Haemolysis during Sample Preparation Alters microRNA Content of Plasma

The presence of cell-free microRNAs (miRNAs) has been detected in a range of body fluids. The miRNA content of plasma/serum in particular has been proposed as a potential source of novel biomarkers for a number of diseases. Nevertheless, the quantification of miRNAs from plasma or serum is made difficult due to inefficient isolation and lack of consensus regarding the optimal reference miRNA. The effect of haemolysis on the quantification and normalisation of miRNAs in plasma has not been investigated in great detail. We found that levels of miR-16, a commonly used reference gene, showed little variation when measured in plasma samples from healthy volunteers or patients with malignant mesothelioma or coronary artery disease. Including samples with evidence of haemolysis led to variation in miR-16 levels and consequently decreased its ability to serve as a reference. The levels of miR-16 and miR-451, both present in significant levels in red blood cells, were proportional to the degree of haemolysis. Measurements of the level of these miRNAs in whole blood, plasma, red blood cells and peripheral blood mononuclear cells revealed that the miRNA content of red blood cells represents the major source of variation in miR-16 and miR-451 levels measured in plasma. Adding lysed red blood cells to non-haemolysed plasma allowed a cut-off level of free haemoglobin to be determined, below which miR-16 and miR-451 levels displayed little variation between individuals. In conclusion, increases in plasma miR-16 and miR-451 are caused by haemolysis. In the absence of haemolysis the levels of both miR-16 and miR-451 are sufficiently constant to serve as normalisers

Antioxidant and anti–glycation activities correlates with phenolic composition of tropical medicinal herbs

AbstractObjectiveTo determine the contribution of total phenolic content (TPC) in glycation inhibitory activity of common tropical medicinal food and spices with potential antioxidative properties.MethodsIn vitro glucose-bovine serum albumin (BSA) assay was used. Ethanolic extracts of ten common household condiments/herbs (Allium sativum, Zingiber officinale, Thymus vulgaris, Petroselinum crispum, Murraya koenigii Spreng, Mentha piperita L., Curcuma longa L., Allium cepa L., Allium fistulosum and Coriandrum sativum L.) were evaluated for antioxidative activity by 2,2-diphenyl-2-picrylhydrazyl (DPPH), and ferric reducing antioxidant power (FRAP) and the TPC, flavonoid and tannins content were determined.ResultsFindings showed good correlation between TPC/DPPH (r = 0.8), TPC/FRAP (r = 0.8), TPC/anti–glycation (r = 0.9), DPPH/anti–glycation (r = 0.6), FRAP/anti–glycation (r = 0.9), Flavonoid/anti–glycation (r = 0.7) and Tannins/anti–glycation (r = 0.8) and relatively fair correlation for TPC/Flavonoids (r = 0.5) and TPC/Tannins (r = 0.5). Results imply that these plants are potential sources of natural antioxidants which have free radical scavenging activity and might be used for reducing oxidative stress.ConclusionsThe positive glycation inhibitory and antioxidative activities of these tropical herbs suggest a possible role in targeting ageing, diabetic complications and oxidative stress related diseases

Abstract P1-05-08: Comprehensive genomic profiling of 8,654 breast carcinoma reveals therapeutically targetable molecular subtypes beyond those defined by hormone-receptor expression

Abstract Background: Breast carcinomas (BC) are commonly classified into 4 subtypes based on hormone receptor expression: basal, luminal A, luminal B, and HER2 overexpressed. Comprehensive genomic profiling (CGP) reveals targetable genomic alterations (GA) across all four mutation classes, as well measuring tumor mutational burden (TMB), and can redefine BC classification into therapeutically relevant subtypes. Testing with immunohistochemistry or hotspot testing can miss a substantial number of targetable alterations and cannot measure TMB. Methods: DNA was extracted from 40 µm of FFPE sections for 8654 consecutive BCs. CGP was performed on hybridization-captured, adaptor ligation-based libraries (mean coverage &amp;gt;500X) for up to 315 cancer-related genes and select introns from up to 28 genes frequently rearranged in cancer. Sequences were analyzed for substitutions, small insertions/deletions, copy number changes, and rearrangements. TMB was determined by counting non-driver, non-germline alterations across 1.1 Mbp of sequenced DNA. Clinically relevant GA (CRGA) are GA linked to therapies on the market or under evaluation in clinical trials. Immunotherapy (IO) sensitivity is defined as TMB &amp;gt;20 mut/Mbp or mutation of specific DNA repair pathways. Results: The table below outlines 7 distinct functional or signal transduction pathways commonly altered in BC. Several are targetable with therapies that are FDA approved for an oncology indication. Mutations can also be found in other targetable kinases such as RET, ROS1, and RAF. 6959 (80.4%) tumors harbor a GA in at least one pathway, and 2697 (31.2%) BC harbor alterations in just one pathway (unique cases). Only 9.8% of BC would be HER2-positive by IHC. Almost 4% (352/8654) of cases harbor rearrangements or gene fusions that may not be detectable with other assays. Mutations in ESR1 characterize an eighth category of tumors with acquired resistance to endocrine therapy; 796/8654 (9%) samples harbor ESR1 alterations. Conclusions: CGP can identify CRGA and TMB that can stratify tumors by predicted sensitivity to a variety of therapies, including HER2- or mTOR-targeted therapies, immunotherapies, and other kinase inhibitors. 80% of BC harbor targetable GA, and 30% of samples harbor mutations in only one pathway. CGP can provide crucial information for identifying which of several treatment modalities is most appropriate for these 30% of patients. High levels of TMB and most GA would not be identified by IHC or hotspot testing, but can be detected by next-generation sequencing. CGP is a powerful tool for guiding treatment across therapeutically distinct, but targetable, pathways.  PI3K/AKT/mTOR pathwayFGFR pathwayCDK pathwayERBB pathwayHR deficientIO sensitiveOther kinasesTotal Cases43752650268512941266419424% Total Cases51%31%31%15%15%5%5%Unique Cases14422262312743094858% Unique Cases17%3%3%3%4%1%1%TherapiesEverolimus, TemsirolimusPazopanib, PonatinbPalbociclibTrastuzumab, Pertuzumab, Afatinib, Lapatinib, NeratinibOlaparibPembrolizumab, Nivolumab, Atezolizumab, IpilumumabSorafenib, Regorafenib, Dabrafenib, Vemurafenib, Crizotinib, Cabozantinib, Sunitinib Citation Format: Ross JS, Gay LM, Elvin JA, Suh J, Vergilio J-A, Ramkissoon S, Schrock A, Ali S, Miller VA, Stephens PJ. Comprehensive genomic profiling of 8,654 breast carcinoma reveals therapeutically targetable molecular subtypes beyond those defined by hormone-receptor expression [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-05-08.</jats:p

Abstract P5-21-20: Integrating comprehensive genomic profiling with treatment decisions – Experience gained while treating 139 advanced breast carcinomas

Abstract Background: Comprehensive Genomic Profiling (CGP) using next-generation sequencing (NGS) technology can provide insight into potentially clinically relevant genomic alterations (CRGA) within a patient's breast cancer. For example, HER2 amplification status and targetable short variants (SV), acquired ESR1 or BRCA1/2 resistance mutations, and the presence of targetable alterations in the PI3K kinase. We retrospectively reviewed CGP results and subsequent outcomes at one cancer center to illustrate the experience of using molecular subtyping to inform treatment decisions. Methods: DNA extracted from FFPE tumor tissue or blood samples obtained during routine clinical care for patients (n=139) with predominantly relapsed, refractory or metastatic breast cancer was analyzed by hybrid-capture, NGS for all classes of GA: 1. base substitutions, 2. insertion and deletions, 3. rearrangements, and 4. copy number changes. Treatment decisions based on comprehensive genomic profiles were captured retrospectively. Tumor mutational burden (TMB), scored as mutations (mut)/Mb, was calculated on 0.8-1.2 Mb of sequenced DNA. Alterations affecting the ERBB family included amplification of or oncogenic mutations in ERBB2 (HER2), ERBB3, and EGFR. Results: From Jan 2013 to May 2017, FFPE tissue samples for 136 patients with advanced breast cancer were analyzed by CGP and 3 additional patients had circulating tumor DNA analyzed for alterations; 11 patients received profiling on multiple biopsies. Tumors analyzed were carcinomas (Ca) NOS (n=84), invasive ductal Ca (n=46), invasive lobular Ca (n=7), a neuroendocrine Ca, and a phyllodes tumor. In total, 118/139 (84.9%) samples harbored CRGA in a targetable pathway: PI3K/MTOR (n=67; 48.2%), CDK cell-cycle (n=40; 28.8%), ERBB family (n=24; 17.3%), FGFR (n=24; 17.3%), ESR1 (n=16; 11.5%), homologous repair (HRD)( n=14; 10.1%), and RAS/RAF/MEK (n=11; 7.9%). Targetable alterations in other cancer-related kinases were found in 10 (7.2%) samples and 10 (7.2%) samples were TMB high (≥20 mut/Mb) or had CD274 (PD-L1) amplification. There were 3 patients (2.1%) with HER2 short variants detected in the absence of ERBB2 amplification; these patients may respond to HER2-targeted therapies but would be HER2-negative by IHC. Many samples had alterations in ≥1 pathway, and overlap is particularly high for the CDK and FGFR pathways (12 samples). Alterations in pathways targeted by MTOR inhibitors, HER2-targeted therapies, or the CDK inhibitors were found in 93/136 (66.9%) tumors. Evaluation of outcomes for these 139 patients is ongoing and will be presented. Conclusions: Genomic profiling of breast carcinomas, using either tissue or liquid biopsies, provides potentially actionable information to guide treatment decisions. Overall, 84.9% of patient samples harbored oncogenic alterations in a targetable pathway, with two-thirds of tumors having alterations in pathways targeted by therapies with FDA approval for breast cancer and 7.2% of patients having high levels of TMB or amplification of PD-L1, suggesting that checkpoint inhibitors may be relevant options. Citation Format: Mahtani R, Gay LM, Chung J, Hartmaier R, Sokol E, Elvin JA, Daniel S, Ramkissoon S, Severson E, Suh J, Vergilio J-A, Stephens PJ, Ross JS. Integrating comprehensive genomic profiling with treatment decisions – Experience gained while treating 139 advanced breast carcinomas [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-21-20.</jats:p

Abstract P1-05-07: Comprehensive genomic profiling of clinically malignant phyllodes tumors of the breast reveals frequent mutation of <i>NF1</i> and other genes associated with PI3K and RAS pathway activation

Abstract Background: Malignant or metastatic breast phyllodes tumors (MPT) are exceptionally rare, and the underlying genomic drivers are still being elucidated. Recent studies report frequent mutations in the RAS and PI3K pathways but have not commonly reported mutations in NF1. Comprehensive genomic profiling (CGP) can measure mutation load (TMB) and identifies all four classes of oncogenic alterations, including rearrangements and copy number loss that commonly affect tumor suppressors such as NF1, and can direct personalized treatment strategies. Methods: CGP using hybridization capture of 3,769 exons from up to 315 cancer-related genes and select introns of 28 genes commonly rearranged in cancer was applied to ≥50ng of DNA extracted from 21 consecutive MPT and sequenced to high, uniform median coverage (&amp;gt;400X). TMB was determined as mutations/Mb on 1.1 Mb of sequenced DNA. Results: The 21 MPT featured a median age of 51 yrs (range 14-70 yrs). CGP was performed on the primary MPT in 15 cases and on metastasis biopsies in 6 cases. TMB for all MPT was low (&amp;lt;10 mut/Mb), and all evaluable tumors (17/21) were microsatellite stable (MSS). The most commonly mutated genes were TP53 (57.1%), TERT (56.3%), NF1 (52.4%), MED12 (38.1%), CDKN2A/B (33.3%), and MLL2 (33.3%). 19/21 (90.5%) MPT harbored clinically relevant genomic alterations (CRGA) associated with therapies available on the market or under investigation in late stage clinical trials. Additional alterations in the PI3K/AKT/MTOR, RAS/RAF/MEK, and FGFR pathways were identified (see table); the PI3K/ATK/MTOR pathway was mutated in 10/21 (47.6%) of samples. Although CDKN2A/B loss was found in 6/11 tumors with NF1 mutation and only 1/10 NF1 wild-type samples, the co-occurrence was not significant (p&amp;lt;0.07). No significant correlation exists between the occurrence of NF1 mutations and mutation of MED12, TERT, the PI3K pathway, or other genes in the RAS/RAF pathway (NRAS, BRAF, EGFR). Targetable KIAA1549-BRAF or FGFR3-TACC3 fusions were identified in 2/21 (9.5%) tumors. Responses to targeted treatments will be presented. Conclusions: More than 90% of MPT feature CRGA, including alteration of NF1, which was by far the most common targetable GA in this study. 52.4% of MPT had alterations predicted to result in loss of NF1 activity. NF1 mutation does not significantly co-occur with mutations in any other gene or pathway commonly altered in MPT. Other tumors with underlying NF1 mutations have responded to the MEK inhibitor selumetinib, suggesting MEK inhibitors may be relevant for the treatment of MPT. Other targetable alterations, including known gene fusions, are common in MPT. Thus, MPT may benefit from combination targeted therapy, warranting further investigation in the clinical trial setting.   Total Mutation NumberPathwayTotal Cases (n=21)Short VariantsCopy NumberRearrangementsRAS/RAF/MEK    NF111 (52.4%)722BRAF3 (14.3%)301NRAS2 (9.5%)300PI3K/AKT/MTOR    PIK3CA4 (19%)310PTEN4 (19%)130STK112 (9.5%)110AKT11 (4.8%)100FBXW71 (4.8%)001TSC21 (4.8%)010PIK3R11 (4.8%)100FGFR    FGFR11 (4.8%)100FGFR31 (4.8%)001Other    EGFR2 (9.5%)020BRCA21 (4.8%)100PDGFRA1 (4.8%)010KIT1 (4.8%)010 Citation Format: Gay LM, Elvin JA, Vergilio J-A, Suh J, Ramkissoon S, Ali S, Schrock A, Hirshfield K, Ganesan S, Miller VA, Stephens PJ, Ross JS. Comprehensive genomic profiling of clinically malignant phyllodes tumors of the breast reveals frequent mutation of NF1 and other genes associated with PI3K and RAS pathway activation [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-05-07.</jats:p

Abstract P2-09-15: <i>NTRK</i> fusions in breast cancer: Clinical, pathologic and genomic findings

Abstract Background: The tropomysin receptor kinase A family includes the 3 NTRK1, NTRK2 and NTRK3 genes and plays major roles in neuronal development. The recent evidence of remarkable efficacy for kinase inhibitors (TKI) targeting NTRK across a wide variety of malignancies that harbor NTRK gene fusions has stimulated great interest in determining the type of cancers driven by these therapy defining NTRK genomic alterations. Methods: A consecutive series of 12,214 locally aggressive, relapsed and metastatic breast malignancies (mBM) were subjected to comprehensive genomic profiling (CGP) using DNA extracted from 40 µm of FFPE sections and adaptor ligation-based libraries to a mean coverage depth 719X for up to 315 cancer-related genes. The results were analyzed for all classes of genomic alterations (GA) including base substitutions, insertions and deletions, select fusions and rearrangements, and copy number changes. Tumor mutational burden (TMB) was determined on 1.1 Mbp of sequenced DNA. Microsatellite instability (MSI) status was calculated by a customized algorithm. Results: 16 (0.13%) mBM (all female) harbored NTRK gene fusions. The median age was 51 years (range 34 to 70 years). There were 9 ductal carcinomas, 2 lobular carcinomas, 3 secretory carcinomas 1 metaplastic carcinoma and 1 angiosarcoma. Tumor stages at the time of sequencing were 12 Stage IV, 1 Stage III and 3 stage I (all 3 secretory carcinomas). In 9 cases, clinical receptor status was known: 3 (33%) were ER+/HER2- and 6 (66%) ER-/HER2- (all TNBC) with all 9 (100%) of cases HER2-. All 3 SCA were TNBC. 10 NTRK fusions involved NTRK1 featuring a variety of fusion partners (CGN, GATAD2B, LMNA, MDM4, PEAR1, and TPM3) and 6 involved NTRK3 (all ETV6 fusions). There were no NTRK2 fusions. NTRK fusion+ mBM featured a mean of 4.25 GA per sample. The most frequent non-fusion partner co-altered genes in this series of NTRK fusion+ mBM were: TP53 at 25%, IKBKE, PIK3C2B CCND1 at 19%, and AKT, PIK3CA, MYC, CDH1, CDKN2A, PTEN, FGF3, FGF4 and FGF19 all at 13%. The median TMB for NTRK fusion+ mBM was 0.9 mutations/Mb and no cases (0%) had a TMB &amp;gt; 10 mutations/Mb and no cases (0%) feared high microsatellite instability (MSI high). Clinical response assessment to NTRK TKI therapies in this series is ongoing. Conclusions: NTRK gene fusions although extremely uncommon in breast malignancies occur across a variety of tumor types, is universally HER2 negative, more frequent in TNBC than in ER+ tumors, is associated with a moderate frequency of additional genomic alterations and a complete absence of either high TMB or high MSI. This study confirms that a CGP assay, when applied to a large cohort of near universal clinically advanced disease can identify extremely rare alterations that can lead a small number of patients to highly effective precision therapies. Citation Format: Ross JS, Chung J, Elvin JE, Vergilio J-A, Ramkissoon S, Suh J, Severson E, Daniel S, Frampton GM, Fabrizio D, Hartmaier RJ, Albacker LA, Ali SM, Schrock AB, Miller VA, Stephens PJ, Gay LM. NTRK fusions in breast cancer: Clinical, pathologic and genomic findings [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-09-15.</jats:p