Formalin-fixed Paraffin-embedded Biopsies Research Articles

Evaluation of molecular and bacteriological detection methods performed on the formalin-fixed paraffin-embedded biopsy samples collected from endometrial and lymph node tuberculosis suspected patients

BackgroundEndometrial Tuberculosis is one of the most common gynecological problems known to have serious implications for the quality of life like infertility. The commonly practiced histopathology solely relies on the suggestive feature of Tuberculosis (TB) with low specificity. Regarding the alternative bacteriological and molecular detection tools, little evidence was generated on their utility in the diagnosis of endometrial tuberculosis in Ethiopia. Therefore, we aim to investigate the detection rate of molecular and bacteriological detection methods on formalin-fixed paraffin-embedded biopsy samples for the diagnosis of endometrial and lymph node TB.MethodsA retrospective cross-sectional study was conducted on 90 formalin fixed paraffin embedded biopsy samples from patients with gynecologic and lymph problems collected between 2018 and 2022 at St. Paul’s Hospital Millennium Medical College, Addis Ababa, Ethiopia. SPSS version 26 was used for statistical analysis. The diagnostic performance was calculated using the histopathology method as the reference standard. Cohen’s Kappa value was used to measure the level of agreement. A test with a P-value of < 0.05 was considered statistically significant.ResultsA total of 90 samples were analyzed in the current study. Auramine O, GeneXpert MTB/RIF assay, and Real-Time PCR tests have shown a detection rate of 32/90 (36%), 43/90 (47.8%), and 54/90 (60%) respectively (P ≤ 0.01). The sensitivity and specificity of AO were 38.1% and 95% respectively. RT PCR showed superior sensitivity followed by GeneXpert MTB/RIF assay, 70% and 58.6%. AO and molecular methods have shown a similarly low level of agreement with histopathology (Kappa value = 0.2).ConclusionsIn a resource-limited setting, the selection of diagnostic tools needs careful attention. Putting the patients on anti-TB treatments based solely on histopathological findings may lead to undesired and adverse complications. Therefore, applying molecular and bacteriological detection methods along with histopathology, could help minimize inappropriate antimicrobial use.

Kidney whole-transcriptome profiling in primary antiphospholipid syndrome reveals complement, interferons and NETs-related gene expression.

Pathogenesis of antiphospholipid syndrome (APS) remains poorly elucidated. We aimed to evaluate for the first time, kidney transcriptome profiles in primary APS vs systemic lupus erythematosus (SLE) and control subjects. We performed RNA-sequencing on archival formalin-fixed paraffin-embedded kidney biopsies from APS (n = 4) SLE (n = 5), and control (n = 3) individuals, differential gene expression analysis (DGEA), and enrichment analysis using gene ontology (GO), and CORUM, KEGG and Reactome pathway databases. Two-dimensional projection showed a distinct gene profile in primary APS vs control kidneys samples, but similar to SLE. DGEA in APS vs controls returned 276 upregulated and 217 downregulated genes, while the comparison between APS and SLE identified 75 upregulated and 111 downregulated genes. In 276 upregulated genes, enriched GO terms were (innate) immune response, inflammatory response, leucocyte and lymphocyte activation, cytokine production and T cell activation. CORUM and KEGG revealed complement-related genes (C3, C4A, C4B). Expression levels showed logFC values of 2.25 (p= 1.58e-05) for C3, 2.17 (p= 2.69e-06) for C4A, and 2.135 (p= 3.7e-06) for C4B in APS vs controls, without differences between APS and SLE. Interferon (IFN) alpha/beta signalling was revealed by Reactome. Expression levels of nine IFN-regulated genes found upregulated in APS vs control kidneys (p-values ≤ 0.001 for all). Examining neutrophil-extracellular traps (NETs)-related gene expression, 13 of 15 upregulated NETs-related genes exhibited higher expression in APS vs controls but not vs SLE. Complement, interferon and NETs-related genes are highly expressed in APS kidney tissues, similarly to SLE, pointing out the role of innate immunity in APS nephropathy pathogenesis and potential treatment targets.

Impact of intra-tumoral immunity on predicting response and survival after neoadjuvant cisplatin-based chemotherapy in patients with muscle invasive bladder cancer.

Neoadjuvant cisplatin-based chemotherapy (NAC) followed by cystectomy is the standard of care for patients with muscle-invasive bladder cancer (MIBC). Pathologic complete response (pCR) is associated with favorable outcomes, but only 30%-40% of patients achieve that response. The aim of this study is to investigate the role played by the Tumor and Immune Microenvironment (TIME) in association with the clinical outcome of patients with MIBC undergoing NAC. Nineteen patients received NAC and were classified as pCR (n = 10) or non-pCR (n = 9). Bulk RNA-seq and immune protein evaluations using Digital Spatial Profiling (DSP) were performed on formalin-fixed paraffin-embedded (FFPE) tumor biopsies collected before NAC (baseline). Immunohistochemistry (IHC) evaluation focused on CD3 and CD20 expression was performed on baseline and end-of-treatment (EOT) FFPEs. Baseline peripheral blood was assessed for lymphocyte and neutrophil counts. Kaplan-Meier analyses and Cox PH regression models were used for survival analyses (OS). In the periphery, pCR patients showed lower neutrophil counts, and neutrophil/ lymphocyte ratio (NLR) when compared to non-pCR patients. In the tumor microenvironment (TME), gene expression analysis and protein evaluations highlighted an abundance of B cells and CD3+ T cells in pCR versus non-pCR patients. On the contrary, increased protein expression of ARG1+ cells, and cells expressing immune checkpoints such as LAG3, ICOS, and STING were observed in the TME of patients with non-pCR. In the current study, we demonstrated that lower NLR levels and increased CD3+ T cells and B cell infiltration are associated with improved response and long-term outcomes in patients with MIBC receiving NAC. These findings suggest that the impact of immune environment should be considered in determining the clinical outcome of MIBC patients treated with NAC.

Expression of cytokeratin 7/20 and Ki67 in Helicobacter pylori-associated gastritis and intestinal metaplasia.

Cytokeratins (CKs) have been associated with precancerous and cancerous gastric lesions in patients with Helicobacter pylori-associated chronic gastritis, making them useful for diagnosing epithelial tumors. A retrospective study was conducted utilizing 200 formalin-fixed paraffin-embedded gastric biopsy samples collected from the lesser curvature of the stomach. Samples from the control group, patients with H. pylori infection, and patients with H. pylori-associated gastritis, with complete and incomplete intestinal metaplasia (IM) were immunostained. Monoclonal antibodies were utilized to determine the expression of CK7, CK20, and Ki-67. Patients infected with H. pylori had strong CK20 expression on the surface, and weak CK7 expression on the surface and deep glands; while non-specific chronic gastritis patients had weak focal CK7 expression and strong CK20 expression. The normal gastric mucosa of patients in the control group had relatively weak CK7 expression, restricted to a few cells in the neck and deep glands. CK20 showed diffuse strong reactivity on the surface. On the other hand, patients with complete IM showed a CK7 staining pattern that was either negative or weakly focal on the surface and crypts associated with diffuse surface CK20 and focal crypt staining corresponding to gastric type IM. The Ki67 proliferating index was low (≤ 15%) in H. pylori infected patients, high (> 30%) in patients with incomplete IM, and intermediate (16-30%) in patients with complete IM. These results indicate a significant link between the expressions of CK7/CK20 and Ki67 in patients afflicted with H. pylori and IM.

Characterisation of HBV and co-infection with HDV and HIV through spatial transcriptomics

Background and aimsThe intrahepatic processes associated with chronic hepatitis B (CHB), especially in the context of hepatitis delta virus (HDV) and HIV co-infection, require a better understanding. Spatial transcriptomics can...

Proteomic Stratification of Prognosis and Treatment Options for Small Cell Lung Cancer.

Small cell lung cancer (SCLC) is a highly malignant and heterogeneous cancer with limited therapeutic options and prognosis prediction models. Here, we analyzed formalin-fixed, paraffin-embedded (FFPE) samples of surgical resections by proteomic profiling, and stratified SCLC into three proteomic subtypes (S-I, S-II, and S-III) with distinct clinical outcomes and chemotherapy responses. The proteomic subtyping was an independent prognostic factor and performed better than current tumor-node-metastasis or Veterans Administration Lung Study Group staging methods. The subtyping results could be further validated using FFPE biopsy samples from an independent cohort, extending the analysis to both surgical and biopsy samples. The signatures of the S-II subtype in particular suggested potential benefits from immunotherapy. Differentially overexpressed proteins in S-III, the worst prognostic subtype, allowed us to nominate potential therapeutic targets, indicating that patient selection may bring new hope for previously failed clinical trials. Finally, analysis of an independent cohort of SCLC patients who had received immunotherapy validated the prediction that the S-II patients had better progression-free survival and overall survival after first-line immunotherapy. Collectively, our study provides the rationale for future clinical investigations to validate the current findings for more accurate prognosis prediction and precise treatments.

An Enhanced Role of Innate Immunity in the Immune Response After Kidney Transplant in People Living With HIV: A Transcriptomic Analysis.

Although kidney transplantation (KT) has become the standard of care for people living with HIV (PLWH) suffering from renal failure, early experiences revealed unanticipated higher rejection rates than those observed in HIV- recipients. The cause of increased acute rejection (AR) in PLWH was assessed by performing a transcriptomic analysis of biopsy specimens, comparing HIV+ to HIV- recipients. An analysis of 68 (34 HIV+, 34 HIV-) formalin-fixed paraffin-embedded (FFPE) renal biopsies matched for degree of inflammation was performed from KT recipients with acute T cell-mediated rejection (aTCMR), borderline for aTCMR (BL), and normal findings. Gene expression was measured using the NanoString platform on a custom gene panel to assess differential gene expression (DE) and pathway analysis (PA). DE analysis revealed multiple genes with significantly increased expression in the HIV+ cohort in aTCMR and BL relative to the HIV- cohort. PA of these genes showed enrichment of various inflammatory pathways, particularly innate immune pathways associated with Toll-like receptors. Upregulation of the innate immune pathways in the biopsies of PLWH with aTCMR and BL is suggestive of a unique immune response that may stem from immune dysregulation related to HIV infection. These findings suggest that these unique HIV-driven pathways may in part be contributory to the increased incidence of allograft rejection after renal transplantation in PLWH.

Diagnostic Utility of Immunofluorescence on Formalin-Fixed Paraffin-Embedded Skin Biopsy.

When immunofluorescence on the frozen section is insufficient or unavailable, salvage immunofluorescence techniques can be used on formalin-fixed, paraffin-embedded tissue. The goal of the current investigation was to evaluate the diagnostic value of paraffin immunofluorescence following proteinase K digestion on skin biopsy samples in comparison to fresh frozen immunofluorescence. It was standardized and compared to the immunofluorescence on fresh frozen tissue (IF-Frozen) for paraffin immunofluorescence by proteinase K digestion of formalin-fixed paraffin-embedded skin biopsies (IF-FFPE). The study included 50 native skin biopsy cases, and fluorescein isothiocyanate-labeled IgA, IgG, IgM, and C3 intensity levels were evaluated in each case. A total of 50 cases of native skin biopsy were included in the study, and their intensities for IgA, IgG, IgM, and C3 antibodies were compared. The average staining intensities in each disease group for the antibodies had equal intensity or had a minor difference (1+)/significant difference (2+). Paraffin immunofluorescence, proteinase K digestion had the best correlation, that is, had either equal or minor difference (1+) with fresh frozen immunofluorescence. The difference of 2+ intensity of antibodies between IF-FFPE and IF-Frozen was noted mainly in C3 antibody on paraneoplastic pemphigus. IF-FFPE showed a sensitivity of 100%, 97.6%, 100%, and 81.6% for IgA, IgG, IgM, and C3, respectively, whereas the specificity was 100% for IgA, IgG, IgM, and C3. Small sample size and and the employment of one method of antigen retrieval in IF-FFPE. Immunofluorescence techniques done on formalin-fixed paraffin-embedded tissue can serve as salvage techniques in cases where immunofluorescence on the frozen section may not be adequate or may not be available.

Deciphering tumor microenvironment dynamics in HER2-amplified refractory metastatic colorectal cancer in the TRIUMPH trial.

3564 Background: Approximately 2-4% of patients with metastatic colorectal cancer (mCRC) harbor HER2 amplification, a subset notorious for poor response rates following conventional treatments. The TRIUMPH trial introduces anti-HER2 therapy, combining trastuzumab and pertuzumab. Despite its potential, the observed response rate stands at 30%, accompanied by an early recurrence within 3.5 months with acquired resistance, which emphasizes the need for further investigations into resistance mechanisms within this patient population. Here, we delineate the dynamic changes in both the tumor and its microenvironment in response to treatment and mechanisms of acquired resistance using spatial transcriptomics. Methods: Within the TRIUMPH trial, a total of 20 formalin-fixed paraffin-embedded (FFPE) biopsy slides from 13 patients were collected before and after treatment or at one time. Utilizing these FFPE slides, we employed imaging-based single-cell spatial transcriptomics, using a custom panel tailored for the tumor and its microenvironment. This panel includes gene sets related to tumor growth, drug resistance, and immune response. Results: To comprehend the complexities of the tumor and its surroundings, we categorized tumor tissue into distinct sections, encompassing tumor, stromal, and immune-enriched areas based on their neighbor cells. Remarkably, we noted a substantial rise in the infiltration of T cells and myeloid cells into tumor-enriched regions post-treatment. T cells increased from a median of 1.5% to 2.2%, and myeloid cells from 4.3% to 7.4%, particularly in patients showing a response. Additionally, ERBB2 expression in tumor cells was high, whereas the non-response group exhibited lower expression levels. Prior to treatment, fibroblast and endothelial cells were intricately intertwined with tumor cells. However, subsequent to treatment, these cells segregated into stromal regions, exhibiting an increase in their proportions. Notably, angiogenic tumor-associated macrophage (angio-TAM), expressing SPP1 and VEGFA, were localized at the interface of tumors and stromal cells and proliferated following disease progression. This spatial reorganization signifies dynamic alterations within the tumor microenvironment following treatment. Conclusions: Our study on HER2-amplified metastatic colorectal cancer in the TRIUMPH trial uncovers intriguing spatial changes post anti-HER2 therapy. Notably, dynamic reorganization of fibroblast and endothelial cells into stromal regions suggest complex treatment responses. Identification of angio-TAM expressing SPP1 and VEGFA adds depth to understanding the contribution of tumor microenvironment to the resistance mechanism. These findings highlight the intricate tumor microenvironment shifts, urging further exploration for optimized therapeutic strategies.

A Fast, Affordable, and Minimally Invasive Diagnostic Test for Cancer of Unknown Primary Using DNA Methylation Profiling

Currently, we cannot provide a conclusive diagnosis for 3% to 5% of people who are confronted with cancer. These patients have cancer of unknown primary (CUP), ie, a metastasized cancer for which the tissue of origin cannot be determined. Studies have shown that the DNA methylation profile is a unique “fingerprint” that can be used to classify tumors. Here we used cell-free reduced representation bisulfite sequencing (cfRRBS), a technique that allows us to identify the methylation profile starting from minimal amounts of highly fragmented DNA, for CUP diagnosis on formalin-fixed paraffin-embedded (FFPE) tissue and liquid biopsies. We collected 80 primary tumor FFPE samples covering 16 tumor entities together with 15 healthy plasma samples to use as a custom cfRRBS reference data set. Entity-specific methylation regions are defined for each entity to build a classifier based on nonnegative least squares deconvolution. This classification framework was tested on 30 FFPE, 19 plasma, and 40 pleural and peritoneal effusion samples of both known metastatic tumors and clinical CUPs for which pathological investigation finally resulted in a cancer diagnosis. Using this framework, 27 of 30 FFPE (all CUPs) and 16 of 19 plasma samples (10/13 CUPs) obtained an accurate diagnosis, with a minimal DNA input of 400 pg. Diagnosis of the 40 pleural and peritoneal effusion samples is possible in 9 of 27 samples with negative/inconclusive cytology (6/13 CUPs), showing that cell-free DNA (cfDNA) methylation profiling could complement routine cytologic analysis. However, a low “cfDNA – high-molecular weight DNA ratio” has a considerable impact on the prediction accuracy. Moreover, the accuracy improves significantly if the predicted tumor percentage is >7%. This proof-of-concept study shows the feasibility of using DNA methylation profiling on FFPE and liquid biopsy samples such as blood, ascites, and pleural effusions in a fast and affordable way. Our novel RRBS-based technique requires minimal DNA input, can be performed in <1 week, and is highly adaptable to specific diagnostic problems as we only use 5 FFPE references per tumor entity. We believe that cfRRBS methylation profiling could be a valuable addition to the pathologist’s toolbox in the diagnosis of CUPs.

#1169 Class-specific gene expression profiling of glomerulus of lupus nephritis by spatial transcriptomics

Abstract Background and Aims Lupus nephritis exhibit various clinical features and subclassifications. Subclassification-specific alteration of transcriptomic profile of lupus nephritis needs additional study which may provide new insights regarding the subclass-specific pathophysiologic mechanism. Method Spatial transcriptomic profiling was conducted using GeoMx Digital Spatial Profiler on formalin-fixed paraffin-embedded kidney biopsy specimens obtained from control (n = 7), LN class I to V (n = 24). We also profiled native kidney primary glomerulonephritis (e.g. anti-PLA2R-associated MN) as additional disease control group. Subclass-specific transcriptomic alteration of glomerulus was compared between the subclasses and primary glomerulonephritis cases. Gene Ontology (GO) terms for DEGs were annotated using the ToppGene suite. Results We first focused on the glomerular transcriptomic alteration in class IV lupus nephritis, which was the most active form with high degree of proteinuria. We found 104 upregulated DEGs and 166 downregulated DEGs which were specifically different in the class IV lupus nephritis cases but not in other subclasses of the disease. The upregulated GO terms included interleukin 10 receptor activity, T cell activation, leukocyte activation, and vesicle membrane. On the other hand, the downregulated DEGs were mostly annotated with GO terms of double-stranded DNA binding domain. In our analysis regarding class V lupus nephritis, total of 128 DEGs were downregulated in class V LN compared to MN. These genes were also downregulated in class V LN when compared to control samples, while no significant expression differences were observed in other LN classes compared to controls. Eleven of 128 downregulated DEGs were annotated with biological process GO terms related to Golgi vesicle transport. Regarding cellular component GO terms, Golgi apparatus (30/128 DEGs), endoplasmic reticulum (ER) membrane (19/128 DEGs), and ER-Golgi intermediate compartment (6/128 DEGs) were among the annotated GO terms associated with the DEGs. DEGs in annotation included GOLGA3, TMED7, BET1L, and RAB10. Conclusion We profiled the class-specific transcriptomic changes in glomerulus of lupus nephritis. Class IV LN was associated upregulation of interleukin 10-mediated inflammatory pathway and downregulation of double stranded-DNA binding domain. The ER-Golgi network and related molecular pathway may be involved in the distinct pathogenesis and clinical features of class V LN. The findings provide novel view of the class-specific pathophysiologic mechanism of lupus nephritis.

Stage-related increase in PIM2 expression in mycosisfungoides.

The oncogene PIM2 is upregulated in several malignancies but has never been investigated in mycosis fungoides (MF), the most common type of cutaneous T-cell lymphoma (CTCL). PIM2 is a well-known oncogene and is regulated by cell signaling pathways like the JAK/STAT- and NF-kB-pathway, key regulators in the pathogenesis of CTCL. The aim of this study was to examine the role of PIM2 in MF. PIM2 gene expression was measured in 81 formalin-fixed paraffin-embedded skin biopsies from patients with MF and 46 control biopsies from healthy skin (HS) and benign inflammatory skin disease (BID). Validation of PIM2 protein expression was performed on selected biopsies with immunohistochemical staining. We found a significant difference in gene expression levels between both early stage MF and HS (p < 0.0001), and BID (p < 0.0001). In addition, the PIM2 gene expression was higher in advanced-stage MF compared to early stage disease (p = 0.0001). No significant difference in gene expression levels was found between patients with and without disease progression. In conclusion, we found PIM2 expression is significantly increased in MF compared to controls, and in advanced-stage MF compared to early stage MF. These findings could potentially have diagnostic value in discriminating early stage MF from BID.

Abstract PO3-01-09: Results of a prospective observational study evaluating the impact of the Prosigna assay on neoadjuvant treatment decision-making in patients with early-stage HR+/HER2- breast cancer

Abstract Background. Prosigna (PAM50) is an FDA-approved prognostic test that measures the expression of 50 classifier genes in breast cancer tissue samples. It provides intrinsic subtyping, which classifies the biology of an individual patient's tumor, as well as a risk of recurrence (ROR) score that predicts the probability of distant recurrence over 10 years. Currently, the Prosigna assay is only approved for use in postoperative specimens. Given studies demonstrating that Prosigna can identify tumors that are more likely to benefit from chemotherapy, this decision-impact study sought to evaluate whether the assay could influence physicians' choices and patients' confidence in the neoadjuvant treatment plan. Methods. This prospective observational study was conducted at a single institution and included patients with HR+/HER2- breast cancer measuring ≥ 0.5 cm, any nodal status, who were deemed candidates for neoadjuvant systemic treatment based on physician’s choice. Formalin-fixed paraffin-embedded core biopsy specimens were centrally analyzed using Prosigna. Patients' tumors were classified according to intrinsic tumor subtype (Luminal A, Luminal B, HER2-enriched, Basal-like) and ROR score (low, intermediate, or high). Physicians and patients were surveyed before and after performing Prosigna, regarding neoadjuvant therapy recommendations. The primary endpoint was assessment of the effect of Prosigna on oncologists' treatment recommendations and the actual treatment received (neoadjuvant hormonal therapy [NAHT], neoadjuvant chemotherapy [NACT], or upfront surgery). Secondary endpoints included determining whether treatment changes were based on ROR score, intrinsic subtype, or both, as well as assessing physicians' and patients' confidence in the treatment plan. Results. A total of 54 patients were enrolled in the study between March 2019 and April 2023. The distribution of intrinsic tumor subtypes was as follows: 15 patients (28%) had a Luminal A subtype, 34 (63%) had a Luminal B subtype, 3 (6%) had a HER2-enriched subtype, and 2 (4%) had a Basal-like subtype. Thirty-three patients (61%) were classified as ROR-low, 11 (20%) as ROR-intermediate, and 10 (19%) as ROR-high. Out of the 43 patients who had both pre- and post-assay survey results available, a change in the treatment decision was observed in 28% of cases. Specifically, 14% of patients transitioned from NAHT to NACT, 7% from NACT to NAHT, and 5% from NACT to upfront surgery, as shown in the Table. Treatment changes were based on the ROR score, intrinsic subtype, and both in 33%, 3%, and 64% of cases, respectively. Overall, 45% of physicians experienced an increase in confidence for the treatment plan after Prosigna testing, while 16% reported a decrease and 39% maintained the same level of confidence. Sixty-two percent of patients experienced a reduction in anxiety about treatment. Associations of ROR score and intrinsic subtypes will be presented at the meeting. Conclusion. Prosigna performed on presurgical core biopsies influenced oncologists' treatment recommendations regarding neoadjuvant treatment and reduced patient anxiety about treatment among patients with early-stage HR+/HER2- breast cancer. Table. Neoadjuvant treatment recommendations before and after Prosigna assay, overall and according to nodal status. Data are presented as frequencies (%). Abbreviations: NACT, neoadjuvant chemotherapy; NAHT, neoadjuvant hormonal therapy. Citation Format: Chiara Corti, Xiangying Chu, Pedro Exman, Danielle Kline, Nolan Priedigkeit, Nabihah Tayob, Erica Mayer, Adrienne Waks, Melissa Hughes, Antonio Giordano, Giuseppe Curigliano, Nancy Lin, Tari King, Rinath Jeselsohn, Deborah Dillon, Elizabeth Mittendorf, Sara Tolaney. Results of a prospective observational study evaluating the impact of the Prosigna assay on neoadjuvant treatment decision-making in patients with early-stage HR+/HER2- breast cancer [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 PO3-01-09.

Molecular analysis of archival diagnostic prostate cancer biopsies identifies genomic similarities in cases with progression post-radiotherapy, and those with de novo metastaticdisease.

It is important to identify molecular features that improve prostate cancer (PCa) risk stratification before radical treatment with curative intent. Molecular analysis of historical diagnostic formalin-fixed paraffin-embedded (FFPE) prostate biopsies from cohorts with post-radiotherapy (RT) long-term clinical follow-up has been limited. Utilizing parallel sequencing modalities, we performed a proof-of-principle sequencing analysis of historical diagnostic FFPE prostate biopsies. We compared patients with (i) stable PCa (sPCa)postprimary or salvage RT,(ii) progressing PCa (pPCa) post-RT,and (iii) de novo metastatic PCa(mPCa). A cohort of 19 patients with diagnostic prostate biopsies (n = 6 sPCa, n = 5 pPCa, n = 8 mPCa) and mean 4 years 10 months follow-up (diagnosed 2009-2016) underwent nucleic acid extraction from demarcated malignancy. Samples underwent 3'RNA sequencing (3'RNAseq) (n = 19), nanoString analysis (n = 12), and Illumina 850k methylation (n = 8) sequencing. Bioinformatic analysis was performed to coherently identify differentially expressed genes and methylated genomic regions (MGRs). Eighteen of 19 samples provided useable 3'RNAseq data. Principal component analysis (PCA) demonstrated similar expression profiles between pPCa and mPCa cases, versus sPCa. Coherently differentially methylated probes between these groups identified ~600 differentially MGRs. The top 50 genes with increased expression in pPCa patients were associated with reduced progression-free survival post-RT (p < 0.0001) in an external cohort. 3'RNAseq, nanoString and 850k-methylation analyses are each achievable from historical FFPE diagnostic pretreatment prostate biopsies, unlocking the potential to utilize large cohorts of historic clinical samples. Profiling similarities between individuals with pPCa and mPCa suggests biological similarities and historical radiological staging limitations, which warrant further investigation.

Abstract 5660: Gene expression signatures from FFPE to predict recurrent metastatic nasopharyngeal cancer

Abstract Nasopharyngeal cancer (NPC) is an EBV-driven epithelial cancer endemic to Southeast Asia, Southern China and the Middle East. Accurate profiling of the gene expression of NPC has been challenging because of its intense inflammatory infiltrate, as well as the 3 - 4mm size of biopsies limiting the amount of available material. Here we present a large cohort of 339 micro-dissected gene expression libraries from formalin-fixed paraffin-embedded (FFPE) biopsies from NPC patients treated at the National University Health System, Singapore, as well as healthy controls. NPC tumors from patients who subsequently developed recurrent or metastatic disease (Group A), and patients who did not develop recurrent or metastatic disease (Group B) were profiled. Tumor epithelial and microenvironment compartments were separately obtained using laser-capture microdissection, followed by library preparation performed for RNA-Seq using an in-house specialised technique. Gene signatures for NPC tumor epithelial and microenvironment content were used to validate the purity of gene expression libraries. Our preliminary analysis revealed that primary, pre-treatment tumors from patients who subsequently developed recurrence (Group A) showed downregulation of processes related to interferon gamma and interferon alpha response (p-adj &amp;lt;0.001 for both), while processes related to epithelial-mesenchymal transition (p-adj &amp;lt; 0.05) were enriched compared to patients who remained healthy (Group B). In contrast to primary tumors, recurrent tumors in Group A were enriched for cellular respiration including oxidative phosphorylation (p-adj &amp;lt; 0.0001). Our approach highlights the utility of whole transcriptome profiling from small quantities of archival FFPE material. The gene signatures identified from the primary tumors of high-risk patients are biologically relevant and have the potential to be used in precision medicine to guide additional targeted intervention. Citation Format: Joshua K. Tay, Wei Keat Teo, Joseph W. Foley, Luvita Suryani, Bing Cheng Wu, Chor Hiang Siow, Kwok Seng Loh. Gene expression signatures from FFPE to predict recurrent metastatic nasopharyngeal cancer [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 5660.

Proteomic analysis of breast cancer based on immune subtypes

BackgroundImmunotherapy is applied to breast cancer to resolve the limitations of survival gain in existing treatment modalities. With immunotherapy, a tumor can be classified into immune-inflamed, excluded and desert based on the distribution of immune cells. We assessed the clinicopathological features, each subtype’s prognostic value and differentially expressed proteins between immune subtypes.MethodsImmune subtyping and proteomic analysis were performed on 56 breast cancer cases with neoadjuvant chemotherapy. The immune subtyping was based on the level of tumor-infiltrating lymphocytes (TILs) and Klintrup criteria. If the level of TILs was ≥ 10%, it was classified as immune-inflamed type without consideration of the Klintrup criteria. In cases of 1–9% TIL, Klintrup criteria 1–3 were classified as the immune-excluded subtype and Klintrup criteria not available (NA) was classified as NA. Cases of 1% TILs and Klintrup 0 were classified as the immune-desert subtype. Mass spectrometry was used to identify differentially expressed proteins in formalin-fixed paraffin-embedded biopsy tissues.ResultsOf the 56 cases, 31 (55%) were immune-inflamed, 21 (38%) were immune-excluded, 2 (4%) were immune-desert and 2 (4%) were NA. Welch’s t-test revealed two differentially expressed proteins between immune-inflamed and immune-excluded/desert subtypes. Coronin-1A was upregulated in immune-inflamed tumors (adjusted p = 0.008) and α-1-antitrypsin was upregulated in immune-excluded/desert tumors (adjusted p = 0.008). Titin was upregulated in pathologic complete response (pCR) than non-pCR among immune-inflamed tumors (adjusted p = 0.036).ConclusionsCoronin-1A and α-1-antitrypsin were upregulated in immune-inflamed and immune-excluded/desert subtypes, respectively. Titin's elevated expression in pCR within the immune-inflamed subtype may indicate a favorable prognosis. Further studies involving large representative cohorts are necessary to validate these findings.

A reliable transcriptomic risk-score applicable to formalin-fixed paraffin-embedded biopsies improves outcome prediction in localized prostate cancer

BackgroundClinical manifestation of prostate cancer (PCa) is highly variable. Aggressive tumors require radical treatment while clinically non-significant ones may be suitable for active surveillance. We previously developed the prognostic ProstaTrend RNA signature based on transcriptome‐wide microarray and RNA-sequencing (RNA-Seq) analyses, primarily of prostatectomy specimens. An RNA-Seq study of formalin-fixed paraffin-embedded (FFPE) tumor biopsies has now allowed us to use this test as a basis for the development of a novel test that is applicable to FFPE biopsies as a tool for early routine PCa diagnostics.MethodsAll patients of the FFPE biopsy cohort were treated by radical prostatectomy and median follow-up for biochemical recurrence (BCR) was 9 years. Based on the transcriptome data of 176 FFPE biopsies, we filtered ProstaTrend for genes susceptible to FFPE-associated degradation via regression analysis. ProstaTrend was additionally restricted to genes with concordant prognostic effects in the RNA-Seq TCGA prostate adenocarcinoma (PRAD) cohort to ensure robust and broad applicability. The prognostic relevance of the refined Transcriptomic Risk Score (TRS) was analyzed by Kaplan–Meier curves and Cox-regression models in our FFPE-biopsy cohort and 9 other public datasets from PCa patients with BCR as primary endpoint. In addition, we developed a prostate single-cell atlas of 41 PCa patients from 5 publicly available studies to analyze gene expression of ProstaTrend genes in different cell compartments.ResultsValidation of the TRS using the original ProstaTrend signature in the cohort of FFPE biopsies revealed a relevant impact of FFPE-associated degradation on gene expression and consequently no significant association with prognosis (Cox-regression, p-value > 0.05) in FFPE tissue. However, the TRS based on the new version of the ProstaTrend-ffpe signature, which included 204 genes (of originally 1396 genes), was significantly associated with BCR in the FFPE biopsy cohort (Cox-regression p-value < 0.001) and retained prognostic relevance when adjusted for Gleason Grade Groups. We confirmed a significant association with BCR in 9 independent cohorts including 1109 patients. Comparison of the prognostic performance of the TRS with 17 other prognostically relevant PCa panels revealed that ProstaTrend-ffpe was among the best-ranked panels. We generated a PCa cell atlas to associate ProstaTrend genes with cell lineages or cell types. Tumor-specific luminal cells have a significantly higher TRS than normal luminal cells in all analyzed datasets. In addition, TRS of epithelial and luminal cells was correlated with increased Gleason score in 3 studies.ConclusionsWe developed a prognostic gene-expression signature for PCa that can be applied to FFPE biopsies and may be suitable to support clinical decision-making.

Intrahepatic Transcriptomics Differentiate Advanced Fibrosis and Clinical Outcomes in Adults With Fontan Circulation

BackgroundThe molecular mechanisms underlying Fontan-associated liver disease (FALD) remain largely unknown. ObjectivesThis study aimed to assess intrahepatic transcriptomic differences among patients with FALD according to the degree of liver fibrosis and clinical outcomes. MethodsThis retrospective cohort study included adults with the Fontan circulation. Baseline clinical, laboratory, imaging, and hemodynamic data as well as a composite clinical outcome (CCO) were extracted from medical records. Patients were classified into early or advanced fibrosis. RNA was isolated from formalin-fixed paraffin-embedded liver biopsy samples; RNA libraries were constructed with the use of an rRNA depletion method and sequenced on an Illumina Novaseq 6000. Differential gene expression and gene ontology analyses were performed with the use of DESeq2 and Metascape. ResultsA total of 106 patients (48% male, median age 31 years [IQR: 11.3 years]) were included. Those with advanced fibrosis had higher B-type natriuretic peptide levels and Fontan, mean pulmonary artery, and capillary wedge pressures. The CCO was present in 23 patients (22%) and was not predicted by advanced liver fibrosis, right ventricular morphology, presence of aortopulmonary collaterals, or Fontan pressures on multivariable analysis. Samples with advanced fibrosis had 228 upregulated genes compared with early fibrosis. Samples with the CCO had 894 upregulated genes compared with those without the CCO. A total of 136 upregulated genes were identified in both comparisons and were enriched in cellular response to cytokine stimulus or oxidative stress, VEGFA-VEGFR2 signaling pathway, TGF-β signaling pathway, and vasculature development. ConclusionsPatients with FALD and advanced fibrosis or the CCO exhibited upregulated genes related to inflammation, congestion, and angiogenesis.

Identification of inflammatory biomarkers in IgA nephropathy using the NanoString technology: a validation study in Caucasians

Objective and designImmunoglobulin A nephropathy (IgAN) is a kidney disease characterized by the accumulation of IgA deposits in the glomeruli of the kidney, leading to inflammation and damage to the kidney. The inflammatory markers involved in IgAN remain to be defined. Gene expression analysis platforms, such as the NanoString nCounter system, are promising screening and diagnostic tools, especially in oncology. Still, their role as a diagnostic and prognostic tool in IgAN remains scarce. In this study, we aimed to validate the use of NanoString technology to identify potential inflammatory biomarkers involved in the progression of IgAN.SubjectsA total of 30 patients with biopsy-proven IgAN and 7 cases of antineutrophil cytoplasmic antibody (ANCA)-associated pauci-immune glomerulonephritis were included for gene expression measurement. For the immunofluorescence validation experiments, a total of 6 IgAN patients and 3 controls were included.MethodsTotal RNA was extracted from formalin-fixed paraffin-embedded kidney biopsy specimens, and a customized 48-plex human gene CodeSet was used to study 29 genes implicated in different biological pathways. Comparisons in gene expression were made between IgAN and ANCA-associated pauci-immune glomerulonephritis patients to delineate an expression profile specific to IgAN. Gene expression was compared between patients with low and moderate risk of progression. Genes for which RNA expression was associated with disease progression were analyzed for protein expression by immunofluorescence and compared with controls.ResultsIgAN patients had a distinct gene expression profile with decreased expression in genes IL-6, INFG, and C1QB compared to ANCA patients. C3 and TNFRSF1B were identified as potential biomarkers for IgAN progression in patients early in their disease course. Protein expression for those 2 candidate genes was upregulated in IgAN patients compared to controls. Expression of genes implicated in fibrosis (PTEN, CASPASE 3, TGM2, TGFB1, IL2, and TNFRSF1B) was more pronounced in IgAN patients with severe fibrosis compared to those with none.ConclusionsOur findings validate our NanoString mRNA profiling by examining protein expression levels of two candidate genes, C3 and TNFRSF1B, in IgAN patients and controls. We also identified several upregulated mRNA transcripts implicated in the development of fibrosis that may be considered fibrotic markers within IgAN patients.

VEDOLIZUMAB RESPONSE AND NON-RESPONSE IN ULCERATIVE COLITIS: INSIGHTS FROM SPATIAL TRANSCRIPTOMICS

Abstract BACKGROUND Vedolizumab (VDZ) is an anti-a4b7 integrin monoclonal antibody effective for treating ulcerative colitis (UC). The cellular and genetic factors that mediate VDZ response and non-response remain unclear. Our previous data employing different multi-omics revealed an impact of VDZ on myeloid dendritic cells (mDCs) and mononuclear phagocytes (MNPs), in addition to lymphocytes, influencing stromal and epithelial compartments. METHODS We performed a mass cytometry (CyTOF) experiment on peripheral blood and colon biopsies in healthy controls (HC, n=6), patients with UC on anti-TNF or 5-ASA (UC, n=5/6) and patients with UC on VDZ (UC-VDZ, n=5). Subsequently, we carried out a retrospective longitudinal spatial analysis using 1000-plex RNA and 62-plex protein CosMx on archived formalin-fixed paraffin-embedded (FFPE) biopsies before and after treatment in VDZ responders (VDZ-R) and non-responders (VDZ-NR) (Fig.1). Lastly, gene set enrichment analysis (GSEA) using VDZ response and non-response signatures obtained from our longitudinal spatial transcriptomic data were validated in an external publicly available dataset. RESULTS CyTOF results confirmed that VDZ is associated with a shift of a4b7+ cells from tissue to blood across multiple subsets. Spatial data showed elevated activated MNPs in UC compared to HC and UC-VDZ. Following treatment, these levels decreased in VDZ-R but increased in VDZ-NR. Neighborhood enrichment analysis revealed a trend toward increased proximity of activated fibroblasts and activated MNPs in UC, which was reduced following treatment. Furthermore, to explore differences pre-treatment, we performed pseudobulk differentially expressed (DE) gene analysis comparing VDZ-NR versus VDZ-R. Stromal and MNP-expressed genes including MMP1, MMP2, and THBS1 were among the top DE genes in VDZ-NR, while genes associated with the IEC crypt base including REG1A, OLFM4, AGR2, SPINK1, and LYZ were related to VDZ-R. GSEA confirmed significant enrichment of the identified VDZ-NR and VDZ-R gene signatures in patients pre-VDZ, aligning with their outcome. The VDZ-R signature was specific to VDZ and was not associated with anti-TNF response. The VDZ-NR signature was also associated with anti-TNF non-response. CONCLUSION Longitudinal spatial transcriptomics of archived FFPE specimens highlighted potential pathways associated with VDZ responsiveness, identifying epithelial-, MNP-, and fibroblast-enriched genes. Validation in an external dataset emphasized the specificity of the VDZ-associated response signature. Fig1. a, Schematic of retrospective, longitudinal analysis of archived FFPE specimens using 1000-plex CosMx spatial transcriptomics of 126,368 cells. b, Dot plot representation of a subset of genes from pseudobulk DE gene analysis for the indicated subsets.