Whole mitochondrial genome sequencing highlights mitochondrial impact in gastric cancer

Abstract Mitochondria are organelles that perform major roles in cellular operation. Thus, alterations in mitochondrial genome (mtGenome) may lead to mitochondrial dysfunction and cellular deregulation, influencing carcinogenesis. Gastric cancer (GC) is one of the most incident and mortal types of cancer in Brazil, particularly in the Amazon region. Here, we sequenced and compared the whole mtGenome extracted from FFPE tissue samples of GC patients (tumor and internal control – IC) and cancer-free individuals (external control – EC) from this region. We found 3-fold more variants and up to 9-fold more heteroplasmic regions in tumor when compared to paired IC samples. Moreover, tumor presented more heteroplasmic variants when compared to EC, while IC and EC showed no significant difference when compared to each other. Tumor also presented substantially more variants in the following regions: MT-RNR1, MT-ND5, MT-ND4, MT-ND2, MT-DLOOP1 and MT-CO1. In addition, our haplogroup results indicate an association of Native American ancestry (particularly haplogroup C) to gastric cancer development. To the best of our knowledge, this is the first study to sequence the whole mtGenome from FFPE samples and to apply mtGenome analysis in association to GC in Brazil.

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High sensitivity sanger sequencing detection of BRAF mutations in metastatic melanoma FFPE tissue specimens

Scientific Reports ◽

10.1038/s41598-021-88391-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Lauren Y. Cheng ◽

Lauren E. Haydu ◽

Ping Song ◽

Jianyi Nie ◽

Michael T. Tetzlaff ◽

...

Keyword(s):

Sanger Sequencing ◽

Limit Of Detection ◽

False Negative ◽

High Sensitivity ◽

Ffpe Tissue ◽

Braf Mutations ◽

Tissue Samples ◽

Ffpe Samples ◽

Droplet Pcr ◽

Tissue Specimens

AbstractMutations in the BRAF gene at or near the p. V600 locus are informative for therapy selection, but current methods for analyzing FFPE tissue DNA generally have a limit of detection of 5% variant allele frequency (VAF), or are limited to the single variant (V600E). These can result in false negatives for samples with low VAFs due to low tumor content or subclonal heterogeneity, or harbor non-V600 mutations. Here, we show that Sanger sequencing using the NuProbe VarTrace BRAF assay, based on the Blocker Displacement Amplification (BDA) technology, is capable of detecting BRAF V600 mutations down to 0.20% VAF from FFPE lymph node tissue samples. Comparison experiments on adjacent tissue sections using BDA Sanger, immunohistochemistry (IHC), digital droplet PCR (ddPCR), and NGS showed 100% concordance among all 4 methods for samples with BRAF mutations at ≥ 1% VAF, though ddPCR did not distinguish the V600K mutation from the V600E mutation. BDA Sanger, ddPCR, and NGS (with orthogonal confirmation) were also pairwise concordant for lower VAF mutations down to 0.26% VAF, but IHC produced a false negative. Thus, we have shown that Sanger sequencing can be effective for rapid detection and quantitation of multiple low VAF BRAF mutations from FFPE samples. BDA Sanger method also enabled detection and quantitation of less frequent, potentially actionable non-V600 mutations as demonstrated by synthetic samples.

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CDK5RAP3 as a Novel Biomarker Signature Predicting Survival and Adjuvant Chemotherapeutic benefit in Gastric Cancer

10.21203/rs.3.rs-130268/v1 ◽

2020 ◽

Author(s):

Jia-Bin Wang ◽

You-Xin Gao ◽

Ning-Zi Lian ◽

Yu-Bin Ma ◽

Ping Li ◽

...

Keyword(s):

Gastric Cancer ◽

Adjuvant Chemotherapy ◽

Cancer Patients ◽

Cox Model ◽

External Validation ◽

Tumour Tissue ◽

Tissue Samples ◽

Significant Difference ◽

Validation Set ◽

Gastric Cancer Patients

Abstract Background: We previously demonstrated that CDK5RAP3 acts as a tumour suppressor in gastric cancer through negative regulation of the Wnt/β-catenin signalling pathway, but its function in chemotherapeutic responsiveness of gastric cancer has not been investigated. In this study, we aimed to examine the clinical significance of CDK5RAP3 to predict chemotherapeutic responsiveness in gastric cancer.Methods: A collection of 188 pairs of tumour tissue microarray specimens from Fujian Medical University were employed for the discovery set, and 310 tumour tissue samples of gastric cancer patients were employed for the internal validation set. Eight-five tumour tissue samples from Qinghai University Hospital were used as the external validation set 1. Transcriptomic and clinical data of 299 gastric cancer patients from TCGA were used as the external validation set 2. CDK5RAP3 expression, microsatellite instability (MSI) status, and tumour-infiltrating lymphocytes (TIL) were examined with immunohistochemistry. Clinical outcomes of patients were compared with Kaplan-Meier curves and the Cox model.Results: In a multi-centre evaluation, increased CDK5RAP3 indication of better prognosis depends mainly on MSI-L/MSS status or TILhigh. High CDK5RAP3 expression predicts sensitive therapeutic responsiveness to postoperative adjuvant chemotherapy in gastric cancer. In a stratification analysis based on CDK5RAP3 combined with TIL or MSI status, patients with CKD5RAP3low TILlow showed no significant difference in prognosis after receiving chemotherapy, whereas patients with CKD5RAP3low TILhigh, CKD5RAP3high TILlow, and CKD5RAP3high TILhigh had better responsiveness to chemotherapy. In addition, patients with CKD5RAP3high MSI-L/MSS status benefitted the most from adjuvant chemotherapy among all patients evaluated. Conclusions: CKD5RAP3 can be used as an effective marker to evaluate individualized chemotherapy regimens in gastric cancer patients dependent on their TIL and MSI status.

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Genotoxic risk in health-care professionals occupationally exposed to low doses of ionizing radiation

Toxicology and Industrial Health ◽

10.1177/0748233720932081 ◽

2020 ◽

Vol 36 (5) ◽

pp. 356-370 ◽

Cited By ~ 2

Author(s):

Flávio Manoel Rodrigues da Silva-Júnior ◽

Ronan Adler Tavella ◽

Caroline Lopes Feijo Fernandes ◽

Alexandra Silveira Mortola ◽

Gianni Goulart Peraza ◽

...

Keyword(s):

Occupational Exposure ◽

Ionizing Radiation ◽

Internal Control ◽

Health Care Professionals ◽

Skin Color ◽

Lifestyle Factors ◽

Exposed Group ◽

External Control ◽

X Rays ◽

Significant Difference

The purpose of this study was to evaluate the potential influence of occupational ionizing radiation (IR) exposure on health professionals, assessing DNA damage using the comet and micronucleus (MN) assays and analyzing relative risks, correlations, and associated factors between outcomes and socioeconomic and lifestyle factors. Blood and buccal samples were collected from 36 workers, who actively participated in an imaging sector of a hospital, who were either exposed to IR directly or indirectly (9 internal control and 27 exposed), and 27 individuals living in the same city but with no occupational exposure (external control, unexposed/healthy). All radiation dosages performed on the 36 workers were less than 20 mSv/y, not exceeding the effective dose limit for occupational exposure. A questionnaire identified socioeconomic and lifestyle factors associated with the outcomes. The results of the MN assay showed a significant difference between both internal control and the exposed group when compared to the external control. For the comet assay, there were significant differences between the percent of tail DNA of the exposed group and external controls, but no difference was found between the exposed group and internal controls. Relative risk associations were found in time of exposure, hours worked per week, and perceived stress. Correlations were found between the outcomes and age, consumption of alcohol, and frequencies of X-rays during life. Variables that showed to be significant in the adjusted analysis were skin color and recent exposure to radiation. Albeit limited, the findings of this study suggest genotoxicity in both blood and buccal mucosa cells of workers exposed directly or indirectly to IR and that lifestyle and socioeconomic factors are associated and correlated with the risk of developing these outcomes.

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Evaluation of the quality of RNA extracted from archival FFPE glioblastoma and epilepsy surgical samples for gene expression assays

Journal of Clinical Pathology ◽

10.1136/jclinpath-2017-204969 ◽

2018 ◽

Vol 71 (8) ◽

pp. 695-701 ◽

Cited By ~ 6

Author(s):

Harry R Haynes ◽

Clare L Killick-Cole ◽

Kelly M Hares ◽

Juliana Redondo ◽

Kevin C Kemp ◽

...

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Ffpe Tissue ◽

Tissue Samples ◽

Total Rna ◽

Expression Studies ◽

Significant Difference ◽

Gene Expression Analyses ◽

Gene Expression Studies ◽

And Control

AimsHistopathological tissue samples are being increasingly used as sources of nucleic acids in molecular pathology translational research. This study investigated the suitability of glioblastoma and control central nervous system (CNS) formalin-fixed paraffin embedded (FFPE) tissue-derived RNA for gene expression analyses.MethodsTotal RNA was extracted from control (temporal lobe resection tissue) and glioblastoma FFPE tissue samples. RNA purity (260/280 ratios) was determined and RNA integrity number (RIN) analysis was performed. RNA was subsequently used for RT-qPCR for two reference genes,18SandGAPDH.ResultsReference gene expression was equivalent between control and glioblastoma tissue when using RNA extracted from FFPE tissue, which has key implications for biological normalisation for CNS gene expression studies. There was a significant difference between the mean RIN values of control and glioblastoma FFPE tissue. There was no significant correlation between 260/280 or RIN values versus total RNA yield. The age of the tissue blocks did not influence RNA yield, fragmentation or purity. There was no significant correlation between RIN or 260/280 ratios and mean qPCR cycle threshold for either reference gene.ConclusionsThis study showed that routinely available CNS FFPE tissue is suitable for RNA extraction and downstream gene expression studies, even after 60 months of storage. Substantial RNA fragmentation associated with glioblastoma and control FFPE tissue blocks did not preclude downstream RT-qPCR gene expression analyses. Cross validation with both archival and prospectively collated FFPE specimens is required to further demonstrate that CNS tissue blocks can be used in novel translational molecular biomarker studies.

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High Sensitivity Sanger Sequencing Detection of BRAF Mutations in Metastatic Melanoma FFPE Tissue Specimens

10.21203/rs.3.rs-109681/v1 ◽

2020 ◽

Author(s):

Lauren Cheng ◽

Lauren Haydu ◽

Ping Song ◽

Jianyi Nie ◽

Michael Tetzlaff ◽

...

Keyword(s):

Sanger Sequencing ◽

Limit Of Detection ◽

False Negative ◽

High Sensitivity ◽

Ffpe Tissue ◽

Braf Mutations ◽

Tissue Samples ◽

Ffpe Samples ◽

Droplet Pcr ◽

Tissue Specimens

Abstract Mutations in the BRAF gene at or near the p. V600 locus are informative for therapy selection, but current methods for analyzing FFPE tissue DNA generally have a limit of detection of 5% variant allele frequency (VAF), or are limited to the single variant (V600E). These can result in false negatives for samples with low VAFs due to low tumor content or subclonal heterogeneity, or harbor non-V600 mutations. Here, we show that Sanger sequencing using the NuProbe VarTraceTM BRAF assay, based on the Blocker Displacement Amplification (BDA) technology, is capable of detecting BRAF V600 mutations down to 0.26% VAF from FFPE lymph node tissue samples. Comparison experiments on adjacent tissue sections using BDA Sanger, immunohistochemistry (IHC), digital droplet PCR (ddPCR), and NGS showed 100% concordance among all 4 methods for samples with BRAF mutations at ≥1% VAF, though ddPCR did not distinguish the V600K mutation from the V600E mutation. BDA Sanger, ddPCR, and NGS (with orthogonal confirmation) were also pairwise concordant for lower VAF mutations down to 0.26% VAF, but IHC produced a false negative. Thus, we have shown that Sanger sequencing can be effective for rapid detection and quantitation of multiple low VAF BRAF mutations from FFPE samples. BDA Sanger method also enabled detection and quantitation of less frequent, potentially actionable non-V600 mutations as demonstrated by synthetic samples.

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Nanomechanical Hallmarks of Helicobacter pylori Infection in Pediatric Patients

International Journal of Molecular Sciences ◽

10.3390/ijms22115624 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5624

Author(s):

Piotr Deptuła ◽

Łukasz Suprewicz ◽

Tamara Daniluk ◽

Andrzej Namiot ◽

Sylwia Joanna Chmielewska ◽

...

Keyword(s):

Gastric Cancer ◽

Mechanical Properties ◽

Helicobacter Pylori ◽

Mechanical Response ◽

Cellular Level ◽

Cancer Development ◽

Tissue Samples ◽

Diagnostic Measures ◽

Gastric Cells ◽

H Pylori

Background: the molecular mechanism of gastric cancer development related to Helicobacter pylori (H. pylori) infection has not been fully understood, and further studies are still needed. Information regarding nanomechanical aspects of pathophysiological events that occur during H. pylori infection can be crucial in the development of new prevention, treatment, and diagnostic measures against clinical consequences associated with H. pylori infection, including gastric ulcer, duodenal ulcer, and gastric cancer. Methods: in this study, we assessed mechanical properties of children’s healthy and H. pylori positive stomach tissues and the mechanical response of human gastric cells exposed to heat-treated H. pylori cells using atomic force microscopy (AFM NanoWizard 4 BioScience JPK Instruments Bruker). Elastic modulus (i.e., the Young’s modulus) was derived from the Hertz–Sneddon model applied to force-indentation curves. Human tissue samples were evaluated using rapid urease tests to identify H. pylori positive samples, and the presence of H. pylori cells in those samples was confirmed using immunohistopathological staining. Results and conclusion: collected data suggest that nanomechanical properties of infected tissue might be considered as markers indicated H. pylori presence since infected tissues are softer than uninfected ones. At the cellular level, this mechanical response is at least partially mediated by cell cytoskeleton remodeling indicating that gastric cells are able to tune their mechanical properties when subjected to the presence of H. pylori products. Persistent fluctuations of tissue mechanical properties in response to H. pylori infection might, in the long-term, promote induction of cancer development.

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Beliefs in Internal-External Control of Reinforcement and Participation in Group and Individual Sports

Perceptual and Motor Skills ◽

10.2466/pms.1969.29.2.551 ◽

1969 ◽

Vol 29 (2) ◽

pp. 551-553 ◽

Cited By ~ 15

Author(s):

Robert W. Lynn ◽

Joseph G. Phelan ◽

Vernon L. Kiker

Keyword(s):

Internal Control ◽

Self Esteem ◽

External Control ◽

Basketball Players ◽

Significant Difference ◽

Group A ◽

Group B ◽

Individual Sports ◽

The Relationship ◽

School Activity

Three groups of 30 12- to 15-yr.-old males, students of California junior high schools, matched for age and IQ, were tested on the Rotter I-E Scale. Group A (school basketball players) were considered to be group sports participants, Group B (gymnasts) individual sportsmen, and Group C non-participants in any school activity. A significant difference in the direction of internal control for group sports participants was noted. Individual sports participants and non-participants were alike in being significantly higher in external control than group players. Further research on the relationship between internal control and self-esteem seems warranted for group sports participants.

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High-throughput proteomic analysis of FFPE tissue samples facilitates tumor stratification

10.1101/667394 ◽

2019 ◽

Author(s):

Yi Zhu ◽

Tobias Weiss ◽

Qiushi Zhang ◽

Rui Sun ◽

Bo Wang ◽

...

Keyword(s):

Biomarker Discovery ◽

Temporal Stability ◽

B Cell Lymphoma ◽

Ffpe Tissue ◽

Tissue Samples ◽

Pattern Similarity ◽

Ffpe Samples ◽

Fresh Frozen ◽

Peroxidase Enzyme ◽

High Degree

AbstractFormalin-fixed, paraffin-embedded (FFPE), biobanked tissue samples offer an invaluable resource for clinical and biomarker research. Here we developed a pressure cycling technology (PCT)-SWATH mass spectrometry workflow to analyze FFPE tissue proteomes and applied it to the stratification of prostate cancer (PCa) and diffuse large B-cell lymphoma (DLBCL) samples. We show that the proteome patterns of FFPE PCa tissue samples and their analogous fresh frozen (FF) counterparts have a high degree of similarity and we confirmed multiple proteins consistently regulated in PCa tissues in an independent sample cohort. We further demonstrate temporal stability of proteome patterns from FFPE samples that were stored between one to 15 years in a biobank and show a high degree of the proteome pattern similarity between two types histological region of small FFPE samples, i.e. punched tissue biopsies and thin tissue sections of micrometer thickness, despite the existence of certain degree of biological variations. Applying the method to two independent DLBCL cohorts we identified myeloperoxidase (MPO), a peroxidase enzyme, as a novel prognostic marker. In summary, this study presents a robust proteomic method to analyze bulk and biopsy FFPE tissues and reports the first systematic comparison of proteome maps generated from FFPE and FF samples. Our data demonstrate the practicality and superiority of FFPE over FF samples for proteome in biomarker discovery. Promising biomarker candidates for PCa and DLBCL have been discovered.

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CDK5RAP3 as a Novel Biomarker Signature Predicting Survival and Adjuvant Chemotherapeutic Benefit in Gastric Cancer

10.21203/rs.3.rs-95106/v1 ◽

2020 ◽

Author(s):

Jia-Bin Wang ◽

You-Xin Gao ◽

Ning-Zi Lian ◽

Yu-Bin Ma ◽

Ping Li ◽

...

Keyword(s):

Gastric Cancer ◽

Adjuvant Chemotherapy ◽

Cancer Patients ◽

External Validation ◽

University Hospital ◽

Tumour Tissue ◽

Tissue Samples ◽

Significant Difference ◽

Validation Set ◽

Gastric Cancer Patients

Abstract BackgroundWe previously demonstrated that CDK5RAP3 acts as a tumour suppressor in gastric cancer through negative regulation of the Wnt/β-catenin signalling pathway, but its function in chemotherapeutic responsiveness of gastric cancer has not been investigated. MethodA collection of 188 pairs of tumour tissue microarray specimens from Fujian Medical University were employed for the discovery set, and 310 tumour tissue samples of gastric cancer patients were employed for the internal validation set. Eight-five tumour tissue samples from Qinghai University Hospital were used as the external validation set 1. Transcriptomic and clinical data of 299 gastric cancer patients from TCGA were used as the external validation set 2. CDK5RAP3 expression, microsatellite instability (MSI) status, and tumour-infiltrating lymphocytes (TIL) were examined with immunohistochemistry. Clinical outcomes of patients were compared with Kaplan-Meier curves and the Cox model.ResultsIn a multi-centre evaluation, increased CDK5RAP3 indication of better prognosis depends mainly on MSI-L/MSS status or TILhigh. High CDK5RAP3 expression predicts sensitive therapeutic responsiveness to postoperative adjuvant chemotherapy in gastric cancer. In a stratification analysis based on CDK5RAP3 combined with TIL or MSI status, patients with CKD5RAP3low TILlow showed no significant difference in prognosis after receiving chemotherapy, whereas patients with CKD5RAP3low TILhigh, CKD5RAP3high TILlow, and CKD5RAP3high TILhigh had better responsiveness to chemotherapy. In addition, patients with CKD5RAP3high MSI-L/MSS status benefitted the most from adjuvant chemotherapy among all patients evaluated. ConclusionsCKD5RAP3 can be used as an effective marker to evaluate individualized chemotherapy regimens in gastric cancer patients dependent on their TIL and MSI status.

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Using Targeted RNA Next Generation Sequencing and Differential Expression to Compare Primary Central Nervous System Lymphoma with CD+ DLBCL, Nodal DLBCL and EBV+ DLBCL

Blood ◽

10.1182/blood-2020-142235 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 22-22

Author(s):

Haifaa Abdulhaq ◽

Maher Albitar

Keyword(s):

Next Generation Sequencing ◽

Differential Expression ◽

Central Nervous System Lymphoma ◽

Ffpe Tissue ◽

Next Generation ◽

Advisory Committees ◽

Tissue Samples ◽

T Score ◽

Significant Difference ◽

Generation Sequencing

Introduction:Primary central nervous system lymphoma (PCNSL) is a diffuse large B-cell lymphoma (DLBCL) with a tropism for the CNS microenvironment. Limited studies suggested differential expression of multiple extracellular matrix (ECM) and adhesions-related genes in PCNSL as compared to nodal DLBCL. We used next generation sequencing to evaluate expression profiles of 1408 genes and and performed differential expression profiling to compare PCNSL with nodal DLBCL, CD5+ DLBCL and EBV+ DLBCL. CD5+ DLBCL was selected because it is frequently ABC subtype. We also selected EBV+ DLBCL due to its poor outcome, similar to PCNSL. Methods:RNA was extracted from 30 formalin-fixed paraffin-embedded (FFPE) tissue samples from PCNSL patients and 23 FFPE tissue samples from cases with lymph node DLBCL-NOS, 8 samples CD5+DLBCL, 14 samples EBV+ DLBCL.. We sequenced the RNA using a 1408 gene panel (Illumina). Next Generation Sequencing was based on hybrid capture methodology. Total number of reads per sample must exceed 5 million to be accepted. RNA levels were quantified using FPKM. Levels of expression of each of the 1408 genes were compared between groups. The T-score is used to show differences. Considering the number of analyzed samples and to correct for multiple testing, only T-score less or greater than 3 was considered clinically significant. Results:PCNSL showed significant overexpression in 25 genes as compared with CD5+ DLBCL, while only 5 genes were highly expressed in PCNSL as compared with nodal DLBCL and 8 when compared with EBV+ DLBCL (Table below). Four genes were significantly less expressed in PCNSL as compared with CD5+ DLBCL, and only one gene when compared to nodal and EBV+ DLBCL. There was no significant difference in RNA expression of MYD88, CD79B, CARD11, KMT2D or SPP1 between PCNSL and DLBC-NOS, CD5+ DLBCL and EBV+ DLBCL . There was no significant difference between PCNSL and the other sub types in MYC, Ki67, BCL2, CD44, or CD274 (PD-L1) expression. Several genes emerged as highly expressed in PCNSL as compared with other subtypes of DLBCL (negative T-score). However, CLTC (Clathrin Heavy Chain) gene is uniquely highly overexpressed in PCNSL and not in any of the other 3 types of lymphoma. The CLTC gene is involved in anaplastic lymphoma as a partner gene for ALK (CLTC-ALK) in t(2,17) translocation. The SEPT2 (SEPTIN2) gene is uniquely highly expressed in PCNSL as compared with nodal and EBV DLBC lymphoma. SEPT2 gene plays significant role in Hodgkin lymphoma and in maintaining Reed-Stenberg cells . In contrast, VEGFC was significantly lower in PCNSL compared with nodal DLBCL. Conclusions:RNA molecular profiling suggests significant difference between PCNSL and CD5+ DLBCL and less difference when compared with nodal and EBV+ DLBCL. The strikingly high expression of CLTC gene in PCNSL suggests possible targeting of this gene might present a specific therapeutic approach for PCNSL. Figure Disclosures Abdulhaq: Novartis:Consultancy, Honoraria, Speakers Bureau;Astellas:Honoraria, Speakers Bureau;Amgen:Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau;Morphosys:Membership on an entity's Board of Directors or advisory committees, Research Funding;Oncopeptide:Research Funding.

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