Paraffin-embedded Biopsy Specimens Research Articles

Purity Independent Subtyping of Tumors (PurIST) Pancreatic Cancer Classifier: Analytic Validation of a 16-RNA Expression Signature Distinguishing Basal and Classical Subtypes

The two major molecular subtypes of pancreatic adenocarcinoma reportedly have differential response to FOLFIRINOX-based therapy. To promote rapid assignment of basal versus classical subtypes, an array-based single-sample classifier assay was developed and applied to 74 formalin-fixed, paraffin-embedded biopsy or resection specimens of known subtype based on transcriptomics. The Purity Independent Subtyping of Tumors (PurIST) algorithm assigns subtype based on relative expression of 16 RNAs counted by RNA sequencing (RNAseq) versus more practical array-based NanoString nCounter Elements XT technology. Subtype calls were largely concordant between RNAseq and array methods (72/74, 97% agreement). Compared with the lengthy RNAseq protocol, the array-based assay takes just 3 working days to analyze, permitting rapid reporting of tumor subtype. In conclusion, the PurIST pancreatic cancer classifier has robust performance to classify pancreatic adenocarcinoma into basal versus classical subtypes. Clinical validation studies are underway to evaluate outcome in patients whose standard-of-care chemotherapy regimen is selected on the basis of rapid subtype assignment (NCT04683315).

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

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

Abstract PO5-06-06: Tumor/stromal expression of CD3/CD8/PD-1/PD-L1 and overall survival (OS) in patients (pts) with metastatic (met) HER2+ breast cancer (BC)

Abstract Background: The “Thousand Patient HER-2 database” project at Saint Vincent’s University Hospital (SVUH) Dublin contains a cohort of met HER2+ BC pts treated with trastuzumab, with OS ranging from short term to durable complete response (never relapsed). While the majority of pts progress on treatment, higher stromal tumor immune infiltrate has been associated with longer OS in met HER2+ BC. Using the SVUH database, we have identified a cohort of met HER2+ BC pts that received trastuzumab and have a broad OS range (0.3 months (mos) to 200.9 mos). This study examines the tumor and stromal levels of pan T cell marker CD3, cytotoxic T cell marker CD8, and immune checkpoints PD-L1 and PD-1 by immunohistochemistry (IHC) in primary and met samples from this cohort. Methods: Clinico-pathological data was available for formalin-fixed, paraffin-embedded biopsy specimens (n=45 primary, n=25 matched met biopsies). IHC staining was conducted for CD3 (Agilent IR50361-2), CD8 (Agilent IR62361-2), PD-L1, (Agilent M365329-2), and PD-1 (Roche 07099029001). A DAKO Link 48 Autostainer was utilised for staining, incorporating positive (tonsil tissue) and negative controls (isotype controls). Slide processing used an Aperio AT2 Digital Slide Scanner (Leica Biosystems), Aperio ImageScope 12.4 software (Leica Biosystems) and QuPath analysis software (University of Edinburgh). Following annotation of tumor areas, an algorithm was trained to identify immune cells and designate them as tumor or stromal. Data was expressed as the number of positively stained cells/mm2 tissue. A median cut-off was applied to denote “high” and “low” expression for CD3 and CD8. For PD-1 and PD-L1, samples with ≥ 1 stained cell/mm2 were designated as positive (pos) and samples with zero stained cells as negative (neg). The Kaplan Meier method was utilised for survival studies. A paired Student’s t test was utilised for primary vs metastatic site comparisons. Results: 51.1% of primary samples displayed high CD3 (23/45) and CD8 (23/45) expression in both the stromal and tumor compartments. Within the tumor compartment, 18.6% (8/43) of samples were pos for PD-L1 expression, and 57.8% (26/45) were pos for PD-1 expression. In the stromal compartment, 30.2% (13/43) of samples were pos for PD-L1 expression, and 68.9% (31/45) were pos for PD-1 expression. High tumor levels of CD3 (median OS CD3 high 113.4 mos vs CD3 low 7.9 mos, HR 0.2938 (95% CI 0.130-0.618), p=0.0012) and CD8 (median OS CD8 high 73.9 mos vs CD8 low 10.8 mos, HR 0.4764 (95% CI 0.238-0.954), p=0.0365), but not stromal levels of these markers, were associated with improved OS. There were no significant differences in CD3, CD8, PD-1 and PD-L1 levels between matched primary and met samples (p >0.05). Tumor, but not stromal, PD-L1 expression was associated with longer OS (HR 0.3120 (95% CI 0.146-0.667), p=0.0027). Interestingly, stromal PD-1 expression, but not tumor PD-1 expression, was also associated with longer OS (median OS PD-1 pos 64.7 mos vs. PD-1 neg 5.6 mos, HR 0.158 (95% CI 0.060-0.411), p=0.0002). Conclusion: Our results report a link between OS and tumor (CD3+, CD8+ and PD-L1+)/stromal (PD-1+) immune cell infiltrate in met HER2+ BC patients treated with trastuzumab. Further expansion of this preliminary dataset is warranted. Citation Format: Denis Collins, Janet McCormack, Laura Ivers, Jose Javier Berenguer-Pina, Jo Ballot, Cecily Quinn, Darko Skrobo, Alex Eustace, Naomi Walsh, Aurelie Fabre, John Crown. Tumor/stromal expression of CD3/CD8/PD-1/PD-L1 and overall survival (OS) in patients (pts) with metastatic (met) HER2+ breast cancer (BC) [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 PO5-06-06.

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

Abstract Background. Prosigna (PAM50) is an FDA-approved prognostic test that measures the expression of 50 classifier genes in breast cancer tissue samples. It provides intrinsic subtyping, which classifies the biology of an individual patient's tumor, as well as a risk of recurrence (ROR) score that predicts the probability of distant recurrence over 10 years. Currently, the Prosigna assay is only approved for use in postoperative specimens. Given studies demonstrating that Prosigna can identify tumors that are more likely to benefit from chemotherapy, this decision-impact study sought to evaluate whether the assay could influence physicians' choices and patients' confidence in the neoadjuvant treatment plan. Methods. This prospective observational study was conducted at a single institution and included patients with HR+/HER2- breast cancer measuring ≥ 0.5 cm, any nodal status, who were deemed candidates for neoadjuvant systemic treatment based on physician’s choice. Formalin-fixed paraffin-embedded core biopsy specimens were centrally analyzed using Prosigna. Patients' tumors were classified according to intrinsic tumor subtype (Luminal A, Luminal B, HER2-enriched, Basal-like) and ROR score (low, intermediate, or high). Physicians and patients were surveyed before and after performing Prosigna, regarding neoadjuvant therapy recommendations. The primary endpoint was assessment of the effect of Prosigna on oncologists' treatment recommendations and the actual treatment received (neoadjuvant hormonal therapy [NAHT], neoadjuvant chemotherapy [NACT], or upfront surgery). Secondary endpoints included determining whether treatment changes were based on ROR score, intrinsic subtype, or both, as well as assessing physicians' and patients' confidence in the treatment plan. Results. A total of 54 patients were enrolled in the study between March 2019 and April 2023. The distribution of intrinsic tumor subtypes was as follows: 15 patients (28%) had a Luminal A subtype, 34 (63%) had a Luminal B subtype, 3 (6%) had a HER2-enriched subtype, and 2 (4%) had a Basal-like subtype. Thirty-three patients (61%) were classified as ROR-low, 11 (20%) as ROR-intermediate, and 10 (19%) as ROR-high. Out of the 43 patients who had both pre- and post-assay survey results available, a change in the treatment decision was observed in 28% of cases. Specifically, 14% of patients transitioned from NAHT to NACT, 7% from NACT to NAHT, and 5% from NACT to upfront surgery, as shown in the Table. Treatment changes were based on the ROR score, intrinsic subtype, and both in 33%, 3%, and 64% of cases, respectively. Overall, 45% of physicians experienced an increase in confidence for the treatment plan after Prosigna testing, while 16% reported a decrease and 39% maintained the same level of confidence. Sixty-two percent of patients experienced a reduction in anxiety about treatment. Associations of ROR score and intrinsic subtypes will be presented at the meeting. Conclusion. Prosigna performed on presurgical core biopsies influenced oncologists' treatment recommendations regarding neoadjuvant treatment and reduced patient anxiety about treatment among patients with early-stage HR+/HER2- breast cancer. Table. Neoadjuvant treatment recommendations before and after Prosigna assay, overall and according to nodal status. Data are presented as frequencies (%). Abbreviations: NACT, neoadjuvant chemotherapy; NAHT, neoadjuvant hormonal therapy. Citation Format: Chiara Corti, Xiangying Chu, Pedro Exman, Danielle Kline, Nolan Priedigkeit, Nabihah Tayob, Erica Mayer, Adrienne Waks, Melissa Hughes, Antonio Giordano, Giuseppe Curigliano, Nancy Lin, Tari King, Rinath Jeselsohn, Deborah Dillon, Elizabeth Mittendorf, Sara Tolaney. Results of a prospective observational study evaluating the impact of the Prosigna assay on neoadjuvant treatment decision-making in patients with early-stage HR+/HER2- breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-01-09.

A Preliminary Study Assessing a Transfer Learning Approach to Intestinal Image Analysis to Help Determine Treatment Response in Canine Protein-Losing Enteropathy.

Dogs with protein-losing enteropathy (PLE) caused by inflammatory enteritis, intestinal lymphangiectasia, or both, have a guarded prognosis, with death occurring as a result of the disease in approximately 50% of cases. Although dietary therapy alone is significantly associated with a positive outcome, there is limited ability to differentiate between food-responsive (FR) PLE and immunosuppressant-responsive (IR) PLE at diagnosis in dogs. Our objective was to determine if a transfer learning computational approach to image classification on duodenal biopsy specimens collected at diagnosis was able to differentiate FR-PLE from IR-PLE. This was a retrospective study using paraffin-embedded formalin-fixed duodenal biopsy specimens collected during upper gastrointestinal tract endoscopy as part of the diagnostic investigations from 17 client-owned dogs with PLE due to inflammatory enteritis at a referral teaching hospital that were subsequently classified based on treatment response into FR-PLE (n = 7) or IR-PLE (n = 10) after 4 months of follow-up. A machine-based algorithm was used on lower magnification and higher resolution images of endoscopic duodenal biopsy specimens. Using the pre-trained Convolutional Neural Network model with a 70/30 training/test ratio for images, the model was able to differentiate endoscopic duodenal biopsy images from dogs with FR-PLE and IR-PLE with an accuracy of 83.78%. Our study represents an important first step toward the use of machine learning in improving the decision-making process for clinicians with regard to the initial treatment of canine PLE.

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

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

Investigating comparative polymerase chain reaction for antigen receptor rearrangement analysis in different types of feline lymphoma samples.

Cats have the highest incidence of lymphoma among all animal species. Lymphoma accounts for 41% of all malignant tumors in cats and is responsible for 90% of hematopoietic tumors in felines. Biopsies are considered the gold standard for diagnosis. Polymerase chain reaction (PCR)-based clonality assessment of antigen receptor gene rearrangements can be a valuable complementary tool for identifying infiltrating B-and T-lymphocyte clones. Many studies have focused on intestinal cases but few have addressed mediastinal lymphoma. This study aims to: (1) investigate the clonality patterns of lymphoma samples from various anatomical sites, with a particular focus on mediastinal lymphoma, and (2) evaluate the sensitivity and specificity of the clonality analysis of pleural effusion samples in comparison with cytology, histology, immunohistochemistry, and immunocytochemistry for diagnosing mediastinal lymphoma. There were 82 cases, divided into 49 formalin-fixed and paraffin-embedded biopsy specimens (FFPE), 22 cell pellets, and 11 fresh tissue. This study examined the sensitivity and specificity of PCR for antigen receptor rearrangement (PARR) compared to immunohistochemistry (IHC) and immunocytochemistry. For T-cell receptor gamma chain genes, PARR demonstrated a sensitivity of 58.33% for both fresh tissue and FFPE samples, with a specificity of 100%. Cell pellet analysis exhibited a sensitivity of 64.71% and maintained 100% specificity. A combined analysis of fresh tissue and FFPE with cell pellets showed a sensitivity of 62.07%. For IGH, the sensitivity for fresh tissue and FFPE samples was 56.25%, while cell pellet analysis showed a sensitivity of 62.50%. When considering fresh tissue and FFPE samples, the sensitivity was 57.14%. In conclusion, molecular techniques have emerged as valuable tools for detecting lymphoma, especially in cases where traditional diagnostic methods yield inconclusive results, such as mediastinal lymphoma. While biopsy may not always be feasible, cytology and cell pellets obtained from pleural effusion offer alternative immunocytochemistry and molecular analysis samples, provided they are of sufficient quality and quantity. All sample types considered in this study were suitable for PARR to aid in cases with inconclusive results. Therefore, the sample selection should be tailored to the clinical situation.

Prevalence of high-risk human papillomavirus in oral squamous cell carcinoma with or without chewing habits.

Oral cancer (OC) is the most common cancer in Pakistani males and the second most common in females. Major risk factors include peculiar chewing habits, human papillomavirus (HPV) infection and molecular pathways. However, less data is available for this avertible cancer regarding its association with high-risk HPV (HR-HPV) and chewing habits in this region. Therefore, this study was done to determine the prevalence of HR-HPV in oral squamous cell carcinoma (OSCC) and its correlation with p16 and chewing habits. Formalin-fixed paraffin-embedded (FFPE) biopsy specimens of 186 samples were tested for HR-HPV type 16/18 by PCR, followed by p16 immunostaining (IHC) in a subset of cases (n = 50). Appropriate statistical tests were applied to find the association between HR-HPV/p16 and peculiar chewing habits with significance criteria of p<0.05 with 95% CI. HR-HPV (type 16 &18) was present in seven out of 186 cases (3.8%). Of these seven cases, five were positive for HPV16, whereas two were positive for HPV16/18. The overall expression of p16 protein in 50 samples was 38% (n = 19), and among these 19-IHC positive samples, 26% were positive for HR-HPV DNA. No significant association was found between HR-HPV positivity and p16 and chewing habits (p>0.05). It was concluded that HR-HPV prevalence in OSCC was very low in our population, with no statistically significant correlation with p16 and chewing habits. These results suggest the role of HR-HPV as an independent risk factor in OSCC in the local setting.

Beyond 2D: A scalable and highly sensitive method for a comprehensive 3D analysis of kidney biopsy tissue.

The spatial organization of various cell populations is critical for the major physiological and pathological processes in the kidneys. Most evaluation of these processes typically comes from a conventional 2D tissue cross-section, visualizing a limited amount of cell organization. Therefore, the 2D analysis of kidney biopsy introduces selection bias. The 2D analysis potentially omits key pathological findings outside a 1- to 10-μm thin-sectioned area and lacks information on tissue organization, especially in a particular irregular structure such as crescentic glomeruli. In this study, we introduce an easy-to-use and scalable method for obtaining high-quality images of molecules of interest in a large tissue volume, enabling a comprehensive evaluation of the 3D organization and cellular composition of kidney tissue, especially the glomerular structure. We show that CUBIC and ScaleS clearing protocols could allow a 3D analysis of the kidney tissues in human and animal models of kidney disease. We also demonstrate that the paraffin-embedded human biopsy specimens previously examined via 2D evaluation could be applicable to 3D analysis, showing a potential utilization of this method in kidney biopsy tissue collected in the past. In summary, the 3D analysis of kidney biopsy provides a more comprehensive analysis and a minimized selection bias than 2D tissue analysis. Additionally, this method enables a quantitative evaluation of particular kidney structures and their surrounding tissues, with the potential utilization from basic science investigation to applied diagnostics in nephrology.

Cellular Interactions within Clonal Dendritic Cell Aggregates Drive Immune Tolerance in Chronic Myelomonocytic Leukemia Bone Marrow Microenvironment

Introduction: Clonal dendritic cell (DC) aggregates in chronic myelomonocytic leukemia (CMML) are seen in approximately 20-30% patients at diagnosis. Previously, these were thought to be comprised of CD123+ plasmacytoid DCs but our data has suggested that these are heterogenous and comprise of myeloid DCs, monocytes, and myeloid-derived suppressor cells. Furthermore, these aggregates uniformly express immune checkpoints such as indoleamine 2,3-dioxygenase-1 (IDO), associate with an IDO-specific systemic impact (increased trytophan catabolism), and expanded regulatory T cell population in CMML, suggesting a role in inducing immune tolerance. DC aggregates are also associated with increased risk of acute myeloid leukemia (AML) transformation and resistance to hypomethylating agents, highlighting that they are important for disease progression in CMML (Mangaonkar A et al. Blood Adv 2020). We performed this study to decipher the architecture of a CMML DC aggregate to understand the complex inter-cellular interactions, and assess their impact on the adaptive immune system. Methods: In this study, we used archival fresh-frozen paraffin-embedded (FFPE) bone marrow biopsy specimens obtained from CMML patients, and isolated monocytes (CD14+ magnetic bead sorting) and lymphocytes (CD14-ve fraction) from untreated CMML and age-matched normal control (NC) peripheral blood mononuclear cells (PBMCs). DCs were cultured from monocytes using a commercial protocol. Spatial architecture of the CMML BM microenvironment was interrogated through multiplex imaging of FFPE samples by co-detection by indexing (CODEX) using 30 markers (CCR6, CD107a, CD11c, CD20, CD21, CD31, CD3e, CD4, CD44, CD45, CD45RO CD56, CD68, CD8, E-Cadherin, FoxP3, HLA-DR, CD14, CD16, CD11b, CD33, CD15, CD66b, CD123, CD303, CD34, CD163 and IDO). Single-cell transcriptomic signatures were analyzed using the 10X single cell Fixed RNA profiling platform, and validated using bulk RNA seq (Illumina stranded mRNA prep) from isolated and cultured cells. Impact of DCs on lymphocytes was assessed via autologous mixed DC-T culture experiments. Results: BM FFPE samples from 5 CMML cases (3 with DC aggregates) were included for multiplex imaging via CODEX. Results confirm the significant interactions of CD8+ and CD4+ T cells with CD123+/HLA-DR+ DCs within a CMML DC aggregate (Fig. 1A). Upon quantitative assessment of cell-cell interactions, IDO+/CD8+T ( P=0.006), IDO+/MDSC ( P=0.005), and IDO+/macrophage ( P=0.001) interactions were significantly increased in CMML samples with versus without DC aggregates . Specific findings were validated using single-stain immunohistochemistry and multiplex immunoflourescence staining. Next, in order to assess transcriptomic signatures within BM at the single-cell level, we chose two cases; CMML (BM aspirate blast% 1, WBC 49.6 x 10 9/L, ASXL1/GATA2/SETBP1/SRSF2 mutations) and AML transformed from CMML (BM aspirate blast% 81, WBC 152 x 10 9/L, NPM1/DNMT3A/TET2 mutations). Approximately, 10,000 cells were obtained and analyzed with cell type annotation through human PBMC reference (Azimuth). Results show an expanded regulatory T cell population in AML compared to CMML (UMAP plot, Fig. 1B), suggestive of immune tolerance. In order to validate findings and perform functional experiments, we used sorted monocytes and cutured DCs from CMML and NC. DC phenotype was confirmed both through visual inspection and flow cytometry, and clonality was confirmed through Sanger sequencing (for previously identified mutations). Differential gene expression analysis through bulk RNAseq showed significant differences between CMML and NC lymphocytes with immunoinhibitory receptor, LILRB2 (log2fold change=7.05, adjusted P=0.0002) being one of the top five significantly upregulated genes, further confirming immune tolerance. Finally, mixed autologous DC-lymphocyte reaction (1:4 ratio) experiments demonstrated that DCs induce T cell tolerance in CMML (expansion of exhausted PD1/TIM3/LAG3+ CD8 T cells) when compared to NC. Conclusion: Clonal dendritic cell aggregates within the CMML bone marrow microenviroment interact with T cells and induce immune tolerance. This represents a novel mechanism of disease progression in CMML with potential therapeutic targets (IDO, LILRB2, and/or CD123).

Prognostic Value of Hypoxia Gene Expression Signature in Early-Stage Non-Small Cell Lung Cancer Patients Treated with Stereotactic Body Radiation Therapy

Stereotactic body radiation therapy (SBRT) is an effective alternative to surgery in the treatment of early-stage non-small cell lung cancer (NSCLC). However, disease recurrence occurs in approximately 20-30% of patients. Hypoxia is a well-known factor promoting solid tumor progression, radiation resistance, and immune evasion. In this study, we utilize a previously published hypoxia gene expression signature (Buffa) and measure its association with clinical outcomes in patients with NSCLC treated with SBRT. Our study focused on patients with localized, node-negative NSCLC treated with SBRT, and we identified 92 patients treated at our institution between 2008 and 2018 with available gene expression data. Total RNA from formalin-fixed paraffin-embedded archival biopsy specimens (pre-therapy) was isolated and mRNA expression was analyzed using an assay for human samples. For each gene in the Buffa signature, the top 50% of mRNA abundance values were given a score of +1, and the bottom 50% were given a score of -1 to generate a tumor hypoxia score. High scores suggest a hypoxic tumor and low scores imply normoxia. Kaplan-Meier curves were used to assess overall survival, disease-free survival, local recurrence, regional nodal recurrence, and distant recurrence. Cox proportional hazards were performed to account for confounding due to age, T stage, ECOG performance status, biologically effective dose, patient sex, and histology (squamous vs non-squamous). The patient's gene expression data were dichotomized based on their median hypoxia score (Table 1). Median follow-up was 23.9 months (95% CI 20.6 - 26.6). A high hypoxia score was associated with squamous histology (p = 0.003). On univariate analysis, a high hypoxia score was significantly associated with local recurrence (hazard ratio [HR] = 5.63; 95% CI 1.03 - 30.78; p = 0.046) and distant recurrence (HR = 1.85; 95% CI 1.14 - 3.02; p = 0.013). There was no significant difference in overall survival (HR = 1.93; 95% CI 0.98 - 3.82; p = 0.058), disease-free survival (HR = 2.13; 95% CI 0.93 - 4.89; p = 0.074), or regional nodal recurrence (HR = 1.45; 95% CI 0.41 - 5.14; p = 0.57). On multivariate analysis, a high hypoxia score was associated with worse overall survival (HR = 2.73; 95% CI 1.23 - 6.07; p = 0.014), local recurrence (HR = 12.85; 95% CI 1.08 - 152.49; p = 0.043) and distant recurrence (HR = 1.91; 95% CI 1.13 - 3.22; p = 0.016). Our findings demonstrate that a hypoxia gene signature is significantly associated with worse survival and increased local and distant recurrence after SBRT for localized NSCLC. This hypoxia signature may be useful in prognostication, selecting patients for surgery versus radiation, or selecting patients for treatment intensification with radiation or other systemic agents, and should be prospectively validated.

A PI3K gene expression signature predicts for recurrence in early-stage non-small cell lung cancer treated with stereotactic body radiation therapy.

Increasingly, early-stage non-small cell lung cancer (NSCLC) is treated with stereotactic body radiation therapy (SBRT). Although treatment is generally effective, a small subset of tumors will recur because of radioresistance. Preclinical studies suggested PI3K-AKT-mTOR activation mediates radioresistance. This study sought to validate this finding in tumor samples from patients who underwent SBRT for NSCLC. Patients with T1-3N0 NSCLC treated with SBRT at our institution were included. Total RNA of formalin-fixed paraffin-embedded tumor biopsy specimens (pretherapy) was isolated and analyzed using the Clariom D assay. Risk scores from a PI3K activity signature and four published NSCLC signatures were generated and dichotomized by the median. Kaplan-Meier curves and Cox regressions were used to analyze their association with recurrence and overall survival (OS). The PI3K signature was also tested in a data set of resected NSCLC for additional validation. A total of 92 patients were included, with a median follow-up of 18.3months for living patients. There was no association of any of the four published gene expression signatures with recurrence or OS. However, high PI3K risk score was associated with higher local recurrence (hazard ratio [HR],11.72; 95% CI, 1.40-98.0; p=.023) and worse disease-free survival (DFS) (HR,3.98; 95% CI, 1.57-10.09; p=.0035), but not OS (p=.49), regional recurrence (p=.15), or distant recurrence (p=.85). In the resected NSCLC data set (n=361), high PI3K risk score was associated with decreased OS (log-rank p=.013) but not DFS (p=0.54). This study validates that higher PI3K activity, measured by gene expression, is associated with local recurrence and worse DFS in early-stage NSCLC patients treated with SBRT. This may be useful in prognostication and/or tailoring treatment, and merits further validation.

Interim analysis examining the feasibility of incorporating laser microdissection enrichment of tumor specimens and reverse phase protein array analysis with next generation sequencing into a molecular tumor board.

e15068 Background: Since next generation sequencing (NGS) based profiling does not provide measures of protein abundance or activation state, clinical integration of reverse phase protein array (RPPA) with NGS could be useful for improving selection of targeted cancer therapy. We incorporated biopsy testing and proteogenomic analyses in an institutional Molecular Tumor Board (MTB) for cancer patients using CLIA-certified commercially available multi-omic platforms, including a commercially available CLIA RPPA functional protein drug target activation mapping platform. We present an interim analysis of our experience integrating functional phosphoprotein/protein based drug target activity data with NGS from 117 patients with metastatic solid tumors. Methods: Formalin-fixed, paraffin-embedded (FFPE) tumor biopsy specimens (n = 166) were obtained prospectively from patients as part of an IRB approved MTB. A representative H&amp;E-stained tissue section for each specimen was reviewed by a board-certified surgical pathologist. Specimens with sufficient total tumor cellularity to achieve protein lysate input requirements for RPPA were thin sectioned onto laser microdissection (LMD) membrane slides for selective harvest of tumor epithelium by LMD and RPPA analysis. In parallel, specimens from each case were also submitted for NGS analysis. The multi-omic data for each patient was reviewed by a panel of clinicians and scientists as part of the Inova Institutional MTB for clinical decision-making. Results: Patient tissue specimens (median = 1 per patient; range 1-5) were reviewed to assess feasibility of enriching tumor areas via LMD prior to RPPA analysis. Specimens from 64/117 patients were used for LMD, RPPA, and NGS. 48/117 patients were dropped due to inadequate tumor cellularity, patient death, or referral cases with inadequate specimen. At this time, 7/117 patients remained in the consenting-LMD-RPPA workflow. The median time from patient consent to RPPA analysis completion was 47.4 days. During this period, specimens were outside of the standard hospital-NGS workflow (ie, utilized for sectioning and review for LMD-RPPA) for ~10 days (median). Integrated review of the RPPA and NGS data by the MTB supported a clinical recommendation change for 34/64 patients (53%) overall, with some dependency by primary disease site. Clinical trial information: U20-11-4308.Conclusions: Incorporation of RPPA analysis of LMD enriched tumor samples with NGS was feasible in this pan-cancer cohort, with the mean time to results being less than 7 weeks. RPPA data provided additional treatment considerations for 59% of patients, the outcomes for whom continue to be monitored. [Table: see text]

Assessing Oral Epithelial Dysplasia Risk for Transformation to Cancer: Comparison Between Histologic Grading Systems Versus S100A7 Immunohistochemical Signature-based Grading.

While a 3-tier oral epithelial dysplasia grading system has been utilized for decades, it is widely recognized as a suboptimal risk indicator for transformation to cancer. A 2-tier grading system has been proposed, although not yet validated. In this study, the 3-tier and 2-tier dysplasia grading systems, and an S100A7 immunohistochemical signature-based grading system were compared to assess prediction of risk of transformation to oral cancer. Formalin-fixed, paraffin-embedded biopsy specimens with known clinical outcomes were obtained retrospectively from a cohort of 48 patients. Hematoxylin and eosin-stained slides were used for the 2- and 3-tier dysplasia grading, while S100A7 for biomarker signature-based assessment was based on immunohistochemistry. Inter-observer variability was determined using Cohen's kappa ( K ) statistic with Cox regression disease free survival analysis used to determine if any of the methods were a predictor of transformation to oral squamous cell carcinoma. Both the 2- and 3-tier dysplasia grading systems ranged from slight to substantial inter-observer agreement ( Kw between 0.093 to 0.624), with neither system a good predictor of transformation to cancer (at least P =0.231; ( P >>>0.05). In contrast, the S100A7 immunohistochemical signature-based grading system showed almost perfect inter-observer agreement ( Kw =0.892) and was a good indicator of transformation to cancer ( P =0.047 and 0.030). The inherent grading challenges with oral epithelial dysplasia grading systems and the lack of meaningful prediction of transformation to carcinoma highlights the significant need for a more objective, quantitative, and reproducible risk assessment tool such as the S100A7 immunohistochemical signature-based system.

Abstract P4-09-10: Spatial protein and RNA expression in tumor, immune and stromal cells in BRCA1/2 mutated metastatic breast cancer

Abstract Background: Spatial transcriptomics and proteomics are cutting-edge techniques that allow for quantification of RNA and protein expression within separate cell populations or regions of interest in a tissue biopsy specimen. We aimed to independently characterize the tumor cells and immune infiltrate in metastatic site biopsy specimens from patients with somatic or germline BRCA1/2 mutations. Methods: We performed spatial transcriptomics and proteomics using the Nanostring GeoMx Digital Spatial Profiler (DSP) on 9 formalin-fixed paraffin-embedded (FFPE) tissue biopsy specimens from 5 patients with metastatic breast cancer and a known somatic or germline BRCA1/2 mutation. Each patient had 1-3 separate metastatic site tissue biopsy specimens. Cell populations of interest were delineated with fluorescently labeled antibodies to pan-cytokeratin on tumor cells, CD45 of immune cells, and a nuclear stain. On each sample, 4-5 regions of interest (ROIs) were selected near the interface of the metastatic tumor deposit and surrounding normal tissue. Each ROI was segmented into 3 areas of interest (AOIs): pan-cytokeratin-positive tumor cells, CD45-positive immune cells, and the remaining stromal cells negative for both pan-cytokeratin and CD45. Data was background corrected and normalized with internal negative and positive internal controls. Relative abundance of immune cell subtypes was derived from CD45+ cell gene expression data using the TIMER algorithm. Results: The expression of 57 proteins and 84 RNAs was quantified in each of the 396 AOIs. Separate analysis of CD45+ cells, pan-cytokeratin cells, and stromal cell populations confirmed expected differential expression of immune, tumor and stromal markers, respectively. Protein expression of the estrogen receptor (ER) and progesterone receptor (PR) on pan-cytokeratin-positive tumor cells using the DSP correlated with pathologist reviewed immunohistochemistry (IHC) for ER and PR. Significant differential expression was found within each cell population between hormone receptor (HR) positive and negative cohorts, including enrichment for S100B in HR negative samples. Comparison will be shown for expression of 14 overlapping genes/proteins. Immune cell compartment gene expression deconvolution showed heterogeneity in the relative proportion of infiltrating immune cell subtypes between samples and also between ROIs within a sample. Changes in gene and protein expression from a patient with 3 serial biopsy specimens can be tracked with substantial heterogeneity across distinct AOIs within individual specimens and across metastatic sites.. Conclusions: Spatial transcriptomics and proteomics is feasible for concurrent, relative quantification of protein and gene expression in tumor, immune, and stromal cells in FFPE tissue biopsies from patients with metastatic breast cancer harboring germline or somatic BRCA1 or BRCA2 mutations. Heterogeneity within individual samples and across serial time points offers opportunity for methods development in the analyses and interpretation of spatial transcriptomic/proteomic data. Citation Format: Katharine A. Collier, Katherine Miller, Zaibo Li, Jesse Westfall, David Tallman, Mark Vater, Gabriel Tinoco, Elaine Mardis, Daniel Stover. Spatial protein and RNA expression in tumor, immune and stromal cells in BRCA1/2 mutated metastatic breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-09-10.

Abstract P5-02-11: Immune cell profile of tumors from patients with metastatic (met) HER2+ breast cancer (BC) with &amp;lt; 30 months overall survival (OS)

Abstract Background: The “Thousand Patient HER-2 database” project at Saint Vincent’s University Hospital (SVUH) Dublin has been used to identify HER2+ BC patients with durable complete response (never relapsed) to trastuzumab-based therapy. ~10% of met HER2+ BC patients achieve a durable complete response to trastuzumab, meaning the majority of patients progress on treatment. Higher stromal tumour immune infiltrate has been associated with longer OS in met HER2+ BC. There is limited tumor immune profile and PD-1 expression data available for patients with met HER2+ BC with short OS. Using the SVUH database, we have identified a preliminary cohort of 21 met HER2+ BC patients that received trastuzumab and had an OS &amp;lt; 30 months. This study examines the levels of pan T cell marker CD3, cytotoxic T cell marker CD8, Natural Killer (NK) cell marker CD56 and immune checkpoint PD-1 by immunohistochemistry (IHC) in this preliminary cohort. Methods: Formalin-fixed, paraffin-embedded (FFPE) biopsy specimens (n=21 primary, n=7 matched metastatic biopsies) and associated clinico-pathological data were curated. Tumor biopsies were processed for IHC staining of CD8 (Agilent IR62361-2), CD3 (Agilent IR50361-2), CD56 (Agilent IR62861-2) and PD-1 (Roche 07099029001). PD-1 staining was available for 20/21 samples. Staining was performed using the DAKO Link 48 Autostainer as per the manufacturer’s instructions using positive (tonsil tissue) and negative controls (isotype controls). Slides were processed using the Aperio AT2 Digital Slide Scanner (Leica Biosystems), reviewed using Aperio ImageScope 12.4 software (Leica Biosystems) and analyzed in QuPath (University of Edinburgh). Images were annotated to outline tumor areas and an algorithm was trained to identify cells and classify them as either tumor or stromal. Data was expressed as number of positively stained cells/mm2 breast tumor or stromal tissue. Survival studies utilized the Kaplan Meier method. The paired Student’s T test was utilized for primary vs metastatic site comparisons. Results: Designating samples with &amp;gt; 1 stained cell/mm2 breast tumor as positive (pos) and zero stained cells as negative (neg), 19/21 (90.5%) primary samples were pos for CD3, 15/21 (71.4%) for CD56, 14/21 (66.6%) for CD8, and 10/20 (50%) for PD-1. Within the stromal compartment, 20/21 (95.2%) primary samples were pos for CD8, 18/21 (85.7%) for CD56, 16/21 (76.2%) for CD3 and 8/20 (40%) for PD-1. PD-1 expression in the primary tumor (median OS PD-1pos 7.85 mo vs PD-1neg 5.39 mo, hazard ratio (HR) 0.642 (95% CI 0.256-1.613), p=0.346) or the stroma (median OS PD-1pos 8.84 mo vs PD-1neg 5.39 mo, HR 0.495 (95% CI 0.197-1.244), p=0.135) was not significantly associated with OS. When comparing matched primary and metastatic samples (n=7), increased stromal levels of CD3 (4/7), CD8 (4/7), CD56 (5/7) and PD-1 (4/7) were observed. Increased levels of CD3 and CD8 were observed for 2/7 samples, and increased levels of CD56 and PD-1 for 4/7 samples. With the exception of tumor CD8 levels which decreased, mean values for tumor and stromal CD3, CD56, PD-1 and stromal CD8 levels were higher in metastatic sites but all differences were not found to be significant (p&amp;gt;0.05). Conclusions: Our results suggest that met HER2+ BC patients with &amp;lt; 30 months OS have significant T cell and NK cell presence in the tumor and stromal compartments in both primary and metastatic sites. Further expansion of this limited dataset is planned to gain greater insight in to the immune cell profiles and PD-1 status of met HER2+ BC patients with short OS. Citation Format: Denis M. Collins, Janet McCormack, Laura P. Ivers, Jose Javier Berenguer Pina, Jo Ballot, Cecily Quinn, Darko Skrobo, Alex J. Eustace, Naomi Walsh, Aurelie Fabre, John Crown. Immune cell profile of tumors from patients with metastatic (met) HER2+ breast cancer (BC) with &amp;lt; 30 months overall survival (OS). [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-11.

Diagnostic utility of special stains in defining the spectrum of maxillofacial pathologies.

The Fine needle aspiration cytology (FNAC) is considered as a valuable and distinguished diagnostic test in the initial assessment of the patients presenting with a mass in the head and neck region or when a recurrence is suspected after previous treatment. This study was therefore designed to elucidate the efficacy of FNAC as an alternate diagnostic tool to histopathology in head and neck swellings and evaluation of staining efficacy of PAP and MGG stain over Haematoxylin and eosin (H and E) in routine cytopathological smears. The study was conducted in the Department of Oral and Maxillofacial Pathology, where FNAC samples were collected from 150 patients with head and neck swellings. All the slides were stained with H and E, Papanicolaou (PAP), and May Grunewald Giemsa (MGG) stains. The cytopathological diagnosis was compared with histopathological diagnosis based on H and E stained sections obtained from paraffin-embedded formalin-fixed biopsy specimen of benign and malignant neoplasms. The resulting data were analyzed using SPSS software version 19. Differences between the variables were analyzed using Pearson Chi-square test and Kruskal-Wallis test wherever applicable. The FNAC as a diagnostic tool has sensitivity of 84.8%, 72.72%, and 78.78%, specificity of 62.5%, 75%, and 75%, and accuracy of 80.48%, 73.14%, and 78.04% in H and E, MGG, and PAP stain, respectively. PAP stain was the most efficient stain when all qualitative parameters are taken into consideration with maximum sensitivity and specificity for achieving definitive cytodiagnosis. The FNAC is an inexpensive and minimally invasive technique to diagnose different types of head and neck swellings and complement histopathological diagnosis.

Special Anatomical Subtypes of Diffuse Large B-Cell Lymphoma Have Distinct Tumor Microenvironments

Background The heterogeneity of diffuse large B-cell lymphoma (DLBCL) and its diverse clinical course encouraged the World Health Organization (WHO) to recognize specific subtypes based on anatomical location. These anatomical subtypes are accompanied by specific mutational profiles and gene-expression patterns. For example, the poor-prognostic primary DLBCL of immune-privileged sites (IP-DLBCL) originating in the central nervous system or testis have frequent NF-κB-activating mutations and a predominantly activated B-cell (ABC) phenotype. In contrast, we recently showed that good-prognostic bone DLBCL (B-DLBCL) have frequent mutations in immune-modulating and epigenetic regulating genes and a germinal center B-cell (GCB) phenotype (PMID: 34478526). We hypothesize that these remarkable anatomical site-specific enrichments for tumors with a distinct molecular signature are associated with different local anti-tumor immune responses. Here, IP-DLBCL and B-DLBCL could be considered opposing ends of a spectrum and we compared the composition of the tumor microenvironment (TME) of these entities to each other and to DLBCL with nodal localization only (nodal-DLBCL). Method This retrospective study used formalin fixed and paraffin embedded biopsy specimens that were selected from clinically, histologically and molecularly well-annotated cases of IP-DLBCL (n=29), B-DLBCL (n=21) and nodal-DLBCL (n=18). All cases were diagnosed according to the revised 2016 WHO classification. High grade B-cell lymphomas (double/triple hit) were excluded. Imaging mass cytometry (Hyperion) was used to define the TME at single-cell resolution and with intact spatial relationships. The panel comprised 41 antibody-markers for deep phenotyping of four major cellular compartments: tumor cells, tumor-infiltrating lymphocytes, myeloid- and stromal cells. Image output was processed to create single-cell data, as described before (PMID: 34196108). Cell phenotyping was performed by supervised clustering after dimension reduction using opt-SNE algorithm (OMIQ software). Cluster abundances were depicted as median (±IQR) per thousand of total cells per patient(‰). Kruskal-Wallis testing was used to determine statistical differences between abundances. Results In total 1,7M single cells (range 7-49k) were obtained and 90 unique cellular phenotypes were defined. Differences in composition of the four cellular compartments were compared between IP-DLBCL and B-DLBCL (Figure 1). The lymphocyte compartment of IP-DLBCL was significantly enriched with granzyme B expressing lymphocytes, mainly cytotoxic T cells (IP 9,4‰ ± 8,8 versus B-DLBCL 2,7‰ ± 3,8; p<0,01), including activation related markers like CCR4 and Ki-67. In contrast, B-DLBCL was characterized by a substantial influx of T cells, more specifically T-helper cells (B-DLBCL 49,9‰ ± 32,5 versus IP 35,4‰ ± 24,6; p<0,01), including T-regulatory cells (B-DLBCL 2,7‰ ± 4,4 versus IP 0,9‰ ± 1,5; p<0,01) and cells expressing ICOS. T-helper 1 cells, expressing TBET, were less abundant (B-DLBCL 0,3‰ ± 0,5 versus IP 1,0‰ ± 0,6; p<0,01). The myeloid compartment was generally increased in B-DLBCL and comprised of HLADR and/or CD11c expressing dendritic cells (B-DLBCL 84,3‰ ± 39,8 vs IP 32,5‰ ± 31,9; p<0,01). In the stromal compartment, a significantly higher expression of activation markers related to the "stromal1" gene signature (PMID: 34597589) in B-DLBCL was identified (B-DLBCL 15,4‰ ± 12,4 vs IP 10,98‰ ± 9,0; p=0,04). Immune checkpoint markers like PD1, LAG3, TIM3, TIGIT and VISTA were generally low in expression within the entire cohort. In nodal-DLBCL both IP- as well as B-DLBCL-like phenotypes were identified. Conclusion This study shows significant differences in the tumor microenvironment of DLBCL subtypes based on anatomical site. In IP-DLBCL, substantial presence of cytotoxic T cells-with activated phenotypes-was identified. In contrast, in bone-DLBCL the TME was generally non-cytotoxic and exhibited immune regulatory elements. Altogether, knowledge on these distinct tumor microenvironments may help understanding heterogeneity between these subtypes and advocates correlation with efficacy of emerging treatments like bispecific antibodies (for the cytotoxic TME) and CAR T-cell therapy (for the non-cytotoxic/immune regulatory TME). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Comparison between Colorimetric In Situ Hybridization, Histopathology, and Immunohistochemistry for the Diagnosis of New World Cutaneous Leishmaniasis in Human Skin Samples.

New world cutaneous leishmaniasis (NWCL) is an anthropozoonosis caused by different species of the protozoan Leishmania. Colorimetric in situ hybridization (CISH) was shown to satisfactorily detect amastigote forms of Leishmania spp. in animal tissues, yet it was not tested for the diagnosis of human NWCL. The aim of this study was to compare CISH, histopathology (HP), and immunohistochemistry (IHC) techniques to diagnose NWCL in human cutaneous lesions. The sample comprised fifty formalin-fixed, paraffin-embedded skin biopsy specimens from patients with NWCL caused by L. (V.) braziliensis. These specimens were analyzed by CISH, using a generic probe for Leishmania, IHC, and HP to assess the sensitivity of these methods by using a parasitological culture as a standard reference. Additional specimens from three patients diagnosed with cutaneous mycoses were also included to evaluate cross-reactions between CISH and IHC. The sensitivities of IHC, CISH, and HP for detecting amastigotes was 66%, 54%, and 50%, respectively. IHC, unlike CISH, cross-reacted with different species of fungi. Together, these results demonstrate that CISH may be a complementary assay for the detection of amastigote in the laboratorial diagnosis routine of human NWCL caused by L. (V.) braziliensis.

A PI3Kinase/Akt/mTOR Gene Expression Signature Predicts for Recurrence in Early-Stage Non-Small Cell Lung Cancer Patients Treated with Stereotactic Body Radiation Therapy

<h3>Purpose/Objective(s)</h3> Stereotactic body radiation therapy (SBRT) has emerged as an effective alternative to surgery in the treatment of early-stage non-small cell lung cancer (NSCLC). Despite this, disease progression occurs in over 20-30% of patients. Previous work established AKT/mTOR signaling as a pathway modulating response to radiation for NSCLC. In this study, we sought to validate a previously published mRNA signature that measures PI3K/Akt/mTOR pathway activation and measure its association with clinical outcomes in patients with early-stage NSCLC treated with SBRT. <h3>Materials/Methods</h3> We queried our institutional dataset for patients with T1-3N0 NSCLC treated with SBRT. RNA of formalin fixed paraffin embedded biopsy specimens (pre-therapy) was isolated and mRNA expression was analyzed using the Clariom D^TM assay. We derived a t score, defined as the two-sided t statistic comparing the average of the PI3K-induced genes with that of repressed genes within each tumor after centering log-transformed values on the median across samples. Cox proportional hazards was performed to account for age, sex, ECOG performance status, biological effective dose, and histology. <h3>Results</h3> We identified 92 patients with NSCLC treated between 2008 and 2018 with available gene expression and dichotomized patients by their median PI3K pathway score (<i>Table 1</i>). Median follow up was 24.0 months. Higher PI3k score was associated with worse progression-free survival (PFS) (HR= 3.98; 95% CI 1.57–10.09; p=0.0035) and local recurrence (LR) (HR= 11.72; 95% CI 1.40–98.0; p=0.023). There was no significant association with overall survival (p=0.49), regional nodal recurrence (p=0.15), or distant recurrence (p=0.85). On multivariable analysis, PI3K score remained independently associated with worse PFS (HR= 3.03; 95% CI 1.13–8.13; p=0.028) and LR (HR= 17.15; 95% CI 1.50–195.59; p=0.022). Worse performance status (HR= 2.58; 95% CI 1.06–6.27) and younger age (HR= 0.94; 95% CI 0.88–0.999) were also associated with worse PFS. <h3>Conclusion</h3> Our findings demonstrate that degree of PI3K pathway activation is associated with PFS and LR after SBRT for early-stage NSCLC. This requires validation in an independent dataset but may be useful in prognostication or in selecting patients for treatment intensification.