Significance Analysis Of Microarrays Research Articles

B-274 Determining Novel Biomarker Candidates Associated With Heroin and Cocaine Exposure Using Urine Metabolomics

Abstract Background Substance abuse poses an escalating health risk in the United States. While acute drug exposure can be tracked using drugs and their metabolites as biomarkers in clinical settings, there is a notable absence of biomarkers for chronic drug exposure or addiction. The development and availability of such biomarkers could significantly enhance clinicians’ ability to identify patients at high risk of addiction, and more accurately predict disease progression and treatment responses. Methods Our study aimed to discover novel biomarkers associated with substance abuse by analyzing raw mass spectrometry (MS) data from urine comprehensive drug screening (UCDS) conducted at the University of Pittsburgh Medical Center (UPMC). The MS data, acquired through untargeted data collection using LC-qToF-MS at the UPMC Clinical Toxicology Laboratory, was initially preprocessed (including peak identification, alignment, and putative annotation) using MS-DIAL to generate a data matrix. This matrix, consisting of objects (cases) and features (m/z and retention time), was further normalized and analyzed using MetaboAnalyst. A combination of statistical methods, including point biserial correlation analysis, volcano plot, significance analysis of microarrays and metabolites (SAM), empirical Bayesian analysis of microarrays and metabolites (EBAM), partial least squares discriminant analysis (PLS-DA), and random forest (RF), were employed to select features significantly associated with the EIA-results. These features were subsequently evaluated for analyte identification using MS-FINDER. Results Our analysis identified nine significant features associated with 6MAM-EIA for heroin abuse, including 6-monoacetylmorphine itself and norfentanyl. For features associated with cocaine metabolite-EIA, 37 significant features were selected, including multiple cocaine metabolites, nicotine metabolites, and norfentanyl. Some of these selected features are likely products of in-source fragmentation or endogenous metabolites. Conclusions Our study has identified a set of potential biomarker candidates for illicit drug exposure. Notably, norfentanyl was found to be significantly associated with both 6MAM-EIA and cocaine metabolite-EIA, reflecting current trends in illicit drug use. Further chemical identification of these biomarkers is planned for future work.

P-029 Genome-wide microRNA expression profiling in human spermatozoa and its relation to sperm quality

Abstract Study question Does the microRNA (miRNA) expression profile in human spermatozoa change with their morphological appearance? Summary answer This study revealed miRNA expression associated with good-quality sperm classified based on their morphology. These miRNAs could represent efficient biomarkers to evaluate male fertility status. What is known already We previously published a scoring scale where the spermatozoa with a Score 6 (Good spermatozoon) display normal head shape with symmetrical nuclear no extrusion and/or no invagination of the nuclear membrane, without any vacuole and normal base. The spermatozoa with a Score 0, display a nuclear-shape disorder with an abnormal base and at least one large vacuole (Bad spermatozoon). Despite the great magnitude of studies investigating the miRNA role in spermatogenesis, limited work has been done to evaluate it as a potential biomarker for sperm quality in relation to their morphology. Study design, size, duration In a prospective study from March 2023 to May 2023, twenty semen samples were collected. The samples were categorized according to their scoring morphology into Score 6 and Score 0. Ten samples (five with a Score 6 and five with a Score 0) were used for the analysis by microarray and ten (five with a Score 6, and five with a Score 0) for the miRNA expression analysis by RT-qPCR. Participants/materials, setting, methods Semen samples were scored according to their high-magnification morphology and stored at -80 °C. We used miRNA microarray chip to analyze miRNA profile. Statistical significance was evaluated by Significance Analysis of Microarrays, screened by Fold change>2 and p-values ≤ 0.05. The correlation between miRNAs and their corresponding mRNA targets was analyzed using in silico prediction algorithms. The methylome was studied using the DNA sequencing method. TaqMan miRNA assays were used to confirm the miRNA results. Main results and the role of chance We perform a robust transcriptomic analysis to define unique miRNA signatures able to identify the spermatozoa that have high scores regardless of morphological appearance at high magnification. There were 86 significantly overexpressed in Score 6 and 21 miRNAs in Score 0. Score 6-spermatozoa had significantly higher levels of hsa-miR-132 (FCx41, p=1.68E-8), hsa-miR-1973 (FCx32, p=6.19E-8), hsa-miR-34 (FCx30, p=8.43E-8), hsa-miR-25 (FCx27, p=1.30E-6), hsa-miR-342 (FCx24, p=1.57E-5), hsa-miR-15 (FCx14, p=8.56E-6), hsa-miR-125 (FCx13, p=6E-4), hsa-miR-30 (FCx12, p = 3.75E-6), than Score 0. The miRNAs overexpressed in Score 6 are predicted to target 910 messenger RNA involved in biological functions of the spermatozoa and several signaling pathways, such as mTOR, p53, and MAPK signaling pathways, as well as cell Intrinsic pathway for apoptosis, cellular senescence, and oxidative stress. Whereas genes targeted by score 0-miRNAs were implicated in the regulation of cellular growth and proliferation. Our data indicate a difference in methylation of the promoter region of miRNAs, and their validated target genes were observed between score 6 and score 0. Aberrant expression of the Score 6-miRs or their targets could be due to the methylation status of the promoter region and indicative of a defect in spermatogenesis. Limitations, reasons for caution This work provides a foundation for the interpretation of miRNA changes associated with spermatozoa morphological appearance. The data must be validated by TaqMan miRNA assays in a higher number of semen samples. Moreover, the molecular network of miRNA-targeted gene pathways also merits investigation. These experiments are currently ongoing. Wider implications of the findings The profiling of miRNAs repertoire in scored spermatozoa morphology opens new perspectives to explore male fertility status and provides a biomarker panel for sperm analysis during the ART procedure. Larger well-controlled investigations employing microRNAome approaches are needed to validate these conclusions. Trial registration number not applicable

Human epicardial adipose tissue contains innate and adaptive lymphoid cells and a higher proportion of innate type 2 lymphoid cells compared to other adipose tissues

ImportanceEpicardial adipose tissue (EAT) is a biologically active organ surrounding myocardium and coronary arteries that has been associated with coronary artery disease (CAD) and atrial fibrillation. Previous work has shown that EAT exhibits beige features. ObjectiveOur objective was to determine whether the stromal vascular fraction of the human EAT contains innate or adaptive lymphoid cells compared to thoracic subcutaneous (thSAT), visceral abdominal (VAT) and subcutaneous abdominal (abSAT). ParticipantsNew pangenomic microarray analysis was performed on previous transcriptomic dataset using significance analysis of microarray and ingenuity pathway analysis (n=41) to identify specific immune signature and its link with browning genes. EAT, thSAT, VAT and abSAT samples from explanted patients with severe cardiomyopathies and multi-organ donor patients (n=17) were used for flow cytometry (FC) immunophenotyping assay. Patients were on average 55±16 years-old; 47% had hypertension and 6% CAD. Phenotypic adaptive and innate immune profiles were performed using a TBNK panel and a specific ILC1-2-3 panel including CD127, CD117, CRTH2 (CD294) and activation markers such as CD25 and CD69. ResultsTranscriptomic analysis showed a significant positive correlation between the TH2 immune pathway (IL-4, IL-5, IL-13, IL-25, IL-33) and browning genes (UCP-1, PRDM16, TMEM26, CITED1, TBX1) in EAT versus thSAT (R=0.82, P<0.0001). Regarding adaptive immune cells, a preponderance of CD8T cells, a contingent of CD4T cells, and a few B cells were observed in all ATs (P<0.0001). In innate lymphoid cells (ILCs), an increase was observed in visceral ATs (i.e. EAT; VAT 35±8ILCs/g of tissue) compared to their subcutaneous counterpart (i.e. thSAT+abSAT: 8±3 ILCs/g of AT, P=0.002), with a difference in the proportion of the 3 subtypes of ILCs (ILC1>ILC3>ILC2). In addition, we observed an increase in EAT-ILC2 compared to other ATs and almost all these EAT-ILC2 expressed CD69 and/or CD25 activation markers (99.75±0.16%; P<0.0001). We also observed more NKs in EAT and VAT (1520±71 cells/g of AT) than in SATs (562±17 cells/g of AT); P=0.01. ConclusionThis is the first study to provide a comparison between innate and adaptive lymphoid cells in human epicardial versus abdominal or thoracic adipose tissues. Further studies are ongoing to decipher whether these cells could be involved in EAT beiging. Trial RegistrationCODECOH No. DC-2021-4518 The French agency of biomedicine PFS21-005.

Abstract PO2-01-13: Clinico-Pathological and Molecular Characterization of Early Hormone Receptor-Positive Breast Cancer in Young Women

Abstract Introduction Premenopausal women diagnosed with hormone receptor-positive (HR+)/HER2-negative (HER2-) early breast cancer (EBC) face a significantly higher risk of cancer recurrence and mortality compared to older women, particularly those diagnosed under the age of 40. The underlying causes behind this disparity remain largely unknown. Material and methods We retrospectively select tumour samples from patients (pts) diagnosed with HR+/HER2- EBC at Hospital Clinic (Barcelona, Spain) from May 2014 to March 2020 who had a PAM50/Prosigna assay (Veracyte) previously performed. This assay examines the expression of 50 genes involved in important breast cancer signaling pathways, as well as the intrinsic subtypes (IS) and the Risk of Recurrence (ROR) score. Our primary objective was to compare the distinct clinicopathological and molecular characteristics of HR+/HER2- EBC between two age groups: patients under 40 years old (Young EBC or YEBC) and those 40 years old and above (Older EBC or OEBC). Disease-free survival (DFS) was defined as the time between diagnosis and the first recurrence. Comparisons between groups were carried out using chi-square tests (categorical variables). Additionally, we performed a two-class unpaired Significance Analysis of Microarrays (SAM) with a false discovery rate (FDR) less than 5% to identify genes with significantly different expression between YEBC and OEBC. The statistical significance level was set at less than 0.05. Results A total of 441 tumor samples from 420 patients were included in the analysis, with 5% (22/441) corresponding to YEBC. With a median follow-up of 64.5 months, there were 20 breast cancer (BC) recurrences (4.8%), 12 second cancers (2.9%) and 5 BC-related deaths (1.2%), all occurring in the OEBC group. Table 1 provides an overview of the main clinico-pathological and molecular characteristics. YEBC tended to have larger tumors, with 45.5% (10/22) measuring over 2 cm compared to 36.1% (151/419) in OEBC (p=0.376). YEBC also had a higher proportion of grade 3 tumors (18.18% versus 3.1% in OEBC, p=0.418) and lower expression of estrogen receptor (ER) as determined by immunohistochemistry (mean of 72.28% vs 88.36%, p=0.004). Regarding the PAM50 IS, there was a difference observed between YEBC and OEBC (p &amp;lt; 0.001). Specifically, YEBC patients exhibited a tendency towards a higher proportion of Luminal B tumors (54.6% versus 38.9% in OEBC, p=0.144), as well as non-Luminal subtypes such as Basal-like (18.2% versus 0.95% in OEBC, p&amp;lt; 0.001) and HER2E (4.6% versus 0.7% in OEBC, p=0.186). YEBC tended to have higher ROR (p=0.167), and to receive chemotherapy in a higher proportion, especially in the intermediate-risk group (p=0.004). In YEBC tumours, SAM analysis (p &amp;lt; 0.05) showed an overexpression of genes mainly related with proliferation (i.e., RRM2, MELK, MIK67), cell cycle progression (i.e., KNTC2, CDC20, MYBL2) and apoptosis inhibition (i.e., BIRC5). In OEBC there were an overexpression of genes related with the oestrogen receptor-signalling pathway (i.e., ESR1, NAT1, FOXA1, PGR). Luminal B tumours in YEBC showed an upregulation of genes related to proliferation, migration, and poor prognosis (i.e., KRT17, KRT5) compared to OEBC. However, since there were no recurrences in the YEBC group, differences in terms of DFS between the groups could not be calculated. Principio del formulario Final del formulario Conclusions YEBC exhibited a more advanced stage and a more aggressive intrinsic phenotype compared to OEBC. Additionally, these tumors showed a tendency to upregulate genes involved in proliferation, cell cycle progression, and survival, while downregulating genes related to hormone signaling pathways. These molecular characteristics may help explain the worse prognosis of YEBC compared to OEBC. However, further research is necessary to delve into the differential genomic characteristics that contribute to the unfavorable prognosis observed in patients diagnosed with HR+ EBC before the age of 40. Key clinical and molecular features in 441 biopsies Citation Format: Francisco Javier Muñoz-Carrillo, José Antonio Sola, Carme Crous, Esther Sanfeliu, Isabel Garcia-Fructuoso, Elia Seguí, Teresa Gorría, Raquel Gómez-Bravo, Barbara Adamo, Tomás Pascual, Olga Martínez-Sáez, Francesco Schettini, Pablo Rivera, Rosalía Cayuela, Montserrat Muñoz, Núria Chic, Maria Vidal. Clinico-Pathological and Molecular Characterization of Early Hormone Receptor-Positive Breast Cancer in Young Women [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-01-13.

Abstract PO3-14-07: Characterizing Immune Infiltration in Metastatic Hormone Receptor-Positive/HER2-Negative Breast Cancer: A Comprehensive Translational Analysis from four SOLTI Clinical Trials

Abstract Background: Hormone Receptor-positive (HR+)/HER2-negative (HER2-) metastatic breast cancer (BC) is a highly heterogenous disease. Non-luminal subtypes within HR+/HER2- BC have been linked to poor survival in the metastatic setting. However, these subtypes might exhibit higher expression of immune-related genes, tumor-infiltrating lymphocytes (TILs), and APOBEC signatures. This study aimed to investigate the presence of tertiary lymphoid structures (TLS), TILs and immune gene expression (GE) across PAM50 intrinsic subtypes (IS) in metastatic samples from patients (pts) with HR+/HER2- BC. Methods: We included pts with HR+/HER2- metastatic BC who participated in the molecular prescreening of the SOLTI-1904-ACROPOLI (NCT04802876), SOLTI-1502-ARIANNA (NCT04142060), SOLTI-1718-NEREA (NCT04460430) and SOLTI-1716-TATEN (NCT04251169) studies, providing metastatic samples for analysis. The proportion of TILs was centrally evaluated by a single pathologist using standard criteria. GE analysis was performed on the same FFPE tumor block using a panel of 72 genes, including 10 immune genes and the PAM50 IS. In a subset of patients, we conducted additional GE analysis using a panel of 192 genes, including the 14-gene B-cell/IgG (IGG) signature, while also retrospectively assessing the presence of TLS. Descriptive statistics, significance analysis of microarrays (using False Discovery Rate [FDR]) and logistic regressions were used to investigate the association between TLS, TILs, immune GE and PAM50 IS. Results: A total of 401 pts were included. Distribution across IS was: 53.4% Luminal B (n=197), 27.1% Luminal A (n=100), 16% HER2-enriched (n=59), 2.7% basal-like (n=10) and 0.8% normal-like (n=3). TILs were evaluated in 375 samples (93.5%). Median (m) TILs proportion was 2% (IQR: 1-4, range 0-80). Non-luminal subtypes exhibited a higher enrichment of TILs compared to luminal subtypes (m=3% vs m=1%, p=0.002). Moreover, significant differences of TILs were also seen according to the site of metastasis (M1): m=6.5% in lymph nodes vs m=4.5% in lung M1 vs m=1% in liver M1 (p&amp;lt; 0.001). Immune GE was significantly higher in samples with a high proportion of TILs ( &amp;gt;10%), although the correlation between TILs and immune genes was moderate (e.g, coefficient=0.44, p&amp;lt; 0.001, between CD8A and TILs). TLS were evaluated in 147 samples and detected in 16 pts (10.9%). Compared to TLS-negative tumors, TILs were significantly higher in TLS-positive (TLS+) tumors (m=1.5% vs m=10%, p&amp;lt; 0.001). In addition, the presence of TLS was significantly higher in non-luminal subtypes compared to luminal subtypes (18% (11/61) vs 5.8% (5/86), p=0.019). TLS+ tumors had a significant enrichment of genes related to activated B-cells (CD19, CD79A), plasma cells (CD27), immunoglobulins (IGKC, IGLV), and T-cells (CD8A, PDCD1). Notably, the IGG signature as a continuous variable demonstrated a significant association with TLS presence, independently of the PAM50 IS and TILs (p=0.033, ROC AUC=0.817). Finally, higher expression of some immune genes was observed in non-luminal subtypes vs luminal subtypes (e.g., CD19 (p=0.016) and CD274/PDL1 (p&amp;lt; 0.001)). Conclusions: Our translational analysis reveals that non-luminal subtypes within metastatic HR+/HER2- BC exhibit a higher enrichment of TILs, immune GE, and presence of TLS. Importantly, we identified the IGG signature as a robust immune GE pattern that is strongly associated with TLS across PAM50 IS, independent of TILs. These findings highlight the potential significance of the IGG signature as a novel biomarker for precisely measuring TLS and selecting pts who might be more likely to derive benefit from immunotherapy. Citation Format: Elia Seguí, Esther Sanfeliu, Fara Brasó-Maristany, Blanca González-Farré, Fernando Salvador, Laura Angelats, Lorea Villanueva, Alejandra Espinosa, Patricia Galván, Esther Fernández, Xavier Gonzalez-Farré, Santiago Escrivá-de-Romaní, Núria Chic, Eva Ciruelos, Cristina Saura, Luis Manso, Mafalda Oliveira, Josep Tabernero, Aleix Prat, Tomás Pascual. Characterizing Immune Infiltration in Metastatic Hormone Receptor-Positive/HER2-Negative Breast Cancer: A Comprehensive Translational Analysis from four SOLTI Clinical Trials [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-14-07.

Serum Metabolites as Diagnostic Biomarkers in Patients with Endometriosis.

Endometriosis diagnosis is usually delayed. The gold standard for diagnosing endometriosis is laparoscopy, which is invasive and accompanied by several risks. Currently, there are no effective non-invasive biomarkers for diagnosing endometriosis. Here, we investigated whether metabolites whose levels are altered in patients with endometriosis hold potential as diagnostic biomarkers for the disease. This case-control study involved 32 patients with endometriosis and 29 patients with other benign gynecological disease. The diagnosis of all patients was confirmed through postoperative histopathological examination, and the patients were divided into two groups: an endometriosis group (EM) and a control group. Fasting blood was collected and used for non-targeted metabolomic-based detection. The data were processed through principal component analysis, orthogonal partial least squares discriminant analysis, and significance analysis of microarrays. A univariate receiver operating characteristic curve was used to evaluate the diagnostic value of the metabolites. The metabolite profiles of patients with endometriosis were markedly different compared with those of the controls. In addition, several metabolic pathways, including biosynthesis of unsaturated fatty acids, arginine biosynthesis, and glutathione metabolism, were altered. Ornithine and medorinone showed better potential as biomarkers for endometriosis diagnosis than CA125. We analyzed the altered metabolic profiles in patients with endometriosis and found ornithine and medorinone as potential non-invasive biomarkers for endometriosis diagnosis, whereas the combined ornithine-medorinone diagnosis is more valuable. These findings may help advance research on non-invasive diagnostic biomarkers for endometriosis. Further research with an improved study design and a larger cohort should be performed to confirm the diagnostic potential and clinical application of these biomarkers.

Abstract 2493: Prospective evaluation of predictive biomarkers for Palbociclib (P) and endocrine therapy (ET) benefit in hormone receptor (HR)-positive [+]/human epidermal growth factor receptor 2 (HER2)-negative [-] advanced breast cancer (ABC) patients (pts) from PARSIFAL clinical trial: The transFAL sub-study

Abstract BACKGROUND Combining cyclin-dependent kinases 4 and 6 inhibitors (CDK4/6i) with first-line ET is the standard of care for pts with HR[+]/HER2[-] ABC. PARSIFAL was a randomized, open-label, phase II trial that evaluated the efficacy and safety of first line P plus fulvestrant (F) or letrozole (L) in this pts population. Both treatments had comparable efficacy and safety results (JAMAOncol. 2021;7(12):1791-1799). Although multiple CDK4/6i clinical outcomes biomarkers have been reported, the only validated predictive factor for response is HR positivity. TransFAL, a translational study of PARSIFAL, assessed a comprehensive set of biomarkers of sensitivity/resistance to P plus ET. MATERIALS AND METHODS TransFAL employed a sample biorepository from PARSIFAL comprising formalin-fixed paraffin-embedded (FFPE) tissue and blood samples obtained from pts included in either study arm. Tumor samples (N=33) derived from the primary tumor at initial diagnosis or from primary tumor or metastatic lesions at study entry. Blood samples were obtained at baseline (N=73) and disease progression (N=22). Pts were categorized as sensitive or resistant based on progression-free survival (PFS). CDK4/6 pathway activation was assessed by immunohistochemistry for CDK4, CDK6, Rb, pRb, ER, PgR, and Ki67. Proteins extracted from FFPE slides were analyzed using hierarchical clustering, significance analysis of microarrays, and probabilistic graphical models. RNA expression and circulating tumor DNA (ctDNA) analysis were assessed by next-generation sequencing (NGS) and analysis of panel of 56 genes/gene regions, respectively. RESULTS A worse median PFS was found in pts with CDK6 positive (≥ 1% positive cells; p &amp;lt; 0.001) and high Ki67 (≥ 10% positive cells; p = 0.013)at baseline. ctDNA density at baseline was significantly higher for resistant (PFS &amp;lt; 9 months; 17.1 ng/mL) pts compared to sensitive (PFS &amp;gt; 31 months; 11.3 ng/mL) pts (p &amp;lt; 0.001). Most pts (83%) presented a genomic alteration detected in ctDNA, TP53 (28%) and PIK3CA (28%) being the most frequently mutated genes. Pts (9%) who harbored ARID1A mutation had lower PFS (p = 0.052). ESR1 mutations at progression were numerically higher in the L arm (37.5%) compared to F arm (14.3%); (p=0.31), but numbers were low. Proteomic and RNA analyses did not yield confirmable distinctive expression pattern between resistant/sensitive pts… CONCLUSIONS High Ki67 levels and CDK6 expression in the tumor and high ctDNA density, but not proteomic or RNA sequencing data, were associated to poor outcome in this analysis of pts treated with CDK4/6i and ET. This data adds to the growing evidence of poor prognosis biomarkers in this setting, highlighting the need of novel strategies for these pts. Citation Format: Joan Albanell, Angelo Gámez Pozo, Carlos L. Arteaga, Meritxell Bellet, Federico Rojo, Abel González, Beatriz Bellosillo, Violeta Serra, Petra Gener, José Antonio Guerrero, Laura López-Montero, Mario Mancino, Jose Rodríguez-Morató, Leonardo Mina, José Manuel Pérez-García, Javier Cortés, Antonio Llombart-Cussac. Prospective evaluation of predictive biomarkers for Palbociclib (P) and endocrine therapy (ET) benefit in hormone receptor (HR)-positive [+]/human epidermal growth factor receptor 2 (HER2)-negative [-] advanced breast cancer (ABC) patients (pts) from PARSIFAL clinical trial: The transFAL sub-study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2493.

Abstract 5141: Autoantibody profiling for predictive biomarkers for immune-related adverse events and clinical benefit in rare tumors treated with anti-PD-1 therapy

Abstract Background: Immune checkpoint inhibitors (ICIs) have changed the cancer treatment paradigm; however, with high frequency of immune-related adverse events (irAEs) and limited response rates in select patients. This underscores the unmet need for the development of biomarkers predictive for the development of irAEs and ICI response. Herein, we conducted autoantibody (AAb) profiling to assess baseline (BL) AAb associations with clinical benefit (CB) and irAEs in patients (pts) with rare tumors (RT) treated with programmed cell death protein 1 (anti-PD-1) therapy. Methods: AAbs against 1168 antigens were analyzed using the SeroTag antibody discovery technology. The study included 41 pts with RT who received anti-PD-1 therapy. We conducted a Significance Analysis of Microarray (SAM) to analyze the association between BL AAbs with irAEs and CB (defined as pts achieving complete response (CR), partial response (PR), or stable disease (SD) for ≥4 months by Response Evaluation Criteria in Solid Tumors (RECIST) v1.1). AAb biomaker candidates were discovered based on filtering for SAM hits with p &amp;lt; 0.05 and a delta score |D| &amp;gt;2. Results: Table 1 outlines patients’ demographics. CB and irAEs were observed in 34% and 39% of patients, respectively. Eighteen BL AAbs were significantly associated with CB such as DNA ligase III (LIG3) and small nuclear ribonucleoprotein polypeptide (SNRPA) (p = 0.002 and p = 0.003, respectively). Likewise, 13 BL AAbs showed significant associations with any-grade irAEs, particularly AAbs to breast cancer 2 (BRCA2) DNA repair associated antigen and chromatin-modifying protein 4A (CHMP4A) (p = 0.001 and p = 0.003, respectively). Conclusion: The predictive potential of autoantibodies as biomarkers is promising. Further evaluation in larger cohorts is needed for validation of our findings and understanding the underlying mechanism. Table 1. Patients’ demographics Total patients n=41 Median age in years (range) 66 (35-84) Male n=19 Female n=22 Tumor type Carcinoma of unknown primary n=13 Squamous cell carcinoma of the skin n=9 Small cell malignancies of non-pulmonary origin n=7 Adrenocortical carcinoma n=6 Vascular sarcoma n=5 Other rare histology n=1 Clinical benefit CR, PR, or SD ≥ 4 months n=14 SD &amp;lt;4 months or progressive disease (PD) n=23 Not evaluable n=4 irAEs Yes n=16 No n=25 Citation Format: Mohamed H. Derbala, Joud Hajjar, Bettzy Stephen, Serdar A. Gurses, Evan Kwiatkowski, Petra Budde, Hans-Dieter Zucht, Manuel Bräutigam, Ann-Sophie Schubert, Behnaz Ahangarianabhari, Enedelia Rodriguez, Mohamed Gouda, Lilibeth Castillo, Abdulrazzak Zarifa, Jeffrey A. How, Justin T. Moyers, David S. Hong, Funda Meric-Bernstam, Aung Naing. Autoantibody profiling for predictive biomarkers for immune-related adverse events and clinical benefit in rare tumors treated with anti-PD-1 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5141.

Machine Learning Methods for Gene Selection in Uveal Melanoma.

Uveal melanoma (UM) is the most common primary intraocular malignancy with a limited five-year survival for metastatic patients. Limited therapeutic treatments are currently available for metastatic disease, even if the genomics of this tumor has been deeply studied using next-generation sequencing (NGS) and functional experiments. The profound knowledge of the molecular features that characterize this tumor has not led to the development of efficacious therapies, and the survival of metastatic patients has not changed for decades. Several bioinformatics methods have been applied to mine NGS tumor data in order to unveil tumor biology and detect possible molecular targets for new therapies. Each application can be single domain based while others are more focused on data integration from multiple genomics domains (as gene expression and methylation data). Examples of single domain approaches include differentially expressed gene (DEG) analysis on gene expression data with statistical methods such as SAM (significance analysis of microarray) or gene prioritization with complex algorithms such as deep learning. Data fusion or integration methods merge multiple domains of information to define new clusters of patients or to detect relevant genes, according to multiple NGS data. In this work, we compare different strategies to detect relevant genes for metastatic disease prediction in the TCGA uveal melanoma (UVM) dataset. Detected targets are validated with multi-gene score analysis on a larger UM microarray dataset.

Transcriptomic Signature of a Patient with Chronic Lymphocytic Leukemia Exhibiting Profound Fluctuations in the Lymphocytosis during Nutrition and Prolonged Fasting Periods

Introduction This study aimed to investigate the underlying reasons behind sustained significant continuous deep fluctuations in the absolute lymphocyte count (ALC) in an untreated patient with Chronic Lymphocytic Leukemia (CLL), who has maintained a favorable prognosis since the time of diagnosis in 2016. The patient has voluntarily chosen to adopt a mainly vegetarian and fruitarian diet, including annual periods of prolonged total fasting (lasting from 4 to 39 days). Methods For this purpose, we decided to conduct a comprehensive analysis of the whole transcriptome profiling of peripheral blood (PB) CD19+ cells from patient (#1) at different time-points, in comparison to the corresponding cells from 5 other untreated CLL patients who followed a varied diet. As a result, the CLL patients were classified as follows: the 1st group consisted of patient #1 at 20 different time-points (16 time-points during nutrition and 4 time-points during fasting), whereas the 2nd group included only one time point for each of the patients (#2, #3, #4, #5, and #6) due to their varied dietary practices. We have performed microarray experiments on the Peripheral Bone Marrow Cells (PBMCs) CD19+ cells utilizing the Affymetrix Human Clariom™ D Pico Assay, a powerful tool for generating high-fidelity biomarker signatures. The data was preprocessed and normalized using the Robust Multiarray Average (RMA) method. Significance Analysis of Microarrays (SAM version 3.0) was utilized to assess significant differentially expressed genes between the 1st group and 2nd group of CLL patients. The Storey and Tibshirani procedure was applied to control the False Discovery Rate (FDR). Results The lymphocytosis trend of patient #1 displayed recurring significant deep fluctuations since the time of diagnosis. Interestingly, we observed that approximately 4-6 weeks after the conclusion of fasting periods, the absolute lymphocyte count was reduced by about half (Figure 1A). On the other hand, in the 2nd group, all patients did not show fluctuations in the absolute lymphocyte count, but instead demonstrated a progressive increase in lymphocytes over time. Differential expression analysis, after correction for multiple testing, identified 18 differentially expressed genes (DEGs) in the comparison between CD19+ cells of patient #1 and patients #2, #3, #4, #5, and #6. Among them, our focus was on 9 genes with functional annotation: 4 genes were downregulated ( IGLC3, RPS26, CHPT1, and PCDH9), and 5 genes were upregulated ( IGHV3-43, IGKV3D-20, PLEKHA1, CYBB, and GABRB2) in the 1st group (patient #1) vs. the 2nd group of patients (#2, #3, #4, #5, and #6), respectively. In particular, IGLC3, RPS26, PLEKHA1, CYBB, and GABRB2 showed high Fold Change (FC) &amp;gt; 3. Additionally, we generated a heat map on log2 expression of the 9 differentially expressed genes across different patients/conditions (Figure 1B). Furthermore, hierarchical clustering analysis revealed that patient #1 during nutrition and fasting (1st group) clustered distinctly from all CLL patients (2nd group), indicating a gene expression signature that differentiated patient #1 from patient #2, #3, #4, #5, and #6. Conclusions In conclusion, our transcriptomic study revealed a small set of 9 genes that distinguished an untreated CLL patient who underwent a prolonged periods of total fasting and maintained a slow-growing tendency of lymphocytosis, in comparison to 5 untreated CLL patients with a varied diet. Future investigations of patient #1 could demonstrate the potential role of prolonged periodic fasting and the involvement of the 9 genes in sustaining the trend of lymphocytosis and the benign course of the disease.

Identification of circulating miRNA as early diagnostic molecular markers in malignant glioblastoma base on decision tree joint scoring algorithm.

The lack of clinical markers prevents early diagnosis of glioblastoma (GBM). Many studies have found that circulating microRNAs (miRNAs) can be used as early diagnostic markers of malignant tumours. Therefore, the identification of novel circulating miRNA biomolecular markers could be beneficial to clinicians in the early diagnosis of GBM. We developed a decision tree joint scoring algorithm (DTSA), systematically integrating significance analysis of microarray (SAM), Pearson hierarchical clustering, T test, Decision tree and Entropy weight score algorithm, to screen out circulating miRNA molecular markers with high sensitivity and accuracy for early diagnosis of GBM. DTSA was developed and applied for GBM datasets and three circulating miRNA molecular markers were identified, namely, hsa-miR-2278, hsa-miR-555 and hsa-miR-892b. We have found that hsa-miR-2278 and hsa-miR-892b regulate the GBM pathway through target genes, promoting the development of GBM and affecting the survival of patients. DTSA has better classification effect in all data sets than other classification algorithms, and identified miRNAs are better than existing markers of GBM. These results suggest that DTSA can effectively identify circulating miRNA, thus contributing to the early diagnosis and personalised treatment of GBM.

Self-Supporting Human Acute Myeloid Leukemia (AML) Bone Marrow Organoids Established from Patient-Derived Xenografts Mimic the Human AML Microenvironment, with Distinct Cytokine Kinetics Correlating with Dynamic Cell Proliferation and Metabolic Shifts

Hallmarks of acute myeloid leukemia (AML) include (1) formation of a self-supporting and self-regulating tumor microenvironment (TME) in the bone marrow (BM) rendering pathological niches that orchestrate retention, proliferation and survival of AML blasts, and (2) a dynamic metabolism that promotes therapy resistance and disease progression. Efforts to understand AML biology and enhance current therapies are hampered by the paucity of state-of-the-art human models that can recreate the three-dimensional (3D) BM TME and recapitulate the heterogeneity, complexity, dynamics, and metabolic shifts observed in human disease. Previously, we described the development of the first long-term self-propagating serum- and cytokine-free human BM organoid generated from an AML patient-derived xenograft (PDX) that captured metabolic shifts during AML progression. Here, we characterize the dynamic changes observed in the self-constituted TME with respect to AML proliferation, metabolism, and upon reconstitution of secondary organoids. BM was collected from NSG mice engrafted with human primary AML cells (PDX-AML model) or naïve control mice and seeded into polyurethane scaffolds in serum- and cytokine-free medium at 4e6 cells per scaffold, optimized previously. Scaffolds were recharged with autologous PDX-AML BM on day (D) 13 and cultured until D70. Organoids were assessed for (1) inter- and intra-scaffold AML kinetics, (2) the TME, (3) metabolic composition and (4) propagation into secondary organoids. AML cell kinetics from organoid culture supernatants reflected those of scaffold-extracted populations (n=3). CD33 +CD44 + AML cells expanded and/or were maintained from 7% (D0) to &amp;gt;10% (D70), being &amp;gt;15% at most culture timepoints. Confocal microscopy revealed reconstitution of typical AML-TME niche interactions within organoids, including expression of fibronectin, VCAM-1 and N-Cadherin, and formation of proliferative niches composed of AML cells with heterogeneous Osteopontin- and Osterix-expressing support cells. Human AML-supportive cytokines were consistently observed in organoid supernatants (N=3), with SDF-1, IL-1α, IL-8, GM-CSF, FGF-2 and Osteopontin present throughout the culture; some cytokines had biphasic kinetics with factors critical for TME formation present early (e.g. Osteopontin, GM-CSF), and those required for AML proliferation dominant later (e.g. FLT3L, IL-8), coinciding also with metabolic shifts during culture. Despite the absence of IL-6, single molecule RNA fluorescence in situ hybridization confirmed IL-6 expression by niche-resident AML blasts suggesting autocrine signaling networks beyond those identified in supernatants. Cells from D70 organoids were able to re-establish secondary organoids (n=2) which also exhibited long-term (D70) AML maintenance (&amp;gt;11% blasts), and regenerated the AML TME. Previously, we identified 3 culture stages defined by metabolic shifts: 1) pre/early recharge (D4-D19), (2) intermediate (D25-D40) and, (3) late (from D55) where organoids exhibited highest proliferation. To further characterize these shifts, intracellular metabolites were extracted from organoids (n=5) at D10, D25, D40, D55 and D70, analyzed with gas-chromatography mass spectrometry (GC-MS) and evaluated through unsupervised hierarchical clustering and multivariate analysis. Significance Analysis of Microarrays (SAM) analysis demonstrated dysregulation of alanine, serine, isoleucine, glycolate, hexanoic acid and glycolysis when comparing stage 1 with stage 2, and in isoleucine, leucine, serine, succinate and tryptophan metabolism when comparing stage 2 with stage 3. When proliferative and non-proliferative timepoints were compared, amino acid and glycolysis-driven metabolic switches were identified, typical of AML metabolism. We have characterized a long-term 3D human AML BM biomimicry in serum- and cytokine-free conditions with a self-organizing TME that recapitulates many features of AML BM, including robust human AML cell maintenance within niches, cytokine and metabolic kinetics to support different phases of progression, and the ability to propagate into secondary organoids. This dynamic, physiologically-relevant ex vivo platform can enable studies in human AML pathogenesis/progression and is poised to test novel therapeutics directed towards AML cells, the TME and metabolic targets.

O-062 MicroRNAs in Blastocoel Fluid: a molecular signature for predicting human embryo implantation potential

Abstract Study question Can microRNA expression in blastocoel fluid (BF) predict embryo implantation potential? Summary answer Up-regulation of miR-106, miR-373, miR-301, miR-320 and miR-525-3p represents the molecular signature characterizing the embryos with high implantation capability. What is known already The discovery of DNA within the BF and in spent embryo culture media, has caused increased interest in the non-invasive preimplantation testing application for monogenic disorders and aneuploidies. Aneuploid embryos have a lower implantation potential, however, chromosomal abnormalities represent only a tiny percentage of the causes of implantation failure. MiRNAs can control all cellular pathways and are involved in pluripotency, self-renewal, and stemness, and their altered regulation affects different human diseases. Study design, size, duration From September 2018 to March 2022, 112 BFsampleswere collected from human embryos on the fifth day of development, before the blastocyst cryopreservation. The samples were classified according to blastocyst grade and the data on implantation outcome and term births were collected for the transferred embryos. We compared the expression profiles of 89 miRNAs, previously identified in BF, between 33 BF from implanted embryos and 30 from non-implanted ones, regardless of blastocyst grade. Participants/materials, setting, methods By custom-designed TaqMan Low-Density Array card (TLDA), we analyzed the expression of 89 miRNAs in 4 different BF samples simultaneously. Differentially expressed (DE) miRNAs were identified by Volcano plot and Significance Analysis of Microarrays (SAM) tests. Bioinformatic analysis was performed to identify the biological role of the DE miRNAs. To evaluate miRNA’s ability for predicting implantation, Pearson’s correlation analyses, classical univariate Receiver Operator Characteristic (ROC) curve analysis, and the optimal cut-off value determination were performed. Main results and the role of chance We found five miRNAs, miR-106, miR-373, miR-301, miR-320 and miR-525-3pup-regulated in BF from implanted blastocysts. The identified miRNAs perform an important role during the first phases of embryo development suggesting that their up-regulation may reflect embryo health. Moreover, four of the five miRNAs showed significant correlation coefficients in both implanted and non-implanted blastocysts, indicating that their expression changes in the same way in the single sample and reflects the potentiality of the embryo to implant. Finally, ROC curve analysis confirmed that our miRNAs could be considered potential biomarkers for implantation. Limitations, reasons for caution Successful implantation requires a close dialogue between the embryo and the endometrium, mediated by different proteins and miRNAs produced by both the embryo and maternal tissues. Determining the quality of the embryo and its implantation potential is not sufficient to predict successful pregnancy outcomes. Wider implications of the findings This study represents the first report correlating miRNA profiles in BFand implantation and suggests that miRNA signature could become an accurate tool to evaluate embryo quality. It could be associated or replaced with the PGT-A to choose the most competent embryo and improve the outcome of assisted reproduction cycles. Trial registration number The study has been approved by theEthical Committee of Azienda Ospedaliero Universitaria Policlinico &amp;quot;G.Rodolico -San Marco&amp;quot; Catania.

P-098 Cryopreservation process deregulates the expression of pathways important for fertility in human spermatozoa

Abstract Study question Does cryopreservation impact on transcriptome of conventional frozen human spermatozoa? Summary answer The cryopreservation of human sperm alters pathways important for fertility, although the upregulation of some genes can compensate for the harmful effects of freezing. What is known already Sperm cryopreservation is a widely used procedure for storing gametes for later use, for preserving fertility in patients before gonadotoxic treatments or surgery, and for sperm donation programs. The cryopreservation technique can cause damage to sperm through induction of DNA alterations, fragmentation and oxidation, and altered motility, mitochondrial activity and morphology. Transcriptomic alterations have been reported in cryopreserved sperm from various animal species, but no experimental data are available on the effect of cryopreservation on the transcriptome-wide profile of human sperm. Study design, size, duration Semen samples were obtained from 20 normospermic men from April to May 2022. Each sample was divided in two aliquots. From one aliquot total RNA was immediately extracted. The second aliquot was slowly cryopreserved and after a week of storage in liquid nitrogen total RNA was extracted. A total of 13 paired RNA samples passed quality control and were analyzed after randomization into 4 pools, each of 6 patients. Participants/materials, setting, methods The men who participated in this study had a median age of 35.0 years (range: 29.0-46.0). RNA was extracted by miRNeasy Micro Kit (Qiagen), analyzed by High Sensitivity RNA on 2200 Tape Station system (Agilent), amplified by Ovation Pico WTA SystemV2 (Nugen), labeled and hybridized on GE 4x44K v2 microarrays (Agilent). The paired Significance Analysis of Microarray (SAM) was performed using the limma hgug4112a.db and samr packages in R/BioConductor. Main results and the role of chance The expression profiles of cryopreserved sperm significantly differed from those of fresh sperm in 219 down-regulated and 28 up-regulated unique transcripts. The gene ontology analysis disclosed that cryopreservation downregulates genes involved in sperm motility by the mitochondria function (CABS1, SPATA19) and the correct organization of the sperm midpiece and flagellum cytoskeleton (ACTL7A, AKAP4, C9ORF24, CAPZB, CCIN, IQCG, ODF2, SPATA6, SPATA6L, TBC1D21), in fertilization (ACTL7A, AKAP4, PLCZ1, PRSS37, TUBGCP3), early embryo development (AGT, CLMN, DCC, DHRS3, EGFLAM, FAM20A, FREM1, GPI, HEMGN, KRTDAP, IFT172, MICAL2, MLF1, NF2, PGRMC2, PHACTR1, POPDC3, RO60, ROBO1, RORA, SGCA, SPRR2D, TCF4, TNC, TNNI3), oxidant detoxification (TXNDC29) and DNA repair (BRIP1, ERCC6, TRIP12), calcium ion binding and homeostasis (AMY1C, ANO1, ANO2, ASGR1, CABS1, CPNE9, FREM1, KCNIP2, ITPR3, PKD2L1, PLCZ1, SELENOK, SYN3, TNNI3). Upregulated genes were enriched in pathways related to the negative regulation of DNA damage response (ALOX15B, CD74, RPL10, SNAI1, THBS1), cellular response to calcium ion (ALOX15B, FOSB, HLA-DQB1, S100A4, THBS1), regulation of transcription (CD74, FOSB, HLA-DRB1, MAMLD1, RASD1, RPL10, SNAI1, TCF15, TLE1), protein stabilization (CD74), protein ubiquitination (BAG1, DCAF6, ERCC6, HERPUD2, KLHL7, MARCHF8, NF2, PSMA6, RNF133, SERGEF, TRIP12, UBE2DNL, UBL3, UBQLN3, ZNRF3) and ubiquitin protein ligase binding (GPI, HSPA1L, PRKAR2A, STX8). Limitations, reasons for caution Data validation in a larger sample cohort and by quantitative PCR would be useful, although we have applied stringent criteria for gene selection (FDR = 0). Further research should be undertaken to experimentally test whether the length of cryostorage can have any effect on the gene expression profile of sperm. Wider implications of the findings Specific pathways are deregulated by cryopreservation, in accordance with altered sperm morphology, motility and molecular integrity reported in literature. Functional enrichment analyses also revealed gene expression changes to compensate for the sperm cryoinjury. This finding is noteworthy for safety issue of sperm banking i.e., fertility preservation, gamete donation. Trial registration number not applicable

P-064 MicroRNAs as potential biomarkers for male infertility related to Testicular Germ Cell Tumors

Abstract Study question Could microRNAs represent efficient biomarkers to evaluate male infertility and the tendency of develop testicular germ cell tumors (TGCTs)? Summary answer The up-regulation of miR-193a-5p, miR-93-5p and let-7c-5p in TGCT patients with impaired spermiogram could represent a molecular signature of male infertility related to TGCTs. What is known already Testicular germ cell tumors (TGCTs) are the most recurrent tumors in young men with the highest incidence between the ages of 20 and 40 years and represent more than 60% of all cancers diagnosed in this age range. Several studies have shown a correlation between TGCTs and infertility, not only because infertility could be a consequence of testicular damage due to TGCTs but also because infertility may represent a risk factor for TGCTs. Therefore, it is important to find valid biomarkers that could be used to specifically identify infertility related to an increased risk of developing TGCTs. Study design, size, duration From March 2021 to January 2023, we collected 44 seminal plasma samples: 17 from TGCT patients who were undergoing sperm cryopreservation prior to chemotherapy, 14 with impaired spermiogram (IS) and 3 with normal spermiogram (NS) and 27 from control patients, 16 with IS and 11 with NS, undergoing assisted fertilization techniques. We evaluated the potential role of miRNAs as non-invasive biomarkers, analyzing the differential expression of 84 miRNAs in relation to cancer, infertility and both. Participants/materials, setting, methods Semen samples were placed 30’/37 °C, seminal plasma was purified using density-gradient centrifugation and stored at -80 °C. RNA was purified by Qiagen miRNeasy Serum/Plasma Kit and analyzed by miRCURY LNA miRNA SYBR®Green PCR_SerumPlasma, 96-well plate. We applied the 2−ΔΔCT method and statistical significance was evaluated by Significance Analysis of Microarrays, screened by p-values ≤ 0.05. We performed a Pearson correlation analysis applying two-sided p-values. miRTarBASE, MIENTURNET and Cytoscape were used for bioinformatics analysis. Main results and the role of chance In the four comparisons, we found 9 differentially expressed microRNAs. In particular, miR-221-3p, miR-222-3p, miR-204-5p and miR-205-5p down regulation would appear to be related to infertility, regardless to the cancer. Conversely, all TGCT patients shown an up-regulation of miR-376c-3p. Interestingly, up-regulation of miR-193a-5p, miR-93-5p and let-7c-5p specifically discriminates the infertile cancer patients vs other categories. Moreover, these three miRNAs showed a significant positive correlation both in cancer patients with impaired spermiogram and in infertile controls. This correlation demonstrates that their expression changes in the same way in the single samples and strongly suggests their role as early biomarkers of TGCT patients with impaired spermiogram. From the KEGG pathway analysis has emerged that DE miRNAs are involved in several signaling pathway, such as MAPK, mTOR, p53, PI3K-Akt, AMPK, FoxO, JAK-STAT, Ras, Estrogen signaling pathways, as well as cell cycle and cellular senescence. Moreover, from the Diseases Ontology analysis we found than those miRNAs are involved in germ cell tumor, male reproductive organ cancer, prostate cancer, male infertility and azoospermia. Limitations, reasons for caution The data must be validated by single assays in higher number of samples. These experiments are currently ongoing. Wider implications of the findings Three of the DE miRNAs are overexpressed in TGCT with impaired spermiogram patients. Specifically, they are over-expressed in TGCT with impaired spermiogram compared to infertile controls, so they may represent specific biomarkers able to discriminate, among the young men with infertility, those with higher risk of develop testicular cancer. Trial registration number Not applicable

Baseline autoantibody profiling in patients with NSCLC with pre-existing autoimmune diseases or who had received prior anti-PD-1 therapy before enrolling in the TAIL study.

2512 Background: Patients (pts) with pre-existing autoimmune diseases (AID) and prior immune checkpoint inhibitor (ICI) experience were historically excluded from studies with ICIs. Moreover, about 50% pts with pre-existing autoimmunity have a flare up when given ICI therapies (1). In this post-hoc exploratory analysis, we characterized the baseline autoantibody (AAb) profiles and evaluated its correlation with efficacy and safety outcomes in NSCLC pts with either pre-existing AID or who had prior ICI therapy and received atezolizumab in the TAIL study (2). Methods: Plasma samples were obtained from 57 NSCLC pts prior to receiving atezolizumab (TAIL study, NCT03285763). AAb testing was performed using tailored immuno-oncology bead-based antigen arrays comprising 1,340 tumor, AID and immune pathway antigens (3, 4). The cohort includes 39 pts with prior ICI therapy or prior AID (experimental group) and 18 pts without prior ICI therapy or AID (control group). Differences in IgG AAbs were compared between the experimental and control group in pts with or w/o irAE and overall survival (OS) longer or shorter than 11.1 months (median OS in overall population in TAIL). Furthermore, the AAb profiles of a subgroup of pts with therapeutic benefit (OS ≥ 11.1 months) and no or low grade irAEs (standard toxicity grade ≤ 2) were analyzed. Statistical analysis included 2-way permutation-based ANOVA (factors: outcome, study group) and Significance Analysis of Microarrays (SAM). Results: While pre-treatment AAbs were detected in the overall cohort, significant differences in AAb were observed between the experimental and control group at baseline, which were associated with OS and irAEs. In experimental group pts, pre-existing AAbs to AQP4 and NCOA1 predicted the development of irAEs. Differences were also observed in AAbs predicting longer OS. AAbs to CENPT were specifically identified in pts in the experimental group with longer OS, whereas AAbs to SOX5 were elevated in pts of the control group with longer OS. In the subgroup of pts with therapeutic benefit and no or low risk of irAE, AAbs to IL6, CENPT and the cancer testis antigens NY-ESO-2 and MAGEB2 were elevated in the experimental group. Conclusions: This AAb analysis provides important preliminary data in helping to understand the drivers of clinical outcomes with ICIs in pts with pre-existing AID and prior ICI therapy. The findings seem biologically plausible as some of the identified antigens refer to the breach of tolerance to classical AAb targets e.g. CENPT, AQP4 and to an immune response to classic tumor antigens e.g. NY-ESO-2 and MAGEB2. Further validation of the AAb profiling approach is warranted to shed insights on potential therapeutically actionable antigens and generate data orthogonal to efforts to identify biomarkers that are predictive of efficacy and safety outcomes with ICI therapies.

Integrative computational approach for gene expression profiling of metastatic breast cancer

Background: Breast cancer (BC) is one of the most common cancers in women worldwide, including an estimated 570,000 deaths in 2015. More than 1.5 million women (25% of all women with cancer) are diagnosed with BC every year worldwide. BC is a metastatic cancer and usually spreads to distant organs such as bone, liver, lungs and the brain. Early detection of the disease can lead to better prognosis and higher survival rates. After genome sequencing, DNA microarray analysis has become the most widely used source of genome scale data in life sciences. Microarray's definition studies produce a large number of genetic expression and other genomics performance data, which promises to provide key information on genetic functioning and interaction within and within metabolic pathways. Aim: Prediction and functional enrichment of significant genes expressed in Breast Cancer. Materials and Methods: Microarray data for this study were downloaded from NCBI gene expression omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo), with accession number of GSE157737. It includes four different kind sets of samples, i.e., healthy control neutrophils, healthy donor monocytes, metastatic BC patient G-myeloid-derived suppressor cell (G-MDSCs) and sepsis patient G-MDSCs. The statistical method was applied to identify the differentially expressed gene (DEGs) using Limma package and significant analysis of microarray (SAM) test. Results: The top ten DEGs were identified between different sample sets using Limma package. Out of 32,321 genes, 93 genes were identified to be the significant genes through SAM test. The functional annotation of significant genes was done using different databases such as gene ontology, Kyoto Encyclopedia of Genes and Genomes and Database for Annotation, Visualisation and Integrated Discovery. Significant genes were found to be enriched in haematopoietic cell lineage pathway and the cancer-causing pathway. Conclusions: This research identifies the important genes that have function in BC and regulation of such genes and pathway can be a potential treatment for the BC and prevent the tumour growth.

Host-microbiome interactions in apical periodontitis: The endodontic microbiome in relation to circulatory immunologic markers.

To explore microbial differences in the endodontic infection of teeth with primary or secondary apical periodontitis (AP), with or without symptomatology. Additionally, to investigate if these differences are depicted in immunologic markers in blood. Twenty-nine teeth with primary or secondary AP were extracted and cryo-pulverized. Blood was drawn from the subjects at three different time-points before and three time-points after the extraction in a time period of four months. The V4 hypervariable region of the 16S rRNA gene was sequenced using Illumina MiSeq. The microbial profiles were ordinated using principal component analysis and tested for differences between groups with permutational multivariate analysis of variance using the Bray-Curtis distance. If significantly different, the microbial profiles were further analysed using the LDA effect size (LEfSe) biomarker discovery tool. A broad panel of inflammatory mediators in blood was examined longitudinally in all subjects during the six visits with mixed models. The Spearman correlation between these mediators and the zOTUs was calculated, and significant correlations (p < .05) were used as input for significant analysis of microarrays (SAM) using MeV. After subsampling, the 467 zOTUs were classified into 9 phyla and 99 genera or higher level taxa. The predominant genus in the entire sample set was Fusobacterium with a relative abundance of 12.3%, followed by Prevotella (9.9%), Actinomyces (7.7%) and Streptococcus (6.7%). The microbiomes of the endodontic infections were significantly associated with endodontic status (primary/secondary infection; p=.015) as well as with the presence or absence of pain (p=.011). There was also a difference in the concentration of inflammatory mediators, namely, C-reactive protein, Interleukin (IL)-8, IL-10, IL-12p70, RANKL and TNF-α, depending on the existence of pain. In addition, the presence of specific bacteria (zOTUs) was correlated, positively or negatively, with the expression of several circulating inflammatory markers. The microbial profiles and the concentration-time relationship of systemic inflammatory mediators of primary endodontic infection differed from those of secondary, and of symptomatic from those of asymptomatic cases. The fingerprint of associations between the immunological and microbiological profiles differed between asymptomatic and symptomatic patients.

Abstract P1-13-16: Dissecting the biological activity of different CDK4/6 inhibitors (CDK4/6i) in hormone receptor-positive/HER2-negative (HR+/HER2-) breast cancer (BC)

Abstract Background: Palbociclib might be less effective than ribociclib in HR+/HER2- and HER2-enriched BC. This hypothesis is currently being tested in the HARMONIA prospective phase III clinical trial (NCT05207709). Here, we evaluated the downstream biological effects of both drugs using cell lines and patient tumor samples. Methods: Three HR+ BC cell lines (i.e., MCF7, T47D and BT474) were treated at 2 different dose levels of palbociclib or ribociclib (i.e., 100 nM and 500 nM) +/- fulvestrant (1 nM) for 72 hours (h). PAM50 gene signatures were determined on the nCounter, as well as phosphorylation of RB (p-RB) by Western Blot and senescence-associated β-galactosidase activity by FACS. In vitro experiments were performed in triplicates. PAM50 gene signatures were obtained from 49 paired baseline versus week-2 samples and 49 paired baseline versus surgery samples of the CORALLEEN phase II study (Prat, Lancet Oncol. 2020), which treated 49 women with PAM50 Luminal B HR+/HER2- early BC with neoadjuvant ribociclib (600 mg daily) plus endocrine therapy (ET). PAM50 signature scores were also evaluated in publicly available data from 23 paired baseline versus week-2 samples and 16 paired baseline versus surgery samples of the NEOPALANA phase II trial (Ma, Clin Cancer Res. 2017) which treated 50 patients with HR+/HER2- early BC with palbociclib (125 mg daily) plus anastrozole. Changes in PAM50 signatures upon CDK4/6i were determined by paired t-tests and significant analysis of microarray (SAM). Results: Across all cell lines, both palbociclib and ribociclib statistically significantly reduced p-RB at 72h with both doses (i.e., 100 and 500 nM) compared to non-treated cells (p&amp;lt; 0.001). Senescence was also observed at 72h with both doses. Both drugs +/- ET significantly increased the Luminal A signature and decreased Luminal B and proliferation signatures with both doses. However, the HER2-enriched signature was only significantly reduced when both CDK4/6 inhibitors were given at 500 nM. In tumor samples from the CORALLEEN and NEOPALANA phase II studies, a similar change in PAM50 biology was observed with both drugs, namely an increase in Luminal A signature and a decrease in Luminal B and proliferation signatures after 2 weeks of treatment and at surgery. At 2 weeks of treatment, the HER2-enriched signature was significantly decreased in both studies with ribociclib (p&amp;lt; 0.001) and palbociclib (p&amp;lt; 0.001). However, the decrease in the HER2-enriched signature was only observed in surgical samples of patients treated with ribociclib (p&amp;lt; 0.001), but not palbociclib (p=0.194). A difference in sample size could explain this result. Nevertheless, in CORALLEEN, the median number of days between the last dose of ribociclib and surgery was 13.1 days (range: 1-78). In NEOPALANA, the median number of days between the last dose of palbociclib and surgery was 29 days (range: 8-49), except for 8 patients who received additional 10-12 days of palbociclib immediately before surgery (Ma, Clin Cancer Res. 2017). In patients who underwent surgery at 8 days or before, the HER2-enriched signature was significantly decreased for both ribociclib (p&amp;lt; 0.001) and palbociclib (p=0.013). Interestingly, in patients that underwent surgery after &amp;gt;8 days from the last dose, a significant reduction of the HER2-enriched signature was only observed with ribociclib (p&amp;lt; 0.001), but not with palbociclib (p=0.500). Conclusions: Both palbociclib and ribociclib have similar effects on PAM50 biology when given at the same dose. However, in clinical practice, palbociclib is given at a lower dose than ribociclib, and although HER2-enriched signature is significantly decreased in tumors after 2 weeks of CDK4/6i+ET, this effect is only maintained at later time points with ribociclib, indicating a dose-dependent efficacy of CDK4/6i in this biologically aggressive subtype. Citation Format: Natàlia Lorman-Carbó, Olga Martínez-Sáez, Aranzazu Fernandez-Martinez, Patricia Galván, Nuria Chic, Barbara Adamo, Maria Vidal, Montserrat Muñoz, Charles M. Perou, Joaquín Gavilá, Tomás Pascual, Aleix Prat, Fara Brasó-Maristany. Dissecting the biological activity of different CDK4/6 inhibitors (CDK4/6i) in hormone receptor-positive/HER2-negative (HR+/HER2-) breast cancer (BC) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-16.

Abstract P4-02-30: Association between tumor infiltrating lymphocytes (TILs) and the HER2DX assay in early-stage of HER2-positive (HER2+) breast cancer

Abstract Background: The HER2DX assay is a genomic test in early-stage HER2-positive (HER2+) breast cancer that provides prognostic and predictive information. HER2DX is a supervised learning algorithm incorporating tumor size, nodal staging, and 4 gene expression signature scores (immune/IGG, tumor cell proliferation, luminal differentiation and the expression of the HER2 amplicon). Among them, the IGG signature is associated with both overall survival and probability of achieving a pathologic complete response (pCR). Here, we studied the association of percentage (%) of tumor infiltrating lymphocytes (TILs) with HER2DX scores, immune genes and other breast cancer-related genes. Methods: HER2DX and %TILs were evaluated in 670 formalin-fixed paraffin-embedded (FFPE) samples of HER2+ breast cancer, including in 3 clinical studies SHORTHER (n=437), PAMELA (n=86), a cohort of patients treated with anti-HER2 therapy plus chemotherapy at Hospital Clínic of Barcelona (n=147). The %TILs were quantified by histological evaluation with hematoxylin eosin staining according to International TILs Working Group guidelines. The nCounter platform determined the expression of 192 genes and HER2DX scores. Pearson correlations (Cor) and Significance Analysis of Microarrays (SAM) with a false-discovery rate (FDR) &amp;lt; 5% assessed the association between %TILs and the expression of individual genes or HER2DX signature scores. Results: A moderate correlation was observed between %TILs and the immune IGG signature (Cor=0.56, p&amp;lt; 0.001). Of note, 171 (25.52%) cases had low %TILs (&amp;lt; 30%) and high IGG score, while 1 (0.15%) case had high %TILs (≥30%) and low IGG score. The %TILs were significantly associated with the expression of immune genes, ERBB2, IGG signature and HER2 amplicon score, and negatively associated with the expression of luminal genes (i.e., ESR1, PRG and BCL2). An unclear relationship between TILs and proliferation genes was observed. Finally, moderate correlations were observed between %TILs and HER2DX pCR score (Cor=0.48, p&amp;lt; 0.001) and between %TILs and HER2DX risk score (Cor=0.33, p&amp;lt; 0.001). Conclusions: Important differences exist between the %TILs and the HER2DX IGG signature in early-stage HER2+ breast cancer. The %TILs should not be used to predict the HER2DX scores. Biologically, a higher %TILs indirectly capture a higher ERBB2 expression and a lower expression of luminal genes, both associated with response to anti-HER2 treatment. Citation Format: Esther Sanfeliu, Fara Brasó-Maristany, Maria Vittoria Dieci, Mercedes Marín-Aguilera, Blanca González-Farré, Gaia Griguolo, Tomas Pascual, Patricia Galván, Laura Angelats, Oleguer Castillo, Paula Blasco, Valeria Sirenko, Pedro Jares, Joan Antón Puig-Butillé, Laia Paré, Antonio Martínez, Antonio Llombart-Cussac, Javier Cortés, Ana Vivancos, Patricia Villagrasa, Joel S Parker, Charles M Perou, Aleix Prat, PierFranco Conte, Valentina Guarneri. Association between tumor infiltrating lymphocytes (TILs) and the HER2DX assay in early-stage of HER2-positive (HER2+) breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-02-30.