Quantitative RT-PCR assay of HER2 mRNA expression in formalin-fixed and paraffin-embedded breast cancer tissues

Detection of human epidermal growth factor receptor 2 gene (HER2, also known as erbB2) expression is a preparatory process to decide a treatment strategy for breast cancer patients. 20-30% of breast cancer patients have HER2 overexpression, and they usually show poor recovery rate. For detection of HER2 expression, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) methods are conventionally used. Although these methods are accurate and reliable, their time-consuming process and high cost need a concise method with high sensitivity and accuracy. As a complementary method to the current IHC/FISH standard techniques, PCR-based methods have been developed. Here we employed a quantitative PCR method to detect HER2 expression in one hundred ninety nine formalin-fixed and paraffin-embedded (FFPE) breast cancer tissue samples from the patients treated over two years at the Yonsei University Severance Hospital, Republic of Korea. Relative expression of HER2 mRNA in the FFPE samples was analyzed using a quantitative RT-PCR (RT-qPCR) method and the obtained HER2 expression levels were compared with those from IHC/FISH methods. Our results show that the RT-qPCR method was highly concordant with IHC/FISH methods for detecting HER2 expression. Overall sensitivity and specificity of the BrightGen HER2 RT-qDx assay kit (Syantra, Calgary, Canada), which is a kit we used for RT-qPCR analyses, were 93.0% and 89.8% (P < 0.0001), respectively. The diagnostic cut-off value of HER2 RT-qDx for the clinical samples was determined by likelihood ratio, among which the highest likelihood ratio of relative HER2 mRNA levels was over 105.5 (AUC = 0.9466) with the highest sensitivity and specificity. Our study indicates that quantification of HER2 mRNA expression with the RT-qPCR could be an alternative method of conventional IHC/FISH methods.     

Formalin-fixed paraffin-embedded clinical tissues show spurious copy number changes in array-CGH profiles

Formalin-fixed paraffin-embedded (FFPE) archival clinical specimens are invaluable in discovery of prognostic and therapeutic targets for diseases such as cancer. However, the suitability of FFPE-derived genetic material for array-based comparative genomic hybridization (array-CGH) studies is underexplored. In this study, genetic profiles of matched FFPE and fresh-frozen specimens were examined to investigate DNA integrity differences between these sample types and determine the impact this may have on genetic profiles. Genomic DNA was extracted from three patient-matched FFPE and fresh-frozen clinical tissue samples. T47D breast cancer control cells were also grown in culture and processed to yield a fresh T47D sample, a fresh-frozen T47D sample and a FFPE T47D sample. DNA was extracted from all the samples; array-CGH conducted and genetic profiles of matched samples were then compared. A loss of high molecular weight DNA was observed in the FFPE clinical tissues and FFPE T47D samples. A dramatic increase in absolute number of genetic alterations was observed in all FFPE tissues relative to matched fresh-frozen counterparts. In future, alternative fixation and tissue-processing procedures, and/or new DNA extraction and CGH profiling protocols, may be implemented, enabling identification of changes involved in disease progression using stored clinical specimens.     

Evaluation of a quantitative RT-PCR assay to detect HER2 mRNA overexpression for diagnosis and selection of trastuzumab therapy in breast cancer tissue samples

Breast cancer patients who have a positive result for HER2 overexpression are commonly treated with Herceptin, a HER2-targeted therapy. In the present study, the BrightGen HER2 RT-qDx (Syantra, Calgary, Canada), which is based on a one-tube nested RT-qPCR method that detects HER2 mRNA overexpression, was clinically evaluated in a total of 237 formalin-fixed paraffin-embedded (FFPE) tissue samples from breast cancer patients. Among the 38 HER2 positive samples, which were determined via IHC/FISH methods, 13 samples out of 16 (81.3%) that were IHC2 +/FISH + and 22 samples out of 22 (100%) that were IHC3 + have been decided positive for HER2 expression via the RT-qPCR method. The true positivity and false positivity results for the RT-qPCR were 92% (35/38) and 2% (1/65), respectively. The concordance between RT-qPCR and IHC results and RT-qPCR and IHC/FISH was 87.2% and 92.1%, respectively. Conclusively, the BrightGen HER2 RT-qDx may be a reliable and convenient method that can supplement traditional IHC and FISH methods for efficient use of trastuzumab.     

Expression of thymidylate synthase in primary colorectal adenocarcinoma.

Thymidylate synthase (TS) is a key regulatory enzyme in the cellular pathway of de novo pyrimidine synthesis and a target enzyme for 5-fluorouracil (5-FU). Most clinical studies have shown that high levels of TS in tumors are associated with decreased sensitivity to 5-FU treatment. In this study TS expression was assessed at DNA, RNA, and protein levels. The study included 69 tumors from patients with primary colorectal adenocarcinoma. At the DNA level TS enhancer polymorphism was measured on whole blood by PCR. At the RNA level TS mRNA expression was measured on formalin-fixed paraffin-embedded tumor tissue and on fresh-frozen tumor tissue by real-time RT-PCR. Protein expression was assessed by IHC. Correlation was found between TS mRNA expression in fresh-frozen tumor tissue and formalin-fixed paraffin-embedded tissue (R=0.71). TS enhancer 3/3 had significantly higher protein levels as assessed by IHC than the TS enhancer 2/2 (P=0.02), although there was no statistically significant correlation between TS enhancer polymorphism and TS mRNA expression. An interesting observation not previously reported is that the predominant IHC reaction pattern in tumors from patients with the TS enhancer genotype 3/3 is different in tumors from patients with genotypes 2/2 and 2/3. The results indicate that clinical studies of the significance of TS with regard to 5-FU-based chemotherapy should be based on assessment of TS activity at DNA, RNA, and protein levels.     

A novel approach for reliable microarray analysis of microdissected tumor cells from formalin-fixed and paraffin-embedded colorectal cancer resection specimens

We present a novel approach for microarray analysis of RNA derived from microdissected cells of routinely formalin-fixed and paraffin-embedded (FFPE) cancer resection specimens. Subsequent to RNA sample preparation and hybridization to standard GeneChips (Affymetrix), RNA samples yielded 36.43 ± 9.60% (FFPE), 49.90 ± 4.43% (fresh-frozen), and 53.9% (cell line) present calls. Quality control parameters and Q-RT-PCR validation demonstrated reliability of results. Microarray datasets of FFPE samples were informative and comparable to those of fresh-frozen samples. A systematic measurement difference of differentially processed tissues was eliminated by a correction step for comparative unsupervised data analysis of fresh-frozen and FFPE samples. Within FFPE samples, unsupervised clustering analyses clearly distinguished between normal and malignant tissues as well as to further separate tumor samples according to histological World Health Organization (WHO) subtypes. In summary, our approach represents a major step towards integration of microarrays into retrospective studies and enables further investigation of the relevance of microarray analysis for clinico-pathological diagnostics. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

Evaluation of DNA and RNA quality from archival formalin‐fixed paraffin‐embedded tissue for next‐generation sequencing – Retrospective study in Japanese single institution

Genetic analysis on formalin‐fixed paraffin‐embedded (FFPE) tissue specimens has become a mainstream method, from conventional direct sequencing to comprehensive analysis using next‐generation sequencing (NGS). In this study, we evaluated the quality of DNA and RNA extracted from FFPE sections, derived from surgical specimens of different tumor types. Electrophoresis was performed using a 4200 TapeStation to evaluate DNA and RNA fragmentation. DNA Ct values were higher and significantly increased over a period of 4 years compared with those from cell lines or frozen tissues. The RNA integrity number equivalent (RIN) ranged from 1 to 4.1 and DV200 ranged from 7.3 to 81%. Twelve of the 108 cases were analyzed by NGS using the AmpliSeq Cancer HotSpot Panel v2 on a Miniseq system. A sufficient number of reads and coverage were obtained in all cases. Our results revealed that NGS analysis was sufficient for FFPE‐derived DNA within 4 years of preservation. Conversely, approximately 20% of the RNA derived from FFPE within 4 years from the collection could be inappropriate for gene analysis based on RIN and DV200. It was suggested that FFPE would be adequate for genetic analysis, although it is desirable to store frozen specimens for the tumor tissues to be subjected to genetic analysis.     

db/db小鼠肝脏中实时定量逆转录PCR内参照基因及标准化方法的评估与整合（英文）

目的:实时定量逆转录PCR(RT-q PCR)结果的标准化对于保证最终结果的准确性尤其重要。常用的标准化方法包括用加入的核酸量校正(ΔCt法)、用单个内参照基因校正(ΔΔCt法)和用统计学软件计算多个内参照基因的几何平均值进行校正。我们在db/db小鼠肝脏中对各种校正方法进行评估。方法:用ge Norm和NormFinder两种软件评估ACTB、e IF5、GAPDH、HMBS、HPRT1、Polr2A和RPLP0共7个内参照基因,以肝脏脂质合成相关基因Thrsp、SCD、SREBP1c和FAS作为目的基因。结果:应用ΔCt法,db/db小鼠肝脏中所有目的基因及GAPDH的表达显著升高(P0.05)。ge Norm计算认为ACTB和HMBS最稳定。Norm Finder计算认为ACTB最稳定,而GAPDH和RPLP0为最佳组合。以单个基因ACTB或RPLP0,以及ACTB与HMBS,或GAPDH与RPLP0的几何平均值进行校正,db/db小鼠肝脏中除SREBP1c以外,Thrsp、SCD1和FAS的表达均升高(P0.05)。结论:用ΔCt法校正RTq PCR的结果稳定并具有生物学意义。统计学软件ge Norm或Norm Finder应与ΔCt法整合使用。     

Genome-wide copy number alterations detection in fresh frozen and matched FFPE samples using SNP 6.0 arrays

SNP arrays offer the opportunity to get a genome-wide view on copy number alterations and are increasingly used in oncology. DNA from formalin-fixed paraffin-embedded material (FFPE) is partially degraded which limits the application of those technologies for retrospective studies. We present the use of Affymetrix GeneChip SNP6.0 for identification of copy number alterations in fresh frozen (FF) and matched FFPE samples. Fifteen pairs of adenocarcinomas with both frozen and FFPE embedded material were analyzed. We present an optimization of the sample preparation and show the importance of correcting the measured intensities for fragment length and GC-content when using FFPE samples. The absence of GC content correction results in a chromosome specific "wave pattern" which may lead to the misclassification of genomic regions as being altered. The highest concordance between FFPE and matched FF were found in samples with the highest call rates. Nineteen of the 23 high level amplifications (83%) seen using FF samples were also detected in the corresponding FFPE material. For limiting the rate of "false positive" alterations, we have chosen a conservative False Discovery Rate (FDR). We observed better results using SNP probes than CNV probes for copy number analysis of FFPE material. This is the first report on the detection of copy number alterations in FFPE samples using Affymetrix GeneChip SNP6.0.     

Multilayer spectrum of intratumoral heterogeneity in basal bladder cancer

Basal/squamous (Ba/Sq) subtype represents an intrinsic and robust group in the consensus molecular classification of muscle-invasive bladder cancer (MIBC), with poor outcome and controversial chemosensitivity. We aimed to investigate the spectrum of intratumor heterogeneity (ITH) in the Ba/Sq subtype. First, we validated a 29-gene NanoString CodeSet to predict the Ba/Sq subtype for FFPE samples. We identified heterogeneous Ba/Sq tumors in a series of 331 MIBC FFPE samples using dual GATA3/KRT5/6 immunohistochemistry (IHC). Heterogeneous regions with distinct immunostaining patterns were studied separately for gene expression using the 29-gene CodeSet, for mutations by targeted next-generation sequencing, and for copy number alteration (CNA) by microarray hybridization. Among 83 Ba/Sq tumors identified by GATA3/KRT5/6 dual staining, 19 tumors showed heterogeneity at the IHC level. In one third of the 19 cases, regions from the same tumor were classified in different distinct molecular subtypes. The mutational and CNA profiles confirmed the same clonal origin for IHC heterogeneous regions with possible subclonal evolution. Overall, two patterns of intratumoral heterogeneity (ITH) were observed in Ba/Sq tumors: low ITH (regions with distinct immunostaining, but common molecular subtype and shared CNA) or high ITH (regions with distinct immunostaining, molecular subtype, and CNA). These results showed multilayer heterogeneity in Ba/Sq MIBC. In view of personalized medicine, this heterogeneity adds complexity and should be taken into account for sampling procedures used for diagnosis and treatment choice. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

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

Formalin-fixed paraffin-embedded (FFPE) tissues are invaluable sources of biological material for research and diagnostic purposes. In this study, we aimed to identify biological and technical variability in RT-qPCR TaqMan® assays performed with FFPE-RNA from lymph nodes of classical Hodgkin lymphoma samples. An ANOVA-nested 6-level design was employed to evaluate BCL2, CASP3, IRF4, LYZ and STAT1 gene expression. The most variable genes were CASP3 (low expression) and LYZ (high expression). Total variability decreased after normalization for all genes, except by LYZ. Genes with moderate and low expression were identified and suffered more the effects of the technical manipulation than high-expression genes. Pre-amplification was shown to introduce significant technical variability, which was partially alleviated by lowering to a half the amount of input RNA. Ct and Cy₀ quantification methods, based on cycle-threshold and the kinetic of amplification curves, respectively, were compared. Cy₀ method resulted in higher quantification values, leading to the decrease of total variability in CASP3 and LYZ genes. The mean individual noise was 0.45 (0.31 to 0.61 SD), indicating a variation of gene expression over ~ 1.5 folds from one case to another. We showed that total variability in RT-qPCR from FFPE-RNA is not higher than that reported for fresh complex tissues, and identified gene-, and expression level-sources of biological and technical variability, which can allow better strategies for designing RT-qPCR assays from highly degraded and inhibited samples.     

Integrating Molecular Subclassification of Medulloblastomas into Routine Clinical Practice: A Simplified Approach

Medulloblastoma (MB) is composed of four molecular subgroups viz. WNT, SHH, groups 3 and 4, identified using various high‐throughput methods. Translation of this molecular data into pathologist‐friendly techniques that would be applicable in laboratories all over the world is a major challenge. Ninety‐two MBs were analyzed using a panel of 10 IHC markers, real‐time PCR for mRNA and miRNA expression, and FISH for MYC amplification. β‐catenin, GAB1 and YAP1 were the only IHC markers of utility in classification of MBs into three subgroups viz. WNT (9.8%), SHH (45.6%) and non‐WNT/SHH (44.6%). mRNA expression could further classify some non‐WNT/SHH tumors into groups 3 and 4. This, however, was dependent on integrity of RNA extracted from FFPE tissue. MYC amplification was seen in 20% of non‐WNT/SHH cases and was associated with the worst prognosis. For routine diagnostic practice, we recommend classification of MBs into three subgroups: WNT, SHH and non‐WNT/SHH, with supplementation by prognostic markers like MYC for non‐WNT/SHH tumors. Using this panel, we propose a new three‐tier risk stratification system for MBs. Molecular subgrouping with this limited panel is rapid, economical, works well on FFPE tissue and is reliable as it correlates significantly with clinicopathological parameters and patient survival.     

Fixation of Brain Tumor Biopsy Specimens with RCL2 Results in Well‐Preserved Histomorphology,Immunohistochemistry and Nucleic Acids

RCL2 is an alcohol‐based fixative reported to preserve histomorphology and nucleic acids in non‐CNS neoplasms. We compared histomorphology, immunohistochemistry, DNA and RNA in brain tumor specimens preserved frozen at −80°C, and after formalin or RCL2 fixation. RCL2‐fixed and paraffin‐embedded (RCLPE) samples showed well‐preserved histomorphology and specific immunoreactivity comparable to formalin‐fixed and paraffin‐embedded (FFPE) specimens testing a broad panel of antibodies. In all the analyzed cases, high‐molecular weight DNA (up to a fragment length of 600 bp) was amplifyable from RCLPE samples, even after prolonged fixation times. Beta‐actin (ACTB) and O6‐methylguanine‐methyltransferase (MGMT) gene concentrations were significantly higher in DNA isolated from RCLPE specimens as compared with FFPE specimens. Testing of MGMT promoter methylation status using methylation‐specific polymerase‐chain reaction (MSP) yielded conclusive results in 8/8 analyses in RCLPE and 6/8 analyses in FFPE material, respectively. Amplification of three reference genes (ABL, RAR‐alpha, BCR‐1) from cDNA showed good RNA preservation in frozen and RCLPE tissue specimens and significant RNA degradation in all FFPE samples. In conclusion, RCL2 fixation of brain tumor biopsies does not seem to significantly compromise histological tumor typing or immunohistochemistry and preserves nucleic acids (DNA and RNA) at a better quality than formalin fixation.     

Comparison of HER2 status between surgically resected specimens and matched biopsy specimens of gastric intestinal-type adenocarcinoma

HER2 protein overexpression and gene amplification are important biomarkers for identifying gastric cancer patients who may respond to HER2-targeted therapy using trastuzumab. The aim of this study was to evaluate the concordance between HER2 protein expression and gene amplification in both surgically resected tumors and matched biopsy specimens of gastric cancer. Formalin-fixed, paraffin-embedded sections of 207 surgically resected tumors and 158 biopsy specimens from 207 cases of invasive intestinal-type gastric cancer were analyzed. Protein expression was assessed using immunohistochemistry and graded by the modified scoring criteria for gastric cancer. Gene amplification was evaluated by fluorescence in situ hybridization (FISH). HER2 overexpression was observed in 17 % of both surgically resected tumors (35/207) and biopsy specimens (26/158). HER2 gene amplification was detected in 31 % (61/200) of surgically resected tumors and 32 % (47/147) of biopsy specimens. Except for immunohistochemistry (IHC) equivocal (2+) cases, the concordance rates between IHC and FISH was 90.9 % in surgically resected tumors and 90.2 % in biopsy specimens. In IHC 2+ cases, the rate of HER2 gene amplification was 56 and 38 % in surgically resected tumors and biopsy specimens, respectively. IHC-FISH discordance was mainly due to intratumoral heterogeneity and low-level gene amplification. The concordance rate of IHC results between surgically resected specimens and the corresponding biopsy specimen was 57.0 % (κ?=?0.224), and in discordant cases, HER2 positivity in biopsies and HER2 negativity in surgically resected tumors were most common. The concordance rate of FISH results between surgically resected tumors and biopsy specimens was 72.7 % (κ?=?0.313). Polysomy 17 was detected in 5.5 and 7.5 % of surgically resected tumors and biopsy specimens and significantly correlated with IHC score, but polysomy 17 could explain one IHC score 3+ and FISH-negative tumor only. Although high concordance rates between HER2-protein expression and gene amplification were observed in both surgically resected tumors and biopsy specimens, the agreement levels were evaluated to be fair. Polysomy 17 was infrequent and seemed to have limited impact on gastric HER2 testing. Further investigations are required for an appropriate biopsy method to reduce false results of HER2 testing and to clarify the clinical significance of intratumoral heterogeneity in HER2 status.     

Sample parameters affecting the clinical relevance of RNA biomarkers in translational breast cancer research

In the frame of translational breast cancer research, eligibility criteria for formalin-fixed paraffin-embedded tissue (FFPE) material processing for gene expression studies include tumor cell content (TCC) and sample site (primary vs metastatic tumors). Herein we asked whether the observed differences in gene expression between paired samples with respect to TCC and sample site also have different clinical significance. We assessed ESR1, ERBB2, MAPT, MMP7, and RACGAP1 mRNA expression with real time PCR in paired samples before (NMD) and after macrodissection (MD) from 98 primary tumors (P_MD, P_NMD) and 72 metastatic lymph nodes (LN_MD, LN_NMD), as well as from 93 matched P (mP) and LN (mLN). TCC range was 2.5–75 % in the NMD series and 28–98 % in the MD and in the mP/mLN series. The prognostic effect of these markers, individually or in clusters, remained stable between paired P_MD/NMD. In comparison, cluster classification failed in the LN_NMD group with lower TCC. In the mP/mLN cohort, RACGAP1 mRNA expression was of prognostic significance when tested in mLN samples (p?<?0.001). Similarly, luminal B, HER2, and triple negative tumors were of dismal prognosis when classified in the LN component of the same series (mLN, overall survival: p?=?0.013, p?=?0.034, and p?=?0.007, respectively). In conclusion, the clinical relevance of the RNA markers examined may be affected by TCC in metastatic LN samples but not in primary tumors, while it differs between primary tumors and matched metastases. These data will facilitate the design of translational studies involving FFPE sample series.     

Stability of oestrogen and progesterone receptor antigenicity in formalin‐fixed paraffin‐embedded breast cancer tissue over time

Use of archived formalin‐fixed paraffin‐embedded (FFPE) tissue is a standard method for evaluation of proposed prognostic and predictive tumour markers. However, little is known of the preservation of biomarker expression in old FFPE tumour blocks. We investigate the quality of immunohistochemical (IHC) oestrogen (ER) and progesterone receptor (PR) evaluation in FFPE tissue over time (1978–2000) using a large breast cancer tissue microarray (N = 573) with access to receptor analyses in cytosol (CYT) at diagnosis, coexpression of other biomarkers and follow‐up data. We found a good correlation between ER analysed with CYT at diagnosis and ER analysed with IHC in archived FFPE tissue from the same tumour. ER evaluation did not seem to be affected by tissue storage time. Nor was there any time‐dependent difference in ER_IHC correlation with other biomarkers (HER2, Ki67) or survival. Discordant cases were more often classified as ER‐positive with IHC than with CYT. For PR, however, we found an increased correlation between methods in more recent time periods. This may possibly be explained by more reliable PR_IHC results in newer samples, although other explanations may also contribute. Our results indicate stable ER expression in FFPE tissue archived for up to 40 years.