Pharmacogenomic analysis of needle biopsies obtained before preoperative docetaxel/capecitabine/FEC (TX/FEC) chemotherapy for breast cancer

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 10595-10595 ◽  
Author(s):  
F. A. Holmes ◽  
J. A. O’Shaughnessy ◽  
B. Hellerstedt ◽  
J. Pippen ◽  
S. Vukelja ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Expression Profiles ◽  
Rna Extraction ◽  
Tumor Biology ◽  
Pathologic Complete Response ◽  
Research Network ◽  
Community Practice ◽  
Oncology Research

10595 Background: Our goal was to evaluate the feasibility of obtaining fine needle biopsies, for pharmacogenomic analysis, in community based oncology practices and develop gene expression-based predictors of pathologic complete response (pCR) to preoperative sequential docetaxel/capecitabine and 5-fluorouracil, epirubicin, cyclophosphamide chemotherapy. Methods: One hundred seventy-five patients were accrued at 29 sites in the US Oncology Research network. FNA specimens were mailed to a central laboratory (MDACC) and gene expression profiling was performed on Affymetrix U133A chips. Results: RNA extraction was started on 140 specimens, 112 of these (80%) yielded ≥1 μg total RNA, 69 were hybridized and 65 (94%) gene expression profiles have passed quality control as of abstract submission date. The analysis plan is to develop a multigene predictor of pCR from the first 80 cases and test its performance independently in the remaining cases. Conclusions: Collection of mandatory research FNA biopsies for pharmacogenomic research is feasible in community practice. Approximately 80% of biopsies yield sufficient RNA for gene expression profiling. In 20% of patients, either technical factors, which can be addressed, or tumor biology (necrotic, rapidly growing tumors) were limiting. Supported by Roche Laboratories, Inc., Nutley, NJ; Pfizer, New York, NY; and Precision Therapeutics, Pittsburgh, PA. [Table: see text]

scholarly journals Three Years Follow-Up Study Showed Prenatal Methamphetamine Exposure May Cause Chronic Abnormalities In Transcriptomic Pattern

2021 ◽  
Author(s):  
Arvin Haghighatfard ◽  
Soha Seifollahi ◽  
Pegah Rajabi ◽  
Niloofar Rahmani ◽  
Rojin Ghannadzadeh
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Rna Extraction ◽  
Childbearing Age ◽  
Methamphetamine Use ◽  
Methamphetamine Use Disorder

Abstract Background: The high rate of methamphetamine use disorder among young adults and women of childbearing age makes it imperative to clarify the long-term effects of Methamphetamine exposure on the offspring. Behavioral and cognitive problems had been reported in children with parental Methamphetamine exposure (PME). The present study aimed to assess the acute and chronic effects of PME in molecular regulations and gene expression profiles of children during their first years of life.Methods: All subjects were recruited before birth, and sampling was conducted from the first ten days of birth, twelve months, twenty months, and thirty-six months of age. Finally, 2658 children with PME and 3573 normal children had been finished the follow-up. RNA extraction was operated from blood samples and gene expression profiling was conducted by using the Affymetrix GeneChip Human Genome U133 plus 2.0 Array Platform. Gene expression data were confirmed by Real-time PCR. Results: Gene expression profiling during thirty-six months showed several constant mRNA level alterations in children with PME compared with normal. These genes are involved in several gene ontologies and pathways involved with the immune system, neuronal functions, and bioenergetic metabolism. It seems that Methamphetamine use disorder before and during the pregnancy period may affect the expression profile of children, and these changes could remain years after birth. Affected genes have some similarities with the gene expression patterns of addiction, psychiatric disorders, neurodevelopmental disabilities, and immune deficiencies. Conclusion: Findings may shed light on the molecular effects of prenatal methamphetamine exposure and may lead to new psychological and somatic caring protocols for these children based on their potential abnormalities.

scholarly journals Effects of Storage, RNA Extraction, Genechip Type, and Donor Sex on Gene Expression Profiling of Human Whole Blood

2007 ◽  
Vol 53 (6) ◽  
pp. 1038-1045 ◽  
Author(s):  
Sung Jae Kim ◽  
David J Dix ◽  
Kary E Thompson ◽  
Rachel N Murrell ◽  
Judith E Schmid ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Whole Blood ◽  
Expression Profiling ◽  
Expression Profiles ◽  
Rna Extraction ◽  
Extraction Technique ◽  
Interindividual Differences ◽  
Human Whole Blood ◽  
Rna Quality

Abstract Background: Gene expression profiling of whole blood may be useful for monitoring toxicological exposure and for diagnosis and monitoring of various diseases. Several methods are available that can be used to transport, store, and extract RNA from whole blood, but it is not clear which procedures alter results. In addition, characterization of interindividual and sex-based variation in gene expression is needed to understand sources and extent of variability. Methods: Whole blood was obtained from adult male and female volunteers (n = 42) and stored at various temperatures for various lengths of time. RNA was isolated and RNA quality analyzed. Affymetrix GeneChips (n = 23) were used to characterize gene expression profiles (GEPs) and to determine the effects on GEP of storage conditions, extraction techniques, types of GeneChip, or donor sex. Hierarchical clustering and principal component analysis were used to assess interindividual differences. Regression analysis was used to assess the relative impact of the studied variables. Results: Storage of blood samples for >1 week at 4 °C diminished subsequent RNA quality. Interindividual GEP differences were seen, but larger effects were observed related to RNA extraction technique, GeneChip, and donor sex. The relative importance of the variables was as follows: storage < genechip < extraction technique < donor sex. Conclusion: Sample storage and extraction methods and interindividual differences, particularly donor sex, affect GEP of human whole blood.

scholarly journals Prenatal Methamphetamine Exposure could Cause Chronic Abnormalities in Transcriptomic Pattern; an Extended Follow-up Cohort Study

2021 ◽  
Author(s):  
Arvin Haghighatfard ◽  
Soha Seifollahi ◽  
Pegah Rajabi ◽  
Niloofar Rahmani ◽  
Rojin Ghannad zadeh
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Rna Extraction ◽  
High Rate ◽  
Childbearing Age ◽  
Methamphetamine Abuse

Abstract BackgroundThe high rate of methamphetamine abuse among young adults and women of childbearing age makes it imperative to clarify the long-term effects of Methamphetamine exposure on the offspring. Behavioral and cognitive problems had reported in children with parental Methamphetamine exposure (PME). The present study aimed to assess the acute and chronic effects of PME in molecular regulations and gene expression profiles of children during their first years of life.ResultsAll subjects were recruited before birth, and sampling was conducted from the first ten days of birth, twelve months, twenty months and thirty-six months of age. Finally, 2658 children with PME and 3573 normal children had been finished the follow-up. RNA extraction was operated from blood samples and gene expression profiling was conducted by using the Affymetrix GeneChip Human Genome U133 plus 2.0 Array Platform. Gene expression data were confirmed by Real-time PCR. Gene expression profiling during thirty-six months showed several constant mRNA level alterations in children with PME compared with normal. These genes are involved in several gene ontology and pathways involved with the immune system, neuronal functions and bioenergetic metabolism. It seems that Methamphetamine abuse before and during the pregnancy period may affect the expression profile of children, and these changes could be remain years after birth. Affected genes have some similarities to the gene expression patterns of addiction, psychiatric disorders, neurodevelopmental disabilities and immune deficiencies. ConclusionFindings may shed light on the molecular effects of prenatal methamphetamine exposure and may lead to new psychological and somatic caring protocols for these children based on their potential abnormalities.

scholarly journals Review of: Gene expression profiling identifies molecular subtypes of inflammatory breast cancer

2006 ◽  
Vol 9 (1) ◽  
pp. 1-3
Author(s):  
P. E. Lønning
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Gene Expression Profiling ◽  
Inflammatory Breast Cancer ◽  
Expression Profiling ◽  
Molecular Subtypes ◽  
Expression Profiles ◽  
Molecular Taxonomy ◽  
Pathologic Complete Response ◽  
Normal Breast

Citation of original article:F. Bertucci, P. Finetti, J. Rougemont, E. Charafe-Jauffret, N. Cervera, C. Tarpin,et al. Gene expression profiling identifies molecular subtypes of inflammatory breast cancer.Cancer Research2005;65(6): 2170–8.Abstract of the original articleBreast cancer is a heterogeneous disease. Comprehensive gene expression profiles obtained using DNA microarrays have revealed previously indistinguishable subtypes of non-inflammatory breast cancer (NIBC) related to different features of mammary epithelial biology and significantly associated with survival. Inflammatory breast cancer (IBC) is a rare, particular, and aggressive form of disease. Here we have investigated whether the five molecular subtypes described for NIBC (luminal A and B, basal, ERBB2 overexpressing, and normal breast-like) were also present in IBC. We monitored the RNA expression of approximately 8,000 genes in 83 breast tissue samples including 37 IBC, 44 NIBC, and 2 normal breast samples. Hierarchical clustering identified the five subtypes of breast cancer in both NIBC and IBC samples. These subtypes were highly similar to those defined in previous studies and associated with similar histoclinical features. The robustness of this classification was confirmed by the use of both alternative gene set and analysis method, and the results were corroborated at the protein level. Furthermore, we show that the differences in gene expression between NIBC and IBC and between IBC with and without pathologic complete response that we have recently reported persist in each subtype. Our results show that the expression signatures defining molecular subtypes of NIBC are also present in IBC. Obtained using different patient series and different microarray platforms, they reinforce confidence in the expression-based molecular taxonomy but also give evidence for its universality in breast cancer, independently of a specific clinical form.

Association of gene expression profile in metastatic melanoma and survival to a dendritic cell-based vaccine

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 9002-9002 ◽  
Author(s):  
T. Gajewski ◽  
Y. Zha ◽  
B. Thurner ◽  
G. Schuler
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Clinical Outcome ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Rna Extraction ◽  
Tumor Biology ◽  
Gene Expression Signature ◽  
Short Survival ◽  
Long Survival

9002 Background: Emerging data suggests that features of the melanoma tumor microenvironment may determine the clinical outcome to immunotherapies. We recently have observed a gene expression signature that correlated with a favorable clinical outcome in response to an IL-12-based melanoma vaccine. Increased expression of chemokine genes and T cell transcripts, and decreased expression of genes associated with aggressive tumor biology, were observed in the favorable group. To determine whether these patterns were reproducible, gene expression profiling was performed from an independent vaccine clinical trial. Methods: Patients with advanced melanoma were treated with autologous, mature monocyte-derived dendritic cells loaded with a combination of melanoma antigen peptides. Pretreatment biopsies were cryopreserved for RNA extraction and gene expression profiling. Patients were categorized into “long survival” (> 24 months) or “short survival” outcomes. Supervised hierarchical clustering was performed to identify genes differentially expressed in the two outcome groups. Results: RNA that passed quality control was obtained from 17 stage IV patients, 5 with a short survival and 12 with a long survival. 408 genes were differentially at least 2- fold. Consistent with previous observations, tumors from favorable outcome patients expressed higher levels of several T cell-specific genes, including Thy1 and CD28; chemokines, including CCL19, CXCL12, and CXCL14; and other immune genes, including LTβ, IL-1R, IFNαR2, IL27R, CD69, and FcRs. Conversely, tumors from unfavorable outcome patients expressed higher levels of pro- angiogeneic genes, including Flt1; anti-apoptotic genes, including SerpinH1 and Serpine1; and multiple collagens. Conclusions: Our results confirm that a subset of transcripts expressed in melanoma metastases may be useful as a predictive biomarker for response to melanoma vaccines. The categories of genes identified point toward new opportunities for overcoming resistance mechanisms. Future studies should integrate gene expression profiling of pre-treatment biopsies as a stratification or enrichment factor in immunotherapy trials. No significant financial relationships to disclose.

scholarly journals 256CONSTRUCTION OF A BOVINE CDNA ARRAY TO MONITOR GENE EXPRESSION PROFILES IN BOVINE PREIMPLANTATION EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 248
Author(s):  
C. Wrenzycki ◽  
T. Brambrink ◽  
D. Herrmann ◽  
J.W. Carnwath ◽  
H. Niemann
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Expression Profiles ◽  
Cdna Array ◽  
Preimplantation Embryos ◽  
Average Insert Size ◽  
Rt Pcr ◽  
Public Data

Array technology is a widely used tool for gene expression profiling, providing the possibility to monitor expression levels of an unlimited number of genes in various biological systems including preimplantation embryos. The objective of the present study was to develop and validate a bovine cDNA array and to compare expression profiles of embryos derived from different origins. A bovine blastocyst cDNA library was generated. Poly(A+)RNA was extracted from in vitro-produced embryos using a Dynabead mRNA purification kit. First-strand synthesis was performed with SacIT21 primer followed by randomly primed second-strand synthesis with a DOP primer mix (Roche) and a global PCR with 35 cycles using SacIT21 and DOP primers. Complementary DNA fragments from 300 to 1500bp were extracted from the gel and normalized via reassoziation and hydroxyapatite chromatography. Resulting cDNAs were digested with SacI and XhoI, ligated into a pBKs vector, and transfected into competent bacteria (Stratagene). After blue/white colony selection, plasmids were extracted and the inserts were subjected to PCR using vector specific primers. Average insert size was determined by size idenfication on agarose gels stained with ethidium bromide. After purification via precipitation and denaturation, 192 cDNA probes were double-spotted onto a nylon membrane and bound to the membrane by UV cross linking. Amplified RNA (aRNA) probes from pools of three or single blastocysts were generated as described recently (Brambrink et al., 2002 BioTechniques, 33, 3–9) and hybridized to the membranes. Expression profiles of in vitro-produced blastocysts cultured in either SOF plus BSA or TCM plus serum were compared with those of diploid parthenogenetic ones generated by chemical activation. Thirty-three probes have been sequenced and, after comparison with public data bases, 26 were identified as cDNAs or genes. Twelve out of 192 (6%) seem to be differentially expressed within the three groups;; 7/12 (58%) were down-regulated, 3/12 (25%) were up-regulated in SOF-derived embryos, and 2/12 (20%) were up-regulated in parthenogenetic blastocysts compared to their in vitro-generated counterparts. Three of these genes involved in calcium signaling (calmodulin, calreticulin) and regulation of actin cytoskeleton (destrin) were validated by semi-quantitative RT-PCR (Wrenzycki et al., 2001 Biol. Reprod. 65, 309–317) employing poly(A+) RNA from a single blastocyst as starting material. No differences were detected in the relative abundance of the analysed gene transcripts within the different groups. These findings were confirmed employing the aRNA used for hybridization in RT-PCR and showed a good representativity of the selected transcripts. Results indicate that it is possible to construct a homologous cDNA array which could be used for gene expression profiling of bovine preimplantation embryos. Supported by the Deutsche Forschungsgemeinschaft (DFG Ni 256/18-1).

scholarly journals Effectiveness of Gene Expression Profiling for Response Prediction of Rectal Adenocarcinomas to Preoperative Chemoradiotherapy

2005 ◽  
Vol 23 (9) ◽  
pp. 1826-1838 ◽  
Author(s):  
B. Michael Ghadimi ◽  
Marian Grade ◽  
Michael J. Difilippantonio ◽  
Sudhir Varma ◽  
Richard Simon ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Response Prediction ◽  
Preoperative Chemoradiotherapy ◽  
Response To Therapy ◽  
Phase Iii ◽  
Correct Prediction

Purpose There is a wide spectrum of tumor responsiveness of rectal adenocarcinomas to preoperative chemoradiotherapy ranging from complete response to complete resistance. This study aimed to investigate whether parallel gene expression profiling of the primary tumor can contribute to stratification of patients into groups of responders or nonresponders. Patients and Methods Pretherapeutic biopsies from 30 locally advanced rectal carcinomas were analyzed for gene expression signatures using microarrays. All patients were participants of a phase III clinical trial (CAO/ARO/AIO-94, German Rectal Cancer Trial) and were randomized to receive a preoperative combined-modality therapy including fluorouracil and radiation. Class comparison was used to identify a set of genes that were differentially expressed between responders and nonresponders as measured by T level downsizing and histopathologic tumor regression grading. Results In an initial set of 23 patients, responders and nonresponders showed significantly different expression levels for 54 genes (P < .001). The ability to predict response to therapy using gene expression profiles was rigorously evaluated using leave-one-out cross-validation. Tumor behavior was correctly predicted in 83% of patients (P = .02). Sensitivity (correct prediction of response) was 78%, and specificity (correct prediction of nonresponse) was 86%, with a positive and negative predictive value of 78% and 86%, respectively. Conclusion Our results suggest that pretherapeutic gene expression profiling may assist in response prediction of rectal adenocarcinomas to preoperative chemoradiotherapy. The implementation of gene expression profiles for treatment stratification and clinical management of cancer patients requires validation in large, independent studies, which are now warranted.

Gene Expression Profiling Reveals Fundamental Differences between Leukemic Stem Cells (Lin-CD34+CD38−) and Mature Blasts (Lin−CD34+CD38+) of Acute Myeloid Leukaemia (AML) Patients.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1377-1377
Author(s):  
Kazem Zibara ◽  
Daniel Pearce ◽  
David Taussig ◽  
Spyros Skoulakis ◽  
Simon Tomlinson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Cell Populations ◽  
Leukemic Stem Cells ◽  
Significant Differential Expression ◽  
New Genes

Abstract The identification of LSC has important implications for future research as well as for the development of novel therapies. The phenotypic description of LSC now enables their purification and should facilitate the identification of genes that are preferentially expressed in these cells compared to normal HSC. However, gene-expression profiling is usually conducted on mononuclear cells of AML patients from either peripheral blood and/or bone marrow. These samples contain a mixture of blasts cells, normal hematopoietic cells and limited number of leukemic stem cells. Thus, this results in a composite profile that obscure differences between LSC and blasts cells with low proliferative potential. The aim of this study was to compare the gene expression profile of highly purified LSC versus leukemic blasts in order to identify genes that might have important roles in driving the leukemia. For this purpose, we analyzed the gene expression profiles of highly purified LSCs (Lin−CD34+CD38−) and more mature blast cells (Lin−CD34+CD38+) isolated from 7 adult AML patients. All samples were previously tested for the ability of the Lin−CD34+CD38− cells but not the Lin−CD34+CD38+ fraction to engraft using the non-obese diabetic/severe combined immuno-deficiency (NOD-SCID) repopulation assay. Affymetrix microarrays (U133A chip), containing 22,283 genes, were used for the analysis. Comparison of Lin-CD34+CD38- cell population to the Lin−CD34+CD38+ cell fraction showed 5421 genes to be expressed in both fractions. Comparative analysis of gene-expression profiles showed statistically significant differential expression of 133 genes between the 2 cell populations. Most of the genes were downregulated in the LSC-enriched fraction, compared to the more differentiated fraction. Gene ontology was used to determine the categories of the up-regulated transcripts. These transcripts, which are selectively expressed, include a number of known genes (e.g., receptors, signalling genes, proliferation and cell cycle genes and transcription factors). These genes play important roles in differentiation, self-renewal, migration and adhesion of HSCs. Among the genes showing the highest differences in expression levels were the following: ribonucleotide reductase M2 polypeptide, thymidylate synthetase, ZW10 interactor, cathepsin G, azurocidin 1, topoisomerase II, CDC20, nucleolar and spindle associated protein 1, Rac GTPase activating protein 1, leukocyte immunoglobulin-like receptor, proliferating cell nuclear antigen, myeloperoxidase, cyclin A1 (RRM2, TYMS, ZWINT, CTSG, AZU1, TOP2A, CDC20, NUSAP1, RACGAP1, LILRB2, PCNA, MPO, CCNA1). Some transcripts detected have not been implicated in HSC functions, and others have unknown function so far. This work identifies new genes that might play a role in leukemogenesis and cancer stem cells. It also leads to a better description and understanding of the molecular phenotypes of these 2 cell populations. Hence, in addition to being a more efficient way to further understand the biology of LSC, this should also provide a more efficient way of identifying new therapeutics and diagnostic targets.

Gene Expression Profiling of Paired Pre- and Post-Prednisolone (PRED) BM Samples from Childhood ALL Identifies Robust Signatures for PRED Response and Eventual Outcome.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 222-222 ◽  
Author(s):  
Yi Lu ◽  
Huiqing Liu ◽  
Ying Xu ◽  
Pei Lin Koh ◽  
Ariffin Hany ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Expression Profiles ◽  
Response To Therapy ◽  
Test Set ◽  
Childhood All ◽  
Blast Count ◽  
Highly Correlated ◽  
Eventual Outcome

Abstract Early response to therapy is the most important prognostic factor for childhood ALL. CCG investigators have shown that Day-7 and Day-14 BM blast counts were prognostically important although there is great inter-observer variability. BFM group have shown that day 8 prednisolone (PRED) response is highly predictive of the treatment outcome. While gene expression profiling (GEP) of diagnostic marrow can discern a pattern of PRED sensitivity as determined by in vitro MTT assay, the accuracy was low at only 70%. We hypothesized that changes in global GEP after therapy have a higher likelihood to predict response as the signatures of sensitivity and resistance may be unmasked during the therapy. We prospectively studied the changes in GEP using Affymetrix HG-U133A or Plus 2 chips on paired BM samples before and after 7-day course of PRED and one dose IT MTX in 58 patients with newly diagnosed or relapsed ALL. Unsupervised hierarchical clustering revealed that pre- and post- PRED samples in the patients still tended to cluster together, indicating that expression profiles of molecular subgroups were still most important. To remove intrinsic influence of molecular subtypes and identify potential signatures independent of genetic abnormalities, we subtracted Day-0 GEP from its paired Day-8 profile and retained probe sets with significant changes (≥ 10-fold). To avoid the ambiguity of variation in BM blast counting at Day-8, we divided the samples into a stringently reproducible group where “Good” PRED response was defined as that Day-8 blast count in PBL < 109/L and BM lymphoblasts ≤ 30% (n=16). “Poor” response was when Day 8 PBL ≥ 109/L (n=11). This stringently reproducible group (n=27) formed the training group to help define a distinct signature while the rest (n=31 pairs) were used as a blinded test set. 54 and 19 discriminating genes were identified by 2 independent statistical methods respectively, and an integrated predictor model was constructed based on shortlisted entries. This model predicted the PRED response with 100% accuracy for the training set using the leave-one-out cross validation but was less accurate in predicting the BM blast count in blinded test set. But intriguingly, in the blinded test set, this model predicted correctly 19 out of 21 reliable “Good” PRED responses are in CCR (91%), while among 8 predicted as “Poor” responses, only 2 are in CCR (25%). This suggests that as gene expression profiling as early as day 8 of PRED could discern the beginning of leukaemia cell death even before morphological changes are discernable and is highly correlated to eventual outcome. In conclusion, we have shown that analyses on the relative changes of gene expression profile can identify real genetic signatures indicating the sensitivity to PRED administration which is highly correlated with outcome.

Intraplatform Reproducibility and Technical Precision of Gene Expression Profiling in Four Laboratories Investigating 112 Leukemia Samples: The DACH Study.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2383-2383
Author(s):  
Alexander Kohlmann ◽  
Elisabeth Haschke-Becher ◽  
Barbara Wimmer ◽  
Ariana Huber-Wechselberger ◽  
Sandrine Meyer-Monard ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Mononuclear Cells ◽  
Expression Profiles ◽  
Control Group ◽  
Unsupervised Hierarchical Cluster ◽  
Daily Routine ◽  
Total Rna

Abstract Gene expression profiling has the potential to offer consistent objective diagnostic test results once a standardized protocol is established. We investigated the robustness, precision, and reproducibility of this technology and present data that complements the Microarray Innovations in LEukemia study (MILE study). In four laboratories, located in Germany (D), Austria (A), and Switzerland (CH) (DACH study), replicates of 112 patient samples were analyzed using the AmpliChip Leukemia research test. Patient samples were centrally collected and diagnosed in daily routine at the Munich Leukemia Laboratory and represented 8 distinct classes of acute and chronic leukemias, with non-leukemia as control group. After purification of the mononuclear cells by Ficoll density centrifugation, 4 × 5 million cells were frozen in lysis buffer and stored at −80°C. Equipped with identical instruments, software, and reagents, study operators were trained on the microarray sample preparation protocol using total RNA from commercially available cell lines. Upon receipt of the frozen lysates each of the four laboratories purified the total RNA from the 112 technical quadruplicates. 99.3% (445/448) of the sample preparations were successfully performed. On average, 8.4 μg, 7.2 μg, 7.4 μg, or 7.5 μg of total RNA, respectively, were isolated from the mononuclear cells from the four laboratories. In three samples less than 1.0 μg of total RNA was obtained and thus the preparation failed. Bland-Altman plots of agreement showed that any two centers were unlikely to have more than an 8.3 μg difference in yield of total RNA from the same sample. On average there was between 0.1 μg to 1.2 μg difference in total RNA yield from the same sample. Further processing of the 445 samples resulted in 437 (98.2%) successfully performed in vitro transcription reactions, i.e. obtained cRNA yield of &gt;8.0 μg. On average there was between 0.4 μg to 7.4 μg difference in cRNA yield from the same sample. After hybridization to microarrays on average, 46.1%, 48.6%, 46.5%, and 47.3% of probe sets were detected as present with mean scaling factors of 4.3, 2.9, 3.9, and 3.7, respectively. The mean values and standard deviations of distributions of the coefficient of variation (CV) within each site over all the probe sets of the quantile normalized signals on the chip were 27.2% (StdDev: 12.3%), 27.0% (StdDev: 12.3%), 27.3% (StdDev: 12.3%), 26.9% (StdDev: 12.4%), respectively. Furthermore, in unsupervised hierarchical cluster and principal component analyses replicates from the same patient always clustered closely together, with no indications of association between gene expression profiles due to different operators or laboratories. In conclusion, we demonstrated that microarray analysis can be performed with remarkably high inter-laboratory reproducibility and with comparable quality and high technical precision across laboratories.

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