Formalin-fixed Paraffin-embedded Tumor Samples Research Articles

Altered extracellular matrix correlates with an immunosuppressive tumor microenvironment and disease progression in younger adults with oral cavity squamous cell carcinoma.

Oral cavity squamous cell carcinoma (OSCC) occurs most frequently in patients >60 years old with a history of tobacco and alcohol use. Epidemiological studies describe increased incidence of OSCC in younger adults (<45 years). Despite its poor prognosis, knowledge of OSCC tumor microenvironment (TME) characteristics in younger adults is scarce and could help inform possible resistance to emerging treatment options. Patients with OSCC were evaluated using TCGA-HNSC (n=121) and a stage and subsite-matched institutional cohort (n=8) to identify differential gene expression focusing on the extracellular matrix (ECM) and epithelial-mesenchymal transition (EMT) processes in younger (≤45 years) vs. older adults (≥60 years). NanoString nCounter analysis was performed using isolated total RNA from formalin-fixed paraffin-embedded (FFPE) tumor samples. Stained tumor slides from young and old OSCC patients were evaluated for CD8+ T-cell counts using immunohistochemistry. Younger OSCC patients demonstrated significantly increased expression of ECM remodeling and EMT process genes, as well as TME immunosuppression. Gene set enrichment analyses demonstrated increased ECM pathways and concurrent decreased immune pathways in young relative to old patients. Transcripts per million of genetic markers involved in ECM remodeling including LAMB3, VCAN, S100A9, COL5A1, and ITGB2 were significantly increased in tumors of younger vs. older patients (adjusted p-value < 0.10). Young patient TMEs demonstrated a 2.5-fold reduction in CD8+ T-cells as compared to older patients (p < 0.05). Differential gene expression impacting ECM remodeling and TME immunosuppression may contribute to disease progression in younger adult OSCC and has implications on response to evolving treatment modalities, such as immune checkpoint inhibitor therapy.

Microsatellite Instability Testing and Prognostic Implications in Colorectal Cancer.

Given the crucial predictive implications of microsatellite instability (MSI) in colorectal cancer (CRC), MSI screening is commonly performed in those with and at risk for CRC. Here, we compared results from immunohistochemistry (IHC) and the droplet digital PCR (ddPCR) MSI assay on formalin-fixed paraffin-embedded tumor samples from 48 patients who underwent surgery for colon and rectal cancer by calculating Cohen's kappa measurement (k), revealing high agreement between the methods (k = 0.915). We performed Kaplan-Meier survival analyses and univariate and multivariate Cox regression to assess the prognostic significance of ddPCR-based MSI and to identify clinicopathological features associated with CRC outcome. Patients with MSI-high had better overall survival (OS; p = 0.038) and disease-free survival (DFS; p = 0.049) than those with microsatellite stability (MSS). When stratified by primary tumor location, right-sided CRC patients with MSI-high showed improved DFS, relative to those with MSS (p < 0.001), but left-sided CRC patients did not. In multivariate analyses, MSI-high was associated with improved OS (hazard ratio (HR) = 0.221, 95% confidence interval (CI): 0.026-0.870, p = 0.042), whereas the loss of DNA mismatch repair protein MutL homolog 1 (MLH1) expression was associated with worse OS (HR = 0.133, 95% CI: 0.001-1.152, p = 0.049). Our results suggest ddPCR is a promising tool for MSI detection. Given the opposing effects of MSI-high and MLH1 loss on OS, both ddPCR and IHC may be complementary for the prognostic assessment of CRC.

The analysis of transcriptomic signature of TNBC—searching for the potential RNA-based predictive biomarkers to determine the chemotherapy sensitivity

AbstractNeoadjuvant chemotherapy is the foundation treatment for triple-negative breast cancer (TNBC) and frequently results in pathological complete response (pCR). However, there are large differences in clinical response and survival after neoadjuvant chemotherapy of TNBC patients. The aim was to identify genes whose expression significantly associates with the efficacy of neoadjuvant chemotherapy in patients with TNBC. Transcriptomes of 46 formalin-fixed paraffin-embedded (FFPE) tumor samples from TNBC patients were analyzed by RNA-seq by comparing 26 TNBCs with pCR versus 20 TNBCs with pathological partial remission (pPR). Subsequently, we narrowed down the list of genes to those that strongly correlated with drug sensitivity of 63 breast cancer cell lines based on Dependency Map Consortium data re-analysis. Furthermore, the list of genes was limited to those presenting specific expression in breast tumor cells as revealed in three large published single-cell RNA-seq breast cancer datasets. Finally, we analyzed which of the selected genes were significantly associated with overall survival (OS) in TNBC TCGA dataset. A total of 105 genes were significantly differentially expressed in comparison between pPR versus pCR. As revealed by PLSR analysis in breast cancer cell lines, out of 105 deregulated genes, 42 were associated with sensitivity to docetaxel, doxorubicin, paclitaxel, and/or cyclophosphamide. We found that 24 out of 42 sensitivity-associated genes displayed intermediate or strong expression in breast malignant cells using single-cell RNAseq re-analysis. Finally, 10 out of 24 genes were significantly associated with overall survival in TNBC TCGA dataset. Our RNA-seq-based findings suggest that there might be transcriptomic signature consisted of 24 genes specifically expressed in tumor malignant cells for predicting neoadjuvant response in FFPE samples from TNBC patients prior to treatment initiation. Additionally, nine out of 24 genes were potential survival predictors in TNBC. This group of 24 genes should be further investigated for its potential to be translated into a predictive test(s).

Personalized circulating tumor DNA monitoring improves recurrence surveillance and management after curative resection of colorectal liver metastases: a prospective cohort study.

Approximately 60% of patients with colorectal liver metastases (CRLM) experience relapse within 2 years after radical resection, previous studies have proven that repeat local treatment (LT) could prolong survival, however, it is difficult to seize the window for LT due to the lack of a high-sensitive surveillance method. In this study, the authors aim to examine the value of longitudinal circulating tumor DNA (ctDNA) in guiding adjuvant chemotherapy, optimizing clinical surveillance strategy, and thereby improving CRLM outcomes. The authors conducted a prospective clinical trial using a personalized, tumor-informed ctDNA assay to monitor 60 CRLM patients undergoing resection with curative intent. Formalin-fixed paraffin-embedded tumor samples were collected after surgery. Blood samples were collected before surgery, 30 days after surgery (post-OP), and every third month until relapse or up to 2 years. A total of 394 plasma samples from 60 eligible patients were analyzed, with a median follow-up time of 31.3 months. Landmark analyses revealed that detectable ctDNA at post-OP (HR, 4.8), postadjuvant chemotherapy (HR, 6.0), and end-of-treatment (HR, 5.6) were associated with higher recurrence risk ( P <0.001). Post-OP ctDNA positivity served as the only independent prognostic marker in the multivariant analysis (HR, 5.1; P <0.001). Longitudinal ctDNA analysis identified relapsed patients at both sensitivity and specificity of 100%. Most (75%) patients were found with radiological relapse within 6 months after the first detectable ctDNA with a median lead time of 3.5 months. In relapsed patients, 73.2% had oligometastatic disease and 61% were liver-restricted, of which 72.0% received repeat LTs, and 60.0% achieved a secondary no evidence of disease status. Longitudinal ctDNA monitoring assists in early prediction of relapse, and thereby improves survival of CRLM patients by increased secondary resection rate and secondary no evidence of disease rate.

Microsatellite Instability Detection in Cancer: A Multiplex qPCR Approach that Obviates the Need for Matching Normal Samples.

Microsatellite instability (MSI) indicates DNA mismatch repair deficiency in certain types of cancer, such as colorectal cancer. The current gold standard technique, PCR-capillary electrophoresis (CE), requires matching normal samples and specialized instrumentation. We developed VarTrace, a rapid and low-cost quantitative PCR (qPCR) assay, to evaluate MSI using solely the tumor sample DNA, obviating the requirement for matching normal samples. One hundred and one formalin-fixed paraffin-embedded (FFPE) tumor samples were tested using VarTrace and compared with the Promega OncoMate assay utilizing PCR-CE. Tumor percentage limit of detection was evaluated on contrived samples derived from clinical high MSI (MSI-H) samples. Analytical sensitivity, specificity, limit of detection, and input requirements were assessed using synthetic commercial reference standards. VarTrace successfully analyzed all 101 clinical FFPE samples, demonstrating 100% sensitivity and 98% specificity compared to OncoMate. It detected MSI-H with 97% accuracy down to 10% tumor. Analytical studies using synthetic samples showed a limit of detection of 5% variant allele frequency and a limit of input of 0.5 ng. This study validates VarTrace as a swift, accurate, and economical assay for MSI detection in samples with low tumor percentages without the need for matching normal DNA. VarTrace's capacity for highly sensitive MSI analysis holds potential for enhancing the efficiency of clinical work flows and broadening the availability of this test.

Abstract 4869: Efficient study design for the discovery of a gene expression signature predicting metastasis in cutaneous squamous cell carcinoma

Abstract Background: The StepIdent study aims to develop a gene signature predicting metastasis in patients with cutaneous squamous cell carcinoma (cSCC) to improve risk stratification, thus enabling personalized decisions about follow-up schedules and treatment options. Here we describe the unique characteristics, challenges, and best practices for an efficient design of a discovery cohort for a rare outcome (metastasis prevalence: 2-5%); for retrieving, curating, and linking the clinical and pathological data through nationwide databases; and for measuring gene expression through sequencing of archived Formalin-Fixed Paraffin-Embedded (FFPE) primary tumor samples. Methods: Following a predefined protocol, we identified a nested-case control cohort (NCC) of 305 cases and 305 controls from a nationwide cohort of 19,120 patients with a first cSCC in the Netherlands from 2007 to 2009, followed up until 2020. We chose an NCC design since it is an efficient study design in a rare outcome setting (weighting is needed to accommodate the under-sampling of the controls). Patients were identified from the Dutch National Cancer Registry (NCR) and the clinical information was retrieved from the NCR which is linked to the nationwide registry of histo- and cytopathology (PALGA). Tumor blocks were requested from PALGA, and pathological characteristics were assessed by dermatopathologists. We matched controls to cases, based on a risk score estimated by a clinicopathological model. Gene expression was measured using the Illumina RNA Prep with Enrichment kit combined with the whole exome panel and paired-end sequenced on the NextSeq 550. Results: Tissue slides for 541 samples were retrieved for sequencing. 151 samples were excluded after pathology review or due to low pre-library concentration. The final cohort includes 195 case-control pairs (n=390). The median sequencing depth was 43M (Q1-Q3: 35-52M); the median Q30 was 85% (Q1-Q3: 83-87%); the median GC content was 51% (Q1-Q3: 50-52%); a median of 1.8% of base pairs (Q1-Q3: 1.4-2.1%) was trimmed prior to the mapping/alignment; a median of 69% (Q1-Q3: 65-74%) of reads were aligned as protein-coding and a median of 7% (Q1-Q3: 6-10%) as rRNA; a median of 95% (Q1-Q3: 93-96%) of reads were aligned by STAR. Two samples were excluded based on quality control. Conclusion: We described an efficient design and implementation of a nationwide discovery study in cSCC, involving the retrieval of clinicopathological data, the collection of FFPE materials, and the execution of omics measurements. This study presents the largest cohort to date, incorporating omics measurements of primary cSCC samples, combined with simultaneous access to well-curated clinical and pathological information and follow-up data. Our findings can provide guidance for similar studies involving a rare clinical endpoint, where an efficient study design is a necessity. Citation Format: Barbara Rentroia-Pacheco, Lara Pozza, Yan Ting Chen, Daphne Huigh, Celeste J. Eggermont, Olivia FM Steijlen, Sheril Alex, Jvalini Dwarkasing, Domenico Bellomo, Harmen JG van de Werken, Antien L. Mooyaart, Marlies Wakkee, Loes M. Hollestein. Efficient study design for the discovery of a gene expression signature predicting metastasis in cutaneous squamous cell carcinoma [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 4869.

Abstract 326: Matched fresh frozen and FFPE patient tissues reveal the enhanced sensitivity and data quality of a novel DNA library prep method

Abstract In cancer genomics, a common source of DNA is formalin-fixed, paraffin-embedded (FFPE) tissue from patient surgical samples, where in most cases high quality fresh or frozen tissue samples are not available. FFPE DNA poses many notable challenges for preparing NGS libraries, including low input amounts and highly variable damage from fixation, storage, and extraction methods. It is difficult to obtain libraries with sufficient coverage and the sequencing artifacts arising from damaged DNA bases confound somatic variant detection. Additionally, many laboratories process FFPE tumor samples alongside matched, high quality, normal DNA and many library prep workflows are not readily compatible with both sample types. We developed a novel NGS library prep method compatible with both high quality and very low quality FFPE DNA samples, employing a novel enzymatic DNA repair mix, enzymatic fragmentation mix, and PCR master mix. To validate this workflow on real patient sample sets, we obtained DNA extracted from matched tumor and normal tissue of various tissue types preserved by both fresh frozen and FFPE methods. The FFPE DNA samples ranged in quality from DNA integrity number (DIN) 1.5 to 6.8, including samples stored for nearly 3 decades. We prepared libraries using this method compared against other library prep workflows and sequenced them by WGS and target capture. We assessed the performance of these workflows by library yield, library quality metrics, depth and evenness of coverage, and somatic variant calling with fresh frozen-extracted DNA providing the gold standard for library quality and mutation content. This new enzymatic fragmentation-based library prep workflow not only reduced the false positive rate in somatic variant detection by repairing damage-derived mutations in FFPE DNA samples but also improved the library yield, library quality metrics (including mapping, chimeras, and properly-paired reads), library complexity, coverage depth and uniformity, and hybrid capture library quality metrics. Comparing the variant calls from matched FFPE and frozen tissues revealed an improved sensitivity and accuracy of variant calling using this library prep method compared to mechanical shearing and other enzymatic fragmentation library prep approaches. This new suite of enzyme mixes allows even highly damaged FFPE samples to achieve high quality libraries with a greater sensitivity for somatic variant identification. The workflow is robust and flexible, compatible with both FFPE DNA and matched high quality DNA samples as well as automation-friendly for convenience in sample processing. Citation Format: Margaret R. Heider, Jian Sun, Brittany Sexton, Bradley W. Langhorst, Laura Blum, Pingfang Liu. Matched fresh frozen and FFPE patient tissues reveal the enhanced sensitivity and data quality of a novel DNA library prep method [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 326.

Abstract 1017: Characterizing immune cell infiltration in colorectal cancer tumor microenvironment among Native Hawaiians

Abstract Background Colorectal cancer (CRC) is recognized as a heterogeneous disease, ranking third in new cancer diagnoses and second in cancer-related deaths globally and nationally. CRC incidence is higher in the state of Hawaii compared to the U.S. overall. This study focuses on profiling immune cell infiltration in Native Hawaiian(NH)-specific CRC tumor microenvironments. Identifying the race-specific tumor infiltrating microenvironment may uncover potential mechanisms for CRC development in the NH population. Methods Deidentified, archival formalin-fixed paraffin-embedded tumor samples from Native Hawaiian patients diagnosed with primary CRC between 1990 and 2006 were obtained from the Hawaii Tumor Registry. RNA was extracted from tumor and adjacent normal tissues. RNA-seq data from Caucasian American (CA) CRC samples were obtained from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO). Differentially expressed genes for each cohort were identified via DESeq2 with Benjamini-Hochberg multiple testing q-value&amp;lt;0.05 and a cutoff of 2-fold change. From the gene expression profiles, we applied the ESTIMATE algorithm to calculate the overall immune cell infiltration score for each sample. Based on a deconvolution algorithm, known as CIBERSORT to estimate the abundance of 22 immune cell types, we profiled the tumor-infiltrating immune cells present in each cohort’s paired samples, respectively. To improve the accuracy, p-value and root mean squared error were counted for each sample. Data with p&amp;lt;0.05 were filtered and selected for the next analysis. The fraction of immune cell subpopulations was evaluated and compared between paired tumor and adjacent normal tissues in NH and CA cohorts. Results In the NH cohort, we found the overall immune cell infiltration score was significantly lower in cancer tissues than the adjacent normal tissues (p&amp;lt;0.05), and there were significantly higher fractions of activated dendritic cells, resting natural killer (NK), and follicular helper T cells in tumor tissues compared to adjacent non-tumor tissues (p&amp;lt;0.05). Furthermore, the fractions of activated NK cells and plasma cells were significantly lower in tumor tissues than in adjacent non-tumor tissues in NH (p&amp;lt;0.05). Resting NK cells are upregulated in both the NH and the CA cohorts, while plasma cells are the only immune cell type downregulated in either cohort. In the NH cohort, activated dendritic cells and follicular helper T cells are exclusively upregulated, while activated NK cells are downregulated relative to the CA cohort. Conclusion This study demonstrates that the NH cohort displays distinctive immunological variances between tumor and adjacent non-tumor tissues which distinguishes them from the CA cohort. This emphasizes the significance of acknowledging population-specific genetic variations which may help understand race specific difference in CRC. Citation Format: Yuanyuan Fu, Gino Quintal, Masaki Nasu, Mayumi Jijiwa, Sudhir K. Rai, Isam Mohd-Ibrahim, Yu Chen, Hua Yang, Zhanwei Wang, Christina Wai, Owen Chan, Brenda Y. Hernandez, Youping Deng. Characterizing immune cell infiltration in colorectal cancer tumor microenvironment among Native Hawaiians [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 1017.

Quantification of CD3, FoxP3, and granzyme B immunostaining in canine renal cell carcinoma

Tumor-infiltrating lymphocyte (TIL) density plays an important role in anti-tumor immunity and isassociated with patient outcome in various human and canine malignancies. As a first assessment of the immune landscape of the tumor microenvironment in canine renal cell carcinoma (RCC), we retrospectively analyzed clinical data and quantified CD3, FoxP3, and granzyme B immunostaining in formalin-fixed paraffin-embedded tumor samples from 16 dogs diagnosed with renal cell carcinoma treated with ureteronephrectomy. Cell density was low for all markers evaluated. Increased numbers of intratumoral FoxP3 labelled (+) cells, as well as decreased granzyme B+: FoxP3+ TIL ratio, were associated with poor patient outcomes. Our initial study of canine RCC reveals that these tumors are immunologically cold and Tregs may play an important role in immune evasion.

Immunohistochemistry assessment of tissue neutrophil-to-lymphocyte ratio predicts outcomes in melanoma patients treated with anti-programmed cell death 1 therapy.

Elevated neutrophil-to-lymphocyte ratio (NLR) is associated with diminished immunotherapy response in metastatic melanoma. Although NLR assessment in peripheral blood is established, tissue dynamics remain insufficiently explored. This study aimed to evaluate tissue NLR (tNLR)'s predictive potential through immunohistochemistry in immunotherapy-treated melanoma. Fifty melanoma patients who underwent anti-programmed cell death 1 (PD-1) therapy were assessed. Hematological, clinical and tumor features were collected from medical records. Responses were categorized using the Response Evaluation Criteria in Solid Tumors for immunotherapy (iRECIST) guidelines. Immunohistochemistry for tumor-infiltrating T cells (cluster differentiation 3) and neutrophils (myeloperoxidase) was performed on formalin-fixed paraffin-embedded tumor samples. NLR, derived NLR (dNLR) and tNLR were calculated. Overall survival (OS) and survival following immunotherapy (SFI) were calculated from diagnosis or immunotherapy start to loss of follow-up or death. Patients with high tNLR presented improved OS ( P = 0.038) and SFI with anti-PD-1 therapy ( P = 0.006). Both NLR and dNLR were associated with OS ( P = 0.038 and P = 0.046, respectively) and SFI ( P = 0.001 and P = 0.019, respectively). NLR was also associated with immunotherapy response ( P = 0.007). In conclusion, tNLR emerged as a novel potential biomarker of enhanced survival post anti-PD-1 therapy, in contrast to classical NLR and dNLR markers.

Pediatric spinal ependymoma with chromothripsis of chromosome 6: a case report and review of the literature

BackgroundEpendymomas are the third most common central nervous system tumor in the pediatric population; however, spinal ependymomas in children are rare. Ependymomas affecting the spinal cord most frequently occur in adults of 20–40 years of age. The current World Health Organization classification system for ependymomas is now composed of ten different entities based on histopathology, location, and molecular studies, with evidence that the new classification system more accurately predicts clinical outcomes.Case presentationWe present the case of a 16-year-old Caucasian female patient with a history of type 2 neurofibromatosis with multiple schwannomas, meningioma, and spinal ependymoma. Chromosome analysis of the harvested spinal ependymoma tumor sample revealed a 46,XX,−6,+7,−22,+mar[16]/46,XX[4] karyotype. Subsequent OncoScan microarray analysis of the formalin-fixed paraffin-embedded tumor sample confirmed + 7, −22 and clarified that the marker chromosome represents chromothripsis of the entire chromosome 6 with more than 100 breakpoints. Fluorescent in situ hybridization and microarray analysis showed no evidence of MYCN amplification. The final integrated pathology diagnosis was spinal ependymoma (central nervous system World Health Organization grade 2 with no MYCN amplification.ConclusionThis case adds to the existing literature of pediatric patients with spinal ependymomas and expands the cytogenetic findings that may be seen in patients with this tumor type. This case also highlights the value of cytogenetics and microarray analysis in solid tumors to provide a more accurate molecular diagnosis.

Hepatocellular Carcinoma Epigenetic Patterns Correspond to Differences in Ethnoracial Status and Treatment Response in a Single-Center Retrospective Study

PurposeTo correlate epigenetic patterns with ethnoracial status and locoregional therapy (LRT) response in patients with hepatocellular carcinoma (HCC). Materials and MethodsDNA and RNA were extracted from 47 distinct formalin-fixed paraffin-embedded tumor samples from 42 patients with HCC (n = 14 Black, n = 19 White, n = 9 Hispanic). LRT response was determined using computed tomography (CT) or magnetic resonance (MR) imaging 3 months posttreatment of 35 tumors (n = 22 complete response, n = 13 retreatment candidates). RNA expression and DNA methylation were used to stratify patients by ethnoracial status and treatment response using partial least-squares discriminant analysis (PLS-DA). Results were validated using hierarchical clustering. Ingenuity pathway analysis was performed to identify upstream regulators and pathways. ResultsPLS-DA identified 100 genes and 12 methylated regions that differentiated tumors from Black from White/Hispanic patients. Hierarchical clustering clustered samples with the top 16 genes or the top 5 methylation regions. Dysregulated pathways included adrenomedullin pathway (P = .030), EIF2 signaling (P = .007), and several metabolic pathways. AGTR1 (log2fold = 1.59) and GSTM3 (log2fold = 2.53) represented potential differentially expressed therapeutic targets. PLS-DA identified 100 genes and 150 methylation regions that differentiated between complete responders and retreatment candidates. Hierarchical clustering clustered samples with the top 30 genes or the top 13 methylation regions. Dysregulated pathways included metabolic and DNA repair–related pathways. ASAP2 (log2fold = 0.29) and RAD50 (log2fold = 0.22) represented potential differentially expressed therapeutic targets. ConclusionsVariation in gene expression and DNA methylation patterns in patients with HCC corresponded to ethnoracial status and LRT response. These initial results suggest tumor profiling has the potential to close ethnoracial disparities and improve treatment stratification.

Molecular and clinicopathological features of KIT/PDGFRA wild-type gastrointestinal stromal tumors.

Approximately 10% of gastrointestinal stromal tumors (GISTs) harbor reportedly no KIT and PDGFRA mutations (wild-type GISTs). The clinicopathological features and oncologic outcomes of wild-type GISTs based on molecular profiles are unknown. We recruited 35 wild-type GIST patients from the two registry studies of high-risk GISTs between 2012 and 2015 and primary GISTs between 2003 and 2014. Molecular profiling of wild-type GISTs was performed by targeted next-generation sequencing (NGS) using formalin-fixed paraffin-embedded tumor samples. Among 35 wild-type GISTs, targeted NGS analysis detected NF1, SDH, or BRAF mutation: 16 NF1-GISTs with various NF1 mutations, 12 SDH-GISTs (4 with SDHA mutations, 4 with SDHB mutations, and 4 with SDHB-negative staining), and 5 BRAF-GISTs with the V600E mutation. Two GISTs showed no mutations based on our targeted NGS analysis. Additional gene mutations were infrequent in primary wild-type GISTs and found in TP53, CREBBP, CDKN2A, and CHEK2. Most NF1-GISTs were located in the small intestine (N = 12; 75%) and showed spindle cell features (N = 15; 94%) and multiple tumors (N = 6, 38%) with modest proliferation activities. In contrast, SDH-GISTs were predominantly found in the stomach (N = 11; 92%), exhibiting epithelioid cell (N = 6; 50%) and multiple (N = 6, 50%) features. The overall survival of patients with SDH-GISTs appeared to be better than that of BRAF-GISTs (p = 0.0107) or NF1-GISTs (p = 0.0754), respectively. In conclusion, major molecular changes in wild-type GISTs include NF1, SDH, and BRAF. NF1-GISTs involved multifocal spindle cell tumors in the small intestine. SDH-GISTs occurred in young patients and were multifocal in the stomach and clinically indolent.

Investigation of the tumor microenvironment, hypoxia and angiogenesis by immunohistochemical and histopathological methods in canine mammary tumors

The tumor microenvironment (TME) is an important component for studying tumor behavior in several cancers in human beings. However, little information regarding the role of the TME in canine mammary tumors (CMTs) is available compared to humans. In this study, the aim was to investigate the relationship between the TME, hypoxia and angiogenesis through CD31, VEGF, HIF-1a, CD68 and CD163 expression by using immunohistochemical (IHC) methods in formalin-fixed paraffin-embedded canine mammary tumor samples [(n=34: malignant (n=28) and benign (n=6)], to compare them with the clinicopathological features of tumors, and to analyze the relationship between them. There was no significant relationship between CD31, VEGF, HIF-1a, CD68 and CD163 expression in malignant tumors compared to benign tumors (P&gt;0.05). There was an association between microvessel density and clinicopathological variables (the tumor size P=0.013, the presence of necrosis P=0.022) and individual histological grade (G2 vs. G3 P=0.028) in malignant tumors. While there was a positive correlation between CD68 and CD163 in malignant tumors in the dogs (P&lt;0.01), no correlation was determined between other antibodies. Immunohistochemical determination of the level of angiogenesis in the TME may give further useful information about the angiogenic potential and grading of the clinical aggressiveness of some CMTs.

Challenges to the effectiveness of next-generation sequencing in formalin-fixed paraffin-embedded tumor samples for non-small cell lung cancer

IntroductionNext-generation sequencing (NGS) of Formalin-Fixed and Paraffin-Embedded (FFPE) specimens is routine in precision oncology practice. However, results are not always conclusive, and it is important to identify which factors may influence FFPE tumor sequencing success. Materials and methodsHere, we evaluated the influence of pre-analytical factors on 705 samples of non-small cell lung cancer specimens that underwent NGS testing. Factors such as tumor site, tumor cell percentage, fragment size, primary tumor or metastasis, presence of necrosis, DNA purity, DNA concentration, sample origin and year of testing. ResultsThe overall NGS success rate was 84.9 % (n = 599). Bone site specimens had a very low success rate (42.1 %), differing from lung samples (79.8 %) (P < 0.05). Samples with tumor percentages <5 % (success rate of 44.4 %) represented 14.1 % of failed sequencings. Moreover, samples with tumor percentages >10 %–20 % (82 %) did not differ from those with >30 % (88.9 %) on sequencing outcomes (P = 0.086). Specimens that provided DNA concentrations >2.0 ng/uL, 1.0–2.0 ng/uL, 0.5–1.0 ng/uL and <0.5 ng/uL had success rates of 92 %, 77.1 %, 61.3 % and 20.4 %, respectively. Small fragments (≤0.2 cm2) had a success rate of 74.7 % and were more prevalent in the unsuccessful group (P < 0.05). ConclusionsOur results suggest that tumor percentage, fragment size, decalcified bone specimens, and DNA concentration are potential modifiers of NGS success rates. Interestingly, specimens with tumor percentages between 10 % and 20 % have the same sequencing outcome than specimens with >30 %. These results can strengthen the understanding of factors that lead to NGS success variability.

Performance of non-formalin fixed paraffin embedded samples in hybrid capture and amplicon next-generation sequencing panels.

Genomic profiling using next-generation sequencing (NGS) is fundamental for driving prognostic and therapy in cancer. Formalin-fixed paraffin embedded (FFPE) tissue is the widely used material, whereas non-FFPE may represent an alternative. However, studies comparing the NGS performance of non-FFPE materials to FFPE are still lacking in the literature. The objective of this study was to characterize in non-FFPE preparations the nucleic acid yield and NGS performance on both a capture-based and an amplicon-based NGS platform. NGS quality metrics obtained from non-FFPE preparations were compared to FFPE. We analyzed the cellularity and nucleic acid yield in 111 tumors from non-FFPE preparations. In addition, comprehensive hybrid capture panel sequencing metrics obtained from DNA and RNA libraries were compared between independent non-FFPE and FFPE samples. A paired comparison between non-FFPE and FFPE samples was performed to analyze concordance in mutant allele detection using an amplicon panel. The mean target coverage from DNA libraries was 2× higher in non-FFPE samples than in FFPE. The detection of exogenous DNA was 2.5× higher in non-FFPE than in FFPE. Conversely, a lower performance was observed in non-FFPE RNA libraries in comparison to FFPE DNA libraries with no impact in minimum standard cutoffs. The variant allele detection in non-FFPE was found to be comparable to that of FFPE tumor samples in matched samples. Non-FFPE was demonstrated to be a suitable material for DNA and RNA library preparations using a comprehensive NGS panel. This is the first study reporting library quality metrics according to the TSO500 analysis pipeline.

Abstract A124: Beyond PD-L1: Unraveling the enigma of immunotherapy response in PD-L1 negative (&amp;lt;1%) NSCLC patients through quantification of PD-1/PD-L1 engagement in the tumor microenvironment

Abstract Profound advances in cancer patient treatments have come with the advent of immunotherapies directed at blocking immune-suppressive ligand-receptor interactions. However, while there has been success in the use of these immune checkpoint interventions, there has been limited success in stratifying patients for these interventions with response rates at approximately 14%.To address this issue of patient stratification, we have determined the extent of the molecular target, intercellular PD-1/PD-L1 interaction, in formalin-fixed paraffin embedded tumor samples from non–small cell lung carcinoma patients, using a high-throughput automated quantitative imaging platform (QF-Pro®). The multi-site blinded analysis across a cohort of 188 IO-treated patients (treated with nivolumab, pembrolizumab, atezolizumab or durvalumab) demonstrated the intra- and inter-tumoral heterogeneity of the PD-1/PD-L1 immune checkpoint engagement and notably showed no correlation between the extent of PD-1/PD-L1 interaction and PD-L1 expression (rs=0.05, P=0.55). Importantly, PD-L1 expression scores used clinically to stratify the patients, correlated poorly with overall survival. By contrast, patients showing a high PD-1/PD-L1 interaction had significantly better responses to anti-PD-1/PD-L1 treatments, as evidenced by increased OS (median 31 months vs 10 months). This relationship was particularly strong in the setting of first line treatments. Even more critically, in a subset of patients determined as being PD-L1 negative (PD-L1 TPS &amp;lt;1%), response to treatment was still observed in those patients with PD-1/PD-L1 interactions (median &amp;gt;60 months vs 14 months). This therefore may yield a mechanism to successfully treat those patients who, under current guidelines, are not be considered for these therapies, despite the therapeutic target existing within their tumor microenvironment. In conclusion using the functional read out of this immune checkpoint engagement (PD-1/PD-L1 interaction) as a predictive biomarker for the stratification of NSCLC patients instead of PD-L1 expression should significantly improve the response rates to immunotherapy. This would both capture patients excluded from checkpoint immunotherapy (high PD-1/PD-L1 interaction but low PD-L1 expression, 24% of patients), and additionally avoid the treatment of patients resistant to this type of treatment (low PD-1/PDL1 interaction but high PD-L1 expression) who may benefit- from alternative cancer therapeutics, such as other immunotherapies. Even at this early stage, QF-Pro® has set an unbiased quantitative cut-off to be considered for anti-PD-1/PD-L1 therapies which can be deployed directly into clinical practice. Citation Format: Juan Gumuzio, Veronique Calleja, James Miles, Lissete Sancehz-Magraner, Fernando Aguirre. Beyond PD-L1: Unraveling the enigma of immunotherapy response in PD-L1 negative (&amp;lt;1%) NSCLC patients through quantification of PD-1/PD-L1 engagement in the tumor microenvironment [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A124.

Abstract B070: Characterizing breast cancer etiologic subtypes in the Ghana Breast Health Study

Abstract Background: Breast cancer subtyping has largely been based on immunohistochemical markers of estrogen receptor status. However, RNA-based intrinsic subtypes, including luminal (hormone receptor-positive) and non-luminal (hormone receptor-negative) have been shown to be etiologically relevant, with studies also suggesting other tumor markers (e.g., TP53 status) to be important contributors to etiologic heterogeneity. Compared to luminal tumors, non-luminal tumors are more likely to be TP53 mutant than wildtype-like. TP53 mutant breast cancers are associated with more aggressive tumor features and these mutations are more common among cases of African descent. However, few RNA-based subtyping studies have described etiologic heterogeneity in indigenous African populations. Therefore, we evaluated associations between established breast cancer risk factors and risk of PAM50 intrinsic subtypes and tumor subtypes defined by RNA-based TP53 functional status in the population-based Ghana Breast Health Study (GBHS). Methods: We analyzed data from 600 invasive breast cancer cases and 2,528 controls in the GBHS. RNA was extracted from formalin-fixed paraffin-embedded tumor samples and run on the nCounter® Breast Cancer 360™ Panel. Samples were then classified for intrinsic subtype (N=285 luminal, N=315 non-luminal) using a research version of the PAM50 assay and for TP53 functional status (N=333 wildtype-like, N=300 mutant-like) using a validated RNA signature. Odds ratios (OR) and 95% confidence intervals (CI) were estimated for risk factors using polytomous logistic regression models adjusted for potential confounders. Results: About 1 in 4 tumors classified as ER-positive by immunohistochemistry were non-luminal by RNA expression. Similarly, about 1 in 5 ER-negative tumors were reclassified as luminal. Etiologic heterogeneity was observed by luminal subtype for certain reproductive factors. Higher parity (≥3 vs. &amp;lt;3 births) was associated with lower risk of luminal tumors (OR [95% CI] = 0.68 [0.49, 0.92]), but not with non-luminal tumors (0.95 [0.69, 1.30]). Likewise, later age at first birth (≥26 vs. &amp;lt;19 years) was more strongly associated with risk of luminal than non-luminal tumors (2.16 [1.37, 3.40] and 1.30 [0.84, 2.00], respectively). Consistent with previous findings, TP53 status was also an important etiologic factor and risk factor patterns mirrored those for luminal/non-luminal subtypes. Finally, we evaluated cross-classification of these two etiologic markers to identify risk factors that represent the dual action of both an estrogenic and a DNA-repair pathway. Age at first birth seems to suggest dual action. Conclusions: This study used high-quality RNA expression data to show that intrinsic subtype and TP53 status are useful breast tumor markers for describing etiologic heterogeneity, with cross-classification leading to improved understanding of the etiologic underpinnings of certain risk factors. This work has important implications for the use of gene-expression-based subtyping for breast cancer classification in this population. Citation Format: Amber N. Hurson, Ebonee N. Butler, Alina M. Hamilton, Bernard Petershie, Thomas U. Ahearn, Melissa A. Troester, Jonine Figueroa, Nicolas Titiloye, Montserrat Garcia-Closas, Mustapha Abubakar. Characterizing breast cancer etiologic subtypes in the Ghana Breast Health Study [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr B070.

Abstract C078: Pembrolizumab response in melanoma patients: an analysis of the tumor microenvironment

Abstract Background: Melanoma has been associated with poor prognosis and limited treatment options. Immunotherapy, particularly the use of immune checkpoint inhibitors, has revolutionized the treatment landscape for advanced melanoma. Pembrolizumab, a PD-1 inhibitor, has shown promising results; however, the underlying mechanisms of its efficacy and the influence of the tumor microenvironment (TME) on treatment response remain incompletely understood. This study aims to investigate the response to pembrolizumab in melanoma patients and analyse the TME using a multiplexed immunofluorescence (mIF) spatial tissue analysis to gain insights into potential predictive factors and therapeutic targets. Methods: A retrospective analysis was conducted on formalin-fixed paraffin-embedded (FFPE) tumour samples from baseline melanoma patient samples, classified as responders (n = 6) or non-responders (n = 4) based on the response at 3 months post pembrolizumab treatment. Analysis was done using the mIF platform MultiOmyx™, a proprietary, immunofluorescence (IF) multiplexing assay that enables visualization and characterization of up to 60 biomarkers on a single FFPE section. Image segmentation, cell phenotyping, and spatial analytics were performed using our AI-powered image analytics software, NeoLYTX™. Results: Analysis of the 10 FFPE samples from metastatic melanoma patients treated with pembrolizumab revealed significant differences in immune cell populations between non-responders and responders. Patients who responded favorably to pembrolizumab exhibited increased infiltration of cytotoxic T lymphocytes (CTLs) and tumor-associated macrophages within the TME. This finding suggests that an immune-inflamed microenvironment, characterized by high levels of tumor-infiltrating lymphocytes may be indicative of a robust antitumor immune response. Moreover, immune checkpoint molecules PD-1, CTLA-4, LAG3, and TIM3 were also significantly increased in responding patients, suggesting potential therapeutic targets to enhance pembrolizumab response, including combination strategies targeting multiple immune checkpoints. When spatially characterizing T cell activation and exhaustion status in different regions of the TME we found an increase in checkpoint molecules among intra-tumoral CTLs as compared to intra-stromal CTLs. In particular, the fraction of CTLs with quadruple-positive CTLA4+LAG3+PD1+TIM3+ expression was doubled. This suggests an immunosuppressive TME in which CTLs are rapidly exhausted upon reaching the tumor. Work is now ongoing to characterize how this cell population spatially interacts with other immune and tumor cells in the TME. Conclusions: This study provides valuable insights into the response to pembrolizumab in melanoma patients and highlights the crucial role of the TME in determining treatment outcomes. Identification of immune cell subsets, and immunosuppressive mechanisms associated with pembrolizumab response may guide the development of novel therapeutic approaches to improve patient outcomes in advanced melanoma. Citation Format: Anna Juncker-Jensen, Juliana Wortman, Nigel Beaton, Kristina Beeler, Tobias Treiber, Mitchell Levesque, Julia Martinez Gomez, Jakob Vowinckel, Harry Nunns. Pembrolizumab response in melanoma patients: an analysis of the tumor microenvironment [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C078.

Unraveling the Transcriptional Landscape of Nodular Lymphocyte-Predominant Hodgkin Lymphoma and T-Cell/Histiocyte Rich Large B-Cell Lymphoma: Impact of Tumor Microenvironment and Checkpoint Gene Expression

Introduction: Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a rare B-cell malignancy with a paucity of malignant cells embedded in a diverse tumor microenvironment (TME). The histological composition of the TME is known to influence outcomes, with nodular B-cell rich TME (classical histology, Fan patterns A&amp;B) associated with indolent clinical course, while T-cell infiltration or diffuse growth (variant histology, Fan patterns C-F) may resemble aggressive T/cell-histiocyte rich large B-cell lymphoma (THRLBCL) to which NLPHL can transform. Molecular features of NLPHL and THRLBCL remain to be discovered. To gain further insights into the biology of these lymphomas, we recruited NLPHL and THRLBCL cases as part of the Atlas of Blood Cancer Genomes (ABCG) initiative, a consortium consisting of 26 institutions. Design: We collected comprehensive clinicopathological, treatment and outcome data from 154 NLPHL and 21 THRLBCL patients, with centralized pathology review performed by a panel of dedicated hematopathologists. Fan patterns of the NLPHL samples were documented in all samples where possible. RNA sequencing was performed on formalin-fixed paraffin-embedded (FFPE) diagnostic tumor samples (n=129 NLPHL; n=17 THRLBCL). Tumor-infiltrating immune cell compositions were enumerated using FARDEEP with signature matrix LM22 from CIBERSORT. Results: Median age at diagnosis for the patients with THRLBCL and NLPHL was 54 years (interquartile ratio [IQR] 43-65) and 40 years (IQR 24-53), respectively. The majority of patients with NLPHL had limited stage (74%), whereas the patients with THRLBCL predominantly had advanced stage at diagnosis (95%). In both diseases, most patients were males (NLPHL, 69%; THRLBCL, 76%). Spleen involvement was present in 11 NLPHL patients (8%) and histological growth pattern was documented in 112 patients (73%). Out of those, 47 (42%) presented with variant histology and 65 with classical histology. Variant histology was enriched in NLPHL patients with splenic involvement. During a median follow-up of 6.4 years (IQR 3.8-10.1), 12 NLPHL patients and 9 THRLBCL patients died. Patients with THRLBCL had worse 10-year overall survival (OS, 31% vs 89%; p&amp;lt;0.001) and progression-free survival (PFS, 30% vs 66%; p=0.002) compared to patients with NLPHL. Moreover, NLPHLs with variant histology had inferior 10-year OS compared to NLPHLs with classical histology (81% vs 91%; P=0.03; Figure 1). According to in silico immunophenotyping, THRLBCL cases had the highest median proportions of M1-macrophages (11.0%) and CD8+ T-cells (17.0%), followed by NLPHLs with variant histology (M1-macrophages, 6.5%; CD8+ T-cells, 14.5%), with the lowest proportion observed in NLPHLs with classical histology (M1-macrophages, 5.2%; CD8+ T-cells, 10.9%). In contrast, the median proportions of naïve B cells followed an opposite trend across subtypes (NLPHL classical histology, 15.5%; NLPHL variant histology, 11.0%; THRLBCL, 2.0%), whereas the median proportion of CD4+ T-cells was similar across both lymphomas (NLPHL classical histology, 33.9%; NLPHL variant histology, 33.9%; THRLBCL, 32.4%). Despite differences in the overall transcriptomic landscape, gene expression patterns posited THRLBCL and NLPHL as comprising a biological continuum. THRLBCL shared more similarities with NLPHL with variant histology, while NLPHLs with classical histology exhibited upregulation of genes related to cell growth and proliferation through the MYC pathway (P&amp;lt;0.001). Finally, we identified a strong association between THRLBCL and upregulation of checkpoint genes (P&amp;lt;0.001) and gene expression related to inflammatory response (P&amp;lt;0.001) (Figure 2). Furthermore, NLPHLs with a high checkpoint score (above the 75th percentile) were associated with spleen involvement (P&amp;lt;0.001) and worse outcome (5-year PFS 61% vs 87%; P=0.02). Conclusion: Our study provides the largest comprehensive clinical and transcriptomic analyses of NLPHL and THRLBCL to-date. The findings suggest that the inferior outcomes observed in THRLBCL and in a subset of NLPHL are attributable to a higher degree of inflammation in the TME, increased expression of checkpoint genes, and a high proportion of macrophages.