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.

Unicentric Castleman's disease imitating metastatic melanoma: A case report.

INTRODUCTION AND IMPORTANCE: This case report shows a unique case of Castleman's disease in the context of histopathological diagnosis of cutaneous melanoma where clinical and radiological features of Castleman's disease were masked by presumptive diagnosis of metastatic melanoma. The disease is part of a group of lymphoproliferative disorders with characteristic histopathological features that can occur in any lymph node in the body, characterised by slow growing painless masses which are asymptomatic until mass effect occurs. This case highlights the need for caution when considering management of lymphadenopathy with clinically/radiologically suspicious nodes. PRESENTATION OF CASE: A 65 year old man with metastatic melanoma of the left elbow presented for axillary sentinel node mapping and was found to have a hypoechoic enlarged node on ultrasound. This was further investigated and found to be a lymphoproliferative growth pathognomonic for Castlemans disease. DISCUSSION: Whilst clinically detected lymphadenopathy in the draining node basin of a primary cutaneous melanoma is highly suspicious for nodal metastasis, it is sometimes not possible to confirm or exclude this diagnosis without complete histological examination of the node. Multidisciplinary input from the surgeon, histopathologist and radiologist is a key step in confirming diagnosis. CONCLUSION: Alternative diagnoses must be considered in the context of lymphadenopathy, even in the context of malignant melanoma.

Neurotoxicity assessment of QoI strobilurin fungicides azoxystrobin and trifloxystrobin in human SH-SY5Y neuroblastoma cells: Insights from lipidomics and mitochondrial bioenergetics.

Strobilurin fungicides are quinone outside inhibitors (QoI) used to treat fungal pathogens for agricultural and residential use. Here, we compared the potential for neurotoxicity of the widely used strobilurins, azoxystrobin (AZS) and trifloxystrobin (TFS), in differentiated human SH-SY5Y cells. Fungicides did not include cytotoxicity up to 200 µM but both induced loss of cell viability at 48 h, with TFS showing slightly higher toxicity that AZS. Caspase 3/7 activity was induced in SH-SY5Y cells by both fungicides at 48 h (50 µM for AZS and 25 µM for TFS). ATP levels were reduced following a 24-hour exposure to > 25 µM AZS and > 6.25 µM TFS and both fungicides rapidly impaired oxidative respiration (~12.5 µM for AZS and ~3.125 µM TFS) and decreased oligomycin-induced ATP production, maximal respiration, and mitochondrial spare capacity. AZS at 100 µM showed a continual impairment of mitochondrial membrane potential (MMP) between 4 and 48 h while TFS at > 50 µM decreased MMP at 24 h. Taken together, TFS exerted higher mitochondrial toxicity at lower concentrations compared to AZS in SH-SY5Y cells. To discern toxicity mechanisms of strobilurin fungicides, lipidomics was conducted in SH-SY5Y cells following exposure to 6.25 µM and 25 µM AZS, and a total of 1595 lipids were detected, representing 49 different lipid classes. Lipid classes with the largest proportion of lipids detected in SH-SY5Y cells included triglycerides (17%), phosphatidylethanolamines (8%), ether-linked triglycerides (8%), phosphatidylcholines (7%), ether-linked phosphatidylethanolamines (6%), and diacylglycerols (5%). Together, these 5 lipid classes accounted for over 50% of the total lipids measured in SH-SY5Y cells. Lipids that were increased by AZS included acyl carnitine, which plays a role in long chain fatty acid utilization for mitochondrial ß-oxidation, as well as non-modified, ether linked, and oxidized triacylglycerols, suggesting compensatory upregulation of triglyceride biosynthesis. The ceramide HexCer-NS, linked to neurodegenerative diseases, was decreased in abundance following AZS exposure. In summary, strobilurin fungicides rapidly inhibit mitochondrial oxidative respiration and alter the abundance of several lipids in neuronal cells, relevant for understanding environmental exposure risks related to their neurotoxicity.

A single-cell and spatially resolved atlas of human breast cancers.

Breast cancers are complex cellular ecosystems where heterotypic interactions play central roles in disease progression and response to therapy. However, our knowledge of their cellular composition and organization is limited. Here we present a single-cell and spatially resolved transcriptomics analysis of human breast cancers. We developed a single-cell method of intrinsic subtype classification (SCSubtype) to reveal recurrent neoplastic cell heterogeneity. Immunophenotyping using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) provides high-resolution immune profiles, including new PD-L1/PD-L2+ macrophage populations associated with clinical outcome. Mesenchymal cells displayed diverse functions and cell-surface protein expression through differentiation within three major lineages. Stromal-immune niches were spatially organized in tumors, offering insights into antitumor immune regulation. Using single-cell signatures, we deconvoluted large breast cancer cohorts to stratify them into nine clusters, termed 'ecotypes', with unique cellular compositions and clinical outcomes. This study provides a comprehensive transcriptional atlas of the cellular architecture of breast cancer.

Stromal cell diversity associated with immune evasion in human triple-negative breast cancer.

The tumour stroma regulates nearly all stages of carcinogenesis. Stromal heterogeneity in human triple-negative breast cancers (TNBCs) remains poorly understood, limiting the development of stromal-targeted therapies. Single-cell RNA sequencing of five TNBCs revealed two cancer-associated fibroblast (CAF) and two perivascular-like (PVL) subpopulations. CAFs clustered into two states: the first with features of myofibroblasts and the second characterised by high expression of growth factors and immunomodulatory molecules. PVL cells clustered into two states consistent with a differentiated and immature phenotype. We showed that these stromal states have distinct morphologies, spatial relationships and functional properties in regulating the extracellular matrix. Using cell signalling predictions, we provide evidence that stromal-immune crosstalk acts via a diverse array of immunoregulatory molecules. Importantly, the investigation of gene signatures from inflammatory-CAFs and differentiated-PVL cells in independent TNBC patient cohorts revealed strong associations with cytotoxic T-cell dysfunction and exclusion, respectively. Such insights present promising candidates to further investigate for new therapeutic strategies in the treatment of TNBCs.

Faculty Perspectives Regarding Day One-Ready Examination Items.

The objective of this mixed-methods, cross-sectional study was to evaluate faculty perspectives regarding Day One-Ready (DOR) content on examination questions given to students at a veterinary medical college and to elucidate whether differing viewpoints on what information constitutes DOR knowledge exist among different veterinary disciplines. Twelve faculty members at a veterinary medical college from three different disciplines (small animal internal medicine, surgery, and primary care) reviewed examination questions given to veterinary students, answered the questions, and stated whether they tested DOR information. After elimination of items not answered by all respondents and after reviewing for question quality, 103 questions remained for analysis. An evaluator from each discipline participated in a discussion about DOR content. Of the questions, 30% were unanimously considered to assess DOR information. No association was found between type of question (medicine, surgery, uncategorized) and whether it was considered DOR. Primary care doctors assessed more questions as testing DOR information than either type of specialist. Questions answered correctly were more likely to be assessed as DOR. During discussion, themes identified with DOR information included common conditions, practical diagnostics, critical knowledge, and discriminating between differential diagnoses. Specialists and primary care doctors differed in their assessment of DOR questions. Veterinary faculty should carefully consider whether examination questions contain DOR information and are appropriate for testing knowledge of the entry-level veterinarian.

ABVD Without Radiation for Newly Diagnosed Pediatric and Young Adult Patients With Hodgkin Lymphoma: A Single Center Retrospective Analysis of 28 Consecutive Patients.

Hodgkin lymphoma (HL) is the most common malignancy affecting adolescents and young adults. Treatment with a combination of chemotherapy and radiation results in cure rates of >90%. However, radiation therapy causes significant late effects and avoiding radiation entirely for patients who respond to chemotherapy is an accepted strategy. Since 2011, 28 consecutive patients diagnosed with classic HL have been treated with doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) for 4 to 6 cycles. Patients who achieved a complete metabolic response (CMR) as assessed by [F] fluorodeoxyglucose positron emission tomography by the end of chemotherapy did not receive radiation. Among the 27 evaluable patients, 26/27 (96.2%) achieved a CMR with ABVD alone with 24/27 (88.9%) having achieved a CMR after 2 cycles. Event-free survival at 5 years is 90.5% and overall survival is 100% with a median follow-up time of 22.4 and 22.1 months, respectively. Treating pediatric and young adult HL patients with ABVD alone results in CMRs in >95% of patients. Patients who were refractory to ABVD or relapsed after treatment eventually achieved remission with a combination of standard and novel salvage therapies. This regimen demonstrates the feasibility of avoiding upfront radiation in newly diagnosed pediatric HL patients.

Myoepithelial cell-specific expression of stefin A as a suppressor of early breast cancer invasion.

Mammography screening has increased the detection of early pre-invasive breast cancers, termed ductal carcinoma in situ (DCIS), increasing the urgency of identifying molecular regulators of invasion as prognostic markers to predict local relapse. Using the MMTV-PyMT breast cancer model and pharmacological protease inhibitors, we reveal that cysteine cathepsins have important roles in early-stage tumorigenesis. To characterize the cell-specific roles of cathepsins in early invasion, we developed a DCIS-like model, incorporating an immortalized myoepithelial cell line (N1ME) that restrained tumor cell invasion in 3D culture. Using this model, we identified an important myoepithelial-specific function of the cysteine cathepsin inhibitor stefin A in suppressing invasion, whereby targeted stefin A loss in N1ME cells blocked myoepithelial-induced suppression of breast cancer cell invasion. Enhanced invasion observed in 3D cultures with N1ME stefin A-low cells was reliant on cathepsin B activation, as addition of the small molecule inhibitor CA-074 rescued the DCIS-like non-invasive phenotype. Importantly, we confirmed that stefin A was indeed abundant in myoepithelial cells in breast tissue. Use of a 138-patient cohort confirmed that myoepithelial stefin A (cystatin A) is abundant in normal breast ducts and low-grade DCIS but reduced in high-grade DCIS, supporting myoepithelial stefin A as a candidate marker of lower risk of invasive relapse. We have therefore identified myoepithelial cell stefin A as a suppressor of early tumor invasion and a candidate marker to distinguish patients who are at low risk of developing invasive breast cancer, and can therefore be spared further treatment. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Programmed death ligand 1 expression in triple-negative breast cancer is associated with tumour-infiltrating lymphocytes and improved outcome.

AIMS: Triple-negative breast cancer (TNBC) patients generally have a poor outcome; there is a pressing need to identify more effective therapeutic strategies. Clinical trials targeting programmed death 1/programmed death ligand 1 (PD1/PDL1) in melanoma and non-small-cell lung cancer have reported high response rates, and tumoral PDL1 expression has been suggested as a potential biomarker to enrich for patient response to these treatments. There are only very limited data to date reporting the expression of PDL1 in TNBC. METHODS AND RESULTS: PDL1 immunohistochemistry was performed on 161 primary TNBCs and assessed in the tumour as well as immune cells in the stromal compartment. PDL1 expression was very common in TNBC, expressed in the tumour cell membrane (64%), cytoplasm (80%) and stromal (93%) cellular compartments. Cytoplasmic tumoral expression of PDL1 was associated with a lower risk of breast cancer-specific death [hazard ratio (HR) 0.45, P = 0.035] while stromal PDL1 expression was associated with a lower rate of deaths from all causes (HR 0.305, P = 0.0042). Membranous expression of PDL1 was not associated with outcome. While both PDL1 expression and tumour-infiltrating lymphocytes were associated with a better outcome, only lymphovascular invasion and high tumour-infiltrating lymphocytes were independently prognostic for breast cancer-specific death. CONCLUSION: While PDL1 expression is frequent in TNBC, it was not independently prognostic. There were differences in outcome depending on the cellular compartment of PDL1 expression. These data provide further impetus for investigating the utility of immune checkpoint therapies in TNBC, given the clinical significance of tumour-infiltrating lymphocytes (TILs) and PDL1 expression in this cohort.

Superselective intra-arterial melphalan therapy for newly diagnosed and refractory retinoblastoma: results from a single institution.

BACKGROUND: Intra-arterial administration of melphalan chemotherapy has shown promise in the treatment of retinoblastoma. This report describes our results using superselective intra-arterial melphalan in patients with newly diagnosed retinoblastoma and those who were treated for progression after systemic chemotherapy. METHODS: This is a retrospective review of all retinoblastoma patients treated with intra-arterial melphalan at the University of California, San Francisco from March 2010 to August 2012. Twenty eyes (16 patients) underwent 40 intra-arterial melphalan infusions, and dose was determined by age. Patients were treated at monthly intervals and received a range of 1-5 treatments. Response to therapy, toxicity, and procedural radiation exposure was assessed. RESULTS: All patients are alive without metastatic disease at a median follow-up of 14.5 (1-29) months. Treatment with enucleation or external beam radiation was avoided in 11/20 eyes (55%) overall [6/12 (50%) in newly diagnosed eyes and 5/8 (63%) in refractory/relapsed eyes]. Response rates (per the International Classification of Retinoblastoma) were as follows: 6/7 (86%) in groups A-C and 5/13 (38%) in groups D and E. Nonhematologic and hematologic toxicities were minimal and comparable with those in previous reports. The mean procedural radiation dose was 20.2 ± 11.9 mGy per eye per procedure. CONCLUSION: Superselective intra-arterial melphalan therapy is effective for less advanced eyes but further modifications to therapy are required to improve results in eyes with advanced retinoblastoma.

Primary mixed malignant tumor of bone in an 18-year-old male: Report of a case with radiologic-pathologic correlation.

We report a case of primary malignant mixed tumor (MMT) of bone in an 18-year-old boy with X-ray, CT, MR, scintigraphic, FDG PET, and pathologic correlation. Primary MMT of bone is a highly aggressive tumor and presents both a diagnostic and clinical treatment challenge. This tumor is extremely rare and to the best of our knowledge, this is the first report of the diagnostic imaging findings for primary MMT arising from bone in a patient of this age group.

Relationship between loneliness and proangiogenic cytokines in newly diagnosed tumors of colon and rectum.

OBJECTIVE: To investigate the association of serum levels of proangiogenic cytokines with different indices of social support and loneliness by measuring the levels of expression of two important proangiogenic cytokines, vascular endothelial growth factor (VEGF), and interleukin-6 in tumors of colon and rectum. Lack of social support has been prospectively associated with cancer progression. METHODS: Fifty-one newly diagnosed patients with colorectal tumors (mean age, 68.3 years) completed two measures of loneliness 1 to 2 days before their surgical treatment. The first was an explicit self-report questionnaire, which tapped into negative feelings as a result of low social support. The second was a standardized computer-based task, which measured loneliness implicitly. Immunohistochemical analyses were performed on tumor tissues post surgery to determine the expression of cytokines. RESULTS: Logistic regression showed that higher levels of implicit loneliness independently predicted stronger expression of VEGF, controlling for Dukes stage and explicit loneliness, both of which were nonsignificant predictors. No significant relationships were found between the loneliness measures and interleukin-6. CONCLUSIONS: The results of this study suggest VEGF to be an angiogenic mechanism through which loneliness may lead to worse cancer-related outcomes. Implications are discussed in terms of devising targeted psychosocial and immunotherapeutic interventions for cancer patients with low social support.

Radiation risks from imaging studies in children with cancer.

Individuals exposed to high doses of ionizing radiation are known to be at risk of developing cancer. Recent reviews about the late effects of ionizing radiation indicate that even low doses of radiation can cause cancer. Significant radiation exposure occurs with commonly used imaging studies including CT scans, PET scans, and bone scans. Children with cancer, particularly those children with solid tumors, are exposed to frequent low doses of ionizing radiation from multiple imaging studies performed at initial diagnosis, throughout treatment, and during the follow-up period. This review provides estimates of cumulative radiation doses from imaging studies in children undergoing chemotherapy and/or surgery for various malignancies. Current risk estimates indicate that the radiation exposure from these imaging studies may be sufficient to increase the lifetime risk of cancer.

Pediatric acute blastic natural killer cell leukemia.

The goal of this report is to describe a rare case of pediatric blastic natural killer (NK) cell leukemia and to compare pediatric blastic NK cell leukemia/lymphoma to other reported cases of pediatric NK cell leukemia. The patient, a 9-year-old girl, presented with acute leukemia with a phenotype similar to adult blastic NK cell leukemia/lymphoma. The blasts were agranular and expressed CD7, 45, 56, and HLA-DR, but not CD3, 11c, 13, 33, or TdT. She had a complete response to ALL-directed chemotherapy, but had multiple relapses involving the cerebrospinal fluid, nasal sinus, lymph node and skin. In addition to the reported case, a review of the literature identified 9 previously reported cases of NK cell leukemia in patients 18 years of age or less. Cases were subdivided into blastic, acute/aggressive, and myeloid precursor NK cell leukemia based upon CD13/33 expression and morphologic characteristics. Compared to pediatric acute/aggressive NK cell leukemia, children with blastic NK cell leukemia showed greater variation in age and race. Prognosis was poor for all groups. Pediatric blastic NK cell leukemia is a distinct clinicopathologic entity which differs from other types of pediatric NK cell leukemia.