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Invasive lobular carcinoma (ILC) is the most common of the breast cancer special types, accounting for up to 15% of all breast cancer cases. ILCs are noted for their lack of E-cadherin function, which underpins their characteristic discohesive growth pattern, with cells arranged in single file and dispersed throughout the stroma. Typically, tumours are luminal in molecular subtype, being oestrogen and progesterone receptor positive, and HER2 negative. Since last reviewing the lobular literature (McCart Reed et al., Breast Cancer Res 17:12, 2015), there has been a considerable increase in research output focused on this tumour type, including studies into the pathology and management of disease, a high-resolution definition of the genomic landscape of tumours as well as the evolution of several potential therapeutic avenues. There abounds a huge amount of new data, which we will review herein.

Invasive lobular carcinoma of the breast is the most common ‘special’ morphological subtype of breast cancer, comprising up to 15% of all cases. Tumours are generally of a good prognostic phenotype, being low histological grade and low mitotic index, hormone receptor positive and HER2, p53 and basal marker negative, and with a generally good response to endocrine therapy. Despite this, clinicians face countless challenges in the diagnosis and long-term management of patients, as they encounter a tumour that can be difficult to detect through screening, elicits a very invasive nature, a propensity for widespread metastatic colonisation and, consequently, in some studies a worse long-term poor outcome compared with invasive carcinoma of no special type. Here we review the morphological and molecular features that underpin the disparate biological and clinical characteristics of this fascinating tumour type.

Spatial transcriptomics (ST) links tissue morphology with gene expression values, opening new avenues for digital pathology. Deep learning models are used to predict gene expression or classify cell types directly from images, offering significant clinical potential but still requiring improvements in interpretability and robustness. We present STimage as a comprehensive suite of models to predict spatial gene expression and classify cell types directly from standard H&E images. STimage enhances robustness by estimating gene expression distributions and quantifying both data-driven (aleatoric) and model-based (epistemic) uncertainty using an ensemble approach with foundation models. Interpretability is achieved through attribution analysis at single-cell resolution integrated with histopathological annotations, functional genes, and latent representations. We validated STimage across diverse datasets, demonstrating its performance across various platforms. STimage-predicted gene expression can stratify patient survival and predict drug response. By enabling molecular and cellular prediction from routine histology, STimage offers a powerful tool to advance digital pathology. Spatial transcriptomics (ST) technologies link tissue morphology with gene expression, but remain expensive to use; furthermore, models that predict ST data from histopathology images possess considerable limitations. Here, the authors develop STimage, a deep learning probabilistic framework for ST prediction from histopathology images while prioritising robustness and interpretability.

Background Metaplastic breast carcinoma encompasses a heterogeneous group of tumours with differentiation into squamous and/or spindle, chondroid, osseous or rhabdoid mesenchymal-looking elements. Emerging immunotherapies targeting Programmed Death Ligand 1 (PD-L1) and immune-suppressing T cells (Tregs) may benefit metaplastic breast cancer patients, which are typically chemo-resistant and do not express hormone therapy targets. Methods We evaluated the immunohistochemical expression of PD-L1 and FOXP3, and the extent of tumour infiltrating lymphocytes (TILs) in a large cohort of metaplastic breast cancers, with survival data. Results Metaplastic breast cancers were significantly enriched for PD-L1 positive tumour cells, compared to triple-negative ductal breast cancers ( P  < 0.0001), while there was no significant difference in PD-L1 positive TILs. Metaplastic breast cancers were also significantly enriched for TILs expressing FOXP3, with FOXP3 positive intra-tumoural TILs (iTILs) associated with an adverse prognostic outcome ( P  = 0.0226). Multivariate analysis identified FOXP3 iTILs expression status as an important independent prognostic factor for patient survival. Conclusions Our findings indicate the clinical significance and prognostic value of FOXP3, PD-1/PD-L1 checkpoint and TILs in metaplastic breast cancer and confirm that a subset of metaplastics may benefit from immune-based therapies.

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.

Background Lung cancer is a heterogeneous disease and the primary cause of cancer-related mortality worldwide. Somatic mutations, including large structural variants, are important biomarkers in lung cancer for selecting targeted therapy. Genomic studies in lung cancer have been conducted using short-read sequencing. Emerging long-read sequencing technologies are a promising alternative to study somatic structural variants, however there is no current consensus on how to process data and call somatic events. In this study, we preformed whole genome sequencing of lung cancer and matched non-tumour samples using long and short read sequencing to comprehensively benchmark three sequence aligners and seven structural variant callers comprised of generic callers (SVIM, Sniffles2, DELLY in generic mode and cuteSV) and somatic callers (Severus, SAVANA, nanomonsv and DELLY in somatic modes). Results Different combinations of aligners and variant callers influenced somatic structural variant detection. The choice of caller had a significant influence on somatic structural variant detection in terms of variant type, size, sensitivity, and accuracy. The performance of each variant caller was assessed by comparing to somatic structural variants identified by short-read sequencing. When compared to somatic structural variants detected with short-read sequencing, more events were detected with long-read sequencing. The mean recall of somatic variant events identified by long-read sequencing was higher for the somatic callers (72%) than generic callers (53%). Among the somatic callers when using the minimap2 aligner, SAVANA and Severus achieved the highest recall at 79.5% and 79.25% respectively, followed by nanomonsv with a recall of 72.5%. Conclusion Long-read sequencing can identify somatic structural variants in clincal samples. The longer reads have the potential to improve our understanding of cancer development and inform personalized cancer treatment.

Background Breast cancers acquire aggressive capabilities via epithelial to mesenchymal transition (EMT), in which various integrins/integrin-linked kinase signalling are upregulated. Methods We investigated this in two patient-derived xenografts (PDXs) developed from breast-to-bone metastases, and its functional significance in a breast cancer cell line system. ED03 and EDW01 PDXs were grown subcutaneously in immunocompromised SCID mice through 11 passages and 7 passages, respectively. Tumour tissue was assessed using immunohistochemistry (IHC) for oestrogen receptor (ER)-alpha, E-cadherin, vimentin, Twist1, beta-catenin, P120-RasGAP, CD44, CD24 and Ki67, and RT-qPCR of EMT-related factors ( CDH1 , VIM , CD44 , CD24 ), integrins beta 1 ( ITGB1 ), alpha 2 ( ITGA2 ) and ILK . Integrin and ILK expression in epidermal growth factor (EGF)-induced EMT of the PMC42-ET breast cancer cell line was assessed by RT-qPCR and Western blotting, as were the effects of their transient knockdown via small interfering RNA +/− EGF. Cell migration, changes in cell morphology and adhesion of siRNA-transfected PMC42-ET cells to various extracellular matrix (ECM) substrates was assessed. Results The ED03 (ER+/PR−/HER2−/lobular) and EDW01 (ER+/PR−/HER2−/ductal) PDXs were both classified as molecular subtype luminal A. ED03 xenografts exhibited mutated E-cadherin with minimal expression, but remained vimentin-negative across all passages. In EDW01, the hypoxic indicator gene CAIX and Twist1 were co-ordinately upregulated at passages 4–5, corresponding with a decrease in E-cadherin. At passages 6–7, VIM was upregulated along with ITGB1 and ITGA2 , consistent with an increasing EMT. The ED03 PDX displayed minimal change over passages in mice, for all genes examined. ILK , ITGB1 and ITGA2 mRNAs were also increased in the EGF-induced EMT of PMC42-ET cells (in which CDH1 was downregulated) although siRNA against these targets revealed that this induction was not necessary for the observed EMT. However, their knockdown significantly reduced EMT-associated adhesion and Transwell migration. Conclusion Our data suggest that despite an increase in ITGA2 and ITGB1 gene expression in the EMT exhibited by EDW01 PDX over multiple generations, this pathway may not necessarily drive the EMT process.

Background: Next-generation sequencing (NGS) in lung cancer specimens from endobronchial ultrasound transbronchial needle aspiration (EBUS-TBNA) is usually performed on formalin-fixed paraffin-embedded cell block material. Objectives: Since DNA can be damaged by this process, we investigated the potential of using DNA extracted from Diff-Quik cytology smears made for rapid on-site evaluation during EBUS-TBNA. Methods: In a prospective study, 67 patients undergoing diagnostic EBUS-TBNA were ana­lysed. We compared cell blocks and smears for DNA yields and sequencing (TruSeq Amplicon Cancer Panel) outcomes. Smears were also evaluated for tumour cell fraction and overall cellularity (cell count). Results: Primary lung cancer was diagnosed in 64 patients and metastatic malignancy in 3 patients. The DNA yield from smears was significantly higher than that obtained from matched cell blocks (mean 1,740 vs. 434 ng; p = 0.001). For 33 cases with matched smears and cell blocks the mutation profiles were similar. Smears with abundant malignant cells (using a cut-off of > 25% tumour cell fraction and > 1,000 cells) accurately predicted high (> 50 ng) DNA yield and therefore success in triaging samples to sequencing. In terms of tissue workflow, using only smears as source DNA for sequencing was an improvement in the use of only cell blocks (54/67 [80.6%] vs. 41/67 [61.2%]); however, the use of cell blocks when smears were not available or did not yield sufficient DNA further improved the success rate to 62/67 (92.5%) cases. Conclusion: We recommend smears in laboratory workflows as the primary source of DNA for NGS following an EBUS procedure.

Background Basal-like breast cancer (BLBC) is a poorly characterised, heterogeneous disease. Patients are diagnosed with aggressive, high-grade tumours and often relapse with chemotherapy resistance. Detailed understanding of the molecular underpinnings of this disease is essential to the development of personalised therapeutic strategies. Inhibitor of differentiation 4 (ID4) is a helix-loop-helix transcriptional regulator required for mammary gland development. ID4 is overexpressed in a subset of BLBC patients, associating with a stem-like poor prognosis phenotype, and is necessary for the growth of cell line models of BLBC through unknown mechanisms. Methods Here, we have defined unique molecular insights into the function of ID4 in BLBC and the related disease high-grade serous ovarian cancer (HGSOC), by combining RIME proteomic analysis, ChIP-seq mapping of genomic binding sites and RNA-seq. Results These studies reveal novel interactions with DNA damage response proteins, in particular, mediator of DNA damage checkpoint protein 1 (MDC1). Through MDC1, ID4 interacts with other DNA repair proteins (γH2AX and BRCA1) at fragile chromatin sites. ID4 does not affect transcription at these sites, instead binding to chromatin following DNA damage. Analysis of clinical samples demonstrates that ID4 is amplified and overexpressed at a higher frequency in BRCA1 -mutant BLBC compared with sporadic BLBC, providing genetic evidence for an interaction between ID4 and DNA damage repair deficiency. Conclusions These data link the interactions of ID4 with MDC1 to DNA damage repair in the aetiology of BLBC and HGSOC.

Mammosphere and breast tumoursphere culture have gained popularity as in vitro assays for propagating and analysing normal and cancer stem cells. Whether the spheres derived from different sources or parent cultures themselves are indeed single entities enriched in stem/progenitor cells compared to other culture formats has not been fully determined. We surveyed sphere-forming capacity across 26 breast cell lines, immunophenotyped spheres from six luminal- and basal-like lines by immunohistochemistry and flow cytometry and compared clonogenicity between sphere, adherent and matrigel culture formats using in vitro functional assays. Analyses revealed morphological and molecular intra- and inter-sphere heterogeneity, consistent with adherent parental cell line phenotypes. Flow cytometry showed sphere culture does not universally enrich for markers previously associated with stem cell phenotypes, although we found some cell-line specific changes between sphere and adherent formats. Sphere-forming efficiency was significantly lower than adherent or matrigel clonogenicity and constant over serial passage. Surprisingly, self-renewal capacity of sphere-derived cells was similar/lower than other culture formats. We observed significant correlation between long-term-proliferating-cell symmetric division rates in sphere and adherent cultures, suggesting functional overlap between the compartments sustaining them. Experiments with normal primary human mammary epithelia, including sorted luminal (MUC1(+)) and basal/myoepithelial (CD10(+)) cells revealed distinct luminal-like, basal-like and mesenchymal entities amongst primary mammospheres. Morphological and colony-forming-cell assay data suggested mammosphere culture may enrich for a luminal progenitor phenotype, or induce reversion/relaxation of the basal/mesenchymal in vitro selection occurring with adherent culture. Overall, cell line tumourspheres and primary mammospheres are not homogenous entities enriched for stem cells, suggesting a more cautious approach to interpreting data from these assays and careful consideration of its limitations. Sphere culture may represent an alternative 3-dimensional culture system which rather than universally 'enriching' for stem cells, has utility as one of a suite of functional assays that provide a read-out of progenitor activity.