A Method to Correlate mRNA Expression Datasets Obtained from Fresh Frozen and Formalin-Fixed, Paraffin-Embedded Tissue Samples: A Matter of Thresholds

Mustafa, Dana A. M.; Sieuwerts, Anieta M.; Smid, Marcel; Weerd, Vania de; Weiden, Marcel van der; Gelder, Marion E. Meijer Van; Martens, John W.M.; Foekens, John A.; Kros, Johan M.

doi:10.1371/journal.pone.0144097

Cited by 6 publications

(6 citation statements)

References 32 publications

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“…The WG-DASL assay was performed according to the manufacturer’s instructions with an input of 500 ng total RNA. To monitor the assay performance and to evaluate the inter-assay BeadChip variability between the experiments, an inner-assay control consisting of 500 ng total RNA pooled from RNA isolated from several cultured breast cancer cell lines was used in each experiment [ 36 ].…”

Section: Methodsmentioning

confidence: 99%

T lymphocytes facilitate brain metastasis of breast cancer by inducing Guanylate-Binding Protein 1 expression

et al. 2018

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The discovery of genes and molecular pathways involved in the formation of brain metastasis would direct the development of therapeutic strategies to prevent this deadly complication of cancer. By comparing gene expression profiles of Estrogen Receptor negative (ER-) primary breast tumors between patients who developed metastasis to brain and to organs other than brain, we found that T lymphocytes promote the formation of brain metastases. To functionally test the ability of T cells to promote brain metastasis, we used an in vitro blood–brain barrier (BBB) model. By co-culturing T lymphocytes with breast cancer cells, we confirmed that T cells increase the ability of breast cancer cells to cross the BBB. Proteomics analysis of the tumor cells revealed Guanylate-Binding Protein 1 (GBP1) as a key T lymphocyte-induced protein that enables breast cancer cells to cross the BBB. The GBP1 gene appeared to be up-regulated in breast cancer of patients who developed brain metastasis. Silencing of GBP1 reduced the ability of breast cancer cells to cross the in vitro BBB model. In addition, the findings were confirmed in vivo in an immunocompetent syngeneic mouse model. Co-culturing of ErbB2 tumor cells with activated T cells induced a significant increase in Gbp1 expression by the cancer cells. Intracardial inoculation of the co-cultured tumor cells resulted in preferential seeding to brain. Moreover, intracerebral outgrowth of the tumor cells was demonstrated. The findings point to a role of T cells in the formation of brain metastases in ER- breast cancers, and provide potential targets for intervention to prevent the development of cerebral metastases.Electronic supplementary materialThe online version of this article (10.1007/s00401-018-1806-2) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

T lymphocytes facilitate brain metastasis of breast cancer by inducing Guanylate-Binding Protein 1 expression

et al. 2018

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“…From 52 patients matched formalin‐fixed paraffin embedded (FFPE) tissue from the primary tumor with at least 30% invasive tumor cells was available (median [range]; 60% [30%–85%]). RNA from the FFPE samples was isolated with the High‐Pure RNA Paraffin Kit (Roche Applied Science, Penzberg, Germany) and the quality and quantity of the RNA was checked with the Nanodrop 1,000‐v.3.7 (Thermo Scientific, Wilmington), the MultiNA Microchip Electrophoresis system (Shimadzu, Kyoto, Japan), and three reference genes ( GUSB, HMBS and HPRT1 ) as described before …”

Section: Methodsmentioning

confidence: 99%

“…RNA from the FFPE samples was isolated with the High-Pure RNA Paraffin Kit (Roche Applied Science, Penzberg, Germany) and the quality and quantity of the RNA was checked with the Nanodrop 1,000-v.3.7 (Thermo Scientific, Wilmington), the MultiNA Microchip Electrophoresis system (Shimadzu, Kyoto, Japan), and three reference genes (GUSB, HMBS and HPRT1) as described before. 24 Normalization and AR cut-off Expression levels of the 93 genes in the CTC and tumor samples were normalized by the average C q value of three reference genes (ΔC q ). If the average C q value of the reference genes (GUSB, HMBS and HPRT1) was >27.5 in a sample, this sample was considered to have insufficient RNA quantity and/or quality.…”

Section: Sample Processing For Gene Expression Profilingmentioning

confidence: 99%

Androgen receptor expression in circulating tumor cells of patients with metastatic breast cancer

Kruijff

Sieuwerts

Onstenk

et al. 2019

Intl Journal of Cancer

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The androgen receptor (AR) has potential clinical relevance in metastatic breast cancer (mBC) since it might be a treatment target and has been associated with endocrine resistance. A minimal‐invasive way to determine AR expression on metastatic tumor cells is by characterization of circulating tumor cells (CTCs). Here, we assessed AR mRNA expression in CTCs (CTC‐AR) and in matched primary tumor samples from mBC patients representing different breast cancer subtypes. In addition, we explored CTC‐AR‐status in relation to outcome on endocrine therapy. AR, and 92 AR or estrogen receptor (ER) related genes, were measured in CellSearch‐enriched CTCs from 124 mBC patients and in 52 matched FFPE primary tissues using quantitative reverse‐transcriptase PCR. AR in CTCs was considered positive if the expression was 1 standard deviation higher than the expression measured in 11 healthy blood donors. A total of 31% of the mBC patients had AR‐positive (AR+) CTCs. 58% of the matched CTC and primary tumor samples were discordant with respect to AR status, observing both switches from AR+ to AR‐negative (AR‐) and vice versa. There was no statistically significant difference in progression‐free survival for patients treated with ER‐targeting drugs and CTC‐AR‐status (13 AR+/ 37 AR‐ cases, p = 0.28). Thus, AR can be determined in RNA isolated from CTCs, with in our set 31% AR‐positive samples. Given the discordance between AR status in CTC samples and corresponding primary tumors, determination of AR expression in CTCs might be a promising tool to select mBC patients for AR inhibiting agents.

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“…Following isolation, RNA was stored in RNase/DNase‐free water at −80 °C. Quality control was performed as previously described (Mustafa et al., 2015).…”

Section: Methodsmentioning

confidence: 99%

“…Following paraffin removal with xylene the high-pure RNA paraffin kit was used according the supplier's instructions (Roche, Mannheim, Germany). Following isolation, RNA was stored in RNase/ DNase-free water at À80 C. Quality control was performed as previously described (Mustafa et al, 2015).…”

Section: Rna Extraction and Purificationmentioning

confidence: 99%

mRNA expression profiles of colorectal liver metastases as a novel biomarker for early recurrence after partial hepatectomy

et al. 2016

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SurgeryBiomarker mRNA Prognostic value A B S T R A C TBackground: Identification of specific risk groups for recurrence after surgery for isolated colorectal liver metastases (CRLM) remains challenging due to the heterogeneity of the disease. Classical clinicopathologic parameters have limited prognostic value. The aim of this study was to identify a gene expression signature measured in CRLM discriminating early from late recurrence after partial hepatectomy. Methods: CRLM from two patient groups were collected: I) with recurrent disease 12 months after surgery (N ¼ 33), and II) without recurrences and disease free for !36 months (N ¼ 30). The patients were clinically homogeneous; all had a low clinical risk score (0e2) and did not receive (neo-) adjuvant chemotherapy. Total RNA was hybridised to Illumina arrays, and processed for analysis. A leave-one-out cross validation (LOOCV) analysis was performed to identify a prognostic gene expression signature. Results: LOOCV yielded an 11-gene profile with prognostic value in relation to recurrent disease 12 months after partial hepatectomy. This signature had a sensitivity of 81.8%, with a specificity of 66.7% for predicting recurrences ( 12 months) versus no recurrences for at least 36 months after surgery (X 2 P < 0.0001). Conclusion:The current study yielded an 11-gene signature at mRNA level in CRLM discriminating early from late or no relapse after partial hepatectomy.

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A Method to Correlate mRNA Expression Datasets Obtained from Fresh Frozen and Formalin-Fixed, Paraffin-Embedded Tissue Samples: A Matter of Thresholds

Cited by 6 publications

References 32 publications

T lymphocytes facilitate brain metastasis of breast cancer by inducing Guanylate-Binding Protein 1 expression

T lymphocytes facilitate brain metastasis of breast cancer by inducing Guanylate-Binding Protein 1 expression

Androgen receptor expression in circulating tumor cells of patients with metastatic breast cancer

mRNA expression profiles of colorectal liver metastases as a novel biomarker for early recurrence after partial hepatectomy

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