Qualification of a multiplexed tissue imaging assay and detection of novel patterns of HER2 heterogeneity in breast cancer.

Emerging data suggests that HER2 intratumoral heterogeneity (ITH) is associated with therapy resistance, highlighting the need for new strategies to assess HER2 ITH. A promising approach is leveraging multiplexed tissue analysis techniques such as cyclic immunofluorescence (CyCIF), which enable visualization and quantification of 10-60 antigens at single-cell resolution from individual tissue sections. In this study, we qualified a breast cancer-specific antibody panel, including HER2, ER, and PR, for multiplexed tissue imaging. We then compared the performance of these antibodies against established clinical standards using pixel-, cell- and tissue-level analyses, utilizing 866 tissue cores (representing 294 patients). To ensure reliability, the CyCIF antibodies were qualified against HER2 immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) data from the same samples. Our findings demonstrate the successful qualification of a breast cancer antibody panel for CyCIF, showing high concordance with established clinical antibodies. Subsequently, we employed the qualified antibodies, along with antibodies for CD45, CD68, PD-L1, p53, Ki67, pRB, and AR, to characterize 567 HER2+ invasive breast cancer samples from 189 patients. Through single-cell analysis, we identified four distinct cell clusters within HER2+ breast cancer exhibiting heterogeneous HER2 expression. Furthermore, these clusters displayed variations in ER, PR, p53, AR, and PD-L1 expression. To quantify the extent of heterogeneity, we calculated heterogeneity scores based on the diversity among these clusters. Our analysis revealed expression patterns that are relevant to breast cancer biology, with correlations to HER2 ITH and potential relevance to clinical outcomes.

Clinical and molecular characteristics of RNF43 mutations as promising prognostic biomarkers in colorectal cancer.

Background: Transmembrane E3 ubiquitin ligase (RNF43) mutations are present in approximately 6-18% of colorectal cancers (CRC) and could enhance Wnt/ß-catenin signaling, which is emerging as a promising therapeutic target. This study aims to investigate the clinical and molecular characteristics and potential heterogeneity of RNF43-mutant CRC. Methods: A total of 78 patients with RNF43-mutant CRC were enrolled from July 2013 to November 2022. Demographic data, clinical characteristics, treatment regimens used, and survival outcomes were collected and analyzed. Results: Our study uncovered that patients with RNF43 mutations in the N-terminal domain (NTD; n = 50) exhibited shorter overall survival (OS; median months, 50.80 versus not reached; p = 0.043) compared to those in the C-terminal domain (CTD; n = 17). Most RNF43 mutations in NTD had positive primary lymph node status, low tumor mutation burden (TMB-L), and correlated with proficient mismatch repair (pMMR)/microsatellite stable (MSS) status. By contrast, RNF43 mutations in CTD were significantly enriched in deficient MMR (dMMR)/microsatellite instability (MSI-H) tumors with high TMB (TMB-H). N-terminal RNF43-mutated tumors harbored a hotspot variant (RNF43 R117fs), which independently predicted a significantly worse outcome in pMMR/MSS CRC with a median OS of 18.9 months. Patients with RNF43 mutations and the BRAF V600E alterations demonstrated sensitivity to BRAF/EGFR inhibitors. Moreover, we observed that pMMR/MSS patients with RNF43 R117fs mutation had a higher incidence of stage IV, ⩾2 metastatic sites, low TMB, and none of them received PD-1/PD-L1 inhibitor therapy. Conclusion: Our findings provide the first evidence that RNF43 mutations in NTD and the R117fs variant correlate with a poorer prognosis in CRC patients, providing strategies for Wnt-targeted therapy to improve clinical efficacy.

Comprehensive analysis reveals potential therapeutic targets and an integrated risk stratification model for solitary fibrous tumors.

Solitary fibrous tumors (SFTs) are rare mesenchymal tumors with unpredictable evolution and with a recurrence or metastasis rate of 10-40%. Current medical treatments for relapsed SFTs remain ineffective. Here, we identify potential therapeutic targets and risk factors, including IDH1 p.R132S, high PD-L1 expression, and predominant macrophage infiltration, suggesting the potential benefits of combinational immune therapy and targeted therapy for SFTs. An integrated risk model incorporating mitotic count, density of Ki-67+ cells and CD163+ cells, MTOR mutation is developed, applying a discovery cohort of 101 primary non-CNS patients with negative tumor margins (NTM) and validated in three independent cohorts of 210 SFTs with the same criteria, and in 36 primary CNS SFTs with NTM. Compared with the existing models, our model shows significantly improved efficacy in identifying high-risk primary non-CNS and CNS SFTs with NTM for tumor progression.Our findings hold promise for advancing therapeutic strategies and refining risk prediction in SFTs.

Targeted demethylation of the CDO1 promoter based on CRISPR system inhibits the malignant potential of breast cancer cells.

BACKGROUND: Cysteine dioxygenase 1 (CDO1) is frequently methylated, and its expression is decreased in many human cancers including breast cancer (BC). However, the functional and mechanistic aspects of CDO1 inactivation in BC are poorly understood, and the diagnostic significance of serum CDO1 methylation remains unclear. METHODS: We performed bioinformatics analysis of publicly available databases and employed MassARRAY EpiTYPER methylation sequencing technology to identify differentially methylated sites in the CDO1 promoter of BC tissues compared to normal adjacent tissues (NATs). Subsequently, we developed a MethyLight assay using specific primers and probes for these CpG sites to detect the percentage of methylated reference (PMR) of the CDO1 promoter. Furthermore, both LentiCRISPR/dCas9-Tet1CD-based CDO1-targeted demethylation system and CDO1 overexpression strategy were utilized to detect the function and underlying mechanism of CDO1 in BC. Finally, the early diagnostic value of CDO1 as a methylation biomarker in BC serum was evaluated. RESULTS: CDO1 promoter was hypermethylated in BC tissues, which was related to poor prognosis (p < .05). The CRISPR/dCas9-based targeted demethylation system significantly reduced the PMR of CDO1 promotor and increased CDO1 expression in BC cells. Consequently, this leads to suppression of cell proliferation, migration and invasion. Additionally, we found that CDO1 exerted a tumour suppressor effect by inhibiting the cell cycle, promoting cell apoptosis and ferroptosis. Furthermore, we employed the MethyLight to detect CDO1 PMR in BC serum, and we discovered that serum CDO1 methylation was an effective non-invasive biomarker for early diagnosis of BC. CONCLUSIONS: CDO1 is hypermethylated and acts as a tumour suppressor gene in BC. Epigenetic editing of abnormal CDO1 methylation could have a crucial role in the clinical treatment and prognosis of BC. Additionally, serum CDO1 methylation holds promise as a valuable biomarker for the early diagnosis and management of BC.

Monitoring tumour resistance to the BRAF inhibitor combination regimen in colorectal cancer patients via circulating tumour DNA.

AIMS: This study aimed to identify mechanisms of drug resistance to the combination of vemurafenib, irinotecan, and cetuximab (VIC) in BRAFV600E metastatic colorectal cancer (mCRC). METHODS: Forty-one patients with BRAFV600E mCRC from July 2018 and June 2020 were evaluated, with tissue and/or plasma samples collected. We profiled tissue and plasma samples using whole-exome sequencing and targeted sequencing of 425 cancer-relevant genes. Clinical cohort analysis from published studies was performed to consolidate our findings. RESULTS: BRAF mutant in baseline plasma and its dynamics are significantly associated with VIC-related response, and concurrent RNF43 mutation significantly sensitises tumour to VIC treatment. VIC resistance frequently involves genes in PI3K, MAPK pathway, and several novel resistance mechanisms such as TGFBR2 and SMAD4 mutations, and copy-number gains in PTK2, MYC, and GATA6 have been identified. We also firstly describe acquired altered genes in DNA damaging repair pathway, occurring in 33 % of patients after VIC treatment, and particularly, patients with this pre-treatment resistance subclones developed inferior responses, along with higher tumour mutation burden both at baseline and progression plasma. CONCLUSION: Analysis of ctDNA can provide novel insights into molecular resistance mechanisms to VIC in BRAFV600E mCRC patients, allowing accurate guidance for clinicians in personalised treatment strategies.

Targeted demethylation at ZNF154 promotor upregulates ZNF154 expression and inhibits the proliferation and migration of Esophageal Squamous Carcinoma cells.

Zinc finger protein 154 (ZNF154) is hypermethylated at the promoter in many epithelial-derived solid tumors. However, its methylation status and function in esophageal squamous carcinoma (ESCC) are poorly understood. We found that the ZNF154 promoter is hypermethylated in ESCC and portends poor prognosis. In addition, ZNF154 functions as a tumor suppressor gene (TSG) in ESCC, and is downregulated by promoter hypermethylation. We established a targeted demethylation strategy based on CRISPR/dCas9 technology and found that the hypermethylation of ZNF154 promoter repressed ZNF154 induction, which in turn promoted the proliferation and migration of ESCC cells in vitro and in vivo. Finally, high-throughput CUT&Tag analysis, GEPIA software and qPCR were used to revealed the role of ZNF154 as a transcription factor to upregulate the expression of ESCC-associated tumor suppressor genes. Taken together, hypermethylation of the ZNF154 promoter plays an important role in the development of ESCC, and epigenetic editing is a promising tool for inhibiting ESCC cells with aberrant DNA methylation.

Alpha-Fetoprotein Ratio Predicts Alpha-Fetoprotein Positive Hepatocellular Cancer Patient Prognosis after Hepatectomy.

BACKGROUND: This study was conducted to investigate the effect of alpha-fetoprotein (AFP) ratio on the prognosis of AFP-positive hepatocellular carcinoma (HCC) patients after hepatectomy. METHODS: We retrospectively included 879 HCC patients with AFP-positive who underwent hepatectomy from February 2012 to October 2017 and randomly divided into training cohort and validation cohort. AFP ratio was equal to the AFP level within one week before hepatectomy to AFP level within 20-40 days after surgery. The end point of follow-up was disease-free survival (DFS) and overall survival (OS). RESULTS: AFP ratio was not associated with clinical characteristics in training cohort and validation cohort. According to the X-tile software, the optimum cut-off point was 17.8 for AFP ratio. Significant differences between AFP ratio high and AFP ratio low were observed in DFS and OS in both cohort (p < 0.05). Kaplan-Meier curves and receiver-operating curves were showed that AFP ratio was better than AFP level preoperation in predicting the prognosis of AFP-positive HCC patients after hepatectomy. The multivariate analysis demonstrated that AFP ratio was a significant independent risk factor for both OS and DFS in HCC patients with AFP-positive. CONCLUSIONS: AFP ratio might be a prognosis predictor for HCC patients with AFP-positive after hepatectomy.

Activity of PD-1 blockade with nivolumab among patients with recurrent atypical/anaplastic meningioma: phase II trial results.

BACKGROUND: Programmed death ligand 1 (PD-L1) contributes to tumor immunosuppression and is upregulated in aggressive meningiomas. We performed a phase II study of nivolumab, a programmed death 1 (PD-1) blocking antibody among patients with grade ≥2 meningioma that recurred after surgery and radiation therapy. METHODS: Twenty-five patients received nivolumab (240 mg biweekly) until progression, voluntary withdrawal, unacceptable toxicity, or death. Tumor mutational burden (TMB) and quantification of tumor-infiltrating lymphocytes (TIL) were evaluated as potential immunocorrelative biomarkers. Change in neurologic function was prospectively assessed using the Neurologic Assessment in Neuro-Oncology (NANO) scale. RESULTS: Enrolled patients had multiple recurrences including ≥3 prior surgeries and ≥2 prior courses of radiation in 60% and 72%, respectively. Nivolumab was well tolerated with no unexpected adverse events. Six-month progression-free survival (PFS-6) rate was 42.4% (95% CI: 22.8, 60.7) and the median OS was 30.9 months (95% CI: 17.6, NA). One patient achieved radiographic response (ongoing at 4.5 years). TMB was >10/Mb in 2 of 15 profiled tumors (13.3%). Baseline TIL density was low but increased posttreatment in 3 patients including both patients with elevated TMB. Most patients who achieved PFS-6 maintained neurologic function prior to progression as assessed by NANO. CONCLUSION: Nivolumab was well tolerated but failed to improve PFS-6, although a subset of patients appeared to derive benefit. Low levels of TMB and TIL density were typically observed. NANO assessment of neurologic function contributed to outcome assessment. Future studies may consider rationally designed combinatorial regimens.

Multimodal platform for assessing drug distribution and response in clinical trials.

BACKGROUND: Response to targeted therapy varies between patients for largely unknown reasons. Here, we developed and applied an integrative platform using mass spectrometry imaging (MSI), phosphoproteomics, and multiplexed tissue imaging for mapping drug distribution, target engagement, and adaptive response to gain insights into heterogeneous response to therapy. METHODS: Patient-derived xenograft (PDX) lines of glioblastoma were treated with adavosertib, a Wee1 inhibitor, and tissue drug distribution was measured with MALDI-MSI. Phosphoproteomics was measured in the same tumors to identify biomarkers of drug target engagement and cellular adaptive response. Multiplexed tissue imaging was performed on sister sections to evaluate spatial co-localization of drug and cellular response. The integrated platform was then applied on clinical specimens from glioblastoma patients enrolled in the phase 1 clinical trial. RESULTS: PDX tumors exposed to different doses of adavosertib revealed intra- and inter-tumoral heterogeneity of drug distribution and integration of the heterogeneous drug distribution with phosphoproteomics and multiplexed tissue imaging revealed new markers of molecular response to adavosertib. Analysis of paired clinical specimens from patients enrolled in the phase 1 clinical trial informed the translational potential of the identified biomarkers in studying patient's response to adavosertib. CONCLUSIONS: The multimodal platform identified a signature of drug efficacy and patient-specific adaptive responses applicable to preclinical and clinical drug development. The information generated by the approach may inform mechanisms of success and failure in future early phase clinical trials, providing information for optimizing clinical trial design and guiding future application into clinical practice.

Circulating PD-L1 is associated with T cell infiltration and predicts prognosis in patients with CRLM following hepatic resection.

BACKGROUND: Exosomal PD-L1 (exoPD-L1) could induce immunosuppression functionally, thus impairing patients' survival in melanoma, NSCLC, and gastric cancer. However, no evidence demonstrates the feasibility of circulating exoPD-L1 and soluble PD-L1 (sPD-L1) as biomarkers for prognosis and early recurrence in colorectal liver metastasis (CRLM) patients following hepatectomy or their association with T cell infiltration at liver metastases. METHODS: In cohort 1, exoPD-L1 and sPD-L1 were preoperatively tested using ELISA. CD3, CD8, granzyme B (GB) and PD1 expressed at liver metastases were evaluated using immunohistochemistry. In cohort 2, exoPD-L1 and sPD-L1 were detected at baseline, before hepatectomy, after hepatectomy, and after disease progression. RESULTS: In cohort 1, higher preoperative exoPD-L1 or sPD-L1 significantly impaired RFS (exoPD-L1, P = 0.0043; sPD-L1, P = 0.0041) and OS (exoPD-L1, P = 0.0034; sPD-L1, P = 0.0061). Furthermore, preoperative exoPD-L1 was negatively correlated with CD3 + T-lymphocytes infiltrated at tumor center (CT), and GB and PD1 were expressed at tumor invasive margin (IM). Preoperative sPD-L1 was negatively correlated with CD3 + and CD8 + T-lymphocytes' infiltration at IM and CT, GB and PD1 expression at IM. In cohort 2, exoPD-L1 and sPD-L1 levels decreased following hepatectomy but increased when tumor progressed. Moreover, higher postoperative exoPD-L1 and sPD-L1 or a small reduction in exoPD-L1 and sPD-L1 levels after hepatectomy suggested higher early recurrence rate. CONCLUSIONS: Both preoperative exoPD-L1 and sPD-L1 had promising prognostic values and were associated with T cell infiltration at liver metastases in CRLM patients following hepatectomy. Dynamically tracking exoPD-L1 and sPD-L1 levels could monitor disease status and detect early recurrence.

Delineating the molecular landscape of different histopathological growth patterns in colorectal cancer liver metastases.

Background: Histopathological growth patterns (HGPs) have shown important prognostic values for patients with colorectal cancer liver metastases, but the potential molecular mechanisms remain largely unknown. Methods: We performed an exploratory analysis by conducting the RNA sequencing of primary colorectal lesions, colorectal liver metastatic lesions and normal liver tissues. Findings: We found that desmoplastic HGPs of the metastatic lesions were significantly enriched in EMT, angiogenesis, stroma, and immune signaling pathways, while replacement HGPs were enriched in metabolism, cell cycle, and DNA damage repair pathways. With the exception of immune-related genes, the differentially expressed genes of the two HGPs from colorectal liver metastases were mostly inherited from the primary tumor. Moreover, normal liver tissue in the desmoplastic HGP subgroup was markedly enriched in the fibrinous inflammation pathway. Conclusions: We surmised that HGPs are observable morphological changes resulting from the regulation of molecular expressions, which is the combined effect of the heterogeneity and remodeling of primary tumors seeds and liver soils.

COVID-19 induces new-onset insulin resistance and lipid metabolic dysregulation via regulation of secreted metabolic factors.

Abnormal glucose and lipid metabolism in COVID-19 patients were recently reported with unclear mechanism. In this study, we retrospectively investigated a cohort of COVID-19 patients without pre-existing metabolic-related diseases, and found new-onset insulin resistance, hyperglycemia, and decreased HDL-C in these patients. Mechanistically, SARS-CoV-2 infection increased the expression of RE1-silencing transcription factor (REST), which modulated the expression of secreted metabolic factors including myeloperoxidase, apelin, and myostatin at the transcriptional level, resulting in the perturbation of glucose and lipid metabolism. Furthermore, several lipids, including (±)5-HETE, (±)12-HETE, propionic acid, and isobutyric acid were identified as the potential biomarkers of COVID-19-induced metabolic dysregulation, especially in insulin resistance. Taken together, our study revealed insulin resistance as the direct cause of hyperglycemia upon COVID-19, and further illustrated the underlying mechanisms, providing potential therapeutic targets for COVID-19-induced metabolic complications.

The Prognostic Value of Locoregional Interventions for BRAF V600E Metastatic Colorectal Cancer: A Retrospective Cohort Analysis.

The prognostic heterogeneity in patients with BRAF V600E metastatic colorectal cancer (mCRC) remains poorly defined. Real-world data of 93 BRAF V600E mCRC patients from Sun Yat-sen University Cancer Center were evaluated using the prognostic factors affecting overall survival (OS). Treatment of metastases served as an independent prognosticator, where curative locoregional interventions (LRIs) were associated with superior clinical outcomes (adjusted hazard ratio (HR): 0.46, 95% confidence interval (CI): 0.22-0.98; p = 0.044). The LRIs group showed an improved median OS of 49.4 months versus 18.3 months for the palliative treatments (PTs) group. The median OS of patients with colorectal liver metastasis (CRLM) was significantly prolonged after undergoing LRIs (42.4 vs. 23.7 months; HR: 0.11, 95% CI: 0.01-1.22; p = 0.030), and patients in the LRIs plus liver-limited or lung-limited metastasis (LLM) group benefited more than those in the LRIs plus non-LLM group when compared to the PTs group (LLM from LRIs vs. PTs, HR: 0.16, 95% CI: 0.04-0.68; p = 0.006. Non-LLM from LRIs vs. PTs, HR: 0.47, 95% CI: 0.21-1.05; p = 0.074). In conclusion, we confirmed the positive prognostic value of LRIs in BRAF V600E mCRC, particularly in patients with CRLM or LLM.

A Prehepatectomy Circulating Exosomal microRNA Signature Predicts the Prognosis and Adjuvant Chemotherapeutic Benefits in Colorectal Liver Metastasis.

BACKGROUND: The clinical risk score (CRS) for prediction and treatment decision in colorectal liver metastasis (CRLM) is important, but imprecise. Exosomal miRNAs play critical roles in CRLM-related biological behavior. However, an exosomal miRNA score system for predicting posthepatectomy survival and the adjuvant chemotherapy benefit of CRLM remains elusive. METHODS: miRNA sequencing was used to identify differentially expressed miRNAs, and the LASSO model was used to select miRNAs to construct the intent model. The predictive performance of the model was evaluated by the area under the ROC curve (AUC) in the training, internal validation, and external validation cohorts. RESULTS: Sixteen differentially expressed exosomal miRNAs were identified, and four miRNAs were selected for model construction. Our model performed well in predicting prognosis with five-year AUCs of 0.70 (95% CI: 0.59-0.81), 0.70 (0.61-0.81), and 0.72 (057-0.86) in the training, internal, and external validation cohorts, respectively. miRNA classifier high-risk patients had better survival benefit from adjuvant chemotherapy regardless of CRS. All four miRNAs target signaling molecules play crucial roles in colorectal cancer metastasis, vesicle-related processing, and T cell activation. It also negatively correlated with the liver metastasis Immunoscore. CONCLUSION: We developed a circulating exosomal miRNA signature that can predict the prognosis and guide adjuvant chemotherapy decisions after hepatectomy in CRLM.

Smoking status combined with tumor mutational burden as a prognosis predictor for combination immune checkpoint inhibitor therapy in non-small cell lung cancer.

BACKGROUND: This study aimed to explore the prognostic value of tumor mutational burden (TMB) combined with smoking status in advanced non-small cell lung cancer (NSCLC) patients who received immune checkpoint inhibitor therapy (anti PD-1/PD-L1 therapy) combined with chemotherapy or anti-angiogenesis therapy. METHODS: We conducted a retrospective analysis of NSCLC patients who underwent next-generation sequencing test (either 295-gene panel NGS or 1021-gene panel NGS) from September 2017 to November 2020. The relationship between TMB and smoking status was investigated. Kaplan-Meier survival analysis was used to compare progression-free survival (PFS) of the NSCLC patients who received combination immunotherapy grouped by TMB value and smoking status. RESULTS: We enrolled 323 cases and 388 cases of NSCLC patients in the 295-gene panel cohort and 1021-gene panel cohort, respectively. Positive correlation between TMB and smoking status was found in lung adenocarcinoma, but not in lung squamous cell carcinoma. Participants with both high TMB and smoking status who received immune checkpoint therapy combined with chemotherapy or anti-angiogenesis therapy had longer PFS than other participants (p < 0.05). CONCLUSIONS: The combination of TMB with smoking status might be a potential predictor for the efficacy of combination immunotherapy in advanced NSCLC.

ELF3 activated by a superenhancer and an autoregulatory feedback loop is required for high-level HLA-C expression on extravillous trophoblasts.

HLA-C arose during evolution of pregnancy in the great apes 10 to 15 million years ago. It has a dual function on placental extravillous trophoblasts (EVTs) as it contributes to both tolerance and immunity at the maternal-fetal interface. The mode of its regulation is of considerable interest in connection with the biology of pregnancy and pregnancy abnormalities. First-trimester primary EVTs in which HLA-C is highly expressed, as well as JEG3, an EVT model cell line, were employed. Single-cell RNA-seq data and quantitative PCR identified high expression of the transcription factor ELF3 in those cells. Chromatin immunoprecipitation (ChIP)-PCR confirmed that both ELF3 and MED1 bound to the proximal HLA-C promoter region. However, binding of RFX5 to this region was absent or severely reduced, and the adjacent HLA-B locus remained closed. Expression of HLA-C was inhibited by ELF3 small interfering RNAs (siRNAs) and by wrenchnolol treatment. Wrenchnolol is a cell-permeable synthetic organic molecule that mimics ELF3 and is relatively specific for binding to ELF3's coactivator, MED23, as our data also showed in JEG3. Moreover, the ELF3 gene is regulated by a superenhancer that spans more than 5 Mb, identified by assay for transposase-accessible chromatin using sequencing (ATAC-seq), as well as by its sensitivity to (+)-JQ1 (inhibitor of BRD4). ELF3 bound to its own promoter, thus creating an autoregulatory feedback loop that establishes expression of ELF3 and HLA-C in trophoblasts. Wrenchnolol blocked binding of MED23 to ELF3, thus disrupting the positive-feedback loop that drives ELF3 expression, with down-regulation of HLA-C expression as a consequence.

Diagnostic and Prognostic Characteristics of Circulating Free DNA Methylation Detected by the Electrochemical Method in Malignant Tumors.

Prior research has established an electrochemical method based on the differential adsorption capacity of gold surfaces with different methylated DNA degrees and found that this method might be valuable for cancer diagnosis by detecting circulating free DNA methylation. However, further investigation on the underlying mechanism and validation of its diagnostic and prognostic values in a large cohort of malignant tumors was limited. We found that DNA with different methylation levels formed particles of diverse sizes on the gold surface. Hydrophobic bonds played a significant role in the binding process of methylated DNA to the gold surface. The detection condition of an adsorption time of 10 min and temperature of 20 °C was optimal. In a large cohort of plasma samples from the patients with different malignant tumors, as well as normal individuals, we found that the electrochemical detection method based on the differential adsorption capacity of methylated DNA degree on a gold surface could be used as a noninvasive tool for malignant tumor diagnosis and prognostic evaluation. The diagnostic efficiency of this method in malignant tumors was even slightly better than that of the current tumor biomarkers widely used in routine clinical practice (circulating free DNA (cfDNA) vs. carcinoembryonic antigen (CEA), 0.8131 vs. 0.7191 and cfDNA vs. CA19-9, 0.7687 vs. 0.6693).

Highly multiplexed immunofluorescence images and single-cell data of immune markers in tonsil and lung cancer.

In this data descriptor, we document a dataset of multiplexed immunofluorescence images and derived single-cell measurements of immune lineage and other markers in formaldehyde-fixed and paraffin-embedded (FFPE) human tonsil and lung cancer tissue. We used tissue cyclic immunofluorescence (t-CyCIF) to generate fluorescence images which we artifact corrected using the BaSiC tool, stitched and registered using the ASHLAR algorithm, and segmented using ilastik software and MATLAB. We extracted single-cell features from these images using HistoCAT software. The resulting dataset can be visualized using image browsers and analyzed using high-dimensional, single-cell methods. This dataset is a valuable resource for biological discovery of the immune system in normal and diseased states as well as for the development of multiplexed image analysis and viewing tools.

Qualifying antibodies for image-based immune profiling and multiplexed tissue imaging.

Multiplexed tissue imaging enables precise, spatially resolved enumeration and characterization of cell types and states in human resection specimens. A growing number of methods applicable to formalin-fixed, paraffin-embedded (FFPE) tissue sections have been described, the majority of which rely on antibodies for antigen detection and mapping. This protocol provides step-by-step procedures for confirming the selectivity and specificity of antibodies used in fluorescence-based tissue imaging and for the construction and validation of antibody panels. Although the protocol is implemented using tissue-based cyclic immunofluorescence (t-CyCIF) as an imaging platform, these antibody-testing methods are broadly applicable. We demonstrate assembly of a 16-antibody panel for enumerating and localizing T cells and B cells, macrophages, and cells expressing immune checkpoint regulators. The protocol is accessible to individuals with experience in microscopy and immunofluorescence; some experience in computation is required for data analysis. A typical 30-antibody dataset for 20 FFPE slides can be generated within 2 weeks.