Distant Normal Tissues Research Articles

Abstract 5665: Native nanopore sequencing of multiple tumor sites reveals genetic and epigenetic intra-tumor heterogeneity in canine osteosarcoma

Abstract Osteosarcoma (OS) is an aggressive bone malignancy affecting children and young adults. Implementation and optimization of neoadjuvant chemotherapy led to significant improvements in outcome, but patients with recurrence or metastases at diagnosis continue to have poor prognosis. Whole genome sequencing (WGS) revealed that a hallmark of OS is a highly rearranged genome with complex copy number aberrations rather than point mutations. A better understanding of the key genomic and epigenetic factors driving clonal evolution may provide insights into the tumor progression and the therapy response. Canine OS recapitulates the majority of the hallmark genetic and molecular factors of human OS and is recognized as an informative model. A significant difference is that initial surgery in canine OS allows us to explore tumor evolution unperturbed by treatment, providing invaluable resources to study the genetic and molecular landscape of tumor evolution. Here, we present the first study to date in which we performed bulk WGS of multiple tumor sites and distal normal tissue in two dogs using nanopore long-read sequencing to analyze the genetic and epigenetic evolution of OS. In both subjects, somatic structural variation detection using Severus revealed chromothripsis-like rearrangements in chr11, leading to the deletion of Cdkn2a, a known cell cycle regulator and tumor suppressor. Interestingly, in one of the subjects, the majority of the SVs (n = 131) shared across all the samples were localized on chr5, coinciding with perturbed copy number profiles and bi-allelic frequency of the SNVs, with all mutations identified using complementary bioinformatics analysis of the same native nanopore reads. The multi-site sample analysis identified subclonal structural alterations in TP53 and ATRX genes. With phased nanopore reads, we observed subject-specific copy-neutral loss-of-heterozygosity events and long identical-by-descent blocks. Haplotype-specific copy-number analysis of tumors showed different aneuploidy patterns in both subjects, highlighted clonal compositions of samples from distinct sites, and identified regions of copy-number heterogeneity. Furthermore, phased nanopore reads also revealed haplotype-specific 5mC and 5hmC methylation differences across tumor samples, where samples from different sites of the same tumor demonstrate distinct methylation patterns that overlap DLG2, RUNX2, KDR, and other genes known as mutational hotspots in canine/human OS. Overall, we showcase how a single sequencing assay allows for a joint analysis of genetic and epigenetic tumor alterations, thus providing a richer understanding of the drivers of somatic evolution and their association with the molecular landscape in the canine OS. These findings can be translated into human disease and shed light on tumor progression and therapy response. Citation Format: Ayse Keskus, Sergey Aganezov, Tanveer Ahmad, Xiaoguang Dai, Robert Walker, John Beaulaurier, Scott Hickey, Sissel Juul, Amy Kathleen LeBlanc, Paul S. Meltzer, Mikhail Kolmogorov. Native nanopore sequencing of multiple tumor sites reveals genetic and epigenetic intra-tumor heterogeneity in canine osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5665.

The interplay of PTEN and AKT nexus in breast cancer: a molecular perspective.

Breast cancer is a highly prevalent and life-threatening ailment that is commonly detected among the females. The downregulation of PTEN in breast cancer is associated with a poor prognosis, aggressive tumor type, and metastasis to lymph nodes, as it activates the pro-survival pathway PI3K/AKT, which is considered the ultimate proliferative pathway. The mRNA expression of PTEN and AKT genes was investigated using RT-qPCR and TaqMan primer probe chemistry. Moreover DNA was also isolated from the same tissue samples and exonic regions of both genes were amplified for mutational analysis. The proteins expression of PTEN and AKT from seven human breast cancer cell lines was checked through western blot experiments. The study revealed a decrease in PTEN expression in 73.3% of the samples, whereas an increase in AKT expression in 40% of samples was observed when compared to the distant normal breast tissue. Conversely, the remaining 60% of samples exhibited a decrease in AKT mRNA expression. There was no observed alteration in the genetic sequence of AKT and PTEN within the targeted amplified regions ofbreast cancer samples. The high levels of PTEN protein in T-47D and MDA-MB-453 resulted in a lower p-AKT. Two cell lines ZR-75-1 and MDA-MB-468 appeared to be PTEN negative on western blot but mRNA was detected on RT-qPCR. In breast cancer the status/expression of PTEN & AKT at mRNA and protein level might be obliging in forecasting the path of disease progression, treatment and prognosis.

The protective role of chemokines 12 and chemokines 4 by mediating interleukin-6 in delayed diabetic foot wound healing

Refractory diabetic foot ulcer is commonly encountered in clinical practice and it is hardly cured with a long duration of treatment and a high expense, as such problem involves multiple disciplines. Therefore, in case of refractory wounds, it is necessary to first analyze the causes, general or local. Immunohistochemistry (IHC) was conducted to determine α-SMA, chemokines 12 (CXCL12) and chemokines 4 (CXCR4) levels in pancreatic tissues and distant normal pancreatic tissues. Cells were treated with recombinant human CXCL12 (rhCXCL12) or the CXCR4 antagonist AMD3100. After treatment, Western blot determined FAK-AKT and ERK1/2 expression in islet cells, whilst ELISA detected the content of IL-6 and IL-8. rhCXCL12 increased the expression and secretion of Interleukin-6 (IL-6) time- and dose-dependently. But the advent of CXCR4 antagonist abrogated the protective effect of rhCXCL12. rhCXCL12 exhibited a protective effect on apoptosis, but this effect was abrogated by down-regulation of IL-6 with AMD3100. In addition, rhCXCL12 increased the phosphorylation of FAK, ERK1/2, AKT, and P38 and inhibition of FAK inhibited IL-6 expression. FAK inhibition almost completely blocked CXCL12-induced activation. This study demonstrates that CXCL12/CXCR4 pathway mediates the expression of IL-6 to enhance healing of refractory diabetic foot wounds by down-regulating the FAK pathway.

M6A methylation reader IGF2BP2 activates endothelial cells to promote angiogenesis and metastasis of lung adenocarcinoma

BackgroundLung adenocarcinoma (LUAD) is a common type of lung cancer with a high risk of metastasis, but the exact molecular mechanisms of metastasis are not yet understood.MethodsThis study acquired single-cell transcriptomics profiling of 11 distal normal lung tissues, 11 primary LUAD tissues, and 4 metastatic LUAD tissues from the GSE131907 dataset. The lung multicellular ecosystems were characterized at a single-cell resolution, and the potential mechanisms underlying angiogenesis and metastasis of LUAD were explored.ResultsWe constructed a global single-cell landscape of 93,610 cells from primary and metastatic LUAD and found that IGF2BP2 was specifically expressed both in a LUAD cell subpopulation (termed as LUAD_IGF2BP2), and an endothelial cell subpopulation (termed as En_IGF2BP2). The LUAD_IGF2BP2 subpopulation progressively formed and dominated the ecology of metastatic LUAD during metastatic evolution. IGF2BP2 was preferentially secreted by exosomes in the LUAD_IGF2BP2 subpopulation, which was absorbed by the En_IGF2BP2 subpopulation in the tumor microenvironment. Subsequently, IGF2BP2 improved the RNA stability of FLT4 through m6A modification, thereby activating the PI3K-Akt signaling pathway, and eventually promoting angiogenesis and metastasis. Analysis of clinical data showed that IGF2BP2 was linked with poor overall survival and relapse-free survival for LUAD patients.ConclusionsOverall, these findings provide a novel insight into the multicellular ecosystems of primary and metastatic LUAD, and demonstrate that a specific LUAD_IGF2BP2 subpopulation is a key orchestrator promoting angiogenesis and metastasis, with implications for the gene regulatory mechanisms of LUAD metastatic evolution, representing themselves as potential antiangiogenic targets.

A comparison of the microbiome composition in lower respiratory tract at different sites in early lung cancer patients.

Lung microbiome dysbiosis has been associated with lung carcinogenesis. However, the differences in the microbiome composition at different lung sites of lung cancer patients remain little understood. Studying the whole lung microbiome in cancer patients could provide new insights for interpreting the complex interplay between the microbiome and lung cancer and finding new targets for more effective therapies and preventive measures. A total of 16 patients with non-small cell lung cancer (NSCLC) were recruited for this study. Samples were obtained from four sites, including lung tumor tissues (TT), para-tumor tissues (PT), distal normal lung tissues (DN), and bronchial tissues (BT). The DNA was isolated from the tissues, and the V3-V4 regions were amplified. Sequencing libraries were generated and sequenced on an Illumina NovaSeq6000 platform. The richness and evenness of the microbiome were generally consistent among the TT, PT, DN, and BT groups in lung cancer patients. Principal coordinate analysis (PCoA) and nonmetric multidimensional scaling (NMDS) based on Bray-Curtis, weighted and unweighted UniFrac distance showed no distinct separation trend among the four groups. Proteobacteria, Firmicutes, Bacteroidota, and Desulfobacterota were the most common phyla in all four groups, while TT showed the highest abundance of Proteobacteria and the lowest abundance of Firmicutes. At the genus level, Rubellimicrobium and Fictibacillus were higher in the TT group. In the predicted functional analysis by PICRUSt, there were no specifically discrepant pathways among the four groups. In addition, an inverse relationship between body mass index (BMI) and alpha diversity was observed in this study. A non-significant result was obtained from the microbiome diversity comparison between different tissues. However, we demonstrated that lung tumors were enriched with specific bacterial species, which might contribute to tumorigenesis. Moreover, we found an inverse relationship between BMI and alpha diversity in these tissues, providing a new clue for deciphering the mechanisms of lung carcinogenesis.

Localization of Fusobacterium nucleatum in oral squamous cell carcinoma and its possible directly interacting protein molecules: A case series.

While 15 to 20% of cancers are associated with microbial infection, the relationship between oral microorganisms and oral squamous cell carcinoma (OSCC) remains unclear. The location of bacteria in a tumor is closely related to its carcinogenic mechanism. The aim of this study was to analyse bacterial diversity in clinical OSCC tissue samples and tumor distant normal tissues, locate target bacteria, and search for proteins that may interact with target bacteria. The 16S rDNA method was used to analyse bacterial diversity in clinical OSCC tissue samples and tumor distant normal tissues. Correlations between Fusobacterium abundance and clinicopathological characteristics were analysed using the χ2 test. The position of target bacteria was analysed by fluorescence in situ hybridization (FISH), and the expression of CK, CD31, CD45, CD68, cyclin D1, β-catenin, E-cadherin, NF-κB, and HIF-1α was analysed by immunohistochemistry (IHC) in OSCC tumor tissues and tumor distant normal tissues. The 16S rDNA results showed that the detected amount of Fusobacterium in OSCC tumor tissues was significantly larger than that in tumor distant normal tissues. High expression of Fusobacterium was significantly correlated with the lifestyle-related oral risk habits, including smoking (p=0.036) and alcohol consumption (p=0.022), but did not correlate with patient sex, age, tumor laterality, tumor size, grade or TNM stage. Fusobacterium nucleatum was enriched in tumor stroma, where CD31+ blood vessels and inflammatory cells (including CD45+ leukocytes and CD68+ macrophages) were densely distributed. Cyclin D1 was mainly expressed in the nucleus of tumor cells. β-catenin was expressed in the tumor cell membrane and was positively expressed in tumor interstitial vascular endothelial cells. E-cadherin was mainly expressed in tumor cell membranes. NF-κB was positively expressed in the cytoplasm of tumor cells, tumor interstitial cells and myo-fibrocytes. HIF-1α was mainly expressed in the cytoplasm of tumor interstitial cells. HIF-1α was highly expressed where Fusobacterium nucleatum was densely distributed. According to our study, the detected amount of Fusobacterium in OSCC tumor tissues was significantly larger than that in tumor distant normal tissues, and Fusobacterium nucleatum might aggravate inflammation and hypoxia by interacting with NF-κB and HIF-1α in OSCC.

Single-cell transcriptome sequencing reveals potential novel combination of biomarkers for antibody-based cancer therapeutics in hepatocellular carcinoma.

Background: Antibody-based cancer therapeutics is developing rapidly in recent years for its advantages in precisely targeting the tumor cells. However, tumor-specific cell surface antigens are still lacking, and the heterogeneity of tumor mass greatly impeded the development of effective drugs. Methods: In the present study, single-cell RNA sequencing was used to dissect tumor heterogeneity in human hepatocellular carcinoma (HCC). Tissues from different spatial regions including the tumor, para-tumor, and distant normal liver tissues were dissociated into single cells, and the gene expressions were compared in a different subpopulation of cells from these regions and validated in independent cohorts. Results: A total of 28 cell clusters with different distribution patterns and gene expression profiles were identified within a heterogenous tumor and its paired liver tissues. Differentially expressed genes encoding the plasma membrane in cells with hepatic lineage were further extracted from single-cell transcriptome sequencing and validated in TCGA database. A 3-gene signature was identified to be significantly upregulated in dominant HCC tumor cell subpopulations with prognostic significance and validated in multiple independent patient cohorts. Conclusion: The composition of the three plasma membrane proteins on the surface of HCC tumor cells within a heterogenous tumor might indicate poor prognostic tumor subpopulations during cancer evolution and potential therapeutic targets.

Abstract 2485: Applications of multimodality ex-vivo tissue imaging to improve breast cancer diagnosis and treatment

Abstract Breast cancer is the most common malignancy affecting women worldwide. Existing models being used to study breast cancer include the Patient-Derived Tumor Organoid (PDTO) Model and the Patient-Derived Tumor Xenograft (PDTX) Model. Both models involve the surgical removal of a tumor from a patient which then undergoes processing to grow an organoid or injected into mice to test different therapies outside the body. However, these models not only take a considerable amount of time to develop properly, but in many cases aren’t representative of the most aggressive tumor cells present within a heterogeneous tissue sample in order to draw the most clinically accurate conclusions of a tumor's response to therapy. In order to advance clinical research, more efficient culture systems that are faster growing and more representative of biological characteristics in-vivo are needed. Previous work from the Karczmar Lab discovered that high-resolution magnetic resonance imaging (MRI) can detect regions of aggressive cancer in biopsy specimens from mouse models, and give additional information on characteristics including cell density, tissue composition, and cancer stage. With this recent discovery, it is hypothesized that ex-vivo imaging could be used to identify aggressive tumor cells in human heterogeneous breast cancer tissue samples to produce higher quality and faster-growing models for testing new therapies and guiding individual patient therapy for breast cancer. The primary aim for this study was to specifically identify image-based markers for regions of aggressive cancer by comparing ex-vivo high-resolution MRI with histology and immunohistochemistry in 3D. Distant normal (DN) and tumor (Tu) tissue samples from breast cancer patients were obtained and imaged using T2-weighted 3D Rapid Imaging with Refocused Echoes (RARE) and stained using hematoxylin and eosin (H&E), commonly used by pathologists to diagnose cancers. Then, the ex-vivo T2-weighted 3D RARE images were correlated to the histopathological H&E staining and confirmed by breast radiologists and pathologists. Preliminary results revealed that high-resolution MR imaging detects mammary glands, ducts, and white adipose tissue in DN tissue samples and regions of invasive cancer, including ductal carcinoma in-situ (DCIS) and necrosis, in Tu tissue. Based on these results, aggressive cancer regions were proven to be identified using image-based guidance and potentially be extracted using core-needle biopsy to improve PDTO and PDTX models for studying breast cancer and advancing individual patient therapy. Citation Format: Corazon Avila, Gregory Karczmar, Devkumar Mustafi. Applications of multimodality ex-vivo tissue imaging to improve breast cancer diagnosis and treatment [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 2485.

Single-nucleus RNA-sequencing analysis reveals transcriptome differences in TIGIT+ exhausted T-cell associated with poor prognosis in muscle-invasive bladder cancer (MIBC).

e16544 Background: Compared with single-cell RNA-sequencing (scRNA-seq), Single-nuclei RNA-sequencing(snRNA-seq) can detect more genes with higher sensitivity, and it is more robust in identifying cellular markers. TIGIT+ exhausted T cells were reported significantly associated with poor prognosis in muscle invasive bladder cancer(MIBC) by flow cytometry. We aimed to evaluate unique markers of TIGIT+ exhausted T cells in MIBC via snRNA-seq and to construct a potential prognostic survival model. Methods: We collected snRNA-seq data from 25 MIBC and 4 non-MIBC patients in GSE169379, including 57 cancer tissues and 4 distant normal tissues. Meanwhile, expression profiling data and clinical information of 402 Bladder Urothelial Carcinoma(BLCA) patients were collected in The Cancer Genome Atlas (TCGA). Firstly, the snRNA-seq data was preprocessed and filtered. Next, We re-clustered T-cell subsets and abstracted TIGIT+ exhausted T cells (TIGIT+Tex), then screened out the genes uniquely expressed in TIGIT+Tex subsets by differential expression analysis. We further evaluate the correlations between uniquely expressed genes and the exhausted T cell markers, identified significantly correlated differential expressed genes, constructed a prognostic model based on multivariate cox regression analysis and exploited the patient's overall survival and disease-free survival to confirm our findings. Results: Top 10 genes (CNTN1, CTPS1, EYA1, GRIK2, FOXP2, YBX1, DACH1, SYT1, NOL4, TRIT1) of unparalleled differential up-regulation were identified from TIGIT+ exhausted T cells via snRNA-seq data. Based on the correlation analysis of TCGA expression profile data, six genes (CNTN1, CTPS1, EYA1, GRIK2, FOXP2, YBX1) that were significantly positively correlated with exhausted T cells were obtained. Next, we constructed a regression prediction model consisting of 5 genes (CNTN1, CTPS1, EYA1, GRIK2, FOXP2) through multivariate cox regression analysis based on patients’ clinical information. The model was associated with significantly poor prognosis overall survival (OS) (P < 0.01,HR = 2) and disease-free survival (DFS) (P < 0.01,HR = 1.6) in BLCA patients. Conclusions: In our study, Single-nuclei RNA expression profiling data revealed transcriptome differences between TIGIT+ exhausted T cells and other types of T cells. The regression model for potential prognostic survival was constructed using TCGA expression profiling data. These results demonstrated that distinctive differentially high expressed genes associated with TIGIT+ exhausted T cells may act as potential markers for predicting prognosis outcomes in bladder cancer patients.

Clinical and genomic analyses of neuroendocrine neoplasms of the breast

Breast neuroendocrine neoplasms (NENs) constitute a rare histologic subtype that includes both neuroendocrine tumors (NETs) and neuroendocrine carcinomas (NECs). In this study, we aimed to gain insight into the clinical and molecular characteristics of NENs of the breast. NEN and paired distant normal fresh tissues and clinicopathological data were obtained from 17 patients with NENs, and clinicopathological data were collected from 755 patients with invasive breast carcinomas of no special type (IBCs-NST). We compared the clinicopathological characteristics of NENs and IBCs-NST and performed whole-exome sequencing (WES) of both NEN and paired normal tissues. Compared with the IBC-NST patients, the NEN patients had a higher mean age, lower clinical stage, and lower pathological nodal (pN) stage (P < 0.001, P < 0.001, and P = 0.017, respectively). The most frequently mutated gene in NENs was KMT2C (3/17, 17.6%). NENs had copy number variations (CNVs) of 8q, 11q, and 17q amplification and 17q and 11q deletion and harbored the following specific genes related to tumorigenesis: (i) suppressor genes with loss of heterozygosity (LOH) such as ACE (2/17, 11.8%); (ii) tumor driver genes such as GATA3 (2/17, 11.8%); and (iii) susceptibility genes such as MAP3K4 (17/17, 100%) and PDE4DIP (17/17, 100%). The oncogenic/likely oncogenic mutations of NETs in PI3K pathway genes (50.0%, 18.2%; P < 0.001) and MAPK signaling pathway genes (83.3%, 18.2%; P = 0.035) affected higher proportions than those of NECs. In conclusion, this study provides certain clinical and molecular evidence supporting NENs as a distinct subtype of breast cancer and provides some potential molecular features for distinguishing NETs from NECs.

The Cole Relaxation Frequency as a Parameter to Identify Cancer in Lung Tissue: Preliminary Animal and Ex Vivo Patient Studies.

Lung cancer is the world's leading cause of cancer deaths, and diagnosis remains challenging. Lung cancer starts as small nodules; early and accurate diagnosis allows timely surgical resection of malignant nodules while avoiding unnecessary surgery in patients with benign nodules. The Cole relaxation frequency (CRF) is a derived electrical bioimpedance signature, which may be utilized to distinguish cancerous tissues from normal tissues. Human testing ex vivo was conducted with NoduleScan in freshly resected lung tissue from 30 volunteer patients undergoing resection for nonsmall cell lung cancer. The CRF of the tumor and the distant normal lung tissue relative to the tumor were compared to histopathology specimens to establish a potential algorithm for point-of-care diagnosis. For animal testing in vivo, 20 mice were implanted with xenograft human lung cancer tumor cells injected subcutaneously into the right flank of each mouse. Spectral impedance measurements were taken on the tumors on live animals transcutaneously and on the tumors after euthanasia. These CRF measurements were compared to healthy mouse lung tissue. For porcine lung testing ex vivo, porcine lungs were received with the trachea. After removal of the vocal box, a ventilator was attached to pressurize the lung and simulate breathing. At different locations of the lobes, the lung's surface was cut to produce a pocket that could accommodate tumors obtained from in vivo animal testing. The tumors were placed in the subsurface of the lung, and the electrode was placed on top of the lung surface directly over the tumor but with lung tissue between the tumor and the electrode. Spectral impedance measurements were taken when the lungs were in the deflated state, inflated state, and also during the inflation-deflation process to simulate breathing. Among 60 specimens evaluated in 30 patients, NoduleScan allowed ready discrimination in patients with clear separation of CRF in tumor and distant normal tissue with a high degree of sensitivity (97%) and specificity (87%). In the 25 xenograft small animal model specimens measured, the CRF aligns with the separation observed in the human in vivo measurements. The CRF was successfully measured of tumors implanted into ex vivo porcine lungs, and CRF measurements aligned with previous tests for pressurized and unpressurized lungs. As previously shown in breast tissue, CRF in the range of 1kHz-10MHz was able to distinguish nonsmall cell lung cancer versus normal tissue. Further, as evidenced by in vivo small animal studies, perfused tumors have the same CRF signature as shown in breast tissue and human ex vivo testing. Inflation and deflation of the lung have no effect on the CRF signature. With additional development, CRF derived from spectral impedance measurements may permit point-of-care diagnosis guiding surgical resection.

A standalone approach to utilize telomere length measurement as a surveillance tool in oral leukoplakia.

Oral squamous cell carcinoma (OSCC) is often preceded by a white patch on a surface of the mouth, called oral leukoplakia (OL). As accelerated telomere length (TL) shortening in dividing epithelial cells may lead to oncogenic transformation, telomere length measurement could serve as a predictive biomarker in OL. However, due to high variability and lack of a universal reference, there has been a limited translational application. Here, we describe an approach of evaluating TL using paired peripheral blood mononuclear cells (PBMC) as an internal reference and demonstrate its translational relevance. Oral brush biopsy and paired venous blood were collected from 50 male OL patients and 44 male healthy controls (HC). Relative TL was measured by quantitative PCR. TL of each OL or healthy sample was normalized to the paired PBMC sample (TL ratio). In OL patients, the mean TL ratio was significantly smaller not only in the patch but also in distal normal oral tissue, relative to healthy controls without a high‐risk oral habit. Dysplasia was frequently associated with a subgroup that showed a normal TL ratio at the patch but significantly smaller TL ratio at a paired normal distal site. Our data suggest that evaluation of TL attrition using a paired PBMC sample eliminates the requirement of external reference DNA, makes data universally comparable and provides a useful marker to define high‐risk OL groups for follow‐up programs. Larger studies will further validate the approach and its broader application in other premalignant conditions.

Association between γδ T cells and clinicopathological features of breast cancer

BackgroundThe roles of γδ T cells in patients with breast cancer (BC) have not been fully clarified, although the efficacy of gamma delta (γδ) T cells, which combine both innate and adaptive potential have extraordinary properties, such as recognizing tumor cells without the need for major histocompatibility complex (MHC) antigen presentation, as well as killing a broad range of tumor cells through their strong cytotoxic and pro-inflammatory activity, has been suggested in the majority of patients with some certain cancers. PurposeTo understand and dissect the association between γδ T cells and the clinical pathology of BC by measuring and analyzing the γδ T cell populations in the patients with BC. MethodsOn the one hand, γδ T cells were measured and analyzed by extracting from peripheral blood mononuclear cells (PBMC) of patients with BC. On the other hand, γδ T cells were measured and analyzed by performing enzymatic digestion in primary BC tissues, cancer adjacent tissues, and distant normal breast tissues from patients with different stages of cancer progression. In addition, patients with breast benign tumors and healthy volunteers were also collected as controls for this study. ResultsThe proportion of γδ T cells in PBMC was significantly correlated with tumor histological grade, ER status, the proportional value of Ki-67, and lymph node (LN) metastasis. The decrease and exhaustion of γδ T cells were a general feature in the PBMC of BC patients. In addition, the primary BC tissue infiltration of γδ T cells was significantly associated with tumor histological grade, ER status, the proportional value of Ki-67, and LN metastasis. ConclusionThese findings suggest that γδ T cell populations are crucial for understanding the clinicopathological features of patients with BC, and may serve as a valuable and independent biomarker, as well as a potential therapeutic target for human BC.

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In This Issue

DNA methylation patterns at and beyond the histological margin of early-stage invasive lung adenocarcinoma radiologically manifested as pure ground-glass opacity

BackgroundEarly-stage lung cancers radiologically manifested as ground-glass opacities (GGOs) have been increasingly identified, among which pure GGO (pGGO) has a good prognosis after local resection. However, the optimal surgical margin is still under debate. Precancerous lesions exist in tumor-adjacent tissues beyond the histological margin. However, potential precancerous epigenetic variation patterns beyond the histological margin of pGGO are yet to be discovered and described.ResultsA genome-wide high-resolution DNA methylation analysis was performed on samples collected from 15 pGGO at tumor core (TC), tumor edge (TE), para-tumor tissues at the 5 mm, 10 mm, 15 mm, 20 mm beyond the tumor, and peripheral normal (PN) tissue. TC and TE were tested with the same genetic alterations, which were also observed in histologically normal tissue at 5 mm in two patients with lower mutation allele frequency. According to the difference of methylation profiles between PN samples, 2284 methylation haplotype blocks (MHBs), 1657 differentially methylated CpG sites (DMCs), and 713 differentially methylated regions (DMRs) were identified using reduced representation bisulfite sequencing (RRBS). Two different patterns of methylation markers were observed: Steep (S) markers sharply changed at 5 mm beyond the histological margin, and Gradual (G) markers changed gradually from TC to PN. S markers composed 86.2% of the tumor-related methylation markers, and G markers composed the other 13.8%. S-marker-associated genes enriched in GO terms that were related to the hallmarks of cancer, and G-markers-associated genes enriched in pathways of stem cell pluripotency and transcriptional misregulation in cancer. Significant difference in DNA methylation score was observed between peripheral normal tissue and tumor-adjacent tissues 5 mm further from the histological margin (p < 0.001 in MHB markers). DNA methylation score at and beyond 10 mm from histological margin is not significantly different from peripheral normal tissues (p > 0.05 in all markers).ConclusionsAccording to the methylation pattern observed in our study, it was implied that methylation alterations were not significantly different between tissues at or beyond P10 and distal normal tissues. This finding explained for the excellent prognosis from radical resections with surgical margins of more than 15 mm. The inclusion of epigenetic characteristics into surgical margin analysis may yield a more sensitive and accurate assessment of remnant cancerous and precancerous cells in the surgical margins.

The downregulation of NCXs is positively correlated with the prognosis of stage II\u2013IV colon cancer

PurposeColon cancer (CC) is a very common gastrointestinal tumor that is prone to invasion and metastasis in the late stage. This study aims to observe the expression of Na+/Ca2+ exchangers (NCXs) and analyze the correlation between NCXs and the prognosis of CC.MethodsSpecimens of 111 stage II–IV CC patients were collected. We used western blotting, qPCR, and immunohistochemical staining to observe the distributions and expression levels of NCX isoforms (NCX1, NCX2, and NCX3) in CC and distal normal tissues. Cox proportional hazards models were used to assess prognostic factors for patients.ResultsThe expression of NCXs in most tumor specimens was lower than that in normal tissues. The NCX expression levels in tumor tissues from the primary tumor, local lymph node metastasis sites, and distant liver metastasis sites were increasingly significantly lower than those in normal tissues. The results of the Kaplan-Meier survival curves showed that the downregulation of any NCX isoform was closely related to the worse prognosis of advanced CC.ConclusionNCXs can be used as independent prognostic factors for CC. Our research results are expected to provide new targets for the treatment of CC.

CD163+ macrophages suppress T cell response by producing TGF-β in pediatric colorectal polyps

The local immune response plays an important role in the pathogenesis of colorectal carcinoma. Patients with colorectal polyps are at increased risk of colorectal cancer. However, the immunoregulation of early-stage colorectal polyps remain unknown. In the study, 202 biopsy samples from 80 pediatric patients with colorectal polyps and from 42 normal controls were collected. We found that the number of CD4+, CD8+T cells and CD19+B cells were reduced, whereas CD68+macrophages (Mϕ) were increased in colorectal polyps compared to the distal normal tissue from the same patients and the tissue from healthy donors. The frequency of Mϕwas negatively correlated with the number of CD4+ and CD8+T cells but not CD19+B cells in colorectal polyps. We further identified that CD163 was highly expressed on Mϕϕ from colorectal polyps compared to those from normal controls. Furthermore, real-time PCR revealed that TGF-β, but not IL-10 and IL-4, was increased in colorectal polyps. Immunofluorescence and flow cytometry showed that TGF-β was predominantly produced by CD163+Mϕ. In vitro experiments demonstrated that the supernatant from cultured polyps induced CD163 expression and TGF-β production in blood-derived Mϕ. A co-culture experiment revealed that purified Mϕ from colorectal polyps suppressed T cell proliferation. Based on these results, we hypothesized that abundant CD163+Mϕ may promote the progression of colorectal polyps by inhibiting the local T cell response through TGF-β production.

Boron Neutron Capture Therapy: A Review of Clinical Applications.

Boron neutron capture therapy (BNCT) is an emerging treatment modality aimed at improving the therapeutic ratio for traditionally difficult to treat tumors. BNCT utilizes boronated agents to preferentially deliver boron-10 to tumors, which, after undergoing irradiation with neutrons, yields litihium-7 and an alpha particle. The alpha particle has a short range, therefore preferentially affecting tumor tissues while sparing more distal normal tissues. To date, BNCT has been studied clinically in a variety of disease sites, including glioblastoma multiforme, meningioma, head and neck cancers, lung cancers, breast cancers, hepatocellular carcinoma, sarcomas, cutaneous malignancies, extramammary Paget’s disease, recurrent cancers, pediatric cancers, and metastatic disease. We aim to provide an up-to-date and comprehensive review of the studies of each of these disease sites, as well as a review on the challenges facing adoption of BNCT.

Dosimetric evaluation of a rotating gamma-ray system for stereotactic body radiation therapy

Gamma-ray stereotactic radiosurgery systems available commercially contain either stationary or rotational source configuration. Rotational systems reduce the number of sources utilized and can also provide beneficial dosimetric properties. In this study, we explore the dosimetric advantage of a novel rotating gamma-ray design for stereotactic body radiotherapy (SBRT) as compared to the well-established CyberKnife system. CybeRay (OUR United Corp., Xian, China) is a rotating gamma-ray system (RGS). Its treatment head can rotate 360° in the axial plane and swing 35° in the superior direction. It includes 13 Co-60 sources focusing on the isocenter. The RT Pro planning system (Prowess, Concord, CA) was used for CybeRay treatment planning, while the Multiplan software (Accuray, Sunnyvale, CA) was used for CyberKnife treatment planning. Twelve SBRT patients previously treated with CyberKnife were re-planned for CybeRay, and their treatment plans were compared based on isodose distributions, dose volume histograms, conformity index (CI), and the estimated treatment time. The planning target volume ranged from 4.4 to 124.2 cc. Both treatment systems provided excellent SBRT plans that meet our clinical acceptance criteria. The mean value of CI was 1.25 for both CyberKnife and CybeRay (p = 0.03). The estimated treatment delivery time for the CyberKnife plans ranged from 13 to 53 min as compared to 6 to 54 min with CybeRay (p = 0.02). CybeRay was superior concerning peripheral doses appearing as excessive isodose lines extending to distal normal tissues. The new CybeRay machine showed promising dosimetric capabilities for stereotactic radiosurgery and radiotherapy of extra- and intra-cranial lesions.

Abstract 1518: A single-cell transcriptomic atlas of lung adenocarcinoma and adjacent normal-appearing tissue

Abstract Lung adenocarcinoma (LUAD) is the most frequently diagnosed histological subtype of lung cancer and accounts for most smoking-related cancer deaths, warranting strategies for early intervention. Earlier work revealed genome-wide aberrations in LUADs and the adjacent premalignant field, known as “field carcinogenesis”, that are pertinent to LUAD pathogenesis. Yet, we still poorly understand the cellular and molecular architecture of LUAD and its nearby “field”. To fill this void, we performed, using the 10X Genomics system and NovaSeq 6000 platform, single-cell RNA sequencing (scRNA-seq) analysis of 4 early-stage resected LUADs as well as 11 matched normal lung tissues with differing spatial proximity from the tumors. We first analyzed 15,739 cells from a LUAD and matched tumor-adjacent and -distant normal tissues from patient 1. The resulting fraction of EPCAM+ cells from this analysis was approximately 4%. For deeper single-cell resolution of LUAD, we thus performed scRNA-seq of separately sorted/enriched epithelial cells (EPCAM+; n = 50,883) and non-epithelial cells (EPCAM-; n= 91,093) from patients 2, 3 and 4, each with an early-stage LUAD and three matched spatially-distributed normal-appearing tissues. Overall, we achieved on average approximately 150,000 reads and 1,956 genes detected per cell. Divergent populations of malignant and non-malignant cells and multiple epithelial and immune subsets clustered in an overall spatially modulated pattern according to proximity to the tumor. Hierarchical clustering revealed multiple distinct populations of airway lineage cells, including alveolar type 1 (AT1), alveolar type 2 (AT2), AT1/AT2 bipotential cells, club, goblet, basal and ciliated cells, with cells from the resected LUAD present in some but not all clusters. By analysis of epithelial-enriched fractions, we were also able to identify and interrogate rare cell subpopulations including CFTR-expressing ionocytes. By inference of copy number variation along with analysis of key oncogene expression patterns, we also identified epithelial cells that represent potential tumor cells-of-origin. Spatial reprogramming of field carcinogenesis encompassed immune cell populations including tumor-exclusive FOXP3+ regulatory T cells with marked over-expression of the major immune checkpoints CTLA4 and TIGIT. In contrast, for at least some of the cases, tumors were nearly devoid of natural killer cells, and abundance of CD8+ T cells dampened with increasing proximity to the tumor. Our single-cell surveys offer insights into novel cues in LUAD pathogenesis. Efforts are underway to interrogate the single-cell epithelial and immune landscape of additional LUADs and matched nearby normal-appearing lung, including those from non-smokers, to better understand the evolution of the disease and, thus, identify a low-hanging fruit of targets for early management of this fatal malignancy. Citation Format: Ansam Sinjab, Guangchun Han, Warapen Treekitkarnmongkol, Patrick Brennan, Kieko Hara, Kyle Chang, Elena Bogatenkova, Beatriz Sanchez-Espiridion, Carmen Behrens, Jianjun Zhang, Boris Sepesi, Tina Cascone, Don L. Gibbons, Jichao Chen, George Eapen, Edwin J. Ostrin, Junya Fujimoto, Avrum E. Spira, Paul A. Scheet, Ignacio I. Wistuba, Linghua Wang, Humam Kadara. A single-cell transcriptomic atlas of lung adenocarcinoma and adjacent normal-appearing tissue [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1518.