Tumor Tissue Specimens Research Articles

Efficacy and potential resistance mechanisms of afatinib in advanced non-small cell lung cancer patients with EGFR G719X/L861Q/S768I.

Afatinib is the only currently approved EGFR-tyrosine kinase inhibitors for advanced non-small cell lung cancer (NSCLC) patients with EGFR G719X/L861Q/S768I. However, there are limited real-world data concerning the benefits and resistance mechanisms of afatinib in patients with these nonclassical mutations. To fill this gap, the present study was conducted. All NSCLC patients treated with afatinib were screened, and patients with EGFR G719X/L861Q/S768I were enrolled into the analysis. Either tumor tissue or blood specimens were detected by the commercial next-generation sequencing (NGS) panels or amplification-refractory mutation system (ARMS)-polymerase chain reaction (PCR) to figure out the mutation genotype. A total of 106 advanced NSCLC patients with EGFR G719X/L861Q/S768I received afatinib treatment. The benefits of afatinib exhibited heterogeneity in different mutation genotypes. Notably, at baseline, NGS testing was performed in 59 patients, and TP53 was the most frequently coexisting mutation. Patients with TP53 mutations obtained fewer survival benefits than those with TP53 wild-type. A total of 68 patients ultimately experienced progression, and 27 patients received NGS testing to clarify the potential resistance mechanisms. EGFR-T790M, CDK4 amplification, FGFR1 amplification, PIK3CA, MET amplification, RET fusions, HER2, and BRAF mutations were identified in three (11.1%), three (11.1%), three (11.1%), three (11.1%), three (11.1%), one (3.7%), one (3.7%), and one (3.7%) of the cases, respectively. Five patients underwent ARMS-PCR testing for detecting EGFR-T790M mutation, and only one patient was T790M-positive. The present study elucidated the differential benefits of afatinib within different mutation genotypes and first revealed the spectrum of potential resistance mechanisms in patients with EGFR G719X/L861Q/S768I. The results of this study may provide practical clinical information that can guide optimal treatment in this setting.

OS01.7.A Detection of methylation-based prognostic signatures in liquid biopsy specimens from patients with meningiomas

Abstract Background Detection of distinct epigenetic biomarkers in circulating cell-free DNA (cfDNA) of liquid biopsy (LB) specimens (e.g. blood) fosters opportunity for prognostication of central nervous system (CNS) tumors and has not been thoroughly explored in patients with meningiomas. Material and Methods We profiled the cfDNA methylome (EPIC array) in serum specimens from patients with meningiomas (MNG; n= 63) and harnessed internal and external meningioma tissue methylome data with reported follow up (n=48). To predict recurrence risk (RR), we consolidated a tissue cohort with at least 5 years of follow up and divided them into confirmed recurrence (CR; either reported progressive disease in post-surgical imaging, or additional resections following initial surgery) and confirmed no-recurrence (CNR: no confirmed disease progression w/in at least 5-years of follow-up). Then through application of an iterative process consisting of multiple tissue- and serum-based supervised analyses, we identified risk-specific methylation markers with serum specific features which, when inputted into a random forest algorithm allowed for segregation of both tumor tissue and liquid biopsy specimens according to recurrence risk. We estimated immune cell composition using MethylCIBERSORT, where a reference methylome atlas of chosen immune cell types was utilized to deconvolute the MNG samples. Results The resulting recurrence risk classifier demonstrated an appreciable predictive power in classifying samples as high or low recurrence risk across the tumor tissue cohort (ACC: 87.5%, CUI+: 85.2%). When compared to another classifier, our model demonstrated statistically significant agreement across primary meningioma samples (κ=0.269, p=0.002), and more accurately predicted samples to recur across an expanded time window (time to recurrence >5yrs). Across resulting liquid biopsy classifications, recurrence risk subgroups were analogous with reported risk factors, including WHO grade, extent of resection, and tumor location. Recurrence risk subgroups (high and low) also demonstrated differential estimated immune cell contributions, with low-risk samples exhibiting a “hot” profile, or enrichment of B-Cells, CD56- and CD4 T-Cells, and natural killer cells. Notably, the estimated neutrophil to lymphocyte ratio, previously purported to be relevant to tumor prognosis, was appreciably higher for those meningioma samples with the highest recurrence risk. Conclusion DNA methylation markers identified in the serum are suitable for the development of machine learning-based models which present high predictive power to prognosticate patients with meningioma and estimate a differential immune profile across recurrence risk groups. After validation in an external cohort, this noninvasive approach may improve the presurgical therapeutic management of patients with meningiomas.

1198TiP A phase Ib/II open-label, nonrandomized study of FGFR inhibitor futibatinib in combination with MEK inhibitor binimetinib in patients with advanced KRAS-mutant cancer

Treatment options for patients (pts) with KRAS-mutant (KRASmt) cancer, including non–small cell lung cancer (NSCLC), are limited. Combination of MEK inhibitors (MEKi) plus futibatinib had synergistic antitumor activity in different KRASmt NSCLC cell lines. An open-label, nonrandomized phase 1b/2 study (NCT04965818) is being conducted to assess the safety and preliminary antitumor activity of futibatinib plus MEKi binimetinib in pts with KRASmt advanced NSCLC. This phase 1b/2 study contains 2 parts. In the phase Ib dose escalation part, the recommended phase II dose (RP2D) and regimen of futibatinib plus binimetinib in pts with advanced cancer (part 1) will be determined. In the phase II part, pts with KRASmt advanced NSCLC will be enrolled in a cohort of 18 pts to evaluate the preliminary antitumor activity of futibatinib plus binimetinib at the RP2D and regimen (part 2). Pts eligible for part 1 may have any type of advanced cancer for which no other treatment options are available. Pts with KRASmt advanced NSCLC in part 2 must have their KRAS mutation determined by local results from a tumor tissue specimen and have had disease progression on prior standard of care therapy including anti–PD-(L)1 therapy and platinum-based chemotherapy. Key exclusion criteria include any history of known phosphate homeostasis disorder and current evidence of any clinically significant retinal or corneal disorder and any history of retinal vein occlusion. The primary objective is to determine an RP2D based on safety, preliminary activity, and PK/PD results (part 1) and to assess the preliminary antitumor activity of the combination in pts with KRASmt advanced NSCLC based on objective response rate by RECIST 1.1 (part 2). Secondary endpoints include pharmacokinetic parameters, duration of response, disease control rate at 24 weeks, and progression-free survival. Exploratory objectives include correlative analysis to explore biomarkers related to tumor biology and their potential associations with antitumor activity and resistance to the study medications (including circulating tumor DNA). The study was initiated September 2021 and is ongoing. NCT04965818. Professional medical writing and editorial assistance were provided by Tracy Diaz, PhD, and Jennifer Robertson, PhD, at Ashfield MedComms, an Ashfield Health company, and funded by Taiho Oncology, Inc. Taiho Oncology, Inc.

Comprehensive analysis of T cell receptor repertoire in patients with KRAS mutant non-small cell lung cancer.

BackgroundKirsten rat sarcoma viral oncogene homolog (KRAS) is one of the most frequently mutated oncogenes in non-small cell lung cancer (NSCLC). The administration of immunotherapy has demonstrated significant efficacy in prolonging the overall survival of patients with KRAS mutation in recent years. However, the efficacy of immunotherapy in KRAS mutant NSCLC is variable. Analysis of T cell receptor (TCR) repertoire may contribute to a better understanding of the mechanisms behind such differential outcomes.MethodsA total of 47 patients with KRAS mutant NSCLC were enrolled in this study. Deep sequencing of the TCR β chain complementarity-determining regions in tumor tissue and paired peripheral blood specimens was conducted. Comprehensive analysis of TCR repertoire metrics was performed with different KRAS mutation subtypes and concomitant mutations. Moreover, the associations between TCR repertoire metrics and tumor mutation burden (TMB), as well as programmed death-ligand 1 were explored, respectively.ResultsTCR repertoire metrics, including Shannon index, Clonality, and Morisita index (MOI), showed no significant differences among different KRAS mutation subtypes. The similar results were observed between patients with tumor protein p53 (TP53) mutation and those with wild-type TP53. In contrast, although no significant differences were found in Shannon index and Clonality, patients with KRAS/serine/threonine kinase 11 (STK11) comutation showed a significantly higher MOI compared to their STK11 wild-type counterparts (P=0.012). In addition, TCR repertoire metrics were neither associated with TMB nor programmed death-ligand 1 expression in KRAS mutant NSCLC.ConclusionsThis retrospective study comprehensively described the TCR repertoire in KRAS mutant NSCLC. A higher MOI represented more overlap of the TCR repertoire between tumor tissue and paired peripheral blood, indicating distinctive immunological features in NSCLC with KRAS/STK11 comutation.

In-house testing for homologous recombination repair deficiency (HRD) testing in ovarian carcinoma: a feasibility study comparing AmoyDx HRD Focus panel with Myriad myChoiceCDx assay.

Homologous recombination repair (HRR) is the main mechanism of repair of DNA double-strand breaks. Its deficiency (HRD) is a common feature of epithelial ovarian cancers (EOCs). BRCA1/2 mutations and/or other aberrations in genes of HRR are well known causes of HRD and genomic instability. Poly ADP-ribose polymerase inhibitors (PARPi) have revolutionized the management of BRCA mutant EOCs and demonstrated activity in HRD tumor cells. Determining HRD status can provide informations on the magnitude of benefit for PARPi therapy. Myriad MyChoice CDx is a next generation sequencing- based in vitro diagnostic test that assesses the Genomic Instability Score (GIS) which is an algorithmic measurement of loss of heterozygosity, telomeric allelic imbalance, and large-scale state transitions using DNA isolated from formalin-fixed paraffin embedded tumor tissue specimens. However Myriad MyChoice CDx, is a centrally performed and costly assay, with no reimbursement scheduled, at least in Italy. In this report, we described our experience in performing the HRD Focus AmoyDx (Amoy Diagnostics Ltd, Xiamen, Fujian, China) on the same samples of EOCs evaluated with Myriad MyChoiceCDx assay. The overall percent agreement between AmoyDx and Myriad was 87.8% (65 of 74 tumors tested). All the 36 AmoyDx negative cases were confirmed to be negative by Myriad (negative predictive value, 100%). The concordance of the results with the gold standard Myriad MyChoice CDx assay suggest the feasibility and reliability of HRD testing in diagnostic laboratories with high-throughput NGS platforms and qualified personnel.

Epidermal Growth Factor Receptor immunohistochemical expression in hepatocellular carcinoma without Epidermal Growth Factor Receptor exons 18-21 mutations.

Introduction: EGFR targeted therapies, have been proved beneficial for patients with HCC, nevertheless additional research on EGFR immunoexpresion and EGFR mutations is still needed, especially in population in which it has not been done yet. The aim of this study is to evaluate EGFR immunoexpression in HCC without EGFR exons 18-21 mutations and to evaluate its influence on survival in HCC patients in North Macedonia. Methods: We studied 31 cases of HCC for EGFR immunohistochemical expression and EGFR exons 18-21 mutations. The following clinical parameters were analyzed: Hepatitis B and C virus infection, presence of cirrhosis, tumor size, enlarged lymph nodes, metastases, alpha fetoprotein level and overall survival. Presence of the EGFR immunosignal (membranous and cytoplasmic) and the percentage of positive tumor cells in the entire tumor tissue specimen were semi-quantitatively determined. Results: Hepatitis B and C virus infection, tumor size, metastatic disease and EGFR immunoexpression have influence on patient's survival. No EGFR exons 18-21 mutations were detected in this group of HCCs. EGFR expression of 61%-80% in tumor tissue significantly influenced survival of the patients (p < 0.01). Multiple Cox regression confirmed tumor size of 5-10 cm (p < 0.05), tumor size > 10 cm (p < 0.01) and EGFR expression in range of 61% to 80% (p < 0.05) as independent survival predictors in patients with HCC. Conclusion: EGFR overexpression in range of 61% to 80% was an independent survival predictor in patients with HCC, implying that these patients could benefit from EGFR inhibition. However, the absence of EGFR mutations in exons 18-21 in any of the cases of this study suggest that single drug EGFR targeted therapy in patients with HCC may be insufficient.

Association of rs9679162 Genetic Polymorphism and Aberrant Expression of Polypeptide N-Acetylgalactosaminyltransferase 14 (GALNT14) in Head and Neck Cancer.

Simple SummaryNeoadjuvant chemotherapy was performed before surgery. Because the tumor itself and the surrounding vascular bed were not damaged, the chemotherapy we performed could have good drug delivery. After the operation, the volume of the tumor can be reduced to facilitate surgery or radiotherapy. However, neoadjuvant chemotherapy also delays the patient’s time to receive main therapy. The physician must make sure that it has a good response and does not allow disease progression in the patient during neoadjuvant chemotherapy. Therefore, predicting the treatment response of neoadjuvant chemotherapy can shorten the treatment time, reduce the harm of chemotherapy side effects, and avoid the occurrence of drug resistance. The results of this study showed that GALNT14-rs9679162 and mRNA expression were associated with post-treatment survival in head and neck cancer. It can be used as an indicator to predict the treatment response of neoadjuvant chemotherapy.The polypeptide N-Acetylgalactosaminyltransferase 14 (GALNT14) rs9679162 and mRNA expression were associated with treatment outcome in various cancers. However, the relation of GALNT14 and head and neck cancer were nuclear. A total of 199 patients with head and neck squamous cell carcinoma (HNSCC) were collected in this study, including oral SCC (OSCC), oropharyngeal SCC (OPSCC), laryngeal SCC (LSCC), and others. The DNA and RNA of cancer tissues were extracted using the TRI Reagent method. The rs9679162 was analyzed using polymerase chain reaction (PCR) and sequencing methods in 199 DNA specimens, and the mRNA expression was analyzed using quantitative reverse transcription PCR (RT-qPCR) methods in 68 paired RNA specimens of non-cancerous matched tissues (NCMT) and tumor tissues. The results showed that the genotype of TT, TG, and GG appeared at 30%, 44%, and 26%, respectively. Non-TT genotype or G alleotype were associated with alcohol, betel nut, and cigarette using among patients with OSCC, and it also affected the treatment and survival of patients with OSCC and LSCC. High GALNT14 mRNA expression levels increased lymphatic metastasis of patients with HNSCC, and treatment and survival in patients with OPSCC. Overall, the GALNT14-rs9679162 genotype and mRNA expression level can be used as indicators of HNSCC treatment prognosis.

In silico and in vivo analysis of TIPE1 expression in diffuse large B cell lymphoma.

TIPE1 is a gene in the TNFAIP8 family involved in immune regulation and tumorigenesis. Although previous studies demonstrated that TIPE1 might play different roles in different tumors, its expression and role in lymphoma are unclear. Here we observed TIPE1 expression in diffuse large B cell lymphoma (DLBCL). Two microarrays containing 96 tumor tissue specimens were obtained from the Affiliated Hospital of Nantong University biobank. All specimens came from patients with a clear pathological diagnosis of lymphoma, lymphadenitis, breast cancer, or bladder cancer, and we performed immunohistochemical experiments on these tissue specimens. GEPIA and TIMER platforms were used for bioinformatic analyses. We found higher TIPE1 expression in tumor tissues from patients with lymphoma compared with those with lymphadenitis, breast cancer, or bladder cancer. The GEPIA and TIMER analyses revealed that TIPE1 was upregulated in DLBCL tissues but not in invasive breast carcinoma, urothelial bladder carcinoma, or liver hepatocellular carcinoma tissues. TIPE1 expression was irrelevant for pathological stage, overall survival, or DLBCL immune infiltration levels. However, TIPE1 expression was correlated with MKI67 expression in DLBCL. Overall, TIPE1’s high expression levels in DLBCL may contribute to tumor growth in DLBCL.

SMAR1 inhibits proliferation, EMT and Warburg effect of bladder cancer cells by suppressing the activity of the Wnt/β-catenin signaling pathway

ABSTRACT This study aimed to investigate the effects of scaffold matrix attachment region binding protein 1 (SMAR1) on the development of bladder cancer (BCa). SMAR1 expression in paired tumor and corresponding adjacent normal tissues from 55 BCa patients was detected by quantitative reverse transcription-polymerase chain reaction. BCa cells were transfected to regulate SMAR1 expression. BCa cells were treated with XAV-939, LiCl and 2-deoxyglucose. The effect of SMAR1 on the viability, proliferation, migration, invasion and Warburg effect of BCa cells was researched by counting kit-8, colony formation assay, Transwell and aerobic glycolysis assays. Western blot was performed to detect protein expression. BCa cell growth in vivo was recorded in nude mice. Immunohistochemical staining was performed for clinical and xenografted tumor tissue specimens. SMAR1 expression was down-regulated in BCa patients, associating with worse prognoses. SMAR1 knockdown enhanced the viability, proliferation, migration, invasion, EMT and Warburg effect of BCa cells. The opposite effect was found in the SMAR1 overexpression BCa cells. XAV-939 treatment reversed the elevation of β-catenin, c-Myc and Cyclin D1 proteins expression and Warburg effect in Bca cells post-SMAR1 knockdown. LiCl treatment abrogated the inhibition of β-catenin, c-Myc and Cyclin D1 proteins expression and Warburg effect proteins due to SMAR1 overexpression in BCa cells. SMAR1 overexpression inhibited the growth of BCa cells in vivo. SMAR1 might suppress the Wnt/β-catenin signaling pathway activity to inhibit the progression of BCa. It might be an effective treatment target for BCa.

Associations of C-X-C motif chemokine ligands 1/2/8/13/14 with clinicopathological features and survival profile in patients with colorectal cancer.

The present study aimed to assess the correlation of C-X-C motif chemokine ligand (CXCL)1, CXCL2, CXCL8, CXCL13 and CXCL14 with clinicopathological features and survival profile in patients with colorectal cancer (CRC). Patients with primary CRC (n=232) were retrospectively reviewed, with their tumor tissue specimens acquired from the Department of Pathology (The First Hospital of Jilin University, Changchun, China), their demographic data and preoperative tumor features collected from the hospital database, and their survival data obtained from the follow-up documents. Tumor CXCL expression was detected by immunohistochemistry (IHC). Based on the total IHC score, the expression of CXCL1, CXCL2, CXCL8, CXCL13 and CXCL14 was categorized as low expression (IHC score ≤3) and high expression (IHC score >3). CXCL1 (51.3% high and 48.7% low), CXCL2 (59.9% high and 40.1% low), CXCL8 (44.4% high and 55.6% low), CXCL13 (40.9% high and 59.1% low) and CXCL14 (31.0% high and 69.0% low) were expressed in CRC tumor tissues, and their expression levels were correlated with each other, except between CXCL8 and CXCL14, and between CXCL13 and CXCL14. CXCL1 was associated with a larger tumor size, and an advanced T stage, N stage and Tumor-Node-Metastasis (TNM) stage. CXCL2 was associated with an advanced N stage and TNM stage, and CXCL8 was associated with a greater T stage and TNM stage. CXCL13 was associated with a greater T stage, N stage and TNM stage, while CXCL14 was not associated with any clinical characteristics. As for survival, CXCL1, CXCL2, CXCL8 and CXCL13, but not CXCL14, were associated with poor overall survival (OS) rate, and further multivariate Cox's regression model analysis revealed that CXCL1 independently predicted unfavorable OS in patients with CRC. Overall, CXCL1, CXCL2, CXCL8 and CXCL13 have good potential as an indicator for tumor features and survival in patients with CRC.

Identification of new aptamer BC-3 targeting RPS7 from rapid screening for bladder carcinoma

Aptamers, short single DNA or RNA oligonucleotides, have shown immense application potential as molecular probes for the early diagnosis and therapy of cancer. However, conventional cell-SELEX technologies for aptamer discovery are time-consuming and laborious. Here we discovered a new aptamer BC-3 by using an improved rapid X-Aptamer selection process for human bladder carcinoma, for which there is no specific molecular probe yet. We show that BC-3 exhibited excellent affinity in bladder cancer cells but not normal cells. We demonstrate that BC-3 displayed high selectivity for tumor cells over their normal counterparts in vitro, in mice, and in patient tumor tissue specimens. Further endocytosis pathway analysis revealed that BC-3 internalized into bladder cancer cells via clathrin-mediated endocytosis. Importantly, we identified ribosomal protein S7 (RPS7) as the binding target of BC-3 via an integrated methodology (mass spectrometry, colocalization assay, and immunoblotting). Together, we report that a novel aptamer BC-3 is discovered for bladder cancer and its properties in the disease are unearthed. Our findings will facilitate the discovery of novel diagnostic and therapeutic strategies for bladder cancer.

Characterization of novel small non-coding RNAs and their modifications in bladder cancer using an updated small RNA-seq workflow.

Background: Bladder cancer (BLCA) is one of the most common cancer types worldwide. The disease is responsible for about 200,000 deaths annually, thus improved diagnostics and therapy is needed. A large body of evidence reveal that small RNAs of less than 40 nucleotides may act as tumor suppressors, oncogenes, and disease biomarkers, with a major focus on microRNAs. However, the role of other families of small RNAs is not yet deciphered. Recent results suggest that small RNAs and their modification status, play a role in BLCA development and are promising biomarkers due to their high abundance in the exomes and body fluids (including urine). Moreover, free modified nucleosides have been detected at elevated levels from the urine of BLCA patients. A genome-wide view of small RNAs, and their modifications, will help pinpoint the molecules that could be used as biomarker or has important biology in BLCA development. Methods: BLCA tumor tissue specimens were obtained from 12 patients undergoing transurethral resection of non-muscle invasive papillary urothelial carcinomas. Genome-wide profiling of small RNAs less than 40 bases long was performed by a modified protocol with TGIRT (thermostable group II reverse transcriptase) to identify novel small RNAs and their modification status. Results: Comprehensive analysis identified not only microRNAs. Intriguingly, 57 ± 15% (mean ± S.D.) of sequencing reads mapped to non-microRNA-small RNAs including tRNA-derived fragments (tRFs), ribosomal RNA-derived fragments (rRFs) and YRNA-derived fragments (YRFs). Misincorporation (mismatch) sites identified potential base modification positions on the small RNAs, especially on tRFs, corresponding to m1A (N1-methyladenosine), m1G (N1-methylguanosine) and m2 2G (N2, N2-dimethylguanosine). We also detected mismatch sites on rRFs corresponding to known modifications on 28 and 18S rRNA. Conclusion: We found abundant non-microRNA-small RNAs in BLCA tumor samples. Small RNAs, especially tRFs and rRFs, contain modifications that can be captured as mismatch by TGIRT sequencing. Both the modifications and the non-microRNA-small RNAs should be explored as a biomarker for BLCA detection or follow-up.

Arginine ADP-ribosyltransferase 1 Regulates Glycolysis in Colorectal Cancer via the PI3K/AKT/HIF1α Pathway

ObjectiveArginine ADP-ribosyltransferase 1 (ART1) is involved in the regulation of a diverse array of pathophysiological processes, including proliferation, invasion, apoptosis, autophagy and angiogenesis of colorectal cancer (CRC) cells. However, how ART1 regulates glycolysis in CRC remains elusive.MethodsTo elucidate the role of ART1 in glycolysis in CRC, we assessed the protein level of ART1, hypoxia-inducible factor 1α (HIF1α), and glucose transporter type 1 (GLUT1) in 61 CRC tumor tissue specimens obtained from patients with different 2-[18F]fluoro-2-deoxy-D-glucose (18F-FDG) uptake as analyzed by PET/CT before surgery. Colon adenocarcinoma CT26 cells with ART1 knockdown and overexpression were established, respectively, and the molecular mechanism underlying the effect of ART1 on glycolysis in CRC was determined both in vivo and in vitro.ResultsThe expression of ART1 and GLUT1 was significantly associated with FDG uptake (P=0.037 and P=0.022, respectively) in CRC tissues. Furthermore, the expression of hexokinase 2 (HK2) and lactate dehydrogenase (LDH) was upregulated in ART1-overexpressed CT26 cells, but was downregulated in ART1-knockdown CT26 cells. The volume and weight of subcutaneously transplanted tumors were markedly increased in the ART1-overexpressed BALB/c mice group and decreased in the ART1-knockdown group. In CT26 cells, the overexpression of ART1 promoted the expression levels of HK2 and LDH, and knockdown of ART1 suppressed them in the CT26 tumors. In both normal and hypoxic conditions, ART1 expression was associated with the protein level of phospho-serine/threonine kinase (p-AKT), HIF1α, and GLUT1 but not with that of AKT in CT26 cells and subcutaneous transplanted tumors.ConclusionART1 plays a crucial role in the elevation of glucose consumption in CT26 cells and may regulate GLUT1-dependent glycolysis in CRC via the PI3K/AKT/HIF1α pathway.

P5-8 Current status and issues of cancer genome profiling tests for insurance at our hospital

Cancer genome profiling tests using tumor tissue specimens performed 78 cases at our hospital. So, we summarized the cases of them performed under insurance medical treatment at our hospital and examined the usefulness and problems of them.

Elevated AKIP1 expression is associated with tumor invasion, shorter survival time and decreased chemosensitivity in endometrial carcinoma.

A-kinase-interacting protein 1 (AKIP1), as a recently discovered oncoprotein, promotes cell malignant behaviors in gynecological malignancies. To the best of our knowledge, no study reports its clinical value in patients with endometrial carcinoma. The present study aimed to explore the association between AKIP1 expression and clinical features and survival in patients with endometrial carcinoma, and to assess the effect of AKIP1 knockdown on the regulation of chemosensitivity in vitro. The tumor and adjacent tissue specimens from 101 patients with endometrial carcinoma were retrieved for AKIP1 protein expression analysis using an immunohistochemistry (IHC) assay. Meanwhile, specimens from 54 patients with endometrial carcinoma were analyzed for AKIP1 mRNA expression using reverse transcription-quantitative PCR. Furthermore, an in vitro experiment was conducted in the Ishikawa cell line to determine the effect of AKIP1 modification on the chemosensitivity of cisplatin and paclitaxel. AKIP1 IHC score (P<0.001) and mRNA expression levels (P<0.001) were increased in tumor tissues compared with those in adjacent tissues. Moreover, increased AKIP1 IHC score was associated with lymphovascular invasion (P=0.007), advanced International Federation of Gynecology and Obstetrics (FIGO) stage (P=0.002) and shorter overall survival (OS) time (P=0.035) in the patients with endometrial carcinoma. Meanwhile, upregulated AKIP1 mRNA expression levels were associated with lymphovascular invasion (P=0.020) and advanced FIGO stage (P=0.027) in the patients with endometrial carcinoma. Multivariate Cox regression showed that tumor AKIP1 protein expression (high vs. low) independently predicted a shorter OS time (P=0.036). Silencing of AKIP1 decreased Ishikawa cell viability when treated with 5, 10, 20 and 40 µM cisplatin (all P<0.05) and decreased the half maximal inhibitory concentration value of cisplatin (P=0.003), whereas its effect on paclitaxel chemosensitivity was less obvious. Overall, elevated AKIP1 expression was associated with tumor invasion, shorter survival time and decreased chemosensitivity in endometrial carcinoma.

Abstract 5415: Glypican-3 immunohistochemistry precision, validation, and prevalence in selected solid tumors to identify target populations for CAR T cell therapy

Abstract Background: Glypican-3 (GPC3) is an oncofetal tumor antigen that is an attractive target for chimeric antigen receptor (CAR) T cell therapy due to its highly restricted expression on normal tissue and high prevalence in several adult and pediatric solid tumors. Aberrant GPC3 expression is implicated in tumorigenesis, and GPC3+ cancers are characterized by a highly immunosuppressive landscape which induces exhaustion in tumor-resident T cells. We sought to assess the validity of a GPC3 antibody clone in immunohistochemistry (IHC) and then measure GPC3+ prevalence in selected solid tumors. Methods: We evaluated GPC3 transcripts in human normal and tumor tissue specimens from The Cancer Genome Atlas (TCGA) and the Genome-Tissue Expression Project. GPC3 IHC was performed on formalin fixed paraffin embedded tumor tissues: hepatocellular (HCC), squamous cell lung (SCC), colorectal (CRC), serous ovarian (OC), Merkel cell carcinomas (MCC) and liposarcoma (LS) using antibody clone GC33 and scored using H-score (range 0-300), with appropriate positive and negative controls. Accuracy and precision of GPC3 antibody clone GC33 were tested in GPC3+ high prevalence tumor types compared to a validated anti-GPC3 clone (1G12). GPC3+ cutoff was set as H-score ≥ 30 (GC33) compared to &amp;gt;20 (1G12). The prevalence of GPC3 expression measured by IHC was also used to calculate the theoretical mRNA cutoff value from the TCGA data for GPC3+ indications represented in the database. Results: GPC3 mRNA was expressed at low levels predominantly in lung, adipose, tibial nerve, kidney and breast. GPC3 IHC of normal tissue showed predominantly faint, cytoplasmic staining in a few tissues including heart, kidney and stomach and no expression in lung or breast. HCC, SCC, LS, CRC, and OC showed high and varying levels of GPC3 mRNA expression in TCGA samples. The theoretical mRNA expression cutoff value based on IHC were HCC=1396 RSEM, SCC= 2675 RSEM, Sarcoma= 4126 RSEM; and OC 4486 RSEM; respectively. A cutoff for CRC could not be determined because the prevalence of GPC3 by IHC was &amp;lt;10%. GPC3 IHC using GC33 showed a higher prevalence of positive cases in myxoid/round cell LS (MRCLS) relative to other LS subtypes (15/45 vs. 8/75). HCC (n=110), SCC (n=73), MRCLS (n=45), and MCC (n=20) had a prevalence of 69%, 33%, 33%, and 70%; respectively. However, &amp;lt;10% of CRC (n=80) or OC (n=31) were GPC3+. GPC3 IHC assay using clone GC33 was fully validated for use as a lab-developed test by assessing accuracy, sensitivity, specificity, and precision in SCC, HCC, MCC and LS specimens. Conclusions: GPC3 has a high prevalence in selected tumors with low level expression in some normal tissues, making it an attractive target for CAR T cell therapy. The GC33 clone performs similarly to 1G12 and could be used for IHC screening for CAR T cell therapy. MRCLS has higher prevalence of GPC3+ expression relative to other LS subtypes. Citation Format: Glen J. Weiss, Amy Jensen-Smith, Sujatha Muralidharan. Glypican-3 immunohistochemistry precision, validation, and prevalence in selected solid tumors to identify target populations for CAR T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5415.

Abstract 1539: LKB1 down-modulation by miR-17 biologically and clinically mirrors LKB1-mutated NSCLC

Abstract Introduction: In non-small cell lung cancer (NSCLC) LKB1 mutations combined with KRAS hyperactivation define a poor responsive, aggressive and disseminating phenotype. We recently unveiled the vulnerability of KRAS/LKB1 co-mutated (KL) patient-derived xenografts (PDXs) to metabolic stress-based treatments, and a clinical trial to test the efficacy of metformin and fasting-mimicking diet on KL NSCLC is currently ongoing in our Institute. Besides inactivating mutations, LKB1 functionality may be impaired by post-translation regulation. Because miR-17 is a potential epigenetic regulator of LKB1,we hypothesized that wild-type LKB1 (LKB1WT) NSCLC with high miR-17 expression may biologically and clinically mirror LKB1 mutated tumors. Methods: NSCLC cell lines with different combinations of KRAS mutation and LKB1 deletion were selected, together with a panel of PDXs with high (LKB1WT/miR-17 high) or low (LKB1WT/miR-17 low) miR-17 expression. Taking advantage of series of NSCLC patients and PDXs, we retrospectively evaluated LKB1 and miR-17 expression levels in tumor tissue specimens. In addition, the TCGA dataset was interrogated for miR-17 expression and potential correlation with lung cancer clinical features. Results: Direct targeting of miR-17 to LKB1 3’UTR was confirmed by luciferase reporter assay. We found that miR-17 overexpression in LKB1WT cell lines functionally impaired the LKB1/AMPK pathway, and prompted apoptotic response to metformin. Of note, LKB1WT/miR-17 high PDXs tumor cells showed a similar behavior upon metformin treatment. A retrospective analysis in patients with NSCLC revealed an inverse correlation between miR-17 and LKB1 expression, and highlighted a prognostic role of miR-17 expression in LKB1WT patients, further confirmed by TCGA data analysis. Conclusions: We validated miR-17 as epigenetic regulator of LKB1 expression in NSCLC tumors. We propose a miR-17 expression score potentially exploitable to discriminate LKB1WT NSCLC patients with impaired LKB1 expression and poor outcome, eligible for energy-stress-based treatments. Citation Format: Cristina Borzi, Monica Ganzinelli, Elisa Caiola, Marika Colombo, Giovanni Centonze, Mattia Boeri, Laura Caleca, Ugo Pastorino, Mirko Marabese, Massimo Milione, Massimo Broggini, Marina C. Garassino, Gabriella Sozzi, Massimo Moro. LKB1 down-modulation by miR-17 biologically and clinically mirrors LKB1-mutated NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1539.

Abstract 5764: Genomic profiling of cutaneous, acral and mucosal melanomas in Chinese populations

Abstract Background: Targeted therapies and immunotherapies have significantly improved the survival of patients with cutaneous melanoma. Nevertheless, patients with acral and mucosal melanomas show limited benefit. The proportion of acral and mucosal subtypes are much higher in Chinese populations than Caucasians. Therefore, it is of great interest to detail the genomic features of acral and mucosal melanomas and compare these with cutaneous melanoma. Methods: A total of 111 Chinese patients with melanoma treated in Union Hospital, Tongji Medical College, Huazhong University of Science and Technology were enrolled in this study, comprising 32 cutaneous(28.8%), 41 acral(36.9%) and 26 mucosal(23.4%) subtypes. NGS-based genomic profiling of major cancer-related genes was performed on formalin-fixed, paraffin-embedded (FFPE) tumor tissue specimens with matched blood samples. Tumor mutational burden (TMB) was calculated based on somatic nonsynonymous missense mutations. Results: The most commonly mutated genes of cutaneous melanoma include BRAF(48%), CDKN2A(41%), CDKN2B(41%), NRAS(22%) and PTEN(22%). In acral melanoma patients, the mutation rate of BRAF was 10% while NRAS(31%), CDKN2A(21%), EMSY(21%), KIT(18%), CCND1(18%)) and NF1(18%) were frequently mutated. Mucosal melanomas were found most commonly to have mutations in CDKN2A(36%), KIT(32%), CDKN2B(28%), PDGFRA(24%), KDR(20%) and BRAF(16%). In acral and mucosal subtypes, the overall mutation rates of BRAF were significantly lower while the relative proportions of non-V600 mutations were higher as compared to cutaneous subtype. These may explain the less significant survival benefit of BRAFi+/-MEKi in acral and mucosal melanomas. In contrast, KIT mutations including SNVs and amplification, were enriched in acral and mucosal melanomas. Acral melanomas had lower mutation load than mucosal melanomas(2.42 Muts/Mb vs 4.84 Muts/Mb, p=0.0002), while cutaneous melanomas(4.03 Muts/Mb) did not differ from other subtypes in mutation burden. Additionally, patients with TERT, APC and FAT1 gene mutations had a significantly higher TMB than patients with wild-type genes respectively, indicating more activated anti-tumor immune profiles and suggesting therapeutic benefit from immune checkpoint inhibitors. Conclusions: We conducted a direction comparison of comprehensive genomic profiles of different melanoma subtypes among Chinese patients. The genomic alterations identified in our study may provide reference to discover potential strategies for targeted therapy and immunotherapy in acral and mucosal melanomas. Citation Format: Jing Chen, Ting Ye, Li Fan, Jieying Zhang, Rubo Cao, Yanshen Liu. Genomic profiling of cutaneous, acral and mucosal melanomas in Chinese populations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5764.

Abstract 5795: Validation of FDA approved oncoReveal Dx lung and colon cancer assay (oRDx-LCCA)

Abstract Introduction: The oncoReveal™ Dx Lung and Colon Cancer Assay (oRDx-LCCA) is an amplicon-based targeted next generation sequencing (NGS), in vitro diagnostic test that detects DNA variants across 22 genes, including single nucleotide variants (SNVs) and insertions and deletions (INDELs) in KRAS and EGFR, from DNA isolated from formalin-fixed paraffin-embedded (FFPE) non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) tumor tissue specimens. It is approved by the FDA as a companion diagnostic (CDx) for identifying approved therapeutic treatments for KRAS G12/G13 wild-type variants in CRC, and EGFR L858R variants and exon 19 in-frame deletions for NSCLC. oRDx-LCCA can detect non-CDx variants such as EGFR T790M, G719X and exon 20 insertions in NSCLC and KRAS Q61X and A146T and BRAF V600E in CRC. Variants are detected using Pillar Biosciences’ proprietary PiVAT® (Pillar Variant Analysis Toolkit) software. Methods: DNA input range was evaluated at 6 concentrations between 5 - 160ng in duplicate using DNA extracted from 15 clinical FFPE samples containing SNVs and INDELs from representative targeted mutations. Accuracy was determined by comparing detected variants with an externally validated comparator (CompO) across 208 samples (84 CRC, 124 NSCLC). Limit of detection (LoD) was determined by serial dilution of KRAS, EGFR, or BRAF positive FFPE samples in normal DNA to detect variant allele frequency (VAF) between 1 - 10%. Reproducibility tests were performed across 3 sites, 9 runs, 6 operators, 3 reagent lots and 3 thermocyclers, resulting in a total of 360 libraries. The non-inferiority (NI) statistical test was used to compare the oRDx-LCCA with an FDA approved CDx assay for EGFR exon 19 deletions and L858R mutations (CompQ) and for KRAS G12/G13 mutations (CompC). Synthetic FASTQ files were created at VAFs above and below the variant detection threshold for CDx mutations and used to validate quality control parameters, mean coverage, and on-target rate. Results: Effective DNA input range was determined to be between 10 - 80ng. LoD’s for KRAS G12/G13, EGFR L858 and exon 19 deletion, and BRAF V600E were established at &amp;lt; 4%. Positive percent agreement (PPA) and negative percent agreement (NPA) between oRDx-LCCA and CompO were &amp;gt; 99% each. Across all variables tested for reproducibility, the average percent positive agreement (APA) and average percent negative agreement (ANA) were each &amp;gt; 95%. NI margin was calculated to be &amp;lt; 10% between oRDx-LCCA and CompQ across all NSCLC samples and between oRDx-LCCA and CompC across all CRC samples. A total of 61 synthetic FASTQ datasets were created to validate the functionality and accuracy of PiVAT including VAF thresholds for 126 SNV and INDEL CDx variants. Conclusion: oRDx-LCCA is a highly accurate FDA approved IVD assay and PiVAT is a powerful FDA approved software tool for the detection of clinically relevant KRAS variants in CRC and EGFR variants in NSCLC and determination of approved therapy. Citation Format: Gloria Chan Johnson, Jordan Aldersley, Nicholas Lodato, Jamie Lim, Ian Flockhart, Mark Urbin, Yue Ke, Sean Polvino, Martin Zillmann, Gang Song, Zhaohui Wang. Validation of FDA approved oncoReveal Dx lung and colon cancer assay (oRDx-LCCA) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5795.

Abstract 57: Patients with germline ATM mutations develop clonal hematopoiesis characterized by co-occurrence of multiple somatic ATM alterations

Abstract BACKGROUND The DNA repair gene ATM is remarkable for its roles across the spectrum of neoplastic biology, including inherited risk of multiple malignancies, somatic tumor biology, and clonal hematopoiesis (CH). We hypothesized that comparison of liquid biopsy (LB) versus tumor tissue (TT) from germline ATM pathogenic mutation carriers (gATM+) might offer additional insights into the role of ATM in CH. METHODS 34,825 LB samples and 384,847 TT specimens were sequenced for up to 324 genes during routine clinical care and analyzed for all classes of genomic alterations (Frampton 2013, Woodhouse 2020). Individual variants were assessed for germline versus somatic origin using the validated somatic-germline zygosity algorithm (Sun 2018). RESULTS Germline ATM mutations were observed in 0.9% (113/12,217) of LB and 0.8% (3,029/384,847) of TT (P&amp;lt;0.001). Up to 27 ATM mutations were identified per gATM+ sample, with a range of 1-27 in LB and 1-8 in TT. Co-occurrence of 3+ ATM mutations in a single sample was 18-fold higher among gATM+ subjects undergoing LB versus TT (23.9% vs. 1.2%, P&amp;lt;0.001). In LB, the incidence of 3+ ATM mutations increased with age for all groups, but the frequency in gATM+ subjects diverged as patient age advanced. In the age 80+ subgroup, 57.1% (28/49) of gATM+ subjects had 3+ ATM mutations, compared to 3.3% (133/4,055) of gATM− subjects (P&amp;lt;0.001). A significant difference was maintained after correcting for the contribution of the germline ATM mutation to the total number of ATM mutations in gATM+ subjects. We hypothesized that multiple ATM variants per sample could be caused by high TMB or positive selection for multiple subclonal hits in the ATM gene. In TT, observation of 3+ ATM mutations was associated with high TMB in 80.5% (513/637) of cases. In contrast, 88.5% (555/627) of LB samples with 3+ ATM mutations had low TMB (Pdiff&amp;lt;0.001), a pattern consistent with positive selection in LB. LB from gATM+ cases with 3+ ATM mutations all had one variant with ~50% variant allele fraction (VAF), while 80.0% (546/682) of the co-mutations had VAF &amp;lt;1%. Co-mutations included 32.0% (793/2,485) truncating alterations and 68.1% (1,692/2,485) missense alterations. Low VAF missense co-mutations were clustered in the FAT and PIKKc domains, which are known pathogenic missense mutation hotspots. This distribution suggests that some missense alterations within these hotspots that are currently classified as VUS are under positive selection and therefore warrant consideration for reclassification. CONCLUSION The finding of numerous ATM mutations under positive selection in gATM+ subjects was specific to LB, increased in incidence with age, and co-mutations tended to have low VAF. All these features are consistent with polyclonal hematopoiesis (polyCH) involving ATM co-mutations in gATM+ subjects. Additional investigation is needed to extend our understanding of this phenomenon. Citation Format: Brennan Decker, Angela A. Kou, Tyler Janovitz, Douglas A. Mata, Ethan S. Sokol, Dexter X. Jin, Hanna Tukachinsky, Jo-Anne Vergilio, Julia A. Elvin, Geoffrey R. Oxnard. Patients with germline ATM mutations develop clonal hematopoiesis characterized by co-occurrence of multiple somatic ATM alterations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 57.