IHC Data Research Articles

Clinical significance of ALDH1A1 expression and its association with E-cadherin and N-cadherin in resected large cell neuroendocrine carcinoma

BackgroundThe roles of cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT) in solid tumors are well established. However, the interaction between CSCs and EMT in pulmonary large cell neuroendocrine carcinoma (LCNEC) remains unknown. The aim of this study was to investigate the expression and clinical significance of a CSC marker (ALDH1A1) and its correlation with Epithelial-like phenotype marker (E-cadherin) and Mesenchymal-like phenotype marker (N-cadherin) in LCNEC patients. MethodsImmunohistochemistry (IHC) for ALDH1A1, E-cadherin and N-cadherin expression was conducted on tissue microarrays made from 79 resected LCNEC patient samples. ALDH1A1 protein expression was evaluated by the IHC score, and its correlations with the expression of E-cadherin, N-cadherin and clinicopathological features were determined based on IHC data. Survival analyses were also performed. ResultsALDH1A1 was positively expressed in 75.9% (60/79 cases) of LCNEC patients. No significant difference in clinicopathological variables was observed between the ALDH1A1-negative and ALDH1A1-positive groups. However, ALDH1A1 expression was positively correlated with E-cadherin (Spearman's rho = 0.229, p-value = 0.007), which represents the epithelial-like phenotype, but not with N-cadherin. Patients with expression of ALDH1A1 had significantly longer disease-free survival (DFS) and overall survival (OS) than those who were ALDH1A1 negative (median DFS: 52 vs 12 months, p = 0.028; median OS: not reached; p = 0.027). Multivariate analysis showed that ALDH1A1 was an independent favorable prognostic factor for DFS (p = 0.032, HR: 0.438, 95% CI: 0.206–0.932) and OS (p = 0.025, HR: 0.279, 95% CI: 0.091–0.852) in LCNEC patients. ConclusionThis study suggests that ALDH1A1 can act as a favorable independent prognostic factor for LCNEC, which related to the epithelioid phenotype in EMT, and its internal mechanism needs further study.

Immune characterization of urothelial bladder carcinoma integrating transurethral resection of bladder tumor (TURBT) samples and serum: A feasibility study.

538 Background: Urothelial bladder cancer (UBC) is characterized by high immunogenicity. Multiple immunotherapies both in the perioperative and advanced setting are being developed. However, we lack good predictive biomarkers of response to immunotherapy in UBC. We aimed to characterize the immune profile of UBC patients using TURBT and serum samples. Methods: 13 UBC biopsies from TURBT samples were analyzed along with sera of these patients. Biopsy samples were divided in two equal parts. One part was formalin-fixed paraffin embedded and stained for immune histochemistry [IHC] for the following markers (CD3, CD4, CD8, CD20, CD68 and PD-L1 using SP142 antibody) and results expressed in percentages. The other part was homogenized and processed for immune cell isolation (human tumor isolation Miltenyi Kit). Tumor cells were stained with PHK and double positive CD8 and PHK were isolated using a MACSTM procedure. Different immune cell populations were analyzed by flow cytometry for the following markers (CD3, CD4, CD8, CD20 and CD68) and results expressed in percentages. Cytokines were examined in sera using the Luminex multiparametric procedure and results were expressed as pg/mL. Results: IHC and flow cytometry data were comparable and identified three groups according to the immune cells associated with the tumors (Table). Groups 1 and 2 showed high and moderate levels of tumor activated CD8+ T cells (1-4%) along with a higher expression of PD-L1 (40-20%). Group 3 lacked tumor activated CD8+ T cells and show lower expression of PD-L1 (15%). All groups showed significant levels of Th2 cytokines (IL-4, IL-6 or IL-10) while very low or lack of Th1 cytokines (IFN or IL-2) was observed except in group 2. Conclusions: TURBT biopsies and serum analysis of cytokines could represent a valid approach to further evaluate potential predictive biomarkers of response to immunotherapy in UBC. Different immune populations subgroups were identified in our series and could potentially predict for diverse responses to treatment. Further prospective validation in larger series is ongoing to confirm these results.[Table: see text]

High-plex proteomic prognostic marker discovery for patients with pancreatic cancer adenocarcinoma using digital spatial profiling.

611 Background: The long-term outcomes following surgical resection for Pancreatic Ductal Adenocarcinoma (PDAC) remain poor, with only 20% of patients surviving 5 years after pancreatectomy. With its immune-privileged nature, starting from the early pre-neoplastic state, it appears to escape easily from the antitumor immune response. Despite rationale for targeting immune pathways in PDAC, there has been little benefit observed at this point. The aim of the current study was to interrogate immune landscape of PDAC utilising the Nanostring GeoMx Digital Spatial Profiler (DSP), a state-of-the art analysis platform enabling Hi-plex proteomic characterisation whilst maintaining tumor microenvironment (TME) topographical features. Methods: We assessed Formalin Fixed Paraffin Embedded (FFPE) tumor samples from 28 treatment-naive PDAC cases represented in a multi-regional tissue microarray (TMA) for which extensive IHC, molecular, genomic characterisation and clinicopathological follow-up data is available. Following multiplex IHC staining for DAPI, panCK, aSMA and CD3 to guide region selection, we employed the GeoMx DSP system (NanoString) to select regions within multiple TMA cores. We quantified 60 immune markers simultaneously in multiple tissue compartments defined by immunofluorescence co-localization including (tumor [panCK+ve], immune stroma (PanCK-ve]. Data analysis was performed by a combination of DSP analysis suite and custom R pipeline. Results: The spatially informed variable assessment by DSP was validated by both regression and variable prognostication compared with IHC for stromal CD3, CD8 CD68 in near serial TMA PDAC sections. Unsupervised analysis of DSP proteome data in the panCK-negative regions identified an immune poor group associated with shorter Overall Survival (OS) (13.0 versus 31.1months, P = 0.005). When transcriptomic subtype was considered, the checkpoint inhibitor B7-H3 was significantly upregulated in the squamous subtype tumours versus the classical group (Log2: 1.63, P = 0.001). Patients with high B7-H3 expression, using a median expression cut-off, were associated with shorter OS on multivariate analysis (Hazard Ratio: 4.16, P = 0.01) including lymph node and resection margin status, a finding that was validated in an external cohort at the transcriptome level. Conclusions: This pilot scale discovery study shows the potential of the Nanostring DSP technology in the identification of spatially-informed biomarkers with prognostic relevance in biopsy sized samples from treatment-naive PDAC. We identified a number of relevant candidate immune predictors in spatial context that are currently undergoing validation in larger independent cohorts and the neoadjuvant setting. Future studies will apply this technology to pre- and post-treatment biopsy samples.

DNA Methylation Profiles of Ovarian Clear Cell Carcinoma.

Ovarian clear cell carcinoma (OCCC) is a rare ovarian cancer histotype that tends to be resistant to standard platinum-based chemotherapeutics. We sought to better understand the role of DNA methylation in clinical and biological subclassification of OCCC. We interrogated genome-wide methylation using DNA from fresh frozen tumors from 271 cases, applied nonsmooth nonnegative matrix factorization (nsNMF) clustering, and evaluated clinical associations and biological pathways. Two approximately equally sized clusters that associated with several clinical features were identified. Compared with Cluster 2 (N = 137), Cluster 1 cases (N = 134) presented at a more advanced stage, were less likely to be of Asian ancestry, and tended to have poorer outcomes including macroscopic residual disease following primary debulking surgery (P < 0.10). Subset analyses of targeted tumor sequencing and IHC data revealed that Cluster 1 tumors showed TP53 mutation and abnormal p53 expression, and Cluster 2 tumors showed aneuploidy and ARID1A/PIK3CA mutation (P < 0.05). Cluster-defining CpGs included 1,388 CpGs residing within 200 bp of the transcription start sites of 977 genes; 38% of these genes (N = 369 genes) were differentially expressed across cluster in transcriptomic subset analysis (P < 10-4). Differentially expressed genes were enriched for six immune-related pathways, including IFNα and IFNγ responses (P < 10-6). DNA methylation clusters in OCCC correlate with disease features and gene expression patterns among immune pathways. This work serves as a foundation for integrative analyses that better understand the complex biology of OCCC in an effort to improve potential for development of targeted therapeutics.

KIF4A Regulates the Progression of Pancreatic Ductal Adenocarcinoma through Proliferation and Invasion.

Background Pancreatic cancer is a malignant tumor of the digestive tract, which is difficult to diagnose and treat due to bad early diagnosis. We aimed to explore the role of kinesin superfamily 4A (KIF4A) in pancreatic ductal adenocarcinoma (PDAC). Methods We first used the bioinformatic website to screen the data of pancreatic cancer in TCGA, and KIF4A protein was detected among the 86 specimens of patients in our hospital combined with clinic-pathological characteristics and survival analysis. KIF4A loss-expression cell lines were established by RNA interference (RNAi). In addition, we performed in vitro cell assays to detect the changes in cell proliferation, migration, and invasion. The proteins involved in the proliferation and metastasis of cancer cells were also detected by western blot. The above results could be proved in vivo. Further, the correlation between KIF4A and CDC5L was analyzed by TCGA and IHC data. Results We first found a high expression of KIF4A in pancreatic cancer, suggesting a role of KIF4A in the development of pancreatic cancer. KIF4A was found to be differentially expressed (P < 0.05) among the 86 specimens of patients in our hospital and was significantly associated with PDAC TNM stages and tumor size. High KIF4A expression also significantly worsened overall survival (OS) and disease-free survival rate (DFS) (P < 0.05, respectively). In addition, cell proliferation, migration, and invasion were inhibited by the KIF4A-shRNA group compared with the control (P < 0.05, respectively). In the end, knockdown of KIF4A could inhibit tumor development and metastasis in vivo. Further, the positive correlation between KIF4A and CDC5L existed, and KIF4A might promote pancreatic cancer proliferation by affecting CDC5L expression. Conclusion In conclusion, the high expression level of KIF4A in PDAC was closely related to poor clinical and pathological status, lymphatic metastasis, and vascular invasion. KIF4A might be involved in promoting the development of PDAC in vitro and in vivo, which might be a new therapeutic target of PDAC.

A Phase 1 Dose Escalation Study of Igm-2323, a Novel Anti-CD20 x Anti-CD3 IgM T Cell Engager (TCE) in Patients with Advanced B-Cell Malignancies

A Phase 1 Dose Escalation Study of Igm-2323, a Novel Anti-CD20 x Anti-CD3 IgM T Cell Engager (TCE) in Patients with Advanced B-Cell Malignancies

Clinical significance of novel identified high-frequency tumor-specific peptides associated signature in predicting disease status of gastric cancer patients.

The effectively early detection and determination of disease progression of gastric cancer (GC) are still required. An emerging demand for identifying the novel targets adherent to cancer cells has been still challenged since those valuable profilings not only could act as for early gastric tumor discovery but also being potential therapeutic views. We have retrospectively analyzed GC biopsies to identify those specific target peptides in association with disease progression. We have detected the polypeptide by liquid mass technology initiated BIO-HIGH innovational assay technology for tumor-specific target peptide identification. We have validated the accessibility and feasibility of multiple target cytotoxic T-lymphocyte for the assessment of potential molecular markers by equally comparing the frequencies of tumor peptides' loci identified in 138 GC patients. The aim was to separate peripheral blood lymphocytes by density gradient centrifugation and use specific target peptides in in vitro culture of lymphocytes. The Cell Counting Kit-8 assay was set up to prove the lymphocytes' proliferation stimulated by identified peptides. Both of GC-specific peptide and shared peptide were detected in the peripheral blood, and the frequencies and quantities were correlated with disease status and cancer differentiation, in which BHGa1510 (78%), BHGa1310 (66%), BHGa0910 (57%), BHGa0310 (54%), BHGa0210 (40%), BHGa0810 (35%), BHGa0110 (33%), and BHGa1410 (30%) were apparently scoped out as high-frequency (HF) peptides could be potentially specific tumor markers. Moreover, BHGa1410 was significantly associated with cancer progression, and BHGa0910 and BHGa0210 were significantly associated with TNM stage. The IHC data have shown that both the HF BHGa1510 and HF BHGa1310 were expressions by 100% in contrast with paracancerous tissues of 40% (p < 0.05) and 33%, respectively (p < 0.05). Those specific peptide pools could be valued in assessment of advanced tumor and differential status in GC patients.

Abstract CT127: Tumor response and MET-detection methods exploratory biomarker analysis of Part B of the Ph 1b TATTON study

Abstract Background/Purpose Following disease progression on osimertinib, a third generation (3G), irreversible, oral epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI), ≤22% of patients (pts) with EGFR mutation positive (EGFRm) non-small cell lung cancer (NSCLC) develop MET-amplification as a resistance mechanism to treatment. In the Ph1b TATTON (NCT02143466) Part B expansion cohort, pts with MET-amplified EGFRm advanced NSCLC and progression on a prior EGFR-TKI received osimertinib + savolitinib, an oral, potent and highly selective MET TKI. Previous analyses have compared MET patient selection assays and found that different methods identify different but partially overlapping proportions of pts with MET amplification or overexpression. Here, we report the response rates observed in the subgroups defined by the different central MET assays in TATTON Part B. Methods MET testing (central, or local with central confirmation) was performed on tumor tissue collected after the most recent therapy. MET assays included FISH (MET gene copy number ≥5 or MET-CEP7 ratio ≥2) and IHC (MET +3 expression in ≥50% of tumor cells). TATTON Part B was split into 3 pt cohorts: pts previously treated with a 3G EGFR TKI and not previously treated with a 3G EGFR TKI (T790M positive/negative). This analysis uses the final study data (cutoff 4 March 2020) to compare the objective response rate (ORR) of subgroups identified by each assay. Results For pts who had received prior treatment with a 3G EGFR TKI, ORR by MET assay was as follows: FISH-positive overall (n=53) 30%; FISH polysomy (n=18) 28%; FISH amplification (n=35) 31%; IHC (n=13) 46%. For FISH-positive tumors, ORR trended higher for tumors with ≥10 MET gene copies (n=29, 35%) vs tumors with 5-9 copies (n=24, 25%). For pts with no prior treatment with a 3G EGFR TKI (T790M negative), ORR was as follows: FISH-positive overall (n=34) 65%; FISH polysomy (n=14) 57%; FISH amplification (n=20) 70%; IHC (n=4) 75%. For FISH-positive tumors, ORR trended higher for tumors with ≥10 MET gene copies (n=14, 79%) vs tumors with 5-9 copies (n=20, 55%). In tumors with both central FISH and IHC data available (n=34), IHC positivity (n=20) and FISH positivity (n=27) were correlated, specifically IHC and FISH amplification: 13/16 cases of FISH amplification were also IHC positive. In FISH-positive tumors IHC expression tended to increase with increasing gene copy number (≥10), but outcomes were largely comparable. Conclusions Overall, the MET assays were comparable with regards to ORR, thus supporting continued use of both FISH and IHC for detection of MET in pts following disease progression on any 3G EGFR TKI, though IHC data were limited. Further study is warranted in this area: the SAVANNAH (NCT03778229; FISH and IHC) and ORCHARD (NCT03944772; NGS) studies will provide opportunity for further analyses. Citation Format: Ryan Hartmaier, Ji-Youn Han, Byoung Chul Cho, Aleksandra Markovets, Nisha Kurian, Mireille Cantarini, Pasi A. Janne. Tumor response and MET-detection methods exploratory biomarker analysis of Part B of the Ph 1b TATTON study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT127.

Abstract 492: CNTN4 is a novel immune checkpoint protein that inhibits proliferation of T cell by interacting with APP

Abstract Contactin 4 (CNTN4, alias BIG-2) is a glycosylphosphatidylinositol (GPI)-anchored neuronal membrane protein that functions as cell adhesion, playing a key role in maintaining the mechanical integrity and signaling properties of the synapse. However, its function and crosstalk with immune cells have not been investigated. Therefore, we tried to find out what role CNTN4 plays in cell adhesion between T cells and antigen-presenting cells (APC). CNTN4 has several binding partners such as the amyloid precursor protein (APP), amyloid-like protein 1 (APLP1), and protein tyrosine phosphatase receptor γ (PTPRγ). In previous papers, the APP was reported to be expressed in T cells. We investigated whether CNTN4 can bind to T cells. The degree of binding of CNTN4 to T cells varied depending on the expression level of APP, and that the binding between these two proteins was in the form of trans-binding. And it was confirmed that the direct binding is achieved through fibronectin 1~2 domain of CNTN4 and KPI (Kunitz protease inhibitor) domain of APP. Furthermore, we investigated whether CNTN4 has any effect on T cell proliferation. T cell proliferation and cytokine secretion assays by using CNTN4 recombinant protein showed that CNTN4 significantly inhibits proliferation and cytokine secretion (IL-2, IFN-γ, TNF-α) of T cells more than PD-L1. Although the expression of CNTN4 in immune cells has not yet been clearly confirmed, the expression of CNTN4 in various carcinomas have confirmed by using western, IHC, and bioinformatic data analysis. In particular, the higher the expression of CNTN4 in tumor tissues of gastric cancer patients, the worse the prognosis is in the survival curve. Our findings suggest that CNTN4 may behave as an immune checkpoint protein similar to other B7 family members. Further investigation of the CNTN4 signaling pathway and its receptors are warranted by our data and may lead to novel therapeutic interventions. Citation Format: Mi Young Cha, Bu-Nam Jeon, Areum Jeong, Joo-Yeon Chung, Hyunsuk Yang, Kyung Mi Park, Hansoo Park. CNTN4 is a novel immune checkpoint protein that inhibits proliferation of T cell by interacting with APP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 492.

Abstract 23: Validation of a 27-gene immuno-oncology algorithm in metastatic urothelial carcinoma treated with an immune checkpoint inhibitor

Abstract Background Prognosis for patients with metastatic urothelial carcinoma (mUC) remains poor with fewer than 10% of patients surviving two years after diagnosis. The IMvigor210 was a multicenter, single-arm, phase 2 trial in patients with mUC treated by the immune checkpoint inhibitor (ICI) atezolizumab. RNA expression, overall survival, tumor mutation burden (TMB), and PD-L1 IHC data from this trial are publicly available.The 27-gene immuno-oncology (IO) algorithm has previously demonstrated significant association with ICI response in NSCLC and TNBC using three different ICI agents. Importantly, the algorithm and threshold for positivity was established prior to testing in ICI treated cohorts. Methods The prospectively defined 27-gene IO algorithm and threshold (IO Score) were applied to classify patients as likely responders or non-responders using available RNA expression data from 348 patients in the IMvigor210 trial. Cox proportional hazard models for overall survival was performed and the coefficient for the IO Score was determined in both a univariate and a multivariate equation with PD-L1 IHC and TMB. Results In total, 142 patients were positive by the IO Score (41%). Positivity was significantly associated with response (HR=0.61; 95% CI = 0.46 to 0.80; p &amp;lt; 0.001) and was significant in multivariate analysis with PD-L1 and TMB (HR=0.64; 95% CI = 0.45 to 0.92; p &amp;lt; 0.02). While both PD-L1 IHC staining and TMB were significant in univariate analysis, only TMB maintained significance in the multivariate equation. Conclusions These data demonstrate the association of the 27-gene IO algorithm and binary IO Score with response to ICI treatment in a third tissue indication (TNBC, NSCLC, and mUC) including four different ICI agents (two anti-PD-L1 and two anti-PD1). The algorithm and classification threshold were established prior to testing in all three studies and was not changed between the different tissue indications. Furthermore, in all three indications, only the IO Score consistently maintained significance in multivariate analysis with PD-L1 IHC and TMB. Taken together, these data begin to establish the 27-gene IO signature as a pan-cancer predictor of response to ICI therapy. Citation Format: Robert S. Seitz, Douglas T. Ross, Tyler J. Nielsen, David R. Hout, Brock L. Schweitzer. Validation of a 27-gene immuno-oncology algorithm in metastatic urothelial carcinoma treated with an immune checkpoint inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 23.

Serum total periostin is an independent marker of overall survival in bone metastases of lung adenocarcinoma

More than 35% of lung adenocarcinoma patients have bone metastases at diagnosis and have a poor survival. Periostin, a carboxylated matrix protein, mediates lung cancer cell dissemination by promoting epithelial-mesenchymal transition, and is involved in bone response to mechanical stress and bone formation regulation. This suggests that periostin may be used as a biomarker to predict survival in lung cancer patients.Serum periostin was assessed at diagnosis in a prospective cohort of 133 patients with lung adenocarcinoma of all stages. Patients were divided into localized and bone metastatic groups. Both groups were matched to healthy controls. Survival analysis and Cox proportional hazards models were conducted in the total population and in bone metastatic group.The median serum periostin level was higher in bone metastatic (n = 67; median: 1752 pmol/L) than in the localized group (n = 66; 861 pmol/L; p < 0.0001). Patients with high periostin (>median) had a poorer overall survival in the whole population (33.3 weeks vs. NR; p < 0.0001) and the bone metastatic group (24.4 vs. 66.1 weeks; p < 0.001). In multivariate analysis, patients with high periostin had increased risk of death (HR = 2.09, 95%CI [1.06–4.13]; p = 0.03). This was also found in the bone metastatic group (HR = 3.62, 95%CI [1.74–7.52]; p = 0.0005). Immunohistochemistry on bone metastasis biopsies showed periostin expression in the bone matrix and nuclear and cytoplasmic staining in cancer cells.Serum periostin was an independent survival biomarker in all-stage and in bone metastatic lung adenocarcinoma patients. IHC data suggest that periostin might be induced in cancer cells in bone metastatic niche in addition to bone microenvironment expression.

The process of urinary bladder cancer recurrence in 33 patients with 113 tumors, analyzed by changes in IHC features of 80 pairs of subsequent tumors.

e16507 Background: The aim was to define IHC changes between the two subsequent urinary bladder cancers (UBC). Methods: IHC data on EGFR, HER2, HER3, Ki-67, MLH1, MSH2, MSH6 and PMS2 in 113 UHC from 24 male and 9 female patients (1 to six recurrences) were used. Except for the Ki-67 value, other markers were stratified: “0” for no positive cells; “1” &lt; = 10% positive cells; “2” 1%-30% positive cells; and “3” 31%-100% positive cells. Data of consecutive tumors were paired in 80 processes of recurrence (PoR). Changes between the latter and the former tumor were calculated: in +/- % for Ki-67 values, and as integer sums of absolute changes in expression for HER markers and for Lynch markers. EM clustering was applied for recognition of relevant IHC changes. Results: Three aspects were tested: the speed of recurrence, was it the first, or later recurrence and whether PoRs depended on the total number of tumors in that patient. Early and late PoRs clustered along the Lynch score, while the intermediate clustered along the delta Ki-67 value. The first PoRs clustered along the HER score and all subsequent PoRs depended on the Lynch score. Conclusions: Even in this limited group of patients Lynch and HER markers showed complex differences between early and late recurrent tumors. The speed of the recurrence and changes of IHC features depended mainly on the rate of change in Lynch markers, suggesting that they should be tested as predictors of UBC recurrence.[Table: see text]

Evaluation of expression and serum concentration of anti-Mullerian hormone as a follicle growth marker following consumption of fennel and flaxseed extract in first-generation mice pups

BackgroundThe aim of the present study was to assess the expression and serum level of AMH in first-generation female mice pups following fennel and flaxseed consumption.MethodsTwenty pregnant NMRI mice were allocated into four groups including control (CTL), fennel (FV), flaxseed (LU) and FV+ LU. Sixty-four female offsprings after lactation period, received the same regimen as their mothers for 56 and 240 days. The ovarian follicles development, serum concentration of AMH, as well as gene and protein expression of AMH were evaluated in the female offsprings at post-natal day 56 (PND56) and 240 (PND240).ResultsThe number of total growing follicles were raised in the FV group in compression to the all experimental groups. In contrast, LU group showed a marked decrease in their numbers. The highest level of serum AMH was seen in the FV-diet mice, whereas LU negatively affected it. The expression level of AMH also increased in the FV and FV + LU groups, while a reduction was observed in the LU group. As well, IHC data showed that the number of AMH-positive cells in almost ovarian follicles of FV and FV + LU-treated mice was in compared to those of the LU group.ConclusionsThe overall effect of fennel treatment (alone and in combination with flaxseed) on ovary might be maintain primordial follicle storage through increased expression and serum level of AMH.

Diagnosis prediction of tumours of unknown origin using ImmunoGenius, a machine learning-based expert system for immunohistochemistry profile interpretation

BackgroundImmunohistochemistry (IHC) remains the gold standard for the diagnosis of pathological diseases. This technique has been supporting pathologists in making precise decisions regarding differential diagnosis and subtyping, and in creating personalized treatment plans. However, the interpretation of IHC results presents challenges in complicated cases. Furthermore, rapidly increasing amounts of IHC data are making it even harder for pathologists to reach to definitive conclusions.MethodsWe developed ImmunoGenius, a machine-learning-based expert system for the pathologist, to support the diagnosis of tumors of unknown origin. Based on Bayesian theorem, the most probable diagnoses can be drawn by calculating the probabilities of the IHC results in each disease. We prepared IHC profile data of 584 antibodies in 2009 neoplasms based on the relevant textbooks. We developed the reactive native mobile application for iOS and Android platform that can provide 10 most possible differential diagnoses based on the IHC input.ResultsWe trained the software using 562 real case data, validated it with 382 case data, tested it with 164 case data and compared the precision hit rate. Precision hit rate was 78.5, 78.0 and 89.0% in training, validation and test dataset respectively. Which showed no significant difference. The main reason for discordant precision was lack of disease-specific IHC markers and overlapping IHC profiles observed in similar diseases.ConclusionThe results of this study showed a potential that the machine-learning algorithm based expert system can support the pathologic diagnosis by providing second opinion on IHC interpretation based on IHC database. Incorporation with contextual data including the clinical and histological findings might be required to elaborate the system in the future.

Expression of VWF and Interferon γ in renal allograft biopsies and correlation with inflammatory cells; Single center experience

Objective: Gene expression profiling by microarrays or RT PCR had been studied in certain western centers to enhance the diagnostic accuracy of allograft biopsy, however, such sophisticated tests are difficult to apply in developing countries. This study was conducted to evaluate the expression of von Willebrand factor (VWF) and Interferon-gamma (IFNγ) as an end PCR product in renal allograft biopsy with different pathological categories.&#x0D; Methods: Forty-nine indicated renal allograft biopsies were analyzed histologically by the Banff 2017 classification, inflammatory cell infiltration was analyzed by immunohistochemistry study for CD4, CD8, CD16, and CD68 markers, and a fresh tissue used for molecular study.&#x0D; Results: The biopsy findings were acute T-cell mediated rejection (A- TCMR) 30.6%, interstitial fibrosis and tubular atrophy (IF/TA) 22.4%, C4d-Transplant glomerulopathy (TG) 12.2%, calcineurin inhibitor toxicity (CNI) 10.2%, C4d+antibody mediated rejection (AMR) 10.2% and normal histology 14.3%. The only significant difference in VWF expression was between acute TCMR and normal, P=0.01, Spearman's correlation also showed a significant relationship between VWF and acute TCMR (r=0.53, P=0.01), and VWF was found to correlate with the numbers of interstitial CD4+ (r=0.29, P=0.03) and CD68+ (r=0.37, P=0.007) cells. IFNγ expression was significant in acute TCMR versus normal, P=0.009. Spearman's pair-wise testing showed that INFγ correlated with CD8 in both the glomerular (r=0.38, P=0.006) and interstitial (r=0.30, P=0.04) compartments and with CD16+ interstitial cells (r=0.36, P=0.01).&#x0D; Conclusion: our molecular and IHC data distinguish acute TCMR from other forms of transplant pathology and mildly dysfunctional kidneys with normal histology.

Abstract PS4-01: Clinical and genomic correlation of a CLIA certified organoid based functional test in breast cancer patients

Abstract The goal of precision medicine is to match the right drug to the right patient. However, every individual cancer carries a unique and complex mosaic of genetic and molecular changes making it difficult to identify the right drug based solely on genomic analysis. We developed a CLIA-certified functional drug assay (PARIS® test) for solid tumors which provides an actionable report of tumor derived organoid sensitivities to targeted, endocrine and chemotherapy agents as a tool for clinical therapeutic decisions. Objectives:1.To establish the concordance between organoid drug sensitivity with well-known genomic or immunohistochemical IHC biomarkers 2.To correlate organoid drug sensitivity with clinical outcomes.Methods: From 2015 to 2020, organoids from 410 tumor samples were subjected to functional testing at SEngine Precision Medicine, including 61 breast tumor samples from 48 patients. Fresh samples of tumor cells from core biopsies, surgical excisions, or from fluids arrived &amp;lt;48 hrs following collection and were cultured as 3D organoids. Samples were evaluated using a multi-dose drug response format with a library of up to 130 oncology drugs. Drug sensitivity was quantified using the SPM score (1-15) that combines sensitivity and personalization of each patient’s response relative to a reference population. Known genomic anchors and IHC subtypes were compared to drug sensitivity to determine concordance.Results: 61 breast cancer samples from 48 patients were analyzed. The median age of patients was 53.4 (r26-76). 65 drugs on average were tested per patient with a mean turnaround time of 21 days (r9 -37). A mean of 6 drugs per patient were identified as top scoring sensitive drugs. In 42 patients with genomic or IHC data, we found high concordance of drug sensitivity with known biomarkers (e.g., HER2+ or ERBB2 amplification:HER2 or EGFR inhibitor, BRCA1 mutation: PARP inhibitor, FGFR1-2 mutation or amplification: FGFR inhibitor, PIK3CA mutation:PI3K inhibitor), measured as sensitivity to the cognate targeted drugs. For PIK3CAmut we found an 80% correlation of organoid sensitivity to alpelisib and taselisib. For HER2/ EGFRinh the correlation was 100%. We also found organoid sensitivity to targeted agents in the absence of known genomic or IHC biomarkers, for example tamoxifen and fulvestrant sensitivity in triple negative breast tumors, or HER2 inhibitor sensitivity in HER2 IHC negative tumors or PARP inhibitor sensitivity with BRCA2 variant of unknown significance (VUS). In a cohort of 18 analyzable patients, the retrospective and prospective correlation between organoid based drug sensitivity and clinical outcome was &amp;gt;90%.Conclusions: Organoid based drug testing exhibits strong concordance with genomic or IHC biomarkers and clinical response. In addition, functional testing identifies candidate therapies in patients lacking biomarkers and can nominate variants of unknown significance as candidate biomarkers. This study highlights the utility of functional assays to support clinical decision making in a genetically heterogenous cancer such as breast cancer. Citation Format: Astrid Margossian, Anne Richardson, Madison Pollastro, Michael Churchill, Franz Schaub, Shalini Pereira, Payel Chatterjee, Rachele Rosati, Lauren Appleyard, Grace Durenberger, Alex Federation, G. Adam Whitney, Hallie Swan, Trevor Ainge, Robert Diaz, Natasha Hunter, Eric Gamboa, Chris Kemp, Vijayakrishna Gadi, Carla Grandori. Clinical and genomic correlation of a CLIA certified organoid based functional test in breast cancer patients [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-01.

Abstract PS13-28: Pre-treating TNBC with docetaxel and il-12 enhances anti-PD1 efficacy

Abstract Introduction: Amongst all breast cancers, Triple Negative Breast Cancer (TNBC) account for 15-20% of all the cases. TNBC affect younger patients, and is more prevalent in African-American women. The poor prognosis for this very aggressive tumor subtype is exacerbated by the lack of specific targeted therapy against the disease. Although TNBC initially respond very well to chemotherapy, paradoxically the disease-free survival is very short. It has been showed that TNBC have higher rates of CD8+ T-cells infiltration, and express high level of PD-L1. Together, these data provide a strong rationale for the combination of chemotherapy and immunotherapy to treat TNBC patients. In this study, we investigated the response to pre-priming the tumor with one round of docetaxel and IL-12, followed by anti-PD1 maintenance in mouse E0771 and 4T1 TNBC syngeneic models. We hypothesized that docetaxel will promote the release of neo-antigens, while IL-12 will activate immunity specific to these antigens and anti-PD1 therapy prevent the exhaustion of those T-cells. Materials/Methods: Mouse TNBC E0771 and 4T1 cell lines were injected in the mammary fat pad of C57BL/6, and Balb/c mice respectively. On day 1, the mice received a single dose (20mg/kg) of docetaxel and one intratumoral injection (1.25x109) of mAdv.IL-12, a replication defective adenoviral vector containing mIL-12 (mouse) cDNA under the transcriptional control of Rous sarcoma virus long terminal repeat (provided by Dr. Chen, HMRI). Anti-PD1 (InVivoMab anti-mouse PD-1 CD279) was administered 3 times a week (2 cycles) starting 5 days post docetaxel and Il-12 treatment. At the end of the study, IFN-gamma levels were measured from blood and tumor samples; tumor sizes were compared between treatment groups (Control/mAdv.IL-12/anti-PD1/and various Combination), as well as survival curves. The metastatic burden to the lungs (H&amp;E), as well as the apoptosis in the tumor (TUNNEL) were assessed by IHC. Results: In both 4T1 and E0771 tumor models, triple combination of docetaxel + IL-12 followed by anti-PD1 significantly reduced tumor size compared to both single agents, and double combination. In the Triple Combo group, 1 mice had lung metastasis vs all of them in the other treatment groups. IHC data indicate a higher level of TILs in the treatment groups, with a statistically significant difference in the combination groups compared to single agents. There was more apoptosis in the triple combo group as indicated by TUNNEL. Conclusion:Our preliminary data strongly supports that treating TNBC models with docetaxel and mAdv.IL-12 followed by anti-PD1 significantly slows down tumor growth, and decrease lung metastasis incidence. We are actively investigating the mechanism through which the response is achieved. Citation Format: Ann Cassany Anselme, Wei Qian, Jianying Zhou, Roberto R. Rosato, Jenny C. Chang. Pre-treating TNBC with docetaxel and il-12 enhances anti-PD1 efficacy [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS13-28.

Pan-Cancer Analysis of Ligand–Receptor Cross-talk in the Tumor Microenvironment

Signaling between cancer and nonmalignant (stromal) cells in the tumor microenvironment (TME) is a key to tumor progression. Here, we deconvoluted bulk tumor transcriptomes to infer cross-talk between ligands and receptors on cancer and stromal cells in the TME of 20 solid tumor types. This approach recovered known transcriptional hallmarks of cancer and stromal cells and was concordant with single-cell, in situ hybridization and IHC data. Inferred autocrine cancer cell interactions varied between tissues but often converged on Ephrin, BMP, and FGFR-signaling pathways. Analysis of immune checkpoints nominated interactions with high levels of cancer-to-immune cross-talk across distinct tumor types. Strikingly, PD-L1 was found to be highly expressed in stromal rather than cancer cells. Overall, our study presents a new resource for hypothesis generation and exploration of cross-talk in the TME. SIGNIFICANCE: This study provides deconvoluted bulk tumor transcriptomes across multiple cancer types to infer cross-talk in the tumor microenvironment.

High expression of vinculin predicts poor prognosis and distant metastasis and associates with influencing tumor-associated NK cell infiltration and epithelial-mesenchymal transition in gastric cancer.

In the process of epithelial-mesenchymal transition (EMT), epithelial cancer cells transdifferentiate into mesenchymal-like cells with high motility and aggressiveness, resulting in the spread of tumor cells. Immune cells and inflammation in the tumor microenvironment are the driving factors of EMT, but few studies have explored the core targets of the interaction between EMT and tumor immune cells. We analyzed thousands of cases of gastric cancer and gastric tissue specimens of TCGA, CPTAC, GTEx and analyzing QPCR and IHC data of 56 gastric cancer patients in SYSU Gastric Cancer Research Center. It was known that EMT has an important connection with the infiltration of NK cells, and that the expression of vinculin may be the target of the phenomenon. The increased expression of vinculin is closely related to the aggressiveness and distant metastasis of cancer, which affects the survival prognosis of the patient. Moreover, through in vitro experiments under 3D conditions, we found that vinculin, cell invasion and metastasis are clearly linked. VCL can affect EMT and tumor immunity by regulating EPCAM gene expression. The role and mechanism of action of vinculin have been controversial, but this molecule may downregulate EpCAM (epithelial cellular adhesion molecule) and its own role in gastric cancer through DNA methylation, causing NK cells to enrich into tumor cells and kill tumor cells. At the same time, it promotes the occurrence of EMT, which in turn causes tumor metastasis and thus poorer prognosis.

High nuclear expression of APE1 correlates with unfavorable prognosis and promotes tumor growth in hepatocellular carcinoma.

APE1 is a multifunctional protein that plays important roles in cancer development. However, the association between APE1 expression and the clinicopathological parameters of HCC patients has not been fully characterized. In this study, bioinformatics analysis of APE1 was performed in several databases, including the TCGA, GeneCard, Human Protein Atlas and Ualcan databases. The relationship between APE1 mRNA expression and several attributes of liver cancer patients in TCGA was investigated. Then, the protein expression of APE1 was detected by IHC analysis in 95 HCC samples and the association between APE1 expression and the clinicopathological parameters of HCC patients was explored. GSEA-KEGG analysis was performed to predict the potential signaling pathways that associated with APE1 expression. Then the siRNA-mediated knockdown model of APE1 was constructed in HCC cell line to further detect the detailed function of APE1 in HCC development in vitro and in vivo. The results of the bioinformatics analysis showed that APE1 expression was primarily located in the cell nucleus. APE1 mRNA expression was substantially correlated with pathological grade and T status in TCGA database. Elevated APE1 expression was observed in HCC samples and was associated with unfavorable survival time in liver cancer patients. IHC data demonstrated that the nuclear expression of APE1 in HCC tissues was significantly higher than that in noncancerous tissues. The expression level of the APE1 protein in HCC was strongly associated with tumor diameter and overall survival. Survival analysis indicated that APE1 nuclear expression is an independent prognostic marker for the overall survival of HCC patients. GSEA-KEGG results confirmed that APE1 associated with the base excision repair signaling pathway. The data of phenotypic experiments indicated that APE1 remarkably promoted tumor growth both in HCC cells and xenografts. The findings firstly imply that nuclear expression of APE1 is a valuable prognostic marker for HCC. APE1 significantly facilitate HCC development and targeting APE1 may be a promising strategy for HCC treatment.