Clinical relevance of DNA microarray analyses using archival formalin-fixed paraffin-embedded breast cancer specimens

Gene profiling is beginning to have an impact on personalized breast cancer care [1, 2]. Gene expression profiling of breast cancers using DNA microarray technology is able to classify breast tumors into distinct biological subgroups and has been shown to predict treatment response and prognosis in several studies [3–9]. This high-throughput molecular technique requires fresh bio-specimens to allow extraction of RNA of sufficient quantity and quality for analysis. There are however limitations to the collection of fresh samples for prospective studies including time-sensitive tissue processing, lengthy patient accrual and follow-up; and bio-banks are not always readily available as a source of fresh frozen samples. Also, a requirement for fresh tissue to be used inevitably leads to a bias towards only larger tumors being studied. To overcome some of these critical shortcomings of prospective studies, the use of archival formalin-fixed paraffin-embedded (FFPE) samples offers a potential solution as most hospitals worldwide have collections of FFPE tumor specimens dating back many years. FFPE is the most widely used standard of practice for tissue fixation for the purpose of diagnostic histology and long-term storage.

The FFPE tissue preserving process was developed long before molecular biologists were concerned with the preservation of RNA. FFPE samples have not been considered a reliable source of RNA due to the tissue processing-associated degradation and chemical modifications of RNA. Formalin fixation creates cross-linking between nucleic acids and proteins and adds mono-methylol to amino groups on all four RNA bases [10]. Thus, a number of recent studies have started to look into the prospect of overcoming the RNA quality issues in FFPE specimens. Several studies used modification of standard RNA extraction methods to generate RNA of sufficient quality and quantity for DNA microarray analysis [11–14]. Some innovations in DNA microarray techniques were also reported [15, 16]. A major breakthrough has been a new microarray technique developed by Illumina Inc. (San Diego, Ca), which involves cDNA-mediated annealing, selection, extension, and ligation (DASL), as well as random priming for detection of degraded RNA from FFPE samples [16–22]. This DNA microarray technique improved detection of fragmented RNA compared with conventional techniques. If the technique for gene profiling using FFPE specimens becomes sufficiently reliable, we anticipate that prognostic and predictive gene signatures can be identified using the vast available libraries of archival FFPE specimens with long-term treatment outcomes. In addition, any biomarker developed from FFPE samples could be more readily translated into clinical practice.

In this study, we aimed 1) to explore the feasibility of obtaining reliable microarray data from archival FFPE samples; 2) to compare gene expression profiles of FFPE samples with those of matched samples obtained from the same patients by FNAB; 3) to test the reproducibility of such experiments using quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR); and 4) to correlate the gene profiling of FFPE specimens with clinical outcome data using published microarray data sets.

Since Rupp and Locker [26] reported their first successful RNA extraction from FFPE specimens in 1988, significant strides have been made to enable RNA profiling from FFPE tissues, including efforts to standardize tissue handling and fixation procedures and improving RNA extraction methodologies [27, 28]. Very few technologies have emerged despite these advancements that are capable of whole transcriptome profiling from archived FFPE material [24, 29, 30]. Initial attempts at DNA microarray analyses using FFPE samples yielded poor reproducibility [31], or loss of detection of gene signatures when compared with matched fresh samples [32]. Several commercial and academic endeavours have recently become more successful in their ability to retrieve meaningful biological information from degraded FFPE-derived RNAs [15, 21, 22, 24, 33–36].

The DASL assay incorporates random priming during cDNA synthesis, and therefore does not depend solely on the polyA/oligo-dT based priming process used in conventional DNA microarray methods. In addition, the assay requires a relatively short target sequence of about 50 nucleotides to query oligonucleotide annealing; which improved its ability to quantify fragmented RNA species [37]. Using these technologies, we evaluated the feasibility of using FFPE samples for DNA microarray analyses. Initially, as a pilot study, RNA from FFPE blocks of human mammary reduction mammoplasty tissues were extracted and used to generate DNA microarray data using DASL assay and Illumina HumanRef8 BeadChip, a genome-wide gene panel that contained over 24 thousands genes. Within eight replicates of the same type of specimens, the average correlation coefficient of array expression signals was very high at 0.96 ± 0.02 which showed an impressive technological reproducibility. This is similar to a recent study by Ton et al who reported a high correlation (r = 0.98) among 12 technical replicates by using the DASL technology [21].

We carried out our current study using human breast cancer specimens to test the feasibility of performing such experiments with a future plan to extend the technology to a large clinical library of breast cancers. Using RNA extraction methods described recently [23], we found that over 80% of the RNA extracted from archival FFPE tumor samples could be used for subsequent DASL analyses and produced DNA microarray data that were informative and could be validated and compared to that obtained from fresh FNAB specimens collected from the same patients.

In this study, we demonstrated a high reproducibility of the DNA microarray data when comparing the array signals within the FNAB or FFPE tumor specimens (r = 0.87). The comparison between the two specimen types yielded lower correlation coefficients (r = 0.45), which was likely due to dominant effects on the RNA related to the tissue processing for FFPE specimens and the different hybridization techniques in DNA microarray protocols (Figure 1C). Nevertheless, some clinically relevant microarray data remained reproducible as illustrated by a very strong correlation between the microarray expression levels of ESR1 and ERBB2 to the clinical status of ER and Her2 respectively in both FFPE and FNAB samples, which were also validated with RT-qPCR in FFPE specimens. This result was consistent with two recent studies by Ton et al [21] and Mulligan et al [22]. The gene profiling of FFPE samples suffered from a higher ratio of noise-to-signal (Figure 1B) and thus detected a smaller number of differentially expressed genes compared to FNAB samples (258 vs. 485). Therefore, we anticipate that the identification of subtle changes in expression levels in FFPE samples will remain challenging using current technologies.

To date, high-throughput gene expression profiling has demonstrated the potential uses of gene profiling as molecular subtype classifiers [6], prognostic indicators [7, 8], and treatment predictors [9] by using fresh breast cancer specimens. If gene profiling becomes a standard part of the pathological assessment of a tumor in the future, the use of FFPE material would be advantageous, as it obviates the need for fresh bio-specimens that can be very time sensitive and often impractical to collect in routine clinical settings. In certain clinical situations, including recurrent diseases and evaluation of long-term endocrine therapy, when there is no fresh bio-specimen from the original primary tumors; gene profiling using FFPE may provide an alternative to allow evaluation of the prognostic or predictive gene profiles.

The use of FFPE samples for gene profiling studies has been considered suboptimal in the past due to concerns about RNA degradation and less than 5% of all microarray studies to date have been reported using archival tumor tissues that were formalin-fixed and paraffin-embedded. Very few microarray studies have been conducted to investigate the gene profiling of FFPE samples and compared the results with their matched fresh frozen tissue. Some data is available in carcinomas of colon, liver and breast [4, 5, 15, 35]. In breast cancer, Da Silva et al [20] used FFPE samples from invasive lobular carcinoma only to check the expression profile of E-cadherin. Ravo et al. [17] used RNA from cultured cell lines, cryopreserved tumors and FFPE samples from breast cancers. Waddell et al. [19] and Bibikova et al. [16] included both fresh frozen and FFPE samples in their studies to compare the gene profiling of breast cancers in both type of tissues. However, their studies focused only on the technical aspect without any clinical correlations. We have correlated our gene profiling results to clinical receptor status, and demonstrated the ability to use gene expression profiling from FFPE to differentiate tumors from 4 distinct tumor subtypes. We identified a set of 38 genes that were reproducibly able to identify the different clinical groups in both FFPE and FNAB specimens. These findings suggested that there are subsets of genes in FFPE specimens that could maintain their clinical relevance despite the tissue processing effects. Interestingly, subsets of these genes were correlated to clinical outcomes as demonstrated in our study using publicly available microarray validation databases.

Although we are the first to show that the over-expression of ANLN and KIF2C, and the under-expression of MAPT predict for poor survival in patients with breast cancer (Figures 4 and 5), there is some evidence that supports the correlation of these three genes with prognosis and carcinogenesis in other cancers, and treatment in breast cancers. The over-expression of ANLN has been reported to be a biomarker for pancreatic carcinoma [38], and predicted for poor survival in early lung cancers [39]. Shimo et al reported that the over-expression of KIF2C might be involved in breast carcinogenesis and is a therapeutic target for breast cancers [40]. The expression of MATP has been correlated to the sensitivity to chemotherapies in breast cancer [41, 42]. These results strongly support that ANLN, KIF2C, and MAPT could play a role in the carcinogenesis, treatment and prognosis of breast cancers.

FFPE samples are widely available and can be linked to clinical outcome information, often available through institutional or provincial cancer registries. The availability of large libraries of archival FFPE tissue samples could potentially solve some of the most critical challenges that investigators face when using prospectively collected specimens, namely, patient recruitment and expensive long-term follow-up. Validation studies can also be done fairly quickly by selecting an independent patient population annotated with critical long-term clinical outcome data. Once the technology for gene profiling using FFPE matures, it will likely play an important role in the clinical management of breast cancer patients.

There is tremendous potential in using FFPE specimens for gene profiling, especially in breast cancer, in which there are existing data to support the prognostic and predictive implications of tumor gene profiles. Based on the comparison of gene profiling on FFPE breast cancer specimens and matched fresh specimens, we can conclude that important clinically relevant information can be identified using FFPE specimens and we even demonstrated the potential for using the microarray data to be used as a prognostic tool. Further improvements in current DNA microarray technologies will likely to bring gene profiling of breast cancer into routine clinical practice as we move towards personalized breast cancer care.