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.
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.
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.
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.
The disciplines of oncology and pathology are at present experiencing a wave of changes as precision medicine becomes embedded as standard-of-care. Consequently, the need to assess increasing numbers ...of biomarkers simultaneously has become more urgent and recognising the vast intra-tumoural heterogeneity, including within the microenvironment, requires a complex dimensional understanding of the localisation of the biomarker expression. Digital spatial profiling (DSP; nanoString™) technology spatially resolves and digitally quantifies proteins in a highly multiplexed assay, underpinned by the nCounter® barcoding platform. We present the application of this technology to breast cancer samples. Applying the ‘off the shelf’ cancer panel and a custom-conjugated E-cadherin antibody, we quantify vast intra-tumoural heterogeneity in immunological and tumour markers, and demonstrate a need for focussed selection of target cell populations. The technology offers enormous potential not only for making research advances but also for improving standard operating procedures in diagnostic applications.
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.
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.
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.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Normal human tissues do not express glycans terminating with the sialic acid N-glycolylneuraminic acid (Neu5Gc), yet Neu5Gc-containing glycans have been consistently found in human tumor tissues, ...cells and secretions and have been proposed as a cancer biomarker. We engineered a Neu5Gc-specific lectin called SubB2M, and previously reported elevated Neu5Gc biomarkers in serum from ovarian cancer patients using a Surface Plasmon Resonance (SPR)-based assay. Here we report an optimized SubB2M SPR-based assay and use this new assay to analyse sera from breast cancer patients for Neu5Gc levels.
To enhance specificity of our SPR-based assay, we included a non-sialic acid binding version of SubB, SubB
, to control for any non-specific binding to SubB2M, which improved discrimination of cancer-free controls from early-stage ovarian cancer. We analysed 96 serum samples from breast cancer patients at all stages of disease compared to 22 cancer-free controls using our optimized SubB2M-
-SPR assay. We also analysed a collection of serum samples collected at 6 monthly intervals from breast cancer patients at high risk for disease recurrence or spread.
Analysis of sera from breast cancer cases revealed significantly elevated levels of Neu5Gc biomarkers at all stages of breast cancer. We show that Neu5Gc serum biomarker levels can discriminate breast cancer patients from cancer-free individuals with 98.96% sensitivity and 100% specificity. Analysis of serum collected prospectively, post-diagnosis, from breast cancer patients at high risk for disease recurrence showed a trend for a decrease in Neu5Gc levels immediately following treatment for those in remission.
Neu5Gc serum biomarkers are a promising new tool for early detection and disease monitoring for breast cancer that may complement current imaging- and biopsy-based approaches.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
Abstract Objective Endometrial cancer patients may benefit from systemic adjuvant chemotherapy, alone or in combination with targeted therapies. Prognostic and predictive markers are needed, however, ...to identify patients amenable for these therapies. Methods Primary endometrial tumors were genotyped for > 100 hot spot mutations in genes potentially acting as prognostic or predictive markers. Mutations were correlated with tumor characteristics in a discovery cohort, replicated in independent cohorts and finally, confirmed in the overall population ( n = 1063). Results PIK3CA , PTEN and KRAS mutations were most frequently detected, respectively in 172 (16.2%), 164 (15.4%) and 161 (15.1%) tumors. Binary logistic regression revealed that PIK3CA mutations were more common in high-grade tumors (OR = 2.03; P = 0.001 for grade 2 and OR = 1.89; P = 0.012 for grade 3 compared to grade 1), whereas a positive TP53 status correlated with type II tumors (OR = 11.92; P < 0.001) and PTEN mutations with type I tumors (OR = 19.58; P = 0.003). Conversely, FBXW7 mutations correlated with positive lymph nodes (OR = 3.38; P = 0.045). When assessing the effects of individual hot spot mutations, the H1047R mutation in PIK3CA correlated with high tumor grade and reduced relapse-free survival (HR = 2.18; P = 0.028). Conclusions Mutations in PIK3CA , TP53 , PTEN and FBXW7 correlate with high tumor grade, endometrial cancer type and lymph node status, whereas PIK3CA H1047R mutations serve as prognostic markers for relapse-free survival in endometrial cancer patients.
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.
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.
•First reported case of metastasising Lipoblastoma-like tumour of the vulva (LLTV).•LLTV is a rare soft tissue neoplasm with reported benign clinical behaviour.•LLTV arose in a 16 year old female who ...subsequently developed histologically identical lesions in the breast, chest wall and retroperitoneum, over the course of three and a half years.•Whole exome sequencing revealed a complex genome with striking similarity between the tumours.•MTOR mutation identified, which enabled tailored patient treatment with a specific inhibitor (everolimus).
Lipoblastoma-like tumour of the vulva (LLTV) is a rare soft tissue neoplasm with only nineteen reported cases to date. The clinical behaviour in all cases has been benign, with a small number of local recurrences and no incidences of metastasis described. We present an unusual case of LLTV that arose in a 16-year-old female who subsequently developed histologically identical lesions in the breast, chest wall and retroperitoneum, over the course of three and a half years. Our comprehensive histopathological analysis and molecular genetic studies supported the diagnosis of LLTV, with absence of DDIT3 rearrangement and absence of other specific diagnostic genetic events for any other soft tissue neoplasm. Whole exome sequencing was performed on the vulva, vulva recurrence, breast, and lung lesions and revealed a complex genome with striking similarity between the tumours. Multiple somatic mutations of interest were identified, including in MTOR and the TERT promoter, which enabled patient treatment with a specific inhibitor. To our knowledge, this is the first reported case of metastasising LLTV, and the first comprehensive genetic analysis of such a tumour.
The heterogeneity of tumor infiltrating lymphocytes (TILs) is not well characterized in brain metastasis. To address this, we performed a targeted analysis of immune-cell subsets in brain metastasis ...tissues to test immunosuppressive routes involved in brain metastasis. We performed multiplex immunofluorescence (mIF), using commercially available validated antibodies on formalin-fixed paraffin embedded whole sections. We quantitated the subsets of immune-cells utilizing a targeted panel of proteins including PanCK, CD8, CD4, VISTA and IBA-1, and analyzed an average of 15,000 cells per sample. Classifying tumors as either high (>30%) or low (<30%) TILs, we found that increased TILs density correlated with survival. Phenotyping these TILs we found tumors with low TILs had significantly higher expression of the immune-checkpoint molecule VISTA in tumor cells (
< 0.01) as well as in their microenvironment (
< 0.001). Contrastingly, the tumors with high TILs displayed higher levels of microglia, as measured by IBA-1 expression. Low TILs-tumors displayed CD8+ T-cells that co-express VISTA (
< 0.01) significantly more compared to high TILs group, where CD8+cells significantly co-express IBA-11 (
< 0.05). These results were supported by RNA analysis of a publicly available, independent cohort. Our work contributes to a growing understanding of the immune surveillance escape routes active in brain metastasis.
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