Paraffin-embedded Tumor Specimens Research Articles

Abstract 5183: Retrospective analysis of CCNG1 expression in tumors, a novel biomarker in development for partnering with DRG-101, a tumor targeted retrovector encoding a CCNG1 inhibitor gene

Abstract Background: CCNG1, a non-canonical cyclin, is a novel biomarker in development for partnering with DRG-101 (DeltaRex-G), a tumor targeted retrovector encoding a CCNG1 inhibitor gene. Expanded access using DRG-101 is currently open for an intermediate-size population of advanced pancreatic cancer, sarcoma and carcinoma of breast (NCT04091295). Therefore, it is important to identify patients who might respond favorably to DRG-101 gene therapy. Previously, we reported that CCNG1 expression is enhanced in sarcoma tumors (Ann Oncol 34, 1980P, 2023). In this report, we have expanded the analysis to 137 patients with all cancer types and describe patients with known CCNG1 expression levels who have been treated with DRG-101. Methods: Archived formalin-fixed paraffin-embedded (FFPE) tumor specimens (n=137) from patients who were followed at the Sarcoma Oncology Research Center were collected, processed, and subjected to RNA sequencing. Briefly, RNA-seq libraries were sequenced to generate 50 million reads, aligned using Kallisto v0.42.4 to GENCODE v23 transcripts with default parameters. Only protein-coding, IGH/K/L- and TCR-related transcripts were retained for downstream processing, resulting in 20,062 protein-coding genes. Gene expression levels in patients of the reference cohorts are categorized as low/medium/high, defined by Low = < 17%; Medium-high = 50%-83%; Medium-low = 17%-49%; High = >83%. Results: Seventy women and sixty-seven men, ranging in age from 16 to 87 years, were studied. All tumors showed enhanced CCNG1 expression. Thirty (22%) tumors exhibited high CCNG1 expression, 53 (39%) showed medium-high CCNG1 expression, 48 (35%) displayed medium-low CCNG1 expression, and six (4%) had low CCNG1 expression. One hundred (73%) patients had metastatic disease, and 37 (27%) had localized disease. There was no correlation between CCNG1 expression and disease stage. As a result of these findings, the FDA-CBER authorized the use of DRG-101 in an Expanded Access program as platform therapy upon which one or more FDA approved cancer drugs/immunotherapies may be added. Historically, one patient with metastatic pancreatic cancer, one with early stage, HR+ HER2+ invasive breast cancer and one with early stage triple negative breast cancer are alive 15, 3 and 2.5 years in sustained remission with DRG-101 therapy, and had 24%, 23% and 74% CCNG1 tumor expression levels respectively. Five patients with advanced pancreatic cancer, sarcoma and breast cancer and known CCNG1 expression levels are currently being treated with DRG-101. Conclusions:(1) 100% of tumors tested showed enhanced CCNG1 expression at varying levels, and (2) the use of DRG-101 in combination with FDA approved cancer drugs/immunotherapies is on-going for an intermediate size population of advanced pancreatic cancer, sarcoma and carcinoma of breast. Citation Format: Nadezhda Omelchenko, Vaishali Kumar, Samantha Jeffrey, Grace Haroun, Don Brigham, William H. Isacoff, Sant Chawla. Retrospective analysis of CCNG1 expression in tumors, a novel biomarker in development for partnering with DRG-101, a tumor targeted retrovector encoding a CCNG1 inhibitor gene [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 5183.

Hypoxia-associated genetic signature in ovarian steroid cell tumor NOS.

Steroid cell tumors, not otherwise specified (SCT-NOS), are uncommon ovarian neoplasms accompanied by virilization symptoms due to hyperandrogenism, which are malignant in approximately one-third of the cases. Given the rarity of SCT-NOS, their molecular underpinnings have not yet been studied in depth. In this case series, we performed the first comprehensive analysis of the genetic landscape of this rare ovarian tumor. A detailed clinicopathological description of an index case is also provided. Over a 20-year period, a total of eight patients were seen at our institution. Total nucleic acids (RNA and DNA) were extracted from evaluable formalin-fixed, paraffin-embedded tumor specimens (n = 7) and subjected to TruSight Oncology 500 testing and/or exome sequencing. The results identified pathogenic variants in several hypoxia-related genes - including HIF1A, VHL, SDHB, SRC, IDH2, and FOXO4. As the first comprehensive genetic analysis of SCT-NOS, this study shows that dysregulation in the hypoxia signaling pathway is a key molecular feature of this rare tumor. Clinically, long-term follow-up with periodic measurements of androgen levels should be pursued in all cases since recurrences may occur several years after the initial diagnosis.

359. NFE2L2 MUTATIONS ARE PREDICTIVE AND PROGNOSTIC OF TREATMENT OUTCOME IN RESECTABLE ESOPHAGEAL SQUAMOUS CELL CARCINOMA PATIENTS

Abstract Background The poor prognosis of resectable esophageal squamous cell carcinoma (ESCC) poses an unmet need to identify genomic early predictive and prognostic biomarkers to improve treatment outcome and risk stratification. Methods Retrospective targeted sequencing was performed on 80 ESCC formalin-fixed, paraffin-embedded (FFPE) tumor specimens from 37 good responders and 43 poor responders receiving neoadjuvant treatment. Somatic mutations and copy number variations (CNVs) were detected in 130 cancer druggable genes with TOMA Os-seq. Kaplan Meier curve and COX regression analysis were used to estimate the risk of relapse and survival. Results Twelve patients (12/80, 15%) with hot spot NFE2L2 mutations occurred with significantly higher frequency in poor responders (p = 0.0003) and in patients with non-complete pathological response (p = 0.007) in resectable ESCC treated by chemo-radiotherapy (CRT). These patients also had poorer prognosis with higher risk of death (HR = 1.942, p = 0.039) and relapse (HR = 1.817, p = 0.063) by COX analysis. The independent prognostic role of hot spot NFE2L2 mutations was validated by the presence of recurrent plasma cell-free (ct)-DNA NFE2L2 mutations in an independent prospective cohort of resectable ESCC patients. Conclusions In summary, our study now demonstrates the clinical usefulness of hot spot NFE2L2 mutations, as a potential predictive and prognostic biomarker associated with pre-surgery CRT responses, and provides novel insights for future therapeutic options targeting the NRF signaling in resectable ESCC treated by neoadjuvant CRT. The novel prognostic role of hot spot NFE2L2 mutations may be useful markers to guide clinicians for treatment decisions in resectable ESCC patients following pre-surgery treatment.

Genomic predictors for early recurrence of hepatocellular carcinoma (HCC) after curative resection.

157 Background: Early recurrence of HCC after curative resection is common. However, the association between genetic alterations and early HCC recurrence, especially in Chinese patients (pts), remains largely unknown. Methods: We retrospectively analyzed 214 formalin-fixed paraffin-embedded (FFPE) tumor specimens of HCC patients that were collected from June 2016 to October 2021. Clinical outcomes related to frequently mutated genes were evaluated in 176 pts with a median follow-up time of 21.9 months. Tumor tissue samples were detected by OncoDrug-Seq 603-gene panel assay through next-generation sequencing. Early recurrence was defined as the appearance of an intra- or extrahepatic tumor nodule within 1 year of curative resection. Results: In multivariate analysis, three tumor suppressor genes related to early recurrence, namely PRDM1, NOTCH3, and MGA. PRDM1 mutations and NOTCH3 mutations were the risk factor for early recurrence (＜1 y after surgical resection) (hazard ratio (HR), 4.22; 95% confidence interval (CI), 1.96-9.06; p＜0.001 ) (HR, 2.96; 95% CI, 1.43-6.13; p=0.003). These two mutated genes were also significantly associated with overall survival (HR, 3.39; 95%CI, 1.23-9.34; p=0.018) (HR, 5.22; 95%CI, 2.10-12.99; p＜0.001). We found a trend toward an increased risk of early recurrence in pts with MGA mutation. (HR, 2.06; 95%CI, 1.05-4.05; p=0.027). Conclusions: Mutations in these three tumor suppressor genes were shown to be shorter time intervals to relapse after curative resection. We also demonstrated that pts who have these markers require more frequent monitoring and follow-up visits.

PI3K-CCL2-CCR2-MDSCs axis: A potential pathway for tumor Clostridia-promoted CD 8+ T lymphocyte infiltration in bile tract cancers

BackgroundMost patients with resected bile tract cancers (BTCs) survive for less than 5 years; however, some achieve better prognosis. The tumor microbiome can improve survival by regulating the tumor immune microenvironment. However, whether the tumor microbiome promotes immune cell infiltration in BTCs is unknown. This study aimed to determine the association between CD8+ T lymphocyte infiltration and the tumor microbiome in patients with resected BTCs. MethodsArchived formalin-fixed paraffin-embedded tumor specimens were collected from patients with resected BTCs and analyzed using 16S rRNA gene sequencing to identify that prognosis-related and significantly differentially enriched taxa. Gene ontology (GO) analysis of the differentially enriched taxa was used to assess how CD8+ T lymphocyte infiltration is affected by the tumor microbiome of BTCs. ResultsWe enrolled 32 patients with resected BTCs. The high CD8+ lymphocyte-infiltration (CD8hi) group had four significantly enriched taxa, and in the low CD8+ lymphocyte-infiltration (CD8low) group comprised one significantly enriched taxon. Patients with higher Clostridia abundance (enriched in the CD8hi group) experienced longer overall survival than those with lower abundance. The enrichment of Clostridia in the CD8hi group corresponded with lower CCL2 expression and downregulation of phosphatidylinositol 3-kinase activity, which might decrease myeloid-derived suppressor cell recruitment to the tumor milieu, thus increasing CD8+ lymphocyte infiltration in BTCs. ConclusionsThe tumor microbiome is related to CD8+ T lymphocyte infiltration in patients with resected BTCs. The relationship between tumor Clostridia and high infiltration of CD8+ T lymphocytes might reflect decreased recruitment of myeloid-derived suppressor cells via the PI3K-CCL2-CCR2 axis.

Detailed characterization of PD-1/PD-L1 and CTLA4 expression and tumor-infiltrating lymphocytes in yolk sac tumors

BackgroundImmune checkpoint blockade (ICB) is considered as a promising approach for cancer treatment. However, the potency of ICB therapy in yolk sac tumors (YSTs) has not been confirmed, and the comprehensive analysis of tumor immune microenvironment and the expression of PD-1/PD-L1 and CTLA4 were also not thoroughly evaluated. MethodsImmunohistochemistry was performed in formalin-fixed, paraffin-embedded tumor specimens from 23 YSTs patients to detect the density and distribution of tumor-infiltrating T cells, tertiary lymphoid structures (TLSs), as well as the expression of PD-1/PD-L1 and CTLA4. ResultsOverall, more than half (61 %) of all patients exhibited an immune-desert phenotype based on CD3+ T cells. PD-1 expression was identified in five tumor samples (21.7 %), and PD-L1 expression exhibited a different positive rate in tumor cells (TCs) and tumor-infiltrating lymphocytes (TILs) (39.1 % and 17.4 %). Noteworthily, the rate of positive CTLA4 expression in both TCs and TILs was markedly higher (69.6 % and 56.5 %) than those of PD-1 and PD-L1 expression. Furthermore, TLSs were observed in 21.74 % of all tissues, and samples with TLSs exhibited significantly higher densities of TILs and higher expression of immune checkpoint molecules, particularly PD-1/PD-L1. In addition, tumors located in testes also exhibited a higher density of TILs and higher expression of immune checkpoint molecules. ConclusionGenerally a high frequency of CTLA4 expression was found, PD-1/PD-L1 expression, the immune-inflamed phenotype, and TLSs were low frequency in YSTs, however, YSTs in testes showed a higher density of TILs and higher expression of immune checkpoint molecules.

Concordance in detection of microsatellite instability by PCR and NGS in routinely processed tumor specimens of several cancer types.

Microsatellite instability (MSI) occurs in several cancer types and is commonly used for prognosis and as a predictive biomarker for immune checkpoint therapy. We analyzed n = 263 formalin-fixed paraffin-embedded (FFPE) tumor specimens (127 colorectal cancer (CRC), 55 endometrial cancer (EC), 33 stomach adenocarcinoma (STAD), and 48 solid tumor specimens of other tumor types) with a capillary electrophoresis based multiplex monomorphic marker MSI-PCR panel and an amplicon-based NGS assay for microsatellite instability (MSI+). In total, n = 103 (39.2%) cases with a known defect of the DNA mismatch repair system (dMMR), determined by a loss in protein expression of MSH2/MSH6 (n = 48, 46.6%) or MLH1/PMS2 (n = 55, 53.4%), were selected. Cases with an isolated loss of MSH6 or PMS2 were excluded. The overall sensitivity and specificity of the NGS assay in comparison with the MSI-PCR were 92.2% and 98.8%. With CRC cases a nearly optimal concordance was reached (sensitivity 98.1% and specificity 100.0%). Whereas EC cases only show a sensitivity of 88.6% and a specificity of 95.2%, caused by several cases with instability in less than five monomorphic markers, which could be difficult to analyze by NGS (subtle MSI+ phenotype). MSI analysis of FFPE DNA by NGS is feasible and the results show a high concordance in comparison with the monomorphic marker MSI-PCR. However, cases with a subtle MSI+ phenotype, most frequently manifest in EC, have a risk of a false-negative result by NGS and should be preferentially analyzed by capillary electrophoresis.

Predictive multigenic scale. Analysis of own results in metastatic breast cancer

Background. Breast cancer is one of the most common female malignancies. Molecular diagnostic methods of tumor profiling allow us to analyze individual tumor characteristics, identify new prognostic and predictive markers. Aim. To increase the efficacy of systemic therapy for breast cancer and reduce inappropriate prescriptions using the data on individual molecular tumor characteristics; to develop a polygenic panel to ensure a tailored approach to systemic therapy for breast cancer. Materials and methods. We analyzed 84 tumor tissue samples from pre- and postmenopausal women with metastatic breast cancer who were treated and followed-up in 6 healthcare institutions. We assessed expression of genes involved in breast cancer. In a pilot study, we analyzed archived paraffin-embedded tumor specimens form 12 out of 1,216 patients with T1–2N0M0 breast cancer included into retrospective analysis. Gene expression was assessed using the nCounter technology based on direct digital detection of targets using fluorescent barcodes (nCounter Analysis System; NanoString Technologies, USA). Tumor tissue (biopsy and surgical specimens) was analyzed. The choice of genes was based on the literature data and experience in the development of other polygenic panels, as well as clinical significance of markers of prognostic scales. Gene mutations were confirmed by next generation sequencing and reverse transcription-polymerase chain reaction. Results. We analyzed the expression of 28 genes with a high predictive value that have been substantially studied (including ESR1, PGR, PIK3CA, BCAR4, BCAS2, CCND1, CCND2, CCND3, FOXA1, Erb2, EGFR, CDH3, FOXC1, KRT14, KRT5, CD274, CDK4, CDK6, P53, PTEN, BRCA1, BRCA2, CHEK2, CLDN3, CLDN7, AR, TOP2a, TUBBIII). We identified 29 cases of discrepancy (29 / 84; 34.5 %) in tumor subtype, including 11 cases of luminal A and B breast cancer, which might potentially affect the choice of the treatment regimen. In 18 cases, there were some principal discrepancies in the tumor subtype that implied totally different treatment regimens. The proposed polygenic signature allows accurate identification of the tumor subtype in patients with metastatic breast cancer and choice of an optimal treatment strategy. Conclusion. We have developed a 100-gene signature including molecular subtypes of breast cancer (luminal A, luminal B, basal, claudin-like) and treatment-oriented clusters. Molecular tumor profiling using this polygenic signature is an accurate method for determining tumor subtype in patients with breast cancer, which enables a tailored approach to therapy.

Spatial genomics to reveal an immunosuppressive tumor microenvironment after chemoradiation in head and neck cancer patients.

6059 Background: Although chemoradiotherapy (CRT) remains the cornerstone of management for locoregionally advanced head and neck squamous cell carcinomas (HNSCC), recurrence occurs in ~50% of patients. We sought to characterize the impact of CRT on the tumor microenvironment (TME) and identify factors associated with progression. Emerging spatial biology technologies, such as NanoString GeoMx Digital Spatial Profiler (DSP), enable spatially defined whole-transcriptomic profiling of cells along the tumor-stromal interface in tumor biopsies to resolve immune cell identities, activation states and signaling pathways in adjacent tumor cells. We used a multimodal approach including immunohistochemistry (IHC), bulk tumor gene expression and spatial genomics to characterize paired pre- and post-CRT tumor samples from HNSCC patients. Methods: 20 patients with locoregionally advanced HNSCC who had undergone curative intent CRT and developed subsequent recurrence requiring salvage surgery from June 14, 2010 to June 14, 2020 were retrospectively identified at Duke University Medical Center. Matched formalin-fixed paraffin embedded tumor specimens collected prior to CRT and at salvage surgery were compared via IHC for markers of immune infiltration and PDL1 expression, bulk tumor whole transcriptomic profiling, and spatial transcriptomic analysis of tumor and stroma using DSP were further examined in “early” progressors ( < 6 months after CRT) and “late” progressors (≥6 months after CRT). Results: IHC analyses demonstrated significant reductions in T-cell immune populations (total CD3+, CD8+, 4-1BB+ cell density [ p < 0.001]) after CRT. PDL1+ expression was similar before and after CRT. Neither T-cell infiltrate nor tumor PDL1 levels at surgery were associated with progression. Bulk tumor gene expression analysis demonstrated that macrophage and fibroblast markers were increased post-CRT. In early progressors, molecular dissection of the tumor-stromal interface using DSP expression profiling revealed macrophage associated genes ( CD68, MRC1/CD206, CD163, F13A1) at salvage surgery had a more pronounced increase in macrophage markers in stroma with enriched M2 genes compared to late progressors. Conclusions: Integrated spatial analysis with high resolution transcriptomics and IHC suggest CRT induces an immunosuppressive environment characterized by decreased T-cell infiltration and an influx of putative immunosuppressive macrophages. We hypothesize that tumor associated macrophages and/or fibroblasts may contribute to immunosuppression due to “reprogramming” of the TME after CRT. This exploratory study suggests that patients with progressive disease after chemoradiation may benefit from additional immunoregulatory therapies that ameliorate the immunosuppressive TME. These findings will be further validated in a larger cohort of patients.

A master screening study to determine biomarker status and trial eligibility for patients with malignant tumors.

TPS8608 Background: Targeting of oncogenic driver alterations has proven to be an effective clinical approach across many tumor types. As a result of the approval of targeted therapies against oncogenic driver alterations, molecular biomarker testing is commonly recommended for patients with advanced/metastatic non-small cell lung cancer (NSCLC) before initiation of first-line treatment. Although there are emerging clinical trial data using targeted therapies in patients with early-stage NSCLC, currently molecular biomarker testing is not universally performed as part of routine clinical practice in the early-stage setting. Screening platforms can be utilized to provide comprehensive, validated biomarker testing to facilitate access to clinical trials for appropriate investigational therapies, based on biomarker status. This type of approach aims to be more efficient and to streamline patient recruitment and improve patient access to clinical trials where biomarker testing is not currently performed as part of routine care, especially when the biomarkers of interest are at low prevalence. Methods: This global, non-Investigational Medicinal Product (non-IMP), multicenter master screening study (NCT05419375) provides centralized tissue-based testing to determine patients’ biomarker eligibility for linked Roche clinical trials. Currently this study is only open to screening patients for a linked platform study in unresectable stage III NSCLC (NCT05170204). However, it is envisaged that this screening study will be expanded to support additional clinical trials and indications in the future. Eligible patients must be aged ≥18 years with locally advanced, unresectable stage III NSCLC (according to AJCC 8th edition), an ECOG PS of 0–2, confirmed availability of a formalin-fixed paraffin-embedded tumor specimen obtained prior to chemoradiation, and adequate hematologic and end-organ function. If a patient is found to have an oncogenic driver mutation and is considered a good candidate for a linked clinical trial, the investigator may propose that they be screened into the respective study. Tumor tissue samples will be centrally tested by validated molecular tests for the presence of specific genetic alterations: EGFR, ALK, ROS1, KRAS, BRAF, HER2, RET, MET, and NTRK1/2/3. Immunohistochemistry may also be performed to determine PD-L1 expression as required by the linked trial eligibility criteria. The primary objective of this screening trial is to determine the biomarker status of patients and their eligibility to participate in a linked clinical trial. Exploratory objectives include characterization of biomarker profiles and patients’ subsequent treatment. No therapeutic intervention will be administered in this study. Enrolment is ongoing across 51 sites in ten countries, with a target enrolment of 15,000 patients. As of 9 February 2023, 27 patients have been enrolled. Clinical trial information: NCT05419375 .

B7H3 expression in sarcoma.

e23517 Background: Targeted therapy development in sarcoma has been burdened by the heterogeneity of this rare group of tumors. B7H3 is an immune checkpoint molecule in the same family as PD-L1 (also known as B7H1). Its limited expression in non-cancerous tissues and over-expression in numerous tumor types makes it an attractive target for cancer immunotherapy. Available data suggests high expression of B7H3 in certain sarcoma subtypes, including rhabdomyosarcoma, osteosarcoma, liposarcoma, Ewing sarcoma, synovial sarcoma and chondrosarcoma. This study was conducted to further explore B7H3 expression patterns across sarcoma subtypes and the relationship, if any, with PD-1 and PD-L1 expression. Methods: This single-institution, retrospective analysis evaluated soft tissue sarcoma Formalin-Fixed Paraffin-Embedded tumor specimens in patients with a variety of different subtypes. Specimens were evaluated for expression of B7H3, vessel positivity for B7H3, B7H3 staining pattern, and expression of PD-L1 and PD-1. The association between B7H3 expression and baseline and tumor factors was tested using the Mantel Haenszel test or two-sample T-test. Overall survival was estimated using the Kaplan-Meier method where survival time was calculated from diagnosis to death (or last follow-up for those last known to be alive). Results: 156 sarcoma specimens encompassing 16 subtypes were included. B7H3 was broadly expressed across sarcoma subtypes, with some degree of expression found in 91% of samples and 61% demonstrating high levels of B7H3 expression. All tumor samples expressing B7H3 exhibited the biomarker in at least 80% of tumor cells per sample (with a mean of 92.34% of cells testing positive for B7H3). All samples positive for B7H3 showed both cytoplasmic and membranous expression, and B7H3 vessel positivity was found in 90% (137/152) of samples. There were no significant differences in expression or level of expression when stratified by sarcoma subtype, prior treatment(s), tumor size, tumor grade or patient age. No significant difference in overall survival was seen regardless of B7H3 expression or expression levels across sarcoma subtypes. There was no correlation between B7H3 expression and expression of PD-L1 or PD-1 in tumor cells. Conclusions: This is the most comprehensive analysis of B7H3 expression in sarcoma to date. This data shows high levels of B7H3 expression across sarcoma subtypes, suggesting its feasibility as a therapeutic target for future sarcoma treatments. Future clinical trials are needed to evaluate its numerous actions and if manipulation of B7H3 can be employed to improve outcomes in sarcoma. [Table: see text]

Analytical validation of the 7-gene biosignature for prediction of recurrence risk and radiation therapy benefit for breast ductal carcinoma in situ.

Ductal carcinoma in situ (DCIS), is a noninvasive breast cancer, representing 20-25% of breast cancer diagnoses in the USA. Current treatment options for DCIS include mastectomy or breast-conserving surgery (BCS) with or without radiation therapy (RT), but optimal risk-adjusted treatment selection remains a challenge. Findings from past and recent clinical trials have failed to identify a 'low risk' group of patients who do not benefit significantly from RT after BCS. To address this unmet need, a DCIS biosignature, DCISionRT (PreludeDx, Laguna Hills, CA), was developed and validated in multiple cohorts. DCISionRT is a molecular assay with an algorithm reporting a recurrence risk score for patients diagnosed with DCIS intended to guide DCIS treatment. In this study, we present results from analytical validity, performance assessment, and clinical performance validation and clinical utility for the DCISionRT test comprised of multianalyte assays with algorithmic analysis. The analytical validation of each molecular assay was performed based on the Clinical and Laboratory Standards Institute (CLSI) guidelines Quality Assurance for Design Control and Implementation of Immunohistochemistry Assays and the College of American Pathologists/American Society of Clinical Oncology (CAP/ASCO) recommendations for analytic validation of immunohistochemical assays. The analytic validation showed that the molecular assays that are part of DCISionRT test have high sensitivity, specificity, and accuracy/reproducibility (≥95%). The analytic precision of the molecular assays under controlled non-standard conditions had a total standard deviation of 6.6 (100-point scale), where the analytic variables (Lot, Machine, Run) each contributed <1% of the total variance. Additionally, the precision in the DCISionRT test result (DS) had a 95%CI ≤0.4 DS units under controlled non-standard conditions (Day, Lot, and Machine) for molecular assays over a wide range of clinicopathologic factor values. Clinical validation showed that the test identified 37% of patients in a low-risk group with a 10-year invasive IBR rate of ~3% and an absolute risk reduction (ARR) from RT of 1% (number needed to treat, NNT=100), while remaining patients with higher DS scores (elevated-risk) had an ARR for RT of 9% (NNT=11) and 96% clinical sensitivity for RT benefit. The analytical performance of the PreludeDx DCISionRT molecular assays was high in representative formalin-fixed, paraffin-embedded breast tumor specimens. The DCISionRT test has been analytically validated and has been clinically validated in multiple peer-reviewed published studies.

Abstract 6485: Oxidative phosphorylation-associated radiomic feature and survival of women with serous ovarian cancer

Abstract Novel biomarkers that can be utilized for clinical decision support to improve patient outcomes are critically needed for ovarian cancer. At present, there are no standard of care biomarkers to inform the first-line treatment regimen (neoadjuvant chemotherapy vs. upfront surgical debulking) best suited for each patient or which patients are at the highest risk of recurrence. To address this unmet need, we identified pre-treatment computed tomography (CT) image-based radiomic features predictive of outcomes and primary treatment response among 298 women diagnosed with serous ovarian cancer from 2008 to 2019. A decision tree analysis identified a single volumetric feature, region of interest (ROI) volume center of mass (CoM) in the X direction, as the most informative radiomic feature that stratified patients with upfront surgery into high- and low-risk groups for overall survival. The high-risk group consists of patients with higher values of the radiomic feature. In the training (N=91) and test (N=91) cohorts of women treated with upfront surgery, high-risk patients had worse survival compared to low-risk patients (HR=2.01, 95% CI=1.07, 3.77 and HR=2.23, 95% CI=1.19, 4.17, respectively). This radiomic feature was not associated with survival among women treated with neoadjuvant chemotherapy (N=116; HR=1.23, 95% CI=0.73, 2.07). To reveal potential underlying biology of this radiomic feature, we performed RNAseq gene expression profiling on 47 formalin-fixed paraffin-embedded tumor specimens and correlated gene expression with the radiomic feature using DESeq2 from RSEM estimates. Dichotomized analysis (high- vs. low-risk) yielded 7 significant genes and continuous analysis yielded 15 significant genes (adjusted p&amp;lt;0.05), with both approaches identifying AKT2 and PSMC4. Gene set enrichment analysis (GSEA) was used to identify enriched gene sets from continuous associations using the MSigDB Hallmarks pathways (adjusted p&amp;lt;0.05). GSEA identified the oxidative phosphorylation (OXPHOS) pathway as one of the gene sets most negatively associated with the predictive radiomic feature (Hallmark Normalized Enrichment Score=-2.28, adjusted p&amp;lt;0.001). We derived a PCA-based gene signature from significantly associated OXPHOS genes (p&amp;lt;0.01) resulting in a negative correlation with the radiomic feature (R=-0.65, p&amp;lt;0.001). Prior studies have shown that high OXPHOS ovarian tumors are associated with an increased response to conventional chemotherapy, suggesting that OXPHOS may be a key pathway for chemoresistance in ovarian cancer. In summary, we identified an OXPHOS-associated radiomic feature predictive of survival among women with serous ovarian cancer treated with upfront surgery. Further research is needed to elucidate the biologic and mechanistic underpinnings of the identified radiomic feature and to validate these findings in a larger cohort of women with ovarian cancer. Citation Format: Christelle Colin-Leitzinger, Jaileene Perez-Morales, Steven Eschrich, Jamie K. Teer, Sweta Sinha, Melissa J. McGettigan, Daniel K. Jeong, Olya Stringfield, Mahmoud A. Abdalah, Natarajan Raghunand, Robert J. Gillies, Jing-Yi Chern, Matthew Schabath, Lauren Cole Peres. Oxidative phosphorylation-associated radiomic feature and survival of women with serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6485.

One Third of Malignant Pleural Mesothelioma Shows High Immunohistochemical Expression of MSLN or CXCR4 Which Indicates Potent Candidates for Endo-Radiotherapy

Malignant pleural mesothelioma (MPM) is a mainly asbestos-related tumour associated with a very poor prognosis. Therapeutic approaches include multimodal therapy and chemotherapeutics, with cisplatin being the drug of choice, but response rates of only up to 14% indicate very poor outcomes. Effective treatment options are lacking. Besides the diagnostic usage of radioligands in positron emission tomography (PET)/computed tomography (CT), the endo-radioligand therapy with Lu177 has been proven as a powerful tool in cancer therapy. Mesothelin (MSLN) and C-XC chemokine receptor 4 (CXCR4) are membrane-bound proteins, expressed in certain cancers, and thus are promising targets for endo-radiotherapy. A significant portion of high MSLN- or CXCR4-expressing tumors within the MPM may open the field for this sophisticated treatment approach in the near future. Formalin-fixed, paraffin-embedded (FFPE) tumour specimens from 105 patients suffering from MPM and treated at the Lung Cancer Centre of Essen and at the Helios Klinikum Emil von Behring Berlin were screened. The tumour samples were arranged in tissue microarrays. We immunohistochemically stained the tumour samples against MSLN and CXCR4. The protein expressions of the stainings were scored by a pathologist by using a semiquantitative method. The data obtained were correlated with the clinical outcome. Overall, 77.1% of the analysed tumours showed CXCR4 protein expression (25.7% of them at high expression level (Score 3)). 48.6% of all samples showed an overall strong staining (Score ≥ 2), 59% of the investigated tumours showed MSLN protein expression (10.5% of them at high expression (Score 3)), and 36.2% of all samples showed an overall strong staining (Score ≥ 2). Our results show significant tissue expression levels, for both CXCR4 and MSLN protein, in a major portion of clinical MPM samples. One-third of patients showed outstanding immunoexpression of at least one of these markers, making them interesting candidates for radioligand-based PET/CT diagnostics and follow-up and furthermore may profit from endo-radiotherapy.

Profile of MicroRNAs Associated with Death Due to Disease Progression in Metastatic Papillary Thyroid Carcinoma Patients.

Papillary thyroid carcinoma (PTC) is the most common neoplasm of the endocrine system and has an excellent long-term prognosis, with low rates of distant metastatic disease. Although infrequent, there are cases of deaths directly related to PTC, especially in patients with metastatic disease, and the factors that could be associated with this unfavorable outcome remain a major challenge in clinical practice. Recently, research into genetic factors associated with PTC has gained ground, especially mutations in the TERT promoter and BRAF gene. However, the role of microRNAs remains poorly studied, especially in those patients who have an unfavorable outcome at follow-up. This paper aims to evaluate molecular markers related to the different pathological processes of PTC, as well as the histological characteristics of the neoplasm, and to compare this profile with prognosis and death from the disease using an analysis of patients treated for metastatic disease in a single tertiary cancer center. Evaluation of microRNA expression in paraffin-embedded tumor specimens was carried out by quantitative PCR using the TaqMan® Low Density Array (TLDA) system. Metastatic patients who died from progression of PTC had higher expressions of miR-101-3p, miR-17-5p, and miR-191-5p when compared to patients with stable metastatic disease. These findings are of great importance but should be considered as preliminary because of the small sample.

Clinical Evaluation of IDH Mutation Status in Formalin-Fixed Paraffin-Embedded Tissue in Gliomas.

Determination of isocitrate dehydrogenase (IDH) 1/2 mutational status is crucial for a glioma diagnosis. It is common for IDH mutational status to be determined via a two-step algorithm that utilizes immunohistochemistry studies for IDH1 R132H, the most frequent variant, followed by next-generation sequencing studies for immunohistochemistry-negative or immunohistochemistry-equivocal cases.The objective of this study was to evaluate addinga rapid real-time polymerase chain reaction (RT-PCR) assay to the testing algorithm. METHODS: We validated a modified, commercial, qualitative, RT-PCR assay with the ability to detect 14 variants in IDH1/2 in formalin-fixed paraffin-embedded glioma tumor specimens. The assay was validated using 51 tumor formalin-fixed paraffin-embedded specimens. During clinical implementation of this assay, 48 brain tumor specimens were assessed for IDH result concordance and turnaround time to result. Concordance between the RT-PCR and sequencing and IHC studies was 100%. This RT-PCR assay also showed concordant results with IHC for IDH1 R132H for 11 of the 12 (92%) tumor specimens with IDH mutations. The RT-PCR assay yielded faster results (average 2.6 days turnaround time) in comparison to sequencing studies (17.9 days), with complete concordance. In summary, we report that this RT-PCR assay can reliably be performed on formalin-fixed paraffin-embedded specimens and has a faster turnaround time than sequencing assays and can be clinically implemented for determination of IDH mutation status for patients with glioma.

Development of a Prognostic Scoring System using MYC Expression and Soluble Interleukin Receptor -2 level for Diffuse Large B-cell Lymphoma.

Diffuse large B-cell lymphoma, not otherwise specified (DLBCL-NOS), is the most frequent type of lymphoid neoplasm. We investigated the relationships between clinical factors of DLBCL-NOS and MYC immunohistochemistry (IHC) staining. A total of 110 patients diagnosed with DLBCL-NOS from 2012 to 2020 at Tottori University Hospital and treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP) chemotherapy were included. IHC staining of MYC in formalin-fixed, paraffin-embedded tumor specimens was performed, and ROC-curve analysis revealed the cut-off value of the MYC positive rate as 55%. The 2-year overall survival (OS) rates of the MYC-negative and -positive groups were 84.7% vs 57.7% (P = 0.0091), and the progression-free survival rates were 77.8% vs 54.7% (P = 0.016), respectively. Multivariate analysis for OS showed prognostic significance of MYC positivity [hazards ratio (HR): 2.496; P = 0.032], and serum levels of soluble interleukin-2 receptor (sIL-2R) > 2000 U/mL (HR: 3.950; P = 0.0019), as well as age > 75 (HR: 2.356; P = 0.068). The original scoring system was developed based on these findings. By assigning one point to each item, age (> 75), MYC positivity, and sIL-2R level (> 2000), all patients were classified into three risk categories: group 1 (0 points), group 2 (1 point), and group 3 (2-3 points). The 2-year survival rates were 100%, 83.0%, and 47.1% for the groups 1, 2, and 3, respectively (P < 0.0001). We suggest that a prognostic scoring system using MYC expression and soluble interleukin receptor -2 level is useful for the prediction of prognosis, contributing to further stratification in DLBCL-NOS.

NGS-based targeted gene mutational profiles in Korean patients with pancreatic cancer

According to molecular profiling studies, a considerable number of patients with pancreatic cancer harbor potentially actionable mutations. However, there are limited relevant data from the Korean population. We assessed the molecular profiles of patients with pancreatic cancer in Korea. This study collected molecular profiling data from patients with pancreatic cancer who visited Seoul National University Bundang Hospital between March 2018 and August 2020. Formalin-fixed, paraffin-embedded tumor specimens were sequenced using a targeted next-generation sequencing (NGS) platform. Cancer-associated mutations were analyzed, and potentially actionable mutations were identified. Potentially actionable mutations were classified into “highly actionable” and “modifies options” based on the Know Your Tumor registry study. In total, 87 patients with NGS tumor panel data were identified. Sixty-one patients (70.1%) had metastatic disease at the time of tissue acquisition. Tissues were obtained from the primary tumors and metastatic sites in 41 (47.1%) and 46 (52.9%) patients, respectively. At least one pathogenic mutation was reported in 86 patients (98.9%). The frequencies of four common mutations in our cohort were similar to those in The Cancer Genome Atlas data. Potentially actionable mutations were identified in 27 patients (31.0%). Of these, mutations categorized as highly actionable and modifies options were identified in 12 (13.8%) and 18 patients (20.7%), respectively. The most frequent highly actionable mutations were located in DNA damage response genes, such as BRCA1, BRCA2, or ATM (n = 6, 6.9%). Two patients with germline BRCA1 mutations received maintenance poly(adenosine diphosphate-ribose) polymerase inhibitor therapy. One patient has been receiving maintenance treatment for 18 months while remaining in radiologically complete remission. Mutational profiles using targeted NGS in Korean patients with pancreatic cancer were similar to those in Western patients. The present study supports the clinical potential and possible expanded clinical use of genetic profiling.

FGFR2-amplified tumor clones are markedly heterogeneously distributed in carcinomas of the upper gastrointestinal tract

BackgroundFGFR2 is a therapy-relevant target in tumors of the upper gastrointestinal tract (GIT), and clinical trials are currently underway to test the efficacy of FGFR2 inhibitors. Tumor heterogeneity is one of the relevant causes of treatment failure. Almost nothing is known about the heterogeneous distribution of FGFR2-amplified clones in adenocarcinomas of the upper GIT.Patients and methodsTo assess FGFR2 gene copy number alteration and intratumoral heterogeneity of upper GIT adenocarcinomas, we analyzed 893 patient-derived formalin-fixed paraffin-embedded tumor specimens, including primary operated and neoadjuvant-treated tumors (462 gastric carcinomas and 429 esophageal adenocarcinomas) as well as complementary lymph node and distant metastasis by fluorescence in situ hybridization.ResultsTwenty-six gastric tumors (5.6%) and 21 esophageal adenocarcinomas (4.9%) showed FGFR2 amplification. Overall, 93% of gastric carcinomas and 83% of esophageal carcinomas showed heterogeneous amplification. FGFR2 amplification was found in different histological growth patterns, including intestinal and diffuse type according to the Lauren classification. In the primary gastric carcinoma group, FGFR2 amplification was associated with poor prognosis (p = 0.005).ConclusionHomogeneous FGFR2 amplification in tumors of the upper GIT is the exception. This has highly relevant implications in the nature of FGFR2 diagnostics (sufficient tumor cell number, determination of amplification at metastasis versus primary tumor, etc.) and on the response probability of appropriate inhibitors. It is relevant that the often poorly treatable and aggressive subtype of diffuse carcinomas (poorly cohesive carcinomas) also shows FGFR2 amplification and that an individualized therapy option with FGFR2 inhibitors could be an option in this group.

Comparative Assessment of DNA Extraction Techniques From Formalin-Fixed, Paraffin-Embedded Tumor Specimens and Their Impact on Downstream Analysis.

Good-quality nucleic acid extraction from formalin-fixed, paraffin-embedded (FFPE) specimens remains a challenge in molecular-oncopathology practice. This study evaluates the efficacy of an in-house developed FFPE extraction buffer compared with other commercially available kits. Eighty FFPE specimens processed in different surgical pathology laboratories formed the study sample. DNA extraction was performed using three commercial kits and the in-house developed FFPE extraction buffer. DNA yield was quantified by a NanoDrop spectrophotometer and Qubit Fluorometer, and its purity was measured by the 260/280-nm ratio. A fragment analyzer system was used for accurate sizing of DNA fragments of FFPE DNA. The downstream effects of all extraction methods were evaluated by polymerase chain reaction (PCR) and Sanger sequencing. In comparison with the commercial kits, the in-house buffer yielded higher DNA quantity and quality number (P < .0001). In addition, DNA integrity and fragment size were preserved in a significantly greater number of samples isolated with the in-house buffer (P < .05). The target PCR amplification rate with the in-house buffer extracted samples was also significantly higher, with 98% of the samples showing interpretable sequencing results. The in-house developed FFPE extraction buffer performed superior to other methods in terms of suitability for downstream applications, time, cost-efficiency, and ease of performance.