Mapping the cited evidence of ductal carcinoma in situ from the 5th edition of the World Health Organisation classification of tumours of the breast.

AIMS: Ductal carcinoma in situ (DCIS) is recognised by the World Health Organisation (WHO) Classification of Tumours (WCT) as a non-invasive neoplastic epithelial proliferation confined to the mammary ducts and lobules. This report categorises the references cited in the DCIS chapter of the 5th edition of the WCT (Breast Tumours) according to prevailing evidence levels for evidence-based medicine and the Hierarchy of Evidence for Tumour Pathology (HETP), identifying potential gaps that can inform subsequent editions of the WCT for this tumour. METHODS AND RESULTS: We included all citations from the DCIS chapter of the WCT (Breast Tumours, 5th edition). Each citation was appraised according to its study design and evidence level. We developed our map of cited evidence, which is a graphical matrix of tumour type (column) and tumour descriptors (rows). Spheres were used to represent the evidence, with size and colour corresponding to their number and evidence level respectively. Thirty-six publications were retrieved. The cited literature in the DCIS chapter comprised mainly case series and were regarded as low-level. We found an unequal distribution of citations among tumour descriptors. 'Pathogenesis' and 'prognosis and prediction' contained the most references, while 'clinical features', 'aetiology' and 'diagnostic molecular pathology' had only a single citation each. 'Prognosis and prediction' had the greatest proportion of moderate- and high-levels of evidence. CONCLUSION: Our findings align with the disposition for observational studies inherent in the field of pathology. Our map is a springboard for future efforts in mapping all available evidence on DCIS, potentially augmenting the editorial process and future editions of WCTs.

Differential response of placental cells to high D-glucose and its impact on extracellular vesicle biogenesis and trafficking via small GTPase Ras-related protein RAB-7A.

Placental extracellular vesicles (EVs) can be found in the maternal circulation throughout gestation, and their concentration, content and bioactivity are associated with pregnancy outcomes, including gestational diabetes mellitus (GDM). However, the effect of changes in the maternal microenvironment on the mechanisms associated with the secretion of EVs from placental cells remains to be fully established. Here, we evaluated the effect of high glucose on proteins associated with the trafficking and release of different populations of EVs from placental cells. BeWo and HTR8/SVneo cells were used as placental models and cultured under 5-mM D-glucose (i.e. control) or 25-mM D-glucose (high glucose). Cell-conditioned media (CCM) and cell lysate were collected after 48 h. Different populations of EVs were isolated from CCM by ultracentrifugation (i.e. pellet 2K-g, pellet 10K-g, and pellet 100K-g) and characterised by Nanoparticle Tracking Analysis. Quantitative proteomic analysis (IDA/SWATH) and multiple reaction monitoring protocols at high resolution (MRMHR) were developed to quantify 37 proteins related to biogenesis, trafficking/release and recognition/uptake of EVs. High glucose increased the secretion of total EVs across the pellets from BeWo cells, an effect driven mainly by changes in the small EVs concentration in the CCM. Interestingly, no effect of high glucose on HTR8/SVneo cells EVs secretion was observed. High glucose induces changes in proteins associated with vesicle trafficking in BeWo cells, including Heat Shock Protein Family A (Hsp70) Member 9 (HSPA9) and Member 8 (HSPA8). For HTR8/SVneo, altered proteins including prostaglandin F2α receptor regulatory protein (FPRP), RAB5A, RAB35, RAB5B, and RB11B, STAM1 and TSG101. These proteins are associated with the secretion and trafficking of EVs, which could explain in part, changes in the levels of circulating EVs in diabetic pregnancies. Further, we identified that proteins RAB11B, PDCD6IP, STAM, HSPA9, HSPA8, SDCBP, RAB5B, RAB5A, RAB7A and ERAP1 regulate EV release in response to high and low glucose when overexpressed in cells. Interestingly, immunohistochemistry analysis of RAB7A revealed distinct changes in placental tissues obtained from women with normal glucose tolerance (NGT, n = 6) and those with GDM (n = 6), influenced by diet or insulin treatment. High glucose regulation of proteins involved in intercellular dynamics and the trafficking of multivesicular bodies to the plasma membrane in placental cells is relevant in the context of GDM pregnancies.

Dataset for reporting of the invasive carcinoma of the breast: recommendations from the International Collaboration on Cancer Reporting (ICCR).

BACKGROUND AND OBJECTIVES: Current national or regional guidelines for the pathology reporting on invasive breast cancer differ in certain aspects, resulting in divergent reporting practice and a lack of comparability of data. Here we report on a new international dataset for the pathology reporting of resection specimens with invasive cancer of the breast. The dataset was produced under the auspices of the International Collaboration on Cancer Reporting (ICCR), a global alliance of major (inter-)national pathology and cancer organizations. METHODS AND RESULTS: The established ICCR process for dataset development was followed. An international expert panel consisting of breast pathologists, a surgeon, and an oncologist prepared a draft set of core and noncore data items based on a critical review and discussion of current evidence. Commentary was provided for each data item to explain the rationale for selecting it as a core or noncore element, its clinical relevance, and to highlight potential areas of disagreement or lack of evidence, in which case a consensus position was formulated. Following international public consultation, the document was finalized and ratified, and the dataset, which includes a synoptic reporting guide, was published on the ICCR website. CONCLUSIONS: This first international dataset for invasive cancer of the breast is intended to promote high-quality, standardized pathology reporting. Its widespread adoption will improve consistency of reporting, facilitate multidisciplinary communication, and enhance comparability of data, all of which will help to improve the management of invasive breast cancer patients.

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.

A dedicated structured data set for reporting of invasive carcinoma of the breast in the setting of neoadjuvant therapy: recommendations from the International Collaboration on Cancer Reporting (ICCR).

AIMS: The International Collaboration on Cancer Reporting (ICCR), a global alliance of major (inter-)national pathology and cancer organisations, is an initiative aimed at providing a unified international approach to reporting cancer. ICCR recently published new data sets for the reporting of invasive breast carcinoma, surgically removed lymph nodes for breast tumours and ductal carcinoma in situ, variants of lobular carcinoma in situ and low-grade lesions. The data set in this paper addresses the neoadjuvant setting. The aim is to promote high-quality, standardised reporting of tumour response and residual disease after neoadjuvant treatment that can be used for subsequent management decisions for each patient. METHODS: The ICCR convened expert panels of breast pathologists with a representative surgeon and oncologist to critically review and discuss current evidence. Feedback from the international public consultation was critical in the development of this data set. RESULTS: The expert panel concluded that a dedicated data set was required for reporting of breast specimens post-neoadjuvant therapy with inclusion of data elements specific to the neoadjuvant setting as core or non-core elements. This data set proposes a practical approach for handling and reporting breast resection specimens following neoadjuvant therapy. The comments for each data element clarify terminology, discuss available evidence and highlight areas with limited evidence that need further study. This data set overlaps with, and should be used in conjunction with, the data sets for the reporting of invasive breast carcinoma and surgically removed lymph nodes from patients with breast tumours, as appropriate. Key issues specific to the neoadjuvant setting are included in this paper. The entire data set is freely available on the ICCR website. CONCLUSIONS: High-quality, standardised reporting of tumour response and residual disease after neoadjuvant treatment are critical for subsequent management decisions for each patient.

Detailed DNA methylation characterisation of phyllodes tumours identifies a signature of malignancy and distinguishes phyllodes from metaplastic breast carcinoma.

Phyllodes tumours (PTs) are rare fibroepithelial lesions of the breast that are classified as benign, borderline, or malignant. As little is known about the molecular underpinnings of PTs, current diagnosis relies on histological examination. However, accurate classification is often difficult, particularly for distinguishing borderline from malignant PTs. Furthermore, PTs can be misdiagnosed as other tumour types with shared histological features, such as fibroadenoma and metaplastic breast cancers. As DNA methylation is a recognised hallmark of many cancers, we hypothesised that DNA methylation could provide novel biomarkers for diagnosis and tumour stratification in PTs, whilst also allowing insight into the molecular aetiology of this otherwise understudied tumour. We generated whole-genome methylation data using the Illumina EPIC microarray in a novel PT cohort (n = 33) and curated methylation microarray data from published datasets including PTs and other potentially histopathologically similar tumours (total n = 817 samples). Analyses revealed that PTs have a unique methylome compared to normal breast tissue and to potentially histopathologically similar tumours (metaplastic breast cancer, fibroadenoma and sarcomas), with PT-specific methylation changes enriched in gene sets involved in KRAS signalling and epithelial-mesenchymal transition. Next, we identified 53 differentially methylated regions (DMRs) (false discovery rate < 0.05) that specifically delineated malignant from non-malignant PTs. The top DMR in both discovery and validation cohorts was hypermethylation at the HSD17B8 CpG island promoter. Matched PT single-cell expression data showed that HSD17B8 had minimal expression in fibroblast (putative tumour) cells. Finally, we created a methylation classifier to distinguish PTs from metaplastic breast cancer samples, where we revealed a likely misdiagnosis for two TCGA metaplastic breast cancer samples. In conclusion, DNA methylation alterations are associated with PT histopathology and hold the potential to improve our understanding of PT molecular aetiology, diagnostics, and risk stratification. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

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.

Branchioma: immunohistochemical and molecular genetic study of 23 cases highlighting frequent loss of retinoblastoma 1 immunoexpression.

Branchioma is an uncommon benign neoplasm with an adult male predominance, typically occurring in the lower neck region. Different names have been used for this entity in the past (ectopic hamartomatous thymoma, branchial anlage mixed tumor, thymic anlage tumor, biphenotypic branchioma), but currently, the term branchioma has been widely accepted. Branchioma is composed of endodermal and mesodermal lineage derivatives, in particular epithelial islands, spindle cells, and mature adipose tissue without preexistent thymic tissue or evidence of thymic differentiation. Twenty-three branchiomas were evaluated morphologically. Eighteen cases with sufficient tissue were assessed by immunohistochemistry, next-generation sequencing (NGS) using the Illumina Oncology TS500 panel, and fluorescence in situ hybridization (FISH) using an RB1 dual-color probe. All cases showed a biphasic morphology of epithelial and spindle cells with intermingled fatty tissue. Carcinoma arising in branchioma was detected in three cases. The neoplastic cells showed strong AE1/3 immunolabeling (100%), while the spindle cells expressed CD34, p63, and SMA (100%); AR was detected in 40-100% of nuclei (mean, 47%) in 14 cases. Rb1 showed nuclear loss in ≥ 95% of neoplastic cells in 16 cases (89%), while two cases revealed retained expression in 10-20% of tumor cell nuclei. NGS revealed a variable spectrum of likely pathogenic variants (n = 5) or variants of unknown clinical significance (n = 6). Loss of Rb1 was detected by FISH in two cases. Recent developments support branchioma as a true neoplasm, most likely derived from the rudimental embryological structures of endoderm and mesoderm. Frequent Rb1 loss by immunohistochemistry and heterozygous deletion by FISH is a real pitfall and potential confusion with other Rb1-deficient head and neck neoplasms (i.e., spindle cell lipoma), especially in small biopsy specimens.

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.

Robust mapping of spatiotemporal trajectories and cell-cell interactions in healthy and diseased tissues.

Spatial transcriptomics (ST) technologies generate multiple data types from biological samples, namely gene expression, physical distance between data points, and/or tissue morphology. Here we developed three computational-statistical algorithms that integrate all three data types to advance understanding of cellular processes. First, we present a spatial graph-based method, pseudo-time-space (PSTS), to model and uncover relationships between transcriptional states of cells across tissues undergoing dynamic change (e.g. neurodevelopment, brain injury and/or microglia activation, and cancer progression). We further developed a spatially-constrained two-level permutation (SCTP) test to study cell-cell interaction, finding highly interactive tissue regions across thousands of ligand-receptor pairs with markedly reduced false discovery rates. Finally, we present a spatial graph-based imputation method with neural network (stSME), to correct for technical noise/dropout and increase ST data coverage. Together, the algorithms that we developed, implemented in the comprehensive and fast stLearn software, allow for robust interrogation of biological processes within healthy and diseased tissues.

Predicting breast cancer-specific survival in metaplastic breast cancer patients using machine learning algorithms.

Metaplastic breast cancer (MpBC) is a rare and aggressive subtype of breast cancer, with data emerging on prognostic factors and survival prediction. This study aimed to develop machine learning models to predict breast cancer-specific survival (BCSS) in MpBC patients, utilizing a dataset of 160 patients with clinical, pathological, and biological variables. An in-depth variable selection process was carried out using gain ratio and correlation-based methods, resulting in 10 variables for model estimation. Five models (decision tree with bagging; logistic regression; multilayer perceptron; naïve Bayes; and, random forest algorithms) were evaluated using 10-fold cross-validation. Despite the constraints posed by the absence of therapeutic information, the random forest model exhibited the highest performance in predicting BCSS, with an ROC area of 0.808. This study emphasizes the potential of machine learning algorithms in predicting prognosis for complex and heterogeneous cancer subtypes using clinical datasets, and their potential to contribute to patient management. Further research that incorporates additional variables, such as treatment response, and more advanced machine learning techniques will likely enhance the predictive power of MpBC prognostic models.

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.

Improved concordance of challenging human epidermal growth factor receptor 2 dual in-situ hybridisation cases with the use of a digital image analysis algorithm in breast cancer.

AIMS: Accurate assessment of human epidermal growth factor receptor 2 (HER2) expression by HER2 immunohistochemistry and in-situ hybridisation (ISH) is critical for the management of patients with breast cancer. The revised 2018 ASCO/CAP guidelines define 5 groups based on HER2 expression and copy number. Manual pathologist quantification by light microscopy of equivocal and less common HER2 ISH groups (groups 2-4) can be challenging, and there are no data on interobserver variability in reporting of these cases. We sought to determine whether a digital algorithm could improve interobserver variability in the assessment of difficult HER2 ISH cases. METHODS AND RESULTS: HER2 ISH was evaluated in a cohort enriched for less common HER2 patterns using standard light microscopy versus analysis of whole slide images using the Roche uPath HER2 dual ISH image analysis algorithm. Standard microscopy demonstrated significant interobserver variability with a Fleiss's kappa value of 0.471 (fair-moderate agreement) improving to 0.666 (moderate-good) with the use of the algorithm. For HER2 group designation (groups 1-5), there was poor-moderate reliability between pathologists by microscopy [intraclass correlation coefficient (ICC) = 0.526], improving to moderate-good agreement (ICC = 0.763) with the use of the algorithm. In subgroup analysis, the algorithm improved concordance particularly in groups 2, 4 and 5. Time to enumerate cases was also significantly reduced. CONCLUSION: This work demonstrates the potential of a digital image analysis algorithm to improve the concordance of pathologist HER2 amplification status reporting in less common HER2 groups. This has the potential to improve therapy selection and outcomes for patients with HER2-low and borderline HER2-amplified breast cancers.

Redefining normal breast cell populations using long noncoding RNAs.

Single-cell RNAseq has allowed unprecedented insight into gene expression across different cell populations in normal tissue and disease states. However, almost all studies rely on annotated gene sets to capture gene expression levels and sequencing reads that do not align to known genes are discarded. Here, we discover thousands of long noncoding RNAs (lncRNAs) expressed in human mammary epithelial cells and analyze their expression in individual cells of the normal breast. We show that lncRNA expression alone can discriminate between luminal and basal cell types and define subpopulations of both compartments. Clustering cells based on lncRNA expression identified additional basal subpopulations, compared to clustering based on annotated gene expression, suggesting that lncRNAs can provide an additional layer of information to better distinguish breast cell subpopulations. In contrast, these breast-specific lncRNAs poorly distinguish brain cell populations, highlighting the need to annotate tissue-specific lncRNAs prior to expression analyses. We also identified a panel of 100 breast lncRNAs that could discern breast cancer subtypes better than protein-coding markers. Overall, our results suggest that lncRNAs are an unexplored resource for new biomarker and therapeutic target discovery in the normal breast and breast cancer subtypes.

COMMD3 loss drives invasive breast cancer growth by modulating copper homeostasis.

BACKGROUND: Despite overall improvement in breast cancer patient outcomes from earlier diagnosis and personalised treatment approaches, some patients continue to experience recurrence and incurable metastases. It is therefore imperative to understand the molecular changes that allow transition from a non-aggressive state to a more aggressive phenotype. This transition is governed by a number of factors. METHODS: As crosstalk with extracellular matrix (ECM) is critical for tumour cell growth and survival, we applied high throughput shRNA screening on a validated '3D on-top cellular assay' to identify novel growth suppressive mechanisms. RESULTS: A number of novel candidate genes were identified. We focused on COMMD3, a previously poorly characterised gene that suppressed invasive growth of ER + breast cancer cells in the cellular assay. Analysis of published expression data suggested that COMMD3 is normally expressed in the mammary ducts and lobules, that expression is lost in some tumours and that loss is associated with lower survival probability. We performed immunohistochemical analysis of an independent tumour cohort to investigate relationships between COMMD3 protein expression, phenotypic markers and disease-specific survival. This revealed an association between COMMD3 loss and shorter survival in hormone-dependent breast cancers and in particularly luminal-A-like tumours (ER+/Ki67-low; 10-year survival probability 0.83 vs. 0.73 for COMMD3-positive and -negative cases, respectively). Expression of COMMD3 in luminal-A-like tumours was directly associated with markers of luminal differentiation: c-KIT, ELF5, androgen receptor and tubule formation (the extent of normal glandular architecture; p < 0.05). Consistent with this, depletion of COMMD3 induced invasive spheroid growth in ER + breast cancer cell lines in vitro, while Commd3 depletion in the relatively indolent 4T07 TNBC mouse cell line promoted tumour expansion in syngeneic Balb/c hosts. Notably, RNA sequencing revealed a role for COMMD3 in copper signalling, via regulation of the Na+/K+-ATPase subunit, ATP1B1. Treatment of COMMD3-depleted cells with the copper chelator, tetrathiomolybdate, significantly reduced invasive spheroid growth via induction of apoptosis. CONCLUSION: Overall, we found that COMMD3 loss promoted aggressive behaviour in breast cancer cells.

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.

BRCA1 and BRCA2 carriers with breast, ovarian and prostate cancer demonstrate a different pattern of metastatic disease compared with non-carriers: results from a rapid autopsy programme.

AIM: To catalogue and compare the pattern of metastatic disease in germline BRCA1/2 pathogenic mutation carriers and non-carriers with breast, ovarian and prostate cancer from a rapid autopsy programme. METHODS AND RESULTS: The number of metastases in the major body systems and the proportion of participants with metastases were documented in 50 participants (19 germline mutation carriers). Analysis was conducted on the participants' pattern of disease for the different cancers and mutation subgroups. The four commonly affected organ systems were the digestive (liver only) (82%), respiratory (76%), gastrointestinal (65%) and reticuloendothelial (42%). There were significant differences in the pattern of metastatic breast cancer in BRCA1/2 germline carriers compared with non-carriers. Breast cancer carriers had significantly fewer organ systems involved (median n = 3, range = 1-3) compared with non-carriers (median n = 9, range = 1-7) (P = 0.03). BRCA1/2 carriers with ovarian carcinomas had significantly more organ systems with metastatic carcinoma (median n = 10, range = 3-8) than non-carriers (median n = 5, range = 3-5) (P < 0.001). There were no significant differences in the number of involved systems in BRCA2 carriers compared with non-carriers with prostate cancer (P = 1.0). There was an absence of locoregional disease (6.5%) compared with distant disease (93.5%) among the three cancer subtypes (P < 0.001). The majority of metastatic deposits (97%) collected during the autopsy were identified by recent diagnostic imaging. CONCLUSION: Even though a major limitation of this study is that our numbers are small, especially in the breast cancer carrier group, the metastatic patterns of breast and ovarian cancers may be impacted by BRCA1/2 carrier status, suggesting that tumours derived from patients with these mutations use different mechanisms of dissemination. The findings may focus clinical diagnostic imaging for monitoring metastases where whole-body imaging resources are scant.