Biology subjects

Shivalingam, B.

Publications and source records attributed to Shivalingam, B..

3 recordsLinked to original sources

bioRxiv2020

A comprehensive proteomic SWATH-MS workflow for profiling blood extracellular vesicles: a new avenue for glioma tumour surveillance

There is a real need for biomarkers that can indicate glioma disease burden and inform clinical management, particularly in the recurrent glioblastoma (GBM; grade IV glioma) setting where treatment-associated brain changes can confound current and expensive tumour surveillance methods. In this regard, extracellular vesicles (EVs; 30-1000 nm membranous particles) hold major promise as robust tumour biomarkers. GBM-EVs encapsulate molecules that reflect the identity and molecular state of their cell-of-origin and cross the blood-brain-barrier into the periphery where they are readily accessible. Despite the suitability of circulating-EVs for GBM biomarker discovery, sample complexity has hindered comprehensive quantitative proteomic studies. Here, sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) was used in conjunction with a targeted data extraction strategy to comprehensively profile circulating-EVs isolated from plasma. Plasma-EVs sourced from pre-operative glioma II-IV patients (n=41) and controls (n=11) were sequenced by SWATH-MS, and the identities and absolute quantities of the proteins were extracted by aligning the SWATH-MS data against a custom glioma spectral library comprised of 8662 high confidence protein species. Overall, 4054 plasma-EV proteins were quantified across the cohorts, and putative circulating-EV biomarker proteins identified (adjusted p-value<0.05) included previously reported GBM-EV proteins identified in vitro and in neurosurgical aspirates. Principle component analyses showed that plasma-EV protein profiles clustered according to glioma subtype and WHO-grade, and plasma-EV proteins reflected the extent of glioma aggression. Using SWATH-MS, we describe the most comprehensive proteomic plasma-EV profiles for glioma and highlight the promise of this approach as an accurate and sensitive tumour monitoring method. Objective blood-based measurements of glioma tumour activity will support the implementation of next-generation, patient-centred therapies and are ideal surrogate endpoints for recurrent progression that would allow clinical trial protocols to be more dynamic and adapt to the individual patient and their cancer.

pathology↗

bioRxiv2020

Hotspot KRAS mutations in brain metastases at the first metastatic recurrence of cutaneous melanoma

IMPORTANCEBrain metastases occur in 60% of patients with advanced melanoma and are a major cause of melanoma-related mortality and morbidity. Although our understanding of the molecular alterations associated with melanoma progression is improving, there are currently no validated biomarkers which might help identify those patients at highest risk of developing brain metastases. OBJECTIVETo examine the somatic mutational and copy-number landscape of brain metastases that develop as the isolated first visceral site of recurrence - "early brain-metastasis" compared to extracranial melanoma metastases. DESIGN, SETTING AND PARTICIPANTSWhole-exome sequencing of 50 tumors from patients undergoing surgical resection of one or more brain metastasis occurring as the first site of visceral relapse were identified from prospectively maintained databases in Sydney, Wellington, New York and Cambridge. Whole exome sequencing analyses allowed mutational profiles to be compared to cutaneous melanomas in The Cancer Genome Atlas (SKCM-TCGA; n=358) and the Memorial Sloan Kettering (SKCM-MSK-IMPACT; n=186) datasets. An external dataset comprising a further 18 patients with surgically resected early brain metastasis from two additional academic centers served as an independent validation cohort. MAIN OUTCOMES AND MEASURESTo assess the frequency of driver mutations in early brain metastasis and their influence on survival. RESULTSIn concordance with the landmark melanoma sequencing studies, we identified mutations in BRAF (21/50, 42%), NRAS (14/50, 28%) and NF1 (11/50, 22%) as the most frequently mutated melanoma driver genes. When compared to the mutational landscape of cutaneous melanomas in TCGA (SKCM-TCGA), KRAS was the most significantly enriched driver gene, with 5/50 (10%) of brain metastases harboring non-synonymous mutations, of which 4/5 (80%) were in the hotspot positions of codons 12 and 61. This was significantly higher than the corresponding frequency of KRAS-mutations within the entire SKCM-TCGA (2% (7/358), p=0.009, Fishers Exact Test) as well as the SKCM-MSK-IMPACT cohort (1.6% (3/186), p=0.016). Variants in KRAS were mutually exclusive from BRAFV600, NRAS and HRAS mutations and were associated with a significantly reduced overall survival from resection of brain metastasis (relative to KRAS-wild type brain metastases) in multivariate Cox proportional hazard models (HR 1.80, 95% CI 1.46-24.89, p=0.013). Mutations in KRAS were also clonal and concordant with extracranial disease, which suggests these mutations are present within the primary tumor CONCLUSIONS AND RELEVANCEOur analysis, the largest to date, suggests that early metastases to the brain (presenting as the first site of visceral relapse) are characterized by significant enrichment of hotspot KRAS mutations, potentially implicating constitutive RAS-driven cellular programs in neurotropic metastatic behavior in these cases. Based on these data, we suggest that screening for KRAS mutations might help identify those patients with primary melanoma at higher risk of brain metastases or poor survival, and could help inform future surveillance strategies. Key PointsO_ST_ABSQuestionC_ST_ABSWhat is the frequency of driver mutations in early melanoma brain metastases? FindingsIn this study of 50 patients with melanoma metastasizing first to the brain, KRAS mutations were the most significantly enriched driver gene (n=5, 10% of patients) when compared to landmark cutaneous melanoma studies. The high KRAS mutation frequency was also observed in an external validation cohort of 18 patients with early brain metastases. Mutations in KRAS were mutually exclusive from mutations in the key RAS signaling genes and conferred a worse overall survival from resection of brain metastasis. MeaningHotspot KRAS mutations could help identify those patients with primary melanoma at higher risk of brain metastases that may benefit from more intensive, protracted surveillance as well as earlier use of adjuvant therapy.

genomics↗

bioRxiv2018

Deep sequencing of circulating exosomal microRNA allows non-invasive glioblastoma diagnosis

Exosomes are nano-sized extracellular vesicles released by many cells that contain molecules characteristic of their cell-of-origin, including microRNA. Exosomes released by glioblastoma cross the blood-brain-barrier into the peripheral circulation, and carry molecular cargo distinct to that of free-circulating miRNA. In this pilot study, serum exosomal-microRNAs were isolated from glioblastoma (n=12) patients and analyzed using unbiased deep sequencing. Results were compared to sera from age- and gender-matched healthy controls, and to grades II-III (n=10) glioma patients. Significant differentially expressed microRNAs were identified, and the predictive power of individual and subsets of microRNAs were tested using univariate and multivariate analyses. Additional sera from glioblastoma patients (n=4) and independent sets of healthy (n=9) and non-glioma (n=10) controls were used to further test the specificity and predictive power of this unique exosomal-microRNA signature. Twenty-six microRNAs were differentially expressed in serum exosomes from glioblastoma patients relative to healthy controls. Random forest modeling and data partitioning selected seven miRNAs (miR-182-5p, miR-328-3p, miR-339-5p, miR-340-5p, miR-485-3p, miR-486-5p and miR-543) as the most stable for classifying glioblastoma. Strikingly, within this model, six iterations of these miRNA classifiers could distinguish glioblastoma patients from controls with perfect accuracy. The seven-miRNA panel was able to correctly classify all specimens in validation cohorts (n=23). Also identified were 23 dysregulated miRNAs in IDHMUT gliomas, a partially overlapping yet distinct signature of lower grade glioma. Serum exosomal-miRNA signatures can accurately diagnose glioblastoma preoperatively. miRNA signatures identified are distinct from previously reported free-circulating miRNA studies in GBM patients, and appear to be superior.

pathology↗