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 Intrahepatic Cholangiocarcinoma: Genomic Heterogeneity Between Eastern and Western Patients

2020 ◽  
pp. 557-569 ◽  
Author(s):  
Jingyu Cao ◽  
Jing Hu ◽  
Siqin Liu ◽  
Funda Meric-Bernstam ◽  
Reham Abdel-Wahab ◽  
...  
Keyword(s):  
Dna Repair ◽  
Intrahepatic Cholangiocarcinoma ◽  
Checkpoint Inhibitors ◽  
Asian Population ◽  
Asian Patients ◽  
Formalin Fixed Paraffin ◽  
Mutation Burden ◽  
Formalin Fixed Paraffin Embedded ◽  
Comprehensive Genomic Profiling ◽  
Ngs Data

PURPOSE Intrahepatic cholangiocarcinoma (IHCCA), a global health problem, is increasing in incidence and has differing etiologies worldwide. Next-generation sequencing (NGS) is rapidly being incorporated into the clinical management of biliary cancers. IHCCA is enriched with actionable mutations, and there are several promising targeted therapies under development. NGS data from Asia, where IHCCA is most prevalent, are limited. METHODS Comprehensive genomic profiling of formalin-fixed paraffin-embedded tumor tissue from 164 Asian and 283 Western patients with IHCCA was performed using NGS. We measured the distribution of DNA repair genetic aberrations (GAs) in IHCCA, along with actionable mutations. Also, we evaluated the association between DNA repair GAs and tumor mutation burden (TMB). Based on the TMB status, patients were distinguished into 3 levels: low (< 6 mut/Mb), intermediate (6-10 mut/Mb), and high (TMB-H; ≥ 10 mut/Mb). RESULTS Seventy-two percent of Asian patients had ≥ 1 actionable GA, with a significantly higher frequency in KMT2C , BRCA1/2, and DDR2 compared with Western patients ( P = .02, .003, and .003, respectively); 60.9% of Western patients had ≥ 1 actionable GA and higher frequency of CDKN2A/B and IDH1/2 GAs ( P = .0004 and < .001, respectively). GAs in nuclear factor kappa B pathway regulators and DNA repair genes occurred more frequently in Asian patients ( P = .006 and .001, respectively). There was a higher frequency of TMB-H in Asian compared with the Western cohort (12.2% v 5.9%; P = .07). CONCLUSION A higher burden of DNA repair mutations and frequency of patients with TMB-H in the Asian IHCCA cohort compared with the Western patients suggests a potential role for DNA repair and immune checkpoint inhibitors in the Asian population. Future clinical trials should account for this genetic heterogeneity.

scholarly journals Automated Extraction of DNA and RNA from a Single Formalin-Fixed Paraffin-Embedded Tissue Section for Analysis of Both Single-Nucleotide Polymorphisms and mRNA Expression

2010 ◽  
Vol 56 (12) ◽  
pp. 1845-1853 ◽  
Author(s):  
Guido Hennig ◽  
Mathias Gehrmann ◽  
Udo Stropp ◽  
Hiltrud Brauch ◽  
Peter Fritz ◽  
...  
Keyword(s):  
Extraction Method ◽  
Nucleotide Polymorphisms ◽  
Automated Extraction ◽  
Single Nucleotide ◽  
Normal Tissues ◽  
Dna And Rna ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Rna And Dna ◽  
Formalin Fixed

BACKGROUND There is an increasing need for the identification of both DNA and RNA biomarkers from pathodiagnostic formalin-fixed paraffin-embedded (FFPE) tissue samples for the exploration of individualized therapy strategies in cancer. We investigated a fully automated, xylene-free nucleic acid extraction method for the simultaneous analysis of RNA and DNA biomarkers related to breast cancer. METHODS We copurified both RNA and DNA from a single 10-μm section of 210 paired samples of FFPE tumor and adjacent normal tissues (1–25 years of archival time) using a fully automated extraction method. Half of the eluate was DNase I digested for mRNA expression analysis performed by using reverse-transcription quantitative PCR for the genes estrogen receptor 1 (ESR1), progesterone receptor (PGR), v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian) (ERBB2), epoxide hydrolase 1 (EPHX1), baculoviral IAP repeat-containing 5 (BIRC5), matrix metallopeptidase 7 (MMP7), vascular endothelial growth factor A (VEGFA), and topoisomerase (DNA) II alpha 170kDa (TOP2A). The remaining undigested aliquot was used for the analysis of 7 single-nucleotide polymorphisms (SNPs) by MALDI-TOF mass spectrometry. RESULTS In 208 of 210 samples (99.0%) the protocol yielded robust quantification-cycle values for both RNA and DNA normalization. Expression of the 8 breast cancer genes was detected in 81%–100% of tumor tissues and 21%–100% of normal tissues. The 7 SNPs were successfully genotyped in 91%–97% of tumor and 94%–97% of normal tissues. Allele concordance between tumor and normal tissue was 98.9%–99.5%. CONCLUSIONS This fully automated process allowed an efficient simultaneous extraction of both RNA and DNA from a single FFPE section and subsequent dual analysis of selected genes. High gene expression and genotyping detection rates demonstrate the feasibility of molecular profiling from limited archival patient samples.

scholarly journals KIT Somatic Mutations and Immunohistochemical Expression in Canine Oral Melanoma

Animals ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 2370
Author(s):  
Ginevra Brocca ◽  
Beatrice Poncina ◽  
Alessandro Sammarco ◽  
Laura Cavicchioli ◽  
Massimo Castagnaro
Keyword(s):  
Pcr Amplification ◽  
Mean Value ◽  
Single Nucleotide ◽  
Oral Melanoma ◽  
Pathway Activation ◽  
Formalin Fixed Paraffin ◽  
Frequent Event ◽  
Formalin Fixed Paraffin Embedded ◽  
Exon 11 ◽  
Formalin Fixed

Canine oral melanoma (COM) is an aggressive neoplasm with a low response to therapies, sharing similarities with human mucosal melanomas. In the latter, significant alterations of the proto-oncogene KIT have been shown, while in COMs only its exon 11 has been adequately investigated. In this study, 14 formalin-fixed, paraffin-embedded COMs were selected considering the following inclusion criteria: unequivocal diagnosis, presence of healthy tissue, and a known amplification status of the gene KIT (seven samples affected and seven non-affected by amplification). The DNA was extracted and KIT target exons 13, 17, and 18 were amplified by PCR and sequenced. Immunohistochemistry (IHC) for KIT and Ki67 was performed, and a quantitative index was calculated for each protein. PCR amplification and sequencing was successful in 97.62% of cases, and no single nucleotide polymorphism (SNP) was detected in any of the exons examined, similarly to exon 11 in other studies. The immunolabeling of KIT was positive in 84.6% of the samples with a mean value of 3.1 cells in positive cases, yet there was no correlation with aberration status. Our findings confirm the hypothesis that SNPs are not a frequent event in KIT activation in COMs, with the pathway activation relying mainly on amplification.

scholarly journals Co-stimulatory and co-inhibitory immune markers in solid tumors with MET alterations

2021 ◽  
Vol 7 (2) ◽  
pp. FSO662
Author(s):  
Karen L Reckamp ◽  
Jasmine A McQuerry ◽  
Isa Mambetsariev ◽  
Rebecca Pharaon ◽  
Susan E Yost ◽  
...  
Keyword(s):  
Solid Tumors ◽  
Checkpoint Inhibitors ◽  
Splice Site Mutation ◽  
Immune Markers ◽  
Site Mutation ◽  
Met Amplification ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Immune Profiling ◽  
Formalin Fixed

The implication of MET alterations in solid tumors and the immune microenvironment remains elusive. Formalin-fixed, paraffin-embedded samples of 21 patients with solid tumors harboring MET alterations were used for immunohistochemical staining. Extracted RNA was analyzed with the NanoString nCounter human PanCancer immune profiling panel (NanoString Technologies, Inc., WA, USA). Patients were diagnosed with lung (n = 10), breast (n = 5), genitourinary (n = 3) or colorectal cancer (n = 3). Eleven had a MET missense mutation, four had an exon 14 splice site mutation and six had MET amplification. CD6, CCL19, CD40LG, XCR1, MAGEA1, ATM and CCL19 genes were significantly differentially expressed in MET-altered cancers. MET alterations may have a role in various solid tumors as potential therapeutic targets and combination therapy candidates with immune checkpoint inhibitors.

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.

Sign in / Sign up

Export Citation Format

Share Document