Formalin-fixed Paraffin-embedded Slides Research Articles

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 < 0.001) and high Ki67 (≥ 10% positive cells; p = 0.013)at baseline. ctDNA density at baseline was significantly higher for resistant (PFS < 9 months; 17.1 ng/mL) pts compared to sensitive (PFS > 31 months; 11.3 ng/mL) pts (p < 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 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 6193: Exploring interplay between tumor intrinsic features and immune cell phenotypes in multi-tumor tissue microarrays by a single step 15-plex fluorescence immunohistochemistry and imaging on the Orion platform

Abstract Purpose: Deeper understanding of the intricacies between tumor microenvironments and immune cell infiltrates is critical for creation of next generation therapies. However, current immunohistochemistry protocols in clinical practice are restricted to one to six markers (cell types) and rely on multiple rounds of antigen retrieval and sequential staining over two days. This current workflow has high risk for tissue and signal loss. Hence, we explored utility of a single step staining workflow using 15 distinct antibodies conjugated novel flours (Argo Dye System) of formalin fixed paraffin embedded (FFPE) tumor microarrays and imaged on the Orion microscope (Rarecyte). Fidelity of the staining was assessed by comparing data from the Orion platform to internally developed clinical grade assays. The implementation of higher plex imaging will enable greater insight into immune surveillance, mechanisms of resistance and patient stratification while minimizing patient tissue required for analysis. Study Design: We utilized a custom panel developed by RareCyte (Hoescht, CD3e, CD4, CD8a, CD20, CD68, CD163, FOXP3, Ki67, Pan-CK, PD1, PDL1, CD45, Granzyme B, LAG3) to evaluate expression of immune markers on a pan-tumor and melanoma TMA including Hodgkin’s lymphoma, ovarian cancer, esophageal carcinoma, hepatic cell carcinoma, head and neck cancer. Serial sections were stained with 6-plex clinical grade assays, and correlation scores were determined between the 15-plex Orion and 6-plex assays utilizing TSA-based amplification. Results: In this study, the performance of Orion platform was assessed by quantitative and qualitative measures. Linear regression between the 6-plex clinical grade assays and 15-plex Orion workflow were calculated for a panel of biomarkers including CD3, CD4, CD8, CD68, CD163, CK, FOXP3, GZMB, Ki67, and PD1. Our results demonstrated that the Orion performed similarly to clinical grade assays both in terms of proportion of cells and fluorescence intensity (R2 ~ 0.7-0.98, Slope ~ 0.7-1.1). Conclusion: Our results suggest that the Orion platform offers a reliable and efficient solution for comprehensive biomarker analysis. Our results demonstrate that this platform can successfully capture the expression of 15 biomarkers on a single FFPE slide which offers workflow advantages over traditional TSA-based fIHC multiplexing that is limited to detection of 6 biomarkers on a single slide. The ability to analyze a wider range of biomarkers through a single platform has the potential to enhance the efficiency of biomarker analysis, leading to improved diagnostic and therapeutic outcomes in clinical practice. Citation Format: Richard Van Krieken, Jehovana O. Bender, Sophia Kekchidou, Michelle Burgin, Kristen Ruma, Naveen Dakappagari, Jennifer Bordeaux. Exploring interplay between tumor intrinsic features and immune cell phenotypes in multi-tumor tissue microarrays by a single step 15-plex fluorescence immunohistochemistry and imaging on the Orion platform [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 6193.

Abstract 4413: Identifying genome-wide histone profiles in patient derived models of AML for predictive biomarker development

Abstract Acute myeloid leukemia (AML) is a complex disease with highly individualized treatment plans for patients. Mostly these are based on genetic and phenotypic markers of the disease, but the patient’s overall condition is playing a significant role as well. To develop innovative drugs for a broad patient cohort, it is essential to understand the biological implications of these differences and to model them preclinically. In this study, we are assessing the utility of EpiCypher’s CUT&Tag-FFPE platform to characterize the epigenome of AML PDX samples. CUT&Tag-FFPE is an epigenome profiling assay that is compatible with Formalin-Fixed Paraffin-Embedded (FFPE) samples, enabling robust genome-wide profiling of histone modifications from limited clinical samples. We derived PDX model LEXFAM 2799 (NPM1 wt; FLT3 wt, t(8;21)) and LEXFAM 2966 (NPM1 mut; FLT3 wt) from treatment naïve patients and established in immunocompromised NSG mice. LEXFAM 2799 is a fast-growing model with passaging times of 33 days, whereas LEXFAM 2966 displays a passaging time of 140 days. We next treated with Standard of Care (SoC) agents Decitabine, Cytarabine and all-trans-retinoic acid (ATRA): LEXF 2966 was highly sensitive towards Decitabine leading to complete remission. Cytarabine induced a partial remission and ATRA had only minor effect on tumor growth. In contrast LEXFAM 2799 was resistant towards treatment with ATRA and showed only partial remission under treatment with Cytarabine or Decitabine. Using the CUT&Tag-FFPE platform we are analyzing FFPE slides from the above mentioned two AML PDX models to investigate differences in the genome-wide histone profile that relate to the different growth behavior as well as sensitivity towards SoC treatment. The goal of these studies is to identify tumor model specific profiles that correlated specifically with the sensitivity towards the hypomethylating agent Decitabine. In addition, we plan to analyze additional untreated as well as treated AML PD models to validate the identified signatures confirming them as predictive biomarkers for sensitivity towards current standard of care and prospectively as well innovative compounds. Citation Format: Alysha Simmons, Eva Oswald, Georg Kuales, Michael Keogh, Vishnu U. Kumary, Eva Brill, Martis W. Cowles, Bryan Venters, Julia B. Schueler. Identifying genome-wide histone profiles in patient derived models of AML for predictive biomarker development [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 4413.

Characterizing immune-genomic signatures associated with response to immune checkpoint inhibitors (ICI) in patients with metastatic urothelial carcinoma (mUC).

e16582 Background: Response rates with ICI are modest at around 20% in mUC and there is a lack of validated biomarkers to select pts who are likely to derive benefit from ICI. There is an unmet need to develop biomarkers of response and resistance to avoid unnecessary treatment (Tx) and spare physical and financial toxicity. Here we report tumor immune-genomic characteristics associated with response and resistance to ICI in mUC. Methods: We selected 58 mUC pts who received at least 2 cycles of ICI (atezolizumab or pembrolizumab) between 2015-2020 and had pre-Tx archival formalin fixed paraffin-embedded (FFPE) tissues available. Pts were categorized by their response to ICI as responders (R) (Complete Response (CR), Partial Response (PR), and Stable Disease (SD)) (N = 30) or non-responders (NR) (Progressive Disease (PD)) (N = 28). Tumor was macrodissected from FFPE slides and RNA extraction was done using Qiagen Rneasy FFPE kit. 100ng of RNA was used for transcriptomic analysis using the Nanostring PanCancer IO 360 panel. Data normalization and analysis was carried out using nSolver 4.0. Results: Differential expression analysis comparing (R) vs (NR) displayed upregulation of gene signatures associated with Immunoproteasome, Antigen presenting machinery (APM), IFN Gamma, PD-L1, and IFN downstream. Additionally, the tumor inflammation signature (TIS), which is trained to predict response to ICI, was upregulated in (R) vs (NR) (Table 1). Single gene analysis showed trend towards upregulation of ISG15 (logFC = 0.7821, p = 0.00337), TAP1(logFC = 0.7649, p = 0.00422), and STAT1 (logFC = 0.6053, p = 0.00510) in (R), P-values, were above significance threshold after adjustment for multiple comparisons. Receiver operator characteristic (ROC) analysis with area-under the curve (AUC) scores assess capacity of gene signatures to predict response to ICI (Table 1). In our cohort, the median overall survival for (R) was 44.1 months compared to 24.5 months in NR (p = 0.0100). Conclusions: In our cohort of mUC pts treated with ICI, we found an association between signatures of tumor immunogenicity (APM and Immunoproteosome) and anti-tumor immune activity (IFN Gamma, IFN Downstream, TIS) in pre-Tx tissue with favorable response to therapy (including SD). These findings are hypothesis generating and need to be further validated in prospective trials. Ongoing genomic and immunologic correlative studies in our cohort will further help understand comprehensive biomarkers of response and resistance to ICI in pts with mUC. [Table: see text]

Artificial intelligence-augmented histopathologic review using image analysis to optimize DNA yield from formalin-fixed paraffin-embedded slides

To achieve minimum DNA input requirements for next-generation sequencing (NGS), pathologists visually estimate macrodissection and slide count decisions. Unfortunately, misestimation may cause tissue waste and increased laboratory costs. We developed an artificial intelligence (AI)-augmented smart pathology review system (SmartPath) to empower pathologists with quantitative metrics for accurately determining tissue extraction parameters. SmartPath uses two deep learning architectures, a U-Net based network for cell segmentation and a multi-field-of-view convolutional network for tumor area segmentation, to extract features from digitized H&E-stained formalin-fixed paraffin-embedded slides. From the segmented tumor area, SmartPath suggests a macrodissection area. To predict DNA yield per slide, the extracted features from within the macrodissection area are correlated with known DNA yields to fit a regularized linear model (R = 0.85). Then, a pathologist-defined target yield divided by the predicted DNA yield per slide gives the number of slides to scrape. Following model development, an internal validation trial was conducted within the Tempus Labs molecular sequencing laboratory. We evaluated our system on 501 clinical colorectal cancer slides, where half received SmartPath-augmented review and half traditional pathologist review. The SmartPath cohort had 25% more DNA yields within a desired target range of 100–2000 ng. The number of extraction attempts was statistically unchanged between cohorts. The SmartPath system recommended fewer slides to scrape for large tissue sections, saving tissue in these cases. Conversely, SmartPath recommended more slides to scrape for samples with scant tissue sections, especially those with degraded DNA, helping prevent costly re-extraction due to insufficient extraction yield. A statistical analysis was performed to measure the impact of covariates on the results, offering insights on how to improve future applications of SmartPath. With these improvements, AI-augmented histopathologic review has the potential to decrease tissue waste, sequencing time, and laboratory costs by optimizing DNA yields, especially for samples with scant tissue and/or degraded DNA.

Transcriptomic Comparison of Non-Hodgkin Lymphomas in Relapsed/Refractory Versus Newly Diagnosed Patients Using Single FFPE Slides

Background Patients newly diagnosed with non-Hodgkin Lymphomas (NHLs) have been historically treated with B-cell depleting antibodies and chemotherapy. Standard of care (SoC) for patients with refractory or relapsed (R/R) disease has rapidly evolved over the past decade to incorporate chimeric antigen receptor T-cell therapy (CAR T), lenalidomide, antibody-directed chemotherapies and stem cell transplantation. This diverse treatment landscape can transform the tumor microenvironment and drive heterogenous tumor escape mechanisms. Understanding this varied tumor contexture will help inform efficacy results from NHL therapeutic studies. Core needle biopsies are used to sample tumor from R/R NHL patients; the bulk of tissue may be consumed for histopathology testing, leaving insufficient sample for transcriptomic profiling. We have shown that HTG EdgeSeq is useful for single-slide analysis of formalin fixed, paraffin embedded (FFPE) tissues (Loya et. al., JCOASCO Suppl. 2022). Here, we use this technique to compare tumors of R/R and newly diagnosed patients with Diffuse Large B-cell Lymphoma (DLBCL) and Follicular Lymphoma (FL). MethodsSamples and Transcriptomics: NHL tumor samples from newly diagnosed, treatment naïve patients (DLBCL n = 99, FL n = 19) and lymph node controls (n = 18) were sourced commercially. Tumor samples from a Phase 1/2a trial of epcoritamab in R/R patients (NCT03625037) were collected prior to treatment (DLBCL n = 38, FL n = 5). Single FFPE slides were processed using the EdgeSeq platform (19,000 gene HT panel, HTG Molecular). Tumors originated from lymphoid organs, gastrointestinal tract, testes, and the pleural cavity. Immunohistochemistry imaging for CD3, PAX5, CD20 was also performed (CellCarta, Belgium). Bioinformatics: Differential expression analysis was performed using linear models in R. Gene set enrichment analysis (GSEA) was performed with GSVA in R (Hänzelmann et. al., BMC Bioinformatics. 2013) using published signature sets (mSigDB: Liberzon et al., Cell Syst. 2015, XCell: Aran, et. al., Genome Biol. 2017). Results Single 5μm slides were analyzed with an average tissue size of 8mm2 for core needle biopsies and 40mm2 for resections. The number of detected genes was comparable to RNAseq, suggesting the HTG platform provides comprehensive data using core needle biopsies obtained during routine histopathology. We compared DLBCL tumor to normal lymph nodes and derived a gene set that was highly correlated to pathologist derived tumor purity. This quantitative proxy allows automated identification of samples that may contain source organ tissue. We applied differential expression analysis to identify gene, pathway and immune cell signatures contrasting newly diagnosed and R/R DLBCL. Top genes upregulated in R/R DLBCL tumors included B-cell markers as expected, T-cell function (CD25/IL2RA), and an intriguing epigenetic mechanism (YTHDC1; a N6-methyladenosine reader with a role in lymphoma proliferation [Liu et al., Front Onco 2022]). MYC pathway signaling was enriched in R/R tumors; consistent with known mutational risk factors for progression (Bisso et. al., Immunol Rev. 2019). Th2 polarized CD4+ T-cell signature was higher in treatment naïve DLBCL compared to R/R and normal lymph nodes. Fibroblast gene signatures were higher in R/R DLBCL, potentially a result of microenvironment remodeling due to chemotherapy or tumor driven adaptation (Dumontet et. al., Front Immunol. 2021). Comparison of DLBCL to FL was also performed in both R/R and untreated patient cohorts. Immune cell signatures for Tregs and CD4+ naïve T-cells were enriched in FL, whereas M1 macrophage signatures were enriched in DLBCL (findings were similar in both R/R and treatment naïve groups). Conclusions The EdgeSeq platform enabled whole-transcriptome profiling from very limited tissue samples in lymphoma clinical trials. This study highlights the importance of characterizing the transcriptional heterogeneity of NHL in relation to genetic alterations and T-cell immunity. Ongoing analysis with large patient cohorts in epcoritamab clinical trials will add statistical power to uncover associations with International Prognostic Score, Cell of Origin, effects of different treatment combinations, and genomic alterations on clinical outcomes.

LB921 Role of VivaScope ® 2500 in skin pathology: Advantages, limitations, and future prospects

The purpose is to highlight the advantages, limitations and future prospects of a novel cutaneous imaging device (VIvaScope 2500) that was used in a clinical setting for 6 months. This device was used as an adjunct device in a dermatology clinic. This device was used to view margins of excised cutaneous cancers while performing Mohs surgeries; this device was also used to image many excisions that were performed in the clinic before sending them to the histology lab. The staining protocol that we used for freshly excised tissues included 1)Dipping in 20 % Acetic Acid for 30 secs , followed by 2) Dipping in 0.5 mM Acridine Orange solution for 30 secs, followed by 3) Dipping in Normal saline to remove excess dye. Ex Vivo Microscopy (EVM) expedites the process to view skin pathology. It takes approximately 5 minutes to view the excised tissue under VivaScope as compared to frozen sectioning which takes 20 minutes on average. EVM is an ideal device to view margins of cutaneous tumors. This feature allows it to be an excellent adjunct tool to be used clinically to expedite Mohs surgeries. EVM can serve as an adjunct tool to confirm the diagnosis of clinically benign lesions. The use of ex vivo microscopy is not without limitation , compared to traditional formalin-fixed paraffin-embedded (FFPE) tissue sections, the image's resolution is still incomparable. Despite good nuclear contrast, there is still a considerable gap in resolution for ex vivo imaging. In diagnostic pathology, a subtle change in the nucleus can be interpreted as mild dysplasia, which is evident in FFPE slides, not ex vivo microscopic (EVM) images. There is great potential in this new ex vivo imaging tool. It allows real time quasi histologic microscopic examination of freshly excised cutaneous tissue and does not require traditional tissue embedding, processing and sectioning.

Molecular predictors of response among patients with MMRd tumors treated on NCI-MATCH Arm Z1D.

2616 Background: On arm Z1D of the NCI-MATCH trial, the PD-1 inhibitor nivolumab was found to have activity among patients with mismatch repair-deficient (MMRd) tumors as defined by complete loss of MLH1 or MSH2 nuclear expression determined by immunohistochemistry, with 6-month progression free survival of 51%. We aimed to identify molecular predictors of response in this population. Methods: Among patients treated on NCI-MATCH Z1D, we evaluated genomic and tissue predictors of clinical benefit (CB), defined as patients with RECIST v1.1 complete or partial response or stable disease for ≥ 6months. WES files were processed and filtered using GATK best practices preceding TMB and MSI calculations according to MSI sensor score, a WES-based MSI rating system. Cutoffs were set to define TMB (TMB-Low: ≤10 mutation/Mb; TMB-High: >10) and MSI (MSS: ≤10% unstable loci; MSI-Low: 10 > x ≤ 20; MSI-High: >20). Multiplex immunofluorescence (mIF) used formalin-fixed paraffin-embedded slides stained using a BOND RX automated stainer. Expression analyses followed normalization in DEseq2's median of ratios method. Gene set enrichment analysis was conducted by “empirical phenotype-based permutation test.” Additional RNA, WES, and mIF comparisons used the Wilcoxon rank-sum test. Results: Among 36 patients accrued to NCI-MATCH Z1D with pretreatment correlative samples available, 7 were unevaluable for response, and 1 was misclassified as having an MMRd tumor. Of the remaining 28, 15 had CB (2 CR, 10 PR, 3 SD ≤ 6 months) and correlative data were available for 26 (WES), 27 (RNAseq), and between 10-20 for mIF based on the marker assessed. According to MSI-sensor score, 11 were MSI-high, 8 were MSI-low, and 7 were MSS. MSI-sensor status, but not TMB was associated with CB (p=0.037 and p=0.185, respectively). Similar results were seen when using CR+PR vs SD+PD evaluation. Using RNAseq gene set enrichment analyses, CB patients had increased expression of interferon alpha (p=0.01), interferon gamma (p=0.03), PI3K-AKT-mTOR (p=0.02), cytotoxicity (p=0.05) and antigen processing (p=0.01) gene sets, while hedgehog signaling genes were increased in non-CB patients (p=0.04). The ESTIMATE immune index and infiltration of CD4+/PD1+/Ki67+ cell populations as determined by mIF were nominally higher in patients with CB (p=0.051 and p=0.075). Conclusions: Among patients with MMRd tumors treated with PD-1 checkpoint blockade, correlative analyses demonstrate associations between CB and MSI-sensor score as well as biomarkers indicative of immune infiltration and antigen presentation. This suggests that these measures may help differentiate patient response in MSI tumors. Clinical trial information: NCT02465060.

Tumor Infiltration Levels of CD3, Foxp3 (+) Lymphocytes and CD68 Macrophages at Diagnosis Predict 5-Year Disease-Specific Survival in Patients with Oropharynx Squamous Cell Carcinoma.

Simple SummaryHead and neck cancer (HNC) is the sixth most common cancer worldwide, with a general prognosis of 50% disease-specific survival (DSS). The subgroup of oropharyngeal (OP) cancers are of interest because HPV infection is one of several causative agents and carries favorable prognosis. Influxes of inflammatory cells into tumors may vary with prognosis. T lymphocytes are important regarding specific immune defense. Within the immune system T regulatory cells (Foxp3 positive) co-governs this process. We have therefore primarily studied levels of Foxp3 (+) cells in malignant tumors from 170 patients related to prognosis of the patients. Higher levels of T lymphocyte Foxp3 (+) cells predicted better 5-year DSS. This case was unique relative to age, gender, TNM stage, and HPV infection; but more so among tumor HPV (+) than HPV (−) patients. The results encourage further study into the use of immune-based therapy in HNC patients.Head and neck cancer (HNC) is the sixth most common cancer worldwide. Oropharyngeal (OP) cancers are of special interest because of possible underlying HPV infection which is tied to prognosis. Influxes of inflammatory cells into tumors may vary with prognoses. We wanted to study whether the number of tumor-infiltrating lymphocytes (TIL) and tumor-associated macrophages (TAM) in tumors correlated to HPV status and predicted 5-year disease-specific survival (DSS). Formalin-fixed paraffin-embedded (FFPE) biopsies cut sections from 170 patients treated for OP cancer were stained by immunohistochemistry and evaluated for the number of CD68 (+) TAMs, CD3 (+), and Foxp3 (+) (T regulatory) TILs. From FFPE slides HPV by PCR and p16 by immunohistochemistry were established. From FFPE Hematoxylin-Eosin slides, levels of tumor nuclear polymorphism, tumor invasion, desmoplasia, and inflammation were determined as previously published. Levels of TIL CD3 (+) and TIL Foxp3 (+) were increased among the HPV (+) compared to the HPV (−) patients. High levels of TIL Foxp3 (+) and CD68 (+) macrophages predicted better 5-year DSS. TIL Foxp3 (+) levels predicted independent of age, gender, TNM stage, and HPV infection as well as level of stromal desmoplasia, tumor invasion, and nuclear polymorphism, but more pronounced among tumor HPV (+) than HPV (−) patients.

FOXP3+ T-cell infiltration is associated with improved outcomes in metastatic urothelial carcinoma (mUC) treated with immune-checkpoint inhibitors (ICI).

549 Background: Given that ICIs benefit a minority of mUC patients and are associated with significant costs, biomarkers are necessary to optimally utilize them in the clinic. Although FOXP3+ T-cells have been associated with an immune-cold environment in many cancers, studies in urothelial carcinoma have shown an opposite trend. Methods: Formalin-fixed paraffin-embedded slides from tumor specimens were collected for patients with mUC treated with ICI at Dana-Farber Cancer Institute. A novel multiplex immunofluorescence (mIF) panel, ImmunoProfile, was performed for PD-L1, PD-1, FOXP3, CD8 and DAPI, then scanned by a Vectra Polaris platform. Regions of interest were defined and used for quantitative analysis using PerkinElmer/Akoya. Intratumoral (IT) and tumor-stroma interface (TSI) density (cells/mm2) of each cell type was calculated. Clinical data was collected through chart review, and associations between cell density and response were assessed. Wilcoxon Rank-Sum test between responders (CR/PR) and non-responders (SD/PD) was used to generate p-values, followed by Benjamini-Hochberg correction. Receiver-operating curve (ROC) and area-under-curve (AUC) calculations were performed to determine the optimal cutoff (OC) differentiating responders from non-responders. Cox proportional hazards models were used to estimate OS and PFS, accounting for type of therapy (single vs. combination ICI), baseline neutrophil-to-lymphocyte ratio (NLR), PD-L1 CPS, prior therapy, non-urothelial component, ECOG-PS and liver metastases (mets). Results: Of 35 patients assessed by ImmunoProfile, 32 were evaluable for response. Most patients (88%) were male and the median age at ICI start was 73 years. Median number of prior lines of therapy was 1 (range 0 – 3), and the majority (72%) was treated with single-agent ICI. Eight patients (25%) had CR/PR, eight had SD and 16 had PD as best response. Of all IF stains assessed, IT-TSI FOXP3 was the strongest predictor of objective response (q-value = 0.006), followed by IT-CD8 (q = 0.014). ROC analysis yielded an AUC of 0.812 (0.656 – 0.969) and the optimal cutpoint was set at 75 IT-TSI-FOXP3 cells/mm2. ORR was 46% in FOXP3-high ( > 75/mm2) vs 14% in FOXP3-low mUCs. A combined model using IT-TSI-FOXP3 and clinical covariates (NLR, ECOG-PS, line of therapy, histology, and liver mets) had an AUC of 0.929. PFS was significantly longer in the FOXP3-high group (7.9 [5.7 – NR] months) compared to the FOXP3-low group (2.3 [2.1 – 6.1] months) on multivariable analysis (p = 0.007). OS also showed the same trend (p = 0.1). Conclusions: In this pilot study of ImmunoProfile, a novel mIF panel, higher FOXP3+ infiltration in tumors was associated with better outcomes and a composite clinico-IHC panel exhibited robust prognostic impact in mUC pts treated with ICI. Further study of this mIF panel is warranted to implement it in routine practice.

Assessment of Molecular Residual Disease Using Circulating Tumor DNA to Identify Multiple Myeloma Patients at High Risk of Relapse.

BackgroundDespite treatment with high-dose chemotherapy followed by autologous stem cell transplantation (AHCT), patients with multiple myeloma (MM) invariably relapse. Molecular residual disease (MRD)-negativity post-AHCT has emerged as an important prognostic marker predicting the duration of remission. Current techniques for MRD assessment involve bone marrow (BM) aspirate sampling, which is invasive, subject to sample variability and is limited by spatial heterogeneity. We compared the performance of a non-invasive, circulating tumor DNA (ctDNA)-based MRD assay with multiparameter flow cytometry (MFC) of marrow aspirate to predict relapse in AHCT recipients with MM.MethodsMRD assessment using ctDNA was retrospectively analyzed on 80 plasma samples collected at different time points from 28 patients, post-AHCT. MFC was used to assess MRD from BM biopsy. Individual archived BM aspirate slides or formalin-fixed paraffin-embedded slides from the time of MM diagnosis and matched blood were used to assess MRD at 3 months, post-AHCT, using a personalized, tumor-informed ctDNA assay.ResultsctDNA was detectable in 70.8% (17/24) of pre-AHCT patients and 53.6% (15/28) of post-AHCT patients (3-month time point). Of the 15 post-AHCT ctDNA-positive patients, 14 relapsed on follow-up. The median PFS for ctDNA-positive patients was 31 months, and that for ctDNA-negative patients was 84 months (HR: 5.6; 95%CI: 1.8-17;p=0.0003). No significant difference in PFS was observed in patients stratified by MFC-based MRD status (HR 1.2; 95%CI: 0.3-3.4;p=0.73). The positive predictive value for ctDNA was also significantly higher than MFC (93.3% vs. 68.4%).ConclusionsThis study demonstrates tumor-informed ctDNA analysis is strongly predictive of MM relapse.

Prognostic Features of Near-Infrared Spectroscopy Following Primary Radical Prostatectomy.

Simple SummaryUp to 53% of patients with localized/locally advanced prostate cancer (PCa) will be confronted with biochemical recurrence (BCR) at some point after primary radical prostatectomy. No robust prognostic biomarker exists in this patient setting. The aim of this retrospective study was therefore to determine if near-infrared (NIR) spectroscopy, an easy-to-use technique, could be an asset as a prognostic biomarker for PCa patients after radical prostatectomy. In a cohort of 82 patients, we confirmed that changes in the NIR spectrum of PCa specimens, obtained via radical prostatectomy, can be prognostic for BCR-free survival. The clinical implementation of NIR spectroscopy can therefore be a great asset as it can aid in the management of PCa patients following primary radical prostatectomy. Further elaboration is recommended.Background: Only a few biomarkers have been evaluated for their prognostic value with regard to biochemical recurrence (BCR) following primary radical prostatectomy. We explored the possibilities of using near-infrared (NIR) spectroscopy as a prognostic biomarker for BCR-free survival (BCR-FS). Methods: Tissue specimens from 82 prostate cancer patients were obtained. Formalin-fixed paraffin-embedded slides (hematoxylin–eosin-stained) were analyzed using NIR spectroscopy. Prognostic features for BCR-FS were determined following normalization of the spectra. Results: Several differences were found throughout the NIR spectrum for the patients with or without BCR, for both the first derivative and second derivative of the NIR spectrum. Following categorization and Cox regression analysis, spectral regions at 5236 cm−1 (first derivative; median BCR-FS not reached versus 3.2 years; HRhigh = 0.18 [0.08–0.39]; and p < 0.0001) and at 5956 cm−1 (second derivative; median BCR-FS not reached versus 3.8 years; HRlow = 0.22 [0.10–0.48]; and p = 0.0002) showed prognostic properties for BCR-FS. The combination of both parameters further increased the prognostic value of NIR (p < 0.0001). Conclusions: We demonstrated NIR spectral variations between patients with or without BCR, which have been shown to have prognostic value. This easy-to-use technique could possibly further improve post-primary radical prostatectomy monitoring and swift referral to adjuvant local therapies. Further elaboration is highly recommended to fully elucidate these variations and to gain a deeper insight into the changing chemical and physical compositions of the prostate tumor architecture.

H&amp;E-like histology of unstained fresh and formalin fixed breast tissue with Photo Acoustic Remote Sensing (PARS) microscopy.

e12590 Background: Approximately 30% of breast cancer patients undergoing breast-conserving surgery (BCS) require re-excision(s) to obtain clear margins, causing delays in adjuvant treatment, poor aesthetic results, and increased infection rates, costs, and patient anxiety. Frozen section pathology assessment (FSA) reduces re-excision rates from 27% to 6%, but FSA extends operative times and has a false negative rate of 17%. Photo-Acoustic Remote Sensing (PARS) is a new laser-based light microscope that permits non-contact cellular resolution imaging of unstained tissues. Methods: We used an ultraviolet reflection-mode PARS microscope to study i) formalin-fixed, paraffin embedded (FFPE) breast tissue blocks, ii) unstained thin sections of FFPE invasive ductal carcinoma and ductal carcinoma in situ, iii) formalin fixed bulk surgical specimens of benign and malignant human breast tissue, and iv) benign and malignant fresh rodent mammary tissue without fixation, processing, or staining. Fresh tissues were imaged after a range of warm ischemic times and across a range of cold ischemic times in neutral phosphate buffered saline at 4°C. For each condition, an ultraviolet laser was used to excite DNA and other cellular components in these samples, and signals were captured using a continuous-wave detection laser. The PARS signal was false-colored to closely replicate traditional hematoxylin and eosin staining. Results: In all fixed tissues, PARS virtual histology images were of diagnostic quality, permitted margin assessment, and analogous to transmission light microscopy of standard hematoxylin and eosin-stained FFPE slides, achieved without tissue sectioning or tissue staining. Furthermore, PARS microscopy also provided cellular level virtual histology images in fresh breast tissue with warm and cold ischemic times ranging from twenty minutes to two hours; these images have no available clinical comparators as non-contact cellular level imaging of unprocessed fresh tissue has not previously been reported. In aggregate, we demonstrate the feasibility of PARS to provide diagnostic and margin assessment images across a range of tissues including formalin fixed and freshly resected, unstained, unprocessed breast tissue. Conclusions: PARS is a new microscope technology addressing the practical needs of intraoperative margin assessment during BCS: i) no requirement for tissue staining, ii) rapid acquisition of hematoxylin and eosin-like images without the requirement for tissue freezing, embedding, or sectioning, iii) diagnostic quality cellular resolution, and iv) assessment of resected bulk tissue margins. In principle, this technology may also permit label-free non-contact intraoperative margin assessment of the surgical cavity. These data support the clinical development and evaluation of PARS microscopic intraoperative assessment of BCS margins.

FP07.14 Implementation of a Lung Biopsy Histology Protocol Supports Successful Cancer Biomarker Testing

Non-small cell lung cancer (NSCLC) management increasingly relies on biomarker testing, which presents a logistical challenge for utilization of lung biopsy specimens beyond routine diagnosis. Appropriate allocation of tissue from small biopsies is critical in order to permit for accurate histologic diagnosis while preserving tissue for ancillary studies such as PD-L1 testing and next generation sequencing (NGS). International surveys show that satisfaction with molecular testing is less than ideal, driven in part by inadequacy of formalin-fixed paraffin embedded (FFPE) tumor tissue for biomarker testing in over a quarter of clinical cases by some reports. We developed a histology protocol (LUNGCOR) that generates multiple unstained FFPE slides up front in order to increase tissue yields for pathologic diagnosis and ancillary testing. We examined the efficacy of this protocol in regards to molecular testing success rates. Biopsy samples (e.g. transbronchial or fine needle biopsies) were flagged for the LUNGCOR protocol based on a clinical indication of "lung mass" or need for genomics. Proceduralists were requested to prospectively specify when samples were obtained for the purposes of biomarker testing. Per protocol, after embedding, 2 H&E slides with 18 intervening 4-micron unstained FFPE slides were prepared and reserved for immunohistochemistry or molecular studies. We performed a retrospective review of consecutive LUNGCOR specimens within the pathology information system and electronic medical record to determine the usage patterns for the unstained slides and success rates of molecular testing. This analysis is restricted to the first 2 years (2015-2016) following protocol initiation. 300 specimens were designated for the LUNGCOR protocol in this time period. Of these specimens, 84% resulted in a malignant diagnosis and 175 (58%) were non-squamous NSCLC. Of these, 87 (50%) of NSCLC underwent molecular profiling of some kind, either in house NGS, single gene PCR assays, or send-out NGS. For cases undergoing in house next generation sequencing (447-gene panel), 61 of 69 (88%) cases generated complete genotyping for all required and emerging biomarkers for targeted therapy. Sequencing failures were most commonly due to inadequate tumor content in the biopsy specimen, rather than inadequate size of the overall sample. Generation of unstained slides during upfront histologic processing of known or suspected NSCLC increases availability of tissue for molecular testing and reduces risk of sample inadequacy due to specimen loss during routine handling steps, such as "refacing" the block. This straightforward process improvement can improve the yield of invasive biopsies for essential biomarker testing and contribute to improved patient outcomes.

Large scale, robust, and accurate whole transcriptome profiling from clinical formalin-fixed paraffin-embedded samples

Transcriptome profiling can provide information of great value in clinical decision-making, yet RNA from readily available formalin-fixed paraffin-embedded (FFPE) tissue is often too degraded for quality sequencing. To assess the clinical utility of FFPE-derived RNA, we performed ribo-deplete RNA extractions on > 3200 FFPE slide samples; 25 of these had direct FFPE vs. fresh frozen (FF) replicates, 57 were sequenced in 2 different labs, 87 underwent multiple library analyses, and 16 had direct microdissected vs. macrodissected replicates. Poly-A versus ribo-depletion RNA extraction methods were compared using transcriptomes of TCGA cohort and 3116 FFPE samples. Compared to FF, FFPE transcripts coding for nuclear/cytoplasmic proteins involved in DNA packaging, replication, and protein synthesis were detected at lower rates and zinc finger family transcripts were of poorer quality. The greatest difference in extraction methods was in histone transcripts which typically lack poly-A tails. Encouragingly, the overall sequencing success rate was 81%. Exome coverage was highly concordant in direct FFPE and FF replicates, with 98% agreement in coding exon coverage and a median correlation of whole transcriptome profiles of 0.95. We provide strong rationale for clinical use of FFPE-derived RNA based on the robustness, reproducibility, and consistency of whole transcriptome profiling.

Clinical Application of Multiple Reaction Monitoring-Mass Spectrometry to Human Epidermal Growth Factor Receptor 2 Measurements as a Potential Diagnostic Tool for Breast Cancer Therapy.

Human epidermal growth factor receptor 2 (HER2) is often overexpressed in breast cancer and correlates with a worse prognosis. Thus, the accurate detection of HER2 is crucial for providing the appropriate measures for patients. However, the current techniques used to detect HER2 status, immunohistochemistry and fluorescence in situ hybridization (FISH), have limitations. Specifically, FISH, which is mandatory for arbitrating 2+ cases, is time-consuming and costly. To address this shortcoming, we established a multiple reaction monitoring-mass spectrometry (MRM-MS) assay that improves on existing methods for differentiating HER2 status. We quantified HER2 expression levels in 210 breast cancer formalin-fixed paraffin-embedded (FFPE) tissue samples by MRM-MS. We aimed to improve the accuracy and precision of HER2 quantification by simplifying the sample preparation through predicting the number of FFPE slides required to ensure an adequate amount of protein and using the expression levels of an epithelial cell-specific protein as a normalization factor when measuring HER2 expression levels. To assess the correlation between MRM-MS and IHC/FISH data, HER2 quantitative data from MRM-MS were divided by the expression levels of junctional adhesion molecule A, an epithelial cell-specific protein, prior to statistical analysis. The normalized HER2 amounts distinguished between HER2 2+/FISH-negative and 2+/FISH-positive groups (AUROC = 0.908), which could not be differentiated by IHC. In addition, all HER2 status were discriminated by MRM-MS. This MRM-MS assay yields more accurate HER2 expression levels relative to immunohistochemistry and should help to guide clinicians toward the proper treatment for breast cancer patients, based on their HER2 expression.

Platform comparison of HTG EdgeSeq and RNA-Seq for gene expression profiling of tumor tissue specimens.

3566 Background: Clinical biomarker studies are often hindered by the availability of tissue specimens of sufficient quality and quantity. While RNA-Seq is often considered the gold standard for measuring mRNA expression levels in cancer tissue, it typically requires multiple formalin-fixed paraffin-embedded (FFPE) tissue sections to extract a sufficient amount of quality RNA for subsequent gene expression profiling analysis. The HTG EdgeSeq technology is a gene expression profiling platform that combines quantitative nuclease protection assay technology with next-generation sequencing detection. Unlike RNA-Seq, the HTG EdgeSeq technology does not require RNA extraction, and can use small amounts of tissue material, typically several mm2, to generate reproducible gene expression profiles. Methods: This study compares the performance of RNA-Seq and HTG's profiling panel, the HTG EdgeSeq Precision Immuno-Oncology Panel (PIP), which is designed to measure expression levels of 1,392 genes focused on tumor/immune interaction. Approximately 1,200 samples from three tumor indications (gastric cancer, colorectal cancer and ovarian cancer) were tested using both technologies. Results: Up to four FFPE slides were used for RNA extraction to support RNA-Seq testing; out of the 1,202 samples processed, 1,099 generated extracted RNA of sufficient quality and quantity (as measured by RNA concentration, RIN score and %DV200) to proceed to sequencing, which resulted in a pass rate of 91.4% for RNA-Seq. The HTG EdgeSeq PIP panel resulted in a pass rate of 97.3% (samples passing QC metrics) when the same 1,200 samples were tested, and required only a single FFPE section owing to the small sample requirement. The t-SNE (a non-linear dimensionality reduction method) analysis of the common 1,358 genes revealed similar clustering of the three cancer indications between the two methods. Correlations across individual genes by sample resulted in the mean Spearman correlation coefficient of 0.73 (95% confidence interval of 0.61 - 0.80). Additionally, gene-wise comparisons across all samples were also evaluated. Conclusions: These data demonstrate that HTG EdgeSeq gene expression panels can be used as a competitive alternative to RNA-Seq, generating equivalent gene expression results, while offering the added benefits of a small sample size requirement, lack of RNA extraction bias, and fully automated data analysis pipeline.

19. The many faces of colonic malakoplakia: A case series and review of the literature

Background: Malakoplakia is a rare inflammatory response to Gram-negative bacteria, involving intracellular deposition of iron and calcium. It is associated with immunosuppression, and occurs most often in the bladder. Although rare, the colon is the most common extra-genitourinary site. Aims and methods: To identify cases of colonic malakoplakia, the Royal Prince Alfred Hospital histopathology data base was searched from 2009. A literature review was performed using PubMed with the term ‘malakoplakia’. Formalin-fixed paraffin-embedded slides were stained with haematoxylin and eosin, PAS and von Kossa. Results and conclusions: A 10-year case file review identified four cases of colonic malakoplakia. Ages ranged from 50–79 with 2 males and 2 females. Clinical presentation and endoscopic appearances varied widely with an incidental polyp, mucosal changes mimicking inflammatory bowel disease, and a pelvic mass lesion with overlying normal mucosa seen. In all cases the biopsies revealed numerous histiocytes and chacteristic Michaelis-Gutmann bodies highlighted with PAS and von Kossa stains diagnostic of malakoplakia. A literature review identified 31 published case reports in adults and 10 in children of colonic malakoplakia. These cases highlight the protean manifestations of colonic malakoplakia. Consideration should be given to the diagnosis in histiocytic infiltrates in the large bowel to facilitate appropriate clinical management.

Tumour mutational burden ring trial: Evaluation of targeted next-generation sequencing platforms for implementation in clinical practice

Abstract Background Tumour mutational burden (TMB) is a measurement of DNA variants in a tumour and is a potential biomarker of response to Immune Checkpoint Inhibitor (ICI) therapy. Patients with non-small cell lung cancer (NSCLC) whose tumours have a high TMB (≥10 mutations/Mb) might benefit from upfront ICI combination therapy. Clinical trials have measured TMB using Whole Exome Sequencing (WES) and/or the FoundationOne CDx (F1CDx) assay. In parallel, several commercial next-generation sequencing (NGS) assays have become available. Before implementation of TMB testing in clinical practice, technical and clinical validation are needed. Hence, a multi-center study was organised to establish concordance between several TMB assays. Methods Fifteen resection NSCLC formalin-fixed paraffin-embedded (FFPE) samples with broad TMB range were selected. Each participant received extracted DNA and was asked to report TMB values using their own protocol. In parallel, FFPE slides were analysed with F1CDx. Eight labs participated using five different methods: Oncomine TML assay (Thermofisher; n = 4), TSO500 assay (Illumina; n = 1), NEOplus assay (NEO New Oncology; n = 1), a 0,4 Mb targeted resequencing lab-developed assay (LDT; n = 1) and WES using three different TMB calculation methods (n = 1). Correlations between each platform and F1CDx were calculated. Also, the reported TMB category (high vs low) of the different platforms in comparison to F1CDx was evaluated for the fifteen samples. Results Assessment of TMB values obtained by the platforms and F1CDx demonstrated a high correlation (R2 between 0.81 and 0.94), except for the smaller LDT (R2 = 0.53). The TMB category (high vs low) reported by each platform showed concordance with the F1CDx category for eleven (73%) to thirteen (93%) of the fifteen samples. From the fifteen samples, the same category was reported by all different platforms for seven (47%) samples. Conclusion Our data show that assays from different providers can be used to predict TMB. However, samples with a TMB value around the cut-off of 10 mut/Mb are challenging and interpretation should occur with caution. Further studies are required before implementing these assays in routine clinical diagnosis. Legal entity responsible for the study The authors. Funding Bristol-Myers Squibb. Disclosure S. Lambin: Research grant / Funding (institution): Bristol-Myers Squibb. All other authors have declared no conflicts of interest.