Formalin-fixed Paraffin-embedded Tissue Samples Research Articles

[Quantitative HER2 mRNA assay in breast cancer with HER2 immunohistochemistry 0].

Objective: To investigate HER2 mRNA expression in breast cancer with HER2 immunohistochemistry (IHC) 0 and to analyze the feasibility of distinguishing between the tumor with HER2 μltra-low expression and the one without expression of HER2 (no staining by IHC) by HER2 mRNA level preliminarily. Methods: HER2 mRNA was analyzed by reverse transcription digital PCR in 41 cases of formalin-fixed paraffin-embedded surgical tissue samples of invasive breast cancer obtained between January 2020 and March 2023 at Peking Union Medical College Hospital. The cohort included 21 HER2 IHC 1+ and 20 IHC 0 (12 ultra-low and 8 non-expression of HER2). HER2 mRNA expression level was quantitatively evaluated by the FAM (HER2)/VIC (reference gene) ratio. Results: The expression of HER2 mRNA for the cases with 1+, ultra-low, and non-expression of HER2 by IHC was 0.30 to 1.78 (average 0.90, median 0.82), 0.55 to 1.51 (average 0.93, median 0.90) and 0.22 to 0.78 (average 0.41, median 0.36), respectively. For the mean and median HER2 mRNA levels, there was no significant difference between HER2 IHC 1+ and HER2 ultra-low expression diseases (P=0.757). A remarkable difference in HER2 gene expression was found between the tumors with 1+ and non-expression of HER2 by IHC (P=0.002). And, HER2 ultra-low cases contained statistically higher levels of HER2 mRNA compared with non-expression of HER2 subgroup by IHC (P=0.001). Conclusions: Based on HER2 mRNA, HER2 non-expression and HER2 weak expression (including HER2 IHC 1+ and ultra-low) belong to two different types of the tumor and the disease with HER2 IHC 1+ and HER2 ultra-low expression may be the same. It is necessary to further test the performance of HER2 mRNA detection for stratifying the HER2 weak expression subgroup and to determine the threshold.

Abstract PO5-01-14: Genomic risk analyses in patients with clinical low risk ER-positive HER2-negative early breast cancer developing an early metastatic event

Abstract Background: Over the past decade, there have been notable changes in the definition of risk estimation in patients with early-stage breast cancer (EBC). Specifically, within the timeframe of 2000 to 2017, following Estrogen Receptor (ER) positive and HER2-negative EBC cases were classified as low risk according to the University Hospitals of Leuven (UHL) criteria of that time: if ER H-score was >200: all patients with tumors N0 and Grade (G) 1, ≤pT2; or G2-3 pT1a-b; in patients ≥40 years with tumors N0 and G2 pT1c-2 or G3 pT1c; in patients ≥ 55 years with tumors N0 G3 pT2 or N1 any Grade, pT≤2. For lower H-scores, other criteria applied. However, despite undergoing endocrine therapy, a subset of these patients with clinically low-risk tumors unexpectedly experienced metastasis within five years of initial diagnosis. This unforeseen outcome may indicate potential undertreatment, as these patients did not receive adjuvant chemotherapy. We have conducted a case-cohort study of those patients with early relapse and performed the current clinical risk assessment methods and genomic risk analyses. Methods: In this study, we included patients with ER-pos, HER2-neg early-stage breast cancer diagnosed between 1-1-2000 and 31-12-2017, who were classified as low-risk tumors by UHL guidelines at that time, who were older than 35 but younger than 71 years, had an unilateral, unifocal breast tumor with tumor size up to 5 cm. These patients did not receive adjuvant chemotherapy. We selected the patients that experienced early distant recurrence, defined as distant recurrence (DR) within 5 years after the initial diagnosis. To ensure comparability for the analyses they were matched to controls for grade, menopausal status, tumor size, lymphovascular invasion, progesterone receptor status, and the year of diagnosis. For each patient, we have calculated the clinical risk score according to the criteria outlined by Mymammaprint.com, based on the modified Adjuvant Online tool used in the MINDACT trial. For the analysis, formalin-fixed paraffin-embedded (FFPE) breast tumor tissue samples were subjected to in-house validated targeted RNA-based Mammaprint(MP) analysis by NGS (MP Agendia). Results: Of the 3190 patients with EBC at UHL during the study period, 2476 patients were considered for this analysis and fitted the clinical low-risk criteria defined low-risk at UHL. Among those, only 42 or 1.7% of the eligible cohort had developed metastasis within 5 years. Tissue for MP analysis was available for 35 of these cases and for their matched control. Due to technical failure, the 1:1 matching was lost and MP results were known for 28 cases and 27 controls. The patient and tumor characteristics of both groups were comparable. In this UHL clinical low-risk group, 21/28 (75%) cases would be identified as clinical high-risk by MyMammaprint.com and 18/27 (67%) controls. We identified 13 patients (46.43%) with high genomic risk within the group with DR and 9 (33.33%) within the control group. There was no statistically significant difference (p=0.412) in the proportion of genomically high-risk patients within the group with DR compared to the control group. Conclusions: In a database of patients with EBC of whom 98% did not develop metastasis within 5 years, we observed in a small series of chemotherapy naïve patients with early DR and UHL-defined clinical low-risk tumors, that nearly half had a genomically high risk of distant metastasis. Albeit numerically higher than the matched-control group, this observation was not significant and would warrant a larger sample size and power to confirm these results. Table. Genomic versus Clinical Risk by MyMammaprint.com Citation Format: Josephine Van Cauwenberge, Hava Izci, Hans Wildiers, Sileny Han, Christine Desmedt, Giuseppe Floris, Sara Vander Borght, Ann Smeets, Ines Nevelsteen, Isabelle Vanden Bempt, Patrick Neven. Genomic risk analyses in patients with clinical low risk ER-positive HER2-negative early breast cancer developing an early metastatic event [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 PO5-01-14.

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.

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.

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.

Enhancing Specific Fluorescence In Situ Hybridization with Quantum Dots for Single-Molecule RNA Imaging in Formalin-Fixed Paraffin-Embedded Tumor Tissues.

Single-molecule fluorescence in situ hybridization (smFISH) represents a promising approach for the quantitative analysis of nucleic acid biomarkers in clinical tissue samples. However, low signal intensity and high background noise are complications that arise from diagnostic pathology when performed with smFISH-based RNA imaging in formalin-fixed paraffin-embedded (FFPE) tissue specimens. Moreover, the associated complex procedures can produce uncertain results and poor image quality. Herein, by combining the high specificity of split DNA probes with the high signal readout of ZnCdSe/ZnS quantum dot (QD) labeling, we introduce QD split-FISH, a high-brightness smFISH technology, to quantify the expression of mRNA in both cell lines and clinical FFPE tissue samples of breast cancer and lung squamous carcinoma. Owing to its high signal-to-noise ratio, QD split-FISH is a fast, inexpensive, and sensitive method for quantifying mRNA expression in FFPE tumor tissues, making it suitable for biomarker imaging and diagnostic pathology.

DETERMINING GOOD DNA QUALITY OF FFPE CRC TISSUES VIA A MACRODISSECTION-DNA EXTRACTION PROTOCOL

A good DNA quality and quantity from a formalin-fixed paraffin-embedded (FFPE) tissue sample is crucial for a better downstream genomic analysis. Previous research has focused on comparing FFPE DNA extraction kits, but the improvement in kit outcome has yet to be studied. This study aimed to make some modifications to a selected Qiagen DNA extraction manual protocol using a manual macrodissected FFPE colorectal cancer (CRC) tissue sample. A variety of DNA extraction protocol-related variables, such as washing steps, tissue sections, and FFPE tissue age have been investigated to determine the DNA quality and quantity of the FFPE tissues. The prechilled absolute ethanol washing step showed the highest DNA concentration with good purity ratios. One, two, or four tissue sections using the washing step were sufficient to obtain adequate DNA concentrations with acceptable DNA purity ratios. 1% or 2% agarose gel electrophoresis with the pre-casting method allowed better visualisation of FFPE DNA. Based on the ideal quality and quantity, we chose a manual macrodissected DNA extraction protocol employing 4 x 10 µm FFPE tissue sections generated in recent years with an optimised prechilled absolute ethanol washing step.

Multi-omic profiling of simultaneous ductal carcinoma in situ and invasive breast cancer.

The progression of ductal carcinoma in situ (DCIS) to invasive breast carcinoma (IBC) in humans is highly variable. To better understand the relationship between them, we performed a multi-omic characterization of co-occurring DCIS and IBC lesions in a cohort of individuals. Formalin-fixed paraffin-embedded tissue samples from 50 patients with co-occurring DCIS and IBC lesions were subjected to DNA-seq and whole transcriptome RNA-seq. Paired DCIS and IBC multi-omics profiles were then interrogated for DNA mutations, gene expression profiles and pathway analysis. Most small variants and copy number variations were shared between co-occurring DCIS and IBC lesions, with IBC exhibiting on average a higher degree of additional mutations. However, 36% of co-occurring lesions shared no common mutations and 49% shared no common copy number variations. The most frequent genomic variants in both DCIS and IBC were PIK3CA, TP53, KMT2C, MAP3K1, GATA3 and SF3B1, with KMT2C being more frequent in DCIS and TP53 and MAP3K1 more frequent in IBC, though the numbers are too small for definitive conclusions. The most frequent copy number variations were seen in MCL1, CKSB1 and ERBB2. ERBB2 changes were not seen in IBC unless present in the corresponding DCIS. Transcriptional profiles were highly distinct between DCIS and IBC, with DCIS exhibiting upregulation of immune-related signatures, while IBC showed significant overexpression in genes and pathways associated with cell division and proliferation. Interestingly, DCIS and IBC exhibited significant differential expression of different components of extracellular matrix (ECM) formation and regulation, with DCIS showing overexpression of ECM-membrane interaction components while IBC showed upregulation of genes associated with fibronectin and invadopodia. While most co-occurring DCIS and IBC were mutationally similar and suggestive of a common clonal progenitor, transcriptionally the lesions are highly distinct, with IBC expressing key pathways that facilitate invasion and proliferation. These results are suggestive of additional levels of regulation, epigenetic or other, that facilitate the acquisition of invasive properties during tumor evolution.

Abstract 6840: Automated preparation of single nuclei from FFPE samples for snRNA-Seq

Abstract S2 Genomics developed the Singulator™ 100 and 200 Systems to automate the processing of fresh, frozen, or OCT tissues into single cell or single nuclei suspensions using disposable cartridges, reagents, and customizable protocols. The systems have been used to prepare cells or nuclei from a wide range of tissues and organisms, including human, mouse, chicken, insects, snails, zebrafish, and plants. Formalin-fixed, paraffin-embedded (FFPE) tissues are the preferred format for pathologists and as many as one billion FFPE slides are in repositories. FFPE tissue samples have traditionally been difficult to study with molecular techniques due to the chemical modifications caused by the formalin fixation, which can lead to degradation and sequestration of RNA and DNA. Recent advances in single cell/nuclei RNA-Seq library preparation technology have made it possible to overcome these challenges and generate high-quality single cell or nuclei genomic data from FFPE samples. Protocols have been released for processing previously deparaffinized and rehydrated FFPE samples on the Singulator 100 or 200 into single nuclei. We now report the development of a fully automated workflow for the deparaffinization and rehydration of FFPE tissues in about 40 min, with an optional enzymatic treatment of 45 min, and processing of FFPE tissues into singulated cells or nuclei on prototype Singulator 200 systems. Data will be presented for the optimization of process parameters for cell or nuclei recovery and RNA quality for six tissues. Finally, single nuclei sequencing data will be presented for snRNA-Seq libraries from healthy and cancerous human FFPE samples including glioblastomas and colorectal cancer. These data demonstrate the successful recovery of genomic materials from FFPE samples using an automated sample preparation system for single cell/nuclei transcriptomics. Citation Format: Stevan Jovanovich, Nate Pereira, Danielle Meyer, Catherine Snopkowski, Ignas Masilionis, Brigita Meškauskaitė, Meril Takizawa, Zaki Abou-Mrad, Kenny Yu, Viviane Tabar, Kathleen Luckett, Karuna Ganesh, Nabiha Khan, Ronan Chaligne. Automated preparation of single nuclei from FFPE samples for snRNA-Seq [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 6840.

Abstract 6399: Real-world experience of an Asian pan-cancer combined DNA/RNA next generation sequencing (NGS) tissue biopsy assay

Abstract Introduction: Comprehensive molecular profiling with combined DNA-RNA next-generation sequencing (NGS) on tumor tissue is now increasingly being used as standard-of-care for sequence therapy selection to improve cancer survival. Combined DNA-RNA NGS is also considered standard-of-care for central nervous system (CNS) tumor classification. Despite the genetic heterogeneity and diversity of cancers, most commercial combined DNA/RNA NGS assays are validated in Western populations, resulting in knowledge gaps in patient outcomes. We report here for the first time a range of actionable outcomes from a comprehensive DNA/RNA tissue NGS assay designed specifically to probe Asian-prevalent cancers and biomarkers in an international multicenter real-world study in Asia. Methods: 1,002 formalin-fixed paraffin-embedded tissue samples from 995 Asian cancer patients across &amp;gt;70 centers in 7 countries and/or territories underwent real-world DNA/RNA NGS testing in a CAP-accredited CLIA-certified laboratory. Genomic alterations including SNVs, INDELS, gene fusions, CNVs, TMB, and MSI were analyzed using an Asian-centric pan-cancer hybrid-capture NGS panel that comprehensively profiles 572 cancer-related genes and 91 RNA fusion partners (UNITED). Biomarkers were considered actionable if the therapeutic indication in any solid tumor or hematological cancer was FDA-approved, guidelines-recommended, or supported by well-powered phase III clinical trials. Results: 27 cancer types were represented in 1,002 tissue samples, with lung, breast, colorectal, and prostate cancers comprising 45.5% of clinical volume. 79.5% of samples harbored ≥1 actionable biomarker, with the most frequently altered genes being TP53, KRAS, PIK3CA, EGFR, and NF1. Overall, 42.6% of samples had ≥1 biomarker with an indication in the patient’s cancer type, including 81.5% of lung, 59.0% of breast, 61.8% of colorectal, and 42.1% of prostate cancers. Across the pan-cancer cohort, 13.3% was TMB-high and 1.9% was MSI-high. 26.3% of samples were from cancer types outside the original assay design; the top 3 were pancreatic cancer (7.3%), sarcoma (3.3%), and CNS tumor (3.1%). Cancers of unknown primary (CUP) also constituted a significant proportion (3.8%). In CNS tumors, 38.7% harbored an actionable mutation in IDH1, BRAF, or H3-3A. In CUP, 78.8% harbored an actionable mutation; 50% of samples harbored biomarkers that additionally informed potential tumor origin, including mutations in EGFR, MET, ALK, ROS1, and BRCA1. Conclusion: The use of an Asian-focused pan-cancer DNA/RNA NGS panel resulted in detection of actionable biomarkers in 79.5% of clinical cases. These findings support the utility of DNA/RNA NGS assays in informing standard-of-care therapeutic decisions in Asian cancer patients for maximizing cancer survival. Citation Format: Jason Yongsheng Chan, Jing Yi Lee, Zi Yi Wan, Abner Herbert Lim, Tun Kiat Ko, Cedric Chuan-Young Ng, Donald Poon, Jens Samol, Tsz Him So, Su Pin Choo, Ravindran Kanesvaran, Cheng-Vai Hui, Joseph Siu-Kie Au, Aya El Helali, Timothy Yip, Vikneswari Rajasegaran, Sandy Lim, Ruifen Weng, Puay Hoon Tan, Bin Tean Teh, Min-Han Tan, Jonathan Poh. Real-world experience of an Asian pan-cancer combined DNA/RNA next generation sequencing (NGS) tissue biopsy assay [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 6399.

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

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

Abstract 7028: Optimization of DNA extraction methods and DNA methylation array quality from FFPE prostate tumor tissues to identify DNA methylation biomarkers

Abstract Prostate cancer is the second leading cause of cancer related deaths among men in the United States. Health disparities in men diagnosed with prostate cancer are observed between patients of African and those of European ancestry. To elucidate molecular mechanisms behind prostate tumorigenesis and racial disparities, researchers have identified genetic alterations such as gene fusions, deletions, and mutations that occur at varying frequencies among different ethnic groups. However, results are inconsistent across studies, suggesting that racial disparities may be multifactorial. Our approach to further understand the mechanisms underlying racial disparities is to compare DNA methylation profiles between cancers from different ethnic groups using formalin-fixed paraffin-embedded (FFPE) prostate cancer tissue samples. For this study, FFPE prostate cancer specimens from 32 Black men and 30 age-matched White men were obtained in the form of tissue sections on glass slides. To profile DNA methylation in these samples, tumor and normal tissue regions (in a subset of cases) were outlined on a hematoxylin &amp; eosin (H&amp;E) reference slide and the respective regions were macro-dissected off the corresponding unstained slides from the same block. The areas of macro-dissected regions were measured using ImageJ, and the number of slides were taken to normalize DNA yield. Next, we compared magnetic bead and column based DNA extraction methods. We evaluated them on their relative DNA yield per tissue size, and identified that the bead-based extraction method had both higher and more consistent DNA yield. In total, we used 95 unique tissue regions to profile global DNA methylation levels using Illumina Epic array. We found that a subset of samples harbored an aberrant DNA methylation pattern. Therefore, we extracted DNA from additional FFPE tissue slides using the bead-based method and repeated the assay with a larger amount of DNA, successfully rescuing the majority of the aberrant samples. Next, we will evaluate associations between DNA methylation levels in different ethnic groups, clinicopathological features, and follow-up data to help identify potential DNA methylation biomarkers and therapeutic targets linked to prostate cancer from different ethnicities. Citation Format: Colton Duran Stensrud, Leonardo Gonzalez-Smith, Claire Stevens, Huan Cao, Jenaye Mack, Olumide Arigbede, Daniel Weisenberger, Sarah G. Buxbaum, Sara M. Falzarano, Suhn Rhie. Optimization of DNA extraction methods and DNA methylation array quality from FFPE prostate tumor tissues to identify DNA methylation biomarkers [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 7028.

Abstract 1154: Spatial profiling of the tumor microenvironment in head and neck squamous cell carcinoma revealed immune cell type infiltration into the TME as a predictor of immunotherapy response

Abstract Background: Immunotherapy has shown promising results in advanced head and neck cancer patients, however, only a subset of patients are responsive to immune checkpoint blockade therapy. The molecular and cellular characterization of the tumor microenvironment (TME) may reveal clues as to why some head and neck tumors are responsive or resistant to the therapy. Methods: Formalin-fixed paraffin-embedded (FFPE) tissue samples were collected from 34 patients at two major Queensland Hospitals with advanced head and neck squamous cell carcinoma (HNSCC), prior to immunotherapy. To investigate the cellular and molecular characteristics of the HNSCC TME, we used the Nanostring GeoMx Digital Spatial Profiler (DSP) spanning modules in an immune-oncology panel, which included immune cell typing and profiling, cell death, PI3K/AKT signaling, drug target, immune activation, and pan tumor protein biomarkers. A deeper structural analysis was performed using the Nanostring whole transcriptome atlas (WTA) panel of tertiary lymphoid structures (TLSs) and germinal centers (GCs). Results: Out of 34 patients, 3 had a complete response (CR), 7 had a partial response (PR), 5 had stable disease (SD), and 19 had progressive disease (PD), based on the Response Evaluation Criteria in Solid Tumors (RECIST). When we compared patient responders (patients with CR, PR, and SD) to patient non-responders (patients with PD), we found that the former had a higher infiltration of immune cell type markers representatives of CD8+ T cells, dendritic cells, and macrophages, into their tumor microenvironment than the latter. Then, to investigate differentially expressed genes between patients with complete response and patients with partial response to immunotherapy, we performed DE analyses and we found that patients with PR had higher Ki-67 expression than those with CR. Spatial transcriptomic mapping of TLS’s (peripheral, intra-tumoral) and normal germinal centers revealed interferon pathway enrichment in the TLS’s. In a subset, these appear to be linked with HPV viral infection. Conclusion: In this study, we found that immune cell type infiltration into the tumor microenvironment of head and neck cancers could be predictive of response to immunotherapy. Furthermore, it was revealed that patients with a high expression of Ki-67 might be less likely to respond completely to immunotherapy. Spatially resolved whole transcriptome analysis of HNSCC tissues revealed the presence of tertiary lymphoid structures with a higher enrichment of interferon pathway genes. Citation Format: Habib Sadeghirad, Chin Wee Tan, Ning Liu, James Monkman, Caroline Cooper, Ken O’Byrne, Melissa Davis, Brett Hughes, Arutha Kulasinghe. Spatial profiling of the tumor microenvironment in head and neck squamous cell carcinoma revealed immune cell type infiltration into the TME as a predictor of immunotherapy response [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 1154.

Abstract 6149: Polygenic risk score and lung adenocarcinoma risk among never-smokers by EGFR mutation status

Abstract Background: Lung cancer is the leading cause of cancer mortality worldwide, and incidence rates for the disease in never-smokers is among the highest in East Asian (EAS) women. Epidermal growth factor receptor (EGFR) is a transmembrane protein that regulates cellular proliferation and apoptosis, and mutations in the EGFR gene have been found to be a defining hallmark of lung adenocarcinoma (LUAD). We investigated if overall genetic susceptibility to LUAD, defined as a polygenic risk score (PRS), is differentially associated with LUAD by EGFR mutation status. Methods: The study consists of 998 female never-smoking histologically confirmed LUAD cases with data on EGFR mutation status and 4,544 female never-smoking controls from the Female Lung Cancer Consortium in Asia. Germline DNA samples were genotyped using the 370K, 610Q, or 660W microarrays. Genomic DNA extracted from fresh, frozen or formalin-fixed paraffin-embedded tumor tissue samples of LUAD cases were genotyped for EGFR mutations on exons 19 and 21. We defined cases with EGFR mutation on either exon as EGFR+ (n=518) and cases without such EGFR mutation as EGFR- (n=480). Weights from 942,592 single nucleotide variants derived from a previously conducted genome-wide association study were used in a Bayesian-based approach, LDpred2, to derive a PRS for all participants. We estimated the odds ratios (OR) and 95% confidence intervals (CI) for the association between continuous PRS, PRS quartiles and tumor EGFR mutation status using logistic regression models in a case-case analysis, as well as using a multinomial logistic regression treating controls as the reference. All models were adjusted for age, study and the top 10 principal components. Results: In case-case comparisons, compared to EGFR- LUAD, we found a positive association between continuous PRS and risk of EGFR+ LUAD (OR=1.17, 95% CI: 1.02, 1.34), as well as a dose-response relationship between quartiles of the PRS and EGFR+ LUAD (p-trend=0.003). We further found that the association between the fourth quartile of the PRS with EGFR+ LUAD (OR=8.63, 95% CI: 5.67, 13.14) was significantly higher than the association between the fourth quartile of the PRS with EGFR- LUAD (OR=3.50, 95% CI: 2.44, 5.00) compared to controls (p-heterogeneity=0.004). Conclusions: We found that the PRS developed for LUAD in EAS individuals was more strongly associated with EGFR+ LUAD compared to EGFR- LUAD, suggesting that germline genetic susceptibility may be differentially associated with LUAD in never-smoking female EAS patients depending on the cancer’s mutation status. Given that patients with LUAD respond differently to treatments that are used as targeted therapy depending on their EGFR mutation status, our findings may have important public health and clinical implications, which may guide risk stratification, screening, and treatment. Citation Format: Batel Blechter, Chao Agnes Hsiung, Keitaro Matsuo, Kouya Shiraishi, Kevin Wang, Haoyu Zhang, Wei Jie Seow, Jianxin Shi, Nilanjan Chatterjee, Jason Y.Y. Wong, Juncheng Dai, H. Dean Hosgood, I-Shou Chang, Jiyeon Choi, Wei Hu, Wei Zheng, Young Tae Kim, Xiao-Ou Shu, Qiuyin Cai, Pan-Chyr Yang, Dongxin Lin, Kexin Chen, Yi-Long Wu, Hongbin Shen, Takashi Kohno, Stephen J. Chanock, Nathaniel Rothman, Qing Lan. Polygenic risk score and lung adenocarcinoma risk among never-smokers by EGFR mutation status [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 6149.

Abstract 6169: High throughput in-situ spatial sequencing of proteins and RNA in FFPE tissue

Abstract We have developed an high throughput high resolution in situ multi-omic platform to enable studies in the rapidly evolving landscape of oncology and immunology research. Despite the potential of existing tools, the speed and throughput of current methodologies pose significant limitations. Here, we introduce a novel spatial sequencing platform employing a rapid 4-color SBS chemistry with sub-micron resolution imaging and an ultra-high throughput capacity. The platform simultaneously profiles RNA transcripts and proteins within formalin-fixed paraffin-embedded (FFPE) tissues. With up to 40 cm^2 of tissue area imaging within a 24-hour timeframe, the platform can power extensive, multi-omic studies in cancer research. To evaluate the performance of the platform, we prepared 5 um serial sections from healthy and tumor kidney FFPE block and used a novel tool to precisely position multiple tissue sections on a slide. Following probe binding and amplification, we used SBS readout to profile 105 transcripts and 6 proteins (via DNA-conjugated antibodies). Images were analyzed using a custom pipeline for feature detection and decoding, and cell segmentation using Cellpose. Results showed strong correlation to single-cell transcription profiles and cell type signatures defined by scRNA-Seq. Additionally, we see good agreement between single cell transcript and protein profiles. Existing spatial biology platforms are known to struggle with degraded and challenging clinical sample types (e.g. decalcified bone marrow), due to RNA fragmentation. We successfully profiled immune cells in FFPE-embedded bone marrow biopsies, measuring 143 transcript targets and 8 proteins. We believe this represents a significant step forward for in-situ analysis of bone marrow samples, and could be valuable for studying the pathology and response to treatment in blood cancers such as AML and CML. In-situ sequencing of antibody genes and T-cell receptors provides a deeper view of cellular immune response. As a step towards this goal, we demonstrated in-situ sequencing of antibody heavy and light chains within a B cell line, resulting in accurate readout of 28 bases within the CDR3 regions. In conclusion, we present an SBS-based approach for in-situ detection of targeted gene transcripts and proteins at an unprecedented throughput, and its application to the analysis of human kidney and bone marrow FFPE tissue samples. The capability to deeply profile tissues at high throughput could enable larger translational studies, and assembly of 3D maps of gene and protein expression at sub-cellular resolution. Citation Format: Michael Lawson, Yuji Ishitsuka, Andrew Pawlowski, Zhenmin Hong, Jake Koh, Kenneth Gouin, Nathan Ing, Richard Que, Yeoan Youn, Sara Ding, Tony Facchini, Ryan Costello, Katelyn Nelson, Jamie Stover, Howon Lee, Sarthak Duggal, Taylor Plaziak, Kaitlin Cameron, Mimi Abdu, Daan Witters, Martin Fabani, Eli Glezer. High throughput in-situ spatial sequencing of proteins and RNA in FFPE tissue [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 6169.

Abstract 1160: Investigating spatial gene expression profiling in colorectal cancer: Tumor and stroma comparison between primary lesions and matched liver metastases

Abstract The nanoString GeoMx® Digital Spatial Profiling (DSP) enables the investigation of spatial assessment of tumors through high-plex profiling at the RNA and protein levels. Recent genomic analyses have revealed the intertumor heterogeneity between primary and metastatic lesions in colorectal cancer (CRC) patients. In CRC with liver metastases, current treatment strategies are mainly based on the parameters of primary tumors, and metastatic heterogeneity is a challenge since molecular heterogeneity contributes to therapeutic resistance. Furthermore, spatial intratumor heterogeneity exists within a single tumor between cancer cells (tumor) and their microenvironment (stroma) in human cancers. However, whether there are distinct spatial gene expression patterns of tumor and stroma between primary lesions and liver metastases remains unclear in CRCs. We examined 24 formalin-fixed paraffin-embedded tissue samples, including primary lesions and matched liver metastases from 12 patients (6 with synchronous and 6 with metachronous metastases) using NanoString GeoMx® DSP. The best regions of interest (ROI) with the invasive front boundary of the tumor were selected by pathologists. The ROI was segmented into PanCK-positive (tumor) and PanCK-negative (stroma), followed by a collection of indexed oligonucleotides and sequencing on Illumina instrument. Differential expression and pathways enrichment analyses were performed using R BioConductor package standR, limma, and GSEABase. Statistical significances were based on |Log2 fold change| &amp;gt; 1 and Benjamini-Hochberg-corrected P &amp;lt; 0.05. Immune cell abundance was estimated using the SpatialDecon package. In 12 metastatic patients (mean age, 61 years ± 7 [standard deviation]), liver metastatic stroma was associated with 68 upregulated and 93 downregulated genes compared to the primary stroma, with enrichment of 'Immune response' terms in gene set enrichment analysis (GSEA). A higher proportion of CD4 T memory cells and increased expression of cytotoxic genes (IL7R [2.02-fold], CD3E [1.75-fold], KLRB1 [1.68-fold], and GZMK [1.54-fold]) were observed in liver stroma compared to primary stroma. Conversely, liver metastatic tumors were associated only with 23 upregulated and 6 downregulated genes compared to primary tumors, with 'humoral response' terms enriched in GSEA. Similar patterns were observed in stratified analyses with synchronous and metachronous metastases, although a more prominent ‘immune response’ was observed in synchronous metastatic stroma compared to the primary stroma. We revealed that higher immune cell proportion and cytotoxic activity were observed in liver metastatic stroma compared to CRC stroma, providing novel insights into a unique etiology and may yield clinical implications for developing targeted treatment modalities for liver metastatic CRC patients. Citation Format: Jongwon Lee, Yeseul Kim, Hyo Seon Ryu, Jongmin Sim, Chungyeul Kim, Jong Min Park, Ah-Reum Lim, Jung Sun Kim, Hwa Jung Sung, Xingyi Guo, Jungmin Choi, Jungyoon Choi. Investigating spatial gene expression profiling in colorectal cancer: Tumor and stroma comparison between primary lesions and matched liver metastases [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 1160.

Prevalence of BK and JC Polyomaviruses among Patients with Breast Cancer.

Breast cancer, a pervasive invasive carcinoma among women globally, afflicts approximately 12% of women worldwide. Previous studies have indicated that certain viruses, including oncogenic viruses such as polyomaviruses BK and JC, may play a role in the development of breast cancer. In light of this, the present study endeavors to assess the incidence of BKV and JCV virus in breast cancer patients. One hundred formalin-fixed paraffin-embedded tissue samples were procured and subjected to deparaffinize by xylene, followed by DNA extraction through the phenol-chloroform methodology. Detection and genotyping of BKV and JCV were carried out utilizing specific primers via PCR analysis. Merely 2 out of 100 (2%) ductal carcinoma in situ with grade 2 specimens exhibited positivity for BK virus genotype IV, whereas JC virus DNA was not discerned across all the samples. The findings of the current investigation demonstrate that there was an absence of JC virus detection in the breast biopsy. Additionally, a small fraction of patients diagnosed with ductal carcinoma exhibited a low prevalence of genotype IV polyomavirus BK at a rate of 2%. However, in order to gain a more comprehensive understanding of the incidence of BKV and JCV in breast cancer, a substantial number of breast samples must undergo investigation.

Using formalin fixed paraffin embedded tissue to characterize the microbiota in p16-positive and p16-negative tongue squamous cell carcinoma: a pilot study

BackgroundTongue squamous cell carcinoma (TSCC) is the most common oral cavity cancer, and p16 immunohistochemistry is an exact and available tool in the prognostic and predictive characterization of squamous cell cancers in the head and neck. Microorganisms have a close relationship with the development of TSCC. However, the association between oral bacteria and p16 status has not been well defined in the case of TSCC. Compared with traditional clinical microbial collection methods, formalin-fixed paraffin-embedded (FFPE) tissue samples have several advantages.MethodsTo compare the microbiota compositions between p16-positive and p16-negative patients with TSCC, we performed a small pilot study of microbiological studies of TSCC by paraffin tissue. DNA from FFPE tissue blocks were extracted and microbiomes were profiled by sequencing the 16 S-rRNA-encoding gene (V1–V2/V3-V4/V4 regions). Alterations in the functional potential of the microbiome were predicted using PICRUSt, Tax4Fun, and BugBase.ResultsA total of 60 patients with TSCC were enrolled in the study, however, some challenges associated with DNA damage in FFPE tissues existed, and only 27 (15 p16-positive and 12 p16-negative) passed DNA quality control. Nevertheless, we have tentatively found some meaningful results. The p16 status is associated with microbiota diversity, which is significantly increased in p16-positive patients compared with p16-negative patients. Desulfobacteria, Limnochordia, Phycisphaerae, Anaerolineae, Saccharimonadia and Kapabacteria had higher abundances among participants with p16-positive. Moreover, functional prediction revealed that the increase of these bacteria may enhance viral carcinogenesis in p16-positive TSCC.ConclusionsBacterial profiles showed a significant difference between p16-positive TSCC and p16-negative TSCC. These findings may provide insights into the relationship between p16 status and the microbial taxa in TSCC, and these bacteria may provide new clues for developing therapeutic targets for TSCC.

Genetic landscape of breast cancer subtypes following radiation therapy: insights from comprehensive profiling.

In breast cancer, in the era of precision cancer therapy, different patterns of genetic mutations dictate different treatments options. However, it is not clear whether the genetic profiling of breast cancer patients undergoing breast-conserving surgery is related to the adverse reactions caused by radiotherapy. We collected formalin-fixed paraffin-embedded (FFPE) tumor tissue samples from 54 breast cancer patients treated with radiation after breast-conserving surgery and identified comprehensive molecular information in hundreds of cancer-associated genes by FoundationOne CDx (F1CDx), a next-generation sequencing (NGS)-based assay. Among our cohort of 54 breast cancer patients, we found high-frequency mutations in cancer-related genes such as TP53 (56%), RAD21 (39%), PIK3CA (35%), ERBB2 (24%), and MYC (22%). Strikingly, we detected that the WNT pathway appears to be a signaling pathway with specific high-frequency mutations in the HER2 subtype. We also compared the mutation frequencies of the two groups of patients with and without cutaneous radiation injury (CRI) after radiotherapy and found that the mutation frequencies of two genes, FGFR1 and KLHL6, were significantly higher in patients with CRI : No subgroup than in those with CRI : Yes. Different breast cancer subtypes have their own type-specific mutation patterns. FGFR1 and KLHL6 mutations are protective factors for radiation-induced skin toxicity in breast cancer patients.

Expression of PD-L1 clones (22C3 and 28-8) in hepatocellular carcinoma: a tertiary cancer care hospital experience

BackgroundHepatocellular carcinoma (HCC) is a highly aggressive and rapidly progressing form of cancer with a poor prognosis. Recent advances in the management of HCC focused on the novel immunotherapeutic modalities for patients with advanced disease. PD-L1 has emerged as a promising immunotherapeutic approach for HCC. The evaluation of PD-L1 expression aids in identifying patients who can derive maximum benefits from these therapies. This study aims to examine and compare the expression of PD-L1 using two clones (22C3 and 28-8) in HCC patients.MethodsForty-six patients with HCC were selected between 2005 and 2022 from the Shaukat Khanum Memorial Cancer Hospital and Research Centre (SKMCH&RC) in Lahore, Pakistan. The patients' formalin-fixed paraffin-embedded (FFPE) tissue samples were retrieved from the department of pathology to conduct immunohistochemical analysis. Moreover, the clinicopathological data of these patients were gathered from the hospital information system (HIS). To assess the relationship between variables, bivariate analysis was carried out using either the chi-square test or Fisher exact test when necessary.ResultsAmong the 46 tissue specimens analyzed, the presence of clone 22C3 was detected in 20 HCC patients, with 10 patients showing high expression (21.7%) and another 10 patients showing low expression (21.7%). 22C3 expression was not observed in 26 patients (56.5%). On the other hand, clone 28-8 was expressed in 10 patients, all of whom exhibited low expression (21.7%), while no expression of clone 28-8 was observed in 36 patients (78.3%). An association was found between the expression of 22C3 and 28-8 PD-L1 clones (p-value 0.01). Furthermore, upon closer examination, it was revealed that 12 cases exhibited positive results for 22C3 but negative results for 28-8. Interestingly, two cases displayed positive results for 28-8 but negative results for 22C3.ConclusionWe obserevd that the PD-L1 clones, 22C3 and 28-8, are comparable. If PD-L1 expression using 22C3 is negative, considering the use of 28-8 for evaluating expression in HCC patients may be beneficial. However, further validation in a larger cohort is necessary.