Formalin-fixed Paraffin-embedded Tissue Research Articles

Granulomatous lymphadenitis in Taiwan: Unraveling infantile peak and Bacillus Calmette-Guérin lymphadenitis

BackgroundGranulomatous lymphadenitis, a histopathological diagnosis, often indicates infections, such as those caused by mycobacterial and fungal agents. MethodsWe conducted an analysis of 1098 granulomatous lymphadenitis cases, examining age distribution, lymph node locations, and laterality. Molecular detection of Bacillus Calmette-Guérin (BCG) was performed on archived formalin-fixed paraffin-embedded tissue specimens. ResultsOur analysis revealed a bimodal age distribution, notably with a minor peak in infants. These infantile cases predominantly featured axillary involvement, frequently occurring on the left side. Positive rates of BCG identification decreased with age: <1 year, 71%; 1–2 year, 33%; 2–3 year, 13%; 3–4 year, 0%. Remarkably, only one of the 14 cases with molecularly confirmed BCG lymphadenitis had comments regarding BCG in the pathological report. Compared with patients born after 2016 (BCG at 5–8 months), those born before 2016 (BCG at birth) developed BCG lymphadenitis at a wider age range with right skewness (before 2016, 13 ± 11 months [range, 3–33 months] vs. after 2016, 10 ± 2 months [range, 8–13 months]). Four of the 14 BCG-positive cases had congenital heart disease. Seven patients received anti-tuberculosis drugs following surgical excision. No surgical complications were reported. ConclusionsBCG lymphadenitis constitutes a distinctive minor peak within the spectrum of granulomatous lymphadenitis in Taiwan. Pathologists should consider the possibility of BCG infection, especially in cases of infantile axillary, supraclavicular, neck lymphadenopathies on the left side. Moreover, BCG administration at 5–8 months may reduce delayed-onset BCG lymphadenitis.

Extent of Resection Thresholds in Molecular Subgroups of Newly Diagnosed Isocitrate Dehydrogenase-Wildtype Glioblastoma.

Maximizing the extent of resection (EOR) improves outcomes in glioblastoma (GBM). However, previous GBM studies have not addressed the EOR impact in molecular subgroups beyond IDH1/IDH2 status. In the current article, we evaluate whether EOR confers a benefit in all GBM subtypes or only in particular molecular subgroups. A retrospective cohort of newly diagnosed GBM isocitrate dehydrogenase (IDH)-wildtype undergoing resection were prospectively included in a database (n = 138). EOR and residual tumor volume (RTV) were quantified with semiautomated software. Formalin-fixed paraffin-embedded tumor tissues were analyzed by targeted next-generation sequencing. The association between recurrent genomic alterations and EOR/RTV was evaluated using a recursive partitioning analysis to identify thresholds of EOR or RTV that may predict survival. The Kaplan-Meier methods and multivariable Cox proportional hazards regression methods were applied for survival analysis. Patients with EOR ≥88% experienced 44% prolonged overall survival (OS) in multivariable analysis (hazard ratio: 0.56, P = .030). Patients with alterations in the TP53 pathway and EOR <89% showed reduced OS compared to TP53 pathway altered patients with EOR>89% (10.5 vs 18.8 months; HR: 2.78, P = .013); however, EOR/RTV was not associated with OS in patients without alterations in the TP53 pathway. Meanwhile, in all patients with EOR <88%, PTEN -altered had significantly worse OS than PTEN -wildtype (9.5 vs 15.4 months; HR: 4.53, P < .001). Our results suggest that a subset of molecularly defined GBM IDH-wildtype may benefit more from aggressive resections. Re-resections to optimize EOR might be beneficial in a subset of molecularly defined GBMs. Molecular alterations should be taken into consideration for surgical treatment decisions in GBM IDH-wildtype.

Prognostic implications of STK11 with different mutation status and its relationship with tumor-infiltrating immune cells in non-small cell lung cancer.

Mutations in STK11 (STK11Mut) gene may present a negative impact on survival in Non-small Cell Lung Cancer (NSCLC) patients, however, its relationship with immune related genes remains unclear. This study is to unveil whether overexpressed- and mutated-STK11 impact survival in NSCLC and to explore whether immune related genes (IRGs) are involved in STK11 mutations. 188 NSCLC patients with intact formalin-fixed paraffin-embedded (FFPE) tissue available for detecting STK11 protein expression were included in the analysis. After immunohistochemical detection of STK11 protein, patients were divided into high STK11 expression group (STK11High) and low STK11 expression group (STK11Low), and then Kaplan-Meier survival analysis and COX proportional hazards model were used to compare the overall survival (OS) and progression-free survival (PFS) of the two groups of patients. In addition, the mutation data from the TCGA database was used to categorize the NSCLC population, namely STK11 Mutated (STK11Mut) and wild-type (STK11Wt) subgroups. The difference in OS between STK11Mut and STK11Wt was compared. Finally, bioinformatics analysis was used to compare the differences in IRGs expression between STK11Mut and STK11Wt populations. The median follow-up time was 51.0 months (range 3.0 - 120.0 months) for real-life cohort. At the end of follow-up, 64.36% (121/188) of patients experienced recurrence or metastasis. 64.89% (122/188) of patients ended up in cancer-related death. High expression of STK11 was a significant protective factor for NSCLC patients, both in terms of PFS [HR=0.42, 95% CI= (0.29-0.61), P<0.001] and OS [HR=0.36, 95% CI= (0.25, 0.53), P<0.001], which was consistent with the finding in TCGA cohorts [HR=0.76, 95%CI= (0.65, 0.88), P<0.001 HR=0.76, 95%CI= (0.65, 0.88), P<0.001]. In TCGA cohort, STK11 mutation was a significant risk factor for NSCLC in both lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD) histology in terms of OS [HR=6.81, 95%CI= (2.16, 21.53), P<0.001; HR=1.50, 95%CI= (1.00, 2.26), P=0.051, respectively]. Furthermore, 7 IRGs, namely CALCA, BMP6, S100P, THPO, CGA, PCSK1 and MUC5AC, were found significantly overexpressed in STK11-mutated NSCLC in both LUSC and LUAD histology. Low STK11 expression at protein level and presence of STK11 mutation were associated with poor prognosis in NSCLC, and mutated STK11 might probably alter the expression IRGs profiling.

Ultrasensitive and Multiplexed Protein Imaging with Clickable and Cleavable Fluorophores.

Single-cell spatial proteomic analysis holds great promise to advance our understanding of the composition, organization, interaction, and function of the various cell types in complex biological systems. However, the current multiplexed protein imaging technologies suffer from low detection sensitivity, limited multiplexing capacity, or are technically demanding. To tackle these issues, here, we report the development of a highly sensitive and multiplexed in situ protein profiling method using off-the-shelf antibodies. In this approach, the protein targets are stained with horseradish peroxidase (HRP) conjugated antibodies and cleavable fluorophores via click chemistry. Through repeated cycles of target staining, fluorescence imaging, and fluorophore cleavage, many proteins can be profiled in single cells in situ. Applying this approach, we successfully quantified 28 different proteins in human formalin-fixed paraffin-embedded (FFPE) tonsil tissue, which represents the highest multiplexing capacity among the tyramide signal amplification (TSA) methods. Based on their unique protein expression patterns and their microenvironment, ∼820,000 cells in the tissue are classified into distinct cell clusters. We also explored the cell-cell interactions between these varied cell clusters and observed that different subregions of the tissue are composed of cells from specific clusters.

Investigating the role of Epstein-Barr virus and human papillomavirus types 16 and 18 co-infections in cervical disease of Iranian women.

High-risk human papillomaviruses (HR-HPVs) are known to contribute to cervical cancer (CC), but the role of Epstein-Barr virus (EBV) in this process remains unclear, despite EBV's widespread detection in premalignant and malignant cervical tissues. In this cross-sectional study of 258 cervical samples, including both formalin-fixed paraffin-embedded (FFPE) and fresh cervical tissues, the presence and viral load of HR-HPVs (HPV-16 and HPV-18) and EBV were evaluated in Iranian women with cervical intraepithelial neoplasia (CIN), squamous cell carcinoma (SCC), and a cervicitis control group using real-time PCR. The study revealed a significant correlation between disease severity and both increased HPV-16 positivity and HPV-16 and HPV-18 co-infection (p<0.001). Interestingly, the control group had a higher frequency of EBV-positive cases than SCC/CIN groups (p<0.001). HPV-16 DNA load increased with disease severity (P<0.001), while HPV-18 showed no significant difference (P=0.058). The control group had a higher EBV DNA load compared to SCC/CIN groups (P=0.033). HPV-16 increased the risk of CIN II, CIN III, and SCC, while HPV-18 increased the risk of CIN II and CIN III. Notably, EBV was associated with a lower risk of CIN groups and SCC. No significant difference in EBV co-infection with HPV-16/18 was found, failing to support the hypothesis that EBV is a cofactor in CC. However, high EBV viral load in the control group suggests a potential "hit and run hypothesis" role in CC progression. This hypothesis suggests that EBV may contribute briefly to the initiation of CC with an initial impact but then becomes less actively involved in its ongoing progression.

Resolving Discrepancies in Idylla BRAF Mutational Assay Results Using Targeted Next-Generation Sequencing.

BRAF mutation identification is important for the diagnosis and treatment of several tumor types, both solid and hematologic. Rapid identification of BRAF mutations is required to determine eligibility for targeted BRAF inhibitor therapy. The Idylla BRAF mutation assay is a rapid, multiplex allele-specific PCR test designed to detect the most common oncogenic BRAF V600 mutations in formalin-fixed paraffin-embedded (FFPE) tissue samples. Here, we describe the validation of the Idylla BRAF mutation assay in our laboratory. During routine clinical practice, we noticed cases in which BRAF V600 mutations were identified with unusual amplification curves, with three cases displaying a delayed amplification within a double amplification pattern and two false-positive calls. We therefore initiated a quality improvement effort to systematically and retrospectively evaluate next-generation sequencing (NGS)-tested cases with BRAF mutations identified within five amino acids of BRAF codon V600 and did not identify additional false-positive cases. We hypothesize that late amplification in a double amplification pattern may represent non-specific amplification, whereas cases displaying single delayed amplification curves may stem from the presence of either non-V600 variants, very low-level V600 variants, cytosine deamination artifacts, and/or non-specific amplification by an allele-specific PCR primer. Regardless, we recommend that Idylla BRAF cases with non-classical amplification curves undergo reflex NGS testing. These findings are likely relevant for other Idylla assays interrogating hotspot mutations in genes such as EGFR, IDH1/2, KRAS, and NRAS.

Reliable Method for Estimating Nerve Fiber Density in Epidermis Using Routine Histopathologic Tissue Preparation: A Promising Diagnostic Tool for Small Fiber Neuropathy.

Practical yet reliable diagnostic tools for small-fiber neuropathy are needed. We aimed to establish a histopathologic protocol for estimating intraepidermal nerve fiber density (eIENFD) on formalin-fixed, paraffin-embedded tissue (FFPE), evaluate its reliability through intraobserver and interobserver analyses, and provide normative reference values for clinical use. Sixty-eight healthy participants underwent nerve conduction studies and quantitative sensory testing. Skin biopsies from the distal and proximal leg were taken and processed using routine immunohistochemistry (anti-PGP9.5 antibodies) on thin 5 µm sections. eIENFD was assessed with a modified counting protocol. Interobserver and intraobserver reliabilities were excellent (ICC=0.9). eIENFD was higher in females than males (fibers/mm, 14.3±4.4 vs. 11.6±5.8, P <0.05), decreased with age ( r s =-0.47, P <0.001), and was higher proximally than distally (15.0±5.5 vs. 13.0±5.3, P =0.002). Quantile regression equations for the fifth percentile of distal and proximal eIENFD were presented: 13.125-0.161×age (y)-0.932×sex (male=1; female=0) and 17.204-0.192×age (y)-3.313×sex (male=1; female=0), respectively. This study introduces a reliable and reproducible method for estimating epidermal nerve fiber density through immunostaining on 5-µm thin FFPE tissue samples. Normative data on eIENFD is provided. Regression equations help identify abnormal decreases in small nerve fiber density.

Effect of formalin-fixation and paraffin-embedded tissue storage times on RNAscope in situ hybridization signal amplification.

RNAscope in situ hybridization (ISH) detects target RNA in formalin-fixed, paraffin-embedded (FFPE) tissues. Protocols suggest that prolonged FFPE storage and formalin fixation may impact signal detection, potentially limiting the utility of RNAscope ISH in retrospective studies. To develop parameters for RNAscope use with archived specimens, we evaluated the effect of formalin-fixation time by measuring the signal of a reference gene (16srRNA) in selected tissues fixed in 10% neutral-buffered formalin for 1, 2, 3, 5, 7, 10, 14, 21, 28, 60, 90, 180, and 270 d. The signal intensity and percent area of signal decreased after 180 d. Tissues had detectable signal at 180 d but not at 270 d of formalin fixation. To assess target detection in paraffin blocks, we qualitatively compared the signal of canine distemper virus (CDV) antigen via immunohistochemistry and CDV RNA via RNAscope ISH in replicate sections from blocks stored at room temperature for 6 mo, 1, 3, 6, 8, 11, 13, and 15 y; RNA was detected in FFPE tissues stored for up to 15 y. Our results demonstrate that RNAscope ISH can detect targets in tissues with prolonged paraffin storage intervals and formalin-fixation times.

Molecular characterization of the evolution of premalignant lesions in the upper aerodigestive tract.

Early relapse and development of metastatic disease are some of the primary reasons for the poor prognosis of patients with head and neck squamous cell carcinoma (HNSCC). HNSCC is a heterogeneous disease which may develop in large premalignant fields of genetically altered cells. Yet knowing which individuals will progress and develop clinically significant cancers during their lifetimes remains one of the most important challenges of reducing HNSCC morbidity and mortality. To further elucidate the molecular mechanisms, we performed a focused analysis of the genome and immune microenvironment from multiple, matched normal squamous tissue, premalignant lesions, as well as primary and recurrent tumors from seven patients with p16-negative HNSCC. We performed targeted panel Next Generation Sequencing (161 genes) to analyze somatic variants from sequentially collected, matched formalin-fixed paraffin-embedded tissue (normal, premalignant, HNSCC) from two patients. These samples plus samples from five additional patients were analyzed with the Nanostring PanCancer Immune Panel. In addition, we performed shallow whole genome sequencing (0.5x coverage on average) on samples from three of these patients. Patients were, apart from one case, primarily treated with curative-intent surgery, and received subsequent adjuvant treatment, if indicated. The most frequently mutated genes were TP53 and NOTCH1. Other mutated genes included NOTCH3 and CDKN2A, among others. A significant number of mutations were private to dysplasia and invasive carcinoma, respectively, however, almost 20% were shared between them. Increasing genomic instability was observed when comparing histologically normal squamous mucosa with higher levels of dysplasia. High-grade dysplasia showed similarly rearranged genomes as invasive carcinoma. Pathways related to interferon alpha and gamma response were upregulated even in moderate dysplastic lesions with increasing expression in higher grades of dysplasia and carcinoma. SPINK5, a known tumor suppressor gene in HNSCC, was already downregulated in low-grade dysplastic lesions, indicating an early deactivation in the evolution of the disease. Genomic alterations as well as aberrant immune gene expression can be observed early in the evolution of tumors of the upper aerodigestive tract, highlighting the potential for targeting early mechanisms of disease progression.

Walking a thin line between fixation and epitope binding - characterization of antigen retrieval methods suitable for eosinophil and HSV-2 staining in formalin-fixed female reproductive tissue.

Antibody-based fluorescence analysis of female reproductive tissues in research of sexually transmitted diseases allows for an in-depth understanding of protein localization, interactions, and pathogenesis. However, in many cases, cryosectioning is not compatible with biosafety regulations; at all times, exposure of lab personnel and the public to potentially harmful pathogens from biological infectious material must be avoided; thus, formaldehyde fixation is essential. Due to formaldehyde's cross-linking properties, protein detection with antibodies can be impeded. To allow effective epitope binding during immunofluorescence of formalin-fixed paraffin-embedded vaginal tissue, we investigated two antigen retrieval methods. We tested these methods regarding their suitability for automated image analysis, facilitating reproducible quantitative microscopic data acquisition in sexually transmitted disease research. Heat-based retrieval at 80°C in citrate buffer proved to increase antibody binding to eosinophil protein and HSV-2 visibly and tissue morphology best, and was the most efficient for sample processing and quantitative analysis.

Identification of New Pathogenic Variants of Hereditary Diffuse Gastric Cancer.

Hereditary diffuse gastric cancer (HDGC) presents a significant genetic predisposition, notably linked to mutations in the CDH1 and CTNNA1. However, the genetic basis for over half of HDGC cases remains unidentified. The aim of this study is to identify novel pathogenic variants in HDGC and evaluate their protein expression. Among 20 qualifying families, two were selected based on available pedigree and DNA. Whole genome sequencing (WGS) on DNA extracted from blood and whole exome sequencing on DNA from formalin-fixed paraffin-embedded tissues were performed to find potential pathogenic variants in HDGC. After selection of a candidate variant, functional validation, and enrichment analysis were performed. As a result of WGS, three candidate germline mutations (EPHA5, MCOA2, and RHOA) were identified in one family. After literature review and in-silico analyses, the RHOA mutation (R129W) was selected as a candidate. This mutation was found in two gastric cancer patients within the family. In functional validation, it showed RhoA overexpression and a higher GTP-bound state in the RhoaR129W mutant. Decreased phosphorylation at Ser127/397 suggested altered YAP1 regulation in the Rho-ROCK pathway. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses linked RhoaR129W overexpression to changed migration/adhesion in MKN1 cell line. However, this RHOA mutation (R129W) was not found in index patients in other families. The RHOA mutation (R129W) emerges as a potential causative gene for HDGC, but only in one family, indicating a need for further studies to understand its role in HDGC pathogenesis fully.

Identification of neutrophils and eosinophils in upper airway mucosa with immunofluorescence multiplex image cytometry.

Characterization of inflammation in chronic rhinosinusitis with (CRSwNP) and without nasal polyps (CRSsNP) is an ongoing research process. To overcome limitations of current cytologic techniques, we investigated whether immunofluorescence multiplex image cytometry could quantify intact neutrophils, eosinophils, and other immune cells in solid upper airway mucosa. We used a four-channel immunofluorescence-microscopy technique for the simultaneous detection of the leukocyte marker CD45, the neutrophil marker myeloperoxidase, two eosinophil markers, i.e., major basic protein and eosinophil peroxidase, and DAPI (4',6-diamidin-2-phenylindole), in formalin-fixed paraffin-embedded upper airway tissue samples of patients with CRSwNP and CRSsNP, as well as of patients free of CRS with inferior turbinate hypertrophy (controls). Image acquisition and analysis were performed with TissueFAXS and StrataQuest (TissueGnostics, Vienna, Austria), respectively. Positive and negative immunostaining were differentiated with a specific fluorescence signal/background signal ratio. Isotype controls were used as negative controls. In six controls, nine patients with CRSsNP, and 11 patients with CRSwNP, the median area scanned and median cell count per patient were 14.2mm2 and 34,356, respectively. In CRSwNP, the number of eosinophils was three times higher (23%) than that of neutrophils (7%). Three times more immune cells were encountered in CRSwNP (33%) compared to CRSsNP (11%). In controls, inflammation was balanced between the epithelial layer and lamina propria, in contrast to CRS (three times more pronounced inflammation in the lamina propria). The quantification of intact neutrophils, eosinophils, and other immune cells in solid tissue with undisrupted architecture seems feasible with immunofluorescence multiplex image cytometry.

Mutational and transcriptional landscape of pediatric B-cell precursor lymphoblastic lymphoma

AbstractPediatric B-cell precursor (BCP) lymphoblastic malignancies are neoplasms with manifestation either in the bone marrow or blood (BCP acute lymphoblastic leukemia [BCP-ALL]) or are less common in extramedullary tissue (BCP lymphoblastic lymphoma [BCP-LBL]). Although both presentations are similar in morphology and immunophenotype, molecular studies have been virtually restricted to BCP-ALL so far. The lack of molecular studies on BCP-LBL is due to its rarity and restriction on small, mostly formalin-fixed paraffin-embedded (FFPE) tissues. Here, to our knowledge, we present the first comprehensive mutational and transcriptional analysis of what we consider the largest BCP-LBL cohort described to date (n = 97). Whole-exome sequencing indicated a mutational spectrum of BCP-LBL, strikingly similar to that found in BCP-ALL. However, epigenetic modifiers were more frequently mutated in BCP-LBL, whereas BCP-ALL was more frequently affected by mutation in genes involved in B-cell development. Integrating copy number alterations, somatic mutations, and gene expression by RNA sequencing revealed that virtually all molecular subtypes originally defined in BCP-ALL are present in BCP-LBL, with only 7% of lymphomas that were not assigned to a subtype. Similar to BCP-ALL, the most frequent subtypes of BCP-LBL were high hyperdiploidy and ETV6::RUNX1. Tyrosine kinase/cytokine receptor rearrangements were detected in 7% of BCP-LBL. These results indicate that genetic subtypes can be identified in BCP-LBL using next-generation sequencing, even in FFPE tissue, and may be relevant to guide treatment.

CORRELATION OF TUMOR MARKERS HER2 NEU ER PR AND KI 67 IN BREAST CANCER PATIENTS

: Every year, more than a million women are diagnosed with breast cancer, and over 700.000 of them have positive for hormone receptors (FIR). The expression of key markers, such as ER and PP, and clinical circumstances show specific biological traits. Risk factors may have a role in the development of some hormone receptor-positive breast cancers. Objective: The objective of this study was to investigate the correlations between key tumor markers, including HER2, ER, PR, ALP, and KI67, in hormone receptor-positive breast cancers. Methods: Tumour tissue samples were collected for histopathology (10% neutral buffered Formalin-fixed paraffin-embedded tissue specimens) during Trucut biopsy or mastectomy at the oncology department of a tertiary care hospital in Karachi from October 2022 to July 2023. Tissues were analyzed under a light microscope for tumor hormone receptors at a tertiary care hospital in Karachi. Results: There were several significant correlations observed between HER 2 NUE, KI 67, ER, and PR. Specifically, a weak negative correlation was noted between HER 2 NUE and KI 67, while weak positive correlations were observed between HER 2 NUE and both ER and PR. Additionally, a weak positive correlation was found between ER and HER 2 NUE, PR, and KI 67, and a weak positive correlation existed between PR and ER, HER 2 NUE, and KI 67. Finally, KI 67 displayed a weak positive correlation with HER 2 NUE, PR, and ER. Conclusions: Her two nurses, ER, ALP, KI 67, and breastfeeding, are significantly and directly related. PR is not significantly related to other mentioned tumor markers.

Abstract LB432: Identification of novel drug targets using the ADAPT biotargeting system on FFPE tissue from patients with glioblastoma multiforme

Abstract Glioblastoma Multiforme (GBM) is one of the most aggressive and lethal brain tumor types with a five-year survival rate of 6.9%. In 2023, more than 14,490 Americans were expected to receive a diagnosis. Although great progress has been achieved in protein target identification and treatment over the last few years, current treatment options are limited, and tumor recurrence occurs at a high rate due to treatment resistance. Aptamers, short single-stranded oligonucleotide ligands with high-affinity binding to molecular targets, are well suited tools for exploring novel targets in highly heterogeneous tumors with complex etiologies like GBM, where clinically relevant biomarkers are limited. In this study, aptamer-based cancer-specific drug target identification was performed by Adaptive Dynamic Artificial Polyligand Targeting (ADAPT) Biotargeting System. In short, a GBM-specific library was enriched through a Systematic Evolution of Ligands by EXponential enrichment (SELEX) process using micro dissected Formalin-Fixed Paraffin-Embedded (FFPE) tissues from GBM patients, along with whole body normal tissue for counterselection. Microdissection of tissue allows the partitioning of different types of cells to focus on specific cells of interest. The enriched libraries have been evaluated on clinical GBM and normal brain control samples through aptamer mediated tissue staining, resulting in preferential binding of enriched libraries compared to controls in the vast majority of GBM patients with little binding to normal brain samples. The final library was subsequently applied in an in-house developed library-based affinity pull-down assay using lysates from FFPE tissue of the same group of GBM patients and normal brain samples, followed by unbiased Mass Spectrometry for protein target identification. Combining the results from this proteomics analysis and data from the internal Whole Transcriptome Sequencing (WTS) database, we were able to identify several targets that are highly expressed in GBM and other cancer lineages. Antibody staining provided further validation of the expression levels and the cellular localization. The targets identified in this study include not only potential novel therapeutic targets, but also well-studied biomarkers and targets with available drugs already in clinical trials, demonstrating the platform has great potential for identifying high affinity binding and selectivity compared to traditional proteomics and other methods in drug target discovery. Citation Format: Xixi Wei, Myunggi An, Radhika Santhanam, Barbara Richardson, Lucas Vu, Justin Saul, Anna Walton, Michael Gee, Daniel Martin, Amanda Hubbard, Alexis Rodin, Matthew Rosenow, Tassilo Hornung. Identification of novel drug targets using the ADAPT biotargeting system on FFPE tissue from patients with glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB432.

Abstract LB373: Pilot whole-exome sequencing data of formalin-fixed paraffin embedded endometrial tumors in the Epidemiology of Endometrial Cancer Consortium (E2C2)

Abstract Background: Endometrial cancer (EC) is the most common gynecologic cancer in the U.S.: incidence and mortality are rising. Black women face 2-fold higher mortality compared with other racial groups. To understand the role of tumor biology underlying these health inequities, we are sequencing the tumor exomes of 1,000 Black and 2,000 non-Black women in the Epidemiology of Endometrial Cancer Consortium. To inform this effort, we have completed whole-exome tumor sequencing in 35 EC patients. We present early sample performance and mutational insights of this pilot. Methods: We extracted DNA from formalin-fixed paraffin-embedded (FFPE) tumor tissues (cores, scroll) and matched normal specimens (blood, tissue) from 35 women diagnosed with EC in the Louisiana Tumor Registry, Nurses’ Health Study, and Polish Endometrial Cancer Study. Whole-exome sequencing was conducted by the Center for Inherited Disease Research at Johns Hopkins University, which called somatic alterations using matched normal samples as the germline profile (NovaSeq6000). We excluded two contaminated core samples and one paired scroll with extremely low coverage, leaving 33 women in our analytic sample. We examined the mutational landscape of EC overall and separately in 7 Black and 26 non-Black patients. For 23 women, we obtained both an FFPE scroll of the full block and FFPE tumor cores enabling a direct comparative analysis of DNA extraction yield and sequencing performance by sample source. We ran a published allele-specific copy number and clonal heterogeneity analysis tool (FACETS) in these 23 women to calculate tumor purity by tissue type. Results: Results from our pilot study revealed the most frequently mutated genes in our pilot population were PTEN (61%), ARID1A (48%), and PIK3CA (39%). We observed similar mutation frequencies for PIK3CA in both non-Black and Black women (38% vs 43%). TP53 was more frequently mutated in Black women (57% vs 16%), while ARID1A mutation was more common in non-Black women (58% vs 14%). 10 (30%) patient samples had chromosome 10 copy-neutral loss of heterozygosity. Of these, 8 (80%) had PTEN bi-allelic alterations. Of participants with both core and scroll tissue available, both sample types were successfully sequenced, although cores showed modestly better performance: 74% of core samples (vs 26% for scrolls) had a higher percentage of bases read at a target depth of 100x, and the median tumor purity for cores estimated using FACETS was higher than scrolls (53% vs 42%). Tumor purity could not be calculated for seven tissue samples due to either tumor diploidy or purity being too low: of these samples the majority were scrolls (86%). We observed greater concordance between core and scroll variant allele frequency when tumor purity was ≥30%: the benefits of cores vs scrolls matter at lower tumor purity. ConclusionsIn line with TCGA data, these pilot findings suggest potential differences in tumor mutational landscape by race and we will be interrogating these in our larger study population. FFPE core tissue generally performed better than FFPE scroll tissue in whole-exome sequencing, but our data suggest scrolls remain a viable option for sequencing when cores are unavailable. This has the potential to offer valuable insight for conducting population-based next-generation studies, which mostly rely on FFPE-derived tissues. Citation Format: Noah C. Peeri, Megan A. Clarke, Immaculata De Vivo, Mengmeng Du, Lucio Miele, Jeffin Naduparambil, Kathryn L. Penney, Venkat Seshan, Ronglai Shen, Veronica W. Setiawan, Nicolas Wentzensen, Xiao-Cheng Wu, Arnold Zea, Wenyu Zhang. Pilot whole-exome sequencing data of formalin-fixed paraffin embedded endometrial tumors in the Epidemiology of Endometrial Cancer Consortium (E2C2) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB373.

Diagnosing adult and pediatric extrapulmonary tuberculosis by MPT64 antigen detection with immunohistochemistry and immunocytochemistry using reproduced polyclonal antibodies.

Diagnosing extrapulmonary tuberculosis (EPTB) is challenging. Immunohistochemistry or immunocytochemistry has been used to diagnose tuberculosis (TB) by detection of MPT64 antigen from various extrapulmonary specimens and has shown good diagnostic performance in our previous studies. The test can distinguish between disease caused by Mycobacterium tuberculosis (Mtb) complex and nontuberculous mycobacteria and can be applied on formalin-fixed paraffin-embedded tissue. As the antibodies previously used were in limited supply, a new batch of polyclonal antibodies was developed for scale-up and evaluated for the first time in this study. Our aim was to assess the diagnostic accuracy of the MPT64 test with reproduced antibodies in the high burden settings of Pakistan and India. Patients were enrolled prospectively. Samples from suspected sites of infection were collected and subjected to histopathologic and/or cytologic evaluation, routine TB diagnostics, GeneXpert MTB/RIF (Xpert), and the MPT64 antigen detection test. Patients were followed until the end of treatment. Based on a composite reference standard (CRS), 556 patients were categorized as TB cases and 175 as non-TB cases. The MPT64 test performed well on biopsies with a sensitivity and specificity of 94% and 75%, respectively, against a CRS. For cytology samples, the sensitivity was low (36%), whereas the specificity was 81%. Overall, the MPT64 test showed higher sensitivity (73%) than Xpert (38%) and Mtb culture (33%). The test performed equally well in adults and children. We found an additive diagnostic value of the MPT64 test in conjunction with histology and molecular tests, increasing the yield for EPTB. In conclusion, immunochemical staining with MPT64 antibodies improves the diagnosis of EPTB in high burden settings and could be a valuable addition to routine diagnostics.

Clinical Validation of a Targeted Next-Generation Sequencing Panel for Lymphoid Malignancies

Lymphoid malignancies are a heterogeneous group of hematological disorders characterized by a diverse range of morphologic, immunophenotypic, and clinical features. Next-generation sequencing (NGS) is increasingly being applied to delineate the complex nature of these malignancies and identify high-value biomarkers with diagnostic, prognostic, or therapeutic benefit. However, there are various challenges in using NGS routinely to characterize lymphoid malignancies, including pre-analytic issues, such as sequencing DNA from formalin-fixed, paraffin-embedded tissue, and optimizing the bioinformatic workflow for accurate variant calling and filtering. This study reports the clinical validation of a custom capture-based NGS panel to test for molecular markers in a range of lymphoproliferative diseases and histiocytic neoplasms. The fully validated clinical assay represents an accurate and sensitive tool for detection of single-nucleotide variants and small insertion/deletion events to facilitate the characterization and management of patients with hematologic cancers specifically of lymphoid origin.

VISTA/CTLA4/PD1 coexpression on tumor cells confers a favorable immune microenvironment and better prognosis in high-grade serous ovarian carcinoma.

Immunotherapy by blocking immune checkpoints programmed death/ligand (PD1/PDL1) and cytotoxic T-lymphocyte-associated protein 4(CTLA4) has emerged as new therapeutic targets in cancer. However, their efficacy has been limited due to resistance. A new- checkpoint V-domain Ig-containing suppressor of T cell activation (VISTA) has appeared, but the use of its inhibition effect in combination with antibodies targeting PDL1/PD1and CTLA4 has not been reported in ovarian cancer. In this study, we investigated the expressions of VISTA, CTLA4, and PDL1 using immunohistochemistry (IHC)on 135 Formalin-Fixed Paraffin-Embedded (FFPE)tissue samples of High-grade serous carcinoma (HGSOC). VISTA, CTLA4, PDL1, PD1, CD8, CD4, and FOXP3 mRNA extracted from 429 patients with ovarian cancer in the Cancer Genome Atlas (TCGA) database was included as a validation cohort. Correlations between these checkpoints, tumor-infiltrating- lymphocytes (TILs), and survival were analyzed. CTLA4 was detectable in 87.3% of samples, VISTA in 64.7%, PD1 in 56.7%, and PDL1 in 48.1%. PDL1 was the only tested protein associated with an advanced stage (p=0.05). VISTA was associated with PDL1, PD1, and CTLA4 expressions (p=0.005, p=0.001, p=0.008, respectively), consistent with mRNA level analysis from the TCGA database. Univariate analyses showed only VISTA expression (p=0.04) correlated with overall survival (OS). Multivariate analyses showed that VISTA expression (p=0.01) and the coexpression of VISTA+/CTLA4+/PD1+ (p=0.05) were associated with better OS independently of the clinicopathological features. Kaplan-Meier analysis showed that the coexpression of the VISTA+/CTLA4+/PDL1+ and VISTA+/CTLA4+/PD1+ checkpoints on tumor cells (TCs)were associated with OS (p=0.02 and p<0.001; respectively). VISTA+/CTLA4+/PD1+ in TCs and CD4+/CD8+TILswere associated with better 2-yer OS. This correlation may refer to the role of VISTA as a receptor in the TCs and not in the immune cells. Thus, targeting combination therapy blocking VISTA, CTLA4, and PD1 could be a novel and attractive strategy for HGSOC treatment, considering the ambivalent role of VISTA in the HGSOC tumor cells.

Investigation of Epstein-Barr virus, Cytomegalovirus, Human herpesvirus 6, and Polyoma viruses (JC virus, BK virus) among Gastric cancer patients: A cross sectional study.

Gastric cancer is a significant global issue with a high death rate. This malignancy could be associated with several viral agents such as EBV, CMV, HHV-6, JCV, and BKV. Evaluation of EBV, CMV, HHV-6,and JCV, BKV frequency among gastric cancer patients. In this cross-sectional study, a total number of 60 gastric cancer specimens (32 male, 28 female) were retrieved from the pathology lab. Formalin-fixed paraffin-embedded tissue was used for molecular testing. DNA was extracted from samples, according to protocol, and used for PCR reaction. Polymerase chain reactions were used to assess CMV, EBV, HHV-6, JCV, and BKV frequency. The mean age of the participants was 61 years and 53.3% (32) of the participants were Male. A total number of 5 samples (8.34%) were infected with viral agents. Four male gastric samples were infected with EBV (6.67%) and only one female sample contained the BKV genome (1.67%). Totally 8.34% of the samples were infected with EBV and BKV. The CMV, HHV-6, and JCV genome was not detected in the samples. In conclusion, the presence of two viral agents including EBV and BKV among male and female samples respectively, and the genome of other viruses were not detected.