B7-H3 Expression in Breast Cancer and Brain Metastasis.

Brain metastasis is a significant challenge for some breast cancer patients, marked by its aggressive nature, limited treatment options, and poor clinical outcomes. Immunotherapies have emerged as a promising avenue for brain metastasis treatment. B7-H3 (CD276) is an immune checkpoint molecule involved in T cell suppression, which is associated with poor survival in cancer patients. Given the increasing number of clinical trials using B7-H3 targeting CAR T cell therapies, we examined B7-H3 expression across breast cancer subtypes and in breast cancer brain metastases to assess its potential as an interventional target. B7-H3 expression was investigated using immunohistochemistry on tissue microarrays of three clinical cohorts: (i) unselected primary breast cancers (n = 347); (ii) brain metastatic breast cancers (n = 61) and breast cancer brain metastases (n = 80, including a subset of 53 patient-matched breast and brain metastasis cases); and (iii) mixed brain metastases from a range of primary tumours (n = 137). In primary breast cancers, B7-H3 expression significantly correlated with higher tumour grades and aggressive breast cancer subtypes, as well as poorer 5-year survival outcomes. Subcellular localisation of B7-H3 impacted breast cancer-specific survival, with cytoplasmic staining also correlating with a poorer outcome. Its expression was frequently detected in brain metastases from breast cancers, with up to 90% expressing B7-H3. However, not all brain metastases showed high levels of expression, with those from colorectal and renal tumours showing a low frequency of B7-H3 expression (0/14 and 2/16, respectively). The prevalence of B7-H3 expression in breast cancers and breast cancer brain metastases indicates potential opportunities for B7-H3 targeted therapies in breast cancer management.

Evaluation of Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration (EBUS-TBNA) Samples from Advanced Non-Small Cell Lung Cancer for Whole Genome, Whole Exome and Comprehensive Panel Sequencing.

Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is often the only source of tumor tissue from patients with advanced, inoperable lung cancer. EBUS-TBNA aspirates are used for the diagnosis, staging, and genomic testing to inform therapy options. Here we extracted DNA and RNA from 220 EBUS-TBNA aspirates to evaluate their suitability for whole genome (WGS), whole exome (WES), and comprehensive panel sequencing. For a subset of 40 cases, the same nucleic acid extraction was sequenced using WGS, WES, and the TruSight Oncology 500 assay. Genomic features were compared between sequencing platforms and compared with those reported by clinical testing. A total of 204 aspirates (92.7%) had sufficient DNA (100 ng) for comprehensive panel sequencing, and 109 aspirates (49.5%) had sufficient material for WGS. Comprehensive sequencing platforms detected all seven clinically reported tier 1 actionable mutations, an additional three (7%) tier 1 mutations, six (15%) tier 2-3 mutations, and biomarkers of potential immunotherapy benefit (tumor mutation burden and microsatellite instability). As expected, WGS was more suited for the detection and discovery of emerging novel biomarkers of treatment response. WGS could be performed in half of all EBUS-TBNA aspirates, which points to the enormous potential of EBUS-TBNA as source material for large, well-curated discovery-based studies for novel and more effective predictors of treatment response. Comprehensive panel sequencing is possible in the vast majority of fresh EBUS-TBNA aspirates and enhances the detection of actionable mutations over current clinical testing.

Synthesis and cellular uptake of neutral rhenium(I) morpholine complexes.

Morpholine motifs have been used extensively as targeting moieties for lysosomes, primarily in fluorescence imaging agents. Traditionally these imaging agents are based on organic molecules which have several shortcomings including small Stokes shifts, short emission lifetimes, and susceptibility to photobleaching. To explore alternative lysosome targeting imaging agents we have used a rhenium based phosphorescent platform which has been previously demonstrated to have an improved Stokes shift, a long lifetime emission, and is highly photostable. Rhenium complexes containing morpholine substituted ligands were designed to accumulate in acidic compartments. Two of the three complexes prepared exhibited bright emission in cells, when incubated at low concentrations (20 µM) and were non-toxic at concentrations as high as 100 µM, making them suitable for live cell imaging. We show that the rhenium complexes are amenable to chemical modification and that the morpholine targeted derivatives can be used for live cell confocal fluorescence imaging of endosomes-lysosomes.

Discrepancies in tumor mutation burden reporting from sequential endobronchial ultrasound transbronchial needle aspiration samples within single lymph node stations - brief report.

Introduction: Tumour Mutation Burden (TMB) is a potential biomarker for immune cancer therapies. Here we investigated parameters that might affect TMB using duplicate cytology smears obtained from endobronchial ultrasound transbronchial needle aspiration (EBUS TBNA)-sampled malignant lymph nodes. Methods: Individual Diff-Quik cytology smears were prepared for each needle pass. DNA extracted from each smear underwent sequencing using large gene panel (TruSight Oncology 500 (TSO500 - Illumina)). TMB was estimated using the TSO500 Local App v. 2.0 (Illumina). Results: Twenty patients had two or more Diff-Quik smears (total 45 smears) which passed sequencing quality control. Average smear TMB was 8.7 ± 5.0 mutations per megabase (Mb). Sixteen of the 20 patients had paired samples with minimal differences in TMB score (average difference 1.3 ± 0.85). Paired samples from 13 patients had concordant TMB (scores below or above a threshold of 10 mutations/Mb). Markedly discrepant TMB was observed in four cases, with an average difference of 11.3 ± 2.7 mutations/Mb. Factors affecting TMB calling included sample tumour content, the amount of DNA used in sequencing, and bone fide heterogeneity of node tumour between paired samples. Conclusion: TMB assessment is feasible from EBUS-TBNA smears from a single needle pass. Repeated samples of a lymph node station have minimal variation in TMB in most cases. However, this novel data shows how tumour content and minor change in site of node sampling can impact TMB. Further study is needed on whether all node aspirates should be combined in 1 sample, or whether testing independent nodes using smears is needed.

Targeted Hyperbranched Nanoparticles for Delivery of Doxorubicin in Breast Cancer Brain Metastasis.

Breast cancer brain metastases (BM) are associated with a dismal prognosis and very limited treatment options. Standard chemotherapy is challenging in BM patients because the high dosage required for an effective outcome causes unacceptable systemic toxicities, a consequence of poor brain penetration, and a short physiological half-life. Nanomedicines have the potential to circumvent off-target toxicities and factors limiting the efficacy of conventional chemotherapy. The HER3 receptor is commonly expressed in breast cancer BM. Here, we investigate the use of hyperbranched polymers (HBP) functionalized with a HER3 bispecific-antibody fragment for cancer cell-specific targeting and pH-responsive release of doxorubicin (DOX) to selectively deliver and treat BM. We demonstrated that DOX-release from the HBP carrier was controlled, gradual, and greater in endosomal acidic conditions (pH 5.5) relative to physiologic pH (pH 7.4). We showed that the HER3-targeted HBP with DOX payload was HER3-specific and induced cytotoxicity in BT474 breast cancer cells (IC50: 17.6 µg/mL). Therapeutic testing in a BM mouse model showed that HER3-targeted HBP with DOX payload impacted tumor proliferation, reduced tumor size, and prolonged overall survival. HER3-targeted HBP level detected in ex vivo brain samples was 14-fold more than untargeted-HBP. The HBP treatments were well tolerated, with less cardiac and oocyte toxicity compared to free DOX. Taken together, our HER3-targeted HBP nanomedicine has the potential to deliver chemotherapy to BM while reducing chemotherapy-associated toxicities.

Deep spatial-omics analysis of Head & Neck carcinomas provides alternative therapeutic targets and rationale for treatment failure.

Immune checkpoint inhibitor (ICI) therapy has had limited success (<30%) in treating metastatic recurrent Head and Neck Oropharyngeal Squamous Cell Carcinomas (OPSCCs). We postulate that spatial determinants in the tumor play a critical role in cancer therapy outcomes. Here, we describe the case of a male patient diagnosed with p16+ OPSCC and extensive lung metastatic disease who failed Nivolumab and Pembrolizumab/Lenvatinib therapies. Using advanced integrative spatial proteogenomic analysis on the patient's recurrent OPSCC tumors we demonstrate that: (i) unbiased tissue clustering based on spatial transcriptomics (ST) successfully detected tumor cells and enabled the investigation of phenotypic traits such as proliferation or drug-resistance genes in the tumor's leading-edge and core; (ii) spatial proteomic imagining used in conjunction with ST (SpiCi, Spatial Proteomics inferred Cell identification) can resolve the profiling of tumor infiltrating immune cells, (iii) ST data allows for the discovery and ranking of clinically relevant alternative medicines based on their interaction with their matching ligand-receptor. Importantly, when the spatial profiles of ICI pre- and post-failure OPSCC tumors were compared, they exhibited highly similar PD-1/PD-L1low and VEGFAhigh expression, suggesting that these new tumors were not the product of ICI resistance but rather of Lenvatinib dose reduction due to complications. Our work establishes a path for incorporating spatial-omics in clinical settings to facilitate treatment personalization.

Taming the Biological Activity of Pd(II) and Pt(II) Complexes with Triazolato "Protective" Groups: 1H, 77Se Nuclear Magnetic Resonance and X-ray Crystallographic Model Studies with Selenocysteine to Elucidate Differential Thioredoxin Reductase Inhibition.

The biological activity of Pd(II) and Pt(II) complexes toward three different cancer cell lines as well as inhibition of selenoenzyme thioredoxin reductase (TrxR) was modulated in an unexpected way by the introduction of triazolate as a "protective group" to the inner metal coordination sphere using the iClick reaction of [M(N3)(terpy)]PF6 [M = Pd(II) or Pt(II) and terpy = 2,2':6',2″-terpyridine] with an electron-poor alkyne. In a cell proliferation assay using A549, HT-29, and MDA-MB-231 human cancer cell lines, the palladium compound was significantly more potent than the isostructural platinum analogue and exhibited submicromolar activity on the most responsive cell line. This difference was also reflected in the inhibitory efficiency toward TrxR with IC50 values of 0.1 versus 5.4 µM for the Pd(II) and Pt(II) complexes, respectively. UV/Vis kinetic studies revealed that the Pt compound binds to selenocysteine faster than to cysteine [k = (22.9 ± 0.2)·10-3 vs (7.1 ± 0.2)·10-3 s-1]. Selective triazolato ligand exchange of the title compounds with cysteine (Hcys) and selenocysteine (Hsec)─but not histidine (His) and 9-ethylguanine (9EtG)─was confirmed by 1H, 77Se, and 195Pt NMR spectroscopy. Crystal structures of three of the four ligand exchange products were obtained, including [Pt(sec)(terpy)]PF6 as the first metal complex of selenocysteine to be structurally characterized.

Whole Genome Sequencing in Advanced Lung Cancer can be Performed Using Diff-Quik Cytology Smears Derived from Endobronchial Ultrasound, Transbronchial Needle Aspiration (EBUS TBNA).

INTRODUCTION: Maximising alternative sample types for genomics in advanced lung cancer is important because bronchoscopic samples may sometimes be insufficient for this purpose. Further, the clinical applications of comprehensive molecular analysis such as whole genome sequencing (WGS) are rapidly developing. Diff-Quik cytology smears from EBUS TBNA is an alternative source of DNA, but its feasibility for WGS has not been previously demonstrated. METHODS: Diff-Quik smears were collected along with research cell pellets. RESULTS: Tumour content of smears were compared to research cell pellets from 42 patients, which showed good correlation (Spearman correlation 0.85, P < 0.0001). A subset of eight smears underwent WGS, which presented similar mutation profiles to WGS of the matched cell pellet. DNA yield was predicted using a regression equation of the smears cytology features, which correctly predicted DNA yield > 1500 ng in 7 out of 8 smears. CONCLUSIONS: WGS of commonly collected Diff-Quik slides is feasible and their DNA yield can be predicted.

Obesity-associated changes in molecular biology of primary breast cancer.

Obesity is associated with an increased risk of developing breast cancer (BC) and worse prognosis in BC patients, yet its impact on BC biology remains understudied in humans. This study investigates how the biology of untreated primary BC differs according to patients' body mass index (BMI) using data from >2,000 patients. We identify several genomic alterations that are differentially prevalent in overweight or obese patients compared to lean patients. We report evidence supporting an ageing accelerating effect of obesity at the genetic level. We show that BMI-associated differences in bulk transcriptomic profile are subtle, while single cell profiling allows detection of more pronounced changes in different cell compartments. These analyses further reveal an elevated and unresolved inflammation of the BC tumor microenvironment associated with obesity, with distinct characteristics contingent on the estrogen receptor status. Collectively, our analyses imply that obesity is associated with an inflammaging-like phenotype. We conclude that patient adiposity may play a significant role in the heterogeneity of BC and should be considered for BC treatment tailoring.

Australian Genomics: Outcomes of a 5-year national program to accelerate the integration of genomics in healthcare.

Australian Genomics is a national collaborative partnership of more than 100 organizations piloting a whole-of-system approach to integrating genomics into healthcare, based on federation principles. In the first five years of operation, Australian Genomics has evaluated the outcomes of genomic testing in more than 5,200 individuals across 19 rare disease and cancer flagship studies. Comprehensive analyses of the health economic, policy, ethical, legal, implementation and workforce implications of incorporating genomics in the Australian context have informed evidence-based change in policy and practice, resulting in national government funding and equity of access for a range of genomic tests. Simultaneously, Australian Genomics has built national skills, infrastructure, policy, and data resources to enable effective data sharing to drive discovery research and support improvements in clinical genomic delivery.

Evaluating Diff-Quik cytology smears for large-panel mutation testing in lung cancer-Predicting DNA content and success with low-malignant-cellularity samples.

BACKGROUND: Cytology smears are commonly collected during endobronchial ultrasound-guided transbronchial needle aspiration (EBUS TBNA) procedures but are rarely used for molecular testing. Studies are needed to demonstrate their great potential, in particular for the prediction of malignant cell DNA content and for utility in molecular diagnostics using large gene panels. METHODS: A prospective study was performed on samples from 66 patients with malignant lymph nodes who underwent EBUS TBNA. All patients had air-dried, Diff-Quik cytology smears and formalin-fixed, paraffin-embedded cell blocks collected for cytopathology and molecular testing. One hundred eighty-five smears were evaluated by microscopy to estimate malignant cell percentage and abundance and to calculate smear size and were subjected to DNA extraction. DNA from 56 smears from 27 patients was sequenced with the TruSight Oncology 500 assay (Illumina). RESULTS: Each microscopy parameter had a significant effect on the DNA yield. An algorithm was developed that predicted a >50-ng DNA yield of a smear with an area under the curve of 0.86. Fifty DNA samples (89%) with varying malignant yields were successfully sequenced. Low-malignant-cell content (<25%) and smear area (<15%) were the main reasons for failure. All standard-of-care mutations were detected in replicate smears from individual patients, regardless of malignant cell content. Tier 1/2 mutations were discovered in two cases where standard-of-care specimens were inadequate for sequencing. Smears were scored for tumor mutation burden. CONCLUSIONS: Microscopy of Diff-Quik smears can triage samples for comprehensive panel sequencing, which highlights smears as an excellent alternative to traditional testing with cell blocks.

Prospective Optimization of Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration Lymph Node Assessment for Lung Cancer: Three Needle Agitations Are Noninferior to 10 Agitations for Adequate Tumor Cell and DNA Yield.

Introduction: Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS TBNA) is an important means of obtaining a tissue for advanced lung cancer. Optimizing the EBUS TBNA needling technique is important to maintain procedural simplicity and maximize sample quality for emerging molecular diagnostics. Methods: We prospectively explored three versus 10 agitations of the needle in sequential passes into the lymph node using separate needles. Resulting Diff-Quik cytology smears were quantitatively assessed using microscopic (tumor cell cellularity, abundance scores, erythrocyte contamination) and DNA yields. Microscopy was reported by two cytopathologists, and an inter-rater assessment was made by four additional cytopathologists. Results: In 86 patients confirmed as having malignant disease by EBUS TBNA (45 males, 41 females), a mean of 5.3 smears were made per patient with a total of 459 smears scored by pathologists and 168 paired smears extracted for DNA. There was no significant difference between three versus 10 agitations for smear cellularity (p = 0.44), DNA yield (p = 0.84), or DNA integrity (p = 0.20), but there was significantly less contamination by erythrocytes from three agitations (chi-square p = 0.008). There was significantly more DNA in the first pass into the node using three agitations than with other passes and with 10 agitations (pass × agitations interaction, p = 0.031). Reviewing pathologists correctly classified smears as more than or equal to 25% cellularity 86.3% of the time (κ = 0.63 [95% confidence interval: 0.55-0.71]). Conclusions: Three agitations are noninferior to 10 agitations for overall abundance of malignant cells and DNA content on smears. A smear with adequate DNA for panel sequencing could almost always be made with the first needle pass using three agitations.

Rhenium(I) conjugates as tools for tracking cholesterol in cells.

Cholesterol is vital to control membrane integrity and fluidity, but is also a precursor to produce steroid hormones, bile acids, and vitamin D. Consequently, altered cholesterol biology has been linked to many diseases, including metabolic syndromes and cancer. Defining the intracellular pools of cholesterol and its trafficking within cells is essential to understand both normal cell physiology and mechanisms of pathogenesis. We have synthesized a new cholesterol mimic (ReTEGCholestanol), comprising a luminescent rhenium metal complex and a cholestanol targeting unit, linked using a tetraethylene glycol (TEG) spacer. ReTEGCholestanol demonstrated favourable imaging properties and improved water solubility when compared to a cholesterol derivative, and structurally related probes lacking the TEG linker. A non-malignant and three malignant prostate cell lines were used to characterize the uptake and intracellular distribution of ReTEGCholestanol. The ReTEGCholestanol complex was effectively internalized and mainly localized to late endosomes/lysosomes in non-malignant PNT1a cells, while in prostate cancer cells it also accumulated in early endosomes and multivesicular bodies, suggesting disturbed cholesterol biology in the malignant cells. The ReTEGCholestanol is a novel imaging agent for visualizing endosomal uptake and trafficking, which may be used to define cholesterol related biology including membrane integration and altered lipid trafficking/processing.

2,7- and 4,9-Dialkynyldihydropyrene Molecular Switches: Syntheses, Properties, and Charge Transport in Single-Molecule Junctions.

This paper describes the syntheses of several functionalized dihydropyrene (DHP) molecular switches with different substitution patterns. Regioselective nucleophilic alkylation of a 5-substituted dimethyl isophthalate allowed the development of a workable synthetic protocol for the preparation of 2,7-alkyne-functionalized DHPs. Synthesis of DHPs with surface-anchoring groups in the 2,7- and 4,9-positions is described. The molecular structures of several intermediates and DHPs were elucidated by X-ray single-crystal diffraction. Molecular properties and switching capabilities of both types of DHPs were assessed by light irradiation experiments, spectroelectrochemistry, and cyclic voltammetry. Spectroelectrochemistry, in combination with density functional theory (DFT) calculations, shows reversible electrochemical switching from the DHP forms to the cyclophanediene (CPD) forms. Charge-transport behavior was assessed in single-molecule scanning tunneling microscope (STM) break junctions, combined with density functional theory-based quantum transport calculations. All DHPs with surface-contacting groups form stable molecular junctions. Experiments show that the molecular conductance depends on the substitution pattern of the DHP motif. The conductance was found to decrease with increasing applied bias.

Epigenome erosion and SOX10 drive neural crest phenotypic mimicry in triple-negative breast cancer.

Intratumoral heterogeneity is caused by genomic instability and phenotypic plasticity, but how these features co-evolve remains unclear. SOX10 is a neural crest stem cell (NCSC) specifier and candidate mediator of phenotypic plasticity in cancer. We investigated its relevance in breast cancer by immunophenotyping 21 normal breast and 1860 tumour samples. Nuclear SOX10 was detected in normal mammary luminal progenitor cells, the histogenic origin of most TNBCs. In tumours, nuclear SOX10 was almost exclusive to TNBC, and predicted poorer outcome amongst cross-sectional (p = 0.0015, hazard ratio 2.02, n = 224) and metaplastic (p = 0.04, n = 66) cases. To understand SOX10's influence over the transcriptome during the transition from normal to malignant states, we performed a systems-level analysis of co-expression data, de-noising the networks with an eigen-decomposition method. This identified a core module in SOX10's normal mammary epithelial network that becomes rewired to NCSC genes in TNBC. Crucially, this reprogramming was proportional to genome-wide promoter methylation loss, particularly at lineage-specifying CpG-island shores. We propose that the progressive, genome-wide methylation loss in TNBC simulates more primitive epigenome architecture, making cells vulnerable to SOX10-driven reprogramming. This study demonstrates potential utility for SOX10 as a prognostic biomarker in TNBC and provides new insights about developmental phenotypic mimicry-a major contributor to intratumoral heterogeneity.

Advanced molecular imaging for the characterisation of complex medicines.

A growing number and diversity of complex medicines is in development and reaching the market, with many of these medicines utilising innovative delivery technology to achieve appropriate biodistribution and exposure. Accurate assessment of biodistribution, cell penetration, internalised form, cargo release and efficacy are essential for the development of these medicines. Advanced imaging technologies, deploying different labelling techniques that allow the assessment of both carrier and cargo, are enabling in-depth analysis and providing a mechanistic understanding of each step in the drug delivery pathway. Translation across cell, tissue and whole-body settings using multiple imaging methods can provide decision-making information that is critical for clinical phase selection and for the development of complex medicines.

Blood-Derived Extracellular Vesicle-Associated miR-3182 Detects Non-Small Cell Lung Cancer Patients.

With five-year survival rates as low as 3%, lung cancer is the most common cause of cancer-related mortality worldwide. The severity of the disease at presentation is accredited to the lack of early detection capacities, resulting in the reliance on low-throughput diagnostic measures, such as tissue biopsy and imaging. Interest in the development and use of liquid biopsies has risen, due to non-invasive sample collection, and the depth of information it can provide on a disease. Small extracellular vesicles (sEVs) as viable liquid biopsies are of particular interest due to their potential as cancer biomarkers. To validate the use of sEVs as cancer biomarkers, we characterised cancer sEVs using miRNA sequencing analysis. We found that miRNA-3182 was highly enriched in sEVs derived from the blood of patients with invasive breast carcinoma and NSCLC. The enrichment of sEV miR-3182 was confirmed in oncogenic, transformed lung cells in comparison to isogenic, untransformed lung cells. Most importantly, miR-3182 can successfully distinguish early-stage NSCLC patients from those with benign lung conditions. Therefore, miR-3182 provides potential to be used for the detection of NSCLC in blood samples, which could result in earlier therapy and thus improved outcomes and survival for patients.

Whole genome deep sequencing analysis of cell-free DNA in samples with low tumour content.

BACKGROUND: Circulating cell-free DNA (cfDNA) in the plasma of cancer patients contains cell-free tumour DNA (ctDNA) derived from tumour cells and it has been widely recognized as a non-invasive source of tumour DNA for diagnosis and prognosis of cancer. Molecular profiling of ctDNA is often performed using targeted sequencing or low-coverage whole genome sequencing (WGS) to identify tumour specific somatic mutations or somatic copy number aberrations (sCNAs). However, these approaches cannot efficiently detect all tumour-derived genomic changes in ctDNA. METHODS: We performed WGS analysis of cfDNA from 4 breast cancer patients and 2 patients with benign tumours. We sequenced matched germline DNA for all 6 patients and tumour samples from the breast cancer patients. All samples were sequenced on Illumina HiSeqXTen sequencing platform and achieved approximately 30x, 60x and 100x coverage on germline, tumour and plasma DNA samples, respectively. RESULTS: The mutational burden of the plasma samples (1.44 somatic mutations/Mb of genome) was higher than the matched tumour samples. However, 90% of high confidence somatic cfDNA variants were not detected in matched tumour samples and were found to comprise two background plasma mutational signatures. In contrast, cfDNA from the di-nucleosome fraction (300 bp-350 bp) had much higher proportion (30%) of variants shared with tumour. Despite high coverage sequencing we were unable to detect sCNAs in plasma samples. CONCLUSIONS: Deep sequencing analysis of plasma samples revealed higher fraction of unique somatic mutations in plasma samples, which were not detected in matched tumour samples. Sequencing of di-nucleosome bound cfDNA fragments may increase recovery of tumour mutations from plasma.