Analysis of biological and technical variability in gene expression assays from formalin-fixed paraffin-embedded classical Hodgkin lymphomas

Vera‐Lozada, Gabriela; Scholl, Vanesa; Barros, Mário Henrique M.; Sisti, Davide; Guescini, Michele; Stocchi, Vilberto; Stefanoff, Cláudio Gustavo; Hassan, Rocı́o

doi:10.1016/j.yexmp.2014.09.014

Cited by 6 publications

(11 citation statements)

References 36 publications

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“…To be considered as a tool for clinical prognostication or therapy individualization, a chosen set of assays should render reproducible results in independent patient series. In the present study of pediatric patients with cHL, despite using similar methodological approaches (same primers/probe) and carefully validated preanalytical and analytical methodological strategies, we were not able to validate the 11‐gene score proposed by Sanchez‐Espiridión et al to be applied in the our study group.…”

Section: Discussionmentioning

confidence: 61%

“…8,9 To be considered as a tool for clinical prognostication or therapy individualization, a chosen set of assays should render reproducible results in independent patient series. In the present study of pediatric patients with cHL, despite using similar methodological approaches (same primers/probe) and carefully validated preanalytical and analytical methodological strategies, 17 we were not able to validate the 11-gene score proposed Moreover, the identification of subpopulations responsible for CASP3 expression in the TME will help to understand the immune balances in cHL and, perhaps, will shed a light on the intriguing poor and good prognostic associations of cytotoxic and regulatory T cells, respectively, that have been described in cHL. 15,29 In the present work, we also disclosed a good prognosis associated with high expression of LYZ and poor outcome marginally associated with high expression of STAT1 mRNA, mainly in the unfavorable-risk patients.…”

Section: Discussionmentioning

confidence: 99%

“…The expression levels of the 11 genes of interest (GOI: CENPF , CDK1 , CCNA2 , CCNE2, HMMR, BCL2 , BCL2L1 , CASP3 , LYZ , STAT1 , and IRF4 ) were quantified with TaqMan ® predesigned assays (Applied Biosystems) by reverse transcription quantitative real‐time PCR (RT‐qPCR) . GUSB and HMBS were used as reference genes (RG) because of their uniform expression in cHL tumor samples . Each measurement was performed in duplicate and quantified by Cq values with fixed thresholds.…”

Section: Methodsmentioning

confidence: 99%

“…Total RNA was extracted from FFPE lymph node sections with the Master Pure™ RNA Purification Kit (Epicenter) as described. 17 Firststrand cDNA was synthesized from 0.5 μg of total RNA using the High Capacity cDNA Archive Kit (Applied Biosystems). In all cases, a preamplification step (Applied Biosystems) helped to improve the sensitivity of the assays.…”

Section: Rna Extraction and Cdna Synthesismentioning

confidence: 99%

“…8,17 GUSB and HMBS were used as reference genes (RG) because of their uniform expression in cHL tumor samples. 7,17 Each measurement was performed in duplicate and quantified by Cq values with fixed thresholds. Relative gene expression was calculated as log2 of 2 −ΔCq after normalization with the RG means.…”

Section: Gene Expression Analysismentioning

confidence: 99%

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Pathway‐focused gene expression profiles and immunohistochemistry detection identify contrasting association of caspase 3 (CASP3) expression with prognosis in pediatric classical Hodgkin lymphoma

Vera‐Lozada

Segges

Stefanoff

et al. 2018

Hematological Oncology

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The search for clinically relevant molecular markers in classical Hodgkin lymphoma (cHL) is hampered by the histopathological complexity of the disease, resulting from the admixture of a small number of neoplastic Hodgkin and Reed-Sternberg (H-RS) cells with an abundant and heterogeneous microenvironment. In this study, we evaluated gene expression profiles of 11 selected genes previously proposed as a molecular score for adult cHL, aiming to validate its application in the pediatric setting. Assays were performed by RT-qPCR from formalin-fixed paraffin-embedded (FFPE) lymph nodes in 80 patients with cHL. Selected genes were associated with cell cycle (CENPF, CDK1, CCNA2, CCNE2, and HMMR), apoptosis (BCL2, BCL2L1, and CASP3), and monocytes/macrophages (LYZ and STAT1). Despite using controlled preanalytical and analytical strategies, we were not able to validate the 11-gene score to be applied in pediatric cHL. Principal component analysis (PCA) disclosed 3 components that accounted for 65.7% of the total variability. The second PC included microenvironment and apoptosis genes, from which CASP3 expression was associated with a short time of progression-free survival, which impact was maintained in the unfavorable risk group, Epstein-Barr virus-negative cases, and multivariate analysis (P < .05). Because this is a counterintuitive association, CASP3 active expression was assessed at the protein level in H-RS cells by double immunohistochemistry. In contrast to the association of mRNA levels with a poor therapeutic response, a high number of cleaved CASP3+ cells were associated with longer progression-free survival (P = .03) and overall survival (P = .002). Our results demonstrate the feasibility of using FFPE samples as RNA source for molecular prognostication, but argue against the concept of direct and wide applicability of molecular scores in cHL. We reinforce the potential of CASP3 as an interesting target to be explored in adult and pediatric cHL, and alert for its dual biological role in H-RS cells and tumor microenvironment.

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Section: Discussionmentioning

confidence: 61%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Rna Extraction and Cdna Synthesismentioning

confidence: 99%

Section: Gene Expression Analysismentioning

confidence: 99%

See 3 more Smart Citations

Pathway‐focused gene expression profiles and immunohistochemistry detection identify contrasting association of caspase 3 (CASP3) expression with prognosis in pediatric classical Hodgkin lymphoma

Vera‐Lozada

Segges

Stefanoff

et al. 2018

Hematological Oncology

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Straightforward and sensitive RT-qPCR based gene expression analysis of FFPE samples

Zeka

Vanderheyden

Smet

et al. 2016

Sci Rep

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Fragmented RNA from formalin-fixed paraffin-embedded (FFPE) tissue is a known obstacle to gene expression analysis. In this study, the impact of RNA integrity, gene-specific reverse transcription and targeted cDNA preamplification was quantified in terms of reverse transcription polymerase chain reaction (RT-qPCR) sensitivity by measuring 48 protein coding genes on eight duplicate cultured cancer cell pellet FFPE samples and twenty cancer tissue FFPE samples. More intact RNA modestly increased gene detection sensitivity by 1.6 fold (earlier detection by 0.7 PCR cycles, 95% CI = 0.593–0.850). Application of gene-specific priming instead of whole transcriptome priming during reverse transcription further improved RT-qPCR sensitivity by a considerable 4.0 fold increase (earlier detection by 2.0 PCR cycles, 95% CI = 1.73–2.32). Targeted cDNA preamplification resulted in the strongest increase of RT-qPCR sensitivity and enabled earlier detection by an average of 172.4 fold (7.43 PCR cycles, 95% CI = 6.83–7.05). We conclude that gene-specific reverse transcription and targeted cDNA preamplification are adequate methods for accurate and sensitive RT-qPCR based gene expression analysis of FFPE material. The presented methods do not involve expensive or complex procedures and can be easily implemented in any routine RT-qPCR practice.

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Interleukin 10(IL10) proximal promoter polymorphisms beyond clinical response in classical Hodgkin lymphoma: Exploring the basis for the genetic control of the tumor microenvironment

et al. 2018

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Interleukin-10 (IL10) is an immune regulatory cytokine. Single nucleotide polymorphisms (SNPs) in IL10 promoter have been associated with prognosis in adult classical Hodgkin lymphoma (cHL). We analyzed IL10 SNPs −1082 and −592 in respect of therapy response, gene expression and tumor microenvironment (TME) composition in 98 pediatric patients with cHL. As confirmatory results, we found that −1082AA/AG; −592CC genotypes and ATA haplotype were associated with unfavourable prognosis: Progression-free survival (PFS) was shorter in −1082AA+AG (72.2%) than in GG patients (100%) (P = 0.024), and in −592AA (50%) and AC (74.2%) vs. CC patients (87.0%) (P = 0.009). In multivariate analysis, the −592CC genotype and the ATA haplotype retained prognostic impact (HR: 0.41, 95% CI 0.2–0.86; P = 0.018, and HR: 3.06 95% CI 1.03–9.12; P = 0.044, respectively). Our analysis further led to some new observations, namely: (1) Low IL10 mRNA expression was associated with −1082GG genotype (P = 0.014); (2) IL10 promoter polymorphisms influence TME composition;−1082GG/−592CC carriers showed low numbers of infiltrating cells expressing MAF transcription factor (20 vs. 78 and 49 vs. 108 cells/mm2, respectively; P< 0.05); while ATA haplotype (high expression) associated with high numbers of MAF+ cells (P = 0.005). Specifically, −1082GG patients exhibited low percentages of CD68+MAF+ (M2-like) intratumoral macrophages (15.04% vs. 47.26%, P = 0.017). Considering ours as an independent validation cohort, our results give support to the clinical importance of IL10 polymorphisms in the full spectrum of cHL, and advance the concept of genetic control of microenvironment composition as a basis for susceptibility and therapeutic response.

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Analysis of biological and technical variability in gene expression assays from formalin-fixed paraffin-embedded classical Hodgkin lymphomas

Cited by 6 publications

References 36 publications

Pathway‐focused gene expression profiles and immunohistochemistry detection identify contrasting association of caspase 3 (CASP3) expression with prognosis in pediatric classical Hodgkin lymphoma

Pathway‐focused gene expression profiles and immunohistochemistry detection identify contrasting association of caspase 3 (CASP3) expression with prognosis in pediatric classical Hodgkin lymphoma

Straightforward and sensitive RT-qPCR based gene expression analysis of FFPE samples

Interleukin 10(IL10) proximal promoter polymorphisms beyond clinical response in classical Hodgkin lymphoma: Exploring the basis for the genetic control of the tumor microenvironment

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