scholarly journals Higher Mutation Burden in High Proliferation Compartments of Heterogeneous Melanoma Tumors

2021 ◽  
Vol 22 (8) ◽  
pp. 3886
Author(s):  
Tomasz M. Grzywa ◽  
Agnieszka A. Koppolu ◽  
Wiktor Paskal ◽  
Klaudia Klicka ◽  
Małgorzata Rydzanicz ◽  
...  
Keyword(s):  
Single Nucleotide Variants ◽  
Primary Cutaneous Melanoma ◽  
Ki 67 ◽  
Formalin Fixed Paraffin ◽  
Mutation Burden ◽  
Formalin Fixed Paraffin Embedded ◽  
Cause Of Failure ◽  
Tumor Types ◽  
Next Generation Sequencing Ngs

Melanoma tumors are the most heterogeneous of all tumor types. Tumor heterogeneity results in difficulties in diagnosis and is a frequent cause of failure in treatment. Novel techniques enable accurate examination of the tumor cells, considering their heterogeneity. The study aimed to determine the somatic variations among high and low proliferating compartments of melanoma tumors. In this study, 12 archival formalin-fixed paraffin-embedded samples of previously untreated primary cutaneous melanoma were stained with Ki-67 antibody. High and low proliferating compartments from four melanoma tumors were dissected using laser-capture microdissection. DNA was isolated and analyzed quantitatively and qualitatively. Libraries for amplicon-based next-generation sequencing (NGS) were prepared using NEBNext Direct Cancer HotSpot Panel. NGS detected 206 variants in 42 genes in melanoma samples. Most of them were located within exons (135, 66%) and were predominantly non-synonymous single nucleotide variants (99, 73.3%). The analysis showed significant differences in mutational profiles between high and low proliferation compartments of melanoma tumors. Moreover, a significantly higher percentage of variants were detected only in high proliferation compartments (39%) compared to low proliferation regions (16%, p < 0.05). Our results suggest a significant functional role of genetic heterogeneity in melanoma.

scholarly journals Tumor mutation burden testing: a survey of the International Quality Network for Pathology (IQN Path)

2021 ◽  
Author(s):  
Francesca Fenizia ◽  
Nicola Wolstenholme ◽  
Jennifer A. Fairley ◽  
Etienne Rouleau ◽  
Melanie H. Cheetham ◽  
...  
Keyword(s):  
Current Practice ◽  
Checkpoint Inhibitors ◽  
Testing Methods ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Tumor Mutation Burden ◽  
Formalin Fixed Paraffin ◽  
Mutation Burden ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

AbstractWhile tumour mutation burden (TMB) is emerging as a possible biomarker for immune-checkpoint inhibitors (ICI), methods for testing have not been standardised as yet. In April 2019, the International Quality Network for Pathology (IQN Path) launched a survey to assess the current practice of TMB testing. Of the 127 laboratories that replied, 69 (54.3%) had already introduced TMB analysis for research purposes and/or clinical applications. Fifty laboratories (72.5%) used targeted sequencing, although a number of different panels were employed. Most laboratories tested formalin-fixed paraffin-embedded material (94.2%), while 18/69 (26%) tested also cell-free DNA. Fifty-five laboratories used both single nucleotide variants and indels for TMB calculation; 20 centers included only non-synonymous variants. In conclusion, the data from this survey indicate that multiple global laboratories were capable of rapidly introducing routine clinical TMB testing. However, the variability of testing methods raises concerns about the reproducibility of results among centers.

scholarly journals Comprehensive NGS Panel Validation for the Identification of Actionable Alterations in Adult Solid Tumors

2021 ◽  
Vol 11 (5) ◽  
pp. 360
Author(s):  
Paula Martínez-Fernández ◽  
Patricia Pose ◽  
Raquel Dolz-Gaitón ◽  
Arantxa García ◽  
Inmaculada Trigo-Sánchez ◽  
...  
Keyword(s):  
Solid Tumors ◽  
Limit Of Detection ◽  
Copy Number Variants ◽  
Hybridization Capture ◽  
Formalin Fixed Paraffin ◽  
Ffpe Tumor ◽  
Formalin Fixed Paraffin Embedded ◽  
Tumor Types ◽  
Next Generation Sequencing Ngs ◽  
Formalin Fixed

The increasing identification of driver oncogenic alterations and progress of targeted therapies addresses the need of comprehensive alternatives to standard molecular methods. The translation into clinical practice of next-generation sequencing (NGS) panels is actually challenged by the compliance of high quality standards for clinical accreditation. Herein, we present the analytical and clinical feasibility study of a hybridization capture-based NGS panel (Action OncoKitDx) for the analysis of somatic mutations, copy number variants (CNVs), fusions, pharmacogenetic SNPs and Microsatellite Instability (MSI) determination in formalin-fixed paraffin-embedded (FFPE) tumor samples. A total of 64 samples were submitted to extensive analytical validation for the identification of previously known variants. An additional set of 166 tumor and patient-matched normal samples were sequenced to assess the clinical utility of the assay across different tumor types. The panel demonstrated good specificity, sensitivity, reproducibility, and repeatability for the identification of all biomarkers analyzed and the 5% limit of detection set was validated. Among the clinical cohorts, the assay revealed pathogenic genomic alterations in 97% of patient cases, and in 82.7%, at least one clinically relevant variant was detected. The validation of accuracy and robustness of this assay supports the Action OncoKitDx’s utility in adult solid tumors.

scholarly journals Plasma DNA-Based Molecular Diagnosis, Prognostication, and Monitoring of Patients With EWSR1 Fusion-Positive Sarcomas

2017 ◽  
pp. 1-11 ◽  
Author(s):  
Neerav N. Shukla ◽  
Juber A. Patel ◽  
Heather Magnan ◽  
Ahmet Zehir ◽  
Daoqi You ◽  
...  
Keyword(s):  
Integrated Approach ◽  
Patient Specific ◽  
Plasma Dna ◽  
Hybridization Capture ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Clinically Significant ◽  
Tumor Types ◽  
Next Generation Sequencing Ngs ◽  
Digital Polymerase Chain Reaction

Purpose Ewing sarcoma (ES) and desmoplastic small round cell tumors (DSRCTs) are aggressive sarcomas molecularly characterized by EWSR1 gene fusions. As pathognomonic genomic events in these respective tumor types, EWSR1 fusions represent robust potential biomarkers for disease monitoring. Methods To investigate the feasibility of identifying EWSR1 fusions in plasma-derived cell-free DNA (cfDNA) from patients with ES and DSRCT, we evaluated two complementary approaches in samples from 17 patients with radiographic evidence of disease. The first approach involved identification of patient-specific genomic EWSR1 fusion breakpoints in formalin-fixed, paraffin-embedded tumor DNA using a broad, hybridization capture-based next-generation sequencing (NGS) panel, followed by design of patient-specific droplet digital polymerase chain reaction (ddPCR) assays for plasma cfDNA interrogation. The second approach used a disease-tailored targeted hybridization capture-based NGS panel applied directly to cfDNA, which included EWSR1 as well as several other genes with potential prognostic use. Results EWSR1 fusions were identified in 11 of 11 (100%) ES and five of six (83%) DSRCT cfDNA samples by ddPCR, whereas 10 of 11 (91%) and four of six (67%) were identified by NGS. The ddPCR approach had higher sensitivity, ranging between 0.009% and 0.018%. However, the hybrid capture–based NGS assay identified the precise fusion breakpoints in the majority of cfDNA samples, as well as mutations in TP53 and STAG2, two other recurrent, clinically significant alterations in ES, all without prior knowledge of the tumor genotype. Conclusion These results provide a compelling rationale for an integrated approach using both NGS and ddPCR for plasma cfDNA-based biomarker evaluations in prospective cooperative group studies.

An analysis of ERBB2 alterations (amplifications and mutations) found by next-generation sequencing (NGS) in 2000+ consecutive solid tumor (ST) patients.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 11000-11000
Author(s):  
Gary A. Palmer ◽  
Kai Wang ◽  
Vincent A. Miller ◽  
Roman Yelensky ◽  
Phil Stephens ◽  
...  
Keyword(s):  
Solid Tumor ◽  
Treatment Options ◽  
Genomic Changes ◽  
Activating Mutations ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
High Level ◽  
Tumor Types ◽  
Next Generation Sequencing Ngs ◽  
Erbb2 Amplification

11000 Background: Testing for ERBB2 amplification by FISH and IHC is routine in breast and gastro-esophageal cancer. There are 3 approved and multiple targeted therapies in clinical trials that rely on the results of these tests. ERBB2 amplification and activation by mutation/fusion has also been described in a wide variety of other ST. As these alterations are not routinely tested for but may predict response to anti-ERBB2 agents we sought to determine their frequency in an unselected cohort of specimens from advanced ST patients. Methods: We reviewed genomic profiles from the first 2,223 formalin-fixed, paraffin-embedded specimens received and analyzed by our CLIA-certified lab (Foundation Medicine) with our NGS platform. 3,230 exons in 182 cancer-related genes and 14 genes frequently rearranged were assayed for base pair substitutions, small insertions/deletions (indels), amplifications, and rearrangements. Results: 110/2,223 (4.9%) specimens had 116 ERBB2 alterations: 67 (58%) amplifications, 29 (25%) substitutions, 16 (14%) indels, 2 (2%) splice site variants and 2 (2%) translocations, including a potential fusion. Six samples (5%) had multiple alterations, and two had both ERBB2 substitution and amplification. 14 ST types had evidence of ERBB2 alterations including 29% of esophageal, 20% of uterine, 14% of breast, and 12% of stomach carcinomas. 6% of all lung cancer samples had ERBB2 alterations. Amplifications predominated, but lung specimens had predominantly indels. Durable responses exist to anti-ERBB2 agents in STs with activating ERBB2 mutations. Conclusions: Use of a broad NGS panel identifies an unprecedented number of actionable genomic changes including a significant rate of ERBB2 alterations across 14 different solid tumor types. The discovery of unanticipated ERBB2 amplifications and activating mutations in a wide variety of ST highlights the need to study a broad range of genes at a high level of sensitivity and specificity when searching for novel targets of therapy. Widespread use of this approach could provide more treatment options and enable more rapid accrual to ongoing and planned trials of agents targeting pathways under study.

scholarly journals TERT promoter mutations in penile squamous cell carcinoma: high frequency in non-HPV-related type and association with favorable clinicopathologic features

2021 ◽  
Vol 147 (4) ◽  
pp. 1125-1135
Author(s):  
Sang Kyum Kim ◽  
Jang-Hee Kim ◽  
Jae Ho Han ◽  
Nam Hoon Cho ◽  
Se Joong Kim ◽  
...  
Keyword(s):  
Squamous Cell ◽  
Malignant Neoplasm ◽  
Clinicopathologic Features ◽  
Ki 67 ◽  
Tissue Samples ◽  
Formalin Fixed Paraffin ◽  
Tert Promoter Mutations ◽  
Formalin Fixed Paraffin Embedded ◽  
Penile Squamous Cell Carcinoma ◽  
Promoter Mutations

Abstract Purpose Penile carcinoma is a rare malignant neoplasm with a largely unknown molecular pathogenesis. Telomerase reverse transcriptase promoter (TERT-p) mutations have been detected in several types of human malignancies. The aim of this study was to investigate the presence of TERT-p mutations in penile squamous cell carcinomas (SCCs) and their associations with clinicopathologic features. Methods In this retrospective study, Sanger sequencing was performed to detect TERT-p mutations in formalin-fixed paraffin-embedded tissue samples from 37 patients with penile SCC, 16 patients with cutaneous SCC, and 4 patients with non-neoplastic penile/skin tissue. The expression of p16INK4a and Ki-67 was investigated via immunohistochemistry. Associations of TERT-p mutation with clinicopathological factors, immunohistochemical results, and clinical outcome were statistically analyzed. Results Recurrent TERT-p mutations were identified in 18 out of 37 (48.6%) penile SCCs, including all 3 carcinoma in situ cases. TERT-p mutations were significantly more frequent in non-human papilloma virus (HPV)-related penile SCC types than in non-HPV-related penile SCC based on both histologic classification and p16INK4a immunoreactivity. Furthermore, TERT-p mutation was associated with a low histologic grade, low mitotic count, absence of necrosis, low Ki-67/MIB-1 labeling index, and absence of lymph node or distant metastasis. Conclusion Our study shows TERT-p mutations are the most frequent somatic mutations in penile SCC. In addition, TERT-p mutations are far more frequent in non-HPV-related penile SCC than in HPV-related penile SCC, indicating TERT-p mutations may have a role in tumorigenesis distinct from HPV-related penile SCC.

scholarly journals Higher EU-TIRADS-Score Correlated with BRAF V600E Positivity in the Early Stage of Papillary Thyroid Carcinoma

2021 ◽  
Vol 10 (11) ◽  
pp. 2304
Author(s):  
Karolina Skubisz ◽  
Joanna Januszkiewicz-Caulier ◽  
Patrycja Cybula ◽  
Elwira Bakuła-Zalewska ◽  
Krzysztof Goryca ◽  
...  
Keyword(s):  
Early Stage ◽  
Braf V600e ◽  
P Value ◽  
Dna And Rna ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Ion Proton ◽  
Indolent Disease ◽  
Next Generation Sequencing Ngs ◽  
Formalin Fixed

The data demonstrating a correlation between sonographic markers of malignancy of thyroid cancer (TC) and its genetic status are scarce. This study aimed to assess whether the addition of genetic analysis at the preoperative step of TC patients’ stratification could aid their clinical management. The material consisted of formalin-fixed paraffin-embedded tumor fragments of 49 patients who underwent thyroidectomy during the early stages of papillary TC (PTC). Tumor DNA and RNA were subjected to next-generation sequencing (NGS) on Ion Proton using the Oncomine™ Comprehensive Assay panel. We observed a significant correlation between BRAF V600E and a higher EU-TIRADS score (p-value = 0.02) with a correlation between hypoechogenicity and taller-than-wide tumor shape in analysed patients. There were no other significant associations between the identified genetic variants and other clinicopathological features. For TC patient’s stratification, a strong suspicion of BRAF V600E negativity in preoperative management of TC patients could limit the over-treatment of asymptomatic, very low-risk, indolent disease and leave room for active surveillance.

Expression of proliferation genes in formalin-fixed paraffin-embedded (FFPE) tissue from breast carcinomas. Feasibility and relevance for a routine histopathology laboratory

2016 ◽  
Vol 70 (1) ◽  
pp. 25-32 ◽  
Author(s):  
Carla Thomas ◽  
Cleo Robinson ◽  
Ben Dessauvagie ◽  
Benjamin Wood ◽  
Greg Sterrett ◽  
...  
Keyword(s):  
Gene Expression ◽  
Breast Carcinoma ◽  
Expression Profiling ◽  
Proliferative Activity ◽  
Breast Cancers ◽  
Breast Carcinomas ◽  
Ki 67 ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

AimBreast carcinoma proliferative activity, histological grade and commercial molecular tests are all important in prognostication and treatment. There is a particular need for improved, standardised techniques for subclassification of grade 2 breast cancers into low-risk and high-risk prognostic groups. In this study we investigated whether gene expression profiling of five proliferation genes was feasible using breast cancer tissue in a clinical setting and whether these profiles could enhance pathological assessment.MethodsExpression of five proliferation gene mRNAs; Ki-67, STK 15, CCNB1, CCND1 and MYBL2, was quantified in 27 breast carcinomas and compared with Ki-67 proliferation index (PI) and Nottingham mitotic score.ResultsExpression of Ki-67, STK15 and MYBL2 mRNA showed moderate Spearman's correlation with Ki-67 PI (p<0.01), but CCND1 and CCNB1 showed weak, non-significant correlation. Individual gene expression did not associate with mitotic score but combined mRNA expression correlated with both Ki-67 PI (p=0.018) and mitotic score (p=0.03; 0.007).ConclusionsThis study confirms mRNA analysis in breast carcinoma formalin-fixed, paraffin-embedded samples is feasible and suggests gene expression profiling, using a small set of five proliferation genes, has potential in aiding histological grading or assessment of proliferative activity of breast cancers. To fully evaluate the clinical applicability of this approach, a larger cohort study with long-term follow-up data is required.

Antitumor immune response is associated with favorable survival in GEP-NEN G3

2021 ◽  
Vol 28 (10) ◽  
pp. 683-693
Author(s):  
Vivian Rosery ◽  
Henning Reis ◽  
Konstantinos Savvatakis ◽  
Bernd Kowall ◽  
Martin Stuschke ◽  
...  
Keyword(s):  
Immune Response ◽  
Immune Cell ◽  
Spatial Interaction ◽  
Antitumor Immune Response ◽  
Cytotoxic T Cell ◽  
Neuroendocrine Neoplasms ◽  
Ki 67 ◽  
Tissue Samples ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded

The tumor immune microenvironment (TME) represents a key determinant for responses to cancer treatment. However, the immune phenotype of highly proliferative gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN) is still largely elusive. In this retrospective study, we characterized the TME of high-grade (G3, Ki-67 > 20%) GEP-NEN. We analyzed formalin-fixed paraffin-embedded samples from 37 patients with GEP-NEN G3 by immunohistochemistry and multiplex immunofluorescence to address the abundance and spatial interaction of relevant immune subsets. We focused on the expression of immune checkpoint molecules PD-1 and PD-L1, the cytotoxic T-cell marker CD8, and the tumor-associated macrophage marker CD206. Findings were correlated with overall survival (OS) from the date of a cancer diagnosis. Patients with PD-L1-positive tumors (CPS ≥ 1) and intense PD-1+CD8+ immune cell infiltration showed the most favorable median OS. Multiplex immunofluorescence staining of ten representative tissue samples illustrated intratumoral heterogeneity of PD-L1 expression. Dense PD-1+CD8+ immune cell infiltrates were observed in PD-L1-positive tumor regions but not in PD-L1-negative regions. Proximity analysis revealed a spatial interaction between PD-1+CD8+ cells and PD-L1-positive cells. Our data suggest a pre-existing antitumor immune response in the TME in a subgroup of GEP-NEN G3. This supports a targeted clinical exploration of immunotherapeutic approaches.

scholarly journals Improved next-generation sequencing pre-capture library yields and sequencing parameters using on-bead PCR

BioTechniques ◽  
2020 ◽  
Vol 68 (1) ◽  
pp. 48-51 ◽  
Author(s):  
Christopher R McEvoy ◽  
Timothy Semple ◽  
Bhargavi Yellapu ◽  
David Y Choong ◽  
Huiling Xu ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Variant Calling ◽  
Limiting Factor ◽  
Next Generation ◽  
Tumor Biopsy ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Next Generation Sequencing Ngs ◽  
Tumor Dna ◽  
Generation Sequencing

Tumor DNA sequencing results can have important clinical implications. However, its use is often limited by low DNA input, owing to small tumor biopsy size. To help overcome this limitation we have developed a simple improvement to a commonly used next-generation sequencing (NGS) capture-based library preparation method using formalin-fixed paraffin-embedded-derived tumor DNA. By using on-bead PCR for pre-capture library generation we show that library yields are dramatically increased, resulting in decreased sample failure rates. Improved yields allowed for a reduction in PCR cycles, which translated into improved sequencing parameters without affecting variant calling. This methodology should be applicable to any NGS system in which input DNA is a limiting factor.

SOX2 Expression in Canine Neoplasia

2020 ◽  
pp. 030098582096013
Author(s):  
Ileana C. Miranda ◽  
Andrew D. Miller
Keyword(s):  
Rational Design ◽  
Prognostic Indicator ◽  
Screening Methods ◽  
Tumor Type ◽  
Sox2 Expression ◽  
Formalin Fixed Paraffin ◽  
Normal Expression ◽  
Formalin Fixed Paraffin Embedded ◽  
Stem Cell Pluripotency ◽  
Tumor Types

SOX2 is a major transcriptional regulator of stem cell pluripotency and self-renewability. Its expression in cancer stem cells from several different tumor types in humans and rodent models directly implicates SOX2 in tumorigenicity, metastasis, drug resistance, recurrence, and poor survival. Our objective was to investigate the expression of SOX2 in canine neoplasia. Immunohistochemistry for SOX2 was performed in sets of 10 archived formalin-fixed paraffin-embedded tissues from 45 distinct canine neoplasms. Normal expression of SOX2 was evaluated in a canine tissue microarray. Strong and diffuse SOX2 intranuclear immunolabeling was consistently found in the majority of ectodermal (13/15) and endodermal tumors (5/7). Negative, variable, or inconsistent SOX2 intranuclear immunolabeling was detected in the majority of mesodermal tumors (10/16) and in tumors with dual or uncertain origin (5/7). Although further studies are necessary to understand mechanistically how SOX2 contributes to the biology of each tumor type, this study demonstrates the expression of SOX2 in a wide variety of canine cancers. In the future, screening methods based on cellular plasticity and pluripotency biomarkers may provide avenues for the rational design of therapeutic strategies that target vulnerable signals upstream or downstream of SOX2 in different cancers, and possibly offer novel clinical applications for SOX2 as a prognostic indicator.

Sign in / Sign up

Export Citation Format

Share Document