 96.73%. In the assessment of the PT test set, our model achieved an overall 99.62% specificity, 92.31% accuracy and 89.79% sensitivity, exhibiting performance comparable to similar models in other studies. Additionally, our model accurately traced 91% of metastatic tumors in the MTP test set to the prostate, surpassing previous lines of work by a large margin. Conclusions: Gene expression patterns reveal organ-specific characteristics that could be used to identify tumor origin. The combination of a condensed yet comprehensive gene expression panel with a robust machine learning model serves as a promising tool for tumor diagnosis. Ongoing correlative studies aim to extend predictions from tissue organs to cancer subtypes.

Analysis of the sensitivity to endocrine therapy (SET) assay in the PALLAS adjuvant trial of palbociclib in HR+/HER2- breast cancer (ABCSG-42/AFT-05/BIG-14-13).

538 Background: The phase 3 PALLAS trial compared 2 years of the CDK4/6 inhibitor palbociclib with endocrine therapy of provider choice, versus endocrine therapy alone, as adjuvant treatment for patients (pts) with Stage II-III hormone receptor-positive HER2-negative (HR+/HER2-) breast cancer. The SET2,3 index adjusts the measurement of SETER/PR index of endocrine receptor-related transcriptional activity with a baseline prognostic index (BPI) derived from tumor size (T), number of involved lymph nodes (N) and gene expression levels of ESR1, PGR, ERBB2 and AURKA (RNA4 subtype score). High SET2,3 (index ≥ 2.10) represents high endocrine sensitivity with favorable prognosis. Clinical outcome results from PALLAS were previously published, and the trial now has 5-year median follow-up. Methods: Participants in PALLAS provided a tumor tissue block prior to randomization. RNA was extracted from samples with sufficient tumor tissue and cellularity (>25mm2 with ≥20% cancer nuclei). SET2,3 index was measured from 300ng total RNA using the QuantiGene Plex platform (ThermoFisher, Waltham, MA) following the manufacturer’s protocol and using pre-defined quality control (QC) parameters and cut point. Results from primary tumor samples prior to systemic therapy were analyzed. Invasive disease-free survival (iDFS) was summarized with Kaplan-Meier plots using log-rank test. Cox models were adjusted for treatment arm assignment, with a treatment arm by SET index value interaction term to test for prediction of palbociclib benefit. The pre-defined level of significance was a two-sided 0.05. Results: There were 4075 pts from the total PALLAS population (N=5796) who provided a primary tumor sample that met pre-analytical requirements to perform the SET assay, from which 3388 (83%) results passed quality control, including 3093 from a sample obtained prior to receiving systemic therapy (1559 in palbociclib arm and 1534 in the control arm). SET2,3 index could not be calculated for 12 pts due to missing tumor or nodal information. There was no evidence of an interaction between treatment arm and SET indices (continuous) for iDFS: SET2,3 index interaction p=0.12 and SETER/PR index interaction p=0.92. Estimated 5-year iDFS was superior for those with high SET2,3: iDFS was 91.4% (95%CI 89.9–92.8) in 1719 (55.8%) pts with high SET2,3, versus 78.5% (95%CI 76.0–80.8) in 1362 (44.2%) pts with low SET2,3 (HR 0.38, 95%CI 0.31–0.47, p<0.0001). The SETER/PR index was also significantly associated with iDFS (HR 0.70 per 0.5 units, 95%CI 0.65–0.77, p<0.0001). Conclusions: There was no evidence that the SET2,3 or SETER/PR index of endocrine-related transcriptional activity predicts benefit from palbociclib treatment. SET2,3 index was strongly prognostic for pts with Stage II-III breast cancer, with excellent 5-year IDFS for pts classified as SET2,3 high. Clinical trial information: NCT02513394 .

Association of tumor-infiltrating lymphocytes (TILs) and immune signatures with PAM50 intrinsic subtyping hormone receptor-positive (HR+)/HER2-negative (HER2-) early breast cancer (BC): A translational analysis of two multicentric neoadjuvant trials.

558 Background: HR+/HER2− BCs are generally described as immunologically cold, with low levels of TILs and PD-L1. Nevertheless, the addition of immunotherapy (ICI) to chemo has been reported to increase pCR rates in high-risk early HR+/HER2– BCs. Assessing the interaction between immune features and tumor biology may identify of a subset of patients (pts) with HR+/HER2− BC that would be the ideal candidates for the combination of chemo and ICI. Methods: Gene-expression data from two multicentric neoadjuvant phase II trials (GIADA, LETLOB) enrolling stage II-IIIA HR+HER2- BC pts was retrieved (Dieci, CCR 2022; Guarneri, JCO 2014). In the GIADA trial, 758 genes were assessed on baseline tumor samples by nCounter (N=43). In the LETLOB trial, gene-expression data (Affymetrix platform) from baseline core-biopsies was available for 66 pts. Intrinsic subtype was assigned using a research-based PAM50 predictor. TILs were evaluated following guidelines. Association of TILs and immune signatures with intrinsic subtyping was assessed by Kruskal-Wallis test. Results: PAM50 subtype distribution (N=109) was 44% Luminal B (LumB), 33% Luminal A (LumA), 18% Basal-like, 5% HER2-enriched (HER2-E). TILs were assessed on 101 samples: median 2 (range 0-100). Basal-like BCs showed higher TILs than other subtypes (p=0.008). No significant difference was observed between LumA and LumB BCs (Table). Basal-like tumors showed higher levels of previously published immune signatures associated with CD8 T-cells (p<0.001), Cytotoxic cells (p<0.001), IFN-gamma (p<0.001), and response to ICI in the GeparNuevo trial (p=0.003) (N=109). PD-1 (p=0.002), PD-L1 (p=0.001), and PD-L2 (p=0.001) genes were also more expressed in Basal-like BCs (evaluable in 43 samples). No significant difference was observed between LumA and LumB tumors. A significant weak to moderate positive correlation was observed between continuous TILs and immune signatures and basal-like PAM50 signature (p<0.001, Spearman coefficient=0.372 for TILs, from 0.354 to 0.648 for immune signatures). Conclusions: PAM50 intrinsic subtyping and TILs/immune signatures capture only partially overlapping information on the biology of HR+/HER2- BCs. Pts with HR+/HER2- BCs simultaneously showing features of biological aggressiveness and enhanced immunogenicity may represent the ideal candidates for the combination of ICI and chemo. [Table: see text]

Expression of alternatively spliced PD-L1 RNA with skipping of transmembrane domain.

e14596 Background: Programmed death-ligand 1 (PD-L1) is a transmembrane ligand for the programmed cell death protein 1 (PD-1), a receptor that inhibits T-cell activity. The PD-L1/PD-1 immune checkpoint axis has been successfully targeted to enhance antitumor immune responses. This gene is composed of seven exons. Exons 1-4 are responsible for the extracellular domain, exon 5 for the transmembrane domain, and exons 6-7 for the intracellular domain. Emerging data indicates that alternative splicing in genes is an important mechanism for expression of different isoforms that may influence antibody-based therapies. We explored the potential of the presence of alternative splicing in PD-L1 transcripts via sequencing of PD-L1 RNA in patients with various type of solid tumor cancers. Methods: RNA was extracted from 3443 FFPE solid tumor samples including lung, colon, pancreas, breast, and other cancer types. In addition, cfRNA was extracted from 532 peripheral blood plasma samples from patients with solid tumor cancers. RNA was sequenced using a hybrid capture targeted RNA panel on FFPE samples and peripheral blood with analysis focused on PD-L1 gene transcript. Quantification of RNA transcript was done using Salmon algorithm. Results: Of the 3443 FFPE samples, 38 (1.2%) samples showed alternative splicing involving deletion of exon 5 (PD-L1∆Ex5). The alternative splicing was in 2.10% of lung cancer, 2% of colon cancer, 0.70% of pancreatic cancer, 1.40% of gastric cancer, 0.38% of breast cancer, 0.30% of ovarian cancer, 1.10% of sarcoma cases, 1.50% of glioblastomas, 3.30% of thyroid cancer and 2% of urothelial cancer. Of the 532 cfRNA samples, 20 (3.7%) showed PD-L1∆Ex5. The median percentage of PD-L1∆Ex5 transcripts was 2.2% of total PD-L1 transcripts in tissue samples as compared with 6.6% in cfDNA samples. In tissue samples, there was a correlation between levels of PD-L1 and PD-L1∆Ex5 (r = .567, p < .001). In contrast, cfRNA showed no significant correlation between levels of PD-L1 and levels of PD-L1∆Ex5 (r = .268). Conclusions: PD-L1 exon 5 skipping was detected in a number of patients with different types of solid tumor cancers. The percentage of skipping transcripts is low in cells and relatively higher in cfRNA. Since exon 5 skipping deletes the transmembrane domain, the PD-L1 protein may remain in the cytoplasm or perhaps is secreted as cell-free PD-L1 protein. The demonstration of relatively higher levels of PD-L1∆Ex5 in cfRNA is likely reflecting higher turnover of cells and raises the possibility that cells with this isoform of PD-L1 are more aggressive than cells without the expression of this isoform. Further studies are needed to explore the clinical relevance of the expression of such abnormal PD-L1 protein on treatment with immune checkpoint inhibitors.

Genomic alterations in DNA damage response (DDR) genes in HR+/HER2- metastatic breast cancer (mBC) and impact on clinical efficacy with sacituzumab govitecan (SG): Biomarker results from TROPICS-02 study.

1075 Background: In the phase 3 TROPiCS-02 study, SG, a Trop-2–directed antibody-drug conjugate coupled to SN-38 as the payload, demonstrated clinically meaningful improvement in survival outcomes over chemotherapy in patients (pts) with pretreated HR+/HER2- mBC. SN-38 leads to double-stranded DNA damage, therefore we hypothesized that in tumors with defective DDR machinery, SG may provoke synthetic lethality. We report on comprehensive genomic analysis of DDR gene variants and the impact on SG clinical efficacy in this patient population. Methods: Pts with HR+/HER2– mBC who received prior taxane, endocrine therapy (ET), CDK4/6 inhibitor, and 2-4 prior lines of chemotherapy were randomized to receive SG (10 mg/kg IV days 1 and 8, every 21 days) or treatment of physician’s choice (TPC) until disease progression or unacceptable toxicity. DDR gene deleterious variants were identified using whole exome sequencing (WES) on archival or screening tumor tissues based on 142 DDR pathway genes annotated in the Kyoto Encyclopedia Genes and Genomes (KEGG) database and Human DNA Repair Gene database; association between these variants and clinical outcomes was evaluated using Cox regression model as Hazard Ratio (HR) with 95% Confidence Intervals (CI). Results: Of 543 patients included in the intent-to-treat (ITT) population, WES data was available for 195 (36%) – clinicopathological features were similar between ITT and WES dataset (median age = 57 vs 58 for SG and 55 vs 55 for TPC; prior lines of chemotherapy >2 lines = 59% vs 57% for SG and 58% vs 55% for TPC). Estrogen Receptor (ER)>10% and prior lines of chemotherapy were comparable between WT and MUT in SG arm and in TPC arm. Overall, 114 (58%) of tumor samples had ≥1 deleterious alteration with BRCA2, PRKDC, ATM being the most common alterations. The primary results are included (Table). Although all patients demonstrated improved efficacy with SG vs TPC, patients with DDR mutant MBC had numerically greater benefit in PFS vs WT (MUT HR= 0.61; WT HR= 0.76) and OS (MUT HR = 0.68; WT HR= 0.82). Conclusions: While SG benefit over TPC was observed in both DDR WT and DDR MUT HR+/HER2– mBC, numerically greater benefit was observed for patients with DDR deficient tumors, suggesting possible synergy between the DDR pathway and SG’s anti-tumor effect. Further study of the synergistic effects of SG in combination with agents targeting DDR pathway are warranted. Clinical trial information: NCT03901339 . [Table: see text]

Phase 1b/2a study evaluating the combination of MN-166 (ibudilast) and temozolomide in patients with newly diagnosed and recurrent glioblastoma (GBM).

2016 Background: Ibudilast is a selective inhibitor of macrophage inhibitory factor (MIF) activity, which is upregulated in GBM and promotes stem cell growth. It also disrupts the interaction between MIF and CD74. When combined with temozolomide (TMZ) in preclinical studies, it attenuates the immunosuppressive properties of myeloid-derived suppressor cells and enhances tumor regression. This is a phase 1b/2a single-center, open-label, dose escalation study evaluating the safety, tolerability, and efficacy of the combination of Ibudilast and TMZ in newly diagnosed (nGBM) and recurrent (rGBM) GBM was initiated (NCT03782415). Methods: We included patients with nGBM who had completed concurrent chemoradiation, and patients with first GBM relapse who had a measurable enhancing disease. Patients were treated with monthly cycles of temozolomide (5 days of a 28-day cycle) and daily ibudilast, with a starting dose of 30 mg twice daily (BID), and escalated to a maximal dose of 50 mg BID. The primary objectives were to evaluate the safety and tolerability of the combination and determine the phase 2 recommended dose (R2PD) for phase 1b, and to evaluate the efficacy of the combination, determined by the 6-month progression-free survival rate (PFS-6) for phase 2a. A standard 3+3 design was used for phase 1b. For the rGBM cohort, a sample size of 30 provided 80% power to discriminate between historical 15% and 35% PFS-6 rates for rGBM patients. The trial would be considered successful if at least 8 patients were progression free at 6 months. Secondary objectives included overall survival (OS). Immunohistochemistry (IHC) studies were performed on archival tumor samples to evaluate factors of the tumor immune microenvironment. Results: 36 patients with nGBM and 26 patients with rGBM were included; 61% were males, with median age of 59 years. Ibudilast 50 mg BID was deemed to be the R2PD. PFS-6 was 44% (16 patients) in nGBM, and 31% (8 patients) in rGBM. The median OS was 21.0 months (17.7, 23.1) for nGBM and 8.6 months (7.8, 10.5) for rGBM. IHC evaluation on the original tumor tissue for patients with rGBM revealed a significantly higher CD3 positive cells infiltration, and elevated CD74 expression in tumors of patients who progressed at 5 months compared to those who did not. Conclusions: This study met its primary endpoint for rGBM, as the combination of ibudilast and TMZ was associated with a higher PFS-6 rate than historical controls. CD3 expression was a good predictor for tumor progression at 5 months in patients with rGBM. Encouraging preclinical studies suggest enhanced efficacy of ibudilast in GBM when combined to immune checkpoint blockade agents. Clinical trial information: NCT03782415 .

The prognostic impact of transforming growth factor beta 2 (TGFB2) mRNA levels, in conjunction with interferon-gamma receptor activation of interferon regulatory factor 5 (IRF5) and expression of CD276/B7-H3 in low-grade gliomas.

e14564 Background: Low-grade glioma (LGG) tumors are characterized by a low infiltration of immune cells, requiring therapeutic interventions to boost the immune response. Methods: We analyzed mRNA expression datasets from the UCSC Xena web platform to compare normal brain tissue with LGG tumor samples to screen for upregulated genes. We also used cBioportal to determine the relationship between mRNA expression levels of 513 LGG patients and their overall survival (OS) outcomes. Results: Tumor-associated macrophage (TAM) markers, MSR1/CD204, CD86, and CD68, exhibited a 6-fold (P<0.0001), 8.9-fold (P<0.0001), and 15.6-fold increases in mRNA expression levels, respectively in LGG tumors. Both TGFB1 (4.1-fold increase, P<0.0001) and TGFB2 (2.2-fold increase, P<0.0001) were upregulated in brain tumors, suggesting that TGFB ligands are pivotal in establishing an immunosuppressive TME. In addition, mRNA upregulation of interferon-gamma receptors, IFNGR1 and IFNGR2, and signaling molecules STAT1, IRF1, and IRF5, pointed to an essential role for IFN-γ mediated remodeling of the TME. A tumor-associated antigen, CD276/B7-H3, mRNA expression showed a significant (P<0.0001) 4.03-fold increase in tumor tissue. Multivariate Cox proportional hazards models investigating the interaction of TGFB2 and activation of IFNGR2, STAT1, IRF1, or IRF5 showed that the prognostic impact of high mRNA levels (25 percentile cut-off) of TGFB2high was independent of IFNGR2, STAT1, IRF1, or IRF5 mRNA levels (TGFB2high HR (95% CI) = 4.07 (2.35-7.06), 6 (3.62-10.11), 4.38 (2.67-7.17), and 4.48 (2.82-7.12) for models with IFNGR2, STAT1, IRF1, or IRF5 respectively). Patients with high levels of TGFB2 were overrepresented by LGG patients with IDH wild-type mutation status. The prognostic impact of TGFB2high and IDH wild-type observed by the increases in hazard ratios for TGFB2 (HR (95% CI range) = 2.02 (1.05-3.89)) and IDH wild-type (HR (95% CI range) = 4.44 (1.9-10.4)) were independent predictors of survival requiring risk stratification of patients identify LGG patients with IDH wild-type and TGFB2high in the design of clinical trials. The ratio of TGFB1:TGFB2 expression levels was also a prognostic indicator for OS, whereby patients who exhibited a 2.5-fold greater level of TGFB1 compared to TGFB2 mRNA levels experienced improved survival outcomes. A 1.8-fold higher TGFB2 levels, the mRNA expression corresponded to worse OS. Conclusions: The specific abrogation of TGFB2 and not TGFB1 by OT101, a TGFB2-specific synthetic phosphorothioate antisense oligodeoxynucleotide, is a prerequisite to employing a strategy to target TGFB pathways. IRF5 and CD276/B7-H3 as prognostic markers are potential targets for combination therapies with TGFB2 inhibitors.

Comparative analysis of actionable gene reporting in targeted panels versus comprehensive NGS testing for solid tumor samples.

11185 Background: The landscape of actionable genes significantly influences clinical decisions in cancer diagnosis, prognosis, and treatment planning. In a clinical context, the selection of NGS panels hinges on striking a balance among informative data, insurance coverage, and the preferences of patients and healthcare providers. Molecular pathology reference laboratories commonly employ large-scale next-generation sequencing (NGS) testing, curating smaller, disease-specific targeted panels. This study investigates the prevalence of unreported actionable genes in real-world clinical samples subjected to disease-specific panels. Methods: We analyzed SNV/InDel DNA variants in 795 clinical solid tumor samples using the Illumina TSO 500 platform for comprehensive NGS panel testing of 517 genes. Comprehensive NGS panel-tested samples were intersected with disease-specific targeted NGS panels for breast (54 genes), brain (62 genes), colorectal (36 genes), and lung (44 genes). An artificial filter was applied to align targeted panels with patient disease. Variants were classified based on pathogenicity, and benign variants were filtered out. Detected variants were assessed for actionable mutations as defined by the FDA recognized OncoKB actionable gene database criteria. Additionally, 1484 lung patient samples tested with a lung-specific targeted panel were de-identified and unmasked to the comprehensive NGS tumor profiling to investigate the prevalence of actionable alteration beyond those covered by the disease-specific panel. Results: The study revealed that 3.2% of breast, 0% of brain, 66.2% of colorectal, and 2.7% of lung samples had additional actionable mutations undisclosed by the targeted panels. The number of actionable genes beyond those included in the targeted panels were 14 for breast, 2 for brain, 25 for lung, and 41 for colorectal cancer. This underscores the importance of comprehensive testing to fully capture each patient tumor’s actionable mutation profile. Conclusions: Our findings detect masked actionable mutations in down-sampled targeted panels and highlight the need for comprehensive testing to accurately identify actionable mutations in various cancer types. Reporting actionable mutations can significantly influence clinical decision-making, emphasizing the importance of NGS test selection in routine molecular diagnostics as well as the impact of integrating comprehensive NGS testing.

Peony exploratory analyses of biomarker changes: From baseline to surgical samples in patients with HER2+ breast cancer.

e15136 Background: PEONY (NCT02586025) is a randomized, multicenter, double-blind, placebo-controlled, phase III trial evaluating neoadjuvant and adjuvant pertuzumab/placebo with trastuzumab and docetaxel in HER2-positive early/locally advanced breast cancer. We previously reported that pertuzumab showed an improved efficacy vs. placebo in terms of total pathological complete response (tpCR), event-free survival and disease-free survival (DFS), as well as in pre-defined biomarker subgroups. Limited data are available on biomarker changes after neoadjuvant treatment in the Asian population. Thus, we aim to (1) describe the biomarker changes from baseline to surgical samples after HER2 targeted neoadjuvant therapy, and (2) explore the association of surgical biomarkers and DFS. Methods: This study will focus on biomarkers of tumor tissue samples collected at baseline and surgery among 218 non-tpCR patients, due to limited samples collected at disease progression. Samples were tested using Immunohistochemistry (IHC) for HER3, PTEN, CD8 and PD-L1. Both HER2/HER3 mRNA expression and PIK3CA mutation were assessed using a PCR based methodology. The statistical analyses were using R (4.1.3). Biomarkers were categorized as high/low either by median derived from baseline samples, or with well-established cutoffs, e.g. HER2 IHC of 3+ vs. 1/2+ and PD-L1 at 1%. The changes of biomarkers were examined among the paired samples using Fisher’s exact test and depicted by alluvial plots. Cox proportional hazard regression analysis was performed to examine the association between surgical biomarkers and DFS in pooled arms. Results: Since the surgical biomarkers are only evaluable among patients with residual disease, patients in this analysis had a higher stage, and were more likely to be node+. Of 166 paired samples, more patients show a decrease in HER2 and HER3 mRNA/IHC, PTEN IHC from baseline to surgery. PIK3CA mutation status remained the same among the majority of patients (n = 154, 95%). Immune-related biomarkers, PD-L1 and CD8, are more enriched in surgical samples compared to baseline. All these observations are similar between the two arms. No clear association has been shown in most of the biomarkers, except for PIK3CA, with DFS. Surgical tumor samples with PIK3CA mutation detected trended with a worse DFS than those with no PIK3CA mutation detected [HR: 1.81 (95% CI: 0.94 – 3.49)]. Conclusions: Of baseline and surgery paired samples, most biomarkers showed a change, except for PIK3CA mutation status. In pooled analyses, we only observed a trend of PIK3CA mutation detected in surgical samples with a worse DFS, which is in line with the association observed with baseline PIK3CA mutation status. No other associations were found between surgical biomarkers and DFS. These findings may suggest that despite biomarker changes patients continue to benefit From HER2 therapies and dual blockade. Clinical trial information: NCT02586025 .

Closing classification gaps in luminal breast cancer with single-cell RNA-seq insights from normal breast lineages.

e13168 Background: Almost two decades ago, invasive breast cancer was classified into five subgroups based on expression patterns, each with distinct biological and prognostic characteristics. Advances in single-cell RNA sequencing offers insights into the expression profiles of normal luminal epithelial cells. Here, we investigate the connection between breast cancer (BC) classification and luminal expression profiles. Methods: We applied single-cell data from recent studies (Table) to compile gene signatures corresponding to expression profiles of luminal epithelial cell clusters in normal breast tissue. Normalized enrichment scores (ESs) of these signatures were calculated for bulk RNA-seq gene expression data from open source cohorts (TCGA-BRCA, SCAN-B, METABRIC). The BC molecular subtype of each tumor sample was predicted based on a modified PAM50 classification using gene expression profiling (1); LumA and LumB subtypes were combined into Luminal. ESs of signatures were compared between the BC subtypes (Table). Results: The ESs of all signatures differed significantly across BC subtypes (p < 0.001, Kruskal-Wallis test, Table 1). The ESs of signatures for LumSec-major and Luminal progenitor clusters are the highest in subtypes HER2-low and Basal-like (p < 0.001, Dwass-Steel-Critchlow-Fligner test [DSCF]). Basal-like also had the highest enrichment of the ductal signature (p < 0.001, DSCF). Luminal subtype showed the highest ESs of signatures corresponding to HR+ clusters of mature luminal cells and of the TDLU signature (p < 0.001, DSCF). HER2-low subtype showed the highest ES of the LumSec-lac signature (p < 0.001, DSCF). Conclusions: Our analyses challenge traditional classifications by showing that luminal breast cancers are less similar to most luminal epithelial clusters in normal breast tissue than other breast cancer subtypes. These findings suggest traditional expression classification may not fully capture the complexity of this largest subset of luminal-type breast cancers. 1. Turova et al., SABCS, 2023. [Table: see text]

Representation of genomic intratumor heterogeneity in multi-region non-small cell lung cancer patient-derived xenograft models

Patient-derived xenograft (PDX) models are widely used in cancer research. To investigate the genomic fidelity of non-small cell lung cancer PDX models, we established 48 PDX models from 22 patients enrolled in the TRACERx study. Multi-region tumor sampling increased successful PDX engraftment and most models were histologically similar to their parent tumor. Whole-exome sequencing enabled comparison of tumors and PDX models and we provide an adapted mouse reference genome for improved removal of NOD scid gamma (NSG) mouse-derived reads from sequencing data. PDX model establishment caused a genomic bottleneck, with models often representing a single tumor subclone. While distinct tumor subclones were represented in independent models from the same tumor, individual PDX models did not fully recapitulate intratumor heterogeneity. On-going genomic evolution in mice contributed modestly to the genomic distance between tumors and PDX models. Our study highlights the importance of considering primary tumor heterogeneity when using PDX models and emphasizes the benefit of comprehensive tumor sampling.

Low GPR81 in ER+ breast cancer cells drives tamoxifen resistance through inducing PPARα-mediated fatty acid oxidation

AimsThe intricate molecular mechanisms underlying estrogen receptor-positive (ER+) breast carcinogenesis and resistance to endocrine therapy remain elusive. In this study, we elucidate the pivotal role of GPR81, a G protein-coupled receptor, in ER+ breast cancer (BC) by demonstrating low expression of GPR81 in tamoxifen (TAM)-resistant ER+ BC cell lines and tumor samples, along with the underlying molecular mechanisms. Main methodsFatty acid oxidation (FAO) levels and lipid accumulation were explored using MDA and FAβO assay, BODIPY 493/503 staining, and Lipid TOX staining. Autophagy levels were assayed using CYTO-ID detection and Western blotting. The impact of GPR81 on TAM resistance in BC was investigated through CCK8 assay, colony formation assay and a xenograft mice model. ResultsAberrantly low GPR81 expression in TAM-resistant BC cells disrupts the Rap1 pathway, leading to the upregulation of PPARα and CPT1. This elevation in PPARα/CPT1 enhances FAO, impedes lipid accumulation and lipid droplet (LD) formation, and subsequently inhibits cell autophagy, ultimately promoting TAM-resistant BC cell growth. Moreover, targeting GPR81 and FAO emerges as a promising therapeutic strategy, as the GPR81 agonist and the CPT1 inhibitor etomoxir effectively inhibit ER+ BC cell and tumor growth in vivo, re-sensitizing TAM-resistant ER+ cells to TAM treatment. ConclusionOur data highlight the critical and functionally significant role of GPR81 in promoting ER+ breast tumorigenesis and resistance to endocrine therapy. GPR81 and FAO levels show potential as diagnostic biomarkers and therapeutic targets in clinical settings for TAM-resistant ER+ BC.

Spatially resolved tissue imaging to analyze the tumor immune microenvironment: beyond cell-type densities

IntroductionThe tissue immune microenvironment is associated with key aspects of tumor biology. The interaction between the immune system and cancer cells has predictive and prognostic potential across different tumor types....

PIK3CA Hotspot Mutations in Saliva as a Diagnostic Marker in Oral Squamous Cell Carcinoma Patients.

This study aimed at the analogous detection of PIK3CA mutations, common in oral squamous cell carcinoma (OSCC), in matched tumor and saliva samples. Tissue and saliva samples were obtained from 29 patients diagnosed with primary OSCC. Saliva samples were obtained preoperatively; tissue specimens were acquired during tumor resection. Tumor DNA was extracted from both tissue and saliva samples. All samples were controlled for DNA quantity and quality and genetic matching of sample pairs was confirmed using the iPlex Pro Exome QC Panel. Variant detection was performed using the MassARRAY® System, a mass-spectrometry based detection system. Mutational analysis in tissue tumor DNA was made using the multiplexed ClearSEEK™ PIK3CA v1.0 Panel covering 20 hotspot mutations in PIK3CA. In saliva samples, variants were analyzed using both the ClearSEEK™ and the UltraSEEK® Lung v1.1 Panel, with a higher limit of detection but covering less PIK3CA variants. Overall, a PIK3CA variant was found in seven of the 29 tumor tissue samples (24%) by ClearSEEK™; UltraSEEK® additionally confirmed the variant in four of these seven positive samples. Of the three variants not detected by UltraSEEK®, two were not included in the panel and one was included but not detected. Of the seven variants found in tissue, five could also be detected in the matching saliva samples (71%), either by utilizing ClearSEEK™ or UltraSEEK® Conclusion: The detection of PIK3CA hotspot mutations in OSCC and their simultaneous occurrence in saliva underline the potential benefit of liquid biopsies for non-invasive cancer detection and follow-up care of OSCC patients.

Identification of PATL1 as a prognostic and immunotherapeutic predictive factor for nasal-type natural killer/T-cell lymphoma and head and neck squamous cell carcinoma

This research examines the function of protein associated with topoisomerase II homolog 1 (PATL1) in nasal-type natural killer/T-cell lymphoma (NKTCL) and head and neck squamous cell carcinoma (HNSCC). We analyzed bulk RNA-seq data from NKTCL, nasal polyps, and normal nasal mucosa, identifying 439 differentially expressed genes. Machine learning algorithms highlighted PATL1 as a hub gene. PATL1 exhibited significant upregulation in NKTCL and HNSCC tumor samples in comparison to normal tissues, showing high diagnostic accuracy (AUC = 1.000) for NKTCL. Further analysis of local hospital data identified PATL1 as an independent prognostic risk factor for NKTCL. Data analysis of TCGA and GEO datasets revealed that high PATL1 expression correlated with poorer prognosis in HNSCC patients (p < 0.05). We also constructed a PATL1-based nomogram, which emerged as an independent prognostic predictor for HNSCC after addressing missing values. Additionally, we found a strong correlation between PATL1 and various immune cell infiltrates (e.g., activated.CD4 T cell), and a significant association with the expression of 37 immune checkpoints genes (e.g., CTLA4, PDCD1) and 20 N6-methyladenosine-related genes (e.g., ZC3H13, METTL3) (all p < 0.05). Both TCIA and TIDE algorithms suggested that PATL1 could potentially predict immunotherapy efficacy (p < 0.05). Cellular experiments demonstrated that transfection with a silencing plasmid of PATL1 significantly inhibited the malignant behaviors of SNK6 and FaDu cell lines(p < 0.05). In conclusion, our findings suggest that PATL1 may serve as a valuable prognostic and predictive biomarker in NKTCL and HNSCC, highlighting its significant role in these cancers.

Genetic Alterations of SMYD4 in Solid Tumors Using Integrative Multi-Platform Analysis.

SMYD4 is a member of the SMYD family that has lysine methyltransferase function. Little is known about the roles of SMYD4 in cancer. The aim of this study is to investigate genetic alterations in the SMYD4 gene across the most prevalent solid tumors and determine its potential as a biomarker. We performed an integrative multi-platform analysis of the most common mutations, copy number alterations (CNAs), and mRNA expression levels of the SMYD family genes using cohorts available at the Cancer Genome Atlas (TCGA), cBioPortal, and the Catalogue of Somatic Mutations in Cancer (COSMIC). SMYD genes displayed a lower frequency of mutations across the studied tumors, with none of the SMYD4 mutations detected demonstrating sufficient discriminatory power to serve as a biomarker. In terms of CNAs, SMYD4 consistently exhibited heterozygous loss and downregulation across all tumors evaluated. Moreover, SMYD4 showed low expression in tumor samples compared to normal samples, except for stomach adenocarcinoma. SMYD4 demonstrated a frequent negative correlation with other members of the SMYD family and a positive correlation between CNAs and mRNA expression. Additionally, patients with low SMYD4 expression in STAD and LUAD tumors exhibited significantly poorer overall survival. SMYD4 demonstrated its role as a tumor suppressor in the majority of tumors evaluated. The consistent downregulation of SMYD4, coupled with its association with cancer progression, underscores its potential usefulness as a biomarker.