Relative quantification based on logistic models for individual polymerase chain reactions

Chervoneva, Inna; Li, Yanyan; Iglewicz, Boris; Hyslop, Terry

doi:10.1002/sim.3127

Cited by 23 publications

(33 citation statements)

References 21 publications

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“…Target transcripts for guanylin were quantified relative to the intestinal epithelial cell marker villin (VIL) by RT-qPCR employing relative logistic regression models fit to fluorescence data from each well for replicate amplification reactions (27). Villin was chosen for normalization because it is a selective product of epithelial, compared to other cells, in intestine and is persistently and reliably expressed during tumorigenesis (28–30).…”

Section: Methodsmentioning

confidence: 99%

The Paracrine Hormone for the GUCY2C Tumor Suppressor, Guanylin, Is Universally Lost in Colorectal Cancer

Wilson

Lin

et al. 2014

Cancer Epidemiology, Biomarkers &Amp; Prevention

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Background: Although colorectal cancer is a disease characterized by sequential accumulation of mutations in epithelial cells, mechanisms leading to genomic vulnerability contributing to tumor initiation remain undefined. GUCY2C has emerged as an intestine-specific tumor suppressor controlling epithelial homeostasis through circuits canonically disrupted in cancer. Surprisingly, the GUCY2C tumor suppressor is universally overexpressed by human colorectal cancer cells. This apparent paradox likely reflects silencing of GUCY2C through loss of its paracrine hormone guanylin. Here, we quantified expression of guanylin mRNA and protein in tumors and normal epithelia from patients with colorectal cancer.Methods: Guanylin mRNA was quantified in tumors and normal adjacent epithelia from 281 patients by the reverse transcriptase-polymerase chain reaction. Separately, the guanylin protein was quantified by immunohistochemistry in 54 colorectal tumors and 30 specimens of normal intestinal epithelium.Results: Guanylin mRNA in colorectum varied more than a 100-fold across the population. Guanylin mRNA was reduced 100-to 1,000-fold in >85% of tumors compared with matched normal adjacent mucosa (P < 0.001). Loss of guanylin mRNA was greatest in tumors from patients <50 years old (P < 0.005) and with the highest expression in normal adjacent mucosa (Spearman correlation coefficient ¼ 0.61; P < 0.001). In a separate validation cohort, guanylin protein was detected in all 30 normal colorectal mucosa specimens, but in none of 54 colorectal tumors.Conclusions: Colorectal cancer may initiate as a disease of paracrine hormone insufficiency through loss of guanylin expression, silencing the GUCY2C tumor suppressor and disrupting homeostatic mechanisms regulating colorectal epithelia cells.Impact: Intestinal tumorigenesis may be prevented by oral GUCY2C hormone replacement therapy. Cancer Epidemiol Biomarkers Prev; 23(11); 2328-37. Ó2014 AACR.

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Section: Methodsmentioning

confidence: 99%

The Paracrine Hormone for the GUCY2C Tumor Suppressor, Guanylin, Is Universally Lost in Colorectal Cancer

Wilson

Lin

et al. 2014

Cancer Epidemiology, Biomarkers &Amp; Prevention

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“…25, 29 Statistical methods for estimating GUCY2C and β-actin mRNA expression by logistic regression analysis have been described. 18, 30 A linear mixed effects model of GUCY2C relative expression across all nodes from eligible patients included random effect of patient, and fixed effects of center, and race. This linear model was used to determine differences in GUCY2C expression in patient lymph nodes based on race, after adjusting for potential center-to-center differences and for repeated measures within patient.…”

Section: Methodsmentioning

confidence: 99%

Occult tumor burden contributes to racial disparities in stage‐specific colorectal cancer outcomes

Hyslop

Weinberg

Schulz

et al. 2011

Cancer

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Background There are differences in outcomes in blacks, compared to whites, with lymph node-negative (pN0) colorectal cancer. Recurrence in pN0 patients suggests the presence of occult metastases undetected by conventional approaches. This study explores the association of racial differences in outcomes with occult tumor burden in regional lymph nodes. Methods Lymph nodes (range: 2-159) from 282 prospectively enrolled pN0 colorectal cancer patients followed for a median of 24 months (range: 2-63) were subjected to molecular analysis. Occult tumor burden was estimated by quantifying the expression of the biomarker GUCY2C, a biomarker for metastatic colorectal cancer cells. Risk categories defined using occult tumor burden was the primary outcome measure. Association of prognostic variables and risk were defined by multivariate polytomous logistic regression. Results Occult tumor burden stratified this cohort of 259 whites and 23 blacks into categories with low (60%; recurrence rate (RR)=2.3% [95%CI 0.1-4.5%]), intermediate (31%; RR=33.3% [23.7%-44.1%]), and high (9%; RR=68.0% [46.5%-85.1%], p<0.001) risk. Blacks, compared to whites, exhibited 4-fold greater occult metastases in individual nodes (p<0.001). Multivariate analysis revealed that race (p=0.02), T stage (p=0.02), and number of nodes collected (p=0.003) were independent prognostic markers of risk category. Blacks, compared to whites, were more likely to harbor levels of occult tumor burden, associated with the highest recurrence risk (adjusted odds ratio=5.08 [1.69-21.39]; p=0.007). Conclusions Racial disparities in stage-specific outcomes in colorectal cancer are associated with differences in occult tumor burden in regional lymph nodes.

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“…Logistic models imply that efficiency is declining through the course of reaction, although in the early cycles it stays approximately constant at the maximum (Figure 2). Hence, the estimated maximum may be used as PCR efficiency [9]. Logistic models are implemented in the R package qpcR [18], which is the most comprehensive library implementing standard methods for qRT-PCR quantification.…”

Section: Relative Quantification Of Mrna Using Qrt-pcrmentioning

confidence: 99%

“…The standard methods of estimating individual PCR reaction efficiency are based on models for fluorescence intensity F ( x ) as a function of PCR cycle x . The majority of these methods employ either an exponential growth model [8], which is valid only in the early exponential phase, or logistic/sigmoid models [5, 9, 10]. …”

Section: Introductionmentioning

confidence: 99%

“…the point of the second derivative maximum). The use of the maximum efficiency is justified when it is reasonable to assume that the growth of the PCR product is approximately exponential up to the threshold cycle (see 2 and section 2.2 for definition) that is used for computing the relative expression [9]. But if the efficiency decrease by the threshold cycle is substantial, then different choices of a constant efficiency estimate at some point along the fluorescence intensity curve do not have any theoretical justification.…”

Section: Introductionmentioning

confidence: 99%

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Modeling qRT-PCR dynamics with application to cancer biomarker quantification

Chervoneva

Freydin

Hyslop

2017

Stat Methods Med Res

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Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is widely used for molecular diagnostics and evaluating prognosis in cancer. The utility of mRNA expression biomarkers relies heavily on the accuracy and precision of qRT-PCR quantification, which is still challenging for low abundance transcripts. The critical step for qRT-PCR quantification is accurate estimation of efficiency needed for computing relative qRT-PCR expression. We propose a new approach to estimating qRT-PCR efficiency based on modeling dynamics of PCR amplification. In contrast, only models for fluorescence intensity as a function of PCR cycle have been used so far for relative qRT-PCR quantification. The dynamics of qRT-PCR efficiency is modeled using an ordinary differential equation (ODE) model, and the fitted ODE model is used to obtain effective PCR efficiency estimates needed for efficiency-adjusted relative qRT-PCR quantification. The proposed new qRT-PCR efficiency estimates were used to quantify GUCY2C mRNA expression in the blood of colorectal cancer patients. Time to recurrence and GUCY2C expression ratios were analyzed in a joint model for survival and longitudinal outcomes. The joint model with GUCY2C quantified using the proposed PCR efficiency estimates provided clinically meaningful results for association between time to recurrence and longitudinal trends in GUCY2C expression.

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Relative quantification based on logistic models for individual polymerase chain reactions

Cited by 23 publications

References 21 publications

The Paracrine Hormone for the GUCY2C Tumor Suppressor, Guanylin, Is Universally Lost in Colorectal Cancer

The Paracrine Hormone for the GUCY2C Tumor Suppressor, Guanylin, Is Universally Lost in Colorectal Cancer

Occult tumor burden contributes to racial disparities in stage‐specific colorectal cancer outcomes

Modeling qRT-PCR dynamics with application to cancer biomarker quantification

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