Proteomic analysis of urine to identify breast cancer biomarker candidates using a label-free LC-MS/MS approach

Introduction: Breast cancer is a complex heterogeneous disease and is a leading cause of death in women. Early diagnosis and monitoring progression of breast cancer are important for improving prognosis. The aim of this study was to identify protein biomarkers in urine for early screening detection and monitoring invasive breast cancer progression. Method: We performed a comparative proteomic analysis using ion count relative quantification label free LC-MS/MS analysis of urine from breast cancer patients (n = 20) and healthy control women (n = 20). Results: Unbiased label free LC-MS/MS-based proteomics was used to provide a profile of abundant proteins in the biological system of breast cancer patients. Data analysis revealed 59 urinary proteins that were significantly different in breast cancer patients compared to the normal control subjects (p3). Thirty-six urinary proteins were exclusively found in specific breast cancer stages, with 24 increasing and 12 decreasing in their abundance. Amongst the 59 significant urinary proteins identified, a list of 13 novel up-regulated proteins were revealed that may be used to detect breast cancer. These include stage specific markers associated with pre-invasive breast cancer in the ductal carcinoma in-situ (DCIS) samples (Leucine LRC36, MAST4 and Uncharacterized protein CI131), early invasive breast cancer (DYH8, HBA, PEPA, uncharacterized protein C4orf14 (CD014), filaggrin and MMRN2) and metastatic breast cancer (AGRIN, NEGR1, FIBA and Keratin KIC10). Preliminary validation of 3 potential markers (ECM1, MAST4 and filaggrin) identified was performed in breast cancer cell lines by Western blotting. One potential marker MAST4 was further validated in human breast cancer tissues as well as individual human breast cancer urine samples with immunohistochemistry and Western blotting, respectively. Conclusions: Our results indicate that urine is a useful non-invasive source of biomarkers and the profile patterns (biomarkers) identified, have potential for clinical use in the detection of BC. Validation with a larger independent cohort of patients is required in the following study

Multiplexed immunofluorescence identifies high stromal CD68+PD‑L1+ macrophages as a predictor of improved survival in triple negative breast cancer

Triple negative breast cancer (TNBC) comprises 10–15% of all breast cancers and has a poor prognosis with a high risk of recurrence within 5 years. PD-L1 is an important biomarker for patient selection for immunotherapy but its cellular expression and co-localization within the tumour immune microenvironment and associated prognostic value is not well defined. We aimed to characterise the phenotypes of immune cells expressing PD-L1 and determine their association with overall survival (OS) and breast cancer-specific survival (BCSS). Using tissue microarrays from a retrospective cohort of TNBC patients from St George Hospital, Sydney (n = 244), multiplexed immunofluorescence (mIF) was used to assess staining for CD3, CD8, CD20, CD68, PD-1, PD-L1, FOXP3 and pan-cytokeratin on the Vectra Polaris™ platform and analysed using QuPath. Cox multivariate analyses showed high CD68+PD-L1+ stromal cell counts were associated with improved prognosis for OS (HR 0.56, 95% CI 0.33–0.95, p = 0.030) and BCSS (HR 0.47, 95% CI 0.25–0.88, p = 0.018) in the whole cohort and in patients receiving chemotherapy, improving incrementally upon the predictive value of PD-L1+ alone for BCSS. These data suggest that CD68+PD-L1+ status can provide clinically useful prognostic information to identify sub-groups of patients with good or poor prognosis and guide treatment decisions in TNBC

TILs immunophenotype in breast cancer predicts local failure and overall survival : analysis in a large radiotherapy trial with long-term follow-up

Aim: To determine the prognostic significance of the immunophenotype of tumour-infiltrating lymphocytes (TILs) within a cohort of breast cancer patients with long-term follow-up. Methods: Multiplexed immunofluorescence and automated image analysis were used to assess the expression of CD3, CD8, CD20, CD68, Fox P3, PD-1 and PD-L1 in a clinical trial of local excision and radiotherapy randomised to a cavity boost or not (n = 485, median follow-up 16 years). Kaplan–Meier and Cox multivariate analysis (MVA) methodology were used to ascertain relationships with local recurrence (LR), overall survival (OS) and disease-free survival (DFS). NanoString BC360 gene expression panel was applied to a subset of luminal patients to identify pathways associated with LR. Results: LR was predicted by low CD8 in MVA in the whole cohort (HR 2.34, CI 1.4–4.02, p = 0.002) and luminal tumours (HR 2.19, CI 1.23–3.92, p = 0.008) with associations with increased stromal components, decreased Tregs (FoxP3), inflammatory chemokines and SOX2. Poor OS was associated with low CD20 in the whole cohort (HR 1.73, CI 1.2–2.4, p = 0.002) and luminal tumours on MVA and low PD-L1 in triple-negative cancer (HR 3.44, CI 1.5–7, p = 0.003). Conclusions: Immunophenotype adds further prognostic data to help further stratify risk of LR and OS even in TILs low-luminal tumours

Cryopreservation of human cancers conserves tumour heterogeneity for single-cell multi-omics analysis

Background: High throughput single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for exploring cellular heterogeneity among complex human cancers. scRNA-Seq studies using fresh human surgical tissue are logistically difficult, preclude histopathological triage of samples, and limit the ability to perform batch processing. This hindrance can often introduce technical biases when integrating patient datasets and increase experimental costs. Although tissue preservation methods have been previously explored to address such issues, it is yet to be examined on complex human tissues, such as solid cancers and on high throughput scRNA-Seq platforms. Methods: Using the Chromium 10X platform, we sequenced a total of ~ 120,000 cells from fresh and cryopreserved replicates across three primary breast cancers, two primary prostate cancers and a cutaneous melanoma. We performed detailed analyses between cells from each condition to assess the effects of cryopreservation on cellular heterogeneity, cell quality, clustering and the identification of gene ontologies. In addition, we performed single-cell immunophenotyping using CITE-Seq on a single breast cancer sample cryopreserved as solid tissue fragments. Results: Tumour heterogeneity identified from fresh tissues was largely conserved in cryopreserved replicates. We show that sequencing of single cells prepared from cryopreserved tissue fragments or from cryopreserved cell suspensions is comparable to sequenced cells prepared from fresh tissue, with cryopreserved cell suspensions displaying higher correlations with fresh tissue in gene expression. We showed that cryopreservation had minimal impacts on the results of downstream analyses such as biological pathway enrichment. For some tumours, cryopreservation modestly increased cell stress signatures compared to freshly analysed tissue. Further, we demonstrate the advantage of cryopreserving whole-cells for detecting cell-surface proteins using CITE-Seq, which is impossible using other preservation methods such as single nuclei-sequencing. Conclusions: We show that the viable cryopreservation of human cancers provides high-quality single-cells for multiomics analysis. Our study guides new experimental designs for tissue biobanking for future clinical single-cell RNA sequencing studies

The tale of TILs in breast cancer : a report from The International Immuno-Oncology Biomarker Working Group

The advent of immune-checkpoint inhibitors (ICI) in modern oncology has significantly improved survival in several cancer settings. A subgroup of women with breast cancer (BC) has immunogenic infiltration of lymphocytes with expression of programmed death-ligand 1 (PD-L1). These patients may potentially benefit from ICI targeting the programmed death 1 (PD-1)/PD-L1 signaling axis. The use of tumor-infiltrating lymphocytes (TILs) as predictive and prognostic biomarkers has been under intense examination. Emerging data suggest that TILs are associated with response to both cytotoxic treatments and immunotherapy, particularly for patients with triple-negative BC. In this review from The International Immuno-Oncology Biomarker Working Group, we discuss (a) the biological understanding of TILs, (b) their analytical and clinical validity and efforts toward the clinical utility in BC, and (c) the current status of PD-L1 and TIL testing across different continents, including experiences from low-to-middle-income countries, incorporating also the view of a patient advocate. This information will help set the stage for future approaches to optimize the understanding and clinical utilization of TIL analysis in patients with BC

A case of amoebic colitis following remote historical exposure

A 67-year-old male with a background history of diverticular disease, atrial fibrillation and renal stones presented with dull lower abdominal pain associated with bloody diarrhoea following an episode of bilateral hip pain. He denied fevers, chills, pre-syncope, recent travel, gastroenteritis contacts and trauma. His medications included warfarin, metoprolol and rosuvastatin. He had not undergone prior colonoscopy or surgery. He had emigrated to Australia from Macedonia over 10 years ago

Tumour stroma ratio assessment using digital image analysis predicts survival in triple negative and luminal breast cancer

We aimed to determine the clinical significance of tumour stroma ratio (TSR) in luminal and triple negative breast cancer (TNBC) using digital image analysis and machine learning algorithms. Automated image analysis using QuPath software was applied to a cohort of 647 breast cancer patients (403 luminal and 244 TNBC) using digital H&E images of tissue microarrays (TMAs). Kaplan–Meier and Cox proportional hazards were used to ascertain relationships with overall survival (OS) and breast cancer specific survival (BCSS). For TNBC, low TSR (high stroma) was associated with poor prognosis for both OS (HR 1.9, CI 1.1–3.3, p = 0.021) and BCSS (HR 2.6, HR 1.3–5.4, p = 0.007) in multivariate models, independent of age, size, grade, sTILs, lymph nodal status and chemotherapy. However, for luminal tumours, low TSR (high stroma) was associated with a favourable prognosis in MVA for OS (HR 0.6, CI 0.4–0.8, p = 0.001) but not for BCSS. TSR is a prognostic factor of most significance in TNBC, but also in luminal breast cancer, and can be reliably assessed using quantitative image analysis of TMAs. Further investigation into the contribution of tumour subtype stromal phenotype may further refine these findings

Primary renal neuroblastoma in adults

A 79-year-old woman was admitted to the hospital with a closed intertrochanteric femoral neck fracture after a fall and underwent dynamic hip screw fixation. Postoperatively, she developed painless hematuria but was hemodynamically stable. She had no significant medical history, apart from hypertension, which had been managed with antihypertensive medications for the previous 20 years. Her blood test and urine cytology results did not show any obvious abnormality. However, computed tomography of the abdomen and pelvis showed an enhancing mass measuring 5.4 x 6.1x 6.0 cm in the lower pole of the left kidney . She underwent additional staging examinations with computed tomography of the chest and a bone scan, the findings of which were unremarkable

Molecular assays in breast cancer pathology

Recent advances in understanding the molecularpathology of breast cancer offer significantpotential to identifypatients who may benefit from adjuvant therapies. To date, few of these advances are utilised in a routine setting. We review molecular assays that are currently in use or are in the advanced stages of development, which may be used aspredictive orprognostic biomarkers in breast cancer. The only widely used breast cancer molecular assay is in situ hybridisation (ISH) for human epidermal growth factor receptor 2 (HER2) gene amplification and we highlight key issues with the interpretation of this assay, withparticular attention to the difficulties of the equivocal category. New molecular assays such as ISH for the topoisomerase II alpha (TOP2A) gene and for the aberrations in the copy number of the centromeric region of chromosome 17 are readilyperformed in a standard histopathology laboratory, but to date there are insufficient data to support their routine use.We also review the current data on two commercially available multigene expression assays, Oncotype DX and MammaPrint and discuss theirpotential use. Overall, while new molecular assays have significant potential to improve patient selection for therapy, well-performed histopathology with reliable interpretation of standard hormone and HER2 assays provides the most important predictive and prognostic information in early breast cancer

Proteomics for breast cancer urine biomarkers

Although the survival of breast cancer (BC) patients has increased over the last two decades due to improved screening programs and postoperative adjuvant systemic therapies, many patients die from metastatic relapse. Current biomarkers used in the clinic are not useful for the early detection of BC, or monitoring its progression, and have limited value in predicting response to treatment. The development of proteomic techniques has sparked new searches for novel protein markers for many diseases including BC. Proteomic techniques allow for a high-throughput analysis of samples with the visualization and quantification of thousands of potential protein and peptide markers. Human urine is one of the most interesting and useful biofluids for routine testing and provides an excellent resource for the discovery of novel biomarkers, with the advantage over tissue biopsy samples due to the ease and less invasive nature of collection. In this review, we summarize the results from studies where urine was used as a source for BC biomarker research and discuss urine sample preparation, its advantage, challenges, and limitation. We focus on the gel-based proteomic approaches as well as the recent development of quantitative techniques in BC urine biomarker detection. Finally, the future use of modern proteomic techniques in BC biomarker identification will be discussed